Non-Visual Opsins & Vasoregulation: Implications for Vascular Therapy

非视觉视蛋白

• 批准号: 9264005
• 负责人: DAN E BERKOWITZ
• 金额: $ 40.29万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-01 至 2019-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9264005
• 关键词: ADRBK1 gene; Adenoviruses; Adrenergic Agents; Arrestins; Arteries; Blood; Blood Circulation; Blood Vessels; Blood flow; Cells; Cephalic; Circadian Rhythms; Complement; Coronary Arteriosclerosis; Cutaneous; Cyclic GMP-Dependent Protein Kinases; Data; Disease; Dominant-Negative Mutation; Dose; Electrodes; Electrophysiology (science); Endothelium; Erectile dysfunction; Exhibits; Fingers; Functional disorder; G protein coupled receptor kinase; G-Protein-Coupled Receptors; G-substrate; GTP-Binding Proteins; Grant; Health; Human; Hypertension; Immunohistochemistry; Impairment; Injury; Invertebrate Photoreceptors; Invertebrates; Lasers; Light; Measurement; Mediating; Membrane Potentials; Microdialysis; Molecular; Mus; Muscle Cells; Myography; Nitric Oxide Synthase; Opsin; Pain; Paroxetine; Pathway interactions; Patients; Pharmacology; Phosphotransferases; Physiological; Potassium Channel; Process; Protein Isoforms; Proteins; Publications; Rattus; Raynaud Disease; Recording of previous events; Regulation; Relaxation; Retinal Ganglion Cells; Reverse Transcriptase Polymerase Chain Reaction; Role; Scleroderma; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Skin; Sleep; Smooth Muscle Myocytes; Soluble Guanylate Cyclase; Stimulus; Stroke; Tail; Techniques; Testing; Tissues; Universities; Vascular Diseases; Vascular Smooth Muscle; Vasodilation; Vasomotor; Vertebrates; Visual; base; beta-adrenergic receptor; constriction; desensitization; experimental study; in vivo; inhibitor/antagonist; insight; knock-down; light intensity; melanopsin; mouse model; mutant; new therapeutic target; novel; overexpression; phosphodiesterase 6; prevent; public health relevance; receptor; response; small hairpin RNA; small molecule inhibitor; targeted treatment; vascular bed; vasoconstriction

 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.

 描述（由申请人提供）：由神经体液信号传导紊乱介导的血管舒缩功能受损和/或内皮功能受损是血管疾病（例如高血压、冠状动脉疾病、中风、勃起功能障碍）和特定血管病（例如雷诺现象（RP））的直接原因。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。 Melanospin (Opn4) 通常存在于视网膜神经节细胞中，调节昼夜节律和睡眠。我们在许多哺乳动物物种的血管和许多血管床中发现了一种非视觉光（视蛋白）受体 Opn4。这些受体介导特定波长（蓝色 430-460nM）光的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 蛋白偶联受体受到调节，因为 G 蛋白受体激酶的抑制可防止刺激依赖性脱敏，并显着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。显着增加小鼠尾动脉的血流量。我们假设：1）Opn4是血管舒张功能的重要调节因子，介导体内生理功能； 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”感光器； 3) 通过GRK2/arrestin机制发生脱敏/调节； 4) 在 NO 信号传导（血管舒张）受损且血管收缩增强的疾病（例如 RP）中，该通路上调。在本提案中，我们计划：1）利用Opn4-/-小鼠和新发现的Opn4抑制剂opsinamides，使用离体血管肌动描记法和颅窗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) 使用特异性抑制剂和通路蛋白的 shRNA 敲低，通过尖锐电极测量分离血管中的膜电位，确定从受体和 G 蛋白到通道的信号转导机制； 3) 使用GRK2、帕罗西汀、血管平滑肌选择性GRK2-/-和抑制蛋白-/-小鼠的新型药理学抑制剂，以及分离血管中GRK2和抑制蛋白shRNA腺病毒敲低来了解受体调节。 4) 确定 Opn4 受体作为 RP 小鼠模型中靶标的作用，并使用激光流多普勒和皮肤微透析探索健康人和原发性 RP 患者皮肤循环中介导体内光松弛的机制。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，并确定其作为新治疗靶点的潜力。