Diffusion delays and unstirred layer effects at monolayer cultures of Chinese hamster ovary cells |
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Authors: | Charles E Spivak Murat Oz Carol L Beglan Richard I Shrager |
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Institution: | (1) Cellular Neurobiology Branch, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Department of Health and Human Services, 21224 Baltimore, MD;(2) Center for Information Technology, Mathematical and Statistical Computing Laboratory, National Institutes of Health, Department of Health and Human Services, 20892 Bethesda, MD |
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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. |
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Keywords: | μ Opioid receptor confocal microscopy binding kinetics nonspecific binding cerebroside sulfate mathematical modeling Crank-Nicolson method |
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