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Leukotoxin-activated Human Pulmonary Artery Endothelial Cell Produces Nitric Oxide and Superoxide Anion
Pulmonary Pharmacology & Therapeutics (2002) 15, 25–33 doi:10.1006/pupt.2001.0322, available online at http://www.idealibrary.com on
PULMONARY PHARMACOLOGY & THERAPEUTICS
Leukotoxin-activated Human Pulmonary Artery Endothelial Cell Produces Nitric Oxide and Superoxide Anion Seitaro Okamura, Shingo Ameshima, Yoshiki Demura, Takeshi Ishizaki, Shigeru Matsukawa∗, Isamu Miyamori Department of Internal Medicine and ∗Central Research Laboratory, Fukui Medical University, Fukui, Japan
SUMMARY: To provide evidence that pulmonary endothelial cells exposed to 9,10-epoxy-12-octadecenoate (Lx) produce nitric oxide (NO) and superoxide anion (O2• −), we measured NO production, using a NO chemiluminescence analyzer, and nitric oxide synthase (NOS) activity, monitoring the conversion of L- [14C] arginine to L- [14C] citrulline, and O2• − by a fluorescence assay using a fluorescence spectrophotometer with hydroethidine (HE) in human pulmonary artery endothelial cells (HPAEC). NO production and eNOS were increased significantly when HPAEC were incubated with 10 M Lx, and this effect was inhibited by L-NMMA or in the absence of extracellular Ca2+. Addition of 10 mM HE to the cell suspension spontaneously and continuously caused a subtle increase in fluorescence intensity, due to intracellular oxidation of HE to ethidium bromide (EB). Treatment of the cell suspension with Lx after the addition of HE exerted a dose-dependent increase in intracellular EB fluorescence. Pre-treatment with allopurinol, a xanthine oxidase inhibitor, decreased the intracellular EB fluorescence by 54% in HPAEC incubated with 100 M Lx. These results show that Lx induces NO production via activation of eNOS and O2• − production in endothelial cells via activation of cellular xanthine oxidase. Thus, Lx is a bioactive lipid. 2002 Elsevier Science Ltd.
KEY WORDS: Endothelial nitric oxide synthase (eNOS), Human pulmonary artery endothelial cells, Leukotoxin (Lx), Nitric oxide, Superoxide anion.
nitric oxide (• NO) and its derivative, peroxynitrite, may be responsible for the induction of acute lung injury.5–10 Ishizaki et al11,12 considered that • NO and O2• − may be involved in Lx-induced lung injury in isolated perfused rat lungs, and Ishizaki et al13 and Nakanishi et al14 demonstrated NO-dependent vasodilation following Lx administration to isolated rat pulmonary artery rings. However, at that time, it was unclear which cell(s) are responsible for NO production by Lx: though circumstantial evidence suggests pulmonary arterial endothelial cells which are initially injured in the response to Lx. We now examined nitric oxide (NO) production and nitric oxide synthase activity in response to Lx in human pulmonary artery endothelial cells, using a NO analyzer and whole cell assay, respectively. We also investigated the effect of Lx on the production of O2• − by an endothelial cell line15 using a fluorescence assay with hydroethidine (HE).
INTRODUCTION Leukotoxin, 9,10-epoxy-12-octadecenoate (Lx), a linoleate epoxide which is synthesized by neutrophils,1 was found to be increased in the bronchoalveolar lavage fluid from patients with the adult respiratory distress syndrome (ARDS) and to cause ARDS-like acute pulmonary vascular injury when injected into rats.2 Lx also caused edema of alveolar walls3 and cellular mitochondrial damage in isolated perfused rat lungs.4 However, the mechanism of pulmonary vascular injury induced by Lx have not been entirely elucidated. Recently, it has been reported that reactive oxygen species, such as superoxide anion (O2• −), hydrogen peroxide, and hydroxyl radical, as well as Offprint requests to: Seitaro Okamura, M.D., Department of Internal Medicine, Fukui Medical University, Fukui 910-11, Japan. Tel: +81-776-61-3111. Fax: +81-776-61-8111. E-mail: [email protected] 1094–5539/02/$35.00/0
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2002 Elsevier Science Ltd. All rights reserved.
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Fig. 1a Effect of Lx on NOS activity in a whole-cell assay. The effect of Lx on the conversion of L- [14C] citrulline by NOS was measured in the HPAEC culture. Vehicle or various concentrations of Lx were added to the medium 5 min before termination of the assay. Lx dose-dependently activated NOS. Lx (100 M) caused less activation of NOS than the lower dose of Lx (10 M). Data are means ± SE, n=6. ∗P<0.01 vs control. †P<0.05 vs Lx 10 M.
Fig. 1b The effect of L-NMMA or on activation of NOS by Lx. 200 M L-NMMA was added to the medium 3 h before the experiment. NOS activation was significantly inhibited by L-NMMA. Data are means ± SE, n=6. ∗P<0.01 vs control.
Human pulmonary artery endothelial cells
Fig. 2 Effect of Lx on NOS activity in HPAEC with (closed bar) or without (hatched bar) extra cellular Ca. When HPAEC were incubated with Ca free buffer, Lx did not increase the production of L- [14C] citrulline significantly. Data are means ± SE, n=4. ∗P<0.01 vs control.
Fig. 3 Effect of Lx on extracted NOS activity. Low dose of Lx (0.1–10 M) did not activate extracted NOS, and high dose of Lx (100 M) suppress NOS activity. Data are means ± SE, n=4. ∗P<0.05 vs control.
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Fig. 4 The effect of L-NMMA on production of NOx by Lx. 200 M L-NMMA was added to the medium 3 h before the experiment. NOx production was significantly inhibited by L-NMMA. Data are means ± SE, n=6. ∗P<0.05 vs control.
MATERIALS AND METHODS Materials Lx was purchased from Cayman Chemical Co. (Ann Arbor, MI, USA) and its purity was identified by high performance liquid chromatography (HPLC). Ethidium bromide (EB) and Hank’s balanced salt solution (HBSS) were purchased from Wako Pure Chemical Ltd. (Osaka, Japan). Hydroethidine (HE), Linoleic acid, Allopurinol, indomethacin, catalase, and superoxide dismutase (SOD) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). NGmonomethyl L-arginine (L-NMMA) was purchased from Calbiochem. (La Jolla, CA, USA). Apocynin, an NADPH oxidase inhibitor, was from Ardrich Chemical (Tokyo, Japan). L- [U- 14C] arginine (292317 mCi/mmol) were purchased from Amersham (Buckinghamshire, England). AG50W-X8 (Na+ form, 200-400 mesh) was purchased from Bio-Rad (Hercules, CA, USA). Cell culture Human pulmonary arterial endothelial cells (HPAEC) from an established cell line (Endocell-PA) and Humedia EG medium were obtained from KURABOU Ltd (Osaka, Japan). HPAEC were grown Humedia EG medium containing 2% fetal bovine serum, 50 g/
ml gentamicin, 50 ng/ml amphotericin-B, 10 ng/ml recombinant human EGF, 1 mg/ml hydrocortisone, and 0.4% bovine brain extract (BBE). Cells were cultured in 75 cm2 tissue culture flasks (Corning, NY, USA) and those reaching confluence at the 5th-10th passage were used after trypsinization. Measurement of NOS activity in HPAEC Whole cell assay: HPAEC were suspended in E-GM UV medium at a cell density of 2×105/ml, and then 2 ml of the cell suspension was added to each well of a six-well flat-bottom culture plate (Corning, NY). After 24 h, all cells were adherent to the bottom and had reached confluency. Cells were kept in HBSS buffer for 4 h before the assay. The conversion assay using whole intact cells was performed in KrebsHenseleit buffer (composition; 121 mM NaCl, 5.4 mM KCl, 1.2 mM MgSO4, 1.2 mM NaH2PO4, 15 mM NaHCO3, 2.5 mM CaCl2, 11.5 mM glucose, adjusted to pH 7.4) according to the method of Lamas et al16 with some modifications.17 Briefly, L- [14C] arginine was added at a concentration of 0.5 Ci/ml as indicated. Cells were incubated in 95% air and 5% CO2 at 37 °C for 3 hrs, then Lx or the vehicle was added 5 min before termination of the experiment. The reaction was stopped by addition of 1 ml of ice-cold 0.6 N perchloric acid. The acid soluble extracts were neutralized with 120 l of 5 M K2CO3. The neutralized
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Fig. 5a Intracellular EB fluorescence (O2• − production) in human pulmonary artery endothelial cells by a fluorescence spectrophotometer versus time in min. The cell suspension was warmed to 37 °C in a cylindrical glass cuvette and then 10 mM HE was added 1 min after data collection was started. EB was excited at 488 nm and emission collected at 610 nM. Ten min after the addition of HE, Lx was added to the suspension. The increase in intracellular EB fluorescence 5 min after the addition of Lx was defined as DF.
cell extract and culture medium were collected and then applied to a 700-l AG50 W-X8 column (Na+form, Bio-Rad) that had been preequilibrated with stop buffer (10 mM EDTA, 100 mM HEPES at pH5.5). The void eluate and washes were collected in scintillation vials, to which was added 5 ml of ACSII (Amersham) scintillation fluid. The amount of L[14C] citrulline produced was determined with an Aloka liquid scintillation counter (Tokyo, Japan), and the formation of L- [14C] citrulline from L- [14C] arginine was confirmed by thin-layer chromatography, as described previously.17 Cell-free assay HPAEC were suspended in sterile-filtered PBS and spun at 1200 rpm for 5 min at 4°C. The supernatant was discarded, and the cell pellet was washed twice with PBS and resuspended in an ice-cold homogenization buffer consisting of 50 mM HEPES (pH 7.40), 250 mM sucrose, 1 mM DTT, 0.1 mM EDTA, 0.1 mM EGTA, leupeptin (10 g/ml), pepstatin A (10 g/ml), and PMSF (100 g/ml). The cells were then homogenized with a glass homogenizer.
NO synthase activity was measured by monitoring the conversion of L- [14C] arginine to L- [14C] citrulline, as previously described.18 Briefly, 10 l of crude cell extract was added to a 1.5 ml tube containing final concentrations of 10 g/ml calmodulin, 10 M BH4, 1 mM CaCl2, 1 mM NADPH, 1 mM DTT, 10 M FAD, 50 mM HEPES (pH 7.80), 150 nM L- [14C] arginine, and various concentrations of Lx. Thereafter, the whole reaction mixture was incubated for 5 min at 37°C and the reaction was terminated by addition of 200 l of stop buffer (10 mM EDTA, 100 mM HEPES at pH 5.5). The total volume (300 l) was then applied to a 700 l AG50 W-X8 column as described for the whole-cell assay and L- [14C] citrulline was confirmed by thin-layer chromatography, as described previously. Measurement of perfusate NOx concentration For quantification of the NO produced by HPAEC, cells in T-25 flasks were washed three times with Krebs–Henseleit buffer and incubated in the same buffer in 5% CO2 at 37°C. Cells were stimulated with 0.1 to 10 M Lx in 2 ml Krebs–Henseleit buffer
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Fig. 5b Effects of Lx (10, 30, 100 M) on the production of intracellular superoxide anion. 10 M (n=4), 30 M (n=4), and 100 M (n=7) significantly induced a dose-dependent increase in intracellular O2• − production. ∗P<0.01 vs Lx 10 M. Values are mean ± SE.
containing 1 mM L-arginine, at pH 7.4, for 5 min. After this, 1 ml of the aliquot was collected and assayed for NOx (nitric oxide, nitrite and nitrate), using a catalytic method for reduction of oxidation products of NO to NO gas. The samples were injected into a refluxing glass reaction chamber containing vanadium (III)-HCl (2N) at 85 °C and NO gas was detected by a NO analyzer (FES-450 NO analyzer, Scholartec, Osaka).19,20 Detection of intracellular superoxide anion HE has been conventionally used to evaluate O2• − production during the respiratory burst in neutrophils and monocytes.21,22 HE is a fluorogenic compound which freely permeates cells and can be directly oxidized to EB by O2• − produced by cells. Intracellular EB combines with nuclear or mitochondrial DNA, and the resultant complex emits fluorescent light with a maximum intensity of 610 nm when excited with 488 nm light. Carter et al23 used this technique to evaluate the production of intracellular O2• − by endothelial cells. They confirmed that stable intracellular HE fluorescence was evident at approximately 5 min after the addition of HE to an endothelial cell suspension. We modified their method to evaluate the continuous production of O2• − in HPAEC induced by Lx.
Cells were suspended in sterile-filtered HBSS buffer to a density of 1 × 106/ml. An aliquot (0.5 ml) of the cell suspension in a cylindrical glass cuvette (7 mm × 50 mm; MC medical, Tokyo, Japan) was warmed to 37°C by circulation of thermostatically controlled water and mixed by a magnetic stirrer attached to the bottom of a cell holder in a fluorescence spectrophotometer (RF5000; SHIMADZU, Tokyo, Japan). Ten micromoles of HE were then added to the cuvette 1-min after data collection had been started. Excitation was performed by a 488 nM light with a 5-nM slit, and emission fluorescence intensity was recorded at 610 nm with a 20-nM slit for detection of the EB-DNA complex. Ten min after the addition of HE, Lx was added to a final concentration of 10 per 100 M. For inhibitor studies, 1 mM allopurinol, 1 mM indomethacin, 1 mM apocynin, 1000 U/ml SOD, or 100 M L-NMMA was added to the cell suspension 30 min before Lx addition. The increase in intracellular EB fluorescence observed for 5 min after the addition of Lx was defined as F. Statistical analysis Results are expressed as means ± SE. Statistical analysis was performed with the use of one-way analysis of variance with Bonferroni for multiple
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Fig. 5c Relative percentage of intracellular EB fluorescence (O2• − measurement) in HPAEC 5 min after stimulation with 100 M Lx. 1 mM allopurinol, 1 mM indomethacin, 1 mM apocynin, 1000 U/ml SOD, and 100 mM L-NMMA were incubated with the endothelial cells for 15 min at 37 °C prior to the addition of HE, and 100 M Lx was added to the cell suspension 10 min after the addition of HE. Allopurinol and SOD significantly decreased intracellular O2• −. ∗P<0.05 vs control, without pretreatment. Data are means ± SE, n=4.
comparisons. Comparisons were considered statistically significant when P<0.05.
RESULTS Effect of Lx on NOS activity When we examined the effect of Lx on NOS activity in HPAEC we found that the production of L- [14C] citrulline in HPAEC was increased by Lx in a concentration-dependent manner. When HPAEC were incubated with 10 M Lx, the production of L- [14C] citrulline was maximally increased (Fig. 1a). This effect was inhibited by 200 M L-NMMA (Fig. 1b). When HPAEC were incubated with Ca free buffer, the production of L- [14C] citrulline was not increased by Lx (Fig. 2). When we examined the direct effect of Lx on crude cell extract, Lx did not increase the production of L- [14C] citrulline and 100 M Lx decreased it (Fig. 3). Linoleic acid, a parent compound of Lx, did not affect the NOS activity in HPAEC (data not shown).
Effect of Lx on NO production To confirm that the activation of NOS by Lx actually reflects NO production, we measured NOx production of the monolayers of HPAEC. NOx production of HPAEC treated with Lx at 10 M (we used this dose of Lx since 10 M of Lx maximally stimulated NO production by HPAEC as shown in Fig. 1a) was significantly increased and this effect was inhibited by 200 M L-NMMA (Fig. 4). Detection of intracellular superoxide anion in HPAEC Addition of HE to the cell suspension spontaneously and continuously exhibited a subtle increase in fluorescence intensity due to formation of an EB-DNA complex, suggesting intracellular accumulation of O2• − in the cells while in a resting state. Treatment of cells with Lx 10 min after the addition of HE exerted a rapid and marked increase in EB fluorescence, as shown in Figure 5a. Lx induced a dose-dependent increase in fluorescence intensity (Fig. 5b).
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Effects of pre-treatment with various inhibitors on the 100 M Lx-induced increase in intracellular EB fluorescence were examined for the same period of incubation. Allopurinol at 1 mM significantly decreased intracellular EB fluorescence ( F) by 54% in Lx-stimulated HPAEC (Fig. 5c). Allopurinol at 5 mM, however, did not show a further suppressive effect above that observed with 1 mM. In contrast, indomethacin (1 mM) and apocynin (1 mM) and little effect. SOD (1000 U/ml) significantly decreased F by 37% in Lx-stimulated cells. Endothelial cells pretreated with 100 M L-NMMA exhibited a 114% increase in intracellular EB fluorescence. Endothelial cells incubated with allopurinol, indomethacin, apocynin, SOD, or L-NMMA exhibited no marked changes in intracellular EB fluorescence when Lx was not added to the cells (data not shown) Discussion In the present study, we demonstrated in cultured HPAEC that Lx increased the production of NO via NOS activation. The activation of NOS and production of NO were observed within 5 minutes; these events were inhibited when HPAEC were cultured with a Ca-free buffer. These results indicate that Lx does indeed activate eNOS in HPAEC, probably via mobilization of extracellular Ca2+. Our findings also demonstrated that Lx rapidly increased O2• − production in cultured human pulmonary artery endothelial cells (HPAEC). Even if one considers that the stoichiometry for the reaction of HE with superoxide to form EB may be qualitative rather than quantitative for superoxide24 since HE catalyzes the dismutation of superoxide and also can be oxidized by cytochrome C, our results indicate that Lx is a powerful O2• − generator in cultured human endothelial cells. The O2• − production by Lx was significantly suppressed in the presence of allopurinol, an xanthine oxidase inhibitor, but not in the presence of indomethacin or apocynin, suggesting that Lx stimulates O2• − production via the activation of cellular xanthine oxidase but not of cyclooxygenase or NADPH oxidase. As to NADPH oxidase, which is dependent upon two membrane-bound proteins, however, we cannot rule out the possibility of protein digestion during the cell treatment with trypsin. Overproduction of O2• − at a Lx concentration of 100 M or because of formation of peroxynitrite25 may suppress the NOS activity (Fig.1a). From these results it appears that the cytotoxic action of a higher dose of Lx on HPAEC is more complex than that of a lower dose.26,27 Our experimental results, thus, are interesting with respect to the study by Toborek et al28,29 who noted that linoleic acid, an unsaturated fatty acid, disturbs endothelial function via increased oxidant stress, increased
intracellular Ca2+, although the authors did not refer the involvement of NO and did not characterize the responsible active oxygen species. The action of linoleate also differs from the action of Lx in that linoleate did not affect mitochondrial function.4 One can therefore say that Lx has its own biologic-toxic cellular effect distinguishable from the effect of linoleate. Although the precise local concentration of Lx in the clinical setting of acute lung injury is not known, estimated levels from BALF samples in a report by Ozawa et al2 and from a blood samples of another one of their reports30 reached 0.45–6.2 M. Taken together with a recent report that the Lx-induced diol had a greater cytotoxicity31 and that Lx and its diol has a potent human neutrophil32 chemoattractant activity, Lx and its metabolite apparently act synergistically. Therefore, the doses of Lx used in our study may not have been supraphysiologic. In summary, our results indicate that Lx augments NO production via activation of eNOS and O2• − production in endothelial cells likely via activation of xanthine oxidase. The production of O2• − may contribute to Lx-induced cell injury and suggests that Lx, linoleate metabolites, similar to 13-hydroperoxyoctadecadienoic acid, an oxidized form of linoleate33,34 exerts the profile of a biologically active lipid.
ACKNOWLEDGEMENTS We thank Dr. Norbert F Voelkel (Pulmonary Hypertension Center, University of Colorado Health Sciences Center) for his critical comments and reading. This work was supported by a Grant-in-Aid for Encouragement of Young Scientists (No.07770420) and by Grants-in-Aid for Scientific Research (No 07457145 and No 10670536) from the Ministry of Education, Science and Culture of Japan.
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22. Rothe G, Valet G. Flow cytometric analysis of respiratory burst activity in phagocytes with hydroethidine and 2′, 7′dichlorofluorescin. J Leukoc Biol 1990; 47: 440–448. 23. Carter W O, Narayanan P K, Robinson J P. Intracellular hydrogen peroxide and superoxide anion detection in endothelial cells. J Leukoc Biol 1994; 55: 253–258. 24. Benov L, Szteinberg L and Fridovich I. Critical evaluation of the use of hydroethidine as a measure of superoxide anion radical. Free Radic Biol Med 1998; 25: 826–831. 25. Robinson V K, Sato E, Nelson D K, Camhi S L, Robbins R A, Hoyt J C. Peroxynitrite inhibits inducible (Type2) nitric oxide synthase in murine lung epithelial cells in vitro. Free Radic Biol Med 2001; 30: 986–991. 26. Ishizaki T, Ameshima S, Matsukawa S. Are leukotoxins toxic? Nature Med 1997; 3: 592. 27. Ishizaki T, Ozawa T, Voelkel N F. Leukotoxins and the lung. Pul Pharmacol Therapeutics 1999; 12: 145–155. 28. Toborek M, Barger S W, Mattson M P, Barve S, McClain C J, Henning B. Linoleic acid and TNF-alpha cross-amplify oxidative injury and dysfunction of endothelial cells. J Lipid Res 1996; 37: 123–135. 29. Toborek M, Blanc E M, Kaiser S, Mattson M P, Henning B. Linoleic acid potentiates TNF-mediated oxidative stress, disruption of ancium homeostasis, and apoptosis of cultured vascular endothelial cells. J Lipid Res 1997; 38:2155–2167. 30. Ozawa T, Sugiyama S, Hayakawa M, Taki F. ARDS and Leukotoxin. Nippon-kyoubu-Sikkann-Gakkaishi 1991; 29: 159–164. 31. Moghaddam M F, Grant D F, Cheek J M, Greene J F, Williamson K C, Hammock B D. Bioactivation of leukotoxin to their toxic diols by epoxide hydrolase. Nature Med 1997; 3: 562–566. 32. Totani Y, Saito Y, Ishizaki T, Sasaki F, Ameshima S, Miyamori I. Leukotoxin and its diol induce neutrophil chemotaxis through signal transudation different from that of fMLP. Eur Respir J 2000; 15: 75–79. 33. Ramasamy S, Parthasarathy S, Harrison, D G. Regulation of endothelial nitric oxide synthase gene expression by oxidized linoleic acid. J lipid Res 1998; 39: 268–276. 34. DeMeyer G R Y, Bult H, Verbeuren TJ, Herman A G. The role of endothelial cells in the relaxations induced by 13hydroxy-and 13-hydroperoxylinoleic acid in canine arteries. Br J Pharmacol 1992; 107: 597–603.
Abbreviation List ARDS=adult respiratory distress syndrome; BBE=bovine brain extract; cNOS=constitutive nitric oxide synthase; DMPO=5, 5-dimethyl-1-pyrroline- N-oxide; EB=ethidium bromide; EGF=endothelial growth factor; ESR=electron spin resonance; HBSS=Hank’s balanced salt solution; HE=hydroethidine; HPAEC=human pulmonary artery endothelial cells; HPLC=high performance liquid chromatography; L-NMMA=NG-monomethyl L-arginine; Lx=leukotoxin, 9,10-epoxy-12-octadecenoate; SOD=superoxide dismutase.
Date received: 27 June 2001. Date accepted: 24 September 2001.
PULMONARY PHARMACOLOGY & THERAPEUTICS
Leukotoxin-activated Human Pulmonary Artery Endothelial Cell Produces Nitric Oxide and Superoxide Anion Seitaro Okamura, Shingo Ameshima, Yoshiki Demura, Takeshi Ishizaki, Shigeru Matsukawa∗, Isamu Miyamori Department of Internal Medicine and ∗Central Research Laboratory, Fukui Medical University, Fukui, Japan
SUMMARY: To provide evidence that pulmonary endothelial cells exposed to 9,10-epoxy-12-octadecenoate (Lx) produce nitric oxide (NO) and superoxide anion (O2• −), we measured NO production, using a NO chemiluminescence analyzer, and nitric oxide synthase (NOS) activity, monitoring the conversion of L- [14C] arginine to L- [14C] citrulline, and O2• − by a fluorescence assay using a fluorescence spectrophotometer with hydroethidine (HE) in human pulmonary artery endothelial cells (HPAEC). NO production and eNOS were increased significantly when HPAEC were incubated with 10 M Lx, and this effect was inhibited by L-NMMA or in the absence of extracellular Ca2+. Addition of 10 mM HE to the cell suspension spontaneously and continuously caused a subtle increase in fluorescence intensity, due to intracellular oxidation of HE to ethidium bromide (EB). Treatment of the cell suspension with Lx after the addition of HE exerted a dose-dependent increase in intracellular EB fluorescence. Pre-treatment with allopurinol, a xanthine oxidase inhibitor, decreased the intracellular EB fluorescence by 54% in HPAEC incubated with 100 M Lx. These results show that Lx induces NO production via activation of eNOS and O2• − production in endothelial cells via activation of cellular xanthine oxidase. Thus, Lx is a bioactive lipid. 2002 Elsevier Science Ltd.
KEY WORDS: Endothelial nitric oxide synthase (eNOS), Human pulmonary artery endothelial cells, Leukotoxin (Lx), Nitric oxide, Superoxide anion.
nitric oxide (• NO) and its derivative, peroxynitrite, may be responsible for the induction of acute lung injury.5–10 Ishizaki et al11,12 considered that • NO and O2• − may be involved in Lx-induced lung injury in isolated perfused rat lungs, and Ishizaki et al13 and Nakanishi et al14 demonstrated NO-dependent vasodilation following Lx administration to isolated rat pulmonary artery rings. However, at that time, it was unclear which cell(s) are responsible for NO production by Lx: though circumstantial evidence suggests pulmonary arterial endothelial cells which are initially injured in the response to Lx. We now examined nitric oxide (NO) production and nitric oxide synthase activity in response to Lx in human pulmonary artery endothelial cells, using a NO analyzer and whole cell assay, respectively. We also investigated the effect of Lx on the production of O2• − by an endothelial cell line15 using a fluorescence assay with hydroethidine (HE).
INTRODUCTION Leukotoxin, 9,10-epoxy-12-octadecenoate (Lx), a linoleate epoxide which is synthesized by neutrophils,1 was found to be increased in the bronchoalveolar lavage fluid from patients with the adult respiratory distress syndrome (ARDS) and to cause ARDS-like acute pulmonary vascular injury when injected into rats.2 Lx also caused edema of alveolar walls3 and cellular mitochondrial damage in isolated perfused rat lungs.4 However, the mechanism of pulmonary vascular injury induced by Lx have not been entirely elucidated. Recently, it has been reported that reactive oxygen species, such as superoxide anion (O2• −), hydrogen peroxide, and hydroxyl radical, as well as Offprint requests to: Seitaro Okamura, M.D., Department of Internal Medicine, Fukui Medical University, Fukui 910-11, Japan. Tel: +81-776-61-3111. Fax: +81-776-61-8111. E-mail: [email protected] 1094–5539/02/$35.00/0
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2002 Elsevier Science Ltd. All rights reserved.
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Fig. 1a Effect of Lx on NOS activity in a whole-cell assay. The effect of Lx on the conversion of L- [14C] citrulline by NOS was measured in the HPAEC culture. Vehicle or various concentrations of Lx were added to the medium 5 min before termination of the assay. Lx dose-dependently activated NOS. Lx (100 M) caused less activation of NOS than the lower dose of Lx (10 M). Data are means ± SE, n=6. ∗P<0.01 vs control. †P<0.05 vs Lx 10 M.
Fig. 1b The effect of L-NMMA or on activation of NOS by Lx. 200 M L-NMMA was added to the medium 3 h before the experiment. NOS activation was significantly inhibited by L-NMMA. Data are means ± SE, n=6. ∗P<0.01 vs control.
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Fig. 2 Effect of Lx on NOS activity in HPAEC with (closed bar) or without (hatched bar) extra cellular Ca. When HPAEC were incubated with Ca free buffer, Lx did not increase the production of L- [14C] citrulline significantly. Data are means ± SE, n=4. ∗P<0.01 vs control.
Fig. 3 Effect of Lx on extracted NOS activity. Low dose of Lx (0.1–10 M) did not activate extracted NOS, and high dose of Lx (100 M) suppress NOS activity. Data are means ± SE, n=4. ∗P<0.05 vs control.
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Fig. 4 The effect of L-NMMA on production of NOx by Lx. 200 M L-NMMA was added to the medium 3 h before the experiment. NOx production was significantly inhibited by L-NMMA. Data are means ± SE, n=6. ∗P<0.05 vs control.
MATERIALS AND METHODS Materials Lx was purchased from Cayman Chemical Co. (Ann Arbor, MI, USA) and its purity was identified by high performance liquid chromatography (HPLC). Ethidium bromide (EB) and Hank’s balanced salt solution (HBSS) were purchased from Wako Pure Chemical Ltd. (Osaka, Japan). Hydroethidine (HE), Linoleic acid, Allopurinol, indomethacin, catalase, and superoxide dismutase (SOD) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). NGmonomethyl L-arginine (L-NMMA) was purchased from Calbiochem. (La Jolla, CA, USA). Apocynin, an NADPH oxidase inhibitor, was from Ardrich Chemical (Tokyo, Japan). L- [U- 14C] arginine (292317 mCi/mmol) were purchased from Amersham (Buckinghamshire, England). AG50W-X8 (Na+ form, 200-400 mesh) was purchased from Bio-Rad (Hercules, CA, USA). Cell culture Human pulmonary arterial endothelial cells (HPAEC) from an established cell line (Endocell-PA) and Humedia EG medium were obtained from KURABOU Ltd (Osaka, Japan). HPAEC were grown Humedia EG medium containing 2% fetal bovine serum, 50 g/
ml gentamicin, 50 ng/ml amphotericin-B, 10 ng/ml recombinant human EGF, 1 mg/ml hydrocortisone, and 0.4% bovine brain extract (BBE). Cells were cultured in 75 cm2 tissue culture flasks (Corning, NY, USA) and those reaching confluence at the 5th-10th passage were used after trypsinization. Measurement of NOS activity in HPAEC Whole cell assay: HPAEC were suspended in E-GM UV medium at a cell density of 2×105/ml, and then 2 ml of the cell suspension was added to each well of a six-well flat-bottom culture plate (Corning, NY). After 24 h, all cells were adherent to the bottom and had reached confluency. Cells were kept in HBSS buffer for 4 h before the assay. The conversion assay using whole intact cells was performed in KrebsHenseleit buffer (composition; 121 mM NaCl, 5.4 mM KCl, 1.2 mM MgSO4, 1.2 mM NaH2PO4, 15 mM NaHCO3, 2.5 mM CaCl2, 11.5 mM glucose, adjusted to pH 7.4) according to the method of Lamas et al16 with some modifications.17 Briefly, L- [14C] arginine was added at a concentration of 0.5 Ci/ml as indicated. Cells were incubated in 95% air and 5% CO2 at 37 °C for 3 hrs, then Lx or the vehicle was added 5 min before termination of the experiment. The reaction was stopped by addition of 1 ml of ice-cold 0.6 N perchloric acid. The acid soluble extracts were neutralized with 120 l of 5 M K2CO3. The neutralized
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Fig. 5a Intracellular EB fluorescence (O2• − production) in human pulmonary artery endothelial cells by a fluorescence spectrophotometer versus time in min. The cell suspension was warmed to 37 °C in a cylindrical glass cuvette and then 10 mM HE was added 1 min after data collection was started. EB was excited at 488 nm and emission collected at 610 nM. Ten min after the addition of HE, Lx was added to the suspension. The increase in intracellular EB fluorescence 5 min after the addition of Lx was defined as DF.
cell extract and culture medium were collected and then applied to a 700-l AG50 W-X8 column (Na+form, Bio-Rad) that had been preequilibrated with stop buffer (10 mM EDTA, 100 mM HEPES at pH5.5). The void eluate and washes were collected in scintillation vials, to which was added 5 ml of ACSII (Amersham) scintillation fluid. The amount of L[14C] citrulline produced was determined with an Aloka liquid scintillation counter (Tokyo, Japan), and the formation of L- [14C] citrulline from L- [14C] arginine was confirmed by thin-layer chromatography, as described previously.17 Cell-free assay HPAEC were suspended in sterile-filtered PBS and spun at 1200 rpm for 5 min at 4°C. The supernatant was discarded, and the cell pellet was washed twice with PBS and resuspended in an ice-cold homogenization buffer consisting of 50 mM HEPES (pH 7.40), 250 mM sucrose, 1 mM DTT, 0.1 mM EDTA, 0.1 mM EGTA, leupeptin (10 g/ml), pepstatin A (10 g/ml), and PMSF (100 g/ml). The cells were then homogenized with a glass homogenizer.
NO synthase activity was measured by monitoring the conversion of L- [14C] arginine to L- [14C] citrulline, as previously described.18 Briefly, 10 l of crude cell extract was added to a 1.5 ml tube containing final concentrations of 10 g/ml calmodulin, 10 M BH4, 1 mM CaCl2, 1 mM NADPH, 1 mM DTT, 10 M FAD, 50 mM HEPES (pH 7.80), 150 nM L- [14C] arginine, and various concentrations of Lx. Thereafter, the whole reaction mixture was incubated for 5 min at 37°C and the reaction was terminated by addition of 200 l of stop buffer (10 mM EDTA, 100 mM HEPES at pH 5.5). The total volume (300 l) was then applied to a 700 l AG50 W-X8 column as described for the whole-cell assay and L- [14C] citrulline was confirmed by thin-layer chromatography, as described previously. Measurement of perfusate NOx concentration For quantification of the NO produced by HPAEC, cells in T-25 flasks were washed three times with Krebs–Henseleit buffer and incubated in the same buffer in 5% CO2 at 37°C. Cells were stimulated with 0.1 to 10 M Lx in 2 ml Krebs–Henseleit buffer
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Fig. 5b Effects of Lx (10, 30, 100 M) on the production of intracellular superoxide anion. 10 M (n=4), 30 M (n=4), and 100 M (n=7) significantly induced a dose-dependent increase in intracellular O2• − production. ∗P<0.01 vs Lx 10 M. Values are mean ± SE.
containing 1 mM L-arginine, at pH 7.4, for 5 min. After this, 1 ml of the aliquot was collected and assayed for NOx (nitric oxide, nitrite and nitrate), using a catalytic method for reduction of oxidation products of NO to NO gas. The samples were injected into a refluxing glass reaction chamber containing vanadium (III)-HCl (2N) at 85 °C and NO gas was detected by a NO analyzer (FES-450 NO analyzer, Scholartec, Osaka).19,20 Detection of intracellular superoxide anion HE has been conventionally used to evaluate O2• − production during the respiratory burst in neutrophils and monocytes.21,22 HE is a fluorogenic compound which freely permeates cells and can be directly oxidized to EB by O2• − produced by cells. Intracellular EB combines with nuclear or mitochondrial DNA, and the resultant complex emits fluorescent light with a maximum intensity of 610 nm when excited with 488 nm light. Carter et al23 used this technique to evaluate the production of intracellular O2• − by endothelial cells. They confirmed that stable intracellular HE fluorescence was evident at approximately 5 min after the addition of HE to an endothelial cell suspension. We modified their method to evaluate the continuous production of O2• − in HPAEC induced by Lx.
Cells were suspended in sterile-filtered HBSS buffer to a density of 1 × 106/ml. An aliquot (0.5 ml) of the cell suspension in a cylindrical glass cuvette (7 mm × 50 mm; MC medical, Tokyo, Japan) was warmed to 37°C by circulation of thermostatically controlled water and mixed by a magnetic stirrer attached to the bottom of a cell holder in a fluorescence spectrophotometer (RF5000; SHIMADZU, Tokyo, Japan). Ten micromoles of HE were then added to the cuvette 1-min after data collection had been started. Excitation was performed by a 488 nM light with a 5-nM slit, and emission fluorescence intensity was recorded at 610 nm with a 20-nM slit for detection of the EB-DNA complex. Ten min after the addition of HE, Lx was added to a final concentration of 10 per 100 M. For inhibitor studies, 1 mM allopurinol, 1 mM indomethacin, 1 mM apocynin, 1000 U/ml SOD, or 100 M L-NMMA was added to the cell suspension 30 min before Lx addition. The increase in intracellular EB fluorescence observed for 5 min after the addition of Lx was defined as F. Statistical analysis Results are expressed as means ± SE. Statistical analysis was performed with the use of one-way analysis of variance with Bonferroni for multiple
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Fig. 5c Relative percentage of intracellular EB fluorescence (O2• − measurement) in HPAEC 5 min after stimulation with 100 M Lx. 1 mM allopurinol, 1 mM indomethacin, 1 mM apocynin, 1000 U/ml SOD, and 100 mM L-NMMA were incubated with the endothelial cells for 15 min at 37 °C prior to the addition of HE, and 100 M Lx was added to the cell suspension 10 min after the addition of HE. Allopurinol and SOD significantly decreased intracellular O2• −. ∗P<0.05 vs control, without pretreatment. Data are means ± SE, n=4.
comparisons. Comparisons were considered statistically significant when P<0.05.
RESULTS Effect of Lx on NOS activity When we examined the effect of Lx on NOS activity in HPAEC we found that the production of L- [14C] citrulline in HPAEC was increased by Lx in a concentration-dependent manner. When HPAEC were incubated with 10 M Lx, the production of L- [14C] citrulline was maximally increased (Fig. 1a). This effect was inhibited by 200 M L-NMMA (Fig. 1b). When HPAEC were incubated with Ca free buffer, the production of L- [14C] citrulline was not increased by Lx (Fig. 2). When we examined the direct effect of Lx on crude cell extract, Lx did not increase the production of L- [14C] citrulline and 100 M Lx decreased it (Fig. 3). Linoleic acid, a parent compound of Lx, did not affect the NOS activity in HPAEC (data not shown).
Effect of Lx on NO production To confirm that the activation of NOS by Lx actually reflects NO production, we measured NOx production of the monolayers of HPAEC. NOx production of HPAEC treated with Lx at 10 M (we used this dose of Lx since 10 M of Lx maximally stimulated NO production by HPAEC as shown in Fig. 1a) was significantly increased and this effect was inhibited by 200 M L-NMMA (Fig. 4). Detection of intracellular superoxide anion in HPAEC Addition of HE to the cell suspension spontaneously and continuously exhibited a subtle increase in fluorescence intensity due to formation of an EB-DNA complex, suggesting intracellular accumulation of O2• − in the cells while in a resting state. Treatment of cells with Lx 10 min after the addition of HE exerted a rapid and marked increase in EB fluorescence, as shown in Figure 5a. Lx induced a dose-dependent increase in fluorescence intensity (Fig. 5b).
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Effects of pre-treatment with various inhibitors on the 100 M Lx-induced increase in intracellular EB fluorescence were examined for the same period of incubation. Allopurinol at 1 mM significantly decreased intracellular EB fluorescence ( F) by 54% in Lx-stimulated HPAEC (Fig. 5c). Allopurinol at 5 mM, however, did not show a further suppressive effect above that observed with 1 mM. In contrast, indomethacin (1 mM) and apocynin (1 mM) and little effect. SOD (1000 U/ml) significantly decreased F by 37% in Lx-stimulated cells. Endothelial cells pretreated with 100 M L-NMMA exhibited a 114% increase in intracellular EB fluorescence. Endothelial cells incubated with allopurinol, indomethacin, apocynin, SOD, or L-NMMA exhibited no marked changes in intracellular EB fluorescence when Lx was not added to the cells (data not shown) Discussion In the present study, we demonstrated in cultured HPAEC that Lx increased the production of NO via NOS activation. The activation of NOS and production of NO were observed within 5 minutes; these events were inhibited when HPAEC were cultured with a Ca-free buffer. These results indicate that Lx does indeed activate eNOS in HPAEC, probably via mobilization of extracellular Ca2+. Our findings also demonstrated that Lx rapidly increased O2• − production in cultured human pulmonary artery endothelial cells (HPAEC). Even if one considers that the stoichiometry for the reaction of HE with superoxide to form EB may be qualitative rather than quantitative for superoxide24 since HE catalyzes the dismutation of superoxide and also can be oxidized by cytochrome C, our results indicate that Lx is a powerful O2• − generator in cultured human endothelial cells. The O2• − production by Lx was significantly suppressed in the presence of allopurinol, an xanthine oxidase inhibitor, but not in the presence of indomethacin or apocynin, suggesting that Lx stimulates O2• − production via the activation of cellular xanthine oxidase but not of cyclooxygenase or NADPH oxidase. As to NADPH oxidase, which is dependent upon two membrane-bound proteins, however, we cannot rule out the possibility of protein digestion during the cell treatment with trypsin. Overproduction of O2• − at a Lx concentration of 100 M or because of formation of peroxynitrite25 may suppress the NOS activity (Fig.1a). From these results it appears that the cytotoxic action of a higher dose of Lx on HPAEC is more complex than that of a lower dose.26,27 Our experimental results, thus, are interesting with respect to the study by Toborek et al28,29 who noted that linoleic acid, an unsaturated fatty acid, disturbs endothelial function via increased oxidant stress, increased
intracellular Ca2+, although the authors did not refer the involvement of NO and did not characterize the responsible active oxygen species. The action of linoleate also differs from the action of Lx in that linoleate did not affect mitochondrial function.4 One can therefore say that Lx has its own biologic-toxic cellular effect distinguishable from the effect of linoleate. Although the precise local concentration of Lx in the clinical setting of acute lung injury is not known, estimated levels from BALF samples in a report by Ozawa et al2 and from a blood samples of another one of their reports30 reached 0.45–6.2 M. Taken together with a recent report that the Lx-induced diol had a greater cytotoxicity31 and that Lx and its diol has a potent human neutrophil32 chemoattractant activity, Lx and its metabolite apparently act synergistically. Therefore, the doses of Lx used in our study may not have been supraphysiologic. In summary, our results indicate that Lx augments NO production via activation of eNOS and O2• − production in endothelial cells likely via activation of xanthine oxidase. The production of O2• − may contribute to Lx-induced cell injury and suggests that Lx, linoleate metabolites, similar to 13-hydroperoxyoctadecadienoic acid, an oxidized form of linoleate33,34 exerts the profile of a biologically active lipid.
ACKNOWLEDGEMENTS We thank Dr. Norbert F Voelkel (Pulmonary Hypertension Center, University of Colorado Health Sciences Center) for his critical comments and reading. This work was supported by a Grant-in-Aid for Encouragement of Young Scientists (No.07770420) and by Grants-in-Aid for Scientific Research (No 07457145 and No 10670536) from the Ministry of Education, Science and Culture of Japan.
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Abbreviation List ARDS=adult respiratory distress syndrome; BBE=bovine brain extract; cNOS=constitutive nitric oxide synthase; DMPO=5, 5-dimethyl-1-pyrroline- N-oxide; EB=ethidium bromide; EGF=endothelial growth factor; ESR=electron spin resonance; HBSS=Hank’s balanced salt solution; HE=hydroethidine; HPAEC=human pulmonary artery endothelial cells; HPLC=high performance liquid chromatography; L-NMMA=NG-monomethyl L-arginine; Lx=leukotoxin, 9,10-epoxy-12-octadecenoate; SOD=superoxide dismutase.
Date received: 27 June 2001. Date accepted: 24 September 2001.