beta-Adrenergic agonists stimulate Mg2+ uptake in mouse distal convoluted tubule cells

beta-Adrenergic agonists influence electrolyte reabsorption in the proximal tubule, loop of Henle, and distal tubule. Although isoproterenol enhances magnesium absorption in the thick ascending limb, it is unclear what effect , if any, beta-adrenergic agonists have on tubular magnesium handling. The effects of isoproterenol were studied in immortalized mouse distal convolut ed tubule (MDCT) cells by measuring cellular cAMP formation with radioimmun oassays and Mg2+ uptake with fluorescence techniques. Intracellular free Mg 2+ concentration ([Mg2+](i)) was measured in single MDCT cells by using mic rofluorescence with mag-fura-2. To assess Mg2+ uptake, MDCT cells were firs t 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 [Mg2+](i) were determ ined. [Mg2+](i) returned to basal levels, 0.53 +/- 0.02 mM, with a mean ref ill rate, d([Mg2+](i))/dt, of 168 +/- 11 nM/s. Isoproterenol stimulated Mg2 + entry in a concentration-dependent manner, with a maximal response of 252 +/- 11 nM/s, at a concentration of 10(-7) M, that represented a 50 +/- 7% increase in uptake rate above control values. This was associated with a si xfold increase in intracellular cAMP generation. Isoproterenol-stimulated M g2+ uptake was completely inhibited with RpcAMPS, a protein kinase A inhibi tor, and U-73122, a phospholipase C inhibitor, and partially blocked by RO 31-822, a protein kinase C inhibitor. Accordingly, isoproterenol-mediated M g2+ entry rates involve multiple intracellular signaling pathways. Aldoster one potentiated isoproterenol-stimulated Mg2+ uptake (326 +/- 31 nM/s), whe reas elevation of extracellular Ca2+ inhibited isoproterenol-mediated cAMP accumulation and Mg2+ uptake, 117 +/- 37 nM/s. These studies demonstrate th at isoproterenol stimulates Mg2+ uptake in a cell line of mouse distal conv oluted tubules that is modulated by hormonal and extracellular influences.