Adenosine modulates Mg2+ uptake in distal convoluted tubule cells via A1 and A2 purinoceptors

Hyung Sub Kang, Dirk Kerstan, Long-Jun Dai, Gordon Ritchie, and Gary A. Quamme

Department of Medicine, University of British Columbia, Vancouver Hospital and Health Sciences Centre, Vancouver, British Columbia, Canada V6T 1Z3


    ABSTRACT
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tk;1Adenosine plays a role in the control of water and electrolyte reabsorption in the distal tubule. As the distal convoluted tubule is important in the regulation of renal Mg2+ balance, we determined the effects of adenosine on cellular Mg2+ uptake in this segment. The effect of adenosine was studied on immortalized mouse distal convoluted tubule (MDCT) cells, a model of the intact distal convoluted tubule. The rate of Mg2+ uptake was measured with fluorescence techniques using mag-fura 2. To assess Mg2+ uptake, MDCT cells were first Mg2+ depleted to 0.22 ± 0.01 mM by being cultured in Mg2+-free media for 16 h and then placed in 1.5 mM MgCl2; next, changes in intracellular Mg2+ concentration ([Mg2+]i) were determined. [Mg2+]i returned to basal levels, 0.53 ± 0.02 mM, with a mean refill rate, d([Mg2+]i)/dt, of 137 ± 16 nM/s. Adenosine stimulates basal Mg2+ uptake by 41 ± 10%. The selective A1 purinoceptor agonist N6-cyclopentyladenosine (CPA) increased intracellular Ca2+ and decreased parathyroid hormone (PTH)-stimulated cAMP formation and PTH-mediated Mg2+ uptake. On the other hand, the selective A2 receptor agonist 2-[p-(2-carbonyl-ethyl)-phenylethylamino]-5'-N-ethylcarboxamidoadenosine (CGS) stimulated Mg2+ entry in a concentration-dependent fashion. CGS increased cAMP formation and the protein kinase A inhibitor RpcAMPS inhibited CGS-stimulated Mg2+ uptake. Selective inhibition of phospholipase C, protein kinase C, or mitogen-activated protein kinase enzyme cascades with U-73122, Ro-31-8220, and PD-98059, respectively, diminished A2 agonist-mediated Mg2+ entry. Aldosterone potentiated CGS-mediated Mg2+ entry, and elevation of extracellular Ca2+ diminished CGS-responsive cAMP formation and Mg2+ uptake. Accordingly, MDCT cells possess both A1 and A2 purinoceptor subtypes with intracellular signaling typical of these respective receptors. We conclude that adenosine has dual effects on Mg2+ uptake in MDCT cells through separate A1 and A2 purinoceptor pathways.

intracellular magnesium; fluorescence; intracellular calcium transients; intracellular adenosine 3',5'-cyclic monophosphate; immortalized mouse distal convoluted tubule cells


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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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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 Gi, leading to inhibition of adenylate cyclase, and to Gq, resulting in activation of phospholipase C, intracellular Ca2+ release, and an increase in protein kinase C activity (15). A2 receptors are coupled, through Gq, to stimulate adenylate cyclase (15). The A3 receptors, like the A1 receptor subtypes, are coupled to a Gi and Gq 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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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.

Epifluorescence microscopy was used to monitor changes in the mag-fura 2 or fura 2 fluorescence of single MDCT cells cultured in monolayers. The chamber was mounted on an inverted Nikon Diaphot-TMD microscope, with a Fluor ×100 objective, and fluorescence within a single cell was monitored under oil immersion over the course of the study. Fluorescence was recorded at 1-s intervals using a dual-excitation wavelength spectrofluorometer (Delta-scan, Photon Technologies, Princeton, NJ) with excitation for mag-fura 2 at 335 and 385 nm, for fura 2 at 340 and 380 nm (chopper speed set at 100 Hz/s), and emission at 505 nm. All experiments were performed at 21°C because the mag-fura 2 and fura 2 responses were found to be identical at room temperature and 37°C. Media changes were made without an interruption in recording.

Free [Mg2+]i and [Ca2+]i were calculated from the ratio of the fluorescence at the two excitation wavelengths as described using a dissociation constant (Kd) of 1.4 mM and 224 nM, respectively, for the mag-fura 2-Mg2+ and fura 2-Ca2+ complexes (2). The minimum (Rmin) and maximum (Rmax) ratios were determined for the cells at the end of each experiment using 20 µM digitonin.

Rmax for mag-fura 2 was found by the addition of 50 mM MgCl2 in the absence of Ca2+, and Rmin was obtained by removal of Mg2+ and addition of 100 mM EDTA, pH 7.2. The excitation spectrum of the cellular mag-fura 2 under these conditions was similar to that of free mag-fura 2 in the same solutions. Rmax and Rmin for fura 2 were obtained with Ca2+ and EGTA by previously published techniques (2).

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.


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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).

Adenosine stimulated Mg2+ uptake in Mg2+-depleted MDCT cells by 41 ± 10% of control values (Fig. 1). The adenosine/P1 receptor family in epithelial cells comprises A1 and A2 adenosine receptors that have been identified by molecular and pharmacological studies (15). We used N6-cyclopentyladenosine (CPA), a selective A1 agonist, and 2-[p-(2-carbonyl-ethyl)-phenylethylamino]-5'-N-ethylcarboxamidoadenosine (CGS), an A2 agonist, to determine the P1 subtype by which adenosine alters Mg2+ entry. The agonists, CPA and CGS, were from RBI (Sigma, St. Louis, MO). The A1 agonist, CPA (10 µM) did not alter basal Mg2+ uptake, 159 ± 17 nM/s, n = 5, but the A2 receptor agonist CGS nearly doubled the entry rate to 252 ± 18 nM/s, n = 5. 


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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.

The relatively selective antagonist of A1 receptors, 8-cyclopentyl-1,3-diproxylxanthine (DPCPX), and of A2 receptors, 3,7-dimethyl-1-propargylxanthine (DMPX), were used to confirm that adenosine stimulated Mg2+ entry by A2 purinoceptors. The antagonists were from RBI. The A2 receptor antagonist DMPX inhibited adenosine-stimulated Mg2+ uptake from 252 ± 18 to 120 ± 15 nM/s, n = 4; the latter value was not different from basal values (Fig. 2). DPCPX did not alter adenosine responses (Fig. 2). These findings support the conclusion that adenosine increases Mg2+ entry via A2 purinoceptors.


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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.

Next, we determined some of the receptor-mediated signaling mechanisms involved with the adenosine-mediated Mg2+ uptake. The most commonly recognized signal transduction mechanism for the A2 receptor is activation of adenylate cyclase. This implies coupling with the G protein Gs, although other G proteins may also be involved (15). A1 purinoceptors, on the other hand, mediate a broad range of signaling responses caused by its coupling to different G proteins within the Gi/o family (15). This signaling pathway leads to diminished cAMP, activation of phospholipase C, which, in turn, leads to membrane phosphoinositide metabolism and increased production of inositol triphosphate and Ca2+ mobilization. First, we determined receptor-mediated [Ca2+]i with fluorescence. Adenosine resulted in a transient increase in Ca2+ concentration, from basal concentrations of 95 ± 7 to 516 ± 35 nM, which is typical of receptor-mediated intracellular Ca2+ release (Table 1). The A1 agonist CPA also initiated a transient increase in intracellular Ca2+ of a magnitude similar to that for adenosine. However, the A2 agonist CGS did not illicit large changes in Ca2+ signaling (129 ± 6 nM). The observation that CPA induced Ca2+ signaling supports the idea that there are A1 purinoceptors present in MDCT cells. Second, we determined the effect of the A1 and A2 receptor agonists on intracellular cAMP formation. Adenosine and CGS increased intracellular cAMP production by about threefold, from 20 ± 2 to 54 ± 9 and 65 ± 10 pmol · mg protein-1 · 5 min-1, respectively, whereas CPA had no effect (25 ± 2 pmol · mg protein-1 · 5 min-1) (Table 2). PTH stimulated cAMP in control cells (42 ± 6 pmol · mg protein-1 · 5 min-1). CPA diminished PTH-stimulated cAMP (35 ± 2 pmol · mg protein-1 · 5 min-1), whereas the A2 receptor agonist CGS increased PTH-stimulated cAMP formation (69 ± 9 pmol · mg protein-1 · 5 min-1), which was similar to the results for CGS alone; i.e., no additive effect. These findings indicate that MDCT cells possess both A1 and A2 purinoceptors that have the classic signaling pathways of each of the respective receptor families.

                              
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Table 1.   P1 receptor agonists stimulate cytosolic Ca2+ transients in MDCT cells


                              
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Table 2.   P1 receptor agonists alter intracellular cAMP accumulation

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.


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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, 10-7 M) or the A2 agonist CGS (10 µM) 5 min before the addition of PTH. Values are means ± SE for 3-5 observations. *, +, open circle : 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.


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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.

To characterize some of the A2-mediated signaling pathways that adenosine uses to stimulate Mg2+ uptake, we pretreated the MDCT cells with a number of well-known inhibitors of kinases involved in G protein transduction. RpcAMPS, an inhibitor of protein kinase A, inhibited CGS agonist-stimulated Mg2+ entry rates (143 ± 16 nmol/s, n = 3), supporting the above observation that receptor-mediated cAMP formation is involved in A2-receptor agonist-mediated actions (Fig. 5). Pretreatment of MDCT cells with the phospholipase C inhibitor U-73122 inhibited A2-adrenergic agonist-stimulated Mg2+ uptake (124 ± 25 nmol/s, n = 5), and the protein kinase C inhibitor Ro-31-822 diminished CGS agonist-stimulated uptake, from 252 ± 18, n = 5, to 178 ± 18 nmol/s, n = 4 (Fig. 5). Inhibition of the MAP kinase cascade (extracellular-signal-regulated kinase, c-jun NH2-terminal kinase, p38) with PD-98059 also decreased Mg2+ uptake (125 ± 22 nmol/s, n = 4). Accordingly, A2 receptors act through a number of receptor-mediated signaling pathways to affect changes in Mg2+ transport. These findings are similar to those we have reported for hormone (PTH, glucagon, AVP) receptor-mediated Mg2+ transport in MDCT cells (4). Accordingly, we would expect that aldosterone and high extracellular Ca2+ concentration might alter CSG-stimulated Mg2+ uptake.


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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. dagger  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 protein-1 · 5 min-1), suggesting that the actions are downstream of the generation of this second message (Fig. 6).


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Fig. 6.   Aldosterone (Aldo) potentiates CGS-mediated Mg2+ uptake. MDCT cells were incubated for 16 h in magnesium-free buffer solution containing aldosterone (10-7 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. *, +, open circle , : 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 protein-1 · 5 min-1, 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 Galpha i proteins, which is consistent with the conclusion that CGS responses in MDCT cells are dependent, in part, on cAMP-mediated signaling pathways.


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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 beta ,gamma -methylene-ATP (beta ,gamma -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 beta ,gamma -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.


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Fig. 8.   Extracellular ATP inhibits A2 receptor agonist-stimulated Mg2+ uptake in MDCT cells. P2X purinoceptor agonists, such as beta ,gamma -methylene-ATP ( beta ,gamma -Me-ATP) inhibit hormone-mediated Mg2+ uptake (unpublished observations). The more selective P2X agonist, beta ,gamma -Me-ATP (10-4 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+beta ,gamma -Me-ATP, respectively.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 Gi-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 Gs-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 Galpha s , Galpha i, Gbeta , Ggamma , 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.

These studies indicate that MDCT cell Mg2+ uptake is regulated at two levels: first by membrane-receptor (adenosine) signaling and, second, by nuclear transcription-dependent receptor (aldosterone) signaling.

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 beta ,gamma -Me-ATP inhibited basal and hormone-stimulated Mg2+ uptake by 32%. In the present study, beta ,gamma -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.


    ACKNOWLEDGEMENTS

We thank Dr. Peter A. Friedman for providing the MDCT cell line.


    FOOTNOTES

Dr. Hyung Sub Kang is a Postdoctoral Fellow of the Korean Science and Engineering Foundation. This work was supported by research grants from the Canadian Institutes of Health Research (MT-5793) and Kidney Foundation of Canada.

Address for reprint requests and other correspondence: G. A. Quamme, Dept. of Medicine, Vancouver Hospital and Health Sciences Centre, Koerner Pavilion, 2211 Wesbrook Mall, Vancouver, BC, Canada V6T 1Z3 (E-mail:quamme{at}interchange.ubc.ca).

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

Received 16 March 2001; accepted in final form 25 July 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Bapty, BW, Dai LJ, Ritchie G, Canaff L, Hendy GN, and Quamme GA. Activation of Mg2+/Ca2+-sensing inhibits hormone-stimulated Mg2+ uptake in mouse distal convoluted tubule cells. Am J Physiol Renal Physiol 275: F353-F360, 1998[Abstract/Free Full Text].

2.   Dai, L-J, Bapty BW, Ritchie G, and Quamme GA. Glucagon and arginine vasopressin stimulates Mg2+ uptake in mouse distal convoluted tubule cells. Am J Physiol Renal Physiol 274: F328-F335, 1998[Abstract/Free Full Text].

2a.   Dai, L-J, Kang HS, Kerstan D, Ritchie G, and Quamme GA. ATP inhibits Mg2+ uptake in MDCT cells via P2X purinoceptors. Am J Physiol Renal Physiol 281: F833-F840, 2001[Abstract/Free Full Text].

3.   Dai, L-J, Ritchie G, Bapty B, and Quamme GA. Aldosterone potentiates hormone-stimulated Mg2+ uptake in distal convoluted tubule cells. Am J Physiol Renal Physiol 274: F336-F341, 1998[Abstract/Free Full Text].

4.   Dai, L-J, Ritchie G, Kerstan D, Kang HS, Cole DEC, and Quamme GA. Magnesium transport in the renal distal convoluted tubule. Physiol Rev 81: 51-84, 2001[Abstract/Free Full Text].

5.   Edwards, RM, and Spielman WS. Adenosine A1 receptor-mediated inhibition of vasopressin action in inner medullary collecting duct. Am J Physiol Renal Fluid Electrolyte Physiol 266: F791-F796, 1994[Abstract/Free Full Text].

6.   Fredholm, BB. Adenosine receptors in the central nervous system. News Physiol Sci 10: 122-128, 1995[Abstract/Free Full Text].

7.   Friedman, PA, and Gesek FA. Calcium transport in renal epithelial cells. Am J Physiol Renal Fluid Electrolyte Physiol 264: F181-F198, 1993[Abstract/Free Full Text].

8.   Hayslett, JP, Macala LJ, Smallwood JI, Kalghatgi L, Gasalla-Herraiz J, and Isales C. Adenosine stimulation of Na+ transport is mediated by an A1 receptor and a [Ca2+]i-dependent mechanism. Kidney Int 47: 1576-1584, 1995[ISI][Medline].

9.   Hoenderop, JGJ, Hartog A, Willems PHGM, and Bindels RJM Adenosine-simulated Ca2+ reabsorption is mediated by apical A1 receptors in rabbit cortical collecting system. Am J Physiol Renal Physiol 274: F736-F743, 1998[Abstract/Free Full Text].

10.   Lang, MA, Preston AS, and Handler JS. Adenosine stimulates sodium transport in kidney A6 epithelia in culture. Am J Physiol Cell Physiol 249: C330-C336, 1985[Abstract].

11.   Morris, AJ, and Malbon CC. Physiological regulation of G protein-linked signaling. Physiol Rev 79: 1373-1430, 1999[Abstract/Free Full Text].

12.   Moyer, BD, McCoy DE, Lee B, Kizer N, and Stanton BA. Adenosine inhibits arginine vasopressin-stimulated chloride secretion in a mouse IMCD cell line (mIMCD-K2). Am J Physiol Renal Fluid Electrolyte Physiol 269: F884-F891, 1995[Abstract/Free Full Text].

13.   Pizzonia, JH, Gesek FA, Kennedy SM, Coutermarsh BA, Bacskai BJ, and Friedman PA. Immunomagnetic separation, primary culture, and characterization of cortical thick ascending limb plus distal convoluted tubule cells from mouse kidney. In Vitro Cell Dev Biol 27A: 409-416, 1991[ISI].

14.   Quamme, GA. Renal magnesium handling: new insights in understanding old problems. Kidney Int 52: 1180-1195, 1997[ISI][Medline].

15.   Ralevic, V, and Burnstock G. Receptors for purines and pyrimidines. Pharmacol Rev 50: 413-492, 1998[Abstract/Free Full Text].

16.   Rubera, I, Barrière H, Tauc M, Bidet M, Verheecke-Mauze C, Poujeol C, Cuiller B, and Poujeol P. Extracellular adenosine modulates a volume-sensitive-like chloride conductance in immortalized rabbit DC1 cells. Am J Physiol Renal Physiol 280: F126-F145, 2001[Abstract/Free Full Text].

17.   Schwiebert, EM, Karlson KH, Friedman PA, Dietl P, Spielman WS, and Stanton BA. Adenosine regulates a chloride channel via protein kinase C and a G protein in a rabbit cortical collecting duct cell line. J Clin Invest 89: 834-841, 1992[ISI][Medline].

18.   Yagil, C, Katni G, and Yagil Y. The effect of adenosine on transepithelial resistance and sodium uptake in the inner medullary collecting duct. Pflügers Arch 427: 225-232, 1994[ISI][Medline].


Am J Physiol Renal Fluid Electrolyte Physiol 281(6):F1141-F1147
0363-6127/01 $5.00 Copyright © 2001 the American Physiological Society




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