Taurocholate Transport by Rat Liver Sinusoidal Membrane Vesicles: Evidence of Sodium Cotransport

Abstract

To elucidate the first step in the vectorial transport of bile acids by the liver, plasma membrane vesicles were isolated from rat liver by differential and sucrose-Ficoll density gradient centrifugation. The membranes were selectively enriched 20-fold in Na',K'-ATPase activity, a marker of sinusoidal plasma membranes. Electron microscopy of pellets from sinusoidal membrane fraction did not reveal other organelles.The initial rate of taurocholate uptake by the membrane vesicles was stimulated by a sodium gradient but not by a potassium gradient (Naouteide > Nainside). Sodium-dependent uptake of taurocholate was inhibited at low temperature (O"C), reduced by decreasing intravesicular space, and showed transstimulation in tracer exchange experiments indicating that uptake into vesicles, rather than binding to membranes, was occurring. Sodium-dependent taurocholate transport by the vesicles exhibited saturation kinetics with respect to taurocholate (apparent K, = 56 pM, V , , = 0.65 nmoles per mg X 15 sec at 100 mM NaN03 and 25°C) and sodium (apparent K, = 48 mM at 50 taurocholate). Other cations, such as lithium and choline, did not replace sodium in its stimulatory action. Sodium-dependent taurocholate uptake was selectively inhibited by cholic acid and probenecid. When the electrical potential difference across the vesicle membrane was altered by anion replacement, a more negative intravesicular potential inside the vesicles stimulated and a more positive potential inhibited sodium-dependent taurocholate transport. These data indicate the presence of a taurocholate-sodium cotransport system in the sinusoidal membranes of rat hepatocytes. This sodium-cotransport system probably participates in sodium-dependent uptake of bile acids into the hepatocyte. Bile acid secretion in the liver can be classified as secondary active transport.Transport of organic anions, such as bile acids and bilirubin, is an important function of the liver. Based on kinetic analysis in intact animals (1-4), isolated perfused liver (5-9), and hepatocyte suspensions (10-14), carriermediated, sodium-dependent hepatic uptake of bile acids has been postulated. Vectorial transport of bile acids from plasma into bile is a sequence of many steps including uptake at the sinusoidal membrane, interaction with Received November 16, 1981; accepted April 27, 1982. This work was supported by NIH Grants AM 02019 and GM 27859. cytoplasmic binding proteins, intracellular conjugation, and excretion at the canalicular membrane. Studies of these complex systems provide limited information about the transmembane transport mechanisms since intracelluar events cannot be separated from processes occurring at the plasma membrane. Isolated plasma membrane vesicles which retain transport activity provide a simpler system in which separation can be achieved. Kinetic analysis of transport by sinusoidal membrane vesicles should elucidate the mechanism of the first step in the vectorial transport of bile acids across the hepatocyte.We describe isolation of a plasma mem...

Estrogen-Induced Cholestasis: Clues to Pathogenesis and Treatment

“…Bile salt uptake across the sinusoidal membrane involves a sodium cotransport, carrier-mediated process. The transcellular sodium gradient is the major driving force for influx (79)(80)(81)(82)(83). This gradient depends upon (Na+,K+)ATPase which, based on cytochemical and cell fractionation techniques, is believed to be located on the sinusoidal or basolateral membrane surface (82,84,85).…”

Electrophysiological characterization of human Na+/taurocholate cotransporting polypeptide (hNTCP) heterologously expressed in Xenopus laevis oocytes

“…However, earlier investigations of TCA transport via Ntcp with isolated hepatocytes and liver basolateral membrane vesicles yielded controversial results in favor of either an electroneutral or electrogenic transport mechanism [26][27][28][29][30][31][32]. These studied focused on measurements of radio isotopic flux and provided only indirect evidence for electrogenicity due to TCA uptake.…”

“…Although substrate transport via NTCP unequivocally exhibits an obligatory Na + dependency, the electrogenicity of the NTCP transport cycle remains controversial [26][27][28][29][30][31][32].…”

The evolution of the OATP hepatic uptake transport protein family in DMPK sciences: from obscure liver transporters to key determinants of hepatobiliary clearance

“…Initially vesicles were isolated from membranes of hepatocytes and enriched fractions with high concentrations of basolateral transporter inside out vesicles produced (Takahaski et al, 1997). These sinusoidal membrane vesicles have been utilized to study hepatic uptake transporters such as NTCP (Inoue et al, 1982) but these enriched membrane fraction vesicles were technically challenging to produce and generally yields were low, so they have not been used extensively in drug discovery and development. Vesicle based assays are currently used mainly to study efflux transporters interactions, as recently reviewed by Glavinas et al, 2008. …”