pH, morphology, and diffusion in lateral intercellular spaces of epithelial cell monolayers

Abstract

The lateral intercellular spaces (LIS) of reabsorptive epithelia are the site of the proposed local osmotic gradient responsible for transepithelial transport. We developed techniques for loading the LIS of living cultured renal cells (MDCK and LLC-PK1) with the fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF), visualizing LIS geometry, measuring pH, and determining the BCECF diffusion coefficient within the LIS. The LIS pH was remarkably constant and differed substantially from that of the superfusate in both the presence and absence of HCO3 or CO2. The LIS of MDCK cells had a pH of 7.66 +/- 0.04 in bicarbonate-free solutions of pH 7.0, 7.4, or 7.8. In bicarbonate-containing solutions, MDCK LIS pH was acidic to the superfusate by 0.3-0.4 units. In the absence of bicarbonate, the LIS of LLC-PK1 cells was markedly acidic (6.83 +/- 0.05), becoming alkaline by approximately 0.25 units in the presence of bicarbonate. Gradients in pH or dye concentration were not detected within the LIS. The diffusion coefficient of BCECF within the LIS was approximately equal to that seen in free solution.

Water does not flow across the tight junctions of MDCK cell epithelium

“…We recently measured the diffusion coefficients of two solutes by using photo-activated fluorescence and high-speed photometry in the LIS of MDCK cells grown on glass coverslips (10). The diffusivity within the LIS of a small fluorescent solute, hydroxypyrene-1,3,6-trisulfonic acid (524 M r ), was indistinguishable from that in free solution, an observation in agreement with an earlier study of BCECF diffusivity by fluorescence recovery after photobleaching (12). Although the rate of diffusion of 70,000 M r fluorescein dextran could not be measured because of limited solubility of the caged dye, the diffusivity of 10,000 M r fluorescein dextran within the LIS was determined.…”

“…As shown in Fig. 4a, BCECF, a fluorescein derivative of molecular weight 520 with a measured diffusion coefficient in the LIS 9.8 times that of the 70,000 M r dextran (10,12), does not show any significant gradient in fluorescence intensity along the LIS under isotonic conditions. This observation is in good agreement with a previous study from our laboratory using wide-field fluorescence microscopy showing that gradients in BCECF concentration along the LIS did not occur (8).…”

Extracellular pH regulation in microdomains of colonic crypts: effects of short-chain fatty acids.

“…Dual-emission ratios of SNARF-1 fluorescence were used to estimate extracellular pH (21) in the crypt lumen and submucosal tissue directly surrounding crypts. Lateral intercellular spaces may function as a distinct extracellular pH microdomain (16,17). However, in our preparation, lateral spaces had insufficient signal for reliable ratio calculations in most experiments and therefore were not analyzed.…”

“…At present there is no consensus whether this space is regulated by luminal fluid pH or [SCFA] (13)(14)(15). Recent information also shows that lateral intercellular spaces between cultured renal cells (Madin-Darby canine kidney) maintain a pH distinct from bulk perfusates (16,17). A similar basolateral pH microenvironment has been suggested for enterocytes, on the basis of mathematical modeling of transepithelial SCFA fluxes (18,19).…”

Extracellular pH in Restricted Domains as a Gating Signal for Ion Channels Involved in Transepithelial Transport

“…5,6) It has also been proposed that epithelia through the process of absorption or secretion of acid or base generate pH o domains, separate from bulk medium, at the apical surface, lateral intercellular spaces, and basal surface. [7][8][9][10][11][12] An acid microclimate has been found in the vicinity of the apical surface that is postulated to be important in the transport of nutrients, micronutrients and drugs coupled to H ϩ fluxes. 13) The absorption of short chain fatty acids (SCFA) coupled with sodium across colon epithelium is thought to produce an acidification of the lateral intercellular spaces.…”