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Production of eicosanoids in response to endothelin-1 and identification of specific endothelin-1 binding sites in airway epithelial cells. 1993

T Wu, and R D Rieves, and P Larivee, and C Logun, and M G Lawrence, and J H Shelhamer
Critical Care Medicine Department, National Institutes of Health, Bethesda, Maryland 20892.

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)

UI MeSH Term Description Entries
D007700 Kinetics The rate dynamics in chemical or physical systems.
D008401 Gas Chromatography-Mass Spectrometry A microanalytical technique combining mass spectrometry and gas chromatography for the qualitative as well as quantitative determinations of compounds. Chromatography, Gas-Liquid-Mass Spectrometry,Chromatography, Gas-Mass Spectrometry,GCMS,Spectrometry, Mass-Gas Chromatography,Spectrum Analysis, Mass-Gas Chromatography,Gas-Liquid Chromatography-Mass Spectrometry,Mass Spectrometry-Gas Chromatography,Chromatography, Gas Liquid Mass Spectrometry,Chromatography, Gas Mass Spectrometry,Chromatography, Mass Spectrometry-Gas,Chromatography-Mass Spectrometry, Gas,Chromatography-Mass Spectrometry, Gas-Liquid,Gas Chromatography Mass Spectrometry,Gas Liquid Chromatography Mass Spectrometry,Mass Spectrometry Gas Chromatography,Spectrometries, Mass-Gas Chromatography,Spectrometry, Gas Chromatography-Mass,Spectrometry, Gas-Liquid Chromatography-Mass,Spectrometry, Mass Gas Chromatography,Spectrometry-Gas Chromatography, Mass,Spectrum Analysis, Mass Gas Chromatography
D011863 Radioimmunoassay Classic quantitative assay for detection of antigen-antibody reactions using a radioactively labeled substance (radioligand) either directly or indirectly to measure the binding of the unlabeled substance to a specific antibody or other receptor system. Non-immunogenic substances (e.g., haptens) can be measured if coupled to larger carrier proteins (e.g., bovine gamma-globulin or human serum albumin) capable of inducing antibody formation. Radioimmunoassays
D002415 Cats The domestic cat, Felis catus, of the carnivore family FELIDAE, comprising over 30 different breeds. The domestic cat is descended primarily from the wild cat of Africa and extreme southwestern Asia. Though probably present in towns in Palestine as long ago as 7000 years, actual domestication occurred in Egypt about 4000 years ago. (From Walker's Mammals of the World, 6th ed, p801) Felis catus,Felis domesticus,Domestic Cats,Felis domestica,Felis sylvestris catus,Cat,Cat, Domestic,Cats, Domestic,Domestic Cat
D002478 Cells, Cultured Cells propagated in vitro in special media conducive to their growth. Cultured cells are used to study developmental, morphologic, metabolic, physiologic, and genetic processes, among others. Cultured Cells,Cell, Cultured,Cultured Cell
D002851 Chromatography, High Pressure Liquid Liquid chromatographic techniques which feature high inlet pressures, high sensitivity, and high speed. Chromatography, High Performance Liquid,Chromatography, High Speed Liquid,Chromatography, Liquid, High Pressure,HPLC,High Performance Liquid Chromatography,High-Performance Liquid Chromatography,UPLC,Ultra Performance Liquid Chromatography,Chromatography, High-Performance Liquid,High-Performance Liquid Chromatographies,Liquid Chromatography, High-Performance
D004848 Epithelium The layers of EPITHELIAL CELLS which cover the inner and outer surfaces of the cutaneous, mucus, and serous tissues and glands of the body. Mesothelium,Epithelial Tissue,Mesothelial Tissue,Epithelial Tissues,Mesothelial Tissues,Tissue, Epithelial,Tissue, Mesothelial,Tissues, Epithelial,Tissues, Mesothelial
D006893 Hydroxyeicosatetraenoic Acids Eicosatetraenoic acids substituted in any position by one or more hydroxy groups. They are important intermediates in a series of biosynthetic processes leading from arachidonic acid to a number of biologically active compounds such as prostaglandins, thromboxanes, and leukotrienes. HETE,Acids, Hydroxyeicosatetraenoic
D000818 Animals Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA. Animal,Metazoa,Animalia
D014132 Trachea The cartilaginous and membranous tube descending from the larynx and branching into the right and left main bronchi. Tracheas

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