REGULATING PRESSURE DEPENDENT AUTOREGULATION OF CEREBRAL MICROVASCULAR BLOOD FLOW

该计划项目补助金的总体目标是定义一些 调节营养性血流分布的基本机制 到大脑。在这方面，项目1内的研究将侧重于 分子、细胞和信号转导事件介导 营养性脑血流量（CBF）的自动调节 跨壁压逐步升高。我们最近克隆了 对分离的前毛细管内的P4504 A ω-羟化酶cDNA进行测序 小动脉肌细胞，其表达催化形成 20-羟基二十碳三烯酸（20-HETE）从花生四烯酸（AA）中分离。 对ω-羟化酶的抑制消除了正常的，几乎完美的， 激光多普勒测量血流的自动调节， 大鼠顶叶皮层的颅窗。我们已经证明，20- HETE是在小动脉肌肉中内源性产生的， （<10/-10M）抑制大电导Ca/2+激活的K+ 通道（K/Ca），使质膜去极化，增加[Ca/2+]i， 激活可收缩的元素。初步研究结果显示，20-HETE 也直接增强膜片L型Ca/2+通道电流， 夹住小动脉肌细胞。信号转导级联 介导20-HETE的收缩作用涉及PKC的激活 如在PKC存在下其作用的抑制所证明的 假底物抑制剂Myr psiPKC-I（19-27）。另外我们有 初步数据显示，肉豆蔻酰化丙氨酸的磷酸化- 富C激酶底物（MARCKS）以20-HETE依赖性方式发生在 脑动脉肌细胞的原代培养物。内部协议 项目1将集中于进一步定义的分子表达 P4504 A在脑微循环中的定位 大脑中的ω-羟化酶亚型，生理意义 P4504 A过表达和AA ω-羟化酶上调 酶通过调节底物的可用性。我们将关注的基质 将是分子氧。我们最近证明， P4504 A ω-羟化酶对氧的Km约为60 乇当PO/2福尔斯从100降到200 Ω时， 20-HETE的形成表明P4504 A酶可能作为一种蛋白酶发挥作用。 大脑中的氧感。我们将定义大脑自动调节 在缺氧和高氧条件下的能力。概述的研究 本项目中的其他项目与本项目中的其他项目密切相关， 这个计划的项目。该项目和整个项目将 大大推进了我们对分子机制的认识 调节毛细血管前小动脉口径，以及这些机制如何 与大脑的血流分布有关， 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.