APOPTOSIS IN AN INTERLEUKIN-2-DEPENDENT CYTOTOXIC T-LYMPHOCYTE CELL-LINE IS ASSOCIATED WITH INTRACELLULAR ACIDIFICATION - ROLE OF THE NA+ H+-ANTIPORT/

Apoptosis is a form of cell death associated with DNA fragmentation an d chromatin condensation. We recently established that intracellular a cidification occurred during apoptosis following cytotoxic insult. The current studies were designed to determine whether intracellular acid ification was more generally associated with apoptosis, specifically i n a model of growth factor withdrawal. Upon withdrawal of interleukin- 2, CTLL-2 cells accumulated in the G(1) phase of the cell cycle and st arted to fragment their DNA around 12 h concurrent with both decreased pH and increased Ca2+. Chelation of Ca2+ did not inhibit DNA digestio n, whereas incubation with a calcium ionophore pre vented both acidifi cation and DNA digestion Hence,. acidification rather than increased C a2+ was associated with apoptosis. The acidified cells represented a d iscrete population up to 0.7 pH units below normal. The extent of acid ification depended upon the extracellular pH; above pH 6.3, intracellu lar pH was significantly below extracellular pH, whereas below pH 6.3, the cells still regulated their pH. inhibition of the Na+/H+-antiport prevented the apoptotic cells from regulating their intracellular pH under these acidic conditions. These results demonstrate that apoptoti c cells retain a functional antiport but that its set-point has change d, Many survival factors are known to phosphorylate and activate the a ntiport, hence apoptosis is likely to be associated with dephosphoryla tion. Although acidification always occurred during apoptosis, maintai ning intracellular pH above 7.2 did not prevent apoptosis, suggesting that an acid pH is not essential for apoptosis, We hypothesize that ot her critical regulators of apoptosis must be subject to dephosphorylat ion.