Abstract
In human, OCTN2 (SLC22A5) and ATB0,+ (SLC6A14) transporters mediate the uptake of L-carnitine, essential for the transport of fatty acids into mitochondria and the subsequent degradation by β-oxidation. Aim of the present study was to characterize L-carnitine transport in EpiAirway™, a 3D organotypic in vitro model of primary human tracheal-bronchial epithelial cells that form a fully differentiated, pseudostratified columnar epithelium at air-liquid interface (ALI) condition. In parallel, Calu-3 monolayers grown at ALI for different times (8d or 21d of culture) were used as comparison. OCTN2 transporter was equally expressed in both models and functional at the basolateral side. ATB0,+ was, instead, highly expressed and active on the apical membrane of EpiAirway™ and only in early-cultures of Calu-3 (8d but not 21d ALI). In both cell models, L-carnitine uptake on the apical side was significantly inhibited by the bronchodilators glycopyrrolate and tiotropium, that hence can be considered substrates of ATB0,+; ipratropium was instead effective on the basolateral side, indicating its interaction with OCTN2. Inflammatory stimuli, such as LPS or TNFα, caused an induction of SLC6A14/ATB0,+ expression in Calu-3 cells, along with a 2-fold increase of L-carnitine uptake only at the apical side; on the contrary SLC22A5/OCTN2 was not affected. As both OCTN2 and ATB0,+, beyond transporting L-carnitine, have a significant potential as delivery systems for drugs, the identification of these transporters in EpiAirway™ can open new fields of investigation in the study of drug inhalation and pulmonary delivery.
Highlights
L-Carnitine (β-hydroxy-γ-trimethylaminobutyrate) is a small, highly polar zwitterionic molecule, essential in the transfer of activated long-chain fatty acids across the inner mitochondrial membrane, for their degradation through β-oxidation [1]
In a previous contribution we addressed L-carnitine transport in undifferentiated human airway epithelial cells and we demonstrated that OCTN2 is the only transporter active in A549 and BEAS-2B cells, while both OCTN2 and ATB0,+ are operative in Calu-3 and NCl-H441 [12]
L- carnitine transport measured at the apical side was higher than in Calu-3 cultured for 21d and comparable to that of EpiAirwayTM, with the same inhibition pattern; data obtained at the basolateral side overlapped with those of primary normal cells (Fig 2, panel B)
Summary
L-Carnitine (β-hydroxy-γ-trimethylaminobutyrate) is a small, highly polar zwitterionic molecule, essential in the transfer of activated long-chain fatty acids across the inner mitochondrial membrane (the so-called “carnitine shuttle”), for their degradation through β-oxidation [1]. OCTN2 expression has been demonstrated in liver, heart, testis, skeletal muscle, lung and brain, sustaining a role for the transporter in the systemic distribution of carnitine [8]; the transporter has been found in human macrophages, where it has been identified as a novel target gene of the mTOR-STAT3 axis [9]. In polarized epithelia, such as intestine and kidney, OCTN2 is located in the apical membrane of the cells [10], where it is involved in intestinal absorption and renal reabsorption. OCTN2 has been included in the catalog of transporters responsible for the interaction with drugs by the International Transporter Consortium (ITC) [16]; its involvement in the transport of bronchodilators has been suggested [17,18]
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