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Published on: September 26, 2015
Ethanol-induced modulation of inducible nitric-oxide synthase activity in human A172 astrocytoma cells
Randall L Davis1, Janet Dertien, Peter J Syapin
1Alchohol and Brain Research Laboratory, Department of Pharmacology, Texas Tech University Health Sciences Center, Lubbock, Texas 79430, USA.
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-100beta. The ability of A172 cells to express iNOS was assessed by stimulating cells with interferon-gamma (IFNgamma), tumor necrosis factor-alpha (TNFalpha), interleukin-1beta (IL-1beta), 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 [IFNgamma (100 ng/ml) + TNFalpha (30 ng/ml) + IL-1beta (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.
