We have investigated the effects of hypotonic stress on intracellular calci
um concentration ([Ca2+](i)) in bovine aortic endothelial cells. Reducing e
xtracellular osmolarity by 5% to 40% elicited a steep Ca2+ transient both i
n normal Krebs and Ca2+-free solutions. The hypotonic stress-induced Ca2+ t
ransient was inhibited by phospholipase C inhibitors (neomycin and U-73122)
, a P-2-receptor antagonist (suramin), and an ATP-hydrolyzing enzyme (apyra
se), suggesting that the hypotonic stress-induced Ca2+ transient is mediate
d 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 grad
ual [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 obse
rvations indicate that hypotonic stress induces a dual Ca2+ response in bov
ine aortic endothelial cells, i.e., an ATP-mediated fast Ca2+ transient and
an arachidonic acid-mediated gradual Ca2+ increase, the former being the p
redominant response in normal conditions.