PRODUCTION OF EICOSANOIDS IN RESPONSE TO ENDOTHELIN-1 AND IDENTIFICATION OF SPECIFIC ENDOTHELIN-1 BINDING-SITES IN AIRWAY EPITHELIAL-CELLS

The effect of endothelin-1 (ET-1) on arachidonate metabolism in the re spiratory epithelium was investigated in primary cultures of feline tr acheal epithelial cells. Subconfluent epithelial cell cultures were st imulated by ET-1, and eicosanoid generation was determined by high per formance liquid chromatography (HLPC) of H-3-labeled arachidonic acid (AA) metabolites and by radioimmunoassay (RIA) of corresponding nonrad iolabeled HPLC elution. The HPLC chromatograms of [H-3]AA-prelabeled s amples revealed that ET-1 (10(-5) M) augmented the release of prostagl andin (PG) E2, 12-hydroxyeicosatetraenoic acid (HETE), PGF2alpha, and AA. RIA of corresponding nonradiolabeled HPLC elution demonstrated a s ignificantly increased release of PGE2, PGF2alpha, and 12-HETE as well as 5-HETE in response to ET-1 stimulation. 5-HETE release from ET-1-s timulated cells was further identified by gas chromatography/mass spec trometry (GC/MS). The stimulating effect of ET-1 on AA metabolism was dose dependent (10(-5) to 10(-7) M) and peaked within 1 h with a progr essive decline over the subsequent hours. Using I-125-labeled ET-1 as radioligand, the presence of specific binding sites for ET-1 was demon strated in cultured feline tracheal epithelial cells. ET-1 binding rea ched equilibrium within 1 h at 37-degrees-C. Scatchard analysis sugges ted the existence of two saturable binding sites, with the estimated e quilibrium dissociation constant (K(d)) of 35.3 pM and maximal binding capacity (Bmax) of 15.0 fmol/10(7) cells for the higher affinity bind ing site and K(d) of 205.9 pM and Bmax of 35.0 fmol/10(7) cells for th e lower affinity binding site. Thus ET-1 binds to receptors on airway epithelial cells and initiates the production of a variety of AA metab olites that may modulate airway inflammatory responses.