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 respir atory 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 transdu ction were assessed. 2 Specific [I-125]-ET-1 binding in ovine trachea increased in a time- and concentration-dependent manner. Autoradiograp hic studies demonstrated that significant binding was associated with airway smooth muscle, although higher densities of specific binding we re 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 bi nding to airway smooth muscle. Quantitative analyses of autoradiograph ic data describing the time-dependence of specific [I-125]-ET-1 bindin g in ovine airway smooth muscle in the presence and absence of BQ 123 or sarafotoxin S6c, revealed a homogeneous population of ET(A) recepto rs. BQ 123 (1 mu M) also abolished specific binding to structures asso ciated with submucosal glands, whereas the ET(B) receptor selective ag onist, sarafotoxin S6c (100 nM) had little effect on this binding, ind icating the predominance of ET(A) receptors at these sites. In contras t, ET(B) receptors predominated in the lamina propria, since sarafotox in S6c abolished specific binding in this tissue. 3 High levels of spe cific [I-125]-ET-1 binding were also detected in the alveoli and in th e walls of blood vessels and small airways in ovine peripheral lung. S pecific 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 eq ual proportions in this tissue. In contrast, specific binding to blood vessels and to peripheral bronchial smooth muscle was abolished in th e 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 a lso caused concentration-dependent contraction of ovine lung parenchym a strips. In contrast, the ET(B) receptor-selective agonists, sarafoto xin S6c and BQ 3020, were virtually inactive as spasmogens in both tra cheal smooth muscle and lung strip preparations. Thus contraction was mediated by ET(A) receptors in ovine tracheal smooth muscle and this i s consistent with binding and autoradiographic data demonstrating a ho mogeneous population of these binding sites in this tissue. Contractio n of parenchymal lung strip preparations to ET-1 was mediated via non- ET(B) receptors, presumably ET(A) receptors, with contributions to thi s response perhaps coming from airway and vascular smooth muscle and f rom alveolar wall contractile cells. 5 ET-1-induced contraction of tra cheal smooth muscle was not Significantly altered in the presence of i ndomethacin (5 mu M), indicating that cyclo-oxygenase metabolites of a rachidonic acid were not involved in this response. Contraction induce d by ET-1 was virtually abolished in Ca2+-free medium containing 0.1 m M EGTA, indicating that this response was dependent upon the influx of extracellular Ca2+. Contraction was inhibited by about 50% in the pre sence of nicardipine (1 mu M), indicating that a significant component of this response was mediated via the activation of L-type Ca2+ chann els.