Characterization of a novel JNK-mediated mechanism of cannabinoid tolerance

描述(申请人提供)：这项研究将调查对？9-THC耐受的细胞、分子和生理机制。我们已经产生了突变的小鼠(S426A/S430A)，表达了一种对大麻素受体1(CB)脱敏耐受的形式，表现出对？9-THC的延迟耐受。1然而，S426A/S430A突变体最终对9-THC完全耐受。用c-jun氨基末端激酶(JNK)抑制剂处理S426A/S430A突变小鼠可消除对β9-THC镇痛作用的耐受性，提示该信号通路可能与S426A/S430A突变小鼠的残留耐受有关。通过测定野生型、S426A/S430A x JNK1基因敲除(KO)和S426A/S430A x JNK2 KO双突变小鼠对？9-THC的镇痛、低温和触觉作用的耐受性，确定参与大麻类药物耐受的JNK亚型。在S426A/S430A单个突变小鼠中，将检测SP600125(JNK抑制剂)对低温、过敏性和止痛耐受性的预防作用的剂量-反应曲线，以确定该抑制剂的最佳剂量。基因芯片分析将检测Vehicle和SP600125处理的S426A/S430A突变体以及S426A/S430A单突变体与S426A/S430A x JNK1和S426A/S430A x JNK2 KO双突变体之间的基因表达差异，以确定JNK介导的大麻类药物耐受的分子靶点。所有通过微阵列分析确定的假定JNK靶标都将使用实时定量聚合酶链式反应进行验证。本研究的目的是确定JNK的形式，并确定JNK对大麻素类药物耐受的分子和生化靶点。

DESCRIPTION (provided by applicant): This study will investigate the cellular, molecular, and physiological mechanisms responsible for tolerance to ?9-THC. We have produced mutant mice (S426A/S430A) expressing a desensitization- resistant form of the cannabinoid receptor 1 (CB) that exhibit delayed tolerance for ?9-THC. 1 However S426A/S430A mutants eventually become completely tolerant to ?9-THC. Treatment of S426A/S430A mutant with an inhibitor of c-Jun N-terminal kinase (JNK) eliminates tolerance to the analgesic effects of ?9-THC suggesting that this signaling pathway might be responsible for the residual tolerance observed in S426A/S430A mutant mice. The specific JNK isoform involved in cannabinoid tolerance will be determined by measuring tolerance for the analgesic, hypothermic, and cataleptic effects of ?9-THC in wild-type, S426A/S430A x JNK1 knockout (KO), and S426A/S430A x JNK2 KO double mutant mice. A dose response curve for the preventative effects of SP600125 (JNK inhibitor) on hypothermic, cataleptic, and analgesic tolerance will be examined in S426A/S430A single mutant mice to determine an optimal dosage for this inhibitor. Microarray analyses examining differences in gene expression between vehicle and SP600125-treated S426A/S430A mutants as well between S426A/S430A single mutants and S426A/S430A x JNK1 and S426A/S430A x JNK2 KO double mutants will be done to determine the molecular targets responsible for JNK-mediated cannabinoid tolerance. All putative JNK targets identified by microarray analysis will be validated using quantitative real-time PCR. The goal of this study is to determine the form of JNK responsible for mediating tolerance to the analgesic effects of ?9-THC and also to identify the molecular and biochemical targets of JNK that are responsible for cannabinoid tolerance.