The mechanism of gentisic acid-induced relaxation of the guinea pig isolated trachea: the role of potassium channels and vasoactive intestinal peptide receptors

We examined some of the mechanisms by which the aspirin metabolite and the naturally occurring metabolite gentisic acid induced relaxation of the guin ea pig trachea in vitro. In preparations with or without epithelium and con tracted by histamine, gentisic acid caused concentration-dependent and repr oducible relaxation, with mean EC,, values of 18 muM and E-max of 100% (N = 10) or 20 muM and E-max of 92% (N = 10), respectively. The relaxation caus ed by gentisic acid was of slow onset in comparison to that caused by norep inephrine, theophylline or vasoactive intestinal peptide (VIP). The relativ e rank order of potency was: salbutamol 7.9 > VIP 7.0 > gentisic acid 4.7 > theophylline 3.7. Gentisic acid-induced relaxation was markedly reduced (2 4 +/- 7.0, 43 +/- 3.9 and 78 +/- 5.6%) in preparations with elevated potass ium concentration in the medium (20, 40 or 80 mM, respectively). Tetraethyl ammonium (100 muM), a nonselective blocker of the potassium channels, parti ally inhibited the relaxation response to gentisic acid, while 4-AP (10 muM ), a blocker of the voltage potassium channel, inhibited gentisic acid-indu ced relaxation by 41 +/- 12%. Glibenclamide (1 or 3 muM), at a concentratio n which markedly inhibited the relaxation induced by the opener of ATP-sens itive K+ channels, levcromakalim, had no effect on the relaxation induced b y gentisic acid. Charybdotoxin (0.1 or 0.3 muM), a selective blocker of the large conductance Ca2+-activated K+ channels, caused rightward shifts (6- and 7-fold) of the gentisic acid concentration-relaxation curve. L-N-G-nitr oarginine (100 muM), a NO synthase inhibitor, had no effect on the relaxant effect of gentisic acid, and caused a slight displacement to the right in the relaxant effect of the gentisic acid curve at 300 muM, while methylene blue (10 or 30 muM) or ODQ (1 muM), the inhibitors of soluble guanylate cyc lase, all failed to affect gentisic acid-induced relaxation. D-p-Cl-Phe(6), Leu(17)[VIP] (0.1 muM), a VIP receptor antagonist, significantly inhibited (37 +/- 7%) relaxation induced by gentisic acid, whereas CGRP (8-37) (0.1 m uM), a CGRP antagonist, only slightly enhanced the action of gentisic acid. Taken together, these results provide functional evidence for the direct a ctivation of voltage and large-conductance Ca2+-activated K+ channels, or i ndirect modulation of potassium channels induced by VIP receptors and accou nts for the predominant relaxation response caused by gentisic acid in the guinea pig trachea.