Department of Pharmacology, Keio University, Tokyo, Japan.
Department of Physiology, School of Medical Sciences, New Zealand National Eye Centre, University of Auckland, New Zealand.
Exp Eye Res. 2019 Sep;186:107707. doi: 10.1016/j.exer.2019.107707. Epub 2019 Jun 20.
The transient receptor protein vanilloid channels, TRPV1 and TRPV4, have recently been shown to be mechanosensors in the ocular lens that act to transduce physical changes in lens volume and internal hydrostatic pressure into the activation of signalling pathways in lens epithelial cells. These pathways in turn regulate ion and water transport to ensure that the optical properties of the lens remain constant. Despite the functional evidence that implicate the roles of TRPV1 and TRPV4 in the lens, their respective cellular expression patterns in the different regions of the lens has to date not been fully characterised. Using Western blotting we have confirmed that TRPV1 and TRPV4 are expressed throughout all regions (epithelium, outer cortex, inner cortex/core) of the adult mouse lens. Subsequent immunolabeling of lens cryosections confirmed that TRPV1 and TRPV4 are expressed throughout all regions of the lens, but revealed differentiation-dependent differences in the subcellular expression of the two channels in the different regions. In the epithelium and outer cortex, intense TRPV1 and TRPV4 labeling was predominately associated with the cytoplasm. In a discrete zone in the inner cortex, labeling for both proteins was greatly diminished, but could be enhanced by incubating sections with the detergent Triton X-100 to reveal TRPV1 and TRPV4 labelling that was associated with the membrane. This suggests that in this region of the lens there is a potential interacting protein that masks the binding of the TRPV1 and TRPV4 antibodies to their respective epitopes in the lens inner cortex. In the core of the lens, which contains the embryonic nucleus, TRPV1 and TRPV4 labelling was associated exclusively with fibre cell membranes. This labelling in the lens core of the adult mouse lens appeared to originate in early development as a similar membrane labelling was observed at embryonic day 10 (E10) of the cells in the lens vesicle that subsequently forms the embryonic nucleus in the adult lens. During subsequent stages of embryonic development TRPV1 and TRPV4 remained membranous in the inner cortex and core, while showing labelling that was associated with the cytoplasm in the superficial outer cortical region. The extent of cytoplasmic labelling for TRPV4, but not TRPV1, in this cortical region could however be dynamically regulated by cutting the zonules that normally attach the lens to the ciliary body. We have shown an early onset and continuous expression of TRPV1 and TRPV4 across all lens regions, and that TRPV4 can be dynamically trafficked into the membranes of differentiating fibre cells, results that suggests that these mechanosensitive channels may also be functionally active in lens fibre cells.
瞬时受体蛋白香草素通道 TRPV1 和 TRPV4 最近被证明是眼部晶状体中的机械感受器，可将晶状体体积和内部静水压力的物理变化转导为晶状体上皮细胞中信号通路的激活。这些途径反过来又调节离子和水的运输，以确保晶状体的光学性质保持不变。尽管有功能证据表明 TRPV1 和 TRPV4 在晶状体中起作用，但它们在晶状体不同区域的各自细胞表达模式迄今尚未完全描述。通过 Western blot 分析，我们已经证实 TRPV1 和 TRPV4 均在成年小鼠晶状体的所有区域（上皮、外皮层、内皮层/核）表达。晶状体冷冻切片的免疫标记进一步证实 TRPV1 和 TRPV4 均在晶状体的所有区域表达，但揭示了在不同区域中两种通道的亚细胞表达存在分化依赖性差异。在上皮和外皮层，强烈的 TRPV1 和 TRPV4 标记主要与细胞质相关。在内皮层的一个离散区域中，两种蛋白质的标记均大大减少，但用去污剂 Triton X-100 孵育切片可增强标记，从而揭示与膜相关的 TRPV1 和 TRPV4 标记。这表明在晶状体的这个区域存在一种潜在的相互作用蛋白，该蛋白掩盖了 TRPV1 和 TRPV4 抗体与其在晶状体内皮层中的相应表位的结合。在晶状体的核（包含胚胎核）中，TRPV1 和 TRPV4 标记仅与纤维细胞膜相关。成年小鼠晶状体核心中的这种标记似乎起源于早期发育，因为在晶状体囊泡中的细胞（随后在成年晶状体中形成胚胎核）中观察到类似的膜标记，该囊泡在胚胎第 10 天 (E10)。在随后的胚胎发育阶段，TRPV1 和 TRPV4 在晶状体内皮层和核中保持膜性，而在外皮层浅层区域中则与细胞质相关。在这个皮质区域中，TRPV4 的细胞质标记程度（但不是 TRPV1）可以通过切割通常将晶状体连接到睫状体的晶状体悬韧带来动态调节。我们已经证明 TRPV1 和 TRPV4 在所有晶状体区域中的早期出现和持续表达，并且 TRPV4 可以动态地运输到分化纤维细胞的膜中，这一结果表明这些机械敏感通道在晶状体纤维细胞中也可能具有功能活性。
