Neural Regulation of Pancreatic Function

描述(申请人提供)：急性胰腺炎是一种炎症性疾病，死亡率为5%-10%。严重的腹痛是最突出的症状，然而胰腺炎性疼痛的机制尚不清楚，因此治疗策略还不成熟。这项建议的长期目标是确定胰腺神经源性炎症和疼痛的主要介质，并为这些疾病开发新的治疗方法。我们建议研究在胰腺炎性疼痛的起始和终止过程中涉及的通路。虽然已知瞬时受体电位(TRP)家族的离子通道在躯体模型中促进炎症和疼痛，我们已经证明TRPV1通过在胰腺和脊髓中释放神经肽来介导胰腺炎性疼痛，但新的离子通道TRPV4和TRPA1是否参与胰腺炎症和疼痛尚不清楚。TRPV4对机械应激、渗透性变化以及炎症过程中释放的某些花生四烯酸代谢产物作出反应。TRPV4是由胰腺感觉神经元表达的，我们的初步结果表明，由于TRPV4激动剂注入胰管或引发实验性胰腺炎导致野生型小鼠脊髓伤害性神经元的激活(用FOS表达来评估)，而在TRPV4-/-动物中的作用减弱，因此TRPV4参与了伤害感受。此外，胰蛋白酶等在炎症的胰腺中过早激活并可以裂解和激活蛋白酶激活的受体2(PAR2)的蛋白酶，使TRPV4敏感。实验旨在确定炎症介质敏化胰腺神经元中TRPV4的机制，以促进导致神经源性炎症和疼痛的神经肽的释放。我们最近发现，4-羟基壬烯醛(HNE)是一种内源性的TRPA1激动剂，它是由活性氧自由基(ROS)作用于膜脂类产生的，可引起肽释放、神经源性炎症和疼痛。TRPA1促进胰腺炎性疼痛，因为我们的初步结果显示，导管内激动剂和实验性胰腺炎刺激TRPA1野生型小鼠的Fos表达和炎症，而在TRPA1-/-动物中的作用明显减弱。本实验旨在明确TRPA1在胰腺神经元中的表达，确定HNE等脂质过氧化产物在神经源性胰腺炎症和伤害性反应中的作用，确定促炎肽缓激肽如何增敏TRPA1，并探讨TRPA1在几种实验性胰腺炎模型中对神经源性炎症和疼痛的作用。最后，我们将通过检测金属内肽酶的作用来研究终止胰腺炎症的途径，金属内肽酶能降解细胞表面和内吞体内的致炎性多肽。我们将确定中性内肽酶(NEP)是否可以减轻胰腺的炎症和疼痛，并评估重组人NEP是否是一种新型的抗炎和止痛剂。中性内肽酶是一种细胞表面酶，可降解至少11种多肽，包括P物质和缓激肽。我们最近发现，内皮素转换酶-1(ECE1)能降解内皮体中的致炎性多肽，从而促进内毒素受体的再循环和再增敏。实验旨在确定是否通过阻止受体循环，抑制胰腺神经源性炎症的再敏化，从而成为新的抗炎药。公共卫生相关性：胰腺炎症导致发病率和死亡率，并伴有严重的顽固性疼痛。急性胰腺炎和胰腺炎疼痛的发病机制目前知之甚少。我们建议的总体目标是确定胰腺炎症和疼痛的关键介质，并评估针对这些疾病的新疗法。具体地说，我们将确定新发现的离子通道在胰腺感觉神经上的作用。一旦在炎症的胰腺中被激活或敏化，这些通道就会诱导胰腺和脊髓中的感觉神经释放神经肽，这些神经肽分别引起神经源性炎症和疼痛。因此，这些通道的拮抗剂可以预防胰腺炎性疼痛。我们还将评估能降解促炎性和伤害性多肽的重组人肽酶是否能缓解炎症性疼痛，从而成为新的治疗药物。我们确定调节神经肽受体循环和再敏化的内容体中的肽酶抑制剂是否可以阻止这些肽的持续信号传递，从而缓解神经源性炎症和疼痛。

DESCRIPTION (provided by applicant): Acute pancreatitis is an inflammatory condition with 5-10% mortality rate. Severe abdominal pain is the most prominent symptom, however the mechanisms of pancreatic inflammatory pain are poorly understood and consequently the therapeutic strategies are unsophisticated. The long term goal of this proposal is to identify the major mediators of pancreatic neurogenic inflammation and pain and to develop novel therapies for these conditions. We propose to study pathways involved both in the initiation and termination of inflammatory pain in the pancreas. Although ion channels of the transient receptor potential (TRP) family are known to promote inflammation and pain in somatic models, and we have shown that TRPV1 mediates pancreatic inflammatory pain via the release of neuropeptides both in the pancreas and spinal cord, whether the newer ion channels TRPV4 and TRPA1 participate in pancreatic inflammation and pain is unknown. TRPV4 responds to mechanical sheer stress, osmotic changes, as well as certain arachidonic acid metabolites released during inflammation. TRPV4 is expressed by pancreatic sensory neurons and our preliminary results show that it mediates nociception since either injection of TRPV4 agonists into the pancreatic duct or initiation of experimental pancreatitis result in activation of nociceptive neurons in the spinal cord (assessed by fos expression) in wild-type mice with diminished effects in TRPV4-/- animals. Moreover, proteases such as trypsin, that are prematurely-activated in the inflamed pancreas and can cleave and activate protease activated receptor 2 (PAR2), sensitize TRPV4. Experiments are designed to define the mechanisms by which inflammatory mediators sensitize TRPV4 in pancreatic neurons to promote the release of neuropeptides that cause neurogenic inflammation and pain. We have recently discovered that 4-hydroxynonenal (HNE), an aldehyde generated by the action of reactive oxygen species (ROS) on membrane lipids, is an endogenous agonist of TRPA1 which causes peptide release, neurogenic inflammation and pain. TRPA1 promotes pancreatic inflammatory pain since our preliminary results show that intraductal agonists and experimental pancreatitis stimulate fos expression and inflammation in TRPA1 wild-type mice with markedly reduced effects in TRPA1-/- animals. Experiments are designed to define the expression of TRPA1 in pancreatic neurons, to determine the effects of lipid peroxidation products such as HNE on neurogenic pancreatic inflammation and nociception, to determine how the pro-inflammatory peptide bradykinin sensitizes TRPA1, and to investigate the contribution of TRPA1 to neurogenic inflammation and pain in several models of experimental pancreatitis. Finally we will investigate pathways that terminate pancreatic inflammation by examining the role of metalloendopeptidases that degrade proinflammatory peptides on the cell surface and in endosomes. We will determine whether neutral endopeptidase (NEP), a cell surface enzyme that degrades at least 11 peptides including substance P and bradykinin, attenuates pancreatic inflammation and pain, and to evaluate whether administration of recombinant human NEP is a novel anti-inflammatory and analgesic agent. We have recently discovered that endothelin converting enzyme-1 (ECE-1) degrades pro-inflammatory peptides in endosomes to thereby promote recycling and resensitization of endocytosed receptors. Experiments are designed to determine whether ECE-1 inhibitors, by preventing receptor recycling, inhibit resensitization of neurogenic inflammation in the pancreas, and are thus novel anti-inflammatory agents. PUBLIC HEALTH RELEVANCE: Pancreatic inflammation results in morbidity and mortality, with severe intractable pain. The mechanisms of acute pancreatitis and pancreatitis pain are very poorly understood. The overall objective of our proposal is to identify key mediators of pancreatic inflammation and pain, and to evaluate novel therapies for these conditions. Specifically, we will determine the role of newly-identified ion channels on pancreatic sensory nerves. Once activated or sensitized in the inflamed pancreas, these channels induce the release of neuropeptides from sensory nerves in the pancreas and the spinal cord, where peptides cause neurogenic inflammation and pain, respectively. Thus, antagonists of these channels may prevent pancreatic inflammatory pain. We will also evaluate whether recombinant human peptidases, which degrade proinflammatory and nociceptive peptides, ameliorate inflammatory pain, and are thus novel therapeutic agents. We determine whether inhibitors of peptidases in endosomes, that regulate the recycling and resensitization of neuropeptide receptors, prevent the sustained signaling of these peptides and thereby ameliorate neurogenic inflammation and pain.