MECHANISMS FOR TOLERANCE TO ACTIONS OF A2 AGONISTS

α 2肾上腺素能受体激动剂用于手术患者麻醉管理的临床效用已得到验证。现在，α 2激动剂的管理正在考虑用于慢性疼痛的管理和机械通气患者的长期镇静。然而，动物研究表明，α 2肾上腺素能激动剂的镇痛和镇静/催眠作用随着时间的推移而减弱，这是一种称为耐受性的生物学现象。这项研究旨在确定对α 2肾上腺素能激动剂这两种行为特性的耐受性的诱导和表达机制。将使大鼠耐受右旋美托咪定（一种强效和高选择性α 2激动剂）的催眠和/或镇痛特性，并将在从蓝斑（催眠作用部位）和脊髓（镇痛作用部位）获得的组织中确定推定的“耐受级联反应”中关键分子组分的功能。还将使用NG 108-15细胞在细胞培养范例中研究耐受性“表型”，以更直接地定义分子机制。我们认为，通过NMDA型谷氨酸受体复合物和一氧化氮合酶（NOS）的参与诱导的推定的耐受级联反应，而持久的耐受表达涉及cAMP依赖性蛋白激酶（PKA）及其磷酸化底物，L型钙通道（LTCC）和cAMP反应元件结合蛋白（CREB）。LTCC的表达由磷酸化CREB诱导，并且磷酸化LTCC变得容易对α 2激动剂的阻断具有相对抗性，这是对α 2激动剂的行为反应转导中的关键步骤。我们将通过评估慢性暴露于α 2激动剂+/-阻断单个组分后的表达来确定单个组分是否是耐受性发展所必需的。通过在存在阻断剂的情况下重复关键分子组分的生化分析，我们将建立“阻断”组分与耐受性级联中其他分子组分的顺序关系。为了证明功能改变的分子组分是否足以产生耐受性，我们将在幼稚范例中模拟生物化学改变并确定耐受性表型是否可以再现。这些研究的数据可用于开发预防或逆转耐受性的治疗策略。

The clinical utility of alpha2 adrenergic agonists for the anesthetic management of the surgical patient has been validated. Now the administration of alpha2 agonists is being considered for the management of chronic pain and for prolonged sedation of mechanically-ventilated patients. However, animal studies reveal that the analgesic and sedative/hypnotic effects of alpha2 adrenergic agonists diminish over time, a biologic phenomenon known as tolerance. The work proposed in this grant application seeks to define the mechanisms responsible for the induction and expression of tolerance to these two behavioral properties of alpha2 adrenergic agonists. Rats will be rendered tolerant to the hypnotic and/or analgesic properties of dexmedetomidine, a potent and highly-selective alpha2 agonist, and the function of key molecular components in a putative "tolerance cascade" will be determined in tissue obtained from the locus coeruleus (site of hypnotic action) and the spinal cord (site of analgesic action). The tolerance "phenotype" will also be studied in a cell culture paradigm using NG 108-15 cells to define the molecular mechanisms more directly. We propose that the putative tolerance cascade induced through the participation of the NMDA-type glutamate receptor complex and nitric oxide synthase (NOS) while the long-lasting expression of tolerance involve cAMP-dependent protein kinase (PKA) and it phosphorylated substrates, L-type Ca2+ channel (LTCC) and cAMP- responsive element binding protein (CREB). The expression of LTCC is induced by phosphoCREB and phosphorylated LTCC becomes facilitated an relatively resistant to blockade by alpha2 agonists, a key step in the transduction of the behavioral response to alpha2 agonists. We will determine whether the individual components are necessary for the development of tolerance by assessing its expression following chronic exposure to alpha2 agonists +/- blockade of the individual components. By repeating the biochemical analysis of the pivotal molecular components in the presence of the blocker we will establish the sequential relationship of the "blocked" component to the other molecular components within the tolerance cascade. To demonstrate whether a functionally-altered molecular component is sufficient to produce tolerance, we will mimic the biochemical alteration in naive paradigms and determine whether the tolerance phenotype can be reproduced. Data from these studies can be used to develop therapeutic strategies to prevent or reverse tolerance.