Effect of Guanylate Cyclase Inhibition on the Concentration of Nitrite in Culture Medium of Vascular Smooth Muscle Cell

ABSTRACT

PURPOSE: Nitric oxide (NO) exerts the relaxant effect in vascular smooth muscle cells (VSMC) by activating soluble guanylate cyclase (sGC), which produces cyclic guanidine monophosphate (cGMP) in the cell. This study was undertaken to investigate the mechanism of the inhibitory actions of sGC inhibitors, LY 83583 and methylene blue in the VSMC. METHODS: VSMC was primarily cultured from rat aorta and confirmed by immunocytochemistry of anti-smooth muscle myosin antibody. Bacterial lipopolysacchride (LPS), an inducer of inducible nitric oxide synthase (iNOS) and sodium nitroprusside (SNP), an NO donor, were uesd to increase NO within VSMC. The changes in concentrations of nitrite in culture media by an addition of LPS or SNP with a pretreatment of LY 83583 or methylene blue were measured by the spectrophotometry with griess regent and absorbance at 550 nm. Western blot and RT-PCR for iNOS and iNOS mRNA, respectively were performed. RESULTS: LPS and SNP increased nitrite concentration. LY 83583 potentiated the increase in nitrite concentration by LPS and SNP. LY 83583 also increased expressions of iNOS protein and mRNA induced by LPS. Methylene blue has no effect on nitrite concentration increased by LPS or SNP, and it did not affect the expressions of iNOS protein or mRNA induced by LPS. CONCLUSION: These results suggest that the mechanism of inhibitory actions of LY83583 and methylene blue on sGC are different each other LY83583 interferes the interaction of sGC and NO resulting positive feedback increase in iNOS gene expression, but methylene blue eliminates NO from cytosol inducing no compensatory effect.