Previous studies have shown that acetylcholine (ACh) evokes an increase in short circuit current (I_sc) and transepithelial potential difference (PD) across isolated distal bronchi that is unaffected by inhibition of either Cl^– or HCO₃^– secretion but is blocked if both pathways are inhibited (Inglis et al. 1996). These data suggest that ACh stimulates both Cl^– and HCO₃^– secretion and that the independent inhibition of secretion of one anion augments secretion of the other. The aim of the present study was to investigate whether ACh-induced anion secretion in tracheal epithelium is similar to that described in bronchi.

Tracheas were removed from humanely killed pigs and the epithelial layer dissected from underlying cartilage. Epithelia were mounted in modified Ussing chambers and bathed in HCO₃^– buffered solution, gassed with 95 % O₂-5 % CO₂ at 37°C. Serosal ACh (10 µM) stimulated a peak increase in I_SC (17.7 ± 2.9 µA cm^-2, mean ± S.E.M., n = 5) that slowly decayed with time. The response of paired epithelia bathed in Hepes-buffered solution containing acetazolamide (10 mM), to inhibit carbonic anhydrase, was not significantly different (20.3 ± 1.6 µA cm^-2, Student’s paired t test used throughout, P = 0.55), showing that inhibition of HCO₃^– secretion alone has no effect on the response to ACh. Bumetanide (10 µM, serosal), however, significantly inhibited the response to ACh (81.7 ± 7.4 %, n = 5, P < 0.05), demonstrating that inhibition of Cl^– secretion alone is sufficient to block the response to ACh in tracheal epithelium. Apical addition of the anion channel blockers DPC (1 mM) and NPPB (300 µM) also significantly reduced the response to ACh (by 60.1 ± 14.8 % (n = 9) and 80.1 ± 10.5 % (n = 7) respectively, P < 0.05), whilst DIDS had no effect (P > 0.05, n = 7). Basolateral addition of DIDS, however, significantly reduced the response to ACh (33.1 ± 12.3 %, n = 10, P < 0.05), whilst DNDS had no significant effect (n = 7, P > 0.05). These results suggest that the majority of the electrogenic response to ACh in tracheal epithelium results from stimulation of Cl^– secretion. Involvement of HCO₃^– secretion is suggested, however, by the inhibitory effect of DIDS, which may block HCO₃^– uptake across the basolateral membrane via anion exchange. To investigate this further, we measured the effect of ACh on the pH of HCO₃^–-free, unbuffered solution placed in the luminal side of the Ussing chamber. The mean rate of secretion of base equivalents into the luminal chamber was significantly increased by ACh addition from -0.44 ± 0.09 µmol h^-1 for the 10 min prior to ACh addition to 0.59 ± 0.13 µmol h^-1 for 10 min after ACh addition (n = 13, P < 0.05). This is consistent with ACh-evoked stimulation of HCO₃^– secretion into the luminal chamber.

In summary, ACh stimulates I_SC across porcine tracheal epithelium. In contrast to its effect on bronchial epithelium, the majority of this I_SC results from stimulation of Cl^– secretion. HCO₃^– secretion is also evoked by ACh, and this may partly be via an electroneutral mechanism.

This work was supported by the Wellcome and Tenovus Trusts