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多种组织类型和器官中的平滑肌收缩至关重要。然而，人们对尿道平滑肌（维持尿失禁的关键器官）的功能和潜在机制知之甚少。本提案旨在了解肌浆网中 2 型兰尼碱受体 (RyR2) 自发开放导致的高度局部化和短暂的 Ca2+ 瞬变（“Ca2+ 火花”），以及尿道质膜中的离子通道目标平滑肌（USM）。我们的初步研究表明，(1) USM 中的 Ca2+ 火花仅激活 ANO1（一种 Ca2+ 激活的 Cl- (ClCa) 通道），产生自发瞬态内向电流 (STIC) 并使膜充分去极化，从而开启 L 型电压依赖型Ca2+通道； (2) RyR2 R176Q 突变（导致人类儿茶酚胺能多形性室性心动过速）的敲入小鼠的 USM 在接触咖啡因（一种 RyR 激动剂）和去氧肾上腺素 (PE)（一种 α1 肾上腺素受体激动剂）时产生的力比正常RyR2小鼠； (3) ANO1基因缺失导致Ca2+火花无法激活STIC，并降低新生小鼠咖啡因刺激下的尿道收缩； (4) PE 增加 STIC，而一氧化氮 (NO)（一种气体松弛剂）抑制 STIC。因此，我们认为USM中的RyR2和ANO1对于控制尿道肌源性和神经源性张力至关重要，它们的功能障碍可能导致尿道功能障碍和尿失禁。为了测试这一中心假设，我们将采用一种综合方法，使用高速 Ca2+ 成像，同时进行膜片钳、2D 和 3D 蛋白质定位、单细胞缩短和组织收缩生物测定、体内尿动力学测试和转基因（敲入和条件敲除）小鼠。具体来说，我们将通过使用 RyR2 R176Q 突变小鼠和正常小鼠控制整体 [Ca2+]I、膜电位和尿道张力，确定在 USM 中 Ca2+ 火花充当收缩机制，而不是像膀胱和血管平滑肌那样作为松弛机制。小鼠（目标 1）。全身性ANO1-/-小鼠在很年轻时就会死亡，因此在成熟小鼠和体内研究ANO1在尿道中的作用一直很困难。我们获得了一系列存活至成熟的平滑肌特异性 ANO1-/- 小鼠。通过该敲除系，我们将确定在成熟小鼠中，ANO1 对于维持尿道收缩和压力至关重要，其缺失可能会导致尿失禁（目标 2）。我们将通过 3D 成像、通道生物物理学和反应扩散模型进一步揭示 RyR2 激活 ANO1 的机制（目标 2）。最后，基于我们对 PE 和 NO 影响的初步结果，我们将确定 PE 和 NO 差异调节 RyR2 和 ANO1，分别导致尿道收缩和松弛（目标 3）。我们期望这些研究不仅将显着增进我们对 RyR2 和 ANO1 在尿道生理学中的作用的理解，而且还将确定新的分子靶点，用于开发有效且特异性的尿失禁治疗方法。