Abstract

Binding of idazoxan (IDA) to imidazoline receptors of the I₂ subtype in astrocytes influences astroglial gene expression as evidenced by increased expression of glial fibrillary acidic protein and mRNA. To determine whether IDA affected glial inflammatory gene expression, we tested the effects of IDA on astroglial nitric oxide synthase type-2 (NOS-2) expression. NOS-2 was induced in primary rat astrocytes and C6 glioma cells by incubation with 1 μg/ml lipopolysaccharide (LPS) plus three cytokines (tumor necrosis factor-α, interleukin-1β, and interferon-γ) or three cytokines alone. Cells were incubated with 1–100 μM IDA, and at 24 h NOS-2 expression assessed. In astrocytes and C6 cells, preincubation with IDA dose-dependently inhibited nitrite accumulation (IC₅₀ ∼25 μM), accompanied by a reduction in NOS-2 protein levels and l-citrulline synthesis activity in cell lysates. IDA also inhibited nitrite production in LPS stimulated RAW 264.7 macrophages. In astrocytes, but not C6 cells, longer preincubation times with IDA yielded significantly greater suppression, and maximal suppression (>90%) was achieved after a 8 h preincubation in 100 μM IDA. The degree of inhibition was diminished whether IDA was added after LPS plus cytokine mixture. In contrast to NE, continuous incubation with IDA was required to achieve suppression. IDA reduced induction of NOS-2 protein levels, steady state NOS-2 mRNA levels, and activity of a NOS-2 promoter construct stably transfected in C6 cells. These results show that IDA inhibits NOS-2 activity and protein expression in glial cells and macrophages, and suggest that this occurs by decreasing transcription from the NOS-2 promoter.