Novel magnesium influx pathways in isolated renal thick ascending limb cells

This study was performed to identify the specific magnesium influx pathways in single cortical thick ascending limb (cTAL) cells of the loop of Henle. The segment of thick ascending limb of the Henle's loop plays an important role in renal magnesium homeostasis. Up to 50-60% of the filtered magnesium is reabsorbed in this part and various hormones control the balance of magnesium in the same level. Primary cell cultures were prepared from porcine kidneys using a double antibody technique. Intracellular free magnesium concentration ([Me]i) was determined using magnesium fluorescent dye, mag-fura-2 and the measurement of magnesium refill rates (d[Me] jdt) was used as a means to characterize the magnesium pathway in the plasma membrane. Isolated cTAL cells were pretreated with Me-free media for 16-24 hours and d[Me];/dt was tested in magnesium-rich buffer solution. Basal [Me]; was 0.52±0.02 mM (mean ± S.E.) in normal cells and 0.27±0.03mM in magnesium-depleted cells. Cells cultured (16 h) in high magnesium media (5 mM) maintained basal [Me];, 0.48± 0.02 mM, in the normal range. The decrease of [Mg"Ji inMg-depleted cells was associated with a significant increase in net magnesium transport,(control, 0.19±0.03 and low Mg , 0.35 ±0.01 nmol.mg1 protein.minl as assessed by 28Mguptake. [Me], returned to normal basal levels, 0.53±0.07 mM, with a refill rate of 210±25nM.s-i. Me entry was not changed by 5.0 mM Ca" or 2 mM Sr", Cd", Co", nor Ba" but was inhibited by MezLa":-.1Gd"k:Ni"^Medicine, Faculty ofMedicine, Department ofExperimental Medicine, Division ofGraduat