A xanthine-based epithelium-dependent airway relaxant KMUP-3 (7-[2-[4-(4-nitrobenzene)piperazinyl]ethyl]-1,3-dimethylxanthine) increases respiratory performance and protects against tumor necrosis factor-α-induced tracheal contraction, involving nitric oxide release and expression of cGMP and protein kinase G

KMUP-3 (7-[2-[4-(4-nitrobenzene)piperazinyl]ethyl]-1,3-dimethyl-xanthine) was investigated in guinea pig tracheal smooth muscle. Intratracheal instillation of tumor necrosis factor (TNF)-alpha (0.01 mg/kg/300 mu l) induced bronchoconstriction, increases of lung resistance, and decreases of dynamic lung compliance. Instillation of KMUP-3 (0.5-2.0 mg/kg) reversed this situation. In isolated trachea precontracted with carbachol, KMUP-3 (10-100 mu M)-caused relaxations were attenuated by epithelium removal and by pretreatments with an inhibitor of K+ channel, tetraethylammonium (10 mM); K-ATP channel, glibenclamide (1 mu M); voltage-dependent K+ channel, 4- aminopyridine (100 mu M); Ca2+-dependent K+ channel, charybdotoxin (0.1 mu M) or apamin (1 mu M); soluble guanylate cyclase (sGC), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 1 mu M); nitric-oxide (NO)synthase, N-omega-nitro-L-arginine methyl ester (L-NAME, 100 mu M); and adenylate cyclase, SQ 22536 [9-(terahydro-2-furanyl)-9H-purin-6-amine] (100 mu M). KMUP-3 0.01-100 mu M) induced increases of cGMP and cAMP in primary culture of tracheal smooth muscle cells (TSMCs). The increase in cGMP by KMUP-3 was reduced by ODQ and L-NAME; the increase in cAMP was reduced by SQ 22536. Western blot analysis indicated that KMUP-3 (1 mu M) induced expression of protein kinase A (PKA)(RI) and protein kinase G (PKG)(1 alpha 1 beta) in TSMCs. SQ 22536 inhibited KMUP-3-induced expression of PKA(RI). On the contrary, ODQ inhibited KMUP-3-induced expression of PKG(1 alpha 1 beta). In epithelium-intact trachea, KMUP-3 increased the NO release. Activation of sGC, NO release, and inhibition of phosphodiesterases in TSMCs by KMUP-3 may result in increases of intracellular cGMP and cAMP, which subsequently activate PKG and PKA, efflux of K+ ion, and associated reduction in Ca2+ influx in vitro, indicating the action mechanism to protect against TNF-alpha-induced airway dysfunction in vivo.