Ionic mechanisms of toluene cerebrovascular actions

甲苯脑血管作用的离子机制

• 批准号: 10627927
• 负责人: Anna Bukiya
• 金额: $ 43.22万
• 依托单位: UNIVERSITY OF TENNESSEE HEALTH SCI CTR
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-25 至 2027-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10627927
• 关键词: Ablation; Acute; Address; Affinity; Aging; Animals; Area; Arteries; Binding; Biological; Biotinylation; Blood; Blood flow; Brain; Brain Ischemia; Brain region; Cells; Cephalic; Cerebrum; Cessation of life; Circle of Willis; Complementary DNA; Complex; Coupling; Data; Diabetes Mellitus; Diameter; Drug Targeting; Early Intervention; Electrophysiology (science); Electroporation; Endothelium; Engineering; Environment; Exposure to; Genetic; Human; Hypertension; In Vitro; Inhalation; Inhalation Drug Administration; Intoxication; Ion Channel; Irrigation; Ischemia; Knowledge; Lead; Lipid Bilayers; Lipids; Mediating; Membrane; Metabolism; Minor; Molecular; Molecular Target; Mus; Muscle Contraction; Neuroglia; Neurologic Deficit; Neurons; Neurotoxins; Neurotransmitters; Organ; Perfusion; Pharmaceutical Preparations; Phenotype; Physiology; Population; Potassium Channel; Process; Proteins; Public Health; Publishing; Rattus; Recombinant DNA; Recombinants; Role; Signal Transduction; Signs and Symptoms; Site; Smooth Muscle; Smooth Muscle Myocytes; Stroke; Stupor; Sudden Death; Tail; Testing; Tissues; Toluene; Toxicology; Variant; Vasodilation; Vasodilator Agents; Voltage-Gated Potassium Channel; Western Blotting; Work; animal data; base; blood perfusion; brain circulation; cellular targeting; cerebral artery; cerebrovascular; clinically relevant; constriction; dimer; drug action; drug of abuse; human data; hypoperfusion; in vivo; interdisciplinary approach; large-conductance calcium-activated potassium channels; middle cerebral artery; neurotoxicity; neurovascular; novel; patch clamp; pharmacologic; phenomenological models; receptor; side effect; single photon emission computed tomography; therapeutic target; tool; vapor; voltage

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.

甲苯急性中毒是一个世界性的公共卫生问题。人和动物数据 表明急性Tol中毒与脑灌注不足有关。血流量的减少是一种 Tol诱导的长期神经功能缺损和灾难性急性情况的重要决定因素，包括 死亡值得注意的是，Tol诱导的脑缺血再灌注减少的生物学靶点和机制可能与Tol诱导的脑缺血再灌注损伤有关。 灌注未知。我们对大鼠和小鼠的初步数据显示，与低灌注一致， 急性暴露于中毒浓度的Tol导致体外和活体脑动脉收缩 动物因此，我们将涵盖目前的知识差距，在神经血管毒理学离开所有 以前的工作，集中在Tol对中枢神经元离子通道的影响，以解决这一总体问题 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）本身中。这种药物作用是 通过引起SM BK表型的两个BK亚基对Tol的不同感知来确定：通道- 形成cbv 1，其通过其胞质尾部结构域实现药物作用，以及SM丰富的调节性β1， 其下调Tol对通道和脑动脉功能的作用。我们将讨论三个 概念上相关，但可独立测试的具体目标（SA）：SA 1（现象学）将建立， 在急性中毒期间血液和脑中达到的Tol水平会收缩脑动脉， 抑制全身代谢、循环或内皮因子，但主要通过抑制BK，其仅需要 裸脂质环境中的两个SM BK亚基。SA 2（药物作用机制）将确定具体的 cbv 1、β1和决定Tol对BK活性和脑动脉作用的变构门控过程的作用 直径. SA 3（翻译方面）将证明β1水平的天然变异决定了 脑动脉分支对Tol诱导的收缩的不同脆弱性，而该亚基可用于 作为选择性小分子药物的治疗靶点，以抵消Tol对脑血管的作用。为了测试拟议的 目的，我们将使用多学科的方法，包括Tol蒸汽暴露范例和颅 体内窗口，体外肌原性张力测定，新的和选择性的药理学工具， 基因工程小鼠，重组DNA和基因工程BK亚单位，电穿孔组织与外源 cDNA、生物素化和蛋白质印迹、脂质双层和膜片钳电生理学以及变构 门控分析我们希望揭示介导Tol的细胞靶点和分子机制， 诱导脑血管收缩，并提供新的选择性药理学工具，用于早期干预， Tol诱导的脑缺血，同时对其他器官有轻微的副作用。