Chronic inflammation is a characteristic feature of asthma. Multiple inflammatory mediators are released within the asthmatic lung, some of which may have detrimental effects on signal transduction pathways in airway smooth muscle. The effects of tumor necrosis factor (TNF)-alpha on the expression and function of muscarinic receptors and guanine nucleotide-binding protein (G protein) alpha-subunits were examined in human airway smooth muscle cells. Cultured human airway smooth muscle cells were incubated in serum-free culture medium for 72 h in the presence and absence of 10 ng/ml of TNF-alpha, after which the cells were lysed and subjected to electrophoresis and Galphai-2, Gqalpha, and Gsalpha protein subunits were detected by immunoblot analysis with specific antisera. TNF-alpha treatment for 72 h significantly increased the expression of Galphai-2 and Gqalpha proteins and enhanced carbachol (10(-7) M)-mediated inhibition of adenylyl cyclase activity and inositol phosphate synthesis. These data provide new evidence demonstrating that TNF-alpha not only increases expression of Galphai-2 and Gqalpha proteins but also augments the associated signal transduction pathways that would facilitate increased tone of airway smooth muscle.