| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Unité Mixte de Recherche, Centre National de la Recherche Scientifique 6548, Université de Nice-Sophia Antipolis, O6108 Nice Cedex 2, France
 |
ABSTRACT |
|---|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
Cl
conductances were
studied in cultured rabbit proximal convoluted tubule (PCT) epithelial
cells and compared with those measured in cultured distal bright
convoluted tubule (DCTb) epithelial cells. Using the whole cell
patch-clamp technique, three types of
Cl conductances were
identified in DCTb cultured cells. These consisted of volume-sensitive,
Ca2+-activated, and
forskolin-activated Cl
currents. In PCT cultured cells, only volume-sensitive and
Ca2+-activated
Cl currents were recorded.
The characteristics of
Ca2+-activated currents in PCT
cells closely resembled those in DCTb cells. Volume-sensitive
Cl currents could be
elicited both in PCT and in DCTb cells by hypotonic stress. The
pharmacological profile of this conductance was established for both
cell types. Forskolin activated a linear
Cl current in DCTb cells
but not in PCT cells. This conductance was insensitive to DIDS and
corresponds to cystic fibrosis transmembrane conductance regulator
(CFTR)-like channels. Quantitative measurements of SPQ fluorescence
showed that only the apical membrane of DCTb cells possessed a
Cl pathway that was
sensitive to forskolin. RT-PCR experiments showed the presence of CFTR
mRNA in both cultures, whereas immunostaining experiments revealed the
expression of CFTR in DCTb cells only. The physiological role of the
different types of channels is discussed.
whole cell; cystic fibrosis transmembrane conductance regulator; renal epithelium
| |
INTRODUCTION |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
REABSORPTION OF THE filtered load of NaCl is the major
transport function of the kidney. The fact that <1% of the filtered load of NaCl is excreted in the final urine clearly illustrated the
efficiency of the transport mechanisms responsible for NaCl transport
along the nephron. Approximately 70% of the filtered load of
Cl is reabsorbed by the
proximal tubule and 20% from the loop of Henle, whereas only
5-9% is reabsorbed along the different segments of terminal
nephron (i.e., early distal tubule, connecting tubule, and collecting
ducts). In this complex structure, any segment of the nephron may
transport Cl by a mechanism
different from that of other segments, and this transport may be
regulated via different pathways. Ion channels play an essential role
among the various mechanisms of transcellular Cl transport. As in
secretory epithelia, where they have been extensively studied,
Cl channels with very
diverse and distinct properties have been described for the kidney. For
example, it has been demonstrated that rabbit proximal convoluted
tubule (PCT) cells in culture possess an apical outwardly rectifying
Cl channel activated by
parathyroid hormone via the protein kinase A (PKA) and protein kinase C
(PKC) pathways (37). In rat PCT cells grown in primary culture, a more
recent study described at least four different
Cl-selective channels
located in the apical membrane and modulated by cyclic nucleotides,
Ca2+, or voltage (9). In
microdissected thick ascending limbs of Henle's loop from the mouse,
basolateral Cl channels of
40-pS conductance regulated by arginine vasopressin via the PKA pathway
have been recorded (12, 13). In the bright part of the distal
convoluted tubule (DCTb), we have shown the presence of three different
Cl conductances, regulated
by cAMP, cytosolic Ca2+, or by
cell swelling (4, 34, 42). A cAMP-sensitive
Cl channel has also been
recorded in the apical membrane of cultured epithelial cells from the
cortical collecting tubule (19) and from the inner medullary collecting
tubule (45). An interesting feature identified in these tight epithelia
was that the Cl channel
could be controlled by polypeptide hormones (28, 40) and other
effectors (32) coupled to adenylate cyclase.
Of the cAMP-activated Cl
channels found in the apical membrane of the terminal nephron, some
have been shown to exhibit characteristics similar to those of the
cystic fibrosis transmembrane conductance regulator (CFTR). In the
DCTb, patch-clamp and whole cell studies (30, 42) strongly suggest that
the CFTR may play a role in transepithelial
Cl movements. Moreover, in
primary cultures of medullary and cortical collecting ducts, a
cAMP-activated Cl
conductance resembling CFTR conductance has also been demonstrated (14,
19). The presence of CFTR channels in the proximal tubule is more
controversial than in distal tubule. Although CFTR mRNAs are present in
the S1, S2, and S3 segments of the proximal tubule and CFTR expression
can be located by immunostaining of the apical membrane, it has not yet
been established whether this CFTR is functional in terms of
Cl channels in this
structure. At present, none of the cAMP-activated Cl channels described in
the proximal tubule behave exactly as the CFTR channels that have been
identified in secretory epithelia or transfected into other cell types.
In view of these results, we have carried out whole cell patch-clamp
and fluorescence experiments to further investigate the Cl permeability of the
S1-S2 segments of the proximal tubule in primary culture. Because
we have previously demonstrated that primary cultures of DCTb cells
express different types of
Cl conductance, one of
which is a CFTR-like Cl
channel, these experiments were conducted in parallel in cultured DCTb
cells. Moreover, in an attempt to investigate the existence of the CFTR
in monolayers of both cell types, we used molecular biological
approaches. We found that cultured PCT and DCTb cells exhibited both
Ca2+-activated and
swelling-sensitive Cl
conductance, whereas only DCTb displayed forskolin-activated Cl conductance. This last
conductance correlates well with the presence of CFTR in the apical membrane.
| |
MATERIALS AND METHODS |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
Primary Cultures
The primary cell culture technique has been described in detail in previous papers (3, 23). The PCT and the rabbit DCTb were microdissected under sterile conditions from the kidneys of 4- to 5-wk-old male New Zealand rabbits. The kidneys were perfused with Hanks' solution (GIBCO) containing 600-700 U/ml collagenase (Worthington) and were then cut into small pieces that were incubated in medium containing 150 U/ml collagenase. The tubules were seeded in collagen-coated 35-mm petri dishes or in collagen-coated polycarbonate filters filled with a culture medium composed of equal quantities of DMEM and Ham's F-12 (GIBCO), containing 15 mM NaHCO3, 20 mM HEPES, pH 7.5, 2 mM glutamine, 5 µg/ml insulin, 50 nM dexamethasone, 10 ng/ml epidermal growth factor, 5 µg/ml transferrin, 30 nM sodium selenite, and 10 nM triiodothyronine. Cultures were maintained at 37°C in a 5% CO2-95% air-water saturated atmosphere. The medium was changed 4 days after seeding and every 2 days thereafter.Identification of CFTR mRNA
Reverse transcription and PCR amplification were performed using standard protocols in a thermal cycler (Techne). Total RNA was prepared from primary cultures of rabbit proximal and distal tubules (2 × 106 cells) by using a micro RNA isolation kit (Stratagene) according to the manufacturer's recommendations. Primers were chosen to amplify a sequence of 359 bp localized in exon 13 of rabbit CFTR. Reverse transcription was accomplished with recombinant Moloney murine leukemia virus reverse transcriptase (RT-MLV; Strata Script; Stratagene). The RNAs were reverse transcribed into cDNAs. RNA (100 ng) was dissolved in 25 µl of buffer containing 20 mM Tris · HCl, pH 8.3, 50 mM KCl, 4 mM MgCl2, 1 mg/ml gelatin, 0.8 mM dNTP, 10 mM dithiothreitol, and 10 pmol oligo 5'-TCGCCTCTCCCTGTTCTGAATCT-3' (oligo A). The mixture was heated 2 min at 80°C, and the reaction was incubated for 45 min at 42°C after addition of 200 units reverse transcriptase. The reaction was then heated at 96°C during 30 s and cooled at 80°C before the addition of 25 µl of PCR mixture containing 20 mM Tris · HCl, pH 8.3, 50 mM KCl, 4 mM MgCl2, 1 mg/ml gelatin, 10 pmol oligo 5'-GAAGGCAGCAGCTATTTTTATGG-3' (oligo B), and 1.25 units Taq polymerase (Stratagene). The conditions for amplification were as follows: each cycle consisted of incubation at 94°C for 30 s, 52°C for 30 s, and 72°C for 40 s for a total of 30 cycles. At the end of this series, the reaction was incubated at 72°C for 5 min. Mineral oil (100 µl) was overlaid to prevent evaporation during thermocycling. Controls were performed without RT-MLV and also without RNA. All buffers were prepared in diethyl pyrocarbonate-treated water. After RT-PCR, 10-20 µl of each reaction mixture was subjected to electrophoresis on a 0.8% agarose gel to size fractionate the RT-PCR products. The PCR-amplified fragments were subsequently cloned in the pGEM vector using Promega pGEM-T easy cloning kit. Plasmid DNA containing the 380-bp insert was then sequenced according to Sanger et al. (35) using oligonucleotides A and B (see above) as sequencing primers.Whole Cell Experiments
Whole cell currents were recorded from cultured cells (12-22 days of age) grown on collagen-coated supports and maintained at 33°C throughout the experiments. The ruptured-patch whole cell configuration of the patch-clamp technique was used. Patch pipettes were made from borosilicate capillary tubes (1.5 mm OD, 1.1 mm ID; Clay Adams) using a two-stage vertical puller (PP 83; Narishige) and filled with N-methyl-D-glucamine (NMDG)-Cl solution. The
pipettes had resistance ranging from 2 to 3 M
. Cells were observed
by using an inverted microscope, the stage of which was equipped with a
water robot micromanipulator (WR 89; Narishige). The patch pipette was
connected via an Ag/AgCl wire to the head stage of a RK 400 patch
amplifier (Biologic). After the formation of a gigaohm seal, the fast
compensation system of the amplifier was used to compensate for the
head-stage intrinsic input capacitance and the pipette capacitance. The
membrane was ruptured by additional suction to achieve the conventional
whole cell configuration. At this stage, the cell capacitance was
compensated for using a facility provided on the RK 400 amplifier. No
series resistance compensation was applied, but experiments in which the series resistance was higher than 20 M were discarded.
Extracellular test solutions were perfused into the bath using a
four-channel glass pipette, the tip of which was placed as near as
possible to the clamped cell.
Voltage clamp commands, data acquisition, and data analysis were
controlled by a computer (ERN) equipped with a Digi data 1200 interface
(Axon Instruments). pClamp software (versions 5.51 and 6.0; Axon
Instruments) was used to generate whole cell current-voltage (I-V)
relationships. Membrane currents resulting from voltage stimuli were
filtered at 1 kHz, sampled at a rate of 2560/s, and stored directly
onto the hard disk. Cells were held at a holding potential of 
50
mV and 40-ms pulses from 100 to +120 mV were applied in
increments of 20 mV every 2 s.
Fluorescence Experiments
Intracellular Cl measurement.
Cultures (12-22 days of age) grown on collagen-coated filters were
loaded for 12-16 h at 37°C, with 5 mM
6-methoxy-1-(3-sulfonatopropyl)quinolinium (SPQ) added to the culture
medium. Confluent cultures growing on filters were carefully rinsed
with an NaCl solution (containing in mM: 140 NaCl, 5 KCl, 1 CaCl2, 1 MgSO4, 5 glucose, and 20 HEPES, pH
7.4) and mounted in an Ussing chamber (aperture 7 mm2) with the apical face
directed downward. This chamber was then placed in a perfusion chamber
installed on the stage of an inverted microscope. The perfusion chamber
permitted the independent perfusion of the apical and the basolateral
membranes of the culture (3).
F/dt)/F0 /min,
where F/dt is the initial rate of fluorescence change
upon addition or removal of
Cl, and
F0 is the SPQ fluorescence in the
presence of 140 mM potassium thiocyanate.
Cl efflux was induced by
replacement of the NaCl solution with an isoosmotic
NaNO3 solution containing (in mM)
140 NaNO3, 5 KNO3, 3 calcium gluconate, 1 MgSO4, 5 glucose, and 20 HEPES, pH
7.4. To determine the background fluorescence, cultured cells were
incubated at the end of each experiment in 140 mM KSCN, which rapidly
quenched SPQ fluorescence.
Intracellular
Ca2+
measurements.
Confluent monolayers (12-22 days of age) were loaded for 30 min at
37°C, with a solution of 4 µM indo 1-AM containing 0.02% pluronic acid. The cells were then washed with NaCl solution and mounted as described in Intracellular Cl
measurement. Fluorescence measurements were performed
using the laser cytometer. The excitation of indo 1 was carried out by
means of the UV wavelength light of the argon laser (351-363 nm).
Emission was detected at 405 and 485 nm. The intracellular
Ca2+ concentration was calculated
from the dual-wavelength fluorescence ratio.
Labeling of Primary Cultures With Monoclonal Antibodies
Primary cultures of PCT and distal convoluted tubule (DCT) grown on collagen-coated filters were fixed for 30 min at 4°C with 4% paraformaldehyde in phosphate-buffered saline solution. After rinsing overnight, cultures were gradually dehydrated in ethanol and finally in xylene. Afterward, cultures were embedded in paraffin. Sections of thickness 10 µm were incubated overnight at 4°C with MATG 1061 (Transgène, Strasbourg France) diluted at 4 µg/ml in PBS-0.5% BSA. After several washings, cultures were incubated for 2 h in fluorescent rabbit anti-mouse IgG diluted 1:200 in PBS-0.5% BSA.Chemicals
Forskolin (Sigma) was prepared as 10 mM stock solution in ethanol and dissolved at 10 µM in buffer solutions. Ionomycin (Sigma) was dissolved at 2 mM in ethanol and used at 2 µM in final solutions. 5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) from Calbiochem was prepared at 100 mM in DMSO and used at 0.1 mM in final solutions. Diphenylamine-2-carboxylate (DPC) from Aldrich was prepared as 1 M stock solution in DMSO and dissolved at 1 mM in incubation medium. DIDS from Sigma was directly dissolved at a final concentration of 1 mM. SPQ from Calbiochem was directly dissolved at 5 mM in final solution. Indo 1-AM from Molecular Probes was dissolved at 10 mM in DMSO and used at 4 µM in buffer solution containing 0.02% pluronic acid. All other products were from Sigma.Solutions
The composition of the different solutions used in these experiments are given in Table 1.
|
| |
RESULTS |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
Identification of Transcripts Encoding the Rabbit CFTR Sequence by RT-PCR in Primary Cultures of PCT and DCTb Cells
PCT and DCTb total RNA was reverse transcribed and amplified by PCR using A and B primers. These primers amplify a product of 382 bp encoding for a part of the exon 13 of the rabbit CFTR. The analysis of the RT-PCR products by electrophoresis on agarose gel stained with ethidium bromide revealed only one product of ~380 bp (Fig. 1) both in PCT and DCT RNA extracts. Identical analysis without prior reverse transcriptase of the RNA sample revealed no amplification of any product.
|
The PCR product obtained from cultured PCT or DCTb was sequenced and, of the 300 bases read, was found to share 100% identity with the region on the rabbit CFTR mRNA.
Labeling of Primary Cultures of PCT and DCTb With MATG 1061
The indirect immunofluorescence technique was used to localize CFTR in primary cultures of PCT and DCTb cells. MATG 1061 (Monoclonal Antibody Transgène) was used on paraffin sections of 15-day-old PCT and DCTb cells grown on collagen-coated filters. As shown in Fig. 2, cultures presented with an epithelial morphology. The antibody revealed an intense labeling in cultured DCT, the localization of which was diffuse among the cells (Fig. 2a). In the PCT monolayer (Fig. 2b), only a few cells exhibited a very weak labeling. Control experiments in the absence of the first antibody showed negligible background labeling (Fig. 2c).
|
Electrophysiological Studies
Whole cell currents were recorded under several experimental conditions to identify Cl conductances
in primary cultures of PCT and DCTb cells. Volume-sensitive and
Ca2+-dependent
Cl currents were identified
in both PCT and DCTb monolayers, whereas forskolin-activated
Cl currents were only
recorded in cultured DCTb cells.
Ca2+-induced
Cl currents.
Whole cell currents were recorded with
Ca2+-free (1 mM EGTA) solutions
containing NMDG-Cl as the
major cation in the pipette and with the extracellular solutions
containing NaCl. The osmotic pressure of the extracellular solution was
adjusted to 350 mosmol/l with mannitol to avoid inducing volume-activated currents. Two different methods were used to increase
the intracellular Ca2+
concentration. In a first series of experiments, the control macroscopic currents were recorded and then 2 µM ionomycin was added
to the bathing NaCl solution. Stimulated currents were recorded after 1 min. Under these conditions, the cytoplasmic free
Ca2+ rose to 1.00 ± 0.19 µM
(n = 13). In a second set of
experiments, the total Ca2+
concentration in the pipette solution was raised to 0.1 mM in the
presence of 0.15 mM EGTA, and currents recorded 0.5 to 1 min after the
whole cell configuration were obtained. Because the kinetics of the
currents recorded using the two methods were not different, the data
were pooled. Figure
3A shows
the currents recorded in PCT and DCTb monolayers. In the presence of
ionomycin, the currents increased during depolarizing voltage pulses.
The kinetics of the macroscopic current were clearly time dependent for
depolarizing potentials with a slowly developing component. The
corresponding
I-V
relationships for steady-state activated currents are given in Fig.
3C. In cultured PCT cells, currents reversed at 1.8 ± 0.8 mV (n = 7). Instantaneous currents were linear: the inward current at
100 mV was 477 ± 61 pA, and the outward current at +100 mV
was 505 ± 65 pA. The steady-state current exhibited a marked
outward rectification with an inward current at 100 mV of 357 ± 46 pA and an outward current at +100 mV of 743 ± 127 pA
(n = 7). When the steady-state current
measurements were used to calculate the chord conductance, the maximal
outward conductance was significantly different from the maximal inward conductance (14.8 ± 3.9 vs. 3.94 ± 0.31 nS,
P < 0.02, n = 7). Concerning cultured DCTb
cells, the data obtained in the present study
(n = 7) confirm those described in a
preceding paper (4). As illustrated in Fig. 3,
A and
B, the
Cl currents induced by
ionomycin in cultured DCTb strongly resembled those induced in cultured
PCT cells. The anion permeability of the PCT cell membrane after
application of ionomycin was studied by replacing all except 2 mM of
the Cl in the bath solution
with Br,
I, or glutamate. Table
2 summarizes the reversal potentials
(Erev) as well
as the calculated permeability ratios for each anion. {The
estimated relative permeability
PX/PCl
was calculated in Tables 2 and 3 using the equation
Erev = 58
log [([Cl]o × PCl + [X]o × PX)/([Cl]i × PCl + [X]i × PX)],
where [Cl]o is extracellular
Cl concentration; PCl is chloride
permeability; [X]o is extracellular concentration of the
replacing anion; PX is permeability of the replacing anion; [Cl]i is intracellular
Cl concentration; and [X]i is
intracellular concentration of the replacing
anion.} Replacing external
Cl with glutamate markedly
reduced the outward current and moved the
Erev toward more
positive voltages. In the presence of
I or
Br, the
Erev shifted
toward the negative values. Finally, the sequence for the
ionomycin-sensitive conductance was
I > Br >>
Cl >> glutamate.
|
|
Cl currents induced by a hypotonic
shock.
To study the effects of changes in osmotic pressure on the development
of Cl conductance, currents
were induced by osmotic shock. In these experiments, whole cell
currents were recorded with
Ca2+-free (5 mM EGTA) pipette
solutions containing
NMDG-Cl and maintained at
an osmolarity of 290 mosmol/l. Moreover, to eliminate any participation
of cations in the inward current, experiments were carried out after
replacing Na+ in the bath solution
with NMDG+. Figure
4A
shows Cl currents observed
in cultured PCT and DCTb cells with an extracellular solution
osmolarity of 350 mosmol/l. The voltage step protocol elicited small
time-independent currents that changed linearly with the membrane
voltage and had a
Erev of
1.2 ± 0.4 mV (n = 9) for
PCT cells and of 2.9 ± 1.6 mV
(n = 4) for DCTb cells. Because of
their small amplitude, the nature of these currents was not analyzed
further.
|
100 mV. These large, outward rectifying currents
showed time-dependent inactivation at depolarizing potentials 
60 mV
in cultured PCT cells and 40 mV in cultured DCTb cells. In most
cases, the time course of this inactivation could be well fitted with a
single exponential irrespective of the recording time. When the cells
were reexposed to the hyperosmotic solution, the currents returned to
the control level within 2-3 min (Fig.
4C). The currents induced by
hypotonicity were analyzed at their maximal values, and the
I-V
relationships for initial currents are shown in Fig.
4D. In cultured DCTb cells, it was found that the Cl currents
were very similar to those previously described in this nephron segment
(34). Thus no further characterization was carried out in the present
series.
|
currents recorded in
cultured PCT cells, the anion permselectivity of the cell membrane after the hypotonic shock was determined by replacing all except 2 mM
of the Cl in the bath with
I,
Br, or glutamate. The
Erev was obtained
from the
I-V
relationship. Table 3 summarizes the
results with the calculated anion permeability sequence being I Br > Cl > glutamate.
|
conductances were
tested. The data from these experiments are summarized in Table
4. The application to the bath of 1 mM DPC
or 0.1 mM NPPB strongly decreased the amplitude of the
Cl current induced by
osmotic shock at both positive and negative potentials, whereas the
addition of 1 mM DIDS rapidly reduced the
Cl current. The block by
DIDS was less efficient than that of NPPB or DPC. Moreover, this
blocking effect was voltage dependent, with the outward current being
inhibited to a much greater extent than the inward current. Tamoxifen
was the most potent blocker used, but its action was slower than that
of the other inhibitors. In fact, it was necessary to incubate the
cells for 5 min in the presence of the drug before exposing them to the
osmotic shock. The cells treated with 5 µM tamoxifen never developed
Cl currents in response to
hypotonicity.
|
Cl currents activated by
forskolin.
Experiments were performed in an hyperosmotic extracellular solution
(350 mosmol/l) to characterize Cl currents activated
by forskolin in DCTb and PCT cells. Under these
conditions, volume-activated
Cl currents could not be
detected. Figure
6A shows
that the voltage-step protocol elicited small currents that changed
linearly with the membrane potential
(Em)
in both PCT and DCTb cell cultures. In PCT cells, the slope conductance
was 1.2 ± 0.2 nS, and the
Erev was 0.2 ± 0.3 mV, n = 23. In DCTb cells,
the slope conductance was 0.8 ± 0.4 nS, and the
Erev was 1.8 ± 0.3 mV, n = 5. The exposure of
cultured DCTb cells to 10 µM forskolin induced an increase in
membrane current amplitudes (Fig. 6, B and
C) that reached a peak value
3-4 min after the beginning of the perfusion. Figure 6C shows that the activated currents
exhibited a linear
I-V
relationship with a
Erev of
1.4 ± 0.9 mV and a conductance of 6.4 ± 0.4 nS (n = 5). In contrast, the application
of forskolin did not modify the currents recorded in cultured PCT cells
(Fig. 6A). The unstimulated whole
cell current in these cells reversed at 1.6 ± 0.8 mV with a
slope conductance of 1.8 ± 0.3 nS,
n = 23.
|
concentrations in the
presence of 5 mM EGTA in the pipette to rule out any involvement of
intracellular Ca2+. The
forskolin-stimulated Cl
currents recorded in DCTb cells had the same characteristics as we have
previously described (42). However, to further characterize these
properties, additional ion substitution and inhibition experiments were
performed. The anion permeability sequence of these channels was
studied by replacing all except 2 mM of the
Cl in the bath solution
with I,
Br, or glutamate. The
results for these experiments are illustrated in Fig.
7. Currents were measured at
Em = +100 mV and
at the maximum of the response. The results are expressed as a
percentage of the maximal response obtained in the
NMDG-Cl solution. Replacing
external Cl with
I or glutamate strongly
inhibited the Cl currents
activated by forskolin and caused the
Erev to shift toward the positive values
(Erev of
I = 18.6 ± 3.6, n = 7;
Erev of glutamate = 33.4 ± 1.3 mV, n = 5). In
contrast, the replacement of
Cl with
Br led to a negative
Erev
(Erev of
Br = 12.4 ± 0.9, n = 9) . Finally, the permeability
sequence derived from these
Erev values is
Br > Cl >>
I > glutamate for
forskolin-stimulated currents.
|
Fluorescence Studies
Effects of forskolin on apical membrane
Cl permeability.
The Cl permeability of
apical membranes of DCTb and PCT cells was estimated by the measurement
of intracellular SPQ fluorescence on confluent monolayers. Confluency
was checked by the measurement of transepithelial voltage and
resistance (23). The flow of Cl across the apical
membrane was assessed by the addition or removal of
Cl from the apical
solutions. The cell monolayer was first perfused with NaCl solution in
both apical and basolateral compartments. After 5 min, the apical
Cl was replaced by an
NaNO3 solution, and
104 M NPPB was added to the
basolateral NaCl solution to block basolateral Cl permeability. Figure
8 shows the time course of intracellular SPQ fluorescence determined in seven different PCT monolayers and in
eight different DCTb monolayers. Upon apical
Cl removal, the relative
SPQ fluorescence increased slowly due to Cl efflux. The fluorescence
then fell to around its initial level when
NO3 was replaced by
Cl. In PCT cells, the
addition of forskolin did not modify the rates of
Cl efflux and influx
previously induced by the removal and addition of
Cl, whereas in DCTb cells
the addition of forskolin promptly increased the
Cl fluxes. The initial
rates of relative Cl efflux
are shown in Fig. 9. This figure confirms
that forskolin had no effect on the relative
Cl efflux in PCT cells,
whereas it strongly increased the relative Cl efflux in DCTb cells.
Moreover, in PCT cells, the control efflux was significantly impaired
after the apical addition of NPPB or 103 M DPC. Figure 9 also
shows that 105 M NPPB
completely blocked the forskolin-induced increase of
Cl efflux in DCTb
monolayers.
|
|
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
The aim of this study was to investigate the properties of the
Cl currents in PCT cells
and to compare them with the properties of the
Cl currents found in DCT
cells. The experiments were simultaneously conducted in primary
cultures of epithelial cells from the early proximal tubule and the
DCTb microdissected from the rabbit kidney. These cultures were
obtained as described previously (23, 42). Using the patch-clamp
technique to measure whole cell conductance, several
Cl conductances were
identified. Three distinct types of
Cl channels were found in
cultured DCTb cells. These consisted of forskolin-activated,
volume-sensitive, and
Ca2+-activated
Cl conductances. The main
features of these currents were identical to those reported in previous
papers (see Ref. 11 for review). In PCT cells, we found
volume-sensitive and
Ca2+-activated conductances.
The volume-sensitive Cl
current was developed in cells exposed to a hypotonic shock. Control
currents were obtained by maintaining the cells in a bath that was 50 mosmol/l hypertonic to the pipette solution. When the osmolarity of the
bath solution was lowered to 290 mosmol/l, the whole cell conductance
increased ~10-fold within 5-6 min. Once maximally developed, the
current remained very stable and could be rapidly inhibited by
reexposing the cells to the hypertonic bath solution.
The biophysical and the pharmacological characteristics of the
Cl conductances induced by
hypotonic stress in PCT cells show strong similarities with the
properties of swelling-activated
Cl currents described in
many other epithelial cells, including DCTb in primary culture (11,
37). Our findings clearly demonstrate that the major part of the
swelling-induced current was
Cl selective. However, when
glutamate was substituted for
Cl in the bathing solution,
the Erev was
shifted to just 30 mV. Because the theoretical
Erev for
glutamate substitution was over +100 mV, the
Cl channel could be
slightly permeable to glutamate. A volume-sensitive anion channel with
a high relative permeability to glutamate, aspartate, and taurine has
already been reported in Madin-Darby canine kidney (MDCK) cells (1).
Even so, the recent findings of Boese et al. (5) suggest that the
hypotonic stress would activate a common pathway for conductive
Cl and amino acids (as
glutamate or taurine).
The halide selectivity sequence permits the different types of
Cl channels to be
identified. In DCTb cells, the sequence for the hypotonicity-induced
Cl current was
I = Br > Cl. In PCT cells,
swelling-induced Cl
channels have been found to be more permeable to
I than to
Br. Such small differences
in I permeability do not
necessarily indicate that the channels are of a different nature. In
fact, literature data show that the selectivity sequence for the
volume-activated channel could be slightly different from one cell type
to another (6, 46).
Sensitivity to various anion channel blockers also helps one to
distinguish the type of Cl
channel under investigation. For this purpose, we examined the effects
of NPPB, DPC, DIDS, and tamoxifen on the swelling-induced Cl currents. In DCTb cells,
NPPB and DIDS mainly modified the outward current, whereas DPC strongly
blocked both outward and inward currents. In PCT cells, the application
of NPPB and DPC to the bath completely suppressed the
Cl current at both positive
and negative Em
values, whereas DIDS inhibited outward currents to a much greater
extent than inward currents. Compared with DCTb, the whole cell
Cl current in PCT cells was
less sensitive to DIDS. Various reports in the literature show that the
voltage dependence of the blocking effect of DIDS and the voltage
independence of the inhibitory effect of both DPC and NPPB are common
characteristics of swelling-activated Cl currents.
The effect of the anti-estrogen compound tamoxifen, which has been
reported to block volume-activated
Cl current in various cell
types (44, 46), was also tested. In both cultured PCT and DCTb cells,
tamoxifen strongly inhibited the volume-sensitive
Cl conductance. In contrast
to the inhibitory effect of NPPB, DIDS, or DPC, tamoxifen required
preincubation periods of 5 min to impair the development of swelling
currents. Moreover, tamoxifen failed to produce significant inhibition
of preactivated currents. Valverde et al. (43) have reported that
tamoxifen blocks the P-glycoprotein, such that the action of tamoxifen
in PCT and DCTb cells could be consistent with an involvement of
P-glycoprotein in the control of the swelling-activated
Cl channels.
As we previously discussed (34) for DCTb cells, it is very likely that
the Cl conductive pathway
activated by hypotonicity is implicated in the regulatory volume
decrease (RVD) process. Concerning the more distal segments of the
nephron, a large-conductance
Cl channel was identified
in the apical membrane of isolated rabbit cortical collecting duct (18)
and in RCCT-28A cells derived from rabbit collecting duct (36). This
channel could mediate Cl
efflux during RVD (24, 38). In PCT cells, this RVD process was
extensively studied, with several reports showing increases in
basolateral K+ and
Cl conductances when
isolated proximal tubules (2, 20, 47) or cultured proximal cells (17)
are swollen by exposure to a hypotonic shock. Together with a
volume-sensitive K+ conductance,
the Cl conductance that we
found in PCT could well contribute to the RVD phenomenon.
Cultured DCTb and PCT cells also exhibited a
Ca2+-dependent increase in whole
cell Cl conductance. The
extracellular application of ionomycin rapidly activated currents in
44% of the DCTb monolayers and in 70% of the PCT monolayers. This
Ca2+-sensitive conductance was
strikingly similar to that previously described in DCTb cells under
identical experimental conditions (4). The biophysical features of this
conductance in PCT cells resembled those recorded in DCTb cells.
However, substitution experiments indicated that the magnitude of the
glutamate permeability in PCT cells was smaller than that in DCTb
cells. In contrast, the relative halide permeability did not
significantly differ between the two types of epithelia, with the
sequence being I > Br > Cl. The ionomycin-sensitive
Cl current described here
in cultured DCTb and PCT cells closely resembled the
Ca2+-dependent
Cl currents seen in
Cl-secreting epithelia
(11).
The effect of forskolin on
Cl conductance was also
tested in both PCT and DCTb cells. For this purpose, swelling-activated currents were blocked by exposing the cells to an hyperosmotic solution, and Ca2+-activated
conductances were impaired by the use of high EGTA concentrations in
the pipette solution. In DCTb, the external application of forskolin
activated a linear Cl
current. The halide selectivity sequence was consistent with a low
relative iodide permeability and with an inhibitory effect of
I. Moreover this
forskolin-stimulated conductance was quite insensitive to DIDS.
Additional characterization of
Cl permeability was
performed using the technique of SPQ quantitative fluorescence
measurement. The results clearly showed that forskolin increases the
Cl permeability in the
apical membrane. These characteristics are very similar to those
reported previously in identical DCTb cultures (42). As we have already
concluded in this first study, the channels involved in the
Cl currents activated by
forskolin in DCTb may be the small-conductance channel corresponding to
CFTR-like channels.
Surprisingly, the application of forskolin did not increase
Cl currents in primary
cultures of PCT cells. The absence of such an effect was confirmed by
fluorescence experiments. To further investigate the presence of CFTR
in both types of primary cultures, the expression of CFTR mRNA was
investigated using RT-PCR. The two primers yielded a RT-PCR product of
expected size in both DCTb and PCT cells. This PCR product was indeed a
portion of the CFTR because it exhibited 100% homology with the rabbit
sequence. However, mapping the CFTR protein expression with a synthetic monoclonal antibody raised against the nucleotide binding
domain demonstrated high expression in cultured DCTb cells but no
significant expression in cultured PCT cells.
Literature data regarding the terminal nephron indicate that whole cell
Cl currents stimulated by
forskolin or cAMP are correlated with the expression of CFTR mRNA (14,
26, 45) and with the immunolocalization of the CFTR protein in the
apical domain (8, 26). Concerning the proximal tubule, there is no
clear-cut evidence for a correlation between CFTR expression and
Cl conductance. In
agreement with the present results, CFTR mRNA has been detected in rat
proximal tubules (26). Nevertheless, some results obtained by
immunostaining conflict with the present data because they demonstrate
that CFTR is abundant in the apical membrane of human adult proximal
tubules (8). In contrast, CFTR expression is not detectable in LLC-PK
cells, which originate from proximal tubules (21). Interestingly, MDCK
type II cells, which are also thought to originate from proximal
tubules (31), lack CFTR protein expression and cAMP-stimulated
Cl conductance, whereas
MDCK type I cells, which originated from the distal tubule, possess
CFTR expression and cAMP-stimulated anion conductance in their apical
membrane (25).
In spite of the presence of CFTR transcripts, we did not detect CFTR
expression along with forskolin-induced
Cl conductance. This
observation could signify that the final maturation of the protein was
impaired. It is now well demonstrated that there is a
differentiation-dependent cellular location of CFTR. For example,
undifferentiated colonic cells retain the protein in a perinuclear zone
(27). Moreover, recent experiments show that CFTR expression patterns
vary during nephrogenesis (10). In light of these observations, we must
question whether the tissue culture conditions we used could have
altered the insertion of CFTR into the apical membrane of PCT cells.
Because the whole cell patch-clamp experiments were performed on cells
that were grown on collagen-coated petri dishes, it would be possible
that this support prevents the cells from assuming their correct
polarization. Two observations deserve this proposition:
1) distal cells have been grown in
exactly the same experimental conditions and have exhibited CFTR
expression (30, 42); and 2)
forskolin did not increase apical
Cl permeability during
fluorescence experiment conducted with PCT cells, even though the cells
were grown on permeable support that provides good conditions for
epithelium polarization.
Several cAMP-sensitive Cl
channels have been reported to exist in the renal proximal tubule (9,
39, 41). Some characteristics of these channels are dissimilar to those
commonly reported for CFTR channels. Thus the question arises as to
whether the 30-pS channels previously described in the apical membrane
of rabbit and rat PCT are homologous to the CFTR channel. In this way,
a recent study performed using renal proximal brush-border membrane reached the conclusion that the intrinsic mouse brush-border membrane vesicle Cl
conductance is composed of a multiplicity of
Cl conductances, including
a minor component associated with CFTR expression (see Ref. 16). If
this hypothesis is true, it explains why we were not able to detect
CFTR conductance and CFTR expression modulated by forskolin even though
we were able to identify CFTR transcripts in primary culture of PCT cells.
The possibility still exists that cAMP could control the other types of
Cl channels in the proximal
tubule. For the moment, we have not tested this line of thought because
the volume-dependent and the Ca2+-activated
Cl currents must be blocked
to study the putative CFTR channels. As discussed above,
the Ca2+-activated
Cl conductance is found in
a variety of epithelial cells and it is not sensitive to PKA (see Ref.
11 for review). In contrast, the swelling-activated currents measured
in different tissues (22, 29) could be activated by cAMP. Suzuki et al.
(41) have proposed that a PKA- and PKC-activated
Cl channel measured in PCT
cells may contribute to cell volume regulation. Whether or not this
channel corresponds to the swelling-activated channel that we have
recorded in PCT cells remains to be determined. However, it can be
recalled that the swelling-activated
Cl conductance in cultured
DCTb was shown to be activated by PKC (34) and insensitive to cAMP
(33).
In conclusion, the present study demonstrates that PCT and DCT in
primary culture manifest Cl
currents induced by swelling and cytosolic
Ca2+. The swelling-activated
Cl conductance may play a
role in cell volume regulation in both epithelial types. In DCTb, the
Ca2+-induced conductance is
located in the basolateral membrane and could represent the route
whereby Cl exits the cell
under resting conditions (4). Further experiments are necessary to
identify the physiological role of the
Ca2+-induced conductance of the
PCT. The data obtained for DCTb in this study strengthen the hypothesis
that CFTR mediates the forskolin-activated Cl currents in the apical
membrane. Despite the presence of CFTR mRNA in PCT cells, there was no
detectable CFTR expression and no forskolin-activated
Cl conductance, indicating
that CFTR could be differentially regulated along the different parts
of the nephron. In relating these results to the cystic fibrosis
condition, no detectable alteration of renal function in cystic
fibrosis patients has been reported. This observation raises the issue
of the role of CFTR in controlling Cl transport along the
terminal nephron. According to several authors, it is possible that the
lack of cAMP-activated Cl
conductance in cystic fibrosis might be compensated by an increase in
another type of Cl channel
(7, 15). This could be the
Ca2+-sensitive channel present in
both PCT and DCTb cell cultures.
| |
FOOTNOTES |
|---|
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. §1734 solely to indicate this fact.
Address for reprint requests: P. Poujeol, UMR CNRS 6548, Bâtiment Sciences Naturelles, Université de Nice-Sophia Antipolis, Parc Valrose, O6108 Nice Cedex 2, France.
Received 17 February 1998; accepted in final form 25 June 1998.
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
1.
Banderali, U.,
and
G. Roy.
Anion channels for amino acids in MDCK cells.
Am. J. Physiol.
263 (Cell Physiol. 32):
C1200-C1207,
1992
2.
Beck, J. S.,
and
D. J. Potts.
Cell swelling, co-transport activation and potassium conductance in isolated perfused rabbit kidney proximal tubules.
J. Physiol. (Lond.)
425:
369-378,
1990
3.
Bidet, M.,
M. Tauc,
N. Koechlin,
and
P. Poujeol.
Videomicroscopy of intracellular pH in primary cultures of rabbit proximal and early distal tubules.
Pflügers Arch.
416:
270-280,
1990[Medline].
4.
Bidet, M.,
M. Tauc,
I. Rubera,
G. De Renzis,
C. Poujeol,
M. T. Bohn,
and
P. Poujeol.
Calcium-activated chloride currents in primary cultures of rabbit distal convoluted tubule.
Am. J. Physiol.
271 (Renal Fluid Electrolyte Physiol. 40):
F940-F950,
1996
5.
Boese, S. H.,
F. Wehner,
and
R. K. Kinne.
Taurine permeation through swelling-activated anion conductance in rat IMCD cells in primary culture.
Am. J. Physiol.
271 (Renal Fluid Electrolyte Physiol. 40):
F498-F507,
1996
6.
Cahalan, M. D.,
G. R. Ehring,
Y. V. Osipchuk,
and
P. E. Ross.
Volume-sensitive Cl channels in lymphocytes and multidrug-resistant cell lines.
Jpn. J. Physiol.
44:
S25-S30,
1994.
7.
Clarke, L. L.,
B. R. Grubb,
J. R. Yankaskas,
C. U. Cotton,
A. McKenzie,
and
R. C. Boucher.
Relationship of a non-cystic fibrosis transmembrane conductance regulator-mediated chloride conductance to organ-level disease in Cftr(/) mice.
Proc. Natl. Acad. Sci. USA
91:
479-483,
1994
8.
Crawford, I.,
P. C. Maloney,
P. L. Zeitlin,
W. B. Guggino,
S. C. Hyde,
H. Turley,
K. C. Gatter,
A. Harris,
and
C. F. Higgins.
Immunocytochemical localization of the cystic fibrosis gene product CFTR.
Proc. Natl. Acad. Sci. USA
88:
9262-9266,
1991
9.
Darvish, N.,
J. Winaver,
and
D. Dagan.
Diverse modulations of chloride channels in renal proximal tubules.
Am. J. Physiol.
267 (Renal Fluid Electrolyte Physiol. 36):
F716-F724,
1994
10.
Devuyst, O.,
C. R. Burrow,
E. M. Schwiebert,
W. B. Guggino,
and
P. D. Wilson.
Developmental regulation of CFTR expression during human nephrogenesis.
Am. J. Physiol.
271 (Renal Fluid Electrolyte Physiol. 40):
F723-F735,
1996
11.
Frizzell, R. A.,
and
A. P. Morris.
Chloride conductance of salt-secreting epithelial cells.
In: Current Topics in Membranes: Chloride Channels, edited by W. B. Guggino. New York: Academic, 1994, p. 173-214.
12.
Guinamard, R.,
A. Chraibi,
and
J. Teulon.
A small-conductance Cl channel in the mouse thick ascending limb that is activated by ATP and protein kinase A.
J. Physiol. (Lond.)
485:
97-112,
1995
13.
Guinamard, R.,
M. Paulais,
and
J. Teulon.
Inhibition of a small-conductance cAMP-dependent Cl channel in the mouse thick ascending limb at low internal pH.
J. Physiol. (Lond.)
490:
759-765,
1996
14.
Husted, R. F.,
K. A. Volk,
R. D. Sigmund,
and
J. B. Stokes.
Anion secretion by the inner medullary collecting duct. Evidence for involvement of the cystic fibrosis transmembrane conductance regulator.
J. Clin. Invest.
95:
644-650,
1995.
15.
Johnson, L. G.,
S. E. Boyles,
J. Wilson,
and
R. C. Boucher.
Normalization of raised sodium absorption and raised calcium-mediated chloride secretion by adenovirus-mediated expression of cystic fibrosis transmembrane conductance regulator in primary human cystic fibrosis airway epithelial cells.
J. Clin. Invest.
95:
1377-1382,
1995.
16.
King, N.,
W. H. Colledge,
R. Ratcliff,
M. J. Evans,
and
N. L. Simmons.
The intrinsic Cl conductance of mouse kidney cortex brush-border membrane vesicles is not related to CFTR.
Pflügers Arch.
434:
575-580,
1997[Medline].
17.
Le Maout, S.,
M. Tauc,
N. Koechlin,
and
P. Poujeol.
Polarized 86Rb+ effluxes in primary cultures of rabbit kidney proximal cells: role of calcium and hypotonicity.
Biochim. Biophys. Acta
1026:
29-39,
1990[Medline].
18.
Light, D. B.,
E. M. Schwiebert,
G. Fejes-Toth,
A. Naray-Fejes-Toth,
K. H. Karlson,
F. V. McCann,
and
B. A. Stanton.
Chloride channels in the apical membrane of cortical collecting duct cells.
Am. J. Physiol.
258 (Renal Fluid Electrolyte Physiol. 27):
F273-F280,
1990
19.
Ling, B. N.,
K. E. Kokko,
and
D. C. Eaton.
Prostaglandin E2 activates clusters of apical Cl channels in principal cells via a cyclic adenosine monophosphate-dependent pathway.
J. Clin. Invest.
93:
829-837,
1994.
20.
Macri, P.,
S. Breton,
J. S. Beck,
J. Cardinal,
and
R. Laprade.
Basolateral K+, Cl, and HCO3 conductances and cell volume regulation in rabbit PCT.
Am. J. Physiol.
264 (Renal Fluid Electrolyte Physiol. 33):
F365-F376,
1993
21.
Marshall, J.,
S. Fang,
L. S. Ostedgaard,
C. R. O'Riordan,
D. Ferrara,
J. F. Amara,
H. T. Hoppe,
R. K. Scheule,
M. J. Welsh,
A. E. Smith,
and
S. H. Cheng.
Stoichiometry of recombinant cystic fibrosis transmembrane conductance regulator in epithelial cells and its functional reconstitution into cells in vitro.
J. Biol. Chem.
269:
2987-2995,
1994
22.
Meng, X. J.,
and
S. A. Weinman.
cAMP- and swelling-activated chloride conductance in rat hepatocytes.
Am. J. Physiol.
271 (Cell Physiol. 40):
C112-C120,
1996
23.
Merot, J.,
M. Bidet,
B. Gachot,
S. Le Maout,
N. Koechlin,
M. Tauc,
and
P. Poujeol.
Electrical properties of rabbit early distal convoluted tubule in primary culture.
Am. J. Physiol.
257 (Renal Fluid Electrolyte Physiol. 26):
F288-F299,
1989
24.
Mills, J. W.,
E. M. Schwiebert,
and
B. A. Stanton.
Evidence for a role of actin filaments in regulating cell swelling.
J. Exp. Zool.
268:
111-120,
1994[Medline].
25.
Mohamed, A.,
D. Ferguson,
F. S. Seibert,
H. M. Cai,
N. Kartner,
S. Grinstein,
J. R. Riordan,
and
G. L. Lukacs.
Functional expression and apical localization of the cystic fibrosis transmembrane conductance regulator in MDCK I cells.
Biochem. J.
322:
259-265,
1997.
26.
Morales, M. M.,
T. P. Carroll,
T. Morita,
E. M. Schwiebert,
O. Devuyst,
P. D. Wilson,
A. G. Lopes,
B. A. Stanton,
H. C. Dietz,
G. R. Cutting,
and
W. B. Guggino.
Both the wild type and a functional isoform of CFTR are expressed in kidney.
Am. J. Physiol.
270 (Renal Fluid Electrolyte Physiol. 39):
F1038-F1048,
1996
27.
Morris, A. P.,
S. A. Cunningham,
A. Tousson,
D. J. Benos,
and
R. A. Frizzell.
Polarization-dependent apical membrane CFTR targeting underlies cAMP-stimulated Cl secretion in epithelial cells.
Am. J. Physiol.
266 (Cell Physiol. 35):
C254-C268,
1994
28.
Nagy, E.,
A. Naray-Fejes-Toth,
and
G. Fejes-Toth.
Vasopressin activates a chloride conductance in cultured cortical collecting duct cells.
Am. J. Physiol.
267 (Renal Fluid Electrolyte Physiol. 36):
F831-F838,
1994
29.
Oz, M. C.,
and
S. Sorota.
Forskolin stimulates swelling-induced chloride current, not cardiac cystic fibrosis transmembrane-conductance regulator current, in human cardiac myocytes.
Circ. Res.
76:
1063-1070,
1995
30.
Poncet, V.,
M. Tauc,
M. Bidet,
and
P. Poujeol.
Chloride channels in apical membrane of primary cultures of rabbit distal bright convoluted tubule.
Am. J. Physiol.
266 (Renal Fluid Electrolyte Physiol. 35):
F543-F553,
1994
31.
Richardson, J. C.,
V. Scalera,
and
N. L. Simmons.
Identification of two strains of MDCK cells which resemble separate nephron tubule segments.
Biochim. Biophys. Acta
673:
26-36,
1981[Medline].
32.
Rocha, A. S.,
and
L. H. Kudo.
Atrial peptide and cGMP effects on NaCl transport in inner medullary collecting duct.
Am. J. Physiol.
259 (Renal Fluid Electrolyte Physiol. 28):
F258-F268,
1990
33.
Rubera, I.,
M. Tauc,
F. Michel,
C. Poujeol,
and
P. Poujeol.
Simultaneous functional expression of swelling and forskolin-activated chloride currents in primary cultures of rabbit distal convoluted tubule.
C. R. Acad. Sci. III
320:
223-232,
1997[Medline].
34.
Rubera, I.,
M. Tauc,
C. Poujeol,
M. T. Bohn,
M. Bidet,
G. De Renzis,
and
P. Poujeol.
Cl and K+ conductances activated by cell swelling in primary cultures of rabbit distal bright convoluted tubules.
Am. J. Physiol.
273 (Renal Fluid Electrolyte Physiol. 42):
F680-F697,
1997
35.
Sanger, F.,
S. Nicklen,
and
A. R. Coulson.
DNA sequencing with chain-terminating inhibitors.
Proc. Natl. Acad. Sci. USA
74:
5463-5467,
1977
36.
Schwiebert, E. M.,
D. B. Light,
G. Fejes-Toth,
A. Naray-Fejes-Toth,
and
B. A. Stanton.
A GTP-binding protein activates chloride channels in a renal epithelium.
J. Biol. Chem.
265:
7725-7728,
1990
37.
Schwiebert, E. M.,
A. G. Lopes,
and
W. B. Guggino.
Chloride channels along the nephron.
In: Current Topics in Membranes: Chloride Channels, edited by W. B. Guggino. New York: Academic, 1994, p. 265-315.
38.
Schwiebert, E. M.,
J. W. Mills,
and
B. A. Stanton.
The actin-based cytoskeleton regulates a chloride channel and cell volume in renal cortical collecting duct cell line.
J. Biol. Chem.
269:
7081-7089,
1994
39.
Seki, G.,
H. Yamada,
S. Taniguchi,
S. Uwatoko,
K. Suzuki,
and
K. Kurokawa.
Mechanism of anion permeation in the basolateral membrane of isolated rabbit renal proximal tubule S3 segment.
Am. J. Physiol.
272 (Cell Physiol. 41):
C837-C846,
1997
40.
Shindo, M.,
N. L. Simmons,
and
M. A. Gray.
Characterization of whole cell chloride conductances in a mouse inner medullary collecting duct cell line mIMCD-3.
J. Membr. Biol.
149:
21-31,
1996[Medline].
41.
Suzuki, M.,
T. Morita,
K. Hanaoka,
Y. Kawaguchi,
and
O. Sakai.
A Cl channel activated by parathyroid hormone in rabbit renal proximal tubule cells.
J. Clin. Invest.
88:
735-742,
1991.
42.
Tauc, M.,
M. Bidet,
and
P. Poujeol.
Chloride currents activated by calcitonin and cAMP in primary cultures of rabbit distal convoluted tubule.
J. Membr. Biol.
150:
255-273,
1996[Medline].
43.
Valverde, M. A.,
M. Diaz,
F. V. Sepulveda,
D. R. Gill,
S. C. Hyde,
and
C. F. Higgins.
Volume-regulated chloride channels associated with the human multidrug-resistance P-glycoprotein.
Nature
355:
830-833,
1992[Medline].
44.
Valverde, M. A.,
G. M. Mintenig,
and
F. V. Sepulveda.
Differential effects of tamoxifen and I on three distinguishable chloride currents activated in T84 intestinal cells.
Pflügers Arch.
425:
552-554,
1993[Medline].
45.
Vandorpe, D.,
N. Kizer,
F. Ciampollilo,
B. Moyer,
K. Karlson,
W. B. Guggino,
and
B. A. Stanton.
CFTR mediates electrogenic chloride secretion in mouse inner medullary collecting duct (mIMCD-K2) cells.
Am. J. Physiol.
269 (Cell Physiol. 38):
C683-C689,
1995
46.
Verdon, B.,
J. P. Winpenny,
K. J. Whitfield,
B. E. Argent,
and
M. A. Gray.
Volume-activated chloride currents in pancreatic duct cells.
J. Membr. Biol.
147:
173-183,
1995[Medline].
47.
Welling, P. A.,
and
M. A. Linshaw.
Importance of anion in hypotonic volume regulation of rabbit proximal straight tubule.
Am. J. Physiol.
255 (Renal Fluid Electrolyte Physiol. 24):
F853-F860,
1988
This article has been cited by other articles:
|
|
 |
|
  Y. Xie and J. A. Schafer Inhibition of ENaC by intracellular Cl- in an MDCK clone with high ENaC expression Am J Physiol Renal Physiol, October 1, 2004; 287(4): F722 - F731. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Laverty, C. McWilliams, A. Sheldon, and S. S. Arnason PTH stimulates a Cl--dependent and EIPA-sensitive current in chick proximal tubule cells in culture Am J Physiol Renal Physiol, May 1, 2003; 284(5): F987 - F995. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Barriere, R. Belfodil, I. Rubera, M. Tauc, C. Poujeol, M. Bidet, and P. Poujeol CFTR null mutation altered cAMP-sensitive and swelling-activated Cl- currents in primary cultures of mouse nephron Am J Physiol Renal Physiol, April 1, 2003; 284(4): F796 - F811. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Belfodil, H. Barriere, I. Rubera, M. Tauc, C. Poujeol, M. Bidet, and P. Poujeol CFTR-dependent and -independent swelling-activated K+ currents in primary cultures of mouse nephron Am J Physiol Renal Physiol, April 1, 2003; 284(4): F812 - F828. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Bens, J.-P. D. Van Huyen, F. Cluzeaud, J. Teulon, and A. Vandewalle CFTR disruption impairs cAMP-dependent Cl- secretion in primary cultures of mouse cortical collecting ducts Am J Physiol Renal Physiol, September 1, 2001; 281(3): F434 - F442. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Andreasen, B. L. Jensen, P. B. Hansen, T.-H. Kwon, S. Nielsen, and O. Skott The alpha 1G-subunit of a voltage-dependent Ca2+ channel is localized in rat distal nephron and collecting duct Am J Physiol Renal Physiol, December 1, 2000; 279(6): F997 - F1005. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
X 174 + Hae III) were run in parallel at the
right edge of the agarose gel. Lanes marked +RT analyzed products
obtained from 25, 100, and 500 ng total RNA. Lanes marked 
, Control conditions; 
, ionomycin in the bath
(n = 7 cells from 3 monolayers).
Relationships were obtained from the same cell at rest and during
stimulation. Proximal, n = 8 cells
obtained from 3 different monolayers. Distal,
n = 7 cells obtained from 4 different
monolayers.
, rinsing. Relationships were obtained from
the same cell during control conditions, during hypotonic shock, and
after returning to control conditions. Proximal,
n = 9 cells obtained from 3 different
monolayers. Distal, n = 4 cells obtained from 3 different monolayers.
) and DCTb (