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Angiotensin II-Type 1 Receptor Blockade with Losartan Affects the Expression of Enzymes Involved in Vascular Reactivity and Balance of Vascular Oxidative Stress of Sprague-Dawley Rats

Introduction: It is well known that RAS (Renin- angiotensin system) mediates its signal transduction and functions via ANGII and ANGII receptors interaction and suppression of ANG II levels with high salt diet leads to increased vascular oxidative stress. Our recent study in healthy rats showed that losartan (angiotensin II type 1 (AT-1R) receptor antagonist) impaired vasodilator response of middle cerebral arteries, increased vascular and systemic oxidative stress, decreased activity of antioxidative enzymes and increased antioxidative enzymes gene expression in vessels. Present study evaluated the effects of losartan on protein expression of AT1 and AT2 (angiotensin II type 1 and 2) receptors, enzymes involved in the mechanisms of vascular reactivity (iNOS, COX-1, COX-2), antioxidative enzymes (SOD-1, SOD-2, SOD-3, GPx4, CAT) and NOX1. Methods: Healthy male Sprague-Dawley 9–11 weeks old rats (n = 36) were divided to a control (LS, n = 18) and LS+Losartan group (n = 18), both fed with standard rat chow with 0, 4% NaCl. Rats from LS+Losartan group received 40 mg of Losartan (daily dose) in drinking water for 7 days. Following dietary protocol, rats were anesthetized with ketamine (75 mg/kg) and midazolam (2.5 mg/kg) and sacrificed by decapitation. Protein expressions were determined by Western blot in surface cerebral vessels. Due to small amount of tissue, brain blood vessels from two animals were pooled for one sample. Images were processed and analyzed with ImageJ software, data were analyzed by t- test and presented as mean±SD. All experimental procedures are conformed to the European Guidelines for the Care and Use of Laboratory Animals (directive 86/609) and were approved by the local and national Ethical Committee (No.525-10/0255-15- 6). Results: Protein expression of GPx4, AT1 and iNOS was significantly decreased in LS+losartan group (GPx4 0.48 ± 0.34, AT1 0.58 ± 0.13, iNOS 0.84 ± 0.20) vs. LS group (GPx4 1.03 ± 0.46, AT1 1.44 ± 0.43, iNOS 1.18 ± 0.23). Expression of COX-2 and NOX1 was significantly increased in LS+losartan group (COX-2 2.99 ± 0.79, NOX1 1.14 ± 0.19) compared to LS (COX-2 1.92 ± 0.96, NOX1 0.92 ± 0.17). Conclusions: This data confirmed our previous observations of increased oxidative stress (possible due the increased NOX1 expression and activity) and impaired vascular reactivity (decreased iNOS expression). Acknowledgement: Supported by Croatian Science Foundation under the project #IP-2014-09-6380: “Impaired Vasorelaxation and Endothelial Leukocyte Interaction (ELI) in Development of Atherosclerotic Lesions – V-ELI Athero”.

angiotensin II type 1 receptors ; losartan ; vascular reactivity ; oxidative stress ; Sprague-Dawley rats

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