Low Environmental pH Is Responsible for the Induction of Nitric-oxide Synthase in Macrophages

Abstract

Stimulation of macrophages with endotoxin and/or cytokines is responsible for the expression of the inducible isoform of nitric oxide synthase (iNOS). Because macrophages are exposed to low pH within the microenvironment of inflammatory lesions, the potential role of acidic pH as an additional regulator of iNOS was investigated. Substitution of the culture medium of rat peritoneal macrophages at pH 7.4 with medium at pH 7.0 up-regulated iNOS activity, as reflected by a 2.5-fold increase in nitrite accumulation. The increase in iNOS activity was associated with a similar increase in iNOS mRNA expression that reflected an increase in iNOS mRNA synthesis rather than stability. Low environmental pH-induced iNOS gene transcription involved the activation of nuclear factor-B (NF-B) transcription factor since exposure of macrophages to low environmental pH both increased NF-B binding activity in the nucleus and enhanced NF-B-driven reporter gene expression. In addition, treatment of macrophages with pyrrolidine dithiocarbamate or n-acetyl-leucinyl-leucinyl-norleucinal, two drugs preventing NF-B translocation to the nucleus, canceled low pH-induced nitrite accumulation. The overall mechanism required the synthesis of tumor necrosis factor ␣ (TNF␣). Indeed, 1) elevated TNF␣ bioactivity was observed in the medium of macrophages exposed to pH 7.0, and 2) incubation of macrophages with a neutralizing anti-TNF␣ antibody impaired both NF-B activation and nitrite accumulation in response to acid challenge. In summary, exposure of macrophages to acidic microenvironment in inflammatory lesions leads to the up-regulation of iNOS activity through the activation of NF-B.Acidosis is a hallmark of both ischemia and inflammation processes. The decrease of pH in tissue ischemia is secondary to the release of H ϩ during ATP hydrolysis and to the accumulation of CO 2 (1). The acidic environment in inflammatory lesions and abscesses (2) is due to increased metabolic acid generation during cell activation. This originates primarily from the hexose monophosphate shunt, by the dissociation of hydrated CO 2 (3).In most cases, acidosis occurs along with nitric oxide (NO) 1 generation. In ischemia, NO generation is due in one part to the acidification and reduction of the large pool of nitrite present within the tissue (4). In inflammatory processes, macrophage exposure to bacterial lipopolysaccharide (LPS) or cytokines such as tumor necrosis factor ␣ (TNF␣) and interferon-␥ (IFN-␥) causes the expression of the inducible isoform of NO synthase (NOS II or iNOS) that is responsible for high output production of NO (5). The expression of iNOS is regulated mainly at the transcriptional level. Analyses of the murine iNOS promoter have shown the presence of numerous consensus sequences for the binding of transcription factors (6, 7), of which nuclear factor-B (NF-B) (8), interferon regulatory factor-1 (IRF-1) (9), and signal transducer and activator of transcription (STAT) 1␣ (10) are functionally important for iNOS induction. NF-B is composed of a p...

Somatostatin Increases Glucocorticoid Binding and Signaling in Macrophages by Blocking the Calpain-specific Cleavage of Hsp 90

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Acidic Extracellular pH Induces Matrix Metalloproteinase-9 Expression in Mouse Metastatic Melanoma Cells through the Phospholipase D-Mitogen-activated Protein Kinase Signaling

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