Characterization of Calu-3 cell monolayers as a model of bronchial epithelial transport: organic cation interaction studies.
BACKGROUND
To fully exploit organic cation transporters for targeted drug delivery in the lung, the use of a readily available and well-characterized tissue culture model and cheap easily detectable substrates is indispensable.


OBJECTIVE
To investigate the suitability of Calu-3 as tissue model for characterizing organic cation permeation across the bronchial cells using a fluorescent dye, 4-(4-(Dimethylamino)styryl)-N-methylpyridinium iodide (4-DI-1-ASP).


METHODS
Substrate uptake, inhibition, and transport were performed to establish active transport mechanism. Organic cation transporter expression was determined with quantitative polymerase chain reaction (qPCR), immune-histochemistry, and fluorescent microscopy.


RESULTS
4-Di-1-ASP uptake in Calu-3 cells was concentration (K(m) = 2.7 ± 0.3 mM, V(max) = 4.6 ± 2.6 nmol/µg protein/30 min), temperature (uptake at 37°C>>4°C), and pH dependent (higher uptake at pH ≥ 7). L-carnitine, verapamil, and corticosterone significantly inhibited its uptake with IC(50) of 28.2, 0.81, and 0.12 mM, respectively. Transport of the dye across the cells was polarized (AP→BL transport was 2.5-fold > BL→AP), saturable (Km = 43.9 ± 3.2) (µM; Vmax =0.0228 ± nmol/cm(2)/sec) and reduced 3-fold by metabolic inhibition. The expression pattern of the organic cation transporters (OCT) and carnitine/organic cation transporter (OCTN) isoforms was: OCT1<<OCT3 <OCTN1<OCTN2; OCT2 was not detected.


CONCLUSIONS
Based on qPCR, immunohistochemistry, uptake and transport data, the Calu-3 cells can be used as a model for not only studying strategies for optimizing the effect of inhaled organic cations, but also for cross-validating newly-developed respiratory cell lines.
Biopharmaceutics and Drug Delivery Laboratory, Dalhousie University, Halifax, NS, Canada.
Chris
C
Macdonald
Di
D
Shao
Angus
A
Oli
Remigius U
RU
Agu
D001693:Biological Transport, Active; D001980:Bronchi / Q000166:cytology / Q000378:metabolism; D002331:Carnitine / Q000008:administration & dosage / Q000494:pharmacology; D002460:Cell Line; D003345:Corticosterone / Q000008:administration & dosage / Q000494:pharmacology; D004847:Epithelial Cells / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000493:pharmacokinetics; D005786:Gene Expression Regulation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D020128:Inhibitory Concentration 50; D008744:Methylamines / Q000493:pharmacokinetics*; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D027701:Organic Cation Transport Proteins / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011726:Pyridinium Compounds / Q000493:pharmacokinetics*; D013696:Temperature; D014700:Verapamil / Q000008:administration & dosage / Q000494:pharmacology
C517027:4-(4-(dimethylamino)styryl)-1-methylpyridinium iodide; D005456:Fluorescent Dyes; D008744:Methylamines; D027701:Organic Cation Transport Proteins; D011726:Pyridinium Compounds; D014700:Verapamil; D002331:Carnitine; D003345:Corticosterone