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 bovine aortic endothelial cells (BAECs). We have investigated the cellular mechanism of the hypotonic stress-induced ATP release. 2. Hypotonic stress induced tyrosine phosphorylation of at least two proteins, of 110 and 150 kDa. Inhibition of tyrosine kinase by the tyrosine kinase inhibitors herbimycin A and tyrphostin 46 prevented ATP release and ATP-mediated Ca²⁺ oscillations induced by hypotonic stress. 3. ATP release was also inhibited by the pretreatment of the cells with botulinum toxin C3, and augmented by lysophosphatidic acid. Furthermore, pre-treating the cells with Y-27632, a selective inhibitor of Rho-kinase, also suppressed the hypotonic stress-induced ATP release and Ca²⁺ oscillations, indicating 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 cytoskeleton, which was suppressed by the tyrosine kinase inhibitors Y-27632 and cytochalasin B. However, pretreatment of the cell with cytochalasin B inhibited neither the hypotonic stress-induced ATP release nor the Ca²⁺ oscillations. 5. These results indicate that tyrosine kinase and the Rho-Rho-kinase pathways are involved in hypotonic stress-induced ATP release and actin rearrangement, but actin polymerization is not required for ATP release in BAECs.