Characterization of oxidized guanosine 5′-triphosphate as a viable inhibitor of soluble guanylyl cyclase

The guanine base is prone to oxidation by free radicals regardless of the cellular moiety it is bound to. However, under conditions of oxidative stress, 8-oxoguanosine triphosphate (oxo8GTP) formation has been shown to occur without oxidation of the guanine base in DNA. In vitro studies have suggested that oxo8GTP could impact G-protein signaling and RNA synthesis. Whether increased levels of oxo8GTP translate into cellular malfunction is unknown. Data presented herein show that oxo8GTP is formed in cell-free preparations as well as in PC12 cells after exposure to physiologically relevant oxidative conditions generated with 10 μM copper sulfate and 1 mM l-ascorbic acid (Cu/Asc). We also determined that oxo8GTP has biological activity as a potent inhibitor of nitric oxide-stimulated soluble guanylyl cyclase (sGC). The increase in oxo8GTP formation in purified GTP and PC12 cells exposed to Cu/Asc caused a significant reduction in the product of sGC activity, cGMP. This oxidation of GTP was attenuated by the addition of reduced glutathione under these same Cu/Asc conditions, thus preventing the decrease in sGC activity. This suggests that oxo8GTP is produced by free radicals in vivo and could have significant impact on cell functions regulated by sGC activity such as synaptic plasticity in the central nervous system.