ENDOTHELIN-1 (ET-1)-INDUCED CONTRACTION IN RAT ISOLATED TRACHEA - INVOLVEMENT OF ETA AND ETB RECEPTORS AND MULTIPLE SIGNAL-TRANSDUCTION SYSTEMS

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 [I-125]-ET-1 to tracheal smooth muscle was inhibited by at least 40% in the presence of either the ET(A) receptor selective ligand BQ-123 (1 mum) or the ET(B) receptor-selective ligand sarafotoxin S6c (30 nm), indicating the presence of both ET(A) and ET(B) 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 mum), but were markedly attenuated in tissue previously exposed to 100 nm sarafotoxin S6c to induce ET(B) receptor desensitization. ET-1-induced contractions were, at most, only partially attenuated either by blocking the ET(A) receptor-effector system (with 10 muM BQ-123) or by desensitizing the ET(B) 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 ET(A) or ET(B) receptors. 4 ET-1 (10 mum) induced a 7 fold increase in intracellular [H-3]-inositol phosphate accumulation over basal levels in rat isolated tracheal smooth muscle. In contrast, sarafotoxin S6c (2.5 mum) increased intracellular [H-3]-inositol phosphate accumulation by only 2 fold. ET-1-induced accumulation of [H-3]-inositol phosphates was abolished by 10 muM 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% C(max), n = 5-7). This presumed intracellular Ca2+-dependent phase of contraction induced by ET-1 was significantly inhibited by 10 mum BQ-123 (7.5 +/- 1.0% C(max)). 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 in magnitude to that induced by sarafotoxin S6c (63.6 +/- 4.5% C(max) versus 58.0 +/- 3.7% C(max)) 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, ET(A) and ET(B) receptors coexist in rat isolated tracheal smooth muscle and stimulation of both receptor subtypes contributes to ET-1-induced contraction in this tissue. However, stimulation of these receptor subtypes appears to induce contraction by activating different second messenger pathways; ET(A) receptor stimulation induces phosphoinositide turnover and subsequent release of intracellular Ca2+ whereas stimulation of ET(B) receptors facilitates the influx of extracellular Ca2+.