A novel signaling pathway of ADP-ribosyl cyclase activation by angiotensin II in adult rat cardiomyocytes

ADP-ribosyl cyclase (ADPR-cyclase) produces a Ca(2+)-mobilizing second messenger, cADP-ribose (cADPR), from NAD(+). In this study, we investigated the molecular basis of ADPR-cyclase activation in the ANG II signaling pathway and cellular responses in adult rat cardiomyocytes. The results showed that ANG II generated biphasic intracellular Ca(2+) concentration increases that include a rapid transient Ca(2+) elevation via inositol trisphosphate (IP(3)) receptor and sustained Ca(2+) rise via the activation of L-type Ca(2+) channel and opening of ryanodine receptor. ANG II-induced sustained Ca(2+) rise was blocked by a cADPR antagonistic analog, 8-bromo-cADPR, indicating that sustained Ca(2+) rise is mediated by cADPR. Supporting the notion, ADPR-cyclase activity and cADPR production by ANG II were increased in a time-dependent manner. Application of pharmacological inhibitors and immunological analyses revealed that cADPR formation was activated by sequential activation of Src, phosphatidylinositol 3-kinase (PI 3-kinase)/protein kinase B (Akt), phospholipase C (PLC)-gamma1, and IP(3)-mediated Ca(2+) signal. Inhibitors of these signaling molecules not only completely abolished the ANG II-induced Ca(2+) signals but also inhibited cADPR formation. Application of the cADPR antagonist and inhibitors of upstream signaling molecules of ADPR-cyclase inhibited ANG II-stimulated hypertrophic responses, which include nuclear translocation of Ca(2+)/calcineurin-dependent nuclear factor of activated T cells 3, protein expression of transforming growth factor-beta1, and incorporation of [(3)H]leucine in cardiomyocytes. Taken together, these findings suggest that activation of ADPR-cyclase by ANG II entails a novel signaling pathway involving sequential activation of Src, PI 3-kinase/Akt, and PLC-gamma1/IP(3) and that the activation of ADPR-cyclase can lead to cardiac hypertrophy.     

Extracellular NAD(+) induces calcium signaling and apoptosis in human osteoblastic cells

ADP-ribosyl cyclase/CD38 is a bifunctional enzyme that catalyzes at its ectocellular domain the synthesis from NAD(+) (cyclase) and the hydrolysis (hydrolase) of the calcium-mobilizing second messenger cyclic ADP ribose (cADPR). Furthermore, CD38 mediates cADPR influx inside a number of cells, thereby inducing Ca(2+) mobilization. Intracellularly, cADPR releases Ca(2+) from ryanodine-sensitive pools, thus activating several Ca(2+)-dependent functions. Among these, the inhibition of osteoclastic-mediated bone resorption has been demonstrated. We found that HOBIT human osteoblastic cells display ADP-ribosyl cyclase activity and we examined the effects of CD38 stimulation on osteoblasts function. Extracellular NAD(+) induced elevation of cytosolic calcium due to both Ca(2+) influx from the extracellular medium and Ca(2+) release from ryanodine-sensitive intracellular stores. Culturing these cells in the presence of NAD(+) caused a complete growth arrest with a time-dependent decrease of cell number and the appearance of apoptotic nuclei. The first changes could be observed after 24 h of treatment and became fully evident after 72-96 h. We propose a role of extracellular NAD(+) in bone homeostatic control.     

Angiotensin II-induced hypertrophy is potentiated in mice overexpressing p22phox in vascular smooth muscle

Increased reactive oxygen species (ROS) are implicated in several vascular pathologies associated with vascular smooth muscle hypertrophy. In the current studies, we utilized transgenic (Tg) mice (Tg(p22smc)) that overexpress the p22(phox) subunit of NAD(P)H oxidase selectively in smooth muscle. These mice have a twofold increase in aortic p22(phox) expression and H(2)O(2) production and thus provide an excellent in vivo model in which to assess the effects of increased ROS generation on vascular smooth muscle cell (VSMC) function. We tested the hypothesis that overexpression of VSMC p22(phox) potentiates angiotensin II (ANG II)-induced vascular hypertrophy. Male Tg(p22smc) mice and negative littermate controls were infused with either ANG II or saline for 13 days. Baseline blood pressure was not different between control and Tg(p22smc) mice. ANG II significantly increased blood pressure in both groups, with this increase being slightly exacerbated in the Tg(p22smc) mice. Baseline aortic wall thickness and cross-sectional wall area were not different between control and Tg(p22smc) mice. Importantly, the ANG II-induced increase in both parameters was significantly greater in the Tg(p22smc) mice compared with control mice. To confirm that this potentiation of vascular hypertrophy was due to increased ROS levels, additional groups of mice were coinfused with ebselen. This treatment prevented the exacerbation of hypertrophy in Tg(p22smc) mice receiving ANG II. These data suggest that although increased availability of NAD(P)H oxidase-derived ROS is not a sufficient stimulus for hypertrophy, it does potentiate ANG II-induced vascular hypertrophy, making ROS an excellent target for intervention aimed at reducing medial thickening in vivo.     

ADP-ribosyl cyclase couples to cyclic AMP signaling in the cardiomyocytes

ADP-ribosyl cyclase (ADPR-cyclase) produces a Ca(2+)-mobilizing second messenger cyclic ADP-ribose (cADPR) from beta-NAD(+). In this study, we examined the molecular basis of which beta-adrenergic receptor (betaAR) stimulation induces cADPR formation and characterized cardiac ADPR-cyclase. The results revealed that isoproterenol-mediated increase of [Ca(2+)](i) in rat cardiomyocytes was blocked by pretreatment with a cADPR antagonistic derivative 8-Br-cADPR, a PKA inhibitor H89 or high concentration of ryanodine. Moreover, incubation of ventricular lysates with isoproterenol, forskolin or cAMP resulted in activation of ADPR-cyclase that was inhibited by pretreatment with H89. Supporting the observations, the cADPR antagonist and H89 blocked 8-CPT-cAMP, a cell-permeant cAMP analog-induced increase in [Ca(2+)](i) but not cGMP-mediated increase. Characterization of partially purified cardiac ADPR-cyclase showed a molecular mass of approximately 42 kDa and no cross-activity with CD38 antibodies, and the enzyme activity was inhibited by Zn(2+) but not dithiothreitol. Microinjection of the enzyme into rat cardiomyocytes increased the level of [Ca(2+)](i) in a concentration-dependent manner. The enzyme-mediated increase of [Ca(2+)](i) was blocked by the cADPR antagonist. These findings suggest that betaAR-mediated regulation of [Ca(2+)](i) in rat cardiomyocytes is primed by activation of cardiac ADPR-cyclase via cAMP/PKA signaling and that cardiac ADPR-cyclase differs from CD38 in biochemical and immunological properties.     

Cyclic ADP-ribose as a universal calcium signal molecule in the nervous system

beta-NAD(+) is as abundant as ATP in neuronal cells. beta-NAD(+) functions not only as a coenzyme but also as a substrate. beta-NAD(+)-utilizing enzymes are involved in signal transduction. We focus on ADP-ribosyl cyclase/CD38 which synthesizes cyclic ADP-ribose (cADPR), a universal Ca(2+) mobilizer from intracellular stores, from beta-NAD(+). cADPR acts through activation/modulation of ryanodine receptor Ca(2+) releasing Ca(2+) channels. cADPR synthesis in neuronal cells is stimulated or modulated via different pathways and various factors. Subtype-specific coupling of various neurotransmitter receptors with ADP-ribosyl cyclase confirms the involvement of the enzyme in signal transduction in neurons and glial cells. Moreover, cADPR/CD38 is critical in oxytocin release from the hypothalamic cell dendrites and nerve terminals in the posterior pituitary. Therefore, it is possible that pharmacological manipulation of intracellular cADPR levels through ADP-ribosyl cyclase activity or synthetic cADPR analogues may provide new therapeutic opportunities for treatment of neurodevelopmental disorders.     

Increase of intracellular Ca(2+) during ischemia/reperfusion injury of heart is mediated by cyclic ADP-ribose

While the molecular mechanisms by which oxidants cause cytotoxicity are still poorly understood, disruption of Ca(2+) homeostasis appears to be one of the critical alterations during the oxidant-induced cytotoxic process. Here, we examined the possibility that oxidative stress may alter the metabolism of cyclic ADP-ribose (cADPR), a potent Ca(2+)-mobilizing second messenger in the heart. Isolated heart perfused by Langendorff technique was subjected to ischemia/reperfusion injury and endogenous cADPR level was determined using a specific radioimmunoassay. Following ischemia/reperfusion injury, a significant increase in intracellular cADPR level was observed. The elevation of cADPR content was closely correlated with the increase in ADP-ribosyl cyclase activity. Inclusion of oxygen free radical scavengers, 2,2,6,6-tetramethyl-1-piperidinyloxy and mannitol, in the reperfusate prevented the ischemia/reperfusion-induced increases in cADPR level and the ADP-ribosyl cyclase activity. Exposure of isolated cardiomyocytes to t-butyl hydroperoxide increased the ADP-ribosyl cyclase activity, cADPR level, and intracellular Ca(2+) concentration ([Ca(2+)](i)) and consequently resulting in cell lethal damage. The oxidant-induced elevation of [Ca(2+)](i) as well as cell lethal damage was blocked by a cADPR antagonist, 8-bromo-cADPR. These results provide evidence for involvement of cADPR and its producing enzyme in alteration of Ca(2+) homeostasis during the ischemia/reperfusion injury of the heart.     

Cyclic ADP-ribose as a potential second messenger for neuronal Ca2+ signaling

Cyclic ADP-ribose (cADPR), a known endogenous modulator of ryanodine receptor Ca2+ releasing channels, is found in the nervous system. Injection of cADPR into neuronal cells primarily induces a transient elevation of intracellular Ca2+ concentration ([Ca2+]i), and/or secondarily potentiates [Ca2+]i increases that are the result of depolarization-induced Ca2+ influx. Acetylcholine release from cholinergic neurons is facilitated by cADPR. cADPR modifies K+ currents or elicits Ca2+-dependent inward currents. cADPR is synthesized by both membrane-bound and cytosolic forms of ADP-ribosyl cyclase in neuronal cells. cADPR hydrolase activity is weak in the membrane fraction, but high in the cytoplasm. Cytosolic ADP-ribosyl cyclase activity is upregulated by nitric oxide/cyclic GMP-dependent phosphorylation. Stimulation of muscarinic and beta-adrenergic receptors activates membrane-bound ADP-ribosyl cyclase via G proteins within membranes of neuronal tumor cells and cortical astrocytes. These findings strongly suggest that cADPR is a second messenger in Ca2+ signaling in the nervous system, although many intriguing issues remain to be addressed before this identity is confirmed.     

Connexin-43 hemichannels mediate cyclic ADP-ribose generation and its Ca2+-mobilizing activity by NAD+/cyclic ADP-ribose transport

The ADP-ribosyl cyclase CD38 whose catalytic domain resides in outside of the cell surface produces the second messenger cyclic ADP-ribose (cADPR) from NAD(+). cADPR increases intracellular Ca(2+) through the intracellular ryanodine receptor/Ca(2+) release channel (RyR). It has been known that intracellular NAD(+) approaches ecto-CD38 via its export by connexin (Cx43) hemichannels, a component of gap junctions. However, it is unclear how cADPR extracellularly generated by ecto-CD38 approaches intracellular RyR although CD38 itself or nucleoside transporter has been proposed to import cADPR. Moreover, it has been unknown what physiological stimulation can trigger Cx43-mediated export of NAD(+). Here we demonstrate that Cx43 hemichannels, but not CD38, import cADPR to increase intracellular calcium through RyR. We also demonstrate that physiological stimulation such as Fcγ receptor (FcγR) ligation induces calcium mobilization through three sequential steps, Cx43-mediated NAD(+) export, CD38-mediated generation of cADPR and Cx43-mediated cADPR import in J774 cells. Protein kinase A (PKA) activation also induced calcium mobilization in the same way as FcγR stimulation. FcγR stimulation-induced calcium mobilization was blocked by PKA inhibition, indicating that PKA is a linker between FcγR stimulation and NAD(+)/cADPR transport. Cx43 knockdown blocked extracellular cADPR import and extracellular cADPR-induced calcium mobilization in J774 cells. Cx43 overexpression in Cx43-negative cells conferred extracellular cADPR-induced calcium mobilization by the mediation of cADPR import. Our data suggest that Cx43 has a dual function exporting NAD(+) and importing cADPR into the cell to activate intracellular calcium mobilization.     

NADP+-Dependent internalization of recombinant CD38 in CHO cells

CD38 is a 46-kDa type II transmembrane glycoprotein that catalyses the synthesis of cyclic ADP-ribose (cADPR) from NAD+. cADPR is a second messenger known to regulate intracellular Ca2+-induced Ca2+-release (CICR). A recent study has revealed that CD38 in Namalwa B cells undergoes internalization upon exposure to external NAD+. In this study, recombinant rat CD38 was expressed in Chinese hamster ovary (CHO) cells and the possibility of the protein to undergo internalization upon exposure to a substrate analog NADP+ was examined. It was found that such treatment of CHO cells resulted in a decrease of ADP-ribosyl cyclase activity, as well as immunofluorescence of CD38 on the cell surface. The same treatment of CHO cells also resulted in intracellular clustering of CD38 molecules as revealed by confocal microscopic analysis. The internalized CD38 was purified using a streptavidin/biotin-based method and was found to exhibit both ADP-ribosyl cyclase and cADPR hydrolase activities. On immunoblot, the internalized CD38 appeared as a monomer of 46 kDa under reducing condition of SDS-PAGE. Our data demonstrate that NADP+ can efficiently induce internalization of CD38, a process that may be important in the production of cADPR intracellularly to regulate CICR.     

Sex differences in angiotensin signaling in bulbospinal neurons in the rat rostral ventrolateral medulla

Sex differences may play a significant role in determining the risk of hypertension. Bulbospinal neurons in the rostral ventrolateral medulla (RVLM) are involved in the tonic regulation of arterial pressure and participate in the central mechanisms of hypertension. Angiotensin II (ANG II) acting on angiotensin type 1 (AT(1)) receptors in RVLM neurons is implicated in the development of hypertension by activating NADPH oxidase and producing reactive oxygen species (ROS). Therefore, we analyzed RVLM bulbospinal neurons to determine whether there are sex differences in: 1) immunolabeling for AT(1) receptors and the key NADPH oxidase subunit p47 using dual-label immunoelectron microscopy, and 2) the effects of ANG II on ROS production and Ca(2+) currents using, respectively, hydroethidine fluoromicrography and patch-clamping. In tyrosine hydroxylase-positive RVLM neurons, female rats displayed significantly more AT(1) receptor immunoreactivity and less p47 immunoreactivity than male rats (P < 0.05). Although ANG II (100 nM) induced comparable ROS production in dissociated RVLM bulbospinal neurons of female and male rats (P > 0.05), an effect mediated by AT(1) receptors and NADPH oxidase, it triggered significantly larger dihydropyridine-sensitive long-lasting (L-type) Ca(2+) currents in female RVLM neurons (P < 0.05). These observations suggest that an increase in AT(1) receptors in female RVLM neurons is counterbalanced by a reduction in p47 levels, such that ANG II-induced ROS production does not differ between females and males. Since the Ca(2+) current activator Bay K 8644 induced larger Ca(2+) currents in females than in male RVLM neurons, increased ANG II-induced L-type Ca(2+) currents in females may result from sex differences in calcium channel densities or dynamics.     

Assay for ADP-ribosyl cyclase by reverse-phase high-performance liquid chromatography.

Cyclic ADP-ribose (cADPR), a natural metabolite of beta-NAD(+), is a second messenger for Ca(2+) signaling in T cells. As a tool for purification and identification of ADP-ribosyl cyclase(s) in T cells, a sensitive and specific enzymatic assay using 1,N(6)-etheno-NAD(+) as substrate was developed. A major problem-the sensitivity of 1,N(6)-etheno-cADPR toward the extraction medium perchloric acid-was solved by replacing the perchloric acid extraction procedure of nucleotides by a filtration step. Standard compounds for the HPLC analysis of ADP-ribosyl cyclases and NAD(+)-glycohydrolases, e.g., 1,N(6)-etheno-cADPR, 1,N(6)-etheno-ADPR, and 1,N(6)-etheno-AMP, were produced by ADP-ribosyl cyclase from Aplysia californica and dinucleotide pyrophosphatase. The assay was applied to subcellular fractions prepared from human Jurkat T cells. As a result ADP-ribosyl cyclase and NAD(+)-glycohydrolase activity could be detected and precisely quantified in different subcellular fractions indicating the presence of different isoenzymes in T cells.     

Angiotensin II induces MMP-2 in a p47phox-dependent manner

Activated matrix metalloproteinases (MMPs) in patients with acute coronary syndromes may contribute to plaque destabilization. Since reactive oxygen species (ROS) induce MMP-2 and angiotensin II (ANG II) enhances NADPH-oxidase-dependent ROS formation, we assessed whether ANG II induces MMP-2 in a NADPH-oxidase-dependent manner. MMP-2 mRNA expression and activity were analyzed in wildtype and p47phox-deficient (p47phox-/-) murine smooth muscle cells (SMC). To address a clinical implication, sections of human atherosclerotic arteries were stained for MMP-2, p47phox, ANG II, AT1-receptor, and alpha-smooth muscle cell actin (alpha-SMC actin). MMP-2 protein expression and activity from these arteries were compared to those without atherosclerosis. ANG II enhances mRNA synthesis and activity of MMP-2 in a p47phox-dependent manner. Immunohistochemical analyses revealed a co-localization of MMP-2 with p47phox, ANG II, AT1-receptor, and alpha-SMC actin. MMP-2 protein expression and gelatinolytic activity are increased in atherosclerotic arteries. Thus, activation of the renin-angiotensin system may contribute to plaque destabilization via ROS-dependent induction of MMP-2.     

Angiotensin II-induced Akt activation is mediated by metabolites of arachidonic acid generated by CaMKII-stimulated Ca2(+)-dependent phospholipase A2

Angiotensin II (ANG II) promotes vascular smooth muscle cell (VSMC) growth, stimulates Ca(2+)-calmodulin (CaM)-dependent kinase II (CaMKII), and activates cytosolic Ca(2+)-dependent phospholipase A2 (cPLA2), which releases arachidonic acid (AA). ANG II also generates H2O2 and activates Akt, which have been implicated in ANG II actions in VSMC. This study was conducted to investigate the relationship of these signaling molecules to Akt activation in rat aortic VSMC. ANG II increased Akt activity, as measured by its phosphorylation at serine-473. ANG II (200 nM)-induced Akt phosphorylation was decreased by extracellular Ca2+ depletion and calcium chelator EGTA and inhibitors of CaM [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide] and CaMKII [(2-[N-(2-hydroxyethyl)]-N-(4-me-thoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzyl-amine)]. cPLA2 inhibitor pyrrolidine-1, antisense oligonucleotide, and retroviral small interfering RNA also attenuated ANG II-induced Akt phosphorylation. AA increased Akt phosphorylation, and AA metabolism inhibitor 5,8,11,14-eicosatetraynoic acid (ETYA) blocked ANG II- and AA-induced Akt phosphorylation (199.03 +/- 27.91% with ANG II and 110.18 +/- 22.40% with ETYA + ANG II; 405.00 +/- 86.22% with AA and 153.97 +/- 63.26% with ETYA + AA). Inhibitors of lipoxygenase (cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate) and cytochrome P-450 (ketoconazole and 17-octadecynoic acid), but not cyclooxygenase (indomethacin), attenuated ANG II- and AA-induced Akt phosphorylation. Furthermore, 5(S)-, 12(S)-, 15(S)-, and 20-hydroxyeicosatetraenoic acids and 5,6-, 11,12-, and 14,15-epoxyeicosatrienoic acids increased Akt phosphorylation. Catalase inhibited ANG II-increased H2O2 production but not Akt phosphorylation. Oleic acid, which also increased H2O2 production, did not cause Akt phosphorylation. These data suggest that ANG II-induced Akt activation in VSMC is mediated by AA metabolites, most likely generated via lipoxygenase and cytochrome P-450 consequent to AA released by CaMKII-activated cPLA2 and independent of H2O2 production.     

A unified mechanism of enzymatic synthesis of two calcium messengers: cyclic ADP-ribose and NAADP.

Cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) mobilize Ca2+ from two different types of intracellular stores and through completely independent mechanisms. The two Ca2+ messengers are also structurally distinct. cADPR is a cyclic nucleotide derived from NAD, while NAADP is a linear metabolite of NADP. Systems responsive to these two novel signaling molecules are widespread among eukaryotes and include protozoan, plant, invertebrate, mammalian as well as human cells. Despite their functional and structural differences, cADPR and NAADP are sibling messengers synthesized by a single enzyme, ADP-ribosyl cyclase. In this article the recent progress in understanding the physiological roles of cADPR and NAADP is briefly reviewed. A unified mechanism of catalysis is also proposed, which takes into consideration the crystallographic structure of ADP-ribosyl cyclase and accounts for its novel multi-functionality.     

The CD38-cyclic ADP-ribose signaling system in insulin secretion

Glucose induces an increase in the intracellular Ca2+ concentration in pancreatic -cells to secrete insulin. CD38 occurs in -cells and has both ADP-ribosyl cyclase, which catalyzes the formation of cyclic ADP-ribose (cADPR) from NAD+, and cADPR hydrolase, which converts cADPR to ADP-ribose. ATP, produced by glucose metabolism, competes with cADPR for the binding site, Lys-129, of CD38, resulting in the inhibition of the hydrolysis of cADPR and thereby causing cADPR accumulation in -cells. Cyclic ADP-ribose then binds to FK506-binding protein 12.6 in the ryanodine receptor Ca2+ channel (RyR), dissociating the binding protein from RyR to induce the release of Ca2+ from the endoplasmic reticulum. Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) phosphorylates RyR to sensitize and activate the Ca2+ channel. Ca2+, released from the RyR, further activates CaM kinase II and amplifies the process. Thus, cADPR acts as a second messenger for Ca2+ mobilization to secrete insulin. The novel mechanism of insulin secretion described above is different from the conventional hypothesis in which Ca2+ influx from extracellular sources plays a role in insulin secretion by glucose.     

Angiotensin II-induced negative inotropy in rat ventricular myocytes: role of reactive oxygen species and p38 MAPK

The octapeptide angiotensin II (ANG II) can modulate cardiac contractility and is increased in heart failure, where contractile function is impaired. In rat cardiac myocytes, 1 microM of ANG II produces a negative inotropic effect (NIE) (24.6 +/- 5% reduction). However, the subcellular signaling involved in this effect remains elusive. We examined the mechanisms and signaling events involved in the reduction in contractile function induced by the peptide in indo-1-loaded rat cardiomyocytes. The results showed that the NIE of ANG II was not associated with a parallel decrease in the intracellular Ca2+ transient, indicating that a decrease in myofilament responsiveness to Ca2+ underlies the reduction in contractility. We assessed the role of PKC, tyrosine kinases, reactive oxygen species (ROS), and mitogen-activated protein kinases (MAPKs) in the NIE of the peptide. Pretreatment of cells with the NAD(P)H oxidase inhibitor diphenyleneiodonium chloride or with the superoxide scavenger 4,5-dihydroxy-1,3-benzene-disulfonic acid did not affect the ANG II-induced NIE. Moreover, ANG II-induced ROS production, after 20 min of incubation with the peptide, could not be detected with the use of either the fluorophore 5-(6)-chloromethyl-2',7'-dichlorodihydrofluorecein diacetate or lucigenin-enhanced chemiluminescence. In contrast, the ANG II-induced NIE was abrogated by the inhibitors of PKC (calphostin C), tyrosine kinase (genistein), and p38 MAPK (SB-202190). Furthermore, the NIE was significantly exacerbated (60 +/- 10% reduction) by p38 MAPK overexpression. These results exclude the participation of ROS in the NIE of the peptide and point to PKC and tyrosine kinase as upstream mediators. Furthermore, they reveal p38 MAPK as the putative effector of the reduction in myofilament responsiveness to Ca2+ and the decrease in contractility induced by the peptide.     

Enhanced angiotensin II-mediated central sympathoexcitation in streptozotocin-induced diabetes: role of superoxide anion

Studies have shown that the superoxide mechanism is involved in angiotensin II (ANG II) signaling in the central nervous system. We hypothesized that ANG II activates sympathetic outflow by stimulation of superoxide anion in the paraventricular nucleus (PVN) of streptozotocin (STZ)-induced diabetic rats. In α-chloralose- and urethane-anesthetized rats, microinjection of ANG II into the PVN (50, 100, and 200 pmol) produced dose-dependent increases in renal sympathetic nerve activity (RSNA), arterial pressure (AP), and heart rate (HR) in control and STZ-induced diabetic rats. There was a potentiation of the increase in RSNA (35.0 ± 5.0 vs. 23.0 ± 4.3%, P < 0.05), AP, and HR due to ANG II type I (AT(1)) receptor activation in diabetic rats compared with control rats. Blocking endogenous AT(1) receptors within the PVN with AT(1) receptor antagonist losartan produced significantly greater decreases in RSNA, AP, and HR in diabetic rats compared with control rats. Concomitantly, there were significant increases in mRNA and protein expression of AT(1) receptor with increased superoxide levels and expression of NAD(P)H oxidase subunits p22(phox), p47(phox), and p67(phox) in the PVN of rats with diabetes. Pretreatment with losartan (10 mg·kg(-1)·day(-1) in drinking water for 3 wk) significantly reduced protein expression of NAD(P)H oxidase subunits (p22(phox) and p47(phox)) in the PVN of diabetic rats. Pretreatment with adenoviral vector-mediated overexpression of human cytoplasmic superoxide dismutase (AdCuZnSOD) within the PVN attenuated the increased central responses to ANG II in diabetes (RSNA: 20.4 ± 0.7 vs. 27.7 ± 2.1%, n = 6, P < 0.05). These data support the concept that superoxide anion contributes to an enhanced ANG II-mediated signaling in the PVN involved with the exaggerated sympathoexcitation in diabetes.     

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.     

Nitric oxide inhibits ADP-ribosyl cyclase through a cGMP-independent pathway in airway smooth muscle

There is evidence for a role of cyclic ADP-ribose (cADPR) in intracellular Ca2+ regulation in smooth muscle. cADPR is synthesized and degraded by ADP-ribosyl cyclase and cADPR hydrolase, respectively, by a bifunctional protein, CD38. Nitric oxide (NO) inhibits intracellular Ca2+ mobilization in airway smooth muscle. The present study was designed to determine whether this inhibition is due to regulation of ADP-ribosyl cyclase and/or cADPR hydrolase activity. Sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine, NO donors, produced a concentration-dependent decrease in ADP-ribosyl cyclase, but not cADPR hydrolase, activity. The NO scavenger carboxy-PTIO prevented and reversed, and reduced glutathione prevented, the inhibition of ADP-ribosyl cyclase by SNP, suggesting S-nitrosylation by NO as a mechanism. N-ethylmaleimide, which covalently modifies protein sulfhydryl groups, making them incapable of nitrosylation, produced a marked inhibition of ADP-ribosyl cyclase, but not cADPR hydrolase, activity. SNP and N-ethylmaleimide significantly inhibited the ADP-ribosyl cyclase activity in recombinant human CD38 without affecting the cADPR hydrolase activity. These results provide a novel mechanism for differential regulation of CD38 by NO through a cGMP-independent pathway involving S-nitrosylation of thiols.     

Role of cyclic ADP-ribose-Ca2+ signaling in mediating renin production and release in As4.1 cells.

The present study was designed to test the hypothesis that cyclic-ADP-ribose (cADPR) serves as a novel second messenger to mediate intracellular Ca(2+) concentration in As4.1 cells, a prototype of renal juxtaglomerular cells, and thereby regulates the renin production and release. Western blot analysis showed that CD38, an enzyme responsible for the production of cADPR, was abundant in As4.1 cells. Using cADPR cycling assay, it was found that NaCl stimulated cADPR production in these cells, which was blocked by inhibition of ADP-ribosyl cyclase with nicotinamide. HPLC analysis showed that the conversion rate of beta-NGD into cGDPR was dramatically increased by NaCl, which was attenuated by nicotinamide. Using fluorescent microscopic imaging analysis, NaCl (100 mM) was demonstrated to stimulate a rapid Ca(2+) increase from the endoplasmic reticulum (ER), which was inhibited by a cADPR antagonist, 8-bromo-cADPR (30 microM), an inhibitor of ADP-ribosyl cyclase, nicotinamide (6 mM), the ryanodine receptors blocker, ryanodine (30 microM), or a Ca(2+)-induced Ca(2+) release inhibitor, tetracaine (10 microM) by 70-90%. Finally, NaCl was found to significantly lower the renin production and release levels in As4.1 cells, which was accompanied by decreases in renin mRNA levels. Pretreatment of these cells with various inhibitors or blockers above significantly blocked the inhibitory effect of NaCl on renin production and release. These results indicate that cADPR-mediated Ca(2+) signaling pathway is present in As4.1 cells and that this signaling pathway may play a contributing role in the regulation of renin production and release.