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Department of Experimental Pathology, The William Harvey Research Institute, St. Bartholomew’s and the Royal London School of Medicine and Dentistry, London, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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NO generated from ecNOS has been reported to have both pro- and anti-inflammatory properties. Under physiological conditions, NO released from the endothelium regulates vascular tone and maintains vessel patency by helping prevent platelet aggregation and down-regulating adhesion molecule expression (3). However, mediators released during the acute phase of inflammation, including histamine, 5-hydroxytryptamine, bradykinin, platelet-activating factor, and substance P, evoke the release of endothelial NO, causing vasodilatation and vascular permeability, thus facilitating edema formation and trafficking of inflammatory cells (4).
L-arginine analogues are the pharmacological agents most commonly used to inhibit NO production, but have poor selectivity between NOS isoforms, thereby inhibiting iNOS and ecNOS during inflammatory insult, the latter resulting in a reduction of basal blood flow. In the majority of experiments, L-arginine analogues were given systemically, resulting in an amelioration of inflammation (5, 6, 7), but in some cases these anti-inflammatory actions were reversed by vasodilators (8, 9). Therefore, we suspect that NOS inhibitors, administered systemically, will inhibit ecNOS, resulting in vasoconstriction, followed by a reduction in blood delivery to the inflamed site. This sequence of events may ultimately lead to reduced cellular diapedesis and exudation and thus be interpreted as anti-inflammatory.
To test the hypothesis that NOS inhibitors will have different effects on inflammation depending on their route of administration, we used the carrageenin-induced pleurisy. This is a well-characterized model of acute inflammation in which iNOS activity, protein expression, and nitrite production peak between 1 and 6 h after carrageenin injection (10). The present study was designed to investigate the effects on inflammation of NOS inhibitors administered locally to the inflammatory site and to compare their effects with NOS inhibitors given systemically. To this end, we used S-(2-aminoethyl) isothiourea (AE-ITU) (11) and N-(3-(aminomethyl)-benzyl) acetamidine (1400W) (12), which are structurally different and the most selective iNOS inhibitors commercially available, as well as the more selective ecNOS inhibitor, L-N5(1-iminoethyl)-ornithine (L-NIO) (13). These drugs were administered into the pleural or peritoneal cavity immediately before the establishment of a carrageen-induced pleurisy. Edema formation, infiltrating inflammatory cell numbers, and nitrite levels in exudates were measured at various times throughout the inflammatory process.
It was found that NOS inhibitors administered locally exacerbated inflammation and prolonged resolution as a result of an increase in the proinflammatory mediators histamine, leukotriene B4 (LTB4), O2-, and cytokine-induced neutrophil chemoattractant (CINC), suggesting that the local production of NO is protective. By contrast, however, giving NOS inhibitors systemically ameliorated inflammation, thereby showing differential anti-inflammatory properties of NOS inhibitors depending on their route of administration.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male Wistar rats (180 ± 20 g; T. Tuck and Sons, Batties Bridge, U.K.) were housed with a 12-h light-dark cycle and allowed access to food and water ad libitum. Animals were anesthetized with halothane and injected with 0.15 ml, 1% carrageenin in saline into the pleural cavity. Pleural exudates were collected with or without lavage (0.5 ml of 3.15% tri-sodium citrate in saline) at various time points postcarrageenin injection. Blood-contaminated exudates were rejected. Exudate volumes were quantified and inflammatory cells counted (Dn coulter-counter; Coulter Electronics, Luton, U.K.). Samples were centrifuged (800 x g, 10 min, 4°C) to separate inflammatory cells from exudate, both of which were then stored at -70°C for subsequent procedures.
Drug administration
Animals (n = 6–8 per group) were injected intrapleurally with either 3,3'-hydroxynitrosohydrazino]bis-1-propanamine (DPTA) NoNoate, AE-ITU, 1400W, L-NIO, or saline immediately before intrapleural injection of carrageenin. For experiments examining the systemic effects of NOS inhibitors, AE-ITU or NG-monomethyl-L-arginine (L-NMMA) was injected into the peritoneal cavity immediately before carrageenin injection into the pleural cavity.
Mast cell depletion
Rats were depleted of mast cells using compound 48/80 CMP 48/80), as previously described (14). Briefly, CMP 48/80 (0.1% w/v) was injected i.p. twice daily for 4 days (0.6 mg/kg for the first six injections and 1.2 mg/kg for the final two). Carrageenin-induced pleurisy was induced 6 h after the last injection of CMP 48/80.
Western blot analysis
Cell pellets were sonicated on ice in protease-inhibitory buffer (0.05 M Tris, 1 mM PMSF, 1.5 mM pepstatin A, 0.2 mM leupeptin) and centrifuged (4000 x g, 5 min, 4°C). Protein concentrations of the supernatants were determined by the Bradford assay. Samples were equilibrated for protein (1 mg/ml), mixed 1:1 with 2x Laemmli buffer (125 mM Tris base, 2 mM EDTA, 10% mercaptoethanol, 4% SDS, 20% glycerol, and 0.1% Coomassie brilliant blue, pH 6.8), and boiled for 5 min. Samples (30 µg/lane) and m.w. color markers were resolved by gel electrophoresis on 7.5% SDS-polyacrylamide gels and transferred onto nitrocellulose membranes. iNOS protein was detected using a specific polyclonal rabbit anti-mouse Ab (1:5000), which does not cross-react with either ecNOS or neuronal NOS (15). The signal was amplified with a HRP-linked goat anti-rabbit polyclonal Ab and visualized on x-ray film using ECL chemiluminescence reagents.
iNOS activity in inflammatory cells
iNOS activity was measured as the ability of inflammatory cells to catalytically convert [3H]L-arginine to [3H]L-citrulline. The reaction mixture consisted of NADPH (1 mM), tetrahydrobiopterin (5 µM), calmodulin (300 U/ml), EGTA (1 mM), and valine (1 mM). Samples were homogenized and added to each reaction tube. The reaction was initiated by the addition of 10 µl of L-arginine/[3H]L-arginine at a final concentration of 10 µM and at a ratio of 1000:3 pmol of L-arginine:[3H]L-arginine. Samples were incubated at 37°C for 30 min, and the reaction was terminated by the addition of ice-cold stop buffer (2 mM EGTA, 2 mM EDTA in 20 mM HEPES, pH 5.5). Samples were eluted through Dowex cationic exchange resin columns with stop buffer and collected in scintillation vials. Scintillation fluid was added to each vial, and the amount of radioactive L-citrulline was determined in a liquid scintillation counter. The protein concentration of samples was measured and NOS activity expressed as pmol L-citrulline/mg protein/30 min.
Cyclooxygenase (COX) activity in inflammatory cells
Cell pellets were sonicated on ice in protease-inhibitory buffer and incubated (37°C, 30 min) in the presence of arachidonic acid (30 µM), glutathione (5 mM), and adrenalin (5 mM). The reaction was terminated by boiling, and samples were then centrifuged (10,000 x g, 30 min). PGE2 in the supernatant was measured by RIA, and the results were expressed as ng PGE2/mg protein/30 min.
Measurement of PGE2[infi], LTB4, 6-keto
PGF1
, histamine, and CINC in cell-free exudates
PGE2 and LTB4 were
measured by enzyme immunoassay and 6-keto PGF1
(as the stable breakdown product of PGI2) by RIA.
Results were expressed as quantity (pg) of each eicosanoid in the total
volume of cell-free exudate. Histamine levels were measured by RIA and
results expressed as total histamine (ng) in the total volume of
exudate. CINC levels were measured using a mouse
KC ELISA that had cross-reactivity
with rat rCINC-1 protein, and results expressed as total amount of CINC
(pg) in total volume of exudate.
Nitrite levels in cell-free exudates
Nitrite formation was measured by a modification of the Greiss reaction. Briefly, 10 µl of NADPH (10 µM), glucose-6-phosphate (5 mM), glucose-6-phosphate dehydrogenase (0.16 U), and PBS (10 mM) were added to cell-free exudate in a 96-well plate. Nitrate was converted to nitrite by addition of 10 µl (0.08 U) nitrate reductase and incubated for 45 min. A total of 200 µl of Greiss reagent (equal volumes of 1% w/v sulfanilamide in 5% H3PO4 and 0.1% w/v N(1-napthyl)ethylenediamine) was added and incubated for an additional 10 min. Nitrite concentrations were determined at 570 nm with a reference filter at 620 nm and results expressed as µM nitrite in cell-free exudate.
Total antioxidant status in cells and cell-free exudate
Exudate and plasma total antioxidant status (TAOS) was measured as previously described (16). Briefly, the reaction mixture consisted of (final concentration): 20 µl 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS; 2 mM), 10 µl HRP (30 mU/ml), 20 µl H2O2 (0.1 mM), and 40 µl PBS (pH 7.4), and 10 µl sample to make a total volume of 100 µl in a 96-well plate. The reaction was initiated by the addition of H2O2 and conducted at 37°C. The increase in absorbance at 405 nm, a reflection of 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) radical cation (ABTS+) accumulation, was determined using an Anthos Labtech type plate reader (Austria). Experiments were conducted in triplicate on both cell-free inflammatory exudate as well as inflammatory cells. For the latter determinant, values were adjusted for protein concentration.
Superoxide-scavenging activity in cells and cell-free exudate
Superoxide-scavenging activity (SSA) was measured as previously described (17). Briefly, the reaction mixture consisted of (final concentration): 50 µl ferricyctochrome c (50 µM), 10 µl xanthine oxidase (20 mU/ml), 20 µl hypoxanthine (100 µM), and 5–10 µl sample with the volume being made up to 100 µl with PBS (pH 7.4). The reaction was initiated by the addition of hypoxanthine and conducted at 37°C. The increase in OD at 550 nm was measured over a 3-min period at 30-s intervals using an Anthos Labtech type plate reader (Salzburg, Austria). All determinants of initial reaction rates were made with and without sample at least in triplicate.
MTT assay
Cell viability was assessed as dehydrogenase activity, measured as the dehydrogenation of MTT to form the formazan product. A total of 50 µl of MTT was added to make 0.5 mg/ml in each well and incubated until the control lanes were developed (20 min). The cells were then washed twice in Dulbecco’s PBS A, the formazan product was dissolved by adding 100 µl DMSO to each well, and the plates were read at 570 nm. All samples were measured in duplicate.
Exudate cell smears
Exudate smears (n = 4 per group, 6 h) were stained with Harris’ hematoxylin and eosin, and differential cell counts were performed in drug-treated and control animals. Mast cell numbers were assessed in drug-treated and control animals at 1 h in exudate smears stained with toluidine blue.
Materials
Carrageenin (Viscarin) was obtained from FMC (Rockland, ME);
LTB4, PGE2 enzyme
immunoassay kits,
[3H]PGE2,
[3H]6-keto PGF1, and
[3H]L-arginine were purchased from
Amersham International (Buckinghamshire, U.K.). Mouse
KC Quantikine immunoassay kit was
obtained from R&D Systems (Oxon, U.K.). AE-ITU, 1400W, and
L-NIO were purchased from Alexis (Nottingham,
U.K). iNOS Ab was obtained from Santa Cruz Biotechnology (Santa Cruz,
CA). All other chemicals were obtained from Sigma (Poole, U.K.).
Data analysis
Results are expressed as the mean ± SEM. Statistical
analysis was performed using a one-way Kruskal-Wallis nonparametric
test, followed by a Mann-Whitney U test. A p
value of 
0.05 was considered to be statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this model, polymorphonuclear cells (PMNs) are the principal
cell type up to 12 h after carrageenin injection and are replaced
by phagocytosing mononuclear cells that predominate up to resolution at
48 h (18). NO, as determined by measuring nitrite in
the cell-free inflammatory exudate, was initially high 0.5–1 h after
carrageenin injection (Fig. 1
a). Thereafter, NO levels
declined but increased again at 6 h in parallel with inflammatory
cell iNOS enzyme activity. Neither NO nor iNOS activity was detectable
during resolution at 48 h. In the inflammatory cells from the
pleural cavity, there was a peak in iNOS protein expression at 6
h, mirroring iNOS activity (Fig. 1b), which gradually
declined but increased again at inflammatory resolution at 48 h.
This finding is similar to the recent report that COX 2 is also
expressed in inflammatory mononuclear cells at resolution in this model
(18), and further studies are being conducted to examine
the relevance of this apparently enzymatically inactive iNOS during
resolution.
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As the initial peak in exudate nitrite levels was detectable at
1 h, this would suggest that the principal source of NO at this
time was from the postcapillary endothelial cells lining the pleural
cavity and not from an inducible source. For our first experiments, to
determine the contribution of NO to the developing inflammatory
response, we investigated the effects on inflammation of administering
NOS inhibitors directly into the pleural cavity immediately before
carrageenin injection. We found that AE-ITU at 3 and 10 mg/kg
significantly increased exudate volume and inflammatory cell influx
compared with untreated controls (Fig. 2, a and b). 1400W also significantly elevated
exudate volume, but was without effect on cell numbers (Fig. 2, a and b). Both inhibitors reduced nitrite levels
significantly at the higher dose used (Fig. 2c). Given their
lack of selectivity, it is likely that AE-ITU and 1400W also inhibited
ecNOS at the dosing levels used. Finally, in addition to reducing
exudate nitrite levels, L-NIO (10 mg/kg)
increased exudate volume compared with controls, whereas inflammatory
cell numbers were unchanged (Fig. 2a–c).
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From these studies, it appears that the initial peak of NO
generation at 1 h is anti-inflammatory. We therefore proceeded
to examine whether the second peak of NO generation, apparently from
iNOS, at 6 h has a similarly protective role. To this
end, the effects of intrapleural injection of AE-ITU, 1400W,
and L-NIO immediately before the establishment of
a carrageenin pleurisy were determined. These experiments also showed
that at doses that inhibited NO generation, NOS inhibitors
significantly worsened inflammation by increasing pleural exudates and
leukocyte influx into the pleural cavity in comparison with saline
controls (Fig. 3, a–c). By
contrast, in a parallel experiment, it was found that a slow-releasing
NO donor (DPTA NoNoate), administered locally at the time of
carrageenin injection, reduced inflammatory cell influx (Fig. 4).
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). Thus, from these experiments it
appears that NO plays a critical role in determining the outcome of an
inflammatory response.
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The fundamental question addressed in this study is whether there
is a differential effect on inflammation by administering NOS
inhibitors locally vs systemically. To determine this, we examined the
effects on inflammation at 6 and 36 h of AE-ITU and
L-NMMA when injected into the peritoneal cavity at the time
of carrageenin injection. In keeping with the bulk of data published on
the systemic effects of NOS inhibitors on inflammation, both drugs
tested reduced inflammation at 6 h (Fig. 6, a and b) at
doses that inhibited inflammatory exudate nitrite levels (Fig. 6c) and at 36 h (Fig. 7, a and b), in which nitrite was undetectable. In
contrast, AE-ITU and L-NMMA, when injected
locally into the pleural cavity immediately before carrageenin
injection, increased exudate volume and inflammatory cell number at
6 h (Fig. 2
, a and b) and 36 h (Fig. 8, a and b). These
findings present highly conflicting outcomes on inflammation depending
on whether NOS inhibitors are administered either locally or
systemically.
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To show that AE-ITU (3 mg/kg) and 1400W (10 mg/kg) did not exacerbate inflammation simply as a result of direct irritation, we injected these drugs, as well as saline, into the pleural cavity in the absence of carrageenin. Neither drug caused measurable exudate formation or an increase in cellular influx (data not shown). Moreover, cationic compounds, including NOS inhibitors, may cause nonspecific mast cell degranulation. Therefore, NG-nitro-D-arginine-methyl ester, the inactive enantiomer of the nonspecific NOS inhibitor L-NAME, was injected intrapleurally at the same molarity as AE-ITU (10 mg/kg) and caused no increase in inflammatory parameters (data not shown). As a final control experiment, using the MTT assay for the assessment of cell viability, we found that neither AE-ITU nor L-NMMA, when injected intrapleurally, caused toxicity to influxing inflammatory cells in the pleural cavity (data not shown).
Mechanisms by which NOS inhibition may exacerbate inflammation
In an attempt to elucidate the mechanisms by which inhibition of
NO synthesis worsens inflammation, we measured levels of the most
likely proinflammatory mediators after treatment with AE-ITU. The first
candidate for investigation, histamine, was measured in cell-free
exudates at 1 h, and levels were correlated with mast cell numbers
(the cellular source of histamine) in pleural exudates. AE-ITU (10
mg/kg) significantly increased histamine (Fig. 9a), while reducing mast cell
numbers by 72%, presumably as a consequence of increased cellular
degranulation. Moreover, in animals previously depleted of mast cells
using CMP 48/80, the increase in exudate volume observed at 1 h
after treatment with AE-ITU was partially attenuated (compare Fig. 2a with 9b) at dosing levels that significantly
reduced exudate nitrite levels (Fig. 9d). Although there was
an expected reduction in exudate formation in animals treated with CMP
48/80 alone (Fig. 9b), there was no change in cell numbers
(Fig. 9c). This observation is consistent with the fact that
histamine mediates edema formation and not cell migration.
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a), the intrapleural
injection of AE-ITU (3, 10 mg/kg) caused a significant increase in CINC
in cell-free pleural exudates (Fig. 10b). An additional
explanation for exacerbated inflammation after NOS inhibition could be
enhanced eicosanoid synthesis, particularly as NO has been suggested to
modulate COX activity (for review, see Ref. 20). Thus,
given the proinflammatory properties of eicosanoids and their abundance
in the carrageenin-induced pleurisy up to 6 h (10),
we examined the effects of NOS inhibition on inflammatory cell COX
activity as well as cell-free exudate levels of
PGE2, 6-keto PGF1 (Table I), and the neutrophil chemoattractant,
LTB4. Surprisingly, AE-ITU (3 mg/kg) had no
effect on either COX activity in inflammatory cell pellets or levels of
PGE2 and 6-keto PGF1.
Interestingly, however, exudate levels of LTB4
were significantly increased in comparison with controls (Table I).
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In a series of final experiments, TAOS and SSA activity were
measured as an indirect indication of
O2- and other oxidant species
formation. These assays are based on the principle that oxidants formed
during inflammation (21) react with antioxidants such as
glutathione, ascorbic acid, and -tocopherol and reduce the
inflammatory exudates antioxidant capacity (22). These two
parameters may provide additional support for the concept that during
inflammation NO may indeed have a protective role. Administered
immediately before the induction of inflammation, AE-ITU (3, 10 mg/kg)
significantly reduced both TAOS (Table II) and SSA (Table III) in cells and cell-free exudates at 1
and 6 h in comparison with saline controls.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The vast majority of reports have shown that NOS inhibitors are considered anti-inflammatory in models of both acute and chronic inflammation. In a model of trinitrobenzene sulfonic acid-induced colitis in rats, L-NAME reduced neutrophil and macrophage influx (23), while in a carrageenin-induced pleurisy, L-NMMA also significantly reduced exudate formation and cellular influx (6). In these studies, however, NOS inhibitors were administered at high doses away from the site of inflammation, i.e., orally or i.p. Given their lack of selectivity, it is conceivable that systemic administration may also inhibit ecNOS remote from the inflammatory locus, resulting in vasoconstriction, reduced blood delivery to the inflamed site, and, thus, a reduction in inflammation. Indeed, it was shown that the anti-inflammatory effects of L-arginine analogues, given systemically, could be reversed by vasodilators (8, 9, 24). In agreement with these studies, we also found that when NOS inhibitors were injected i.p., immediately before intrapleural injection of carrageenin, pleural exudates were significantly reduced. Therefore, to bypass the systemic effects of NOS inhibitors, we administered AE-ITU, 1400W, and L-NIO locally and found them to exert a proinflammatory effect, without altering inflammatory cell viability. In support of our findings, others have also reported a protective role for NO. For instance, acetic acid-induced colitis in iNOS-deficient mice resulted in increased PMN-associated tissue damage in comparison with wild-type animals (25). A similar inflammatory cell accumulation in hepatic microvasculature was also observed in LPS-treated iNOS knockout mice (26). Thus, the protective effects of NO shown in these genetically modified animals may arise from the absence of only iNOS at the inflammatory site with ecNOS being functionally active, thereby avoiding systemic perturbations.
To elucidate the mechanism by which inhibition of NO generation exacerbates inflammation, we measured levels of a number of candidate proinflammatory mediators. As shown by others (27), mast cell-derived histamine peaked in this model between 0.5 and 1 h after carrageenin injection. Given that histamine mediates edema formation (27) and that NO stabilizes mast cells, thereby preventing histamine release (28), levels of this acute inflammatory mediator were measured after NOS inhibition. We found that while histamine was increased, mast cell numbers decreased in pleural exudates. Additional experiments showed that prior depletion of mast cells with CMP 48/80 attenuated plasma exudation observed with NOS inhibitors without altering the increase in inflammatory cells. In support of this observation, others have shown that inhibition of NO in mesenteric postcapillary venules increased inflammatory cell emigration, plasma extravasation, free radical-mediated tissue damage, mast cell degranulation, and histamine release (29).
As well as histamine, levels of CINC were also determined. CINC, which
is induced through a NF-
B pathway in response to inflammatory
stimulation (30), mediates PMN chemotaxis in the rat
(19) by up-regulating PMN CD11/18 expression, thus
facilitating PMN diapedesis (31). NOS inhibition increased
levels of CINC in cell-free exudates and, in addition to histamine, may
be a contributing factor to the increased inflammatory cell numbers
recorded after NOS inhibition. Levels of lipid-derived mediators were
also measured particularly as inhibition of NO synthesis increases
endothelial PGI2 release (32) and
PGE2, PGI2, and
LTB4 facilitate edema formation and inflammatory
cell influx. Treatment with NOS inhibitors locally at a time when iNOS
protein expression and enzyme activity are maximal had no affect on
either PGE2 or PGI2, but
significantly increased LTB4. Previous
experiments have also shown that NO donors dose dependently reduce
LTB4 production from activated PMNs
(33). Interestingly, differential cell counts revealed
that AE-ITU at 6 h caused an increase in the proportion of PMNs,
presumably as a result of an increase in CINC and
LTB4. Collectively, these mechanistic studies
demonstrate that NO generated at the inflammatory site critically
regulates the severity of the inflammatory response by keeping in check
levels of such potently proinflammatory mediators as histamine, CINC,
and LTB4.
As a final mechanism, we examined levels of
O2- after NOS inhibition
indirectly as a reduction in TAOS and SSA, and found that these indices
of O2- and other oxidant
species formation were reduced, suggesting that in the absence of NO,
O2- generation was enhanced.
O2- is produced by PMNs and
macrophages from the enzyme activity of NADPH oxidase and xanthine
oxidase at inflammatory sites. Both enzyme systems contain a heme
prosthetic group with which NO can react to inhibit
O2- release (34).
Therefore, inhibiting NO removes the brakes on
O2- production. In support of
this notion, others have shown that NO generation reduces
O2- levels, while its
inhibition increases O2-
production both in vitro and in vivo (35, 36, 37). As
O2- has been associated with
tissue damage and loss of function during inflammatory episodes
(38), it is conceivable that one of the contributors to an
enhanced inflammatory response, consequent to NOS inhibition, is
O2- generation. Indeed,
elevated levels of O2- increase
histamine release from mast cells (39), as well as
LTB4 and PMN accumulation in a model of
pancreatitis through a platelet-activating factor-dependent mechanism
(40). In addition, raised levels of
O2- activate NF-B
(41), which may account for an elevated production of the
chemokine CINC. Although no direct effect of
O2- on the up-regulation of
CINC has been demonstrated, an
O2- scavenger
N-acetylcysteine significantly reduced NF-B DNA binding
and CINC mRNA expression in inflamed lungs (42).
Therefore, a disturbance in the balance between NO and
O2- production may lead to an
increase in proinflammatory mediators and provides a possible mechanism
for the exacerbation of inflammation observed in this study.
In conclusion, in the rat carrageenin-induced pleurisy, inhibition of NO at the inflammatory site exacerbates inflammation and prolongs the pathology, suggesting a protective role for NO in this model. Moreover, NOS inhibitors appear to have differential effects on inflammation depending on their route of administration.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Derek W. Gilroy, Department of Experimental Pathology, The William Harvey Research Institute, St. Bartholomew’s and the Royal London School of Medicine and Dentistry, Charterhouse Square, London, EC1 M 6BQ, U.K.
3 Abbreviations used in this paper: NOS, NO synthase; AE-ITU, S-(2-aminoethyl) isothiourea; CINC, cytokine-induced neutrophil chemoattractant; CMP 48/80, compound 48/80; COX, cyclooxygenase; DPTA NoNoate, 3,3'-[hydroxynitrosohydrazino]bis-l-propanamine; ecNOS, endothelial cell NOS; iNOS, inducible NOS; L-NIO, L-N5(1-iminoethyl)-ornithine; L-NMMA, NG-monomethyl-L-arginine; LTB4, leukotriene B4; O2-, superoxide; PMN, polymorphonuclear cell; SSA, superoxide-scavenging activity; TAOS, total antioxidant status; 1400W, N-(3-(aminomethyl)-benzyl) acetamidine.
Received for publication June 28, 2000. Accepted for publication October 11, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B activation. Am. J. Respir. Cell Mol. Biol. 11:464.[Abstract]
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) in the cell-free
inflammatory exudate was measured by the Greiss reaction, while iNOS
enzyme activity (
) was determined by the catalytic conversion of
[3H]L-arginine to
[3H]L-citrulline by inflammatory cells.
b, Inflammatory cell iNOS protein expression was
determined by Western blotting, with densitometric analysis shown
beneath a representative blot. Data are expressed as mean ± SEM
(n = 6–8 per time point).
