Inflammation,caveolae and CD38-mediated calcium regulation in human airway smooth muscle

The pro-inflammatory cytokine tumor necrosis factor-alpha (TNFα) increases expression of CD38 (a membrane-associated bifunctional enzyme regulating cyclic ADP ribose), and enhances agonist-induced intracellular Ca^2 + ([Ca^2 +]_i) responses in human airway smooth muscle (ASM). We previously demonstrated that caveolae and their constituent protein caveolin-1 are important for ASM [Ca^2 +]_i regulation, which is further enhanced by TNFα. Whether caveolae and CD38 are functionally linked in mediating TNFα effects is unknown. In this regard, whether the related cavin proteins (cavin-1 and -3) that maintain structure and function of caveolae play a role is also not known. In the present study, we hypothesized that TNFα effects on CD38 expression and function in human ASM involve caveolae. Caveolar fractions from isolated human ASM cells expressed CD38 and its expression was upregulated by exposure to 20 ng/ml TNFα (48 h). ASM cells expressed cavin-1 and cavin-3, which were also upregulated by TNFα. Knockdown of caveolin-1, cavin-1 or cavin-3 (using siRNA) all significantly reduced CD38 expression and ADP-ribosyl cyclase activity in the presence or absence of TNFα. Furthermore, caveolin-1, cavin-1 and cavin-3 siRNAs reduced [Ca^2 +]_i responses to histamine under control conditions, and blunted the enhanced [Ca^2 +]_i responses in TNFα-exposed cells. These data demonstrate that CD38 is expressed within caveolae and its function is linked to the caveolar regulatory proteins caveolin-1, cavin-1 and -3. The link between caveolae and CD38 is further enhanced during airway inflammation demonstrating the important role of caveolae in regulation of [Ca^2 +]_i and contractility in the airway.     

Adenoviral mediated anti-sense CD38 attenuates TNF-alpha-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.     

Tyrosine kinase-dependent calcium signaling in airway smooth muscle cells

Contractile agonists may stimulate mitogenic responses in airway smooth muscle by mechanisms that involve tyrosine kinases. The role of contractile agonist-evoked activation of tyrosine kinases in contractile signaling is not clear. We addressed this issue using cultured rat airway smooth muscle cells. In these cells, serotonin (5-HT, 1 microM) caused contraction (quantitated by a decrease in cell area), which was blocked by the tyrosine kinase inhibitor genistein (40 microM). Genistein and tyrphostin 23 (40 and 10 microM, respectively) significantly decreased 5-HT-evoked peak Ca(2+) responses, and the effect of genistein could be observed in the absence of extracellular Ca(2+). The specific inhibitor of mitogen-activated protein kinase kinase PD-98059 (30 microM) had no significant effect on peak Ca(2+) levels. Western analysis of cell extracts revealed that 5-HT caused a significant increase in tyrosine phosphorylation of proteins with molecular masses of approximately 70 kDa within 10 s of stimulation but no measurable tyrosine phosphorylation of the gamma isoform of phospholipase C (PLC-gamma). Tyrosine phosphorylation was inhibited by genistein. Furthermore, genistein (40 microM) significantly attenuated 5-HT-induced inositol phosphate production. We conclude that in airway smooth muscle contractile agonists acting on G protein-coupled receptors may activate tyrosine kinase(s), which in turn modulate calcium signaling by affecting, directly or indirectly, PLC-beta activity. It is unlikely that PLC-gamma or the mitogen-activated protein kinase pathway is involved in Ca(2+) signaling to 5-HT.     

Autocrine regulation of asthmatic airway inflammation: role of airway smooth muscle

Chronic airway inflammation is one of the main features of asthma. Release of mediators from infiltrating inflammatory cells in the airway mucosa has been proposed to contribute directly or indirectly to changes in airway structure and function. The airway smooth muscle, which has been regarded as a contractile component of the airways responding to various mediators and neurotransmitters, has recently been recognised as a rich source of pro-inflammatory cytokines, chemokines and growth factors. In this review, we discuss the role of airway smooth muscle cells in the regulation and perpetuation of airway inflammation that contribute to the pathogenesis of asthma.     

Autocrine regulation of asthmatic airway inflammation: role of airway smooth muscle

Chronic airway inflammation is one of the main features of asthma. Release of mediators from infiltrating inflammatory cells in the airway mucosa has been proposed to contribute directly or indirectly to changes in airway structure and function. The airway smooth muscle, which has been regarded as a contractile component of the airways responding to various mediators and neurotransmitters, has recently been recognised as a rich source of pro-inflammatory cytokines, chemokines and growth factors. In this review, we discuss the role of airway smooth muscle cells in the regulation and perpetuation of airway inflammation that contribute to the pathogenesis of asthma.     

Subcellular localization of cyclic ADP-ribosyl cyclase and cyclic ADP-ribose hydrolase activities in porcine airway smooth muscle

Recent studies have provided evidence for a role of cyclic ADP-ribose (cADPR) in the regulation of intracellular calcium in smooth muscles of the intestine, blood vessels and airways. We investigated the presence and subcellular localization of ADP-ribosyl cyclase, the enzyme that catalyzes the conversion of beta-NAD(+) to cADPR, and cADPR hydrolase, the enzyme that degrades cADPR to ADPR, in tracheal smooth muscle (TSM). Sucrose density fractionation of TSM crude membranes provided evidence that ADP-ribosyl cyclase and cADPR hydrolase activities were associated with a fraction enriched in 5'-nucleotidase activity, a plasma membrane marker enzyme, but not in a fraction enriched in either sarcoplasmic endoplasmic reticulum calcium ATPase or ryanodine receptor channels, both sarcoplasmic reticulum markers. The ADP-ribosyl cyclase and cADPR hydrolase activities comigrated at a molecular weight of approximately 40 kDa on SDS-PAGE. This comigration was confirmed by gel filtration chromatography. Investigation of kinetics yielded K(m) values of 30.4+/-1.5 and 695. 3+/-171.2 microM and V(max) values of 330.4+/-90 and 102.8+/-17.1 nmol/mg/h for ADP-ribosyl cyclase and cADPR hydrolase, respectively. These results suggest a possible role for cADPR as an endogenous modulator of [Ca(2+)](i) in porcine TSM cells.     

Caveolins and intracellular calcium regulation in human airway smooth muscle

Regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) is a key factor in airway smooth muscle (ASM) tone. In vascular smooth muscle, specialized membrane microdomains (caveolae) expressing the scaffolding protein caveolin-1 are thought to facilitate cellular signal transduction. In human ASM cells, we tested the hypothesis that caveolae mediate Ca(2+) responses to agonist stimulation. Fluorescence immunocytochemistry with confocal microscopy, as well as Western blot analysis, was used to determine that agonist receptors (M(3) muscarinic, bradykinin, and histamine) and store-operated Ca(2+) entry (SOCE)-regulatory mechanisms colocalize with caveolin-1. Although caveolin-2 coexpressed with caveolin-1, caveolin-3 was absent. In fura 2-loaded ASM cells, [Ca(2+)](i) responses to 1 microM ACh, 10 microM histamine, and 10 nM bradykinin, as well as SOCE, were attenuated (each to a different extent) after disruption of caveolae by the cholesterol-chelating drug methyl-beta-cyclodextrin. Transfection of ASM cells with 50 nM caveolin-1 small interfering RNA significantly weakened caveolin-1 expression and blunted [Ca(2+)](i) responses to bradykinin and histamine, as well as SOCE, but the response to ACh was less intense. These results indicate that caveolae are present in ASM and that caveolin-1 contributes to regulation of [Ca(2+)](i) responses to agonist.     

Cyclic ADP-ribose requires CD38 to regulate the release of ATP in visceral smooth muscle

It is well established that the intracellular second messenger cADP-ribose (cADPR) activates Ca(2+) release from the sarcoplasmic reticulum through ryanodine receptors. CD38 is a multifunctional enzyme involved in the formation of cADPR in mammals. CD38 has also been reported to transport cADPR in several cell lines. Here, we demonstrate a role for extracellular cADPR and CD38 in modulating the spontaneous, but not the electrical field stimulation-evoked, release of ATP in visceral smooth muscle. Using a small-volume superfusion assay and an HPLC technique with fluorescence detection, we measured the spontaneous and evoked release of ATP in bladder detrusor smooth muscles isolated from CD38(+/+) and CD38(-/-) mice. cADPR (1 nM) enhanced the spontaneous overflow of ATP in bladders isolated from CD38(+/+) mice. This effect was abolished by the inhibitor of cADPR receptors on sarcoplasmic reticulum 8-bromo-cADPR (80 μM) and by ryanodine (50?μm), but not by the nonselective P2 purinergic receptor antagonist pyridoxal phosphate 6-azophenyl-2',4'-disulfonate (30 μM). cADPR failed to facilitate the spontaneous ATP overflow in bladders isolated from CD38(-/-) mice, indicating that CD38 is crucial for the enhancing effects of extracellular cADPR on spontaneous ATP release. Contractile responses to ATP were potentiated by cADPR, suggesting that the two adenine nucleotides may work in synergy to maintain the resting tone of the bladder. In conclusion, extracellular cADPR enhances the spontaneous release of ATP in the bladder by influx via CD38 and subsequent activation of intracellular cADPR receptors, probably causing an increase in intracellular Ca(2+) in neuronal cells.     

Mechanism-based inhibitors of CD38: a mammalian cyclic ADP-ribose synthetase

The soluble domain of human CD38 catalyzes the conversion of NAD(+) to cyclic ADP-ribose and to ADP-ribose via a common covalent intermediate [Sauve, A. A., Deng, H. T., Angelletti, R. H., and Schramm, V. L. (2000) J. Am. Chem. Soc. 122, 7855-7859]. Here we establish that mechanism-based inhibitors can be produced by chemical stabilization of this intermediate. The compounds nicotinamide 2'-deoxyriboside (1), 5-methylnicotinamide 2'-deoxyriboside (2), and pyridyl 2'-deoxyriboside (3) were synthesized and evaluated as inhibitors for human CD38. The nicotinamide derivatives 1 and 2 were inhibitors of the enzyme as determined by competitive behavior in CD38-catalyzed conversion of nicotinamide guanine dinucleotide (NGD(+)) to cyclic GDP-ribose. The K(i) values for competitive inhibition were 1.2 and 4.0 microM for 1 and 2, respectively. Slow-onset characteristics of reaction progress curves indicated a second higher affinity state of these two inhibitors. Inhibitor off-rates were slow with rate constants k(off) of 1.5 x 10(-5) s(-1) for 1 and 2.5 x 10(-5) s(-1) for 2. Apparent dissociation constants K(i(total)) for 1 and 2 were calculated to be 4.5 and 12.5 nM, respectively. The similar values for k(off) are consistent with the hydrolysis of common enzymatic intermediates formed by the reaction of 1 and 2 with the enzyme. Both form covalently attached deoxyribose groups to the catalytic site nucleophile. Chemical evidence for this intermediate is the ability of nicotinamide to rescue enzyme activity after inactivation by either 1 or 2. A covalent intermediate is also indicated by the ability of CD38 to catalyze base exchange, as observed by conversion of 2 to 1 in the presence of nicotinamide. The deoxynucleosides 1 and 2 demonstrate that the chemical determinants for mechanism-based inhibition of CD38 can be satisfied by nucleosides that lack the 5'-phosphate, the adenylate group, and the 2'-hydroxyl moiety. In addition, these compounds reveal the mechanism of CD38 catalysis to proceed by the formation of a covalent intermediate during normal catalytic turnover with faster substrates. The covalent 2'-deoxynucleoside inactivators of CD38 are powerful inhibitors by acting as good substrates for formation of the covalent intermediate but are poor leaving groups from the intermediate complex because hydrolytic assistance of the 2'-hydroxyl group is lacking. The removal of the adenylate nucleophile required for the cyclization reaction provides slow hydrolysis as the only exit from the covalent complex.     

CD38 signaling in B lymphocytes is controlled by its ectodomain but occurs independently of enzymatically generated ADP-ribose or cyclic ADP-ribose

CD38 is a type II transmembrane glycoprotein that is expressed by many cell types including lymphocytes. Signaling through CD38 on B lymphocytes can mediate B cell activation, proliferation, and cytokine secretion. Additionally, coligation of CD38 and the B cell Ag receptor can greatly augment B cell Ag receptor responses. Interestingly, the extracellular domain of CD38 catalyzes the conversion of NAD+ into nicotinamide, ADP-ribose (ADPR), and cyclic ADPR (cADPR). cADPR can induce intracellular calcium release in an inositol trisphosphate-independent manner and has been hypothesized to regulate CD38-mediated signaling. We demonstrate that replacement of the cytoplasmic tail and the transmembrane domains of CD38 did not impair CD38 signaling, coreceptor activity, or enzyme activity. In contrast, independent point mutations in the extracellular domain of CD38 dramatically impaired signal transduction. However, no correlation could be found between CD38-mediated signaling and the capacity of CD38 to catalyze an enzyme reaction and produce cADPR, ADPR, and/or nicotinamide. Instead, we propose that CD38 signaling and coreceptor activity in vitro are regulated by conformational changes induced in the extracellular domain upon ligand/substrate binding, rather than on actual turnover or generation of products.     

Acetylcholine-induced calcium signaling and contraction of airway smooth muscle cells in lung slices

The Ca(2+) signaling and contractility of airway smooth muscle cells (SMCs) were investigated with confocal microscopy in murine lung slices (approximately 75-microm thick) that maintained the in situ organization of the airways and the contractility of the SMCs for at least 5 d. 10--500 nM acetylcholine (ACH) induced a contraction of the airway lumen and a transient increase in [Ca(2+)](i) in individual SMCs that subsequently declined to initiate multiple intracellular Ca(2+) oscillations. These Ca(2+) oscillations spread as Ca(2+) waves through the SMCs at approximately 48 microm/s. The magnitude of the airway contraction, the initial Ca(2+) transient, and the frequency of the subsequent Ca(2+) oscillations were all concentration-dependent. In a Ca(2+)-free solution, ACH induced a similar Ca(2+) response, except that the Ca(2+) oscillations ceased after 1--1.5 min. Incubation with thapsigargin, xestospongin, or ryanodine inhibited the ACH-induced Ca(2+) signaling. A comparison of airway contraction with the ACH-induced Ca(2+) response of the SMCs revealed that the onset of airway contraction correlated with the initial Ca(2+) transient, and that sustained airway contraction correlated with the occurrence of the Ca(2+) oscillations. Buffering intracellular Ca(2+) with BAPTA prohibited Ca(2+) signaling and airway contraction, indicating a Ca(2+)-dependent pathway. Cessation of the Ca(2+) oscillations, induced by ACH-esterase, halothane, or the absence of extracellular Ca(2+) resulted in a relaxation of the airway. The concentration dependence of the airway contraction matched the concentration dependence of the increased frequency of the Ca(2+) oscillations. These results indicate that Ca(2+) oscillations, induced by ACH in murine bronchial SMCs, are generated by Ca(2+) release from the SR involving IP(3)- and ryanodine receptors, and are required to maintain airway contraction.     

Deficit of CD38/cyclic ADP-ribose is differentially compensated in hearts by gender

To elucidate whether myocardial CD38/cyclic ADP-ribose (cADPR) signaling plays a physiological role, we investigated the heart of CD38 knockout mice (CD38KO). In CD38KO, the myocardial cADPR content was reduced by 85% compared with wild-type mice (WT). Cardiac hypertrophy developed only in males. At 36 degrees C, none of the parameters for Ca(2+) transients and forces of the papillary muscles differed between WT and CD38KO. In contrast, at 27 degrees C, at which cADPR does not work, the peak [Ca(2+)](i) was increased and the decline in [Ca(2+)](i) was accelerated in CD38KO compared with WT. In CD38KO, the protein expression of SR Ca(2+) ATPase type2 (SERCA2) and the SERCA2-to-phospholamban ratio were increased compared with WT. The ryanodine receptor protein was increased only in female CD38KO compared with WT. These data suggest that the CD38/cADPR signaling plays an important role in intracellular Ca(2+) homeostasis in cardiac myocytes in vivo. Its deficiency was compensated differentially according to gender.     

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.     

Enhanced calcium signaling to bradykinin in airway smooth muscle from hyperresponsive inbred rats

Inbred Fischer 344 rats display airway hyperresponsiveness (AHR) in vivo compared with the normoresponsive Lewis strain. Fischer AHR has been linked with increased airway smooth muscle (ASM) contraction ex vivo and enhanced ASM cell intracellular Ca(2+) mobilization in response to serotonin compared with Lewis. To determine the generality of this association, we tested whether bradykinin (BK) also stimulates greater contraction of Fischer airways and greater Ca(2+) mobilization in Fischer ASM cells. Explants of Fischer intraparenchymal airways constricted faster and to a greater degree in response to BK than Lewis airways. BK also evoked higher Ca(2+) transients in Fischer than in Lewis ASM cells. ASM cell B(2) receptor expression was similar between the two strains. BK activated both phosphatidylinositide-specific phospholipase C (PI-PLC) and phosphatidylcholine-specific PLC to mobilize Ca(2+) in Fischer and Lewis ASM cells. PI-PLC activity, as measured by inositol polyphosphate accumulation, was similar in the two strains. PKC inhibition with GF109203X, Go6973, or Go6983 attenuated BK-mediated Ca(2+) transients in Fischer cells, whereas GF109203X potentiated while Go6976 and Go6983 did not affect Ca(2+) transients in Lewis cells. Enhanced Ca(2+) mobilization in ASM cells can arise from variations in PKC and may be an important component of nonspecific, innate AHR.     

Resting calcium influx in airway smooth muscle

Plasma membrane Ca2+ leak remains the most uncertain of the cellular Ca2+ regulation pathways. During passive Ca2+ influx in non-stimulated smooth muscle cells, basal activity of constitutive Ca2+ channels seems to be involved. In vascular smooth muscle, the 3 following Ca2+ entry pathways contribute to this phenomenon: (i) via voltage-dependent Ca2+ channels, (ii) receptor gated Ca2+ channels, and (iii) store operated Ca2+ channels, although, in airway smooth muscle it seems only 2 passive Ca2+ influx pathways are implicated, one sensitive to SKF 96365 (receptor gated Ca2+ channels) and the other to Ni2+ (store operated Ca2+ channels). Resting Ca2+ entry could provide a sufficient amount of Ca2+ and contribute to resting intracellular Ca2+ concentration ([Ca2+]i), maintenance of the resting membrane potential, myogenic tone, and sarcoplasmic reticulum-Ca2+ refilling. However, further research, especially in airway smooth muscle, is required to better explore the physiological role of this passive Ca2+ influx pathway as it could be involved in airway hyperresponsiveness.     

NO responsiveness in pulmonary artery and airway smooth muscle: the role of cGMP regulation

The purpose of this study was to assess intrinsic smooth muscle mechanisms contributing to greater nitric oxide (NO) responsiveness in pulmonary vascular vs. airway smooth muscle. Canine pulmonary artery smooth muscle (PASM) and tracheal smooth muscle (TSM) strips were used to perform concentration response studies to an NO donor, (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO). PASM exhibited a greater NO responsiveness whether PASM and TSM were contracted with receptor agonists, phenylephrine and acetylcholine, respectively, or with KCl. The >10-fold difference in NO sensitivity in PASM was observed with both submaximal and maximal contractions. This difference in NO responsiveness was not due to differences in endothelial or epithelial barriers, since these were removed, nor was it due to the presence of cGMP-independent NO-mediated relaxation in either tissue. At equal concentrations of NO, the intracellular cGMP concentration ([cGMP]i) was also greater in PASM than in TSM. Phosphodiesterase (PDE) inhibition using isobutylmethylxanthine indicated that the greater [cGMP]i in PASM was not due to greater PDE activity in TSM. Expression of soluble guanylate cyclase (sGC) subunit mRNA (2 +/- 0.2 and 1.3 +/- 0.2 attomol/microg total RNA, respectively) and protein (47.4 +/- 2 and 27.8 +/- 3.9 ng/mg soluble homogenate protein, respectively) was greater in PASM than in TSM. sGCalpha1 and sGCbeta1 mRNA expression was equal in PASM but was significantly different in TSM, suggesting independent regulation of their expression. An intrinsic smooth muscle mechanism accounting for greater NO responsiveness in PASM vs. TSM is greater sGC activity.