Mechanisms and Modulation of Opsin 3 Mediated Airway Smooth Muscle Relaxation

项目摘要/摘要 最近已经证实了光介导的平滑肌松弛。非视觉光学系统 在平滑肌上的表达被认为是通过可见光进行松弛的关键媒介 频谱。然而，放松的机制还没有完全被理解。我们小组已经证明了 这种现象也存在于由视蛋白调节的光介导的松弛的气道平滑肌中 受体偶联到G蛋白信号通路。在初步研究中，我们证明了呼吸道畅通 肌肉光松弛是波长和强度依赖的，光在肌肉之间诱导相互作用 免疫共沉淀法检测到视蛋白3受体和GαS的表达。这与以下情况有关： 细胞内cAMP，已知的呼吸道平滑肌松弛的关键第二信使。我们建议 确定视蛋白受体亚型(S)、G蛋白(S)和细胞信号通路(如cAMP、cGMP、 和调节呼吸道顺畅的细胞骨架调节蛋白(RhoA、CPI-17和肌球蛋白轻链-20) 肌肉光松弛。我们还将探索通过调制光波长来激活的机制 气道平滑肌的敏感性。正常情况下，视素受体结合11-顺式视网膜(一个发色团和联合 具有光子的激活剂)以形成席夫碱。11顺式视网膜允许光异构化改变 视蛋白受体的构象，它诱导下游的细胞信号。视网膜的衍生物有 已经被证明可以将视蛋白的敏感度转移到更长的波长。在我们的提案中，我们演示了使用 11顺式2，3双脱氢视网膜使气道平滑肌的敏感性向更长的波长移动。我们建议 在细胞、体内和体外模型中评估额外的视网膜衍生物，以确定可以 改变内源性视蛋白的波长敏感性，以响应更长波长的光， 穿透身体。在眼睛中，对不同波长的光的敏感性不仅是特定的 配体也是视蛋白受体亚型的一种功能表达。对OPN1变种的转基因将使 不同波长光作用下激活细胞的气道平滑肌特异性配体的评价 信号与放松是一致的。我们将探索胡萝卜素对视蛋白受体的直接激活。 在没有光的情况下的代谢物。我们将证明胡萝卜素的内源性代谢物β-紫罗兰酮， 是一种植物化学物质，在没有光的情况下可以直接激活视蛋白3。我们将展示同样的专业- 当-3直接激活视蛋白3受体时，松弛细胞信号发生在气道平滑肌中。 离子与发色团/光激活的比较。这些拟议的研究将允许更好的 对先前在呼吸道表达的未知受体的激活和信号传递的理解 调节前松弛信号通路的平滑肌肉。

Project Summary/ Abstract Light-mediated relaxation of smooth muscle has been recently demonstrated. Non-visual opsins expressed on smooth muscle have been implicated as the key mediator of relaxation via light within the visible spectrum. However the mechanism of relaxation has yet to be fully understood. Our group has demonstrated that this phenomenon is also present in airway smooth muscle with light-mediated relaxation modulated by opsin receptors coupled to G protein signaling pathways. In preliminary studies, we demonstrate that airway smooth muscle photorelaxation is wavelength- and intensity-dependent and that light induces an interaction between the opsin 3 receptor and Gαs demonstrated by co-immunoprecipitation. This was associated with an increase in intracellular cyclic AMP, a known key second messenger of airway smooth muscle relaxation. We propose to identify the opsin receptors subtype(s), the G protein(s) and the cellular signaling pathways (e.g. cAMP, cGMP, and cytoskeletal regulatory proteins (RhoA, CPI-17 and myosin light chain-20) that modulate airway smooth muscle photorelaxation. We will also explore the mechanism of activation by modulating the light wavelength sensitivity of airway smooth muscle. Normally opsin receptors bind 11-cis-retinal (a chromophore and co- activator with photons of light) to form a Schiff base. The 11-cis-retinal allows for photoisomerization that changes the conformation of the opsin receptor, which induces downstream cellular signaling. Derivatives of retinal have been shown to shift the sensitivity of opsins to longer wavelengths. In our proposal we demonstrate that the use of 11-cis 2,3 didehydroretinal shifts airway smooth muscle sensitivity to longer wavelengths. We propose to evaluate additional retinal derivatives in cellular, in vivo and ex vivo models to identify chromophores that can shift the wavelength sensitivity of endogenous opsins to respond to longer wavelengths of light that may penetrate the body. In the eye, the sensitivity to different wavelengths of light is not only a function of the specific ligand but also a function of the subtype of opsin receptor expressed. Transfection of variants of OPN1 will allow evaluation of specific ligands at varying wavelengths of light in airway smooth muscle that activate cellular signaling congruent with relaxation. We will explore the direct activation of opsin receptors by carotene metabolites in the absence of light. We will demonstrate that the endogenous metabolite of carotenes, β-ionone, is a phytochemical that can directly activate opsin 3 in the absence of light. We will show that the same pro- relaxant cellular signaling occurs in airway smooth muscle when the opsin 3 receptor is directly activated by - ionine as compared to activation by chromophores/light. These proposed studies will allow for a better understanding of the activation and signaling of a previously uncharacterized receptor expressed in airway smooth muscle that modulates pro-relaxant signaling pathways.