Title Page
Contents
한글초록 15
Background 19
Chapter 1. Cationic amino acids transport characteristics and neuroprotective effects in NSC-34 cell lines 25
Ⅰ. Introduction 25
Ⅱ. Materials 31
1. Chemical reagents 31
2. Instruments 34
3. Cell lines 35
Ⅲ. Methods 36
1. Cell culture 36
2. [³H]L-Lysine and [³H]L-arginine uptake study in NSC-34 cell lines 36
3. Small interfering RNA (siRNA) transfection and Real-time PCR analysis 37
4. Pretreatment with inflammatory cytokines in ALS model cell lines 37
5. Statistical analysis of data 38
Ⅳ. Results and Discussion: Part I 41
1. [³H]L-Lysine uptake in a time dependent manner in NSC-34 cell lines 41
2. Transport kinetic characteristics of [³H]L-lysine uptake in the ALS model cell lines 43
3. Effect of several amino acids and transporter inhibitors on [³H]L-lysine uptake by NSC-34 cell lines 48
4. The CAT-1/Slc7a1/ system y⁺ mRNA expression level and effect of Slc7a1 siRNA on and uptake and mRNA expression of [³H]L-lysine in ALS model cell lines 51
5. Effect of drugs and kinetics of drug-induced inhibition on [³H]L-lysine uptake and by NSC-34 cell lines 53
6. Effect of lysine on the ALS cell lines pretreated with glutamate and other pro-inflammatory cytokines 56
Ⅳ. Results and Discussion: Part II 59
1. Kinetics of [³H]L-arginine is altered in NSC-34 cell lines 59
2. Differential analysis technique for the determination of relative contribution of transporters for the of arginine transport in NSC-34 Cell Lines 63
3. Effect of numerous amino acids and transport inhibitors on [³H]L-arginine uptake in ALS model cell Lines 66
4. Effect of the [³H]L-arginine uptake by therapeutic compounds and Lineweaver Burk plot analysis in NSC-34 cell lines 69
5. Arginine pretreatment effect on neuronal nitric oxide synthase (nNOS) mRNA expression in NSC-34 Cell Lines 73
6. Protective effects of arginine against H₂O₂ induced motor neuronal cytotoxicity 75
Ⅴ. Conclusions 77
Chapter 2. Characterization of essential nutrient, choline transport via choline like transport (CTL-1) and effect against inflammatory mediators in ALS model cell lines 78
Ⅰ. Introduction 78
Ⅱ. Materials 82
1. Chemical compounds 82
2. Equipment 83
3. NSC-34 cell lines 83
Ⅲ. Methods 84
1. NSC-34 cell culture 84
2. [³H]Choline uptake study in NSC-34 cell lines 84
3. Uptake study under sodium- and potassium-free conditions 84
4. Kinetic parameter estimation of [³H]choline uptake in NSC-34 cell lines 85
5. Real-time PCR analysis 85
6. Pretreatment of nerve growth factor and inflammatory cytokines pretreatment methods 86
Ⅳ. Results and Discussion 88
1. [³H]Choline transport was sodium, time and pH dependent in ALS model cell lines 88
2. Concentration dependent transport of [³H]choline in NSC-34 cell lines 92
3. CTL-1 gene expression in NSC-34 cell lines 96
4. Effects of therapeutic compounds on [³H]choline uptake by ALS model cell lines 98
5. IC₅₀ of drugs in disease model cell lines 100
6. Uptake of [³H]choline increases with NGF 102
7. Effect of choline against inflammatory conditions in ALS model cell lines 104
8. Cell viability analysis by MTT assay 106
Ⅴ. Conclusions 109
Chapter 3. Antioxidant effect of pharmacological compound paeonol and neuroprotective effect against inflammatory states in ALS model cell lines 110
Ⅰ. Introduction 110
Ⅱ. Materials 115
1. Chemical reagents 115
2. Equipment 116
3. NSC-34 cell lines 116
Ⅲ. Methods 117
1. Cell culture method 117
2. In vitro uptake study of [³H]paeonol in ALS model cell line 117
3. MTT assay 118
4. Analysis and calculation of data 118
5. Gene knockdown via siRNA study in NSC-34 cell lines 118
Ⅳ. Results and Discussion 119
1. Characteristics of [³H]paeonol uptake in NSC-34 cell lines 119
2. Kinetic parameters of [³H]paeonol uptake in motor neurons like ALS cell lines 122
3. Effects of pharmacological drugs on the paeonol transport in ALS model cell lines 126
4. SiRNA transfection and gene regulation of Pmat, Octn1, and Oat1 128
5. Effect of Oat-1 substrates/ inhibitors on paeonol uptake in NSC-34 cell lines 131
6. Determination of minimal inhibitory concentration (IC₅₀) of drugs in disease model cell lines 133
7. Neuroprotective effect of paeonol in ALS model cell lines 135
Ⅴ. Conclusions 141
Summary 142
References 144
Abstract in English 161
Table 1. Saturation kinetics of [³H]L-lysine uptake in ALS model cell lines 46
Table 2. Slc7a1 siRNA transfection of NSC-34 cell lines 52
Table 3. Lineweaver-Burk plot analysis of various drugs in ALS model cell lines 55
Table 4. Pretreatment effect on [³H]L-lysine uptake by NSC-34 cell lines 57
Table 5. Pharmacokinetic parameters of [³H]L-arginine uptake in ALS cell lines. 62
Table 6. Transporters involved for the [³H]L-arginine transport in NSC-34 cell lines 65
Table 7. Kinetic inhibition constant for [³H]L-arginine uptake in NSC-34 cell lines 72
Table 8. Expression of nNOS mRNA levels in NSC-34 cell lines 74
Table 9. Effect of H₂O₂ on the uptake of [³H]L-arginine and relative Slc7a1 mRNA expression in NSC-34 cell lines. 76
Table 10. Uptake of choline in a pH dependent manner in ALS model cell lines. 90
Table 11. Ion dependency of [³H]choline in NSC-34 cell lines. 91
Table 12. Kinetics of [³H]choline transport in ALS model cell lines. 95
Table 13. [³H]Choline uptake and CTL-1 mRNA followed by Slc44a1 siRNA in NSC-34 cell lines. 97
Table 14. Effect of drugs on the [³H]choline uptake in NSC-34 cell lines 99
Table 15. Dose response relation of [³H]choline uptake in MT cell line 101
Table 16. NGF pretreatment effect in NSC-34 cell lines 103
Table 17. Cell viability after inducing inflammatory conditions in MT cell line. 108
Table 18. Effect of pH on the uptake of [³H]paeonol in ALS model cell lines 121
Table 19. [³H]Paeonol uptake kinetic parameters in ALS model cell lines. 125
Table 20. Effects of Oat1 substrate/inhibitors on the uptake of [³H]paeonol. 132
Table 21. Dose–response effect of [³H]paeonol uptake in the disease model cell line. 134
Figure 1. Time course study on [³H]L-lysine uptake by NSC-34 cell lines. Uptake of [³H]L-lysine in (A) Wild type (WT) open circle (o) and (B) Mutant type (MT) close... 42
Figure 2. Concentration dependent uptake of [³H]L-lysine in WT cell line. Concentrations ranges 0–1 mM of unlabeled L-lysine were used for [³H]L-lysine uptake... 44
Figure 3. Concentration dependent uptake of [³H]L-lysine in MT cell line. Concentrations ranges 0–1 mM of unlabeled L-lysine were used for [³H]L-lysine uptake... 45
Figure 4. Effect of ions on the uptake of [³H]L-lysine in ALS model cell lines. (A) WT and (B) MT cell lines. Uptake was performed at 37℃ for 5 min at pH 7.4 using... 47
Figure 5. Effect of various amino acids on the uptake of [³H]L-lysine in NSC-34 cell lines. Uptake was measured in the absence or presence of 2 mM of various amino acids... 49
Figure 6. Effect of various transporter inhibitors on the uptake of [³H]L-lysine in NSC-34 cell lines. Uptake was carried at 37℃ for 5 min at pH 7.4 in (A) WT and (B) MT cell... 50
Figure 7. Inhibition analog study of [³H]L-lysine in ALS model cell lines. Uptake was measured either in the presence 0.5-2 mM of pharmacological drug solutions or in the... 54
Figure 8. Expression of Slc7a1 mRNA levels in MT cell line. Each value represents the mean ± S.E.M. (n=3-4). ***p 〈0.001, **p 〈0.01, *p 〈0.05, significant difference vs... 58
Figure 9. Concentration dependent uptake of [³H]L-arginine in WT cell line. Concentration of unlabeled L-arginine varies from 0-1 mM were used for [³H]L-... 60
Figure 10. Concentration dependent uptake of [³H]L-arginine in MT cell line. Concentration of unlabeled L-arginine varies from 0-1 mM were used for [³H]L-... 61
Figure 11. Effect of ion and membrane potential on [³H]L-arginine uptake in NSC-34 cell lines. (A) WT and (B) MT cell lines. Uptake was performed for 5 min at 37℃ and... 64
Figure 12. Effect of numerous amino acids on the uptake of [³H]L-arginine in ALS model cell lines. Uptake was measured either in the presence or absence of 2 mM of... 67
Figure 13. Effect of numerous transport inhibitors on the uptake of [³H]L-arginine in ALS model cell lines. Uptake was performed in the absence (control) or presence of... 68
Figure 14. Analog inhibition study of [³H]L-arginine in NSC-34 cell lines. (A) WT and (B) MT NSC-34 cell lines. Uptake was carried either in the absence or presence 0.5-2... 71
Figure 15. Time dependent uptake of [³H]choline in ALS model cell lines. (A) WT with open circle (o) and (B) MT with close circle (●). Uptake was performed at pH 7.4 at... 89
Figure 16. Concentration-dependency of [³H]choline uptake in WT cell line. Cold choline concentrations from 0-1 mM was used and uptake was carried under... 93
Figure 17. Concentration-dependency of [³H]choline uptake in MT cell line. Cold choline concentrations from 0-1 mM was used and uptake was carried under... 94
Figure 18. Pro-inflammatory cytokines pretreatment effects in the disease model of ALS. (A) MT cell line was treated with choline (1 mM) and LPS and TNF-α (20 ng/ml) in the... 105
Figure 19. Cell viability images after inducing inflammatory conditions in MT cell line. Images obtained via the EVOS XL cell imaging system (100x) in disease model of ALS... 107
Figure 20. Time dependent uptake of [³H]paeonol in NSC-34 cell lines. Uptake was observed for 0–60 min at pH 7.4 at 37℃ using ECF buffer solution in both WT and MT... 120
Figure 21. [³H]Paeonol concentration-dependent uptake in WT cell lines. Uptake was carried out under physiological pH 7.4 and temperature 37℃ either in the absence or... 123
Figure 22. [³H]Paeonol concentration-dependent uptake in MT cell line. Uptake was carried out under physiological pH 7.4 and temperature 37℃ either in the absence or... 124
Figure 23. Analog study of various compounds on [³H]paeonol uptake in NSC-34 cell lines. Uptake was carried in the absence (control) or presence of 1 mM inhibitor... 127
Figure 24. SiRNA transfection study on [³H]paeonol uptake in ALS model cell lines. Cells were transfected using lipofectamine with 200 nM control, Octn1, Octn2, MATE,... 130
Figure 25. Cell viability images of WT cell line exposed to inflammatory conditions. Images of NSC-34 WT cell lines exposed to inflammatory states and effect of paeonol... 137
Figure 26. Cell viability images of MT cell line exposed to inflammatory conditions. Images of NSC-34 MT cell lines exposed to inflammatory cytokines and neuroprotective... 138
Figure 27. Cell viability using MTT assay in NSC-34 cell lines. Cells were pretreated for 24 h with glutamate (2 mM), H₂O₂ (300 μM) and LPS (20 ng/mL) with or without... 139
Figure 28. Restoration effect of paeonol against inflammatory conditions in ALS model cell lines. (A) Uptake of [³H]paeonol in WT cell line pretreated for 24 h with H₂O₂ (300... 140
Scheme 1. Pathophysiology of ALS disease. 22
Scheme 2. Diagrammatic representation of the NSC-34 cell lines. 23
Scheme 3. Schematic presentation of research design. 24
Scheme 4. Molecular structure of cationic amino acids 28
Scheme 5. Molecular structure of pharmacological drugs 29
Scheme 6. Possible mechanism of arginine and NO signaling. 30
Scheme 7. Procedure for uptake study. 39
Scheme 8. Quantitative Real-Time PCR analysis. 40
Scheme 9. Molecular structure and formula of choline. 80
Scheme 10. Transport of choline via different transporters. 81
Scheme 11. MTT assay 87
Scheme 12. Molecular structure of Paeonol. 113
Scheme 13. Actions of paeonol. 114