A STUDY ON THAPSIGARGIN-INDUCED CALCIUM-ION AND CATION INFLUX PATHWAYS IN VASCULAR ENDOTHELIAL-CELLS

To investigate Ca2(+)/cation entry pathway in vascular endothelial cel ls, we examined the effects of thapsigargin on [Ca2+](i) and Mn2+ entr y in cultured porcine aortic endothelial cells. Thapsigargin inhibits the activity of endoplasmic reticulum (ER, intracellular Ca2+ pool) Ca 2+-ATPase, and stimulates Ca2+ entry from extracellular space by deple ting intracellular Ca2+ pool. Cultured endothelial cells were loaded w ith fura-2/AM, and [Ca2+](i) was measured by the ratios of fluorescenc e at 340/380 nm excitation, and Mn2+ entry was observed by the quenchi ng of fluorescence at 360 nm excitation. Thapsigargin elevated [Ca2+]( i) in a time- and dose-dependent manner. The increase in [Ca2+](i) cau sed by thapsigargin was lowered in Ca2+-free solution containing 3 mM EGTA. Verapamil(10(-5) M) and equimolar replacement of extracellular N aCl by LiCl had no effects on the maximum elevation of [Ca2+](i) by th apsigargin. The increase in [Ca2+](i) by thapsigargin was significantl y inhibited by either NiCl2 (10(-3) M) or membrane depolarization usin g 50 mM KCl. Thapsigargin stimulated Mn2+ entry concomitantly with the increase in [Ca2+](i). Mn2+ entry was augmented in Ca2+-free solution . These results suggested that (1) the increase in [Ca2+](i) by thapsi gargin consisted of both Ca2+ release from ER and Ca2+ entry from extr acellular space, and (2) thapsigargin also stimulated Mn2+ entry, whic h was interfered with in the presence of extracellular Ca2+.