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- Identification of Potassium-Dependent and -Independent Components: DISCUSSION(4)

To determine whether the effect of potassium on inter-nucleosomal DNA cleavage in ovarian cells occurs at the level of a direct suppression of endonucleolytic activity, we used a number of previously characterized cell-free assays to monitor activity of nuclear extract-derived or purified nucleases. The results from these experiments demonstrated that potassium directly suppresses activity of the nucle-ase(s) responsible for internucleosomal DNA cleavage that is present in granulosa cell nuclei, a finding consistent with data derived from comparable analyses of potassium effects on internucleosomal DNA-cleaving nucleases involved in thymocyte or S49-Neo lymphocyte apoptosis.

Furthermore, the present results extend these previous reports by showing that the activities of two purified nucleases in particular, DNase-I and DNase-II, both of which have been implicated in chromatin degradation during apoptosis, are directly inhibited by potassium. The present results differ, however, from data derived using thymocytes in which large-fragment DNA generation is blocked by potassium with a similar IC50 as that for the inhibition of internucleosomal DNA cleavage (unpublished results). In the present studies, the generation of 50-kb chromatin fragments was not affected by 150 mM potassium either in the culture medium (intact cells) or in the autodigestion reaction (cell-free assays). In thymocytes, a potential large-frag-ment cleavage enzyme has been identified as NUC18 (cy-clophilin), but in vitro studies have shown this nuclease to be sensitive to potassium-mediated inhibition. The present results therefore suggest that oocytes and granulosa cells possess a potassium-independent pathway leading to the activation of a large DNA fragment-generating nuclease or nucleases distinct from NUC18/cyclophilin.

October 22, 2013 Cells
Tags: apoptosis follicle granulosa cells ovum