INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

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 (OK_LC and OK_HC) 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 (OK_HC 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 OK_HC cells are endowed with Na⁺-K⁺-ATPase and Na⁺/H⁺ exchanger activities greater than those in OK_LC cells (13). The characteristics of OK_HC 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 OK_HC and OK_LC cells. To allow a more precise characterization of OK_HC and OK_LC cells, we also measured the activities of other transporters [organic ions, -methyl-D-glucoside (-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.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Cell culture. OK cells (ATCC 1840-HTB) were maintained in a humidified atmosphere of 5% CO₂-95% air at 37°C. OK cells (OK_LC, passages 52-65, and OK_HC 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).

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.

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 (pH_i) 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 pH_i recovery periods. The concentration response relationship of the initial rate of pH_i 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.

Electrogenic ion transport. Cell monolayers were continuously monitored for changes in short-circuit current (I_sc; 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 ( · 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 P_i 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 P_i 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 -MG. Transport of -MG was initiated by adding Hanks' medium containing -[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 -[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 -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). -[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.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Cell morphology. Cell morphology of OK_HC and OK_LC 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 OK_HC and OK_LC 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, OK_LC and OK_HC 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).

View larger version (155K): [in this window] [in a new window]   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.

View larger version (167K): [in this window] [in a new window]   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).

Na+/H⁺ exchanger activity. Na⁺/H⁺ exchanger activity was assayed as the initial rate of pH_i 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 pH_i in OK_HC cells was steeper than that observed in OK_LC cells. Table 1 depicts the pH_i recovery rates (in pH units/s) during the linear phase of pH_i recovery after intracellular acidification. To define whether the steeper Na⁺-dependent recovery of pH_i in OK_HC cells was related to increases in maximal activity of the transporter or enhanced affinity for Na⁺, pH_i recovery was evaluated at increasing extracellular Na⁺ concentrations (0-140 mM). As shown in Fig. 4, the recovery of pH_i was clearly an Na⁺-dependent process in both OK_LC and OK_HC cells. However, the maximal rate at which the pH_i recovery occurred in OK_HC cells was greater than that in OK_LC cells. This is also evidenced by the fact that V_max values (in pH units/s) for Na⁺-dependent pH_i recovery in OK_HC cells (0.00521 ± 0.0004) were twice (P < 0.05) those in OK_LC (0.00202 ± 0.0001), with similar K_m values (in mM) for Na⁺ (OK_LC, 21.0 ± 5.5, and OK_HC, 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 OK_LC and OK_HC 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 OK_LC [amiloride, IC₅₀ =48 (26, 89) µM; EIPA, IC₅₀ =1.8 (0.7, 4.8) µM] and OK_HC 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).

View larger version (11K): [in this window] [in a new window]   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.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Na+ dependence of Na+/H+ exchanger activity in OKLC and OKHC cells. Values are means ± SE of 8 experiments/group.

View larger version (16K): [in this window] [in a new window]   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 I_sc 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 I_sc 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 I_sc was greater in OK_HC cells than in OK_LC cells. To confirm that the difference in the amphotericin B-induced increase in I_sc between OK_HC and OK_LC 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 OK_HC cells than in OK_LC 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 I_sc by amphotericin B or the Na⁺-dependent recovery of pH_i in OK_HC and OK_LC cells (data not shown).

View larger version (14K): [in this window] [in a new window]   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 OK_LC and OK_HC 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 OK_LC and OK_HC cells. In agreement with the functional data, the abundance of NHE3 in cell membranes was greater in OK_HC than in OK_LC cells; the relative density (% area) of the bands in three independent experiments was 58 ± 2 and 42 ± 1 in OK_HC and OK_LC cells, respectively. The presence of Na⁺-K⁺-ATPase was also evaluated in cell membranes from OK_LC and OK_HC cells. As shown in Fig. 7B, the antibody raised against the -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 OK_HC than in OK_LC cells, with relative density of the bands of 70 ± 3 and 30 ± 3%, respectively.

View larger version (14K): [in this window] [in a new window]   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 OK_LC, OK_HC, and LLC-PK₁ cells. As shown in Fig. 8, the accumulation of cAMP cells was markedly higher in OK_HC cells (E_max= 1,402 ± 84 fmol/well) than in OK_LC (E_max= 301 ± 11 fmol/well); LLC-PK₁ cells were unresponsive to PTH. The EC₅₀ values for cAMP accumulation by PTH in OK_HC cells [10.2 (3.1, 33.7) nM] and OK_LC 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).

View larger version (16K): [in this window] [in a new window]   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 OK_HC 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, OK_LC cells transported [³H]PAH quite efficiently, and both the cell accumulation and transport were markedly (P < 0.05) reduced by nonlabeled PAH (Fig. 9).

View larger version (19K): [in this window] [in a new window]   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 · cm² · min¹) was considerably greater (P < 0.05) in LLC-PK₁ cells (84.3 ± 4.1) than in OK cells (OK_LC, 27.0 ± 9.1, and OK_HC, 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.

View larger version (21K): [in this window] [in a new window]   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.

-MG. The transport of -MG was initiated by adding Hanks' medium containing -[¹⁴C]MG (10 µM) to the apical side of the monolayers. As shown in Fig. 11, the transport of -[¹⁴C]MG (in pmol · cm² · min¹) was considerably greater (P < 0.05) in LLC-PK₁ cells (58.5 ± 9.8) than in OK cells (OK_LC, 3.8 ± 0.1, and OK_HC, 9.0 ± 0.7). The addition of phlorizin markedly reduced both the accumulation and transport of -[¹⁴C]MG in LLC-PK₁ cells only.

View larger version (21K): [in this window] [in a new window]   Fig. 11.   Effect of phlorizin (50 µM) on cell accumulation (A) and apical-to-basal transport (B) of 10 µM -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 (K_m and V_max) 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 K_m values.

View larger version (18K): [in this window] [in a new window]   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.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The present study describes characteristic features of two clonal subpopulations of OK cells (OK_LC and OK_HC) that are functionally different but morphologically identical. The most impressive difference between OK_HC and OK_LC 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 pH_i 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, OK_HC and OK_LC cells were identical at both day 2 and day 5 after initial seeding, as observed by optical microscopy. However, both OK_HC and OK_LC 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 OK_HC and OK_LC 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 OK_LC and OK_HC cells, the most interesting aspect concerns differences in Na⁺ handling. Na⁺/H⁺ exchanger and Na⁺-K⁺-ATPase activities and expression in OK_HC cells were markedly higher than in OK_LC 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 OK_HC 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 OK_HC 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 OK_HC cells express more NHE3 and are endowed with greater Na⁺/H⁺ exchanger activity than OK_LC cells. It should be stressed that the enhanced Na⁺/H⁺ exchanger activity in OK_HC 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 OK_HC cells was greater than in OK_LC 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 OK_HC cells resembles that observed in renal tubular cells from spontaneously hypertensive rats (10, 11, 25, 45). OK_HC 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 OK_LC and OK_HC 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 OK_LC and OK_HC 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 OK_LC cells were able to transport the organic anion PAH. Although the response of OK_HC cells to PTH was greater than in OK_LC 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 OK_HC cells [10.2 (3.1, 33.7) nM] did not differ from those in OK_LC 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 OK_LC and OK_HC 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 -MG. In contrast to that reported for OK cells (27), both OK_LC and OK_HC cells were found to transport -MG in a phlorizin-insensitive manner. On the other hand, the phlorizin-sensitive apical transport of -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 OK_LC and OK_HC 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 OK_LC and OK_HC cells. Similarly, no significant differences in AADC and COMT activities were observed between OK_LC and OK_HC 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 OK_LC and OK_HC 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 OK_HC and OK_LC 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.