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 μmol/L ryanodine and was unaffected by buffer Ca²⁺ removal. After depiction of an intracellular Ca²⁺ store by the irreversible endoplasmic reticulum Ca²⁺-ATPase inhibitor thapsigargin (1 μmol/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 depiction of ryanodine- and histamine-sensitive Ca²⁺ stores or after pretreatment with the endoplasmic reticulum Ca²⁺-ATPase inhibitor cyclopiazonic acid (10 μmol/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 depiction 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.