Reduced Activity of the Aortic Gamma-Glutamyltransferase Does Not Decrease S-Nitrosoglutathione Induced Vasorelaxation of Rat Aortic Rings

Abstract

Aims: Gamma-glutamyl transferase (GGT), an enzyme present on the endothelium, is involved in the release of nitric oxide (NO) from S-nitrosoglutathione (GSNO) and in the GSNO-induced vasodilation. Endogenous GSNO is a physiological storage form of NO in tissues while exogenous GSNO is an interesting candidate for compensating for the decreased NO bioavailability occurring during cardiovascular diseases. We investigated in a rat model of human hypertension, the spontaneous hypertensive rat (SHR), submitted or not to high salt diet, whether a decreased vascular GGT activity modifies the vasorelaxant effect of GSNO. Methods: Thoracic aortic rings isolated from male SHR and Wistar Kyoto rats (WKY) aged 20-22 weeks-submitted or not for 8 weeks to a high salt diet (1% w/v NaCl in drinking water) were pre-constricted with phenylephrine then submitted to concentration-vasorelaxant response curves (maximal response: E_max; pD₂) to carbachol or sodium nitroprusside to evaluate endothelial dependent or independent NO-induced vasodilation, or GSNO (exogenous NO vasodilation depending from the endothelial GGT activity). GGT activity was measured using a chromogenic substrate in aortic homogenates. Its role in GSNO-induced relaxation was assessed following inhibition of the enzyme activity (serine-borate complex). That of protein disulfide isomerase (PDI), another redox sensitive enzyme involved in GSNO metabolism, was assessed following inhibition with bacitracin. Results: Aortic GGT activity (18-23 μmol/min/mg of tissue in adult WKY) decreased by 33% in SHR and 45% in SHR with high salt diet. E_max and pD₂ for sodium nitroprusside were similar in all groups. E_max for carbachol decreased by -14%, reflecting slight endothelial NO-dependent dysfunction. The GSNO curve was slightly shifted to the left in SHR and in SHR with high salt diet, showing a small enhanced sensitivity to GSNO. Involvements of GGT, as that of PDI, in the GSNO effects were similar in all groups (pD₂ for GSNO -0.5 to -1.5 following enzymatic inhibition). Conclusion: Hypertension is associated with a decreased aortic GGT activity without decreasing the vasorelaxant effects of GSNO, whose bioactivity may be supplemented through the alternative enzymatic activity of PDI.

Keywords: NO-dependent vasorelaxation; S-nitrosoglutathione; Spontaneous Hypertensive Rat; aortic ring; gamma-glutamyltransferase.

Figure 1

Influence of hypertension and/or salt diet on aortic GGT activity and aortic glutathione content. Thoracic aortic GGT activity (A,B) and aortic glutathione content (C,D) in WKY/SHR rats submitted (SHR-S/WKY-S; B,D) or not (A,C) to high-salt diet 1% (w/v) during 8 weeks. ^*p < 0.05 Student t-test vs. WKY of the same series. n = 3–4 aortic rings per group, isolated from 4 different rats per group.

Figure 2

Influence of hypertension and/or salt diet on the vasorelaxant responses to carbachol. Cumulative concentration response curves to carbachol of phenylephrine pre-constricted aortic rings isolated from: (A) adult SHR/WKY rats and (B) adult SHR/WKY rats submitted to high-salt diet 1% (w/v) during 8 weeks (SHR-S/WKY-S). ^*p < 0.05 Student t-test vs. WKY or WKY-S of the same series following the Hill analysis. ^#p < 0.05 vs. WKY or WKY-S at the same concentration, two-way analysis of variance (ANOVA) + Bonferroni post-test. n = number of aortic rings per group, isolated from 4 to 8 different rats per group.

Figure 3

Influence of hypertension and/or salt diet on the vasorelaxant responses to GSNO. Cumulative concentration response curves to GSNO of phenylephrine pre-constricted aortic rings isolated from: (A) adult SHR/WKY rats and (B) adult SHR/WKY rats submitted to high-salt diet 1% (w/v) during 8 weeks (SHR-S/WKY-S). ^*p < 0.05 Student t-test vs. WKY or WKY-S of the same series following the Hill analysis. ^#p < 0.05 vs. WKY or WKY-S at the same concentration, two-way analysis of variance (ANOVA) + Bonferroni post-test. n = number of aortic rings per group, isolated from 6 different rats per group.

Figure 4

Impact of serine-borate complex and bacitracin on GSNO-induced vasorelaxations. Cumulative concentration response curves to GSNO in the presence of serine-borate complex (20 mM) or bacitracin (200 μM) in phenylephrine pre-constricted aortic rings isolated from (A) SHR/WKY rats and (B) SHR/WKY rats submitted to high-salt diet 1% (w/v) during 8 weeks (SHR-S/WKY-S). ^*p < 0.05 Student t-test vs. WKY or WKY-S of the same series following the Hill analysis. ^#p < 0.05 vs. GSNO alone of the same group, Student t-test, n = number of aortic rings per group, isolated from 4 to 11 different rats per group.