Influence of the epithelium on acetylcholine release from parasympathetic nerves of the rat trachea

Abstract

1 The present study was undertaken to investigate the influence of the airway epithelium on the release of acetylcholine (ACh) from parasympathetic nerves of the rat trachea. Epithelium-intact and epithelium-denuded preparations of rat trachea were incubated with [³H]-choline to incorporate [³H]-ACh into the cholinergic transmitter stores. Release of radiolabelled transmitter ACh was evoked by electrical field stimulation (60 s trains of 1 ms pulses, 5 Hz, 15 V).

2 Field stimulation both of epithelium-intact and epithelium-denuded radiolabelled tracheal preparations evoked an increase in the efflux of radioactivity; however, the mean stimulation-induced (S-I) efflux from epithelium-denuded preparations (2932 ± 190 d.p.m., n=9) was approximately 60% of that from epithelium-intact preparations (4802 ± 820 d.p.m., n=11). We have shown previously that, in epithelium-intact (but not epithelium-denuded) tracheal preparations, a substantial proportion of the S-I efflux is resistant to tetrodotoxin (1 μM) and to the removal of extracellular Ca²⁺, indicating that much of the S-I efflux is not caused by exocytotic release of neuronal [³H]-ACh. In epithelium-denuded tracheal preparations, superfused individually, phosphorylcholine (1 and 100 μM) did not alter S-I efflux. In epithelium-intact tracheal preparations, both in the absence and in the presence of atropine (1 μM), neither N^G-nitro- L-arginine (100 μM), superoxide dismutase (100 units ml⁻¹), indomethacin (10 μM), capsaicin (30 μM) nor α-chymotrypsin (1 unit ml⁻¹) altered S-I efflux.

3 Experiments were also performed using two tracheal preparations superfused in series. When unlabelled epithelium-intact preparations were present in the upper chamber (superfused first), the S-I efflux from radiolabelled epithelium-denuded preparations in the lower chamber (superfused second) did not differ significantly from radiolabelled epithelium-denuded preparations superfused individually. Moreover, there was no significant difference in the S-I efflux from radiolabelled epithelium-denuded preparations in the lower chamber between experiments in which the upper chamber contained epithelium-intact or epithelium-denuded preparations.

4 Field stimulation of epithelium-intact tracheal preparations in the upper chamber with 90, 120 and 300-s periods (trains of 1 ms pulses, 5 Hz, 15 V) did not significantly alter the S-I efflux from radiolabelled epithelium-denuded tracheal preparations in the lower chamber.

5 When introduced into the upper (unlabelled epithelium-intact) and subsequently allowed to superfuse the lower (radiolabelled epithelium-denuded) tracheal preparations, the stable cholinomimetic carbachol (3 μM) markedly reduced the S-I efflux whereas ACh (0.1 and 1 μM) had no significant effect. However, in the presence of the anti-cholinesterase neostigmine (1 μM), ACh (1 μM) significantly reduced S-I efflux, indicating that ACh is subject to rapid hydrolysis by cholinesterase enzymes. When atropine (10 μM) was only exposed to radiolabelled epithelium-denuded preparations in the lower chamber, the inhibitory effects of ACh (1 μM) and carbachol (3 μM) on S-I efflux were prevented.

6 In conclusion, the findings of the present study do not support the notion that the airway epithelium exerts an inhibitory influence on ACh release from parasympathetic nerves of the rat trachea. Alternatively, if epithelium-dependent modulation of cholinergic transmission does occur in the rat trachea, then the mechanism does not appear to involve phosphorylcholine, nitric oxide, superoxide radicals, cyclo-oxygenase products of arachadonic acid, capsaicin-sensitive neuropeptides or vasoactive intestinal peptide. Moreover, the inhibitory effect of carbachol and ACh on transmitter ACh release in the rat trachea appears to be due solely to activation of prejunctional inhibitory muscarinic cholinoceptors on parasympathetic nerves and does not involve the liberation of a putative epithelium-derived inhibitory factor.