Received for publication July 7, 2005.
Revised October 14, 2005.
Accepted for publication October 14, 2005.

A xanthine-based epithelium-dependent airway relaxant KMUP-3 increases respiratory performance and protects against TNF--induced tracheal contraction, involving NO release and expression of cGMP and PKG

Abstract

KMUP-3 (7-[2-[4-(4-nitrobenzene)piperazinyl]ethyl]-1,3-dimethylxanthine) was investigated in guinea-pig tracheal smooth muscle (TSM). Intra-tracheal instillation of TNF-(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 (TEA, 10 mM); K_ATP channel, glibenclamide (1 µM); voltage-dependent K⁺ channel, 4-aminopyridine (4-AP, 100 µM); Ca²⁺-dependent K⁺ channel, charybdotoxin (ChTX, 0.1 µM) or apamin (1 µM); soluble guanylate cyclase (sGC), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 1 µM); nitric oxide (NO) synthase, N-nitro-l-arginine methyl ester (L-NAME, 100 µM); adenylate cyclase, 9-(terahydro-2-furanyl) -9H-purin-6-amine (SQ 22536, 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 PKA_RI and PKG₁₁ in TSMC. SQ 22536 inhibited KMUP-3-induced expression of PKA_RI. On the contrary, ODQ inhibited KMUP-3-induced expression of PKG₁₁. In epithelium-intact trachea, KMUP-3 increased the NO release. Activation of sGC, NO release, and inhibition of phosphodiesterases (PDEs) in TSMC 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--induced airway dysfunction in vivo.

Key words: KMUP-3, PDE inhibition, cGMP/cAMP, epithelium-dependent, protein kinase G/protein kinase A, tracheal smooth muscle relaxation

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