Citations

AbstractAirway smooth muscle (ASM) cells modulate the local airway milieu via production of inflammatory mediators and growth factors including classical neurotrophins, such as brain‐derived neurotrophic factor (BDNF). The glial cell‐derived neurotrophic factor (GDNF) family of ligands (GFLs) are nonclassical neurotrophins and their role in the airway is barely understood. The major GFLs, GDNF and Neurturin (NRTN) bind to GDNF family receptor (GFR) α1 and α2 respectively that pair with Ret receptor to accomplish signaling. In this study, we found GDNF is expressed in human lung and increased in adult asthma, while human ASM expresses GDNF and its receptors. Accordingly, we used human ASM cells to test the hypothesis that ASM expression and autocrine signaling by GFLs regulate [Ca2+]i. Serum‐deprived ASM cells from non‐asthmatics were exposed to 10 ng/ml GDNF or NRTN for 15 min (acute) or 24 h (chronic). In fura‐2 loaded cells, acute GDNF or NRTN alone induced [Ca2+]i responses, and further enhanced responses to 1 µM ACh or 10 µM histamine. Ret inhibitor (SPP86; 10 µM) or specific GDNF chelator GFRα1‐Fc (1 µg/ml) showed roles of these receptors in GDNF effects. In contrast, NRTN did not enhance [Ca2+]i response to histamine. Furthermore, conditioned media of nonasthmatic and asthmatic ASM cells showed GDNF secretion. SPP86, Ret inhibitor and GFRα1‐Fc chelator markedly decreased [Ca2+]i response compared with vehicle, highlighting autocrine effects of secreted GDNF. Chronic GDNF treatment increased histamine‐induced myosin light chain phosphorylation. These novel data demonstrate GFLs particularly GDNF/GFRα1 influence ASM [Ca2+]i and raise the possibility that GFLs are potential targets of airway hyperresponsiveness.

Purpose Asthma prevalence has been shown higher in female as compared to male, strongly indicating the involvement of sex steroids in modulating airway hyperreactivity. Airway smooth muscle (ASM) cells are major structural cells of the airway that determine the airway tone. Our recent study demonstrated the presence of estrogen receptors (ERα and ERβ) in ASM cells and that both ER's are significantly upregulated during asthma and/or inflammation with pronounced ERβ expression. We previously reported that estrogen acutely reduces agonist‐induced intracellular calcium ([Ca 2+ ] i ) levels in ASM cells. However, the role of long‐term ER signaling in regulating [Ca 2+ ] i of ASM is unknown, especially during inflammation or in asthma. Accordingly, we hypothesized that estrogen receptor isoforms have differential roles in [Ca 2+ ] i regulation in ASM cells, with enhanced functionality during inflammation. Methods Human ASM cells were enzymatically dissociated from tissues obtained incidental to lung surgery (Mayo Clinic). Asthmatic and non‐asthmatic ASM cells were exposed to pro‐inflammatory cytokines TNFα (20ng/ml) or IL‐13 (50ng/ml) in the presence or absence of 17β‐estradiol (E 2 ), various ERα (PPT, THC) or ERβ (WAY, FERB, DPN) agonists and antagonists (MPP,PHTPP) for 24h. Cells were incubated with calcium indicator dye Fura‐2 AM and [Ca 2+ ] i responses in these cells were measured using various bronchoconstrictors such as bradykinin (BK,10nM), acetylcholine (Ach,1μM), histamine (His,10μM) and KCl (100μM) under Olympus epifluorescence imaging system. Results In both asthmatic and non‐asthmatic human ASM cells, ERβ agonists reduced [Ca 2+ ] i response to BK, His, Ach as compared to vehicle. Conversely E 2 and ERα agonists showed no change or a slight increase in [Ca 2+ ] i response compared to vehicle. The effectiveness of ERβ in reducing the [Ca 2+ ] i in human ASM cells was further confirmed by ERβ siRNA and ERβ overexpression studies. In the presence of inflammation induced by TNFα or IL‐13, ERβ agonists significantly reduced [Ca 2+ ] i response, while E 2 and ERα agonists did not draw out any notable changes. To understand the mechanistic basis of the ER specific signaling in [Ca 2+ ] i regulation, we measured L‐type calcium channel activity (using L‐type channel blocker nifedipine, 1μM) with KCl. ERβ agonist reduced KCl induced [Ca 2+ ] i increase, which was comparable to the effect of nifedipine. [Ca 2+ ] i reuptake pump (SERCA) which sequesters the cytosolic calcium into sarcoplasmic reticulum showed increased expression and activity in ASM cells with ERβ activation during inflammation. Conclusion Overall, our data highlights the presence of divergent ER signaling in [Ca 2+ ] i handling in ASM. Specifically ERβ activation is more effective in reducing [Ca 2+ ] i during inflammation, through both L‐type channel inhibition and increased SERCA activity. These varied signaling mechanisms could play a crucial role in smooth muscle contractility and tone in asthma. Support or Funding Information Supported by NIH grants R01 HL123494 (Venkatachalem), and R01 HL088029 (Prakash). This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Background The pathophysiology of asthma involves inflammation and altered structure and function of airway smooth muscle (ASM). There is emerging evidence that asthma affects adult women disproportionately with more severity than men. Also, our recent findings suggest that differential estrogen receptors (ER), ERα and ERβ expression and function are present in asthmatic ASM cells. Due to high oxygen environment in the lungs and the presence of inflammation, asthmatic ASM cells undergo oxidative stress. Mitochondrial dysfunction due to this oxidative stress plays a crucial role in the bioenergetics pathways involved in asthma. In this study, we hypothesize that differential ER signaling affects mitochondrial dynamics (fission/fusion) in ASM, which subsequently affects reactive oxygen species (ROS) generation and intracellular Ca 2+ ([Ca 2+ ] i ) buffering capacity of mitochondria. Methods Human ASM cells were enzymatically dissociated from lung samples of non‐asthmatics undergoing thoracic surgery at Mayo Clinic. Cells were grown onto 8‐well labtek plates and treated with pro‐inflammatory cytokine, TNFα (20 mg/mL) in the presence or absence of 1 nM 17β‐estradiol (E 2 ) which is a nonselective ER agonist, 10 nM PPT (ERα agonist) or 10 nM WAY (ERβ agonist) for 24h. For morphology studies, the cells were loaded with Mitotracker Green and then imaged on the Bio‐Tek Lionheart FX Live Cell Imager. Form factor and aspect ratio are used as parameters for determining fission vs fusion in mitochondria and these were calculated using MATLAB. Western blot and qPCR techniques were performed to access the protein and gene expression of the fission and fusion proteins Dynamin Related Protein1 (Drp1) and Mitofusion2 (Mfn2). ROS measurement was performed by loading cells with MitoSOX Red and their fluorescence intensity was recorded on the Live Cell Imager. Results In mitotracker loaded ASM cells, ERβ agonist WAY was effective in increasing the form factor and aspect ratio as compared to vehicle. In addition, ERβ was effective in reducing TNFα induced reduction in form factor and aspect ratio. However, both E 2 and ERα agonist, PPT, did not show any changes in form factor and aspect ratio of TNFα exposed ASM cells. Western analysis and gene expression studies showed higher expression of Drp1 and lower expression of Mfn2 in TNFα treated cells. Pre‐treatment with ERβ‐agonist reversed the altered expression of Drp1 and Mfn2 in TNFα treated cells. Using MitoSOX Red, it was observed that ERβ agonist WAY treated cells showed lower ROS generation as compared to TNFα. Conclusion Our data suggests a novel estrogen signaling in ASM cells for maintaining the structure of mitochondria. ERβ is effective in ameliorating the inflammation due to TNFα and hence preserves the morphology of mitochondria during inflammation. Further studies will confirm the subsequent alterations in mitochondrial ROS generation and [Ca 2+ ] i buffering capacity due to this altered mitochondrial morphology. Support or Funding Information Supported by the UGSRF fellowship by APS (Jesme) and NIH grants R01 HL123494, (Venkatachalem), and R01 HL 088029 (Prakash) This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Neurotrophins are growth factors traditionally associated with neuronal structure and function; however, neurotrophins and their receptors have been found throughout the lung, including airway, pulmonary artery and endothelium although their roles within the lung are still under investigation. We tested the hypothesis that brain derived neurotrophic factor (BDNF) enhances pulmonary artery constriction. In endothelium‐intact arterial rings, BDNF (acute 10nM, 1h; chronic 1nM, 18h) did not affect phenylephrine‐induced contraction, but enhanced subsequent ACh‐induced relaxation. These effects were blunted by endothelium‐denudation. In fura‐2 loaded human pulmonary artery smooth muscle cells, acute and chronic BDNF decreased the Ca 2+ responses to 10 μM bradykinin. In human pulmonary artery exposed to TNFα (50 ng/mL, 18h), BDNF levels were decreased, compared to human airway, where previous studies under identical treatment resulted in increased levels. These data refute our hypothesis and suggest that neurotrophins, working via endothelial cells, enhance vasodilation in the pulmonary vasculature. Supported by FAMRI, NIH grant UL1RR024150, and the Department of Anesthesiology, Mayo Clinic.

Gender disparity in incidence and severity of asthma is clinically recognized. While estrogen‐induced vasodilation is well‐known, effects on airway smooth muscle (ASM) are controversial. In human ASM, we found that both estrogen receptors (ERs), ERα‐66 and ERβ‐47 are expressed, as are two truncated ERα isoforms (ERα‐36 and ERα‐47). In fura‐2 loaded human ASM cells, acute exposure to 17β‐estradiol (17βE 2 ; 1nM) blunts intracellular calcium ([Ca 2+ ] i ) response to ACh and histamine, effects prevented by ER antagonist. We tested the hypothesis that ERα is the main receptor involved in nongenomic Ca 2+ regulation. Real‐time calcium imaging showed greater decrease in [Ca 2+ ] i with ERα specific agonist R,R THC (10nM) compared to 17βE 2 . TNFα (50ng/ml; 24h) upregulated ERα‐66 but downregulated ERα‐36 expression. TNFα‐induced increase in its receptor TNFRI was blunted by 17βE 2 . Finally, TNFα‐induced enhancement of [Ca 2+ ] i response to histamine was attenuated by ERα activation. These data suggest that in human ASM: 1) nongenomic [Ca 2+ ] i regulation by E 2 involves ERα; b) ERα activation may be beneficial in attenuating inflammatory response to TNFα. Overall, ERs may be a novel therapeutic target in airway diseases such as asthma. Supported by Mayo Graduate School and NIH grant UL1RR024150.

TSLP is a newly‐identified cytokine thought to be involved in airway inflammation. TSLP levels have been shown to significantly increase in airway smooth muscle (ASM) with chronic obstructive pulmonary disease and other inflammatory diseases. This study evaluated the expression of TSLP in human ASM cells and tissues exposed to cigarette smoke extract (CSE) or to pro‐inflammatory cytokines TNF‐α and IL‐13. In parallel studies, TSLP expression was evaluated in house dust mite sensitized mice with or without cigarette smoke exposure. The presence of TSLP and TSLP‐receptors were verified using Western blot analysis. Exposure to TNF‐α and IL‐13 (50 ng/mL each) for 24 hours resulted in increased TSLP and TSLP‐R expression. Simultaneous exposure to functional TSLP antibody blunted cytokine induced TSLP‐R expression. Interestingly, acute CSE (30 min) down‐regulated TSLP expression, while chornic exposure increased expression. Similarly, [Ca 2+ ] i responses of human ASM to histamine were blunted with chronic CSE exposure, effects reversed by TSLP and TSLP‐R antibodies. Sensitized mice demonstrated an increase in TSLP expression in whole lung samples. This effect was even further enhanced in dust mite sensitized mice exposed to chronic cigarette smoke. These results indicate a complex role for TSLP in ASM inflammation with or without cigarette smoke exposure. Supported by NIH grants HL088029, HL090595, RR024150, and the Flight Attendants Medical Research Institute (FAMRI). Additional funding from the Department of Anesthesiology, Mayo Clinic.

Airway inflammation leads to increased intracellular Ca 2+ ([Ca 2+ ] i ) in airway smooth muscle (ASM). There is now growing evidence that the bidrectional plasma membrane Na + /Ca 2+ exchange (NCX) may contribute to ASM [Ca 2+ ] i regulation. However, the roles of NCX‐mediated Ca 2+ influx vs. efflux, or the role of NCX in airway inflammation are largely unknown. In the present study, we examined NCX expression and function in human ASM exposed to the pro‐inflammatory cytokines TNFα and IL‐13. Western blot analysis of human ASM tissue showed significant expression of the NCX1 isoform, with substantial increase in expression following overnight cytokine exposure. In fura‐2 loaded human ASM cells, NCX‐mediated Ca 2+ influx as well as efflux (measured as slopes of changes in [Ca 2+ ] i ) was elicited using techniques previously described for cardiac muscle. Contribution of NCX was verified by measuring [Na + ] i using the fluorescent Na + indicator SBFI. NCX‐mediated influx was verified by demonstrating prevention of rising [Ca 2+ ] i or falling [Na + ] i in the presence of the NCX inhibitor KBR7943. In second set of studies, NCX siRNA transfection of human ASM substantially blunted efflux and influx mode of NCX (siRNA effect verified by Western blot). Exposure to TNF‐α as well as IL‐13 significantly enhanced influx mode of NCX. Although both cytokines also increased efflux mode of NCX, these effects were relatively smaller. These data suggest that NCX may be one mechanism contributing to inflammation‐induced enhancement of [Ca 2+ ] i. Supported by NIH grants HL74309, RR024150, and the Flight Attendants Medical Research Institute (FAMRI). Additional funding from the Department of Anesthesiology, Mayo Clinic.

In airway smooth muscle (ASM), the state of sarcoplasmic reticulum (SR) Ca 2+ stores is an important determinant of [Ca 2+ ] i response to agonist. We have previously shown that acetylcholine (ACh) induces sustained [Ca 2+ ] i oscillations representing repetitive SR Ca 2+ release and reuptake. Thus, SR refilling is a key component in refilling of SR stores. We hypothesized that, like cardiac and vascular smooth muscle, ASM SR refilling is regulated by calmodulin (CaM)‐CaM kinase (CaMKII) dependent phosphorylation of phospholamban (PLB). In ASM cells from fresh porcine trachea, effects of W7 (CaM antagonist) and KN‐93/KN‐62 (CaMKII antagonists) on [Ca 2+ ] i responses to ACh were measured using fluorescence microscopy. Separately, SR‐enriched membranes were isolated to monitor ATP‐energized Ca 2+ uptake and in vitro phosphorylation of PLB in the presence of CaMKII inhibitors. KN‐93 significantly slowed decay of [Ca 2+ ] i responses, and slowed or inhibited ongoing [Ca 2+ ] i oscillations, with visible slowing of fall‐times of individual oscillations. Pre‐exposure to W7 or KN‐62 did not prevent initiation of oscillations. In SR‐enriched membranes, addition of KN‐93/KN‐62 resulted in significantly slower Ca 2+ uptake, accompanied by a matching decrement in the in vitro phosphorylation of Thr 17 ‐PLB, indicating the influence of CaM‐CaMKII on this protein. These data suggest that agonist‐induced [Ca 2+ ] i regulation in ASM involves the CaM‐CaMKII pathway which indirectly influences Ca 2+ release and reuptake, likely through PLB. Supported by NIH grant HL74309 (GCS)

Asthma is a disease characterized by airway hyperresponsiveness and inflammation. One‐third of pregnant women with pre‐existing disease experience a decrease in symptoms during the third trimester, when estrogen levels are highest. Previous studies have shown that estrogens relax vascular smooth muscle, however, little is known about estrogen effects on airway smooth muscle (ASM). We tested the hypothesis that estrogen modulates human ASM contractility. Western blot analysis and immunohistochemical studies showed the presence of both full‐length and truncated isoforms of ERα and β. Furthermore, ER isoforms were localized to caveolae. Caveolin‐1 siRNA downregulated ERα isoform in human ASM. Exposure to the pro‐inflammatory cytokine TNFα substantially increased ERα expression in ASM. In fura‐2 loaded human ASM cells, acute exposure to biologically‐active 17β‐estradiol decreased the intracellular Ca 2+ response to contractile agonists such as histamine and bradykinin; this effect was blunted by the ER antagonist ICI 182, 780. Overall, these data suggest that estrogens may have a bronchodilatory effect. Accordingly, increased ER expression with airway inflammation may actually be protective during pregnancy in that bronchodilation resulting from higher estrogen levels (acting via ERs) may alleviate the increased ASM contractility of asthma. Supported by the Mayo Graduate School and NIH grant UL1RR024150‐01(Mayo Clinic CTSA awards to YSP and CMP)

Store operated calcium entry (SOCE) is important for regulation of intracellular Ca 2+ ([Ca 2+ ] i ) in neurons. Recent studies in other cell type suggest that stromal interaction molecule (STIM1) triggers SOCE. We examined the role of STIM1 in the sensitivity of SOCE to temperature, pH and glutamate in differentiated, fura‐2 loaded rat pheochromocytoma (PC12) cells, a model for neuronal cells. In STIM1 siRNA‐transfected vs. control cells, SOCE was triggered by introduction of extracellular Ca 2+ following endoplasmic reticulum depletion via cyclopiazonic acid in zero Ca 2+ . Cells were washed and exposed to altered pH (6.0–8.0), altered temperature (34–40 o C) or different concentrations of glutamate. In controls, SOCE was progressively inhibited by decreased pH or temperature, while alkalosis and increased temperature stimulated Ca 2+ influx. Increasing glutamate concentrations progressively inhibited SOCE. siRNA inhibition of STIM1 substantially blunted both temperature sensitivity (especially increased temperature) and pH sensitivity (especially acidic pH) of SOCE. These data show that SOCE in PC12 cells is susceptible to acidosis, temperature, and agonist stimulation: factors that can adversely affect neuronal function under pathological conditions. In this regard, the SOCE triggering protein STIM1 may play a role in such susceptibility. This work has been supported by NIH grants UL1RR024150‐01(CR to CMP, CR20 and Early Career Development Award to YSP), and the Department of Anesthesiology (CMP, YSP), Mayo Clinic College of Medicine, Rochester, MN