REGULATING PRESSURE DEPENDENT AUTOREGULATION OF CEREBRAL MICROVASCULAR BLOOD FLOW

本计划项目赠款的总体目标是定义一些 调节营养性血流分布的基本机制 对大脑的影响。在这方面，项目1的研究将集中在 分子、细胞和信号转导事件 营养性脑血流量(CBF)的自动调节 跨壁压阶梯升高。我们最近克隆了 分离毛细管前体内P4504A omega-羟基酶基因的序列分析 小动脉肌细胞表达一种催化形成的酶 从花生四烯酸(AA)中分离得到20-羟基二十碳四酸(20-HETE)。 抑制omega-羟基酶可以废除正常的，近乎完美的， 自动调节激光多普勒测量的血流量通过记录 大鼠顶叶皮质的颅窗。我们已经证明了20- HETE是在小动脉肌中内源性产生的，在那里它有效地( (&lt；10/-10M)抑制大电导Ca/2+激活K+的活性 通道(K/Ca)，使质膜去极化，增加[Ca/2+]i和 激活收缩元素。初步研究结果显示，20-HETE 还可直接增强膜片内向L型钙通道电流。 夹住的小动脉肌细胞。信号转导级联 介导20-HETE的收缩作用涉及PKC的激活 在PKC存在的情况下抑制其作用证明了这一点 假底物抑制剂Myr psiPKC-I(19-27)。此外，我们还有 初步数据显示，肉豆蔻化丙氨酸的磷酸化- 富C激酶底物(Marcks)以20-HETE依赖的方式存在于 大脑动脉肌细胞的原代培养。协议范围内 项目1将专注于进一步定义分子表达 P4504A在脑微循环中的定位 脑内omega-羟基酶亚型的生理学意义 P4504A的过度表达和AA omega-羟基酶的上调 酶通过调节底物的利用率。我们将重点关注底物 这将是分子氧。我们最近已经证明了 P4504A omega-羟基酶对氧的Km约为60 托尔。当PO/2从100托下降到200托时，线性减少 20-HETE的形成提示P4504A酶可能作为一种 大脑中的氧气感受器。我们将定义大脑自动调节 低氧和高氧条件下的能力。概述的研究 在本项目中与其他项目相关并紧密结合在一起 本计划的项目。本项目和整个计划将 极大地提高了我们对分子机制的了解 调节毛细血管前小动脉管径及其作用机制S 与血液流向大脑的完整状态有关 动物。

The overall goal of this Program Project Grant is to define some of the fundamental mechanisms regulating distribution of nutritive blood flow to the brain. In this regard, studies within Project 1 will focus on the molecular, cellular and signal transduction events mediating autoregulation of nutritive cerebral blood flow (CBF) in response to step elevations in transmural pressure. We have recently cloned and sequenced a P4504A omega-hydroxylase cDNA within isolated pre-capillary arteriolar muscle cells which expresses an enzyme catalyzing formation of 20-hydroxyeicosetrinoic acid (20-HETE) from arachidonic acid (AA). Inhibition of omega-hydroxylases abolishes the normal, nearly perfect, autoregulation of laser-Doppler measured blood flow recorded via a cranial window in the rat parietal cortex. We have demonstrated that 20- HETE IS endogenously produced in arteriolar muscle where it potently ( (<10/-10M) inhibits activity of the large conductance Ca/2+ activated K+ channel (K/Ca), depolarizes the plasma membrane increases [Ca/2+]i and activates contractile elements. Preliminary findings show that 20-HETE also directly enhances inward L-type Ca/2+ channel current in patch- clamped arteriolar muscle cells. The signal transduction cascade mediating the contractile action of 20-HETE involves activation of PKC as evidenced by inhibition of its action in the presence of a PKC pseudosubstrate inhibitor Myr psiPKC-I(19-27). In addition, we have preliminary data showing that phosphorylation of myristolylated alanine- rich C kinase substrate (MARCKS) occurs in a 20-HETE dependent manner in primary cultures of cerebral arterial muscle cells. Protocols within Project 1 will focus on further defining the molecular expression of P4504A within the cerebral microcirculation, localization of P4504A omega-hydroxylase isoforms in the brain, the physiological significance of P4504A over-expression, and up-regulation of the AA omega-hydroxylase enzyme by regulating substrate availability. The substrate we will focus on will be molecular oxygen. We have recently demonstrated that the P4504A omega-hydroxylase possesses a Km for oxygen of approximately 60 torr. As PO/2 falls from 100 to 200 torr, there is a linear reduction of 20-HETE formation suggesting that P4504A enzymes may function as an oxygen sensory in the brain. We will define cerebral autoregulatory capacity under hypoxic and hyperoxic conditions. The studies as outlined in this Project relate to and are closely integrated into the other Projects of this Program. This Project, and the Program as a whole will significantly advance our knowledge regarding the molecular mechanisms regulating pre-capillary arteriolar caliber and how these mechanism s relate to the distribution of blood flow to the brain in the intact animal.