Direct determination of the driving forces for taurocholate uptake into rat liver plasma membrane vesicles.
Direct determination of the driving forces for taurocholate uptake into rat liver plasma membrane vesicles.
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直接测定大鼠肝质膜囊泡摄取牛磺胆酸盐的驱动力。
DOI：
10.1172/jci111103
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发表时间：
1983
期刊：
影响因子：
--
通讯作者：
J. Boyer
中科院分区：
文献类型：
--
作者：
M. Duffy;B. Blitzer;J. Boyer
为了直接确定胆汁酸进入肝细胞的驱动力，研究了大鼠肝质膜囊泡对[3 H]牛磺胆酸的摄取。膜制剂主要含有右侧外囊泡，并且高度富集质膜标记酶。平衡状态下牛磺胆酸盐的摄取与介质渗透压浓度呈负相关，表明转运进入了一个敏感的空间。在一个向内指向钠梯度（氯化钠或葡萄糖酸钠）的存在下，摄取的初始速率是快速的，牛磺胆酸盐瞬时积累在浓度的两倍，在平衡（过冲）。其他向内定向的阳离子梯度（K+，Li+，胆碱+）或钠的存在下，梯度（Na+平衡）的情况下，导致在一个较慢的初始摄取速率，并没有维持过冲。胆汁酸抑制钠依赖性牛磺胆酸盐摄取，而溴磺酞抑制钠依赖性和钠非依赖性摄取，D-葡萄糖对摄取没有影响。吸收是温度依赖性的，在25 ℃时发生最大超调。在没有钠梯度的情况下，跨囊泡施加质子梯度（pH小于pHi）未能增强牛磺胆酸盐摄取，表明双离子交换（Na+-H+，OH-阴离子）是不可能的。通过改变伴随的阴离子或通过缬氨霉素诱导的K+扩散电位产生负的囊内电位并没有增强牛磺胆酸盐的摄取，这表明了电中性转运机制。牛磺胆酸盐摄取的动力学表现出饱和性，米氏常数为52 μ M，最大速率为4.5 nmol X mg-1 X蛋白X min-1。这些研究为肝牛磺胆酸盐摄取的钠梯度依赖性、载体介导的电中性转运机制提供了明确的证据。这些发现与胆汁分泌模型一致，其中基底外侧酶Na+，K+-ATP酶通过建立跨膜钠梯度为“上坡”胆汁酸转运提供驱动力。
To determine directly the driving forces for bile acid entry into the hepatocyte, the uptake of [3H]taurocholic acid into rat liver plasma membrane vesicles was studied. The membrane preparation contained predominantly right-side-out vesicles, and was highly enriched in plasma membrane marker enzymes. The uptake of taurocholate at equilibrium was inversely related to medium osmolarity, indicating transport into an osmotically sensitive space. In the presence of an inwardly directed sodium gradient (NaCl or sodium gluconate), the initial rate of uptake was rapid and taurocholate was transiently accumulated at a concentration twice that at equilibrium (overshoot). Other inwardly directed cation gradients (K+, Li+, choline+) or the presence of sodium in the absence of a gradient (Na+ equilibrated) resulted in a slower initial uptake rate and did not sustain an overshoot. Bile acids inhibited sodium-dependent taurocholate uptake, whereas bromsulphthalein inhibited both sodium-dependent and sodium-independent uptake and D-glucose had no effect on uptake. Uptake was temperature dependent, with maximal overshoots occurring at 25 degrees C. Imposition of a proton gradient across the vesicle (pHo less than pHi) in the absence of a sodium gradient failed to enhance taurocholate uptake, indicating that double ion exchange (Na+-H+, OH- -anion) is unlikely. Creation of a negative intravesicular potential by altering accompanying anions or by valinomycin-induced K+-diffusion potentials did not enhance taurocholate uptake, suggesting an electroneutral transport mechanism. The kinetics of taurocholate uptake demonstrated saturability with a Michaelis constant at 52 microM and maximum velocity of 4.5 nmol X mg-1 X protein X min-1. These studies provide definitive evidence for a sodium gradient-dependent, carrier-mediated, electrically neutral transport mechanism for hepatic taurocholate uptake. These findings are consistent with a model for bile secretion in which the basolateral enzyme Na+,K+-ATPase provides the driving force for "uphill" bile acid transport by establishing a trans-membrane sodium gradient.