EA3452 CITHEFOR, Drug Targets, Formulation And Preclinical Assessment, Faculté de Pharmacie, Université de Lorraine, Nancy, France.
PLoS One. 2012;7(9):e43190. doi: 10.1371/journal.pone.0043190. Epub 2012 Sep 11.
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.
S-亚硝基谷胱甘肽 (GSNO) 参与一氧化氮 ((•)NO) 的储存和运输，在血管稳态中发挥重要作用。GSNO 的分解可以被 γ-谷氨酰转移酶 (GGT) 催化。我们研究了血管 GGT 是否会影响 GSNO 在离体大鼠主动脉中的血管舒张作用。通过使用显色底物在切片中和主动脉匀浆中分别进行组织化学定位和测定其活性，来研究 GGT 的作用。通过测量 GSNO 消耗率（334nm 处吸光度衰减）来评估 GGT 在 GSNO 代谢中的作用，使用荧光探针 4,5-二氨基荧光素二乙酸酯可视化和定量 (•)NO 释放。使用离体大鼠主动脉环（存在或不存在内皮细胞）测定 GSNO 的血管舒张作用。通过用共底物甘氨酰甘氨酸刺激酶活性以及使用两种独立的抑制剂，竞争性丝氨酸硼酸复合物和非竞争性 acivicin，来评估 GGT 的作用。特异性 GGT 活性在组织化学上定位于内皮细胞。在存在丝氨酸硼酸复合物和甘氨酰甘氨酸的情况下，GSNO 的消耗和游离 (•)NO 的释放分别减少和增加。在含有完整内皮的主动脉的血管舒张实验中，GSNO 的半最大有效浓度（EC50 = 3.2 ± 0.5.10(-7) M）在两种不同的 GGT 抑制剂丝氨酸硼酸复合物（1.6 ± 0.2.10(-6) M）和 acivicin（8.3 ± 0.6.10(-7) M）存在时增加，而在甘氨酰甘氨酸（4.7 ± 0.9.10(-8) M）存在时减少。在内皮细胞去除的主动脉中，GSNO 单独的 EC(50) 增加到 2.3 ± 0.3.10(-6) M，而在丝氨酸硼酸复合物存在时没有变化。这些数据表明，在生理条件下，内皮细胞 GGT 活性在介导 GSNO 对大鼠主动脉的血管舒张作用中发挥重要作用。由于基于 GSNO 的治疗方法目前正在开发中，因此应该从药理学角度考虑这种涉及 GSNO 血管作用的内皮依赖性机制。
