Endothelin Mechanisms in Metastic Prostate Cancer Pain

描述(申请人提供)：内源性多肽-1(ET-1)对疼痛以及在神经损伤、切开、炎症和癌症后疼痛纤维对非伤害性刺激的敏化是必不可少的。在这项建议中，我们试图了解损伤后皮肤中ET-1的升高和炎症与随后的疼痛之间的关系。最近的研究表明，ET-1通过作用于神经末梢周围的组织而不是直接作用于神经，导致外周疼痛加剧；ET-1及其同源物、GPC受体ETA和ETB在痛觉过敏和痛觉异常的情况下升高。ET受体的拮抗剂能够预防或逆转这些疼痛，证明内源性ET-1在驱动慢性疼痛状态中起着重要作用。目前的证据表明，ET-1‘S在皮肤损伤/炎症过程中的作用与另外三个分子/受体密切相关：TRPV1(辣椒的受体，也可以被热和H+激活)、谷氨酸和CGRP。在很短的时间内，TRPV1的S参与了ET-1或CFA注入大鼠足爪引起的皮肤疼痛，并且TRPV1在皮肤神经纤维中的免疫细胞化学染色增加了5-10倍。在注射ET-1或CFA后约30分钟，谷氨酸和降钙素基因相关肽参与了ET-1‘S的痛敏效应；这些物质也被培养的感觉神经元释放出来，并被ET-1增强。这项建议包含4个具体的目标，以解决这些变化背后的过程：1.确定注射ET-1后表皮伤害性感受器TRPV1迅速增加和局部炎症的机制。2.探讨皮肤中ETA受体分布的快速变化(炎症和ET-1释放后)与局部ET-1反应脱敏之间的时间关系。3.确定皮肤细胞中的ET受体是否具有调节角蛋白TRPV1和其他Trp通道(TRPV3、TRPV4)的功能。4.探讨ET-1诱导的皮肤谷氨酸、降钙素基因相关肽和三磷酸腺苷的释放在ET-1引起的痛觉过敏中的作用。实验将综合行为学、免疫细胞化学、生化和细胞生理学技术的结果，确定不同途径和物质在ET-1诱导的疼痛中的重要性，定量ET-1诱导的皮肤TRPV受体和ET受体的变化，建立角质形成细胞中ET-Rs调节这些细胞中TRPV受体的能力，并检测ET-1‘S刺激皮肤释放疼痛诱导物质的能力。

DESCRIPTION (provided by applicant): The endogenous peptide endothelin-1 (ET-1) is essential for pain and the sensitization of pain fibers to non-noxious stimuli after nerve injury, incision, inflammation and in cancer. In this proposal we seek to understand the relationship between the elevation of ET-1 in skin after injury and inflammation and the ensuing pain. Recent work suggests that ET-1 causes elevated pain in the periphery by acting on tissues that surround nerve endings, rather than directly on nerves; ET-1 and its cognate, GPC receptors ETA and ETB are elevated in conditions of hyperalgesia and allodynia. Antagonists of the ET receptors are able to prevent or reverse these pains, testifying to an important role for endogenous ET-1 in driving chronic pain states. Current evidence indicates that ET-1's actions during cutaneous injury/inflammation is closely linked to three other molecules/ receptors; TRPV1 (the receptor for hot peppers that is also activated by heat and by a H+), glutamate, and CGRP. TRPV1's participation in cutaneous pain from ET-1 or CFA injection into the rat's paw is evident within a very short time and immunocytochemical staining of TRPV1 in cutaneous nerve fibers increases 5-10-fold. In ~30 min after ET-1 or CFA injection, glutamate and CGRP contribute to ET-1's pain sensitizing effects; these substances are also released by cultured sensory neurons in a way that is potentiated by ET-1. This proposal contains 4 Specific Aims to address the processes underlying these changes: 1. To determine the mechanism for the rapid increase in TRPV1 in epidermal nociceptors following injection of ET-1 and local inflammation. 2. To investigate the temporal relationship between the rapid changes in ETA receptor distribution in skin (after inflammation and ET- 1 delivery) and the resulting desensitization of responses to local ET-1. 3. To determine if the ET receptors in skin cells are able to modulate keratinocye TRPV1, and other TRP channels (TRPV3, TRPV4). 4. To evaluate the role of ET-1-induced release of cutaneous glutamate, CGRP and ATP in ET-1- induced algesia and allodynia. Experiments will integrate the results from behavioral, immunocytochemical, biochemical and cell physiological techniques, for determining the importance of different pathways and substances for ET-1-induced pain, for quantitating ET-1-induced changes in TRPV receptors and ET receptors in skin, for establishing the ability of ET-Rs in keratinocytes to modulate TRPV receptors in those cells, and for examining ET-1's ability to stimulate release of pain-inducing substances from the skin.