Nüsing Rolf M, Schweer Horst, Fleming Ingrid, Zeldin Darryl C, Wegmann Markus
Institute of Clinical Pharmacology, Johann Wolfgang Goethe University, Theodor Stern Kai 7, 60590 Frankfurt am Main, Germany.
Am J Physiol Renal Physiol. 2007 Jul;293(1):F288-98. doi: 10.1152/ajprenal.00171.2006. Epub 2007 May 9.
We investigated the effects of epoxyeicosatrienoic acids (EETs) on ion transport in the polarized renal distal tubular cell line, Madin-Darby canine kidney (MDCK) C7. Of the four EET regioisomers (5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET) studied, only apical, but not basolateral, application of 5,6-EET increased short-circuit current (I(sc)) with kinetics similar to those of arachidonic acid. The ion transport was blocked by preincubation with the cyclooxygenase inhibitor indomethacin or with the chloride channel blocker NPPB. Furthermore, both a Cl(-)-free bath solution and the Ca(2+) antagonist verapamil blocked 5,6-EET-induced ion transport. Although the presence of the PGE(2) receptors EP2, EP3, and EP4 was demonstrated, apically added PGE(2) was ineffective and basolaterally added PGE(2) caused a different kinetics in ion transport compared with 5,6-EET. Moreover, PGE(2) synthesis in MDCK C7 cells was unaffected by 5,6-EET treatment. GC/MS/MS analysis of cell supernatants revealed the presence of the biologically inactive 5,6-dihydroxy-PGE(1) in 5,6-EET-treated cells, but not in control cells. Indomethacin suppressed the formation of 5,6-dihydroxy-PGE(1). 5,6-Epoxy-PGE(1), the precursor of 5,6-dihydroxy-PGE(1), caused a similar ion transport as 5,6-EET. Cytochrome P-450 enzymes homolog to human CYP2C8, CYP2C9, and CYP2J2 protein were detected immunologically in the MDCK C7 cells. Our findings suggest that 5,6-EET affects Cl(-) transport in renal distal tubular cells independent of PGE(2) but by a mechanism, dependent on its conversion to 5,6-epoxy-PGE(1) by cyclooxygenase. We suggest a role for this P450 epoxygenase product in the regulation of electrolyte transport, especially as a saluretic compound acting from the luminal side of tubular cells in the mammalian kidney.
我们研究了环氧二十碳三烯酸（EETs）对极化的肾远端小管细胞系——马-达二氏犬肾（MDCK）C7细胞离子转运的影响。在所研究的四种EET区域异构体（5,6-EET、8,9-EET、11,12-EET和14,15-EET）中，仅顶端而非基底外侧施加5,6-EET可增加短路电流（I(sc)），其动力学与花生四烯酸相似。离子转运可通过用环氧化酶抑制剂吲哚美辛或氯离子通道阻滞剂NPPB预孵育来阻断。此外，无Cl(-)的浴液和Ca(2+)拮抗剂维拉帕米均可阻断5,6-EET诱导的离子转运。尽管已证实存在前列腺素E2（PGE2）受体EP2、EP3和EP4，但顶端添加的PGE2无效，且基底外侧添加的PGE2与5,6-EET相比，在离子转运方面引起不同的动力学。此外，5,6-EET处理对MDCK C7细胞中PGE2的合成无影响。细胞上清液的气相色谱/串联质谱（GC/MS/MS）分析显示，在5,6-EET处理的细胞中存在生物活性不高的5,6-二羟基-PGE1，而在对照细胞中则不存在。吲哚美辛可抑制5,6-二羟基-PGE1的形成。5,6-环氧-PGE1是5,6-二羟基-PGE1的前体，其引起的离子转运与5,6-EET相似。在MDCK C7细胞中通过免疫检测到与人CYP2C8、CYP2C9和CYP2J2蛋白同源的细胞色素P-450酶。我们的研究结果表明，5,6-EET影响肾远端小管细胞中的Cl(-)转运，其不依赖于PGE2，但通过一种依赖于其被环氧化酶转化为5,6-环氧-PGE1的机制。我们认为这种细胞色素P-450环氧化酶产物在电解质转运调节中起作用，尤其是作为一种从哺乳动物肾脏肾小管细胞管腔侧起作用的利钠化合物。
