Characterization of Soluble Guanylate Cyclase in NO-Induced Increases in Aqueous Humor Outflow Facility and in the Trabecular Meshwork
Characterization of Soluble Guanylate Cyclase in NO-Induced Increases in Aqueous Humor Outflow Facility and in the Trabecular Meshwork
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DOI：
10.1167/iovs.08-2750
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发表时间：
2009-04-01
影响因子：
4.4
通讯作者：
Chokshi, Binna M.
中科院分区：
文献类型：
--
作者：
Ellis, Dorette Z.;Dismuke, William M.;Chokshi, Binna M.
目的.一氧化氮（NO）增加了房水排出眼睛的速率;然而，可溶性鸟苷酸环化酶（sGC）的参与尚不清楚。本研究探讨了sGC在介导NO诱导的流出设施增加中的作用。应用眼前节器官培养灌流系统测量猪眼的流出功能。通过酶免疫测定法测定低传代猪和人小梁网（TM）细胞和转化的人TM细胞中cGMP的产生来评估sGC活性。使用蛋白质印迹分析测定sGC α和sGC β同种型表达。sGC的激活对于NO诱导的流出设施的增加是必要的（0.3215 μ L/min/mm Hg [基线流出设施] +/- 0.0837 [SEM]）。1H-[1，2，4]恶二唑并[4，3-a]喹喔啉-1-1（ODQ）可抑制NO诱导的sGC活性。总蛋白的Western印迹分析表明sGC α和sGC β亚基表达的当量比。然而，在转化的细胞组分中，胞质sGC α亚基的表达水平与低传代人TM细胞相比有所降低。sGC的激活参与了NO诱导的流出功能的增加。α和β sGC亚基以相等比例的表达将表明功能性sGC异源二聚体，因为α-NO增加了低传代人和猪TM细胞中的cGMP水平。然而，β-NO不能引起转化的TM细胞中cGMP水平的增加表明，尽管sGC异二聚体是必需的，但它是不够的，并且可能需要转化细胞中不存在的其他因子。（Invest Ophthalmol维斯科学。2009; 50：1808-1813）DOI：10.1167/iovs.08-2750
PURPOSE. Nitric oxide (NO) increases the rate at which aqueous humor exits the eye; however, the involvement of soluble guanylate cyclase (sGC) is unknown. This study investigated the role of sGC in mediating the NO-induced increases in outflow facility.METHODS. Outflow facility was measured in porcine eyes using the anterior segment organ culture perfusion system. sGC activity was assessed by cGMP production in low-passage porcine and human trabecular meshwork (TM) cells and transformed human TM cells, as measured by enzyme immunoassay. sGC alpha and sGC beta isoform expression were determined using Western blot analysis.RESULTS. Activation of sGC is necessary for the NO-induced increases in outflow facility (0.3215 mu L/min per mm Hg [baseline outflow facility] +/- 0.0837 [SEM]). NO resulted in increased sGC activity that was abolished by 1H-[1,2,4] oxadiazolo[ 4,3-a] quinoxalin-1-1 (ODQ). Western blot analysis of total protein demonstrated an equivalent ratio of sGC alpha and sGC beta subunit expression. In transformed cell fractions, however, the level of cytoplasmic sGC alpha subunit expression was decreased compared with low-passage human TM cells.CONCLUSIONS. Activation of sGC is involved in the NO-induced increases in outflow facility. The expression of alpha and beta sGC subunits in an equivalent ratio would suggest a functional sGC heterodimer because DETA-NO increased cGMP levels in low-passage human and porcine TM cells. However, the inability of DETA-NO to cause increases in cGMP levels in transformed TM cells suggests that though the sGC heterodimer is necessary, it is not sufficient and may require other factors not present in transformed cells. (Invest Ophthalmol Vis Sci. 2009; 50: 1808-1813) DOI: 10.1167/iovs.08-2750