Zhang Min, Rao P Vasantha
Department of Ophthalmology, Duke University School of Medicine, Durham, NC 27710, USA.
Invest Ophthalmol Vis Sci. 2005 Nov;46(11):4130-8. doi: 10.1167/iovs.05-0164.
To investigate the specific role of myosin II, a critical biochemical determinant of cellular contraction, in modulation of aqueous humor outflow facility through the trabecular meshwork (TM) pathway.
Expression of the nonmuscle myosin II heavy chains (IIA, IIB, and IIC) in human TM and ciliary body (CB) cells was determined by RT-PCR analyses. The effects of inhibition of myosin II on cell morphology, actomyosin organization, and cell adhesions were evaluated in porcine TM and CB cells treated with blebbistatin, a cell-permeable, specific inhibitor of myosin II adenosine triphosphatase (ATPase) activity. Changes in aqueous humor outflow facility were determined in enucleated porcine eyes by using a constant-pressure Grant perfusion model system. Ultrastructural integrity of the outflow pathway in drug-perfused eyes was analyzed by transmission electron microscopy.
Expression of nonmuscle myosin IIA and IIB was confirmed in both human TM and CB cells. Confluent cultures of primary porcine TM and CB cells treated with blebbistatin in the presence of serum revealed dose (10-200 microM)-dependent changes in cell morphology, decreases in actin stress fiber content and in focal adhesions and adherens junctions. These changes were found to be reversible within 24 hours of drug withdrawal from the cell culture media. Blebbistatin did not affect the status of myosin light chain phosphorylation in TM cells. Perfusion of enucleated porcine eyes for 5 hours with 100 and 200 microM blebbistatin produced a significant increase (P < 0.01, n = 7) in aqueous outflow facility (53% and 64%, respectively) from the baseline facility, compared with a 21% facility increase in sham control specimens. The integrity of the inner wall of aqueous plexi in drug-perfused porcine eyes was found to be intact, and TM cell morphology appeared to be similar to that noted in sham-treated eyes.
These data demonstrate that selective inhibition of myosin II in the aqueous humor outflow pathway leads to increased aqueous outflow facility, suggesting a critical role for myosin II in the regulation of aqueous humor outflow facility. This study also suggests myosin II as a potential therapeutic target for lowering intraocular pressure in patients with glaucoma.
研究肌球蛋白II（细胞收缩的关键生化决定因素）在通过小梁网（TM）途径调节房水流出易度中的具体作用。
通过逆转录聚合酶链反应（RT-PCR）分析确定非肌肉肌球蛋白II重链（IIA、IIB和IIC）在人TM和睫状体（CB）细胞中的表达。在用肌球蛋白II特异性细胞渗透性抑制剂blebbistatin处理的猪TM和CB细胞中，评估肌球蛋白II抑制对细胞形态、肌动球蛋白组织和细胞黏附的影响。通过使用恒压格兰特灌注模型系统，在摘除眼球的猪眼中确定房水流出易度的变化。通过透射电子显微镜分析药物灌注眼流出途径的超微结构完整性。
在人TM和CB细胞中均证实了非肌肉肌球蛋白IIA和IIB的表达。在血清存在下用blebbistatin处理的原代猪TM和CB细胞汇合培养物显示出细胞形态的剂量（10 - 200 microM）依赖性变化，肌动蛋白应力纤维含量以及粘着斑和黏附连接减少。发现这些变化在从细胞培养基中撤出药物后24小时内是可逆的。Blebbistatin不影响TM细胞中肌球蛋白轻链磷酸化状态。用100和200 microM blebbistatin对摘除眼球的猪眼灌注5小时，与假手术对照标本中房水流出易度增加21%相比，房水流出易度从基线易度显著增加（P < 0.01，n = 7）（分别为53%和64%）。发现药物灌注猪眼中房水丛内壁的完整性完好，且TM细胞形态似乎与假手术处理眼中的相似。
这些数据表明，在房水流出途径中选择性抑制肌球蛋白II会导致房水流出易度增加，提示肌球蛋白II在调节房水流出易度中起关键作用。本研究还表明肌球蛋白II作为降低青光眼患者眼压的潜在治疗靶点。
