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发表于 2009-4-21 13:37 |只看该作者 |倒序浏览 |打印

作者：HaoHe, TiinaPodymow, JosephZimpelmann,  Kevin D.Burns作者单位：Department of Medicine, Ottawa Hospital, and the KidneyResearch Centre, Ottawa Health Research Institute, University ofOttawa, Ottawa, Ontario, Canada K1H 8L6


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      【摘要】
   Nitric oxide (NO) exerts directeffects on nephron transport. We determined the effect of NO onNa -K -2Cl cotransport in a cellline (MMDD1) with properties of macula densa.Na -K -2Cl cotransport wasmeasured as bumetanide-sensitive 86 Rb uptakein the presence of ouabain. MMDD1 cells expressed mRNA for the neuronalisoform of nitric oxide synthase, as well as NKCC1 and NKCC2(B)isoforms of the Na -K -2Cl cotransporter. Preincubation of cells with the NO donors sodium nitroprusside (SNP) or S -nitroso- N -acetylpenicillamine (SNAP) causedconcentration-dependent inhibition ofNa -K -2Cl cotransport. Bothapical and basolateral Na -K -2Cl cotransport was inhibited by NO donors. SNP or SNAP had no significant effect on cellular levels of cGMP, cAMP, cytosolic calcium, or phosphorylation of ERK1 and ERK2. In contrast, the inhibitors ofcytochrome P -450, 1-aminobenzotriazole (ABT;10 3 M) or ketoconazole (1.5 × 10 5 M),completely reversed the inhibitory effect of SNAP on apical orbasolateral Na -K -2Cl cotransport [apical: control 1.18 ± 0.15 vs. SNAP(10 4 M) 0.41 ± 0.05 pmol · mg 1 · 5 min 1; P 4 M) ABT 1.32 ± 0.10 pmol · mg 1 · 5 min 1; P = not significant vs. control; n = 5]. The cytochrome P -450 epoxyeicosatrienoic acid (EET) metabolite 14,15-EET (5 × 10 7 M) inhibited both apical and basolateral cotransport,whereas 8,9-EET and 11,12-EET had no significant effect. Although20-hydroxyeicosatetraenoic acid inhibited apical cotransport, theinhibitor of -hydroxylase activity HET0016 did not reverseSNAP-mediated inhibition of apical cotransport. These data indicatethat NO inhibits apical and basolateral Na -K -2Cl cotransport in MMDD1cells. The results suggest that the inhibitory pathway is independentof cGMP and might involve stimulation of a cytochrome P -450-dependent pathway.
      【关键词】 NKCC NKCC nitric oxide synthase epoxyeicosatrienoic acids
   INTRODUCTION  ^( M& L2 Y' f3 ?$ S

THE LABILE GAS nitricoxide (NO) is generated in the kidney and exerts direct effects ontransport in several nephron segments. In proximal tubule cells, NO hasbeen shown to inhibit both apical Na -H exchange ( 32 ) and Na -K -ATPaseactivity ( 22 ) via pathways involving generation of cGMP. Thick ascending limb apical and basolateralNa -H exchange is inhibited by NO, as iscortical collecting duct transport of sodium and water ( 9, 35 ). The inhibitory actions of NO on sodium and water transportalong the nephron suggest that NO generated locally could promotenatriuresis and diuresis. Consistent with this view, recent studies inthe isolated perfused rat thick ascending limb showed that the NO donorspermine NONOate blocks chloride reabsorption by selectively inhibitingthe apical Na -K -2Cl cotransporter, with noeffect on Na -K -ATPase activity orapical K permeability ( 28 ). In isolated ratcortical thick ascending limbs, inhibition of endogenous NO productionby the NO synthase (NOS) inhibitor N G -nitro- L -arginine has beenobserved to stimulate chloride transport, suggesting that thick limb NOgeneration directly regulates cotransport ( 30 ).) G) ^# S0 [6 s8 E  f7 T

Regulation of apical Na -K -2Clcotransport is of critical importance in cells of the macula densa,which are involved in the tubuloglomerular feedback (TGF) response.Macula densa cells express high levels of the neuronal isoform of NOS(nNOS) ( 25 ), and several studies indicate that NO releasedby macula densa nNOS inhibits TGF ( 13, 16, 40 ). In rabbitafferent arterioles with attached macula densae, NO has been shown toact directly on macula densa cells to block TGF responses in acGMP-dependent fashion ( 31, 39 ). Because NO donorssignificantly inhibit theNa -K -2Cl cotransporter in ratthick ascending limb ( 28 ), one possibility for theattenuating effect of NO on TGF is via inhibition of the apicalNa -K -2Cl cotransporter (NKCC2)( 41 ) in macula densa.
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The purpose of the present studies was to investigate the effectsof NO on Na -K -2Cl cotransportand signaling pathways for NO in a renal epithelial cell line (MMDD1)with properties of macula densa cells ( 42 ). We determinedthat these cells express both NKCC1 and NKCC2 isoforms of theNa -K -2Cl cotransporter, and wedemonstrated that NO potently inhibits both apical and basolateralcotransport. Furthermore, we uncovered a novel role for cytochrome P -450 (also referred to as P -450) in mediatingresponses to NO in these cells.
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MATERIALS AND METHODS2 o" I2 I4 y. Y4 T9 p( P  m8 y: l

Cell culture. A renal epithelial cell line, MMDD1, with properties of macula densacells, was used for these studies, kindly provided by Dr. J. Schnermann(National Institutes of Health, Bethesda, MD). These cells were derivedfrom SV40 transgenic mice, using fluorescent-activated cell sorting ofrenal tubular cells labeled with segment-specific lectins( 42 ). The cell line has been shown to express well-known markers of macula densa, such as cyclooxygenase 2 (COX-2), nNOS, ROMK,and NKCC2. In the present studies, MMDD1 cells at passages 5 - 20 were routinely trypsinized and suspended in DMEMnutrient mixture-Ham's F-12 (DMEM/F-12) supplemented with 10% fetalbovine serum, penicillin (100 U/ml), and streptomycin (100 µg/ml).The cells were plated onto six-well culture dishes and incubated at 37°C in a humidified atmosphere of 95% room air-5% CO 2.For experiments involving measurement of apical and basolateralNa -K -2Cl cotransport, cellswere plated onto six-well cell culture inserts (0.4-µm pore size,Falcon, VWR Canlab, Mississauga, ON, Canada). The media was changedevery 2 days, and once the cells reached confluence (typically in3-4 days), the cells were maintained in serum-free DMEM/F-12 for24 h before experiments.0 h5 O) _8 C6 c# M8 l, u4 c
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RT-PCR for nNOS, NKCC1, NKCC2, and cytochrome P-450 2J5. Total RNA was isolated from MMDD1 cells, using a commercial kit(RNeasy, Qiagen, Chatsworth, CA). RNA (1 µg) was treated with DNase Iand then reverse-transcribed using random hexamers (2.5 µM) andmurine leukemia reverse transcriptase (RT; 2.5 U/µl) (Gene Amp RNAPCR core kit, Applied Biosystems Roche Molecular System, Branchburg,NJ). To control for possible genomic DNA contamination, all experimentsincluded a reaction in which RT was omitted from the transcriptionbuffer. PCR amplification was carried out in 100 µl of a solutioncontaining 2.5 U ampli Taq DNA polymerase, 2 mMMgCl 2, 1× PCR buffer II (Gene Amp PCR core kit), and 1 µM of the sense and antisense oligonucleotide DNA primers for the cDNA of interest. For nNOS, the sense primer was5'-ACTGCTCAAAGAGATAGAACC-3' and the antisense primer was5'-GGTAATTCTCCAAACCGAG-3', corresponding to nucleotides 695-716and 1201-1219 of the murine nNOS cDNA, respectively( 26 ), and predicting amplification of a 525-bp cDNAproduct. For NKCC1, the sense primer was 5'-CAGAGACAATTTGAAGACC-3' andthe antisense primer was 5'-CCAACGTCAGCATGAGAT-3', corresponding tonucleotides 5305-5323 and 5968-5985 of the murine NKCC1 cDNA, respectively, and predicting a PCR product of 681 bp ( 6 ).For NKCC2, the sense primer was 5'-CCCGCTCTCTTGGATATATAAC-3' and the antisense primer was 5'-GCTCCGAACAAATTCTTCCG-3', corresponding tonucleotides 2301-2321 and 2790-2809 of the murine NKCC2 cDNA, respectively ( 14 ), and predicting a PCR product of 509 bp.RT-PCR specific for the B isoform of NKCC2 [found in macula densa and thick ascending limb ( 41 )] was performed with senseprimer 5'-GTGTGATTATCATCGGCTTAGC-3' and antisense primer5'-ATCAAGCCTATTGCACCACC-3', corresponding to nucleotides 853-874and 977-1006 of the B isoform of murine NKCC2 and predicting a PCRproduct of 154 bp ( 14 ). For cytochrome P -450isoform CYP2J5, the sense primer was 5'-ATCAGAGAAGCGAAAAGAATGTAG-3' andthe antisense primer was 5'-TACTCAGTCTTAGTCTCCTTTACC-3', corresponding to nucleotides 1549-1572 and 1810-1833 of the murinecytochrome P -450 2J5 cDNA, respectively, and predicting aPCR product of 285 bp ( 21 ). PCR was routinely performedfor 35 cycles, with a hot-start at 95°C for 5 min, followed by cyclesat 95°C for 60 s, 58°C for 30 s, and 72°C for 60 s, followed by extension at 72°C for 10 min. DNA samples wereelectrophoresed on 2% agarose gels stained with ethidium bromide. DNAsequencing was performed by the DNA Sequencing Facility, University of Ottawa.
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Measurement ofNa -K -2Cl cotransport activity. The activity of Na -K -2Cl cotransport was measured by the uptake of 86 Rb  , in the presence of ouabain and in thepresence or absence of the inhibitor bumetanide, essentially asdescribed ( 12 ). Briefly, confluent cells were rinsed twicein Earle's salt solution consisting of (in mM) 140 NaCl, 5 KCl, 1 MgSO 4, 1.8 CaCl 2, 5 glucose, and 25 HEPES (pH7.40 with Tris), and then incubated in this solution for 30 min at37°C. Cells were then incubated for an additional 15 min in thissolution, supplemented with or without agonists/antagonists, at roomtemperature. For assay ofNa -K -2Cl cotransport, theincubation solution was then changed to one supplemented with 1 µCi/ml 86 Rb (Amersham, Oakville, ON,Canada), with 2 mM ouabain, and with or without 6 µM bumetanide(Sigma, St. Louis, MO), at room temperature. After 5 min, the uptakewas terminated by removal of the solution, followed by four rapidwashings with ice-cold 100 mM MgCl 2, buffered with 10 mMHEPES (pH 7.4 with Tris). Culture dishes were air-dried, and cells weresolubilized in 1 N NaOH with 0.1% SDS, followed by measurement ofradioactivity by liquid scintillation spectrometry. For cells grown onsix-well cell culture inserts, activity of apical or basolateralNa -K -2Cl cotransport wasdetermined by adding 86 Rb to the apical orbasolateral surfaces, respectively, in the presence or absence ofbumetanide (6 µM). Bumetanide (6 µM) was added and maintained inthe media bathing the opposing cell membrane. Protein content wasmeasured by the Bradford assay method, using BSA as standard (Bio-Rad,Montreal, QC, Canada). Activity ofNa -K -2Cl cotransport is theouabain-insensitive, bumetanide-sensitive component of 86 Rb uptake and is expressed as picomoles of 86 Rb per milligram of protein per 5 min, withall experiments performed in duplicate. In preliminary experiments,Na -K -2Cl cotransport activitywas linear for 30 min under these assay conditions.$ \  r' F  E0 n9 M
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Measurement of cAMP and cGMP. MMDD1 cells were grown to confluence on 24-well plastic dishes and thenincubated for 24 h in serum-free DMEM/F-12 medium. For assays ofcAMP or cGMP, cells were incubated at 37°C for 15 min in DMEM/F-12,supplemented with 3-isobutyl-1-methylxanthine (5 × 10 4 M), 0.5% BSA, in the presence or absence ofagonists. Medium was then aspirated and replaced with ice-cold 10%trichloroacetic acid (TCA; vol/vol). After a further 30 min, sampleswere extracted four times with 4 vol of water-saturated ether andbrought to pH 7.0 with Tris. Aliquots were assayed for cAMP or cGMP,using radioligand competitive binding assay kits, containing[ 3 H]cAMP (Intermedico, Markham, ON, Canada) or[ 3 H]cGMP (Amersham), as we performed previously ( 5, 32 ).
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Measurement of cytosolic calcium concentration. Cytosolic calcium concentration ([Ca 2  ] i ) wasmeasured in MMDD1 cells, essentially as we described ( 5 ).Cells were grown to confluence on glass coverslips and loaded with 5 µM of fura 2-acetoxymethyl ester (fura 2-AM) in the presence of0.005% Pluronic-F127 for 45 min at room temperature. Cells were thencontinuously perfused at 37°C with a solution consisting of (in mM)105 NaCl, 24 NaHCO 3, 2 Na 2 HPO 4, 5 KCl, 1.0 MgSO 4, 1.5 CaCl 2, 4 lactic acid, 5 glucose, 1 alanine, and 10 HEPES (pH 7.3), as well as 0.2% BSA, and NO donors were added at various times. Fluorescence was measured from dualmonochromators set at 340 and 380 nm, using a computer-linked analyticsystem (Photon Technology International, South Brunswick, NJ).Fluorescence emission was measured by photon counting, and thecorrected fluorescence emission intensity ratio, from 340- and 380-nmexcitation, was monitored continuously in selected cells and used as anindicator of [Ca 2  ] i.
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Western blotting of ERK1 and ERK2. Phosphorylation of ERK1 (p44) and ERK2 (p42) was measured by Westernblotting. After incubation with or without donors of NO for 15 min,cells were lysed in an ice-cold buffer consisting of 50 mMTris · HCl (pH 8.0), 150 mM NaCl, 1% NonidetP-40, 1 mM EDTA, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 µg/mlaprotinin, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodiumorthovanadate, and 1 mM sodium fluoride. After quantification ofproteins, equal amounts of protein lysates (10 µg) were run on 15%SDS-polyacrylamide gels and transferred to nitrocellulose membranes(Bio-Rad). The membranes were incubated in TBS-T buffer [10 mM Tris(pH 7.6), 150 mM NaCl, 0.05% Tween 20, 0.5% skim milk] for 1 hat room temperature. The membranes were then incubated for 16 h at4°C with 1:500 dilution polyclonal antibody to phosphorylated ratERK1 and ERK2 (Santa Cruz Biotechnology, Santa Cruz, CA), followed byincubation with 1:1,000 dilution of anti-rat secondary antibodyconjugated to horseradish peroxidase (Amersham). After membranes werewashed, phosphorylated proteins were detected by enhancedchemiluminescence (Amersham) on Hyperfilm (Amersham). Prestainedstandards were used as molecular weight markers (Bio-Rad), and tocontrol for protein loading, all membranes were stripped and reprobedwith polyclonal antibody to unphosphorylated rat ERK1 and ERK2 (Santa Cruz Biotechnology). Signals on Western blots were quantified bydensitometry and corrected for unphosphorylated ERK1 and ERK2 levels,using an image analysis software program (Kodak Densitometer 1S440CF).; x8 z5 J0 w3 D2 N, U" K
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Eicosanoids. The epoxyeicosatrienoic acids (EETs) and 20-hydroxyeicosatetraenoicacid (20-HETE) were obtained from Sigma as HPLC-purified compounds.% ]. \" Z0 |, e7 L1 P

Data analysis. Results are presented as means ± SE of experiments performed induplicate. Significance was determined by Student's t -test and by ANOVA for cases with multiple comparisons. Significance isconsidered as P- m4 i) v- R  ?5 y2 p
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RESULTS
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Characterization of MMDD1 cells. The cell line used in these studies has previously been shown toexpress a number of specific markers for macula densa, including nNOS,NKCC2, and COX-2. In our laboratory, we confirmed expression of nNOS byRT-PCR, with restriction endonuclease digestion with Bgl IIgenerating fragments of the expected sizes (400 and 125 bp) (Fig. 1 A ). RT-PCR also revealed thepresence of NKCC2 (Fig. 1 B ). DNA sequencing of the NKCC2 PCRproduct confirmed its identity to the mouse NKCC2 isoform( 14 ). RT-PCR was also performed using primers thatgenerated a 154-bp product specific for the B isoform of murine NKCC2(not shown) ( 14 ). DNA sequencing of this product confirmedthe presence of the B isoform in MMDD1 cells. In contrast, RT-PCR using5'-primers specific for the A and F isoforms of NKCC2 did not generatePCR products, although both products were amplified from mouse kidneyRNA (not shown). RT-PCR also demonstrated the presence of NKCC1 mRNA inthese cells (Fig. 1 C ), confirmed by DNA sequencing." c. o" v7 Y( J) q& r" `

Fig. 1. Expression of neuronal nitric oxide synthase (nNOS),NKCC1, and NKCC2 mRNA in MMDD1 cells by RT-PCR. A :representative agarose gel showing DNA size ladder ( lane 1 ),525-bp nNOS PCR product ( lane 2 ), and 400- and 125-bp nNOSPCR products following digestion with Bgl II. B :representative agarose gel depicting DNA size ladder ( lane1 ), 509-bp PCR product for NKCC2 in MMDD1 cells ( lane2 ), reaction lacking reverse transcriptase as negative control( lane 3 ), 509-bp PCR product for NKCC2 in mouse kidneycortex as positive control ( lane 4 ), and correspondingreaction lacking reverse transcriptase ( lane 5 ). C : representative gel showing DNA size ladder ( lane1 ), 681-bp product for NKCC1 in MMDD1 cells ( lane 2 ),corresponding reaction lacking reverse transcriptase ( lane3 ), 681-bp PCR product for NKCC1 in mouse kidney cortex (positivecontrol) ( lane 4 ), and corresponding reaction lackingreverse transcriptase ( lane 5 ).& Y2 M  J$ g0 `" H
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Effect of NO onNa -K -2Cl cotransport. NO has been shown to inhibitNa -K -2Cl activity in the ratthick ascending limb ( 28 ) and to inhibit theNa H exchanger in proximal tubule and thickascending limb ( 9, 32 ). We first determined the effects ofNO donors on Na -K -2Cl activityin MMDD1 cells grown on plastic dishes. Both sodium nitroprusside (SNP)and S -nitroso- N -acetylpenicillamine (SNAP) causedconcentration-dependent inhibition of ouabain-insensitive, bumetanide-sensitive 86 Rb uptake (Fig. 2 ). Significant inhibition was observedat concentrations of SNP greater than or equal to 10 6 Mand at concentrations of SNAP greater than or equal to10 7 M. SNP or SNAP had no significant effect on thebumetanide-insensitive component of 86 Rb uptake (not shown).4 K2 n9 |2 h: e8 H

Fig. 2. Dose-dependent inhibition ofNa -K -2Cl cotransport by sodiumnitroprusside (SNP) and S -nitroso- N -acetylpenicillamine (SNAP) inMMDD1 cells. Cells were grown to confluence on plastic culture dishesand incubated with varying concentrations of SNAP ( A ) or SNP( B ), followed by assay of bumetanide-sensitive 86 Rb uptake, in the presence of ouabain.Results are means ± SE of experiments performed in duplicate.* P P n = 6.# B' E/ ]( Z# p9 m3 [
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These data suggested that NO inhibitsNa -K -2Cl cotransport in MMDD1cells. To indicate whether the effects of NO donors were indeed due torelease of NO, and not nonspecific, cells were preincubated withhemoglobin (5 × 10 5 M), a scavenger of NO( 11 ), before incubation with SNP or SNAP. As shown in Fig. 3, the inhibitory effects of SNP or SNAPon Na -K -2Cl cotransport werecompletely abolished by scavenging of NO [control 8.31 ± 0.40 vs. SNP (10 4 M) 2.50 ± 0.64 pmol · mgprotein 1 · 5 min 1; P 4 M) hemoglobin(5 × 10 5 M) 8.69 ± 0.77 pmol · mgprotein 1 · 5 min 1; P = not significant (NS) vs. control, n = 4]. By contrast, incubation of cells with hemoglobin alone had nosignificant effect on Na -K -2Cl cotransport activity.- M2 A# U8 k% i5 E) \
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Fig. 3. Effect of scavenging of nitric oxide (NO) with hemoglobin (HB) onSNP- and SNAP-mediated inhibition of cotransport. Bumetanide-sensitive 86 Rb uptake, in the presence of ouabain, wasmeasured in cells grown on plastic dishes. A : effect ofpreincubation with HB (5 × 10 5 M) on SNAP(10 4 M)-mediated inhibition of cotransport. B :effect of HB (5 × 10 5 M) on SNP (10 4 M)-mediated inhibition of cotransport. Results are means ± SE ofexperiments performed in duplicate. * P n = 4.
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Because the MMDD1 cells express both NKCC1 and NKCC2, we determinedeffects of NO on apical and basolateralNa -K -2Cl cotransport, usingmeasurements in cells grown on cell culture inserts. BasolateralNa -K -2Cl cotransport exceededapical cotransport by ~6- to 10-fold. Incubation of cells with SNAP(10 4 M) significantly inhibited both apical andbasolateral Na -K -2Cl cotransport (Fig. 4 ).+ G, t# I& x' P5 F0 v% I

Fig. 4. Inhibition of apical and basolateralNa -K -2Cl cotransport by SNAP inMMDD1 cells. Cells were grown to confluence on cell culture inserts,followed by measurement of apical or basolateral bumetanide-sensitive 86 Rb uptake, in the presence of ouabain and inthe presence or absence of SNAP (10 4 M). A :apical cotransport activity ( n = 4). B :basolateral cotransport activity ( n = 5). Results aremeans ± SE of experiments performed in duplicate.* P1 y) }3 b. P8 N1 n8 G; c

Because MMDD1 cells express nNOS, we determined the role of endogenousNO production on Na -K -2Cl cotransport. Incubation of cells with the inhibitor of nNOS, 7-nitroindazole (7-NI; 10 6 M), caused small butsignificant increases in activity of both apical and basolateralcotransport, suggesting that NO exerts autocrine, tonic inhibition ofcotransport in these cells (Fig. 5 ).- q- W( ]; @2 @" r" A9 O- N  k/ U& O

Fig. 5. Effect of the inhibitor of nNOS, 7-nitroindazole (7-NI), on apicaland basolateral Na -K -2Cl cotransport. MMDD1 cells were grown on cell culture inserts andincubated with the nNOS inhibitor 7-NI (10 6 M) beforemeasurement of bumetanide-sensitive 86 Rb uptake. A : apical cotransport activity. B :basolateral cotransport activity. Results are means ± SE ofexperiments performed in duplicate. * P P n = 4.
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Signaling pathways for inhibition ofNa -K -2Cl cotransport by NO. In many cells, NO stimulates soluble guanylate cyclase, leading toelevation of cGMP levels. In MMDD1 cells grown on regular plasticdishes, SNP or SNAP had no significant effect on cGMP levels, atconcentrations up to 10 3 M. As a positive control, atrialnatriuretic peptide (ANP; 10 6 M) caused an eightfoldincrease in cGMP release in these cells (Fig. 6 A ). Similarly, neither NOdonor had any significant effect on cellular levels of the secondmessenger cAMP (Fig. 6 B ).3 G% f" a+ g& W6 m- {, i

Fig. 6. Effect of NO donors on cGMP and cAMP in MMDD1 cells. A :cells were incubated with varying concentrations of SNP or SNAP for 15 min, followed by assay of cGMP levels. As a positive control, cellswere incubated with atrial natriuretic peptide (ANP; 10 6 M). B : cells were incubated with SNP or SNAP(10 3 and 10 4 M) for 15 min, followed byassay of cAMP levels. As a positive control, cells were incubated withforskolin, an activator of adenylate cyclase (10 5 M).Results are means ± SE of experiments performed in duplicate( n = 4). * P1 c# M5 z* l' j1 o+ d. _; p$ J6 C

In vascular smooth muscle cells, NO modulates the levels of cytosoliccalcium ( 43 ), thereby regulating signaling pathways linkedto calcium transients. In MMDD1 cells loaded with the calcium-sensitive dye fura 2-AM, however, we observed no significant effect of SNP orSNAP (10 3 or 10 4 M) on cytosolic calciumlevels. By contrast, bradykinin (10 8 M) stimulatedsignificant increases in calcium (Fig. 7 ).
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Fig. 7. SNAP has no effect on cytosolic calcium concentration inMMDD1 cells. Cells were loaded with fura-2 AM as described in MATERIALS AND METHODS, followed by administration of SNAP(10 4 M) at the indicated point. As a positive control,bradykinin (10 8 M) caused a significant increase incytosolic calcium. Shown is a representative tracing of experiments inwhich either SNP or SNAP was administered ( n = 4).
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Recent studies also suggested a role for NO in modulating the activityof mitogen-activated protein kinase (MAP kinase) in mesangial cells( 15 ). To determine whether MAP kinase was affected by NO,MMDD1 cells were incubated with NO donors, followed by immunoblotanalysis for the phosphorylated MAP kinase substrates ERK1 and ERK2(p44 and p42, respectively). As shown in Fig. 8, SNP or SNAP had no significant effecton phosphorylation of ERK or ERK2. Furthermore, immunoblot analysisrevealed no effect of NO on the levels of nonphosphorylated ERK1 andERK2 (not shown).

Fig. 8. SNP and SNAP do not affect phosphorylation of ERK1 andERK2. Shown is a representative immunoblot ( n = 4) forphosphorylated ERK1 (44 kDa) and ERK2 (42 kDa) from cell extracts(10 5 g) derived from cells incubated with SNP or SNAP(10 4 M). No significant effect on phosphorylation wasobserved, and there was also no effect on levels of nonphosphorylatedERK1 or ERK2 by immunoblot analysis (not shown).
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Modulation of cytochrome P -450 by NO has been suggested byrecent in vivo studies in rats ( 20 ). To determine whetherthe inhibition of Na -K -2Cl cotransport activity in MMDD1 cells might involve this pathway, cellswere preincubated with the suicide inhibitor of cytochrome P -450, 1-aminobenzotriazole (ABT; 10 3 M)( 36 ), before determination of the effects of NO donors on cotransport activity. ABT completely reversed the inhibitory effects ofboth SNP and SNAP on either apical or basolateralNa -K -2Cl cotransport [apical:control 1.18 ± 0.15 vs. SNAP (10 4 M) 0.41 ± 0.05 pmol · mgprotein 1 · 5 min 1; P 4 M) ABT (10 3 M) 1.32 ± 0.10 pmol · mgprotein 1 · 5 min 1; P = NS vs. control, n = 5; Fig. 9 A ]. Incubation of cells with ABT alone did not significantly affect cotransporter activity. In MMDD1cells grown on plastic dishes, ABT (10 3 M) completelyreversed the inhibitory effect of either SNP or SNAP (10 4 M) on total Na -K -2Cl cotransport activity (not shown). The inhibitor of cytochrome P -450 ketoconazole (1.5 × 10 5 M)( 4 ) also completely blocked the inhibitory effect of SNAP on apical or basolateralNa -K -2Cl cotransport (Fig. 9, C and D ).4 e) P: w- W& O) ]/ l
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Fig. 9. Effect of inhibition of cytochrome P -450 onSNAP-mediated inhibition of cotransport. Cells were preincubated withthe P -450 inhibitors 1-aminobenzotriazole (ABT;10 3 M; A and B ) or ketoconazole(Keto; 1.5 × 10 5 M; C and D )before determination of the effect of SNAP (10 4 M) oncotransport. A and C : apical cotransportactivity. B and D : basolateral cotransportactivity. Results are means ± SE of experiments performed induplicate. * P P n = 5 (ABT) or n = 3-4 (Keto)." m3 {. ^2 p8 q9 z5 x0 s
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Effect of EET and 20-HETE onNa -K -2Cl cotransport. To determine possible mediators of the effect of cytochrome P -450 on inhibition of cotransport by NO, cells wereincubated for 15 min with metabolites of P -450. The EETs8,9-EET and 11,12-EET (5 × 10 6 M) had nosignificant effect on apical or basolateral cotransport activity (Fig. 10 ). In contrast, in cells grown onplastic, incubation with 14,15-EET caused a concentration-dependentinhibition of total Na -K -2Cl cotransport (Fig. 11 A ). Incells grown on cell culture inserts, both apical and basolateralcotransport was also inhibited by preincubation with 14,15-EET (5 × 10 7 M) (Fig. 11, B and C ).
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Fig. 10. Effect of 8,9-epoxyeicosatrienoic acid (EET) and 11,12-EET onNa -K -2Cl cotransport. MMDD1cells were grown on cell culture inserts and were incubated for 30 minwith 8,9-EET or 11,12-EET (5 × 10 6 M) before assayof cotransport activity. Cells were incubated with SNAP(10 4 M) as a positive control. A : apicalcotransport activity. B : basolateral cotransport activity.Results are means ± SE of experiments performed in duplicate.* P n = 3.
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Fig. 11. Inhibition ofNa -K -2Cl cotransport by14,15-EET. A : dose-dependent inhibition of cotransport inMMDD1 cells grown on plastic dishes. Cells were incubated with varyingconcentrations of 14,15-EET for 30 min before assay of cotransport. B : effect of 14,15-EET (5 × 10 7 M) onapical cotransport in cells grown on culture inserts. C :effect of 14,15-EET (5 × 10 7 M) on basolateralcotransport in cells grown on culture inserts. Results are means ± SE of experiments performed in duplicate. * P P n = 3 ( A ) and n = 8 ( B and C ).( _2 l+ @1 c* l2 ?
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The product of arachidonic acid -hydroxylase activity, 20-HETE, hasbeen shown to inhibit apicalNa -K -2Cl cotransport in thickascending limb ( 7 ). In MMDD1 cells grown on cell cultureinserts, 20-HETE caused a significant inhibition of apicalNa -K -2Cl cotransport but had noeffect on basolateral cotransport (Fig. 12 ). However, the inhibitor of -hydroxylase activity, HET0016 (10 5 M)( 23 ), had no significant effect on SNAP-mediatedinhibition of apical cotransport (Fig. 13 )." u" l: l. N7 j% A7 _' z' F

Fig. 12. Effect of 20-HETE onNa -K -2Cl cotransport in MMDD1cells. Assay of Na -K -2Cl cotransport was performed on confluent cells grown on cell cultureinserts following preincubation with 20-HETE (5 × 10 6 M). A : apical cotransport activity. B : basolateral cotransport activity. Results are means ± SE of experiments performed in duplicate. * P n = 8., e1 m5 X, d) d5 x

Fig. 13. Effect of the -hydroxylase inhibitor HET0016 onSNAP-mediated inhibition of apicalNa -K -2Cl cotransport.Bumetanide-sensitive 86 Rb uptake was measuredin confluent cells grown on cell culture inserts after preincubationwith the selective -hydroxylase inhibitor HET0016 (10 5 M), with or without SNAP (10 4 M). Results are means ± SE of experiments performed in duplicate. There was no significantdifference between the effect of SNAP and SNAP HET0016; n = 8. * P P
9 o0 I3 S% b* V( y) m) F  `. J5 x9 m
To determine if MMDD1 cells express a cytochrome P -450isoform that is abundant on mouse kidney, RT-PCR for the CYP2J5 isoform ( 21 ) was performed on RNA derived from these cells. Asshown in Fig. 14, the CYP2J5 isoform isexpressed in this cell line, with mouse kidney used as a positivecontrol.' X( v3 r& ~2 D2 @* {

Fig. 14. Expression of cytochrome P -450 (CYP) 2J5 inMMDD1 cells by RT-PCR. Representative agarose gel showing DNA 100-bpladder ( lane 1 ), 285-bp CYP 2J5 PCR product from MMDD1 cells( lane 2 ), corresponding reaction lacking reversetranscriptase ( lane 3 ), 285-bp PCR product for CYP 2J5 frommouse liver ( lane 4 ), and corresponding reactionlacking reverse transcriptase ( lane 5 ).* i+ O7 {/ h8 d0 K& i6 \
5 k% T+ ^2 B5 v5 d: \8 O1 [' }" h
DISCUSSION# S3 a7 B( q5 q: J1 {% }
- p% O* X# ~% U
Two isoforms of the electroneutral, loop diuretic-sensitiveNa -K -2Cl cotransporter havebeen identified and are encoded by separate genes. The NKCC1 isoform isfound on the basolateral membranes of secretory epithelia, and in thekidney it is localized to outer and inner medullary collecting ducts,glomerular and extraglomerular mesangium, and to afferent arteriole( 18 ). The NKCC2 isoform is found exclusively in thekidney, and in the rabbit and mouse it consists of splice variants (A,B, F), with the B variant expressed specifically on the apical surfaceof cells of the cortical thick ascending limb and macula densa( 41 ). The mouse renal epithelial cell line (MMDD1) used inthe present study was derived from SV40 transgenic mice and retainssome well-known properties of macula densa, such as expression andactivity of NKCC2, COX-2, and nNOS ( 42 ). We confirmedthese properties, and we demonstrated expression of the B variant ofNKCC2 in these cells, and not the A or F variants, suggestingderivation of these cells from macula densa or cortical thick ascendinglimb ( 42 ). We also demonstrated that these cells expressNKCC1. When MMDD1 cells were grown on cell supports,bumetanide-sensitive 86 Rb transport waspresent on apical and basolateral membranes, indeed with a relativeabundance of basolateral cotransport. Our major finding is that NOinhibits apical and basolateralNa -K -2Cl cotransport activityin these cells. The inhibitory mechanism does not involve generation ofcGMP, but our data suggest a novel pathway, with a requirement forcytochrome P -450 epoxygenase activity.2 O3 _' n/ J) W

In macula densa, transport through the apical membraneNa -K -2Cl cotransporter isthought to be the initiator of the TGF response, resulting in afferentarteriolar constriction. The nNOS isoform is highly expressed in maculadensa, and studies show that NO produced in the macula densa blunts TGF( 13, 16, 37, 40 ). Because our data show that the NKCC2 Bisoform is present in MMDD1 cells, the results are consistent with thehypothesis that NO derived from macula densa or adjacent thick limbcells in vivo could act in an autocrine or paracrine fashion to inhibitapical cotransporter activity. In recent studies in rabbit afferentarterioles with attached macula densae, inhibition of macula densa nNOSwith 7-NI increased TGF responses, and it was also suggested that NO derived from the thick ascending limb could act as a paracrine factorto inhibit TGF at the macula densa ( 39 ). Similarly, our data in MMDD1 cells indicate that inhibition of nNOS with 7-NI caused asmall but significant increase in cotransporter activity. Autocrineinhibition of apical cotransport by NO could therefore represent amechanism for precise modulation of the TGF mechanism, independently ofthe direct inhibitory effects of NO on afferent arteriolar tone.

Although previous studies examined effects of NO donors and endogenousNO on apical Na -K -2Cl cotransport (NKCC2), there is little information on the effects of NOon basolateral cotransport via NKCC1. In rat aortic smooth muscle, NOinhibits basal NKCC1 activity and reduces its phosphorylation ( 1 ). In our studies, MMDD1 cells expressed abundantbasolateral cotransport activity. Earlier studies by Lapointe andcolleagues ( 3, 19 ) did not identify basolateralcotransport activity in rabbit macula densa. Although the presence ofNKCC1 on the basolateral membrane of murine macula densa cells ispossible, it is surprising that basolateral cotransport activity farexceeded apical cotransport in our study. Accordingly, it is possiblethat the MMDD1 cells have undergone dedifferentiation, with newexpression of NKCC1, or they may contain nonepithelial cells or cellsfrom tubular segments known to express NKCC1.3 B" B* C1 o! d( H2 Q

In many cell types, including proximal tubule, thick ascending limb,and cortical collecting duct, NO stimulates soluble guanylate cyclase,leading to production of cGMP ( 8, 27, 32, 34 ). In thesesegments, inhibition of transport pathways by NO is at least partlydependent on generation of cGMP, and recent in vivo studies in ratsindicate that increases in renal interstitial cGMP levels promotenatriuresis ( 17 ). In thick ascending limb, NO inhibitschloride reabsorption by activation of cGMP-stimulated phosphodiesterase, which decreases cAMP levels ( 27 ). Incontrast, our data indicate that NO had no effect on cGMP or cAMPlevels in MMDD1 cells. ANP significantly stimulated cGMP production. The effects of ANP on juxtaglomerular cell renin release have beenstudied in animal models ( 10, 38 ), although there is presently no information on direct effects of ANP on macula densa function. Our data suggest that MMDD1 cells express ANP receptors andalso contain the particulate, but not the soluble, guanylate cyclase.In contrast, studies in an isolated rabbit afferent arteriole preparation by Ren et al. ( 31, 39 ) showed that NO produced by the macula densa acts on macula densa cells to inhibit TGF, via apathway involving activation of guanylate cyclase and generation ofcGMP. It is possible that macula densa responses to NO may be speciesspecific or that MMDD1 cells may have reduced expression of guanylatecyclase. However, the potent inhibition of cotransport by NO in thepresent study does not exclude the possibility that in vivo cGMPgeneration may also induce inhibition.

There has been recent interest in the effects of NO that areindependent of cGMP production, including effects of NO metabolites such as peroxynitrite. In the present studies, NO did not alter celllevels of calcium. Of interest, previous studies suggest that theB 2 isoform of the bradykinin receptor is not expressed inhuman macula densa ( 33 ). However, MMDD1 cells demonstrated reproducible calcium transients in response to bradykinin, suggesting expression of bradykinin B 1 or B 2 receptors inthese cells. In mesangial cells in culture, NO inhibits mechanicalstretch-induced ERK activity and nuclear translocation( 15 ). Although we did not measure ERK activity directly,we observed no effect of NO on basal phosphorylation of ERK1 or ERK2 inMMDD1 cells.

Our studies uncovered a unique signaling pathway for NO in MMDD1 cells.ABT, an agent that blocks the renal metabolism of arachidonic acid toEETs and 20-HETE by cytochrome P -450 ( 36 ), completely reversed the inhibitory effect of exogenous NO on apical andbasolateral Na -K -2Cl cotransport, suggesting that cytochrome P -450 is involved in NO signaling. Similarly, the inhibitor of P -450 epoxygenaseketoconazole reversed the inhibitory effect of SNAP. Neither ABT norketoconazole alone affected cotransport activity. Because incubationwith 7-NI caused small but significant increases in apical andbasolateral cotransport, this suggests that either ABT or ketoconazoledoes not completely inhibit P -450-mediated production ofEETs at baseline, induced by endogenous NO, but instead blocksNO-stimulated P -450 activity. In this regard, incubation ofrat renal microsomes with ABT at concentrations of 10 mM or greater wasrequired to induce 80% inhibition of P -450 in vitro( 24 ). An alternate possibility is that basal NO productionin these cells inhibits cotransport activity via a pathway separatefrom that involving activation of P -450 by exogenous NO.8 B$ t- @6 E4 w

Although both apical and basolateral cotransport activities wereinhibited by NO and this was reversed by inhibition of cytochrome P -450, and mimicked by 14,15-EET, only apical cotransportwas inhibited by the arachidonic acid -hydroxylase product 20-HETE. In rabbit thick ascending limb, 20-HETE has also been shown to inhibitNa -K -2Cl cotransport( 7 ). However, in MMDD1 cells, the inhibitor of -hydroxylase HET0016 did not have a significant effect onSNAP-mediated inhibition of apicalNa -K -2Cl cotransport, whereasABT and ketoconazole completely blocked the inhibitory effect of SNAP.Taken together, these data suggest that the inhibitory effect of NO maybe mediated by selective activation of cytochrome P -450epoxygenase activity in these cells, with no effect on the -hydroxylase activity. The data also suggest that NKCC1 and NKCC2have distinct regulatory mechanisms with regards to these arachidonicacid metabolites.2 b# l$ `( Z/ I. g$ T$ \7 d

Along the nephron, P -450-derived eicosanoids, including EETsand 20-HETE, have been shown to influence sodium and water transport. NO has been shown to inhibit cytochrome P -450 isoenzymes ofthe 1A, 2B1, 3C, and 4A classes by forming iron-nitrosyl complexes atthe heme binding sites ( 2, 29 ). Recent in vivo studies inrats suggest that the renal vascular effects of NO are mediated viainhibition of P -450 and activation of guanylate cyclase,whereas the natriuretic effects are independent of P -450( 20 ). Our data suggest that in MMDD1 cells, NO may indeedactivate a cytochrome P -450 isozyme with selectiveinhibition of the cotransporter by 14,15-EET.3 N5 g2 _( t+ @. d7 z

Although kidney microsomal content of P -450 activity issignificant, knowledge of kidney P -450 isoforms isincomplete. In mouse kidney, the CYP2J5 isoform is expressed inabundance in proximal tubules and collecting ducts ( 21 ).This isoform is associated with production of EETs, of which the14,15-EET is predominant ( 21 ). Our studies demonstratethat mRNA for this isoform is present in MMDD1 cells. Further studiesare required to determine whether the CYP2J5 isoform is expressed inmacula densa in vivo and whether NO activates this enzyme, leading to production of EETs.5 A: t, H, S/ S7 T+ v. n

In summary, we showed that NO inhibits apical and basolateralNa -K -2Cl cotransport activityin MMDD1 cells, independently of guanylate cyclase activation. Theinhibitory mechanism may involve a cytochrome P -450-dependent pathway, possibly mediated by production of14,15-EET.

ACKNOWLEDGEMENTS

This work was supported by grants from the Canadian Institutes ofHealth Research and the Kidney Foundation of Canada (to K. D. Burns).
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