TRP Channel-Dependent Regulation of Arterial Tone

TRP 通道依赖性动脉张力调节

• 批准号: 9244824
• 负责人: Scott Earley
• 金额: $ 35.88万
• 依托单位: UNIVERSITY OF NEVADA RENO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-01-01 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9244824
• 关键词: Affect; Angiotensinogen; Animals; Ankyrins; Arteries; Atherosclerosis; Biological Assay; Blood Vessels; Blood coagulation; Blood flow; Brain; Calcium; Cardiovascular Diseases; Cardiovascular system; Cations; Cell membrane; Cerebral Arterial Diseases; Cerebrovascular Circulation; Cerebrovascular Disorders; Cerebrovascular system; Cerebrum; Coronary; Data; Disease; Electrophysiology (science); Endothelial Cells; Endothelium; Enzymes; Frequencies; Generations; Goals; Heart; Human; Hypertension; Image; Intestines; Ion Channel; Kidney; Knock-out; Knockout Mice; Ligation; Lipid Peroxidation; Lipid Peroxides; Mediating; Membrane; Membrane Potentials; Mesentery; Methods; Microelectrodes; Mus; Muscle Cells; Myography; NADPH Oxidase; Organ; Outcome Study; Oxidases; Oxidative Stress; Pattern; Physiological; Play; Property; Proteins; Reactive Oxygen Species; Regulation; Relaxation; Renin; Research; Resolution; Role; Sequence Homology; Severities; Signal Pathway; Signal Transduction; Skeletal Muscle; Skin; Smooth Muscle Myocytes; Sodium; Speed; Stroke; Stroke prevention; Structure; TRPA1 Channel; Testing; Thinness; Tissues; Total Internal Reflection Fluorescent; Transgenic Model; Vasodilation; Vasomotor; autocrine; base; cerebral artery; design; drug development; endothelial dysfunction; experimental study; gene product; in vivo; innovation; member; novel; patch clamp; pressure; prevent; public health relevance; receptor; renal artery; response; selective expression; therapeutic target; therapy development; tool; vascular bed

DESCRIPTION (provided by applicant): The major objective of this competitive renewal application is to elucidate the physiological significance of the sole member of the ankyrin (A) TRP subfamily, TRPA1, in endothelium-dependent regulation of cerebral arteries. Preliminary findings indicate that TRPA1 is present in the endothelium of human and mouse cerebral arteries, but is not present in other vascular beds, suggesting that TRPA1 plays a critical but unknown role in the regulation of the cerebral vasculature. Therefore, the overall goal of the proposed research is to determine the channel's function in healthy and diseased cerebral arteries. The goal of Specific Aim 1 is to identify endogenous activators of TRPA1 in the endothelium. This Aim will test the hypothesis that lipid peroxidation products (LPP) generated by NADPH oxidase (NOX) activity stimulate TRPA1 in the endothelium in an autocrine manner. We created the first endothelial cell-specific TRPA1 knockout (eTRPA1-/-) mice as a powerful experimental tool for these studies. Patch-clamp electrophysiology and an innovative total internal reflection fluorescent microscopy (TIRFM) method that we developed will be used to record the effects of endogenously-produced LPP on TRPA1 activity in native endothelial cells isolated from eTRPA1-/- and control mice. The goal of Specific Aim 2 is to elucidate the signaling pathways responsible for vasodilation evoked by endogenous TRPA1 activators. This Aim will test the hypothesis that LPP generation localized to myoendothelial junctions (MEJs) of cerebral arteries causes dilation by activating TRPA1 to initiate endothelium-dependent hyperpolarization (EDH). This hypothesis is supported by preliminary data showing that TRPA1 channels, NOX, and LPP are concentrated in MEJs, and that endogenously-produced LPP dilate cerebral arteries from control, but not eTRPA1-/- mice. Experiments for this Aim will use pressure myography and intracellular microelectrode recordings to examine the effects of exogenous and endogenously-generated LPP on arteries from control and eTRPA1-/- mice. The detailed structure of MEJs in cerebral arteries will be elucidated by immunolabeling experiments and proximity ligations assay. Subcellular Ca2+ signaling associated with TRPA1 activity will be recorded from arteries isolated from mice expressing the genetically-encoded Ca2+ indicator protein GCaMP2 selectively in the endothelium. The goal of Specific Aim 3 is to test the hypothesis that TRPA1-mediated EDH of cerebral arteries opposes elevated smooth muscle cell contractility associated with hypertension. We will test the hypothesis that basal TRPA1 activity is elevated in arteries from hypertensive animals vs. controls. Further, we will test the hypothesis that eTRPA1-/- mice will suffer stroke at higher frequency during hypertension compared with controls. These experiments will utilize the established renin-angiotensinogen (R+A+) transgenic model of hypertension. Proposed studies are expected to have substantial impact because they will elucidate the physiological function of TRPA1 in the endothelium of cerebral arteries and determine if TRPA1 is a potential therapeutic target for cerebrovascular diseases.

描述（由申请人提供）：这种竞争性更新应用的主要目的是阐明在依赖大脑动脉的内皮依赖性调节中，arnkyrin（a）trp trpA1的唯一成员TRPA1的生理意义。初步发现表明，TRPA1存在于人和小鼠脑动脉的内皮中，但在其他血管床中不存在，这表明TRPA1在调节大脑血管的调节中起着至关重要但未知的作用。因此，拟议的研究的总体目标是确定渠道在健康和患病的脑动脉中的功能。特定目标1的目的是鉴定内皮中TRPA1的内源激活剂。该目标将检验以下假设：NADPH氧化酶（NOX）活性产生的脂质过氧化产物（LPP）以自分泌方式刺激内皮中的TRPA1。我们创建了第一个内皮细胞特异性TRPA1敲除（ETRPA1 - / - ）小鼠，作为这些研究的强大实验工具。我们开发的斑块钳电生理学和创新的总内反射荧光显微镜（TIRFM）方法将用于记录从ETRPA1-/ - 和对照小鼠中分离出的天然内皮细胞中内生生产的LPP对TRPA1活性的影响。特定目标2的目的是阐明负责由内源性TRPA1激活剂引起的血管舒张的信号传导途径。该目的将检验以下假设：LPP产生局部局部到脑动脉的肌膜内皮连接（MEJ），通过激活TRPA1启动内皮依赖性超极化（EDH）引起扩张。该假设得到了初步数据的支持，表明TRPA1通道，NOX和LPP集中在MEJS中，并且内生产生的LPP从对照中进行了脑动脉，但没有ETRPA1 - / - 小鼠。此目标的实验将使用压力密码和细胞内微电极记录来检查外源和内源性生成的LPP对对照和ETRPA1 - / - 小鼠的动脉的影响。通过免疫标记实验和接近诱因测定法，将阐明MEJ在脑动脉中的详细结构。与TRPA1活性相关的亚细胞Ca2+信号传导将记录在从内皮中选择性地表达遗传编码的Ca2+指示剂蛋白GCAMP2的小鼠中分离出的动脉。特定目的3的目的是检验以下假设：脑动脉的TRPA1介导的EDH反对与高血压相关的平滑肌细胞收缩力升高。我们将检验以下假设：高血压动物与对照组的动脉中基础TRPA1活性升高。此外，我们将检验以下假设：与对照组相比，在高血压期间，ETRPA1 - / - 小鼠将在较高的频率下遭受中风。这些实验将利用已建立的肾素 - 血管紧张素原（R+A+）的转基因模型。预计拟议的研究将产生重大影响，因为它们将阐明脑动脉内皮中TRPA1的生理功能，并确定TRPA1是否是脑血管疾病的潜在治疗靶点。