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（TOL）急性中毒构成了全球公共卫生问题。人类和动物数据 证明急性TOR插入与大脑灌注不足有关。血流的减少是 明确确定了TOL诱导的长期神经系统缺陷和灾难性的急性场景，包括 死亡。值得注意的是，生物学靶和机制降低了大脑的降低 灌注是未知的。我们来自大鼠和鼠标的初步数据表明，与灌注不足一致， 急性暴露于陶醉的TOL浓度会导致体外和活体内的脑动脉收缩 动物。这，我们将通过从所有人开始涵盖神经血管毒理学的当前知识差距 以前的工作着重于对中央神经离子渠道的影响，以解决这一总体 假设：急性TOL暴露对脑动脉的收缩主要是由于药物抑制 BK类型的钾通道本身存在于动脉平滑肌（SM）中。这种毒品动作是 通过两个BK亚基对TOL的明显感应，从而导致SM BK表型：通道 - 形成CBV1，可以通过其胞质尾部结构域进行药物作用，并具有SM含量的调节性β1， 它下调了在通道和脑动脉功能上的TOL作用。我们将解决三个 从概念上相关但可以独立测试的特定目的（SA）：SA1（现象学）将确定这一点 急性中毒期间血液和大脑达到的水平的TOR缩减了脑动脉独立于 TOL全身代谢，循环或内皮因素，但通过主要抑制BK，仅需要 在裸露的脂质环境中，两个SM BK亚基。 SA2（药物作用机制）将确定特定 CBV1，β1和变构门控过程的作用，确定对BK活性和脑动脉的作用 直径。 SA3（翻译方面）将证明β1水平的自然发生变化决定了 大脑伪影对TOL引起的收缩的差异脆弱性，而该亚基可以使用 作为选择性小型药物的治疗靶标，可以抵消对脑血管的TOL作用。测试拟议的 目的，我们将使用一种多学科方法，其中包括TOL蒸气暴露范式和颅骨 体内窗户，体外肌原张力测定，新颖和选择性的药物工具， 工程鼠标，重组DNA和工程BK亚基，外国组织的电穿孔 cDNA，生物素化和蛋白质印迹，脂质双层和斑块钳电生理学以及变构学 门控分析。我们期望揭示介导Tol-tol-tol-tol的细胞靶标和分子机制 诱发脑血管收缩，并提供新的选择性药物工具，以提早干预 TOL诱导的脑缺血，同时在其他器官中具有较小的副作用。