PREDOMINANCE OF ENDOTHELIN(A) (ET(A)) RECEPTORS IN OVINE AIRWAY SMOOTH-MUSCLE AND THEIR MEDIATION OF ET-1-INDUCED CONTRACTION

1 Autoradiographic studies were conducted to investigate the receptor subtypes for endothelin-1 (ET-1) that were present in the ovine respiratory tract. In addition, the receptor subtypes mediating contraction of airway smooth muscle and the possible involvement of extracellular Ca2+ and inositol phosphate generation in intracellular signal transduction were assessed. 2 Specific [I-125]-ET-1 binding in ovine trachea increased in a time- and concentration-dependent manner. Autoradiographic studies demonstrated that significant binding was associated with airway smooth muscle, although higher densities of specific binding were associated with submucosal glands and with cells immediately below the epithelial basement membrane (lamina propria). The ET(A) receptor-selective antagonist, BQ 123 (1 mu M), virtually abolished specific binding to airway smooth muscle. Quantitative analyses of autoradiographic data describing the time-dependence of specific [I-125]-ET-1 binding in ovine airway smooth muscle in the presence and absence of BQ 123 or sarafotoxin S6c, revealed a homogeneous population of ET(A) receptors. BQ 123 (1 mu M) also abolished specific binding to structures associated with submucosal glands, whereas the ET(B) receptor selective agonist, sarafotoxin S6c (100 nM) had little effect on this binding, indicating the predominance of ET(A) receptors at these sites. In contrast, ET(B) receptors predominated in the lamina propria, since sarafotoxin S6c abolished specific binding in this tissue. 3 High levels of specific [I-125]-ET-1 binding were also detected in the alveoli and in the walls of blood vessels and small airways in ovine peripheral lung. Specific binding associated with alveoli was reduced to similar extents by BQ 123 (1 mu M; 54%) and sarafotoxin S6c (100 nM; 40%), suggesting the coexistence of both ET(A) and ET(B) receptors in approximately equal proportions in this tissue. In contrast, specific binding to blood vessels and to peripheral bronchial smooth muscle was abolished in the presence of BQ 123 (1 mu M), but was unaffected by sarafotoxin S6c, indicating the presence of only ET(A) receptors at these sites. 4 ET-1 caused concentration-dependent contractions of ovine tracheal smooth muscle which were inhibited in the presence of BQ 123 (1 mu M). ET-1 also caused concentration-dependent contraction of ovine lung parenchyma strips. In contrast, the ET(B) receptor-selective agonists, sarafotoxin S6c and BQ 3020, were virtually inactive as spasmogens in both tracheal smooth muscle and lung strip preparations. Thus contraction was mediated by ET(A) receptors in ovine tracheal smooth muscle and this is consistent with binding and autoradiographic data demonstrating a homogeneous population of these binding sites in this tissue. Contraction of parenchymal lung strip preparations to ET-1 was mediated via non-ET(B) receptors, presumably ET(A) receptors, with contributions to this response perhaps coming from airway and vascular smooth muscle and from alveolar wall contractile cells. 5 ET-1-induced contraction of tracheal smooth muscle was not Significantly altered in the presence of indomethacin (5 mu M), indicating that cyclo-oxygenase metabolites of arachidonic acid were not involved in this response. Contraction induced by ET-1 was virtually abolished in Ca2+-free medium containing 0.1 mM EGTA, indicating that this response was dependent upon the influx of extracellular Ca2+. Contraction was inhibited by about 50% in the presence of nicardipine (1 mu M), indicating that a significant component of this response was mediated via the activation of L-type Ca2+ channels.