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Muscarinic Receptor-mediated Dual Regulation of ADP-ribosyl Cyclase in NG108-15 Neuronal Cell Membranes

1997; Elsevier BV; Volume: 272; Issue: 50 Linguagem: Inglês

10.1074/jbc.272.50.31272

1083-351X

Haruhiro Higashida, Shigeru Yokoyama, Minako Hashii, Megumi Taketo, Masaharu Higashida, Tatsunori Takayasu, Tohru Ohshima, Shin Takasawa, Hiroshi Okamoto, Mami Noda,

Ion channel regulation and function

Cyclic ADP-ribose (cADP-ribose) is an endogenous modulator of ryanodine-sensitive Ca2+ release channels. An unsolved question is whether or not cADP-ribose mediates intracellular signals from hormone or neurotransmitter receptors. The first step in this study was to develop a TLC method to measure ADP-ribosyl cyclase, by which conversion of [3H]NAD+ to [3H]cADP-ribose was confirmed in COS-7 cells overexpressing human CD38. A membrane fraction of NG108-15 neuroblastoma × glioma hybrid cells possessed ADP-ribosyl cyclase activity measured by TLC. Carbamylcholine increased this activity by 2.6-fold in NG108-15 cells overexpressing m1 or m3 muscarinic acetylcholine receptors (mAChRs), but inhibited it by 30–52% in cells expressing m2 and/or m4 mAChRs. Both of these effects were mimicked by GTP. Pretreatment of cells with cholera toxin blocked the activation, whereas pertussis toxin blocked the inhibition. Application of carbamylcholine caused significant decreases in NAD+ concentrations in untreated m1-transformed NG108-15 cells, but an increase in cholera toxin-treated cells. These results suggest that mAChRs couple to ADP-ribosyl cyclase within cell membranes via trimeric G proteins and can thereby control cellular function by regulating cADP-ribose formation. Cyclic ADP-ribose (cADP-ribose) is an endogenous modulator of ryanodine-sensitive Ca2+ release channels. An unsolved question is whether or not cADP-ribose mediates intracellular signals from hormone or neurotransmitter receptors. The first step in this study was to develop a TLC method to measure ADP-ribosyl cyclase, by which conversion of [3H]NAD+ to [3H]cADP-ribose was confirmed in COS-7 cells overexpressing human CD38. A membrane fraction of NG108-15 neuroblastoma × glioma hybrid cells possessed ADP-ribosyl cyclase activity measured by TLC. Carbamylcholine increased this activity by 2.6-fold in NG108-15 cells overexpressing m1 or m3 muscarinic acetylcholine receptors (mAChRs), but inhibited it by 30–52% in cells expressing m2 and/or m4 mAChRs. Both of these effects were mimicked by GTP. Pretreatment of cells with cholera toxin blocked the activation, whereas pertussis toxin blocked the inhibition. Application of carbamylcholine caused significant decreases in NAD+ concentrations in untreated m1-transformed NG108-15 cells, but an increase in cholera toxin-treated cells. These results suggest that mAChRs couple to ADP-ribosyl cyclase within cell membranes via trimeric G proteins and can thereby control cellular function by regulating cADP-ribose formation. Cyclic ADP-ribose (cADP-ribose) 1The abbreviations used are: cADP-ribose, cyclic ADP-ribose; [NAD+] i , intracellular NAD+ concentration; mAChR, muscarinic acetylcholine receptor; HPLC, high pressure liquid chromatography; kb, kilobase; COS-CD38 cells, CD38-transfected COS-7 cells; PBS, phosphate-buffered saline; CCh, carbamylcholine; GTPγS, guanosine 5′-O-(3-thiotriphosphate); CTx, cholera toxin; PTx, pertussis toxin.1The abbreviations used are: cADP-ribose, cyclic ADP-ribose; [NAD+] i , intracellular NAD+ concentration; mAChR, muscarinic acetylcholine receptor; HPLC, high pressure liquid chromatography; kb, kilobase; COS-CD38 cells, CD38-transfected COS-7 cells; PBS, phosphate-buffered saline; CCh, carbamylcholine; GTPγS, guanosine 5′-O-(3-thiotriphosphate); CTx, cholera toxin; PTx, pertussis toxin. is synthesized from β-NAD+, an abundant intracellular substrate, by ADP-ribosyl cyclase in sea urchin eggs and in mammalian cells (1Rusinko N. Lee H.C. J. Biol. Chem. 1989; 264: 11725-11731Abstract Full Text PDF PubMed Google Scholar, 2Kim H. Jacobson E.L. Jacobson M.K. Science. 1993; 261: 1330-1333Crossref PubMed Scopus (233) Google Scholar). Pharmacological studies suggest that cADP-ribose is an endogenous modulator of ryanodine-sensitive Ca2+ release channels (3Mészáros L.G. Bak J. Chu A. Nature. 1993; 364: 76-79Crossref PubMed Scopus (317) Google Scholar, 4White A.M. Watson S.P. Galione A. FEBS Lett. 1993; 318: 259-263Crossref PubMed Scopus (101) Google Scholar, 5Takasawa S. Nata K. Yonekura H. Okamoto H. Science. 1993; 259: 370-373Crossref PubMed Scopus (401) Google Scholar, 6Thorn P. Gerasimenko O. Petersen O.H. EMBO J. 1994; 13: 2038-2043Crossref PubMed Scopus (187) Google Scholar, 7Hua S.-Y. Tokimasa T. Takasawa S. Furuya Y. Nohmi M. Okamoto H. Kuba K. Neuron. 1994; 12: 1073-1079Abstract Full Text PDF PubMed Scopus (126) Google Scholar, 8Lee H.C. Aarhus R. Graeff R. Gurnack M.E. Walseth T.F. Nature. 1994; 370: 307-309Crossref PubMed Scopus (208) Google Scholar, 9Kuemmerle J.F. Makhlouf G.M. J. Biol. Chem. 1995; 270: 25488-25494Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 10Lee H.C. Aarhus R. Graeff R.M. J. Biol. Chem. 1995; 270: 9060-9066Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). If cADP-ribose acts as an intracellular second messenger, ADP-ribosyl cyclase, as an effector enzyme, should be activated or inhibited in response to stimulation by hormones or neurotransmitters, which should simultaneously be associated with a transient decrease in the intracellular NAD+ concentration ([NAD+] i ) and an increase in cADP-ribose concentration (11Berridge M.J. Nature. 1993; 365: 388-389Crossref PubMed Scopus (290) Google Scholar). ADP-ribosyl cyclase seems to be present in both cytosolic and membrane-bound forms (1Rusinko N. Lee H.C. J. Biol. Chem. 1989; 264: 11725-11731Abstract Full Text PDF PubMed Google Scholar, 2Kim H. Jacobson E.L. Jacobson M.K. Science. 1993; 261: 1330-1333Crossref PubMed Scopus (233) Google Scholar, 12Lee H.C. Aarhus R. Cell Regul. 1991; 2: 203-209Crossref PubMed Scopus (322) Google Scholar). The mammalian membrane-bound form of ADP-ribosyl cyclase has been identified as a cell-surface antigen, CD38 (13Jackson D.G. Bell J.I. J. Immunol. 1990; 144: 2811-2815PubMed Google Scholar, 14Howard M. Grimaldi J.C. Bazan J.F. Lund F.E. Santos-Argumedo L. Parkhouse R.M.E. Walseth T.F. Lee H.C. Science. 1993; 262: 1056-1059Crossref PubMed Scopus (670) Google Scholar, 15Tohgo A. Takasawa S. Noguchi N. Koguma T. Nata K. Sugimoto T. Furuya Y. Yonekura H. Okamoto H. J. Biol. Chem. 1994; 269: 28555-28557Abstract Full Text PDF PubMed Google Scholar, 16Lund F. Solvason N. Grimaldi J.C. Parkhouse R.M.E. Howard M. Immunol. Today. 1995; 16: 469-473Abstract Full Text PDF PubMed Scopus (104) Google Scholar, 17Kato I. Takasawa S. Akabane A. Tanaka O. Abe H. Takamura T. Suzuki Y. Nata K. Yonekura H. Yoshimoto T. Okamoto H. J. Biol. Chem. 1995; 270: 30045-30050Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 18Prasado G.S. McRee D.E. Stura E.A. Levitt D.G. Lee H.C. Stout C.D. Nat. Struct. Biol. 1996; 3: 957-964Crossref PubMed Scopus (140) Google Scholar, 19Inageda K. Takahashi K. Tokita K. Nishina H. Kanaho Y. Kukimoto I. Kontani K. Hoshino S. Katada T. J. Biochem. (Tokyo). 1995; 117: 125-131Crossref PubMed Scopus (66) Google Scholar) and BST-1 (20Hirata Y. Kimura N. Sato K. Ohsugi Y. Takasawa S. Okamoto H. Ishikawa J. Kaisho T. Ishihara K. Hirano T. FEBS Lett. 1997; 356: 244-248Crossref Scopus (152) Google Scholar).Recently, it has been shown that the formation of cADP-ribose is regulated by nitric oxide or cGMP (21Galione A. White A. Willmott N. Turner M. Potter B.V.L. Watson S.T. Nature. 1993; 365: 456-459Crossref PubMed Scopus (259) Google Scholar, 22Willmott N. Sethi J.K. Walseth T.F. Lee H.C. White A.M. Galione A. J. Biol. Chem. 1996; 271: 3699-3705Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 23Clementi E. Riccio M. Sciorati C. Nistico G. Meldolesi J. J. Biol. Chem. 1996; 271: 17739-17745Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar) and that nitric oxide or cGMP is increased by stimulation with agonists (24Mathes C. Thompson S.H. J. Neurosci. 1996; 16: 1702-1709Crossref PubMed Google Scholar, 25Matsuzawa H. Nirenberg M. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 3472-3476Crossref PubMed Scopus (163) Google Scholar). These findings suggest the hypothesis that the regulation of the cADP-ribose level is located far downstream in the signal transduction cascade from receptors (11Berridge M.J. Nature. 1993; 365: 388-389Crossref PubMed Scopus (290) Google Scholar). An alternative hypothesis is that the cADP-ribose formation is regulated by ADP-ribosyl cyclase through the direct action of G proteins activated by receptors within the surface membrane, as already shown for the formation of cyclic AMP, inositol 1,4,5-trisphosphate, and diacylglycerol (26Gilman A.G. Cell. 1984; 36: 577-579Abstract Full Text PDF PubMed Scopus (1102) Google Scholar, 27Berridge M.J. Irvine R.F. Nature. 1984; 312: 315-321Crossref PubMed Scopus (4221) Google Scholar, 28Nishizuka Y. Nature. 1984; 308: 693-698Crossref PubMed Scopus (5741) Google Scholar). To test this hypothesis, we used NG108-15 neuroblastoma × glioma hybrid cells (29Nirenberg M. Wilson S. Higashida H. Rotter A. Krueger K. Busis N. Ray R. Kenimer J.G. Adler M. Science. 1983; 222: 794-799Crossref PubMed Scopus (237) Google Scholar), in which signal transduction from receptors to effectors has been extensively characterized (29Nirenberg M. Wilson S. Higashida H. Rotter A. Krueger K. Busis N. Ray R. Kenimer J.G. Adler M. Science. 1983; 222: 794-799Crossref PubMed Scopus (237) Google Scholar, 30Noda M. Ishizaka N. Yokoyama S. Hoshi N. Kimura Y. Hashii M. Taketo M. Egorova A. Knijnik R. Fukuda K. Morikawa H. Brown D.A. Higashida H. J. Lipid Mediators Cell Signalling. 1996; 14: 175-185Crossref PubMed Scopus (15) Google Scholar). In particular, in NGPM1-27 cells (31Fukuda K. Higashida H. Kubo T. Maeda A. Akiba I. Bujo H. Mishina M. Numa S. Nature. 1988; 335: 355-358Crossref PubMed Scopus (181) Google Scholar), which overexpress muscarinic acetylcholine receptors (mAChRs), it has been shown that intracellular NAD+ or NAD+metabolites are involved in signal transduction from m1 mAChRs to K+ channels (32Higashida H. Robbins J. Egorova A. Noda M. Taketo M. Ishizaka N. Takasawa S. Okamoto H. Brown D.A. J. Physiol. (Lond.). 1995; 482: 317-323Crossref Scopus (49) Google Scholar, 33Higashida H. Egorova A. Hoshi N. Noda M. FEBS Lett. 1996; 379: 236-238Crossref PubMed Scopus (13) Google Scholar). In this context, such neuronal cell lines have advantages for analyzing receptor-ADP-ribosyl cyclase coupling in detail.For measurement of ADP-ribosyl cyclase, high pressure liquid chromatography (HPLC) is commonly used to separate cADP-ribose-related compounds (1Rusinko N. Lee H.C. J. Biol. Chem. 1989; 264: 11725-11731Abstract Full Text PDF PubMed Google Scholar, 2Kim H. Jacobson E.L. Jacobson M.K. Science. 1993; 261: 1330-1333Crossref PubMed Scopus (233) Google Scholar, 8Lee H.C. Aarhus R. Graeff R. Gurnack M.E. Walseth T.F. Nature. 1994; 370: 307-309Crossref PubMed Scopus (208) Google Scholar, 14Howard M. Grimaldi J.C. Bazan J.F. Lund F.E. Santos-Argumedo L. Parkhouse R.M.E. Walseth T.F. Lee H.C. Science. 1993; 262: 1056-1059Crossref PubMed Scopus (670) Google Scholar, 15Tohgo A. Takasawa S. Noguchi N. Koguma T. Nata K. Sugimoto T. Furuya Y. Yonekura H. Okamoto H. J. Biol. Chem. 1994; 269: 28555-28557Abstract Full Text PDF PubMed Google Scholar, 17Kato I. Takasawa S. Akabane A. Tanaka O. Abe H. Takamura T. Suzuki Y. Nata K. Yonekura H. Yoshimoto T. Okamoto H. J. Biol. Chem. 1995; 270: 30045-30050Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 19Inageda K. Takahashi K. Tokita K. Nishina H. Kanaho Y. Kukimoto I. Kontani K. Hoshino S. Katada T. J. Biochem. (Tokyo). 1995; 117: 125-131Crossref PubMed Scopus (66) Google Scholar, 34Takasawa S. Tohgo A. Noguchi N. Koguma T. Nata K. Sugimoto T. Yonekura H. Okamoto H. J. Biol. Chem. 1993; 268: 26052-26054Abstract Full Text PDF PubMed Google Scholar, 35Tohgo A. Munakata H. Takasawa S. Nata K. Akiyama T. Hayashi N. Okamoto H. J. Biol. Chem. 1997; 272: 3879-3882Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). However, since it takes 30–60 min to process one sample, it is essential to develop a much more rapid method that can allow processing of multiple samples at once. There are two papers that describe ADP-ribosyl cyclase assay by TLC (21Galione A. White A. Willmott N. Turner M. Potter B.V.L. Watson S.T. Nature. 1993; 365: 456-459Crossref PubMed Scopus (259) Google Scholar, 36Mészáros V. Socci R. Mészáros L.G. Biochem. Biophys. Res. Commun. 1995; 210: 452-456Crossref PubMed Scopus (22) Google Scholar), in which NAD+ migrates faster than cADP-ribose. The methods used in those reports seem to be affected by large amounts of radiolabeled substrates. We here developed a TLC method that overcomes this problem and allows separation of cADP-ribose in up to 19 samples within 40–50 min. Our TLC method was first tested on COS-7 cells overexpressing human CD38 and was shown to be applicable for measuring ADP-ribosyl cyclase activity. We demonstrate that crude cell membranes of NG108-15 cells possess ADP-ribosyl cyclase activity and that such activity is activated or inhibited in a mAChR subtype-specific manner in NG108-15 cells overexpressing distinct mAChR subtypes. Furthermore, to ascertain the intracellular role of the catalytic activity of ADP-ribosyl cyclase in neuronal cell membranes, the time course of [NAD+] i after extracellular application of acetylcholine to these cells was investigated.DISCUSSIONThe chromatographic procedure used here resulted in a resolution sufficient to measure 3H accumulation in the cADP-ribose fraction originating from adenine-labeled [3H]NAD+, and it was free from interference by the labeled substrate. One possible limitation could be that the cADP-ribose fraction was contaminated by ∼15% of the newly synthesized [3H]ADP-ribose (Fig. 2 A). However, the risk that we measured the ADP-ribose count in the cADP-ribose fraction seems to be negligible since different patterns of3H accumulation in ADP-ribose and cADP-ribose fractions were obtained (Fig. 4), reflecting that distinct materials are accumulated with different rates of formation and degradation. The autoradiograms for COS-CD38 and NGPM1-27 cells (Fig. 3) clearly demonstrated the overall fate of NAD+ in that the majority of NAD+ was converted to either ADP-ribose or cADP-ribose or both in two or more enzyme reaction steps, probably ADP-ribosyl cyclase and cADP-ribose hydrolase.The NAD+ concentration used in our reaction mixture (2 μm) was much lower than values (∼100 μm) used in other studies (1Rusinko N. Lee H.C. J. Biol. Chem. 1989; 264: 11725-11731Abstract Full Text PDF PubMed Google Scholar, 2Kim H. Jacobson E.L. Jacobson M.K. Science. 1993; 261: 1330-1333Crossref PubMed Scopus (233) Google Scholar, 5Takasawa S. Nata K. Yonekura H. Okamoto H. Science. 1993; 259: 370-373Crossref PubMed Scopus (401) Google Scholar, 15Tohgo A. Takasawa S. Noguchi N. Koguma T. Nata K. Sugimoto T. Furuya Y. Yonekura H. Okamoto H. J. Biol. Chem. 1994; 269: 28555-28557Abstract Full Text PDF PubMed Google Scholar). However, our concentration is very similar to the estimated K m value (3–5 μm) for ADP-ribosyl cyclase in NG108-15 cell membranes. 2H. Higashida, S. Yokoyama, M. Higashida, and M. Noda, unpublished data. Under such conditions, [3H]cADP-ribose accumulated linearly for 2–4 min and [3H]ADP-ribose for 16 min, suggesting that it is not an insufficient dose. Thus, our TLC method was used for measurement of ADP-ribosyl cyclase, at least during the first few minutes. We do not know whether or not this TLC method is applicable for measurement of other recently found enzyme reactions in ADP-ribosyl cyclase, such as production of cyclic 2′-phosphoadenosine diphosphoribose (41Vu C.Q. Lu P.-J. Chen C.-S. Jacobson M.K. J. Biol. Chem. 1996; 271: 4747-4754Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), nicotinic acid adenine 5′-diphophate ribose (42Genazzani A.A. Mezna M. Summerhill R.J. Galione A. Michelangeli F. J. Biol. Chem. 1997; 272: 7669-7675Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar), and dimeric ADP-ribose (43De Flora A. Guida L. Franco L. Zocchi E. Bruzzone S. Benatti U. Damonte G. Lee H.C. J. Biol. Chem. 1997; 272: 12945-12951Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). It remains to be tested how our TLC method differs in accuracy from HPLC and fluorometric measurement of cyclic GDP-ribose formation (20Hirata Y. Kimura N. Sato K. Ohsugi Y. Takasawa S. Okamoto H. Ishikawa J. Kaisho T. Ishihara K. Hirano T. FEBS Lett. 1997; 356: 244-248Crossref Scopus (152) Google Scholar, 44Graeff R.M. Walseth T.F. Fryxell K. Branton W.D. Lee H.C. J. Biol. Chem. 1994; 269: 30260-30267Abstract Full Text PDF PubMed Google Scholar, 45De Flora A. Guida L. Franco L. Zocchi E. Pestarino M. Usai C. Marchetti C. Fedele E. Fontana G. Raiteri M. Biochem. J. 1996; 320: 665-672Crossref PubMed Scopus (52) Google Scholar).The results provide the first evidence that cholinergic signals are transduced from mAChRs to ADP-ribosyl cyclase within the membrane in a receptor subtype-specific fashion (Figs. 5 and 6). The finding that the inhibition of ADP-ribosyl cyclase via m2/m4 mAChRs is mediated through a PTx-sensitive G protein resembles the inhibitory signal transduction known for adenylate cyclase in NG108-15 cells (29Nirenberg M. Wilson S. Higashida H. Rotter A. Krueger K. Busis N. Ray R. Kenimer J.G. Adler M. Science. 1983; 222: 794-799Crossref PubMed Scopus (237) Google Scholar, 30Noda M. Ishizaka N. Yokoyama S. Hoshi N. Kimura Y. Hashii M. Taketo M. Egorova A. Knijnik R. Fukuda K. Morikawa H. Brown D.A. Higashida H. J. Lipid Mediators Cell Signalling. 1996; 14: 175-185Crossref PubMed Scopus (15) Google Scholar, 31Fukuda K. Higashida H. Kubo T. Maeda A. Akiba I. Bujo H. Mishina M. Numa S. Nature. 1988; 335: 355-358Crossref PubMed Scopus (181) Google Scholar). Coupling of m1/m3 mAChRs to ADP-ribosyl cyclase is relatively resistant to PTx, but highly sensitive to CTx, which seems to be different from the signal pathway to phospholipase Cβ (30Noda M. Ishizaka N. Yokoyama S. Hoshi N. Kimura Y. Hashii M. Taketo M. Egorova A. Knijnik R. Fukuda K. Morikawa H. Brown D.A. Higashida H. J. Lipid Mediators Cell Signalling. 1996; 14: 175-185Crossref PubMed Scopus (15) Google Scholar, 31Fukuda K. Higashida H. Kubo T. Maeda A. Akiba I. Bujo H. Mishina M. Numa S. Nature. 1988; 335: 355-358Crossref PubMed Scopus (181) Google Scholar). Thus, the coupling from mAChRs to ADP-ribosyl cyclase via G proteins would be a new mode of signal transduction, in parallel with the known pathways to adenylate cyclase and phospholipase C in NG108-15 cells.The simplest explanation for our [NAD+] i data in NGPM1-27 cells (Fig. 7) could be that NAD+ consumption is accelerated by the activation with the agonist of a membrane-bound form of ADP-ribosyl cyclase whose catalytic site resides in the cell interior. This speculation, however, would require a different topology from that proposed for the CD38 cell-surface antigen, whose catalytic sites of ADP-ribosyl cyclase (15Tohgo A. Takasawa S. Noguchi N. Koguma T. Nata K. Sugimoto T. Furuya Y. Yonekura H. Okamoto H. J. Biol. Chem. 1994; 269: 28555-28557Abstract Full Text PDF PubMed Google Scholar, 18Prasado G.S. McRee D.E. Stura E.A. Levitt D.G. Lee H.C. Stout C.D. Nat. Struct. Biol. 1996; 3: 957-964Crossref PubMed Scopus (140) Google Scholar, 45De Flora A. Guida L. Franco L. Zocchi E. Pestarino M. Usai C. Marchetti C. Fedele E. Fontana G. Raiteri M. Biochem. J. 1996; 320: 665-672Crossref PubMed Scopus (52) Google Scholar), ADP-ribose hydrolase (15Tohgo A. Takasawa S. Noguchi N. Koguma T. Nata K. Sugimoto T. Furuya Y. Yonekura H. Okamoto H. J. Biol. Chem. 1994; 269: 28555-28557Abstract Full Text PDF PubMed Google Scholar,46Zocchi E. Franco L. Guida L. Benatti U. Bargellesi A. Malavasi F. Lee H.C. De Flora D. Biochem. Biophys. Res. Commun. 1993; 196: 1459-1465Crossref PubMed Scopus (261) Google Scholar), and NAD+ glycohydrolase (19Inageda K. Takahashi K. Tokita K. Nishina H. Kanaho Y. Kukimoto I. Kontani K. Hoshino S. Katada T. J. Biochem. (Tokyo). 1995; 117: 125-131Crossref PubMed Scopus (66) Google Scholar) are located at the extracellular side. Thus, ADP-ribosyl cyclase activity detected in the intracellular side in NG108-15 cells could not be the same as CD38, but a neuronal isoform of ADP-ribosyl cyclase. The degree of identity between ADP-ribosyl cyclase in NG108-15 neuronal cells and that in ovotestis of Aplysia (12Lee H.C. Aarhus R. Cell Regul. 1991; 2: 203-209Crossref PubMed Scopus (322) Google Scholar) or CD38 should be further examined.cADP-ribose formation was not affected by cGMP in NG108-15 cell membranes. This conclusion, however, does not exclude the regulatory mechanism in the cytoplasm, where cytosolic ADP-ribosyl cyclase could be activated through the nitric oxide/cGMP-dependent cascade (11Berridge M.J. Nature. 1993; 365: 388-389Crossref PubMed Scopus (290) Google Scholar). In summary, our results suggest that stimulation of conventional neurotransmitter receptors can regulate cellular function through cADP-ribose as a second messenger, independently from or in concert with other second messengers. Cyclic ADP-ribose (cADP-ribose) 1The abbreviations used are: cADP-ribose, cyclic ADP-ribose; [NAD+] i , intracellular NAD+ concentration; mAChR, muscarinic acetylcholine receptor; HPLC, high pressure liquid chromatography; kb, kilobase; COS-CD38 cells, CD38-transfected COS-7 cells; PBS, phosphate-buffered saline; CCh, carbamylcholine; GTPγS, guanosine 5′-O-(3-thiotriphosphate); CTx, cholera toxin; PTx, pertussis toxin.1The abbreviations used are: cADP-ribose, cyclic ADP-ribose; [NAD+] i , intracellular NAD+ concentration; mAChR, muscarinic acetylcholine receptor; HPLC, high pressure liquid chromatography; kb, kilobase; COS-CD38 cells, CD38-transfected COS-7 cells; PBS, phosphate-buffered saline; CCh, carbamylcholine; GTPγS, guanosine 5′-O-(3-thiotriphosphate); CTx, cholera toxin; PTx, pertussis toxin. is synthesized from β-NAD+, an abundant intracellular substrate, by ADP-ribosyl cyclase in sea urchin eggs and in mammalian cells (1Rusinko N. Lee H.C. J. Biol. Chem. 1989; 264: 11725-11731Abstract Full Text PDF PubMed Google Scholar, 2Kim H. Jacobson E.L. Jacobson M.K. Science. 1993; 261: 1330-1333Crossref PubMed Scopus (233) Google Scholar). Pharmacological studies suggest that cADP-ribose is an endogenous modulator of ryanodine-sensitive Ca2+ release channels (3Mészáros L.G. Bak J. Chu A. Nature. 1993; 364: 76-79Crossref PubMed Scopus (317) Google Scholar, 4White A.M. Watson S.P. Galione A. FEBS Lett. 1993; 318: 259-263Crossref PubMed Scopus (101) Google Scholar, 5Takasawa S. Nata K. Yonekura H. Okamoto H. Science. 1993; 259: 370-373Crossref PubMed Scopus (401) Google Scholar, 6Thorn P. Gerasimenko O. Petersen O.H. EMBO J. 1994; 13: 2038-2043Crossref PubMed Scopus (187) Google Scholar, 7Hua S.-Y. Tokimasa T. Takasawa S. Furuya Y. Nohmi M. Okamoto H. Kuba K. Neuron. 1994; 12: 1073-1079Abstract Full Text PDF PubMed Scopus (126) Google Scholar, 8Lee H.C. Aarhus R. Graeff R. Gurnack M.E. Walseth T.F. Nature. 1994; 370: 307-309Crossref PubMed Scopus (208) Google Scholar, 9Kuemmerle J.F. Makhlouf G.M. J. Biol. Chem. 1995; 270: 25488-25494Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 10Lee H.C. Aarhus R. Graeff R.M. J. Biol. Chem. 1995; 270: 9060-9066Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). If cADP-ribose acts as an intracellular second messenger, ADP-ribosyl cyclase, as an effector enzyme, should be activated or inhibited in response to stimulation by hormones or neurotransmitters, which should simultaneously be associated with a transient decrease in the intracellular NAD+ concentration ([NAD+] i ) and an increase in cADP-ribose concentration (11Berridge M.J. Nature. 1993; 365: 388-389Crossref PubMed Scopus (290) Google Scholar). ADP-ribosyl cyclase seems to be present in both cytosolic and membrane-bound forms (1Rusinko N. Lee H.C. J. Biol. Chem. 1989; 264: 11725-11731Abstract Full Text PDF PubMed Google Scholar, 2Kim H. Jacobson E.L. Jacobson M.K. Science. 1993; 261: 1330-1333Crossref PubMed Scopus (233) Google Scholar, 12Lee H.C. Aarhus R. Cell Regul. 1991; 2: 203-209Crossref PubMed Scopus (322) Google Scholar). The mammalian membrane-bound form of ADP-ribosyl cyclase has been identified as a cell-surface antigen, CD38 (13Jackson D.G. Bell J.I. J. Immunol. 1990; 144: 2811-2815PubMed Google Scholar, 14Howard M. Grimaldi J.C. Bazan J.F. Lund F.E. Santos-Argumedo L. Parkhouse R.M.E. Walseth T.F. Lee H.C. Science. 1993; 262: 1056-1059Crossref PubMed Scopus (670) Google Scholar, 15Tohgo A. Takasawa S. Noguchi N. Koguma T. Nata K. Sugimoto T. Furuya Y. Yonekura H. Okamoto H. J. Biol. Chem. 1994; 269: 28555-28557Abstract Full Text PDF PubMed Google Scholar, 16Lund F. Solvason N. Grimaldi J.C. Parkhouse R.M.E. Howard M. Immunol. Today. 1995; 16: 469-473Abstract Full Text PDF PubMed Scopus (104) Google Scholar, 17Kato I. Takasawa S. Akabane A. Tanaka O. Abe H. Takamura T. Suzuki Y. Nata K. Yonekura H. Yoshimoto T. Okamoto H. J. Biol. Chem. 1995; 270: 30045-30050Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 18Prasado G.S. McRee D.E. Stura E.A. Levitt D.G. Lee H.C. Stout C.D. Nat. Struct. Biol. 1996; 3: 957-964Crossref PubMed Scopus (140) Google Scholar, 19Inageda K. Takahashi K. Tokita K. Nishina H. Kanaho Y. Kukimoto I. Kontani K. Hoshino S. Katada T. J. Biochem. (Tokyo). 1995; 117: 125-131Crossref PubMed Scopus (66) Google Scholar) and BST-1 (20Hirata Y. Kimura N. Sato K. Ohsugi Y. Takasawa S. Okamoto H. Ishikawa J. Kaisho T. Ishihara K. Hirano T. FEBS Lett. 1997; 356: 244-248Crossref Scopus (152) Google Scholar). Recently, it has been shown that the formation of cADP-ribose is regulated by nitric oxide or cGMP (21Galione A. White A. Willmott N. Turner M. Potter B.V.L. Watson S.T. Nature. 1993; 365: 456-459Crossref PubMed Scopus (259) Google Scholar, 22Willmott N. Sethi J.K. Walseth T.F. Lee H.C. White A.M. Galione A. J. Biol. Chem. 1996; 271: 3699-3705Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 23Clementi E. Riccio M. Sciorati C. Nistico G. Meldolesi J. J. Biol. Chem. 1996; 271: 17739-17745Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar) and that nitric oxide or cGMP is increased by stimulation with agonists (24Mathes C. Thompson S.H. J. Neurosci. 1996; 16: 1702-1709Crossref PubMed Google Scholar, 25Matsuzawa H. Nirenberg M. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 3472-3476Crossref PubMed Scopus (163) Google Scholar). These findings suggest the hypothesis that the regulation of the cADP-ribose level is located far downstream in the signal transduction cascade from receptors (11Berridge M.J. Nature. 1993; 365: 388-389Crossref PubMed Scopus (290) Google Scholar). An alternative hypothesis is that the cADP-ribose formation is regulated by ADP-ribosyl cyclase through the direct action of G proteins activated by receptors within the surface membrane, as already shown for the formation of cyclic AMP, inositol 1,4,5-trisphosphate, and diacylglycerol (26Gilman A.G. Cell. 1984; 36: 577-579Abstract Full Text PDF PubMed Scopus (1102) Google Scholar, 27Berridge M.J. Irvine R.F. Nature. 1984; 312: 315-321Crossref PubMed Scopus (4221) Google Scholar, 28Nishizuka Y. Nature. 1984; 308: 693-698Crossref PubMed Scopus (5741) Google Scholar). To test this hypothesis, we used NG108-15 neuroblastoma × glioma hybrid cells (29Nirenberg M. Wilson S. Higashida H. Rotter A. Krueger K. Busis N. Ray R. Kenimer J.G. Adler M. Science. 1983; 222: 794-799Crossref PubMed Scopus (237) Google Scholar), in which signal transduction from receptors to effectors has been extensively characterized (29Nirenberg M. Wilson S. Higashida H. Rotter A. Krueger K. Busis N. Ray R. Kenimer J.G. Adler M. Science. 1983; 222: 794-799Crossref PubMed Scopus (237) Google Scholar, 30Noda M. Ishizaka N. Yokoyama S. Hoshi N. Kimura Y. Hashii M. Taketo M. Egorova A. Knijnik R. Fukuda K. Morikawa H. Brown D.A. Higashida H. J. Lipid Mediators Cell Signalling. 1996; 14: 175-185Crossref PubMed Scopus (15) Google Scholar). In particular, in NGPM1-27 cells (31Fukuda K. Higashida H. Kubo T. Maeda A. Akiba I. Bujo H. Mishina M. Numa S. Nature. 1988; 335: 355-358Crossref PubMed Scopus (181) Google Scholar), which overexpress muscarinic acetylcholine receptors (mAChRs), it has been shown that intracellular NAD+ or NAD+metabolites are involved in signal transduction from m1 mAChRs to K+ channels (32Higashida H. Robbins J. Egorova A. Noda M. Taketo M. Ishizaka N. Takasawa S. Okamoto H. Brown D.A. J. Physiol. (Lond.). 1995; 482: 317-323Crossref Scopus (49) Google Scholar, 33Higashida H. Egorova A. Hoshi N. Noda M. FEBS Lett. 1996; 379: 236-238Crossref PubMed Scopus (13) Google Scholar). In this context, such neuronal cell lines have advantages for analyzing receptor-ADP-ribosyl cyclase coupling in detail. For measurement of ADP-ribosyl cyclase, high pressure liquid chromatography (HPLC) is commonly used to separate cADP-ribose-related compounds (1Rusinko N. Lee H.C. J. Biol. Chem. 1989; 264: 11725-11731Abstract Full Text PDF PubMed Google Scholar, 2Kim H. Jacobson E.L. Jacobson M.K. Science. 1993; 261: 1330-1333Crossref PubMed Scopus (233) Google Scholar, 8Lee H.C. Aarhus R. Graeff R. Gurnack M.E. Walseth T.F. Nature. 1994; 370: 307-309Crossref PubMed Scopus (208) Google Scholar, 14Howard M. Grimaldi J.C. Bazan J.F. Lund F.E. Santos-Argumedo L. Parkhouse R.M.E. Walseth T.F. Lee H.C. Science. 1993; 262: 1056-1059Crossref PubMed Scopus (670) Google Scholar, 15Tohgo A. Takasawa S. Noguchi N. Koguma T. Nata K. Sugimoto T. Furuya Y. Yonekura H. Okamoto H. J. Biol. Chem. 1994; 269: 28555-28557Abstract Full Text PDF PubMed Google Scholar, 17Kato I. Takasawa S. Akabane A. Tanaka O. Abe H. Takamura T. Suzuki Y. Nata K. Yonekura H. Yoshimoto T. Okamoto H. J. Biol. Chem. 1995; 270: 30045-30050Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 19Inageda K. Takahashi K. Tokita K. Nishina H. Kanaho Y. Kukimoto I. Kontani K. Hoshino S. Katada T. J. Biochem. (Tokyo). 1995; 117: 125-131Crossref PubMed Scopus (66) Google Scholar, 34Takasawa S. Tohgo A. Noguchi N. Koguma T. Nata K. Sugimoto T. Yonekura H. Okamoto H. J. Biol. Chem. 1993; 268: 26052-26054Abstract Full Text PDF PubMed Google Scholar, 35Tohgo A. Munakata H. Takasawa S. Nata K. Akiyama T. Hayashi N. Okamoto H. J. Biol. Chem. 1997; 272: 3879-3882Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). However, since it takes 30–60 min to process one sample, it is essential to develop a much more rapid method that can allow processing of multiple samples at once. There are two papers that describe ADP-ribosyl cyclase assay by TLC (21Galione A. White A. Willmott N. Turner M. Potter B.V.L. Watson S.T. Nature. 1993; 365: 456-459Crossref PubMed Scopus (259) Google Scholar, 36Mészáros V. Socci R. Mészáros L.G. Biochem. Biophys. Res. Commun. 1995; 210: 452-456Crossref PubMed Scopus (22) Google Scholar), in which NAD+ migrates faster than cADP-ribose. The methods used in those reports seem to be affected by large amounts of radiolabeled substrates. We here developed a TLC method that overcomes this problem and allows separation of cADP-ribose in up to 19 samples within 40–50 min. Our TLC method was first tested on COS-7 cells overexpressing human CD38 and was shown to be applicable for measuring ADP-ribosyl cyclase activity. We demonstrate that crude cell membranes of NG108-15 cells possess ADP-ribosyl cyclase activity and that such activity is activated or inhibited in a mAChR subtype-specific manner in NG108-15 cells overexpressing distinct mAChR subtypes. Furthermore, to ascertain the intracellular role of the catalytic activity of ADP-ribosyl cyclase in neuronal cell membranes, the time course of [NAD+] i after extracellular application of acetylcholine to these cells was investigated. DISCUSSIONThe chromatographic procedure used here resulted in a resolution sufficient to measure 3H accumulation in the cADP-ribose fraction originating from adenine-labeled [3H]NAD+, and it was free from interference by the labeled substrate. One possible limitation could be that the cADP-ribose fraction was contaminated by ∼15% of the newly synthesized [3H]ADP-ribose (Fig. 2 A). However, the risk that we measured the ADP-ribose count in the cADP-ribose fraction seems to be negligible since different patterns of3H accumulation in ADP-ribose and cADP-ribose fractions were obtained (Fig. 4), reflecting that distinct materials are accumulated with different rates of formation and degradation. The autoradiograms for COS-CD38 and NGPM1-27 cells (Fig. 3) clearly demonstrated the overall fate of NAD+ in that the majority of NAD+ was converted to either ADP-ribose or cADP-ribose or both in two or more enzyme reaction steps, probably ADP-ribosyl cyclase and cADP-ribose hydrolase.The NAD+ concentration used in our reaction mixture (2 μm) was much lower than values (∼100 μm) used in other studies (1Rusinko N. Lee H.C. J. Biol. Chem. 1989; 264: 11725-11731Abstract Full Text PDF PubMed Google Scholar, 2Kim H. Jacobson E.L. Jacobson M.K. Science. 1993; 261: 1330-1333Crossref PubMed Scopus (233) Google Scholar, 5Takasawa S. Nata K. Yonekura H. Okamoto H. Science. 1993; 259: 370-373Crossref PubMed Scopus (401) Google Scholar, 15Tohgo A. Takasawa S. Noguchi N. Koguma T. Nata K. Sugimoto T. Furuya Y. Yonekura H. Okamoto H. J. Biol. Chem. 1994; 269: 28555-28557Abstract Full Text PDF PubMed Google Scholar). However, our concentration is very similar to the estimated K m value (3–5 μm) for ADP-ribosyl cyclase in NG108-15 cell membranes. 2H. Higashida, S. Yokoyama, M. Higashida, and M. Noda, unpublished data. Under such conditions, [3H]cADP-ribose accumulated linearly for 2–4 min and [3H]ADP-ribose for 16 min, suggesting that it is not an insufficient dose. Thus, our TLC method was used for measurement of ADP-ribosyl cyclase, at least during the first few minutes. We do not know whether or not this TLC method is applicable for measurement of other recently found enzyme reactions in ADP-ribosyl cyclase, such as production of cyclic 2′-phosphoadenosine diphosphoribose (41Vu C.Q. Lu P.-J. Chen C.-S. Jacobson M.K. J. Biol. Chem. 1996; 271: 4747-4754Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), nicotinic acid adenine 5′-diphophate ribose (42Genazzani A.A. Mezna M. Summerhill R.J. Galione A. Michelangeli F. J. Biol. Chem. 1997; 272: 7669-7675Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar), and dimeric ADP-ribose (43De Flora A. Guida L. Franco L. Zocchi E. Bruzzone S. Benatti U. Damonte G. Lee H.C. J. Biol. Chem. 1997; 272: 12945-12951Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). It remains to be tested how our TLC method differs in accuracy from HPLC and fluorometric measurement of cyclic GDP-ribose formation (20Hirata Y. Kimura N. Sato K. Ohsugi Y. Takasawa S. Okamoto H. Ishikawa J. Kaisho T. Ishihara K. Hirano T. FEBS Lett. 1997; 356: 244-248Crossref Scopus (152) Google Scholar, 44Graeff R.M. Walseth T.F. Fryxell K. Branton W.D. Lee H.C. J. Biol. Chem. 1994; 269: 30260-30267Abstract Full Text PDF PubMed Google Scholar, 45De Flora A. Guida L. Franco L. Zocchi E. Pestarino M. Usai C. Marchetti C. Fedele E. Fontana G. Raiteri M. Biochem. J. 1996; 320: 665-672Crossref PubMed Scopus (52) Google Scholar).The results provide the first evidence that cholinergic signals are transduced from mAChRs to ADP-ribosyl cyclase within the membrane in a receptor subtype-specific fashion (Figs. 5 and 6). The finding that the inhibition of ADP-ribosyl cyclase via m2/m4 mAChRs is mediated through a PTx-sensitive G protein resembles the inhibitory signal transduction known for adenylate cyclase in NG108-15 cells (29Nirenberg M. Wilson S. Higashida H. Rotter A. Krueger K. Busis N. Ray R. Kenimer J.G. Adler M. Science. 1983; 222: 794-799Crossref PubMed Scopus (237) Google Scholar, 30Noda M. Ishizaka N. Yokoyama S. Hoshi N. Kimura Y. Hashii M. Taketo M. Egorova A. Knijnik R. Fukuda K. Morikawa H. Brown D.A. Higashida H. J. Lipid Mediators Cell Signalling. 1996; 14: 175-185Crossref PubMed Scopus (15) Google Scholar, 31Fukuda K. Higashida H. Kubo T. Maeda A. Akiba I. Bujo H. Mishina M. Numa S. Nature. 1988; 335: 355-358Crossref PubMed Scopus (181) Google Scholar). Coupling of m1/m3 mAChRs to ADP-ribosyl cyclase is relatively resistant to PTx, but highly sensitive to CTx, which seems to be different from the signal pathway to phospholipase Cβ (30Noda M. Ishizaka N. Yokoyama S. Hoshi N. Kimura Y. Hashii M. Taketo M. Egorova A. Knijnik R. Fukuda K. Morikawa H. Brown D.A. Higashida H. J. Lipid Mediators Cell Signalling. 1996; 14: 175-185Crossref PubMed Scopus (15) Google Scholar, 31Fukuda K. Higashida H. Kubo T. Maeda A. Akiba I. Bujo H. Mishina M. Numa S. Nature. 1988; 335: 355-358Crossref PubMed Scopus (181) Google Scholar). Thus, the coupling from mAChRs to ADP-ribosyl cyclase via G proteins would be a new mode of signal transduction, in parallel with the known pathways to adenylate cyclase and phospholipase C in NG108-15 cells.The simplest explanation for our [NAD+] i data in NGPM1-27 cells (Fig. 7) could be that NAD+ consumption is accelerated by the activation with the agonist of a membrane-bound form of ADP-ribosyl cyclase whose catalytic site resides in the cell interior. This speculation, however, would require a different topology from that proposed for the CD38 cell-surface antigen, whose catalytic sites of ADP-ribosyl cyclase (15Tohgo A. Takasawa S. Noguchi N. Koguma T. Nata K. Sugimoto T. Furuya Y. Yonekura H. Okamoto H. J. Biol. Chem. 1994; 269: 28555-28557Abstract Full Text PDF PubMed Google Scholar, 18Prasado G.S. McRee D.E. Stura E.A. Levitt D.G. Lee H.C. Stout C.D. Nat. Struct. Biol. 1996; 3: 957-964Crossref PubMed Scopus (140) Google Scholar, 45De Flora A. Guida L. Franco L. Zocchi E. Pestarino M. Usai C. Marchetti C. Fedele E. Fontana G. Raiteri M. Biochem. J. 1996; 320: 665-672Crossref PubMed Scopus (52) Google Scholar), ADP-ribose hydrolase (15Tohgo A. Takasawa S. Noguchi N. Koguma T. Nata K. Sugimoto T. Furuya Y. Yonekura H. Okamoto H. J. Biol. Chem. 1994; 269: 28555-28557Abstract Full Text PDF PubMed Google Scholar,46Zocchi E. Franco L. Guida L. Benatti U. Bargellesi A. Malavasi F. Lee H.C. De Flora D. Biochem. Biophys. Res. Commun. 1993; 196: 1459-1465Crossref PubMed Scopus (261) Google Scholar), and NAD+ glycohydrolase (19Inageda K. Takahashi K. Tokita K. Nishina H. Kanaho Y. Kukimoto I. Kontani K. Hoshino S. Katada T. J. Biochem. (Tokyo). 1995; 117: 125-131Crossref PubMed Scopus (66) Google Scholar) are located at the extracellular side. Thus, ADP-ribosyl cyclase activity detected in the intracellular side in NG108-15 cells could not be the same as CD38, but a neuronal isoform of ADP-ribosyl cyclase. The degree of identity between ADP-ribosyl cyclase in NG108-15 neuronal cells and that in ovotestis of Aplysia (12Lee H.C. Aarhus R. Cell Regul. 1991; 2: 203-209Crossref PubMed Scopus (322) Google Scholar) or CD38 should be further examined.cADP-ribose formation was not affected by cGMP in NG108-15 cell membranes. This conclusion, however, does not exclude the regulatory mechanism in the cytoplasm, where cytosolic ADP-ribosyl cyclase could be activated through the nitric oxide/cGMP-dependent cascade (11Berridge M.J. Nature. 1993; 365: 388-389Crossref PubMed Scopus (290) Google Scholar). In summary, our results suggest that stimulation of conventional neurotransmitter receptors can regulate cellular function through cADP-ribose as a second messenger, independently from or in concert with other second messengers. The chromatographic procedure used here resulted in a resolution sufficient to measure 3H accumulation in the cADP-ribose fraction originating from adenine-labeled [3H]NAD+, and it was free from interference by the labeled substrate. One possible limitation could be that the cADP-ribose fraction was contaminated by ∼15% of the newly synthesized [3H]ADP-ribose (Fig. 2 A). However, the risk that we measured the ADP-ribose count in the cADP-ribose fraction seems to be negligible since different patterns of3H accumulation in ADP-ribose and cADP-ribose fractions were obtained (Fig. 4), reflecting that distinct materials are accumulated with different rates of formation and degradation. The autoradiograms for COS-CD38 and NGPM1-27 cells (Fig. 3) clearly demonstrated the overall fate of NAD+ in that the majority of NAD+ was converted to either ADP-ribose or cADP-ribose or both in two or more enzyme reaction steps, probably ADP-ribosyl cyclase and cADP-ribose hydrolase. The NAD+ concentration used in our reaction mixture (2 μm) was much lower than values (∼100 μm) used in other studies (1Rusinko N. Lee H.C. J. Biol. Chem. 1989; 264: 11725-11731Abstract Full Text PDF PubMed Google Scholar, 2Kim H. Jacobson E.L. Jacobson M.K. Science. 1993; 261: 1330-1333Crossref PubMed Scopus (233) Google Scholar, 5Takasawa S. Nata K. Yonekura H. Okamoto H. Science. 1993; 259: 370-373Crossref PubMed Scopus (401) Google Scholar, 15Tohgo A. Takasawa S. Noguchi N. Koguma T. Nata K. Sugimoto T. Furuya Y. Yonekura H. Okamoto H. J. Biol. Chem. 1994; 269: 28555-28557Abstract Full Text PDF PubMed Google Scholar). However, our concentration is very similar to the estimated K m value (3–5 μm) for ADP-ribosyl cyclase in NG108-15 cell membranes. 2H. Higashida, S. Yokoyama, M. Higashida, and M. Noda, unpublished data. Under such conditions, [3H]cADP-ribose accumulated linearly for 2–4 min and [3H]ADP-ribose for 16 min, suggesting that it is not an insufficient dose. Thus, our TLC method was used for measurement of ADP-ribosyl cyclase, at least during the first few minutes. We do not know whether or not this TLC method is applicable for measurement of other recently found enzyme reactions in ADP-ribosyl cyclase, such as production of cyclic 2′-phosphoadenosine diphosphoribose (41Vu C.Q. Lu P.-J. Chen C.-S. Jacobson M.K. J. Biol. Chem. 1996; 271: 4747-4754Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), nicotinic acid adenine 5′-diphophate ribose (42Genazzani A.A. Mezna M. Summerhill R.J. Galione A. Michelangeli F. J. Biol. Chem. 1997; 272: 7669-7675Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar), and dimeric ADP-ribose (43De Flora A. Guida L. Franco L. Zocchi E. Bruzzone S. Benatti U. Damonte G. Lee H.C. J. Biol. Chem. 1997; 272: 12945-12951Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). It remains to be tested how our TLC method differs in accuracy from HPLC and fluorometric measurement of cyclic GDP-ribose formation (20Hirata Y. Kimura N. Sato K. Ohsugi Y. Takasawa S. Okamoto H. Ishikawa J. Kaisho T. Ishihara K. Hirano T. FEBS Lett. 1997; 356: 244-248Crossref Scopus (152) Google Scholar, 44Graeff R.M. Walseth T.F. Fryxell K. Branton W.D. Lee H.C. J. Biol. Chem. 1994; 269: 30260-30267Abstract Full Text PDF PubMed Google Scholar, 45De Flora A. Guida L. Franco L. Zocchi E. Pestarino M. Usai C. Marchetti C. Fedele E. Fontana G. Raiteri M. Biochem. J. 1996; 320: 665-672Crossref PubMed Scopus (52) Google Scholar). The results provide the first evidence that cholinergic signals are transduced from mAChRs to ADP-ribosyl cyclase within the membrane in a receptor subtype-specific fashion (Figs. 5 and 6). The finding that the inhibition of ADP-ribosyl cyclase via m2/m4 mAChRs is mediated through a PTx-sensitive G protein resembles the inhibitory signal transduction known for adenylate cyclase in NG108-15 cells (29Nirenberg M. Wilson S. Higashida H. Rotter A. Krueger K. Busis N. Ray R. Kenimer J.G. Adler M. Science. 1983; 222: 794-799Crossref PubMed Scopus (237) Google Scholar, 30Noda M. Ishizaka N. Yokoyama S. Hoshi N. Kimura Y. Hashii M. Taketo M. Egorova A. Knijnik R. Fukuda K. Morikawa H. Brown D.A. Higashida H. J. Lipid Mediators Cell Signalling. 1996; 14: 175-185Crossref PubMed Scopus (15) Google Scholar, 31Fukuda K. Higashida H. Kubo T. Maeda A. Akiba I. Bujo H. Mishina M. Numa S. Nature. 1988; 335: 355-358Crossref PubMed Scopus (181) Google Scholar). Coupling of m1/m3 mAChRs to ADP-ribosyl cyclase is relatively resistant to PTx, but highly sensitive to CTx, which seems to be different from the signal pathway to phospholipase Cβ (30Noda M. Ishizaka N. Yokoyama S. Hoshi N. Kimura Y. Hashii M. Taketo M. Egorova A. Knijnik R. Fukuda K. Morikawa H. Brown D.A. Higashida H. J. Lipid Mediators Cell Signalling. 1996; 14: 175-185Crossref PubMed Scopus (15) Google Scholar, 31Fukuda K. Higashida H. Kubo T. Maeda A. Akiba I. Bujo H. Mishina M. Numa S. Nature. 1988; 335: 355-358Crossref PubMed Scopus (181) Google Scholar). Thus, the coupling from mAChRs to ADP-ribosyl cyclase via G proteins would be a new mode of signal transduction, in parallel with the known pathways to adenylate cyclase and phospholipase C in NG108-15 cells. The simplest explanation for our [NAD+] i data in NGPM1-27 cells (Fig. 7) could be that NAD+ consumption is accelerated by the activation with the agonist of a membrane-bound form of ADP-ribosyl cyclase whose catalytic site resides in the cell interior. This speculation, however, would require a different topology from that proposed for the CD38 cell-surface antigen, whose catalytic sites of ADP-ribosyl cyclase (15Tohgo A. Takasawa S. Noguchi N. Koguma T. Nata K. Sugimoto T. Furuya Y. Yonekura H. Okamoto H. J. Biol. Chem. 1994; 269: 28555-28557Abstract Full Text PDF PubMed Google Scholar, 18Prasado G.S. McRee D.E. Stura E.A. Levitt D.G. Lee H.C. Stout C.D. Nat. Struct. Biol. 1996; 3: 957-964Crossref PubMed Scopus (140) Google Scholar, 45De Flora A. Guida L. Franco L. Zocchi E. Pestarino M. Usai C. Marchetti C. Fedele E. Fontana G. Raiteri M. Biochem. J. 1996; 320: 665-672Crossref PubMed Scopus (52) Google Scholar), ADP-ribose hydrolase (15Tohgo A. Takasawa S. Noguchi N. Koguma T. Nata K. Sugimoto T. Furuya Y. Yonekura H. Okamoto H. J. Biol. Chem. 1994; 269: 28555-28557Abstract Full Text PDF PubMed Google Scholar,46Zocchi E. Franco L. Guida L. Benatti U. Bargellesi A. Malavasi F. Lee H.C. De Flora D. Biochem. Biophys. Res. Commun. 1993; 196: 1459-1465Crossref PubMed Scopus (261) Google Scholar), and NAD+ glycohydrolase (19Inageda K. Takahashi K. Tokita K. Nishina H. Kanaho Y. Kukimoto I. Kontani K. Hoshino S. Katada T. J. Biochem. (Tokyo). 1995; 117: 125-131Crossref PubMed Scopus (66) Google Scholar) are located at the extracellular side. Thus, ADP-ribosyl cyclase activity detected in the intracellular side in NG108-15 cells could not be the same as CD38, but a neuronal isoform of ADP-ribosyl cyclase. The degree of identity between ADP-ribosyl cyclase in NG108-15 neuronal cells and that in ovotestis of Aplysia (12Lee H.C. Aarhus R. Cell Regul. 1991; 2: 203-209Crossref PubMed Scopus (322) Google Scholar) or CD38 should be further examined. cADP-ribose formation was not affected by cGMP in NG108-15 cell membranes. This conclusion, however, does not exclude the regulatory mechanism in the cytoplasm, where cytosolic ADP-ribosyl cyclase could be activated through the nitric oxide/cGMP-dependent cascade (11Berridge M.J. Nature. 1993; 365: 388-389Crossref PubMed Scopus (290) Google Scholar). In summary, our results suggest that stimulation of conventional neurotransmitter receptors can regulate cellular function through cADP-ribose as a second messenger, independently from or in concert with other second messengers. We thank Tatsuya Haga for discussion and Hugh Robinson for critical reading of the manuscript.