Blebbistatin, a novel inhibitor of myosin II ATPase activity, increases aqueous humor outflow facility in perfused enucleated porcine eyes
Blebbistatin, a novel inhibitor of myosin II ATPase activity, increases aqueous humor outflow facility in perfused enucleated porcine eyes
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DOI：
10.1167/iovs.05-0164
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发表时间：
2005-11-01
影响因子：
4.4
通讯作者：
Rao, PV
中科院分区：
文献类型：
--
作者：
Zhang, M;Rao, PV
目的。目的探讨细胞收缩的关键生化决定因子肌球蛋白II在小梁细胞(TM)房水流出调节中的特殊作用。方法：采用RT-PCR方法检测非肌型肌球蛋白II重链(IIA、IIB和IIC)在人眼小梁细胞和睫状体(CB)细胞中的表达。研究了肌球蛋白II对肌球蛋白II腺苷三磷酸酶(ATPase)活性的抑制作用对猪TM和CB细胞的细胞形态、肌动蛋白组织和细胞黏附的影响。用恒压Grant灌流模型系统测定摘除眼球后房水流出设施的变化。结果：非肌肉肌球蛋白IIA和IIB在人TM和CB细胞中均有表达。原代培养的猪TM和CB细胞在含血清的条件下经Blebbistatin处理后，细胞形态发生了剂量依赖性的改变(10-200 mU M)，肌动蛋白应激纤维含量减少，局部粘连和粘连连接减少。这些变化在药物从细胞培养液中撤除后24小时内被发现是可逆的。灯盏花素不影响TM细胞肌球蛋白轻链的磷酸化状态。用100mU和200mU的M blbbistatin灌流去核猪眼5h，房水流出设施中的房水流出设施(分别为53%和%)显著增加(P<；0.01，n=7)，而假对照标本的设施设施增加了21%。在药物灌流的猪眼中发现房水丛内壁的完整性是完整的，TM细胞的形态似乎与假治疗眼相似。结论：这些数据表明选择性地抑制房水流出通路中的肌球蛋白II导致房水流出设施的增加，提示肌球蛋白II在房水流出设施的调节中起着关键作用。这项研究还表明，肌球蛋白II是降低青光眼患者眼压的潜在治疗靶点。
PURPOSE. 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.METHODS. 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.RESULTS. 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 mu M)-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 mu M 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.CONCLUSIONS. 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.