Citations

Abstract Plasma membrane Ca2+ influx, especially store-operated Ca2+ entry triggered by sarcoplasmic reticulum (SR) Ca2+ release, is a key component of intracellular calcium concentration ([Ca2+]i) regulation in airway smooth muscle (ASM). Agonist-induced Ca2+ oscillations in ASM that involve both influx and SR mechanisms have been previously demonstrated. In nonexcitable cells, [Ca2+]i oscillations involve Ca2+ influx via arachidonic acid (AA) –stimulated channels, which show similarities to store-operated Ca2+ entry, although their molecular identity remains undetermined. Little is known about AA-regulated Ca2+ channels or their regulation in ASM. In enzymatically dissociated human ASM cells loaded with the Ca2+ indicator, fura-2, AA (1–10 μM) triggered [Ca2+]i oscillations that were inhibited by removal of extracellular Ca2+. Other fatty acids, such as the diacylglycerol analog, 1-oleoyl-2-acetyl-SN-glycerol, oleic acid, and palmitic acid (10 μM each), failed to elicit similar [Ca2+]i responses. Preincubation with LaCl3 (1 μM or 1 mM) inhibited AA-induced oscillations. Inhibition of receptor-operated channels (SKF96,365 [10 μM]), lipoxygenase (zileuton [10 μM]), or cyclooxygenase (indomethacin [10 μM]) did not affect oscillation parameters. Inhibition of SR Ca2+ release (ryanodine [10 μM] or inositol 1,4,5-trisphosphate receptor inhibitor, xestospongin C [1 μM]) decreased [Ca2+]i oscillation frequency and amplitude. Small interfering RNA against caveolin-1, stromal interaction molecule 1, or Orai3 (20 nM each) reduced the frequency and amplitude of AA-induced [Ca2+]i oscillations. In ASM cells derived from individuals with asthma, AA increased oscillation amplitude, but not frequency. These results are highly suggestive of a novel AA-mediated Ca2+–regulatory mechanism in human ASM, reminiscent of agonist-induced oscillations. Given the role of AA in ASM intracellular signaling, especially with inflammation, AA-regulated Ca2+ channels could potentially contribute to increased [Ca2+]i in diseases such asthma.

Pulmonary arterial hypertension (PAH), a form of pulmonary hypertension, is a complex disease of multifactorial origin. While new developments regarding pathophysiological features and therapeutic options in PAH are being reported, one important fact has emerged over the years: there is a sex difference in the incidence of this disease such that while there is a higher incidence in females, disease outcomes are much worse in males. Accordingly, recent attention has been focused on understanding the features of sex differences in the pulmonary circulation and the contributory mechanisms, particularly sex hormones and their role in the pathological and pathophysiological features of PAH. However, to date, there is no clear consensus whether sex hormones (particularly female sex steroids) are beneficial or detrimental in PAH. In this review, we highlight some of the most recent evidence regarding the influence of sex hormones (estrogen, testosterone, progesterone, dehydroepiandrosterone) and estrogen metabolites on key pathophysiological features of PAH such as proliferation, vascular remodeling, vasodilation/constriction, and inflammation, thus setting the stage for research avenues to identify novel therapeutic target for PAH as well as potentially other forms of pulmonary hypertension.

The balance between mitochondrial fission and fusion is crucial for mitochondria to perform its normal cellular functions. We hypothesized that cigarette smoke (CS) disrupts this balance and enhances mitochondrial dysfunction in the airway. In nonasthmatic human airway smooth muscle (ASM) cells, CS extract (CSE) induced mitochondrial fragmentation and damages their networked morphology in a concentration-dependent fashion, via increased expression of mitochondrial fission protein dynamin-related protein 1 (Drp1) and decreased fusion protein mitofusin (Mfn) 2. CSE effects on Drp1 vs. Mfn2 and mitochondrial network morphology involved reactive oxygen species (ROS), activation of extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt), protein kinase C (PKC) and proteasome pathways, as well as transcriptional regulation via factors such as NF-κB and nuclear erythroid 2-related factor 2. Inhibiting Drp1 prevented CSE effects on mitochondrial networks and ROS generation, whereas blocking Mfn2 had the opposite, detrimental effect. In ASM from asmatic patients, mitochondria exhibited substantial morphological defects at baseline and showed increased Drp1 but decreased Mfn2 expression, with exacerbating effects of CSE. Overall, these results highlight the importance of mitochondrial networks and their regulation in the context of cellular changes induced by insults such as inflammation (as in asthma) or CS. Altered mitochondrial fission/fusion proteins have a further potential to influence parameters such as ROS and cell proliferation and apoptosis relevant to airway diseases.

Abstract Inflammation elevates intracellular Ca2+ ([Ca2+]i) concentrations in airway smooth muscle (ASM). Store-operated Ca2+ entry (SOCE) is an important source of [Ca2+]i mediated by stromal interaction molecule–1 (STIM1), a sarcoplasmic reticulum (SR) protein. In transducing SR Ca2+ depletion, STIM1 aggregates to form puncta, thereby activating SOCE via interactions with a Ca2+ release-activated Ca2+ channel protein (Orai1) in the plasma membrane. We hypothesized that STIM1 aggregation is enhanced by inflammatory cytokines, thereby augmenting SOCE in human ASM cells. We used real-time fluorescence microscopic imaging to assess the dynamics of STIM1 aggregation and SOCE after exposure to TNF-α or IL-13 in ASM cells overexpressing yellow fluorescent protein–tagged wild-type STIM1 (WT-STIM1) and STIM1 mutants lacking the Ca2+-sensing EF-hand (STIM1-D76A), or lacking the cytoplasmic membrane binding site (STIM1ΔK). STIM1 aggregation was analyzed by monitoring puncta size during the SR Ca2+ depletion induced by cyclopiazonic acid (CPA). We found that puncta size was increased in cells expressing WT-STIM1 after CPA. However, STIM1-D76A constitutively formed puncta, whereas STIM1ΔK failed to form puncta. Furthermore, cytokines increased basal WT-STIM1 puncta size, and the SOCE triggered by SR Ca2+ depletion was increased in cells expressing WT-STIM1 or STIM1-D76A. Meanwhile, SOCE in cells expressing STIM1ΔK and STIM1 short, interfering RNA (siRNA) was decreased. Similarly, in cells overexpressing STIM1, the siRNA knockdown of Orai1 blunted SOCE. However, exposure to cytokines increased SOCE in all cells, increased basal [Ca2+]i, and decreased SR Ca2+ content. These data suggest that cytokines induce a constitutive increase in STIM1 aggregation that contributes to enhanced SOCE in human ASM after inflammation. Such effects of inflammation on STIM1 aggregations may contribute to airway hyperresponsiveness.

Abstract Enhanced airway smooth muscle (ASM) contractility contributes to increased resistance to airflow in diseases such as bronchitis and asthma that occur in passive smokers exposed to secondhand smoke. Little information exists on the cellular mechanisms underlying such airway hyperreactivity. Sputum samples of patients with chronic sinusitis, bronchitis, and asthma show increased concentrations of growth factors called neurotrophins, including brain-derived growth factor (BDNF), but their physiological significance remains unknown. In human ASM, we tested the hypothesis that BDNF contributes to increased contractility with cigarette smoke exposure. The exposure of ASM to 1% or 2% cigarette smoke extract (CSE) for 24 hours increased intracellular calcium ([Ca2+]i) responses to histamine, and further potentiated the enhancing effects of a range of BDNF concentrations on such histamine responses. CSE exposure increased the expression of the both high-affinity and low-affinity neurotrophin receptors tropomyosin-related kinase (Trk)–B and p75 pan-neurotrophin receptor, respectively. Quantitative ELISA showed that CSE increased BDNF secretion by human ASM cells. BDNF small interfering (si)RNA and/or the chelation of extracellular BDNF, using TrkB-fragment crystallizable, blunted the effects of CSE on [Ca2+]i responses as well as the CSE enhancement of cell proliferation, whereas TrkB siRNA blunted the effects of CSE on ASM contractility. These data suggest that cigarette smoke is a potent inducer of BDNF and TrkB expression and signaling in ASM, which then contribute to cigarette smoke–induced airway hyperresponsiveness.

Clinically observed differences in airway reactivity and asthma exacerbations in women at different life stages suggest a role for sex steroids in modulating airway function although their targets and mechanisms of action are still being explored. We have previously shown that clinically relevant concentrations of exogenous estrogen acutely decrease intracellular calcium ([Ca2+]i) in human airway smooth muscle (ASM), thereby facilitating bronchodilation. In this study, we hypothesized that estrogens modulate cyclic nucleotide regulation, resulting in decreased [Ca2+]iin human ASM. In Fura-2-loaded human ASM cells, 1 nM 17β-estradiol (E2) potentiated the inhibitory effect of the β-adrenoceptor (β-AR) agonist isoproterenol (ISO; 100 nM) on histamine-mediated Ca2+entry. Inhibition of protein kinase A (PKA) activity (KT5720; 100 nM) attenuated E2effects on [Ca2+]i. Acute treatment with E2increased cAMP levels in ASM cells comparable to that of ISO (100 pM). In acetylcholine-contracted airways from female guinea pigs or female humans, E2potentiated ISO-induced relaxation. These novel data suggest that, in human ASM, physiologically relevant concentrations of estrogens act via estrogen receptors (ERs) and the cAMP pathway to nongenomically reduce [Ca2+]i, thus promoting bronchodilation. Activation of ERs may be a novel adjunct therapeutic avenue in reactive airway diseases in combination with established cAMP-activating therapies such as β2-agonists.

Maintenance of blood oxygen saturation dictates supplemental oxygen administration to premature infants, but hyperoxia predisposes survivors to respiratory diseases such as asthma. Although much research has focused on oxygen effects on alveoli in the setting of bronchopulmonary dysplasia, the mechanisms by which oxygen affects airway structure or function relevant to asthma are still under investigation. We used isolated human fetal airway smooth muscle (fASM) cells from 18–20 postconceptual age lungs (canalicular stage) to examine oxygen effects on intracellular Ca 2+ ([Ca 2+ ] i ) and cellular proliferation. fASM cells expressed substantial smooth muscle actin and myosin and several Ca 2+ regulatory proteins but not fibroblast or epithelial markers, profiles qualitatively comparable to adult human ASM. Fluorescence Ca 2+ imaging showed robust [Ca 2+ ] i responses to 1 μM acetylcholine (ACh) and 10 μM histamine (albeit smaller and slower than adult ASM), partly sensitive to zero extracellular Ca 2+ . Compared with adult, fASM showed greater baseline proliferation. Based on this validation, we assessed fASM responses to 10% hypoxia through 90% hyperoxia and found enhanced proliferation at <60% oxygen but increased apoptosis at >60%, effects accompanied by appropriate changes in proliferative vs. apoptotic markers and enhanced mitochondrial fission at >60% oxygen. [Ca 2+ ] i responses to ACh were enhanced for <60% but blunted at >60% oxygen. These results suggest that hyperoxia has dose-dependent effects on structure and function of developing ASM, which could have consequences for airway diseases of childhood. Thus detrimental effects on ASM should be an additional consideration in assessing risks of supplemental oxygen in prematurity.

Caveolae are flask-shaped plasma membrane invaginations expressing the scaffolding caveolin proteins. Although caveolins have been found in endothelium and epithelium (where they regulate nitric oxide synthase activity), their role in smooth muscle is still under investigation. We and others have previously shown that caveolae of human airway smooth muscle (ASM), which express caveolin-1, contain Ca 2+ and force regulatory proteins and are involved in mediating the effects of inflammatory cytokines such as TNF-α on intracellular Ca 2+ concentration responses to agonist. Accordingly, we tested the hypothesis that in vivo, absence of caveolin-1 leads to reduced airway hyperresponsiveness, using a knockout (KO) (Cav1 KO) mouse and an ovalbumin-sensitized/challenged (OVA) model of allergic airway hyperresponsiveness. Surprisingly, airway responsiveness to methacholine, tested by use of a FlexiVent system, was increased in Cav1 KO control (CTL) as well as KO OVA mice, which could not be explained by a blunted immune response to OVA. In ASM of wild-type (WT) OVA mice, expression of caveolin-1, the caveolar adapter proteins cavins 1–3, and caveolae-associated Ca 2+ and force regulatory proteins such as Orai1 and RhoA were all increased, effects absent in Cav1 KO CTL and OVA mice. However, as with WT OVA, both CTL and OVA Cav1 KO airways showed signs of enhanced remodeling, with high expression of proliferation markers and increased collagen. Separately, epithelial cells from airways of all three groups displayed lower endothelial but higher inducible nitric oxide synthase and arginase expression. Arginase activity was also increased in these three groups, and the inhibitor nor-NOHA ( N-omega-nor-l-arginine) enhanced sensitivity of isolated tracheal rings to ACh, especially in Cav1 KO mice. On the basis of these data disproving our original hypothesis, we conclude that caveolin-1 has complex effects on ASM vs. epithelium, resulting in airway hyperreactivity in vivo mediated by altered airway remodeling and bronchodilation.

Regulation of cytosolic Ca2+ concentration ([Ca2+]cyt) in airway smooth muscle (ASM) is a key aspect of airway contractility and can be modulated by inflammation. Mitochondria have tremendous potential for buffering [Ca2+]cyt, helping prevent Ca2+ overload, and modulating other intracellular events. Here, compartmentalization of mitochondria to different cellular regions may subserve different roles. In the present study, we examined the role of Ca2+ buffering by mitochondria and mitochondrial Ca2+ transport mechanisms in the regulation of [Ca2+]cyt in enzymatically dissociated human ASM cells upon exposure to the proinflammatory cytokines TNF-α and IL-13. Cells were loaded simultaneously with fluo-3 AM and rhod-2 AM, and [Ca2+]cyt and mitochondrial Ca2+ concentration ([Ca2+]mito) were measured, respectively, using real-time two-color fluorescence microscopy in both the perinuclear and distal, perimembranous regions of cells. Histamine induced a rapid increase in both [Ca2+]cyt and [Ca2+]mito, with a significant delay in the mitochondrial response. Inhibition of the mitochondrial Na+/Ca2+ exchanger (1 μM CGP-37157) increased [Ca2+]mito responses in perinuclear mitochondria but not distal mitochondria. Inhibition of the mitochondrial uniporter (1 μM Ru360) decreased [Ca2+]mito responses in perinuclear and distal mitochondria. CGP-37157 and Ru360 significantly enhanced histamine-induced [Ca2+]cyt. TNF-α and IL-13 both increased [Ca2+]cyt, which was associated with decreased [Ca2+]mito in the case of TNF-α but not IL-13. The effects of TNF-α on both [Ca2+]cyt and [Ca2+]mito were affected by CGP-37157 but not by Ru360. Overall, these data demonstrate that in human ASM cells, mitochondria buffer [Ca2+]cyt after agonist stimulation and its enhancement by inflammation. The differential regulation of [Ca2+]mito in different parts of ASM cells may serve to locally regulate Ca2+ fluxes from intracellular sources versus the plasma membrane as well as respond to differential energy demands at these sites. We propose that such differential mitochondrial regulation, and its disruption, may play a role in airway hyperreactivity in diseases such as asthma, where [Ca2+]cyt is increased.