Department of Physiology, University of Arizona, Tucson, 85724, USA.
Am J Physiol Cell Physiol. 2012 Jun 15;302(12):C1751-61. doi: 10.1152/ajpcell.00010.2012. Epub 2012 Apr 4.
In several tissues, transient receptor potential vanilloid 4 (TRPV4) channels are involved in the response to hyposmotic challenge. Here we report TRPV4 protein in porcine lens epithelium and show that TRPV4 activation is an important step in the response of the lens to hyposmotic stress. Hyposmotic solution (200 mosM) elicited ATP release from intact lenses and TRPV4 antagonists HC 067047 and RN 1734 prevented the release. In isosmotic solution, the TRPV4 agonist GSK1016790A (GSK) elicited ATP release. When propidium iodide (PI) (MW 668) was present in the bathing medium, GSK and hyposmotic solution both increased PI entry into the epithelium of intact lenses. Increased PI uptake and ATP release in response to GSK and hyposmotic solution were abolished by a mixture of agents that block connexin and pannexin hemichannels, 18α-glycyrrhetinic acid and probenecid. Increased Na-K-ATPase activity occurred in the epithelium of lenses exposed to GSK and 18α-glycyrrhetinic acid + probenecid prevented the response. Hyposmotic solution caused activation of Src family kinase and increased Na-K-ATPase activity in the lens epithelium and TRPV4 antagonists prevented the response. Ionomycin, which is known to increase cytoplasmic calcium, elicited ATP release, the magnitude of which was no greater when lenses were exposed simultaneously to ionomycin and hyposmotic solution. Ionomycin-induced ATP release was significantly reduced in calcium-free medium. TRPV4-mediated calcium entry was examined in Fura-2-loaded cultured lens epithelium. Hyposmotic solution and GSK both increased cytoplasmic calcium that was prevented by TRPV4 antagonists. The cytoplasmic calcium rise in response to hyposmotic solution or GSK was abolished when calcium was removed from the bathing solution. The findings are consistent with hyposmotic shock-induced TRPV4 channel activation which triggers hemichannel-mediated ATP release. The results point to TRPV4-mediated calcium entry that causes a cytoplasmic calcium increase which is an essential early step in the mechanism used by the lens to sense and respond to hyposmotic stress.
在几种组织中，瞬时受体电位香草醛 4（TRPV4）通道参与了对低渗挑战的反应。在这里，我们报告了猪晶状体上皮中的 TRPV4 蛋白，并表明 TRPV4 的激活是晶状体对低渗应激反应的重要步骤。低渗溶液（200mosM）可从完整晶状体中诱发出 ATP 释放，而 TRPV4 拮抗剂 HC 067047 和 RN 1734 可阻止这种释放。在等渗溶液中，TRPV4 激动剂 GSK1016790A（GSK）可引发 ATP 释放。当在灌流液中存在碘化丙啶（PI）（MW 668）时，GSK 和低渗溶液都会增加 PI 进入完整晶状体上皮的摄取。PI 摄取和对 GSK 和低渗溶液的反应所引起的 ATP 释放，均被阻断连接蛋白和半通道的混合剂、18α-甘草次酸和丙磺舒所抑制。在暴露于 GSK 和 18α-甘草次酸+丙磺舒的晶状体上皮中，Na-K-ATP 酶活性增加，而该反应可被丙磺舒所抑制。低渗溶液引起 Src 家族激酶在晶状体上皮中的激活并增加了 Na-K-ATP 酶的活性，而 TRPV4 拮抗剂则可阻止这种反应。已知可增加细胞质钙的离子霉素可诱发出 ATP 释放，而当晶状体同时暴露于离子霉素和低渗溶液时，其释放量并无增加。在无钙介质中，离子霉素诱导的 ATP 释放明显减少。在 Fura-2 加载的培养晶状体上皮中检测 TRPV4 介导的钙内流。低渗溶液和 GSK 均增加了细胞质钙，而 TRPV4 拮抗剂则可阻止这一作用。当从灌流液中去除钙时，对低渗溶液或 GSK 的反应引起的细胞质钙升高会被消除。这些发现与低渗休克诱导的 TRPV4 通道激活一致，该激活触发了半通道介导的 ATP 释放。结果表明，TRPV4 介导的钙内流导致细胞质钙增加，这是晶状体感知和响应低渗应激的机制中的一个基本早期步骤。
