Measurement of Hepatobiliary Transport

Nazzareno Ballatori1

1 University of Rochester School of Medicine, Rochester, New York
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 14.5
DOI:  10.1002/0471140856.tx1405s19
Online Posting Date:  May, 2004
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Abstract

A major hepatic function involves the removal of xenobiotics from portal blood and their subsequent storage, metabolism, and excretion into bile, or delivery of various products back into the bloodstream. Transport across the liver cell basolateral (or sinusoidal) plasma membrane is a key step in the overall metabolism of many compounds, whereas transport across the apical (or canalicular) plasma membrane into bile is often the principal pathway for their elimination. Although significant progress has been made in the molecular identification of the transport proteins that mediate these processes, much remains to be learned about regulation and physiological integration. The critical limiting factor in studying biliary excretion is that the bile canaliculus is very small (∼1 µm in diameter) and is generally inaccessible to sampling by conventional approaches. The bile canalicular membrane is a specialized part of the hepatocellular plasma membrane, such that bile is separated from blood plasma only by the tight junctions that encircle each hepatocyte. Because hepatocyte polarity is rapidly lost during cell isolation, most cell culture models provide only limited information on mechanisms of biliary excretion. Thus, biliary secretion has been studied using four major experimental models: canalicular plasma membrane vesicles, cultured hepatocyte couplets, perfused liver, and in vivo (bile duct‐cannulated animals). This unit describes basic methods for collecting bile from anesthetized mice and rats, for carrying out isolated perfused rat liver studies, and for the simultaneous isolation of plasma membrane vesicles derived from the basolateral and canalicular domains of rat liver.

Keywords: Hepatic transport; bile secretion; perfused rat liver; canalicular membrane vesicles; basolateral membrane vesicles

     
 
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Table of Contents

  • Basic Protocol 1: Bile Collection in Anesthetized Rats and Mice
  • Basic Protocol 2: Preparing an Isolated Perfused Rat Liver
  • Basic Protocol 3: Simultaneous Isolation of Rat Canalicular and Basolateral Liver Plasma Membrane Vesicles
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Bile Collection in Anesthetized Rats and Mice

  Materials
  • Rat or mouse
  • 50 mg/ml pentobarbital sodium
  • Flexible temperature probe (rectal probe; e.g., Yellow Springs Instrument)
  • Thermostat‐controlled heat lamp or heat pad
  • Surgical instruments, sterile, including:
    • Scalpel or blunt‐end scissors
    • Hemostats
    • Forceps, fine
    • Iridectomy scissors (i.e., fine, sharp‐pointed scissors)
  • Suture, size 3‐0
  • PE‐10 tubing (polyethylene tubing), ≥10 cm long with beveled end (e.g., Clay Adams)
  • Surgical clamps, optional
  • 1‐ml microcentrifuge tubes

Basic Protocol 2: Preparing an Isolated Perfused Rat Liver

  Materials
  • KH buffer (see recipe)
  • 1000 U/ml heparin
  • D‐Glucose, aliquots of 90.1 mg stored at room temperature
  • pH 7 buffer (for pH meter calibration)
  • Rat
  • 50 mg/ml pentobarbital sodium
  • 12 × 75–mm test tubes or 1‐ml microcentrifuge tubes
  • Perfusion setup (Fig. ), including:
    • Two pumps capable of delivering 10 to 50 ml/min, preferably with minimal pulsatile flow
    • 250‐ml Erlenmeyer flask
    • Water bath, 37°C
    • Tank of 95% O 2/5% CO 2
    • Glass humidifier and dispersion tube
    • Tygon tubing: 0.12‐in. (0.32‐cm) inner diameter (i.d.) and 0.25‐in. (0.64‐cm) outer diameter (o.d.)
    • In‐line filter (Millipore XX43‐047‐00) containing a 1.2‐µm filter (Millipore RAWP‐047‐00) and a prefilter (Millipore AP25‐042‐00)
    • Gas exchange mechanism: 0.5‐liter jar containing 7.7 m gas‐permeable silastic tubing (Dow Corning 602‐235) attached to y‐shaped connector
    • Perfusion dish: 10‐cm plastic Petri dish with a 1‐cm round hole cut in the center
    • 100‐ml, 3‐neck perfusate reservoir (e.g., Kontes Glass 606020‐0224), with a plastic funnel over the central neck of this reservoir
    • pH meter and electrode
    • Thermometer
    • Small heater and blower
  • Operating tray
  • Surgical instruments, sterile, including:
    • Hemostats, two
    • Iridectomy scissors (i.e., fine, sharp‐pointed scissors)
    • Forceps, two
    • Heavy scissors
    • Blunt‐end scissors or scalpel
  • Suture, 3‐0: one 30‐cm length, folded in half, and four 15‐cm lengths
  • Bile cannula: 16‐cm length of PE‐10 tubing, beveled at one end
  • Portal vein cannula: 14‐ or 16‐G stainless steel, blunt‐end needle with slight bevel forged at the end, connected to a three‐way stopcock
  • Thoracic vena cava cannula: 2.5‐cm length of PE‐205 tubing, beveled at one end
  • Temperature monitor with small flexible temperature probe
  • Dewar flask, tall
  • Two 1‐ml syringes attached to PE‐90 tubing

Basic Protocol 3: Simultaneous Isolation of Rat Canalicular and Basolateral Liver Plasma Membrane Vesicles

  Materials
  • 1 mM NaHCO 3, pH 7.4, ice cold
  • Male Sprague‐Dawley rats, 200 to 250 g
  • 50 mg/ml pentobarbital sodium, to anesthetize rats
  • Sucrose solutions: 8.1%, 31%, 34%, 36.5%, 38%, 44%, and 56% sucrose (see recipe), ice cold
  • Resuspension buffer: e.g., 250 mM sucrose/20 mM KCl/0.2 mM CaCl 2, buffered with 10 mM HEPES/TRIZMA, pH 7.5 (see recipe)
  • 100‐ml and 2‐liter plastic beakers
  • 250‐ml and 1‐liter glass beakers
  • 7‐, 40‐, and 100‐ml glass‐glass Dounce homogenizers with loose‐fitting type A and tight‐fitting type B pestles
  • 2‐liter Erlenmeyer flasks
  • Guillotine
  • Scissors
  • Cheesecloth
  • 250‐ml plastic centrifuge tubes with caps
  • Superspeed centrifuge (e.g., Sorvall RC‐5B), 4°C
  • Centrifuge rotors: Sorvall TZ‐28‐Zonal, GSA, TH‐641, and T‐865 (Sorvall Instruments), or equivalent, 4°C
  • Peristaltic pump
  • 16 × 125–mm test tubes
  • 10‐ml plastic Pasteur pipets or transfer pipets
  • Ultracentrifuge (e.g., Sorvall OTD55B), 4°C
  • Spectrophotometer (700‐nm wavelength)
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Figures

Videos

Literature Cited

Literature Cited
   Ballatori, N. and Truong, A.T. 1989. Relation between biliary glutathione excretion and bile acid‐independent bile flow. Am. J. Physiol. 256:G22‐G30.
   Ballatori, N. and Truong, A.T. 1992. Glutathione as a primary osmotic driving force in hepatic bile formation. Am. J. Physiol. 263:G617‐G624.
   Ballatori, N., Jacob, R., and Boyer, J.L. 1986. Intrabiliary glutathione hydrolysis—a source of glutamate in bile. J. Biol. Chem. 261:7860‐7865.
   Ballatori, N., Jacob, R., Barrett, C., and Boyer, J.L. 1988. Biliary catabolism of glutathione and differential reabsorption of its amino acid constituents. Am. J. Physiol. 254:G1‐G7.
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   Meier, P.J. and Boyer, J.L. 1990. Preparation of basolateral (sinusoidal) and canalicular plasma membrane vesicles for the study of hepatic transport processes. Methods Enzymol. 192:534‐545.
   Meier, P.J., Sztul, E.S., Reuben, A., and Boyer, J.L. 1984. Structural and functional polarity of canalicular and basolateral plasma membrane vesicles isolated in high yield from rat liver. J. Cell Biol. 98:991‐1000.
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