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Angiotensin II Supplementation by Miniosmotic Pumps Reduces the Level of Oxidative Stress Induced by High Dietary Salt Intake in Rat Microcirculation

Introduction: Previously, we demonstrated that suppressed levels of angiotensin II by high salt diet (HS) impaired endothelium-dependent dilation and reduced nitric oxide (NO) bioavailability due to increased endothelial levels of reactive oxygen species (ROS) in middle cerebral arteries (MCAs) of rats (J Physiol 2016 ; 594(17):4917–31 ; AJP 2018 ; 315(3):H718– H730). Present study aimed to determine the effect of angiotensin II supplementation on vascular NO and superoxide/ROS production and vascular antioxidative enzymes’ mRNA expression. Methods: 9–11 weeks old healthy male Sprague- Dawley rats were randomly assigned to 3 groups (n = 6–8 rats/group): low saltdiet group (LS group, 0.4% NaCl in rat chow) ; HS group (4% NaCl in rat chow for 1 week) and HS+angiotensin II group (HS diet for 7 days, additionally 4th-7th day subpressor doses of angiotensin II via osmotic minipump (100 ng/kg/min/3 days). Following dietary protocol, rats were anesthetized with ketamine (75 mg/kg) and midazolam (2.5 mg/kg) and sacrificed by decapitation. MCA were isolated and cannulated on pressure myograph, under flow (Δ80 mm Hg) or no flow conditions. Endothelial NO and superoxide/ ROS production (determined by the nonfluorescent 4, 5-diaminofluorescein and with dihydroethidine, respectively) were measured by direct flourescence microscopy. In a separate group of experiments, all surface brain blood vessels (BBVs) were isolated and collected for rtPCR expression measurements of mRNA of Cu/Zn SOD, MnSOD, EC-SOD, GPx1 and GPx4 and catalase (CAT). All experimental procedures were approved by the local Ethical Committee and conformed to the EU Directive 86/609. Data were analyzed using One-Way ANOVA, presented as mean±SD. p < 0.05 was considered significant. Results: Basal NO production in no-flow condition was similar among groups. L-NAME blocked the production of NO in each group (basal vs. L-NAME LS:35.4 ± 7.09 vs. 22.8 ± 8.01 ; HS: 32.4 ± 14.5 vs. 14.2 ± 2.58 ; HS+ANG II 35.22 ± 4.67 vs. 12.23 ± 3.27). Flow-induced NO production was significantly lower in HS group (21.34 ± 1.37) compared to LS (31.26 ± 7.20) and HS+angiotensin II (40.80 ± 8.22) groups. L-NAME blocked flow- induced NO production similarly among groups (p > 0.05). No-flow superoxide/ ROS levels in the BBVs endothelium were not significantly different among the groups (p > 0.05). However, flow increased level of superoxide/ROS in the HS group (9.16 ± 1.52) compared with the other groups (LS 7.30 ± 1.13 and HS+angiotensin II 6.44 ± 1.56). There was significantly higher expression of only of EC-SOD and GPx4 in HS+angiotensin II (EC-SOD 1.75 ± 0.43, GPx4 2.15 ± 0.41) group compared to LS (EC-SOD 0.60 ± 0.20, GPx4 0.82 ± 0.17) and HS group (EC-SOD 0.51 ± 0.11, GPx4 0.64 ± 0.07). Conclusions: Suppressor doses of angiotensin II restored NO production by decreasing flow-induced superoxide/ROS production in MCAs, due to increased antioxidant capacity of vasculature.

high salt diet ; oxidative stress ; angiotensin II ; Sprague-Dawley rats

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