Nitric oxide (NO)-nucleophile adducts are compounds of the general structure [XN(O)NO]-, where X is a nucleophile residue, and they release NO spontaneously in aqueous solution. This study determined the effects of two NO-nucleophile adducts [diethylaminodiazen-1-ium-1,2-diolate (DEA-NO) and sperminediazen-1-ium-1,2-diolate (DEA-NO) and sperminediazen-1-ium-1,2-diolate (spermine-NO)] on isometric force and the cytosolic concentrations of cyclic GMP ([cGMP]1), cyclic AMP ([cAMP]i) and calcium in canine tracheal smooth muscle. The ratio of fura-2 emission fluorescence intensities with excitation at 340- and 380-nm wavelengths (F340/ F380) was used as an index of cytosolic [Ca+2] concentration. Both DEA-NO and spermine-NO caused a concentration-dependent and reversible decrease in force (EC50 of 0.13 +/- 0.02 microM and 4.1 +/- 0.3 microM, respectively) and F340/F380, a concentration-dependent increase in [cGMP]i and no change in [cAMP]i. There were no significant differences in the relationship between [cGMP]l and the percentage decrease in force or in the relationship between the percentage decrease in F340/ F380 and the percentage decrease in force between tissues relaxed with DEA-NO or spermine-NO. Oxyhemoglobin increased the EC50 for both DEA-NO (from 0.13 +/- 0.03 microM to 8.1 +/- 0.2 microM) and spermine-NO (from 3.9 +/- 0.3 microM to 81.6 +/- 6.4 microM) and completely scavenged NO released by 0.13 (EC50) and 1 (EC100)microM DEA-NO. These results suggest that both DEA-NO and spermine-NO generate NO extracellularly and relax airway smooth muscle in association with an increase in [cGMP]i and a decrease in cytosolic Ca+2 concentration. NO-nucleophile adducts may serve as vehicles for the controlled delivery of NO into biological systems and thus provide a useful tool by which the physiology of NO can be investigated.