SWELLING-ACTIVATED K+ FLUXES IN VASCULAR ENDOTHELIAL-CELLS - ROLE OF INTRACELLULAR CA2+

Swelling of bovine aortic endothelial cells activates Ca2+-dependent K+ channels. To determine the role of Ca2+ in this response, we examined the effect of cell swelling on intracellular Ca2+ concentration ([Ca2+](i)), and the role of [Ca2+](i) in swelling-activated K+ efflux. Basal [Ca2+](i), measured by fura 2 fluorescence, was 62 nM and increased by 36 nM in hypotonic medium (220 mosmol/l) compared with a 277 nM increase in response to extracellular ATP. In cells loaded with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), the increases induced by swelling and by ATP were reduced to 13 and 20 nM, respectively. Exposure to hypotonic medium (220 mosmol/kg) or to the Ca2+ ionophore A-23187 stimulated a furosemide-insensitive Rb-86 efflux consistent with activation of K+ channels. The swelling-activated efflux was inhibited 16% by 5 mM tetraethylammonium and 24% by 23 mM tetrabutylammonium, but not by 100 mu M quinidine, a pattern similar to that previously observed for swelling-activated K+ channels in cell-attached patches. The effects of A-23187 and hypotonic swelling on Rb-86 efflux were completely additive, suggesting Ca2+-independent activation by cell swelling. Removal of Ca2+ from the external medium or loading of cells with BAPTA to buffer intracellular Ca2+ blocked the activation of Rb-86 efflux by A-23187, but not by hypotonic swelling. Hypertonic medium (440 mosmol/kg by the addition of sucrose) attenuated the increased Rb-86 efflux in response to A-23187. We conclude that the activation of K+ efflux in swollen endothelial cells occurs independently of changes in [Ca2+](i). Furthermore, the results suggest that Ca2+-dependent K+ channels in aortic endothelial cells are under dual and independent regulation by intracellular Ca2+ and cell volume.