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

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Abstract
1 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. 2 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. 3 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). 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 (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). 5 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). 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.