Characterization of PMCA isoforms and their contribution to transcellular Ca2+ flux in MDCK cells

Sertac N. Kip and Emanuel E. Strehler

Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota 55905


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Plasma membrane Ca2+ ATPases (PMCAs) are ubiquitous in Ca2+-transporting organs, including the kidney. Using RT-PCR, we detected PMCA1b, PMCA2b (rare), and PMCA4b in Madin-Darby canine kidney (MDCK) cells. At the protein level, only PMCA1 and PMCA4 were readily detected and were highly enriched in the basolateral membrane. The Na+/Ca2+ exchanger NCX1 was also detected at the transcript and protein level. A functional assay measuring 45Ca2+ flux across MDCK cell monolayers under resting conditions indicated that two-thirds of apicobasolateral Ca2+ transport was provided by Na+/Ca2+ exchanger and one-third by PMCAs, as determined in Na+-free media and using various PMCA inhibitors (La3+, vanadate, calmidazolium, and trifluoroperazine). The importance of PMCA4b for basolateral Ca2+ efflux was demonstrated by overexpression of PMCA4b or antisense knockdown of endogenous PMCA4b. Overexpression of PMCA4b increased apicobasolateral Ca2+ transport to ~140%, whereas antisense treatment reduced Ca2+ flux ~45% compared with controls. The MDCK system is thus an ideal model for functional studies of the specific role and regulation of PMCA isoforms in Ca2+ reabsorption in the distal kidney.

calcium transport; kidney distal tubules; Madin-Darby canine kidney; sodium/calcium exchanger; plasma membrane calcium ATPase


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CA2+ is an essential mineral in animals, where it plays a crucial role in processes ranging from the formation and maintanance of the skeleton to the temporal and spatial regulation of neuronal function. Overall Ca2+ homeostasis must therefore be under tight and finely tuned control. Ca2+ uptake from the environment largely occurs through the intestine, whereas Ca2+ loss occurs mainly via the kidney. The overall balance between Ca2+ uptake and loss is under multiple hormonal control and is dictated by the body's changing needs for this mineral element. As the primary organ responsible for Ca2+ excretion, the kidney has a preeminent role in regulating Ca2+ homeostasis. Filtered Ca2+ in the urine is reabsorbed throughout the kidney, with the bulk being handled by the proximal tubules and only ~10% by the distal convoluted tubules (19, 39). Although most of the Ca2+ reabsorption in the proximal nephron occurs via a passive, paracellular pathway, the reuptake of Ca2+ in the distal tubules occurs mainly via an active transcellular pathway that moves Ca2+ against its electrochemical gradient. This is achieved by Ca2+ influx through specific channels (such as ECaC; see Ref. 23) in the apical plasma membrane and active Ca2+ extrusion by Ca2+ pumps and Na+/Ca2+ exchangers (NCXs) in the basolateral plasma membrane of the distal tubule epithelial cells (7). Because even small changes in the retention or loss of Ca2+ in the distal kidney have large effects on the overall Ca2+ homeostasis in the body, the reabsorption of Ca2+ in the distal tubules is tightly regulated and under multiple hormonal controls (parathyroid hormone, calcitonin, and vitamin D3). Studies of the diverse transporters involved in vectorial Ca2+ flux are therefore of particular interest, because changes in their expression, localization, and function are likely the primary targets of regulation.

The plasma membrane Ca2+ ATPases (PMCAs) are high-affinity Ca2+ efflux pumps found in virtually all eukaryotic cells, wherein they are responsible for the maintenance and resetting of the resting intracellular Ca2+ levels (12). Four genes encode separate isoforms called PMCA1-4; in addition, alternative splicing of the transcripts yields a large variety of splice variants differing mainly in their COOH-terminal amino acid sequence (38). Using a PMCA-specific antibody that recognizes all isoforms, the presence of the PMCA in the kidney of several 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 tissue microdissection techniques indicate that all PMCA isoforms are expressed in the rat kidney; however, they are expressed with distinct isoform-specific expression patterns and variable abundance along the different regions of the nephron (13, 25). Other studies using RT-PCR on whole kidney RNA, as well as studies at the protein level, have been controversial, suggesting that PMCA1 and PMCA4 are the major isoforms expressed in the kidney and that PMCA2 and PMCA3 may be minor components of this tissue, if expressed at all (11, 36, 37). The expression level and localization of the NCX along the nephron have also been a matter of debate. Of the three major isoforms of the exchanger, NCX1-3, only NCX1 appears to be expressed in significant amounts in the kidney (47). As expected for a transporter involved in active expulsion of Ca2+ into the interstitial space during urinary Ca2+ reabsorption, the exchanger has been localized mainly to the basolateral membrane, at least in the distal tubule and connecting tubule (9, 33). However, the relative contribution of the NCX to vectorial Ca2+ extrusion in the distal kidney (relative to that by the PMCA) remains to be established.

Madin-Darby canine kidney (MDCK) cells are a well-established model for distal tubule epithelial cells (2, 22, 34). On confluence, they form sheets of polarized cell monolayers that reproduce many physiological parameters of the distal transporting kidney epithelium. When grown on semipermeable filter inserts, the apical and basolateral membrane domains can be separately accessed and represent the urinary lumen and interstitial compartments, respectively. Vectorial transcellular ion flux studies can be conducted if the cell monolayer is not leaky, i.e., if tight junctions have formed between adjacent cells. The tightness of the monolayer can be assessed by measurements of transepithelial electrical resistance (TEER), and the net transcellular flux of ions such as Ca2+ can be determined after correction for paracellular transport (2, 6, 29).

Here, we demonstrate that the MDCK system is an attractive model for the study of transcellular Ca2+ transport by the PMCAs in the distal kidney. Using RT-PCR, immunoblotting, and immunolocalization techniques, we show that PMCA4b and PMCA1b are the major pump isoforms in MDCK cells, whereas full-length PMCA2b is detected only in small amounts. 45Ca2+ flux studies using different pharmacological blockers reveal that one-third of the apical-to-basolateral Ca2+ flux in resting MDCK cells is handled by the PMCAs, whereas a Na+-dependent Ca2+ extrusion mechanism is responsible for the remaining two-thirds. The importance of PMCA4b in transcellular Ca2+ flux is further emphasized by transport studies conducted in transfected cells with altered Ca2+ pump expression.


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Materials. MDCK type I cells were obtained from the American Type Culture Collection (Manassas, VA). Trypsin-EDTA, DMEM, FBS, L-glutamine, Na+ pyruvate, and antibiotics/antimycotics were bought from Invitrogen (Carlsbad, CA). RT-PCR reagents and enzymes were purchased from Roche-Boehringer Mannheim (Indianapolis, IN). All other chemicals were from Sigma (St. Louis, MO). Monoclonal (5F10, JA9) and polyclonal antibodies against PMCAs were generously provided by Dr. John T. Penniston and Adelaida G. Filoteo (Mayo Clinic, Rochester, MN), and a polyclonal antibody against SAP97 was a gift from Dr. Craig C. Garner (University of Alabama, Birmingham, AL). The characterization and specificity of these antibodies have been described (14, 16, 30). Primary antibodies against the exchanger NCX1 and beta -actin and all secondary antibodies were products of Sigma, whereas an antibody against Na+-K+-ATPase was purchased from Affinity Bioreagents (Golden, CO). X-ray films were from Eastman Kodak (Rochester, NY), and 45Ca2+ was obtained from PerkinElmer Life Sciences (Boston, MA). The expression constructs for full-length PMCA4b (pMM24b) and for antisense PMCA4 (pCIneo-AS4) have been described previously (1, 20).

Cell culture. MDCK cells were propagated in DMEM containing 10% (vol/vol) FBS, 2 mM L-glutamine, 1 mM Na+ pyruvate, 50 µg/ml gentamycin sulfate, 100 U/ml penicillin, and 100 U/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO2.

RT-PCR. Total RNA was isolated from MDCK cells by using the TRIzol reagent (Invitrogen), as specified by the manufacturer. Briefly, cells grown to 80-90% confluence on 175-mm collagen-coated flasks were washed twice with PBS and lysed by adding 5 ml of TRIzol reagent. The lysates were 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 and centrifuged for 15 min at 12,000 rpm at 4°C. After removal of the aqueous phase and addition of 5 ml of isopropanol, samples were incubated 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 for 10 min before being dissolved in diethyl pyrocarbonate-treated water and stored at -70°C.

Reverse transcription was carried out by using 5 µg RNA in 28 µl PCR-grade water containing 5 µM hexanucleotide random primer. The mixture was incubated at 65°C for 6 min and cooled to room temperature before being mixed with 22 µl of a solution to yield a final 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 then incubated at 37°C for 50 min, heated at 99°C for 5 min, and stored at -70°C. The reverse transcription reaction (5 µl) was used to perform 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 following amplification profiles were used: 5 min of initial denaturation at 94°C followed by 35 cycles of 1 min of denaturation at 94°C, 1 min of 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 extension at 72°C and soak at 4°C. The only exception to this profile was for PMCA2, for which the cycle number was 40. The location of primers relative to the PMCA coding sequence and their sequences and origins are 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 ethidium bromide-containing 1.8% agarose gel, along with a molecular weight marker (100-bp ladder; Bio-Rad). The bands of expected size were excised from the gel, purified with the Qiaquick Gel Extraction Kit (Qiagen, Valencia, CA), and subjected to sequencing in the Mayo Molecular Biology Core Facility.


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Fig. 1.   Scheme of the major COOH-terminal plasma membrane Ca2+ ATPase (PMCA) splice variants and primers used for RT-PCR. A: intron-exon structures of each PMCA gene around splice site C are shown schematically (top), and the two major splice variants a and b generated by alternative splicing are indicated (bottom). Size of alternatively spliced exons is indicated in base pairs. For a more complete description of PMCA alternative splicing options, see Ref. 38. B: source and sequence of the primers for each PMCA isoform and for Na+/Ca2+ exchanger 1 (NCX1) are listed, and the start and end position of each primer pair within its cognate cDNA is indicated by the nucleotide number (nt). The expected sizes of amplification products corresponding to the a and b splice variants of the PMCAs are given in the last column, as is the expected product for NCX1. F, forward; R, reverse.

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, cells were thawed and lysed in lysis buffer (HEPES, pH 7.5, 0.1% Nonidet P-40, 0.5% deoxycholate, 1 mM EDTA, 150 mM NaCl, and 0.1 mM Na3VO4) containing a cocktail of protease inhibitors (aprotinin, leupeptin, pefabloc, and pepstatin). The cells were sonicated at 4°C twice for 7 s and incubated on ice for 10 min before precipitation with ice-cold 5% TCA. The precipitates were then spun at 4°C for 15 min at 12,000 g, and the pellets were resuspended in Krebs-Ringer-HEPES (KRH) solution containing (in mM) 130 NaCl, 5 KCl, 20 HEPES, 1.2 KH2PO4, 1 CaCl2, 1 MgSO4, and 10 glucose, as well as 1 ml/l DMEM, pH 7.4, and were homogenized by aspiration with a Hamilton syringe.

For total membrane preparations, the cells were lysed in lysis buffer containing protease inhibitors, and undisrupted cells and nuclear debris 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 resuspended in KRH solution (35).

To generate a purified mixed crude plasma membrane (MCPM) fraction, the thawed cell pellet was sonicated (2 bursts, 7 s each) in 10 vol/wt of 0.3 M sucrose containing protease inhibitors by using a Sonifier Cell Disrupter (Heat Systems-Ultrasonic, Plainview, NY). While vortexing, 1.43 vol of 2 M sucrose were added to the suspension, and the mixture was transferred to a TI70 ultracentrifuge tube (Beckman Instruments). The sucrose cushion was overlaid with 0.3 M sucrose and subjected to isopycnic centrifugation for 1 h at 240,000 g (56,000 rpm). The membrane band was removed and diluted with ice-cold dH2O 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, and centrifuged at 90,000 g (27,000 rpm) for 3 h in an SW 28 rotor (Beckman Instruments) (42).

The MCPM was further separated into distinct apical and basolateral plasma membrane domains on a three-step sucrose gradient (38, 34, and 31% wt/wt). For this, the MCPM band was diluted with 4 vol of 1 mM NaHCO3, 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 pellet was 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 gradient consisting 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% sucrose layer and at the 31/34 and 34/38% interfaces were collected as apical plasma membrane, a combination of apical plasma membrane plus basolateral 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 M sucrose and pelleted at 40,000 rpm for 1 h in an SW41 rotor. The resulting pellets were resuspended in KRH buffer by suction through a 25-gauge needle (20 times in and out) and stored at -70°C until further use. All procedures were carried out at 4°C.

Domain-specific assays were performed on the apical plasma membrane and basolateral plasma membrane to determine enrichment of the bands with apical and basolateral plasma membrane markers, respectively. Alkaline phosphatase, a commonly used marker for the apical plasma membrane domain (28) was assayed biochemically by using commercially available enzyme kits according to the supplier's instructions (Sigma). Immunoblotting for Na+-K+-ATPase was performed to confirm enrichment of the basolateral plasma membrane domain.

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 of total cell lysate, 6 µg of total cell membranes, and 1-2 µg of distinct plasma membrane domains were mixed with NuPAGE electrophoresis buffer in the presence of reducing agents and antioxidants and heated to 70°C for 15 min before being separated in denaturing NuPAGE 4-12% 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.

Immunoblotting was performed with standard Western blotting techniques (3). Nitrocellulose membranes were blocked in TBST (50 mM Tris · HCl, pH 7.4, 150 mM NaCl, and 0.05% Tween 20)+10% milk for 1 h at room temperature before exposure to primary antibodies for 1 h at room temperature. Primary antibodies were as follows. For the detection of all PMCAs and of PMCA4, mouse monoclonal 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 was detected with a commercially available monoclonal antibody diluted at 1:1,000. In addition, the same blots were reprobed with an anti-Na+-K+-ATPase alpha 1-antibody (1:500), as a plasma membrane marker, or anti-beta -actin antibody (1:1,000), as a cytosolic housekeeping protein, to standardize each lane and ensure equal protein loading. After exposure to primary antibodies, the blots were washed three times for 5 min in TBST and incubated in peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG (1:5,000) for 1 h at room temperature. Before immunodetection with the Renaissance chemiluminescence detection system (PerkinElmer Life Sciences), the membranes were washed three times for 10 min in TBST. Immunoreactive bands on the resulting autoradiographs were determined by using a model GS-700 imaging densitometer, and Molecular Analyst software (Bio-Rad) was utilized to calculate the ratio of PMCA reactivity to that of beta -actin and Na+-K+-ATPase. Western blots were repeated at least three times, and the imaging data were averaged.

Ca2+ efflux across monolayers of MDCK cells. Two hundred fifty thousand MDCK cells were seeded on permeable inserts (24.5-mm diameter; Costar, Cambridge MA) and grown as described above but in the absence of phenol red. Cells were maintained at 37°C in an environment of 5% CO2-95% air, and the media (2 ml) in both the top and the bottom compartments were changed on alternate days between 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 was noted to gradually increase by day 10, stabilize between days 10 and 15, and then decline after 2 wk in culture.

Transepithelial transport of 45Ca2+ was measured on day 15, when the cells were fully differentiated and polarized and formed tight monolayers. Cells were rinsed twice with wash buffer [Dulbecco's PBS containing (in mM) 138 NaCl, 8 Na2HPO4, 2.7 KCl, and 1.5 KH2PO4], and the inserts were transferred to fresh six-well cluster dishes that contained nonradiolabeled transport medium [(in mM) 140 NaCl, 5.8 KCl, 0.34 Na2HPO4, 0.44 KH2PO4, 0.8 MgSO4, 20 HEPES, 4 glutamine, 0.5 CaCl2, 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 45Ca2+ 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, duplicate 200-µl aliquots were removed from the bottom compartment and read in a scintillation counter to assess total transport of 45Ca2+ from the apical toward the basolateral compartment.

To estimate the paracellular transport of Ca2+ and the tightness of the monolayers, phenol red transport was measured in aliquots 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, the absorbance was read at 560 nm, and the concentration of phenol red appearing in the bottom compartment was determined by comparing the reading to that of the known standards. The percentage of phenol red transport was calculated, and an equivalent amount of Ca2+ was subtracted from the total Ca2+ transport to derive the transcellular Ca2+ transport. After termination of the transport reaction by ice-cold wash buffer, the MDCK cells were washed twice and incubated with 1 ml 0.5 N NaOH at 70°C to dissolve the protein off the inserts. An aliquot was assayed for protein concentration, and the transport of transcellular Ca2+ was normalized for protein content of each insert (cpm × min-1 × µg protein-1). An aliquot of the solubilized cells was also processed for scintillation counting to determine the Ca2+ content of the cells.

To determine the transcellular Ca2+ transport caused by NCX activity, NaCl present in the transport buffer was exchanged with the same molarity of choline. To dissect the transcellular Ca2+ 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 Ca2+ transport was also determined in MDCK cells transiently transfected with both sense and antisense PMCA cDNA expression constructs (see below).

Transfections. MDCK cells were plated at a density of 2.5 × 105/insert and grown for 12 days as described above. Three days before the Ca2+ flux assays, the cells were transfected with plasmid constructs expressing full-length PMCA4b (pMM24b) or antisense PMCA4 (pCIneo-AS4), by using LipofectAMINE 2000 Plus (Invitrogen) according to the manufacturer's instructions. Briefly, 1 µg of plasmid DNA was mixed with 100 µl of Opti-MEM, and 8 µl of LipofectAMINE were added in a separate tube to 100 µl of Opti-MEM. After incubation for 5 min, the contents of the two tubes were mixed and further incubated at room temperature for 30 min. During this incubation period, the culture medium on the cells was removed, 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 cells were incubated for 10 h at 37°C. One milliliter of medium containing 2× FBS but no antibiotics was then added and incubation continued for 24 h. The next day, the medium was removed, cells were washed, and the medium was replaced by the maintenance medium containing 1× FBS and antibiotics. The transfected MDCK cells were cultured for an additional day before the functional assays were performed.

Immunofluorescence confocal microscopy. MDCK cells grown to confluence on glass coverslips were washed with PBS plus Ca2+ and Mg2+ (PBS+CM) and fixed for 5 min at room temperature in 4% paraformaldehyde (Tousimis, Rockville, MD) diluted in PBS+CM. After three washes of 2 min each, cells were further fixed and permeabilized in ice-cold methanol for 15 min at -20°C. The cells were blocked for 1 h at room temperature in PBS+CM containing 5% normal goat serum and 1% bovine serum albumin and were then incubated for 1 h at room temperature with monoclonal pan-anti-PMCA antibody 5F10 and polyclonal anti-SAP97 antibodies diluted 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+-ATPase antibody was used at a 1:500 dilution. After a washing three times for 5 min in PBS+CM, cells were incubated for 1 h at room temperature in darkness with secondary antibodies, either anti-mouse Alexa 488 or anti-rabbit Alexa 594 (Molecular Probes, Eugene, OR), each diluted 1:600 in blocking buffer. After incubation, cells were washed three times for 5 min with PBS+CM and coverslips were mounted onto slides by using Prolong mounting media (Molecular Probes). Confocal micrographs were taken on a Zeiss LSM 510 with an Apochromat ×63 oil-immersion objective and captured by using Zeiss LSM 510 software.

Statistical analysis. Each experiment was done in triplicate, and all data were expressed as means ± SE. Statistical differences were analyzed by Student's t-test using StatView, and results were considered to be statistically significant at P < 0.05.


    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 the four PMCA isoforms in MDCK cells. The primers were chosen to amplify the region of splice site C to allow the simultaneous identification of the alternative splice variants at site C. The primers (Fig. 1) were derived from rat (PMCA1, PMCA3, and PMCA4) and human (PMCA2) sequences because dog PMCA cDNAs were not yet known. The identity of the PCR fragments was determined by sequencing. The overall genomic structure of the PMCA genes and the two major described splice variants at site C are illustrated in Fig. 1. For PMCA1, RT-PCR amplification of MDCK RNA yielded a single band of ~400 bp corresponding to the PMCA1b variant (Fig. 2A). PMCA2 cDNA amplification by PCR yielded a band of very low intensity (~500 bp) in MDCK cells (Fig. 2A), which on sequencing was found to correspond to PMCA2b. This band was only observed after 40 PCR cycles, indicating that the PMCA2 transcripts are rare in MDCK cells. PMCA3 primers did not amplify any PMCA fragments of expected size, suggesting that PMCA3 is not expressed in MDCK cells. By contrast, when the presence of PMCA4 was analyzed, a single amplicon of ~350 bp was readily obtained (Fig. 2A), demonstrating the existence of PMCA4b and absence of other splice site C variants. Although these RT-PCR assays are not quantitative, the data clearly show that PMCA1b, PMCA2b (likely at low levels), and PMCA4b transcripts are expressed in MDCK cells.


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Fig. 2.   Madin-Darby canine kidney (MDCK) cells express transcripts for PMCA1b, PMCA2b, PMCA4b, and NCX1. RT-PCR products obtained with MDCK cell RNA by using primers for the splice site C region of PMCA1, PMCA2, and PMCA4 (A) and for a conserved region of NCX1 (B) were separated by agarose gel electrophoresis and stained with ethidium bromide. The identity of the PMCA1b (393 bp), PMCA2b (452 bp), PMCA4b (352 bp), and NCX1 (712 bp) amplicons was confirmed by comparison with PCR products obtained from positive controls (+C) and sequencing. Lane 1: 100-bp size standard ladder (M). Lane 2: negative control PCR without cDNA input. Forty PCR cycles were performed for the detection of PMCA2 and 35 cycles for all other amplifications.

Detection of NCX1 transcripts in MDCK cells. Using primers flanking a conserved region of NCX1 known to amplify a PCR product of ~700 bp from kidney mRNA (45), we were able to demonstrate the presence of transcripts for this additional Ca2+ transport protein in MDCK cells (Fig. 2B).

PMCA1 and PMCA4 are the major isoforms in MDCK cells and are enriched in the basolateral membrane. When Western blots of total cell lysates were probed with an antibody recognizing all PMCAs, a band of the expected size of ~140 kDa was readily observed (Fig. 3A, first row). Similarly, antibodies against PMCA1 and PMCA4 recognized bands of around 140 kDa in the total cell lysates (Fig. 3A). By contrast, NR2 antibody specific for PMCA2 (16) detected only a faint band of the appropriate size in the total cell lysates and instead reacted with a distinct band at around 90-100 kDa (Fig. 3A, third row). This band was not detected on the blots when the NR2 antibody was preabsorbed with the peptide used as antigen (data not shown), suggesting that the 100-kDa band corresponds to a proteolytic breakdown fragment of PMCA2 or to an unrelated protein specifically cross-reacting with the antibody.


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Fig. 3.   Expression of PMCA isoforms and NCX in MDCK cells. A: representative immunoblots of 25 µg total cell lysate (TCL), 6 µg crude total plasma membrane (PM), 2 µg apical (Api), 2 µg mixed apical and basolateral (M), and 1.5 µg basolateral plasma (BL) membranes probed with antibodies recognizing all PMCAs or specific for PMCA1, PMCA2, and PMCA4 as indicated. Mature PMCA1, PMCA2, and PMCA4 migrate at ~140 kDa (right); faster migrating bands of ~100 kDa likely correspond to breakdown products. PMCAs are enriched in the plasma membrane and especially in the basolateral membrane. Full-length 140-kDa PMCA2 is only weakly detected in total cell lysate, whereas a <100-kDa cross-reacting band is present throughout. B: immunoblot of 25 µg total cell lysate and 6 µg crude total plasma membrane showing the presence of NCX1 mature protein 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 crude plasma membrane and in purified subfractions enriched in apical or basolateral plasma membrane. The enrichment for apical and basolateral membranes in the corresponding fractions was demonstrated by assaying for known apical (alkaline phosphatase) and basolateral (Na+-K+-ATPase) marker proteins (data not shown). As expected, the antibody against all PMCAs reacted strongly with a 140-kDa band in total plasma membrane. The same antibody, when used on the plasma membrane subfractions, revealed that the PMCA was highly enriched in the basolateral membrane (Fig. 3A, first row). However, some PMCA was also detected in the apical domain as well as in the intermediary fraction consisting of mixed apical and basolateral membranes. An essentially identical pattern of distribution was seen when the antibody against PMCA4 was used; i.e., PMCA4 was concentrated in the plasma membrane and highly enriched in the basolateral membrane fraction (Fig. 3A, fourth row). The antibody against PMCA1 detected the expected 140-kDa band in total plasma membrane, but this band did not appear to be enriched with respect to the total cell lysate (Fig. 3A, second row). Instead, an additional band of ~110 kDa was detected that likely corresponded to a proteolytic fragment of the pump. As found for total PMCA and PMCA4, PMCA1 also was enriched in the basolateral membrane, with little if any detectable in the apical domain (Fig. 3A, second row). Finally, the antibody against PMCA2 did not detect any significant amount of full-length pump in any of the plasma membrane fractions. The additional 90- to 100-kDa immunoreactive band detected by this antibody was still apparent but was not enriched in the plasma membrane compared with the total cell lysate. However, this band was slightly enriched in the basolateral fraction but was also detected in the apical and intermediate fractions (Fig. 3A, third row).

Detection of NCX1 in MDCK total cell lysates and the basolateral plasma membrane. Expression of the exchanger NCX1 at the protein level was observed in total cell lysates and the plasma membrane domain of MDCK cells (Fig. 3B) by using a commercially available monoclonal antibody that detected the mature protein at 125 kDa as well as a proteolytic fragment at around 85 kDa. In the plasma membrane fraction, the 125-kDa band was weaker than in the total cell lysate, whereas the 85-kDa band appeared more prominent. This likely reflects increased proteolysis due to the additional steps required to obtain the plasma membrane-enriched fraction.

Immunofluorescence localization of PMCA isoforms in polarized MDCK cells. Immunofluorescence confocal microscopy was performed on polarized MDCK cells grown in monolayers. In agreement with the biochemical data showing enrichment of the PMCAs in the basolateral plasma membrane, immunocytochemical localization with the pan-PMCA antibody 5F10 or the PMCA4-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 in immunostaining for the PMCAs (all PMCAs or PMCA4 alone) and SAP97, a scaffolding protein associated with the basolateral plasma membrane in epithelial cells (30). The antibody against PMCA1 showed only faint plasma membrane staining and, in addition, yielded some nuclear staining (Fig. 4). However, this staining is likely an artifact due to the relatively high concentration of antibody used (1:200 dilution). In fact, the NR1 antibody has been shown to be of low sensitivity (16) and hence may not be ideally suited for immunocytochemistry on cells with a low level of PMCA1 expression. As expected from the transcript and (full-length) protein expression data, no specific immunofluorescence staining was observed for PMCA2 in the MDCK cells. Taken together, the immunolocalization results support the biochemical fractionation data and show that most of the PMCA in polarized MDCK cells is found in the basolateral membrane. They also support 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 of MDCK cells. Fluorescent confocal microscopy images showing coimmunolocalization of all PMCAs or of PMCA4 and PMCA1 alone (red) with SAP97 or Na+-K+-ATPase (green), at the basolateral 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 immunostained for SAP97 and all PMCAs confirms the high degree of overlap between SAP97 (a basolateral membrane marker) and the PMCAs.

Tightness of MDCK cell monolayers. Before we embarked on functional Ca2+ flux studies, it was important to determine the tightness and paracellular transport properties of MDCK cells grown on permeable filter inserts. The transport of phenol red, known to cross cell monolayers by the paracellular route rather than by permeation through the cells, was assessed in the presence and absence of a cell monolayer. TEER measurements were performed as described in MATERIALS AND METHODS to check for the tightness of the monolayers. MDCK cells started to be virtually impermeable to phenol red by around day 12 in culture. At this time, they demonstrated <0.5% of phenol red transport across the monolayer within a period of 1 h in contrast to >35% of transport observed for inserts without MDCK cells. The phenol red transport data correlated well with the TEER values, which started to peak around day 10, reached a plateau by day 13, and began to decline if the cells were kept in culture for >2 wk (data not shown).

Contribution of PMCAs and NCX to transcellular Ca2+ flux in MDCK cells. The transcellular Ca2+ flux from the apical to the basolateral chamber across a tight monolayer of MDCK cells under resting (basal) conditions was determined as described in MATERIALS AND METHODS. The transport of 45Ca2+ was measured after 60 min of incubation and, after correction for the paracellular transport, amounted to 150 cpm · µg protein-1 · well-1 in control cells (Fig. 5A). To determine the contribution of the NCX to the overall Ca2+ transport, the flux studies were performed in transport media in which Na+ was isosmotically replaced by choline. Because the NCX requires Na+ as the counterion for net transport of Ca2+, substitution of extracellular Na+ by choline effectively blocks Ca2+ extrusion by the NCX. Transcellular Ca2+ flux across MDCK cells under these conditions was decreased by ~67%, indicating that two-thirds of the entire Ca2+ flux were dependent on Na+-dependent transport. We next used a variety of agents known to inhibit the PMCAs to determine the contribution of the pumps to transcellular Ca2+ flux. PMCA activation is dependent on Ca2+-calmodulin, and inhibition of calmodulin is known to prevent PMCA stimulation. Trifluoroperazine (50 µM) and calmidazolium (145 nM), two inhibitors of calmodulin, decreased the Ca2+ flux by 45 and 33%, respectively (Fig. 5A). The PMCAs can also be nonspecifically blocked by La3+ (0.25 mM) as well as vanadate (5 mM), which inhibits all P-type ATPases. Addition of these inhibitors reduced the transcellular Ca2+ flux by 43 and 15%, respectively (Fig. 5A). The comparatively minor inhibition of Ca2+ flux in the presence of vanadate is likely due to its poor membrane permeability and incomplete access to the intracellular ATP-binding site of the PMCAs. On the other hand, using both choline and La3+ together significantly potentiated the inhibition of transcellular Ca2+ flux (77% inhibition; Fig. 5A), although it was impossible to inhibit 100% of Ca2+ flux with any combination of the blocking agents. This residual Ca2+ transport may be due to incomplete inhibition of the PMCAs and/or the NCX or to additional extrusion/leak mechanisms operating to eliminate intracellular Ca2+ under conditions whereby the major pumps and exchangers are blocked. Regardless, our data using a functional Ca2+ flux assay show that the PMCAs contribute about one-third and Na+/Ca2+ exchange about two-thirds toward the total vectorial Ca2+ transport across polarized MDCK cells under resting conditions.


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Fig. 5.   Importance of the NCX and PMCAs for transcellular Ca2+ flux in polarized MDCK cells. A: functional 45Ca2+ efflux assays performed in tight layers of polarized MDCK cells grown in the absence and presence of various inhibitors to determine the contribution of the NCX and PMCAs to vectorial Ca2+ flux. Isosmotic replacement of Na+ by choline (CH) indicates that two-thirds of the 45Ca2+ efflux is dependent on NCX. Trifluoroperazine (TFP), calmidazolium (Cal), lanthanum (Lan), and vanadate (Van) block PMCA activity and demonstrate that about one-third of the 45Ca2+ efflux depends on the pumps. The combined use of choline and lanthanum (CH+Lan) potentiates the inhibition and blocks almost 80% of transcellular 45Ca2+ efflux. The statistical significance (P value) of each treatment with respect to the untreated conrol (C) is indicated above each data bar, and the number of independent measurements is also indicated. B: effect of expression of PMCA4b sense (s4) and antisense (as4) cDNAs on the transcellular 45Ca2+ flux across MDCK monolayers. Cells transfected with PMCA4b and antisense PMCA4 show significantly enhanced and decreased capacity of transcellular 45Ca2+ efflux, respectively, compared with untreated control cells, indicating the important role of this pump isoform in transcellular 45Ca2+ transport.

Finally, to demonstrate the importance of the contribution of the PMCAs, and specifically of the PMCA4b isoform, to transcellular Ca2+ transport in MDCK cells, we transfected cells with an expression construct for full-length PMCA4b or with a plasmid-generating antisense RNA to PMCA4. As shown in Fig. 5B, the transcellular Ca2+ transport in PMCA4b overexpressing cells increased to ~140% of control values, whereas antisense treatment reduced the Ca2+ flux to 53% of the control. Thus manipulation of the expression level of the PMCAs [at least of the major isoform (PMCA4b) of MDCK cells] has a significant effect on the transcellular Ca2+ transport across these cells.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The MDCK cell system represents a model for the transporting epithelium of kidney distal tubules. The cells form electrically resistant tight layers on semipermeable filter supports and differentiate into polarized structures with clearly separated apical and basolateral membrane domains that are able to sustain the vectorial transport of ions and other solutes. Studies of the transport processes involved in Ca2+ reabsorption are therefore feasible with this system, e.g., as demonstrated in an analysis of the effects of parathyroid hormone (24) and in a recent report on the role of the Ca2+-sensing receptor in these cells (6). To date, however, there has been no detailed study examining the expression of different PMCA isoform transcripts and proteins in MDCK cells. Similarly, the cellular distribution of the PMCAs and their contribution to Ca2+ expulsion in these cells has not yet been investigated. Although previous studies indicated that all four PMCA isoforms are expressed in the rat kidney at the transcript level (13, 21), the data at the protein level suggested that neither PMCA2 nor PMCA3 is abundant pump isoforms in rat and human kidneys (36). Moreover, the presence and abundance of the second major Ca2+-expulsion system, i.e., the NCX, in the distal tubule of the kidney has been a matter of dispute (see Ref. 7 for a recent review).

Using the sensitive RT-PCR technique, we detected PMCA1b, PMCA2b, and PMCA4b in MDCK cells, but only PMCA1b and PMCA4b amplicons were readily obtained after 35 PCR cycles. By contrast, using equal amounts of input cDNA, detection of PMCA2b required 40 cycles. Because of potentially different primer and amplification efficiencies, these RT-PCR assays are not quantitative. Nevertheless, the results suggest that PMCA2 is not a major pump isoform in MDCK cells. On the other hand, the data confirm the earlier detection of PMCA2 transcripts in total rat kidney and microdissected distal convoluted tubules (13, 25). By contrast, 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 in microdissected distal convoluted tubules. Our data also fit well with a very recent report on the PMCA isoform distribution in mouse distal convoluted tubule cells (26), wherein only PMCA1b and PMCA4b were detected at the transcript level.

In agreement with the RT-PCR results, the Western blotting data showed that PMCA4 and PMCA1 are the major PMCA isoforms in MDCK cells. By contrast, full-length PMCA2 is virtually undetectable in these cells. In fact, using the high-affinity antibody NR2 (16), it was not possible to detect a band of the appropriate size in any of the membrane subfractions. Although the ~100-kDa band detected by this antibody in Western blots of total cell lysates and the membrane subfractions could potentially correspond to proteolytically truncated PMCA2, we favor the notion that this band represents an unrelated cross-reacting protein. This is supported by the absence of any distinct membrane staining when the NR2 antibody was used for immunolocalization in MDCK cells. Obviously, a quantitative comparison among PMCA1, PMCA2, and PMCA4 based solely on Western blot data is not permissible because of the different affinities and specificities of the antibodies used. However, the combined data from RT-PCR, Western blotting, and immunolocalization suggest that PMCA4 (splice variant b) is the major isoform in MDCK cell membranes. For example, the amount of full-length PMCA1, expected to run at 135-140 kDa, appeared to be lower in total plasma membrane and in the plasma membrane subfractions compared with the mature PMCA4 protein. On the other hand, potential cleavage products of PMCA1 (around 100 kDa) were more prominent in the plasma membrane, apical, and basolateral membrane subfractions than in the total cell lysates. These cleavage products might have arisen during sample handling necessary to prepare the membrane subfractions, although protease inhibitors were present throughout the procedure and similar cleavage products were not observed for PMCA4. Alternatively, the PMCA1, and potentially PMCA2, fragments in the plasma membrane subfractions may reflect physiological events taking place in these cells, because observation of such cleavage products is not a rare event for some pumps and cell types (16, 32). Regardless, given the lack of enrichment of full-length PMCA1 (and PMCA2) in the plasma membrane subfractions, PMCA4 appears to be the major pump isoform in the MDCK cell membranes.

We have previously shown that endogenous PMCA is almost exclusively localized to the basolateral membrane in polarized MDCK cells (15). These results were confirmed in the present study by using antibodies against the PMCAs and a basolateral marker protein (SAP97) for coimmunolocalization. The same conclusion was reached by Magyar et al. (26), who recently found that the PMCAs (as well as NCX1) were confined to the basolateral membrane domain in polarized mouse kidney distal tubule cells. In addition, we now provide corroborating biochemical evidence, by combining plasma membrane subfractionations with Western blot analyses. Judging from the band intensities of Western blots (see Fig. 3A, top) probed with an antibody recognizing all PMCAs, the pumps were enriched at least 10-fold in the basolateral plasma membrane of MDCK cells (1.5 µg of protein loaded/lane) compared with the amount in total cell lysates (25 µg protein/lane).

The predominantly basolateral localization of the PMCAs in polarized MDCK cells predisposes these pumps to contribute substantially to vectorial, transcellular Ca2+ transport from the apical to the basolateral side. In the distal convoluted tubule of the intact kidney, this is the major direction of Ca2+ flux during active Ca2+ reabsorption. Both PMCA and NCX have been shown to be involved in active Ca2+ reabsorption in the distal kidney (18, 44), but the relative contribution of these two mechanisms for "uphill" Ca2+ transport remains poorly understood. For example, although functional measurements indicate a large role for the NCX in the kidney distal convoluted tubule, connecting tubule, and cortical collecting duct (40, 46), immunocytochemical localization data have yielded controversial results, suggesting that NCX is only abundant in the basolateral membrane of connecting tubule cells (10, 33). On the other hand, using RT-PCR, Yu et al. (47) readily identified NCX1 in the distal convoluted tubule. Our RT-PCR and Western blot data on MDCK cells clearly show that NCX1 is expressed in these cells. Moreover, using a transcellular Ca2+ flux assay similar to that previously employed to determine Ca2+ transport in other cell types (4, 5, 17), we determined for the first time the contribution of the NCX and PMCA toward apical to basolateral Ca2+ transport in resting MDCK cells. Interestingly, about two-thirds of this transport are not due to PMCA activity but rather depend on a Na+-dependent Ca2+ exchange. The PMCA is responsible for the remaining one-third of Ca2+ flux under control conditions. These results are in excellent agreement with transcellular Ca2+ flux studies on cultured rabbit kidney cells isolated from connecting tubules and cortical collecting ducts (5). This report showed a strong dependence of transcellular Ca2+ flux on basolateral Na+, with up to 67% of transport inhibitable by isosmotic Na+ substitution. Although not shown directly, the remaining 30% of transport was suggested to be handled by a different, Na+-independent Ca2+ extrusion system, likely the PMCA (5). Although our data on the role of the PMCA for basolateral Ca2+ extrusion in MDCK cells are consistent with a recent report on the effect of the Ca2+ receptor on the PMCA in these cells (6), the major role played by the NCX (and/or additional Na+-dependent Ca2+ extrusion systems) in MDCK cells has not previously been appreciated.

Regardless of the contribution of the NCX, the significant role of the PMCA, especially of PMCA4, in vectorial Ca2+ transport was further demonstrated by overexpression and knockdown experiments in MDCK cells. The expression constructs for PMCA4b and an antisense RNA to PMCA4 have previously been used for functional overexpression and knockdown of this isoform, respectively (20, 43). Although Western blot analyses of total cell lysates from transfected MDCK cells showed only 1.2- to 1.5-fold differences of PMCA protein intensities (after standardization to actin; data not shown), the functional impact was remarkable. Overexpression of PMCA4b resulted in a 1.4-fold stimulation of Ca2+ flux, whereas antisense inhibition of PMCA4 decreased the transport by up to 47%. Because transfection efficiencies of the MDCK cells were at best 30-40%, it is clear that even moderate changes in PMCA4b expression have a significant effect on the overall transcellular Ca2+ transport capacity of these cells. Future studies using viral expression vectors to obtain virtually 100% transfection efficiencies may be needed to determine the full extent of the contribution of PMCA to transcellular Ca2+ flux in this cell system.

Finally, it should be noted that a small fraction of the PMCAs (both PMCA1 and PMCA4) were also found in the apical membrane domain of polarized MDCK cells. Although the physiological relevance of this apical fraction of the pump is not clear, it is possible that the distribution of the PMCAs between the apical and basolateral membrane domains is dynamic and that the relative distribution of the pumps among the different membrane domains is specifically regulated to meet the changing demands on transcellular Ca2+ flux. The establishment of the MDCK cell system for functional Ca2+ transport studies and the characterization of its major Ca2+ export mechanisms pave the way for detailed studies of the hormonal and pharmacological regulation of Ca2+ reabsorption (24) via the diverse Ca2+ transporting systems in this distal kidney tubule model.


    ACKNOWLEDGEMENTS

We thank Adelaida Filoteo and John T. Penniston for the gift of PMCA-specific antibodies, Craig C. Garner for the antibody against SAP97, Amy S. Lienhard for technical assistance, and Michael C. Chicka for help with the confocal microscopy.


    FOOTNOTES

This work was supported by National Institute of General Medical Sciences Grant GM-58710 and the Mayo Foundation for Medical Education and Research.

Address for reprint requests and other correspondence: E. E. Strehler, Dept. of Biochemistry and Molecular Biology, Mayo Clinic, 200 First St. SW, Rochester, MN 55905 (E-mail: strehler.emanuel{at}mayo.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

August 21, 2002;10.1152/ajprenal.00161.2002

Received 26 April 2002; accepted in final form 5 August 2002.


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