Regulation of cAMP-dependent chloride channels in DC1 immortalized rabbit distal tubule cells in culture

Isabelle Rubera, Michel Tauc, Catherine Verheecke-Mauze, Michel Bidet, Chantal Poujeol, Nicolas Touret, Béatrice Cuiller, and Philippe Poujeol

Unite Mixte de Recherche Centre National de la Recherche Scientifique 6548, Université de Nice-Sophia Antipolis, O6108 Nice Cedex 2, France

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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) (GTPgamma 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

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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).

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 MOmega ) were made from borosilicate capillary tubes (1.5 mm OD, 1.1 mm ID, Clay-Adams) using a two-stage vertical puller (model PP 83; Narishige, Tokyo, Japan) and filled with an N-methyl-D-glucamine chloride (NMDG-Cl) solution. Cells were observed by using an inverted microscope, the stage of which was equipped with a water robot micromanipulator (model WR 89, Narishige). The patch pipette was connected via an Ag/AgCl wire to the head stage of a patch amplifier (model RK 400, Biologic). After 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 (Cm) was compensated for by using settings available on the RK 400 amplifier. No series resistance compensation was applied, but experiments in which the series resistance was higher than 20 MOmega were discarded. Solutions were perfused in the extracellular bath by using a four-channel glass pipette, the tip of which was placed as close as possible to the clamped cell.

Data 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) of -50 mV, and 400-ms pulses from -100 to +120 mV were applied with increments of 20 mV every 2 s.

125I- Efflux from DC1 Monolayers

125I- efflux experiments were performed on DC1 cells grown on collagen-coated 35-mm petri dishes and on collagen-coated permeable filters. Cells (3-5 days of age) were loaded with 125I- (10 µCi /ml) for 3-4 h at room temperature in RPMI 1640 medium (Life Technologies) devoid of sodium bicarbonate, buffered with 10 mM HEPES at pH 7.4, and supplemented with 10 mM NaI. After a rinse in unlabeled RPMI to remove unloaded isotope from the extracellular space, apical and basolateral 125I- effluxes were measured simultaneously. Every 2 min, all of the external medium (2 ml of apical and 2 ml of basolateral RPMI) was collected and replaced by fresh medium. The remaining radioactivity in the epithelium at the end of the experiments was determined by counting the cells solubilized with 1% Triton X-100.

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.
(<IT>K</IT><SUB>a</SUB>)<SUB><IT>t</IT></SUB> = <FR><NU>(C<SUB>a</SUB>)<SUB><IT>t</IT></SUB></NU><DE>C<SUB>ep</SUB> + <FENCE><LIM><OP>∑</OP><LL><IT>i</IT> = <IT>t</IT></LL><UL><IT>t</IT> + 1</UL></LIM> [(C<SUB>a</SUB>)<SUB><IT>i</IT></SUB> + (C<SUB>b</SUB>)<SUB><IT>i</IT></SUB>]</FENCE><FENCE> + 1/2[(C<SUB>a</SUB>)<SUB><IT>t</IT></SUB> + (C<SUB>b</SUB>)<SUB><IT>t</IT></SUB>]</FENCE></DE></FR> ⋅ (1/<IT>T</IT> )
(<IT>K</IT><SUB>b</SUB>)<SUB><IT>t</IT></SUB> = <FR><NU>(C<SUB>b</SUB>)<SUB><IT>t</IT></SUB></NU><DE>C<SUB>ep</SUB> + <FENCE><LIM><OP>∑</OP><LL><IT>i</IT> = <IT>t</IT></LL><UL><IT>t</IT> + 1</UL></LIM> [(C<SUB>a</SUB>)<SUB><IT>i</IT></SUB> + (C<SUB>b</SUB>)<SUB><IT>i</IT></SUB>]</FENCE> + 1/2[(C<SUB>a</SUB>)<SUB><IT>t</IT></SUB> + (C<SUB>b</SUB>)<SUB><IT>t</IT></SUB>]</DE></FR> ⋅ (1/<IT>T</IT> )
where (Ka)t and (Kb)t are, respectively, the apical and basolateral efflux rate constants at time t; (Ca)t and (Cb)t are, respectively, the radioactivity lost from the apical and basolateral sides at time t and during the period T. Cep is the radioactivity remaining in the epithelium at the conclusion of measurements.

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: (Delta F/dt)/F0 · min-1, where Delta 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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Table 1.   Composition of solutions used in whole cell clamp experiments

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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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Fig. 1.   RT-PCR products using primers specific for rabbit cystic fibrosis transmembrane conductance regulator (CFTR) generated from cDNA from cultures of DC1 cells. Exon 13 primers (primers A and B) generated a band of 382 bp both in proximal convoluted tubule (PCT) and distal convoluted tubule (DCT). Molecular weight markers (phi X174/Hae III) were run in parallel at the left edge of the agarose gel. Lanes marked +RT correspond to products obtained from 100 and 500 ng total RNA. Lanes marked -RT provide a control in absence of initial reverse transcription for contaminating genomic DNA.

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

Whole cell experiments. Whole cell currents were recorded with Ca2+-free pipette solutions containing 140 mM NMDG-Cl, while hyperosmotic extracellular solutions contained 140 mM NaCl and 50 mM mannitol. After successful gigaohm seal formation, the whole cell configuration was obtained in 25% of cases. Voltage-clamp experiments were performed by holding the membrane potential at -50 mV and applying voltage steps of 400-ms duration every 2 s from -100 to 120 mV in 20-mV increments. In a large majority of cells, the voltage step protocol elicited small currents (Fig. 2A) that changed linearly with the membrane voltage and had a slope conductance of 0.78 ± 0.14 nS and a reversal potential of -1.8 ± 0.9 mV (Fig. 2F). The amplitude of the currents was 47 ± 8 pA (n = 22) at +80 mV. Because of its small amplitude, the nature of the current was not analyzed further, although its reversal potential indicates that Cl- may well be the charge carrier.


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Fig. 2.   Characteristics of forskolin-induced whole cell Cl- currents. With a hyperosmotic NaCl solution in bath and N-methyl-D-glucamine chloride (NMDG-Cl) solution in pipette, membrane potential was held at -50 mV and stepped to test potential values between -100 and +120 mV in 20-mV increments. Whole cell currents were measured from unstimulated DC1 cells (A) or in presence in bath solution of 10 µM forskolin alone (B) or in presence of ADO with Br- (C), I- (D), or glutamate (E). F: average current-voltage relationships measured 390 ms after onset of pulse, obtained from same cell at rest, during forskolin (FK) stimulation alone, and after chloride substitution. Each value is mean ± SE of n cells obtained from 12 monolayers.

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 10-5 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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Table 2.   Effect of substitution of extracellular Cl- by various anions on Erev in the presence of forskolin, adenosine, or NECA in cultured DC1 cells

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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Fig. 3.   Effect of chloride channel inhibitors on forskolin-activated whole cell currents. Whole cell currents were measured in presence in bath solution of 10 µM forskolin (FK) alone (A) or in presence of FK with extracellular perfusion of 1 mM DIDS (B), 0.1 mM 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, C), or 1 mM diphenylamine-2-carboxylate (DPC, D). E: average current-voltage relationships measured 390 ms after onset of voltage pulse, obtained from same cell at rest, during forskolin stimulation alone, and after blocker application. Each value is mean ± SE of n cells obtained from 7 different monolayers.

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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Fig. 4.   Effect of forskolin on 125I- effluxes. A: experiments were performed with cells growing on collagen-coated petri dishes. After an initial control period, effector was added at the time indicated by arrow. Total effluxes were measured in a bath medium containing either 10 µM forskolin alone (n = 12 monolayers) or with 1 mM DIDS (n = 3) or 1 mM DPC (n = 3). B: experiments were performed with cells growing on permeable Millipore supports. After an initial control period, apical and basolateral effluxes were measured in a bath medium containing 10 µM forskolin (n = 12 monolayers). K represents the efflux rate constant.

In a second series of experiments, DC1 cells were grown on permeable Millipore supports that permitted separate measurements to be made of 125I- effluxes across the apical and the basolateral membranes. Figure 4B shows the 125I- effluxes across both membranes. Under control conditions, (i.e., during the first 4 min prior to the application of forskolin) the apical and basolateral 125I- effluxes were independent of time. The basolateral efflux rate constant (6.49 ± 0.24 × 10-2 min-1; n = 12) exceeded by a factor of 1.25 the apical rate constant (5.15 ± 0.17 × 10-2 min-1; n = 12). Exposure of the monolayer to forskolin led to an increase in 125I- loss through the apical membrane only. Basolateral 125I- efflux was not significantly modified by the application of forskolin (Fig. 4B).

SPQ fluorescence experiments. The Cl- permeability of apical membranes of DC1 cells was estimated by the measurement of intracellular SPQ fluorescence on confluent monolayers. The passage of Cl- across the apical membrane was assessed by the addition or removal of Cl- from the bath solution. The cell monolayer was first perfused with a NaCl solution over both apical and basolateral compartments. After 5 min, the apical Cl- was replaced by a NaNO3 solution (see MATERIALS AND METHODS). The inset in Fig. 5 shows a typical recording of the time course of intracellular SPQ fluorescence determined in a DC1 monolayer. The same protocol was performed on three different monolayers. Upon apical Cl- removal, the relative SPQ fluorescence increased slowly due to Cl- efflux and then fell to near control levels when NO-3 was replaced once again by Cl-. The addition of forskolin promptly increased the rates of Cl- efflux and influx previously induced by the Cl- removal and addition. The initial rates of relative Cl- efflux and influx are shown in Fig. 5, which provides a comparison between DC1 and primary cultures of DCTb cells. In DC1 cells, exposure to forskolin elicited a significant increase in Cl- permeability, with Cl- efflux increasing fivefold and Cl- influx increasing fourfold. In primary cultures of DCTb cells, the effect of forskolin to increase Cl- fluxes was of the same order of magnitude.


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Fig. 5.   6-Methoxy-1-(3-sulfonatopropyl)quinolinium (SPQ) fluorescence experiments. Effects of forskolin on relative Cl- efflux and influx through apical membrane of SPQ-loaded distal bright convoluted tubule (DCTb) and DC1 cells. Cl- efflux was induced by replacement of apical NaCl solution with NaNO3 solution, and Cl- influx was induced by removal of apical NaNO3 solution. Basolateral compartment was continuously perfused with NaCl solution containing 0.1 mM NPPB. Open columns represent values of Cl- fluxes in control conditions. Solid columns represent experimental conditions. Values (units/min) are means ± SE of n monolayers. Inset: representative tracings of effects of forskolin on intracellular SPQ fluorescence (sequence of buffer substitution in apical compartment is indicated at top). Forskolin (10 µM) was added to both compartments 2 min prior to the second NaCl substitution. Basolateral compartment was continuously perfused with NaCl solution containing 0.1 mM NPPB. ** P < 0.02, significantly different from control values (paired Student's t-test).

Effects of Adenosine on Cl- Permeability in Cultured DC1 Cells

Because Cl- secretion is regulated by adenosine in several types of epithelia including epithelia of the kidney (6, 7, 24, 30), and as it is known that the stimulation of A2 adenosine receptors enhances cAMP production (5); experiments were therefore conducted to determine whether adenosine activates Cl- currents in 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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Fig. 6.   Characteristics of adenosine-induced whole cell Cl- currents. With a hyperosmotic NaCl solution in bath and NMDG-Cl solution in pipette, membrane potential was held at -50 mV and stepped to test potential values between -100 and +120 mV in 20-mV increments. Whole cell currents were measured from unstimulated DC1 cells (A) or in presence in bath solution of 10 µM adenosine (ADO) alone (B) or in presence of ADO with Br- (C), glutamate (D), or I- (E). F: average current-voltage relationships measured 390 ms after onset of pulse, obtained from same cell at rest, during forskolin stimulation alone, and after chloride substitution. Each value is mean ± SE of n cells obtained from 46 monolayers.


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Fig. 7.   Effect of chloride channel inhibitors on adenosine-activated whole cell currents. Whole cell currents were measured in presence in bath solution of 10 µM adenosine (ADO) alone (A) or in presence of ADO with extracellular perfusion of 1 mM DIDS (B), 0.1 mM NPPB (C), or 1 mM DPC (D). E: average current-voltage relationships measured 390 ms after onset of pulse, obtained from same cell at rest, during adenosine stimulation alone, and after application of blocker. Each value is mean ± SE of n cells obtained from 8 different monolayers.

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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Fig. 8.   Characteristics of 5'-(N-ethylcarboxamido)adenosine (NECA)-induced whole cell Cl- currents. With a hyperosmotic NaCl solution in bath and NMDG-Cl solution in pipette, membrane potential was held at -50 mV and stepped to test potential values between -100 mV and +120 mV in 20-mV increments. Whole cell currents were measured from unstimulated DC1 cells (A) or in presence in bath solution of 10 µM NECA alone (B) or in presence of NECA with Br- (C) or I- (D). E: average current-voltage relationships measured 390 ms after onset of pulse, obtained from same cell at rest, during NECA stimulation alone, and after chloride substitution. Each value is mean ± SE of n cells obtained from 7 monolayers.

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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Fig. 9.   Regulation of Cl- conductance in DC1 cells as induced by different drugs. Currents were recorded at +80 mV, 390 ms after onset of pulse. A: effect of 10 µM 8-cyclopentyl-1,3-diproxylxanthine (DPCPX) on Cl- current induced by 10 µM adenosine (n = 6). B: effect of 10 µM protein kinase A inhibitor (PKI) in pipette solution on Cl- current induced by 10 µM CGS-21680 (n = 6). C: activation of linear Cl- current by 100 µM guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S) in pipette solution. D: effect of 100 µM guanosine 5'-O-(2-thiodiphosphate) (GDPbeta S) in pipette solution on Cl- current induced by 10 µM CGS-21680 (n = 8).

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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Fig. 10.   Characteristics of CGS-21680-induced whole cell Cl- currents. With a hyperosmotic NaCl solution in bath and NMDG-Cl solution in pipette, membrane potential was held at -50 mV and stepped to test potential values between -100 and +120 mV in 20-mV increments. Whole cell currents were measured from unstimulated DC1 cells (A) or in presence in bath solution of 10 µM CGS-21680 alone (B) or in presence of CGS with 1 mM DIDS (C) or after substitution of bath Cl- by I- (D). E: average current-voltage relationships measured 390 ms after onset of pulse, obtained from same cell at rest, during CGS stimulation alone, in presence of DIDS, and after chloride substitution. Each value is mean ± SE of n cells obtained from 7 monolayers.

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 (GTPgamma S) was checked. GTPgamma 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) (GDPbeta S) (100 µM) in the pipette solution. GDPbeta S is an analog of GDP that prevented G proteins from activating effectors. As shown in Fig. 9D, GDPbeta 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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Fig. 11.   Effect of adenosine on 125I- effluxes. Experiments were performed on cells growing on collagen-coated petri dishes. After an initial control period, effector was added at the time indicated by arrow. A: effluxes were measured in a bath medium containing either 10 µM adenosine alone (n = 4 monolayers) or adenosine with either 1 mM DIDS (n = 3) or 1 mM DPC (n = 3). B: effluxes were measured in a bath medium containing 10 µM N6-cyclopentyladenosine (CPA; n = 4 monolayers) or 10 µM NECA (n = 10 monolayers).

To determine the location of adenosine receptors, 125I- efflux experiments were performed on DC1 cells grown on permeable Millipore supports. Figure 12 shows the 125I- effluxes across both membranes when adenosine was applied either to the apical (Fig. 12A) or to the basolateral (Fig. 12B) side of the monolayer. Adenosine induced an increase in apical 125I- efflux only when added to the basolateral bathing medium. Moreover, basolateral efflux was never modified by adenosine.


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Fig. 12.   Polarity of adenosine receptor. Effluxes of 125I were measured in cells growing on permeable Millipore supports. After an initial control period, apical and basolateral effluxes were measured in presence of 10 µM adenosine in apical solution (A) or in basolateral solution (B). Each experiment was performed on 3 different monolayers.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 GTPgamma S and GDPbeta 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

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.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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