The temperature-signaling cascade in sponges involves a heat-gated cation channel, abscisic acid, and cyclic ADP-ribose.

Sponges (phylum Porifera) are the phylogenetically oldest metazoan animals, their evolution dating back to 600 million years ago. Here we demonstrate that sponges express ADP-ribosyl cyclase activity, which converts NAD(+) into cyclic ADP-ribose, a potent and universal intracellular Ca(2+) mobilizer. In Axinella polypoides (Demospongiae, Axinellidae), ADP-ribosyl cyclase was activated by temperature increases by means of an abscisic acid-induced, protein kinase A-dependent mechanism. The thermosensor triggering this signaling cascade was a heat-activated cation channel. Elucidation of the complete thermosensing pathway in sponges highlights a number of features conserved in higher organisms: (i) the cation channel thermoreceptor, sensitive to heat, mechanical stress, phosphorylation, and anesthetics, shares all of the functional characteristics of the mammalian heat-activated background K(+) channel responsible for central and peripheral thermosensing; (ii) involvement of the phytohormone abscisic acid and cyclic ADP-ribose as its second messenger is reminiscent of the drought stress signaling pathway in plants. These results suggest an ancient evolutionary origin of this stress-signaling cascade in a common precursor of modern Metazoa and Metaphyta.

Heat stress-activated, calcium-dependent nitric oxide synthase in sponges.

The presence of Ca(2+)-dependent, heat-stress-activated nitric oxide synthase (NOS) activity in peculiarly shaped, fusiform, and dendritic sponge cells is described for the first time. The NOS activity was evidenced evaluating the conversion of radioactive citrulline from [(14)C]arginine in intact cells from two different species that are phylogenetically unrelated in the class of Demospongiae: Axinella polypoides and Petrosia ficiformis. The production of nitrogen monoxide (NO) was confirmed by electron paramagnetic resonance analysis, and the histochemistry technique of NADPH diaphorase showed a specific localization of NOS activity in a particular network of dendritic cells in the sponge parenchyma. Sponges are the most primitive metazoan group; their evolution dates back 600 million years. The presence of environmental stress-activated NOS activity in these organisms may prove to be the most ancient NO-dependent signaling network in the animal kingdom.

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 [Ca(2+)](i) levels as a result of reduced intracellular conversion of NAD(+) to cADPR. A sharp inverse correlation emerged between [Ca(2+)](i) levels and NAD(+) transport (measured as influx into cells and as efflux therefrom), both in the CD38(+) cells (high [Ca(2+)](i), low transport) and in the CD38(-) fibroblasts (low [Ca(2+)](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 Ca(2+)-stimulated protein kinase C (PKC). Thus, a self-regulatory loop emerged in CD38(+) fibroblasts whereby high [Ca(2+)](i) restricts further Ca(2+) 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 [Ca(2+)](i).

Evidence of a role for cyclic ADP-ribose in calcium signalling and neurotransmitter release in cultured astrocytes.

Astrocytes possess different, efficient ways to generate complex changes in intracellular calcium concentrations, which allow them to communicate with each other and to interact with adjacent neuronal cells. Here we show that cultured hippocampal astrocytes coexpress the ectoenzyme CD38, directly involved in the metabolism of the calcium mobilizer cyclic ADP-ribose, and the NAD+ transporter connexin 43. We also demonstrate that hippocampal astrocytes can release NAD+ and respond to extracellular NAD+ or cyclic ADP-ribose with intracellular calcium increases, suggesting the existence of an autocrine cyclic ADP-ribose-mediated signalling. Cyclic ADP-ribose-induced calcium changes are in turn responsible for an increased glutamate and GABA release, this effect being completely inhibited by the cyclic ADP-ribose specific antagonist 8-NH2-cADPR. Furthermore, addition of NAD+ to astrocyte-neuron co-cultures results in a delayed intracellular calcium transient in neuronal cells, which is strongly but not completely inhibited by glutamate receptor blockers. These data indicate that an astrocyte-to-neuron calcium signalling can be triggered by the CD38/cADPR system, which, through the activation of intracellular calcium responses in astrocytes, is in turn responsible for the increased release of neuromodulators from glial cells.

Paracrine roles of NAD+ and cyclic ADP-ribose in increasing intracellular calcium and enhancing cell proliferation of 3T3 fibroblasts.

CD38 is a bifunctional ectoenzyme synthesizing from NAD(+) (ADP-ribosyl cyclase) and degrading (hydrolase) cyclic ADP-ribose (cADPR), a powerful universal calcium mobilizer from intracellular stores. Recently, hexameric connexin 43 (Cx43) hemichannels have been shown to release cytosolic NAD(+) from isolated murine fibroblasts (Bruzzone, S., Guida, L., Zocchi, E., Franco, L. and De Flora, A. (2001) FASEB J. 15, 10-12), making this dinucleotide available to the ectocellular active site of CD38. Here we investigated transwell co-cultures of CD38(+) (transfected) and CD38(-) 3T3 cells in order to establish the role of extracellular NAD(+) and cADPR on [Ca(2+)](i) levels and on proliferation of the CD38(-) target cells. CD38(+), but not CD38(-), feeder cells induced a [Ca(2+)](i) increase in the CD38(-) target cells which was comparable to that observed with extracellular cADPR alone and inhibitable by NAD(+)-glycohydrolase or by the cADPR antagonist 8-NH(2)-cADPR. Addition of recombinant ADP-ribosyl cyclase to the medium of CD38(-) feeders induced sustained [Ca(2+)](i) increases in CD38(-) target cells. Co-culture on CD38(+) feeders enhanced the proliferation of CD38(-) target cells over control values and significantly shortened the S phase of cell cycle. These results demonstrate a paracrine process based on Cx43-mediated release of NAD(+), its CD38-catalyzed conversion to extracellular cADPR, and influx of this nucleotide into responsive cells to increase [Ca(2+)](i) and stimulate cell proliferation.

Modified in vitro conditions for cord blood-derived long-term culture-initiating cells.

OBJECTIVE: The aim of this study was to compare the in vitro growth of cord blood-derived progenitors with that of bone marrow and peripheral blood. MATERIALS AND METHODS: We analyzed 192 umbilical cord blood (UCB), 35 normal bone marrow (NBM), and 35 granulocyte colony-stimulating factor (G-CSF)-primed normal peripheral blood (NPB) samples. Standard clonogenic assays (colony-forming unit granulocyte-macrophage [CFU-GM], burst-forming unit erythroid [BFU-E], CFU-granulocyte erythroid megakaryocyte macrophage [GEMM]) and standard long-term culture-initiating cell (LTC-IC) assay were performed. LTC-IC frequency also was tested under modified culture conditions. The variables tested were incubation temperature (37 degrees C and 33 degrees C) and supportive stromal cell lines (NIH3T3 and M210-B4). RESULTS: The CFU-GM and CFU-GEMM frequencies of UCB samples were similar to NPB and higher compared to NBM samples (p < 10(-4) and p < 0.007 respectively). On the other hand, the BFU-E frequency was lower in cord blood samples (5.2 +/- 5.6/10(4) MNC) compared to bone marrow (7 +/- 3.8/10(4) MNC; p < 0.005) and peripheral blood (15.2 +/- 11.1/10(4) MNC; p < 10(-4)). All colony types (CFU-GM, BFU-E, CFU-GEMM) generated from cord blood progenitors were larger with respect to the other tissues. The LTC-IC frequency was markedly decreased (8.8 +/- 3.8/10(6) MNC) in cord blood with respect to bone marrow (40.7 +/- 7.4/10(6) MNC; p < 10(-4)) and peripheral blood (28.8 +/- 3.8/10(6) MNC; p < 0.04). However, when culture conditions (temperature, stromal layers) were modified, UCB-LTC-IC frequency significantly increased, while the growth of early progenitors derived from adult tissues (BM and PB) did not show any variation. Whatever culture conditions were used, the proliferative potential of UCB LTC-IC was significantly higher with respect to bone marrow and G-CSF-primed PB (10.6 +/- 7.7 colonies vs. 5.9 +/- 5 vs 3.2 +/- 2.2 colonies; p < 0.02 and p < 0.001 respectively). CONCLUSIONS: Optimal conditions for estimation of the LTC-IC frequency in cord blood samples seem to be different from those usually applied to PB and BM progenitors. Although UCB hemopoietic progenitors have a higher proliferative potential than those from bone marrow and G-CSF-primed peripheral blood, their quantitation depends on the culture conditions, which makes it difficult to establish their exact number. This problem and the fact that a significant proportion of UCB samples grew poorly in culture make it necessary to develop suitable and standardized functional assays to test UCB progenitor content before the transplantation procedure.

Extracellular cyclic ADP-ribose potentiates ACh-induced contraction in bovine tracheal smooth muscle.

Cyclic ADP-ribose (cADPR), a universal calcium releaser, is generated from NAD(+) by an ADP-ribosyl cyclase and is degraded to ADP-ribose by a cADPR hydrolase. In mammals, both activities are expressed as ectoenzymes by the transmembrane glycoprotein CD38. CD38 was identified in both epithelial cells and smooth myocytes isolated from bovine trachea. Intact tracheal smooth myocytes (TSMs) responded to extracellular cADPR (100 microM) with an increase in intracellular calcium concentration ([Ca(2+)](i)) both at baseline and after acetylcholine (ACh) stimulation. The nonhydrolyzable analog 3-deaza-cADPR (10 nM) elicited the same effects as cADPR, whereas the cADPR antagonist 8-NH(2)-cADPR (10 microM) inhibited both basal and ACh-stimulated [Ca(2+)](i) levels. Extracellular cADPR or 3-deaza-cADPR caused a significant increase of ACh-induced contraction in tracheal smooth muscle strips, whereas 8-NH(2)-cADPR decreased it. Tracheal mucosa strips, by releasing NAD(+), enhanced [Ca(2+)](i) in isolated TSMs, and this increase was abrogated by either NAD(+)-ase or 8-NH(2)-cADPR. These data suggest the existence of a paracrine mechanism whereby mucosa-released extracellular NAD(+) plays a hormonelike function and cADPR behaves as second messenger regulating calcium-related contractility in TSMs.

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 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.

G(s) protein dysfunction in allergen-challenged human isolated passively sensitized bronchi.

We studied the intracellular mechanisms of allergen-induced beta(2)-adrenoceptor dysfunction in human isolated passively sensitized bronchi. Sensitization was obtained by overnight incubation of bronchial rings with serum containing a high specific IgE level to Dermatophagoides but a low total IgE level. Allergen challenge was done by incubation with a Dermatophagoides mix. The G(s) protein stimulant cholera toxin (2 microg/ml) displaced the carbachol (CCh) concentration-response curves of control and sensitized but not of challenged rings to the right. Cholera toxin (10 microg/ml) displaced the concentration-response curves to CCh of control, sensitized, and challenged rings to the right, but this effect was less in challenged rings. The effects of the G(i) protein inhibitor pertussis toxin (250 ng/ml or 1 microg/ml) on salbutamol concentration-relaxation curves did not differ significantly between challenged and sensitized rings. The adenylyl cyclase activator forskolin and the Ca(2+)-activated K(+)-channel opener NS-1619 relaxed CCh-contracted bronchial rings without significant differences between control, sensitized, and challenged rings. Neither G(i) nor G(s) alpha-subunit expression differed between control, sensitized, and challenged tissues. We conclude that G(s) protein dysfunction may be a mechanism of allergen-induced beta(2)-adrenoceptor dysfunction in human isolated passively sensitized bronchi.

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 microM 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.

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 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.

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.

Dimeric and tetrameric forms of catalytically active transmembrane CD38 in transfected HeLa cells.

CD38, a type II transmembrane glycoprotein, behaves as a catalytically active transporter responsible for ectocellular generation of cyclic ADP-ribose (cADPR) from NAD+ and for subsequent influx of cADPR across membranes [Franco, L., Guida, L., Bruzzone, S., Zocchi, E., Usai, C. and De Flora, A. (1998) FASEB J. in press]. cADPR regulates intracellular calcium homeostasis by releasing calcium from responsive stores. The cADPR-transporting function of CD38 requires channel-generating oligomeric forms of the protein rather than the 46 kDa monomers that have been described so far in CD38+ cells. Here we demonstrate that CD38, both in reconstituted proteoliposomes and in CD38-transfected HeLa cells, is a mixture of catalytically active monomers, homodimers and homotetramers. A soluble recombinant form of CD38 corresponding to its ectocellular region proved to be monomeric. Thus, association of native CD38 with either artificial or natural membranes seems to result in a reversible juxtaposition of monomers suitable to cADPR-transporting activity.

Characterization of a CD38-like 78-kilodalton soluble protein released from B cell lines derived from patients with X-linked agammaglobulinemia.

Studies on murine B lymphocytes showed that Bruton's tyrosine kinase mediates signal transduction induced via CD38, a nonlineage-restricted 45-kD ectoenzyme. This signaling is defective in B cells from X-linked immunodeficient mice affected with the analogue of human X-linked agammaglobulinemia (XLA). We performed a structural and functional analysis of CD38 in XLA and other immunodeficiencies, using EBV-immortalized B cells derived from such patients. Membrane CD38 was not significantly different from controls in structure, epitope density, enzymatic activity, and internalization upon binding of agonistic mAbs. Meanwhile, an increased release of soluble CD38 from XLA cells was observed: immunoprecipitation from XLA culture media yielded a protein of approximately 78 kD (p78), reacting also in Western blot and displaying both enzymatic activities and a peptide map similar to membrane CD38. Soluble forms and homotypic aggregations of CD38 were documented in different cell models and by crystallographic analysis of the Aplysia ADP-ribosyl cyclase, the ancestor of human CD38. p78 might represent the product of an altered turn-over of membrane CD38, a starting point for studying its association with Bruton's tyrosine kinase and its role in XLA and other B cell immunodeficiencies.

Expression of CD38 increases intracellular calcium concentration and reduces doubling time in HeLa and 3T3 cells.

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. The ectocellular localization of the cyclase activity, however, contrasts with the intracellular site of action of cADPR. Here we demonstrate that ectocellular expression of human CD38 in CD38(-) HeLa and 3T3 cells results in intracellular CD38 substrate (NAD+ + NADH) consumption and product (cADPR) accumulation. Furthermore, a causal relationship is established between presence of intracellular cADPR, partial depletion of thapsigargin-sensitive calcium stores, increase in basal free cytoplasmic calcium concentration, and decrease of cell doubling time. The significant shortening of the S phase in CD38(+) HeLa cells, as compared with controls, demonstrates an effect of intracellular cADPR on the mammalian cell cycle.

Ectocellular CD38-catalyzed synthesis and intracellular Ca(2+)-mobilizing activity of cyclic ADP-ribose.

CD38 is a type-II transmembrane glycoprotein occurring in several hematopoietic and mature blood cells as well as in other cell types, including neurons. Although classified as an orphan receptor, CD38 is also a bifunctional ectoenzyme that catalyzes both the conversion of NAD+ to nicotinamide and cyclic ADP-ribose (cADPR), via an ADP-ribosyl cyclase reaction, and also the hydrolysis of cADPR to ADP-ribose (hydrolase). Major unresolved questions concern the correlation between receptor and catalytic properties of CD38, and also the apparent contradiction between ectocellular generation and intracellular Ca(2+)-mobilizing activity of cADPR. Results are presented that provide some explanations to this topological paradox in two different cell types. In cultured rat cerebellar granule neurons, extracellular cADPR (either generated by CD38 or directly added) elicited an enhanced intracellular Ca(2+)-response to KCl-induced depolarization, a process that can be qualified as a Ca(2+)-induced Ca2+ release (CICR) mechanism. On the other hand, in the CD38+ human Namalwa B lymphoid cells, NAD+ (and thiol compounds as well) induced a two-step process of self-aggregation followed by endocytosis of CD38, which resulted in a shift of cADPR metabolism from the cell surface to the cytosol. Both distinctive types of cellular responses to extracellular NAD+ seem to be suitable to elicit changes in the intracellular Ca2+ homeostasis.

CD38 and ADP-ribosyl cyclase catalyze the synthesis of a dimeric ADP-ribose that potentiates the calcium-mobilizing activity of cyclic ADP-ribose.

CD38, a lymphocyte differentiation antigen, is also a bifunctional enzyme catalyzing the synthesis of cyclic ADP-ribose (cADPR) from NAD+ and its hydrolysis to ADP-ribose (ADPR). An additional enzymatic activity of CD38 shared by monofunctional ADP-ribosyl cyclase from Aplysia californica is the exchange of the base group of NAD+ (nicotinamide) with various nucleophiles. Both human CD38 (either recombinant or purified from erythrocyte membranes) and Aplysia cyclase were found to catalyze the exchange of ADPR with the nicotinamide group of NAD+ leading to the formation of a dimeric ADPR ((ADPR)2). The dimeric structure of the enzymatic product, which was generated by recombinant CD38 and by CD38(+) Namalwa cells from as low as 10 microM NAD+, was demonstrated using specific enzyme treatments (dinucleotide pyrophosphatase and 5'-nucleotidase) and mass spectrometry analyses of the resulting products. The linkage between the two ADPR units of (ADPR)2 was identified as that between the N1 of the adenine nucleus of one ADPR unit and the anomeric carbon of the terminal ribose of the second ADPR molecule by enzymatic analyses and by comparison with patterns of cADPR cleavage with Me2SO:tert-butoxide. Although (ADPR)2 itself did not release Ca2+ from sea urchin egg microsomal vesicles, it specifically potentiated the Ca2+-releasing activity of subthreshold concentrations of cADPR. Therefore, (ADPR)2 is a new product of CD38 that amplifies the Ca2+-mobilizing activity of cADPR.

The CD38/cyclic ADP-ribose system: a topological paradox.

CD38 was first identified as a lymphocyte differentiation antigen that showed typical properties of an orphan receptor involved in many programs of cell proliferation and activation. However, CD38 proved also to be a bifunctional ectoenzyme that catalyzes the transient formation of cyclic ADP-ribose (cADPR) in a variety of cell types. This property raises many intriguing and so far unanswered questions, since cADPR is a new second messenger molecule directly involved in the control of calcium homeostasis by means of receptor-mediated release of calcium from ryanodine-sensitive intracellular stores. The relationship between receptor-like and enzymatic properties of CD38 is still unknown. The apparent topological paradox of ectocellular synthesis and intracellular activity of cADPR might be explained by: (a) influx of cADPR across the plasma membrane to reach its target stores, as suggested by experiments on cerebellar granule cells; and (b) NAD(+)-induced internalization, following membrane oligomerization, of CD38 with consequent partial import of cADPR metabolism to an intracellular compartment, as recently observed in lymphoid B cells. These two distinct mechanisms and other potential ones (e.g. binding of ectocellularly formed cADPR to cell surface receptors and initiation of signal-transducing pathways across the plasmamembrane) seem to be paradigmatic of processes affecting different types of cells. Although in some biological systems, such as Aplysia and sea urchin egg, cADPR metabolism is restricted to the intracellular environment, in mammalian cells the CD38/cADPR system provides new challenges in terms of subcellular compartmentation and qualifies as an unusual example of "ectobiochemistry" with potential, still unrecognized, properties of cellular regulation.