INTRODUCTION

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ADENOSINE MODULATES A VARIETY of transport functions in the distal tubule. Adenosine increases transepithelial resistance and diminishes osmotic water absorption and Na⁺ transport in inner medullary collecting duct (IMCD) cells in culture and Cl secretion in distal and collecting tubule cells (5, 15, 17, 18). On the other hand, adenosine stimulates Na⁺ transport in amphibian kidney A6 cells, a model of the distal tubule (8, 10), and Cl conductance in rabbit distal convoluted tubule cells (16). Adenosine also stimulates Ca²⁺ reabsorption in a mixture of rabbit primary connecting tubule and cortical collecting duct cells (10). The diversity of responses might be due to expression of different purine receptors in cells comprising the distal tubule. Three receptor subtypes (A₁, A₂, A₃) have been identified, all of which are coupled to G proteins. The A₁ receptor is coupled to G_i, leading to inhibition of adenylate cyclase, and to G_q, resulting in activation of phospholipase C, intracellular Ca²⁺ release, and an increase in protein kinase C activity (15). A₂ receptors are coupled, through G_q, to stimulate adenylate cyclase (15). The A₃ receptors, like the A₁ receptor subtypes, are coupled to a G_i and G_q but are only found in the heart and nervous system (6). Thus adenosine may have diverse effects on electrolyte reabsorption in the distal tubule. The convoluted portion of the distal tubule provides the final control of urinary Mg²⁺ excretion, as there is no Mg²⁺ reabsorption beyond this segment (14). Accordingly, any influence of adenosine on distal magnesium transport would be expected to alter renal Mg²⁺ excretion.

In the present studies, we determined the effect of adenosine on Mg²⁺ uptake into immortalized mouse distal convoluted tubule (MDCT) cells, a model we have extensively used to study Mg²⁺ handling in the distal convoluted tubule (4). The distal convoluted tubule has not been extensively studied because performing in vivo or in vitro perfusion experiments is difficult. Our studies using MDCT cells suggest that the rate of Mg²⁺ entry reflects overall transepithelial reabsorption (4). The MDCT cell line possesses many of the properties of the intact distal convoluted tubule, including many hormone receptors and extracellular divalent cation-sensing receptors (CaSR). We have reported that hormones such as parathyroid hormone (PTH), glucagon, and arginine vasopressin (AVP) stimulate Mg²⁺ uptake (2, 4). Aldosterone potentiates hormone-mediated Mg²⁺ entry (3) and high extracellular Ca²⁺ and Mg²⁺ levels inhibit hormone-responsive uptake (1). Accordingly, MDCT cells are a useful model to study controls of Mg²⁺ transport. In the present study, we show that adenosine may stimulate Mg²⁺ entry or inhibit hormone-mediated Mg²⁺ uptake in MDCT cells via A₂ and A₁ receptors, respectively. We infer from these studies that adenosine might modulate Mg²⁺ transport in the intact distal convoluted tubule.

	MATERIALS AND METHODS

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Cell culture. Distal convoluted tubule cells were isolated from mice and immortalized by Pizzonia et al. (13) and functionally characterized as described by Friedman and Gesek and their colleagues (7). The MDCT cell line was grown on 60-mm plastic culture dishes (Corning Glass Works, Corning Medical and Scientific, Corning, NY) in basal DMEM-Ham's F-12, 1:1, media (GIBCO) supplemented with 10% fetal calf serum (Flow Laboratories, McLean, VA), 1 mM glucose, 5 mM L-glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin in a humidified environment of 5% CO₂-95% air at 37°C. For the fluorescence studies, confluent cells were washed three times with PBS containing 5 mM EGTA, trypsinized, and seeded on glass coverslips. Aliquots of harvested cells were allowed to settle onto sterile glass coverslips in 100-mm Corning tissue culture dishes, and the cells were grown to subconfluence over 1-2 days in supplemented media as described above. The normal media contained 0.6 mM Mg²⁺ and 1.0 mM Ca²⁺. In the experiments indicated, MDCT cells were cultured in Mg²⁺-free media (<0.01 mM) for 16-24 h before study. Other constituents of the Mg²⁺-free culture media were similar to the complete media. Customized Mg²⁺-free media were purchased from Stem Cell Technologies (Vancouver, BC). These media contained 0.2% bovine serum albumin rather than fetal calf serum.

Cytoplasmic Mg²+ and Ca²⁺ measurements. Coverslips were mounted in a perfusion chamber, and intracellular Mg²⁺ and Ca²⁺ concentration ([Mg²⁺]_i and [Ca²⁺]_i, respectively) were determined with the use of the Mg²⁺- and Ca²⁺-sensitive fluorescent dyes mag-fura 2 and fura 2, respectively (Molecular Probes, Eugene, OR). The cell-permeant acetoxymethyl ester (AM) form of the dye was dissolved in DMSO to a stock concentration of 5 mM and then diluted to 5 or 10 µM fura 2-AM in media for 20 min at 37°C. Cells were subsequently washed three times with buffered salt solution containing (in mM) 145 NaCl, 4.0 KCl, 0.8 Na₂HPO₄, 0.2 KH₂PO₄, 1.0 CaCl₂, 5 glucose, and 20 HEPES/Tris, at pH 7.4. The MDCT cells were incubated for a further 20 min to allow for complete deesterfication and washed once before measurement of fluorescence.

Determination of cAMP concentration. cAMP was determined in confluent MDCT cell monolayers cultured in 24-well plates in DMEM-Ham's F-12 media without serum but with 0.1% BSA. The media contained 0.6 mM or 0 Mg²⁺ where indicated. After addition of the agonist to be tested, MDCT cells were incubated at 37°C for 5 min. cAMP was extracted with 5% trichloroacetic acid, which was removed with ether, and the extract was acidified with 0.1 N HCl. The aqueous phase was dried, dissolved in Tris-EDTA buffer, and then cAMP was measured with a radioimmunoassay kit (Diagnostic Products, Los Angeles, CA).

Statistical analysis. Representative tracings of fluorescent intensities are given, and significance was determined by Student's t-test or Tukey's analysis of variance as appropriate. All results are expressed as means ± SE where indicated.

	RESULTS

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Adenosine alters Mg²+ uptake into Mg²⁺-depleted MDCT cells. Because there is not an appropriate radioisotope for Mg²⁺ to directly measure Mg²⁺ transport rates, we developed the following model to assess Mg²⁺ influx into single MDCT cells (4). Subconfluent MDCT monolayers were cultured in Mg²⁺-free medium for 16 h. These cells possessed a significantly lower [Mg²⁺]_i, 0.22 ± 0.01 mM, than that observed in normal MDCT cells, 0.53 ± 0.02 mM. When the Mg²⁺-depleted MDCT cells were placed in a bathing solution containing 1.5 mM MgCl₂, [Mg²⁺]_i increased with time and plateaued at 0.50 ± 0.07 mM, n = 7, which was similar to that observed in normal cells (4). The mean rate of refill, d([Mg²⁺]_i)/dt, measured as the change in [Mg²⁺]_i with time, was 137 ± 16 nM/s, n = 7, experiments, as determined over the first 500 s after addition of Mg²⁺. We have previously reported data that indicate the Mg²⁺ uptake is concentration dependent and selective for Mg²⁺ (4).

View larger version (33K): [in this window] [in a new window]   Fig. 1.   Adenosine alters Mg2+ uptake in Mg2+-depleted mouse distal convoluted tubule (MDCT) cells. MDCT cells were cultured in Mg2+-free media (<0.01 mM) for 16 h. Fluorescence studies were performed in buffer solutions in absence of external magnesium, and where indicated, MgCl2 (1.5 mM final concentration) was added to observe changes in intracellular Mg2+ concentration ([Mg2+]i). The buffer solutions contained (in mM) 145 NaCl, 4.0 KCl, 0.8 K2HPO4, 0.2 KH2PO4, 1.0 CaCl2, 5.0 glucose, and 10 HEPES/Tris, pH 7.4, with and without 1.5 mM MgCl2. N6-cyclopentyladenosine (CPA), a selective A1 agonist, and 2-[p-(2-carbonyl-ethyl)-phenylethylamino]-5'-N-ethylcarboxamidoadenosine (CGS), an A2 agonist, were added at concentrations of 10 µM. Fluorescence was measured at 1 data point/s with 25-point signal averaging, and the tracing was smoothed according to methods previously described (2). Values are means ± SE for 5-7 cells. *P < 0.01, Mg2+ entry rates vs. control values.

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Antagonists of A2 purinoceptors inhibit adenosine-mediated Mg2+ uptake. Relatively selective antagonists of A1 receptors, 8-cyclopentyl-1,3-diproxylxanthine (DPCPX), and A2 receptors, 3,7-dimethyl-1-propargylxanthine (DMPX), were added where indicated 15 min before the addition of adenosine at concentrations of 10 µM. *, +: P < 0.01, Mg2+ entry rates vs. control and adenosine values, respectively.

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A₁ purinoceptor agonists inhibit receptor-stimulated Mg²+ uptake. As the A₁ purinoceptor agonists inhibit PTH-stimulated cAMP formation (Table 2), we tested whether CPA might inhibit hormone-mediated Mg²⁺ uptake. Pretreatment of MDCT cells with CPA diminished PTH-stimulated Mg²⁺ entry rate by 27 ± 9% (Fig. 3). The pretreatment of MDCT cells with CPA also inhibited CSG-stimulated Mg²⁺ uptake from 252 ± 18, n = 4, to 196 ± 14, n = 3, nM/s (Fig. 3). These observations indicate that A₁ purinoceptor agonists inhibit hormone-stimulated Mg²⁺ entry and modulate the actions of A₂ purinoceptor agonists in MDCT cells.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   A1 purinoceptors inhibit parathyroid hormone (PTH)- and A2 agonist-mediated cAMP formation and Mg2+ uptake in MDCT cells. Cells were pretreated with the A1 agonist CPA (10 µM, 107 M) or the A2 agonist CGS (10 µM) 5 min before the addition of PTH. Values are means ± SE for 3-5 observations. *, +, : P < 0.01, mean Mg2+ entry rates and cAMP determinations of either PTH or CGS vs. respective control values, of PTH+CPA vs. PTH alone, and of CGS+CPA vs. CGS alone, respectively.

Characterization of A₂ purinoceptor agonist-stimulation of Mg²+ uptake in MDCT cells. CGS increased Mg²⁺ uptake in a concentration-dependent fashion with the maximal dose of ~10 µM (Fig. 4). These data for selective P1 receptor agonists indicate that adenosine stimulates Mg²⁺ uptake by the A₂ purinoceptor.

View larger version (31K): [in this window] [in a new window]   Fig. 4.   Concentration dependence of CGS-stimulation of Mg2+ entry in MDCT cells. The rate of Mg2+ influx as determined by uptake rate {d([Mg2+]i)/dt} was measured with the CGS concentrations shown, using fluorescence techniques performed according to procedures given in legend to Fig. 1. d([Mg2+]i)/dt values were determined over the first 500 s of fluorescence measurements. Values are means ± SE for 3-6 cells. Cont, control. *P < 0.01 for Mg2+ entry rates.

View larger version (30K): [in this window] [in a new window]   Fig. 5.   CGS stimulates Mg2+ uptake though A2 receptor-mediated signaling pathways. Inhibitors for protein kinase A [Rp-cAMPS (0.5 µM)], phospholipase C [U-73122 (15 µM)], and protein kinase C [Ro-31-822 (0.1 µM)] were added to Mg2+-depleted MDCT cells 5 min before the addition of CGS (10 µM). The MAP kinase inhibitor PD-98059 (1.0 µM) was added 10 min before CGS. Values are means ± SE for 3-5 cells. * P < 0.001, CGS vs. control uptake rates.  P < 0.01, inhibitor + CGS vs. CGS alone.

Aldosterone potentiates A₂ receptor agonist-stimulated Mg²+ uptake in MDCT cells. We have previously shown that aldosterone, applied 16 h before experimentation, increases PTH-, glucagon-, and AVP-mediated cAMP generation and potentiates hormone-mediated Mg²⁺ uptake (4). Although the cellular mechanisms are not known, it has been speculated that aldosterone-induced proteins modulate receptor signaling in epithelial cells (11). In the present study, we determined whether pretreatment of MDCT cells with aldosterone for 16 h potentiated the actions of the A₂ receptor agonist CGS. Treatment of cells with aldosterone, for 16 h before the study, did not significantly affect basal Mg²⁺ uptake (142 ± 11 nM/s, n = 3) but potentiated CGS-stimulated Mg²⁺ entry, from 251 ± 18 nM/s, n = 4, to 305 ± 17 nM/s, n = 6 (Fig. 6). Interestingly, aldosterone did not potentiate CGS-responsive cAMP production (62 ± 16 pmol · mg protein¹ · 5 min¹), suggesting that the actions are downstream of the generation of this second message (Fig. 6).

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Aldosterone (Aldo) potentiates CGS-mediated Mg2+ uptake. MDCT cells were incubated for 16 h in magnesium-free buffer solution containing aldosterone (107 M). CGS (10 µM) was added where indicated, and Mg2+ uptake was determined after 500 s in 1.5 mM MgCl2 or cAMP was measured after 5 min. Values are means ± SE for 4-7 observations. *, +, , : P < 0.01, mean Mg2+ entry rates and cAMP determinations vs. respective control values, CGS+aldosterone vs. CGS, CPA+CGS vs. CGS, and aldosterone+CPA+CGS vs. aldosterone+CGS, respectively.

Elevation of extracellular Ca²+ inhibits A₂ receptor agonist-stimulated cAMP generation and Mg²⁺ uptake. MDCT cells possess an extracellular CaSR that, on activation with polyvalent cations such as Ca²⁺, Mg²⁺, or neomycin, inhibits PTH-, glucagon-, and AVP-mediated cAMP generation and glucagon- and AVP-stimulated Mg²⁺ uptake (1). To determine whether activation of the CaSR alters A₂ receptor agonist actions, we pretreated cells for 5 min with 5.0 mM CaCl₂ before the addition of CGS. Elevation of extracellular Ca²⁺ did not have any effects on basal Mg²⁺ entry (147 ± 10 nM/s, n = 4) but abolished CGS stimulation of cAMP generation (21 ± 2 pmol · mg protein¹ · 5 min¹, n = 4) and Mg²⁺ uptake (128 ± 13 nM/s, n = 5) (Fig. 7). The mechanisms by which the CaSR inhibits CGS actions remain unclear, but the receptor is coupled to G_i proteins, which is consistent with the conclusion that CGS responses in MDCT cells are dependent, in part, on cAMP-mediated signaling pathways.

View larger version (17K): [in this window] [in a new window]   Fig. 7.   Summary of the effects of extracellular Ca2+ on CGS-stimulated cAMP formation and Mg2+ uptake. cAMP was measured by radioimmunoassay, and d([Mg2+]i)/dt was determined with 1.5 mM extracellular Mg2+ in the absence and presence of 5.0 mM CaCl2 as indicated. CaCl2 was added 5 min before the addition of 10 µM CGS. d([Mg2+]i)/dt was determined over the initial 500 s after addition of CGS. Values are means ± SE for 4-7 cells. *, +: P < 0.01, mean Mg2+ entry rates and cAMP determinations vs. respective control values and CGS vs. CGS+5.0 mM extracellular Ca2+, respectively.

ATP inhibits A₂ receptor agonist-stimulated Mg²+ uptake in MDCT cells. The relationship between the purines ATP and adenosine is complex. We have recently determined that ATP inhibits basal and hormone-stimulated Mg²⁺ transport by 21% in MDCT cells (2a). Our studies showed that this inhibition was via P2X purinoceptors as the selective P2X agonist ,-methylene-ATP (,-Me-ATP) inhibited Mg²⁺ uptake, but the more P2Y selective agonists UTP, ADP, and 2-methylthio ATP were without effect. Accordingly, it was of interest to see whether ATP would have any effect on Mg²⁺-conserving actions of adenosine. Pretreatment of MDCT cells with ,-Me-ATP prevented the stimulation of Mg²⁺ uptake by the A₂ receptor agonist (160 ± 9 nM/s, n = 4) (Fig. 8). These observations suggest that the purines may modulate Mg²⁺ uptake in MDCT cells by diverse receptor-mediated mechanisms.

View larger version (18K): [in this window] [in a new window]   Fig. 8.   Extracellular ATP inhibits A2 receptor agonist-stimulated Mg2+ uptake in MDCT cells. P2X purinoceptor agonists, such as ,-methylene-ATP ( ,-Me-ATP) inhibit hormone-mediated Mg2+ uptake (unpublished observations). The more selective P2X agonist, ,-Me-ATP (104 M) was added 5 min before 10 µM CGS where indicated. Values are means ± SE for 4-7 cells. *, +: P < 0.01, mean Mg2+ entry rates and cAMP determinations vs. respective control values and CGS vs. CGS+,-Me-ATP, respectively.

	DISCUSSION

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The findings in this study indicate that adenosine modulates Mg²⁺ uptake in MDCT cells, a model for the intact distal convoluted tubule. Our data show that both A₁ and A₂ purinoceptor subtypes are present in MDCT cells. Adenosine and the selective A₂ agonist CPA elicited receptor-mediated intracellular Ca²⁺ signaling, whereas adenosine and the A₁ receptor agonist CGS increase cAMP formation (Tables 1 and 2). CPA inhibited PTH- and CGS-stimulated cAMP formation typical of A₁ purinoceptor-induced signaling involving G_i-coupled proteins (Fig. 3). This was associated with diminished Mg²⁺ uptake (Fig. 3). On the other hand, CGS stimulated cAMP formation in a manner characteristic of A₂ purinoceptor signal transduction. CGS increased Mg²⁺ entry (Fig. 3). Moreover, CGS-stimulate Mg²⁺ uptake was decreased by protein kinase A inhibition, supporting the notion that A₂ receptors modulate Mg²⁺ entry in MDCT cells via the G_s-coupled proteins (Fig. 6). A₁ and A₂ receptors are often polarized to either the apical or basolateral membrane so that adenosine may have divergent effects depending on the concentration at these two sides (15). Unfortunately, we are not able to determine the polarity of A₁ and A₂ receptors in MDCT cells; nevertheless, these observations demonstrate the multipotency of the effect of adenosine on Mg²⁺ transport. Polarization of the purinoceptors remains to be determined in the intact distal convoluted tubule.

Aldosterone modulates adenosine-stimulated Mg²+ entry in MDCT cells. We have shown that aldosterone potentiates hormone-responsive Mg²⁺ transport in MDCT cells (3). The prominent mechanism of steroids, which operate through nuclear receptors, is to control transcriptional regulation, expression, and posttranslational targeting of heterotrimeric G proteins such as G_s , G_i, G, G, and phospholipase C (11). Pretreatment of MDCT cells with aldosterone potentiated CGS-stimulated Mg²⁺ entry (Fig. 6). Aldosterone may increase any of the above pathways or others that ultimately lead to increased adenosine-stimulated Mg²⁺ entry in MDCT cells. In support of this notion, aldosterone potentiates hormone-stimulated Mg²⁺ uptake without increasing cAMP formation so that other processes downstream of cAMP generation are involved.

Extracellular Ca²+ affects A₂ agonist-mediated Mg²⁺ uptake in MDCT cells. The CaSR within the distal tubule is important in controlling Mg²⁺ entry in MDCT cells (1, 4). The extracellular Ca²⁺- and Mg²⁺-sensing mechanisms provide a negative-feedback loop to diminish the renal conserving actions of the circulating hormones like PTH, glucagon, and AVP (4). We have reported that elevation of extracellular Ca²⁺ or Mg²⁺, or the addition of the polyvalent cation neomycin, inhibits peptide hormone-stimulated cAMP formation and hormone-responsive Mg²⁺ uptake in MDCT cells (1). Activation of CaSR inhibits A₂ agonist stimulation of Mg²⁺ uptake in MDCT cells (Fig. 7). The responses likely involve diminished A₂ agonist-mediated cAMP formation, phospholipase C, protein kinase C, or MAP kinase cascades (Fig. 5). These findings show that adenosine-mediated effects may be modulated by extracellular Ca²⁺ and Mg²⁺ concentration.

ATP inhibits adenosine-stimulated Mg²+ uptake. We have shown that ATP inhibits hormone-stimulated Mg²⁺ via P2X purinoceptors (2a). These receptors are coupled to ATP-gated channels that activate nonselective cation channels. In these studies, the selective P2X receptor agonist ,-Me-ATP inhibited basal and hormone-stimulated Mg²⁺ uptake by 32%. In the present study, ,-Me-ATP inhibited adenosine-stimulated Mg²⁺ uptake in MDCT cells (Fig. 8). The pathophysiological implications of these interactions are unclear, but autocrine or paracrine secretion or tissue damage leading to cellular ATP release and its degradation to adenosine may be sufficient to alter Mg²⁺ handling in the distal tubule.

Role of adenosine in distal tubular Mg²+ handling. We infer from our data that adenosine modulates magnesium transport in the distal convoluted tubule of the nephron. Adenosine via A₁ receptors may inhibit hormone-stimulated Mg²⁺ uptake or, via A₂, may stimulate Mg²⁺ entry. Accordingly, hormones such as PTH, vasopressin, and calcitonin stimulate distal Mg²⁺, in part, through intracellular generation of cAMP that may be metabolized to 5'-AMP and adenosine within the cell (17). Adenosine is transported out of the cell by a nucleoside transporter. In addition, intracellular 5'-AMP may be transported out of the cell by a nucleotide transporter and further metabolized to adenosine by a membrane ecto-5'-nucleotidase, as summarized by Schwiebert et al. (17). Extracellular adenosine may act at cell-surface A₁ receptors to diminish hormone-mediated cAMP formation, leading to termination of the hormone stimulus. Alternatively, extracellular adenosine may act at cell-surface A₂ receptors to further increase cAMP, leading to a propagation of the hormone stimulus so that adenosine may act as an autocoid to stimulate Mg²⁺ reabsorption in conjunction with the known Mg²⁺-conserving circulating hormones (4). Further studies are required to identify and locate nucleotide transporters and cell-surface purinoceptors in the distal convoluted tubule.