Nitrate tolerance in vivo is not associated with depletion of arterial or venous thiol levels.

Abstract

Results from in vitro experiments suggest that development of nitrate tolerance is due to a depletion of vascular thiol compounds (ie, cysteine and glutathione [GSH]) necessary for the bioconversion of organic nitrates. However, it is unknown whether in vivo tolerance development is associated with changes in thiol levels. This study measures plasma and vessel tissue GSH and cysteine levels in nontolerant rats, nitratetolerant rats, and rats treated with the two characteristically different thiol donors N-acetyl-L-cysteine and L-2-oxothiazolidine-4-carboxylic acid (OXO). Chronically catheterized conscious rats received an intravenous infusion of either nitroglycerin (NTG, 0.2 mg/h) or matching placebo for 3 days. At day 3, the hypotensive effect of 2.5 mg NTG/kg was decreased by 74+6% (mean±SEM, P<.05) (n=6, P>.05). Hemodynamic tolerance is not associated with changes in aorta cysteine or GSH levels as compared with the placebo group (cysteine, 77±14 versus 57±11 [mean+SEM] nmol/g; GSH, 414±62 versus 399±89 nmol/g; P>.05). However, the increase in vascular thiol levels seen after OXO treatment in nontolerant rats is completely absent in nitratetolerant animals. The results suggest that (1) depletion of vascular cysteine and/or GSH is apparently not the mechanism underlying development of nitrate tolerance in vivo, and (2) the metabolic handling of thiol compounds and/or the activity of enzymes that act on these thiols is altered during the development of nitrate tolerance in vivo. (Circ Res. 1994;74: 115-120.) Key Words * nitroglycerin * cysteine * glutathionenitrate tolerance known whether "thiol depletion" occurs during the development of tolerance in vivo.The present study was carried out to determine whether or not tolerance to NTG is associated with changes in thiol levels in vivo. Plasma and vascular cysteine and GSH levels in normal and nitrate-tolerant rats were compared. In addition, the effects of two potent, but characteristically different, thiol donors on thiol levels and NTG hemodynamics were also investigated. Material and Methods GSH and Thiol MetabolismGSH is found almost exclusively intracellularly, where it constitutes the major nonprotein thiol pool. GSH is synthesized intracellularly from cysteine, glutamate, and glycine by the consecutive actions of y-glutamylcysteine synthetase and GSH synthetase.9 Cysteine is rapidly metabolized, and GSH serves as a storage and transport form of cysteine.10 y-Glutamylcysteine synthetase is the rate-limiting step in GSH synthesis and is feedback-inhibited by the product of the pathway, GSH.9,11 Availability of substrates, especially cysteine, may also regulate cellular GSH levels.Cysteine and GSH levels may be increased by several methods. N-Acetyl-L-cysteine (NAC) leads to increased plasma and cellular cysteine levels after deacetylation.9'12 L-2-Oxothiazolidine-4-carboxylic acid (OXO) is a nonthiol nontoxic cysteine delivery drug that is readily transported into cells and converted into cysteine by 5-oxoprolinase.9 13Thus, whereas NAC has both intra...

Identification of the enzymatic mechanism of nitroglycerin bioactivation

“…It has been suggested that thiols potentiate GTN activity through formation of bioactive SNOs and͞or protect against nitrate tolerance that results from depletion of intracellular thiols. However, the formation of SNO and depletion of intracellular thiol have not been confirmed (28), and alternative hypotheses, such as oxygen radical scavenging by thiol (29), have more recently found favor. We found that the purified mtALDH could catalyze 1,2-GDN formation only when DTT or 2-mercaptoethanol (2ME) were present (although these concentrations of thiol had no effect on their own).…”

Effect of antioxidant treatments on nitrate tolerance development in normotensive and hypertensive rats

“…Although NAC has been shown, in some studies, to reverse nitrate tolerance, controversies still remain about its ef®cacy in preventing nitrate tolerance development under different pathological conditions [25,26]. Results from in vivo experiments in normotensive animals demonstrated that the depletion of vascular thiols, previously thought to be one of the mechanisms responsible for the nitrate tolerance, is not involved, but that an alteration in the cellular thiol turnover could be implicated [27]. Apart from the early studies by Needleman et al [28] who ®rst proposed the presence of thiol as a necessary mechanism for NO donor-mediated vasodilation, little is known about the enzymatic or alternative pathways for formation of in vivo S-nitrosothiols, which are the likely intermediate metabolites responsible for the guanylate cyclase activation [29].…”

Smooth muscle cell expression of type I cyclic GMP-dependent protein kinase is suppressed by continuous exposure to nitrovasodilators, theophylline, cyclic GMP, and cyclic AMP.

“…In several reports, administration of N-acetylcysteine may reverse nitrovasodilator resistance (38)(39)(40)(41)(42). However, other studies failed to demonstrate benefit of sulfhydryl repletion (44)(45)(46), and when tissue thiol levels have been evaluated directly, no deficiency was observed (47). The clinically beneficial effect of thiol administration may represent a compensation, not a correction, of the underlying defect.…”