Nitric oxide differentially regulates induction of type II nitric oxide synthase in rat vascular smooth muscle cells versus macrophages

Hanfang Zhang, Connie Snead, John D. Catravas

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

We studied effects of nitric oxide (NO) released by different NO donors on induction of inducible NO synthase (iNOS) in rat aortic smooth muscle cells (RASMC) and rat macrophage cell line NR8383. iNOS protein expression induced by a CM (interleukin-1β 250 U/mL, interferon-γ 150 U/mL, and tumor necrosis factor-α 150 U/mL) was not affected by the NO donor SNAP (0.2 to 1 mmol/L) in RASMC at 24 hours of incubation but was dose-dependently decreased by SNAP in macrophages (maximal 60% inhibition). A fully functional -3.2-kb rat iNOS promoter was transfected into RASMC and macrophages. The CM-induced promoter activity in transfected macrophages was inhibited by SNAP (maximal 67% inhibition), but this inhibitory effect by SNAP was not observed in transfected RASMC. Electrophoretic mobility-shift assays demonstrated that nuclear factor-κB (NF-κB) binding patterns were different in 2 cell types and that the ratio of p50:p65 subunits was significantly lower in macrophages than in RASMC. Furthermore, NF-κB activity was not affected by SNAP in RASMC but was reduced by SNAP in macrophages. Another putative NO donor, NOR3 (1 mmol/L), completely inhibited iNOS induction by CM in RASMC, but this was accompanied by severe cytotoxicity, which resulted in cell death. Similar concentrations of SNAP did not exhibit cytotoxicity in RASMC, whereas macrophages demonstrated 88% viability compared with cells without SNAP. NO synthase inhibitor Ng-monomethyl-L-arginine significantly inhibited CM-induced nitrite production in both cell types and stimulated iNOS protein expression in macrophages but did not affect iNOS expression in RASMC. These data strongly suggest that NO may affect transcriptional regulation of iNOS differently in RASMC versus macrophages, possibly by means of regulation of NF-κB activation.

Original languageEnglish (US)
Pages (from-to)529-535
Number of pages7
JournalArteriosclerosis, thrombosis, and vascular biology
Volume21
Issue number4
DOIs
StatePublished - Jan 1 2001

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Nitric Oxide Synthase Type II
Vascular Smooth Muscle
Smooth Muscle Myocytes
Nitric Oxide
Macrophages
Nitric Oxide Synthase
Nitric Oxide Donors
Electrophoretic Mobility Shift Assay
Nitrites
Interleukin-1
Interferons
Arginine
Proteins
Cell Death
Tumor Necrosis Factor-alpha

Keywords

  • Gene induction
  • Macrophage
  • Muscle, smooth
  • Nitric oxide donors
  • Nitric oxide synthase
  • Nuclear factor-κB

ASJC Scopus subject areas

  • Cardiology and Cardiovascular Medicine

Cite this

Nitric oxide differentially regulates induction of type II nitric oxide synthase in rat vascular smooth muscle cells versus macrophages. / Zhang, Hanfang; Snead, Connie; Catravas, John D.

In: Arteriosclerosis, thrombosis, and vascular biology, Vol. 21, No. 4, 01.01.2001, p. 529-535.

Research output: Contribution to journalArticle

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abstract = "We studied effects of nitric oxide (NO) released by different NO donors on induction of inducible NO synthase (iNOS) in rat aortic smooth muscle cells (RASMC) and rat macrophage cell line NR8383. iNOS protein expression induced by a CM (interleukin-1β 250 U/mL, interferon-γ 150 U/mL, and tumor necrosis factor-α 150 U/mL) was not affected by the NO donor SNAP (0.2 to 1 mmol/L) in RASMC at 24 hours of incubation but was dose-dependently decreased by SNAP in macrophages (maximal 60{\%} inhibition). A fully functional -3.2-kb rat iNOS promoter was transfected into RASMC and macrophages. The CM-induced promoter activity in transfected macrophages was inhibited by SNAP (maximal 67{\%} inhibition), but this inhibitory effect by SNAP was not observed in transfected RASMC. Electrophoretic mobility-shift assays demonstrated that nuclear factor-κB (NF-κB) binding patterns were different in 2 cell types and that the ratio of p50:p65 subunits was significantly lower in macrophages than in RASMC. Furthermore, NF-κB activity was not affected by SNAP in RASMC but was reduced by SNAP in macrophages. Another putative NO donor, NOR3 (1 mmol/L), completely inhibited iNOS induction by CM in RASMC, but this was accompanied by severe cytotoxicity, which resulted in cell death. Similar concentrations of SNAP did not exhibit cytotoxicity in RASMC, whereas macrophages demonstrated 88{\%} viability compared with cells without SNAP. NO synthase inhibitor Ng-monomethyl-L-arginine significantly inhibited CM-induced nitrite production in both cell types and stimulated iNOS protein expression in macrophages but did not affect iNOS expression in RASMC. These data strongly suggest that NO may affect transcriptional regulation of iNOS differently in RASMC versus macrophages, possibly by means of regulation of NF-κB activation.",
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