Conversion of glyceryl trinitrate to nitric oxide in tolerant and non‐tolerant smooth muscle and endothelial cells

Abstract

Exposure of smooth muscle cells (SMC) to glyceryl trinitrate (GTN, 75–600 μm) for 30 min led to a concentration‐dependent increase in nitrite (NO2−), one of the breakdown products of nitric oxide (NO). This was not affected by 30 min pretreatment of the cells with 0.5 mm of sulphobromophthalein (SBP) an inhibitor of glutathione‐S‐transferase (GST), by metyrapone or SKF‐525A inhibitors of cytochrome P450. These experiments were confirmed by organ bath studies using rabbit aortic strips denuded of endothelium and contracted with phenylephrine. Thus, a 30 min incubation of the strips with 0.5 mm SPB, metyrapone or SKF‐525A did not affect the relaxations in response to GTN (10−10−10−6 m). Potentiation of the anti‐platelet effect of GTN (44 μm) by endothelial cells (EC, 40 × 103 cells) was not affected by prior incubation of EC with SBP, metyrapone or SKF‐525A (all at 0.5 mm). Potentiation of the antiplatelet activity of GTN (11–352 μm) by small numbers of SMC (24 × 103 cells) or EC (40 × 103 cells) treated with indomethacin (10 μm) was attenuated when the SMC or EC were treated in culture with a high concentration of GTN (600 μm) for 18 h beforehand (referred to as ‘tolerant’ cells). In addition, tolerant SMC produced far less NO2− than non‐tolerant SMC. Exposure of non‐tolerant SMC or EC (105 cells) to GTN (200 μm) for 3 min increased (3–4 fold) the levels of guanosine 3′:5′‐cyclic monophosphate (cyclic GMP). This increase was much less (≤ 1 fold) in the tolerant SMC or EC (105 cells). The basal levels of cyclic GMP were similar in normal or tolerant SMC or EC. Sodium nitroprusside (80 μm) or atrial natriuretic factor (ANF, 10−7 m) increased the levels of cyclic GMP in normal or tolerant SMC or EC to the same extent. The anti‐platelet effects of GTN (44 μm) were potentiated by the sulphydryl donor N‐acetylcysteine (NAC, 0.5 mm). Incubation of GTN (150–1200 μm) for 30 min with NAC (0.1–1 mm) led to a concentration‐dependent increase in NO2− formation. The reduced ability of tolerant SMC or EC to potentiate the anti‐platelet activity of GTN was restored by NAC (0.5 mm). These anti‐aggregatory effects were abolished by concurrent co‐incubation with oxyhaemoglobin (10 μm) indicating that they were due to NO release. Thus, in SMC or EC, metabolism of GTN to NO does not depend on glutathione‐S‐transferase or the cytochrome P450 system. Furthermore, when compared to normal cells, tolerant SMC or EC metabolize GTN to NO less effectively as assessed by the reduced capacity to potentiate the antiplatelet effects of GTN, to release NO2− and to increase the level of cyclic GMP. This decrease in NO formation shows that tolerance to GTN is mainly due to impaired biotransformation of GTN to NO. NAC, by directly forming NO from GTN, compensates for this failing mechanism.

Influence of S‐nitrosothiols and nitrate tolerance in the rat gastric fundus

“…Although not all experimental findings support it (Bassenge & Zanzinger, 1992;Fung et al, 1992), the most widespread concept to explain the selective organic nitrate tolerance is that intracellular thiols are required to liberate NO from organic nitrates; prolonged exposure to an organic nitrate will lead to depletion of the intracellular thiol pool and tolerance . Decreased bioconversion of GTN to NO in tolerant vascular smooth muscle cells has been described (Salvemini et al, 1993). Although the mechanism of NO release from S-nitrosothiols is not completely clear, intracellular penetration of extracellular S-nitrosothiols does not seem required to induce relaxation.…”

Direct gas measurements indicate that the novel cyclooxygenase inhibitor AZD3582 is an effective nitric oxide donor in vivo

“…Glutathione‐ S ‐transferases can potentially be inhibited by metabolites formed during their transformation of NO donors to NO (e.g. Lee & Fung, 2003), but this group of enzymes might be of less importance in NO donation in blood vessels as compared with in the liver (Salvemini et al ., 1993). Another possibility is cofactor depletion during transformation of the organic nitrate to NO by xanthine oxidoreductase (Harrison, 2002).…”

Vascular and anti‐platelet actions of 1,2‐ and 1,3‐glyceryl dinitrate

“…TSA produced equal amounts of N02-to those produced by GSH, but in contrast to GSH it was as effective as NAC (which produced less N02-than TSA) in potentiating the anti-platelet effects of the GDNs and thus in releasing NO. This indicates that as for GTN (Chung & Fung, 1991;Salvemini et al, 1993), there is a preferential release of NO from the molecule of the GDNs and this preferential release depends on the type of thiol used. Our study has therefore raised the possibility that bioconversion of the GDNs in EC or SMC to form the active species NO may contribute in part to the pharmacological actions of GTN.…”