ACTIVATION OF CYCLIC-GMP FORMATION IN MOUSE NEUROBLASTOMA-CELLS BY A LABILE NITROXYL RADICAL - AN ELECTRON-PARAMAGNETIC RESONANCE SPIN TRAPPING STUDY

The receptor-mediated generation of an endothelial-derived relaxing factor (EDRF)-free radical intermediate in a neuronal cell line detected by spin trapping techniques has been reported. Here we report the time course of the appearance of the 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS) spin adduct and cyclic GMP formation following addition of carbamylcholine to suspensions of cultured mouse neuroblastoma cells (clone N1E-115). The time course of the appearance of the DBNBS spin adduct shows that spin adduct formation decreases possibly reaching a minimum approximately between 35 and 40 s. This is inversely proportional to cGMP formation which reaches a maximum at approximately 40 s after carbamylcholine activation. In addition, the inhibitory effect of N(G)-monomethyl-L-arginine (NMMA), potassium ferricyanide, K3Fe(CN)6 and methylene blue in cytosol preparation was investigated. A mechanism is proposed that essentially accounts for the combined results observed by spin trapping/electron paramagnetic resonance (EPR) study providing direct evidence for the muscarinic receptor-mediated formation of a labile, diffusible precursor of nitric oxide (NO.) derived from L-arginine that activates soluble guanylate cyclase.