Characterization of endothelin-converting enzyme activities in ARPE-19 cells, a human retinal pigmented epithelial cell line

Adnan Dibas, Ganesh Prasanna, Thomas Yorio

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3 Citations (Scopus)

Abstract

Elevated endothelin-1 (ET-1) levels are detected in patients with glaucoma. ET-1 is produced from its precursor, Big ET-1, by endothelin-converting enzyme (ECE). Characterization of ET-1 secretion and ECE activity was performed in ARPE-19 cells, a human retinal pigmented epithelial cell-line. The ET B receptor but not the ETA receptor was detected by immunoblotting and cross-linking using 125I-ET-1 at the plasma membrane (PM). Tumor necrosis factor-α (10 nmol/L) induced a 700% increase in ET-1 levels and such an effect was further potentiated by BQ788, an ET B receptor antagonist, suggesting the involvement of ETB receptor in ET-1 clearance. Big ET-1-converting activities were detected in both the PM and cytosol. Phosphoramidon, thiorphan, acidification, and phenanthroline inhibited PM ECE activity; the cytosolic ECE activity was not affected by phenanthroline but was inhibited by the others. In contrast, ECE cytosolic activities were activated by acidification (pH 6.4), suggesting the involvement of ECE-2 or cathepsin-like activity. Pepstatin, a potent inhibitor of cathepsins, and phosphoramidon, a potent inhibitor of ECE, inhibited the cytosolic conversion of Big ET-1 peptide by 46% and 35%, respectively, whereas the combination of both inhibited the cytosolic activity by 93%. Based on immunoblotting, ECE-1 was detected only at the PM, whereas ECE-2 and cathpesins B and D were detected in the cytosol. In summary, ET-1 production in RPE is regulated by at least two isoforms of ECE, (cytosolic and PM) as well as cathepsins.

Original languageEnglish
Pages (from-to)196-204
Number of pages9
JournalJournal of Ocular Pharmacology and Therapeutics
Volume21
Issue number3
DOIs
StatePublished - 1 Jun 2005

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Endothelin-1
Epithelial Cells
Cell Line
Cathepsins
Cell Membrane
Phenanthrolines
Immunoblotting
Cytosol
Thiorphan
Endothelin-Converting Enzymes
Endothelin A Receptors
Glaucoma
Protein Isoforms
Tumor Necrosis Factor-alpha
Peptides

Cite this

@article{6f6eb506635e45868df47457a9c48151,
title = "Characterization of endothelin-converting enzyme activities in ARPE-19 cells, a human retinal pigmented epithelial cell line",
abstract = "Elevated endothelin-1 (ET-1) levels are detected in patients with glaucoma. ET-1 is produced from its precursor, Big ET-1, by endothelin-converting enzyme (ECE). Characterization of ET-1 secretion and ECE activity was performed in ARPE-19 cells, a human retinal pigmented epithelial cell-line. The ET B receptor but not the ETA receptor was detected by immunoblotting and cross-linking using 125I-ET-1 at the plasma membrane (PM). Tumor necrosis factor-α (10 nmol/L) induced a 700{\%} increase in ET-1 levels and such an effect was further potentiated by BQ788, an ET B receptor antagonist, suggesting the involvement of ETB receptor in ET-1 clearance. Big ET-1-converting activities were detected in both the PM and cytosol. Phosphoramidon, thiorphan, acidification, and phenanthroline inhibited PM ECE activity; the cytosolic ECE activity was not affected by phenanthroline but was inhibited by the others. In contrast, ECE cytosolic activities were activated by acidification (pH 6.4), suggesting the involvement of ECE-2 or cathepsin-like activity. Pepstatin, a potent inhibitor of cathepsins, and phosphoramidon, a potent inhibitor of ECE, inhibited the cytosolic conversion of Big ET-1 peptide by 46{\%} and 35{\%}, respectively, whereas the combination of both inhibited the cytosolic activity by 93{\%}. Based on immunoblotting, ECE-1 was detected only at the PM, whereas ECE-2 and cathpesins B and D were detected in the cytosol. In summary, ET-1 production in RPE is regulated by at least two isoforms of ECE, (cytosolic and PM) as well as cathepsins.",
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Characterization of endothelin-converting enzyme activities in ARPE-19 cells, a human retinal pigmented epithelial cell line. / Dibas, Adnan; Prasanna, Ganesh; Yorio, Thomas.

In: Journal of Ocular Pharmacology and Therapeutics, Vol. 21, No. 3, 01.06.2005, p. 196-204.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

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AU - Dibas, Adnan

AU - Prasanna, Ganesh

AU - Yorio, Thomas

PY - 2005/6/1

Y1 - 2005/6/1

N2 - Elevated endothelin-1 (ET-1) levels are detected in patients with glaucoma. ET-1 is produced from its precursor, Big ET-1, by endothelin-converting enzyme (ECE). Characterization of ET-1 secretion and ECE activity was performed in ARPE-19 cells, a human retinal pigmented epithelial cell-line. The ET B receptor but not the ETA receptor was detected by immunoblotting and cross-linking using 125I-ET-1 at the plasma membrane (PM). Tumor necrosis factor-α (10 nmol/L) induced a 700% increase in ET-1 levels and such an effect was further potentiated by BQ788, an ET B receptor antagonist, suggesting the involvement of ETB receptor in ET-1 clearance. Big ET-1-converting activities were detected in both the PM and cytosol. Phosphoramidon, thiorphan, acidification, and phenanthroline inhibited PM ECE activity; the cytosolic ECE activity was not affected by phenanthroline but was inhibited by the others. In contrast, ECE cytosolic activities were activated by acidification (pH 6.4), suggesting the involvement of ECE-2 or cathepsin-like activity. Pepstatin, a potent inhibitor of cathepsins, and phosphoramidon, a potent inhibitor of ECE, inhibited the cytosolic conversion of Big ET-1 peptide by 46% and 35%, respectively, whereas the combination of both inhibited the cytosolic activity by 93%. Based on immunoblotting, ECE-1 was detected only at the PM, whereas ECE-2 and cathpesins B and D were detected in the cytosol. In summary, ET-1 production in RPE is regulated by at least two isoforms of ECE, (cytosolic and PM) as well as cathepsins.

AB - Elevated endothelin-1 (ET-1) levels are detected in patients with glaucoma. ET-1 is produced from its precursor, Big ET-1, by endothelin-converting enzyme (ECE). Characterization of ET-1 secretion and ECE activity was performed in ARPE-19 cells, a human retinal pigmented epithelial cell-line. The ET B receptor but not the ETA receptor was detected by immunoblotting and cross-linking using 125I-ET-1 at the plasma membrane (PM). Tumor necrosis factor-α (10 nmol/L) induced a 700% increase in ET-1 levels and such an effect was further potentiated by BQ788, an ET B receptor antagonist, suggesting the involvement of ETB receptor in ET-1 clearance. Big ET-1-converting activities were detected in both the PM and cytosol. Phosphoramidon, thiorphan, acidification, and phenanthroline inhibited PM ECE activity; the cytosolic ECE activity was not affected by phenanthroline but was inhibited by the others. In contrast, ECE cytosolic activities were activated by acidification (pH 6.4), suggesting the involvement of ECE-2 or cathepsin-like activity. Pepstatin, a potent inhibitor of cathepsins, and phosphoramidon, a potent inhibitor of ECE, inhibited the cytosolic conversion of Big ET-1 peptide by 46% and 35%, respectively, whereas the combination of both inhibited the cytosolic activity by 93%. Based on immunoblotting, ECE-1 was detected only at the PM, whereas ECE-2 and cathpesins B and D were detected in the cytosol. In summary, ET-1 production in RPE is regulated by at least two isoforms of ECE, (cytosolic and PM) as well as cathepsins.

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