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Conditions to Study Nitric Oxide Generation by Polymorphonuclear Cells from an Inflammatory Exudate in Rats
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
Vol. 327, No. 2, March 15, pp. 292–294, 1996 Article No. 0124
Conditions to Study Nitric Oxide Generation by Polymorphonuclear Cells from an Inflammatory Exudate in Rats Jesu´s Ro´denas, Teresa Carbonell, and M. Teresa Mitjavila1 Departament de Fisiologia, Facultat de Biologia, Universitat de Barcelona, Avda. Diagonal 645, 08028 Barcelona, Spain
Received September 6, 1995, and in revised form December 27, 1995
Superoxide and nitric oxide release by leukocytes has been usually performed after exposure to a particular stimulus. We measured the generation of superoxide and nitric oxide by cells isolated from an inflammatory exudate of rats in either the absence or the presence of a variety of stimuli. Nonstimulated leukocytes generated superoxide radical (1.2 nmolr106 cells01) and nitric oxide (3.8 nmolr106cells01) after 2 h incubation. When cells were incubated with lipopolysaccharides, opsonized zymosan or phorbol 12-myristate 13-acetate, superoxide level increased while nitric oxide decreased. Phorbol 12-myristate 13-acetate (100 ng/ml) induced a decrease of 0.88 nmolr106cells01 compared with nonstimulated cells, and incubation with N-iminoethyl-L-ornithine increased superoxide production by 0.81 nmolr106cells01. These results provide clear evidence that cells from an inflammatory exudate which are already triggered are able to generate a considerable amount of nitric oxide and in less proportion superoxide, that the measure of nitric oxide must be performed without a further stimulus, and that both molecules react in an equimolar proportions to give peroxynitrite anion. q 1996 Academic Press, Inc. Key Words: nitric oxide; superoxide anion; peroxynitrite; polymorphonuclear leukocytes; inflammation; rats.
Activated phagocytes release highly reactive species such as superoxide anion (O•0 2 ), hydrogen peroxide, hypochlorous acid (1–3), and nitric oxide (•NO) (4, 5). The formation of •NO from L-arginine (6) is a tool of considerable interest owing to its wide biochemical activity, which includes cytotoxic activity of stimulated 1 To whom correspondence should be addressed. Fax: (343) 4110358.
macrophages (7). This has engendered studies on •NO reaction with the O•0 2 , because of the high rate constant in aqueous solution (6.7 1 109 M01s01) (8). Both radicals can be simultaneously produced by the same cell type (4), thus favoring the generation of peroxynitrite, known to decompose to nitrogen dioxide radical and a strong oxidant (9). However, the same experimental conditions are usually used in studies on the release • of O•0 2 and NO. Because of the reaction between both radicals, the real production of •NO measured by the HbO2 method can be higher than that previously reported, as the •NO detected may give a measure of the • NO that has not reacted with O•0 2 . The aim of our study was to evaluate the effects of several stimulants on the generation of these radicals using inflammatory cells and to assess interactions between them, in order to establish the best conditions for an independent measure. Cells isolated from an inflammatory exudate in rats were chosen, since they were already triggered by inflammatory mediators in vivo. As experimental inflammatory model we used the carrageenin-induced granuloma in rats. The induction of the granuloma and the procedure for isolating leukocytes from the exudate have been previously described (10). Cells were counted and the volume was adjusted to give 1 1 107 cells/ml. Differential cell counts were performed microscopically after nonspecific esterase staining, giving 90% of polymorphonuclear (PMN).2 At the beginning of the experiment, viability was above 95% as assessed by the Trypan blue exclusion test. Animal care standards and experimental protocols were in accordance with the EC guidelines. In order to determine the ability of inflammatory
2 Abbreviations used: SOD, superoxide dismutase; LPS, lipopolysaccharides; PMA, phorbol 12-myristate 13-acetate; L-NNA, NG-nitro-L-arginine; L-NIO, N-iminoethyl-L-ornithine; PMN, polymorphonuclear.
292
0003-9861/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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AP: Archives
CONDITIONS TO STUDY NITRIC OXIDE BY POLYMORPHONUCLEAR LEUKOCYTES
FIG. 1. Superoxide (O2•0) and nitric oxide (•NO) production by inflammatory cells in the absence or in the presence of stimulants. Inflammatory cells (1 1 106 cells/tube) from the exudate were incubated in PBS, pH 7.4, with 2 mM Ca2/ and 0.5 mM Mg2/. The following final concentration of stimulants was used: 1 mg/ml opsonized zymosan, 1 mg/ml lipolysaccharides (LPS), 100 or 500 ng/ml phorbol 12myristate 13-acetate (PMA) (Sigma, St. Louis, MO). O2•0 production was determined as superoxide dismutase (SOD)-inhibitable reduction of 0.15 mM cytochrome c (11), using 60 U/ml SOD (Sigma) at 550 nm (Dj Å 21 mM01rcm01). •NO was determined by the HbO2 method (12) as modified by Murphy et al. (13). After washing, cells were incubated in 1 ml PBS containing 15 mM HbO2 , 100 U/ml catalase (Sigma), 60 U/ml SOD, and 0.6 mM L-arginine (Sigma). After 2 h of incubation at 377C in a water bath, the reaction was stopped by immersing the tubes in ice and cold centrifugation. The supernatant was used to measure O2•0, and •NO formation. For •NO measure a difference spectrum vs sample preincubated for 30 min with 0.6 mM L-NIO (Cookson Chemicals LTD, Southampton, UK) was recorded. The HbO2 –MetHb conversion (indicating •NO formation) was measured by change in the absorbance at 578 nm vs 592 nm (Dj Å 11.2 mM01rcm01) (13). O2•0 and •NO formation are expressed as nmolr2 h01r106cells01. Values are the mean { SEM of six separate experiments performed in triplicate. Data were analyzed by Student’s t test: ***P õ 0.001 vs O2•0 in nonstimulated cells; /P õ 0.05 vs •NO in nonstimulated cells.
• cells to generate O•0 2 and NO and to study the reaction between both radical species, the cells were incubated in either the absence or the presence of different stimulants. The superoxide dismutase (SOD)-inhibitable reduction of cytochrome c represented approximately 85% of the total O•0 2 generated in all cases, and Fig. 1 shows that nonstimulated PMN generated 1.2 nmol 6 01 O•0 2 r10 cells . PMN stimulated with 1 mg/ml lipopolysaccharides (LPS) or with 1 mg/ml zymosan produced 6 01 2.4 nmolr106cells01 and 3.8 nmol O•0 2 r10 cells , respectively, and with 100 or 500 ng/ml phorbol 12-myristate 13-acetate (PMA) production reached 7.4 nmol
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6 01 • O•0 2 r10 cells . The production of NO (Fig. 1) by nonstimulated leukocytes detected by the HbO2 –MetHb conversion was 3.5 nmolr106cells01. A reduction of 0.6 nmolr106cells01 was found in cells incubated with zymosan, and with 100 ng/ml PMA the reduction was 0.9 nmolr106cells01, which means a decrease of 25%. When cells were stimulated with 100 ng/ml PMA in the presence of an inhibitor of NO synthase such as NG-nitroL-arginine (L-NNA) or N-iminoethyl-L-ornithine (LNIO) at 0.6 mM (Fig. 2), a variable and small degree of inhibition was detected. However, preincubation for 30 min with L-NNA or with L-NIO reduced •NO production by 69 and 93% respectively (Fig. 2). These results show a different behavior of O•0 and •NO simultane2 ously generated by PMN from an inflammatory exudate of rats. First, •NO was released even in nonstimulated leukocytes, when low levels of O•0 were found. 2 Probably cells were already triggered by inflammatory mediators generated in response to carrageenin. Moreover, rat PMN exposed to LTB4 synthesize more nitric oxide (14). Our results agree with those found by Ischiropoulos et al. (4), who reported that freshly isolated rat alveolar macrophages have a relatively high background of nitrite plus nitrate production. Second, the • • stimulus that increases O•0 2 decreases NO. NO detection by HbO2 –MetHb conversion has recently been • questioned (15) in situations in which O•0 2 and NO are
FIG. 2. Nitric oxide (•NO) production by inflammatory cells in the absence or in the presence of inhibitors of NO synthase. Conditions were as shown in Fig. 1, but cells were stimulated with 100 ng/ml phorbol 12-myristate 13-acetate (PMA) either with 0.6 mM NG-nitroL-arginine (L-NNA) (Sigma, St. Louis, MO) or 0.6 mM N-iminoethylL-ornithine (L-NIO) (Cookson Chemicals LTD, Southampton, UK) as inhibitors of NO synthase with or without preincubation. Values are the mean { SEM of three separate experiments performed in triplicate. Data were analyzed by Student’s t test: *P õ 0.05, **P õ 0.01, ***P õ 0.001 vs cells without inhibitor.
AP: Archives
´ DENAS, CARBONELL, AND MITJAVILA RO
294 TABLE I
Superoxide Production by Inflammatory Cells in the Presence of an Inhibitor of NO Synthase (L-NIO) O2•0 production nmolr106 cells01 Zymosan (1 mg/ml) Zymosan (1 mg/ml) / L-NIO (0.6 mM) PMA (100 ng/ml) PMA (100 ng/ml) / L-NIO (0.6 mM)
5.82 6.41 7.28 8.09
{ { { {
0.10 0.23* 0.11 0.18**
Note. Conditions were as shown in Fig. 1 for superoxide (O2•0) determination, but cells were stimulated with either 1 mg/ml zymosan or 100 ng/ml phorbol 12-myristate 13-acetate (PMA) (Sigma, St. Louis, MO). 0.6 mM N-iminoethyl-L-ornithine (L-NIO) (Cookson Chemicals LTD, Southampton, UK) was used as inhibitor of NO synthase with preincubation. Values are mean { SEM of three separate experiments performed in triplicate. Data were analyzed by Student’s t test. * P õ 0.05. ** P õ 0.01.
PMN at a basal level in the presence of L-arginine, with, at present, an unknown role. When phagocytes are stimulated to produce O•0 2 , which is unable to initiate lipid peroxidation (19), peroxynitrite, a more potent oxidant, is formed, which may explain the cytotoxicity associated with •NO. It is concluded that two aspects should be taken into account when measuring O•0 and •NO produced by 2 phagocytes: in order to avoid interference cells should be incubated with a potent inhibitor of NO synthase • for O•0 2 detection. Second, when studying NO release, cells should be incubated with SOD, avoiding the use of any stimulant of O•0 2 production and using a blank preincubated with an inhibitor of •NO synthase. However, studies on peroxynitrite production will require the use of a stimulant at such concentration that would • give the same amount of O•0 2 as NO. ACKNOWLEDGMENTS The authors thank Robin Rycroft for his help in the English editing of the manuscript. This study was supported by Grant FIS 94/0733.
simultaneously generated, as HbO2 can also be oxidized by peroxynitrite. Although a more specific technique for •NO has been developed (15), in our conditions we found a maximum of only 14–23% of peroxynitrite generated compared with •NO and this peroxynitrite is not detected by HbO2 ; otherwise, the level of •NO would remain constant. To assess whether the decrease in •NO level after stimulating the cells was due to a reaction with O•0 2 , cells were incubated in the presence of 0.6 mM L-NIO for O•0 2 determination in the absence of L-arginine. Leukocytes stimulated with zymosan (1 mg/ml) or PMA (100 ng/ml) displayed an increase in O•0 2 production of 0.6 and 0.8 nmol, respectively (Table I). It can be deduced that 10% of PMN-derived O•0 reacts with •NO 2 which is about the same order as the O•0 non-SOD 2 inhibited. So, the concentrations of SOD generally used to inhibit cytochrome c reduction by O•0 2 , as in our case, are not sufficient to prevent the reaction between •NO • and O•0 2 . NO is produced by NO synthase, a cytosolic enzyme (16), and must diffuse through the plasma membrane, as •NO is soluble in both water and the lipids (17) where NADPH oxidase is located (18). So, it will react with O•0 2 in a fast reaction provided enough O•0 is produced. If O•0 production is not stimulated, 2 2 • less NO will react to give peroxynitrite and more •NO will be detected. The increases in O•0 generation are 2 of the same order as the decreases in •NO observed when incubating cells with zymosan (0.6 nmol) or PMA (0.9 nmol), which confirms that both radicals react in equimolar proportions. The different response in O•0 and •NO levels with 2 the stimulants tested led us to speculate that they may have different roles in vivo. •NO may be produced by
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REFERENCES 1. Babior, B. M., Kipnes, R. S., and Curnutte, J. T. (1973) J. Clin. Invest. 52, 741–744. 2. Biemond, P., van Eijk, H. G., Swaak, A. J. G., and Koster, J. F. (1984) J. Clin. Invest. 73, 1576–1579. 3. Halliwell, B. (1982) Biol. Int. Rep. 6, 529–542. 4. Ischiropoulos, H., Zhu, L., and Beckman, J. S. (1992) Arch. Biochem. Biophys. 298, 446–451. 5. Carreras, M. C., Pargament, G. A., Catz, S. D., Poderoso, J. J., and Boveris, A. (1994) FEBS Lett. 341, 65–68. 6. Palmer, R. M. J., Ashton, D. S., and Moncada, S. (1988) Nature 333, 664–666. 7. Liew, F. Y., Millot, S., Parkinson, C., Palmer, R. M. J., and Moncada, S. (1990) J. Immunol. 144, 4794–4797. 8. Huie, R. E., and Padmaja, S. (1993) Free Rad. Res. Commun. 18, 195–199. 9. Beckman, J. S., Beckman, T. W., Chen, J., Marshall, P. A., and Freeman, B. A. (1990) Proc. Natl. Acad. Sci. USA 87, 1620– 1624. 10. Ro´denas, J., Mitjavila, M. T., and Carbonell, T. (1995) Free Rad. Biol. Med. 18, 869–875. 11. Johnston, R. B., Godzik, C. A., and Cohn, Z. A. (1978) J. Exp. Med. 148, 115–119. 12. Feelisch, M., and Noack, E. A. (1987) Eur. J. Pharmacol. 139, 19–30. 13. Murphy, M. E., Piper, H. M., Watanabe, H., and Sies, H. (1991) J. Biol. Chem. 266, 19378–19383. 14. McCall, T. B., Boughton-Smith, N. K., Palmer, R. M. J., Whittle, B. J. R., and Moncada, S. (1989) Biochem. J. 261, 293–296. 15. Schmidt, K., Klatt, P., and Mayer, B. (1994) Biochem. J. 301, 645–647. 16. McCall, T. B., Palmer, R. M. J., and Moncada, S. (1991) Eur. J. Immunol. 21, 2523–2527. ¨ ngga`rd, E. (1994) Lancet 343, 1199–1206. 17. A 18. Babior, B. M. (1984) Blood 64, 959–966. 19. Winterbourn, C. C. (1979) Biochem. J. 182, 625–628.
AP: Archives
Vol. 327, No. 2, March 15, pp. 292–294, 1996 Article No. 0124
Conditions to Study Nitric Oxide Generation by Polymorphonuclear Cells from an Inflammatory Exudate in Rats Jesu´s Ro´denas, Teresa Carbonell, and M. Teresa Mitjavila1 Departament de Fisiologia, Facultat de Biologia, Universitat de Barcelona, Avda. Diagonal 645, 08028 Barcelona, Spain
Received September 6, 1995, and in revised form December 27, 1995
Superoxide and nitric oxide release by leukocytes has been usually performed after exposure to a particular stimulus. We measured the generation of superoxide and nitric oxide by cells isolated from an inflammatory exudate of rats in either the absence or the presence of a variety of stimuli. Nonstimulated leukocytes generated superoxide radical (1.2 nmolr106 cells01) and nitric oxide (3.8 nmolr106cells01) after 2 h incubation. When cells were incubated with lipopolysaccharides, opsonized zymosan or phorbol 12-myristate 13-acetate, superoxide level increased while nitric oxide decreased. Phorbol 12-myristate 13-acetate (100 ng/ml) induced a decrease of 0.88 nmolr106cells01 compared with nonstimulated cells, and incubation with N-iminoethyl-L-ornithine increased superoxide production by 0.81 nmolr106cells01. These results provide clear evidence that cells from an inflammatory exudate which are already triggered are able to generate a considerable amount of nitric oxide and in less proportion superoxide, that the measure of nitric oxide must be performed without a further stimulus, and that both molecules react in an equimolar proportions to give peroxynitrite anion. q 1996 Academic Press, Inc. Key Words: nitric oxide; superoxide anion; peroxynitrite; polymorphonuclear leukocytes; inflammation; rats.
Activated phagocytes release highly reactive species such as superoxide anion (O•0 2 ), hydrogen peroxide, hypochlorous acid (1–3), and nitric oxide (•NO) (4, 5). The formation of •NO from L-arginine (6) is a tool of considerable interest owing to its wide biochemical activity, which includes cytotoxic activity of stimulated 1 To whom correspondence should be addressed. Fax: (343) 4110358.
macrophages (7). This has engendered studies on •NO reaction with the O•0 2 , because of the high rate constant in aqueous solution (6.7 1 109 M01s01) (8). Both radicals can be simultaneously produced by the same cell type (4), thus favoring the generation of peroxynitrite, known to decompose to nitrogen dioxide radical and a strong oxidant (9). However, the same experimental conditions are usually used in studies on the release • of O•0 2 and NO. Because of the reaction between both radicals, the real production of •NO measured by the HbO2 method can be higher than that previously reported, as the •NO detected may give a measure of the • NO that has not reacted with O•0 2 . The aim of our study was to evaluate the effects of several stimulants on the generation of these radicals using inflammatory cells and to assess interactions between them, in order to establish the best conditions for an independent measure. Cells isolated from an inflammatory exudate in rats were chosen, since they were already triggered by inflammatory mediators in vivo. As experimental inflammatory model we used the carrageenin-induced granuloma in rats. The induction of the granuloma and the procedure for isolating leukocytes from the exudate have been previously described (10). Cells were counted and the volume was adjusted to give 1 1 107 cells/ml. Differential cell counts were performed microscopically after nonspecific esterase staining, giving 90% of polymorphonuclear (PMN).2 At the beginning of the experiment, viability was above 95% as assessed by the Trypan blue exclusion test. Animal care standards and experimental protocols were in accordance with the EC guidelines. In order to determine the ability of inflammatory
2 Abbreviations used: SOD, superoxide dismutase; LPS, lipopolysaccharides; PMA, phorbol 12-myristate 13-acetate; L-NNA, NG-nitro-L-arginine; L-NIO, N-iminoethyl-L-ornithine; PMN, polymorphonuclear.
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0003-9861/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
/ 6b14$$9340
02-12-96 09:58:49
arca
AP: Archives
CONDITIONS TO STUDY NITRIC OXIDE BY POLYMORPHONUCLEAR LEUKOCYTES
FIG. 1. Superoxide (O2•0) and nitric oxide (•NO) production by inflammatory cells in the absence or in the presence of stimulants. Inflammatory cells (1 1 106 cells/tube) from the exudate were incubated in PBS, pH 7.4, with 2 mM Ca2/ and 0.5 mM Mg2/. The following final concentration of stimulants was used: 1 mg/ml opsonized zymosan, 1 mg/ml lipolysaccharides (LPS), 100 or 500 ng/ml phorbol 12myristate 13-acetate (PMA) (Sigma, St. Louis, MO). O2•0 production was determined as superoxide dismutase (SOD)-inhibitable reduction of 0.15 mM cytochrome c (11), using 60 U/ml SOD (Sigma) at 550 nm (Dj Å 21 mM01rcm01). •NO was determined by the HbO2 method (12) as modified by Murphy et al. (13). After washing, cells were incubated in 1 ml PBS containing 15 mM HbO2 , 100 U/ml catalase (Sigma), 60 U/ml SOD, and 0.6 mM L-arginine (Sigma). After 2 h of incubation at 377C in a water bath, the reaction was stopped by immersing the tubes in ice and cold centrifugation. The supernatant was used to measure O2•0, and •NO formation. For •NO measure a difference spectrum vs sample preincubated for 30 min with 0.6 mM L-NIO (Cookson Chemicals LTD, Southampton, UK) was recorded. The HbO2 –MetHb conversion (indicating •NO formation) was measured by change in the absorbance at 578 nm vs 592 nm (Dj Å 11.2 mM01rcm01) (13). O2•0 and •NO formation are expressed as nmolr2 h01r106cells01. Values are the mean { SEM of six separate experiments performed in triplicate. Data were analyzed by Student’s t test: ***P õ 0.001 vs O2•0 in nonstimulated cells; /P õ 0.05 vs •NO in nonstimulated cells.
• cells to generate O•0 2 and NO and to study the reaction between both radical species, the cells were incubated in either the absence or the presence of different stimulants. The superoxide dismutase (SOD)-inhibitable reduction of cytochrome c represented approximately 85% of the total O•0 2 generated in all cases, and Fig. 1 shows that nonstimulated PMN generated 1.2 nmol 6 01 O•0 2 r10 cells . PMN stimulated with 1 mg/ml lipopolysaccharides (LPS) or with 1 mg/ml zymosan produced 6 01 2.4 nmolr106cells01 and 3.8 nmol O•0 2 r10 cells , respectively, and with 100 or 500 ng/ml phorbol 12-myristate 13-acetate (PMA) production reached 7.4 nmol
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6 01 • O•0 2 r10 cells . The production of NO (Fig. 1) by nonstimulated leukocytes detected by the HbO2 –MetHb conversion was 3.5 nmolr106cells01. A reduction of 0.6 nmolr106cells01 was found in cells incubated with zymosan, and with 100 ng/ml PMA the reduction was 0.9 nmolr106cells01, which means a decrease of 25%. When cells were stimulated with 100 ng/ml PMA in the presence of an inhibitor of NO synthase such as NG-nitroL-arginine (L-NNA) or N-iminoethyl-L-ornithine (LNIO) at 0.6 mM (Fig. 2), a variable and small degree of inhibition was detected. However, preincubation for 30 min with L-NNA or with L-NIO reduced •NO production by 69 and 93% respectively (Fig. 2). These results show a different behavior of O•0 and •NO simultane2 ously generated by PMN from an inflammatory exudate of rats. First, •NO was released even in nonstimulated leukocytes, when low levels of O•0 were found. 2 Probably cells were already triggered by inflammatory mediators generated in response to carrageenin. Moreover, rat PMN exposed to LTB4 synthesize more nitric oxide (14). Our results agree with those found by Ischiropoulos et al. (4), who reported that freshly isolated rat alveolar macrophages have a relatively high background of nitrite plus nitrate production. Second, the • • stimulus that increases O•0 2 decreases NO. NO detection by HbO2 –MetHb conversion has recently been • questioned (15) in situations in which O•0 2 and NO are
FIG. 2. Nitric oxide (•NO) production by inflammatory cells in the absence or in the presence of inhibitors of NO synthase. Conditions were as shown in Fig. 1, but cells were stimulated with 100 ng/ml phorbol 12-myristate 13-acetate (PMA) either with 0.6 mM NG-nitroL-arginine (L-NNA) (Sigma, St. Louis, MO) or 0.6 mM N-iminoethylL-ornithine (L-NIO) (Cookson Chemicals LTD, Southampton, UK) as inhibitors of NO synthase with or without preincubation. Values are the mean { SEM of three separate experiments performed in triplicate. Data were analyzed by Student’s t test: *P õ 0.05, **P õ 0.01, ***P õ 0.001 vs cells without inhibitor.
AP: Archives
´ DENAS, CARBONELL, AND MITJAVILA RO
294 TABLE I
Superoxide Production by Inflammatory Cells in the Presence of an Inhibitor of NO Synthase (L-NIO) O2•0 production nmolr106 cells01 Zymosan (1 mg/ml) Zymosan (1 mg/ml) / L-NIO (0.6 mM) PMA (100 ng/ml) PMA (100 ng/ml) / L-NIO (0.6 mM)
5.82 6.41 7.28 8.09
{ { { {
0.10 0.23* 0.11 0.18**
Note. Conditions were as shown in Fig. 1 for superoxide (O2•0) determination, but cells were stimulated with either 1 mg/ml zymosan or 100 ng/ml phorbol 12-myristate 13-acetate (PMA) (Sigma, St. Louis, MO). 0.6 mM N-iminoethyl-L-ornithine (L-NIO) (Cookson Chemicals LTD, Southampton, UK) was used as inhibitor of NO synthase with preincubation. Values are mean { SEM of three separate experiments performed in triplicate. Data were analyzed by Student’s t test. * P õ 0.05. ** P õ 0.01.
PMN at a basal level in the presence of L-arginine, with, at present, an unknown role. When phagocytes are stimulated to produce O•0 2 , which is unable to initiate lipid peroxidation (19), peroxynitrite, a more potent oxidant, is formed, which may explain the cytotoxicity associated with •NO. It is concluded that two aspects should be taken into account when measuring O•0 and •NO produced by 2 phagocytes: in order to avoid interference cells should be incubated with a potent inhibitor of NO synthase • for O•0 2 detection. Second, when studying NO release, cells should be incubated with SOD, avoiding the use of any stimulant of O•0 2 production and using a blank preincubated with an inhibitor of •NO synthase. However, studies on peroxynitrite production will require the use of a stimulant at such concentration that would • give the same amount of O•0 2 as NO. ACKNOWLEDGMENTS The authors thank Robin Rycroft for his help in the English editing of the manuscript. This study was supported by Grant FIS 94/0733.
simultaneously generated, as HbO2 can also be oxidized by peroxynitrite. Although a more specific technique for •NO has been developed (15), in our conditions we found a maximum of only 14–23% of peroxynitrite generated compared with •NO and this peroxynitrite is not detected by HbO2 ; otherwise, the level of •NO would remain constant. To assess whether the decrease in •NO level after stimulating the cells was due to a reaction with O•0 2 , cells were incubated in the presence of 0.6 mM L-NIO for O•0 2 determination in the absence of L-arginine. Leukocytes stimulated with zymosan (1 mg/ml) or PMA (100 ng/ml) displayed an increase in O•0 2 production of 0.6 and 0.8 nmol, respectively (Table I). It can be deduced that 10% of PMN-derived O•0 reacts with •NO 2 which is about the same order as the O•0 non-SOD 2 inhibited. So, the concentrations of SOD generally used to inhibit cytochrome c reduction by O•0 2 , as in our case, are not sufficient to prevent the reaction between •NO • and O•0 2 . NO is produced by NO synthase, a cytosolic enzyme (16), and must diffuse through the plasma membrane, as •NO is soluble in both water and the lipids (17) where NADPH oxidase is located (18). So, it will react with O•0 2 in a fast reaction provided enough O•0 is produced. If O•0 production is not stimulated, 2 2 • less NO will react to give peroxynitrite and more •NO will be detected. The increases in O•0 generation are 2 of the same order as the decreases in •NO observed when incubating cells with zymosan (0.6 nmol) or PMA (0.9 nmol), which confirms that both radicals react in equimolar proportions. The different response in O•0 and •NO levels with 2 the stimulants tested led us to speculate that they may have different roles in vivo. •NO may be produced by
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REFERENCES 1. Babior, B. M., Kipnes, R. S., and Curnutte, J. T. (1973) J. Clin. Invest. 52, 741–744. 2. Biemond, P., van Eijk, H. G., Swaak, A. J. G., and Koster, J. F. (1984) J. Clin. Invest. 73, 1576–1579. 3. Halliwell, B. (1982) Biol. Int. Rep. 6, 529–542. 4. Ischiropoulos, H., Zhu, L., and Beckman, J. S. (1992) Arch. Biochem. Biophys. 298, 446–451. 5. Carreras, M. C., Pargament, G. A., Catz, S. D., Poderoso, J. J., and Boveris, A. (1994) FEBS Lett. 341, 65–68. 6. Palmer, R. M. J., Ashton, D. S., and Moncada, S. (1988) Nature 333, 664–666. 7. Liew, F. Y., Millot, S., Parkinson, C., Palmer, R. M. J., and Moncada, S. (1990) J. Immunol. 144, 4794–4797. 8. Huie, R. E., and Padmaja, S. (1993) Free Rad. Res. Commun. 18, 195–199. 9. Beckman, J. S., Beckman, T. W., Chen, J., Marshall, P. A., and Freeman, B. A. (1990) Proc. Natl. Acad. Sci. USA 87, 1620– 1624. 10. Ro´denas, J., Mitjavila, M. T., and Carbonell, T. (1995) Free Rad. Biol. Med. 18, 869–875. 11. Johnston, R. B., Godzik, C. A., and Cohn, Z. A. (1978) J. Exp. Med. 148, 115–119. 12. Feelisch, M., and Noack, E. A. (1987) Eur. J. Pharmacol. 139, 19–30. 13. Murphy, M. E., Piper, H. M., Watanabe, H., and Sies, H. (1991) J. Biol. Chem. 266, 19378–19383. 14. McCall, T. B., Boughton-Smith, N. K., Palmer, R. M. J., Whittle, B. J. R., and Moncada, S. (1989) Biochem. J. 261, 293–296. 15. Schmidt, K., Klatt, P., and Mayer, B. (1994) Biochem. J. 301, 645–647. 16. McCall, T. B., Palmer, R. M. J., and Moncada, S. (1991) Eur. J. Immunol. 21, 2523–2527. ¨ ngga`rd, E. (1994) Lancet 343, 1199–1206. 17. A 18. Babior, B. M. (1984) Blood 64, 959–966. 19. Winterbourn, C. C. (1979) Biochem. J. 182, 625–628.
AP: Archives