Konieczko E M, Ralston A K, Crawford A R, Karpen S J, Crawford J M
Department of Pathology, Yale University School of Medicine, New Haven, CT 06520-8023, USA.
Hepatology. 1998 Jan;27(1):191-9. doi: 10.1002/hep.510270130.
Although bile salts are toxic to the liver at high plasma concentrations, the effects of physiological concentrations of bile salts on normal hepatic function are poorly understood. We examined the effect of taurocholate (TC) on the basolateral uptake of [3H]TC in WIF-B cells, a hybrid cell line stably exhibiting in vitro the structural and functional polarity of hepatocytes. Cells were grown in the absence or presence of TC (50 micromol/L) over 12 days, and then incubated with [3H]TC concentrations ranging from 1 to 250 micromol/L. For both control and TC-grown cells, uptake of [3H]TC was linear over 2 minutes. In control cells, the Km for [3H]TC Na+-dependent uptake over 1 minute was 6 +/- 5 micromol/L, and the Vmax was 45 +/- 6 pmol TC/mg protein/min (+/- SEM). TC-grown cells exhibited no significant change in Km but showed a doubling of Vmax to 87 +/- 6 pmol TC/mg protein/min (P < .005). In both control and TC-grown cells, maximal uptake of [3H]TC occurred following 10 to 12 days in culture, with TC-grown cells consistently showing greater rates of [3H]TC uptake from 4 to 14 days in culture. Western blots immunostained for the basolateral Na+-dependent plasma membrane protein, ntcp, revealed the appropriate approximately 50-kd band in control and TC-grown cells, and confocal immunofluorescence microscopy demonstrated staining along the basolateral plasma membrane. Northern blots hybridized with a cDNA probe directed against ntcp indicated a modest TC-induced increase in mRNA levels. Reverse-transcriptase polymerase chain reaction (RT-PCR) using RNA isolated from WIF-B cells and oligonucleotide primers specific for rat ntcp or human NTCP transcripts revealed only the presence of the rat ntcp transcript. We conclude that bile salts, at concentrations normally found in mammalian portal blood, may be capable of promoting enhanced hepatocellular bile salt uptake via an increase in basolateral Na+-dependent plasma membrane transport capacity.
尽管胆汁盐在高血浆浓度时对肝脏有毒性，但生理浓度的胆汁盐对正常肝功能的影响却知之甚少。我们研究了牛磺胆酸盐（TC）对WIF - B细胞中[3H]TC基底外侧摄取的影响，WIF - B细胞是一种稳定表现出肝细胞结构和功能极性的杂交细胞系。细胞在无TC（50微摩尔/升）或有TC的情况下培养12天，然后与浓度范围为1至250微摩尔/升的[3H]TC孵育。对于对照细胞和经TC培养的细胞，[3H]TC的摄取在2分钟内呈线性。在对照细胞中，1分钟内[3H]TC钠依赖性摄取的Km为6±5微摩尔/升，Vmax为45±6皮摩尔TC/毫克蛋白/分钟（±SEM）。经TC培养的细胞Km无显著变化，但Vmax增加了一倍，达到87±6皮摩尔TC/毫克蛋白/分钟（P <.005）。在对照细胞和经TC培养的细胞中，[3H]TC的最大摄取在培养10至12天后出现，经TC培养的细胞在培养4至14天期间[3H]TC摄取率始终更高。针对基底外侧钠依赖性质膜蛋白ntcp进行免疫染色的蛋白质印迹显示，对照细胞和经TC培养的细胞中出现了合适的约50 kDa条带，共聚焦免疫荧光显微镜显示沿基底外侧质膜有染色。与针对ntcp的cDNA探针杂交的Northern印迹表明，TC诱导mRNA水平适度增加。使用从WIF - B细胞分离的RNA和针对大鼠ntcp或人NTCP转录本的寡核苷酸引物进行逆转录聚合酶链反应（RT - PCR），结果仅显示存在大鼠ntcp转录本。我们得出结论，哺乳动物门静脉血中正常浓度的胆汁盐可能能够通过增加基底外侧钠依赖性质膜转运能力来促进肝细胞对胆汁盐的摄取增强。
