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

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 项目三：该提案的长期目标是证明胃窦平滑肌细胞中的细胞内Ca2+信号通过CaM激酶II受磷蛋白磷酸化调节，并且这种调节胃中肌源性兴奋性的新途径被糖尿病破坏。所提出的研究的总体假设是受磷蛋白和CaM激酶II通过SERCA调节肌浆网Ca 2+摄取和释放，是胃窦平滑肌兴奋性的关键决定因素，受磷蛋白、CaM激酶II、和SERCA触发病理性重构，导致糖尿病性胃窦平滑肌营养不良和胃肌运动丧失。本申请的目的是（i）检验受磷蛋白和CaM激酶II对细胞内Ca2+的调节调节胃窦平滑肌兴奋性的假设。为了评估这一点，将利用受磷蛋白-/-小鼠来显示受磷蛋白通过调节肌浆网Ca2+摄取来影响平滑肌膜电位。(ii)检验肌浆网靶向CaM激酶II磷酸化受磷蛋白并调节胃窦平滑肌细胞内Ca2+水平的假设。我们建议，钙调素激酶II锚定的肌浆网膜可能是一种机制，以实现受磷蛋白磷酸化的时空特异性。(iii)验证受磷蛋白和SERCA表达改变以及CaM激酶II引起的受磷蛋白磷酸化触发导致糖尿病胃窦平滑肌营养不良的分子重塑的假设。将采用几种技术，包括胃窦平滑肌的机械测量、SDS-PAGE和蛋白质印迹、CaM激酶II测定、STOC的穿孔斑全细胞记录、膜电位的锐电极记录和荧光Ca2+指示剂染料，以测量胃窦平滑肌细胞中的细胞内Ca2+瞬变和[SR Ca2+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.