UNIVERSITY OF IBADAN LIBRARY * • . X *'*-' INTERACTION OF PROSTAGLANDIN E (PGE ) WITH NORADRENALINE AND ITS ANTAGONISTS IN THE ISOLATED MESENTERIC ARTERY OE RAT BY AYOTUNDE SAMUEL CKE ADEAGBO BSc. (EONS.) IBADAN A THESIS IN THE DEPARTMENT OF PHARMACOLOGY AND THERAPEUTICS SUBMITTED TO THE] FACULTY OF MEDICINE IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE CF DOCTOR OF PHILOSOPHY OF TIIB UNIVERSITY OF IBADAN, IBADAN, NIGERIA. JULY 1980 UNIVERSITY OF IBADAN LIBRARY - P D E D I C A T I O N This thesis is dedicated to Temitayo Omoteleola whom God has given as consolation for valuable research period lost during transition to nayrital status. UNIVERSITY OF IBADAN LIBRARY C O N T E N T S PACES CERTIFICATION ACKNOWLEDGEMENTS ii ABSTRACT iv INTRO TITCTICN VASCULAR SMOOTH MUSCLE ___ 1 Excitation - contraction coupling ... 2 Evidence for the involvement of Ca 2+ ... in the E - C coupling .... 4 Forms of activator Ca 2+ .... ... 7 Sites of cellular Ca 2+ .... ... 9 Electrical and non-electrical activation 12 ANTAGONISTS OF NORADRENALINE-INDUCED E - C COUPLING ___ ___ ___ 16 2 - substituted imidazolines ,... 16 Yohimbine .... .... •.•. 17 Phenoxybenzamine .... .... .... 19 Prazosin .... .... .. •« 23 Cinnarizine and Verapamil .... .... 23 Indomethacin and sodium meclofenamate.... 25 SUPERSENSITIVITY TO SYMPATHOMIMETIC AMINES. ___ ___ ___ ___ 27 Mechanisms of supersensitivity .... 37 Supersensitivity and metabolizing enzymes 38 UNIVERSITY OF IBADAN LIBRARY c CONTENTS (CONTD.) PAGES Supersensitivity and NA content .... 41 Deviation of transmitter substance from its site of loss to the receptors .... 44 Mechanisms involving the post sjmaptic sites 50 DIVALENT CATION IONOPHORE A23187 .... ___ 56 PROSTAGLANDINS ... ............. 59 Formation .... .... .... 59 Sites and mode of release .... .... 59 Prostacyclin (PG^) •••• •••• 63 Smooth muscle actions of PGS’s and P G ^ 64- AIM AND SCOPE OF PRESENT WORK ___ ___ 68 CHAPTER 2 MATERIALS AND METHODS ___ ___ ___ 70 Animals .... .... .... 70 Physiological solutions used .... 70 (a) Krebs solution .... .... 70 (b) Calcium-free Krebs solution .... 70 (c) Depolarizing Krebs solution .... 70 (d) Tyrode solution (mMol/litre) .... 71 Preparation of mesenteric artery for perfusion 71 Experimental procedure .... .... 73 Ascertaining the potency of prostacyclin (p0Io) 74 UNIVERSITY OF IBADAN LIBRARY CONTENTS (CCNTD. ) PAGES Metabolism of prostaglandins in rat mesenteric and guinea-pig pulmonary vasculature .... .... .... ... 75 (a) Inactivation of prostaglandins on perfusion through the rat mesenteric artery ... 75 00 Inactivation of prostaglandins in the pulmonary, vascular bed of the guinea-pig lung 76 Investigations on a possible influence of endogenous NA on prostaglandin action .... .... ... ; 77 Effect of prostaglandins on NA uptake .... ... 77 Measurement of the degree of potentiation ... 78 Determination of the degree of antagonism ... 79 Mesenteric artery preparation as a model for studies on sources of Ca 2+ used during vascular muscle contractions .... .... .... ... 80 Drugs used, sources and methods of preparation ... 81 Statistical analysis .... .... ... 82 CHAPTER 5 RESULTS Vasoconstrictor responses of the rat mesenteric arteiy to HA in normal krebs .... .... 83 Interactions of exogenously administered PGE0, and PGI2 with NA 83 Anti-aggregatory action of PGI? 87 UNIVERSITY OF IBADAN LIBRARY CONTENTS ( CONTI).) PAGES Inactivation of prostaglandins in rat mesenteric vascular bed .... .... .... .... 87 Interactions of prostaglandins with cocaine and methoxamine .... .... .... .... 90 Interactions of prostaglandins vrith HA in reserpinized rats and in Krebs solution containing bretylium ;... .... .... .... 92 HA antagonism by phentolamine, tolazoline, yohimbine and phenoxybenzamine (PB2) .... 95 Effects of PGS^ on <*.- adrenoceptor blockade .... 97 Interactions of indomethacin and prazosin with HA and the effect of PGE^ on ths HA block produced 106 Effect of PGE^ on NA block produced by cinnarizine and verapamil .... .... .... .... 111 Calcium ionophore A23187 influence on the basal perfusion pressure of the rat mesentery .... 111 Interaction of A25187 with NA and methoxamine 114 Influence of A23187 on adrenoceptor block by phentolamine, tolazoline and yohimbine .... 114 Interaction of A2J187 with NA in reserpinized rats 118 Effect of c<- adrenoceptor antagonists, prostaglan­ din synthetase inhibitors (pGSIs), and Ca antagonists on A23187 - induced contractions of the rat 118 UNIVERSITY OF IBADAN LIBRARY CONTENTS (CONTD.) PAGES Vasocontrictor responses of the rat mesentery to potassium chloride .... .... .... 120 Effect of E - C coupling antagonists on K+~ induced vasoconstriction .... .... .... 123 Effect of PGE^ on K4- induced vasoconstriction 123 Effect of external Ca2+ (a) on the vasoconstriction induced by NA and K+ 127 (h) on the potentiation of NA by PGE„, PC? and Ca2r ionophore, A23187 .... .... 127 (c) on NA - blockade by o4- adrenoceptor antagonists and on reversal by PSE2 •••• 131 Effect of variation in external Ca^+ on antagonism by indomethacin and prazosin .... .... 137 Effect of cinnarizine and verapamil on PGE^- induced potentiation of NA .... .... .... 139 Blockade by indomethacin, of Ca 2+- induced vasoconstriction in the mesenteric artery perfused with Ca 2+— free* depolarizing Knots solution # .. » 139 2-j. Effect of Ca on the mesenteric artery perfused with Ca 2+ containing depolarizing solution.... 141 CHAPTER 4 DISCUSSION ___ ___ ___ .... 145 Mechanism of prostaglandin- induced potentiation of NA 146 UNIVERSITY OF IBADAN LIBRARY CONTESTS (CONTQ.) PACES The role of external Ca^+ .... .... .... 150 Effect of high K+ ___ .... --- 152 Mode of action of the Ca 2+’ ionophore A2J187 .... 154 Types and mechanisms of blockade of vasoconstric­ tion in rat mesentery .... .... • •. • 157 Action of prazosin .... .... .... 161 Cinnarizine and verapamil .... .... .... 165 Mechanism of prostaglandin reversal of NA antagonism .... .... .... . ... 1 64 CHAPTER 5 SUMMARY AMD CONCLUSION --- --- --- 169 REFERENCES ___ ___ ___ ___ 172 UNIVERSITY OF IBADAN LIBRARY C BRTIFICATIOU PAGE I certify that this work was carried out "by Mr. A.3.0. Adeagbo in the Department of Pharmacology and Therapeutics, University of Ibadan, Nigeria. "tyfrUlL SUPERVISOR), D.T. CKPAKO, B.Pham. (Lond.), Ph.D. (Brad.) II.I. Biol Professor and Head of Department of Pharmacology and Therapeutics, University of Ibadan, Ibadan, UNIVERSITY OF IBADAN LIBRARY - ii - A ' c n o n E K S M i n I wi3h to express my deepest gratitude to my supervisor* Professor D.T. Okpako who is also the head of department, for his great interest, active support, and many invaluable and inspiring discussions during the whole course of the present study. I must confess that the Professor was exceptionally open- minded and on all olccasions, gave me full opportunity and encourage- ment to test n$r ideas in the laboratory. I further wish to thank my c\ olleagues especially Dr S.O. Fagbemi, Kiss T.O.C. Sokunbi and '‘Messrs S.'S. Chia and L. Adenekan whose vigorous pursuit of their various research programmes was a beacon and a constant source of inspiration for greater aspiration throughout the studies. Doctors F.M. Tayo, and K.A. Oriowo will for long be remembered for their crititism which has helped towards a 3peedy and precise final presentation of thi3 work. I am also veiy grateful to Messrs Luke Odueze, Yisau Mohammed, P.0. Akinniyi and Mrs P.0. Ajayi for their technical assistance at various stages of the study. Professor S.M. Essien of Haematology Department and his laboratory technologists are gratefully acknowledged for making all the necessary facilities available in his laboratory for the prostacyclin woife. I also extend my gratitude to the Rockefeller Foundation, U.3.A. for the award of a research fellowship during the period of my training. UNIVERSITY OF IBADAN LIBRARY / - iii - I am gratefully indebted to my uncle, Professor 3.A. Agboola whose sober and disciplined moral and academic life has been a very inspiring model for me. I am further indebted to Mrs V.I. -agade for the generosity and goodwill extended to me by the free access to the xeroxing machine in her office. I am no less grateful to my parents, Abosede and Ojo Adeagbo who have always (since my youth) expressed their wish to see me through the citadel of learning. I have now come to I r'ealise that the Ph.D* in view is actually not a ci! tadel i" n itself but a mere f*oundation for it. To my beloved friends* Messrs Biodun Olatunbode, Dapo Eplawole, Niyi Kolade, Tunde Awofisoye and Dr Olusola Oni; your words of encouragement in times of difficulty and your time-to-time unflinching willingness to assist me in achieving my aim(s) is by no means a small factor to the overall success of the study. Messrs J.A. Ogunleye and J.O. Osasona are gratefully acknowledged for their co-operation during the typing of this thesis. May God bless you all. Ayotunde 3.0. Adeagbo ' July 1980 UNIVERSITY OF IBADAN LIBRARY ABSTRACT The effect of PGE^, P5P?a and PGI0 on tilu lion induced by different mechanisms was studied in the isolated rat mesenteric arteiy as described by McGregor (1965) Vasoconstriction was induced by mechanisms involving dii modes of calcium utilization viz: (i) Pharmaccnechanical pathway by low doses of the adrenergic neurotransm.itter, noradrenaline acting at - receptor; (ii) electromechanical pathway by 1.1 b potassium-and” (iip.) agents which facilitate Ca influx e.g. A23187. ! The prostaglandins potentiated the vasoconstrictor effect of NA. Potentiation factors calculated from different doses of the prostaglandins showed the effects of the prostaglandins to be dose - dependent and FGE^ to be significantly more potent (P>0.005) than PGE,,a and PGI^* The prostaglandins failed to potentiate high potassium - induced vasoconstriction. PChi^ also failed to potentiate NA if the vasoconstrictor effects were evoked in Ca~ - free Krebs solution; but the degree -of potentia tion increased with increase in the concentration of Ca 2+ ions in the perfusion fluid. This result suggested strongly that the potentiation was associated with external calcium. Evidence is presented to show that potentiation was not prejunctional since cocaine, bretylium and reserpine pretreatment did not materially alter the effect of It was concluded that prostaglandins potentiated HA vasoconstriction by facilitating Ca influx. UNIVERSITY O IBADAN LIBRARY - XX - The mechanism 'of this facilitation is discussed. ITA vasoconstriction was competitively antagonised by adrenoceptor antagonists-phentolamine, tolazoline, yohimt• and phenoxybenzamine (in low concentrations). The blockade caused by these antagonists was reversed by PGE?. By comparing NA dose-ratios in the presence of antagonist with dose-ratios in the nresence of antagonists plus different doses of PGB^0, I showed 1hat the degree of reversal was related to the dose of PGE. For example, the NA dose - ratio for yohimbine (1.28 x ICT^I) \ o was reduced from 26.6 + 0.9 to ^.7 + 0.1 when PGE^ (2.8 x 10 M) was included in the perfusion fluid with the antagonist. The reveio_< of antagonism was not due to a change in the binding characteristics of the a j - adrenoceptor since pA^ values for the antagonist were not significantly different (P^.0.05) when PGS was included with the antagonists. Evidence is presented which suggests that reversal of antagonism involved utilization of internally bound calcium since reversal of antagonism occured even after the omission of Ca 2+ from the external medium. In this sense, the mechanism of reversal was different from that of potentiation. Furthermore, the degree of reversal (measured as-reversal factor) was quantitatively greater than would be the nans- if reversal was simply a reflection of the e n h g r e s p o n s i v e n e s s of the vascular muscle to NA. UNIVERSITY OF IBADAN LIBRARY iii - In contrast to the "competitive” - adrenoceptor antagonists, did not reverse the block of ITA vasoconstriction caused by phenoxybenzanine (high doses); verapamil, cinnarizine or prazosin. All these agents caused blockade of NA that was not competitive in'nature. Since noneof the competitive & Beth, (1973); the initial fast response following HA administration t 2-l appears to be caused by Ca of intracellular origin, and a i maintained slow response ajjpears to be caused by Ca ?+ from the fi in some manner dependent upon the presence of an adequate concentration of ionised intracellular Ca ?+ . This ionized Ca 2 i + is derived from the tissue bound sites which is maintained by the extracellular Ca 2+, '2+ Sites of cellular Ca { There are three possible storage sites for cellular calcium?- sarcoplasmic reticulum, mitochondria, and plasma membrane with its surface vesicles, In recent" years three types of studies have furnished evidence about these possible sources of activator Ca : (1) electron microscope studies, (2) cell fragment studies, Or and (3) studies employing Ca - blocking agents. Devine, Somlyo and Somlyo (1972), have demonstrated, by electron microscopic* studies, that the sarcoplasmic reticulum occupies an appreciable part of the vascular smooth muscle cell, ranging from over 3/1 of the volume in the aorta and the main pulmonary artery to approximately j 2/C in the portal, anterior mesenteric vein and the mesenteric artery. i These authors made the interesting corollary observation that UNIVERSITY OF IBADAN LIBRARY cxiootli ixuscles conciining tile larger amounts ox Gc.rcoplo.ai.ixc roticu maintain their contractions better in a Ga 2+ - free en' vi, ron­ ment than do muscles with relatively little intracellular sarcoplasmic reticulum. They showed that the extracellular markers, ferritin and lanthanum, do not enter the sarcoplasmic reticulum but that the sarcoplasmic reticulum does accumulate the bivalent cation marker, strontium. These authors also observed that the mitochondria of the vascular smooth muscle cell accumulate barium and strontium, and, therefore, are possible sequestration sites' for the bivalent cation calcium. Both the sarcoplasmic reticulum and the mitochondria make close contact with the plasma membrane and the surface vesicles. The latter are differentiated from the sarcoplasmic reticulum by the fact that they are open to the extracellular markers. The mitochondria and the microsomal fractions fxca the vascular smooth muscle have been shown to be capable of energy dependent sequestration of Ca^+ (Fitzpatrick, Landon, Debbas & Hurwitz, 1972; Baudouin, Keyer, Fermandjian & Morgat, 1972). Although, details of the control of activator Ca^+ is still obscure nevertheless, the studies of the uptake and the release of Ca 2+ from cell fragments promises valuable insight, since, for instance, angiotensin in physiologically active concentrations accelerates 'the release of bound Ca^+ from the microsomal fraction (Baudouin e_t al, 1972)* UNIVERSITY OF IBADAN LIBRARY - 11 Agents which effectively decrease membrane permeability to Ca 2+ have been valuable tools for studying the source of activator Ca 2+. At least four such agents have been-extensively studied: cinnarizine (Godfraird& Kaba, • 1969), 3KF 525A (Kalsner, Nickerson & Boyd, 1970), verapamil (Haousler, 1972; Peiper, ■> z Nende, 1971; Golenhofen & Lammel, 1972); and lanthanum (van Breemen et al, 1972;. Yar-"ilar smooth muscle incubated xn a Ca 2+ -,free potassium sulphate depolarizing solution can be j caused to contract by the addition of low calcium concentrations to the muscle bath. This response presumably occurs because the added Ca 2+ passes through the\' membrane which has been made highly permeable by the potassium sulphate - induced depolarization. Various 0a ‘ blocking agents eliminate or greatly reduce this contractile response. These agents are much less effective in eliminatin'- the response produced by agonists such as adrenaline,, noradrenaline and angiotensin, suggesting that the contractile responses initiated by these agonists are less dependent on the passage of extracellular Ca^+ through the plasma membrane than is the contractile response initiated by potassium sulphate - induced depolarization. .Van Breemen e_t _al (1972) demonstrated that lanthanum blocks the passage of Ca 2+ across the cell membrane of vascu lar smooth muscle and that it replaces all Ca^+ bound to extracellular UNIVERSITY OF IBADAN LIBRARY - 12 - structures. Ey measuring the Ga + remaining in segments of O t rabbit aorta, these researchers estimated intracellular C a^' content. Using this technique, they observed that there were parallel rates of increase in tension and in intracellular Ca + content in response to activation by potassium sulphate - induced depolarization or by lithium substitution for sodium. However, HA (10 ?M) caused a maximum increase in tension with no increase in li. ntracellular Ga 2+ concentration, which again indicates that the activator Ca^ involved in contraction in response to alpha-adrenergic stimulation must have its origin primarily in an intracellular pool. When this pool is discharged by NA, adrenaline or artgiotensin after the muscle has been in a Ca^+ - free lanthanum containing medium for 30 minutes, a single contraction occurs, but the muscle will not respond subsequently to stimulation with any of the three agonists. Therefore, each of the three agents appearstc use the same- Ca"‘+ pool. However, in spite of the enormous biochemical, histological and pharmacological evidence about the three candidates for the site of the cellular calcium pool, their relative significance as sources of Ca 2+ for the physiological response of adrenaline, angiotensin and NA remains unclear. Electrical and non-electrical activation In mammalian fast striated muscle fibres, the initiation of contraction by a chemical stimulus (acetylcholine) is mediated UNIVERSITY OF IBADAN LIBRARY 13 - by the action potential which triggers the release of Ca from the sarcoplasmic reticulum raising free rayoplasmic Ga 2+ to a critical level required for activation of the actomyosin system (Davies, 1963; Sandow, 1965). In smooth muscle systems, the mechanism by which various stimulatory agents initiate contractile responses appear to be diverse. Bozler (1946); Burnstock et al (1963), using intestinal smooth muscle for their studies have established a compelling case for a primary spike - mediated mechanism of excitation - contraction coupling, which differ only in minor details from that of striated muscle. Marshall (1962) demonstrated a similar behaviour for uterine smooth muscle. However, the case for the action potential being the sole B-G coupling process of visceral smooth muscle was weakened by the demonstration by Evans, Schild & Thesleff (1958) that drugs can produce contraction of depolarized smooth muscle. The contraction of the vascular smooth muscle may be initiated by either electrical or non-electiical activation. The classical model used for study of electrical activation is the portal vein. Smooth muscle in this vessel has spontaneous action potentials which cause the delivery of activator Ga 2+ into the myoplasm, and, hence, cause contraction of the muscle. However, this muscle and other vascular smooth muscles can be made to contract in response to NA and other agonists when the UNIVERSITY OF IBADAN LIBRARY - 14 - call membrane has been completely depdlarized by isotonic potassium sulphate (Somlyo& Somlyo, 1968). Under these conditions, the response to the constrictor a g o n is t o c c u r in the absence of a change in membrane potential,and therefore, is reasonably called ncn-electrical activation - also termed pnarmacomechanical coupling (Somlyo & Sonlyo,] 963). Conversely, physiological responses that involves depolarization of the cell membrane or brings about changes in membrane potential are defined as Electrical activattion (or electromechanical coupling Somlyo Sc Somlyo, 1968). Micr^electrode 3tudie3 support the possibility that NA usually causes constriction of smooth muscle in large arteries without the occurrence of action potentials. Haeusler (1972) reported that in over 200 microelectrode penetra­ tions of. at least 10 seconds each in the smooth muscle cells of the rabbit pulmonary artery contracting in response to NA (10 M), he observed no action potentials. He did find that NA reduced membrane potential from a mean of 58.4 mV to 43«5mV. Kekata & Niu, (1972), using lower concentrations of .adrenaline on the common carotid artery of the rabbit, observed contraction of this vascular smooth muscle without action potentials or a change in membrane potential. These studies agree with the earlier observations by Somlyo & Somlyo (1968) that the same pattern of inequality of maximal responses to adrenaline, angiotensin and UNIVERSITY OF IBADAN LIBRARY - 15 - vasopressin is obtained whether the smooth muscle is polarized or depolarised. This observation indicates that the inequality in response in the polarized state is. not due to differences in the electrical phenomena of the membrane and that factors which determine the magnitude of non-electrical activation are important when the cell is polarized. Electrical and non electrical modes of activation have been explained in t■ erms of differential pathways of Ca 2+ leakage into the contractile proteins. In their review article Rasmussen & Goodman, (1977) noted that the regulation of the free Ca ion concentration in the cell cytosol is achieved by pump-leak systems at both the plasma membrane and at the inner mitochondrial membrane. It is the view of these authors that there is an inward leak of Ca down its concentration gradient and an outward pump in the plasma membrane. At least, two separate channels by which Ca leak into the cell has been described. The first is a relatively specific Ca channel that is independent of the membrane potential and that may well be altered when certain hormones interact with their receptors but do not lead to membrane depolarization (pharmaccmochanical pathway). The second is a potential -dependent Ca^+ permeability channel (electrome­ chanical pathway). This process is also referred to as the "late Ca^+ channel". In addition to these two pathways, some UNIVERSITY OF IBADAN LIBRARY - 16 - Ca 2+ may also enter the cell after membrane depolarization, via the sodium channel and is called "early Ca* 2+ channel" in nerve. The relative importance of these two (or three) different processes by which the entry of Ca 2+ into the cell is regulated varies from one cell to another, and different ones are of major importance in the activation of particular cells by extracellularmessangers.- Evaluation of the relative importance of the channels can be obtained by the ufee of agents that block specific ion channels. The early Ca^+ entry via the ITa+ channel can be blocked by tetrodotoxin and the late, pote' ntial-dependent entry of Ca.2 + can be blocked by verapamil, D600 (a methoxy derivative of verapamil) or K,,n 2+ Ok ITOiLUdlEIIALINE - El DUG ED 5 - C COUPLIITC 2 - substituted imidazolines The 2 - substituted imidazolines have a wide range of pharmacological actions including adrenergic blocking, antihyper­ tensive, sympathomimetic, antihistaminic, histamine-like, and cholinomimetic; slight changes in structure may make one or another of these properties dominant. At the present time, members of the series are marketed for each of the first, four properties listed. The structural formulas of tolazoline (2-benzyl-2-imidazoline) and phentolamine are shown in fig. 1a and b respectively. UNIVERSITY OF IBADAN LIBRARY UNIVERSITY OF IBADAN LIBRARY UNIVERSITY OF IBADAN LIBRARY - 18 - Tolazoline and phentolamine produce a moderately effective competitive alpha-adrenergic blockade that is relatively transient. That is, the reaction of the antagonist with specific receptors usually exhibit mass-action characteristics, and the blockade produced is a measure of competition between the agonist and antagonist for receptor occupancy. Thus, both phentolamine and tolazoline can correctly be referred to as classical competitive antagonists (Nickerson, 1959).| Apart from the receptor blocking effect, both phentolamine and tolazoline have important direct actions on cardiac and smooth muscle that may be divided into three classes: (l) ’'sympathomimetic" including cardiac stimulation, (2) parasympathomi­ metic, including gastrointestinal tract stimulation that is blocked by atropine; and (3) histamine-like, including stimulation of gastric secretion and peripheral vasodilatation (Ahlquist,Huggin & Woodbury, 1947; Nickerson, 1949). Both phentolamine and tolazoline were found to be ineffective against the metabolic effects of adrenaline. Yohimbine - Yohimbine is an alkaloid obtained from a West African tree, yohimbehe. It is Closely related chemically to the rauwolfia alkaloids. Despite the fact that yohimbine’s adrenergic blocking action has been known since 1925 (Raymond-Hamet), it has had only limited use as a laboratory tool and has not been employed therapeutically as a blocking agent. However, fo11owing about UNIVERSITY OF IBADAN LIBRARY c - 19 - a decade old postulate that the adrenergic neurotransmitter, NA, in the peripheral nervous system, regulates its own release through a negative feedback mechanism mediated by prejunctional ot-adrenoceptors (Langer, Bnero and Stefano, 1971; McCulloch, Rand and Story, 1972; Rand, Story, Allen, Glover and McCulloch, 1973), yohimbine has been recognised as a predominant presy-paptic fit-adrenoceptor antagonist. The evidence for t£is is based on selective antagonism of presynap- t;Lcaily acting fit-agonists like clonidine, naphazoline and oxymetazoline by yohimbine (Drew, 1976, 1977).\ Post synaptically acting \ Ct-adrenoceptor agonists like phenylephrine and methoxamine were not antagonized by yohimbine or any other relatively selective presynaptic fiC-antagcnist. Thus, yohimbine, will cause an overflow of neurotransmitter substance by shutting up the auto-regulatory pathway of release mediated by presynaptic fit-adrenoceptors. Yohimbine is a classical competitive antagonist of the presynaptic Ct-adrenoceptor site. However, it is not devoid ox post-synaptic antiadrenergic activity. Phenoxybenzamine (FEZ) Phenoxybenzamine is a member of the B-haloalkylamine blocking agents. It differs from dibenamine (an older member of the group) only in the replacement of one benzyl group by a phenoxyisopropyl moiety. ?ig.1 (c) represents its structural formula. The haloalky- lamine adrenergic blocking agents are closely related chemically to the nitrogen mustards; like the latter, the tertiary amine UNIVERSITY OF IBADAN LIBRARY - 20 - cyclizes to form a reactive ethylenimonium inteimediate. The molecular configuration directly responsible for blockade is probably a highly reactive carbonium ion formed when the three- membered ring breaks (Belleau & Triggle, 1962). The relatively slow onset of action, even after intravenous injection, is probably due to the time required for the formation of these reactive intermediates, which then act es alkylating agent (Harvey & Nickerson, 1954). It has been shown (Nickerson, 1956) that PBZ (its reactive J ' intermediate) combines with the same receptors as the agonist, as in classical competitive antagonism,\ but then reacts with the receptor or some adjacent group(s) to form a relatively stable chemical bond. Such a reaction precludes further mass-action "competition" and effectively reduces the number of available receptors. PBZ has thus been described as a NONEQUILIBRINK antagonist. This type of blockade has also been referred to as "irreversible competitive" (Purchgott, 1955) and "unsurmountable" (Gaddum, Hameed, Eathway & Stephens, 1955). The exact nature of.the groupings on or near 0(.-adrenergic receptors with which the haloalkylamine react has not been determined. It was anticipated that a labelled haloalkylamine could be used in combination with agonist protection of specific receptors to identify and characterize the £t-adrenergic receptors. However, experiments of this type have been beset by numerous complications, and the only valid conclusion that has been drawn UNIVERSITY OF IBADAN LIBRARY - 21 to date appears to be that these receptors are protein in nature (Lewis & Hiller, 1966; Yong & Marks, 1969). This is in agreement with the experimental observation that trypsin can reverse the blockade of 06-adrenergic receptors by dibenamine (Graham & A1 Katib, 1966). In addition to producing fit-adrenergic blockade, PBZ is also thought to exert important effects on catecholamine metabolism. Dibenamine and PBZ increase the amount of NA released in the venou3 effluent per stimulus delivered to the splenic nerves at low but not at high frequencies. This view was first attributed to blockade of Ck-adrenoceptors and later to inhibition of NA inactivation. Prevention of access to inactivating enzymes was thought to be the most important component of this effect but prevention of NA uptake is also known to be involved (Brown & Gillespie, 1957; Kalsner & Nickerson, 1969). A more up-to-date explanation has been presented. This view states that the increase in NA overflow obtained in the presence of the 01-blocker PBZ or phentolamine, represents an actual increase in transmitter release and is not due to blockade of sites of loss for the released transmitter. The reason for this is that, these effects are obtained with concentrations of the drug which do not inhibit neuronal or extraneuronal uptake (Starice, Montel & Schumann, 1971; Longer, 1974; Alder-Graschinsky & Langer, 1974). Further evidence in support of the view that ot-receptcT' UNIVERSITY OF IBADAN LIBRARY - 0? ■blocking agents actually increase transmitter release during nerve stimulation was obtained by Cubeddu, Barnes, Langer & b'einer (1974). These authors found an increase in the release of d o p a m in e-B -h yd ro xy la se when th e s y m p a th e tic n e r v e s o f th e p e r fu s e d sp le e n were s t im u la te d in the p re se n c e o f th e CLr-z’e c e p to r b lo c k in g agent. Farah & Langer, (1974) have concluded that phentolamine and PBZ increase transmitter release by acting on the same prejunctional (^adrenoceptors. Furthermore, PBZ and other haloalkylamine3 can inhibit responses to 5-HT, histamine, and acetylcholine, Blockade of these other types of agonist has the same general p h arm a co lo g ica l characteristics as does the adrenergic blockade. Effective blockade of responses to acetylcholine usually require relatively high dose of haloalky- laaines. Owing to this non-selectivity in the type of receptor antagonism by PBZ, the latter has also been thought to act at a point beyond the receptor level but which is common to all the agonists (probably a specific process between receptor activation and the contractile response). PBZ is thought t.o .in.hi.bi t Ca 2+ fluxes across cell membranes. This has been supported by the observation that PBZ also antogonized the contractile response induced by potassium chloride. Such contraction is exclusively said to be due to calcium influx from the extracellular sources (Bevan & 3u, 1965; Hudgin & Yfeiss?, 1968; Kalsner & Nickerson, 1969). UNIVERSITY OF IBADAN LIBRARY - 23 - Prazosin Compounds having the quinazoline ring system have been reported to have diverse biological activities (Armarego, 1963). Cne of these compounds, prazosin, was found to possess significant antihy­ pertensive effect which was believed to be due to direct relaxation of the arteriolar wall and to blockade of iJC— adrenoceptors in the • peripheral vascular wall (Constantine, 1973). As an ^-antagonist, prazosin exhibits properties which are not * typical of conventional ct-blocking agents. Thus,it lowers blood pressure without causing tachycardia, does not cause renin release and reverses the sustained tachycardia induced by hydralazine therapy in hypertensive dogs (Constantine, Hcsh^ne, Scriabane &Hess, 1973; Constantine, 1974; Massingham & Hayden, 1975). Cinnarizine and Verapamil Cinnarizine (l-benzhydrxl-4-cinnamyl piperazine dihydrochloride) (-stugeron) is an antihistamine (il^-receptor antagonist) and also has vasodilating properties. It antagonizes adrenaline, angiotensin and 5-HT. It is used clinically for the treatment of peripheral arterial disease and disorders of balance. Cinnarizine inhibits the contractile response to Ca and induces relaxation of depolarized O c muscle previously contracted by Ca~ . Its antagonism of adrenaline responses occurs nnly in polarizing solution./-nd not In UNIVERSITY OF IBADAN LIBRARY - 24 - Ca^+-free depolarizing solution. Based on these observations, the two most likely sites for cinnarizine action in arterial smooth muscle would seem to be the membrane of the muscle fibril (where the drug would reduce the availability of ^a ions to the contractile mechinery of the depolarized muscle) or the contractile machinery itself (where it would interfere with the binding of Ca 24"). The latter mechanism is unlikely since cinnarizine did not affect .__-- / adrenaline induced contractions in Ca -free depolarizing solution I (GodfrainiS Kaba, 1969, 1972'). Cinnarizine has been shown to be about four times more potent th■ an chlorpromazine as Ca 2+ antagonist and several times more potent than each of the other Ca antagonists (procaine, papaverine and manganese ions). However, the potencjr of these agents as Ca^+ antagonists is usually determined by the extent to which they antagonise calcium chloride - induced contractions. Verapamil (iproveratiil) i3 another example of antagonist of excitation - contraction coupling. Its mode of action is similar to that of cinnarizine and has therefore been used in laboratories as a Ca antagonist (Golenhofen & Lammel, 1972; Godfrain'& Kaba, 1972). This agent has been shown to exhibit some selectivity in its mode of Ca"-4" antagonism. According to Rasmussen & Goodman (1977) verapamil antagonises specifically the Ca 2+ influx indiiced as a result of membrane depolarization (i.e. it blocks the electromechanical pathway). UNIVERSITY OF IBADAN LIBRARY - 25 - Indomethacin and sodium meclofenamate These two compounds belong to the seine group of drug generally classified as non steroidal anti-inflammatory drugs (NSAIDS). They have been most extensively studied and used to investigate the functional role of the prostaglandins in physio­ logical and pathological systems. Both indomethacin and sodium meclofenamate have been shown to reduce or abolish responses to lit and angiotensin in the rat mesenteric vascular bed (Horrobin, Manku, Karmali, Nasser & Davies, 1974; Malik & McGiff, 1974; Mtabaji, Manku & Horrobin, 1976). The effects of these drugs cculdnot b ascribed specifically'to endogenous prostaglandin synthesis inhibition This idea is widely based on the non-specific nature of the antagonism. For instance, in the rat mesenteric artery preparation, indomethacin inhibited the pressor stimuli to adrenaline, calcium chloride and barium chloride (Northover, 1968, 1971, 1977); and to vasopressin and angiotensin (Horrobin et al. 1974; Manku & Horrobin, 1976). Similarly, contractile responses of the intestinal smooth muscle to prostaglandins are also inhibited by indomethacin (Sorrentino, Capasso, & Dirosa, 1972). It has thus been thought that indomethacin and other NSAIDS may be affecting a common process crucial to the manner in which all the agonists cause contraction (i.e. the excitation-contraction coupling process). Evidence in support of this view is as follows: (a) the observation by North over,(1 968) that indomethacin was equally effective in blocking .adrenaline vasoconstriction in arteries perfused with normal salt, against UNIVERSITY OF IBADAN LIBRARY - 26 - p_j_ p . vasoconstriction produced by Ca or Ba ^ in the artery perfused with , . . -f. a high A depolarizing solution. This will suggest that the antagonist (indomethacin) was not preventing Ca2+ entry; (b) the observation that indomethacin rapidly relaxed the spasm observed in an artery previously perfused for a long time with Ca2+ - depolarizing solution i.e. in a situation in which Ca 2+ ion must have already gained entrance to the vicinity of the contractile proteins (Northover, 1968). The contractile proteins of vascular smooth muscle are thought to respond to Ca'2 + ions as a result of the activation of an adenosine triphosphatase enzyme (Somlyo & Somlyo, 1968; 1970) and since indome­ thacin did not reduce creatine phosphate and adenosine triphosphate (ATP) content of the muscle ( Northover, 1971)/ it was concluded that the contraction inhibiting effect of indomethacin was due to some other disturbance of the excitation-contraction coupling. Two other suggestions advanced by Northover {1971 ) are that NSAIDS could either cause (a) failure of Ca2+ .ions to reach the contractile proteins or (b) failure of the contractile proteins to respond to 0a2+ ions. Based on the observation that indomethacin did not inhibit contraction oi glyce- rinated smooth (i.e. smooth muscle preserved for 1 week at 0 G in a 2+ 24- solution of glycerol) elicited by a mixture of ATP, Ca ions and Mg ions, it was concluded by exclusion, that indomethacin distrupts e - c 2+ coupling mainly by reducing the availability of Ca ions within the muscle. Direct evidence in support of this conclusion was obtained by measuring the Ca2+ content of strips of smooth muscle subjected to electrical stimulation in vitro. The rise in Ca content is.reduced by treatment with indomethacin in concentrations similar to those UNIVERSITY OF IBADAN LIBRARY reqrj^-£?d to i n h i b i t in f lam m a tio n (No 1971, 1972). I t has a ls o been shown (Flower, 1974) that indomethacin in concentrations higher tnan 5 ug/ml can di3trupt metabolic processes including inhibition of oxidative phosohorylation. Other effects of indomethacin at high concentrations include inhibition of proteolytic endues -e.nu .inuŝ -— ference with various biological member ^Famaey, Fontaine & Reusse, 1 97?). In his review article of 19t>3, Trendelenburg outlined physiological and pharmacological procedures that can induce sup^rsensitivity to sympathomimetic aminos in the cat nictitating membrane as follows (i) administration of cocaine (ii) chronic pretreatment with reserpine (iii) decentralization and (iv) denervation. Procedures (i) and (ii) are characteristically pharmacological in nature while (iii) and (iv) are physiological in nature. The sensitizing effect of cocaine seems to be restricted to sympathomimetic amines. Acetylcholine is^for instance^not potentiated by cocaine. 5- hydroxytryptamine which has been shown to be potentiated by cocaine was also shown to stimulate the adrenal medulla (Lecomte, 1955) and the superior cervical ganglion (Trendelenburg, 1956) in addition to its direct stimulant action on the muscle of the nictita­ ting membrane. Notwithstanding, 3 ~HT is only potentiated by cocaine when resting tension rises - an observation compatible with the view that the endogenous noradrenaline exert3 an additive effect. It was thus concluded by Trendelenburg (l963;that the sensitizing action UNIVERSITY OF IBADAN LIBRARY - 28 of cocaine on the nictitating membrane is highly selective and seems to be especially pronounced for all sympathomimetic amines possessing a phenolic hydroxyl group in the meta-position. The basis for this conclusion was derived from the experiments of Trendelenburg et al (1962) where they compared the of sixteen different sympathomimetic amines before and after the administration of cocaine, in acutely reserpinised animals. Acute reserpinization eliminated the indirect component of the action of these amines. The effect of pretreatment with reserpine depends on the schedule employed for the pretreatment. Short-term pretreat­ ment (24 hours) causes depletion of the NA stores of the caiN}, * nictitating membrane with no concomitant supersensitivity to ITA (Fleming & Trendelenburg, 1961). However, when a small amount of reserpine (0.1 mg^kg) was injected daily for 3, 7 and 14 days, supersensitivity of the nictitating membrane to NA was first observed after 7 days and becomes more pronounced after 14 days. Fleming (1963) in his own studies extended the pretreatment period to 28 days and found no further increase in response to NA after 23 days. These observations indicate that pretreat­ ment with reserpine is able to cause supersensitivity to NA but that time is essential for the development of this supersen3itivity. The time factor varies from organ to organ. For example, supersensitivity of the cardiovascular system develops after 2 days of pretreatment with 2.5 - 5 mg reserpine U IVERSITY OF IBADAN LIBRARY - 29 - per kilogram per day (Burn & Rand, 1958) and also within 3 days of Ion*-term pretreataont whereas supersensitivity of the nictitating membrane developed only after 7 days (Fleming1 Sz Trendelenburg, 1961)* Quite different results have been reported after acute injections of reserpine into spinal cats. A short time after such an injection, the response to HA was enhanced and was observed to remain so for a few hours (innes, 1960; Hakamura & Shimamoto, 1960; Schmitt & Schmitt, 1955) - The time course of the development of this potentiation was found to be similar to the time course of the elevation of the plasma level of catecholamines after intravenous injections of reserpine (Kuscholl & Vogt, 1957). This observation was explained as being due to an addition of the effects of endogenous HA (increased in the proximity of the receptors because of the releasing action of reserpine) to the action of injected sympathomimetic amines - a view that was supported by the finding that tyraaine is not only effective but actually more effective than normally, on the nictitating membrane during the first few hours after the intravenous injections of reserpine. Other explanations are (1) that the reserpine - induced release of HA saturated the non-specific x’eceptor sites (as defined by Koelle, 1559) and that consequently, a larger proportion of the injected amines reaches their respective sites of action UNIVERSITY OF IBADAN LIBRARY 30 - and (2) that the increased response to tyranine was due to the summation of the releasing action of re sorpine and that of tyramine. Decentralisation, otherwise known as chronic preganglionic denervation, is well knovm to cause supersen^itivity of the nictitating membrane to various substances. It causes a characteristic type of supersensitivity which is (a) of moderate degree, (b) not accompanied by a loss of HA stores of the nictitating membrane, (c) non-specific in that it is as prominent with acetylcholine as it is with NA, and (d) not associated with subsensitivity to any of the sympathomimetic amines. The non-specificity is striking, since supersensitivity after decentralization is of similar magnitude for directly and for indirectly acting amines, and it is of similar magnitude for m - OH compounds and their p - OH analogues (a.g. phenylephrine and synephrine). According to the conclusion of Trendelenburg & Weiner (1962), the most important factor responsible for the development of this type of supersensitivity seems to be a prolonged functional interruption of the pathway between the central nervous system and the nictitating membrane. Thus, procedures or pharmacological agents which produce this effect also produce this type of supersensitivity. This view agrees with the extensive studies of Emelin (1961) who reached similar conclusions on the basis of experiments on the salivary UNIVERSITY OF IBADAN LIBRARY - 31 - glands of the cat. This type of supersensitivity ig termed "pharmacological denervation" (Hnmelin, 1961) or "pharmacological decentralization" (Trendelenburg 1963). Supersensitivity after decentralization develops slowly and reaches its maximum about two weeks after the section of the preganglionic nerve (Hampel, 1935); this characteristic time course is similar to that for the development of supersonsitivity after lorg-tem pretreatmen!t with reserpine. Slow developmen0t of supersensitivity has also been observed by Smnelin and his group (1961) in their studies of the supersensi*tivity of the salivary glands of the cat. In order to obtain more information about thi3 time factor, Trendelenburg & Weiner (1962) determined dose - response curves on the nictitating membrane of the spinal cat for three test substances (iTA, tyramine and acetylcholine) in normal preparations as well as after various pretreatment schedules designed to interrupt the normal pathway between the higher centres and the nictitating membranes. These pretreatments were: (a) 7 days of denervation; (b) 7 days of decentralization; (c) 7 days of ganglion block (produced by two daily injections of ohlorisondamine); (d) 7 days of prevention of the release of HA from its peripheral stores (produced by daily injections of 34 10); (e) 7 days of depletion of the NA stores (produced by daily injections of a small amount, 0.1 mg/kg, of reserpine); (f) 7 days of blockade of the HA receptors UNIVERSITY OF IBADAN LIBRARY - 32 - (produced by daily injections of phenoxybenzamine). After some of these prolonged protreatments, the catecholamine content of the nictitating membrane was determined fluorimetrically. • Short tern pretreatment with chiorisondamine, I'M 10, or reserpine (agents c, d and e above) had no effect on the sensitivity of the nictitating membrane to h'A and acetylcholine, but long-term pretreatment with these drugs caused uniformly a moderate supersensitivity to both substances similar to that observed after decentralization. Denervation, on the other hand, caused the well-known pronounced supersensitivity to HA, but its sensitizing effect in regard to acetylcholine was not much stronger than that of decentralization. It was thus concluded that there is a correlation between the development of supersensitivity and the time factor, IIo correlation was found between the development of supersensitivity to HA and the DA content of the nictitating membrane; however, the response to tyramine clearly depends on the presence of these stores. The similarity of the sensitizing effect on the nictitating membrane of chronic post ganglionic. denervation (i.e. dener­ vation) and of the administration of cocaine is very pronounced and has been observed by numerous workers. However, this supersensitivity develops rather slowly after denervation and reaches its maximum after about 2 weeks (Hampell, 1935), whereas UNIVERSITY OF IBADAN LIBRARY the sensitising effect of cocaine is observed within a few minutes after the intravenous injection of this substance (Trendelenburg, 1959). The close similarity is convincingly demonstrated on the nictitating membrane by a quantitative study of the influence of denervation (Pleckenstein & Bum, 1955) and of cocaine (Pleckenstein & Bass, 1955; Pleckenstein & Stoclcle, 1955) on the dose - response curves of a large number of sympathomimetic amines. Fleckenstain & Bass (1955J, concluded from this 3tudy that the administration of cocaine causes "pharmacological denervation", Pleckenstein & Stockle (1955) on their own part found the correlation between the effects of cocaino and of denervation to extend to their desensitising effects towards amines with indirect actions. Also, the sensitization of the nictitating membrane caused by denervation exhibits specificity in the sympathomimetic amines as was found for cocaine (Trendelenburg et al, 1962). However, as closely similar a3 the effects of cocaine and denervation appear to be, they have been classified as different forms of supersensitivity based on the substantial differences between the two types. In two successive publications, Trendelenburg (1962), and Trendelenburg et al (1962) came to the conclusion that chronic - denervation -- induced type of supersensitivity is not only different from cocaine - induced type, but that it is actually UNIVERSITY OF IBADAN LIBRARY 34 - equal to the sum of the sensitisation caused by decentralize— I tion, plus the highly selective supersensitivity similar to that produced by cocaine, plus the effect of depletion of the KA stores. The evidence for this includes the fact that (i) admini­ stration of cocaine fails to cause supersensitivity of the nictitating membrane to acetylcholine whereas denervation does cause supersensitivity to this substance; (ii) denervation shifted fee di6se - response curves of all of thirteen directly ~ or indirectly - acting sympathomimetic amines more to the left than by cocaine. This effect was proved to be a qualitative \ one since cocaine interaction with indirectly - acting sympa­ thomimetic amine e.g. tyramine or ephedrine is antagonistic and moreover since cocaine did not potentiate the direct component (obtained by pretreatment with reserpine) of the action of ephedrine. A plot (on log - scales) of the shifts of the dose - response curves of thirteen sympathomimetic amines as observed after denervation of the nictitating membrane (ordinates) against the shifts due to the administration of cocaine (abscissa) though gave regression co-efficient of nearly unity (1 .095) but the'regression line was displaced in such a way that at no point 'on* the line were the values for cocaine and denervation the same. This finding was taken to be consistent with the view that supersensitivity caused by denervation also comprises a moderate and non-specific type of supersensitivity similar to that' produced by decentralization. UNIVERSITY OF IBADAN LIBRARY - 35 In his concluding remark, Trendelenburg (1963) identified two separate and distinct forms of supersensitivity in adrenergically innervated structures denervated post ganglio- nically. The terms presynaptic and post synaptic (Trendelenburg, 1956) or prejunctional and postjunctional (Fleming, HcPhillips & Westfall, 1973) have been used to describe the two forms ox supersensitivity. Prejunctional supersensitivity does not involve a change in the ability of the target cells to respond. Rather, it is the loss of some process, such as neuronal amine transport, which normally diverts specific drugs away from their site of action. Such supersensitivity is characterised by rapid onset and high specificity. Sensitization of the same characteristics as stated above was classified "type 1 sensiti­ zations" by Kalsner (1974). In contrast, postjunctional supersensitivity is a true change in the sensitivity of the target cells. It usually comes on slovfly (days to weeks, depending on the typo of target cells) and is generally quite non-specific, the sensitivity being increased to a variety of unrelated drugs and ions. Kalsner (1974) described supersensitivity of this nature "type II sensitizations". The characteristics of this type II sensitizations are similar to those described for post junctional supersensitivity. Other t oms used to denote postjunctional type of supersensitivity include non­ UNIVERSITY OF IBADAN LIBRARY - % - specific supersensitivity (Fleming, 1953) and disuse supersensitivity (sharpiecs, 1965). these terns have their set backs. For instance, supersensitivity cannot yet be correctly classified on th e basis of specificity since no one is sure that all types of postjunctional supersensitivity have been tested for specificity. Also, the cuestion of whether "disuse" (i,.e end organ inactivity) or los3 of trophic factor released by nerves is responsible for supersensitivity after interruption of the innervation has not been adequately explored in smooth muscles. Furthermore, under certain circumstances, extraneuronal uptake (including uptake into the effector cells themselves) and subsequent intracellular metabolism divert a portion ox the administered dose of a drug from its sito of action. Inhibition of these processes can produce supersensitivity (Kalsner & Nickerson, 1969). 'This phenomenon does not readily fit into the classification of pro- and post - junctional. This is because, its location is postjunctional (i.e. in the effector cells) but its mechanism (i.e. an increase in the percent of administered drug that reaches the receptors) is essentially the same as prejunctional supersensitivity. However, in a recent symposium on supersensitivity in smooth muscle, Fleming, (1975) described as premature any effort to choose the best terms by which types of supersensitivity may be classified. / UNIVERSITY OF IBADAN LIBRARY - 37 - He classified sraooth muscle supersensitivity into two as follows:- I "Deviation" supersensitivity or sensitivity due to changes in uptake and/or metabolism of the drug i.e. functionally similar to prejunctional supersensitxvi i;y. (a ) Decreased adrenergic neuronal uptake of specific syapathomimetics (cocaine, desmethy- limipramine, destruction of adrenergic neurones). (b ) Decreased cholinesterase activity (super­ sensitivity to cholinesterase sensitive choline esters, as caused by cholinesterase inhibitors or destruction of cholinergic neurones). (c) Decreased extraneuronal uptake and metabolism. II "Non-deviation" supersensitivity or enhanced responsiveness of the target cells. This term infers that there is no change in the portion of an administered dose of a drug that reaches the receptors. Bather, the responsiveness of the target cell3 Is increased. UNIVERSITY OF IBADAN LIBRARY of supersensitivity. These authors raised the following important points (a) that there must be more than one mechanism underlying supersensitivity, (b) that one of these mechanisms probably involved changes in the removal, by metabolism or storage, or normal transmitters, and (c) that in view of the non-specific characteristics of soma examples of supersensitivity, another mechanism may involve a change in the physiological characteristics of the responding cells, such as an altered permeability of the membrane to ions, Sunersensitivitv and metabolising enzymes: The potentiation of catecholamine effects by cocaine is accompanied by raised blood levels of the catecholamines, (Trendelenburg, 1959; Huscholl, 1961), This is an indication that cocaine probably inhibited the inactivation of catecholamines. Philpot (1940) provided an experimental evidence that cocaine inhibits the enzyme monoamine oxidase (MO), This enzyme, found in almost all body tissues (Blaschko, 1952) has been shown in vitro to oxidise the side chain of many amines, for example adrenaline, 1TA, tyramine and 5 - Hf. For some time, the enzyme was thought to be mainly responsible for inactivation of catecholamines in the body. It was suggested that the enzyme played a role at the sympathetic nerve endings analogous to that of acetylcholinesterase at cholinergic nerve endings (Bum,. 1953). Burn & Robinson (1953) demonstrated a diminution UNIVERSITY OF IBADAN LIBRARY - 39 - ' in the amount of amine oxidase after denervation and degeneration of the sympathetic nerve fibres. Thi3 observation was later confirmed by Stromblad (1965) in studies of the enzyme content of salivary glands. B u m & Hutcheon and Stromblad (1952) independently found a correlation between the fall in amine oxidase content and the development of supersensitivity and suggested that the supersensitivity was due to the fall in KAO content. i B u m & Rand (1958, 1959) abandoned this suggestion on the grounds that reduction in enzyme level was not sufficient to change the response of the membrane sd greatly as denervation does. In 20 out of 25 experiments of B u m & Hutcheon (1952) the amine oxidase in the denervated nictitating membrane was not reduced below of the amount in the normal membrane. In addition, reduction in enzyme activity fails to explain the increased sensitivity of the denervated structure- to agents other than the chemical mediator. B u m & Rand (1958, 1959) therefore concluded that the supersensitivity to HA and adrenaline which develops in sympathetically innervated tissues after degeneration of these nerves is attributed to the disappearance of stores of NA in these tissues. This conclusion was justified by the observation that there was a simultaneous decline of the HA stores of the iris and the spleen and increased sensitivity to NA after degeneration of the sympathetic nerves. Furthermore, UNIVERSITY OF IBADAN LIBRARY - 40 - i administration of the potent amine oxidase inhibitors, iproniazid to animals failed to cause potentiation of adrenaline, A 1TA, and 5 - HT (Griesemer et _al , 1933; Poster, Ing & Veragic, 1935; C o m o & Graham, 1957; Vane, 1959). This finding has been widely supported by other workers (Brown & Gillespie, 1957; Herttin, & Axelrod, 1961; Kopin, 1964). Gvidence is therefore lacking that KAO plays, a major role in the inactivation of adrenaline and NA. Consequently, inhibition of this enzyme is unlikely to be the major cause of supersensitivity to these amines. The discovery of derivVatives of adrenaline and ITA methylated in the hydroxyl position of the phenyl ring (Armstrong et al, 1957; Axelrod, 1957; Axelrod et al, 1959) led to a detailed study of the fate of the amines in man and of the methylating enzyme. The involvement of the enzyme, catechol-o-rnethy 1- transferase (COIIf), in the catabolism of injected NA led to the postulate that this enzyme terminates the active life of ITA and that blockade of its activity could explain the phenomenon of supersensitivity. liuscholl (i960, 1961), Stromblad & ITicIrerson (1961) have independently demonstrated an appreciable accumulation of ITA and adrenaline in bhe rats heart and salivary glands by using fluorinetrie assay techniques. Lavollay (1941) showed that pyrogallol, quercitin and similar compounds, found later to be COI-IT inhibitors would potentiate slightly the biological UNIVERSITY OF IBADAN LIBRARY - 41 effects of adrenaline and NA. Pyrogallol (Wylie, Archer j & Arnold, 1960) and 4 - me thyltro polone (Murnaglan & Kazurkiewicz, 1963) also prolonged tbfe pressor effects of injected catecholamine in the cat only slightly. But, Lund (.1931); Celander,(1954) showed that intravenously administered adrenaline and ITA disappear very rapidly from the plasma and this rapid inactivation process was not markedly retarded by inhibition.' of either KAO or COAT or even by the simultaneous \ inhibition of the two enzymes (Crout, 1961). COhf inhibition as the sole mechanism of su\ persensitivity was farther turned down on the following additional grounds : (.a) that cocaine (an agent that can cause supersensitivity) fails to block catechol-o-methyl-transferase ('iylie et al, I960) and (b)that cocaine produces supersensitivity to amines which are not substrates of COAT e.g, phenylephrine (Axelrod & fomchick, 1958) Supersensitivity and ITA content: Denervation is known to cause a considerable fall in the NA content of the denerv'ated organ ( B u m & Rand, 1939; Cooper, Gilbert, Blood well & Crout, 1961; Kirpekar, Cervioni Sz Furchgott 1962; Stroablad, I960). Pleckenstein Sz Bass (1953) observed that cocaine prevented tlie release of NA from nerve terminals following postganglionic stimulation. On the strength of this observation, these authors postulated that cocaine, by virtue • of its local anaesthetic action can block the postganglionic UNIVERSITY OF IBADAN LIBRARY - 42 - fibres and thus cause "pharmacological denervation" since cocaine and denervation caused very similar types of supersensitivity. Macmillan, (1939) likewise postulated that cocaine interferes with the release of HA from the stores. However, explanation of the phenomenon of supersensitivity based on this concept is untenable sinco (a) there is evidence that cocaine does not impair the release of HA on nerve stimulation (Xirpekar & Cervioni, 1962; Trendelenburg, 1959); (b) compounds known to block the release of HA from nerve endings on nerve stimulation (such 03 TH 10 and brotyliuia) caused only a very slight supersensitivity to HA, which is quite different from that produced by either denervation or the administration of cocaine (Hxley, 1957; Boura, Green, McCou’orey, Lawrence, Moulton & Hosenhein, 1959); and (c) other compounds which are at least as potent local anaesthetics as cocaine do not cause the typical cocaine - like supersensitivity. When resorpine became available as a pharmacological tool for depleting the HA stores, experiments seemed to indicate that pretreatment with reserpine (like denervation and cocaine) always led to supersensitivity to HA, when depletion of the stores was achieved. Therefore, B u m & Rand, (1959) postulated that the sensitivity of an organ is inversely related to its HA content. However, this hypothesis is untenable since the following experimental observations cannot be explained on its basis: ■ UNIVERSITY OF IBADAN LIBRARY A '5 - (a) decentralisation, a process known not to deplete the scores (ifirpekar ot al, 1962; Helm, 1953) caused supersensitivity to 1TA; (b) short-terra pretreatment with reserpine causes depletion of the HA stores without concomitant supersensitivity to HA (Crout e;t al, 1962; Fleming & Trendelenburg, 1961; Krayer, Alper, Peasonen, 1962; Trendelenburg, 1961; Krayer, Alper, Peasonen, 1962; Trendelenburg & Weiner, 1962); (c) prolonged ganglion block causes supersensitivity to HA ’without reducing the HA content of the tissues (Trendelenburg & Weiner, 1962); (dj prolonged treatment with TM 10 or bretyliun causes super­ sensitivity to HA (Smiaelin 8: Sngstrom, 1961; Trendelenburg & Weiner, 1962) with very little change in the HA content of the tissue (Boura et al, 1959); (e) prolonged pretreatment with very small amount of reserpine causes supersensitivity to NA although the 3toreo of HA are not completely depleted, as judged by the reduced (but by no means abolished) response of the nictitating membrane to nerve stimulation (Fleming & Trendelenburg, 1961). Apparently in favour of this hypothesis of an inverse relation­ ship between the sensitivity of an organ and HA content is the observation that an infusion of HA into a reserpine pretreated preparation not only increases its response to tyramine but also reduces its sensitivity to HA (Bejrablaya, Burn & Walker, 1958j B u m & Rand, 1958), This pehnomenon has been interpreted as a "normalization" in so far as the infusion of HA refills the UNIVERSITY OF IBADAN LIBRARY - 44 - originally depleted stores and reduces to normal the originally increased sensitivity of the organ. However, repetitive - stimulation, of the post ganglionic fibres of previously decentralized nictitating membrane also caused a reduction of the supersensitivity of this organ to injected NA (Wolff & Cattell, 1937); although decentralization is known not to affect the NA content of this organ (Kirpekar et ai_ 1962;. Moreover, observations with the isolated guinea-pig atria likewise do not support this hypothesis. After short-term pretreatment with reserpine the HA content of the atria was only l̂s of normal, and they failed to respond to tyranine, but their sensitivity to NA was normal; although exposure to NA then restored their response to ty ranine to 70?j of normal, their sensitivity to NA remained unchanged after the "refilling" of the stores (Grout st al, 1962). Deviation of transmitter substance from its site of loss to the receptors The site of loss of NA, may be taken to include uptake of the transmitter into the NA stores, temporary hinding to proteins ("silent receptors"), enzymatic catabolism and diffusion from the receptor. By far the most popular hypothesis concerned with the phenomenon of supersensitivity are those which consider supersensi­ tivity to be the result of a deviation of liberated or injected NA from its usual "site of loss" to the pharmacological receptor. This concept implies that the supersensitive effector organ is not UNIVERSITY OF IB DAN LIBRARY 45 - changed, at all but that the effective concentration of the injected agonist at the receptor ia increased by the sensitizing agent or procedure. According to the uptake theory, the sensitivity of an effector organ is determined by the rate with, which uptake into the nerve te r m in a ls rem oves th e 1IA from th e n e ig h b o u rh o o d o f th e receptors. In the presence of normal uptake, the concentration of injected Ha at the receptors remain low, under such conditions, the sensitivity to NA is low. Thus, blockade of the "site of loss" (uptake system for adrenergic transmitter) results in a large number of NA (or other catecholamine) molecules in the vicinity of adrenergic receptors to produce the exaggerated and prolonged pharmacological responses of the effector organ i.e. in supersensitivity. This mechanism of action was first proposed by Macmillan (1959)• The supporting experimental evidence includes the demonstration that cocaine delayed the disappearance of injected NA from the circulation and that the drug inhibited uptake of NA into various peripheral tissues (v/hitby, 1960), Denger ds Titus (1961) confirmed the ability of cocaine to 3 inhibit NA uptake in tissue slices incubated with H -IIA. The same effect of cocaine lias been demonstrated by Lindmar & Muscholl (1962, 1964) in perfused rabbit and rat heart where the drug more than doubled the overflow of NA following sympathetic nerve stimulation. In addition, Muscholl (1961) found'a quantitative correlation between the changes in uptake and in sensitivity. UNIVERSITY OF IBADAN LIBRARY This author determined, (i) the response of the blood pressure to a 3nall dose of L-l.'A before and after the intravenous injection of 10-20 mg/kg of cocaine; (ii) the NA content of the heart after an i.v. infusion of 20 ug of NA and found an inverse linear correlation between the NA content of the heart and the sensitivity of the blood pressure to this amine* In other words, the more pronounced the impairment of the uptake of NA, the more pronounced was the increase in sensitivity of the blood pressure to HA - a correlation that is consistent with the uptake theory, Furthermore, administration of cocaine (or denervation) was observed to sensitive an effector organ much more to an amine that is taken up rapidly than to an amine that is taken up slowly. For example, the relative rates of uptake of the d - isomer3 of HA and adrenaline are : 1 “ HA 1 - adrenaline d - isomers (Iversen, 1363; Kaickel, Beaven, & Brodie, 1963) and cocaine was found to increase the sensitivity of the nictitating membrane of the spinal cat to these amines by factors of 23, 5 and 2.5 respectively (Trendelenburg, 1965). Also, the amine, isopreneline is not uptaken by the heart of the rat (HerttiAg, 1964) and neither cocaine nor denervation caused supersensitivity to its effects on. blood pressure (Maxwell, Daniel, Sheppard & Zimmerman, 1962); and nictitating membrane (Smith, 1963). UNIVERSITY OF IBADAN LIBRARY - 47 - Supersensitivity due to neuronal uptake blockade has been substantiated yet in another way. It was recently postulated that the release of NA by nerve stimulation is regulated through a negative feedblack control mechanism mediated by presynaptic a - adrenoceptors (Langer, Alder, Enero & otefano, 1971; Starke, 1972; T'cCulloch, !>ar»d -p- Story, 1972: Enero •*•*. . • - A V - m UNIVERSITY OF IBADAN LIBRARY vasoactive substances » Ferreira & Vane (1967) identified PGs in dog splenic venous blood when the spleen contracted. Gilmore, Vane & Wyllie (1968) and Davies, Horton & Withrington (1968) independently detected PGs in the effluent blood from the dog spleen after splenic nerve stimulation; and also during adrenaline - induced contraction. PGF„ê ci and PGS,<.L were identified in the perfusate by thin layer chromatography and bioassay techniques. The PGs so released were not of nervous origin because : (a) adrenaline also induced release, (b) - adrenoceptor antagonists prevented splenic contraction and adrenaline - induced prostaglandin release, and (c) the quantities of PGS2 and PGF2a released during adrenaline stimulation of denervated and innervated preparations were not significantly different (Gilmore et al, 1968). Release of prostaglandins have also been reported from many other tissues of various animal species, Shaw & Ramwell (1968) reported prostaglandin release from rat epididymal fat pad on nervous and humoral (hi) stimulations; Dunham & Zimmerman (1970), Kcgiff, Crowshaw, Teiragno, Malik & Lonigro (1972) demonstrated a basal release of material identified as PGS from the dog kidney. It was observed that the release was markedly enhanced during periods of renal vascular constrictions induced by either nerve stimulation or noradrenaline (h a ) infusion. In 1972, Davis & Horton, reported the mass spectroneti-ic identification of PGS^ and PGPoa ill rabbit renal venous blood. This basal output was greatly UNIVERSITY OF IBADAN LIBRARY - 6 2 - increased by renal nerve stimulation. Indomethacin (10 mg/kg) injected intravenously reduced the basal PG output into renal Q venous blood and also prevented the increase in output f i x renal nerve stimulation. *bOTl'b i/O Trhich ‘\r* SwlOO'bll CTn.SClG tto.X̂ ‘pr.T’i/l. in the generation of PG detected in the outflow from organs treated with vasoconstrictor substances has stimulated much interest in recent years. Hedqvist (1972) observed PG in the venous effluent of blood perfused hindlinb of the cat after HA infusion and sympathetic nerve stimulation and proposed a possibility, of an intramural generation of prostaglandins by contracting blood vessels, Aikens (1974) made a similar observation. Gryglewski & Korbut (1975) reported that the vasoconstriction induced by HA in the perfused rabbit ear is accompanied by a release of prostaglandin-like substance. Since exogenous prostaglandin .s antagonized the vasoconstriction action of HA, the authors concluded that the peripheral function of the PG was to attenuate HA. In preparations pretreated with indomethacin (5 ug/ml), HA infusion did not release any substance capable of contracting biological assay tissues. Also, - receptor blocking drug (phanoxybenzamine) completely blocked the pressor response to HA and also the release of prostaglandin like substance into the efflaunt UNIVERSITY OF IBADAN LIBRARY - 63 - Grodzinska, Panczenko & Gryglewski (1976) demonstrated simultaneous increase in perfusion pressure and release of PCS - like material after infusing HA into the mesenteric vascular preparation of rabbit. Indomethacin prevented, whereas arachidonic acid (0.2 ugAnl) augmented the HA - evoked Pq release. The authors noted no prostaglandin output from perfused or 3uperfused rabbit arteries unless the small distal vessels were left in the vascular preparation and therefore proposed the az’teriolar wall or precapillary vessels as the site of prostaglandin generation. Other site at which PG - like substances are released in response to nerve stimulation or humoral substances include rat diaphragm (Ramwell, Shaw & Eucharski, 1965; Laity, 1969); frog skin (Ramwell & Shaw, 1970); guinea-pig vas deferens (Ambache & Zar, 1970) and cat spleen (Hodqvist, Sjarne & Wennmakn, 1971) • The ubiquity of prostaglandin is therefore not in doubt. Despite this ubiquitous nature however, PG3 have not been proved to be transmitters at naive endings although they are known to modify synaptic functions. Their release, especially from muscles, is thought to be transynaptic in mechanism i.e. as a secondary but simultaneous action of the humoral substances. Prostacyclin (PGI0) Prostacyclin is formed by microsomes of several organs such as pig and rabbit aorta (Gryglewski, Bunting, Koncada, UNIVERSITY OF IBADAN LIBRARY - 64 Flower & Vane, 1976); rat stomach fundus, pig mesenteric arteries, and venous tissues incubated with ?G endoperoxides, file PG endoperoxides are then enzymically converted to an unstable product (PGX) which relaxes arterial strips and prevents platelet aggregation. Ilicroconos from rat stomach corpus, rat liver, rabbit lungs, rabbit 3pleen, brain, kidney medulla, ram seminal vesicle as well as particulate fractions of rat skin homogenenate transform PG endoperoxides to PGB and PGP rather than PGX (Grygle.wski et al, 1976). Prostacyclin is the primary metabolite of hrachidonic acid in blood vessels of all species so far tested including man (Bunting, Giygl wski, Moncada & Vane, 1976). This prostaglandin, originally called PGX, was later identified as 5 2 - 5 , 6 - didehydro-9 Jr̂ eox3r-6-9 - epoxyprostaglandin F and renamed Prostacyclin by Johnson, Horton, Kiruier, Gorman, HcGuire, Sim, Whitacre, Bunting, Salmon, Moncada & Vana (1976) or simply PCJI2* Smooth muscle actions of PG51s and PGIo. Prostaglandins are known to produce two kinds of effects on smooth muscle. First, the direct effect which may manifest as contraction or relaxation (Hall & Pickles, 1962; Strong & Bohr, 1967; Mark, Schmid, Eckstein & Wendhing,i9?1), Secondly, they may indirectly inhibit or enhance responses to other agonists. For instance, PGS-] and POE2 can induce a persistent non-specific UNIVERSITY OF IBADAN LIBRARY - 65 - increase in sensitivity to agonists such as adrenaline, NA, tyramine, acetylcholine and serotonin in rat and guinea-pig seminal vesicles (Blliason & Risley, 1966), in rat uterus and guinea-pig ileum (Clegg, Hall & Pickles, 1966; Hall & Pickles, 1963), Also, PCS^ enhances the responses of the rat stomach fundus to acetylcholine (Goceani & i/olfe, 196p) . Prostaglandins S-j and have been shown to enhance contractile responses to nerve stimulation and catecholamines in the guinea-pig vas deferens while having no effect on responses in the cat and actually depressing responses to both nerve stimulation and catecholamines in the rabbit vas deferens (Graham & Al Katih, 1967; Naimzada, 1969 a, b), Euler & Hedqvist, (1969) i Hedqvist & Euler, (1972) reported that low and high doses of PGE inhibited and potentiated respectively the contractile response of the guinea-pig vas deferens to post­ ganglionic nerve stimulation. Exogenous NA was also shown to be consistently potentiated by high dose of the PGSs. They concluded that the potentiation by high doses of PGrJ3.j was post­ junctional since it was greatly or completely antagonised by compound SC 19220 (a PG receptor antagonist). The inhibitory effect, on the other hand has been shown to be a prejunctional effect where the postaglandin modulates the release of the adrenergic transmitter (Hedqvist, 1972, 1974). UNIVERSITY OF IBADAN LIBRARY -66.- The effect of postaglandins -on the isolated vascular smooth muscle in vitro varies according to the animal 6pecie3, the vascular tissue under stud;/, the existing tone of the tissue and the concentration ofpibstaglandins used in the study. potentiates vasoconstrictor responses to exogenous 1TA in the rabbit aorta and mesenteric artery in vitro (Strong <1 Chandler, 1972; Tobian & Yiet, 1970). PC30 on the other hand has been shown to i facilita-te the vaso-con3triction induced by nerve stimulation without affecting the responses to NA in the dog mesenteric arteries and veins (Kadowits, Sweet & Broody, 1976). However, in the anaesthesized rat, PC3.S and PCI^ are hypotensive in vivo. According to Armstrong, Lettimer, Koncada & Vane (1978), intravenous PCrl^ produced hypotension which is 4 “ 8 tines and about 128 tines more than that produced by PCH^ a^d 6-oxo-PCP-ja respectively in rats. AI30, the potency of P m e a s u r e d by its hypotensive effect wa3 2 times that of PCC^ end about 250 times more potent than 6-oxo-PGF,1 a in rabbit. It has been hypothesized(Horton, 1979) that PCs act on receptors that can be distinguished from those for other agonists by actions of selective antagonists. In support of this is the fact that the actions of PCs on smooth muscle are not blocked by atropine, methysergide, mepyramine, propranolol, phenoxybenzanine or hexame- thonium. Three groups of PC receptor antagonists have been described and are represented by (i) polyphloretin phosphate, (ii) mie UNIVERSITY OF IBADAN LIBRARY - 67 - dibenzoxazepirie, SC 19220, and (iii) the 7 - oxapro staglandins. ITone of these antagonists is either very potent or highly selective (Satins 3: Sanner, 1972). Pickles (1967) had put forward evidence for the existence of mol's than two prostaglandin receptors. Many smooth muscle actions of PCs have also been associated with a change in tissue levels of one of the cyclic nucleotides (i.e. either adenylate cyclase or guanylate cyclase systems), further­ more , events at theI cellular (including cell membrane) level have received some attention. Contraction of smooth muscle b y PCs is associated with depolarization alone (e.g. main pulmonary artery, Kitazrura, Suzuki & Kuriyana, 1976) or with'depolarisation and with increased frequency and length of action potential bursts (e.g. rat uterus, Kuriyana & Suzuki, 1976). Relaxation of smooth muscle on other ' hand, has been described as associated with hyperpolarisation of the membrane (Stomach, Hischina & Euriyama, 1976). An increase in membrane conductance has been found both during excitatory and during inhibitory smooth muscle responses to prostaglandins. According to ?Leiner & Iiarshall (1976), it appears there is an action potential - dependent mechanism of tension gene rati on., which can be triggered by P3s(and by other stimulants) which can be blocked by Ca 2+ antagonist verapamil and a more sustained contractile component which does not involve action potential and is resistant to blockade by verapamil. Thtts, as elaborate as the volume of the -li te nature - - on smooth-muscle actions of Pds is, very little UNIVERSITY OF IBADAN LIBRARY - 68 - measure of consistence ha3 been attained as to the mechanism of the various actions. ADI AITD SCOPd 0? PPAISSITT ITOkK The present investigation is aimed at finding out how PC-T^, P ^ 2 a ^ ^ 2 i*1';erac^ with adrenergic neurotransmitter substance, ITA, and agents that antagonize its vasoconstrictor action. The isolated rat mesenteric artery was chosen for this investigation because the aim was to study the interaction between prostaglandins, NA and UA antagonists in arterial smooth muscle in which ITA produces y a purely vasoconstrictor effect. The preparation as it is made completely excludes the venous side. The study was prompted by the finding that prostaglandins of the S series can potentiate agonist action on smooth muscle by facilitating 0a2+ influx (Pickles, 1966, hagling, _et a^ 197s). Prostaglandins are therefore potentially useful agents for investigating ITA supersensitivity mechanisms which occur entirely postjunctionally since prostaglandins do not appear to influence uptake or other inactivating mechanisms of ITA (Adeagbo & Okpako, 1380). This kind of study seem3 worthwhile because it has been suggested that prostaglandins mediate or amplify the vasoconstrictor action of ITA in mesenteric arteries (Horrobin et al. 1974; Coupar & I-IcLennan, 1973; Coupar, 193.0). UNIVERSITY OF IBADAN LIBRARY - 69 - Qjho interaction of with antagonists o f Ilk was also invest!rented, Pô * the jHirpose of the scudy^ antagonists of HA have been classified into three as follows a) o L - adrenoceptor antagonists - phentolamine, tolazoline, yohimbine, phenoxybenzamine and prazosin; b) Non-steroidal anti-inflammatory drugs - indonethacin and sodium meclofenamate. c) antagonists interfering with Ca 2+ i. ons — cm. nari.z i.ne and verapamil UNIVERSITY OF IBADAN LIBRARY M A T E R I A L S A N D M E T H O D S UNIVERSITY OF IBADAN LIBRARY - 70 - MATERIALS AND METHODS Animals Albino rats (sprague Dudley strain) and guinea-pigs (bred from a strain developed in Yom, Nigeria) were used throughout this study. All animals were bred locally in the departmental animal house. The house was adequately ventilated. Rats were fed on standard livestock cubes (Pfizer Nigeria Ltd.) while guinea-pigs were fed on standard livestock pellets supplemented with green grass. All animals had free access to water. Physiological solutions used \\ The physiological solutions used and their composition were as follows:- (a) Krebs solution (mH per litre): Sodium chloride, 113.0; potassium chloride, 4.7; calcium chloride, 2.5; sodium dihvdrogen phosphate, 1.2; magnesium chloride, 1.2; sodium bicarbonate, 25.0 and glucose, 11.5. (b ) Calcium - free Krebs solution This solution has the same composition as Krebs solution described above except that calcium chloride was completely omitted. ' (c) ' Depolarizing krebs solution This solution has the same composition asK:rebs solution described in (a) above except that sodium chloride and sodium bicarbonate were replaced by potassium sulphate and potassium UNIVERSITY OF IBADAN LIBRARY - 71 bicarbonate respectively. Depolarizing Krebs solution used in this study is of the following composition (in ml-'ol per litre):- potassium sulphate, 92; potassium chloride, 10; sodium dihydrogen phosphate, 1.2; potassium bicarbonate, 10; magnesium chloride, 1.2 and glucose, 11.5. When calcium chloride was to be used as agonist, it was omitted in the preparation of the depolarizing solution. (d) Tyrode solution (ml'ol/litre). Sodium chloride,- 156.8; potassium chloride, 2.7; calcium chloride, 1.8; sodium dihydrogen phosphate, 0.3; sodium \ hydrogen carbonate, 11.9; magnesium chloride, 0.9 and glucose, 5.6. Krebs solution was gassed with 57-> Co^ and 95/» 0^ mixture and Tyrode solution gassed with air. Preparation of mesenteric artery for -perfusion Surgical operations were carried out according to the method of Hcgregor (1965). Adult male rats weighing 250g,and above were anaesthesized by diethyl ether or chloroform. The abdomen was opened and the pancreatico-duodenal, ileo-colic and colic branches of the superior mesenteric artery were all tied off. The dorsal aorta was ligated a few millimeters anteriorly and posteriorly from its junction with the superior mesenteric artdry. The latter was then isolated by cutting round the intestinal borders of the mesentery (Fig. 4). Thereafter, the whole preparation was* quickly transferred UNIVERSITY OF IBADAN LIBRARY PX (JURE 4 Tiie preparation of 'mesenteric arteries’ stained with sudan black. The preparation consists of superior mesenteric, jttjunal, and ileal arteries, (a) adipose tissue attached, (b) adipose tissue and veins removed. (Reproduced from "Blood vessels" JJ5 : 279 - 292) WSI: R.A. tTAiiXB co B.B. Panl-il (197o ̂ Isolation and characterization of plasma membrane from rat mesenteric arteries Blood vessels (1976) J!:279-292. UNIVERSITY OF IBADAN LIBRARY UNIVERSITY OF IBADAN LIBRARY - 73 - unto the surface of a conical flask containing water at 37°G. The artery was cannulated and perfused with Krebs solution whose composition is given above. The solution was bubbled with 5/= Co^ and 0 ^ mixture and was perfused through the tissue at a constant flow race of 4 ml min ̂ with a ¥atson*-.Marlow constant flow inducer (type KHR3-88,'. The preparation was carefully arranged on a blotting paper moistened with krebs solution which was placed on the surface of a 250 ml conical flask in which water at 37°C ;i circulated. The preparation was lightly covered with moist cotton wool which was periodically wette\ d with warm Krebs solution from a pipette. An angle poised lamp'arranged above the flask ensured that the whole preparation was maintained at 37°G throughout the experiment. Changes in perfusion pressure were recorded by a Bell & Eowell pressure transducer (type 4-327-L -223) on a devices M.19 recorder. All arteries set up were allowed to equilibrate for at least 30 min. before the commencement of the experiment. Experimental Procedure Drugs we re injected through a pressure tubing placed just before the constant flow inducer in volumes not exceeding 0.2ml and at 5 min. intervals. In order to ensure that the tissue responses are reasonably constant, two or three ^ose responses were recorded following injections of geometrically increasing doses of the agonist. The time interval between agonist injection was 5 min. and responses UNIVERSITY OF IBADAN LIBRARY - 74 - obtained in this manner are termed 'control responses'. In cases where the artery was perfused with prostaglandin or antagonist or any other test dmig or vehicle, the Kreb3 solution weu» prepared to contain the desired concentrations of the drugs. In such a medium, the artery was allowed to equilibrate for at least 10 min. before the next addition of the agonist. Ascertaining the potency of prostacvlin (PGI^) The half-lifd (tj) of PGI2 at pH 7.48 is between 3.5 min and 10.5 min. at 25 C depending on buffer concentration, but with shorter at higher buffer concentration. At pH 12, t? is approximately 6 days at 25 C (Cho & Allen, in the manufacturers ©annual). The biological activity was sustained at pH 9*37 (o.05K tr is-buffer) for 48 hrs. when stored at 0°C. Under our working conditions, ¥ sodium carbonate solution pH 10 was used to dissolve PGI2. In view of the intermediate position and owing to the relative unstable nature of PGI2, it was important to ascertain on a daily basis the retention of potency of an aliquot of the prostacyclin before U3e. The model U3ed for this purpose was the inhibition by FGI2, of the aracnidonic acid-induced human platelet aggregation. Platelets obtained from the blood o: voluntary donors in the University Teaching Hospital, Ibadan, aggregated in the presence of low concentrations of aracfcidonic acid (a a ). The aggregoneter used was Payton module made by Payton Associates, Scarborough, Canada. Low concentrations of PGI2 would prevent UNIVERSITY OF IBADAN LIBRARY - 75 - this action, and the ability to do so is taken as a means of ascertaining the potency of various aliquots of the prostacyclin. A particular POI^ aliquot is assuned to have lost potency when it can no longer prevent AA - induced human platelet aggregation or when the concentration at which it does so has become considerably high. Hetabolism of ra-ostnvlnndins in rat mesenteric and minea-ni? pulmonary vasculature . _ ' f In order toideteimine whether the effects of the infused i .prostaglandins was due to the primary prostaglandins or their 'k metabolites, the following experiments wore performed: v (a) Inactivation of -rostaglandins on rerfusion throu-h the rat mesenteric artery Different concentrations of PGH^ an-0.005 ** Statistically significant p > 0.005 UNI —J k 0 I» V C T i ERSITY OF IBADAN LIBRARY pisuihi 7 Prostaglandin - induced potentiation of noradrenaline vasoconstrictor responses in relation to .prostaglandin dose, in rat mesenteric artery. (0) prostaglandin (PGE ); { 0----0) P0F2p and (®) prostacyclin (PCJI ). Each point is a mean of measurements frora 8 separate preparations. Vertical bars are s.e mean, UNIVERSITY OF IBADAN LIBRARY t.-.: - . -■ - ; „' *S I - V-i.r-i-: :■ • • *■ •: — -■ 7r- ..• .-‘ . ... - : -H^' ; . » • • : . .■ y : * : « . - • • . • V'' ... • \ S * ■ , V. 7 : -- -••••- : ̂ 1 t « t - -r-v - - ' • , ‘ • 4. - T■"f* * -s'.«- u., - t1 . . i : -^-7 ■ ri. 1. •;.-:* 5 :■*:***::', • F ; V \ "T vi /’ . ’-: ■ r '- • • ' ' - - - r. T .v..-' F- ' 5 **-. •. « :■y ,-r- .% - • •-.* -- $ r.. • • ^ ;t. ,fgr>•;£?;& ^■ : 'T.' -*T '~.•*■>>* ': -- c .*f t/,■;' m : : & m ■ ; ■ . . ■ ' ^ -tV, feg 7’ • ■ m : T '7 '■ . . • > m 's '■ ‘ - , .7 ■ M ' '■ '■ - ; :' ;7 ;;v; 7 : ■: • ■ ; ■ : ? .% 7;: \ ' w 77' - ,. •" ' .7 - . -.Jut A: ^ -TrI ' • ?*' *•» «—•A -/,f . '#-f• k . ! i , . - - * • - • ' - i - ' . - ? L7 r. . ftiv'■ ■ ] •-/ V -l - jS-i > .**--? 7-7 7 7 l7:7. •.7 75VX '.7 .. - ^ :-..7 .*. -77C‘77 i. . . . W.. 'j.t ; , 6- i. ^ 4- H+ i- •» t ;7C ' V; >2- -X'.• i 2- ------- - ? **?'•*’• 0 10- 9 r8 10 •7 -61 0 ' 1 0 ? *»•::k /V : . r > . • PROSTAGLANDINS ! if ' ,*.y j :< i7v :•■ ■C- v >•' } • V « i t . - : r * * S• .. . . . <• .' r >r7-V; ?7.‘5-irs :iVAt’f-v7l'^ /l’ •5 ? “ 'fi--v-v ;....v. . ■ ■ . 7 , ' 7 y-- r' ■ f' T i . . • 5 . i ^ ■ - r,-• * • W v ' ; . . . . • » . ' .• • ■ . . • : •- r- • • >.■> ,:7.;., r.- /■ •7. ; • ■ • r . • • . ■ : • •' . . . . . \ l<' *• . ’ . V ‘ ‘ -.*-v ■ - . ■ i .;:*t i U P.F (SGGNIVERSITY OF IBADAN LIBRARY - 88 - Anti-aggregatory action of POI Platelet rich human plasma is turbid and almost opaque in nature. When caliberating the aggreyometer, the zero margin was set by slotting a micro tube containing high concentration of platelets while the upper (100/5) margin on the aggregometer chart was set using clear tris buffer solution. Following addition of low concentration of arachidonic acid (a a ) there was a gentle rise of the recording-pen towards 100̂ 5 level - an indication of aggregation. This was proceeded by a short latent period. Addition of PGI^ before AA prevented the aggregation (Fig. 8). The anti- aggregatory property was due to the PGI,-, and not the vehicle (llâ Co^ solution) since the latter, added before AA did not prevent platelet aggregation. Inactivation of -prostaglandins in rat mesenteric vascular bed It was important to know whether the effects of the prostaglandin seen in these experiments was due to the primary prostaglandins or their metabolites since it was possible that the potentiating effect of the prostaglandins was caused by metabolites. PGF^ or PGF?rN at a concentration of 10 g ml" wa3 therefore perfused through the preparation and the effluent assayed against the parent solution as described in methods. Percentage inactivation of the prostaglandins was calculated as follows (PG concentration infused) - (pG concentration in nerfusate)x 100 PG concentration infused 1 UNIVERSITY OF IBADAN LIBRARY FIGURE 8 Effect of prostac; clinCPGI^) on aracliicloiiic acid (m ) - induced aggregation of hunan platelet - rich plasma, platelets were incubated in an aggregometer at 37 oC with constant stirring for; 2 min. with either tris buffer (pH 7.4) (control) or tris buffer to which prostacyclin (13.5 mil) has been added. The dose of AA used to induce platelet aggregation was 0.5 Eli. UNIVERSITY OF IBADAN LIBRARY UNIVERSITY OF IBADAN LIBRARY The r e s u l t s a r e sum m arized in T ab le 2 . I t can be se e n from th e table that only about 20fo of POE C. and PGF_C. & was inactivated during perfixsion. Table 2 I~ nactivation ~o f PGE„ aTn d ~PGF„_ in rat meartery~ ‘ s.e.n..t.e.ri~rc" Cone, of prostaglandin Kean fo inactivation of in perfusion medium added prostaglandin - S.E.M. g ml"1 p g e 2 P® 2 a To-6 20.6 - 3.1 23.0 - 3.9 (n = 8) (n = 8) 10"7 19.0 - 2.1 20.0 - 2.5 (n = 6) (n - 6) This degree of inactivation was considered substantial enough. It was therefore desirable to establish whether the metabolites of PGE2 and PGF2a had NA potentiating actions in the mesenteric artery. Thus, concent rations of PGS2 and PG?2a causing NA potentiation were inactivated by passage through the guinea-pig lung pulmonary vasculature, and the effect of the perfusate effluent on the vasoconstrictor responses to NA investigated. When 10 -8 and 10~7 g ml-1 PGE2 and PGF2a were perfused through guinea-pig lungs, no activity in the effluent was detected on the assay tissues UNIVERSITY OF IBADAN LIBRARY (rat stomach strips for PGE0 and rat colon for PGF0 ). The sensitivity of the assay tissues was good enough to detect 0.5 ng ml ̂ (Pig. 9). The effect of the effluent used immediately after collection on NA vasoconstrictor responses in the rat mesenteric artery are shown in table 3. Only the effluent from 10-7 g ml-1 showed slight potentiation. Table 3 The e..f...f...e...c..t... o.f1. two conc..e ntrI a.t...i Io. n| s. oI.f PGE„ an1.d I P0F„ after passage through ?uinea-pig -pulmonary- circulation on vasoconstrictor responses to HA in the rat mesenteric artery Cone, of prostaglandin in ❖lean P.F. due to lung perfusate Krebs solution perfusing the lungs g ml 1 p g e 2 PGF2a 10"8 1.1 - 0.2 Not done (n = 8) IQ"7 1.4 - 0.3 1.3 t 0.2 (n = 6) (n =8) Interactions of prostag;!andins with cocaine and methoxamine Cocaine is an established inhibitor of neuronal uptake of the adrenergic transmitter substance (iversen, 1967). In order to find out whether prostaglandins potentiate NA by inhibition of uptake, maximal potentiating concentration of cocaine was obtained as described under methods; and was used separately and in combination UNIVERSITY OF IBADAN LIBRARY FIGURE! 9 Assay of guinea-pig lung perfusate on rat stomach strip (.RSS)' and rat colon (RC). The biological activities of PGF„2 a and PCS-2 were detected more specifically by RC and RSS respectively. Doses under the dots are in ng while *3* in either case represents the guinea-pig lung effluent. UNIVERSITY OF IBADAN LIBRARY UNIVERSITY OF IBADAN LIBRARY _ 09 ~ with submaximal potentiating concentration of P3E„ (found, to be 10 ^g ml on the same preparations. The principle of this test is based on the idea that if PGE2 is producing potentiation by- blocking uptake (as assumed for cocaine), then it should not work in a situation where uptake is completely blocked e.$. by a dose of cocaine which produces maximum potentiation. The results are presented in (fig. 10) . Prom the figure, the P.P. value due to the submaximal potentiating concentration of PGE2(a ) was 5*2^ 0.1 (n=6) and that due to the maximal potentiating concentration of cocaine (b ) 5.6 +0.1 ( n = 6 ) while that due to the combination of the former procedures ( c ) was 5*9 - 0 . 1 (n = 6). B and C are significantly different p / 0 . 0 5 . Hethoxamine caused vasoconstriction of the rat mesenteric artery similar to vasoconstrictor response of NA (although less potent than the latter) (see Pig. 5). Dose - response curves for methoxamine were obtained in the absence and presence of PGE2 and PGF2a and results compared (Pig. 1l). Methoxamine which is not a substrate for the uptake process (iversen, 1967; Trendelenburg et al, 1970) was potentiated by both prostaglandins. Interactions of prostaglandins with PA in reserninized rats and in Krebs solution containing bretylium The mesenteric artery is innervated by a meshwork of adrenergic neurones (Furness & Marshall, 1974). In order to examine whether Prostaglandin - induced supersensitivity was mediated by release UNIVERSITY OF IBADAN LIBRARY FI GPRS 10 Perfused rat mesenteric artery: effect of cocaine and PG2 on ITA vasoconstrictor responses. Shown in the figure are the dose - response curves to ITA (x) alone; (f) in the presence of 10 ng/nl FG^j (o) in the presence of 100 ns/ml cocaine; and (A) in the presence of 10 ng/ml PGGp and 100 ng/nl cocaine combined. Standard errors are small and completely masked by the symbols. Rach point on the graph represents a mean ox 6 - 8 experiments. UNIVERSITY OF IBADAN LIBRARY ~ • i < • • • * * .,_***» 'v.- ■ ' $ - - - ^ • •• . ' • T ‘V- • : .?fK* v v ; - a . ' ■ ■ ;.'S • • ' H ' • i? ; *. * !• t , • . ■’ •. .• ! = • • *. -•- . UNIVERSITY OF IBADAN LIBRARY FIGURE 11 Potentiation of methoxamine - evoked vasoconstrictor responses by prostaglandin 2̂ (PGB^) an^ PCS?2a ra^ mesenteric artery. The first set of responses in each panel are control responses, PG-S^ (a ) or P G F ^ (b ) , in the concentrations shown was introduced at the arrow. Kethoxamine responses were repeated 5 min. after adding a prostaglandin. The doses of methoxanine under the dots are in ug. (a ) and (b) are from separate mesenteric artery. UNIVERSITY OF IBADAN LIBRARY tf 2 E2 _ 10ng/m| LPGF?n1 0 n g / m | UNIVERSITY OF IBADAN LIBRARY - 9 5 - of transmitter substance, from the adrenergic nerve endings, 6 rats were given a daily dose of reserpine (10 mg/kg) for 3 days before isolating and setting up the artery as described earlier. The potentiation factors due to PG32 and PGF2a (10— 8 g ml —1) in each case on the vasoconstrictor action of NA in control preparations were 5.0 + 0.9 (n = 6), and 2.2 + 0.1 (n = 6) respectively. In reserpinized rats, P.P due to PG52 was 9.6 +1.1 (n = 6), a value that is significantly higher than control (analysis of variance p / 0.005). Pig. 12 shows a comparison of ED_q to NA obtained in the control and reserpinized rats. It can be observed from the figure that animals pretreated consecutively for 3 days with reserpine were more sensitive to exogenous NA. No such effect was observed in mesenteries isolated from rats pretreated for 1 or 2 days before setting up or when reserpine was administered in vitro (i.e. added to the perfusion medium). In preparations perfused with Krebs solution containing bretylium (10 ^g ml )̂, the P.P. values for FG32 and PG?0a were 3.0 + 0.6 and 1.7 + 0.6 respectively (n = 6) in either case). These * values are significantly lower than the controls (i.e... the potentia­ tion induced by the two prostaglandins were reduced in the presence * of bretylium). NA antagonism by phentolamine, tolazoline. yohimbine and rhenoxybenzanine (F3Z As would be expected, phentolamine ( 3. 2x 10 —8 - 2 5 . 2 x 1 0 *8M); tolazoline (2.5 x 10 ^ _ 4.0 10 ^M) and yohimbine (5.0 x 10"”® - 12.8 x 10 —T M) blocked NA in a competitive manner; (a') the NA dose - UNIVERSITY OF IBADAN LIBRARY :Ci#: FIGURE 12 Comparison of the ED_q of 3SA in control (a ) and reserpinized rats. (b ). In B, x~-----x, 0------0; and 0--------- 0 represent responses to NA 24 , 48 and 72 hours respectively after reserpine pretreatment. ED20 in control rats = 0.38 ug after 24 hrs of reserpinization = 0.4 ug " 48 hrs " t t = 0.36 ug i t i t ^2 !f f t = 0.20 ug. EDp.Q in control rats were statistically higher than those obtained in the rats 72 hours after reserpinization (p / 0.005: analysis of variance). UNIVERSITY OF IBADAN LIBRARY Response (mm Hg) UNIVERSITY OF IBADAN LIBRARY - 97 - response curve in the presence of the antagonist was shifted rightwards parallel to the control curve; and (b) the slopes of the A - 3 plots 0.96 - 0.04 (phentolamine); 1.03 - 0.04 (tolazoline) and 0.85 - 0.02 (yohimbine) (n = 8 in each case) were not signifi­ cantly different from 1 (p>0.05). In bbs 0̂39 ts.tit'Q (7. 4 x 10 — 2*9 ^ **) PB2j block of* NA was surmountable by increasing the dose of NA (Fig. 13) and the block exhibited characteristics of competitive antagonism viz, parallel rightward shift of the NA dose-response line; the slope of the A-S line plotted with dose ratios obtained in the presence of three different concentrations of PBZ was 0.87 - 0.01. This value was not significantly different from 1. Furthermore, when pA£ was calculated from the equation = log (DR-l) - Log(l) where (i) = concentration of PBZ, the. values obtained with three different concentrations of PBZ were not significantly different from one another as indicated by zero regression of the plot of -pk^ versus Log(l) (Fig. 14). This suggests competitive antagonism (Mackay, 1978). At higher concentrations of PBZ, antagonism of NA vasocon­ striction was not competitive. In concentrations higher than 2.9 x 10~^ M, the block of NA could not be overcome by increasing the dose of NA. Effects of PGE„ on &C- adrenocentor blockade Antagonism of NA vasoconstriction caused by phentolamine, tolazoline and yohimbine were consistently reversed by PCS- (Figs 15 a & UNIVERSITY OF IBADAN LIBRARY F I3 U R B 13 The block of NA vasoconstriction by phenozybenzanine in rat mesentery. The first set of responses in each, panel are the control responses. The second set were obtained in the presence of the stated doses of PBZ. Doses indicated by dots are in ug. (a ), (b) and (c) are from separate artery preparations. UNIVERSITY OF IBADAN LIBRARY UNIVERSITY OF IBADAN LIBRARY FIGURE 14 The figure shows a plot of pA2 against antagonist concentration. The pAp values were calculated from the equation pl^ = Log (DR-1) - Log (i) where (l) = cone, of PBS (according.to Mackey, 1978). Each point on the graph represents the near, of 6 - 3 experiments. Vertical bars represent s.e mean. UNIVERSITY OF IBADAN LIBRARY UNIVERSITY OF IBADAN LIBRARY - Log. Molar Cone. PBZ Reversal of phento laiaino antagonism on HA vasoconstrictor responses by PCH0 in rat mesenteric artery preparation. Roses of 1TA indicated by the dots are in £g. Responses in the second panel v;ore obtained in the presence of 3.2 x 1CT% phentolanine while those in the third panel were in the presence of phentolamine and (2.8 x 1CT^k ) combined. UNIVERSITY OF IBADAN LIBRARY - 100 - 12-8x1Q~8M RGB 3:2x10 8M Phentolamine -— — ■ • > UNIVERSITY OF IBADAN LIBRARY Reversal o f phentolaraine (3 .2 x 10 H ); phenoxy- ben.zer.inc (2 .9 x 10 ^ l ) ; tolazoline (4 x 10 ^M) and —7 yohimbine, (12.8 x 10 'll) antagonism on 1TA vasoconstrictor, responses by PC^ in rat mesenteric artery preparation. (ft) control responses to NA, (o) responses to HA in the presence o f each o f the antagonists and (0-------0) responses to HA in the presence o f each o f the antagonists and 2.8 x 10"6!! P(JE2. Each point is a mean o f 10 ~ 12 experiments and vertica l bars represent 8.E .II. UNIVERSITY OF IBADAN LIBRARY 101 PHENTOLAMINE YOHIMBINE NA(Ug, 0.1Log. scale) • 10 .100 PHENOXYBENZAMINE. 10 100 Response (mm Hg) UNIVERSITY OF IBADAN LIBRARY 102 - the degree or reversal being d irectly proportional to the concentra­ tion o f the prostaglandin. Antagonism o f NA vasoconstriction by PBZ in the concentration range (7.4 x 10 11 - 2.9 x 10 1°M) in which the block was competitive, was also reversed by PGE2 (Fig. 16). Block by PBZ at higher concentration were not reserved by PGE2. The degree o f reversal by FSB o f the block by each o f the antagonists was estimated by determining dose-ratios with the antagonist before and in the presence o f d ifferen t concentrations o f PGÊ . The results are shown in Table 4. Concentrations o f PCŜ ranging from 2.8 x 10 ̂ - 2.8 x 10 greatly reduced the NA dose ratios obtained with the four antagonists. The a b ility to PGÊ to reverse competitive type block o f £>(- adrenoceptor may be taken to suggest that PGE2 interferred with antagonist - receptor interaction. I f this were so, different- binding characteristics for the antagonist might be expected. To test this possib ility , p for each antagonist was determined -8 against NA before and in the presence o f 2.8 x 10 M PGE_. The results, presented in Table 5, show that PGÊ does not reduce the a ffin ity o f antagonist fo r the receptor. Indeed, in the case o f phentolamine, and tolazoline, PGS2 sign ifican tly increased the pa2 values. PGF„ was tried against phentolamine block and was found / not to cause any change in pA2 value o f the antagonist although ' ̂ reversed its blockade o f NA e ffec t. UNIVERSITY OF IBADAN LIBRARY Several o f phenoxybeiizaraine antagonism on IT A vasoconstriction, by ^ rat mesenteric artery. Doses o f 1TA indicated by the dots are in ug. Responses in the second panel were obtained in the presence o f 2.9 x 10 "'Si PBS while those in the third panel were in the presence of PBS and PGUg (10 ag/ml) combined UNIVERSITY OF IBADAN LIBRARY { - 1C3 - FIG 16 10nq/ml PGF 2.9x 10~10M PR 200mmHg UNIVERSITY OF IBADAN LIBRARY fa - 104 - Table 4 O ffse t o f P05 (2 .8 n I 0~ai.i) on H\ blockade caxtsed bv various *K. - adrenoceptor antagonists ~ in iso la ted Toa.rfu.3ed r 4 mesenteric a rtery , The Bean viylues are derived from 6 - 9 exixeriments. Goncentx-ation HA dose-ratio HA dose - x-atio HA dose - AHTAGOITIS'1' o f antagonist in the presence in the presence r-bio in of antagonist o f antagonist + - m APS the presence alone (l®^) PSS2 O a^ j,,) ("i?.eversal factor” ) of i a (J -O1/ 2 ■ . EH2H TOLAI-IINE 3.2 z 10-3I-I 13.9 + 1.0 2.5 ± 0.1 11.4 + 0.8 T0LAZ0LINE 4.1 z 10” 5 22.0+ 1.0 5 . 9 + 0.4 16.1 0.9 YOHIMBINE 1.28 2: 10~6H 26.6 +_ 0.9 1.7 + 0.1 24.9 + 0.9 FHSHOXIBENZ-U'IIIfE 2.9 x l0~1Ck 17.9 + 1.0 8.4 + 0.8 9.5 + 0.7 __________________ i 1 1 r 1 1 UNIVERSITY OF IBADAN LIBRARY tv» 0 +! 0 !A - 105 - Table 5 Effect of ?SS0 on Pi - adrenoceptor antn/ronisn in isolated rat mesenteric artorv N0RI-1AL iUiHBS LUAIN} 2.S x 10- 31 PaE? FEHiUSlON ANTAGONIST pa2 SLOPE OF SLOPS OF A - S PLOT A - 3 PLOT ffiENTOLAHINE 8.58 +0.11 0.96 + 0.04 * 8.95 ± 0.14 ** 0.92 + O.O; U = 8) in •- 8) TOLAZOLINE 5.69 + 0,01 1 .Op hh 0.04 •* * 6.15 + 0.01 ** 0.97 + 0.0; (n = 8) in = 8) YOHIMBINE 7.48 + 0.07 0.85 + 0.02 ** -7.54 + 0.04 ** 0.83 + 0.0 (n = 8) (n = 8) PHEWOXYBEN ZAMHIE 10.84 + 0.01 0.87 + 0.01 ** -.10.55 + 1.10 ** 0.83 + 03 (PBZ) (n = 6) (n = 6) * Significantly hij^ier than controls (p ^O.OOo). ** Not significantly different from controls (p^.0.05). UNIVERSITY OF IBADAN LIBRARY CM Ph — 106 - Interactions of i ndomethacin and -prazosin with NA and the e ffec t o f PGEp on the HA block -produced The inclusion in the perfusion flu id , o f indomethacin 2.8x1 Q~7M-2.8x 10~^M 0r prazosin ( l -3 x 10"1°M - 5.3 x 10“ 1°M) 7 greatly inhibited the vasoconstrictor action o f NA. The threshold dose was not appreciably changed in the presence o f each o f these antagonists. The magnitude o f block in either case increased with time (F ig. 17). Also, the usual characteristics o f the response to NA ( i . e . quick contractile and relaxant phases) was changed by indomethacin and prazosin. The relaxant phase became XSa If vp-py ■ prolonged (F ig . 18). Thus, the drugs increased the duration o f action o f each dose administered. The block was rapidly over- come by washing out the antagonists. PGEp 2.8x 10 .M, reversed -7 completely the indomethacin block due to low doses '2.8 x 10 M and 2.8x10"fk-However, with 2.8x 10 \ indomethacin, reversal by PGEp was inconsistent, occuring only in 17 out o f every 25 experiments (Figs 19 and 20). Prazosin block o f NA on the other hand, was consistently irreversib le by PGEp (F ig. 20) even up to concentration /* rj —6 o f 2.8 x 10 M. The block produced by 2.8 x 10 - 2.8 x 10 M indomethacin and 1.5 x 10 - 2.6x10 ̂ M prazosin, was surmoun­ table by increasing the dose o f NA. However, there was l i t t l e reduction o f the maximal response (to about 80£a le v e l) when indome- thacin and prazosin concentrations were increased to 2.8x10 -5M and 5.3x10 respectively. The suppression was more severe when the concentration of the antagonists was further increased. pAp values UNIVERSITY OF IBADAN LIBRARY I 1 2 M J J Z Perfused rat mesenteric artery preparation; Increasing HA dose ratio in the presence of 2.8 s 10_5I-I indomethacin (o) and 2.5 x 10”""' prazosin (* ) . Each point ox* the graph is a mean of 6 experiments on separa tissue preparations. Vertical bars are s.e mean. UNIVERSITY OF IBADAN LIBRARY NA Dose ratio UNIVERSITY OF IBADAN LIBRARY ' i n n ; 18 Isolated rat mesenteric artery prei paration; e ffe c t o f prazosin on the nature o f 111 Yasoconstrictor responses. Poses indicated by the dot: 3 are in up. Responses to HA in the presence o f indcir.ethacin are similar, UNIVERSITY OF IBADAN LIBRARY Fig. 18 - 103 - UNIVERSITY OF IBADAN LIBRARY FI1UR3 19 The block of UA vasoconstriction by indomethacin and its reversal by PSE0. .The f ir s t panel are the control responses; the second are responses in the presence of 2.8 x 10- 5M indonethacin while the third panel represents responses in the presence of indonethacin and PGĤ (10 ny/r.ii) combined. Doses of NA indicated by the dots are in vig. UNIVERSITY OF IBADAN LIBRARY V\ - 109 - FIG 19 m5min . * M L -M * G - M - . & £ O O O O n o O t * w o ^ r i ^ O O O - T ^ U J c n f s j J > - C O C D g o o ^ - S ^Go Vcn" h O N J Q O c n | s j 10ng ml"1 PGjE? 7 .8 x 10~5 M Indomethacin UNIVERSITY OF IBADAN LIBRARY FIGURE 20 Interaction o f FG32 with indomethacin ( a ) and prasosin ( b ) . (z ) control responses to UAj (c) -8 responses to NA in the presence o f 2.8 - 10 M indo- methacin in A and 1.3 x 10~1°K prazosin in B; and (© ) responses to NA in the presence of the antagonists and P032 (2.8 x 10 M). Each point on the graph is a neon of 6 - 8 experiments. Vertical bars re present S.3.M. UNIVERSITY OF IBADAN LIBRARY i o o 0-1 i o o iS o U g NA ( Log scale ) UNIVERSITY OF IBADAN LIBRARY 111 (see chapter 2) were measured at any three concentrations at which maximum response to NA was not below 80^. When such values were p lotted against antagonist concentrations, the resultant line was not linear indicating that the block was hcn-competitive in nature (F ig. 21). E ffec t o f PTE ̂ on ITA block produced by cinnarizine and veranamil Both cinnarizine and verapamil are potent antagonists o f the excitation - contraction coupling system in the vascular smooth muscle (Codfraindd: Kaba, 1972). Neither o f these two substances has been shown to possess 0(, -'adrenoceptor blocking a c t iv ity \ although i t has been observed in the course o f this study that both compounds are potent antagonists o f NA -induced vasoconstrictor responses o f the rat mesentery. NA block by these a.gents was always accompanied by severe depression o f the maximum response i . e . the block was non-ccmpetitive in nature. Neither PGE2„ nor PGF~2a in terferred with the blockade (F ig. 22). Calcium ionowhore A25187' influence on the basal perfusion pressure o f the rat mesentery _7 In concentrations lower than 9.6x10 M A23137 did not a lte r the basal perfusion pressure o f the rat mesenteric artery. But, ~ _7 g l l concentrations above 9.6 x10 K consistently induced a characteristica lly slow contraction o f the artery. The slow vasoconstrictor response was characteristic because i t occured only on the f i r s t contact o f the ionophore with the tissue. The UNIVERSITY OF IBADAN LIBRARY FIOUR3 21 Plot o f pA2 values against concentrations of indometliacin ( l e f t panel) and prazosin (righ t panel). Bach point on the graph is derived from 6 - 8 experiments. Vertical bars are the S.e mean. UNIVERSITY OF IBADAN LIBRARY UNIVERSITY OF IBADAN LIBRARY In teraction of PGÊ (10 nr/nl) with the block o f HA by cinnarizine and verapamil, (ft) represents control responses to HA; (o) responses in the presence o f the stated doses o f cinnarizine or verapamil, §---- ^ responses in the presence o f cinnarizine or verapamil plus PGED̂ combined. Each point on the ^raph is derived from the mean o f 8 - 12 experiments. V ertica l bars represent S.E. mean. UNIVERSITY OF IBADAN LIBRARY Fig. 22 - 113 - ClNN4Ri2INE(,0-6gmr') VERAPAMIL ( Sx|0'7g m[R f Response (mm Hg.) UNIVERSITY OF IBADAN LIBRARY contraction faded away until base line pressure wa3 again attained. Subsequent increase in concentration o f innophore in the perfusion medium did not induce vasoconstriction. However, using d ifferen t tissue preparations fo r each concentration o f ionophore in the perfusion medium, the transient vasoconstrictor e ffect seemed to increase with increasing dose of A23187 (F ig. 23). I nteraction of A23187 ’with NA and methoxamine A23187, in concentrations ranging from 1 .9x10 -9 M - 9.6x10 -7M did not evoke any contractile response but greatly potentiated NA - and methoxamine - induced vasoconstrictor responses in the rat mesenteric artery. The magnitude o f the potentiation faded with time (Fig. 24). Influence of A23187 on <%- adrenoceptor block by ohertolamine. tolazoline and yohimbine Like PGÊ , A23187 potentiated NA vasoconstriction and also reversed PC-adrenoceptor blockade over a wide range o f doses. 1.9x10 K̂- A23187 potentiated NA nearly nine-fold. The e ffec t o f this concentration on £?C-adrenoceptor blockade by phentolamine, tolazoline and yohimbine was investigated since one possible way o f overcoming antagonism might be by fa c ilita t ion of excitation - contraction coupling. The results are shown in table 6. UNIVERSITY OF IBADAN LIBRARY FIGURE 25 Vasoconstrictor action of increasing doses o f A2J187 in rat mesenteric artery preparation, fhe doses indicated by dots are in Molar (m) , Each response is obtained from a separate artery preparation. UNIVERSITY OF IBADAN LIBRARY 115 FIG 23 .© 7 9.6x10' 1-9x10'u 9-5x10 (M ) A23187 UNIVERSITY OF IBADAN LIBRARY FIGURE 24 The relationship of the potentiating e ffec t o f A23187 with time. Each point on the graph is a mean o f observations derived from 8 experiments. Vertical bars represent Q.e. mean. UNIVERSITY OF IBADAN LIBRARY V UNIVERSITY OF IBADAN LIBRARY - 117 - TABLE 6 Effect o f A231S7 ( l «6 x 10 m̂) on HA blockade caused by various Cf'— adrenoceptor antagonists in isolated perfused rat mesenteric artery. Bach mean value is derived from six experiments ANTAGONIST NA dose-ratio NA dose ratio in NA dose-ratio in the presence the presence o f M A " DRA+A23187 in the presence o f antagonist antagonist +A23187 ("Reversal \ac to )o f A23187 alone alone (DR^) TVD A +A23187 BRP PHENTOLAMINE 16.1 i 1.3 6.7 - 0.6 9.4 + 1.1 (3.2x 10^M ) | TOLAZOLINE 22.3 + 1.0 14.3 + 1 .4 8.1 + 0.9 8.7 + 1.0 [4.1 x io “ 5m) YOHIMBINE 24.8 + 0.4 17*0 + 0*8 7.3 + 0,5 [1.28 x 10_6t.l) UNIVERSITY OF IBADAN LIBRARY I t can be seen that A23187 reduced considerably the NA dose-ratios obtained in the presence o f the three antagonists. Interaction o f A23187 with NA in reserpinized rats In order to determine whether A23187 - induced potentiation was due to release o f NA from the adrenergic nerve endings, 6 rats were injected intraperitoneally with reserpine (5 mg/kg) for two consecutive days. A.bout 12 hrs af ter the second injection, the mesenteric arteries were isolated and set up as described under methods. Short-term reserpinization has been shown in an earlier part of this thesis not to sensitize mesenteric artery to NA. Reserpine pretreatment abolished the potentiating e ffec t o f A23187 (P ig. 25). Effec t of vj - adrenoceptor antagonists, prostaglandin synthetase inhibitors (PGSIs), and Ga-?+ antagonists on A25187 - induced contractions of the rat mesentery The mechanism by which A23187 induced contraction o f the mesenteric vascular bed was investigated. Three d ifferent classes o f antagonists were tested. The f ir s t were the adrenoceptor antagonists phentolamine and yohimbine; the second were the PGSIs, indomethacin and sodium rneclofenamate; and the third category were the Ca +̂ antagonists, cinnarizine and verapamil. Since A23187- induced vasoconstriction was tachyphylactic, the control and the test arterieswere from different rats. The height o f contraction due to A23187 was measured in a set o f arteries and compared with UNIVERSITY OF IBADAN LIBRARY FIGURE 25 / -6 * Interaction of A23187 (1.9 x 10 H) with NA in untreated ( l e f t ) and reserpine pretreated (righ t) rats. (x) represents control responses in each of the panels while (o) represents the responses in the presence o f A23187. Each point on the graph is a mean o f 8 - 10 experiments. UNIVERSITY OF IBADAN LIBRARY Fig. 25 119 - (Log scale) { ! Response (mm Hg) UNIVERSITY OF IBADAN LIBRARY - 120 - • the height o f contraction by the same dose o f A2318'7 in another set o f arteries perfused with Krebs solution containing a desired concentration o f the test drug ( P ig .26). The fo inhibition was calculated from the relationship* HA, - HA,.t x 100 'where = height o f A23187 - induced vasoconstriction . , - ■' tin Krebs solution, HA.t, = iheight o f A23187 - induced vasoconstriction during perfusion with Krebs solution containing the test drug! V The results are summarized in Table 7. I t can be seen from the table that neither phentolanine nor yohimbine up to 1.6 x 10 -5M and 2.6 x 10 -5K respectively, inhibited A23187 - induced contraction appreciably. On the other hand, indomethacin, sodium meclofenamate, cinnarizine and verapamil inhibited and almost completely abolished A23187 - evoked contractions. Vasoconstrictor responses o f the rat mesentery to potassium chloride High concentrations o f potassium chloride (KCl) caused an increase in perfusion pressure - an indication o f a rteria l vasocons­ tr ic t ion . The vasoconstrictor e f f ec t was c lea rly dose-dependent and rapidly reversible (see P ig. 5)• Since K - induced contraction o f this tissue is unaffected by short-term reserpinization o f the experimental animals, the responses are not thought to be due to UNIVERSITY OF IBADAN LIBRARY FIGUR3 26 A23137 - induced vasoconstriction in rat mesenteric artery. I,( > 12* arui are responses to A23187 in the presence of increasing concentrations o f indomethacin. B+ and V. are responses in tho presence of phentolamine and verapamil respectively. UNIVERSITY OF IBADAN LIBRARY UNIVERSITY OF IBADAN LIBRARY - 122 - TABL3 7 Antagonism o f A25187-induced contractions o f the rat mesentery bv gi- adrenoceptor antagonists (phentolaming and yohimbine) : POSTs ( indoi^thacin and sodium meclofenamate) and Ca2+ antagonists (cinnarizine and verapamil) Antagonist Concentration ( ) fj inhibition o fm A23187-evoked contraction 3.2 x 10"7 0 Phentolamine -6 3.2 x 10 8.0 + 0.5 1.6 x 1C'5 25.0 + 3.0 2.6 x 10-7 0 Yohimbine -6 2.6 x 10 5.0 + 1.0 2.6 x 10"5 18.0 + 2.5 2.8 x 10"7 15.0 + 1 .0 Indomethacin c2.3 x 10" 68.0 + 3.5 2.8 x 10"5 82.0 + 1.5 2.5 x 10"7 20.0 + 2.5 Sodium f. Meclofenamate 2.5 x 10" 80.5 + 5.5 1.2 x 10"5 90.0 + 5.0 2.7 x 10"7 19.0 + 1.8 Cinnarizine 2.7 x 10"6 50.5 + 5.0 1.4 x IQ"5 95.0 + 3.0 2 x 10"7 20.0 + 2.0 Verapamil -6 2 x 10 65.0+ 4.0 1 x 1C"5 98.0 + 4.5 UNIVERSITY OF IBADAN LIBRARY - 123 - catecholamine release (Fig. 27), rather they could be due to massive influx consequent upon the membrane depolarizing action o f potassium. Effect o f E - g coupling antagonists on K ' ■ - induced vasoconstriction The adrenoceptor antagonist, phentolamine, up to the concentration o f 3.2 x 10- 5 M, did not inhibit K +- induced vaso­ constriction o f the rat mesentery. Also, prazosin, a very potent NA antagonist, did not antagonize responses toECl even when its concentration in the perfusion medium was as high as 2.6 x 10~̂ M. Similarly, indomethacin, up to 2.8 x 10 M did not affect K - \ induced vasoconstriction. On the* other hand, P3Z in doses that have been shown to block NA non-competitively ( i . e . above 2.9 x 10 1 k ) and low concentrations o f cinnarizine and verapamil were potent antagonists of the K+- induced vasoconstriction. The order o f potency being cinnarizine = verapamil > FEZ. The results are summarized in (F ig. 28). Another figure,■■(Fig. 29) compares these antagonists against NA ana KOI.’ It can be observed fronr the la tte r figure that the potent antagonists o f NA-induced vasoconstric­ tion, prazosin, phentolamine and indomethacin in that order were inactive against K+- induced vasoconstriction. E ffect of PGEL on K+- induced vasoconstriction I t was o f interest to determine the e ffec t o f PGŜ on K -induced vasoconstriction. Doses o f PGÊ which have been shown to potentiate NA were tested against K+- induced vasoconstriction. The results UNIVERSITY OF IBADAN LIBRARY F I3 U R 3 27 Isolated rat mesenteric artery preparation ; Dose - response curves to potassiun chloride in normal (x) and reserpinized (o ) rats. Each point on the graph represents measurements from 6 - 8 separate artery preparations. Vertical bars represent s .e. mean. UNIVERSITY OF IBADAN LIBRARY F ig 27 CD XUNIVERSITY OF IBADAN LIBRARY FIGUR5 28 Antagonisn of potassium chloride vasoconstrictor responses by cinnarizine ( A ) ; verapaail (©) ; phenoxy- benzamine (x ) ' and indomethacin ( O ) in the isolated rat mesenteric artery preparation, Sacli point on the graph is a mean o f measurements from 8 - 1 0 separate artery preparations. 3.3 nean are small and cannot be represented due to large symbols used orx the graph. UNIVERSITY OF IBADAN LIBRARY -155- FIG .28 - . - l ; A n ta g o n is t Cone. ( X 10” ^ M ) Log sca le % Inhibition of K+ resp. UNIVERSITY OF IBADAN LIBRARY 2+ Comparative e ffe c t o f c<~ receptor antagonists, Ca / antagonists; and indomethacin on NA - ( a ) and Kcl - ( b ) evoked vasoconstrictor responses in rat mesenteric artery preparation. Responses in the presence o f prazosin (o); phenoxybonzamine ( x ) ; phentolanine (© ); cinnurizine {£$ ; verapamil (@) and indomethacin ( o ) are shown on the graphs Prazosin and phentolanine did not antagonize Kcl - evoked contractions at a l l concentrations used. Each point on the graphs is a mean o f 6 - 9 experiments. S.S mean are small and Qanuot be represented UNIVERSITY OF IBADAN LIBRARY Antagonist Cone. (X IC f^M ) Log scale Inhibition of response UNIVERSITY OF IBADAN LIBRARY - 127 - are presented in Fig. 30. I t can be observed from the figure that PGÊ did not potentiate K+- induced vasoconstrictor e ffe c t in the rat mesentery. EFFECT OF EXTERNAL Ca2+ I t is known that NA and K+ u t i l iz e d ifferen t sources of activator Ca 2+ fo r contraction. Therefore,' i t was important to know the influence o f external Ca 2+: ( a) on the vasoconstriction induced by these aronists ( i . e . NA and K+) Towards this end, experiments, were carried out to study the e ffe c t o f omitting Ca^+ from the perfuming Krebs solution on NA - and K Cl. - y induced vasoconstrictor e ffe c ts . A fte r allowing the preparation to equ ilibrate fo r 30 min. in Câ + - free Krebs, 0.8 ug NA or 16 mg KC1 was injected at 5 min. in terval over a period o f 1 hr. I t was observed that NA action was substantially sustained over this period o f time while responses to kcl were d rastica lly reduced within the f i r s t 15 min. (Figs 31 a and 31b). I t was thus evident that external Ca^+ played a greater role in K+- induced contraction than i t did in NA - induced contraction. pi (b) on the potentiation o f NA by PCE ,̂ PCF and Ca ionophone, A25187 A possible involvement o f Ca 2+ on the potentiation o f NA induced by prostaglandins or A23187 was investigated by elim inating pi Ca ions from the krebs solution perfusing the artery. F irs t, the e ffe c t o f omitting Ca^+ on dose response curves to NA was studied. UNIVERSITY OF IBADAN LIBRARY FISORg 30 E ffect o f prostaglandin (PGEp, 1° ng/nl) on vasoconstrictor responses o f rat mesenteric artery to potassium chloride: (o) control responses; # responses in the presence of PGEp. Each point is a mean of measurements from at least 6 separate preparations. Vertical bars represent s .e . mean. UNIVERSITY OF IBADAN LIBRARY FIG 30 UNIVERSITY OF IBADAN LIBRARY KCI (mg) Rat mesenteric artery preparation: E ffect o f 2+. C& omission from the Krebs solution perfusing the mesentery on HA vasoconstrictor responses, The dot indicate responses to 8 ug HA injected at 5 min. intervals over a period o f 1 hr. UNIVERSITY OF IBADAN LIBRARY UNIVERSITY OF IBADAN LIBRARY 200mm Hg -gi.sraa 31b Respon3e( s) of the perfused rat mesenteric artery preparation to potassium chloride (16 mg) m normal Krebs solution ( a ) . The responses in B, wore induced to Kcl during Ca2+ emission, at 5 min. intervals a fter allowing the artery to equilibrate for 30 min. in Ca2+ - free Krebs. UNIVERSITY OF IBADAN LIBRARY - ?.-• • -v *> -A f ’ /'A, • ■ a .> V */;< i.. i V - •• ' J\W- v..<<; C * V? • - 1 , 5 0 » * ' * ,• « ! > * 1 * - * v r ’v ' * . . . •• •• **. • ■ ‘ . 7 - f » 'A*> -• • < . * ’ ’ 'll’ :•;*_.*« ^ifv' • ? -{ f • .,*• tV.V .tfV.v'" •. . .*V .' -M- » w- *. v.*..'-V. —..V f,*V *• / ***-»-■ f . ’ *■:•ov -'.-v A*h.*,■• •»'»*- ; • - i ••v»\ •. •• ■ <| • '• 1 •S ' « " - - . « v j , ■ ^ ~rr- •*■ <. .v- ■ « . . » “«■ ; , " , . 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V ; . , : F F ■ •B. i m :: V . . . • -•••• 1 *. v ■ * •* >v •t : ■ , ' . !.* . t • V . ’ .* 1 «* .* :7.:. f $ r . 3:7'*7 • . . . f v ' ; ' V V ' ‘ k* T* j- ' v’.. ' "7 ‘.J > J/y.. ..*••. . ••Vf..- ________ • • • -■"‘W'.fMV »' . . ■ 7 'V r 'A v • i7 'f- ri f •■ t -• • • H .• «*x,r ■ t; ,■ • • ? • ■ ••••■• '.•••■ •' • - • »• s 1 t r f o i ? ! 'v;’7 *»••';• . v “t',;.. ‘ 7Vf -' ... • vs. ■ •.. •'•. .. •. • . . • .• . . i ; V • ' • " ‘ 4~ r'- - ■ - , ‘ • . i / *■ . ' . |.f ■' - • . / - v s !.. •: T 'V ’l v - ' ' ' ' .• ' • V > . ■•••,-. .V--:- . . . .. ' v - ; 7 \ . - • ' ' V . - v r • 1 . . .• ;>• • • • •:. ... .1 - ' • ::vi . ..' Ji-.-.V A tfv -f, •»'• ^ ...v : .■ ... ; - v f . V ..v.. r - ; : • . . , y ■?. J I ’ A - - •• .- •••• ,v ‘ . t f . -Z G 7.> • . . •• .. ■*) . ’r . • • r/•• . .... •- ,7 -*■ ' V 'f ' ̂ * * m * ;* n V*;,V J**V i*'-' »**-:•*' ■- -j'... I 'l. '. V|.. I. . ’ /V ': / v 4*1 S. vt UNIVERSITY OF IBADAN LIBRARY - 131 Control dose-response curves were f i r s t obtained in normal Krebs', then the tissues were exposed to Ca2 -free Krebs fo r 30 min. Dose - response curves to NA constructed within 60 min. o f Ca2+ removal were not s ign ifican tly d ifferen t from.control curves constructed in Krebs containing 2.5 mM Ca - (F ig . 32). The e ffec ts o f rrostaglandins or A23187 on NA vasoconstriction in . 2+ Ca free Krebs were studied by constructing dose—response curves to NA in the absence and presence o f prostaglandins or A23187. The results are shown in Figs 33 and 34 for PCE0 and PGF respectively and F ig. 35 fo r A23187. From the graphs, i t can be seen that prostaglandin - or A23187 - induced potentiation o f NA 2+V was completely abolished in Ca -free Krebs solution. In another set o f experiments, the relationship between prostaglandin - or A23187 - induced potentiation and concentration 2+ o f Ca in the perfusion medium was examined. This was done by Obtaining PF values in Krebs solution containing d ifferen t concen­ trations o f Ca2+ ions. A separate preparation wa3 used at each Ca 2+ ion concentration. I t can be seen from Fig. 36 that the PF values increased with increasing Ca2+ concentration up to (2.5mM fo r PGE2 ; 1.25mM fo r A23137) and decreased on further -increasing 2-l the Ca" ions in Krebs solution. This results showed that external r\ Ca + was crucial fo r the potentiating e ffe c t o f PGÊ and A23187. ( c ) on NA- blockade by o i - adrenocentor antagonists and on reversal by FGŜ NA caused vasoconstriction by stimulating p adrenoceptors UNIVERSITY OF IBADAN LIBRARY FIGUR3 32 Vasoconstrictor responses to HA in normal Krebs Uf )\ &nd in Ga 2*'4 * - free Krebs solution (o). Responses in calcium - free Krebs were obtained within 45 min. 2+ o f Ga - free perfusion of the artery. Each point on the graph is a mean of observations from 6 - 8 separate artery preparations. Vertical bars represent s .e . mean. UNIVERSITY OF IBADAN LIBRARY FIG F. .-r-Gv’ \ ; F- •" ‘ ' 1 . .. (• * ■ •• • • , ■ • V.i ; l Z - 1 3 2 - UNIVERSITY OF IBADAN LIBRARY .FXGIIIE.,12 E ffec t o f POE ̂ on NA vasoconstrictor responses in rat mesenteric arte iy perfused with normal ( l e f t panel) and Ca ■ free (r igh t panel) Krebs solution. The curves represent dose response to NA (x) alone, and (o) in the presence o f 10 ng/ml PGE .̂ -Each point on the graph represents the mean + 3.B. o f 6 - 8 experiments. UNIVERSITY OF IBADAN LIBRARY FIG 33 - 1 3 3 - . Uq- NA ( LOG SC A LE) -R ESPO N SE (m m ) UNIVERSITY OF IBADAN LIBRARY 5TCTJRS 34 Effect o f PGF ̂ on NA vasoconstrictor responses in rat mesenteric artery perfused with normal ( le f t panel) and Ca2+- free (r igh t panel) Krebs solution. The curves represent dose responses to NA (x) alone, and (0) ?n the presence o f 10 ng/ml PG?2a . Each point on the graph represents the mean + S.B o f 6 experiments. UNIVERSITY OF IBADAN LIBRARY FIG 34 '-•134 - 1b - Ug. N A . (LOG. SCALE) liUMMa—ii i. UNIVERSITY OF IBADAN LIBRARY Figure 35 Effect of A2J187 ( l .9 x 10 °Il) on NA vasoconstrictor responses in rat mesenteric artery perfused with normal ( l e f t panel ) and Câ +- free (right panel) Krebs solution. The curves represent dose responses to NA (X) alone, and (0) in the presence o f 1.9 x 10~6K A23187. Each point on the graph represents the mean + S.S o f 6 experiments. UNIVERSITY OF IBADAN LIBRARY Fig- 35 - 155 - jug N A (LOG SCALE) Response (mm Hg) UNIVERSITY OF IBADAN LIBRARY FI Q-URS 36 Effect o f increasing calcine, chloride concentration on noradrenaline vasoconstrictor potentiation (PF) caused by 2.8 x 10 "'ll prostaglandin E0 ( l e f t panel) and 1.9 x10 A23187 (r igh t panel) in rat mesenteric artery. Facia poin is a mean of measurements made in 7 - 8 preparations. Vertical bars represent S.e. mean. UNIVERSITY OF IBADAN LIBRARY '&$$ UNIVERSITY OF IBADAN LIBRARY - 137 - o f the mesenteric artery. The responses to NA were potentiated by prostaglandins in normal Krebs but not when Ca2+ was omitted from the perfusion medium. NA vasoconstriction was blocked by adrenoceptor antagonists phentolanine, to lazoline and yohimbine in normal Krebs and the blockade can be reversed by low concentra­ tions o f -PC-Eg. I t was important to know ( i ) the e ffe c t o f Ca2-f omission on the adrenoceptor block by these antagonists, and ( i i ) how prostaglandins ( i f at a l l ) interact with the blockade 2+ during Ga omission. 'The aim o f this experiment was to determine / whether the mechanism by which PGEg potentiated JJA vasoconstriction was the same as that by which(i t reversed 0̂ - adrenoceptor blockade. The adrenoceptor block indufted by phentolamine, tolazoline and yohimbine wa3 unaffected by Ga 2+ omission although FGG, _o 2+ (2.8 x 10 M) which did not potentiate HA in Ga - freeK.rebs, i reversed the cfe~adrenoceptor blockade due to each o f the antagonists during Ca2+ emission to about the same extent as in normal -Krebs, E ffec t o f variation in external Ga 2+ on antagonism by indomethacin and prazosin 24. I f an inh ibitor o f constriction prevented the entry o f Ca 2+ . ions into the muscle c e l l , the concentration o f Ca ions in the perfusing flu id might be expected to influence the action o f tne inh ib itor. Indomethacin and prazosin were tested against NA in normal Krebs solution. Cinnarisine and verapamil were not tested since both agents have been conclusively shown to antagonize Ca2+ in flux across c e l l membranes (Godfraindeb KaDa, 1972). The results summarized in Table 9 show that' variation ol the concen­ tration o f CaClp in the external medium did not appear to a ffe c t ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ rd o m ^ h a c m ^ n ^ p ra zo s m ^ r^ h ^ ^ d ^ n e s e n te ry . UNIVERSITY OF IBADAN LIBRARY 138 - 'T)I1 8 I solated Rat 1.13 sent a ric artery: or?feet of PQ8.0 C2 ~q on'~~rTlo ̂ :'aao~ "cl^rTgd by y .rious" Chapter 1. The low doses o f ITA used in this work are comparable with those used by Casteels Droogaans (1976) and have been shown not to cause depolarization o f the vascular smooth muscle c e ll and w il l thus serve as a suitable example fo r pharmacomechanical pathway o f m obilizing Ca 24- fo r contraction. Bolus in jections o f hiyh doses o f potassium chloride on the other hand, have been shown to cause vasoconstriction by a mechanism which u t iliz e s extrace llu lar Ca"+ (Bevan, Oshar & 3u, 1963; HudyLn d: V/eiss, 1968). Xdl does not act on any spec ific receptoi’ s ite but causes depolari­ zation o f the c o ll membrane tiros rendering the la tte r permeable to extrace llu lar Ca 2+ whose in flux causes the contraction. Vascular contraction induced this way represents an "electromechanical" pathway fo r Ca 24- . UNIVERSITY OF IBADAN LIBRARY - 146 — Mechanism of prostaglandin - induced potentiation, of ITA» Exogenously administered prostaglandins had no action on its ox-m in the isolated mesenteric artery preparation, but greatly potentiated the vasoconstrictor action o f the neurohumoral trasraitter substance, noradrenaline, . (na) used as an agonist in this study. For PGE2> the potentiation is dose - related at least up to 10“ 7 gml- 1 whereas PGF and PSI? , there was no clear cut dose -/relationship (Table l ) . This result is in agreement with results obtained in other vascular smooth muscle preparations such as rabbit aorta and mesenteric artery in v itro (Strong & Chandler, 1972; Tobian & V iet, 1970); rabbit mesenteric artery (Maliic et a l , 1972) ; canine saphenous veins and hindpaw resistance vessels (Kadowitz, Sweet & Broody, 1971; Kadoxzitz, et a l . 1971; Brody & Kadoxzitz, 1974) but d ifferent from those o f Coupar & McLennan, (1973). The la tte r authors had observed that PGA and PGSj but not P0E2 potentiated NA in rat mesenteric artery prepara­ tion. The fa ilure o f these authors to observe potentiation with PGE2 may be due to the high doses o f NA and also o f PGS2 used in their studies. In our experiments, the PGE2 - dose potentiation curve was b e ll shaped (See F ig, 7). Prostaglandin - induced supersensitivity was probably due to the perfused prostaglandins 3ince according to the results expressed in Table'2, only about UNIVERSITY OF IBADAN LIBRARY - 1 4 7 - 2C$ of the PG32 and. P̂ ]?2a infused into the mesenteric artery was inactivated. Prostaglandin metabolites possess a variety of biological activ ities (Anggard, 1966; Pike, Kupiecld. & Weeks, 1967; Crutchley & Piper, 1975) on a variety o f smooth muscle preparations. I t is furthermore, clear that products o f PGE2 or PGI1̂ metabolism did not contribute to enhancement of IIA vasoconstriction since tlie perfusate o f the prostaglandin a fter passage through the guinea- pig lung pulmonary artery did not display any HA - potentiating e ffec t. PG3d„ and PGP cJX undergo more than 9Cfi> inactivation on single passage through the guinea-pig pulmonary vasculature (.Piper, Vane&Wyllie, 1970; Crutchley & Piper, 1975). There is evidence that the innervation o f the mesenteric artery is adrenergic in nature. The abolition of responses to stimulation by bretylium and by guanethidine, and also the reduction or abolition of responses to nerve stimulation by pretreatment with reserpine, together with the lack of e ffect of atropine and physostigmine, are a l l evidence supporting the fact that vasoconstriction caused by stimulation was due to excitation of adrenergic fibres (McGregor, 1965). Further support is derived from the observation of Furness & Marshall (1974) that the principal and small arteries and terminal arterioles of the mesenteric vascular bed are a l l innervated by a network of adrenergic fibres and a ll constrict in response to stimulation UNIVERSITY OF IBADAN LIBRARY - 148 - of paravascular nerves and exogenous HA. Prostaglandin - induced supersensitivity may therefore be pre - or post - synaptically mediated. Presynaptically, i t could be due to release or fa c ilita t ion o f release o f transmitter substance from the nerve endings since there is evidence that PGSg and PGF^ fa c ilita tes ITA release in vascular tissue. For example, in the hindlimb of the dog, PSF„ causes venoconotriction which is abolished a fte r cJX denervation (bucharme, Weeks & Montgomery, 1968), Also, both f t PGS and PQS have been shown to se lective ly fa c ilita te the C. C.c l responses to nerve stimulation in the resistance vessels o f the dog* hindpaw (gadowitz et a l, 1971; Brody & Kadowitz, 1974). Our results show that both P13„ and PIP_ s t i l l potentiated HA even c. c., presence of excess Ca in the perfusion medium (5.0 mM Ca ) , indomethacin block of NA was drastically reduced. This situation w ill be expected i f the membrane s tab iliz in g action o f high doses of indomethacin (Horthover, 1971, 1972; Flower, 1974; Famaey, Fontaine & Reusse, 1977) was prevented or reduced by excess Ca^+ . The vasoconstrictor response to A23187, shown earlie r in this thesis to be due to massive release o f Ca from bound in tracellu lar sites was almost completely abolished by UNIVERSITY OF IBADAN LIBRARY 1 61 2.8 x 10 n indomethacin (Table 7 ) . This observation can be taken to mean that the s ite of indomethacin action is intracellu lar preventing release from the mitochondria or the sarcoplasmic r e t i­ culum, This suggestion is compatible with that o f Northover,( 1977) who reported that indomethacin did not block responses o f glycerinated smooth muscle strips made to contract by the addition o f a mixture of adenosine triphosphate (ATP; , Ca 2+ ions and Mg 2+ ions. Action o f Prazosin Prazosin (1.3 x 1O’" — 5.3 x 10-1 K) potently inhibited HA vasoconstriction in the rat mesentery. Like indomethacin, there was prolongation o f the duration o f the agonist action and severe depression of the maximal responses to the agonist by high concentrations o f the antagonist. Also, its blockade of NA can be overcome by increasing agonist concentration. However, prazosin block of HA in rat mesentery is non-competitive in nature since the values of "plo" obtained with prazosin vary with varying concentrations of the antagonist. This conclusion contradicts ea rlie r reports by Gavero (1976), Gavero et a l (197S) that prazosin is a competitive post-synaptic cK- adrenoceptor blocking agent in vascular smooth muscles. The la tte r conclusion has been based on receptor characterization experiments in anaesthesized animals UNIVERSITY OF IBADAN LIBRARY - 1 6 2 - (ra ts and dogs). The magnitude o f prazosin block was not affected by changes in external Ca2+ concentration up to 2.5 aM Ca 2+ « Doubling the external Ca 2+ concentration did not a ffec t the block. These put together w ill suggest that prazosin was not preventing Ca influx across c e ll membrane. In contrast to indomethacin, prazosin block was i f anything only p a rtia lly reversed by (? ig . 20'). Prazosin block appears very selective . to KA action. At the concentration o f 2.6 x 10 ^ II in the perfusion medium, 1IA dose ratio was 11.2 + 0.4» -whereas up to a \ concentration o f 1,3 z 10 ^ M , '( i .e . one m illion fo ld increase in concentration), prazosin did not show any antagonistic e ffec t on vasoconstriction induced by KC-1. This interesting observation suggests that prazosin demonstrates some spec ific ity fo r the pharmacomechanical pathway o f vasoconstriction. However, since prazosin is not a competitive c<- adrenoceptor antagonist ( present work) i t s inhibitory action i 3 probably on the events following receptor stimulation ( i . e . block Ca 2+ channels mobilized spec ifica lly by pharmacomechanical coupling). This is consistent with the conclusions of Constantine et a l (1973) that prazosin caused functional blockade o f vasoconstriction at a point d ista l to the C<.~ adrenoceptors UNIVERSITY OF IBADAN LIBRARY 163 - Cinnarizine and 7erapag.il Cinnarizine and verapamil antagonised both ITA - and Kgl — induced vasoconstriction in rat mesentery. Both agents were c learly more potent against 1̂ *1 - induced vasoconstriction than HA vasoconstx'iction (F ig . 29) . Both cinnarizine and verapamil blocked ITA in a non-conpe tit iv e manner with the magnitude o f the maximal responses to ITA always suppressed. This is expected since both compounds hav> e been shown to prevent Ca 2+ in flux across .b io logical membranes (Codfrairrl& Kaba, 1969, 1972). This conclusion is supported by our,observation that concentrations o f these agent, lower than required to block ITA vasoconstriction, completely abolished PQgp - induced potentiation o f ITA (F ig . 37 ) shown ea rlie r in the thesis to be dependent on external Ca^+. Furthermore, cinnarizine and verapamil were potent antagonists o f A23187 ~ induced vasoconstriction of the rat mesentery; suggesting that the s ite o f action o f these two compounds may extend beyond the membrane leve l. They could be preventing the mobilization of Ca 2+ from bound sites or preventing u tiliza tion o f free Ca 2+ by the contractile machine. Godfraihdd Kaba (.1969) had ruled unlikely the probability that cinnarizine was acting d irectly on the contractile proteins based on the observation that the compound did not modify the contraction o f isolated rabbit, mesenteric artery evoked by adrenaline in Ca 2+ - free depolarizing solution. I t can therefore be suggested by exclusion, that UNIVERSITY OF IBADAN LIBRARY - 1 64 ~ cinnarizins prevents the mobilisation o f sequestered. Ga 2+ required for ITA action. Yerapatail probably acts by the same mechanism. This conclusion is in addition to the fact that both compounds prevents Ca^+ influx, a fact vindicated in this study by the observed inhibition of K - induced vasoconstriction. Mechanism o f -prostaglandin reversal o f ITA - antagonism The possib ility that prostaglandins may act by in terfering with tho pharmacological receptors was considered. In a study of the interaction o f PGEp with phentolamine, to lazoline, yohimbine and phenoxybenzamine, i t was observed that the blockade by the la tte r was consistently and dose - dependently reversed by PGBg* The responses were reversed almost to control levels when the block was of the competitive type, PG'I^j sim ilarly reversed the block by phentolamine, tolazoline and yohimbine. The block by cinnarizine, verapamil and prazosin were unaffected by doses of PGEp a3 high as 2.8 x 10 ̂M. These results are similar to those o f Maxwell, Plummer, Povalski Schneider & Coombs (1959) who showed that cocaine reversed the blocking action o f surmountable antagonists o f IA but did not a ffec t the blocking action of an unsurmountable antagonist. They suggested that cocaine caused supersensitivity by producing a change in the configuration o f the receptor ( i . e . "deforming" the receptor). I f this were so, a d ifferent binding characteristic^ for the receptor antagonists mignt be UNIVERSITY OF IBADAN LIBRARY - 165 - ex pec bed. since according to Green & Pleming, (1967); Taylor & Green (1971), any changes in the nature o f the adrenoceptor might be expected to lead to changes in the binding characteristics of the receptor with its specific antagonist. In the present experiments, PGG„ could not be said to reverse 0^- adrenoceptor blockade by a lter ing ;he receptor in such a way as to reduce its a f f in it y fo r the antagonist, since pA/> values determined in the presence o f were, i f anything, increased, 'The reason fo r th is apparent increase in the a f f in it y o f the antagonist fo r the receptor in the presence o f exogenous PG22 is not c lear. A similar apparent increase in the effectiveness o f phentolamine blockade at A - adrenoceptors has been observed in rabbit ear artery made supersensitive by prior reserpine treatment (Okpako, personal communication). As reported ea r lie r in this thesis, (p ig . 56 .), PG^ potentiates IT A vasoconstriction in the rat mesenteric artery preparation by a mechanism which involved u tilisa tio n o f external Ca^+ , Part o f the evidence was that potentiation was absent in Ga 2+ - free Krebs and increased in proportion to Ga 2+ in the external medium. Several o f antagonism described here appears to be more complex than can bo accounted fo r in terns o f enhancement o f NA vasoconstriction caused by PGS^. In the f i r s t place, NA potentiation is not observed in Ga 2+ - free Krebs UNIVERSITY OF IBADAN LIBRARY 166 - (Adeagbo & Okpako, 19CO and F ig . 33)» but lid antagonism by 2+ phentolamine, tolasoline and yohimbine in Ga - free Krebs was reversed by PGOg about the same extent as in normal Krebs. This suggests that external Ga 2+ m y not be involved in blockade reversal by PGE/j. Secondly, i f enhancement of HA vasoconstriction by PG32 would account for the reversal, then the following relationship should hold for a given dose o f PGihg DR (Potentiation factor) = DR -̂ DR̂ p,-, (Reveral factor) where QR̂ = NA dose - ratio in the presence o f PGE2 DR̂ = NA dose ~ ratio in the presence o f antagonist. DR,-op = .HA dose - ra tio , in the presence o f antagonist A x v j + PGE2. For a l l the antagonists (Table 4) the reversal factors were sign ifican tly higher (p Z. 0.C05) than the potentiation factor obtained for HA ’with 2.8 x 10- 8 M PGEg. This would suggest that the reversal o f antagonism by PGS2 cannot be accounted for in terms of simple enhancement of NA vasoconstriction. In contrast, the partial reversal by A23187 of NA antagonism appeared to be due to its a b ility to enhance HA vasoconstriction, since A23187 reversed NA blockade by the same order of magnitude as that by which i t potentiated NA ( ‘Table 6) • This compound fa c ilita tes Ca^+ influx(Reed, 1968; Reed & Lardy, 1972; Pressman, 1976) which would account for potentiation o f HA vasoconstriction. UNIVERSITY OF IBADAN LIBRARY - 167 - The "s e lec tiv ity " o f PG3 in its mode of blockade reversal is noteworthy and interesting. HA antagonistic actions o f cinnarizine, PBZ (above 2.9 x 10 M; and verapamil were unaffected by PGE0, A ll these agents have been shown to block excitation — contraction (9 — C) coupling. For instance, verapamil has been shown to block 3 - C coupling in the heart - an action which has been attributed to Ga antagonism (Fleckenstein, fr it th a r t , Fleckenstein, Herbst '& Gran, 1969) and/or specific blocking e ffec t o f potential dependent Ca^+ permeability channels (Rasmussen & Goodman, 1977). Sim ilarly, cinnarizine (Gcdfrain & Kaba, 1963, 1972) and PBZ in high doses (Bevan, Osher & Su, 1963; Shibata & Carrier, 1967) have been shown to be Ga 2+ antagonists. In contrast, non o f the so - called competitive antagonists at the Ĉ. - adrenoceptor s ite have been shown to be a Ga 2+ antagonist. Catecholamine - o L - receptor interaction has been shown to in itia te the mobilization of a d irectly proportional O i number of Ca ions to generate a proportionate response (Moran, Swamray & Triggle, 1970). Competitive c< - adrenoceptor blockers therefore reduce the amount of bound Ga mobilized by the agonist. Consequently, the response is reduced. I t thus seems, that probably augments the mobilization o f internal Ca^’ to induce blockade reversal. PG3p would therefore not be expected to reverse the HA antagonistic actions of cinnarizine, UNIVERSITY OF IBADAN LIBRARY - 168 - PBZ (in high doses) and verapamil since these agents are blockers o f the mobilizable Ca 2+ ions. PCS ̂ is thus a useful too l fo r distinguishing between types o f (A. - adrenoceptor blockade UNIVERSITY OF IBADAN LIBRARY CHAPTER FIVE SUMMARY AHD CONCLUSION UNIVERSITY OF IBADAN LIBRARY - 169 - SUf-STARY AND CONCLUSION PGS ̂ causes potentiation o f HA vasoconstriction fcv a mechanism which involves u tiliza tion o f external Ga 2+ The evidence for th is includes (a ) potentiation is d irec tly related to the concentratiion 2+ . o f Ca in the external medium and i t is absent when no Ga 2+ is present in external medium; (b ) e ffects o f PGE£ is additive with cocaine and reserpine, two agents reported to cause potentiation by th e ir e ffec ts on calcium movement (Summers & Tillman, 1979; Carrier & Hester, 1976 fo r cocaine and reserpine respective ly ); (c ) potentiation was blocked by bretylium, cinnarizine, chlorpromazine and verapamil in doses at which these substances did not a ffec t the responses to NA. These substances are known to block Ca 2+ in flux. High K+ vasoconstriction shown in this study to depend sole ly on external Ca +̂ was not potentiated by PGE .̂ Sim ilarly, PGÊ did not potentiate NA when mesenteric artery was perfused with Ca 2+ - free depolarizing krebs solution. Ca2' + ionophore, A23187, enhanced HA vasoconstric. HA vasoconstric- tion by a mechanism which involves Ca 2+ - dependent neuro­ transmitter release from adrenergic nerve endings in the isolated UNIVERSITY OF IBADAN LIBRARY - 170 - rat mesentery. This is evident because A23187 induced enhancement o f NA was absent in reserpine pretreated rats, and also during perfusion o f the artery with Ca 2+ - free krebs solution. 4. Hi-h doses o f A23187 caused vasoconstriction which wa3 not blocked by oi - adrenoceptor antagonists but was almost completely abolished by putative Câ + antagonists. The vasoconstriction was sustained in Câ + - free krebs solution. 5. Blockade o f NA vasoconstriction caused by "competitive" cL~ adrenoceptor agents such as phentolamine, tolazoline and yohimbine was reversed by low doses o f PGE2» The reversal can be demonstrated in Ca 2+- free krebs where NA potentiation is absent, and the extent o f reversal ("reversal fa c to r") o f the blockade is greater than could be expected from simple potentiation. 6. The block produced by the so-called non-competitive antagonists such as high doses o f phenoxybenzamine, cinnarizine, and verapamil was not reversed by PGS .̂ The block caused by these agents was not of the competitive type and may have involved Câ + antagonism. 7. Prazosin block o f NA was also non-competitive and not reversed by PG32. UNIVERSITY OF IBADAN LIBRARY - '71 - 8 . The results showed IGE ̂ to be a useful tool for d ifferen tia­ ting between types o f - adrenoceptor blockade. Furthermore, the finding that blockade caused by phentolamine, tolazoline and yohimbine which are not known to be prostaglandin synthetase inhibitor can be reversed by low doses o f PGÊ shows that this prostaglandin can reverse blockade even i f the mechanism o f inhibition did not involve prostaglandin inhibition in the f ir s t place. The following mechanism o f PGE -̂ induced potentiation o f NA have therefore been proposed based on the fact that NA causes ♦ vasoconstriction by activating the pharmacomechanical pathway (Somlyo & Somlyo, 1968) - in the dose range used in this study. The contraction following this pathway does not require external Ca +̂ - a view supported by the finding that NA dose - response curves with or without external Ca^+ were superimposable (present study). Thus, presence o f pGs ̂ in perfusion medium enables NA to cause depolarization o f the vascular smooth muscle, thereby activating the electromechanical pathway in addition to the pharmaco­ mechanical pathway. On the other hand, the mechanism by which PGÊ reversed '’competitive” cK.- adrenoceptor block is not fu lly clear but appears to be due to enhanced mobilization o f sequestered Câ + by the prostaglandin. UNIVERSITY OF IBADAN LIBRARY - 1 7 2 - R E F E R E N C E S ADEASBO, A.3.0. & 0KPAK0, D.T. (1980): Mechanism o f noradrenaline potentiation by PGE£ in rat mesenteric artery. Br. J. Pharmc. (in press). AHLQUIST, R.P? HUGGINS, R.A.; & WOODBURY, R-. (1947) s Pharmacology o f benzyl imidazoline (PRI3C0L). J. Pharmac. exp. Ther. 82, 271 - 228. AIKENS, J.W. (1974): Effects o f prostaglandin synthesis inhibitors . on angiotensin tachyphylaxis in the isolated coeliac and mesenteric arteries o f the rabbit. Pol. J. Pharmac. Pharm. 26s 217 - 227. AKBACiTE, N. (1946)tJ Interaction: o f drugs and the e ffec t of cooling on the isolated mammalian intestine. J. Physiol. ' JO4: 226 - 287. I AKBACHE, N. & ZAR, II.A. (1970): An inhibitory e ffect of prostaglandin on neuromuscular transmission in the guinea-pig vas diferens. J. Physiol. 208: 30 - 32. ANGGARD, E. (1966): The biological activ ities o f three metabolites o f prostaglandin . Acta. Physiol. Scand. 66: 509. ARHARSGO, W.L.F. (1963): Quinazolines. Advances in Heterocyclic chemistry J_: 253 - 309. ARMSTRONG, J.M.: LATTIKER, N.s MONCADA, S. & VANS, J.R. (1978): Comparison of the vasodepressor effects of prostacyclin and 6-oxo-PGE. with those o f PGÊ in rabbits and rats. Br. J. Pharmac. '§6: 125 - 130. ARUNLAK3HANA, 0. & 3CHLLD, H.O. (1959): Some quantitative uses o f drug antagonists. Br. J. Pharmac. J_4_: 48 - 58. - AXELROD, J. & TONCHICX, R. (1958): Enzymatic O-methylation o f epinephrine and other cathechols. J. Biol. chem. 233: 702 - 705. AXELROD, J ., WPIL-MALHE3BE, H: & TOKCHICK, R: (1959)s The physiological disposition o f 3H-norepinephrine and its metabolites metanephrine. J. Pharmac. 127: 251 - 256. UNIVERSITY OF IBADAN LIBRARY - 173 - BARANY, M. (1967): ATPase a c tiv ity o f myosin correlated with speed o f shortening. J. Gen. Physiol. jjO: 197 - 218. BARNETT, A: GREENHOUSE, D.D. & TABER, R .I. (1968): A new type o f drug enhancement: increased maximum response to cummulative NA in the isolated rat vas deferens. Br. J. Pharmac. 15: BEJRABI4V», U; BURN, J.A. WAXKER, J.M. (1 <358) : The action o f sympathomimetic amines in heart rate in relation to the e ffe c t o f reserpine. Br. J. Pharmac. 461 - 466. B5LLEAU, B. ; & TRIGGLE, D.J. (1962) : Blockade o f adrenergic - receptors by a carbonium ion. J. Mednl. Phaimac. Chem. jp: 636 - 639. BENEY, B.G. & GRILLC, S.H. (1963)2 Antagonism o f acetylcholine by adrenaline antagonists. Br. J. Pharmac. 20: 528 - 533. BENNETTE, A; ELEY, K.G. & 3T0CKLEY, H.L. (1975): The e ffe c t o f prostaglandins on guinea-pig isolated intestine and their possible contribution to muscle a c t iv ity and tone. Br. J. Pharmac. _£4: 197. BEVAN, J .A .; OSEER, J.V. & SU, C. (1963): Responses o f vascular smooth muscle potassium and its antagonism by phenoxyben- zamine. J. Pharmac. exp. Ther. 139: 216 - 221. BEVAN, J.A. & VERITY, M.A. (1967): Sympathetic nerve free vascular muscle. J. Pharmac. exp. Ther. 157: 117. BIRMINGHAM, A.T.; PATERSON, G. ftWOJCICKI, I . (1970) - A comparison o f the sen s itiv ities o f innervated and denervated rat vasa deferentia to agonist drugs. Br. J. Pharmac. _2£: 754 - 784. BITO, L.3. & DAWSON, M.J. (1970): The s ite and mechanism o f control o f cholinergic sen s itiv ity . J. Pharmac. exp. Ther. 175: 673 - 684. >*■ UNIVERSITY OF IBADAN LIBRARY - 174 - BLASCHKO, H. (1952): Amine oxidase and amine metabolism. Phaimae. Rev. 4: 415 - 458. BOHR, D.F. (1973): Vascular smooth muscle updated. Circ. Rea. 22: 665 - 672. T?r.TTO * ' T A . r r y r f r ^ r /, , * V*f,rT rP'31G,V A * r »TT’ T̂T’TJf!? T* 7} • MOULTON, R. & ROSENHEIM, M.L. (1959)s Darenthin, hypotensive agent of new type. Lancet 2: 17 21. BRIGGS, A.H. (1962): Da].GiUni movements during potassium contracture in isolated rabbit aortic strips. Am. J. Physiol. 205: 849 - 852. BRIGGS, A.H. & MELVIN, S. (1961): Ion movements in isolated rabbit aortic strips. Am. J. Physiol. 201; 365 - 368. BROWN, G.L. & GILLESPIE, T.S. (1957)s The output o f sympathetic transmitter from the splean o f the cat. J. Physiol. 138: 81 - 102. BULBRING, E. & KURIYAMA, H. (1963)s Effect o f changes in ionic environment on the action o f acetylcholine and adrenaline on the smooth muscle ce lls o f guinea-pig taenia co li. J. Physiol. 166: 59 - 74. BUNTING, S.; GRYOLNWSKI, R .; MONCADA, 3.; & VANE, J.R. (1976): Arterial walls generate from prostaglandin, endoperoxides a substance (prostaglandin x) which relaxes strips o f mesenteric and coeliac arteries and inhibits platelet aggregation. Prostaglandins J_2: 897 - 913. BURKS, T.F.; WHITACRE, T.S. & LONG, J.P. ( 1967): E ffect o f calcium deficient perfusion on isolated mesenteric arteries. Arch, int. pharmacodyn. 168: 304 -311. UNIVERSITY OF IBADAN LIBRARY 175 - BUlilT, J .TI. (1952): The entries at sympathetic nerve endings. Br. Med. J. U 784 - 787. BURN, J.H. & RAND, M.J. (1958): The action of sympathomimetic amines in animals treated with reserpine. J. Physiol. 144: 314 - 336. BURN, J.H. & RAND, M.J. (1959)? The cause of supersensitivity o f smooth muscle to noradrenaline a fte r sympathetic degeneration. J. Physiol. 147: 135 - 143. BURN, J.H. & ROBINSON, J. (1S53): Hypersensitivity o f the denervated n ictita ting membrane and amine oxidase. J. Physiol. 120: 224 -*229. BURN3T0CK, G. (1970): Structure o f smooth muscle and its inner­ vation. In smooth muscle, edited by E. Bulbring, A.P. Brading, A.W. Jones and T. Tomita. Baltimore, Williams & Wilkins Company pp 1 - 69. BURNSTOCK, G.; HOLMAN, M.S. & PROSSER, L.L. ( 1963) : Electrophysiology o f smooth muscle. Physiol. Rev. 43: 482 - 527. CALDWELL, P.G. ( 1964) : Factors governing movement and distribution o f inorganic ions in nerve and muscle. Physiol. Rev. 48: 1 - 64. CANNON, W.B.; & LISSAK, K. (1939): Evidence fo r adrenaline in adrenergic neurones. Am. J. Physiol. 125: 765 - 777. CANNON, W.B. & ROSENBLUETH, A. (1949) : The supersensi'tivity of denervated structures. New York: Macmillan, (sited in Fed. proc. (1975) J54: 1969 1970). CARRIER, 0. (1975): Role o f calcium in postjunctional supersen­ s it iv it y . Fed. Proc. J54: 1975 - 1980. UNIVERSITY OF IBADAN LIBRARY - 176 - CARRIER, 0. & HESTER, R.K. (1976): The relationship between calcium and increased sensitiv ity o f the rabbit aorta four hours a fte r reserpine. Br. J. Fhaimac. jjS: 449 - 455. CARRIER, 0. & HOLLAND, J.C. (1965) s Supersensitivity in perfused isolated arteries aft^r reserpine. J. Pharmac. exp. Ther. 149; 212 - 218. . CARRIER, 0. & 2EREVICS, H.A. (1973)s The role o f calcium in "non-specific" supersensitivity o f vascular muscle. J. Pharmac. exp. Ther. 184; 81 - 94. CARRIER, 0. WHITTINGTON-COLEMAN, P .J .; MATH3RY, J. & SHIBATA, S'. (1975); Physical properties o f b iological membranes deter­ mined by the flourescence of the calcium ionophore A23187. Arch. Biochem. Biophys. 162; 174 - 185. CASTEELS, R. & DROOGMANS, G. (1976); Membrane potential and excitation - contraction coupline in the smooth muscle ce lls o f the rabbit ear artery. J. Physiol. 265; 163 - 164P CAVERO, I . (1976): Cardiovascular e ffects o f prazosin in dogs. Clin. Sci. Mol. Med. 51; 609 - 612. CAVERO, I . ; FENARD, S.; COMERI, R .; LEFEVRE, P. & ROACH, A.G. (1978) D ifferential e ffects of prazosin on the pre-and post synaptic £>< - adrenoceptors in the rat and dog. Br. J. Pharmac. 61: 469P. CELANDER, 0. (1954): The range of control exercised by the sympathico-adrenal system. Acta. Physiol. Scand. 3£ Suppl. 116. CEL ANDER, 0. & MSLLANDER, S. (1955): Elimination of adrenaline and noradrenaline from the circulating blood. Nat. 176; 973 - 974. y UNIVERSITY OF IBADAN LIBRARY - 1 7 7 - CHUJYO, N. & HOLLAND, W.C. (1962): Na, K, Ca and Cl exchange in guinea-pig ileum. Am. J. Physiol. 202: 909 - 912. CLEGG, P.C. ; , HALL, W.J. & PICKLES, 7.R. (1966): The action of ketonic prostaglandins on the guinea-pig nyometrium. J. Physiol. 183: 123 - 144. COCEANI, F . & '.iOL?S, L.S. (1965): Prostaglandins in brain and the release o f prostaglandin like compounds from the cat cerebellar cortex. Can. J. Physiol. 4J5: 445. CONSTANTINE, J.N. (1974): An analysis of hypotensive action o f prazosin. In Prazosin - Evaluation o f a new antihypertensive agent. Ed. Cotton, D.W.K. pp: 1 6 -3 6 Excerpta medica Amsterdam. CONSTANTINE, J.W.; KCSHANE, W.K.; 3CRIABANS, A & HESS, H.J. (1973): Analysis of the hypotensive action o f prazosin. In Hyper­ tension: Eechanisms and management, ed. Onesti, G.; Kim, K. E. & Moyer, J.E. pp 429 - 444: New York Granne and Stratton, \ COOPER, T.; GILBERT, J.V . ; BLOOWSLL, R.D. & CROUT, J.R. ( 1961): Chronic extrinsic cardiac denervation by regional neural ablation. Circulation Res. 0- 275 - 281. CORNS, S.J, & GRAHAM, J.D. (1957): The effect of inhibition o f monoamine oxidase in vivo on administered adrenaline, noradrenaline, tyr amine and serotonin. 3. Physiol. A 55: 339 - 349. COUPAR, I.K . (1S80): Prostaglandin action, release and inactivation by rat isolated perfused mesenteric blood vessels. Br. J. Pharmac. 68: 757 - 763 COUPAR, I.K . & MCLENNAN, P.L. (1978): The influence o f prostaglandins on noradrenaline - indiced vasoconstriction in isolated perfused mesenteric blood vessels o f the Br. J. Pharmac. 62: 51 - 59. CRCUT, J.R. (1961): Effect o f inhibiting both catechol-o-methyl- transferase and monoamine oxidase on cardiovascular responses to norepinephrine. Proc. 3oc. exp. Biol. N.Y. 108: 482 - 484. UNIVERSITY OF IBA A LIBRARY - 178 - GROUT, J .R .j KU3KDS, A.J. & TRENDELENBURG, U. (1962): E ffect o f tyramine on isolated guinea-pig atria in relation to their noradrenaline stores. Br. J. Pharmac. 18: 600 -611. CRUNKHORN, P. & WILLIS, A.L. (1971): Cutaneous reactions to intradermal prostaglandins. Br. J. Fharmac. 49 - 56. CRUTCHLSY, D.J. & PIPER, P.J. (1975): Prostaglandin inactivation in guinea-pig lung and its inhibition. Br. J. Pharmac. 52: 197 - 203. CUBEDDU, L .; BARNES, E.M.; LANCER, 3.Z. & WEINER, N .(1974) Release o f norepinephrine and dopamine-R-bydroxylase by nerve stimulation. I : Role o f neuronal and extraneuronal uptake and o f alpha presynaptic receptors. J. Pharmac. exp. Ther. 190: 431. DANIEL, E.E. (1 964): Effects o f drugs on contractions o f vertebrat smooth muscle: Ann. Rev. Pharmac. 189 - 222. DANIEL, E.E.; SEHDBV, H.; & ROBINSON, K. ( 1962) : Mechanisms o f activation o f smooth muscle. Physiol. Rev. $2: (suppl. 5) 228 - 260. DENOLSB, H .J.; SPIEGEL, H.E. & TITUS, E.O. ( 1961) : Effects of drugs on uptake o f isotopic norepinephrine. Nat. 191: 816 - 817. DAVIES, B.N.; HORTON, E.W. & WITHRINGTON, P.G. (1968): The occurence of prostaglandin Ê in splenic venous blood o f the dog following splenic nerve stimulation. Br. J. Pharmac 32: 127. DEVINE, C.E.; SOHLYO, A.V. & 30MLY0, A .P .((l972 ): Sarcoplasmic reticulum and excitation - contraction coupling in mammalian smooth muscles. J. Ce.. B iol. 52: 690 - 718. A UNIVERSITY OF IBADAN LIBRARY - 179 - DOUGLAS, W.W. & RUBIN, R.P. (1964): The e ffec ts o f alkaline earths and other divalent cations. J. Physiol. 175: 231 - 241. DREW, G.M. (1976): E ffects o f adrenoceptor agonists and antagonists on pre- and post-symaptically located - adrenoceptors. Eur. J. Pharmac. 36: 313 - 320. DREU, G.K. (1977): Pharraacological characterization o f the presynaptic - adrenoceptor in the rat vas deferens. Eur. J. Pharmac. 42: 123 - 130. DTJBOCOVICE, M.L. & LANGER, L.Z. (1974-) s Negative feedback regulation o f noradrenaline release by nerve stimulation in the perfused cat spleen: Differences in potency o f phenoxybenzamine in blocking the pre- and post- synaptic adrenergic receptors. J. Physiol. 237: 505 - 519. DUCHARME, D.U.; WEEKS, J.R. & -MONTGOMERY, R.G. (1968): Studies on the mechanism o f the hypertensive e ffe c t o f FGP? . J. Pharmac. exp. Then. 160; 1 - 1 0 . DURBINE, R.P. & J3RKIN30N, D.H. ( 1961a): The e ffe c t of carbachol on the permeability o f depolarized smooth muscle to inorganic ions. J. Physiol. 157: 74 - 89. DURBIN, R.P. & JSNKINSON, D.H. ( 1961b ): The calcium dependence o f tension development in depolarized smooth muscle. J. Physiol. 157: 90 - 96. EAGLING, E.M.; LOVELL, H.G. & PICKLES, V.R. (1972): Interaction of prostaglandin E and calcium in the guinea-pig myometrium. Br. J. Pharmac. _44: 510 - 516. EDM AN, K.A:.P. & SCHILD, H.O. (1962): The need for calcium in the contractile responses induced by acetylcholine and potassium in the rat uterus. J. Physiol. 161: 424 - 441. UNIVERSITY OF IBADAN LIBRARY - 180 - ELLIASON, R. & RISLEY, P.L. (1966): Potentiated responses o f isolated seminal resides to cat echo lanines and acetylcholine in the presence of PGE . Acta. Physiol. Scand. j$7: 253. EMMELIN, N. (1961): Supersensitivity following "pharmacological denervation". Pharmac. Rev. 15: 17 - 37. EMMELIN, N. & ENGSTROM, J. (1961 ): Supersensitivity o f salivary glands following treatment with bretylium or guanethidine. Br. J. Pharmac. 16: 315 - 319. ENERO, M.A. & LARGER, S.Z. (1973): Influence o f reserpine - induced depletion o f noradrenaline on the negative feedback mechanism for transmitter release during nerve stimulation. Br. J. Pharmac. 49*. 214 - 225. EULER, U.3. VON & HEDQUIST, P. (1969) - Inhibitory action o f prostaglandin E„ and E9 on the neuromuscular transmission in the guinea-pig vas deferens. Acta Physiol, scand. 77: 510 - 512. EVANS, D.H.L.; SCHILD, H.O. & THESLEEP, S. (1958) : Effects of drugs on depolarized plain muscle. J. Physiol. 143: 474-485. EXLEY, K.A. (1957): The blocking action of choline 2:6 - xyly l ether bromide on adrenergic nerves. Br. J. pharmac. 12: 297 - 305. FAIRHUR3T, A.S.; JULIE1T, R.M. WHITTAKER, K.L. (1975): E ffect of ionophores A23187 and X537A on brain calcium, catecholamines and exc itab ility . L ife Sci. 1_7: 1433 - 1444. FAKAEY, J .P .; FONTAINE, J. & REU3SE, J. (1977): The e ffec t o f non­ steroidal anti -inflammatory drags on cholinergic- and histamine induced contractions of guinea-pig isolated ileum. Br. J. Pharmac. 60: 165-171. S' UNIVERSITY OF IBADAN LIBRARY 181 FARAH, M.B. & LARGER, 3.Z. (1974): Protection by phentolanine against the e ffects o f phenoxybenzanine on transmitter release e lic ited by nerve stimulation in perfused cat heart. Br. J. Pharmac. _̂ 2: 54 9P FERREIRA, S.H. & VANE, J.R. (1967): Prostaglandins: their disappearance from And release into the circulation. Rat. 216: 863 - 873. FILO, R.3.; BOHR, D.F. & RUHGG, J.C. (1965): Glycerinated skeletal and smooth muscle: calcium and magnesium dependence. Sdience 147: 1581 - 1583. FITZPATRICK, D.F.; LAPDON, E .J.; DEBBAS, G. & HURWITZ, L. (1972>: Calcium pump in vascular smooth muscle. Science 21: 305 - 306. FLECKFiNSTEIN, A. & BURN, J.H. (1953): The e ffec t of denervation on the action of sympathomimetic amines on the n ictitating membrane. Br. J. Pharma. 8: 69 - 78. FLECKEN3TSIN, A.; GRITTHART, H.; FLECKERSTEIN, B.; HERB3T, A. & GRUR, G. ( 1969) : A new group o f competitive calcium antagonists (ip roveratril, D 600, phenylamine) with highly potent inhibitory effects on excitation - contraction coupling im mammalian myocardium. Eur. J. physiol. 307 , R25. FLU-IING, W.W, (1963): Changes in the sensitiv ity of the cat's n ictita ting membrane to norepinephrine, acetylcholine and potassium. Biochem. Pharmac. 1_2 (suppl.) 202. FLEMING, ¥.¥. (1975): Supersensitivity in smooth muscle: Introduction and h istorical perspective. Fed. Proc. 21: 1969 - 1974. FLEMING, W.W.; MCPHILLIP3, J.J. & WESTFALL, D.P. (1973) : Postjunc­ tional supersensitivity and subsensitivity of excitable tissues to drugs. Srgeb. Physiol. Biol. Chem. exp. Pharmac. 68: 55-119. UNIVERSITY OF IBADAN LIBRARY 182 - FLEMING, V.W. & TRENDELENBURG, U. (1961) : The development o f supersensitivity to norepinephrine a fte r pretreatment with reserpine. J. Pharnac. exp. Ther. 133: 41 -5 1 . FLEMING, W.W. & WESTFALL, B.P. (1 975) : Altered resting membrane potential in the supersensitive vas deferens o f the guinea-pig J. Pharmac. exp. Ther. 192: 381 -389 . FLOWER; R.J. (1974): Drugs which inh ibit prostaglandin biosynthesis Pharnac. Rev. 26: 3 3 -6 7 . FURCHGOTT, R.F. (195$): The pharmacology o f vascular smooth muscle. Pharmac. Rev.i 7: 183 - 265. PUHNES3, J.B. & MARSHALL, J.K. (1974): Correlation o f the d irec tly observed responses o f mesenteric vessles o f the rat to nerve stimulation and noradrenaline with the distribution o f adrenergic nerves. J. Physiol. 239: 75 - 88. GADDUI'T, J.H.; HAMEED, K .A .; HATH!'/AY, D.E. & STEPHENS, F.F. (1955): Quantitative studies o f antagonists fo r 5-hydroxytryptamine. Q. J l. exp. Physiol. 40: 49 - 74. GEDULDIG, D. & JUNGE, D. (1 $68) : Sodium and calcium components o f action Potentials in the aplysia giant neurone. J. Phusiol. 199: 747 - 766. GILMORE, N .; VANS, J.R. & m L IE , J.H. (1968) r Prostaglandins released by the spleen. Nat. 218: 1135 - 1140. GLOVER, >/.E. (1978): Potentiation o f vascular responses by 3- and 4 - amino pyridine. Br. J. Pharmac. 63.: 577 - 585. GCDFRAIND, T. & KABA, A. (1969): Blockade or reversal o f contraction in depolarized a r te r ia l smooth muscle. Br. J. Pharmac. jo.: 549 -~560. UNIVERSITY OF IBADAN LIBRARY - 1 8 3 - GODFRAIND, T. & KABA, A (1972)s The role o f calcium in the action o f drugs on vascular smooth muscle. Arch. in t. Pharmacodyn. 196: 35 - 49. GOLENHOFEN, K. & LAKKEL, E. (1972)i Selective suppression o f some components o f spontaneous ac tiv ity in various types o f smooth muscle by Iproveratril (veraoamil). Pflueegers Arch. _231_: 233 - 243. GRAHAM, J.D.P. & AL KATIB, H. (1566): The action o f tiypsin on blockade b y '2 -haloalkylamines: speculation on the nature of the alrhA-receptor fo r catecholamines. Br. J. Pharmac. 28: 1 - 1 4 . . i '\ GRAHAM, J.D.P. & AL KATIB, E. (1967): Adrenolytic and sympatholytic properties o f 2 -halogenoalkylamines in the vas deferens o f the guinea-pig. Br. J. Pharmac. J51.: 42 - 55. GREEN, R.D. & FLEMING, W.N. (1 967): Agonist - antagonist Altera­ tions in the normal and supersensitiviesnictitating membrane o f the 3pinal cat. J. Pharmac. exp. Ther. 156: 207 - 214. GREEN, R.D. & FLEMING, W.W. ( 1968) : Analysis o f supersensitivity in the isolated soleen o f the cat. J. Pharmac. exo. Ther. 162: 254 - 262. GREENBERG, R. & HINES, I .R. (1968): The role o f calcium in cocaine supersensitivity to norepinephrine. Fed. Proc. 27: 599. GREENBERG, R. & HINES, I.R . '(1976): The role of bound calcium in supersen3itivity induced by cocaine. Br. J. Pharmac. £7: 329 - 334. GREENBERG, S .; KADOWITZ, P .J .; DIECKE, F.P.J. & LONG, J.P. (1974): Effect of prostaglandin F on arterial and venous con tractility and 45ca uptake. Arch. aint. Pharmacodyn. Ther. 205: 3S1. UNIVERSITY OF IBADAN LIBRARY - 184 - GREENBERG, 3. & LONG, J.P. (1971): The e ffec ts o f cocaine, norepinephrine and ionic stimulants on the isolated superfused rat vas deferens: antagonism by adrenergic neurone blockers and reserpine. J. Pharmac. exp. Ther. 177: 136 - 145. GRIESEMER, B.C.; BAR3KY, J . ; DRAGSTEDT, G.A.; WELLS, J.A. & ZELLER, E.A. (1963): Potentiating e ffe c t o f iproniazid on the pharmacological action o f sympathomimetic amines. Proc. Soc. exp. Biol. Med. 84 : 699 - 701. GRODZINSKA, L .; PANCZENKO, B. & GRY GLEWSKI, R.J. (1976): Release o f prostaglandin E - lik e material from perfused mesenteric blood vessles o f rabbits. J. Pharm. Pharmac. ,28: 40-43. GRYGLEWSKI, R .J.; BUNTING, 3 .; MCNCADA, 3 .; FLOWER, R.J. & VANE, J.R. (1976): A rteria l walls are protected against deposition o f p la te le t thrombi by a substance (PGX.) which they make from prostaglandin endoperoxides. Prostaglandins 12: 685 - 713. GRYGLEWSKI, R.J. & KORBUT, R. (1975): Prostaglandin feedback mechanism lim its vasoconstrictor action o f norepinephrine in perfused rabbit ear. Experientia 31: 89 -9 1 . HAEU3LER, G. (1972): D ifferen tia l e ffec t o f verapamil on exc ita­ tion - contraction coupling in smooth muscle and on excita­ tion - secretion coupling in adrenergic nerve terminals. J. Pharmac. exp. Ther. 180: 672 - 682. HAGIWARA, S. & NAKAJIMA, 3. (1966): Differences sodium and calcium spikes as examined by application o f tetrodotoxin, procaine and manganese ions. J. Gen. Physiol. 42j 793 - 805. HAMPEL, G.¥. (1935): The e ffe c t o f denervation on the s en s itiv ity to adrenine o f the smooth muscle o f the n ic tita tin g membrane o f the cat. Am. J. Physiol. 111: 611 - 621. UNIVERSITY OF IBADAN LIBRARY - 135 - HARVEY, J. & NICKERSON, !■!. (1954): Reactions o f dibensnine and some congeners with substances o f b io logica l in terest in relation to the mechanism o f adrenergic blockade. J. Pharmac. exp. Ther. 112; 274 - 290. HEDOVIST, P. (1970): Studies on the e ffe c t o f prostaglandins B* ahd on the sympathetic neuromuscular transmission in some animal tissues. Acta physiol, scand. (suppl. 345). 7g* -j _ HEDOVIST, P. (1972) :n Prostaglandin - induced inhibition o f vascular tone ana reac tiv ity in the ca t 's hind le g in vivo. Bur. J. Pharmac. 17: 157 - 162. HEDOVIST, P. (1974): Prostaglandins in adrenergic transmission and their role in cardiovascular system. Pol. J. Pharmac. Pharm. 2_6; 119 - 125. HEDQVIST, P. & EULER, U.S. VON (197?): Prostaglandin controls neuromuscular transmission in guinea-pig vas deferences. Nat. 236: 113 -1 15 . HEDQVIST, P .; 3JARNE, L. & WENNKALM, A. (1971): Facilita tion o f sympathetic neurotransaission in the cat spleen a fte r inhibition o f prostaglandin synthesis. Acta physiol. Scand. 8Jj_ 430 - 432. HBRTTINO, G. & AXELROD, J. ( 1961) : Fate o f tr it ia ted noradrenaline at the sympathetic nerve-endings. Nat. 192: 172 - 173. HINKE, J.A.K. ( 1965) : Calcium requirements fo r noradrenaline and high potassium icn contraction in a r te r ia l smooth muscle In "muscle" W.M. Paul, E.E. Daniel, C.M. Kay & Honckton, G. pg. 269. HORROBIN, D.F.; MANKIT, M.S.; KARMALI, R.A. ; NASSER, B.A. & DAVIES, P.A. (1974)* Aspirin, indomethacin, catecholamine and prostaglandin interaction in rat arterio les and rabbit heart. Nat. 250; 425. S* UNIVERSITY OF IBADAN LIBRARY 106 - HORTON, E.U. (1973): Prostaglandins at adrenergic nerve endings. Br. Med. Bull. 29: 148- 151. HORTON, E.¥. (1979): Prostaglandins and smooth muscle. Br. Med. Bull. 21- 295 - 300. HOD NINE, P.M, & UEISS, *0.3. ( “'968): D ifferen tia l e ffec ts o f calcium removal upon vascular smooth muscle contraction induced by norepinephrine, histamine and potassium. J. Pharnac. exp. Ther. 139: 91 - 97. INNE3, T.R. (i960): Sensitization o f the n ic t ita tin g membrane to sympathomimetic amines by reserpine. Fed. Froc. _1_9: 285. INNES, I.R . & KARR, G.¥. (1971): Protection against induction o f supersensitivity to catecholamines by cocaine. Br. J. Pharmac. 42: 603 - 610. INNES, I.R . & MAILHOT, R. (1973): E ffect o f cocaine on the a f f in ity o f the - adrenoceptors fo r noradrenaline. Br. J. Pharmac. 48: 139 ~ 143. IVERSEN, L.L. (1963): The uptake o f noradrenaline by the isolated perfused rat heart. Br. J. Pharmac. 21: 523 - 537. IVERSEN, L.L. (1967): The uptake and storage o f noradrenaline in sympathetic nerves. London Cambridge University Press. JOHNSON, R.A.; MORTON,: KINNER, : GORMAN, ; MCGUIRE, J.C .; . SUN, F.F . ; WEETACRE, ; BUNTING, S . ; SALMON, ; MONCADA, S. & VANE, J.R. (1976); The chemical structure o f prostaglandin X (prostacyclin ). Prostaglandins J_2 : 915 - 928. KADOWITZ, P .J .; SHEET, C.S. & BROODY, M.J. (1971): 'D ifferen tia l e ffects o f prostaglandins E-j , E2, F^a and F^a on adrenergic vasoconstriction in the dog hinapaw. J. Pharmac. exp. Ther. 177: 461 - 649. UNIVERSITY OF IBADAN LIBRARY KAL3MER, 3. (1974/: A new approach to the measurement and classifica tion o f forms o f supersensitivity o f autonomic e ffec to r responses, Br. J. Pharmac. jjjh 427 - 474. » KAL3NER, 3, & NICKERSON, K. (19 69): Mechanism o f cocaine potentiation o f responses to amines. 3r. J. Pharmac. 35 : 428 - 439. KAL3NER, 3.; NICKERSON, K. & BOYD, B.N. (1970: Selective blockade o f potassium induced contraction o f aortic strips by B - diethylaminoethyIpropylacetate (SKF 525A). J. Pharmac. exp. Ther. 174: 500 1 508. KASUYA, Y. & GOTO, L. (1968): The mechanism o f supersensitivity to norepinephrine induced by cocaine in rat isolated vas deferens. 3ur. J. Pharmac. _4: 355 - 362. KHAIRALLAH, P.A. ; PAGE, I.H. & TURNER, R.K. (1967): Some properties o f prostaglandin E action on muscle. Arch. in t. pharmacodyn. ■Ther. 16°: 328 - 341 . KIRAN, B.K. & KHAIRALLAH, P.A. (1969): Angiotensin and norepine­ phrine e fflu x . Eur. J. Pharmac. 6: 102-108 . KIRPEKAR, 3.1-1. & CBRVONI, P. ( 1 962): E ffect o f cocaine on catecholamine in venous effluents from sympathetically stimulated adrenal and spleen. Fed. proc. 21y. 340. KIRPEKAR, S.I-I.; CERVONI, P. & FURCHGCTT, R.F. (1962): Catechola­ mine content o f the cat n ic t ita tin g membrane following procedures sensitizing i t to norepinephrine. J. Pharmac." 135: 180 - 190. KITAMURA, L; SUZUKI, H. & KURIYAKA, E. (1976): E ffect o f noradrena­ line on the membrane properties and on the contraction o f the rabbit main pulmonary artery. J. Physiol. 265: 164 - 165P. UNIVERSITY OF IBADAN LIBRARY 'rxt . T•?> . v ( i » ̂-j' r rj r)\ \ • t* o .>.3. -i1d^1l.e wn, rerantans lo r Xt'AnV i e> .termination ol the physiological actions o f catecholamines . Pharmac. le v . 21: 331 - 386. I . J . (1364): Storage and. metabolism o f catecholamines; the role o f monoamine oxidase. Pharmac. Rev. J_6: 179 - 191 guanethidine and reserpine upon the iso la ted mammalian heart. J. Pharmac. 135: 164- 173. KURIYAKA, H. & SUZUKI, H. (1976): E ffect 0 f prostaglandin E? and oxytocin on the e le c tr ic a l a c t iv ity o f hormone-treated and pregnant rat myometrium. J. Physiol . 260: 335 - 349. LARGER, 3.Z. (l9 7 4 ): ? re synaptic regulation o f catecholamine release. Biochem. Pharmac. 23: 1793 - 1800. LEC0M7H, J. (l955 ): 5 - hydroxytrypteaine et membrane nictante du chat. Arch, in t phemacodyn. 100 : 457 - 464. LII'TDMAR, R. &KJ3CHCLL, E. (1.962): Die Uirkung von cocain, Guanethidin, Reserpin, Hexamethonium, Tetracain and Psicain auf die IToradrenalin-Freisetzung aus dem Herzen. Arch. exp. Path. Pharmak. 242: 214 - 227. MACKAY, D (1978): How should values o f pA ̂ and a f f in i t y constants fo r phermacological competitive antagonists be estimated? J. Pharm. Pharmac. 30: 312 - 313- MACMILLAN, U.H. (1959): A hypothesis concerning th e 'e f fe c t o f cocain on the action o f sympathomimetic amines. Br. J. Pharmac. 14: 335 - 391. MARKEL, R .P .; BEAVER, M.A. & BRODIE, B.B. ( 1963) : Im plications o f uptake and storage o f norepinephrine by sympathetic nerve endings. L ife Sc. 2 : 953 - 958. UNIVERSITY OF IBADAN LIBRARY KALUv, X.U. HCOIPF., J.C. (1S74): Relationship o f glucose metabolism to adrenergic transmission in rat mesenteric arteries . E ffect o f glucose deprivation, glucose metabolism arid changes in ionic composition on adrenergic mechanisms. Circulation res. 35: 553. HALIK, K.U.; RYAN, P. & MCBIFF, J.C. (1276): Modification by "recta rri nv-.riin m and indomethacin and arachidonic acid o f the vasoconstrictor’ respcnses o f the iso la ted perfused rabbit and rat mesenteric a rteries to adrenergic stim uli. Circulation Res. 39: 163 - 168. KAMCU, M.S. & EORROBIN, D.F. (1976)s Indomethacin in h ib it responses to a l l vasoconstrictors in the rat mesenteric vascular bed: Restoration o f responses by FGE . Prostaglandins 12: 369 - 376. ‘ 2 MARSHALL, J.M. (1962): Regulation o f a c t iv ity in uterine smooth muscle. Physiol. Rev. (suppl. 5) 213 - 227. MARK , A .L .; SCHMID, P.G. ; ECKSTEIN, J.W. & IvERDHING, M. 0. (1971 ) : Venous responses to prostaglandin 7 . Am. J. Physiol. 220: 222. * -a MAXELL, R.A. ; DANIEL, A. I . ; SHEPPARD, H. & ZIMMERMAN, J.H. (1962): Some interaction o f guanethidine, cocaine, methvlphenidate and phenylalkylamines in rabbit aortic s trip s . J. Pharmac. 137: 31 - 38. MAX’ELL, R.A.; PLUMMER, A .J .; F07ALSHI, H. ; .SCHNID3R, P. & COOMBS, H. (1959): A comparison o f some o f the cardiova­ scular actions o f methylphenidate and cocaine. J. Pharmac. _12oj_ 250 - 257. MAXWELL, R.A.; WAS?TLA, U.B.; & HGKHARDT, 3.B. { i 9 6 0 ) : Some factors determining the response o f rabbit aortic strips to dl-norepinerhrine-7-H^ hydrochloride and' the influence o f cocaine, guanethidine and methylphenidate on these factors . J. Pharmac. exp. Ther. 151 : 253 - 261 . UNIVERSITY OF IBADAN LIBRARY 190 - KCGIP? J C • ‘•'’ERR AG3J0, N.A. ; MALIK, K.U. & LCI'IGaC, . . I . (1972): Release o f prostaglandin E - lik e substance from canine kidney by. bradykinin. Giro ill at ion Res. 3Jj 36. MCGREGOR, D.D. ( i 960) : The e ffe c t o f sympathetic nerve stimulation on vasoconstrictor responses in perfused mesenteric blood vessels o f the rat. J. Physiol. 177: 21 - 30. MEKATA, ?. & NITJ, H. (1 972): Biophysical e ffec ts o f adrenaline on the smooth muscle o f the rabbit common carotid artery. J. Gen. Physiol. 59; 92 - 102. MONOADA, 3.; HIGG3, E.A. & VANS, J.R. (1977): Human a r te r ia l and venous tissues generate prostacyclin (prostaglandin x) - a potent inh ib itor o f p la te le t aggregation. Lancet I 18. HORAN, J.P. ; SHAMMY, V.C. & TBIGGLE, D.J. (19T0) : Irrevers ib le antagonism at the adrenergic alpha-receptor: The ro le o f calcium. L ife 3c. 9: 1303 - 1315* KTABAJI , J .P . ; KANKU, M.S. & HORROBIN, D.P. (1976): Vascular actions o f furosemide and bumetanide on the ra t superior mesenteric vascular bed: Interactions with prolactin and prostaglandins. Can. J. Physiol. Pharaac. MUENAGHAN, M.P. &. KXZURKIEtfICZ, I.M. (1963) : Some pharmacological properties o f 4 - methyltronolone. Rev. Can. B io l. 22: 99 - 102. MURPHY, R. A. ; BOHR, D.P. & HENMAN, D.L. ( 1969) : A rteria l actomycsin: Kg, Ca and ATP ion dependencies fo r A’TPase a c t iv ity . Am. J. Physiol. 217: 666 - 673. MU3CH0LL, E. ( i 960) : Die Hemmung der horadrenalin - Autnahme des Herzens durch Reserpin und die Wixkung von Tyramin. Arch. exp. Path. Pharmak. 240: 234 - 241. UNIVERSITY OF IBADAN LIBRARY - 191 MUSCHOLL, S. (l9 6 l): Effect of cocaine and related drugs on the uptake of noradrenaline by heart and spleen. Br. J. Pharaac. 16: 352 - 359. MUSCHOOL, E. & VOGT, M.(1957): The concentration o f adrenaline in the plasma of reserpinized rabbits. Br. J. Pharmac. 12: 532 - 535. NAIKEADA, M.K. (1969a): Effect o f seme naturally occuring prosta­ glandins (PGB, PGÊ .̂ PG and PGP̂ ) on the hypogastric nerve vas deferens arid seminal vesiie preparations o f the guinea-pig. Chimica Ther. 34. NAIMZADA, M.K. (1969b): Effects o f some naturally occuring prosta­ glandins on the isolated hypogastric nerve seminal vesicle preparation o f the guinea-pig. L ife Sci. 8: 49. NAKAMURA, K.S SHIKAMOTO, K. (i960): The effects of reserpine on the responses o f the n ictita ting membrane in the cat. Jap. J. pharmac. 150 - 158. NAKATSU, K & REIFFENSTEIN, R.J. ( 1968) : Increased receptor u tiliza tion : mechanism o f cocaine potentiation. Nat. 217: 1276 - 1277. NICKERSON, M. (1949)s The pharmacology o f adrenergic blockade. Pharmac. gev. J_: 27 - 101. NICKERSON, M. (1956): Receptor occupancy and tissue response. Nat. 178: 697 - 698. NICKERSON, M. (1959): Blockade o f the actions o f adrenaline and noradrenaline. Pharmac. Rev. JJ_: 443 - 461. NORTHOVER, B.J. (1968): The effect of drugs on constriction of isolated depolarized blood vessels in response to calcium and barium. Br. J. Pharmac. 34: 411 - 418. U IVERSITY OF IBADAN LIBRARY NORTHOVir., B.J. (1971): I!©Chanism o f the inhibitory action o f indomethacin on smooth muscle. Br. J. Pharmac. 41: 540-551. NORTHOVER, B.J. (1972): The e ffec t o f indomethacin on calcium, sodium, potassium and magnesium fluxes in various tissues of the guinea-pi^. Br. J. Phamac. 4£: 651 - 659. IK 2 .T J .0 7 2 2 , B.J. (1977): Indoncthacin - a calcium antagonist Gen. Pharmac. 8: 293 - 296. OKPAKO, D.T. (1971): The e ffec t o f graded doses o f antigen on bronchoconstrictor and mediator release responses in anaphylaxis o f the isolated perfused guinea-pig lung. Int. Arch. Allergy 40: 420 - 430. OXPAICO, D.T. & OLADITAN, F.G. (1979): The e ffec t o f varying the concentration o f calcium ions on the actions o f indomethacin and POÊ in guinea-pig ileum. Gen. Fharmac. 10_: 527 - 329. PATOU, D.II. & DA'IBL, B.E. (1967a): On the contractile response o f the isolated rat uterus to prostaglandin S . Gan. J. Physiol. Phanaac. 45.: 795 - 804. PATOU, D.M. DANIEL, 3.B. ( 1967b): Contractile response of uterine horns to prostaglandin . Fed. proc. 26: 736. PATOU, D.M. & BUCKLAND, NICK3, J & JOHNS, A. (1976)t Postjunctional supersensitivity o f the rat. vas deferens and gap junctions. Can. J. Physiol. Pharmac. £4: 412 - 416. PATCH, V/.D.K. (1957): A tiendulum auxotonic lever. J. Physiol. 137: 35. PKITI IS FATHER, J.N. (1976): The e ffec t of cocaine and diphenhy­ dramine upon the reactivity o f rat vas deferens to supramaximal doses o f noradrenaline and o f ether agonists; the mode o f action o f cocaine. Eur. J. Pharmac. 333 - 339* UNIVERSITY OF IBADAN LIBRARY - 193 FiIILPOT, F.J. (1940): The inhibition o f adrenaline oxidation by by local anaesthetics. J. Physiol. 2 h 201 - 207. FICKLE3, V.R. (1967) s The prostaglandins. Biol. Rev. 42: 614 - 652. PIPER, P .J .; VANE, J.R. & WYLLIE, J.H. (1970): Inactivation o f prcs ta ~1 andinc by +he lm - g. Tat. 225: 600 - 604. P03TE, G. & ALLISON, A.G. (1973)s Membrane fusion. Biochem. biophys. Acta 200: 421 - 465. PRESSMAN, 3.C. (1976): Biological applications o f ionophores.' Ann. Rev. Biochem. 45: 501 - 530. FIJBLI COVER, 3.J.; DUNCAN, C.J. & SMITH, J.L. (1978): The use o f A23187 to demonstrate the role of intracellu lar calcium in causing ultra-structural damage in mammalian muscle. J. Neuropath. Exp. Neur. 2Z: 544 - 557. RAH NELL, P.N. & SHAN, J.E. (1970): Biological significance o f the prostaglandins. Recent Prog. Norm. Res. 26: 139. RAMNELL, P.N.; SHAW, J.E. & JUCHARSKI, J. (1965): Prostaglandin release from the rat phrenic nerve - diaphragm preparation Science II.Y. 149: 1390 - 1391. RA3KU33EN, H. & GOODMAN, D.B.P. (1977)s Relationships between calcium and cyclic nucleotides in cell activation. Physiol. Rev. 421 - 509. RAYMON D-HAl-IST (1925): 3ur un nouveau cas d' inv j vs i on des effets adrenaliniques. Corapt. rend. Acad. d. 3e. 180: 2074-2077 REED, P.W. (1968): A23187: A divalent cation ionophore. Fed. proc. 31: 432. UNIVERSITY OF IBADAN LIBRARY REED, P.W. & IiEDY, H.A. (1972): A23187: a divalent cation ionophore. J. B io l. Chem. 247: 6970 - 6977. REHN, N.0. (1958): E ffect o f decentralization on the content and kidney o f the cat. Acta. Physiol. Scand. 42: 309 - 312. ROBE7TS0N, P.A. ( 1960) : Calcium and con trac tility in the depola­ rized smooth muscle. Nat. 166: 316. ROGELI, P. & VANE, J.R. ( 1964) : A sensitive method fo r the assay o f angiotensin. Br. J. Pharmac. 23: 351 - 359. SCHATZI1ANN, H.J. (1961) s Calciumaufnahme undabgabe am Darmmuskel des Meer-Schweinchens. Pflugers Arch. ges. Physiol. 274: 295 - 310. 3HARPLESS, S.K. ( 1965) : Reorganization o f function in the nervous tissue - use and disuse. Ann. Rev. Physiol. 26: 3 57 SHAW, J.E. & RAKWELL, P.W. (196S): Inhibition o f gastric secretion in rats by prostaglandin E . Prostaglandin symposium Worcester Pound. Exp. B iol. Ramwell, P.VJ. & Shaw, J.E. (Eds) pp 55-66 (New York: Intersciences). SHIBATA, 3. (1969): E ffect o f Kn2+ on Ca45 content and potassium- induced contraction o f the aortic s tr ip . Gan. J. Physiol. Phaimac. 47: 827 - 829. SHIBATA, 3. & CARRIER, 0. (1967): Antagonizing action o f chlor^ promazine, dibenamine and phenoxybenzamine on potassium- induced contraction. Gan. J. Physiol. Pharmac. 4£: 587 - 596. SHIBATA, 3.; HATTORI, K .; SUKURA, I . ; MCRI, J. &'PUJIWARA, M.(1971) Adrenergic innervation and cocaine - induced potentiation o f adrenergic responses o f aortic s trip from young and old rabbits. J. Pharmac. exp. Ther. 177: 621 - 632. UNIVERSITY OF IBADAN LIBRARY - 195 SITRIN, K.D. & BOHR. D.F, (1971)5 Ga and Na interaction in vascular smooth muscle contraction. Am. J. Physiol. 220: 1124 - 1128. SOKUNBI, Y.0,0. (1979)t Does a prostaglandin modulate cholinergic transmission in the guinea-pig ileum? Br. J. Pharmac. 67: 464P. SOMLYO, A.P. & SOMLYO, A.7. (1970): Vascular smooth muscle - I I Pharmacology o f normal and hypertensive vessels. Pharmac. Rev. 22: 249 - 355. SOMLYO, A.V. & SOMLYO, A.P. (1968): Electromechanical and pharmacomechanical coupling in vascular smooth muscle J. Pharmac. exp. Ther. 159: 129 - 145. SORRENTINO, L . ; CAPASSO, R. & DIROSA, M. (1972): Indomethacin and prostaglandins. Eur. J. Pharmac. 17: 306 - 308. SPARROW, K .L.; MAXWELL, L.G.; RUEGG, J.G. & BOHR, D.F. (1970): Preparation and properties o f a calcium ion sensitive actomyosin from arteries. Am. J. Physiol. 219: 1366-1372. SPERELAXIS, N. (1962): Ca^ and S r^ movements with contraction o f depolarized smooth muscle. Am. J. Physiol. 205: 860 - 866. STARKE, K, (1972): - sympathomimetic inhibition o f adrenergic and cholinergic transmission in the rabbit heart. Naunyn-schmiedebergs Arch. Pharmac. 274? 18 - 45. STATHAM, H.E.; DUNCAll, C.J. & SMITH, J.L. (1976): The e ffec t o f the ionophcre A23187 on the ultrastructure and e lec tro - physiological properties o f frog skeletal muscle. Cell Tissue Res. 173: 193 - 209. STSIN3LAHD, O.S.; FURCEGOTT, R.F. & KIRPEKAR, S.M. (1973): Biphasic vasoconstriction o f the rabbit ear artery, Circulation Res. 32: 4 9 -5 8 . UNIVERSITY OF IBADAN LIBRARY - 196 - STROMBLAD, E.C.R. (i960): E ffect o f denervation and cocaine on the action o f sympathomimetic amines. Br. J. Pharmac. 15. : 328 - 332. STROMBLAD, B.C.R. & NICKERSON, M. (1961): Accumulation o f epinephrine and norepinephrine by some rat tissues. J. Pharmac. exp. Ther. 134: 154 - 159 STROUCJ, C.G. & BOHR, D.P. (1967): E ffects o f prostaglandin Et E_, A and F. on isolated vascular smooth muscle. Am. J. Physio?. 21_3: 725. STRONG, C.G. & CHANDLER, J.T. (1972): Interactions o f prosta­ glandin E., and catecholamines in isolated vascular snooth muscle. In prostaglandins in c e llu la r Biology, Ramwell, P.lf. & Pharriss, B.B. (eds). pp 369 - 383 New York and London: Plenum Press. 3U, C. & BEVAN, J.A. (1965) : The e lec trica l response o f pulmonary artery muscle to acetylcholine, histamine and senotonin. L ife Sci. ±: 1025 - -1029. SUMMERS, R.J. & TILLMAN, J. (1979): Investigation o f the ro le o f calcium in the supersensitivity produced by cocaine in cat spleen strips. Br. J. Fhamac. _6̂>: 689 - 699. SUN, P .P .; CHAPMAN, J.P. & MCGUIRE, J.C. (1977): Metabolism o f Pa endoperoxidp in animal tissues. Prostaglandins 14: 1055 - 1074. ‘ . TAYLOR, J. & GREEN, R.D. (1971): Analysis o f reserpine - induced supersensitivity in aortic strips o f rabbits. J. Pharmac. exp. Ther. J_72: 127 - 135. UNIVERSITY OF IBADAN LIBRARY THOA, N.B.; CC3TA, J .L .; MCS3, J. & KCPIN, I .J . (1975): Mechanism o f release o f norepinephrine from peripheral adrenergic neurones by the calcium ionophores X537A and A23187. L ife Scit. i i : 1705 - 1719. THOMAS, 0. & WEST, G.3. (1975): Prostaglandins as modulators o f bradylcinin responses. J. Pharm. Pharmac. 25: 747 - 748. THOMAS, 0. & VEST, G.B. (1974): Prostaglandins, kinins, and inflammation in the rat. Br. J. Pharmac. J50: 231 - 235. TOBIAH, L. & VIETS, J. (1970): Potentiation o f in v itro norepine- ohrine vasoconstriction with prostaglandin E. . Fed. proc. h - 387. TRENDELENBURG, U. (1956): The action o f 5 - hydroxytryptomine on the n ictita tin g membrane and on the superior cervical ganglion o f the cat. Br. J. Pharmac. 11 : 74 - 80. TRENDELENBURG, U. (1959): The supersensitivity caused by cocaine. J. Pharmac. 125: 55 - 65. •TRENDELENBURG, U. (1962): Restoration by sympathomimetics o f the response o f isolated a tria o f reserpine pretreated guinea- pigs to tyrarnine and DMPP. Fed. proc. 2J_: 332. TRENDELENBURG, U. ( 1963) : Supersensitivity/and subsensitivity to sympathomimetic amines. Pharmac. Rev. 225 - 276. TRENDELENBURG, U. (1966): Mechanisms o f supersensitivity and subsensitivitv to sympathomimetic amines, pharmac. Rev. J8: 629 - 640. TRENDELENBURG, U .; MAXWELL, R.A. & PLUCKINO, S. (1970). Kethoxamine as a too l to assess the importance o f intraneuronal uptake o f noradrenaline in the cat n ic tita tin g membrane. J. Phaimac. exp. Ther. 172: 91 - 99. UNIVERSITY OF IBADAN LIBRARY TREN DEL ENBNR G, U .; HUSNUS, A.; PL EKING, W.V. & GOMEZ (Alonzo De La Sierra) B. (1962): E ffect o f cocaine, denervation and decentralization on the response o f the n ictita tin g membrane to various sympathomimetic amines. J. Pharmac. 158: 181 - 193. TRENDELENBURG, U. & WEINER, N. ( 1962) : Sensitiv ity o f the n ictita ting membrane a fter various procedures and agents J. Pharmac. exp. Ther. 136: 152 - 161. VAN BREEMEN, C. & DANIEL, E.E. (1S66): The influence o f high potassium depolarization and acetylcholine on calcium exchange in the rat uterus. J. Gen. Physiol. 42: 1299 - 1317. VAN BREEMEN, C.; FARINAS, B.R.; CASTEELS, R .; GERBA, P .; WUYTACK,F. & DBTH, R. (l973). Factors controlling cytoplasmic Ca2+ concentration. Ph il. Trans. R. Soc. B. 265: 57 -7 1 . VAN BREEMEN, C .; FARINAS, B.R.; GERBA, P. & MCNAUGHTON, E.D. (1972) Excitation contraction coupling in rabbit aorta studied by the lanthanum method for measuring cellu lar Ca2+ influx. Circulation Res. j$0: 44 - 54. VANE, J.R, (l957): A sensitive method fo r the assay of 5 - hydroxytryptamine. Br. J. Pharmac. L2: 344 - 349. VANE, J.R. (1959): The re lative activ ities of some tryptamine analogues on the isolated rat stomach strip preparation. Br. J. Pharmac. J_4: 87 - 98. WAN®, W,W. ( 1962) : Role o f calcium in contractile excitation o f vascular smooth muscle by epinephrine and potassium. Circulation Res. JJ_: 927 - 940. WEISS, G.B. & HNRWITZ, L. (1963): Physiological evidence fo r multiole calcium sites in smooth muscle. J. Gen. Physiol. 47: 173 - 187. UNIVERSITY OF IBADAN LIBRARY WESTFALL, D.P.; MCCLURE, D.C. & FLEMING, W.W. (1972)s The effect o f denervation, decentralization and cocaine on the smooth muscle o f the guinea-pig vas deferens to various drugs. J. Pharmac. exp. Ther. 181; 328. WESTFALL, D.P.; LEE, T.J.F. & STITZEL, R.E. (1975)s Korphotogical and biochemical changes in supersensitive smooth muscle. Fed. proc. J4: 1935 -1989. WOLFF, H.C. & CATTELL, M.C.K. (1937): Orr the mechanism o f hypersensitivity produced by denervation. Am. J. Physiol. 119: 422 - 423. WOOTEW, G.F.; THOA, N.B.; KOPIN, I.J . & AXELROD, J. (1973): Enhanced release of dopamine B-hydroxylase and norepine­ phrine from sympathetic nerves by dibutyryl cyclic Adenosine 3 ', 5' - monophosphate and theophylline.Mol. Pharmac. _g: 178 - 183. WYLLIS, D.W.; ARCHER, S. & ARNOLD, A. (i960): Augmentation o f pharmacological properties o f catecholamines by o - methyl- transferase inhibitors. J. Pharmac. 130: 239 - 244. YONG, M.S. & MARKS, G.S. (1969)s Studies of the chemical nature o f the PC- adrenergic receptor. I I I . Further investiga­ tion of the labelling procedure. Biochem. Pharmac. 18: 1609 - 1618. UNIVE SITY OF IBADAN LIBRARY