Unite Mixte de Recherche Centre National de la Recherche Scientifique 6548, Université de Nice-Sophia Antipolis, O6108 Nice Cedex 2, France
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ABSTRACT |
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Cl conductances were
studied in an immortalized cell line (DC1) derived from rabbit distal
bright convoluted tubule (DCTb). The DC1 clone was obtained after
transfection of primary cultures of DCTb with pSV3
neo. RT-PCR experiments showed the
presence of cystic fibrosis transmembrane conductance regulator (CFTR) mRNA in the DC1 cell line. Using the whole cell
patch-clamp technique, we recorded a linear
Cl
conductance activated by
forskolin (FK). This conductance was insensitive to DIDS and
corresponded to a CFTR-like channel conductance. Fluorescence
experiments with 6-methoxy-1-(3-sulfonatopropyl)quinolinium (SPQ)
showed that FK induced an increase in
Cl
efflux and influx in DC1
cells similar to that observed in cultured DCTb cells.
125I
efflux experiments performed on DC1 cells grown on collagen-coated filters showed that exposure of the monolayer to FK led to an increased
125I
loss through the apical membrane only. The addition of 10 µM adenosine activated a linear conductance identical to that recorded with FK and corresponding to the CFTR-like conductance. This
conductance was also activated by
5'-(N-ethylcarboxamido)adenosine and CGS-21680 and
inhibited in the presence of
8-cyclopentyl-1,3-diproxylxanthine (DPCPX). This
Cl
conductance could also
be activated by guanosine 5'-O-(3-thiotriphosphate) (GTP
S). The addition of protein kinase A (PKA) inhibitor to the pipette solution inhibited the development of the current activated by
CGS-21680. Finally,
125I
efflux showed that adenosine induced an apical efflux mediated through
basolateral A2 receptors. Overall,
the data show that the DC1 cell line expressed an apical CFTR
Cl
conductance that could
be activated by adenosine via A2A
receptors located in the basolateral membrane and
involving G protein and PKA pathways.
kidney cell line; cystic fibrosis transmembrane conductance regulator; adenosine
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INTRODUCTION |
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IT IS NOW WELL ESTABLISHED that chloride channels play
an important role in fluid movement across epithelia. As for secretory epithelia, Cl channels with
diverse and distinct properties have been described in the kidney. In
primary cultures of rabbit distal bright convoluted tubule (DCTb)
cells, we have previously shown the presence of three different
Cl
conductances, regulated
by cAMP, cytosolic Ca2+, or by
cell swelling (4, 27, 34). The cAMP-sensitive channel found in the
apical membrane of these cells exhibited characteristics similar to
those of the cystic fibrosis transmembrane conductance regulator
(CFTR). With these results in mind, we have undertaken further
experiments to study the hormonal regulation of the CFTR conductance
and its effect in the overall
Cl
transport across the
distal tubule. There are, however, some disadvantages in using primary
cultures of DCTb cells, which include the low number of cells produced
and their limited life span. To overcome these limitations we set out
to transform primary cultures of DCTb cells using a plasmid containing
the early DNA region of the SV40 virus and the selectable
neo gene as a transforming agent. Of
the different transfected cell lines that we obtained, the DC1 clone
was selected because it expressed CFTR associated with a cAMP-activated
Cl
conductance in the
apical membrane.
In the present study, we have examined the effect of adenosine on the
Cl conductance in the DC1
cell line. Adenosine is a potent modulator of the renal function acting
via interactions with A1 and
A2 receptors. More specifically,
adenosine has been observed to modulate
Cl
and
Na+ transport in microperfused rat
medullary thick ascending limb (3) and in rabbit cortical collecting
duct cell line (30), as well as in mouse inner medullary (24) or A6
cells (7) lines. However, these studies were mainly concerned with the
physiological role of A1
receptors, meaning that little information is currently available on
the physiological role and the exact subtype of
A2 receptor in renal tissue.
In secreting epithelia, it has been demonstrated that the activation of
adenosine A2 receptors coupled to
cAMP production stimulates apical
Cl secretion through the
CFTR Cl
channel. In the
kidney, several Cl
channels
activated by cAMP have already been described in cultured DCTb (25,
34), cortical collecting duct (CCD) (18), and inner medullary
collecting duct (IMCD) (24, 35) cells and in the A6 cell
line (20). On the other hand, A2
receptors have also been found to increase the level of cAMP in primary
cultures of rabbit CCD (1) and rabbit thick ascending limb
of loop of Henle (5). Taken together, these observations indicate that adenosine could regulate cAMP-sensitive
Cl
channels by a mechanism
involving the A2 receptor. In the
present study, we demonstrate that the DC1 cell line immortalized from cultured DCTb cells expresses both
A1 and
A2 receptors in the basolateral
membrane. Adenosine activates an apical CFTR
Cl
conductance by a pathway
involving A2A receptors, G
proteins, adenylate cyclase, and protein kinase A (PKA).
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MATERIALS AND METHODS |
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Primary Cultures of DCTb Cells
The primary cell culture technique has been described in detail in previous studies (21, 22, 25). The bright part of the rabbit distal tubule, the DCTb, was 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.Transformation of Primary Cultures With pSV3 neo
Fifteen-day-old primary cultures of DCT from rabbit kidney were transfected with pSV3 neo using the calcium phosphate technique. Twenty-four hours prior to transfection, monolayers were treated with trypsin and harvested cells were replated at a density of 1-2 × 105 cells/cm2. The calcium phosphate-DNA coprecipitate was prepared as follows: 19 µg of pSV3 neo plasmid in 10 µl Tris-EDTA buffer were dissolved in 490 µl of a 250 mM CaCl2 solution. This solution was added drop-wise with gentle mixing to 500 µl of a solution containing 280 mM NaCl, 1.5 mM Na2HPO4, and 50 mM HEPES, adjusted at pH 7.08 with NaOH. A 160-µl aliquot of this solution was added drop-wise to each petri dish containing 1.6 ml of culture medium (see above). Cells were incubated overnight in this solution at 37°C, rinsed, and incubated in culture medium. After 24 h, selection of the clones was performed by the addition of 500 µg/ml G418. Culture medium containing G418 was changed every day. Isolated clones were subcultured and used after 10 trypsinization steps.Identification of CFTR mRNA
RT-PCR was performed using standard protocols in a thermal cycler (Techne). Total RNA was prepared from DC1 cells (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, StrataScript, 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, 10 pmol oligonucleotide A (5'-TCGCCTCTCCCTGTTCTGAATCT-3'). The mixture was heated 2 min at 80°C, and the reaction was incubated for 45 min at 42°C after addition of 200 U 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 oligonucleotide B (5'-GAAGGCAGCAGCTATTTTTATGG-3'), and 1.25 U 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. Following 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. (28) using oligonucleotides A and B (see above) as sequencing primers.
Whole Cell Experiments
Whole cell currents were recorded from DC1 cells (3-4 days of age) grown on collagen-coated supports maintained at 33°C for the duration of the experiments. The ruptured-patch whole cell configuration of the patch-clamp technique was used. Patch pipettes (resistance 2-3 MData Acquisition and Analysis
Voltage-clamp commands, data acquisition, and data analysis were controlled by a computer 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 relationships, with the membrane currents resulting from voltage stimuli being filtered at 1 kHz, sampled at 2.5 kHz, and stored directly on hard disk. Cells were held at a holding potential (Vhold) of125I
Efflux from DC1 Monolayers
Calculation. From back-addition of the radioactivity in the efflux samples to the radioactivity remaining in the cells, the apical and basolateral efflux rate constants were calculated (according to the following equations), i.e., the fraction of total radioactivity lost per unit time.
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Fluorescence Experiments
Intracellular chloride measurements. Cultures (3-5 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 of the cells 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.Quantitative measurements of SPQ fluorescence were made with the interactive laser cytometer (model ACAS 570; Meridian Instruments, Okemos, MI). The optical system was composed of an Olympus inverted microscope (model IMT2), fitted with a Zeiss ×40 objective (Ph2 LD-Plan 40) for epifluorescence measurements. Excitation wavelengths were obtained with a 5-W argon ion laser that produces illumination in several discrete lines over the 457.9-528.7 nm range and one in the ultraviolet (UV) spectra (351-364 nm range). This latter wavelength was used for the SPQ experiments. The excitation laser beam (0.6 µm diameter) was applied to the cell monolayer through the epifluorescence port of the microscope and a UV filter block mounted in the dichroic cube (350-nm band-pass excitation filter, a 380-nm dichroic mirror, and a 390-nm barrier filter). Images were collected as single frames repeated every 30 s and stored on hard disk. Fluorescent levels were analyzed with the image processing system after a series of frames had been taken. The gray level variations from one frame to another were analyzed in different zones automatically redrawn with the Meridian software. The average of pixel grey level intensities was calculated for each zone, and the data were finally processed with EXCEL software.
Calculation. Relative rates of
Cl influx and efflux were
computed from the time course of intracellular fluorescence and were expressed as relative fluorescence variation using the equation: (
F/dt)/F0 · min
1,
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.
Chloride efflux was induced by replacement of the NaCl solution with an isosmotic NaNO3 solution containing (in mM) 140 NaNO3, 5 KNO3, 3 calcium gluconate, 1 MgSO4, 5 glucose, and 20 HEPES, pH to 7.4 with NaOH. To determine the background fluorescence, cultured cells were incubated at the end of each experiment in 140 mM KSCN, which rapidly quenched SPQ fluorescence.
Chemicals
Forskolin (Sigma) was prepared as a 10 mM stock solution in ethanol and dissolved at 10 µM in buffer 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 the final solutions. Diphenylamine-2-carboxylate (DPC) from Aldrich was prepared as 1 M stock solution in DMSO and dissolved at 1 mM in the incubation medium. DIDS from Sigma was dissolved directly to a final concentration of 1 mM. SPQ from Calbiochem was directly dissolved at a concentration of 5 mM in the final solution. Adenosine was prepared as a 10 mM stock solution in NaCl buffer. 5'-(N-ethylcarboxamido)adenosine (NECA) was prepared as a 10 mM stock solution in ethanol. CGS-21680 from RBI (Natick, MA), N6-cyclopentyladenosine (CPA from Sigma), and 8-cyclopentyl-1,3-diproxylxanthine (DPCPX from Sigma) were prepared as 10 mM stock solutions in DMSO. PKA inhibitor (PKI) from Sigma was used at 10 µM in pipette solution. All other products were from Sigma.Solutions
The compositions of the different solutions used in these experiments are given in Table 1.
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RESULTS |
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Identification of Transcripts Encoding the Rabbit CFTR Sequence by RT-PCR in Cultures of DC1 Cells
DC1 total RNA was reverse transcribed and amplified by PCR using oligonucleotides A and B. These primers amplify a product of 382 bp encoding for a part of exon 13 of the rabbit CFTR. An analysis of the RT-PCR products by electrophoresis on agarose gel stained with ethidium bromide revealed only one product of ~380 bp (Fig. 1) in DC1 RNA extracts. An identical analysis without prior reverse transcription of the RNA sample revealed no amplification of any product.
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The PCR product obtained from cultured DC1 cells was sequenced and, of the 290 bases read, was found to share 100% identity with the region on the rabbit CFTR mRNA.
Effects of Forskolin on
Cl Permeability in Cultured DC1
Cells
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In previous reports (25, 34), we found that cAMP was implicated in the
regulation of chloride channels in cultured DCTb cells, and for this
reason the role of cAMP in the whole cell currents of DC1 cells was
evaluated. Exposure of cells to
105 M forskolin induced an
increase in membrane currents (Fig. 2, B and
F), with the maximum increase
obtained 3-4 min after the onset of perfusion. Figure
2F shows that the activated currents presented with a linear current-voltage relationship that reversed at
2.6 ± 0.6 mV. In the presence of forskolin, the current
amplitude at +80 mV reached 463 ± 53 pA while the slope conductance
was 5.3 ± 0.8 nS (n = 16).
The experiments yielding these data were performed in symmetrical
Cl concentrations, in the
presence of EGTA in the pipette, to avoid involvement of intracellular
Ca2+, and in hyperosmotic bath
solutions, to block swelling-activated currents.The reversal potential
was very close to that of
Cl
, and, in the absence of
permeable cations in the pipette, the outward current was carried by
Cl
. To eliminate any
participation of cations in the inward current, some experiments were
performed after replacing NaCl by NMDG-Cl in the bath solution. This
substitution did not modify the current evoked by forskolin at each
voltage step (data not shown).
To study the anion permeability of the cell membrane after application
of forskolin, all except 2 mM of the
Cl in the bath solution was
replaced with I
or
glutamate. Figure 2,
C-E,
shows typical recordings of the currents obtained in the presence of
Br
,
I
, and glutamate,
respectively. Figure 2F shows
current-voltage relations for these current carriers, and the reversal
potentials as well as the calculated permeability ratios for each anion
are summarized in Table 2. Replacing
external chloride with glutamate or
I
shifted the reversal
potential toward more positive potentials. However,
glutamate decreased the outward currents and had little effect on the
inward ones, whereas I
blocked both the outward and inward currents. In the presence of
Br
, the reversal potential
shifted toward negative values. Finally, the sequence for the linear
forskolin-sensitive conductance was Br
>>
Cl
> I
> glutamate.
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To further characterize the
Cl current induced by
forskolin, we tested three anion channel blockers which were separately added to the bathing solution. Figure 3,
C and
D, shows that inhibition of the whole
cell Cl
current occurred
following the addition of NPPB and DPC. The inhibitory effect of
10
4 M NPPB was 83 ± 6%
(n = 4) and was reversible, and that
of 10
3 M DPC was 97 ± 2% (n = 4) and irreversible. In
contrast, forskolin-stimulated currents were not significantly modified
by exposure to 10
3 M DIDS
(Fig. 3B).
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125I
efflux experiments.
The presence of a Cl
conductance activated by forskolin was also assayed using iodide efflux
measurements. In a first series of experiments, DC1 cells were grown on
collagen-coated petri dishes, and apical effluxes were measured after
loading the cells with
125I
.
Figure 4A
presents the
125I
efflux rate constant (as a percentage of the initial value at time t = 1 min) as a function of time.
Under control conditions, the efflux of
125I
from the monolayer into the bathing solution was independent of time,
with the efflux rate constant being 4.37 ± 0.06 × 10
2
min
1 (mean ± SE,
n = 70). The addition of forskolin to
the control solution caused a rapid increase in
125I
efflux, which reached a maximum value 4 min after the forskolin application. When DPC (1 mM) was added to a medium containing forskolin, no increase in efflux was observed. In contrast, DIDS (1 mM)
did not significantly modified the forskolin-stimulated efflux (Fig.
4A).
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Effects of Adenosine on
Cl Permeability in Cultured
DC1 Cells
Whole cell experiments. As illustrated
in Fig.
6B, the
perfusion of 10 µM adenosine induced a linear
Cl current with a maximum
effect at 2-3 min after onset of the perfusion. The reversal
potential of the stimulated current was 3.6 ± 0.7 mV, with a mean
conductance of 5.1 ± 0.6 nS and the current level at +80 mV of 415 ± 54 pA (n = 26) (Fig.
6F). The adenosine-sensitive Cl
current was further
investigated with anion substitution experiments. The results reported
in Fig. 6 show that I
strongly blocked the current (Fig.
6E), whereas it was increased by
Br
(Fig.
6C). Moreover, glutamate almost
completely suppressed the outward current (Fig.
6D). The estimated relative anion
permeability was Br
> Cl
> I
> glutamate (Table 2).
The sensitivity of the adenosine-induced Cl
currents to NPPB, DPC,
and DIDS was also tested. Exposure of the cells to
standard solutions in the presence of 0.1 mM NPPB or 1 mM DPC inhibited
the activated current by 81 ± 11%
(n = 4) and 88 ± 3%
(n = 3), respectively (Fig.
7,
C-E).
In contrast, the application of
10
3 M DIDS did not
significantly inhibit the activated current (Fig. 7,
B and
E).
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To further analyze the nature of the adenosine receptor implicated in
the increase of Cl
conductance, we tested the effect of NECA. NECA is a potent
A2 agonist that is known to
increase cAMP levels (5) in renal cells. Figure
8B
illustrates the effect of the external perfusion of 10 µM NECA. As
observed for adenosine, the agonist-induced linear
Cl
currents were maximally
developed within 3-4 min. The current-voltage relationship given
in Fig. 9E exhibits a reversal
potential of
0.2 ± 0.2 mV, a current amplitude at +80 mV of
478 ± 53 pA, and a slope conductance of 5.1 ± 0.8 nS
(n = 8), for currents measured in the
presence of NECA. The anion selectivity of the NECA-activated Cl
conductance was examined
by the replacement of anions in the bath solution. Results presented in
Fig. 8, C and
D, and Table 2 show an ion selectivity
sequence of Br
> Cl
> I
, which is similar to that
of the forskolin- or the adenosine-activated Cl
conductance.
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The effect of the adenosine receptor antagonist DPCPX on the
adenosine-stimulated Cl
current was then studied. As shown in the histogram of Fig.
9A, treatment of DC1 cells with 10 µM DPCPX completely inhibited the development of linear Cl
currents first induced by 10 µM adenosine. The action of DPCPX was
fully reversible such than in the presence of adenosine, after washout
of the antagonist, the cells developed a
Cl
conductance identical to
that recorded prior the addition of the antagonist (Fig.
9A).
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The results obtained with NECA and DPCPX confirm that the effect of
adenosine to increase Cl
conductance is mediated via interaction of the nucleoside with specific
adenosine receptors. Because NECA and DPCPX are not completely specific
to A2 receptors (9),
we studied the action of CGS-21680, which is very specific to the
A2A adenosine receptor subtype. As
for adenosine and NECA, 10 µM CGS-21680 increased linear
Cl
currents within 2-3
min (Fig. 10,
A and
B). In this experimental series, the
currents reversed at 0 ± 2.4 mV, the maximal current at +80 mV was
328 ± 58 mV, and the slope conductance was 3.3 ± 0.2 nS
(n = 8) (Fig.
10E). The main characteristics of
this conductance are described in Fig. 10,
C-E.
Substitution of bath Cl
with I
shifted the reversal
potential toward more positive values
[Erev = +21 ± 6 mV; relative I
permeability
(PI/PCl) = 0.43 ± 0.10; n = 3]. The
application of 1 mM DIDS did not modify the activated
Cl
conductance (Fig. 10,
C and
E). Overall, the currents induced by
adenosine, NECA, or CGS-21680 resemble those induced by forskolin. This
observation is consistent with the fact that stimulation of
A2 receptors induced the
production of cAMP via adenylate cyclase activation. Because an
increase in intracellular cAMP activates PKA, the effect of a PKI was
studied. The histogram in Fig. 9B shows current responses of DC1 cells to CGS-21680 in the presence or
absence of 100 µM PKI in the pipette solution. PKI clearly abolished
the activation of Cl
currents induced by CGS-21680.
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Several reports have shown that
Cl channels are also
regulated by G proteins (23, 30). To determine whether these proteins might participate in the transduction signal responsible for increasing linear Cl
currents, the
effect of a nonhydrolyzable GTP analog (GTP
S) was checked. GTP
S
(100 µM) was added to the pipette solution, and currents were
continuously recorded. Immediately after the establishment of the whole
cell recording, small currents were observed (Fig.
9C). These currents increased with
time and reached a peak value after about 6 min (Fig.
9C). The currents developed at this
time presented a linear current-voltage relationship with an
Erev =
0.4 ± 0.7 mV, a current amplitude at +80 mV of 335 ± 50 pA, and a
slope conductance of 3.4 ± 0.7 nS
(n = 6). They were insensitive to the
external application of 1 mM DIDS (Fig. 9C). Replacement of bath
Cl
with glutamate shifted
the reversal potential toward the positive values and strongly
inhibited the outward currents (data not given). In view of the
coupling of A2 adenosine receptors
to G protein, the effect of CGS-21680 was tested in the
presence of guanosine 5'-O-(2-thiodiphosphate) (GDP
S) (100 µM) in the pipette solution. GDP
S is an analog of GDP
that prevented G proteins from activating effectors. As
shown in Fig. 9D, GDP
S completely
prevented the activation of
Cl
currents induced by the
A2 agonist.
125I
efflux experiments.
The iodide efflux technique was subsequently used to examine the
presence of a Cl
conductance activated by adenosine. DC1 cells were grown on
collagen-coated petri dishes, and apical effluxes were measured after
the cells had been loaded with
125I
.
Figure
11A
shows the
125I
efflux rate constant measured as a function of time. The addition of
adenosine evoked a biphasic increase in
125I
efflux. An early transient increase in efflux that occurred within 1 min was followed by a sustained response. Figure
11A also shows that in the presence
of DIDS (1 mM), the transient increase was abolished while the
sustained increase was not significantly modified. In contrast, the
application of DPC (1 mM) completely abolished the adenosine-induced
125I
efflux. Thereafter, various agonists of the adenosine receptors were
also checked. The A1
receptor-selective ligand CPA induced a rapid transient increase in
125I
efflux, whereas NECA, which is more selective toward
A2 receptors, induced a more
gradual increase in
125I
efflux (Fig. 11B).
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DISCUSSION |
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Primary cultures of microdissected nephron segments provide a useful
tool for the study of ion channels and transporters present in apical
membranes. Over the past 10 years or so, we have used this technique to
investigate the nature and the role of several ion channels in proximal
convoluted tubule (22), cortical ascending limb (21), and DCTb (25) in
primary cultures. Notably, our more recent studies have revealed
information concerning the presence of at least three different
Cl channels in cultured
DCTb cells (4, 27, 34). Of these channels, one has been shown to
exhibit characteristics similar to those of the CFTR. However, there
are several disadvantages linked to the use of primary cultures of DCTb
cells, including the fact that the quantity of cells per culture is
relatively low and that the cells have a limited life span. These
features significantly constrain the number of experimental approaches that can be used when attempting to elucidate properties of these cells. Therefore, to further characterize the properties of the renal
CFTR Cl
channel, it was
necessary to overcome these difficulties. For this purpose, we
developed cells lines by transecting cultured DCTb cells with the pSV3
neo plasmid. This plasmid contains the bacterial gene neo that confers
resistance to the antibiotic G418 plus the DNA from the early region of
SV40 (31). The introduction of pSV3
neo by calcium precipitation or
lipofectin techniques enabled the isolation of 20 cells lines that
survived when subcultured in neomycin-containing media. All of these
cells lines presented with an epithelial morphology.
Because we were interested in the expression of CFTR
Cl channels in these cells,
we developed a screening test based on
Cl
efflux determination.
The Cl
permeability of
apical membranes was estimated by the measurement of intracellular SPQ
fluorescence on confluent monolayers. This technique has been described
in detail in previous studies (4, 34). Of the different immortalized
monolayers, the DC1 cell line was particularly interesting, because of
the fact that the application of forskolin strongly increased the
Cl
permeability in the
apical membrane only, indicating the presence of
Cl
channels sensitive to
cAMP. To further investigate the presence of CFTR in DC1 cells, the
expression of CFTR mRNA was investigated using RT-PCR. The two primers
yielded a RT-PCR product of expected size. This PCR product was indeed
a portion of the CFTR, because it exhibited 100% homology with the
rabbit sequence. Moreover, the levels of CFTR mRNA expressed in DC1
cells were comparable to those detected in primary cultures of DCTb cells.
On the basis of these observations, the effect of forskolin on
Cl conductance was tested
in DC1 cells by using the patch-clamp technique to measure whole cell
currents. The data clearly demonstrated that forskolin was able to
induce Cl
currents. In
unstimulated cells, the amplitude of the currents was small, and the
extracellular perfusion of forskolin activated a linear current. Most
of the current recorded in stimulated cells was carried by
Cl
, as confirmed by the
strongly reduced outward current recorded after the removal of
extracellular Cl
.
The halide selectivity sequence makes it possible to recognize the
different types of Cl
channel. In DC1 cells, the sequence for the forskolin-stimulated Cl
current was
Br
> Cl
>>I
. Our data are
consistent with a low iodide relative permeability and with an
inhibitory effect of I
, as
is seen for other Cl
channels including CFTR (8). The sensitivity to various anion channel
blockers is also an indication of the nature of the channels. We
therefore tested the effects of NPPB, DPC, and DIDS. Of the three, DPC
had the greatest inhibitory effect. Finally, the forskolin-stimulated Cl
conductance was quite
insensitive to DIDS. Taken together, these results demonstrate that
forskolin activated a time-independent macroscopic
Cl
current. The linear
current-voltage relationship, the anion selectivity sequence, and the
blocker sensitivity profile strongly suggest that the macroscopic
current recorded in DC1 cells flows through cAMP-activated CFTR
Cl
channels. These
characteristics are very similar to those reported previously in
primary cultures of DCTb cells (25), although the stimulated
Cl
conductance was lower in
DC1 cells. Whether this difference was due to a difference in the
number and/or in the conductance of unitary channels remained
to be determined.
In contrast to primary cultures of microdissected DCTb, DC1 cell line
had the advantage of generating a high yield of cells, thus making it
possible to obtain sufficiently large surface areas of epithelial cell
layers for effective measuring of tracer effluxes. In epithelial cells,
125I
was believed to be a good indicator of
Cl
movements. The
125I
efflux experiments described here provide further evidence that forskolin increased Cl
efflux through the apical membrane only. The stimulated efflux was not
instantaneous but reached a peak value over 3-4 min. The efflux
rate then decreased slowly toward the basal value. This special time
course has also been seen in other CFTR-expressing epithelial cells
(36). The fact that the forskolin-stimulated efflux was completely
blocked by DPC and insensitive to DIDS confirms that this efflux
occurred through a CFTR-like conductance.
Nevertheless, the present results do not prove that
Cl currents or
Cl
effluxes were directly
mediated by CFTR, and additional experiments will be necessary to
confirm the correlation between apical CFTR and the cAMP-activated
Cl
conductance response.
However, it is interesting to note that, of the 20 different cell lines
obtained, only four clones (DC1, DC6, DC7, and DC9) presented an
increase in Cl
efflux after
the application of forskolin and expressed significant levels of CFTR
mRNA. The other clones were not sensitive to forskolin and did not
exhibit positive RT-PCR products. This comparison between functional
data and the PCR results strengthens the possibility that the
cAMP-sensitive Cl
conductance may be via CFTR channels.
Adenosine is an effector molecule that modulates the production of cAMP
in various tissues including kidney (1, 5, 7). We therefore examined
the effect of the nucleoside on the
Cl conductance of DC1
cells. As can be seen from results, exposure to adenosine was followed
within 1-2 min by an increase in whole cell currents. In most
cells, the adenosine-stimulated currents reversed near 0 mV, were time
independent, and exhibited an ohmic current-voltage relationship. These
properties are very similar to those of the forskolin-activated
Cl
current discussed above.
The halide selectivities and blocker effects confirm the identity of
the two types of macroscopic current.
In the present study the activation of a linear
Cl current by adenosine was
mainly due to activation of the adenosine A2
receptor because the agonist NECA was able to mimic the effect of
adenosine. Moreover, according to the classification given by Collis
and Hourani (9), the effect of the agonist CGS-21680
indicated that the receptor was very likely an
A2A subtype. The observation that
the antagonist DPCPX blocked the activation of adenosine-invoked Cl
currents is not contradictory to
the involvement of an A2A
receptor. In fact, DPCPX is a more specific
A1 and A2B
agonist that also antagonizes A2A
receptor when used at high concentrations (9).
PKA was also implicated in the CGS-21680-induced linear
Cl currents, since the PKA inhibitor
PKI-(5
24) completely blocked the effect of the agonist.
Finally, the experiments using GTP
S and GDP
S showed that G
proteins were involved in the control of linear
Cl
conductance induced by
adenosine in DC1 cells. Taken together, these observations are
consistent with the conclusion that adenosine activates the CFTR
Cl
channel by a sequential
pathway involving the A2A
receptor, G proteins, adenylate cyclase, and PKA.
In many epithelia, the regulation of ion transport by adenosine
implicated both A1 and
A2 receptors. In
Cl-secreting tissues, the
activation of A2 adenosine
receptors stimulates Cl
secretion (2, 10, 26), whereas activation of
A1 receptors inhibits
Cl
transport (10, 12, 15,
16). The presence of A1 and
A2 adenosine receptors in renal
tissue has also been demonstrated in the perfused thick ascending limb
(3) and extensively studied in several cell lines or primary cultures
(1, 5, 7, 17, 24, 30, 32). However, the physiological role of these
receptors in the kidney is not fully understood. In rabbit renal CCD
cells (30), as well as in A6 cells (7) in culture, adenosine was found
to activate an apical Cl
channel by a pathway involving A1
receptors, whereas in the rat medullary thick ascending limb (MTAL),
adenosine was described as a potent inhibitor of
Cl
reabsorption (3).
A1 receptors also play an
important role in the regulation of distal nephron
Na+ reabsorption, although
contrasting effects have been reported in the literature. For example,
A1 stimulation by adenosine
increased (19) or did not modify (7)
Na+ reabsorption in A6 cells and
inhibited Na+ reabsorption in
primary cultures of rat IMCD (13). Concerning the
A2 receptors, although their
presence has been demonstrated in cultured MTAL (5) and
CCD (1) cells, few studies have been performed on renal
tissue to elucidate their physiological role. Indeed, the presence of
Cl
channels activated by
cAMP suggests that adenosine could control Cl
movements via
A2 receptors coupled to a cAMP
cascade. However, to the best of our knowledge, the present study is
the first to demonstrate that adenosine may interact with
A2A receptors in the distal tubule
to induce apical Cl
secretion through CFTR-like channels.
The fact that coexpression of A1
and A2 receptors on the same cells
has already been reported for cultured CCD (1) and IMCD (37) cells
and also for the A6 cell line (7) led us to examine
whether this was the case for DC1 cells. In whole cell experiments, 10 µM adenosine induced linear
Cl currents in 40% of the
cells tested but also nonlinear currents in 10% of the cells. The
nonlinear conductance presented an outwardly rectifying current-voltage
relationship and time-dependent inactivation at depolarizing
potentials. These characteristics are typical of volume-sensitive
Cl
currents reported for
epithelial cells (14). In cultured CCD, it has already been postulated
that adenosine activates a
Cl
channel implicated in
volume regulation via a pathway involving the
A1 receptor (30). It could
therefore be possible that the activation of nonlinear currents in DC1
cells was due to A1 receptor stimulation. Our observation that 10 µM NECA induced both types of
currents, whereas 0.1 µM NECA induced only linear current, also
indicates the presence of A1
receptors. In this way, at high NECA concentrations, both
A1 and
A2 receptors will be recruited, whereas at low concentrations only
A2 will be stimulated.
Iodide efflux experiments carried out in this study confirmed the
presence of A1 receptors in DC1
cells. The biphasic increase in
125I
efflux induced by adenosine could be due to the stimulation of both
A1 and
A2 receptors. The transient
increase was blocked by DIDS and as such could correspond to an
increase in Cl
flux through
volume-sensitive Cl
channels controlled by A1
receptors. The A1-selective
agonist CPA reproduced this transient increase, thus confirming the
nature of the receptor. Moreover, in a recent study performed in DC1 cells, we found that adenosine enhanced cytosolic
Ca2+ via the stimulation of
A1 receptor (data not given). The
sustained increase in 125I efflux
was DIDS insensitive and could be due to the activation of
CFTR-Cl
channels controlled
by A2 receptors via the cAMP pathway.
Our findings clearly show that adenosine exerts its effect only when
applied to the basolateral side of the DC1 monolayer, suggesting that
the adenoreceptors implicated in the control of Cl movements are located
mainly in the basolateral membrane. Reports in the literature confirm
that the spatial distribution of
A1 and
A2 receptors in the apical or
basolateral membrane is yet to be fully elucidated. For example,
A2 receptors coupled to cAMP production have been found in the basolateral membrane (7, 26), whereas
A1 receptors have been localized
in apical membrane (7, 24, 30). However, in some studies,
A2 receptors were located in the
apical membrane (19) and A1
receptors in the basolateral membrane (11, 24). The reasons for these
apparent discrepancies are not clear. Moreover, details concerning the exact subtypes of P1 purinoceptors
implicated in renal ion transports are incomplete. At present, at least
two classes of A1 and three classes of A2 receptors have been
found in several cell types (9). It could be hypothesized that for a
given class of receptors, A1 or
A2, the subtype located in the
basolateral membrane differs from that in the apical membrane.
The question arises as to whether the data obtained with DC1 cell line
could be extrapolated to the original DCTb in primary culture.
Concerning CFTR, cultured DC1 and DCTb cells exhibited a cAMP-sensitive
increase in whole cell Cl
conductance in the apical membrane and expressed CFTR transcripts. The
biophysical features of this CFTR-like
Cl
conductance were closely
identical in both cell types. Concerning the role of adenosine, recent
experiments performed on cultured DCTb cells show that extracellular
application of adenosine or NECA activated a linear
Cl
current, the
characteristics of which resembled those observed in DC1 cells (data
not given). Taken together, these observations indicated that the
transfected cells are representative of primary culture of DCTb cells.
The physiological role of an apical
Cl conductance in distal
tubule remains unclear. In vivo, however, NaCl secretion by the very
early portion of distal tubule has been already reported in the rat
(29). Although the pathway for this NaCl secretion has not yet been
addressed, there is the possibility that
Cl
is secreted via a
conductive pathway in the apical membrane. In fact, under normal
physiological conditions, the apical
Cl
concentration in the
distal fluid is low, and cAMP-coupled hormones might induce
Cl
secretion via CFTR-like
Cl
channels. Moreover,
studies using MDCK transfected cells demonstrate that CFTR decreases
the permeability of epithelial Na+
channels by acting as a cAMP-dependent negative regulator (33). In the
distal cells, it is therefore possible that the stimulation of CFTR by
cAMP induced NaCl secretion by increasing the
Cl
secretion and blocking
the Na+ reabsorption across the
apical membrane.
Adenosine has been demonstrated to be released by the kidney during ischemia and sodium loading. According to the above-proposed mechanism, the stimulation of A2 adenosine receptors could induce an increase of NaCl excretion by the distal tubule via the production of cAMP.
In conclusion, we have developed a cell line from primary cultures of
DCTb cells. This cell line expresses characteristics of the native
epithelium and can be used as a model for studying the functional
characteristics of electrolyte transport in the distal tubule. Notably,
CFTR Cl channels are
present in the apical membrane of DC1 cells and are an important
pathway for forskolin-stimulated increases in Cl
conductance. This
conductance could also be enhanced by extracellularly applied adenosine
via A2A receptors located in the
basolateral membrane and coupled to adenylate cyclase.
![]() |
FOOTNOTES |
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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 28 May 1998; accepted in final form 18 September 1998.
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