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Department of Medicine, University of British Columbia, Vancouver Hospital and Health Sciences Centre, Koerner Pavilion, Vancouver, British Columbia, Canada V6T 1Z3
Insulin has been shown to be a
magnesium-conserving hormone acting, in part, through stimulation of
magnesium absorption within the thick ascending limb. Although the
distal convoluted tubule possesses the most insulin receptors, it is
unclear what, if any, actions insulin has in the distal tubule. The
effects of insulin were studied on immortalized mouse distal convoluted
tubule (MDCT) cells by measuring cellular cAMP formation with
radioimmunoassays and Mg2+ uptake with fluorescence
techniques using mag-fura 2. To assess Mg2+ uptake, MDCT
cells were first Mg2+ depleted to 0.22 ± 0.01 mM by
culturing in Mg2+-free media for 16 h and then placed in
1.5 mM MgCl2, and the changes in intracellular
Mg2+ concentration
([Mg2+]i) were measured with
microfluorescence. [Mg2+]i returned
to basal levels, 0.53 ± 0.02 mM, with a mean refill rate,
d([Mg2+]i)/dt, of 164 ± 5 nM/s. Insulin stimulated Mg2+ entry in a
concentration-dependent manner with maximal response of 214 ± 12 nM/s, which represented a 30 ± 5% increase in the mean uptake rate
above control values. This was associated with a 2.5-fold increase in
insulin-mediated cAMP generation (52 ± 3 pmol · mg
protein
1 · 5 min1). Genistein, a tyrosine kinase inhibitor,
diminished insulin-stimulated Mg2+ uptake (169 ± 11 nM/s), but did not change insulin-mediated cAMP formation (47 ± 5 pmol · mg
protein1 · 5 min1). PTH stimulates Mg2+ entry, in
part, through increases in cAMP formation. Insulin and PTH increase
Mg2+ uptake in an additive fashion. In conclusion, insulin
mediates Mg2+ entry, in part, by a genistein-sensitive
mechanism and by modifying hormone-responsive transport. These studies
demonstrate that insulin stimulates Mg2+ uptake in MDCT
cells and suggest that insulin acts in concert with other peptide and
steroid hormones to control magnesium conservation in the distal
convoluted tubule.
intracellular magnesium; fluorescence; insulin; genistein; tyrosine kinase; intracellular adenosine 3',5'-cyclic monophosphate; parathyroid hormone; aldosterone; extracellular calcium sensing; neomycin
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