BLOOD-BRAIN BARRIER ION TRANSPORT IN CEREBRAL ISCHEMIA

该项目的长期目标是确定血脑屏障(BBB)离子转运体 脑缺血引起的脑水肿。在缺血性中风的早期几个小时内，会出现浮肿 一种完整的血脑屏障，通过血脑屏障将钠和氯从血液输送到脑中。我们的研究已经 研究表明，存在于管腔血脑屏障膜上的Na-K-CI共转运受到缺血因素的刺激。 包括低氧、高血糖、加压素(AVP)和抑制共转运体可减轻大鼠的水肿 中风的模型。因此，血脑屏障Na-K-CI共转运体似乎是导致脑缺血的主要因素 浮肿。我们现在已经在初步研究中发现，血脑屏障钠氢交换器似乎也参与了 缺血诱导的水肿形成，这表明额外的BBB靶点可能对 中风早期的浮肿减轻。我们的假设是，除了共转运蛋白， 在缺血期间，腔内血脑屏障钠氢交换器被激活，以增加钠从血液到体内的转运 大脑。第一个目的是确定管腔血脑屏障膜上是否存在Na/H交换 受到缺血因素的刺激。我们将使用免疫电子显微镜来评估血脑屏障Na/H交换 蛋白原位和微量荧光分光光度法评价缺血因子对CMEC Na/H交换的影响 活动。第二个目的是确定抑制血脑屏障Na/H交换器是否能减轻缺血 浮肿。在这里，我们将研究血脑屏障Na/H交换抑制对缺血诱导的影响 用核磁共振方法观察大鼠脑组织钠和水的变化。我们亦会评估 Na/H交换和Na-K-CI共转运体抑制剂减轻脑水肿的疗效 在缺血发作后给药。这个项目的第三个目标是评估信号通路。 通过缺血刺激血脑屏障、Na-K-CI共转运体和Na/H交换器活性。我们将从 AMP、p38MAP和细胞内[Ca]在低氧、高血糖和AVP诱导中的作用 由于提案中详细说明的原因，对共转运体和交换器的刺激。然而，我们的 研究还将包括对蛋白激酶C、ERK1/2蛋白激酶和JNK蛋白的初步评估 激活剂。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 激酶的激活(通过Western印迹)和激酶抑制剂对缺血(低氧， 血糖和AVP)刺激共转运体活性(放射性同位素通量)和Na/H交换活性 (荧光分光光度和放射性同位素通量)。脑缺血引起的水肿是脑损伤的一个主要原因。 中风是美国的主要死亡原因。这项拟议的研究将揭示是否具有治疗作用 防止Na-K-CI共转运体和/或Na/H交换的缺血刺激途径 活动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.