多巴胺受体和脂筏对高血压患者细胞NADPH氧化酶的作用

Dopamine receptor and raft lipids regulate NADPH oxidase activity in hypertensive renal proximal tubule cells

目的探讨还原型烟酰胺腺嘌呤二核苷酸磷酸氧化酶(NADPH氧化酶即Nox)亚单位在高血压患者肾脏近曲小管细胞中的表达及其活性变化,以及多巴胺受体和脂筏在其中的调节作用。方法细胞分为正常组和高血压组,未经任何药物刺激的两组细胞分别作为正常对照组和高血压对照组,采用葡萄糖浓度梯度超速离心法提取细胞膜的脂筏和非脂筏区蛋白,经Western blot检测Nox亚单位蛋白的表达,光泽精化学发光法动态测定细胞膜Nox的活性。结果多巴胺受体激动剂fenoldopam明显减少gp91phox在正常对照组[(17±3.3)%]和高血压对照组[(20±3.4)%,P<0.05]细胞膜脂筏区域的表达,降低正常对照组p22phox[(15±2.0)%,P<0.05]、p67phox、rac1在脂筏区的表达,但不能减少高血压对照组p22phox、p67phox、rac1蛋白的表达;胆固醇耗竭剂β-CD减少正常对照组gp91phox、p22phox在脂筏区的表达,不能减少高血压对照组Nox亚单位的表达;高血压对照组Nox的基础活性是正常对照组的5倍。结论高血压患者肾脏近曲小管细胞具有较高的Nox亚单位的活性,多巴胺受体和脂筏对Nox亚单位的抑制作用减弱。

Objective To investigate the expression and activity of NADPH oxidase （Nox） subunit in hypertensive renal proximal tubule cells （HT） and the regulatory role of dopamine receptors and lipid boat. Methods Cells were seperated into normotensive group（NT） and hypertensive group （HT）, and their respective control group was established by leafing the cells intact. Glucose concentration gradient was used to extract cell membrane lipid rafts and non-lipid rafts region. The expression levels of Nox subunits were detected by Western blot, and NADPH oxidase activity were measured by Lucigenin Chemiluminescence. Results Compared with control group, dopamine receptor agonist of fenoldopam significantly reduced gp91 expression in membrane lipid raft regions both in NT[ （17 ±3.3）%] and in HT[ （20 ±3.4）% , P〈0.05, n=4], also reducedp22[（15±2.0）%, P〈0.05, n=3], p67, racl expression in lipid rafts in NT, but didn＇t reduce the p22, p67, racl protein expression in HT; Cholesterol depleting agent β-CD reduced gp91, p22 expression in lipid rafts in NT, but didn＇t reduce Nox expression in the area of lipid rafts of hypertension group; The basic Nox activity was 4-fold higher in HT than in NT. Conclsion Nox subunits expression are significantly high in HT, and the regulatary effect of dopamine receptor and lipid rafts on these expressions are weakened.