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Epoxyeicosatrienoic acids affect electrolyte transport in renal tubular epithelial cells: dependence on cyclooxygenase and cell polarity

¹Institute of Clinical Pharmacology, Johann Wolfgang Goethe University, Frankfurt; ²Department of Pediatrics, Philipps University, Marburg; ³Vascular Signalling Group, Institute of Cardiovascular Physiology, Johann Wolfgang Goethe-University, Frankfurt, Germany; and ⁴Division of Intramural Research, National Institutes of Health/National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina

Submitted 17 May 2006 ; accepted in final form 2 May 2007

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
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 Ca²⁺ antagonist verapamil blocked 5,6-EET-induced ion transport. Although the presence of the PGE₂ receptors EP2, EP3, and EP4 was demonstrated, apically added PGE₂ was ineffective and basolaterally added PGE₂ caused a different kinetics in ion transport compared with 5,6-EET. Moreover, PGE₂ sythesis 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-PGE₁ in 5,6-EET-treated cells, but not in control cells. Indomethacin suppressed the formation of 5,6-dihydroxy-PGE₁. 5,6-Epoxy-PGE₁, the precursor of 5,6-dihydroxy-PGE₁, 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 PGE₂ but by a mechanism, dependent on its conversion to 5,6-epoxy-PGE₁ 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.

kidney; CYP450; prostaglandin; collecting duct

In this study, we characterized the effects of EETs on ion transport in a hormone-sensitive model of collecting duct principal cells using polarized Madin-Darby canine kidney (MDCK) C7 cells (12). In electrophysiological Ussing experiments, we found that 5,6-EET to activate a transient electrogenic Cl^– transport. We provide evidence that this ion transport is exclusively activated from the apical (luminal) membrane of the epithelial cell and is dependent on conversion of 5,6-EET by COX to an active metabolite, most likely 5,6-epoxy-PGE₁.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cell culture. MDCK cells of the C7 subtype between passages 74 to 80 were used. They were kindly provided by Dr. M. Gekle (Physiologisches Institut, Universität Würzburg, Germany) and have properties of distal tubular principal cells (12). The cells were passaged twice a week after reaching confluence and were grown under standard cell culture conditions (37°C and 5% CO₂ atmosphere) in Earle's MEM medium (PAA Laboratories, Cölbe, Germany) supplemented with L-glutamine, 10% fetal calf serum (Greiner, Frickenhausen, Germany), 100 U/ml penicillin, 100 µg/ml streptomycin, and 1 mM sodium bicarbonate (PAA). For electrophysiological experiments, 5 x 10⁵ cells were seeded in 12-mm culture plate well inserts (Millicell-HA, Millipore, Eschborn, Germany) and medium was replaced twice a week. Under these conditions, MDCK C7 cells form a polarized, high-resistance epithelial monolayer on the Millicell microporous membrane within 5–7 days. For cell culture experiments, cells were seeded in six-well plates and cultured until confluent. Cells were washed and experiments were performed in PBS. Inhibitors were added to the cell culture 30 min before addition of EETs.

Ussing experiments. Millicell inserts were mounted in specially designed Ussing-type chambers (Feinmechanische Werkstatt, Universität Marburg) exposing 0.6 cm² surface area. The cells were bathed on both sides with 8 ml of bath solution (37°C) circulated by a bubble-lift of carbogen (95% O₂-5% CO₂) and maintained at pH 7.4. Short-circuit current (I_sc), transepithelial potential difference (PD), and tissue resistance (R_TE) measurements were performed as previously described (42) using a computer-controlled CVC6 voltage clamp device (Fa. Fiebig, Berlin, Germany). All measurements were corrected for bath resistance. Agar-saline bridges and Ag/AgCl-electrodes (Argenthal Elektrolyt 9823, Fa. Ingold, Germany) served as electrical connections between the Ussing chamber and the voltage clamp device. Tissue equilibration time of 20 min was sufficient to obtain stable baseline currents and only C7 cells exhibiting a transepithelial resistance of more than 1,000 ·cm2 were used throughout the experiments.

Solutions and drugs. All experiments were carried out in a bath solution containing 140.0 mmol/l Na⁺, 123.8 mmol/l Cl^–, 5.4 mmol/ l K⁺, 2.4 mmol/l H₂PO₄^–, 1.2 mmol/l Ca²⁺, 0.6 mmol/l H₂PO₄^–, 1.2 mmol/l Mg²⁺, 21.0 mmol/l HCO₃^–, and 10 mmol/l glucose. For Cl^–-free experiments, Cl^– was substituted with glucuronic acid in equimolar concentrations. AA (in ethanol) and EETs (in acetonitrile) were obtained from Cayman Chemical (Ann Arbor, MI); all other drugs came from Sigma Chemicals (Deisenhofen, Germany) and were dissolved in bath solution.

Sample preparation for LC/MS. For determination of the four EETs and the corresponding DHETs by LC/MS analysis, samples were prepared as described recently (25).

Sample preparation for GC/MS/MS. For determination of 5,6-EET metabolites, MDCK C7 cells were grown in 145-mm tissue culture dishes (Greiner). After reaching confluence, 5,6-EET (0.3 µM in acetonitrile) or acetonitrile (control) were added to the medium. Following an additional 2 min (the time when maximal electrophysiological effects were observed), the reaction was stopped by adding a 1:1 mixture of ice-cold PBS and ethyl acetate. Likewise, cells were grown in 60-mm dishes to evaluate possible effects of 5,6-EET on intracellular and extracellular PGE₂ levels. Medium (extracellular PGE₂) was collected before (control), 2, and 5 min after addition of 1 µM 5,6-EET to the medium. The reaction was stopped by adding a 1:1 mixture of ice-cold PBS and ethyl acetate. The cells were collected, briefly vortexed, and prepared for gas chromatography-triple stage quadrupole mass spectrometry (GC/MS/MS) determination of 5,6-EET metabolites and intracellular PGE₂. Briefly, the samples were acidified with formic acid (10%) to pH 3.2 and O-methylhydroxylamine hydrochloride (130 mg) in 1.5 ml acetate buffer (1.5 M, pH 5) was added to form the methoxime. After acidification with formic acid to pH 2.5, the prostanoid derivatives were extracted twice with 3 ml ethyl acetate-hexane (70:30 vol/vol). After evaporation of the solvent, acetonitrile (80 µl), pentafluorobenzyl bromide (7 µl), and N,N-diisopropylethylamine (25 µl) were added. The mixture was allowed to react at 40°C for 25 min. The dry sample was purified by TLC (developing solvent: ethyl acetate-hexane 90:10 vol/vol). A broad zone (RF 0.03–0.39) was eluted with the TLC developing solvent (800 µl) and water (50 µl) was added. After centrifugation, the ethyl acetate phase was withdrawn, the solvent was evaporated, and the prostanoids were derivatized with BSTFA (25 µl; 40°C, 1 h). A 2-µl aliquot of this solution was injected.

GC/MS/MS analysis. A Finnigan MAT TSQ700 tandem mass spectrometer equipped with a Varian 3400 gas chromatograph and a CTC A200S autosampler was employed. Gas chromatography of prostanoid derivatives was carried out on a J&W DB-1 (15 m, 0.25-mm ID, 0.25-µm film thickness) capillary column (Carlo Erba, Hofheim, Germany) in the splitless mode at an inlet pressure of 100 kPa. The oven temperature program for all prostanoids analyzed was as follows: initial temperature of 100°C was held for 2 min, then increased at 30°C per min to 280°C and at 5°C per min to 310°C. This temperature was held for 2 min. Mass spectrometer conditions were as follows: interface temperature 300°C, source temperature 150°C, methane CI gas pressure 50 Pa, electron energy 70 eV, emission current 0.4 mA, conversion dynode 20 kV, and electron multiplier 1,800 V. Collision-induced decompostion spectra of the [M-PFB]^–-ion of the 5,6-dihydroxy-PGE₁ derivative (m/z 702) were taken at a collision cell pressure of 0.2 Pa and a collision energy of 15 eV.

Preparation of 5,6-epoxy-PGE₁. The 5,6-epoxy-PGE₁ was obtained exactly as described by Sakairi et al. (37) using PGE₂ methyl ester (Cayman Chemicals) dissolved in anhydrous CH₂Cl₂ and to which in situ prepared dimethyldioxirane (28) was added. Structure was confirmed by GC/MS/MS.

Preparation of canine kidney sections. We used canine kidney tissue to look for expression and localization of an EET-forming epoxygenase. We focused on CYP2J protein which has already been reported to be expressed in mouse, rat, and human kidney (22). The fresh kidney of a male Rottweiler was obtained after euthanization because of fatal hip dysplasia. Normal renal tissue was snap-frozen in liquid nitrogen and stored at –80°C. For cryosectioning, specimens were embedded in tissue adhesive medium (Tissue-Tek, Sakura Finetek, Torrence, CA). Slices of 5-µm thickness were cut using a Leica CM1900 cryostat and were thaw mounted onto poly-L-lysine-coated slides.

Immunohistochemistry. For CYP2J immunostaining, the avidin:biotinylated enzyme complex (ABC) method was used following the manufacturer's protocol (VECTASTAIN Elite ABC-Peroxidase Kit, Vector Laboratories, Burlingame, CA). Slides were fixed in acetone at 4°C for 10 min and then air-dried at room temperature. For quenching of endogenous peroxidase, slides were incubated in 0.3% H₂O₂ in methanol for 30 min. Overnight incubation using a polyclonal anti-human CYP2J2 antibody (1:200 dilution in PBS/10% goat serum) was followed by successive incubation at 37°C with a biotinylated secondary antibody, VECTASTAIN Elite ABC Reagent, and peroxidase substrate solution. Control experiments without primary antibody showed no specific immunostaining. The CYP2J2 antibody was prepared by immunizing rabbits with a partially purified preparation of recombinant CYP2J2 as previously described (44). The antibody has been previously shown to immunoreact with CYP2J subfamily P450s in rabbit, mouse, and rat, but does not cross-react with non-CYP2J subfamily P450s including members of the CYP1A, CYP2A, CYP2B, CYP2C, CYP2D, CYP2E, and CYP4A subfamilies (22, 29, 44).

Immunoblotting. MDCK C7 cell pellets and human and rat kidney tissue specimens were disrupted and homogenized using a Polytron homogenizer (Kinematica, Lucerne, Switzerland) and a Potter-Elvehjem homogenizer (Kobe, Marburg, Germany). Total protein was extracted and protein concentrations were determined by the bicin choninic acid method according to the manufacturer's protocol (Pierce, Rockford, IL) using bovine serum albumin as protein standard. Samples of 100 µg protein were separated by SDS-PAGE on 8–16% polyacrylamide minigels (Gradigel, Frenchs Forest, Australia) and transferred to nitrocellulose membranes (Hybond C extra, Amersham, Little Chalfont, UK). The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline (TBS) and incubated with 5% goat serum (in TBS) for 30 min. After being washed with 0.1% Tween 20/TBS (TTBS), the primary antibodies [anti-human EP1 to EP4 receptor from Cayman Chemicals, anti-human CYP2C8 (26), anti-human CYP2C9 (25), anti-human sEH and anti-human CYP2J2 antibody] were applied for 2 h, followed by incubation with donkey anti-rabbit IgG peroxidase-conjugate (1:10,000). After several washing steps, sites of antibody-antigen reaction were visualized by enhanced chemiluminescence technique (ECL plus, Amersham) with exposure times of 5 s to 2 min on autoradiographic film (Hyperfilm ECL, Amersham). In control experiments without primary antibody, no protein bands were detected after the staining procedure.

PCR analysis of EP receptor mRNA. mRNA expression of the EP1 to EP4 receptor in cultured MDCK C7 cells was examined by PCR as described (3).

Statistics. Data are presented as mean values ± SE. Student's t-test (paired or unpaired, as appropriate) was used to compare data between two groups. To compare data between various groups, statistical analysis was performed by one-way ANOVA followed by Bonferroni's multiple comparison test. Values of P < 0.05 were considered statistically significant.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Effects of AA on MDCK monolayers. MDCK C7 cells seeded in Millicell HA culture plate well inserts form a tight and polarized epithelial monolayer within several days. In a first approach, we studied the effect of AA on ion transport. In Ussing chamber experiments (Table 1), basolateral AA (1 µM) had no significant effect on I_sc, PD, or R_TE. In contrast, addition of AA to the apical bath solution rapidly increased basal I_sc. The corresponding PD decreased while R_TE remained unchanged. As depicted in an original trace recording (Fig. 1), the maximum effect occurred within 3 min and returned to baseline values within 10 min of adding AA. Preincubation of MDCK cells with P450 epoxygenase inhibitors ketoconazole (10 µM), clotrimazole (10 µM), or SKF525A (100 µM) for 30 min markedly attenuated the effect of AA (Table 1). The electrogenic ion transport was completely blocked by 10 µM indomethacin (Table 1), suggesting that a COX-dependent AA metabolite mediated the observed effect.

View larger version (15K): [in this window] [in a new window]   Fig. 1. Original trace recording showing the effect of 1 µM arachidonic acid on short-circuit current (Isc) of indomethacin-pretreated (10 µM, chambers 1–3) and control (chambers 4–6) Madin-Darby canine kidney (MDCK) C7 monolayers. Note time course and cyclooxygenase (COX) sensitivity of ion transport. Cells were seeded on microporous membranes until forming a high-resistance epithelial monolayer. Membranes were mounted in Ussing-type chambers and bathed on both sides. Time point of addition of indomethacin and arachidonic acid is indicated in the figure.

View larger version (8K): [in this window] [in a new window]   Fig. 2. Original trace recording showing the effect of basolateral PGE2 (1 µM) on Isc of MDCK C7 monolayers. Note different time course compared with arachidonic acid (AA; Fig. 1), and 5,6-EET (Fig. 4B), as well as sensitivity of ion transport to epithelial sodium channel blocker, amiloride, but not Cl– channel blocker, 5-nitro-2-(3-phenylpropyl-amino)benzoic acid (NPPB). Cells were seeded on microporous membranes until forming a high-resistance epithelial monolayer. Membranes were mounted in Ussing-type chambers and bathed on both sides. Time point of addition of PGE2, NPPB, and amiloride is indicated in the figure. A representative of 4 independent experiments is shown.

View larger version (18K): [in this window] [in a new window]   Fig. 3. Expression of EP receptor mRNA and protein in MDCK C7 cells. mRNA expression (top) was analyzed by PCR using EP receptor-specific oligonucleotides and EP receptor protein (bottom) expression was studied by immunoblotting using specific EP receptor antibodies. The observed apparent molecular weights are 62, 65, and 72 kDa for the EP2, EP3, and EP4 receptor, respectively. Note that neither EP1 mRNA nor EP1 protein was detected.

View larger version (18K): [in this window] [in a new window]   Fig. 4. A: effect of apical application of 5,6-EET (0.3 µM) on Isc. Cells were seeded on microporous membranes until forming a high-resistance epithelial monolayer. Membranes were mounted in Ussing-type chambers and bathed on both sides. Time point of apical addition of 5,6-EET is indicated in the figure. Representative 3 stimulations (chamber 1–3) out of n = 6 are shown. B: effect of indomethacin (10 µM), diclofenac (10 µM), the EP1 antagonist ONO-8711 (10 µM), and the EP4 antagonist ONO-AE3-208 (10 µM) on 5,6-EET induced ion transport. The inhibitors and the antagonists, respectively, were added to apical bath solution 20 min before 5,6-EET addition (n = 6). *P < 0.05.

View larger version (15K): [in this window] [in a new window]   Fig. 5. Characterization of the effect of 0.3 µM 5,6-EET on Isc of MDCK C7 cell monolayers using established ion transport inhibitors/blockers. Cells were seeded on microporous membranes until forming a high-resistance epithelial monolayer. Membranes were mounted in Ussing-type chambers and bathed on both sides. Bars indicate apical 5,6-EET alone (n = 7) as control (100%), and after preincubation for 10 min with 0.3 mM furosemide (furo; n = 4), 0.1 mM hydrochlorothiazide (HCT; n = 4), 10 µM amiloride (ami; n = 4), 10 µM NPPB (n = 3), 100 µM verapamil (vera; n = 4), and use of chloride-free bath solution (Cl– free, n = 4). *P < 0.05.

View larger version (22K): [in this window] [in a new window]   Fig. 6. A: formation of the 4 EETs, their hydration products DHETs, and of PGE2 following addition of 100 µM AA. MDCK C7 cells were cultured until confluent, washed, and AA was added with buffer for 30 min. Thereafter, supernatants were removed and EETs, DHETs, and PGE2 were quantified by LC/MS (n = 4). B: effect of 5,6-EET (1 µM) on intracellular and extracellular PGE2 levels of MDCK C7 cells as determined in duplicate by GC/MS/MS. MDCK C7 cells were cultured until confluent. Cells and medium were collected before (0 min), and after apical addition of 5,6-EET for 2 and 5 min, respectively, according to the maximal effect on ion transport (n = 3 for each group). C: product ion spectrum of the GC peak observed in 5,6-EET-treated MDCK C7 cells. The fragments identified this metabolite as 5,6-dihydroxy-PGE1. TMS, trimethylsilylether; PFB, pentafluorobenzylester; parent ion [P]– = [M – PFB]–; TMSOH = (CH3)3SIOH. D: inhibition of 5,6-dihydroxy-PGE1 (5,6-dh-PGE1) formation from AA by SKF525A (SKF; 100 µM) and from 5,6-EET by indomethacin (indo; 10 µM). MDCK C7 cells were cultured until confluent, washed, and AA or 5,6-EET were added with buffer for 2 min. The supernatants were removed and prepared for MS analysis (n = 3). *P < 0.05.

1

To further study the metabolism of 5,6-EET in this system, MDCK C7 monolayers were exposed to either solvent or 5,6-EET. In contrast to control cells, MDCK C7 cells treated with apical 5,6-EET for 2 min showed a specific peak in the gas chromatogram identified by its product ion spectrum as 5,6-dihydroxy-PGE₁, the stable hydrolysis product of 5,6-epoxy-PGE₁ (Fig. 6C). Following incubation of the EET-treated cells with the COX inhibitor indomethacin, the 5,6-dihydroxy-PGE₁ peak disappeared (Fig. 6D). When MDCK cells were stimulated with AA in the presence of the P450 inhibitor SKF525A, the 5,6-dihydroxy-PGE₁ peak was also diminished (Fig. 6D). However, electrophysiological experiments with 5,6-dihydroxy-PGE₁ revealed that it exhibits no electrogenic activity (Table 3). Therefore, 5,6-epoxy-PGE₁, the unstable precursor of 5,6-dihydroxy-PGE₁, was synthesized and analyzed by Ussing technique. Following apical addition, a significant increase in I_SC was observed with a similar pattern of ion transport as 5,6-EET (Table 3).

View larger version (90K): [in this window] [in a new window]   Fig. 7. A: immunoblot of CYP2J2 (top) CYP2C8, CYP2C9 (middle), and sEH (bottom) immunoreactive proteins in MDCK C7 cells (MDCK). Recombinant protein standards for CYP2C8 (2C8), CYP2C9 (2C9), and sEH are indicated. For CYP2J2 protein, the expression in human and rat kidney homogenates is also shown (top). B: localization of CYP2J2 immunoreactive protein in canine kidney sections by immunocytochemistry. Positive staining was seen in blood vessels (thin arrows) and also in cortical collecting ducts (thick arrows). Magnification: x100 (A) and x400 (B). GLOM, glomerulus; PT, proximal tubule.

View larger version (17K): [in this window] [in a new window]   Fig. 8. Effect of the sEH inhibitors AUDA [12-(3-adamantan-1-yl-ureido)dodecanoic acid] and CDU (1-cyclohexyl-3-dodecylurea) on formation of DHETs. Cells were grown until confluent, washed, and then preincubated with AUDA or CDU (each at 100 µM) for 30 min in renewed buffer. Thereafter, 5,6-EET or 14,15-EET were added to the culture medium and supernatants were removed after 15 min of incubation. The amount of 5,6-DHET and 14,15-DHET was determined in the supernatants by LC/MS/MS (n = 5). Without EET addition, no DHETs were detected (data not shown). *P < 0.05.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Under our experimental conditions, 5,6-EET and its methyl ester, but not 8,9-, 11,12-, or 14,15-EET, exerted strong electrogenic effects and only apical application of 5,6-EET was able to stimulate ion transport. We excluded PGE₂ as a mediator of the observed 5,6-EET-stimulated effects for five reasons. First, compared with 5,6-EET, the apical application of PGE₂ induced only a marginal increase in I_sc. Second, the I_sc kinetics evoked by PGE₂ was quite different from the kinetics elicited by AA or 5,6-EET. In particular, there was a strict polarity in the actions of these eicosanoids; the apical action of 5,6-EET was in contrast to the basolateral action of PGE₂. Third, the effect of PGE₂ was sensitive to amiloride (10 µM), indicating the involvement of Na⁺ fluxes, most likely via Na⁺ channels [IC₅₀ of amiloride for Na⁺ channel is 0.1–0.5 µM, for the Na⁺/H⁺ antiporter 5–50 µM, and for the Na⁺/Ca²⁺ exchanger 1,100 µM (39)]. In contrast, the effect of 5,6-EET was insensitive to amiloride. Fourth, 5,6-EET had no effect on the cellular formation of PGE₂; moreover, 5,6-EET also does not provoke the formation of other EETs. Fifth, 5,6-epoxy-PGE₁, a cyclooxygenase product of 5,6-EET, evoked a similar change in ion transport as AA or 5,6-EET.

Our experiments using different ion transport inhibitors indicate that 5,6-EET increases apical Cl^– secretion in MDCK C7 cells suggesting that this eicosanoid has saluretic properties in the distal tubule. It is not known whether 5,6-EET also demonstrates saluretic properties in vivo; however, 5,6-EET-induced saluresis would fit well with the proposed antihypertensive role EETs. Excess dietary salt induces a P450 epoxygenase expression in rat kidneys and treatment with the epoxygenase inhibitor, clotrimazole, produces significant increases in mean arterial blood pressure (24). It has been suggested that the salt-inducible renal epoxygenase pathway may be one of the functionally significant components of the adaptive response to increased salt intake. In support of this assumption, 5,6-EET has been shown to be a powerful inhibitor of Na⁺ absorption in the distal nephron (20). Interestingly, elevated levels of 8,9-DHET and 11,12-DHET, the stable metabolites of 8,9-EET and 11,12-EET, were found in urine samples from patients with pregnancy-induced hypertension (8). Although it is unknown whether increases in EET levels are causative or compensatory with respect to hypertension, it might be possible that different EET isoforms are involved in different aspects of blood pressure regulation.

In the rat kidney, 5,6-EET has been shown to mediate the renal vasodilation to AA. This effect was unmasked by inhibition of COX, which abolished the PGH₂-mediated vasoconstrictor effect (33). This indicates that 5,6-EET itself and not a COX product of 5,6-EET mediates the reported vasodilator effect. In our model, 5,6-EET itself did not affect Cl^– efflux while its COX product, 5,6-epoxy-PGE₁, appears to be the active agent. Importantly, both effects (vasodilation and electrogenic effects) would be expected to lower blood pressure.

Electrogenic ion transport stimulated by 5,6-EET, but not by other EETs, has been demonstrated in isolated perfused collecting ducts from rabbits (37) and 5,6-EET depolarized transepithelial voltage (V_T) in association with an increase in [Ca²⁺]_i. However, there are three striking differences in that study compared with our findings. First, the authors observed that 5,6-EET stimulated PGE₂ production severalfold in the perfused collecting ducts and therefore indomethacin completely blocked the effect of 5,6-EET on V_T. Second, both addition of 5,6-EET from luminal and basolateral affected [Ca²⁺]_i. Third, the authors oberserved that PGE₂ caused a similar effect on V_T as 5,6-EET and that 5,6-epoxy-PGE₁ was ineffective under their experimental setting. In our study, the electrogenic effect of 5,6-EET in MDCK C7 cells was also sensitive to COX inhibition; however, PGE₂ levels were not altered following 5,6-EET addition. Moreover, PGE₂ affected V_T different to 5,6-EET. Therefore, we conclude that in MDCK C7 cells ion transport evoked by 5,6-EET (or AA) is dependent on formation of another metabolite generated by COX, 5,6-epoxy-PGE₁. Its hydration product 5,6-dihydroxy-PGE₁ was detected in the supernatants of 5,6-EET (or AA)-treated cells. Furthermore, only the apical application of 5,6-EET induced Cl^– secretion. This is in direct contrast to the observations that exogenous PGE₂ induces a distinct I_SC pattern and that only following its basolateral application. This might explain why 5,6-EET in the basolateral bathing solution did not affect Cl^– transport in the microperfused medullary TAL of the rat (15). Interestingly, 5,6-EET also requires its conversion by COX to exert its vasoactivity in the rat caudal artery (6). The renal vasodilator effects of 5,6-EET, on the other hand, have been attributed to both the COX-dependent conversion of 5,6-EET to 5,6-epoxy-PGE₁ as well as the stimulation of PGE₂ generation by 5,6-EET (5).

The mechanism by which 5,6-EET increases Cl^– secretion is currently unknown. Several investigations point to an increase in [Ca²⁺]_i as part of the signaling mechanism involved (14, 16, 23). Accordingly, preventing influx of extracellular Ca²⁺ by the voltage-gated Ca²⁺ channel blocker verapamil strongly inhibited the effects of 5,6-EET in our study. The activation of Ca²⁺-dependent K⁺ channels might also be involved in mediating the actions of 5,6-EET (1, 31). In the rat colon, we observed that 5,6-EET indirectly induces Cl^– secretion via a mechanism dependent on the activation of basolateral K_Ca channels (Wegmann M, unpublished observation). Of note, K_Ca channels are known to be located in the apical membrane of collecting tubule principal cells (13). Further studies are necessary to identify the precise molecular mechanism by which 5,6-EET affects ion transport in MDCK C7 cells.

CYP2C and 2J generate EETs and are abundantly expressed within tubules of the mouse renal cortex and outer medulla (22) and increased CYP2J expression and EET formation was observed in the spontaneously hypertensive rat kidney (45). Immunohistochemical analysis and Western blotting studies demonstrated abundant expression of CYP2J-immunoreactive protein in canine kidney blood vessels and in the cortical collecting duct, as well as in the MDCK C7 cell line. In addition, we observed expression of two enzymes of the CYP2C subfamily, CYP2C8 and CYP2C9 in MDCK C7 cells. Although other P450s may also contribute to EET biosynthesis, we speculate that CYP2J- and/or CYP2C-derived EETs may play an important role in regulation of renal tubular ion transport. Since renal tissue express both COX and P450 epoxygenases (22), the metabolism of AA to 5,6-EET and further conversion to 5,6-epoxy-PGE₁ seems likely. We propose that rapid nonenzymatic hydrolysis to the stable inactive 5,6-dihydroxy-PGE₁ (7, 37) may represent a "switch-off" mechanism for the 5,6-EET signal.

It remains unknown whether 5,6-epoxy-PGE₁ acts directly on channel activity or indirectly as a second messenger. As an example for the latter, 14,15-EET serves as an intracellular second messenger for EGF involving the activation of kinase-associated mitogenic pathways in the renal LLCPKc14 cells (9). Another possibility would be the binding to specific EET receptors, although the identity of these putative receptors remains unknown. We exclude the activation of PGE₂ receptors as PGE₂ evoked a Na⁺ channel dependent of ion transport with different kinetics compared with 5,6-EET. The strict basolateral action indicates that the relevant PGE₂ receptors are located on the basolateral side of the cells, whereas 5,6-EET acts on the apical membrane. Although we demonstrated the presence of EP2, EP3, and EP4 receptor in MDCK C7 cells, at least EP1 and EP4 antagonists were unable to block the effects of 5,6-EET. Interestingly, PGE₁ does not differ from PGE₂ but does differ from 5,6-EET with regard to stimulation of amiloride-sensitive Na⁺ reabsorption in MDCK C7 cells (data not shown).

In conclusion, apical application of 5,6-EET induces Cl^– transport in MDCK C7 cells, a model of renal collecting duct principal cells. We provide evidence that 5,6-EET is a substrate for COX forming the Cl^– transport-activating metabolite 5,6-epoxy-PGE₁, which is then further metabolized to the corresponding inactive 5,6-dihydroxy-PGE₁. The role of 5,6-EET might have been underestimated due to its rapid in vivo decomposition and in light of difficulties in its detection. Since EETs are also found in human urine (8), the possibility of a saluretic compound acting predominantly from the luminal side of tubular cells is intriguing.

	FOOTNOTES

 

Address for reprint requests and other correspondence: R. M. Nüsing, Institute of Clinical Pharmacology, Bldg. 75, Johann Wolfgang Goethe-Univ., Theodor Stern Kai 7, 60590 Frankfurt am Main, Germany (e-mail: r.m.nuesing{at}med.uni-frankfurt.de)

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	REFERENCES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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