Characterization of protein factor(s) in rat bronchoalveolar lavage fluid that enhance insulin transport via transcytosis across primary rat alveolar epithelial cell monolayers

Abstract

The aim of this study was to characterize factor(s) in rat bronchoalveolar lavage fluid (BALF) that enhance(s) insulin transport across primary rat alveolar epithelial cell monolayers (RAECM) in primary culture. BALF was concentrated 7.5-fold using the Centricon device and the retentate was used to characterize the factor(s) involved in enhancing apical-to-basolateral transport of intact 125I-insulin across various epithelial cell monolayers. These factor(s) enhanced transport of intact insulin across type II cell-like RAECM (3-fold increase) and type I cell-like RAECM (2-fold increase), but not across Caco-2 or MDCK cell monolayers. The insulin transport-enhancing factor(s) were temperature- and trypsin-sensitive. The mechanism of enhancement did not seem to involve paracellular transport or fluid-phase endocytosis, since fluxes of sodium fluorescein and FITC-dextran (70 kDa) were not affected by the factor(s) in the apical bathing fluid. BALF enhancement of intact 125I-insulin transport was abolished at 4 degrees C and in the presence of monensin, suggesting involvement of transcellular pathways. Sephacryl S-200 purification of BALF retentate, followed by LC-MS/MS, indicated that the high molecular weight (>100 kDa) fractions (which show some homology to alpha-1-inhibitor III, murinoglobulin gamma 2, and pregnancy-zone protein) appear to facilitate transcellular transport of insulin across RAECM.

Fig. 1

Transport of insulin across RAECM-II and -I. Apical-to-basolateral transport of intact insulin, when dosed apically at 5 μg/mL in either KRP or 7.5x BALF across RAECM-II and -I. Data represent mean ± SD (n=3). * = significantly different (p<0.05) compared to KRP across RAECM-II; ‡ = significantly different (p<0.05) compared to KRP across RAECM-I; and † = significantly different (p<0.05) compared to BALF across RAECM-I.

Fig. 2

Transport of insulin across RAECM-II and MDCK and Caco-2 monolayers. Apical-to-basolateral transport of intact insulin, when dosed apically at 5 μg/mL in either KRP or 7.5x BALF retentate or 7.5x BALF filtrate across RAECM-II and MDCK and Caco-2 cell monolayers. Data represent mean ± SD (n=3). * = significantly different (p<0.05) from all others.

Fig. 3

Effects of temperature and monensin on enhanced insulin transport across RAECM-II. Apical-to-basolateral transport of intact insulin, when dosed apically at 5 μg/mL in KRP or 7.5x BALF across RAECM-II at 37 and 4°C after 1 h and with or without 60 μM monensin at 37°C after 1 h. Data represent mean ± SD (n=4). * = significantly different (p<0.05) from all others.

Fig. 4

Elution profile and SDS-PAGE of BALF proteins. BALF was concentrated 20-fold using TFF system, and purified by applying to a Sephacryl S-200 column (1 cm x 40 cm, PBS (pH 7.4)). A: Absorbance profile (◆) at 280 nm of fractions (1 mL) from purified 20x BALF. B: Apical-to-basolateral transport of intact insulin, when dosed apically at 10 μg/mL in fractions #22 and #29, and control (PBS) across RAECM-II. Data represent mean ± SD (n=3). * = significantly different (p<0.05) from both the transport observed under control conditions (PBS) and that observed with dosing in fraction #29. C: BALF protein detected by 7.5% SDS-PAGE. Lane 1: fraction #22 of purified 20x BALF. Lane 2: rainbow molecular weight markers (myosin, 220,000; phosphorylase b, 97,000; bovine serum albumin 66,000).