Hemichannels, TRPV4 and a mechanosensitive form of autocrine regulation in the NPE

我们有证据表明，无色素睫状体上皮(NPE)中的TRPV4通道与 连接蛋白-50形成一种对机械刺激作出反应的机制。NPE细胞的失真是 能够引起TRPV4通道激活，这进而导致在 NPE面向房水的表面。这意味着睫状体，它分泌 房水，有一种机制，能够感知和响应由 眼压升高。我们的工作假设是机械敏感的半通道 开放和ATP释放是降低Na，K-ATPase活性的自分泌反馈循环中的步骤 在NPE里。在这里，我们建议研究半通道开放机制如何意义和 对机械刺激作出反应(目标1)。我们将描述半通道机制是如何 以TRPV4激活为中心，连接蛋白-50和pAnnexin-1的作用，TRPV4通道如何响应 机械刺激(细胞肿胀和拉伸)以及细胞的电导信号 半条腿。AIM 2研究将检查半通道开放如何使ATP离开细胞，然后 激活受体和信号通路，以自分泌的方式改变Na，K-ATPase的活性。 我们还将研究cAMP和褪黑素通过半隧道释放到房水中。研究 在目标3中，将确定眼压对非晶状体上皮细胞半隧道机制的影响。 体外动脉灌注眼准备及检测半隧道封闭的效果 分子和TRPV4相互作用的药物对房水形成速度的影响。这一概念 NPE中Na，K-ATPase活性的机械敏感反馈调节意义重大，因为 Na，K-ATPase活性为房水分泌提供动力。NPE形成了一个 血液和水之间的细胞屏障在以下情况下会受到改变的物理力 眼压变化与睫状突间质液静压的关系。

We have evidence that TRPV4 channels in the nonpigmented ciliary epithelium (NPE) interact with connexin-50 to form a mechanism that responds to mechanical stimuli. Distortion of NPE cells is capable of causing TRPV4 channel activation which in turn causes hemichannel opening at the aqueous humor-facing surface of the NPE. What this means is the ciliary body, which secretes aqueous humor, has a mechanism capable to sensing and responding to distortion caused by an increase of intraocular pressure. Our working hypothesis is that mechanosensitive hemichannel opening and ATP release are steps in an autocrine feedback loop that reduces Na,K-ATPase activity in the NPE. Here, we propose studies on how the hemichannel opening mechanism senses and responds to a mechanical stimulus (Aim 1). We will characterize how the hemichannel mechanism pivots on TRPV4 activation, the role of connexin-50 vs pannexin-1, how TRPV4 channels respond to mechanical stimuli (cell swelling and stretch), and the electrical conductance signal of the hemichannels. Aim 2 studies will examine how hemichannel opening allows ATP to exit the cell then activate receptors and signaling pathways that change Na,K-ATPase activity in an autocrine fashion. We also will study cAMP and melatonin release into the aqueous humor via hemichannels. Studies in Aim 3 will determine the effect of intraocular pressure on the NPE hemichannel mechanism in an ex vivo arterially perfused eye preparation and test whether the effect of hemichannel blocking molecules and TRPV4-interacting drugs on the rate of aqueous humor formation. The concept of mechanosensitive feedback regulation of Na,K-ATPase activity in the NPE is significant because Na,K-ATPase activity provides the driving force for aqueous humor secretion. The NPE forms a cellular barrier between blood and aqueous and so is subjected to altered physical forces when intraocular pressure changes in relation to hydrostatic pressure in the ciliary process stroma.