Molecular and functional characterization of choline transporter in human colon carcinoma HT-29 cells |
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Authors: | Hironobu Kouji Masato Inazu Tomoko Yamada Tatsuya Aoki |
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Affiliation: | a Department of Pharmacology, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan b Department of Preventive Medicine (SONOKO), Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan c Third Department of Surgery, Tokyo Medical University, 6-7-1 Nishi-shinjuku, Shinjuku-ku, Tokyo 160-0023, Japan |
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Abstract: | We examined the molecular and functional characterization of choline uptake in human colon carcinomas using the cell line HT-29. Furthermore, we explored the possible correlation between choline uptake and cell proliferation. Choline uptake was saturable and mediated by a single transport system. Interestingly, removal of Na+ from the uptake buffer strongly enhanced choline uptake. This increase in component of choline uptake under Na+-free conditions was inhibited by a Na+/H+ exchanger 1 (NHE1) inhibitor. Collapse of the plasma-membrane H+ electrochemical gradient by a protonophore inhibited choline uptake. Choline uptake was inhibited by the choline analogue hemicholinium-3 (HC-3) and various organic cations, and was significantly decreased by acidification of the extracellular medium and by intracellular alkalinization. Real-time PCR revealed that choline transporter-like protein 1 (CTL1), CTL2, CTL4 and NHE1 mRNA are mainly expressed in HT-29 cells. Western blot and immunocytochemical analysis indicated that CTL1 protein was expressed in plasma membrane. The biochemical and pharmacological data indicated that CTL1 is functionally expressed in HT-29 cells and is responsible for choline uptake in these cells. We conclude that choline transporters, especially CTL1, use a directed H+ gradient as a driving force, and its transport functions in co-operation with NHE1. Finally, cell proliferation was inhibited by HC-3 and tetrahexylammonium chloride (THA), which strongly inhibits choline uptake. Identification of this novel CTL1-mediated choline uptake system provides a potential new target for therapeutic intervention. |
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Keywords: | Choline Transporter Na+/H+ exchanger Colon carcinoma Cell proliferation |
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