Expression and function of sodium transporters in two opossum kidney cell clonal sublines

Pedro Gomes1, Jing Xu2, Paula Serrão1, Sofia Dória3, Pedro A. Jose2, and Patrício Soares-da-Silva1

1 Institute of Pharmacology and Therapeutics and 3 Department of Genetics, Faculty of Medicine, 4200-319 Porto, Portugal; and 2 Department of Pediatrics and Department of Physiology and Biophysics, Georgetown University Medical Center, Washington, District of Columbia 20007


    ABSTRACT
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ABSTRACT
INTRODUCTION
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The present study describes characteristic features of two clonal subpopulations of opossum kidney (OK) cells (OKLC and OKHC) that are functionally different but morphologically identical. The most impressive differences between OKHC and OKLC cells are the overexpression of Na+-K+-ATPase and type 3 Na+/H+ exchanger by the former, accompanied by an increased Na+-K+-ATPase activity (57.6 ± 5.6 vs. 30.0 ± 0.1 nmol Pi · mg protein-1 · min-1); the increased ability to translocate Na+ from the apical to the basolateral surface; and the increased Na+-dependent pHi recovery (0.254 ± 0.016 vs. 0.094 ± 0.011 pH units/s). Vmax values (in pH units/s) for Na+-dependent pHi recovery in OKHC cells (0.00521 ± 0.0004) were twice (P < 0.05) those in OKLC (0.00202 ± 0.0001), with similar Km values (in mM) for Na+ (OKLC, 21.0 ± 5.5; OKHC, 14.0 ± 5.6). In addition, we measured the activities of transporters (organic ions, alpha -methyl-D-glucoside, L-type amino acids, and Na+) and enzymes (adenylyl cyclase, aromatic L-amino acid decarboxylase, and catechol-O-methyltransferase). The cells were also characterized morphologically by optical and scanning electron microscopy and karyotyped. It is suggested that OKLC and OKHC cells constitute an interesting cell model for the study of renal epithelial physiology and pathophysiology, namely, hypertension.

sodium-hydrogen exchanger; sodium-potassium adenosine 5'-triphosphatase; hypertension


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INTRODUCTION
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ESTABLISHED CELL LINES OF renal origin are frequently used for analyzing renal transport functions and their regulation. A porcine renal tubular cell line, LLC-PK1, and a canine renal tubular cell line, Madin-Darby canine kidney, are examples of well-characterized renal cell lines, which are often employed as model systems for the proximal (32-35) and distal tubules (20), respectively. However, LLC-PK1 cells, in contrast to proximal tubular epithelial cells, do not express the organic anion transporter (22), and the Na+-dependent phosphate transporter is not under the control of parathyroid hormone (PTH) or cAMP (26). In fact, LLC-PK1 cells contain few or no PTH receptors (4, 30). In contrast, opossum kidney (OK) cells, which also express renal transport systems that are characteristic of the proximal tubule, are the only renal epithelial cell line possessing high-affinity PTH receptors coupled to both the activation of adenylyl cyclase and the inhibition of Na+-dependent phosphate cotransporter (5, 7, 26, 42). Although certain properties of OK cells are consistent with a proximal tubular site of origin, this cell line was derived from the whole kidney (24). Other characteristics of OK cells, such as the presence of receptors for vasopressin, prostaglandin E1, and vasoactive intestinal peptide (5, 8, 43), suggest that the cells were derived from other regions of the nephron. Three clonal subpopulations of OK cells obtained by limiting dilution have been reported (9). These three clonal subpopulations of OK cells are morphologically and functionally distinct from the parental (OK/P) cell line.

More recently, two clonal subpopulations of OK cells (OKLC and OKHC) with origins in the same batch [F-12476 at passage 36; American Type Culture Collection (ATCC), Rockville, MD] were isolated in our laboratory. The first evidence indicating differences between the two clones of OK cells, which are morphologically identical, was of the functional type and concerned their ability to take up L-3,4-dihydrophenylalanine (L-DOPA) (13). The cells with the highest capacity to take up L-DOPA (OKHC cells) were those in which changes in transepithelial flux of Na+ more importantly affected the uptake of L-DOPA (13). Subsequently, it was also found that OKHC cells are endowed with Na+-K+-ATPase and Na+/H+ exchanger activities greater than those in OKLC cells (13). The characteristics of OKHC cells are of interest because some of these phenotypes have been described in renal proximal tubule cells from humans and rodents with genetic hypertension (10, 11, 25, 45). Salt-sensitive hypertensive patients have been suggested to take up less L-DOPA and synthesize less dopamine at the kidney level (12, 39), whereas spontaneous hypertensive rats appear to be endowed with an enhanced ability to take up L-DOPA and Na+ (36, 38, 48).

Because the relationship between the ability of renal epithelial cells to take up L-DOPA and Na+ is not yet clearly defined, especially in hypertension, it was believed worthwhile to evaluate in more detail the function and expression of Na+ transporters (Na+-K+- ATPase and Na+/H+ exchanger) in OKHC and OKLC cells. To allow a more precise characterization of OKHC and OKLC cells, we also measured the activities of other transporters [organic ions, alpha -methyl-D-glucoside (alpha -MG) and L-type amino acids] and enzymes [adenylyl cyclase, aromatic L-amino acid decarboxylase (AADC), and catechol-O-methyltransferase (COMT)]. The cells were also characterized morphologically by optical and scanning electron microscopy and karyotyped. In some of these assays, LLC-PK1 cells were used for comparison.


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Cell culture. OK cells (ATCC 1840-HTB) were maintained in a humidified atmosphere of 5% CO2-95% air at 37°C. OK cells (OKLC, passages 52-65, and OKHC cells, passages 53-74) were grown in minimal essential medium (Sigma, St. Louis, MO) supplemented with 100 U/ml penicillin G, 0.25 µg/ml amphotericin B, 100 µg/ml streptomycin (Sigma), 10% fetal bovine serum (Sigma), and 25 mM HEPES (Sigma).

LLC-PK1 cells (ATCC CRL 1392, passages 198-206) were maintained in a humidified atmosphere of 5% CO2-95% air at 37°C and grown in Medium 199 (Sigma) supplemented with 100 U/ml penicillin G, 0.25 µg/ml amphotericin B, 100 µg/ml streptomycin (Sigma), 3% fetal bovine serum (Sigma), and 25 mM HEPES (Sigma).

For subculturing, the cells were dissociated with 0.05% trypsin-EDTA, split 1:4, and subcultured in flasks with 75- or 162-cm2 growth areas (Costar, Badhoevedorp, The Netherlands). For uptake studies, the cells were seeded in collagen-treated 24-well plastic culture clusters (16-mm internal diameter; Costar) at a density of 40,000 cells/well or onto collagen-treated 0.2-µm polycarbonate filter supports (12-mm internal diameter; Transwell, Costar) at a density of 13,000 cells/well (2.0 × 104 cells/cm2). The cell medium was changed every 2 days, and the cells reached confluence after 3-5 days of incubation. For 24 h before each experiment, the cells were maintained in fetal bovine serum-free medium. Experiments were generally performed 2-3 days after cells reached confluence and 6-8 days after the initial seeding; each square centimeter contained ~80-100 µg of cell protein.

Morphology. Cells used in optical microscopy studies were cultured in plastic petri dishes with 21-cm2 growth areas (Costar). Cells were photographed with a Nikon Plan Fluor DL ×10 objective (0.30 numerical aperature) on the stage of an inverted microscope (Nikon Diaphot) at days 2 and 5 after the initial seeding.

Cells prepared for scanning electronic microscopy were cultured in collagen-coated glass coverslips (1 cm2) and fixed 24 h after plating. Cells were prepared by immersion fixation in 3.5% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.4) at room temperature for 3 h. After glutaraldehyde fixation, the specimens were washed in phosphate buffer and then treated with 1.0% osmium tetroxide for 2 h. After being washed again in phosphate buffer, the specimens were dehydrated in a graded series of ethanol. The specimens were then examined in a scanning electron microscope (model JSM-6301F, JEOL, Tokyo, Japan) at 15 keV.

Karyotype. Standard methods for air-dried slide preparations were used for karyotyping. Cultured cells were inoculated with sterile colcemide solution (10 µg/ml) and incubated for 4 h at 37°C. Thereafter, cells detached from the petri dishes with 0.4% trypsin-EDTA for 10 min at 37°C were transferred to centrifuge tubes. The cells were lysed by prewarmed (37°C) distilled water and fixed with 3:1 methanol-acetic acid solution. Several air-dried slides were prepared, and some were stained with Giemsa.

Na+/H+ exchanger activity. Na+/H+ exchanger activity was assayed as the initial rate of intracellular pH (pHi) recovery after an acid load imposed by 10 mM NH4Cl, followed by removal of Na+ from the Krebs modified buffer solution [(in mM) 140 NaCl, 5.4 KCl, 2.8 CaCl2, 1.2 MgSO4, 0.3 NaH2PO4, 0.3 KH2PO4, 10 HEPES, and 5 glucose, pH = 7.4, adjusted with Tris base] in the absence of CO2/HCO3 (15, 21). In these experiments, NaCl was replaced by an equimolar concentration of tetramethylammonium chloride. Test compounds were added to the extracellular fluid during the acidification and Na+-dependent pHi recovery periods. The concentration response relationship of the initial rate of pHi recovery for extracellular Na+ was evaluated by bathing the apical side of the monolayers with a modified Krebs-Hensleit solution over a range of Na+ concentrations from 0 to 143 mM (NaCl replaced with tetramethylammonium chloride) without affecting the concentrations of other ions.

For pHi measurement experiments, OK cells were grown in 10-mm-wide collagen-coated glass coverslips. pHi was measured as previously described (14). At days 6-8 after being seeded, the glass coverslips were incubated at 37°C for 40 min with 5 µM of 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-AM. Coverslips were then washed twice with prewarmed dye-free modified Krebs buffer before initiation of the fluorescence recordings. Cells were mounted diagonally in a 1 × 1-cm acrylic fluorometric cuvette inserted in a PerkinElmer cuvette holder (model LS 50) and subsequently placed in the sample compartment of a FluoroMax-2 spectrofluorometer (Jobin Yvon-SPEX, Edison, NJ). The cuvette volume of 3.0 ml was constantly stirred and perfused at 5.0 ml/min with modified Krebs buffer prewarmed to 37°C. Under these conditions, the cuvette medium was replaced within 150 s. After 5 min, fluorescence was measured every 5 s, alternating between 440- and 490-nm excitation (1-nm slit size) at 525-nm emission (3-nm slit size). The ratio of intracellular BCECF fluorescence at 490 and 440 nm was converted to pHi values by comparison with values from an intracellular calibration curve using the nigericin (10 µM) and high-K+ method (14).

Electrogenic ion transport. Cell monolayers were continuously monitored for changes in short-circuit current (Isc; µA/cm2) after the addition of amphotericin B to the apical-side reservoir, to increase the Na+ delivered to Na+-K+-ATPase at the half-saturating level (14, 44). Under short-circuit current conditions, the resulting current is due to the transport of Na+ across the basolateral membrane mediated by Na+-K+-ATPase, as indicated by complete inhibition of transport by ouabain (100 µM) and removal of Na+ from the medium bathing the apical side of the monolayer. OK cells grown on polycarbonate filters (Snapwell; Costar) were mounted in Ussing chambers (1.0-cm2 window area) equipped with water-jacketed gas lifts bathed on both apical and basolateral sides with 10 ml of Krebs-Hensleit solution [(in mM) 118 NaCl, 4.7 KCl, 25 NaHCO3, 1.2 KH2PO4, 2.5 CaCl2, and 1.2 MgSO4; pH was adjusted to 7.4 after gassing with 95% O2-5% CO2], gassed with 95% O2-5% CO2, and maintained at 37°C. Monolayers were continuously voltage clamped to zero potential differences by application of external current, with compensation for fluid resistance, by means of an automatic voltage-current clamp (DVC 1000; World Precision Instruments, Sarasota, FL). Transepithelial resistance (Omega  · cm2) was determined by altering the membrane potential stepwise (±3 mV) and applying the ohmic relationship. The voltage-current clamp unit was connected to a PC by means of a BIOPAC MP1000 data-acquisition system (BIOPAC Systems, Goleta, CA). Data analysis was performed by using AcqKnowledge 2.0 software (BIOPAC Systems).

Na+-K+-ATPase activity. Na+-K+-ATPase activity in OK cells was measured by the method of Quigley and Gotterer (31) with minor modifications. Briefly, OK cells in suspension were permeabilized by rapid freezing in dry ice-acetone and thawing. The reaction was initiated by the addition of 4 mM ATP. For determination of ouabain-sensitive ATPase, NaCl and KCl were omitted, and Tris · HCl (150 mM) and ouabain (100 µM) were added to the incubation medium. After incubation at 37°C for 15 min, the reaction was terminated by the addition of 50 µl of ice-cold trichloroacetic acid. Samples were centrifuged (3,000 rpm), and liberated Pi in supernatant was measured by spectrophotometry at 740 nm. Na+-K+-ATPase activity, determined as the difference between total and ouabain-insensitive ATPase, was expressed as nanomoles Pi per milligram protein per minute.

Transport of p-aminohippurate. Transport of p-aminohippurate (PAH) was initiated by adding Hanks' medium containing [3H]PAH (3 µM) to the basal side of the monolayers. [14C]sorbitol (3 µM) was used to estimate paracellular fluxes and extracellular trapping of [3H]PAH. For the measurement of transepithelial transport, the medium in the apical side was collected after incubation for the specified period of time, and the radioactivity was counted. In time course studies, an aliquot of the medium (100 µl) was collected every 15 min over a period of 60 min, and the aliquot was replaced with an equal volume of Hanks' medium. The data at 30, 45, and 60 min represent cumulative values. The monolayers were agitated every 5 min during transport measurement. In some experiments, cell monolayers were incubated in the presence of unlabeled PAH (1 mM) added from the basal side. At the end of the transport experiment, the medium was immediately aspirated, and the filter was washed three times with ice-cold Hanks' medium. Subsequently, the cells were solubilized with 0.1% vol/vol Triton X-100 (dissolved in 5 mM Tris · HCl, pH 7.4), and radioactivity was measured by liquid scintillation counting.

Transport of tetraethylammonium. Transport of tetraethylammonium (TEA) was initiated by adding Hanks' medium containing [14C]TEA (50 µM) to the basal side of the monolayers. [3H]Sorbitol (0.2 µM) was used to estimate paracellular fluxes and extracellular trapping of [14C]TEA. The transport studies were conducted similar to those described for PAH.

Transport of alpha -MG. Transport of alpha -MG was initiated by adding Hanks' medium containing alpha -[14C]MG (10 µM) to the apical side of the monolayers. [3H]sorbitol (0.2 µM) was used to estimate paracellular fluxes and extracellular trapping of alpha -[14C]MG. The transport studies were conducted in a similar fashion to those described for PAH.

Transport of L-[14C]leucine. On the day of the experiment, the growth medium was aspirated and the cells were washed with Hanks' medium; thereafter, the cell monolayers were preincubated for 15 min in Hanks' medium at 37°C. Time course studies were performed in experiments in which cells were incubated with 0.25 µM substrate for 1, 3, 6, 12, 30, and 60 min. Saturation experiments were performed in cells incubated for 6 min with 0.25 µM L-[14C]leucine in the absence and presence of increasing concentrations of L-leucine (1-3,000 µM). Test substances were only applied at the apical side and were only present during the incubation period. During preincubation and incubation, the cells were continuously shaken at 37°C. Apical uptake was initiated by the addition of 2 ml Hanks' medium with a given concentration of the substrate. Uptake was terminated by the rapid removal of uptake solution followed by a rapid wash with cold Hanks' medium and the addition of 250 µl of 0.1% vol/vol Triton X-100 (dissolved in 5 mM Tris · HCl, pH 7.4). Radioactivity was measured by liquid scintillation counting.

cAMP measurement. cAMP was determined with an enzyme immunoassay kit (Amersham Pharmacia Biotech, Little Chalfont, UK), as previously described (6). Cells were preincubated for 15 min at 37°C in Hanks' medium [(in mM) 137 NaCl, 5 KCl, 0.8 MgSO4, 0.33 Na2HPO4, 0.44 KH2PO4, 0.25 CaCl2, 1.0 MgCl2, 0.15 Tris · HCl, and 1.0 sodium butyrate, pH 7.4], containing 100 µM IBMX, a phosphodiesterase inhibitor. Cells were then incubated for 15 min with increasing concentrations of PTH (1-100 nM). At the end of the experiment, cells were lysed by the addition of 200 µl of lysis reagent. Aliquots were taken for the measurement of total cAMP content.

AADC activity. AADC activity was evaluated by the ability of cells to decarboxylate L-DOPA to dopamine, as previously described (40, 41). The growth medium was aspirated, and the cells were washed with Hanks' medium at 4°C; thereafter, the cell monolayers were preincubated for 30 min in Hank's medium at 37°C. The incubation medium contained pyridoxal phosphate (120 µM) as well as tolcapone (1 µM) and pargyline (100 µM) to inhibit the enzymes COMT and monoamine oxidase, respectively. After preincubation, cells were incubated for 6 min in Hanks' medium with increasing concentrations of L-DOPA (10-1,000 µM). The reaction was terminated by the addition of 250 µl of 0.2 M perchloric acid. The acidified samples were stored at 4°C until the assay of dopamine by HPLC with electrochemical detection.

COMT activity. COMT activity was evaluated by the ability of cells to methylate epinephrine to metanephrine, as previously described (17). The growth medium was aspirated, cells were washed with phosphate buffer (0.5 mM) at 4°C, and cell monolayers were preincubated for 30 min in phosphate buffer (0.5 mM) at 37°C. Thereafter, the cells were incubated for 30 min with increasing concentrations of adrenaline (1-300 µM) in the presence of a saturating concentration of the methyl donor (100 µM S-adenosyl-L-methionine); the incubation medium also contained pargyline (100 µM), MgCl2 (100 µM), and EGTA (1 mM). The reaction was terminated by the addition of 250 µl of 0.2 mM perchloric acid. The acidified samples are stored at 4°C until the assay of metanephrine by high-pressure liquid chromatography with electrochemical detection.

Assay of catechol derivatives. L-DOPA, dopamine, and metanephrine were quantified by means of high-pressure liquid chromatography with electrochemical detection, as previously reported (40). The HPLC system consisted of a pump (model 302, Gilson Medical Electronics, Villiers le Bel, France) connected to a manometric module (model 802 C, Gilson) and a stainless-steel 5-µm ODS column (Biophase, Bioanalytical Systems, West Lafayette, IN) of 25 cm in length; samples were injected by means of an automatic sample injector (model 231, Gilson) connected to a dilutor (model 401, Gilson). The mobile phase was a degassed solution of citric acid (0.1 mM), sodium octylsulfate (0.5 mM), sodium acetate (0.1 M), EDTA (0.17 mM), dibutylamine (1 mM), and methanol (8% vol/vol) that was adjusted to pH 3.5 with perchloric acid (2 M) and pumped at a rate of 1.0 ml/min. The detection was carried out electrochemically with a glass carbon electrode, Ag-AgCl reference electrode, and amperometric detector (model 141, Gilson); the detector cell was operated at 0.75 V. The current produced was monitored with Gilson 712 HPLC software. The lower limits for detection of L-DOPA, dopamine, and metanephrine ranged from 350 to 500 fmol.

Immunoblotting. The cells, cultured to 90% confluence, were washed with PBS two or three times, lysed by brief sonication (15 s) in PBS, and centrifuged at 20,000 g in an Eppendorf tabletop refrigerated centrifuge. The pellets were resuspended with ice-cold lysis buffer (10 mM Tris · HCl, pH 8.0, 150 mM NaCl, 1% Nonidet 40, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin and leupeptin for NHE3 studies or 10 mM Tris · HCl, pH 8.0, 150 mM NaCl, 1% Nonidet 40, 0.5% Na-deoxycholate, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin and leupeptin for Na+-K+-ATPase), sonicated briefly, and incubated on ice for 1 h. After centrifugation (14,000 rpm × 30 min; Eppendorf tabletop refrigerated centrifuge), the supernatant was mixed in 6× sample buffer (0.27 M SDS, 0.6 M dithiothreitol, 0.18 M bromphenol blue in 7 ml of 0.5 M Tris · HCl, pH 6.8, and 3 ml glycerol) and boiled for 5 min. The proteins were subjected to SDS-PAGE (8% SDS-polyacrylamide gel) and electrophoretically transferred onto nitrocellulose membranes. The transblot sheets were blocked with 5-10% nonfat dry milk in 25 mM Tris · HCl, pH 7.5, 150 mM NaCl, and 0.1% Tween 20 overnight at 4°C. Then, the membranes were incubated with appropriately diluted antibodies or antisera, and the reaction was detected by a peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and enhanced chemiluminescence (Amersham Life, Arlington Heights, IL). Specificity of the affinity-purified NHE3 antibody was determined by the use of preimmune sera or antibody preadsorbed with immunizing peptide, as previously described (45). Monoclonal antibodies to the purified rabbit alpha -subunit of Na+-K+-ATPase were obtained from Upstate Biotechnology (Lake Placid, NY). The densities of the appropriate bands were determined by using Quantiscan (Biosoft, Ferguson, MO). Protein concentration was measured with the DC protein assay kit (Bio-Rad Laboratories, Hercules, CA) and bovine serum albumin as the standard.

Drugs. S-Adenosyl-L-methionine, adrenaline, amiloride, PAH, amphotericin B, benserazide, L-DOPA, dopamine, ouabain, metanephrine, pargyline, phlorizin, and TEA were purchased from Sigma. BCECF-AM, ethylisopropylamiloride, and nigericin were obtained from Molecular Probes (Eugene, OR). Tolcapone was kindly donated by the late Professor Mosé Da Prada (Hoffman La Roche, Basel, Switzerland). alpha -[14C]MG, specific activity 316 mCi/mmol; [14C]TEA, specific activity 2.4 mCi/mmol; [3H]PAH, specific activity 3.25 Ci/mmol; [3H]sorbitol, specific activity 12.9 Ci/mmol; and [14C]sorbitol, specific activity 256 mCi/mmol, were purchased from New England Nuclear (Boston, MA). L-[14C]Leucine, specific activity 303 mCi/mmol, was purchased from Amersham.


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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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Cell morphology. Cell morphology of OKHC and OKLC cells was of the epithelial type and identical, as revealed by optical microscopy, at days 2 and 5 after the initial seeding (Fig. 1). However, their morphological appearance was markedly different from that of LLC-PK1 cells. From days 2-5, both OKHC and OKLC cells changed from an elongated oval cell shape to a polygonal cell shape, whereas LLC-PK1 cells largely maintained their oval cell shape. For scanning electron microscopy, cells were cultured in collagen-coated glass coverslips (1 cm2) and fixed 24 h after plating. As shown in Fig. 2, OKLC and OKHC cells expressed microvilli that covered most of the apical membrane. This profile is similar to that described by others in OK/P cells but differed from that in the parental cell line in which the cell population was markedly heterogeneous; some of the cells expressed apical microvilli only at cell borders (9).


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Fig. 1.   Optical photographs of opossum kidney (OK)LC (A and D), OKHC (B and E), and LLC-PK1 cell monolayers (C and F) at days 2 (A-C) and 5 (D-F) in culture.



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Fig. 2.   Scanning electron microscopy of OKLC (A-C) and OKHC (D-F) cells 24 h after seeding. Side (B and E) and top (C and F) views of apical villi are shown. Magnification ×20,000 (B, C, E, and F); ×2,500 (A and D).

Karyotype evaluation in OKLC and OKHC cells was obtained at passages 56 and 72, respectively. One or more distinctive, large metacentric chromosomes was observed. OKLC and OKHC cells showed a unimodal distribution, with cells having between 22 and 26 chromosomes. However, the majority of cells had 23 or 24 chromosomes. No significant differences in chromosomal number were observed in OKLC and OKHC cells. LLC-PK1 cells showed a unimodal distribution, with the majority of cells having 38 pairs of chromosomes.

Na+/H+ exchanger activity. Na+/H+ exchanger activity was assayed as the initial rate of pHi recovery measured after an acid load imposed by 10 mM NH4Cl followed by removal of Na+ from the Krebs modified buffer solution, in the absence of CO2/HCO3 (Fig. 3). As shown in Fig. 3, the Na+-dependent recovery of pHi in OKHC cells was steeper than that observed in OKLC cells. Table 1 depicts the pHi recovery rates (in pH units/s) during the linear phase of pHi recovery after intracellular acidification. To define whether the steeper Na+-dependent recovery of pHi in OKHC cells was related to increases in maximal activity of the transporter or enhanced affinity for Na+, pHi recovery was evaluated at increasing extracellular Na+ concentrations (0-140 mM). As shown in Fig. 4, the recovery of pHi was clearly an Na+-dependent process in both OKLC and OKHC cells. However, the maximal rate at which the pHi recovery occurred in OKHC cells was greater than that in OKLC cells. This is also evidenced by the fact that Vmax values (in pH units/s) for Na+-dependent pHi recovery in OKHC cells (0.00521 ± 0.0004) were twice (P < 0.05) those in OKLC (0.00202 ± 0.0001), with similar Km values (in mM) for Na+ (OKLC, 21.0 ± 5.5, and OKHC, 14.0 ± 5.6). The sensitivity of the Na+/H+ exchanger to inhibition by amiloride and ethylisopropyl amiloride (EIPA) was also evaluated. As indicated in Fig. 5, both amiloride and EIPA produced marked inhibition of Na+/H+ exchanger activity in OKLC and OKHC cells, but EIPA is considerably more potent than amiloride. Differences in IC50 values for inhibition of Na+/H+ exchanger activity by amiloride and EIPA between OKLC [amiloride, IC50 =48 (26, 89) µM; EIPA, IC50 =1.8 (0.7, 4.8) µM] and OKHC cells [amiloride, IC50 =125 (46, 339) µM; EIPA, IC50 =1.9 (1.0, 3.6) µM] failed to attain statistical significance. Differences in sensitivity to amiloride and EIPA are in agreement with the observation that OK cells mainly express the type 3 Na+/H+ exchanger (NHE3) (29).


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Fig. 3.   Assessment of Na+/H+ exchanger activity under Vmax conditions as the initial rate of Na+-dependent intracellular pH recovery after an acid load imposed by exposure to NH4Cl followed by Na+ removal of the perfusion medium in OKLC (A) and OKHC cells (B). Values are means of 5 experiments. TMA, tetramethylammonium chloride.


                              
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Table 1.   Na+/H+ and Na+-K+-ATPase activities in OKLC and OKHC cells



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Fig. 4.   Na+ dependence of Na+/H+ exchanger activity in OKLC and OKHC cells. Values are means ± SE of 8 experiments/group.



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Fig. 5.   Concentration-dependent effect of amiloride and ethylisopropylamiloride (EIPA) on Na+/H+ exchanger activity in OKLC and OKHC cells. Values are means ± SE of 4-8 experiments/group.

Na+-K+-ATPase activity. To study Na+-K+-ATPase activity in OK cells, it was decided to use an eletrophysiological method in which cell monolayers were continuously monitored for changes in Isc after the addition of amphotericin B to the apical cell side, to increase the Na+ delivered to Na+-K+-ATPase to the saturating level. As shown in Fig. 6, addition of amphotericin B increased Isc in a concentration-dependent manner. This effect is due to the transport of Na+ across the basolateral membrane mediated by Na+-K+-ATPase, as indicated by complete inhibition of activity by ouabain (100 µM) and removal of Na+ from medium bathing the apical side of the monolayer (14). As shown in Fig. 6, the amphotericin B-induced increase in Isc was greater in OKHC cells than in OKLC cells. To confirm that the difference in the amphotericin B-induced increase in Isc between OKHC and OKLC cells corresponded to a difference in Na+-K+-ATPase activity, the enzyme was assayed with the use of a biochemical method. Basal Na+-K+-ATPase activity was significantly greater (P < 0.05) in OKHC cells than in OKLC cells (Table 1). In some experiments, amphotericin B (0.25 µg/ml) was omitted from the culture medium, but this did not affect the increase in Isc by amphotericin B or the Na+-dependent recovery of pHi in OKHC and OKLC cells (data not shown).


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Fig. 6.   Amphotericin B-induced increases in short-circuit current (Isc, µA/cm2) across monolayers of OKLC and OKHC cells mounted in Ussing chambers. Values are means ± SE of 6-10 experiments/group. * P < 0.05 vs. OKLC cells.

Immunoblotting. Because there were marked differences in Na+/H+ exchanger and Na+-K+-ATPase activities between OKLC and OKHC cells, it was decided to quantify the abundance of both proteins by means of Western blotting. The presence of the Na+/H+ exchanger was performed by using an antibody raised against the rat NHE3 (25, 45). As shown in Fig. 7, this antibody recognizes the presence of NHE3 in cell membranes from both OKLC and OKHC cells. In agreement with the functional data, the abundance of NHE3 in cell membranes was greater in OKHC than in OKLC cells; the relative density (% area) of the bands in three independent experiments was 58 ± 2 and 42 ± 1 in OKHC and OKLC cells, respectively. The presence of Na+-K+-ATPase was also evaluated in cell membranes from OKLC and OKHC cells. As shown in Fig. 7B, the antibody raised against the alpha -subunit of rabbit Na+-K+-ATPase revealed the presence of Na+-K+ ATPase in cell membranes. The abundance of Na+-K+-ATPase in cell membranes was greater in OKHC than in OKLC cells, with relative density of the bands of 70 ± 3 and 30 ± 3%, respectively.


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Fig. 7.   Abundance of type 3 Na+/H+ exchanger (NHE3; A)and Na+-K+-ATPase (B) in membrances of OKLC and OKHC cells. Each lane contains equal amounts of protein (50 µg). Western blot analysis was repeated 2-3 times. Values are means ± SE of 3-4 separate experiments. Columns, relative density. * P < 0.05 vs. OKLC cells.

PTH. High-affinity PTH receptors have been identified in OK cells (43), and occupancy of the receptors by PTH produces concentration-dependent activation of adenylyl cyclase (5, 7, 26). We compared the ability of PTH to stimulate cAMP accumulation in OKLC, OKHC, and LLC-PK1 cells. As shown in Fig. 8, the accumulation of cAMP cells was markedly higher in OKHC cells (Emax= 1,402 ± 84 fmol/well) than in OKLC (Emax= 301 ± 11 fmol/well); LLC-PK1 cells were unresponsive to PTH. The EC50 values for cAMP accumulation by PTH in OKHC cells [10.2 (3.1, 33.7) nM] and OKLC cells [5.4 (1.3, 23.4) nM] were similar to that described in OK/P cells (9). The forskolin-stimulated increase in cAMP accumulation was of similar magnitude in all three types of cells (Fig. 8B).


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Fig. 8.   Effect of parathyroid hormone (PTH; A) and forskolin (B) on cAMP accumulation in OKLC, OKHC, and LLC-PK1 cells. Values are means ± SE of 4 separate experiments. * P < 0.05 vs. corresponding controls.

PAH. The transepithelial transport and accumulation of [3H]PAH in LLC-PK1 and OKHC cells were close to those of [14C]sorbitol (data not shown), indicating that the apparent accumulation and transport of [3H]PAH represented nonspecific transfer and/or trapping (Fig. 9). The basal-to-apical transport and cell accumulation of [3H]PAH and [14C]sorbitol were not affected by unlabeled PAH (Fig. 9). By contrast, OKLC cells transported [3H]PAH quite efficiently, and both the cell accumulation and transport were markedly (P < 0.05) reduced by nonlabeled PAH (Fig. 9).


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Fig. 9.   Effect of unlabeled p-aminohippurate (PAH; 1 mM) on cell accumulation (A) and basal-to-apical transport (B) of 3 µM [3H]PAH across OKLC, OKHC, and LLC-PK1 cell monolayers. Values are means ± SE of 4 separate experiments. * P < 0.05 vs. corresponding controls.

TEA. The transport of TEA was initiated by adding Hanks' medium containing [14C]TEA (50 µM) to the basal side of the monolayers. As shown in Fig. 10, the accumulation of [14C]TEA in LLC-PK1 cells was lower than in OK cells. By contrast, the transport of [14C]TEA (in pmol · cm-2 · min-1) was considerably greater (P < 0.05) in LLC-PK1 cells (84.3 ± 4.1) than in OK cells (OKLC, 27.0 ± 9.1, and OKHC, 44.7 ± 3.8). The addition of nonlabeled TEA (2.5 mM) markedly reduced the accumulation of [14C]TEA in all three types of cells but failed to affect the basal-to-apical transport of [14C]TEA.


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Fig. 10.   Effect of unlabeled tetraethylammonium (TEA; 2.5 mM) on cell accumulation (A) and basal-to-apical transports (B) of 50 µM [14C]TEA across OKLC, OKHC, and LLC-PK1 cell monolayers. Values are means ± SE of 4 separate experiments. * P < 0.05 vs. corresponding controls.

alpha -MG. The transport of alpha -MG was initiated by adding Hanks' medium containing alpha -[14C]MG (10 µM) to the apical side of the monolayers. As shown in Fig. 11, the transport of alpha -[14C]MG (in pmol · cm-2 · min-1) was considerably greater (P < 0.05) in LLC-PK1 cells (58.5 ± 9.8) than in OK cells (OKLC, 3.8 ± 0.1, and OKHC, 9.0 ± 0.7). The addition of phlorizin markedly reduced both the accumulation and transport of alpha -[14C]MG in LLC-PK1 cells only.


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Fig. 11.   Effect of phlorizin (50 µM) on cell accumulation (A) and apical-to-basal transport (B) of 10 µM alpha -methyl-D-[14C]glucoside across OKLC, OKHC, and LLC-PK1 cell monolayers. Values are means ± SE of 4 separate experiments. * P < 0.05 vs. corresponding controls.

L-Leucine uptake. To determine initial rates of L-leucine uptake, cells were incubated with a nonsaturating (0.25 µM) concentration of L-[14C]leucine for 1, 3, 6, 12, 30, and 60 min. In all three types of cells, uptake of a nonsaturating concentration of the substrate was linear with time for up to 30 min of incubation (Fig. 12A). In a subsequent set of experiments designed to determine the kinetics of the L-type amino acid transporter, cells were incubated for 6 min with L-[14C]leucine (0.25 µM) in the absence or presence of increasing concentrations (1-3,000 µM) of nonlabeled L-leucine (Fig. 12B). Kinetic parameters of L-[14C] leucine uptake (Km and Vmax) were determined by nonlinear analysis of inhibition curves for L-leucine and are given in Table 2. As shown in the table, the affinity of the transporter for L-leucine was higher in LLC-PK1 cells, as evidenced by lower Km values.


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Fig. 12.   Time course (A) and concentration-dependent accumulation (B) of L-[14C]leucine in OKLC, OKHC, and LLC-PK1 cells. In time course studies, cells were incubated at 37°C with 0.25 µM L-[14C]leucine applied from the apical cell border; whereas, in saturation experiments, cells were loaded with 0.25 µM L-[14C]leucine plus increasing concentrations (10-3,000 µM) of the nonlabeled substrate for 6 min. Values are means ± SE of 4 experiments/group.


                              
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Table 2.   Km and Vmax values for saturable component of L-leucine uptake in OKLC, OKHC, and LLC-PK1 cells

AADC activity was determined in cells incubated with L-DOPA (10-1,000 µM). The decarboxylation of L-DOPA into dopamine was found to be linear up to 100 µM L-DOPA and became saturated at high concentrations of the substrate (250-1,000 µM L-DOPA). AADC activity was significantly higher in LLC-PK1 cells than in OK cells. Kinetic parameters of the saturation curves are given in Table 3.

                              
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Table 3.   Kinetic parameters of aromatic L-aminoacid decarboxylase activity in OKLC, OKHC, and LLC-PK1 cells

COMT activity was determined by the ability of cells to convert adrenaline (1-300 µM) into metanephrine. The highest COMT activity was found in OKLC cells, followed by OKHC and LLC-PK1 cells. Kinetic parameters of the saturation curves are given in Table 4.

                              
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Table 4.   Kinetic parameters of catechol-O-methyltransferase activity in monolayers of OKLC, OKHC, and LLC-PK1 cells


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present study describes characteristic features of two clonal subpopulations of OK cells (OKLC and OKHC) that are functionally different but morphologically identical. The most impressive difference between OKHC and OKLC cells was that the former overexpressed Na+-K+-ATPase accompanied by increased Na+-K+-ATPase activity and increased ability to translocate Na+ from the apical to the basolateral cell side. This feature was accompanied by increased expression and activity of the Na+/H+ exchanger, as assessed by Na+-dependent pHi recovery measured after an acid load. Other important differences between these two clonal subpopulations concerned their ability to respond to PTH and transport PAH.

Morphologically, OKHC and OKLC cells were identical at both day 2 and day 5 after initial seeding, as observed by optical microscopy. However, both OKHC and OKLC cells changed from an elongated oval cell shape at day 2 to a polygonal cell shape at day 5 and expressed microvilli that covered most of the apical membrane. The morphological appearance and karyotype in OKHC and OKLC cells were markedly different from those of LLC-PK1 cells. The latter cells maintained their oval cell shape at confluence and had the expected number of chromosomes (38 pairs) in the species of origin (the pig).

Despite the morphological similarity between OKLC and OKHC cells, the most interesting aspect concerns differences in Na+ handling. Na+/H+ exchanger and Na+-K+-ATPase activities and expression in OKHC cells were markedly higher than in OKLC cells. It is not apparent from these studies of the relationship between the two events; i.e., it is not clear whether the increase in Na+-K+-ATPase activity in OKHC cells is related to increases in the ability to take up Na+ from the apical cell border through the Na+/H+ exchanger. Na+-K+-ATPase in the basolateral domain of epithelial cells provides the driving force for active Na+ and K+ translocation and for the secondary active transport of other solutes across the renal tubules (3). Transient increases in intracellular Na+ in OK cells were found to result in inhibition of the Na+/H+ exchanger (14). However, inhibition of the Na+/H+ exchanger reduced intracellular Na+, which was accompanied by decreases in Na+-K+-ATPase activity (14). Thus increased basolateral Na+-K+-ATPase activity may be responsible for the increases in apical-to-basal Na+ flux and increased Na+/H+ exchange in OKHC cells. Increased activity and overexpression of the Na+/H+ exchanger after acid stress or proton suicide have been described in renal cells, namely, LLC-PK1 (19, 37) and OK cells (28, 46). Other maneuvers that also cause increases in Na+/H+ exchanger activity and expression in OK/P cells include chronic hypertonicity (1) and incubation in a low-K+ medium (2). More recently, two mechanisms have been suggested to cause acid-induced increases in NHE3 activity (47). Initially, there is an increase in apical membrane NHE3 that is due to stimulated exocytic insertion and is required for increased Na+/H+ exchanger activity. At a later stage, there is an additional increase in total cellular NHE3 (47). In this respect, it is interesting to observe that OKHC cells express more NHE3 and are endowed with greater Na+/H+ exchanger activity than OKLC cells. It should be stressed that the enhanced Na+/H+ exchanger activity in OKHC cells is not accompanied by differences in the affinity for Na+ or sensitivity to inhibition by amiloride and EIPA. Considering that the expression of Na+-K+-ATPase in OKHC cells was greater than in OKLC cells, it is likely that changes in Na+/H+ exchanger activity in the former cell type are a consequence of enhanced Na+-K+-ATPase expression and activity. The enhanced transport of Na+ in OKHC cells resembles that observed in renal tubular cells from spontaneously hypertensive rats (10, 11, 25, 45). OKHC cells are also endowed with the highest capacity to take up L-DOPA (13), a particularity that is also observed in spontaneously hypertensive rats (36, 38, 48). Altogether, it is suggested that these cell lines may be of value in studying the association between enhanced Na+ handling and the formation of dopamine, an issue of particular relevance in hypertension.

The purpose of the subsequent functional characterization performed on OKLC and OKHC cells was basically to explore some of the unique functional characteristics attributed to OK cells, such as responses to PTH and transport of organic anions, organic cations, carbohydrates, and amino acids. High-affinity PTH receptors coupled to adenylyl cyclase (5, 7, 26) and the transporter for organic anions are present in OK cells (22), whereas the transporter for organic cations is present in LLC-PK1 cells. Indeed, LLC-PK1 cells contain no or few PTH receptors (4, 30). In addition, LLC-PK1 cells, in contrast to proximal tubular epithelial cells and OK cells, do not express the organic anion transporter (22), and the Na+-dependent phosphate transport is not under the control of PTH or cAMP (26). By contrast, LLC-PK1 cells are endowed with the H+/organic cation antiport system (23), the primary structure and functional expression of which have been recently reported (16). The data presented here on these three features are in agreement with those in the literature; LLC-PK1 cells do not increase cAMP accumulation in response to PTH and do not transport the organic anion PAH but were able to transport the organic cation TEA. Both OKLC and OKHC cells were found to transport the organic cation TEA, the magnitude of which was similar to that observed in LLC-PK1 cells. However, the data obtained on response to PTH and transport of PAH in OK cells reveal some heterogeneity. Only OKLC cells were able to transport the organic anion PAH. Although the response of OKHC cells to PTH was greater than in OKLC cells, the affinity of PTH receptors for the agonist was of similar magnitude in both cell lines. Indeed, EC50 values for cAMP accumulation by PTH in OKHC cells [10.2 (3.1, 33.7) nM] did not differ from those in OKLC cells [5.4 (1.3, 23.4) nM] and were similar to those described in OK/P cells (3.0 ± 0.7 nM) (9). On the other hand, both OKLC and OKHC cells were found to respond to forskolin with increases in cAMP, the magnitude of the responses being similar in all three cell lines. These findings are in agreement with those reported in the literature while showing that there is some heterogeneity in the responses of clonal subpopulations of OK cells to PTH (9). It has been suggested that some of these clonal subpopulations might have a defective coupling of the PTH receptor to adenylyl cyclase (9). The finding that LLC-PK1 cells do not respond to PTH is in agreement with the suggestion that these cells may not express PTH receptors (4, 30). Another finding in agreement with the view that OK cells may constitute a heterogeneous population is the apical transport of alpha -MG. In contrast to that reported for OK cells (27), both OKLC and OKHC cells were found to transport alpha -MG in a phlorizin-insensitive manner. On the other hand, the phlorizin-sensitive apical transport of alpha -MG in LLC-PK1 cells reported here was similar to that described earlier (33). Another major difference between LLC-PK1 and OK cells concerned the transport of the neutral amino acid L-leucine. The transport of L-[14C]leucine in both OKLC and OKHC cells was greater than in LLC-PK1 cells. However, the affinity of the leucine transporter was slightly higher in LLC-PK1 cells than in OK cells. No differences in L-[14C]leucine accumulation were observed between OKLC and OKHC cells. Similarly, no significant differences in AADC and COMT activities were observed between OKLC and OKHC cells. However, the differences in AADC and COMT activities between LLC-PK1 and OK cells are in agreement with that described in the literature. OK cells are endowed with low AADC activity (41), and the affinity of COMT for the substrate is greater in OK cells than in pig kidneys (18). Altogether, our results indicate clear differences between OK and LLC-PK1 cells, some of which may be related to species difference. Another aspect that emerges from these studies concerns the marked differences between OKLC and OKHC cells, which may be related to the heterogeneity among OK cells and their propensity to give rise to clonal subpopulations on the basis of limiting dilution processes (9).

In conclusion, we have isolated and characterized two clonal subpopulations of OK cells that are morphologically similar but clearly exhibit different functional properties. The most impressive difference between OKHC and OKLC cells is that the former overexpress Na+-K+-ATPase and NHE3, accompanied by similar increases in Na+-K+ ATPase and Na+/H+ exchanger activities. Other differences concern their ability to respond to PTH and transport PAH. These cell lines may be valuable in studying the association between enhanced Na+ handling and the formation of dopamine, an issue of particular relevance in hypertension.


    ACKNOWLEDGEMENTS

This study was supported by Foundation for Science and Technology (Portugal) Grant POCTI/35474/FCB/2000 and National Institutes of Health Grants DK-39308 and HL-23081.


    FOOTNOTES

Address for reprint requests and other correspondence: P. Soares-da-Silva, Institute of Pharmacology and Therapeutics, Faculty of Medicine, 4200-319 Porto, Portugal (E-mail: patricio.soares{at}mail.telepac.pt).

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.

First published January 29, 2002;10.1152/ajprenal.00340.2001

Received 13 November 2001; accepted in final form 25 January 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Ambuhl, P, Amemiya M, Preisig PA, Moe OW, and Alpern RJ. Chronic hyperosmolality increases NHE3 activity in OKP cells. J Clin Invest 101: 170-177, 1998[Abstract/Free Full Text].

2.   Amemiya, M, Tabei K, Kusano E, Asano Y, and Alpern RJ. Incubation of OKP cells in low-K+ media increases NHE3 activity after early decrease in intracellular pH. Am J Physiol Cell Physiol 276: C711-C716, 1999[Abstract/Free Full Text].

3.   Bertorello, AM, and Katz AI. Short-term regulation of renal Na-K-ATPase activity: physiological relevance and cellular mechanisms. Am J Physiol Renal Fluid Electrolyte Physiol 265: F743-F755, 1993[Abstract/Free Full Text].

4.   Bringhurst, FR, Juppner H, Guo J, Urena P, Potts JT, Jr, Kronenberg HM, Abou-Samra AB, and Segre GV. Cloned, stably expressed parathyroid hormone (PTH)/PTH-related peptide receptors activate multiple messenger signals and biological responses in LLC-PK1 kidney cells. Endocrinology 132: 2090-2098, 1993[Abstract].

5.   Caverzasio, J, Rizzoli R, and Bonjour JP. Sodium-dependent phosphate transport inhibited by parathyroid hormone and cyclic AMP stimulation in an opossum kidney cell line. J Biol Chem 261: 3233-3237, 1986[Abstract/Free Full Text].

6.   Cheng, L, Precht P, Frank D, and Liang CT. Dopamine stimulation of cAMP production in cultured opossum kidney cells. Am J Physiol Renal Fluid Electrolyte Physiol 258: F877-F882, 1990[Abstract/Free Full Text].

7.   Cole, JA, Eber SL, Poelling RE, Thorne PK, and Forte LR. A dual mechanism for regulation of kidney phosphate transport by parathyroid hormone. Am J Physiol Endocrinol Metab 253: E221-E227, 1987[Abstract/Free Full Text].

8.   Cole, JA, Forte LR, Eber S, Thorne PK, and Poelling RE. Regulation of sodium-dependent phosphate transport by parathyroid hormone in opossum kidney cells: adenosine 3',5'-monophosphate-dependent and -independent mechanisms. Endocrinology 122: 2981-2989, 1988[Abstract].

9.   Cole, JA, Forte LR, Krause WJ, and Thorne PK. Clonal sublines that are morphologically and functionally distinct from parental OK cells. Am J Physiol Renal Fluid Electrolyte Physiol 256: F672-F679, 1989[Abstract/Free Full Text].

10.   Dagher, G, and Sauterey C. H+ pump and Na+-H+ exchange in isolated single proximal tubules of spontaneously hypertensive rats. J Hypertens 10: 969-978, 1992[ISI][Medline].

11.  Garg LC and Narang N. Differences in renal tubular Na-K-adenosine triphosphatase in spontaneously hypertensive and normotensive rats. J Cardiovasc Pharmacol: 186-189, 1986.

12.   Gill, JR, Jr, Gullner HG, Lake R, Lakatua D, and Lan G. Plasma and urinary catecholamines in salt-sensitive idiopathic hypertension. Hypertension 11: 312-319, 1988[Abstract].

13.   Gomes, P, and Soares-da-Silva P. Transepithelial flux of sodium and handling of L-DOPA in renal epithelial cells. FASEB J 14: A349, 2000[ISI].

14.   Gomes, P, Vieira-Coelho MA, and Soares-da-Silva P. Ouabain-insensitive acidification by dopamine in renal OK cells: primary control of the Na+/H+ exchanger. Am J Physiol Regulatory Integrative Comp Physiol 281: R10-R18, 2001[Abstract/Free Full Text].

15.   Gore, J, and Hoinard C. Na+/H+ exchange in isolated hamster enterocytes. Its major role in intracellular pH regulation. Gastroenterology 97: 882-887, 1989[ISI][Medline].

16.   Grundemann, D, Babin-Ebell J, Martel F, Ording N, Schmidt A, and Schomig E. Primary structure and functional expression of the apical organic cation transporter from kidney epithelial LLC-PK1 cells. J Biol Chem 272: 10408-10413, 1997[Abstract/Free Full Text].

17.   Guimaraes, JT, Vieira-Coelho MA, Serrão MP, and Soares-da-Silva P. Opossum kidney (OK) cells in culture synthesize and degrade the natriuretic hormone dopamine: a comparison with rat renal tubular cells. Int J Biochem Cell Biol 29: 681-688, 1997[ISI][Medline].

18.   Guldberg, H, and Marsden C. Catechol-O-methyl transferase: pharmacological aspects and physiological role. Pharmacol Rev 27: 135-206, 1975[ISI][Medline].

19.   Haggerty, JG, Agarwal N, Cragoe EJ, Jr, Adelberg EA, and Slayman CW. LLC-PK1 mutant with increased Na+-H+ exchange and decreased sensitivity to amiloride. Am J Physiol Cell Physiol 255: C495-C501, 1988[Abstract/Free Full Text].

20.   Herzlinger, DA, Easton TG, and Ojakian GK. The MDCK epithelial cell line expresses a cell surface antigen of the kidney distal tubule. J Cell Biol 93: 269-277, 1982[Abstract].

21.   Hoinard, C, and Gore J. Cytoplasmic pH in isolated rat enterocytes. Role of Na+/H+ exchanger. Biochim Biophys Acta 941: 111-118, 1988[ISI][Medline].

22.   Hori, R, Okamura M, Takayama A, Hirozane K, and Takano M. Transport of organic anion in the OK kidney epithelial cell line. Am J Physiol Renal Fluid Electrolyte Physiol 264: F975-F980, 1993[Abstract/Free Full Text].

23.   Inui, K, Saito H, and Hori R. H+-gradient-dependent active transport of tetraethylammonium cation in apical-membrane vesicles isolated from kidney epithelial cell line LLC-PK1. Biochem J 227: 199-203, 1985[ISI][Medline].

24.   Koyama, H, Goodpasture C, Miller MM, Teplitz RL, and Riggs AD. Establishment and characterization of a cell line from the American opossum (Didelphys virginiana). In Vitro 14: 239-246, 1978[ISI][Medline].

25.   Li, XX, Xu J, Zheng S, Albrecht FE, Robillard JE, Eisner GM, and Jose PA. D1 dopamine receptor regulation of NHE3 during development in spontaneously hypertensive rats. Am J Physiol Regulatory Integrative Comp Physiol 280: R1650-R1656, 2001[Abstract/Free Full Text].

26.   Malmstrom, K, and Murer H. Parathyroid hormone inhibits phosphate transport in OK cells but not in LLC-PK1 and JTC-12.P3 cells. Am J Physiol Cell Physiol 251: C23-C31, 1986[Abstract/Free Full Text].

27.   Malstrom, K, Stange G, and Murer H. Identification of proximal tubular transport functions in the established kidney cell line, OK. Biochim Biophys Acta 902: 269-277, 1987[ISI][Medline].

28.   Moe, OW, Miller RT, Horie S, Cano A, Preisig PA, and Alpern RJ. Differential regulation of Na/H antiporter by acid in renal epithelial cells and fibroblasts. J Clin Invest 88: 1703-1708, 1991[ISI][Medline].

29.   Noel, J, and Pouyssegur J. Hormonal regulation, pharmacology, and membrane sorting of vertebrate Na+/H+ exchanger isoforms. Am J Physiol Cell Physiol 268: C283-C296, 1995[Abstract/Free Full Text].

30.   Pizurki, L, Rizzoli R, Moseley J, Martin TJ, Caverzasio J, and Bonjour JP. Effect of synthetic tumoral PTH-related peptide on cAMP production and Na-dependent Pi transport. Am J Physiol Renal Fluid Electrolyte Physiol 255: F957-F961, 1988[Abstract/Free Full Text].

31.   Quigley, JP, and Gotterer GS. Distribution of Na,K-stimulated ATPase activity in rat intestinal mucosa. Biochim Biophys Acta 173: 456-468, 1969[ISI][Medline].

32.   Rabito, CA. Phosphate uptake by a kidney cell line (LLC-PK1). Am J Physiol Renal Fluid Electrolyte Physiol 245: F22-F31, 1983[Abstract/Free Full Text].

33.   Rabito, CA, and Ausiello DA. Na+-dependent sugar transport in a cultured epithelial cell line from pig kidney. J Membr Biol 54: 31-38, 1980[ISI][Medline].

34.   Rabito, CA, and Karish MV. Polarized amino acid transport by an epithelial cell line of renal origin (LLC-PK1). The basolateral systems. J Biol Chem 257: 6802-6808, 1982[Abstract/Free Full Text].

35.   Rabito, CA, and Karish MV. Polarized amino acid transport by an epithelial cell line of renal origin (LLC-PK1). The apical systems. J Biol Chem 258: 2543-2547, 1983[Abstract/Free Full Text].

36.   Racz, K, Kuchel O, Buu NT, and Tenneson S. Peripheral dopamine synthesis and metabolism in spontaneously hypertensive rats. Circ Res 57: 889-897, 1985[Abstract].

37.   Reilly, RF, Haggerty JG, Aronson PS, Adelberg EA, and Slayman CW. Increased Na+-H+ antiporter activity in apical membrane vesicles from mutant LLC-PK1 cells. Am J Physiol Cell Physiol 260: C738-C744, 1991[Abstract/Free Full Text].

38.   Sanada, H, Watanabe H, Shigetomi S, and Fukuchi S. Gene expression of aromatic L-amino acid decarboxylase mRNA in the kidney of normotensive and hypertensive rats. Hypertens Res 18, Suppl1: S179-S181, 1995[Medline].

39.   Soares-da-Silva, P, Pestana M, Ferreira A, Damasceno A, Polonia J, and Cerqueira-Gomes M. Renal dopaminergic mechanisms in renal parenchymal diseases, hypertension, and heart failure. Clin Exp Hypertens 22: 251-268, 2000[ISI][Medline].

40.   Soares-da-Silva, P, Serrão MP, and Vieira-Coelho MA. Apical and basolateral uptake and intracellular fate of dopamine precursor L-dopa in LLC-PK1 cells. Am J Physiol Renal Physiol 274: F243-F251, 1998[Abstract/Free Full Text].

41.   Soares-da-Silva, P, Vieira-Coelho MA, and Serrão MP. Uptake of L-3,4-dihydroxyphenylalanine and dopamine formation in cultured renal epithelial cells. Biochem Pharmacol 54: 1037-1046, 1997[ISI][Medline].

42.   Teitelbaum, AP, Nissenson RA, Zitzner LA, and Simon K. Dual regulation of PTH-stimulated adenylate cyclase activity by GTP. Am J Physiol Renal Fluid Electrolyte Physiol 251: F858-F864, 1986[Abstract/Free Full Text].

43.   Teitelbaum, AP, and Strewler GJ. Parathyroid hormone receptors coupled to cyclic adenosine monophosphate formation in an established renal cell line. Endocrinology 114: 980-985, 1984[Abstract].

44.   Vieira-Coelho, MA, Gomes P, Serrão MP, and Soares-da-Silva P. D1-like dopamine receptor activation and natriuresis by nitrocatechol COMT inhibitors. Kidney Int 59: 1683-1694, 2001[ISI][Medline].

45.   Xu, J, Li XX, Albrecht FE, Hopfer U, Carey RM, and Jose PA. Dopamine(1) receptor, G(salpha), and Na(+)-H(+) exchanger interactions in the kidney in hypertension. Hypertension 36: 395-399, 2000[Abstract/Free Full Text].

46.   Yamaji, Y, Amemiya M, Cano A, Preisig PA, Miller RT, Moe OW, and Alpern RJ. Overexpression of csk inhibits acid-induced activation of NHE-3. Proc Natl Acad Sci USA 92: 6274-6278, 1995[Abstract].

47.   Yang, X, Amemiya M, Peng Y, Moe OW, Preisig PA, and Alpern RJ. Acid incubation causes exocytic insertion of NHE3 in OKP cells. Am J Physiol Cell Physiol 279: C410-C419, 2000[Abstract/Free Full Text].

48.   Yoshimura, M, Ikegaki I, Nishimura M, and Takahashi H. Role of dopaminergic mechanisms in the kidney for the pathogenesis of hypertension. J Auton Pharmacol 10: s67-s72, 1990[ISI][Medline].


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