Calcium Signaling in Cerebral Arteries

描述(申请人提供)：阻力大小动脉和微动脉直径决定脑灌注压和血流量。脑动脉直径的一个主要调节因素是心肌细胞内钙离子浓度([Ca2+]i)。整体[Ca~(2+)]i升高刺激血管收缩，而整体[Ca~(2+)]i降低则导致血管扩张。局部(火花)和传播(波)的钙信号也出现在心肌细胞中，可以直接和间接地调节全局[钙]i，导致动脉直径的变化。尽管过去十年的研究已经揭示了动脉肌细胞局部和全局钙信号的一些调节机制和生理功能，但仍有许多不清楚的地方。与磷脂酶C偶联受体结合的血管收缩药可升高肌醇1，4，5-三磷酸(IP3)，从而激活位于IP3受体(IP3R)的肌浆网(SR)，导致钙释放和[Ca2+]i升高。我们实验室的初步数据表明，除了动员肌浆网钙离子外，IP3还激活不依赖于肌浆网释放的质膜非选择性阳离子电流(ICAT)，从而导致血管收缩。这项建议将集中于研究IP3调节大脑动脉心肌细胞内钙信号和动脉收缩的机制。我们将调查三个具体目标。目的1验证IP3激活心肌细胞ICAT，导致膜去极化、电压依赖性钙通道激活、整体[钙]i升高和血管收缩的假说。目的2探讨瞬时受体电位(Trp)通道参与IP3诱导的ICAT和血管收缩的假说。目的3将评估IP3Rs在IP3诱导的ICAT激活、局部和全局细胞内钙信号和收缩中的作用。为了研究这些目标，我们将使用各种技术，包括激光扫描共聚焦和传统的钙成像、膜片钳和细胞内电生理学、加压动脉直径测量、RNA干扰和IP3R缺陷小鼠。这一建议将使我们更好地理解血管系统中的主要信号信使IP3调节动脉钙信号和血管直径的机制。由此产生的数据将提高我们对调节脑血流的生理过程的了解，脑血流的变化可能导致脑血管疾病，包括高血压和中风。 公共卫生相关性通常认为，在动脉平滑肌细胞中，三磷酸肌醇(IP3)激活IP3受体，导致储存的钙(Ca~(2+))释放和细胞内钙离子浓度([Ca~(2+)]i)升高，刺激收缩。我们的建议表明，在血管紧张剂诱导的IP3也激活IP3受体的新机制中，通过一种不需要肌浆网钙离子释放的机制，这导致了质膜阳离子电流的刺激，TRPC3通道参与其中。随后的膜去极化激活电压依赖性钙通道，导致[Ca~(2+)]i升高和收缩。

DESCRIPTION (provided by applicant): Resistance-size artery and arteriole diameter determines cerebral perfusion pressure and blood flow. A major regulator of cerebral artery diameter is the myocyte intracellular calcium (Ca2+) concentration ([Ca2+]i). An elevation in global [Ca2+]i stimulates vasoconstriction, whereas a reduction in global [Ca2+]i leads to vasodilation. Local (sparks) and propagating (waves) Ca2+ signals also occur in myocytes and can directly and indirectly modulate global [Ca2+]i, leading to changes in arterial diameter. Although research over the past decade has revealed some regulatory mechanisms and physiological functions of local and global Ca2+ signals in arterial myocytes, much remains unclear. Vasoconstrictors that bind to phospholipase C-coupled receptors elevate inositol 1,4,5-trisphophate (IP3) which activates sarcoplasmic reticulum (SR) located IP3 receptors (IP3R), leading to Ca2+ release and an [Ca2+]i elevation. Preliminary data from our laboratory suggest that in addition to mobilizing SR Ca2+, IP3 activates a plasma membrane non-selective cation current (ICat) independently of SR release, and this leads to vasoconstriction. This proposal will focus on investigating the mechanisms by which IP3 regulates cerebral artery myocyte intracellular Ca2+ signaling and arterial contractility. We will investigate 3 specific aims. Aim 1 will test the hypothesis that IP3 activates ICat in myocytes, leading to membrane depolarization, voltage-dependent Ca2+ channel activation, a global [Ca2+]i elevation, and vasoconstriction. Aim 2 will investigate the hypothesis that transient receptor potential (TRP) channels contribute to the IP3-induced ICat and vasoconstriction. Aim 3 will evaluate the contribution of IP3Rs to IP3-induced ICat activation, local and global intracellular Ca2+ signaling and constriction. To investigate these aims, we will use a wide variety of techniques, including laser-scanning confocal and conventional Ca2+ imaging, patch clamp and intracellular electrophysiology, pressurized artery diameter measurements, RNA interference, and IP3R deficient mice. This proposal will provide a better understanding of the mechanisms by which IP3, a principal signaling messenger in the vasculature, regulates arterial Ca2+ signaling and diameter. The resulting data will improve our knowledge of physiological processes that regulate cerebral blood flow, the alteration of which can lead to cerebrovascular pathologies, including hypertension and stroke. PUBLIC HEALTH RELEVANCE It is generally considered that in arterial smooth muscle cells, inositol trisphosphate (IP3) activates IP3 receptors, leading the release of stored calcium (Ca2+) and an elevation in intracellular Ca2+ concentration ([Ca2+]i) that stimulates constriction. Our proposal suggests the novel mechanism that in smooth muscle cells, vasoconstrictor-induced IP3 also activates IP3 receptors, and through a mechanism that does not require sarcoplasmic reticulum Ca2+ release, this leads to the stimulation of a plasma membrane cation current to which TRPC3 channels contribute. The ensuing membrane depolarization activates voltage-dependent calcium channels, leading to an [Ca2+]i elevation, and constriction.