Lifa Scieaces Vol . 19, pp . 1061-1066, 1976 . Priated in tha ü .S .A .

Pargamon Prass

DOPAMINE-SENSITIVE ADENYLATE CYCLA3E AND CAMP PHOSPH~IESTERASE IN 3UBSTANTIA NI(;RA AND CORPUS STRIATUM OF RAT BRAIN L. J. Traßcante, E. Friedman, M. A. Oleehansl~, and S. Gershan Department of Psychiatry, New York University School of Medicine, 550 First Avenue, New York, N. Y. 10018 (Becaived in final form August 16, 1976)

Summary A dopamine-sensitive adenylate cyclase has been identified in substantia nigra a~f the rat brain. Low conce~rations a~f dopamine markedly increased the accumulation of cyclic AMP while 1-narepinephrhne and isoproterenol had Sale effect at cancentratians up to 100 pM. The phenothiazine, trifluoperazine, is a potent inhibitor od the s, nigral adulate cyclase (~. C. ~ of 0. 1 1~, while the adrenergic receptor blocking ages prapranolol and phentolamine are ineffective. Basal activity a~f striatal adeny]ate cyclase and cAMP phosphodiesterase is approximately 3-fold higher than that found in substantia nigra. A dopamine-sensitive adenylate cyc]ase has been demonstrated in the corpus striatum of rat brain (1). The striatum contains terminals of dopaminergic neurons which originate in peri~rya locaSzed in the zona com pacta od the substantia nigra (3) . Anatomical studies of dendritic processes of the s, nigra have revealed vesicular structures which have been shown to contain (5, 6) and release (7, ß) dopamine . in the present communication, we have ~demanstrated a dopamine-sensitive adenylate cyclase which may mediate the postulated actions of dopamine in this brain raglan (8). In addition, a preliminary investigation of cA1ViP phosphodiesterase in striatum and s, nigra has been undertaken. Methods Male, Sprague-Dawley rats, 150-800 g, were killed by decapitation and the brains rapidly excised and dissected at 0-4° C . For the assay of adenylate cyclase, pairs aß s, nigra from each rat were pooled and homo genized gently by hand in a Kontes glass-teflon ha~nogenizer in 30 volumes (w/v) of ice-cold 8mM Tris-base containing 0. 8 mM EGTA brought to pH 7. 4 by titration with malefic acid. The standard assay mixture (ßnal volume 380 ul) contained in mmol/Yiter : Tris-base 80; Trie-ATP, 0. 5; MgSO,i, $. 0; theophylline, 10. 0; E(ITA, 0. 8; (pH adjusted to 7. 4 with malefic acid), 0. OS ml aß tissue homogenate plus test substances as indicated . Pre-incubation was carried out for 30 min at 0° C with all ca~mponents of the standard assay 1061

losz

A. Cyclasa and

rAMD

Phoaphodieaterase

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system except ATP; the reaction mixture was placed in a water bath at 30° C for 5 min at the e~ of which the ruction was commenced by the addition oaf ATP (SO pl). The system was allowed to incubate for 2. 5 min at 30 ° C and the reaction terminated by placing the tubes in a boiling water bath for 2 . 5 min. Tubes were then centrifuged and the supernatant fluid assayed for cyclic AMP by the method ad Brown et al (9) . Away of adenylate cyclase in rat striatum was carried out as described above for s, nigra except that the tissue was homogenized in 75 volumes a~f homogenization buffer . Phosphodiesterase was assayed by the one-step method of Brooker et al (10) as modified by Füburn and Barn (11). Incubfttlans were carried oûf"for 8 min. Results 3timulatian a~f adenylate cyclase in haanogenates od rat s, nigra and striatum by dopamine are presented in Figs. lA and B. The addition a~f dopamine to the incubation mixture produced a marked enhancement of adenylate cyclaee activity in a dose-related maser. A half-ma~dmal increase in enzyme activity was observed in both brain regions with $. 8 yiM dopamine. 11Rsiimal stimulation occurred in the presence of a5-50 pM dopamine. Basal activity in the s, nigra (7. 5 f S. 8 p moles CAMP/mg tissue/$. 5 min) was considerably lower than in striatum (S~ . 5 f 5 p moles cAMP/mg tissue/$. 6 min); haavever, overall percent etimulaüan was similar . We examined the effect ad trifluoperazine, propranolol and phentolamine on the dopamine-sensitive adealylate cyclase in s, migra (Fig. a) . Trifluoperazine produced a 50~ bloctsade a~! dopamine-induced production of CAMP at a concentration a~f 0. 1 ~u1VI and 100~ô inhibition at S . 5 ubl . Both propranolol and phentolamine had little effect an enzyme stimulation with ccnce~ratians ad up to 10 and 80 pM, respectively, The effect of various cancentratians ad the catecholamines 1-norepinephrine and isoproterenol cn adenylate cyclase activity of s, nigra homogenates is shown in Fig. 9 . Norepinephrine (12, 6 >~ produced a 33~ increase in enzyme activity ; isoproterenol (18, 5 ~ addition enhanced enzyme activity by lad. The same concentration of dopamine produced a 198 activation ad adenylate cyclaee (Fig. lA) . Cyclic AMP phosphodiesteraee activity was measured at substrate conc~tratians a~f 100 and 3 ~ in homogenates of e, nigra and striatum . A twoto threefold higher specific activity for the enzyme was obtained in striatum than in s, nigra (Table 1).

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TABLE 1 cAMP Phosphodiesterase Activity in Rat Striatum and S. Nigra

Tissue

-

-

n-moles CAMP hydrolyzed/mg protein/min, f 3, E. M. (4) 100 p1VI CAMP s~McAMp

striatum

sa . ~ ± a, ~

s, o t o, a

S, Nigra

18. 9 ± 0, 9

1. 3 ± 0. 1

PDE was assayed as described in Methode, r

A

3odo,~

B ô 1QfJ

y DOPAMINE (uM) FIG. 1 Effect oaf d ins oa adenylate cyclase activity in a homogenate of rat striatum (A~and substantia nigra (H) .

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100 0

~- SO z O m z

40 20 0 DRUG CONCENTRATION (~M) FIG .

a

Inhibition of dopamine-stimulated adenylate cyclase activity in homogenates of rat substantia nigra. Homogenates were incubated with and without 85 pM dopamine and various concentrations of test drugs. Inhibition is eapreseed as the percent of drug-induced reduction in dopamine-stimulated sdenylate cyclase activity. (e) trifluoperazine, (o) propranolol, U~hentolamine. Discussion The present data demonstrates the existence of a specific dopamineseneitive adenylate cyclase in s, nigra ad rat brain. The specificity of the enzyme for dopamine resembles that of rat etriatal adenylate cyclase (1) . It exhibits greater stimulation to dopamine than to 1-norepinephrine or isoproterenol and more marked inhibition of dopamine stimulation with trifluoperazine, a dopaminergic blocking aged, than to d, 1-propranolol or phentolamine, q and ~3-adrenergic blocking agea~ts, respectively. Our Eindinge are consistent with those recently reported bq Phillipson et al (1S). The finding of a dopamine-sensitive adenylate cyclase in a region rich in dopamine cell bodies is consistent with recent work demonstrating dendritic vesicles (3, 4) and release and uptake a~f dopamine (5, 8, ~ in the s, nigra . A role a~f dopamine as an auto-regulator at the level of the cell body as well as the dopaminergic neuron terminals has been postulated previously (8, 18). Our ßnding that basal adenylate cyclase activity is louver in s. nigra than striatum directed us to measure cA1Vlp phoephodiesterase activity in these iwo areas (14). An investigation of cAMP phosphodiesteraee reviled

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clearly lover values far e, nigra thaw striatum . Thin would not account for the regional differences in basal cAMP accumulation observed . Whether these differences in basal adenylate cyclase and cAMP phosphodiesterase activities can be correlated with functional dopaminergic input, as previously postulated for ~ -adrenerglc activation in pineal, is currently being studied (15).

CATECHOLAMINE(uM) FIG. 3 Effect of 1-norepinephrhne and isoproterenol an adenylate cyclase activity in a ha~mogenate of rat eubstaatia nigra. (~) 1-norepinephrine, (o) ieoproterenol .

References 1. $. 9. 4.

J. W. I~HABIAN, G. PETZOLD, and P . GREENGARD, Proc. Natl. Acsd. Sci . U 8. A. , 89, 3146 (1972). Ac~a Physiol. Scared. S~; 1. , 387, 1-87 (1971) . E.ra RIIdVIK and I. ~ , n es. , ~ 229-348 (1970). F. HAJDU, R. EASSLER, a . . eII~orschung, 148, 207-331 (1979).

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O. BJORKLiJND and O. LIIVDVALL, Brain Res. , 83, 591-537 (1975). J. I~RF, M. ZIELEMAN, and B. H.Z~~RII~K, Nature, 980, 957-958 (1978). 7. L. H. GEFFEN, T. M. JE88ELL, A. C. CUELLO, and L. L. IVERB~T, Nature 980 958-980 (1978). 8. ~~( EB, C. J. WIL90N, 3. J. YOLAG, and G. V. REHEC, Sci ., 190, 599-599 (1975). 9. Ï~ L. -$ROWN, J. D. M. ALBANS, R. P. EI~T3, and A. M. SQHENZI, Biochem . J., 191, 581-589 (1971). 10 . ~I~, L. J. THOMAS, and M. M. APPELMAN, Biochem . 181, 4177-4181(1988) . 11. Zr`-R. FILHURN and J. KARN, Anal. Biochem ., 59, 505-518 (1979). antiers in Catecholamine 19 . G. K. AGHAJANiAN s~ H . 3. ~ , er, ergama~n, Research 843-848), Eds. : E. Usdin, . . R~r~~1979) . 13. O. T. PHILLIP3~T and A. 3. HORN, Nature, 981, 418-490 (1978). 14. M. A. OLE5HAN3KY and N. H. NEFF~L~iéSc~ 17, 1499-149a (1975) . olOf`f`g6armacol., il, 559-557 15 . M. A. OLE3HAN3KY and N . H. NEFF, (1975). 5. 8.

Dopamine-sensitive adenylate cyclase and cAMP phosphodiesterase in substantia nigra and corpus striatum of rat brain.

Lifa Scieaces Vol . 19, pp . 1061-1066, 1976 . Priated in tha ü .S .A . Pargamon Prass DOPAMINE-SENSITIVE ADENYLATE CYCLA3E AND CAMP PHOSPH~IESTERAS...
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