A previously unrecognized passive transport for pyridine dinucleotides has been described recently in the plasmamembrane of several mammalian cells. Despite elucidation of some functional and kinetic properties of this transport system, it is still undefined at the molecular level. Therefore, we have addressed the molecular characterization of the NAD+ transporter and identified it as connexin 43 (Cx43). This is a structural component of hexameric hemichannels that, when juxtaposed on adjacent cells, builds up intercellular gap junctions and mediates exchange of molecules between cells. However, the role of connexin hemichannels as potential pores in individual, noncoupled cells remains elusive. Bidirectional NAD+ transport in isolated Cx43-expressing mur ine 3T3 fibroblasts was affected by known modulators of connexin-mediated intercellular coupling and was completely inhibited by treatment of the cells with a Cx43-antisense oligonucleotide. NAD+ transport in proteoliposomes reconstituted with 3T3 membrane proteins was inhibited in the presence of a monoclonal anti-Cx43 antibody. Finally, Cx43 immunopurified to homogeneity was reconstituted in unilamellar proteoliposomes, which displayed full NAD+-transporting activity. This finding is the first evidence that connexin hemichannels can mediate transmembrane fluxes of a nucleotide in whole cells: The pleiotropy of NAD+-dependent cellular events, including redox reactions, signaling, and DNA repair, implicates Cx43 hemichannels in intercellular NAD+ trafficking, which suggests new paracrine functions of NAD.

inflammation ͉ intracellular calcium

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CD38 is a bifunctional ectoenzyme, predominantly expressed on hematopoietic cells during differentiation, that catalyzes the synthesis (cyclase) and the degradation (hydrolase) of cyclic ADP-ribose (cADPR), a powerful calcium mobilizer from intracellular stores. Due to the well established role of calcium levels in the regulation of apoptosis, proliferation, and differentiation, the CD38/cADPR system seems to be a likely candidate involved in the control of these fundamental processes. CD38 is a type II transmembrane glycoprotein predominantly expressed on lymphocytes (1) but also present in a number of different cell types, including erythrocytes (2), hematopoietic progenitor cells (1), ␤-pancreatic cells (3), and cerebral (4) and cerebellar (5) neurons. Immunologically, CD38 can be defined as an "orphan receptor" since its binding by specific monoclonal antibodies directed against ectocellular epitopes elicits cellular responses in lymphocytes, including proliferation, activation, and rescue from apoptosis (6, 7). The signal transduction pathways implicated in these events are under study, but phosphorylation/dephosphorylation reactions of target kinases have been already demonstrated (6,8,9). Biochemically, CD38 is a bifunctional ectoenzyme that catalyzes the synthesis of cADPR 1 from NAD ϩ and also its hydrolysis to ADPR (2, 10). Cyclic ADPR is a potent Ca 2ϩ mobilizer from intracellular stores, in invertebrate as well as in mammalian cells (11), and its presence has been described in most mammalian tissues (12).The widespread tissue distribution of the CD38/cADPR system suggests its involvement in the control of pivotal Ca 2ϩ -controlled functions like contraction, secretion, cell proliferation/differentiation, and apoptosis. However, the topological paradox of the ectocellular production of an intracellular Ca 2ϩ mobilizer has raised questions on both the immunological and the biochemical functions of the CD38/cADPR system (13, 14). Cell death has been advocated as a means for local increase in extracellular NAD ϩ concentrations, sufficient to elicit the production of cADPR by the ectoenzyme CD38; in this respect, nanomolar concentrations of NAD ϩ have been detected in plasma (15) and cerebellar interstitial fluid (5) suggesting the theoretical possibility of an extracellular production of cADPR by CD38 "in vivo." In few selected cell systems, i.e. murine B-lymphocytes (10), rat cerebellar granule cells (5), and rat osteoclasts (16), extracellular, exogenously added cADPR was demonstrated to elicit functional responses in intact cells, but most reported effects of cADPR on cellular functions require permeabilization of target cells to ensure binding of the nucleotide to its intracellular receptor(s).Internalization of membrane-bound CD38, as observed in human Namalwa B-lymphocytes upon incubation with NAD ϩ or thiol reagents, is followed by an increase of intracellular cyclase activity (insensitive to protein synthesis inhibitors) and of intracellular cADPR concentration ([cADPR] i ) (17), suggesting that...

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