INTRODUCTION

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THE DISTAL TUBULE of the nephron normally reabsorbs ~10% of the filtered magnesium (23). As there is little or no magnesium beyond the distal tubule, this segment is important in determining renal magnesium balance. Magnesium absorption within this nephron segment is influenced by a number of hormones including glucagon and arginine vasopressin (AVP) (23). Using an immortalized mouse distal convoluted cell (MDCT) line, we have shown that glucagon and AVP stimulates Mg²⁺ uptake, suggesting that among the segments composing the distal tubule, the convoluted portion is a site of hormone action (2). The distal convoluted segment also possesses mineralocorticoid receptors (7, 9, 10, 15). Although there is some dispute concerning their functional purpose within this segment (14, 21), there is evidence that mineralocorticoid hormones enhance the expression of both Na⁺ conductance and NaCl cotransport in distal convoluted tubule cells (1, 27). Accordingly, mineralocorticoids may alter Mg²⁺ entry by acting through changes in ionic transport (4). Alternatively, mineralocorticoids may affect hormone-mediated control of Mg²⁺ transport within the distal tubule. Doucet et al. (6) have shown that adrenalectomy reduced glucagon-stimulated adenylate cyclase activity in thick ascending limbs; this reduced cAMP response was prevented with aldosterone administration (6). Other investigators have shown that AVP-sensitive adenylate cyclase is diminished in rat and rabbit collecting tubules harvested from adrenalectomized animals (8, 13, 20, 26). Accordingly, mineralocorticoids could alter hormonal control of transport in the distal tubule through its actions on receptor-mediated signaling pathways.

In an accompanying report (2), we reported that glucagon and AVP increases cAMP accumulation in MDCT cells. As cAMP also stimulates Mg²⁺ entry, these hormones may act, in part, through cAMP-mediated responses. It was therefore of interest to examine the effects of aldosterone on Mg²⁺ transport in distal convoluted tubule cells and the interactions with hormone-stimulated Mg²⁺ uptake. In the present studies, it was shown that aldosterone potentiates glucagon- and AVP-stimulated intracellular cAMP accumulation and Mg²⁺ uptake in MDCT cells. The potentiation of hormone actions by mineralocorticoids may have significant effects on renal magnesium balance.

	METHODS

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Materials. Basal Dulbecco's minimal essential medium (DMEM) and Ham's F-12 media were purchased from GIBCO. Customized Mg²⁺-free media were purchased from Stem Cell Technologies (Vancouver, BC, Canada). Fetal calf serum was from Flow Laboratories (McLean, VA). Mag-fura 2-AM was obtained from Molecular Probes (Eugene, OR). Glucagon, AVP, aldosterone, and other materials were from Sigma Chemical (St. Louis, MO).

Cell culture. Distal convoluted tubule cells were isolated from mice, immortalized, and characterized as previously described (12, 17). The MDCT cell line was grown on 60-mm plastic culture dishes (Corning Glass Works, Corning Medical and Scientific, Corning, NY) in DMEM-F12 (1:1) media supplemented with 10% fetal calf serum, 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 phosphate-buffered saline containing 5 mM ethylene glycol-bis(-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 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, the cells were cultured in Mg²⁺-free media (<0.01 mM) for 16-24 h prior to study. Other constituents of the Mg²⁺-free were similar to the complete media. The Mg²⁺-free media contained 0.2% bovine serum albumin rather than fetal calf serum.

Cytosolic Mg²⁺ measurements. Coverslips were mounted into a perfusion chamber, and intracellular free Mg²⁺ concentration ([Mg²⁺]_i) was determined with the use of the Mg²⁺-sensitive fluorescent dye, mag-fura 2 (18). The cell-permeant acetoxymethyl ester (AM) form of the dye was dissolved in dimethyl sulfoxide to a stock concentration of 5 mM and then diluted to 5 µM mag-fura 2-AM in media for 20 min at 23°C. Cells were subsequently washed three times with buffered salt solution containing (in mM) 145 NaCl, 4.0 KCl, 0.8 K₂HPO₄, 0.2 KH₂PO₄, 1.0 CaCl₂, 5 glucose, and 20 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-tris(hydroxymethyl)aminomethane (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.

	RESULTS

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Mg²⁺ entry into MDCT cells. Since 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. Subconfluent MDCT monolayers were cultured in Mg²⁺-free medium for 8-16 h. These cells possessed a significantly lower and reproducible [Mg²⁺]_i, 0.22 ± 0.01 mM, as indicated in Fig. 1. When the Mg²⁺-depleted MDCT cells were placed in a bathing solution containing 1.5 mM MgCl₂, [Mg²⁺]_i increased with time and leveled at a [Mg²⁺]_i value of 0.52 ± 0.02 mM (n = 6), which was similar to normal cells (0.53 ± 0.03 mM, n = 6). The rate of refill, d([Mg²⁺]_i)/dt, measured as the change in [Mg²⁺]_i with time, was 164 ± 5 nM/s (n = 6), as determined over the first 500 s following addition of magnesium.

View larger version (13K): [in this window] [in a new window]   View larger version (15K): [in this window] [in a new window]   Fig. 1.   Aldosterone potentiates glucagon- and arginine vasopressin (AVP)-stimulated Mg2+ uptake in Mg2+-depleted mouse distal convoluted tubule (MDCT) cells. Confluent MDCT cells were cultured in Mg2+-free media (<0.01 mM) for 16-20 h. Fluorescence studies were performed in buffer solutions in absence of Mg2+, and, as indicated, MgCl2 (1.5 mM final concentration) was added to observe changes in intracellular Mg2+ concentration ([Mg2+]i). 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. Glucagon, 107 M (A), and AVP, 3 × 107 M (B), were added to this buffer solution where indicated. Where indicated, MDCT cells were treated with aldosterone (Aldo), 107 M, for 16 h prior to fluorescence. Fluorescence was measured at 1 data point/s with 25-point signal averaging, and tracing was smoothed according to methods previously described (18).

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Aldosterone potentiates hormone-stimulated Mg²⁺ uptake. Next, we determined the effects of mineralocorticoids on Mg²⁺ entry into distal convoluted tubule cells. The addition of aldosterone, 10⁷ M, 20 min prior to the measurement of Mg²⁺ entry did not alter the rate of Mg²⁺ uptake (189 ± 21 nM/s, n = 3). Incubation of MDCT cells with aldosterone, 10⁷ M, for 16 h prior to determination of Mg²⁺ entry also had no effect on basal Mg²⁺ uptake (172 ± 8 nM/s, n = 3). However, pretreatment of MDCT cells with aldosterone potentiated both glucagon- and AVP-stimulated Mg²⁺ uptake rates (Fig. 1). Hormone-stimulated Mg²⁺ refill was enhanced with aldosterone, but aldosterone treatment had no effect on the final [Mg²⁺]_i attained following refill; this first concentration was not different from control values (0.52 mM). Furthermore, aldosterone potentiated glucagon and AVP at all hormone concentrations above 10⁹ M (Figs. 2 and 3). The glucagon concentration required to produce half-maximal stimulation of Mg²⁺ uptake in aldosterone-treated cells was ~5 × 10⁷ M, which was not different from control cells. Unlike glucagon, AVP stimulates Mg²⁺ uptake after a latent period of 5-10 min (Fig. 1B). Pretreatment of cells with aldosterone appeared to shorten the duration of this latent period (Fig. 1B). The AVP concentrations for half-maximal stimulated Mg²⁺ uptake was not changed (Fig. 3).

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Aldosterone potentiates glucagon-stimulated Mg2+ entry in MDCT cells. MDCT cells were incubated with and without aldosterone, 107 M, for 16 h prior to determination of Mg2+ entry. Rate of Mg2+ uptake, d([Mg2+]i)/dt, was determined over the initial 500 s following addition of the hormone and MgCl2. Glucagon was added at concentrations indicated. Values without aldosterone are from Ref. 2. Values are means ± SE for 3-6 cells. * P < 0.05, significantly different for Mg2+ uptake with glucagon vs. aldosterone + glucagon.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Aldosterone potentiates AVP-stimulated Mg2+ uptake in MDCT cells. Fluorescence determinations were performed as described in Fig. 1. AVP was added at concentrations indicated. Cells were incubated with and without aldosterone, 107 M, for 16-20 h prior to uptake measurements. Mg2+ uptake rates were determined 6 min following addition of AVP. Tracings are representative of 4-6 cells. * P < 0.05, significantly different for Mg2+ uptake with AVP vs. aldosterone + AVP.

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Aldosterone potentiation of hormone-stimulated Mg2+ entry is through dihydropyridine-sensitive pathways. MDCT cells were treated with aldosterone (Aldo), 107 M, for 16 h prior to determining hormone-responsive Mg2+ uptake. Nifedipine, 10 µM, was added with the 1.5 mM MgCl2 refill solution. Values are means ± SE for 3-6 cells. * Significantly different from control values. + Significantly different for aldosterone plus hormone vs. respective glucagon (Glu) or AVP values.

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View larger version (10K): [in this window] [in a new window]   Fig. 5.   Aldosterone potentiates glucagon-stimulated intracellular cAMP accumulation in a concentration-dependent manner. MDCT cells were incubated for 16 h in Mg2+-free buffer solution containing the indicated aldosterone concentrations. Glucagon, 107 M, was added, and cAMP was measured following 5 min in presence of 3-isobutyl-1-methylxanthine. Values are means ± SE for 3-5 observations. * P < 0.05, significantly different from control values.

Aldosterone potentiates hormone-stimulated Mg²⁺ uptake and cAMP generation through de novo protein synthesis. As it has been well established that mineralocorticoids act, in part, through translational processes, we tested whether potentiation of hormone action may be through induced proteins. In this study, we Mg²⁺ depleted the MDCT cells for 16 h prior to the addition of aldosterone with and without cycloheximide. The cells were incubated for a further 3 h, and fluorescence measurements were performed in the presence of 1.5 mM MgCl₂ to determine Mg²⁺ uptake. Mg²⁺ uptake was 244 ± 14 nM/s in the presence of aldosterone plus glucagon. Addition of cycloheximide with aldosterone, 3 h prior to fluorescence determination, resulted in the inhibition of aldosterone potentiation of glucagon-stimulated Mg²⁺ entry (Table 2). Cycloheximide did not have any effect on glucagon-stimulated Mg²⁺ entry in the absence of mineralocorticoids (Table 2). These observations indicate that aldosterone may potentiate hormone actions through mechanisms dependent on de novo protein synthesis.

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	DISCUSSION

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Mineralocorticoid receptors are present in distal convoluted tubule cells, which are thought to be involved in enhanced expression of NaCl cotransport, Na⁺ conductance, and sodium pump activity (1, 7, 9, 10, 15, 21, 25). Reports of the effects of adrenal steroids on renal magnesium conservation are equivocal (reviewed in Refs. 16 and 19). On balance, acute administration of aldosterone has little or no effect on renal magnesium conservation, whereas chronic hyperaldosteronism is associated with renal magnesium wasting (16). The latter has been explained by mineralocorticoid-induced volume expansion leading to diminished proximal and loop magnesium reabsorption. Alternatively or in conjunction with volume expansion, cellular potassium depletion may provide the basis of renal magnesium wasting. We have shown that Mg²⁺ entry into potassium-depleted MDCT cells is significantly diminished compared with control cells (3). Alteration of sodium absorption in the distal tubule may also affect Mg²⁺ transport in this segment. The present studies suggest that aldosterone may have little effect on distal Mg²⁺ transport by itself but potentiates the actions of magnesium-conserving hormones, such as glucagon and AVP. Glucagon- and AVP-stimulated Mg²⁺ entry was greater in MDCT cells pretreated with aldosterone relative to control values (Fig. 1). These actions may be through an augmentation of hormone-mediated signaling pathways.

A number of studies have shown that mineralocorticoids enhance hormone-stimulated cAMP generation. Adrenal insufficiency is associated with impairment of urinary diluting and concentrating capacity (13, 26; reviewed in Ref. 6). Rajerison et al. (20) demonstrated that adrenalectomy reduced AVP-stimulated adenylate cyclase activity in membrane fractions prepared from rat kidney medulla. Doucet et al. (6) have shown that glucagon-, calcitonin-, and AVP-responsive adenylate cyclase activity is diminished in thick ascending limb and collecting tubule segments harvested from adrenalectomized rats compared with animals treated with aldosterone (6). They used segments within 2.5 h (8). These workers postulate that aldosterone induces a protein(s) that stimulates hormone-sensitive adenylate cyclase activity. Their studies with kidney membrane fractions and isolated segments demonstrated that an improvement of coupling between hormone receptors and adenyl cyclase catalytic units was responsible for enhanced cAMP generation in the presence of aldosterone (8). The mechanism(s) through which steroids control G_s proteins (synthesis and/or degradation vs. activity of each unit) is not known (6). Mineralocorticoids may also increase the number of hormone receptors expressed on the cell surface, thereby amplifying hormonal actions. The present studies suggest that aldosterone potentiates glucagon- and AVP-stimulated cAMP generation in distal convoluted tubule cells. This was associated with an increase in Mg²⁺ entry rate in response to these hormones. We have previously shown that cAMP increases Mg²⁺ uptake in MDCT cells; accordingly, aldosterone may enhance hormone-stimulated Mg²⁺ entry by increases in hormone-mediated cAMP release or by amplification of cAMP-mediated actions (2). As there is no association of Mg²⁺ uptake rate with hormone-induced cAMP release, the latter actions may be valid (2). Further research is warranted to determine the signaling pathways whereby hormones mediate the control of Mg²⁺ transport.

Aldosterone potentiates AVP-stimulated Mg²⁺ entry in MDCT cells. Although the physiological role of this response to AVP is not clear, stimulation of Mg²⁺ reabsorption by AVP may be necessary to maintain normal Mg²⁺ balance when salt and water flow in the distal tubule is reduced during antidiuresis. Mineralocorticoid potentiation would increase AVP-stimulated Mg²⁺ reabsorption commensurate with enhanced salt and water retention. The importance of potentiation of glucagon effects is also not clear, but a similar rationale may hold as for AVP, because de Rouffignac (22) has postulated that a number of hormones including glucagon and AVP act in combination to maintain salt and water equilibrium. Accordingly, aldosterone potentiation of these hormones is associated with increased Mg²⁺ conservation along with salt and water retention.

In summary, these studies show that aldosterone potentiates glucagon- and AVP-stimulated Mg²⁺ uptake into MDCT cells. From these observations, we infer that mineralocorticoids may have important effects on hormone regulation of magnesium absorption within the distal convoluted tubule. Aldosterone acts through de novo protein synthesis on nifedipine-sensitive pathways. However, the intracellular pathways involved in stimulation of Mg²⁺ uptake remain to be described.