BLOOD-BRAIN BARRIER ION TRANSPORT IN CEREBRAL ISCHEMIA

本项目的长期目标是确定血脑屏障（BBB）离子转运蛋白，介导 缺血性脑水肿。在缺血性中风的早期，水肿在以下情况下形成： 通过涉及血脑屏障将Na和Cl从血液转运到脑中的过程形成完整的血脑屏障。我们的研究 显示存在于腔BBB膜中的Na-K-Cl共转运受到缺血因子的刺激， 包括缺氧、无血糖、加压素（AVP），并且抑制协同转运蛋白减少大鼠水肿 中风模型因此，BBB Na-K-Cl协同转运蛋白似乎是缺血诱导的脑缺血的主要贡献者。 水肿我们现在已经在初步研究中发现，BBB Na/H交换器似乎也参与了 缺血诱导的水肿形成，这表明一个令人兴奋的可能性，额外的血脑屏障的目标， 减少中风早期的水肿。我们的假设是，除了协同转运蛋白， 在局部缺血期间刺激腔BBB Na/H交换器以增加Na从血液转运到 个脑袋第一个目的是确定Na/H交换是否存在于腔BBB膜处，并且是否存在于腔BBB膜处。 由缺血性因素刺激。我们将使用免疫电镜来评估BBB Na/H交换 蛋白质原位和显微荧光光谱法评估缺血因子对CMEC Na/H交换的影响 活动第二个目的是确定BBB Na/H交换器的抑制是否减弱缺血诱导的脑缺血。 水肿在这里，我们将研究BBB Na/H交换抑制对缺血诱导的脑缺血的影响。 用核磁共振方法测定大鼠脑钠和水的变化。我们亦会评估 Na/H交换和Na-K-Cl共转运抑制剂对减轻脑水肿有效性 在缺血发作后施用。本项目的第三个目的是评估信号通路 缺血刺激血脑屏障Na-K-Cl协同转运体和Na/H交换体活性。我们将首先 探讨AMP激酶、p38 MAP激酶和细胞内[Ca] 2+在缺氧、缺糖和AVP诱导的心肌缺血中的作用。 由于提案中详述的原因，刺激协同转运蛋白和交换蛋白。但我们的 研究还将包括对蛋白激酶C、ERK 1/2 MAP激酶和JNK MAP的初步评估。 激酶。在这些研究中，我们将使用脑微血管内皮细胞来评估缺血诱导的 激酶的活化（通过Western印迹）和激酶抑制剂对缺血（缺氧， 高血糖和AVP）刺激协同转运蛋白活性（放射性同位素通量）和Na/H交换活性 （荧光分光光度法和放射性同位素通量）。缺血引起的水肿是脑损伤的主要原因， 中风，在美国的主要死因，拟议的研究将揭示是否治疗 旨在防止Na-K-Cl协同转运蛋白和/或Na/H交换的缺血刺激的方法 活性可能对减轻中风引起的脑水肿有价值。

The long term goal of this project is to identify blood-brain barrier (BBB) ion transporters that mediate ischemia-induced brain edema. During the early hours of ischemic stroke, edema forms in the presence of an intact BBB by a process involving BBB transport of Na and Cl from blood into brain. Our studies have shown that Na-K-CI cotransport, present in the luminal BBB membrane, is stimulated by ischemic factors, including hypoxia, aglycemia, vasopressin (AVP) and that inhibiting the cotransporter reduces edema in a rat model of stroke. Thus, the BBB Na-K-CI cotransporter appears to be a major contributor to ischemia-induced edema. We have now found in preliminary studies that a BBB Na/H exchanger also appears to participate in ischemia-induced edema formation which suggests the exciting possibility of an additional BBB target for reduction of edema during the early hours of stroke. Our hypothesis is that, in addition to the cotransporter, a luminal BBB Na/H exchanger is stimulated during ischemia to increase transport of Na from blood into brain. The first aim is to determine whether Na/H exchange is present at the luminal BBB membrane and is stimulated by ischemic factors. We will use immunoelectron microscopy to evaluate BBB Na/H exchange protein in situ and microspectrofluorometry to assess ischemic factor effects on CMEC Na/H exchange activity. The second aim is to determine whether inhibition of the BBB Na/H exchanger attenuates ischemiainduced edema. Here, we will examine the effect of BBB Na/H exchange inhibition on ischemia-induced changes in rat brain Na and water, using nuclear magnetic resonance methods. We will also assess the efficacy of Na/H exchange and Na-K-CI cotransport inhibitors for reduction of cerebral edema when administered after the onset of ischemia. The third aim of this project is to evaluate the signaling pathways by which ischemia stimulates BBB Na-K-CI cotransporter and Na/H exchanger activities. We will start by evaluating the roles of AMP kinase, p38 MAP kinase and intracellular [Ca] in hypoxia, aglycemia and AVPinduced stimulation of the cotransporter and exchanger for reasons detailed in the proposal. However, our studies will also include an initial assessment of protein kinase C, ERK1/2 MAP kinase and JNK MAP kinase. For these studies we will use cerebral microvascular endothelial cells to evaluate ischemia-induced activation of the kinases (by Western blot) and the effects of kinase inhibitors on ischemia (hypoxia, aglycemia and AVP) stimulated cotransporter activity (radioisotope flux) and Na/H exchanger activity (spectrofluorometry and radioisotope flux). Ischemia-induced edema is a major cause of brain damage in stroke, a leading cause of death in the U.S. The proposed studies will reveal whether therapeutic approaches aimed at preventing ischemia stimulation of Na-K-CI cotransporter and/or Na/H exchange activity may be of value for attenuating stroke-induced brain edema.