Analysing of possible involvement of rho-kinase in ouabain mediated cellular responses by proteomics method

Na+/K+-ATPase is an integral membrane protein that maintains ionic balance in the cells. Ouabain, a cardiac glycoside, is a specific inhibitor of the pump. It has been shown that prolonged exposure to cells to ouabain results in an apoptotic cell death. However the exact mechanism of this effect was not completely understood. Rho kinases (Rho kinase-1 and Rho kinase-2), one of the most studied effectors of small G protein Rho, involve in several biochemical and morphological events in apoptosis. In the current work, we evaluated possible involvement of rho kinase in the ouabain-induced apoptotic cell death using a proteomic approach.Human umbilical vein endothelial cells were exposed to ouabain (10 µM) for 16 hours in the presence and absence of the specific rho kinase inhibitor Y-27632. Cell homogenates were separated by 2D gel electrophoresis and stained with coomassie blue. It was detected that 54 spots were changed with the ouabain treatment. Seven spots, changed with ouabain plus Y-27632 treatment, were excised and subjected to MALDI-TOF MS to indentify the proteins of interest. Caspase-7 and protein unc-119 homolog B proteins, increased with the ouabain treatment, were decreased by the pretreatment with Y-27632. In addition, neurensin-2, C-type lectin domain family-2 member D and putative FERM domain-containing protein FKSG43 proteins decreased with ouabain treatment, were increased by the pretreatment with Y-27632.These results indicate that rho kinase pathway may involve in the expression changes of these proteins. However, further studies are needed to confirm our results and to determine the importance of these proteins.