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

描述（由申请人提供）：纤维肌痛（FM）和颞下颌关节紊乱病（TMD）等复杂的持续性疼痛疾病无法有效治疗，因为潜在的分子机制在很大程度上仍然未知。我们小组和其他人的工作表明，这些疾病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）的活性降低，导致儿茶酚胺水平升高和β 2/3-肾上腺素能受体（β 2/3AR）活性增加。然而，β 2/3AR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，β 2/3AR的激活可能通过增加下游信号分子的表达来增加疼痛敏感性。我们表明，COMT抑制导致促炎细胞因子肿瘤坏死因子-α（TNF-1），白细胞介素-12（IL-12），白细胞介素-6（IL-6）以及一氧化氮（NO）的表达增加，这种增加被2AR拮抗剂阻断。额外的数据进一步表明，COMT依赖性疼痛部分由外周肾上腺素能系统介导，因为缺乏外周肾上腺素的肾上腺切除大鼠表现出降低的COMT依赖性疼痛敏感性。本申请提出通过在由持续COMT抑制产生的持续性疼痛的新动物模型中应用不同的方法来扩展这项工作，以阐明β AR在细胞和系统水平上驱动持续性疼痛中发挥的作用。首先，我们将应用行为药理学方法在完整的大鼠，肾上腺切除大鼠，和COMT基因敲除小鼠，以确定β-肾上腺素能系统驱动持续COMT依赖性疼痛的作用部位。其次，我们将应用免疫细胞化学技术来确定β AR在持续COMT抑制后介导神经元、小胶质细胞和星形胶质细胞活化中的作用。第三，我们将应用分子生物学方法来确定β AR在持续COMT抑制后介导促炎细胞因子和NO表达中的作用。我们假设持续性COMT依赖性疼痛通过外周、脊髓和中枢β 2/3AR产生细胞活性和促炎细胞因子及NO表达的长期变化。这些研究的新方法将1）鉴定导致持续性疼痛病症（如FM和TMD）的β AR的亚型和位置，2）表征持续β AR激活对位于脊髓和脑区域中的神经元和神经胶质的长期后果，所述神经元和神经胶质传递疼痛信息，3）表征持续2AR激活对促炎细胞因子和NO的长期后果，其代表伤害感受的有效标记，和4）确定β 2/3AR拮抗剂抑制伤害感受信息传递的能力。这些研究的结果将为适应不良疼痛状况的潜在机制提供新的见解，并有助于识别以前未开发的靶点（例如，β 2和β 3 AR），用于开发针对持续性疼痛患者的有效疗法。]]

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.]]