Insulin stimulates Mg2+ uptake in mouse distal convoluted tubule cells

Insulin has been shown to be a magnesium-conserving hormone acting, in part , through stimulation of magnesium absorption within the thick ascending li mb. Although the distal convoluted tubule possesses the most insulin recept ors, 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 radioimmunoa ssays and Mg2+ uptake with fluorescence techniques using mag-fura 2. To ass ess 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 m M, with a mean refill rate, d([Mg2+](i))/dt, of 164 +/- 5 nM/s. Insulin sti mulated Mg2+ entry in a concentration-dependent manner with maximal respons e of 214 +/- 12 nM/s, which represented a 30 +/- 5% increase in the mean up take rate above control values. This was associated with a 2.5-fold increas e in insulin-mediated cAMP generation (52 +/- 3 pmol mg protein-l 5 min(-1) ). Genistein, a tyrosine kinase inhibitor, diminished insulin-stimulated Mg 2+ uptake (169 +/- 11 nM/s), but did not change insulin-mediated cAMP forma tion (47 +/- 5 pmol.mg.protein(-1).5 min(-1)). PTH stimulates Mg2+ entry, i n part, through increases in cAMP formation. Insulin and PTH increase Mg2uptake in an additive fashion. In conclusion, insulin mediates Mg2+ entry, in part, by a genistein-sensitive mechanism and by modifying hormone-respon sive transport. These studies demonstrate that insulin stimulates Mg2+ upta ke in MDCT cells and suggest that insulin acts in concert with other peptid e and steroid hormones to control magnesium conservation in the distal conv oluted tubule.