Dr. Long Jun Dai

Dai LJ, Raymond L, Friedman PA, and Quamme GA: Mechanisms of amiloride stimulation of  Mg²⁺ uptake in immortalized mouse distal convoluted tubule cells. American Journal of Physiology 272:F249-F256, 1997.

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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.

Dai LJ, Friedmam PA, and Quamme GA:  Acid-base changes alter Mg²⁺ uptake in mouse distal  convoluted tubule cells. American Journal of Physiology 272:F759-F766, 1997.

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Abstract Metabolic alkalosis leads to renal magnesium conservation, whereas metabolic acidosis is associated with urinary magnesium wasting. Micropuncture studies suggest that these actions affect magnesium transport in the distal tubule. The cellular mechanisms of acidbase changes were investigated in an immortalized mouse distal convoluted tubule (MDCT) cell line. Intracellular free Mg2+ concentration ([Mg2+]i) was determined by microfluorescence using the Mg2+- responsive dye, mag-fura 2. Mg2+ transport was assessed as a function of change in [Mg2+]i with time following placement of Mg2+-depleted cells into a buffer containing 1.5 mM magnesium. The uptake rate of Mg2+, d( [Mg2+]i)ldt 9 into Mg 2+-depleted cells determined with a buffer solution of pH 7.4 was 178 t 21 nM/s. Mg2+ uptake at pH 8.0 was markedly increased 278 2 35 r&I/s, whereas transport at pH 6.0 was significantly reduced to 121 ? 15 nM/s. Mg2+ uptake at pH 7.4 was not stimulated with 20 or 40 mM bicarbonate, nor were the differences in Mg2+ uptake with pH associated with changes in membrane voltage. Mg2+ uptake was stimulated with membrane hyperpolarization at pH 6.0 but not at pH 8.0. Chlorothiazide (10m4 M), which stimulates Mg2+ uptake by hyperpolarizing the membrane voltage, increased uptake at pH 6.0,59 t 14%, but decreased it at alkaline pH of 8.0, -55 t 3%. Accordingly, MDCT cells become refractory to the stimulating effects of hyperpolarization at alkaline pH values. These studies show that protons may directly affect Mg2+ transport in MDCT cells.