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) |
|---|---|
| Pages (from-to) | 155-162 |
| Number of pages | 8 |
| Journal | Regulatory Peptides |
| Volume | 66 |
| Issue number | 3 |
| DOIs | |
| State | Published - Oct 22 1996 |
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Keywords
- A-10 cell
- Degradation
- Endothelin-1
- Internalization
- Lysosome
ASJC Scopus subject areas
- Biochemistry
- Physiology
- Endocrinology
- Clinical Biochemistry
- Cellular and Molecular Neuroscience
Cite this
Endothelin degradation by vascular smooth muscle cells. / Bermek, Hakan; Peng, Kou Cheng; Angelova, Krassimira; Ergul, Adviye; Puett, David.
In: Regulatory Peptides, Vol. 66, No. 3, 22.10.1996, p. 155-162.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Endothelin degradation by vascular smooth muscle cells
AU - Bermek, Hakan
AU - Peng, Kou Cheng
AU - Angelova, Krassimira
AU - Ergul, Adviye
AU - Puett, David
PY - 1996/10/22
Y1 - 1996/10/22
N2 - 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.
AB - 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.
KW - A-10 cell
KW - Degradation
KW - Endothelin-1
KW - Internalization
KW - Lysosome
UR - http://www.scopus.com/inward/record.url?scp=0030598198&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0030598198&partnerID=8YFLogxK
U2 - 10.1016/S0167-0115(96)00094-8
DO - 10.1016/S0167-0115(96)00094-8
M3 - Article
VL - 66
SP - 155
EP - 162
JO - Regulatory Peptides
JF - Regulatory Peptides
SN - 0167-0115
IS - 3
ER -