Diffusion of Small Solutes in the Lateral Intercellular Spaces of MDCK Cell Epithelium Grown on Permeable Supports

Abstract

The diffusion coefficients of four solutes ranging in molecular weight from 238 to 10,000 in the lateral intercellular spaces (LIS) of cultured kidney cells (MDCK) grown on permeable supports were determined from the spread of fluorescence produced after the release of caged compounds by a pulse from a UV laser. Two types of experiments were performed: measurement of the rate of change of fluorescence after releasing a caged fluorophore, and measurement of the change in fluorescence of a relatively static fluorescent dye produced by the diffusion of an uncaged ligand for the dye. Fluorescence intensity was determined by photon-counting the outputs of a multichannel photomultiplier tube. Diffusion coefficients were determined in free solution as well as in the LIS of MDCK cells grown on permeable supports and the hindrance factor, theta, determined from the ratio of the free solution diffusivity to that in the LIS. The hindrance factors for 3000-MW dextran, 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS, MW 524) and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES, MW 238) were not significantly different from 1. The diffusion of 10,000-MW dextran was substantially reduced in the LIS with a theta of 5.6 +/- 0.3. Enzymatic digestion by neuraminidase of the sialic acid residues of the glycosylation groups in the LIS increased the diffusivity of the 10,000-MW dextran 1.8-fold indicating hindrance by the glycocalyx. We conclude that small solutes, such as Na(+) and Cl(-), would not be significantly restricted in their diffusion in the LIS and that solute concentration gradients could not develop along the LIS under physiologic conditions.

A guide to Ussing chamber studies of mouse intestine

“…However, the safe handling and clean up of radioactive materials adds a laborious aspect to their use. Present interests focused on intestinal permeability in disease have resulted in an increasing number of studies that use fluorescent tracers often of specific size or composition to probe the permeability characteristics of the paracellular pathway (16,23).…”

Role of villus microcirculation in intestinal absorption of glucose: coupling of epithelial with endothelial transport

“…The diffusion coefficients of small hydrophilic solutes through intercellular channels of cultured Madin-Derby canine kidney (MDCK) cells are not significantly different from their free diffusion coefficients (Kovbasnjuk et al 2000). We therefore assume that the same is true of glucose in dilated channels of absorbing jejunal enterocytes.…”

Computational Modeling of Extracellular Mechanotransduction

“…In the LIS of MDCK epithelial cells, which share a similar architecture with human bronchial epithelial cells, large molecules experience hindered diffusion due to the protrusion of a glycocalyx into the LIS (6). Specifically, while a 3-kDa molecule diffuses in the LIS of MDCK cells as if in free solution, a significant decrease in diffusion coefficient is observed for a 10-kDa molecule relative to that for free diffusion (Fig.…”

“…Cells express a variety of autocrine mediators with a range of diffusivities dependent on molecular size and charge characteristics (6,7). These mediators are likely released at various rates, further influencing their kinetics within the LIS.…”

“…Diffusivities on the order of 100 mm 2 /s characterize free diffusion of smaller molecules whose molecular weight ranges from ;0.1-10 kDa, whereas D on the order of 10 mm 2 /s corresponds to free diffusion of larger molecules 10-1000 kDa. The cases of D ¼ 0.1 and 1 mm 2 /s represent hindered diffusion of large molecules (6). We further assumed equal diffusivities inside and outside of the LIS, D LIS ¼ D out (this assumption will be addressed later).…”