METHYLENE-BLUE INHIBITS NITROVASODILATOR-DERIVED AND ENDOTHELIUM-DERIVED RELAXING FACTOR-INDUCED CYCLIC-GMP ACCUMULATION IN CULTURED PULMONARY ARTERIAL SMOOTH-MUSCLE CELLS VIA GENERATION OF SUPEROXIDE ANION

The mechanism of modulation of cyclic guanosine monophosphate (CGMP) accumulation by methylene blue (MB), a putative inhibitor of soluble guanylate cyclase, was investigated in cultured rabbit pulmonary arterial smooth muscle cells (RPASM). Control or MB-pretreated RPASM were stimulated with sodium nitroprusside (SNP), nitrosothiols or endothelium-derived relaxing factor (EDRF) released basally from bovine pulmonary arterial endothelial cells, in short-term co-cultures. The putative EDRF, S-nitroso-L-cysteine (CYSNO), a stable deaminated analog of CYSNO, S-nitroso-3-mercaptoproprionic acid (MPANO) and SNP produced concentration-dependent (1-100 muM) increase (1.5- to 12-fold) in RPASM cGMP levels. MB pretreatment inhibited CYSNO and SNP-induced cGMP accumulation by 51 % to 100%, but MPANO-mediated responses were not altered by MB. The inhibition profile of MB on nitrovasodilator-induced cGMP accumulation was quantitatively reproduced by extracellular generation of superoxide anion with xanthine (100 muM) and xanthine oxidase (5 mU). Similarly to MB pretreatment, superoxide anion generation had no effects on base-line cGMP levels or cGMP responses elicited by MPANO. Furthermore, MB induced a dose- and time-dependent generation of superoxide anion from RPASM, as evidenced from spectrophotometric determination of cytochrome c reduction. Inhibition of cGMP accumulation in response to CYSNO and SNP by MB was completely prevented by superoxide dismutase but not catalase. Selective pretreatment of endothelial cells with MB before co-culture with untreated RPASM produced a reduction in RPASM cGMP levels of a magnitude comparable with that seen in co-cultures of MB-pretreated RPASM with untreated endothelial cells, and which was partially prevented by superoxide dismutase. This study suggests that inactivation of guanylate cyclase likely plays a minor role in mediating the effects of MB, in culture, whereas a major mechanism of inhibition appears to be superoxide inactivation of EDRF and nitrovasodilator-released nitric oxide.