KMUP-1, a xanthine derivative, induces relaxation of guinea-pig isolated trachea: The role of the epithelium, cyclic nucleotides and K + channels

Abstract

1. 7-[2-[4-(2-chlorophenyl)piperazinyl]ethyl]-1,3-dimethylxanthine (KMUP-1) produces tracheal relaxation, intracellular accumulation of cyclic nucleotides, inhibition of phosphodiesterases (PDEs) and activation of K + channels. 2. KMUP-1 (0.01-100 μM) induced concentration-dependent relaxation responses in guinea-pig epithelium-intact trachea precontracted with carbachol. Relaxation responses were also elicited by the PDE inhibitors theophylline, 3-isobutyl-1-methylxanthine (IBMX), milrinone, rolipram and zaprinast (100 μM), and a K ATP channel opener, levcromakalim. 3. Tracheal relaxation induced by KMUP-1 was attenuated by epithelium removal and by pretreatment with inhibitors of soluble guanylate cyclase (sGC) (1H-[1,2,4]oxadiazolo[4,3-a]quinox-alin-1-one (ODQ), 1 μM), nitric oxide synthase (N ω-nitro-L-arginine methyl ester, 100 μM), K + channels (tetraethylammonium, 10 mM), K ATP channels (glibenclamide, 1 μM), voltage-dependent K + channels (4-aminopyridine, 100 μM) and Ca 2+-dependent K + channels (charybdotoxin, 0.1 μM or apamin, 1 μM). 4. Both KMUP-1 (10 μM) and theophylline nonselectively and slightly inhibited the enzyme: activity of PDE3, 4 and 5, suggesting that they are able to inhibit the metabolism of adenosine 3′,5′-cyclic monophosphate (cyclic AMP) and guanosine 3′,5′-cyclic monophosphate (cyclic GMP). Likewise, the effects of IBMX were also measured and its IC 50 values for PDE3, 4 and 5 were 6.5±1.2, 26.3±3.9 and 31.7±5.3 μM, respectively. 5. KMUP-1 (0.01-10 μM) augmented intracellular cyclic AMP and cyclic GMP levels in guinea-pig cultured tracheal smooth muscle cells. These increases in cyclic AMP and cyclic GMP were abolished in the presence of an adenylate cyclase inhibitor SQ 22536 (100 μM) and an sGC inhibitor ODQ (10 μM), respectively. 6. KMUP-1 (10 μM) increased the expression of protein kinase A (PKA RI) and protein kinase G (PKG 1α1β) in a time-dependent manner, but this was only significant for PKG after 9h. 7. Intratracheal administration of tumour necrosis factor-α (TNF-α, 0.01 mg kg -1) induced bronchoconstriction and exhibited a time-dependent increase in lung resistance (R L) and decrease in dynamic lung compliance (C dyn). KMUP-1 (1.0 mg kg -1), injected intravenously for 10 min before the intratracheal TNF-aα reversed these changes in R L and C dyn. 8. These data indicate that KMUP-1 activates sGC, produces relaxation that was partly dependent on an intact epithelium, inhibits PDEs and increases intracellular cyclic AMP and cyclic GMP, which then increases PKA and PKG, leading to the opening of K + channels and resulting tracheal relaxation.