MECHANISMS FOR TOLERANCE TO ACTIONS OF A2 AGONISTS

α2肾上腺素能激动剂在外科病人麻醉管理中的临床效用已得到验证。现在正在考虑使用Alpha2激动剂来治疗慢性疼痛和延长机械通气患者的镇静时间。然而，动物研究表明，α2肾上腺素能激动剂的止痛和镇静/催眠作用会随着时间的推移而减弱，这是一种被称为耐受的生物现象。在这项拨款申请中提出的工作试图确定对α2肾上腺素能激动剂这两种行为特性的耐受诱导和表达的机制。大鼠将对右美托咪定的催眠和/或止痛特性产生耐受性，这是一种有效和高选择性的α2激动剂，从蓝斑(催眠作用部位)和脊髓(止痛作用部位)获得的组织中将确定推测的“耐受级联反应”中关键分子成分的功能。耐受性“表型”也将在使用NG 108-15细胞的细胞培养范例中进行研究，以更直接地定义分子机制。我们认为，NMDA型谷氨酸受体复合体和一氧化氮合酶参与诱导耐受级联反应，而耐受的持久表达涉及cAMP依赖的蛋白激酶及其磷酸化底物、L型钙通道和cAMP反应元件结合蛋白。LTCC的表达是由磷酸化的CREB诱导的，磷酸化的LTCC对α2激动剂的阻断具有相对抵抗力，这是转导对α2激动剂的行为反应的关键步骤。我们将通过评估其在长期暴露于α2激动剂+/-阻断个别成分后的表达来确定个别成分是否对形成耐受是必要的。通过在阻滞剂存在的情况下重复关键分子成分的生化分析，我们将建立耐受级联中被阻断的成分与其他分子成分的顺序关系。为了证明一个功能改变的分子成分是否足以产生耐受性，我们将模拟朴素范例中的生化变化，并确定耐受性表型是否可以复制。来自这些研究的数据可用于制定预防或逆转耐受的治疗策略。

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.