Persistent COMT-dependent Pain: Role of beta-adrenergic Receptors

描述(申请人提供)：复杂的持续性疼痛情况，如纤维肌痛(FM)和颞下颌关节紊乱病(TMD)，由于潜在的分子机制仍然很大程度上仍不清楚，治疗效果不佳。我们和其他人的工作表明，这些情况在很大程度上是由于儿茶酚-O-甲基转移酶(COMT；一种代谢儿茶酚胺的酶)活性降低，导致儿茶酚胺水平升高和β2/3肾上腺素能受体(Beta2/3ARs)活性增加。然而，β2/3ARs介导COMT依赖性疼痛的确切机制尚不清楚，需要进一步研究。初步数据显示，β2/3ARs的激活可能通过增加下游信号分子的表达而增加痛觉敏感性。我们发现COMT抑制导致促炎细胞因子肿瘤坏死因子-α(TNF1)、白介素12(IL-12)、白介素6(IL-6)以及一氧化氮(NO)的表达增加，这种增加可被2AR拮抗剂阻断。[更多数据进一步表明，COMT依赖的疼痛部分是由外周肾上腺素能系统介导的，因为缺乏外周肾上腺素的肾上腺切除大鼠表现出COMT依赖的疼痛敏感性降低。本申请建议通过在持续COMT抑制产生的持续性疼痛的新型动物模型中应用不同的方法来扩展这项工作，以阐明β受体在细胞和系统水平上驱动持续性疼痛中所起的作用。首先，我们将在正常大鼠、肾上腺切除大鼠和COMT基因敲除小鼠中应用行为药理学方法来确定β-肾上腺素能系统驱动持续性COMT依赖疼痛的作用部位。其次，我们将应用免疫细胞化学技术来确定在COMT持续抑制后，β受体在介导神经元、小胶质细胞和星形胶质细胞激活中的作用。第三，我们将应用分子生物学方法来确定BetaARs在调节促炎细胞因子和持续COMT抑制后NO表达中的作用。我们假设持续性COMT依赖疼痛通过外周、脊髓和中枢β2/3ARs产生细胞活性和促炎细胞因子和NO表达的长期变化。这些研究的新方法将1)确定导致FM和TMD等持续性疼痛的BetaAR的亚型和位置，2)表征持续的BetaAR激活对脊髓和大脑中传递疼痛信息的神经元和神经胶质细胞的长期后果，3)表征持续的2AR激活对促炎细胞因子和NO的长期影响，它们代表伤害性感受的有效标志，以及4)确定Beta2/3AR拮抗剂抑制伤害性信息传递的能力。这些研究的结果将对适应不良疼痛状况的潜在机制提供新的见解，并有助于确定以前未开发的靶点(例如，β2和β3AR)，以开发针对持续性疼痛状况的有效治疗方法。]]

DESCRIPTION (provided by applicant): Complex persistent pain conditions, such as fibromyalgia (FM) and temporomandibular disorder (TMD), are ineffectively treated because the underlying molecular mechanisms remain largely unknown. Work by our group and others suggests that these conditions are due, in large part, to diminished activity of catechol-O- methyltransferase (COMT; an enzyme that metabolizes catecholamines), which results in elevated levels of catecholamines and increased activity of beta2/3-adrenergic receptors (beta2/3ARs). However, the exact mechanisms whereby beta2/3ARs mediate COMT-dependent pain are unclear and necessitate further study. Preliminary data show that activation of beta2/3ARs may increase pain sensitivity by increasing the expression of downstream signaling molecules. We show that COMT inhibition results in increased expression of the proinflammatory cytokines tumor necrosis factor-alpha (TNF1), interleukin-12 (IL-12), and interleukin-6 (IL-6) as well as nitric oxide (NO), and that this increase is blocked by 2AR antagonists. [[Additional data further suggest that COMT- dependent pain is mediated in part by peripheral adrenergic systems as adrenalectomized rats lacking peripheral epinephrine exhibit reduced COMT-dependent pain sensitivity. The present application proposes to extend this work by applying diverse methodologies in a novel animal model of persistent pain produced by sustained COMT inhibition in order to elucidate the role that betaARs play in driving persistent pain at cellular and systems levels. First, we will apply behavioral pharmacologic methods in intact rats, adrenalectomized rats, and COMT knockout mice to determine the site of action whereby beta-adrenergic systems drive persistent COMT-dependent pain. Second, we will apply immunocytochemical techniques to determine the role of betaARs in mediating the activation of neurons, microglia, and astrocytes following sustained COMT inhibition. Third, we will apply molecular biologic methods to determine the role of betaARs in mediating the expression of proinflammatory cytokines and NO following sustained COMT inhibition. We hypothesize that persistent COMT-dependent pain produces long-term changes in cellular activity and expression of proinflammatory cytokines and NO by way of peripheral, spinal, and central beta2/3ARs. The novel approach of these studies will 1) identify the subtype and location of betaARs that contribute to persistent pain conditions such as FM and TMD, 2) characterize the long-term consequences of sustained betaAR activation on neurons and glia located in spinal and brain regions that relay pain information, 3) characterize the long-term consequences of sustained 2AR activation on proinflammatory cytokines and NO, which represent validated markers of nociception, and 4) determine the ability of beta2/3AR antagonists to suppress the transmission of nociceptive information. The outcome of these studies will provide new insights into mechanisms underlying maladaptive pain conditions as well as contribute to the identification of previously unexploited targets (e.g., beta2- and beta3ARs) for development of effective therapies for patients with persistent pain conditions.]]