Cerebral Artery Alpha1 Adrenergic and PKC Regulatory Mechanisms

描述（申请人提供）：在生命的每个阶段，脑血管张力和血流（CBF）的调节都是至关重要的。许多新生儿，特别是早产儿，在调节流向大脑的血液方面存在严重问题。这种失调可能对脑室内和生发基质出血造成严重后果，并伴有长期神经系统后遗症。目前的研究试图了解成熟发育如何改变胎儿/早产儿和成人脑血管的基本信号转导机制。这个项目基础广泛，多学科，垂直整合了生理学、细胞学、生物化学和分子方法。基于几十年的研究结果，我们将验证成熟发育与脑动脉（CA）收缩反应的显著变化有关，这些变化继发于α 1-肾上腺素能受体（1-AR）亚型和/或特异性蛋白激酶C异构体（PKC）介导的下游Ca2+依赖和Ca2+独立信号转导途径的改变。一个相关的假设是，与成人的“收缩”表型相比，发育显著改变了1- ar亚型和特异性PKC同功酶介导的原癌基因和代表血管平滑肌“合成”和/或“增殖”表型的基因的表达。具体目标如下。1)特异性1- ar亚型和下游效应蛋白在信号转导中的作用是什么？2)特异性PKC异构体、细胞外信号调节激酶（ERKs）、Rho A/Rho激酶和相关激酶在信号转导中的作用是什么？3)特异性1-AR亚型和PKC亚型在血管表型发育的基因调控中的作用是什么？4)其他信号转导蛋白在这些信号转导和基因调控途径中的作用是什么？在绵羊胎儿、新生儿和成人CA中，我们将进行激动剂诱导的收缩性和细胞内[Ca2+]测量、Western免疫印迹、RT- PCR、共聚焦显微镜、流式细胞术、2d凝胶-质谱、双链RNA或morpholinos基因沉默、基因上调、基因微阵列/途径分析和基因/蛋白质发现。从科学上讲，这些研究将促进我们对脑血管在胎儿、新生儿和成人发育过程中表型和功能变化的基本机制的理解。在临床上，这些研究涉及了解胎儿和/或早产儿的脑血管张力、压力和血流调节的基础，以及其失调导致脑出血和严重的神经系统后遗症。

DESCRIPTION (provided by applicant): At every stage of life the regulation of cerebral vascular tone and blood flow (CBF) is of vital importance. Many newborn infants, particularly those that are premature, have serious problems in the regulation of blood flow to their brains. This dysregulation may have serious consequences with intraventricular and germinal matrix hemorrhage with long-term neurological sequelae. The present studies seek to understand whereby maturational development alters fundamental signal transduction mechanisms in the cerebrovasculature of the fetus/premature newborn and the adult. This project is broadly based, multidisciplinary, and vertically integrated using physiologic, cellular, biochemical, and molecular approaches. Based on several decades of research findings, we shall test the overall hypothesis that maturational development is associated with significant changes in cerebral artery (CA) contractile responses secondary to altered alpha1-adrenergic-receptor (1-AR) subtype and/or specific protein kinase C isoform (PKC)-mediated downstream Ca2+-dependent and Ca2+-independent signal transduction pathways. An associated hypothesis is that development significantly alters 1-AR-subtype- and specific PKC isozyme-mediated expression of proto-oncogenes and genes representing vascular smooth muscle "synthetic" and/or "proliferative" phenotypes, as compared to adult "contractile" phenotype. Four Specific Aims are as follows. 1) What is the role of specific 1-AR subtypes and downstream effector proteins in signal transduction? 2) What is the role of specific PKC isoforms, extracellular signal regulated kinases (ERKs), Rho A/Rho kinases, and related kinases in signal transduction? 3) What is the role of specific 1-AR subtypes and PKC isoforms in gene regulation of developing vascular phenotypes? 4) What is the role of other signal transduction proteins presently poorly described in these signal transduction and gene regulation pathways? In ovine fetal, newborn, and adult CA, we will perform agonist-induced contractility and intracellular [Ca2+] measurements, Western immunoblots, RT- PCR, confocal microscopy, flow cytometry, 2D-gel-mass spectroscopy, gene silencing by double stranded RNA or morpholinos, gene upregulation, gene microarray/pathway analysis, and gene/protein discovery. Scientifically, the studies will advance our understanding of basic mechanisms whereby cerebral vessels change phenotypically and functionally with development from fetus, to newborn, to adult. Clinically, the studies relate to understanding the basis of the regulation of cerebral vascular tone, pressure, and blood flow in the fetus and/or premature newborn infant, and its dysregulation that results in intracerebral hemorrhage and serious neurologic sequelae.