Capacitative Ca2+ entry in vascular endothelial cells is mediated via pathways sensitive to 2 aminoethoxydiphenyl borate and xestospongin C

Abstract

1. Agonists increase endothelial cell intracellular Ca2+, in part, by capacitative entry, which is triggered by the filling state of intracellular Ca2+ stores. It has been suggested that depletion of endoplasmic reticulum (ER) Ca2+ stores either leads to a physical coupling between the ER and a plasma membrane channel, or results in production of an intracellular messenger which affects the gating of membrane channels. As an axis involving the IP3 receptor has been implicated in a physical coupling mechanism the aim of this study was to examine the effects of the putative IP3 receptor antagonists/modulators, 2 aminoethoxydiphenyl borate (2APB) and xestospongin C, on endothelial cell Ca2+ entry. 2. Studies were conducted in fura 2 loaded cultured bovine aortic endothelial cells and endothelial cells isolated from rat heart. 3 2APB (30-300 μM) inhibited Ca2+ entry induced by both agonists (ATP 1 μM, bradykinin 0.1 μM) and receptor-independent mechanisms (thapsigargin 1 μM, ionomycin 0.5 and 5 μM). 2APB did not diminish endothelial cell ATP-induced production of IP3 nor effect in vitro binding of [3H]-IP3 to an adrenal cortex binding protein. Capacitative Ca2+ entry was also blocked by disruption of the actin cytoskeleton with cytochalasin (100 nM) while the initial Ca2+ release phase was unaffected. 4. Similarly to 2APB, xestospongin C (3-10 μM) inhibited ATP-induced Ca2+ release and capacitative Ca2+ entry. Further, xestospongin C inhibited capacitative Ca2+ entry induced by thapsigargin (1 μM) and ionomycin (0.5 μM). 5. The data are consistent with a mechanism of capacitative Ca2+ entry in vascular endothelial cells which requires (a) IP3 receptor binding and/or an event distal to the activation of the ER receptor and (b) a spatial relationship, dictated by the cytoskeleton, between Ca2+ release and entry pathways.