Characterization of protein factor(s) in rat bronchoalveolar lavage fluid that enhance insulin transport via transcytosis across primary rat alveolar epithelial cell monolayers.
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.
Bahhady
Rana
R
Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90089-9121, USA.
Kim
Kwang-Jin
KJ
Borok
Zea
Z
Crandall
Edward D
ED
Shen
Wei-Chiang
WC
D000042:Absorption; D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects; D001992:Bronchoalveolar Lavage Fluid / Q000737:chemistry*; D018938:Caco-2 Cells; D002460:Cell Line; D002478:Cells, Cultured; D030541:Databases, Genetic; D003911:Dextrans; D004285:Dogs; D004705:Endocytosis / Q000187:drug effects*; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives; D005456:Fluorescent Dyes; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D013058:Mass Spectrometry; D010455:Peptides / Q000302:isolation & purification / Q000494:pharmacology*; D011650:Pulmonary Alveoli / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013268:Stimulation, Chemical; D013696:Temperature; D014357:Trypsin / Q000494:pharmacology
D003911:Dextrans; D005456:Fluorescent Dyes; D007328:Insulin; D010455:Peptides; C015219:fluorescein isothiocyanate dextran; D014357:Trypsin; D016650:Fluorescein-5-isothiocyanate