Sex-dependent expression and utilization of spinal mu- and kappa-opioid systems

描述(由申请人提供)：以前定义阿片类药物抗伤害性感觉的性依赖的研究没有准备好揭示相关的机制基础和生物底物。本申请建议建立脊髓阿片类抗伤害性作用的性别依赖药理学特征与(1)脊髓阿片受体的性别二形性和(2)鞘内(I.T.)脊髓阿片类药物释放的性别相关性之间的因果关系。吗啡。大量的初步数据支持脊髓β-阿片受体(MOR)、内吗啡2(EM2)、β-阿片受体(KOR)和强啡肽1-17(Dyn)之间的性二型性关系，这一假设得到了我们先前的I.T.在男性中，吗啡引起的主要是吗啡偶联的脊髓抗伤害性感受，而在女性中，I.T.吗啡招募了一个更具包容性的综合系统，需要脊髓MOR和KOR的伴随活动。脊髓MOR剪接变异体和MOR KOR异源二聚体的性二态表达水平被认为是女性对脊髓吗啡的表型反应的分子基础。与假设的脊髓阿片受体的性二态组织相平行，我们假设对脊髓阿片类药物释放和镇痛反应的性别依赖性调节是性别依赖性脊髓阿片类药物抗伤害性感觉的组成部分。具体地说，目标1将检验这样一个假设，即IT产生的抗伤害性感觉。吗啡需要男性脊髓释放EM2，但女性不需要。目标2将检验这一假设，即IT。与女性相比，EM2在男性中引起更强的抗伤害性感受，这加剧了I.T.对脊髓EM2的性别依赖性释放的后果。吗啡。我们推测，这至少部分是由脊柱MOR剪接变异体的性别依赖性表达所介导的。确认I.T.释放脊髓EM2。吗啡是其在男性身上产生的脊髓抗伤害性感觉的先决条件，而不是女性，不会完全揭示脊髓吗啡抗伤害性感觉背后的性别依赖景观。要做到这一点，需要了解I.T.招募的止痛药底物。女性体内的吗啡。因此，目标3将检验这一假设，即在女性中，I.T.吗啡的镇痛作用是通过释放Dyn而不是EM2来实现的。目的4将验证一种假设，即女性对脊髓吗啡抗伤害作用的同时激活脊髓MOR和KOR的要求明显反映了女性脊髓中MOR KOR异源二聚体的表达水平显著高于男性。我们将使用多维方法来实现这些相互关联的目标，该方法集成了行为、药理学、分子和基于免疫的生化水平的分析。这些将被用作交叉验证调查结果的补充措施。由于疼痛和成瘾具有共同的底物，预期结果将揭示新的药物靶点，这些靶点不仅与优化男性和女性的疼痛管理有关，而且还与以性别依赖的方式治疗阿片类药物的戒断和复发有关。

DESCRIPTION (provided by applicant): Previous studies that defined sex dependency of opioid antinociception were not poised to reveal relevant mechanistic underpinnings and biological substrates. The present application proposes to establish a causal association between sex-dependent pharmacological characteristics of spinal opioid antinociception and (1) the sexual dimorphic nature of spinal opioid receptors and (2) the sex-dependent release of spinal opioids by intrathecal (i.t.) morphine. Hypothesized sexual dimorphic relationships among spinal ?-opioid receptors (MOR), endomorphin 2 (EM2), ?-opioid receptors (KOR) and dynorphin 1-17 (Dyn) are supported by substantial preliminary data, which is undergirded by our previous demonstrations that i.t. morphine elicits predominantly MOR-coupled spinal antinociception in males, whereas in females i.t. morphine recruits a more inclusive integrative system that requires concomitant activity of spinal MOR and KOR. Sexual dimorphic expression levels of spinal MOR splice variants and MOR KOR heterodimers are postulated to be molecular underpinnings of the female phenotypic response to spinal morphine. In parallel with the postulated sexually dimorphic organization of spinal opioid receptors, we hypothesize that the sex-dependent regulation of the release of and analgesic responsiveness to spinal opioids are integral component of sex-dependent spinal opioid antinociception. Specifically, Aim 1 will test the hypothesis that the antinociception produced by i.t. morphine, requires the release of EM2 from the spinal cord of males, but not females. Aim 2 will test the hypothesis that i.t. EM2 elicits greater antinociception in males vs. females, which exacerbates the consequences of the sex-dependent release of spinal EM2 by i.t. morphine. We postulate that this is mediated, at lest in part, by the sex-dependent expression of spinal MOR splice variants. Validation that release of spinal EM2 by i.t. morphine is a prerequisite for the spinal antinociception it produces in males, but not females, would not fully reveal the sex-dependent landscape that underlies spinal morphine antinociception. To do so requires knowledge of the analgesic substrates recruited by i.t. morphine in females. Accordingly, Aim 3 will test the hypothesis that in females, i.t. morphine analgesia is mediated via the release of Dyn, not EM2. Aim 4 will test the hypothesis that the requirement in females for the concomitant activation of spinal MOR and KOR for spinal morphine antinociception to be manifest reflects the presence of significantly higher expression levels of MOR KOR heterodimers in spinal cord of females vs. males. We will pursue these interrelated Aims using a multi-dimensional approach that integrates behavioral, pharmacological, molecular and immuno-based biochemical levels of analyses. These will be used as complementary measures to cross validate findings. Since pain and addiction share common substrates, expected outcomes will reveal novel drug targets that are not only relevant to optimizing pain management in men and women but also to treating opioid withdrawal and relapse in a sex-dependent fashion.