IONIC REGULATION OF ENDONUCLEASE ACTIVITY IN PC12 CELLS

We have investigated the Ca2+ dependency of DNA degradation into nucle osome-sized fragments in intact chromaffin-like PC12 cells and PC12 nu clear fractions. In intact cells we were unable to trigger DNA fragmen tation by inducing either transient or sustained elevations of cytopla smic Ca2+ ([Ca2+](i)) with the Ca2+ ionophore ionomycin. On the contra ry, DNA fragmentation was induced ill intact cells by the intracellula r Zn2+ chelator NNN'N'-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN ). To characterize further PC12 cell endonuclease activity, we then in vestigated digestion by purified PC12 cell fractions of exogenously ad ded plasmids. In nuclear fractions two endonuclease activities were id entified: an acidic (pH 5.0) endonuclease activity that was fully Ca2- and Mg2+-independent; and a neutral (pH 7.6) endonuclease activity t hat was Ca2+-independent but Mg2+-dependent. Both endonuclease activit ies were inhibited by Zn2+ Nuclear membrane permeabilization greatly e nhanced plasmid digestion at pH 7.6, but not at pH 5.0. This suggests that neutral endonuclease was located in a membrane-bound compartment, whereas acidic endonuclease was freely accessible to the substrate ev en in the presence of an intact nuclear membrane. In intact nuclei, di gestion of genomic DNA could not be triggered by increasing the bivale nt cation composition of the medium. On the contrary, in hypotonic med ium we observed a large spontaneous nucleolytic DNA degradation that w as increased by Zn2+ chelation. However, an acidic pH shift was a pote nt stimulus for DNA fragmentation in isotonic as well as hypotonic med ium.