Phenylarsine oxide inhibits nitric oxide synthase in pulmonary artery endothelial cells

Yunchao Su, Edward R. Block

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

The role of protein tyrosine phosphorylation during regulation of NO synthase (eNOS) activity in endothelial cells is poorly understood. Studies to define this role have used inhibitors of tyrosine kinase or tyrosine phosphatase (TP). Phenylarsine oxide (PAO), an inhibitor of TP, has been reported to bind thiol groups, and recent work from our laboratory demonstrates that eNOS activity depends on thiol groups at its catalytic site. Therefore, we hypothesized that PAO may have a direct effect on eNOS activity. To test this, we measured (i) TP and eNOS activities both in total membrane fractions and in purified eNOS prepared from porcine pulmonary artery endothelial cells and (ii) sulfhydryl content and eNOS activity in purified bovine aortic eNOS expressed in Escherichia coli. High TP activity was detected in total membrane fractions, but no TP activity was detected in purified eNOS fractions. PAO caused a dose-dependent decrease in eNOS activity in total membrane and in purified eNOS fractions from porcine pulmonary artery endothelial cells, even though the latter had no detectable TP activity. PAO also caused a decrease in sulfhydryl content and eNOS activity in purified bovine eNOS. The reduction in eNOS sulfhydryl content and the inhibitory effect of PAO on eNOS activity were prevented by dithiothreitol, a disulfide-reducing agent. These results indicate that (i) PAO directly inhibits eNOS activity in endothelial cells by binding to thiol groups in the eNOS protein and (ii) results of studies using PAO to assess the role of protein tyrosine phosphorylation in regulating eNOS activity must be interpreted with great caution. Copyright (C) 2000 Elsevier Science Inc.

Original languageEnglish (US)
Pages (from-to)167-173
Number of pages7
JournalFree Radical Biology and Medicine
Volume28
Issue number2
DOIs
StatePublished - Jan 15 2000
Externally publishedYes

Fingerprint

Endothelial cells
Nitric Oxide Synthase
Pulmonary Artery
Tyrosine
Endothelial Cells
Phosphoric Monoester Hydrolases
Sulfhydryl Compounds
Phosphorylation
Membranes
Swine
Proteins
oxophenylarsine
Dithiothreitol
Reducing Agents
Disulfides
Protein-Tyrosine Kinases
Escherichia coli
Catalytic Domain

Keywords

  • Endothelial cell
  • Free radicals
  • Nitric oxide synthase
  • Phenylarsine oxide
  • Pulmonary

ASJC Scopus subject areas

  • Medicine(all)
  • Toxicology
  • Clinical Biochemistry

Cite this

Phenylarsine oxide inhibits nitric oxide synthase in pulmonary artery endothelial cells. / Su, Yunchao; Block, Edward R.

In: Free Radical Biology and Medicine, Vol. 28, No. 2, 15.01.2000, p. 167-173.

Research output: Contribution to journalArticle

@article{7cfc7da9b0ca496681a7a64234bc7bcb,
title = "Phenylarsine oxide inhibits nitric oxide synthase in pulmonary artery endothelial cells",
abstract = "The role of protein tyrosine phosphorylation during regulation of NO synthase (eNOS) activity in endothelial cells is poorly understood. Studies to define this role have used inhibitors of tyrosine kinase or tyrosine phosphatase (TP). Phenylarsine oxide (PAO), an inhibitor of TP, has been reported to bind thiol groups, and recent work from our laboratory demonstrates that eNOS activity depends on thiol groups at its catalytic site. Therefore, we hypothesized that PAO may have a direct effect on eNOS activity. To test this, we measured (i) TP and eNOS activities both in total membrane fractions and in purified eNOS prepared from porcine pulmonary artery endothelial cells and (ii) sulfhydryl content and eNOS activity in purified bovine aortic eNOS expressed in Escherichia coli. High TP activity was detected in total membrane fractions, but no TP activity was detected in purified eNOS fractions. PAO caused a dose-dependent decrease in eNOS activity in total membrane and in purified eNOS fractions from porcine pulmonary artery endothelial cells, even though the latter had no detectable TP activity. PAO also caused a decrease in sulfhydryl content and eNOS activity in purified bovine eNOS. The reduction in eNOS sulfhydryl content and the inhibitory effect of PAO on eNOS activity were prevented by dithiothreitol, a disulfide-reducing agent. These results indicate that (i) PAO directly inhibits eNOS activity in endothelial cells by binding to thiol groups in the eNOS protein and (ii) results of studies using PAO to assess the role of protein tyrosine phosphorylation in regulating eNOS activity must be interpreted with great caution. Copyright (C) 2000 Elsevier Science Inc.",
keywords = "Endothelial cell, Free radicals, Nitric oxide synthase, Phenylarsine oxide, Pulmonary",
author = "Yunchao Su and Block, {Edward R.}",
year = "2000",
month = "1",
day = "15",
doi = "10.1016/S0891-5849(99)00231-2",
language = "English (US)",
volume = "28",
pages = "167--173",
journal = "Free Radical Biology and Medicine",
issn = "0891-5849",
publisher = "Elsevier Inc.",
number = "2",

}

TY - JOUR

T1 - Phenylarsine oxide inhibits nitric oxide synthase in pulmonary artery endothelial cells

AU - Su, Yunchao

AU - Block, Edward R.

PY - 2000/1/15

Y1 - 2000/1/15

N2 - The role of protein tyrosine phosphorylation during regulation of NO synthase (eNOS) activity in endothelial cells is poorly understood. Studies to define this role have used inhibitors of tyrosine kinase or tyrosine phosphatase (TP). Phenylarsine oxide (PAO), an inhibitor of TP, has been reported to bind thiol groups, and recent work from our laboratory demonstrates that eNOS activity depends on thiol groups at its catalytic site. Therefore, we hypothesized that PAO may have a direct effect on eNOS activity. To test this, we measured (i) TP and eNOS activities both in total membrane fractions and in purified eNOS prepared from porcine pulmonary artery endothelial cells and (ii) sulfhydryl content and eNOS activity in purified bovine aortic eNOS expressed in Escherichia coli. High TP activity was detected in total membrane fractions, but no TP activity was detected in purified eNOS fractions. PAO caused a dose-dependent decrease in eNOS activity in total membrane and in purified eNOS fractions from porcine pulmonary artery endothelial cells, even though the latter had no detectable TP activity. PAO also caused a decrease in sulfhydryl content and eNOS activity in purified bovine eNOS. The reduction in eNOS sulfhydryl content and the inhibitory effect of PAO on eNOS activity were prevented by dithiothreitol, a disulfide-reducing agent. These results indicate that (i) PAO directly inhibits eNOS activity in endothelial cells by binding to thiol groups in the eNOS protein and (ii) results of studies using PAO to assess the role of protein tyrosine phosphorylation in regulating eNOS activity must be interpreted with great caution. Copyright (C) 2000 Elsevier Science Inc.

AB - The role of protein tyrosine phosphorylation during regulation of NO synthase (eNOS) activity in endothelial cells is poorly understood. Studies to define this role have used inhibitors of tyrosine kinase or tyrosine phosphatase (TP). Phenylarsine oxide (PAO), an inhibitor of TP, has been reported to bind thiol groups, and recent work from our laboratory demonstrates that eNOS activity depends on thiol groups at its catalytic site. Therefore, we hypothesized that PAO may have a direct effect on eNOS activity. To test this, we measured (i) TP and eNOS activities both in total membrane fractions and in purified eNOS prepared from porcine pulmonary artery endothelial cells and (ii) sulfhydryl content and eNOS activity in purified bovine aortic eNOS expressed in Escherichia coli. High TP activity was detected in total membrane fractions, but no TP activity was detected in purified eNOS fractions. PAO caused a dose-dependent decrease in eNOS activity in total membrane and in purified eNOS fractions from porcine pulmonary artery endothelial cells, even though the latter had no detectable TP activity. PAO also caused a decrease in sulfhydryl content and eNOS activity in purified bovine eNOS. The reduction in eNOS sulfhydryl content and the inhibitory effect of PAO on eNOS activity were prevented by dithiothreitol, a disulfide-reducing agent. These results indicate that (i) PAO directly inhibits eNOS activity in endothelial cells by binding to thiol groups in the eNOS protein and (ii) results of studies using PAO to assess the role of protein tyrosine phosphorylation in regulating eNOS activity must be interpreted with great caution. Copyright (C) 2000 Elsevier Science Inc.

KW - Endothelial cell

KW - Free radicals

KW - Nitric oxide synthase

KW - Phenylarsine oxide

KW - Pulmonary

UR - http://www.scopus.com/inward/record.url?scp=0033960738&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0033960738&partnerID=8YFLogxK

U2 - 10.1016/S0891-5849(99)00231-2

DO - 10.1016/S0891-5849(99)00231-2

M3 - Article

VL - 28

SP - 167

EP - 173

JO - Free Radical Biology and Medicine

JF - Free Radical Biology and Medicine

SN - 0891-5849

IS - 2

ER -