cyclic-gmp and 7-(2-(4-(4-nitrobenzene)piperazinyl)ethyl)-1-3-dimethylxanthine

KMUP-3 (7-[2-[4-(4-nitrobenzene)piperazinyl]ethyl]-1,3-dimethylxanthine) was investigated in guinea pig tracheal smooth muscle. Intratracheal instillation of tumor necrosis factor (TNF)-alpha (0.01 mg/kg/300 microl) 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 microM)-caused relaxations were attenuated by epithelium removal and by pretreatments with an inhibitor of K(+) channel, tetraethylammonium (10 mm); K(ATP) channel, glibenclamide (1 microM); voltage-dependent K(+) channel, 4-aminopyridine (100 microM); Ca(2+)-dependent K(+) channel, charybdotoxin (0.1 microM) or apamin (1 microM); soluble guanylate cyclase (sGC), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1one (ODQ, 1 microM); nitric-oxide (NO) synthase, N(omega)-nitro-L-arginine methyl ester (L-NAME, 100 microM); and adenylate cyclase, SQ 22536 [9-(terahydro-2-furanyl)-9H-purin-6-amine] (100 microM). KMUP-3 (0.01-100 microM) 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 microM) induced expression of protein kinase A (PKA)(ri) and protein kinase G (PKG)(1alpha 1beta) in TSMCs.SQ 22536 inhibited KMUP-3-induced expression of (PKA)(ri). On the contrary, ODQ inhibited KMUP-3-induced expression of PKG(1alpha 1beta) 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 Ca(2+) influx in vitro, indicating the action mechanism to protect against TNF-alpha-induced airway dysfunction in vivo.

Topics: Adenylyl Cyclases; Animals; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Guinea Pigs; In Vitro Techniques; Male; Muscle Contraction; Muscle, Smooth; Nitric Oxide; Phosphoric Diester Hydrolases; Piperidines; Potassium Channels; Respiration; Trachea; Tumor Necrosis Factor-alpha; Xanthines

The cellular mechanisms of vasorelaxant effects of newly synthesized KMUP-3 and KMUP-4 were investigated in rat aortic smooth muscle (RASM). KMUP-3 (7-[2-[4-(4-nitrobenzene)piperazinyl]ethyl]-1,3-dimethylxanthine) and KMUP-4 (7-[2-[4-(2-nitrobenzene)piperazinyl]ethyl]-1,3-dimethylxanthine) elicited concentration-dependent relaxation of endothelium-intact and denuded RASM precontracted with phenylephrine. Relaxant responses were also produced by the PDE inhibitors theophylline, milrinone, rolipram, and zaprinast (1 nM-100 microM). The relaxant responses of KMUP-3 and KMUP-4 were reduced by endothelium removal and by the presence of the NOS inhibitor L-NAME (100 microM), the sGC inhibitor ODQ (1 microM), the adenylyl cyclase (AC) inhibitor SQ 22536 (100 microM), and the prostaglandin inhibitor indomethacin (10 microM). Additionally, the vasorelaxations of both agents were also attenuated by pretreatment with the nonselective K+ channel blocker TEA (10 mM), the KATP channel blocker glibenclamide (1 microM), the voltage-dependent K+ (KV) channel blocker 4-AP (100 microM), and Ca(2+)-dependent K+ (KCa) channel blockers apamin (1 microM) and charybdotoxin (ChTX, 0.1 microM). In addition, elevated extracellular K+ (80 mM) interferes with KMUP-3- and KMUP-4-induced vasorelaxations. Preincubation with both agents (1 microM) significantly enhanced the dilator responses of isoproterenol and SNP. KMUP-3 and KMUP-4 inhibited PDE activities and increased cAMP and cGMP levels in primary culture of RASM that were inhibited by SQ 22536 and ODQ, respectively. In cultured HUVECs, KMUP-3 and KMUP-4 (0.1 microM), more potent than YC-1, significantly increased the expression of eNOS protein. In summary, KMUP-3 and KMUP-4 induce aortic relaxations through both endothelium-dependent and -independent mechanisms. Mechanisms of vasorelaxation induced by both compounds involve multiple processes, such as accumulation of cyclic nucleotides partly as a result of PDE inhibition, K-channel activation, and indomethacin-sensitive endothelium function.

Topics: Animals; Aorta; Cell Line; Cyclic AMP; Cyclic GMP; Endothelium, Vascular; In Vitro Techniques; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Phosphoric Diester Hydrolases; Piperazines; Piperidines; Potassium Channels; Rats; Rats, Wistar; Vasoconstrictor Agents; Vasodilator Agents; Xanthines