探討內皮素對角膜內皮細胞及角膜纖維母細胞的影響與調節機轉

Effects and regulatory mechanisms of endothelins on cultured corneal keratocytes and endothelial cells

吳國揚(Kwou-Yeung Wu)

指導教授 ： 王惠珠

高雄醫學大學/醫學院/醫學研究所/博士(2004年)

摘要

報告顯示內皮素可能會改變角膜的生理特性。因此本論文主要是探討內皮素對角膜纖維母細胞及角膜內皮細胞的作用與調節機轉。以10-7、10-8、10-9 M內皮素處理牛角膜內皮細胞株BCE C/D-1b細胞，結果發現內皮素-1（ET-1）會隨著劑量加大而增加角膜內皮細胞對3H-thymidine、3H-leucine及3H-uridine的攝取量，其50%有效劑量（EC50）分別是10-8.78 M、10-8.53 M及10-8.04 M。而內皮素-2（ET-2）及內皮素-3（ET-3）對角膜內皮細胞的3H-thymidine攝取量卻沒有影響。ET-1會隨著劑量加大而增加角膜內皮細胞內cAMP及cGMP的濃度。ET-1也會增加角膜內皮細胞內IPl、IP2及IP3的濃度。螢光免疫染色法顯示ET-1會增加角膜內皮細胞fibronectin蛋白質的量及分佈。在含鈣的溶液中，10-7、10-8、10-9 M ET-1會使角膜內皮細胞內游離的鈣離子濃度增加。在不含鈣的緩衝溶液中，ET-1也會使角膜內皮細胞內游離鈣離子濃度增加。內皮素-1會減少thapsigargin（TG）或carbonylcyanide m-chlorophenylhydrazone（CCCP）所誘發角膜內皮細胞內鈣離子的增加，而TG或CCCP也會減少ET-1所誘發角膜內皮細胞內鈣離子的增加。ET-1誘發角膜內皮細胞內鈣離子增加的作用會部分被U73122（phospholipase C抑制劑）所抑制。在含鈣的緩衝溶液中，內皮素A受體（ETA receptor）拮抗劑（BQ123）及內皮素B受體（ETB receptor）拮抗劑（BQ788）會部分抑制ET-1所誘發的角膜內皮細胞內鈣離子增加。Nifedipine及La3+也會部分抑制ET-1所誘發的角膜內皮細胞內鈣離子增加。在不含鈣的溶液中若先以內皮素-1將角膜內皮細胞內鈣離子由細胞內胞器釋出後，再加入5 mM CaCl2會促使更多細胞外鈣離子流向細胞內（capacitative calcium influx）。另一實驗顯示，將培養的豬角膜纖維母細胞經10-7 M、10-8 M及10-9 M內皮素-1處理兩小時後，細胞對3H-thymidine、3H-leucine及3H-uridine攝取量明顯受到抑制，其50%抑制劑量（ID50）分別是10-7 M、10-0.52 M及10-11.8 M。以MTT染色法測試細胞粒線體的活性時，發現內皮素-1對角膜纖維母細胞的50%抑制劑量是10-5.1 M。而內皮素-1對角膜纖維母細胞移動性的50%抑制劑量是10-7.86 M。以雷射掃描細胞儀（laser scanning cytometer）觀察時發現ET-1會快速改變角膜纖維母細胞的形態並降低細胞的附著力。而且ET-1會對角膜纖維母細胞產生慢性凋亡的效果，因為利用TUNEL染色法可以很明顯的染出細胞DNA的碎片。內皮素-1也會使角膜纖維母細胞的fibronectin產生斷裂的現象。由以上結果顯示內皮素-1會增加角膜內皮細胞增生，並且增加細胞內cAMP、cGMP、phosphoinositides的濃度及fibronectin蛋白質的分佈。內皮素-1也會透過活化ETA受體、ETB受體、La3+-sensitive Ca2+ pump、L-type voltage-operated Ca2+ channel、phospholipase C等來增加角膜內皮細胞內游離鈣離子，也會促使鈣離子由內質網及粒線體游離出並且進一步促進capacitative 鈣離子進入細胞內。但是內皮素-1反而會對角膜纖維母細胞造成慢性凋亡的作用，導致角膜纖維母細胞在形態及功能的喪失。

關鍵字

內皮素 ； 角膜內皮細胞 ； 角膜纖維母細胞

並列摘要

It is indicated that endothelin may change the physiological characteristics of cornea. Thus, the effects and regulatory mechanism of endothelins on cultured corneal keratocytes and endothelial cells were investigated in the present study. It was found that ET-1 remarkably affected the increase of corneal endothelial cells on 3H-thymidine, 3H-leucine, and 3H-uridine uptakes in a dose-dependent manner. The 50% effective concentration (EC50) for ET-1, as measured by 3H-thymidine, 3H-uridine, 3H-leucine uptake, were 10-8.78 M, 10-8.53 M and 10-8.04 M, respectively. The 3H-thymidine uptake was enhanced by ET-1 significantly, whereas ET-2 and ET-3 had no effect. The cellular IPl, IP2, IP3, cAMP and cGMP concentrations were also stimulated in the presence of ET-1 in a dose dependent manner. Immunofluorescent staining revealed that ET-1 increased the fibronectin protein concentration and changed the fibronectin distribution in corneal endothelial cells. Endothelin-1 of 10-7，10-8，10-9 M increased intracellular calcium concentration in corneal endothelial cells by being preloaded with fura-2-AM and was assayed by spectrophotometry. In Ca2+-containing buffer and Ca2+--free buffer, ET-1 induced a significant rise in [Ca2+]i in corneal endothelial cells at concentrations from 10-9 to 10-7 M. In Ca2+--free buffer, pretreatment of the corneal endothelial cells with ET-1 inhibited thapsigargin-induced [Ca2+]i increase and carbonylcyanide m-chlorophenylhydrazone (CCCP)-induced Ca2+ release. Pretreatment of the corneal endothelial cells with thapsigargin or CCCP also inhibited ET-1-induced [Ca2+]i increase. In Ca2+-containing buffer, the ETA receptor antagonist (BQ123) and ETB receptor antagonist (BQ788) partially inhibited ET-1-induced [Ca2+]i increase in corneal endothelial cells. Nifedipine and La3+ also inhibited ET-1-induced [Ca2+]i increase in corneal endothelial cells. The intracellular calcium release caused by ET-1 was partially inhibited by phospholipase C inhibitor (U73122) in corneal endothelial cells. After incubation of the corneal endothelial cells with ET-1 in Ca2+-free buffer, the addition of 5 mM CaCl2 increased Ca2+ influx. It implied that release of Ca2+ from internal stores caused by ET-1 could further induce capacitative Ca2+ entry. The effect of ET-1 on cultured porcine corneal keratocytes was investigated. The results indicate that ET-1 has remarkable effects to inhibit corneal keratocytes on 3H-thymidine, 3H-leucine, 3H-uridine uptakes and cellular migration. It is in a dose-dependent manner at concentrations ranging from 10-7 to 10-9 M. The 50% inhibitory dose (ID50) for ET-1, as measured by 3H-thymidine uptake, 3H-uridine uptake and 3H-leucine uptake, were 10-7 M, 10-0.52 M and 10-11.8 M, respectively. In the presence of ET-1, the cellular MTT values of keratocytes were also decreased. The ID50 for ET-1 with cell migration assay and MTT assay were measured at 10-7.86 M and 10-5.1 M. Endothelin-1 (10-6 M) promptly changed cellular morphology and attenuated adhesion observed with laser scanning cytometer in corneal keratocytes. Endothelin-1-induced characteristic apoptosis of corneal keratocytes were observed by using a TUNEL assay that detected fragmented DNA of apoptosis. Western blot assay revealed that endothelin-1 induced proteolysis and decreased in fibronectin protein in corneal keratocytes. These findings indicate that endothelin-1 increases the corneal endothelial cell proliferation and biological changes may be involved in changing intracellular calcium mobility, increasing intracellular phosphoinositides, enhancing intracellular cGMP and cAMP accumulation and fibronectin protein synthesis. ET-1 also induces [Ca2+]i increase in corneal endothelial cells. The regulatory mechanisms of calcium mobility are mediated by ETA receptor, ETB receptor, La3+-sensitive Ca2+ pump and L-type voltage-operated Ca2+ channel, that leading to Ca2+ influx. ET-1 also increases the internal Ca2+ release from endoplasmic reticulumn and mitochondria Ca2+ stores followed by capacitative Ca2+ entry. Furthermore, ET-1-induced intracellular Ca2+ release is modulated by phospholipase C-coupled event. However, endothelin-1 may lead corneal keratocytes to death resulting from induction of apoptosis and functional loss.

並列關鍵字

endothelins ； corneal endothelial cells ； corneal keratocytes

參考文獻

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