Production of Eicosanoids in Response to Endothelin-1 and Identification of Specific Endothelin-1 Binding Sites in Airway Epithelial Cells
| Autor: | James H. Shelhamer, R. D. Rieves, Pierre Larivée, Tong Wu, Marion G. Lawrence, Carolea Logun |
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| Rok vydání: | 1993 |
| Předmět: |
Pulmonary and Respiratory Medicine
Clinical Biochemistry Radioimmunoassay Prostaglandin Tritium Epithelium Gas Chromatography-Mass Spectrometry chemistry.chemical_compound Hydroxyeicosatetraenoic Acids Animals Binding site Molecular Biology Cells Cultured Chromatography High Pressure Liquid Arachidonic Acid Receptors Endothelin Endothelins Cell Biology Endothelin 1 Molecular biology Trachea Dissociation constant Kinetics chemistry Eicosanoid Biochemistry Cats Eicosanoids Respiratory epithelium Arachidonic acid |
| Zdroj: | American Journal of Respiratory Cell and Molecular Biology. 8:282-290 |
| ISSN: | 1535-4989 1044-1549 |
| DOI: | 10.1165/ajrcmb/8.3.282 |
| Popis: | 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) |
| Databáze: | OpenAIRE |
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