Author: Ugwu Malachy Chigozie
Department: Pharmaceutical Microbiology and Biotechnology
Affiliation: Nnamdi Azikiwe University Awka

To fully exploit membrane 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 are indispensable. Human multidrug and toxin extrusion proteins (hMATEs) are organic cation /H+ antiporters, which have been identified to be involved in the secretory transport of various organic cations across the cell membranes. Fluorescent dyes are important class of sub-cellular probes which allow the examination of cellular processes in real-time with minimal impact upon the processes. They are, therefore widely used in bio-analysis. One of primary objectives of the present study is therefore to screen for protein expression, localization and transport characteristics of hMATEs in Calu-3 cell line using a fluorescent cationic dye: 4,6 di-amidino-2-phenyhlindole (DAPI). The study equally evaluated the suitability and relevance of four fluorescent dyes as non-radioactive probes for characterizing organic cation transporters using fluorometry. Also the suitability of human nasal epithelial (HNE_248 ) cell as a physiologically adequate and relevant cell culture model for investigating the interactions of inhaled drugs with the organic cations transporters (OCT) in the respiratory epithelium was evaluated. Substrate uptake, inhibition, and transport were performed to establish active transport mechanisms. MATEs and OCTs genes expression were also determined with quantitative polymerase chain reaction (qPCR). PCR studies revealed the expression of hMATE1 gene in Calu-3 cells. The uptake of DAPI followed Michealis-Menteen kinetics. It was concentration (Vmax=6.663  2.610M/mg protein/ 5mins, Km =2252  1632M), temperature (uptake at 37 C4C) and pH (highest uptake at an alkaline pH of 8)-dependent. Cimetidine, famotidine, and propidium iodide (PI ) significantly inhibited DAPI uptake with IC50 of 204.1M, 287.2M and 324.6M respectively while probenecid had no observable inhibition on the dye’s uptake. Transport of the dye across the cells was polarized {apicalbasolateral (APBL) transport was 3fold  BLAP), saturable (Km = 44.76  39.41 μM, Vmax = 0.0647  0.01451 nmol/cm2/s.}. Intracellular uptake of the cell-permeant fluorescent dyes was demonstrated to be enhanced in Chinese hamster ovary (CHO) cells stably expressing hOCT2 when compared with control CHO-wild cells. This increase of dyes influxes was found to be concentration –dependent and a saturable carriermediated process (except for rhodamine 123) .Their kinetic parameters were: Km = 414.6  141M, Vmax=0.0219  0.005 M/mg protein/ 7 mins (4-Di-1-ASP), Km = 72.63  12.02M, Vmax=0.131 0.007M/mg protein/ 7 mins (amiloride) and Km = 82.47  29.15M, Vmax=0.952  0.17 M/mg protein/ 7 mins (rhodamine 6G) respectively. Their uptake was pH -dependent (higher uptake at alkaline pH range of 8.0 – 8.5 and the uptake of dyes was significantly (P < 0.05) decreased in acidic conditions in all the transfected cells.The uptake was reduced by various known inhibitors of hOCT2 activity. Quinine, verapamil, corticosterone and quinidine markedly reduced the uptakes of the dyes across board. RT-PCR analysis showed high mRNA levels for two polyspecific organic cation/carnitine transporters, OCT3 and OCTN2, in the human nasal epithelia of adenocystic fibrotic (HNE-248) cells. The uptake of the dye was saturable/ concentration- dependent and with a Km of 2640 ± 224 M and a Vmax of 0 .0753 ± 0.008 M/ mg protein/15 min. Significantly Statistical difference was observed between total uptake at 37°C and 4°C (p < 0.05). The intracellular accumulation of the compound was inhibited by organic cation transporters (OCTs) and carnitine/organic cation transporter (OCTNs) inhibitors {quinine,quinidine &verapamil with IC50s of 226,1306 and 1131M respectively.}. The uptake mechanism was found to be independent of sodium and glucose.

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