231
European Journal of Pharmacology, 52 (1978) 231--234 © Elsevier/North-Holland Biomedical Press
Short communication I N H I B I T I O N O F D O P A M I N E - S T I M U L A T E D A D E N Y L A T E C Y C L A S E A C T I V I T Y BY PHENOXYBENZAMINE * KENNETH G. WALTON, PETER LIEPMANN and ROSS J. BALDESSARINI Laboratories for Psychiatric Research, Mailman Research Center, McLean Division of Massachusetts General Hospital, and Department of Psychiatry, Harvard Medical School, Belmont, Massachusetts 021 78, U.S.A.
Received 18 August 1978, accepted 12 September 1978
K.G. WALTON, P. LIEPMANN and R.J. BALDESSARINI, Inhibition ofdopamine-stimulated adenylate cyclase activity by phenoxybenzamine, European J. Pharmacol. 52 (1978) 231--234. Phenoxybenzamine at a concentration of 2 x 10 -6 M produced half-maximal inhibition of dopamine-stimulated adenylate cyclase activity in homogenates of rat striatum. Inhibition was sharply dependent on time and temperature of preincubation with the inhibitor. When included in the preincubation medium, dopamine was nearly 8 times more effective than norepinephrine at protecting against this inhibition, whereas neither isoproterenol nor methoxamine appeared to protect at all. These results suggest a direct, and possibly irreversible, interaction of phenoxybenzamine with the dopamine-binding component of the adenylate cyclase. ~-Adrenergic receptors Adenylate cyclase
Dopamine receptors Catecholamines
1. I n t r o d u c t i o n D e s p i t e i n d i c a t i o n s t h a t s u p p o s e d l y selective a - a d r e n e r g i c r e c e p t o r a n t a g o n i s t s c a n have effects on a variety of hormone and drug r e c e p t o r sites, including t h o s e for h i s t a m i n e , serotonin and acetylcholine (Nickerson and Collier, 1 9 7 5 ) , p h e n o x y b e n z a m i n e a n d p h e n t o l a m i n e c o n t i n u e t o be used t o h e l p establish the m e d i a t i o n o f various e f f e c t s b y a - a d r e n e r gic r e c e p t o r s (e.g., B o r i s o n a n d D i a m o n d , 1 9 7 8 ; G o l d a n d S t e r n b e r g , 1978). This use o f such " a - b l o c k e r s " has b e e n b a s e d o n t h e i m p r e s s i o n t h a t t h e y are q u i t e selective a m o n g c a t e c h o l a m i n e r e c e p t o r s ( N i c k e r s o n a n d Colllier, 1 9 7 5 ) . H o w e v e r , t h e r e c e n t e m e r g e n c e o f
* Supported in part by U.S. Public Health Service Grants: MH-30511, MH-31154, the Scottish Rite Benevolent Foundation, Northern Masonic Jurisdiction of the U.S.A., N.I.H. Career Research Scientist Award MH-47370 (RJB) and a Fellowship from The Charles A. King Trust, Boston (KGW).
Phenoxybenzamine
Striatum
d o p a m i n e r e c e p t o r s as a n e w class o f r e c e p t o r f o r c a t e c h o l a m i n e s raises t h e q u e s t i o n of w h e t h e r t h e classical a - a n t a g o n i s t s m i g h t i n t e r a c t at these sites as well. A l t h o u g h t h e r e has b e e n s o m e discussion as to w h e t h e r t h e r e are r e c e p t o r s f o r d o p a m i n e e n t i r e l y s e p a r a t e f r o m the classical a - a n d / 3 - a d r e n e r g i c r e c e p t o r s (Harris, 1 9 7 6 ) , t h e e v i d e n c e in s u p p o r t o f such a s e p a r a t e class is increasingly c o m p e l l i n g (see reviews b y Iversen, 1 9 7 5 and G o l d b e r g , 1975). O n e t e c h n i q u e t h a t is c o m m o n l y e m p l o y e d to s t u d y d o p a m i n e r e c e p t o r - m e d i a t e d e f f e c t s is b a s e d o n t h e t h e o r y t h a t d o p a m i n e - s t i m u l a t e d a d e n y l a t e cyclase is a p a r t o f t h e recept o r - e f f e c t o r s y s t e m f o r this c a t e c h o l a m i n e ( K e b a b i a n et al., 1972). We have, a c c o r d i n g l y , used this r e a c t i o n to evaluate a possible antid o p a m i n e r g i c a c t i o n o f t h e classical aadrenergic antagonist, phenoxybenzamine, a n d h a v e f o u n d t h a t such an e f f e c t occurs at pM c o n c e n t r a t i o n s o f t h e drug. We also p r e s e n t e v i d e n c e suggesting t h a t this b l o c k a d e
232
is irreversible. Our results thus emphasize the risks that could be encountered in using the effects of such non-selective a-antagonists to support the nature of the receptor involved in a pharmacological effect, even among catecholamine receptors.
K.G. W A L T O N ET AL.
chloride (Vasoxyl) was the generous gift of Burroughs-Wellcome. All other reagents were of certified or reagent grade. Catecholamines were dissolved in cold, nitrogen-gassed deionized water just before use.
3. Results 2. Materials and methods The striata of brains from 200 g male Sprague-Dawley rats were dissected immediately following sacrifice by decapitation, as described previously (Walton and Baldessarini, 1976). Striata from 2 or 3 rats were pooled and weighed in a preweighed beaker containing 2.0 ml of ice-cold 2 mM Tris-(hydroxymethyl)aminomethane maleate (Tris-maleate), pH 7.4, in 2 mM EGTA; the volume of buffer was then adjusted to give 30 times the tissue weight. Striata and buffer were transferred to an ice-cold glass Potter--Elvejhem homogenizer, homogenized by hand with a Teflon pestle, and stored in ice until use. The assay of adenylate cyclase activity was as described previously (Walton and Baldessarini, 1976) except for slight modifications made necessary by the time-dependent nature of the inhibition by phenoxybenzamine. Thus, phenoxybenzamine (with or without a potential competitor) was added to each assay tube containing homogenate in the usual assay buffer minus ATP. Following a preincubation period (conditions indicated in the figure legends), a final concentration of 250 #M dopamine was added to some of the tubes and the reaction was started by addition of ATP. Incubation was for 2.5 min at 37°C in a total volume of 500 pl. Cyclic AMP values were determined from aliquots of the assay mixture using the adrenal binding-protein method. Phenoxybenzamine hydrochloride (Dibenzyline; kindly donated by Smith, Kline and French) was dissolved in acidified ethanol and added to the incubation mixture so that the concentration of ethanol never exceeded 1%. An equal volume of acidified ethanol was added to controls. Methoxamine hydro-
In preliminary experiments we found that preincubation of striatel homogenates with micromotar concentrations of phenoxybenzamine reduced the subsequent stimulation of adenylate cyclase which was produced by excess dopamine (250 pM). Furthermore, we found that when this concentration of dopamine was added prior to or along with phenoxybenzamine, the inhibition did not occur. We then extended these investigations in more detailed experiments. Fig. 1 shows the effects of various times
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Fig. 1. Effect of phenoxybenzamine on dopaminestimulated adenyiate cyclase activity. Homogenates o f rat s t r i a t u m , p r e i n c u b a t e d w i t h p h e n o x y b e n z a m i n e in t h e a b s e n c e o f A T P (times a n d t e m p e r a t u r e s indicated), were i n c u b a t e d 2.5 m i n a t 37°C following t h e a d d i t i o n o f A T P (1.5 m M ) + - d o p a m i n e ( 2 5 0 p M ) . O t h e r details are given in Materials a n d M e t h o d s . O n l y t w o e x p e r i m e n t s involving p r e i n c u b a t i o n at 0 - - 2 ° C were carried o u t ; t h e m e a n s are s h o w n . All o t h e r values a n d bars r e p r e s e n t m e a n s _+ S.E.M. for 3--6 s e p a r a t e e x p e r i m e n t s , each w i t h 3--5 replicate samples per c o n d i t i o n . Ordinate: % inhibition; abscissa: c o n c e n t r a t i o n o f p h e n o x y b e n z a m i n e (pM).
PHENOXYBENZAMINE INHIBITION OF DOPAMINE RECEPTORS
and temperatures of preincubation with phenoxybenzamine on subsequent stimulation by excess dopamine. At ice temperature, inhibition required high concentrations of phenoxybenzamine even when preincubation was for 90 min. However, at 37°C the amount of inhibition was clearly dependent on the time of preincubation with phenoxybenzamine as well as on the concentration of this agent, with half-maximal inhibition occurring at about 2 pM when preincubation was for 10 min. Fig. 2 shows the results of competition experiments. Four drugs were tested for their abilities to prevent the inhibition by phenoxybenzamine. Different concentrations of each agonist were added along with phenoxybenzamine at the start of the preincubation period. Dopamine was the most effective at reducing the inhibition (ECs0 ~ 20 pM). Norepinephrine was 7--8 times less effective than dopamine (ECs0 ~ 150 pM), and neither the a-adrenergic agonist methoxamine nor the fl-adrenergic agonist isoproterenol appeared to have any effect at concentrations up to 500 pM.
METHOXAMINE
,oo
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Fig. 2. P r o t e c t i o n of d o p a m i n e - s t i m u l a t e d a d e n y l a t e cyclase f r o m i n h i b i t i o n b y p h e n o x y b e n z a m i n e . T h e agents i n d i c a t e d were a d d e d a few s e c o n d s prior t o p h e n o x y b e n z a m i n e , a n d s a m p l e s were p r e i n c u b a t e d for 10 m i n at 37°C. O t h e r details are as d e s c r i b e d in fig. 1 a n d Materials a n d m e t h o d s . Ordinate: % inhibit i o n b y p h e n o x y b e n z a m i n e ; abscissa: c o n c e n t r a t i o n of agonist (pM).
233
4. Discussion Early behavioral investigations which suggested that dopaminergic neurotransmission might involve a unique catecholamine receptor system have now been supplemented by studies of dopamine-stimulated adenylate cyclase activity as well as by studies of the direct binding of labeled pharmacological agonists and antagonists of dopamine. Our present studies indicate that phenoxybenzamine can inhibit dopamine-stimulated adenylate cyclase at quite high potency (ICs0 ~ 2 pM). Others have shown that this agent, but not ~-adrenergic receptor blockers, can block both the behavioral effects presumably mediated by nigro-striatal dopamine projections in the rat (And~n et al., 1966) and the specific binding of [3H]haloperidol to striatal membrane preparations (Burt et al., 1976). Furthermore, phentolamine, another a-adrenergic antagonist, has been shown to block both the dopamine-stimulated adenylate cyclase activity (Kebabian et al., 1972) and the direct binding of [3H]haloperidol (Howlett and Nahorski, 1978). Thus, the combined results using these three methods of receptor evaluation appear to strongly support the conclusion that a-blockers can interact with dopamine receptors. Although the potency with which phenoxybenzamine blocks the dopamine-stimulated adenytate cyctase is less than that %ypicatly reported in the blockade of a-adrenergic receptors by this drug, the effects occur at sufficiently low concentrations as to generate possible confusion in receptor identification. (Moreover, the potency appears to increase with increasing time of exposure to the drug (fig. 1), providing the possibility that an even greater potency might prevail in vivo). Whether or not the various methods of studying dopamine receptors are, in fact, reflecting the same populations of receptors, the evidence is compelling that unless experimental conditions permit a careful evaluation of the potencies, blockade of an effect by these two a-blockers would not constitute
234
sufficient evidence to infer a rote of a-adrenergic receptors. Two out of the many examples of experiments in which consideration of the susceptibility of dopaminergic receptors to inhibition by these a-blockers might have altered the conclusions are represented by recent studies on phenylethylamineinduced stereotyped behavior in rats (Borison and Diamond, 1978} and studies concerning the ability of phenoxybenzamine to attenuate the retrograde amnesia produced by several treatments in the rat (Gold and Sternberg, 1978). The present finding of a sharp dependence of inhibition on time and temperature of preincubation with phenoxybenzamine, along with the ability of dopamine to protect against inhibition only when added during (not after) the preincubation, suggests the following interpretation of the results. Phenoxybenzamine competes with dopamine for its initial interaction with the dopaminebinding site. During the 10-min preincubation in the absence of dopamine, phenoxybenzamine first binds to this specific site, then alkylates a reactive group within or very close to the site so that the inhibition is essentially irreversible. The observations that dopamine affords far better protection against inhibition than does norepinephrine, and that other, supposedly selective, a- and fi-adrenergic agonists seem to provide no protection at all, argues against the possibility that phenoxybenzamine might exert its effects on the dopamine receptor indirectly through either an a- or a fi-adrenergic system. These competition experiments, in fact, appear to reinforce other studies which have indicated that the properties of the dopamine-stimulated adenylate cyclase are distinguishable from those of the a- and ~adrenergic receptor systems (Kebabian et al., 1972; and reviews by Goldberg, 1975 and Iversen, 1975). The failure of methoxamine, a putatively selective a-adrenergic agonist, to
K.G. WALTON ET AL.
protect against phenoxybenzamine inhibition of the dopamine-stimulated adenylate cyclase may also suggest that the impression of a close similarity between dopamine receptors and a-adrenergic receptors could be due merely to a lack of specificity of the antagonists used. References And~n, N.E., A. Dahlstr6m, K. Fuxe and K. Larsson, 1966, Functional role of the nigro-neostriatal dopamine neurons, Acta Pharmacol. Toxicol. 24, 263. Borison, R.L. and B.I. Diamond, 1978, A new animal model forschizophrenia: interactions with adrenergic mechanisms, Biol. Psychiat. 13,217. Burt, D.R., I. Creese and S.H. Snyder, 1976, Properties of [3H]haloperidol and [3H]dopamine binding associated with dopamine receptors in calf brain membranes, Mol. Pharmacol. 12, 800. Gold, P.E. and D.B. Sternberg, 1978, Retrograde amnesia produced by several treatments: evidence for a common neurobiological mechanism, Science 201,367. Goldberg, L.I., 1975, The dopamine vascular receptor, Biochem. Pharmacol. 24,651. Howlett, D.R. and S.R. N'ahorski, 1978, A comparative study of [3H]haloperidol and [3H]spiroperidol binding to receptors on rat cerebral membranes, F.E.B.S. Lett. 87, 152. Harris, J.E.: 1976; ~-Adrenergic receptor-mediated adenosine cyclic 3',5'-monophosphate accumulation in the rat corpus striatum, Mol. Pharmacol. 12, 546. Iversen, L.L., 1975, Dopamine receptors in the brain, Science 188, 1084. Kebabian, J.W., G.L. Petzold and P. Greengard, 1972, Dopamine-sensitive adenylate cyclase in caudate nucleus of rat brain, and its similarity to the "dopamine receptor", Proc. Nat. Acad. Sci. U.S.A. 69, 2145. Nickerson, M. and B. Collier, 1975, Drugs inhibiting adrenergic nerves and structures innervated by them, in: The Pharmacological Basis of Therapeutics, eds. L.S. Goodman and A. Gilman (MacMilland Co., New York) p. 533. Walton, K.G. and R.J. Baldessarini, 1976, Effects of Mn 2+ and other divalent cations on adenylate cyclase activity in rat brain, J. Neurochem. 27, 557.
European Journal of Pharmacology, 52 (1978) 231--234 © Elsevier/North-Holland Biomedical Press
Short communication I N H I B I T I O N O F D O P A M I N E - S T I M U L A T E D A D E N Y L A T E C Y C L A S E A C T I V I T Y BY PHENOXYBENZAMINE * KENNETH G. WALTON, PETER LIEPMANN and ROSS J. BALDESSARINI Laboratories for Psychiatric Research, Mailman Research Center, McLean Division of Massachusetts General Hospital, and Department of Psychiatry, Harvard Medical School, Belmont, Massachusetts 021 78, U.S.A.
Received 18 August 1978, accepted 12 September 1978
K.G. WALTON, P. LIEPMANN and R.J. BALDESSARINI, Inhibition ofdopamine-stimulated adenylate cyclase activity by phenoxybenzamine, European J. Pharmacol. 52 (1978) 231--234. Phenoxybenzamine at a concentration of 2 x 10 -6 M produced half-maximal inhibition of dopamine-stimulated adenylate cyclase activity in homogenates of rat striatum. Inhibition was sharply dependent on time and temperature of preincubation with the inhibitor. When included in the preincubation medium, dopamine was nearly 8 times more effective than norepinephrine at protecting against this inhibition, whereas neither isoproterenol nor methoxamine appeared to protect at all. These results suggest a direct, and possibly irreversible, interaction of phenoxybenzamine with the dopamine-binding component of the adenylate cyclase. ~-Adrenergic receptors Adenylate cyclase
Dopamine receptors Catecholamines
1. I n t r o d u c t i o n D e s p i t e i n d i c a t i o n s t h a t s u p p o s e d l y selective a - a d r e n e r g i c r e c e p t o r a n t a g o n i s t s c a n have effects on a variety of hormone and drug r e c e p t o r sites, including t h o s e for h i s t a m i n e , serotonin and acetylcholine (Nickerson and Collier, 1 9 7 5 ) , p h e n o x y b e n z a m i n e a n d p h e n t o l a m i n e c o n t i n u e t o be used t o h e l p establish the m e d i a t i o n o f various e f f e c t s b y a - a d r e n e r gic r e c e p t o r s (e.g., B o r i s o n a n d D i a m o n d , 1 9 7 8 ; G o l d a n d S t e r n b e r g , 1978). This use o f such " a - b l o c k e r s " has b e e n b a s e d o n t h e i m p r e s s i o n t h a t t h e y are q u i t e selective a m o n g c a t e c h o l a m i n e r e c e p t o r s ( N i c k e r s o n a n d Colllier, 1 9 7 5 ) . H o w e v e r , t h e r e c e n t e m e r g e n c e o f
* Supported in part by U.S. Public Health Service Grants: MH-30511, MH-31154, the Scottish Rite Benevolent Foundation, Northern Masonic Jurisdiction of the U.S.A., N.I.H. Career Research Scientist Award MH-47370 (RJB) and a Fellowship from The Charles A. King Trust, Boston (KGW).
Phenoxybenzamine
Striatum
d o p a m i n e r e c e p t o r s as a n e w class o f r e c e p t o r f o r c a t e c h o l a m i n e s raises t h e q u e s t i o n of w h e t h e r t h e classical a - a n t a g o n i s t s m i g h t i n t e r a c t at these sites as well. A l t h o u g h t h e r e has b e e n s o m e discussion as to w h e t h e r t h e r e are r e c e p t o r s f o r d o p a m i n e e n t i r e l y s e p a r a t e f r o m the classical a - a n d / 3 - a d r e n e r g i c r e c e p t o r s (Harris, 1 9 7 6 ) , t h e e v i d e n c e in s u p p o r t o f such a s e p a r a t e class is increasingly c o m p e l l i n g (see reviews b y Iversen, 1 9 7 5 and G o l d b e r g , 1975). O n e t e c h n i q u e t h a t is c o m m o n l y e m p l o y e d to s t u d y d o p a m i n e r e c e p t o r - m e d i a t e d e f f e c t s is b a s e d o n t h e t h e o r y t h a t d o p a m i n e - s t i m u l a t e d a d e n y l a t e cyclase is a p a r t o f t h e recept o r - e f f e c t o r s y s t e m f o r this c a t e c h o l a m i n e ( K e b a b i a n et al., 1972). We have, a c c o r d i n g l y , used this r e a c t i o n to evaluate a possible antid o p a m i n e r g i c a c t i o n o f t h e classical aadrenergic antagonist, phenoxybenzamine, a n d h a v e f o u n d t h a t such an e f f e c t occurs at pM c o n c e n t r a t i o n s o f t h e drug. We also p r e s e n t e v i d e n c e suggesting t h a t this b l o c k a d e
232
is irreversible. Our results thus emphasize the risks that could be encountered in using the effects of such non-selective a-antagonists to support the nature of the receptor involved in a pharmacological effect, even among catecholamine receptors.
K.G. W A L T O N ET AL.
chloride (Vasoxyl) was the generous gift of Burroughs-Wellcome. All other reagents were of certified or reagent grade. Catecholamines were dissolved in cold, nitrogen-gassed deionized water just before use.
3. Results 2. Materials and methods The striata of brains from 200 g male Sprague-Dawley rats were dissected immediately following sacrifice by decapitation, as described previously (Walton and Baldessarini, 1976). Striata from 2 or 3 rats were pooled and weighed in a preweighed beaker containing 2.0 ml of ice-cold 2 mM Tris-(hydroxymethyl)aminomethane maleate (Tris-maleate), pH 7.4, in 2 mM EGTA; the volume of buffer was then adjusted to give 30 times the tissue weight. Striata and buffer were transferred to an ice-cold glass Potter--Elvejhem homogenizer, homogenized by hand with a Teflon pestle, and stored in ice until use. The assay of adenylate cyclase activity was as described previously (Walton and Baldessarini, 1976) except for slight modifications made necessary by the time-dependent nature of the inhibition by phenoxybenzamine. Thus, phenoxybenzamine (with or without a potential competitor) was added to each assay tube containing homogenate in the usual assay buffer minus ATP. Following a preincubation period (conditions indicated in the figure legends), a final concentration of 250 #M dopamine was added to some of the tubes and the reaction was started by addition of ATP. Incubation was for 2.5 min at 37°C in a total volume of 500 pl. Cyclic AMP values were determined from aliquots of the assay mixture using the adrenal binding-protein method. Phenoxybenzamine hydrochloride (Dibenzyline; kindly donated by Smith, Kline and French) was dissolved in acidified ethanol and added to the incubation mixture so that the concentration of ethanol never exceeded 1%. An equal volume of acidified ethanol was added to controls. Methoxamine hydro-
In preliminary experiments we found that preincubation of striatel homogenates with micromotar concentrations of phenoxybenzamine reduced the subsequent stimulation of adenylate cyclase which was produced by excess dopamine (250 pM). Furthermore, we found that when this concentration of dopamine was added prior to or along with phenoxybenzamine, the inhibition did not occur. We then extended these investigations in more detailed experiments. Fig. 1 shows the effects of various times
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i
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Fig. 1. Effect of phenoxybenzamine on dopaminestimulated adenyiate cyclase activity. Homogenates o f rat s t r i a t u m , p r e i n c u b a t e d w i t h p h e n o x y b e n z a m i n e in t h e a b s e n c e o f A T P (times a n d t e m p e r a t u r e s indicated), were i n c u b a t e d 2.5 m i n a t 37°C following t h e a d d i t i o n o f A T P (1.5 m M ) + - d o p a m i n e ( 2 5 0 p M ) . O t h e r details are given in Materials a n d M e t h o d s . O n l y t w o e x p e r i m e n t s involving p r e i n c u b a t i o n at 0 - - 2 ° C were carried o u t ; t h e m e a n s are s h o w n . All o t h e r values a n d bars r e p r e s e n t m e a n s _+ S.E.M. for 3--6 s e p a r a t e e x p e r i m e n t s , each w i t h 3--5 replicate samples per c o n d i t i o n . Ordinate: % inhibition; abscissa: c o n c e n t r a t i o n o f p h e n o x y b e n z a m i n e (pM).
PHENOXYBENZAMINE INHIBITION OF DOPAMINE RECEPTORS
and temperatures of preincubation with phenoxybenzamine on subsequent stimulation by excess dopamine. At ice temperature, inhibition required high concentrations of phenoxybenzamine even when preincubation was for 90 min. However, at 37°C the amount of inhibition was clearly dependent on the time of preincubation with phenoxybenzamine as well as on the concentration of this agent, with half-maximal inhibition occurring at about 2 pM when preincubation was for 10 min. Fig. 2 shows the results of competition experiments. Four drugs were tested for their abilities to prevent the inhibition by phenoxybenzamine. Different concentrations of each agonist were added along with phenoxybenzamine at the start of the preincubation period. Dopamine was the most effective at reducing the inhibition (ECs0 ~ 20 pM). Norepinephrine was 7--8 times less effective than dopamine (ECs0 ~ 150 pM), and neither the a-adrenergic agonist methoxamine nor the fl-adrenergic agonist isoproterenol appeared to have any effect at concentrations up to 500 pM.
METHOXAMINE
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Fig. 2. P r o t e c t i o n of d o p a m i n e - s t i m u l a t e d a d e n y l a t e cyclase f r o m i n h i b i t i o n b y p h e n o x y b e n z a m i n e . T h e agents i n d i c a t e d were a d d e d a few s e c o n d s prior t o p h e n o x y b e n z a m i n e , a n d s a m p l e s were p r e i n c u b a t e d for 10 m i n at 37°C. O t h e r details are as d e s c r i b e d in fig. 1 a n d Materials a n d m e t h o d s . Ordinate: % inhibit i o n b y p h e n o x y b e n z a m i n e ; abscissa: c o n c e n t r a t i o n of agonist (pM).
233
4. Discussion Early behavioral investigations which suggested that dopaminergic neurotransmission might involve a unique catecholamine receptor system have now been supplemented by studies of dopamine-stimulated adenylate cyclase activity as well as by studies of the direct binding of labeled pharmacological agonists and antagonists of dopamine. Our present studies indicate that phenoxybenzamine can inhibit dopamine-stimulated adenylate cyclase at quite high potency (ICs0 ~ 2 pM). Others have shown that this agent, but not ~-adrenergic receptor blockers, can block both the behavioral effects presumably mediated by nigro-striatal dopamine projections in the rat (And~n et al., 1966) and the specific binding of [3H]haloperidol to striatal membrane preparations (Burt et al., 1976). Furthermore, phentolamine, another a-adrenergic antagonist, has been shown to block both the dopamine-stimulated adenylate cyclase activity (Kebabian et al., 1972) and the direct binding of [3H]haloperidol (Howlett and Nahorski, 1978). Thus, the combined results using these three methods of receptor evaluation appear to strongly support the conclusion that a-blockers can interact with dopamine receptors. Although the potency with which phenoxybenzamine blocks the dopamine-stimulated adenytate cyctase is less than that %ypicatly reported in the blockade of a-adrenergic receptors by this drug, the effects occur at sufficiently low concentrations as to generate possible confusion in receptor identification. (Moreover, the potency appears to increase with increasing time of exposure to the drug (fig. 1), providing the possibility that an even greater potency might prevail in vivo). Whether or not the various methods of studying dopamine receptors are, in fact, reflecting the same populations of receptors, the evidence is compelling that unless experimental conditions permit a careful evaluation of the potencies, blockade of an effect by these two a-blockers would not constitute
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sufficient evidence to infer a rote of a-adrenergic receptors. Two out of the many examples of experiments in which consideration of the susceptibility of dopaminergic receptors to inhibition by these a-blockers might have altered the conclusions are represented by recent studies on phenylethylamineinduced stereotyped behavior in rats (Borison and Diamond, 1978} and studies concerning the ability of phenoxybenzamine to attenuate the retrograde amnesia produced by several treatments in the rat (Gold and Sternberg, 1978). The present finding of a sharp dependence of inhibition on time and temperature of preincubation with phenoxybenzamine, along with the ability of dopamine to protect against inhibition only when added during (not after) the preincubation, suggests the following interpretation of the results. Phenoxybenzamine competes with dopamine for its initial interaction with the dopaminebinding site. During the 10-min preincubation in the absence of dopamine, phenoxybenzamine first binds to this specific site, then alkylates a reactive group within or very close to the site so that the inhibition is essentially irreversible. The observations that dopamine affords far better protection against inhibition than does norepinephrine, and that other, supposedly selective, a- and fi-adrenergic agonists seem to provide no protection at all, argues against the possibility that phenoxybenzamine might exert its effects on the dopamine receptor indirectly through either an a- or a fi-adrenergic system. These competition experiments, in fact, appear to reinforce other studies which have indicated that the properties of the dopamine-stimulated adenylate cyclase are distinguishable from those of the a- and ~adrenergic receptor systems (Kebabian et al., 1972; and reviews by Goldberg, 1975 and Iversen, 1975). The failure of methoxamine, a putatively selective a-adrenergic agonist, to
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protect against phenoxybenzamine inhibition of the dopamine-stimulated adenylate cyclase may also suggest that the impression of a close similarity between dopamine receptors and a-adrenergic receptors could be due merely to a lack of specificity of the antagonists used. References And~n, N.E., A. Dahlstr6m, K. Fuxe and K. Larsson, 1966, Functional role of the nigro-neostriatal dopamine neurons, Acta Pharmacol. Toxicol. 24, 263. Borison, R.L. and B.I. Diamond, 1978, A new animal model forschizophrenia: interactions with adrenergic mechanisms, Biol. Psychiat. 13,217. Burt, D.R., I. Creese and S.H. Snyder, 1976, Properties of [3H]haloperidol and [3H]dopamine binding associated with dopamine receptors in calf brain membranes, Mol. Pharmacol. 12, 800. Gold, P.E. and D.B. Sternberg, 1978, Retrograde amnesia produced by several treatments: evidence for a common neurobiological mechanism, Science 201,367. Goldberg, L.I., 1975, The dopamine vascular receptor, Biochem. Pharmacol. 24,651. Howlett, D.R. and S.R. N'ahorski, 1978, A comparative study of [3H]haloperidol and [3H]spiroperidol binding to receptors on rat cerebral membranes, F.E.B.S. Lett. 87, 152. Harris, J.E.: 1976; ~-Adrenergic receptor-mediated adenosine cyclic 3',5'-monophosphate accumulation in the rat corpus striatum, Mol. Pharmacol. 12, 546. Iversen, L.L., 1975, Dopamine receptors in the brain, Science 188, 1084. Kebabian, J.W., G.L. Petzold and P. Greengard, 1972, Dopamine-sensitive adenylate cyclase in caudate nucleus of rat brain, and its similarity to the "dopamine receptor", Proc. Nat. Acad. Sci. U.S.A. 69, 2145. Nickerson, M. and B. Collier, 1975, Drugs inhibiting adrenergic nerves and structures innervated by them, in: The Pharmacological Basis of Therapeutics, eds. L.S. Goodman and A. Gilman (MacMilland Co., New York) p. 533. Walton, K.G. and R.J. Baldessarini, 1976, Effects of Mn 2+ and other divalent cations on adenylate cyclase activity in rat brain, J. Neurochem. 27, 557.
