INTRACELLULAR MG2+ AND MAGNESIUM DEPLETION IN ISOLATED RENAL THICK ASCENDING LIMB CELLS
INTRACELLULAR MG2+ AND MAGNESIUM DEPLETION IN ISOLATED RENAL THICK ASCENDING LIMB CELLS
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DOI：
10.1172/jci115429
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发表时间：
1991-10-01
影响因子：
15.9
通讯作者：
QUAMME, GA
中科院分区：
文献类型：
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作者：
DAI, LJ;QUAMME, GA
肾脏内的镁重吸收和调节主要发生在Henle袢的皮质厚升支（cTAL）细胞内。 使用染料mag-fura-2的荧光测定法来表征单个cTAL细胞中的细胞内Mg 2+浓度（[Mg 2 +]i）。 使用双抗体技术（山羊抗人Tamm-Horsfall和兔抗山羊IgG抗体）从猪肾脏制备原代细胞培养物。 基础[Mg 2 +]i为0.52 +/-0.02 mM，约为总细胞Mg的2%。 在高镁培养基（5 mM）中培养（16小时）的细胞维持基础[Mg 2 +]i，0.48 +/-0.02，在正常范围内。 然而，在标称无镁培养基中培养的细胞具有[Mg 2 +]i，0.27 +/- 0.01 mM，这与净Mg转运的显著增加相关，（对照，0.19 +/- 0.03和低Mg，0.35 +/- 0.01 nmol.mg-1蛋白.min-1），如通过Mg-28摄取所评估的。 随后将Mg 2+耗尽的细胞置于高Mg溶液（5 mM）中，并通过荧光评估Mg 2+再填充速率。 [Mg2+]i恢复到正常基础水平，0.53 +/- 0.03 mM，再填充率为257 +/- 37 nM/s。 5.0 mM Ca 2+或2 mM Sr 2+、Cd 2+、Co 2+和Ba 2+均不改变Mg 2+的内流，但在2 mM时，Mn 2 +-La 3 +-Gd 3 +-Ni 2 +-Zn 2 +-Be 2+抑制了Mg 2+的内流。这表明该条目相对特异于Mg 2+。 硝苯地平（117 +/- 20 nM/s）、维拉帕米（165 +/- 34 nM/s）和地尔硫卓（194 +/- 19 nM/s）抑制Mg 2+摄取，但二氢吡啶类似物Bay K 8644（366 +/- 71 nM/s）增强Mg 2+摄取。 这些拮抗剂和激动剂是可逆的去除和[Mg 2 +]i随后恢复到正常的基础水平。 Mg 2+进入率是浓度和电压依赖性的，在无镁培养基中4小时后刺激最大。 细胞镁耗竭导致Mg 2+再填充速率增加，这部分依赖于从头蛋白质合成。 这些数据为cTAL细胞中新的Mg 2+进入途径提供了证据，该途径对Mg 2+具有特异性并且高度受调节。 这些进入途径可能与肾脏Mg 2+稳态有关。
Magnesium reabsorption and regulation within the kidney occur principally within the cortical thick ascending limb (cTAL) cells of the loop of Henle. Fluorometry with the dye, mag-fura-2, was used to characterize intracellular Mg2+ concentration ([Mg2+]i) in single cTAL cells. Primary cell cultures were prepared from porcine kidneys using a double antibody technique (goat anti-human Tamm-Horsfall and rabbit anti-goat IgG antibodies). Basal [Mg2+]i was 0.52 +/- 0.02 mM, which was approximately 2% of the total cellular Mg. Cells cultured (16 h) in high magnesium media (5 mM) maintained basal [Mg2+]i, 0.48 +/- 0.02, in the normal range. However, cells cultured in nominally magnesium-free media possessed [Mg2+]i, 0.27 +/- 0.01 mM, which was associated with a significant increase in net Mg tranport, (control, 0.19 +/- 0.03 and low Mg, 0.35 +/- 0.01 nmol.mg-1 protein.min-1) as assessed by Mg-28 uptake. Mg2+-depleted cells were subsequently placed in high Mg solution (5 mM) and the Mg2+ refill rate was assessed by fluorescence. [Mg2+]i returned to normal basal levels, 0.53 +/- 0.03 mM, with a refill rate of 257 +/- 37 nM/s. Mg2+ entry was not changed by 5.0 mM Ca2+ or 2 mM Sr2+, Cd2+, Co2+, nor Ba2+ but was inhibited by Mn2+ congruent-to La3+ congruent-to Gd3+ congruent-to Ni2+ congruent-to Zn2+ congruent-to Be2+ at 2 mM. Intracellular Ca2+ and Ca-45 uptake was not altered by Mg depletion or Mg2+ refill, indicating that the entry is relatively specific to Mg2+. Mg2+ uptake was inhibited by nifedipine (117 +/- 20 nM/s), verapamil (165 +/- 34 nM/s), and diltiazem (194 +/- 19 nM/s) but enhanced by the dihydropyridine analogue, Bay K 8644 (366 +/- 71 nM/s). These antagonists and agonists were reversible with removal and [Mg2+]i subsequently returned to normal basal levels. Mg2+ entry rate was concentration and voltage dependent and maximally stimulated after 4 h in magnesium-free media. Cellular magnesium depletion results in increases in a Mg2+ refill rate which is dependent, in part, on de novo protein synthesis. These data provide evidence for novel Mg2+ entry pathways in cTAL cells which are specific for Mg2+ and highly regulated. These entry pathways are likely involved with renal Mg2+ homeostasis.