Endothelin degradation by vascular smooth muscle cells.
The mechanism(s) of degradation of the potent vasoconstrictor endothelin-1 (ET-1) by rat vascular smooth muscle A-10 cells, which possess the ETA receptor subtype, was investigated by incubating [125I]ET-1 (0.1 nM) with cells for 0-4 h at 37 degrees C in the presence and absence of lysosomal enzyme inhibitors, NH4Cl and chloroquine, and a neutral endopeptidase inhibitor, phosphoramidon. The assay buffer and cell extracts were analyzed by reverse-phase HPLC, and the radioactivity in the fractions was measured. In the absence of inhibitors, most of the radioactivity in the medium was in the form of [125I]Tyr after a 4 h incubation. When [125I]ET-1 was incubated with A-10 cells at 4 degrees C, six radiolabeled peaks, including some [125I]Tyr and about 30% of the original [125I]ET-1, were present in the medium. In the presence of 5 microM chloroquine there was no [125I]Tyr peak in the medium, indicating that internalization and putative lysosomal degradation of ET-1 were blocked. NH4Cl (50 and 100 mM) also reduced the amount of [125I]Tyr formed. The presence of ET-1 fragments indicated that, in addition to lysosomal degradation, some of the ligand is metabolized by enzymes located on the cell membrane; we demonstrated, however, that secreted proteases from A-10 cells are not involved in the degradation of ET-1. The neutral endopeptidase inhibitor, phosphoramidon, did not completely inhibit the metabolism of [125I]ET-1 to [125I]Tyr. These results establish that various cell-associated enzymes are capable of degrading ET-1 in A-10 cells. Moreover, analysis of the cell lysates indicated the presence of a relatively stable pool of ET-1-occupied receptors or compartmentalized ET-1, protected from cell proteases, which may contribute to the potent contractility of ET-1.
Department of Biochemistry & Molecular Biology, University of Georgia, Athens 30602, USA.
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Bermek
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Peng
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Angelova
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Puett
D000818:Animals; D002460:Cell Line; D002738:Chloroquine / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D019332:Endothelin-1 / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D011869:Radioligand Assay; D051381:Rats
D019332:Endothelin-1; D004791:Enzyme Inhibitors; D002738:Chloroquine