ENDOTHELIN-1-INDUCED [H-3] INOSITOL PHOSPHATE ACCUMULATION IN RAT TRACHEA

1 The effects of endothelin-1 (ET-1) and of the muscarinic cholinoceptor agonist, carbachol, on [H-3]-inositol phosphate ([H-3]-InsP) accumulation and smooth muscle contraction were determined in rat isolated tracheal tissue. 2 ET-1 (1-mu-M) and carbachol (10-mu-M) induced significant accumulation of [H-3]-InsPs in myo-[2-H-3]-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 [H-3]-InsPs in response to ET-1 and carbachol. Following stimulation with ET-1, a greater proportion of tracheal [H-3]-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 [H-3]-InsPs was greatest in the airway smooth muscle band. The tracheal epithelium did not appear to generate [H-3]-InsPs in response to ET-1 or modulate either basal or ET-1-induced accumulation of [H-3]-InsPs in rat tracheal segments. 3 In the rat tracheal smooth muscle band, ET-1 caused a time- and concentration-dependent accumulation of [H-3]-InsPs. Concentrations of ET-1 as low as 10 nM produced significant accumulation of [H-3]-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-mu-M, the highest concentration used, ET-1 produced similar levels of [H-3]-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 [H-3]-InsPs was not significantly affected by indomethacin (5-mu-M), nordihydroguaiaretic acid (NDGA, 10-mu-M), WEB 2086 (10-mu-M) or phosphoramidon (10-mu-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 (EC50) was 31 nM (95% confidence limits, 20-49 nM, n = 12). The presence of an intact tracheal epithelium, indomethacin (5-mu-M), WEB 2086 (10-mu-M) and phosphoramidon (10-mu-M) had no significant effect on the mean EC50 for ET-1-induced contraction (n = 5). In contrast, NDGA (10-mu-M) 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 [H-3]-InsPs accumulation were similar, although the EC50 for [H-3]-InsP accumulation was 2.9 fold greater than that for smooth muscle contraction. For carbachol, the EC50 for [H-3]-InsP accumulation (mean EC50 = 5.0-mu-M, 1.2-21-mu-M, n = 4) was 25 fold greater than that for smooth muscle contraction (mean EC50 = 0.20-mu-M, 0.17-0.24-mu-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.