Pabelick, Christina M.

Supplemental oxygen places the immature airway at risk for bronchopulmonary dysplasia and asthma. Hyperoxia enhances intracellular Ca2+ ([Ca2+]i) and contractility of airway smooth muscle (ASM), and impairs relaxation (potentially by altering cyclic nucleotide levels). The neurotrophin brain‐derived neurotrophic factor (BDNF) enhances [Ca2+]i in adult ASM. We tested the hypothesis that hyperoxia‐induced airway hyperreactivity involves cyclic nucleotide‐mediated enhancement of BDNF release in human fetal (18–22 weeks) ASM cells exposed to 21% (normoxia) or 80% oxygen in the presence/absence of 1mM dibutyryl cAMP (DBcAMP) or 8‐Br‐cGMP. BDNF release (ELISA) was increased by hyperoxia (143% of normoxia). DBcAMP enhanced BDNF release with normoxia (242% of baseline) and even more with hyperoxia (280%), while 8‐Br‐cGMP blunted BDNF release in normoxia (57% of baseline) but to a lesser extent with hyperoxia (68% of baseline). Measurements of [Ca2+]i in fetal ASM cells showed DBcAMP increasing [Ca2+]i responses to histamine or ACh (effects enhanced with hyperoxia). Further, chelation of released BDNF using chimeric TrkB‐Fc (1?g/ml) reversed DBcAMP effects and blunted [Ca2+]i responses to below controls. We conclude that an imbalance between cAMP vs. cGMP enhances BDNF release in the developing airway, resulting in greater airway reactivity with hyperoxia. Supported by NIH grants HL056470 and HL088029

Women have a higher incidence and severity of asthma exacerbations compared to men. Asthma symptoms fluctuate with hormonal status suggesting a role for sex steroids in modulating bronchoconstriction. We have previously shown that acute estrogen decreases intracellular calcium in human airway smooth muscle thereby facilitating bronchodilation, similar to estrogen‐induced vasodilation. Akin to endothelium, airway epithelium is a source of nitric oxide (NO), a bronchodilator. We hypothesized that estrogens facilitate bronchodilation by generating NO in bronchial epithelium. In acutely dissociated human bronchial epithelial cells, 5–10 min exposure to 1 μM ACh or 17β‐estradiol (E2) (1 and 10 nM) resulted in increased DAF‐2 fluorescence (NO indicator). Both ER specific agonists THC (ERα) and DPN (ERβ) (10 nM,) induced NO production comparably. E2 effects were abrogated by the estrogen receptor (ER) antagonist ICI 182,780 (Faslodex®). eNOS phosphorylation was enhanced by E2 while E2‐induced NO production was attenuated by Ca2+ chelation (BAPTA; 20 μM, 30 min) and eNOS inhibition (L‐NAME). Overall, these data indicate a role for airway epithelium‐derived NO in the bronchodilatory effects of estrogens in human ASM. Supported by NIH grants HL090595 (CMP), HL088029 & HL56470 (YSP), and Mayo Graduate School (EAT).

Traditionally associated with neuronal structure and function, neurotrophins (NTs; e.g. brain derived neurotrophic factor (BDNF), neurotrophin‐3 (NT3 )) and their receptors (tropomyosin related kinase; Trk) have been found in the adult lung, pulmonary artery and airway; however, their roles remain unclear. Based on previous findings of pulmonary hypertension in NT receptor knockout mice, we hypothesized that NTs induce production of nitric oxide (NO) in pulmonary endothelial cells (EC), thereby modulating pulmonary vascular tone. In human pulmonary artery EC loaded with the NO sensitive fluorescent dye DAF‐2, 50 ƒÝM ATP and both BDNF and NT3 (1 and 10 nM) acutely (<5 min) and substantially in increased fluorescence levels. Addition of the NO scavenger PTIO reversed these changes. Specificity of DAF2 for NO was tested using the short‐acting NO donor MAHMA‐NONOate, and the nitrous oxide donor Sulfa‐NONOate. Furthermore, NT‐induced elevation in NO was significantly reduced by pre‐exposure to the tyrosine kinase inhibitor K252A (100 nM, 20 min), the NOS inhibitors L‐NMMA or L‐NAME, and the Ca2+ chelator BAPTA. Acute NT exposure also increased eNOS phosphorylation. These results indicate that NT¡¦s such as BDNF and NT3 can acutely modulate endothelial NO production, and may contribute to pulmonary vasodilation. Supported by NIH grants HL088029, (YSP), HL56470 (YSP), and Mayo Graduate School (LWM).

Regulation of intracellular Ca2+ in human airway smooth muscle (ASM) involves sarcoplasmic reticulum (SR) Ca2+ release/reuptake and plasma membrane (PM) Ca2+ influx, including store‐operated Ca2+ entry (SOCE) following SR Ca2+ depletion. Studies in human ASM have shown that siRNA suppression of stromal interaction molecule 1 (STIM1) and Orai1 decrease SOCE; however, their exact role in regulating Ca2+ are still under investigation. In human ASM, STIM1 and STIM2 were found exclusively in SR membrane, and Orai1 only in PM (especially caveolae). GFP‐tagged STIM1 transfection showed intracellular/SR localization at baseline, with increased peri‐PM aggregation following store depletion with 10ƒÝM cyclopiazonic acid (CPA). Such aggregation was prevented by microtubule inhibition (nocodazole), but not actin inhibition (phalloidin). FRET analysis revealed co‐localization of STIM1 with TRPC3 and Orai1 in CPA‐stimulated cells. In fura‐2 loaded ASM cells, SOCE was significantly reduced by STIM1 and Orai1 siRNA (less so by STIM2 siRNA). However, basal Ca2+ and agonist (ACh, histamine)‐induced Ca2+ responses were substantially modulated by STIM2 suppression. These results indicate that in human ASM, STIM1 mediates SR‐PM interactions in regulating SOCE, while STIM2 modulates sub‐maximal agonist‐induced changes in Ca2+. Supported by NIH grant HL74309 (GCS), HL090595 (CMP) and HL088029 (YSP).

Plasma membrane Ca 2+ influx and sarcoplasmic reticulum (SR) Ca 2+ release are key components of intracellular Ca 2+ regulation in airway smooth muscle (ASM). We and others have previously found that ASM Ca 2+ regulation involves propagating Ca 2+ oscillations via these mechanisms. Ca 2+ oscillations in non‐excitable cells involve influx via arachidonic acid (AA) regulated channels (ARC); however, little is known about ARC in ASM. In human ASM cells loaded with fura2 (5 μM), we found that 10 μM AA triggered Ca 2+ oscillations. OAG, oleic acid and palmitic acid (10 μM) failed to elicit Ca 2+ responses. Pre‐incubation with LaCl 3 1 μM and 1mM; 5 min) inhibited AA‐induced oscillations. Inhibition of SR Ca 2+ release (ryanodine 10 μM and IP3 channel inhibitor Xestospongin C 10 μM; 30 min), decreased Ca 2+ oscillation frequency and amplitude. Inhibitors of receptor‐operated channels (SKF‐96365; 10 μM), lipoxygenase (zileuton; 10 μM), or cyclooxygenase (indomethacin; 1 μM), did not affect oscillations. Finally, siRNA for Caveolin‐1, STIM1 and Orai3 (25nM) reduced frequency and amplitude of AA‐induced oscillations. These results indicate a novel caveolar Ca 2+ influx mechanism in human ASM mediated via AA that interacts with SR to produce repetitive SR Ca 2+ release. Such AA‐mediated influx could potentially contribute to increased Ca 2+ in ASM during inflammation. Supported by NIH grants HL090595 (CMP) and HL088029 (YSP)

Enhanced airway contractility by inflammatory cytokines involves increased intracellular Ca 2+ levels in airway smooth muscle (ASM). In this study, we examined the role of the bidirectional plasma membrane Na + /Ca 2+ exchanger (NCX) in human ASM Ca 2+ regulation following exposure to the pro‐inflammatory cytokines tumor necrosis factor alpha (TNFα) or interleukin‐13 (IL‐13). Both cytokines significantly increased NCX1 expression, with these effects inhibited by dexamethasone (NFκB inhibitor) or PD98059 (MAPK inhibitor). In human ASM cells loaded with the Ca 2+ indicator fura‐2, or the intracellular Na + indicator SBFI, NCX‐mediated Ca 2+ influx (with simultaneous Na + efflux) was enhanced by both cytokines. NCX‐mediated Ca 2+ efflux (with simultaneous Na + influx) was also enhanced, but not to the same extent. The NCX inhibitor KBR7943 as well as NCX1 siRNA inhibited cytokine enhancement of both modes NCX and of Ca 2+ responses to bronchoconstrictor agonists. These data indicate that a) NCX normally operates in human ASM; b) cytokines increase NCX expression, leading to greater NCX‐mediated Ca 2+ influx, thus contributing to enhanced Ca 2+ in inflamed ASM. Supported by NIH grants HL74309 (GCS), HL090595‐S2 (VS) and HL090595 (CMP).