Research reportAgmatine suppresses nitric oxide production in microglia
Introduction
Nitric oxide (NO) functions as an intercellular messenger in the immune, cardiovascular and central nervous system [39]. NO is enzymatically formed from a terminal guanidino-nitrogen of l-arginine by the NO synthase (NOS) [25]. Among isoforms of NOS, the macrophage-type, inducible NOS (iNOS) is expressed only when the cells are exposed to a bacterial endotoxin lipopolysaccharide (LPS) or cytokines such as interferon-γ, tumor necrosis factor-α and interleukin-1. In the brain, astrocytes and microglia are known to express iNOS [11], [21], [29], [38]. The production of NO is higher in microglia than in astrocytes [8], [15], [20]. Furthermore, NO produced by glial cells has been implicated in the neuropathogenesis of various diseases, including Gram-negative bacterial meningitis, multiple sclerosis, acquired immunodeficiency syndrome, Parkinson’s disease, Huntington’s disease and Alzheimer’s disease [7], [9], [26], [37], [41]
Agmatine is an endogenous substance synthesized from arginine by arginine decarboxylase [22], [23], [28], and is present in the brain of mammals including the rat, bovine [22] and human [24], [32]. The level of agmatine in rat brain has been reported as 0.2–0.4 μg/g tissue by mass spectroscopy [22] and as 0.331–1.105 μg/g tissue by high-performance liquid chromatography [10]. The presence of agmatine in astrocytes and neurons has been demonstrated by immunohistochemical examination with anti-agmatine antibody [30], [33]. Electron microscopic examination has demonstrated that agmatine is present in axon terminals associated with synaptic vesicles [30], [35]. In addition, agmatine is released from synaptosomes or brain slice in response to depolarizing stimuli [34] and is taken up into synaptosomes via a Na+-independent transport system [36]. These observations suggest that agmatine functions as a neurotransmitter or neuromodulator in the brain.
Since agmatine is structurally analogous to the NOS substrate l-arginine, it is possible that agmatine modulates the production of NO. Indeed, agmatine has been reported to inhibit the activity of iNOS, but not constitutive NOS, in rat aorta [5], to inhibit the activity of NOS purified from brain, macrophages and endothelial cells [12], or to activate NOS in endothelial cells [27]. However, the impact of agmatine on microglial NO production was unknown. Therefore, in the present study, we investigated its effect on iNOS expression and NO production by using cultured microglia obtained from the cerebral cortex of neonatal rats.
Section snippets
Chemicals and antibodies
Agmatine sulfate was purchased from Research Biochemicals Inc. (Natick, MA, USA). Amyloid β protein fragment 1–40 (Aβ1-40) was purchased from Bachem Inc. (Torrance, CA, USA) and dissolved in distilled water, as described previously [19]. Recombinant rat interferon-γ was purchased from Gibco-BRL (Gaithersburg, MD, USA). Rabbit anti-iNOS antibody was purchased from Affinity BioReagents, Inc. (Golden, CO, USA). Diphenyleneiodonium chloride (DPI) and horseradish peroxidase-conjugated anti-rabbit
Effects of agmatine on viability of microglia and neurons
Agmatine (1–300 μM) had no effect on the viability of cultured microglia (Fig. 1A) and cultured rat cortical neurons (Fig. 1B).
Effect of agmatine on NO production in microglia
Neither iNOS expression or nitrite accumulation was detected in intact microglial cultures. Addition of LPS (1 μg/ml) induced iNOS expression and nitrite accumulation in microglia cultures. The LPS-induced iNOS expression occurred within 12 h and reached a peak at 24 h (Fig. 2A), while the nitrite accumulation increased linearly with time (Fig. 2B). Agmatine had no
Discussion
In the present study, we found that agmatine does not affect cell viability and suppresses LPS-induced NO production in cultured microglia. Furthermore, we employed co-cultures of neuron and microglia, and demonstrated that agmatine was effective in attenuating the increase of NO production and the decrease of neuron viability induced by a combination of microglia and LPS. The potency of agmatine in preventing the neuronal death was very consistent with that in suppressing the NO production in
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