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Modulation of human myometrial ANO1 attenuates contractile agonist induced f-actin formation and myosin light chain phosphorylation.
Abstract Number: O2-05
Abstract Type: Original Research
Calcium-activated chloride channel anoctamin 1 (ANO-1) leads to significant attenuation of human uterine smooth muscle (USM) contractility in vitro and completely abolishes intracellular calcium elevations in human myometrial cells. At a cellular level, USM contractions mechanistically result from actin-myosin cytoskeletal effects including phosphorylation of myosin light chain (MLC20) and increases in thin filamentous actin (f-actin) formation. In this study, we questioned if ANO1 antagonism modulates contractile agonist-induced f-actin formation and if siRNA targeted genetic knockdown of ANO1 similarly leads to an alteration in the contractile-regulatory protein MLC20 phosphorylation state.
Primary human myometrial cells (HuUSM) were grown to 70% confluence on sterile coated coverslips and transfected with a red fluorescent indicator (pCMVLifeAct-TagRFP) of f-actin formation according to manufacturer’s recommendations. Subsequent live cell imaging utilizing confocal microscopy allowed for real-time quantitative measurement of changes in f-actin (555/584 nm) evoked by contractile agonist (oxytocin 1uM) in the presence or absence (vehicle controls) of the ANO1 antagonist benzbromarone (100uM). In parallel studies, HuUSM cells underwent genetic editing with siRNA directed at ANO1 or scrambled siRNA (negative control) according to manufacturer’s recommendations. Treated cells were subsequently challenged with oxytocin and assessed for MLC20 phosphorylation (procontractile cellular signaling event) by protein immunoblotting. Data was compiled and analyzed by ANOVA or student t-test, and p<0.05 was taken as significant.
The ANO-1 antagonist benzbromarone (100uM) suppressed oxytocin mediated elevations in f-actin fluorescence [TRITC] by 95.1% + 6.3% (p < 0.001, N=7) compared to vehicle controls (N=5). In separate experiments, siRNA induced knock down of ANO1 reduces oxytocin-induced MLC20 phosphorylation by 59% + 5% (n=3, p < 0.05) compared to human USM cells treated with scrambled siRNA.
ANO1 channel modulation exerts positive pro-relaxant effects on two critical components of the contractile apparatus in human myometrial cells. Our results provide exciting evidence that ANO-1 antagonism exerts a pro-relaxant effect at the level of cellular actin-cytoskeletal regulatory mechanisms.
Taggart MJ, Morgan KG, Regulation of the uterine contractile apparatus and cytoskeleton. Semin Cell Dev Biol. 2007 June ; 18(3): 296–304.