Abstract
As part of the enterohepatic circulation, hepatocytes take up bile acids from the intestines via the hepatic portal blood using a sodium-dependent carrier mechanism and resecrete the bile acids into the bile. In order to assess whether intracellular organelles are involved in the transcellular secretion of bile acids, we measured directly the ability of purified subcellular fractions of rat liver to take up taurocholate using a Millipore filtration assay. Two distinct uptake mechanisms can be discerned, one localized in the plasma membranes and the other in the Golgi and smooth microsomal fractions. Plasma membranes prepared by the method of Fleischer and Kervina (Fleischer, S., and Kervina, M. (1974) Methods Enzymol. 31, 6) take up taurocholate in a saturable manner with an apparent Vmax of 2.4 nmol min-1 mg protein-1 and a Km of 190 microM at 37 degrees C. After preincubation of the membranes with K+ ions, a sodium gradient (100 mM outside) stimulates the uptake rate by 90% with the observed Km unchanged. The stimulation is inhibited by phalloidin but not by bromosulfophthalein. Bile canalicular plasma membranes made according to Kramer et al. (Kramer, W., Bickel, U., Buscher, H. P., Gerok, W., and Kurz, G. (1982) Eur. J. Biochem. 129, 13-24) do not take up taurocholate. The transport by Golgi vesicles and smooth microsomes differs from that in the plasma membrane fraction in that it is not stimulated by a sodium gradient, has a Vmax of 12 nmol min-1 mg protein-1 and a Km of 440 microM at 37 degrees C, and is inhibited by bromosulfophthalein but not by phalloidin. Taurocholate uptake into smooth microsomes is abolished by filipin, an antibiotic that complexes with cholesterol to disrupt the membrane. This suggests that taurocholate uptake occurs into a nonendoplasmic reticulum subfraction since endoplasmic reticulum membranes contain negligible amounts of cholesterol. Little uptake was observed using rough microsomes or mitochondria. A model of transhepatic transport compatible with our observations is that taurocholate uptake into the cytoplasm occurs via the plasma membranes on the sinusoidal side of the hepatocyte; taurocholate is then taken up into smooth vesicles and the Golgi complex and is secreted into the bile by exocytosis as the vesicles fuse with the canalicular plasma membranes.
Highlights
As part of the enterohepatic circulation, hepatocytes The mammalian liver serves a major role in the excretion take up bile acids from the intestines via the hepatic of organic anions such as antibiotics, dyes, and the degradaportal blood using a sodium-dependent carrier mecha- tionproducts of bilirubin, byway of the bile [1, 2]
The total plasma membrane preparation contains plasmalemma derived from all three domains of the hepatocyte surface including sinusoidal, contiguous, andbile canalicular plasma membranes [22], each domain having distinct physiological roles [23]
Similar uptake rates were obtained when concentrated plasma membranes were first preincubated with 0.1 M KC1 and diluted into 0.1 M NaCl containing 300 p~ [3H]taurocholate. the totaulptake in the presence of a sodium gradient was not stimulated by an internal K' gradient
Summary
In determining the taurocholate uptake rate into smooth microsomes orGolgi complex, the amount of nonspecific background absorption of taurocholate to the subcellular fractions and filter can be significantly reduced (-50%) by the addition of bromosulfophthalein and nonradioactive taurocholate to thestop solution to give final concentrations of 60 and 420 pM, respectively. This reduces the background without significantly affecting the observed rates or kinetic parameters for taurocholate uptakeinto either smooth microsome or Golgi fractions
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