Download paper

Abstract 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 Mg21 uptake with fluorescence techniques using mag-fura 2. To assess Mg21 uptake, MDCT cells were first Mg21 depleted to 0.22 6 0.01 mM by culturing in Mg21-free media for 16 h and then placed in 1.5 mM MgCl2, and the changes in intracellular Mg21 concentration ([Mg21]i) were measured with microfluorescence. [Mg21]i returned to basal levels, 0.53 6 0.02 mM, with a mean refill rate, d([Mg21]i)/dt, of 164 6 5 nM/s. Insulin stimulated Mg21 entry in a concentration-dependent manner with maximal response of 214 6 12 nM/s, which represented a 30 6 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 6 3 pmol·mg protein21 ·5 min21). Genistein, a tyrosine kinase inhibitor, diminished insulin-stimulated Mg21 uptake (169 6 11 nM/s), but did not change insulin-mediated cAMP formation (47 6 5 pmol·mg protein21 ·5 min21). PTH stimulates Mg21 entry, in part, through increases in cAMP formation. Insulin and PTH increase Mg21 uptake in an additive fashion. In conclusion, insulin mediates Mg21 entry, in part, by a genistein-sensitive mechanism and by modifying hormone-responsive transport. These studies demonstrate that insulin stimulates Mg21 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.

]]> http://jamesdai.com/longjundai/2009/09/insulin-stimulates-mg2-uptake-in-mouse-distal-convoluted-tubule-cells-2/feed/ 0 http://jamesdai.com/longjundai/2009/09/cyclic-nucleotides-alter-intracellular-free-mg2-in-renal-epithelial-cells/ http://jamesdai.com/longjundai/2009/09/cyclic-nucleotides-alter-intracellular-free-mg2-in-renal-epithelial-cells/#comments Sat, 12 Sep 2009 00:45:58 +0000 greedy http://jamesdai.com/longjundai/?p=66 Dai LJ, and Quamme GA: Cyclic nucleotides alter intracellular free Mg²⁺ in renal epithelial  cells. American Journal of Physiology 262:F1100-1104, 1992.

Download Paper

Abstract Cyclic nucleotides alter intracellular free Mg2+ in renal epithelial cells. Am. J. Physiol. 262 (Renal Fluid Electrolyte Physiol. 31): FllOOF1104,1992.- Intracellular Mg”+ plays an important role in cell physiology. Studies were performed on MDCK cells and primary cortical thick ascending limb (CTAL) cells to determine hormonal influences on intracellular Mg2+ control. Free Mg2+ ( [Mg2+]i) was measured by fluorescence with mag-fura-2. Addition of 8bromoguanosine 3’,5’-cyclic monophosphate @-BrcGMP, 10e4 M) to subconfluent MDCK cells resulted in rapid increases in [Mg2+]i from basal levels of 552 it 6 PM to peak concentrations of 682 t 5 PM, whereas 8BrcAMP (10v4 M) led to significant decreases in [Mg2+]i from 538 of 5 to 362 t 17 PM. These effects of cyclic nucleotides were dose dependent with half-maximal concentrations (EC,,) of – 10e5 M for both increments in [Mg2+]i with cGMP and decrements in [Mg”+]i with CAMP. Atria1 natriuretic peptide (ANP) and cGMP increased Mg2+ in porcine primary CTAL cells from 525 t 12 to 592 & 18 PM and from 538 & 8 to 609 t 18 PM, respectively. The increment in [Mg2+]i with ANP was dose responsive with EC& values of – lo-l1 M suggesting that these effects may be of physiological importance. Parathyroid hormone and calcitonin and their second messenger, CAMP, diminished Mg2+ by -80 PM. The EC& value for calcitonin was in the order of 10mg M. The changes in [Mg2+]i, whether increases with ANP or cGMP and decreases with PTH, calcitonin, or CAMP, were rapid in nature and independent of changes in intracellular free Ca2+ concentration. These data indicate that [Mg”+]i is influenced by peptide hormones and their second messengers likely through activation of appropriate protein kinases. The functional role of these changes remains to be determined.