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 guinea pig trachea in vitro. In preparations with or without epithelium and contracted by histamine, gentisic acid caused concentration-dependent and reproducible 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 caused by gentisic acid was of slow onset in comparison to that caused by norepinephrine, theophylline or vasoactive intestinal peptide (VIP). The relative 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 (24 +/- 7.0, 43 +/- 3.9 and 78 +/- 5.6%) in preparations with elevated potassium concentration in the medium (20, 40 or 80 mM, respectively). Tetraethylammonium (100 muM), a nonselective blocker of the potassium channels, partially inhibited the relaxation response to gentisic acid, while 4-AP (10 muM), a blocker of the voltage potassium channel, inhibited gentisic acid-induced relaxation by 41 +/- 12%. Glibenclamide (1 or 3 muM), at a concentration which markedly inhibited the relaxation induced by the opener of ATP-sensitive K+ channels, levcromakalim, had no effect on the relaxation induced by 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-nitroarginine (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 cyclase, 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 muM), a CGRP antagonist, only slightly enhanced the action of gentisic acid. Taken together, these results provide functional evidence for the direct activation of voltage and large-conductance Ca2+-activated K+ channels, or indirect modulation of potassium channels induced by VIP receptors and accounts for the predominant relaxation response caused by gentisic acid in the guinea pig trachea.