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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(申请人提供)：钙信号和离子通道在控制几种组织类型和器官的平滑肌收缩中是必不可少的。然而，它们在维持尿控的关键器官--来自尿道的平滑肌中的功能和潜在机制却知之甚少。本研究旨在了解由肌浆网中2型Ryanodine受体(RyR2)的自发开放引起的高度局部化和短暂的钙瞬变(“钙火花”)，以及它们在尿路平滑肌(USM)质膜上的离子通道靶标。我们的初步研究表明：(1)超声心动图中的钙火花仅激活钙离子激活的氯离子(CLCA)通道ANO_1，产生自发的瞬时内向电流(STIC)，并使膜去极化，从而开启L电压依赖性钙离子通道；(2)RyR2 R176Q突变(可导致人类儿茶酚胺能多形性室性心动过速)敲入小鼠的超声心动图在咖啡因(RyR激动剂)和苯肾上腺素(PE)(α1肾上腺素能受体激动剂)作用下产生的作用力比正常RyR2小鼠要小；(3)ANO1基因缺失导致钙离子火花不能激活STICs，并降低咖啡因刺激下新生小鼠的尿路收缩；(4)PE增加STICs，而一氧化氮(NO)，一种气体松弛药抑制STICs。因此，我们认为USM中的RyR2和ANO1对调节尿路肌源性和神经源性张力是必不可少的，它们的功能障碍可能导致尿失禁和尿失禁。为了验证这一中心假设，我们将使用一种集成的方法，使用高速钙离子成像，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。具体地说，我们将利用RyR2 R176Q突变小鼠和正常小鼠(目标1)，通过控制整体[Ca2+]i、膜电位和尿道张力，确定在USM中，钙火花作为一种收缩机制，而不是像在膀胱和血管平滑肌中那样的一种松弛机制。全身性ANO1-/-小鼠很早就死亡，因此很难研究ANO1在成年小鼠和体内的尿路中的作用。我们已经获得了一株存活到成熟的平滑肌特异的ANO1-/-小鼠。有了这个基因敲除系，我们将确定在成年小鼠中，ANO1对于维持尿路收缩和压力至关重要，并且它的缺失可能导致尿失禁(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的潜在机制(目标2)。最后，基于我们关于PE和NO作用的初步结果，我们将确定PE和NO对RyR2和ANO1的不同调制，分别导致尿路的收缩和松弛(目标3)。我们期望这些研究不仅能显著提高我们对RyR2和ANO1在尿路生理学中的作用的了解，还能为开发有效和特异的尿失禁治疗方法寻找新的分子靶点。