Intracellular potassium levels inhibit apoptotic dna degradation
Degradation of chromatin into oligonucleosomal and/or large =3050 Kb fragments during apoptosis can be recapitulated in vitro in isolated rat thymocyte nuclei (i.e. autodigestion). We have used this model to assess the effects of K+ concentration on apoptotic DNA degradation. Briefly, thymocyte nuclei were incubated for 90 min (25 C) in 2 mM MgCl2, 1 mM CaCl2, 50 mM Tris (pH 7.4) ± ions and subsequently processed for pulsed field (large fragments) and conventional (oligonucleosomal fragments) agarose electrophoresis. KC1, in a dose-dependent manner, inhibited the generation of small and large DNA fragments, reaching maximal inhibition at 150 mM and 200 mM, respectively. Similar results were seen with other monovalent cations (Na+, Cs+, Li+, Rb+). Addition of K+ after various periods of time demonstrated that the degree of chromatin degradation was directly dependent on the period of time without K+. Finally, culture of intact thymocytes in isosmotic media containing 150 mM K+ extracellularly (essentially negating the normal IC gradient), significantly reduced both spontaneous and steroid-induced apoptosis. Because K+ is the only ion present intracellularly in such concentrations, these results suggest that a sustained decrease in K+ levels must occur within a cell undergoing apoptosis to allow for nuclease activation and DNA degradation. Furthermore, this decrease appears !o be aided by the normal K+ gradient across the membrane. Overall, these results demonstrate the importance of regulating ion levels, particularly K+, in apoptosis.
Hughes, FM; Purdy, GD; Cidlowski, JA
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