ADP-ribosyl Cyclase Activity Research Articles

Extracellular NAD+ metabolism modulates osteoclastogenesis

Changes in NAD+ and its metabolites contribute to longevity and age-associated diseases. The role of NAD+ metabolism in bone diseases has however not been investigated, despite the fact that osteoporosis is a leading cause of morbidity in old age. TRAP(+) osteoclast formation from C57 Bl/6J mice was assessed after the addition of varying concentrations of NAD+ metabolites or exogenous ADPribosyl cyclase and NADase enzymes. The NAD+ metabolite cyclic ADPribose (cADPr) or exogenous addition of the enzyme ADPribosyl cyclase stimulated osteoclast formation. Blocking cADPr action with the antagonist 8-Br-cADPr potently inhibited osteoclast formation. In contrast to cADPr, its noncyclized derivative ADPribose (ADPr) or the exogenous addition of NADase both inhibited osteoclastogenesis. As CD38 is the major NAD+-degrading enzyme present in the bone marrow, these results suggest that CD38-mediated inhibition of osteoclastogenesis is related to its NADase activity, not its ADPribosyl cyclase activity.

Bradykinin activates ADP-ribosyl cyclase in neuroblastoma cells: Intracellular concentration decrease in NAD and increase in cyclic ADP-ribose

ADP-ribosyl cyclase activity in the crude membrane fraction of neuroblastoma × glioma NGPM1-27 hybrid cells was measured by monitoring [ 3H] cyclic ADP-ribose (cADPR) formation from [ 3H] NAD +. Bradykinin (BK) at 100 nM increased ADP-ribosyl cyclase activity by about 2.5-fold. Application of 300 nM BK to living NGPM1-27 cells decreased NAD + to 78% of the prestimulation level at 30 s. In contrast, intracellular cADPR concentrations were increased by 2–3-fold during the period from 30 to 120 s after the same treatment. Our results suggest that cADPR is one of the second messengers downstream of B 2 BK receptors.

Transcriptional regulation of CD38 expression by tumor necrosis factor‐α in human airway smooth muscle cells: role of NF‐κB and sensitivity to glucocorticoids

The transmembrane glycoprotein CD38 catalyzes the synthesis of the calcium mobilizing molecule cyclic ADP-ribose from NAD. In human airway smooth muscle (HASM) cells, the expression and function of CD38 are augmented by the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha), leading to increased intracellular calcium response to agonists. A glucocorticoid response element in the CD38 gene has been computationally described, providing evidence for transcriptional regulation of its expression. In the present study, we investigated the effects of dexamethasone, a glucocorticoid, on CD38 expression and ADP-ribosyl cyclase activity in HASM cells stimulated with TNF-alpha. In HASM cells, TNF-alpha augmented CD38 expression and ADP-ribosyl cyclase activity, which were attenuated by dexamethasone. TNF-alpha increased NF-kappaB expression and its activation, and dexamethasone partially reversed these effects. TNF-alpha increased the expression of IkappaBalpha, and dexamethasone increased it further. An inhibitor of NF-kappaB activation or transfection of cells with IkappaB mutants decreased TNF-alpha-induced CD38 expression. The results indicate that TNF-alpha-induced CD38 expression involves NF-kappaB expression and its activation and dexamethasone inhibits CD38 expression through NF-kappaB-dependent and -independent mechanisms.

The CD38-independent ADP-ribosyl cyclase from mouse brain synaptosomes: a comparative study of neonate and adult brain

cADPR (cADP-ribose), a metabolite of NAD+, is known to modulate intracellular calcium levels and to be involved in calcium-dependent processes, including synaptic transmission, plasticity and neuronal excitability. However, the enzyme that is responsible for producing cADPR in the cytoplasm of neural cells, and particularly at the synaptic terminals of neurons, remains unknown. In the present study, we show that endogenous concentrations of cADPR are much higher in embryonic and neonate mouse brain compared with the adult tissue. We also demonstrate, by comparing wild-type and Cd38-/- tissues, that brain cADPR content is independent of the presence of CD38 (the best characterized mammalian ADP-ribosyl cyclase) not only in adult but also in developing tissues. We show that Cd38-/- synaptosome preparations contain high ADP-ribosyl cyclase activities, which are more important in neonates than in adults, in line with the levels of endogenous cyclic nucleotide. By using an HPLC method and adapting the cycling assay developed initially to study endogenous cADPR, we accurately examined the properties of the synaptosomal ADP-ribosyl cyclase. This intracellular enzyme has an estimated K(m) for NAD+ of 21 microM, a broad optimal pH at 6.0-7.0, and the concentration of free calcium has no major effect on its cADPR production. It binds NGD+ (nicotinamide-guanine dinucleotide), which inhibits its NAD+-metabolizing activities (K(i)=24 microM), despite its incapacity to cyclize this analogue. Interestingly, it is fully inhibited by low (micromolar) concentrations of zinc. We propose that this novel mammalian ADP-ribosyl cyclase regulates the production of cADPR and therefore calcium levels within brain synaptic terminals. In addition, this enzyme might be a potential target of neurotoxic Zn2+.

Enhancement of Streptolysin O Activity and Intrinsic Cytotoxic Effects of the Group A Streptococcal Toxin, NAD-Glycohydrolase

Streptolysin O (SLO) is a cholesterol-dependent cytolysin produced by the important human pathogen, group A Streptococcus (Streptococcus pyogenes or GAS). In addition to its cytolytic activity, SLO mediates the translocation of GAS NAD-glycohydrolase (NADase) into human epithelial cells in vitro. Production of both NADase and SLO is associated with augmented host cell injury beyond that produced by SLO alone, but the mechanism of enhanced cytotoxicity is not known. We have now shown that expression of NADase together with SLO dramatically enhanced the lytic activity of GAS culture supernatants for erythrocytes but had no effect on SLO-mediated poration of synthetic cholesterol-rich liposomes. This result revealed a previously unknown contribution of NADase to the cytolytic activity associated with GAS production of SLO. Purified recombinant SLO bound NADase in vitro, supporting a specific, physical interaction of the two proteins. Exposure of human keratinocytes to wild-type GAS, but not to a NADase-deficient mutant strain, resulted in profound depletion of cellular NAD+ and ATP. Furthermore, expression of recombinant GAS NADase in yeast, in the absence of SLO, induced growth arrest, depletion of NAD+ and ATP, and cell death. These findings have provided evidence that the augmentation of SLO-mediated cytotoxicity by NADase is a consequence of depletion of host cell energy stores through the enzymatic action of NADase. Together, the results have provided mechanistic insight into the cytotoxic effects of a unique bipartite bacterial toxin.

Extracellular NAD+ regulates intracellular calcium levels and induces activation of human granulocytes

Beta-NAD+e (extracellular beta-NAD+), present at nanomolar levels in human plasma, has been implicated in the regulation of [Ca2+]i (the intracellular calcium concentration) in various cell types, including blood cells, by means of different mechanisms. Here, we demonstrate that micromolar NAD+e (both the alpha and the beta extracellular NAD+ forms) induces a sustained [Ca2+]i increase in human granulocytes by triggering the following cascade of causally related events: (i) activation of adenylate cyclase and overproduction of cAMP; (ii) activation of protein kinase A; (iii) stimulation of ADP-ribosyl cyclase activity and consequent overproduction of cADP-ribose, a universal Ca2+ mobilizer; and (iv) influx of extracellular Ca2+. The NAD+e-triggered [Ca2+]i elevation translates into granulocyte activation, i.e. superoxide and nitric oxide generation, and enhanced chemotaxis in response to 0.1-10 microM NAD+e. Thus extracellular beta-NAD+e behaves as a novel pro-inflammatory cytokine, stimulating human granulocytes and potentially recruiting them at sites of inflammation.

Novel localization of CD38 in perivascular sympathetic nerve terminals

Using high performance liquid chromatography fraction analysis we have recently established that numerous smooth muscle preparations, including the canine mesenteric artery and vein, release β-nicotinamide adenine dinucleotide upon short-pulse electrical field stimulation in tetrodotoxin- and ω-conotoxin GVIA-sensitive manners [ Smyth LM, Bobalova J, Mendoza MG, Lew C, Mutafova-Yambolieva VN (2004) Release of beta-nicotinamide adenine dinucleotide upon stimulation of postganglionic nerve terminals in blood vessels and urinary bladder. J Biol Chem 279:48893–48903.]. The β-nicotinamide adenine dinucleotide metabolites ADP-ribose and cyclic ADP-ribose are also present in the tissue superfusates. CD38 is a multifunctional enzyme involved in the degradation of β-nicotinamide adenine dinucleotide to ADP-ribose and cyclic ADP-ribose. Western immunoblot analysis revealed that CD38 is expressed in both artery and vein. Confocal laser scanning microscopy established colocalization of CD38 with tyrosine hydroxylase, synaptotagmin and synaptic vesicle protein in both blood vessels. High performance liquid chromatography with fluorescence detection demonstrated that whole tissue segments metabolize 1, N 6-etheno-nicotinamide adenine dinucleotide to 1, N 6-etheno-ADP-ribose and nicotinamide-guanine dinucleotide to cyclic GDP-ribose, suggesting the presence of both nicotinamide adenine dinucleotide-glycohydrolase and ADP-ribosyl cyclase activities in these blood vessels. Both enzymes appear to be associated with the membrane fraction, and therefore might be attributed to CD38. These data demonstrate a previously uncharacterized localization of CD38 in perivascular autonomic nerve terminals. Therefore, the β-nicotinamide adenine dinucleotide/CD38 system may provide new mechanisms in autonomic neurovascular control.

Altered CD38 expression in thioacetamide‐induced rat model of liver cirrhosis

Cirrhosis is a gradually developing, chronic disease which involves the whole liver. Here, we have shown that CD38 undergoes altered expression upon thioacetamide-induced cirrhosis in rats. CD38 is a type II transmembrane glycoprotein that exhibits ADP-ribosyl cyclase and cADPR hydrolase activities. In this study, the gene and protein expressions of CD38 were investigated in a thioacetamide-induced rat model of cirrhosis. CD38 expression was studied by using real-time RT-PCR, immunohistochemistry, and immunoblotting. cADPR content in liver was measured using cycling assay. There was a significant increase in CD38 mRNA and protein expressions as well as ADP-ribosyl cyclase activity in cirrhotic liver compared to the control liver. cADPR level was found to be modestly but significantly augmented in cirrhotic liver. These results raised the possibility that altered CD38 expression and a concomitant elevation of the enzymatic activity as well as cADPR may be involved in the pathogenesis of liver cirrhosis.

Cyclic ADP ribose-mediated Ca2+signaling in mediating endothelial nitric oxide production in bovine coronary arteries

The present study tested the hypothesis that cyclic ADP ribose (cADPR) serves as a novel second messenger to mediate intracellular Ca2+ mobilization in coronary arterial endothelial cells (CAECs) and thereby contributes to endothelium-dependent vasodilation. In isolated and perfused small bovine coronary arteries, bradykinin (BK)-induced concentration-dependent vasodilation was significantly attenuated by 8-bromo-cADPR (a cell-permeable cADPR antagonist), ryanodine (an antagonist of ryanodine receptors), or nicotinamide (an ADP-ribosyl cyclase inhibitor). By in situ simultaneously fluorescent monitoring, Ca2+ transient and nitric oxide (NO) levels in the intact coronary arterial endothelium preparation, 8-bromo-cADPR (30 microM), ryanodine (50 microM), and nicotinamide (6 mM) substantially attenuated BK (1 microM)-induced increase in intracellular [Ca2+] by 78%, 80%, and 74%, respectively, whereas these compounds significantly blocked BK-induced NO increase by about 80%, and inositol 1,4,5-trisphosphate receptor blockade with 2-aminethoxydiphenyl borate (50 microM) only blunted BK-induced Ca2+-NO signaling by about 30%. With the use of cADPR-cycling assay, it was found that inhibition of ADP-ribosyl cyclase by nicotinamide substantially blocked BK-induced intracellular cADPR production. Furthermore, HPLC analysis showed that the conversion rate of beta-nicotinamide guanine dinucleotide into cyclic GDP ribose dramatically increased by stimulation with BK, which was blockable by nicotinamide. However, U-73122, a phospholipase C inhibitor, had no effect on this BK-induced increase in ADP-ribosyl cyclase activity for cADPR production. In conclusion, these results suggest that cADPR importantly contributes to BK- and A-23187-induced NO production and vasodilator response in coronary arteries through its Ca2+ signaling mechanism in CAECs.

A multifunctional enzyme adds to its repertoire

ADP-ribosyl cyclases are a family of multifunctional enzymes that appear to be expressed ubiquitously in eukaryotic systems (reviewed in refs. 1 and 2). These enzymes are known to metabolize NAD+ and produce at least three molecules that have been demonstrated to be involved in calcium signaling. These enzymes are named for their ability to convert NAD+ to cyclic ADP-ribose (cADPR), a signaling molecule that has been shown to mobilize calcium from intracellular stores by means of a ryanodine receptor/calcium channeldependent system (reviewed in refs. 3 and 4). In addition to cADPR, ADP-ribosyl cyclases have been shown to produce ADP-ribose (ADPR) and nicotinic acid adenine dinucleotide 2′-phosphate (NAADP) (1, 2, 5). These molecules are also involved in regulating calcium signaling: ADPR has been shown to regulate plasma membrane LTRPC2 calcium channels in mammalian cells (6), and NAADP induces calcium mobilization from intracellular stores in several systems (7). In mammalian systems, the ectoenzymes CD38 and CD157 appear to be the major enzymes with ADP-ribosyl cyclase activity (2), although a non-CD38/CD157 ADP-ribosyl cyclase has been detected recently in brains from CD38 knockout mice (8). The work of Basile et al. (9) in a recent issue of PNAS demonstrates that, in addition to producing cADPR, ADPR, and NAADP, ADP-ribosyl cyclases can also generate three adenine dinucleotides (9). Interestingly, these adenine dinucleotides are shown to exist naturally and to have biological activity (9). These findings have several important implications, including expanding the signaling capabilities of the ADP-ribosyl cyclase …

Crystal Structure of Human CD38 Extracellular Domain

Human CD38 is a multifunctional protein involved in diverse functions. As an enzyme, it is responsible for the synthesis of two Ca2+ messengers, cADPR and NAADP; as an antigen, it is involved in regulating cell adhesion, differentiation, and proliferation. Besides, CD38 is a marker of progression of HIV-1 infection and a negative prognostic marker of B-CLL. We have determined the crystal structure of the soluble extracellular domain of human CD38 to 1.9 A resolution. The enzyme's overall topology is similar to the related proteins CD157 and the Aplysia ADP-ribosyl cyclase, except with large structural changes at the two termini. The extended positively charged N terminus has lateral associations with the other CD38 molecule in the crystallographic asymmetric unit. The analysis of the CD38 substrate binding models revealed two key residues that may be critical in controlling CD38's multifunctionality of NAD hydrolysis, ADP-ribosyl cyclase, and cADPR hydrolysis activities.

ADP-ribosyl cyclase and GDP-ribosyl cyclase activities are not always equivalent: Impact on the study of the physiological role of cyclic ADP-ribose

ADP-ribosyl cyclase and GDP-ribosyl cyclase activities are not always equivalent: Impact on the study of the physiological role of cyclic ADP-ribose

Acetylcholine stimulates cyclic ADP-ribose formation via M1 muscarinic receptors in rat superior cervical ganglion

The role of cyclic ADP-ribose (cADPR) as the downstream signal of neuronal muscarinic acetylcholine receptors (mAChRs) and the enzyme responsible for its synthesis, ADP-ribosyl cyclase, were examined in the rat superior cervical ganglion (SCG). Application of acetylcholine or other mAChR agonists increased the ADP-ribosyl cyclase activity by about 250–300% in crude membrane fractions from the SCG of 14-day-old rats. This effect was inhibited by atropine or by the M1-mAChR antagonist, pirenzepine, and was mimicked by GTP. These results indicate that the M1 mAChRs couple to the membrane-bound form of ADP-ribosyl cyclase and suggest that cADPR is a second messenger of M1 mAChR signaling in nervous tissue.

Adenoviral mediated anti-sense CD38 attenuates TNF-α-induced changes in calcium homeostasis of human airway smooth muscle cells

CD38 is a membrane-bound protein involved in the synthesis and degradation of cyclic-ADP-ribose (cADPR). cADPR mobilizes calcium from intracellular stores in airway smooth muscle cells. To determine the role of CD38/cADPR signaling in calcium regulation in human airway smooth muscle (HASM) cells, we downregulated CD38 expression using a recombinant replication-defective adenovirus with anti-sense human CD38 (Ad-asCD38). CD38 expression was determined by RT-PCR and real-time quantitative PCR, and ADP-ribosyl cyclase (cyclase) activity was determined by competitive binding assay. In HASM cells infected with Ad-asCD38, TNF-alpha-induced, augmented-CD38 expression and cyclase activity were significantly lower than in TNF-alpha-treated cells. The net intracellular calcium responses to 10 nmol/L bradykinin were measured in HASM cells by fluorescence imaging. In cells infected with Ad-asCD38 in the presence of TNF-alpha, the net intracellular Ca2+ responses were significantly lower than in cells treated with TNF-alpha in the presence of the control vector (p < 0.001). These results provide evidence for the feasibility of using adenoviral vectors for gene transfer to down regulate gene expression, and confirm the role of CD38 in calcium homeostatis in ASM cells.

Ryanodine receptor subtype 2 encodes Ca2+ oscillations activated by acetylcholine via the M2 muscarinic receptor/cADP-ribose signalling pathway in duodenum myocytes

In this study, we characterized the signalling pathway activated by acetylcholine that encodes Ca2+ oscillations in rat duodenum myocytes. These oscillations were observed in intact myocytes after removal of external Ca2+, in permeabilized cells after abolition of the membrane potential and in the presence of heparin (an inhibitor of inositol 1,4,5-trisphosphate receptors) but were inhibited by ryanodine, indicating that they are dependent on Ca2+ release from intracellular stores through ryanodine receptors. Ca2+ oscillations were selectively inhibited by methoctramine (a M2 muscarinic receptor antagonist). The M2 muscarinic receptor-activated Ca2+ oscillations were inhibited by 8-bromo cyclic adenosine diphosphoribose and inhibitors of adenosine diphosphoribosyl cyclase (ZnCl2 and anti-CD38 antibody). Stimulation of ADP-ribosyl cyclase activity by acetylcholine was evaluated in permeabilized cells by measuring the production of cyclic guanosine diphosphoribose (a fluorescent compound), which resulted from the cyclization of nicotinamide guanine dinucleotide. As duodenum myocytes expressed the three subtypes of ryanodine receptors, an antisense strategy revealed that the ryanodine receptor subtype 2 alone was required to initiate the Ca2+ oscillations induced by acetylcholine and also by cyclic adenosine diphosphoribose and rapamycin (a compound that induced uncoupling between 12/12.6 kDa FK506-binding proteins and ryanodine receptors). Inhibition of cyclic adenosine diphosphoribose-induced Ca2+ oscillations, after rapamycin treatment, confirmed that both compounds interacted with the ryanodine receptor subtype 2. Our findings show for the first time that the M2 muscarinic receptor activation triggered Ca2+ oscillations in duodenum myocytes by activation of the cyclic adenosine diphosphoribose/FK506-binding protein/ryanodine receptor subtype 2 signalling pathway.

Role of NAADP and cADPR in the Induction and Maintenance of Agonist-Evoked Ca2+ Spiking in Mouse Pancreatic Acinar Cells

Nicotinic acid adenine dinucleotide phosphate (NAADP) and cyclic adenosine diphosphate ribose (cADPR) were first demonstrated to mobilize Ca2+ in sea urchin eggs. In the absence of direct measurements of these messengers, pharmacological studies alone have implicated these molecules as intracellular second messengers for specific cell surface receptor agonists. We now report that in mouse pancreatic acinar cells, cholecystokinin, but not acetylcholine, evokes rapid and transient increases in NAADP levels in a concentration-dependent manner. In contrast, both cholecystokinin and acetylcholine-mediated production of cADPR followed a very different time course. The rapid and transient production of NAADP evoked by cholecystokinin precedes the onset of the Ca2+ signal and is consistent with a role for NAADP in the initiation of the Ca2+ response. Continued agonist-evoked Ca2+ spiking is maintained by prolonged elevations of cADPR levels through sensitization of Ca2+ -induced Ca2+ -release channels. This study represents the first direct comparison of NAADP and cADPR measurements, and the profound differences observed in their time courses provide evidence in support of distinct roles of these Ca2+ -mobilizing messengers in shaping specific Ca2+ signals during agonist stimulation.

The expression of messenger RNA for ADP‐ribosyl cyclase in aldosterone‐producing adenomas

Cyclic ADP-ribose (cADPR) has been reported to be as potent and powerful at releasing intracellular Ca(2+) as inositol triphosphate (IP(3)). To determine whether the cADPR system plays a signalling role in angiotensin II (Ang II)-induced aldosterone synthesis in the human adrenal gland, we investigated the effects of Ang II on ADP-ribosyl cyclase activity in human adrenal cortical tissue. In addition, the expression of ADP-ribosyl cyclase messenger RNA was evaluated in aldosterone-producing adenomas (APAs) and compared with normal adrenal tissue and nonfunctioning adenomas. ADP-ribosyl cyclase activity was measured in crude membrane fractions of human adrenocortical tissues with Ang II or with Ang II plus the Ang II receptor (AT(1)R) antagonist losartan or plus the AT(2)R antagonist PD123319. The effect of 8-bromo-cADPR on Ang II-induced aldosterone production from adrenal tissue was estimated. The expression of ADP-ribosyl cyclase, CYP11B2 and AT(1)R mRNA was measured in APAs, nonfunctioning adenomas, adjacent adrenal tissue and normal adrenal tissue. ADP-ribosyl cyclase activity was measured by using high-performance liquid chromatography (HPLC) and [(3)H] NAD(+). mRNA expression was measured by competitive reverse-transcription polymerase chain reaction (RT-PCR). Results Ang II (10(-7) and 10(-8) mol/l) significantly increased ADP-ribosyl cyclase activity in a dose-dependent manner. This increase was inhibited by pretreatment with losartan but not with PD123319. Treatment with 8-bromo-cADPR (50 micromol/l) reduced Ang II-induced aldosterone secretion. ACTH did not significantly increase the enzyme activity. The expression of ADP-ribosyl cyclase, CYP11B2 and AT(1)R mRNA was increased in APAs compared with that of nonfunctioning adenomas, adjacent adrenal tissue or normal adrenal tissue. These results demonstrated the existence of a signalling pathway from the Ang II receptor to ADP-ribosyl cyclase in the human adrenal gland and suggest that the cADP-ribose signalling system might play an important role in the pathogenesis of APAs.

Changes in NAD/ADP-ribose metabolism in rectal cancer

The extent of ADP-ribosylation in rectal cancer was compared to that of the corresponding normal rectal tissue. Twenty rectal tissue fragments were collected during surgery from patients diagnosed as having rectal cancer on the basis of pathology results. The levels of ADP-ribosylation in rectum cancer tissue samples (95.9 +/- 22.1 nmol/ml) was significantly higher than in normal tissues (11.4 +/- 4 nmol/ml). The level of NAD+ glycohydrolase and ADP-ribosyl cyclase activities in rectal cancer and normal tissue samples were measured. Cancer tissues had significantly higher NAD+ glycohydrolase and ADP-ribosyl cyclase activities than the control tissues (43.3 +/- 9.1 vs 29.2 +/- 5.2 and 6.2 +/- 1.6 vs 1.6 +/- 0.4 nmol mg(-1) min(-1)). Approximately 75% of the NAD+ concentration was consumed as substrate in rectal cancer, with changes in NAD+/ADP-ribose metabolism being observed. When [14C]-ADP-ribosylated tissue samples were subjected to SDS-PAGE, autoradiographic analysis revealed that several proteins were ADP-ribosylated in rectum tissue. Notably, the radiolabeling of a 113-kDa protein was remarkably greater than that in control tissues. Poly(ADP)-ribosylation of the 113-kDa protein in rectum cancer tissues might be enhanced with its proliferative activity, and poly(ADP)-ribosylation of the same protein in rectum cancer patients might be an indicator of tumor diagnosis.

Dendritic cells and follicular dendritic cells express a novel ligand for CD38 which influences their maturation and antibody responses

CD38 is a cell surface molecule with ADP-ribosyl cyclase activity, which is predominantly expressed on lymphoid and myeloid cells. CD38 has a significant role in B-cell function as some anti-CD38 antibodies can deliver potent growth and differentiation signals, but the ligand that delivers this signal in mice is unknown. We used a chimeric protein of mouse CD38 and human immunogobulin G (IgG) (CD38-Ig) to identify a novel ligand for murine CD38 (CD38L) on networks of follicular dendritic cells (FDCs) as well as dendritic cells (DCs) in the spleen. Flow-cytometry found that all DC subsets expressed cytoplasmic CD38L but only fresh ex vivo CD11c+ CD11b- DCs had cell surface CD38L. Anti-CD38 antibody blocked the binding of CD38-Ig to CD38L, confirming the specificity of detection. CD38-Ig immuno-precipitated ligands of 66 and 130 kDa. Functional studies found that CD38-Ig along with anti-CD40 and anti-major histocompatibility complex (MHC) class II antibody provided maturation signals to DCs in vitro. When CD38-Ig was administered in vivo with antigen, IgG2a responses were significantly reduced, suggesting that B and T cells expressing CD38 may modulate the isotype of antibodies produced through interaction with CD38L on DCs. CD38-Ig also expanded FDC networks when administered in vivo. In conclusion, this study has identified a novel ligand for CD38 which has a role in functional interactions between lymphocytes and DCs or FDCs.

Abscisic Acid Signaling through Cyclic ADP-ribose in Hydroid Regeneration

Cyclic ADP-ribose (cADPR) is an intracellular calcium (Ca(2+)(i)) mobilizer involved in fundamental cell functions from protists to higher plants and mammals. Biochemical similarities between the drought-signaling cascade in plants and the temperature-sensing pathway in marine sponges suggest an ancient evolutionary origin of a signaling cascade involving the phytohormone abscisic acid (ABA), cADPR, and Ca(2+)(i). In Eudendrium racemosum (Hydrozoa, Cnidaria), exogenously added ABA stimulated ADP-ribosyl cyclase activity via a protein kinase A (PKA)-mediated phosphorylation and increased regeneration in the dark to levels observed under light conditions. Light stimulated endogenous ABA synthesis, which was conversely inhibited by the inhibitor of plant ABA synthesis Fluridone. The signal cascade of light-induced regeneration uncovered in E. racemosum: light --> increasing ABA --> PKA --> cyclase activation --> increasing [cADPR](i) --> increasing [Ca(2+)](i) --> regeneration is the first report of a complete signaling pathway in Eumetazoa involving a phytohormone.