Non-Visual Opsins & Vasoregulation: Implications for Vascular Therapy

 描述(由申请人提供)：神经体液信号传导中的微管介导的血管运动功能受损和/或内皮功能受损是导致高血压、冠状动脉疾病、中风、勃起功能障碍等血管疾病和雷诺现象(RP)等特殊血管疾病的直接原因。调节血管张力的新途径的发现为血管疾病的治疗提供了新的靶点。我们最近发现了一种调节血管张力的新机制。黑素蛋白(Opn4)典型地存在于视网膜神经节细胞中，在那里它们调节昼夜节律和睡眠。我们已经在许多哺乳动物的血管和许多血管床中发现了一种非视觉光(Opsin)受体Opn4。这些受体介导强度依赖的血管对特定波长(蓝430-460 nm)光的松弛。初步数据表明，S的信号转导机制涉及可溶性鸟苷环化酶和磷酸二酯酶6，而不涉及蛋白激酶G。光激活导致血管超极化，这一过程涉及K+通道。该受体可能是一个经典的G蛋白偶联受体，因为抑制G蛋白受体激酶可以防止刺激依赖的脱敏，并显著降低产生松弛反应所需的光强度。最后，在体内，蓝光能够引起生理反应，显著增加小鼠尾部动脉的血流量。我们假设：1)Opn4是血管扩张功能的重要调节因子，并在体内介导一种生理功能；2)血管中的信号转导机制模仿脊椎动物的视觉视蛋白或无脊椎动物的“简单”光感受器；3)通过GRK2/arrestin机制发生脱敏/调节；以及4)这一途径在没有信号(血管扩张)受损而血管收缩增强的疾病中上调，如RP。在这个建议中，我们计划：1)利用Opn4-/-小鼠和新发现的Opn4抑制剂opsinamide，进一步表征opn4在血管调节中的潜在生理作用，使用分离血管的肌肉造影和颅窗激光多普勒作为活体终点；2)确定受体和G蛋白到通道的信号转导机制/S使用特定的抑制剂和shRNA敲除途径蛋白，以了解受体的调节；3)使用新型药理抑制剂GRK2，帕罗西汀，血管平滑肌选择性GRK2-/-和arrestin-/-小鼠，以及GRK2和arrestin-/-小鼠，以及GRK2和arrestin腺病毒敲除分离的血管。4)确定Opn4受体在RP小鼠模型中的作用，并利用激光血流多普勒和皮肤微透析技术探讨活体介导正常人和原发性RP患者皮肤循环中光松弛的机制。通过这种方式，我们希望深入了解这一新途径在健康和疾病中的功能和功能障碍，并确定其作为新的治疗靶点的潜力。

 DESCRIPTION (provided by applicant): Impaired vasomotor function mediated by pertubations in neurohumoral signaling and/or impaired endothelial function is the proximate cause of vascular diseases such as hypertension, coronary artery disease, stroke, erectile dysfunction, and specific vasculopathies such as Raynaud's phenomenon (RP). The identification of novel pathways that regulate vascular tone provides new targets for treatment of vascular disorders. We have recently identified a novel mechanism that modulates vascular tone. Melanospin (Opn4) are classically found in the retinal ganglion cells where they regulate circadian rhythm and sleep. We have identified a non-visual light (opsin) receptor, Opn4, in blood vessels from a number of mammalian species, and from number of vascular beds. These receptors mediated intensity-dependent vasorelaxation to light of a specific wavelength (blue 430-460nM). Preliminary data suggest that signal transduction mechanism(s) involve soluble guanylate cyclase and phosphodiesterase 6 but not protein kinase G. Light activation leads to vascular hyperpolarization a process that involves K+ channels. The receptor is likely regulated as a classic G-protein coupled receptor in that inhibition of G-protein receptor kinase prevents stimulus-dependent desensitization and significantly decrease the light intensity needed to produce a relaxation response. Finally, in vivo, blue light is able evoke a physiologic response; significantly increasing blood flow in the mouse tail artery. We hypothesize that 1) Opn4 is an important regulator of vasodilatory function and mediates a physiologic function in vivo; 2) the signal transduction mechanism in vessels mimics visual opsins of vertebrates or "simple" photoreceptors of invertebrates; 3) desensitization/regulation occurs by a GRK2/arrestin mechanism; and 4) this pathway is upregulated in diseases in which NO signaling (vasodilation) is impaired and vasocontriction is enhanced such as RP. In this proposal, we plan to: 1) Further characterize the potential physiologic role of Opn 4 in vasoregulation utilizing Opn4-/- mice and newly discovered Opn4 inhibitors, opsinamides, using myography in isolated vessels and laser doppler in cranial windows as endpoints in vivo; 2) Determine signal transduction mechanism/s from receptor and G protein to channel with sharp electrode measurement of membrane potential in isolated vessels using specific inhibitors and shRNA knockdown of pathway proteins; 3) Understand receptor regulation using novel pharmacologic inhibitor of GRK2, paroxetine, vascular smooth muscle-selective GRK2-/-, and arrestin-/- mice, as well as GRK2 and arrestin shRNA adenovirus knockdown in isolated vessels. 4) Determine the role of Opn4 receptors as a target in mouse models of RP and explore mechanisms mediating photorelaxation in vivo in the cutaneous circulation of healthy humans and those with primary RP using laser flow doppler and skin microdialysis. In this way we hope to gain insight into the function and dysfunction of this novel pathway in health and disease, and determine its potential as a novel therapeutic target.