REGULATION OF MEMBRANE CHLORIDE CURRENTS IN RAT BILE-DUCT EPITHELIAL-CELLS
REGULATION OF MEMBRANE CHLORIDE CURRENTS IN RAT BILE-DUCT EPITHELIAL-CELLS
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DOI：
10.1172/jci116188
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发表时间：
1993-01-01
影响因子：
15.9
通讯作者：
COHN, JA
中科院分区：
文献类型：
--
作者：
FITZ, JG;BASAVAPPA, S;COHN, JA
这项研究检查了从胆管肝内部分分离的细胞的质膜的导电特性。用全细胞膜片钳记录技术测定单个细胞的膜氯电导，用氯-36和I-125外排法测定短期培养细胞的膜氯电导。分离的钙依赖和cAMP依赖的氯电流被识别出来。依赖于Ca~(2+)的Cl-电流表现出电流-电压关系的外向整流，去极化电位下依赖时间的激活，以及在平衡电位附近的翻转。在5：7细胞中，洛诺霉素(2um)使该电流从357+/-72pA增加到1192+/-414pA(+80 mV)，并在15：15实验中刺激I-125和氯-36的外流。氯离子通道阻断剂4，4‘-二异硫氰基-2，2’-二苯乙烯二磺酸(DIDS)(1.5微米)可抑制洛诺霉素的外排。CAMP激活的氯离子电流呈线性电流-电压关系，不存在时间依赖性。在11：16细胞中，Forskolin(10微米)或CPT-cAMP(500微米)使这一电流从189+/-50 pA增加到784+/-196 pA(+80 mV)，并在16：16实验中刺激氯-36和I-125的外流。DIDS对cAMP刺激的外排无影响。由于cAMP刺激的氯离子电导类似于囊性纤维化跨膜电导调节器(CFTR)，CFTR是一种可能的氯离子通道蛋白，因此用免疫印迹分析方法检测了CFTR在大鼠肝脏中的存在。在导管细胞数量增加的样本中，CFTR被检测到为150-165-kD的蛋白。免疫过氧化物酶染色证实CFTR定位于胆管细胞，而不是肝细胞。这些结果提示，钙离子和cAMP调节的氯离子通道可能参与了肝内胆管上皮细胞分泌液体和电解质的调控，cAMP调节的电导与内源性CFTR的表达有关。胆管分泌异常可能参与了囊性纤维化胆汁淤积性肝病的发病机制。
This study examines the conductive properties of the plasma membrane of cells isolated from the intrahepatic portion of bile ducts. Membrane Cl- conductance was measured in single cells using whole-cell patch clamp recording techniques and in cells in short-term culture using Cl-36 and I-125 efflux. Separate Ca2+- and cAMP-dependent Cl currents were identified. Ca2+-dependent Cl- currents showed outward rectification of the current-voltage relation, time-dependent activation at depolarizing potentials, and reversal near the equilibrium potential for Cl-. lonomycin (2 muM) increased this current from 357+/-72 pA to 1,192+/-414 pA (at +80 mV) in 5:7 cells, and stimulated efflux of I-125 > Cl-36 in 15:15 studies. lonomycin-stimulated efflux was inhibited by the Cl- channel blocker 4,4'-diisothio-cyano-2,2'-stilbene disulfonic acid (DIDS) ( 1 50 muM). A separate cAMP-activated Cl- current showed linear current-voltage relations and no time dependence. Forskolin (10 muM) or cpt-cAMP (500 muM) increased this current from 189+/-50 pA to 784+/-196 pA (at +80 mV) in 11:16 cells, and stimulated efflux of Cl-36 > I-125 in 16:16 studies. cAMP-stimulated efflux was unaffected by DIDS. Because the cAMP-stimulated Cl- conductance resembles that associated with cystic fibrosis transmembrane conductance regulator (CFTR), a putative Cl- channel protein, the presence of CFTR in rat liver was examined by immunoblot analyses. CFTR was detected as a 150-165-kD protein in specimens with increased numbers of duct cells. Immunoperoxidase staining confirmed localization of CFTR to bile duct cells but not hepatocytes. These findings suggest that Ca2+- and cAMP-regulated Cl- channels may participate in control of fluid and electrolyte secretion by intrahepatic bile duct epithelial cells, and that the cAMP-regulated conductance is associated with endogenous expression of CFtR. Abnormal ductular secretion may contribute to the pathogenesis of cholestatic liver disease in cystic fibrosis.