Multiple types of chloride channels in bovine pulmonary artery endothelial cells

We have characterized two different types of Cl- currents in calf pulmonary artery endothelial (CPAE) cells by using a combined patch-clamp and Fura-2 microfluorescence technique to measure simultaneously ionic currents and the intracellular Ca2+ concentration. [Ca2+](i). Exposure of CPAE cells to 28% hypotonic solution induces cell swelling without a change in membrane capacitance and [Ca2+](i), and concomitantly activates a current, This current, I-Cl,I-vol, is closely correlated with the changes in cell volume and shows a modest outward rectification, It slowly inactivates at potentials more positive than +60 mV but is time- and voltage-independent at other potentials. Increase in [Ca2+](i) by different maneuvers, such as application of vasoactive agonists (ATP), ionomycin, or loading of the cells directly with Ca2+ also activates a Cl- current, I-Cl,I-Ca. This current slowly activates at positive potentials, inactivates quickly at negative potentials and shows strong outward rectification, A time-independent component of the current activated by elevation of [Ca2+](i) alone can be inhibited by cell shrinking by exposing the cells to hypertonic solution, indicating that an increase in [Ca2+](i) also co-activates I-Cl,I-vol. Forskolin or cAMP never activated a current in CPAE cells, which indicates the lack of cAMP-activated channels in these cells, There is also no evidence for the existence of voltage-gated Cl- channels in resting, nonstimulated cells, Challenging a cell with elevated [Ca2+](i) and hypotonic solutions activated I-Cl,I-vol on top of I-Cl,I-Ca, suggesting that I-Cl,I-Ca and I-Cl,I-vol are different channels, We conclude that CPAE cells do not express voltage-gated (ClC-type) or cAMP-gated (CFTR-type) Cl- channels, but activate large Cl- currents after volume (mechanical?) or chemical (Ca2+) stimulation.