Knecht Wolfgang, Cottrell Graeme S, Amadesi Silvia, Mohlin Johanna, Skåregärde Anita, Gedda Karin, Peterson Anders, Chapman Kevin, Hollenberg Morley D, Vergnolle Nathalie, Bunnett Nigel W
Molecular Pharmacology and Lead Generation, AstraZeneca Research and Development, Mölndal 431 83, Sweden.
J Biol Chem. 2007 Sep 7;282(36):26089-100. doi: 10.1074/jbc.M703840200. Epub 2007 Jul 10.
Although principally produced by the pancreas to degrade dietary proteins in the intestine, trypsins are also expressed in the nervous system and in epithelial tissues, where they have diverse actions that could be mediated by protease-activated receptors (PARs). We examined the biological actions of human trypsin IV (or mesotrypsin) and rat p23, inhibitor-resistant forms of trypsin. The zymogens trypsinogen IV and pro-p23 were expressed in Escherichia coli and purified to apparent homogeneity. Enteropeptidase cleaved both zymogens, liberating active trypsin IV and p23, which were resistant to soybean trypsin inhibitor and aprotinin. Trypsin IV cleaved N-terminal fragments of PAR(1), PAR(2), and PAR(4) at sites that would expose the tethered ligand (PAR(1) = PAR(4) > PAR(2)). Trypsin IV increased Ca(2+) in transfected cells expressing human PAR(1) and PAR(2) with similar potencies (PAR(1), 0.5 microm; PAR(2), 0.6 microm). p23 also cleaved fragments of PAR(1) and PAR(2) and signaled to cells expressing these receptors. Trypsin IV and p23 increased Ca(2+) in rat dorsal root ganglion neurons that responded to capsaicin and which thus mediate neurogenic inflammation and nociception. Intraplantar injection of trypsin IV and p23 in mice induced edema and granulocyte infiltration, which were not observed in PAR (-/-)(1)(trypsin IV) and PAR (-/-)(2) (trypsin IV and p23) mice. Trypsin IV and p23 caused thermal hyperalgesia and mechanical allodynia and hyperalgesia in mice, and these effects were absent in PAR (-/-)(2) mice but maintained in PAR (-/-)(1) mice. Thus, trypsin IV and p23 are inhibitor-resistant trypsins that can cleave and activate PARs, causing PAR(1)- and PAR(2)-dependent inflammation and PAR(2)-dependent hyperalgesia.
尽管胰蛋白酶主要由胰腺产生以降解肠道中的膳食蛋白质，但它也在神经系统和上皮组织中表达，在这些组织中，其具有多种可能由蛋白酶激活受体（PARs）介导的作用。我们研究了人胰蛋白酶IV（或中胰蛋白酶）和大鼠p23（胰蛋白酶的抗抑制剂形式）的生物学作用。胰蛋白酶原IV和前p23酶原在大肠杆菌中表达并纯化至表观均一。肠肽酶切割这两种酶原，释放出对大豆胰蛋白酶抑制剂和抑肽酶具有抗性的活性胰蛋白酶IV和p23。胰蛋白酶IV在可暴露拴系配体的位点切割PAR(1)、PAR(2)和PAR(4)的N端片段（PAR(1) = PAR(4) > PAR(2)）。胰蛋白酶IV以相似的效力增加表达人PAR(1)和PAR(2)的转染细胞中的Ca(2+)（PAR(1)，0.5微摩尔；PAR(2)，0.6微摩尔）。p23也切割PAR(1)和PAR(2)的片段并向表达这些受体的细胞发出信号。胰蛋白酶IV和p23增加对辣椒素产生反应的大鼠背根神经节神经元中的Ca(2+)，这些神经元因此介导神经源性炎症和伤害感受。在小鼠足底注射胰蛋白酶IV和p23会诱导水肿和粒细胞浸润，而在PAR (-/-)(1)（胰蛋白酶IV）和PAR (-/-)(2)（胰蛋白酶IV和p23）小鼠中未观察到这种情况。胰蛋白酶IV和p23在小鼠中引起热痛觉过敏以及机械性异常性疼痛和痛觉过敏，这些效应在PAR (-/-)(2)小鼠中不存在，但在PAR (-/-)(1)小鼠中持续存在。因此，胰蛋白酶IV和p23是抗抑制剂的胰蛋白酶，它们可以切割并激活PARs，引起PAR(1)和PAR(2)依赖性炎症以及PAR(2)依赖性痛觉过敏。
