Identification of alpha adrenoceptor subtypes in dog arteries by (3H) yohimbine and (3H) prazosin

Life Sciences, Vol. 30, pp. 219-228 Printed in the U.S.A.

Pergamon Press

IDENTIFICATION OF ALPHAADRENOCEPTORSUBTYPES IN DOG ARTERIES BY (3H) YOHIMBINEAND (3H) PRAZOSIN A. Bobik

Baker Medical Research Institute, Alfred Hospital, Commercial Road, Prahran, Victoria, 3181, Australia (Received in final form November 24, 1981)

Summary Binding of the alpha adrenergic antagonists (3H)prazosin and (3H) yohimbine to membranes of dog arteries exhibit the characteristics expected of alpha adrenoceptors. Binding of both ligands is saturable with dissociation constants of O.19nM and 1.15nM for (3H)prazosin and (3H)yohimbine respectively. A series of catecholamines i n h i b i t binding of both ligands with a potency in the order epinephrine > norepinephrine >> isoproterenol, corresponding with the a c t i v i t y of these agents at alpha adrenoceptors in blood vessels. Competition for binding in both instances is stereoselective. ~-Phenylephrinehas similar potencies in inhibiting (3H)prazosin and (3H)yohimbine specific binding whilst the imidazoline related partial alpha adrenergic agonists clonidine and guanfacine are more potent in inhibiting (3H) yohimbine specific binding. The a f f i n i t y of prazosin for the (3H)yohimbine binding site is approximately 2500 times less than for the (3H)prazosin site whilst yohimbine is approximately 150 times more potent in inhibiting (3H)yohimbine than (3H)prazosin specific binding. Non-selective alpha adrenergic antagonists have similar a f f i n i t i e s for both binding sites. The concentrations of (3H)yohimbine binding sites in different arteries vary about two fold whilst for (3H)prazosin the variation was about three fold. These results indicate that there are two discrete noradrenergic binding sites in the major arteries of dog which have binding properties expected of alpha1 and alpha2 adrenoceptors. Alpha adrenergic receptors appear to exist in many tissues as a heterogenous population (1). On the basis of the differential potencies of certain adrenergic agonists and antagonists they have been generally divided into two major subgroups, namely alphal and alpha2-adrenoceptors (2,3). Both alpha1 and alpha2-adrenoceptors are thought to mediate post synaptic alpha adrenergic effects (3) whilst only alpha2 adrenoceptors appear to be located presynaptically and have been suggested to be involved in modulating norepinephrine release (4). On the basis of functional pharmacological studies evidence has recently been presented that both alpha1 and alpha2 adrenoceptors are located in blood vessels of several mammalian species (3,5,6). However, demonstration of the presence of alphal and alpha2 adrenoceptor binding sites in homogenate subfractions of blood vessels using alpha1 selective adrenergic antagonist (3H)WB4101 and the alpha2 selective adrenergic agonists (3H) clonidine and (3H)aminoclonidine were unsuccessful (1,7). More recently, alphal-adrenoceptors have been identified in rat mesenteric arteries using (3H) dihydroergocryptine (8) and (3H)WB4101 (9). In the dog (3H)dihydroergocryptine 0024-3205/82/030219-10503.00/0 Copyright (c) 1982 Pergamon Press Ltd.

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has been used to label alpha adrenoceptors in a o r t i c membranes (10). In the l a t t e r report, no subclassification of alpha adrenoceptors was attempted. This paper reports the presence of both alphal- and alpha2 adrenoceptor binding sites in four major a r t e r i e s in the dog. Alphal-adrenoceptor binding sites were characterized with the specific alphal-adrenergic antagonist (3H) prazosin ( I I ) w h i l s t alpha2-adrenoceptor binding sites were i d e n t i f i e d with the selective alpha2-adrenergic antagonist (3H)yohimbine (12). Materials and Methods Tissue Preparation. The aorta, femoral, renal and mesenteric a r t e r i e s were removed from dogs following i . v . administration of an overdose of butabarbital. Vessels were immediately frozen in l i q u i d nitrogen in which they were stored for up to one week. Membranes from the vessels were prepared as follows: thawed blood vessels were homogenized in about I0 volumes of ice-cold 50 mM sodium phosphate buffer pH 7.5 at 23°C with a Polytron PT20 homogenizer (speed I0, 2 x I0 seconds). A f t e r f i l t r a t i o n through two layers of gauze the homogenate was centrifuged at 300 xg for I0 min. The r e s u l t i n g homogenate was c a r e f u l l y removed and centrifuged at 135,000 xg f o r 60 min. The resulting p e l l e t was resuspended in approximately I0 ml of 2M sucrose- 50mM phosphate pH 7.5 b u f f e r , transferred to an u l t r a c e n t r i f u g e tube and overlayed with 8 ml 50 mM sodium phosphate buffer pH 7.5. A f t e r centrifugation at 135,000 xg for 2h the p a r t i c u l a t e f r a c t i o n c o l l e c t i n g at the interface of the two buffers was collected, diluted with 50 mM phosphate buffer pH 7.5 to 0.25 M sucrose concentration and recentrifuged at 190,000 xg for 30 minutes. The resulting p e l l e t was resuspended in 50 mM sodium phosphate buffer pH 7.5 and r e c e n t r i f uged. The f i n a l p e l l e t was suspended at a protein concentration between 1.5 3 mg/ml in 50 mM sodium phosphate buffer pH 7.5. Protein concentrations were determined by the method of Lowry et al. (13). Norepinephrine Analyses. Tissue norepinephrine analyses were performed on the i n i t i a l tissue homogenate using high pressure l i q u i d chromatographic assay (14). Radioligands and Drugs. All radioligands were obtained from New England Nuclear Corp. and stored at -20°C. The radiochemical p u r i t i e s of (3H)prazosin (17.1Ci/mmole) and (3H)yohimbine (81.4 Ci/mmoles) were greater than 98 percent in both instances. Radiochemical purity of (3H)prazosin and (3H)yohimbine were routinely checked by thin layer chromatography on Merck S i l i c a Gel 60 F-254 as directed by the manufacturer. Drugs used in displacement experiments were obtained from the companies of o r i g i n w h i l s t other biochemicals were obtained from the Sigma Chemical Co., St. Louis, M.O.. Other chemicals used were of analytical grade. Binding Assays. Membranes (0.2 - 0.4 mg protein) were incubated in 50 mM sodium phosphate buffer pH 7.5 (containing 95mM Na+) in a f i n a l assay volume of 0.40 ml which contained increasing concentrations of e i t h e r (3H)prazosin or (3H)yohimbine. Separate incubations were carried out in the presence of 300 ~M(-)-norepinephrine to determine the 'non s p e c i f i c ' binding of each ligand. Incubations were carried out at 25°C f o r 30 minutes and terminated by rapid d i l u t i o n with 3 ml 50 mM sodium phosphate buffer pH 7.5 at 25°C, f i l t r a t i o n through Whatman GF/C f i l t e r s under vacuum followed by 2 x 10 ml rinses of the f i l t e r s with the same buffer. Radioactivity collected on each f i l t e r was determined by l i q u i d s c i n t i l l a t i o n spectrometry in i0 ml Instagel (Packard Instrument Co., Downes Grove, I I i . U.S.A.) at an e f f i c i e n c y of 40 percent. In both instances s p e c i f i c binding of the radioligands was taken as the d i f f e r ence between total binding and that in the presence of 300 mM(-)-norepinephrine.

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During the 30 min incubation period no reduction in noradrenaline concentration is apparent (15). In displacement experiments (3H)prazosin (final concentration 0.60 nM) and (3H)yohimbine (0.91 nM) were added to membrane preparations containing various concentrations of drugs (see Results) in a final volume of 0.40 ml. Results i . Saturation of (3H)prazosin and (3H)yohimbine binding. Specific binding of both (3H)prazosin and (3H)yohimbine to high a f f i n i t y sites on membranes prepared from the aorta, femoral, renal and mesenteric arteries is saturable. Specific high a f f i n i t y binding for (3H)prazosin to membranes from all four vessels is half maximal between 0.i and 0.2 nM (3H)prazosin, whilst at i nM specific binding is near saturated. Scatchard analysis of the binding isotherms indicate that (3H)prazosin binds to a single site with a mean dissociation constant (KD) of 0.19 ± 0.03 (mean ± standard error of 5 experiments). A H i l l plot of the data is linear with an average H i l l coefficient of 0.97 ± 0.12 indicating an apparent absence of co-operative interactions in (3H)prazosin binding ( f i g . 1).

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FIG. 1 (3H)Prazosin binding to aortic membranes A. Total binding of (3H)prazosin (&) and non-specific binding in the presence of 300 ~M ~-norepinephrine (e). Each value is the mean ± standard deviation of t r i p l i c a t e determinations. B. Specific (3H)prazosin binding determined by subtracting total from non-specific binding. C. Scatchard analysis of specific binding isotherm (Regression coefficient = 0.948). D. H i l l plot of specific binding isotherm with slope, NH = 1.13 and regression coefficient = 0.955. The data shown is typical of five such experiments.

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(3H)Yohimbine specific binding to membranes of all four vessels was a saturable process whose binding isotherms yield linear Scatchard plots ( f i g . 2). The mean dissociation constant (KD) for (3H)yohimbine binding averaged 1.15 ± 0.12 nM (mean ± standard error of 5 experiments). H i l l plots of specific (3H)yohimbine binding isotherms y i e l d a slope of 1.04 ± 0.03. A

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FIG. 2 (3H)Yohimbine binding to aortic membranes A. Total binding of (3H)yohimbine (&) and non-specific binding in the presence of 300 ~M ~-norepinephrine (e). Each value is the mean ± standard deviation of t r i p l i c a t e determinations. B. Specific (3H)yohimbine binding to aortic membranes. C. Scatchard analysis of (3H)yohimbine specific binding isotherm (regression coefficient = 0.994). D. H i l l plot of specific binding isotherm. The slope (NH) is 1.08 and regression coefficient is 0.992. The data shown is typical of five such experiments. 2. Kinetics of (3H)prazosin and (3H)yohimbine specific bindin 9. The rate of association of (3H)prazosin to specific binding sites on membranes prepared from aorta is rapid with half maximal binding occuring in less than 30 seconds at 25°C. The bimolecular rate constant (KI) for this association process was calculated according to Williams et al. (16) and averaged 0.21 min-lnM-l. The rate of dissociation of (3H)prazosin specific binding was examined by incubating (3H)prazosin and membranes to equilibrium at 25°C and then adding i00 ~M (-)-norepinephrine to prevent rebinding of the dissociated (3H)prazosin ( f i g . 3), The rate constant for the dissociation ( k - I ) of (3H)prazosin averaged 0.0259 min-l. The dissociation constant calculated from the ratio of (k-1/kl) was 0.12 nM which is in good agreement with the Scatchard analysis of specific binding isotherms determined from equilibrium binding experiments.

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FIG. 4 Binding kinetics of (3H)yohimbine to aortic membranes. A. Time course of specific yohimbine binding to aortic membranes at 25°C at a (3H)ligand concentration of 1.0 nM. Insert:- pseudof i r s t order association plot of the data. Xeq is the amount bound at equilibrium and Xt the amount bound at each time. B. First order plot of the dissociation of specific binding. Data is typical of three such experiments performed in duplicate. Experiments with (3H)yohimbine indicate that its rate of association to specific binding sites on membranes from dog aorta is slower than for prazosin. Equilibrium is attained by about 20 min (fig. 4). The bimolecular rate constant for this association process is 0.050 min-lnM-1. The rate of dissociation of (3H)yohimbine from specific binding sites was more rapid than for (3H)prazosin specific binding. (3H)Yohimbine dissociated from its specific

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binding site with a f i r s t order rate constant of 0.102 min-l. The dissociation constant (KD) calculated from these kinetic experiments ( k - I / k l ) was 2.01 nM, in close agreement with the saturation experiments.

3.

Influence of adrenergic agonists and antagonists on (3H)prazosin and (3H)yohimbine specific bindin 9. A variety of catecholamines compete for (3H)yohimbine specific binding sites (Table I ) . In both instances the order of potency of adrenergic catecholamines was k-epinephrine > ~-norepinephrine >> ~-isoproterenol at both specific binding sites. In addition binding to each site was stereospecific. The a f f i n i t y of ~-epinephrine at both sites was approximately f o r t y times that of the dextro isomer. ~-Phenylephrine binds to (3H)prazosin and (3H)yohimbine specific binding sites with approximately equal a f f i n i t y . Dopamine which is devoidofaB-hydroxyl group when compared with norepinephrine has a reduced a f f i n i t y for both binding sites compared to norepinephrine. TABLE I I n h i b i t i o n constants (Ki) of various drugs i n h i b i t i n g (3H)prazosin and (3H)yohimbine specific binding to dog aortic membranes.

Ki (M)*t Drug Z-Epinephrine d-Epinephrine Z-Norepinephrine £-Isoproterenol Dopamine £-Phenylephrine Guanfacine Clonidine Tolazoline Dihydroergotamine Phentolamine Prazosin Yohimbine Serotonin

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*Average of two to four experiments performed in duplicate. I I n h i b i t i o n constants Ki were determined from the equation Ki=ICso/(I + L/KD) where IC50 is the molar concentration of the agent causing 50% inhibition of specific binding of the radioligand. L is the concentration of radioligand used in the experiments and KD is i t s dissociation constant determined from equilibrium binding data. All three imidazoline related derivatives, guanfacine, clonidine and tolazoline i n h i b i t specific binding of (3H)prazosin and (3H)yohimbine. Guanfacine was most selective in inhibiting (JH)yohimbine specific binding followed by clonidine whilst tolazoline displayed least s e l e c t i v i t y in i t s a b i l i t y to i n h i b i t (3H)yohimbine specific binding compared to (3H)prazosin. The alpha

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adrenergic antagonists phentolamine and dihydroergotamine displaY3similar r e l a t i v e potencies in t h e i r a b i l i t y to i n h i b i t (3H)prazosin and ( H)yohimbine s p e c i f i c binding. S e l e c t i v i t y in i n h i b i t i n g specific binding of the radioligands was displayed by the two adrenergic antagonists prazosin and yohimbine. Prazosin was approximately 2500 times less potent in displacing (3H)yohimbine specific binding w h i l s t yohimbine was about 150 times less potent in displacing (3H)prazosin specific binding. 4.

Relation between a r t e r i a l alpha adrenoceptor concentration and norepinephrine concentration. The concentration of (3H)prazosin binding sites differed about two f o l d in the four a r t e r i e s ( f i g . 5). Highest concent r a t i o n s were found in the aorta and lowest were observed in the femoral and mesenteric a r t e r i e s . Norepinephrine concentrations were highest in the mesenteric a r t e r i e s and lowest in the femorals. The r a t i o of norepinephrine concentration to (3H)prazosin sites is greatest in the mesenteric (1.88), followed by the renal a r t e r i e s (0.92) and lowest in the aorta (0.39) and femoral a r t e r i e s (0.41). The concentration of (3H)yohimbine binding sites differed from those i d e n t i f i e d by (3H)prazosin binding. Highest concentrations of (3H)yohimbine sites were found in the renal a r t e r i e s and lowest in the femoral a r t e r i e s . The r a t i o of norepinephrine concentration to (3H)yohimbine sites was greatest in the mesenteric a r t e r i e s (2.34) w h i l s t this r a t i o is closely s i m i l a r in the aorta ( I . 0 0 ) , femoral (0.76) and renal a r t e r i e s (1.19).

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FIG. 5 Concentration of norepinephrine, (3H)prazosin and (3H)yohimbine specific binding sites in dog a r t e r i e s . Results are the mean ± s.e.m, f o r four dogs. Discussion This study has i d e n t i f i e d two d i s t i n c t alpha adreneroic binding sites on membranes from dog a r t e r i e s . The sites i d e n t i f i e d by (3H)prazosin and (3H) yohimbine had the binding properties expected of alpha adrenoceptors with respect to s a t u r a b i l i t y , s t e r e o s p e c i f i c i t y , r e v e r s i b i l i t y and binding k i n e t i c s . Differences between binding properties of the two sites were most apparent from the d i f f e r e n t i a l potencies of the imidazoline related selective alpha2 p a r t i a l agonists guanfacine and clonidine (17,18) and the selective alpha~ and alpha 2 antagonists prazosin and yohimbine in i n h i b i t i n g radioligand speclfic binding.

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Guanfacine and clonidine preferentially inhibited (3H)yohimbine specific binding. The s e l e c t i v i t y of clonidine for this site was approximately eight times greater than for the (3H)prazosin site which is similar to the potency r a t i o reported for clonidine at pre- and postsynaptic receptors in pulmonary artery (19). This degree of s e l e c t i v i t y is however somewhat less than reported by Greenberg et al. (1) in rat cerebral cortex for i t s a b i l i t y to i n h i b i t (3H) clonidine specific binding compared with i t s a b i l i t y to i n h i b i t binding of the selective alpha I adrenergic antagonist WB4101. The difference is probably due to the inclusion of sodium ions in my incubations. Sodium ions increase the a f f i n i t y of clonidine and other adrenergic agents for alphal receptors in brain (20). The present estimates of the i n h i b i t i o n constants for the catecholamine adrenergic agonists, phenylephrine and clonidine are in good agreement with those reported by Glossman and Hornung (20) in rat brain membranes using similar incubation conditions. A surprising result from the displacement studies is the apparently equal a f f i n i t y of phenylephrine for the two adrenergic binding sites. On t h e b a s i s o f functional pharmacological studies, phenylephrine is thought to be a selective alphal-adrenergic agonist having only weak alpha2-agonist a c t i v i t y (21). However, recently phenylephrine has been Shown to be a potent alpha2-adrenoceptor antagonist in human platelets (22). Whether or not phenylephrine is also an alpha2-adrenoceptor antagonist in blood vessels remains to be determined. Greatest differences in the properties of the two sites were observed with respect to prazosin and yohimbine i n h i b i t i o n constants. Prazosin was approximately 2500 times less potent in i n h i b i t i n g (3H)yohimbine specific binding whilst yohimbine was approximately 150 times less potent at the prazosin site. On the basis of the d i f f e r e n t i a l i n h i b i t i o n potencies of the imidazoline related adrenergic partial agonists and the selective alpha adrenergic antagonist, I suggest that the (3H)prazosin and (3H)yohimbine sites represent alphal and alpha2 adrenoceptor binding sites in blood vessel. The a f f i n i t i e s of (3H)prazosin and (3H) yohimbine for alpha I and alpha 2 adrenoceptors respectively are 15 to 30 times greater than those calculated from functional pharmacological studies (23,24). Differences between the two methods can be most probably accounted for by the d i f f e r e n t temperatures used in the respective proceedures and/or extensive non-specific tissue binding of prazosin and yohimbine in classical pharmacological tests which would markedly reduce the free concentration of antagonist. The relative contribution of these factors in accounting for the discrepancies between the two methods clearly requires further study. Vascular smooth muscle alpha I adrenoceptors are located post-synaptically in close proximity to sympathetic nerve terminals and are predominately activated by neurally released norepinephrine (3,5,6). I f one assumes that the norepinephrine content of blood vessels determined in my experiments is a measure of the degree of sympathetic innervation in the vessels, then the ratio of norepinephrine concentration to prazosin specific binding sites should represent the relative degree of sympathetic innervation of these receptors. Differences in the r a t i o of norepinephrine content to prazosin specific binding sites in the four vessels varied approximately five fold. Highest r a t i o was observed in the mesenteric artery whilst lowest ratios were observed in the aorta and femoral arteries. Differences in the relative degree of sympathetic innervation of alpha I adrenoceptors in the mesenteric and femoral arteries are in agreement with the known magnitude of influence of sympathetic nerves on these vessels. The tone of the mesenteric arteries is predominately controlled by sympathetic nerves whilst the femoral is influenced both by sympathetic neural a c t i v i t y and circulating catecholamines (25). While there is much evidence to suggest that vascular post-synaptic alphal adrenoceptors identified by (3H)prazosin specific binding are probably involved in smooth muscle contracture (3,5), i t is more d i f f i c u l t to postulate both the location and role of alpha2 adrenoceptor binding sites identified by

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(3H)yohimbine specific binding. The close similarity in the ratio of blooa vessel norepinephrine content to alpha2 adrenoceptor concentration in the aorta, femoral and renal arteries despite an approximate four fold variation in norepinephrine content might suggest that these receptors are located presynaptically on sympathetic nerve terminals. However, this close association between norepinephrine content and alpha2 adrenoceptor concentration does not hold for the mesenteric artery. Whether the alpha2 adrenoceptor binding sites identified in canine arteries are located pre- or postsynaptically remains to be determined. Acknowledgements This research was supported by the National Heart Foundation of Australia. I thank Dr. Jim Angus for his interest in this work and helpful suggestions during its progress. The technical assistance of Ms. Pam Scott is greatly appreciated. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

D.C. U'PRICHARD and S.H. SNYDER, Life Sci. 24, 79-88, (1979) S. BERTHELSENand W.A. PETTINGER, Life S c i . ~ l , 595-606, (1977) J.R. DOCHERTYand J.C. McGRATH, Naunyn-Schmiedeberg's Arch. Pharmacol. 312, 107-116, (1980) K. STARKE, Rev. Physiol. Biochem. and Pharmacol. 77, 1-124, (1977) P.B.M.W.M. TIMMERMANS, H.Y. KWA and P.A. van ZWIETEN, NaunynSchmiedeberg's Arch. Pharmacol. 310, 189-193, (1979) S . Z . LANGER, R. MASSINGHAMand N.B. SHEPPERSON, Clin. Sci. 59, 225S-228S, (1980) B.R. ROUOTand S.H. SNYDER, Life Sci. 25, 769-774, (1979) W.S. COLUCCI, M.A., GIMBRONEand R.W. ALEXANDER, Hypertension 2, 149-155, (1980) W.S. COLUCCI, M.A., GIMBRONEand R.W. ALEXANDER, Circ. Res. 48, 104-111, (1981) B.S. TSAI and R.J. LEFKOWITZ, J. Pharmacol. Exp. Ther. 204, 606-614, (1978) M.J. DAVEY, J. Cardiovasc. Pharmacol. 2 (suppl.3), $287-$301, (1980) R. WEITZELL, T. TANKAand K. STARKE, ~unyn-Schiedeberg's Arch. Pharmacol. 308, 127-136, (1979) O.H. LOWRY, N.J. ROSEBROUGH,A.L. FARRand R.J. RANDALL, J. Biol. Chem. 193, 265-275, (1951) G.P. JACKMAN, V.J. CARSON,A. BOBIK and H. SKEWS, J. Chromat. 182, 277284, (1980) A. BOBIK, J.H. CAMPBELL, V. CARSONand G.R. CAMPBELL, J. Cardiovasc. Pharmacol. 3, 541-553, (1981) L.T. WILLIES, D. MULLIKIN and R.J. LEFKOWITZ, J. Biol. Chem. 251, 69156923, (1976) R.R. RUFFOLO, E.L. ROSINGand J.E. WADDELL, J. Pharmacol. Exp. Ther. 209, 429-436, (1979) B. JARROTT, R. SUMMERS, A.J. CULVENORand W.J. LOUIS, Circ. Res. 46 (supple.i), 15-20,(1980) K. STARKE, T. ENDOand H.D. TAUBE, Naunyn-Schiedeberg's Arch. Pharmacol. 291, 55-78, (1975) H. GLOSSMANand R. HORNUNG,Eur. J. Pharmacol. 6_11,407-408, (1980) K. STARKE, Rev. Physiol. Biochem. Pharmacol. 88, 199-236, (1981) K.H. JAKOBS, Nature 274, 819-820, (1978) M.J. DAVEY, J. Cardiovasc. Pharmacol. 2, $287-$301, (1980)

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J.C. DOXEYand A.G. ROACH, J. Auto. Pharmac. 1, 73-99, (1981) P.I. KORNER, In MTP International Review of Science, Physiology, ed. A.C. Guyton and C.E. Jones, Series I , vol 1, pp 123-162, University Park Press, Baltimore, 1974