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

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. 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. 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. 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. 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. Mechanical stress stimulates the production of biologically active mediators, gene expression and cellular alignment in vascular endothelial cells (for a review see Davies, 1995). In particular, the ‘immediate’ endothelial responses to mechanical stress (Takahashi et al. 1997), including alterations in cellular messengers, such as Ca2+ transient (Oike et al. 1994a) and the production of nitric oxide (NO) (Korenaga et al. 1994), have a significant importance for the real-time regulation of vascular tonus. We have used hypotonic stress as an in vitro example of mechanical stress in this study. Though hypotonic stress does not correspond to a particular in vivo mechanical stress loaded onto the endothelium, hypotonic cell swelling would generate some stress on the cell membrane. Actually, reported hypotonic stress-induced endothelial responses share some common characteristics with shear stress-induced ones. For instance, both of these stresses induce ATP release in endothelium (Bodin et al. 1991; Oike et al. 2000). Transient reorganization of actin cytoskeleton induced by hypotonic stress (Oike et al. 1994b) is quite similar to that induced by shear stress (Knudsen & Frangos, 1997). Furthermore, shear stress has been reported in aortic endothelium to activate chloride current (Barakat et al. 1999), which is similar to hypotonic stress-activated volume-regulated anion channel (VRAC) current (Nilius et al. 1996). Therefore, we consider that investigation of hypotonic stress-induced responses would provide significant information about endothelial mechanosensitivity. We have observed that hypotonic stress induced Ca2+ oscillations in bovine aortic endothelial cells (BAECs), which were inhibited by phospholipase C inhibitors (neomycin or U-73122), P2 antagonist (suramin) and ATP-hydrolysing agent (apyrase). We showed that ATP release of 91.5 amol cell−1 (10 min)−1 was induced by 40 % hypotonic stress (Oike et al. 2000). We have also reported that the hypotonic stress-induced, ATP release-mediated Ca2+ transient leads to NO production in BAECs (Kimura et al. 2000), thereby suggesting the potential role of this mechanism in the regulation of vascular tonus. However, the cellular mechanism by which ATP is released by hypotonic stress from endothelium has not been clarified yet. Tyrosine kinase has been reported to regulate hypotonic stress-induced activation of VRACs in bovine pulmonary endothelium by Voets et al. (1998). The same group also reported the involvement of Rho, a small G-protein, and Rho-activated protein kinase (Rho-kinase), a serine/threonine kinase, in the activation of VRACs (Nilius et al. 1999). Hypotonic stress has also been reported in epithelial cells to activate phosphatidylinositol 3-kinase (PI 3-kinase) (Tilly et al. 1996a) and mitogen-activated protein (MAP) kinase (Tilly et al. 1996b). Therefore, it would be of interest to examine the possible involvement of these intracellular signalling pathways in hypotonic stress-induced ATP release. Furthermore, the mechanism by which hypotonic stress is sensed by endothelium has not been fully clarified yet. Oike et al. (1994b) have shown the possibility that hypotonic stress-induced generation of arachidonic acid is related to the rearrangement of actin cytoskeleton in human umbilical cord endothelial cells (HUVECs). The cytoskeleton, especially actin filaments, is involved in various cellular signalling in many cell types (for a review, see Janmey, 1998). Therefore, the possible role of the actin cytoskeleton as a stress sensor in hypotonic stress-induced ATP release should be considered as well. In this study, we examined the signalling pathways that are involved in hypotonic stress-induced ATP release in BAECs. Furthermore, the possible contribution of the actin cytoskeleton to these signalling pathways was also investigated. The results show for the first time that hypotonic stress-induced ATP release is mediated by the tyrosine kinase and Rho-Rho-kinase pathways.