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3H-catecholamine binding to α-receptors in rat brain: Enhancement by reserpine
European Journal of Pharmacology, 51 (1978) 145--155
145
© Elsevier/North-Holland Biomedical Press
3H-CATECHOLAMINE BINDING TO a-RECEPTORS IN RAT BRAIN: ENHANCEMENT BY RESERPINE DAVID C. U'PRICHARD and SOLOMON H. SNYDER Departments of Pharmacology and Experimental Therapeuritcs and Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, U.S.A.
Received 28 February 1978, revised MS received 8 May 1978, accepted 9 June 1978
D.C. U'PRICHARD and S.H. SNYDER, 3H-Catecholamine binding to a-receptors in rat brain: enhancement by reserpine, European J. Pharmacol. 51 (1978)145--155. (±)-3H-Epinephrine and (--)-3H-norepinephrine bind to rat cortex membranes in a saturable manner with dissociation constants ~of 16.7 and 27 nM respectively. The maximum number of 3H-catecholamine binding sites, 10--12 pmoles/g tissue, and the pharmacological characteristics of (±)-3H-epinephrine binding, indicate that the catecholamines label the same a-noradrenergic receptor in the rat as does 3H-clonidine. At 25 °, (±)-3H-epinephrine binding associates rapidly to equilibrium, and dissociates in a biphasic manner. The affinities of a-agonists at the 3H-eatecholamine binding site are 2--4 fold weaker in the rat than in the calf cortex under the same experimental conditions. Ergot alkaloids and phenoxybenzamine have similar affinities in the two tissues, whereas phentolamine and WB-4101 are 8--10 times weaker in the rat. Reserpine (0.25 mg/kg s.c. per day for 3 weeks) causes 25 and 46% increases in the numbers of (-+)-3H-epinephrine and 3H-WB-4101 a-receptor binding sites respectively, and a 51% increase in the number of 3H-dihydroalprenolol fi-recepto~ sites, in rat forebrain. Reserpine pretreatment does not alter the affinities of either a- or fi-receptor 3H-ligands. Catecholamines
Adrenoceptor binding
Rat brain
1. Introduction a-Noradrenergic receptor sites in brain and peripheral tissues can be labelled by a variety of ligands, including 3H~lihydroergokryptine (Williams and Lefkowitz, 1976; Greenberg and Snyder, 1977; Kafka et al., 1977), the antagonist 3H-WB-4101 (2-( [ 2',d'-dimethoxy]p h e n o x y e t h y l a m i n o ) m e t h y l b e n z o d i o x a n ) (U'Prichard et al., 1977a) and the agonists 3Hclonidine (U'Prichard et ai., 1977a) (--)-3Hnorepinephrine and (+)-3H-epinephrine (U'Prichard and Snyder, 1977a). Several binding studies in rats show that the numbers o f noradrenergic receptors can be altered in a variety of tissues by chronic treatment with certain drugs and hormones (Williams and Lefkowitz, 1977a, b; Glaubiger and Lefkowitz, 1977). Reserpine causes postjunctional supersensi-
Reserpine
tivity in the nictitating membrane (Trendelenburg, 1966), brain (Ungerstedt et al., 1975) and cardiovascular tissue (Carrier, 1975). Reports have differed as to whether reserpine supersensitivity is receptor specific (Broadley and Lumley, 1977) or a non-specific calcium mobilization effect (Carrier, 1975). In the brain, chronic reserpine treatment increases adenylate cyclase stimulation by norepinephfine and isoproterenol (Dismukes and Daly, 1974; Williams and Pitch, 1974; Palmer et ai., 1976) but not by dopamine (Rotrosen et al., 1975), although Burr et al. (1977) reported an increased number o f dopamine receptor binding sites labeled by 3H-haioperidol following reserpine. We sought to determine if chronic reserpine similarly increased a-noradrenergic receptor sites. Properties of a-receptors in terms of relative drug potencies vary depending on whether
146 receptor sites are labelled with 3H-agonists or 3H-antagonists (U'Prichard et al., 1977a; U'Prichard and Snyder, 1977b; Greenberg and Snyder, 1978; Peroutka et al., 1978). For studies of agonist-preferring sites, 3H-catecholamines are often preferable to the use of 3Hclonidine, because the 3H-catecholamines are more readily available commercially, and because the absolute amount of specific binding, and the ratio of specific to non-specific binding, is greater with these compounds than with 3H-clonidine (U'Prichard and Snyder, 1977b). Relative potencies of drugs in competing for (+)-3H-epinephrine and (--)-3Hnorepinephrine binding to a-receptors in calf brain membranes differ somewhat from their effects on rat brain a-receptor sites as labeled by 3H-clonidine. Since studies of the regulation in vivo of neurotransmitter receptors are most efficiently conducted in rodents, we desired to compare properties of (+)-3Hepinephrine and (--)-3H-norepinephrine binding to a-receptors in rats with properties previously described in calf (U'Prichard and Snyder, 1977b). In the present study we characterize (--)-3H-norepinephrine and (+)3H-epinephrine labeling of a-receptor in rat cerebral cortex and report differences between receptor properties in rat and calf. Additionally, we report an enhancement of a - a n d t3receptor binding following chronic reserpine treatment.
2. Materials and methods 2.1. Measurement o f (+)-3H-epinephrine and (--)-3H-norepinephrine binding in rat cortex
Fresh cerebral cortices from adult male Sprague-Dawley rats were homogenized in 20 volumes of ice-cold 50 mM Tris-HC1 buffer, pH 7.7 at 25°C, with a Brinkmann Polytron PT-10. The homogenates were centrifuged twice at 50,000 × g for 10 min, with intermediate rehomogenization in fresh buffer. Final pellets were resuspended in 22 volumes of Tris-HC1 buffer, pH 7.7 at 25°C, and incu-
D.C. U'PRICHARD, S.H. SNYDER bated with 1.0 pM pargyline for 15 min at 25°C. Triplicate incubation tubes received 20 pl of (+)-3H-epinephrine (D,L-[7-3H]epi nephrine L-bitartrate, 13.8 Ci/mmole, New England Nuclear Corp.) or (--)-3H-norepinephrine (L-[7,8-3H]norepinephrine, 24 Ci/ mmole, New England Nuclear), 10pl of various concentrations of drugs, 20 pl of a solution containing 50 mM pyrocatechol, 0.05% ascorbic acid, 5 mM disodium EDTA, and 0.5 mM dithiothreitol, and 0.45 ml of freshly resuspended tissue. Radioactive ligands and added drugs were all prepared in a 0.1% ascorbic acid solution. The final concentration of (+)-3H-epinephrine and (--)-3H-norepinephrine was 5 nM, and the final incubation volume was 0.5 ml. Tubes were incubated at 25°C for 60 min, and subsequently filtered under vacuum through Whatman GF/B filters presoaked in 1 mM pyrocatechol. The filters were rinsed with 15 ml of ice-cold 50 mM Tris-HC1 buffer, pH 7.7 at 25°C, containing 1 mM pyrocatechol and 0.1% ascorbic acid, and were counted by liquid scintillation spectrometry at 37% efficienty. Saturable of specific binding of (+_)-3H-epinephrine and (--)-3H-norepinephrine was defined as the excess over blanks containing 2.0 pM oxymetazoline. Incubation of cortex membranes ( 2 0 m g tissue, original weight) with 5 nM of either 3H-catecholamine, the routine concentration used, gave 2000 cpm total binding and 1000 cpm non-specific binding. In general, since there was relatively more non-specific binding of 3H-cateeholamines in rat than in calf cortex, the present alterations for rat cortex of the assay method previously used in calf cortex (U'Prichard and Snyder, 1977b) were designed to minimize nonspeeific binding. 3H-Catecholamines were stored and diluted as described previously (U'Prichard and Snyder, 1977b). 2.2. Chronic reserpine treatments
Adult male Sprague-Dawley rats, 150-175 g, were injected with 0.25 mg/kg reserpine (Serpasil, Ciba) s.c. daily for 3 weeks.
RESERPINE ENHANCES BRAIN NE RECEPTOR BINDING
Control animals received equivalent volumes of saline. The animals were killed on the day after the last injection, and the forebrains (area rostral to the colliculi) frozen rapidly and stored at --20°C. For binding assays, suspensions of forebrain tissue were prepared as described above, except that the final resuspension was in 44 volumes of Tris-HC1 buffer, pH 7.7 at 25°C, and 10 mg tissue, original wet weight, was added to the assay. (+)-all-epinephrine binding to a-receptors was assayed as above, and aH-WB-4101 and 3H-dihydroergokryptine binding to a-receptors and 3Hdihydroalprenolol binding to r-receptors was determined in the same samples as described previously (U'Prichard et al., 1977a; Greenberg and Snyder, 1977; Bylund and Snyder, 1976). Compared to fresh tissue controls, frozen rat forebrains showed a loss in affinity for (-+)-all-epinephrine, but not for aH-WB4101 or aH-dihydroalprenolol. 2.3. Drugs WB-4101 was custom tritiated to a specific activity of 11.7 Ci/mmole at New England Nuclear (U'Prichard et al., 1977a). 3H Dihydroalprenolol, 48--50 Ci/mmole, was obtained from New England Nuclear. Catecholamines and phenylephrine were donated by SterlingWinthrop, clonidine by Boehringer-Ingelheim, oxymetazoline by Schering, ergot alkaloids by Sandoz, phentolamine by Ciba, WB-4101 by Ward Blenkinsop, phenoxybenzamine by Smith Kline and French, and indoramin by Wyeth. Other drugs and compounds were obtained from the pharmaceutical company of origin or commercial sources.
3. Results
3.1. Saturation and kinetics o f (+_)-3H-epinephrine and (--)-aH-norepinephrine binding to rat cerebral cortex membranes The specific binding of (+)-3H-epinephrine to rat cerebral cortex membranes was a satur-
147
able process. At concentrations up to 10 nM, specific binding of (+)-3H-epinephrine equalled non-specific binding, while at higher concentrations non-specific binding increased more rapidly. There was a linear relationship between the amount of nonspecific binding and concentration of (+)-3H-epinephrine (fig. 1). Specific (+)-all-epinephrine binding plateaued at 25--40 nM concentration and reached half maximal binding at approximately 10 nM. Scatchard analysis (line of best fit determined by linear regression analysis) revealed a single population of binding sites with a dissociation constant ( K D ) of 10.7 nM and a maximal number of binding sites (Brnax) of 11.6 pmoles/g original wet weight of tissue (fig. 1). In three similar experiments, the mean values +S.E.M. for KD and Bmax were 16.7 +- 5.6 nM and 12.0 + 0.4 pmoles/g respectively. In general it was found that, whereas the Bmax values determined by regression analysis were relatively invariant from experiment to experiment, the K D v a l u e s were more ~z)oo
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[aH]-epinephrine bound (cpm); abscissa: [3H]-epinephrine concentration (nM). (+)3H-Epinephrine binding in the rat cortex as a function of increasing concentrations of (-+)-3H-epinephrine. Homogenates (20 mg tissue, original weight) were incubated for 60 min at 25°C as described in Materials and methods with varying concentrations of (+)-all-epinephrine. Non-specific binding was determined b y the addition of 2.0 ~ oxymetazoline. Points shown are from one o f three similar experiments, each performed in triplicate. Inset: Scatchard plot of binding data was determined by linear regression analysis. Fig. 1.
Ordinate:
148
variable, reflecting the variation in stability of the (+)-3-epinephrine. Specific (--)-3H-norepinephrine binding in the rat cortex displayed saturation characteristics similar to that of (+)-3H-epinephrine. Ratios of specific and non-specific binding at various concentrations of (--)-3H-norepinephrine were similar to (+)-3H-epinephrine. Scatchard analysis o f (--)-3H-norepinephrine saturation data indicated a single population of sites, with a KD value for (--)-3H-norepinephrine binding of 27 nM, while the Bmax value was 10 pmole/g. Since a-receptor binding of (±)-3H-epinephrine and (--)-3H-norepinephrine was stereospecific and (--)-3H-norepinephrine employed, while the racemic form of 3H-epinephrine was utilized, the actual affinity of norepinephrine was only about one third of that of epinephrine for a-receptors in rat brain. In other experiments the kinetic properties of (+)-3H-epinephrine binding in rat cortex were examined. At 25°C, (+)-3H-epinephrine specific binding to cerebral cortex membranes of the rat increased to a plateau at about 200 min. Half maximal binding was obtained at about 3 min (fig. 2A). Nonspecific (+_)-3H-epinephrine binding also displayed a gradual time course which increased to a plateau at about 50 min. Thus in routine experiments, an incubation time of 60 min at 25°C was used to ensure steady-state conditions for both specific and nonspecific binding. Association of specific (±)-3H-epinephrine binding was hyperbolic and followed pseudo-first order kinetics. The observed rate of association (kob) was 0 . 1 9 r a i n -1. The calculated rate constant of association (k~) was 0.008 nM -1 min -~. These properties differ from the specific binding of (±)-3H-epinephrine at 25°C to calf cerebral cortex membranes (U'Prichard and Snyder, 1977b). In the calf, association of specific binding is nonhyperbolic, and at 25°C is biphasic when plotted on a semilogarithmic scale. The value of kl for association of (+)-3H-epinephrine binding in rat cerebral cortex could not be determined using the equation k~ = (kob--
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[3H]-epinephrine bound (cpm); abscissa: time (min). (A) Association at 25°C of (_+).3H-epinephrine binding to rat cortex homogenates (20 mg tissue, original weight). Specific and nonspecific binding, as defined in Materials and methods, Ordinate:
was determined at various time intervals following the addition of 5 nM (±)-3H-epinephrine. Points shown are from one of three similar experiments, each performed in triplicate. Inset: pseudo-first order kinetic plo_t of initial (+-)-3H-epinephrine binding. B and Be are the amounts of (-+)-3H-epinephrine specifically bound at time t and at equilibrium respectively. Slope is equal to kob, the observed rate constant. (B) Ordinate: [3H]-epinephrine specifically bound (B/B0 × 100); abscissa: time (min). Dissociation at 25°C of (±)-3H-epinephrine binding, after incubation to equilibrium for 60 min. Specific binding was determined at various time intervals following the addition of 2.0 pM oxymetazoline at time zero. Points shown are from one of three experiments, each performed in triplicate. k 1)/[(+)-3H-epinephrine], since kob was less than the initial k_ 1. Association at time points of less than one minute were not examined in the rat cortex, and possible the kob for initial association may be greater if the semilogarithmic plot were nonlinear, with a steep initial slope. Assuming the irrevers-
RESERPINE ENHANCES BRAIN NE RECEPTOR BINDING
ibility of the forward reaction, kl could be determined using the equation k~ = [1/t(F--R)]ln[R(F--B)/F(R--B)] (see table 1).
TABLE 1 Comparison of binding constants of 3H-catecholamines labeling a-noradrenergic receptors in rat and calf cerebral cortex membranes at 25°C. The constants for S H-catecholamine binding to rat cortex homogenates (20 rag, original tissue weight) were obtained as described in the text. Constants for calf cortex binding were obtained from U'Prichard and Snyder (1977b). (±).3 H-Epinephrine Rat
(_).3 HNorepinephrine
Calf Rat Calf
kl (nM -I rain -1 ) ~ tl/2 (association) min k_ 1 ( m i n - I ) 2 tl / 2 (dissociation) (rain) 2 K D (kinetic) (nM) 3 K D (equilibrium) (nM) 4 Bmax (pmol/g tissue)
0.008 3 0.35, 0.031
0.04 5 0.24
----
0.06 5 0.24
2, 24 44, 4
3.3 6.2
---
2 4.0
17 12
6.5 8.9
27 10
9.7 7.0
I The association rate constant for (±)-3H-epinephrine binding in the rat cortex was determined by fitting the equation kl = [ 1 / t ( F - - R)] In [R(F -- B ) / F ( R - - B)], where F is the initial concentration o f (±)-3H-epinephrine, R is the receptor concentration and B is the concentration o f specifically bound (_+)-3H-epinephrine at time t. The equation kl = (kob -- k-1 )/[(+).3 H-epinephrine], which takes ongoing ligand dissociation into account and was used to calculate the calf association rate constants, cound not be successfully fitted to the rat data. 2 Dissociation of 3 H-eatecholamine specific binding to a-receptors at 25°C was biphasic for both rat and calf cortex. The first k_ 1 and t l / 2 values are for the initial phase of dissociation, and the second values are for the slow dissociation phase, 3 The KD values obtained from kinetic experiments equal k_] (fast)/kl and k-1 (slow)/kt. 4 Obtained from saturation experiments.
149
The value thus obtained is very approximate, since considerable dissociation does occur during the rising portion of the association curve (fig. 2). The apparent association constant for (+)-3H-epinephrine binding to areceptors is four times smaller in the rat cortex than in the calf cortex (table 1). To examine the dissociation of specifically bound (+)-3H-epinephrine, rat cerebral cortex membranes were labelled with (+)-3H-epinephrine at 25°C for 60 min, at which time 2.0 /~M oxymetazoline was added and binding was examined at subsequent time intervals. When plotted on a semilogarithmic scale the dissociation rate was biphasic (fig. 2B). For the initial phase of dissociation, during which, in repeated experiments, 50--70% of the ligand was released, the half life was 2 min and the calculated rate constant for dissociation (k_l) was 0.35 min -1. The slower phase of dissociation had a half life of about 23 min, and a k I of 0.031 min -1. These findings resemble results in calf cerebral cortex membranes, in which dissociation of specifically bound (+)-3H-epinephrine at 25°C, after labelling at 25°C, also was biphasic with a similar initial phase k_~ (table 1). The dissociation constants ( K D ) for the fast and slow dissociating phases of (_+)-all-epinephrine binding to rat cortex a-receptors, derived from kinetic experiments (k_~/k~), were 44 nM and 4 nM. The equilibrium value for K D lies midway between these values (table 1). Such biphasic dissociation plots have also been observed with other labelled agonist ligands, such as 3H-protaglandin E, (Lefkowitz et al., 1977). Both here and for calf brain 3H-catecholamine a-receptor binding (U'Prichard and Snyder, 1977b), it has been suggested that the 3H-agonist, during the course of receptor labelling, converts some receptors to a higheragonist-affinity (desensitized) state. If the higher- and lower-affinity forms are interconvertible, the Scatchard plot would be expected to yield a single line, as seen in the present study. The apparent KD thus obtained, however, would be a composite value from the high and low-affinity forms (Lefkowitz et al.,
150
D.C. U~PRICHARD, S.H. SNYDER
TABLE 2 Inhibition of (+).3 H-epinephrine binding to a-receptors in rat cortex membranes at 25 °C; comparison with calf cortex values for (_+).3H-epinephrine binding 1. Rat cortex homogenates were incubated with 5 nM (+_)-3H-epinephrine for 60 min at 25°C, together with 4--6 concentrations of unlabeled drugs, under standard assay conditions. IC50 values were determined by log-probit analysis, and apparent K i values calculated from the equation Ki = iCs0/( 1 + [(_+).3 H-epinephrine]/K D ), where the K D value used was 16.7 nM. Values given are means +- S.E. of 3 experiments, each conducted in triplicate. Drug
Rat, K i (nM)
Calf, Ki (37°C) (nM)
Calf, K i (25°C) (nM)
a-Agonists (--)-Epinephrine (+)-Epinephrine (--)-a-Me-Norepinephrine (+)-a-Me-Norepinephrine (--)-Norepinephrine (+)-Norepinephrine Dopamine (--)-Phenylephrine Clonidine Oxymetazoline
5.2 92 11.5 8,400 20 2,200 200 580 3.0 3.0
+ + _+ +_ -+ _+ + +-+ +-
0.8 15 1.8 900 5 200 40 130 1.6 0.2
6.5 97 14 16,000 24 1,000 310 370 3.3 3.3
2.1 26 3.9 4,600 6.4 260 87 140 1.3 4.7
6.3 7.1 1,400 19 340 490 36,000
+ 1.5 +1.2 +_ 200 + 6 _+ 60 _+ 140 + 13,000
5.8 8.3 3,800 1.2 16 200 12,000
9.6 5.4 5,600 2.0 41 1,100 11,000
11,000 2,800 25,000 17,000
-+ 4,000 -+ 200 +_ 4,000 +- 6,000
5,700 3,900 -21,000
6,600 1,100 -19,000
a-Antagonists Ergotamine Dihydro-~-ergokryptine Ergonovine Phentolamine WB-4101 Phenoxybenzamine Indoramin
Other drugs Serotonin (--)-Isoproterenol (+_)-Propranolol (--)-Alprenolol
1 Calf cortex values were obtained from U'Prichard and Snyder (1977b).
1 9 7 7 ) , a n d s h o u l d t h u s f a l l b e t w e e n t h e KD values obtained kineticaUy from the slow and f a s t d i s s o c i a t i o n p h a s e s , as is s e e n i n t h e p r e s e n t s t u d i e s ( t a b l e 3). T h e s p e c i f i c b i n d i n g of low (2 nM) or high (50 nM) concentrations of (+-)-3H-epinephrine shows the same a-receptor characteristics.
3.2. Influence o f drugs on specific (+)-3H-epinephrine binding to rat cortex a-receptor sites A variety of a-agonists, antagonists and other durgs were examined for their influence upon specific (+)-3H-epinephrine binding to r a t c e r e b r a l c o r t e x m e m b r a n e s (fig. 3, t a b l e
RESERPINE ENHANCES BRAIN NE RECEPTOR BINDING 200C 41,
TABLE 3
0 <'TOTAL
Effect of chronic reserpine on (_+).3H-epinephrine and 3 H-WB-4101 binding to a-receptors, and 3 H-dihydroalprenolol binding to/~-receptors, in rat forebrain. Rats were injected with reserpine (Serpasil, 0.25 rag/ kg) daily for three weeks and were killed one day after the last dose. Frozen forebrains from these animals were subsequently thawed and assayed for a-receptor binding with four concentrations of (_+)3H-epinephrine (2.5--70 nM) or 3H-WB-4101 (0.1-1.5 nM), and for ~-receptor binding with 3 H-dihydroalprenolol (0.3--3.0 nM), as described in Materials and methods. Forebrains from control rats receiving saline injections were assayed in parallel. Scatchard plots from the saturation curves for each ligand in each forebrain were obtained by linear regression analysis to determine the apparent dissociation constants (K D ) and maximum number of binding sites (Bmax). Values given are means_+ S.E. for 5 rats in each group. Values of P were determined by Student's t-test (two-tailed). 3 H-ligand
Control
Reserpine % change
31.5 _+11.3 8.5 +0.9
28.0 _+2.9 10.6 -+0.3 1
0.36 _+0.05 6.7 _+0.9
0.43 _+0.08 9.8 _+0.02 2
1.43 _+0.21 8.5
1.81 _+0.15 12.8
_+1.0
_+ 1.0 i
(+)_3H-Epinephrine K D (nM) Bmax (pmol/g tissue)
Bmax (pmol/g tissue)
+46
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151
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1 Significantly different from control, P ~ 0.05. 2 Significantly different from control, P ~ 0.01.
2). Initial experiments showed that (--)epinephrine, oxymetazoline and WB-4101 maximally inhibited (+)-all-epinephrine binding to the same extent, at concentrations of 2.0 pM or higher. All combinations of 2.0/~M concentrations of these three inhibitors caused no further loss of binding than any of the inhibitors alone. (+)-3H-Epinephrine binding was stereospecific, with (--)-epinephrine dis-
playing 18 times greater affinity than (+)epinephrine, while (--)-isomers of a-methylnorepinephrine and norepinephrine were 700 and 100 times more potent respectively than the corresponding (+)-isomers. (--)-Epinephrine displayed about four times greater affinity than (--)-norepinephrine while dopamine was only 2.6% as potent as (--)-epinephrine. Under the same incubation conditions at 25°C, (--)-epinephrine and (--)-norepinephrine were 2--3 times more potent in competing for (+)3H-epinephrine binding in calf than in rat (table 2). This agrees with the approximate three fold greater affinities of (+)-all-epinephrine and (--)-3H-norepinephrine in calf than rat membranes in saturation experiments (table 1). Other a-agonists such as phenylephrine, clonidine and oxymetazoline were also 2--3 times more potent in calf than rat membranes at 25°C. The differences in affinities of agonists for (+)-all-epinephrine binding sites in rat and calf membranes appeared to be characteristic for a-receptors in the two
152
species rather than for (+)-3-epinephrine binding alone. Thus affinities of these agents for 3H¢lonidine binding sites in rat cerebral cortex membranes were less than for 3H-clonidine binding sites in calf cerebral cortex membranes, but closely resembled affinities of the same drugs for (+)3H-epinephrine binding sites in rat membranes (U'Prichard et al., 1977a; U'Prichard and Snyder, 1977b). Among a-blocking drugs, phentolamine and WB-4101 were 8--10 times more potent in competing for (+-)3H-epinephrine binding in calf than in rat, under the same 25°C conditions. As in the case of the agonists, this difference was species rather than drug specific, as both of these agents were substantially more potent in competing for 3H-clonidine binding in calf than in rat as well. The irreversible antagonist phenoxybenzamine failed to display consistent differences in potencies between calf and rat in competing for binding of 3H-clonidine or (+)3H-epinephrine. For phenoxybenzamine, ligand-specific differences in potencies were observed. Both in calf and rat phenoxybenzamine was 7--8 times more potent in competing for 3Hclonidine than (+)3H-epinephrine binding. The ergot agents, ergotamine, dihydroergokryptine and ergonovine displayed similar potencies in competing for (+)3H-epinephrine and 3H-clonidine binding in rat and calf. It had been previously shown that the affinities of 3H-catecholamines and other aagonists for calf cortical a-receptors decreased 2--4 fold when the incubation temperature was raised from 25 to 37°C. Conversely, the affinities of some a-antagonists increased several fold (U'Prichard and Snyder, 1977b). Comparison between the pharmacological properties of (+)3H-epinephrine binding in the rat at 25°C and the calf at 37°C showed that under these experimental conditions, the affinities of agonists for the a-sites in the two tissues were essentially identical (tabel 2), suggesting that the receptor is in the same conformation in the rat cortex at 25°C and the calf cortex at 37°C. However, the differences in the affinities of the antagonists phentol-
D.C. U~PRICHARD, S.H. SNYDER
amine and WB-4101 for the rat and calf areceptors were more pronounced in these conditions, phentolamine being 16 times, and WB-4101 21 times, more potent in the calf than the rat. While a-receptor agonists and antagonists were potent competitors for specific (+)3Hepinephrine binding in rat cortex,/~-receptor agonists and antagonists as well as 5-hydroxytryptamine were much weaker, an observation which supports the specificity of labelling of a-receptors by (+)-3H-epinephrine. The slopes of displacement curves for aagonists and antagonists in competing for specific (+)3H-epinephrine binding were parallel and displayed log-logit slopes of 1.0 indicating the absence of cooperative interactions (fig. 3).
3.3. Enhancement of a-noradrenergic and [3noradrenergic receptor binding in rat forebrain by chronic reserpine treatment Depletion of catecholamines by reserpine elicits biochemical evidence of receptor supersensitivity as measured by adenylate cyclase activity of pineal gland (Deguchi and Axelrod, 1973) and brain homegenates or slices (Palmer et al., 1976). Binding of 3H-haloperidol associated with dopamine receptors in corpus striatum membrane of the rat is also enhanced by reserpine treatment (Butt et al., 1977). We examined the influence of chronic administration of reserpine, 0.25 mg/ kg s.c. per day for 3 weeks, upon specific binding in rat forebrain of (+)-3H-epinephrine, 3H-WB-4101 and 3H-dihydroergokryptine associated with a-receptors and of 3H~lihydroalprenolol to H-receptor sites. Preliminary experiments using a single concentration of each ligand showed significant increases of 23+ 10, 43+ 5, 28+ 5 and 46+ 11% in the specific binding of 3H-WB-4101, 3Hdihydroalprenolol, '3H-dihydroergokryptine and (+)3H-epinephrine respectively, in the forebrains of 6 reserpine-treated rats compared to matched control rats. To determine whether the increased binding was due to
RESERPINE ENHANCES BRAIN NE RECEPTOR BINDING increased receptor number, or altered ligand affinity, we examined the saturation parameters for a- and fl-receptor binding in reserpine-treated and control rats. These assays were performed as paired experiments, with binding to control and reserpine-pretreated membranes run in parallel, at each of the different concentrations of (+)-3H-epinephrine, 3H-WB-4101 and 3H
4. Discussion
The present studies establish the feasibility of labelling a-noradrenergic receptors in rat brain with the agonists (+)-3H~epinephrine and (--)-3H-norepinephrine. Earlier investigations involved labelling agonist-preferring sites of the a-receptor in the rat with 3H-clonidine. (+)-3H-Epinephrine is more readily available commercially, and at higher specific activity than 3H-clonidine. In typical experiments, using concentrations of the 3H-ligand one half the KD value, we obtained with 3H-clonidine 150 specific cpm and 150 non-specific cpm, while with (+)-3H-epinephrine corresponding values were 1,000 cpm specific binding and 1,000 cpm nonspecific binding. These studies employed racemic 3H-epinephrine. If (--)-3Hepinephrine were available, one would anticipate an enhancement of the ratio of specific to non-specific binding. Certain properties of agonist.preferring
153
states o f a-noradrenergic receptors appear to differ between rat and calf. Under equivalent incubation conditions at 25°C, most agonists are substantially less potent in competing for specific (+)-3I-I-epinephrine and 3H-clonidine binding in rat than in calf, though their potencies in competing for 3HH-WB-4101 binding to antagonist preferring sites, or for the binding of SH-dihydroergokryptine to a-noradrenergic receptors, are the same in rat and calf cortex. Certain antagonists, especially phentolamine and WB-4101, are 8--10 times weaker in competing for specific (+)-3H-epinephrine and 3H-clonidine binding in rat than in calf. No such differences for antagonists are apparent between rat and calf in competing for 3H,WB-4101 and 3I-I
154 labelled either with (+-)-3H-epinephrine or 3HWB-4101. Th e augmentation in 3H-WB-4101 binding was greater than that of (-+)-3H-epinephrine binding, again supporting the n o t i o n that these ligands label different a-receptors. T h e increased binding o f b o t h a- and/3-1igands indicates th at reserpine supersensitivity is due to an effect at th e level o f t he receptor, perhaps caused b y n o r e p i n e p h r i n e depletion, rather than a non-specific postsynaptic ion mobilization effect. The increased binding of the three ligands in reserpine-pretreated membranes could conceivably be due to nonspecific increases in a m o u n t s of m e m b r a n e protein. In this c o n n e c t i o n , it is n o t e w o r t h y t h a t nonspecific binding at each concentration o f each ligand, which m a y be an indicator o f general m e m b r a n e protein concentration, was no different in reserpine-pretreated than in c o n t r o l membranes. A n o t h e r possible explanation of the data, depletion by reserpine o f endogenous norepinephrine tightly b o u n d to t he adrenergic receptors, c a n n o t however be ruled out. T h e increase in the n u m b e r o f binding sites supports the data of Broadley and L u m l e y (1977) which suggested t ha t in guinea-pig atria, reserpine supersensitivity was specific to the/3-receptor and involved an increased number of receptors. T he increase in norepinepho fine-stimulated cyclic AMP p r o d u c t i o n in rat c o rt e x after chronic reserpine appears to be linked p r e d o m i n a n t l y to /3-receptor supersensitivity {Palmer et al., 1876). T he present results may support this finding, in t hat the n u m b e r o f 3H-dihydroalprenolol sitesincreased 51%, whereas the n u m b e r of (+)-3H-epinephrine sites increased only 25%. Elsewhere we present evidence that the (+)-all-epinephrine, b u t n o t th e aH-WB-4101, a - r e c e pt or binding site m ay be co u pl ed t o adenylate cyclase (U'Prichard and Snyder, 1978). The present observations resemble t he e n h a n c e m e n t o f d o p a m i n e r e c e p t o r binding in rat corpus striatum after chronic reserpine t r e a t m e n t . These findings might provide an explanation for behavioral evidence o f supersensitivity o f catecholamine receptors after
D.C. U'PRICHARD, S.H. SNYDER chronic reserpine t r e a t m e n t (Williams and Pirch, 1974; Tarsy and Baldessarini, 1973; Ungerstedt et al., 1975).
Acknowledgements
We thank Mrs. Linda Hester and Mr. Peter Sheehan for their excellent technical assistance, and Miss Pamela Morgan for manuscript preparation. This research was supported by USPHS grant MH-18501 to S.H.S., USPHS fellowship award MH--5105 to D.C.U'P., and by grants from the McKnight and John A. Hartford Foundation, Inc.
References
Broadley, K.J. and P. Lumley, 1977, Selective reserpine-induced supersensitivity of the positive inotropic and chronotropic responses to isoprenaline and salbutamol in guinea-pig isolated atria, Brit. J. Pharmacol. 59, 51. Burt, D.R., I. Creese and S.H. Snyder, 1977, Antischizophrenic drugs: chronic treatment elevates dopamine receptor binding in brain, Science 169, 326. Bylund, D.B. and S.H. Snyder, 1976, ~-Adrenergic receptor binding in membrane preparations from mammalian brain, Mol. Pharmacol. 12, 568. Carrier, O., 1975, Role of calcium in postjunctional supersensitivity, Federation Proc. 34, 1975. Deguchi, T. and J. Axelrod, 1973, Supersensivitiy and subsensitivity of the 13-adrenergic receptor in pineal gland regulated by catecholamine transmitter, Proc. Nat. Acad. Sci, U.S.A. 70, 2411. Dismukes, K. and J.W. Daly, 1974, Norepinephrinesensitive systems generating adenosine 3',5'monophosphate: increased responses in cerebral cortical slices from reserpine-treated rats, Mol. Pharmacol. 10, 933. Glaubiger, G. and R.J. Lefkowitz, 1977, Elevated fladrenergic receptor number after chronic propranolol treatment, Biochem. Biophys. Res. Commun. 78,720. Greenberg, D.A. and S.H. Snyder, 1977, Selective labeling of a-noradrenergic receptors in rat brain with aH-dihydroergokryptine, Life Sci. 20, 927. Greenberg, D.A. and S.H. Snyder, 1978, Pharmacologic properties of [aH]dihydroergokryptine binding sites associated with a-noradrenergic receptors in rat brain membranes, Mol. Pharmacol.
(in press). Kafka, M.S., J.F. Tallman and C.C. Smith, 1977, aAdrenergic receptors on human platelets, Life Sci. 21, 1429.
RESERPINE ENHANCES BRAIN NE RECEPTOR BINDING Lefkowitz, R.J., D. Mullikin, C.L. Wood, T.B. Gore and C. Mukherjee, 1977, Regulation of prostaglandin receptors by prostaglandins and guanine nucleotides in frog erythrocytes, J. Biol. Chem. 252, 5292. Palmer, G.C., H.R. Wagner and R.W. Putman, 1976, Neuronal localization of the enhanced adenylate cyclase responsiveness to catecholamines in the rat cerebral cortex following reserpine injections, Neuropharmacology 15, 695. Peroutka, S.J., D.A. Greenberg, D.C. U'Prichard and S.H. Snyder, 1978, Regional variations in anoradrenergic receptor interactions of 3H-dihydroergokryptine in calf brain: implications for a twostate model of a-receptor function, Mol. Pharmacol. 14,403. Rotrosen, J., E. Friedman and S. Gershon, 1975, Striatal adenylate cyclase activity following reserpine and chronic chlorpromazine administration in rats, Life Sci. 17,563. Tarsy, D. and R.J. Baldessarini, 1973, Pharmacologically induced behavioral supersensitivity of apomorphine, Nature (New Biol.) 245,262, Trendelenburg, U., 1966, Mechanisms of supersensitivity and subsensitivity to sympathomimetic amines, Pharmacol. Rev. 18, 629. Ungerstedt, U., T. Ljungberg, B. Holler and G. Siggins, 1975, Dopaminergic supersensitivity in the striatum, Advan. Neurol. 9, 57. U'Prichard, D.C. and S.H. Snyder, 1977a, 3H-Epinephrine and 3H-norepinephrine binding to a-
155
noradrenergic receptors in calf brain membranes, Life Sci. 20, 527. U'Prichard, D.C. and S.H. Snyder, 1977b, Binding of 3H-catecholamines to a-noradrenergic receptor sites in calf brain, J. Biol. Chem. 252, 6450. U'Prichard, D.C. and S.H. Snyder, 1978, Guanyl nucleotide influences on 3H-ligand binding to anoradrenergic receptors in calf brain membranes, J. Biol. Chem. 235, 3444. U'Prichard, D.C., D.A. Greenberg and S.H. Snyder, 1977a, Binding characteristics of a radiolabeled agonist and antagonist at central nervous system a-noradrenergic receptors, Mol. Pharmacol. 13, 454. U'Prichard, D.C., D.A. Greenberg, P. Sheehan and S.H. Snyder, 1977b, Regional distribution of anoradrenergic receptor binding in calf brain, Brain Res. 138, 151. Williams, B.J. and J.H. Pirch, 1974, Correlation between brain adenyl cyclase activity and spontaneous motor activity in rats after chronic reserpine treatment, Brain Res. 68, 227. Williams, L.T. and R.J. Lefkowitz, 1976, a-Adrenergic receptor identification by [3H]dihydroergokryptine binding, Science 192, 791. Williams, L.T. and R.J. Lefkowitz, 1977a, Regulation of rabbit myometrial alpha adrenergic receptors by estrogen and progesterone, J. Clin. Invest. 60, 815. Williams, L.T. and R.J. Lefkowtiz, 1977b, Thyroid hormone regulation of/3-adrenergic receptor number, J. Biol. Chem. 252, 2787.
145
© Elsevier/North-Holland Biomedical Press
3H-CATECHOLAMINE BINDING TO a-RECEPTORS IN RAT BRAIN: ENHANCEMENT BY RESERPINE DAVID C. U'PRICHARD and SOLOMON H. SNYDER Departments of Pharmacology and Experimental Therapeuritcs and Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, U.S.A.
Received 28 February 1978, revised MS received 8 May 1978, accepted 9 June 1978
D.C. U'PRICHARD and S.H. SNYDER, 3H-Catecholamine binding to a-receptors in rat brain: enhancement by reserpine, European J. Pharmacol. 51 (1978)145--155. (±)-3H-Epinephrine and (--)-3H-norepinephrine bind to rat cortex membranes in a saturable manner with dissociation constants ~of 16.7 and 27 nM respectively. The maximum number of 3H-catecholamine binding sites, 10--12 pmoles/g tissue, and the pharmacological characteristics of (±)-3H-epinephrine binding, indicate that the catecholamines label the same a-noradrenergic receptor in the rat as does 3H-clonidine. At 25 °, (±)-3H-epinephrine binding associates rapidly to equilibrium, and dissociates in a biphasic manner. The affinities of a-agonists at the 3H-eatecholamine binding site are 2--4 fold weaker in the rat than in the calf cortex under the same experimental conditions. Ergot alkaloids and phenoxybenzamine have similar affinities in the two tissues, whereas phentolamine and WB-4101 are 8--10 times weaker in the rat. Reserpine (0.25 mg/kg s.c. per day for 3 weeks) causes 25 and 46% increases in the numbers of (-+)-3H-epinephrine and 3H-WB-4101 a-receptor binding sites respectively, and a 51% increase in the number of 3H-dihydroalprenolol fi-recepto~ sites, in rat forebrain. Reserpine pretreatment does not alter the affinities of either a- or fi-receptor 3H-ligands. Catecholamines
Adrenoceptor binding
Rat brain
1. Introduction a-Noradrenergic receptor sites in brain and peripheral tissues can be labelled by a variety of ligands, including 3H~lihydroergokryptine (Williams and Lefkowitz, 1976; Greenberg and Snyder, 1977; Kafka et al., 1977), the antagonist 3H-WB-4101 (2-( [ 2',d'-dimethoxy]p h e n o x y e t h y l a m i n o ) m e t h y l b e n z o d i o x a n ) (U'Prichard et al., 1977a) and the agonists 3Hclonidine (U'Prichard et ai., 1977a) (--)-3Hnorepinephrine and (+)-3H-epinephrine (U'Prichard and Snyder, 1977a). Several binding studies in rats show that the numbers o f noradrenergic receptors can be altered in a variety of tissues by chronic treatment with certain drugs and hormones (Williams and Lefkowitz, 1977a, b; Glaubiger and Lefkowitz, 1977). Reserpine causes postjunctional supersensi-
Reserpine
tivity in the nictitating membrane (Trendelenburg, 1966), brain (Ungerstedt et al., 1975) and cardiovascular tissue (Carrier, 1975). Reports have differed as to whether reserpine supersensitivity is receptor specific (Broadley and Lumley, 1977) or a non-specific calcium mobilization effect (Carrier, 1975). In the brain, chronic reserpine treatment increases adenylate cyclase stimulation by norepinephfine and isoproterenol (Dismukes and Daly, 1974; Williams and Pitch, 1974; Palmer et ai., 1976) but not by dopamine (Rotrosen et al., 1975), although Burr et al. (1977) reported an increased number o f dopamine receptor binding sites labeled by 3H-haioperidol following reserpine. We sought to determine if chronic reserpine similarly increased a-noradrenergic receptor sites. Properties of a-receptors in terms of relative drug potencies vary depending on whether
146 receptor sites are labelled with 3H-agonists or 3H-antagonists (U'Prichard et al., 1977a; U'Prichard and Snyder, 1977b; Greenberg and Snyder, 1978; Peroutka et al., 1978). For studies of agonist-preferring sites, 3H-catecholamines are often preferable to the use of 3Hclonidine, because the 3H-catecholamines are more readily available commercially, and because the absolute amount of specific binding, and the ratio of specific to non-specific binding, is greater with these compounds than with 3H-clonidine (U'Prichard and Snyder, 1977b). Relative potencies of drugs in competing for (+)-3H-epinephrine and (--)-3Hnorepinephrine binding to a-receptors in calf brain membranes differ somewhat from their effects on rat brain a-receptor sites as labeled by 3H-clonidine. Since studies of the regulation in vivo of neurotransmitter receptors are most efficiently conducted in rodents, we desired to compare properties of (+)-3Hepinephrine and (--)-3H-norepinephrine binding to a-receptors in rats with properties previously described in calf (U'Prichard and Snyder, 1977b). In the present study we characterize (--)-3H-norepinephrine and (+)3H-epinephrine labeling of a-receptor in rat cerebral cortex and report differences between receptor properties in rat and calf. Additionally, we report an enhancement of a - a n d t3receptor binding following chronic reserpine treatment.
2. Materials and methods 2.1. Measurement o f (+)-3H-epinephrine and (--)-3H-norepinephrine binding in rat cortex
Fresh cerebral cortices from adult male Sprague-Dawley rats were homogenized in 20 volumes of ice-cold 50 mM Tris-HC1 buffer, pH 7.7 at 25°C, with a Brinkmann Polytron PT-10. The homogenates were centrifuged twice at 50,000 × g for 10 min, with intermediate rehomogenization in fresh buffer. Final pellets were resuspended in 22 volumes of Tris-HC1 buffer, pH 7.7 at 25°C, and incu-
D.C. U'PRICHARD, S.H. SNYDER bated with 1.0 pM pargyline for 15 min at 25°C. Triplicate incubation tubes received 20 pl of (+)-3H-epinephrine (D,L-[7-3H]epi nephrine L-bitartrate, 13.8 Ci/mmole, New England Nuclear Corp.) or (--)-3H-norepinephrine (L-[7,8-3H]norepinephrine, 24 Ci/ mmole, New England Nuclear), 10pl of various concentrations of drugs, 20 pl of a solution containing 50 mM pyrocatechol, 0.05% ascorbic acid, 5 mM disodium EDTA, and 0.5 mM dithiothreitol, and 0.45 ml of freshly resuspended tissue. Radioactive ligands and added drugs were all prepared in a 0.1% ascorbic acid solution. The final concentration of (+)-3H-epinephrine and (--)-3H-norepinephrine was 5 nM, and the final incubation volume was 0.5 ml. Tubes were incubated at 25°C for 60 min, and subsequently filtered under vacuum through Whatman GF/B filters presoaked in 1 mM pyrocatechol. The filters were rinsed with 15 ml of ice-cold 50 mM Tris-HC1 buffer, pH 7.7 at 25°C, containing 1 mM pyrocatechol and 0.1% ascorbic acid, and were counted by liquid scintillation spectrometry at 37% efficienty. Saturable of specific binding of (+_)-3H-epinephrine and (--)-3H-norepinephrine was defined as the excess over blanks containing 2.0 pM oxymetazoline. Incubation of cortex membranes ( 2 0 m g tissue, original weight) with 5 nM of either 3H-catecholamine, the routine concentration used, gave 2000 cpm total binding and 1000 cpm non-specific binding. In general, since there was relatively more non-specific binding of 3H-cateeholamines in rat than in calf cortex, the present alterations for rat cortex of the assay method previously used in calf cortex (U'Prichard and Snyder, 1977b) were designed to minimize nonspeeific binding. 3H-Catecholamines were stored and diluted as described previously (U'Prichard and Snyder, 1977b). 2.2. Chronic reserpine treatments
Adult male Sprague-Dawley rats, 150-175 g, were injected with 0.25 mg/kg reserpine (Serpasil, Ciba) s.c. daily for 3 weeks.
RESERPINE ENHANCES BRAIN NE RECEPTOR BINDING
Control animals received equivalent volumes of saline. The animals were killed on the day after the last injection, and the forebrains (area rostral to the colliculi) frozen rapidly and stored at --20°C. For binding assays, suspensions of forebrain tissue were prepared as described above, except that the final resuspension was in 44 volumes of Tris-HC1 buffer, pH 7.7 at 25°C, and 10 mg tissue, original wet weight, was added to the assay. (+)-all-epinephrine binding to a-receptors was assayed as above, and aH-WB-4101 and 3H-dihydroergokryptine binding to a-receptors and 3Hdihydroalprenolol binding to r-receptors was determined in the same samples as described previously (U'Prichard et al., 1977a; Greenberg and Snyder, 1977; Bylund and Snyder, 1976). Compared to fresh tissue controls, frozen rat forebrains showed a loss in affinity for (-+)-all-epinephrine, but not for aH-WB4101 or aH-dihydroalprenolol. 2.3. Drugs WB-4101 was custom tritiated to a specific activity of 11.7 Ci/mmole at New England Nuclear (U'Prichard et al., 1977a). 3H Dihydroalprenolol, 48--50 Ci/mmole, was obtained from New England Nuclear. Catecholamines and phenylephrine were donated by SterlingWinthrop, clonidine by Boehringer-Ingelheim, oxymetazoline by Schering, ergot alkaloids by Sandoz, phentolamine by Ciba, WB-4101 by Ward Blenkinsop, phenoxybenzamine by Smith Kline and French, and indoramin by Wyeth. Other drugs and compounds were obtained from the pharmaceutical company of origin or commercial sources.
3. Results
3.1. Saturation and kinetics o f (+_)-3H-epinephrine and (--)-aH-norepinephrine binding to rat cerebral cortex membranes The specific binding of (+)-3H-epinephrine to rat cerebral cortex membranes was a satur-
147
able process. At concentrations up to 10 nM, specific binding of (+)-3H-epinephrine equalled non-specific binding, while at higher concentrations non-specific binding increased more rapidly. There was a linear relationship between the amount of nonspecific binding and concentration of (+)-3H-epinephrine (fig. 1). Specific (+)-all-epinephrine binding plateaued at 25--40 nM concentration and reached half maximal binding at approximately 10 nM. Scatchard analysis (line of best fit determined by linear regression analysis) revealed a single population of binding sites with a dissociation constant ( K D ) of 10.7 nM and a maximal number of binding sites (Brnax) of 11.6 pmoles/g original wet weight of tissue (fig. 1). In three similar experiments, the mean values +S.E.M. for KD and Bmax were 16.7 +- 5.6 nM and 12.0 + 0.4 pmoles/g respectively. In general it was found that, whereas the Bmax values determined by regression analysis were relatively invariant from experiment to experiment, the K D v a l u e s were more ~z)oo
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[aH]-epinephrine bound (cpm); abscissa: [3H]-epinephrine concentration (nM). (+)3H-Epinephrine binding in the rat cortex as a function of increasing concentrations of (-+)-3H-epinephrine. Homogenates (20 mg tissue, original weight) were incubated for 60 min at 25°C as described in Materials and methods with varying concentrations of (+)-all-epinephrine. Non-specific binding was determined b y the addition of 2.0 ~ oxymetazoline. Points shown are from one o f three similar experiments, each performed in triplicate. Inset: Scatchard plot of binding data was determined by linear regression analysis. Fig. 1.
Ordinate:
148
variable, reflecting the variation in stability of the (+)-3-epinephrine. Specific (--)-3H-norepinephrine binding in the rat cortex displayed saturation characteristics similar to that of (+)-3H-epinephrine. Ratios of specific and non-specific binding at various concentrations of (--)-3H-norepinephrine were similar to (+)-3H-epinephrine. Scatchard analysis o f (--)-3H-norepinephrine saturation data indicated a single population of sites, with a KD value for (--)-3H-norepinephrine binding of 27 nM, while the Bmax value was 10 pmole/g. Since a-receptor binding of (±)-3H-epinephrine and (--)-3H-norepinephrine was stereospecific and (--)-3H-norepinephrine employed, while the racemic form of 3H-epinephrine was utilized, the actual affinity of norepinephrine was only about one third of that of epinephrine for a-receptors in rat brain. In other experiments the kinetic properties of (+)-3H-epinephrine binding in rat cortex were examined. At 25°C, (+)-3H-epinephrine specific binding to cerebral cortex membranes of the rat increased to a plateau at about 200 min. Half maximal binding was obtained at about 3 min (fig. 2A). Nonspecific (+_)-3H-epinephrine binding also displayed a gradual time course which increased to a plateau at about 50 min. Thus in routine experiments, an incubation time of 60 min at 25°C was used to ensure steady-state conditions for both specific and nonspecific binding. Association of specific (±)-3H-epinephrine binding was hyperbolic and followed pseudo-first order kinetics. The observed rate of association (kob) was 0 . 1 9 r a i n -1. The calculated rate constant of association (k~) was 0.008 nM -1 min -~. These properties differ from the specific binding of (±)-3H-epinephrine at 25°C to calf cerebral cortex membranes (U'Prichard and Snyder, 1977b). In the calf, association of specific binding is nonhyperbolic, and at 25°C is biphasic when plotted on a semilogarithmic scale. The value of kl for association of (+)-3H-epinephrine binding in rat cerebral cortex could not be determined using the equation k~ = (kob--
D.C. U'PRICH)kRD, S.H. SNYDER I000
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[3H]-epinephrine bound (cpm); abscissa: time (min). (A) Association at 25°C of (_+).3H-epinephrine binding to rat cortex homogenates (20 mg tissue, original weight). Specific and nonspecific binding, as defined in Materials and methods, Ordinate:
was determined at various time intervals following the addition of 5 nM (±)-3H-epinephrine. Points shown are from one of three similar experiments, each performed in triplicate. Inset: pseudo-first order kinetic plo_t of initial (+-)-3H-epinephrine binding. B and Be are the amounts of (-+)-3H-epinephrine specifically bound at time t and at equilibrium respectively. Slope is equal to kob, the observed rate constant. (B) Ordinate: [3H]-epinephrine specifically bound (B/B0 × 100); abscissa: time (min). Dissociation at 25°C of (±)-3H-epinephrine binding, after incubation to equilibrium for 60 min. Specific binding was determined at various time intervals following the addition of 2.0 pM oxymetazoline at time zero. Points shown are from one of three experiments, each performed in triplicate. k 1)/[(+)-3H-epinephrine], since kob was less than the initial k_ 1. Association at time points of less than one minute were not examined in the rat cortex, and possible the kob for initial association may be greater if the semilogarithmic plot were nonlinear, with a steep initial slope. Assuming the irrevers-
RESERPINE ENHANCES BRAIN NE RECEPTOR BINDING
ibility of the forward reaction, kl could be determined using the equation k~ = [1/t(F--R)]ln[R(F--B)/F(R--B)] (see table 1).
TABLE 1 Comparison of binding constants of 3H-catecholamines labeling a-noradrenergic receptors in rat and calf cerebral cortex membranes at 25°C. The constants for S H-catecholamine binding to rat cortex homogenates (20 rag, original tissue weight) were obtained as described in the text. Constants for calf cortex binding were obtained from U'Prichard and Snyder (1977b). (±).3 H-Epinephrine Rat
(_).3 HNorepinephrine
Calf Rat Calf
kl (nM -I rain -1 ) ~ tl/2 (association) min k_ 1 ( m i n - I ) 2 tl / 2 (dissociation) (rain) 2 K D (kinetic) (nM) 3 K D (equilibrium) (nM) 4 Bmax (pmol/g tissue)
0.008 3 0.35, 0.031
0.04 5 0.24
----
0.06 5 0.24
2, 24 44, 4
3.3 6.2
---
2 4.0
17 12
6.5 8.9
27 10
9.7 7.0
I The association rate constant for (±)-3H-epinephrine binding in the rat cortex was determined by fitting the equation kl = [ 1 / t ( F - - R)] In [R(F -- B ) / F ( R - - B)], where F is the initial concentration o f (±)-3H-epinephrine, R is the receptor concentration and B is the concentration o f specifically bound (_+)-3H-epinephrine at time t. The equation kl = (kob -- k-1 )/[(+).3 H-epinephrine], which takes ongoing ligand dissociation into account and was used to calculate the calf association rate constants, cound not be successfully fitted to the rat data. 2 Dissociation of 3 H-eatecholamine specific binding to a-receptors at 25°C was biphasic for both rat and calf cortex. The first k_ 1 and t l / 2 values are for the initial phase of dissociation, and the second values are for the slow dissociation phase, 3 The KD values obtained from kinetic experiments equal k_] (fast)/kl and k-1 (slow)/kt. 4 Obtained from saturation experiments.
149
The value thus obtained is very approximate, since considerable dissociation does occur during the rising portion of the association curve (fig. 2). The apparent association constant for (+)-3H-epinephrine binding to areceptors is four times smaller in the rat cortex than in the calf cortex (table 1). To examine the dissociation of specifically bound (+)-3H-epinephrine, rat cerebral cortex membranes were labelled with (+)-3H-epinephrine at 25°C for 60 min, at which time 2.0 /~M oxymetazoline was added and binding was examined at subsequent time intervals. When plotted on a semilogarithmic scale the dissociation rate was biphasic (fig. 2B). For the initial phase of dissociation, during which, in repeated experiments, 50--70% of the ligand was released, the half life was 2 min and the calculated rate constant for dissociation (k_l) was 0.35 min -1. The slower phase of dissociation had a half life of about 23 min, and a k I of 0.031 min -1. These findings resemble results in calf cerebral cortex membranes, in which dissociation of specifically bound (+)-3H-epinephrine at 25°C, after labelling at 25°C, also was biphasic with a similar initial phase k_~ (table 1). The dissociation constants ( K D ) for the fast and slow dissociating phases of (_+)-all-epinephrine binding to rat cortex a-receptors, derived from kinetic experiments (k_~/k~), were 44 nM and 4 nM. The equilibrium value for K D lies midway between these values (table 1). Such biphasic dissociation plots have also been observed with other labelled agonist ligands, such as 3H-protaglandin E, (Lefkowitz et al., 1977). Both here and for calf brain 3H-catecholamine a-receptor binding (U'Prichard and Snyder, 1977b), it has been suggested that the 3H-agonist, during the course of receptor labelling, converts some receptors to a higheragonist-affinity (desensitized) state. If the higher- and lower-affinity forms are interconvertible, the Scatchard plot would be expected to yield a single line, as seen in the present study. The apparent KD thus obtained, however, would be a composite value from the high and low-affinity forms (Lefkowitz et al.,
150
D.C. U~PRICHARD, S.H. SNYDER
TABLE 2 Inhibition of (+).3 H-epinephrine binding to a-receptors in rat cortex membranes at 25 °C; comparison with calf cortex values for (_+).3H-epinephrine binding 1. Rat cortex homogenates were incubated with 5 nM (+_)-3H-epinephrine for 60 min at 25°C, together with 4--6 concentrations of unlabeled drugs, under standard assay conditions. IC50 values were determined by log-probit analysis, and apparent K i values calculated from the equation Ki = iCs0/( 1 + [(_+).3 H-epinephrine]/K D ), where the K D value used was 16.7 nM. Values given are means +- S.E. of 3 experiments, each conducted in triplicate. Drug
Rat, K i (nM)
Calf, Ki (37°C) (nM)
Calf, K i (25°C) (nM)
a-Agonists (--)-Epinephrine (+)-Epinephrine (--)-a-Me-Norepinephrine (+)-a-Me-Norepinephrine (--)-Norepinephrine (+)-Norepinephrine Dopamine (--)-Phenylephrine Clonidine Oxymetazoline
5.2 92 11.5 8,400 20 2,200 200 580 3.0 3.0
+ + _+ +_ -+ _+ + +-+ +-
0.8 15 1.8 900 5 200 40 130 1.6 0.2
6.5 97 14 16,000 24 1,000 310 370 3.3 3.3
2.1 26 3.9 4,600 6.4 260 87 140 1.3 4.7
6.3 7.1 1,400 19 340 490 36,000
+ 1.5 +1.2 +_ 200 + 6 _+ 60 _+ 140 + 13,000
5.8 8.3 3,800 1.2 16 200 12,000
9.6 5.4 5,600 2.0 41 1,100 11,000
11,000 2,800 25,000 17,000
-+ 4,000 -+ 200 +_ 4,000 +- 6,000
5,700 3,900 -21,000
6,600 1,100 -19,000
a-Antagonists Ergotamine Dihydro-~-ergokryptine Ergonovine Phentolamine WB-4101 Phenoxybenzamine Indoramin
Other drugs Serotonin (--)-Isoproterenol (+_)-Propranolol (--)-Alprenolol
1 Calf cortex values were obtained from U'Prichard and Snyder (1977b).
1 9 7 7 ) , a n d s h o u l d t h u s f a l l b e t w e e n t h e KD values obtained kineticaUy from the slow and f a s t d i s s o c i a t i o n p h a s e s , as is s e e n i n t h e p r e s e n t s t u d i e s ( t a b l e 3). T h e s p e c i f i c b i n d i n g of low (2 nM) or high (50 nM) concentrations of (+-)-3H-epinephrine shows the same a-receptor characteristics.
3.2. Influence o f drugs on specific (+)-3H-epinephrine binding to rat cortex a-receptor sites A variety of a-agonists, antagonists and other durgs were examined for their influence upon specific (+)-3H-epinephrine binding to r a t c e r e b r a l c o r t e x m e m b r a n e s (fig. 3, t a b l e
RESERPINE ENHANCES BRAIN NE RECEPTOR BINDING 200C 41,
TABLE 3
0 <'TOTAL
Effect of chronic reserpine on (_+).3H-epinephrine and 3 H-WB-4101 binding to a-receptors, and 3 H-dihydroalprenolol binding to/~-receptors, in rat forebrain. Rats were injected with reserpine (Serpasil, 0.25 rag/ kg) daily for three weeks and were killed one day after the last dose. Frozen forebrains from these animals were subsequently thawed and assayed for a-receptor binding with four concentrations of (_+)3H-epinephrine (2.5--70 nM) or 3H-WB-4101 (0.1-1.5 nM), and for ~-receptor binding with 3 H-dihydroalprenolol (0.3--3.0 nM), as described in Materials and methods. Forebrains from control rats receiving saline injections were assayed in parallel. Scatchard plots from the saturation curves for each ligand in each forebrain were obtained by linear regression analysis to determine the apparent dissociation constants (K D ) and maximum number of binding sites (Bmax). Values given are means_+ S.E. for 5 rats in each group. Values of P were determined by Student's t-test (two-tailed). 3 H-ligand
Control
Reserpine % change
31.5 _+11.3 8.5 +0.9
28.0 _+2.9 10.6 -+0.3 1
0.36 _+0.05 6.7 _+0.9
0.43 _+0.08 9.8 _+0.02 2
1.43 _+0.21 8.5
1.81 _+0.15 12.8
_+1.0
_+ 1.0 i
(+)_3H-Epinephrine K D (nM) Bmax (pmol/g tissue)
Bmax (pmol/g tissue)
+46
3H-Dihydroalprenolol K D (nM) Bmax (pmol/g tissue)
'
"~x
'
"~V
' 9
'
(-)-
I
"j~,o
~d9
Fig. 3. I n h i b i t i o n
I
I
~o-8 of
~o-7
I
pal6
{
,o-S
(+)-3H-epinephrine
I
~o-"
Jo-3
binding
in
rat cortex by a- and ~-agents. Homogenates (20 mg tissue, original weight) were incubated at 25°C for 60 rain with 5 nM (+)-3H-epinephrine and various concentrations of unlabelled drugs. ICs0 values were derermined as the concentration of drug which inhibited 50% of (~)-3H-epinephrine specific binding (binding displaceable by 2.0 pM oxymetazoline). T o t a l (-+)-3H-epinephrine binding, in the absence of any inhibitor, is shown in the top left. Points shown are from one experiment, performed in triplicate. Ordinate: [3H]-epinephrine bound (cpm).
+25
3H-WB-4101 KD (nM)
151
+51
1 Significantly different from control, P ~ 0.05. 2 Significantly different from control, P ~ 0.01.
2). Initial experiments showed that (--)epinephrine, oxymetazoline and WB-4101 maximally inhibited (+)-all-epinephrine binding to the same extent, at concentrations of 2.0 pM or higher. All combinations of 2.0/~M concentrations of these three inhibitors caused no further loss of binding than any of the inhibitors alone. (+)-3H-Epinephrine binding was stereospecific, with (--)-epinephrine dis-
playing 18 times greater affinity than (+)epinephrine, while (--)-isomers of a-methylnorepinephrine and norepinephrine were 700 and 100 times more potent respectively than the corresponding (+)-isomers. (--)-Epinephrine displayed about four times greater affinity than (--)-norepinephrine while dopamine was only 2.6% as potent as (--)-epinephrine. Under the same incubation conditions at 25°C, (--)-epinephrine and (--)-norepinephrine were 2--3 times more potent in competing for (+)3H-epinephrine binding in calf than in rat (table 2). This agrees with the approximate three fold greater affinities of (+)-all-epinephrine and (--)-3H-norepinephrine in calf than rat membranes in saturation experiments (table 1). Other a-agonists such as phenylephrine, clonidine and oxymetazoline were also 2--3 times more potent in calf than rat membranes at 25°C. The differences in affinities of agonists for (+)-all-epinephrine binding sites in rat and calf membranes appeared to be characteristic for a-receptors in the two
152
species rather than for (+)-3-epinephrine binding alone. Thus affinities of these agents for 3H¢lonidine binding sites in rat cerebral cortex membranes were less than for 3H-clonidine binding sites in calf cerebral cortex membranes, but closely resembled affinities of the same drugs for (+)3H-epinephrine binding sites in rat membranes (U'Prichard et al., 1977a; U'Prichard and Snyder, 1977b). Among a-blocking drugs, phentolamine and WB-4101 were 8--10 times more potent in competing for (+-)3H-epinephrine binding in calf than in rat, under the same 25°C conditions. As in the case of the agonists, this difference was species rather than drug specific, as both of these agents were substantially more potent in competing for 3H-clonidine binding in calf than in rat as well. The irreversible antagonist phenoxybenzamine failed to display consistent differences in potencies between calf and rat in competing for binding of 3H-clonidine or (+)3H-epinephrine. For phenoxybenzamine, ligand-specific differences in potencies were observed. Both in calf and rat phenoxybenzamine was 7--8 times more potent in competing for 3Hclonidine than (+)3H-epinephrine binding. The ergot agents, ergotamine, dihydroergokryptine and ergonovine displayed similar potencies in competing for (+)3H-epinephrine and 3H-clonidine binding in rat and calf. It had been previously shown that the affinities of 3H-catecholamines and other aagonists for calf cortical a-receptors decreased 2--4 fold when the incubation temperature was raised from 25 to 37°C. Conversely, the affinities of some a-antagonists increased several fold (U'Prichard and Snyder, 1977b). Comparison between the pharmacological properties of (+)3H-epinephrine binding in the rat at 25°C and the calf at 37°C showed that under these experimental conditions, the affinities of agonists for the a-sites in the two tissues were essentially identical (tabel 2), suggesting that the receptor is in the same conformation in the rat cortex at 25°C and the calf cortex at 37°C. However, the differences in the affinities of the antagonists phentol-
D.C. U~PRICHARD, S.H. SNYDER
amine and WB-4101 for the rat and calf areceptors were more pronounced in these conditions, phentolamine being 16 times, and WB-4101 21 times, more potent in the calf than the rat. While a-receptor agonists and antagonists were potent competitors for specific (+)3Hepinephrine binding in rat cortex,/~-receptor agonists and antagonists as well as 5-hydroxytryptamine were much weaker, an observation which supports the specificity of labelling of a-receptors by (+)-3H-epinephrine. The slopes of displacement curves for aagonists and antagonists in competing for specific (+)3H-epinephrine binding were parallel and displayed log-logit slopes of 1.0 indicating the absence of cooperative interactions (fig. 3).
3.3. Enhancement of a-noradrenergic and [3noradrenergic receptor binding in rat forebrain by chronic reserpine treatment Depletion of catecholamines by reserpine elicits biochemical evidence of receptor supersensitivity as measured by adenylate cyclase activity of pineal gland (Deguchi and Axelrod, 1973) and brain homegenates or slices (Palmer et al., 1976). Binding of 3H-haloperidol associated with dopamine receptors in corpus striatum membrane of the rat is also enhanced by reserpine treatment (Butt et al., 1977). We examined the influence of chronic administration of reserpine, 0.25 mg/ kg s.c. per day for 3 weeks, upon specific binding in rat forebrain of (+)-3H-epinephrine, 3H-WB-4101 and 3H-dihydroergokryptine associated with a-receptors and of 3H~lihydroalprenolol to H-receptor sites. Preliminary experiments using a single concentration of each ligand showed significant increases of 23+ 10, 43+ 5, 28+ 5 and 46+ 11% in the specific binding of 3H-WB-4101, 3Hdihydroalprenolol, '3H-dihydroergokryptine and (+)3H-epinephrine respectively, in the forebrains of 6 reserpine-treated rats compared to matched control rats. To determine whether the increased binding was due to
RESERPINE ENHANCES BRAIN NE RECEPTOR BINDING increased receptor number, or altered ligand affinity, we examined the saturation parameters for a- and fl-receptor binding in reserpine-treated and control rats. These assays were performed as paired experiments, with binding to control and reserpine-pretreated membranes run in parallel, at each of the different concentrations of (+)-3H-epinephrine, 3H-WB-4101 and 3H
4. Discussion
The present studies establish the feasibility of labelling a-noradrenergic receptors in rat brain with the agonists (+)-3H~epinephrine and (--)-3H-norepinephrine. Earlier investigations involved labelling agonist-preferring sites of the a-receptor in the rat with 3H-clonidine. (+)-3H-Epinephrine is more readily available commercially, and at higher specific activity than 3H-clonidine. In typical experiments, using concentrations of the 3H-ligand one half the KD value, we obtained with 3H-clonidine 150 specific cpm and 150 non-specific cpm, while with (+)-3H-epinephrine corresponding values were 1,000 cpm specific binding and 1,000 cpm nonspecific binding. These studies employed racemic 3H-epinephrine. If (--)-3Hepinephrine were available, one would anticipate an enhancement of the ratio of specific to non-specific binding. Certain properties of agonist.preferring
153
states o f a-noradrenergic receptors appear to differ between rat and calf. Under equivalent incubation conditions at 25°C, most agonists are substantially less potent in competing for specific (+)-3I-I-epinephrine and 3H-clonidine binding in rat than in calf, though their potencies in competing for 3HH-WB-4101 binding to antagonist preferring sites, or for the binding of SH-dihydroergokryptine to a-noradrenergic receptors, are the same in rat and calf cortex. Certain antagonists, especially phentolamine and WB-4101, are 8--10 times weaker in competing for specific (+)-3H-epinephrine and 3H-clonidine binding in rat than in calf. No such differences for antagonists are apparent between rat and calf in competing for 3H,WB-4101 and 3I-I
154 labelled either with (+-)-3H-epinephrine or 3HWB-4101. Th e augmentation in 3H-WB-4101 binding was greater than that of (-+)-3H-epinephrine binding, again supporting the n o t i o n that these ligands label different a-receptors. T h e increased binding o f b o t h a- and/3-1igands indicates th at reserpine supersensitivity is due to an effect at th e level o f t he receptor, perhaps caused b y n o r e p i n e p h r i n e depletion, rather than a non-specific postsynaptic ion mobilization effect. The increased binding of the three ligands in reserpine-pretreated membranes could conceivably be due to nonspecific increases in a m o u n t s of m e m b r a n e protein. In this c o n n e c t i o n , it is n o t e w o r t h y t h a t nonspecific binding at each concentration o f each ligand, which m a y be an indicator o f general m e m b r a n e protein concentration, was no different in reserpine-pretreated than in c o n t r o l membranes. A n o t h e r possible explanation of the data, depletion by reserpine o f endogenous norepinephrine tightly b o u n d to t he adrenergic receptors, c a n n o t however be ruled out. T h e increase in the n u m b e r o f binding sites supports the data of Broadley and L u m l e y (1977) which suggested t ha t in guinea-pig atria, reserpine supersensitivity was specific to the/3-receptor and involved an increased number of receptors. T he increase in norepinepho fine-stimulated cyclic AMP p r o d u c t i o n in rat c o rt e x after chronic reserpine appears to be linked p r e d o m i n a n t l y to /3-receptor supersensitivity {Palmer et al., 1876). T he present results may support this finding, in t hat the n u m b e r o f 3H-dihydroalprenolol sitesincreased 51%, whereas the n u m b e r of (+)-3H-epinephrine sites increased only 25%. Elsewhere we present evidence that the (+)-all-epinephrine, b u t n o t th e aH-WB-4101, a - r e c e pt or binding site m ay be co u pl ed t o adenylate cyclase (U'Prichard and Snyder, 1978). The present observations resemble t he e n h a n c e m e n t o f d o p a m i n e r e c e p t o r binding in rat corpus striatum after chronic reserpine t r e a t m e n t . These findings might provide an explanation for behavioral evidence o f supersensitivity o f catecholamine receptors after
D.C. U'PRICHARD, S.H. SNYDER chronic reserpine t r e a t m e n t (Williams and Pirch, 1974; Tarsy and Baldessarini, 1973; Ungerstedt et al., 1975).
Acknowledgements
We thank Mrs. Linda Hester and Mr. Peter Sheehan for their excellent technical assistance, and Miss Pamela Morgan for manuscript preparation. This research was supported by USPHS grant MH-18501 to S.H.S., USPHS fellowship award MH--5105 to D.C.U'P., and by grants from the McKnight and John A. Hartford Foundation, Inc.
References
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(in press). Kafka, M.S., J.F. Tallman and C.C. Smith, 1977, aAdrenergic receptors on human platelets, Life Sci. 21, 1429.
RESERPINE ENHANCES BRAIN NE RECEPTOR BINDING Lefkowitz, R.J., D. Mullikin, C.L. Wood, T.B. Gore and C. Mukherjee, 1977, Regulation of prostaglandin receptors by prostaglandins and guanine nucleotides in frog erythrocytes, J. Biol. Chem. 252, 5292. Palmer, G.C., H.R. Wagner and R.W. Putman, 1976, Neuronal localization of the enhanced adenylate cyclase responsiveness to catecholamines in the rat cerebral cortex following reserpine injections, Neuropharmacology 15, 695. Peroutka, S.J., D.A. Greenberg, D.C. U'Prichard and S.H. Snyder, 1978, Regional variations in anoradrenergic receptor interactions of 3H-dihydroergokryptine in calf brain: implications for a twostate model of a-receptor function, Mol. Pharmacol. 14,403. Rotrosen, J., E. Friedman and S. Gershon, 1975, Striatal adenylate cyclase activity following reserpine and chronic chlorpromazine administration in rats, Life Sci. 17,563. Tarsy, D. and R.J. Baldessarini, 1973, Pharmacologically induced behavioral supersensitivity of apomorphine, Nature (New Biol.) 245,262, Trendelenburg, U., 1966, Mechanisms of supersensitivity and subsensitivity to sympathomimetic amines, Pharmacol. Rev. 18, 629. Ungerstedt, U., T. Ljungberg, B. Holler and G. Siggins, 1975, Dopaminergic supersensitivity in the striatum, Advan. Neurol. 9, 57. U'Prichard, D.C. and S.H. Snyder, 1977a, 3H-Epinephrine and 3H-norepinephrine binding to a-
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noradrenergic receptors in calf brain membranes, Life Sci. 20, 527. U'Prichard, D.C. and S.H. Snyder, 1977b, Binding of 3H-catecholamines to a-noradrenergic receptor sites in calf brain, J. Biol. Chem. 252, 6450. U'Prichard, D.C. and S.H. Snyder, 1978, Guanyl nucleotide influences on 3H-ligand binding to anoradrenergic receptors in calf brain membranes, J. Biol. Chem. 235, 3444. U'Prichard, D.C., D.A. Greenberg and S.H. Snyder, 1977a, Binding characteristics of a radiolabeled agonist and antagonist at central nervous system a-noradrenergic receptors, Mol. Pharmacol. 13, 454. U'Prichard, D.C., D.A. Greenberg, P. Sheehan and S.H. Snyder, 1977b, Regional distribution of anoradrenergic receptor binding in calf brain, Brain Res. 138, 151. Williams, B.J. and J.H. Pirch, 1974, Correlation between brain adenyl cyclase activity and spontaneous motor activity in rats after chronic reserpine treatment, Brain Res. 68, 227. Williams, L.T. and R.J. Lefkowitz, 1976, a-Adrenergic receptor identification by [3H]dihydroergokryptine binding, Science 192, 791. Williams, L.T. and R.J. Lefkowitz, 1977a, Regulation of rabbit myometrial alpha adrenergic receptors by estrogen and progesterone, J. Clin. Invest. 60, 815. Williams, L.T. and R.J. Lefkowtiz, 1977b, Thyroid hormone regulation of/3-adrenergic receptor number, J. Biol. Chem. 252, 2787.