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Abstract The distal convoluted tubule reabsorbs – 10% of the filtered magnesium, which is -70% of that delivered to it from the loop of Henle. The cellular mechanisms of magnesium transport in the distal convoluted tubule are not known. Amiloride has been reported to promote magnesium conservation. Studies were performed on immortalized mouse distal convoluted tubule (MDCT) cells to characterize distal magnesium transport and the effects of amiloride. Intracellular free Mg”+ concentration ( [Mg2+]i) was determined on single MDCT cells using microfluorescence with mag-fura 2. Basal [Mg”+]i was 0.53 t 0.01 mM, which is -2% of the total cellular magnesium. To assess Mg2+ uptake, MDCT cells were first Mg2+ depleted (0.22 t 0.01 mM) by culturing in Mg2+-free media for 8-16 h and then placed in 5 mM MgC12, and the [Mg2+]i was determined. [Mg2+]i returned to basal levels (0.50 t 0.04 mM) with refill rate, d([Mg2+]i)ldt, of 181 t 33 r&I/s. Mg2+ entry rate was concentration dependent; a concentration of -0.1 mM resulted in half-maximal uptake rates. Mg”+ uptake was inhibited by La3+ (36 t 17 nM/s), Mn2+ (56 t 25 r&I/s), and nitrendipine (52 t 18 nM/s), but not Ca2+ (225 t 70 nnlvs). Mg2+ uptake was influenced by the transmembrane voltage; hyperpolarization either with the addition of valinomycin or the substitution of bath NaCl with NaSCN stimulated Mg2+ influx (205 ? 3 and 561 t 54 r&I/s, respectively). Depolarization with external KC1 diminished Mg2+ uptake (57 t 25 r&I/s). These data provide evidence for novel Mg2+ entry pathways in MDCT cells that are specific for Mg2+ and activated by an increase in transmembrane voltage. Because amiloride leads to a hyperpolarization of the apical membrane, we postulated that amiloride may enhance Mg2+ transport by influencing the membrane voltage. Amiloride (50 pM) increased Mg2+ uptake (235 t 79 nM/s) in a concentration.-dependent manner (half-maximal concentration of 35 uM amiloride). Accordingly, the distal diuretic, amiloride, inhibits Na+ transport, hyperpolarizes the apical membrane, and results in a stimulation of Mg2+ uptake in MDCT cells. These results provide the cellular basis for the clinical use of amiloride to bring about renal magnesium conservation.

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Abstract Basal free Mg2’ concentration was 0.49 t 0.03 mM in normal single Madin-Darby canine kidney (MDCK) cells as measured by fluorescence with the aid of mag-fura-2. Accordingly, Mg2+ may enter the cell down a transmembrane electrical gradient. The present study describes some aspects of Mg2+ entry into the established MDCK cell line. MDCK cells were Mg2’-depleted (0.26 t 0.01mM) by culturing in Mg2’-free media for 16-20 h. Cells were subsequently exposed to 5 mM MgCL,, and intracellular M$+ concentration ( [ Mg2+] i) was monitored with fluoresence. [Mg+]i returned to normal basal levels, 0.56 t 0.05 mM, with a refill rate of 272 t 39 nM/s, n = 4. M$+ entry was not changed by 5.0 mM external Ca2+ but was completely inhibited with 5.0 mM La3+. Intracellular Ca2+ concentration was not altered by Mg+ depletion or during M$+ repletion. Mg2+ uptake was inhibited by verapamil (0 t 27 nM/s, n = 3), was inhibited less so by diltiazem (141 t 34 nM/s, n = 3), and was not affected by nifedipine (300 t 53 nM/s, n = 6). These inhibitors were fully reversible on removal, and [Mg”‘]i returned to normal levels. These data indicate the presence of a unique M$+ entry pathway in MDCK cells that may be important in M$+ homeostasisT. he model of Mg2+ refill into M$+- depleted cells may be useful in other cell types.