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2-hydroxymethyl-1-naphthol diacetate (TAC) suppresses the superoxide anion generation in rat neutrophils
Free Radical Biology & Medicine, Vol. 26, Nos. 7/8, pp. 1010 –1018, 1999 Copyright © 1999 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/99/$–see front matter
PII S0891-5849(98)00288-3
Original Contribution 2-HYDROXYMETHYL-1-NAPHTHOL DIACETATE (TAC) SUPPRESSES THE SUPEROXIDE ANION GENERATION IN RAT NEUTROPHILS JIH-PYANG WANG,*† LO-TI TSAO,* AI-YU SHEN,‡ SHUE-LING RAUNG,*
and
LING-CHU CHANG*
†
*Department of Medical Research, Taichung Veterans General Hospital, Taichung, Taiwan, Graduate Institute of Pharmaceutical Chemistry, China Medical College, Taichung, Taiwan, ‡Department of Biomedical Science, Foo Yin Institute of Technology, Kaohsiung, Taiwan (Received 8 May 1998; Revised 9 October 1998; Accepted 9 October 1998)
Abstract—We have investigated the inhibitory effect of 2-hydroxymethyl-1-naphthol diacetate (TAC) on the respiratory burst of rat neutrophils and the underlying mechanism of action was also assessed in this study. TAC caused concentration-related inhibition of the formylmethionyl-leucyl-phenylalanine (fMLP) plus dihydrocytochalasin B (CB)and phorbol 12-myristate 13-acetate (PMA)-induced superoxide anion (O2•2) generation (IC50 10.2 6 2.3 and 14.1 6 2.4 mM, respectively) and O2 consumption (IC50 9.6 6 2.9 and 13.3 6 2.7 mM, respectively) of neutrophils. TAC did not scavenge the generated O2•2 during dihydroxyfumaric acid autoxidation. TAC inhibited both the transient elevation of [Ca21]i in the presence or absence of [Ca21]o (IC50 75.9 6 8.9 and 84.7 6 7.9 mM, respectively) and the generation of inositol trisphosphate (IP3) (IC50 72.0 6 9.7 mM) in response to fMLP. Cytosolic phospholipase C (PLC) activity was also reduced by TAC at a same range of concentrations. The PMA-induced PKC-b associated to membrane was attenuated by TAC (about 80% inhibition at 30 mM). Upon exposure to fMLP, the cellular cyclic AMP level was decreased in neutrophils pretreated with TAC. TAC attenuated fMLP-induced phosphorylation of mitogen-activated protein kinase (MAPK) p42/44 (IC50 17.4 6 1.7 mM), but not p38. The cellular formation of phosphatidic acid (PA) and, in the presence of ethanol, phosphatidylethanol (PEt) induced by fMLP was inhibited by TAC in a concentrationdependent manner (IC50 25.4 6 2.4 and 25.9 6 1.4 mM, respectively). TAC had no effect on the O2•2 generation of PMA-stimulated and arachidonic acid (AA)-stimulated NADPH oxidase preparations. However, TAC caused concentration-related decrease of the membrane associated p47phox in PMA-stimulated neutrophils (about 80% inhibition at 30 mM). We conclude that inhibition by TAC of the neutrophil respiratory burst is probably attributable to the blockade of the p42/44 MAPK and phospholipase D (PLD) pathways, the membrane translocation of PKC, and to the failure in assembly of a functional NADPH oxidase complex. Blockade of the PLC pathway by TAC probably plays a minor role. © 1999 Elsevier Science Inc. Keywords—TAC, Neutrophil, Respiratory burst, NADPH oxidase, Protein kinase C, Phospholipase D, Mitogenactivated protein kinase, Translocation, Phospholipase C, Inositol phosphate, Intracellular Ca21 concentration
INTRODUCTION
known as the NADPH oxidase. This non-mitochondrial O2 consumption process is known as the respiratory burst [2]. Reactive O2 species produced during the respiratory burst are believed to serve as bactericidal agents, as evidenced by the susceptible to recurrent infections by patients with chronic granulomatous disease [2]. Under certain circumstances, the excessive or inappropriate release of these highly reactive O2 species can result in undesirable tissue damage. This is probably involved in the pathogenesis of many diseases [3]. Therefore, a drug that would inhibit the generation of toxic O2 metabolites could terminate this tissue damage.
Neutrophils play an essential role in the body’s defense against bacterial infection. Upon exposure to a pathogenic stimulus, neutrophil becomes activated and generates superoxide anion (O2•2) and other potent toxic O2 metabolites [1]. The generation of these oxidants accompanied by the increase of O2 uptake from surrounding medium by neutrophils occurs through the activation of a membrane-associated complex Address correspondence to: Jih-Pyang Wang, Department of Medical Research, Taichung Veterans General Hospital, 160, Chung-Kung Road, Sec. 3, Taichung, Taiwan 407; Fax: (886) 4-359-2705 1010
TAC inhibits neutrophil O2•2 generation
In resting cells, NADPH oxidase is dormant, the components of the NADPH oxidase are segregated into cytosolic and membrane compartments. On neutrophil activation, the cytosolic components of NADPH oxidase (mainly p47phox and p67phox) translocate to the plasma membrane where they associate with a heterodimeric cytochrome b558, composed of gp91phox and p22phox, resulting a functional complex responsible for the production of oxygen radicals in neutrophils [4]. Intracellular signaling events used in the response of neutrophils to external stimuli are complicated and not clearly defined. Stimulation of neutrophils by receptor-binding ligands results in an intracellular signaling cascade including the activation of phospholipase C (PLC) that releases inositol trisphosphate (IP3) and diacylglycerol, which, in turn, increases intracellular Ca21 concentration and activates protein kinase C (PKC), respectively [5]. The two pathways function synergistically for O2•2 generation. Phospholipase D (PLD) activation is also functionally linked to O2•2 generation in neutrophils [6]. PLD acts upon phosphatidylcholine to release phosphatidic acid (PA), which is then converted into diradylglycerol [7]. In addition, it has been recently suggested that mitogenactivated protein kinase (MAPK) may also participate in the activation of neutrophil respiratory burst [8 –10]. 2-Hydroxymethyl-1-naphthol diacetate (TAC), has been found to possess cytotoxicity against several human carcinoma cell lines and potent antimicrobial activity [11]. TAC was also found through preliminary in vitro tests to inhibit O2•2 generation from activated neutrophils. The primary objective of the present study was to identify the sites and mechanisms of the inhibitory effect of TAC on the respiratory burst in rat peripheral neutrophils.
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inghamshire, UK). Mouse monoclonal antibodies to PKC-b, p38 MAPK, ERK1, and ERK2, and rabbit polyclonal anti-p47phox antibody (Transduction Lab., Lexington, KY). Rabbit polyclonal antibodies to phospho-p38 MAPK and phospho-p42/44 MAPK (New England Biolabs., Beverly, MA). Isolation of neutrophils Rat blood was collected from the abdominal aorta and the neutrophils were purified by dextran sedimentation, hypotonic lysis of erythrocytes, and centrifugation through Ficoll-Hypaque [12]. Purified neutrophils containing .95% viable cell were normally resuspended in Hanks’ balanced salt solution (HBSS) containing 10 mM HEPES, pH 7.4, and 4 mM NaHCO3, and kept in an ice-bath before use. Measurement of O2•2 generation and O2 consumption The O2•2 generation in neutrophil suspension was determined by the superoxide dismutase (SOD)-inhibitable ferricytochrome c reduction as previously described [12,13]. O2•2 generation during dihydroxyfumaric acid autoxidation was determined by the reduction of nitroblue tetrazolium as previously described [14]. Absorbance changes of the reduction of ferricytochrome c and nitroblue tetrazolium were continuously monitored in a double-beam spectrophotometer. Whole cell O2 consumption was continuously measured with a Clark-type oxygen electrode using a YSI biological oxygen monitor [15]. Measurement of [Ca21]i
MATERIALS AND METHODS
Reagents TAC was synthesized as the described previously [11]. All chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) except for the following: dextran T-500 (Pharmacia Biotech., Uppsala, Sweden). Hanks’ balanced salt solution (Gibco BRL Co., Gaithersburg, MD). Diphenylene iodonium (DPI) (Research Biochemicals International, Natick, MA). U73122 (Biomol Research Lab. Inc., Plymouth Meeting, PA). Fura-2 AM and fluo-3 AM (Molecular Probes, Eugene, OR). L-a-Posphatidyl-D-myo-inositol-4,5-bisphosphate (PIP2) (Boehringer Mannheim, Mannheim, Germany). myo-[3H]Inositol, L-3-phosphatidyl[2-3H]-inositol-4,5-bisphosphate ([3H]PIP2), 1-O[3H]octadecyl-sn-glycero-3-phosphocholine, cyclic AMP assay kit, [g-32P]ATP and enhanced chemiluminescence reagent (Amersham International plc., Buck-
Neutrophils (1 3 107 cells/ml) were suspended in HEPES buffer A (composition in mM: NaCl 124, KCl 4, Na2HPO4 0.64, KH2PO4 0.66, NaHCO3 15.2, dextrose 5.56 and HEPES 10, pH 7.4), and loaded with 5 mM fura-2 AM as previously described [13] or loaded with 5 mM fluo-3 AM at 37°C for 45 min. After being washed, cells were resuspended in HEPES buffer A with 0.05% (w/v) bovine serum albumin. The fluorescence of fura2-loaded cells was monitored by a double-wavelength fluorescence spectrophotometer (PTI, Deltascan 4000) at 510 nm with excitation 340 and 380 nm in the ratio mode. Calibration of the excitation ratio in terms of Ca21 concentration was performed as previously described [16]. The fluorescence of fluo-3-loaded cells was monitored at 525 nm with excitation 488 nm. [Ca21]i was calibrated from the fluorescence intensity as follows: [Ca21]i 5 Kd 3 [(F 2 Fmin)/(Fmax 2 F)], where Kd was taken as 400 nM [17].
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Determination of inositol phosphate levels Neutrophils (3 3 107 cells/ml) were loaded with myo-[3H]inositol (83 Ci/mmol) at 37°C for 2 h [13]. Ten seconds after the stimulation with formylmethionylleucyl-phenylalanine (fMLP), reactions were stopped by adding CHCl3:CH3OH (1:1, v/v) mixture and 2.4 M HCl. The aqueous phase was removed and neutralized by 0.4 M NaOH, and then applied to an AG 1-X8 resin (formate) column (Bio-Rad). Inositol monophosphate (IP), inositol bisphosphate (IP2), and IP3 were eluted sequentially by using 0.2, 0.4, and 1.0 M ammonium formate, respectively, in 0.1 M formic acid as eluents, and then counted as previously described [13]. Cytosolic PLC activity Neutrophil cytosolic PLC was isolated and assayed as previously described [18]. Substrate stock was prepared by mixing PIP2 and 5 mCi [3H]PIP2 (1 Ci/mmol) in solvent mixture (20% sodium cholate, 250 mM 2-mercaptoethanol, 2.5 M NaCl, 1 M PIPES, pH 6.8) to produce a 0.361 mM PIP2 solution, sonicated on ice, and then stored at 220°C. PLC activity was assayed by measuring the hydrolysis of PIP2 into inositol phosphates. PKC membrane translocation For the analysis of subcellular distribution of PKC, neutrophils (4 3 107 cells/ml) were stimulated with 0.2 mM phorbol 12-myristate 13-acetate (PMA) for 5 min at 37°C. Reactions were stopped by addition of 4 volume of ice-cold HBSS, and then resuspended in disruption solution (0.34 M sucrose, 10 mM Tris-HCl, pH 7.0, 1 mM phenylmethylsulphonyl fluoride, 1 mM EGTA, 10 mM benzamidine, 10 mg/ml of leupeptin and antipain). After sonication, lysate was centrifuged to remove the unbroken cells, and then further centrifuged at 100,000 g for 30 min at 4°C. Pellet (as membrane fraction) and supernatant (as cytosol fraction) were boiled in Laemmli sample buffer, subjected to SDS-PAGE, transferred to Immobilon-P membrane (Millipore), blocked with 5% non-fat milk in TST buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl and 0.05% Tween-20) and probed with mouse monoclonal antibody to PKC-b (1:500 dilution in TST buffer with 0.5% non-fat milk) [19]. Detection was made using the enhanced chemiluminescence reagent. Measurement of cellular cyclic AMP level Cyclic AMP was determined as described [20]. Neutrophils were preincubated with test drugs for 9.5 min at 37°C. Thirty seconds after addition of fMLP, reaction
mixture was immediately added to 1.0 ml of 0.05 M acetate buffer, pH 6.2, containing 0.05 mM 3-isobutyl1-methylxanthine. After boiling for 5 min, suspension was kept in ice, sonicated and followed by sedimentation. The supernatants were acetylated by addition of 0.025 volume of triethylamine:acetic anhydride (2:1, v/v). Cyclic AMP content in aliquots of the acetylated samples were assayed by using enzyme immunoassay kit.. Western blot analysis of cellular MAPK Neutrophils (1 3 107 cells/ml) in HBSS were preincubated with dimethylsulfoxide (DMSO) or test drugs at 37°C for 5 min before initiating the reaction by adding fMLP plus dihydrocytochalasin B (CB). One minute later, reactions were quenched by addition of stopping solution (20% trichloroacetic acid, 1 mM phenylmethylsulphonyl fluoride, 2 mM N-ethylmaleimide, 100 mM NaF, 2 mM Na3VO4, 10 mM p-nitrophenyl phosphate, 7 mg/ml of leupeptin and pepstatin). Proteins were separated through SDS-PAGE as described above, and probed with rabbit polyclonal anti-phospho-p38 MAPK antibody (1:2000 dilution in TST buffer with 1% BSA) or with rabbit polyclonal anti-phospho-p42/44 MAPK antibody (1:2000 dilution in TST buffer with 0.5% nonfat milk). Detection was made using the enhanced chemiluminescence reagent. To confirm that the same amount of cellular proteins was loaded on each lane, the membranes were stripped, after detection of phospho-p38 MAPK and phospho-p42/44 MAPK, with stripping buffer (100 mM 2-mercaptoethanol, 2% SDS, and 62.5 mM Tris-HCl, pH 6.8) for 30 min at 50°C, and then reprobed with mouse monoclonal anti-p38 MAPK antibody (1:250 dilution in TST buffer with 0.5% non-fat milk), or with mouse monoclonal anti-ERK1 or antiERK2 antibodies (1:1000 to 1:2500 dilution in TST buffer with 0.5% non-fat milk), respectively. Detection was made using the enhanced chemiluminescence reagent. Measurement of PLD activity Neutrophils (5 3 107 cells/ml) were loaded with 10 mCi 1-O-[3H]octadecyl-sn-glycero-3-phosphocholine (150 Ci/mmol) at 37°C for 75 min [21]. Thirty seconds after stimulation with fMLP/CB in the presence or absence of 0.5% ethanol, the lipids in reaction mixture were extracted, dried, and separated on silica gel 60 plate. The plates were developed halfway by using the solvent system consisting of hexane:diethyl ether:methanol:acetic acid (90:20:3:2, v/v/v/v), dried and then developed again to the top using the upper phase of the
TAC inhibits neutrophil O2•2 generation
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Table 1. Concentration Dependence of TAC Inhibition of O2•2 Generation and O2 Consumption in Formylmethionyl-leucyl-phenylalanine (fMLP)- and Phorbol 12-myristate 13-acetate (PMA)-stimulated Neutrophils O2•2 generation (nmol/10 min per 106 cells)
Control TAC
3 mM 10 mM 30 mM
O2 consumption (nmol/5 min per 2 3 106 cells)
fMLP
PMA
fMLP
PMA
6.5 6 0.5 4.7 6 0.3 2.3 6 0.5* 0.5 6 0.2*
16.4 6 1.3 12.9 6 2.1 7.2 6 1.8* 3.5 6 0.4*
14.4 6 0.5 10.9 6 1.0 4.6 6 1.5* 0.5 6 0.5*
39.3 6 1.3 32.5 6 2.8 21.4 6 2.8* 4.4 6 0.5*
Neutrophils were preincubated with DMSO (vehicle control) or TAC for 3 min before stimulation with 0.3 mM fMLP plus 5 mg/ml of CB and 1 nM PMA for O2•2 generation, or 1 mM fMLP plus 5 mg/ml of CB and 3 nM PMA for O2 consumption. Values are expressed as mean 6 SEM for four to five separate experiments. *P , .01 compared to the corresponding control values.
solvent system consisting of ethylacetate:isooctane:acetic acid:water (110:50:20:100, v/v/v/v). The lipids were located by staining with iodine vapor. The radioactivity of [3H] products were directly quantified with a PhosphorImager (Molecular Dynamics 445 SI) using ImageQuaNT software. phox
NADPH oxidase activity and p47 translocation
membrane
For the arachidonic acid (AA)-induced NADPH oxidase activation, the subcellular fractions of neutrophils were prepared as previously described [21]. PMA-stimulated particulate NADPH oxidase was isolated as previously described [13]. The NADPH oxidase activity was measured spectrophotometrically at 28°C by detecting the SOD-inhibitable ferricytochrome c reduction as previously described [21]. For the analysis of the subcellular distribution of p47phox, neutrophils (2 3 107 cells/ml) in HBSS were preincubated with DMSO or test drugs at 37°C for 5 min before stimulation with 0.2 mM PMA. Five minutes later, four volume of ice-cold HBSS was added. Cells were disrupted by sonication, layered on discontinuous sucrose gradients, and centrifuged at 100,000 g for 60 min [22]. The band between 20 and 50% sucrose was collected, washed with 2 volume of 0.45 M NaCl, and centrifuged at 10,000 g for 10 min. The supernatant was further centrifuged at 100,000 g for 60 min. The NaCl-washed membrane pellet was then boiled in Laemmli sample buffer. Proteins were separated through SDS-PAGE as described above, and then probed with rabbit polyclonal anti-p47phox antibody (1: 500 dilution in TST buffer with 0.5% non-fat milk). Statistical analysis Statistical analyses were performed using the Bonferroni t test method after analysis of variance. A P value less than 0.05 was considered significant for all tests.
Analysis of the regression line test was used to calculated IC50 values. Means are represented as mean 6 standard error of the mean (SEM). RESULTS
Neutrophils exposed to fMLP/CB demonstrate a rapid and transient O2•2 generation and O2 consumption, whereas, PMA evoked a slow and long lasting O2•2 generation and O2 consumption. Concentration-dependent inhibition of O2•2 generation and O2 consumption were observed in cells pretreated with TAC for 3 min prior to stimulation with fMLP/CB or PMA (Table 1). Inhibition of fMLP/CB- and PMA-induced O2•2 generation with IC50 values of 10.2 6 2.3 and 14.1 6 2.4 mM, respectively, and O2 consumption with IC50 values of 9.6 6 2.9 and 13.3 6 2.7 mM, respectively. More than 95% viability was observed with trypan blue exclusion in cells treated with 100 mM TAC for 10 min. The scavenging ability of drug was assessed by using a cell-free oxygen radical generating system. Unlike SOD, which significantly reduced the O2•2 generation during dihydroxyfumaric acid autoxidation [13], TAC had no effect in this respect (0.060 6 0.004 DA560 as control vs 0.060 6 0.011 DA560 at 100 mM TAC, P . 0.05). Addition of fMLP to fura-2-loaded cells in the presence of [Ca21]o or to fluo-3-loaded cells in the absence of [Ca21]o, both induced rapid and transient elevation of [Ca21]i, however, much less extensive [Ca21]i changes were observed in the absence of [Ca21]o. Fura-2 can be used in the ratio mode, making the measurement of [Ca21]i independent of several artifacts. However, fluo-3 undergo a greater enhancement of emission intensity upon Ca21 binding, making fluo-3 useful for measuring the kinetics of Ca21 transient at lower levels. Pretreatment of cells with a PLC inhibitor U73122 (1 mM) abolished the fMLP-induced [Ca21]i changes in the presence or absence of [Ca21]o [21,23]. TAC also concentration-dependently inhibited the [Ca21]i changes of
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Fig. 1. Effect of TAC on formylmethionyl-leucyl-phenylalanine (fMLP)-induced [Ca21]i changes in (A) fura-2-loaded neutrophils in the presence of 1 mM CaCl2 in medium or (B) fluo-3-loaded neutrophils in the presence of 1 mM EDTA in medium. Cell suspension was preincubated with DMSO (vehicle control) or 30 –200 mM TAC at 37°C for 3 min before addition of 0.1 mM fMLP. [Ca21]i levels were monitored in the excitation ratio 340/380 nm mode with emission wavelength at 510 nm for fura-2-loaded cells, or in the excitation and emission wavelength at 488 and 525 nm, respectively, for fluo-3-loaded cells. The results shown are representative of four to five separate experiments.
fMLP-stimulated neutrophils whether [Ca21]o was present or not (IC50 values 75.9 6 8.9 and 84.7 6 7.9 mM, respectively) (Fig. 1). Myo-[3H]inositol-loaded neutrophils exposed to fMLP evoke a significant increase of cellular IP2 and IP3 (both P , 0.01) levels. Pretreatment of cells with U73122 and TAC reduced the fMLP-induced IP2 and IP3 formation in a concentration-dependent manner (Fig. 2A,B). IC50 values of U73122 and TAC for the inhibition of IP3 formation were estimated to be 13.4 6 0.8 and 72.0 6 9.7 mM, respectively. Upon exposure to CaCl2 and PIP2, neutrophil cytosolic PLC was activated and used PIP2 as substrate to produce inositol phosphate. U73122 (10 mM) greatly reduced PLC activity (68.2 6 5.5% inhibition). Concentration-related inhibition of PLC activity was also observed in TAC-treated reaction mixture (41.9 6 6.1% inhibition at 100 mM TAC) (Fig. 2C). Subcellular distribution of PKC in activated neutrophils was assessed by immunoblotting with a specific monoclonal anti-PKC-b antibody. In the resting cells, PKC-b was enriched in the cytosol fraction. Upon exposure to PMA, PKC-b was translocated from the cytosol to the membrane. This enzyme translocation can be used as an index of the enzyme activation. Pretreatment of PMA-stimulated cells with TAC reduced the membrane associated PKC-b (about 80% inhibition at 30 mM TAC) (Fig. 3), whereas TAC alone had no effect on the membrane translocation of PKC-b. Analysis of the cyclic AMP levels in neutrophils showed that a significant increase was observed in cells treated with 10 mM forskolin (2.79 6 0.47 vs 0.61 6 0.11 pmol per 2 3 106 cells, P , 0.01), an adenylate cyclase activator [24]. On the contrary, TAC decreased
Fig. 2. Effect of TAC on formylmethionyl-leucyl-phenylalanine (fMLP)-induced cellular inositol phosphate formation and on cytosolic PLC activity. Cellular (A) IP2 and (B) IP3 levels were determined in myo-[3H]inositol-loaded cells pretreated with DMSO (vehicle control), 10 –100 mM TAC (E) or 0.3-30 mM U73122 (h) in the presence of 10 mM LiCl at 37°C for 3 min prior to stimulation with 0.3 mM fMLP. The resting levels of IP2 and IP3 were measured from the cells which exposed to DMSO without fMLP challenge (●). (C) Cytosolic PLC was preincubated with DMSO (vehicle control), 10 –100 mM TAC or 10 mM U73122 for 3 min at 37°C before addition of 100 mM CaCl2 and 48 mM PIP2/[3H]PIP2. Reaction was stopped by addition of ice-cold CHCl3:CH3OH:HCl (50:50:1, v/v/v) mixture. Inositol phosphate formation in the upper aqueous phase was determined. Values are expressed as mean 6 SEM for four to five separate experiments. *P , .01 compared to the corresponding control values.
the cellular cyclic AMP level (0.16 6 0.10 pmol per 2 3 106 cells at 30 mM TAC, P , 0.01). Moreover, TAC (up to 100 mM) did not affect the porcine heart protein kinase A activity in the presence or absence of cyclic AMP (data not shown). Immunoblot analysis with specific anti-phosphop42/44 or anti-phospho-p38 MAPK antibody was performed to determine the effect of TAC on MAPK pathways in neutrophils. Following stimulation with fMLP/ CB, as shown in Figure 4, cellular p42/44 and p38 MAPKs become phosphorylated and hence activated. TAC specifically inhibited p42/44 but not p38 MAPK in a concentration dependent manner (IC50 about 17.0 6 1.0 mM). However, TAC alone at high concentration (100 mM) significantly increased the phosphorylation of p38 MAPK (Fig. 4B). The differences observed for the induced protein phosphorylation of cellular MAPKs did not result from differences in loading or from cellular protein digestion. The anti-ERK1 that we used also recognizes ERK2 (p42). Upon exposure to fMLP/CB, a significantly increased (P , 0.01) the formation of PA and, in the presence of 0.5% ethanol, the corresponding phosphatidylethanol (PEt)
TAC inhibits neutrophil O2•2 generation
Fig. 3. Effect of TAC on phorbol 12-myristate 13-acetate (PMA)induced PKC-b membrane translocation. Neutrophils were preincubated with DMSO (lane 2) or 10 –30 mM TAC (lanes 3– 4) for 5 min at 37°C and then stimulated with 0.2 mM PMA for another 5 min. Cells may also react with DMSO (lane 1) or 10 –30 mM TAC (lanes 5– 6) alone for total 10 min reaction time. After termination of reaction, cells were disrupted, and then the membrane and cytosol proteins were subjected to SDS-PAGE. Each lane contains sample protein 50 mg. Analysis was performed by immunoblotting with a monoclonal antibody to PKC-b. The results shown are representative of three separate experiments.
was observed in [3H]alkyl-lysophosphatidylcholine-loaded neutrophil suspension. The cellular levels of PA and PEt in response to fMLP/CB were reduced by genistein, a tyrosine kinase inhibitor [25], in parallel. In addition, TAC concentration-dependently inhibited the fMLP/CB-induced PA and PEt formation with IC50 values of 25.4 6 2.4 and 25.9 6 1.4 mM, respectively (Fig. 5). In PMA-stimulated neutrophil particulate NADPH oxidase preparation, addition of NADPH to initiate O2•2 generation by detecting the reduction of ferricytochrome c and diphenylene iodonium (DPI), an inhibitor of NADPH oxidase [26], inhibited O2•2 generation. In an AA-stimulated cell-free system, addition of AA induced the assembly of the cytosolic and membrane components of NADPH oxidase and then generation of O2•2 in the presence of NADPH. Trifluoperazine (TFP), an inhibitor of NADPH oxidase [27], inhibited O2•2 generation in this cell-free system (Table 2). In contrast, TAC (up to 100 mM) failed to reduce the O2•2 generation of either system. In PMA-stimulated neutrophils, the membrane associated p47phox was increased as assessed by immunoblotting with a polyclonal anti-p47phox antibody (Fig. 6, lanes 1–2, arrow). TAC concentration-dependently inhibited the p47phox membrane translocation in neutrophils in response to PMA (about 80 and 90% inhibition at 30 and 100 mM TAC, respectively) (Fig. 6).
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Fig. 4. Effect of TAC on formylmethionyl-leucyl-phenylalanine (fMLP)-induced the activation of MAPKs in rat neutrophils. Neutrophils were preincubated with DMSO (lane 2) or 3–100 mM TAC (lanes 3– 6) for 5 min at 37°C and then stimulated with 0.1 mM fMLP plus 5 mg/ml of CB (lanes 2– 6) for 1 min. Effect of DMSO (lane 1) or 100 mM TAC (lane 7) alone on the cells was also studied for total 6 min reaction time. After termination of reaction, cells were rapidly sedimented, boiled in Laemmli sample buffer, and subjected to SDSPAGE. Each lane contains sample protein 60 mg. Analysis was performed by immunoblotting with (A) anti-phospho-p42/44, anti-ERK1, anti-ERK2, and (B) anti-phospho-p38 or anti-p38 MAPK antibody. The results shown are representative of four separate experiments.
DISCUSSION
We have shown that the fMLP- and PMA-induced neutrophil O2•2 generation and O2 consumption are inhibited in a concentration-dependent manner by TAC. Using a cell-free oxygen radical generation system to assess the scavenging effect of TAC, we did not observe a significant reduction of the O2•2 generation by TAC, preclude the possibility that TAC acts as a O2•2 scavenger but probably as an inhibitor of certain signal transduction steps that follows after activation of neutrophils. Pharmacologically increase cellular cyclic AMP level is one recognized means to down-regulated O2•2 generation in neutrophils [28]. The inhibitory action of cyclic AMP on neutrophils appears to depend on the activation of protein kinase A. TAC did not increase the porcine heart protein kinase A activity whether the cyclic AMP was present or not. In addition, the cellular cyclic AMP level was not increased by TAC. Thus, there is no indications that inhibition of respiratory burst by TAC is through the activation of protein kinase A. fMLP- but not PMA-induced O2•2 generation is a 21 Ca -dependent process [29]. Unlike the PLC inhibitor
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Fig. 5. Effect of TAC on PLD activity of rat neutrophils. 1-O-[3H]octadecyl-sn-glycero-3-phosphocholine-loaded cells were pretreated with DMSO (vehicle control), 3–100 mM TAC or 30 mg/ml of genistein for 3 min at 37°C in the presence of 0.5% ethanol before addition of 1 mM fMLP plus 5 mg/ml of CB. The lipids were extracted from the reaction mixture, separated on silica gel 60 plates, and the phosphatidic acid (PA) (open column) and phosphatidylethanol (PEt) (crosshatched column) were quantified by phosphor screen autoradiography. The level of PA and PEt in fMLP/CB-stimulated cells in the absence of 0.5% ethanol was also shown (the 1st pair of columns). Values are expressed as mean 6 SEM for four separate experiments. *P , .05, **P , .01 compared to the corresponding control values.
U73122 [23], TAC attenuated fMLP-induced [Ca21]i changes in the presence and absence of [Ca21]o. In the presence of EDTA to remove extracellular Ca21 and to diminish the Ca21-influx during cells activation, TAC inhibited the fMLP-induced [Ca21]i elevation over the same range of concentrations as its used in the presence of [Ca21]o. In addition, we have demonstrated that both the formation of IP2 and IP3 in intact cells and the neutrophil cytosolic PLC activity are inhibited by TAC Table 2. Effect of TAC on NADPH Oxidase Activity in the Cell-free Systems NADPH oxidase activity
Control TAC DPI TFP
100 mM 3 mM 100 mM
PMA-stimulated (nmol O2•2/10 min per 6 3 106 cells eq.)
AA-stimulated (nmol O2•2/10 min per 107 cells eq.)
2.78 6 0.18 2.60 6 0.08 0.93 6 0.22* —
3.29 6 0.11 3.10 6 0.52 — 1.27 6 0.31*
Values are expressed as mean 6 SEM for four to five separate experiments. *P , .01 compared to the corresponding control values. —, not determined.
Fig. 6. Effect of TAC on phorbol 12-myristate 13-acetate (PMA)induced p47phox membrane translocation. Neutrophils were preincubated with DMSO (lane 2), 10-100 mM TAC (lanes 3–5) for 5 min at 37°C and then stimulated with 0.2 mM PMA for another 5 min. Cells may also react with DMSO (lane 1) or 100 mM TAC (lane 6) alone for total 10 min reaction time. After termination of reaction, cells were disrupted, and the membrane proteins were subjected to SDS-PAGE. Each lane contains sample protein 50 mg. Analysis was performed by immunoblotting with a polyclonal anti-p47phox antibody. The results shown are representative of three separate experiments.
in a same range of concentrations as its used in [Ca21]i assay providing strong evidence for the blockade of PLC pathway by TAC in fMLP-stimulated neutrophils. Since higher concentrations of TAC had to be used to inhibit PLC pathway than respiratory burst, suggesting the blockade of this signaling pathway plays a minor role in the inhibition of O2•2 generation by TAC. Rat neutrophil contains PKC isoforms of four categories: conventional PKCs (a, b, and g), novel PKCs (d, e, and u), atypical PKCs (i, l, and z), and PKC-m, although PKC-l and -z are barely detected [19]. However, the isoform specific regulation and function remain unclear yet. Endogenous PKC activator diacylglycerol is generated follows after activation of fMLP-receptor, whereas PMA bypass the membrane receptor and directly activate PKC [30]. It has been reported that PKC-b plays a role in the phosphorylation of membranes associated p47phox and the assembly or maintenance of an active NADPH oxidase [31]. Through the use of monoclonal antibody that specifically bind to PKC-b, we have shown that PKC-b is primarily cytosolic in unstimulated neutrophils but become associate with the membrane fraction after PMA treatment reconciling the earlier report [32]. TAC reduced the immunoreactivity at the membrane fraction of PMA-stimulated neutrophils indicates that the activation of PKC in intact cells might be suppressed by TAC. MAPKs have been demonstrated to play an important role in mediating intracellular signal transduction and regulating cellular functions. However, the roles of MAPKs in neutrophils are not well understood yet. It has been reported that activation of cellular p42/44 and p38
TAC inhibits neutrophil O2•2 generation
MAPKs are probably indispensable for the fMLP-induced O2•2 generation [8 –10], although the correlation between p42/44 MAPK activation and activation of oxidase has not been observed by all investigators. Since both p38 and p42/44 MAPKs phosphorylated p47phox at the same site and at similar rates [33], MAPKs might have a role in the translocation of this oxidase component to the plasma membrane and activation of the respiratory burst. TAC attenuated fMLP-induced phosphorylation of p42/44 MAPK, but did not reduce the immunoreactivity of phospho-p38 MAPK, with an IC50 value closed to that of the inhibition of O2•2 generation. Several lines of evidence indicate that PLD activation is functionally linked to the O2•2 generation in neutrophils [6,34]. PLD catalyzes the hydrolysis primarily of phosphatidylcholine to produce PA. In the presence of ethanol, PA via a transphosphatidylation reaction yield PEt. PA could act on the respiratory burst through the activation of PKC or NADPH oxidase [22,35]. TAC and genistein suppressed both of the PA and PEt formation in neutrophils in response to fMLP. Because the inhibition of PLD pathway and respiratory burst by TAC over the same range of concentrations, blockade of this signaling pathway might play an important role in the inhibition of respiratory burst by TAC. The O2•2generating NADPH oxidase complex in neutrophils is constituted of a heterodimeric cytochrome b558 and cytosolic factors, mainly p47phox and p67phox [4]. Upon activation, p47phox is phosphorylated, the polyproline motif of p47phox would then be accessible to the C-terminal SH3 domain of p67phox [36]. This new interaction changes the overall structure of the complex and makes it able to recognize the membrane cytochrome b558 through the polyproline motif of p22phox and one of the SH3 domains of p47phox [37], favorable to electron transport, and thereby proceeding to the univalent reduction of O2 [4]. TAC decreased the membrane associated p47phox in neutrophils in response to PMA. In contrast, TAC affected the oxidase activity neither in PMA-stimulated neutrophil particulate NADPH oxidase preparation, in which a functional oxidase complex has been already formed, nor in an AA-stimulated cell-free system, in which oxidase complex is assembled through a PKC-independent manner [38]. These results indicate that TAC did not influence the assembly of a functional NADPH oxidase complex in vitro, nor suppressed the activity of assembled oxidase. The inhibition of O2•2 generation by TAC seems not simply through its 1-naphthol structure because 1-naphthol did not affect the fMLP-induced rise of [Ca21]i and 1,4-naphthoquinone, the active metabolite of 1-naphthol, prevents the assembly of a functional NADPH oxidase in the cell-free system [39]. In summary, we demonstrated that TAC is capable of
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inhibiting the respiratory burst (IC50 about 10-14 mM) in neutrophils in response to fMLP as well as to PMA. The underlying mechanism of action was also assessed. TAC acts as neither an O2•2scavenger nor a cyclic AMP-elevating agent. Inhibition of respiratory burst in neutrophils by TAC is probably attributable mainly to the blockade of p42/44 MAPK (IC50 about 17 mM), PKC (IC50 # 30 mM) and PLD (IC50 about 25 mM) pathways, and thereby, influence of the NADPH oxidase assembly (IC50 # 30 mM). Whereas, the blockade of PLC/Ca21 pathway (IC50 about 72–85 mM) may play a minor role in the mechanisms of inhibition by TAC of neutrophil respiratory burst. Acknowledgements — This work was supported by grants from the National Science Council of the Republic of China (NSC-87-2314-B075A-013).
REFERENCES [1] Badwey, J. A.; Karnovsky, M. L. Active oxygen species and the functions of phagocytic leukocytes. Annu. Rev. Biochem. 49:695– 726; 1980. [2] Smith, R. M.; Curnutte, J. T. Molecular basis of chronic granulomatous disease. Blood 77:673– 686; 1991. [3] Halliwell, B.; Gutteridge, J. M. Role of free radicals and catalytic metal ions in human disease: An overview. Methods Enzymol. 186:1– 85; 1990. [4] Segal, A. W.; Abo, A. The biochemical basis of the NADPH oxidase of phagocytes. Trends Biochem. Sci. 18:43– 47; 1993. [5] Berridge, M. J. Inositol trisphosphate and diacylglycerol: Two interacting second messengers. Annu. Rev. Biochem. 56:159 –193; 1987. [6] Yasui, K.; Yamazaki, M.; Miyabayashi, M.; Tsuno, T.; Komiyama, A. Signal transduction pathway in human polymorphonuclear leukocytes for chemotaxis induced by a chemotactic factor: Distinct from the pathway for superoxide anion production. J. Immunol. 152:5922–5929; 1994. [7] Billah, M. M.; Eckel, S.; Mullmann, T. J.; Egan, R. W.; Siegel, M. I. Phosphatidylcholine hydrolysis by phospholipase D determines phosphatidate and diglyceride levels in chemotactic peptide-stimulated human neutrophils: Involvement of phosphatidate phosphohydrolase in signal transduction. J. Biol. Chem. 264: 17069 –17077; 1989. [8] Zu, Y. L.; Qi, J.; Gilchrist, A.; Fernandez, G. A.; Vazquez-Abad, D.; Kreutzer, D. L.; Huang, C. K.; Sha’afi, R. I. p38 Mitogenactivated protein kinase activation is required for human neutrophil function triggered by TNF-a or FMLP stimulation. J. Immunol. 160:1982–1989; 1998. [9] Downey, G. P.; Butler, J. R.; Tapper, H.; Fialkow, L.; Saltiel, A. R.; Rubin, B. B.; Grinstein, S. Importance of MEK in neutrophil microbicidal responsiveness. J. Immunol. 160:434 – 443; 1998. [10] Schnyder, B.; Meunier, P. C.; Car, B. D. Inhibition of kinases impairs neutrophil activation and killing of Staphylococcus aureus. Biochem. J. 331:489 – 495; 1998. [11] Shen, A. Y.; Hwang, M. H.; Roffler, S.; Chen, C. F. Cytotoxicity and antimicrobial activity of some naphthol derivatives. Arch. Pharm. (Weinheim) 328:197–201; 1995. [12] Wang, J. P.; Raung, S. L.; Kuo, Y. H.; Teng, C. M. Daphnoretininduced respiratory burst in rat neutrophils is, probably, mainly through protein kinase C activation. Eur. J. Pharmacol. 288:341– 348; 1995. [13] Wang, J. P.; Raung, S. L.; Hsu, M. F.; Chen, C. C. Inhibition by gomisin C (a lignan from Schizandra chinensis) of the respiratory burst of rat neutrophils. Br. J. Pharmacol. 113:945–953; 1994. [14] Goldberg, B.; Stern, A. The role of the superoxide anion as a toxic
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AA—Arachidonic acid CB—Dihydrocytochalasin B DMSO—Dimethylsulfoxide DPI—Diphenylene iodonium FMLP—Formylmethionyl-leucyl-phenylalanine IP3—Inositol trisphosphate MAPK—Mitogen-activated protein kinase PA—Phosphatidic acid PEt—Phosphatidylethanol PIP2—Phosphatidylinositol-4,5-bisphosphate PLC—Phospholipase C PLD—Phospholipase D PMA—Phorbol 12-myristate 13-acetate PKC—Protein kinase C SOD—Superoxide dismutase TAC—2-Hydroxymethyl-1-naphthol diacetate TFP—Trifluoperazine
PII S0891-5849(98)00288-3
Original Contribution 2-HYDROXYMETHYL-1-NAPHTHOL DIACETATE (TAC) SUPPRESSES THE SUPEROXIDE ANION GENERATION IN RAT NEUTROPHILS JIH-PYANG WANG,*† LO-TI TSAO,* AI-YU SHEN,‡ SHUE-LING RAUNG,*
and
LING-CHU CHANG*
†
*Department of Medical Research, Taichung Veterans General Hospital, Taichung, Taiwan, Graduate Institute of Pharmaceutical Chemistry, China Medical College, Taichung, Taiwan, ‡Department of Biomedical Science, Foo Yin Institute of Technology, Kaohsiung, Taiwan (Received 8 May 1998; Revised 9 October 1998; Accepted 9 October 1998)
Abstract—We have investigated the inhibitory effect of 2-hydroxymethyl-1-naphthol diacetate (TAC) on the respiratory burst of rat neutrophils and the underlying mechanism of action was also assessed in this study. TAC caused concentration-related inhibition of the formylmethionyl-leucyl-phenylalanine (fMLP) plus dihydrocytochalasin B (CB)and phorbol 12-myristate 13-acetate (PMA)-induced superoxide anion (O2•2) generation (IC50 10.2 6 2.3 and 14.1 6 2.4 mM, respectively) and O2 consumption (IC50 9.6 6 2.9 and 13.3 6 2.7 mM, respectively) of neutrophils. TAC did not scavenge the generated O2•2 during dihydroxyfumaric acid autoxidation. TAC inhibited both the transient elevation of [Ca21]i in the presence or absence of [Ca21]o (IC50 75.9 6 8.9 and 84.7 6 7.9 mM, respectively) and the generation of inositol trisphosphate (IP3) (IC50 72.0 6 9.7 mM) in response to fMLP. Cytosolic phospholipase C (PLC) activity was also reduced by TAC at a same range of concentrations. The PMA-induced PKC-b associated to membrane was attenuated by TAC (about 80% inhibition at 30 mM). Upon exposure to fMLP, the cellular cyclic AMP level was decreased in neutrophils pretreated with TAC. TAC attenuated fMLP-induced phosphorylation of mitogen-activated protein kinase (MAPK) p42/44 (IC50 17.4 6 1.7 mM), but not p38. The cellular formation of phosphatidic acid (PA) and, in the presence of ethanol, phosphatidylethanol (PEt) induced by fMLP was inhibited by TAC in a concentrationdependent manner (IC50 25.4 6 2.4 and 25.9 6 1.4 mM, respectively). TAC had no effect on the O2•2 generation of PMA-stimulated and arachidonic acid (AA)-stimulated NADPH oxidase preparations. However, TAC caused concentration-related decrease of the membrane associated p47phox in PMA-stimulated neutrophils (about 80% inhibition at 30 mM). We conclude that inhibition by TAC of the neutrophil respiratory burst is probably attributable to the blockade of the p42/44 MAPK and phospholipase D (PLD) pathways, the membrane translocation of PKC, and to the failure in assembly of a functional NADPH oxidase complex. Blockade of the PLC pathway by TAC probably plays a minor role. © 1999 Elsevier Science Inc. Keywords—TAC, Neutrophil, Respiratory burst, NADPH oxidase, Protein kinase C, Phospholipase D, Mitogenactivated protein kinase, Translocation, Phospholipase C, Inositol phosphate, Intracellular Ca21 concentration
INTRODUCTION
known as the NADPH oxidase. This non-mitochondrial O2 consumption process is known as the respiratory burst [2]. Reactive O2 species produced during the respiratory burst are believed to serve as bactericidal agents, as evidenced by the susceptible to recurrent infections by patients with chronic granulomatous disease [2]. Under certain circumstances, the excessive or inappropriate release of these highly reactive O2 species can result in undesirable tissue damage. This is probably involved in the pathogenesis of many diseases [3]. Therefore, a drug that would inhibit the generation of toxic O2 metabolites could terminate this tissue damage.
Neutrophils play an essential role in the body’s defense against bacterial infection. Upon exposure to a pathogenic stimulus, neutrophil becomes activated and generates superoxide anion (O2•2) and other potent toxic O2 metabolites [1]. The generation of these oxidants accompanied by the increase of O2 uptake from surrounding medium by neutrophils occurs through the activation of a membrane-associated complex Address correspondence to: Jih-Pyang Wang, Department of Medical Research, Taichung Veterans General Hospital, 160, Chung-Kung Road, Sec. 3, Taichung, Taiwan 407; Fax: (886) 4-359-2705 1010
TAC inhibits neutrophil O2•2 generation
In resting cells, NADPH oxidase is dormant, the components of the NADPH oxidase are segregated into cytosolic and membrane compartments. On neutrophil activation, the cytosolic components of NADPH oxidase (mainly p47phox and p67phox) translocate to the plasma membrane where they associate with a heterodimeric cytochrome b558, composed of gp91phox and p22phox, resulting a functional complex responsible for the production of oxygen radicals in neutrophils [4]. Intracellular signaling events used in the response of neutrophils to external stimuli are complicated and not clearly defined. Stimulation of neutrophils by receptor-binding ligands results in an intracellular signaling cascade including the activation of phospholipase C (PLC) that releases inositol trisphosphate (IP3) and diacylglycerol, which, in turn, increases intracellular Ca21 concentration and activates protein kinase C (PKC), respectively [5]. The two pathways function synergistically for O2•2 generation. Phospholipase D (PLD) activation is also functionally linked to O2•2 generation in neutrophils [6]. PLD acts upon phosphatidylcholine to release phosphatidic acid (PA), which is then converted into diradylglycerol [7]. In addition, it has been recently suggested that mitogenactivated protein kinase (MAPK) may also participate in the activation of neutrophil respiratory burst [8 –10]. 2-Hydroxymethyl-1-naphthol diacetate (TAC), has been found to possess cytotoxicity against several human carcinoma cell lines and potent antimicrobial activity [11]. TAC was also found through preliminary in vitro tests to inhibit O2•2 generation from activated neutrophils. The primary objective of the present study was to identify the sites and mechanisms of the inhibitory effect of TAC on the respiratory burst in rat peripheral neutrophils.
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inghamshire, UK). Mouse monoclonal antibodies to PKC-b, p38 MAPK, ERK1, and ERK2, and rabbit polyclonal anti-p47phox antibody (Transduction Lab., Lexington, KY). Rabbit polyclonal antibodies to phospho-p38 MAPK and phospho-p42/44 MAPK (New England Biolabs., Beverly, MA). Isolation of neutrophils Rat blood was collected from the abdominal aorta and the neutrophils were purified by dextran sedimentation, hypotonic lysis of erythrocytes, and centrifugation through Ficoll-Hypaque [12]. Purified neutrophils containing .95% viable cell were normally resuspended in Hanks’ balanced salt solution (HBSS) containing 10 mM HEPES, pH 7.4, and 4 mM NaHCO3, and kept in an ice-bath before use. Measurement of O2•2 generation and O2 consumption The O2•2 generation in neutrophil suspension was determined by the superoxide dismutase (SOD)-inhibitable ferricytochrome c reduction as previously described [12,13]. O2•2 generation during dihydroxyfumaric acid autoxidation was determined by the reduction of nitroblue tetrazolium as previously described [14]. Absorbance changes of the reduction of ferricytochrome c and nitroblue tetrazolium were continuously monitored in a double-beam spectrophotometer. Whole cell O2 consumption was continuously measured with a Clark-type oxygen electrode using a YSI biological oxygen monitor [15]. Measurement of [Ca21]i
MATERIALS AND METHODS
Reagents TAC was synthesized as the described previously [11]. All chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) except for the following: dextran T-500 (Pharmacia Biotech., Uppsala, Sweden). Hanks’ balanced salt solution (Gibco BRL Co., Gaithersburg, MD). Diphenylene iodonium (DPI) (Research Biochemicals International, Natick, MA). U73122 (Biomol Research Lab. Inc., Plymouth Meeting, PA). Fura-2 AM and fluo-3 AM (Molecular Probes, Eugene, OR). L-a-Posphatidyl-D-myo-inositol-4,5-bisphosphate (PIP2) (Boehringer Mannheim, Mannheim, Germany). myo-[3H]Inositol, L-3-phosphatidyl[2-3H]-inositol-4,5-bisphosphate ([3H]PIP2), 1-O[3H]octadecyl-sn-glycero-3-phosphocholine, cyclic AMP assay kit, [g-32P]ATP and enhanced chemiluminescence reagent (Amersham International plc., Buck-
Neutrophils (1 3 107 cells/ml) were suspended in HEPES buffer A (composition in mM: NaCl 124, KCl 4, Na2HPO4 0.64, KH2PO4 0.66, NaHCO3 15.2, dextrose 5.56 and HEPES 10, pH 7.4), and loaded with 5 mM fura-2 AM as previously described [13] or loaded with 5 mM fluo-3 AM at 37°C for 45 min. After being washed, cells were resuspended in HEPES buffer A with 0.05% (w/v) bovine serum albumin. The fluorescence of fura2-loaded cells was monitored by a double-wavelength fluorescence spectrophotometer (PTI, Deltascan 4000) at 510 nm with excitation 340 and 380 nm in the ratio mode. Calibration of the excitation ratio in terms of Ca21 concentration was performed as previously described [16]. The fluorescence of fluo-3-loaded cells was monitored at 525 nm with excitation 488 nm. [Ca21]i was calibrated from the fluorescence intensity as follows: [Ca21]i 5 Kd 3 [(F 2 Fmin)/(Fmax 2 F)], where Kd was taken as 400 nM [17].
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J-P. WANG et al.
Determination of inositol phosphate levels Neutrophils (3 3 107 cells/ml) were loaded with myo-[3H]inositol (83 Ci/mmol) at 37°C for 2 h [13]. Ten seconds after the stimulation with formylmethionylleucyl-phenylalanine (fMLP), reactions were stopped by adding CHCl3:CH3OH (1:1, v/v) mixture and 2.4 M HCl. The aqueous phase was removed and neutralized by 0.4 M NaOH, and then applied to an AG 1-X8 resin (formate) column (Bio-Rad). Inositol monophosphate (IP), inositol bisphosphate (IP2), and IP3 were eluted sequentially by using 0.2, 0.4, and 1.0 M ammonium formate, respectively, in 0.1 M formic acid as eluents, and then counted as previously described [13]. Cytosolic PLC activity Neutrophil cytosolic PLC was isolated and assayed as previously described [18]. Substrate stock was prepared by mixing PIP2 and 5 mCi [3H]PIP2 (1 Ci/mmol) in solvent mixture (20% sodium cholate, 250 mM 2-mercaptoethanol, 2.5 M NaCl, 1 M PIPES, pH 6.8) to produce a 0.361 mM PIP2 solution, sonicated on ice, and then stored at 220°C. PLC activity was assayed by measuring the hydrolysis of PIP2 into inositol phosphates. PKC membrane translocation For the analysis of subcellular distribution of PKC, neutrophils (4 3 107 cells/ml) were stimulated with 0.2 mM phorbol 12-myristate 13-acetate (PMA) for 5 min at 37°C. Reactions were stopped by addition of 4 volume of ice-cold HBSS, and then resuspended in disruption solution (0.34 M sucrose, 10 mM Tris-HCl, pH 7.0, 1 mM phenylmethylsulphonyl fluoride, 1 mM EGTA, 10 mM benzamidine, 10 mg/ml of leupeptin and antipain). After sonication, lysate was centrifuged to remove the unbroken cells, and then further centrifuged at 100,000 g for 30 min at 4°C. Pellet (as membrane fraction) and supernatant (as cytosol fraction) were boiled in Laemmli sample buffer, subjected to SDS-PAGE, transferred to Immobilon-P membrane (Millipore), blocked with 5% non-fat milk in TST buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl and 0.05% Tween-20) and probed with mouse monoclonal antibody to PKC-b (1:500 dilution in TST buffer with 0.5% non-fat milk) [19]. Detection was made using the enhanced chemiluminescence reagent. Measurement of cellular cyclic AMP level Cyclic AMP was determined as described [20]. Neutrophils were preincubated with test drugs for 9.5 min at 37°C. Thirty seconds after addition of fMLP, reaction
mixture was immediately added to 1.0 ml of 0.05 M acetate buffer, pH 6.2, containing 0.05 mM 3-isobutyl1-methylxanthine. After boiling for 5 min, suspension was kept in ice, sonicated and followed by sedimentation. The supernatants were acetylated by addition of 0.025 volume of triethylamine:acetic anhydride (2:1, v/v). Cyclic AMP content in aliquots of the acetylated samples were assayed by using enzyme immunoassay kit.. Western blot analysis of cellular MAPK Neutrophils (1 3 107 cells/ml) in HBSS were preincubated with dimethylsulfoxide (DMSO) or test drugs at 37°C for 5 min before initiating the reaction by adding fMLP plus dihydrocytochalasin B (CB). One minute later, reactions were quenched by addition of stopping solution (20% trichloroacetic acid, 1 mM phenylmethylsulphonyl fluoride, 2 mM N-ethylmaleimide, 100 mM NaF, 2 mM Na3VO4, 10 mM p-nitrophenyl phosphate, 7 mg/ml of leupeptin and pepstatin). Proteins were separated through SDS-PAGE as described above, and probed with rabbit polyclonal anti-phospho-p38 MAPK antibody (1:2000 dilution in TST buffer with 1% BSA) or with rabbit polyclonal anti-phospho-p42/44 MAPK antibody (1:2000 dilution in TST buffer with 0.5% nonfat milk). Detection was made using the enhanced chemiluminescence reagent. To confirm that the same amount of cellular proteins was loaded on each lane, the membranes were stripped, after detection of phospho-p38 MAPK and phospho-p42/44 MAPK, with stripping buffer (100 mM 2-mercaptoethanol, 2% SDS, and 62.5 mM Tris-HCl, pH 6.8) for 30 min at 50°C, and then reprobed with mouse monoclonal anti-p38 MAPK antibody (1:250 dilution in TST buffer with 0.5% non-fat milk), or with mouse monoclonal anti-ERK1 or antiERK2 antibodies (1:1000 to 1:2500 dilution in TST buffer with 0.5% non-fat milk), respectively. Detection was made using the enhanced chemiluminescence reagent. Measurement of PLD activity Neutrophils (5 3 107 cells/ml) were loaded with 10 mCi 1-O-[3H]octadecyl-sn-glycero-3-phosphocholine (150 Ci/mmol) at 37°C for 75 min [21]. Thirty seconds after stimulation with fMLP/CB in the presence or absence of 0.5% ethanol, the lipids in reaction mixture were extracted, dried, and separated on silica gel 60 plate. The plates were developed halfway by using the solvent system consisting of hexane:diethyl ether:methanol:acetic acid (90:20:3:2, v/v/v/v), dried and then developed again to the top using the upper phase of the
TAC inhibits neutrophil O2•2 generation
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Table 1. Concentration Dependence of TAC Inhibition of O2•2 Generation and O2 Consumption in Formylmethionyl-leucyl-phenylalanine (fMLP)- and Phorbol 12-myristate 13-acetate (PMA)-stimulated Neutrophils O2•2 generation (nmol/10 min per 106 cells)
Control TAC
3 mM 10 mM 30 mM
O2 consumption (nmol/5 min per 2 3 106 cells)
fMLP
PMA
fMLP
PMA
6.5 6 0.5 4.7 6 0.3 2.3 6 0.5* 0.5 6 0.2*
16.4 6 1.3 12.9 6 2.1 7.2 6 1.8* 3.5 6 0.4*
14.4 6 0.5 10.9 6 1.0 4.6 6 1.5* 0.5 6 0.5*
39.3 6 1.3 32.5 6 2.8 21.4 6 2.8* 4.4 6 0.5*
Neutrophils were preincubated with DMSO (vehicle control) or TAC for 3 min before stimulation with 0.3 mM fMLP plus 5 mg/ml of CB and 1 nM PMA for O2•2 generation, or 1 mM fMLP plus 5 mg/ml of CB and 3 nM PMA for O2 consumption. Values are expressed as mean 6 SEM for four to five separate experiments. *P , .01 compared to the corresponding control values.
solvent system consisting of ethylacetate:isooctane:acetic acid:water (110:50:20:100, v/v/v/v). The lipids were located by staining with iodine vapor. The radioactivity of [3H] products were directly quantified with a PhosphorImager (Molecular Dynamics 445 SI) using ImageQuaNT software. phox
NADPH oxidase activity and p47 translocation
membrane
For the arachidonic acid (AA)-induced NADPH oxidase activation, the subcellular fractions of neutrophils were prepared as previously described [21]. PMA-stimulated particulate NADPH oxidase was isolated as previously described [13]. The NADPH oxidase activity was measured spectrophotometrically at 28°C by detecting the SOD-inhibitable ferricytochrome c reduction as previously described [21]. For the analysis of the subcellular distribution of p47phox, neutrophils (2 3 107 cells/ml) in HBSS were preincubated with DMSO or test drugs at 37°C for 5 min before stimulation with 0.2 mM PMA. Five minutes later, four volume of ice-cold HBSS was added. Cells were disrupted by sonication, layered on discontinuous sucrose gradients, and centrifuged at 100,000 g for 60 min [22]. The band between 20 and 50% sucrose was collected, washed with 2 volume of 0.45 M NaCl, and centrifuged at 10,000 g for 10 min. The supernatant was further centrifuged at 100,000 g for 60 min. The NaCl-washed membrane pellet was then boiled in Laemmli sample buffer. Proteins were separated through SDS-PAGE as described above, and then probed with rabbit polyclonal anti-p47phox antibody (1: 500 dilution in TST buffer with 0.5% non-fat milk). Statistical analysis Statistical analyses were performed using the Bonferroni t test method after analysis of variance. A P value less than 0.05 was considered significant for all tests.
Analysis of the regression line test was used to calculated IC50 values. Means are represented as mean 6 standard error of the mean (SEM). RESULTS
Neutrophils exposed to fMLP/CB demonstrate a rapid and transient O2•2 generation and O2 consumption, whereas, PMA evoked a slow and long lasting O2•2 generation and O2 consumption. Concentration-dependent inhibition of O2•2 generation and O2 consumption were observed in cells pretreated with TAC for 3 min prior to stimulation with fMLP/CB or PMA (Table 1). Inhibition of fMLP/CB- and PMA-induced O2•2 generation with IC50 values of 10.2 6 2.3 and 14.1 6 2.4 mM, respectively, and O2 consumption with IC50 values of 9.6 6 2.9 and 13.3 6 2.7 mM, respectively. More than 95% viability was observed with trypan blue exclusion in cells treated with 100 mM TAC for 10 min. The scavenging ability of drug was assessed by using a cell-free oxygen radical generating system. Unlike SOD, which significantly reduced the O2•2 generation during dihydroxyfumaric acid autoxidation [13], TAC had no effect in this respect (0.060 6 0.004 DA560 as control vs 0.060 6 0.011 DA560 at 100 mM TAC, P . 0.05). Addition of fMLP to fura-2-loaded cells in the presence of [Ca21]o or to fluo-3-loaded cells in the absence of [Ca21]o, both induced rapid and transient elevation of [Ca21]i, however, much less extensive [Ca21]i changes were observed in the absence of [Ca21]o. Fura-2 can be used in the ratio mode, making the measurement of [Ca21]i independent of several artifacts. However, fluo-3 undergo a greater enhancement of emission intensity upon Ca21 binding, making fluo-3 useful for measuring the kinetics of Ca21 transient at lower levels. Pretreatment of cells with a PLC inhibitor U73122 (1 mM) abolished the fMLP-induced [Ca21]i changes in the presence or absence of [Ca21]o [21,23]. TAC also concentration-dependently inhibited the [Ca21]i changes of
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Fig. 1. Effect of TAC on formylmethionyl-leucyl-phenylalanine (fMLP)-induced [Ca21]i changes in (A) fura-2-loaded neutrophils in the presence of 1 mM CaCl2 in medium or (B) fluo-3-loaded neutrophils in the presence of 1 mM EDTA in medium. Cell suspension was preincubated with DMSO (vehicle control) or 30 –200 mM TAC at 37°C for 3 min before addition of 0.1 mM fMLP. [Ca21]i levels were monitored in the excitation ratio 340/380 nm mode with emission wavelength at 510 nm for fura-2-loaded cells, or in the excitation and emission wavelength at 488 and 525 nm, respectively, for fluo-3-loaded cells. The results shown are representative of four to five separate experiments.
fMLP-stimulated neutrophils whether [Ca21]o was present or not (IC50 values 75.9 6 8.9 and 84.7 6 7.9 mM, respectively) (Fig. 1). Myo-[3H]inositol-loaded neutrophils exposed to fMLP evoke a significant increase of cellular IP2 and IP3 (both P , 0.01) levels. Pretreatment of cells with U73122 and TAC reduced the fMLP-induced IP2 and IP3 formation in a concentration-dependent manner (Fig. 2A,B). IC50 values of U73122 and TAC for the inhibition of IP3 formation were estimated to be 13.4 6 0.8 and 72.0 6 9.7 mM, respectively. Upon exposure to CaCl2 and PIP2, neutrophil cytosolic PLC was activated and used PIP2 as substrate to produce inositol phosphate. U73122 (10 mM) greatly reduced PLC activity (68.2 6 5.5% inhibition). Concentration-related inhibition of PLC activity was also observed in TAC-treated reaction mixture (41.9 6 6.1% inhibition at 100 mM TAC) (Fig. 2C). Subcellular distribution of PKC in activated neutrophils was assessed by immunoblotting with a specific monoclonal anti-PKC-b antibody. In the resting cells, PKC-b was enriched in the cytosol fraction. Upon exposure to PMA, PKC-b was translocated from the cytosol to the membrane. This enzyme translocation can be used as an index of the enzyme activation. Pretreatment of PMA-stimulated cells with TAC reduced the membrane associated PKC-b (about 80% inhibition at 30 mM TAC) (Fig. 3), whereas TAC alone had no effect on the membrane translocation of PKC-b. Analysis of the cyclic AMP levels in neutrophils showed that a significant increase was observed in cells treated with 10 mM forskolin (2.79 6 0.47 vs 0.61 6 0.11 pmol per 2 3 106 cells, P , 0.01), an adenylate cyclase activator [24]. On the contrary, TAC decreased
Fig. 2. Effect of TAC on formylmethionyl-leucyl-phenylalanine (fMLP)-induced cellular inositol phosphate formation and on cytosolic PLC activity. Cellular (A) IP2 and (B) IP3 levels were determined in myo-[3H]inositol-loaded cells pretreated with DMSO (vehicle control), 10 –100 mM TAC (E) or 0.3-30 mM U73122 (h) in the presence of 10 mM LiCl at 37°C for 3 min prior to stimulation with 0.3 mM fMLP. The resting levels of IP2 and IP3 were measured from the cells which exposed to DMSO without fMLP challenge (●). (C) Cytosolic PLC was preincubated with DMSO (vehicle control), 10 –100 mM TAC or 10 mM U73122 for 3 min at 37°C before addition of 100 mM CaCl2 and 48 mM PIP2/[3H]PIP2. Reaction was stopped by addition of ice-cold CHCl3:CH3OH:HCl (50:50:1, v/v/v) mixture. Inositol phosphate formation in the upper aqueous phase was determined. Values are expressed as mean 6 SEM for four to five separate experiments. *P , .01 compared to the corresponding control values.
the cellular cyclic AMP level (0.16 6 0.10 pmol per 2 3 106 cells at 30 mM TAC, P , 0.01). Moreover, TAC (up to 100 mM) did not affect the porcine heart protein kinase A activity in the presence or absence of cyclic AMP (data not shown). Immunoblot analysis with specific anti-phosphop42/44 or anti-phospho-p38 MAPK antibody was performed to determine the effect of TAC on MAPK pathways in neutrophils. Following stimulation with fMLP/ CB, as shown in Figure 4, cellular p42/44 and p38 MAPKs become phosphorylated and hence activated. TAC specifically inhibited p42/44 but not p38 MAPK in a concentration dependent manner (IC50 about 17.0 6 1.0 mM). However, TAC alone at high concentration (100 mM) significantly increased the phosphorylation of p38 MAPK (Fig. 4B). The differences observed for the induced protein phosphorylation of cellular MAPKs did not result from differences in loading or from cellular protein digestion. The anti-ERK1 that we used also recognizes ERK2 (p42). Upon exposure to fMLP/CB, a significantly increased (P , 0.01) the formation of PA and, in the presence of 0.5% ethanol, the corresponding phosphatidylethanol (PEt)
TAC inhibits neutrophil O2•2 generation
Fig. 3. Effect of TAC on phorbol 12-myristate 13-acetate (PMA)induced PKC-b membrane translocation. Neutrophils were preincubated with DMSO (lane 2) or 10 –30 mM TAC (lanes 3– 4) for 5 min at 37°C and then stimulated with 0.2 mM PMA for another 5 min. Cells may also react with DMSO (lane 1) or 10 –30 mM TAC (lanes 5– 6) alone for total 10 min reaction time. After termination of reaction, cells were disrupted, and then the membrane and cytosol proteins were subjected to SDS-PAGE. Each lane contains sample protein 50 mg. Analysis was performed by immunoblotting with a monoclonal antibody to PKC-b. The results shown are representative of three separate experiments.
was observed in [3H]alkyl-lysophosphatidylcholine-loaded neutrophil suspension. The cellular levels of PA and PEt in response to fMLP/CB were reduced by genistein, a tyrosine kinase inhibitor [25], in parallel. In addition, TAC concentration-dependently inhibited the fMLP/CB-induced PA and PEt formation with IC50 values of 25.4 6 2.4 and 25.9 6 1.4 mM, respectively (Fig. 5). In PMA-stimulated neutrophil particulate NADPH oxidase preparation, addition of NADPH to initiate O2•2 generation by detecting the reduction of ferricytochrome c and diphenylene iodonium (DPI), an inhibitor of NADPH oxidase [26], inhibited O2•2 generation. In an AA-stimulated cell-free system, addition of AA induced the assembly of the cytosolic and membrane components of NADPH oxidase and then generation of O2•2 in the presence of NADPH. Trifluoperazine (TFP), an inhibitor of NADPH oxidase [27], inhibited O2•2 generation in this cell-free system (Table 2). In contrast, TAC (up to 100 mM) failed to reduce the O2•2 generation of either system. In PMA-stimulated neutrophils, the membrane associated p47phox was increased as assessed by immunoblotting with a polyclonal anti-p47phox antibody (Fig. 6, lanes 1–2, arrow). TAC concentration-dependently inhibited the p47phox membrane translocation in neutrophils in response to PMA (about 80 and 90% inhibition at 30 and 100 mM TAC, respectively) (Fig. 6).
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Fig. 4. Effect of TAC on formylmethionyl-leucyl-phenylalanine (fMLP)-induced the activation of MAPKs in rat neutrophils. Neutrophils were preincubated with DMSO (lane 2) or 3–100 mM TAC (lanes 3– 6) for 5 min at 37°C and then stimulated with 0.1 mM fMLP plus 5 mg/ml of CB (lanes 2– 6) for 1 min. Effect of DMSO (lane 1) or 100 mM TAC (lane 7) alone on the cells was also studied for total 6 min reaction time. After termination of reaction, cells were rapidly sedimented, boiled in Laemmli sample buffer, and subjected to SDSPAGE. Each lane contains sample protein 60 mg. Analysis was performed by immunoblotting with (A) anti-phospho-p42/44, anti-ERK1, anti-ERK2, and (B) anti-phospho-p38 or anti-p38 MAPK antibody. The results shown are representative of four separate experiments.
DISCUSSION
We have shown that the fMLP- and PMA-induced neutrophil O2•2 generation and O2 consumption are inhibited in a concentration-dependent manner by TAC. Using a cell-free oxygen radical generation system to assess the scavenging effect of TAC, we did not observe a significant reduction of the O2•2 generation by TAC, preclude the possibility that TAC acts as a O2•2 scavenger but probably as an inhibitor of certain signal transduction steps that follows after activation of neutrophils. Pharmacologically increase cellular cyclic AMP level is one recognized means to down-regulated O2•2 generation in neutrophils [28]. The inhibitory action of cyclic AMP on neutrophils appears to depend on the activation of protein kinase A. TAC did not increase the porcine heart protein kinase A activity whether the cyclic AMP was present or not. In addition, the cellular cyclic AMP level was not increased by TAC. Thus, there is no indications that inhibition of respiratory burst by TAC is through the activation of protein kinase A. fMLP- but not PMA-induced O2•2 generation is a 21 Ca -dependent process [29]. Unlike the PLC inhibitor
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Fig. 5. Effect of TAC on PLD activity of rat neutrophils. 1-O-[3H]octadecyl-sn-glycero-3-phosphocholine-loaded cells were pretreated with DMSO (vehicle control), 3–100 mM TAC or 30 mg/ml of genistein for 3 min at 37°C in the presence of 0.5% ethanol before addition of 1 mM fMLP plus 5 mg/ml of CB. The lipids were extracted from the reaction mixture, separated on silica gel 60 plates, and the phosphatidic acid (PA) (open column) and phosphatidylethanol (PEt) (crosshatched column) were quantified by phosphor screen autoradiography. The level of PA and PEt in fMLP/CB-stimulated cells in the absence of 0.5% ethanol was also shown (the 1st pair of columns). Values are expressed as mean 6 SEM for four separate experiments. *P , .05, **P , .01 compared to the corresponding control values.
U73122 [23], TAC attenuated fMLP-induced [Ca21]i changes in the presence and absence of [Ca21]o. In the presence of EDTA to remove extracellular Ca21 and to diminish the Ca21-influx during cells activation, TAC inhibited the fMLP-induced [Ca21]i elevation over the same range of concentrations as its used in the presence of [Ca21]o. In addition, we have demonstrated that both the formation of IP2 and IP3 in intact cells and the neutrophil cytosolic PLC activity are inhibited by TAC Table 2. Effect of TAC on NADPH Oxidase Activity in the Cell-free Systems NADPH oxidase activity
Control TAC DPI TFP
100 mM 3 mM 100 mM
PMA-stimulated (nmol O2•2/10 min per 6 3 106 cells eq.)
AA-stimulated (nmol O2•2/10 min per 107 cells eq.)
2.78 6 0.18 2.60 6 0.08 0.93 6 0.22* —
3.29 6 0.11 3.10 6 0.52 — 1.27 6 0.31*
Values are expressed as mean 6 SEM for four to five separate experiments. *P , .01 compared to the corresponding control values. —, not determined.
Fig. 6. Effect of TAC on phorbol 12-myristate 13-acetate (PMA)induced p47phox membrane translocation. Neutrophils were preincubated with DMSO (lane 2), 10-100 mM TAC (lanes 3–5) for 5 min at 37°C and then stimulated with 0.2 mM PMA for another 5 min. Cells may also react with DMSO (lane 1) or 100 mM TAC (lane 6) alone for total 10 min reaction time. After termination of reaction, cells were disrupted, and the membrane proteins were subjected to SDS-PAGE. Each lane contains sample protein 50 mg. Analysis was performed by immunoblotting with a polyclonal anti-p47phox antibody. The results shown are representative of three separate experiments.
in a same range of concentrations as its used in [Ca21]i assay providing strong evidence for the blockade of PLC pathway by TAC in fMLP-stimulated neutrophils. Since higher concentrations of TAC had to be used to inhibit PLC pathway than respiratory burst, suggesting the blockade of this signaling pathway plays a minor role in the inhibition of O2•2 generation by TAC. Rat neutrophil contains PKC isoforms of four categories: conventional PKCs (a, b, and g), novel PKCs (d, e, and u), atypical PKCs (i, l, and z), and PKC-m, although PKC-l and -z are barely detected [19]. However, the isoform specific regulation and function remain unclear yet. Endogenous PKC activator diacylglycerol is generated follows after activation of fMLP-receptor, whereas PMA bypass the membrane receptor and directly activate PKC [30]. It has been reported that PKC-b plays a role in the phosphorylation of membranes associated p47phox and the assembly or maintenance of an active NADPH oxidase [31]. Through the use of monoclonal antibody that specifically bind to PKC-b, we have shown that PKC-b is primarily cytosolic in unstimulated neutrophils but become associate with the membrane fraction after PMA treatment reconciling the earlier report [32]. TAC reduced the immunoreactivity at the membrane fraction of PMA-stimulated neutrophils indicates that the activation of PKC in intact cells might be suppressed by TAC. MAPKs have been demonstrated to play an important role in mediating intracellular signal transduction and regulating cellular functions. However, the roles of MAPKs in neutrophils are not well understood yet. It has been reported that activation of cellular p42/44 and p38
TAC inhibits neutrophil O2•2 generation
MAPKs are probably indispensable for the fMLP-induced O2•2 generation [8 –10], although the correlation between p42/44 MAPK activation and activation of oxidase has not been observed by all investigators. Since both p38 and p42/44 MAPKs phosphorylated p47phox at the same site and at similar rates [33], MAPKs might have a role in the translocation of this oxidase component to the plasma membrane and activation of the respiratory burst. TAC attenuated fMLP-induced phosphorylation of p42/44 MAPK, but did not reduce the immunoreactivity of phospho-p38 MAPK, with an IC50 value closed to that of the inhibition of O2•2 generation. Several lines of evidence indicate that PLD activation is functionally linked to the O2•2 generation in neutrophils [6,34]. PLD catalyzes the hydrolysis primarily of phosphatidylcholine to produce PA. In the presence of ethanol, PA via a transphosphatidylation reaction yield PEt. PA could act on the respiratory burst through the activation of PKC or NADPH oxidase [22,35]. TAC and genistein suppressed both of the PA and PEt formation in neutrophils in response to fMLP. Because the inhibition of PLD pathway and respiratory burst by TAC over the same range of concentrations, blockade of this signaling pathway might play an important role in the inhibition of respiratory burst by TAC. The O2•2generating NADPH oxidase complex in neutrophils is constituted of a heterodimeric cytochrome b558 and cytosolic factors, mainly p47phox and p67phox [4]. Upon activation, p47phox is phosphorylated, the polyproline motif of p47phox would then be accessible to the C-terminal SH3 domain of p67phox [36]. This new interaction changes the overall structure of the complex and makes it able to recognize the membrane cytochrome b558 through the polyproline motif of p22phox and one of the SH3 domains of p47phox [37], favorable to electron transport, and thereby proceeding to the univalent reduction of O2 [4]. TAC decreased the membrane associated p47phox in neutrophils in response to PMA. In contrast, TAC affected the oxidase activity neither in PMA-stimulated neutrophil particulate NADPH oxidase preparation, in which a functional oxidase complex has been already formed, nor in an AA-stimulated cell-free system, in which oxidase complex is assembled through a PKC-independent manner [38]. These results indicate that TAC did not influence the assembly of a functional NADPH oxidase complex in vitro, nor suppressed the activity of assembled oxidase. The inhibition of O2•2 generation by TAC seems not simply through its 1-naphthol structure because 1-naphthol did not affect the fMLP-induced rise of [Ca21]i and 1,4-naphthoquinone, the active metabolite of 1-naphthol, prevents the assembly of a functional NADPH oxidase in the cell-free system [39]. In summary, we demonstrated that TAC is capable of
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inhibiting the respiratory burst (IC50 about 10-14 mM) in neutrophils in response to fMLP as well as to PMA. The underlying mechanism of action was also assessed. TAC acts as neither an O2•2scavenger nor a cyclic AMP-elevating agent. Inhibition of respiratory burst in neutrophils by TAC is probably attributable mainly to the blockade of p42/44 MAPK (IC50 about 17 mM), PKC (IC50 # 30 mM) and PLD (IC50 about 25 mM) pathways, and thereby, influence of the NADPH oxidase assembly (IC50 # 30 mM). Whereas, the blockade of PLC/Ca21 pathway (IC50 about 72–85 mM) may play a minor role in the mechanisms of inhibition by TAC of neutrophil respiratory burst. Acknowledgements — This work was supported by grants from the National Science Council of the Republic of China (NSC-87-2314-B075A-013).
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AA—Arachidonic acid CB—Dihydrocytochalasin B DMSO—Dimethylsulfoxide DPI—Diphenylene iodonium FMLP—Formylmethionyl-leucyl-phenylalanine IP3—Inositol trisphosphate MAPK—Mitogen-activated protein kinase PA—Phosphatidic acid PEt—Phosphatidylethanol PIP2—Phosphatidylinositol-4,5-bisphosphate PLC—Phospholipase C PLD—Phospholipase D PMA—Phorbol 12-myristate 13-acetate PKC—Protein kinase C SOD—Superoxide dismutase TAC—2-Hydroxymethyl-1-naphthol diacetate TFP—Trifluoperazine