Masyuk A I, Gong A Y, Kip S, Burke M J, LaRusso N F
Center for Basic Research in Digestive Disease, Division of Gastroenterology and Hepatology, Mayo Medical School, Clinic and Foundation, Rochester, Minnesota 55905, USA.
Gastroenterology. 2000 Dec;119(6):1672-80. doi: 10.1053/gast.2000.20248.
BACKGROUND & AIMS: We report a novel approach to study biliary water, bile acid, and HCO(3)(-) transport: the microperfusion of intrahepatic bile duct units (IBDUs) isolated from normal rat liver.
To study water transport, IBDUs were perfused in vitro with a membrane-impermeant fluorescent volume marker, fluorescein sulfonate; net water movement (J(v)) and osmotic water permeability (P(f)) were then calculated. To study solute transport, IBDUs were perfused with taurocholic acid (TCA) and bile acid uptake was calculated from the concentrations of TCA in the perfused and collected solutions. To study ion transport, IBDUs were perfused with the cell-impermeant pH-sensitive dye BCECF dextran; luminal pH was determined from fluorescence excitation ratios.
When inward (secretory) or outward (absorptive) osmotic gradients were established across IBDUs, water movement was observed from bath to lumen (i.e., secretion) and from lumen to bath (i.e., absorption). The perfused IBDUs absorbed TCA in a saturable, sodium-dependent manner; in addition, TCA absorption was blocked in a dose-dependent fashion by S0960, a specific inhibitor of the Na(+)/bile acid cotransporter. Addition of forskolin to HCO(3)(-)-containing (but not HCO(3)(-)-free) bath buffer resulted in lumen alkalinization reflecting HCO(3)(-) transport into the lumen of perfused IBDUs.
The results provide direct functional evidence that intrahepatic bile ducts both secrete and absorb water in response to osmotic gradients, actively absorb bile acid, and transport HCO(3)(-).
我们报告了一种研究胆汁水、胆汁酸和HCO₃⁻转运的新方法：对从正常大鼠肝脏分离出的肝内胆管单位（IBDUs）进行微灌注。
为研究水转运，在体外向IBDUs灌注一种不能透过细胞膜的荧光体积标记物，荧光素磺酸盐；然后计算净水移动（J(v)）和渗透水通透性（P(f)）。为研究溶质转运，用牛磺胆酸（TCA）灌注IBDUs，并根据灌注液和收集液中TCA的浓度计算胆汁酸摄取量。为研究离子转运，用不能透过细胞的pH敏感染料BCECF葡聚糖灌注IBDUs；根据荧光激发比率测定管腔pH值。
当在IBDUs上建立内向（分泌性）或外向（吸收性）渗透梯度时，观察到水从浴液向管腔移动（即分泌）以及从管腔向浴液移动（即吸收）。灌注的IBDUs以饱和的、钠依赖的方式吸收TCA；此外，Na⁺/胆汁酸共转运体的特异性抑制剂S0960以剂量依赖方式阻断TCA的吸收。向含HCO₃⁻（但不含HCO₃⁻的）浴液缓冲液中添加福斯可林导致管腔碱化，反映HCO₃⁻转运到灌注的IBDUs管腔中。
这些结果提供了直接的功能证据，表明肝内胆管对渗透梯度有反应，既能分泌又能吸收水，能主动吸收胆汁酸，并能转运HCO₃⁻。
