Guanylyl cyclase inhibitors NS2028 and ODQ and protein kinase G (PKG) inhibitor KT5823 trigger apoptotic DNA fragmentation in immortalized uterine epithelial cells: Anti-apoptotic effects of basal cGMP/PKG

Abstract

The cGMP/protein kinase G (PKG) signalling pathway, at basal levels, has anti-apoptotic/pro-survival effects in certain neural cells. The present study determined apoptosis-regulating effects of basal cGMP/PKG in an immortalized uterine epithelial cell line, HRE-H9 cells, using two soluble guanylyl cyclase (sGC) inhibitors, NS2028 and ODQ, and a PKG inhibitor, KT5823. A new quantitative, ultrasensitive technique using capillary electrophoresis with laser-induced fluorescent detector (CE-LIF), recently developed in our laboratory, was used to quantify levels of apoptotic DNA fragmentation. NS2028 and ODQ increased apoptotic DNA fragmentation by 3.5- and 9-fold respectively, suggesting that lowering basal cGMP levels causes spontaneous apoptosis. 8-Br-cGMP, a cell-permeable cGMP analogue that directly activates PKG, reduced ODQ-induced apoptosis by 81%, indicating that replacement of lowered cGMP with a direct PKG activator prevents apoptosis. Western blot analysis, using PKG type I (PKG-I)-specific antibody, indicated that HRE-H9 cells express PKG-I at moderate levels. Inhibiting basal PKG activity with KT5823 increased apoptotic DNA fragmentation by 9.8-fold. Overall, the data show that inhibitors of basal sGC and PKG activities in immortalized uterine epithelial cells cause apoptosis, suggesting that normal basal levels of cGMP and PKG activity may be necessary to prevent a spontaneous development of apoptosis in these cells.