P-glycoprotein efflux pump expression and activity in Calu-3 cells

Hamilton, Karen O.; Bäckström, Gunilla; Yazdanian, Mehran; Audus, Kenneth L.

doi:10.1002/1520-6017(200105)90:5<647::aid-jps1021>3.0.co;2-g

Cited by 85 publications

(15 citation statements)

References 34 publications

(32 reference statements)

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“…*Significantly different from the Rh123 flux measured in the ab direction (Kennedy and Mangini 2002;Mi et al 2001), the presence of multiple bands for P-gp (especially evident with lower protein quantities). These bands are consistent with the wide and varied reports for P-gp migration (and hence molecular weight) on Western blots (from 150 to 200 kDa; Greiner et al 1999;Demeule et al 2000Demeule et al , 2001Hamilton et al 2001;Axiotis et al 1995) and with the observed single or multiple bands that are probably dependent upon the varied glycosylation products of this protein.…”

Section: Discussionsupporting

confidence: 91%

P-glycoprotein (MDR1) functional activity in human alveolar epithelial cell monolayers

et al. 2006

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The distribution of the P-glycoprotein (P-gp/MDR1) efflux transporter at mucosal barriers has defined it as a functionally important element in limiting drug absorption into the systemic circulation. However, little is known about the distribution and functionality of P-gp/MDR1 in the human lung. Here, the presence of P-gp/MDR1 was investigated immunohistochemically in distal human lung tissue and at mRNA and protein levels in human alveolar epithelial cells (hAEpC) in primary culture. We studied the presence and activity of P-gp/MDR1 in hAEpC monolayers by Western blotting, by immunofluorescence microscopy and by conducting bi-directional transport studies employing a P-gp substrate (rhodamine 123) with and without a P-gp inhibitor (verapamil). The flux of fluorescein sodium was also examined as a paracellular transport marker. Alveolar tissue specimens showed P-gp localised at the luminal membranes of type I pneumocytes. Reverse transcription-polymerase chain reaction revealed the presence of mRNA encoding for P-gp/MDR1 in freshly isolated (i.e. type II) hAEpC and in monolayers of hAEpC cultured for 8 days (i.e. type I-like morphology). At the protein level, P-gp could be detected in hAEpC monolayers after 8 days in culture but not in freshly isolated type II pneumocytes. The flux of rhodamine 123 across hAEpC monolayers on day 8 in culture exhibited net secretion, which disappeared in the presence of verapamil. Fluorescein sodium fluxes showed no distinct directionality. Our findings indicate that P-gp is functionally active in the human alveolar airspace and that hAEpC monolayers might provide a suitable in vitro model for studying P-gp function mechanistically in the distal human lung.

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Section: Discussionsupporting

confidence: 91%

P-glycoprotein (MDR1) functional activity in human alveolar epithelial cell monolayers

et al. 2006

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“…Experiments were carried out with or without the P-gp inhibitor, Verapamil, in both apical and basolateral fluids. A significant net secretion of Rh123 was observed, which was decreased in the presence of the inhibitor P-gp (MDR1), LPR, and caveolin-1 have been reported to be expressed in both the normal human bronchus (LechaptZalcman et al 1997;Rybarova et al 2001;Newman et al 1999) and in cultures of continuously growing bronchial epithelial cells (Ehrhardt et al 2003;Hamilton et al 2001). Immunocytochemistry has indicated that the level of expression of LRP in the CFBE41o-cell line is slightly higher compared with previously reported data obtained in the bronchial epithelial cell line 16HBE14o- (Ehrhardt et al 2003), whereas the expression pattern of caveolin-1 is similar in both cell lines.…”

Section: Discussionmentioning

confidence: 99%

Towards an in vitro model of cystic fibrosis small airway epithelium: characterisation of the human bronchial epithelial cell line CFBE41o-

et al. 2005

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The CFBE41o- cell line was generated by transformation of cystic fibrosis (CF) tracheo-bronchial cells with SV40 and has been reported to be homozygous for the DeltaF508 mutation. A systematic characterisation of these cells, which however, is a pre-requisite for their use as an in vitro model, has not been undertaken so far. Here, we report an assessment of optimal culture conditions, the expression pattern of drug-transport-related proteins and the stability/presence of the CF transmembrane conductance regulator (CFTR) mutation in the gene and gene product over multiple passages. The CFBE41o- cell line was also compared with a wild-type airway epithelial cell line, 16HBE14o-, which served as model for bronchial epithelial cells in situ. The CFBE41o- cell line retains at least some aspects of human CF bronchial epithelial cells, such as the ability to form electrically tight cell layers with functional cell-cell contacts, when grown under immersed (but not air-interfaced) culture conditions. The cell line is homozygous for DeltaF508-CFTR over multiple passages in culture and expresses a number of proteins relevant for pulmonary drug absorption (e.g. P-gp, LRP and caveolin-1). Hence, the CFBE41o- cell line should be useful for studies of CF gene transfer or alternative treatment with small drug molecules and for the gathering of further information about the disease at the cellular level, without the need for primary culture.

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“…46 Western blot analysis however revealed that cell line expresses P-gp. 39,40,42,47 Functional studies in Calu-3 layers using rhodamine 123, 40 cyclosporine 47 and digoxin 9 as P-gp substrates showed a polarised transport in the basolateral to apical (B-A) direction, indicating an apical localisation of the efflux pump on the cell membrane. By contrast, P-gp was localised in immunofluorescence on the basolateral side of the cell layer and flunisolide transport was enhanced in the absorptive direction in the study by Florea et al 39 Those conflicting results can likely be explained by differences in cell culture conditions and by the use of nonspecific P-gp substrates and inhibitors.…”

Section: P-gp In Respiratory Cell Culture Models In Vitromentioning

confidence: 99%

Drug transporters in the lung—do they play a role in the biopharmaceutics of inhaled drugs?

Bosquillon

2010

Journal of Pharmaceutical Sciences

133

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P-glycoprotein efflux pump expression and activity in Calu-3 cells

Cited by 85 publications

References 34 publications

P-glycoprotein (MDR1) functional activity in human alveolar epithelial cell monolayers

P-glycoprotein (MDR1) functional activity in human alveolar epithelial cell monolayers

Towards an in vitro model of cystic fibrosis small airway epithelium: characterisation of the human bronchial epithelial cell line CFBE41o-

Drug transporters in the lung—do they play a role in the biopharmaceutics of inhaled drugs?

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