Pretreatment Effect of Inflammatory Stimuli and Characteristics of Tryptophan Transport on Brain Capillary Endothelial (TR-BBB) and Motor Neuron Like (NSC-34) Cell Lines
Abstract
:1. Introduction
2. Materials and Methods
2.1. Materials
2.2. Cell Culture
2.3. [3H]l-Tryptophan Uptake by NSC-34 and TR-BBB Cells
2.4. Transfection with rLAT1 and rCAT1 Small Interfering RNA (siRNA)
2.5. Pretreatment Effect of Pro-Inflammatory Cytokines on the Uptake of [3H]l-Tryptophan
2.6. Data Analysis
3. Results
3.1. Characteristics of [3H]l-Tryptophan Transport in TR-BBB Cell Lines
3.2. LAT1 and CAT1 siRNA Transfection Study in TR-BBB Cell Lines
3.3. Effect of Pharmacological Drugs on the Uptake of [3H]l-Tryptophan by TR-BBB Cell Lines
3.4. Pretreatment Effect of Inflammatory Stimuli on [3H]l-Tryptophan Uptake by TR-BBB Cell Lines
3.5. Changes in [3H]l-Tryptophan Uptake by NSC-34 Cell Lines.
3.6. Effect of LAT1 siRNA Transfection on NSC-34 Cell Lines
3.7. Pretreatment Effect of Inflammatory Stimuli on [3H]l-Tryptophan Uptake by NSC-34 Cell Lines
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
ALS | Amyotrophic lateral sclerosis |
AD | Alzheimer’s disease |
5-HT | 5-Hydroxytryptamine; serotonin |
BBB | Blood-brain barrier |
BCH | 2-Aminobicyclo heptane-2-carboxylic acid |
CAT1 | Cationic amino acid transporter 1 |
CNS | Central nervous system |
ECF | Extracellular fluid |
IDO | Indoleamine 2, 3-dioxygenase |
IF-γ | Interferon-γ |
LNAA | Large neutral amino acids |
LAT1 | l-type amino acid transporter 1 |
MeAIB | α-Methylaminoisobutyric acid |
NAA | Neutral amino acids |
NMG | N-methyl d-glucamine |
ND | Neurodegenerative disease |
PD | Parkinson’s disease |
RT-PCR | Real-time polymerase chain reaction |
TDO | Tryptophan 2, 3-dioxygenase |
TNF-α | Tumor necrosis factor α |
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Cell lines | WT (NSC-34 hSOD1WT) | MT (NSC-34 hSOD1G93A) | TR-BBB |
---|---|---|---|
Km (µM) | 33.1 ± 1.2 | 101 ± 2 | 121 ± 11 |
Vmax (nmol/mg protein/min) | 2.10 ± 0.27 | 4.07 ± 0.42 | 4.69 ± 0.20 |
Kd (µL/mg protein/min) | 0.805 | 1.46 | 3.66 |
Vmax/Km (µL/mg protein/min) | 0.063 | 0.041 | 0.704 |
Compounds | Concentration | Relative Uptake |
---|---|---|
mM | (% of Control) | |
Control | 100 ± 4 | |
+ BCH | 2 | 21.0 ± 1.3 *** |
+ d-Methionine | 2 | 30.8 ± 3.4 *** |
+ Methylmaleimide | 2 | 60.4 ± 1.2 *** |
+ Homoarginine | 2 | 88.4 ± 3.4 |
+ MeAIB | 2 | 96.8 ± 2.7 |
+ Harmaline | 2 | 108 ± 5 |
Compounds | Concentration | Relative Uptake |
---|---|---|
mM | (% of Control) | |
Control | 100 ± 4 | |
+ Tryptophan | 2 | 7.27 ± 0.9 *** |
+ Phenylalanine | 2 | 8.68 ± 0.3 *** |
+ Tyrosine | 2 | 14.1 ± 1.0 *** |
+ Methionine | 2 | 10.3 ± 7.4 *** |
+ Glutamine | 2 | 19.4 ± 1.2 *** |
+ Citrulline | 2 | 27.3 ± 1.4 *** |
+ Valine | 2 | 38.4 ± 1.8 *** |
+ Cysteine | 2 | 41.3 ± 1.3 *** |
+ Lysine | 2 | 53.1 ± 4.1 *** |
+ Serine | 2 | 58.6 ± 1.5 *** |
+ Aspartic acid | 2 | 90.2 ± 3.4 |
+ Glutamic acid | 2 | 97.4 ± 3.8 |
+ Taurine | 2 | 102 ± 5 |
+ Choline | 2 | 110 ± 4 |
Drugs | Concentration | Relative Uptake |
---|---|---|
mM | (% of Control) | |
Control | 100 ± 4 | |
+ Verapamil | 0.5 | 15.0 ± 0.9 *** |
+ l-Dopa | 0.5 | 18.6 ± 1.6 *** |
+ Gabapentin | 0.5 | 46.0 ± 0.5 *** |
+ Baclofen | 2 | 73.1 ± 5.3 ** |
+ Quinidine | 0.5 | 78.5 ± 5 ** |
+ Dopamine | 2 | 94.4 ± 3.3 |
+ Donepezil | 2 | 97.1 ± 7.6 |
+ Riluzole | 0.5 | 102 ± 8 |
+ Amiloride | 2 | 111 ± 6 |
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Gyawali, A.; Kang, Y.-S. Pretreatment Effect of Inflammatory Stimuli and Characteristics of Tryptophan Transport on Brain Capillary Endothelial (TR-BBB) and Motor Neuron Like (NSC-34) Cell Lines. Biomedicines 2021, 9, 9. https://doi.org/10.3390/biomedicines9010009
Gyawali A, Kang Y-S. Pretreatment Effect of Inflammatory Stimuli and Characteristics of Tryptophan Transport on Brain Capillary Endothelial (TR-BBB) and Motor Neuron Like (NSC-34) Cell Lines. Biomedicines. 2021; 9(1):9. https://doi.org/10.3390/biomedicines9010009
Chicago/Turabian StyleGyawali, Asmita, and Young-Sook Kang. 2021. "Pretreatment Effect of Inflammatory Stimuli and Characteristics of Tryptophan Transport on Brain Capillary Endothelial (TR-BBB) and Motor Neuron Like (NSC-34) Cell Lines" Biomedicines 9, no. 1: 9. https://doi.org/10.3390/biomedicines9010009