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 (oxo(8)GTP) formation has been shown to occur without oxidation of the guanine base in DNA. In vitro studies have suggested that oxo(8)GTP could impact G-protein signaling and RNA synthesis. Whether increased levels of oxo(8)GTP translate into cellular malfunction is unknown. Data presented herein show that oxo(8)GTP is formed in cell-free preparations as well as in PC12 cells after exposure to physiologically relevant oxidative conditions generated with 10 microM copper sulfate and 1 mM L-ascorbic acid (Cu/Asc). We also determined that oxo(8)GTP has biological activity as a potent inhibitor of nitric oxide-stimulated soluble guanylyl cyclase (sGC). The increase in oxo(8)GTP 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 oxo(8)GTP 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.
Bolin
Celeste
C
Department of Pharmaceutical Sciences, Center for Environmental Health Sciences, University of Montana, 32 Campus Drive, Missoula, MT, USA.
Cardozo-Pelaez
Fernando
F
D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology; D002457:Cell Extracts; D002460:Cell Line; D002490:Central Nervous System / Q000201:enzymology* / Q000473:pathology; D002851:Chromatography, High Pressure Liquid; D019327:Copper Sulfate / Q000494:pharmacology; D006152:Cyclic GMP / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects*; D005609:Free Radicals / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D006162:Guanylate Cyclase / Q000037:antagonists & inhibitors*; D066298:In Vitro Techniques; D009569:Nitric Oxide / Q000378:metabolism; D010084:Oxidation-Reduction / Q000187:drug effects; D018384:Oxidative Stress; D010673:Pheochromocytoma / Q000201:enzymology* / Q000473:pathology; D051381:Rats
D002457:Cell Extracts; D005609:Free Radicals; C057674:8-hydroxyguanosine triphosphate; D009569:Nitric Oxide; D006160:Guanosine Triphosphate; D006162:Guanylate Cyclase; D006152:Cyclic GMP; D019327:Copper Sulfate; D001205:Ascorbic Acid