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Ingoglia F.,University of Parma | Visigalli R.,University of Parma | Rotoli B.M.,University of Parma | Barilli A.,University of Parma | And 3 more authors.
Biochimica et Biophysica Acta - Biomembranes | Year: 2015

Organic cation transporters (OCT1-3) mediate the transport of organic cations including inhaled drugs across the cell membrane, although their role in lung epithelium hasn't been well understood yet. We address here the expression and functional activity of OCT1-3 in human airway epithelial cells A549, Calu-3 and NCl-H441. Kinetic and inhibition analyses, employing [3H]1-methyl-4-phenylpyridinium (MPP+) as substrate, and the compounds quinidine, prostaglandine E2 (PGE2) and corticosterone as preferential inhibitors of OCT1, OCT2, and OCT3, respectively, have been performed. A549 cells present a robust MPP+ uptake mediated by one high-affinity component (Km ~ 50 μM) which is identifiable with OCT3. Corticosterone, indeed, completely inhibits MPP+ transport, while quinidine and PGE2 are inactive and SLC22A3/OCT3 silencing with siRNA markedly lowers MPP+ uptake. Conversely, Calu-3 exhibits both a high (Km < 20 μM) and a low affinity (Km > 0.6 mM) transport components, referable to OCT3 and OCT1, respectively, as demonstrated by the inhibition analysis performed at proper substrate concentrations and confirmed by the use of specific siRNA. These transporters are active also when cells are grown under air-liquid interface (ALI) conditions. Only a very modest saturable MPP+ uptake is measurable in NCl-H441 cells and the inhibitory effect of quinidine points to OCT1 as the subtype functionally involved in this model. Finally, the characterization of MPP+ transport in human bronchial BEAS-2B cells suggests that OCT1 and OCT3 are operative. These findings could help to identify in vitro models to be employed for studies concerning the specific involvement of each transporter in drug transportation. © 2015 Elsevier B.V. All rights reserved. Source


Ingoglia F.,University of Parma | Visigalli R.,University of Parma | Rotoli B.M.,University of Parma | Barilli A.,University of Parma | And 3 more authors.
Biochimica et Biophysica Acta - Biomembranes | Year: 2016

Carnitine plays a physiologically important role in the β-oxidation of fatty acids, facilitating the transport of long-chain fatty acids across the inner mitochondrial membrane. Distribution of carnitine within the body tissues is mainly performed by novel organic cation transporter (OCTN) family, including the isoforms OCTN1 (SLC22A4) and OCTN2 (SLC22A5) expressed in human. We performed here a characterization of carnitine transport in human airway epithelial cells A549, Calu-3, NCl-H441, and BEAS-2B, by means of an integrated approach combining data of mRNA/protein expression with the kinetic and inhibition analyses of L-[3H]carnitine transport. Carnitine uptake was strictly Na+-dependent in all cell models. In A549 and BEAS-2B cells, carnitine uptake was mediated by one high-affinity component (Km < 2 μM) identifiable with OCTN2. In both these cell models, indeed, carnitine uptake was maximally inhibited by betaine and strongly reduced by SLC22A5/OCTN2 silencing. Conversely, Calu-3 and NCl-H441 exhibited both a high (Km ~ 20 μM) and a low affinity (Km > 1 mM) transport component. While the high affinity component is identifiable with OCTN2, the low affinity uptake is mediated by ATB0,+, a Na+, and Cl--coupled transport system for neutral and cationic amino acids, as demonstrated by the inhibition by leucine and arginine, as well as by SLC6A14/ATB0,+ silencing. The presence of this transporter leads to a massive accumulation of carnitine inside the cells and may be of peculiar relevance in pathologic conditions of carnitine deficiency, such as those associated to OCTN2 defects. © 2015 Elsevier B.V. Source

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