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CELLULAR AND MOLECULAR
Laboratory of Pathophysiological Biochemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, Miyagi, Japan (J.H., Y.N., A.Y., K.I., K.O.); and Laboratory of Pathophysiology, College of Pharmacy, Sookmyung Women's University, Seoul, Korea (J.H., S.K., Y.-S.K.)
Received June 12, 2006; accepted August 7, 2006.
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B and activator protein-1 and decreased the level of IB-
and increased that of phosphorylated c-Jun N-terminal kinase (JNK). NO production induced by these V-ATPase inhibitors was suppressed by the NF-B inhibitor Bay 11-7082 [(E)3-[(4-methylphenyl)sulfonyl])-2-propenenitrile] and the JNK inhibitor SP600125 [anthra[1,9-cd]pyrazol-6(2H)-one] in parallel with the partial alleviation of the V-ATPase inhibitor-induced decrease in MTT response. The Na+,K+-ATPase inhibitor dibucaine and the F-ATPase inhibitor oligomycin did not induce NO production at which concentrations the MTT response was decreased. The NO donor S-nitroso-N-acetyl-DL-penicillamine further lowered the V-ATPase inhibitor-induced decrease in the MTT response, and the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, sodium salt (carboxy-PTIO) alleviated it partially. Mitochondrial depolarization, an index of apoptosis, was induced by bafilomycin A1 and concanamycin A. On treatment with the nitric-oxide synthase inhibitor NG-monomethyl-L-arginine acetate, the disruption of mitochondrial membrane potential induced by bafilomycin A1 and concanamycin A was alleviated partially in parallel with the decrease in NO production. Carboxy-PTIO also alleviated it partially. Our findings suggest that the V-ATPase inhibitors bafilomycin A1 and concanamycin A similarly induce NO production and the newly produced NO participates partially in the V-ATPase inhibitor-induced apoptosis in RAW 264.7 cells.
) induces apoptosis in the mouse leukemic monocyte cell line RAW 264.7 (Hong et al., 2003, 2005b) and in the human promyelocytic leukemia cell line HL-60 (Hong et al., 2005a). In addition, we have clarified that apicularen A induces production of nitric oxide (NO), and the newly produced NO partially participates in apicularen A-induced apoptosis in RAW 264.7 cells (Hong et al., 2005b). Structurally, apicularen A has a benzolactone enamide core like the vacuolar-type (H+)-ATPase (V-ATPase) inhibitors salicylihalamide A, lobatamides A to F, and oximidines I and II (Boyd et al., 2001). Therefore, it was possible that apicularen A also inhibits V-ATPase. Previously, we have suggested that apicularen A inhibits V-ATPase in HL-60 cells as revealed by the vital staining of intracellular organelles with acridine orange (Hong et al., 2005a) and have recently demonstrated that apicularen A inhibits the ATP-dependent proton transport into inside-out microsome vesicles and inhibits the V-ATPase inhibitor bafilomycin A1sensitive ATP hydrolysis in mouse peritoneal macrophages, indicating that apicularen A inhibits V-ATPase (Hong et al., 2006). From our findings, we have speculated that V-ATPase inhibitors other than apicularen A also induce NO production in RAW 264.7 cells. Therefore, in this study, we intended to clarify whether the commercially available V-ATPase inhibitors bafilomycin A1 and concanamycin A also have an activity to induce NO production and compared the effect of these inhibitors with that of the Na+,K+-ATPase inhibitor dibucaine (Hudgins and Bond, 1984), the F-ATPase inhibitor oligomycin (Lardy et al., 1958), and the endomembrane Ca2+-ATPase inhibitor thapsigargin (Thastrup et al., 1990). We also analyzed the role of the newly produced NO in the V-ATPase inhibitor-induced decrease in cell growth and survival because it is reported that NO is a potent inducer of apoptosis in various types of cells in vitro (Shimaoka et al., 1995; Lincoln et al., 1996; Leist et al., 1997) including RAW 264.7 cells (Messmer et al., 1995; Jun et al., 1999; Gotoh et al., 2002). |
Materials and Methods |
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B- phosphorylation, Bay 11-7082, and the inhibitor of c-Jun N-terminal kinase (JNK), SP600125, were purchased from Calbiochem (Darmstadt, Germany). All of the drugs, with the exception of LPS, L-NMMA, and dibucaine hydrochloride, were dissolved in dimethyl sulfoxide (DMSO). LPS, L-NMMA, and dibucaine hydrochloride were dissolved in the medium. An aliquot of each solution was added to the medium, and the final concentration of DMSO in the medium was adjusted to 0.1%. The control medium contained the same amount of the vehicle. Cell Culture. RAW 264.7 cells were obtained from RIKEN Gene Bank (Tsukuba, Japan) and cultured at 37°C under 5% CO2-95% air in Eagle's minimal essential medium (Nissui, Tokyo, Japan) containing 10% heat-inactivated (56°C, 30 min) fetal bovine serum (Sigma), 1% nonessential amino acid solution (Sigma), penicillin G potassium (18 µg/ml), and streptomycin sulfate (50 µg/ml) (Meiji Seika, Tokyo, Japan). The cells at passage number 10 or lower were used for experiments.
Measurement of Nitrite. RAW 264.7 cells (2.5 x 105 cells) were incubated for 24 h at 37°C in 0.5 ml of medium. The cells were then washed three times with medium and further incubated for the specified period at 37°C in 0.5 ml of medium in the presence or absence of drugs. After incubation, nitrite levels in the conditioned medium were determined using Griess reagent (Green et al., 1982).
Measurement of Cell Growth and Survival. RAW 264.7 cells (2.5 x 105 cells) were incubated for 24 h at 37°C in 0.5 ml of medium. The cells were washed three times with medium and further incubated for 20 h at 37°C in 0.5 ml of medium containing various drugs. Then, 10 µl of phosphate-buffered saline containing 3-(4,5-dimethyl(thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma) (5 mg/ml) was added, and the cells were further incubated for 4 h at 37°C. After the removal of the medium, 100 µl of DMSO was added, and the absorbance at 595 nm was measured (Mosmann, 1983).
Western Blotting Analysis. RAW 264.7 cells (1 x 106 cells) were incubated for 24 h at 37°C in 2 ml of medium. The cells were then washed three times with medium and further incubated at 37°C for 24 h for the detection of iNOS and for various periods for the detection of IB-, actin, and JNK in 2 ml of medium in the presence or absence of drugs. After incubation, the cells were washed three times with PBS, dipped in 150 µl of ice-cold lysis buffer (20 mM HEPES, pH 7.4, 1% Triton X-100, 10% glycerol, 1 M sodium fluoride, 2.5 mM p-nitrophenylene phosphate, 10 µg/ml phenylmethylsulfonylfluoride, 1 mM Na3VO4, 5 µg/ml leupeptin, and 1 mM EDTA) for 15 min, and disrupted with a Handy Sonic Disruptor (UR-20P; Tomy, Tokyo, Japan). The lysis buffer containing the disrupted cells was centrifuged at 13,000g and 4°C for 20 min. The supernatant obtained was boiled for 5 min in 3x sample buffer (50 mM Tris, pH 7.4, 4% SDS, 10% glycerol, 4% 2-mercaptoethanol, and 0.05 mg/ml bromphenol blue) at a ratio of 2:1 (v/v), loaded on an acrylamide gel (8 or 10%), and subjected to electrophoresis (150 min at 125 V). Each antibody for iNOS, IB-, actin, and JNK was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and Western blotting was carried out as described previously (Hong et al., 2005b). The levels of each protein were quantified by scanning densitometry, and the individual band density value for each point was expressed as the relative density signal.
Semiquantitation of the RNA Level of iNOS by Reverse Transcription-Polymerase Chain Reaction. RAW 264.7 cells (1 x 106 cells) were incubated for 24 h at 37°C in 2 ml of medium. The cells were then washed three times with medium and further incubated for 6 h at 37°C in 2 ml of medium in the presence or absence of drugs. The cells were washed with ice-cold PBS, and the total RNA was extracted using a GenElute Mammalian Total RNA Miniprep Kit (Sigma). The yield of RNA extracted was determined by spectrophotometry. One microgram of each sample was reverse-transcribed for 1 h at 37°C in 20 µl of buffer (50 µM Tris-HCl, pH 8.3, 75 mM KCl, and 3 mM MgCl2) containing 5 µM random hexamer oligonucleotides (Invitrogen, Carlsbad, CA), 500 µM 2'-deoxynucleotide 5'-triphosphate (Takara Bio Inc., Shiga, Japan), and 10 mM dithiothreitol (Takara Bio Inc.). The sequences of the primers for iNOS were forward, 5'-GTGTTCCACCAGGAGATGTTG-3', and reverse, 5'-CTCCTGCCCACTGAGTTCGTC-3', which amplify a 576-base pair fragment of iNOS (Hong et al., 2005b). Polymerase chain reaction (PCR) mixtures consisted of 10 µl of the reverse-transcribed RNA solution and 40 µl of PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, and 1.5 mM MgCl2) containing 0.2 µM of each primer, 200 mM 2'-deoxynucleotide 5'-triphosphate, and 1.25 U of Taq polymerase (Takara Bio Inc.). PCR was performed for 27 cycles; 30-s denaturation at 94°C, 1-min annealing at 54°C, and 1-min extension at 72°C, using a thermal cycler (PCR Thermal Cycler SP; Takara Bio Inc.). The level of mRNA for rat glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was determined as an internal control. The PCR primers for rat GAPDH were forward, 5'-TGATGACATCAAGAAGGTGGTGAAG-3', and reverse, 5'-TCCTTGGAGGCCATGTAGGCCAT-3', which amplify a 249-bp GAPDH fragment (Hong et al., 2005b). PCR was performed for 24 cycles: 30-s denaturation at 94°C, 1-min annealing at 57°C, and 1-min extension at 72°C. After PCR, 10 µl of the reaction mixture was loaded onto a 1.5% agarose minigel, and the PCR products were visualized by ethidium bromide staining after electrophoresis. The levels of mRNA for iNOS and GAPDH were quantified by scanning densitometry, and the ratio of iNOS mRNA to GAPDH mRNA was calculated.
Preparation of Nuclear Extract. RAW 264.7 cells (2 x 106 cells) were incubated for 4 h at 37°C in 4 ml of EMEM containing 10% fetal bovine serum in the presence or absence of drugs. After incubation, the cells were scraped off the plate using a cell scraper and centrifuged at 2500g and 4°C for 5 min. The cells were suspended in 400 µl of Tris-buffered KCl solution (20 mM Tris-HCl, pH 7.8, 50 mM KCl, 10 µg/ml leupeptin, 0.1 mM dithiothreitol, and 1 mM phenyl methylsulfonyl fluoride) and lysed by the addition of the same volume of Tris-buffered KCl solution containing 1.2% Nonidet P-40 (Sigma) with vigorous mixing for 10 s. The homogenate was centrifuged at 4°C and 15,000g for 30 s, and the nuclear pellet was suspended in 30 µl of ice-cold Tris-buffered KCl solution by mixing at 4°C for 15 min. The suspension was then centrifuged at 4°C and 15,000g for 20 min, and the resultant supernatant (nuclear extract fraction) was stored at–80°C before use (Hong et al., 2005b).
Electrophoretic Mobility Shift Assay. Electrophoretic mobility shift assay (EMSA) was carried out according to the protocol accompanying the Gel Shift Assay System (Promega, Madison, WI). In brief, the double-stranded oligonucleotide probes containing NF-B- and AP-1-binding sequences were end-labeled with 1.85 MBq of [
-32P]ATP (111 TBq/nmol; PerkinElmer Life Sciences, Boston, MA) using T4 polynucleotide kinase. The nuclear extract (4 µg) was incubated at room temperature for 20 min with 4 µl of 32P-labeled probe in a binding buffer [50 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 2.5 mM EDTA, 2.5 mM dithiothreitol, 250 mM NaCl, 0.25 mg/ml poly(dI-dC), and 20% glycerol]. DNA/nuclear protein complexes were separated from the DNA probe by electrophoresis on a native 4% acrylamide gel, and the gel was vacuum-dried and visualized with a GS-250 Molecular Imager (Bio-Rad, Hercules, CA) (Hong et al., 2005b).
Detection of Mitochondrial Membrane Potential. To analyze mitochondrial depolarization, a marker of apoptosis, RAW 264.7 cells were stained with the mitochondrial membrane potential-dependent dye DePsipher (Trevigen, Inc., Gaithersburg, MD). RAW 264.7 cells (1 x 106 cells) were incubated for 24 h at 37°C in 2 ml of medium. The cells were then washed three times with medium and further incubated for 24 h at 37°C in 2 ml of medium containing bafilomycin A1 (100 nM) or concanamycin A (100 nM) in the presence or absence of L-NMMA (100 µM) or carboxy-PTIO (100 µM). For the microscopic observation, the cells were washed three times with PBS and stained using a DePsipher Kit (Trevigen, Inc.) and observed under a fluorescence microscope (IX70; Olympus, Tokyo, Japan) (Hong et al., 2005b). For the flow-cytometric analysis, the cells were washed three times with PBS, scraped off the plate, and stained using a DePsipher Kit (Trevigen, Inc.). Subsequently, the intensities for green fluorescence (FL1; a maximal emission at 530 nm) and red fluorescence (FL2; a maximal emission at 590 nm) were analyzed by flow cytometry using FACScan (Becton Dickinson, San Jose, CA), and the percentage of the cells with decreased mitochondrial membrane potential (
m) was calculated (Hong et al., 2005b).
Statistical Analysis. The statistical significance of the results was analyzed using Dunnett's test for multiple comparisons and Student's t test for unpaired observations.
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Effects of L-NMMA on Nitrite Production Induced by Bafilomycin A1 and Concanamycin A. RAW 264.7 cells were incubated at 37°C for 24 h in medium containing bafilomycin A1 (100 nM) or concanamycin A (100 nM) in the presence or absence of the NOS inhibitor L-NMMA. Nitrite production at 24 h induced by bafilomycin A1 or concanamycin A was suppressed by L-NMMA at 10 and 100 nM (Fig. 2), indicating that NOS is responsible for the nitrite production induced by these compounds. The LPS (0.1 µg/ml)-induced nitrite production was also suppressed by L-NMMA at 10 and 100 µM (Fig. 2).
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Effects of Bafilomycin A1 and Concanamycin A on Cell Growth and Survival. RAW 264.7 cells were incubated at 37°C for 24 h in medium containing various concentrations of bafilomycin A1 or concanamycin A. Significant inhibition of cell growth and survival as determined by MTT assay was induced by bafilomycin A1 at 10 and 100 nM in a concentration-dependent manner (Fig. 4). Concanamycin A (1 nM) suppressed the MTT response slightly but significantly, whereas the inhibitory effect of 10 nM concanamycin A was almost the same as that of 100 nM bafilomycin A1 (Fig. 4). In the case of concanamycin A, the inhibitory effect reached a plateau at 10 nM. LPS (0.1 µg/ml) also inhibited the MTT response to almost the same level as 100 nM bafilomycin A1 and 10 and 100 nM concanamycin A (Fig. 4).
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B Inhibitor Bay 11-7082 and the JNK Inhibitor SP600125 on the Decrease in Cell Growth and Survival and the Increase in Nitrite Production Induced by Bafilomycin A1 and Concanamycin A. The decrease in cell growth and survival as determined by MTT assay at 24 h induced by bafilomycin A1 (100 nM) and concanamycin A (100 nM) was partially recovered by Bay 11-7082 in a concentration-dependent manner (Fig. 5A). The increase in nitrite production at 24 h induced by bafilomycin A1 (100 nM) and concanamycin A (100 nM) was partially suppressed by Bay 11-7082 in a concentration-dependent manner (Fig. 5B). The decrease in MTT response and the increase in nitrite production induced by LPS (0.1 µg/ml) were also counteracted by Bay 11-7082 in a concentration-dependent manner (Fig. 5, A and B). SP600125 had the same effect on the decrease in MTT response (Fig. 6A) and the increase in nitrite production (Fig. 6B) induced by bafilomycin A1 (100 nM), concanamycin A (100 nM), and LPS (0.1 µg/ml).
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Combined treatment with Bay 11-7082 (3 µM) and SP600125 (3 µM) additively counteracted the effects of bafilomycin A1 (100 nM), concanamycin A (100 nM), and LPS (0.1 µg/ml) on MTT response (Fig. 7A) and nitrite production (Fig. 7B). These findings suggest that the activation of NF-B and AP-1 contributed to the decrease in cell growth and survival and the increase in nitrite production induced by bafilomycin A1 and concanamycin A.
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B and AP-1 by Bafilomycin A1 and Concanamycin A and Effects of Bay 11-7082 and SP600125. To clarify the mechanism of action of bafilomycin A1 (100 nM) and concanamycin A (100 nM) for the expression of iNOS mRNA, effects of bafilomycin A1 (100 nM) and concanamycin A (100 nM) on the activation of NF-B and AP-1 were examined by EMSA. Treatment with bafilomycin A1 (100 nM) or concanamycin A (100 nM) for 4 h increased the activation of both NF-B and AP-1 (Fig. 8, A and B). LPS (0.1 µg/ml) treatment also activated NF-B and AP-1 (Fig. 8, A and B). Treatment with Bay 11-7082 (10 mM) suppressed the NF-B activation at 4 h induced by bafilomycin A1 (100 nM) or concanamycin A (100 nM) (Fig. 8A), and treatment with SP600125 (10 µM) suppressed the AP-1 activation at 4 h induced by bafilomycin A1 (100 nM) or concanamycin A (100 nM) (Fig. 8B). These findings suggest that the expression of iNOS mRNA by bafilomycin A1 and concanamycin A (Fig. 3B) is induced through the activation of NF-B and AP-1, and the inhibition of the bafilomycin A1 (100 nM)- and concanamycin A (100 nM)-induced nitrite production by Bay 11-7082 and SP600125 is due to the inhibition of iNOS mRNA expression through the inhibition of the activation of NF-B and AP-1, respectively.
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Effects of Bafilomycin A1 and Concanamycin A on the Level of IB- and Phosphorylation of JNK. Incubation of RAW 264.7 cells with bafilomycin A1 (100 nM) or concanamycin A (100 nM) decreased the level of IB- at 40 min (Fig. 9A), whereas LPS (0.1 µg/ml) decreased the level of IB- at 20 min (Fig. 9A). In addition, bafilomycin A1 (100 nM) and concanamycin A (100 nM) induced phosphorylation of JNK at 45 min (Fig. 9B). LPS (0.1 µg/ml) also phosphorylated JNK at 30 min (Fig. 9B). These findings indicate that bafilomycin A1 and concanamycin A activated NF-B by decreasing the level of IB- and activated AP-1 through phosphorylation of JNK.
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Effects of the Na+,K+-ATPase Inhibitor Dibucaine, the F-ATPase Inhibitor Oligomycin, and the Endomembrane Ca2+-ATPase Inhibitor Thapsigargin on Cell Growth and Survival and Nitrite Production. RAW 264.7 cells were incubated at 37°C for 24 h in the presence of various concentrations of dibucaine, oligomycin, or thapsigargin, and MTT response and nitrite production were examined. Dibucaine (0.03–1 mM) did not increase nitrite production (Fig. 10B) but decreased the MTT response at 0.1 mM and above (Fig. 10A). Oligomycin did not increase nitrite production at 1 to 30 ng/ml (Fig. 10D) but inhibited the MTT response at 3 to 30 ng/ml (Fig. 10C). In contrast, thapsigargin induced nitrite production (Fig. 10F) and inhibited the MTT response at 10 to 100 nM (Fig. 10E).
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Effects of L-NMMA and Carboxy-PTIO on Bafilomycin A1- and Concanamycin A-Induced Disruption of Mitochondrial Membrane Potential. After incubation for 24 h at 37°C in the presence of bafilomycin A1 (100 nM) or concanamycin A (100 nM), fluorescence microscopic analysis demonstrated that the number of adherent cells was decreased, and many cells lost mitochondrial membrane potential as shown by the increased number of cells having green fluorescence (Fig. 12). The disruption of mitochondrial membrane potential by bafilomycin A1 (100 nM) and concanamycin A (100 nM) was partially recovered by L-NMMA (100 µM) (Fig. 12) and carboxy-PTIO (100 µM) (Fig. 12). The flow cytometry analysis also demonstrated that bafilomycin A1 (100 nM) and concanamycin A (100 nM) disrupted the mitochondrial membrane potential at 24 h (Fig. 13), which was partially recovered by L-NMMA (100 µM) and carboxy-PTIO (100 µM) (Fig. 13). These findings suggest that the NO produced by bafilomycin A1 and concanamycin A partially participates in the bafilomycin A1- and concanamycin A-induced apoptosis in RAW 264.7 cells.
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). It was reported that the V-ATPase inhibitor bafilomycin A1 (Bowman et al., 1988) induces apoptosis in the human pancreatic cancer cell line Capan-1 (Ohta et al., 1998) and in RAW 264.7 cells (Xu et al., 2003), and concanamycin A (Dröse et al., 1993) induced apoptosis in B cell hybridoma cell line HS-72 (Ishisaki et al., 1999). However, the mechanism underlying the V-ATPase inhibitor-induced apoptosis still remains to be elucidated. We reported that apicularen A induces apoptosis in RAW 264.7 cells as evidenced by a decrease in cell growth and survival determined with the MTT assay, the condensation of chromatin, the formation of DNA ladder, an increase in the percentage of annexin V-positive and propidium iodide-negative cells, and the activation of caspase (Hong et al., 2003). Because apicularen A has a benzolactone enamide core like the V-ATPase inhibitors salicylihalamide A, lobatamides A to F, and oximidines I and II (Boyd et al., 2001), it was suggested that apicularen A also inhibits V-ATPase. Our finding that apicularen A inhibited the vital staining with acridine orange in cultures of the human promyelocytic leukemia cell line HL-60 (Hong et al., 2005a) also supported that apicularen A inhibits V-ATPase. Recently, we have demonstrated that apicularen A inhibits V-ATPase by determining the inhibition of ATP-dependent proton transport into inside-out microsome vesicles and the inhibition of bafilomycin A1-sensitive ATP hydrolysis in mouse peritoneal macrophages (Hong et al., 2006). Huss et al. (2005) also demonstrated that apicularen A inhibits V-ATPase purified from the midgut of the tobacco hornworm, Manduca saxta. In addition, we reported that apicularen A induces NO production through the expression of iNOS mRNA and its protein, and the newly produced NO participates in apicularen A-induced apoptosis in RAW 264.7 cells because the inhibition of NO production by the NOS inhibitor L-NMMA partially alleviated the apicularen A-induced apoptosis (Hong et al., 2005b). Based on our findings, we speculated that the V-ATPase inhibitors commonly induce nitrite production in RAW 264.7 cells. Therefore, in this study, we examined the effect of the commercially available V-ATPase inhibitors bafilomycin A1 and concanamycin A on nitrite production in RAW 264.7 cells and compared it with the effect of other ATPase inhibitors such as the Na+,K+-ATPase inhibitor dibucaine (Hudgins and Bond, 1984), the F-ATPase inhibitor oligomycin (Lardy et al., 1958), and the endomembrane Ca2+-ATPase inhibitor thapsigargin (Thastrup et al., 1990).
As shown in Fig. 1A, both the V-ATPase inhibitors induced nitrite production in a concentration-dependent manner. Concanamycin A was more potent than bafilomycin A1. The time course of changes in nitrite production (Fig. 1B) indicated that a longer time lag is necessary for the bafilomycin A1- and concanamycin A-induced nitrite production than it is for the LPS-induced nitrite production. Therefore, the possibility remained that bafilomycin A1 and concanamycin A induce some cytokine such as interleukin-1
, which in turn induces NO production. Both bafilomycin A1 and concanamycin A increased the level of iNOS mRNA (Fig. 3B) and induced iNOS protein expression (Fig. 3A), and the bafilomycin A1- and concanamycin A-induced NO production was inhibited by the NOS inhibitor L-NMMA (Fig. 2). These findings indicated that the bafilomycin A1- and concanamycin A-induced NO production is due to the expression of the iNOS gene. The expression of the iNOS gene is regulated by the binding of transcription factors to several consensus sequences of the gene. The promoter region of the iNOS gene contains the binding sites for NF-B, AP-1, and CCAAT/enhancer-binding protein, and the component essential for the expression of the iNOS gene is NF-B (Xie et al., 1994). In our previous article (Hong et al., 2005b), we described that apicularen A induced iNOS gene expression by the activation of NF-B and AP-1 in RAW 264.7 cells. In mouse peritoneal macrophages, NF-B is activated by the V-ATPase inhibitor bafilomycin A1 (Conboy et al., 1999). In this study, we showed that bafilomycin A1 and concanamycin A activated NF-B and AP-1 through the degradation of IB- and phosphorylation of JNK, respectively (Fig. 8). Furthermore, the NO production induced by bafilomycin A1 and concanamycin A was inhibited by the NF-B inhibitor Bay 11-7082 and the JNK inhibitor SP600125 (Figs. 5,6,7), and the bafilomycin A1- and concanamycin A-induced activation of NF-B and AP-1 was inhibited by Bay 11-7082 and SP600125, respectively (Fig. 8). Therefore, it is possible that the increase in the level of iNOS mRNA caused by the V-ATPase inhibitors bafilomycin A1 and concanamycin A (Fig. 3B) is due to the activation of NF-B and AP-1. Other ATPase inhibitors such as the Na+,K+-ATPase inhibitor dibucaine and the F-ATPase inhibitor oligomycin did not induce nitrite production (Fig. 10, B and D). On the other hand, the endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin induced NO production (Fig. 10F). Induction of NO production by thapsigargin is also reported in rat mesangial cells (Rodriguez-Lopez et al., 1999) and in Jurkat T cells (Srivastava et al., 1999). However, the induction of NO production by thapsigargin is due to the increase in intracellular free Ca2+ (Srivastava et al., 1999), the mechanism being different from that of the V-ATPase inhibitors.
NO, a radical produced from L-arginine by NOS, plays a significant role as a cellular second messenger (Palmer et al., 1988), and iNOS produces a high level of NO, whereas constitutively expressed NOS generates a low level of physiologically active NO (Kubes, 2000). It is possible that a high level of NO is associated with apoptosis, anticancer, bactericidal, and antiparasitic effects probably due to the formation of reactive radicals including peroxynitrite (Szabo and Ohshima, 1997). However, NO seems to be a bifunctional regulator of apoptosis. It exerts an antiapoptotic action in splenocytes (Genaro et al., 1995), vascular endothelial cells (Dimmeler et al., 1997), hepatocytes (Kim et al., 1997), and eosinophils (Hebestreit et al., 1998). On the other hand, it induces apoptosis in macrophages (Albina et al., 1993; Shimaoka et al., 1995), pancreatic islet cells (Heller et al., 1995), thymocytes (Fehsel et al., 1995), vascular endothelial cells (Lincoln et al., 1996), and neurons (Leist et al., 1997). In RAW 264.7 cells, it is also reported that NO induces apoptosis (Messmer et al., 1995; Jun et al., 1999; Gotoh et al., 2002). Therefore, it is possible that NO produced by bafilomycin A1 and concanamycin A also participates in bafilomycin A1- and concanamycin A-induced apoptosis in RAW 264.7 cells. On the other hand, depending on surrounding cell environment, cells undergo oxidative stress, which induces apoptosis in a variety of cells, when levels of reactive oxygen species exceed the counter-regulatory antioxidant capacity of the cell (Martindale and Holbrook, 2002). Therefore, participation of reactive oxygen species in the bafilomycin A1- and concanamycin A-induced apoptosis in RAW 264.7 cells remains to be elucidated.
As determined by the MTT assay, cell growth and survival were decreased by treatment with bafilomycin A1 and concanamycin A (Fig. 4). Treatment with bafilomycin A1 (100 nM) or concanamycin A (100 nM) for 24 h increased the percentage of annexin V-positive and propidium iodide-negative cells (data not shown), suggesting that both the V-ATPase inhibitor induce apoptosis in RAW 264.7 cells. Induction of apoptosis in RAW 264.7 cells by bafilomycin A1 was also reported by Xu et al. (2003). The bafilomycin A1- and concanamycin A-induced decrease in cell growth and survival was augmented by combined treatment with the NO donor SNAP (Fig. 11A). It is reported that SNAP induces apoptosis in RAW 264.7 cells and that NO-induced apoptosis is mediated by an endoplasmic reticulum stress pathway involving p50ATF6 and CCAAT/enhancer-binding protein homologous protein in RAW 264.7 cells (Gotoh et al., 2002). In addition, the bafilomycin A1- and concanamycin A-induced decrease in cell growth and survival was partially recovered by combined treatment with the NO scavenger caroxy-PTIO (Fig. 11B). It is also reported that the apoptosis determined by DNA fragmentation induced by the NO-generating agent, NOC 18, was suppressed by carboxy-PTIO in HL-60 cells (Yabuki et al., 1997). In addition, bafilomycin A1- and concanamycin A-induced disruption of mitochondrial membrane potential, an index of apoptosis, was partially recovered by L-NMMA (Figs. 12 and 13) in parallel with the inhibition of NO production (Fig. 2). The NO scavenger carboxy-PTIO also alleviated partially the disruption of mitochondrial membrane potential induced by the V-ATPase inhibitors bafilomycin A1 and concanamycin A (Figs. 12 and 13). In contrast, the Na+,K+-ATPase inhibitor dibucaine and the F-ATPase inhibitor oligomycin decreased cell growth and survival, although they did not induce NO production (Fig. 10). Therefore, the mechanism for the dibucaine- and oligomycin-induced decrease in cell growth and survival is different from that for the V-ATPase inhibitor-induced decrease in cell growth and survival.
In this study, we indicated for the first time that the V-ATPase inhibitors bafilomycin A1 and concanamycin A, but not the Na+,K+-ATPase inhibitor dibucaine and the F-ATPase inhibitor oligomycin, produce NO and suggested that the newly produced NO participates partially in the V-ATPase inhibitor-induced apoptosis in RAW 264.7 cells.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: NO, nitric oxide; V-ATPase, vacuolar-type (H+)-ATPase; LPS, lipopolysaccharide; NOS, nitric-oxide synthase; L-NMMA, NG-monomethyl-L-arginine acetate; SNAP, S-nitroso-N-acetyl-DL-penicillamine; carboxy-PTIO, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, sodium salt; Bay 11-7082, (E)3-[(4-methylphenyl)sulfonyl])-2-propenenitrile; JNK, c-Jun N-terminal kinase; SP600125, anthra[1,9-cd]pyrazol-6(2H)-one; DMSO, dimethyl sulfoxide; MTT, 3-(4,5-dimethyl(thiazol-2-yl)-2,5-diphenyltetrazolium bromide; iNOS, inducible nitric oxide synthase; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; EMSA, electrophoretic mobility shift assay; NF, nuclear factor; AP-1, activator protein-1; NOC 18, 1-hydroxy-2-oxo-3,3-bis (2-aminoethyl)-1-triazene.
Address correspondence to: Dr. Kazuo Ohuchi, Laboratory of Pathophysiological Biochemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aoba Aramaki, Aoba-ku, Sendai, Miyagi 980-8578, Japan. E-mail: ohuchi-k{at}mail.pharm.tohoku.ac.jp
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m) was then calculated. Values are the means from four samples, with the S.E.M. shown by vertical bars. Statistical significance: ***, p < 0.001 versus the nonstimulated control; and ###, p < 0.001 versus the corresponding control group.