7,7,

A STUDY OF THE ACTIONS OF ET ILEFR INE ON SYMPATHET IcALLY INNERVAÍeo BLOOD VESSELS

Thesis submitted to the University of Adelaide fon the degree of Docton of Philosophy

by

BARRY RAYMOND FROST

(a.sc. Hons)

Depantment of Clinical and Experimental Phanmacology, The Univensity of Adelaide, South Austnalia.

November 1980 CONTENTS

SUMMARY

DECLARAT I ON ilt

PUBLICATIONS IV

ACKNOWLEDGEMENTS

CHAPTER 1 lntnoduction 1.1-1 .21

CHAPTER 2 Genenal Methods 2.1 -2.12

CHAPTER 3 The Effects of Etilefrine on 3. 1 -3.9 Blood Vessels in the Rat Tail

CHAPTER 4 The Effects of Agents Modifying 4.1-4.6 Sympathetic Nenve Function on the Response of the lsolated Rat Tail Arteny to Etilefr ine and Tynamine

CHAPTER 5 A Study of the Vasculan Effects 5. 1 -5.6 of Etí lef rine Using the lsolated Rabbit Ear Anteny

CHAPTER 6 The lndinect Sympathomimetic 6. 1 -6.8 Activity of Etilefrine - A Comparison with Tyramine and Ephedr ine Using 3H-Noradrenaline

CHAPTER 7 The Effect of Etilefnine and Othen 7 .1-7 .9 Sympathomimetic Agents on Noradrenal i ne and Metabol i te Effl ux f rom the Rat Tail Arter-y

CHAPTER 8 Genenal Discussion 8. 1 -8.8

APPEND I X A. 1 -4.7

BIBLIOGRAPHY B. I -8. 12 AUVWWNS 1 . The a im of th is study was to exam i ne the ef f ects of etilefnine on blood vessels and to compane its actions on vasculan smooth muscle with those of othen sympathomimetic amines. ln panticulan, it was proposed to detenmine the mode of action of etilefnine and to ascentain whether it intenacted with othen substances which modif ied nonadrenaline disposition via neuronal and extraneunonal mechanisms. 2. The vasculan model used in this study was the ventnal caudal antery of the nat tail. This artery was used rrin situ'l and also as an isolated pneparation. ln some parts of this investigation the isolated nabbit ear anteny was also used.

3. Etilefrine was found to exent a vasoconstnicton action on the ventnal tail artery rrin siturr, which was in pant mediated by noradnenaline release from sympathetic nenve endings in the vessel. The response of etilefnine was, enhanced by pnetneatment of the antery with nonadnenaline or cocaine and abolished by phentolamine. The sympatholytic agents neserpine, guanethídine and 6-hydnoxydopamine decreased the nesponse of the antery to etilefrine. The potency of etilefnine on this vessel was less than that of noradrenaline, adnenaline and phenylephnine but similan to metanaminol .

4. ln the isolated tail antery similan findings were observed as in the tail artery rrin siturr. Pnetneatment with nesenpine on guanethidine decneased the response to etilef rine and thís panalleled the decnease in tissue catecholamines obsenved. Pnetreatment with the monoamine oxidase inhibitor iproniazid elevated tissue catecholamine levels but panadoxical ly decreased the etilef nine nesponse. 5. The response to etilef nine in the rabbit ean anteny wãs analagous to that obsenved in the ventral tail anteny. Sungical denervation of the ean arteny decreased the response to etilefrine but d¡d not alter the nesponse to nonadrenaline. Tneatment of the anteny with 9-alpha-fluonohydnocontisone did not affect the eti lefnine nesponse. 6. When the ventral tail anteny was incubated with tnitiated noradrenaline, etilefr ine was found to enhance the eff lux of nadio- label f nom this vessel . This enhanced eff lux was companab le with that of ephedrine but less than that induced by tynamine. I ?. The metabolite content of the enhanced efflux fnom the ventnal tail antery was also assessed. The sympathomimetic amines etilefnine, ephednine, tyramine and REN-293 wene found to increase the eff lux of tnítiated nonadnenaline and dihydnoxy- phenylglycol to different degrees. These nesults provided, good evidence that etilefrine has an indinect sympathomimetic component to i ts act ion . ttt

DECLARATION

I declane that this thesis contains no material which has been accepted fon the awand of any othen degnee on diploma in any University, and to the best of my knowledge contains no matenial pneviously published by anothen penson, except whene

due ref erence is made in the text .

BARRY RAYMOND FROST

-.i IV

PUBLICATIONS

Pant of the material pnesented in this thesis has been: (1) published in the following jounnals - Journal of Pharmacy and Phanmacology,

29, 272-275, 1977; Austnalian Journal of Exber'¡mental Biology and Medical Science, 56r.499-505, 1978; Jounnal of Phanmacy and Phanmacology,

30, 628-631, 1978; Austnalian Journal of Experimental Biology and Medical Science, 57, 443-446, 1979;

(21 accepted fon publication in the Eunopean Journal of Pharmacology. ACKNOWLEDGEMENTS

Ithank the Depantment of Rehabilitation Medicine, College of Physicians and Surgeons of Columbia University, New York, through the ausp ices of Dn J A Downey, for a Reseanch Fel low- ship for the yeans 1976-1979. I wish to expness my sincene thanks to Dn D. B. Fnewin, Reader in the Department of Clinical and Expenimental Phar maco- logy, University of Adelaide, who supenvised my studies and experimental wonk and whose constnuctive advice and encourage- ment was greatly appreciatejd. I would also like to thank Dr J. Tindale, Medical Directon of Boehringen lngelheim Pty Ltd, fon financial assistance and also fon the supplies of etilefrine and REN-293 used duning this project. The expert secnetanial assistance of Mns L. L. Kingston and the 'photographic expertise of Mn D. Caville ane also gratefully acknow I edged . I would also like to thank my wife Linda, who helped in the pneliminany pnepanation of this thesis. I U:lIdVHJ T 1.1

I NTRODUCT I ON

The concept of neunohumoral tnansmission originated in the late 1890's and eanly 1900's when Lewandowsky (1898) and Langley (1901 ) obsenved that injections of adnenal gland extnact mimicked the action of the sympathetic nenves. The possibility that the effects of stimulating the sympathetic nenve to an organ might be due to the libenation of a chemical substance f rom the nenve ending was suggested by the work of Lewandowsky in 'l 899. ln that yean, Abel named the active pnesson compound in adnenal gland extracts rradnenalinerr. ln 1905, it was suggested by T. R. Elliott that stimulation of sympathetic nerves neleased small amounts of an adnenaline-like substance into immediate contact with effecton cells. However, despite the close similarity between the sympathetic trans- mitten and adnenaline in both chemical tests and biological action, ther^e wene centain differences to be obsenved in most situations.

These differ ences wene commented on by Bar gen and Dale (lgtO), who investigated the phanmacological activity of a large numben of synthetic amines and tenmed thein action rrsympathomimeticrr. They pointed out that the action of amino and ethy lamino bases of the catechol gnoup conresponded more closel y wi th that of the sympathetic nenves than did that of adrenaline. ln 1921 , Loewi, and independently Cannon and Unidil, neponted the nelease of an adrenaline-like substance f rom sympathetic nenves. Cannon cal led this substance I'sympathin". This substance was similar to adnen- aline in its pharmacological and chemical pnopenties. Howeven, when both compounds were i njected i nto the body, adnena I i ne was found to have excitatony and inhibîtony effects, while rrsympathinrl 1.2

showed only excitatony effects. Bacq, in 1934, suggested that the effects of sympathetic nenve stimulation wene due to the libenation from nenve endings of a substance which behaved like nonadnen- aline nather than adnenaline, an obsenvation made by Bangen and Dale over twenty yeans pneviously.

ln 1939' Lissak made the f irst extnactions f nom sympathetic nenves and showed that such extnacts had an adnenal ine-l ike action; howeven, he was not able to def ine the pnecise natune of these extracted substances. ln 1946, u. s. von Eulen and his coll- eagues devised r ef ined methods fon pur if ication and biological and chemical analysis of catecholamines and found that they could extract fnom the hearts of cattle, honses and cats a substance identical with noradnenaline in all its chanactenistics. Many wonkers have confinmed and extended these obsenvations indicating that nonadnenaline is the pnedominant .sympathomimetic substance in post-ganglionic sympathetic nerves and is libenated by their st imu I at ion .

wonk penfonmed in concurrence with the fonegoing, by Tainten and chang (1927 ) and Bunn and rainter- (,l93.| ), showed that cocaine or chnonic denervation neduced the response of effector ongans to administered tyramine and ephednine but enhanced the effects of adnenaline. This led to the suggestion that adnenaline had a direct action on the effecton cell, while ephedrine and tynamine had an indirect effect by acting on the nerve terminals. Subsequent to the discoveny by Bertler et al. (1956) tnat resenpine depleted tissues of nonadnenaline, carlsson et al. (1952) showed that tynamine was without activity in cats tneated with nesenpine. ln 1958, Bunn and Rand extended this finding, and showed that 1.3

nesenpìne depleted periphenal nenve stores of noradnenaline and that

the effects of indirectly acting sympathomimetic amines were abolished oi' decneased, but could be temponarily nestoned by inf us-

ion of nonadrenaline (Burn and Rand, 1960) .

some yeans eanl ier, Ah lquist (1949) suggested that thene were

two types of adnenoreceptors on vascu lan smooth muscle based on the nesponses of a numben of sympathomimetic amines. He hypo- thesised that alpha-neceptons in smooth muscle wene mostly assoc- iated with excitatony functions, €.g.¡ vasoconstniction, while beta- neceptons wene mainly involved with inhibitony phenomena, e .g. ¡ vasod i latat ion .

Tnendelenbung (1963) reviewed the actions of many sympatho- mimetics and showed that they could be loosely classified into dinect acting, mixed acting and indinect acting amines. Howeven, a complete range of actions is possible in diffenent animals and tissues. Trendelenbung (1972) states that "Sympathomimetic amines should be regarded as a gnoup of agents which share centain basic pnoperties but which may differ quantitatively in their- affinities to the vanious mechanisms involved în thein action.r lt would seem that the dinect effects of a sympathomimetic would be expected to be observed when the affinity of the amine fon adrenoceptons is high. lf an amine has a low affinity fon adnenoceptors then any indinect effects should appean. He goes on to suggest that an evaluation of the actions of dinectly and indirectly acting sympa- thomimetics should take the following factons into considenation.

1. Aff inity of alpha-neceptor fon sympathomimetic amines ln determining the relative potency of sympathomimetic 1.4

amines, two factors must be considered: firstly, the affinity of alpha-neceptors for the agent and, secondly, the inactivation mech- anisms responsible fon its nemoval. Tnendelenburg et al. (1970) and Tnendelenburg et al. (1971), using the cat nictitating rnembrane, shovred that the r elative potencies of nonadnenaline arrd methoxamine changed when noradnenaline inactivation mechanisms were elimin- ated. Since methoxamine is not a substnate fon eithen monoamine oxidase (t4AO) on catechol-O-methyl tnansferase (COÀ4T) and is not taken up intnaneuronal ly, it was found to be mone potent than nor- adrenaline unden nonmal cìrcumstances. Howeven, this situation was nevensed when the i nact i vat ion of noradrena I i ne was pneventec . 5 6 s( 1 /3 L cH NH

-cH I 3 Fig. 1 .1 Stnuctunal skeleton of a sympathomimetic amine. ln evaluating critenîa fon alpha-receptor aff inity, Tnendelen- burg (1962) showed Lhat the following stnuctunal changes were important. Firstly, offiission cf the beta-hydroxyl gnoup or intno- duction of an alpha-methyl gnoup decreases potency. Secondly, any group I arger than a methy I group on the n i tnogen atom or O-methylation decneases potency. Thindly, the rneta hydroxyl group is of greater importance than the para hydroxyI group. To neveaI the indirect action of a sympathomimetic, Trendelenbung (1972) suggesis that the amine must have a low aff in ity for a lpha-neceptors and also be ,3 substrate for uptake (acr^oss the neuronal membrane). Evidence that amines rvith weak direct effects tend to exhibit strong indirect effects has been reported (fi4arley, 1962; Tnendelenbur^g et 1.5

al., 1962; Schmidt and Fleming, 1963; Tsai et al., 1967\.

2. Uptake of sympathomimetic am¡nes across the neuronal banrien

Fon an indinect symoathomimetic to exert its action, it must possess the ability to be taken up into the nenve tenminal. The drugrs aff inity for this uptake not only determines its capacity to release neunonal nonadnenaline, but also its potential as a compet- itive inhibiton of noradnenaline ne-uptake. Burgen and lvensen (1965) examined a number of sympatho- mimetic amines fon thein ability to inhibit nonadnenaline uptake in rat heant. Thein nesuIts showed that centain stnuctunaI nequire- ments wene necessany for inhibition. Howeven, the study did not differentiate between those compounds which acted as competitive substrates for uptake and those which inhibited the uptake pnocess but wene not tnansponted into the neurone. ln addition, these amîneg may have had more than one sîte of action; fon example, in competing fon vesiculan uptake and binding nathen than just membnanal uptake. Muscholl and Weber- (lg0S) detenmined the arterio-venous differ ences in nabbit heants penfused with alpha- methyl-nonadnenaline. Their f indings, using a number of symoatho- mimetics, agreed with the nesu lts of Bungen anC lvensen (1965) and this indicated that it was membranal uptake that was measuned in the latten study.

3. Uptake into vesicles containing neunotransmitten

As a requi nement for vesicu l ar nonadrenaline nelease, the indinectly acting sympathomimetic rnust have the capability of 1.6

being taken up by the intnaneunonal stonage vesicle. This uptake system has been shown to have a high degree of steneospecificity (von Eulen and Lishajko, 1964; stjanne and von Eulen, 1966) with a preference fon the (-) isomen.

Two uptake systems exist for the accumu lation of amines into the storage vesicle: one is dependent on ATp-Mg++, is neserpine sensitive, and is nesponsible fon uptake of adnenaline and non- adnenaline (canlsoon et al., 1963). These authons also showed that anothen uptake exists which is not inhibited by nesenpine non is ++ reliant on ATP-Mg In conclusion, the latter uptake is thought to be the means by which the indir-ectly acting sympatho- mimetics, such as tynamine, enten the vesicle and exent their act ion .

4. Stonage in neurotransmitter vesicles

I t has been shown (Musacchio et al ., 1 965) that stonage vesicles ane able to bind sympathomimetics which are catechols on have a beta-hydnoxyl gnoup. Nash et al. (1968) showed that vesi- culan binding may be a detenminant fon the dr ug to displace nor- adnenaline. Howeven, Musacchio et al. (tgos) and Kopin et al. (1965) showed that a high affinity fon vesicular binding was not necessary fon a sympathomimetic to have an indinect action. They neponted that although tynamine was convented intnavesicular-ly to octopamíne, this was not a pr erequisite for its indir-ect action.

5. Effect of monoamine oxidase

It has been suggested that alpha-methylated sympathomimetics may exert a stnongen indirect action than their non-methylated 1.7

analogues (Tsai, 1967). This was postulated since alpha-methy lated compounds ane not substrates for MAo and so, following membranal uptake, they would achieve higher cytoplasmic concentnations.

Thenefone, it might be expected that MAo not only govenns the inactivation of cytoplasmic nonadrenaline neleased by the indirect sympathomimetic, but also detenmines the cytoplasmic levels of the neleasing amine. Smith (1966) showed that with tyramine (a sub- stnate fon l'4Ao), inhibition of this enzyme potentiated the indìnect sympathomimetic action.

6. Structure of synapse

de I a Lande and Watenson ( 1 968) , usi ng the nabbi t ear antery, showed that tynamine administened extnaluminally was mone potent as a vasoconstnicton than when it was intnaluminally admin- istered. Their findings indicated that the indir-ect component of tynaminers action was mone evident when the dnug was adrninistened extnaluminally, since denervation on tneatment with cocaine tended to make the noutes equipotent.

This study provided evidence that in this vessel ther e is an appreciable gap between the nerve endings at the medio-adventitial bonden and the smooth muscle cells in the media. Van orden et al. (1967) found in the cat nictitating membnane that the adnen- ergic innenvation is in closer proximity to the smooth muscle than it is in vasculan smooth muscle.

The Vasculan Model

Many different experimental model systems have been used to examine and categorise the effects and modes of act i on of the 1.8

various sympathomimetics. These systems may be ci ivided into vascular and non-vasculan models. The latten is nepnesented by systems such as rat vas defenens (Barrett et al., 1969)r 9ut (lnnes and Kohli, 1969), cat iris (lvlarley, 1962\, and cat nictitating mem- bnane (Fleming and Tnendelenbung, 196'l ; Dnaskoczy and Tnendelen- bur-g, 1970).

The vascu lan and cardiac models ane exemp I if ied by the iso- Iated penfused nat heant ( Ivensen, 1963; Burgen and Iversen, 1965), guinea pig atrium (Furchgott and Garcia, 1968), nabbit aonta (Maengwyn-Davies and Kopoonyi, 1966), nabbit ean arteny (de la

Lande and Watenson, 1968), and the nat tail artery (Nicholas, 1969).

The ventral caudal antery of the nat has been used by a numben of wonkens as a vasculan resistance model for examining the actions of vasoactive substances. Nicholas (1969) used the isolated nat tail antery in an organ bath and found it nes,ponded to nonadnenaline, electrical stimulation, tynamine, angiotensin and vasopnessi n. He suggested that the nat taî I antery wou I d be a suitable pnepanation fon the assay of sympathomimetic amines. Wade and Beilin (lgZO) studíed the effects of a numben of vaso- active drugs using the isolated perfused nat tail. The whole r at tail penfused via the ventral antery was subject to oedema and consequently an incnease in resting penfusion pnessune. Addition of bovine senum albumin eliminated this problem but complicated the system by intr oducing a potent drug binding pnotein. ln this thesis, a pnoportion of the work has been carnied out using a seg- ment of the isolated nat tai I (Frost et al., 19761, thereby over- coming the problem encountened by Wade and Beilin (1970) . ln severaI othen expeniments, the ventnaI arteny was removed fnom the 1.9

tail and set up in an organ bath fon penfusion as descnibed by de la Lande et al. (1966). It has been established by de la Lande et al. (lg6Za) ttrat the penivasculan nenve plexus surnounding the smooth muscle of smallen anteries (Waterson and Smale, 1967; 1'{atenson and de la Lande, 1967) plays a substantial nole in contnolling the sensitivity of the antery to catecholamines. lt was also neported by Fnewin et al. (1971b) that thene is a close similarity between the histology and innenvation of the human digital antery and that of the rabbit ean arteny. The nabbit ean arteny has been shown by de la Lande and Rand (lg0S) and \l/aterson and Smale (lS0Z) to be a small artery consisting of a thick smooth muscle laver which is bounded on the luminal sunface by the folded intima and on its outen aspect by loose adventitial tissue. Hodge and Robinson (1972) showed that the nat tail anteny in section has a smallen diameter than the nabbit ear arteny but is similan in other- charactenistics. They showed that both nat tail and r abbit ear antenies have a zone of specific catecholamine f luonescence at the medio-adventitial bor der and that this fluonescence nepnesents the site of the symoathetic nerve varicosities. The specific fluonescence in nabbit ean arteries was eliminated by cervical ganglionectomy. On this basis, the nabbit ean arteny would seem to be a usef ul pneparation to examine the effects of sympathom imet ic am i nes. The isolated centnal ar-teny of the nabbit ean, as described by de la Lande and Rand (1965) and penfused by the method of de

la Lande, Cannel I and \t/atenson ( 1966) , was thus used as the test vessel in some of these experiments. The isolated rat tail anteny has not been as exhaustively studied as the nabbit ean antery, 1.10

but on the basis of the afonementioned wonk can be considered to be a simi lan vascu lar model .

Mechanisms Available fon the lnactivation of Nonadrenaline in Vasculan Tissue

Neunonal uptake (Uptake 1) One of the earl iest studies which indicated that adnenergic nerves could accumulate adnenaline was that of Burn (1932). He f ou nd that the nesponse to symoathet ic nenve st i mu I at i on was impnoved by pnion inf usion of adrenal ine. He also suggested that the am íne was stoned tn the tissue fnom which it could be subse- quently neleased.

Eanly studies on neuronal uptake wene cannied out by Axelnod et al. (1959) and Hentting et al. (1961), who showed that ,tissues wene able to netain infused radiolabelled noradrenaline for long per iods and this ability was lost if the adrenengic nenves were destnoyed. Whitby et al. (1961) found that tissues f nom the cat accumulated tnitiated nonadnenaline and that this accumulation was greatest in tissues with high sympathetic innenvation, such as the heant. Hentting and Axelr-od (1961 ) also showed that this nadio- labelled noradnenaline could be neleased by adnenergic stimulation. lvensen (tSeS, 1965b) and Bur-gen and lversen (1965), using

the i sol ated penfused nat heant, demonstrated that the neurona I uptake pnocess had stereochemical and stnuctunal specificity. They neponted that (-) noradnenaline and (-) adrenaline wene taken up mone rapidly than the cornesponding (+) isomens. Thene ane prob- lems involved in determining the steneospecificity of membranal 1.11

uptake, since both vesiculan uptake (von Eulen and Lishajko, 1967) and MAO deamination (Blashko, Richter and Schlossmann, 1937; Giachetti and Shore, 1966a) ane highly steneoselective for the (-) nonadnenaline isomen. Hence the stereoselectivity obtained in accumulation expeniments is influenced by these secondary mechan- isms, To eliminate these pnoblems, expeniments ane penfonmed in nesenpine pretneated animals and in the pnesence of a monoamine oxidase inhibiton which has no effect on membranal uptake. Ecker t et al. (lgZ6a, b) and Henseling and Trendelenbung (1978) showed in rabbit aortic strips that axoplasmic uptake of noradrenaline showed no pnonounced steneoselectivity, wheneas vesiculan accumul- atîon and netention exhibited steneoselectivity when MAO was inhibited, with pnefenence fon the (-) isomer. They also showed that noradrena I i ne uptake was greaten than that of adnena I i ne, wh ich was in tunn gneaten than that of igopnena I ine. It4uscholl (1960), Axelrod, r,l\/hitby and Hentting (1961) and Dengler et al. (1962) demonstrated both in vivo and in vitro that pnetneatment with nesenpine greatly neduced the accumulation of exogenously administer-ed noradnenaline. ln 1962, it was found by Kopin and Gordon that most of the tnitiated nonadnenaline depleted from tissues by nesenpine was deaminated by N4AC. This suggested that the nonadnenaline was deaminated and lost f nom the tissues instead of being bound. Lindmar and Muscholl (1964) showed that af ten pnetneatment with nesenpine, perfused nabbit heant still nemoved noradnenaline f nom the penf usate, but no net uptake could be observed. ,A combination of nesenpine pnetneatment and inhibit- ion of ÀaAO by ipnoniazid revealed that the amine was still nemoved fnom the penfusate but that, now, a si gn ificant net uptake 1.12

occunned. They also showed that this uptake was gneatly dimin- ished by treatment with either cocaine on guanethidine. Lindman and Muscholl concluded that rrthere ane two mechanisms which concentrate nonadrenal i ne: ( 1 ) tnanspont acnoss cel I membranes; (2) uptake of nonadnenaline into storage sites, and pnetreatment with neserpine will block the second.rl Hambengen et al. (1964), using a f luorescent histochemîcal technique, conf irmed that resenpine impaired vesiculan nonadnenaline stonage. Waterson and de la Lande (1974) and de la Lande et al. (19?4) demonstnated that the perivasculan nenve plexus of the nabbit ean anteny was still capable of taking LJp noradnenaline in nesenpine pnetreated rabbits. This is in agneement with the find- ings of Lindman and lr4uscholl (1964). Henseling et al. (lgZøa, b) and Eckent et al. (lgZOa) have shown that intnaneunonal accumulation -of noradnenaline involves at least two compantments. These two compantments are neferned to as axoplasmic and vesiculan. Hence, neuronal uptake can be taken to mean the tnansmembnanal influx into the neurone. Tainter and Chang (192?) neponted that cocaine administered systemically in a vaniety of animals inhibited the response of tyramine and potentiated the response of adnenaline. Burn and

Tainten (1931 ) extended these f indings, and showed that the actions of tyr.amine and ephedrine were attenuated by cocaine, whereas adrenaline was potentiated. lt was also found that chronic dener'- vatîon of the pupi I of the cat eye sensitised this organ to adnen- aline but abolished the effects of tynamine and ephedrine. Burn and Rand (1958) showed that tynamine was without action in resen- pine tneated animals and that tynaminers pressor nesponse could 1.13

be nestoned following an infusion of nonadnenaline. Tnendelenburg (1959) suggested that cocaine delayed the inactivation of nonadren-

aline and adnenaline, leading to a highen concentnation at necepton

sites and hence an augmented nesponse. Macmiilan (195g), as a result of obsenvations made in isolatecl nabbit atnia, suggested that cocaine pnevented the uptake of nonadnenaline by the stonage sites.

Henttinq et a I . (l SOI ) found that tyr-amine neduced the amount of

tritiated nonadrenal ine in the heant when given eithen befone on af ten a norad.enaline infusion. lt was then suggested that tyna- mine was neleasing bound noradrenarine f nom its stonage sites. Falck and Tor-p (1962), using a new histochemical technique, were the f ir st to conclusively show that uptake of nor-adnenaline was directly into adnenengic nerves and not into othen ancillary sítes. Hillar p and Malmfo¡ s (lgO¿), also using histof luonescence techniques, showed that onetneatment with cocaine blocked the accumulation of noradnenaline by ttre adnenergic nerves in the nat iris' This pnovided direct evidence that adr-enengic nenves had an eff icient uptake system in the cell membrane and that this mechan- ism was inhibited by cocaine. de ra Lande et al. (r96za, b) arso suggested a pnejunctional action for cocaine in inhibiting neunonal uptake in the nabbit ean antery.

Nícholas (1969) showed in the perf used rat tai r arteny that cocaine incneased the sensitivity to nonadnenaline. subsequently, wyse (1976), using rat tail arter-y stnips, suggested that both exogenous and neunonal nonadrenaline ar e pnefenentially inactivated by neuronal uptake and stonage. venning (lgzg) found that the sensitivity of the tail anteny to extraluminal nonadrenaline was greatly incneased in the presence of cocaine. Vanhoutte and \{ebb 1 .14

( 1 979), a l so usi ng nat tai l str ios, suggested that neunonal uptake of nonadnenaline following electnical stimulation was an impontant disposition mechanism for the transmitter in the nat ta i I antery .

Extnaneuronal uptake (Uptat

lvensen (lg0Sa) showed that when the isolated rat heart was perf used with high concentnations of adnenaline on nonadnenaline, satunation of Uptake 1 occunned. once satunation of uptake t had occurned, a sudden and lange incnease in tissue uptake followed.

lversen tenmed this uptake 2, and suggested that this uptake was effective only at high concentrations of adnenaline on nonadren- aline. Lightman and lvensen (1969) showed that when rat heant was incubated rryith low concentnations of catecholamines in the presence of MAO and COMT inhibitons, the nonmal levels of metab- ol ites wene substituted by unchanged amine. This suggested the vaniability in uptake pneviously obsenved may be due to vanying degrees of metabolism and inf luences on uotake by nonmetanephnine.

Howeven, in this study, metabolites were measuned only in the heart tissue, not in the penfusion fluid. lversen subsequently nevised his theory and suggested that extnaneunonal uptake may occur at all catecholamine concentrations. Bevan and su (1973) also showed that uptake does occur in nat heart at concentnations of amine which would occur physiologically both at nerve terminals and in circulating blood. This uptake was also shown to be satun- able, with a highen capacity and a lowen aff inity for nonadrenaline than neunonal uptake, but a higher affinity than neunonal uptake for isopnenaline. Gillespie and Towart (1973), using the rabbit ear arteny, and Lightman and lvensen (1969), in studies involving 1.15

penfused nat heant, showed that the uptake system in smooth muscle has a low affinity and a high capacity. lversen (1965a), Gillespie (1923) and Henseling and Trendel_ enbung (1978) have demonstnated that uptake 2 has no pronounced stereoselectivity and is inhibited by vanious stenoids, e.g. ¡ conti- costenone (lversen and salt, 1970), haloalkylaminesr €.g.r phenoxy- benzamine (lvensen et al., 1972\, and O-methylated metabolites of catecholamines, e.9.r nonmetanephr ine (lvensen, ,l965a; Mineylees and Foster, 1973). Avakian and Gillespie (1968) found accumulation of nonadnenaline in smooth muscle cells of the rabbit ean anteny, as well as binding by collagen and elastin following incubation with high concentnations of nonadnenaline. This accumulation of nonadrenaline in smooth muscle cells was found to be abolished by phenoxybenzamine and nonmetanephrine. On the basis of this find- ing, Gillespie postulated that this smooth muscle uptake was simîlar to lvensenrs uptake 2. Gillespie and Muin (l9zo) found a lange vaniability in the ability of anterial smooth muscle f nom diffenent species and differ ent smooth muscle in one species to accumulate noradnenaline. Evidence for conf inming that uptake 2 is a very significant mechanism fon tnansmitten inactivation in eithen the nabbit ean antery on rat tail anteny is scar-ce. de la Lande et al. (1974) showed thai in neserpine onetreated and MAO inhibited anteries, nestoration of noradrenaline f luonescence could not be obtained by intnaluminal noradnenaline perf usion unless metaneph- nine was present. This contnasted with napid nestoration of fluo- nescence without metanephr ine present when noradr-enaline was appl ied extral uminal I y.

Wyse (1976) suggested that the nat ta i I antery may have a 1 .16

nelatively modest extnaneunonal uptake system which could only be demonstnated when high concentnations of noradnenaline and combin- at ions of uptake and metabol ism inh ib i tors wene used . Venn ing and de la Lande (1979) also showed in the perfused nat tail anteny that deoxyconticostenone acetate caused only a small increase in sensitiv- ity to intnaluminal adnenaline which was not signif icant at low dose levels. Modif ications of the uptake 2 system have been suggested.

Evidence now pr oposes that two extnaneunonal compantments may exist (Kaumann, 1972; Eckent et al., 19?6a, b; Henseling et al., 1975a).

Funthenmore, Powis (1973) showed that nonadnenaline would bind to collagen and elastin and that this binding was sensitive to tetnacyclines. He found that in the rabbit ean arteny and nat tai I arteny (tissues with high propor-tions of connective tissue), oxytetracycline enhanced the nesponse to noradrenaline ten ,times.

He suggested that th is may be of some importance in sma I I smooth musculan antenies. ln summany, it would seem from the fonegoing that extnaneunonal uptake openates in the nat tai I anteny and nabbit ean antery, but is comparatively insignificant when cornpaned with neunonal uptake mechanisms.

Monoamine oxidase (MAO) Jarnott (1971a) has suggested that thene ane at least two fonms of MAC in sympathetically innenvated tissues with different substrate nequinements. These two types can be broadly classified as neunonal and extnaneunonal. Gnaefe and Eckent (lszz) and Henseling and Tnendelenburg (1978) have shown in the nabbit aonta that both these fonms ( tfra t is, neunona I and extnaneunona I MAO ) 1.17

are steneoselective with a preference fon the (-) isomer^. de la Lande et al. (lgZO) and Honita and Lowe (1972) demonstnated the pnesence of extnaneunonal MAO in nabbit ean antery and rat heant, nespectively. Jannott and Langer (l9Z.l ), using cat nictitating

membrane, Jannott and lvensen (1921 ), on nat, guinea piS and

nabbit vas defenens, and coquir et ar. (tszs¡, using r-at femonal and mesentenic antenies, neported a decnease in MAO activity follow- ing surgical denenvation. These f indings suggest the presence of neunonal MAO.

Biochemical studies by Jarr ott (lSZl a) on rat heant and other tissues, and histochemical studies on nabbit ean anteny by de la Lande et al. (lgZOa) ¿¡¿ not show a decnease in MAO activity

following denervation. lt was suggested by Jannott that the amount of neuronal t\4Ao may be much lowen than extraneunonal N4Ao in

these tissues. lt would seem, on the basis of these f indings, that extraneunonal and intraneuronal f\4AO are pnesent in differ ent tissues, although their nelative functions and physìological roles are yet to be fully defined. de la Lande and Johnson (1972,) showed that extnaneunona I

MAo inactivated high concentnations of nonadrenaline. lt was suggested that this extraneunonal MAO activity may account for the fact that intnaluminal tyramine nesponses in the nabbit ear anteny ane lowen than extnaluminal nesponses (de la Lande and waterson, 1968; de la Lande et al., l97oa). ln a laten study, de la Lande and Jellett (1972) showed that inhibition of MAO caused an incr-ease in sensitivity of the nabbit ean antery to noradnenaline but only if the nonadnenaline was app lied extnaluminally. de la Lande et al. (lgZ+) supponted these f indîn9s histochemically and suggested 1 .18

that neunonal MAO was pnobably mone impontant in determining the response of the rabbit ean anteny to exogenous nonadrenaline. This may be attributed to highen concentnations of noradnenaline achieved intnaneunonally by an eff icient neunonal uptake system. These observations ane in agreement with those of Funchgott and Garcia (1968) using the isolated guinea piS atnium. de la Lande (1975) suggested that the influence the neur-onal uptake process has in determining the concentnation of nonadnenaline at the receptot s is controlled by the rate of inactivation of noradnenaline by intnaneunonal MAO.

Catechol-O-methyl tnansfenase (COMT) Studies have demonstrated that in some tissues - for example, cat heant, nabbit and nat vas defenens - COMT activity is decreased by denenvation (Cnout and Cooper, 1962 Jarnott, 19?1b; Janrott and I vensen, 1971 ) . Convensely, in othen tissues, such as guinea piS vas deferens and nabbit ean anteny, no such decneases in COMT levels following denervation wene obsenved (Jannott, 1971b; Jarr-ott and I vensen, 1971; Head et al., 1974, 1977b) . Head et al. (1975) showed that neither denenvation nor cocaine pretneatment inhibited the production of the majon COMT metabolite subsequent to incub- ation with tr itiated nonadnenaline. On the basis of this f inding, de la Lande (1975) su5lgested that COMT was probably distr ibuted extnaneunonaIly in the rabbit ear anteny. Johnson and de la Lande (1977), using the rabbit ean antery, indicated that in the pnesence of cocaine (inhibiting neunonal uptake), COMT inhibition by UO52l caused a potentiation of the response to catecholamines. This potentiation was blocked by the Uptake 2 inhibitor deoxy- 1.19

coÌ ticostenone acetate.

The fonegoing results agnee with the findings of Eisenfeld et al. (1967), Bonisch et al. (1974), Graefe and Tnendelenburg (i974\ , and Eckent et al. (1976b), who confirmed that the nate of extna- neunonal tnansport appears to detenmine the nate of O-methylation of nonadnenaline. Eckent (tgZOU) also showed that CO[\4T is far more important than MAO as a detenminant of extnaneunonal metab- olism in nenve-f nee nabbit aontic strips.

Kalsnen ( 1969) tra¿ also pneviously shown that inhibitors of COMT or extnaneunonal uptake blocking agents potentiated the res- ponses to adnenaline and nonadnenaline, and these effects wene not additive. No evidence is available to show whethen on not COMT is pnesent in the rat tail anteny; howeven, it would not seem unneasonableì to assume that, with its similanity to the rabbit ean artery, COMT probably has some nole in this vessel.

Scope of Study

The pnesent study was initiated to examine in detail the mechanism of action of the sympathomimetic agent etilefrine on blood vessels. Fnom here, the pnoject evolved to become a companative study, which examined the vasculan effects of etilef r-ine and a numben of othen sympathomimetics, including a new sympathomimetic agent, REN-293 (Z-amino-3[3,5-dihydr-oxyphenyl]-l-propanol). As part of the over^all pnotocol, the interactions between etilef r ine and several othen agents used in treating the clinical syndrome of dysautonomia wene also investigated. 1 .20

Etilefrine has been used pnimarily in the tneatment of patients with onthostatic hypotension. The benef iciar effect of dnug ther apy in these cases has been attr-ibuted to the similar action etilef nine has to nonadrenarine (Miiler, wienen and Broomf ierd, 1gr3). Mellander (lg0o) demonstr'ated that etilefnine had a local constnictor effect which was pnoduced by arpha-neceptor occupation, and arso a distant dilator effect which was mediated via the sympathetic nenvous system. He funther showed that etilefr ine caused a pattenn of response which was quantitatively similan to that of nonadnen- aline. An impontant considenation in the selection of a sympatho_ mimetic agent to tneat onthostatic hypotension is the ability the dnug has to exent a dinect constnictor effect on vasculan smooth muscle' Parks, sandison et ar. (1961), in neviewing the dnug therapy of postural hypotension, showed that in patients with auto- nomic degenenation, the indinectly acting .sympathomimetics ephednine and methy lamphetamine had no constricton effect on denenvated blood vessels. Nanda et al. (lgzz) citea a case whene orthostatic hypotension was due to impairment of nonadnenaline nelease and showed that tynam i ne had no pnesson nesponse; however, the dinectly acting sympathomimetics pheny lephnine and noradr.enaline exhibited manked pnessor nesponses in this situation. With etil- efnine, and any othen sympathomimetic having a nore in treating orthostatic hypotension, it is thus impontant to know the extent to which the dnug nelies on the sympathetic nenves for its constnicton action.

The expeniments to be described in this thesis seek to examine the effects of two sympathomimetics (etirefr ine and REN-293) on small musculan antenies and to elucidate their possible mechanisms 1 .21

of action. ln addition, these investigations considened some aspects of the influence that neuronal uptake, extraneunonal uptake, and metabolism by monoamine oxidase (MAO) and catechol-O-methyl transferase (COI4T) had on the actions of these sympathomimetics. Z USIdVHf, 2.1

GENERAL METHODS

This chapten describes the general methodology used in the penfusion and efflux experiments that wene cannied out duning this project. The following methods and techniques ane described.

The technique used fon the penfusion of the nat tail segment via the ventnal caudal antery. 2 The technique used fon the perfusion of both the isolated

ventnal tail anteny and the centnal ar teny of the r abbit ear. 3. The autoanalysen assay fon catechoramines which was to con- finm the success of sungical on chemical sympathetic denerv- ation.

4 The technîque of sungical denenvation of the nabbit ean arteny. 3H-no".dnenaline 5 The pnotocol used fon detenmining eff lux from rat tail antenies and the modif ication of this technique for 3H-nonadnenarine the punpose of detenmining metabolites¡ 3H-noradnenaIine 6 The pnocedune used fon separating and its metabol ites.

7 Tests of statistical significance.

All other expenÌmental pnocedunes ane descnibed in the aopro- priate sect ions of each chapten. A list of drugs used thnoughout the pnoject is included in the Appendix. '))

1 . Penf usion of rat tail segment

ft4a le a lb ino Sprague-Daw ley rats ( weigh in9 250_350 9 ) wene

stunned and exsangu inated. The ta i I was severed f rom the tr-unk and pnoximal the section of the tail then divided into two 3 cm segments. The proximal eñd of the ventnal anteny was located in each segment and cannulated under a dissecting microscope using a Stenivac No.2 heat-dnawn polythene cannula (extennal diameten

2 mm, intennal diameter 1 mm). The cannulated segments wene placed in a wanming chamben at 3zoc and penfused with Krebs- bica.bonate solution (see Appendix 1a) containing EDTA (lo u M). Funchgott (1955) has neponted that concentnations of EDTA of this orden of magnitude neither inhibit non potentiate the effects of adrenaline and nelated compounds on vasculan smooth muscre. The appanatus used is shown in Figune 2.1. The penfusion f luid was gassed with 95% o z, 5% ,oz (carbogen)- and then pumped thnough a constant volume nollen pump (see Appendix 1b) at a f low nate of 4-5 ml/min. Pnessure changes wene measured using a Statham P23GC on P23DC pnessure tnansducen and neconded on a Rikadenki pen neconCer (see Appendix 1c). The penfusion f luid f lowed into the segment via the cannura in the ventnar arteny (Fig. z.z) and then thr ough the severed end of the tail, being f inally nemoved fnom the wanming chamber vía the overflow escape. The penfusion fluid was wa.med in a bath and again pne-warmed just befone it entened the anteny. The wanming chamben (F ig. 2.3) was a pen_ spex box containing a brass tank heated to 3zo c by cír culating waten. This chamben was covened by a penspex l¡d during the expeniment to maintain high humidity.

A f ive-channel sequential timen (see Appendix 1d) was used PERFUSI ON WARMING CHAMBER FLUI D PUM P

RAT TAI L -+ S EGME NT 95"Â 0^ soh coz -Þ + OVERFLOW ¡ t ,}

o o a o a I TRANSDUCER

Fig.2.1 The perfusion appanatus used in the study of rat tail segments. Fis. 2.2 The nat tail segment showing the cannula in the ventnal ar tery Fis.2.3 The wanming chamben used in the study showing the rat tail segment rrin situ.rl 2.3

to change the penfusing drug solutions in some expeniments and thus cumulative dose nesponses could be obtained. The tail seg- ments were allowed to perfuse with Knebs for 30 minutes befone any dnugs were administered. This nesulted in a resting penfusion pnessune of approximately 30 mmHg. All drugs wene added to the penf usion f luid and were thus applied intnaluminally. Each test dnug was maintained in contact with the intnaluminal sunface of the vessel until a maximum nesponse was obtaîned.

2a. Penfusion of the isolated rat tail antery Male albino Spnague-Dawley nats (weighing 250-350 9) wene stunned and exsangu i nated. The tai I was then severed from the trunk and the ventnal caudal anteny isolated. A oiece of this antery, aporoximately 3 cm in length, was nemoved fnom the pnox- imal end. A Ster ivac No. 3 heat-dnawn polythene cannula was insented into the pnoximal end w¡th the aîd of a dissecting micro- scope, and a finen cannula (Stenivac No. 2) was insented and tied into the distal end of the antery. Appnoximately 1.5 to 2 cm of anteny nemained between the tips of the cannulae. The anteny was then quickly set up in a ventical, double-jacketed ongan bath (lS ml capacity), and bathed extnaluminally and penfused intnalumin- ally with Krebs-bicarbonate solution at 37oC. The uppen distal cannula was held at a tension of 1 g. Figure 2.4 is an illustrat- ion of the penfusion appanatus, wlrich was similan to that descnibed by de la Lande et al. (1966). The perf usion f luid was gassed with 95% OZ, 5% CO, (Canbogen) and then pumped thnough a con- stant volume nollen pump at a f low rate of 4-5 ml/min. Pressune changes wene measuned using a Statham P23GC on P23DC pnessure TENSI ON I 95% 0 2---+ 5010 c0 2 15mt ORGAN BATH PUMP L 0 0 \ o ù 95% 0 -4 RTERYO 50h c

E XTRALUMINAL FL UID

TRANSDUCER

PERFUS ION FLUID Fis.2.4 The penfusìon apparatus used in the studies involving isolated rat tail anteries and nabbit ear arteries. 2.4

tnansducen and neconded on a Rikadenki pen necorden. Two seg- ments of anteny fnom the same animal wene always used and per- fused simultaneously. The vessel was allowed to equilibrate fon 30 minutes and the reconded resting perfusion pressune was approx- imately 30 mmHg. Absence of leakage between the extnaluminal and intnaluminal f luid compar tments was noutinely tested at the begin- ning of each expeniment by observing the level of the extnaluminal f luid in the ongan bath. On some occasions, at the end of the expeniment, leakage was also tested by penfusing the arteny intra- luminally with 1% Evans Blue dye and examining the extraluminal fluid fon the appearance of the dye. Appnoximately one antery in six displayed evidence of leakage, usually through a side bnanch not detected duning dissection; such anteries wene nejected. Dnugs wene administened to the antenies eithen extnalurninally on intna- luminally and maintained in contact with - the vessel until a m,aximal nesponse was obtained.

2a. Penfusion of the rabbit ean antery Semi-lop-eaned nabbits, br ed at the Centnal Animal House, Waite Agricultunal Reseanch lnstitute, Univensity of Adelaide, wene used in all penfusion expeniments and genenally weighed between

1.5 lo 2.5 Kg. Rabbits wene f inst stunned and exsanguinateC. The ovenlying skin at the base of the ear was then nemoved. A 3-4 cm segment of antery up to the finst majon bnanch was dissected clear of adher ing¡ tissue (FiS. 2.5). Tissues were kept .oi=t with Krebs-bicanbonate solution (see Appendix 1a), and a Stenivac No. 3 heat-drawn polythene cannula was insented into the pr-oximal end of the arteny and tied into position. A finer cannula (Sterivac NERV E

1 AR ERY V E IN ts.TA I L OF RABB IT

Fìg.2.5 The rabbit ear showing the anatomical location of the nabbit ean antery used in some of the studies. 2.5

No. 2l was inserted and tied into the distal end of the arteny. Approximately 1.5 to 2 cm of anteny nemained between the tips of the cannulae. The anteny was then quickly set up in a ventical, double-jacketed ongan bath (lS ml capacity), bathed extnaluminally anC penf used intnaluminally with Knebs-bicarbonate solution at 3?o C. The uppen distal cannula was held at a tension of 1g. FiS- ure 2.4 is an illustration of the penf usion appanatus, which was similar to that descnibed by de la Lande et al. (1966). The per-fusion f luid was gassed with 95% OZ, 5% CoZ (Canbogen) and then pumped through a constant volume rollen pump (see Appendix) at a f low rate of 4-5 ml/min. Pressune changes wene measured using a Statham P23GC on P23DC pressure transducen ancl neconded on a Rikadenki pen neconder (see Appendix 1c) . Two anteries f nom the same an ima I wene a lways perf used and mon itoned simu I tan- eously. The penf usate exiting f nom the distal cannula was dis- carded. Dnugs could be added independently to eithen the Krebs solution bathing the adventitia (extnaluminal application) o" the intennal penfusate (intraluminal application). Leaks from small side bnanches wene rare and easily detected at the commencement of each experiment due to a spontaneous increase in the volume of the extnaluminal bath. Leaking anteries wene discanded.

3. Autoanalyser catecholamine assay Estimations of tissue catecholamine content wene made by the catecholamine assay technique descnibed by Head et al. (1977).

A Technicon Autoanalysen tnain was used, which consisted of a samp len, pr^opont ion ing pump and f luonometen The necorden was a Rikadenki Model B1G1 potentiometnic necorden, which was matched 2.6

to the f luonometen by installation of a tnansiston emitten followen ci ncu i t. The fl uonometen was set at maxi mum sensi ti v i ty, and height of responses (detenmined by catecholamine content in assay) was controlled by a mi llivolt attenuaton on the necorden. The auto- analysen f unctioned by (1) sampling unknown and standard solut- ions of catecholamine automatically; (21 neutralising these solutions to pH 5.0-6.0; (3) oxidising catecholamines with potassîum fenni- cyanide; (4) fonming f luorescent tni-hydnoxyindole (TH I ) der ivatives by treating the oxidised catecholamine with sodium hydr oxide in the pnesence of asconbic acid (¡s/nciO); (5) estimating the f luones- cence pnoduced by THI derivatives.

The f luorescence was continuously measured at the following wavelengths z 410 nm fon activation (interference f ilten) and emission at gneaten than 485 nm (Wratten No. I shar p cut f ilter ).

The f low diagnam of the system is shown in Figur e 2.6., The catecholamine content of the antenies was measuned by removing the arteries f nom the tails, weighing them, and placing them into tubes containing 1 ml of EDTA (30 uM) in HCL (100 ml'vl). The tissues wene lef t 18-24 hours at 4oC, and the catecholamine content was determined from tlris clear acid extr act by using the autoanalyser system described above. Successful denervation of the tail antery was confinmed by detecting a substantial decrease in the catechol- amine content of the sungically treated vessel by comparison with normal contnol antenies.

4 Sungical sympathetic denenvation of the rabbit ear artery

It was found by de la Lande and Rand ( 1 965 ) that remova I of the homolatenal superion cervical ganglion caused denervation WASH WE L L

r--l l¡ 5oo mmol l-1 Hct , 1. ô rnl min-l

200 mmol Phosphate + 500 mg r3fe(CN)6 L pH=ô'5 0'6 ml min -1 sample 1.0 mI min-l SMC Air 0.42 mI min*l I t4 Phosphate Buffen pH-ô.5 DMC 1 [1 Na0H 0.8 ¡rI min-1 c Air 0.42 mI min-l

2.5 l.4 Na0H 1.6 ml min-l

0.2% Ascorbic Aeid WASTE t.faste 1.6 ml min-1 DMC I L J

MC PUMP FLUOROMET ER

REC ORDER

Fis.2.6 Flow diagram for the autoanalyzer when used for the assay of catecholamines. The reagents, thein concentnations and flow nates ane shown . SMC and DMC nefer to sing le mixing coi I and double mixing coil, respectively. 2.7

of the rabbit ean antery. Rabbits vvere anaesthetised with pento- banbitone (t:S dilution with stenile salîne) injected into the marginal ear vein (Mundock, 1969). The dose of barbitunate vanied considerably but was usually within the range of 30-50 mg/Kg. lnjection at a slow nate continued until the toe nef lex was weak and nespination was decneased in rate but incneased in depth. The neck was extended, shaved and cleansed with Savlon 3% in 70% ethanol. A midline incision through skin and muscle was made to expose the tnachea. The lef t canotid anteny was located and the supenior cenvical ganglion identif ied adjacent to the thynoid canti- lage and under the angle of the mandible. The ganglion was nemoved with appnoximately 1cm of tissue attached on eithen side to ensune that the pne- and post-ganglionic f ibres had been taken. The wound was closed with silk sutures. Antibiotic coven vvas not routinely administened since the sungeny was conducted using an aseptic technique. Experiments using denervated anteries wene cannied out as close to 21 days following ganglionectomy as pnact- icable.

5. 3H-no".drenaline eff lux protocol

Irla le Spr ague-Daw ley nats (250-350 g ) were stunned and exsanguinated. The tail was then sevened f nom the tnunk and the ventral antery isolated. A piece of this artery, measuning appnox- imately 3 cm, was removed and placed in Knebs-bicarbonate solution. The anteny was blotted dny (with the intnaluminal fluid being care- ful ly expnessed) and then weighed. The vessel was then pne- incúbated fon 30 minutes in 1ml Krebs-bîcanbonate solutîon with

EDTA and ascor bic acid (see Appendix 1e) at 37o C and bubbled 2.8

w¡th 95% O Z, 5% CO Z before being incubated in either a f unthen 1 3H-nonadnenaline ml of asconbic Krebs containing 11¡ 1.18 uM or 14C-sonbitol 1.1 uM for 30 minutes. The equipment used in the eff lux studies (designed and built by the authot and illustnated in Figure 2.7\ pnovided a neliable and efficient method for- continuously washing the antenies with 1 ml aliquots of asconbic Krebs. Following incubation, the anteny segment was tnansfenred to the syringe containing 1 ml of ascorbic Kr ebs bubbled with 95%

O 5% ,O, (Canbogen). Af ter 1 minute, the wash cycle was init- Z, iated. Relay l was openated by timen 1pr'oviding a sufficient peniod fon the nelay which clamped thin silicone tubing to nemain open and allow the 1 ml wash to dnain into a collection vial. Timer l then tniggered timer 2, which caused a closing of relay 1 and an opening of nelay 2, thus allowing a volume of 1, ml of ascorbic Krebs to be dispensed fnom reservoir 1. This Krebs solut- ion was kept wanmed to 37oC and gassed with Canbogen. Timen 2 then tniggened timer 3, which closed nelay 2 and opened relay 3, which in tunn al lowed a quantity of asconbic Krebs to be released into resenvoir 1 from resenvoin 2 until ovenf!ow occut ned. To ensune that a constant volume of 1 ml was always dispensed into the 1 ml syringe, a constant pnessune head had to be maintained

as wel I as accunacy of timing (provided by timer Z). Variation in volume nanely exceeded 1-2%, i.e., 0.01-0.02 ml. Timer^ 3 then tni ggered timer 4, which closed nelay 3. Timer 4 provided a time buffer to obtain an intenval of l minute between washes. Timen

4 then tr igger ed timen I, which opened nelay 1 and nepeated the cycle. This pnocess continued fon 20 minutes. Each 1ml wash RESERVOIR 2 (100nt)

950 oz 5o/o C 0 2

RESERVOI R 1 c +- 0 (20mt) o I OV ERFLOW 0 0 TO WASTE 0

95% 0 2 50 co 2

WARMING lmt CHAMBER SYR INGE

AR TE Y

T1 T2 T3 T¿

COLLECTI ON V IAL

Fis. 2.7 The system used to wash the tail artenies is shown in this diagnam. Each tîmer T1-T3 was responsible for open- ing and closing relays R1-R3, thus allowing aliquots of Knebs to wash the antery and be subsequently collected. 2.9

collected cuning this period was retained and added to 10 ml of scintillation f luid (see Appendix 1f ). The samples wene counted using a Packand 2425 liquid scintillation spectnometen and connected for efficiency using an automatic external standand. Results were expressed as p.ot 1t-u A/s/min.

6a. 3 H-noradrenaline and metabolite efflux pnotocol Nlale Spnague-Dawley nats (250-350 g) wene stunned and exsanguinateC. The tail was then sevened f nom the tnunk and the ventnal anteny isolated. A piece of arteny measuring appnoximately

7 cm was removed and placed in Knebs-bicarbonate solution. The arteny was blotted dry (with the intnaluminal f luid being car efully expnessed) and then weighed. lVeights vanied from 14.0 T.o 21 .O mg. The vessel was then pr e-incubated fon 30 minutes in Krebs-bicar b- onate soluticn with EDTA and ascor bic acid (asconbic Kr-ebs) (see

Appendix 1e) at 37o C and bubbted with 95% OZ, 5% CO, befone 3H-nonadnenaline being incubated in (-) (3 U-N¡) l.tB u M for 30 minutes. The incubation f luid was sampled (0. t ml) befone and af ten each incubation. Following the incubation, the ar-teny vJas nemoved and immersed in a 1 ml aliquot of asconbic Knebs fon 5 minutes. This pnocedure was nepeated six times, with a f nesh ali- quot of asconbic Knebs each time, using the equipment descnibed in the pnevious methods section (Fig. 2.7\. Dr ugs wene introduced into the immension (wash) tlu¡A at the commencement of the third 5 minute peniod and maintained in contact with the segment for all subsequent washes up to the 35 minute maximum. The two 0.1 ml samples of incubation fluid and the seven 1ml washes were then assessed for 3 H-Na and 3 H metabolite content using alumina and 2.10

Dowex columns (Gnaefe et al., 1973). A conrection was made for crossoven and necoveny of NA metabol ites (t4onris and lrvine, pensonal communication; see Appendix 1S)' 3H-N¡ Af ter the and the vanious metabolites had been sepan- atecì fnom each wash aliquot, a unit volume (1 ml) of each eluate (see on eff luent was sampled and added to 10 ml scintillation f luìd Packard Appendix 1f ) . The samples wene counted using either a 2425 or 3310 liquid scÎntillation spectnometen and conrected fon efficiency using an automatic external stancjard on by obtaining a channels ratio.

6b . Metabol i te seParation The method used was similan to that descnîbed by Gnaefe et al. (1973). The 1 ml tnitiated wash volume fnom the artery or the 0.1 ml sample from the incubation fluid (to which was added a further 0.9 ml Krebs-bicanbonate to keep the volumes consistent) was collected in a chilled vial containing o.2 ml 0.1 N HCI and 0.1 ml l% ascorbic acid. These samples wene kept f roz.e¡ until aSSay. lmmediately pr^ion to loading on to the alumina column, the following solutions wene added to each sample - the Tris-buffen added last: (1) 0.1 ml 1% asconbic acid; (2) O'1 ml 12'5% solution NarSO3; (3) 0.1 ml 1A% EDTA; (4) O.01 ml of a cold carnien . containing a mixtune of nonadrenaline metabolites, each at a con- and centnation of 1 mgf ml , i .e. , VMA' MOPEG, DOMA' DOPEG' Nt'¡N NA; (5) 0.5 ml 1N Tris-buffer at pH 8.4. The total volume thus loaded on to the alumina column (see Appendix th) was 2'11 ml' ThecolumnWasthenwashedwith4mlofwater.Thiseffluent was allowed to nun on to a Dowex SOlVx4 column (see Appendix 1i)r 2.11

which was subsequently washed with 1 ml waten. The final volume of the eff luent was thenefone 7.11 ml. This eff luent contained O- methylated deamînated nonadnenaline metaboliies (OMDA), i.e., methoxyhydr-oxyphenylglycol (MOPEG) and methoxyhycìnoxymandelic acid (VfúA). No attempt was made to funthen separate these metab- olites, and a 1 ml aliquot was added to 10 ml scintillation fluid and counted. The Dowex column was then washed with 2 ml of a

6 N HCI-ethanol mixtui e (natio 1 :1). This eluate contained nonmeta- nephrine (Nl4N) and 1 ml of this solution was also counted. The alumina column was then washed with 2 ml O.2 N acetic acid, and this wash allowed to nun on to a seconci Dowex column and collected. This second Dowex column was washed with 2 ml waten, making a f inal volume of 4 ml. This eluate contained dihydnoxyphenylglycol (DOPEG). Again, 1 ml was sampled and counted as before. This second Dowex column was now washed with 2 ml 6 N HCI-ethanol (natio 1 :1 ) . This eluate contained the nonadnenaline f raction and 1 ml of this was sampled and counted. The alumina column was next washed with 4 ml 0.5 N acetic acid and this was discarded. As the final step, the alumina was washed wíth 2 ml O.2 N HCI and this wash was collected. ln this final wash, dihydnoxymandelic acid (DOMA) had been eluted, and 1 ml was again sampled and counted as befone (Fig. 2.8).

7. Tests of signif icance When companing the effects of diffenent dnugs and these effects under different conditions, a Student's t-test was penformed as def ined by Runyon and Haber (1968). Pained on unpained t-tests were perfonmed, depending on which one of these was appnopniate. E (pH 8.4 ) WITH L mts *rO

A E f f tuent 2 ELuo t e

2 4 mts 2 mls 0.2N OH 02N HCr DOVIEX 'ilt::""' 1.5x 0.5 cm H*- Fo rm 'pH 2 Eff tuent Eluot e Efftuent Etuote 1mt H^0 2 mls 2 mls H^0 2 mts Hz 0 2mts 6 N HCI-EtOH 1z 6 N HCI- Et OH I Woste l' I I OMDA NMN DOPEG NA DOMA

Fis.2.8 3 The column sepanation technique used in this study to sepanate H-NA and its various metabol ites. 2.12

Whene dinectional hypotheses were assented (i.e., rrthe nesponse was decreasedrr or rrthe nesponse was enhanced" ) then one-tai led pnob- ability levels wene applied to'the t-values. Significance levels are genenally indicated throughout the text; howeven, whene convenient, diffenences between obsenvations ane descnibed as ilsignificantrl (p <0.05) or rrnot signif icantrr (p> 0.05). Tests of signif icance for diffenences between dose-response cunves wene general ly made at individual dose points and the overall signif icance indicated in the text.

,:< e U:lIdVHJ 3.1

THE EFFECTS OF ETILEFRINE ON BLOOD VESSELS IN THE RAT TAIL

lntnoduction

Etilefnine is a sympathomimetic amine which has been used in the treatment of patients with onthostatic hypotension (Miller et al., 1973). Offermeien and Dneyer (1970) demonstr ated that etil- efnine has both alpha- and beta-stimulant effects on the nat vas defenens and the nat atnium, nespectively. Ir4ellanden (1966), using a cat calf muscle pneparation, suggested that etilefrine had rra local constrictor^ effect which was mediateC by alpha-receptorsrl and also a rrcl istantrr dilaton effect which was activated via the sympathetic nenvous systenr. He further showeC that etilefnine caused a pattenn of nesponse which was quantitatively símilar to that of nonadnenaline. Colernan et al. (1975) showed in a clinical tr-ial that etilef rine caused a positive chnonotnopic effect and also

incneased per iphenal vascular nesistance. They concluded that etil- ef rine has both a lpha- and beta-adrenergic eff ects in man. The expenimental drug RF-N-293 (2-amino-3-[3-5-dihydroxy- pheny l]-l-pnopanol ) has also been developed as an agent for use in the treatment of or-thostatic hypotension. This compounC has been shown to have alpha-recepton activity in the nat vas deferens (personal communication - Boehr inger lngelheim). Their studies showeC that neserpine pretreatment inhibiteci the contractile nesponse to REN-293 and that repeated administration of this agent resulted in tachyphylaxis. An impontant considenation in the selection of a sympatho- 3.2

mimetic agent to tneat onthostatic hypotension is the ability the dnug has to exent a dinect constnicton effect on vasculan smooth muscle. Par ks et al. (1961 ), in neviewing the dnug thenapy of postunal hypotension, showed that in patients with autonomic degen- eration, the indinectly acting sympathomimetics, ephednine and methylamphetamine, had no constricton effect on denenvated blood vessels. ln this chapten, an attempt has been made to detenmine the mechanism of action of etilef nine and REN-293 using a sympa- thetically innenvated vasculan bed, i.e., a perfused rat tail seg- ment, and to compane the eff ects of these dnugs with othen sym- pathomimetics on the same pnepanation.

Methods

The nat tail arteny segment was single cannulated in this senies of expeniments and per fused with Knebs-bicarbonate solution as descnibed in Chapter 2. Using the protocol outlined in that chapter, the following series of dnug applications wene carnied out. 1. lncneasing doses of adrenal ine, nonadnenal ine, phenyl- ephnine, metaraminol, etilef nine, REN-293, tyramine and ephednine wene applied to the artery and dose nesponse cunves constructed in a mannen descnibed by de la Lande, Glover. and Head (1967). The r-elative constnictor activity of the dnugs was detenmined by constnucting a mean dose nesponse curve for each agent and fnom

¡t calculating the appnopr-iate dose pnoducing pressune nesponses of 50, 100, 150 and 2OO mmHg. A mean was then obtained fnom the fou n ava i I ab le measu remen ts and the sh if t to the ni gh t of the 3.3

adnenaline cunve expressed as a natio of I :mean shif t (i .e., a necipnocal was obtained). 2. Etilef nine and noradnenaline wene administened to the tail anteny segments fnom nats which had been pretneated with guaneth- idine (25- ms/Kg intnapenitoneally on altennate days fon a peniod of seven weeks).

3. The effects of etilefnine (S.S u M), REN-293 (91 lr M) and nonadnenaline (148 nM) infusions wene examined on tail segments fnom nats which had been pnetneated with resenpine. The latter was injected intnapenitoneally at a dose of 2.5 mg/Kg, 24 hours befone the expeninrent. 4. The effect of the alpha-recepton blocking dnug phentolamine on the vasoconstnictor nesponse of eti lefnine, phenylephrine, non- adrenaline, ad¡ enaline and REN-293 was examined. The doses of these agents wene selected so as to give equivalent vasoconstniction on the rat tai I segments, and the phentolamine ( I . zg u M) was administened fon ten minutes befone and also duning the testing of these dr^ugs.

5. The nesponse to an infusion of etilefnine (5.5 u M) was detenmined befor e and aften a f ive minute infusion of each of the following drugs: (a) nonadrenaline (148 nM), (b) tynamine (1.45 mM) and (c) cocaine (zg.s uM). ln this senîes, etilef nine was also administened to a contnol anteny segment with the same time sequence as that used in (a), (b) and (c) so as to moniton any spontaneous changës in vasculan sensitivity which may have occunned duning the experiment.

6. The nesponse to an infusion of REN-293 (gl u M) was detenmined befone and aften a five minute infusion of each of the 3.4

following dr ugs: (a) tynamine (1.45 mM), and (b) cocaine (29.5

uM) . ln this series, REN-293 was also administened to a contnol antery segment with the same time sequence as that used in (a) and (b) so as to monitor any spontaneous changes in vascular sensitivity which may have occunred duning the experiment.

7. The nesponse to 5.5 u M inf usion of etilef nine was deten- mined in anteny segments fnom rats which had been pretneated with 6-hydnoxydopamine. The 6-hydnoxydopamine was administened in a dose of 100 mg/KS intnaperitoneally on day 1 and on day 2, and

the arteries then tested on day 3. At least f ive rat tail artery pnepanations were used in each test series. Mean values wene obtained for all nesponses and the S.E.Ms calculated and included in the figunes wheneven appropriate.

Resu lts

Figure 3.1 shows the dose nesponse cunves to adnenal ine,

noradrenaline, phenylephrine, metanaminol, etilef nine and REN-293

as obtained f rom the nat tai I arteny segment. (Although tynamine

and ephecì nine wene also tested on the prepanation, the nesults for

these agents are not shown in Figure 3.1 . ) The nesponses for tynamine and ephedrine ane shown in Table 3.1. Adnenaline was found to be the most active agent on the prepanation and ephednine the least active. The mean natio of activity of the dnugs compared

to adrenal ine was adnenal ine : nonadnenal ine : pheny lephrine : metanaminol : etilefrine, i.e., 1 : 0.38 : 0.06 : O.O2 : 0.01. With REN-293, tynamine and ephednine it was not possible to obtain ¿0

300

;- E 3 200 trJ ztn o À Ø u.l É.

100

0.1 1.0 10 100

D0SE (¡rM )

Fis. 3. 1 Comparative dose response curves fon adnenal ine (o), nonadnenaline (A), phenylephnine (¡), metaraminol (o), etil- efrine (¡) and REN-293 (O) on the nat tail anteny. Each

point repnesents the mean ! S.E.M. of the nesults of at I east five experiments. 3.5

pnessure nesponses above 100 mmHg. Tyramine exh ibited tachy- phylaxis and ephedr ine, too, showed veny weak activity. Thus the calculation of shif ts in dose response cunve as descnibed earlien cou ld not be imp lemented . The shape of the standard etilefnine response on the rat tail arteny is shown in Figune 3.2a and companed with the nesponse to the drug in a segment pretreated with 6-hydnoxydopamine (FiS. 3.2b) and the vascular nesponse to nonadrenaIine (Fig. 3.2c). The etilef rine nesponse demonstrated a pnompt initial phase which was followed by a slowen nise in pnessure, till a maximum was reached in approximately ten minutes. ln contrast, the nonadnen- aline nesponse was monophasic, with the maximum constniction being generated within a shont time of the drug being applied to the pnepanation. 6-Hydnoxydopamîne pnetreatment completely abolished the "slow-nise" phase of the etilef nine nesponse. The nesponse to nonadnenaline in the 6-hyci noxydopamine pnetneated segment was within the nonmal range. REN-293, when administered as a 15 minute inf usion at a dose of 91 ü M, also exhibited a biphasic res- ponse similar to etilef r-ine. Figune 3.3 shows the nesponse of tail antery segments fnom nats which had been chnonically pretreated with guanethidine. The vasoconstriction produced by nonadrenaline nemained unaltened, while the magnitude of the nesponse to etil- efrine was signif icantly reduced. Resenpine pnetreatment signif i- cantly reduced the vasoconstriction pnoduced by etilefrine on the nat tail arteny segment (p<0.005). The vasculan nesponse to non- adnenaline was unaffected, while that of REN-293 was completely abolished. Phentolamine, the alpha-neceptor antagonist, blocked the (a) 50

0

; 50 I E

5 0 (b) l¿l Ø (c) z. Ào tn UJ É.

0

0 5 10 15

TIME (mins )

Fis.3.2 The actual vasculan response (mmHg) obtained to (a) a 5.5 uM inf usion of etilefnine in an untneated anteny; (b) a 5.5 uM inf usion of etilefnine in an arteny pretneated with 6-OH dopamine; and (c) a 148 nM inf usion of noradnenaline in an untreated antery. 300

-Ol É E - 200 tr, an z. ùo vt lrl É.

100

0.1 10 10

D0 SE (pM )

Fis. 3.3 companative dose-response cunves to nonadnenaline in tail antery segments of untneated nats ( o ) and in nats pretneated with guanethidine ( r ). companative dose-nêsponse curves to etilef rine on the same blood vessels ane also shown in untneated anteries (f ) and pretr eated artenies (o). 3.6

constricton response to etilef nine, adrenaline, nonadrenaline, phenylephnine and REN-293 on the nat tail arteny.

Following a 148 nM infusion of noradnenaline fon f ive min- utes, the response to etilefnine was signif icantly augmented (Fig. 3.4a). Following a 1.45 nM infusion of tynamine fon the same time period, the etilef rine response was signif icantly decneased (Fig.

3.4b). Cocaine (29.5 u M) enhanced the nesponse of the rat tail segment to etilef nine (FiS. 3.4c) . The mean nesponse to etilef rine in the time contnol nemained unchanged from the initial value (FiS.

3.4d) . An infusion of tynamine (1 .45 mM) fon f ive minutes signif i- cantly decreased the nesponse to RFN-293 (FiS. 3.5a). Cocaine

(29.5 pM) also significantly

D iscuss ion

The nesults of this chapter indicate that etilefrine possesses a significant indinect sympatlromimetic action on the nat tail antery. This statement is based on sevenal findings.

Guanethidine has been shown (Lindmar and Muscholl, 1966) to be taken up into adrenergic neunones by the same uptake involved with nonadnenaline accumulation and to displace intna- neunonal noradnenaline from stonage granules. Chronic auanethidine r50

cn :r 1oo E 5 UJ an oz. fL 50 an UJ É.

a) (b) (c) (d)

Fig. 3.4 The open columns nepnesent the mean t S.E.¡¡. nesponse to a 5.5 ¡rM inf usion of etilef nine befone treatment with noradnen-. aline, tynamine and cocaine. The hatched columns show the nesponse to a 5.5 uM infusion of etilefrine following an inf usion of (") 148 nM noradrenali.re, (b) 1.45 mt¡1 tyramine, and (.) 29.5 u¡¡ co:aine. The cnoss hatched column (d) represents the response to etilef r ine given to a control nat tail segment with the same time sequence as the second etilefr ine dose in the treated antenies. This was to moniton any spontaneous change in vasculan sensitivity with time ;s0 - E 5

LU U)z o 25 o- (n tlJ É.

(o) (b) (c)

Fis.3.5 The open columns represent the mean t 5.E.M. nesponse to a 91 u M inf usion of REN-293 befone tneatment with tyramine and cocaine. The hatche.J columns show the nesponse to a 91 uM infusion of REN-293 following the infusion of (a) 1.45 mM tynamine and (b) 29.5 uM cocaine. The cnoss hatched column (c) nepresents the response to REN-293 given to a control nat tai I segment with the same time sequence as the second REN-293 dose in the tneated antenies. This was to moniton any spontaneous change in vasculan sensitivity with time. 3.7

administration causes depletion of nonadnenaline and an enhancement of the sensitivity of effector cells to catecholamines similan to that

obsenved following sympathetic denenvation. ln the senies of

experiments using guanethidine, the results showed that the nes- ponse to etilef nine was decneased, indicating that etilef rine is in part dependent on the integnity of the sympathetic nenves fon its

action. No supersensitivity to exogenous nonadnenaline was observed with guanethidine pnetreatment, which did not alten the

noradnenaline nesponse, except at the highest dose used, where a decnease in nesponse was observed. Reserpine has also been shown to deplete neuronal nonadren- aline stores by inhibiting the uptake of tnansmitten into the intr-a- neuronal storage vesicles (notrr and Stone, l96g). Reserpine differs from guanethidine in thai it is not taken up by the neuronal uptake mechanism and has a slowen nate of entry. The results show that the etilefrine nesponse is signif icantly decneased following pnetneatment with reserpine and that of REN-293 is completely abol- ished. The f inding that resenpine completely eliminates the response to REN-293 is in agneement with pneclinical data pnovided by the pharmaceutical company (Boehringen lngelheim). Following pretneatment with 6-hydroxydopamine, which has been shown to selectively destroy sympathetic nenve endings (Thoenen and rranzer-,

1968), a neduction in the nesponse to etilef nine was obsenved. 6- Hydroxydoparnine inhibits the action of indirectly acting sympatho- mimetics due to its depletion of neuronal noradnenaline. on the basis of the nesu lts using these thnee agents which compnomise adnenengic neunone function, it seems likely that etil- efnine has an indinect sympathomimetic component to its action. 3.8

The pnior administration of tynamine (an indirectly acting sym- pathomimetic - Fleckenstein and Burn, 1953) significantly neduced the magnitude of the eti lefnine response. Both these agents ane pnesumably acting on a common pool of neleasable nonadnenaline and tend to mod if y the vascu I ar ef f ects caused by each othen. V/hen tynamine is administened befone the addition of REN-293, the response to REN-293 is also depressed. This is similar to that obsenved for etilefnine, except the depnession of the REN-293 nes- ponse is gneaten, indicating the greaten indinect action of the latten. The depnession of the REN-293 nesponse following cocaine was due to the inhibition of REN-293 uptake. Burgen anC lversen (1965) suggested centain cnitenia fon a sympathornimetic amine to be a substnate of neunonal uptake. Based on these critenia, REN- 293 would be a good substrate for uptake, and hence be blocked by cocaine, thus decneasing REN-293 uptake and hence dep¡essing its nonadrenaline neleasing potential. The vascular nesponse to etilefrine, which showed a secondany "slow niserr phase and was sîmi lan to that obsenved with REN-293, stnongly contrasted with the nesponse to noradnenaline, suggesting an indinect sympathomimetic component. Additional suppor tive evidence fon such a component came from the enhancement of the etilef nine nesponse by both noradnenaline and cocaine. The fonmen observation pr obably repnesents an augmentation of the neleasable neuronal nonadrenaline pool by infusion of this agent, while the latter f inding suggests a block of re-uptake of noradnenaline released by etilef rine in the pnesence of cocaine (de la Lande et al ., 1967) . The comp lete abol ition of the nesponse to etilef rine, REN-293 and the othen sympathcrnimetic amines pnoduced by 3.9

phentolamine indicates that the vasoconstricton effects of these dnugs on the nat tail arteny wene totally mediated by alpha-

adnenonecepton stimu I ation.

An add it iona I f ind ing noted in the pnesent study was when tynamine was administered to the vessel and a maximal response obtained, discontinuance of the tynamine infusion caused a funthen

incnease in penfusion pressune. This ,wash out, nesponse was only observed with tynamine and its onigin is uncertain. The rrwash outrrresponse could be blocked by adding phentolamine at the commencement of the wash period. Farmen (1969) obsenved that tyramine had a biphasic response in the nabbit ean entery when injected into the perfusion fluid befone it entened the anteny. de la Lande and watenson (1968) suggested that this was due to the fact that in Fanmenrs study intnaluminal f luid was allowed to mix wîth extnaluminal fluid and the biphasic response nepresented the dinect action of tynamine intnaluminally on smooth muscle and then the indinect action on sympathetic nenves neached mone effectively fnom the extraluminal side. de la Lande and watenson (,l96g) then suggested that the biphasic nesponse was due to the diffenence in tyraminers potency when admínistened eithen extraluminally oi^ intnaluminally. 17 USIdVHJ 4.1

THE EFFECTS OF AGENTS MODIFYING SYMPATHETIC

NERVE FUNCTION ON THE RESPONSE OF THE ISOLATED RAT TAIL ARTERY TO ETILEFRINE AND TYRAMINE

lntroduction

ln the previous chapter, it was found that etilefnine possessed a significant indirect sympathomimetic component to its action. Since this previous series waS cannied out on the nat tail segment, it was decided to ne-examine the effects of etile- fni ne on the isol ated ventnal cauda l antery of the rat i n the pnesent chapten fon two neasons. Finstly, the fact that the ventnal artery has antenio-venous anastomoses with the tail vein seemed an important considenation in tenms of drug distnibutÎon; for this neason, it was essential to ascertain the effects of the dr ug on one isolated artery as compared with those on the whole vasculan bed. Secondly, it was decided to detenmine the r elative nesponse to etilef r-ine administened intra- and extraluminally to the vessel. A special attribute of the ventnal caudal anteny is the nich sympathetic innenvation ¡t possesses (Hodge and Robinson, 1972) which is veny similan in distribution to that seen in small human artenies (Fnewin et al., 197 1a, b); it thus senves as a good model fon testing sympathomimetic agents. An additional .pant of the study involved the use of the indirectly acting sympatho- mimetic agent, tynamine, as a standand for comparison with etilefrine. 4.2

Methods

ln this senies of expeniments, the rat tail anteny was double cannulated and per fused according to the method described in Chapten 2. Dnugs wene administered to the anteries either extraluminally on intnaluminally and maintained in contact with the vessel until a maximal nesponse was obtained. The nespect- ive concentnations of these agents in either the extennal bathing medium on in the per fusion f luid ane refenned to in panentheses. Single adrninistnations wene used in this study, rathen than complete dose nesponse cunves, due to the prominent indinect sympathomimetic action of the agents tested. The sensitivity of the nabbit ear anteny to extnaluminal tyramine has been shown to clecrease w i th nepeated adm in istnat ion of th is agen t ( de I a Lande and Watenson, 1968), and this p.henomenon was also,noted in preliminary experiments with etilef nine and tyramine on the ventnal antery of the rat tail. The test concentnations of etil- efnine and tynamine selected wene those which lay about the middle of the dose nesponse cunve and which wene veny repnoduc- ible on nepeated dnug administration. Etilefrine (5.52 uM) and tyramine (1 .45 mM) wene each administered internally and externally to anteries fnom untneated rats on nats pretreated wíth (a) iproniazid (200 mS/Kg i.p. each day fon 2 days), (b) nesenpine (2.5 ms/Kg i.p. each day for 2 days), (c) guanethidine (to ms/Ks i.p. daÌly fön 4 weeks), in the manner described by de la Lande and Watenson (1968). The extraluminal and intnaluminal response to etilef rine (5.52 uM) was also determined in the pnesence of cocaine (29.5 u M) and 4.3

and nialamide (¡SS uM). The catecholamine content of segments of untneated and pne- tneated artenies adjacent to those used in the perfusion experi- ments wene detenmined using the fluorimetnic assay as descnibed in the general methods chapten. At least f ive nat tail anteny pnepanations were used in each senies. Mean nesponses were obtained and the 5.E.l¡'ls calculated and included in the figur es whereven appnopniate. Statistical analysis was penfonmed on gnoup data using a Studentrs t-test.

Resu lts

The mean nesponse to etilefrine (5.52 u M) administened intnaluminally (intennally) and extraluminally (extennally) in nonmal antenies and arteries tneated with nesenpine, guanethidine, ipnoniazid and cocaine is shown in Figur e 4.1. Reserpine, guan- ethidine and iproniazid pnetneatment all signif icantly decreased the r'esponse to both inter nally and extennally applied etilefnine (p <0.05). The extennal nesponse was decneased to a gneater extent in al I cases. This is contnast to the normal antenies, whene the extennal nesponse was obsenved to be greaten than the internal nesponse (p < 0.05). The nesponse to etilef nine in the pnesence of nialamide was similan to that seen in those artenies pretneated with ipnoniazid. Although the extennal nesponse to etilef rine was augmented in the presence of cocaine in some prepanations, fon the group, the mean value was not signifi- cantly diffenent fnom the contnol due to a lange variance in the 150 ñ lnternot

E Ex ternol

TO) 100 E : o û C o o- .n 50 (I, É.

(o b c d o

Fig. 4.1 The response to etilefrine 5.52 uM in nonmal arteries ("), and antenies pretneated with neserpine (b), guanethidine (c), ipnoniazid (d) and cocaine ("). The respective S.E.M's ane al so shown. Sl lntraluminal (internal), tr Extnaluminal (external). 4.4

nesults (F¡g. 4.1). Figure 4.2 shows the nesponse to tyramine (1 .45 mM) admin- inistened internally and externally in nonmal anteries and antenies pnetreated with nesenpine, guanethidine and ipnoniazid. Resen- pine and guanethidine pnetneatment completely abolished the nesponse to tyramine both intennally and extennally. lpnoniazid caused a depression in the nesponse to tynamine, but the values obtained were not significantly different fnom the values in Figur-e 4.2a. The catecholamine content of the artenies was also detenmined and the results ane nepresented in Figure 4.3. Resen- pine and guanethidine both caused a significant decrease in catecholamine content when companed with the untneated vessels. The monoamine oxidase (MAO) intribitor, ipnoniazid, caused a significant incnease in the catecholamine content of the antenies.

Discussion

Fnom the nesults obtained in this chapten ¡t would seem that etilef r ine does indeed possess a signif icant indirect sympatho- mimet ic component to its action . I n the pnesent study, the response of the isolated arteny to the drug was of a simi lar magnitude to that obtained when etilef nine was administened to the tail segment. ln the presence of agents depleting the neuronal nonadnenaline content in the artenies (e.S., nesenpine and guanethidine), both tyramine and eti lefnine exhibited an attenuated effect. The nesponse to tyramine was signif icantly gneater (p< 0.025) 150

('l - 1 N lnternol E 5 D Externol o o C o o- û 5 o É.

(o) (b) (c) (d)

Fis. 4.2 The response to a 1.45 mM dose of tynamine in normal anteries (a), and antenies pnetreated with resenpine (b)' guan- ethidine (c), and ipnoniazid (d). The respective S.E.M's ane also shown. Slntennal, gExternal. 15

O)

Ct) 3 10 C o C o O

L) 5

(o ( b c (d )

Fis.4.3

The mean catecho I anr i ne con ten t i n segmen ts of nonma I artenies (a), and of those pnetreated with nesenpine (b), guan- ethidíne (c), and iproniazid (d). The respective S.E.Mrs ane also shown. 4.5

when applied extnaluminally than when applied intraluminally (Fis. 4.2). This is in agreement with the findings of de la

Lande and Watenson ( 1 968) in the nabbi t ear antery . Those authors also found that chronic denenvation, which caused the noradrenengic stonage structunes to disappean, reduced the potency of extnaluminal tyramine mone than that of ìntnaluminal tynamine. ln the nat tail arteny, it was found that the intna- luminal and extraluminal tynamine nesponse was completely abol- ished by neserpine and guanethidine pnetneatment. This indicated that the nesponse to tynamine in this vessel is totally dependent on its nonadrenaline-neleasing potential . The fact that the etil- efnine nesponse was significantly attenuated by both pnetneatments (with the extennal nesponse being affected to a gneaten degnee than the intennal response) is thus indicative of an indirect component to its action. Since the nesponse to the dnug was not completely abolished in the presence of the sympatholytic agents' it would seem that etilef nine also has a dinect component to its vasculan effect. Cocaine, in blocking neuronal nonadrenaline re- uptake, appeaned to incnease the extennal (as compared with the inter nal) response to etilefrine, but the f inding was not signif i- cant at the 5% level due to the lange vaniance in the nesponse. The catecholamine content of the arteries cornelated well with the perfusíon results in the nesenpine and guanethidine tneated vessels, i.e., a decneased catecholamine content was associated with an ahtenuation of the vasculan nesponse to the sympathom imet ic . lpnoniazid etevated the catecholamine level in the vessels as expected, bu t the decnease Ï n resÉ\onse to eti lef r ine îs i n 4.6

contrast to what was anticipated. A similar tnend was seen with tynamine, although the result was not signif icant at Lhe 5% level.

Th is depnession of the nesponse due to the MAO inh ib itot', ipno- niazid, is diff icult to intenpret. MAO is believed to negulate the levels of tnansmitten monoamines which diffuse fnom thein neunonal stonage vesicles into the neunoplasm. lnhibition of MAO has been shown (Spector, Hirsch and Brodie, 1963) to elevate neuronal catecholarnine levels. Weinen and Bjun (1972) suggested that intnaneunonal MAO may negulate noradrenaline synthesis by pneventing elevations in the unbound nonadnenaline pool . These findings may in pant explain why there were incneased levels of catecholamines in the tissue but a decnease in indinect sympatho- mimetic agents due to the decrease in neleasable vesicular nonadrenaline. However, the fact nemains that etilef nine, which has a langer dinect component to its v-asoconstnicton action, than does tyr-amine, was depnessed to a greaten degnee. This may be due to a depnessant effect on the vascu lan smooth muscle caused by an intenaction between etilefr'Îne and the MAO inhibiton. A series of experiments using nialamide was also canried out to test the specif icity of the depressant effect in nelation to iproniazid. This pnoduced nesults which wene similan to those obtained with ipnoniazid. The pnecise mechanism fon this f inding still nemains to be resolved. 9 Uf l.dVHJ \ a.)

5. I

A STUDY OF THE VASCULAR EFFECTS OF ET¡LEFRINE

USING THE ISOLATED RABBIT EAR ARTERY

lntnoduction

The suggested use of etilef nine in the treatment of patients with orthostatic hypotension is based on the premise that it has an action similar to that of nonadrenaline (tvtitlen et ê1., 1973). ln the two previous chaptens it was demonstrated that etilef rine has a substantial indirect sympathomimetic action on the ventnal antery of the nat tai I . The pnesent chapter examînes the eff ects of etilef nine on a similan blood vessel, namely, the central antery of the nabbit ean. This vessel is known to have a nich sympa- thetîc innenvation (de la Lande, Frewin and Watenson, 1967 ) and has the added advaniage that it can be sungically denei^vated by removal of the homolatenal supenior cervical ganglion (de la Lande and Rand, 1965). ln addition, the effects of , and intenactions w¡th, other agents commonly employed in the therapy of onthostatic hypo- tension have been examined.

Methods

The centnal artery of the nabbit ear was double cannulated and oenfused w i th Krebs-bicanbonate sol ut ion as described in

Chapten 2. Using the general pnotocol descr ibed in that chaPter', the following series of expeniments were carried out. Dnugs were administered to the artenies intr aluminally and maintained in 5.2

contact with the vessel until a maximal nesponse was obtained. 1. lncneasing doses of nonadnenaline, phenylephnine, etilef nine and ephednine wene applied to the arteny and dose nesponse cunves constructed in a manner descnibed by de la Lande, Glover and Head

(1967). The nelative constnicton activity of the dr ugs was deten- mined by constructing a mean dose response curve fon each agent and from this calculaiing the dose pnoducing pressure nesponses of 50, 100, 150 and 2AO mmHg. A mean was then obtained fnorn the foun available measurements and the shif t to the night of the nor- adnenaline curve expnessed as a ratio of 1 :mean shif t (i.e., a neciprocal was obtaîned). 2. Etilef nine was administered to the anteny before and during an infusion of 9-alpha-fluorohydnocontisone at a dose of 22.8 nM or 22.8 uM. The 9-alpha-fluonohydrocontisone perfusion was com- menced 10 minutes before the etilef nine was added. ln concunnence with the fonegoing pnocedune, etilefrine was administened with the same time sequence to a vessel untreated with the mineralocorticoid so as to moniton any spontaneous change in vascular sensitivity with time, i.e., a iltime contro|rwas employed. The response of the anteny to nonadnenaline was also examined before and duning an infusion of 22.8 nM of 9-alpha-fluonohydnocortisone, which was commenced 10 m inutes befone the add it ion of the nonadnena I ine. rrTime contnolsrr wene used as before to moniton any changes in vasculan sensitivity with time. 3. Dose resoonse curves to etilefnine and noradrenaline were obtained on artenies fnom nabbíts in which the superion cenvical ganglion had been surgically nemoved on one side thnee weeks previously. This procedure caused the sympathetic denenvation of 5.3

the respective arteny. The response to the indinectly acting amine ephednine and to electnical stimulation was also examined to confirm denervation. Electnical stimulation was given at a level which caused release of neuronal noracrenaline, but did not stimulate the smooth muscle directly, i.e., at ?O volts, 38 pulses/sec for 5 seconds (de la Lande - pensonal commun ication ) ' 4. The catecholamine content of the denenvated artenies and normal antenies was also deter mined using the f luorometnic catechol- amine assay descnibed in the general methods. At least five artenies were used in each senies. Mean res- ponses were obtained and the S.E.Ms calculated and included in the figures rvheneven appnopriate. Statistical analysis was pen- fonmed on group data using a Studentrs t-test'

Resu I ts

Figure 5.1 shows the dose response cunves to nonadrenaline, phenylephnine, etilefnine and ephedrine in the rabbit ear anteny. Nonadrenaline was found to be the most active agent on the pnepar- ation and ephedrine the least active. The mean natio of activìty of the dnugs companed to nonadnenal ine was noradrenal ine : phenylephrine : etilefrine : ephednine = 1.0 : 0'5 : 0'07 : 0'003' When etilef rine was added to the rabbit ean anteny before and duning an infusion of 9-alpha-fluorohydrocontisone (22.8 nM or 22.8 UM) no significant increase in nesponse was obsenved at all the doses used. The time contnols also shov.'ed no signif icant diffenence over the dunation of the expeniment. The dose response TABLE 5.1

Catecholamine levels (nS,/S) in the

normal and denenvated nabbit ean anteny segments (n = 5).

X + SEM

Ì< Denenvated o.16 ng/g 0.01

Nonma I 0.33 ns/s 0.03

signif icantty neduced (p<0.001 ). 5.4

curves to noradnenaline befone and duning an infusion of 9-alpha- fluonohydnocontisone (22.8 nM) showed no significant diffenence. The time contnols fnom this senies exhibited no change dur ing the expeniment oven the entine dose nange. Figure 5.2 shows the response to etilef nine in symoathecto- mised and non-sympathectomised ean antenies obtained fnom the same rabbit. The denenvated vessels showed a signif icant decrease in response for all the doses of etilefrine used. Figure 5.3 shows the nesponse to noradnenaI ine in these antenies. Thene was no significant diffenence between the denervated and the nonmal anteries as a gnoup; howeven, two of the f ive vessels examined showed an incnease in the noradnenaline nesponse. The denervated vessels also showed a gneatly reduced response to the indinectly acting amine ephednine (p<0.05) and no nesponse to electnical stim- ulation. The catecholamine levels in the denenvated artenies wene decneased (Table 5.1 ) .

Discussion

The nesults of the pnesent study on the centnal anteny of the rabbit ean suppont the findings in the previous chapters of this thesis that etilefrine relies heavily on sympathetic nerves for its vasoconstnictor effect. The advantage of using the nabbit ean antery prepanation is that ¡t can be sungically sympathectomised, wheneas the nat tail artery (used in the pnevious studies) cannot be successfully tneated in this fashion. Denenvation was substan- tiated by testing with electrical stimulation, assaying catecholamine 300 r; E J 200 l¡¡ tn oz fL tt lr, É.

100

0.1 't.0 t0 100 't000

DOSE t¡rM I

Fis. 5.1 Dose response cunves to nonadnenal ine ( o ), phenylephnine ( r), eti lefrine ( f ), and ephedrine (o ) . Nonadnenal ine was found to be the most active and ephedrine the least active on the isolated nabbit ear arteny. The dose refers to the pM concentnation of dnug perfusing the ar teny. 300

200 6 - E E az Ào vl LlJ É. 100

1.0 10 DOSE ( pM I

Fis.5.2 Dose-response cunves to etilefnine in sympathectomised (O ) and non-sympathectomised (o) artenies. Sympathectomy decneased the response to etilefnine. 200

-Ot E s

LIJ an z. o 100 o_ r/', tr,Í.

0l 1.0 ( DOSE ¡rM )

Fis. 5.3 Dose nesponse cunves to nonadnenaline in sympathectomised (o) and non-sympathectomised (f) antenies. 5.5

levels in the preparation and testing with the sympathomimetic amine ephedrine. ln the face of the foregoing, it was shown that etilefnine's constnictor action was signif icantly attenuated in sympathectomised ear anteries, and the nesu lts demonstnated the strong dependence of the dnug on these nerve endings to exent its pharmacological action.

The minenaloconticoid 9-alpha-f luorohydnocontisone is fr equently used in the thenapy of patients with postuna I hypotension (Corbett,

1976). Since etilefnine's pnoposed thenapeutic nole is in the same clinical situation, ¡t seemed appnopniate to examine potential inten-

actions between these two agents. The mineralocorticoid was used at two different dose levels, the low dose to approximate the cir cu-

lating blood level of the drug during thenapy and the high dose

to appnoximate the dose of deoxyconticostenone acetate used to block extnaneuronal nonadnenaline uptake. Johnson and de la

Lande (1978) showed that deoxycorticosterone acetate is a b locken of extnaneuronal uptake in the rabbit ear anteny. This is in agree-

ment with the findings of lvensen and salt (1920), who showed

that corticostenone is an inhibitor- of extnaneunonal uptake. The potentiation of the nonadnenaline nesponse by 9-alpha-f luorohydno- contisone has been reported by Schmidt et al. (1966). The neason fon the failure of 9-alpha-f luonohydrocontísone to potentiate etil- efnine is difficult to determine from the present nesults. The potentiation was expected to occun by inhibition of extnaneuronal uptake of released nonadnenaline.

Phenylephnine and ephedrine wene included in this study as two othen sympathomimetic amines that have been used in treating postunal hypotension (corbett, 1926l' . A diffenence in potency of 5.6

nonadrenaline, pheny lephnine, eti lef nine and ephednine was obsenved between the rat tail antery used in Chapten 3 and the nabbit ean anteny used in this chapten. It was found that nonadnenaIine was more potent in the tai I arteny and pheny lephnine and ephednine wene mone potent in the ean antery. Etilefnine was equipotent in

the two vesse I s .

,ß 9 Uf tdvHJ 6.1

THE INDIRECT SYMPATHOMIMETIC ACTIVITY OF ETILEFRINE - A COMPARISON WITH TYRAMINE 3 AND EpHEDR rNE usrNG H-¡¡oRaoRENAL rNE

lntroduction

ln view of the f indings of the pnevious chapters, which indicated that etilef nine possesses consídenable indinect sympatho- mimetic activity, it was decided to examine the nonadrenaline neleasing potential of this amine next, using 3H-no".drenaline and the nat tail anteny as the test vessel. Many studies have been cannied out to examine vanious aspects of neuronal and 3H-no".dnenaline extnaneuronal eff lux. Tnendelenburg (1963) neviewed the actions of several sympathomimetic amines on the cat nictitating membrane and othen tissues with nespect to, their dinect and indirect activity based on their phanmacological action. Paton (t9Z3a, b) studìed the effects of vanious sympatho- 3H-no""dnenaline mimetics on the eff lux of f nom reserpine pr.e- tneated and MAo and coMT inhibited nabbit atria. His results indicated that tynamine, metanaminol, amphetamine and noradnen- 3H-no".drenaline aline itself enhanced the neuronal eff lux of fnom this prepanation. Eckent et al . (1976) and Henseling et al . 3H-no""dnenaline (1970a, b) have examined the distnibution in rabbit aortic strips and its modification by inhibitons of noradnen- aline metabolism and uptake. Those authons also studied the steneoselectivity of this distr ibution and subjected the nesultant efflux curves to compantmental analysis. The present chapten examines the action of etilefrine in 6.2

modifying the 3 H-nonadnenaline eff lux fnom rat tail antenies and companes its action with that of tynamine and ephednine. ln addition, the effects of inhibitors of noradrenaline metabolism and uptake on the efflux ane assessed.

Methods

This efflux study was carried out acconding to the pnotocol described in the general methods section (Chapten 2). The initial 14 series of expeniments examined the eff lux pr of ile of C-sonbitol over 20 minutes f rom the nat tail anteny. This eff lux was also examined in the pnesence of (1) etilefr ine 55.2 F M, (2) tyramine

1.45 mM, (3) cocaine 29.5 u M and (+) deoxycorticosterone acetate 26.9 uM with UO521 (55 uM) as an inhibiton of catechol-O-methyl tnansferase (Hensel ing et al ., 1978) . The subsequent experiments 3 examined the eff lux of H-no".drenaline in the pnesence and absence of the following agents: (1) etilefrine 55.2 lrM, (2) tynamine 1 .45 mM, (¡) ephedrine (182 ¡rM), (4) cocaine 29.5 ¡r M,

(5) etilefrine 55.2 u M + cocaine 29.5 u M, and (O) tynamine 1 .45 mM + cocaine 29.5 p M.

On the basis of nesu lts obtained dur ing the series with 3 sorbitol, the expenimental protocol was modif ied fon the H- nonadnenaline study. The modification consisted of an initîal f ive minute wash in asconbic/Krebs solution, followed by a 15 minute wash in asconbic/Krebs containing the individual druS(s).

A f unther series was cannied out using antenies f nom rats that were vaniously tneated. These included: (1) pretreatment of 6.3

nats with the monoamine oxidase inhibiton ipnoniazid (2OO mg/KS i.p. each day fon 2 days) and exposing the segments of the tail antenies during the washout peniod to ascorbic/Knebs containing cocaine 29.5 tM, deoxyconticostenone acetate 26.9 U M and UO521 55 uM; (2) pnetreatment of nats with guanethidine (25 mS/Kg ¡.p. ) on altennate days fon 7 weeks befone nemoving thein caudal arter ies.

The content of compounds wi th a di hydnoxyphenol stnuctune in the efflux was calculated by combining f ive one-minute (i.e., 1 ml) samples and subjecting each to a batch alumina pnocedure (Ae la Lande et al., 1967\ to detenmine what percentage of the tota I nad ioact iv ity was adsonbed on to alumina.

Iüean efflux cunves wene ca lcu lated f rom the nesu lts of at least five individual experiments.

Resu lts

14 C-sorbitol eff lux 14 The nesu I ts of expeniments in wh ich the C-sonbi tol effl ux was measuned indicated that the counts obtained aften five minutes of eff lux had decreased to approximately 5% of the counts 3 eff luxed at the finst minute. Fon this neason, the eff lux of ¡t- noradrenaline fnom the arteny duning the f inst f ive minutes was discarded to avoid complicatíng the overall nesults with those due to th is marked eff lux f rom the extrace I I u I ar space. The nate of 1lL eff lux of 'C-sonbitol was unchanged by the addition of any of the drugs used. 6.4

3 H-nonadrenal ine eff lux

It was found that approximately 90% of the total radio- activity was adsorbed on to alumina, índicating that the collected efflux was predomi nant I y dihydnoxyphenol stnuctuned compounds 3H-no".dnenaline tr-a,4-dihydnoxyphenylglycol - probably on (DOPEG), which ane both of neunonal origin.

2 "H-nonadrenaline eff lux in the pnesence of sympathomimetic amines The comparative nates of eff lux in contnol artenies and artenies treated with etilefr ine, tyramine and ephedrine ane shown in Figune 6.1. The rate of eff lux was in the descending onden tynamine > ephednine = etilefrine > contnol. ln the pnesence of cocaine, no sign if icant díff enence was obsenved between the rates of effl ux fon cocai ne alone and cocai ne and eti I efni ne ( F i S. 6.2a). The efflux rate for tynamine in the pnesence of cocaine was significantly greaten than the efflux curve obtained for cocaine alone, irnespective of whethen tyramine was introduced at the start of minute six on at minute eleven (f ig. 6.2b).

3 H-noradrenaline efflux fnom pretneated antenies

The eff lu>< cunves f rorn antenies which had been p netneated witlr guanethidine ane shown in Figune 6.3. lt can be seen that 3 al though the amount of H-no".dnena I ine ef f I uxed is lowen than the values in the control eff lux cunves (Fig. 6.1), etilefnine has 3H-r,o".dnenaline caused an enhanced effl ux of companed with the values obtained when the vessels were not tneated with etilefnine. ln Figune 6.4, the eff lux curves fnom anteries tr eated with 300 C Ë o)

E 3 x l 200 J lt lr I.lJ otL UJ t- É. 100 T EP ET c

6 10 15 20

TIME (min )

Fis. 6.1 3 Eff lux cunves of U-NR from control (C) antenies and antenies tneated with tyramine (T), ephednine (EP) and etilefrine (ET). Each point represents the mean values t S.E.M of five expeniments. 300

.E2 o) 20 o E ï*COC _o >< l J IJ- LL ul IL 100 o ET + C0C IJ F c0c É.

6 10 15 20 TIME (min)

FiS.6.2a 3U-N¡ Efflux cunves of fnom antenies tneated with cocaine (COC), etilefnine and cocaine (Ef + COC), and tynamine and cocaine (f + COC). Each point nepresents the mean values t S.E.M, of five experiments. ff,. cocaine and tynamine wene administened togethen at minute 5. 300

E E ol 200 o E _o, x l J ¡r l! l¡, o¡¡- 100 lrl F É.

5 10 15 20 25

ïlME f min)

Fig. 6.2b Figure 6.26 shows efflux cunves whene cocaine wa5 added at minute 5 (f ) and also whene cocaine was added at minute 5 and tynamine at minute 10 (o). .ç E

c'r 200 o gE l>< ) lJ- lJ- I.JJ oll- 100 Lt t- ET+G É. G

6 10 15 20 TIME (min)

Fis.6. 3 Efflux cunves of 3 H-u¡ fnom .artenies obtained fnom nats chnonically treated with guanethidine (G), on those pretreated with guanethidine to which etilefnine was added (ef + G). Each point nepnesents the mean values t S.E.M. of five experiments. 300 .c E o) a gE )>< 200 J LL t.L IJ oTL t-lJ-J É. 100 E + IDCU I DCU

6 10 15 20 TIME (min )

Fig.6.4 3 Eff lux cunves of ¡t-X¡ f rom arteries tneated with ipnoniazid to which doca, cocalne and UO521 were added (IDCU) or fnom those to which etilef nine was funthen added (g + IDCU) . Each point nepresents the mean values t'S.E.M. of five expeniments. 6.5

ipr oniazid, cocaine, deoxyconticosterone acetate and UO521 are dep icted . The anteríes treated with etilef nine :show an enhanced rate of eff I ux in r elation to the anteries which received no etil- efnine. The efflux fnom the vessels tneated with the lvlAO inh ib- i tor, coca i ne deoxycorticosterone acetate, and UO52'l was higher than that fnom the contnol untreated anteries shown in Figure 6.1 .

Discussion

The overal l nesults of the pnesent study suppont eanl ien f indings that etilefnine possesses a signif icant indinect sympatho- mirnetíc component to its action. The nesults show that etilefrine 3H-no".dnenaline enhanced the rate of eff lux of f rom the sympa- thetically innenvated rat tail anteny . in companison to contnol values. The nate of efflux was less than that induced by tyramine but companable with that of ephednine. These results ane in agneement with those of Paton (t973a, b), who showed that 3H-no""drenaline tynamine enhanced the eff lux from the rabbit atria. Paton (lgZ¡a) suggested that the ability of some inciirectly- acting sympathomimetics to enhance 3H-.,o""drenaline efflux may be influenced by thein affinities for cannien-dependent influx sites, thein lipid solubilities and thein ability to displace nor- adrenaline f rom resenpine-nesistant intnaneunonal binding sites. It would seem that rnany explanatíons ane possible for the vani- ation in the action of sympathomimetics. This pÌ'esent study diffens from that of Paton (tgZ¡a) in that vesiculan uptake and stonage is intact and may thus nepnesent a langer I'pool" of 6.6

releasable nonadnenaline. The pnedominant component of tyr- aminers sympathomimetic action nelates to nonadnenaline release from sympathetic nerve endings (Bunn and Rand, 1958; Trendel- enburg, 1963) . Tnendelenbung ( 1963) showed that tynamine is an

indinectly acting sympathomimetic, wheneas ephedr ine can be classed as a "mixedrr acting sympathomimetic with both dir ect and indirect actions. The results fnom the present study agnee with these f indings and suggest that etilef r ine may also be a 'rmixed" acting amine.

Pnevious evidence (Tainter and Chang, 1927; Tainten, 1929; Ross and Renyi, 1966) showed that the actions of tyramine and ephedrine wene antagonised by cocaine. ln the pnesent study, 3H-no".drenal the eff lux of ine f rom tynamine treated antenies in the presence of cocaine was sígnif icantly augmented. This aug- mentation occurned when cocaine and tynamine wene added to- gether to the wash fluid and also when tyramine was added five minutes af ten the application of cocaine at the beginning of the eleventh minute. The increase in eff lux of 3 H-.ro,-adrenaline in the presence of cocaine and tynamine may be due to the fact that the tynamine concentration is fan highen than that of cocaine and so this dose of cocaine is ineffective in pneventing uptake of tyramine into the neunone. lf this is the case, then the com- bined neuronal uptake blocking action of cocaine and competition fon uptake with tynamine would pnevent ne-uptake of tynamine neleased noradrenaline and thus account for the enhanced efflux from the tissue into the wash fluid. Anothen possibility is that tynamine is entering the neunone via an uptake system insensitive to cocaine and displacing intraneunonal vesicular nonadnenaline. 6.7

3H-.'o".dnenaline VVith etilef nine, the incnease of eff lux was not significantly diffenent fnom the values obtained with cocaine alone. This may ref lect a blocking of neunonal uptake of etil- efnine by cocaine. Burgen and lvensen (1965), using the per- fused nat heant, evaluatecj a number of sympathomimetics for thein affinity as substnates for neunonal uptake, ín nelation to thein

structure. Based on stnuctunal cniteria, €tilef nine would be expected to have a low affinity for neunonal uptake, and cocaine would be able to inhibit the etilefr ine uptake and thus its non-

adnenaline neleasing potential. These f indings ane in agneement with the perf usion study cannied out in chapten 4. Another- poss-

ibility for- cocainers action is advanced by paton (1923b), who suggests that cocaine decneases the enhanced efflux induced by sympathomimetic arnines by inhibiting 3H-no".drenaline efflux

rathen than the uptake of the sympathomimetic agent. Ho¡,veven, Henseling et al. (1976b) showed, using rabbit aontic strips, that cocaine neduced the neunonal accumulation of 3 H-no"-drenaline

anc enhanceci the eff lux f rom the neunonal compantment, which indicates a diffenence in the action of cocaine on rabbit atnia and nabbit aonta.

The depressed nate of eff lux obtained following guanethidine pretneatment indicates that the stonage potential fon 3H-no"-d"".,- aline has been decneased (pnesumably intnaneunonally). Evidence

fon th is phenomenon comes f rom the wonk of Bísson ancj Muschol I (lgOZ) and Schanken and Monnison (1965), who showed that guan- ethidine is selectively accumulated in sympathetic nenves, thus affecting noradnenaline storage. Etilef nine's action in the guan- ethidine treated ar tenies which had been pne-incubated with 3 tt- 6.8

nonadnenaline appeared unimpained. The difference in effl ux nate when this dnug was added was appanent over vintually the entire washout peniod.

Antenies tneated with ipnoniazid, cocaine, deoxyconticostenone acetate and Uo52l simultanepusly had efflux rates highen than those of control vessels (Fis. 6.1). This was expected, since cocaine and deoxyconticostenone acetate block neuronal and extra- neunonal uptake, respectively, leading to incneased eff lux of 3 H- noradrenaline f rom the antenies. ln addîtion, ipnoníazid and UO521 would tend to decnease the breakdown of 3H-no".drenaline, 3 ther-eby providing more H-no".dnenaline, nathen than its metab- olites, to be available for nelease from the antery. since wyse (1976) and Venning and de ta Lande (tgZg) showed that the extnaneunonal uptake system in the nat tail artery is nelatively unimpontant, this increase above the contnol efflux values may indicate a pnedominant nole fon cocaine on the obsenved efflux. Etilefrine also pnoduced an enhanced efflux above the ipro- niazid, cocaine, DocA and uo521 control values. This efflux was also greater than that obsenved when etilefnine was administered to normal untneated anteries. ¿ uSIdvHS 7.1

THE EFFECT OF ETILEFRINE AND OTHER

SYMPATHOMIMETIC AGENTS ON NORADRENALINE AND

METABOLITE EFFLUX FROM THE RAT TAIL ARTERY

lntroduction

lnthepreviouschapter,¡twasfoundthatetilefnine enhanced the eff lux of nadioactive mater ial f nom nat tail anteries pre-incubated with tnitiated nonadnenaline. This effect was simi lar to that observed with tyramine and ephedrine' lt was suggested that the efflux was pnobably of neunonal onigin in the 3 dihydnoxy- fonm of tnitiated nonadrenaline 1 H-ttl) and tnitiated phenylglycol (3 u-ooprc). lt has been shown (Paton' 1973a' b) thatvarioussympathomimeticaminescanenhancetheeffluxof 3H-NÀ f"o'n nabbit atria. ln those studies, only axoplasmic efflux was examined, since vesicular stonage of noradrenaline was metab- impair ed by pnetreatment with reserpine and noradnenaline olism suppnessed by treatment with pangyline and tropolone' Brandao et al. (1978) found that electnical stimulation and 3 tyramine enhanced the eff lux of H-t1l and its metabol i tes f nom the canine saphenous vein. They also found that electnical stimu- lation primarily enhanced the eff lux of noradrenaline, whereas 3 tyramine enhanced the eff lux of tl-OOpfC and noradrenaline' Thischapterexaminesthecompositionoftheradíoactive by tneatment eff lux f nom the nat tail anteny and its modif ication with etilef rine and sevenal othen sympathomimetîc agents' The effects of pnetneatment with a monoamine oxidase inhibitor' ipro- niazid, and an inhibitor of neuronal uptake, cocaine' ane also 7.2

examined. ln this study, only (-)3H-nonadnenaline was used in onder to avoid any diffenences in the disposition of the isomens (lvensen et al., 1971).

Methods

This eff lux study was cannied out in accordance with the protocol descnibed in the general methods. The initial series of expeniments examined the metabolite eff lux prof ile oven 35 minutes without any dnugs pnesent. Subsequent expeniments investigated the changes in metabolites when the following drugs wene added individually at the commencement of the thind f ive minute period:

(1) eti lefnine 55.2 uM

(2) tynam i ne 1.45 mtul

(3) ephedri ne 182 ufvl

(4) REN-293 227 pM

(5) cocai ne 29.5 uM (6) cocai ne 29.5 ÉM, followed by tyramine 1.45 mM at the start of the founth wash peniod. A senies was also canried out in which the arteries wene pne-

3 incubateC with cocaine 29.5 pM before exposune to H-,''ro"adnena I i ne to detenmine whether cocaine could inhìbit neunonal uptake.

ln a f urthen senies of expeniments, nats wene pretneated with ipr oniazid (ZOO mg/KS ¡.p. ) each day fon two days. The metabolite eff lux pnof iles were examined, as befone, in the presence and absence of etilef r ine 55.2 ul'¡. TABLE 7.1 The mean value ! S.E.M. fon the gnoup of antenies studied is shown.

Metabol i te Eff lux Period Numben 2 345 6 7 Control Efflux NA 48.7!7.3 43 .018.7 36.018.5 33.019.4 34. 3t9.6 40.318.9 38.618.6

DOPEG 28.5!2.5 27.5t2.7 27.5Ð.7 23.3Ð.5 22.0!3.9 23 . 013.0 23.016.5

DOMA 4.0!O.4 3.310.8 3.7Ð.4 4.0Ð.4 4.010. 6 4. 0t0. 5 5.010. 1 OMDA 14.7 !3.7 21 .316.5 29.8!ß.2 36.816.6 35.7!8.2 30. 016.0 29.6!6.2 NMN 2.9Ð.9 4.5tl .3 3.5Ð.9 3.0Ð.7 2.4!O.8 2. 310.6 2.310.8 Eti lefrine Efflux

NA 31 .4!4.3 18.4!2.9 18.613.3 31 .2Ð.4 1 1 .8t2.6 12"2!2.5 12.2!2.2 DOPEG 36.O!2.4 30.0 12. I 44.0!3.6 50.0Ð.8 52.7!4.4 51.813.6 5't .2t3.8 DOMA 4.2!O.2 4.0Ð.3 5.0!0.5 4.2Ð.4 4.O!O.7 4.6!0.4 4.6t0. 5 OMDA 24.8!2.7 44.42.7 29 .0 t5. 8 29 .8f2.O 29.3!4.0 29.4!2.7 30.413.4 NMN 2.7lO.5 3.010.5 3.0fo.4 1.4Ð.2 1.410.3 1 .4!O.2 1 .1 10.09 Tynamine Efflux

NA 49.3!2.4 32.3t4.4 41 .7 4.3 40. 3 16. 1 46.7!5.3 41 .0t6.5 37.616. 1 DOPEG 30.011.2 27.5!4.6 43.7 !4.1 48. 3 !5.4 41 .3!5.2 45.6t5.7 49 .O!5.2 DOMA 4.8!1 .7 5.7 !1 .7 4.311 .4 4.8!1 .4 4.3!1 .6 2.3t0. 3 2.3!O.3 O¡¡DA 14.8t2.1 33.5t5.0 9.711.6 6.8j1.4 6.0t1 .1 7.6!1 .6 8.7!1 .4

NMN 0.510.07 1 .710.3 1.3!0.2 1.310.4 1.1lO.2 2.O!O.4 2 .0!0. 3

The figures in each column nepnesent the pencentage of the total eff lux pen peniod. 7.3

3H-NA, 3 3 The amount of H-oopEc and H-metabolites, i .e., normetanephnine 13H-ruuru), dihydnoxymandelic acid (3 H-ootr¡¡), the

OMDA fraction (containing methoxyhydnoxyphenylglycol - MOPEG, and methoxymandelic acid - vMA) eff luxed pen f ive minute peniod has been expnessed as a pencentage of the efflux over the first five minute peniod which is taken as 1oo%. At each eff lux peniod, the pencentage of the individual metabolites contributing to the total tritium effluxed was also detenmined. This was calculated for the contnol values and also fon the eff luxes following dnug treatment. Appendix 4 shows the stnuctures of the various metab- ol ites and the enzymatic convension pathways.

Resu I ts

Figune 7.1 shows the eff lux pnof ile of 3 H-NA, 3 H-DOPEG and

the othen nonadnenaline metabolites f nom control antenies and artenies tneated with etilefnine and tyramine. ln the untneated 3 artenies, thene was a pnognessive decnease in the eff lux of H- ? NA, "H-DOPEG and the other metabol ites over the 35 minute wash peniod. Both etilef rine and tynamine signif icantly incneased the 3H-OOpEC, efflux of commencing at the third efflux peniod.

Tynamine caused a signif icant incnease i.r 3 H-tlA eff lux. Although 3H-tl¡ a similan tnend in eff lux was observed with etilefrine, the values wene not signif icant at the 5% level. 3H-oolr¡R An incnease in efflux was also obsenved upon the addition of etilef nine or tyramine to the wash medium. Howeven, 3 as shown in Tab le 7 .1 , the H-OOΡ¡ fnaction repnesented only T ABLE 7 .2 The mean value t S.E.M. fon the gnoup of arteries studied is shown.

Metabol i te Eff lux Period Number I 2 345 6 7

REN-293 Efflux NA 12.7!1 .6 10.3!2.2 39.016.9 43.7!7.5 51.0r9.3 52. 31 8 .4 39.31 2.5 DOPEG 35.016.1 27 .OU.3 11.7L3.2 6.5!1 .0 7.010.9 5.510. I 6.0t0.9 DOMA 5.0t1.I 4.8!2.3 3.010.8 4.ot} .7 2.5jo.7 5.2!O.7 5. 3r0.6 OMDA 42.5t6.1 52.0t5.9 41 .O!2.8 38.5t2.9 30.512.0 27.3L1 .5 34.7t5.3

NMN 4.311 .6 5.011 .7 5.01't .0 6.5!1 .5 8.3!1 .8 9. 311 .4 1 4.013.4 Ephedrine Efflux NA 51 .0t5.8 42.3!7.1 46.3ß.2 39.3{3.0 40.2!5.4 39.8!2.7 34. 5r3. 5

DOPEG 33 . 814.6 33.7!6.2 33.514.8 38.O!2.1 37.O!4.1 36.7!2.2 38.513.6 DOMA 4.010.4 4,O!O.7 4.3r0.6 4.310.3 4.8r0.3 3.810.3 4.010.4

OMDA 9. 710. 8 18.8!2.4 14 .8!2.2 14.7 !2.8 1 3.5 !3.4 15.7!1 .7 1 9.3r0.8

NMN 1 .2!0.2 1 .7t0.3 1.310.3 2.810.3 3.510.2 3.310.6 3.510.5

The figures in each column repnesent the pencentage of the totaI efflux pen period.

I 7.4

4.2% and 4.8% of the total tnitium eff lux, respectively. Tynamine 3H-NMN, also induced an incnease in but in this instance also this only repnesented 1.31, of the totar tritium eff luxed.

Figure 7.2 shows the efflux pnofile in the pnesence of REN- 293 and ephedrine. REN-293 caused a lange incnease in 3H-N¡

neleased companed with the untneated antenies and a decnease in 3 3H-ooue the amount of H-oopEc effluxed. The efflux vanied oven the seven peniods, accounting for approximately 4% of the total tnitium effluxed pen peniod. REN-293 also caused an incr.ease 3H-tt¡¡¡tl 3 tn oven the latter f ive eff lux peniods, with H-Ntuil.l com- pnising 14% of the total tnitium effluxed in the final wash peniod. 3 Ephednine slowed the nate of decline of the H-oopgc eff lux compared with the control values for all the time periods. The ) "H-NA efflux in the pnesence of ephedrine was significantly higher than the contnol only duning periods three and, foun. Ephedrine also caused an incnease in the efflux of the 3H-Nt¡l.t fraction, though this was not as manked as that initiated by tynamine and REN-293. From Tabre z.z ¡t can be seen that in 3H-l.tt¡tt the case of ephednine, the fraction contnibuted to appnox- imately 3.5% of the total tritium effluxed duning the latten peníods of efflux.

F i gure 7.3 shows the eff lux f nom anteries tneated with cocaine on cocaine and tynamine. Tyramine again caused a 3 3 3 signif icant eff lux of both u-r.tA and H-oopec. The H-otr¿o¡ fnaction was not changed significantly by the addition of tyn- 3 amine. The enhancement of the H-oot¡¡ and 3 H-tr¡ttrt eff tux caused by the tynamine in the pnesence of cocaine accounted for appnox imate I y 2ls and 1% of the total tritium effluxed, as obsenved TABLE 7.3 The mean value t S.E.¡¡. fon the gnoup of antenies studied is shown.

Metabol i te Eff lux Peniod Numben 2 345 6 7

Cocaine Pre-incubation Efflux NA 21.0t5.0 6.0!1 .O +.5t1.1 3.È0.4 3.510.9 4.0r0.5 2.ÈO.3 DOPEG 23.013.0 1.0r0. t 1.010.2 o.8t0.05 0.810.2 0.6io.1 O. 5r0. l jo.05 DOMA 0.4t0.05 o to 0.5!0.1 0.110.02 0.210.05 0.2 0. 1 10.02 OMDA 41 .5!5.2 92.Otz.0 94.O!2.O 95.5t0.5 94.$!2.2 95 . O J2.0 97.0!3.4 NMN o.7 lo.2 1.OlO.2 0.5Ð.2 0.210.05 0.310.05 0. 1 1O.02 0. 1 !0.02 Cocaine Contnol Efflux

NA jT .O!4.9 10.0t3.2 10.5t3.1 13.2!2.1 10.313.1 10.81 0.8 1 0.514.9 DOPEG 26.3!5.9 28.316.1 23.5!3.8 25.5!3.2 18.0t4.0 24.Oú.1 22.3!6.4 DOMA 2.6!0.8 2.5!1 .O 2.2Ð.4 2.3!O.4 2.7!O.5 2.2Ð .7 2.OtO.4 OMDA 52.519.8 58.818.2 63.3!6.2 58.O!7.2 OZ.5!8.2 61 .Bra.1 64.0!6.3 NMN 0. 5t0.06 0.4!0.05 0.510.1 0.5!0.06 0.510.09 0.610. 1 0.5r0. 1 Tyramine and Cocaine Efflux NA 33.0t4.2 12.8!2.O 8.91:l .3 17.511 .5 18.0t3.0 22.OL4.1 20.013.0 gt3. DOPEG 23.013.0 23.4!2.7 14.4t2.3 27.$!3.5 37.5!2.5 32.013.0 39. 1 DOMA 1.5t0.3 1.5t0.2 1.410.3 2.0!0.1 1.5!0.5 2.Ofr.2 1 . 110.3 OMDA 41 .5r8.1 61 .016.2 74.5!8.4 52.5!2.5 4l.Ot6.O 43.014.5 47.5!2.5 NMN 0.6!0.2 o.6to.1 0.810.2 0.910.05 0.910.07 1.0!0.3 0.7!0. 1

The figures in each column represent the pencentage of the total eff lux per period. 7.5

in Table 7.3

Pne-incubation with cocaine decneased the uptake of 3 H-NA, 3H-NR 3H-oopec so that and eff lux accounted fon only 6% and 1% of the total tr itium eff lux, nespectively (Table 2.3), wheneas in the contnols these values wene 43% and zr% (taute 7.1), taken during the second five minute efflux period.

Figure 7.4 shows the eff lux pr-of iles in antenies pnetneated

with ipr oniazid. Table 7.4 shows that in the ipnoniazid contnol 3H-oopEc group the efflux contnibutes only 4.g% of the total eff lux compared to 28.5% in the untneated gnoup (Table ?.i]}. 3H-Na Etilefnine has now caused an incnease in efflux. An 3H-OOpEC incnease in efflux was also obsenved (as in artenies not tneated with ipnoniazid) tollowing etilef rine administnation (F¡g. 3H-Nlr,tN 3 ?.1). No change was obsenved in on H-olr¿oR eff lux following etilefnine administnation; however, an unexplained fluct- 3H-oot¡¡ uation in occurred in both iproniazid tneated groups with and without etilefrine.

Discussion

The nesults of the pnesent study indicate that the sympatho- mimetic amines examined have quite diffenent effects ón nonadnen- aline and metabolite eff lux fnom a sympathetically innervated blood vessel. Leitz and Stefano (1921), using the rat heart, obsenved no incnease in efflux of deaminated metabolites following exposune to tyramine on amphetamine. Howeven, Bnandao et al. (1978) found that tyramine induced an increase mainly in TABLE 7.4 The mean value t S.E.M. fon the gnoup of anteries studied is shown. Metabol ite Eff lux Peniod Number 1 2 34 5 6

lpnoniazid Control Efflux NA 27.7!3.2 14.8t1.6 12.5!4.0 7.7t2.1 6.æ2.1 7.7!1 .6 DOPEG 4.3Ð.8 5.0!1 .2 4.5Ð.8 4.3Ð.5 3.8i0.4 4.3t0.4

DOMA 0.5t0.1 0.5Ð.10Ð0.3Ð.10.210.05 0.610. 1 OMDA 65.O!7 .2 77.8!8.3 80.5f6.4 86.313.5 87.7t3.5 85.3t2. I

NMN 2.3Ð.2 2.OtO.4 '.l .8Ð.4 1.5Ð.2 1.510.2 1 .5!O.2 lproniazid and Etilefnine Efflux

NA fi.3n.4 40.3!4.4 58.316.9 55.816.9 54.3!7.4 53.517. 1 DOPEG 8.312.0 11.5!2.1 10.312.1 10.0t2.1 11.3!2.6 11 .5!2.1 DOMA 4.310.8 5.3Ð.9 2.7lO.4 4.3!O.7 3.0r0.2 4.3!0.8 OMDA 25.3t2.7 31 .7!4.2 18.812.3 22.3!2.4 24.O!2.7 23.5t2.5 NMN 8.511.6 11.5!2.6 7.0!1 .7 7.311 .6 5.811.5 6.5!t.S

The figunes in each column nepnesent the pencentage of the total efflux pen peniod. 7.6

3 H-DOPEG, whereas electnical stimulation pnedominantly enhanced aa the efflux of "H-NA followed by 'H-DOPEG.

I n the pnev ious chapten, ¡t was shown that et i lef nine increased the eff lux of neunonal tritium (ttl¡ and DOPEG) fr om the rat tail anteny. The pnesent study shows that etilefnine is in fact enhancing the release of 3 H-OOpgC to a largen extent than it is of nonadrenaline. The incnease in 3 H-OOpEC may be due to ne-uptake of released nonadrenal ine and subsequent metabol ism and diffusion into the wash fluid. This is in agneement with the f indings of Vanhoutte and Webb (1979), who showed that neunonal uptake is an impontant mechanism of tnansmitten elimination from the synapse in the nat tail anteny. Bungen and lvensen (1965) suggested centain stnuctural nequinements which need to be ful- f illed for sympathomimetics to be substnates fon neuronal uptake in the nat heant: (a) beta-hydnoxylation caused a decneased affinity; (b) alpha-methylation nesulted in an incneased affinity; (c) phenolic hydnoxyl gnoups, especially in the 3' ,4' posit-

ion, enhanced aff in ity ; (O) N-substitution with bulky groups decreased affinity; (e) O-methylation of phenolic gnoups caused a decnease in affinity for uptake. Based on these cnitenia, etilef nine would be expected to be a poon substnate. Etilefrîne is not a substnate for monoamine oxîdase (MAO) and therefone would not compete with nonadnenaline for this enzyme. Tynamine has been shown to nelease neunonal noradnenaline (Burn and Rand, 1958; Paton, 1973a; Bnandao et al., 1979). NMN 100

50

0

OMDA 100

I I I

0

DOMA o 100 o L É. ul I I À 50 x J. f J t¡- lJ- 0 IJJ J DOPEG g 100 T t- z I ¡ I t \o 50

I

0

NA 100

50

0 23¿56 23¡.56? 23¿567 CONTROL ETI LEFRINE TYRAH INE EFFLUX PERIOD

Fis. 7 .1 The efflux of 3H-NA, 3H-OOpEC and the other metab- olites (expnessed as a pencentage of the amount washed out in the finst wash peniod) fnom the rat tail artery. The effect of etilef nine and tyramine ( introduced at the beginning of the third wash peniod) ¡s companed with control efflux. NHN 100 I I t L I L 50 ¡ I

OMDA 100

50

0

DOMA o 100 C) õ Àlr, x 50 :) J l¡- ll 0 1¡l

J OOPEG 100 =z. Sso

0

NA f00 I

5() ¡

0 1231567 123t,567 REN -2 93 EPHEOR I NE EFFLUX PERI OD

Fis. 7.2 3H-NA, 3U-oOpEO The efflux of and the othen metabol ites (expr^essed as a penðentage of the amount washed out in the finst wash peniod) fnom the nat ta i I anteny . The effect of REN-293 an d ep hed n i ne (introduced at the beginning of the thind wash peniod) is shown. NHN 100

.t

50

¡

0

OMOA 100

T 50 I L J,

0

DOMA o 100 o I É. L Lrj I fL 50 x t f J l! tr 0 t¡J J DO PEG { roo z. J. I Sso

0

NA 100

50

0 123 t,5 6 ? 12 315 6 7 COCAINE CONTROL TYRAMINE ANO COCAINE EFFLUX PERIOO

Fig. 7.3

The i nf I uence of coca i ne and coca i ne/ ty ram i ne on the efflux pattenns from the tail arteny. Cocaine was added at the commencement of the third five minute period and tyramine at the start of the founth five minute wash period. NMN r00

50

0

0l"lDA 100

50

DOMA 100 o a. (L50llJ x =J TL l-L u lr¡ DOPEG J < 100 E z

Sso I

0

NA 100

50 ¡

I

0 123156 123456 IPRONIAZID CONTROL IPRONIAZID AND ETILEFRINE EFFLUX PERIOD

Fis. 7.4 Eff lux patterns f rom rat tai I antenies taken fnom animals pnetneated with ipr oniazid for 48 houns. Etif ef rine now causes a signif icant incnease a in "H-NA efflux. 7.7

Schümann and Philipu (1961) showed that tynamine is taken up into the nerve tenminal, displacing noradrenaline fnom its stonage vesicles into the cytoplasm and f inally into the synapse. Oun 3H-NA 3 results show that thene is an increase of both .nd H-DOPEG efflux in the presence of tyramine. Since tyramine has also been shown to be a good substnate fon neunonal uptake (Bungen and lvensen, 1965), this would explain the ability of tyramine to 3H-Nl¡ enhance nonadnenaline eff lux by nelease of and subsequent inhibition of re-uptake by competing with 3 H-ne fon the Uptake I mechanism. 3 REN-293 induced a large incnease tn H-NA efflux but a a decnease in "H-DOPEG efflux. Pnevious obsenvat i ons (Chapten 3) have shown that REN-293 has a vasoconstricton action in the nat tail anteny which is blocked by phentolamine and abolished by pnetneatmen t with resenpine Togethen, these nesults indicate that REN-293 has a stnong indinect sympathomimetic action mediated via neunonal nonadren- aline release. Applying the cniteria of Burgen and lvensen (1965) once again, REN-293 would seem to be a good substrate fon neunonal uptake and so would have a dual effect on noradnenaline efflux by enhancing nelease and inhibiting ne-uptake. The decnease in 3H-OOpEC is diff icult to explain. Since REN-293 has been shown to be a substnate for MAO (pensonal communication, Boehningen lngelheim), it is possible that 3 H-OOpEC fonmation has been inhibited by REN-293, firstly by inhibition of re-uptake and secondly by competition fon MAO. Howeven, on this basis one would not expect the increase in 3 H-OOpEC obsenved on the addition of tynamine. This diffenence may be explained if REN- 7.8

293 has a higher aff inity than tyramine for eithen neunonal uptake on MAO. lf REN-293 is a betten substnate fon MAO, then the nesultant competition with nonadnenaline fon this enzyme may inhibit the fonmation of H-DOPEG. W¡th tynamine this competition fon MAO may not be as gneat and an increase in 3H-OOpEC fonm- ation would occur.

Ephedni ne tended to decnease the nate of dec I i ne of both 3H-tll 3H-DOPEG ..,d efflux from the anteny when companed with untneated controls. Ephednine has been descnibed as a "mixed acting aminerrby Trendelenbung (1963) and these findings indi- cate a modest indinect action via neunonal nonadnenaline nelease. Based on the cnitenia of Bungen and lvensen (1965), ephednine would appean to have a nelatively low affinity fon neuronal uptake and so would be expected to be a poon neleasen of nonadrenaline and a poor inhibitor of ne-uptake. The pnesent findings ane in agreement with these critenia. 3 3 Cocaine did not cause an enhancement of H-NA on H_DOPEG eff lux, but when tynamine was added in the pnesence of coca î ne, 3H-t\¡ 3 an increase in both and H-oopEc was observed . Th is incnease in the pnesence of cocaine may be due to an incomplete block of neunonal uptake by cocaine at the doses used in this study.

3 I pron i azi d did not modify the tnend in pencentage H-NA 3U-OOpfO efflux, but the levels of expnessed as a percentage of tota I tnitium wene neduced when companed to contnol values. The fact that etilefnine induced a signif icant incnease in 3 H-trt¡ eff lux in the iproniazid treated vessels suggests that inhibition of MAO has pnevented the metabolism of 3 H-*R and shif ted the eff lux 7.9

pattenn in the direction of the latter. Cocaine pnetneatment significantly inhibited the uptake of tnitiated nonadnenaline, so confirming the pnesence of a cocaine sensitive uptake pnocess in the nat ta i I arteny . 8 USIdVHJ 8.1

GENERAL DISCUSSION

The pr oject commenced as a study to examine the effects of

the sympathomimetic amine etilef nine. Etilefrine had been used in the treatment of patients with orthostatic hypotension (Mi¡en

et al ., 1973), and in a pnevious repont Met randen (tgoo) rrao

suggested that etilefnine had rra local constnicton effect mediated by alpha neceptons" and a rrdistantrr dilaton effect activated via

the sympathetic nervous system. w¡th this in mind, it was decided to look at the mechanism of action of etilef nine on blood vessels, to determine whethen or not it had any dinect sympatho-

mimetic effects. This was impontant, since it had been shown (Panks et al ., 1961 ) that in the tneatment of postunal hypotension

the indirectly acting sympathomimetics, such as ephednine and methyl amphetamine, were ineffective in patients with autonomic degenenation.

ln selecting a suitable vascular model to examine the actions of eti lefnine, several factons wene impontant. Finstly, ¡t was considened essential that the model be similan to human artenies with respect to its sympathetic innenvation and, secondly, that it was sensitive to sympathetic amines and exhibited nepr-o- ducible dose nesponse cunves. Hodge and Robinson (1922) had shown that the nat tail arteny had good sympathetic jnnervation and was sim i I an in chanacter ist ics to the nabb i t ean antery , which had been shown previousry (Fnewin et al., 19?1b) to resemble the human digital anteny in its histology and innenv- ation. Nicholas (1969) suggested that the nat taíl artery was a sensitive pnepanation and that ¡t would be suitable fon the assay 8.2

of sympathomimetic amines. These facts, combined with the ease with which the pnepanation could be isolated, wannanted the

selection of the rat tail arteny, and in some expeniments the centnal anteny of the nabbit ean, as vascular models for this study. ln Chapter 3, it was observed that the rat tail artery ttin siturr d¡d nespond well to a numben of dinectly and indinectly act- ing sympathomimetics. Etilefnine caused an incnease in perf usion pnessune which was comparable to that of metanaminol and this

increase was mediated via alpha-adnenengic neceptors as demon- strated by the inhibition of response caused by phentolamine. Of particulan intenest in this chapten was the nesponse characten-

istics exhibited by etilefrine. The etilef rine nesponse was com-

posed of a short initial rise phase, followed by a prolonged slow nise phase. The shape of the response was qualitatively similar

to that of ephednine and contrasted markedly with the nesponse of adnenaline and noradrenaline which typically had a monophasic response with a subsequent plateau. Tnendelenbung (1963) categ- orised ephednine as a trmixed-actingrr amine, i.e., having both direct and indir ect actions. This similanity of etilefnine and ephednine, combined with the suppontive evidence that the slow rise phase of etilef nine was abolished by pnetneatment with 6- hydroxydopamine, indicated that etilef nine possessed some indirect sympathomimetic activity. The sympatholytic agents neserpine and guanethidine also caused a decnease in the etilef nine nesponse. Funther supportive evidence for a nonadnenaline neleasing role came fnom the fact that pnior administnation of a noradren- aline depleting agent (namely, tynamine) depnessed the nesponse 8.3

to etilef nine. Tynamine has been shown to be the classic example

of an indinectly acting sympathomimetic (canlsson et al., 1957; Bunn and Rand, 1958) andr âs such, by depressing the etilefrine

responser a common mechanism of action between these two amines

was speculated. The augmentation of the etilefrine nesponse by cocaine indicated that etilefnine may not be dependent on the

cocaine sensitive uptake and that the enhanced response was due to the inhibition of ne-uptake of nonadrenaline neleased by etil- ef nine. ln Chapten 4, ¡t was decided to examine the effects of etilefnine on the isolated rat tail arteny. This was instigated in an attempt to elucidate furthen the dnugrs mechanism of action by companing the responses of extennally and intennally admin- istened etilefnine. de la Lande and watenson (1969), who invest- igated the action of intnaluminally and extr-aluminally administened

tynamine, postulated that the difference in nesponse to the dr ug

via the two noutes was nelated to tynaminers indir-ect action and

hence its access to intnaneu¡onaI storage sites. Etilefnine showed a larger nesponse when administened extennally to the rat tail arteny, and when the same sympatholytic agents (nesenpine and guanethidine) wene used as in the pnevious chapten, the extennal response was depressed to a greaten extent than the intennal nesponse. The intennal and the extennal nesponses to tyramine wene similan to etilefnine in nonmal arteries, with the extennal response being langer than the intennal nesponse. The gneaten

indirect sympathomimetic activity of tynamine was demonstnated by the complete inhibition of its response by neserpine and guaneth-

idine companed to a partial depression in the nesponse obsenved wi th eti lefrine. 8.4

Chapter 5 examined the actions of some of the sympatho-

mimetic amines used in pnevious chaptens on the rabbit ean

anteny. The selection of the ean anteny as a vascu lan model was

fon two reasons : f irst I y, i t seemed advan tageous to compane the responses obtained in the nat tail arteny with those on anothen vessel and, secondly, the nabbit ean antery could be sungically denervated with relative ease. ln relation to the use of etil-

efnine in orthostatic hypotension, the intenaction of this dnug with 9-alpha-fluorohydnocontisone (one of the main thenapeutic agents used in this condition) was also evaluated. S¡nce 9-alpha-

f luonohydrocontisone is used thenapeutically at a low dose level, a dose was selected which ¡t was thought would approximate physiological levels. ln addition, a higher dose was also selected which was companable to that used with othen mineralo- corticoids for the punpose of inhibiting extraneuronal uptake. Howeven, no change in the etilefnine nesponse was obsenved in the pnesence of either dose of the minenaloconticoid. Funther evidence of the postulated pantial indirect action of etilef rine was suggested when it was found that the etílefrine nesponse was depressed in sungically sympathectomised nabbit ear artenies. To funthen substantiate the phanmacological findings,

the next step was to determ ine whethen or not thene was a mod if - ication of the tritiated nonadnenaline eff lux fnom the tail arteny

in the presence of etilefnine. lt has been shown (Paton, 1973a, b) that vanious sympathomimetic amines can enhance the efflux of tnitiated nonadrenaline fnom rabbit atria. The results of the study in Chapter 6 showed that etilefrine did enhance the trit- iated efflux fnom antenies pne-incubated with tnitiated 8.5

nonadnenaline. The enhancement obsenved was equivalent to that of ephedrine but substantially less than that induced by tynamine. The findings seen with tynamine and ephedrine wene in agreement with the actions of those amines as suggested by Tnendelenbur g (1963). Tyramine was found to cause an incneased efflux in the pnesence of cocaine, and it was suggested that tynamine may enten the neunone via an altennative uptake to that inhibited by cocaine on may just be pnesent in quantities lange enough to render the cocaine block ineffective. Chnonic Auanethidine admin- istnation reduced the capacity of the neunones to store nonadren- aline, but an incnease in tnitium eff lux was st¡ll evident upon administration of etilef nine. lnhibition of the vanious noradrenaline disposition mechan- isms by dnugs inhibiting tnansmitter uptake and metabolism incneased the nate of eff lux with nespect to contnol values. Etil- efrine also caused an enhanced efflux in those antenies which had been tneated with iproniazid, UO52l, cocaine and deoxyconticost- enone acetate. This efflux was gneater than that neleased fnom control anteries and also those vessels tneated with the inhibitons of tnansmitter uptake and metabolism alone. This increased tnitium efflux was thought to be due to the nelease of more un- metabolised nonadnenaline fnom the anteries in the pnesence of etilef nine.

To furthen verif y the composition of the tritiated eff lux f nom the nat tail artery, it was decided to sepanate the various metab- olites within the nadioactive eff lux. This procedure enabled direct detenmination of whether the enhanced efflux induced by the vanious sympathomimetics was due to increased nonadnenalîne 8.6

ot metabolite displacement. ln this investigation, it was found that tyramine again caused an incnease in tritium efflux and 3H-tt¡ 3H-OOpEC this could be attributed to enhanced and efflux. 3 Etilef rine, on the other hand, caused an incnease in H-OOpEC 3 eff lux and only a veny slight, non-signif icant incnease ¡.t H-tttA. Howeven, fol lowing pnetneatment with ipnoniazid, a largen increase in 3H-Nl efflux was observed following etilefnine administnation.

Togethen, these f indings suggested that, although etilefrine 3H-trl¡ caused an increase in release, ne-uptake and subsequent metabolism pnobably occul ned during the f ive minute eff lux 3 peniods monitoned. Thus, the incnease in eff luxed H-ruA was only unmasked when MAO was inhibited by ipnoniazid. Tynamine 3H-NR 3 was again nesponsible fon an incnease in and H-OOpf C eff I ux in the presence of cocai ne. The possi b le reasons fon cocaine being unable to inhibit tynaminers action has been dis- cussed pneviously. The sympathomimetic REN-293 is a drug which is under investigation by the phanmaceutical agency Boehr inger lngelheim as a possible successon to etilefnine. Samples of the dnug wene neceived late in this investigation and, as such, data on this agent ane only included in chapten 3 and chapter' 7. REN-293, on the basis of the wonk carnied out in Chapten 3, appeans to be an indirectly acting sympathomimetic in the rat tail anteny, i.e., via the action of noradrenaline on alpha-receptons. lts indirect effect seems equivalent to that of tynamine as suggested by the tota I suppression of the nesponse of both agents i n neserp i ne pnetreated artenies. These findings ane in agneement with company investigational litenatune (Boehninger lngelheim). Fnom 8.7

the dose nesponse cunves generated in the pnesent study, REN-293 would appear to be mone potent than tyramine in this vessel. The nesponse to REN-293 following tynamine administnation was significantly depressed. This suggested that tachyphylaxis to the REN-293 nesponse occunned following the addition of tyn- amine. This would be expected ¡f the two agents wene acting on a common pool of neleasable nonadnenaline. The inhibition of the REN-293 response by cocai ne suggested that i ts act ion i s dependent to a large extent on the cocaine sensitive uptake. Based on the critenia of Burgen and lvensen (1965), REN-293 would be expected to be a good substnate for neuronal uptake, which pnovides supportive evidence fon the cocaine inhibition. The nesults in Chapter 7 neganding REN-293 show that this agent a does enhance JH-NA nelease from the tail artery. The depnession ) in 'H-DOPEG efflux, however, was postulated to be due to an inhibition of re-uptake by REN-293 and competition for the enzyme

MAO.

ln conclusion, it would seem that etilefrine does have an indirect sympathomimetic component to its action which is compan- able with ephednine but not as gneat as that of tynamine. This is of relevance in the clinical situation where the use of this agent in tr eating postural hypotension has been pnoposed. REN- 293 would appean to have an indirect action similan to that of tynamine and, as such, is of intenest as a pharmacological tool fon examination of sympathetic nerve f unction. REN-293 as a drug of clinical impontance is doubtf ul, since it is f ar less potent than etilef nine and due to its indinect action would be subject to tachyphylaxis and loss of effect in autonomic neuropathy, 8.8

and so be limited in its therapeutic usefulness. It is obvious that the sympathomimetic agents examined in this thesis have varying modes of action on blood vessels. The interplay between the vanious neunonal and extraneuronal dis- position mechanisms with nespect to the actions of these agents is yet to be f u I ly elucidated. As Tnendelenbung (1963) suggests, rrsympathomimetic amines possess a continuous spectnum of actions ranging fnom punely dinect to completely indir ect in a vaniety of species and tissues with¡n each species.rl

):< xroNfddv 4.1

1a. Knebs bicarbonate solution Composition of the Krebrs bicarbonate solution used in this study. Millimolan Consti tuent Grams/Litre Equivalent

NaC I 6.9 120.0

KCI o.29 3.9 CaCl, o.28 2.5

MsC l, 0. t0 1.1

NaHCO 2.10 25,O 3 KH2PO4 0.16 1.0

g I ucose 1 .00 5.5

EDTA 0.0045 0.01

Ail compounds, except CaCl ,, MgCl, gnd ethylenediamine- tetracet i c acid (EDTA), wene dissolved in the nequired volume of distilled waten. CaCl , and MSCI, wene added from standardised 1O% stock solutions. The EDTA was added fnom a stock solution of 30 uM. The fully constituted Krebs solution was f i ltered before use and gassed with 5% canbon dioxide and 95% oxygen

(Canbog.en ) . The pH of thè gassed solution was 7.4. 1b. Perfusion pump

The pump used was a penistaltic roller pump model 131900

(Desaga Ger many). 1c. Pressure necorder

Pnessure changes in the arteries were measuned using a pressure transducen (Statham, P23GC) and neconded on a twin channel potentiometric recorden (Rikadenki model 8-261),. The 5 and l0 mV attenuaton settings were used. A.2

td. Sequential timer A five channel sequential timer, designed and constnucted by the authon, was used to change dnug solutions on wash volumes automatícally. The timen consisted of f ive LM2905 inte- grated cincuit (lC) timers (National Semiconducton Conp.) ¡n panallel. The output from one lC was used to tnigger^ the next. Each individual timen had a continuously variable nange from 0- 20 minutes. The output from each timen was used to tniggen a relay, which in tunn tr iggered a fur ther relay modified to clamp thin silicone nubber tubing and thus prevent the f low of penf us- ion or wash fluid. le. Ascorbic Krebs was Krebs-bicanbonate to which ascorbic acid had been added to give a concentnation of 113 p M to inhibit nonadnenaline oxidation (Hughes and Smith, 1978). lf . Liquid scintillation spectrometry Radioactivity was counted using a Packard 2425 or 3310 Tni- Canb liquid scintil lation spectrometen.

The scintillation f luid had the following composition (g/ litre): PPO (2,s-diphenyloxazole) 8.25, POPOP (1,4-di(2[5-phenvl- oxazolyl]benzene)) 0.25. The PPO and POPOP were dissolved in one litre of toluene to which was added 500 ml of Triton X-100. 1g. Sepanation of unlabelled nonadnenaline metabolites To ver-ify that the Dowex and alumina column technique used 3H-no".dnena in th is thesis was separat ing the vanious I ine metab- ol ites, the necoveries and crossovers of un I abel led metabol i tes wene determined. The technique was that used by Monris and lrvine (personal 4.3

communication). Each metabolite (100 u g in 0.0167 ml), i.e., MOPEG, VMA, NMN, DOPEG, NA and DOMA on a blank containing no metabolites, was individually added to the following: O.2 ml 1% ascorbic acid, o.2 ml 0.1 N Hcl, 0.1 ml 10% EDTA, 0.1 ml 12.5% NarSOr, 1 ml Knebs, 0.5 ml 1 M Tnis (pH 8.4). This mixture was loaded on to an alumina column and the pnocedune followed as described in the 'rmetabolite sepanationrr section in the genenal methods.

The f ract ions el u ted f rom the alumina and Dowex columns, as wel I as the two washes, were then assayed fon native fluo- rescence on a Perkin-Elmer 2O2 f luorometen (excitation 288 îffir em iss ion = 315 nm).

FRACT ION ACTUAL % RECOVERY (Expected Metabolite) MOPEG VMA NMN DOPEG NA DOMÀ

MOPEG+VMA 96.8 92.3 0 0 0

2 NMN 2 0 o.7 't00 0 0.5 0

3 DOPEG 0 1.2 0 95.8 o.2 0

4 NA 1 1 1.1 0 0 95. 1 2.3

5 DOMA 0 00 1.9 1.7 56.3

6 Wash I 0 00 0 00

7 Wash 2 0 4.8 0 2.3 2.5 41.4

On the basis of these resu I ts cnossover correct i ons wene made. lh. Preparation of alumina for columns The pnocedune used was similar to that used by Crout (1961). Alumina (200-300 s) was boiled in one litne of 2 N Hcl fon 30 minutes and the acid decanted. The alumina was then washed twelve times with distilled waten, allowing f ive minutes 4.4

each time for the alumina to settle. When the water was clear, the pH of the mixture was appnoximately 4-5. The alumina was then dried at IOOoC fon two hours and stoned for use. 1¡. Preparation of Dowex for columns The procedune used was similar to that descnibed by Graefe et al. (1973). Dowex 50 Wx4 (200-400 mesh) was washed twice with 2 N sodium hydr oxide (containing 1% di-sodium EDTA) at oC 50 unti I the supennatant was colounless. The Dowex was then washed with distilled waten and subsequently with 2 N HCl. Following a furthen wash with distilled water and equilibration with o.01 N Hcl to pH 2, the Dowex was f inally washed with 6 N

HCI-ethanol (natio 1 :1, v/vl and again equilibrated with 0.01 N HCI to pH 2. The Dowex was then stoned ready for use in 0.01

N HCI. 1j. pH measurements

The pH of solutions was measured with a digital pH meten

Rad iometer (Copenhagen), model PHM62, using a Rad iometen g lass e I ectnode .

I k. Electrical stimulation This was perfonmed using an electrical stimulator (Grass model S4E). The following settings were used: voltage 20 V, pulse delay 0.01 ffisr pulse duration 5 ms, fnequency vanied from 1-20 pulses/sec.

2. Purification of 3 H-noradrenal ine 3 The H-nonadnena I i ne purif ication pnocedure was similar to 3 that descni bed by Head et al . (1977\ . The H-no".dnenal ine

(0. s ml ), as neceived fnom Amensham on New England Nuclear, A.5

was added to 700 mg alumina, 1OO mg ascorbic acid, 100 mg EDTA, 0.'f ml O.25 M sodium acetate and 2 ml 0.001 M HCl. This mix- tune was bubbled with nitnogen and the pH adjusted to 8.4 using 0.1 M sodium carbonate and maintained at this pH for four minutes. The alumina was allowed to settle and the eff luent was removed. The alumina was then washed with two aliquots of 10 ml of distilled water. The 3H-r',o".dnenaline was first eluted with 5 ml 0.3 M acetic acid, then with a further 2 ml 0.3 M acetic acid. The 7 ml of eluate was then stoned at 4oC. A 0.1 ml sample fnom the foregoing was added to 2 ml 0.5 M HCI and assayed for nonadnenal ine content using the fluoro- metric assay technique described in the genenal methods. A 0.1 ml sample of the eluate was also counted fon. nadioactivity and the counts corrected fon efficiency using an automatic extennal standard. This enabled the specific acJivity expnessed in dpm/ng of the punif ied 3 H-no".dnenaline to be determined. The punity vanied fnom 80% to 95%. Unlabelled nonadnenaline was added to the eluate and the specif ic activity nanged from 25,000 to 40,000 dpm/ns. 3 Prior to an expeniment the requined volume of the H- nonadrenaline stock solution was f nozen using a mixtune of solid- if ied CO and ethanol . The samp le was then f reeze-dnied using z^ a Dynavac FDA/24 houn fneeze dnyer and a volume of asconbic- 3 Kr ebs added to give the nequired concentnation of H-.'o"adre.t- aline for the expeniment.

3. Dnugs The following drugs were used: 4.6

etilefrine hydrochlonide (Effonti I ) Boehringer lngelheim cocaine hydroch lonide MacFanlan-Smith tynami ne hydnochloride Koch-Li ght reserpine (Serpasi I ) C iba guanethidine sulphate ( lsmel in) C iba ipnoniazid phosphate (Mansi I id) Roche nialamide Pf i zen

(-)-nonadnenal i ne bi tartnate Koch-Li 9ht

(-)-adrenal ine bitantnate Koch-L i ght pheny lephni ne hydrochlonide Winthrop

(Neo-synephri ne) metaraminol bitartrate (Anamine) Menck, Sharp & Dohme phentolamine mesylate (Regitine) Ciba

6-hydroxydopami ne hydnobromi de Si gma 14C-sorbitol (to mci/mmol) Amersham

(t )3H-noradrenal i ne hydro- Amensham

chloride (10.8-1 2.o ci/mmol ) (-)3H-noradnenal ine hydro- Amensham chlonide (10.8-12.o Ci/mmol)

(1-5 cilmmol ) New England Nuclear ephednine hydnochlonide Knol I uo521 (3'4'-dihydroxy-2- Upjohn methyIpropiophenone) deoxycort i costerone acetate Stena loids

REN-293 (2-amino-3-(3, 5-d¡- Boehringer lngelheim

hydroxypheny I ) -l -pnopanol hydroch loride 4.7

Pneparation of drugs Most dnugs were pnepaned in O.9% sodium chlonide contain- ing ascorbic acid (O.SZ mM), i.e., ascorbic saline. Asconbic/saline. This was a 9 gram/litne solution of NaCl in distilled water to which had been added 1 ml of a 1 gramf 10 ml solution of asconbic acid. The ascorbic saline solution was adjusted to give a final pH of 5.4-5.6. Deoxyconticosterone acetate was pnepaned in a stock solution of 67 mM in ethanol . 9-alpha-fluorohydnocontisone was pnepared in a stock solut- ion of 22.8 mM in ethanol. Nialamide solution was prepaned by dissolving the requined amount of nialamide in 20 ml of waten with the aid of gentle heat. This solution was then made up to the appropriate volume with Krebs solution immediately pnior to.use Concentnations of adnenaline, noradnenaline, phentolamine, nialamide and neserpine refen to the bases. Other dnug concen- tnations nefen to the salts. NA NMN T _NH -- - --l rr-_-_-_- H0 HO -c -l I 2 H H 0 3

DOPEG HO OH HO MOPEG L---5 Þ -ð-tr +-J td I I õ H H o 0 o 3 H. x È

DOMA 0 HO VMA _C_OHll I L - --+, c H <____J I H 0 3

COMT PATHWAY

_ _ _+ MAo PATHWAY

Products formed fnom noradrenaline (NA) by the comblned action of COMT (catechol-O-methyl tnansferase) and MAO (monoamine oxidase). BIBLIOGRAPHY 8.1

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