Mechanisms of amiloride stimulation of mg2+ uptake in immortalized
mouse distal convoluted tubule cells.
Dai, Long-Jun, Lynn Raymond, Peter A. Friedman, and Gary A. Quamme.
Departments of Medicine and Psychiatry, University of British
Columbia Vancouver Hospital and Health Sciences Centre, Vancouver, BC
Canada and Department of Pharmacology and Toxicology, Dartmouth
Medical School, Hanover, NH, U.S.A.
APStracts 3:0195F, 1996.
The distal convoluted tubule reabsorbs approximately 10% of the
filtered magnesium, which is about 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 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 about 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 hr 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 about 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 which 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 [mu]M, increased Mg2+ uptake, 235+/-79 nM/s,
in a concentration-dependent manner (half maximal concentration of 35
[mu]M 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.
Received 30 May 1996; accepted in final form 25 October 1996.
APS Manuscript Number F163-6.
Article publication pending Am. J. Physiol. (Renal Fluid Electrolyte
Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 13 November 1996