Ibuprofen: effect on inducible nitric oxide synthase

Ibuprofen: effect on inducible nitric oxide synthase

Molecular Brain Research 50 Ž1997. 107–112 Research report Ibuprofen: effect on inducible nitric oxide synthase Nancy C. Stratman, Donald B. Carter,...

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Molecular Brain Research 50 Ž1997. 107–112

Research report

Ibuprofen: effect on inducible nitric oxide synthase Nancy C. Stratman, Donald B. Carter, Vimala H. Sethy

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CNS Diseases Research, Pharmacia and Upjohn Inc., Kalamazoo, MI 49001, USA Accepted 22 April 1997

Abstract Treatment with ibuprofen and other non-steroidal anti-inflammatory drugs ŽNSAIDS. has been reported to decrease the incidence as well as slow down the progression of Alzheimer’s disease. Understanding the mechanism of this therapeutic effect would provide a target for development of drugs which may be devoid of side effects observed with NSAIDs. In addition to inhibiting cyclooxygenase ŽCOX., the NSAIDs have recently been shown to decrease inducible nitric oxide synthase ŽiNOS. activity. Ibuprofen and other NSAIDs had no direct effect on catalytic activity of iNOS, but decreased levels of iNOS mRNA. The mechanism of action of ibuprofen on reduction of iNOS activity has been further investigated in the present study using rat primary cerebellar glial cell cultures. In addition, the effect of ibuprofen on COX mRNA expression and prostaglandin formation was also studied. Glial cells treated with E. coli lipopolysaccharide ŽLPS. and interferong ŽINFg . for 16 h expressed iNOS and COX. Ibuprofen did not directly inhibit iNOS activity. However, when ibuprofen was incubated at the same time with LPS and INFg for 16 h, enzyme activity was reduced, with an IC 50 of 0.76 mM. Ibuprofen concentration-dependently decreased iNOS mRNA levels, with an IC 50 ) 2 mM. Thus, there was no correlation between decrease in iNOS activity and reduction in iNOS mRNA levels. Ibuprofen decreased iNOS protein levels, as determined by Western blot, with an IC 50 of 0.89 mM. The data suggest that the reduction in iNOS activity by ibuprofen is due to inhibition of post-transcriptional processing of this enzyme. Ibuprofen had no effect on constitutive COX ŽCOX-1. or inducible COX ŽCOX-2. mRNA expression. However, ibuprofen inhibited PGE 2 formation with an IC 50 of 0.86 mM. The anti-inflammatory actions of ibuprofen have been related to inhibition of COX and, subsequently, reducing prostaglandin formation. Since the potency of ibuprofen for inhibition of PGE 2 formation and reduction in iNOS activity are similar, it is suggested that, at therapeutically effective doses, a decrease in iNOS activity may also occur in vivo. Therefore, reduction in iNOS protein levels in the brain may have a role in preserving the integrity of neurons in individuals susceptible to Alzheimer’s disease. q 1997 Elsevier Science B.V. Keywords: iNOS; Glia; Cultures; Ibuprofen; Neurodegenerative disorder

1. Introduction Daily administration of NSAIDs for treatment of arthritic disorders has been shown to delay the onset andror decrease the risk of Alzheimer’s disease w2,3x. The therapeutic effect of NSAIDs is thought to be due to inhibition of cyclooxygenase enzymes and, subsequently, reducing synthesis of prostaglandins w10,12,17x. Recently, NSAIDs have shown an inhibitory effect on inducible nitric oxide synthase ŽiNOS. w1x. This has significant implications since inhibition of iNOS may prevent immunemediated neurodegeneration by diminishing the synthesis of free radical nitric oxide ŽNO.. Nitric oxide synthase can be induced in many cells, and elevations of NO may have ) Corresponding author. CNS Diseases Research 7251-209-508, Pharmacia and Upjohn Inc., 301 Henrietta St., Kalamazoo, MI 49001, USA. Fax: q1 Ž616. 833-2525.

a role in the pathogenesis of neurodegenerative disorders of the brain w6,13x. Microglia, the resident macrophages in the CNS, can express iNOS in response to bacterial toxins and cytokines, and the generation of NO from these activated cells has been demonstrated to produce neuronal cell injury in culture w4x. Thus, inhibition of iNOS may prevent neuronal injury in brain disorders associated with inflammation. Ibuprofen and other NSAIDs were found to reduce iNOS activity in rat alveolar macrophage cultures when incubated with lipopolysaccharide ŽLPS. and interferon-g ŽIFNg . w1x. However, these agents had no direct effect on iNOS catalytic activity determined by incubating the drugs with the enzyme preparation obtained from cells treated with LPS and IFNg w1x. Furthermore, ibuprofen, at 1 mM, inhibited 30% to 70% of iNOS mRNA in these cells, providing a possible mechanism of reduction in iNOS activity. Our investigations further characterize the mecha-

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nism of action of ibuprofen on iNOS activity by identifying the potency of this drug on Ž1. direct and indirect inhibition of iNOS activity, Ž2. iNOS mRNA expression, and Ž3. iNOS protein expression in primary cerebellar glial cell cultures treated with LPS and IFNg . The effect of ibuprofen on iNOS was also compared to its effect on PGE 2 formation and constitutive and inducible cyclooxygenase ŽCOX-1 and COX-2, respectively. mRNA expression in the same cells since it has been demonstrated that NSAIDs inhibit cyclooxygenase enzymes and then lead to a reduction in prostaglandins in vivo.

2. Materials and methods 2.1. Cell culture Primary cerebellar granule cell cultures were prepared from 7-day old Sprague–Dawley rats as reported previously w14x. Cells were grown in 6-well, 35 mm culture dishes ŽNunc, Denmark. for 8 days, 2 mlrwell at a density of 2 = 10 6 cellsrml, and glial cells were allowed to proliferate by not adding cytosine-arabinofuranoside after plating the cells. On the 8th day, growth media was aspirated off, the content of each well was rinsed twice with 2 ml aliquots of serum free culture media, and then 1.8 ml of the same media was added to each well. Subsequently, 200 m l ibuprofen or vehicle, 20 m l of E. coli lipopolysaccharide ŽLPS; 1 m grml., and 20 m l rat interferon-g ŽINFg ; 10 IUrml. were added to each well and the glial cultures were incubated for 16 h at 378C in a incubator with 5% CO 2 and 95% O 2 w14x. Control cultures were treated with vehicles of drug and iNOS inducers. 2.2. iNOS actiÕity in primary cerebellar cultures Determination of iNOS activity was carried out by the procedure previously described w14x. Incubation of glial cells with ibuprofen or vehicle, LPS, and INFg was terminated by aspiration of media, and cells were frozen at y808C until used for estimation of enzyme activity. On the day of the assay, 250 m l of 20 mM Tris-HCl buffer ŽpH 7.4. containing 2 mM EDTA and 50 m M tetrahydrobiopterin ŽTHB. was added to each well and cells were thawed for 10 min and dislodged using a VirSonic 475

sonicator for 10 srwell. The cell suspensions obtained from cultures treated only with LPS and INFg were used for investigating direct iNOS catalytic activity. Enzymatic activity was determined by incubating 50 m l of cell suspension, 10 m l of L-w 14 Cxarginine Žspecific activity 0.32 Cirmmol, DuPont NEN, Boston, MA. to a give a final concentration of approximately 8.0 m M, 30 m l of incubation buffer Ž20 mM Tris-HCl containing 2 mM EDTA, 50 m M THB, 1 mM NADPH and 50 unitsrml of calmodulin., and 10 m l of ibuprofen Ž0.1 to 6 mM. or incubation buffer. The mixtures were incubated for 15 min at 378C, and the reaction was stopped by adding 3 ml of ice-cold Ž08C. 20 mM HEPES buffer, pH 5.5 containing 2 mM EDTA. 14 14 L-w Cxarginine was separated from L-w Cxcitrulline using 0.5 ml columns of Dowex AG50WX-8 resin ŽNaq form. and the radioactive L-w 14 Cxcitrulline was counted using a liquid scintillation counter. Non-specific enzyme activity was determined using boiled suspensions and represented less than 30% of total enzyme activity. Inhibitions of enzyme activity by ibuprofen was investigated three times at 6–9 concentrations each in triplicate. IC 50 s for each curve were determined by non-linear analysis, and a mean IC 50 was obtained by an average weighting method. For indirect inhibition of iNOS activity, cultures were incubated with various concentrations of ibuprofen along with LPS and IFNg for 16 h at 378C. The reaction was terminated by aspiration of culture media. Procedures for estimation of iNOS activity were followed as described before, except ibuprofen was not added to the incubation mixture. 2.3. Measurement of iNOS, COX-1, and COX-2 mRNA 2.3.1. RNA isolation Glial cells were treated with ibuprofen or vehicle and LPS plus INFg and incubated for 16 h at 378C in a culture incubator as described in the cell culture methods. Incubation was terminated by washing cells twice with phosphate buffered saline. Total RNA extraction was performed using the RNA Stat-60 method ŽTel-Test, Inc., Friendswood, TX., a modification of the Chomczynski and Sacchi single-step procedure w5x. 2.3.2. Specific primer preparation Oligonucleotide primers were chosen from published rat iNOS, COX-1, COX-2, and glyceraldehyde-3-phosphate

Table 1 Primer sequences used for RT-PCR mRNA species

Product size Žbp.

Starting position

Sequence

iNOS, sense iNOS, anti-sense COX-1, sense COX-1, anti-sense COX-2, sense COX-2, anti-sense GAPDH, sense GAPDH, anti-sense

540

901 1440 1433 1851 1200 1525 33 534

5 X 5 X 5 X 5 X 5 X 5 X 5 X 5

419 326 502

X

X

CGG CCA TTA CTG TGT TCC CCC 3 X CAG CTG CAT TGA TCT CGG TGA 3 X CGC CTG CAG TCC TTC AAT GAA TA 3 X GTG GAC GGT CTC ACG AAG ACA G 3 X CAT CCT CCT TGA ACA CGG ACT TGC 3 X TCT CCC CAA AGA TAG CAT CTG GAC G 3 X GGT GAA GGT CGG TGT CAA CGG ATT T 3 X GAT GCC AAA GTT GTC ATG GAT GAC C 3

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dehydrogenase ŽGAPDH. DNA sequences w7–9,16x. Sense and anti-sense primers were purchased from Genosys ŽThe Woodlands, TX.. The dried oligos were dissolved in DEPC-H 2 0 to a concentration of 0.5 m grm l for iNOS and COX-1 and 15 m M for COX-2 and GAPDH. The sequences of the primers are shown in Table 1. 2.3.3. First strand cDNA synthesis For comparison of samples, total RNA was converted to cDNA so that amplification of specific genes could be performed by reverse transcriptase polymerase chain reaction ŽRT-PCR.. First strand cDNA synthesis was performed following the procedures specified by the manufacturer for the following chemicals: RNasin Ribonuclease Inhibitor Ž40 Urm l, Promega, Madison, WI., deoxynucleotide triphosphates ŽPharmacia Biotech, Piscataway, NJ., random primers ŽNew England Biolabs, Beverly, MA., and SuperScript II Reverse Transcriptase Ž200 Urm l, Gibco-BRL, Grand Island, NY.. 2.3.4. Amplification of cDNA Synthesized cDNA was amplified by the polymerase chain reaction ŽPCR. using standard PCR methods with the thermostable Taq DNA Polymerase Ž5 Urm l, GibcoBRL, Grand Island, NY.. The PCR reactions were conducted in a DNA Thermal Cycler ŽPerkin-Elmer Corp., Norwalk, CT. and cycle parameters were as follows: Ž1. Time delay cycle at 948C for 5 min, Ž2. Step cycle for 14 to 30 cycles: Ža. 948C for 1 min, Žb. 608C for 2 min, and Žc. 728C for 3 min, and Ž3. Soak cycle for 48C. 2.3.5. RT-PCR product analysis The reaction products were analyzed by electrophoresis on 1.5% agarose gels. A 100 bp DNA ladder was also run in the gel obtained from Gibco-BRL ŽGrand Island, NY.. Gels were stained with SYBR Green I Nucleic Acid Gel Stain ŽMolecular Probe Inc., Eugene, OR. and were scanned on a Molecular Dynamics Fluroimager using ImageQuaNT Software ŽMolecular Dynamics Corp., Sunnyvale, CA.. In volume integration, ImageQuant sums the pixel values within a present quadrant, subtracting background, and the integrated volume of each RT-PCR enzyme product was normalized to the integrated volume of GAPDH. RT-PCR products were obtained from every other cycle from 14 to 30 cycles performed once and ran on two separate gels. Integrated volume values were analyzed by linear analysis to obtain the optimal cycle numbers to utilize for ibuprofen inhibition studies. The optimal range for quantitative analysis of iNOS, COX-1, and COX-2 RT-PCR products was between 14–22 cycles Ž r s 0.998., 22–28 cycles Ž r s 0.999., and 20–26 cycles Ž r s 0.987., respectively. For ibuprofen inhibitory studies, RTPCR products for iNOS were determined at cycle 20 and 22, from one experiment each, and for COX-1 and COX-2 at cycle 24 and 22, respectively, from two experiments each.

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2.4. Detection of iNOS protein by western blotting For the detection of iNOS protein levels, glial cells were treated with ibuprofen or vehicle and LPS plus INFg and incubated for 16 h at 378C as described in the cell culture methods. Incubation was terminated by washing cells twice with phosphate buffered saline and cells were solubilized in hot Ž808C. sodium dodecyl sulfate ŽSDS. polyacrylamide gel electrophoresis ŽPAGE. sample buffer. Western blotting was carried out by separation of proteins by SDS-PAGE and transferring proteins to nitrocellulose by a Semi-Dry Blotter apparatus ŽEnprotech Integrated Separation Systems, Natick, MA.. Rainbow Coloured Protein Molecular Weight Markers ŽAmersham Corp., Arlington Heights, IL. were also separated and transferred with the protein samples. Immunodetection of iNOS protein was performed by blocking nitrocellulose with 5% non-fat dry milk in TTBS Ž20 mM Tris, 500 mM NaCl, 0.1% Tween-20, pH 7.5. for 60 min, followed by incubation for 60 min with rabbit anti-mouse iNOS antibody Ž1.0 m grml, NOS2 ŽM19., Santa Cruz Biotech. Inc., Santa Cruz, CA.. The nitrocellulose was washed 3 times for 5 min each in TTBS and added to 5% non-fat dry milk in TTBS containing goat anti-rabbit immunoglobulins conjugated to horseradish peroxidase Ž1:5000, DAKO Corp., Carpinteria, CA. and was incubated for 60 min. Following three washes with TTBS for 5 min each, iNOS was detected by enhanced chemiluminescence ŽECL kit from Amersham Corp., Arlington Heights, IL.. ECL-treated nitrocellulose was exposed to Hyperfilm-MP ŽAmersham Corp., Arlington Heights, IL. at different time intervals to determine adequate development. Films were analyzed by obtaining optical densities ŽOD. with the RAS-1000 image analyzer from Loats Associates, Inc. ŽWestminster, MD.. To determine optimal total cell protein concentration needed for inhibition studies, total cell protein was added in increments of 10 m grwell Ž10 to 50 m g., and protein samples were run in duplicate. The best linear correlation from a plot of total cell protein versus OD was seen at a 1 min film exposure giving a correlation coefficient of r s 0.977. The midpoint of the linear curve was at 30 m g. Therefore, for determination of the effect of ibuprofen on iNOS protein expression, 30 m g of total-cell-proteinrwell was used for Western blotting, and ECL-treated nitrocellulose was exposed to Hyperfilm-MP for 1 min. There was no non-specific binding of the secondary antibody determined by incubating nitrocellulose paper without primary antibody. Proteins from each treatment group were run in duplicate, and the IC 50 of ibuprofen was determined by non-linear analysis. 2.5. PGE2 formation Glial cells were treated with ibuprofen or vehicle and LPS plus INFg at the same time, and incubated for 16 h at 378C as described in the cell culture methods. Incubation

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was terminated by washing cells twice with phosphate buffered saline and frozen at y808C until used for the assay. The prostaglandin PGE 2 was measured by radioimmunoassay using the Prostaglandin PGE 2 w125 Ix FlashPlate Assay kit from DuPont NEN ŽBoston, MA.. On the day of assay, 0.4 ml of buffer was added to each well and the cells were allowed to thaw for 10 min. Subsequently, the cell suspension was obtained by sonicating ŽVirSonic 475 sonicator. each well for 30 s. The cell suspension was transferred to microfuge tubes and the samples were centrifuged for 4 min at 12,000 rpm. The supernatant was used for radioimmunoassay. A concentration curve using 1–1000 m g of PGE 2 was run with each assay. The concentration of PGE 2 in the cell suspension was determined by linear regression analysis. Two separate experiments were performed with 6 concentrations each in triplicate. Non-linear analysis was used to determine IC 50 s for each curve, and the mean IC 50 was calculated by an average weighting method.

Fig. 2. Effect of ibuprofen on iNOS mRNA Župper box. and iNOS protein expression Žlower box. in primary glial cell cultures treated at the same time with ibuprofen, LPS ŽL., and INFg ŽI. for 16 h. Bands were observed in appropriate gradient areas for iNOS mRNA Ž540 base pairs. and iNOS protein Ž130 kDa..

3. Results 3.1. iNOS actiÕity, mRNA, and protein analysis Ibuprofen, up to 6 mM, had no effect on catalytic activity of iNOS isolated from glial cells treated with LPS and IFNg for 16 h. However, when glial cells were treated with ibuprofen in combination with LPS and IFNg for 16 h, a concentration-dependent inhibition of iNOS activity was observed, with an IC 50 of 0.76 " 0.01 mM Žmean " SE, Fig. 1.. Glial cells treated with LPS and IFNg demonstrated a 4-fold increase in iNOS mRNA levels as compared to control cell cultures ŽFig. 2.. Ibuprofen concentration-dependently decreased iNOS mRNA levels, and the effect was significant only at 2 mM Ž P - 0.05; Fig. 2., with ibuprofen reducing 33.3 " 8.3% Žmean " SE. of iNOS

Fig. 1. Effect of ibuprofen on iNOS activity Žv ., iNOS protein expression ŽB., and PGE 2 formation Ž'. in primary cerebellar glial cell cultures treated at the same time with ibuprofen, LPS, and INFg for 16 h.

mRNA at this concentration. Thus, the IC 50 of ibuprofen for decreasing iNOS mRNA was ) 2.0 mM. Western blotting detected iNOS protein only in cells treated with LPS and INFg ŽFig. 2., and ibuprofen concentration-dependently decreased iNOS protein expression, giving an IC 50 of 0.89 " 0.05 mM Žmean " SE, Figs. 1 and 2.. 3.2. COX-1 and COX-2 mRNA and PGE2 formation Glial cells treated with LPS and INFg displayed a 4-fold increase in COX-2 mRNA compared to control cell cultures, and there was no change in COX-1 mRNA levels ŽFig. 3.. This is in agreement with previously reported observations w11x. Ibuprofen had no effect on COX-1 or COX-2 mRNA expression Ž P - 0.05; Fig. 3.. LPS and INFg caused a 6-fold increase in PGE 2 formation as compared to control cell cultures and is consistent with that described previously w11x. Ibuprofen concentra-

Fig. 3. Effect of ibuprofen on COX-1 and COX-2 mRNA determined by RT-PCR from primary glial cell cultures treated at the same time with ibuprofen, LPS ŽL., and INFg ŽI. for 16 h. Bands were observed in appropriate gradient areas for COX-1 mRNA Ž419 base pairs. and COX-2 mRNA Ž326 base pairs..

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tion-dependently inhibited PGE 2 formation in treated cells, with an IC 50 of 0.86 " 0.02 mM Žmean " SE, Fig. 1.. The concentration-dependent response curves of ibuprofen on inhibition of indirect iNOS activity, iNOS protein expression, and PGE 2 formation were similar, as were their IC 50 s obtained from these curves ŽFig. 1.. 4. Discussion Ibuprofen reduced iNOS activity in glial cells when incubated with LPS and IFNg for 16 h, and it had no effect on direct catalytic activity of this enzyme. This is consistent with previously reported findings w1x. To understand the mechanism of ibuprofen reduction of iNOS activity, the effect of this drug on iNOS gene and protein expression were investigated. The IC 50 of ibuprofen for reducing iNOS mRNA levels was found to be greater than 2 mM, which does not correlate with the potency of this drug for inhibition of iNOS activity ŽIC 50 s 0.76 mM. or reduction of iNOS protein ŽIC 50 s 0.89 mM.. The similar potencies for decreasing iNOS activity and iNOS protein levels suggest that ibuprofen may inhibit post-transcriptional processing of iNOS enzyme. To determine if the concentration of ibuprofen needed to inhibit iNOS activity could be physiologically relevant in vivo, the effect of ibuprofen on cyclooxygenase was also investigated. Ibuprofen had no effect on either COX-1 or COX-2 mRNA levels. However, ibuprofen concentration-dependently inhibited PGE 2 formation, with an IC 50 of 0.86 mM. The reduction in PGE 2 formation may be due to inhibition of cyclooxygenase, and in support of this observation, Mitchell et al. w12x reported an inhibitory effect of ibuprofen on COX-2 activity, with an IC 50 of 0.70 mM. A phenomena similar to that observed in the present investigation has also been reported with other NSAIDs in which no effect on COX-1 or COX-2 mRNA levels were seen, yet cyclooxygenase enzymatic activity, determined by measuring PGE 2 formation, was inhibited w15x. It is relevant that the potency of ibuprofen for reduction of PGE 2 formation was comparable to the potency of inhibition of iNOS activity observed in our investigations. Since the anti-inflammatory mechanism of ibuprofen and other drugs in its class may be due to inhibition of cyclooxygenase and the potencies of ibuprofen for both PGE 2 formation and iNOS activity are similar, it is likely that ibuprofen would decrease iNOS activity in vivo.

5. Conclusions In conclusion, the reduction of iNOS protein by ibuprofen presents a novel mechanism of therapeutic action of this NSAID in inflammatory disorders. The decrease of iNOS activity in brain following treatment with ibuprofen

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may have a significant impact in the prevention of immune-mediated neurodegenerative disorders by reducing NO-mediated neuronal injury.

Acknowledgements The authors would like to thank R.F. Krzesicki for supplying COX-2 and GAPDH primers and for Fluroimager technical assistance, and E. Dunn for assistance with Western blotting methodology.

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