Calcium sparklets-induced vascular dysfunction during diabetes

描述(由申请人提供)：通过二氢吡啶敏感的电压门控L型钙通道(LTCC)进行的钙内流在调节动脉平滑肌的兴奋性、收缩和基因表达方面起着至关重要的作用。在非胰岛素依赖型糖尿病(NIDDM)中，通过平滑肌LTCCs过度的钙离子内流参与了导致高血糖诱导的血管功能障碍的一系列事件。然而，高血糖和NIDDM期间LTCC活性增加的分子机制仍不清楚。最近，我们发现并表征了一种新的LTCC功能模式，在这种模式下，单个或一小群LTCC通道可以在持续的门控模式下运行，从而在动脉肌细胞中产生几乎持续的钙内流部位(称为持续钙火花)。在生理条件下，持久钙火花活性较低。然而，在本申请中提出的初步数据表明，在高血糖和NIDDM期间，通过一种需要蛋白激酶A(PKA)激活和支架蛋白AKAP150膜靶向该激酶的机制，钙火花活性增加。这项应用的目标是检验中心假设，即持续钙离子火花活性的增加是导致糖尿病期间血管功能障碍的途径中的早期关键事件。中心假设是建立在强大的初步数据基础上的，并将通过追求三个新的具体目标来检验。目的1探讨高血糖和糖尿病时动脉平滑肌钙激活的机制和功能后果。目的2研究急性高血糖和糖尿病时AKAP150和PKA活性在导致钙激活的机制中的作用。目的3验证在急性高血糖和糖尿病时，持续钙离子火花通过激活NFATc3下调K+通道表达的假说。这些假说将使用我们团队开发的一系列新的成像方法与最先进的电生理、细胞和分子生物学方法相结合进行测试。这项拟议的工作具有创新性，因为它的目标是在多个层面上整合导致NIDDM期间血管功能障碍的机制。这些结果将是重要的，因为它们将提供关于NIDDM期间导致血管功能障碍的钙离子火花活性增加的机制的新的基本信息，并可能有助于开发治疗这种病理状况的合理疗法。 公共卫生相关性：大约有1000万美国人患有非胰岛素依赖型糖尿病，如果不治疗，会导致多种心血管并发症，如高血压和中风。本研究项目将确定高血糖诱导血管肌肉细胞中一种新的钙信号通路(例如，持续性钙火花)激活，导致收缩增加，从而导致糖尿病时动脉功能障碍的机制。

DESCRIPTION (provided by applicant): Calcium influx via dihydropyridine-sensitive, voltage-gated L-type calcium channels (LTCC) plays a crucial role in the regulation of excitability, contraction, and gene expression in arterial smooth muscle. Exaggerated Ca2+ influx through smooth muscle LTCCs has been implicated in the chain of events contributing to hyperglycemia- induced vascular dysfunction during non-insulin dependent diabetes mellitus (NIDDM). However, the molecular mechanisms underlying the increase in LTCC activity during hyperglycemia and NIDDM remain poorly defined. Recently, we identified and characterized a novel modality of LTCC function in which a single or a small cluster of these channels can operate in a persistent gating mode that create sites of nearly continual Ca2+ influx (called "persistent Ca2+ sparklets") in arterial myocytes. Under physiological conditions, persistent Ca2+ sparklet activity is low. However, preliminary data presented in this application suggest that Ca2+ sparklet activity increases during hyperglycemia and NIDDM through a mechanism requiring protein kinase A (PKA) activation and membrane targeting of this kinase by the scaffolding protein AKAP150. The goal of this application is to test the central hypothesis that an increase in persistent Ca2+ sparklet activity is an early, critical event in the pathway leading to vascular dysfunction during diabetes. The central hypothesis has been formulated on the basis of strong preliminary data and will be tested by pursuing three novel specific aims. Aim 1 will investigate the mechanisms and functional consequences of increased Ca2+ sparklet activity in arterial smooth muscle during hyperglycemia and diabetes. Aim 2 will determine the role of AKAP150 and PKA activity in the mechanisms leading to increase Ca2+ sparklet activity during acute hyperglycemia and diabetes. Aim 3 will test the hypothesis that persistent Ca2+ sparklets downregulate K+ channel expression through the activation of NFATc3 during acute hyperglycemia and diabetes. These hypotheses will be tested using a series of novel imaging approaches developed by our team in combination with state-of-the-art electrophysiological, cellular, and molecular biological approaches. The proposed work is innovative as it aims to integrate, at multiple levels, the mechanisms contributing to vascular dysfunction during NIDDM. Such outcomes will be significant because they will provide new fundamental information on the mechanisms by which increased Ca2+ sparklet activity underlie vascular dysfunction during NIDDM and may contribute to the development of rational therapies for the treatment of this pathological condition. PUBLIC HEALTH RELEVANCE: Approximately 10 million Americans suffer of non-insulin dependent diabetes, which, if untreated, leads to several cardiovascular complications such as hypertension and strokes. This research project will determine the mechanisms by which hyperglycemia induces the activation of a novel Ca2+ signaling modality (e.g. persistent Ca2+ sparklets) in the muscle cells of blood vessels, causing increased contraction and thereby leading to arterial dysfunction during diabetes.