IN-VIVO DEPLETION OF FREE THIOLS DOES NOT ACCOUNT FOR NITROGLYCERIN-INDUCED TOLERANCE - A THIOL-NITRATE INTERACTION HYPOTHESIS AS AN ALTERNATIVE EXPLANATION FOR NITROGLYCERIN ACTIVITY AND TOLERANCE

The present study investigates the effects of thiol-depleting/modifyin g agents on the activity of and tolerance to nitroglycerin (GTN), sodi um nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP) in a n in vivo rat model. Rats were treated with either vehicle (control), GTN (before and after induction of tolerance), diethyl maleate (a thio l-depleting agent) or N-ethylmaleimide (a thiol-modifying agent). The effects of GTN, SNP and SNAP an vascular cyclic GMP levels were invest igated before and after each treatment. In addition, plasma and tissue thiol concentrations were measured in the same tissues as used for th e determination of cyclic GMP in aorta and inferior vena cava after si ngle and serial i.v. bolus doses of each drug. Depletion of free thiol s (glutathione and cysteine) was not found to accompany tolerance deve lopment in GTN-treated tolerant rats or to significantly enhance toler ance development or augment its magnitude in diethyl maleate-treated r ats. When rats were pretreated with a low single dose of N-ethylmaleim ide, where no significant changes in vascular free thiols were observe d, significant reduction in GTN- and SNP-induced, but not SNAP-induced , vascular cyclic GMP production was obtained. Considering the differe ntial effects of diethyl maleate (mainly free thiol depletion) and N-e thylmaleimide (mainly proteinous thiol-alkylation) on vascular thiols, these results indicate that depletion of sulfhydryl groups other than those from free glutathione and cyst eine seems to be involved in the mechanisms defining GTN and SNP (but not SNAP) action and tolerance. Here we propose that SNAP may act either directly by nitrosation of th e heme moiety of the enzyme or via an S-enzyme-S-drug transnitrosation reaction, whereas GTN and SNP actions are mediated by the formation o f S-nitrosothiol on the enzyme itself, rather than by activation of th e enzyme by free S-nitrosothiols.