Agmatine suppresses nitric oxide production in microglia

Brain Research 872 (2000) 141–148 www.elsevier.com / locate / bres

Research report

Agmatine suppresses nitric oxide production in microglia Kazuho Abe*, Yuzuru Abe, Hiroshi Saito Department of Chemical Pharmacology, Faculty of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113 -0033, Japan Accepted 16 May 2000

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 mM) did not affect viability of cultured microglia. Activation of microglia by lipopolysaccharide (LPS, 1 mg / 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-g (500 U / ml) and amyloid b protein (10 mM). 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.  2000 Elsevier Science B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters, and receptors Topic: Other neurotransmitters Keywords: Agmatine; Nitric oxide; Inducible nitric oxide synthase; Microglia; Neurotoxicity; Culture

1. 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 macrophagetype, inducible NOS (iNOS) is expressed only when the cells are exposed to a bacterial endotoxin lipopolysaccharide (LPS) or cytokines such as interferon-g, tumor necrosis factor-a 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] *Corresponding author. Fax: 181-3-5498-5787. E-mail address: [email protected] (K. Abe)

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 mg / g tissue by mass spectroscopy [22] and as 0.331–1.105 mg / g tissue by highperformance 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 1 -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

0006-8993 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 00 )02517-8

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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.

2. Materials and methods

2.1. Chemicals and antibodies Agmatine sulfate was purchased from Research Biochemicals Inc. (Natick, MA, USA). Amyloid b protein fragment 1–40 (Ab1-40) was purchased from Bachem Inc. (Torrance, CA, USA) and dissolved in distilled water, as described previously [19]. Recombinant rat interferon-g 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 IgG antibody were from Sigma Chemical Co. (St. Louis, MO, USA). LPS (Escherichia coli 026:B6) and other chemicals were from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

2.3. Neuron culture Primary culture of neurons was prepared from the cerebral cortices of 18-day-old embryos of Wistar rats as described previously [1,2]. Briefly, dissociated cortical cells were suspended in a serum-free modified Eagle’s medium containing N1 supplements (5 mg / ml human transferrin, 5 mg / ml bovine insulin, 20 nM progesterone, 100 mM putrescine and 30 nM sodium selenite) and seeded on polylysine-coated 24-well culture plates at a density of 200 000 cells / cm 2 . No mitotic inhibitors were required in these minimal conditions, and .95% of the cells were neuronal as defined by staining with microtubule-associated protein-2. The culture medium was changed to a fresh medium 24 h after the plating, and the cells were cultured for a further 48 h until each assay.

2.4. Co-culture of neuron and microglia Microglia were collected from mixed glial cultures as described above, and seeded onto the microporous membranes of cell culture inserts (for 24-well microtiter plates, 3.0-mm pore, 0.31-cm 2 area; Falcon) with a modified Eagle’s medium containing 10% fetal bovine serum 24 h before the experiment. The cell culture inserts without or with microglia were set in each well of neuron cultures. Test reagents were added in the cell culture inserts. Fortyeight hours after, neuron viability and the concentration of nitrite in neuron culture supernatant were determined.

2.2. Microglia culture 2.5. Determination of cell viability Mixed glial cultures were prepared from the cerebral cortices of 2-day-old neonates of Wistar rats as described previously [4]. Briefly, dissociated cortical cells were suspended in a modified Eagle’s medium containing 30 mM glucose, 2 mM glutamine, 1 mM pyruvate and 10% fetal bovine serum, and plated on uncoated 25 cm 2 flasks at a density of 600 000 cells / cm 2 . The culture medium was changed 24 h after the plating and then every 3–4 days. The cells were grew and became confluent after 10–14 days. Microglial cells were isolated from this mixed glial cultures by the method of Baker and Giulian [6]. The flasks were shaken by an orbital shaker (200 rpm) for 24 h at 378C. Floating cells in the supernatant were plated on an uncoated plastic dish, and left for 30 min at 378C. The dish was rinsed three times with culture medium. Adhered cells were collected with a cell scraper (Nalge Nunc International, Rochester, NY, USA), and suspended in modified Eagle’s medium containing 10% fetal bovine serum. The cells were reseeded on uncoated 96-well or 48-well microtiter plates at a density of 70 000 cells / cm 2 . In this culture, more than 98% of the cells were identified as microglia by immunohistochemical examination with antibodies to the microglia marker Mac-1. Twenty-four hours after the plating, test reagents were added to culture medium.

Cell viability was quantitated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay as described previously [3]. Briefly, the cells were incubated with 0.5 mg / ml MTT for 1 h at 378C and then solubilized by adding a solution containing 50% dimethylformamide and 20% sodium dodecyl sulfate (pH 4.7). The amount of MTT formazan produced was determined by measuring its absorbances at a test wavelength of 570 nm and a reference wavelength of 655 nm. By microscopic examination we confirmed that decrease in MTT reduction was parallel to cell death in all cases.

2.6. Measurement of nitrite accumulation The concentration of nitrite, a stable oxidation product of NO, in the culture medium was determined by a colorimetric method with the Griess reagent [40]. Fifty ml of culture supernatant was transferred to 96-well microtiter plates, and mixed with 50 ml of the Griess reagent (0.1% N-[1-naphthyl]ethylenediamine dihydrochloride and 1% sulfanilamide in 2% phosphate solution). After the reaction had taken place for 10 min at room temperature, the absorbance was measured with a microplate reader at a test wavelength of 540 nm and a reference wavelength of 655

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nm. The standard curve was constructed with the culture medium containing known concentrations of sodium nitrite, and employed to calculate the concentration of nitrite in samples.

2.7. Western blot analysis of iNOS expression Microglial cultures were rinsed twice with ice-cold phosphate-buffered saline (PBS), and the cells were lysed in a buffer containing 2% sodium dodecyl sulfate, 10% glycerol, 50 mM dithiothreitol and 0.1% bromophenol blue. The samples were subjected to 7% sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed by transfer onto a polyvinylidine difluoride membrane filter (Bio-Rad Laboratories, Hercules, CA, U.S.A.) at 1 mA / cm 2 for 90 min at room temperature. The filters were incubated in PBS containing 0.5% Tween 20 (PBS-T) and 1% bovine serum albumin for 1 h at room temperature and then with anti-iNOS antibody (1:2000 dilution in PBS-T) overnight at 48C. The filters were washed in PBS-T (3310 min) at room temperature and further incubated with horseradish peroxidase-conjugated anti-rabbit IgG (1:5000 dilution in PBS-T) for 1 h at room temperature. The blots were washed in PBS-T (3310 min), and immunoreactive proteins were visualized on a film with an enhanced chemiluminescence kit (NEN Life Science Products, Inc., Boston, MA, USA). Optical density on the film was measured with a computer imaging system (Imaging Technology, Inc., Ontario, Canada), and protein level was estimated from the standard curve, which was constructed using several dilutions of samples in each analysis. Molecular size was estimated with molecular mass markers (7.1–209 kDa; Bio-Rad Laboratories, Hercules, CA, USA).

Fig. 1. Effects of agmatine on the viability of cultured rat cortical microglia (A) and cultured rat cortical neurons (B). The cells were exposed to 1–300 mM agmatine for 3 days. Cell viability was quantitated by the MTT reduction assay, and expressed as percentage of the control (no addition of agmatine). The data are represented as the means6S.E.M. of the determinations on five separate cultures.

3.2. Effect of agmatine on NO production in microglia 2.8. Detection of cellular proteins with silver staining The samples were prepared as above, and subjected to 8 or 13% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Proteins on the gel were visualized with a silver staining kit (2D-Silver Stain II ‘Daiichi’; Daiichi Pure Chemicals Co., Ltd., Tokyo, Japan), according to the manufacturer’s instruction.

3. Results

3.1. Effects of agmatine on viability of microglia and neurons Agmatine (1–300 mM) had no effect on the viability of cultured microglia (Fig. 1A) and cultured rat cortical neurons (Fig. 1B).

Neither iNOS expression or nitrite accumulation was detected in intact microglial cultures. Addition of LPS (1 mg / 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 effect on the LPS-induced iNOS expression (Figs. 2A and 3A), but significantly suppressed the LPS-induced nitrite accumulation (Fig. 2B). The suppressive effect of agmatine on nitrite accumulation was concentration dependent in the range of 1–300 mM (Fig. 3B). Agmatine (1–300 mM) alone did not induce iNOS expression or nitrite accumulation in microglia cultures (Fig. 3). To examine if agmatine affects other events induced by LPS, we also checked expression of cellular proteins by silver staining. There was no apparent difference in expression pattern of cellular proteins between intact and agmatine-treated cultures (Fig. 4, lanes

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Fig. 2. Time course of LPS-induced iNOS expression and NO production in the absence or presence of agmatine in cultured rat cortical microglia. The cells were exposed to none (s), 300 mM agmatine (d), 1 mg / ml LPS (n) or 1 mg / ml LPS 1 300 mM agmatine (m) for 12–72 h. (A) The level of iNOS was estimated by Western blot analysis, and normalized by the value of the group treated with LPS alone for 24 h. Representative blots are shown as inset. C: immediately before addition of drugs, L: LPS alone, LA: LPS 1 agmatine. (B) The concentration of nitrite in culture supernatants was measured by using the Griess reaction. All data are represented as the means6S.E.M. of five separate observations. *P,0.01 vs. LPS alone, Duncan’s multiple range test.

1–4). LPS induced changes in the expression level of cellular proteins, especially 210-, 130-, 34- and 33-kDa proteins (Fig. 4, bands I–IV). The 210-kDa protein and the 130-kDa protein, probably iNOS, were not detected in intact microglia, but appeared in LPS-treated microglia (Fig. 4A). The 34-kDa and 33-kDa proteins were slightly detected in intact microglia, and their levels were increased by treatment with LPS (Fig. 4B). Agmatine had no effect on the LPS-induced expression of these proteins (Fig. 4A, B).

Fig. 3. Concentration-dependent effect of agmatine on LPS-induced iNOS expression and NO production in cultured rat cortical microglia. The cells were exposed to none (s), 1–300 mM agmatine (d), 1 mg / ml LPS (n) or 1 mg / ml LPS 1 1–300 mM agmatine (m) for 36 h. (A) The level of iNOS was estimated by Western blot analysis, and normalized by the value of the group treated with LPS alone. (B) The concentration of nitrite in culture supernatants was measured by using Griess reaction. Data are the means6S.E.M. of five separate observations.

To examine if agmatine affects NO production in microglia stimulated with endogenous substances, we also employed interferon-g and Ab. Neither interferon-g (500 U / ml) nor Ab1-40 (10 mM) induced significant increase in the level of nitrite in culture medium by itself (data not shown, n55), but a combination of interferon-g and Ab clearly induced nitrite accumulation, consistent with previous reports [13,16,18,31]. Agmatine significantly suppressed the nitrite accumulation induced by a combination of interferon-g and Ab (Fig. 5). The effect of agmatine against interferon-g and Ab was comparable to that against LPS.

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Fig. 4. Effects of LPS and agmatine on the expression of cellular proteins in cultured rat cortical microglia. The cells were exposed to none (lane 1), agmatine (3, 30 and 300 mM; lanes 2, 3 and 4, respectively), 1 mg / ml LPS (lane 5) or 1 mg / ml LPS 1 agmatine (3, 30 and 300 mM; lanes 6, 7 and 8, respectively) for 36 h. The cell extract was subjected to 8% (upper) or 13% (lower) sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Proteins on the gel were visualized by silver staining.

3.3. Effect of agmatine in co-cultures of neuron and microglia It is supposed that excessive NO production by reactive microglia leads to neuronal death. To test the possibility that agmatine protects neurons from toxicity of microgliaderived NO, we employed co-cultures of neuron and microglia. Cell culture inserts without or with microglia were set in culture wells containing neurons. Insertion of

intact microglia or addition of LPS alone did not affect nitrite accumulation and neuron viability (Fig. 6B). When microglia and LPS were added together, nitrite accumulation was increased, and neuron viability was decreased (Fig. 6B). The nitrite accumulation and neuronal death induced by a combination of microglia and LPS were both blocked by 1 mM DPI, an irreversible NOS inhibitor (Fig. 6B). DPI (1 mM) alone had no effect on neuron viability (data not shown, n55). Agmatine significantly attenuated

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Fig. 5. Effect of agmatine on NO production induced by a combination of interferon-g and Ab1-40 in cultured rat cortical microglia. The cells were exposed to none (s), 1–300 mM agmatine (d), 500 U / ml interferon-g 1 10 mM Ab1-40 (n) or 500 U / ml interferon-g 1 10 mM Ab1-40 1 1–300 mM agmatine (m) for 36 h. The concentration of nitrite in culture supernatants was measured by using the Griess reaction. Data are the means6S.E.M. of five separate observations.

the increase of nitrite accumulation and the decrease of neuron viability induced by a combination of microglia and LPS (Fig. 6C).

4. 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 microglia. Agmatine had no effect on neuron viability in the absence of activated microglia. These data suggest that agmatine is effective in protecting neurons from toxicity of NO derived from activated microglia. Although the mechanism by which agmatine suppresses NO production remains to be elucidated, several possibilities can be argued. First, agmatine had no effect on the expression of iNOS or other cellular proteins in microglia, suggesting that agmatine does not affect signal transduction mechanisms from LPS stimulation to cellular protein expression. Second, agmatine was effective in suppressing the production of NO induced by interferon-g and Ab as well as by LPS, suggesting that agmatine suppresses a mechanism common to NO production induced by LPS, interferon-g and Ab. Agmatine is likely to suppress the production of NO by inhibiting iNOS activity in microglia. There are discrepancies among previous reports demon-

Fig. 6. Effects of agmatine on NO production and neuron viability in co-cultures of rat cortical neurons and microglia. (A) Experimental procedures. Cell culture inserts without or with microglia were set in each well of neuron cultures. Test reagents were added in the cell culture inserts (B,C) Quatitative data. N 1 ins: Cell culture insert without microglia was set in neuron culture. N 1 ins / Mic: Cell culture insert with microglia was set in neuron culture. N 1 ins 1 LPS: Cell culture insert without microglia was set in neuron culture, and 1 mg / ml LPS was added. N 1 ins / Mic 1 LPS: Cell culture insert with microglia was set, and 1 mg / ml LPS was added. N 1 ins / Mic 1 LPS 1 DPI: Cell culture insert with microglia was set, and 1 mg / ml LPS and 1 mM DPI were added. N 1 ins / Mic 1 LPS 1 agmatine: Cell culture insert with microglia was set, and 1 mg / ml LPS and 1–100 mM agmatine were added. Nitrite concentration in culture supernatants (white columns) was determined by the Griess reaction. Neuron viability (gray columns) was determined by the MTT reduction assay, and expressed as percentage of the control (N 1 ins). Data are the means6S.E.M. of five separate observations. *P,0.01 vs. N 1 ins, [P,0.01 vs. N 1 ins / Mic 1 LPS; Duncan’s multiple range test.

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strating possible interaction of agmatine with NOS. Auguet et al. [5] found that agmatine inhibited the activity of iNOS, but not constitutive NOS, in rat aorta. Galea et al. [12] employed NOS isoforms I, II and III purified from brain, macrophages and endothelial cell, and demonstrated that agmatine works as a competitive NOS inhibitor for all of the three NOS isoforms. Morrissey and Klahr [27] observed that agmatine stimulated the production of NO in endothelial cells, probably by activating NOS. The impact of agmatine on the NO production system may be different with experimental conditions or cell types. The effective concentration of agmatine in suppressing microglial NO production was 1–300 mM. The level of agmatine in rat brain has been reported 0.2–0.4 mg / g tissue by mass spectroscopy [22] and 0.331–1.105 mg / g tissue by high-performance liquid chromatography [10]. On the assumption that 1 g of wet weight of tissue corresponds to 1 ml of water, average concentration of agmatine in the brain is estimated at 1.5–8.5 mM. Since agmatine is released from the cells in response to depolarizing stimuli [34], local concentration of agmatine may be higher than these values. Furthermore, it has been reported that the activity of the agmatine-synthesizing enzyme arginine decarboxylase increases during brain development or after ischemia [14] or that plasma agmatine concentrations are elevated in patients with depression [17]. In addition, the Ki value of agmatine for iNOS purified from macrophages has been reported 220 mM [12]. Therefore, the modulation of microglial NO production by agmatine is likely to occur in vivo. In conclusion, agmatine may function as a neuroprotective agent in a pathological condition in which microglia are activated. We are planning in vivo experiments to test this hypothesis.

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