Involvement of Rho-kinase and tyrosine kinase in hypotonic stress-induced ATP release in bovine aortic endothelial cells

1. Hypotonic stress induces ATP release followed by Ca2+ oscillations in bo vine aortic endothelial cells (BAECs). We have investigated the cellular me chanism of the hypotonic stress-induced ATP release. 2. Hypotonic stress induced tyrosine phosphorylation of at least two protei ns, of 110 and 150 kDa. Inhibition of tyrosine kinase by the tyrosine kinas e inhibitors herbimycin A and tyrphostin 46 prevented ATP release and ATP-m ediated Ca2+ oscillations induced by hypotonic stress. 3. ATP release was also inhibited by the pretreatment of the cells with bot ulinum toxin C3, and augmented by lysophosphatidic acid. Furthermore, pre-t reating the cells with Y-27632, a selective inhibitor of Rho-kinase, also s uppressed the hypotonic stress-induced ATP release and Ca2+ oscillations, i ndicating that Rho-mediated activation of Rho-kinase may be involved in the hypotonic: ATP release. 4. Hypotonic stress also induced a transient rearrangement of the actin cyt oskeleton, which was suppressed by the tyrosine kinase inhibitors Y-27632 a nd cytochalasin B. However, pretreatment of the cell with cytochalasin B in hibited neither the hypotonic stress-induced ATP release nor the Ca2+ oscil lations. 5. These results indicate that tyrosine kinase and the Rho-Rho-kinase pathw ays are involved in hypotonic stress-induced ATP release and actin rearrang ement, but actin polymerization is not required for ATP release in BAECs.