DIVERGENT EFFECTS OF EXTRACELLULAR AND INTRACELLULAR ALKALOSIS ON CA2-CELLS( ENTRY PATHWAYS IN VASCULAR ENDOTHELIAL)

Modulation by alkalosis of basal leak Ca2+ entry and store-depletion-i nduced Ca2+ entry was investigated in the vascular endothelial cell li ne ECV 304. Ca2+ entry was monitored as the increase in the intracellu lar free Ca2+ concentration ([Ca2+](i)) induced by elevation of the ex tracellular Ca2+ concentration. When ECV 304 cells were challenged wit h 100 nM thapsigargin in nominally Ca2+-free solution, [Ca2+](i) incre ased transiently, and the increase in [Ca2+](i) during a subsequent cu mulative 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, b ut increased leak Ca2+ entry as well as store-depletion-activated Ca2 entry significantly. Basal Ca2+ leak and store-depletion-activated Ca 2+ 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 extrac ellular K+ (110 mM) solution, suggesting that the effects of alkalosis are independent of membrane potential. The intracellular pH (pH(i)) i ncreased slightly during both acute and chronic extracellular alkalosi s (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 administrat ion of 20 mM NH4Cl failed to stimulate, and in fact inhibited, store-d epletion-activated Ca2+ entry. Our results demonstrate that a decrease in the extracellular but not the intracellular proton concentration p romotes both basal and stimulated Ca2+ entry into endothelial cells.