Mechanisms of amiloride stimulation of Mg2+ uptake in immortalized mouse distal convoluted tubule cells.

Abstract

The distal convoluted tubule reabsorbs approximately 10% of the filtered magnesium, which is approximately 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 ofamiloride. Intracellular free Mg2+ concentration ([Mg2+]i) was determined on single MDCT cells using microfluorescence with mag-fura 2. Basal [Mg2+]i was 0.53 +/- 0.01 mM, which is approximately 2% of the total cellular magnesium. To assess Mg2+ uptake, MDCT cells were first Mg2+ depleted (0.22 +/- 0.01 mM) by culturing in Mg2+-free media for 8-16 h and then placed in 5 mM MgCl2, and the [Mg2+]i was determined. [Mg2+]i returned to basal levels (0.50 +/- 0.04 mM) with refill rate, d([Mg2+]i)/dt, of 181 +/- 33 nM/s. Mg2+ entry rate was concentration dependent; a concentration of approximately 0.1 mM resulted in half-maximal uptake rates. Mg2+ uptake was inhibited by La3+ (36 +/- 17 nM/s), Mn2+ (56 +/- 25 nM/s), and nitrendipine (52 +/- 18 nM/s), but not Ca2+ (225 +/- 70 nM/s). 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 +/- 54 nM/s, respectively). Depolarization with external KCl diminished Mg2+ uptake (57 +/- 25 nM/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 microM) increased Mg2+ uptake (235 +/- 79 nM/s) in a concentration-dependent manner (half-maximal concentration of 35 microM 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.