本研究利用fura-2螢光來偵測新一代的抗憂鬱藥物mirtazapine對攝護腺癌細胞(PC3)與骨肉瘤細胞(MG63)的細胞內鈣傳訊(Ca2+ signaling)之影響。在有細胞外鈣時，mirtazapine可造成攝護腺癌細胞與骨肉瘤細胞的細胞內鈣濃度([Ca2+]i)上升，而且此反應與mirtazapine之濃度呈正相關。在無細胞外鈣時，mirtazapine仍可造成與濃度呈正相關之細胞內鈣上升，但上升之幅度減少。在無鈣溶液中(Ca2+-free medium)，當細胞先以mirtazapine處理時，thapsigargin(內質網鈣幫浦抑制劑)所引起之細胞內鈣上升受抑制；相反地，thapsigargin也減少mirtazapine所造成細胞內鈣增加的幅度，因此內質網中鈣離子儲存(Ca2+ store)在mirtazapine所導致之細胞內鈣改變，扮演重要角色。在攝護腺癌細胞，U73122(磷酯

The effects of the antidepressant mirtazapine on cytosolic Ca2+ concentrations ([Ca2+]i) in human prostate cancer PC3 cells and human osteosarcoma MG63 cells were measured by Ca2+-sensitive fluorescent probe fura-2. In Ca2+-containing medium, mirtazapine induced [Ca2+]i rises in a concentration-dependent manner in both PC3 and MG63 cells. Removal of extracellular Ca2+ inhibited the mirtazapine-induced Ca2+ signal. In Ca2+-free medium, thapsigargin (an inhibitor of the endoplasmic reticulum Ca2+-ATPase pump) induced [Ca2+]i rises by passively depleting the endoplasmic reticulum Ca2+ store, after which the increasing effect of mirtazapine (1.5 mM) on [Ca2+]i was reduced. Conversely, pretreatment with mirtazapine decreased thapsigargin-induced [Ca2+]i rises in PC3 and MG63 cells. When PC3 cells were pretreated with U73122, a phospholipase C inhibitor, mirtazapine-induced [Ca2+]i rises were inhibited by 47%. But in MG63 cells, 2 mM U73122 did not change mirtazapine-induced [Ca2+]i rises. These finding suggest that mirtazapine-induced [Ca2+]i rises were caused both by extracellular Ca2+ influx and intracellular depletion of the endoplasmic reticulum Ca2+ stores. Furthermore, the mechanism of mirtazapine-induced Ca2+ release may be different between PC3 and MG63 cells. Additionally, cell proliferation assays suggest that overnight incubation with higher concentrations of mirtazapine decreased cell viability in a concentration-dependent manner.

誌謝與感言-2
中文摘要-6
Abstract-7
Abbreviations-8
I. Introduction-9
1. General background-9
2. Depressive disorders-10
3. Antidepressants-12
4. Mirtazapine-13
5. Ca2+ as a pivotal intracellular signal in cells-15
6. The role of Ca2+ signals in depressive disorders and antidepressants-18
II. Materials and Methods-20
1. General aims-20
2. Procedures-20
3. Materials-22
(1) Cell culture-22
(2) Solutions-22
(3) [Ca2+]i measurements-23
(4) Cell proliferation assays-24
(5) Chemicals-25
4. Statistics-26
III. Results-27
1. Human prostate cancer PC3 cells-27
(1) Effect of mirtazapine on [Ca2+]i in Ca2+-containing medium-27
(2) Effect of removal of extracellular Ca2+ on mirtazapine-induced [Ca2+]i rises-27
(3) Effects of L-type Ca2+ channel blockers on mirtazapine-induced [Ca2+]i rises-28
(4) Endoplasmic reticulum Ca2+ store and mirtazapine-induced [Ca2+]i rises-28
(5) Effect of inhibiting phospholipase C on mirtazapine-induced [Ca2+]i rises-29
(6) Effect of mirtazapine on the proliferation of PC3 cells-29
2. Human osteosarcoma MG63 cells-30
(1) Effect of mirtazapine on [Ca2+]i in Ca2+-containing medium-30
(2) Effect of removal of extracellular Ca2+ on mirtazapine-induced [Ca2+]i rises-30
(3) Effects of L-type Ca2+ channel blockers on mirtazapine-induced [Ca2+]i rises-31
(4) Endoplasmic reticulum Ca2+ store and mirtazapine-induced [Ca2+]i rises-31
(5) Effect of inhibiting phospholipase C on mirtazapine-induced [Ca2+]i rises-32
(6) Effect of mirtazapine on the proliferation of MG63 cells-33
IV. Discussions-34
V. References-38
VI. Figures-46
1. Human prostate cancer PC3 cells-46
Fig 1. In Ca2+-containing medium, effect of mirtazapine on [Ca2+]i in PC3 cells-46
Fig 2. In Ca2+-free medium, effect of mirtazapine on [Ca2+]i in PC3 cells-47
Fig 3. Concentration-response plots of mirtazapine-induced Ca2+ release in PC3 cells-48
Fig 4. The extracellular Ca2+ influx and mirtazapine-induced [Ca2+]i rises in PC3 cells-49
Fig 5. Effect of thapsigargin on mirtazapine-induced Ca2+ release in PC3 cells-50
Fig 6. Effect of mirtazapine on thapsigargin-induced passive depleting of the endoplasmic reticulum Ca2+ store in PC3 cells-51
Fig 7. In Ca2+-free medium, ATP-induced Ca2+ release in PC3 cells-52
Fig 8. Effect of U73122 on mirtazapine-induced [Ca2+]i rises in PC3 cells-53
Fig 9. Effect of U73122 and U73343 on ATP and mirtazapine-induced [Ca2+]i rises-54
Fig 10. Effect of mirtazapine on the proliferation of PC3 cells-55
2. Human osteosarcoma MG63 cells-56
Fig 11. In Ca2+-containing medium, effect of mirtazapine on [Ca2+]i in MG63 cells-56
Fig 12. In Ca2+-free medium, effect of mirtazapine on [Ca2+]i in MG63 cells-57
Fig 13. Concentration-response plots of mirtazapine-induced Ca2+ release in MG63 cells-58
Fig 14. The extracellular Ca2+ influx and mirtazapine-induced [Ca2+]i rises-59
Fig 15. Effect of thapsigargin on mirtazapine-induced Ca2+ release in MG63 cells-60
Fig 16. Effect of mirtazapine on thapsigargin-induced passive depleting of the endoplasmic reticulum Ca2+ store in MG63 cells-61
Fig 17. In Ca2+-free medium, histamine-induced Ca2+ release in MG63 cells-62
Fig 18. Effect of U73122 on mirtazapine-induced [Ca2+]i rises in MG63 cells-63
Fig 19. Effect of U73122 & U73343 on histamine and mirtazapine-induced [Ca2+]i rises-64
Fig 20. Effect of mirtazapine on the proliferation of MG63 cells-65
Fig 21. In Ca2+-containing medium, effects of L-type Ca2+ channel blockers on mirtazapine-induced [Ca2+]i rises in MG63 cells-66

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