Identification of potassium-dependent and -independent components of the apoptotic machinery in mouse ovarian germ cells and granulosa cells

Recent studies with thymocytes have suggested a critical role for intracell ular potassium in the regulation of apoptosis. In this study, we examined t he pathways of K+ regulation during ovarian cell death. In initial studies, fluorographic analysis demonstrated a significant loss of K+ during apopto sis stimulated by doxorubicin in oocytes and trophic hormone deprivation in granulosa cells. In oocytes, suppression of potassium efflux by potassium- enriched medium prevented condensation, budding, and fragmentation, althoug h it did not block DNA degradation, suggesting the existence of potassium-i ndependent nucleases in oocytes. Culture of granulosa cells in potassium-en riched medium inhibited internucleosomal DNA cleavage, although high-molecu lar weight DNA cleavage was apparent, suggesting that the nuclease or nucle ases responsible for generating 50-kilobase (kb) fragments in these cells i s potassium independent. To address this directly, isolated granulosa cell nuclei were stimulated to autodigest their DNA, and internucleosomal, but n ot large-fragment, cleavage was completely blocked by 150 mM potassium. We next examined whether the proapoptotic caspases are targets for potassium r egulation. In cell-free assays, processing of pro-interleukin-1 beta and pr oteolysis of cellular actin by recombinant caspase-1 and caspase-3, respect ively, were suppressed by the presence of 150 mM potassium. Other monovalen t ions (NaCl, LiCl) exerted a similar effect in these cell-free assays. Thu s, in oocytes and granulosa cells, potassium efflux appears to occur early in the cell death program and may regulate a number of apoptotic events inc luding caspase activity and internucleosomal DNA cleavage. However, there a lso exist novel potassium-independent pathways in both ovarian germ cells a nd somatic cells that signal certain apoptotic events, such as large-fragme nt DNA cleavage.