Function and regulation of elemental Ca2+ signaling in urethral smooth muscle

尿道平滑肌中元素 Ca2 信号的功能和调节

基本信息

批准号：
8482736
负责人：
Ronghua ZhuGe
金额：
$ 16.65万
依托单位：
UNIV OF MASSACHUSETTS MED SCH WORCESTER
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-08-15 至 2015-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8482736
关键词：
Adrenergic Agents Adrenergic Agonists Affect Agonist Animals Biological Biological Assay Biophysics Bladder Bladder Control Blood Vessels Caffeine Cell membrane Cells Characteristics Contracts Coupling Diffusion Disease Electrophysiology (science)Foundations Functional disorder Gases Generations Genetic Histocompatibility Testing Human Image Impairment In Vitro Ion Channel Ion Channel Protein Knock-in Mouse Knock-out Knockout Mice Life Maintenance Measurement Measures Mediating Mediator of activation protein Medical Membrane Membrane Potentials Methodology Modeling Molecular Molecular Target Mus Muscle Muscle Contraction Mutant Strains Mice Mutation National Institute of Diabetes and Digestive and Kidney Diseases Neonatal Neurotransmitters Nitric Oxide Organ Pharmacology Phenylephrine Physiological Physiology Preparation Process Proteins Qualifying Reaction Regulation Relaxation Resolution Role RyR2 Ryanodine Receptors Sarcoplasmic Reticulum Signal Transduction Smooth Muscle Smooth Muscle Myocytes Speed Stress Urinary Incontinence Technology Testing Therapeutic Three-Dimensional Imaging Time Tissues Transgenic Organisms Urethra Urethral sphincter Urinary Incontinence Urination Urine Urodynamics Vascular Smooth Muscle Ventricular Tachycardia Woman adrenergic base cell type emotional distress experience in vivo in vivo Bioassay innovation mouse model novel patch clamp pressure prevent public health relevance receptor respiratory smooth muscle response voltage

项目摘要

DESCRIPTION (provided by applicant): Ca2+ signals and ion channels are essential in controlling the contraction of smooth muscle in several tissue types and organs. However, their functions and underlying mechanisms in smooth muscle from urethra, an organ critical for maintaining urinary continence, are poorly understood. The present proposal seeks to understand highly localized and short-lived Ca2+ transients ("Ca2+ sparks") that result from the spontaneous opening of type 2 ryanodine receptors (RyR2) in the sarcoplasmic reticulum, and their ion channel targets in the plasma membrane in urethral smooth muscle (USM). Our preliminary studies revealed that (1) Ca2+ sparks in USM only activate ANO1, a Ca2+-activated Cl- (ClCa) channel, to produce spontaneous transient inward currents (STICs) and depolarize the membrane sufficiently to turn on L-type voltage-dependent Ca2+ channels; (2) USM from knock-in mice of RyR2 R176Q mutation (which causes catecholaminergic polymorphic ventricular tachycardia in human) generates less force when exposed to caffeine, a RyR agonist, and phenylephrine (PE), an alpha1-adrenergic receptor agonist, than in normal RyR2 mice; (3) genetic deletion of ANO1 causes Ca2+ sparks unable to activate STICs, and decreases the urethral contraction upon stimulation by caffeine in neonatal mice; and (4) PE increases STICs, and nitric oxide (NO), a gas relaxant, inhibits STICs. Thus, we propose that RyR2 and ANO1 in USM are essential for governing urethral myogenic and neurogenic tone, and their malfunction may result in urethral dysfunction and urinary incontinence. To test this central hypothesis we will employ an integrated approach using high-speed Ca2+ imaging with simultaneous patch-clamping, 2D and 3D protein localization, single cell shortening and tissue contraction bioassays, in vivo urodynamics tests, and transgenic (knock-in and conditional knockout) mice. Specifically, we will establish that in USM Ca2+ sparks act as a contractile mechanism, rather than a relaxing mechanism as in bladder and vascular smooth muscle, by controlling global [Ca2+]I, membrane potential, and urethra tone using RyR2 R176Q mutant mice and normal mice (Aim 1). Systemic ANO1-/- mice die very young, so it has been difficult to study the role of ANO1 in urethra in mature mice and in vivo. We have obtained a line of smooth muscle specific ANO1-/- mice which live to maturity. With this knockout line, we will establish that in mature mice ANO1 is critical for the maintenance of urethral contraction and pressure and its deletion likely leads to urinary incontinence (Aim 2). We will further uncover the mechanisms underlying activation of ANO1s by RyR2s with 3D imaging, channel biophysics and reaction-diffusion modeling (Aim 2). Finally, building upon our preliminary results on the effects of PE and NO, we will establish that PE and NO differentially modulate RyR2 and ANO1, resulting in the contraction and relaxation of urethra, respectively (Aim 3). We expect that these studies will not only significantly advance our understanding of the roles of RyR2 and ANO1 in urethral physiology, but also identify novel molecular targets for developing effective and specific treatments for urinary incontinence.

描述（由申请人提供）：CA2+信号和离子通道对于控制几种组织类型和器官的平滑肌收缩至关重要。然而，对尿素的平滑肌的功能和潜在机制，这是维持尿道持续性至关重要的器官，对尿道的理解很少。本提案旨在理解由肌休息性网状中2型ryanodine受体（RYR2）自发开放及其离子通道靶标在尿素平滑肌（USM）中的质膜中产生的高度局部和短寿命的Ca2+瞬变（“ Ca2+ Spark”）。我们的初步研究表明，（1）USM中的Ca2+火花仅激活ANO1，一个Ca2+激活的Cl-（ClCa）通道，以产生自发的瞬时向内电流（Stics），并充分地将膜去极化以打开L型电压电压依赖于L型电压依赖依赖L型CAC2+通道；（2）来自RYR2 R176Q突变的敲击小鼠的USM（引起人类的catecholamin能多态性心心动过速）会产生较少的力，而咖啡因（Ryr a anistist），Ryr a andist，ryr admonist，and andpha1-苯肾上腺素（PE），An alpha1-腺肾上腺素能接受者agonerist agonerist，比正常生成正常。（3）ANO1的遗传缺失会导致Ca2+火花无法激活Stics，并减少咖啡因在新生小鼠中刺激时的尿道收缩；（4）PE增加了Stics和一氧化氮（NO），一种气体松弛剂，抑制了Stics。因此，我们提出USM中的RyR2和ANO1对于尿道肌源性和神经源性语调至关重要，它们的故障可能导致尿道功能障碍和尿失禁。为了检验该中心假设，我们将使用高速Ca2+成像采用集成方法，并同时使用斑块夹，2D和3D蛋白定位，单细胞缩短和组织收缩生物测定，体内尿动力学测试以及转基因（敲除和条件淘汰）小鼠。具体而言，我们将在USM Ca2+火花中确定通过控制全局[Ca2+] I，膜电位和尿道张力，使用RYR2 R176Q突变小鼠和正常小鼠（AIM 1），在USM Ca2+ Sparks中充当收缩机制，而不是像膀胱和血管平滑肌一样放松机制。系统性ANO1 - / - 小鼠死亡很小，因此很难研究成熟小鼠和体内尿道中ANO1的作用。我们获得了一系列平滑肌特异性ANO1 - / - 小鼠，这些小鼠生存至成熟。借助此淘汰线，我们将确定在成熟的小鼠中，ANO1对于维持尿道收缩和压力至关重要，其缺失可能导致尿失禁（AIM 2）。我们将进一步揭示RyR2s通过3D成像，通道生物物理学和反应 - 扩散建模的ANO1激活的机制（AIM 2）。最后，基于我们对PE和NO的影响的初步结果，我们将确定PE和没有差异化的RyR2和ANO1，分别导致尿道的收缩和放松（AIM 3）。我们预计，这些研究不仅会显着提高我们对RYR2和ANO1在尿道生理学中的作用的理解，而且还可以确定新的分子靶标，以开发有效和特定的尿液尿失禁。