Extracellular ATP increases [Ca2+]i in distal tubule cells. II. Activation of a Ca2+-dependent Clminus conductance

Isabelle Rubera, Michel Tauc, Michel Bidet, Catherine Verheecke-Mauze, Guy De Renzis, Chantal Poujeol, Béatrice Cuiller, and Philippe Poujeol

Unité Mixte de Recherche 6548, Centre National de la Recherche Scientifique, Université de Nice-Sophia Antipolis, 06108 Nice Cedex 2, France


    ABSTRACT
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We characterized Cl- conductance activated by extracellular ATP in an immortalized cell line derived from rabbit distal bright convoluted tubule (DC1). 125I- efflux experiments showed that ATP increased 125I- loss with an EC50 = 3 µM. Diphenylamine-2-carboxylate (10-3 M) and NPPB (10-4 M) abolished the 125I- efflux. Preincubation with 10 µM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester or 10-7 M thapsigargin inhibited the effect of ATP. Ionomycin (2 µM) increased 125I- efflux with a time course similar to that of extracellular ATP, suggesting that the response is dependent on the intracellular Ca2+ concentration ([Ca2+]i). The ATP agonist potency order was ATP >=  UTP > ATPgamma S. Suramin (500 µM) inhibited the ATP-induced 125I- efflux, consistent with P2 purinoceptors. 125I- effluxes from cells grown on permeable filters suggest that ATP induced an apical efflux that was mediated via apical P2 receptors. Whole cell experiments showed that ATP (100 µM) activated outwardly rectifying Cl- currents in the presence of 8-cyclopentyl-1,3-dipropylxanthine, excluding the involvement of P1 receptors. Ionomycin activated Cl- currents similar to those developed with ATP. These results demonstrate the presence of a purinergic regulatory mechanism involving ATP, apical P2Y2 receptors, and Ca2+ mobilization for apical Cl- conductance in a distal tubule cell line.

kidney; intracellular calcium


    INTRODUCTION
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INTRODUCTION
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ION CHANNELS IN EPITHELIAL cells play an essential role among various mechanisms of transcellular Cl- transport. As in secretory epithelia, where they have been extensively studied, Cl- channels with diverse and distinct properties have been described for the kidney. In previous papers we have shown that primary cultures of the rabbit distal tubule express three different Cl- conductances regulated by cAMP, cytosolic Ca2+, or by cell swelling (3, 30, 36). Moreover, proof of the existence of both A1 and A2 receptors in the basolateral membrane of distal tubule cells has been made possible by using the DC1 cell line immortalized from primary cultures of these cells (31).

Adenosine activates an apical cystic fibrosis transmembrane conductance regulator (CFTR) Cl- conductance by a pathway involving A2A receptors, G proteins, adenylate cyclase, and protein kinase A (31). It has also been postulated that adenosine induces an increase in calcium influx via A1 receptors, which, in turn, stimulates swelling-activated Cl- channels (29). In attempting to elucidate properties of purinergic receptors in the distal tubule, we were able to identify, in a preceding paper (1), a P2Y2 receptor linked to a Ca2+-dependent signaling transduction mechanism. Consequently, in the present study, we have examined the effect of ATP on the Cl- conductance in the DC1 cell line.

Previous studies in secretory epithelia have shown that extracellular ATP stimulates Cl- secretion across the apical membrane (15, 33, 35). The majority of authors concur with the existence of P2Y2 receptors in secretory epithelia, the stimulation of which increases cytosolic Ca2+, which, in turn, activates Ca2+-sensitive Cl- channels. Various studies performed in cultured epithelial cells of the kidney, such as proximal tubule (5) and collecting duct cells (18), as well as Madin-Darby canine kidney (MDCK) (40), LLC- PK1 (38), and A6 (24, 26) cells, have demonstrated the presence of ATP receptors responsible for the triggering of ion transport mechanisms. This is also the case for isolated Necturus maculosus and rabbit proximal tubules (4, 6). Although it seems clear that the receptor involved is a P2Y-type receptor, conflicting conclusions have arisen concerning the location of this receptor, the location and nature of the channels involved in mediating its effects, and the mechanism of the signaling pathway involved. For example, in N. maculosus proximal tubule (4), a basolateral P2Y1 receptor increases a Ca2+-insensitive basolateral Cl- conductance and mobilizes Ca2+ independently from internal stores. In MDCK cells, two apical subtypes of purinoceptors could control transepithelial ion transport by means of an increase in cytosolic Ca2+ (P2Y1) or prostaglandin synthesis (P2Y2) (39). Moreover, in primary cultures of rabbit cortical collecting and connecting tubules (18), ATP binds to an apical P2Y2 receptor, thereby inhibiting Na+ and Ca2+ reabsorption independently of a Ca2+ signaling mechanism.

In the present study, we demonstrate that DC1 cells, immortalized from cultured DCTb cells, express the P2Y2 receptor in their apical membranes. Stimulation of this receptor by ATP activates a Ca2+-sensitive Cl- conductance in the apical membrane via a pathway involving the liberation of Ca2+ from internal stores.


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Cultures

The DC1 renal cell line was obtained from primary cultures of rabbit distal convoluted tubule after transfection of cells with the pSV3 neo plasmid and G418 selection. The technique of transformation of primary cultures is described in a previous paper (31). 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, 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.

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 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 rinsing 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 RPMI on the apical side and 2 ml on the basolateral side) was collected and replaced by fresh medium. The remaining radioactivity in the epithelium at the end of the experiments was determined by counting the remaining radioactivity in cells which had been solubilized with 1% Triton X-100.

Calculations. From backaddition 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. The equations thus give the fraction of total radioactivity lost per unit time
(K<SUB>a</SUB>)<SUB><IT>t</IT></SUB><IT>=</IT><FR><NU>(C<SUB>a</SUB>)<SUB><IT>t</IT></SUB></NU><DE>C<SUB>ep</SUB><IT>+</IT><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><IT>+</IT>(C<SUB>b</SUB>)<SUB><IT>i</IT></SUB>]</FENCE><IT>+</IT><FR><NU><IT>1</IT></NU><DE><IT>2</IT></DE></FR> [(C<SUB>a</SUB>)<SUB><IT>t</IT></SUB><IT>+</IT>(C<SUB>b</SUB>)<SUB><IT>t</IT></SUB>]</DE></FR><IT> · </IT><FENCE><FR><NU><IT>1</IT></NU><DE><IT>T</IT></DE></FR></FENCE>

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

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) by using a two-stage vertical puller (PP 83, Narishige, Tokyo, Japan) and filled with an N-methyl-D-glucamine chloride (NMDGCl) solution that contained (in mM) 140 NMDGCl, 5 ATP, and 10 HEPES. The extracellular bath solution contained (in mM) 140 NMDGCl, 1 CaCl2, and 10 HEPES. Osmolarity was adjusted to 350 mosmol/kgH2O with mannitol, and pH for both solutions was maintained at 7.4. Cells were observed by using an inverted microscope, the stage of which was equipped with a water robot micromanipulator (WR 89, Narishige). The patch pipette was connected via an Ag/AgCl wire to the 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 (Cm) was compensated for by using settings available on the RK 400 amplifier. No series resistance compensation was applied, but experiments with series resistances >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 via 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 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.

Chemical Compounds

Diphenylamine-2-carboxylate (DPC) from Aldrich was prepared as a 1 M stock solution in DMSO and dissolved at 1 mM in the incubation medium. DIDS from Sigma Chemical was dissolved directly to a final concentration of 1 mM. 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX; Sigma Chemical) was prepared as a 10 mM stock solution in DMSO. Suramin was kindly provided by Bayer-Pharma, France (Puteaux). Ionomycin (Sigma Chemical) was dissolved at 2 mM in ethanol and used at a final concentration of 2 µM in solutions. The following compounds were purchased from Sigma-Aldrich: ATP, adenosine 5'-O-[thiotriphosphate] (ATPgamma S), UTP, and thapsigargin (TG). Charybdotoxin was purchased from Latoxan.


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125I- Efflux Stimulated by Extracellular ATP

Effects of ATP on membrane chloride permeability were assessed by using the 125I- efflux technique. In the first series of experiments, DC1 cells were grown on collagen-coated petri dishes and apical effluxes were measured after the cells had been loaded with 125I-. Figure 1A shows the 125I- efflux rate constant (given as %initial value at time t = 1 min) expressed as a function of time. Under control conditions, the efflux of 125I- from the monolayer into the bathing solution was time independent, with an efflux rate constant of (5.05 ± 0.13) × 10-2 min-1 (n = 46). The addition of ATP (10 µM) to the bathing solution induced an early transient increase in 125I- efflux equal to 275.9 ± 11.3% of control (n = 4) that took place within 1 min (Fig. 1A). The effects of ATP followed a dose-response curve when the external ATP concentration ranged from 0.1 to 50 µM (Fig. 1B). Maximal effects were observed with an ATP concentration higher than 50 µM, with the EC50 being close to 3 µM.


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Fig. 1.   Effect of ATP on 125I- effluxes. Experiments were performed on cells grown on collagen-coated petri dishes. After an initial control period, ATP was added at the time indicated by the arrow. A: 125I- effluxes were measured in a bath medium containing either 1 µM ATP (n = 8 monolayers), 10 µM ATP (n = 4 monolayers), or 50 µM ATP (n = 4 monolayers). B: log concentration-effect relationship of ATP on 125I- effluxes. K, efflux rate constant.

Figure 2 shows that the application of the chloride channel blockers DPC (1 mM) or DIDS (1 mM) completely abolished the ATP-evoked 125I- efflux.


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Fig. 2.   Effect of Cl- channel inhibitors on ATP-stimulated 125I- effluxes. Experiments were performed on cells grown on collagen-coated petri dishes. After an initial control period, ATP was added at the time indicated by the arrow. Effluxes were measured in a bath medium containing either 10 µM ATP only (n = 4 monolayers) or ATP with either 1 mM DIDS (n = 5 monolayers) or 1 mM diphenylamine-2-carboxylate (DPC; n = 3 monolayers).

Involvement of intracellular Ca2+ in the ATP response. We have previously shown that purinoceptor stimulation in DC1 cells increases the intracellular Ca2+ concentration ([Ca2+]i) (1). Consequently, the involvement of cytosolic Ca2+ in the Cl- permeability of DC1 cells was investigated here. Preincubations for 1 h with the membrane-permeable, Ca2+-chelating agent 1,2-bis (2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM; 10 µM) resulted in the complete inhibition of the extracellular ATP-induced rise in 125I- efflux (Fig. 3). TG, a well-known inhibitor of the endoplasmic reticulum Ca2+-ATPase that causes depletion of intracellular Ca2+ stores, was used to further investigate the way in which depleting cytosolic Ca2+ could affect the level of 125I- efflux. Treatment with TG (10-7 M for 30 min) significantly inhibited the ATP-evoked 125I- efflux (Fig. 3), which strongly suggests that the ATP-induced chloride efflux is regulated by [Ca2+]i.


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Fig. 3.   Effect of intracellular Ca2+ clamping on ATP-stimulated 125I- effluxes. ATP (10 µM)-stimulated effluxes were measured in control cells (n = 4 monolayers) or in cells preincubated with 10 µM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM; n = 5 monolayers) or 10-7 M thapsigargin (n = 3 monolayers).

To confirm that the ATP-stimulated Cl- permeability was mediated via an increase in intracellular free Ca 2+, the effect of the calcium ionophore ionomycin was studied next.

Ionomycin (2 µM) increased 125I- efflux with a time course similar to that obtained with extracellular ATP. Within 1 min, the 125I- efflux in the presence of ionomycin was 590.2 ± 77% (n = 6) of that measured under control conditions (Fig. 4A). No increase in efflux was observed when DPC (1 mM) or DIDS (1 mM) was added to the medium containing ionomycin (Fig. 4A).


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Fig. 4.   Effect of ionomycin on 125I- effluxes. Experiments were performed in cells grown on collagen-coated petri dishes. After an initial control period, ionomycin was added at the time indicated by the arrow. A: effluxes were measured in a bath medium containing either 2 µM ionomycin only (n = 6 monolayers) or ionomycin with either 1 mM DIDS (n = 3 monolayers) or 1 mM DPC (n = 4 monolayers). B: effect of K+ channel blockers on ionomycin-stimulated 125I- effluxes. K+ channel blockers Ba2+ (5 mM; n = 3 monolayers) and charibdotoxin (10-7 M; n = 3 monolayers) were added before exposure to 2 µM ionomycin.

The Cl- conductance is not the only conductance in kidney cells that is increased by raised cytosolic Ca2+ levels. One notable example relevant here is the Ca2+-activated K+ channel, which has been described in the terminal nephron (11, 14). The effects of the K+ channel inhibitors barium and charybdotoxin were therefore tested on the activation of the 125I- efflux elicited by ionomycin. As shown in Fig. 4B, neither barium (5 mM) nor charybdotoxin (10-7 M) had any significant effect on the 125I- efflux stimulated by ionomycin. These findings indicate that the Cl- flux induced by ionomycin was independent of the activity of Ca2+-sensitive K+ channels.

Characterization of purinergic receptors. To analyze the nature of the purinoceptor involved in the increase in 125I- efflux described here, the effects of various other nucleotides were tested. The histogram in Fig. 5A shows the magnitude of the 125I- efflux response obtained 1 min after exposure of cells to ATP or ATP analogs. ATPgamma S (10 µM), a nonhydrolyzable ATP analog, enhanced 125I- efflux, although the amplitude of the response was less than that obtained with 10 µM ATP. UTP (50 µM) or ATP at the same concentration was equipotent in stimulating 125I- efflux. The order of agonist potency was thus measured to be ATP >=  UTP > ATPgamma S.


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Fig. 5.   Identification of purinergic receptor subtypes expressed in DC1 cells. Values correspond to the maximal response elicited. A: effects of different agonists. Cells grown on collagen-coated petri dishes were stimulated by exposure to 10 µM ATP (n = 4 monolayers), 10 µM ATPgamma S (n = 4 monolayers), 50 µM ATP (n = 4 monolayers), or 50 µM UTP (n = 4 monolayers). B: effect of 500 µM suramin on ATP-stimulated 125I- effluxes (n = 3 monolayers).

The effects of the nonspecific P2 receptor antagonist suramin on ATP-evoked 125I- effluxes were then studied. As shown in the histogram of Fig. 5B, the treatment of DC1 cells with 500 µM suramin completely inhibited the development of 125I- effluxes induced by 10 µM ATP.

Localization of purinergic receptors and ATP-activated Cl- conductance. In a second set of experiments, DC1 cells were grown on permeable Millipore supports, which permitted separate measurements to be made of 125I- effluxes across the apical and basolateral membranes, as well as determination of the location of P2 receptors. Figure 6 shows the degree of 125I- efflux measured across both membranes. Under control conditions (i.e., during the first 4 min before the application of ATP), apical and basolateral 125I- effluxes were independent of time. The basolateral efflux rate constant [(6.09 ± 0.27) × 10-2 min-1, n = 18] exceeded by a factor of 1.25 the apical rate constant [(4.13 ± 0.28) × 10-2 min-1, n = 18]. Figure 6 shows the 125I- effluxes across both membranes when ATP was applied either to the apical (Fig. 6A) or to the basolateral (Fig. 6B) side of the monolayer. ATP induced an increase in apical 125I- efflux only when added to the apical bathing medium, which is to say that basolateral efflux was not modified by ATP.


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Fig. 6.   Polarity of ATP receptors. 125I- effluxes were measured in cells grown on permeable Millipore supports. After an initial control period, apical and basolateral effluxes were measured in the presence of 50 µM ATP in the apical solution (A) or in the basolateral solution (B). Each experiment was performed in 5 different monolayers (A) and 7 different monolayers (B), respectively.

ATP-Activated Whole Cell Currents

To further characterize the regulation of Cl- permeability by extracellular ATP, macroscopic currents were measured by using the whole cell patch-clamp recording technique.

Whole cell currents were recorded with a low-[Ca2+] pipette solution containing 140 mM NMDGCl (see MATERIALS AND METHODS). Extracellular solutions were made hyperosmotic (140 mM NMDGCl + 50 mM mannitol) to prevent the development of swelling-activated Cl- currents. After successful gigaseal formation, the whole cell configuration was obtained in 10% of cells tested. 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. 7A) that changed linearly with the membrane voltage and reversed near 0 mV (Fig. 7D). The amplitude of the currents was 59 ± 11 pA (n = 8) at +100 mV. Because of its small amplitude, the nature of the current was not analyzed further, although its reversal potential indicates that Cl- may well have been the charge carrier.


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Fig. 7.   Characteristics of ATP-induced whole cell Cl- currents. With N-methyl-D-glucamine (NMDG)-Cl- solution in the bath and in the pipette, membrane voltages were held at -50 mV and stepped to test potential values between -100 and +120 mV in 20-mV increments. A: whole cell currents from unstimulated DC1 cells were recorded with low Ca2+ concentration in the pipette solution (n = 6 cells from 3 monolayers). B and C: whole cell currents were recorded 1 (n = 4 cells from 3 monolayers) and 3 min (n = 4 cells from 3 monolayers) after the application of 100 µM ATP to the bath solution. D: current-voltage relationships measured 360 ms after the onset of the test pulse. Values are means ± SE. DPCPX, 8-cyclopentyl-1,3-dipropylxanthine.

In previous reports (3), it was shown that intracellular Ca2+ was implicated in the regulation of Cl- channels in cultured DCTb cells. For this reason, therefore, the role of ATP and ionomycin in the whole cell currents of DC1 cells was evaluated.

The application of ATP (100 µM) to the bath medium induced an increase in membrane currents within 1 min, even in the presence of DPCPX, which thus excludes the involvement of P1 receptors in this process (Fig. 7B). The kinetics of the macroscopic current were clearly time dependent for depolarizing potentials and exhibited a slowly developing component. The corresponding current-voltage relationships for steady-state activated currents measured at 380 ms are given in Fig. 7D. The ATP-activated currents measured in symmetrical Cl- solutions reversed at -2.59 ± 1.82 mV (n = 4), which is close to the equilibrium potential for Cl-. The steady-state current exhibited marked outward rectification, with an inward current at -100 mV of -53.2 ± 10.8 pA and outward current at +100 mV of 144.1 ± 26.1 pA (n = 4). The currents were transient in nature and returned to control levels within 3 min after the exposure of cells to ATP (Fig. 7C).

To determine the possible role of cytosolic Ca2+ in the development of ATP-induced Cl- currents, experiments were performed by using pipette solutions containing the Ca2+- chelating agent EGTA (5 mM) to suppress intracellular free Ca2+. In 100% of the cells tested, the ATP-induced activation of Cl- currents was blocked (data not shown).

Ionomycin-activated whole-cell currents were also studied. Elevation of [Ca2+]i by the application of 2 µM ionomycin to the bath solution activated outwardly rectifying Cl- currents that exhibited delayed activation kinetics at depolarizing voltages, in a manner very similar to those developed with extracellularly applied ATP (Fig. 8B). The reversal potential of these currents was -1.8 ± 0.9 mV. One minute after the exposure of cells to ionomycin, the amplitude of the steady-state current recorded at +100 mV was 2.7 times the current at -100 mV (i.e., -35.2 ± 12.2 vs. 95.3 ± 8.6 pA at -100 mV and +100 mV, respectively; n = 4). The current amplitude increased 3 min after the commencement of exposure to ionomycin (Fig. 8C).


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Fig. 8.   Characteristics of ionomycin-induced whole cell chloride currents. With NMDG-Cl- solution in the bath and in the pipette, the membrane potential was held at -50 mV and stepped to test potential values between -100 and +120 mV in 20-mV increments. A: whole cell currents from unstimulated DC1 cells were recorded in the absence of Ca2+ in the pipette solution (n = 4 cells from 3 monolayers). B and C: whole cell currents were recorded 1 min (n = 4 cells from 3 monolayers) and 3 min (n = 4 cells from 3 monolayers) after the application of 2 µM ionomycin to the bath solution. D: current-voltage relationships measured 360 ms after the onset of the test pulse. Values are means ± SE.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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In the present study, the effects of extracellular ATP on 125I- efflux and on whole cell Cl- currents in DC1 cells were investigated. Results clearly showed that extracellular ATP stimulated Cl- secretion via the activation of apical P2Y2 receptors and intracellular Ca2+-dependent signaling pathways, which in turn activated Ca2+-dependent Cl- channels in the apical membrane of the DC1 cells.

The activation of Cl- conductances via P2 purinoceptors has been reported for various epithelial tissues such as airway epithelium (34, 41), colonic T84 (10) or HT-29 cells (19), epididymal cells (8), endometrial epithelium (7), and the distal colon (21). Although many studies have been devoted to the characterization and the localization of P2 purinoceptors in renal tissue (4, 6, 18, 39), fewer works have investigated the role of these receptors in the control of transmembrane ion movements. Of the studies that have investigated these effects, data were mainly obtained by using MDCK cells (40), A6 cell lines (24, 26), N. maculosus proximal tubule (4), and cultured rabbit connecting tubules (18). We have previously shown that the DC1 cell line immortalized from rabbit DCTb expresses characteristics of the native epithelium (23). Notably, it was found that DC1 cells exhibited CFTR Cl- channels in the apical membrane and that this conductance could be enhanced by extracellularly applied adenosine acting via A2A receptors located in the basolateral membrane that were coupled to the adenylate cyclase second-messenger system (31).

In a companion paper to this report (1), we have also shown that extracellular ATP raises [Ca2+]i via P2Y2 receptor activation. The present data demonstrate that exposure of 125I--loaded DC1 cells led to a concentration-dependent increase in 125I- efflux with a half-maximal effect at an ATP concentration of ~3 µM. Comparison of the effects of ATP with the effects of other nucleotides yielded the potency sequence UTP >=  ATP > ATPgamma S, which corresponds to the conventional pharmacology of the P2Y2-type receptor (28).

In accordance with literature data (7, 8, 15, 35, 41, 42), our observations suggest that [Ca2+]i-activated signaling pathways were important for stimulation of Cl- conductances by ATP. First, the stimulation of 125I- efflux by ATP was completely inhibited by the pretreatment of cells with BAPTA-AM or TG, suggesting that ATP receptors in DC1 cells utilized Ca2+ as a second messenger. Second, the elevation of [Ca2+]i by ionomycin mimicked the response to ATP. Finally, the order of nucleotide potencies for the elevation of [Ca2+]i and stimulation of 125I- efflux was equivalent.

It could be postulated that the stimulation of Cl- secretion by ATP is mediated by P2Y2-linked Ca2+ mobilization, which in turn activates Ca2+-dependent Cl- channels. This notion is supported by the results obtained from whole cell patch-clamp experiments, which demonstrated that the currents activated by ATP and ionomycin shared similar characteristics. This finding suggests that the two agents act on the same Cl- conductance by a common mechanism, i.e, by raising intracellular Ca2+. The ATP-activated whole cell currents exhibited delayed activation at depolarized voltages and outward rectification. Similar properties have been described for the Ca2+-dependent Cl- channel described in cultured DCTb (3) and in various epithelial cells (12). However, compared with ionomycin-induced currents, ATP-sensitive Cl- currents were of smaller amplitude and decreased rapidly after the nucleotide application. This could be explained by the fact that the rise in cytosolic Ca2+ induced by ATP was transient and probably occurred in a more physiological range than that obtained with ionomycin (1, 30). In contrast to other reports (10, 16), the mechanism underlying Cl- secretion by ATP is probably not related to the activation of Ca2+-sensitive K+ channels because barium and charybdotoxin were not effective in blocking the ATP-induced Cl- secretion.

As stated above, we have previously demonstrated that DC1 cells possess P1 purinoceptors (31). Thus ATP might activate a Cl- conductance either directly by binding to a P2Y2 receptor or indirectly by its conversion by ectoenzymes to adenosine, which binds to P1 receptors. Because the response to ATP is preserved in the presence of DPCPX, an adenosine receptor antagonist, it is unlikely that hydrolysis of ATP to adenosine is responsible for the response initiated by raised [Ca2+]i.

Most of the literature data agree that the increase in Cl- conductance via P2Y2 receptors is mediated by an increase in intracellular Ca2+ (7, 13, 16, 41). However, a study has indicated that such an activation of Cl- conductance is not dependent on an increase in cytosolic Ca2+ (4). Taken together, these results indicate that extracellular ATP might modulate different types of Cl- channels, probably via different mechanisms. For instance, in MDCK cells, Zegarra-Moran et al. (40) suggest that ATP stimulates Cl- secretion via a P2Y1 receptor linked to increased cytoplasmic Ca2+ and via P2Y2 receptors coupled to prostaglandin secretion in the absence of any change in cytosolic Ca2+. In primary cultures of tracheal epithelial cells, Hwang et al. (15) proposed an interesting model in which ATP stimulates Cl- secretion through Ca2+-activated Cl- channels via P2Y2 receptors coupled to pathways, resulting in increased Ca2+. This group also activated CFTR Cl- channels via P2Y3 receptors coupled to a cAMP-dependent pathway. Chan et al. (8) described another model, using epididymal cells, which suggested that both Ca2+- and cAMP-dependent Cl- conductances were activated by extracellular ATP via a unique P2 receptor and that activation of a cAMP cascade by ATP is Ca2+ and calmodulin dependent. These three examples underlie the complexity of the regulation of Cl- transport by extracellular ATP. Although DC1 cells express at least three types of Cl- conductances [i.e., CFTR Cl- channels, volume-sensitive Cl- channels, and Ca2+-dependent Cl- channels (3, 30, 36)], the present data indicate that ATP is involved only in the control of the calcium-sensitive Cl- conductance, probably via the activation of P2Y2 receptors only. In contrast to the studies cited above, an action of ATP on CFTR Cl- channels was never observed, although it can be clearly demonstrated that adenosine activates an apical CFTR Cl- conductance via a pathway involving A2A receptors (31).

In addition to Ca2+-dependent Cl- channels, it has also been postulated that ATP could activate a volume-sensitive Cl- conductance in human bronchial cells (41). This action will be Ca2+ dependent and mediated by the P2Y2 receptor. In DC1 cells, ATP did not stimulate a Cl- conductance with characteristics of volume-sensitive currents, although adenosine acting via A1 receptors is capable of this (29). Thus considering that ectonucleotidase activity exists in many tissues, including kidney (20, 32), one could pose the question of whether some of the effects of ATP on epithelial Cl- conductances reported in the literature could be attributable to adenosine.

One of the findings of the present work was that the action of ATP in increasing Cl- conductance was observed only when ATP was applied to the apical membrane of the cell monolayer. Moreover, ATP triggered a Cl- efflux only through the apical membrane. This result strongly suggests that P2Y2 receptors are located on the apical membrane, together with the Ca2+- sensitive Cl- conductance. The location of P2 receptors in epithelial cells, as judged from literature reports, is not clear-cut. It can be generally concluded that P2Y2 receptors in respiratory cells are located in the apical membrane (9, 15, 22), whereas other subtypes of purinoceptors are probably present in the basolateral membrane (15, 22). In other epithelial cells P2Y2 receptors are found either exclusively in the basolateral membrane (42) or in both the basolateral and apical membranes (7, 13, 16, 18).

In DC1 cells, the apical location of P2Y2 receptors raises the question of the in vivo sources of luminal nucleotides that could bind to apical purinoceptors. An autocrine mechanism can be postulated for the release of ATP from epithelial cells (6, 25, 37). In the kidney, ATP is also produced by paracrine mechanisms, particularly involving macula densa cells. In this case, ATP is released into the interstitial fluid bathing the juxtaglomerular structures (17), and can therefore reach the distal tubule.

The physiological responses induced by ATP on renal function remain largely unexplored. Up until the present time, ATP has been shown to affect renal microvascular function and, more specifically, influences the tubuloglomerular feedback mechanism while indirectly affecting epithelial transport mechanisms. Besides this role, the action of ATP on Cl- secretion in the distal tubule has not yet been placed in a physiological context. However, the bright part of the rabbit distal tubule, which expresses apical CFTR Cl- channels (27, 36) and Na+ channels (23), could behave as a Cl- secretory epithelium (27, 36). Therefore, activation of the apical Ca2+-dependent Cl- conductance would contribute to the regulation of transepithelial Cl- transport. Because these cells possess an apical Cl-/HCO3- exchanger (2), one could postulate that the secretion of Cl- through the apical membrane might indirectly activate the Cl-/HCO3- exchanger by increasing the apical inward Cl- gradient, thereby causing the secretion of HCO3- ions.

In conclusion, the results for these experiments are summarized in the schematic diagram presented in Fig. 9: ATP stimulates apical Ca2+-sensitive Cl- conductances in DC1 cells via an apical P2Y2 receptor, which triggers an increase in cytosolic Ca2+. In addition, basolateral adenosine could also stimulate apical CFTR Cl- channels via an A2 receptor coupled to cAMP production (31).


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Fig. 9.   Model for regulation of Cl- conductances in the DC1 cell line. ATP stimulates apical Ca2+-sensitive Cl- conductances via apical P2Y2 receptors, which triggers an increase in cytosolic Ca2+. Basolateral adenosine could also stimulate apical cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels via an A2 receptor coupled to the production of cAMP. The resulting increase in Cl- secretion across the apical membrane could generate an inwardly directed Cl- gradient, which activates the apical Cl-/HCO3- exchanger. PLC, phospholipase C; AC, adenylate cyclase; NECA, 5'-(N-ethylcarboxamido)adenosine; ER, endoplasmic reticulum; IP3, 1,4,5-inositol trisphosphate. G, G protein.


    FOOTNOTES

Address for reprint requests and other correspondence: P. Poujeol, UMR, CNRS 6548, Bâtiment Sciences Naturelles Université de Nice-Sophia Antipolis, Parc Valrose, 06108 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. §1734 solely to indicate this fact.

Received 20 September 1999; accepted in final form 29 February 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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