The Role Of At1r In Renal Vascular Hypertension Rat Mesenteric Artery Enos Phosphorylation Regulation

Objective To investigate effects of AT1R on regulation of phosphorylation of eNOS in mesenteric arteries of 2-kidney, 1-clip renovascular hypertensive rats. Methods Renovascular hypertensive rat model was established by two-kidney-artery one-clip (2K1C) method with self-made"Ω"shape aluminium clips. 80 male Sprague-Dawley rats were randomly divided into sham-operation-4w group (n=8), 2K1C-4w group (n=8), sham-operation-8w group (n=18), 2K1C-8w group (n=18) and candesartan-treated group (n=18). Systolic blood pressure was measured by tail-cuff method every 2 weeks. Rats were killed after 4 or 8 weeks and mesenteric arteries tissue was separated then preserved at -70℃. The level of total eNOS protein expression, eNOS phosphorylation at Ser¹¹⁷⁹, Ser635 and Thr⁴⁹⁷, as well as level of total Akt expression, Akt phosphorylation at Thr308 and Ser473, expression of PP1 and PP2A in mesenteric arteries tissue were determined by western blotting. The NO production in mesenteric vessel was measured by colorimetric assay. Results Compared with the sham group, systolic blood pressure in 2K1C group was significantly higher after 2 weeks of operation (P<0.05) and continued to rise after 2K1C. Blood pressure in ARB group significantly decreased after treated with candesartan cilexetil tablets, an AT1R blocker, for 2 weeks. Blood pressure fell to normal range after candesartan treatment for 4 weeks. Compared with the sham-4w group, the level of Akt phosphorylation at Ser473 expression was significantly reduced in hypertensive rats (P<0.05), there were no significant differences in total eNOS, eNOS phosphorylation at Ser¹¹⁷⁹, Ser635 ,Thr⁴⁹⁷, total Akt, Akt phosphorylation at Thr308, PP1 and PP2A expression between Sham-4w and 2K1C-4w group. Compared with the sham-8w group, the level of Akt phosphorylation at Ser⁴⁷³ and eNOS phosphorylation at Ser¹¹⁷⁹, Ser635expression was significantly reduced in hypertensive rats (P<0.05) and restored in rats treated with ARB (P<0.05), there were no significant differences in total eNOS, eNOS phosphorylation atThr⁴⁹⁷, total Akt, Akt phosphorylation at Thr³⁰⁸, PP1 and PP2A expression in each group. Compared with the sham-8w group, NO production in mesenteric arteries tissue was significantly reduced in hypertensive rats (P<0.05) and restored in rats treated with ARB (P<0.05). Conclusion Systolic blood pressure increases significantly after 2 weeks of 2K1C operation. The level of Akt phosphorylation at Ser⁴⁷³ expression are significantly reduced in 2K1C-4w hypertensive rats and the level of Akt phosphorylation at Ser⁴⁷³ and eNOS phosphorylation at Ser1179 and Ser⁶³⁵ are significantly reduced in 2K1C-8w hypertensive rats. After treated with ARB, the levels of Akt Ser473, eNOS Ser¹¹⁷⁹ and eNOS Ser⁶³⁵ restore. These data suggested that eNOS phosphorylation both at Ser¹¹⁷⁹and Ser635 decrease in development of hypertension in rat, resulting in lowering eNOS activity, thereby reducing NO produciton. AT1R inhibits the PI3K/Akt signaling pathway, reduced Akt Ser⁴⁷³ leads to eNOS phosphorylation at Ser¹¹⁷⁹ reduction. However the mechanism of eNOS phosphorylation at Ser635 reduction needs further stdudy.