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 respiratory epithelium was investigated in primary cultures of feline tracheal epithelial cells. Subconfluent epithelial cell cultures were stimulated by ET-1, and eicosanoid generation was determined by high performance liquid chromatography (HLPC) of H-3-labeled arachidonic acid (AA) metabolites and by radioimmunoassay (RIA) of corresponding nonradiolabeled HPLC elution. The HPLC chromatograms of [H-3]AA-prelabeled samples revealed that ET-1 (10(-5) M) augmented the release of prostaglandin (PG) E2, 12-hydroxyeicosatetraenoic acid (HETE), PGF2alpha, and AA. RIA of corresponding nonradiolabeled HPLC elution demonstrated a significantly 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-stimulated cells was further identified by gas chromatography/mass spectrometry (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 progressive decline over the subsequent hours. Using I-125-labeled ET-1 as radioligand, the presence of specific binding sites for ET-1 was demonstrated in cultured feline tracheal epithelial cells. ET-1 binding reached equilibrium within 1 h at 37-degrees-C. Scatchard analysis suggested the existence of two saturable binding sites, with the estimated equilibrium dissociation constant (K(d)) of 35.3 pM and maximal binding capacity (Bmax) of 15.0 fmol/10(7) cells for the higher affinity binding site and K(d) of 205.9 pM and Bmax of 35.0 fmol/10(7) cells for the lower affinity binding site. Thus ET-1 binds to receptors on airway epithelial cells and initiates the production of a variety of AA metabolites that may modulate airway inflammatory responses.