Glucocorticoid-induced plasma membrane depolarization during thymocyte apoptosis: association with cell shrinkage and degradation of the Na+/K+ adenosine triphosphatase

Multiple signaling pathways are known to induce apoptosis in thymocytes thr ough mechanisms that include the loss of mitochondrial membrane potential, cell shrinkage, caspase activation, and DNA degradation but little is known about the consequences of apoptosis on the properties of the plasma membra ne. We have previously shown that apoptotic signals, including survival fac tor withdrawal and glucocorticoids, induce plasma membrane depolarization d uring rat thymocyte apoptosis, but the mechanisms involved in this process are unknown. We report here that inhibition of the Na+/K+-adenosine triphos phatase (Na+/K+-ATPase) with ouabain similarly depolarized control thymocyt es and enhanced glucocorticoid-induced membrane depolarization, suggesting a link between Na+/K+-ATPase and plasma membrane depolarization of thymocyt es. To determine whether repression of Na+/K+-ATPase levels within cells ca n account for the loss of plasma membrane potential, we assessed protein le vels of the Na+/K+-ATPase in apoptotic thymocytes. Spontaneously dying thym ocytes had decreased levels of both catalytic and regulatory subunits of Na +/K+-ATPase, and glucocorticoid. treatment enhanced the loss of Na+/K+-ATPa se protein. The pan caspase inhibitor (z-VAD) blocked both cellular depolar ization and repression of Na+/K+-ATPase in both spontaneously dying and glu cocorticoid-treated thymocytes; however, specific inhibitors of caspase 8, 9, and caspase 3 did not. Interestingly, glucocorticoid treatment simultane ously induced cell shrinkage and depolarization. Furthermore, depolarizatio n and the loss of Na+/K+-ATPase protein were limited to the shrunken popula tion of cells. The data indicate an important role for Na+/K+-ATPase in bot h spontaneous and glucocorticoid-induced apoptosis of rat thymocytes.