Wei Yuan, Lin Dao-Hong, Kemp Rowena, Yaddanapudi Ganesh S S, Nasjletti Alberto, Falck John R, Wang Wen-Hui
Department of Pharmacology, New York Medical College, Valhalla, NY 10595, USA.
J Gen Physiol. 2004 Dec;124(6):719-27. doi: 10.1085/jgp.200409140. Epub 2004 Nov 15.
We used the patch-clamp technique to study the effect of arachidonic acid (AA) on epithelial Na channels (ENaC) in the rat cortical collecting duct (CCD). Application of 10 microM AA decreased the ENaC activity defined by NPo from 1.0 to 0.1. The dose-response curve of the AA effect on ENaC shows that 2 microM AA inhibited the ENaC activity by 50%. The effect of AA on ENaC is specific because neither 5,8,11,14-eicosatetraynoic acid (ETYA), a nonmetabolized analogue of AA, nor 11,14,17-eicosatrienoic acid mimicked the inhibitory effect of AA on ENaC. Moreover, inhibition of either cyclooxygenase (COX) with indomethacin or cytochrome P450 (CYP) omega-hydroxylation with N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS) failed to abolish the effect of AA on ENaC. In contrast, the inhibitory effect of AA on ENaC was absent in the presence of N-methylsulfonyl-6-(propargyloxyphenyl)hexanamide (MS-PPOH), an agent that inhibits CYP-epoxygenase activity. The notion that the inhibitory effect of AA is mediated by CYP-epoxygenase-dependent metabolites is also supported by the observation that application of 200 nM 11,12-epoxyeicosatrienoic acid (EET) inhibited ENaC in the CCD. In contrast, addition of 5,6-, 8,9-, or 14,15-EET failed to decrease ENaC activity. Also, application of 11,12-EET can still reduce ENaC activity in the presence of MS-PPOH, suggesting that 11,12-EET is a mediator for the AA-induced inhibition of ENaC. Furthermore, gas chromatography mass spectrometry analysis detected the presence of 11,12-EET in the CCD and CYP2C23 is expressed in the principal cells of the CCD. We conclude that AA inhibits ENaC activity in the CCD and that the effect of AA is mediated by a CYP-epoxygenase-dependent metabolite, 11,12-EET.
我们运用膜片钳技术研究了花生四烯酸（AA）对大鼠皮质集合管（CCD）上皮钠通道（ENaC）的影响。施加10微摩尔/升的AA使由NPo定义的ENaC活性从1.0降至0.1。AA对ENaC作用的剂量反应曲线表明，2微摩尔/升的AA可使ENaC活性抑制50%。AA对ENaC的作用具有特异性，因为AA的非代谢类似物5,8,11,14 - 二十碳四烯炔酸（ETYA）以及11,14,17 - 二十碳三烯酸均未模拟出AA对ENaC的抑制作用。此外，用吲哚美辛抑制环氧化酶（COX）或用N - 甲基磺酰基 - 12,12 - 二溴十二碳 - 11 - 烯酰胺（DDMS）抑制细胞色素P450（CYP）ω - 羟化作用均未能消除AA对ENaC的作用。相反，在存在抑制CYP - 环氧合酶活性的试剂N - 甲基磺酰基 - 6 - （炔丙氧基苯基）己酰胺（MS - PPOH）的情况下，AA对ENaC的抑制作用消失。200纳摩尔/升的11,12 - 环氧二十碳三烯酸（EET）可抑制CCD中的ENaC这一观察结果也支持了AA的抑制作用由CYP - 环氧合酶依赖性代谢产物介导这一观点。相比之下，添加5,6 - 、8,9 - 或14,15 - EET未能降低ENaC活性。同样，在存在MS - PPOH的情况下施加11,12 - EET仍可降低ENaC活性，这表明11,12 - EET是AA诱导的ENaC抑制作用的介质。此外，气相色谱 - 质谱分析检测到CCD中存在11,12 - EET，且CYP2C23在CCD的主细胞中表达。我们得出结论，AA抑制CCD中的ENaC活性，且AA的作用由CYP - 环氧合酶依赖性代谢产物11,12 - EET介导。
