Evidence of P-glycoprotein mediated apical to basolateral transport of flunisolide in human broncho-tracheal epithelial cells (Calu-3).
1. Transepithelial transport of flunisolide was studied in reconstituted cell monolayers of Calu-3, LLC-PK1 and the MDR1-P-glycoprotein transfected LLC-MDR1 cells. 2. Flunisolide transport was polarized in the apical (ap) to basolateral (bl) direction in Calu-3 cells and was demonstrated to be ATP-dependent. In LLC-MDR1 cells, flunisolide was transported in the bl to ap direction and showed no polarization in LLC-PK1 cells. 3. Non-specific inhibition of cellular metabolism at low temperature (4 degrees C) or by 2-deoxy-D-glucose (2-d-glu) and sodium azide (NaN(3)) abolished the polarized transport. Polarized flunisolide transport was also inhibited by the specific Pgp inhibitors verapamil, SDZ PSC 833 and LY335979. 4. Under all experimental conditions and in the presence of all used inhibitors, no decrease in the TransEpithelial Electrical Resistance (TEER) values was detected. From all inhibitors used, only the general metabolism inhibitors 2-deoxy-D-glucose and NaN(3), decreased the survival of Calu-3 cells. 5. Western blotting analysis and confocal laser scanning microscopy demonstrated the presence of MDR1-Pgp at mainly the basolateral side of the plasma membrane in Calu-3 cells and at the apical side in LLC-MDR1 cells. Mass spectroscopy studies demonstrated that flunisolide is transported unmetabolized across Calu-3 cells. 6. In conclusion, these results show that the active ap to bl transport of flunisolide across Calu-3 cells is facilitated by MDR1-Pgp located in the basolateral plasma membrane.
Florea
B I
BI
Division of Pharmaceutical Technology, Leiden/Amsterdam Center for Drug Research, Leiden University, 2300 RA Leiden, The Netherlands.
van der Sandt
I C
IC
Schrier
S M
SM
Kooiman
K
K
Deryckere
K
K
de Boer
A G
AG
Junginger
H E
HE
Borchard
G
G
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D001980:Bronchi / Q000166:cytology / Q000378:metabolism; D002460:Cell Line; D016764:Cell Polarity; D002470:Cell Survival; D003524:Cyclosporins / Q000494:pharmacology; D003847:Deoxyglucose / Q000494:pharmacology; D003986:Dibenzocycloheptenes / Q000494:pharmacology; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D005446:Fluocinolone Acetonide / Q000031:analogs & derivatives* / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D013058:Mass Spectrometry; D018613:Microscopy, Confocal; D011804:Quinolines / Q000494:pharmacology; D019810:Sodium Azide / Q000494:pharmacology; D013696:Temperature; D013997:Time Factors; D014132:Trachea / Q000166:cytology / Q000378:metabolism; D014700:Verapamil / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D003524:Cyclosporins; D003986:Dibenzocycloheptenes; D011804:Quinolines; D005446:Fluocinolone Acetonide; C007734:flunisolide; C095179:zosuquidar trihydrochloride; D019810:Sodium Azide; D003847:Deoxyglucose; D014700:Verapamil; C070272:valspodar