Hypotonic stress-induced dual Ca2+ responses in bovine aortic endothelial cells

We have investigated the effects of hypotonic stress on intracellular calcium concentration ([Ca2+](i)) in bovine aortic endothelial cells. Reducing extracellular osmolarity by 5% to 40% elicited a steep Ca2+ transient both in normal Krebs and Ca2+-free solutions. The hypotonic stress-induced Ca2+ transient was inhibited by phospholipase C inhibitors (neomycin and U-73122), a P-2-receptor antagonist (suramin), and an ATP-hydrolyzing enzyme (apyrase), suggesting that the hypotonic stress-induced Ca2+ transient is mediated by ATP. A luciferin-luciferase assay confirmed that 40% hypotonic stress released 91.0 amol/cell of ATP in 10 min. When the hypotonic stress-induced fast Ca2+ transient was inhibited by neomycin, suramin, or apyrase, a gradual [Ca2+](i) increase was observed instead. This hypotonic stress-induced gradual [Ca2+](i) increase was inhibited by a phospholipase A(2) inhibitor, 4-bromophenacyl bromide. Furthermore, exogenously applied arachidonic acid induced a gradual [Ca2+](i) increase with an ED50 of 13.3 mu M. These observations indicate that hypotonic stress induces a dual Ca2+ response in bovine aortic endothelial cells, i.e., an ATP-mediated fast Ca2+ transient and an arachidonic acid-mediated gradual Ca2+ increase, the former being the predominant response in normal conditions.