IN VITRO MODEL SYSTEM FOR DET REGULATORY MECHANISMS FOR SMOOTH MUSCLE MECHANICS

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 项目3：这项提议的长期目标是证明胃窦平滑肌细胞内的钙信号是通过钙调素蛋白激酶II的磷化蛋白磷酸化来调节的，并且这一调节胃部肌源性兴奋性的新途径被糖尿病破坏。这些研究的总体假设是，磷蛋白和CaM激酶II通过调节SERCA对肌浆网钙的摄取和释放，是胃窦平滑肌兴奋性的关键决定因素，而磷蛋白、CaM激酶II和SERCA的表达或活性的变化触发了病理重塑，从而导致糖尿病胃窦平滑肌营养不良和胃肌动力丧失。该应用的目的是(I)验证磷蛋白和CaM激酶II调节细胞内钙离子调节胃窦平滑肌兴奋性的假说。为了评估这一点，将利用磷蛋白-/-小鼠来证明磷蛋白通过调节肌浆网钙摄取来影响平滑肌膜的潜力。(Ii)验证肌浆网靶向CaM激酶II磷酸化蛋白并调节胃窦平滑肌细胞内钙离子水平的假说。我们认为，CaM激酶II锚定在肌浆网膜上可能是实现磷蛋白磷酸化时空特异性的一种机制。(Iii)验证磷蛋白和SERCA表达的改变，以及CaM激酶II对磷蛋白的磷酸化触发导致糖尿病胃窦平滑肌营养不良的分子重构的假说。将使用多种技术，包括胃窦平滑肌的机械测量、SDS-PAGE和Western blotting、CaM激酶II分析、STOCs的穿孔贴片全细胞记录、膜电位的尖锐电极记录和荧光钙指示剂染色来测量胃窦平滑肌细胞内的钙瞬变和[SR钙水平和释放事件]。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Project 3: The long-term objectives of this proposal are to demonstrate that intracellular Ca2+ signaling in gastric antrum smooth muscle cells is regulated by phospholamban phosphorylation via CaM kinase II and that this novel pathway regulating myogenic excitability in the stomach is disrupted by diabetes. The overall hypothesis of the proposed studies is that phospholamban and CaM kinase II, through their modulation of sarcoplasmic reticulum Ca2+ uptake and release by SERCA, are critical determinants of antrum smooth muscle excitability, and that alterations in the expression or activities of phospholamban, CaM kinase II, and SERCA trigger the pathological remodeling that leads to the diabetic dystrophy of antrum smooth muscles and the loss of gastric muscle motility. The application's aims are to (i) test the hypothesis that regulation of intracellular Ca2+ by phospholamban and CaM kinase II modulates the excitability of gastric antrum smooth muscles. To evaluate this, phospholamban-/- mice will be utilized to show that phospholamban affects smooth muscle membrane potential by modulating sarcoplasmic reticulum Ca2+ uptake. (ii) Test the hypothesis that sarcoplasmic reticulum-targeted CaM kinase II phosphorylates phospholamban and modulates intracellular Ca2+ levels in gastric antrum smooth muscles. We propose that CaM kinase II anchoring to the sarcoplasmic reticulum membrane could be a mechanism to achieve spatio-temporal specificity of phospholamban phosphorylation. (iii) Test the hypothesis that altered phospholamban and SERCA expression, and phospholamban phosphorylation by CaM kinase II trigger the molecular remodeling that leads to the dystrophy of diabetic antrum smooth muscles. Several techniques will be employed, including mechanical measurements of antrum smooth muscles, SDS-PAGE and Western blotting, CaM kinase II assays, perforated-patch whole cell recordings of STOCs, sharp electrode recordings of membrane potential, and fluorescent Ca2+ indicator dyes to measure intracellular Ca2+ transients and [SR Ca2+ levels and release events] in antrum smooth muscle cells.