Calcium sparklets-induced vascular dysfunction during diabetes

描述（由申请人提供）：通过二氢吡啶敏感性电压门控L型钙通道（LTCC）的钙内流在动脉平滑肌兴奋性、收缩和基因表达的调节中起着至关重要的作用。通过平滑肌LTCC的Ca 2+内流增加与非胰岛素依赖型糖尿病（NIDDM）期间高血糖诱导的血管功能障碍的事件链有关。然而，在高血糖和NIDDM期间LTCC活性增加的分子机制仍然不清楚。最近，我们确定并表征了一种新的LTCC功能模式，其中单个或一小簇这些通道可以以持续门控模式操作，在动脉平滑肌中产生几乎连续的Ca 2+内流（称为“持续Ca 2+火花”）。在生理条件下，持续的Ca 2+火花活性较低。然而，本申请中提供的初步数据表明，在高血糖期间，Ca 2+火花活性通过需要蛋白激酶A（PKA）活化和通过支架AKAP 150靶向的机制而增加。本申请的目的是检验中心假设，即持续Ca 2+火花活性的增加是糖尿病期间导致血管功能障碍的途径中的早期关键事件。中心假设是在强有力的初步数据的基础上提出的，并将通过追求三个新的具体目标进行检验。目的1探讨急性高血糖和糖尿病引起动脉平滑肌Ca ~（2+）火花活性增加的机制。目的2将确定是否AKAP 150和PKA活性所需的增加钙火花活性在急性高血糖症和糖尿病。目的3将验证在急性高血糖和糖尿病期间持续的Ca 2+火花通过激活NFATc 3下调K+通道表达的假设。这些假设将使用我们的团队开发的一系列新的成像方法与最先进的电生理学，细胞和分子生物学方法相结合进行测试。本申请中概述的实验是重要的，因为它们将提供关于NIDDM期间血管功能障碍的分子机制的新的基本信息，并且可能有助于开发用于治疗这种病理状况的合理疗法。 大约有1000万美国人患有非胰岛素依赖型糖尿病和高血压，如果不治疗，会导致几种心血管并发症。该研究项目将确定高血糖诱导血管肌细胞中新型Ca 2+信号传导模式（例如Ca 2+火花）激活的机制，导致收缩增加，从而导致糖尿病和高血压期间的动脉功能障碍。

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 smooth muscle. Under physiological conditions persistent Ca2+ sparklet activity is low. However, preliminary data presented in this application suggest that Ca2+ sparklet activity increases during hyperglycemia through a mechanism requiring protein kinase A (PKA) activation and targeting via scaffolding 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 by which acute hyperglycemia and diabetes increases Ca2+ sparklet activity in arterial smooth muscle. Aim 2 will determine whether AKAP150 and PKA activity are required for increased 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 experiments outlined in this application are significant because they will provide new fundamental information on the molecular mechanisms underlying 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 and hypertension, which, if untreated, leads to several cardiovascular complications. This research project will determine the mechanisms by which hyperglycemia induces the activation of a novel Ca2+ signaling modality (e.g. Ca2+ sparklets) in the muscle cells of blood vessels, causing increased contraction and thereby leading to arterial dysfunction during diabetes and hypertension.