Jmirrzal of Nperoclrrriir,ri-?. 1976. Vol 27. pp. lS6S ~ 1 5 6 8 .Pergnmon Press. Printed m Grcnt Britain


A dopamine-stimulated adenylate cyclase in rat substantia nigra (Received 29 April 1976. Accepted 2 July 1976)

THLPKLSENCE of dopamine (DA) receptors in the substanAdenylate cyclase activity was measured by the method er ul. (1972) with minor modifications. After tia nigra was first suggested by AGHAJANIAN& BUNNEY of KEBABIAN (1973) on the basis of microiontophoretic results. These 3 min of incubation the reaction was stopped by boiling authors showed that dopamine and the dopaminemimetic the samples for 3 min. Cyclic A M P (CAMP) was purified compound apomorphine, when applied by microionto- and assayed using the CAMP-dependent protein kinase phoresis to cell bodies of the substantia nigra. are able method described by KUO & GREENGARD (1972). Protein to inhibit the unit firing of these neurons (BUNNEY& was measured according to LOWRVet a/. (1951). AGHAJANIAN, 1975).In addition to these data are the histo& LINDVALL (1975) chemical observations of BJORKLLIND et ul. (1971) on the terminal dendritic proand PARIZEK RESULTS cesses of dopamine cell bodies in the nigra. These authors demonstrated that dopamine is stored in the extensive denPilot experiments showed that in substantia nigra homodritic processes of dopaminergic perikarya. Moreover the genates from rat and calf brains an adenylate cyclase same authors showed that an uptake mechanism for dopa- stimulated by dopamine was present. In order to tcst thc mine is present in the dendrites. More recently GEFFEN specificity of this effect we determined concentration-rcser 01. (1976) and KORFet a/. (1976) have provided evidence ponse curves for different catecholamines. The effects of for the release of DA from dendrites of dopaminergic various concentrations of dopamine. I-norepinephrine and neurons in rat substantia nigra. On the basis of these I-isoproterenol on adenylate cyclase activity in rat substanobservations, it has been proposed that a local dendritic tia nigra homogenates is shown in Fig. I. Dopamine at release of DA may have a number of functions in the sub- a concentration as low as I pM significantly stimulated stantia nigra neuronal network. the formation of cyclic AMP over the basal activity, while In this study we have found that a n adenylate cyclase I-norepinephrine at the same concentration had no etfect. which is preferentially stimulated by dopamine is present O n the other hand, the maximal stimulation of adenylate in the substantia nigra of rat brain. We suggest that the cyclase activity produced by I-norepinephrine was of the dopamine-stimulated adenylate cyclase of substantia nigra same order as that observed with dopamine. However, may be part of a receptor mechanism for the pool of dopa- considerably higher concentrations of I-norepinephrine mine released from the dendrites of dopaminergic neurons. than of dopamine were necessary to obtain maximum Therefore, the dopamine-stimulated adenylate cyclase of stimulation. Moreover. the /I-adrenoceptor agonist l-isothe substantia nigra may provide a highly specific model proterenol had no effect on the adenylate cyclase of subfor studying dopamine receptor function in this area. stantia nigra even at concentrations of 1000 AIM. Apomorphine-an agent that mimics the actions of dopamine in ef a/.. 1969: A N D ~ er N the caudate nucleus (UNGERSTEDT MATERIALS AND METHODS al., 1967) and which has been shown to stimulate adenylate Male Charles River rats, weighing 120-15Og were used cyclase activity in all areas of the brain where dopaminerin our experiments. After decapitation. the brains were gic terminals and dopamine-stimulated adenylate cyclase et nl.. 1972; HORNc’f a[.. quickly removed from the skull and frozen in powdered have been described (KEBABLAN solid CO,. Using a microtome at -1°C. serial sections 1974; TRABUCCHIer d.,1 9 7 6 t w a s also examined for (400 p n thick) were cut. Using the KONIG & KLIPPEL possible effects on adenylate cyclase in substantia nigra (1963)atlas as a guide. it was possible to localize the differ- homogenates. The results are shown in Fig. 2. Apomorent brain nuclei on the various coronal sections. Following phine at concentrations between 1 and 5 0 p i increased the the method of KOSLOWet a/. (1974) we used the decussa- formation of cyclic AMP over the basal activity. The maxition of the anterior commisure as the landmark for mea- mum stimulatory effect was achieved at a concentration suring the distance to the various areas. The nuclei were of 10 PM. However. apomorphine-induced stimulation of punched out with stainless steel tubing (1 mm diameter) cyclic A M P formation in substantia nigra tended to be and transfered to homogenizers (CHENEYer al., 1975). The lower than the maximal stimulation when concentrations wet weight for substantia nigra tissue was 2.8 k 0.2mg. of apomorphine higher than 50 p~ were used. These results et al. (1972) on adenyCalf substmtia nigra was removed within 30 min of parallel those obtained by KEBABIAN slaughter and frozen at -20°C until assayed (up to 3 days). late cyclase activity in rat caudate nucleus and suggest the possibility that in the substantia nigra apomorphinr may also behave as a mixed agonist-antagonist of the dopa1’1 d.. 1975). Ahhrevitrtions used: CAMP, cyclic AMP; DA. dopamine. mine-stimulated adenylate cyclase (IVERSEN 1565

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I566 400






.-'2 300

. E


; a


P 4 .-C

8 100,





0,' c l

U 0









concentration of catecholamine ()IMj

FIG. 1. Effect of dopamine. I-norepinephrine and I-isoproterenol on adenylate cyclase activity in rat substantia nigra homogenates. In absence of added compounds 210 15 pmol/min/mg protein of cAMP was formed. Each point represents the mean S.E.M. of at least 10 determinations.

We have also studied the effects of haloperidol and fluphenazine on the dopamine-stimulated formation of cyclic AMP in rat substantia nigra homogenates. These drugs are specific antagonists of dopamine-stimulated formation of cyclic AMP in all thc dopaminergic areas of the CNS so far examined (CLtmx.r-CoRvirR 1.r ( I ! . . 1974: IVFRSFN et 01.. 1975: MAKMAN et ul.. 1975: TRABWCIJI et a/.. 1976). The results in substantia nigra are summarized in Table I . Haloperidol and fluphenazine at 1 /IM concentrations



were able to block completely the increase in substantia nigra adenylate cyclase activity induced by dopamine. Neither haloperidol nor fluphenazine. at the concentrations used in this study, significantly inhibited basal activity of substantia nigra adenylate cyclase. Furt bermore, the antagonism of cyclic A M P formation induced by dopamine in substantia nigra homogenates was apparently of a competitive type (data not shown). The alpha-adrenoceptor antagonist phentolamine and the beta-adrenoceptor





concentration of Apomorphtne ( p M )

FIG. 2. ERect of aponiorphine on adenylate cyclase activity in rat substantia nigra homogenatcs. I n the absence of apomorphine 205 16 pmol/min/mg protein of cAMP was formed. Each point repS.E.M. of at least 10 determinations. resents the mean




Drug None Haloperidol Fluphenazine Propranolol Phentolamine





CAMP formation (pmol/mg prot/min) Dopamine + Dopamine


205 11 192 f. 15 180 f 22 213 k 18 207 13


410 f.25* 221 f. 16 230 k 21 391 +44* 371 f 37*

Drugs were added at a concentration of 1 p ~ . Dopamine was added at a concentration of 5 p ~ . Values are the mean S.E.M. of at least 12 determinations. (*) P < 0.001 in comparison with samples without DA added.



which the diencephalon was transected at a level anterior to the substantia nigra (A 2970-A 3290) the activity of dopamine-stimulated adenylate cyclase seems to be abolished. Therefore, there is a possible functional role for DA in the coordination and control of excitability both for the neuronal input and output from the substantia nigra. Further studies are in progress in our laboratory on mapping DA-receptor in substantia nigra either through the measurement of dopamine-stimulated adenylate cyclase or through the binding method of BURT et al. (1975). Department of Pharmacology and Pharmacognosy. Unitlersity of Milan Department of Pharmacology, University of Cagliuri Department of Pharmacology and Therapeutics, Universirj, of Brescia. Italy


REFERENCES blocking agent propanolol had no significant effects either on basal activity or on dopamine-stimulated cyclic AMP formation (Table 1).

A N D ~N. N E., RUBENSON A.. FUXE K. & HOKFELT T. (1967) J . Pharm. Pharmac. 19, 627429. ACHAJANIAN G. K. & BUNNEY B. S. (1973) in Froritiers S., eds.) in Catecholaniinr Research (USDINE. & SNYDER DISCUSSION pp. 643448. Pergamon Press, New York. The nigral dopaminergic neurons are so far the only BJORKLUND A. & LINDVALL A. (1975) Brain Res. 83. 531-537. adrenergic neurons in the brain for which storage, uptake, and release of transmitter have been demonstrated in den- BROWNJ. H. & MAKMAN M. H. (1972) Proc. natn Acad. dritic terminals (BJORKLUND & LINDVALL, 1975; KORFet Sci. U . S . A . 69, 539-543. al., 1976; GEFFENet al., 1976). Our results suggest that BUNNEY B. S. & AGHAJANIANG. K . (1975) in Pre- and dopamine released by the processes of dopaminergic nigral Posr~,vnLrpticReceprors (USDINE. & BUNNEY W. E.. eds.) neurons projecting into the pars compacta and the pars pp. 89-120. Dekker, New York. reticulata of the substantia nigra may have its own specific BURTD. R., ENNAS. J.. CREESE I. & SNYDER S. H. (1975) receptor operating through an adenylate cyclase system. Proc. iiatn Acud. Sci U.S.A. 72, 46554659. This hypothesis is supported by previous findings on CHENEY D. L.. LE FEVRE H. F. & RACACNI G. (1975) Neuradenylate cyclase which are specifically stimulated by opharmacology 14, 801-809. dopamine in homogenates of bovine superior cervical CLEMENT CORMIER Y. C., KEBABIAN J. W., PETZOLD G. L. ganglia (KEBABIAN & GREENCARD, 1971). calf and rat retina & GREENCARD P. (1974) Proc. natn Acad. Aci. U.S.A. (BROWN& MAKMAN, 1972). rat caudate nucleus (KEBABIAN 71, 1113-1117. A. C. & IVERSEN et al., 1972). rat nucleus accumbens and tuberculum olfac- GEFFENL. B., JESSELLT. M., CUELLO L. L. (1975) Nature, Lond. 260, 258-260. torium (HORNet al., 1974) and rat entorhinal cortex (TRABUCCHI et al., 1976). These findings have led to the suggesGROVESP. M., WILSONC. J.. YOUNG S. J. & REBEC G. Y. (1975) Science, N.Y 190. 527-529. tion that in these dopaminergic areas the dopamine-stimuHAJDUF., HASSLER R. & BAK I. J. (1973) Z . Zellforsch. lated adenylate cyclase and the ‘do,pamine-receptor’ may mikrosk. Annt. 146, 207-221. be related, and that the physiological effects of dopamine may be mediated by cyclic AMP. On the other hand our HORN A. S.. CUELLOA. C. & MILLERR. J. (1974) J . Neurochem. 22, 265-270. observations raise the questions as to the localization and IVERSEN L. L., HORNA. S. & MILLERR. J. (1975) in Prefunction of dopamine receptors in the substantia nigra. It has been observed by light and electron microscopy W. E., and Postsynaptic Receptors (USDINE. & BUNNEY eds.) pp. 207-243. Dekker, New York. that the perikarya of dopaminergic neurons ending in the striatum are mostly localized in the pars compacta of sub- KEBABIAN J. W. & GREENCARD P. ( 1 971) Science. N . Y 174, stantia nigra (RAMONY CAJAL,1904; HAJDUet al., 1973). 13461349. J. W., PETZOLD G. L. & GREENCARD P. (1972) The majority of the dendritic processes of these perikarya KEBABIAN project to the pars reticulata. whereas a certain portion Proc. natn Acad. Sci. U.S..4. 69. 2145-2149. ends within the pars compacta itself. The hypothesis, sug- KONIGJ. F. R. & KLIPPELR. A. (1963) The Rat Brain: gested also by the studies on the release of DA from dena Stereotaxic Atlas of the Forebrain and Lower Part of the Brain Stem Krieger, Huntington. New York. drites (GEFFENet a / . . 1976). is that DA receptors in the substantia nigra may be located on the DA neurons, as KORF J., ZIELEMAN M. & WESTERINK B. H. C. (1976) suggested also by the recent work of GROVES et al. (1975), Nature, Lond. 260, 257-258. S. H.. RACAGNIG. & COSTAE. (1974) Neuropharor presynaptically on other axon terminals afferent to the KOSLOW macology 13, 1123-1 130. nigra. This last possibility is strongly supported by some P. (1972) in Advances in Cyclic Nupreliminary findings we have obtained in our laboratory. Kuo J. & GREENCARD P., PAOLETTI R. cleotide Research Vol. 2 (GREENGARD Our preliminary data show the DA-stimulated adenylate & ROBINSON G. A,, eds.) pp. 41-50. Raven Press, New cyclase activity is not changed by making lesions in DA York. neurons with 6-hydroxydopamine whereas in animals in


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as Hisro/oyir du b!.sfPme Nerveu.~de I'tiomme et des VerLOWRY 0. H.. ROSEBROLGHN. J.. FARR A. L. & RAYDALL R. J. (1951) J . biol. C h m . 193, 265-275. tehr4.s (Cousejo Superior de Investigaciones Cientificas, MAKMAN M. H..MISHRAR . K. & BROWNJ. H. (1975) Inst. Ramon y Cajal, Madrid, 1955) Vol. 2, 275-278. Adc. Neurol. 9, 213-220. M., GOVONI S., TONONG. C. & SPANOP. F. TRAEUCCHI PARIZEK J., HASSLERH. & BAKI. J. (1971) Z . Zellforsch. (1976) J . Pkarrn. Pharrriac. 28. 244-245. mikrosk. Anat. 115, 137-148. UNGERSTEDTU.. BUTCHER L. L., BUTCHERS. G., ANDEN RAMONY CAJALS. (1904) Estrwturu de/ Sisrrrm~Kcwioso N.E. & FUXEK. (1969) Brain Res. 14, 461471. del Hottibrt. y de la C'rrrehratlos Madrid, 1904. reprinted

A dopamine-stimulated adenylate cyclase in rat substantia nigra.

Jmirrzal of Nperoclrrriir,ri-?. 1976. Vol 27. pp. lS6S ~ 1 5 6 8 .Pergnmon Press. Printed m Grcnt Britain SHORT COMMUNICATION A dopamine-stimulated...
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