Cellular signaling by the potent bronchoconstrictor endothelin-1 in airway smooth muscle

Objective: The objective of this study was to determine whether the potent bronchoconstrictor endothelin-1 was coupled to the activation of the inositol phosphate and/or inhibition of the cyclic adenine monophosphate second messenger pathways in porcine airway smooth muscle. Design: Prospective, controlled, in vitro, nonblinded study. Setting: University biochemical and molecular biological research laboratory. Subjects: Pigs of both genders. Interventions: Airway smooth muscle was dissected from the trachea of pigs exsanguinated under anesthesia. Airway smooth muscle from six animals preloaded with ³H-myoinositol was exposed to endothelin-1, carbachol (positive control) or vehicle for 30 mins. Some tissues were pretreated with antagonists selective for the ET(A) (BQ-485) and ET(B) (BQ-788) endothelin receptor subtypes. Newly synthesized ³H-inositol phosphates were recovered by column chromatography. Airway smooth muscle from an additional 7 pigs was homogenized and incubated in the presence of ³²P-α-adenosine triphosphate, guanosine triphosphate (GTP) and either carbachol or endothelin to measure the inhibitory influence of carbachol (positive control) or endothelin on GTP-stimulated adenylyl cyclase activity. Newly synthesized ³²P-cyclic adenosine monophosphate was isolated by sequential column chromatography over Dowex and alumina. Measurements and Main Results: Total inositol phosphates increased in porcine airway smooth muscle in response to either carbachol or endothelin. The endothelin receptor antagonist BQ-485 (ET(A) selective) but not BQ-788 (ET(B) selective) dose-dependently inhibited endothelin-1 induced inositol phosphate accumulation. In adenylyl cyclase assays, carbachol (positive control), but not endothelin-1, significantly inhibited GTP- stimulated adenylyl cyclase activity. Conclusion: Endothelin-1 couples to the activation of the inositol phosphate pathway via the ETA receptor subtype but does not couple to inhibition of the adenylyl cyclase pathway in porcine airway smooth muscle. The potent bronchoconstrictive effects of endothelin likely involve the acute activation of the inositol phosphate pathway in airway smooth muscle.