FEBS 30727 6 May 2006; Disk Used No. of Pages 9 ARTICLE IN PRESS FEBS Letters xxx (2006) xxx–xxx 2 Fatty acid control of nitric oxide production by macrophages 3 Thais Martins de Limaa,*, Larissa de Sa Limab, Cristoforo Scavoneb, Rui Curia 4 5 6 a b Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, Av. Prof Lineu Prestes, 1524, 05508-900, São Paulo, Brazil Department of Pharmacology, Institute of Biomedical Sciences, University of São Paulo, Av. Prof Lineu Prestes, 1524, 05508-900, São Paulo, Brazil Received 17 April 2006; accepted 26 April 2006 Edited by Sandro Sonnino 30 Keywords: Fatty acid; Nitric oxide; NF-jB; iNOS expression; 31 iNOS activity; Cell death RR 32 33 1. Introduction CO NO produced by activated macrophages has been shown to regulate antimicrobial and antitumor activities. However, NO production in excess causes tissue damage that is associated with acute and chronic inflammation [1]. NO is synthesized from L -arginine by NO synthase (NOS) using NADPH and oxygen as cosubstrates [2]. Macrophages stimulated with lipopolysaccharide (LPS) and pro-inflammatory cytokines such as interferon-c (IFN-c) and tumor necrosis factor a (TNF-a) [3,4] produce large amounts of NO through inducible NOS (iNOS) activity. The expression of iNOS is regulated by the nuclear factor kappa B (NF-jB) in several cell types, including macrophages [5,6]. NF-jB plays an important role in controlling inflammatory gene activation [7]. This transcription factor is usually found in the cytosol as a heterodimer complex with its inhibitory protein, IjB. When cells are stimulated with LPS, phorbol esther or inflammatory cytokines, IjB is phosphorylated by IjB kinase and degraded. IjB phosphorylation UN 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 2. Materials and methods 88 2.1. Reagents RPMI-1640 medium, HEPES, penicillin and streptomycin were purchased from Invitrogen (Carlsbad, CA, USA). Fatty acids, LPS, sulfanilamide, naphthylene diamine dihydrochloride, sodium pyrophosphate and sodium orthovanadate were obtained from Sigma (St. Louis, MO, USA). Ethanol and phosphoric acid were purchased from Merck (Frankfurter, Germany). Sodium bicarbonate and sodium nitrite were purchased from Labsynth products (Diadema, SP, Brazil). DAF-DA was obtained from Molecular Probes (Eugene, OR, USA). 89 90 91 92 93 94 95 96 97 D PR dissociates the dimmer and allows NF-jB to translocate to the nucleus, where it activates target genes, including iNOS [8]. Recent studies have shown that fatty acids (FA) can modulate NF-jB activation. In human monocytic THP-1 cells, linoleic (LA), a-linolenic (ALA) and docosahexaenoic (DHA) acids decreased NF-jB DNA-binding activity [9]. Palmitic acid (PA) increased NF-jB activity in 3T3-L1 adipocytes [10] and pericytes [11]. On the other hand, palmitic (PA), oleic (OL) and linoleic (LA) acids induced iKKb activation and decreased NF-jB activity in endothelial cells [12]. Eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids decreased LPS induced NF-jB activation in human kidney-2 (HK-2) cells [13]. Several authors have shown modulation of macrophage functions by FA [14–17]. The x 6 polyunsaturated FA usually stimulate the inflammatory response, whereas x 3 FA have been considered as anti-inflammatory agents. The effect of FA has been examined on cytokine, eicosanoids and reactive oxygen species production [18–20], and adhesion molecule expression [17,21]. However, the effect of FA on NO production and iNOS expression in macrophages has been poorly examined [22–25]. The studies have shown that x 3 FA, especially DHA, markedly suppress NO production and iNOS expression in murine macrophages. An increase in NO production by macrophages from animals fed x 3 FA rich diets has been also reported [26,27]. Up to now, however, there is no report comparing the effect of the more abundant FA in plasma, palmitic (saturated) and oleic acids (monounsaturated, x 9), with x 3 (DHA and EPA) and x 6 (linoleic and arachidonic) FA. As described above, NO production and iNOS expression are regulated by NF-jB activation, and the activity of this transcription factor can be modulated by FA. This information led us to investigate the effect of various FA on NF-jB activity and NO production in J774 cells (a murine macrophage cell line). The following FA were studied: palmitic (PA), stearic (SA), oleic (OA), linoleic (LA), arachidonic (AA), docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids. TE Abstract Modulation of macrophage functions by fatty acids (FA) has been studied by several groups, but the effect of FA on nitric oxide production by macrophages has been poorly examined. In the present study the effect of palmitic, stearic, oleic, linoleic, arachidonic, docosahexaenoic and eicosapentaenoic acids on NF-jB activity and NO production in J774 cells (a murine macrophage cell line) was investigated. All FA tested stimulated NO production at low doses (1–10 lM) and inhibited it at high doses (50–200 lM). An increase of iNOS expression and activity in J774 cells treated with a low concentration of FA (5 lM) was observed. The activity of NF-jB was time-dependently enhanced by the FA treatment. The inhibitory effect of FA on NO production may be due to their cytotoxicity, as observed by loss of membrane integrity and/or increase of DNA fragmentation in cells treated for 48 h with high concentrations. The results indicate that, at low concentrations FA increase NO production by J774 cells, whereas at high concentrations they cause cell death.  2006 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. EC 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 OO F Available online * Corresponding author. Fax: +55 11 30917285. E-mail address: thais@icb.usp.br (T.M. de Lima). 0014-5793/$32.00  2006 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. doi:10.1016/j.febslet.2006.04.091 FEBS 30727 6 May 2006; Disk Used No. of Pages 9 ARTICLE IN PRESS 2 T.M. de Lima et al. / FEBS Letters xxx (2006) xxx–xxx 2.3. Determination of nitric oxide The content of nitrite was measured in the supernatant of cultured cells based on the method described by Ding et al. [29]. Cells (5 · 105 per well) were seeded in 96 well plates and treated with 2.5 lg per mL LPS and different concentrations of FA for 48 h. At the end of the culture period, 50 lL of the supernatant were removed and incubated with an equal volume of Griess reagent (1% sulfanilamide, 0.1% naphthylene diamine dihydrochloride, 2.5% H3PO4) at room temperature for 10 min. The absorbance was determined at 550 nm. Nitrite concentration was determined by using sodium nitrite as standard. 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 2.4. Determination of iNOS protein expression J774 cells were seeded in 25 cm flasks and treated with 2.5 lg per mL LPS and 5 lM of the FA for 6, 12 and 24 h. At the end of the incubation period, cells were immediately homogenized in 150 lL extraction buffer (100 mM Trizma, pH 7.5; 10 mM EDTA; 10% sodium dodecyl sulfate (SDS); 100 mM NaF; 10 mM sodium pyrophosphate; 10 mM sodium orthovanadate; at 100 C) for 30 s. Samples were boiled for 5 min and centrifuged at 12 000 rpm, for 40 min, at 4 C. Aliquots of supernatants were used for the measurement of total protein content, as described by Bradford [30]. Equal amount of protein of each sample (70 lg) was separated using 6% SDS–polyacrylamide gel. Western blotting was carried out following the method described by Towbin et al. [31]. The proteins of the gel were transferred to a nitrocellulose membrane at 120 V for 1 h. Non-specific bounds were blocked by incubating the membranes with 5% defatted milk in basal solution (10 mM Trizma, pH 7.5; 150 mM NaCl; 0.05% Tween 20) at room temperature, for 2 h. Membranes were washed in basal solution three times for 10 min each and then incubated with anti-iNOS antibody in basal solution containing 3% defatted milk, at room temperature, for 3 h. Membranes were washed three times for 10 min each and incubated with anti-IgG antibody linked to horseradish peroxidase in basal solution containing 1% defatted milk, at room temperature, for 1 h. Following another washing, membranes were incubated with substrate for peroxidase and chemiluminescence enhancer (Amersham Biosciences, Upsalla, SW) for 1 min and immediately exposed to X-ray film for 30 min. Films were then revealed in the conventional manner. Band intensities were analysed using the ScionImage software (Scion Corporation, MD, USA). 153 154 155 156 157 158 159 160 161 162 163 164 165 166 2.5. Measurement of iNOS activity iNOS activity was measured in J774 cells treated for 12, 24 and 48 h with 2.5 lg per mL LPS and 5 lM FA. This assay is based on the biochemical conversion of L -arginine to L -citrulline by iNOS. Cells were homogenized in ice-cold Tris–HCl buffer (20 mM Tris–HCl, 10 mM EDTA, and 10 mM EGTA, pH 7.4) using a Teflon homogenizer. The homogenates were centrifuged at 12 000 · g for 5 min at 4 C. Supernatants were removed and NOS assay was performed by incubating (37 C for 20 min) 150 lg (20 lL) of protein in a final volume of 60 lL of assay mixture containing 50 mM Tris–HCl, 6 lM tetrahydrobiopterin, 2 lM FAD, 2 lM FMN, 10 mM NADPH, 100 mM L -arginine/L -[H3]-arginine (5 lCi/mL), 1 mM EDTA/EGTA. The reaction was stopped with 1 mL of ice-cold stop buffer (50 mM HEPES and 5 mM EDTA, pH 5.5) and 100 lL of cation-exchange resin (Dowex, 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 OO F 115 116 117 118 119 120 121 122 123 124 2.6. Electrophoretic mobility shift assay NFjB activation was evaluated after treatment of the cells for 3, 6, 12, 24 and 48 h with 5 lM fatty acids; concentrations in which a high production of NO was observed. Nuclear extract from J774 cells was obtained as previously described [31]. Double-stranded oligonucleotides containing the NF-jB (5 0 AGTTGAGGGGACTTTCCCAGGC-3 0 ) consensus binding site [33] were end-labeled using T4 PNK and [c-32P]ATP (Amersham Biosciences). Binding reactions of the probes (30 000 cpm) were performed with 10 lg proteins from nuclear extract, at room temperature, for 20 min, in 20 lL of the binding buffer consisting of 20 mM HEPES, pH 7.6, 50 mM KCl, 10% glycerol, 0.2 mM EDTA, 1 mM DTT and 2 lg polydeoxyinosinic–deoxycytidylic acid (poly[dI–dC]). Competitive binding assays were conducted under the same conditions with the addition of 2 pmol (100-fold molar excess) of unlabeled competitor oligonucleotides. The DNA–protein complexes were electrophoresed on 4% non-denaturing polyacrylamide gels, at 4 C, in 45 mM Tris, 45 mM borate and 1 mM EDTA buffer. The gels were dried and subjected to autoradiography. The blots were analysed by scanner densitometry (Image Master 1D, Amersham Biosciences) and the results of the binding activity were expressed as arbitrary units. PR 2.2. Culture conditions and fatty acid treatment J774 cells were grown in RPMI-1640 medium containing 10% fetal calf serum (FCS). This medium was supplemented with glutamine (2 mM), HEPES (20 mM), streptomycin (10 000 lg/mL), penicillin (10 000 UI/mL) and sodium bicarbonate (24 mM). Cells were grown in 75 cm2 flasks containing 0.5–1 · 106 cells per mL. The cells were kept in a humidified atmosphere containing 5% CO2 at 37 C. Cells were treated with various concentrations (1–200 lM) of palmitic (PA), stearic (SA), oleic (OA), linoleic (LA), arachidonic (AA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids for different periods (from 3 up to 48 h). The fatty acids were dissolved in ethanol. The final concentration of ethanol in the culture medium did not exceed 0.5%. This concentration of ethanol is not toxic to the cells as reported by Siddiqui et al. [28]. 167 168 169 170 171 172 173 2.7. Determination of fatty acid cytotoxicity Fatty acid cytotoxicity was assessed by flow cytometry after treatment of the cells for 48 h with high concentrations of fatty acids. At the end of the culture period, 0.5 mL of medium containing cells were used to evaluate the membrane integrity. In this assay, 50 lL of a propidium iodide (PI) solution (100 lg per mL in saline buffer) were added to the cells. Propidium iodide is a highly water-soluble fluorescent compound that cannot pass through intact membranes and is generally excluded from viable cells. It binds to DNA by intercalating between the bases with little or no sequence preference. After 5 min incubation at room temperature, the cells were evaluated in a FACScalibur flow cytometry equipment (Becton Dickinson, CA, USA) by using the Cell Quest software. Fluorescence was measured using the FL2 channel (Orange-red fluorescence – 585/42 nm). Ten thousand events were analysed per experiment. We also determined the percentage of cells with fragmented DNA after the treatments using propidium iodide. In this assay, cells were resuspended in a solution containing detergents that permeabilize the cells, which promptly incorporate the dye into DNA. Briefly, 0.5 mL of medium containing cells were centrifuged at 1000 · g, for 10 min, at 4 C. The pellet was gently resuspended in 300 lL hypotonic solution containing 50 lg/mL propidium iodide, 0.1% sodium citrate, and 0.1% Triton X-100. The cells were then incubated for 2 h at 4 C. Fluorescence was measured and analysed by flow cytometry as described above. Both the lost of membrane integrity and/or DNA fragmentation were considered signs of toxicity, regardless which one was first observed. 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 2.8. Statistical analysis Results are presented as means ± S.E.M. of 6–9 determinations from 2–3 experiments. Comparisons with control were performed by analysis of variance (2-way-ANOVA). Significant differences were analysed by the Bonferroni post-tests (Graph Pad Prism 4 – Graph Pad Software Inc., San Diego, CA, USA). The level of significance was set at P < 0.05. 222 223 224 225 226 227 228 3. Results 229 3.1. Effect of fatty acids on NO production All FA tested stimulated NO production at low doses but showed inhibitory effect at high concentrations (Fig. 1). The 230 231 232 UN CO RR EC TE 101 102 103 104 105 106 107 108 109 110 111 112 113 114 Na+ form, equilibrated with 50 mM HEPES, pH 5.5) was added to each reaction mixture to remove the excess of L -[H3]-arginine. Aliquots were collected into vials, scintillation liquid (6 mL) was added and radioactivity was quantified in a scintillation counter (Packard TRI CARB 2100 TR Counters, Downers Grove, IL, USA). Protein concentrations in samples were determined as described by Bradford [30] with bovine serum albumin as standard. D 98 Reagents for SDS–PAGE and immunoblotting were from Bio-Rad 99 Laboratories (Richmond, CA, USA). Antibodies were obtained from 100 Santa Cruz Biotechnology (Santa Cruz, CA, USA). FEBS 30727 6 May 2006; Disk Used No. of Pages 9 ARTICLE IN PRESS 3 T.M. de Lima et al. / FEBS Letters xxx (2006) xxx–xxx stimulatory effect of the FA was more pronounced at concentrations between 2.5 and 5 lM (P < 0.001). At 5 lM, palmitic acid was the less effective, inducing an increase of 32% in NO production. Arachidonic acid, on the other hand, was the most potent, increasing in 119% the production of NO. The crescent order of stimulatory effect at 5 lM was: PA (32%), SA (60%), DHA (79%), LA (83%), EPA (89%), OA (94%), and AA (117%). PA and EPA inhibited NO production at 50 lM. SA suppressed NO production at 100 lM, AA and DHA at 150 lM, and OA and LA at 200 lM. SA PA 14 14 * * µM nitrite 10 * 12 * 10 8 * 6 4 8 6 4 2 2 0 0 0 1 2.5 5 10 25 0 50 1 14 * 12 * 10 8 * 6 4 0 1 2.5 5 10 25 * * * EC * RR * 0 1 CO µM nitrite AA * 2.5 5 25 50 100 10 25 50 * * LA * * * * * * 8 6 4 * 2 0 50 100 150 200 µM 18 16 14 12 10 8 6 4 2 0 10 TE 2 0 10 D µM nitrite * * 12 µM nitrite 14 16 * 0 1 2.5 5 10 25 50 100 150 200 µM DHA 16 14 * 12 µM nitrite * 5 PR OA * 2.5 * µM µM 16 OO F * µM nitrite 12 * * * * 10 8 6 * 4 2 0 100 150 0 1 2.5 5 10 25 50 100 150 µM µM EPA UN 16 14 * * * * 12 µM nitrite 233 234 235 236 237 * 10 8 * 6 * 4 2 0 0 1 2.5 5 10 25 50 100 µM Fig. 1. Effects of fatty acids on NO production of J774 cells. The concentration (lM) of nitrite measured in the supernatant of cultured cells after treatment for 48 h with palmitic, stearic, oleic, linoleic, arachidonic, docosahexaenoic and eicosapentaenoic acids in the presence of 2.5 lg per mL LPS are shown. The supernatant was incubated with an equal volume of Griess reagent and the absorbance was determined at 550 nm. The values are presented as means ± S.E.M. of 16 determinations from four experiments. * P < 0.001 for comparison with control. 238 239 240 241 242 FEBS 30727 6 May 2006; Disk Used 4 T.M. de Lima et al. / FEBS Letters xxx (2006) xxx–xxx 262 263 264 265 266 267 268 269 3.4. Effect of fatty acids on iNOS activity Cells treated with AA for 12 h showed higher iNOS activity when compared with the correspondent control (ethanol 12 h). Exposure for 24 h to PA also led to an increased of iNOS activity. Cells treated with OA, LA, DHA and EPA presented higher iNOS activity after 48 h treatment. SA did not present significant effect on iNOS activity (Fig. 4). 270 271 272 273 274 275 276 3.5. Effect of fatty acids on cell viability and DNA fragmentation In order to evaluate if the inhibitory effect of the FA on NO production at high concentrations was due to their cytotoxic- 277 278 279 UN CO RR EC TE D 255 3.3. Involvement of NFjB activation on modulation of iNOS 256 expression by FA 257 Treatment of J774 cells with 5 lM FA altered NFjB activa258 tion as observed in Fig. 3. However, the period of incubation 259 for the effect of FA to be observed varied considerably. LPS 260 caused NFjB activation and presented the more potent effect 261 after 3 h treatment (115%) but the addition of ethanol (vehicle) diminished NFjB activation by 16.6 ± 3.2% (means ± S.E.M. of five experiments). PA, DHA and EPA increased NFjB activation after 3 h treatment, when compared to cells treated with LPS and ethanol, by 51%, 52% and 62%, respectively. AA exerted its stimulatory effect after 6 h of treatment (36%) and reached its peak after 24 h (106%). OA increased NFjB activation after 12 h (39%). SA and LA did not affect NFjB activation at any period of treatment tested. OO F 3.2. Effect of fatty acids on iNOS expression In order to investigate the mechanisms involved in the regulation of NO production by FA, we determined iNOS protein expression in J774 cells treated for 6, 12 and 24 h with 5 lM FA in the presence of LPS (2.5 lg/mL). Cells treated with PA did not present changes in iNOS expression when compared with those treated with LPS and ethanol at any period of treatment. SA and OA treated cells presented higher iNOS expression after 12 h (P < 0.001). Cells treated with LA, AA, DHA and EPA showed increased iNOS expression after 6 h of treatment (P < 0.001) and this effect remained up to 24 h (Fig. 2). PR 243 244 245 246 247 248 249 250 251 252 253 254 No. of Pages 9 ARTICLE IN PRESS Fig. 2. Effects of fatty acids on LPS induced iNOS expression. Cells were treated for 6, 12 and 24 h with 2.5 lg per mL LPS and 5 lM of the FA. Whole cell lysates were dissolved in a sample buffer and submitted to 8% SDS–PAGE. Western blotting was performed using mouse anti-iNOS polyclonal antibody. Band intensities were analysed using the ScionImage software (Scion Corporation) and are expressed as relative values compared to the respective control (LPS and ethanol). Controls received an arbitrary value of 1. The values are presented as means ± S.E.M. of three experiments. * P < 0.001, # P < 0.01 for comparison with control. FEBS 30727 6 May 2006; Disk Used No. of Pages 9 ARTICLE IN PRESS 5 T.M. de Lima et al. / FEBS Letters xxx (2006) xxx–xxx 3 hours 4 * 3 * * J K 2 1 A B C D E F G H I OO F 0 6 hours 4 3 ¤ ¤ 2 PR 1 0 A B C D 4 E F G H I J K H I J K 12 hours D 3 1 A B C D E F G 24 hours 4 * RR 3 EC 0 TE ¤ 2 2 0 A B C D E CO 1 F G H I J K G H I J K 48 hours UN 4 3 2 1 0 A B C D E F Fig. 3. Results of the electrophoretic mobility shift assay. (Right) Nuclear extracts prepared from J774 cells that had been treated with 2.5 lg per mL LPS and 5 lM of the FA for 3, 6, 12, 24 and 48 h were used for protein–DNA binding reactions in the presence of the radio-labeled probe (30 000 cpm), as described in Section 2. A: control, B: ethanol, C: LPS 2.5 lg per mL, D: LPS 2.5 lg per mL + ethanol, E: PA 5 lM, F: SA 5 lM, G: OA 5 lM, H: LA 5 lM, I: AA 5 lM, J: DHA 5 lM, K: EPA 5 lM. (Left) Band intensities were analysed using the ScionImage software (Scion Corporation) and are expressed as relative values compared to the respective control. Controls received an arbitrary value of 1. The values are presented as means ± S.E.M. of three experiments. * P < 0.001, P < 0.05 for comparison with cells treated with LPS + ethanol. FEBS 30727 6 May 2006; Disk Used No. of Pages 9 ARTICLE IN PRESS 6 T.M. de Lima et al. / FEBS Letters xxx (2006) xxx–xxx 0.3 # * # ¤ 0.2 * ¤ 0.15 0.1 0.05 0 OH+LPS PA SA OA 24 hours AA 48 hours DHA EPA PR 12 hours LA OO F pmol.mg-1.min-1 0.25 Fig. 4. Effects of fatty acids on iNOS activity. Cells were treated for 12, 24 and 48 h with 2.5 lg per mL LPS and 5 lM of the FA. iNOS activity was determined after quantification of radioactivity of the L -citrulline produced after conversion of L -arginine to L -citrulline by iNOS. The values are presented as means ± S.E.M. of three experiments. * P < 0.001, # P < 0.01, P < 0.05 for comparison with the respective control (ethanol and LPS). EC RR CO 296 4. Discussion 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 D high concentrations. FA toxicity has been reported in several cell types, as a concentration and time-dependent effect of these metabolites [35,36]. The stimulatory effect of FA was also observed by fluorescence microscopy using DAF-DA. Cells treated for 12 and 24 h presented high intracellular NO content, which diminished after 48 h of exposure to FA. These results corroborate with the low level of NO in the medium of cells treated with FA for 24 h (data not shown). High production of NO is achieved when the expression of inducible NOS is stimulated [3]. J774 cells treated with LPS and FA (5 lM) presented higher expression of iNOS protein than cells treated with LPS and ethanol (vehicle). The period of incubations where they exerted their effects varied, and may be related to the activity of the nuclear factor kappa B (NF-jB). Activation of NF-jB can trigger inflammatory responses by transcriptional induction of several pro-inflammatory proteins and enzymes that generate mediators of inflammation (e.g. iNOS) [37,38]. J774 cells presented significant NF-jB activity in basal conditions (absence of LPS and FA) that was increased by two fold after LPS addition. The FA tested, with exception of SA and LA, stimulated NF-jB activation, and this effect occurred prior to or at the same incubation period where an increase in iNOS protein level was observed. Several studies have shown a decrease in NF-jB activation by DHA and EPA [39–44]. However, Maziere et al. [45] observed an increase of NFjB activation in fibroblasts treated with DHA and EPA and Camandola et al. [46] stated that EPA (45 lM) exerts no effect on the nuclear translocation of NF-jB in the human promonocytic cell line U937. In the present study, DHA and EPA stimulated NO production in J774 cells at low concentrations (1–25 lM). Concomitantly, an increase of NF-jB activation was observed at 5 lM, indicating that this effect on NF-jB may raise iNOS protein level and, consequently, NO production. Stimulatory effect of PA, AA and OA on the nuclear translocation of NF-jB has also been observed in different cells TE ity, the percentage of cells with intact cell membrane and with fragmented DNA after the treatment for 48 h was determined (Fig. 5). All FA induced loss of membrane integrity, except AA. Cells treated with PA (50 lM), SA (100 lM), OA (200 lM), LA (200 lM), DHA (150 lM) and EPA (100 lM) presented a significant decrease in cell membrane integrity by 34%, 26%, 12%, 24%, 27% and 18%, respectively. The treatments also induced DNA fragmentation, except for LA. An increase in the percentage of cells with fragmented DNA was observed after treatment with PA (50 lM), SA (100 lM), OA (200 lM), AA (150 lM), DHA (150 lM) and EPA (100 lM) by 2-, 1.9-, 1.7-, 2.1-, 1.7- and 1.6-fold, respectively. Cells treated with ethanol, the vehicle used for FA preparation, did not present loss of membrane integrity or induction of DNA fragmentation, indicating that the concentration of ethanol used (0.5%) is not cytotoxic. The effect of different concentrations of various FA on nitric oxide production by J774 cells and the possible mechanisms involved were investigated in this study. J774 cells cultivated for 48 h with FA and LPS showed high production of NO as compared to control cells, especially at low concentrations (1–10 lM). Cells treated with LA, AA, DHA and EPA showed a stimulatory effect up to 25 lM. On the other hand, higher concentration (50–200 lM) inhibited NO production. These results seem controversial but they may help to explain the discrepancy in the literature. Many studies observed a decrease in NO production by mice macrophages and cell lineages after exposure to FA [22–25], whereas others found an increase [27,34]. In addition to cell type and period of stimulation, these controversial results may be also due to the different concentrations of the FA used. The inhibitory effect of FA on NO production may be due to their cytotoxicity, as observed by loss of membrane integrity and/or increase of DNA fragmentation in cells treated for 48 h with UN 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 FEBS 30727 6 May 2006; Disk Used No. of Pages 9 ARTICLE IN PRESS 7 T.M. de Lima et al. / FEBS Letters xxx (2006) xxx–xxx A 100 * * * * 60 40 20 0 C OH PA SA OA 100µM 200µM LA AA DHA EPA 200µM 150µM 150µM 100µM PR 50µM B * * OO F Percentage (%) of cells with intact membrane 80 60 D * 15 0 OH PA 50µM RR C TE 30 * EC Percentage (%) of cells with fragmented DNA 45 SA 100µM * * * * OA LA AA DHA EPA 200µM 200µM 150µM 150µM 100µM types, such as L6 myotubes, mouse C2C12 myoblasts, skeletal muscle cells, human promonocytic cell line U937, human endothelial cells and mouse macrophages [46–51]. On the other hand, no effect on NF-jB activation and iNOS expression was observed in insulinoma (INS)-1E cells, human fibroblasts and human endothelial cells treated with OA [52,53]. In the periods of incubation studied, LA and SA did not affect NF-jB activation but increased iNOS expression after 6 and 12 h, respectively. Other transcriptional factors are involved in iNOS expression, as the octamer factor (Oct) [54,55], signal transducer and activator of transcription-1a (STAT-1a) [56–58], cAMP-induced transcription factors such as cAMP-responsive element binding protein (CREB) and CCAAT-enhancer box binding protein (C/EBP) [59,60], and activating protein-1 (AP-1) [61]. SA and LA may have activated one of these transcriptional factors and increased iNOS expression. UN 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 CO Fig. 5. Effects of fatty acids on cell membrane integrity and DNA fragmentation. The percentage of cells with intact membrane (A) and fragmented DNA (B) after treatment for 48 h with palmitic (PA), stearic (SA), oleic (OA), linoleic (LA), arachidonic (AA), docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids are shown. Cells were stained with a saline buffer containing propidium iodide to assess membrane integrity. A buffer containing citrate, triton X-100 and propidium iodide was used to assess DNA fragmentation. The values are presented as means ± S.E.M. of 9 determinations from three experiments. * P < 0.001 for comparison with control (ethanol – OH). Three proteins that interact with iNOS and regulate its activity have been recently identified. In the central nervous system, the protein kalirin appears to inhibit iNOS by preventing enzyme dimerization [62]. In murine macrophages, a 110-kDa protein (named NAP110) has been identified that directly interacts with the amino-terminus of iNOS, thereby preventing dimer formation and inhibiting NOS activity [63]. Stable overexpression of Rac2 in RAW 264.7 cells increased LPS-induced nitrite generation and iNOS activity without measurably affecting iNOS protein abundance [64]. Our results suggest that FA are also modulators of iNOS activity in J774 cells. The results presented herein demonstrate that FA stimulate NO production by J774 cells at low concentrations (1–10 lM) and inhibit it at high doses (50–200 lM). The stimulatory effect of most FA on NO production is time dependent, involves NF-jB activation, and increases iNOS expression and activity. 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 FEBS 30727 6 May 2006; Disk Used 8 T.M. de Lima et al. / FEBS Letters xxx (2006) xxx–xxx 391 Acknowledgements: The authors are grateful to the technical assistance 392 of G. de Souza, J.R. Mendonça and E.P. Portioli. This research is sup393 ported by FAPESP, CNPq, and CAPES. 394 References CO RR EC TE [1] MacMicking, J., Xie, Q.W. and Nathan, C. 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