Key result
Extracellular but not intracellular alkalosis promotes basal and stimulated Ca2+ entry into vascular endothelial cells.
Why the study?
The effects of extracellular versus intracellular alkalosis on basal and store-depletion-activated Ca2+ entry in vascular endothelial cells were unclear.
Extracellular and intracellular alkalosis exert divergent effects on calcium entry pathways in vascular endothelial cells, with extracellular alkalosis promoting and intracellular alkalosis inhibiting calcium entry.
Divergent extra- vs intracellular alkalosis effects on endothelial Ca2+ entry; leaves open relevance to vascular tone in vivo.
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 (pHo) 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 pHo from 7.4 to 7.9 or by chronic alkalosis (pHo=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 (pHi) 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 pHi to 7.60+/-0.06 at constant pHo 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.
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Wakabayashi et al. (1997) studied Vascular endothelial cell line ECV 304. Extracellular and intracellular alkalosis vs. Normal pH (pHo 7.4) was evaluated on Ca2+ entry (basal leak and store-depletion-induced). A decrease in extracellular, but not intracellular, proton concentration promotes both basal and stimulated Ca2+ entry into vascular endothelial cells.
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