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 P₂-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 A₂ inhibitor, 4-bromophenacyl bromide. Furthermore, exogenously applied arachidonic acid induced a gradual [Ca²⁺](i) increase with an ED₅₀ of 13.3 μM. These observations indicate that hypotonic stress induces a dual Ca²⁺ response in bovine aortic endothelial ceils, 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.