NG hydroxy L arginine in fMLP stimulated human blood by benbenzhou


NG hydroxy L arginine in fMLP stimulated human blood

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									No detectable NO synthesis from L-arginine or
  NG-hydroxy-L-arginine in fMLP-stimulated human blood
  neutrophils despite production of nitrite, nitrate, and citrulline
  from NG-hydroxy-L-arginine
            Paivi Holm,*† Hannu Kankaanranta,*‡ Simo S. Oja,*§ Richard G. Knowles, and Eeva Moilanen*†
            *Medical School, University of Tampere; †Department of Clinical Chemistry; ‡Department of Respiratory Medicine and
            §Department of Clinical Physiology, Tampere University Hospital, Finland; and Enzyme Pharmacology, Glaxo

            Wellcome Research, Stevenage, SG1 2NY, United Kingdom

Abstract: Nitric oxide (NO) is a well-documented                  production of NO in human neutrophils is controversial and
effector molecule in rodent phagocytes but its                    inducible NO synthase (iNOS) gene in human neutrophils is
synthesis in human neutrophils has been controver-                under very different regulation than the rat gene [2]. Human
sial. In this study, NO production in human neutro-               neutrophils were reported to inhibit platelet aggregation by
phils activated by chemotactic peptide N-formyl-                  releasing a NO-like factor [3–5]. Human neutrophils stimulated
methionyl-leucyl-phenylalanine (fMLP) was measured                with bacteria [6] and cytokines were shown to express iNOS [7].
in the presence of L-arginine (L-Arg) and NG-                     Also, iNOS activity was demonstrated in human neutrophils
hydroxy-L-arginine (OH-L-Arg), the precursor and                  isolated from oral cavity or from urine from patients with
intermediate amino acids in NO synthesis, respec-                 urinary tract infections [8, 9]. However, other studies [2,
tively. Incubation of fMLP-activated neutrophils                  10–12], including our own unpublished observations, have not
with OH-L-Arg resulted in a production of nitrite,                found NO synthesis in human neutrophils.
nitrate, and citrulline that was greater than with
                                                                     NG-hydroxy-L-arginine (OH-L-Arg) is an intermediate in the
unstimulated neutrophils but was not inhibited by
                                                                  biosynthesis of nitric oxide from L-Arg. The second step of the
the NOS inhibitors L-NMMA and L-NIO or the
                                                                  NO synthase reaction results in the oxidation of OH-L-Arg to
cytochrome P450 inhibitor troleandomycin and was
not seen when OH-L-Arg was replaced with L-Arg.                   form citrulline and NO [1, 13] (Fig. 1). An elevated concentra-
This nitrite, nitrate, and citrulline production was              tion of OH-L-Arg in serum has been reported in patients with
not associated with any detectable NO synthesis                   rheumatoid arthritis and systemic lupus erythematosus [14].
because no increases in cyclic GMP were observed                  Increased nitrite production was reported in the absence of
in the presence of phosphodiesterase inhibitors and               NOS activity in rat vascular smooth muscle cells supplemented
in the presence or absence of superoxide dismu-                   with OH-L-Arg [15]. Cytochrome P450 has been shown to
tase. Moreover, no increases in the formation of the              catalyze the oxidation of OH-L-Arg to NO and citrulline in the
reaction product of NO with superoxide, peroxyni-                 liver [16], suggesting another mechanism of NO formation from
trite, were observed on addition of either OH-L-Arg               OH-L-Arg not involving NO synthase. This would permit
or L-Arg to activated neutrophils, as assessed either             formation of OH-L-Arg in one cell type and the uptake and
by dihydrorhodamine oxidation or protein nitra-                   oxidation of this amino acid to NO in another cell type lacking
tion. This suggests that, in spite of the production of           NO synthase. In this work we tested the hypothesis that
nitrite, nitrate, and citrulline, commonly used indi-             activated human neutrophils produce NO from exogenous
cators of NO formation, normal human blood                        OH-L-Arg. The results suggest that OH-L-Arg is metabolized to
neutrophils, are not producing detectable amounts                 nitrite, nitrate, and citrulline in N-formyl-methionyl-leucyl-
of either NO or peroxynitrite when stimulated with                phenylalanine (fMLP)-stimulated neutrophils without detect-
fMLP in the presence of OH-L-Arg. J. Leukoc. Biol.                able NO production.
66: 127–134; 1999.

Key Words: troleandomycin · cytochrome P450 · peroxynitrite

                                                                     Abbreviations: NO, nitric oxide; fMLP, N-formyl-methionyl-leucyl-phenylala-
INTRODUCTION                                                      nine; iNOS, indicible nitric oxide synthase; DPBS, Dulbecco’s phosphate-
                                                                  buffered saline; IBMX, isobutylmethylxanthine; SOD, superoxide dismutase;
                                                                  SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Nitric oxide (NO) is a signaling molecule in the physiology of       Correspondence: Eeva Moilanen, Medical School, University of Tampere,
mammalian cardiovascular, immune, and nervous systems. NO         P. O. Box 607, FIN-33101 Tampere, Finland. E-mail:
is synthesized from L-arginine by NO synthase enzymes [1]. The       Received June 29, 1998; revised March 1, 1999; accepted March 2, 1999.

                                                                       Journal of Leukocyte Biology            Volume 66, July 1999       127
Fig. 1. Biosynthetic pathway for produc-
tion of NO from L-arginine in mammals [1].

METHODS                                                                          Measurement of peroxynitrite-induced oxidation
                                                                                 of dihydrorhodamine 123
Isolation of human neutrophils
                                                                                 The formation of peroxynitrite in stimulated neutrophils was measured by a
Blood was collected by venipuncture from healthy volunteers. A buffy-coat        peroxynitrite-dependent oxidation of dihydrorhodamine 123 to rhodamine 123
preparation was layered on Ficoll-Paque and centrifuged for 10 min at 3400 g.    [21]. Neutrophils in DPBS were incubated in the absence or in the presence of
Red blood cells were removed by dextran sedimentation followed by lysis of the   L-Arg or OH-L-Arg and loaded with dihydrorhodamine 123 (5 µM) for 10 min at
remaining erythrocytes with Tris-buffered 0.15 M NH4Cl. Neutrophils were         37°C. The cells were stimulated with fMLP for 10 min and the fluorescence of
washed twice with Dulbecco’s phosphate-buffered saline (DPBS) [17].              rhodamine 123 produced was measured with a Shimadzu RF-5000 spectrofluo-
                                                                                 rophotometer at an excitation wavelength of 500 nm and an emission
                                                                                 wavelength of 536 nm (both slit widths 3.0).
Neutrophil incubations
The cell suspensions (1 107/mL of DPBS) were incubated for 30 min with           Nitrotyrosine Western blotting
PBS, NG-hydroxy-L-arginine, or L-arginine either in the presence (samples for
                                                                                 After 10 min of incubation with fMLP human neutrophils were centrifuged and
cyclic GMP) or absence of phosphodiesterase inhibitors isobutylmethylxan-
                                                                                 the cell pellets diluted in PBS (1        106 cells/50 µL) and transferred to
thine (IBMX; 250 µM) and zaprinast (10 µM), superoxide dismutase (SOD; 300
                                                                                 Microfuge tubes. Cells were lysed in extraction buffer at pH 7.4 at 4°C (10 mM
U/mL), NG-monomethyl-L-arginine (L-NMMA; 1 mM), and NG-iminoethyl-L-
                                                                                 Tris base, 5 mM ethylenediaminetetraacetate, 50 mM NaCl, 1% Triton X-100,
ornithine (L-NIO; 1 mM). The cells were then activated by adding fMLP (1 µM),
                                                                                 0.5 mM phenylmethylsulfonyl fluoride, 2 mM sodium orthovanadate, 10 µg/mL
incubated for 10 min, and then pelleted by centrifugation (10,000 g, 30 s).
                                                                                 leupeptin, 25 µg/mL aprotinin, 1.25 mM NaF, 1 mM sodium pyrophosphate, 10
                                                                                 mM n-octyl-B-D-glucopyranoside; all from Sigma, St. Louis, MO). After
Measurement of nitrite/nitrate concentrations                                    centrifugation the supernatants were mixed in four volumes of sample buffer
                                                                                 (62.5 mM Tris, pH 6.8, 10% glycerol, 2% sodium dodecyl sulfate, 0.025%
Nitrite was measured as previously described [18] by adding 100 µL of Griess     bromophenol blue, and 5% -mercaptoethanol; all from Sigma), and heated at
reagent to 100-µL samples of medium. The optical density at 540 nm was           100°C for 5 min. The samples were then loaded into sodium dodecyl
measured using a microplate reader. Sodium nitrite was used as a standard. The   sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 10% polyacryl-
total nitrite    nitrate (NOx) concentration in the supernatant was also         amide gels. The gels were run at 160 V for 1 h and then transferred to
determined. For NOx measurements, nitrate in the supernatant was reduced to      nitrocellulose (Hybond-ECL, Amersham International, Buckinghamshire, UK)
nitrite by incubation with nitrate reductase and NADPH at room temperature       for 60 min at 400 mA in trans-blotting buffer (183 mM glycine-HCl, 25 mM Tris
and the nitrite concentration in the samples was measured by the Griess          base, and 20% methanol). The blotted nitrocellulose was washed twice with
reaction [19].                                                                   water and then blocked in PBS containing 3% nonfat dry milk for 20 min at
                                                                                 20°C. The nitrocellulose was incubated overnight at 4°C in the nitrotyrosine
                                                                                 antibody (1 µg/mL) (Upstate Biotechnology, Saranac Lake, NY) diluted in PBS
Measurement of citrulline                                                        containing 3% nonfat dry milk. The blot was washed twice with water and then
The samples of incubation medium were added to 5% sulfosalicylic acid            incubated for 1.5 h with the secondary antibody, anti-rabbit IgG linked to
(containing L-2,4-diaminobutyrate as an internal standard) to precipitate        horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA; diluted
proteins, centrifuged, and mixed with 0.2 M lithium citrate buffer, pH 2.2.      1:3000) in PBS-nonfat dry milk at room temperature. The blot was washed once
Amino acids were separated by ion-exchange chromatography using an               with PBS-0.05% Tween for 5 min and five times with water. Thereafter 10 mL
automated amino acid analyzer Alpha Plus (Pharmacia Biotech, Biochrom Ltd.,      mixed ECL chemiluminescence reagent (Amersham International) was added
Cambridge, UK). The assays were carried out by means of the post-column          for 1 min and the blot was exposed to Kodak Biomax film.
derivatization with o-phthaldialdehyde and subsequent fluorescence detection.
cGMP production                                                                  Results are expressed as means SEM. Statistical significance was calculated
                                                                                 by one-way analysis of variance supported by Bonferroni significance levels.
The neutrophil incubations (see above) were terminated by adding ice-cold        Differences were considered significant when P 0.05.
trichloroacetic acid (final concentration 6%) and samples were incubated on
ice for 30 min. The samples were then sonicated for 1 min and centrifuged at
10,000 g for 5 min. The supernatants were washed four times with water-
saturated ethyl ether and stored at 20°C until assayed for cGMP. For cGMP
determinations the samples were neutralized and dissolved with an equal
volume of 100 mM sodium acetate buffer, pH 6.2. cGMP was measured by             Nitrite and nitrate formation
radioimmunoassay as described earlier [17, 20]. NO donors S-nitroso-N-acetyl-
penicillamine (SNAP; 100 µM) and 1,2,3,4-oxatriazolium, 5-amino-3-(3,4-          When human neutrophils were incubated in the presence of
diphenyl)-chloride (GEA 3162; 0–100 µM) were incubated for 10 min with           increasing concentrations of OH-L-Arg and then stimulated
neutrophils.                                                                     with fMLP, nitrite accumulated into the incubate in a concentra-

128     Journal of Leukocyte Biology           Volume 66, July 1999                                                        
tion-dependent manner (Fig. 2A). NOS inhibitors L-NIO (1                           cGMP production
mM) or L-NMMA (1 mM) did not alter nitrite formation in the
                                                                                   NO activates guanylate cyclase, leading to cGMP production in
presence of OH-L-Arg (Fig. 2A). Because cytochrome P450s3A
                                                                                   target cells even at low physiological concentrations [20], and
has been shown to catalyze oxidation of OH-L-Arg to nitrogen
                                                                                   cGMP assay serves as a sensitive measure of NO production.
oxides and citrulline in the rat liver [22] the effects of P450s3A
                                                                                   When human neutrophils were incubated in the presence of
inhibitor troleandomycin [23] were tested. Troleandomycin did
                                                                                   phosphodiesterase inhibitors to inhibit cGMP metabolism and
not alter nitrite production in fMLP-stimulated neutrophils
                                                                                   then exposed to increasing concentrations of OH-L-Arg and
(Fig. 2A). Unstimulated neutrophils did not produce nitrite
                                                                                   activated by fMLP, no increases in cGMP production were
although exposed to millimolar concentrations of OH-L-Arg.
When neutrophils were incubated with corresponding concen-                         found. Neither L-Arg nor NOS inhibitors L-NIO and L-NMMA
trations of L-Arg instead of OH-L-Arg and then activated by                        had any effects on cGMP production (Table 1). NO donors
                                                                                   SNAP and GEA 3162 were used as positive controls. SNAP
fMLP, no increases in nitrite formation were found (Fig. 2B).
                                                                                   (100 gmM) was shown to increase cGMP production in
   When OH-L-Arg was incubated in DPBS in the presence of
                                                                                   unstimulated as well as in fMLP-stimulated cells (Table 1, top).
activated neutrophils, NOx (nitrite nitrate) was formed in a
                                                                                   The dose-dependent effect of GEA 3162 on cGMP production
dose-dependent manner (Fig. 3A). In the absence of neutro-
                                                                                   is shown in Table 1, bottom.
phils the accumulation of NOx was significantly lower than in
                                                                                      When neutrophils are stimulated by fMLP, the NADPH
the presence of cells. The accumulation of NOx was somewhat
                                                                                   pathway is activated to produce superoxide anion. Superoxide
higher in the suspensions of fMLP-stimulated neutrophils
                                                                                   reacts rapidly with NO to form a reactive oxidant and nitrating
compared with unstimulated cells. The difference in NOx
                                                                                   agent peroxynitrite [24]. This pathway may also serve as an
concentrations (as measured in the presence of 1000 µM
                                                                                   inactivation mechanism for both of these radicals [25]. To
OH-L-Arg) between fMLP-stimulated and unstimulated cells
(5.3      2.6 µM) corresponds to the nitrite production in                         bypass the interaction between superoxide anion and NO, SOD
fMLP-stimulated cells (Fig. 2A), suggesting that nitrite but not                   was added into the incubates. Addition of SOD did not result in
nitrate production is increased by fMLP-stimulation. L-NIO did                     increased cGMP production in neutrophils incubated with
not inhibit NOx formation by activated cells (Fig. 3B). When                       OH-L-Arg or L-Arg, but SOD merely decreased cGMP levels
                                                                                   (Table 1, middle).
OH-L-Arg was replaced with L-Arg (up to 1 mM concentration)
no detectable NOx was found either in unstimulated or
fMLP-stimulated neutrophils.                                                       Peroxynitrite formation
                                                                                   Peroxynitrite is formed in a reaction between NO and superox-
Citrulline formation
                                                                                   ide anion with a high rate constant [24]. The production of
Citrulline was formed in a concentration-dependent manner in                       peroxynitrite was measured by the following two methods:
incubations of activated neutrophils in the presence of OH-L-                      oxidation of dihydrorhodamine 123 to a fluorescein rhodamine
Arg (Fig. 4A) but not in the presence of L-Arg. L-NIO did not                      123 [21] and measurement of the presence of nitrotyrosine in
inhibit citrulline production from OH-L-Arg. Citrulline forma-                     cellular proteins by Western blotting [26].
tion from OH-L-Arg (1 mM) in experiments with unstimulated                            When neutrophils were first loaded with dihydrorhodamine
neutrophils or in the absence of cells was about 50% lower than                    and then stimulated with fMLP some increase in rhodamine
that found in the presence of activated cells (Fig. 4B).                           fluorescence was found compared with unstimulated cells (Fig.

Fig. 2. The effect of (A) NG-hydroxy-L-arginine (OH-L-Arg) and (B) L-arginine (L-Arg) on nitrite formation in fMLP-stimulated and unstimulated (without fMLP)
human neutrophils. Neutrophils were incubated at 37°C for 30 min with OH-L-Arg or L-Arg in the absence (open bars) or presence of NOS inhibitors L-NIO (1 mM;
hatched bars) or L-NMMA (1 mM; cross-hatched bars), or P450 inhibitor troleandomycin (100 µM; filled bars) and then stimulated by fMLP for 10 min. Data represent
means SEM, duplicate experiments with cells from six donors.

                                                                                   Holm et al.    OH-L-Arg metabolism in human neutrophils                 129
5). This increase in fluorescence was not inhibited by L-NIO (1
mM) or SOD (300–1000 U/mL; Fig. 5A), suggesting that it is
due to other reactive oxygen species than peroxynitrite [27].
When activated neutrophils were incubated with the NO-donor
SNAP (100 µM), the fluorescence doubled. This increase was
inhibitable by SOD (1000 U/mL), suggesting that it was due to
formation of peroxynitrite. When activated neutrophils were
incubated with OH-L-Arg or L-Arg (1 mM), no increases in
rhodamine fluorescence were found, suggesting that peroxyni-
trite is not formed in these conditions (Fig. 5B). Moreover,
oxidation of dihydrorhodamine by activated neutrophils was
decreased in the presence of OH-L-Arg but not by L-Arg.
   When unstimulated or stimulated neutrophils were exposed
to peroxynitrite, tyrosine residues in several proteins were

                                                                                   Fig. 4. (A) The production of citrulline from OH-L-Arg by fMLP-stimulated
                                                                                   neutrophils. (B) Citrulline formation by unstimulated and fMLP-stimulated
                                                                                   cells in the presence of 1000 µM OH-L-Arg. Data represent means           SEM,
                                                                                   duplicate experiments with cells from six donors. ns, not significant; ***P

                                                                                   nitrated to nitrotyrosine as detected by Western blotting (Fig.
                                                                                   6). The response resembles that reported earlier in rat brain
                                                                                   homogenates by Beckmann et al. [26]. When activated neutro-
                                                                                   phils were incubated with OH-L-Arg or L-Arg (1 mM) no signs
                                                                                   of nitrated tyrosine were found (Fig. 6), suggesting that the cells
                                                                                   do not produce peroxynitrite at concentrations detectable by
                                                                                   this methodology.

Fig. 3. (A) The production of nitrite          nitrate (NOx) from OH-L-Arg by      DISCUSSION
fMLP-stimulated neutrophils (open bars) and in cell-free buffer (hatched bars).
(B) NOx formation by unstimulated and fMLP-stimulated cells in the presence
of 1000 µM OH-L-Arg. Data represent means             SEM, duplicate experiments   The present data show that OH-L-Arg, an intermediate in the
with cells from six donors. ns, not significant; *P 0.05.                          biosynthesis of NO, is metabolized to nitrite, nitrate, and

130     Journal of Leukocyte Biology             Volume 66, July 1999                                                        
         TABLE 1.      cGMP Production in Human Neutrophils                          The critical step in testing this hypothesis is to confirm
                                                                                  whether NO is produced in these incubation conditions. Our
                                         Cyclic GMP (fmol/106 cells)
                                                                                  results suggest that, although nitrite, nitrate, and citrulline are
                               Unstimulated             fMLP-stimulated           formed, NO is not produced in these reactions. To demonstrate
                                neutrophils               neutrophils
                                                                                  the biological activity of NO we measured cGMP production in
Without SOD                                                                       neutrophils. No increase in cGMP production was found in
  Control                     24.5     5.2               26.6        5.2          activated neutrophils incubated in the presence of OH-L-Arg in
  OH-L-Arg 100 µM             25.7     5.7      ns       26.8        11.7   ns
  OH-L-Arg 300 µM             27.7     5.5      ns       28.8        15.9   ns
                                                                                  conditions where nitrite, nitrate, and citrulline production was
  OH-L-Arg 1000 µM            27.0     7.1      ns       25.5        13.8   ns    evident. The presence of L-Arg in the incubates did not result in
  OH-L-Arg 1000                                                                   increased cGMP production either, whereas the NO-releasing
    µM NIO 1 mM                                          26.3        6.1    ns    compounds SNAP and GEA 3162 activated cGMP production.
  L-Arg 1000 µM               21.3     10.6     ns       24.1        12.3   ns    NO is rapidly converted to peroxynitrite in the presence of
  L-Arg 1000   NIO 1
    mM                                                   24.8        4.2     ns
                                                                                  superoxide anion [24], which is produced by activated neutro-
  SNAP 100 µM                251.0     63.7    ***      274.2        78.1   ***   phils [29]. Therefore cGMP production was also measured in
                                                                                  the presence of SOD, which catalyzes the dismutation of two
With SOD (300 U/mL)
  Control                                            12.0      3.4                molecules of superoxide to hydrogen peroxide and water.
  OH-L-Arg 100 µM                                    12.6      3.4          ns    Addition of SOD into the incubation did not reveal any cGMP
  OH-L-Arg 300 µM                                    13.6      2.4          ns
  OH-L-Arg 1000 µM                                   15.1      3.6          ns    response in OH-L-Arg-treated cells. Production of cGMP is a
  OH-L-Arg 1000 µM without fMLP                      20.4      5.2          ns    sensitive measure of NO synthesis even at the low physiological
  OH-L-Arg 1000 µM NMMA 1 mM                         17.6      5.2          ns    level of NO production [30]. Thus these data suggest that NO
  L-Arg 1000 µM                                      15.6      3.2          ns
  Control without fMLP                               13.5      3.3          ns    was not produced from OH-L-Arg by fMLP-activated neutro-
                                                                                  phils, although nitrite, nitrate, and citrulline were formed.
GEA 3162
  Control                                           18.1       1.3                   Formation of peroxynitrite was used as another measure of
  GEA 3162 1.0 µM                                   33.7       2.2           ns   NO production in these experiments. When human neutrophils
  GEA 3162 10 µM                                   120.5       15.7          *
  GEA 3162 100 µM                                  314.8       86.8         ***   are activated with agonists like fMLP, the superoxide generating
                                                                                  system is activated [31, 32]. NO and superoxide anion form the
  Effects of OH-L-Arg and L-Arg on cyclic GMP production in human
neutrophils in the absence (top) or in the presence of SOD (300 U/mL; middle).
                                                                                  strong biological oxidant and nitrating agent peroxynitrite with
Neutrophils were incubated at 37°C for 30 min with OH-L-Arg, L-Arg, and           a high rate constant [24]. The production of peroxynitrite was
NOS-inhibitors L-NMMA or L-NIO and then activated by fMLP for 10 min.             measured in this study by the following two means: by
Incubations were stopped by the addition of cold trichloroacetic acid.            measuring oxidation of dihydrorhodamine 123 to a fluorescein
Dose-dependent effect of NO-donor 1,2,3,4-oxatriazolium, 5-amino-3-(3,4-
                                                                                  rhodamine 123 [21] and by measuring the nitration of tyrosine
diphenyl)-chloride (GEA 3162) (bottom). Data represent means             SEM,
duplicate experiments with cells from six donors. Difference from correspond-     residues to nitrotyrosine [26]. Neither method demonstrated
ing control value is denoted by ns, not significant; * P 0.05, *** P 0.001.       signs of peroxynitrite production in activated neutrophils incubated
                                                                                  in the presence of OH-L-Arg in experimental conditions where
citrulline in the presence of activated human neutrophils.                        nitrite, nitrate, and citrulline production was evident.
Measurable nitrite production was found only in the experi-                          Oxidation of dihydrorhodamine to fluorescent rhodamine has
ments with activated neutrophils, whereas lower amounts of                        been successfully used as a marker of peroxynitrite in various
citrulline and nitrate were formed also in the presence of                        experiments [21, 33]. This method is not, however, specific for
unstimulated cells and in cell-free experiments. Corresponding                    peroxynitrite but other oxidant species like hydrogen peroxide
concentrations of L-Arg did not result in significant formation of                result from the formation of rhodamine from dihydrorhodamine
nitrite, nitrate, or citrulline. NO is a short-lived mediator and in              [21]. To overcome this problem, the measurements of NOS
aqueous solutions it is rapidly converted to nitrite and nitrate                  inhibitor-inhibitable oxidation of the probe and/or simultaneous
[28]. In the reaction catalyzed by NO synthase L-arginine is                      measurements of other footprints of peroxynitrite (such as
metabolized to NO and citrulline. Therefore the formation of                      nitration of tyrosine) have been recommended [27]. In the
nitrite, nitrate, and citrulline by fMLP-stimulated neutrophils                   present study, activation of neutrophils by fMLP resulted in an
suggests activation of NO synthesis in these cells. Nitrite,                      increase in oxidation of dihydrorhodamine. This increase was
nitrate, and citrulline were formed in cell suspensions incu-                     not inhibited by either L-NIO or SOD, which inhibit the
bated with OH-L-Arg but not with L-Arg. Moreover, the                             formation of NO and accelerate the metabolism of superoxide to
NOS-inhibitors L-NIO and L-NMMA did not inhibit the forma-                        hydrogen peroxide, respectively, and thus reduce the concentra-
tion of these markers of NO synthesis. Because cytochrome                         tions of the components needed in peroxynitrite synthesis.
P450s3A has been shown to catalyze oxidation of OH-L-Arg to                       These results suggest that the fMLP-induced increase in the
nitrogen oxides and citrulline in the rat liver [22] the effects of               oxidation of dihydrorhodamine is not due to formation of
P450s3A inhibitor troleandomycin were tested. Like L-NMMA                         peroxynitrite but to some other oxidants produced by the cells.
and L-NIO, troleandomycin was ineffective. These data raise                       This is consistent with the data of Hendersson and Chappell
the possibility of the presence of a NO-producing mechanism                       [34] showing that in phorbol myristate acetate-activated neutro-
different from the classical L-Arg/NO pathway and the earlier                     phils dihydrorhodamine oxidation reports the presence of
described P450-catalyzed pathway [22] in activated human                          hydrogen peroxide and intracellular peroxidases. They con-
neutrophils.                                                                      cluded that in activated neutrophils hydrogen peroxide is

                                                                                  Holm et al.   OH-L-Arg metabolism in human neutrophils         131
Fig. 5. Peroxynitrite production, as measured by the oxidation of dihydrorhodamine 123 to a fluorescein rhodamine 123. The effects of L-NIO (1 mM), SOD (1000
U/mL), and SNAP (100 µM) on the formation of rhodamine 123 by fMLP-stimulated neutrophils. (B) The formation of rhodamine 123 in fMLP-stimulated neutrophils
in the presence of OH-L-Arg (1000 µM) or L-Arg (1000 µM). Data represent means SEM, duplicate experiments with cells from six donors. ns, not significant, *P

formed from superoxide anion mainly in the extracellular                         was found. This increase was inhibitable by addition of SOD,
space. The oxidation of dihydrorhodamine to rhodamine by                         which blocks the availability of the other component needed for
hydrogen peroxide occurs slowly in extracellular medium.                         peroxynitrite, suggesting that the SNAP-induced oxidation of
However, hydrogen peroxide can cross the plasma membrane                         dihydrorhodamine is due to formation of peroxynitrite in
and the oxidation of dihydrorhodamine is considerably faster as                  fMLP-stimulated cells. This dihydrorhodamine method has
it is catalyzed by cellular peroxidases. Therefore SOD, which                    been successfully used to report peroxynitrite formation in
catalyzes the dismutation of two molecules of superoxide to                      macrophages producing both superoxide and NO [33]. In the
hydrogen peroxide and water, did not inhibit dihydrorhodamine                    present study, addition of OH-L-Arg or L-Arg to the suspensions
oxidation in neutrophils activated by phorbol myristate acetate                  of activated neutrophils did not increase the oxidation of
[34] or fMLP (this study). In our subsequent experiments                         dihydrorhodamine. These results suggest that measurable
activated neutrophils were exposed to exogenous NO released                      amounts of peroxynitrite were not formed in the presence of
from SNAP; a clear increase in oxidation of dihydrorhodamine                     these NO precursors. Merely, the oxidation of dihydrorhoda-
                                                                                 mine was decreased in the presence of OH-L-Arg. This may
                                                                                 suggest that oxidation of OH-L-Arg to citrulline and nitrite
                                                                                 consumes some of the oxidative species, e.g., hydrogen perox-
                                                                                 ide, which are produced by activated neutrophils, and mediates
                                                                                 the peroxynitrite-independent oxidation of dihydrorhodamine.
                                                                                    Nitration of tyrosine residues in cellular proteins was
                                                                                 measured by Western blotting using a commercial antibody to
                                                                                 nitrotyrosine. When activated neutrophils were exposed to
                                                                                 chemically synthesized peroxynitrite a number of proteins
                                                                                 showed immunoreactivity toward nitrotyrosine antibody. The
                                                                                 result was comparable to that found earlier in rat brain
                                                                                 homogenates by Beckmann et al. [26]. OH-L-Arg or L-Arg did
                                                                                 not induce formation of detectable amounts of nitrotyrosine in
Fig. 6. Western blotting of nitrotyrosine in cellular proteins as a marker of    activated neutrophils. These data together with the above-
peroxynitrite production in fMLP-stimulated human neutrophils. The cells were    mentioned results on the lack of cGMP production and
incubated in the presence of L-Arg (1000 µM), OH-L-Arg (1000 µM), or the         oxidation of dihydrorhodamine suggest that fMLP-stimulated
buffer (control) for 30 min and then activated by fMLP for 10 min. Lanes 1 and
2 show the positive controls in which peroxynitrite (ONOO ) was added to
                                                                                 neutrophils do not produce detectable amounts of NO when
fMLP-treated neutrophils. A separate line shows a positive control in which      incubated in the presence of OH-L-Arg or L-Arg, although in
ONOO was added to albumin. One representative experiment of four is shown.       the former case nitrite, nitrate, and citrulline are formed.

132     Journal of Leukocyte Biology           Volume 66, July 1999                                                      
   NO produced by activated phagocytes is an important               ated chemically or produced by activated murine macrophages
effector molecule in the rodent immune response [35–37]. The         [45]. This mechanism is not applicable in the present case
presence of NOS and the role of NO in the functions of human         because NO was generated as an intermediate in the decompo-
phagocytes has been more difficult to demonstrate [38]. Rat          sition of OH-L-Arg by superoxide. This was evidenced by
neutrophils express an inducible calcium-independent form of         cGMP production and NO-hemoglobin complex formation [45].
NOS and produce NO in response to lipopolysaccharide and             Cytochrome P450, particularly the isoenzyme 3A, has been
proinflammatory cytokines [35, 36, 39, 40]. Inflammatory             shown to catalyze the formation of nitrite and citrulline from
stimuli known to induce iNOS in rat neutrophils do not have the      OH-L-Arg in rat liver [16, 22]. UV and EPR measurements
same action in human neutrophils, at least under in vitro            suggest transient formation of P450-Fe(II)-NO and P420-
conditions [2]. However, recent data show that neutrophils           Fe(II)-NO complexes in these reactions [16]. This mechanism
isolated from the oral cavity [8] or from urine from patients with   is not a likely explanation for the present findings because
bacterial cystitis [9] do express iNOS, suggesting that induction    troleandomycin, an inhibitor of P450s3A, was ineffective in our
of iNOS is also involved in antimicrobial mechanisms of human        experiments but abolished nitrite formation in rat liver [16, 22].
neutrophils, but the human iNOS gene is regulated differently        This does not, however, rule out a mechanism involving some
from the rat gene. Our unpublished data show that iNOS protein       other oxidative enzymes activated by fMLP in human neutro-
is not found by Western immunoblot analysis in synovial fluid        phils. Another possible explanation rises from the findings of
cells from patients with rheumatoid arthritis, suggesting that       Clague et al. [46] and Pufahl et al. [47]. They found that NOS
bacterial infection may be needed for induction of iNOS in           catalyzes the oxidation of OH-L-Arg but not L-Arg by hydrogen
human neutrophils.                                                   peroxide. When hydrogen peroxide acts as an oxidant NOS, and
   Several anti-microbial and inflammatory mechanisms are            possibly some other oxidative enzymes, produces cyano-
rapidly activated in human neutrophils in response to immuno-        ornithine and citrulline, and NOx from OH-L-Arg. By contrast,
logical or bacterial agents such as opsonized particles or fMLP.     with NADPH and oxygen, NOS catalyzes the exclusive forma-
We have earlier shown that fMLP increases intracellular free         tion of citrulline and NO from either OH-L-Arg or L-Arg. The
calcium concentrations in experimental conditions correspond-        former reaction might be involved in our experimental system
ing to those used in the present experiments [41]. The               because hydrogen peroxide is produced by activated neutro-
                                                                     phils. This might also explain the present finding that citrulline
constitutive form of NOS is activated in vascular endothelium
                                                                     formation was substoichiometric with respect to the formation of
and other cell types in response to agonist-induced increase in
                                                                     NOx, because our method to analyze amino acids does not
free cellular calcium [1]. In this study we were not able to
                                                                     measure cyano-ornithine. Further experiments are needed to
demonstrate NO production in fMLP-activated human neutro-
                                                                     test whether this hypothesis might explain our observations of
phils in the presence of OH-L-Arg or L-Arg. Neutrophils from
                                                                     OH-L-Arg metabolism in activated neutrophils.
more than 30 different donors were used in these experiments.
                                                                        In conclusion, NO production in fMLP-stimulated human
The lack of NO synthesis in intact neutrophils confirms the
                                                                     neutrophils was measured in the presence of the substrate
earlier findings of Yan et al. [10], Padgedd and Pruett [11],
                                                                     L-Arg or the intermediate OH-L-Arg. No detectable NO produc-
McBride et al. [12] Malawista et al. [6], and Miles et al. [2].
                                                                     tion was found as measured by cGMP production or peroxyni-
However, we found that fMLP-activated neutrophils produce
                                                                     trite formation, whereas commonly used measures of NO
nitrite, nitrate, and citrulline from OH-L-Arg. These data
                                                                     production, nitrite, nitrate, and citrulline were formed from
question the use of these markers as measures of NO synthesis        OH-L-Arg. For studies assessing NO production, our results
unless the method is confirmed with the use of NOS inhibitors        clearly demonstrate the crucial importance of the use of NOS
and/or simultaneous measurements of other markers of NO              inhibitors and additional methods to confirm NO production.
synthesis such as the cGMP response.
   In contrast to our results, some authors have reported a rapid
NO synthesis in human neutrophils activated by receptor-
mediated agonists like fMLP or by the activation of protein
kinase C with phorbol myristate acetate [5, 31, 32, 42, 43]. It is
                                                                     This study was supported by grants from the Academy of
not clear whether this is due to differences in experimental
                                                                     Finland, the University of Tampere, the Medical Research
conditions (including platelet contamination) or between the
                                                                     Fund of Tampere University Hospital, and the Finnish Cultural
blood donors. For instance, Larfards and Gyllenhammar demon-         Foundation. The skillful technical assistance of Ms. Niina
strated a reproducible NO production in fMLP-activated periph-                                                  ¨¨ ¨
                                                                     Railo, Mrs. Raija Repo, and Mrs. Heli Maatta is gratefully
eral blood neutrophils using only one blood donor in their           acknowledged.
studies, leaving open the question of whether the response is
universal or found in some subjects only [42]. The latter idea
could be explained by polymorphism in the gene of the
constitutive NOS responsible for NO production in neutrophils.
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134     Journal of Leukocyte Biology              Volume 66, July 1999                                                            

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