Diferenças sexuais no relaxamento induzido pelo agonista seletivo do GPER em artérias de resistência de ratos gonadectomizados

Abstract

Introduction: Non-genomic effects of estrogen, which include rapid vascular effects, have been attributed to the G protein-coupled estrogen receptor (GPER). However, the mechanisms underlying the vascular effects modulated through GPER are not well known, including in the context of resistance arteries, especially in deprivation of gonadal sex hormones. Thus, we investigated the vascular function of GPER in mesenteric resistance arteries from gonadectomized rats of both sex. Methods: Wistar rats (12 weeks old) of both sexes were used. Gonadectomy was performed. After 21 days, rats were euthanized. Third-order mesenteric arteries were isolated and mounted. Concentration-response curves were obtained by cumulative additions of G-1 agonist (1 nM - 10 μM) or vehicle (dimethyl sulfoxide – DMSO) in vessels pre-contracted with phenylephrine. The vasodilatory effects of G-1 were assessed before and after removal of the endothelium or incubation for 30 min with non-selective inhibitor of the enzyme nitric oxide synthase (NOS), non-selective inhibitor of the cyclooxygenase enzyme (COX), non-specific inhibitor of the cytochrome P450 enzyme (CYP) or H2O2 enzymatic scavenger (L-NAME, INDOMETACIN, CLOTRIMAZOLE and CATALASE, respectively), PI3K-Akt fast signaling pathway inhibitor (LY-294.002), selective iNOS enzyme inhibitor (1400W), selective enzyme inhibitor nNOS (L-NPA), selective GPER antagonist (G36) and simultaneous REα and REβ antagonist (ICI 182,780). Tissue protein expression of endothelial nitric oxide synthase (eNOS), neuronal nitric oxide synthase (nNOS), protein kinase B (Akt 1/2/3) and extracellular signal-regulated kinase (ERK 1/2) was measured by western blotting, with and without stimulus (G-1, 10 μM). Vascular levels of nitric oxide (NO) and hydrogen peroxide (H2O2) were determined in situ using 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM) and 2',7' dichlorodihydrofluorescein-diacetate (DCF-DA), respectively, with and without G-1 stimulation [10 μM] in the presence or not of L-NAME [300 μM] or catalase [1000 unt/ml], respectively. Immunofluorescence for labeling GPER or iNOS was performed in arteries investigated by immunofluorescence. Data were analyzed by one-way or two-way ANOVA, followed by post-hoc Bonferroni/Newman-Keuls or t-test. MannWhitney test was used for non-parametric data. It was considered p < 0.05. The protocols were approved by CEUA-UFES (067/2017). Results: Selective GPER agonist induced concentration-dependent relaxation in mesenteric resistance arteries in both female (93.6 ± 0.8 %) and male (90.0 ± 1.3 %) gonadectomized, without sex difference. In the endothelium absence, the response to G-1 was decreased in both female (54.4 ± 1.6 %) and male (46.9 ± 1.9 %), but was not abolished. GPER marking was similar in the arterial sections of the studied groups, regardless of the vascular layer. Vasorelaxation was attenuated in both groups in the presence of L-NAME (OVX: 68.5 ± 6.6 % vs ORX: 65.3 ± 4.0 %). Local NO production was also similar in the gonadectomized animals. 1400W (OVX: 96.5 ± 1.2 % vs ORX: 63.6 ± 5.4 %*) and LY-294.002 (OVX: 91.7 ± 2.1 % vs ORX: 75.6 ± 3.5 %*) reduced the response to maximum concentration only in males. iNOS labeling was significantly higher in the arterial sections of ORX animals. Protein expression of p-eNOS (OVX: 99.6 ± 7.4 U.A vs ORX: 151.8 ± 5.5 U.A*) and p-Akt 1/2/3 (OVX: 104.3 ± 7.9 U.A vs ORX: 134.5 ± 7.9 U.A*) were higher in the ORX group. eNOS (OVX: 104.2 ± 12.6 U.A vs ORX: 117.1 ± 5.8 U.A), Akt 1/2/3 (OVX: 108.0 ± 6.6 U.A vs ORX: 107.9 ± 14.6 U.A) and ERK 1/2 (OVX: 91.1 ± 2.2 U.A vs ORX: 96.4 ± 1.7 U.A) total were similar between groups. On the other hand, p-ERK 1/2 showed an increase in expression only in the OVX group (OVX: 146.2 ± 2.2 U.A* vs ORX: 97.0 ± 14.5 U.A). L-NPA (OVX: 88.3 ± 2.3 % vs ORX: 84.8 ± 3.3 %) promoted greater vasodilator responsiveness in both sexes, without changing the vasodilator response at maximum concentration. Protein expression of total nNOS was increased, with no difference between groups, after agonist stimulation (OVX: 126.5 ± 5.6 U.A vs ORX: 124.0 ± 17.9 U.A). CATALASE (OVX: 72.1 ± 2.9 %* vs ORX: 90.7 ± 2.8 %) reduced relaxation to maximum concentration only in female. In situ production of H2O2 was also higher in the female arterial sections. INDOMETHACIN (OVX: 97.1 + 1.0 % vs ORX: 95.3 ± 0.6 %) and CLOTRIMAZOLE (OVX: 92.4 ± 2.3 % vs ORX: 92.2 ± 2.1 %) did not reduced vasorelaxation in either group. Conclusion: These findings suggest that GPER modulated vascular relaxing through different endothelial mediators and mechanisms that will vary according to sex. The results obtained in the present study provide new insight into the effects of oestrogen-induced responses via GPER on vascular function in deprivation of gonadal sex hormones.