Leite Letícia N, Gonzaga Natália A, Simplicio Janaina A, do Vale Gabriel T, Carballido José M, Alves-Filho José C, Tirapelli Carlos R
Departamento de Farmacologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo (USP), Ribeirão Preto, SP, Brazil; Laboratório de Farmacologia, DEPCH, Escola de Enfermagem de Ribeirão Preto, USP, Ribeirão Preto, SP, Brazil.
Novartis Institutes for Biomedical Research, Autoimmunity, Transplantation and Inflammation, Basel CH-4002, Switzerland.
Eur J Pharmacol. 2016 Oct 15;789:334-343. doi: 10.1016/j.ejphar.2016.07.045. Epub 2016 Jul 27.
We investigated the mechanisms underlying the vascular effects of succinate. Vascular reactivity experiments were performed in aortic rings isolated from male Wistar rats and C57BL/6 wild type (WT) or GPR91(-/-) mice. Nitrate/nitrite (NOx) was measured colorimetrically whereas 6-keto-prostaglandin F1α (stable product of prostacyclin) was measured by enzyme immunoassay (EIA). Phosphorylation of endothelial nitric oxide synthase (eNOS) was assessed by western immunoblotting. Functional assays revealed that the direct effect of succinate in the vasculature is biphasic. At lower concentrations succinate induced relaxation while at higher concentrations succinate induced vascular contraction. Succinate concentration dependently relaxed rat aortic rings with intact endothelium. Endothelial removal reduced, but not abolished succinate-induced relaxation. Similarly, succinate relaxed endothelium-intact and endothelium-denuded aortas isolated from both C57BL/6 and GPR91(-/-) mice. Pre-incubation of endothelium-intact, but not endothelium-denuded rat aortic rings with l-NAME, indomethacin and tetraethylammonium (TEA) reduced succinate-induced relaxation. In endothelium-intact rings, succinate-induced relaxation was attenuated by ODQ, haemoglobin, Rp-8-Br-Pet-cGMPS, thapsigargin, wortmannin and SC-560. Blockade of K(+) channels with 4-aminopyridine, apamin and charybdotoxin reduced succinate-induced relaxation. Succinate increased the concentration of NOx and 6-keto-prostaglandin F1α as well as eNOS phosphorylation at ser(1177) residue. CaCl2-induced contraction of endothelium-intact or endothelium-denuded aortas was not affected by succinate. The major finding of our study is that it first demonstrates a direct effect of succinate in the vasculature. Succinate displays a biphasic and concentration-dependent effect. The vascular relaxation induced by succinate is partially mediated by endothelial GPR91 receptors via the NO-cGMP pathway, a vasodilator cyclooxygenase (COX) product(s) and the opening of K(+) channels.
我们研究了琥珀酸酯血管效应的潜在机制。在从雄性Wistar大鼠以及C57BL/6野生型（WT）或GPR91（-/-）小鼠分离的主动脉环中进行血管反应性实验。采用比色法测定硝酸盐/亚硝酸盐（NOx），通过酶免疫测定法（EIA）测定6-酮-前列腺素F1α（前列环素的稳定产物）。通过蛋白质免疫印迹法评估内皮型一氧化氮合酶（eNOS）的磷酸化。功能测定显示，琥珀酸酯在脉管系统中的直接作用具有双相性。在较低浓度下，琥珀酸酯诱导舒张，而在较高浓度下，琥珀酸酯诱导血管收缩。琥珀酸酯浓度依赖性地使具有完整内皮的大鼠主动脉环舒张。去除内皮可降低但不能消除琥珀酸酯诱导的舒张。同样，琥珀酸酯使从C57BL/6和GPR91（-/-）小鼠分离的内皮完整和去内皮的主动脉舒张。用L-NAME、吲哚美辛和四乙铵（TEA）预孵育内皮完整而非去内皮的大鼠主动脉环可降低琥珀酸酯诱导的舒张。在内皮完整的环中，ODQ、血红蛋白、Rp-8-Br-Pet-cGMPS、毒胡萝卜素、渥曼青霉素和SC-560可减弱琥珀酸酯诱导的舒张。用4-氨基吡啶、蜂毒明肽和大蝎毒素阻断钾通道可降低琥珀酸酯诱导的舒张。琥珀酸酯增加了NOx和6-酮-前列腺素F1α的浓度以及eNOS在丝氨酸（1177）残基处的磷酸化。氯化钙诱导的内皮完整或去内皮主动脉的收缩不受琥珀酸酯的影响。我们研究的主要发现是，首次证明了琥珀酸酯在脉管系统中的直接作用。琥珀酸酯表现出双相性和浓度依赖性作用。琥珀酸酯诱导的血管舒张部分由内皮GPR91受体通过NO-cGMP途径、一种血管舒张性环氧化酶（COX）产物和钾通道的开放介导。
