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Tumor necrosis factor (TNF)-α, 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-α-induced mucin secretion using guinea pig tracheal epithelial (GPTE) cells in air-liquid interface culture. Exogenously applied TNF-α (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-α 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 μM); and mucin secretion in response to TNF-α or to the cGMP analogue dibutyryl cGMP (100 and 500 μM) was attenuated by the specific PKG inhibitor KT5823 (1 μM). Increased mucin secretion and increased cGMP production in response to TNF-α 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 μg/ml), a specific PC-PLC inhibitor, or by each of three PKC inhibitors: Calphostin C (0.3 and 0.5 μM), bisindoylmaleimide (GF 109203X, Go 6850; 20 nM), or Ro31-8220 (10 μM). Collectively, the results suggest that TNF-α 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.