In the present study, we investigated the mechanisms underlying the differential effects of endothelins (ETs) in the rat trachea. Sarafotoxin-S6c (SRTX-c) and ET-3 were more potent spasmogens to rat tracheal strips than ET-1. The EC50 values were 12, 14.1 and 89.1 nM, respectively. Tension responses to ET-1 and ET-3, but not to SRTX-c, were enhanced by either indomethacin or the ET(A) blocker, BQ-610 (1 microM). In epithelium-intact tracheal rings, both ET-1 and ET-3 activated adenylate cyclase (AC) in a concentration-dependent manner. The activation by ET-1 of AC was significantly higher than that of ET-3. Thus, EC50 values for ET-1 and ET-3 were 71 and 200 nM, and maximal cAMP increments were 196% and 62% above baseline, respectively. SRTX-c, up to 1 microM, did not alter basal cAMP level. Mechanical removal of the epithelium neither had an effect on AC activation by ET-1 or ET-3, nor did it alter the inability of SRTX-c to modulate AC activity. Conversely, pre-incubation of tracheal strips with indomethacin (1 microM) virtually ablated the increments in cAMP by the ETs. Likewise, BQ-610 attenuated AC activation, concentration-dependently (IC50=28.2 nM). Taken together, the present study suggests that ET(A) receptors, from non-epithelial source, are functionally-linked to AC activation via a prostanoid-dependent pathway. This ET(A)-initiated cascade acts to negatively regulate muscle contraction. Such a cross-talk between ET signals most likely accounts for variation of tension responses to ET homologs.