PURPOSE: To characterize factor(s) contained in apical medium of primary cultured rat alveolar epithelial type II cell-like monolayers (RAECM-II) that enhance insulin absorption across alveolar epithelial cells. MATERIALS AND METHODS: Primary rat alveolar epithelial cell monolayers cultured on Transwells in the presence and absence of 10 ng/ml keratinocyte growth factor for 6 days were dosed from the apical compartment with radiolabeled insulin in: newborn bovine serum-containing medium (SM), conditioned medium from apical compartment of rat alveolar epithelial type I cell-like monolayers (RAECM-I) (CMI), or conditioned medium from apical compartment of RAECM-II (CMII). At the end of 2 h incubation, basolateral medium was collected and amounts of transported radiolabeled insulin were determined using a gamma counter. In order to determine the molecular size range of the enhancing factor(s), CMII was centrifuged in 50 kDa molecular weight cut-off Centricon tubes, and both retentate and filtrate were used as separate dosing solutions. Heat denaturation and ammonium sulphate precipitation were used to determine if the involved factor(s) represent proteins or other smaller soluble factors. Transalveolar transport rates of a paracellular marker, (14)C-mannitol, and fluid-phase marker, horseradish peroxidase, were determined in the presence and absence of the factors. Effects of temperature (4, 16 and 37 degrees C) on radiolabeled insulin fluxes were also measured. RESULTS: Conditioned medium obtained from the apical compartment of RAECM-II, CMII, increased transport of insulin across the monolayers when compared to SM or CMI. The enhancing effect of CMII was retained in the precipitate following ammonium sulfate treatment and in the retentate after Centricon filtration. The enhancing effect of CMII was significantly decreased when heated at 80 degrees C for 15 min. CMII did not affect the transport of (14)C-mannitol or HRP, while its effect on insulin transport was decreased by 87% when temperature was lowered to 4 degrees C from 37 degrees C. CONCLUSIONS: Conditioned medium from type II cell-like monolayer cultures appears to contain protein factor(s) which seem to be involved in facilitating active transcellular transport of insulin across primary cultured RAECM-II.
PURPOSE: To characterize factor(s) contained in apical medium of primary cultured rat alveolar epithelial type II cell-like monolayers (RAECM-II) that enhance insulin absorption across alveolar epithelial cells. MATERIALS AND METHODS: Primary rat alveolar epithelial cell monolayers cultured on Transwells in the presence and absence of 10 ng/ml keratinocyte growth factor for 6 days were dosed from the apical compartment with radiolabeled insulin in: newborn bovine serum-containing medium (SM), conditioned medium from apical compartment of rat alveolar epithelial type I cell-like monolayers (RAECM-I) (CMI), or conditioned medium from apical compartment of RAECM-II (CMII). At the end of 2 h incubation, basolateral medium was collected and amounts of transported radiolabeled insulin were determined using a gamma counter. In order to determine the molecular size range of the enhancing factor(s), CMII was centrifuged in 50 kDa molecular weight cut-off Centricon tubes, and both retentate and filtrate were used as separate dosing solutions. Heat denaturation and ammonium sulphate precipitation were used to determine if the involved factor(s) represent proteins or other smaller soluble factors. Transalveolar transport rates of a paracellular marker, (14)C-mannitol, and fluid-phase marker, horseradish peroxidase, were determined in the presence and absence of the factors. Effects of temperature (4, 16 and 37 degrees C) on radiolabeled insulin fluxes were also measured. RESULTS: Conditioned medium obtained from the apical compartment of RAECM-II, CMII, increased transport of insulin across the monolayers when compared to SM or CMI. The enhancing effect of CMII was retained in the precipitate following ammonium sulfate treatment and in the retentate after Centricon filtration. The enhancing effect of CMII was significantly decreased when heated at 80 degrees C for 15 min. CMII did not affect the transport of (14)C-mannitol or HRP, while its effect on insulin transport was decreased by 87% when temperature was lowered to 4 degrees C from 37 degrees C. CONCLUSIONS: Conditioned medium from type II cell-like monolayer cultures appears to contain protein factor(s) which seem to be involved in facilitating active transcellular transport of insulin across primary cultured RAECM-II.