Cell Biochemistry and Biophysics

, Volume 45, Issue 1, pp 43–58 | Cite as

Diffusion delays and unstirred layer effects at monolayer cultures of Chinese hamster ovary cells

Radioligand binding, confocal microscopy, and mathematical simulations
  • Charles E. Spivak
  • Murat Oz
  • Carol L. Beglan
  • Richard I. Shrager
Original Article

Abstract

Cells grown in monolayer culture offer a convenient system for binding and other experiments under conditions that preserve the complexity of the living state. Kinetics experiments, however, may be distorted by the time course of drug penetration into even so simple a “tissue” as the monolayer. The impediments include unstirred layers both above and between the cells, the congregation of receptors within the confined space between cells, and nonspecific binding to membrane components. The contributions of these factors were investigated in cultures of Chinese hamster ovary (CHO) cells either nontransfected or stably transfected with μ opioid receptors. The dissociation of [3H]naloxone was four times faster under displacement than under infinite dilution conditions, clearly demonstrating the “retention effect” of receptors confined in space. Even the penetration of this ligand between nontransfected cells showed salient delays with respect to diffusion into a slab, indicating that nonspecific, low-affinity binding to membrane components was arresting its progress. The optical sectioning capabilities of confocal microscopy demonstrated that the kinetics of two fluorescent antagonists depended on the vertical plane, providing direct evidence for slowed diffusion down a single cell depth. Modeling shows that kinetic errors increase with receptor density, forward rate constant, and the thickness of the unstirred layer.

Index Entries

μ Opioid receptor confocal microscopy binding kinetics nonspecific binding cerebroside sulfate mathematical modeling Crank-Nicolson method 

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Copyright information

© Humana Press Inc. 2006

Authors and Affiliations

  • Charles E. Spivak
    • 1
  • Murat Oz
    • 1
  • Carol L. Beglan
    • 1
  • Richard I. Shrager
    • 2
  1. 1.Cellular Neurobiology Branch, Intramural Research Program, National Institute on Drug AbuseNational Institutes of Health, Department of Health and Human ServicesBaltimore
  2. 2.Center for Information Technology, Mathematical and Statistical Computing LaboratoryNational Institutes of Health, Department of Health and Human ServicesBethesda

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