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

我们有证据表明，在非色素睫状体上皮细胞（NPE）TRPV 4通道相互作用， 连接蛋白-50以形成响应机械刺激的机制。NPE细胞的变形是 能够引起TRPV 4通道激活，这反过来又引起半通道开放， NPE的面向水性体液的表面。这意味着睫状体分泌 水状体，具有能够感测和响应由眼内液引起的变形的机制。 眼内压升高。我们的假设是机械敏感性半通道 开放和ATP释放是降低Na，K-ATP酶活性的自分泌反馈回路中的步骤 在NPE。在这里，我们提出了关于半通道开放机制如何感知和 对机械刺激作出反应（目标1）。我们将描述半通道机制如何 以TRPV 4激活为支点，连接蛋白-50 vs泛连接蛋白-1的作用，TRPV 4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。 半通道目的2研究将检查半通道开放如何允许ATP离开细胞， 激活受体和信号通路，以自分泌方式改变Na，K-ATP酶活性。 我们还将研究cAMP和褪黑激素通过半通道释放到房水中。研究 目的3将确定眼内压对NPE半通道机制的影响， 离体动脉灌注眼准备和测试是否影响半通道阻断 分子和TRPV 4相互作用药物对房水形成速率的影响。的概念 NPE中Na，K-ATP酶活性的机械敏感性反馈调节是重要的， Na，K-ATP酶活性为房水分泌提供驱动力。NPE形成一个 血液和水之间的细胞屏障，因此当 眼内压的变化与睫状突基质中的流体静压有关。

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.