Extracellular adenosine modulates a volume-sensitive-like chloride conductance in immortalized rabbit DC1 cells

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

Unité 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 AND METHODS
RESULTS
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Cl- currents induced by cell swelling were characterized in an immortalized cell line (DC1) derived from rabbit distal bright convoluted tubule by the whole cell patch-clamp techniques and by 125I- efflux experiments. Exposure of cells to a hypotonic shock induced outwardly rectifying Cl- currents that could be blocked by 0.1 mM 5-nitro-2-(3-phenylpropyl-amino)benzoic acid, 1 mM DIDS, and by 1 mM diphenylamine-2-carboxylate. 125I- efflux experiments showed that exposure of the monolayer to a hypotonic medium increased 125I- loss. Preincubation of cells with LaCl3 or GdCl3 prevented the development of the response. The addition of 10 µM adenosine to the bath medium activated outwardly rectifying whole cell currents similar to those recorded after hypotonic shock. This conductance was inhibited by the A1-receptor antagonist 8-cyclopentyl-1,3-diproxylxanthine (DPCPX), LaCl3, or GdCl3 and was activated by GTPgamma S. The selective A1-receptor agonist N6-cyclopentyladenosine (CPA) mimicked the effect of hypotonicity on 125I- efflux. The CPA-induced increase of 125I- efflux was inhibited by DPCPX and external application of LaCl3 or GdCl3. Adenosine also enhanced Mn2+ influx across the apical membrane. Overall, the data show that DC1 cells possess swelling- and adenosine-activated Cl- conductances that share identical characteristics. The activation of both conductances involved Ca2+ entry into the cell, probably via mechanosensitive Ca2+ channels. The effects of adenosine are mediated via A1 receptors that could mediate the purinergic regulation of the volume-sensitive Cl- conductance.

cell volume; kidney; A1 receptor


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MOST ANIMAL CELLS POSSESS volume-regulation mechanisms as a response to changes in extracellular osmolarity (14). After cell swelling, volume is restored by the loss of ions and other osmolytes, whereas the concomitant loss of water induces a regulatory volume decrease (RVD). Swelling-activated Cl- channels have been observed in a wide variety of cell types (6, 9). We have previously demonstrated that proximal and distal convoluted tubules in primary culture exhibit Cl- and K+ currents when exposed to a hypotonic shock. Moreover, these cells are sensitive to changes in osmolarity of the bathing medium and are capable of RVD on exposure to hypotonic conditions (27, 31).

Although most reports in the literature agree that the movement of Cl- across the cell membrane is essential in this regulation, the precise mechanism underlying the activation of swelling-induced Cl- conductances is largely unknown. Of the factors that could modulate these channels, Ca2+ plays an important role, but conflicting results have been reported concerning the origin of Ca2+ in this mechanism. For example, some studies have highlighted a positive role for cytoplasmic Ca2+ due to its release from internal cellular stores (11, 17), whereas other studies have indicated that Ca2+ entry across the plasma membrane could be one of the mediators of the hypotonicity-induced activation of Cl- channels (18, 24).

In distal bright convoluted tubule cells (DCTb) in primary culture, external Ca2+ is clearly implicated in the regulation of volume-sensitive Cl- channels (27). With this in mind, we have undertaken studies to elucidate the mechanisms governing this influx of Ca2+ from the extracellular medium and have used the DC1 cell line immortalized from primary cultures of DCTb for the purpose (28). These cells express both A1 and A2 receptors, and adenosine can be shown to activate an apical CFTR conductance via a pathway involving A2A receptors and adenylate cyclase (28). Moreover, this nucleoside also enhances an increase in calcium influx via A1 receptors (25).

In the kidney, several observations indicate that A1 receptors decrease cAMP production but also enhance the inositol phosphate cascade and the liberation of Ca2+ from internal Ca2+ stores (1). Moreover, according to Schwiebert et al. (29) adenosine could activate a Cl- channel implicated in the cell volume regulation of the cortical collecting ducts. In view of these results, we have carried out whole cell patch-clamp and 125I- efflux experiments to investigate whether the A1 receptor could be implicated in the activation of a swelling-induced Cl- conductance in the DC1 cell line. It can be shown here that DC1 cells exhibit a swelling-activated Cl- conductance and express A1 receptors. Second, stimulation of this receptor by adenosine activates a Cl- current having properties of the swelling-activated Cl- conductance. The mechanism of this stimulation implicates Ca2+ entry into the cell probably via mechanosensitive Ca2+ channels.


    MATERIALS AND METHODS
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Cultures

The DC1 renal cell line was obtained from primary cultures of rabbit distal convoluted tubules after transfection of cells with the PSV3 neoplasmid and G418 selection. The technique of transformation of primary cultures is described in a previous paper (28). Cultures were seeded on collagen-coated 35-mm petri dishes or on collagen-coated permeable Millipore filters filled with a culture medium composed of equal quantities of DMEM and Ham's F-12 (GIBCO BRL, Grand Island, NY). The medium was supplemented with 15 mM NaHCO3, 20 mM HEPES at pH 7.4, 2 mM glutamine, 5 mg/l insulin, 50 nM dexamethasone, 10 µg/l epidermal growth factor, 5 mg/l transferrin, 30 nM sodium selenite, and 10 nM triiodothyronine. Cultures were maintained at 37°C in a 5% CO2-95% air-water-saturated atmosphere. DC1 cells were used between passages 12 and 25.

Whole Cell Patch-Clamp Experiments

Whole cell currents were recorded from DC1 cells (3-4 days of age) grown on collagen-coated supports and 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) by using a two-stage vertical puller (PP 83, Narishige, Tokyo, Japan) and filled with an N-methyl-D-glucamine chloride (NMDGCl) solution. Cells were observed by using an inverted microscope (Zeiss IM35), the stage of which was equipped with a water robot micromanipulator (WR 89, Narishige). The patch pipette was connected via an Ag/AgCl wire to the headstage of an RK 400 patch amplifier (Biologic). After formation of a gigaseal, the fast compensation system of the amplifier was used to compensate for the intrinsic input capacitance of the headstage and the pipette capacitance. The membrane was ruptured by additional suction to achieve the conventional whole cell configuration. The cell capacitance 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. Extracellular solutions delivering pharmacological agents were perfused into the bath medium by using a four-channel glass pipette, the tip of which was placed as close as possible to the clamped cell.

Voltage-clamp commands, data acquisition, and data analysis were controlled via a computer equipped with a Digidata 1200 interface (Axon Instruments). pCLAMP software (versions 5.51 and 6.0; Axon Instruments) was used to generate whole cell current-voltage (I-V) relationships, with the membrane currents resulting from voltage pulse stimuli filtered at 1 kHz, sampled at 2.5 kHz, and stored directly onto the computer's hard disk. Cells were held at a holding potential of -50 mV, and 400-ms pulses from -100 to +120 mV were applied in increments of 20 mV every 2 s.

125I- Efflux from DC1 Monolayers

125I- efflux experiments were performed in DC1 cells grown on collagen-coated 35-mm petri dishes. Cells (3-5 days of age) were loaded with 125I- (10 µCi/ml) for 3-4 h at room temperature in an RPMI 1640 medium (Life Technologies) with no added sodium bicarbonate, buffered with 10 mM HEPES at pH 7.4, and supplemented with 10 mM NaI. After being rinsed in unlabeled RPMI to remove unloaded isotope from the extracellular space, 125I- effluxes were measured simultaneously. Every 2 min, all of the external of medium (2 ml of 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 radioactivity in cells that had been 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, which provide the fraction of total radioactivity lost per unit time
(K)<SUB>t</SUB>=<FR><NU>(C)<SUB><IT>t</IT></SUB></NU><DE>C<SUB>ep</SUB><IT>+</IT>{<IT>&Sgr;</IT><SUP><IT>t+1</IT></SUP><SUB><IT>i=t</IT></SUB> [(C)<SUB><IT>i</IT></SUB>]}<IT> + ½</IT>[(C)<SUB><IT>t</IT></SUB>]</DE></FR><IT>·</IT><FENCE><FR><NU><IT>1</IT></NU><DE><IT>T</IT></DE></FR></FENCE>
where (K)t represent the efflux rate constants respectively at time t, (C)t represents the radioactivity lost at time t and during the period T, and Cep is the radioactivity remaining in the solubilized epithelia at the conclusion of measurements.

Fluorescence Experiments

Image analysis was performed by using an optical system composed of a Zeiss ICM405 inverted microscope and a Zeiss ×40 objective for epifluorescent measurements with a 75-W xenon lamp. The excitation beam was filtered through narrow-band filters (350, 360, and 380 nm, Oriel) mounted in a motorized wheel (Lambda 10-2, Sutter Instrument) equipped with a shutter that controlled exposure times. The incident and emitted fluorescence radiation beams were separated by using a Zeiss chromatic beam splitter. Fluorescence emission was detected via a 510-nm narrow-band filter (Oriel). The transmitted light images were viewed by an 8-bit extended ISIS camera (Extended ISIS, Photonic Science, Sussex, UK) equipped with an integration module to maximize the signal-to-noise ratio. The video signal from the camera was fed into an image processor integrated with a DT2867 image card (Data Translation) installed in a Pentium 100-MHz PC. The processor converted the video signal into 512 lines of 768 square pixels/line and 8 bits/pixel. The 8-bit information obtained for each pixel represented one of a possible 256 gray levels, ranging from 0 (for black) to 255 (for white). Image acquisition and analysis were performed by using AIW software (version 2.1, Axon Instruments). Final calculations were made by using Excel software (Microsoft).

Intracellular Ca2+ measurements were carried out on 4- to 8-day-old DC1 cell monolayers grown in petri dishes and loaded for 45 min at room temperature with a solution of 5 µM fura 2-acetoxymethyl ester (AM) containing 0.01% pluronic acid. The cells were then washed with a NaCl solution containing (in mM) 140 NaCl, 5 KCl, 1 MgSO4, 5 glucose, and 20 HEPES, pH 7.40, with 1 Tris. Cells were successively excited at 350 and 380 nm, with images digitized and stored on the computer hard disk for later analysis. Each raw image was the result of an integration of four to five frames averaged four times. The acquisition rate used was one image every 10 s. For each monolayer, intracellular Ca2+ was monitored in 18-20 random cells. Ca2+ concentrations ([Ca2+]i) were calculated from the dual wavelength-to-fluorescence ratio by using the Grynkiewicz equation (13).

Mn2+ influx measurements were made on 4- to 8-day-old DC1 cell monolayers grown in petri dishes and loaded with fura 2-AM as described above. Fluorescence quenching experiments were carried out by the addition of 50 µM MnCl2 to the NaCl medium. Fluorescent measurements were performed at 360 nm (the isosbestic point for fura 2, where the fluorescence signal is independent of the free calcium concentration). The level of Mg2+ influx was quantified by the slope of the calibration line describing quenching kinetics. Each raw image was the result of an integration of six frames averaged four times. The acquisition rate was one image every 5 s.

Chemicals

Adenosine was prepared as a 10 mM stock solution in NaCl buffer. N6-cyclopentyladenosine (CPA) and 8-cyclopentyl-1,3-diproxylxanthine (DPCPX; Sigma) were prepared as 10 mM stock solutions in DMSO. A stock solution of 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB; Calbiochem) was prepared at 100 mM in DMSO and used at a concentration of 0.1 mM in final solutions. Diphenylamine-2-carboxylate (DPC; Aldrich) was prepared as 1 M stock solution in DMSO and dissolved at 1 mM in the incubation medium. DIDS (Sigma) was dissolved in the final solutions at a concentration of 1 mM. Fura 2-AM (Molecular Probes) was dissolved at 3 mM in DMSO and added to the loading solution at a final concentration of 5 µM plus 0.01% pluronic acid.

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


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Effects of Hypotonic Shock on Cl- Permeability of Cultured DC1 Cells

To study the effect of hypotonicity on Cl- permeability of DC1 cells, two series of experiments were carried out. First, the nature of the currents induced by hypotonic shock was determined by using whole cell patch-clamp experiments. Second, the effects of hypotonic shock on membrane Cl- permeability were assessed by using the 125I- efflux technique. Then, the results obtained by both techniques were compared.

Whole cell experiments. Nature of the Cl- current induced by hypotonicity. Whole cell currents were recorded with Ca2+-free pipette solutions containing 140 mM NMDGCl, whereas hyperosmotic extracellular solutions contained 140 mM NMDGCl and 50 mM mannitol. After successful gigaseal 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 then applying voltage steps of 400-ms duration every 2 s from -100 to 120 mV in 20-mV increments. When the osmolarity of the extracellular solution was 350 mosmol/kgH2O, the voltage-step protocol elicited small currents (Fig 1A) that changed linearly with the membrane voltage and had a slope conductance of 0.57 ± 0.02 nS and a reversal potential of -1.5 ± 0.9 mV (Fig 1D). The amplitude of the currents was 69 ± 11 pA (n = 15) at +100 mV. Because of its small amplitude, the nature of the current was not analyzed further, although its reversal potential suggests that Cl- may well have been the charge carrier.


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Fig. 1.   Characteristics of whole cell Cl- currents induced by exposure of cells to a hypotonic solution. Membrane voltage was held at -50 mV and stepped to test potential values between -100 mV and +120 mV in 20-mV increments. Pipette and bath contained N-methyl-D-glucamine chloride (NMDGCl) solutions. A: control whole cell currents recorded from DC1 cells with an extracellular solution osmolarity of 350 mosmol/kgH2O (n = 14). B: whole cell currents were recorded 6 min after the onset of the hypotonic shock induced with an extracellular solution osmolarity of 290 mosmol/kgH2O (n = 11). C: whole cell currents were recorded 2 min after replacement of the extracellular solution with the 350 mosmol/kgH2O solution (n = 5). D and E: current-voltage relationships measured 11 (D) and 390 ms (E) after onset of pulse. Results are means ± SE of (n) cells from 10 monolayers.

In a previous study, we demonstrated that cultured DCTb cells exhibit swelling-activated whole cell Cl- currents (27). For this reason, the effect on Cl- permeability of DC1 cells after exposure of cells to a hypotonic solution was investigated here. The monolayer was thus perfused with a 290 mosmol/kgH2O solution, after which an increase in the whole cell current was observed within 1 min in ~40% of the cells. The currents reached a maximum amplitude after 6 min (Fig. 1B). At this time, the initial current recorded at a membrane potential of 100 mV was 2.5 times the current recorded at -100 mV (at 100 mV, ICl- = 1,774 ± 187 pA; at -100 mV, ICl- = 715.1 ± 77.1 pA, n = 11).

These large, outwardly rectifying currents showed time-dependent inactivation at depolarizing step potentials. The I-V relationships for initial currents (measured 11 ms after the start of the step pulse) and for steady-state currents (measured at 390 ms) are illustrated in Fig. 1, D and E. The reversal potential was very close to that of Cl- (Erev = -1.54 ± 0.47) and, in the absence of permeable cations in the pipette, the outward current was carried by Cl-. When the cells were reexposed to a hyperosmotic solution, the currents returned to the control levels within 2-3 min (Fig. 1C).

To study the anion permeability of the cell membrane after exposure to a hypotonic solution, all except 2 mM of the Cl- in the bath solution was replaced with I-, Br-, or glutamate. Table 2 summarizes reversal potential values as well as the calculated permeability ratios obtained for a given anion. Replacing external Cl- with I- or Br- shifted the reversal potential toward more negative values. Thus the sequence for this conductance was I- = Br- > Cl-.

                              
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Table 2.   Effect of substitution of extracellular Cl- by various anions on Erev during hypotonic shock

To further characterize the Cl- currents, we tested three anion channel blockers that were separately added to the bathing solution. Figure 2, B-D, shows that inhibition of the whole cell Cl- currents occurred within 2 min after the addition of DIDS, NPPB, or DPC to the bathing solution. The effects of these blockers were reversible on washing (data not shown). The I-V relationships for initial currents are displayed in Fig. 2E. Overall, 1 mM DIDS, 0.1 mM NPPB, or 1 mM DPC reversibly inhibited both initial inward (%inhibition at -100 mV: DIDS = 58.8 ± 7.1, n = 4; NPPB = 79.3 ± 2.7, n = 4; DPC = 69.3 ± 1.0, n = 3) and outward currents (%inhibition at +100 mV: DIDS = 82.2 ± 1.7, n = 4; NPPB = 75.6 ± 4.3, n = 4; DPC = 77.7 ± 4.7, n = 3). The effects of DIDS were therefore voltage dependent.


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Fig. 2.   Effect of Cl- channel inhibitors on hypotonicity-induced Cl- currents. Pipette and bath contained NMDGCl solutions. A: whole cell currents recorded at the maximum of the response (n = 11). B: with extracellular perfusion of 1 mM DIDS (n = 4). C: with extracellular perfusion of 0.1 mM 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB; n = 4). D: with extracellular perfusion of 1 mM diphenylamine-2-carboxylate (DPC; n = 3). E: average current-voltage relationships measured 11 ms after onset of pulse. Values are means ± SE of n cells from 8 monolayers.

To determine whether P1 receptors could be implicated in the control of swelling activated Cl- currents, hypotonic shock was performed in the presence of P1-selective receptor antagonist DPCPX. As shown in Fig. 3, treatment of DC1 cells with 10 µM DPCPX completely inhibited the development of outward Cl- currents induced by hypotonicity.


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Fig. 3.   Regulation of the Cl- conductance induced by hypotonic shock. Initial currents were recorded at 80 mV, 11 ms after the onset of pulse. Each experiment was carried out according the following protocol: control currents were recorded in hypertonic medium, and hypotonic shock was induced. Once the Cl- currents were maximally developed, they were inhibited by replacing the hypotonic medium by a hypertonic medium in the presence of the inhibitors, and a new hypotonic shock was applied and the currents were analyzed. Currents were triggered in cells pretreated with 10 µM nifedipine (n = 4), 50 µM LaCl3 (n = 6), 400 µM GdCl3 (n = 3), or 10 µM 8-cyclopentyl-1,3-diproxylxanthine (DPCPX; n = 3). Cl- currents were also recorded in the absence of external Ca2+ (n = 7). Values are means ± SE.

ROLE OF CA2+ in the regulation of Cl- currents induced by hypotonicity. To eliminate the implication of cytosolic Ca2+ in the development of hypotonicity-induced Cl- currents, the experiments described above were performed by using pipette solutions containing 5 mM EGTA without additional Ca2+.

To further analyze the role of Ca2+, the effects of extracellular Ca2+ on the development of hypotonicity-induced Cl- currents were also tested. The histogram in Fig. 3 shows that, when hypotonic shock was carried out in the absence of bath Ca2+, the development of the Cl- current was significantly impaired. These experiments indicate that extracellular Ca2+ was required to activate the swelling-activated Cl- conductance. To determine whether this activation was related to Ca2+ influx across the cell membrane, we then performed experiments using known channel blockers. Figure 3 clearly shows that, when the hypotonic shock was performed in the presence of nifedipine (10 µM), La3+ (50 µM), or Gd3+ (400 µM), the development of Cl- currents was completely impaired.

To provide further evidence that external Ca2+ entry was implicated in the hypotonic response, we studied the role of ionomycin in the development of the Cl- conductance. In a previous paper (26), we demonstrated that ionomycin induced an increase of divalent cation entry in DCTb cells in primary culture. Interestingly, this entry was strongly reduced in the presence of La3+ (10 µM). Therefore, we took advantage of this property of ionomycin to study its effects on whole cell Cl- currents developed in the presence or the absence of [Ca2+]i. To avoid the apparition of swelling-induced Cl- conductance, the currents were recorded in monolayers continuously perfused with hypertonic NMDG solution. In the experiments in Fig. 4A, whole cell currents were recorded in the absence of EGTA in the pipette solution. After control macroscopic currents were recorded, 2 µM ionomycin was added to the bathing NMDG solution, and the stimulated currents were recorded after 1 min. In these conditions the cytoplasmic free Ca2+ rose to 1.10 ± 0.25 µM (n = 9). Figure 4B shows that in the presence of ionomycin the currents increased during depolarizing voltage pulses. The kinetics of the macroscopic current were clearly time dependent for depolarizing potentials with a slowly developing component. The corresponding I-V relationships for steady-state activated currents are given in Fig. 4C. The average reversal potential of the currents was close to 0 mV (n = 6). The steady-state current presented marked outward rectification with an inward current at -100 mV of -80 ± 2 pA and an outward current at +100 mV of 307 ± 27 pA (n = 5). In the experiments in Fig. 4D, whole cell currents were recorded in the presence 5 mM EGTA in the pipette solution. In five different monolayers, the addition of 2 µM ionomycin induced the activation of Cl- currents within 3-4 min. Under these conditions ionomycin also enhanced divalent-cation influx across the membrane (26) The ionomycin-activated Cl- currents showed time-dependent inactivation at depolarizing steps potentials >60 mV (Fig. 4E) and displayed an outwardly rectified instantaneous I-V plot (Fig. 4F) with a reversal potential close to 0 mV. Overall, these currents were quite similar to those induced by hypotonic shock.


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Fig. 4.   Cl- currents induced by extracellular ionomycin in hypertonic NMDG solution. A and B: whole cell currents were recorded in the absence of EGTA in the pipette solution. C: average current-voltage relationships measured 390 ms after the onset of pulse. Values are means ± SE of 5 cells from 5 different monolayers. D and E: whole cell currents were recorded in the presence of 5 mM EGTA in the pipette solution. F: average current-voltage relationships measured 11 ms after the onset of pulse. Values are means ± SE of 5 cells from 5 different monolayers.

The results of these experiments indicated that the nature of the Cl- currents induced by ionomycin depends on the ability of cells to increase their [Ca2+]i. Moreover, they clearly confirm that an entry of Ca2+ in the presence of intracellular EGTA mimics the effect of an hypertonic shock on the Cl- conductance of DC1 cells.

125I- efflux experiments. Effect of DC1 cell exposure to a hypotonic medium. The presence of a Cl- conductance activated by hypotonic shock was also assayed by using I- efflux measurements. In one set of experiments, DC1 cells were grown on collagen-coated petri dishes, and apical effluxes were measured after the cells were loaded with 125I-. Figure 5 presents the 125I- efflux rate constant (expressed as a percentage of the initial value at time t = 1 min) as a function of time. Initial effluxes, measured in the isotonic control medium, were independent of time, the efflux rate remaining constant at 5.03 ± 0.12 · 10-2/min (mean ± SE, n = 70). 125I- efflux variations were then examined when cultured monolayers were exposed to a hypotonic medium (290 mosmol/kgH2O), which was obtained by diluting the control solution. Figure 5A shows that exposure of the monolayers to a hypotonic medium led to an increase in 125I- efflux, which reached a maximum level 3-4 min after onset of the exposure. The mean increase in the 125I- efflux was 246.9 ± 37.7% (n = 9).


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Fig. 5.   Effect of hypotonic shock on 125I- effluxes. Experiments were performed with cells grown on collagen-coated petri dishes. A: after an initial control period, total effluxes were measured in an isotonic bath medium (n = 3 monolayers) or in a hypotonic bath medium (n = 9 monolayers) in the presence or the absence of either 1 mM DIDS (n = 3 monolayers) or 1 mM DPC (n = 3 monolayers). B: after an initial control period, total effluxes were measured in a hypotonic bath medium containing 10 µM LaCl3 (n = 3 monolayers) or 400 µM gadolinium (n = 4 monolayers). Values are means ± SE.

PHARMACOLOGICAL PROPERTIES OF THE 125I- efflux stimulated by hypotonicity. To characterize the stimulated 125I- efflux under hypotonic conditions, the effects of Cl- channel inhibitors were studied. The action of 1 mM DIDS and 1 mM DPC is shown in Fig. 5A. Both channel blockers almost completely abolished the effect of the hypotonic medium on the 125I- effluxes.

REGULATION OF THE 125I- efflux induced by hypotonic shock. To elucidate the mechanism by which hypotonicity activates 125I- efflux, we tried to determine what role Ca2+ might play in this effect. The effects of extracellular Ca2+ on the development of the hypotonicity-induced 125I- efflux were therefore investigated. In a second set of experiments, the exposure of DC1 cells to a hypotonic medium was carried out in the presence of 10 µM La3+ in the bath solution. This trivalent cation had an inhibitory action on 125I- efflux stimulated by the exposure of cells to the hypotonic medium (Fig. 5B).

Exposure of DC1 cells to Gd3+ (400 µM) prevented the 125I- efflux in response to exposure to a hypotonic medium, suggesting the involvement of a mechanosensitive Ca2+ channel in the hypotonicity-induced response (Fig. 5B).

Measurement of [Ca2+]i During Hypotonic Shock

We reasoned that the presence of EGTA in the pipette solution did not prevent localized increases in Ca2+ in the vicinity of the plasma membrane secondary to a Ca2+ influx. Fluorescence experiments using fura 2-loaded DC1 cells were therefore carried out to follow cytosolic Ca2+ variations during a hypotonic shock. Figure 6 shows the effect of hypotonic solution on intracellular calcium (i.e., [Ca2+]i) under different experimental conditions. When the cells were bathed with an isotonic NaCl solution (300 mosmol/kgH20) containing 1 mM CaCl2, the resting [Ca2+]i averaged 30.8 ± 5.2 nM (n = 288 cells from 16 monolayers). Swelling the DC1 cells with hypotonic NaCl solution (200 mosmol/kgH2O) induced a transient increase in [Ca2+]i, which reached a maximum value of 120.1 ± 25.1 nM and returned close to the control value within 5 min (Fig. 6A). After the cells were rinsed with isotonic NaCl solution, a second hypotonic shock could be reinduced. However, the amplitude of the [Ca2+]i increase was systematically reduced with [Ca2+]i reaching 71.4 ± 10.5 nM (Fig. 6A). Overall, the results in Fig. 6 show that hypotonic shock induced a significant increase in [Ca2+]i in 75 ± 4% of the 230 cells analyzed in 13 different monolayers. In the experiments in Fig. 6, B and C, the second hypotonic shock was performed in the presence of La3+(50 µM) or Gd3+ (400 µM) respectively. Under these conditions, the second shock did not evoke [Ca2+]i changes in all the cells of the three monolayers studied. These observations strongly suggest that the increase in [Ca2+]i induced by hypotonicity required a Ca2+ influx from the extracellular medium. To eliminate the participation of intracellular Ca2+ store release in the swelling-induced [Ca2+]i increase, experiments were then performed in the presence of thapsigargin. As illustrated in Fig. 6D, emptying intracellular Ca2+ pools did not prevent the increase in [Ca2+]i during the hypotonic shock.


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Fig. 6.   Effect of hypotonic shock on intracellular free calcium concentration ([Ca2+]i) of fura 2-loaded DC1 cells. Fura 2 fluorescence was monitored and converted to [Ca2+]i values as described in MATERIALS AND METHODS. A: hypotonic shock was induced by perfusing a hypotonic NaCl solution of 200 mosmol/kgH2O. Monolayers were then perfused with an isotonic NaCl solution of 300 mosmol/kgH2O, and a second hypotonic shock was applied (n = 4 monolayers). B, C, and E: the second hypotonic shock was induced in the presence of 50 µM LaCl3 (n = 3 monolayers; B), 400 µM GdCl3 (n = 3 monolayers; C), and 10 µM DPCPX (n = 3 monolayers; E). D: hypotonic shock was induced in the presence of 10 nM thapsigargin (n = 3 monolayers).

Effects of Adenosine and P1 Purinoceptor Agonists on Cl- Permeability of Cultured DC1 Cells

Cl- secretion is regulated by adenosine in several types of cells, including epithelia of the kidney (7, 21, 29). As we have previously demonstrated that the stimulation of A2 adenosine receptors enhances cAMP production and Cl- conductance in DC1 cells (28), experiments were therefore performed to determine whether adenosine activates other types of Cl- currents in DC1 cells.

Whole cell experiments. Nature of the Cl- current induced by adenosine in cultured DC1 cells. In 40% of the trials carried out, adenosine activated a linear cystic fibrosis transmembrane conductance regulator (CFTR)-like Cl- conductance (28). In 30% of the trials, adenosine (10 µM) activated an outwardly rectifying Cl- conductance with a time-dependent inactivation at depolarizing potentials and with a maximal effect at 3-4 min (Fig. 7B). At this time, the initial current recorded at 100 mV was 1.9 times the current measured at -100 mV (at 100 mV, ICl-= 479.3 ± 27.4 pA; at -100 mV, ICl- = -256.5 ± 16.2 pA, n = 18). The reversal potential of the stimulated current was -1.16 ± 3.4 mV (n = 18). The I-V relationships for initial currents measured 11 ms after the onset of the voltage pulse are illustrated in Fig. 7E.


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Fig. 7.   Characteristics of adenosine-induced whole cell Cl- currents in cultured DC1 cells. Membrane voltage was held at -50 mV and stepped to test potential values between -100 mV and +120 mV in 20-mV increments. Pipette and bath contained NMDGCl solutions. A: whole cell currents from unstimulated DC1 cells (n = 18). B: whole cell currents in the presence of 10 µM adenosine in the bath solution (n = 18). C: whole cell currents recorded during extracellular perfusion of 0.1 mM NPPB (n = 3). D: with extracellular perfusion of 1 mM DPC (n = 3). E: current-voltage relationships measured 11 ms after onset of pulse. Values are means ± SE of (n) cells from 10 monolayers.

The adenosine-sensitive Cl- current was further investigated in a series of anion substitution experiments, the results of which are reported in Table 3. The estimated relative anion permeability recorded was Br- = I- > Cl-. The sensitivity of the adenosine-induced Cl- currents to NPPB and DPC was also tested. NPPB (0.1 mM) and DPC (1 mM) inhibited both initial inward (%inhibition at -100 mV: NPPB = 38.3 ± 24, n = 3; DPC = 70.5 ± 9.8, n = 3) and outward currents (%inhibition at +100 mV: NPPB = 54.6 ± 5.0, n = 3; DPC = 87.7 ± 3.2, n = 3) (Fig. 7, C-E). The characteristics of the adenosine-induced Cl- conductance are similar to those of the hypotonicity-induced Cl- conductance.

                              
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Table 3.   Effect of substitution of extracellular Cl- by various anions on Erev in the presence of extracellular adenosine

To determine whether the response to adenosine was via receptor-mediated mechanisms, we examined the effect of a P1- selective receptor antagonist DPCPX. As shown in the histogram in Fig. 10, treatment of DC1 cells with 10 µM DPCPX completely inhibited the development of outward Cl- currents induced by 10 µM adenosine. The action of DPCPX was fully reversible, such that in the presence of adenosine, after washout of the antagonist, the cells developed a Cl- conductance identical to that recorded before the addition of the antagonist (see Fig. 10).

G proteins have been shown to be involved in Cl- channel regulation in some cell lines (19, 29). To determine whether these proteins might participate in the signal transduction mechanism responsible for increasing outward Cl- currents, the effect of a nonhydrolyzable GTP analog (GTPgamma S) was examined. Representative records, where GTPgamma S (100 µM) was added to the pipette solution and currents were continuously recorded, are shown in Fig. 8. Immediately after establishment of the whole cell recording configuration, currents increased with time and reached a peak value after ~6 min. The currents developed at this time presented an outward I-V relationship with a reversal potential equal to -0.3 ± 0.9 mV, a current amplitude at +100 mV of 1,259 ± 260 pA and at -100 mV of -409 ± 85 pA (n = 6). These currents were sensitive to the external application of 1 mM DIDS (Fig. 8).


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Fig. 8.   Activation of outward Cl- current by 100 µM GTPgamma S in the pipette solution. A: whole cell conductance was monitored by sequentially clamping the cell at 0 and +20 mV for 200 ms from a holding potential voltage of -40 ms (n = 6). DIDS (1 mM) blocked the evoked current (n = 3). B: currents were recorded at +100 mV, 11 ms after the onset of the pulse. Each value represents the mean ± SE of (n) cells obtained from 4 different monolayers.

DCTB CELLS IN PRIMARY CULTURE. In 12% of the trials, adenosine (10 µM) activated an outwardly rectifying Cl- conductance with a time-dependent inactivation at depolarizing potentials and a maximal effect at 3-4 min after onset of the perfusion (Fig. 9B). At this time, the initial current recorded at 100 mV was 865 ± 55 pA; at -100 mV ICl- was -240 ± 31 pA. The reversal potential of the stimulated current was 6.6 ± 2.1 mV. The I-V relationships for initial currents measured 10 ms after the onset of the voltage pulse are illustrated in Fig. 9C.


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Fig. 9.   Characteristics of adenosine-induced whole cell Cl- currents in distal bright convoluted tubule cells (DCTb) cells in primary culture. Membrane voltage was held at -50 mV and stepped to test potential values between -100 mV and +120 mV in 20-mV increments. Pipette and bath contained NMDGCl solutions. A: whole cell currents from unstimulated cells (n = 4). B: whole cell currents in the presence of 10 µM adenosine in the bath solution (n = 3). C: current-voltage relationships measured 10 ms after onset of pulse. Values are means ± SE of (n) cells from 3 monolayers.

ROLE OF CA2+ in the regulation of Cl- currents induced by adenosine. Cl- currents induced by adenosine share identical characteristics with those induced by hypotonic shock. Because this swelling-activated Cl- conductance was sensitive to extracellular Ca2+, it was also interesting to analyze the role of Ca2+ on the development of adenosine-induced Cl- currents. As observed for swelling-activated Cl- currents, the adenosine-activated Cl- currents were completely impaired in the absence of bath Ca2+ or the presence of nifedipine (10 µM), La3+ (50 µM), or Gd3+(400 µM) (Fig. 10).


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Fig. 10.   Regulation of the Cl- conductance induced by adenosine. Treatment of cells with the various compounds was carried out before exposure to adenosine (adeno). Initial currents were recorded at 80 mV, 11 ms after the onset of pulse. Control currents were recorded in hyperosmotic solution and after application of 10 µM adenosine (n = 18). Currents were then triggered in cells pretreated with 10 µM nifedipine (n = 4), 50 µM LaCl3 (n = 6), 400 µM GdCl3 (n = 3), or 10 µM DPCPX (n = 4). Cl- currents were also recorded in the absence of external Ca2+ (n = 7). Values are means ± SE.

125I- efflux experiments. Because we have previously shown that adenosine causes a biphasic increase in 125I- efflux in DC1 cells, probably due to the stimulation of both A1 and A2 receptors (27), it was decided to study the effect of CPA on 125I- efflux. CPA is a metabolically stable adenosine analog that is a selective A1-receptor agonist. The increase in the rate of 125I- efflux induced by CPA was slow, reached a maximum level equal to 166 ± 12% of control (n = 9) after 3-min exposure to the agonist, then slowly returned to the control level within 5 min. (Fig. 11A). Overall, the kinetics of this increase were very similar to those evoked by the exposure of cells to hypotonic medium. The CPA-induced 125I- efflux was inhibited effectively by DIDS (1 mM) and DPC (1 mM) (Fig. 11A).


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Fig. 11.   Effect of N6-cyclopentyladenosine (CPA) on 125I- effluxes. Experiments were performed on cells grown on collagen-coated petri dishes. A: after an initial control period, total effluxes were measured in a bath medium in the absence (n = 3 monolayers) or presence of 10 µM CPA alone (n = 9 monolayers) or in a bath medium containing either CPA with 1 mM DIDS (n = 6 monolayers), or CPA with 1 mM DPC (n = 3 monolayers). B: inhibition of CPA-evoked 125I- effluxes by 10 µM DPCPX. After an initial control period, effluxes were measured in a bath medium containing either 10 µM CPA alone (n = 5 monolayers) or CPA with 10 µM DPCPX (n = 4 monolayers). C: after an initial control period, total effluxes were measured in a bath medium containing either 10 µM CPA alone (n = 4 monolayers), CPA with 10 µM LaCl3 (n = 4 monolayers), or CPA with 400 µM GdCl3 (n = 4 monolayers). Values are means ± SE.

The effects of other compounds on the CPA-induced 125I- efflux were also studied. As shown in Fig. 11B, the treatment of DC1 cells with the adenosine-receptor antagonist DPCPX (10 µM) completely inhibited the development of the 125I- efflux induced by CPA. The effects of extracellular Ca2+ on the development of CPA-induced Cl- currents were also studied, whereby the 125I- efflux was found to be strongly inhibited (Fig. 11C) when cells were stimulated in the presence of 10 µM La3+. As observed with La3+, Gd3+ (400 µM) abolished the action of CPA on 125I- efflux (Fig. 11C).

Measurement of [Ca2+]i During Adenosine Application

Experiments were then undertaken to describe the effect of exogenous adenosine on [Ca2+]i. In these experimental series, the [Ca2+]i of cultured DC1 cells maintained in a bathing solution with 1 mM Ca2+ was 51.9 ± 2.4 nM (n = 720 cells from 37 monolayers). As shown in Fig. 12A, the addition of 10 µM adenosine in NaCl buffer evoked a transient increase in [Ca2+]i, which took place within 20 s (maximum [Ca2+]i = 223.3 ± 6.4 nM, n = 171 cells from 12 monolayers). This response was observed in 80% of the analyzed cells (171 of 214 cells). Ten minutes after adenosine removal, a second application of 10 µM adenosine was unable to induce a second [Ca2+]i increase (Fig 12A). The experiments in Fig. 12, B and C, show that in the presence of La3+(50 µM) or Gd3+ (400 µM), adenosine did not increase [Ca2+]i. As already concluded for the swelling increase in [Ca2+]i, the present results indicate that the adenosine-induced increase in [Ca2+]i was dependent on a Ca2+ influx from the extracellular medium.


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Fig. 12.   Effect of 10 µM adenosine on [Ca2+]i of fura 2-loaded DC1 cells. Fura 2 fluorescence was monitored and converted to [Ca2+]i values as described in MATERIALS AND METHODS. A: 2 successive applications of 10 µM adenosine were performed (n = 3 monolayers). B, C, and D: the first application of 10 µM adenosine was performed in the presence of 50 µM LaCl3 (n = 3 monolayers), 400 µM GdCl3 (n = 3 monolayers), and 10 µM DPCPX (n = 3 monolayers), respectively. E: the first application of 10 µM adenosine was followed by a hypotonic shock (n = 7 monolayers). F: the application of 10 µM adenosine was followed by addition of 10 µM ATP. ATP was washed out and replaced by adenosine (n = 3 monolayers).

To confirm that the stimulatory action of adenosine on [Ca2+]i was mediated by the P1 receptor, additional experiments were performed in the presence of the P1-selective receptor antagonist DPCPX. As shown in Fig. 12D, treatment of the monolayer with DPCPX (10 µM) completely inhibited the action of 10 µM adenosine.

In another series, monolayers were stimulated with 10 µM adenosine then, 7-8 min after removal of the nucleoside from the bath solution, the cells were submitted to an hypotonic shock. Under these conditions, the hypotonic shock did not evoke an increase in [Ca2+]i (Fig. 12E).

To verify that after addition of adenosine cells were still responsive, the effects of 10 µM ATP were examined 30 s after the adenosine was added to the cells. The results in Fig. 12F clearly show that this subsequent addition of ATP induced a rapid and transient increase in [Ca2+]i. This increase was significantly higher than the one measured during adenosine stimulation (adenosine: [Ca2+]i = 193.0 ± 23 nM; ATP: [Ca2+]i = 291.2 ± 29 nM, P < 0.001, n = 54 cells from 3 monolayers).

In a last group of experiments, the stimulatory responses of a lower dose of adenosine were studied. The recordings in Fig. 13A illustrate the [Ca2+]i variations induced by 1 µM adenosine. As observed with higher concentration, the addition of 1 µM adenosine induced a transient increase in [Ca2+]i (maximum [Ca2+]i = 110.4 ± 10.3 nM, n = 102 cells from 6 monolayers). However, a subsequent addition of 1 µM adenosine still induced an increase in [Ca2+]i (maximum [Ca2+]i = 114.0 ± 10.8 nM, n = 102 cells from 6 monolayers). These successive increases were completely abolished by the application of Gd3+ (Fig. 13B) or nifedipine (Fig. 13C). Moreover, as illustrated in Fig. 13D, a first stimulation of [Ca2+]i with 1 µM adenosine could be followed by a stimulation induced by a hypotonic shock and then by a new stimulation with 1 µM adenosine.


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Fig. 13.   Effect of 1 µM adenosine on [Ca2+]i of fura 2-loaded DC1 cells. Fura 2 fluorescence was monitored and converted to [Ca2+]i values as described in MATERIALS AND METHODS. A: 2 successive applications of 1 µM adenosine were performed (n = 6 monolayers). B and C: applications of 1 µM adenosine were performed in the presence of 400 µM GdCl3 (n = 3 monolayers) and 10 µM nifedipine (n = 3 monolayers) D: the first application of 1 µM adenosine was followed by a hypotonic shock and by a second application of 1 µM adenosine ( n = 3 monolayers).

Mn2+ Influx Induced by Adenosine

The experiments described above indicated that extracellular Ca2+ was required to activate adenosine Cl- currents via A1 receptors. To provide evidence that adenosine induced an increase in divalent cation entry, we took advantage of the fact that fura 2 fluorescence can be quenched by Mn2+, a commonly used substitute for Ca2+. The fluorescence at 360 nm (isobestic point for fura 2) is independent of cytosolic Ca2+. Thus only fura 2 quenching due to Mn2+ uptake by the cells modifies the fluorescence signal at this wavelength. When cells were perfused with an NaCl solution containing MnCl2, an immediate decrease in fluorescence (0.12 ± 0.02 arbitrary fluorescence units/s, n = 5) was observed. As shown in Fig. 14, extracellular adenosine accelerated the rate of quenching (0.23 ± 0.04 fluorescence arbitrary units/s, n = 3; P < 0.05), indicating an enhanced influx of divalent cations. This uptake mechanism was blocked by 10 µM La3+ (rate of quenching: 0.12 ± 0.01; n = 3) , 400 µM Gd3+ (rate of quenching: 0.06 ± 0.01; n = 3) and by DPCPX (rate of quenching: 0.09 ± 0.01; n = 9) (Fig 14).


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Fig. 14.   Fura 2 fluorescence quenching by external Mn2+ manganese before and after addition of adenosine. The entry of Mn2+ into the cytoplasm was monitored by fluorescence quenching of fura 2-loaded cultures excited at 360 nm. MnCl2 was maintained in the extracellular medium throughout the experiment. Adenosine was added at the indicated time at a final concentration of 10 µM (n = 3 monolayers), either with 10 µM La3+ (n = 3 monolayers), 400 µM GdCl3 (n = 3), or 10 µM DPCPX (n = 9 monolayers).

Effects of ATP on Cl- Currents in Cultured DC1 Cells

In addition to adenosine, it has been postulated that ATP could activate a volume-sensitive Cl- conductance in human bronchial cells (33). To check for this possibility in DC1 cells, we studied the role of ATP in the control of whole cell Cl- currents developed in the presence or the absence of intracellular free Ca2+. In the experiments in Fig. 15A, whole cell currents were recorded with a low Ca2+ concentration in the pipette solution containing NMDGCl (Table 1). Under these conditions, the application of 10 µM ATP to the bath medium induced an increase in membrane currents within 1 min (Fig. 15B). The kinetics of the macroscopic current was strongly time dependent for depolarizing potentials. The corresponding I-V relationships for steady-state activated currents are given in Fig. 15D. The average reversal potential of the currents was close to 0 mV (n = 6). The steady-state current presented marked outward rectification with an inward current at -100 mV of -217 ± 76 pA and an outward current at +100 mV of 977 ± 173 pA (n = 3). In the experiments in Fig. 15E, whole cell currents were recorded in the presence of 5 mM EGTA in the pipette solution. ATP-activated Cl- currents showed time-dependent inactivation at depolarizing steps potentials of >60 mV (Fig. 15E) and displayed an outwardly rectified instantaneous I-V plot (Fig. 15H) with a reversal potential close to 0 mV. Overall, these currents were quite similar to those induced by hypotonic shock, ionomycin, or adenosine.


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Fig. 15.   Cl- currents induced by extracellular ATP in hypertonic NMDG solution. A, B, C, and D: whole cell currents were recorded in the absence of EGTA in the pipette solution. E, F, G, and H: whole cell currents were recorded in the presence of 5 mM EGTA in the pipette solution. A and E: Cl- currents recorded in control conditions. B and F: currents recorded after perfusion of 10 µM ATP. C and G: currents recorded after perfusion of 10 µM ATP in the presence of 1 mM DIDS. D and H: average current-voltage relationships measured 390 and 11 ms after the onset of pulse, respectively. Values are means ± SE of 3 cells from 3 different monolayers.

To analyze the nature of the receptor implicated in the ATP-induced Cl- currents, experiments were performed in the presence of purinergic receptor antagonists. The results are reported in Fig. 16. When the Cl- currents were measured in the absence of EGTA in the pipette solution, the effect of ATP was completely blocked by 100 µM of the P2-antagonist suramin whereas the action of ATP was not modified by 10 µM of the P1-antagonist DPCPX (Fig. 16A). By contrast, when the Cl- currents were recorded in the presence of 5 mM EGTA in the pipette solution, the effect of ATP was inhibited by DPCPX and not modified by suramin (Fig. 16B). Finally, two types of Cl- currents could be activated by ATP: Ca2+ dependent-like Cl- currents and swelling-sensitive-like Cl- currents.


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Fig. 16.   Effect of DPCPX and suramin on Cl- currents induced by extracellular ATP. Treatment of the cells with antagonists was carried out before exposure to ATP. Initial currents were recorded at 80 mV, 11 ms after the onset of pulse. Control currents were recorded in hyperosmotic solution and after application of 10 µM ATP. Currents were then triggered in cells pretreated with 10 µM DPCPX or 100 µM suramin. A: currents recorded in the presence of 5 mM EGTA in the pipette solution (n = 4). B: currents recorded in the absence of EGTA in the pipette solution (n = 3). Values are means ± SE of (n) cells from 3 or 4 different monolayers.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

An increase in cell volume activates a Cl- current in most mammalian cells. Cell swelling can be induced by challenging cells with either a hypotonic extracellular solution or a hypertonic intracellular solution. The present work describes a hypotonicity-induced Cl- conductance that shares similar biophysical and pharmacological features with that described in primary cultures of DCTb cells (27) and in other epithelial cells (12). Notably, the I-V relationship is outwardly rectifying, and a time-dependent inactivation is observed at the most positive membrane potentials (i.e., +80 mV).

The halide selectivity sequence of this current permits the different types of Cl- channels to be identified. In DC1 cells, the sequence for the hypotonicity-induced Cl- current was I- = Br- > Cl-, which is similar to that found in DCTb cells. The sensitivity to various anion channel blockers is also an indication of the nature of the channels. DIDS, NPPB, and DPC were found to inhibit the hypotonicity-induced Cl- current

In contrast to primary cultures of microdissected DCTb cells, the DC1 cell line has the advantage of generating a high yield of cells, thus making it possible to obtain sufficiently large surface areas of epithelial cell layers for the effective measurement of tracer effluxes. In epithelial cells, 125I- fluxes are believed to be a good indicator of Cl- ion movements across the cell membrane. When cultured monolayers were exposed to a hypotonic medium (290 mosmol/kgH2O), an increase in 125I- efflux, which reached a maximum level 3-4 min after exposure to the solution, was elicited. This increase was inhibited by Cl- channel blockers such as DIDS and DPC. Finally, both experimental approaches lead to clearly demonstrate the existence of swelling-activated Cl- permeability in cultured DC1 cells.

Despite abundant literature, the precise mechanisms underlying the activation of swelling-activated Cl- currents remain unclear. In the present study the finding that nifedipine, La3+, and Gd3+ strongly blocked both the 125I- efflux and the Cl- currents induced by the exposure of cells to a hypotonic solution indicated the involvement of external Ca2+ in the response. Thus it was interesting to check the effect of nucleotides in the development of Cl- currents because these molecules are known to modulate [Ca2+]i by controlling both Ca2+ entry and Ca2+ release from intracellular pools. For example, adenosine is an effector molecule that modulates cAMP production and intracellular Ca2+ in various tissues (25). Moreover, it has been proposed that this nucleoside could participate in the regulation of Cl- secretion in several types of epithelia, including kidney epithelia (5, 21, 29). Notably, we have previously shown the existence of A1 and A2 receptors in the basolateral membrane of DC1 cells, whereas adenosine has also been shown to activate an apical CFTR Cl- conductance in these cells via a pathway involving A2A receptors, G proteins, adenylate cyclase, and protein kinase (28). In this previous work, preliminary experiments clearly indicated that, besides the activation of CFTR Cl- currents, adenosine increased a second type of Cl- conductance, the nature of which remained to be determined. We therefore examined in the present study, the effect of adenosine via A1 receptors on Cl- conductance in DC1 cells. As can be seen from the results presented, the exposure of DC1 cells 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 outwardly rectifying, and exhibited time-dependent inactivation at depolarized membrane potentials. It could be concluded that the activation of an outwardly rectifying Cl- current by adenosine was mainly due to activation of the adenosine A1 receptor because the antagonist DPCPX blocked the activation of adenosine-evoked Cl- currents. Furthermore, these currents were also dependent on Ca2+ influx because they were totally inhibited in the presence of nifedipine, La3+, or Gd3+. I- efflux experiments confirmed the data obtained by using the whole cell patch-clamp technique.

Overall, the present experiments demonstrated that the A1 adenosine receptor could mediate the purinergic regulation of the volume-sensitive Cl- conductance. This hypothesis is strengthened by the following observations: first, the biophysical properties of Cl- permeability induced by adenosine were very similar to those of hypotonicity-activated Cl- currents; second, the A1-receptor antagonist DPCPX blocked the activation of Cl- currents by adenosine and the activation of 125I- efflux by CPA but also the swelling-induced Cl- currents; third, the selective A1-receptor agonist CPA (25) mimicked the effect of hypotonic shock on 125I- efflux; and fourth, adenosine and swelling-activated Cl- currents were mediated by Ca2+ influx through the cell membrane. The presence of A1 and A2 adenosine receptors in renal tissue has been demonstrated in the perfused thick ascending limb (3) and extensively studied in several cell lines or primary cultures (2, 5, 21, 29, 30). However, the physiological role of these receptors in the kidney is not fully understood. In rabbit renal cortical collecting duct cells (29), as well as in A6 cells (5) in culture, adenosine was found to activate an apical Cl- channel via a pathway involving A1 receptors, whereas in the rat medullary thick ascending limb 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 (16) or did not modify (5) Na+ reabsorption in A6 cells, whereas it was reported to inhibit Na+ reabsorption in primary cultures of rat inner medullary connecting tubule (3).

In several studies, an increase in cytosolic Ca2+ has been reported during hypotonic stress or during A1 receptor stimulation by adenosine (17, 18, 25). In the present study, adenosine and hypotonic shock increased [Ca2+]i. This increase was suppressed by Ca2+ channel blockers and by DPCPX, indicating that the action of both stimuli is mainly due to Ca2+ entry into the cell and depends on the integrity of the A1 receptor. On the basis of these results, it could be concluded that the increase in Cl- conductance observed during hypotonicity could be mediated by Ca2+ influx via A1 receptor stimulation. Nevertheless, it may be recalled that in the present study, the activation of a Cl- conductance by hypotonicity or by adenosine took place in the presence of a high concentration of EGTA in the pipette solution. The simplest interpretation of this finding could be that an increase in [Ca2+]i is not necessarily required to activate Cl- currents and that Ca2+ influx is sufficient per se to increase the Cl- conductance during hypotonicity or adenosine stimulation. However, it is also possible that the cells still responded by increasing their [Ca2+]i when the pipette solution contained 5 mM EGTA. The observations of Evans and Marty (8) shed light on this problem by indicating that with EGTA as a buffer a whole region of the cell could escape control by the Ca2+ buffer. Because this region could extend to a large part of the plasma membrane (8), a local transient increase in Ca2+ could arise in the presence of EGTA.

It remains that Ca2+ influx is an essential step in activating swelling- and adenosine-sensitive Cl- channels. To further support this hypothesis, we studied the effects of ionomycin and ATP in the activation of Cl- channels in hypertonic bathing medium. Both molecules are known to induce a strong increase in [Ca2+]i. When applied on DC1 cells, they induced two types of Cl- conductances depending on the presence of high EGTA concentrations in the pipette solution. In the absence of a chelator, the Cl- currents induced by ionomycin or ATP were roughly identical to the Ca2+ dependent Cl- currents previously found in cultured DCTb (3). These currents were likely stimulated by an increase in cytoplasmic Ca2+ due to Ca2+ release from intracellular stores. By contrast, in the presence of EGTA, the application of ionomycin or ATP increased the uptake of Mn2+. This increase is evidence of Ca2+ uptake across the membrane (5, 26). This maneuver induced large Cl- currents, which inactivated during depolarizing voltage steps. Although they are not fully characterized, these currents closely resemble the swelling-activated Cl- currents described in the present study. Finally, these data bring further evidence that Ca2+ entry across the plasma membrane could be one of the mediators of the activation of Cl- currents by hypotonicity. Moreover the fact that Gd3+ completely inhibited Cl- conductance activation and [Ca2+]i increase by exposure to hypotonic solution or adenosine suggests that Ca2+ influx occurs through mechanosensitive channels.

Stretch-activated Ca2+-permeable channels have been described in many epithelial cells (7), including those of the proximal tubule (10). The role of external Ca2+ has already been postulated by McCarty and O'Neil (18), who showed that RVD in proximal straight tubules was highly dependent on the extracellular Ca2+ concentration. Moreover, they concluded that Ca2+ channels could be responsible for a swelling-activated Ca2+ entry. Interestingly, a relationship among mechanical stress, Ca2+ entry, and Cl- currents has been recently demonstrated in cultured endothelial cells (23).

The experiments using GTPgamma S showed that G proteins were involved in the control of the outwardly rectifying Cl- conductance in DC1 cells. This result is in accordance with that of Schwiebert et al. (29), who reported that the stimulation of A1 receptors in a rabbit cortical collecting duct cell line activates a volume-sensitive Cl- channel, via a pathway involving phospholipase C, protein kinase C, and a G protein (29).

Finally, in addition to the mechanism involving adenylate cyclase and cAMP, adenosine might act via an alternative transduction mechanism in DC1 cells to activate a volume-sensitive Cl- conductance. It is possible that a mechanosensitive Ca2+ entry is the pathway for Cl- conductance activation in response to hypotonic shock or A1 purinoceptor stimulation.

If A1 purinergic receptors are linked to swelling-activated Cl- conductance, it remains to be understood how these receptors are stimulated during hypotonic cell swelling. It has been recently postulated that hypotonically induced ATP release could induce an autocrine stimulus for activating ion channels permeable to Cl- and organic osmolytes (19, 22). In the present study, ATP also induced swelling-induced-like Cl- current in the presence of 5 mM EGTA in the pipette solution. Interestingly, these currents were blocked by DPCPX but were insensitive to suramin, indicating that under these conditions the action of ATP was not through P2 receptors. The best explanation is that ATP could be degraded to adenosine, which then acts on the A1 receptors to activate Cl- conductance. This hypothesis matches with that of Musante et al. (22), who have demonstrated that hypotonic shock induces an autocrine release of ATP from airway cells. According to these authors, ATP is then hydrolyzed to adenosine on the extracellular side of the membrane, increasing the activity of volume-sensitive Cl- channels. Such a mechanism could well serve in DC1 cells because renal cells exhibit a high level of ectonucleotidases (15).

In our experiment, external ATP also induced Ca2+-sensitive Cl- currents via P2 receptors. However, although DC1 cells exhibited both A1 and P2 receptors (4, 26), hypotonic shock or adenosine application induced swelling Cl- currents only (data not shown), irrespective of the EGTA concentration in the pipette solution. It is therefore reasonable to postulate that ATP released during shock does not activate Ca2+-sensitive Cl- currents. Finally, in DC1 cells the activation of Ca2+-induced Cl- currents by ATP could be due to the release of Ca2+ from internal stores (4, 26) whereas activation of adenosine or swelling-sensitive Cl- conductance would be mainly due to Ca2+ influx.

In our study, two successive hypotonic shocks induced a [Ca2+]i increase as well as an activation of Cl- conductance. By contrast, a double application of 10 µM adenosine did not evoke a second rise in [Ca2+]i a second Cl- conductance increase. This inability of adenosine (10 µM) to trigger two successive responses could be the consequence of a desensitization of A1 receptors at micromolar nucleotide concentrations (16). However, the possibility of obtaining a second response to a hypotonic shock indicates that a part of the A1 receptor population remained potentially active. We hypothesized therefore that the quantity of adenosine produced during the first hypotonic shock was too weak to saturate the receptors. This idea is further supported by the fact that a second Ca2+ response was restored with a low adenosine concentration (1 µM).

At this stage, the question arises as to whether the data obtained with DC1 cell line could be extrapolated to the original DCTb in primary culture. Concerning volume-sensitive Cl- conductance, cultured DC1 and DCTb cells exhibited a hypotonicity-sensitive increase in whole cell Cl- conductance. The biophysical features of this Cl- conductance were closely identical in both cell types. Concerning the role of adenosine, experiments performed on cultured DCTb cells show that extracellular application of adenosine activated an outwardly rectifying Cl- current in 12% of the trial, the characteristics of which resembled those observed in DC1 cells. Taken together, these observations indicated that the DC1 cell line is representative of a primary culture of DCTb cells.

The physiological role of A1 receptors in the distal tubule has not yet been elucidated. However, adenosine might regulate cell volume by controlling the swelling-activated Cl- conductance. Schwiebert et al. (29) have proposed that, during ischemia, the release of adenosine could play an important role in cell volume regulation in the collecting tubule. This proposal could also be available in the distal tubule because we have demonstrated that distal cells exhibited swelling-activated Cl- and K+ channels implicated in the RVD phenomenon (27).

In conclusion, our study provides evidence that A1 receptors play an important role in the regulation of swelling-induced Cl- currents. It is suggested that hypotonic shock stimulates ATP release from the DC1 cells. A1 receptors are then activated by adenosine generated by the degradation of ATP by membrane ectoenzymes. This stimulation of A1 receptors induces an influx of extracellular Ca2+ through Gd3+-sensitive Ca2+ channels. Finally, the Ca2+ influx activates the Cl- channels. The mechanism of this activation is not fully elucidated. However, according to Verdon et al. (32), the Ca2+ influx during cell swelling would increase the [Ca2+]i in the vicinity of the cell membrane [this region of the cell escapes control by the Ca2+ buffer (8)]. This increase will be sufficient to activate protein kinase C. The protein kinase C, in turn, phosphorylates a regulatory protein. This protein could be the P-glycoprotein which is implicated in the control of swelling-sensitive Cl- conductance. Such a mechanism has been also proposed by Rubera et al. (27) in DCTb cells in primary culture.


    FOOTNOTES

Address for reprint requests and other correspondence: P. Poujeol, Unité Mixte de Recherche Centre National de la Recherche Scientifique 6548, Bâtiment Sciences Naturelles Université de Nice-Sophia Antipolis, Parc Valrose, O6108 Nice Cedex 2, France (E-mail: poujeol{at}unice.fr).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 10 February 2000; accepted in final form 18 September 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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