CD38 inhibition by apigenin ameliorates mitochondrial oxidative stress through restoration of the intracellular NAD+/NADH ratio and Sirt3 activity in renal tubular cells in diabetic rats.

Mitochondrial oxidative stress is a significant contributor to the pathogenesis of diabetic kidney disease (DKD). We previously showed that mitochondrial oxidative stress in the kidneys of Zucker diabetic fatty rats is associated with a decreased intracellular NAD+/NADH ratio and NAD+-dependent deacetylase Sirt3 activity, and increased expression of the NAD+-degrading enzyme CD38. In this study, we used a CD38 inhibitor, apigenin, to investigate the role of CD38 in DKD. Apigenin significantly reduced renal injuries, including tubulointerstitial fibrosis, tubular cell damage, and pro-inflammatory gene expression in diabetic rats. In addition, apigenin down-regulated CD38 expression, and increased the intracellular NAD+/NADH ratio and Sirt3-mediated mitochondrial antioxidative enzyme activity in the kidneys of diabetic rats. In vitro, inhibition of CD38 activity by apigenin or CD38 knockdown increased the NAD+/NADH ratio and Sirt3 activity in renal proximal tubular HK-2 cells cultured under high-glucose conditions. Together, these results demonstrate that by inhibiting the Sirt3 activity and increasing mitochondrial oxidative stress in renal tubular cells, CD38 plays a crucial role in the pathogenesis of DKD.

Implications of NAD+ boosters in translational medicine.

Nicotinamide adenine dinucleotide (NAD+ ) is an essential metabolite in energy metabolism as well as a co-substrate in biochemical reactions such as protein deacylation, protein ADP-ribosylation and cyclic ADP-ribose synthesis mediated by sirtuins, poly (ADP-ribose) polymerases (PARPs) and CD38. In eukaryotic cells, NAD+ is synthesized through three distinct pathways, which offer different strategies to modulate the bioavailability of NAD+ . The therapeutic potential of dietarily available NAD+ boosters preserving the NAD+ pool has been attracting attention after the discovery of declining NAD+ levels in ageing model organisms as well as in several age-related diseases, including cardiometabolic and neurodegenerative diseases. Here, we review the recent advances in the biology of NAD+ , including the salubrious effects of NAD+ boosters and discuss their future translational strategies.

CD157: From Myeloid Cell Differentiation Marker to Therapeutic Target in Acute Myeloid Leukemia.

: Human CD157/BST-1 and CD38 are dual receptor-enzymes derived by gene duplication that belong to the ADP ribosyl cyclase gene family. First identified over 30 years ago as Mo5 myeloid differentiation antigen and 10 years later as Bone Marrow Stromal Cell Antigen 1 (BST-1), CD157 proved not to be restricted to the myeloid compartment and to have a diversified functional repertoire ranging from immunity to cancer and metabolism. Despite being a NAD+-metabolizing ectoenzyme anchored to the cell surface through a glycosylphosphatidylinositol moiety, the functional significance of human CD157 as an enzyme remains unclear, while its receptor role emerged from its discovery and has been clearly delineated with the identification of its high affinity binding to fibronectin. The aim of this review is to provide an overview of the immunoregulatory functions of human CD157/BST-1 in physiological and pathological conditions. We then focus on CD157 expression in hematological tumors highlighting its emerging role in the interaction between acute myeloid leukemia and extracellular matrix proteins and its potential utility for monoclonal antibody targeted therapy in this disease.

Inhibiting the CD38/cADPR pathway protected rats against sepsis associated brain injury.

BACKGROUND: The CD38/cADPR pathway has been found to play roles in various inflammatory conditions. However, whether CD38 plays a protective or detrimental effect in the central nervous system (CNS) is controversial. The aim of this study was to determine the effect of CD38/cADPR pathway in sepsis associated brain injury. MATERIALS AND METHODS: Male Sprague-Dawley rats were undergone cecal ligation and puncture (CLP) or sham laparotomies. NAD+, cADPR and CD38 were measured in the hippocampus of septic rats at 0, 6, 12, 24, and 48h after CLP surgery. Rats were divided into the sham, CLP group, CLP+ CD38 expression lentivirus (CLP+ CD38 LV), CLP+ CD38 interference lentivirus (CLP+ CD38 Ri), CLP+ negative control lentivirus (CLP+NC) and the CLP+8-Br-cADPR groups. The Western blots of Bcl-2, Bax and iNOS, TUNEL assays, malondialdehyde (MDA) and superoxide dismutase (SOD) assays, transmission electron microscope analysis were performed in the hippocampus of rats. RESULTS: NAD+, cADPR and CD38 levels increased significantly in the hippocampus of septic rats as early as 12-24h after CLP surgery. CD38 knockdown or blocking cADPR with 8-Br-cADPR significantly reduced apoptosis, MDA and SOD activity, iNOS expression and ultrastructural morphology damages in the hippocampus of septic rats. CONCLUSIONS: In this study, we found that the CD38/cADPR pathway was activated in sepsis associated brain injury. Blocking this pathway protected the hippocampus from apoptosis, oxidative stress and ultrastructural morphology damages in septic rats.

Targeting CD157 in AML using a novel, Fc-engineered antibody construct.

Antibody-based immunotherapy represents a promising strategy to eliminate chemorefractory leukemic cells in acute myeloid leukemia (AML). In this study, we evaluated a novel Fc-engineered antibody against CD157 (MEN1112) for its suitability as immunotherapy in AML. CD157 was expressed in 97% of primary AML patient samples. A significant, albeit lower expression level of CD157 was observed within the compartment of leukemia-initiating cells, which are supposed to be the major source of relapse. In healthy donor bone marrow, CD157 was expressed on CD34+ cells. In ex vivo assays, MEN1112 triggered natural killer (NK) cell-mediated cytotoxicity against AML cell lines and primary AML cells. Compared to its parental analogue, the Fc-engineered antibody exhibited higher antibody dependent cellular cytotoxicity responses. Using NK cells from AML patients, we observed heterogeneous MEN1112-mediated cytotoxicity against AML cells, most likely due to well-documented defects in AML-NK cells and corresponding inter-patient variations in NK cell function. Cytotoxicity could not be correlated to the time after completion of chemotherapy. In summary, we could demonstrate that CD157 is strongly expressed in AML. MEN1112 is a promising antibody construct that showed high cytotoxicity against AML cells and warrants further clinical testing. Due to variability in NK-cell function of AML patients, the time of application during the course of the disease as well as combinatorial strategies might influence treatment results.

NAD derived second messengers: Role in spontaneous diastolic Ca(2+) transients in murine cardiac myocytes.

Strong ß-adrenergic stimulation induced spontaneous diastolic Ca(2+) transients (SCTs) in electrically paced murine cardiac myocytes [28]. To obtain further insights into the underlying mechanism, we developed a method for a simultaneous analysis, in which the free luminal Ca(2+) concentration in the sarcoplasmic reticulum (SR) ([Ca(2+)]SR) and the free cytosolic Ca(2+) concentration ([Ca(2+)]i) were measured in parallel in the same cell. Each spontaneous diastolic Ca(2+) transient was exactly mirrored by a decrease of [Ca(2+)]SR. Since antagonism of the Ca(2+) mobilizing second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) was shown to block SCTs in single cardiac myocytes [28], we analyzed the effect of the novel ADP-ribosyl cyclase inhibitor SAN4825 on both cytosolic and intra-luminal Ca(2+) transients upon strong ß-adrenergic stimulation. A strong antagonist effect of SAN4825 on SCTs at low micromolar concentrations was observed. Our results suggest that the underlying mechanism of spontaneous diastolic Ca(2+) transients observed upon strong ß-adrenergic stimulation is sensitization of type 2 ryanodine receptor by the Ca(2+) releasing activity of the products of ADP-ribosyl cyclase activity.

Mapping NAD(+) metabolism in the brain of ageing Wistar rats: potential targets for influencing brain senescence.

Over the last decade, the importance of NAD(+) has expanded beyond its role as an essential cofactor for energy metabolism. NAD(+) has emerged as a major signalling molecule that serves as the sole substrate for several enzymatic reactions including the DNA repair enzyme, poly(ADP-ribose) polymerase (PARP), NAD-dependent protein deacetylases or CD38, and transcriptional factors by a new class of histone deacetylases known as sirtuins. NAD(+) levels are regulated by the metabolic status and cellular stress caused by oxidative stress and DNA damage. Since a detailed study of NAD(+) metabolism in the healthy ageing mammalian brain is nascent, we examined the effect of ageing on intracellular NAD(+) metabolism in different brain regions in female Wistar rats in young (3 months), middle aged (12 months) and older adults (24 months). Our results are the first to show a significant decline in intracellular NAD(+) levels and NAD:NADH ratio with ageing in the CNS, occurring in parallel to an increase in lipid peroxidation and protein oxidation (o- and m-tyrosine) and a decline in total antioxidant capacity. Hyperphosphorylation of H2AX levels was also observed together with increased PARP-1 and PARP-2 expression, and CD38 activity, concomitantly with reduced NAD(+) and ATP levels and SIRT1 function in the cortex, brainstem, hippocampus and cerebellum. Reduced activity of mitochondrial complex I-IV and impaired maximum mitochondrial respiration rate were also observed in the ageing rat brain. Among the multiple physiological pathways associated with NAD(+) catabolism, our discovery of CD38 as the major regulator of cellular NAD(+) levels in rat neurons indicates that CD38 is a promising therapeutic target for the treatment of age-related neurodegenerative diseases.

[The role of reactive oxygen species and calcium ions in the implementation of the stress-protective effect of brassinosteroids on plant cells].

The effect of the brassinosteroids (BS) 24-epibrassiniolide and 24-epicastasterone on the thermoresistance of wheat coleoptile (Triticum aestivum L.) and their generation of the superoxide anion radical and antioxidant enzymes activity were investigated. The treatment of coleoptiles with 10 nM solutions of BS caused a transient increase in O2·- generation and a subsequent increase in the activity of superoxide dismutase and catalase and an improvement in heat resistance. Pretreatment of coleoptiles with the NADPH oxidase inhibitor imidazole leveled the increase in production of the superoxide anion radical and prevented an increase in the activity of antioxidant enzymes and the development of cell thermostability. The investigated effects of BS were also depressed by the pretreatment of coleoptile segments with extracellular calcium chelator EGTA and inhibitor of ADP-ribosyl cyclase nicotinamide. A conclusion was made about the participation of calcium ions and reactive oxygen species generated by the action of NADPH oxidase in the implementation of the stress-protective effect of the BS in the cells of wheat coleoptile.

Selective inhibitors of cardiac ADPR cyclase as novel anti-arrhythmic compounds.

ADP-ribosyl cyclases (ADPRCs) catalyse the conversion of nicotinamide adenine dinucleotide to cyclic adenosine diphosphoribose (cADPR) which is a second messenger involved in Ca(2+) mobilisation from intracellular stores. Via its interaction with the ryanodine receptor Ca(2+) channel in the heart, cADPR may exert arrhythmogenic activity. To test this hypothesis, we have studied the effect of novel cardiac ADPRC inhibitors in vitro and in vivo in models of ventricular arrhythmias. Using a high-throughput screening approach on cardiac sarcoplasmic reticulum membranes isolated from pig and rat and nicotinamide hypoxanthine dinuleotide as a surrogate substrate, we have identified potent and selective inhibitors of an intracellular, membrane-bound cardiac ADPRC that are different from the two known mammalian ADPRCs, CD38 and CD157/Bst1. We show that two structurally distinct cardiac ADPRC inhibitors, SAN2589 and SAN4825, prevent the formation of spontaneous action potentials in guinea pig papillary muscle in vitro and that compound SAN4825 is active in vivo in delaying ventricular fibrillation and cardiac arrest in a guinea pig model of Ca(2+) overload-induced arrhythmia. Inhibition of cardiac ADPRC prevents Ca(2+) overload-induced spontaneous depolarizations and ventricular fibrillation and may thus provide a novel therapeutic principle for the treatment of cardiac arrhythmias.

The CD157-integrin partnership controls transendothelial migration and adhesion of human monocytes.

CD157, a member of the CD38 gene family, is an NAD-metabolizing ectoenzyme and a signaling molecule whose role in polarization, migration, and diapedesis of human granulocytes has been documented; however, the molecular events underpinning this role remain to be elucidated. This study focused on the role exerted by CD157 in monocyte migration across the endothelial lining and adhesion to extracellular matrix proteins. The results demonstrated that anti-CD157 antibodies block monocyte transmigration and adhesion to fibronectin and fibrinogen but that CD157 cross-linking is sufficient to overcome the block, suggesting an active signaling role for the molecule. Consistent with this is the observation that CD157 is prevalently located within the detergent-resistant membrane microdomains to which, upon clustering, it promotes the recruitment of ß(1) and ß(2) integrin, which, in turn, leads to the formation of a multimolecular complex favoring signal transduction. This functional cross-talk with integrins allows CD157 to act as a receptor despite its intrinsic structural inability to do so on its own. Intracellular signals mediated by CD157 rely on the integrin/Src/FAK (focal adhesion kinase) pathway, resulting in increased activity of the MAPK/ERK1/2 and the PI3K/Akt downstream signaling pathways, which are crucial in the control of monocyte transendothelial migration. Collectively, these findings indicate that CD157 acts as a molecular organizer of signaling-competent membrane microdomains and that it forms part of a larger molecular machine ruled by integrins. The CD157-integrin partnership provides optimal adhesion and transmigration of human monocytes.

The role of intracellular ion channels in regulating cytoplasmic calciumin pulmonary arterial mmooth muscle: which store and where?

The mobilisation of intracellular Ca(2+) stores plays a pivotal role in the regulation of arterial smooth muscle function, paradoxically during both contraction and relaxation. Moreover, different spatiotemporal Ca(2+) signalling patterns may trigger differential gene expression while mediating the same functional response. These facts alone serve to highlight the importance of the growing body of evidence in support of the view that different Ca(2+) storing organelles may be selected by the discrete or co-ordinated actions of multiple Ca(2+) mobilising messengers. In this respect, it is generally accepted that sarcoplasmic reticulum stores may be mobilised by the ubiquitous messenger inositol 1,4,5 trisphosphate. However, relatively little attention has been paid to the role of Ca(2+) mobilising pyridine nucleotides in arterial smooth muscle, namely cyclic adenosine diphosphate-ribose and nicotinic acid adenine dinucleotide phosphate. This review will, therefore, focus on the role of these novel Ca(2+) mobilising messengers in pulmonary arterial smooth muscle, with particular reference to hypoxic pulmonary vasoconstriction.

Mice lacking the ADP ribosyl cyclase CD38 exhibit attenuated renal vasoconstriction to angiotensin II, endothelin-1, and norepinephrine.

ADP ribosyl (ADPR) cyclases comprise a family of ectoenzymes recently shown to influence cytosolic Ca(2+) concentration in a variety of cell types. At least two ADPR cyclase family members have been identified in mammals: CD38 and CD157. We recently found reduced renal vascular reactivity to angiotensin II (ANG II), endothelin-1 (ET-1), and norepinephrine (NE) in the presence of the broad ADPR cyclase inhibitor nicotinamide. We hypothesized that CD38 mediates effects attributed to ADPR cyclase. We found expression of ADPR cyclases CD38 and CD157 mRNA in spleen, thymus, skin, and preglomerular arterioles of wild-type (WT) animals. Mice lacking CD38 showed decreased CD157 expression in most tissues tested. No difference in systolic or mean arterial pressure was observed between strains in either conscious or anesthetized states, whereas heart rate was reduced 10-20% in CD38-/- animals (P < 0.05). During anesthesia, CD38-/- mice had reduced basal renal blood flow (RBF) and urine excretion (P < 0.05). RBF responses to intravenous injection of ANG II, ET-1, and NE were attenuated approximately 50% in CD38-/- vs. WT mice (P < 0.01 for all). The systemic pressor response to ANG II was decreased in the absence of CD38 (P < 0.01), whereas that to NE was normal (P > 0.05); ET-1 was administered at a nonpressor dose. Nicotinamide effectively inhibited ANG II-induced renal vasoconstriction in WT mice (P < 0.001), but had no effect on renal responses to ANG II in CD38-/- mice (P > 0.5). Overall, our observations indicate the presence of two ADPR cyclase family members in renal preglomerular resistance arterioles and the importance of CD38 participation in acute vascular responses to all three vasoconstrictors in the renal microcirculation.

Inhibition of ADP-ribosyl cyclase attenuates angiotensin II-induced cardiac hypertrophy.

AIMS: Here, we report the discovery of a small molecule inhibitor, 2,2'-dihydroxyazobenzene (DAB), of ADP ribosyl cyclase (ADPR-cyclase) and showed that this inhibitor attenuated angiotensin (Ang) II-induced hypertrophic responses. METHODS: and results The intracellular concentration of free Ca(2+) [Ca(2+)](i) in adult rat cardiomyocytes was measured by using a confocal microscope. Cardiac hypertrophy was induced by the two-kidney one-clip (2K1C) method. Hypertrophy was determined by de novo protein synthesis, cell volume, echocardiography, nuclear translocation of nuclear factor of activated T-cells, and transforming growth factor-beta1 protein expression. Treatment of cardiomyocytes with Ang II generated a biphasic [Ca(2+)](i) increase that included an initial Ca(2+)peak and sustained Ca(2+) rise via inositol trisphosphate and cyclic ADP-ribose (cADPR) formation, respectively. A cADPR antagonistic analogue, 8-Br-cADPR, and an ADPR-cyclase inhibitor, DAB, blocked the sustained Ca(2+) signal, but not the initial Ca(2+) rise. Furthermore, DAB significantly inhibited Ang II-mediated cADPR formation and hypertrophic responses in vitro. Echocardiography and histological examination revealed significant cardiac hypertrophy in 2K1C rats that was potently inhibited by treatment with DAB. In addition, the hypertrophic responses induced by Ang II in vitro were significantly increased by 2K1C, and DAB treatment reversed these hypertrophic responses to the levels of sham Control. CONCLUSION: ADPR-cyclase is an important mediator of cardiac hypertrophy, and inhibition of ADPR-cyclase by DAB may provide a new therapeutic strategy for cardiac diseases.

Role of kidney ADP-ribosyl cyclase in diabetic nephropathy.

The role of ADP-ribosyl cyclases (ADPR-cyclases) in diabetic nephropathy was investigated. ADPR-cyclases synthesize cADP-ribose (cADPR), a Ca(2+)-mobilizing second messenger, and are stimulated by G protein-coupled receptors. We have previously reported that ADPR-cyclases can be activated by ANG II and showed that a specific kidney ADPR-cyclase inhibitor, 4,4'-dihydroxyazobenzene (DHAB), can protect ANG II-mediated mesangial cell growth (Kim SY, Gul R, Rah SY, Kim SH, Park SK, Im MJ, Kwon HJ, Kim UH. Am J Physiol Renal Physiol 294: F982-F989, 2008). In this study, we examined the preventive effect of DHAB on glomerular injury in streptozotocin (STZ)-induced diabetic mice. Male mice were randomly assigned to normal control and diabetic groups of comparable age. A diabetic group received 45 microg/kg of DHAB for 6 wk via daily intraperitoneal injections. Several nephropathy parameters were improved in the DHAB-treated diabetic group compared with the diabetic group, including urinary albumin (diabetic, 44.6 +/- 5.1 vs. treated, 33.9 +/- 3.9 microg/day), creatinine clearance (diabetic, 0.72 +/- 0.03 vs. treated, 0.83 +/- 0.04 ml.min(-1).100 g(-1)), ratio of kidney to body weight (diabetic, 2.5 +/- 0.04 vs. treated, 1.4 +/- 0.04), and mesangial matrix expansion (diabetic, 13.9 +/- 2.2 vs. treated, 8.5 +/- 2.0%). These results indicate that kidney function in STZ-induced diabetes was improved by DHAB administration. Furthermore, DHAB inhibited phosphorylation of Akt and nuclear factor of activated T cell 3 nuclear translocation, as well as ADPR-cyclase activity and cADPR production, which were increased in the kidneys of the diabetic group. In addition, DHAB treatment decreased fibrosis marker protein expression and glomerular hypertrophy in the diabetic kidney. These findings indicate a crucial role that ADPR-cyclase signaling plays in the renal pathogenesis of diabetes and provide a therapeutic tool for the treatment of renal diseases.

Local production of O2- by NAD(P)H oxidase in the sarcoplasmic reticulum of coronary arterial myocytes: cADPR-mediated Ca2+ regulation.

The present study was designed to determine whether the sarcoplasmic reticulum (SR) could locally produce superoxide (O2-) via NAD(P)H oxidase (NOX) in coronary arterial myocytes (CAMs) and to address whether cADPR-RyR/Ca2+ signaling pathway regulates this local O2- production from the SR. Using confocal microscopic imaging analysis in intact single CAMs, a cell-permeable indicator CM-H2DCFDA for dynamic changes in intracellular ROS (in green color) and a highly selective ER-Tracker Red dye for tracking of the SR were found co-localized. A quantitative analysis based on the intensity of different spectra demonstrated a local O2- production derived from the SR. M(1)-receptor agonist, oxotremorine (Oxo) and a Ca2+ ionophore, A23187, time-dependently increased this O2- production colocalized with the SR. NOX inhibitors, diphenylene iodonium (DPI) and apocynin (Apo), or superoxide dismutase (SOD) and catalase, and Nox4 (a major intracellular NOX subunit) siRNA all substantially blocked this local production of O2-, demonstrating an involvement of NOX. This SR-derived O2- production was also abolished by the inhibitors of cyclic ADP-ribose (cADPR)-mediated Ca2+ signaling, such as nicotinamide (Nicot, 6 mM), ryanodine (Rya, 50 muM) or 8-Br-cADPR (30 microM). However, IP3 antagonist, 2-APB (50 microM) had no effect. In CAMs transfected with siRNA of ADP-ribosyl cyclase or RyR, this SR O2- production was attenuated. Electron spin resonance (ESR) spectromic assay in purified SR also demonstrated the production of O2- that was dependent on NOX activity and Ca2+ concentrations. These results provide direct evidence that O2- could be locally produced via NOX on the SR and that this local O2- producing system is controlled by cADPR-RyR/Ca2+ signaling pathway.

Endothelin-A and -B receptors, superoxide, and Ca2+ signaling in afferent arterioles.

It is unknown if endothelin-A and -B receptors (ET(A)R and ET(B)R) activate the production of superoxide via NAD(P)H oxidase and subsequently stimulate the formation of cyclic adenine diphosphate ribose (cADPR) in afferent arterioles. Vessels were isolated from rat kidney and loaded with fura 2. Endothelin-1 (ET-1) rapidly increased cytosolic Ca(2+) concentration ([Ca(2+)](i)) by 303 nM. The superoxide dismutase mimetic tempol, the NAD(P)H oxidase inhibitor apocynin, and nicotinamide, an inhibitor of ADPR cyclase, diminished the response by approximately 60%. The ET(B)R agonist sarafotoxin 6c (S6c) increased peak [Ca(2+)](i) by 117 nM. Subsequent addition of ET-1 in the continued presence of S6c caused an additional [Ca(2+)](i) peak of 225 nM. Neither nicotinamide or 8-bromo- (8-Br) cADPR nor apocynin decreased the [Ca(2+)](i) response to S6c, but inhibited the subsequent [Ca(2+)](i) response to ET-1. The ET(B)R blockers BQ-788 and A-192621 prevented the S6c [Ca(2+)](i) peak and reduced the ET-1 response by more than one-half, suggesting an ET(B)R/ET(A)R interaction. In contrast, the ET(A)R blocker BQ-123 had no effect on the S6c [Ca(2+)](i) peak and obliterated the subsequent ET-1 response. ET-1 immediately stimulated superoxide formation (measured with TEMPO-9-AC, 68 arbitrary units) that was inhibited 95% by apocynin or diphenyl iodonium. S6c or IRL-1620 increased superoxide by 8% of that caused by subsequent ET-1 addition. We conclude that ET(A)R activation of afferent arterioles increases the formation of superoxide that accounts for approximately 60% of subsequent Ca(2+) signaling. ET(B)R activation appears to result in only minor increases in superoxide production. Nicotinamide and 8-Br-cADPR results suggest that ET-1 (and primarily ET(A)R) causes the activation of vascular smooth muscle cell-ADPR cyclase.

CD38 induces apoptosis of a murine pro-B leukemic cell line by a tyrosine kinase-dependent but ADP-ribosyl cyclase- and NAD glycohydrolase-independent mechanism.

Cross-linking of CD38 on hematopoietic cells induces activation, proliferation and differentiation of mature T and B cells and mediates apoptosis of myeloid and lymphoid progenitor cells. In addition to acting as a signaling receptor, CD38 is also an enzyme capable of producing several calcium-mobilizing metabolites, including cyclic adenosine diphosphate ribose (cADPR). It has been previously postulated that the calcium-mobilizing metabolites produced by CD38 may regulate its receptor-based activities. To test this hypothesis, we examined whether the enzyme activity of CD38 controls the apoptosis of an anti-CD38-stimulated leukemic B cell. We show that anti-CD38-induced apoptosis of Ba/F3 cells, a murine pro-B cell line, is not affected by blocking the calcium-mobilizing activity of cADPR or by inhibiting intracellular or extracellular calcium mobilization. In addition, we demonstrate that blocking CD38 enzyme activity with 2'-deoxy-2'-fluoro-nicotinamide arabinoside adenine dinucleotide has no effect on apoptosis and that Ba/F3 cells expressing catalytically inactive mutant forms of CD38 still undergo apoptosis upon CD38 cross-linking. Instead, we find that anti-CD38-induced apoptosis is dependent on tyrosine kinase and caspase activation, and that this process appears to be potentiated by the presence of membrane microdomains. Thus, the receptor-mediated functions of CD38 can be separated from its enzyme activity in a murine leukemic cell line, suggesting that CD38 plays multiple, but independent, biologic roles.

Boric acid inhibits adenosine diphosphate-ribosyl cyclase non-competitively.

Adenosine diphosphate-ribosyl cyclase (ADP-ribosyl cyclase) is a ubiquitous enzyme in eukaryotes that converts NAD+ to cyclic-ADP-ribose (cADPR) and nicotinamide. A quantitative assay for cADPR was developed using capillary electrophoresis to separate NAD+, cADPR, ADP-ribose, and ADP with UV detection (254 nm). Using this assay, the apparent Km and Vmax for Aplysia ADP-ribosyl cyclase were determined to be 1.24+/-0.05 mM and 131.8+/-2.0 microM/min, respectively. Boric acid inhibited ADP-ribosyl cyclase non-competitively with a Ki of 40.5+/-0.5 mM. Boric acid binding to cADPR, determined by electrospray ionization mass spectrometry, was characterized by an apparent binding constant, KA, of 655+/-99 L/mol at pH 10.3.

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+.

Hypoxia stimulates Ca2+ release from intracellular stores in astrocytes via cyclic ADP ribose-mediated activation of ryanodine receptors.

The ability of O(2) levels to regulate Ca(2+) signalling in non-excitable cells is poorly understood, yet crucial to our understanding of Ca(2+)-dependent cell functions in physiological and pathological situations. Here, we demonstrate that hypoxia mobilizes Ca(2+) from an intracellular pool in primary cultures of cortical astrocytes. This pool can also be mobilized by bradykinin, which acts via phospholipase C and inositol trisphosphate production. By contrast, hypoxic Ca(2+) mobilization utilizes ryanodine receptors, which appear to be either present on the same intracellular pool, or on a separate but functionally coupled pool. Hypoxic activation of ryanodine receptors requires formation of cyclic ADP ribose, since hypoxic Ca(2+) mobilization was fully prevented by nicotinamide (which inhibits ADP ribosyl cyclase) or by 8-Br-cADP ribose, an antagonist of cyclic ADP ribose. Our results demonstrate for the first time the involvement of cyclic ADP ribose in hypoxic modulation of Ca(2+) signalling in the central nervous system, and suggest that this modulator of ryanodine receptors may play a key role in the function of astrocytes under conditions of fluctuating O(2) levels.