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 through 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 membrane. We have previously shown that apoptotic signals, including survival factor withdrawal and glucocorticoids, induce plasma membrane depolarization during rat thymocyte apoptosis, but the mechanisms involved in this process are unknown. We report here that inhibition of the Na(+)/K(+)-adenosine triphosphatase (Na(+)/K(+)-ATPase) with ouabain similarly depolarized control thymocytes and enhanced glucocorticoid-induced membrane depolarization, suggesting a link between Na(+)/K(+)-ATPase and plasma membrane depolarization of thymocytes. To determine whether repression of Na(+)/K(+)-ATPase levels within cells can account for the loss of plasma membrane potential, we assessed protein levels of the Na(+)/K(+)-ATPase in apoptotic thymocytes. Spontaneously dying thymocytes had decreased levels of both catalytic and regulatory subunits of Na(+)/K(+)-ATPase, and glucocorticoid treatment enhanced the loss of Na(+)/K(+)-ATPase protein. The pan caspase inhibitor (z-VAD) blocked both cellular depolarization and repression of Na(+)/K(+)-ATPase in both spontaneously dying and glucocorticoid-treated thymocytes; however, specific inhibitors of caspase 8, 9, and caspase 3 did not. Interestingly, glucocorticoid treatment simultaneously induced cell shrinkage and depolarization. Furthermore, depolarization and the loss of Na(+)/K(+)-ATPase protein were limited to the shrunken population of cells. The data indicate an important role for Na(+)/K(+)-ATPase in both spontaneous and glucocorticoid-induced apoptosis of rat thymocytes.
Mann
C L
CL
Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
Bortner
C D
CD
Jewell
C M
CM
Cidlowski
J A
JA
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D061945:Caspase Inhibitors; D020169:Caspases / Q000502:physiology; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology; D048429:Cell Size; D002478:Cells, Cultured; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004594:Electrophysiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005938:Glucocorticoids / Q000494:pharmacology*; D008297:Male; D010042:Ouabain / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism; D013950:Thymus Gland / Q000166:cytology / Q000187:drug effects* / Q000502:physiology*
D000590:Amino Acid Chloromethyl Ketones; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D004791:Enzyme Inhibitors; D005938:Glucocorticoids; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D010042:Ouabain; D020169:Caspases; D000254:Sodium-Potassium-Exchanging ATPase