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Calcium activated chloride channels: critical mediators of calcium dynamics in human myometrial cells
Abstract Number: GM-02
Abstract Type: Original Research
Objective: Myometrial contraction is intimately dependent on calcium. In the pregnant myometrium, an initial intracellular calcium wave is spread throughout the uterus via gap junctions . The resultant increased calcium levels activate myosin light chain kinase and promote myosin-actin bridging, allowing for cytoskeletal contraction. Two mechanisms govern the initial elevation in intracellular calcium: membrane depolarization and GPCR induced sarcoplasmic release. Based on prior observations that calcium activated chloride channels (CaCCs) participate in the propogation of myometrial contractions, and our prior observations that CaCC blockade completely abolishes intracellular calcium elevations in human myometrial cells, we hypothesize that CaCCs are central to modulation of calcium dynamics in the human myometrium. We sought to determine the role CaCCs play in these two integral mechanisms of intracellular calcium handling: cell membrane depolarization, and GPCR-mediated SR calcium release.
Methods: Immortalized human myometrial cells were grown to confluence in 96 well plates. In all experiments, fluorescence was measured on a Flexstation 3 plate reader and drugs were injected in real time. For GPCR-mediated SR release studies, cells were loaded with two fluorescent calcium indicators to obtain differential cytosolic (Fura-2) and sarcoplasmic (Mag-Fluo-4) calcium loading. GPCR agonists (10uM bradykinin or 1uM oxytocin) were injected in the presence and absence of escalating doses of a specific CaCC antagonist (10uM, 25uM, 50uM, and 100uM benzbromarone). For membrane potential studies, cells were loaded with the potential indicator FLIPR and injected with either a CaCC antagonist (50uM benzbromarone) or K-gluconate 40mM (positive depolarizing control) or NS-1619 10uM (positive hyperpolarizaing control). Data is reported as percentage of mean inhibition vs control or mean fluorescence (RFU) and SEM. In all cases p<0.05 was considered significant.
Results: The CaCC antagonist benzbromarone (100uM) suppressed bradykinin mediated peak Fura-2 fluorescence [Cai] by 85.1% (p<0.001, N=7) and nadir Mag-Fluo-4 fluorescence [CaSR] by 92.2% (p<0.001, N=7); 100uM benzbromarone suppressed oxytocin mediated Fura-2 peak by 77.6% (p<0.001; N=5). In FLIPR studies, CaCC modulation resulted in significant changes in membrane potential. Eact significantly depolarized the membrane (25.33±11.6 RFU) paralleling the effect of K-gluconate (98±28.57 RFU) compared to vehicle control (3.2±0.6 RFU, p<0.05). Similarly, benzbromarone hyperpolarized the myometrial cell membrane (-25.67±4.9 RFU; p<0.05) paralleling the effect from NS1619 (-39±1.5 RFU) compared to vehicle control.
Conclusions: CaCCs are integral to the 2 central aspects of human myometrial calcium handling: GPCR-mediated SR calcium release, and membrane potential modulation. Our results suggest the exciting possibility that CaCC antagonisim represents a novel means to achieve tocolysis.