Inhibition of Rho Kinase by Y27632 Increases Low Oxygen Tension‐Induced ATP Release from Human Red Blood Cells (RBCs)

Abstract

Mechanical deformation and low oxygen (O2) tension were reported to stimulate ATP release from RBCs via a signaling pathway that includes the G protein Gi. Stiffening of the RBC with diamide, which promotes cross linking of cytoskeletal proteins, resulted in decreased low O2 tension‐induced ATP release. In the RBC, spectrin molecules must fold for deformation to occur. Phosphorylation of adducin and moesin by Rho kinase may stabilize cytoskeletal‐membrane interactions. Rho kinase signaling has been linked to increased stiffness of fibroblasts, ventricular myocytes, airway smooth muscle cells and endothelial cells. We hypothesized that Rho kinase inhibits low O2 tension‐induced ATP release from RBCs by stiffening the membrane cytoskeleton. We examined the effect of the Rho kinase inhibitor, Y27632 (1 uM), on low O2 tension‐induced ATP release from RBCs. In human RBCs, ATP release was increased in the presence of Y27632 (p<0.01, n=13). To support the hypothesis that ATP release is not the result of a direct effect of Y27632 to inhibit the activity of Gi, RBCs were incubated with Mastoporan 7, a direct Gi activator. Y27632 had no effect on Mastoporan 7‐ induced ATP release (n=3). These results are consistent with the hypothesis that Rho kinase inhibition increases RBC deformability resulting in augmented ATP release in response to exposure of these cells to low O2 tension. (NIH grant HL089094)