Endothelin degradation by vascular smooth muscle cells

Hakan Bermek, Kou Cheng Peng, Krassimira Angelova, Adviye Ergul, David Puett

Research output: Contribution to journalArticle

9 Citations (Scopus)

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 languageEnglish (US)
Pages (from-to)155-162
Number of pages8
JournalRegulatory Peptides
Volume66
Issue number3
DOIs
StatePublished - Oct 22 1996

Fingerprint

Endothelins
Endothelin-1
Vascular Smooth Muscle
Smooth Muscle Myocytes
Muscle
Cells
Degradation
antineoplaston A10
Neprilysin
Radioactivity
Chloroquine
Protease Inhibitors
Peptide Hydrolases
Endothelin A Receptors
Enzyme Inhibitors
Vasoconstrictor Agents
Enzymes
Cell membranes
Cell Extracts
Metabolism

Keywords

  • A-10 cell
  • Degradation
  • Endothelin-1
  • Internalization
  • Lysosome

ASJC Scopus subject areas

  • Biochemistry
  • Physiology
  • Endocrinology
  • Clinical Biochemistry
  • Cellular and Molecular Neuroscience

Cite this

Bermek, H., Peng, K. C., Angelova, K., Ergul, A., & Puett, D. (1996). Endothelin degradation by vascular smooth muscle cells. Regulatory Peptides, 66(3), 155-162. https://doi.org/10.1016/S0167-0115(96)00094-8

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 journalArticle

Bermek, H, Peng, KC, Angelova, K, Ergul, A & Puett, D 1996, 'Endothelin degradation by vascular smooth muscle cells', Regulatory Peptides, vol. 66, no. 3, pp. 155-162. https://doi.org/10.1016/S0167-0115(96)00094-8
Bermek H, Peng KC, Angelova K, Ergul A, Puett D. Endothelin degradation by vascular smooth muscle cells. Regulatory Peptides. 1996 Oct 22;66(3):155-162. https://doi.org/10.1016/S0167-0115(96)00094-8
Bermek, Hakan ; Peng, Kou Cheng ; Angelova, Krassimira ; Ergul, Adviye ; Puett, David. / Endothelin degradation by vascular smooth muscle cells. In: Regulatory Peptides. 1996 ; Vol. 66, No. 3. pp. 155-162.
@article{92ee2ac9cfd946bfa4a55941b1762462,
title = "Endothelin degradation by vascular smooth muscle cells",
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.",
keywords = "A-10 cell, Degradation, Endothelin-1, Internalization, Lysosome",
author = "Hakan Bermek and Peng, {Kou Cheng} and Krassimira Angelova and Adviye Ergul and David Puett",
year = "1996",
month = "10",
day = "22",
doi = "10.1016/S0167-0115(96)00094-8",
language = "English (US)",
volume = "66",
pages = "155--162",
journal = "Regulatory Peptides",
issn = "0167-0115",
publisher = "Elsevier",
number = "3",

}

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 -