Endo T, Kimura O, Sakata M, Shaikh Z A
Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido 1757, Hokkaido, Ishikari-Tobetsu, 061-02, Japan.
Toxicol Appl Pharmacol. 1997 Oct;146(2):294-8. doi: 10.1006/taap.1997.8244.
The purpose of this study was to investigate the mechanism of inorganic mercury (Hg) uptake in LLC-PK1 cells, a renal tubular epithelial cell line, and to compare the results with those reported previously by us in rat renal cortical epithelial (RCE) cells in primary culture. The LLC-PK1 cells were cultured for 3-12 days, incubated with 1 microM HgCl2 in Hanks' balanced salt solution at 4 or 37 degrees C for 30 min, and washed with phosphate-buffered saline containing BAL to remove the cell membrane-bound Hg. The uptake of Hg was higher in nonconfluent cultures than in confluent cultures and higher at 37 than at 4 degrees C. In confluent culture (Day 8) Hg uptake at 4 degrees C was only 27% of that at 37 degrees C. The initial accumulation of Hg (5 min) from different concentrations of HgCl2 (0.5-50 microM) was linear and did not show a tendency toward saturation, suggesting that a carrier-mediated process was not involved. Pretreatment of cells with 10 microM FCCP, a metabolic inhibitor and a proton ionophore, 0.5 mm DIDS, an anion transport inhibitor, or 0.5 mM ouabain, a Na+/K+-ATPase inhibitor, resulted in 72, 60, and 57% reduction in Hg uptake, respectively. Furthermore, replacement of 137 mm NaCl in the incubation medium with 137 mM KCl or LiCl or 274 mM mannitol caused 30, 45, and 87% reduction in Hg uptake, respectively. These results suggest that in LLC-PK1 cells, as in RCE cells, Hg uptake is inversely related to cell density and is influenced by membrane fluidity, membrane potential, and HCO3-/Cl- transporter.
本研究的目的是探究肾小管上皮细胞系LLC-PK1细胞摄取无机汞（Hg）的机制，并将结果与我们之前在原代培养的大鼠肾皮质上皮（RCE）细胞中所报道的结果进行比较。将LLC-PK1细胞培养3至12天，在4℃或37℃下于汉克斯平衡盐溶液中用1μM HgCl₂孵育30分钟，并用含有二巯丙醇（BAL）的磷酸盐缓冲盐水洗涤以去除细胞膜结合的汞。在未汇合培养物中汞的摄取高于汇合培养物，且在37℃时高于4℃时。在汇合培养（第8天）中，4℃时汞的摄取仅为37℃时的27%。来自不同浓度HgCl₂（0.5至50μM）的汞的初始积累（5分钟）呈线性，且未显示出饱和趋势，这表明不涉及载体介导的过程。用10μM羰基氰化物间氯苯腙（FCCP，一种代谢抑制剂和质子离子载体）、0.5mM 4,4'-二异硫氰酸酯-2,2'-二磺酸基苯（DIDS，一种阴离子转运抑制剂）或0.5mM哇巴因（一种Na⁺/K⁺-ATP酶抑制剂）对细胞进行预处理，分别导致汞摄取减少72%、60%和57%。此外，将孵育培养基中的137mM NaCl替换为137mM KCl或LiCl或274mM甘露醇，分别导致汞摄取减少30%、45%和87%。这些结果表明，在LLC-PK1细胞中，与RCE细胞一样，汞的摄取与细胞密度呈负相关，并受膜流动性、膜电位和HCO₃⁻/Cl⁻转运体的影响。
