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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The plant hormone abscisic acid (ABA) is released from glucose-challenged human pancreatic β cells and stimulates insulin secretion. We investigated whether plasma ABA increased during oral and intravenous glucose tolerance tests (OGTTs and IVGTTs) in healthy human subjects. In all subjects undergoing OGTTs (n=8), plasma ABA increased over basal values (in a range from 2- to 9-fold). A positive correlation was found between the ABA area under the curve (AUC) and the glucose AUC. In 4 out of 6 IVGTTs, little or no increase of ABA levels was observed. In the remaining subjects, the ABA increase was similar to that recorded during OGTTs. GLP-1 stimulated ABA release from an insulinoma cell line and from human islets, by ∼10- and 2-fold in low and high glucose, respectively. Human adipose tissue also released ABA in response to high glucose. Nanomolar ABA stimulated glucose uptake, similarly to insulin, in rat L6 myoblasts and in murine 3T3-L1 cells differentiated to adipocytes, by increasing GLUT-4 translocation to the plasma membrane. Demonstration that a glucose load in humans is followed by a physiological rise of plasma ABA, which can enhance glucose uptake by adipose tissues and muscle cells, identifies ABA as a new mammalian hormone involved in glucose metabolism.

CD38, a transmembrane glycoprotein widely expressed in vertebrate cells, is a bifunctional ectoenzyme catalyzing the synthesis and hydrolysis of cyclic ADP-ribose (cADPR). cADPR is a universal second messenger that releases calcium from intracellular stores. Since cADPR is generated by CD38 at the outer surface of many cells, where it acts intracellularly, increasing attention is paid to addressing this topological paradox. Recently, we demonstrated that CD38 is a catalytically active, unidirectional transmembrane transporter of cADPR, which then reaches its receptor-operated intracellular calcium stores. Moreover, CD38 was reported to undergo a selective and extensive internalization through non clathrin-coated endocytotic vesicles upon incubating CD38(+) cells with either NAD+ or thiol compounds: these endocytotic vesicles can convert cytosolic NAD into cADPR despite an asymmetric unfavorable orientation that makes the active site of CD38 intravesicular. Here we demonstrate that the cADPR-generating activity of the endocytotic vesicles results in remarkable and sustained increases of intracellular free calcium concentration in different cells exposed to either NAD+, or GSH, or N-acetylcysteine. This effect of CD38-internalizing ligands on intracellular calcium levels was found to involve a two-step mechanism: 1) influx of cytosolic NAD+ into the endocytotic vesicles, mediated by a hitherto unrecognized dinucleotide transport system that is saturable, bidirectional, inhibitable by 8-N3-NAD+, and characterized by poor dinucleotide specificity, low affinity, and high efficiency; 2) intravesicular CD38-catalyzed conversion of NAD+ to cADPR, followed by outpumping of the cyclic nucleotide into the cytosol and subsequent release of calcium from thapsigargin-sensitive stores. This unknown intracellular trafficking of NAD+ and cADPR based on two distinctive and specific transmembrane carriers for either nucleotide can affect the intracellular calcium homeostasis in CD38(+) cells.

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