title: Pharmacological characterization of the mechanisms underlying the vascular effects of succinate.
abstract: 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.
authors.affiliation: 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.
authors.forename: Letícia N
authors.initials: LN
authors.lastname: Leite
authors.affiliation: 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.
authors.forename: Natália A
authors.initials: NA
authors.lastname: Gonzaga
authors.affiliation: 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.
authors.forename: Janaina A
authors.initials: JA
authors.lastname: Simplicio
authors.affiliation: 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.
authors.forename: Gabriel T
authors.initials: GT
authors.lastname: do Vale
authors.affiliation: Novartis Institutes for Biomedical Research, Autoimmunity, Transplantation and Inflammation, Basel CH-4002, Switzerland.
authors.forename: José M
authors.initials: JM
authors.lastname: Carballido
authors.affiliation: Departamento de Farmacologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo (USP), Ribeirão Preto, SP, Brazil.
authors.forename: José C
authors.initials: JC
authors.lastname: Alves-Filho
authors.affiliation: Laboratório de Farmacologia, DEPCH, Escola de Enfermagem de Ribeirão Preto, USP, Ribeirão Preto, SP, Brazil. Electronic address: crtirapelli@eerp.usp.br.
authors.forename: Carlos R
authors.initials: CR
authors.lastname: Tirapelli
mesh_terms: D015121:6-Ketoprostaglandin F1 alpha / Q000378:metabolism; D000818:Animals; D001013:Aorta, Thoracic / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D002118:Calcium / Q000378:metabolism; D002122:Calcium Chloride / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D008297:Male; D051379:Mice; D009589:Nitrogen Oxides / Q000378:metabolism; D010656:Phenylephrine / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D019802:Succinic Acid / Q000494:pharmacology*; D014661:Vasoconstriction / Q000187:drug effects; D014665:Vasodilator Agents / Q000494:pharmacology*
chemical_list: D009589:Nitrogen Oxides; D014665:Vasodilator Agents; D010656:Phenylephrine; D015121:6-Ketoprostaglandin F1 alpha; D019802:Succinic Acid; D002122:Calcium Chloride; D002118:Calcium
keywords: Aorta; Endothelium; GPR91; Nitric oxide; Prostanoids; Relaxation; Succinate