Capsazepine及壬基苯酚對於犬腎小管細胞質中的游離內鈣的影響是不清楚的。所以我們使用TPRV1 antagonist capsazepine及環境化學因子壬基苯酚來了解不同種類的化學物質對犬腎小管細胞細胞內鈣的變化及細胞存活的影響。
用capsazepine加入於懸浮態的細胞中再以對鈣質敏感的螢光染料 fura-2 來觀察內鈣的變化。發現在10-200microM capsazepine對於細胞內鈣的影響有正相關。事先清除細胞外的鈣質，細胞內鈣增加的幅度減少四成。Capsazepine可以誘發錳離子熄滅fura-2螢光染料，間接証明是有鈣離子的流入。Capsazepine誘發鈣離子流入和Ｌ型鈣離子抑制劑及protein kinase C modulators都沒有相關。在不含鈣離子的細胞培養液中的犬腎小管細胞,若先以內質網鈣離子泵抑制劑thapsigargin處理後，再加入100microM capsazepine發現誘發的鈣離子放出被抑制。以capsazepine 前處理會阻斷thapsigargin引起的鈣離子釋放。以U73122抑制phospholipase C不會改變capsazepine引起的細胞內鈣增加。綜合以上的結果可以發現犬腎小管細胞中，capsazepine會引起細胞內鈣增加，其機轉在於由內質網藉由不需phospholipase C的鈣釋出及非Ｌ型的鈣離子通道的流入。
而壬基苯酚同樣是可以引發的細胞內鈣增加，大部份來源是由細胞內的儲存。同樣的它可以釋放thapsigargin作用後，仍保留於胞器中的鈣離子。利用WST-1螢光法測定壬基苯酚不同藥劑濃度下細胞存活率，顯示壬基苯酚能隨濃度增加造成犬腎小管細胞死亡。
使用兩種不同的藥劑對於同一種犬腎小管細胞作用可以看出兩種藥劑在細胞內鈣作用的影響及其差異：Capsazepine不經由磷脂酶C引起內質網鈣儲存的釋放而細胞外鈣流入不經由L型鈣離子通道。壬基苯酚同樣不經由磷脂酶C引起內質鈣儲存的釋放，但而細胞外鈣流入部份是由蛋白質激酶C調節的通道管控。

The effect of capsazepine and nonylphenol on cytosolic free Ca2+ concentrations ([Ca2+]i) in MDCK renal tubular cells is unclear. This study explored whether capsazepine and nonylphenol changed basal [Ca2+]i levels in suspended MDCK cells by using fura-2 as a Ca2+-selective fluorescent dye. Capsazepine at concentrations between 10 and 200 microM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partially by 40% after removing extracellular Ca2+. Capsazepine induced Mn2+ quench of fura-2 fluorescence, indirectly implicating Ca2+ entry. Capsazepine-induced Ca2+ influx was not changed by L-type Ca2+ entry inhibitors and protein kinase C modulators [phorbol 12-myristate 13-acetate (PMA) and GF109203X]. In Ca2+-free medium, 100microM capsazepine-induced Ca2+ release was substantially suppressed by pretreatment with thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor). Pretreatment with capsazepine nearly abolished thapsigargin-induced Ca2+ release.
Nonylphenol also increased [Ca2+]i in a concentration- dependent manner like capsazepine does. Similar response in [Ca2+]i rise can be found by inhibition of phospholipase C and using thapsigargin. Different from capasazpine, the [Ca2+]i rise was inhibited by PMA. At concentrations between 5 and 100microM, nonylphenol killed cells in a concentration-dependent manner.
Collectively, in MDCK cells, capsazepine induced [Ca2+]i rises by causing phospholipase C-independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via non-L-type Ca2+ channels. Nonylphenol induced [Ca2+]i increase in MDCK cells via evoking Ca2+ entry through protein kinase C-regulated Ca2+ channels, and releasing Ca2+ from endoplasmic reticulum and other cellular storage in a phospholipase C-independent manner.

審定書 ii
誌謝 iii
中文摘要 v
ABSTRACT vii
LIST OF ABBREVIATIONS ix
CHAPTER 1 INTRODUCTION 1
CHAPTER 2 MATERIALS AND METHODS 22
CHAPTER 3 THE EFFECT OF CAPSAZEPINE
ON [Ca2+]i IN MDCK CELLS 26
CHPATER 4 THE EFFECT OF NONYLPHENOL
ON [Ca2+]i AND VIABILITY IN MDCK CELLS 36
CHAPTER 5 DISCUSSION 49
CHAPTER 6 CONCLUSION 56
REFERENCES 57
PUBLICATION 71

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