Calcium Sparklets-Induced Vascular Dysfunction During Diabetes

描述（由申请人提供）：钙通过二氢吡啶敏感、电压门控的l型钙通道（LTCC）内流在动脉平滑肌的兴奋性、收缩性和基因表达调节中起着至关重要的作用。在非胰岛素依赖型糖尿病（NIDDM）期间，通过平滑肌ltcc的过量Ca2+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，在高血糖和NIDDM期间LTCC活性增加的分子机制仍然不明确。最近，我们确定并表征了一种新的LTCC功能模式，其中单个或小簇这些通道可以在持续的门控模式下运作，在动脉肌细胞中产生几乎连续的Ca2+内流位点（称为“持续Ca2+火花”）。生理条件下，持续性Ca2+火花活性较低。然而，在本应用中提出的初步数据表明，Ca2+火花活性在高血糖和NIDDM期间增加，其机制需要蛋白激酶a （PKA）激活和支架蛋白AKAP150对该激酶的膜靶向。本应用程序的目的是测试中心假设，即持续Ca2+火花活性的增加是导致糖尿病期间血管功能障碍途径的早期关键事件。中心假设是在强有力的初步数据的基础上制定的，并将通过追求三个新的具体目标来检验。目的1将研究高血糖和糖尿病期间动脉平滑肌Ca2+火花活性增加的机制和功能后果。Aim 2将确定AKAP150和PKA活性在急性高血糖和糖尿病期间导致Ca2+火花活性增加的机制中的作用。Aim 3将验证在急性高血糖和糖尿病期间，持续的Ca2+火花通过激活NFATc3下调K+通道表达的假设。这些假设将由我们的团队结合最先进的电生理、细胞和分子生物学方法开发的一系列新成像方法进行测试。这项工作具有创新性，因为它旨在从多个层面整合NIDDM期间血管功能障碍的机制。这些结果将具有重要意义，因为它们将为NIDDM期间Ca2+火花活性增加导致血管功能障碍的机制提供新的基础信息，并可能有助于开发合理的治疗方法来治疗这种病理状况。

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.