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(Journal of Leukocyte Biology. 2000;68:260-266.)
© 2000 by Society for Leukocyte Biology

Combined inhibition of indoleamine 2,3-dioxygenase and nitric oxide synthase modulates neurotoxin release by interferon-{gamma}-activated macrophages

Alberto Chiarugi*, Persio Dello Sbarba{dagger}, Alessandro Paccagnini{dagger}, Sandra Donnini*, Sandra Filippi* and Flavio Moroni*

* Department of Preclinical and Clinical Pharmacology and
{dagger} Department of Experimental Pathology and Oncology, University of Florence, Italy

Correspondence: Dr. Flavio Moroni, Dipartimento di Farmacologia Preclinica e Clinica, Università di Firenze, Viale Pieraccini 6, 50139 Firenze, Italy. E-mail: moronif{at}ds.unifi.it


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We evaluated the synthesis of nitric oxide (NO) and of the neurotoxic kynurenine metabolites 3OH-kynurenine and quinolinic acid (QUIN) in interferon-{gamma} (IFN-{gamma})-activated macrophages of the murine BAC1.2F5 cell line with the aim of investigating the roles of mononuclear phagocytes in inflammatory neurological disorders. IFN-{gamma} induced indoleamine 2,3-dioxygenase (IDO) and NO synthase (NOS) and increased the synthesis of 3OH-kynurenine, QUIN, and NO that accumulated in the incubation medium where they reached neurotoxic levels. Macrophage exposure to norharmane, an IDO inhibitor, resulted in a decreased formation of not only the kynurenine metabolites but also NO. The inhibition of NO synthesis could not be ascribed to reduced NADPH availability or decreased NOS induction. Norharmane inhibited NOS activity also in coronary vascular endothelial cells and in isolated aortic rings. Our findings suggest that activated macrophages release large amounts of neurotoxic molecules and that norharmane may represent a prototype compound to study macrophage involvement in inflammatory brain damage.

Key Words: neuroinflammation • kynurenine pathway • nitric oxide • quinolinic acid • 3OH-kynurenine


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The concentration of potentially neurotoxic compounds, such as 3OH-kynurenine, 3-OH-anthranilic acid, and quinolinic acid (QUIN) that are formed along the metabolic pathway leading from tryptophan to NAD (the kynurenine pathway) significantly increases in blood and cerebrospinal fluid of patients affected by a number of inflammatory neurological disorders and in animal models of immune activation [1 , 2 ]. This increase seems to participate in the neurological damage found in AIDS-related dementia complex [3 4 5 ], global or focal brain ischemia [6 , 7 ], spinal cord traumatic injury [8 ], multiple sclerosis [9 , 10 ], and measles or malaria encephalitis [11 , 12 ].

Activated macrophages are an important source of the above-mentioned neurotoxic kynurenine metabolites [13 14 15 ]. In fact, exposure of macrophages to bacterial lipopolysaccharide or interferon-{gamma} (IFN-{gamma}) causes a rapid induction of indoleamine 2,3-dioxygenase (IDO; E.C1.13.11.42), the rate-limiting step enzyme of the kynurenine pathway [16 17 18 19 20 ]. The subsequent accumulation of 3OH-kynurenine and QUIN, an agonist of a subset of NMDA glutamate receptors, may cause neuronal death of either the excitotoxic or apoptotic type [21 22 23 24 ].

Activated macrophages also produce large amounts of nitric oxide (NO) via enhanced expression of the inducible NO-synthase isoform (iNOS) [25 ]. Excessive NO production may play a key role in the pathogenesis of the above-mentioned neurological diseases in which toxic kynurenine metabolites have been shown to accumulate [4 , 26 27 28 29 ]. It is interesting that iNOS is an NADPH-dependent enzyme, and its activity is essentially regulated by cofactor availability [30 , 31 ]. Hence, considering that pyridine nucleotides are the end products of tryptophan metabolism through the kynurenine pathway, it is reasonable to hypothesize that iNOS activity could be regulated by the IDO-dependent synthesis of NADP.

In this study, we tested the hypothesis that, in IFN-{gamma}-activated mononuclear phagocytes, IDO inhibition could decrease the release of neurotoxic kynurenines and NO. We observed that norharmane, an IDO inhibitor [13 , 32 ], decreased the activities of IDO and NOS and significantly reduced the production of the above-mentioned neurotoxic metabolites. These findings represent the basis for further studies on the involvement of 3OH-kynurenine, QUIN, and NO in brain damage in the course of inflammatory neurological disorders.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell and culture conditions
Macrophages of the murine BAC-1.2F5 cell line, which maintains many of the morphological and biochemical properties of tissue macrophages [33 ], were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 2 mM glutamine, 10% fetal bovine serum, antibiotics, and 6 ng/mL murine recombinant and affinity-purified macrophage colony-stimulating factor (M-CSF), which allowed the macrophages to maintain the molecular and functional properties of primary macrophages. Cells were activated by adding 50 units/mL IFN-{gamma} for 24 h before culture medium collection for the quantification of nitrites, 3OH-kynurenine, and QUIN.

Coronary vascular endothelial cells (CVEC) [34 ] were maintained in DMEM supplemented as above (without M-CSF) on gelatin-coated dishes. These cells exhibit a typical post-capillary venular morphology, and their origin was confirmed by immunofluorescence for a factor VIII-related antigen. BAC-1.2F5, and CVEC cells were cultured in a humidified incubator at 37°C under 5% CO2 in air.

Measurement of NO synthesis
The levels of NO accumulation in the culture medium of IFN-{gamma}-activated macrophages were measured using a microtiter plate version of the Greiss reaction [35 ]. Briefly, 100 µL of the medium was added to an equal volume of Greiss reagent (0.1% naphthylenediamine dihydrochloride in water/1% sulfanilamide in 5% phosphoric acid, w/v). After 10 min, the absorbance at 550 nm was measured by means of a Bio Rad microplate reader (model 350 UV). The amount of the nitrite formed was calculated from a calibration curve obtained using sodium nitrite as a standard.

The sensitivity of this approach was not sufficient to evaluate NO synthesis in coronary vascular endothelial cells and in this model we evaluated the amount of neoformed [3H]citrulline from L-[3H]arginine to monitor NOS activity [36 ]. Cells were incubated in HEPES buffer at 37°C for 20 min before the addition of 5 µCi of L-[3H]arginine plus 10 µM L-arginine for 30 min at 37°C. Cells were then washed with 10 mM HEPES at pH 5.5 containing 4 mM EDTA. The buffer was decanted and ethanol added to the cell monolayer and evaporating was allowed. The buffer was then added again (2 mL) and vigorously mixed for 2 min with 2 mL of Dowex AG50WX-8, and the mixture was loaded in a Pasteur pipette equipped with a glass-fiber filter and eluted with water. The radioactivity (corresponding to [3H]citrulline) contained in a 6-mL eluate was measured by liquid scintillation counting. Citrulline synthesis was expressed as counts per minute per milligram cell protein.

Determination of IDO and kynurenine hydroxylase activities
Macrophages were activated with 50 units/mL IFN-{gamma} for 24 h, then scraped and centrifuged at 3000 rpm for 5 min. The cell pellet was resuspended and sonicated in 0.1 M phosphate buffer pH 7.4 containing 1 mM phenylmethylsulfonyl fluoride, and 2 µg/mL aprotinin and leupeptin. The cell lysate was then centrifuged at 12,000 rpm for 10 min at 4°C. The supernatant was used as a cytosol preparation and the resuspended pellet as a crude mitochondria preparation.

IDO activity was evaluated according to Takikawa et al. [16 ]. The reaction mixture consisted of 50 µL of cytosol preparation and 50 µL of 0.1 M phosphate buffer, pH 6.5, containing 20 mM ascorbate, 50 µM methylene blue, 200 µg catalase, and 2 mM tryptophan. After 30 min of incubation at 37°C, the reaction was terminated with 100 µL of 20% (w/v) trichloroacetic acid (TCA), and the mixture centrifuged at 14,000 rpm for 5 min. Aliquots of the supernatant were injected into the high-performance liquid chromatography (HPLC) apparatus to measure kynurenine content.

Kynurenine hydroxylase activity was evaluated according to Carpenedo et al. [37 ]. The reaction mixture was as follows: 50 µL of crude mitochondria preparation and 50 µL of 0.1 M phosphate buffer, pH 8, containing 8 mM NADPH and 2 mM kynurenine. After 30 min of incubation at 37°C, the reaction was terminated with 100 µL of 20% (w/v) trichloroacetic acid, and the samples centrifuged at 14,000 rpm for 5 min. Aliquots of the supernatant were injected into the HPLC apparatus to measure 3OH-kynurenine content.

Measurement of kynurenine metabolites
Kynurenine was measured with HPLC and ultraviolet detection as described by Holmes [38 ]. Briefly, a reverse-phase column (Spherisorb S5 ODS2, 10 cm) and a mobile phase containing 0.1 mM ammonium acetate, 0.1 M acetic acid, and 2% acetonitrile, 1 mL/min flow rate were used. Kynurenine was detected at 365 nm with an ultraviolet detector (Perkin Elmer model LC 90).

3OH-kynurenine was measured by HPLC and electrochemical detection in 100 µL of culture medium deproteinized with an equal volume of 10% (w/v) trichloroacetic acid (TCA) [39 ]. Separation was obtained with a reverse-phase column (Spherisorb S5 ODS2, 25 cm) and a mobile phase (flow rate 1.5 mL/min) composed of 2% acetonitrile, 0.9% triethylamine, 0.59% phosphoric acid, 9 mM heptane sulfonic acid, and 0.25 mM sodium EDTA. Detection was obtained with a coulometric detector (ESA model 5100 A) operating at a preoxidation voltage of 0.03 V and an oxidation voltage of 0.23 V.

QUIN was measured according to a modification of previously reported procedures [40 ]. Briefly, after the addition of [713C]QUIN as an internal standard, 100 µL of culture medium were deproteinized with 10% TCA, dried, and derivatized at 80°C for 1 h with 100 µL of hexafluoro-2-propanol and 100 µL of trifluoroacetyl-imidazole. One hundred microliters of water and 100 µL of heptane were added to the derivatized samples, which were then vigorously mixed and frozen at -80°C. The heptane fraction was collected and injected into a gas chromatography/mass spectrometry system (HP 6890/5973 MSD, HP5973MSD) equipped with an automatic injector. The chromatographic column used was an HP 5MS, 30 mm x 0.25 mm x 0.25 µm. The carrier gas was helium at a constant flow of 1.2 mL/min. The oven temperature was as follows: 1 min at 80°C, raised at a rate of 10°C/min to 135°C and then at a rate of 25°C/min to 300°C. Injector and transfer line temperatures were 230°C and 270°C, respectively. The mass spectrometry detector operated in negative ion chemical ionization mode using methane as a negative gas. The recorded ions were m/z 467 for quinolinate and m/z 474 for [713C]QUIN. The dwell time was 70 ms for each ion.

Western blot analysis
Macrophages were cultured to confluence in 60-mm plates, stimulated for 24 h with 50 units/mL IFN-{gamma}, washed twice with phosphate-buffered saline (PBS), scraped off the plate, and centrifuged at 2,000 rpm for 5 min. The cell pellet was resuspended in 50 µL of 20 mM Tris buffer pH 8 containing 1 mM phenylmethylsulfonyl fluoride, 2 µg/mL aprotinin and leupeptin, and 1% Triton X-100, and the suspension was sonicated three times in ice. Protein content was determined by the Pierce bicinchoninic acid assay kit according to the manufacturer’s instructions, and the samples normalized on the basis of protein content (30 µg protein/sample). Laemmli buffer containing 100 mM 2-mercaptoethanol was then added and the samples boiled for 10 min and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in 8% acrylamide mini-gels (200 V, 45 min). The separated proteins were transferred onto a nitrocellulose membrane (Hybond-ECL, Amersham, Milan, Italy) by electroblotting (100 V, 90 min). To estimate the level of iNOS expression, membranes were blocked (3 h, RT) with PBS containing 0.1% Tween-20 and 5% BSA (T-BPS/5% BSA) and incubated overnight at 4°C in a 1:1000 dilution of anti-iNOS rabbit antibodies (Sigma, St. Louis, MO). Membranes were then incubated for 1 h in a 1:5000 dilution of horseradish peroxidase (HRP)-conjugated anti-rabbit antibodies in T-BPS/2% BSA. After a final wash in T-BPS, membranes were incubated (1 min, RT) in a chemiluminescent reagent (ECL protein detection system, Amersham), and the peroxidase-coated protein bands visualized on Hyperfilm-ECL (Amersham) after a 1-min exposure.

Evaluation of vascular relaxation
The thoracic segment of the aorta was obtained from male New Zealand rabbits (2.5–3 kg) and cut into 3- to 4-mm-wide rings. The preparation was suspended between stainless-steel hooks and mounted in a 10-mL organ bath filled with warm (37°C) and oxygenated (95% O2, 5% CO2) Krebs solution (in mM: NaCl 118, NaHCO3 25, KCl 4.7, KH2PO4 1.2, MgSO4 1.2, CaCl2 2.5, and glucose 10). A tension of 2 g was applied and a transducer recorded isometric contractions. After 1 h equilibration, concentration-response curves were obtained with different concentrations (0.1–1 µM) of (-)arterenol-hydrochloride (NE), and the concentration leading to a 50% contraction was used in the experiment. The vasorelaxing effect of acetylcholine (ACh) and A23187 was then tested in norepinephrine (NE)-precontractured aortic rings incubated for 15 min in the presence of different concentrations of norharmane. At the end of each experiment, the integrity of the NO-vasorelaxing pathway was tested by adding 0.1–10 µM sodium nitroprusside (SNP) to the incubation solution.

Reagents
Murine recombinant IFN-{gamma} was from PeproTech EC (London, UK). [713C]QUIN was kindly provided by Dr. J. F. Reinhard, Jr. (Glaxo-Wellcome, Research Triangle Park, NC). Hexafluor-2-propanol and trifluoroacetyl-imidazole were from Aldrich (Milan, Italy). Diphenyleneiodonium chloride was from Calbiochem (La Jolla, CA). Cell culture media, norharmane, ACh, NE, calcium ionophore A23187, NG-monomethyl-L-arginine (L-NMMA), SNP, and all other chemicals were from Sigma (St. Louis, MO).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Activation of the kynurenine pathway in macrophages
BAC1.2F5 cells cultured in a medium containing 80 µM tryptophan were found to release 3OH-kynurenine and QUIN, suggesting that these cells are able to metabolize tryptophan through the kynurenine pathway (Table 1 ). The addition of IFN-{gamma} (50 units/mL for 24 h) caused an about 10-fold increase in the activities of at least two enzymes of the pathway: IDO and KH. In supernatants of IFN-{gamma}-exposed macrophages, 3OH-kynurenine concentration increased approximately 15 times and that of QUIN 20 times. It is interesting that, in the same supernatants, kynurenine concentration decreased to one-third of controls (Table 1) , suggesting that the stimulatory effect of IFN-{gamma} on the activity of kynurenine-metabolizing enzymes (KH and kynureninase) prevailed on that of IDO.


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  		Table 1. Induction of Kynurenine Pathway Enzymes and Accumulation of Neurotoxic Kynurenine Metabolites in IFN-{gamma}-Treated Macrophages

 
Inhibition by norharmane of IDO activity and release of toxic metabolites
In keeping with previous data showing that norharmane inhibits IDO activity in homogenates of rabbit small intestine or mouse epidydimis [32 ], the compound also inhibited IDO in IFN-{gamma}-treated macrophages (Fig. 1A ). With 50 µM tryptophan as substrate, the IC50 of norharmane on IDO activity was 29 µM (15–43; 95% confidence interval). IDO inhibition corresponded to a reduced accumulation of 3OH-kynurenine and QUIN in culture medium, with norharmane IC50 values of 27 µM (18–35; confidence interval) and 28 µM (13–42), respectively (Fig. 1B) .



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  		Figure 1. (A) Inhibition by norharmane of IDO activity in IFN-{gamma}-treated macrophages. Cells were treated with 50 units/mL IFN-{gamma} for 24 h and lysed (see Materials and Methods). The enzymatic activity measured in crude cytosol extracts was 42 ± 6 pmol/mg protein/min. (B) Inhibition by norharmane of 3OH-kynurenine (3OHKYN) and QUIN release in culture medium of IFN-{gamma}-treated macrophages. In control cultures 3OHKYN and QUIN concentrations were 3075 ± 420 and 682 ± 86 nM, respectively. Values are means ± SEM of three independent experiments, each carried out in duplicate.

 
Inhibition by norharmane of nitrite formation
A reduced synthesis of QUIN is usually associated with a decreased formation of NAD, a QUIN metabolite. Because NAD(PH) is one of the cofactors modulating iNOS activity [30 , 31 ], we evaluated whether norharmane inhibits nitrite formation, which is an indicator of iNOS activity [41 ]. Figure 2A shows that norharmane reduced nitrite accumulation in macrophage supernatants with an IC50 of 75 µM (57–102; 95% confidence interval). Because this finding may be due to the inhibition of IDO by norharmane, we attempted to bypass this inhibition by adding to norharmane-treated cultures kynurenine or nicotinamide, NADPH precursors downstream from IDO activity. As reported in Figure 2B , neither kynurenine (up to 1 mM) nor nicotinamide (up to 200 µM) prevented the inhibition of nitrite formation by norharmane.



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  		Figure 2. (A) Inhibition by norharmane of nitrite release in culture medium of IFN-{gamma}-treated macrophages (100% was 29 ± 6 µM). Values are means ± SEM of three independent experiments, each carried out in duplicate. (B) Effects of kynurenine (1 mM, KYN) and nicotinamide (200 µM, NA) on nitrite release in culture medium of norharmane-exposed (30 µM, NH) cells. Bars show means ± SEM of three independent experiments, each carried out in duplicate; *P < 0.01 vs. controls.

 
Norharmane does not affect iNOS induction in activated macrophages
Because IDO was apparently not involved in the inhibition of nitrite formation by norharmane, we tested whether the compound exerts a direct effect on iNOS and, in particular, whether it inhibits iNOS induction in IFN-{gamma}-exposed macrophages. Under basal conditions, BAC-1.2F5 cells express a minimal amount of iNOS (Fig. 3 ) and, accordingly, the concentration of nitrites in their culture medium is quite low (0.3 ± 0.2 µM). Exposure to IFN-{gamma} (50 units/mL, overnight) caused a marked increase of iNOS expression, paralleled by an increase of nitrite concentration in culture medium (to 29 ± 6 µM). When 100 µM norharmane was present during macrophage activation, iNOS induction did not change, whereas nitrite concentration in culture medium was reduced by approximately 70% (Fig. 2A) .



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  		Figure 3. Western blot determination of iNOS expression in BAC-1.2F5 macrophages. Cells were treated or not with IFN-{gamma} (50 units/mL for 24 h), in the presence or absence of norharmane (NH), lysed, and iNOS detected in lysates. To ensure proper loading and transfer of proteins, the preparations were stained with Ponceau red; no significant differences were found among the lanes.

 
Inhibition by norharmane of constitutive NOS
To test the possibility that norharmane inhibits isoforms of NOS other than iNOS, we studied the effects of the compound on the ACh- or A23187-induced relaxation of NE-precontracted rabbit aortic rings (Fig. 4 ). This relaxation is believed to be mediated by NOS constitutively expressed in endothelia (cNOS) and activated by ACh receptor stimulation or A23187-induced increase in intracellular Ca2+. Preincubation of aortic rings with norharmane for 15 min caused a dose-dependent inhibition of the relaxing effect of either ACh (0.01–1 µM) or A23187 (0.01–3 µM). In this experimental setting, norharmane IC50 was 3.0 µM (1.2–4.2; 95% confidence interval) against 1 µM ACh and 3.1 µM (1.1–4.5) against 3 µM A23187. Complete inhibition of the effects of ACh and A23187 on NE-precontracted aortic rings was reached with 10 µM norharmane. When the relaxation was induced by 3 µM SNP, norharmane was inactive (Fig. 4 , insets).



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  		Figure 4. Inhibition by norharmane (NH) of the ACh- and A23187-induced relaxation of NE-precontracted aortic rings. Insets: dose-response of the NH inhibition of the maximal vasorelaxing effect of ACh (1 µM) and A23187 (3 µM), in the presence or the absence of SNP 3 µM.

 
The effects of norharmane were also evaluated in CVEC, exhibiting cNOS activity [42 ]. Cells were preincubated with L-[3H]arginine and the formation of [3H]citrulline measured as an index of cNOS activity [36 ]. Norharmane (10 and 100 µM) inhibited cNOS activity by about 60%. Under identical experimental conditions, L-NMMA (2 mM) reduced cNOS activity by 98.5% (Table 2 ).


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  		Table 2. Inhibition by Norharmane or L-NMMA of cNOS activity in CVEC

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We showed that IDO and iNOS activities were significantly increased in IFN-{gamma}-activated BAC1.2F5 macrophages, causing accumulation of 3OH-kynurenine, QUIN, and NO to concentrations exceeding the neurotoxic thresholds. The potential neurotoxicity of each of the above-mentioned compounds has been, indeed, shown in a number of studies. Concentrations of 3OH-kynurenine similar to or lower than those found in our supernatants may induce neuronal apoptosis [21 ]. In a similar manner, QUIN, at concentrations similar to those reported here, has been shown to cause excitotoxic neuronal death [43 , 44 ] and NO to cause damage of neuronal and glial cells, demyelinization, plasma extravasation, and recruitment of blood monocytes with a further potentiation of the inflammatory neuronal damage [28 , 45 , 46 ]. It is worth noting that the concentrations of 3OH-kynurenine, QUIN, and NO we measured represent neurotoxins diluted in the culture medium, suggesting that, in the proximity of activated macrophages, neurons may be exposed to even higher neurotoxin concentrations.

Norharmane, a previously described IDO inhibitor, when tested in our experimental model, was able to decrease the formation not only of 3OH-kynurenine and QUIN, but also of NO (Fig. 5 ). To elucidate the involvement of the IDO-dependent synthesis of pyridine nucleotides (essential cofactors for iNOS) in NO formation, we performed experiments aimed at bypassing IDO by incubating the cells with large concentrations of kynurenine or nicotinamide, two NADPH precursors. To our surprise, these treatments did not interfere with the norharmane effects, thus ruling out the possibility that reduced cofactor availability was involved in the inhibition of NO synthesis.



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  		Figure 5. Immune activation within the CNS causes induction of IDO and iNOS in mononuclear cells. This led to increased production and release of 3OHKYN and QUIN, molecules endowed with neurotoxic properties (see text). Furthermore, NO promotes oxidative stress of neurons, demyelinization, leukocyte recruitment, and plasma extravasation, thus boosting the toxic component of the neuroimmune response. Norharmane, by the concomitant inhibition of IDO and NOS, could lead to decreased synthesis of these neurotoxins and may be a tool in clarifying their role in neuroinflammatory disorders.

 
Large concentrations of picolinic acid, another kynurenine metabolite, had been previously shown to significantly potentiate IFN-{gamma}-mediated iNOS expression in macrophages [47 ]. It was therefore reasonable to assume that IDO inhibition by norharmane reduced picolinic acid availability, and that this, in turn, decreased iNOS expression. This alternative hypothesis was addressed with the experiments shown in Figure 3 . Because norharmane did not modify iNOS expression, the possibility that a reduced synthesis of picolinic acid could be responsible for the norharmane effects on NO synthesis was also ruled out. In keeping with this result, kynurenine, a precursor of picolinic acid, did not revert this inhibition (Fig. 2B) .

In mononuclear phagocytes, an increased NO availability has been shown to reduce IDO activity. This effect may be due to either the high affinity of NO for the heme iron present in IDO molecules, or the interaction of NO with superoxide ion, an essential IDO cofactor [48 ]. These data, together with the above-mentioned effect of picolinic acid on NOS expression [47 ], are in line with the concept that the metabolism of the two amino acids arginine and tryptophan is mutually regulated, the functional meaning of which is yet to be clarified.

Norharmane inhibited the activity of not only iNOS but also cNOS because it reduced the formation of [3H]citrulline from [3H]arginine in CVEC (Table 2) and the smooth muscle-relaxing effects of both ACh and A23187 (a Ca2+ ionophore able to activate cNOS in a receptor-independent manner) in NE-precontracted aortic rings (Fig. 4) . The latter experiment suggested that norharmane acted downstream of the ACh receptor-dependent intracellular Ca2+ release. On the other hand, norharmane did not modify the direct muscle relaxation induced by SNP (Fig. 4 , insets), thus ruling out a possible direct action of norharmane on muscle cell targets. Finally, the fact that norharmane inhibition of cNOS was even greater than that of iNOS lends further support to the possibility that the decreased nitrite accumulation in macrophage supernatants was due to an inhibition of enzyme activity and not to a reduction in either NADPH availability or iNOS expression. It is interesting that diphenyleneiodonium, a flavoprotein inhibitor with a molecular structure very similar to that of norharmane, was shown to reduce both cNOS and iNOS activity [49 ]. However, in our experimental setting, diphenyleneiodonium antagonized iNOS at nanomolar concentrations, but did not affect IDO up to millimolar concentrations (data not shown).

A crucial issue in macrophage activation within central nervous system, like other tissues, is the balance between its beneficial and harmful effects [50 51 52 ]. The use of norharmane and related molecules to modulate the synthesis of neurotoxins by activated macrophages could represent a useful strategy to explore the possibility of limiting neuronal damage in a number of inflammatory neurological disorders [53 ].


   ACKNOWLEDGEMENTS
 
This work was supported by grants from Istituto Superiore di Sanità (Multiple Sclerosis research project), the University of Florence, CNR, and the European Union (Biomed 2 BMH4-CT96-0228 and Biotech BIO4-CT96-0049 projects). The authors acknowledge G. Pieraccini for his expertise in gas chromatography/mass spectroscopy procedures.

Received January 14, 2000; revised March 30, 2000; accepted April 10, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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