Proteomic Analysis of Vascular Smooth Muscle Cells Treated With Ouabain

Apoptosis of vascular smooth muscle cells (VSMC) plays an important role in remodeling the vessel walls, one of the major determinants of long-term blood pressure elevation and an independent risk factor for cardiovascular morbidity and mortality. Apoptosis in VSMC can be inhibited by inversion of the intracellular [Na⁺ ]/[K⁺ ] ratio after the sustained blockage of the Na⁺ ,K⁺ -ATPase by ouabain. Using two-dimensional gel electrophoresis followed by tandem mass spectroscopy, we compared proteomes of control VSMC and of those with ouabain-inhibited Na⁺ ,K⁺ -ATPase and found that ouabain treatment led to overexpression of numerous soluble and membrane-bound proteins. Among proteins, which showed the highest level of ouabain-induced expression, we identified mortalin (also known as GRP75 or PBP-74), a member of the heat shock protein 70 superfamily and a marker for cellular mortal and immortal phenotypes. Further experiments showed that mortalin RNA and protein levels are induced in ouabain-treated VSMC, and that transient transfection of cells with mortalin cDNA inhibited serum deprivation-induced apoptosis via inactivation of the tumor suppressor gene, p53 .