Endothelin‐1‐induced [ 3 H]‐inositol phosphate accumulation in rat trachea

1 The effects of endothelin‐1 (ET‐1) and of the muscarinic cholinoceptor agonist, carbachol, on [ 3 H]‐inositol phosphate ([ 3 H]‐InsP) accumulation and smooth muscle contraction were determined in rat isolated tracheal tissue. 2 ET‐1 (1 μ m ) and carbachol (10 μ m ) induced significant accumulation of [ 3 H]‐InsPs in myo ‐[2‐ 3 H]‐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 [ 3 H]‐InsPs in response to ET‐1 and carbachol. Following stimulation with ET‐1, a greater proportion of tracheal [ 3 H]‐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 [ 3 H]‐InsPs was greatest in the airway smooth muscle band. The tracheal epithelium did not appear to generate [ 3 H]‐InsPs in response to ET‐1 or modulate either basal or ET‐1‐induced accumulation of [ 3 H]‐InsPs in rat tracheal segments. 3 In the rat tracheal smooth muscle band, ET‐1 caused a time‐ and concentration‐dependent accumulation of [ 3 H]‐InsPs. Concentrations of ET‐1 as low as 10 n m produced significant accumulation of [ 3 H]‐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 μ m , the highest concentration used, ET‐1 produced similar levels of [ 3 H]‐InsP accumulation (7.03 ± 0.55 fold above basal levels, n = 5) to that produced by a maximally effective concentration of carbachol (10 m m ; 7.97 ± 0.31 fold increase above basal levels, n = 4). ET‐1‐induced accumulation of [ 3 H]‐InsPs was not significantly affected by indomethacin (5 μ m ), nordihydroguaiaretic acid (NDGA, 10 μ m ), WEB 2086 (10 μ m ) or phosphoramidon (10 μ m ). 4 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 (EC 50 ) was 31 n m (95% confidence limits, 20–49 n m , n = 12). The presence of an intact tracheal epithelium, indomethacin (5 μ m ), WEB 2086 (10 μ m ) and phosphoramidon (10 μ m ) had no significant effect on the mean EC 50 for ET‐1‐induced contraction ( n = 5). In contrast, NDGA (10 μ m ) inhibited ET‐1‐induced contractions (4.0 fold increase in mean EC 50 , 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 C 4 and leukotriene D 4 did not contract rat isolated tracheal smooth muscle preparations ( n = 4). 5 The threshold concentrations of ET‐1 that produced increases in smooth muscle contraction and [ 3 H]‐InsPs accumulation were similar, although the EC 50 for [ 3 H]‐InsP accumulation was 2.9 fold greater than that for smooth muscle contraction. For carbachol, the EC 50 for [ 3 H]‐InsP accumulation (mean EC 50 = 5.0 μ m , 1.2–21 μ m , n = 4) was 25 fold greater than that for smooth muscle contraction (mean EC 50 = 0.20 μ m , 0.17–0.24 μ m , n = 12). 6 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.