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Abstract The distal convoluted tubule plays a significant role in renal magnesium conservation. Although the cells of the distal convoluted tubule possess the vitamin D receptor, little is known about the effects of 1a,25-dihydroxyvitamin D [1,25(OH)2D3] on magnesium transport. In this study, we examined the effect of 1,25(OH)2D3 on distal cellular magnesium uptake and the modulation of this response by extracellular Ca21 and Mg21 in an immortalized mouse distal convoluted tubule (MDCT) cell line. MDCTcells possess the divalent cation-sensing receptor (CaSR) that responds to elevation of extracellular Ca21 and Mg21 concentrations to diminish peptide hormone-stimulated Mg21 uptake. Mg21 uptake rates were determined by microfluorescence in Mg21-depleted MDCT cells. Treatment of MDCT cells with 1,25(OH)2D3 for 16–24 h stimulated basal Mg21 uptake in a concentration-dependent manner from basal levels of 164 6 5 to 210611 nM/s, representing a 2863% change. Pretreatment with actinomycin D or cycloheximide abolished 1,25(OH)2D3- stimulated.Mg21 uptake (154 6 18 nM/s), suggesting that 1,25(OH)2D3 stimulates Mg21 uptake through gene activation and protein synthesis. Elevation of extracellular Ca21 inhibited 1,25(OH)2D3-stimulated Mg21 uptake (143 6 5 nM/s). Preincubation of the cells with an antibody to the CaSR prevented the inhibition by elevated extracellular Ca21 of 1,25(OH)2D3-stimulated Mg21 uptake (202 6 8 nM/s). Treatment with an antisense CaSR mRNA oligodeoxynucleotide also abolished the effects of extracellular Ca21 on 1,25(OH)2D3-responsive Mg21 entry. This showed that elevated extracellular calcium modulates 1,25(OH)2D-mediated responses through the CaSR. In summary, 1,25(OH)2D3 stimulated Mg21 uptake in MDCT cells, and this is dependent on de novo protein synthesis. Elevation of extracellular Ca21, acting via the CaSR, inhibited 1,25(OH)2D3-stimulated Mg21 entry. These data indicate that 1,25(OH)2D3 has important effects on the control of magnesium entry in MDCT cells and these responses can be modulated by extracellular divalent cations.

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Abstract Nucleotides have diverse effects on water and electrolyte reabsorption within the distal tubule of the nephron. As the distal tubule is important in control of renal Mg21 balance, we determined the effects of ATP on cellular Mg21 uptake in this segment. The effects of ATP on immortalized mouse distal convoluted tubule (MDCT) cells were studied by measuring Mg21 uptake with fluorescence techniques. The mean basal Mg21 uptake rate was 165 6 6 nM/s. ATP inhibited basal Mg21 uptake and hormone-stimulated Mg21 entry by 40%. Both P2X (P2X1– P2X5 subtypes) and P2Y2 receptor subtypes were identified in MDCT cells using differential RT-PCR. Activation of both receptor subtypes with selective agonists increased intracellular Ca21 concentration, P2X purinoceptors by ionotropicgated channels, and P2Y receptors via G protein-mediated intracellular Ca21 release. The more relatively selective P2X agonists [b,g-methylene ATP (b,g-Me-ATP) and 29- and -39- O-(4-benzoyl-benzoyl)-ATP] inhibited arginine vasopressin (AVP)- and parathyroid hormone (PTH)-mediated Mg21 uptake whereas agonists more selective for P2Y purinoceptors (UTP, ADP, and 2-methylthio-ATP) were without effect. Removal of extracellular Ca21 diminished b,g-Me-ATP-mediated increase in intracellular Ca21 and inhibition of AVPstimulated Mg21 entry. We conclude from this information that ATP inhibited Mg21 uptake in MDCT cells through P2X purinoceptors expressed in this distal convoluted tubule cell line.

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