Horseradish Peroxidase Transport Across Rat Alveolar Epithelial Cell Monolayers

Abstract

Purpose. To evaluate the transport characteristics of horseradish peroxidase (HRP, a nonspecific fluid-phase endocytosis marker) across an in vitro model of tight (> 2,000 ohm-cm²) rat alveolar epithelial cell monolayers grown on tissue culture-treated polycarbonate filters.

Methods. Unidirectional HRP fluxes were estimated from the appearance rate of HRP in the receiver fluid following instillation in the donor fluid as a function of donor [HRP] and temperature. Molecular species present in either bathing fluid were determined at the end of flux experiments using fluorescein isothiocyanate (FITC)-labeled HRP by gel permeation chromatography. Cell-associated HRP activity at the end of the transport experiment was determined, as were the rates of recycling and transcellular movement of HRP. An enzymatic assay was used to quantify HRP activity in the bathing fluid and cells.

Results. Unidirectional HRP fluxes were symmetric and increased linearly with up to 50 µM donor [HRP]. The apparent permeability coefficient of HRP was reduced by 3.5 times upon lowering the temperature from 37 to 4°C. About 50% of the FITC-labeled species present in either receiver fluid was intact HRP. Cell-associated HRP estimated from apical HRP incubation was about 4 times greater than that from basolateral incubation. Recycling into apical fluid of cell-associated HRP following apical incubation occurred rapidly with a half-time (T_1/2) of ~5 min, reaching a plateau at ~67% of the initial cell-associated HRP, while transcellular movement of HRP (into basolateral fluid) took place with a T_1/2 of ~20 min, attaining a steady-state at ~13% of the initial cell-associated HRP. Basolateral recycling of HRP was also rapid (T_1/2 = ~5 min) reaching a steady-state at ~35% of the initial basolaterally-bound HRP. Transcellular movement of HRP following basolateral incubation was slower (T_1/2 = ~70 min), leveling off at 50% of the initial cell-associated HRP.

Conclusions. HRP appears to be transported relatively intact (~50%) across rat alveolar epithelial barrier via nonspecific fluid-phase endocytosis. The transepithelial pinocytotic rate of alveolar epithelial cells is estimated to be about 25 nL/cm²/h.