Mechanism Of BK Ca- Mediated Changes In Mesenteric Artery Relaxation In Vascular Smooth Muscle Of Hypertensive And Elderly Hypertensive Rats

objective:This article aims to investigate the changes in the expression and function of TRPV4 and BK_Ca complexes in rat mesenteric artery smooth muscle and their effects on vasodilation in elderly,hypertensive and elderly with hypertension.Methods:The rats were divided into four groups:WKY（16 weeks Wistar-Kyoto rats,with normal blood pressure）,SHR（16weeks spontaneously hypertension rats,with hypertension）,old WKY（≥18months Wistar-Kyoto rats,with normal blood pressure）and old SHR（≥18months spontaneously hypertension rats,with hypertension）.Blood pressure was measured via Femoral artery cannulation to ensure the successful establishment of the animal model.The association of TRPV4 with BK_Ca was examined by immunofluorescence staining.The third grade branches of the mesenteric artery were isolated and used to test the changes of vasoconstriction induced by phenylephrine（PE）and the GSK1016790A（GSK,TRPV4-specific agonist）-mediated vasodilation by the vascular tension measurement.The adrenergicα1（ARα1）receptor、TRPV4 channel and BK_Ca channel protein expression were detected by Western blotting.Results:（1）The heart rate of rats in each group had no significant difference（P>0.05）.Compared with WKY group and old WKY group,the systolic and diastolic blood pressure in SHR group and old SHR group increased significantly（P<0.05）.The systolic and diastolic blood pressure of WKY group and SHR group were147.51±13.49mmHgvs212.68±20.83mmHg,95.19±10.98mmHgvs145.18±14.77mmHg,respectively（P<0.05）;The systolic and diastolic blood pressures in old WKY and old SHR groups were150.56±11.07mm Hg vs227.04±25.69mmHg,94.75±10.47mmHg vs 156.47±14.19mmHg,respectively（P<0.05）,indicating that the model was established successfully.（2）The results of immunofluorescence showed that TRPV4 co-localized with BK_Ca in mesenteric artery ring、HEK293 cells co-transfected TRPV4 and BK_Ca and rat mesenteric artery smooth muscle cells.（3）The Contraction induced by60mmol/L high K⁺was significantly increased in SHR and old SHR,compared with that in WKY rat.PE induced a concentration-dependent contraction in all groups.compared with WKY group,The sensitivity to PE was decreased in the old SHR group,but the maximal contractility was significantly increased（P<0.05）.（4）TRPV4-specific agonist GSK mediated concentration-dependent relaxation of the mesenteric artery(EC₅₀:57.82 nmol/L),BK_Caa blocker（5mmol/L TEA）can significantly block the GSK-mediated relaxation response（P<0.05）.Compared with WKY group,GSK-mediated relaxation of mesenteric artery was significantly decreased in SHR group、old WKY group and old SHR group(EC₅₀0 were 92.74 nmol/L、120.58 nmol/L and 85.69 nmol/L,respectively,P<0.05),the most obvious reduction was found in old WKY group,with EC₅₀in SHR group 1 time higher than WKY group.（5）Western blotting results showed that the expression of ARα1 receptor protein was significantly downregulated in the mesenteric artery of old WKY group and old SHR group,compared with WKY group（P<0.05）.Compared with WKY group,the expression of TRPV4 and BK_Caβ1 subunit protein were significantly decreased in SHR group、old WKY group and old SHR group（P<0.05）.Compared with WKY group,there was no significant difference in the expression of BK_Caαsubunit in SHR group（P>0.05）,but significantly decreased in old WKY group and old SHR group（P<0.05）.Old WKY group showed the most obvious reduction,with the degree of reduction 1.5 times of WKY group.Conclusions:（1）The sensitivity of mesenteric artery to PE decreased in elderly、hypertension and elderly with hypertension,which was related to the significant down-regulation of ARα1 receptor protein expression.In the hypertension and elderly with hypertension,The maximum contraction induced by 60mm KCl and PE in mesenteric artery increased significantly.（2）The vasodilation of mesenteric artery induced by TRPV4 were weakened and the protein levels of TRPV4 and BK_Ca in mesenteric artery were down-regulated in hypertensive、elderly and elderly with hypertensive.The changes of TRPV4-BK_Ca complex may be related to the decreased relaxant effect in mesenteric artery.objective:The purpose of this study was to investigate the relaxant effect and mechanism of DS-201 on mesenteric artery from SHR,and to provide the experimental basis for the possibility of clinical application of hypertension,and also provide a scientific basis for revealing the basic characteristics and regulation mechanism of the changes of arterial vasomotor activity in the disease state.Methods:SHR and their corresponding control Wistar-Kyoto（WKY）rats,16 weeks old,were used in this study.Endothelium-denuded and the third branches of mesenteric artery were used in arterial tone measurement.Vascular responses to DS-201（20 to 200 μmol/L）were observed following preconstriction with phenylephrine（PE）or the thromboxane mimetic（U46619）.The vasoreactivity of DS-201 was also investigated by incubating the artery rings with 5 mmol/L TEA or 200 nmol/L Ib TX（BKCa channel blocker）for 25 minutes.The expression of BKCa channel in mesenteric artery was detected by immunoblotting.Results:（1）DS-201 relaxed the PE preconstricted mesenteric artery in a concentration-dependent manner（EC50: 62.17±12.5 μmol/L）,200 nmol/L Ib TX treatment significantly blocked the vasodilation of DS-201（P<0.05）.（2）DS-201 had a vasodilatory effect on mesenteric artery rings preconstricted with either PE or U46619 in a concentration-dependent manner,but the vasodilatory effect was significantly weaker in SHR,compared with WKY rat.In PE preconstricted mesenteric artery,the EC50 of DS-201 in WKY rat and SHR were 86.96 ± 8.72 μmol/L and 101.04±10.84 μmol/L,respectively（P<0.05）.TEA（5mmol/L）significantly blocked the relaxant effect of DS-201 on mesenteric arteries preconstricted with PE（P<0.05）.（3）There was no difference in the protein expression of BKCa α subunit,but BKCa β1 subunit was significantly down-regulated in mesenteric artery of SHR.Conclusions:DS-201 has a dose-dependent vascular relaxant effect on mesenteric artery smooth muscle.The vascular relaxant effect is weaker in SHR than WKY rat,which is possibly related to the down-regulation of the β1 subunit of BKCa.