Abstract
The mechanism(s) of degradation of the potent vasoconstrictor endothelin-1 (ET-1) by rat vascular smooth muscle A-10 cells, which possess the ET, receptor subtype, was investigated by incubating [125I]ET-1 (0.1 nM) with cells for 0-4 h at 37°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-I was incubated with A-10 cells at 4°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 μM 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.
Original language | English (US) |
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Pages (from-to) | 155-162 |
Number of pages | 8 |
Journal | Regulatory Peptides |
Volume | 66 |
Issue number | 3 |
DOIs | |
State | Published - Oct 22 1996 |
Keywords
- A-10 cell
- Degradation
- Endothelin-1
- Internalization
- Lysosome
ASJC Scopus subject areas
- Biochemistry
- Physiology
- Endocrinology
- Clinical Biochemistry
- Cellular and Molecular Neuroscience