Endothelin‐1‐induced [3H]‐inositol phosphate accumulation in rat trachea
Authors: Henry Peter J. , Rigby Paul J. , Self Glenn J. , Preuss Janet M. , Goldie Roy G. 
Published: July 2012

The effects of endothelin‐1 (ET‐1) and of the muscarinic cholinoceptor agonist, carbachol, on [3H]‐inositol phosphate ([3H]‐InsP) accumulation and smooth muscle contraction were determined in rat isolated tracheal tissue.

ET‐1 (1 μm) and carbachol (10 μm) induced significant accumulation of [3H]‐InsPs in myo‐[2‐3H]‐inositol‐loaded rat tracheal segments. Several components of the tracheal wall including the airway smooth muscle band, the cartilaginous region and the intercartilaginous region generated significant levels of [3H]‐InsPs in response to ET‐1 and carbachol. Following stimulation with ET‐1, a greater proportion of tracheal [3H]‐InsPs were generated in the intercartilaginous region (49%) than in either the airway smooth muscle band (25%) or cartilaginous region (26%). However, when the respective weights of these regions is taken into account, ET‐1‐induced accumulation of [3H]‐InsPs was greatest in the airway smooth muscle band. The tracheal epithelium did not appear to generate [3H]‐InsPs in response to ET‐1 or modulate either basal or ET‐1‐induced accumulation of [3H]‐InsPs in rat tracheal segments.

In the rat tracheal smooth muscle band, ET‐1 caused a time‐ and concentration‐dependent accumulation of [3H]‐InsPs. Concentrations of ET‐1 as low as 10 nm produced significant accumulation of [3H]‐InsPs (1.23 ± 0.10 fold increase above basal levels of 295 ± 2 d.p.m. mg−1 wet wt., n = 3 experiments). At 10 μm, the highest concentration used, ET‐1 produced similar levels of [3H]‐InsP accumulation (7.03 ± 0.55 fold above basal levels, n = 5) to that produced by a maximally effective concentration of carbachol (10 mm; 7.97 ± 0.31 fold increase above basal levels, n = 4). ET‐1‐induced accumulation of [3H]‐InsPs was not significantly affected by indomethacin (5 μm), nordihydroguaiaretic acid (NDGA, 10 μm), WEB 2086 (10 μm) or phosphoramidon (10 μm).

ET‐1 also produced concentration‐dependent contractions of epithelium‐denuded rat tracheal ring preparations. The mean concentration of ET‐1 producing 50% of the maximum contractile response to carbachol (EC50) was 31 nm (95% confidence limits, 20–49 nm, n = 12). The presence of an intact tracheal epithelium, indomethacin (5 μm), WEB 2086 (10 μm) and phosphoramidon (10 μm) had no significant effect on the mean EC50 for ET‐1‐induced contraction (n = 5). In contrast, NDGA (10 μm) inhibited ET‐1‐induced contractions (4.0 fold increase in mean EC50, P > 0.001, n = 5). However, this effect of NDGA did not appear to be related to inhibition of leukotriene synthesis via lipoxygenase since the leukotriene antagonist SKF 104353 did not affect ET‐1‐induced contractions (n = 5) and moreover, leukotriene C4 and leukotriene D4 did not contract rat isolated tracheal smooth muscle preparations (n = 4).

The threshold concentrations of ET‐1 that produced increases in smooth muscle contraction and [3H]‐InsPs accumulation were similar, although the EC50 for [3H]‐InsP accumulation was 2.9 fold greater than that for smooth muscle contraction. For carbachol, the EC50 for [3H]‐InsP accumulation (mean EC50 = 5.0 μm, 1.2–21 μm, n = 4) was 25 fold greater than that for smooth muscle contraction (mean EC50 = 0.20 μm, 0.17–0.24 μm, n = 12).

It seems likely that ET‐1 has a direct effect on InsP generation in rat tracheal smooth muscle and that this is largely responsible for the spasmogenic actions of this peptide.