Estrogen Receptors Differentially Regulates Intracellular Calcium Handling in Human Asthmatic Airway Smooth Muscle Cells
Authors: Bhallamudi Sangeeta , Connell Jennifer , Thompson Michael , Pabelick Christina , Prakash Y S , Sathish Venkatachalem 
Published: June 2021

PurposeAsthma 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 ([Ca2+]i) levels in ASM cells. However, the role of long‐term ER signaling in regulating [Ca2+]i of ASM is unknown, especially during inflammation or in asthma. Accordingly, we hypothesized that estrogen receptor isoforms have differential roles in [Ca2+]i regulation in ASM cells, with enhanced functionality during inflammation.MethodsHuman 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 (E2), 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 [Ca2+]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.ResultsIn both asthmatic and non‐asthmatic human ASM cells, ERβ agonists reduced [Ca2+]i response to BK, His, Ach as compared to vehicle. Conversely E2 and ERα agonists showed no change or a slight increase in [Ca2+]i response compared to vehicle. The effectiveness of ERβ in reducing the [Ca2+]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 [Ca2+]i response, while E2 and ERα agonists did not draw out any notable changes. To understand the mechanistic basis of the ER specific signaling in [Ca2+]i regulation, we measured L‐type calcium channel activity (using L‐type channel blocker nifedipine, 1μM) with KCl. ERβ agonist reduced KCl induced [Ca2+]i increase, which was comparable to the effect of nifedipine. [Ca2+]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.ConclusionOverall, our data highlights the presence of divergent ER signaling in [Ca2+]i handling in ASM. Specifically ERβ activation is more effective in reducing [Ca2+]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 InformationSupported 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.