Zhong Yisheng, Gao Jian, Ye Wen, Huang Ping, Cheng Yu, Jiao Qin
Department of Ophthalmology, Ruijin Hospital Affiliated with Shanghai Jiaotong University, Shanghai, China.
Ophthalmic Res. 2007;39(4):232-40. doi: 10.1159/000104832. Epub 2007 Jun 29.
To study the interaction between latanoprost and pilocarpine on cultured rabbit ciliary muscle (RCM) cells, and investigate the time courses of the two drugs, when given alone or in combination.
Cultured RCM cells were treated for 24 h with different concentrations of latanoprost acid, pilocarpine and mixtures of latanoprost acid and pilocarpine. RNA was extracted, expressions of matrix metalloproteinase 1 (MMP-1), tissue inhibitor of metalloproteinase 1 (TIMP-1) and TIMP-2 were detected by reverse transcription-polymerase chain reaction (RT-PCR), and the optimum concentrations of those drugs were found. Then the cells were treated with the optimum concentrations of those drugs for various periods. RNA was extracted after the treatment and expressions of MMP-1, TIMP-1 and TIMP-2 were detected by RT-PCR again. Changes in Ca(2+) were estimated by fluorescence measurement using the Ca(2+) indicator Fluo-3 AM with a laser scanning confocal microscope. Ca(2+) of each cell was monitored continually after administration of the drugs. Gray values at 5 s and 2, 4, 6, 8 and 10 min were chosen for statistical analysis, and the influence and time-effect relationship of those drugs on Ca(2+) of the cultured cells were evaluated.
Exposure of the cells to increasing concentrations of latanoprost acid induced increased MMP-1 mRNA and decreased TIMP-1 and TIMP-2 mRNA in a dose-dependent manner. After 24 h of treatment, the optimum concentration of latanoprost acid for maximal changes in MMP-1 and TIMP-2 expression was 2 x 10(-7)M, and for maximal changes in TIMP-1 expression, the optimum concentration was 5 x 10(-7)M. When the optimum concentrations of latanoprost acid were chosen to treat the cells for various periods, the optimum time of the peak MMP-1 expression and trough TIMP-1 expression was 24 h, and of the trough TIMP-2 expression, it was 36 h after initiation of treatment. No significant expression changes of MMP-1, TIMP-1 and TIMP-2 mRNA were found when the cells were treated with pilocarpine at any concentration or at any time. Exposure of the cells to the mixtures of latanoprost acid and pilocarpine induced the same changes and time course of MMP-1, TIMP-1, and TIMP-2 mRNA expression as exposure of the cells to latanoprost acid alone. Exposure of ciliary muscle cells to pilocarpine induced an increase in Ca(2+), with the peak of increase observed at 5 s after initiation of treatment; then Ca(2+) gradually decreased near to baseline level within 10 min. Exposure of the cells to latanoprost acid did not significantly change Ca(2+). Exposure of the cells to the mixtures of latanoprost acid and pilocarpine induced the same Ca(2+) change as exposure to pilocarpine alone.
Latanoprost and pilocarpine have no interaction in their various effects on the cultured RCM cells.
研究拉坦前列素与毛果芸香碱对培养的兔睫状肌（RCM）细胞的相互作用，并探讨两种药物单独或联合使用时的时间进程。
用不同浓度的拉坦前列素酸、毛果芸香碱以及拉坦前列素酸与毛果芸香碱的混合物处理培养的RCM细胞24小时。提取RNA，通过逆转录-聚合酶链反应（RT-PCR）检测基质金属蛋白酶1（MMP-1）、金属蛋白酶组织抑制剂1（TIMP-1）和TIMP-2的表达，找出这些药物的最佳浓度。然后用这些药物的最佳浓度处理细胞不同时间。处理后提取RNA，再次通过RT-PCR检测MMP-1、TIMP-1和TIMP-2的表达。使用Ca(2+)指示剂Fluo-3 AM通过激光扫描共聚焦显微镜荧光测量估计Ca(2+)的变化。给药后持续监测每个细胞的Ca(2+)。选择5秒以及2、4、6、8和10分钟时的灰度值进行统计分析，评估这些药物对培养细胞Ca(2+)的影响和时间效应关系。
细胞暴露于浓度递增的拉坦前列素酸中，MMP-1 mRNA表达增加，TIMP-1和TIMP-2 mRNA表达呈剂量依赖性降低。处理24小时后，MMP-1和TIMP-2表达最大变化时拉坦前列素酸的最佳浓度为2×10(-7)M，TIMP-1表达最大变化时的最佳浓度为5×10(-7)M。当选择拉坦前列素酸的最佳浓度处理细胞不同时间时，MMP-1表达峰值和TIMP-1表达谷值的最佳时间为处理开始后24小时，TIMP-2表达谷值的最佳时间为处理开始后36小时。用任何浓度的毛果芸香碱在任何时间处理细胞时，未发现MMP-1、TIMP-1和TIMP-2 mRNA表达有明显变化。细胞暴露于拉坦前列素酸与毛果芸香碱的混合物中，MMP-1、TIMP-1和TIMP-2 mRNA表达的变化和时间进程与细胞单独暴露于拉坦前列素酸时相同。睫状肌细胞暴露于毛果芸香碱中会导致Ca(2+)增加，在处理开始后5秒观察到增加峰值；然后Ca(2+)在10分钟内逐渐降至接近基线水平。细胞暴露于拉坦前列素酸中未显著改变[Ca(
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