降低眼內壓的eicosanoid衍生藥對角膜內皮細胞的影響

Ocular hypotensive drugs of eicosanoid derivatives on cultured corneal endothelial cells

呂怡萱(Yi-Syuan Lyu)

指導教授 ： 洪秀貞

高雄醫學大學/醫學院/醫學研究所/碩士(2005年)

摘要

目的： 本實驗是探討eicosanoid 衍生的降眼壓藥 bimatoprost、latanoprost、travoprost、unoprostone以及prostaglandin F2α (PGF2α)和防腐劑benzalkonium chloride對豬角膜內皮細胞的生化影響及細胞內第二訊息IP3、cAMP、cGMP和鈣離子濃度的改變。
方法： 利用細胞攝取3H-thymidine、3H-uridine和3H-leucine的變化來觀察藥物對細胞中合成DNA、RNA和protein的影響；利用MTT、3H-proline和western blotting來觀察這些藥物對角膜內皮細胞存活率、collagen以及蛋白質表現的影響。利用細胞攝取3H-inositol、3H-cAMP及3H-cGMP測量角膜內皮細胞內IP3、cAMP、cGMP的變化。利用細胞攝取Fura-2-AM及螢光分光光度計測量細胞內游離鈣離子的流動性變化。實驗中所測試的降眼壓藥濃度是以藥水原濃度，經稀釋成1/100，1/1000及1/10000。
結果： 細胞經藥物培養90分鐘後發現unoprostone (28 μM、2.8 μM和0.28 μM)、latanoprost (1.1 μM和0.11 μM)、travoprost (0.8 μM)和bimatoprost (7.2 μM)都會抑制DNA的合成。而unoprostone (28 μM和2.8 μM)和PGF2α (5.6 μM)則會促進RNA的合成。但是unoprostone (28 μM)、latanoprost (1.1 μM和0.11 μM) 和travoprost (0.8 μM)都會抑制protein合成；unoprostone (28 μM和2.8 μM)、latanoprost (1.1 μM和0.11 μM)、travoprost (0.8 μM和0.08 μM) 和bimatoprost (7.2 μM)都會抑制collagen的形成。另外western blotting結果發現，加藥反應6小時之後，28 μM unoprostone、1.1 μM latanoprost、7.2 μM bimatoprost、和5.6 μM PGF2α可以抑制角膜內皮細胞內type I collagen的表現。而benzalkonium chloride (0.0001%)會抑制DNA、RNA及protein的合成，也會抑制type I collagen的表現。進一步探討這些藥物對細胞訊息影響時發現，28 μM unoprostone會抑制IP3，1.1 μM latanoprost、0.8 μM travoprost、7.2 μM bimatoprost、5.6 μM PGF2α和0.0001% benzalkonium chloride則會增加IP3的量，而只有28 μM unoprostone會增加cAMP的量，7.2 μM bimatoprost會增加cGMP的濃度，其他藥物對於細胞內cAMP和cGMP的變化沒有影響。測量細胞內鈣離子的變化時發現在有鈣的溶液中，unoprostone (28 μM、2.8 μM和0.28 μM)、latanoprost (1.1 μM、0.11 μM和0.011μM)、travoprost (0.8 μM和0.08 μM)和bimatoprost (7.2 μM和0.72 μM)都會引起細胞內鈣離子[Ca2+]i的增加，不過5.6 μM PGF2α和0.0001% benzalkonium chloride則會抑制細胞內[Ca2+]i的增加；而在無鈣的溶液中只有28 μM unoprostone會引起細胞內[Ca2+]i的增加，因此只用28 μM unoprostone再進一步探討對細胞內鈣離子釋放的途徑，結果發現它可以分別釋出thapsigargin -sensitive (內質網)和CCCP-sensitive (粒腺體)中儲存的鈣離子，增加細胞內鈣離子的濃度，並且unoprostone所增加的細胞內鈣離子的濃度的效果會被U73122 (phospholipase C抑制劑)和aristolochic acid (phospholipase A2抑制劑)抑制。
結論： unoprostone及latanoprost會降低角膜內皮細胞存活率、並且抑制細胞的DNA、RNA、protein和collagen合成及表現。travoprost和bimatoprost會抑制細胞的增生及collagen合成。latanoprost、travoprost及bimatoprost會增加IP3，而unoprostone則是降低IP3增加cAMP。unoprostone及latanoprost、travoprost和bimatoprost會增加細胞外鈣離子流入細胞內，而unoprostone可經由phospholipase C和phospholipase A2 pathway調節細胞內鈣離子的濃度。高濃度benzalkonium chloride也會影響細胞的DNA、RNA、protein和collagen合成，因此長期使用這些藥物可能會改變角膜內皮細胞的生理特性。

關鍵字

降眼壓 ； 角膜內皮細胞

並列摘要

Purpose: In the present study, it is investigated the effects of eicosanoid derivatives drugs such as bimatoprost, latanoprost, travoprost, unoprostone and prostaglandin F2α (PGF2α), preservative benzalkonium chloride to the changes of cellular biological responses and signal transductions including IP3, cAMP, cGMP and calcium mobility in cultured porcine corneal endothelial cells.
Methods: It is used 3H-thymidine, 3H-uridine and 3H-leucine uptakes to measure the cellular DNA, RNA and protein syntheses. The MTT value, 3H-proline uptake and western blotting methods were used to estimate the cellular viability, collagen synthesis rate and collagen protein expression. The cellular IP3, cAMP and cGMP concentrations were measured by uptaking the 3H-inositol, 3H-cAMP and 3H-cGMP. Intracellular calcium mobility was measured by spectrofluorometer after laoding fura-2-AM. The antiglaucoma drugs were diluted to 1/100, 1/1000 and 1/10000 from original concentrations.
Results: It was found that cellular DNA synthesis were inhibited by unoprostone (28 μM, 2.8 μM and 0.28 μM)、latanoprost (1.1 μM and 0.11 μM), travoprost (0.8 μM) and bimatoprost (7.2 μM) after 90 min drugs incubation. However, unoprostone (28 μM and 2.8 μM) and PGF2α (5.6 μM) increased the RNA synthesis. In the presence of unoprostone (28 μM), latanoprost (1.1 μM and 0.11 μM) and travoprost (0.8 μM) all suppressed protein synthesis. It showed that unoprostone (28 μM and 2.8 μM), latanoprost (1.1 μM and 0.11 μM), travoprost (0.8 μM and 0.08 μM) and bimatoprost (7.2 μM) all inhibited cellular collagen synthesis rate. Western blotting assay indicated that 28 μM unoprostone, 1.1 μM latanoprost, 7.2 μM bimatoprost and 5.6 μM PGF2α inhbited the collagen type I protein expresssion after 6 hours drugs incubation. In the case of benzalkonium chloride, it was found that only at 0.0001% inhibited the cellular DNA, RNA and protein synthesis and type I collagen expression. We found that 1.1 μM latanoprost, 0.8 μM travoprost, 7.2 μM bimatoprost, 5.6 μM PGF2α and 0.0001% benzalkonium chloride induced IP3 increase except 28 μM unoprostone inhibited IP3 concentrations. The cellular cAMP and cGMP concentrations were only enhanced by 28 μM unoprostone and 7.2 μM bimatoprost respectively. In calcium containing buffer, it was found that unoprostone (28 μM, 2.8 μM and 0.28 μM), latanoprost (1.1 μM, 0.11 μM, 0.011μM), travoprost (0.8 μM and 0.08 μM) and bimatoprost (7.2 μM and 0.72 μM) all increased the intracellular free calcium concentration [Ca2+]i. Neverthesless, 5.6 μM PGF2α and 0.0001% benzalkonium chloride decreased [Ca2+]i after addition of drugs. In the calcium-free containing buffer, only 28 μM unoprostone significantly enhanced [Ca2+]i increase. The endoplasmic reticulum-sensitive calcium and mitochondria-sensitive calcium were released by unoprostone. Moreover, the unoprostone-induced intracellular releases were regulated by phospholipase C and phospholipase A2.
Conclusion: Our results indicate that unoprostone and latanoprost and high concentration of benzalkonium chloride may decrease cellular viability, DNA, RNA, protein syntheses and collagen type 1 expression in porcine corneal endothelial cells. The latanoprost, travoprost, and bimatoprost all increase the IP3 concentrations. Nevertheless, unoprostone decreases cellular IP3 but increase cAMP concentration. The unoprostone, latanoprost, travoprost and bimatoprost all increase the extracellular calcium influx, however, only unoprostone increase the intracellular calcium store rlease and may be mediated by phospholipase C and phopholipase A pathways. Thus, this study shows that long-term use of those eicosanoid derivatives drugs may change the physiological functions and signal transductions in corneal endothelial cells.

並列關鍵字

unoprostone ； corneal endothelial cells ； eicosanoid derivatives ； travoprost ； latanoprost ； bimatoprost

參考文獻

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