Department of Physiology, Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6085, USA.
Invest Ophthalmol Vis Sci. 2011 Oct 10;52(11):7996-8005. doi: 10.1167/iovs.11-8170.
To test whether adenosine triphosphate (ATP) release links cytoskeletal remodeling with release of matrix metalloproteinases (MMPs), regulators of outflow facility and intraocular pressure.
ATP release was measured by luciferin-luciferase. Ecto-ATPases from transformed human trabecular meshwork (TM) cells (TM5) and explant-derived TM cells were identified by RT-PCR. Actin was visualized by phalloidin staining. Cell viability was assayed by lactate dehydrogenase and thiazolyl blue tetrazolium bromide methods and propidium iodide exclusion, gene expression by real-time PCR, and MMP release by zymography. Cell volume was monitored by electronic cell sorting.
Hypotonicity (50%) and mechanical stretch increased ATP release with similar pharmacologic profiles. TM cells expressed ecto-ATPases E-NPP1-3, E-NTPD2, E-NTPD8, and CD73. Prolonged dexamethasone (DEX) exposure (≥ 2 weeks), but not brief exposure (3 days), increased cross-linked actin networks and reduced swelling-triggered ATP release. Cytochalasin D (CCD) exerted opposite effects. Neither DEX nor CCD altered the cell viability, gene expression, or pharmacologic profile of ATP-release pathways. DEX accelerated, and CCD slowed, the regulatory volume decrease after hypotonic exposure. Activating A(1) adenosine receptors (A(1)ARs) increased total MMP-2 and MMP-9 release. DEX reduced total A(1)AR-triggered MMP release, and CCD increased the active form of MMP-2 release. The A(1)AR agonist CHA and the A(1)AR antagonist DPCPX partially reversed the effects of DEX and CCD, respectively.
Cytoskeletal restructuring modulated swelling-activated ATP release, in part by changing the duration of cell swelling after hypotonic challenge. Modifying ATP release is expected to modulate MMP secretion by altering ecto-enzymatic delivery of adenosine to A(1)ARs, linking cytoskeletal remodeling and MMP-mediated modulation of outflow facility.
检测三磷酸腺苷（ATP）释放是否将细胞骨架重塑与细胞外基质金属蛋白酶（MMPs）的释放联系起来，后者是房水流出阻力和眼内压的调节剂。
通过荧光素酶-荧光素法测量 ATP 释放。通过 RT-PCR 鉴定转化的人眼小梁网（TM）细胞（TM5）和组织衍生 TM 细胞中的细胞外 ATP 酶。用鬼笔环肽染色显示肌动蛋白。通过乳酸脱氢酶和噻唑蓝四唑溴盐法和碘化丙啶排斥法检测细胞活力，通过实时 PCR 检测基因表达，通过酶谱法检测 MMP 释放。通过电子细胞分选监测细胞体积。
低渗（50%）和机械拉伸增加了具有相似药理学特征的 ATP 释放。TM 细胞表达细胞外 ATP 酶 E-NPP1-3、E-NTPD2、E-NTPD8 和 CD73。延长地塞米松（DEX）暴露（≥2 周）而不是短暂暴露（3 天）增加了交联肌动蛋白网络并减少了肿胀触发的 ATP 释放。细胞松弛素 D（CCD）则产生相反的效果。DEX 和 CCD 均未改变 ATP 释放途径的细胞活力、基因表达或药理学特征。DEX 加速了低渗暴露后的调节性体积减少，而 CCD 则减缓了该过程。激活 A1 腺苷受体（A1ARs）增加了总 MMP-2 和 MMP-9 的释放。DEX 减少了总 A1AR 触发的 MMP 释放，而 CCD 增加了 MMP-2 活性形式的释放。A1AR 激动剂 CHA 和 A1AR 拮抗剂 DPCPX 分别部分逆转了 DEX 和 CCD 的作用。
细胞骨架重构调节了肿胀激活的 ATP 释放，部分原因是改变了低渗刺激后细胞肿胀的持续时间。改变 ATP 释放有望通过改变细胞外酶促传递腺苷到 A1ARs，将细胞骨架重塑与 MMP 介导的流出阻力调节联系起来，从而调节 MMP 分泌。
