Abstract
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)-α (0.01 mg/kg/300 μl) 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 μM)-caused relaxations were attenuated by epithelium removal and by pretreatments with an inhibitor of K+ channel, tetraethylammonium (10 mm); KATP channel, glibenclamide (1 μM); voltage-dependent K+ channel, 4-aminopyridine (100 μM); Ca2+-dependent K+ channel, charybdotoxin (0.1 μM) or apamin (1 μM); soluble guanylate cyclase (sGC), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1one (ODQ, 1 μM); nitric-oxide (NO) synthase, Nω-nitro-l-arginine methyl ester (l-NAME, 100 μM); and adenylate cyclase, SQ 22536 [9-(terahydro-2-furanyl)-9H-purin-6-amine] (100 μM). KMUP-3 (0.01-100 μM) 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 μM) induced expression of protein kinase A (PKA)ri and protein kinase G (PKG)1α 1β in TSMCs.SQ 22536 inhibited KMUP-3-induced expression of (PKA)ri.On the contrary, ODQ inhibited KMUP-3-induced expression of PKG1α 1β 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-α-induced airway dysfunction in vivo.
Footnotes
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This work was supported by Grants NSC-89-2320-B-037-056-M59 (to I.- J.C.) from the National Science Council, Taiwan and Vital Pharm Co., Ltd. (Kaohsiung, Taiwan).
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doi:10.1124/jpet.105.092171.
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ABBREVIATIONS: PDE, phosphodiesterase; PKG, protein kinase G; TNF, tumor necrosis factor; TSMC, tracheal smooth muscle cell; KMUP-1, 7-[2-[4-(2-chlorophenyl)piperazinyl] ethyl]-1,3-dimethyl xanthine; TSM, tracheal smooth muscle; KMUP-3, 7-[2-[4-(4-nitrobenzene)piperazinyl] ethyl]-1,3-dimethylxanthine; NO, nitric oxide; sGC, soluble guanylyl cyclase; PKA, protein kinase A; IBMX, 3-isobutyl-1-methylxanthine; TEA, tetraethylammonium; 4-AP, 4-aminopyridine; ChTX, charybdotoxin; ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; NOS, nitric-oxide synthase; l-NAME, Nω-nitro-l-arginine methyl ester; AC, adenylate cyclase; SQ 22536, 9-(terahydro-2-furanyl)-9H-purin-6-amine; TBS, Tris-buffered saline; ANOVA, analysis of variance; 8-Br-cGMP; 8-bromo-cGMP.
- Received July 7, 2005.
- Accepted October 14, 2005.
- The American Society for Pharmacology and Experimental Therapeutics
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