In Vivo Ca2+ and Voltage Imaging on The Urinary Bladder

膀胱体内 Ca2 和电压成像

基本信息

批准号：
7618459
负责人：
Michael I. Kotlikoff
金额：
$ 30.92万
依托单位：
CORNELL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-02-01 至 2011-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7618459
关键词：
Affect Bacterial Artificial Chromosomes Bladder Cardiac Cells Complex Coupling Data Development Disease Frequencies Functional disorder Gastrointestinal tract structure Gene Transfer Techniques Generations Genetic Heart Hormonal Hyperactive behavior Hypertrophy Image Imagery Imaging technology In Vitro Incontinence Individual Interstitial Cell of Cajal Life Maps Measurement Measures Methods Molecular Monitor Morbidity - disease rate Mus Muscle Muscle Cells Nature Nerve Noise Obstruction Organ Overactive Bladder Pacemakers Pattern Periodicity Physiological Play Property Quality of life Regulation Resolution Role Ryanodine Receptor Calcium Release Channel Ryanodine Receptors Sarcoplasmic Reticulum Secondary to Signal Transduction Smooth Muscle Smooth Muscle Myocytes Source Surface Symptoms Syndrome Techniques Temperature Testing Transgenic Mice Urge Incontinence Urinary tract Urination base cholinergic cholinergic neuron in vivo micturition urgency molecular scale novel psychologic relating to nervous system selective expression social tool voltage

项目摘要

DESCRIPTION (provided by applicant): Postobstructive urinary tract dysfunction is a disorder with extremely high morbidity and devastating social and psychological consequence, resulting in a complex of symptoms including incontinence, increased frequency of urination, and an inappropriate sense of urinary urgency, all of which appear to result from abnormal contractile activity of the smooth muscle lining the bladder. In this proposal we seek to understand the generation of rhythmic or phasic contractile activity in the normal and postobstructed urinary bladder in vivo. Although the study of the cellular signals underlying phasic activity is extraordinarily difficult in vivo, it is important to understand this activity in the context of neural and hormonal inputs that play an important role in the regulation of contractile activity. We have recently developed methods to monitor intracellular free Ca2+ in vivo through the development of transgenic mice expressing a novel high signal-noise, genetically encoded Ca2+ indicator. GCaMP2, the Ca2+ indicator, is stable at physiological temperatures and approaches the brightness of GFP, enabling sustained, high resolution recording of cellular Ca2+ transients in the living mouse. Mice in which this molecule is highly expressed in urinary bladder smooth muscle have been created and will be used to visualize cellular Ca2+ signals in vivo. We will use these mice to more fully understand the dysfunction associated with urinary tract outlet obstruction. The basis of abnormal contractile activity in postobstructive mice that develop spontaneous bladder overactivity will be studied to understand the cellular and molecular basis for abnormal bladder contractions. We hypothesize that bladder hyperactivity occurs secondary to the development of abnormal pacemaker activity and anomalous Ca2+ waves associated with dysregulated Ca2+ release from the sarcoplasmic reticulum (SR) of smooth muscle or Interstitial Cells of Cajal (ICC). To test this hypothesis, we will image phasic Ca2+ signals in smooth muscle cells under normal conditions, seek to understand the cellular and molecular basis for spontaneous activity, determine the extent to which this pattern is disturbed by sustained outflow obstruction, and test whether abnormal activity arises due to alterations in SR Ca2+ release in muscle or changes in the activity of pacemaker cells in the urinary bladder. These findings should provide a clearer understanding of smooth muscle dysfunction associated with urinary tract outflow obstruction.

描述（由申请人提供）：梗阻后尿路功能障碍是一种发病率极高、具有破坏性社会和心理后果的疾病，会导致一系列复杂的症状，包括失禁、排尿频率增加和不适当的尿急感，所有这些症状似乎是由膀胱内壁平滑肌的异常收缩活动引起的。在本提案中，我们试图了解体内正常膀胱和梗阻后膀胱中节律性或阶段性收缩活动的产生。尽管在体内研究相位活动背后的细胞信号非常困难，但在神经和激素输入的背景下理解这种活动非常重要，神经和激素输入在收缩活动的调节中发挥着重要作用。我们最近开发了通过开发表达新型高信号噪声、基因编码的 Ca2+ 指示剂的转基因小鼠来监测体内细胞内游离 Ca2+ 的方法。 GCaMP2 是 Ca2+ 指示剂，在生理温度下稳定且接近 GFP 的亮度，能够持续、高分辨率地记录活体小鼠细胞 Ca2+ 瞬变。这种分子在膀胱平滑肌中高度表达的小鼠已经被培育出来，并将用于体内观察细胞 Ca2+ 信号。我们将使用这些小鼠来更全面地了解与尿路出口梗阻相关的功能障碍。将研究发生自发性膀胱过度活动的梗阻后小鼠异常收缩活动的基础，以了解异常膀胱收缩的细胞和分子基础。我们假设膀胱过度活动继发于起搏器活动异常和异常 Ca2+ 波的发展，这些异常 Ca2+ 波与平滑肌肌浆网 (SR) 或卡哈尔间质细胞 (ICC) 的 Ca2+ 释放失调相关。为了检验这一假设，我们将对正常条件下平滑肌细胞中的相位 Ca2+ 信号进行成像，寻求了解自发活动的细胞和分子基础，确定这种模式受到持续流出阻塞干扰的程度，并测试是否存在异常活动由于肌肉中 SR Ca2+ 释放的变化或膀胱中起搏细胞活性的变化而产生。这些发现应该可以让我们更清楚地了解与尿路流出道梗阻相关的平滑肌功能障碍。