Fischer B M, Rochelle L G, Voynow J A, Akley N J, Adler K B
Department of Anatomy, Physiological Sciences, and Radiology, College of Veterinary Medicine, North Carolina State University, Raleigh 27606, USA.
Am J Respir Cell Mol Biol. 1999 Mar;20(3):413-22. doi: 10.1165/ajrcmb.20.3.3393.
Tumor necrosis factor (TNF)-alpha, a pluripotent cytokine implicated in the pathogenesis of airway inflammation, has been shown to provoke hypersecretion of mucin by airway epithelial cells in vitro. In this study, we investigated potential signaling pathways mediating TNF-alpha-induced mucin secretion using guinea pig tracheal epithelial (GPTE) cells in air-liquid interface culture. Exogenously applied TNF-alpha (human recombinant) stimulated mucin secretion in a concentration-dependent manner, with maximal effects at 10 to 15 ng/ml (286 to 429 U/ml). The pathway of stimulated secretion appeared to involve generation of intracellular nitric oxide (NO), activation of soluble guanylate cyclase (GC-S), production of cyclic guanosine monophosphate (cGMP), and activation of cGMP-dependent protein kinase (PKG). TNF-alpha increased production of nitrite and nitrate by GPTE cells; both mucin secretion and cGMP production were attenuated by NG-monomethyl-L-arginine (1 mM), a competitive inhibitor of nitric oxide synthase (NOS), or by the GC-S inhibitor LY83583 (50 microM); and mucin secretion in response to TNF-alpha or to the cGMP analogue dibutyryl cGMP (100 and 500 microM) was attenuated by the specific PKG inhibitor KT5823 (1 microM). Increased mucin secretion and increased cGMP production in response to TNF-alpha both appeared to be mediated by a phospholipase C that hydrolyzes phosphatidylcholine (PC-PLC), and by protein kinase C (PKC), since both responses were attenuated by either D609 (10 and 20 microg/ml), a specific PC-PLC inhibitor, or by each of three PKC inhibitors: Calphostin C (0.3 and 0.5 microM), bisindoylmaleimide (GF 109203X, Go 6850; 20 nM), or Ro31-8220 (10 microM). Collectively, the results suggest that TNF-alpha stimulates secretion of mucin by GPTE cells via a mechanism(s) dependent on PC-PLC and PKC, and involving activation of NOS, generation of NO, production of cGMP, and activation of PKG.
肿瘤坏死因子（TNF）-α是一种与气道炎症发病机制相关的多能细胞因子，已证实在体外可促使气道上皮细胞分泌过多黏蛋白。在本研究中，我们利用气液界面培养的豚鼠气管上皮（GPTE）细胞，研究介导TNF-α诱导黏蛋白分泌的潜在信号通路。外源性应用TNF-α（重组人TNF-α）以浓度依赖的方式刺激黏蛋白分泌，在10至15 ng/ml（286至429 U/ml）时达到最大效应。刺激分泌的途径似乎涉及细胞内一氧化氮（NO）的生成、可溶性鸟苷酸环化酶（GC-S）的激活、环磷酸鸟苷（cGMP）的产生以及cGMP依赖性蛋白激酶（PKG）的激活。TNF-α增加了GPTE细胞中亚硝酸盐和硝酸盐的产生；黏蛋白分泌和cGMP产生均被一氧化氮合酶（NOS）的竞争性抑制剂NG-单甲基-L-精氨酸（1 mM）或GC-S抑制剂LY83583（50 μM）减弱；对TNF-α或cGMP类似物二丁酰cGMP（100和500 μM）的黏蛋白分泌反应被特异性PKG抑制剂KT5823（1 μM）减弱。TNF-α诱导的黏蛋白分泌增加和cGMP产生增加似乎均由水解磷脂酰胆碱的磷脂酶C（PC-PLC）和蛋白激酶C（PKC）介导，因为这两种反应均被特异性PC-PLC抑制剂D609（10和20 μg/ml）或三种PKC抑制剂中的每一种减弱：钙泊三醇C（0.3和0.5 μM）、双吲哚马来酰亚胺（GF 109203X，Go 6850；20 nM）或Ro31-8220（10 μM）。总体而言，结果表明TNF-α通过依赖于PC-PLC和PKC的机制刺激GPTE细胞分泌黏蛋白，该机制涉及NOS的激活、NO的生成、cGMP的产生以及PKG的激活。
