Extracellular ATP increases [CA2+]i in distal tubule cells. I. Evidence for a P2Y2 purinoceptor

Michel Bidet, Guy De Renzis, Sonia Martial, Isabelle Rubera, Michel Tauc, 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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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Experiments were performed to characterize the P2 purinoceptor subtype responsible for cytoplasmic calcium mobilization in cells from the initial part of rabbit distal convoluted tubule (DCT). Free calcium concentration was measured in a DCT cell line (DC1) with the probe fura 2. Both ATP and UTP increased cytosolic Ca2+ concentration ([Ca2+]i; EC50 3 and 6 µM, respectively). The order of potency for nucleotide analogs was ATP = UTP > adenosine 5'-O-[thiotriphosphate] ADP > UDP, which is consistent with the pharmacology of the P2Y2 receptor subtype. The increased [Ca2+]i responses to ATP and UTP were strongly inhibited by suramin. Pretreatment of cells with pertussis toxin (PTX) attenuated the action of both nucleotides. Inhibition of phospholipase C with U-73122 totally blocked the [Ca2+]i response to ATP. Thus ATP- and UTP-stimulated [Ca2+]i mobilization in DC1 cells appears to be mediated via the activation of P2Y2 purinoceptors coupled to a G protein mechanism that is partially sensitive to PTX. Calcium flux measurements showed that lanthanum- and nifedipine-sensitive calcium channels are involved in the [Ca2+]i response to ATP.

purinergic receptors; kidney; intracellular calcium; calcium channels


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

NUMEROUS STUDIES PERFORMED in a variety of tissues have suggested that extracellular ATP plays a role in modulating cell function. Because of the existence of extracellular ectonucleotidases, most of the effects of extracellular ATP were thought to be mediated by its metabolite adenosine, via the stimulation of P1 purinoceptors. However, recent studies using agonists or antagonists of P1 demonstrated that ATP could affect cell metabolism independently of adenosine.

Receptors that interact specifically with ATP (classified as P2 purinergic receptors) have been demonstrated in many tissues and cell types (28). Recent studies in molecular biology have indicated the existence of two main subclasses of purinoceptors with different molecular structures. The molecular characterization of these P2 receptors has been used as a framework for a new purinoceptor classification (4, 28). To the present time, seven subtypes for P2X receptors (P2X1-7) and six for P2Y receptor (P2Y1-4, P2Y6, and P2Y11) have been characterized. Purinergic receptors exhibit fundamental differences in their molecular structures. P2X receptors present a structure similar to the epithelial sodium channel with two transmembrane domains. Moreover, P2X receptors are ionotropic receptors with cation channel characteristics (35). The interaction of extracellular nucleotides with P2 purinergic receptors elicits a range of intracellular signaling responses. P2Y receptors have seven transmembrane hydrophobic domains. These metabotropic receptors interact with effectors via a GTP-binding protein. The most common response elicited by ATP is the activation of phospholipase C (PLC) with a subsequent increase in the formation of 1,4,5-inositol trisphosphate (IP3), the production of diacylglycerol, an increased intracellular calcium concentration, and activation of protein kinase C.

An important modulatory function of extracellular ATP has been demonstrated in the kidney. Indeed, ATP has been shown to increase afferent arteriolar resistance, while having little effect on postglomerular vasculature (12). ATP also stimulates prostaglandin synthesis in mesangial cells (24). Several studies have indicated the existence of P2 receptors on renal epithelial cells in culture. Extracellular ATP and UTP also raise cytosolic Ca2+ concentrations ([Ca2+]i) in A6 cells (17), which exhibit morphological characteristics similar to amphibian distal nephron and Madin-Darby canine kidney (MDCK) cells [a cell line derived from the distal tubule of canine kidney (23)], probably via an interaction with P2U receptors. ATP also increases intracellular calcium release in cortical slices (19), cortical tubules in suspension (29), renal tubular epithelial cell lines (23, 40), primary cell cultures of proximal rabbit convoluted tubule (5, 6), and in the cortical thick ascending limb of the mouse kidney (22).

The functional effects of extracellular ATP on ion transport in the kidney have been well documented. For example, it is well known that in cortical collecting tubules or primary cultures of cells from proximal convoluted tubules (5, 6), extracellularly applied ATP increases [Ca2+]i. Increasing [Ca2+]i by several other maneuvers has also been shown to inhibit the action of arginine vasopressin (30).

An increase in [Ca2+]i has also been used to demonstrate the existence of Ca2+-regulated chloride channels in numerous secretory epithelial cells (9). Recently, we have reported that increasing [Ca2+]i by applying extracellular ionomycin or ATP activates Ca2+-regulated chloride channels on the basolateral membrane of rabbit distal convoluted tubule (DCT) cells in primary culture (2). The present study examines the effect of extracellularly applied ATP on [Ca2+]i, in a cell line (named DC1) obtained by transfection of rabbit DCT cells in primary culture with the plasmid pSV3 neo and selected in G418. Because the purpose of this paper was to investigate the P2-linked effects of the exposure of cells to extracellular ATP, experiments were carried out in the presence of a P1 blocker, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). Results show that ATP activates a P2Y2 purinoceptor coupled to Gq/Gi proteins. An increase in [Ca2+]i occurs after the activation of PLC and a Ca2+-dependent transduction mechanism.


    MATERIALS AND METHODS
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Cultures

The DC1 renal cell line established in our laboratory was initiated by infecting, with the wild-type simian virus SV40, primary cultures of distal bright convoluted tubule cells isolated from the kidney of young male New Zealand White rabbits. Cultures were seeded in collagen-coated 35-mm petri dishes 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 and 20 mM HEPES at pH 7.4, together with 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. The medium was changed 4 days after seeding and then every alternate day. DC1 cells were used between passages 12 and 25.

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 Instruments) 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 eight-bit extended ISIS camera (Extended ISIS, Photonic Science, Sussex, UK) equipped with an integration module to maximize 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 1 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). The final calculations were made using Excel software (Microsoft).

Intracellular calcium measurements were carried out in 4- to 8-day-old DC1 cell monolayers grown in petri dishes and loaded with a solution of 5 µM fura 2-acetoxymethyl ester (fura 2-AM) containing 0.01% pluronic acid for 45 min at room temperature. 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 M 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 5 s. Intracellular Ca2+ concentrations were calculated from the dual wavelength-fluorescence ratio by using the Grynkiewicz equation (11).

Manganese influx measurements were made in 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. Fluorescence 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 manganese 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.

Isotopic Calcium Flux Experiments

Measurements of isotopic Ca2+ influx were made on 3- to 6-day-old DC1 cells grown in collagen-coated multiwell plates. The cells were preincubated for 20 min at 37°C in 250 µl of a NaCl medium containing (in mM) 140 NaCl, 5 KCl, 1 CaCl2, 1 MgSO4, 5 glucose, and 20 HEPES, pH 7.40, with 1 M Tris. This medium was then changed to an identical one containing ATP (50 µM final concentration). One minute later, Ca2+ uptake was initiated by adding 10 µCi/ml 45Ca2+ (less than 10 µl) into the wells in the presence or the absence of inhibiting agents (lanthanum, nifedipine). Ca2+ influx was measured, unless otherwise stated, for 1 min at 37°C and was stopped by removing the flux solution and washing the cell layer with ice-cold preincubation medium (1 mM Ca2+). The cells were scraped in 0.1 M NaOH, and the associated 45Ca2+ activity was measured by liquid scintillation. Triplicate measurements were made for each experiment and for each condition.

For Ca2+ efflux studies, 3- to 6-day-old DC1 cells were loaded with 45Ca2+ (10 µCi/ml) for 12 h at 37°C in standard culture medium. The cells were then washed quickly in a large volume of unlabeled solution to remove the isotope from the extracellular space. A 100-µl sample of the solution was first collected every 2 min 30 s for 15 min and then at 1-min intervals after the addition of ATP (50 µM). At the end of the experiment, the remaining radioactivity in the epithelium was determined by counting the cells after exposure to a 1% solution of the solubilizing agent Triton X-100. The 45Ca2+ content of each sample was determined by liquid scintillation. To test the role of intracellular calcium pools in relation to Ca2+ efflux, similar experiments were also carried out after exposure of cells to thapsigargin (TG; 0.5 µM) for 15 min. Results are expressed as picomoles per µg DNA per minute. DNA was measured by the fluorimetric micromethod described by Switzer and Summer (36).

Chemical Compounds

Fura 2-AM (Molecular Probes, Eugene, OR) was dissolved at 10 mM in DMSO. Pluronic acid (Molecular Probes) was dissolved at a final concentration of 20-25% (vol/vol) in DMSO. The following compounds were purchased from Sigma-Aldrich (St. Louis, MO): adenosine, AMP, ADP, ATP, adenosine 5'-O-[thiotriphosphate] (ATPgamma S), alpha ,beta -methylene (Me)-ATP, beta ,gamma -Me-ATP, benzoylbenzoyl (Bz)-ATP, UDP, UTP, pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), cibacron blue 3GA [formally listed as reactive blue 2 (RB-2)], TG, pertussis toxin (PTX), DPCPX, EGTA, ionomycin and valinomycin. 2-Methylthio ATP (2-MeS-ATP) was purchased from ICN Pharmacologicals. 1-[6-((17 beta -3-Methoxyestra-1,3,5-(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5- dione (U-73122) and 1-[6-((17 beta -3-methoxyestra-1,3,5-(10)-trien-17-yl)amino)-hexyl]-2,5-pyrrolidine-dione (U-73343) were purchased from Biomol Research Laboratories. Suramin was kindly donated by Bayer-Pharma (Puteaux). 45Ca2+ was purchased from Amersham. Other chemicals were either from Sigma or from Merck (Darmstadt, Germany).


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MATERIALS AND METHODS
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Resting [Ca2+]i Levels in the DC1 Cell Line

Once loaded with fura 2-AM, DC1 cells were maintained in NaCl buffer containing 1 mM Ca2+. Under these resting conditions, the [Ca2+]i in cells from 86 different petri dishes averaged 73.3 ± 2.3 nM. This value was similar to that obtained in primary cultures of DCTb cells (77.7 ± 2.8 nM, n = 66). The stability of this low [Ca2+]i against a large, inwardly directed electrochemical Ca2+ gradient provided a good indicator of the viability of these cells.

Effects of Phosphorylated Adenosine Compounds on [Ca2+]i in DC1 Cells

Figure 1A shows a typical fluorescence experiment in which the extracellular application of ATP (10 µM) induced a biphasic response of [Ca2+]i in DC1 cells. An early transient increase in [Ca2+]i occurred within 5 s of the addition of ATP to the extracellular medium. Intracellular Ca2+ levels reached an average of 561.3 ± 68.9 nM (n = 14) in these experiments. This transient increase was followed by a sustained response that lasted for 2-4 min. [Ca2+]i gradually returned to the basal level afterward even in the presence of ATP in the medium. However, it must be pointed out that the removal of ATP during the sustained phase caused [Ca2+]i to promptly return to the baseline level (data not shown).


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Fig. 1.   Effect of ATP and adenosine 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: at the indicated time, petri dishes were perfused with NaCl buffer supplemented successively with ATP (10 µM), adenosine (Ado; 10 µM), or ATP (10 µM)+8-cyclopentyl-1,3-dipropylxanthine (DPCPX; P1 blocker; 10 µM). B: relative calcium concentration, expressed as a percentage of the resting value (Init), measured in the presence of ATP (10 µM), Ado (10 µM; open bars), or DPCPX (10 µM)+ATP (10 µM), ADP (10 µM), or AMP (10 µM; hatched bars). The increase in [Ca2+]i induced by Ado was compared with that induced by ATP alone. Responses induced by ADP and AMP+DPCPX were compared with ATP+DPCPX. NS, not significant. * P < 0.01.

In the second part of the above-mentioned experiment (Fig. 1A), adenosine (10 µM) was added to the external bathing medium. The concomitant increase in [Ca2+]i suggests the presence of the A1 subtype of P1 purinoceptors in DC1 cells. To ensure that P1 purinoceptors were not involved in the response of DC1 cells to ATP, P1 activity was blocked with 10 µM DPCPX. As can also be seen in Fig. 1A, the increase in [Ca2+]i induced by ATP was only 10% smaller in the presence of DPCPX than in control conditions [512.1 ± 41.6 (n = 33) vs. 561.3 ± 68.9 nM (n = 14); not significant]. Consequently, all subsequent experiments using ATP and other derivatives of adenosine were carried out in the presence of 10 µM DPCPX.

Figure 1B shows percentage increases in free calcium in DC1 cells induced by ATP (10 µM), adenosine (10 µM), and ATP (10 µM)+DPCPX (10 µM) in the bathing medium. The measured increases were 758.5 ± 90.2 (n = 14), 210.8 ± 28.7 (n = 19), and 665.9 ± 54.7 (n = 32) for each condition, respectively. The effect of exposure of cells to other phosphorylated adenosine derivatives [i.e., ADP (10 µM) and AMP (10 µM)] was also tested in the presence of DPCPX. Figure 1B shows that ADP increased [Ca2+]i by 292.2 ± 95.8% (n = 4, P < 0.01 vs. ATP; P < 0.01 vs. AMP), whereas AMP had no effect on [Ca2+]i. It should be noted that only 60% of the tested cells (27 of 45) responded to ADP. Of those, the average increase in [Ca2+]i was 296.4 ± 43.5%.

Figure 2 shows that the rise in [Ca2+]i induced by ATP was dose dependent. The half-maximal increase was obtained with 3 µM ATP, whereas maximal activation was reached in the presence of 100 µM ATP in the bathing medium. UTP yielded similar results, with an EC50 of 6 µM.


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Fig. 2.   Concentration dependence of [Ca2+]i increase on external ATP. Dose-response relationship for increase in [Ca2+]i caused by externally applied ATP () or UTP (black-triangle). The mean intracellular calcium contents, expressed as percentages of the resting values, are plotted against the NTP concentrations ([NTP]). Each point is calculated from 3-18 experiments.

Effects of Synthetic ATP Analogs and Pyrimidic Nucleotides on [Ca2+]i in DC1 Cells

To examine which class of ATP receptor was responsible for mediating the increase in [Ca2+]i in DC1 cells, the effects of other nucleotides were investigated. Because of the lack of highly specific agonists for P2 purinoceptors, this characterization was mainly based on sensitivities to synthetic analogs. Figure 3 shows average [Ca2+]i increases in response to exposure of cells to alpha ,beta -Me-ATP; beta ,gamma -Me-ATP, Bz-ATP, and 2-MeS-ATP compared with [Ca2+]i changes in response to exposure of cells to ATP. The synthetic nucleotides, tested at a final concentration of 10 µM and in the presence of 10 µM DPCPX, were found to have no effect on average [Ca2+]i levels compared with that seen after exposure of cells to ATP. The poorly hydrolysable ATP analog gamma S-ATP increased [Ca2+]i by 339.8 ± 32.8% (n = 6). UTP also enhanced [Ca2+]i by 629.0 ± 60.8% (n = 17), which was not significantly different from that observed when the purinoceptor was stimulated by ATP. UDP induced a small increase in [Ca2+]i but only in 21 cells of 45, as already seen with ADP. The relative affinity of the receptor for the nucleotides tested was thus ATP = UTP > gamma S-ATP ADP > UDP.


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Fig. 3.   Effect of synthetic ATP analogs and uridine derivatives on [Ca2+]i of DC1 cells. Each agonist was superfused at a final concentration of 10 µM. Results are presented as a percentage of resting values and expressed as means ± SE (n = 4-18). The percent changes observed in the presence of adenosine 5'-O-[thiotriphosphate] (ATPgamma S) or UTP were also compared with that induced by ATP in the presence of DPCPX (* P < 0.01; n = 10). UDP induces a slight but significant increase in [Ca2+]i compared with beta ,gamma -methylene (Me)-ATP, alpha ,beta -Me-ATP, benzoylbenzoyl (BzBz-ATP), or 2-MeS-ATP.

Effects of P2 Purinoceptor Antagonists

The effects of suramin, RB-2, and PPADS were tested on ATP-stimulated [Ca2+]i increases in DC1 cells. As shown in Fig. 4, 100 µM suramin, a nonselective P2 purinoceptor antagonist, reversibly blocked the [Ca2+]i increase induced by 10 µM ATP by 92.5 ± 6.4% (n = 4). The well-known P2Y blocker RB-2 (5 µM) did not affect the ATP-induced increase in [Ca2+]i (Fig. 4B) even after a 2-min preincubation period. When used at a concentration >50 µM, this antagonist provoked a modest rise (157.9 ± 10.2 nM) in [Ca2+]i in 32 cells after a 30- to 60-s exposure and a fall in [Ca2+]i after its removal. As this effect was observed in three different experiments, RB-2 was not tested at higher concentrations. One of the most selective P2X antagonists known, PPADS (21, 28), had no effect on the ATP-induced rise in [Ca2+]i, even when added at a concentration of 100 µM (Fig. 4B). Similar results were obtained after external stimulation by 10 µM UTP (data not shown).


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Fig. 4.   Effect of P2 purinoreceptor antagonists on [Ca2+]i increase induced by ATP. A: typical experiment showing the response to external ATP (10 µM; horizontal bar) in absence or presence of suramin (Sura; 100 µM). Suramin was added 2 min before the addition of ATP, as indicated. The reversibility of inhibition by suramin was tested by adding ATP (10 µM) 1 min after washout of the inhibitor. B: effect of 3 inhibitors: suramin (100 µM), reactive blue 2 (RB-2, 5 µM), and pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS; 100 µM). Values are means ± SE (n > 3). * P < 0.01 for results that were statistically different from those measured after exposure of cells to ATP only (open bars), paired t-test.

Dependence of [Ca2+]i Increases on Intracellular and Extracellular Ca2+: Effect of Calcium Channel Blockers

In different cellular systems, cells respond to ATP stimulation by releasing Ca2+ from internal stores. We thus investigated, using TG, the role of intracellular calcium pools on the response of DC1 cells to the extracellular application of ATP. TG is the well-known irreversible inhibitor of endoplasmic Ca2+ ATPase and was used to empty the intracellular Ca2+ pools by blocking their continuous refilling. In the presence of 0.5 µM TG, [Ca2+]i increased from a basal value of 83.3 ± 5.2 nM to a peak value of 383.4 ± 32.5 nM (n = 5) within 45 s. After 4-5 min, [Ca2+]i returned to a plateau value of 162.6 ± 9.2 nM. These rises and falls in Ca2+ are consistent with the known actions of this drug in various cell types (6). The external application of ATP (10 µM) induced only a slight increase in [Ca2+]i within 5-12 min after TG addition. The further addition of ATP failed to elicit any response 15-20 min after the addition of TG into the incubation medium (data not shown), with the intracellular calcium pools theoretically being empty under these conditions (6). From these results, it can be reasonably deduced that ATP receptors were probably metabotropic in nature, with the release of Ca2+ from intracellular stores being mediated via a second-messenger mechanism.

As mentioned above, the responses of DC1 cells to externally applied ATP were usually biphasic in the presence of extracellular Ca2+. In a separate set of experiments, the effects of ATP in a NaCl buffer without calcium and containing 0.5 mM EGTA were tested. Under this condition, we observed (Fig. 5) that the time course and the magnitude of the initial peak change in [Ca2+]i were relatively unaffected. The sustained response, however, was found to be dependent on the presence of external Ca2+. When cells were maintained in the medium without calcium, a second addition of ATP was only followed by a very small rise in [Ca2+]i. When this step was carried out in the presence of calcium (1 mM), a slight increase in the resting [Ca2+]i was produced. Fifteen minutes later, a third addition of ATP in the presence of external calcium produced an increase in [Ca2+]i similar to that observed under control conditions.


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Fig. 5.   Changes in [Ca2+]i during replenishment of intracellular stores. At the indicated time, the fura 2-loaded cells were perfused with calcium-free medium (EGTA; 0.5 mM) supplemented with ATP (10 µM; horizontal bar). A second addition of ATP while EGTA was maintained in the bathing medium failed to induce a rise in [Ca2+]i. After 15 min, the culture was rinsed with NaCl buffer containing 1 mM CaCl2 for 12.5 min before a third exposure of cells to ATP.

Figure 6A shows the effect of the well-known calcium channel blocker nifedipine on the [Ca2+]i response after the addition of ATP to the extracellular medium. Nifedipine (20 µM) notably modified the sustained phase that followed the rapid and transient increase in [Ca2+]i induced by ATP and brought about the prompt return of [Ca2+]i to baseline levels. Identical results were obtained in the presence of lanthanum (50 µM). To quantify the effects of these inhibitors, calcium concentrations measured between 1 and 2 min after the addition of ATP were averaged for control conditions and in the presence of lanthanum or nifedipine. The results presented in Fig. 6B show that both lanthanum and nifedipine totally abolished the slowly declining phase that followed the ATP-induced increase in [Ca2+]i.


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Fig. 6.   Effect of lanthanum and nifedipine on the sustained phase that followed the rapid transient increase in [Ca2+]i induced by ATP. A: at the indicated time, ATP (10 µM; horizontal bar) was perfused for 2 min in the absence or presence of nifedipine (20 µM). Inhibitors were added 30 s before the addition of ATP. B: means ± SE of the percent changes in [Ca2+]i measured between 1 and 2 min after ATP addition are reported for control conditions (open bars) and in the presence (hatched bars) of lanthanum (50 µM) or nifedipine (20 µM). Experiments were performed at least 3 times. * P < 0.01 for results that were statistically different from those measured under control conditions (paired t-test).

Changes in Ca2+ Permeability During ATP Treatment

To characterize calcium fluxes across the membrane of DC1 cells, isotopic experiments using 45Ca2+ and fura 2 in the presence of extracellular manganese were performed.

Isotopic experiments. 45Ca2+ efflux measurements were undertaken to study transmembrane calcium fluxes. DC1 cells stimulated with ATP presented a twofold increase in the rate of 45Ca2+ efflux under control conditions (data not shown). This increase could be totally abolished after the pretreatment of cells with TG.

45Ca2+ influx. In the presence of 1 mM CaCl2, the intracellular uptake of 45Ca2+ was linear for the first 5 min (data not shown). The 1-min time point was thus taken as the experimental value for subsequent experiments, the results of which are presented in Table 1. The addition of ATP (50 µM) to the extracellular medium 1 min before uptake measurements induced a significant increase in 45Ca2+ influx. Compared with the control influx rate, ATP increased uptake by 265.3 ± 35.8% (n = 21). This increase was significantly reduced in the presence of either lanthanum (20 µM) or nifedipine (20 µM). 45Ca2+ influxes measured in the presence of ATP and the two inhibitors were not significantly different from control.

                              
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Table 1.   45Ca2+ uptake by DC1 cells: effect of Ca2+ channel inhibitors on the ATP-induced calcium influx

Manganese Influx: Effect of P2 Agonists and Calcium Channel Blockers

Manganese is able to enter DC1 cells through the same channels as calcium and quenches fura 2 fluorescence (6). We used these properties to evaluate calcium influx during stimulation by ATP or UTP. Figure 7 shows that ATP increased the basal influx of Mn2+ by 50%, an effect that could be significantly inhibited by both lanthanum (50 µM) and nifedipine (20 µM) (Table 2). Moreover, the subsequent addition of ATP to the extracellular medium had no action on Mn2+ influx. These results demonstrate that both lanthanum and nifedipine blocked the calcium influx that follows the rapid transient increase in [Ca2+]i. Similar results were obtained in the presence of external UTP (data not shown). As shown in Table 2, the effect of external ATP was also tested after a 15-min pretreatment of cells with TG. When intracellular pools were empty, ATP did not induce any change in Mn2+ influx.


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Fig. 7.   Fura 2 fluorescence quenching by external manganese before and after addition of ATP. The entry of manganese 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. ATP was added at the indicated time at a final concentration of 10 µM.


                              
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Table 2.   Fluorescence quenching by external manganese under control conditions, in the presence of inhibitors, and after addition of ATP

How Does ATP Increase [Ca2+]i?: Effect of PTX and U-73122

Figure 8A illustrates the effect of PTX on both ATP- and UTP-stimulated increases in [Ca2+]i. DC1 cells were pretreated for 18 h with 0.1 or 1 µg/ml PTX. Pretreatment with 1 µg/ml PTX did not affect basal [Ca2+]i but inhibited the response to ATP by 63.8 ± 4.4% (n = 39 ) and the response to UTP by 65.4 ± 2.0% (n = 24). In contrast, 0.1 µg/ml PTX had no significant effect. This inhibition by 1 µg/ml PTX suggests that a G protein could be involved in the transduction of the signal that gives rise to the increase in [Ca2+]i. To further investigate this mechanism, we used the aminosteroid U-73122, a PLC inhibitor that is active at micromolar concentrations (34). Figure 8B shows that U-73122 (1 µM) irreversibly blocked any subsequent ATP-induced response, indicating that the ATP effect is likely to be mediated by products generated by the turnover of phosphatidyl inositol. The inactive stereoisomer of U-73122, U-73343 (1 µM), did not affect the response to ATP.


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Fig. 8.   Effect of phospholipase C (PLC) and Gi protein inhibitors on the rise in [Ca2+]i induced by 10 µM ATP or UTP. A: effect of pertussis toxin (PTX). PTX was tested at 2 different concentrations, 0.1 (gray bars) and 1 (solid bars) µg/ml. In experiments with PTX, DC1 cells were preincubated overnight with the toxin. Each experiment was performed at least 3 times. Results are expressed as the percentage of the resting [Ca2+]i value. Statistical comparisons were made vs. control conditions (open bars). * P < 0.01. B: effect of the PLC inhibitor U-73122. U-73122 (gray bars) and its inactive analog U-73343 (solid bars) have been tested at 1 µM. DC1 cells were preincubated 5 min with the toxin. Each experiment was performed 3 times. Results are expressed as the percentage of the resting [Ca2+]i value. Statistical comparisons were made vs. control conditions (open bars). ** P < 0.001.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

It is now well accepted that extracellular ATP regulates a large variety of cell functions (28). In recent years, we and others described P2 purinergic receptors in proximal convoluted tubule (5, 6, 41), Henle's loop (22), and cortical collecting duct (30) of mammalian kidney nephron. Moreover, using DCTb cells, we showed that extracellular ATP increased [Ca2+]i in a manner similar to that described here, suggesting the presence of P2 purinoceptors (2).

In the present paper, we investigated the effects of ATP and its structural analogs in a cell line (DC1) obtained by transfection of DCTb cells with the early region of pSV3 neo plasmid DNA. In fact, as shown by Scott et al. (32) for other kidney cells, DC1 cells retain most of the essential characteristics of the original DCTb cells. In particular, these cells conserve the apical cAMP-sensitive chloride channel that we studied in primary cultures of DCTb cells (31). Moreover, in culture, these cells grow as well-polarized monolayers. From these characteristics, we chose to use DC1 cells as a model of DCTb cells in functional experiments designed to elucidate the mechanisms of Ca2+ reabsorption in this part of the nephron.

The ATP-mediated stimulation of [Ca2+]i was found here to be concentration dependent (Fig. 2), with an EC50 of 3 µM. This value, in the micromolar range, is in agreement with that reported for other epithelial cells from the kidney (17, 22, 41) or in other tissues such as the distal colonic mucosa (14) and the gerbil middle ear epithelium (10).

Pharmacological probing for the P2 receptor subtype with structural analogs of ATP in fura 2 experiments (Fig. 3) revealed a typical rank in order of potency (ATP = UTP > ATPgamma S ADP > UDP), indicating a G protein-coupled P2 receptor. As already mentioned above, these receptors are now known as P2Y, because they are a metabotropic type of receptor. The measured order of potency is in accordance with the characteristics of a P2Y2 receptor subtype (4). P2Y1 has a high affinity for 2-MeS-ATP and no affinity for UTP as reported by Simon and colleagues (33), and P2Y2 has a high affinity for both ATP and UTP (15). P2Y3 is actually an ADP receptor, whereas P2Y4 is a uridine nucleotide receptor. However, it is noticeable that, for some authors (3, 39), P2Y4 shows the same identity profile as P2Y2 (high affinity for ATP and UTP). This observation suggests that DC1 cells may bear P2Y2, P2Y4, or both types of receptors. Hence either type might be involved in the observed response to ATP stimulation. P2Y11 is characterized by a very low affinity for UTP, whereas P2Y6 has a high affinity for UTP and a very poor affinity for ATP. The absence of selective antagonists renders the task of subtype identification of P2 receptors difficult.

The inhibition by suramin of the [Ca2+]i response to ATP and UTP is in agreement with other results obtained in renal cells (5, 7, 20, 22). Such reversibility has already been observed in proximal cells in culture (5). PPADS, a relatively selective antagonist of P2X and P2Y1 receptors (21, 28), had no inhibitory effect on the [Ca2+]i increase induced by ATP or UTP in our experiments (Fig. 4B). As such, it could be concluded that P2X receptors were not involved in the DC1 cell response after exposure to ATP. This conclusion is also confirmed by the observation that the 2-MeS-ATP, alpha ,beta -Me-ATP, beta ,gamma -Me-ATP, and Bz-ATP agonists are totally inactive. The absence of inhibition by RB-2 contradicts the generally observed effect of this substance on renal cell P2Y receptors (7, 41). However, in both cases, this effect was reported on P2Y receptors that exhibit a noncharacteristic P2Y2 agonist profile. For example, this inhibition pattern was found by Bogdanov et al. (3) and by Charlton et al. (8) for P2Y4 receptors. Bogdanov et al. (3) demonstrated that, in rat or human P2Y4 cRNA-injected Xenopus laevis oocytes, UTP-evoked Ca2+ fluxes were reversibly antagonized by RB-2 but insensitive to suramin. Charlton et al. (8) presented similar results in 1,321 N1 cells transfected with cloned P2Y4 receptor. Similar results were recently reported in kidney cells: Nilius et al. (20) showed that, in the A6 cell line, ATP-induced Ca2+ transients were inhibited by suramin. Banderali et al. (1) demonstrated that ATP- and UTP-evoked Cl- currents in the same cells were insensitive to RB-2.

Mobilization of intracellular calcium pools by extracellular P2Y agonists was confirmed with TG. This sesquiterpene lactone is an inhibitor of intracellular calcium-dependent ATPases and, as such, blocks the uptake of calcium into intracellular pools. Under such circumstances, the passive efflux of calcium from these pools leads to their subsequent depletion. Because the preincubation of cells with TG abolished the Ca2+ response to P2Y agonists, it seems clear that Ca2+ signaling pathways were involved in the observed action of ATP on increases in [Ca2+]i.

The significant decrease in [Ca2+]i observed after incubation of DC1 cells with PTX, or the total inhibition of the ATP- or UTP-induced [Ca2+]i increase after incubation with U-73122, is in total agreement with the presence on DC1 cells of metabotropic-like purinoceptors. The partial inhibition induced by PTX is consistent with the model proposed by Lustig et al. (16), in which the nucleotide-activated purinoceptor stimulates the exchange of GDP for GTP on the alpha -subunit of Gq and Gi proteins and causes the dissociation of the GTP-bound alpha -subunit from the beta gamma -complex (blocked by PTX). Either GTP-bound alpha -subunits from Gq or the beta gamma -complex (or both) bind and stimulate PLC. Once activated, PLC hydrolyzes phosphatidylinositol-4,5-bisphosphate to yield IP3, which liberates Ca2+ from intracellular storage sites. This phase can be fully blocked by U-73122, which inhibits the action of PLC. Our observations thus correlate well with the presence of a metabotropic-like purinoceptor in DC1 cells. The existence of such a type of receptor has been frequently reported in relation to kidney proximal tubule cells in primary culture (5, 6), in isolated tubules (41), in slices of rat renal cortex (19), and in mouse cortical thick ascending limb (22). However, Cha et al. (7) presented evidence suggesting that rat DCT lack P2Y purinoceptors, whereas Koster et al. (13) demonstrated the presence of P2Y2-like receptors in both rabbit connecting tubule and cortical collecting duct. This apparent antagonism is not surprising in regard to the strong structural differences between rat and rabbit distal nephron segments (18). Other studies describe P2Y receptors in distal cell lines, especially in the apical membrane of MDCK cells (23, 25). Even though Post et al. (26) show that P2Y1, P2Y2, and P2Y11 purinoceptors are expressed in their MDCK-D1 cells, they conclude that P2Y2 is the main target of ATP.

In our culture conditions, P2Y2 agonists, especially ATP, have always been applied on the luminal side of the epithelium; it is likely that P2Y2 receptors are located in the apical membrane of DC1 cells. This is further demonstrated by our results in DCTb cells growing on permeable filters, where ATP only induces [Ca2+]i increase when applied on the apical side of the epithelium (data not shown). Similarly, as shown in the accompanying paper, ATP only modulates chloride currents when applied on the apical side of DC1 cells. Moreover, Banderali et al. (1) recently presented evidence showing the presence of P2Y1-2 receptors in the apical membrane of A6 distal cells.

We demonstrated here that the increase in [Ca2+]i induced by nucleotides is biphasic. The early transient peak, which corresponds to a release of Ca2+ from intracellular stores, is followed by a sustained phase. The dependence of this sustained phase on external ATP and external Ca2+ suggests that this phase represents a cellular uptake of Ca2+, as has previously been reported for other cell types (37). We have provided direct evidence here, using Mn2+ quenching and 45Ca2+ influx measurements, for an ATP-stimulated entry of divalent cations. These experiments indicated an increase in Ca2+ uptake during the sustained phase. Moreover, this uptake was strongly blocked by either the removal of external Ca2+ or the application of La3+ or nifedipine to the extracellular membrane. Taken together, these results suggest that Ca2+ uptake during the sustained phase occurs through Ca2+ channels. Previous work in our laboratory (27) shows that such Ca2+ channels are present in the apical membrane of DCTb cells in primary culture. From this observation, we deduce that, in DC1 cells, apical Ca2+ channels are involved in the uptake of Ca2+ that occurs under ATP stimulation. These channels could be involved in the replenishment of intracellular calcium pools or in transepithelial calcium reabsorption (27, 38), with Ca2+ entering the cell down its electrochemical gradient.

In summary, the present paper shows that both ATP and UTP induce a biphasic increase in [Ca2+]i. The first part of the signal is the consequence of a Ca2+ release from intracellular stores due to 1) the activation of a P2 purinoceptor, 2) the presence of a GTP-binding protein, and 3) the activation of PLC. The second phase of the [Ca2+]i increase was demonstrated to be due to the entry of Ca2+ via apical calcium channels, which permits the refilling of intracellular calcium stores.


    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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