Multiple Types of Chloride Channels in Bovine Pulmonary Artery Endothelial Cells

Abstract

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, _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_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_Clvol· 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 C^- channels in resting, nonstimulated cells. Challenging a cell with elevated [Ca²⁺]_i and hypotonic solutions activated I_Clvol on top of I_Cl_ca, suggesting that I_{Cl Ca} and I_Clvol are different channels. We conclude that CPAE cells do not express voltage-gated (CIC-type) or cAMP-gated (CFTR-type) Cl^- channels, but activate large Cl^- currents after volume (mechanical?) or chemical (Ca²⁺) stimulation.