HORSERADISH-PEROXIDASE TRANSPORT ACROSS RAT ALVEOLAR EPITHELIAL-CELL MONOLAYERS

Purpose. To evaluate the transport characteristics of horseradish pero xidase (HRP, a nonspecific fluid-phase endocytosis marker) across an i n vitro model of tight (>2,000 ohm-cm(2)) rat alveolar epithelial cell monolayers grown on tissue culture-treated polycarbonate filters. Met hods. Unidirectional HRP fluxes were estimated from the appearance rat e of HRP in the receiver fluid following instillation in the donor flu id as a function of donor [HRP] and temperature. Molecular species pre sent in either bathing fluid were determined at the end of flux experi ments using fluorescein isothiocyanate (FITC)-labeled HRP by gel perme ation chromatography. Cell-associated HRP activity at the end of the t ransport experiment was determined, as were the rates of recycling and transcellular movement of HRP. An enzymatic assay was used to quantif y HRP activity in the bathing fluid and cells. Results. Unidirectional HRP fluxes were symmetric and increased linearly with up to 50 mu M d onor [HRP]. The apparent permeability coefficient of HRP was reduced b y 3.5 times upon lowering the temperature from 37 to 4 degrees C. Abou t 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. Recyc ling into apical fluid of cell-associated HRP following apical incubat ion occurred rapidly with a half-time (T1/2) of similar to 5 min, reac hing a plateau at similar to 67% of the initial cell-associated HRP, w hile transcellular movement of HRP (into basolateral fluid) took place with a T-1/2 of similar to 20 min, attaining a steady-state at simila r to 13% of the initial cell-associated HRP. Basolateral recycling of HRP was also rapid (T-1/2 = similar to 5 min) reaching a steady-state at similar to 35% of the initial basolaterally-bound HRP. Transcellula r movement of HRP following basolateral incubation was slower (T-1/2 = similar to 70 min), leveling off at 50% of the initial cell-associate d HRP. Conclusions. HRP appears to be transported relatively intact (s imilar to 50%) across rat alveolar epithelial barrier via nonspecific fluid-phase endocytosis. The transepithelial pinocytotic rate of alveo lar epithelial cells is estimated to be about 25 nL/cm(2)/h.