Cellular signaling by the potent bronchoconstrictor endothelin-1 in airwaysmooth muscle

Objective: The objective of this study was to determine whether the potent bronchoconstrictor endothelin-l was coupled to the activation of the inosit ol phosphate and/or inhibition of the cyclic adenine monophosphate second m essenger pathways in porcine airway smooth muscle. Design: Prospective, controlled, in vitro, nonblinded study. Setting: University biochemical and molecular biological research laborator y. 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 prelo aded with H-3-myoinositol was exposed to endothelin-l, carbachol (positive control) or vehicle for 30 mins. Some tissues were pretreated with antagoni sts selective for the ETA (BQ-485) and ETB (BQ-788) endothelin receptor sub types. Newly synthesized H-3-inositol phosphates were recovered by column c hromatography. Airway smooth muscle from an additional 7 pigs was homogeniz ed and incubated in the presence of P-32-alpha-adenosine triphosphate, guan osine triphosphate (GTP) and either carbachol or endothelin to measure the inhibitory influence of carbachol (positive control) or endothelin on GTP-s timulated adenylyl cyclase activity. Newly synthesized P-32-cyclic adenosin e monophosphate was isolated by sequential column chromatography over Dowex and alumina, Measurements and Main Results: Total inositol phosphates increased in porci ne airway smooth muscle in response to either carbachol or endothelin. The endothelin receptor antagonist BQ-485 (ETA selective) but not BQ-788 (ETB s elective) dose-dependently inhibited endothelin-l induced inositol phosphat e accumulation, In adenylyl cyclase assays, carbachol (positive control), b ut not endothlein-1, significantly inhibited GTP-stimulated adenylyl cyclas e activity, Conclusion: Endothelin-1 couples to the activation of the inositol phosphat e pathway via the ETA receptor subtype but does not couple to inhibition of the adenylyl cyclase pathway in porcine airway smooth muscle. The potent b ronchoconstrictive effects of endothelin likely involve the acute activatio n of the inositol phosphate pathway in airway smooth muscle.