Juitr,io/ u/ ivuiirocliefiii\r,.!.
1976 Vol 27. pp 1533 1535 Pcrganion PI?%.Printed in Grcal Britain.
SHORT COMMUNICATION
Nucleotide requirement of dopamine sensitive adenylate cyclase in synaptosomal membranes from the striatum of rat brain (Received 20 April 1976. Accepted 2 June 1976)
NUCLEOTIDES, particularly GTP and its non-hydrolyz- by the method of LOWRYrt a/. (1951). using bovine serum able analog GMP-PNP, regulate the hormonal sensitivity albumin as a standard. The synaptosomal samples conof adenylate cyclase in a number of tissues (RODBELLet tained 10-20 pg of protein. The specific activities of basal at., 1 9 7 1 ~ ;RODBELL et al., 1971b; BIRNBAUMER, 1973; and dopamine sensitive adenylate cyclase were increased LONDOSet a/., 1974; BIRNBAUMER et a/., 1974; LONDOS& 2 3 fold and 3-5 fold, respectively, above those in the RODBELL, 1975; PFEUFFER & HELMREICH, 1975; LEFKOWITZ, striatal homogenates. 1974; BIRNBAUMER & YANG,1974; EBERT& SCHWABE, Assay of adenylale cyclase. Adenylate cyclase activity 1974). SHIMIZU (1973) reported that GTP had little effect was assayed essentially as described by CLEMENT-CORMIER on adenylate cyclase in the brain, but recent studies et a/. (1974) in a medium containing 80m~-T'ris-rnaleate showed that GTP inhibited basal adenylate cyclase (TELL buffer (pH 7.4 at 30"C), 6 mM-MgCI,, 0.2 mM-EGTA. er a/., 1975) but enhanced its dopamine sensitivity (CLE- 10 mwtheophylline, 2.5-5 pg of phosphatidyl serine, 10 mM-phosphoenolpyruvate, 4 pg pyruvate kinase. MENT-CORMIER et al.. 1975) in the striatum of rat brain. In view of the potential regulatory role of nucleotides on 15 mM-NH,SO, (from the pyruvate kinase). in a final vol the dopamine sensitivity of adenylate cyclase in the CNS, of 0.5ml. Dopamine and nucleotides were added to the we have investigated both the requirement and specificity synaptosomal membranes in this medium at O'C 10min of the dopamine sensitivity for nucleotides in synaptosomal prior to starting the enzymic reaction by the addition of ATP. The tubes were incubated in a water bath at 3 0 C membranes from the striatum of rat brain. for 2.5 min. The reaction was stopped by heating the tubes for 2.5 min in a boiling water bath. Samples (50pl) were MATERIALS AND METHODS removed for assay of cyclic AMP following centrifugation Preparation of synaptosomal membranes. Synaptosomal for 10min at W"C. Cyclic AMP was determined by the membranes were prepared by adaptation of the method method of BROWNet a/. (1971). A standard curve was of JONES& MATUS(1974). All procedures were carried out obtained with 1-6 pmol of cyclic AMP with each experat 04°C. Striatum (approx 240 mg) was homogenized in iment. 2.3 ml of 100,b sucrose (pH 7.4) containing 0.3 mg of phosMaterials. ATP (disodium salt), GTP, GMP, AMP, phatidyl serine (Sigma Brain Extract. Type 111) with 9-11 guanosine, adenosine, phosphoenolpyruvate (trisodium strokes in a glass-Teflon homogenizer for 2 min at 800 rev./ salt), pyruvate kinase (645 units per mg of protein). theomin. Following centrifugation at 8009 for 20min the phylline, bovine serum albumin, EGTA, dopamine hydrosupernatant was further centrifuged at 9000 g for 20 min, chloride and Tris (Trizma base) were purchased from and the pellet was suspended to 2.3 ml in 5 mM-Tris-HC1 Sigma Chemical Co, (St Louis, MO). Phosphatidyl serine (pH 8) and allowed to stand for 30min at 0°C. After (bovine) was obtained from Applied Science Labs Inc. further hand homogenization (6 strokes) the lysed suspen- (State College, PA). AMP-PNP and GMP-PNP were pursions were centrifuged at 9000 g for 20 min. The pellet was chased from International Chemical and Nuclear Corp suspended to 0.33ml in the Tris buffer and mixed with (Irvine, CA). Cyclic AMP kits were obtained from the 0.67 ml of 48'3%(w/w) sucrose. 1 ml of 28S0,, sucrose was Radiochemical Centre (Amersham, England). layered on top of this suspension in a 2.5 ml polycarbonate RESULTS AND DISCUSSION centrifuge tube and topped with 0.3ml of 10"; sucrose. Following centrifugation at 60,ooOg for llOmin in a Dopamine stimulation of basal adenylate cyclase activity swinging bucket rotor (MSE 43127-104 with 2.5 ml adapin synaptosomal membranes from the rat brain striatum tor) the synaptosomal fraction at the lower sucrose inter- was not observed unless GTP was included in the incubaface was removed and diluted to 1-2ml with distilled tion medium (Fig. 1). This is in contrast to results obtained water. Samples (30 PI) were assayed for adenylate cyclase by us and others in homogenates (CLEMENT-CORMIER et activity immediately. The protein content was determined a/., 1974; KEBABIAN et al., 1972). suggesting that endogenous GTP or other nucleotides were removed during 1979, and Abbreviations used; GMP-PNP, guanyl-5'-yl imidodi- the isolation procedure (LONDOS& RODBELL, phosphate; AMP-PNP, adenyl-5'-yl imidodiphosphate; that GTP may be obligatory for dopamine sensitivity et a/., 1975). Maximum EGTA, ethylene glycol bis(&aminoethyl ether)-N,N'- (RODBELL,1975; CUATRECASAS tetraacetic acid; CDP-choline, cytidine diphosphate cho- slope of the dopamine concentration-response curve was obtained with 10 PM-GTP. line. 1333
1534
Short communication
of a ground state of the enzyme to an active transition state complex (RODBELL. 1975). and that hormones act by enhancing the rate limiting step in the process of nucleoer tide activation of the adenylate cyclase (CUATRECASAS al., 1975).The present results are consistent with these proposals, since GMP-PNP, which produces a persistent & stable activated intermediate in many tissues (PFEUFFER HELMREICH. 1975; RODBELL,1975; CUATRECASAS et a/., 1975; SCHRAMM & RODBELL, 1975) was also the most effective nucleotide in enhancing the adenylate cyclase activity in synaptosomal membranes from the striatum. The molecular mechanism of promotion of formation of the activated transition state complex by nucleotides, and the facilitation of hormone sensitivity, is unknown. In this study dopamine sensitivity was partially supported by monophosphates of adenosine and guanosine, but the corresponding nucleosides were ineffective. The partial effectiveness of purine monophosphates and diphosphates & HELMhas also been observed in other tissues (PFEUFFER 0 1 5 10 20 50 loo REICH, 1975; BIRNBAUMER & YANG, 1974; LEFKOWITZ, 1974), and suggests either that ‘reversible’ binding to a nuDOPAMINE ( p) cleotide regulatory site may partially support promotion FIG. 1. Concentration-response curves for dopamine of formation of the activated state by dopamine. or alternastimulation of adenylate cyclase in synaptosomal mem- tively that nucleotide monophosphates and triphosphates branes prepared from rat brain stnatum. The data rep- act by different mechanisms. It is less likely that monoresents a typical experiment from synaptosomal mem- phosphates are converted to di- and then triphosphates branes prepared from 4 rats. The ATP concentration was by contaminating enzymes in the ATP regenerating system 0.6mM. Dopamine was vaned in the absence of GTP (0) ( L o m s & RODBELL,1979, since the small amounts of and in the presence of GTP; 1 p~ (W); 1 O p ~(0):and additional ATP formed are unlikely to be significant in 100 p~ (A). this system, In contrast to the striatum. the hepatic adenylate cyclase appears to have an absolute specificity for nuThe influence of related nucleotides and nucleosides on cleotide triphosphates (RODBELLer al., 1975). The facilithe basal and dopamine sensitive adenylate cyclase activity tation of dopamine sensitivity by CDP-choline is of potenwas also investigated (Table 1). GMP-PNP, a non-hydro- tial interest in view of the reported efficacy of this agent lyzable analog of GTP, was more effective than G T P in in disorders of consciousness and certain movement diser a/.. 1973). It is stimulating basal adenylate cyclase activity and enhancing orders in Parkinson’s disease (MANAKA its dopamine sensitivity. The observation that GTP inhi- interesting to speculate on the possibility that purine nubited basal activity in homogenates (Roufogalis, Thornton cleotides may modulate dopaminergic transmission or & Wade, submitted; TELLet al., 1975) but slightly stimuTABLE1. EFFECTOF PURINE DERIVATIVES ON BASAL ADENYlated this activity in synaptosomal membranes (Fig. 1, LATE CYCLASE AND DOPAMINE SENSITIVITY IN SYNAPTOSOMAL Table 1) is difficult to explain in terms of removal of endoMEMBRANES FROM THE STRIATUM genous G T P during synaptosomal membrane preparation, although different transient states of the enzymes can be Changcs in adeny late isolated by different preparative procedures (LONDOS& Adenylate cyclase cyclase RODBELL, 1975; RODBELL.1975).G T P inhibition in striatal aciiviiy acti\.ity (pmol!mg, min) Ipmolimgmin) homogenates could have occurred by a transient process that was related to the presence of ATP in the incubation Additions Dopamine Dopaminet medium (RODBELL,1973, whereas in the synaptosomal (10PM) Basal (50 1 1 ~ ) Bad’ (50~ I M ) membranes the predominant effect of GTP may have been 30 330 18 360 + 36 None to promote the transition of the enzyme from the ground GTP 28 365 723 k 45 358 31 state to the activated state. However, other explanations GMP-PNP 286 191 616 808 - 48 69 281 351 AMP-PNP et al., 1975). AMP-PNP, are also possible (CUATRECASAS -35 121 295 416 GMP the corresponding analog of ATP, had little effect on basal AMP 397 -47 115 283 - 16 45 313 3 59 activity and was only weakly effective in enhancing dopa- Guanoune 17 19 347 365 4denoslne mine sensitivity. G M P and AMP had little effect on basal CDP-choline 379 - 50 99 280 adenylate cyclase activity, but they were partially effective Synaptosomal membranes were prepared as described in enhancing dopamine sensitivity. CDP-choline was also partially effective in enhancing dopamine sensitivity. in Methods. The ATP concentration in the assay was whereas adenosine and guanosine were without significant 0.6 mM. The protein content of synaptosomal membranes was approx 0.05 mg/ml. Where errors are shown the values activity. represent the mean of 4 or 5 determinations f S.E.M. The nucleotide requirement for the dopamine sensitivity Otherwise. values are means of duplicate determinations. of adenylate cyclase activity in the striatum is consistent * Refers to the change in basal adenylate cyclase activity with observations made in other tissues. including hepatic in the presence of the nucleotide derivatives. et ul.. and fat cell membranes (RODBELL, 1975; SALOMON t Refers to the change in adenylate cyclase activity on addition of 50pM-dopamine in the presence of the nucleo1975; LIN er a/., 1975: RENDELLet a/.. 1975). It has been tide derivatives. suggested that adenine nucleotides promote the conversion
*
~
Short communication
I535
EBERTR. & SCHWABEU. (1974) Naun?n-Schwiiedeherys .4rchs exp. Path. Pharwiac. 286, 297-313. HORNYKIEWICZ 0. (1973) Fedn Proc. Fedn Am. Socs. rup. Biol. 32, 183-190. JONESD. H. & MATUSA. I. (1974) Biochirn. hiophys. Acta 356, 276-287. KEBABIAN J. W.. PETZOLD G. L. & GREENGARD P. (1972) Proc. nutn. Acail. Sci.. U.S.A. 69, 2145-2149. H. L. (1973) Am. J . Psychiat. 130, 82-86. Acknowledgements-We thank our colleagues Drs. P. KLAWANS LEFKOWITZ R. J. (1974) J. hiol. Chern. 249, 6119-6124. MEARRICK, D. DOUGANand D. BURKITTfor useful discusY., RENDELLM. & RODBELL M. (1975) sions during this study, and Dr. R. DAY for the initial LIN M. S., SALOMON J . biol. Chern. 250, 424W252. suggestion that we examine G T P in this system. LONDOSC. & RODBELLM. (1975) J. biol. Chem. 250, B. D. ROUFOGALIS' 867-876. Department of Clinical P harnwcoloyy. St. Vincent's M. THORNTONLONDOSC., SALOMONY.. LIN M. C.. HARWOODJ. P.. M. (1974) Proc. niifn. SCHRAMM M., WOLFFJ. & RODBELL Hospirrrl. Sydney. N.S.W. 2010. D. N. WADE Acud. Sci.. U.S.A.71. 3087-3090. .4usrrirliri LOWRY0. H., ROSEBROUGH N. J.. FARRA. L. & RANDALL R. J. (1951) J . hid. Chern. 193, 265-275. MANAKA S.. SANOK., FUCHINOUE T. & SEKINOH. (1973) REFERENCES Experientia 30, 179-180. PFEUFFER T. & HELMREICH E. J. M. (1975) J . h i d . Chem. BIRNBAUMER L. (1973) Biochirn. biophys. Acta. 300, 250, 867-876. 129-1 58. RENDELL M., SALOMON Y., LIN M. C., RODBELL M. & BERBIRNBAUMER L. & YANGP.-C. (1974) J. b i d . Chem. 249, MAN M. (1975) J . biol. Chem. 250, 42534260. 7848-7856. RODBELLM. (1975) J. b i d Chem. 250, 58265834. BIRNBAUMER L., NAKAHARA T. & YANG P.-C. (1974) J. RODBELLM., BIRNBAUMER L., POHLS. L. & KRANSH. b i d . Chem. 249, 7857-7866. M. J. (1971~)J . b i d . Chem. 246, 1877-1882. BROWNB. L., ALBANOJ. D. M., EKINSR. P. & SCHERZI RODBELLM., KRANSH. M. J.. POHLS. L. & BIRNBAUMER A. M. (1971) Biochem. J . 121, 561-562. L. (19716) J. b i d . Chern. 246, 1872-1876. CLEMENT-CORMIER Y. C.. KEBABIAN J. W.. PETZOLDG. RODBELLM., LIN M. C.. SALOMON Y., LONDOSC., HARL. & GREENGARD P. (1974) Pi-or. nutn. Acad. Sci. U.S.A. WOOD J. P., MARTIN B. R.. RENDELL M. & BERMAN M. 71, 1113-1117. (1975) in Adtrances in Cyclic Nircleofide Research (DRUMCLEMENT-CORMIER Y. C.. PARRISHR. G., PETZOLDG. L., MOND G. I., GREENGARD P. & ROBISONG. A., eds.) Vol. KEBABIAN J. W. & GREENGARD P. (1975) J. Neurochem. 5, pp. 3-29. Raven Press, New York. 25, 143-149. SALOMON Y., LIN M. C.. LONDOSC., RENDELL M. & RODCUATRECASAS P., JACOBSS. & BENNETTV. (1975) Proc. BELL M. (1975) J. hid. Chem. 250, 42394245. nutn. Acad. Sci., U.S.A. 72, 1739-1743. SCHRAMM M. & RODBELLM. (1975) J. h i d . Chem. 250, 2232-2237. H. (1973) Abst. of 4th Meeting of International On sabbatical leave from the Faculty of Pharrnaceuti- SHIMIZU Society for Neurochemistry, Tokyo. pp. 88-89. cal Sciences, University of British Columbia, Vancouver, G. W. & CUATRECASAS P. (1975) B.C. Canada V6T 1W5. To whom reprint requests should TELLG. P., PASTERNAK FEBS Lett. 51, 242-245. be sent.
homeostasis, either by their release together with neurotransmitters or through purinergic innervation. Changes in the levels of nucleotides could conceivably account for fluctuations in the effectiveness of L-Dopa during the treatment of Parkinson's disease (HORNYKIEWICZ, 1973) or fluctuations in psychotic states of susceptible subjects (KLAWANS, 1973).
1976 Vol 27. pp 1533 1535 Pcrganion PI?%.Printed in Grcal Britain.
SHORT COMMUNICATION
Nucleotide requirement of dopamine sensitive adenylate cyclase in synaptosomal membranes from the striatum of rat brain (Received 20 April 1976. Accepted 2 June 1976)
NUCLEOTIDES, particularly GTP and its non-hydrolyz- by the method of LOWRYrt a/. (1951). using bovine serum able analog GMP-PNP, regulate the hormonal sensitivity albumin as a standard. The synaptosomal samples conof adenylate cyclase in a number of tissues (RODBELLet tained 10-20 pg of protein. The specific activities of basal at., 1 9 7 1 ~ ;RODBELL et al., 1971b; BIRNBAUMER, 1973; and dopamine sensitive adenylate cyclase were increased LONDOSet a/., 1974; BIRNBAUMER et a/., 1974; LONDOS& 2 3 fold and 3-5 fold, respectively, above those in the RODBELL, 1975; PFEUFFER & HELMREICH, 1975; LEFKOWITZ, striatal homogenates. 1974; BIRNBAUMER & YANG,1974; EBERT& SCHWABE, Assay of adenylale cyclase. Adenylate cyclase activity 1974). SHIMIZU (1973) reported that GTP had little effect was assayed essentially as described by CLEMENT-CORMIER on adenylate cyclase in the brain, but recent studies et a/. (1974) in a medium containing 80m~-T'ris-rnaleate showed that GTP inhibited basal adenylate cyclase (TELL buffer (pH 7.4 at 30"C), 6 mM-MgCI,, 0.2 mM-EGTA. er a/., 1975) but enhanced its dopamine sensitivity (CLE- 10 mwtheophylline, 2.5-5 pg of phosphatidyl serine, 10 mM-phosphoenolpyruvate, 4 pg pyruvate kinase. MENT-CORMIER et al.. 1975) in the striatum of rat brain. In view of the potential regulatory role of nucleotides on 15 mM-NH,SO, (from the pyruvate kinase). in a final vol the dopamine sensitivity of adenylate cyclase in the CNS, of 0.5ml. Dopamine and nucleotides were added to the we have investigated both the requirement and specificity synaptosomal membranes in this medium at O'C 10min of the dopamine sensitivity for nucleotides in synaptosomal prior to starting the enzymic reaction by the addition of ATP. The tubes were incubated in a water bath at 3 0 C membranes from the striatum of rat brain. for 2.5 min. The reaction was stopped by heating the tubes for 2.5 min in a boiling water bath. Samples (50pl) were MATERIALS AND METHODS removed for assay of cyclic AMP following centrifugation Preparation of synaptosomal membranes. Synaptosomal for 10min at W"C. Cyclic AMP was determined by the membranes were prepared by adaptation of the method method of BROWNet a/. (1971). A standard curve was of JONES& MATUS(1974). All procedures were carried out obtained with 1-6 pmol of cyclic AMP with each experat 04°C. Striatum (approx 240 mg) was homogenized in iment. 2.3 ml of 100,b sucrose (pH 7.4) containing 0.3 mg of phosMaterials. ATP (disodium salt), GTP, GMP, AMP, phatidyl serine (Sigma Brain Extract. Type 111) with 9-11 guanosine, adenosine, phosphoenolpyruvate (trisodium strokes in a glass-Teflon homogenizer for 2 min at 800 rev./ salt), pyruvate kinase (645 units per mg of protein). theomin. Following centrifugation at 8009 for 20min the phylline, bovine serum albumin, EGTA, dopamine hydrosupernatant was further centrifuged at 9000 g for 20 min, chloride and Tris (Trizma base) were purchased from and the pellet was suspended to 2.3 ml in 5 mM-Tris-HC1 Sigma Chemical Co, (St Louis, MO). Phosphatidyl serine (pH 8) and allowed to stand for 30min at 0°C. After (bovine) was obtained from Applied Science Labs Inc. further hand homogenization (6 strokes) the lysed suspen- (State College, PA). AMP-PNP and GMP-PNP were pursions were centrifuged at 9000 g for 20 min. The pellet was chased from International Chemical and Nuclear Corp suspended to 0.33ml in the Tris buffer and mixed with (Irvine, CA). Cyclic AMP kits were obtained from the 0.67 ml of 48'3%(w/w) sucrose. 1 ml of 28S0,, sucrose was Radiochemical Centre (Amersham, England). layered on top of this suspension in a 2.5 ml polycarbonate RESULTS AND DISCUSSION centrifuge tube and topped with 0.3ml of 10"; sucrose. Following centrifugation at 60,ooOg for llOmin in a Dopamine stimulation of basal adenylate cyclase activity swinging bucket rotor (MSE 43127-104 with 2.5 ml adapin synaptosomal membranes from the rat brain striatum tor) the synaptosomal fraction at the lower sucrose inter- was not observed unless GTP was included in the incubaface was removed and diluted to 1-2ml with distilled tion medium (Fig. 1). This is in contrast to results obtained water. Samples (30 PI) were assayed for adenylate cyclase by us and others in homogenates (CLEMENT-CORMIER et activity immediately. The protein content was determined a/., 1974; KEBABIAN et al., 1972). suggesting that endogenous GTP or other nucleotides were removed during 1979, and Abbreviations used; GMP-PNP, guanyl-5'-yl imidodi- the isolation procedure (LONDOS& RODBELL, phosphate; AMP-PNP, adenyl-5'-yl imidodiphosphate; that GTP may be obligatory for dopamine sensitivity et a/., 1975). Maximum EGTA, ethylene glycol bis(&aminoethyl ether)-N,N'- (RODBELL,1975; CUATRECASAS tetraacetic acid; CDP-choline, cytidine diphosphate cho- slope of the dopamine concentration-response curve was obtained with 10 PM-GTP. line. 1333
1534
Short communication
of a ground state of the enzyme to an active transition state complex (RODBELL. 1975). and that hormones act by enhancing the rate limiting step in the process of nucleoer tide activation of the adenylate cyclase (CUATRECASAS al., 1975).The present results are consistent with these proposals, since GMP-PNP, which produces a persistent & stable activated intermediate in many tissues (PFEUFFER HELMREICH. 1975; RODBELL,1975; CUATRECASAS et a/., 1975; SCHRAMM & RODBELL, 1975) was also the most effective nucleotide in enhancing the adenylate cyclase activity in synaptosomal membranes from the striatum. The molecular mechanism of promotion of formation of the activated transition state complex by nucleotides, and the facilitation of hormone sensitivity, is unknown. In this study dopamine sensitivity was partially supported by monophosphates of adenosine and guanosine, but the corresponding nucleosides were ineffective. The partial effectiveness of purine monophosphates and diphosphates & HELMhas also been observed in other tissues (PFEUFFER 0 1 5 10 20 50 loo REICH, 1975; BIRNBAUMER & YANG, 1974; LEFKOWITZ, 1974), and suggests either that ‘reversible’ binding to a nuDOPAMINE ( p) cleotide regulatory site may partially support promotion FIG. 1. Concentration-response curves for dopamine of formation of the activated state by dopamine. or alternastimulation of adenylate cyclase in synaptosomal mem- tively that nucleotide monophosphates and triphosphates branes prepared from rat brain stnatum. The data rep- act by different mechanisms. It is less likely that monoresents a typical experiment from synaptosomal mem- phosphates are converted to di- and then triphosphates branes prepared from 4 rats. The ATP concentration was by contaminating enzymes in the ATP regenerating system 0.6mM. Dopamine was vaned in the absence of GTP (0) ( L o m s & RODBELL,1979, since the small amounts of and in the presence of GTP; 1 p~ (W); 1 O p ~(0):and additional ATP formed are unlikely to be significant in 100 p~ (A). this system, In contrast to the striatum. the hepatic adenylate cyclase appears to have an absolute specificity for nuThe influence of related nucleotides and nucleosides on cleotide triphosphates (RODBELLer al., 1975). The facilithe basal and dopamine sensitive adenylate cyclase activity tation of dopamine sensitivity by CDP-choline is of potenwas also investigated (Table 1). GMP-PNP, a non-hydro- tial interest in view of the reported efficacy of this agent lyzable analog of GTP, was more effective than G T P in in disorders of consciousness and certain movement diser a/.. 1973). It is stimulating basal adenylate cyclase activity and enhancing orders in Parkinson’s disease (MANAKA its dopamine sensitivity. The observation that GTP inhi- interesting to speculate on the possibility that purine nubited basal activity in homogenates (Roufogalis, Thornton cleotides may modulate dopaminergic transmission or & Wade, submitted; TELLet al., 1975) but slightly stimuTABLE1. EFFECTOF PURINE DERIVATIVES ON BASAL ADENYlated this activity in synaptosomal membranes (Fig. 1, LATE CYCLASE AND DOPAMINE SENSITIVITY IN SYNAPTOSOMAL Table 1) is difficult to explain in terms of removal of endoMEMBRANES FROM THE STRIATUM genous G T P during synaptosomal membrane preparation, although different transient states of the enzymes can be Changcs in adeny late isolated by different preparative procedures (LONDOS& Adenylate cyclase cyclase RODBELL, 1975; RODBELL.1975).G T P inhibition in striatal aciiviiy acti\.ity (pmol!mg, min) Ipmolimgmin) homogenates could have occurred by a transient process that was related to the presence of ATP in the incubation Additions Dopamine Dopaminet medium (RODBELL,1973, whereas in the synaptosomal (10PM) Basal (50 1 1 ~ ) Bad’ (50~ I M ) membranes the predominant effect of GTP may have been 30 330 18 360 + 36 None to promote the transition of the enzyme from the ground GTP 28 365 723 k 45 358 31 state to the activated state. However, other explanations GMP-PNP 286 191 616 808 - 48 69 281 351 AMP-PNP et al., 1975). AMP-PNP, are also possible (CUATRECASAS -35 121 295 416 GMP the corresponding analog of ATP, had little effect on basal AMP 397 -47 115 283 - 16 45 313 3 59 activity and was only weakly effective in enhancing dopa- Guanoune 17 19 347 365 4denoslne mine sensitivity. G M P and AMP had little effect on basal CDP-choline 379 - 50 99 280 adenylate cyclase activity, but they were partially effective Synaptosomal membranes were prepared as described in enhancing dopamine sensitivity. CDP-choline was also partially effective in enhancing dopamine sensitivity. in Methods. The ATP concentration in the assay was whereas adenosine and guanosine were without significant 0.6 mM. The protein content of synaptosomal membranes was approx 0.05 mg/ml. Where errors are shown the values activity. represent the mean of 4 or 5 determinations f S.E.M. The nucleotide requirement for the dopamine sensitivity Otherwise. values are means of duplicate determinations. of adenylate cyclase activity in the striatum is consistent * Refers to the change in basal adenylate cyclase activity with observations made in other tissues. including hepatic in the presence of the nucleotide derivatives. et ul.. and fat cell membranes (RODBELL, 1975; SALOMON t Refers to the change in adenylate cyclase activity on addition of 50pM-dopamine in the presence of the nucleo1975; LIN er a/., 1975: RENDELLet a/.. 1975). It has been tide derivatives. suggested that adenine nucleotides promote the conversion
*
~
Short communication
I535
EBERTR. & SCHWABEU. (1974) Naun?n-Schwiiedeherys .4rchs exp. Path. Pharwiac. 286, 297-313. HORNYKIEWICZ 0. (1973) Fedn Proc. Fedn Am. Socs. rup. Biol. 32, 183-190. JONESD. H. & MATUSA. I. (1974) Biochirn. hiophys. Acta 356, 276-287. KEBABIAN J. W.. PETZOLD G. L. & GREENGARD P. (1972) Proc. nutn. Acail. Sci.. U.S.A. 69, 2145-2149. H. L. (1973) Am. J . Psychiat. 130, 82-86. Acknowledgements-We thank our colleagues Drs. P. KLAWANS LEFKOWITZ R. J. (1974) J. hiol. Chern. 249, 6119-6124. MEARRICK, D. DOUGANand D. BURKITTfor useful discusY., RENDELLM. & RODBELL M. (1975) sions during this study, and Dr. R. DAY for the initial LIN M. S., SALOMON J . biol. Chern. 250, 424W252. suggestion that we examine G T P in this system. LONDOSC. & RODBELLM. (1975) J. biol. Chem. 250, B. D. ROUFOGALIS' 867-876. Department of Clinical P harnwcoloyy. St. Vincent's M. THORNTONLONDOSC., SALOMONY.. LIN M. C.. HARWOODJ. P.. M. (1974) Proc. niifn. SCHRAMM M., WOLFFJ. & RODBELL Hospirrrl. Sydney. N.S.W. 2010. D. N. WADE Acud. Sci.. U.S.A.71. 3087-3090. .4usrrirliri LOWRY0. H., ROSEBROUGH N. J.. FARRA. L. & RANDALL R. J. (1951) J . hid. Chern. 193, 265-275. MANAKA S.. SANOK., FUCHINOUE T. & SEKINOH. (1973) REFERENCES Experientia 30, 179-180. PFEUFFER T. & HELMREICH E. J. M. (1975) J . h i d . Chem. BIRNBAUMER L. (1973) Biochirn. biophys. Acta. 300, 250, 867-876. 129-1 58. RENDELL M., SALOMON Y., LIN M. C., RODBELL M. & BERBIRNBAUMER L. & YANGP.-C. (1974) J. b i d . Chem. 249, MAN M. (1975) J . biol. Chem. 250, 42534260. 7848-7856. RODBELLM. (1975) J. b i d Chem. 250, 58265834. BIRNBAUMER L., NAKAHARA T. & YANG P.-C. (1974) J. RODBELLM., BIRNBAUMER L., POHLS. L. & KRANSH. b i d . Chem. 249, 7857-7866. M. J. (1971~)J . b i d . Chem. 246, 1877-1882. BROWNB. L., ALBANOJ. D. M., EKINSR. P. & SCHERZI RODBELLM., KRANSH. M. J.. POHLS. L. & BIRNBAUMER A. M. (1971) Biochem. J . 121, 561-562. L. (19716) J. b i d . Chern. 246, 1872-1876. CLEMENT-CORMIER Y. C.. KEBABIAN J. W.. PETZOLDG. RODBELLM., LIN M. C.. SALOMON Y., LONDOSC., HARL. & GREENGARD P. (1974) Pi-or. nutn. Acad. Sci. U.S.A. WOOD J. P., MARTIN B. R.. RENDELL M. & BERMAN M. 71, 1113-1117. (1975) in Adtrances in Cyclic Nircleofide Research (DRUMCLEMENT-CORMIER Y. C.. PARRISHR. G., PETZOLDG. L., MOND G. I., GREENGARD P. & ROBISONG. A., eds.) Vol. KEBABIAN J. W. & GREENGARD P. (1975) J. Neurochem. 5, pp. 3-29. Raven Press, New York. 25, 143-149. SALOMON Y., LIN M. C.. LONDOSC., RENDELL M. & RODCUATRECASAS P., JACOBSS. & BENNETTV. (1975) Proc. BELL M. (1975) J. hid. Chem. 250, 42394245. nutn. Acad. Sci., U.S.A. 72, 1739-1743. SCHRAMM M. & RODBELLM. (1975) J. h i d . Chem. 250, 2232-2237. H. (1973) Abst. of 4th Meeting of International On sabbatical leave from the Faculty of Pharrnaceuti- SHIMIZU Society for Neurochemistry, Tokyo. pp. 88-89. cal Sciences, University of British Columbia, Vancouver, G. W. & CUATRECASAS P. (1975) B.C. Canada V6T 1W5. To whom reprint requests should TELLG. P., PASTERNAK FEBS Lett. 51, 242-245. be sent.
homeostasis, either by their release together with neurotransmitters or through purinergic innervation. Changes in the levels of nucleotides could conceivably account for fluctuations in the effectiveness of L-Dopa during the treatment of Parkinson's disease (HORNYKIEWICZ, 1973) or fluctuations in psychotic states of susceptible subjects (KLAWANS, 1973).
