Full text of DOI:10.1038/sj.bjp.0704725

British Journal of Pharmacology (2002) 136, 391 ± 398
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Increased arginase activity underlies allergen-induced de®ciency of
cNOS-derived nitric oxide and airway hyperresponsiveness
1
1
1
1
*,1Herman Meurs, Sue McKay, Harm Maarsingh, Marco A.M. Hamer, Lejla Macic,
1Niek Molendijk & Johan Zaagsma
1
1Department of Molecular Pharmacology, University Centre for Pharmacy, Antonius Deusinglaan 1, Groningen, NL-9713 AV,
The Netherlands
1
A de®ciency of constitutive nitric oxide synthase (cNOS)-derived nitric oxide (NO), due to
reduced availability of L-arginine, importantly contributes to allergen-induced airway hyperrespon-
siveness (AHR) after the early asthmatic reaction (EAR). Since cNOS and arginase use L-arginine as
a common substrate, we hypothesized that increased arginase activity is involved in the allergen-
induced NO de®ciency and AHR.
2
Using a guinea-pig model of allergic asthma, we addressed this hypothesis by examining the
e€ects of the speci®c arginase inhibitor No-hydroxy-nor-L-arginine (nor-NOHA) on the
responsiveness to methacholine of isolated perfused tracheae from unchallenged control animals
and from animals 6 h after ovalbumin challenge. Arginase activity in these preparations was
investigated by measuring the conversion of L-[14C]arginine to [14C]urea.
3
Airways from allergen-challenged animals showed a 2 fold (P50.001) increase in responsiveness
to intraluminal (IL) administration of methacholine compared to controls. A similar hyperrespon-
siveness (1.8 fold, P50.01) was observed in control airways incubated with the NOS inhibitor No-
nitro-L-arginine methyl ester (L-NAME, 0.1 mM, IL), while L-NAME had no further e€ect on the
airways from challenged animals.
4
Remarkably, 5 mM nor-NOHA (IL) normalized the hyperresponsiveness of challenged airways to
basal control (P50.001), and this e€ect was fully reversed again by 0.1 mM L-NAME (P50.05).
Moreover, arginase activity in homogenates of the hyperresponsive airways was 3.5 fold (P50.001)
enhanced compared to controls.
5
The results indicate that enhanced arginase activity contributes to allergen-induced de®ciency of
cNOS-derived NO and AHR after the EAR, presumably by competition with cNOS for the
common substrate, L-arginine. This is the ®rst demonstration that arginase is involved in the
pathophysiology of asthma.
British Journal of Pharmacology (2002) 136, 391 ± 398
Keywords: Arginase; constitutive nitric oxide synthase; nitric oxide; methacholine; No-hydroxy-nor-L-arginine; allergic
asthma; early asthmatic reaction; airway hyperresponsiveness; tracheal perfusion; guinea-pig
Abbreviations: AHR, airway hyperreactivity; cNOS, constitutive nitric oxide synthase; EAR, early asthmatic reaction; EL,
extraluminal; Emax, maximal e€ect; eNOS, endothelial nitric oxide synthase; IL, intraluminal; iNANC,
inhibitory nonadrenergic noncholinergic; iNOS, inducible nitric oxide synthase; KH, Krebs-Henseleit; LAR, late
asthmatic reaction; L-NAME, No-nitro-L-arginine methyl ester; L-NMMA, NG-monomethyl-L-arginine; nNOS,
neuronal nitric oxide synthase; nor-NOHA, No-hydroxy-nor-L-arginine; DP, di€erential (hydrostatic) pressure;
Pinlet, (hydrostatic) pressure at the inlet; Poutlet, (hydrostatic) pressure at the outlet; pEC50, 7log10 of the
concentration causing 50% of the e€ect
Introduction
Characteristic features of allergic asthma are allergen-induced
early (EAR) and late (LAR) asthmatic reactions (Booij-
Noord et al., 1971), in®ltration and activation of in¯amma-
tory cells in the airways (De Monchy et al., 1985) and airway
hyperresponsiveness (AHR), both after the EAR and LAR
(Durham et al., 1988). The mechanisms behind these
pathological features are complex and incompletely under-
stood, involving a variety of neurotransmitters, mediators
and other signalling molecules, including nitric oxide (NO).
NO is synthesized from the semi-essential amino acid L-
arginine both by constitutive (cNOS) and inducible (iNOS)
NO synthase isoforms (Barnes & Belvisi, 1993; Barnes, 1998).
In the airways, cNOS is mainly expressed in inhibitory
nonadrenergic noncholinergic (iNANC) nerves (neuronal
NOS or nNOS), endothelial cells (endothelial NOS or eNOS),
and airway epithelium (nNOS and eNOS) (Tucker et al.,
1990; Fischer et al., 1993; Kobzik et al., 1993; Asano et al.,
1994), and is primarily involved in the regulation of airway
and vascular tone by the local production of small amounts
of NO in response to neurogenic and non-neurogenic stimuli
(Barnes & Belvisi, 1993). The involvement of endogenous
cNOS-derived NO in the regulation of airway tone is
indicated by observations that nonselective NOS inhibitors
such as No-nitro-L-arginine methyl ester (L-NAME) enhanced
contractile agonist-induced airway constriction in vitro
*Author for correspondence; E-mail: h.meurs@farm.rug.nl

392
H. Meurs et al
Arginase, NO and airway hyperresponsiveness
(Nijkamp et al., 1993; De Boer et al., 1996) and in vivo
(Nijkamp et al., 1993; Ricciardolo et al., 1996; Schuiling et
al., 1998b; Taylor et al., 1998).
Using a perfused guinea-pig tracheal tube preparation, we
recently demonstrated that the potent and highly speci®c
arginase inhibitor No-hydroxy-nor-L-arginine (nor-NOHA)
iNOS, producing much larger amounts of NO than cNOS,
may be induced by pro-in¯ammatory cytokines during airway
in¯ammation, particularly in in¯ammatory and epithelial
cells (Hamid et al., 1993; Asano et al., 1994; Barnes, 1998),
and may be involved in mucosal swelling (Kuo et al., 1992),
in®ltration of in¯ammatory cells (Schuiling et al., 1998a) and
epithelial damage (Flak & Goldman, 1996; Schuiling et al.,
(Custot et al., 1997) caused a concentration-dependent
inhibition of methacholine-induced airway constriction,
which was reversed by L-NAME (Meurs et al., 2000). This
indicates that arginase activity in the airways is also involved
in the modulation of airway responsiveness, by limiting
cNOS-derived bronchodilating NO production. For
a
number of cells, it has been reported that the expression of
arginase may be enhanced by Th2 lymphocyte-derived
cytokines such as IL-4, IL-10 and IL-13 and by cyclic
AMP-elevating stimuli such as PGE2 (Modolell et al., 1995;
Munder et al., 1999; Wei et al., 2000). Since these mediators
are crucial in the development of asthmatic airway in¯amma-
tion (Chung & Barnes, 1999), we hypothesized that induction
of arginase activity after allergen challenge is involved in the
reduction of cNOS activity and the initiation of AHR after
the EAR. In the present study, we investigated this
hypothesis by examining the e€ect of the speci®c arginase
inhibitor nor-NOHA on cholinergic AHR in isolated
perfused tracheae from sensitized guinea-pigs at 6 h after
allergen challenge. In addition, we assessed the e€ect of
allergen challenge on arginase activity in these preparations.
1998a). However, iNOS-derived NO may also have
a
bene®cial bronchodilating action (De Gouw et al., 1998;
Schuiling et al., 1998a), indicating its dualistic action in the
airways.
Several studies in animal models (De Boer et al., 1996;
1999; Mehta et al., 1997; Miura et al., 1997; Schuiling et al.,
1998a, b; Samb et al., 2001) and in asthmatic patients
(Ricciardolo et al., 1997; 2001; Silko€ et al., 2000) have
indicated that a de®ciency of cNOS-derived NO may be
importantly involved in the pathophysiology of allergic
asthma. Using
a guinea-pig model of allergic asthma,
characterized by allergen-induced EAR and LAR, we have
established both ex vivo (De Boer et al., 1996) and in vivo
(Schuiling et al., 1998a, b) that a de®ciency of bronchodilat-
ing cNOS-derived NO contributes to the AHR observed after
the EAR, and that this de®ciency is due to limitation of L-
arginine as substrate for cNOS (De Boer et al., 1999).
Di€erent mechanisms could account for this limitation.
One mechanism involves inhibition of cellular uptake of L-
arginine by speci®c cationic amino acid transporters induced
by polycationic proteins, including eosinophil-derived major
basic protein (Hammermann et al., 1999; Meurs et al., 1999).
A second mechanism could be a reduced bioavailability of L-
arginine due to enhanced activity of arginase, which
hydrolyzes L-arginine to L-ornithine and urea. Arginase,
classically an enzyme of the urea cycle in the liver, is also
found in many other cells that do not express a complete urea
cycle, including the lung (Que et al., 1998). Two distinct
isoforms of mammalian arginase ± arginase I and II ± have
been identi®ed (Wu & Morris, 1998), which are constitutively
expressed in the airways, particularly in the bronchial
epithelium and in peribronchial connective tissue ®broblasts
(Que et al., 1998).
The biological function of extrahepatic arginase is largely
unclear, but has been implicated in the regulation of NO
synthesis and cell growth in in¯ammatory conditions. In
activated macrophages various studies have indicated that
arginase activity may limit the utilization of L-arginine by
iNOS and suppress the cytotoxic response by these cells
(Wang et al., 1995; Modolell et al., 1995; Hey et al., 1997),
while concomitant polyamine and proline synthesis from L-
ornithine may cause cell proliferation and repair of
in¯ammatory lesions (Shearer et al., 1997; Wu & Morris,
1998). Recent immunohistochemical studies in rat lung
indicated that the expression of both arginase I and arginase
II in the airways was upregulated during hyperoxic lung
injury and recovery. This was associated with reduced NO
production and increased expression of ornithine decarbox-
ylase, indicating a role for pulmonary arginase in the repair
of hyperoxic lung injury (Que et al., 1998). The role of
arginase in asthmatic airway in¯ammation, however, is
presently unknown.
Methods
Animals
Outbred speci®ed pathogen free guinea-pigs (Harlan, Heath-
®eld, U.K.), weighing 600 ± 800 g, were used in this study.
Animals were actively IgE-sensitized to ovalbumin (OA) at 3
weeks of age as described by Van Amsterdam et al. (1989). In
short, 0.5 ml of an allergen solution containing 100 mg ml71
ovalbumin and 100 mg ml71 Al(OH)3 in saline was injected
intraperitoneally, while another 0.5 ml was divided over seven
intracutaneous injection sites in the proximity of lymphe-
nodes in the paws, lumbar regions and the neck. The animals
were used experimentally in weeks 4 to 8 after sensitization.
The animals were group-housed in individual cages in climate
controlled animal quarters and given water and food ad
libitum, while a 12-h on/12-h o€ light cycle was maintained.
All protocols described in this study were approved by the
University of Groningen Committee for Animal Experimen-
tation.
Allergen provocation
Ovalbumin provocations were performed by inhalation of
aerosolized solutions. The provocations were performed in a
specially designed animal cage, in which the guinea-pigs could
move freely (Santing et al., 1992). The volume of the cage
was 9 l, which ensured fast replacement of the air inside the
cage with aerosol and vice versa. A DeVilbiss nebulizer (type
646, DeVilbiss, Somerset, PA, U.S.A.) driven by an air¯ow of
8 l min71 provided the aerosol required, with an output of
0.33 ml min71. Allergen provocations were performed by
inhalation of an aerosol concentration of 0.05 mg ml71
ovalbumin in saline. Allergen inhalations were discontinued
when the ®rst signs of respiratory distress were observed.
Anti-histamines were not needed to prevent the development
British Journal of Pharmacology vol 136 (3)

H. Meurs et al
Arginase, NO and airway hyperresponsiveness
393
of anaphylactic shock. Previous studies measuring pleural
pressure changes in ovalbumin sensitized, permanently
instrumented, unrestrained guinea-pigs have indicated that
the allergen-induced EAR induced by this procedure is
maximal within 20 min and lasts for up to 5 h (Santing et
al., 1992, 1994b). Six hours after ovalbumin challenge
(between the EAR and LAR; Santing et al., 1992; 1994b),
the guinea-pigs were sacri®ced. The animals were killed by a
sharp blow on the head and exsanguinated. Non-challenged
animals were used as controls.
HCl, 2 mM phenylmethylsulphonyl ¯uoride, pH 7.4 using a
polytron homogenizer (Kinematica GmbH, Luzern, Switzer-
land). The homogenate was centrifuged at 20,0006g for
30 min at 48C and the supernatant was used for arginase
assay. Arginase activity was determined by measuring the
conversion of L-[guanidino-14C]arginine to [14C]urea essen-
tially as described by Custot et al. (1996). In short,
homogenate aliquots (50 ml) were incubated in a ®nal volume
of 150 ml, containing 25 mM Tris HCl, 0.67 mM MnCl2, 1 to
20 mM L-arginine (1.66 mM for standard conditions) and
200,000 d.p.m. of L-[guanidino-14C]arginine (51.5 mCi m-
mol71), pH 7.4, for 20 min at 378C. Speci®c inhibition of
arginase activity was measured in the presence of 0.5 or
5.0 mM nor-NOHA. Reactions were terminated by adding
1 ml of ice-cold stop bu€er, containing 20 mM sodium
acetate, 100 mM urea, 10 mM EDTA and 1 mM L-citrulline,
pH 3.0. Samples were applied to columns containing 2 ml
Dowex AG 50W-X8 (H+ form), preequilibrated with 20 ml
of stop bu€er. Columns were eluted with stop bu€er and
aliquots (0.5 ml) were counted in 4 ml Ultima Gold
scintillation ¯uid using a Beckman LS 1701 liquid scintilla-
tion counter. The protein concentrations of each sample were
determined by the Bradford Coomassie brilliant blue method
(Bradford, 1976), using bovine serum albumin as a standard.
Arginase activity, expressed as pmol urea.mg protein71.-
min71, showed linear enzyme kinetics up to 30 min and was
proportional to protein concentration.
Tracheal perfusion
The tracheas were rapidly removed and placed in Krebs-
Henseleit (KH) solution (378C) of the following composition
(mM): NaCl 117.50, KCl 5.60, MgSO4 1.18, CaCl2 2.50,
NaH2PO4 1.28, NaHCO3 25.00, D-glucose 5.50; gassed with
5% CO2 and 95% O2; pH 7.4. The tracheas were prepared
free of serosal connective tissue and cut into two halves of
approximately 17 mm before mounting in a perfusion setup,
as described previously (De Boer et al., 1996).
To this aim, the tracheal preparations were attached at
each end to stainless steel perfusion tubes ®xed in a Delrin
perfusion holder. The holder with the trachea was then
placed in a water-jacketed organ bath (378C) containing
20 ml of gassed KH (the serosal or extraluminal (EL)
compartment). The lumen was perfused with recirculating
KH from a separate 20 ml bath (mucosal or intraluminal (IL)
compartment) at a constant ¯ow rate of 12 ml min71. Two
axially centred side-hole catheters connected with pressure
transducers (TC-XX,Viggo-Spectramed B.V., Bilthoven, The
Netherlands) were situated at the distal and proximal ends of
the trachealis to measure hydrostatic pressures (Poutlet and
Pinlet, respectively). The signals were fed into a di€erential
ampli®er to obtain the di€erence between the two pressures
(DP = Pinlet ± Poutlet), which was plotted on a ¯atbed chart
recorder (BD 41, Kipp en Zonen, Delft, The Netherlands).
DP re¯ects the resistance of the tracheal segment to perfusion
and is a function of the mean diameter of the trachea
between the pressure taps. The transmural pressure in the
trachea was set at 0 cm H2O. At the perfusion ¯ow rate used,
a baseline DP of 0.1 to 1.0 cm H2O was measured, depending
Data analysis
To compensate for variations in baseline DP and in DP
responses to contractile stimuli due to variation in resting
internal diameter of the preparations used, IL responses of
the tracheal tube preparations to methacholine were
expressed as a percentage of the response induced by EL
administration of 40 mM KCl. The contractile e€ect of
10 mM methacholine (highest concentration) was de®ned as
Emax (Boer et al., 1996; 1999; Meurs et al., 1999; 2000). Using
this Emax, the sensitivity to methacholine was evaluated as
pEC50 (7log10 EC50) value. Kinetic parameters of tracheal
arginase activity and its inhibition by nor-NOHA were
determined by Lineweaver-Burk analysis, by plotting arginase
activity71 versus arginine concentration71 for di€erent L-
arginine and nor-NOHA concentrations. Results are ex-
pressed as means+s.e.mean. Statistical analysis was per-
formed using the Student's t-test for paired or unpaired
observations as appropriate. Di€erences were considered
statistically signi®cant at P50.05.
on the diameter of the preparation. After
a 45 min
equilibration period with three washes with fresh KH (both
IL and EL), 1 mM isoprenaline was added to the EL
compartment for maximal smooth muscle relaxation to assess
basal tone. After three washes during at least 30 min, the
trachea was exposed to EL 40 mM KCl in KH to obtain a
receptor-independent reference response. Subsequently, the
preparation was washed four times with KH during 45 min
until basal tone was reached and a cumulative concentration
response curve (CCRC) was made with IL methacholine.
When used, L-NAME (0.1, 0.5 or 1.0 mM) and nor-NOHA (5
or 10 mM) were applied to the IL reservoir, 40 min prior to
agonist-addition.
Chemicals
Histamine hydrochloride, ovalbumin (grade III), aluminium
hydroxide, (7)-isoprenaline hydrochloride, L-arginine hydro-
chloride, L-citrulline, No-nitro-L-arginine methyl ester and
phenylmethylsulphonyl ¯uoride were obtained from Sigma
Chemical Co. (St. Louis, MO, U.S.A.) and methacholine
chloride from Aldrich (Milwaukee, WI, U.S.A.). Dowex AG
50W-X8 resin was from Bio-Rad Laboratories (Hercules,
CA, U.S.A.). L-[guanidino-14C]arginine (speci®c activity
Arginase assay
Tracheal tissue was snap-frozen in liquid nitrogen and stored
at 7808C until homogenization. The tissue was ground using
a pestle and mortar in the presence of liquid nitrogen prior to
homogenization in approximately 6-vol ice-cold 20 mM Tris
51.5 mCi mmol71
)
was purchased from New England
Nuclear Life Science Products, Inc. (Boston, MA, U.S.A.).
Ultima Gold scintillation ¯uid was obtained from Packard
British Journal of Pharmacology vol 136 (3)

394
H. Meurs et al
Arginase, NO and airway hyperresponsiveness
Bioscience (Groningen, The Netherlands). No-hydroxy-nor-L-
arginine was kindly provided by Dr J.-L. Boucher (Universite
Paris V, Paris).
sensitivity to the agonist; no further e€ect on Emax was
obtained in the presence of 10 mM nor-NOHA (Figure 1b,
Table 1). The normalized responsiveness in the presence of
5 mM nor-NOHA was completely reversed in the additional
presence of 0.1 mM L-NAME (Figure 1b, Table 1). Both in
the unchallenged control preparations and in the prepara-
tions of allergen-challenged animals, L-NAME and nor-
NOHA, alone or in combination, had no e€ect on basal
airway tone (not shown).
Results
In perfused tracheal preparations from unchallenged control
guinea-pigs, the non-selective NOS inhibitor L-NAME
(0.1 mM, IL) caused a signi®cant 1.8 fold increase in the
Emax of IL methacholine (P50.01), without an e€ect on the
sensitivity (pEC50) to this agonist (Figure 1a, Table 1). In the
ovalbumin-challenged group of animals, the DP response to
EL KCl was unchanged compared to the unchallenged
control group (not shown); however, a 2 fold increase in
the Emax to IL methacholine was observed (P50.001),
without a change in pEC50 (Figure 1b, Table 1). This
increase was not further enhanced in the presence of L-
NAME (Figure 1b, Table 1).
IL perfusion of tracheae from unchallenged control
animals with the speci®c arginase inhibitor nor-NOHA
(5 mM) caused a decrease in the Emax of methacholine-
induced airway constriction by 38% (P50.001), while no
e€ect was observed on the pEC50 value for the agonist
(Figure 1a, Table 1). As in a previous study (Meurs et al.,
2000), we demonstrated that the reduced responsiveness in
the presence of nor-NOHA can be concentration-dependently
reversed by L-NAME, to the level of responsiveness of
untreated airways in the presence of the NOS inhibitor
(Figure 1a, Table 1). Remarkably, the enhanced Emax to IL
methacholine at 6 h after allergen challenge was fully restored
to the Emax value of unchallenged control animals by IL
administration of 5 mM nor-NOHA, without an e€ect on the
Arginase activity in normal tracheal homogenates dis-
played Michaelis-Menten kinetics, and Lineweaver-Burk
analysis indicated
protein71.min71 and a Km for L-arginine of 2.74+0.33 mM
at pH 7.4. Lineweaver-Burk plots of arginase inhibition
a Vmax of 2286+596 pmol urea.mg
experiments demonstrated that nor-NOHA acted as
a
competitive inhibitor with respect to L-arginine, with a Ki-
value of 0.23+0.03 mM. The arginase activity in tracheae
from allergen-challenged animals assessed in the presence of
1.66 mM L-arginine was 3.5 fold enhanced compared to
unchallenged controls (2021+273 vs 577+47 pmol urea.mg
protein71.min71, respectively; P50.001). Both in control and
in challenged airways 5 mM nor-NOHA caused approximately
95% inhibition of arginase activity, while the NOS inhibitor
L-NAME (0.1 mM) had no e€ect (Figure 2).
Discussion
The present study demonstrates for the ®rst time the
involvement of arginase in the pathophysiology of allergic
asthma. Using a guinea-pig model of acute allergic asthma,
we con®rmed our previous ®nding that the responsiveness of
isolated intact tracheal tube preparations to methacholine is
Figure 1 Methacholine (MeCh; IL)-induced constriction of intact perfused tracheal preparations obtained from (a) unchallenged
guinea-pigs, in the absence (control) and presence of 0.1 mM L-NAME, 5.0 mM nor-NOHA, and 5.0 mM nor-NOHA plus 0.1, 0.5 or
1.0 mM L-NAME, and (b) ovalbumin (OA)-challenged guinea-pigs, in the absence (control) and presence of 5.0 and 10.0 mM nor-
NOHA, 0.1 mM L-NAME, and 5.0 mM nor-NOHA plus 0.1 mM L-NAME. For comparison, methacholine-induced constriction of
control preparations from unchallenged guinea-pigs is also shown in b. Methacholine, nor-NOHA and L-NAME were all applied to
the IL compartment. Results are means+s.e.mean of 3 ± 14 experiments.
British Journal of Pharmacology vol 136 (3)

H. Meurs et al
Arginase, NO and airway hyperresponsiveness
395
Table 1 E€ects of L-NAME and nor-NOHA on the responsiveness to methacholine of intact perfused tracheae from unchallenged and
ovalbumin-challenged guinea-pigs
Unchallenged
Ovalbumin-challenged
pEC50
Emax
(% KCI)
pEC50
Emax
(% KCI)
(7log M)
2.94+0.08
3.21+0.16
(n)
(14)
(5)
(7log M)
3.00+0.08
3.00+0.10
(n)
(11)
(4)
Control
L-NAME
0.1 mM
51.4+1.6
102.9+2.9*
99.6+16.0
95.6+7.3{
Nor-NOHA
5.0 mM
10.0 mM
2.67+0.19
N.D.
32.0+1.4{
N.D.
(5)
2.98+0.15
2.96+0.12
56.1+ 5.9{,{
51.1+10.5{
(5)
(3)
5.0 mM nor-NOHA+
0.1 mM L-NAME
0.5 mM L-NAME
1.0 mM L-NAME
2.75+0.16
3.10+0.14
3.21+0.19
58.9+7.6}
85.8+8.1{,II
89.8+7.7{II
(5)
(5)
(6)
3.41+0.27
N.D.
N.D.
91.4+9.3{,}
N.D.
N.D.
(4)
Results are means+s.e.mean of n experiments. N.D., not determined. Statistical analysis: *P50.001; {P50.05 compared with
Unchallenged; {P50.001 compared with Control; }P50.05 compared with 0.1 mM L-NAME; IIP50.001; }P50.05 compared with
5.0 mM nor-NOHA.
parations, which was concentration-dependently reversed by
L-NAME, indicating that the reduced airway responsiveness
was caused by enhanced production of NO. In the nor-
NOHA-treated preparations, a higher concentration of L-
NAME (0.5 mM) was required for complete inhibition of the
functional antagonism of cholinergic airway constriction by
agonist-induced NO than in untreated controls (0.1 mM),
indicating that L-NAME had to compete with a higher
concentration of endogenous L-arginine in these preparations.
Since L-arginine is a limiting factor for bronchodilating
NO production in the airways (De Boer et al., 1999), it may
be expected that increased arginase activity is involved in the
allergen-induced AHR and its inhibition by nor-NOHA as
discussed above. Indeed, biochemical measurement of
arginase activity in tracheal homogenates indicated that
allergen challenge caused a 3.5 fold increase in this activity,
which was speci®cally reversed by 5 mM nor-NOHA, the same
concentration as used in the pharmacological experiments.
The No-hydroxy-L-aminoacid nor-NOHA is one of the most
potent arginase inhibitors reported so far, and the observed
Ki value for tracheal homogenates (0.23 mM) corresponds
closely to the Ki of 0.5 mM found for puri®ed rat liver
arginase (Custot et al., 1997).
The sources of NO and constitutive and allergen-induced
arginase activity, as well as the arginase isoforms in our
perfused tracheal tube preparations were not assessed. Both
in control and in challenged airways, L-NAME only
increased the agonist-induced constriction and not basal
tone, indicating that the NO was most likely produced by
cNOS. Previous studies have indicated that agonist-induced
bronchodilating NO is mainly derived from the airway
epithelium (Nijkamp et al., 1993). Since these cells also
constitutively express arginase (type I and type II) (Que et al.,
1998), it can be hypothesized that the epithelium is an
important site of competition between the two enzymes for
their common substrate. This may not only apply to the basal
condition, but also to the reduced cNOS activity after the
allergen challenge. Thus, preliminary data have recently
indicated that Th2 cytokines may cause enhanced expression
of arginase I and arginase II in cultured A549 human lung
epithelial cells (McCluskie et al., 2001). However, it cannot
be excluded that other cell types expressing arginase and/or
cNOS are involved.
Figure 2 Arginase activity in tracheal homogenates from unchal-
lenged and ovalbumin (OA)-challenged guinea-pigs, in the absence
(control) and presence of 0.1 mM L-NAME and 5.0 mM nor-NOHA.
Results are means+s.e.mean of 3 ± 7 experiments. *P50.001,
{P50.0001 compared to unchallenged control; {P50.005 compared
to OA-challenged control.
considerably enhanced at 6 h after allergen challenge (De
Boer et al., 1996). The allergen-induced AHR was closely
mimicked by the administration of L-NAME to unchallenged
control preparations, while, in addition, the challenged
preparations were unresponsive to the NOS inhibitor,
indicating that a de®ciency of agonist-induced airway dilating
NO is a major determinant of the observed hyperresponsive-
ness. Remarkably, the enhanced responsiveness to methacho-
line at 6 h after allergen challenge was fully normalized in the
presence of the speci®c arginase inhibitor nor-NOHA. This
indicates that arginase activity, presumably by competition
with cNOS for the common substrate, L-arginine, contributes
to the de®ciency of NO and subsequent AHR after allergen
challenge. This hypothesis was con®rmed by the observation
that the decrease in AHR by nor-NOHA was reversed by
administration of L-NAME.
As reported in a previous study (Meurs et al., 2000),
endogenous arginase activity is also involved in the regulation
of cholinergic airway tone under basal conditions. Thus, IL
administration of nor-NOHA also reduced methacholine-
induced bronchoconstriction in unchallenged control pre-
British Journal of Pharmacology vol 136 (3)

396
H. Meurs et al
Arginase, NO and airway hyperresponsiveness
Recently, a role for arginase has also been described in the
regulation of inhibitory nonadrenergic noncholinergic (iN-
ANC) nerve-stimulated, cNOS-derived NO-mediated relaxa-
tion of the penile corpus cavernosum smooth muscle (Cox et
al., 1999) and the internal anal s®ncter smooth muscle
(Baggio et al., 1999). Although iNANC nerve activity was
not measured in our tracheal preparations, upregulation of
arginase in airway iNANC nerves could also be of
importance in allergen-induced airway hyperresponsiveness,
since it has been demonstrated that the bronchodilating
iNANC activity is reduced after allergen challenge (Miura et
al., 1997).
Di€erent studies have indicated that the present ex vivo
®ndings are relevant to the development of AHR in vivo.
Using the same guinea-pig model of asthma, we have
recently shown that inhalation of the nonselective NOS
inhibitor L-NAME, but not a selective dose of the speci®c
iNOS inhibitor aminoguanidine, caused AHR to histamine
before allergen challenge, indicating that cNOS-derived NO
counteracts histamine-induced bronchoconstriction under
basal conditions (Schuiling et al., 1998a, b). At 6 h after a
single allergen challenge, both L-NAME and aminoguani-
dine had no e€ect on the allergen-induced AHR to
histamine at this time point, indicating that a de®ciency of
(cNOS-derived) NO contributes to AHR after the EAR
(Schuiling et al., 1998a, b), con®rming the earlier ex vivo
data (De Boer et al., 1996). A de®ciency of cNOS activity
and endogenous bronchodilating NO contributing to AHR
was also demonstrated after repeated allergen challenge of
sensitized guinea-pigs (Mehta et al., 1997; Samb et al.,
2001). Very interestingly, evidence for a de®ciency of cNOS-
derived NO modulating airway reactivity has also been
found in patients with severe asthma treated with
corticosteroids, which suppresses the expression of iNOS
(Ricciardolo et al., 1997). Thus, while the airway reactivity
to bradykinin (and to methacholine) could be signi®cantly
increased by inhalation of NG-monomethyl-L-arginine (L-
NMMA) in patients with mild asthma (Ricciardolo et al.,
1996), a similarly enhanced AHR to bradykinin present in
severe asthmatics was insensitive to the NOS inhibitor,
enhanced in patients with chronic bronchitis (Kochansky et
al., 1980).
Although speculative, the implications of increased argi-
nase activity in the asthmatic lung could be more widespread.
Thus, upregulation of arginase, emerging from Th2 cytokines
and other mediators released during allergic airway in¯am-
mation, could possibly also limit the synthesis of iNOS-
derived-NO known to be present in chronic asthma and to be
induced during the LAR (Kharitonov et al., 1995; Schuiling
et al., 1998a), and therefore protect the airways against
exaggerated NO production and tissue injury. In addition, as
a consequence of increased synthesis of arginase-induced
ornithine as a precursor for polyamines and proline, arginase
could be involved in the rapid repair process of epithelial
damage as observed after allergen challenge in a guinea-pig
model of acute asthma (Erjefalt et al., 1997) and in
in¯ammation-induced airway remodelling in chronic asthma
(Bousquet et al., 2000), by promoting proliferation of
structural subepithelial cells (®broblasts and airway smooth
muscle cells) and collagen deposition. However, the role of
arginase in asthmatic airway in¯ammation and remodelling
remains to be established.
The functional inhibition of AHR to methacholine by nor-
NOHA in the challenged preparations appeared to be maximal
in the presence of 5 mM nor-NOHA, since no additional e€ect
was obtained with 10 mM of the arginase inhibitor. This
maximal inhibitory e€ect did not fully reach the reduced level
of responsiveness observed in the control preparations in the
presence of nor-NOHA, indicating that additional mechan-
isms may be involved in the development of the allergen-
induced NO de®ciency and AHR. One of these mechanisms
could be limitation of L-arginine by reduced cellular uptake of
this amino acid, induced by inhibition of speci®c cationic
amino acid transporters by polycations such as eosinophil-
derived major basic protein (Hammermann et al., 1999; Meurs
et al., 1999). As in humans, eosinophilic polycations have been
demonstrated in the in¯amed guinea-pig airways at 6 h after
allergen challenge (Santing et al., 1994a).
In conclusion, using a guinea-pig model of allergic asthma
we have established that enhanced arginase activity may be
importantly involved in the allergen-induced NO de®ciency
and AHR after the EAR, presumbly by competition with
cNOS for the common substrate, L-arginine. Since reduced
cNOS-derived bronchodilating NO has also been recognized to
contribute to AHR in asthmatics patients, the advent of new
and selective arginase inhibitors could have therapeutic
potential in allergic asthma. However, since enhanced bioavail-
ability of L-arginine may also amplify the in¯ammatory e€ects
of iNOS-derived NO present in most patients (Sapienza et al.,
1998), their usefulness remains to be established.
indicating that
a de®ciency of NO contributed to the
enhanced airway responsiveness in these patients. A recent
study by the same authors suggested that this de®ciency may
indeed be induced by allergen challenge (Ricciardolo et al.,
2001). Accordingly, from the assessment of physical
di€usion parameters of NO by measuring the relationship
between exhaled NO and expiratory ¯ow, a recent study by
Silko€ et al. (2000) suggested that residual airway obstruc-
tion and AHR in asthmatic patients after steroid therapy are
associated with an apparent decrease in activity of cNOS-
derived NO.
Our data correspond with a previous observation by
Kochansky et al. (1980), who found two decades ago that
arginase activity is enhanced in expectorated sputum from
asthmatic patients. The underlying mechanism as well as its
functional relevance have thus far not been investigated, but
could be related to the allergen-induced increase in arginase
activity and AHR found in the present study. Interestingly, in
the same study, arginase activity was also shown to be
The authors wish to thank Drs Fiona Westerhof for expert
technical support and Dr Ad Nelemans for critically reading the
manuscript. We thank Dr Jean-Luc Boucher and Dr Sandrine
Vadon-Le Go€ for providing nor-NOHA and helpfull discussion.
We are indebted to Bartek Szilajtis-Nowicki for translating the
paper by Kochansky et al. (1980) from Polish. We thank the
Netherlands Asthma Foundation for ®nancial support (grant
00.23).
British Journal of Pharmacology vol 136 (3)

H. Meurs et al
Arginase, NO and airway hyperresponsiveness
397
References
ASANO, K., CHEE, C.B., GASTON, B., LILLY, C.M., GERARD, C.,
DRAZEN, J.M. STAMLER, J.S. (1994). Constitutive and
HAMID, Q., SPRINGALL, D.R., RIVEROS, M., V, CHANEZ, P.,
HOWARTH, P., REDINGTON, A., BOUSQUET, J., GODARD, P.,
HOLGATE, S. & POLAK, J.M. (1993). Induction of nitric oxide
synthase in asthma. Lancet, 342, 1510 ± 1513.
&
inducible nitric oxide synthase gene expression, regulation, and
activity in human lung epithelial cells. Proc. Natl. Acad. Sci.
U.S.A., 91, 10089 ± 10093.
HAMMERMANN, R., HIRSCHMANN, J., HEY, C., MOSSNER, J.,
BAGGIO, R., EMIG, F.A., CHRISTIANSON, D.W., ASH, D.E., CHAK-
DER, S. & RATTAN, S. (1999). Biochemical and functional pro®le
of a newly developed potent and isozyme-selective arginase
inhibitor. J. Pharmacol. Exp. Ther., 290, 1409 ± 1416.
BARNES, P.J. (1998). Nitric Oxide. In Asthma: Basic Mechanisms and
Clinical Management. ed. Barnes, P.J., Rodger, I.W. & Thomson,
N.C. pp. 369 ± 388. London: Academic Press Limited.
BARNES, P.J. & BELVISI, M.G. (1993). Nitric oxide and lung disease.
Thorax, 48, 1034 ± 1043.
FOLKERTS, G., NIJKAMP, F.P., WESSLER, I. & RACKE, K.
(1999). Cationic proteins inhibit L-arginine uptake in rat alveolar
macrophages and tracheal epithelial cells. Implications for nitric
oxide synthesis. Am. J. Respir. Cell Mol. Biol., 21, 155 ± 162.
HEY, C., BOUCHER, J.L., VADON, L.G., KETTERER, G., WESSLER, I.
& RACKE, K. (1997). Inhibition of arginase in rat and rabbit
alveolar macrophages by N omega-hydroxy-D,L-indospicine,
e€ects on L-arginine utilization by nitric oxide synthase. Br. J.
Pharmacol., 121, 395 ± 400.
BOOIJ-NOORD, H., ORIE, N.G. & DE VRIES, K. (1971). Immediate
and late bronchial obstructive reactions to inhalation of house
dust and protective e€ects of disodium cromoglycate and
prednisolone. J. Allergy Clin. Immunol., 48, 344 ± 354.
KHARITONOV, S.A., O'CONNOR, B.J., EVANS, D.J. & BARNES, P.J.
(1995). Allergen-induced late asthmatic reactions are associated
with elevation of exhaled nitric oxide. Am. J. Respir. Crit. Care
Med., 151, 1894 ± 1899.
BOUSQUET, J., JEFFERY, P.K., BUSSE, W.W., JOHNSON, M.
&
KOBZIK, L., BREDT, D.S., LOWENSTEIN, C.J., DRAZEN, J., GASTON,
B., SUGARBAKER, D. & STAMLER, J.S. (1993). Nitric oxide
VIGNOLA, A.M. (2000). Asthma. From bronchoconstriction to
airways in¯ammation and remodeling. Am. J. Respir. Crit. Care
Med., 161, 1720 ± 1745.
synthase in human and rat lung: immunocytochemical and
histochemical localization. Am. J. Respir. Cell Mol. Biol., 9,
371 ± 377.
BRADFORD, M.M. (1976). A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing the
principle of protein-dye binding. Anal. Biochem., 72, 248 ± 254.
CHUNG, K.F. & BARNES, P.J. (1999). Cytokines in asthma. Thorax,
54, 825 ± 857.
COX, J.D., KIM, N.N., TRAISH, A.M. & CHRISTIANSON, D.W. (1999).
Arginase-boronic acid complex highlights a physiological role in
erectile function. Nat. Struct. Biol., 6, 1043 ± 1047.
KOCHANSKY, L., KOSSMAN, S., ROGALA, E. & DWORNICKI, J.
(1980). Arginase activity in sputum of patients with bronchial
asthma. Pneum. Pol., 48, 329 ± 332.
KUO, H.P., LIU, S. & BARNES, P.J. (1992). The e€ect of endogenous
nitric oxide on neurogenic plasma exudation in guinea-pig
airways. Eur. J. Pharmacol., 221, 385 ± 388.
MCCLUSKIE, M., MITCHELL, J.A., YACOUB, M.H. & BELVISI, M.G.
(2001). Di€erential gene expression of arginase and NOS in a
human lung epithelial cell line. Am. J. Respir. Crit Care Med.,
163, A946.
MEHTA, S., DRAZEN, J.M. & LILLY, C.M. (1997). Endogenous nitric
oxide and allergic bronchial hyperresponsiveness in guinea pigs.
Am. J. Physiol., 273, L656 ± L662.
MEURS, H., HAMER, M.A., PETHE, S., VADON-LE GOFF, S.,
BOUCHER, J.L. & ZAAGSMA, J. (2000). Modulation of cholinergic
airway reactivity and nitric oxide production by endogenous
arginase activity. Br. J. Pharmacol., 130, 1793 ± 1798.
MEURS, H., SCHUURMAN, F.E., DUYVENDAK, M. & ZAAGSMA, J.
(1999). De®ciency of nitric oxide in polycation-induced airway
hyperreactivity. Br. J. Pharmacol., 126, 559 ± 562.
CUSTOT, J., BOUCHER, J.L., VADON, S., GUEDES, C., DIJOLS, S.,
& MANSUY, D. (1996). N-omega-hydroxy-
DELAFORGE, M.
alpha-amino acids as a new class of very strong inhibitors of
arginases. JBIC, 1, 73 ± 82.
CUSTOT, J., MOALI, C., BROLLO, M., BOUCHER, J.L., DELAFORGE,
M., MANSUY, D., TENU, J.P. & ZIMMERMANN, J.L. (1997). The
new alpha-amino acid N-omega-hydroxy-nor-L-arginine: a high
anity inhibitor of arginase well adapted to bind to its
manganese cluster. J. Am. Chem. Soc., 119, 4086 ± 4087.
DE BOER, J., DUYVENDAK, M., SCHUURMAN, F.E., POUW, F.M.,
ZAAGSMA, J. & MEURS, H. (1999). Role of L-arginine in the
de®ciency of nitric oxide and airway hyperreactivity after the
allergen-induced early asthmatic reaction in guinea-pigs. Br. J.
Pharmacol., 128, 1114 ± 1120.
MIURA, M., YAMAUCHI, H., ICHINOSE, M., OHUCHI, Y., KAGEYA-
DE BOER, J., MEURS, H., COERS, W., KOOPAL, M., BOTTONE, A.E.,
VISSER, A.C., TIMENS, W. & ZAAGSMA, J. (1996). De®ciency of
nitric oxide in allergen-induced airway hyperreactivity to
contractile agonists after the early asthmatic reaction: an ex vivo
study. Br. J. Pharmacol., 119, 1109 ± 1116.
MA, N., TOMAKI, M., ENDOH, N. & SHIRATO, K. (1997).
Impairment of neural nitric oxide-mediated relaxation after
antigen exposure in guinea pig airways in vitro. Am. J. Respir.
Crit. Care Med., 156, 217 ± 222.
MODOLELL, M., CORRALIZA, I.M., LINK, F., SOLER, G.
&
DE GOUW, H.W., GRUNBERG, K., SCHOT, R., KROES, A.C., DICK,
E.C. & STERK, P.J. (1998). Relationship between exhaled nitric
oxide and airway hyperresponsiveness following experimental
rhinovirus infection in asthmatic subjects. Eur. Respir. J., 11,
126 ± 132.
DE MONCHY, J.G., KAUFFMAN, H.F., VENGE, P., KOETER, G.H.,
JANSEN, H.M., SLUITER, H.J. & DE VRIES, K. (1985). Bronch-
oalveolar eosinophilia during allergen-induced late asthmatic
reactions. Am. Rev. Respir. Dis., 131, 373 ± 376.
DURHAM, S.R., CRADDOCK, C.F., COOKSON, W.O. & BENSON, M.K.
(1988). Increases in airway responsiveness to histamine precede
allergen-induced late asthmatic responses. J. Allergy Clin.
Immunol., 82, 764 ± 770.
ERJEFALT, J.S., KORSGREN, M., NILSSON, M.C., SUNDLER, F. &
PERSSON, C.G. (1997). Prompt epithelial damage and restitution
processes in allergen challenged guinea-pig trachea in vivo. Clin.
Exp. Allergy, 27, 1458 ± 1470.
EICHMANN, K. (1995). Reciprocal regulation of the nitric oxide
synthase/arginase balance in mouse bone marrow-derived
macrophages by TH1 and TH2 cytokines. Eur. J. Immunol., 25,
1101 ± 1104.
MUNDER, M., EICHMANN, K., MORAN, J.M., CENTENO, F., SOLER,
G. & MODOLELL, M. (1999). Th1/Th2-regulated expression of
arginase isoforms in murine macrophages and dendritic cells. J.
Immunol., 163, 3771 ± 3777.
NIJKAMP, F.P., VAN DER LINDE, H.J. & FOLKERTS, G. (1993).
Nitric oxide synthesis inhibitors induce airway hyperresponsive-
ness in the guinea pig in vivo and in vitro. Role of the epithelium.
Am. Rev. Respir. Dis., 148, 727 ± 734.
QUE, L.G., KANTROW, S.P., JENKINSON, C.P., PIANTADOSI, C.A. &
HUANG, Y.C. (1998). Induction of arginase isoforms in the lung
during hyperoxia. Am. J. Physiol, 275, L96 ± L102.
RICCIARDOLO, F.L., DI MARIA, G.U., MISTRETTA, A., SAPIENZA,
M.A. & GEPPETTI, P. (1997). Impairment of bronchoprotection
by nitric oxide in severe asthma. Lancet, 350, 1297 ± 1298.
RICCIARDOLO, F.L., GEPPETTI, P., MISTRETTA, A., NADEL, J.A.,
SAPIENZA, M.A., BELLOFIORE, S. & DI MARIA, G.U. (1996).
Randomised double-blind placebo-controlled study of the e€ect
of inhibition of nitric oxide synthesis in bradykinin-induced
asthma. Lancet, 348, 374 ± 377.
FISCHER, A., MUNDEL, P., MAYER, B., PREISSLER, U., PHILIPPIN,
B. & KUMMER, W. (1993). Nitric oxide synthase in guinea pig
lower airway innervation. Neurosci. Lett., 149, 157 ± 160.
FLAK, T.A. & GOLDMAN, W.E. (1996). Autotoxicity of nitric oxide
in airway disease. Am. J. Respir. Crit Care Med., 154, S202 ±
S206.
British Journal of Pharmacology vol 136 (3)

398
H. Meurs et al
Arginase, NO and airway hyperresponsiveness
RICCIARDOLO, F.L., TIMMERS, M.C., GEPPETTI, P., VAN SCHADE-
WIJK, A., BRAHIM, J.J., SONT, J.K., DE GOUW, H.W.F.M.,
HIEMSTRA, P.S., VAN KRIEKEN, H.J.M. & STERK, P.J. (2001).
Allergen-induced impairment of bronchoprotective nitric oxide
synthesis in asthma. J. Allergy Clin. Immunol., 108, 198 ± 204.
SAMB, A., PRETOLANI, M., DINH-XUAN, A.T., OUKSEL, H.,
CALLEBERT, J., LISDERO, C., AUBIER, M. & BOCZKOWSKI, J.
(2001). Decreased pulmonary and tracheal smooth muscle
expression and activity of type 1 nitric oxide synthase (nNOS)
after ovalbumin immunization and multiple aerosol challenge in
guinea pigs. Am. J. Respir. Crit. Care Med., 164, 149 ± 154.
SHEARER, J.D., RICHARDS, J.R., MILLS, C.D. & CALDWELL, M.D.
(1997). Di€erential regulation of macrophage arginine metabo-
lism: a proposed role in wound healing. Am. J. Physiol, 272,
E181 ± E190.
SILKOFF, P.E., SYLVESTER, J.T., ZAMEL, N. & PERMUTT, S. (2000).
Airway nitric oxide di€usion in asthma: Role in pulmonary
function and bronchial responsiveness. Am. J. Respir. Crit. Care
Med., 161, 1218 ± 1228.
TAYLOR, D.A., MCGRATH, J.L., ORR, L.M., BARNES, P.J.
&
O'CONNOR, B.J. (1998). E€ect of endogenous nitric oxide
inhibition on airway responsiveness to histamine and adeno-
sine-5'-monophosphate in asthma. Thorax, 53, 483 ± 489.
TUCKER, J.F., BRAVE, S.R., CHARALAMBOUS, L., HOBBS, A.J. &
GIBSOM, A. (1990). L-NG-nitro arginine inhibits non-adrenergic,
non-cholinergic relaxations of guinea-pig isolated tracheal
smooth muscle. Br. J. Pharmacol., 100, 663 ± 664.
SANTING, R.E., HOEKSTRA, Y., PASMAN, Y., ZAAGSMA, J.
&
MEURS, H. (1994a). The importance of eosinophil activation
for the development of allergen-induced bronchial hyperreactiv-
ity in conscious, unrestrained guinea-pigs. Clin. Exp. Allergy, 24,
1157 ± 1163.
SANTING, R.E., MEURS, H., VAN DER MARK, T.W., REMIE, R.,
OOSTEROM, W.C., BROUWER, F. & ZAAGSMA, J. (1992). A novel
method to assess airway function parameters in chronically
instrumented, unrestrained guinea-pigs. Pulm. Pharmacol., 5,
265 ± 272.
SANTING, R.E., OLYMULDER, C.G., ZAAGSMA, J. & MEURS, H.
(1994b). Relationships among allergen-induced early and late
phase airway obstructions, bronchial hyperreactivity, and
in¯ammation in conscious, unrestrained guinea pigs. J. Allergy
Clin. Immunol., 93, 1021 ± 1030.
VAN AMSTERDAM, R.G., BROUWER, F. & ZAAGSMA, J. (1989).
Analysis of the beta-adrenoceptor mediated inhibition of IgG1
and IgE dependent guinea-pig anaphylactic tracheal smooth
muscle contraction. Agents Actions, 26, 48 ± 51.
WANG, W.W., JENKINSON, C.P., GRISCAVAGE, J.M., KERN, R.M.,
ARABOLOS, N.S., BYRNS, R.E., CEDERBAUM, S.D. & IGNARRO,
L.J. (1995). Co-induction of arginase and nitric oxide synthase in
murine macrophages activated by lipopolysaccharide. Biochem.
Biophys. Res. Commun., 210, 1009 ± 1016.
WEI, L.H., JACOBS, A.T., MORRIS, S.M. & IGNARRO, L.J. (2000). IL-4
and IL-13 upregulate arginase I expression by cAMP and JAK/
STAT6 pathways in vascular smooth muscle cells. Am. J. Physiol.
Cell Physiol., 279, C248 ± C256.
SAPIENZA, M.A., KHARITONOV, S.A., HORVATH, I., CHUNG, K.F. &
BARNES, P.J. (1998). E€ect of inhaled L-arginine on exhaled nitric
oxide in normal and asthmatic subjects. Thorax, 53, 172 ± 175.
SCHUILING, M., MEURS, H., ZUIDHOF, A.B., VENEMA, N.
&
WU, G. & MORRIS, S.M. (1998). Arginine metabolism: nitric oxide
and beyond. Biochem. J., 336, 1 ± 17.
ZAAGSMA, J. (1998a). Dual action of iNOS-derived nitric oxide
in allergen-induced airway hyperreactivity in conscious, unrest-
rained guinea pigs. Am. J. Respir. Crit. Care Med., 158, 1442 ±
1449.
(Received December 3, 2001
Revised March 14, 2002
Accepted March 20, 2002)
SCHUILING, M., ZUIDHOF, A.B., BONOUVRIE, M.A., VENEMA, N.,
ZAAGSMA, J. & MEURS, H. (1998b). Role of nitric oxide in the
development and partial reversal of allergen-induced airway
hyperreactivity in conscious, unrestrained guinea-pigs. Br. J.
Pharmacol., 123, 1450 ± 1456.
British Journal of Pharmacology vol 136 (3)