Effect of cigarette smoke extract on P-glycoprotein function in primary cultured and newly developed alveolar epithelial cells.

The effect of cigarette smoke extract (CSE) on P-glycoprotein (P-gp) function in the distal lung is unclear. In this study, we first examined the expression and function of P-gp and the effect of CSE in rat primary cultured alveolar epithelial cells. The expression of P-gp protein was observed in type I-like cells, but not in type II cells. In type I-like cells, rhodamine 123 (Rho123) accumulation was enhanced by various P-gp inhibitors such as verapamil and cyclosporine A. In addition, the expression of P-gp mRNAs, mdr1a and mdr1b, as well as P-gp activity increased along with the transdifferentiation. When type I-like cells were co-incubated with CSE, P-gp activity was suppressed. Next, we attempted to clarify the effect of CSE on P-gp function in human-derived cultured alveolar epithelial cells. For this purpose, we isolated an A549 clone (A549/P-gp) expressing P-gp, because P-gp expression in native A549 cells was negligible. In A549/P-gp cells, P-gp was functionally expressed, and the inhibitory effect of CSE on P-gp was observed. These results suggested that smoking would directly suppress P-gp activity, and that A549/P-gp cell line should be a useful model to further study the effect of xenobiotics on P-gp function in the alveolar epithelial cells.

Enhancing effect of poly(amino acid)s on albumin uptake in human lung epithelial A549 cells.

The clearance of albumin from the alveolar space is a critical process in the recovery from edema. In this study, we investigated the effect of poly(amino acid)s such as poly-l-ornithine (PLO) on albumin uptake in the cultured lung epithelial cell line A549. FITC-albumin uptake as well as cell surface binding was markedly stimulated by co-incubation with PLO, and there was a good correlation between them. After being taken up by A549 cells, FITC-albumin was predominantly targeted to lysosomes. Interestingly, pretreatment of A549 cells with PLO further stimulated FITC-albumin uptake, even in the absence of PLO in the uptake buffer. FITC-albumin uptake in the presence of PLO was inhibited by a metabolic inhibitor, clathrin-mediated endocytosis inhibitors, and a macropinocytosis inhibitor, indicating the involvement of clathrin-mediated endocytosis and/or macropinocytosis. The effect of PLO on FITC-albumin clearance was also examined in an in vivo pulmonary administration method in rats, and co-administration of PLO enhanced fluorescence elimination from the lungs. These findings suggest that pulmonary administration of poly(amino acid)s such as PLO is a possible strategy for enhancing albumin clearance from the alveolar space, and thereby facilitating the recovery from pulmonary edema.