hrvatski

Short Term Blockade of Angiotensin II Type 1 Receptor by Losartan Effects on Blood Leukocyte Subpopulations’ ROS and RNS Levels in SpragueDawley Rats

Introduction: Endogenously produced reactive oxygen (ROS) and nitrogen (RNS) species in leukocytes primarily act as signalling molecules with the capacity to alter their functional status towards either activation or apoptosis. For example, perturbation of ROS/RNS and antioxidants equilibrium can result in T-cell hyperor hyporesponsiveness, leading to the development of various pathologies. Objective: To determine the level of intracellular ROS and RNS production in various blood leukocyte subpopulations and antioxidant enzymes’ gene expression in cerebral blood vessels (CBV) from Sprague-Dawley (SD) rats following short term blockade of angiotensin II type 1 receptor by losartan. Methods: 10 weeks old, healthy male SD rats were fed low salt diet for 7 days (0.4%NaCl ; LS group) or LS diet+losartan (40 mg/ day in water ad libidum). Following dietary protocol, rats were anesthetized with ketamine (75 mg/kg) and midazolam (2.5 mg/ kg) and sacrificed by decapitation for immediate blood sampling and total surface CBV collection. The intracellular hydrogen peroxide (H2O2) and peroxynitrite (ONOO-) production or superoxide production were determined by flow cytometry with dichlorofluorescein diacetate (DCF-DA) or dihydroethidine (DHE), respectively. mRNA of SOD isoforms (Cu/ZnSOD, MnSOD, ecSOD), glutathione peroxidase 1 and 4 (GPx1, 4) and catalase (CAT) was performed by quantitative rtPCR in CBV. Results are presented as mean ± SD (p < 0.05 was considered significant). All experimental procedures conformed to the European Guidelines for the Care and Use of Laboratory Animals (Directive 86/609) and were approved by the local Ethical Committee. Results: There were no changes in DCF-DA expression in all leukocyte subpopulations except reduced DCF-DA expression in peripheral blood lymphocytes in LS+losartan group compared to LS group (124.9 ± 21.7 vs. 76.7 ± 9.81 ; p = 0.002). In CBV, mRNA levels of SOD1 (1.09 ± 0.18 vs. 0.734 ± 0.20) and GPx1 (1.24 ± 0.38 vs. 0.63 ± 0.25) genes were significantly lower in LS+Losartan group compared to LS group, while CAT (0.87 ± 0.11 vs. 4.89 ± 1.55), SOD2 (0.97 ± 0.21 vs. 2.53 ± 0.66) and SOD3 (0.61 ± 0.20 vs. 1.22 ± 0.34) genes were significantly upregulated in LS+Losartan group, and the GPx4 gene expression remained unchanged. Conclusion: Blockade of angiotensin II type 1 receptors by losartan resulted in alteration of ROS/RNS levels in lymphocytes but no other peripheral blood leukocytes subpopulations. Additionally, antioxidant genes’ expression profile was altered in in CBV. The functional and signalling effect of these changes in peripheral leukocytes and their fine interplay with the endothelium are yet to be elucidated. Grants: This work has been supported by Croatian Science Foundation under the project #IP-2014-09-6380.

angiotensin II type 1 receptor ; oxidative stress ; leukocytes

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