ENDOPLASMIC-RETICULUM CA2+ DEPLETION UNMASKS A CAFFEINE-INDUCED CA2+ INFLUX IN HUMAN AORTIC ENDOTHELIAL-CELLS

Intracellular Ca2+ pools contribute to changes in cytosolic [Ca2+] ([Ca2+](i)), which play an important role in endothelial cell signaling. Recently, endothelial ryanodine sensitive Ca2+ stores were shown to regulate agonist-sensitive intracellular Ca2+ pools. Since caffeine binds the ryanodine Ca2+ release channel on the endoplasmic reticulum in a variety of cell types, we examined the effect of caffeine on [Ca2+](i) in human aortic endothelial cell monolayers loaded with the fluorescent probe indo 1. Under baseline conditions, 10 mmol/L caffeine induced a small increase in [Ca2+](i) from 86+/-10 to 115+/-17 nmol/L (mean+/-SEM); this effect was similar to that of 5 mu mol/L ryanodine and was unaffected by buffer Ca2+ removal. After depletion of an intracellular Ca2+ store by the irreversible endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin (1 mu mol/L), ryanodine did not affect [Ca2+](i). In contrast, caffeine induced a large rapid increase in [Ca2+](i) (176+/-19 to 338+/-35 nmol/L, P<.001) after thapsigargin exposure; this effect of caffeine was only observed when extracellular Ca2+ was present. A similar increase in [Ca2+](i) was induced by caffeine after depletion of ryanodine- and histamine-sensitive Ca2+ stores or after pretreatment with the endoplasmic reticulum Ca2+-ATPase inhibitor cyclopiazonic acid (10 mu mol/L). Thus, under baseline conditions the effect of caffeine on [Ca2+](i) is similar to that of ryanodine and appears to be due to the release of an intracellular store. However, after depletion of an endoplasmic reticulum Ca2+ store, caffeine, but not ryanodine, stimulates Ca2+ influx, resulting in a large increase in [Ca2+](i). The data suggest that caffeine-induced Ca2+ influx is controlled by the status of Ca2+ loading of intracellular Ca2+ stores in human aortic endothelial cells.