Quantitative Determination of Gap Junctional Permeability in the Lens Cortex

Quantitative Determination of Gap Junctional Permeability in the Lens Cortex We have developed a simple dye transfer method, which allows the gap junction permeability of lens fiber cells to be quantified. Two fixable fluorescent dyes (Lucifer yellow and rhodamine-dextran) were introduced into peripheral lens fiber cells via mechanical damage induced by removing the lens capsule. After a defined incubation period, lenses were fixed, sectioned, and the distribution of the dye recorded using confocal microscopy. Rhodamine-dextran and Lucifer yellow both labeled the extracellular space between fiber cells and the cytoplasm of fiber cells that had been damaged by capsule removal. For the gap junctional permeable dye Lucifer yellow, however, labeling was not confined to the damaged cells and exhibited intercellular diffusion away from the damaged cells. The extent of dye diffusion was quantified by collecting radial dye intensity profiles from the confocal images. Effective diffusion coefficients (D eff ) for Lucifer yellow were then calculated by fitting the profiles to a series of model equations, which describe radial diffusion in a sphere. D eff is the combination of dye diffusion through the cytoplasm and through gap junction channels. To calculate the gap junctional permeability (P j ) an estimate of the cytoplasmic diffusion coefficient (D cyt = 0.7 × 10−6 cm2/sec) was obtained by observing the time course of dye diffusion in isolated elongated fiber cells loaded with Lucifer yellow via a patch pipette. Using this approach, we have obtained a value for P j of 31 × 10−5 cm/sec for fiber-fiber gap junctions. This value is significantly larger than the value of P j of 4.4 × 10−6 cm/sec reported by Rae and coworkers for epithelial-fiber junctions (Rae et al., 1996. J. Membrane Biol. 150:89–103), and most likely reflects the high abundance of gap junctions between lens fiber cells. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Membrane Biology Springer Journals

Quantitative Determination of Gap Junctional Permeability in the Lens Cortex

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Publisher
Springer-Verlag
Copyright
Copyright © Inc. by 1999 Springer-Verlag New York
Subject
Life Sciences; Biochemistry, general; Human Physiology
ISSN
0022-2631
eISSN
1432-1424
D.O.I.
10.1007/s002329900521
Publisher site
See Article on Publisher Site

Abstract

We have developed a simple dye transfer method, which allows the gap junction permeability of lens fiber cells to be quantified. Two fixable fluorescent dyes (Lucifer yellow and rhodamine-dextran) were introduced into peripheral lens fiber cells via mechanical damage induced by removing the lens capsule. After a defined incubation period, lenses were fixed, sectioned, and the distribution of the dye recorded using confocal microscopy. Rhodamine-dextran and Lucifer yellow both labeled the extracellular space between fiber cells and the cytoplasm of fiber cells that had been damaged by capsule removal. For the gap junctional permeable dye Lucifer yellow, however, labeling was not confined to the damaged cells and exhibited intercellular diffusion away from the damaged cells. The extent of dye diffusion was quantified by collecting radial dye intensity profiles from the confocal images. Effective diffusion coefficients (D eff ) for Lucifer yellow were then calculated by fitting the profiles to a series of model equations, which describe radial diffusion in a sphere. D eff is the combination of dye diffusion through the cytoplasm and through gap junction channels. To calculate the gap junctional permeability (P j ) an estimate of the cytoplasmic diffusion coefficient (D cyt = 0.7 × 10−6 cm2/sec) was obtained by observing the time course of dye diffusion in isolated elongated fiber cells loaded with Lucifer yellow via a patch pipette. Using this approach, we have obtained a value for P j of 31 × 10−5 cm/sec for fiber-fiber gap junctions. This value is significantly larger than the value of P j of 4.4 × 10−6 cm/sec reported by Rae and coworkers for epithelial-fiber junctions (Rae et al., 1996. J. Membrane Biol. 150:89–103), and most likely reflects the high abundance of gap junctions between lens fiber cells.

Journal

The Journal of Membrane BiologySpringer Journals

Published: May 15, 1999

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