Ethanol-induced modulation of inducible nitric-oxide synthase activity in human A172 astrocytoma cells

Randall Davis, Janet Dertien, Peter J. Syapin

Research output: Contribution to journalArticle

35 Citations (Scopus)

Abstract

Background: Glial cells are critical in the functioning of the central nervous system (CNS), including responsiveness to injury and immunocompetence. The immune and inflammatory response involves the inducible form of nitric-oxide synthase (iNOS), and subsequent nitric oxide (NO) production. Previously, we have demonstrated that ethanol inhibits cytokine-induced iNOS expression and activity in rat glial cells. Evidence of ethanol-induced effects on iNOS in human glial cells is nonexistent. Herein, the conditions necessary for significant iNOS induction in human A172 astrocytoma cells have been characterized, and subsequently, the effects of ethanol on iNOS expression have been investigated. Methods: A172 cells were analyzed immunohistochemically for the astrocyte markers, glial fibrillary acidic protein (GFAP) and S-100β. The ability of A172 cells to express iNOS was assessed by stimulating cells with interferon-γ (IFNγ), tumor necrosis factor-α (TNFα), interleukin-1β (IL-1β), bacterial lipopolysaccharide (LPS), L-arginine, and tetrahydrobiopterin (BH4) in various combinations. Following stimulation, iNOS induction was monitored via measurement of nitrite production and in vitro iNOS enzyme activity. Time-course (6-24 hr) studies assessed the effects of ethanol (50-200 mM) on iNOS induction. Results: Immunohistochemistry analysis confirmed that A172 cells were phenotypically, astrocytic. Induction of nitrite production by a cytomix [IFNγ (100 ng/ml) + TNFα (30 ng/ml) + IL-1β (5 ng/ml)] was differentially enhanced by exposure to supplemental factors including LPS, L-arginine, and BH4. Nitrite production was greatest over the initial 24 hr of stimulation with iNOS enzyme activity peaking at 12 hr. Acute (6-24 hr) exposure of activated cells to 50 mM ethanol enhanced iNOS activity recovered from the cytosol, whereas 200 mM ethanol decreased it. Ethanol had no direct effect on the catalytic activity of the enzyme. Conclusions: The present study is the first published report of ethanol-induced modulation of iNOS expression in human glial cells. The data suggest that ethanol is influencing iNOS enzyme levels most profoundly. Altered astrocyte function may be a point of ethanol-induced perturbation in CNS immune function. These findings should lend insight into the role of ethanol on human CNS immunity and brain injury.

Original languageEnglish
Pages (from-to)1404-1411
Number of pages8
JournalAlcoholism: Clinical and Experimental Research
Volume26
Issue number9
DOIs
StatePublished - 1 Sep 2002

Fingerprint

Astrocytoma
Nitric Oxide Synthase Type II
Human Activities
Nitric Oxide Synthase
Ethanol
Modulation
Neuroglia
Neurology
Nitrites
Central Nervous System
Enzyme activity
Enzymes
Interleukin-1
Astrocytes
Interferons
Lipopolysaccharides
Arginine
Tumor Necrosis Factor-alpha
Nervous System Trauma
Immunocompetence

Keywords

  • Alcohol
  • Cytokines
  • Immune Response
  • Neuroimmunity
  • S-100β

Cite this

@article{1014c8a48a92422ba692ac5c3f02ff50,
title = "Ethanol-induced modulation of inducible nitric-oxide synthase activity in human A172 astrocytoma cells",
abstract = "Background: Glial cells are critical in the functioning of the central nervous system (CNS), including responsiveness to injury and immunocompetence. The immune and inflammatory response involves the inducible form of nitric-oxide synthase (iNOS), and subsequent nitric oxide (NO) production. Previously, we have demonstrated that ethanol inhibits cytokine-induced iNOS expression and activity in rat glial cells. Evidence of ethanol-induced effects on iNOS in human glial cells is nonexistent. Herein, the conditions necessary for significant iNOS induction in human A172 astrocytoma cells have been characterized, and subsequently, the effects of ethanol on iNOS expression have been investigated. Methods: A172 cells were analyzed immunohistochemically for the astrocyte markers, glial fibrillary acidic protein (GFAP) and S-100β. The ability of A172 cells to express iNOS was assessed by stimulating cells with interferon-γ (IFNγ), tumor necrosis factor-α (TNFα), interleukin-1β (IL-1β), bacterial lipopolysaccharide (LPS), L-arginine, and tetrahydrobiopterin (BH4) in various combinations. Following stimulation, iNOS induction was monitored via measurement of nitrite production and in vitro iNOS enzyme activity. Time-course (6-24 hr) studies assessed the effects of ethanol (50-200 mM) on iNOS induction. Results: Immunohistochemistry analysis confirmed that A172 cells were phenotypically, astrocytic. Induction of nitrite production by a cytomix [IFNγ (100 ng/ml) + TNFα (30 ng/ml) + IL-1β (5 ng/ml)] was differentially enhanced by exposure to supplemental factors including LPS, L-arginine, and BH4. Nitrite production was greatest over the initial 24 hr of stimulation with iNOS enzyme activity peaking at 12 hr. Acute (6-24 hr) exposure of activated cells to 50 mM ethanol enhanced iNOS activity recovered from the cytosol, whereas 200 mM ethanol decreased it. Ethanol had no direct effect on the catalytic activity of the enzyme. Conclusions: The present study is the first published report of ethanol-induced modulation of iNOS expression in human glial cells. The data suggest that ethanol is influencing iNOS enzyme levels most profoundly. Altered astrocyte function may be a point of ethanol-induced perturbation in CNS immune function. These findings should lend insight into the role of ethanol on human CNS immunity and brain injury.",
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Ethanol-induced modulation of inducible nitric-oxide synthase activity in human A172 astrocytoma cells. / Davis, Randall; Dertien, Janet; Syapin, Peter J.

In: Alcoholism: Clinical and Experimental Research, Vol. 26, No. 9, 01.09.2002, p. 1404-1411.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Ethanol-induced modulation of inducible nitric-oxide synthase activity in human A172 astrocytoma cells

AU - Davis, Randall

AU - Dertien, Janet

AU - Syapin, Peter J.

PY - 2002/9/1

Y1 - 2002/9/1

N2 - Background: Glial cells are critical in the functioning of the central nervous system (CNS), including responsiveness to injury and immunocompetence. The immune and inflammatory response involves the inducible form of nitric-oxide synthase (iNOS), and subsequent nitric oxide (NO) production. Previously, we have demonstrated that ethanol inhibits cytokine-induced iNOS expression and activity in rat glial cells. Evidence of ethanol-induced effects on iNOS in human glial cells is nonexistent. Herein, the conditions necessary for significant iNOS induction in human A172 astrocytoma cells have been characterized, and subsequently, the effects of ethanol on iNOS expression have been investigated. Methods: A172 cells were analyzed immunohistochemically for the astrocyte markers, glial fibrillary acidic protein (GFAP) and S-100β. The ability of A172 cells to express iNOS was assessed by stimulating cells with interferon-γ (IFNγ), tumor necrosis factor-α (TNFα), interleukin-1β (IL-1β), bacterial lipopolysaccharide (LPS), L-arginine, and tetrahydrobiopterin (BH4) in various combinations. Following stimulation, iNOS induction was monitored via measurement of nitrite production and in vitro iNOS enzyme activity. Time-course (6-24 hr) studies assessed the effects of ethanol (50-200 mM) on iNOS induction. Results: Immunohistochemistry analysis confirmed that A172 cells were phenotypically, astrocytic. Induction of nitrite production by a cytomix [IFNγ (100 ng/ml) + TNFα (30 ng/ml) + IL-1β (5 ng/ml)] was differentially enhanced by exposure to supplemental factors including LPS, L-arginine, and BH4. Nitrite production was greatest over the initial 24 hr of stimulation with iNOS enzyme activity peaking at 12 hr. Acute (6-24 hr) exposure of activated cells to 50 mM ethanol enhanced iNOS activity recovered from the cytosol, whereas 200 mM ethanol decreased it. Ethanol had no direct effect on the catalytic activity of the enzyme. Conclusions: The present study is the first published report of ethanol-induced modulation of iNOS expression in human glial cells. The data suggest that ethanol is influencing iNOS enzyme levels most profoundly. Altered astrocyte function may be a point of ethanol-induced perturbation in CNS immune function. These findings should lend insight into the role of ethanol on human CNS immunity and brain injury.

AB - Background: Glial cells are critical in the functioning of the central nervous system (CNS), including responsiveness to injury and immunocompetence. The immune and inflammatory response involves the inducible form of nitric-oxide synthase (iNOS), and subsequent nitric oxide (NO) production. Previously, we have demonstrated that ethanol inhibits cytokine-induced iNOS expression and activity in rat glial cells. Evidence of ethanol-induced effects on iNOS in human glial cells is nonexistent. Herein, the conditions necessary for significant iNOS induction in human A172 astrocytoma cells have been characterized, and subsequently, the effects of ethanol on iNOS expression have been investigated. Methods: A172 cells were analyzed immunohistochemically for the astrocyte markers, glial fibrillary acidic protein (GFAP) and S-100β. The ability of A172 cells to express iNOS was assessed by stimulating cells with interferon-γ (IFNγ), tumor necrosis factor-α (TNFα), interleukin-1β (IL-1β), bacterial lipopolysaccharide (LPS), L-arginine, and tetrahydrobiopterin (BH4) in various combinations. Following stimulation, iNOS induction was monitored via measurement of nitrite production and in vitro iNOS enzyme activity. Time-course (6-24 hr) studies assessed the effects of ethanol (50-200 mM) on iNOS induction. Results: Immunohistochemistry analysis confirmed that A172 cells were phenotypically, astrocytic. Induction of nitrite production by a cytomix [IFNγ (100 ng/ml) + TNFα (30 ng/ml) + IL-1β (5 ng/ml)] was differentially enhanced by exposure to supplemental factors including LPS, L-arginine, and BH4. Nitrite production was greatest over the initial 24 hr of stimulation with iNOS enzyme activity peaking at 12 hr. Acute (6-24 hr) exposure of activated cells to 50 mM ethanol enhanced iNOS activity recovered from the cytosol, whereas 200 mM ethanol decreased it. Ethanol had no direct effect on the catalytic activity of the enzyme. Conclusions: The present study is the first published report of ethanol-induced modulation of iNOS expression in human glial cells. The data suggest that ethanol is influencing iNOS enzyme levels most profoundly. Altered astrocyte function may be a point of ethanol-induced perturbation in CNS immune function. These findings should lend insight into the role of ethanol on human CNS immunity and brain injury.

KW - Alcohol

KW - Cytokines

KW - Immune Response

KW - Neuroimmunity

KW - S-100β

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