Agmatine suppresses nitric oxide production in microglia

Abstract

We investigated the effect of agmatine, an arginine metabolite synthesized in the brain, in cultured microglia obtained from neonatal rat cerebral cortex. Agmatine (1–300 μM) did not affect viability of cultured microglia. Activation of microglia by lipopolysaccharide (LPS, 1 μg/ml) caused the expression of inducible nitric oxide synthase (iNOS) and the production of nitric oxide (NO) assessed as the accumulation of nitrite in the culture supernatants. Agmatine had no effect on the expression of iNOS, but significantly suppressed the LPS-induced NO production in a concentration-dependent manner. Agmatine was also effective in suppressing the production of NO induced by a combination of interferon-γ (500 U/ml) and amyloid β protein (10 μM). In co-cultures of rat cortical neurons and microglia, LPS caused significant loss of neuron viability. The LPS neurotoxicity was not observed in the absence of microglia, and was completely blocked by the NOS inhibitor diphenyleneiodoium chloride. The neuronal death induced by microglia-derived NO was significantly attenuated by the presence of agmatine. These results suggest that agmatine works to protect neurons by inhibiting the production of NO 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.