| Summary: | 碩士 === 高雄醫學大學 === 醫學研究所碩士班 === 93 === 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.
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