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

Abstract

1. Hypotonic stress induces ATP release followed by Ca 2+ 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 Ca 2+ 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 Ca 2+ 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 Ca 2+ 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.

Thrombin Promotes Release of ATP from Lung Epithelial Cells through Coordinated Activation of Rho- and Ca2+-dependent Signaling Pathways

“…Ito and cowork- ers illustrated that Y26632 impaired lysophosphatidic acidand/or hypotonic challenge-promoted ATP release in human umbilical vein and bovine aortic endothelial cells (61,62). In a recent report, Blum et al (26) illustrated that inactivation of RhoGTPases with Clostridium botulinum C3 exoenzyme impaired thrombin-and lysophosphatidic acid-promoted Ca 2ϩ -dependent ATP release from 1321N1 astrocytoma cells .…”

ATP release from non-excitable cells

“…The major argument was that blockers of VSOAC currents also block ATP release [66]. Interesting follow-up work by the same authors found that swelling-induced endothelial ATP release involves Rhokinase and tyrosine-kinase [74] and suggests the following sequence of events: Cell swelling activates the RhoA/Rhokinase pathway followed by tyrosine phosphorylation of FAK and paxillin, which subsequently leads to ATP release and actin reorganisation [75]. In summary, volumeactivated anion conductances continue to be putative candidates for swelling-induced ATP secretion but this issue is far from being resolved.…”

“…As externally applied ATP is rapidly broken down by ecto-ATPases on the cell plasma membranes, the stable extracellular ATP levels must reflect a steady-state situation of tonic ATP release balanced by ATP degradation at the same rate [19,32,56,74]. Tonic ATP release can be unmasked by nonspecific inhibition of ecto-ATPases.…”

On the role of ATP release, ectoATPase activity, and extracellular ADP in the regulatory volume decrease of Huh-7 human hepatoma cells

“…In principle ATP can be released both by a conductive channel-mediated pathway, driven by the steep electrochemical gradient of ATP through the plasma membrane, and by vesicular transport via exocytosis (19,22,23,40). In liver cells, increases in cell volume of both cholangiocytes and human cholangiocarcinoma trigger parallel changes in vesicular exocytosis and ATP release, whereas in rat hepatoma cells intracellular ATP is localized in discrete vesicles originated from a bafilomycin A1-sensitive pool that are depleted by hypotonic exposure, thus implying an important role of exocytosis in regulated release of ATP from hepatic cells (22).Examples of ATP-induced volume regulation were observed in hepatocytes, hepatoma (21,23,61), endothelial cells, astrocytes, and epithelial cells from human and several other mammalian species (6,7,36). Similarly, swollen cells could be induced to downregulate their volume in the presence of nucleotides other than ATP, such as UTP, UDP, or ATP␥S (but not ADP) in fish hepatocytes (9, 10 17, 49), and UTP in salivary gland duct cells (33).…”

“…Although many intracellular signaling processes of cell volume regulation are now well characterized (29), comparatively little is known regarding the extracellular factors controlling RVD (14,48,49). In this respect, mechanical stimuli such as shear stress (7), mechanical strain (55), and hypotonic stress (45) have been shown to induce the release of ATP in the absence of lysis from a variety of cells (36,44,51,52,61), whereas addition of exogenous ATP to hypotonic cells suspensions was able to stimulate RVD (32,49). In principle ATP can be released both by a conductive channel-mediated pathway, driven by the steep electrochemical gradient of ATP through the plasma membrane, and by vesicular transport via exocytosis (19,22,23,40).…”