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The contractility of airway smooth muscle cells is dependent on dynamic changes in the concentration of intracellular calcium. Signaling molecules such as inositol 1,4,5-trisphosphate and cyclic ADP-ribose play pivotal roles in the control of intracellular calcium concentration. Alterations in the processes involved in the regulation of intracellular calcium concentration contribute to the pathogenesis of airway diseases such as asthma. Recent studies have identified cyclic ADP-ribose as a calcium-mobilizing second messenger in airway smooth muscle cells, and modulation of the pathway involved in its metabolism results in altered calcium homeostasis and may contribute to airway hyperresponsiveness. In this review, we describe the basic mechanisms underlying the dynamics of calcium regulation and the role of CD38/cADPR, a novel pathway, in the context of airway smooth muscle function and its contribution to airway diseases such as asthma.

Cyclic ADP-ribose (cADPR) mobilizes calcium from intracellular stores and contributes to agonist-induced intracellular calcium elevation in airway smooth muscle (ASM). In this study we determined the functional role of CD38/cADPR signaling in the regulation of airway tone using CD38 deficient (cd38(-/-)) mice. The responsiveness to different doses of methacholine, as determined by changes in lung resistance and dynamic compliance, was significantly (P < or = 0.05) lower in cd38(-/-) mice compared with wild-type controls. To determine the mechanism responsible for the reduced responsiveness, we measured the intracellular calcium responses to contractile agonists in ASM cells. In ASM cells isolated from cd38(-/-) mice, the intracellular calcium responses to acetylcholine and endothelin-1 were significantly lower than in controls. Pretreatment of ASM cells with a cADPR antagonist resulted in attenuated intracellular calcium responses to endothelin-1 in cells isolated from wild-type mice, but not in those isolated from the cd38(-/-) mice. Very low cADPR levels and no detectable ADP-ribosyl cyclase activity were observed in lung tissue from cd38(-/-) mice, suggesting that CD38 is a critical source for cADPR synthesis. The results of the present study demonstrate that CD38/cADPR contributes to airway smooth muscle tone and responsiveness through its effects on agonist-induced elevation of intracellular calcium in ASM cells.

Recent evidence suggests that CD38, an ectoenzyme that converts NAD(+) to cyclic ADP-ribose (cADPr), may play a role in cytokine-induced airway smooth muscle (ASM) cell hyper-responsiveness, a key feature associated with chronic asthma. In the present study, we investigated the major signaling pathways by which tumor necrosis factor-alpha (TNFalpha) induces CD38 expression and its role in regulating gene expression in human ASM cells. Using flow cytometry analyses, TNFalpha enhanced CD38 expression in a manner that was time-(0-24 h), concentration-(0.1-40 ng/ml), and protein synthesis-(cycloheximide blockade) dependent. A selective agonistic antibody against tumor necrosis factor receptor (TNFR) 1 also augmented CD38 expression, whereas anti-TNFR2 antagonistic antibody did not prevent the TNFalpha response. Inhibition of the Janus activated kinase/signal transducer and activator of transcription pathways using the soluble inhibitor 2-(1,1-dimethylethyl)-9-fluoro-3,6-dihydro-7H-benz-[h]imidaz[4,5-f]isoquinolin-7-one (DBI) or with neutralizing antibody against interferon beta (IFNbeta) completely abrogated TNFalpha-induced CD38 expression at both protein and mRNA levels. Combining TNFalpha (0.1 and 1 ng/ml) and IFNbeta (100 IU/ml) at concentrations alone that had little effect on CD38 expression induced a robust synergistic induction of CD38 mRNA and protein levels. 8-Bromo-cADPr, a cADPr antagonist, significantly augmented TNFalpha-induced interleukin-6 secretion, whereas regulated on activation normal T cell expressed and secreted secretion was suppressed. 8-Bromo-cADPr, however, did not affect TNFalpha-induced cell surface expression of intercellular adhesion molecule-1. Our study is the first to demonstrate that IFNbeta-dependent activation of CD38 pathway is a novel component by which TNFalpha differentially regulates the expression of inflammatory genes in ASM cells.

Cyclic ADP-ribose (cADPR), synthesized by CD38, regulates intracellular calcium in uterine smooth muscle. CD38 is a transmembrane protein that has both ADP-ribosyl cyclase and cADPR hydrolase enzyme activities involved in cADPR metabolism. CD38 expression and its enzyme activities in uterine smooth muscle are regulated by estrogen. In the present study, we examined CD38 expression, its enzyme activities, and cADPR levels in myometrium obtained from rats at 14-17 days of gestation (preterm) and at parturition (term). CD38 expression, ADP-ribosyl cyclase activity, and cADPR levels were higher in uterine tissues obtained from term rats compared with that of preterm rats, while activity of cADPR hydrolase did not significantly change. In an effort to address whether changes in estrogen: progesterone ratio that occur during pregnancy account for the observed effects on CD38 expression and function, we determined the effect of different doses of progesterone in the presence of estrogen on CD38 expression and its enzyme activities in uterine smooth muscle obtained from ovariectomized rats. In myometrium obtained from ovariectomized rats, estrogen administration caused increased CD38 protein expression and ADP-ribosyl cyclase activity. The estrogen-induced increases in CD38 expression and ADP-ribosyl cyclase activity were inhibited by simultaneous administration of 10 or 20 mg of progesterone. These results indicate that the estrogen:progesterone ratio determines CD38 expression and ADP-ribosyl cyclase activity. These changes in CD38/cADPR pathway may contribute to increased uterine motility and onset of labor.

CD38/cyclic adenosine diphosphate ribose (cADPR) signaling plays an important role in the regulation of intracellular calcium responses to agonists in a variety of cells, including airway smooth muscle (ASM) cells. The present study was aimed at determining the effect of interleukin (IL)-13, a cytokine implicated in the pathogenesis of asthma, on CD38/cADPR signaling and to ascertain the contribution of CD38/cADPR signaling to IL-13-induced airway hyperresponsiveness. Human ASM cells maintained in culture were exposed to 50 ng/ml IL-13 for 22 h and levels of CD38 expression and intracellular calcium responses to agonists were measured. Treatment of human ASM cells with IL-13 resulted in increased CD38 expression as determined by real-time polymerase chain reaction, Western blot analysis, and indirect immunofluorescence. Increased CD38 expression was reflected as increased ADP-ribosyl cyclase activity in the ASM cell membranes. The net intracellular calcium responses to bradykinin, thrombin, and histamine were significantly (P < or = 0.05) higher in cells treated with IL-13 compared with controls. Furthermore, 8-bromo-cADPR, a cADPR antagonist, attenuated IL-13-induced augmented intracellular calcium responses to agonists in human ASM cells. These findings indicate that the CD38/cADPR-dependent pathway has a major role in IL-13-induced modulation of calcium signaling in human ASM.

We previously demonstrated that cyclic ADP-ribose (cADPR) elicits Ca2+ release in airway smooth muscle (ASM) cells through ryanodine receptor channels. CD38 is a cell surface protein that catalyzes the synthesis and degradation of cADPR. In inflammatory diseases such as asthma, augmented Ca2+ responses and Ca2+ sensitivity contribute to increased ASM contractility in response to agonists. In this study, we investigated the regulation of CD38 expression and the role of cADPR-mediated Ca2+ release in airway inflammation. Human ASM cells in culture between the second and fifth passages were exposed to tumor necrosis factor alpha (TNF-alpha), interleukin 1beta, or interferon gamma, or bovine serum albumin (controls). CD38 expression was measured by reverse transcriptase-polymerase chain reaction (RT-PCR), real-time PCR, and Western blot analysis, and ADP-ribosyl cyclase activity was assayed with nicotinamide guanine dinucleotide as the substrate. Ca2+ responses to acetylcholine, bradykinin, and thrombin were measured in fura-2AM-loaded cells by fluorescence microscopy. Cytokines caused significant augmentation of CD38 expression, ADP-ribosyl cyclase activity, and Ca2+ responses to the agonists, compared with the control. TNF-alpha effects were greater than those of the other two cytokines. The cADPR antagonist 8-bromo-cADPR attenuated the Ca2+ responses to the agonists in control and cytokine-treated cells, with the magnitude of inhibition correlating with the level of CD38. This study provides the first demonstration of a role for CD38-cADPR signaling in a model of inflammatory airway disease.

Hormones influence uterine contractility through their effects on intracellular calcium. The regulation of intracellular calcium in uterine smooth muscle is achieved by several mechanisms and includes mobilization from intracellular stores by inositol 1,4,5-trisphosphate and ryanodine-sensitive channels. Cyclic ADP-ribose (cADPR), a metabolite of NAD(+), is known to mediate calcium release through ryanodine receptor channels. A cell surface glycoprotein, CD38, catalyzes the synthesis and breakdown of cADPR and thus possesses bifunctional enzymatic activity. The regulation of cADPR synthesis by ADP-ribosyl cyclase (cyclase) or degradation by cADP-ribose hydrolase (hydrolase) by hormones in the myometrium is poorly understood. We investigated the effects of estradiol-17 beta on CD38 expression and the synthesis and degradation of cADPR in myometrial smooth muscle obtained from ovariectomized rats. CD38 expression was studied by reverse transcription polymerase chain reaction and Western blot analyses. In uterine microsomal fractions, cyclase and hydrolase activities were measured using nicotinamide guanine dinucleotide and [(32)P]cADPR as substrates, respectively. Microsomal proteins subfractionated by SDS-PAGE and gel filtration were used to determine the fractions containing cyclase and hydrolase activities. The results demonstrate that cyclase and hydrolase activities are associated with a single protein fraction, similar to CD38 in uteri from both ovariectomized and estradiol-treated rats, and estradiol-17 beta causes 1) increased CD38 mRNA and protein expression and 2) significantly enhanced cyclase but not hydrolase activity. The differential regulation of CD38 by estradiol-17 beta, resulting in increased cADPR synthesis, would have profound effects on calcium regulation and myometrial contractility.

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.

ADP-ribosyl cyclase synthesizes two Ca(2+) messengers by cyclizing NAD to produce cyclic ADP-ribose and exchanging nicotinic acid with the nicotinamide group of NADP to produce nicotinic acid adenine dinucleotide phosphate. Recombinant Aplysia cyclase was expressed in yeast and co-crystallized with a substrate, nicotinamide. x-ray crystallography showed that the nicotinamide was bound in a pocket formed in part by a conserved segment and was near the central cleft of the cyclase. Glu(98), Asn(107) and Trp(140) were within 3.5 A of the bound nicotinamide and appeared to coordinate it. Substituting Glu(98) with either Gln, Gly, Leu, or Asn reduced the cyclase activity by 16-222-fold, depending on the substitution. The mutant N107G exhibited only a 2-fold decrease in activity, while the activity of W140G was essentially eliminated. The base exchange activity of all mutants followed a similar pattern of reduction, suggesting that both reactions occur at the same active site. In addition to NAD, the wild-type cyclase also cyclizes nicotinamide guanine dinucleotide to cyclic GDP-ribose. All mutant enzymes had at least half of the GDP-ribosyl cyclase activity of the wild type, some even 2-3-fold higher, indicating that the three coordinating amino acids are responsible for positioning of the substrate but not absolutely critical for catalysis. To search for the catalytic residues, other amino acids in the binding pocket were mutagenized. E179G was totally devoid of GDP-ribosyl cyclase activity, and both its ADP-ribosyl cyclase and the base exchange activities were reduced by 10,000- and 18,000-fold, respectively. Substituting Glu(179) with either Asn, Leu, Asp, or Gln produced similar inactive enzymes, and so was the conversion of Trp(77) to Gly. However, both E179G and the double mutant E179G/W77G retained NAD-binding ability as shown by photoaffinity labeling with [(32)P]8-azido-NAD. These results indicate that both Glu(179) and Trp(77) are crucial for catalysis and that Glu(179) may indeed be the catalytic residue.

Mobilization of internal Ca+2 is an important signaling mechanism in cells. In addition to the inositol trisphosphate pathway, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide (NAADP) have been shown to mobilize Ca+2 via independent mechanisms. Although the structures of cADPR and NAADP are totally distinct, both nucleotides can be synthesized by ADP-ribosyl cyclase or CD38, a lymphocyte antigen. Both enzymes cyclize NAD to cADPR. In the presence of nicotinic acid the two enzymes catalyze a base exchange reaction resulting in the synthesis of NAADP from NADP. The switch between these two modes of catalysis is regulated by pH. Furthermore, both enzymes can also cyclize nicotinamide guanine dinucleotide (NGD) to produce a fluorescent product, cyclic GDP-ribose (cGDPR), which has a site of cyclization different from cADPR. A model is proposed to account for the multi-functionality of these enzymes. In order to be able to verify the model, a soluble ADP-ribosyl cyclase has been crystallized and X-ray diffraction shows that it is a dimer. Solution of the crystal structure of the cyclase should provide valuable insight into the structural features necessary for its multiple catalytic functions.

Cyclic adenosine diphosphate ribose (cADPR) is a potent endogenous calcium-mobilizing agent synthesized from beta-NAD+ by ADP-ribosyl cyclases in sea urchin eggs and in several mammalian cells (Galione, A., and White, A. (1994) Trends Cell Biol. 4, 431 436). Pharmacological studies suggest that cADPR is an endogenous modulator of Ca2+-induced Ca2+ release mediated by ryanodine-sensitive Ca2+ release channels. An unresolved question is whether cADPR can act as a Ca2+-mobilizing intracellular messenger. We show that exogenous application of nitric oxide (NO) mobilizes Ca2+ from intracellular stores in intact sea urchin eggs and that it releases Ca2+ and elevates cADPR levels in egg homogenates. 8-Amino-cADPR, a selective competitive antagonist of cADPR-mediated Ca2+ release, and nicotinamide, an inhibitor of ADP-ribosyl cyclase, inhibit the Ca2+-mobilizing actions of NO, while, heparin, a competitive antagonist of the inositol 1,4,5-trisphosphate receptor, did not affect NO-induced Ca2+ release. Since the Ca2+-mobilizing effects of NO can be mimicked by cGMP, are inhibited by the cGMP-dependent-protein kinase inhibitor, Rp-8-pCPT-cGMPS, and in egg homogenates show a requirement for the guanylyl cyclase substrate, GTP, we suggest a novel action of NO in mobilizing intracellular calcium from microsomal stores via a signaling pathway involving cGMP and cADPR. These results suggest that cADPR has the capacity to act as a Ca2+-mobilizing intracellular messenger.

Cyclic ADP-ribose (cADPR) is a Ca(2+)-mobilizing cyclic nucleotide derived from NAD+. Accumulating evidence indicates that it is an endogenous modulator of the Ca(2+)-induced Ca2+ release mechanism in cells. In this study, we show that ADP-ribosyl cyclase catalyzes the cyclization of not only NAD+ but also several of its analogs with various purine bases (guanine, hypoxanthine, or xanthine) substituting for adenine. Unlike cADPR, the resulting cyclic products are fluorescent. Comparisons with various model compounds indicate that only 7-methyl substituted purine nucleosides and nucleotides are fluorescent, and the pH-dependence of their UV spectra is most similar to that of the fluorescent cADPR analogs, indicating that the site of cyclization of these analogs is at the N7-position of the purine ring. This finding is novel since the site of cyclization is at the N1-position for cADPR as determined by X-ray crystallography. That a single enzyme can cyclize a variety of substrates at two different sites has important implications mechanistically, and a model is proposed to account for these novel catalytic properties. Among the analogs synthesized, cyclic GDP-ribose is highly resistant to hydrolysis, while cyclic IDP-ribose can be readily hydrolyzed by CD38, a bifunctional enzyme involved in the metabolism of cADPR. These unique properties of the analogs can be used to develop fluorimetric assays for monitoring separately the cyclization and hydrolytic reactions catalyzed by the metabolic enzymes of cADPR. The convenience of the method in measuring kinetic parameters, pH-dependence, and modulator activity of the metabolic enzymes of cADPR is illustrated.

ADP-ribosyl cyclase catalyzes the cyclization of NAD+ to produce cyclic ADP-ribose (cADPR), which is emerging as an endogenous regulator of the Ca(2+)-induced Ca2+ release mechanism in cells. CD38 is a lymphocyte differentiation antigen which has recently been shown to be a bifunctional enzyme that can synthesize cADPR from NAD+ as well as hydrolyze cADPR to ADP-ribose. In this study, we show that both the cyclase and CD38 can also catalyze the exchange of the nicotinamide group of NADP+ with nicotine acid (NA). The product is nicotinic acid adenine dinucleotide phosphate (NAADP+), a metabolite we have previously shown to be potent in Ca2+ mobilization (Lee, H. C., and Aarhus, R. (1995) J. Biol. Chem. 270, 2152-2157). The switch of the catalysis to the exchange reaction requires acidic pH and NA. The half-maximal effective concentration of NA is about 5 mM for both the cyclase and CD38. In the absence of NA or at neutral pH, the cyclase converts NADP+ to another metabolite, which is identified as cyclic ADP-ribose 2'-phosphate. Under the same conditions, CD38 converts NADP+ to ADP-ribose 2'-phosphate instead, which is the hydrolysis product of cyclic ADP-ribose 2'-phosphate. That two different products of ADP-ribosyl cyclase and CD38, cADPR and NAADP+, are both involved in Ca2+ mobilization suggests a crucial role of these enzymes in Ca2+ signaling.

CD38 is a 42-kilodalton glycoprotein expressed extensively on B and T lymphocytes. CD38 exhibits a structural homology to Aplysia adenosine diphosphate (ADP)-ribosyl cyclase. This enzyme catalyzes the synthesis of cyclic ADP-ribose (cADPR), a metabolite of nicotinamide adenine dinucleotide (NAD+) with calcium-mobilizing activity. A complementary DNA encoding the extracellular domain of murine CD38 was constructed and expressed, and the resultant recombinant soluble CD38 was purified to homogeneity. Soluble CD38 catalyzed the formation and hydrolysis of cADPR when added to NAD+. Purified cADPR augmented the proliferative response of activated murine B cells, potentially implicating the enzymatic activity of CD38 in lymphocyte function.