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title page
Contents
Abbreviations 13
ABSTRACT 15
I. Introduction 19
II. Materials and methods 35
1. Chemicals 35
2. Cell culture 36
3. Determination of dopamine content 37
4. Assay for TH activity 38
5. Assay for AADC activity 39
6. Determination of intracellular L-DOPA content 39
7. Measurement of cell viability: MTT assay 40
8. Flow cytometry analysis 41
9. Measurement of cAMP levels 42
10. Western blot analysis 43
11. Statistical analysis 44
III. Results 45
1. Effects of L-DOPA on dopamine biosynthesis and cell viability in PC12 cells 45
1.1. Time courses of intracellular dopamine content by L-DOPA 46
1.2. Time courses of TH activity by L-DOPA 46
1.3. Time courses of AADC activity by L-DOPA 50
1.4. Time courses of the intracellular L-DOPA content 50
1.5. Time courses of cell viability with L-DOPA treatment 53
1.6. Flow cytometry analysis 55
2. Intracellular mechanisms of dopamine biosynthesis by L-DOPA in PC12 cells 59
2.1. Time courses of cAMP levels by L-DOPA 59
2.2. Effects of D1 receptor antagonist on L-DOPA-induced cAMP and dopamine content in PC12 cells 61
2.3. Effects of L-DOPA on cAMP/PKA and Ca2+/PKC pathways in dopamine biosynthesis in PC12 cells(이미지참조) 64
2.4. Cyclic AMP-mediated activation of PKA by L-DOPA 64
2.5. Stimulation of CREB phosphorylation by L-DOPA 66
2.6. Biosynthesis of TH protein after treatment with L-DOPA 69
3. Signaling pathways of L-DOPA-induced cytotoxicity in PC12 cells 72
3.1. Activation of JNK1/2 by L-DOPA 72
3.2. Activation of p38 by L-DOPA 74
3.3. ERK1/2 was increased with L-DOPA treatment in PC12 cells 74
3.4. Activation of Bad (Ser 155) by PKA phosphorylation with L-DOPA treatment 77
3.5. Activation of Bad (Ser 112) by PKA phosphorylation with L-DOPA treatment 77
3.6. Activation of Bc1-2 by L-DOPA in PC12 cells 80
3.7. Expression of caspase-3 activation by L-DOPA in PC12 cells 80
3.8. Expression of caspase-12 activation by L-DOPA in PC12 cells 80
4. Interrelation of intracellular cAMP levels and cell viability with forskolin, iron and manganese in PC12 cells 85
4.1. Effects of forskolin on intracellular cAMP levels in PC12 cells 85
4.2. Effects of forskolin on dopamine biosynthesis in PC12 cells 87
4.3. Effects of forskolin on cell viability in PC12 cells 87
4.4. Effects of forskolin on L-DOPA-induced cytotoxicity in PC12 cells 90
4.5. Effects of Fe2+ and Mn2+ on cAMP levels in PC12 cells(이미지참조) 90
4.6. Effects of Fe2+ and Mn2+ on cell viability in PC12 cells(이미지참조) 95
IV. Discussion 99
V. Conclusion 118
VI. References 120
요약 144
Acknowledgment 148
Table 1. Intracellular L-DOPA content after treatment with different concentrations of L-DOPA in PC12cells. 52
Table 2. The non-cytotoxic and cytotoxic ranges in response to exposure to L-DOPA for 24 hr and 48 hr in PC12 cells. 58
Fig. 1. Biosynthetic pathway of catecholamines. Dopamine, norepinephrine and epinephrine are derived from the multistep processing of tyrosine in catecholaminergic neurons.... 20
Fig. 2. Changes in intracellular dopamine content at 1, 3, 6, 10, 24 and 48 hr after exposure to L-DOPA in PC12 cells. PC12 cells were treated with L-DOPA (20, 100 and 200 μM), and then incubated at 37℃ for required time.... 47
Fig. 3. Intracellular dopamine content by exposure to L-DOPA for 24 hr (A) and 48 hr (B) in PC12 cells. PC12 cells were treated with L-DOPA (20, 100 and 200 μM), and then incubated at 37℃ for 24 hr and 48 hr.... 48
Fig. 4. Time courses of TH activity by L-DOPA in PC12 cells. TH activity of the control was 3.58 ± 0.6 nmol/min/ng protein. Results were presented as means ± SD of three experiments performed in triplicate dishes.... 49
Fig. 5. Time courses of AADC activity by L-DOPA in PC12 cells. AADC activity of the control was 31.5 ± 0.6 nmol/min/ng protein. Results were presented as means ± SD of three experiments performed in triplicate dishes.... 51
Fig. 6. Effect of L-DOPA (20, 100 and 200 μM) on cell viability. Cell viability was assessed using MTT method, in which viable cells convert the soluble dye, MTT to insoluble blue formazan crystals.... 54
Fig. 7. Flow cytometry histograms of PC12 cells in control and after 24 hr exposure with different concentrations of L-DOPA (20, 100 and 200 μM). After incubation, the cells were harvested and stained with propidium iodide... 56
Fig. 8. Flow cytometry histograms of PC 12 cells in control and after 48 hr exposure with different concentrations of L-DOPA (20, 100 and 200 μM). After incubation, the cells were harvested and stained with propidium iodide... 57
Fig. 9. Time courses of L-DOPA on cAMP levels in PC12 cells. PC12 cells were treated with 20, 100 and 200 μM L-DOPA and incubated at 37℃ for 20 min, 1 hr, 3 hr and 24 hr.... 60
Fig. 10. Effects of D1 receptor antagonist on the enhancement of cAMP levels induced by L-DOPA in PC12 cells. PC12 cells were pretreated with 10 μM SCH23390 for 2 hrs, then treated with L-DOPA for 1 hr.... 62
Fig. 11. Effects of D1 receptor antagonist on the enhancement of dopamine content induced by L-DOPA in PC12 cells. PC12 cells were pretreated with 10 μM SCH23390 for 2 hrs, then treated with L-DOPA for 6 hrs.... 63
Fig. 12. Effects of PKA and PKC inhibitors on L-DOPA-induced dopamine biosynthesis. PC12 cells were pretreated with 2 μM H89 and 2 μM GF109203X for 30min, then treated with 20, 100 and 200 μM L-DOPA for 6 hours.... 65
Fig. 13. Representative blots illustrating the effects of 20, 100 and 200 μM L-DOPA on activation of PKA and phosphor-PKA in PC12 cells. Immunoblots of lysates from treated PC12 cells were probed with phosphor-PKA(upper... 67
Fig. 14. Representative blots illustrating the effects of 20, 100 and 200 μM L-DOPA on activation of CREB and phosphor-CREB (Ser 133) in PC 12 cells. Immunoblots of lysates from treated PC12 cells were probed with phosphor-... 68
Fig. 15. Representative blots illustrating the effects of 20, 100 and 200 μM L-DOPA on TH protein synthesis in PC 12 cells. Immunoblots of lysates from treated PC12 cells were probed with TH (upper panel) and β-actine (low panel)... 71
Fig. 16. Representative blots illustrating the effects of 20, 100 and 200 μM L-DOPA on activation of JNK1/2 and phosphor JNK1/2 in PC12 cells. Immunoblots of lysates from treated PC12 cells were probed with phosphor-... 73
Fig. 17. Representative blots illustrating the effects of 20, 100 and 200 μM L-DOPA on activation of p38 and phosphor-p38 in PC 12 cells. Immunoblots of lysates from treated PC12 cells were probed with phosphor-p38 (upper panel)... 75
Fig. 18. Representative blots illustrating the effects of 20, 100 and 200 μM L-DOPA on activation of ERK1/2 and phosphor ERK1/2 in PC12 cells. Immunoblots of lysates from treated PC12 cells were probed with phosphor-... 76
Fig. 19. Representative blots illustrating the effects of 20, 100 and 200 μM L-DOPA on activation of Bad and phosphor-Bad (Ser 155) in PC12 cells. Immunoblots of lysates from treated PC12 cells were probed with phosphor-... 78
Fig. 20. Representative blots illustrating the effects of 20, 100 and 200 μM L-DOPA on activation of Bad and phosphor-Bad (Ser 112) in PC12 cells. Immunoblots of lysates from treated PC12 cells were probed with phosphor-... 79
Fig. 21. Representative blots illustrating the effects of 20, 100 and 200 μM L-DOPA on activation of Bcl-2 in PC 12 cells. Immunoblots of lysates from treated PC12 cells were probed with Bc1-2 (upper panel) and β-actine (low... 81
Fig. 22. Representative blots illustrating the effects of 20, 100 and 200 μM L-DOPA on activation of cleaved caspase-3 (Asp 175) in PC 12 cells. Immunoblots of lysates from treated PC12 cells were probed with caspase-3... 82
Fig. 23. Representative blots illustrating the effects of 20, 100 and 200 μM L-DOPA on activation of caspase-12 in PC 12 cells. Immunoblots of lysates from treated PC12 cells were probed with caspase-12 (upper panel) and β-actine... 83
Fig. 24. Effects of forskolin on intracellular cAMP levels in PC12 cells. PC12 cells were exposed to different concentrations forskolin (0.01 - 10 μM) for 10 min and 30 min, and then cAMP levels were measured using cAMP assay kit... 86
Fig. 25. Effects of forskolin on dopamine biosynthesis in PC12 cells. PC12 cells were exposed to different for3-12 hr to different concentrations of forskolin (0.01 -10 μM), and then dopamine content was determined by an HPLC system.... 88
Fig. 26. Effects of forskolin (0.01 - 10 μM) on cell viability at 24 hr and 48 hr in PC 12 cells. Cell viability was assessed using MTT method, in which viable cells convert the soluble dye, MTT to insoluble blue formazan crystals.... 89
Fig. 27. Flow cytometry histograms of PC12 cells in control and after 24 hr exposure with concentrations of forskolin (0.1, 1.0 and 10 μM). After incubation, the cells were havested and stained with propidium iodide (PI).... 91
Fig. 28. Flow cytometry histograms of PC12 cells in control and after 48 hr exposure with concentrations of forskolin (0.1, 1.0 and 10 μM). After incubation, the cells were harvested and stained with propidium iodide (PI).... 92
Fig. 29. Effects of forskolin on L-DOPA-induced cytotoxicity for 24 hr in PC12 cells. Cell viability was assessed using MTT method. Results were presented as means ± SD of four experiments performed in triplicate wells.... 93
Fig. 30. Effects of forskolin on L-DOPA-induced cytotoxicity for 48 hr in PC12 cells. Cell viability was assessed using MTT method. Results were presented as means ± SD of four experiments performed in triplicate wells.... 94
Fig. 31. Time courses of the intracellular cAMP levels after treatment with iron and manganese in PC 12 cells. PC12 cells were exposed to different concentrations iron (10-100 μM) and manganese (20-400 μM) for 20 min and... 96
Fig. 32. Effects of iron (10-100 μM) and manganese (20-400 μM) on cell viability at 24 hr and 48 hr in PC12 cells. Cell viability was assessed using MTT method, in which viable cells convert the soluble dye, MTT to insoluble... 97
Scheme 1. Stress mediated apoptosis. 33
Scheme 2. Dopamine biosynthesis pathways. 103
Scheme 3. Mechanisms of dopamine biosynthesis with L-DOPA treatment. 108
Scheme 4. L-DOPA-induced oxidative stress and apoptosis. 113
Scheme 5. Dual effects of cAMP on L-DOPA-induced dopamine biosynthesis and oxidative stress. 114
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