Endothelin‐1 (ET‐1)‐induced contraction in rat isolated trachea: involvement of ETa and ETB receptors and multiple signal transduction systems

Abstract

1. Quantitative autoradiographic, biochemical and functional studies were performed to investigate the endothelin receptor subtypes and signal transduction systems that mediate endothelin-1 (ET-1)-induced contraction in rat isolated tracheal smooth muscle. 2. Specific binding of 0.5 nM [125I]-ET-1 to tracheal smooth muscle was inhibited by at least 40% in the presence of either the ETA receptor selective ligand BQ-123 (1 microM) or the ETB receptor-selective ligand sarafotoxin S6c (30 nM), indicating the presence of both ETA and ETB receptors in this tissue. 3. ET-1 and sarafotoxin S6c were both potent spasmogens of rat isolated tracheal smooth muscle preparations. Sarafotoxin S6c-induced contractions were unaffected in the presence of the ETA receptor antagonist BQ-123 (10 microM), but were markedly attenuated in tissue previously exposed to 100 nM sarafotoxin S6c to induce ETB receptor desensitization. ET-1-induced contractions were, at most, only partially attenuated either by blocking the ETA receptor-effector system (with 10 microM BQ-123) or by desensitizing the ETB receptor-effector system with sarafotoxin S6c. However, ET-1-induced contractions were markedly attenuated by blocking both receptor-effector systems simultaneously. These findings suggest that ET-1 could induce contraction by stimulating either ETA or ETB receptors. 4. ET-1 (10 microM) induced a 7 fold increase in intracellular [3H]-inositol phosphate accumulation over basal levels in rat isolated tracheal smooth muscle. In contrast, sarafotoxin S6c (2.5 microM) increased intracellular [3H]-inositol phosphate accumulation by only 2 fold. ET-1-induced accumulation of [3H]-inositol phosphates was abolished by 10 microM BQ-123. 5. In Ca2+-free Krebs bicarbonate solution, 100 nM ET-1 induced a significantly larger contraction than that induced by 100 nM sarafotoxin S6c (46.6 +/- 5.6% C,., versus 8.8 +/- 2.8% Cmax, n = 5-7). This presumed intracellular Ca2+-dependent phase of contraction induced by ET-1 was significantly inhibited by 10 microM BQ-123 (7.5 +/- 1.0% C.). Subsequent addition of 2.5 mM Ca2+ induced a second phase of contraction. The extracellular Ca2+-dependent phase of contraction induced by ET-1 was similar inmagnitude to that induced by sarafotoxin S6c (63.6 +/- 4.5% C.. versus 58.0 +/- 3.7% C.) and was not inhibited by BQ-123. Sarafotoxin S6c-induced contractions were not inhibited by the L-type Ca2+-channel antagonists, nicardipine or verapamil.6. In summary, ETA and ETB receptors coexist in rat isolated tracheal smooth muscle and stimulation of both receptor subtypes contributes to ET-l-induced contraction in this tissue. However, stimulation of these receptor subtypes appears to induce contraction by activating different second messenger pathways; ETA receptor stimulation induces phosphoinositide turnover and subsequent release of intracellular Ca2+ whereas stimulation of ETB receptors facilitates the influx of extracellular Ca2+.