In the guinea pig bronchus with epithelium, pre-contracted with histamine, bradykinin (BK), lysyl-BK, prostaglandin E2 (PGE2), cromakalim and S-nitroso-N-acetylpenicillamine each caused graded relaxation with mean EC50s of 34 nM, 11 nM, 0.1 nM, 0.3 microM and 3.4 microM, respectively. The addition of NO synthase inhibitors N(W)-nitro-L-arginine (L-NOARG), N(G)-monomethyl-L-arginine or 7-nitroindazole reduced BK-induced relaxation by 41+/-6%, 59+/-4% and 51+/-2%, respectively. The inhibition of BK response caused by L-NOARG was completely reversed by L-, but not by D-arginine. Methylene blue and 6-(phenylamino)-5,8-quinolinedione (LY 83583) inhibited the BK response by 88+/-5% and 64+/-4%, while 1H-[1,2,4]oxadiazolo[4,3-a]quinolaxin- -one (ODQ) had no effect. However, ODQ almost abolished SNAP-induced relaxation. Indomethacin and the cyclo-oxygenase 2 (COX-2) inhibitor 5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulfonyl)phenyl-2(5H)-furanon e (DFU) caused graded inhibtion of BK responses with mean IC50s of 60 nM and 0.6 nM, respectively. Addition of tetraethylammonium (TEA), charybdotoxin (ChTx), or iberotoxin (IbTx) inhibited BK-induced relaxation by 76+/-4%, 30+/-4% and 99+/-1%, respectively, but the relaxations of PGE2 and cromakalim were unaffected. In contrast, 4-aminopyridine, apamin or glibenclamide did not affect BK-induced relaxation. These results indicate that BK-induced epithelium-dependent relaxation in the guinea pig bronchus is partially mediated by release of NO or by NO-related substances, involving an activation of both cyclo-oxygenase 1 (COX-1) and COX-2 enzymes, through a cyclic guanosine monophosphate (cGMP)-independent mechanism. Furthermore, BK-induced relaxation involves an activation of high-conductance Ca(2+)-activated K+ channels highly sensitive to IbTx, and to a lesser extent to ChTx and TEA.