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

The lateral intercellular spaces (LIS) in epithelial tissues are believed to play a role in osmotic gradients that support transepithelial transport. Prior research has shown that LIS are structurally and chemically distinct from surrounding extracellular fluid. However, the exact pH and diffusion properties within LIS remained unclear. Earlier studies focused on bulk extracellular pH and transport without isolating LIS-specific conditions. This gap motivated the development of new techniques to specifically probe LIS in living cells. No prior work had resolved LIS pH in the absence of bicarbonate or CO2. The LIS geometry and dye diffusion within it had not been quantified in real time. This paper introduces methods to load LIS with BCECF and measure pH and diffusion in situ. These findings aim to clarify the LIS as a distinct microenvironment for transport processes.

This study aimed to investigate the pH, morphology, and diffusion characteristics of the lateral intercellular spaces (LIS) in epithelial cell monolayers. The LIS are proposed to be the site of local osmotic gradients that facilitate transepithelial transport. The researchers sought to determine whether LIS pH differs from surrounding extracellular fluid. They also wanted to assess how LIS pH responds to changes in bicarbonate and CO2 availability. The study aimed to visualize LIS geometry using BCECF loading. The purpose included measuring the diffusion coefficient of BCECF within LIS. The researchers also aimed to compare LIS characteristics between MDCK and LLC-PK1 cells. This work addresses the lack of direct measurements of LIS microenvironmental properties. The findings may help clarify how LIS contribute to epithelial transport mechanisms.

The researchers developed a method to load the LIS of cultured renal cells with BCECF, a pH-sensitive fluorescent dye. They used MDCK and LLC-PK1 epithelial cell monolayers for their experiments. The LIS were visualized using fluorescence microscopy after BCECF loading. The pH of the LIS was measured by calibrating BCECF fluorescence against known pH standards. The study tested LIS pH in both bicarbonate-free and bicarbonate-containing solutions. The LIS geometry was analyzed from BCECF fluorescence images. The diffusion coefficient of BCECF was calculated using fluorescence recovery after photobleaching (FRAP). The LIS pH was compared to the superfusate pH in different solution conditions. These methods allowed direct measurement of LIS-specific properties in living cells.

The LIS pH was found to be remarkably constant across different superfusate pH conditions. In bicarbonate-free solutions, MDCK LIS pH was 7.66 ± 0.04 regardless of superfusate pH. In bicarbonate-containing solutions, MDCK LIS pH was 0.3–0.4 units lower than the superfusate. LLC-PK1 LIS pH was acidic at 6.83 ± 0.05 in bicarbonate-free solutions. LLC-PK1 LIS pH increased by approximately 0.25 units in bicarbonate-containing solutions. No pH gradients were detected within the LIS itself. The BCECF diffusion coefficient in LIS was similar to that in free solution. LIS geometry was visualized using BCECF fluorescence imaging. These results suggest LIS maintain a stable pH distinct from surrounding extracellular fluid.

The LIS of epithelial cell monolayers maintain a stable pH that differs from the surrounding extracellular fluid. The LIS pH of MDCK cells was acidic in bicarbonate-containing solutions but not in bicarbonate-free conditions. LLC-PK1 LIS pH was significantly lower in bicarbonate-free solutions and increased with bicarbonate presence. The LIS pH remained uniform without internal gradients. The BCECF diffusion coefficient in LIS was not significantly different from free solution. These findings suggest LIS are a distinct compartment with regulated pH. The study demonstrates the feasibility of measuring LIS-specific properties in living cells. The methods developed allow direct visualization and quantification of LIS characteristics. These results may inform future studies on how LIS contribute to epithelial transport mechanisms.