Functional and molecular analysis of Organic Cation Transporters (OCTs and OCTNs) in human airway epithelial cells and macrophages

Abstract

FIRST ABSTRACT: 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.

SECOND ABSTRACT: 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 expressionwith 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) identifiablewith OCTN2. In both these cellmodels, 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 lowaffinity (Km > 1mM) 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, aswell 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.

THIRD ABSTRACT: L-Carnitine, in addition to playing a fundamental role in the b-oxidation of fatty acids, has been recently identified as a modulator of immune function, although the mechanisms that underlie this role remain to be clarified. In this study, we addressed the modulation of L-carnitine transport and expression of related transporters during differentiation of human monocytes to macrophages. Whereas monocytes display a modest uptake of L-carnitine, GM-CSF–induced differentiation massively increased the saturable Na+-dependent uptake of L-carnitine. Kinetic and inhibition analyses demonstrate that in macrophage L-carnitine transport is mediated by a high-affinity component (Km ;4 mM) that is identifiable with the operation of OCTN2 transporter and a lowaffinity component (Km . 10 mM) that is identifiable with system A for neutral amino acids. Consistently, both SLC22A5/OCTN2 and SLC38A2/SNAT2 are induced during the differentiation of monocytes to macrophages at gene and protein levels. Elucidation of GM-CSF signaling demonstrates that the cytokine causes the activation of mTOR kinase, leading to the phosphorylation and activation of STAT3, which, in turn, is responsible for OCTN2 transcription. SLC22A5/OCTN2 therefore emerges as a novel member of the set of genes markers of macrophage differentiation.