The Ussing Chamber and Measurement of Drug Actions on Mucosal Ion Transport

David R. Brown1, Scott M. O’Grady1

1 University of Minnesota, Departments of Veterinary & Biomedical Sciences (DRB) and Animal Science (SMO), St. Paul, Minnesota
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 7.12
DOI:  10.1002/0471141755.ph0712s41
Online Posting Date:  June, 2008
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Abstract

Ussing chamber technology can be used to investigate the pharmacodynamic and pharmacokinetic properties of drugs and natural substances on polarized epithelia. With this approach, epithelial sheets or confluent epithelial cell monolayers are mounted between two half‐chambers, permitting compound delivery to either the apical or basolateral cell surface. Described in this unit are protocols for determining the effects of test compounds on transepithelial ion transport processes. Curr. Protoc. Pharmacol. 41:7.12.1‐7.12.17. © 2008 by John Wiley & Sons, Inc.

Keywords: epithelium; epithelial cell culture; intestine; airways; skin; transepithelial ion transport; pharmacokinetics

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Determination of Test Compound Actions on Active Ion Transport in Mucosal Tissues by Short‐Circuit Current Analysis
  • Basic Protocol 2: Determination of Test Substance Actions on Active Ion Transport in Mucosal Tissues by Transmural Ion Flux Analysis
  • Basic Protocol 3: Measurement of Ion Transport in Cultured Epithelial Monolayers
  • Basic Protocol 4: Measurements of Apical and Basolateral Membrane Conductances and Pump Currents Using Antibiotic‐Perforated Monolayers
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Determination of Test Compound Actions on Active Ion Transport in Mucosal Tissues by Short‐Circuit Current Analysis

  Materials
  • Carbogen‐bubbled physiological salt solution (e.g., Krebs Ringer physiological salt solution, see recipe; volume needed depends on the number of tissues/chambers used; usually 500 to 1000 ml is sufficient for 8 chambers connected to 10‐ml capacity reservoirs), 37°C
  • Aqueous stock solutions of 1 M D‐glucose and 1 M mannitol
  • Tissue explants
  • Concentrated aliquots of test compound solutions (concentrations should take into account administration of solutions at volumes no greater than 0.1% to 1.0% of total fluid reservoir volume)
  • Ussing chamber system including electrodes (see )
  • Beakers (2) and buckets containing ice for bathing mucosal tissues after isolation and dissection
  • Tissue dissection equipment (surgical scissors, hemostat, small forceps, single‐edge razor blade) for isolating and preparing sheets of mucosal epithelium for mounting in Ussing chambers
  • Lucite board with pins to anchor and dissect tissue specimens (tissue anchoring pins should be positioned to accommodate the average width of the tissue segment to be dissected)

Basic Protocol 2: Determination of Test Substance Actions on Active Ion Transport in Mucosal Tissues by Transmural Ion Flux Analysis

  Materials
  • Radioisotopes (e.g., 22Na, 36Cl)
  • Buffer or scintillation fluid (e.g., Aquasol)
  • Test agent
  • Gamma counter (for 22Na) or liquid scintillation counter (for 36Cl)
  • Additional reagents and equipment for setting up Ussing chamber system, preparing tissue samples, and stabilization of bioelectrical parameters ( protocol 1)

Basic Protocol 3: Measurement of Ion Transport in Cultured Epithelial Monolayers

  Materials
  • Epithelial cells (primary cells, immortalized cells, or tumor cell lines)
  • Physiological salt solution (see )
  • Media, serum, antibiotics, and growth factors required for culturing cells of interest
  • Standard cell culture equipment including:
    • Cell culture hood
    • Low‐speed centrifuge for harvesting cells
    • Vacuum pump/aspirator
    • Inverted tissue culture microscope (phase contrast with 10× and 20× objective lenses)
    • CO 2 incubator with temperature control to 37°C
    • Autoclave
    • Hemacytometer for counting cells
  • Standard cell culture supplies including:
    • Sterile serological pipets (25‐, 10‐, and 2‐ml volumes)
    • Tissue culture flasks (25‐ and 75‐ml volumes)
    • Filter units for filtering media
    • Sterile glass media bottles (100‐, 500‐, and 1000‐ml)
  • Snapwell/Transwell filters of 12‐ to 24‐mm diameter (Corning Life Sciences)
  • Ussing chamber systems (see )
  • Electrodes (see )

Basic Protocol 4: Measurements of Apical and Basolateral Membrane Conductances and Pump Currents Using Antibiotic‐Perforated Monolayers

  Materials
  • Epithelial cell monolayer expressing ENaC channels
  • Modified low chloride‐containing salt solution (see recipe)
  • High K+‐containing salt solution (see recipe)
  • Nystatin or amphotericin B (Sigma)
  • DMSO
  • Amiloride or benzamil (Calbiochem)
  • Ussing chamber system (see )
  • pCLAMP software (Molecular Devices)
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Figures

Videos

Literature Cited

Literature Cited
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   Brown, D.R., Parsons, A.M., and O'Grady, S.M. 1992. Substance P produces sodium and bicarbonate secretion in porcine jejunal mucosa through an action on enteric neurons. J. Pharmacol. Exp. Ther. 261: 1206‐1212.
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