Blocking Cyclic GMP Synthesis Enhances the Pro‐Apoptotic Actions of Nitric Oxide (NO) in the NG108‐15 Cholinergic Neuronal Cell Line

Abstract

Introduction: Little is known about the regulation of apoptosis in sympathetic and parasympathetic nerves that innervate the cardiovascular system. Our previous studies have shown that PC12 cells, a cell culture model of sympathetic nerves, respond to NO or atrial natriuretic peptide (ANP) with increases in production of cyclic GMP (cGMP), resulting in increased levels of cGMP in both the intracellular and extracellular spaces (Fiscus, Robles, Waldman and Murad, J. Neurochem. 48:522-528, 1987). These cGMP elevations resulted from NO- and ANP-induced activation of soluble guanylyl cyclase (sGC) and particulate guanylyl cyclase (pGC), respectively. However, at the time, the biological function of this signaling pathway in neural cells was unknown. In collaboration with Mark Mattson's laboratory at the University of Kentucky, we found that cGMP elevations in hippocampal neurons increased their survival, specifically blocking the cell death induced by stress, such as glutamate toxicity (Barger, Fiscus, Ruth, Hofmann and Mattson, J. Neurochem. 64:2087-2096, 1995). Recently, we showed that cGMP inhibits the onset of apoptosis and prolongs the survival of stressed PC12 cells (Fiscus, Tu and Cheng Chew, NeuroReport (Neurochemistry) 12:185-189, 2001). Specifically, elevating cGMP levels with either ANP or a related peptide, brain natriuretic peptide (BNP), inhibited the apoptotic DNA fragmentation induced by serum deprivation in the PC12 cells. Objectives: We determined if the elevation of cGMP levels induced by an NO donor, S-nitroso-N-acetylpenicillamine (SNAP), has protective (anti-apoptotic) effects in the NG108-15 cholinergic neuronal cell line. Methods: NG108-15 cells were plated at 5 x 106 cells/dish in 100mm culture dishes. After 24 hr, the cells were exposed for 24 hr to SNAP (1-1000 μM), with or without co-incubation with ODQ (40 μM), a selective sGC inhibitor. DNA was extracted from the cells and apoptotic DNA fragmentation (DNA laddering) was analyzed on 2% agarose gels. Results: SNAP, at 500 and 1000 μM, caused apoptotic DNA fragmentation. Inhibition of sGC with ODQ (40 μM) completely blocked the cGMP elevations and exaggerated the pro-apoptotic action of SNAP. Interestingly, ODQ (40 μM), by itself, caused noticeable apoptotic DNA fragmentation, suggesting that even basal levels of cGMP (i.e. in the absence of added NO) may be important for protecting neural cells against onset of apoptosis. Similar results were obtained in 4 other experiments with NG108-15 cells. Conclusions: These data suggest that low basal levels of cGMP (0.1–1 pmol/106 cells) may protect cells against apoptosis and that the elevated levels of cGMP induced by NO may serve as an important counter-balance to the toxic/pro-apoptotic actions of NO. (Supported by a Direct Grant for Research)