In Vivo Ca2+ and Voltage Imaging on The Urinary Bladder

描述(申请人提供)：梗阻后尿路功能障碍是一种发病率极高的疾病，具有破坏性的社会和心理后果，导致一系列症状，包括大小便失禁、尿频增加和不适当的尿紧迫感，所有这些似乎都是由膀胱内平滑肌的异常收缩活动引起的。在这项建议中，我们试图了解在活体内正常和梗阻后膀胱的节律性或相性收缩活动的产生。尽管在活体中研究时相活动背后的细胞信号是非常困难的，但在神经和激素输入的背景下理解这种活动是很重要的，因为神经和激素输入在收缩活动的调节中发挥着重要作用。我们最近开发了一种方法，通过建立表达一种新的高信号噪声、遗传编码的钙指示剂的转基因小鼠，在体内监测细胞内的游离钙离子。钙指示剂GCaMP2在生理温度下稳定，接近GFP的亮度，能够持续、高分辨率地记录活着的小鼠细胞内的钙瞬变。这种分子在膀胱平滑肌中高表达的小鼠已经被创造出来，并将被用来在体内观察细胞内的钙信号。我们将利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。我们推测，膀胱过度活动的发生继发于起搏器活动异常和与Cajal肌浆网(SR)或Cajal间质细胞(ICC)钙释放异常相关的异常钙波的发展。为了验证这一假设，我们将在正常情况下对平滑肌细胞中的时相钙信号进行成像，试图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道梗阻的干扰的程度，并测试是否由于肌肉中SR钙释放的变化或膀胱起搏细胞活性的变化而出现异常活动。这些发现将有助于更清楚地了解与尿路流出道梗阻相关的平滑肌功能障碍。

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.