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 |
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,
-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
 |
INTRODUCTION |
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,
-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.
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METHODS |
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 (
· 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
-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 |
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).
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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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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.
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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
-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.
|
|
-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 · 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
-[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
-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.
|
|
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.
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 |
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
-MG. In contrast to that
reported for OK cells (27), both OKLC and
OKHC 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 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 |
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].
Am J Physiol Renal Fluid Electrolyte Physiol 283(1):F73-F85
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