Epoxyeicosatrienoic acids affect electrolyte transport in renal tubular epithelial cells: dependence on cyclooxygenase and cell polarity.
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.
Nüsing
Rolf M
RM
Institute of Clinical Pharmacology, Johann Wolfgang Goethe University, Theodor Stern Kai 7, 60590 Frankfurt am Main, Germany. r.m.nuesing@med.uni-frankfurt.de
Schweer
Horst
H
Fleming
Ingrid
I
Zeldin
Darryl C
DC
Wegmann
Markus
M
D015126:8,11,14-Eicosatrienoic Acid / Q000494:pharmacology*; D000527:Alprostadil / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000494:pharmacology; D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects; D015153:Blotting, Western; D002460:Cell Line; D016764:Cell Polarity / Q000502:physiology*; D002712:Chlorides / Q000378:metabolism; D002853:Chromatography, Liquid; D015194:Diffusion Chambers, Culture; D015232:Dinoprostone / Q000494:pharmacology; D004285:Dogs; D004573:Electrolytes / Q000378:metabolism*; D004594:Electrophysiology; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D004851:Epoxide Hydrolases / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D007150:Immunohistochemistry; D007536:Isomerism; D007684:Kidney Tubules / Q000187:drug effects / Q000378:metabolism*; D007685:Kidney Tubules, Collecting / Q000187:drug effects / Q000378:metabolism; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
D002712:Chlorides; D004573:Electrolytes; D012333:RNA, Messenger; C044367:5(6)-epoxyprostaglandin E1 alpha; D011451:Prostaglandin-Endoperoxide Synthases; D004851:Epoxide Hydrolases; D000527:Alprostadil; D015126:8,11,14-Eicosatrienoic Acid; D015232:Dinoprostone