Characterization of PMCA isoforms and their contribution totranscellular Ca2 f - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:26 |只看该作者 |倒序浏览 |打印

作者：Sertac N.Kip and EmanuelE.Strehler作者单位：Department of Biochemistry and Molecular Biology, MayoClinic, Rochester, Minnesota 55905


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      【摘要】
   Plasmamembrane Ca 2  ATPases (PMCAs) are ubiquitous inCa 2  -transporting organs, including the kidney. UsingRT-PCR, we detected PMCA1b, PMCA2b (rare), and PMCA4b in Madin-Darbycanine kidney (MDCK) cells. At the protein level, only PMCA1 and PMCA4were readily detected and were highly enriched in the basolateralmembrane. The Na /Ca 2  exchanger NCX1 was alsodetected at the transcript and protein level. A functional assaymeasuring 45 Ca 2  flux across MDCK cellmonolayers under resting conditions indicated that two-thirds ofapicobasolateral Ca 2  transport was provided byNa /Ca 2  exchanger and one-third by PMCAs, asdetermined in Na -free media and using various PMCAinhibitors (La 3 , vanadate, calmidazolium, andtrifluoroperazine). The importance of PMCA4b for basolateralCa 2  efflux was demonstrated by overexpression of PMCA4b orantisense knockdown of endogenous PMCA4b. Overexpression of PMCA4bincreased apicobasolateral Ca 2  transport to ~140%,whereas antisense treatment reduced Ca 2  flux ~45%compared with controls. The MDCK system is thus an ideal model forfunctional studies of the specific role and regulation of PMCA isoformsin Ca 2  reabsorption in the distal kidney.
      【关键词】 calcium transport kidney distal tubules MadinDarby caninekidney sodium/calcium exchanger plasma membrane calcium ATPase* l; M, P8 F' ?' r- |! r
   INTRODUCTION

C A 2 is anessential mineral in animals, where it plays a crucial role inprocesses ranging from the formation and maintanance of the skeleton tothe temporal and spatial regulation of neuronal function. OverallCa 2  homeostasis must therefore be under tight and finelytuned control. Ca 2  uptake from the environment largelyoccurs through the intestine, whereas Ca 2  loss occursmainly via the kidney. The overall balance between Ca 2  uptake and loss is under multiple hormonal control and is dictated bythe body's changing needs for this mineral element. As the primaryorgan responsible for Ca 2  excretion, the kidney has apreeminent role in regulating Ca 2  homeostasis. FilteredCa 2  in the urine is reabsorbed throughout the kidney, withthe bulk being handled by the proximal tubules and only ~10% by thedistal convoluted tubules ( 19, 39 ). Although most of theCa 2  reabsorption in the proximal nephron occurs via apassive, paracellular pathway, the reuptake of Ca 2  in thedistal tubules occurs mainly via an active transcellular pathway thatmoves Ca 2  against its electrochemical gradient. This isachieved by Ca 2  influx through specific channels (such asECaC; see Ref. 23 ) in the apical plasma membrane andactive Ca 2  extrusion by Ca 2  pumps andNa /Ca 2  exchangers (NCXs) in the basolateralplasma membrane of the distal tubule epithelial cells ( 7 ).Because even small changes in the retention or loss of Ca 2  in the distal kidney have large effects on the overall Ca 2  homeostasis in the body, the reabsorption of Ca 2  in thedistal tubules is tightly regulated and under multiple hormonalcontrols (parathyroid hormone, calcitonin, and vitamin D 3 ).Studies of the diverse transporters involved in vectorial Ca 2  flux are therefore of particular interest, becausechanges in their expression, localization, and function are likely theprimary targets of regulation.

The plasma membrane Ca 2  ATPases (PMCAs) are high-affinityCa 2  efflux pumps found in virtually all eukaryotic cells,wherein they are responsible for the maintenance and resetting of the resting intracellular Ca 2  levels ( 12 ). Fourgenes encode separate isoforms called PMCA1-4; in addition,alternative splicing of the transcripts yields a large variety ofsplice variants differing mainly in their COOH-terminal amino acidsequence ( 38 ). Using a PMCA-specific antibody that recognizes all isoforms, the presence of the PMCA in the kidney ofseveral species has been clearly documented, and immunohistochemical studies suggest that the pump is highly concentrated in the basolateral membrane of epithelial cells in distal kidney tubules ( 8 ). Studies at the transcript level using RT-PCR and sophisticated tissuemicrodissection techniques indicate that all PMCA isoforms areexpressed in the rat kidney; however, they are expressed with distinctisoform-specific expression patterns and variable abundance along thedifferent regions of the nephron ( 13, 25 ). Other studies using RT-PCR on whole kidney RNA, as well as studies at theprotein level, have been controversial, suggesting that PMCA1 and PMCA4are the major isoforms expressed in the kidney and that PMCA2 and PMCA3may be minor components of this tissue, if expressed at all ( 11, 36, 37 ). The expression level and localization of the NCX alongthe nephron have also been a matter of debate. Of the three majorisoforms of the exchanger, NCX1-3, only NCX1 appears to beexpressed in significant amounts in the kidney ( 47 ). Asexpected for a transporter involved in active expulsion of Ca 2  into the interstitial space during urinaryCa 2  reabsorption, the exchanger has been localized mainlyto the basolateral membrane, at least in the distal tubule andconnecting tubule ( 9, 33 ). However, the relativecontribution of the NCX to vectorial Ca 2  extrusion in thedistal kidney (relative to that by the PMCA) remains to be established.
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Madin-Darby canine kidney (MDCK) cells are a well-established model fordistal tubule epithelial cells ( 2, 22, 34 ). On confluence,they form sheets of polarized cell monolayers that reproduce manyphysiological parameters of the distal transporting kidney epithelium.When grown on semipermeable filter inserts, the apical and basolateralmembrane domains can be separately accessed and represent the urinarylumen and interstitial compartments, respectively. Vectorialtranscellular ion flux studies can be conducted if the cell monolayeris not leaky, i.e., if tight junctions have formed between adjacentcells. The tightness of the monolayer can be assessed by measurementsof transepithelial electrical resistance (TEER), and the nettranscellular flux of ions such as Ca 2  can be determinedafter correction for paracellular transport ( 2, 6, 29 ).

Here, we demonstrate that the MDCK system is an attractive model forthe study of transcellular Ca 2  transport by the PMCAs inthe distal kidney. Using RT-PCR, immunoblotting, and immunolocalizationtechniques, we show that PMCA4b and PMCA1b are the major pump isoformsin MDCK cells, whereas full-length PMCA2b is detected only in smallamounts. 45 Ca 2  flux studies using differentpharmacological blockers reveal that one-third of theapical-to-basolateral Ca 2  flux in resting MDCK cells ishandled by the PMCAs, whereas a Na -dependentCa 2  extrusion mechanism is responsible for the remainingtwo-thirds. The importance of PMCA4b in transcellular Ca 2  flux is further emphasized by transport studies conducted in transfected cells with altered Ca 2  pump expression./ J( u7 F- z$ j* z, ]( X7 e) u' S

MATERIALS AND METHODS0 ?4 W* D0 V0 F( z, x4 ~, z7 X
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Materials. MDCK type I cells were obtained from the American Type CultureCollection (Manassas, VA). Trypsin-EDTA, DMEM, FBS, L -glutamine, Na pyruvate, andantibiotics/antimycotics were bought from Invitrogen (Carlsbad, CA).RT-PCR reagents and enzymes were purchased from Roche-BoehringerMannheim (Indianapolis, IN). All other chemicals were from Sigma (St.Louis, MO). Monoclonal (5F10, JA9) and polyclonal antibodies againstPMCAs were generously provided by Dr. John T. Penniston and Adelaida G. Filoteo (Mayo Clinic, Rochester, MN), and a polyclonal antibody againstSAP97 was a gift from Dr. Craig C. Garner (University of Alabama,Birmingham, AL). The characterization and specificity of theseantibodies have been described ( 14, 16, 30 ). Primaryantibodies against the exchanger NCX1 and -actin and all secondaryantibodies were products of Sigma, whereas an antibody againstNa -K -ATPase was purchased from AffinityBioreagents (Golden, CO). X-ray films were from Eastman Kodak(Rochester, NY), and 45 Ca 2  was obtained fromPerkinElmer Life Sciences (Boston, MA). The expression constructs forfull-length PMCA4b (pMM 2 4b) and for antisense PMCA4(pCIneo-AS4) have been described previously ( 1, 20 ).9 `4 i; Q7 W- U! V2 E
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Cell culture. MDCK cells were propagated in DMEM containing 10% (vol/vol) FBS, 2 mM L -glutamine, 1 mM Na pyruvate, 50 µg/mlgentamycin sulfate, 100 U/ml penicillin, and 100 U/ml streptomycin at37°C in a humidified atmosphere containing 5% CO 2.

RT-PCR. Total RNA was isolated from MDCK cells by using the TRIzol reagent(Invitrogen), as specified by the manufacturer. Briefly, cells grown to80-90% confluence on 175-mm collagen-coated flasks were washedtwice with PBS and lysed by adding 5 ml of TRIzol reagent. The lysateswere allowed to incubate at room temperature for 5 min. Chloroform (1.2 ml) was added, followed by vigorous vortexing for 15 s.Samples were then incubated for 5 min at room temperature andcentrifuged for 15 min at 12,000 rpm at 4°C. After removal of theaqueous phase and addition of 5 ml of isopropanol, samples wereincubated for 10 min at room temperature and then centrifuged for 15 min at 12,000 rpm at 4°C. The RNA pellets were washed with 5 ml 75%ethanol, sedimented for 5 min at 9,000 rpm at 4°C, and air-dried for10 min before being dissolved in diethyl pyrocarbonate-treated waterand stored at 70°C.) Y+ Q7 [6 q& T

Reverse transcription was carried out by using 5 µg RNA in 28 µlPCR-grade water containing 5 µM hexanucleotide random primer. Themixture was incubated at 65°C for 6 min and cooled to room temperature before being mixed with 22 µl of a solution to yield afinal concentration of 1× first-strand buffer, 10 mM DTT, 40 U RNasin,1 mM of each of the dNTPs, and 5 U MMLV-RT. The samples were thenincubated at 37°C for 50 min, heated at 99°C for 5 min, and storedat 70°C. The reverse transcription reaction (5 µl) was used toperform PCR ( 31 ) in a final volume of 50 µl that contained 1× PCR buffer, 200 µM dNTPs, 10 pmol of each of the primers, and 2.5 U Taq polymerase. The followingamplification profiles were used: 5 min of initial denaturation at94°C followed by 35 cycles of 1 min of denaturation at 94°C, 1 minof annealing at 53, 64, 53, or 54°C for PMCA1-4, respectively,and 1 min of extension at 72°C, followed by a 10-min final extensionat 72°C and soak at 4°C. The only exception to this profile was forPMCA2, for which the cycle number was 40. The location of primersrelative to the PMCA coding sequence and their sequences and originsare indicated in Fig. 1. Negative(exclusion of cDNA) and positive controls (inclusion of brain,testes, and lung cDNA) were included in all experiments. After RT-PCR,10% of the amplicons were electrophoresed on an ethidiumbromide-containing 1.8% agarose gel, along with a molecular weightmarker (100-bp ladder; Bio-Rad). The bands of expected size wereexcised from the gel, purified with the Qiaquick Gel Extraction Kit(Qiagen, Valencia, CA), and subjected to sequencing in the MayoMolecular Biology Core Facility.0 \7 D, \! R+ U/ g" ^9 Y
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Fig. 1. Scheme of the major COOH-terminal plasma membrane Ca 2  ATPase (PMCA) splice variants and primers used for RT-PCR. A : intron-exon structures of each PMCA gene around splicesite C are shown schematically ( top ), and the two majorsplice variants a and b generated by alternative splicing are indicated( bottom ). Size of alternatively spliced exons is indicatedin base pairs. For a more complete description of PMCA alternativesplicing options, see Ref. 38. B : source andsequence of the primers for each PMCA isoform and forNa /Ca 2  exchanger 1 (NCX1) are listed, and thestart and end position of each primer pair within its cognate cDNA isindicated by the nucleotide number (nt). The expected sizes ofamplification products corresponding to the a and b splice variants ofthe PMCAs are given in the last column, as is the expected product forNCX1. F, forward; R, reverse.- d: G/ Z+ h3 w( k$ Y
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Preparation of cell extracts and plasma membranes. Confluent cell cultures were rinsed twice with PBS, trypsinized,pelleted, and stored at 80°C. To prepare total cell lysates, cellswere thawed and lysed in lysis buffer (HEPES, pH 7.5, 0.1% NonidetP-40, 0.5% deoxycholate, 1 mM EDTA, 150 mM NaCl, and 0.1 mMNa 3 VO 4 ) containing a cocktail of proteaseinhibitors (aprotinin, leupeptin, pefabloc, and pepstatin). The cellswere sonicated at 4°C twice for 7 s and incubated on ice for 10 min before precipitation with ice-cold 5% TCA. The precipitates werethen spun at 4°C for 15 min at 12,000 g, and the pelletswere resuspended in Krebs-Ringer-HEPES (KRH) solution containing (inmM) 130 NaCl, 5 KCl, 20 HEPES, 1.2 KH 2 PO 4, 1 CaCl 2, 1 MgSO 4, and 10 glucose, as well as 1 ml/l DMEM, pH 7.4, and were homogenized by aspiration with a Hamilton syringe.
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For total membrane preparations, the cells were lysed in lysis buffercontaining protease inhibitors, and undisrupted cells and nucleardebris were removed by centrifugation at 500 g for 10 min.The supernatant was then centrifuged for 1 h at 100,000 g, and the pellet containing the membranes was resuspendedin KRH solution ( 35 ).

To generate a purified mixed crude plasma membrane (MCPM) fraction, thethawed cell pellet was sonicated (2 bursts, 7 s each) in 10 vol/wtof 0.3 M sucrose containing protease inhibitors by using a SonifierCell Disrupter (Heat Systems-Ultrasonic, Plainview, NY). Whilevortexing, 1.43 vol of 2 M sucrose were added to the suspension, andthe mixture was transferred to a TI70 ultracentrifuge tube (BeckmanInstruments). The sucrose cushion was overlaid with 0.3 M sucrose andsubjected to isopycnic centrifugation for 1 h at 240,000 g (56,000 rpm). The membrane band was removed and dilutedwith ice-cold dH 2 O and spun at 240,000 g for 30 min. To obtain MCPM, the resulting pellet was resuspended in 3 ml KRH solution, layered over a 9-60% linear sucrose gradient, andcentrifuged at 90,000 g (27,000 rpm) for 3 h in an SW28 rotor (Beckman Instruments) ( 42 ).
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The MCPM was further separated into distinct apical and basolateralplasma membrane domains on a three-step sucrose gradient (38, 34, and31% wt/wt). For this, the MCPM band was diluted with 4 vol of 1 mMNaHCO 3, pH 7.5, and sedimented at 7,500 g for 30 min, and the resulting pellet was washed with 10 vol of bicarbonate buffer and recentrifuged at 7,500 g for 15 min. This pelletwas suspended in 0.25 M sucrose to a volume of 3 ml and homogenized with a tight type B glass Dounce homogenizer by 50 up and down strokes.This suspension was layered on top of a three-step sucrose gradientconsisting of 3.8 ml of 38%, 2.1 ml of 34%, and 2.1 ml of 31%sucrose. The tubes were centrifuged at 20,000 g (40,000 rpm)for 3 h in an SW41 rotor (Beckman Instruments). This procedure produced three distinct bands, and the bands on top of the 31% sucroselayer and at the 31/34 and 34/38% interfaces were collected as apicalplasma membrane, a combination of apical plasma membrane plusbasolateral plasma membrane, and basolateral plasma membrane, respectively, as previously characterized and described for different epithelial cell types ( 27, 41 ). The apical plasma membrane and basolateral plasma membrane were each diluted to 10 ml of 0.125 Msucrose and pelleted at 40,000 rpm for 1 h in an SW41 rotor. Theresulting pellets were resuspended in KRH buffer by suction through a25-gauge needle (20 times in and out) and stored at 70°C untilfurther use. All procedures were carried out at 4°C.

Domain-specific assays were performed on the apical plasma membrane andbasolateral plasma membrane to determine enrichment of the bands withapical and basolateral plasma membrane markers, respectively. Alkalinephosphatase, a commonly used marker for the apical plasma membranedomain ( 28 ) was assayed biochemically by usingcommercially available enzyme kits according to the supplier's instructions (Sigma). Immunoblotting forNa -K -ATPase was performed to confirmenrichment of the basolateral plasma membrane domain., {+ P- S% t7 U  ]- s$ g
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Immunoblotting. Protein concentrations of the total cell lysates, total cell membranes,and domain-specific plasma membrane bands were measured spectrophotometrically with the BCA assay (Pierce, Rockwood, IL) following the manufacturer's instructions. Approximately 30 µg oftotal cell lysate, 6 µg of total cell membranes, and 1-2 µg ofdistinct plasma membrane domains were mixed with NuPAGE electrophoresis buffer in the presence of reducing agents and antioxidants and heatedto 70°C for 15 min before being separated in denaturing NuPAGE4-12% precast gradient gels at 200 V for 50 min and transferred onto nitrocellulose membranes (Bio-Rad) for 1 h at 30 V at room temperature.
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Immunoblotting was performed with standard Western blotting techniques( 3 ). Nitrocellulose membranes were blocked in TBST (50 mMTris · HCl, pH 7.4, 150 mM NaCl, and 0.05% Tween20) 10% milk for 1 h at room temperature before exposure toprimary antibodies for 1 h at room temperature. Primary antibodieswere as follows. For the detection of all PMCAs and of PMCA4, mousemonoclonal antibodies 5F10 and JA9 ( 14 ) were used,respectively, at 1:2,000 and 1:400 dilutions. To detect PMCA1-3,polyclonal antibodies NR1-NR3 ( 16 ) were used,respectively, at 1:200, 1:9,000, and 1:500 dilutions. The NCX wasdetected with a commercially available monoclonal antibody diluted at1:1,000. In addition, the same blots were reprobed with ananti-Na -K -ATPase 1 -antibody(1:500), as a plasma membrane marker, or anti- -actin antibody(1:1,000), as a cytosolic housekeeping protein, to standardize eachlane and ensure equal protein loading. After exposure to primaryantibodies, the blots were washed three times for 5 min in TBST andincubated in peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG(1:5,000) for 1 h at room temperature. Before immunodetection withthe Renaissance chemiluminescence detection system (PerkinElmer LifeSciences), the membranes were washed three times for 10 min in TBST.Immunoreactive bands on the resulting autoradiographs were determinedby using a model GS-700 imaging densitometer, and Molecular Analystsoftware (Bio-Rad) was utilized to calculate the ratio of PMCAreactivity to that of -actin and Na -K -ATPase. Western blots were repeated atleast three times, and the imaging data were averaged.
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Ca 2 efflux across monolayers ofMDCK cells. Two hundred fifty thousand MDCK cells were seeded on permeable inserts(24.5-mm diameter; Costar, Cambridge MA) and grown as described abovebut in the absence of phenol red. Cells were maintained at 37°C in anenvironment of 5% CO 2 -95% air, and the media (2 ml) inboth the top and the bottom compartments were changed on alternate daysbetween days 3 and 11 and on a daily basis thereafter.

The TEER across monolayers was measured in cells cultured for up to 20 days by using an epithelial volt-ohmmeter (World Precision Instruments,New Haven, CT) to determine the tightness of the monolayers. TEER wasnoted to gradually increase by day 10, stabilize between days 10 and 15, and then decline after 2 wk in culture.
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Transepithelial transport of 45 Ca 2  wasmeasured on day 15, when the cells were fully differentiatedand polarized and formed tight monolayers. Cells were rinsed twice withwash buffer [Dulbecco's PBS containing (in mM) 138 NaCl, 8 Na 2 HPO 4, 2.7 KCl, and 1.5 KH 2 PO 4 ], and the inserts were transferred tofresh six-well cluster dishes that contained nonradiolabeled transportmedium [(in mM) 140 NaCl, 5.8 KCl, 0.34 Na 2 HPO 4, 0.44 KH 2 PO 4,0.8 MgSO 4, 20 HEPES, 4 glutamine, 0.5 CaCl 2,and 25 glucose, pH 7.4]. After equilibration for 30 min at 37°C, 2 ml of transport buffer containing 0.5 mM phenol red and 1 µCi 45 Ca 2  were added to the top compartment( time 0 ), and the plates were covered and incubated for 30 min at 37°C. At the end of the designated transport period, duplicate200-µl aliquots were removed from the bottom compartment and read ina scintillation counter to assess total transport of 45 Ca 2  from the apical toward the basolateral compartment.
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To estimate the paracellular transport of Ca 2  and thetightness of the monolayers, phenol red transport was measured inaliquots of media drawn from the basolateral compartment at the end of each transport study. After incubation of 200 µl of lower compartment media with 20 µl of 0.1 N NaOH for 5 min at room temperature, theabsorbance was read at 560 nm, and the concentration of phenol redappearing in the bottom compartment was determined by comparing thereading to that of the known standards. The percentage of phenol redtransport was calculated, and an equivalent amount of Ca 2  was subtracted from the total Ca 2  transport to derive thetranscellular Ca 2  transport. After termination of thetransport reaction by ice-cold wash buffer, the MDCK cells were washedtwice and incubated with 1 ml 0.5 N NaOH at 70°C to dissolve theprotein off the inserts. An aliquot was assayed for proteinconcentration, and the transport of transcellular Ca 2  was normalized for protein content of each insert (cpm × min 1 × µg protein 1 ). An aliquot ofthe solubilized cells was also processed for scintillation counting todetermine the Ca 2  content of the cells.1 a0 n; |- E7 O# V1 P3 G( f$ T

To determine the transcellular Ca 2  transport caused by NCXactivity, NaCl present in the transport buffer was exchanged with thesame molarity of choline. To dissect the transcellular Ca 2  efflux via PMCAs, nonspecific inhibitors of the pump, such as calmidazolium (145 nM), trifluoroperazine (50 µM), lanthanum (0.25 mM), and vanadate (5 mM), were used. Transcellular Ca 2  transport was also determined in MDCK cells transiently transfected with both sense and antisense PMCA cDNA expression constructs (see below).# U# W, \, }/ B* E
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Transfections. MDCK cells were plated at a density of 2.5 × 10 5 /insert and grown for 12 days as described above. Threedays before the Ca 2  flux assays, the cells weretransfected with plasmid constructs expressing full-length PMCA4b(pMM 2 4b) or antisense PMCA4 (pCIneo-AS4), by usingLipofectAMINE 2000 Plus (Invitrogen) according to the manufacturer'sinstructions. Briefly, 1 µg of plasmid DNA was mixed with 100 µl ofOpti-MEM, and 8 µl of LipofectAMINE were added in a separate tube to100 µl of Opti-MEM. After incubation for 5 min, the contents of thetwo tubes were mixed and further incubated at room temperature for 30 min. During this incubation period, the culture medium on the cells wasremoved, and the cells were washed twice with PBS and incubated in 0.8 ml of serum-free medium. After the DNA suspension was added, the cellswere incubated for 10 h at 37°C. One milliliter of mediumcontaining 2× FBS but no antibiotics was then added and incubationcontinued for 24 h. The next day, the medium was removed, cellswere washed, and the medium was replaced by the maintenance mediumcontaining 1× FBS and antibiotics. The transfected MDCK cells werecultured for an additional day before the functional assays were performed.
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Immunofluorescence confocal microscopy. MDCK cells grown to confluence on glass coverslips were washed with PBSplus Ca 2  and Mg 2  (PBS CM) and fixed for 5 minat room temperature in 4% paraformaldehyde (Tousimis, Rockville, MD)diluted in PBS CM. After three washes of 2 min each, cells were furtherfixed and permeabilized in ice-cold methanol for 15 min at 20°C.The cells were blocked for 1 h at room temperature in PBS CMcontaining 5% normal goat serum and 1% bovine serum albumin and werethen incubated for 1 h at room temperature with monoclonalpan-anti-PMCA antibody 5F10 and polyclonal anti-SAP97 antibodiesdiluted 1:800 and 1:200, respectively, in blocking buffer. In addition,isoform-specific antibodies NR1, NR2, and JA9, recognizing PMCA1,PMCA2, and PMCA4, were used at dilutions of 1:100, 1:800, and 1:400,respectively, and the anti-Na -K -ATPaseantibody was used at a 1:500 dilution. After a washing three times for5 min in PBS CM, cells were incubated for 1 h at room temperaturein darkness with secondary antibodies, either anti-mouse Alexa 488 oranti-rabbit Alexa 594 (Molecular Probes, Eugene, OR), each diluted1:600 in blocking buffer. After incubation, cells were washed threetimes for 5 min with PBS CM and coverslips were mounted onto slides byusing Prolong mounting media (Molecular Probes). Confocal micrographswere taken on a Zeiss LSM 510 with an Apochromat ×63 oil-immersionobjective and captured by using Zeiss LSM 510 software.
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Statistical analysis. Each experiment was done in triplicate, and all data were expressed asmeans ± SE. Statistical differences were analyzed by Student's t -test using StatView, and results were considered to bestatistically significant at P
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RESULTS
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Analysis of PMCA transcripts in MDCK cells. We used RT-PCR to detect the presence of transcripts for each of thefour PMCA isoforms in MDCK cells. The primers were chosen to amplifythe region of splice site C to allow the simultaneous identification ofthe alternative splice variants at site C. The primers (Fig. 1 ) werederived from rat (PMCA1, PMCA3, and PMCA4) and human (PMCA2) sequencesbecause dog PMCA cDNAs were not yet known. The identity of the PCRfragments was determined by sequencing. The overall genomic structureof the PMCA genes and the two major described splice variants at site Care illustrated in Fig. 1. For PMCA1, RT-PCR amplification of MDCK RNAyielded a single band of ~400 bp corresponding to the PMCA1b variant(Fig. 2 A ). PMCA2 cDNAamplification by PCR yielded a band of very low intensity (~500 bp)in MDCK cells (Fig. 2 A ), which on sequencing was found tocorrespond to PMCA2b. This band was only observed after 40 PCR cycles,indicating that the PMCA2 transcripts are rare in MDCK cells. PMCA3primers did not amplify any PMCA fragments of expected size, suggestingthat PMCA3 is not expressed in MDCK cells. By contrast, when thepresence of PMCA4 was analyzed, a single amplicon of ~350 bp wasreadily obtained (Fig. 2 A ), demonstrating the existence ofPMCA4b and absence of other splice site C variants. Although theseRT-PCR assays are not quantitative, the data clearly show that PMCA1b,PMCA2b (likely at low levels), and PMCA4b transcripts are expressed inMDCK cells.- g3 l8 {, h' K# {! e7 [

Fig. 2. Madin-Darby canine kidney (MDCK) cells expresstranscripts for PMCA1b, PMCA2b, PMCA4b, and NCX1. RT-PCR productsobtained with MDCK cell RNA by using primers for the splice site Cregion of PMCA1, PMCA2, and PMCA4 ( A ) and for a conservedregion of NCX1 ( B ) were separated by agarose gelelectrophoresis and stained with ethidium bromide. The identity of thePMCA1b (393 bp), PMCA2b (452 bp), PMCA4b (352 bp), and NCX1 (712 bp)amplicons was confirmed by comparison with PCR products obtained frompositive controls ( C) and sequencing. Lane 1 : 100-bp sizestandard ladder (M). Lane 2 : negative control PCR withoutcDNA input. Forty PCR cycles were performed for the detection of PMCA2and 35 cycles for all other amplifications.6 f8 Y( F, D+ V* z. [, z

Detection of NCX1 transcripts in MDCK cells. Using primers flanking a conserved region of NCX1 known to amplify aPCR product of ~700 bp from kidney mRNA ( 45 ), we were able to demonstrate the presence of transcripts for this additional Ca 2  transport protein in MDCK cells (Fig. 2 B ).

PMCA1 and PMCA4 are the major isoforms in MDCK cells and areenriched in the basolateral membrane. When Western blots of total cell lysates were probed with anantibody recognizing all PMCAs, a band of the expected size of ~140kDa was readily observed (Fig. 3 A, first row). Similarly, antibodies against PMCA1 and PMCA4 recognized bands of around 140 kDain the total cell lysates (Fig. 3 A ). By contrast, NR2 antibody specific for PMCA2 ( 16 ) detected only a faintband of the appropriate size in the total cell lysates and instead reacted with a distinct band at around 90-100 kDa (Fig. 3 A, third row). This band was not detected on the blots whenthe NR2 antibody was preabsorbed with the peptide used as antigen (datanot shown), suggesting that the 100-kDa band corresponds to aproteolytic breakdown fragment of PMCA2 or to an unrelated proteinspecifically cross-reacting with the antibody.: E2 I3 s0 Q( @8 P: x0 z  C5 K

Fig. 3. Expression of PMCA isoforms and NCX in MDCK cells. A :representative immunoblots of 25 µg total cell lysate (TCL), 6 µgcrude total plasma membrane (PM), 2 µg apical (Api), 2 µg mixedapical and basolateral (M), and 1.5 µg basolateral plasma (BL)membranes probed with antibodies recognizing all PMCAs or specificfor PMCA1, PMCA2, and PMCA4 as indicated. Mature PMCA1, PMCA2, andPMCA4 migrate at ~140 kDa ( right ); faster migrating bandsof ~100 kDa likely correspond to breakdown products. PMCAs areenriched in the plasma membrane and especially in the basolateralmembrane. Full-length 140-kDa PMCA2 is only weakly detected in totalcell lysate, whereas a B : immunoblot of 25 µg total cell lysate and 6 µg crude total plasma membrane showing the presence of NCX1 matureprotein at 125 kDa, as well as its enzymatically cleaved form at 85 kDa, in MDCK cells.

We next analyzed the distribution of the PMCAs in purified total crudeplasma membrane and in purified subfractions enriched in apical orbasolateral plasma membrane. The enrichment for apical and basolateralmembranes in the corresponding fractions was demonstrated by assayingfor known apical (alkaline phosphatase) and basolateral (Na -K -ATPase) marker proteins (data notshown). As expected, the antibody against all PMCAs reacted stronglywith a 140-kDa band in total plasma membrane. The same antibody, whenused on the plasma membrane subfractions, revealed that the PMCA washighly enriched in the basolateral membrane (Fig. 3 A, firstrow). However, some PMCA was also detected in the apical domain as wellas in the intermediary fraction consisting of mixed apical andbasolateral membranes. An essentially identical pattern of distributionwas seen when the antibody against PMCA4 was used; i.e., PMCA4 wasconcentrated in the plasma membrane and highly enriched in thebasolateral membrane fraction (Fig. 3 A, fourth row). Theantibody against PMCA1 detected the expected 140-kDa band in totalplasma membrane, but this band did not appear to be enriched withrespect to the total cell lysate (Fig. 3 A, second row).Instead, an additional band of ~110 kDa was detected that likelycorresponded to a proteolytic fragment of the pump. As found for totalPMCA and PMCA4, PMCA1 also was enriched in the basolateral membrane,with little if any detectable in the apical domain (Fig. 3 A,second row). Finally, the antibody against PMCA2 did not detect anysignificant amount of full-length pump in any of the plasma membranefractions. The additional 90- to 100-kDa immunoreactive band detectedby this antibody was still apparent but was not enriched in the plasma membrane compared with the total cell lysate. However, this band wasslightly enriched in the basolateral fraction but was also detected inthe apical and intermediate fractions (Fig. 3 A, third row).
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Detection of NCX1 in MDCK total cell lysates and the basolateralplasma membrane. Expression of the exchanger NCX1 at the protein level was observed intotal cell lysates and the plasma membrane domain of MDCK cells (Fig. 3 B ) by using a commercially available monoclonal antibodythat detected the mature protein at 125 kDa as well as a proteolyticfragment at around 85 kDa. In the plasma membrane fraction, the 125-kDaband was weaker than in the total cell lysate, whereas the 85-kDa bandappeared more prominent. This likely reflects increased proteolysis dueto the additional steps required to obtain the plasma membrane-enriched fraction." i; M' u; r  O1 b" \* F: Z
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Immunofluorescence localization of PMCA isoforms in polarized MDCKcells. Immunofluorescence confocal microscopy was performed on polarized MDCKcells grown in monolayers. In agreement with the biochemical datashowing enrichment of the PMCAs in the basolateral plasma membrane,immunocytochemical localization with the pan-PMCA antibody 5F10 or thePMCA4-specific antibody JA9 showed the honeycomb pattern typical of(baso)lateral membrane staining (Fig. 4 ).This was further corroborated by the high degree of overlap inimmunostaining for the PMCAs (all PMCAs or PMCA4 alone) and SAP97, ascaffolding protein associated with the basolateral plasma membrane inepithelial cells ( 30 ). The antibody against PMCA1 showedonly faint plasma membrane staining and, in addition, yielded somenuclear staining (Fig. 4 ). However, this staining is likely an artifactdue to the relatively high concentration of antibody used (1:200dilution). In fact, the NR1 antibody has been shown to be of lowsensitivity ( 16 ) and hence may not be ideally suited forimmunocytochemistry on cells with a low level of PMCA1 expression. Asexpected from the transcript and (full-length) protein expression data,no specific immunofluorescence staining was observed for PMCA2 in theMDCK cells. Taken together, the immunolocalization results support thebiochemical fractionation data and show that most of the PMCA inpolarized MDCK cells is found in the basolateral membrane. They alsosupport the notion that PMCA4 is the major PMCA isoform in MDCK cells.
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Fig. 4. PMCAs are mostly localized to the basolateral plasma membrane ofMDCK cells. Fluorescent confocal microscopy images showingcoimmunolocalization of all PMCAs or of PMCA4 and PMCA1 alone (red)with SAP97 or Na -K -ATPase (green), at thebasolateral plasma membrane of MDCK cells. Merged images (Overlay)depict areas of overlap in yellow. A confocal micrograph of a Z -axis section (Z-section) taken through cells immunostainedfor SAP97 and all PMCAs confirms the high degree of overlap betweenSAP97 (a basolateral membrane marker) and the PMCAs.. N0 J- @0 E- G9 D. K/ ~2 O7 e
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Tightness of MDCK cell monolayers. Before we embarked on functional Ca 2  flux studies, it wasimportant to determine the tightness and paracellular transportproperties of MDCK cells grown on permeable filter inserts. Thetransport of phenol red, known to cross cell monolayers by theparacellular route rather than by permeation through the cells, wasassessed in the presence and absence of a cell monolayer. TEERmeasurements were performed as described in MATERIALS AND METHODS to check for the tightness of the monolayers. MDCK cells startedto be virtually impermeable to phenol red by around day 12 in culture. At this time, they demonstrated 35% of transport observed for inserts without MDCK cells. Thephenol red transport data correlated well with the TEER values, which started to peak around day 10, reached a plateau by day 13, and 2 wk (data not shown).
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Contribution of PMCAs and NCX to transcellularCa 2 flux in MDCK cells. The transcellular Ca 2  flux from the apical to thebasolateral chamber across a tight monolayer of MDCK cells underresting (basal) conditions was determined as described in MATERIALS AND METHODS. The transport of 45 Ca 2  was measured after 60 min of incubationand, after correction for the paracellular transport, amounted to 150 cpm · µgprotein 1 · well 1 incontrol cells (Fig. 5 A ). Todetermine the contribution of the NCX to the overall Ca 2  transport, the flux studies were performed in transport media in whichNa was isosmotically replaced by choline. Because the NCXrequires Na as the counterion for net transport ofCa 2 , substitution of extracellular Na bycholine effectively blocks Ca 2  extrusion by the NCX.Transcellular Ca 2  flux across MDCK cells under theseconditions was decreased by ~67%, indicating that two-thirds of theentire Ca 2  flux were dependent onNa -dependent transport. We next used a variety of agentsknown to inhibit the PMCAs to determine the contribution of the pumpsto transcellular Ca 2  flux. PMCA activation is dependent onCa 2  -calmodulin, and inhibition of calmodulin is known toprevent PMCA stimulation. Trifluoroperazine (50 µM) and calmidazolium (145 nM), two inhibitors of calmodulin, decreased the Ca 2  flux by 45 and 33%, respectively (Fig. 5 A ). The PMCAs canalso be nonspecifically blocked by La 3  (0.25 mM) as wellas vanadate (5 mM), which inhibits all P-type ATPases. Addition ofthese inhibitors reduced the transcellular Ca 2  flux by 43 and 15%, respectively (Fig. 5 A ). The comparatively minorinhibition of Ca 2  flux in the presence of vanadate islikely due to its poor membrane permeability and incomplete access tothe intracellular ATP-binding site of the PMCAs. On the other hand,using both choline and La 3  together significantlypotentiated the inhibition of transcellular Ca 2  flux (77%inhibition; Fig. 5 A ), although it was impossible to inhibit100% of Ca 2  flux with any combination of the blockingagents. This residual Ca 2  transport may be due toincomplete inhibition of the PMCAs and/or the NCX or to additionalextrusion/leak mechanisms operating to eliminate intracellularCa 2  under conditions whereby the major pumps andexchangers are blocked. Regardless, our data using a functionalCa 2  flux assay show that the PMCAs contribute aboutone-third and Na /Ca 2  exchange abouttwo-thirds toward the total vectorial Ca 2  transport acrosspolarized MDCK cells under resting conditions.
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Fig. 5. Importance of the NCX and PMCAs for transcellular Ca 2  flux in polarized MDCK cells. A : functional 45 Ca 2  efflux assays performed in tight layersof polarized MDCK cells grown in the absence and presence of variousinhibitors to determine the contribution of the NCX and PMCAs tovectorial Ca 2  flux. Isosmotic replacement ofNa by choline (CH) indicates that two-thirds of the 45 Ca 2  efflux is dependent on NCX.Trifluoroperazine (TFP), calmidazolium (Cal), lanthanum (Lan), andvanadate (Van) block PMCA activity and demonstrate that about one-thirdof the 45 Ca 2  efflux depends on the pumps. Thecombined use of choline and lanthanum (CH Lan) potentiates theinhibition and blocks almost 80% of transcellular 45 Ca 2  efflux. The statistical significance( P value) of each treatment with respect to the untreatedconrol (C) is indicated above each data bar, and the number ofindependent measurements is also indicated. B : effect ofexpression of PMCA4b sense (s4) and antisense (as4) cDNAs on thetranscellular 45 Ca 2  flux across MDCKmonolayers. Cells transfected with PMCA4b and antisense PMCA4 showsignificantly enhanced and decreased capacity of transcellular 45 Ca 2  efflux, respectively, compared withuntreated control cells, indicating the important role of this pumpisoform in transcellular 45 Ca 2  transport.

Finally, to demonstrate the importance of the contribution of thePMCAs, and specifically of the PMCA4b isoform, to transcellular Ca 2  transport in MDCK cells, we transfected cells with anexpression construct for full-length PMCA4b or with aplasmid-generating antisense RNA to PMCA4. As shown in Fig. 5 B, the transcellular Ca 2  transport in PMCA4boverexpressing cells increased to ~140% of control values, whereasantisense treatment reduced the Ca 2  flux to 53% of thecontrol. Thus manipulation of the expression level of the PMCAs [atleast of the major isoform (PMCA4b) of MDCK cells] has a significanteffect on the transcellular Ca 2  transport across these cells.0 N4 E' T+ X' k
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DISCUSSION
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The MDCK cell system represents a model for the transportingepithelium of kidney distal tubules. The cells form electrically resistant tight layers on semipermeable filter supports anddifferentiate into polarized structures with clearly separated apicaland basolateral membrane domains that are able to sustain the vectorialtransport of ions and other solutes. Studies of the transport processes involved in Ca 2  reabsorption are therefore feasible withthis system, e.g., as demonstrated in an analysis of the effects ofparathyroid hormone ( 24 ) and in a recent report on therole of the Ca 2  -sensing receptor in these cells( 6 ). To date, however, there has been no detailed studyexamining the expression of different PMCA isoform transcripts andproteins in MDCK cells. Similarly, the cellular distribution of thePMCAs and their contribution to Ca 2  expulsion in thesecells has not yet been investigated. Although previous studiesindicated that all four PMCA isoforms are expressed in the rat kidneyat the transcript level ( 13, 21 ), the data at the proteinlevel suggested that neither PMCA2 nor PMCA3 is abundant pump isoformsin rat and human kidneys ( 36 ). Moreover, the presence andabundance of the second major Ca 2  -expulsion system, i.e.,the NCX, in the distal tubule of the kidney has been a matter ofdispute (see Ref. 7 for a recent review).

Using the sensitive RT-PCR technique, we detected PMCA1b, PMCA2b, andPMCA4b in MDCK cells, but only PMCA1b and PMCA4b amplicons were readilyobtained after 35 PCR cycles. By contrast, using equal amounts of inputcDNA, detection of PMCA2b required 40 cycles. Because of potentiallydifferent primer and amplification efficiencies, these RT-PCR assaysare not quantitative. Nevertheless, the results suggest that PMCA2 isnot a major pump isoform in MDCK cells. On the other hand, the dataconfirm the earlier detection of PMCA2 transcripts in total rat kidneyand microdissected distal convoluted tubules ( 13, 25 ). Bycontrast, we were unable to detect PMCA3 transcripts in MDCK cells.This compares well with the study by Caride et al. ( 13 ),who detected only spurious amounts of this pump isoform inmicrodissected distal convoluted tubules. Our data also fit well with avery recent report on the PMCA isoform distribution in mouse distalconvoluted tubule cells ( 26 ), wherein only PMCA1b andPMCA4b were detected at the transcript level.
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In agreement with the RT-PCR results, the Western blotting data showedthat PMCA4 and PMCA1 are the major PMCA isoforms in MDCK cells. Bycontrast, full-length PMCA2 is virtually undetectable in these cells.In fact, using the high-affinity antibody NR2 ( 16 ), it wasnot possible to detect a band of the appropriate size in any of themembrane subfractions. Although the ~100-kDa band detected by thisantibody in Western blots of total cell lysates and the membranesubfractions could potentially correspond to proteolytically truncatedPMCA2, we favor the notion that this band represents an unrelatedcross-reacting protein. This is supported by the absence of anydistinct membrane staining when the NR2 antibody was used forimmunolocalization in MDCK cells. Obviously, a quantitative comparisonamong PMCA1, PMCA2, and PMCA4 based solely on Western blot data is notpermissible because of the different affinities and specificities ofthe antibodies used. However, the combined data from RT-PCR, Westernblotting, and immunolocalization suggest that PMCA4 (splice variant b)is the major isoform in MDCK cell membranes. For example, theamount of full-length PMCA1, expected to run at 135-140 kDa,appeared to be lower in total plasma membrane and in the plasmamembrane subfractions compared with the mature PMCA4 protein. On theother hand, potential cleavage products of PMCA1 (around 100 kDa) were more prominent in the plasma membrane, apical, and basolateral membranesubfractions than in the total cell lysates. These cleavage productsmight have arisen during sample handling necessary to prepare themembrane subfractions, although protease inhibitors were presentthroughout the procedure and similar cleavage products were notobserved for PMCA4. Alternatively, the PMCA1, and potentially PMCA2,fragments in the plasma membrane subfractions may reflect physiologicalevents taking place in these cells, because observation of suchcleavage products is not a rare event for some pumps and cell types( 16, 32 ). Regardless, given the lack of enrichment offull-length PMCA1 (and PMCA2) in the plasma membrane subfractions, PMCA4 appears to be the major pump isoform in the MDCK cell membranes.$ l% j1 [8 E9 D/ k2 I' h' e

We have previously shown that endogenous PMCA is almost exclusivelylocalized to the basolateral membrane in polarized MDCK cells( 15 ). These results were confirmed in the present study byusing antibodies against the PMCAs and a basolateral marker protein(SAP97) for coimmunolocalization. The same conclusion was reached byMagyar et al. ( 26 ), who recently found that the PMCAs (aswell as NCX1) were confined to the basolateral membrane domain inpolarized mouse kidney distal tubule cells. In addition, we now providecorroborating biochemical evidence, by combining plasma membranesubfractionations with Western blot analyses. Judging from the bandintensities of Western blots (see Fig. 3 A, top )probed with an antibody recognizing all PMCAs, the pumps were enrichedat least 10-fold in the basolateral plasma membrane of MDCK cells (1.5 µg of protein loaded/lane) compared with the amount in total celllysates (25 µg protein/lane).
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The predominantly basolateral localization of the PMCAs in polarizedMDCK cells predisposes these pumps to contribute substantially tovectorial, transcellular Ca 2  transport from the apical tothe basolateral side. In the distal convoluted tubule of the intactkidney, this is the major direction of Ca 2  flux duringactive Ca 2  reabsorption. Both PMCA and NCX have been shownto be involved in active Ca 2  reabsorption in the distalkidney ( 18, 44 ), but the relative contribution of thesetwo mechanisms for "uphill" Ca 2  transport remainspoorly understood. For example, although functional measurementsindicate a large role for the NCX in the kidney distal convolutedtubule, connecting tubule, and cortical collecting duct ( 40, 46 ), immunocytochemical localization data have yielded controversial results, suggesting that NCX is only abundant in thebasolateral membrane of connecting tubule cells ( 10, 33 ). On the other hand, using RT-PCR, Yu et al. ( 47 ) readilyidentified NCX1 in the distal convoluted tubule. Our RT-PCR and Westernblot data on MDCK cells clearly show that NCX1 is expressed in these cells. Moreover, using a transcellular Ca 2  flux assaysimilar to that previously employed to determine Ca 2  transport in other cell types ( 4, 5, 17 ), we determined for the first time the contribution of the NCX and PMCA toward apicalto basolateral Ca 2  transport in resting MDCK cells.Interestingly, about two-thirds of this transport are not due to PMCAactivity but rather depend on a Na -dependentCa 2  exchange. The PMCA is responsible for the remainingone-third of Ca 2  flux under control conditions. Theseresults are in excellent agreement with transcellular Ca 2  flux studies on cultured rabbit kidney cells isolated from connecting tubules and cortical collecting ducts ( 5 ). This reportshowed a strong dependence of transcellular Ca 2  flux onbasolateral Na  , with up to 67% of transport inhibitableby isosmotic Na substitution. Although not shown directly,the remaining 30% of transport was suggested to be handled by adifferent, Na -independent Ca 2  extrusionsystem, likely the PMCA ( 5 ). Although our data on the roleof the PMCA for basolateral Ca 2  extrusion in MDCK cellsare consistent with a recent report on the effect of theCa 2  receptor on the PMCA in these cells ( 6 ),the major role played by the NCX (and/or additionalNa -dependent Ca 2  extrusion systems) in MDCKcells has not previously been appreciated.% h9 {6 I* ?6 X/ g6 ^- N, }
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Regardless of the contribution of the NCX, the significant role of thePMCA, especially of PMCA4, in vectorial Ca 2  transport wasfurther demonstrated by overexpression and knockdown experiments inMDCK cells. The expression constructs for PMCA4b and an antisense RNAto PMCA4 have previously been used for functional overexpression andknockdown of this isoform, respectively ( 20, 43 ). AlthoughWestern blot analyses of total cell lysates from transfected MDCK cellsshowed only 1.2- to 1.5-fold differences of PMCA protein intensities(after standardization to actin; data not shown), the functional impactwas remarkable. Overexpression of PMCA4b resulted in a 1.4-foldstimulation of Ca 2  flux, whereas antisense inhibition ofPMCA4 decreased the transport by up to 47%. Because transfectionefficiencies of the MDCK cells were at best 30-40%, it is clearthat even moderate changes in PMCA4b expression have a significanteffect on the overall transcellular Ca 2  transport capacityof these cells. Future studies using viral expression vectors to obtainvirtually 100% transfection efficiencies may be needed to determinethe full extent of the contribution of PMCA to transcellularCa 2  flux in this cell system.# W) ~9 ~" R$ R$ X; p. h$ F& ^* j& o

Finally, it should be noted that a small fraction of the PMCAs (bothPMCA1 and PMCA4) were also found in the apical membrane domain ofpolarized MDCK cells. Although the physiological relevance of thisapical fraction of the pump is not clear, it is possible that thedistribution of the PMCAs between the apical and basolateral membranedomains is dynamic and that the relative distribution of the pumpsamong the different membrane domains is specifically regulated to meetthe changing demands on transcellular Ca 2  flux. Theestablishment of the MDCK cell system for functional Ca 2  transport studies and the characterization of its majorCa 2  export mechanisms pave the way for detailed studies ofthe hormonal and pharmacological regulation of Ca 2  reabsorption ( 24 ) via the diverse Ca 2  transporting systems in this distal kidney tubule model.% @  ~; m- G2 |
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ACKNOWLEDGEMENTS

We thank Adelaida Filoteo and John T. Penniston for the gift ofPMCA-specific antibodies, Craig C. Garner for the antibody againstSAP97, Amy S. Lienhard for technical assistance, and Michael C. Chickafor help with the confocal microscopy.& j: \+ ^" `, P, `( F' `
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