Divergent effects of extracellular and intracellular alkalosis on Ca2+ entry pathways in vascular endothelial cells

Modulation by alkalosis of basal leak Ca2+ entry and store-depletion-induced Ca2+ entry was investigated in the vascular endothelial cell line ECV 304. Ca2+ entry was monitored as the increase in the intracellular free Ca2+ concentration ([Ca2+](i)) induced by elevation of the extracellular Ca2+ concentration. When ECV 304 cells were challenged with 100 nM thapsigargin in nominally Ca2+-free solution, [Ca2+](i) increased transiently, and the increase in [Ca2+](i) during a subsequent cumulative elevation of extracellular Ca2+ (from nominally Ca2+-free up to 5 mM) was markedly enhanced compared with non-stimulated cells (i.e. basal Ca2+ leak). Prolonged elevation of the extracellular pH (pH(o)) from 7.4 to 7.9 did not affect resting [Ca2+](i) or the thapsigargin-induced [Ca2+](i) transient evoked in nominally Ca2+ free solution, but increased leak Ca2+ entry as well as store-depletion-activated Ca2+ entry significantly. Basal Ca2+ leak and store-depletion-activated Ca2+ entry were enhanced either by acute elevation of pH, from 7.4 to 7.9 or by chronic alkalosis (pH(o) = 7.9). Stimulation of Ca2+ entry by extracellular alkalosis was observed both in normal and in high extracellular K+ (110 mM) solution, suggesting that the effects of alkalosis are independent of membrane potential. The intracellular pH (pH(i)) increased slightly during both acute and chronic extracellular alkalosis (from 7.22 +/- 0.01 to 7.37 +/- 0.04 and 7.45 +/- 0.05 respectively). Elevation of pH(i) to 7.60 +/- 0.06 at constant pH(o) by administration of 20 mM NH4Cl failed to stimulate, and in fact inhibited, store-depletion-activated Ca2+ entry. Our results demonstrate that a decrease in the extracellular but not the intracellular proton concentration promotes both basal and stimulated Ca2+ entry into endothelial cells.