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

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.

Driving forces and pathways for H+ and K+ transport in insect midgut goblet cells.

In the midgut of larval lepidopteran insects, goblet cells are believed to secrete K+; the proposed mechanism involves an electrogenic K+/nH+ (n > 1) antiporter coupled to primary active transport of H+ by a vacuolar-type ATPase. Goblet cells have a prominent apical cavity isolated from the gut lumen by a valve-like structure. Using H(+)- and K(+)-selective microelectrodes, we showed that electrochemical gradients of H+ and K+ across the apical membrane and valve are consistent with active secretion of both ions into the cavity and that the transapical H+ electrochemical gradient, but not the transapical pH gradient, is competent to drive K+ secretion by a K+/nH+ antiporter. We used 10 mmol l-1 tetramethylammonium ion (TMA+) as a marker for the ability of small cations to pass from the gut lumen through the valve to the goblet cavity, exploiting the high TMA+ sensitivity of 'K(+)-sensitive' microelectrodes. These studies showed that more than half of the cavities were inaccessible to TMA+. For those cavities that were accessible to TMA+, both entry and exit rates were too slow to be consistent with direct entry through the valves. One or more mixing compartments appear to lie between the lumen bathing solution and the goblet cavity. The lateral intercellular spaces and goblet cell cytoplasm are the most likely compartments. The results are not consistent with free diffusion of ions in a macroscopic valve passage; mechanisms that would allow K+ secreted into the goblet cavity to exit to the gut lumen, while preventing H+ from exiting, remain unclear.