MECHANISMS UNDERLYING TNF-ALPHA EFFECTS ON AGONIST-MEDIATED CALCIUM HOMEOSTASIS IN HUMAN AIRWAY SMOOTH-MUSCLE CELLS

We have previously shown that tumor necrosis factor (TNF)-alpha, a cyt okine involved in asthma, enhances Ca2+ responsiveness to bronchoconst rictor agents in cultured human airway smooth muscle (ASM) cells. In t he present study we investigated the potential mechanism(s) by which T NF-alpha modulates ASM cell responsiveness to such agents. In human AS M cells loaded with fura 2, TNF-alpha and interleukin (IL)-1 beta sign ificantly enhanced thrombin-and bradykinin-evoked elevations of intrac ellular Ca2+. In TNF-alpha-treated cells, Ca2+ responses to thrombin a nd bradykinin were 350 +/- 14 and 573 +/- 93 nM vs. 130 +/- 17 and 247 +/- 48 nM in nontreated cells, respectively (P < 0.0001). In IL-1 bet a-treated cells, the Ca2+ response to bradykinin was 350 +/- 21 vs. 12 7 +/- 12 nM in nontreated cells (P < 0.0001). The time course for TNF- alpha potentiation of agonist-induced Ca2+ responses requires a minimu m of 6 h and was maximum after 12 h of incubation. In addition, cycloh eximide, a protein synthesis inhibitor, completely blocked the potenti ating effect of TNF-alpha on Ca2+ signals. We also found that TNF-alph a significantly enhanced increases in phosphoinositide (PI) accumulati on induced by bradykinin. The percentage ofchange in PI accumulation o ver control was 115 +/- 8 to 210 +/- 15% in control cells vs. 128 +/- 10 to 437 +/- 92% in TNF-alpha-treated cells for 3 x 10(-9) to 3 x 10( -6) M bradykinin. The PI turnover to 10 mM NaF, a direct activator of G proteins, was also found to be enhanced by TNF-alpha. The percentage of change in PI accumulation over control increased from 280 +/- 35% in control cells to 437 +/- 92% in TNF-alpha-treated cells. Taken toge ther, these results show that TNF-alpha can potently regulate G protei n-mediated signal transduction in ASM cells by activating pathways dep endent on protein synthesis. Our study demonstrates one potential mech anism underlying the enhanced Ca2+ response to bronchoconstrictor agen ts induced by cytokines in human ASM cells.