Non-Visual Opsins & Vasoregulation: Implications for Vascular Therapy

 描述（由申请人提供）：由神经体液信号传导紊乱介导的血管功能受损和/或内皮功能受损是血管疾病（如高血压、冠状动脉疾病、中风、勃起功能障碍和特定血管病变（如雷诺现象（RP）的近因。对调节血管张力的新途径的鉴定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（Opn 4）通常存在于视网膜神经节细胞中，它们调节昼夜节律和睡眠。我们已经确定了一个非可见光（视蛋白）受体，Opn 4，在血管从一些哺乳动物物种，并从一些血管床。这些受体对特定波长（蓝色430- 460 nM）的光介导强度依赖性血管舒张。初步数据表明，信号转导机制涉及可溶性鸟苷酸环化酶和磷酸二酯酶6，但不涉及蛋白激酶G。光激活导致血管超极化，这是一个涉及K+通道的过程。该受体可能作为经典的G蛋白偶联受体调节，因为G蛋白受体激酶的抑制防止刺激依赖性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;显著增加小鼠尾动脉中的血流量。我们推测：1）Opn 4是血管舒张功能的重要调节因子，在体内介导生理功能; 2）血管中的信号转导机制模拟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GRK 2/arrestin机制发生脱敏/调节;和4）该途径在其中NO信号传导（血管舒张）受损和血管收缩增强的疾病如RP中上调。1）利用Opn 4-/-小鼠和新发现的Opn 4抑制剂，视蛋白酰胺，使用离体血管中的肌造影和颅窗中的激光多普勒作为体内终点，进一步表征Opn 4在血管调节中的潜在生理作用; 2）确定信号转导机制/s从受体和G蛋白到通道，用尖锐电极测量分离的血管中的膜电位，使用特异性抑制剂和途径蛋白的shRNA敲低; 3）理解使用GRK 2的新型药理学抑制剂帕罗西汀、血管平滑肌选择性GRK 2-/-和arrestin-/-小鼠以及GRK 2和arrestin shRNA腺病毒敲低在分离的血管中的受体调节。4)确定Opn 4受体在RP小鼠模型中作为靶点的作用，并使用激光血流多普勒和皮肤微透析在健康人和原发性RP患者的皮肤循环中探索体内介导光松弛的机制。通过这种方式，我们希望深入了解这种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.