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D2 dopamine receptors associated with inhibition of dopamine release from rat neostriatum are independent of cyclic AMP
Neuroscience Letters. 71 (1986) 192 196 Elsevier Scientitic Publishers Ireland Ltd.
192
NSL 04234
D2
dopamine receptors associated with inhibition of dopamine release from rat neostriatum are independent of cyclic AMP
Maurizio M e m o I, Cristina Missale l, Michele O. Carruba 2 and Pier Franco S pano 1 IInstitute q/Pharmacology and E.vperimen tal Therapeutics, School q/ Medicine, Universi O" qf Bres~'ia, 1-25124 Brescia, and-'Department q/' Pharmacology, Therapy and Medical Toxicology. University ff/"Milan, 1-20129 Milan (Italy) (Received 3 June 1986; Revised version received 21 July 1986: Accepted 31 July 1986)
Key words." Dopamine receptor
Adenylate cyclase
Dopamine release
Kainic acid
Striatum
Rat
The ability of the selective D2 dopamine (DA) receptor agonist bromocriptine to inhibit potassiuminduced DA release from striatal slices was measured in rats, which had been unilaterally injected with kainic acid into the left striatum, with the aim of verifying whether the central nervous system contains DA receptors whose stimulation evokes intracetlular events which do not involve cyclic AMP. It was found that increasing concentrations of bromocriptine inhibited the potassium-stimulated DA release from rat striatal slices of the kainic acid-treated side with the same potency as in control slices. On the contrary, bromocriptine and the selective D~ agonist S K F 82526 completely lost the ability to inhibit or stimulate, respectively, striatal adenylate cyclase activity from the lesioned side. Our conclusion asserts that inhibition of DA release from rat striatal slices is mediated by stimulation of D: DA receptors which are fully operative in absence of both DA-stimulated and DA-inhibited adenylate cyclase activity. These data suggest that the intracellular events that follow D2 receptor stimulation in the nigrostriatal nerve terminals may bc regulated by second messengers other than cyclic AMP.
A large body of experimental evidence points to the existence of multiple classes of dopamine (DA) receptors in mammalian brain which can be distinguished by their pharmacological specificity and localization and by their biochemical transduction mechanism [5, 12]. Specifically, two types of DA receptors whose stimulation affects cellular cyclic AMP have been recently characterized. Stimulation of the so-called D i DA receptors increases cyclic AMP formation, whereas stimulation of the D 2 DA receptors reduces the formation of cyclic AMP [8, 13]. This paradigm is currently used for anatomical, pharmacological and biochemical studies of DA receptors. Recent observations, however, raise the question whether the transduction mechanism ('orre.spondence." M. Memo, Institute of Pharmacology and Experimental Therapeutics, School of Medicine, University of Brescia, via Valsabbina 19, 1-25124 Brescia, Italy. 0304-3940/86/$ 03.50 (~) 1986 Elsevier Scientific Publishers Ireland Ltd.
193
of the DA signal may involve second messengers other than cyclic AMP. We have found that DA and various dopaminergic agonists prevent neurotensin-induced prolactin release by interacting with hypophysial D2 DA receptors which are independent from adenylate cylcase but functionally linked with calcium channels [7]. In addition, stimulation of D2 DA receptors in the pituitary reduces phosphatidylinositol breakdown leading to a diminished formation of putative second messengers such as diacyl glycerol and inositol triphosphate [1]. The aim of our study was to verify whether the central nervous system contains DA receptors whose stimulation evokes intracellular events which do not involve cyclic AMP. The functional expression of DA receptor stimulation was assessed by evaluating the ability of the selective D2 agonist bromocriptine to inhibit potassiuminduced DA release from rat striatal slices. Because of its specific toxic properties,
GO0- A
400-
control .-..I
control
200lesioned II,
I~,
lesioned
z
[SKF 8252G]
6
i
[-LOG M]
[BROMOCRIPTIN[]
Fig. 1. Effect of different concentrations of SKF 82526 (A) and bromocriptine (B) on adenylate cyclase activity in control and kainic acid-treated striatal homogenates. Values are expressed as pmol cyclic AMP/ mg prot./min, and are the m e a n + S . E . M , of 3 experiments performed on two tissue preparations. Male Sprague Dawley rats (160-180 g; Charles River, Calco, Italy) were anesthetized with Nembutal; 3 ag kainic acid (Sigma) in 1/A saline were injected stereotaxically during 2.5 min in the left striatum (coordinates: 7.9 A, 2.6 L, 4.8 V). The striatum contralateral to the lesion is referred to as control. Only rats which rotated ipsilaterally to the operated side after 0.25 mg/kg apomorphine injection were used as lesioned rats. Fourteen days after the lesion, the animals were sacrificed by decapitation, the brain was removed and the striatum of each side dissected out. Adenylate cyclase activity was assayed as previously described [2] with minor modifications. Striata were gently homogenized in 10 vols. (w/v) 10 m M Tris-HCl, pH 7.5, containing 1.2 mM EGTA. The incubation medium contained 82.5 mM Tris-HCl buffer (pH 7.4), 4 mM MgSO4, 5 mM theophylline, 0.6 mM EGTA, 0.02% ascorbic acid, 2 m M phosphocreatine, 50 U/ml creatine phosphokinase, 1 mM ATP and an aliquot of homogenate corresponding to I mg of tissue, fresh weight. The mixtures were incubated with or without increasing concentrations of the agonist for 20 min at 30~'C. The reaction was stopped by boiling the samples. The amount of cyclic A M P formed was measured by RIA kit of Du Pont-New England Nuclear.
194 kainic acid was used as a tool to discriminate between striatal DA receptors located on interneurons and on nerve terminals [9]. The functional activity of DA receptors positively and negatively coupled with adenylate cyclase was evaluated by measuring the ability of the selective Dl agonist SKF 82526 [11] and the selective D2 agonist bromocriptine [14] to stimulate or inhibit, respectively, adenylate cyclase activity in striatal homogenates. Fig. 1 shows the effects of intrastriatal injection of kainic acid on SKF 82526-stimulated (Fig. 1A) and bromocriptine-inhibited (Fig. I B) adenylate cyclase activity in striatal homogenates. Basal adenylate cyclase activity on the kainic acid-injected side was reduced to 60% compared to the contralateral side. In striatal homogenates from the control side maximally effective concentrations of SKF 82526 stimulated adenylate cyclase activity by about 95% while maximally effective concentrations of bromocriptine (100/tM) inhibited basal enzyme activity by 35%. The ECs0 values of SKF 82526 for stimulating adenylate cyclase activity were 2.0 + 0.2/tM. The IC50 values of bromocriptine for inhibiting adenylate cyclase activity were 2.5_+0.2 #M. In striatal homogenates from the lesioned side both SKF 82526 and bromocriptine lost their ability to modify adenylate cyclase activity. Fig. 2A shows the inhibitory effects of increasing concentrations of bromocriptine on potassium-stimulated DA release from striatal slices. Incubation of striatal slices with maximally effective concentrations of bromocriptine (100 nM) inhibited potassium-stimulated DA release by about 50%. The inhibitory effect elicited by bromocriptine was dose-dependent (IC50 = 2.5 _+0.5 nM) and mimicked by various dopaminergic agonists such as lisuride and apomorphine (data not shown). The inhibitory effects of bromocriptine were stereospecifically blocked by sulpiride isomers. Preincubation of slice preparations for 10 min with 1/tM (-)-sulpiride, but not with 1/zM (+)-sulpiride, completely prevented the inhibitory action of bromocriptine on potassium-evoked DA release (data not shown). Fig. 2B shows the effects of increasing cencentrations of bromocriptine on potassium-stimulated DA release from striatal slices of rats which have been unilaterally injected with kainic acid into the left striatum. The maximally stimulatory effect induced by 40 mM potassium was similar in striatal slices from the kainic acidtreated (Fig. 2B) and control (Fig. 2A) side. Maximally effective concentrations of bromocriptine (100 nM) inhibited the potassium-stimulated DA release from striatal slices of the kainic acid-treated side (Fig. 2B) with the same potency as in control slices (Fig. 2A). The IC50 values of bromocriptine for inhibiting the potassium-stimulated DA release were slightly increased when compared with those from the control side (2.5 4-0.5 nM for control, 10 4-2 nM for kainic acid-lesioned side). The results of the present study show that inhibition of DA release from rat striatal slices is mediated by stimulation of D2 DA receptors which are fully operative in the absence of both DA-stimulated and DA-inhibited adenylate cyclase activity. These data suggest that the intraceUular events that follow D2 receptor stimulation in the nigrostriatal nerve terminals may be regulated by second messengers other than cyclic AMP. Detailed anatomical studies performed by using radioreceptor binding techniques
195 r=:
];B
A
=4'
4"
r::::
k.a..I
3" I..i.a ,.,1..-
IC50 = 10 nM
IC50 = 3 nM 2-L _, basal II,
2T
,,basal II,
[BROMOCI IPTIN[]
[-log M]
Fig. 2. Effects of different concentrations of bromocriptine on DA release induced by potassium from control (A) and kainic acid-pretreated (B) striatal slices. Animals were unilaterally injected with kainic acid into the left striatum as reported in the legend of Fig. 1. Striatal slices were prepared according to Memo et al. [6]. The slices were preincubated for 20 min at 37°C in Krebs-bicarbonate buffer supplemented with 10 mM dextrose under constant oxygenation (95% 02-5% CO2). Thereafter, the incubation medium was removed from the tubes and the slices were incubated for an additional 20 min in fresh Krebs-bicarbonate buffer containing 0.4 mM pargyline, 1 pM nomifensine, and various concentrations of potassium and bromocriptine as reported in the result section. Each tube contained an aliquot of striatal slices corresponding to about 7 8 mg of fresh tissue. At the end of the incubation, samples were centrifuged at 5000 g for 10 min, the supernatant was collected and DA was extracted by liquid-solid extraction into alumina followed by elution with dilute acid. Endogenous DA levels were determined by HPLC (303 Bioanalytical System employing an amperometric glass carbon detector) as previously described [4]. have s h o w n t h a t within the s t r i a t u m a b o u t 40% o f D2 D A r e c e p t o r s are present on the m e m b r a n e o f intrinsic n e u r o n s while the r e m a i n i n g 60% is l o c a t e d p r e s y n a p t i c a l l y on the nerve terminals o r i g i n a t i n g f r o m the cerebral cortex and, p r e s u m a b l y , the subs t a n t i a nigra [3, 10]. The results o b t a i n e d m e a s u r i n g the functional activity o f D2 D A receptors, i.e. b y a s s a y i n g the ability o f specific D2 r e c e p t o r a g o n i s t s to inhibit cyclic A M P f o r m a t i o n , indicate t h a t the D2 r e c e p t o r s t h a t are negatively c o u p l e d with adenylate cyclase are confined to n e u r o n s intrinsic to the striatum. T a k e n together, these results suggest t h a t only a small p o r t i o n o f D2 D A r e c e p t o r s is negatively c o u p l e d with a d e n y l a t e cyclase while the m a j o r i t y o f them, which are l o c a t e d p r e s y n a p t i c a l l y in the afferents f r o m the s u b s t a n t i a nigra a n d c e r e b r a l cortex, are i n d e p e n d e n t o f the cyclic A M P - g e n e r a t i n g system. This h y p o t h e s i s is c o r r o b o r a t e d by the d a t a s h o w i n g the ability o f b r o m o c r i p t i n e to inhibit p o t a s s i u m - i n d u c e d D A release f r o m k a i n i c a c i d - p r e t r e a t e d striata, e.g. in an e x p e r i m e n t a l c o n d i t i o n where D A - s e n s i t i v e a d e n y l a t e cyclase, t o g e t h e r with the intrinsic neurons, is virtually absent.
196
Preliminary results obtained in our laboratory indicate that bromocriptine prevents the potassium-induced DA release by inhibiting the influx of calcium and the increase in phosphatidylinositol turnover that are both elicited by potassium. These observations lead to the hypothesis of the existence of two transduction mechanisms for D2 DA receptors in rat striatum one of which involves inhibition of adenylate cyctase while the other is possibly linked to calcium. Moreover, the different D2 DA receptors appear to be differentially located; one negatively coupled with adenylate cyclase is restricted to intrinsic neurons, while the other is present on nerve terminals of extrinsic neurons. I Canonico, P.L., Valdenegro, C.A. and MacLeod, R.M., The inhibition of phosphatidylinositol turnover: a possible postreceptor mechanism for the prolactin secretion-inhibiting effect of dopamine, Endocrinology, 113 (1983) 7 14. 2 Clement-Cormier, Y.C., Kcbabian, J.W., Petzold, G.L. and Greengard, P., Dopaminc sensitive adenylate cyclase in mammalian brain: a possible site of action of antipsychotic drugs, Proc. Natl. Acad. Sci. USA, 71(1978) 1113 1117. 3 Garau, L., Govoni, S., Stefanini, E., Trabucchi, M. and Spano, P.F., Dopamine receptors: pharmacological and anatomical evidences indicate that two distinct dopamine receptor populations are present in rat striatum, Life Sci., 23 (1978) 1745 1750. 4 lmperato, A. and Di Chiara, G., Trans-striatal dialysis coupled to reverse phase high performance liquid chromatography with electrochemical detection: a new method for the study of the in vivo release of endogenous dopamine and metabolites, J. Neurosci., 4 (1984) 966 977. 5 Kebabian, J.W. and Calne, D.B., Multiple receptors for dopamine, Nature (London), 277 (1979) 93 96. 6 Memo, M., Lovenberg, W. and Hanbauer, 1., Agonist-induced subsensitivity of adenylate cyclase coupled with a dopamine receptor in slices from rat corpus striatum, Proc. Natl. Acad. Sci. USA, 79 (1982) 4456 4460. 7 Memo, M., Carboni, E., Trabucchi, M., Carruba, M.O. and Spano, P.F., Dopamine inhibition of neurotensin-induced increase in Ca influx into rat pituitary cells, Brain Res., 347 (1985) 253 257. 8 Onali, P.L., Olianas, M.C. and Gessa, G.L., Characterization of dopamine receptors mediating inhibition of adenylate cyclase activity in rat striatum, Mol. Pharmacol., 28 (1985) 138 145. 9 Schwarcz, R. and Coyte, J.T., Striatal lesions with icahlic acid: neurochemical characteristics, Brain Res.. 127 (1977) 235 249. I0 Schwarcz, R., Creese, I., Coyle, J.T. and Snyder, S.H., Dopamine receptors localized on cerebral cortical afferents to rat corpus striatum, Nature (London), 271 (1978) 766- 768. 11 Silbey, D.R., Left, S.E. and Creese, I., Interactions of novel dopaminergic ligands with D-[ and D-2 dopamine receptors, Life Sci., 31 (1982) 637 645. 12 Spano, P.F., Govoni, S. and Trabucchi, M., Studies on the pharmacological properties of dopamine receptors in various areas of the central nervous system, Adv. Biochem. Psychopharmacol., 19 (1978) 155 165. 13 Stool', J.C. and Kebabian, J.W., Opposing roles for D I and D2 dopamine receptors in efflux of cyclic AMP from rat neostriatum, Nature (London), 294 (1981) 366--368. t4 Trabucchi, M., Spano, P.F., Tonon, G.C. and Franola, L., Effects of bromocriptine on central dopaminergic receptors, Life Sci., 19 (1976) 225 232.
192
NSL 04234
D2
dopamine receptors associated with inhibition of dopamine release from rat neostriatum are independent of cyclic AMP
Maurizio M e m o I, Cristina Missale l, Michele O. Carruba 2 and Pier Franco S pano 1 IInstitute q/Pharmacology and E.vperimen tal Therapeutics, School q/ Medicine, Universi O" qf Bres~'ia, 1-25124 Brescia, and-'Department q/' Pharmacology, Therapy and Medical Toxicology. University ff/"Milan, 1-20129 Milan (Italy) (Received 3 June 1986; Revised version received 21 July 1986: Accepted 31 July 1986)
Key words." Dopamine receptor
Adenylate cyclase
Dopamine release
Kainic acid
Striatum
Rat
The ability of the selective D2 dopamine (DA) receptor agonist bromocriptine to inhibit potassiuminduced DA release from striatal slices was measured in rats, which had been unilaterally injected with kainic acid into the left striatum, with the aim of verifying whether the central nervous system contains DA receptors whose stimulation evokes intracetlular events which do not involve cyclic AMP. It was found that increasing concentrations of bromocriptine inhibited the potassium-stimulated DA release from rat striatal slices of the kainic acid-treated side with the same potency as in control slices. On the contrary, bromocriptine and the selective D~ agonist S K F 82526 completely lost the ability to inhibit or stimulate, respectively, striatal adenylate cyclase activity from the lesioned side. Our conclusion asserts that inhibition of DA release from rat striatal slices is mediated by stimulation of D: DA receptors which are fully operative in absence of both DA-stimulated and DA-inhibited adenylate cyclase activity. These data suggest that the intracellular events that follow D2 receptor stimulation in the nigrostriatal nerve terminals may bc regulated by second messengers other than cyclic AMP.
A large body of experimental evidence points to the existence of multiple classes of dopamine (DA) receptors in mammalian brain which can be distinguished by their pharmacological specificity and localization and by their biochemical transduction mechanism [5, 12]. Specifically, two types of DA receptors whose stimulation affects cellular cyclic AMP have been recently characterized. Stimulation of the so-called D i DA receptors increases cyclic AMP formation, whereas stimulation of the D 2 DA receptors reduces the formation of cyclic AMP [8, 13]. This paradigm is currently used for anatomical, pharmacological and biochemical studies of DA receptors. Recent observations, however, raise the question whether the transduction mechanism ('orre.spondence." M. Memo, Institute of Pharmacology and Experimental Therapeutics, School of Medicine, University of Brescia, via Valsabbina 19, 1-25124 Brescia, Italy. 0304-3940/86/$ 03.50 (~) 1986 Elsevier Scientific Publishers Ireland Ltd.
193
of the DA signal may involve second messengers other than cyclic AMP. We have found that DA and various dopaminergic agonists prevent neurotensin-induced prolactin release by interacting with hypophysial D2 DA receptors which are independent from adenylate cylcase but functionally linked with calcium channels [7]. In addition, stimulation of D2 DA receptors in the pituitary reduces phosphatidylinositol breakdown leading to a diminished formation of putative second messengers such as diacyl glycerol and inositol triphosphate [1]. The aim of our study was to verify whether the central nervous system contains DA receptors whose stimulation evokes intracellular events which do not involve cyclic AMP. The functional expression of DA receptor stimulation was assessed by evaluating the ability of the selective D2 agonist bromocriptine to inhibit potassiuminduced DA release from rat striatal slices. Because of its specific toxic properties,
GO0- A
400-
control .-..I
control
200lesioned II,
I~,
lesioned
z
[SKF 8252G]
6
i
[-LOG M]
[BROMOCRIPTIN[]
Fig. 1. Effect of different concentrations of SKF 82526 (A) and bromocriptine (B) on adenylate cyclase activity in control and kainic acid-treated striatal homogenates. Values are expressed as pmol cyclic AMP/ mg prot./min, and are the m e a n + S . E . M , of 3 experiments performed on two tissue preparations. Male Sprague Dawley rats (160-180 g; Charles River, Calco, Italy) were anesthetized with Nembutal; 3 ag kainic acid (Sigma) in 1/A saline were injected stereotaxically during 2.5 min in the left striatum (coordinates: 7.9 A, 2.6 L, 4.8 V). The striatum contralateral to the lesion is referred to as control. Only rats which rotated ipsilaterally to the operated side after 0.25 mg/kg apomorphine injection were used as lesioned rats. Fourteen days after the lesion, the animals were sacrificed by decapitation, the brain was removed and the striatum of each side dissected out. Adenylate cyclase activity was assayed as previously described [2] with minor modifications. Striata were gently homogenized in 10 vols. (w/v) 10 m M Tris-HCl, pH 7.5, containing 1.2 mM EGTA. The incubation medium contained 82.5 mM Tris-HCl buffer (pH 7.4), 4 mM MgSO4, 5 mM theophylline, 0.6 mM EGTA, 0.02% ascorbic acid, 2 m M phosphocreatine, 50 U/ml creatine phosphokinase, 1 mM ATP and an aliquot of homogenate corresponding to I mg of tissue, fresh weight. The mixtures were incubated with or without increasing concentrations of the agonist for 20 min at 30~'C. The reaction was stopped by boiling the samples. The amount of cyclic A M P formed was measured by RIA kit of Du Pont-New England Nuclear.
194 kainic acid was used as a tool to discriminate between striatal DA receptors located on interneurons and on nerve terminals [9]. The functional activity of DA receptors positively and negatively coupled with adenylate cyclase was evaluated by measuring the ability of the selective Dl agonist SKF 82526 [11] and the selective D2 agonist bromocriptine [14] to stimulate or inhibit, respectively, adenylate cyclase activity in striatal homogenates. Fig. 1 shows the effects of intrastriatal injection of kainic acid on SKF 82526-stimulated (Fig. 1A) and bromocriptine-inhibited (Fig. I B) adenylate cyclase activity in striatal homogenates. Basal adenylate cyclase activity on the kainic acid-injected side was reduced to 60% compared to the contralateral side. In striatal homogenates from the control side maximally effective concentrations of SKF 82526 stimulated adenylate cyclase activity by about 95% while maximally effective concentrations of bromocriptine (100/tM) inhibited basal enzyme activity by 35%. The ECs0 values of SKF 82526 for stimulating adenylate cyclase activity were 2.0 + 0.2/tM. The IC50 values of bromocriptine for inhibiting adenylate cyclase activity were 2.5_+0.2 #M. In striatal homogenates from the lesioned side both SKF 82526 and bromocriptine lost their ability to modify adenylate cyclase activity. Fig. 2A shows the inhibitory effects of increasing concentrations of bromocriptine on potassium-stimulated DA release from striatal slices. Incubation of striatal slices with maximally effective concentrations of bromocriptine (100 nM) inhibited potassium-stimulated DA release by about 50%. The inhibitory effect elicited by bromocriptine was dose-dependent (IC50 = 2.5 _+0.5 nM) and mimicked by various dopaminergic agonists such as lisuride and apomorphine (data not shown). The inhibitory effects of bromocriptine were stereospecifically blocked by sulpiride isomers. Preincubation of slice preparations for 10 min with 1/tM (-)-sulpiride, but not with 1/zM (+)-sulpiride, completely prevented the inhibitory action of bromocriptine on potassium-evoked DA release (data not shown). Fig. 2B shows the effects of increasing cencentrations of bromocriptine on potassium-stimulated DA release from striatal slices of rats which have been unilaterally injected with kainic acid into the left striatum. The maximally stimulatory effect induced by 40 mM potassium was similar in striatal slices from the kainic acidtreated (Fig. 2B) and control (Fig. 2A) side. Maximally effective concentrations of bromocriptine (100 nM) inhibited the potassium-stimulated DA release from striatal slices of the kainic acid-treated side (Fig. 2B) with the same potency as in control slices (Fig. 2A). The IC50 values of bromocriptine for inhibiting the potassium-stimulated DA release were slightly increased when compared with those from the control side (2.5 4-0.5 nM for control, 10 4-2 nM for kainic acid-lesioned side). The results of the present study show that inhibition of DA release from rat striatal slices is mediated by stimulation of D2 DA receptors which are fully operative in the absence of both DA-stimulated and DA-inhibited adenylate cyclase activity. These data suggest that the intraceUular events that follow D2 receptor stimulation in the nigrostriatal nerve terminals may be regulated by second messengers other than cyclic AMP. Detailed anatomical studies performed by using radioreceptor binding techniques
195 r=:
];B
A
=4'
4"
r::::
k.a..I
3" I..i.a ,.,1..-
IC50 = 10 nM
IC50 = 3 nM 2-L _, basal II,
2T
,,basal II,
[BROMOCI IPTIN[]
[-log M]
Fig. 2. Effects of different concentrations of bromocriptine on DA release induced by potassium from control (A) and kainic acid-pretreated (B) striatal slices. Animals were unilaterally injected with kainic acid into the left striatum as reported in the legend of Fig. 1. Striatal slices were prepared according to Memo et al. [6]. The slices were preincubated for 20 min at 37°C in Krebs-bicarbonate buffer supplemented with 10 mM dextrose under constant oxygenation (95% 02-5% CO2). Thereafter, the incubation medium was removed from the tubes and the slices were incubated for an additional 20 min in fresh Krebs-bicarbonate buffer containing 0.4 mM pargyline, 1 pM nomifensine, and various concentrations of potassium and bromocriptine as reported in the result section. Each tube contained an aliquot of striatal slices corresponding to about 7 8 mg of fresh tissue. At the end of the incubation, samples were centrifuged at 5000 g for 10 min, the supernatant was collected and DA was extracted by liquid-solid extraction into alumina followed by elution with dilute acid. Endogenous DA levels were determined by HPLC (303 Bioanalytical System employing an amperometric glass carbon detector) as previously described [4]. have s h o w n t h a t within the s t r i a t u m a b o u t 40% o f D2 D A r e c e p t o r s are present on the m e m b r a n e o f intrinsic n e u r o n s while the r e m a i n i n g 60% is l o c a t e d p r e s y n a p t i c a l l y on the nerve terminals o r i g i n a t i n g f r o m the cerebral cortex and, p r e s u m a b l y , the subs t a n t i a nigra [3, 10]. The results o b t a i n e d m e a s u r i n g the functional activity o f D2 D A receptors, i.e. b y a s s a y i n g the ability o f specific D2 r e c e p t o r a g o n i s t s to inhibit cyclic A M P f o r m a t i o n , indicate t h a t the D2 r e c e p t o r s t h a t are negatively c o u p l e d with adenylate cyclase are confined to n e u r o n s intrinsic to the striatum. T a k e n together, these results suggest t h a t only a small p o r t i o n o f D2 D A r e c e p t o r s is negatively c o u p l e d with a d e n y l a t e cyclase while the m a j o r i t y o f them, which are l o c a t e d p r e s y n a p t i c a l l y in the afferents f r o m the s u b s t a n t i a nigra a n d c e r e b r a l cortex, are i n d e p e n d e n t o f the cyclic A M P - g e n e r a t i n g system. This h y p o t h e s i s is c o r r o b o r a t e d by the d a t a s h o w i n g the ability o f b r o m o c r i p t i n e to inhibit p o t a s s i u m - i n d u c e d D A release f r o m k a i n i c a c i d - p r e t r e a t e d striata, e.g. in an e x p e r i m e n t a l c o n d i t i o n where D A - s e n s i t i v e a d e n y l a t e cyclase, t o g e t h e r with the intrinsic neurons, is virtually absent.
196
Preliminary results obtained in our laboratory indicate that bromocriptine prevents the potassium-induced DA release by inhibiting the influx of calcium and the increase in phosphatidylinositol turnover that are both elicited by potassium. These observations lead to the hypothesis of the existence of two transduction mechanisms for D2 DA receptors in rat striatum one of which involves inhibition of adenylate cyctase while the other is possibly linked to calcium. Moreover, the different D2 DA receptors appear to be differentially located; one negatively coupled with adenylate cyclase is restricted to intrinsic neurons, while the other is present on nerve terminals of extrinsic neurons. I Canonico, P.L., Valdenegro, C.A. and MacLeod, R.M., The inhibition of phosphatidylinositol turnover: a possible postreceptor mechanism for the prolactin secretion-inhibiting effect of dopamine, Endocrinology, 113 (1983) 7 14. 2 Clement-Cormier, Y.C., Kcbabian, J.W., Petzold, G.L. and Greengard, P., Dopaminc sensitive adenylate cyclase in mammalian brain: a possible site of action of antipsychotic drugs, Proc. Natl. Acad. Sci. USA, 71(1978) 1113 1117. 3 Garau, L., Govoni, S., Stefanini, E., Trabucchi, M. and Spano, P.F., Dopamine receptors: pharmacological and anatomical evidences indicate that two distinct dopamine receptor populations are present in rat striatum, Life Sci., 23 (1978) 1745 1750. 4 lmperato, A. and Di Chiara, G., Trans-striatal dialysis coupled to reverse phase high performance liquid chromatography with electrochemical detection: a new method for the study of the in vivo release of endogenous dopamine and metabolites, J. Neurosci., 4 (1984) 966 977. 5 Kebabian, J.W. and Calne, D.B., Multiple receptors for dopamine, Nature (London), 277 (1979) 93 96. 6 Memo, M., Lovenberg, W. and Hanbauer, 1., Agonist-induced subsensitivity of adenylate cyclase coupled with a dopamine receptor in slices from rat corpus striatum, Proc. Natl. Acad. Sci. USA, 79 (1982) 4456 4460. 7 Memo, M., Carboni, E., Trabucchi, M., Carruba, M.O. and Spano, P.F., Dopamine inhibition of neurotensin-induced increase in Ca influx into rat pituitary cells, Brain Res., 347 (1985) 253 257. 8 Onali, P.L., Olianas, M.C. and Gessa, G.L., Characterization of dopamine receptors mediating inhibition of adenylate cyclase activity in rat striatum, Mol. Pharmacol., 28 (1985) 138 145. 9 Schwarcz, R. and Coyte, J.T., Striatal lesions with icahlic acid: neurochemical characteristics, Brain Res.. 127 (1977) 235 249. I0 Schwarcz, R., Creese, I., Coyle, J.T. and Snyder, S.H., Dopamine receptors localized on cerebral cortical afferents to rat corpus striatum, Nature (London), 271 (1978) 766- 768. 11 Silbey, D.R., Left, S.E. and Creese, I., Interactions of novel dopaminergic ligands with D-[ and D-2 dopamine receptors, Life Sci., 31 (1982) 637 645. 12 Spano, P.F., Govoni, S. and Trabucchi, M., Studies on the pharmacological properties of dopamine receptors in various areas of the central nervous system, Adv. Biochem. Psychopharmacol., 19 (1978) 155 165. 13 Stool', J.C. and Kebabian, J.W., Opposing roles for D I and D2 dopamine receptors in efflux of cyclic AMP from rat neostriatum, Nature (London), 294 (1981) 366--368. t4 Trabucchi, M., Spano, P.F., Tonon, G.C. and Franola, L., Effects of bromocriptine on central dopaminergic receptors, Life Sci., 19 (1976) 225 232.