Furosemide has recently been shown to be effective in inhibiting various indirect challenges in asthmatic patients, but its mode of action is not yet clear. There is some evidence that furosemide has an inhibitory effect on sensory and cholinergic nerves in the airways. We have investigated the effects of furosemide, bumetanide, and nedocromil sodium on inhibitory nonadrenergic, noncholinergic (iNANC) responses in guinea pig trachea in vitro using electrical field stimulation (50 V, 0.5 ms, 2 to 32 Hz for 30 s) and exogenously applied vasoactive intestinal peptide (VIP) or nitroprusside. In the presence of atropine (1 microM), indomethacin (10 microM), and propranolol (1 microM), both furosemide and bumetanide but not nedocromil sodium produced a concentration-dependent inhibition of the iNANC response (maximum inhibition, 31.2 +/- 5.6% with 100 microM furosemide at 16 Hz and 44.2 +/- 4.1% with 10 microM bumetanide at 4 Hz). Furthermore, after pretreatment of the tissues with L-NG-monomethyl arginine (90 microM), alpha-chymotrypsin (2 U/ml), or both, furosemide and bumetanide produced a further inhibition of the iNANC relaxation. Neither loop diuretic had any effect on the concentration-response curves to exogenous VIP (10(-9) to 10(-7) M) or nitroprusside (10(-8) to 10(-6) M). These results indicate that loop diuretics may inhibit nonadrenergic relaxation in guinea pig trachea in vitro by a prejunctional mechanism, probably through inhibition of nerve activation, the exact mechanism of which is still undefined.