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• W2059354304 abstract "In mammals cyclic ADP-ribose (cADPR), a universal calcium mobilizer from intracellular stores, is generated from NAD+ at the outer cell surface by the multifunctional ectoenzyme CD38 and by related ADP-ribosyl cyclases. Recently, influx of extracellular cADPR has been observed in 3T3 murine fibroblasts, where it elicits Ca2+-mediated enhancement of proliferation. Here we addressed the nature and the properties of cADPR influx into CD38− 3T3 cells, which showed pleiotropic mechanisms of both equilibrative and concentrative transport. Based on selective inhibitors or experimental conditions (e.g.abrogation of Na+-dependent active symport processes and transient transfection experiments) and on reverse transcriptase-polymerase chain reaction analysis of transcripts in 3T3 fibroblasts and comparatively in HeLa cells, we identified cADPR-transporting activities with specific nucleoside transporters (NT), both equilibrative (ENT2) and concentrative (CNT2 and a nitrobenzylthioinosine (NBMPR)-inhibitable NT). A reciprocal inhibition relationship was observed between inosine and cADPR fluxes across these NT species. Concentrative (but not equilibrative) transport of nanomolar extracellular cADPR took place in CD38− 3T3 cells co-cultured for 48 h in transwells on feeders of CD38-transfected, cADPR-generating 3T3 fibroblasts. These results suggest possible, hitherto unrecognized, correlations between ectocellular metabolism of nucleotides/nucleosides and cADPR-mediated regulation of intracellular calcium homeostasis. In mammals cyclic ADP-ribose (cADPR), a universal calcium mobilizer from intracellular stores, is generated from NAD+ at the outer cell surface by the multifunctional ectoenzyme CD38 and by related ADP-ribosyl cyclases. Recently, influx of extracellular cADPR has been observed in 3T3 murine fibroblasts, where it elicits Ca2+-mediated enhancement of proliferation. Here we addressed the nature and the properties of cADPR influx into CD38− 3T3 cells, which showed pleiotropic mechanisms of both equilibrative and concentrative transport. Based on selective inhibitors or experimental conditions (e.g.abrogation of Na+-dependent active symport processes and transient transfection experiments) and on reverse transcriptase-polymerase chain reaction analysis of transcripts in 3T3 fibroblasts and comparatively in HeLa cells, we identified cADPR-transporting activities with specific nucleoside transporters (NT), both equilibrative (ENT2) and concentrative (CNT2 and a nitrobenzylthioinosine (NBMPR)-inhibitable NT). A reciprocal inhibition relationship was observed between inosine and cADPR fluxes across these NT species. Concentrative (but not equilibrative) transport of nanomolar extracellular cADPR took place in CD38− 3T3 cells co-cultured for 48 h in transwells on feeders of CD38-transfected, cADPR-generating 3T3 fibroblasts. These results suggest possible, hitherto unrecognized, correlations between ectocellular metabolism of nucleotides/nucleosides and cADPR-mediated regulation of intracellular calcium homeostasis. Cyclic ADP-ribose (cADPR) 1The abbreviations used are: cADPR, cyclic ADP-ribose; NT, nucleoside transporters; NBMPR, nitrobenzylthioinosine; NAADP+, nicotinic acid adenine dinucleotide phosphate; Cx43, connexin 43; NGD+, nicotinamide guanine dinucleotide; HPLC, high pressure liquid chromatography; RT-PCR, reverse transcriptase-PCR; DIDS, 4–4-di-isothiocyano-2,2-stilbene disulfonic acid; ENT, equilibrative nucleoside transporter; CNT, concentrative nucleoside transporter 1The abbreviations used are: cADPR, cyclic ADP-ribose; NT, nucleoside transporters; NBMPR, nitrobenzylthioinosine; NAADP+, nicotinic acid adenine dinucleotide phosphate; Cx43, connexin 43; NGD+, nicotinamide guanine dinucleotide; HPLC, high pressure liquid chromatography; RT-PCR, reverse transcriptase-PCR; DIDS, 4–4-di-isothiocyano-2,2-stilbene disulfonic acid; ENT, equilibrative nucleoside transporter; CNT, concentrative nucleoside transporter is a potent and universal calcium mobilizer from intracellular stores (1Lee H.C. Walseth T.F. Bratt G.T. Hayes R.N. Clapper D.L. J. Biol. Chem. 1989; 264: 1608-1615Google Scholar, 2Lee H.C. Galione A. Walseth T.F. Vitam. Horm. 1994; 48: 199-257Google Scholar, 3Lee H.C. Physiol. Rev. 1996; 77: 1133-1164Google Scholar, 4Guse A.H. Curr. Mol. Med. 2002; 2: 273-282Google Scholar, 5Higashida H. Hashii M. Yokohama S. Hoshi N. Chen X.L. Egorova A. Noda M. Zhang J.S. Pharmacol. Ther. 2001; 90: 283-296Google Scholar). During phylogenesis, the physiological role of cADPR has evolved in parallel with the increasing complexity of the biological systems where it is present: from cell cycle regulator in Euglena gracilis (6Masuda W. Takenaka S. Inageda K. Nishima H. Takahashi K. Katada T. Tsuyama S. Inui H. Miyatake K. Nakano Y. FEBS Lett. 1997; 405: 104-106Google Scholar), to signal molecule in response to environmental stress in higher plants (7Wu Y. Kuzma J. Marechal E. Graeff R. Lee H.C. Foster R. Chua N.H. Science. 1997; 278: 2126-2130Google Scholar) and in Porifera (8Zocchi E. Carpaneto A. Cerrano C. Bavestrello G. Giovine M. Bruzzone S. Guida L. Franco L. Usai C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14859-14864Google Scholar), or during oocyte fertilization in marine invertebrates (9Lee H.C. Biol. Signals. 1996; 5: 101-110Google Scholar). In mammals, cADPR is involved in a number of Ca2+-dependent cell- and tissue-specific functions, including proliferation, contraction, and secretion (1Lee H.C. Walseth T.F. Bratt G.T. Hayes R.N. Clapper D.L. J. Biol. Chem. 1989; 264: 1608-1615Google Scholar, 2Lee H.C. Galione A. Walseth T.F. Vitam. Horm. 1994; 48: 199-257Google Scholar, 3Lee H.C. Physiol. Rev. 1996; 77: 1133-1164Google Scholar, 10Lee H.C. Annu. Rev. Pharmacol. Toxicol. 2001; 41: 317-345Google Scholar, 11Carafoli E. Santella L. Branca D. Brini M. Crit. Rev. Biochem. Mol. Biol. 2001; 36: 107-260Google Scholar, 12Berridge M.J. Lipp P. Bootman M.D. Nat. Rev. Mol. Cell. Biol. 2001; 1: 11-21Google Scholar).In several mammalian cells and tissue microenvironments, cADPR and its metabolic precursor NAD+ have recently been demonstrated to undergo a variable though potentially intense transmembrane trafficking (13De Flora A. Guida L. Franco L. Bruzzone S. Zocchi E. Lee H.C. Cyclic ADP-ribose and NAADP: Structure, Metabolism and Physiological Functions. Kluwer Acad., Norwell, MS2002Google Scholar). By virtue of these subcellular and intercellular movements, cADPR can access its ryanodine-sensitive intracellular Ca2+stores/channels despite generation of this cyclic nucleotide by ADP-ribosyl cyclases at sites that are topologically opposite to the stores (13De Flora A. Guida L. Franco L. Bruzzone S. Zocchi E. Lee H.C. Cyclic ADP-ribose and NAADP: Structure, Metabolism and Physiological Functions. Kluwer Acad., Norwell, MS2002Google Scholar, 14De Flora A. Franco L. Guida L. Bruzzone S. Usai C. Zocchi E. Chem. Immunol. 2000; 75: 79-98Google Scholar). The most represented member of these cyclases in mammalian cells is CD38 (15Jackson D.G. Bell J.I. J. Immunol. 1990; 144: 2811-2815Google Scholar), a type II glycoprotein involved in several transduction pathways (16Mehta K. Umar S. Malavasi F. FASEB J. 1996; 10: 1408-1417Google Scholar) and a multifunctional ectoenzyme that synthesizes cADPR from NAD+ and also hydrolyzes cADPR to ADP-ribose (17States D.J. Walseth T.F. Lee H.C. Trends Biochem. Sci. 1992; 17: 495Google Scholar, 18Lee H.C. Graeff R.M. Walseth T.F. Adv. Exp. Med. Biol. 1997; 419: 411-419Google Scholar). An important additional activity of CD38 is synthesis of the potent calcium mobilizer NAADP+ from NADP+ and nicotinic acid (18Lee H.C. Graeff R.M. Walseth T.F. Adv. Exp. Med. Biol. 1997; 419: 411-419Google Scholar, 19Aarhus R. Graeff R.M. Dickey D.M. Walseth T.F. Lee H.C. J. Biol. Chem. 1995; 270: 30327-30333Google Scholar).Availability of intracellular NAD+ to the ectocellular active site of CD38 is made possible by its controlled release from cells across an equilibrative transport system, represented by hexameric hemichannels of the gap junctional protein connexin 43 (Cx43) (20Bruzzone S. Guida L. Zocchi E. Franco L. De Flora A. FASEB J. 2001; 15: 10-12Google Scholar). Subsequent cADPR generation at the outer cell surface can be steadily followed by its influx into CD38+ cells because of the peculiar property of transmembrane oligomeric CD38 to couple cADPR synthesis to its active translocation across the cell membrane to reach the Ca2+ stores in the intracellular environment (21Franco L. Guida L. Bruzzone S. Zocchi E. Usai C. De Flora A. FASEB J. 1998; 12: 1507-1520Google Scholar). Such autocrine cross-talk between NAD+-releasing Cx43 hemichannels and CD38 has been demonstrated to finely up-regulate the [Ca2+]i levels in 3T3 murine fibroblasts (22Bruzzone S. Franco L. Guida L. Zocchi E. Contini P. Bisso A. Usai C. De Flora A. J. Biol. Chem. 2001; 276: 48300-48308Google Scholar).However, the peculiar localization and the properties of the catalytic site of CD38 suggest that a significant fraction of ectocellularly produced cADPR might escape transport across CD38 and thus be present in the extracellular environment, especially in close proximity of CD38+ cells. This assumption was strongly supported by the finding of 40–60 nm concentrations of NAD+ in human blood plasma 2T. F. Walseth, personal communication.2T. F. Walseth, personal communication. and by the recent demonstration that nanomolar concentrations of cADPR are consistently detectable in the medium of heterologous transwell co-cultures of CD38+/CD38− 3T3 fibroblasts (23Franco L. Zocchi E. Usai C. Guida L. Bruzzone S. Costa A. De Flora A. J. Biol. Chem. 2001; 276: 21642-21648Google Scholar). Both data raised the question whether extracellular cADPR can permeate across the plasma membrane of cADPR-responsive cells through mechanisms (either diffusion or transport) independent of those featured by CD38 as a catalytically active transporter of cADPR itself (21Franco L. Guida L. Bruzzone S. Zocchi E. Usai C. De Flora A. FASEB J. 1998; 12: 1507-1520Google Scholar). Recently, such CD38-unrelated transport system was reported to be active in native, constitutively CD38−, 3T3 fibroblasts. This process proved to account for the increased [Ca2+]i levels and consequently for the enhanced proliferation of CD38− cells when these are grown on cADPR-generating CD38+ cell feeders (23Franco L. Zocchi E. Usai C. Guida L. Bruzzone S. Costa A. De Flora A. J. Biol. Chem. 2001; 276: 21642-21648Google Scholar). A role for the CD38-independent cADPR transport system can be retrospectively extended to explain the Ca2+-related functional responses elicited by extracellular cADPR in a number of cell types: murine B lymphocytes (24Howard M. Grimaldi J.C. Bazan J.F. Lund F.E. Santos-Argumedo L. Parkhouse R.M. Walseth T.F. Lee H.C. Science. 1993; 262: 1056-1059Google Scholar), rat cerebellar granule cells (25De Flora A. Guida L. Franco L. Zocchi E. Pestarino M. Usai C. Marchetti C. Fedele E. Fontana G. Raiteri M. Biochem. J. 1996; 320: 665-672Google Scholar), smooth myocytes in intact trachea fragments (26Franco L. Bruzzone S. Song P. Guida L. Zocchi E. Walseth T.F. Crimi E. Usai C. De Flora A. Brusasco V. Am. J. Physiol. Lung Cell Mol. Physiol. 2001; 280: L98-L106Google Scholar), and human hemopoietic progenitors (27Podestà M. Zocchi E. Pitto A. Usai C. Franco L. Bruzzone S. Guida L. Bacigalupo A. Scadden D.T. Walseth T.F. De Flora A. Daga A. FASEB J. 2000; 14: 680-690Google Scholar, 28Zocchi E. Podestà M. Pitto A. Usai C. Bruzzone S. Franco L. Guida L. Bacigalupo A. De Flora A. FASEB J. 2001; 15: 1610-1612Google Scholar).These facts prompted us to start a study aiming to elucidate the properties and the nature of the transport system(s) whereby several cells can respond to extracellularly generated cADPR. The results reported here identify specific equilibrative and concentrative nucleoside transporters (NT) responsible for cADPR translocation: these NT are widely though variably expressed in mammalian cells (29Wang J. Schaner M.E. Thomassen S. Su S.F. Piquette-Midler M. Giacomini K.M. Pharmacol. Res. 1997; 14: 1524-1532Google Scholar, 30Cass C.E. Young J.D. Baldwin S.A. Cabita M.A. Graham K.A. Griffiths M. Jennings L.C. Mackey J.R. Ng A.M. Ritzel M.W. Vickers M.F. Yao S.Y. Amidon G.L. Sadee W. Membrane transporters as drug targets. Kluwer Academic/Plenum Publishers, New York1999: 313-352Google Scholar, 31Baldwin S.A. Mackey J.R. Cass C.E. Young J.D. Mol. Med. Today. 1999; 5: 216-224Google Scholar, 32Pastor-Anglada M. Baldwin S.A. Drug Dev. Res. 2001; 52: 431-437Google Scholar, 33Ritzel M.W. Ng A.M. Yao S.Y. Graham K. Loewen S.K. Smith K.M. Hyde R.J. Karpinski E. Cass C.E. Baldwin S.A. Young J.D. Mol. Membr. Biol. 2001; 18: 65-72Google Scholar), accounting for the widespread efficacy of extracellular cADPR observed in several cell types. Accordingly, a functional link is established between nucleosides and cADPR, which may bear novel and unexpected consequences on the control of Ca2+-related cell processes and on possible therapeutic strategies to control them.DISCUSSIONInflux of extracellular cADPR into 3T3 murine fibroblasts occurs both after incubation of intact cells with the cyclic nucleotide and following their co-culture with cADPR-producing, ectocyclase-expressing feeder layers (23Franco L. Zocchi E. Usai C. Guida L. Bruzzone S. Costa A. De Flora A. J. Biol. Chem. 2001; 276: 21642-21648Google Scholar).The results obtained in this study demonstrated that both equilibrative and concentrative nucleoside transporters are responsible for this influx, which had been previously described at a phenomenologic level (23Franco L. Zocchi E. Usai C. Guida L. Bruzzone S. Costa A. De Flora A. J. Biol. Chem. 2001; 276: 21642-21648Google Scholar). Therefore, rather unexpectedly, some of these transmembrane proteins proved to behave as promiscuous transporters beyond the current concept of broad acceptance of both pyrimidine and purine nucleosides, to include also the cyclic nucleotide cADPR. The quite compact structure of cADPR, as revealed by x-ray crystallography (52Lee H.C. Aarhus R. Levitt D. Nat. Struct. Biol. 1994; 1: 143-144Google Scholar), might be responsible for such behavior.The use of two cell types, 3T3 fibroblasts and HeLa cells, that exhibit different nucleoside transport systems, helped discriminate among the cADPR-translocating NT forms. Equilibrative uptake of cADPR occurs through ENT2, whereas ENT1 is not involved. Na+/cADPR symport activities have been detected in 3T3 fibroblasts, but not in HeLa cells. The almost complete inhibition afforded by uridine, guanosine, and inosine identified these activities with nucleoside transport systems. The largest part of Na+-dependent cADPR uptake is susceptible to inhibition by low concentrations of NBMPR (affording 75% inhibition), a property shared with cs and csg (47Paterson A.R.P. Gati W.P. Vijayalakshmi D. Cass C.E. Mant J.J. Young J.D. Belch A.R. Proc. Am. Assoc. Cancer Res. 1993; 34: 14Google Scholar, 48Flanagan S.A. Meckling-Gill K.A. J. Biol. Chem. 1997; 272: 18026-18032Google Scholar, 49Soler C. Felipe A. Mata J.F. Casado F.J. Celada A. Pastor-Anglada M. J. Biol. Chem. 1998; 273: 26939-26945Google Scholar). Whether this NBMPR-sensitive Na+/cADPR symport system is identifiable with either cs or csg is still unknown, however. The residual concentrative transport activity (25%) could be featured by CNT2, which is expressed in 3T3 cells and is not sensitive to NBMPR. This low level might explain the variability of cADPR influx observed in co-cultures where the CD38− 3T3 fibroblasts were pretreated with CNT2 antisense oligodeoxynucleotides (see “Results”). Indeed, transient transfection experiments on COS-7 cells demonstrated that CNT2 has intrinsic cADPR-transporting activity (Fig. 9). Whether additional concentrative Na+/cADPR symport systems exist, as reported earlier for Na+/nucleoside transport in the related COS-1 cell line (41Patel D.H. Crawford C.R. Naeve C.W. Belt J.A. Gene (Amst.). 2000; 242: 51-58Google Scholar), requires further investigations, aimed also to establish whether they are identifiable with, or unrelated to, professional nucleoside transporters.As widely recognized for NT species, also unequivocal identification and characterization of concentrative cADPR transporters seems to be a difficult task because of the following reasons: (i) co-expression of multiple equilibrative and concentrative transporters in the same cell type and their possible distinctive regulation, as that afforded by cytokines on murine macrophages (40Soler C. Garcı̀a-Manteiga J. Valdés R. Xaus J. Comalada M. Casado F.X. Pastor-Anglada M. Celada A. Felipe A. FASEB J. 2001; 15: 1979-1988Google Scholar); (ii) intrinsic variability of expression of NT; (iii) possible interferences between influx and efflux of permeating solutes in the presence of selective inhibitors (53Crawford C.R. Ng C.Y. Belt J.A. J. Biol. Chem. 1990; 265: 13730-13734Google Scholar): a relevant example is the apparently low extent of inhibition of cADPR influx into 3T3 cells by thymidine (Fig. 2), which might in fact be due to blockade of cADPR release from the cells via ENT2 while cADPR is actively pumped into the same cells by CNT forms; and (iv) lack of availability of related transporting proteins, as those corresponding to csg and cs NT (47Paterson A.R.P. Gati W.P. Vijayalakshmi D. Cass C.E. Mant J.J. Young J.D. Belch A.R. Proc. Am. Assoc. Cancer Res. 1993; 34: 14Google Scholar, 48Flanagan S.A. Meckling-Gill K.A. J. Biol. Chem. 1997; 272: 18026-18032Google Scholar, 49Soler C. Felipe A. Mata J.F. Casado F.J. Celada A. Pastor-Anglada M. J. Biol. Chem. 1998; 273: 26939-26945Google Scholar), this hampering their localization in specific cells and tissues.Despite these limitations, the identification in the plasma membrane of 3T3 fibroblasts of pleiotropic cADPR-translocating NT opens new perspectives for elucidating the mechanisms of regulation of intracellular calcium homeostasis in cADPR responsive cells. Our findings cast serious doubts on the possibility of a similar role being played by cADPR-transporting ENT2 (Table III), although extracellular cADPR might reach, in specific districts and under particular conditions (e.g. extensive cell death) levels high enough to determine its equilibrative uptake by neighboring cells. On the contrary, the NT responsible for concentrative cADPR influx proved in the heterologous co-cultures of CD38+/CD38−3T3 fibroblasts to actively accumulate cADPR into the CD38− cells against largely unfavorable concentration gradients. In this experimental system, the eventual, remarkably high gradient between intracellular (almost micromolar) and extracellular (nanomolar) concentrations of cADPR, despite presence of ENT2-mediated equilibrative activity which could dissipate the gradient itself, might be accounted for by progressive sequestration of intracellular cADPR by specific binding proteins, notably by its receptors on the ryanodine stores/channels (54Noguchi N. Takasawa S. Nata K. Tohgo A. Kato I. Ikehata F. Yonekura H. Okamoto H. J. Biol. Chem. 1997; 272: 3133-3136Google Scholar, 55Tang W.X. Chen Y.F. Zou A.P. Campbell W.B. Li P.L. Am. J. Physiol. Heart Circ. Physiol. 2002; 282: H1304-H1310Google Scholar, 56Walseth T.F. Aarhus R. Kerr J.A. Lee H.C. J. Biol. Chem. 1993; 268: 26686-26691Google Scholar, 57Lee H.C. Aarhus R. Graeff R. Gurnack M.E. Walseth T.F. Nature. 1994; 370: 307-309Google Scholar, 58Takasawa S. Ishida A. Nata K. Nakagawa K. Noguchi N. Tohgo A. Kato I. Yonekura H. Fujisawa H. Okamoto H. J. Biol. Chem. 1995; 270: 30257-30259Google Scholar, 59Tanaka Y. Tashjian Jr., A.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3244-3248Google Scholar). Indeed, the balance between kinetics of cADPR generation by CD38+ cells and of its concentrative influx into the cADPR-responsive CD38− cells proved to be functionally adequate to elicit Ca2+-mediated responses in the co-culture conditions (23Franco L. Zocchi E. Usai C. Guida L. Bruzzone S. Costa A. De Flora A. J. Biol. Chem. 2001; 276: 21642-21648Google Scholar, 28Zocchi E. Podestà M. Pitto A. Usai C. Bruzzone S. Franco L. Guida L. Bacigalupo A. De Flora A. FASEB J. 2001; 15: 1610-1612Google Scholar). From a more general standpoint, the principle of concentrative uptake of extracellular cADPR, possibly at sites distant from those of its generation, is reminiscent of endocrine processes in which hormones, still at very low concentrations, regulate a number of functions in target cells quite far from the endocrine tissues that release them in the bloodstream. Interestingly, the cADPR concentration in human plasma was estimated to be around 1 nm (not shown).The concentrative nature of at least two components of cADPR uptake (i.e. CNT2 and the NBMPR-sensitive system identified in 3T3 fibroblasts) bears relevance to “in vivo” events occurring in several tissue microenvironments including intact trachea (26Franco L. Bruzzone S. Song P. Guida L. Zocchi E. Walseth T.F. Crimi E. Usai C. De Flora A. Brusasco V. Am. J. Physiol. Lung Cell Mol. Physiol. 2001; 280: L98-L106Google Scholar), synapses (60Verderio C. Bruzzone S. Zocchi E. Fedele E. Schenk U. De Flora A. Matteoli M. J. Neurochem. 2001; 78: 1-13Google Scholar), and bone marrow (28Zocchi E. Podestà M. Pitto A. Usai C. Bruzzone S. Franco L. Guida L. Bacigalupo A. De Flora A. FASEB J. 2001; 15: 1610-1612Google Scholar), where Ca2+-related cell functions are elicited by protracted generation of extracellular cADPR (13De Flora A. Guida L. Franco L. Bruzzone S. Zocchi E. Lee H.C. Cyclic ADP-ribose and NAADP: Structure, Metabolism and Physiological Functions. Kluwer Acad., Norwell, MS2002Google Scholar, 14De Flora A. Franco L. Guida L. Bruzzone S. Usai C. Zocchi E. Chem. Immunol. 2000; 75: 79-98Google Scholar). The molecular characterization of concentrative cADPR transporter(s) should allow us to elucidate to what extent and through which mechanisms a site-targeted and constant supply of extracellularly generated cADPR can influence Ca2+-related responses. The molecular and functional interactions between NAD+-releasing Cx43 hemichannels and either CD38 or concentrative Na+/cADPR symport systems require challenging “in situ ” studies, whose results would be a biochemical paradigm for many physiological and pharmacological processes involved in the regulation of [Ca2+]i levels.Finally, the present results raise the possibility that several ectoenzymes involved in nucleotide and nucleoside metabolism at the outer surface of different cells have a role, although indirect, in the regulation of [Ca2+]i levels. The site-specific, ectocellular synthesis of a number of nucleosides potentially inhibiting influx of extracellular cADPR into cADPR-responsive cells seems to support this contention. Topological proximity of these ectoenzymes, including CD38, and of the selective transporters of NAD+ and cADPR, as well as possible preferential interactions thereof within the plasma membrane, might confer high efficiency to the related regulatory mechanisms. In any case, the cADPR transporters in the plasma membrane, and especially the concentrative ones, seem to be promising targets for new therapeutic strategies designed to control calcium-related dysfunctions in cADPR-responsive cells and tissues. Cyclic ADP-ribose (cADPR) 1The abbreviations used are: cADPR, cyclic ADP-ribose; NT, nucleoside transporters; NBMPR, nitrobenzylthioinosine; NAADP+, nicotinic acid adenine dinucleotide phosphate; Cx43, connexin 43; NGD+, nicotinamide guanine dinucleotide; HPLC, high pressure liquid chromatography; RT-PCR, reverse transcriptase-PCR; DIDS, 4–4-di-isothiocyano-2,2-stilbene disulfonic acid; ENT, equilibrative nucleoside transporter; CNT, concentrative nucleoside transporter 1The abbreviations used are: cADPR, cyclic ADP-ribose; NT, nucleoside transporters; NBMPR, nitrobenzylthioinosine; NAADP+, nicotinic acid adenine dinucleotide phosphate; Cx43, connexin 43; NGD+, nicotinamide guanine dinucleotide; HPLC, high pressure liquid chromatography; RT-PCR, reverse transcriptase-PCR; DIDS, 4–4-di-isothiocyano-2,2-stilbene disulfonic acid; ENT, equilibrative nucleoside transporter; CNT, concentrative nucleoside transporter is a potent and universal calcium mobilizer from intracellular stores (1Lee H.C. Walseth T.F. Bratt G.T. Hayes R.N. Clapper D.L. J. Biol. Chem. 1989; 264: 1608-1615Google Scholar, 2Lee H.C. Galione A. Walseth T.F. Vitam. Horm. 1994; 48: 199-257Google Scholar, 3Lee H.C. Physiol. Rev. 1996; 77: 1133-1164Google Scholar, 4Guse A.H. Curr. Mol. Med. 2002; 2: 273-282Google Scholar, 5Higashida H. Hashii M. Yokohama S. Hoshi N. Chen X.L. Egorova A. Noda M. Zhang J.S. Pharmacol. Ther. 2001; 90: 283-296Google Scholar). During phylogenesis, the physiological role of cADPR has evolved in parallel with the increasing complexity of the biological systems where it is present: from cell cycle regulator in Euglena gracilis (6Masuda W. Takenaka S. Inageda K. Nishima H. Takahashi K. Katada T. Tsuyama S. Inui H. Miyatake K. Nakano Y. FEBS Lett. 1997; 405: 104-106Google Scholar), to signal molecule in response to environmental stress in higher plants (7Wu Y. Kuzma J. Marechal E. Graeff R. Lee H.C. Foster R. Chua N.H. Science. 1997; 278: 2126-2130Google Scholar) and in Porifera (8Zocchi E. Carpaneto A. Cerrano C. Bavestrello G. Giovine M. Bruzzone S. Guida L. Franco L. Usai C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14859-14864Google Scholar), or during oocyte fertilization in marine invertebrates (9Lee H.C. Biol. Signals. 1996; 5: 101-110Google Scholar). In mammals, cADPR is involved in a number of Ca2+-dependent cell- and tissue-specific functions, including proliferation, contraction, and secretion (1Lee H.C. Walseth T.F. Bratt G.T. Hayes R.N. Clapper D.L. J. Biol. Chem. 1989; 264: 1608-1615Google Scholar, 2Lee H.C. Galione A. Walseth T.F. Vitam. Horm. 1994; 48: 199-257Google Scholar, 3Lee H.C. Physiol. Rev. 1996; 77: 1133-1164Google Scholar, 10Lee H.C. Annu. Rev. Pharmacol. Toxicol. 2001; 41: 317-345Google Scholar, 11Carafoli E. Santella L. Branca D. Brini M. Crit. Rev. Biochem. Mol. Biol. 2001; 36: 107-260Google Scholar, 12Berridge M.J. Lipp P. Bootman M.D. Nat. Rev. Mol. Cell. Biol. 2001; 1: 11-21Google Scholar). In several mammalian cells and tissue microenvironments, cADPR and its metabolic precursor NAD+ have recently been demonstrated to undergo a variable though potentially intense transmembrane trafficking (13De Flora A. Guida L. Franco L. Bruzzone S. Zocchi E. Lee H.C. Cyclic ADP-ribose and NAADP: Structure, Metabolism and Physiological Functions. Kluwer Acad., Norwell, MS2002Google Scholar). By virtue of these subcellular and intercellular movements, cADPR can access its ryanodine-sensitive intracellular Ca2+stores/channels despite generation of this cyclic nucleotide by ADP-ribosyl cyclases at sites that are topologically opposite to the stores (13De Flora A. Guida L. Franco L. Bruzzone S. Zocchi E. Lee H.C. Cyclic ADP-ribose and NAADP: Structure, Metabolism and Physiological Functions. Kluwer Acad., Norwell, MS2002Google Scholar, 14De Flora A. Franco L. Guida L. Bruzzone S. Usai C. Zocchi E. Chem. Immunol. 2000; 75: 79-98Google Scholar). The most represented member of these cyclases in mammalian cells is CD38 (15Jackson D.G. Bell J.I. J. Immunol. 1990; 144: 2811-2815Google Scholar), a type II glycoprotein involved in several transduction pathways (16Mehta K. Umar S. Malavasi F. FASEB J. 1996; 10: 1408-1417Google Scholar) and a multifunctional ectoenzyme that synthesizes cADPR from NAD+ and also hydrolyzes cADPR to ADP-ribose (17States D.J. Walseth T.F. Lee H.C. Trends Biochem. Sci. 1992; 17: 495Google Scholar, 18Lee H.C. Graeff R.M. Walseth T.F. Adv. Exp. Med. Biol. 1997; 419: 411-419Google Scholar). An important additional activity of CD38 is synthesis of the potent calcium mobilizer NAADP+ from NADP+ and nicotinic acid (18Lee H.C. Graeff R.M. Walseth T.F. Adv. Exp. Med. Biol. 1997; 419: 411-419Google Scholar, 19Aarhus R. Graeff R.M. Dickey D.M. Walseth T.F. Lee H.C. J. Biol. Chem. 1995; 270: 30327-30333Google Scholar). Availability of intracellular NAD+ to the ectocellular active site of CD38 is made possible by its controlled release from cells across an equilibrative transport system, represented by hexameric hemichannels of the gap junctional protein connexin 43 (Cx43) (20Bruzzone S. Guida L. Zocchi E. Franco L. De Flora A. FASEB J. 2001; 15: 10-12Google Scholar). Subsequent cADPR generation at the outer cell surface can be steadily followed by its influx into CD38+ cells because of the peculiar property of transmembrane oligomeric CD38 to couple cADPR synthesis to its active translocation across the cell membrane to reach the Ca2+ stores in the intracellular environment (21Franco L. Guida L. Bruzzone S. Zocchi E. Usai C. De Flora A. FASEB J. 1998; 12: 1507-1520Google Scholar). Such autocrine cross-talk between NAD+-releasing Cx43 hemichannels and CD38 has been demonstrated to finely up-regulate the [Ca2+]i levels in 3T3 murine fibroblasts (22Bruzzone S. Franco L. Guida L. Zocchi E. Contini P. Bisso A. Usai C. De Flora A. J. Biol. Chem. 2001; 276: 48300-48308Google Scholar). However, the peculiar localization and the properties of the catalytic site of CD38 suggest that a significant fraction of ectocellularly produced cADPR might escape transport across CD38 and thus be present in the extracellular environment, especially in close proximity of CD38+ cells. This assumption was strongly supported by the finding of 40–60 nm concentrations of NAD+ in human blood plasma 2T. F. Walseth, personal communication.2T. F. Walseth, personal communication. and by the recent demonstration that nanomolar concentrations of cADPR are consistently detectable in the medium of heterologous transwell co-cultures of CD" @default.
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• W2059354304 title "Equilibrative and Concentrative Nucleoside Transporters Mediate Influx of Extracellular Cyclic ADP-Ribose into 3T3 Murine Fibroblasts" @default.
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