Effects of in vivo nitroglycerin treatment on activity and expression of the guanylyl cyclase and cGMP-dependent protein kinase and their downstream target vasodilator-stimulated phosphoprotein in aorta

Background-Chronic in vivo treatment with nitroglycerin (NTG) induces toler ance to nitrates and cross-tolerance to nitrovasodilators and endothelium-d erived nitric oxide (NO). We previously identified increased vascular super oxide formation and reduced NO bioavailability as one causal mechanism. It is still controversial whether intracellular downstream signaling to nitrov asodilator-derived NO is affected as well. Methods and Results-We therefore studied the effects of 3-day NTG treatment of rats and rabbits on activity and expression of the immediate NO target soluble guanylyl cyclase (sGC) and on the cGMP-activated protein kinase I ( cGK-I). Tolerance was induced either by chronic NTG infusion via osmotic mi nipumps (rats) or by NTG patches (rabbits). Western blot analysis. semiquan titative reverse transcription-polymerase chain reaction, and Northern blot analysis revealed significant and comparable increases in the expression o f sGC alpha (1) and beta (1) subunit protein and mRNA. Studies with the oxi dative fluorescent dye hydroethidine revealed an increase in superoxide in the endothelium and smooth muscle. Stimulation with NADH increased superoxi de signals in both layers. Although cGK-I expression in response to low-dos e NTG was not changed, a strong reduction in vasodilator-stimulated phospho protein (VASP) serine239 phosphorylation (specific substrate of cGK-I) was observed in tolerant tissue from rats and rabbits. Concomitant in vivo and in vitro treatment with vitamin C improved tolerance, reduced oxidative str ess, and improved P-VASP. Conclusions-We therefore conclude that increased expression of sGC in the s etting of tolerance reflects a chronic inhibition rather than an induction of the sGC-cGK-I pathway and may be mediated at least in part by increased vascular superoxide.