Glucagon and arginine vasopressin stimulate Mg2+ uptake in mouse distal convoluted tubule cells

Glucagon and arginine vasopressin (AVP) enhance renal magnesium conservation through actions within the loop of Henle and the distal tubule. Studies were performed on an immortalized mouse distal convoluted tubule (MDCT) cell line to characterize the cellular actions of these hormones on Mg(2+) transport in this segment of the distal tubule. Glucagon and AVP increased cellular cAMP concentrations by about fivefold above basal levels in normal and Mg(2-)-depleted cells. Intracellular free Mg(2+) concentration ([Mg(2+)](i)) tvas determined on single MDCT cells using microfluorescence with mag-fura 2. To assess Mg(2+) uptake, MDCT cells were first Mg(2+) depleted (0.22 +/- 0.01 mM) by culturing in Mg(2+)-free media for 16 h and then placed in 1.5 mM MgCl(2), and the [Mg(2+)](i) was determined. [Mg(2+)](i) returned to basal levels, 0.53 +/- 0.02 mM, with a mean refill rate, d([Mg(2+)](i))/dt, of 164 +/- 5 nM/s. Both glucagan and AVP stimulated Mg(2+) uptake into MDCT cells, 196 +/- 11 and 189 +/- 6 nM/s, respectively, at concentrations of 3 X 10(-7) M and 10(-7) M, respectively. Enhanced Mg(2+) uptake for each of the hormones was concentration dependent and inhibited by the channel blocker, nifedipine, Hormone stimulation of Mg(2+) entry was not dependent on protein synthesis. 8-Bromo-cAMP, 10(-4) M, enhanced Mg(2+) uptake (225 +/- 13 nMs), whereas phorbol esters were without effect. Finally, protein kinase A inhibition prevented glucagon and AVP stimulation of Mg(2+) uptake, supporting the notion that the cAMP pathway is important as expected in the hormone action. These studies demonstrate that glucagon and AVP stimulate Mg(2+)-uptake in MDCT cells and suggest that these hormones act to control magnesium conservation in the convoluted segment of the distal tubule.