INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

THE HORMONALLY ACTIVE METABOLITE of vitamin D, 1,25-dihydroxyvitamin D [1,25(OH)₂D₃], has an important role in regulating mineral metabolism at the level of the intestine, bone, and kidney (1, 32, 51). It is clear that 1,25(OH)₂D₃ increases intestinal calcium and magnesium absorption and regulates bone formation, but the actions of 1,25(OH)₂D₃ on the kidney are not as well characterized (1, 31, 51). On balance, clinical and experimental observations support the notion that 1,25(OH)₂D₃ increases calcium and magnesium reabsorption within the kidney (1, 19, 25, 26, 33, 36, 38, 45). Nevertheless, some studies reported little, if any, affect on renal divalent cation conservation (9, 13, 30, 39, 40). This discrepancy may be due to associated vitamin D-induced changes in plasma calcium and magnesium concentrations that affect renal divalent cation handling. An extracellular Ca²⁺/Mg²⁺-sensing receptor (CaSR) that modulates calcium and magnesium reabsorption in the loop of Henle and distal convoluted tubule has been identified along the length of the kidney tubule (27, 43). The distal convoluted tubule plays an important role in renal magnesium conservation as it has final control of urinary magnesium excretion (43). We have shown that elevated extracellular Ca²⁺ or Mg²⁺ inhibits hormone-mediated Mg²⁺ uptake in a distal convoluted tubule cell line (3). Our evidence is that hypercalcemia and hypermagnesemia diminish distal reabsorption, in part, through CaSR inhibition of peptide hormone receptor-mediated responses (3). In the present study, we investigated whether 1,25(OH)₂D₃ stimulates Mg²⁺ uptake into distal convoluted tubule cells and whether elevated extracellular Ca²⁺ or Mg²⁺ may inhibit these responses. Our notion is that 1,25(OH)₂D₃ plays a role in magnesium conservation at both the intestinal and renal level and that the latter is modulated by the prevailing extracellular Ca²⁺ and Mg²⁺ concentrations through the CaSR. Accordingly, increases in 1,25(OH)₂D₃ may lead to enhanced magnesium conservation, no change, or increased urinary excretion, depending on the magnitude of changes in plasma calcium and magnesium concentration.

To date, there are no experiments reporting the direct effects of vitamin D metabolites on cellular magnesium transport. In the present studies, we used mouse distal convoluted tubule (MDCT) cells to investigate the actions of 1,25-(OH)₂D₃ on Mg²⁺ uptake rates. MDCT cells have been extensively used to study cellular mechanisms of calcium and magnesium absorption within the distal tubule (23, 43). Parathyroid hormone (PTH), glucagon, and arginine vasopressin (AVP) stimulate Mg²⁺ uptake in MDCT cells (16, 17). In a recent study, we showed that MDCT cells also possess a polyvalent cation-sensing mechanism that is responsive to extracellular Mg²⁺ and Ca²⁺ (4). We documented the expression of the parathyroid/kidney Ca²⁺-sensing receptor (i.e., CaSR) in MDCT cells and suggested that it is responsible, either entirely or in part, for the extracellular polyvalent cation-sensing mechanism in these cells (4). We also found that activation of this mechanism with either extracellular Ca²⁺, Mg²⁺, or the polyvalent cation neomycin inhibited PTH-, glucagon-, and AVP-stimulated cAMP release (4) as well as hormone-stimulated Mg²⁺ uptake in MDCT cells (3). These studies indicated that this cation-sensing mechanism in immortalized MDCT cells plays an important role in modulating hormone-mediated intracellular signals in response to changes in extracellular Mg²⁺ as well as Ca²⁺. Yang et al. (54) have reported that MDCT cells also express 1,25(OH)₂D₃- and PTH-responsive 25(OH)D 24-hydroxylase (54), and Friedman and Gesek (22) have shown that 1,25(OH)₂D₃ accelerates PTH-dependent Ca²⁺ uptake, suggesting that vitamin D receptors are present in this cell line and play a role in mineral metabolism (22). Accordingly, these cells provide a useful model by which to determine the effects of 1,25(OH)₂D₃ on Mg²⁺ handling in the distal convoluted tubule, which is difficult to study in situ or dissect out for functional measurements. The present studies demonstrate that 1,25(OH)₂D₃ stimulates Mg²⁺ uptake in MDCT cells, providing evidence that the metabolite might affect magnesium absorption in the intact distal tubule. Moreover, elevation of divalent cations, acting through the CaSR, modulates 1,25(OH)₂D₃-mediated Mg²⁺ entry in this cell line.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Basal DMEM and Ham's F-12 media were from GIBCO Laboratories, Grand Island, NY. FCS was from Flow Laboratories (McLean, VA). Mag-fura 2-acetoxymethyl ester (AM) was obtained from Molecular Probes (Eugene, OR). 1,25(OH)₂D₃ and other materials were from Sigma (St. Louis, MO).

Cell preparation. Immortalized MDCT cells were kindly provided by Dr. P. A. Friedman. They were cultured in DMEM-Ham's F-12 media, 1:1, supplemented with 10% FCS, 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 cAMP determinations, the MDCT cells were cultured to confluence in 24-well plastic dishes. Sixteen hours before the cAMP measurements were made, the culture medium was changed to one containing 0.2% BSA rather than FCS. 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 confluence over 4-6 days in supplemented media as described above. The normal media contained 0.6 mM magnesium and 1.0 mM calcium. In the experiments indicated, the cells were cultured in nominally magnesium-free media (<0.01 mM) for 16 h before study (0.2% BSA replaced FCS during this period). Other constituents of the magnesium-free media were identical to those of the complete media.

cAMP measurements. cAMP was determined in confluent MDCT cell monolayers cultured in 24-well plates in DMEM-Ham's F-12 media without serum, as previously reported (4). After addition of various hormones, MDCT cells were incubated at 37°C for 5 min in the presence of 0.1 mM IBMX. cAMP was extracted with 5% trichloroacetic acid, which was removed with ether 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).

Cytoplasmic Mg²+ measurements. Coverslips were mounted within a perfusion chamber, and the attached subconfluent cells were incubated with 5 µM mag-fura 2-AM dissolved in pluronic acid F-127 (0.125%; Molecular Probes) for determination of intracellular Mg²⁺ concentration ([Mg²⁺]_i) in media for 20 min at 37°C. Cells were subsequently washed three times with a buffered salt solution containing (in mM) 145 NaCl, 4.0 KCl, 0.8 Ka₂HPO₄, 0.2 KH₂PO₄, 1.0 CaCl₂, 5.0 glucose, and 20 HEPES-Tris, at pH 7.4. The MDCT cells were incubated for an additional 20 min to allow for complete deesterification and washed once before measurement of fluorescence.

Antibody block of the CaSR in MDCT cells. In those experiments where the CaSR was blocked with a specific antibody, MDCT cells were Mg²⁺ depleted for 16 h and then incubated with the antibody for 1.5-2 h at 36°C. A mouse monoclonal antibody (ADD) raised against a peptide comprising residues 214-236 of the CaSR was used (20). The antibody, which was provided by Drs. P. K. Goldsmith and A. M. Spiegel (National Institutes of Health) and K. V. Rogers (NPS Pharmaceuticals), has been extensively characterized with respect to specificity for the parathyroid/kidney CaSR (24). The antibody was diluted to a final concentration of 1.6 µg/ml in a buffer solution containing 0.02% BSA. The control cells were incubated with mouse IgG. The cells were washed twice in an appropriate buffer solution, and intracellular cAMP content or microfluorescence experiments were performed.

Antisense oligodeoxynucleotides designed to the CaSR in MDCT cells. A second approach was used to block extracellular Ca²⁺/Mg²⁺ sensing in MDCT cells. Oligonucleotide (ODN) antisense methodology, a strategy designed to bind specifically and efficiently to the complementary sequence of a targeted mRNA, has been extensively used in in vitro systems. Twenty-base pair phosphorothioate-derivatized sense and antisense CaSR ODNs were synthesized on an automated solid-phase synthesizer by using standard phosphoramide chemistry. The sequence of the sense ODN was 5'-GAGAAGGCAGAGCCATGGCATGG-3', and that of the antisense ODN was 5'-CCATGCCATGGCTCTGCCTTCTC-3'. The MDCT cells were incubated in buffer containing 0.1% BSA with the ODN, 1.0 nmol/ml, for 48-72 h before fluorescence measurements were performed. Cationic transfection agents, such as lipofectamine, were avoided because they interfere with Mg²⁺ transport, presumably because of membrane damage. Protein was extracted from the cells for Western blot analysis.

Western blots. Cells were lysed in triple detergent buffer [50 mM Tris · HCl (pH 8.0), 150 mM NaCl, 0.02% NaN₃, 0.1% SDS, 1 mM EDTA, 100 µg/ml polymethylsulfonyl fluoride, 2 µg/ml leupeptin, 2 µg/ml aprotonin, 0.1% NP-40, and 0.5% sodium deoxycholate] for 5 min at 0°C. The cell lysates were spun at 1,200 g for 2 min at 4°C, and the supernatants were stored at 80°C. Aliquots were electrophoresed through 8% SDS-polyacrylamide gels and blotted onto polyvinylidene difluoride membranes (Bio-Rad). Membranes were rinsed in TBST (10 mM Tris · HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20), blocked with 5% dried milk powder in TBST for 1-2 h, and incubated with ADD, the antibody against the CaSR (24). As a control, immunoblotting was carried out as described above with the antiserum preabsorbed for 1 h with the peptide (10 µg/ml) against which it was raised. Blots were also incubated with a -tublin monoclonal antibody (Cedarlane Laboratories, Hornby, ON).

Statistical analysis. Representative tracings of fluorescent intensity ratios are given, and all results are means ± SE where indicated. Significance was determined by Tukey's analysis of variance where indicated, and comparisons among groups of data were made by using Student's t-test. A probability of P < 0.05 was taken to be statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

1,25(OH)₂D₃ stimulates Mg²+ entry in MDCT cells. MDCT cells were treated with 1,25(OH)₂D₃ for 16 h before experimentation. During this time they were incubated in media without magnesium to deplete them of intracellular Mg²⁺ (16). The cells were then placed in 1.5 mM MgCl₂, and [Mg²⁺]_i was monitored by fluorescence. Figure 1 shows a typical result of a fluorescence experiment of MDCT cells pretreated with 1,25(OH)₂D₃ compared with control cells. The rate of entry into control cells, measured over the initial 500 s, was 164 ± 5 nM/s, n = 6. Pretreatment with 1,25(OH)₂D₃ increased Mg²⁺ uptake by 59 ± 4% to 260 ± 15 nM/s, n = 5. Next, we determined that vitamin D-stimulated Mg²⁺ uptake required a minimum of 3 h for the largest response and was maintained for 24 h. We have previously shown that the channel blocker nitrendipine inhibits Mg²⁺ uptake into MDCT cells (36). Nitrendipine (50 µM) inhibited Mg²⁺ uptake to a similar extent in 1,25(OH)₂D₃-treated cells (25 ± 5 nM/s, n = 4) as in control cells (24 ± 5 nM/s, n = 5), indicating that the vitamin D metabolite 1,25(OH)₂D₃ stimulated Mg²⁺ entry and not intracellular Mg²⁺ release or inhibition of Mg²⁺ efflux. 1,25(OH)₂D₃ increased Mg²⁺ uptake in a concentration-dependent manner, with half-maximal responses (185 ± 3 nM/s) occurring at ~10⁹ M (Fig. 2). This study indicates that 1,25(OH)₂D₃ stimulates Mg²⁺ entry into MDCT cells.

View larger version (11K): [in this window] [in a new window]   Fig. 1.   1,25-Dihydroxyvitamin D [1,25(OH)2D3] stimulates 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. Where indicated, 107 M 1,25(OH)2D3 was added to the buffer solution from a stock ethanol solution during the 16-h Mg2+-depletion period. Fluorescence studies were performed in buffer solutions in the 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. Fluorescence was measured at 1 data point/s with 25-point signal averaging, and the tracing was smoothed according to methods previously described (17).

View larger version (10K): [in this window] [in a new window]   Fig. 2.   Concentration dependence of 1,25(OH)2D3 stimulation of Mg2+ entry in MDCT cells. Values are means ± SE for 3-6 cells. MDCT cells were treated with the given 1,25(OH)2D3 concentrations for 16 h before fluorescence determinations. The rate of Mg2+ influx, as determined by d([Mg2+]i)/dt, was measured with the given 1,25(OH)2D3 concentrations by using fluorescence techniques described in the legend for Fig. 1. d([Mg2+]i)/dt values were determined over the first 500 s of fluorescence measurements. Significantly different from control values,*P < 0.05.

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View larger version (16K): [in this window] [in a new window]   Fig. 3.   1,25(OH)2D3-stimulated Mg2+ uptake requires transcriptional/translational processes. Values are means ± SE. A: MDCT cells were Mg2+ depleted for 16 h, and 107 M 1,25(OH)2D3 was then added with and without actinomycin D (5.0 µg/ml) as indicated for 4-5 h before fluorescence determinations. Mg2+ uptake was determined with microfluorescence according to methods described in the legend for Fig. 1. B: MDCT cells were treated with 107 M 1,25(OH)2D3 for 16 h before the determination of Mg2+ uptake. In those cells indicated, cycloheximide (Cyclo; 1.0 µg/ml) was added with 1,25(OH)2D3 16 h before Mg2+ uptake and cAMP determinations. Actinomycin D and cyloheximide were added from stock solutions of DMSO; the final DMSO concentration did exceed 0.01% (vol/vol), which had no effect on Mg2+ uptake. Significantly different from control values, *P < 0.01.

Acute effects of polyvalent cations on 1,25(OH)₂D₃-stimulated Mg²+ uptake. We have shown that elevation of extracellular polyvalent cations, neomycin, Mg²⁺, and Ca²⁺ inhibit PTH-, glucagon-, and AVP-mediated cAMP formation and glucagon- and AVP-stimulated Mg²⁺ entry but not basal uptake rates in MDCT cells (3, 4). MDCT cells were treated with 10⁷ M 1,25(OH)₂D₃ for 16 h before the addition of polyvalent cations and determination of Mg²⁺ uptake. Acute (5-min) addition of 50 µM neomycin or elevation of 5.0 mM extracellular Mg²⁺ or 5.0 mM Ca²⁺did not affect Mg²⁺ uptake into 1,25(OH)₂D₃-treated, Mg²⁺-depleted cells (Fig. 4). Accordingly, the acute activation of the CaSR does not alter 1,25(OH)₂D₃-stimulated Mg²⁺ entry in the cells pretreated with the vitamin D metabolite 16 h before experimentation.

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Summary of the acute effects of cation-sensing receptor (CaSR) activation on 1,25 (OH)2D3-stimulated Mg2+ uptake. Values are means ± SE for 3-5 cells. Measurement d([Mg2+]i)/dt was performed in Mg2+-depleted cells treated with or without 1,25(OH)2D3 for 16 h before performance of fluorescence. Mg2+ uptake was determined with 1.5 mM extracellular Mg2+ in the absence or presence of 50 µM extracellular neomycin (Neo); 5.0 mM Mg2+ or 5.0 mM Ca2+ was added 5 min before determination of Mg2+ uptake. In the studies that tested high levels of extracellular Mg2+, 5.0 rather than 1.5 mM MgCl2 was used. The studies were performed as described in the legend for Fig. 1. The Mg2+ uptake rate was determined over 500 s after addition of 1.5 or 5.0 mM Mg2+. Significantly different from control values,*P < 0.05.

Chronic effects of elevated extracellular Ca²+ on 1,25(OH)₂D₃-stimulated Mg²⁺ uptake. Next, we determined the effects of chronic elevation of extracellular Ca²⁺ on 1,25(OH)₂D₃-stimulated Mg²⁺ uptake. A buffer solution containing high CaCl₂ (5.0 mM) was added with 1,25(OH)₂D₃ for 16-20 h before fluorescence measurements were made. In support of our earlier findings, high extracellular Ca²⁺ for 16-20 h did not alter basal Mg²⁺ uptake (165 ± 7 nM/s, n = 4) (Fig. 5). However, high extracellular Ca²⁺ inhibited 1,25(OH)₂D₃-stimulated Mg²⁺ uptake (166 ± 6 nM/s, n = 4). Accordingly, the addition of high extracellular Ca²⁺ with 1,25(OH)₂D₃ diminished vitamin D-mediated Mg²⁺ entry. These findings suggest that signaling via the CaSR may alter 1,25(OH)₂D₃-induced synthesis of proteins involved in Mg²⁺ transport.

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Chronic activation of CaSR-sensing inhibits 1,25(OH)2D3-stimulated Mg2+ uptake. Values are means ± SE for 3-5 cells. MDCT cells were treated with 107 M 1,25(OH)2D3 with and without 5.0 mM CaCl2 for 16 h before microfluorescence measurements. Mg2+ uptake was determined with 1.5 mM extracellular Mg2+ in the absence or presence of 5.0 mM Ca2+ as indicated. We have previously shown that high extracellular Ca2+ does not affect basal Mg2+ uptake; i.e., the entry pathway is selective for Mg2+ (43). The studies were performed as described in the legend for Fig. 1. The Mg2+ uptake rate was determined over 500 s after addition of 1.5 mM MgCl2. Significantly different from control values,*P < 0.01.

CaSR antibody blocks extracellular Ca²+ inhibition of 1,25(OH)₂D₃-stimulated Mg²⁺ uptake. If elevation of extracellular Ca²⁺ inhibits 1,25(OH)₂D₃-stimulated transport through CaSR-mediated transcriptional processes, then blocking this receptor should abolish the chronic effects of extracellular calcium. To do this, MDCT cells were incubated with a specific CaSR antibody. To substantiate that the CaSR was blocked, we measured the effect of extracellular Ca²⁺ and Mg²⁺ on PTH-mediated cAMP formation in control cells and in those pretreated with the CaSR antibody. PTH stimulated intracellular cAMP accumulation by about threefold (72 ± 8 pmol · mg protein¹ · 5 min¹) above control values (25 ± 1 pmol · mg protein¹ · 5 min¹). The presence of 5.0 mM Ca²⁺ or 5.0 mM Mg²⁺ abolished PTH-stimulated cAMP formation (33 ± 2 and 29 ± 1 pmol · mg protein¹ · 5 min¹, respectively) (Fig. 6). Preincubation of MDCT cells with the antibody prevented the effect of extracellular Ca²⁺ (60 ± 5 pmol · mg protein¹ · 5 min¹) or Mg²⁺ (72 ± 8 pmol · mg protein¹ · 5 min¹) on PTH-sensitive cAMP release (72 ± 8 pmol · mg protein¹ · 5 min¹). Accordingly, the antibody blocked the activation of the CaSR and the inhibition of hormone-mediated cAMP formation. With this knowledge, we determined whether antibody blockade of the CaSR would inhibit the effect of elevated Ca²⁺ on 1,25(OH)₂D₃-stimulated Mg²⁺ uptake. We depleted MDCT cells of Mg²⁺ for 16 h before experimentation and incubated them with ADD antiserum to the CaSR for 2 h. The cells were then treated with 1,25(OH)₂D₃ with and without 5.0 mM CaCl₂ for an additional 4-5 h before microfluorescence studies were performed. MDCT cells treated with the antibody responded normally to 1,25(OH)₂D₃ (210 ± 11 nM/s, n = 5) regardless of the presence of elevated extracellular Ca²⁺ (202 ± 8 nM/s, n = 4) (Fig. 7). This study indicates that extracellular Ca²⁺ chronically inhibits 1,25(OH)₂D₃-mediated Mg²⁺ uptake through the activation of CaSR-mediated mechanisms.

View larger version (15K): [in this window] [in a new window]   Fig. 6.   CaSR antibody blocks extracellular polyvalent cation modulation of parathyroid hormone (PTH)-mediated cAMP formation. Values are means ± SE for 10 observations. In the studies indicated, MDCT cells were preincubated with 1.6 µg/ml of a mouse monoclonal antibody (ADD) or 1.6 µg/ml mouse IgG in a buffer solution containing 0.02% BSA and (in mM) 0.5 MgCl2, 1.0 CaCl2, 145 NaCl, 4.0 KCl, 0.8 K2HPO4, 0.2 KH2PO4, 5.0 glucose, and 20 HEPES-Tris, pH 7.4, for 2 h before measurement of intracellular PTH-stimulated cAMP. Where indicated, either 5.0 mM CaCl2 or 5.0 mM MgCl2 was added for 5 min before PTH and cAMP concentrations were measured with radioimmunoassay after a further 5-min incubation period. Significantly different from control values,*P < 0.01.

View larger version (13K): [in this window] [in a new window]   Fig. 7.   Specific antibody to the CaSR blocks extracellular Ca2+ inhibition of 1,25(OH)2D3-stimulated Mg2+entry. Values are means ± SE for 3-5 cells. MDCT cells were Mg2+ depleted for 16 h and incubated with CaSR mouse monoclonal antibody ADD (1.6 µg/ml) for 2 h. The cells were then treated with 107 M 1,25(OH)2D3 with and without 5.0 mM CaCl2 where indicated, and Mg2+ uptake measurement was performed with microfluorescence. The Mg2+ uptake rate was determined over 500 s after addition of 1.5 mM MgCl2. Significantly different from control values,*P < 0.05.

Antisense ODN to the CaSR blocks extracellular Ca²+ inhibition of 1,25(OH)₂D₃-stimulated Mg²⁺ uptake. To substantiate the antibody studies, we used antisense ODN technology to block CaSR receptor expression and its function. The MDCT cells were loaded with sense or antisense CaSR ODNs. Cells with antisense ODN expressed little CaSR as determined by Western analysis (Fig. 8). In the representative experiment shown, the CaSR-to--tublin densitometry ratio was 2.77 for no ODN; 2.6 for sense ODN; and 0.17, for antisense ODN, the last value representing 6.5% of the sense ODN ratio. Extracellular Ca²⁺ did not inhibit PTH-mediated cAMP formation in MDCT cells treated with antisense ODN to the CaSR (67 ± 1 pmol · mg protein¹ · 5 min¹), whereas the response in cells with sense ODN was similar to that observed with control cells (32 ± 2 pmol · mg protein¹ · 5 min¹) (Fig. 9). Accordingly, antisense ODN blocks expression and function of the CaSR. We used this model to test whether extracellular Ca²⁺ diminishes 1,25(OH)₂D₃-stimulated Mg²⁺ uptake through the CaSR. Mg²⁺ uptake in sense ODN-treated cells was 115 ± 2% (n = 5) of control cells (Fig. 10). 1,25(OH)₂D₃ stimulated Mg²⁺ entry by 189 ± 50% (n = 4) in the sense CaSR ODN-treated cells in the presence of normal extracellular Ca²⁺ concentration (1.0 mM). Elevated extracellular Ca²⁺ (5.0 mM) inhibited 1,25(OH)₂D₃-stimulated Mg²⁺ uptake (87 ± 27%, n = 6) in these cells. In contrast, 1,25(OH)₂D₃-stimulated Mg²⁺ uptake was 172 ± 31% (n = 6) in cells loaded with antisense ODN in the presence of normal Ca²⁺ and 181 ± 26% (n = 9) in cells incubated with 5.0 mM Ca²⁺. Accordingly, the antisense CaSR ODN inhibits CaSR expression and mitigates the effect of extracellular Ca²⁺ on 1,25(OH)₂D₃-stimulated Mg²⁺ uptake. These studies, using antibody or antisense ODN technology, clearly indicate that extracellular Ca²⁺ chronically inhibits 1,25(OH)₂D₃-mediated Mg²⁺ uptake through activation of CaSR-mediated mechanisms.

View larger version (75K): [in this window] [in a new window]   Fig. 8.   Antisense oligodeoxynucleotides (ODN) to the CaSR diminish receptor expression in MDCT cells. Western analysis of the CaSR was performed in MDCT cells treated with sense or antisense ODNs at concentrations of 1 nmol/ml for 36-42 h before experimentation. Cell extracts (50 µg protein) were subjected to SDS-PAGE on a 4-12% gradient gel. The blot was stained with CaSR mouse monoclonal antibody, and an identical blot was stained with -tubulin antibody. Lane 1, control MDCT cells (no ODN); lane 2, sense ODN (CaSR sense); lane 3, antisense ODN (CaSR antisense). The CaSR bands were demonstrated to be specific after the blot was stained with the same antiserum preincubated with the peptide against which it was raised (data not shown). Western blots were performed twice with similar results.

View larger version (12K): [in this window] [in a new window]   Fig. 9.   Antisense CaSR ODN diminish CaSR inhibition of PTH-mediated cAMP formation. Values are means ± SE for 6 individual observations. In the studies indicated, MDCT cells were loaded with sense or antisense ODN 72 h before measurement of intracellular PTH-stimulated cAMP. Where shown, 5.0 mM CaCl2 was added for 5 min before PTH and cAMP concentrations were measured with radioimmunoassay after a further 5-min incubation period. Significantly different from control values,*P < 0.01.

View larger version (13K): [in this window] [in a new window]   Fig. 10.   Antisense ODN to the CaSR diminishes extracellular Ca2+ inhibition of 1,25(OH)2D3-stimulated Mg2+entry. Values are means ± SE for 4-8 cells. MDCT cells were loaded with sense (S) or antisense (AS) ODN designed to the CaSR for 72 h before fluorescence studies. The cells were Mg2+ depleted for 16 h, treated with 107 M 1,25(OH)2D3 for 3-4 h with 5.0 mM CaCl2. The Mg2+ uptake rate was determined over 500 s after addition of 1.5 mM MgCl2.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The role of 1,25(OH)₂D₃ in renal magnesium reabsorption. Little is known about the effects of 1,25(OH)₂D₃ on tubular magnesium transport. On balance, urinary clearance data from clinical observations and experimental studies indicate that 1,25(OH)₂D₃ may stimulate renal magnesium handling (1, 13, 25, 26, 30, 33, 36, 38, 39, 45). There have been no micropuncture studies performed to localize the actions of 1,25(OH)₂D₃ on magnesium transport. The data concerning the effects of 1,25(OH)₂D₃ on renal calcium absorption are more substantial but inconclusive (5, 7-9, 31, 49). The most convincing data demonstrating that 1,25(OH)₂D₃ may have a direct effect on calcium transport were obtained by using isolated cells. Bindels et al. (5) and Van Baal et al. (50) showed that 1,25(OH)₂D₃ increased calbindin D_28k protein levels and stimulated transcellular calcium absorption in primary cultures of rabbit distal connecting tubule and cortical collecting duct cells. The maximal transport response occurred at ~48 h posttreatment, suggesting that the response involved initiation of transcriptional processes (5). The response to 1,25(OH)₂D₃ was independent of PTH and not additive to PTH-stimulated calcium transport. The above-mentioned authors also have biochemical evidence, as they showed that 1,25(OH)₂D₃ increased calbindin D_28k mRNA and protein content without a change in Na⁺/Ca²⁺ exchange or Ca²⁺-ATPase RNA or protein in rabbit distal connecting tubule and cortical collecting duct cell preparations (50). Recently, Hoenderop et al. (29) have identified an apical Ca²⁺ channel in 1,25(OH₂)D₃-responsive cells that is responsible for calcium absorption in this segment. They have reported that 1,25(OH)₂D₃ stimulates Ca²⁺ transport in rabbit cortical collecting duct cells (29). The second study to clearly show effects of vitamin D metabolites on calcium transport was performed in MDCT cells by Friedman and Gesek (22). These co-workers reported that 1,25(OH)₂D₃ did not alter basal Ca²⁺ uptake but accelerated PTH-dependent calcium entry rates. This response was rapid, concentration dependent, significant at 2 h, maximal by 5 h, and mediated by translational processes because it was inhibited by cycloheximide (22). The reasons for the discrepancies between the above-mentioned two reports are not known; examples may be the different cell types or the techniques used to measure calcium transport in the two separate studies. Nevertheless, it is clear that 1,25(OH)₂D₃ increases calcium-binding protein in distal tubules, suggesting that it has significant actions on basal or hormone-mediated calcium transport. 1,25(OH)₂D₃ increases divalent cation-binding proteins, such as calbindin D_9K or calbindin D_28k, in distal tubule cells, including convoluted segment, connecting tubule, and collecting duct cells (34, 35, 46, 48). In a preliminary report, we have shown that 1,25(OH)₂D₃ stimulates calbindin D_9K in the MDCT cell line (14) The calbindin D group is thought to be involved, either directly or indirectly, in epithelial calcium transport (6-9, 21, 31, 49, 53). Experimental evidence also supports the notion that these proteins bind magnesium, and some investigators have postulated that they are involved in epithelial magnesium transport (2, 12, 28).

Effect of extracellular Mg²+/Ca²⁺ sensing on 1,25(OH)₂D₃-mediated Mg²⁺ uptake. The extracellular CaSR plays an important role in mineral balance by controlling, among other things, parathyroid gland function and renal calcium and magnesium reabsorption (11, 27, 42). The CaSR mediates a host of intracellular signaling pathways. The CaSR has been best characterized in parathyroid gland cells (11). In the parathyroid, CaSR regulates PTH secretion and activates multiple intracellular pathways involving activation of G-coupled proteins and changes in gene expression among others (11). The ways that CaSR activation influences salt and water transport in the kidney are presently being addressed. Wang et al. (52) showed that the elevation of extracellular calcium inhibits NaCl transport in the thick ascending limb by release of cytochrome P-450 metabolites. We have demonstrated in immortalized MDCT cells that the CaSR initiates intracellular Ca²⁺ transients and inhibits PTH-, glucagon-, and AVP-mediated cAMP formation and magnesium transport, probably by activating G_i-proteins (3, 4, 17). Finally, Sands et al. (47) reported that extracellular calcium inhibits AVP-stimulated cAMP induction of water transport in the inner medullary collecting duct, again likely through G_i-protein mediation. Accordingly, the CaSR may modulate epithelial cell functions in many cell types by different intracellular signaling pathways.

Role of 1,25(OH)₂D₃ in overall renal magnesium balance. 1,25(OH)₂D₃ increases intestinal calcium and magnesium absorption and renal conservation, leading to positive divalent cation balance. Elevation of serum calcium and magnesium concentrations, in turn, diminish renal absorption through the actions of the CaSR on electrolytes in the loop and distal convoluted tubule and water absorption in the medullary collecting ducts (27, 41, 42, 44, 47). Activation of the CaSR modulates the excessive absorption of divalent cations, providing a negative-feedback control to effect normal mineral balance. This may also explain the discrepant reports in the literature concerning the effects of vitamin D metabolites on renal magnesium transport.