Endothelin‐1 (ET‐1)‐induced depolarization in chronically hypoxic (CH) rat pulmonary arterial smooth muscle cells (PASMCs) occurs via activation of Ca2+‐activated Cl− (ClCa) channels

Under normoxic conditions, ET‐1‐induced contraction of rat PASMCs has been shown to be initiated by inhibition of K + channels, leading to depolarization and activation of voltage‐gated Ca 2+ channels. However, no change in membrane potential (E m ) in response to ET‐1 was observed in CH rat PASMCs where E m was measured by whole‐cell patch‐clamp with the addition of BAPTA (Ca 2+ chelator), which buffered intracellular Ca 2+ ([Ca 2+ ] i ). With the use of the E m ‐sensitive dye, DiBac4(3), we observed depolarization in PASMCs from CH rats (10% O 2 ; 3 weeks) in response to ET‐1 (10 −8 M) when [Ca 2+ ] i was not buffered. To test whether this depolarization occurred secondary to an increase in [Ca 2+ ] i , perhaps via activation of Cl Ca channels, we measured the effect of ET‐1 on E m in the presence and absence of extracellular Ca 2+ , BQ‐123, a selective ET A receptor antagonist, or the Cl Ca channel inhibitors, niflumic acid (NA) or DIDS. We found that ET‐1 caused a significant increase in DiBac4(3) fluorescence, similar to that observed with 100 mM KCl. Consistent with our previous results, pretreatment of cells with BAPTA decreased the change in fluorescence in response to ET‐1 but not KCl. Removal of extracellular Ca 2+ or treatment with BQ‐123, DIDS or NA all significantly reduced the ET‐1‐induced change in fluorescence. In contrast, the fluorescence change in response to KCl was unaltered. These data suggest that ET‐1‐induced depolarization of CH PASMCs is dependent upon activation of Cl Ca channels, secondary to Ca 2+ influx initiated by the binding of ET‐1 to the ET A receptor. Funded by: HL67191, HL67919, and HL07963