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 3H-labeled arachidonic acid (AA) metabolites and by radioimmunoassay (RIA) of corresponding nonradiolabeled HPLC elution. The HPLC chromatograms of [3H]AA-prelabeled samples revealed that ET-1 (10(-5) M) augmented the release of prostaglandin (PG) E2, 12-hydroxyeicosatetraenoic acid (HETE), PGF2 alpha, and AA. RIA of corresponding nonradiolabeled HPLC elution demonstrated a significantly increased release of PGE2, PGF2 alpha, 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 125I-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 (Kd) of 35.3 pM and maximal binding capacity (Bmax) of 15.0 fmol/10(7) cells for the higher affinity binding site and Kd of 205.9 pM and Bmax of 35.0 fmol/10(7) cells for the lower affinity binding site.(ABSTRACT TRUNCATED AT 250 WORDS)
Wu
T
T
Critical Care Medicine Department, National Institutes of Health, Bethesda, Maryland 20892.
Rieves
R D
RD
Larivee
P
P
Logun
C
C
Lawrence
M G
MG
Shelhamer
J H
JH
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism*; D002415:Cats; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D015777:Eicosanoids / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D016232:Endothelins / Q000378:metabolism / Q000494:pharmacology*; D004848:Epithelium / Q000187:drug effects / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D006893:Hydroxyeicosatetraenoic Acids / Q000032:analysis / Q000096:biosynthesis; D007700:Kinetics; D011863:Radioimmunoassay; D017466:Receptors, Endothelin / Q000378:metabolism*; D014132:Trachea / Q000378:metabolism*; D014316:Tritium
D015777:Eicosanoids; D016232:Endothelins; D006893:Hydroxyeicosatetraenoic Acids; D017466:Receptors, Endothelin; D014316:Tritium; D016718:Arachidonic Acid; C022022:5-hydroxy-6,8,11,14-eicosatetraenoic acid