Antonio De Flora

Connexin 43 hemi channels mediate Ca2+-regulated transmembrane NAD+ fluxes in intact cells

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 murine 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+.

Autocrine and Paracrine Calcium Signaling by the CD38/NAD+/Cyclic ADP‐Ribose System

Abstract: CD38, a multifunctional enzyme, generates two potent Ca2+‐releasing signal metabolites, cyclic ADP‐ribose (cADPR) and NAADP+, thereby upmodulating many important Ca2+‐mediated cell functions. A topological paradox has long been recognized, as CD38 is an ectoenzyme, or an intravesicularly located enzyme in subcellular membrane vesicles, therefore apparently shielded from its substrate NAD+. Moreover, cADPR generated by CD38 should be unavailable to its target Ca2+ stores, the ryanodine receptors (RyR). We have solved this paradox by identifying some NAD+ and cADPR transmembrane transporters, whose interplay mediates a hitherto‐unrecognized subcellular and intercellular trafficking of nucleotides that enhances intracellular Ca2+ ([Ca2+]i). Connexin 43 (Cx43) hemichannels mediate an equilibrative transport of NAD+ from the cytosol to the active site of CD38 (either ectocellular or intravesicular). Subsequent translocation of in situ‐generated cADPR to reach the RyR is performed, (i) by CD38 itself (concentrative) or (ii) by nucleoside transporters (NT) (one equilibrative and three concentrative). Besides this autocrine mechanism, the same transporters also mediate intercellular (paracrine) trafficking. Thus, Cx43+ and CD38+ cells can provide cADPR to neighboring RyR+ parenchymal cells and enhance their [Ca2+]i levels and Ca2+‐dependent functions accordingly. Examples of cADPR‐responsive cells via paracrine processes include (i) smooth myocytes, (ii) 3T3 murine fibroblasts, (iii) hippocampal neurons, and (iv) human hemopoietic stem cells.

Synthesis of GDP-L-fucose by the Human FX Protein

FX is a homodimeric NADP(H)-binding protein of 68 kDa, first identified in human erythrocytes, from which it was purified to homogeneity. Its function has been unrecognized despite partial structural and genetic characterization. Recently, on the basis of partial amino acid sequence, it proved to be the human homolog of the murine protein P35B, a tumor rejection antigen. In order to address the biochemical role of FX, its primary structure was completed by cDNA sequencing. This sequence revealed a significant homology with many proteins from different organisms. Specifically, FX showed a remarkable similarity with a putative Escherichia coli protein, named Yefb, whose gene maps in a region of E. coli chromosome coding for enzymes involved in synthesis and utilization of GDP-D-mannose. Accordingly, a possible role of FX in this metabolism was investigated. The data obtained indicate FX as the enzyme responsible for the last step of the major metabolic pathway resulting in GDP-L-fucose synthesis from GDP-D-mannose in procaryotic and eucaryotic cells. Specifically, purified FX apparently catalyzes a combined epimerase and NADPH-dependent reductase reaction, converting GDP-4-keto-6-D-deoxymannose to GDP-L-fucose. This is the substrate of several fucosyltranferases involved in the correct expression of many glyconjugates, including blood groups and developmental antigens.

Abscisic acid is an endogenous cytokine in human granulocytes with cyclic ADP-ribose as second messenger

Abscisic acid (ABA) is a phytohormone involved in fundamental physiological processes of higher plants, such as response to abiotic stress (temperature, light, drought), regulation of seed dormancy and germination, and control of stomatal closure. Here, we provide evidence that ABA stimulates several functional activities [phagocytosis, reactive oxygen species and nitric oxide (NO) production, and chemotaxis] of human granulocytes through a signaling pathway sequentially involving a pertussis toxin (PTX)-sensitive G protein/receptor complex, protein kinase A activation, ADP-ribosyl cyclase phosphorylation, and consequent cyclic-ADP-ribose overproduction, leading to an increase of the intracellular Ca2+ concentration. The increase of free intracellular ABA and its release by activated human granulocytes indicate that ABA should be considered as a new pro-inflammatory cytokine in humans. This discovery is an intriguing example of conservation of a hormone and its signaling pathway from plants to humans and provides insight into the molecular mechanisms of granulocyte activation, possibly leading to the development of new antiinflammatory drugs.

The transmembrane glycoprotein CD38 is a catalytically active transporter responsible for generation and influx of the second messenger cyclic ADP-ribose across membranes

CD38 is a type II transmembrane glycoprotein expressed in many vertebrate cells. It is a bifunctional ectoenzyme that catalyzes both the synthesis of Cyclic ADP‐ribose (cADPR) from NAD+ and the degradation of cADPR to ADP‐ribose by means of its ADP‐ribosyl cyclase and cADPR‐hydrolase activities, respectively. The cyclase also converts NGD+ to cyclic GDP‐ribose (cGDPR), which is refractory to cADPR‐hydrolase. cADPR, but not cGDPR, is a potent calcium mobilizer from intracellular stores. It has been demonstrated to be a new second messenger involved in the regulation of calcium homeostasis in many cell types, from plants to mammals. The number of physiological processes shown to be regulated by cADPR is steadily increasing. A topological paradox exists because ectocellularly generated cADPR acts intracellularly. Here we demonstrate that the catalytic functioning of CD38 is accompanied by a cADPR (cGDPR) ‐transporting activity across natural and artificial membranes. In resealed membranes from CD38+ human erythrocytes, transport of catalytically generated cADPR or cGDPR was saturation dependent and occurred against a concentration gradient. Likewise, CD38‐reconstituted proteoliposomes were active in concentrating NAD+ (NGD+) ‐derived cADPR (cGDPR) inside the vesicle compartment. Moreover, the cADPR‐transporting activity in CD38 proteoliposomes prevented the hydrolase‐catalyzed degradation to ADPR that occurs conversely with detergent‐solubilized CD38, resulting in selective influx of cADPR. In the CD38 proteoliposomes, catalytically active CD38 exhibited monomeric, dimeric, and tetrameric structures. In CD38 sense‐ but not in antisense‐transfected HeLa cells, externally added NAD+ resulted in significant, transient increases in cytosolic calcium. These data suggest that transmembrane juxtaposition of two or four CD38 monomers can generate a catalytically active channel for selective formation and influx of cADPR (cGDPR) to reach cADPR‐responsive intracellular calcium stores.—Franco, L., Guida, L., Bruzzone, S., Zocchi, E., Usai, C., De Flora, A. The transmembrane glycoprotein CD38 is a catalytically active transporter responsible for generation and influx of the second messenger cyclic ADP‐ribose across membranes. FASEB J. 12, 1507–1520 (1998)

Extracellular NAD+ Is an Agonist of the Human P2Y11 Purinergic Receptor in Human Granulocytes

Micromolar concentrations of extracellular β-NAD+ (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{NAD}_{e}^{+}\) \end{document}) activate human granulocytes (superoxide and NO generation and chemotaxis) by triggering: (i) overproduction of cAMP, (ii) activation of protein kinase A, (iii) stimulation of ADP-ribosyl cyclase and overproduction of cyclic ADP-ribose (cADPR), a universal Ca2+ mobilizer, and (iv) influx of extracellular Ca2+. Here we demonstrate that exposure of granulocytes to millimolar rather than to micromolar \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{NAD}_{e}^{+}\) \end{document} generates both inositol 1,4,5-trisphosphate (IP3) and cAMP, with a two-step elevation of intracellular calcium levels ([Ca2+]i): a rapid, IP3-mediated Ca2+ release, followed by a sustained influx of extracellular Ca2+ mediated by cADPR. Suramin, an inhibitor of P2Y receptors, abrogated \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{NAD}_{e}^{+}\) \end{document}-induced intracellular increases of IP3, cAMP, cADPR, and [Ca2+]i, suggesting a role for a P2Y receptor coupled to both phospholipase C and adenylyl cyclase. The P2Y11 receptor is the only known member of the P2Y receptor subfamily coupled to both phospholipase C and adenylyl cyclase. Therefore, we performed experiments on hP2Y11-transfected 1321N1 astrocytoma cells: micromolar \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{NAD}_{e}^{+}\) \end{document} promoted a two-step elevation of the [Ca2+]i due to the enhanced intracellular production of IP3, cAMP, and cADPR in 1321N1-hP2Y11 but not in untransfected 1321N1 cells. In human granulocytes NF157, a selective and potent inhibitor of P2Y11, and the down-regulation of P2Y11 expression by short interference RNA prevented \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{NAD}_{e}^{+}\) \end{document}-induced intracellular increases of [Ca2+]i and chemotaxis. These results demonstrate that \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \({\beta}\mathrm{-}\mathrm{NAD}_{e}^{+}\) \end{document} is an agonist of the P2Y11 purinoceptor and that P2Y11 is the endogenous receptor in granulocytes mediating the sustained [Ca2+]i increase responsible for their functional activation.

Ligand-induced internalization of CD38 results in intracellular Ca2+ mobilization: role of NAD+ transport across cell membranes

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 out‐pumping of the cyclic nucleotide into the cytosol and subsequent release of calcium from thapsigarginsensitive 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. —Zocchi, E., Usai, C., Guida, L., Franco, L., Bruzzone, S., Passalacqua, M., De Flora, A. Ligand‐induced internalization of CD38 results in intracellular Ca2+ mobilization: role of NAD+ transport across cell membranes. FASEB J. 13, 273–283 (1999)

A Self-restricted CD38-connexin 43 Cross-talk Affects NAD+ and Cyclic ADP-ribose Metabolism and Regulates Intracellular Calcium in 3T3 Fibroblasts

Connexin 43 (Cx43) hexameric hemichannels, recently demonstrated to mediate NAD+ transport, functionally interact in the plasma membrane of several cells with the ectoenzyme CD38 that converts NAD+ to the universal calcium mobilizer cyclic ADP-ribose (cADPR). Here we demonstrate that functional uncoupling between CD38 and Cx43 in CD38-transfected 3T3 murine fibroblasts is paralleled by decreased [Ca2+] i levels as a result of reduced intracellular conversion of NAD+ to cADPR. A sharp inverse correlation emerged between [Ca2+] i levels and NAD+ transport (measured as influx into cells and as efflux therefrom), both in the CD38+ cells (high [Ca2+] i , low transport) and in the CD38− fibroblasts (low [Ca2+] i , high transport). These differences were correlated with distinctive extents of Cx43 phosphorylation in the two cell populations, a lower phosphorylation with high NAD+ transport (CD38− cells) and vice versa (CD38+ cells). Conversion of NAD+-permeable Cx43 to the phosphorylated, NAD+-impermeable form occurs via Ca2+-stimulated protein kinase C (PKC). Thus, a self-regulatory loop emerged in CD38+ fibroblasts whereby high [Ca2+] i restricts further Ca2+mobilization by cADPR via PKC-mediated disruption of the Cx43-CD38 cross-talk. This mechanism may avoid: (i) leakage of NAD+from cells; (ii) depletion of intracellular NAD+ by CD38; (iii) overproduction of intracellular cADPR resulting in potentially cytotoxic [Ca2+] i .

Abscisic Acid Is an Endogenous Stimulator of Insulin Release from Human Pancreatic Islets with Cyclic ADP Ribose as Second Messenger

Abscisic acid (ABA) is a plant stress hormone recently identified as an endogenous pro-inflammatory cytokine in human granulocytes. Because paracrine signaling between pancreatic β cells and inflammatory cells is increasingly recognized as a pathogenetic mechanism in the metabolic syndrome and type II diabetes, we investigated the effect of ABA on insulin secretion. Nanomolar ABA increases glucose-stimulated insulin secretion from RIN-m and INS-1 cells and from murine and human pancreatic islets. The signaling cascade triggered by ABA in insulin-releasing cells sequentially involves a pertussis toxin-sensitive G protein, cAMP overproduction, protein kinase A-mediated activation of the ADP-ribosyl cyclase CD38, and cyclic ADP-ribose overproduction. ABA is rapidly produced and released from human islets, RIN-m, and INS-1 cells stimulated with high glucose concentrations. In conclusion, ABA is an endogenous stimulator of insulin secretion in human and murine pancreatic β cells. Autocrine release of ABA by glucose-stimulated pancreatic β cells, and the paracrine production of the hormone by activated granulocytes and monocytes suggest that ABA may be involved in the physiology of insulin release as well as in its dysregulation under conditions of inflammation.

Extracellular cyclic ADP-ribose increases intracellular free calcium concentration and stimulates proliferation of human hemopoietic progenitors

Cyclic ADP‐ribose (cADPR) is a universal second messenger that regulates many calcium‐related cellular events by releasing calcium from intracellular stores. Since these events include enhanced cell proliferation and since the bone marrow harbors both ectoenzymes that generate cADPR from NAD+ (CD38 and BST‐1), we investigated the effects of extracellular cADPR on human hemopoietic progenitors (HP). Exposure of HP to 100 μM cADPR for 24 h induced a significant increase in colony output (P<0.01) and colony size (P<0.003). A horizontal expansion of HP, as demonstrated by a markedly increased replating efficiency in semisolid medium (up to 700 times compared to controls), was also observed, indicating that cADPR priming can affect cell growth for multiple generations over several weeks after exposure. Influx of extracellular cADPR into the cells was demonstrated, and a causal relationship between the functional effects and the increase of intracellular free calcium concentration induced by cADPR on HP was established through the use of specific antagonists. Similar effects on HP were produced by nanomolar concentrations of the nonhydrolyzable cADPR analog 3‐deaza‐cADPR. These data demonstrate that extracellular cADPR behaves as a cytokine enhancing the proliferation of human HP, a finding that may have biomedical applications for the ex vivo expansion of hemopoietic cells.—Podestà, 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. Extracellular cyclic ADP‐ribose increases intracellular free calcium concentration and stimulates proliferation of human hemopoietic progenitors. FASEB J. 14, 680–690 (2000)