Ionic mechanisms of toluene cerebrovascular actions

甲苯急性中毒是一个世界性的公共卫生问题。人类和动物数据 证明急性Tol中毒与脑低灌注量有关。血液流量的减少是一种 Tol导致的长期神经缺陷和灾难性急性情景的重要决定因素，包括 死亡。值得注意的是，Tol诱导脑内神经元减少的生物学靶点和机制 血流灌注是未知的。我们来自大鼠和小鼠的初步数据显示，与低灌注率一致， 急性暴露于醉人浓度的Tol在体外和活体均可导致脑动脉收缩 动物。因此，我们将通过背离所有 以前的工作集中在Tol对中枢神经元离子通道的影响，以解决这一突出问题 假设：急性Tol暴露引起的脑动脉收缩主要是由于药物抑制 BK型钾通道存在于动脉平滑肌(SM)本身。这一毒品行动是 由引起SM BK表型的两个BK亚基对Tol的不同感知决定：Channel- 形成CBV1，它通过其胞浆尾部结构域实现药物作用，以及SM丰富的、调节的β1， 其下调Tol对经络和脑动脉功能的作用。我们将解决三个问题 概念上相关，但可独立测试的特定目标(SA)：SA1(现象学)将确立 在急性中毒期间血液和脑中达到的水平的TOL收缩大脑动脉，不依赖于 全身代谢，循环或内皮因子，但主要通过抑制BK，这只需要 裸脂环境中的两个SM BK亚基。SA2(药物作用机制)将识别特定的 CBV1、β1和变构门控过程在决定Tol对BK活性和脑动脉作用中的作用 直径。SA3(翻译方面)将证明β1水平的自然变化决定了 脑动脉分支对Tol引起的收缩的不同易感性，而这个亚单位可以使用 作为选择性小分子药物的治疗靶点，以对抗Tol对脑血管的作用。要测试建议的 目标，我们将使用多学科方法，包括Tol蒸气暴露范例和颅脑 体内窗口，体外肌张力测定，新的和选择性的药理工具， 基因工程小鼠，重组DNA和工程BK亚基，外源基因的组织电穿孔 CDNA、生物素化和Western blotting、脂双层和膜片钳电生理学以及变构 选通分析。我们希望揭示介导Tol-Tol的细胞靶点和分子机制。 诱导脑血管收缩并为早期干预提供新的选择性药理学工具 TOL诱导的脑缺血，同时对其他器官有轻微副作用。

Acute intoxication with toluene (Tol) constitutes a worldwide public health problem. Human and animal data demonstrate that acute Tol intoxication is associated with brain hypoperfusion. The decrease in blood flow is a significant determinant of Tol-induced long-term neurological deficits and catastrophic acute scenarios, including death. Remarkably, the biological targets and mechanisms underlying Tol-induced reduction in cerebral perfusion are unknown. Our preliminary data from rat and mouse show that, consistent with hypoperfusion, acute exposure to intoxicating concentrations of Tol leads to cerebral artery constriction both in vitro and in live animals. Thus, we will cover the current knowledge gap in neurovascular toxicology by departing from all previous work, which focused on Tol effects on central neuron ion channels, to address this overarching hypothesis: constriction of cerebral arteries by acute Tol exposure is primarily due to drug inhibition of potassium channels of the BK type present in the arterial smooth muscle (SM) itself. This drug action is determined by distinct sensing of Tol by the two BK subunits that give rise to the SM BK phenotype: channel- forming cbv1, which enables drug action through its cytosolic tail domain, and the SM-abundant, regulatory β1, which downregulates Tol actions on both channel and cerebral artery function. We will address three conceptually related, yet independently testable specific aims (SA): SA1 (phenomenology) will establish that Tol at levels reached in blood and brain during acute intoxication constricts cerebral arteries independently of Tol systemic metabolism, circulating or endothelial factors but by primarily inhibiting BK, which only requires the two SM BK subunits in a bare lipid environment. SA2 (mechanism of drug action) will identify the specific roles of cbv1, β1, and allosteric gating processes that determine Tol action on BK activity and cerebral artery diameter. SA3 (translational aspects) will prove that naturally occurring variations in β1 levels determine the differential vulnerability of brain arterial branches to Tol-induced constriction, whereas this subunit can be used as therapeutic target of selective small agents to counteract Tol action on brain vessels. To test the proposed aims, we will use a multidisciplinary approach that includes Tol vapor exposure paradigms and a cranial window in vivo, in vitro myogenic tone determinations, novel and selective pharmacological tools, engineered mice, recombinant DNA and engineered BK subunits, electroporation of tissues with foreign cDNAs, biotinylation and Western blotting, lipid bilayer and patch-clamp electrophysiology, and allosteric gating analysis. We expect to unveil the cellular targets and molecular mechanisms that mediate Tol- induced cerebrovascular constriction and to deliver new selective pharmacological tools for early intervention in Tol-induced brain ischemia, while having minor side effects in other organs.