In Vivo Ca2+ and Voltage Imaging on The Urinary Bladder

描述（由申请人提供）：梗阻后尿路功能障碍是一种具有极高发病率和破坏性社会和心理后果的疾病，导致包括失禁、排尿频率增加和不适当的尿急感在内的复杂症状，所有这些似乎都是由膀胱平滑肌的异常收缩活动引起的。在这个建议中，我们试图了解在体内正常和梗阻后膀胱的节律性或阶段性收缩活动的产生。虽然在体内研究阶段性活动的细胞信号是非常困难的，重要的是要了解这种活动的神经和激素的输入，在调节收缩活动中发挥重要作用的背景下。我们最近开发的方法来监测细胞内游离Ca 2+在体内通过发展的转基因小鼠表达一种新的高信号噪声，遗传编码的Ca 2+指标。Ca2+指示剂GCaMP2在生理温度下稳定，接近GFP的亮度，能够持续、高分辨率地记录活小鼠中的细胞Ca2+瞬变。已经建立了这种分子在膀胱平滑肌中高度表达的小鼠，并将用于体内观察细胞Ca2+信号。我们将使用这些小鼠来更充分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中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异常起搏器活动和异常Ca2+波的发展，这些异常Ca2+波与平滑肌或Cajal间质细胞（ICC）的肌浆网（SR）的Ca2+释放失调有关。为了验证这一假设，我们将在正常条件下对平滑肌细胞中的相位Ca2+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阻塞干扰的程度，并测试是否由于肌肉中SR Ca2+释放的改变或膀胱中起搏细胞活性的变化而引起异常活动。这些发现应该提供一个更清楚的了解平滑肌功能障碍与尿路流出道梗阻。

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.