Endothelial γ-glutamyltransferase contributes to the vasorelaxant effect of S-nitrosoglutathione in rat aorta

Abstract

S-nitrosoglutathione (GSNO) involved in storage and transport of nitric oxide ((•)NO) plays an important role in vascular homeostasis. Breakdown of GSNO can be catalyzed by γ-glutamyltransferase (GGT). We investigated whether vascular GGT influences the vasorelaxant effect of GSNO in isolated rat aorta. Histochemical localization of GGT and measurement of its activity were performed by using chromogenic substrates in sections and in aorta homogenates, respectively. The role of GGT in GSNO metabolism was evaluated by measuring GSNO consumption rate (absorbance decay at 334 nm), (•)NO release was visualized and quantified with the fluorescent probe 4,5-diaminofluorescein diacetate. The vasorelaxant effect of GSNO was assayed using isolated rat aortic rings (in the presence or absence of endothelium). The role of GGT was assessed by stimulating enzyme activity with cosubstrate glycylglycine, as well as using two independent inhibitors, competitive serine borate complex and non-competitive acivicin. Specific GGT activity was histochemically localized in the endothelium. Consumption of GSNO and release of free (•)NO decreased and increased in presence of serine borate complex and glycylglycine, respectively. In vasorelaxation experiments with endothelium-intact aorta, the half maximal effective concentration of GSNO (EC50 = 3.2 ± 0.5.10(-7) M) increased in the presence of the two distinct GGT inhibitors, serine borate complex (1.6 ± 0.2.10(-6) M) and acivicin (8.3 ± 0.6.10(-7) M), while it decreased with glycylglycine (4.7 ± 0.9.10(-8) M). In endothelium-denuded aorta, EC(50) for GSNO alone increased to 2.3 ± 0.3.10(-6) M, with no change in the presence of serine borate complex. These data demonstrate the important role of endothelial GGT activity in mediating the vasorelaxant effect of GSNO in rat aorta under physiological conditions. Because therapeutic treatments based on GSNO are presently under development, this endothelium-dependent mechanism involved in the vascular effects of GSNO should be taken into account in a pharmacological perspective.

Figure 1. Histochemical localization of GGT activity in rat aortic tissue.

Typical micrographs (×16) corresponding to histochemical visualization of γ-glutamyltransferase (GGT) activity in endothelium-intact rat aortic rings prepared in the presence of γ-glutamyl-4-methoxy-2-naphthylamide (GMNA, panel A) or in the presence of GMNA and SBC (20 mM, panel B) and in endothelium-denuded aortic rings prepared in the presence of GMNA (panel C). Scale bar, 100 µm.

Figure 2. Effects of modulation of GGT activity on GSNO consumption in rat aorta homogenates.

Consumption rates of GSNO measured in rat aorta homogenates incubated in Tris buffer pH 7.4 with and without SBC (20 mM) or glycylglycine (20 mM). Data are means ± SEM of 3 experiments, * p<0.05 versus substrate alone.

Figure 3. Effects of modulation of GGT activity on GSNO-mediated ^•NO production in aortic tissue.

Panel A: ^•NO production from rat aortic rings exposed to GSNO (1 mM) or GSNO (1 mM) in the presence of SBC (20 mM), glycylglycine (20 mM) or L-NAME (300 µM). Data are means ± SEM of 4–7 experiments, * p<0.05 versus GSNO. Panels B, C, D: representative fluorescence micrographs (×20) of 10-µm sections of rat aortic rings loaded for 2 h with 4,5-diaminofluorescein diacetate (DAF-2 DA, 10 µM) showing ^•NO production following 15-min incubation with GSNO (1 mM, panel C) or GSNO in the presence of SBC (20 mM, panel D) or glycylglycine (20 mM, panel B). Scale bar, 50 µm. All pictures were captured under constant exposure time, gain and offset.

Figure 4. Influence of GGT activity on the vasorelaxant effect of GSNO.

Concentration-dependent vasorelaxation response curves of GSNO (•), GSNO with glycylglycine (20 mM) (Δ), GSNO with acivicin (50 µM) (◊), GSNO with SBC (20 mM) (□), and GSNO with ODQ (5 µM) (○) in endothelium-intact (panel A) and endothelium-denuded (panel B) aortic rings. Panel C: pD2 values (panel C). Data are means ± SEM of 4–12 aortic rings per group. p values (2 ways ANOVA test): p_interaction <0.0001; p_endothelium <0.0001; p_{modulator of GGT} <0.0001. * p<0.05 versus GSNO endothelium-intact; $ p<0.05 versus GSNO endothelium-denuded (post-hoc Bonferroni test). ** p<0.05 versus GSNO in endothelium-intact aortic rings (one way ANOVA test).