RESUMO
Defective solute carrier (SLC) transporters are responsible for neurotransmitter dysregulation, resulting in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). We provided the role and kinetic parameters of transporters such as ASCTs, Taut, LAT1, CAT1, MCTs, OCTNs, CHT, and CTL1, which are mainly responsible for the transport of essential nutrients, acidic, and basic drugs in blood-brain barrier (BBB) and motor neuron disease. The affinity for LAT1 was higher in the BBB than in the ALS model cell line, whereas the capacity was higher in the NSC-34 cell lines than in the BBB. Affinity for MCTs was lower in the BBB than in the NSC-34 cell lines. CHT in BBB showed two affinity sites, whereas no expression was observed in ALS cell lines. CTL1 was the main transporter for choline in ALS cell lines. The half maximal inhibitory concentration (IC50) analysis of [3H]choline uptake indicated that choline is sensitive in TR-BBB cells, whereas amiloride is most sensitive in ALS cell lines. Knowledge of the transport systems in the BBB and motor neurons will help to deliver drugs to the brain and develop the therapeutic strategy for treating CNS and neurological diseases.
RESUMO
Choline is essential for the synthesis of the major membrane phospholipid phosphatidylcholine and the neurotransmitter acetylcholine (ACh). Elevated levels of choline and up-regulated choline kinase activity have been detected in cancer cells. Thus, the intracellular accumulation of choline through choline transporters is the rate-limiting step in phospholipid metabolism and a prerequisite for cancer cell proliferation. However, the uptake system for choline and the functional expression of choline transporters in lung cancer cells are poorly understood. We examined the molecular and functional characterization of choline uptake in the small cell lung carcinoma cell line NCI-H69. 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 choline uptake under the Na(+)-free conditions was inhibited by dimethylamiloride (DMA), a Na(+)/H(+) exchanger (NHE) inhibitor. Various organic cations and the choline analog hemicholinium-3 (HC-3) inhibited the choline uptake and cell viability. A correlation analysis of the potencies of organic cations for the inhibition of choline uptake and cell viability showed a strong correlation (R=0.8077). RT-PCR revealed that choline transporter-like protein 1 (CTL1) mRNA and NHE1 are mainly expressed. HC-3 and CTL1 siRNA inhibited choline uptake and cell viability, and increased caspase-3/7 activity. The conversion of choline to ACh was confirmed, and this conversion was enhanced under Na(+)-free conditions, which in turn was sensitive to HC-3. These results indicate that choline uptake through CTL1 is used for ACh synthesis. Both an acetylcholinesterase inhibitor (eserine) and a butyrylcholinesterase inhibitor (ethopropazine) increased cell proliferation, and these effects were inhibited by 4-DAMP, a mAChR3 antagonist. We conclude that NCI-H69 cells express the choline transporter CTL1 which uses a directed H(+) gradient as a driving force, and its transport functions in co-operation with NHE1. This system primarily supplies choline for the synthesis of ACh and secretes ACh to act as an autocrine/paracrine growth factor, and the functional inhibition of CTL1 could promote apoptotic cell death. Identification of this new CTL1-mediated choline transport system provides a potential new target for therapeutic intervention.