NITRIC OXIDE/20-HETE INTERACTION IN CONTROL OF CEREBRAL VASCULAR TONE

本项目将研究一氧化氮(NO)与20- HETE在调节脑血流方面的作用，因为最近的工作已经 表示这两个系统在 脑血管张力的控制。具体来说，我们将研究 关于NO抑制强效血管收缩物质形成的假说 血管平滑肌细胞内20-HETE及其抑制作用 有助于NO激活K+通道和扩张 大脑小动脉。这一假设是基于我们的初步数据 结果表明：1)NO直接抑制20-HETE的合成； 大鼠脑小动脉对一氧化氮的扩张剂反应 似乎在很大程度上不依赖于cGMP；以及3)防止跌倒 脑小动脉中20-HETE水平减弱K+的激活 大脑小动脉对NO的通道和血管扩张反应 减弱K+通道的激活和血管扩张反应 不是的。在拟议的研究中，不同浓度的NO的影响 刺激鸟苷酸环化酶抑制20-HETE的产生 直接在脑小动脉和血管平滑肌中进行研究 从这些血管中分离出来的细胞。我们将鉴定P4504A亚型 异构体逆转录聚合酶链式反应在大鼠脑血管中的表达 将利用特定的引物和可见光光谱来 NO与相应重组P4504A蛋白结合的研究 利用杆状病毒系统和Sf9细胞表达。一氧化氮对人体健康的影响 脑血管平滑肌细胞的K+通道活性将 用膜片钳技术表征，以及相对的 20-HETE与cGMP水平的变化对这些反应的贡献 将通过改变细胞内20-HETE水平来确定 鸟苷酸环化酶和cGMP依赖的抑制剂的存在或不存在 蛋白激酶。平行研究将在隔离的、灌流的 确定脑小动脉改变的贡献 20-HETE与cGMP的产生对NO改变膜的影响 潜力和血管张力。20-HETE在调解中的意义 NO诱导的脑血流变化将使用激光进行评估 非供体合酶作用下麻醉大鼠的多普勒血流量测定 抑制物，以及促进NO合成的刺激物 在用多种抑制剂阻断P4504A通路后 我们最近开发的受体拮抗剂。鉴于新兴的 20-HETE和NO在脑血流控制中的重要性 有令人信服的理由来更好地理解 这些系统相互作用的细胞和离子机制 调节脑血管张力。

This Project will examine interactions between nitric oxide (NO) and 20- HETE in the regulation of cerebral blood flow, since recent work has indicated that both systems play central but opposing roles in the control of cerebral vascular tone. Specifically, we will examine the hypothesis that NO inhibits the formation of the potent vasoconstrictor 20-HETE in vascular smooth muscle cells and this inhibitory action contributes to the ability of NO to activate K+ channels and dilate the cerebral arterioles. This hypothesis is based on our preliminary data indicating that: 1) NO directly inhibits the synthesis of 20-HETE; 2) the vasodilator response to NO in small cerebral arterioles of the rat appears to be largely cGMP independent; and 3) that preventing the fall in 20-HETE levels in cerebral arterioles attenuates the activation of K+ channels and vasodilatory response to NO in small cerebral arterioles attenuates the activation of K+ channels and vasodilatory response to NO. In the proposed studies, the effects of various concentrations of NO to stimulate guanylyl cyclase and inhibit the production of 20-HETE will be directly studied in cerebral arterioles and vascular smooth muscle cells isolated from these vessels. We will identify the P4504A isoforms expressed in the cerebral vasculature of rats using RT-PCR with isoform specific primers and visible light spectroscopy will be utilized to study the binding of NO to the corresponding recombinant P4504A proteins expressed using a baculovirus system and Sf9 cells. The effects of NO on K+ channel activity in cerebral vascular smooth muscle cells will be characterized using patch-clamp techniques, and the relative contribution of changes in 20-HETE versus cGMP levels to these responses will be determined by altering intracellular levels of 20-HETE in the presence or absence of inhibitors of guanylyl cyclase and cGMP-dependent protein kinase. Parallel studies will be performed in isolated, perfused cerebral arterioles to determine the contribution of changes in the production of 20-HETE versus cGMP on the effects of NO to alter membrane potential and vascular tone. The significance of 20-HETE in mediating NO-induced changes in cerebral blood flow will be evaluated using laser Doppler flowmetry in anesthetized rats treated with NO-donors, synthase inhibitors, and stimuli that promote the synthesis of NO before and after blockade of the P4504A pathway with a variety of inhibitors and receptor antagonists that we have recently developed. Given the emerging importance of 20-HETE and NO to the control of cerebral blood flow, there are compelling reasons to obtain a better understanding of the cellular and ionic mechanisms by which these systems interact to regulate cerebral vascular tone.