Mathur S. Kannan

CD38/cyclic ADP-ribose-mediated Ca2+ signaling contributes to airway smooth muscle hyper-responsiveness

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 α (TNF‐α), interleukin 1β, or interferon γ, 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‐α 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.

IL-13 enhances agonist-evoked calcium signals and contractile responses in airway smooth muscle.

Growing evidence suggests that interleukin (IL)‐13, a Th2‐type cytokine, plays a critical role in the development of bronchial hyper‐responsiveness (BHR), an essential feature of asthma, although the underlying mechanisms remain unknown. In the present study, we investigated whether IL‐13 directly affects airway smooth muscle (ASM) function. In murine tracheal rings, IL‐13 (100 ng ml−1, 24 h) significantly increased both the carbachol‐ and KCl‐induced maximal force generation without affecting ASM sensitivity. In cultured human ASM cells, IL‐13 (50 ng ml−1, 24 h) also augmented cytosolic calcium levels to bradykinin, histamine and carbachol by 60, 35 and 26%, respectively. The present study demonstrates that IL‐13 may promote BHR by directly modulating ASM contractility, an effect that may be due to enhanced G protein‐coupled receptor (GPCR)‐associated calcium signaling.

Role of cyclic ADP-ribose in the regulation of [Ca2+]i in porcine tracheal smooth muscle

The purpose of the present study was to determine whether cyclic ADP-ribose (cADPR) acts as a second messenger for Ca2+ release through ryanodine receptor (RyR) channels in tracheal smooth muscle (TSM). Freshly dissociated porcine TSM cells were permeabilized with β-escin, and real-time confocal microscopy was used to examine changes in intracellular Ca2+ concentration ([Ca2+]i). cADPR (10 nM-10 μM) induced a dose-dependent increase in [Ca2+]i, which was blocked by the cADPR receptor antagonist 8-amino-cADPR (20 μM) and by the RyR blockers ruthenium red (10 μM) and ryanodine (10 μM), but not by the inositol 1,4,5-trisphosphate receptor blocker heparin (0.5 mg/ml). During steady-state [Ca2+]ioscillations induced by acetylcholine (ACh), addition of 100 nM and 1 μM cADPR increased oscillation frequency and decreased peak-to-trough amplitude. ACh-induced [Ca2+]ioscillations were blocked by 8-amino-cADPR; however, 8-amino-cADPR did not block the [Ca2+]iresponse to a subsequent exposure to caffeine. These results indicate that cADPR acts as a second messenger for Ca2+ release through RyR channels in TSM cells and may be necessary for initiating ACh-induced [Ca2+]ioscillations.The purpose of the present study was to determine whether cyclic ADP-ribose (cADPR) acts as a second messenger for Ca2+ release through ryanodine receptor (RyR) channels in tracheal smooth muscle (TSM). Freshly dissociated porcine TSM cells were permeabilized with beta-escin, and real-time confocal microscopy was used to examine changes in intracellular Ca2+ concentration ([Ca2+]i). cADPR (10 nM-10 microM) induced a dose-dependent increase in [Ca2+]i, which was blocked by the cADPR receptor antagonist 8-amino-cADPR (20 microM) and by the RyR blockers ruthenium red (10 microM) and ryanodine (10 microM), but not by the inositol 1,4,5-trisphosphate receptor blocker heparin (0.5 mg/ml). During steady-state [Ca2+]i oscillations induced by acetylcholine (ACh), addition of 100 nM and 1 microM cADPR increased oscillation frequency and decreased peak-to-trough amplitude. ACh-induced [Ca2+]i oscillations were blocked by 8-amino-cADPR; however, 8-amino-cADPR did not block the [Ca2+]i response to a subsequent exposure to caffeine. These results indicate that cADPR acts as a second messenger for Ca2+ release through RyR channels in TSM cells and may be necessary for initiating ACh-induced [Ca2+]i oscillations.

Lymphocyte function-associated antigen 1 is a receptor for Pasteurella haemolytica leukotoxin in bovine leukocytes

ABSTRACT Pasteurella (Mannheimia) haemolytica leukotoxin (Lkt) causes cell type- and species-specific effects in ruminant leukocytes. Recent studies indicate that P. haemolytica Lkt binds to bovine CD18, the common subunit of all β2 integrins. We designed experiments with the following objectives: to identify which member of the β2 integrins is a receptor for Lkt; to determine whether Lkt binding to the receptor is target cell (bovine leukocytes) specific; to define the relationships between Lkt binding to the receptor, calcium elevation, and cytolysis; and to determine whether a correlation exists between Lkt receptor expression and the magnitude of target cell cytolysis. We compared Lkt-induced cytolysis in neutrophils from control calves and from calves with bovine leukocyte adhesion deficiency (BLAD), because neutrophils from BLAD-homozygous calves exhibit reduced β2 integrin expression. The results demonstrate for the first time that Lkt binds to bovine CD11a and CD18 (lymphocyte function-associated antigen 1 [LFA-1]). The binding was abolished by anti-CD11a or anti-CD18 monoclonal antibody (MAb). Lkt-induced calcium elevation in bovine alveolar macrophages (BAMs) was inhibited by anti-CD11a or anti-CD18 MAb (65 to 94% and 37 to 98%, respectively, at 5 and 50 Lkt units per ml; P < 0.05). Lkt-induced cytolysis in neutrophils and BAMs was also inhibited by anti-CD11a or anti-CD18 MAb in a concentration-dependent manner. Lkt bound to porcine LFA-1 but did not induce calcium elevation or cytolysis. In neutrophils from BLAD calves, Lkt-induced cytolysis was decreased by 44% compared to that of neutrophils from control calves (P < 0.05). These results indicate that LFA-1 is a Lkt receptor, Lkt binding to LFA-1 is not target cell specific, Lkt binding to bovine LFA-1 correlates with calcium elevation and cytolysis, and bovine LFA-1 expression correlates with the magnitude of Lkt-induced target cell cytolysis.

Intracellular calcium signaling through the cADPR pathway is agonist specific in porcine airway smooth muscle.

Cyclic ADP‐ribose (cADPR) induces intracellular Ca2+ ([Ca2+]i) release in airway smooth muscle, and the cADPR antagonist, 8‐amino‐cADPR, abolishes [Ca2+]i oscillations elicited by acetylcholine (ACh), suggesting that cADPR is involved during muscarinic receptor activation. Whether the cADPR signaling pathway is common to agonists acting through different G protein‐coupled receptors is not known. Using digital video imaging of Fura2‐AM loaded porcine airway smooth muscle cells, we examined the effects of the membrane‐permeant cADPR antagonist, 8‐bromo‐cADPR (8Br‐cADPR), on the [Ca2+]i responses to ACh, histamine and endothelin‐1 (ET‐1). In cells preincubated with 100 μM 8Br‐cADPR, the [Ca2+]i responses to ACh and ET‐1 were significantly attenuated, whereas responses to histamine were not, suggesting agonist specificity of cADPR signaling. The effects of 8Br‐cADPR were concentration dependent. We further examined whether muscarinic receptor subtypes specifically couple to this pathway, because in porcine airway smooth muscle cells, ACh activates both M2 and M3 muscarinic receptors coupled to Gαi and Gαq, respectively. Methoctramine, an M2‐selective antagonist, attenuated the [Ca2+]i responses to Ach, and there was no further attenuation by 8Br‐cADPR. In airway smooth muscle, the CD38/cADPR signaling pathway is involved in [Ca2+]i responses to contractile agonists in an agonist‐specific manner.

Heterogeneity in dynamic regulation of intracellular calcium in airway smooth muscle cells

Intracellular Ca2+ ([Ca2+]i) regulation in smooth muscle involves multiple mechanisms such as second messengers and ion channels. Intra- and inter-cellular heterogeneities in these mechanisms are likely, and will be reflected by heterogeneities in [Ca2+]i. In the present study, real-time confocal imaging was used to examine intracellular and intercellular heterogeneity in spontaneous Ca2+ sparks and acetylcholine-induced [Ca2+]i oscillations in porcine tracheal smooth muscle (TSM) cells. Ca2+ sparks were highly localized to multiple (2-5) foci in a cell. Individual sparks displayed relatively constant rise times (14.5 +/- 0.3% variance) and amplitudes (11.1 +/- 0.2% variance), but across regions these attributes varied. The incidence of sparks was often coupled across adjacent regions (r2 = 0.93 +/- 0.04). Spark frequency was increased approximately 350% by ryanodine and caffeine, suggesting that they represent unitary Ca2+ release through ryanodine receptor (RyR) channels. In TSM cells, acetylcholine induced [Ca2+]i oscillations that initiated from foci with the highest spark frequency. Results using beta-escin-permeabilized TSM cells indicated that [Ca2+]i oscillations also represent Ca2+ release through RyR channels. [Ca2+]i oscillations displayed intracellular heterogeneity in amplitude (30 +/- 4% variance) and intercellular heterogeneities in amplitude (100-800 nM) and frequency (5-35 per minute). Within a region, the amplitude and frequency of [Ca2+]i oscillations were correlated to both acetylcholine concentration (r = -0.79 +/- 0.04 for amplitude and 0.77 +/- 0.05 for frequency) and basal [Ca2+]i level (r = -0.94 +/- 0.02 for amplitude and 0.84 +/- 0.03 for frequency). Compared with TSM cells, acetylcholine-induced [Ca2+]i oscillations in bronchial cells were slower and lower in amplitude. We conclude that intracellular and intercellular heterogeneity in [Ca2+]i levels in airway smooth muscle reflects heterogeneities in Ca2+ regulatory mechanisms.

Effects of Pasteurella haemolytica A1 leukotoxin on bovine neutrophils: degranulation and generation of oxygen-derived free radicals.

To further define the role of Pasteurella haemolytica A1 leukotoxin in the pathogenesis of bovine pneumonic pasteurellosis, its in vitro effects on bovine neutrophils were investigated. Leukotoxin-containing culture supernatant, from P. haemolytica, stimulated a neutrophil respiratory burst as measured by the generation of oxygen-derived free radicals O2- and H2O2. This effect was immediate because preincubation of neutrophils with the culture supernatant for 5 min or longer substantially suppressed this respiratory burst. This suppression was due to cytolysis of the neutrophils. Prolonged incubation of neutrophils with the same culture supernatant caused further cytolysis and degranulation. Heat-inactivated P. haemolytica culture supernatant that had lost its cytotoxic properties failed to stimulate respiratory burst by neutrophils. Furthermore, the respiratory burst, cytolysis and degranulation were abrogated only by leukotoxin-neutralizing monoclonal and polyclonal antibodies, but not by antibodies against the lipopolysaccharide. These studies show that the leukotoxin component in the culture supernatant was responsible for the generation of oxygen-derived free radicals and proteolytic enzymes from neutrophils which may participate in direct lung injury.

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

The transmembrane glycoprotein CD38 catalyzes the synthesis of the calcium mobilizing molecule cyclic ADP‐ribose from NAD. In human airway smooth muscle (HASM) cells, the expression and function of CD38 are augmented by the inflammatory cytokine tumor necrosis factor‐alpha (TNF‐α), leading to increased intracellular calcium response to agonists. A glucocorticoid response element in the CD38 gene has been computationally described, providing evidence for transcriptional regulation of its expression. In the present study, we investigated the effects of dexamethasone, a glucocorticoid, on CD38 expression and ADP‐ribosyl cyclase activity in HASM cells stimulated with TNF‐α. In HASM cells, TNF‐α augmented CD38 expression and ADP‐ribosyl cyclase activity, which were attenuated by dexamethasone. TNF‐α increased NF‐κB expression and its activation, and dexamethasone partially reversed these effects. TNF‐α increased the expression of IκBα, and dexamethasone increased it further. An inhibitor of NF‐κB activation or transfection of cells with IκB mutants decreased TNF‐α‐induced CD38 expression. The results indicate that TNF‐α‐induced CD38 expression involves NF‐κB expression and its activation and dexamethasone inhibits CD38 expression through NF‐κB‐dependent and ‐independent mechanisms.— Kang, B.‐N., Tirumurugaan, K. G., Deshpande, D. A., Amrani, Y., Panettieri, R. A., Walseth, T. F., Kannan, M. S. Transcriptional regulation of CD38 expression by tumor necrosis factor‐α in human airway smooth muscle cells: role of NF‐κB and sensitivity to glucocorticoids. FASEB J. 20, E170–E179 (2006)

Spatial and temporal aspects of calcium sparks in porcine tracheal smooth muscle cells

Spontaneous, localized intracellular Ca2+concentration ([Ca2+]i) transients (Ca2+ sparks) in skeletal, cardiac, and smooth muscle cells are thought to represent Ca2+ release through ryanodine-receptor (RyR) channels. In porcine tracheal smooth muscle (TSM) cells, ACh induces propagating [Ca2+]ioscillations that also represent Ca2+ release through RyR channels. We used real-time confocal imaging to examine the spatial and temporal relationships of Ca2+ sparks to propagating [Ca2+]ioscillations in TSM cells. Ca2+sparks within an intracellular region displayed different spatial Ca2+ distributions with every occurrence. The amplitudes of Ca2+sparks within a region were approximately integer multiples of the smallest response. However, across different regions, the attributes of Ca2+ sparks varied considerably. Individual sparks were often grouped together and coupled across adjacent regions. Fusion of individual sparks produced large local elevations in [Ca2+]ithat occasionally triggered a propagating [Ca2+]iwave. The incidence of sparks was increased by ryanodine and caffeine but was unaffected by removal of extracellular Ca2+. Exposure to ACh triggered repetitive, propagating [Ca2+]ioscillations that always originated from foci with a high spark incidence. The [Ca2+]ioscillations disappeared with the removal of ACh, and Ca2+ sparks reappeared. We conclude that agonist-induced [Ca2+]ioscillations represent a spatial and temporal integration of local Ca2+-release events through RyR channels in TSM cells.Spontaneous, localized intracellular Ca(2+) concentration ([Ca(2+)](i)) transients (Ca(2+) sparks) in skeletal, cardiac, and smooth muscle cells are thought to represent Ca(2+) release through ryanodine-receptor (RyR) channels. In porcine tracheal smooth muscle (TSM) cells, ACh induces propagating [Ca(2+)](i) oscillations that also represent Ca(2+) release through RyR channels. We used real-time confocal imaging to examine the spatial and temporal relationships of Ca(2+) sparks to propagating [Ca(2+)](i) oscillations in TSM cells. Ca(2+) sparks within an intracellular region displayed different spatial Ca(2+) distributions with every occurrence. The amplitudes of Ca(2+) sparks within a region were approximately integer multiples of the smallest response. However, across different regions, the attributes of Ca(2+) sparks varied considerably. Individual sparks were often grouped together and coupled across adjacent regions. Fusion of individual sparks produced large local elevations in [Ca(2+)](i) that occasionally triggered a propagating [Ca(2+)](i) wave. The incidence of sparks was increased by ryanodine and caffeine but was unaffected by removal of extracellular Ca(2+). Exposure to ACh triggered repetitive, propagating [Ca(2+)](i) oscillations that always originated from foci with a high spark incidence. The [Ca(2+)](i) oscillations disappeared with the removal of ACh, and Ca(2+) sparks reappeared. We conclude that agonist-induced [Ca(2+)](i) oscillations represent a spatial and temporal integration of local Ca(2+)-release events through RyR channels in TSM cells.

Estrogen Increases CD38 Gene Expression and Leads to Differential Regulation of Adenosine Diphosphate (ADP)-Ribosyl Cyclase and Cyclic ADP-Ribose Hydrolase Activities in Rat Myometrium

Abstract 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-17β 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 [32P]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-17β causes 1) increased CD38 mRNA and protein expression and 2) significantly enhanced cyclase but not hydrolase activity. The differential regulation of CD38 by estradiol-17β, resulting in increased cADPR synthesis, would have profound effects on calcium regulation and myometrial contractility.