Proteolytic action of duodenase is required to induce DNA synthesis
in pulmonary artery fibroblasts
A role for phosphoinositide 3-kinase
Alan D. Pemberton
1
, Tatyana S. Zamolodchikova
2
, Cheryl L. Scudamore
3
, Edwin R. Chilvers
4
,
Hugh R. P. Miller
1
and Trevor R. Walker
5
1
Department of Veterinary Studies, University of Edinburgh, Easter Bush Veterinary Centre, Roslin, Edinburgh, UK;
2
Shemyakin-
Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia;
3
Department of Veterinary Pathology,
University of Edinburgh, Easter Bush Veterinary Centre, Roslin, Edinburgh, UK;
4
Respiratory Medicine Unit, Department of
Medicine, University of Cambridge School of Clinical Medicine, Addenbrooke’s and Papworth Hospitals, Cambridge, UK;
5
Rayne Laboratory, Respiratory Medicine Unit, University of Edinburgh Medical School, Edinburgh, UK
Duodenase is a 29-kDa serine endopeptidase that displays
selective trypsin- and c hymotrypsin-like s ubstrate specificity.

(GF109203X) only partially inhibited duodenase-induced
DNA synthesis, but both wortmannin (100 n
M
)and
LY294002 (10 l
M
) inhibited this r esponse completely,
indicating a key role for PtdIns 3-kinase. Furthermore,
duodenase induced a 2.3 ± 0.1-fold increase in PtdIns
3-kinase activity in p85 immu noprecipitates, which was
sensitive t o inhibition by wortmannin. These results suggest
that duodenase can i nduce pulmonary artery fibroblast
DNA synthesis in a PtdIns 3-kinase-dependent manner via a
G-protein-coupled receptor which is activated by a proteo-
lytic m echanism.
Keywords: duodenase; fibroblasts; phosphoinositide 3-kinase;
protease-activated receptor.
Duodenase is a serine endopeptidase, originally isolated
from bovine duodenum, with a dual trypsin-like and
chymotrypsin-like primary substrate specificity, i.e. cleaving
the C-terminal to both basic and hydrophobic amino-acid
residues [1]. The closely related enzyme, sheep mast cell
proteinase-1 (sMCP-1) is 85% identical at the amino-acid
level [2] a nd, due to close similarity of the primary substrate
binding region, has a strikingly similar cleavage specificity
[3]. Duodenase was o riginally immunolocalized to epithelial
cells of Brunner’s glands within the duodenum, and was an
activator of enteropeptidase [4]. Oth er studies employing
esterase staining have provided evidence for the expression
of an enzyme with trypsin-like properties, distinct from

December 20 01)
Eur. J. Biochem. 269, 1171–1180 (2002) Ó FEBS 2002
proteolytic cleavage of the N-terminal exodomain. This
thrombin receptor has since been termed PAR1 (protease-
activated receptor-1) and is known to mediate the actions
of thrombin on platelets and other cell types [10]. Subse-
quently, RT-PCR and Northern analysis have identified
mRNA for three additional members of this receptors
family termed PAR2, PAR3 and PAR4 [11]. Interestingly,
thrombin has now been demonstrated to cleave and activate
PAR1, PAR3 and PAR4 whereas trypsin a nd tryptase
activate PAR2 [12]. Certain other proteases, including
chymotrypsin and cathepsin G, appear to ÔdisarmÕ PAR1 by
cleaving the exodomain of the receptor without inducing
activation and t hus preventing activation by thrombin [13].
All four receptors have a classical heptahelical structure
within the plasma membrane and are known to couple to
both G
q/11
and G
i/o
and stimulate phosphoinositide turn-
over although their other potential downstream signalling
targets h ave not been fully established [ 12]. In this study we
have investigated the ability of duodenase to induce DNA
synthesis in bovine pulmonary artery fibroblasts, attempting
to elucidate which PAR subtype and signalling pathways
may be involved in mediating this effect. We also provide
evidence for an a dditional mast cell o rigin of duodenase,
which has important implications with regard to the

then rechromatographed twice on a M ono-S c olumn
(Pharmacia) using 0.05–0.35
M
NaCl gradients in 20 m
M
Tris/HCl (pH 7.5), 0.1% (v/v) Brij 35, and then 20 m
M
sodium phosphate (pH 7.0), 0.1% (v/v) Brij 35. The final
purification step involved gel fi ltration (Superdex 75, Phar-
macia) in NaCl/P
i
(pH 7.4) containing 0.1% (v/v) Brij 35.
The identity of the product was confirmed by N-terminal
amino-acid sequence analysis (P. Barker, Babraham Insti-
tute, Cambridge, UK), and by comparing its ability to
hydrolyse specific peptide substrates (in 0.1
M
Tris/HCl,
pH 8.0) with duodenase.
Immunohistochemical localization of duodenase
in jejunum and lung
Samples of fresh bovine jejunum were fixed in 10% (v/v)
formalin and 4% (w/v) paraformaldehyde, and processed
into paraffin blocks. Sections (4 lm thick) were stained
using 0 .1% (w/v) toluidine blue (pH 0.5), followed by eosin
counterstain, and duodenase detected using rabbit anti-
duodenase serum (1 : 400), rabbit anti-(sMCP-1) IgG
(1.2 lg ÆmL
)1
) or control rabbit serum (1 : 400) [14], using

5 lgÆmL
)1
, respectively) and amphotericin B ( 2.5 lgÆmL
)1
).
Cells from passages 3–10 were used for all experiments.
Cells were incubated i n serum-free DMEM for 48 h prior to
experimentation.
Assessment of [
3
H]thymidine incorporation
Pulmonary artery fi broblasts at % 80% confluence were
quiesced for 48 h prior to addition of mitogens as indicated.
The cells were then incubated for an additional 20 h, with
[
3
H]thymidine (0.1 lCiÆmL
)1
) added 4 h prior to harvest-
ing. Cells were washed twice with ice-cold NaCl/P
i
, twice
with trichloroacetic acid (5% w/v), twice with ethanol and
finally were solubilized with NaOH (0.3
M
). [
3
H]Thymidine
incorporation was determined by liquid scintillation
counting.

M
NaCl, 3 m
M
MgCl
2
,10l
M
1172 A. D. Pemberton et al. (Eur. J. Biochem. 269) Ó FEBS 2002
GDP with 0.2 n
M
[
35
S]GTPcS and incubating for 60 min at
4 °C. Bound radioactivity was determined by filtration of
membranes onto Whatman GF-B filters using a Brandell
Cell Harvester and counted by scintillation counting.
Nonspecific binding was determined in the presence of
100 l
M
unlabelled GTPcS.
Assay of immunoprecipitated PtdIns 3-kinase
Bovine pulmonary artery fibroblasts were exposed to
mitogens as detailed in the figure legends, and the
reactions were terminated by rapid aspiration of the
media followed by the addition of ice-cold lysis buffer
(50 m
M
Hepes, pH 7.5, 150 m
M
NaCl, 10% v/v glycerol,

phosphatidylserine (3 : 1, v/v, 0.2 mgÆmL
)1
) vesicles and
[c-
32
P]ATP (10 lCiÆpoint
)1
) as substrates [16].
32
P-Labelled phosphoinositide 3-phosphate was then
separated a nd quantified by thin layer chromatography
using a solvent system containing chloroform/methanol/
ammonia/water (20 : 15 : 3 : 5, v/v/v/v) and autoradiog-
raphy;
32
P incorporation w as determined by liquid
scintillation counting.
Ca
2+
measurements using Fura-2
Bovine pulmonary artery fibroblasts (P4-10) w ere g rown to
confluence in supplemented DMEM as d escribed above,
washed with NaCl/P
i
, and gently harvested into a solution
containing BSA (0.2% w /v), glucose ( 0.1% w/v) and CaCl
2
(1 m
M
)inNaCl/P

% 30 min and 2 mL aliquots of cells then used for Ca
2+
measurements over the following 2–3 h. M easurements
were made in 1 · 1 cm quartz cuvettes, equipped with a
magnetic stirrer, using a PerkinElmer LS 50B fluorimeter
with fast-filter accessory. This allowed measurement of
emission at 510 nm for quasi-simultaneous excitation at
340 and 380 nm, for Fura2 bound and unbound to Ca
2+
,
respectively. Additions of agonists (trypsin, thrombin,
duodenase, chymotrypsin and bradykinin) were made in
small volumes (5–20 lL). At the end of each experiment,
the maximum fluorescence was obtained b y disrupting the
cells by addition of 10% (v/v) Triton X-100 (40 lL), and
minimum fluorescence then determined using 20 lLof
0.4
M
EGTA in 3
M
Tris base. Results were analysed, and
conversions to intracellular Ca
2+
concentration p erformed,
using
FL WINLAB
software (PerkinElmer).
In vitro
comparison of PAR2 peptide cleavage
by duodenase, tryptase and trypsin

rat PAR2(5–20) (0.475 mgÆmL
)1
), alanyl-tryptophan (in-
ternal standard, 0.05 mgÆmL
)1
) and 0.13 U of enzyme
(total assay volume 200 lL). Samples (30 lL) were
removed at varying time-points, and reactions terminated
by the addition of 30 lL 10% acetic acid. These samples
were then chilled on ice , and frozen ( )20 °C) prior to
analysis. Intact PAR2(5–20) and internal standard peak
heights were quantified in samples following RP-HPLC
(Jupiter C5 column, Phenomenex) using a water/acetonit-
rile gradient containing 0.1% trifluoroacetic acid. The ratio
of intact PAR2(5–20) to internal standard peak heights
was plotted against time. Fractions collected from some
runs were subjected to mass spectrometry (I. Davidson,
University of Aberdeen, Scotland, UK).
Materials
Anti-duodenase serum and affinity-purified anti-(sMCP-1)
IgG were prepared as described previously [4,8]. Anti-(p85
PtdIns 3-kinase) Ig was obtained from TCS Biologicals
(Botolph Claydon, UK) and [c-
32
P]ATP from Amersham
(Amersham). PAR activating peptides, Ser-Phe-Leu-Leu-
Arg-Asn for PAR1 and Gly-Tyr-Pro-Gly-Lys-Phe for
PAR4 were obtained from Bachem Ltd (Saffron Walden,
Essex, UK) and Ser-Leu-Ile-Gly-Arg-Leu and Ser-Leu-Ile-
Gly-Arg-Leu-NH

Immunolocalization of duodenase
Toluidine blue staining identified abundant spindle or
stellate-shaped m ast cells in bovine jejunum samples. The se
cells were l ocate d principally in the lamina propria (Fig. 1a)
and submucosa (not shown). Immunostaining o f paraform-
aldehyde-fixed sections with rabbit anti-duodenase serum
and affin ity-purified rabbit anti-(sMCP-1) IgG detected cells
only in the lamina propria. These strongly staining cells
showed a similar distribution and morphology to those seen
with toluidine blue within the lamina propria (compare
Fig. 1a with Fig. 1b,d,e). The distribution of positive cells
after labelling with anti-duodenase Ig or anti-(sMCP-1) IgG
was very similar, and in neither instance was there any
labelling of submucosal tissues. Occasional intraepithelial
cells were weakly labelled (Fig. 1d), and the identity of these
toluidine blue negative cells was not confirmed. Tissues fixed
in neutral buffered formalin showed negligible mast cell
staining by comparison, and control rabbit serum was
negative regardless of the fixation procedure (Fig. 1c).
Lungworm-infected lung parenchyma showed the presence
of large numbers of eosinophils and toluidine blue-positive
mast c ells. An example of their distribution around a
bronchiole is shown in Fig. 1h, in which fibrosis and smooth
muscle hyperplasia was also evident. Numerous cells were
also lab elled with duodenase antiserum around bronchioles
(Fig. 1f) and within the alveolar septa (not shown). Their
size and distribution as observed in adjacent sections was
similar to that of toluidine blue-positive cells (compare
Fig. 1f,h). Control r abbit serum gave no labelling (Fig. 1g).
Duodenase induces DNA synthesis in pulmonary artery

[
3
H]thymidine incorporation to a similar extent as addition
of duodenase alone (Fig. 2B), suggesting a rapid signalling
mechanism. Furthermore, conditioned media generated by
this method was used to assess whether duodenase could
cleave and release a cell surface molecule that could interact
Fig. 2. Duodenase induces DNA synthesis in pulmonary artery fibro-
blasts. (A) quiescent cells were treated with duodenase (3–100 n
M
)as
indicated f or 20 h prior to ad dition of [
3
H]thymidine (0.1 lCiÆwell
)1
):
incorporation was assessed after 4 h as detailed in Materials and
methods. (B) [
3
H]Thymidine incorporation tested in c ells treated with
duodenase ( duod, 3 0 n
M
)whichhadbeenpretreatedwithorwithout
soybean trypsin inhibitor (+ STI, 0.2 mgÆmL
)1
) for 15 min. To
examine a role for duo denase-induced release of a mitogenic f actor and
generation of con ditioned media, duodenase was a dded to cells for
10 min prior to addition of soybean trypsin inhibitor for 15 min,
media removed and r eplaced w ith f resh quiescent media (duod + STI

blasts had no significant effect on [
3
H]thymidine in corpor-
ation above control levels (Fig. 2B). Hence duodenase,
purified from bovine jejenum is mitogenic for bovine
pulmonary artery fibroblasts and this effect is dependent
on the direct proteolytic activity of th is enzyme.
As the PARs d escribed to date are activated by cleavage
of trypsin-like primary specificity, and as duodenase, (like
sMCP-1, which is also mitogenic in this system [8]), has a
trypsin-like component, a ctivating peptides selective for
PAR1, PAR2 and PAR4 were used to investigate whether
the mitogenic effect of duodenase was mediated v ia a
known PAR mechanism. Surprisingly, all PAR peptides
were unable to induce [
3
H]thymidine incorporation in
pulmonary artery fibroblasts (Fig. 2C). It should be noted
that two forms of the PAR2 activating peptide were
assessed, the f ree form a nd the a mido form, neither of which
showed ability to induce DNA synthesis ( Fig. 2C). Lack of
activation by these peptides is unlikely to be a c onsequence
of species differences in receptor sequences as Ser-Leu-Ile-
Gly-Arg-Leu (PAR2 activating peptide, mouse-derived
sequence, 100 l
M
) was reported to mobilize Ca
2+
in bovine
coronary artery smooth muscle cells [18]. The PAR1

% 1200 min). Moreover, the
cleavage mixture exhibited HPLC peaks corresponding
both to the activation product (Ser-Leu-Ile-Gly-Arg-Leu-
Asp-Thr-Pro) a nd to other unidentified products, suggesting
multiple sites of cleavage of this substrate.
Together, these results suppo rt the hypothesis that
duodenase acts independently of the known trypsin/
tryptase-sensitive PAR2 receptor.
Duodenase induces GTPcS binding in pulmonary artery
fibroblast membranes
To establish the mechanism of action of duodenase,
[
35
S]GTPcS binding to fibroblast membranes was used as
an index of G protein activation. Duodenase (30 n
M
)
induced a 57.0 ± 2.3% increase in guanine nucleotide
binding to pulmonary artery fibroblast cell membranes
compared to controls, suggesting that the effects of
duodenase are indeed mediated through a G-protein-
coupled rec eptor. Pre-treatment of cells with pertussis toxin
(100 ngÆmL
)1
, 18 h) prior to cell fractionation and
membrane isolation inhibited [
35
S]GTPcS binding by
80.8 ± 10.3%, suggesting that the predominant G-protein
mediating this signal is a member o f the G

transient indicating that these cells were responsive t o
activation through other G-protein-coupled receptors
(Fig. 4). As anticipated, this response to bradykinin could
be desensitized by prior exposure to the agonist (Fig. 4).
These results suggest that this group of proteases do not
appear to cause acute Ca
2+
mobilization or influx in these
cells. Of note, addition of a PAR2-activating peptide or
addition of thrombin, which will act t hrough PAR1, PAR3
and P AR4, all h ad no effect on Ca
2+
mobilization (Fig. 4).
These results demonstrate that Ca
2+
mobilization is
unlikely to be involved in mediating cell growth in
pulmonary artery fibroblasts.
Wortmannin (100 n
M
) and LY294002 (10 l
M
), two
structurally distinct and selective inhibitors of PtdIns
3-kinase, completely blocked duodenase-induced
[
3
H]thymidine incorporation, suggesting a key role for
PtdIns 3-kinase in this response (Fig. 5). In contrast,
PD98059, a MEK1 inhibitor, caused only a partial

i
and G
o
resulting in blockade of G protein
activation, inh ibited duodenase-induced [
3
H]thymidine
incorporation by 52 ± 2.5%, suggesting involvement of
G
i
/G
o
in mediating this component of cell growth
(Fig. 5). In subsequent experiments, duodenase (30 n
M
)
was found to activate p85a-associated PtdIns 3-kinase in
pulmonary artery fibroblasts by 2.28 ± 0.14- fold above
control values, and pretreatment of these cells with
wortmannin (100 n
M
, 20 min) inhibited this activity to
below basal levels (Fig. 6). In combination with the major
inhibitory effects of wortmannin and LY294002 on
duodenase-stimulated [
3
H]thymidine incorporation, these
results indicate a key role for a G-protein-coupled
receptor/PtdIns 3-kinase p athway in mediating duoden-
ase-stimulated DNA synthesis.

H]Thymidine incorporation was assessed as indicated in Methods,
results are expressed as percentage mean ± S EM relative to untreated
cells st imulated with duoden ase. Results are from four independent
experiments e ach performed in triplicate.
Fig. 6. Duodenase a ctivates PtdIn s 3-kinase i n pulmonary artery fibro-
blasts. Pulmonary artery fibroblasts were incubated in the presence
(hatched bars) or absence (open bars) of wortmannin (100 n
M
)for
20 min prior to additio n of du odenase (30 n
M
, duod). Reactions
were terminated and PtdIns 3-kinase activity was assayed in p85a
immunoprecipitates as detailed in Materials and methods. Results are
expressed as mean c .p.m. ± SEM from a single experiment performed
in quadruplicate, representative of two others with s imilar results.
Fig. 4. Effect of duodenase on Ca
2+
mobilization. Pulmonary artery
fibroblasts preloaded with Fura-2 were stimulated with agonists as
indicated. Intracellular Ca
2+
was analysed and plotted over time as
indicated. Traces are representative of three separate experiments
which a ll gave ve ry similar results.
Ó FEBS 2002 Duodenase induces DNA synthesis via PtdIns 3-kinase (Eur. J. Biochem. 269) 1177
5.23 ± 0.47-fold increase in [
3
H]thymidine incorporation
above control levels (Table 1). While pretreatment of cells

The immunolocalization of duodenase to bovine intesti-
nal mucosal mast cells described h ere would suggest that it
too belongs to the ruminant mucosal mast cell proteinase
family, w hich are notable for their dual c hymase and
tryptase-like activities. It was possible to isolate duodenase
from bovine jejunum using methodology identical to that
employed for the purification of sMCP-1 from gastrointes-
tinal tissues. However, duodenase has previously been
localized only to the epithelial cells of Brunner’s glands
located in the duodenal wall [4]. This suggests either that
duodenase is present in both cell types, or that each site
produces distinct enzymes that are nonetheless highly
similar structurally, functionally and immunologically.
Lungworm infection in sheep is known to involve a
pronounced mastocytosis [24], and sMCP-1 is upregulated
in mast c ells recruited t o s ites of allergic lung inflammation
[25]. The current observation of abundant duodenase-
positive mast cells in lungworm-infec ted bovine lung shows
the potential for local duodenase release by mast cells
recruited to i nflammatory sites i n the bovine lung and i s
consistent with a putative role in tissue modelling.
In this study, we have shown t hat the similarity between
duodenase and sMCP-1 e xtends to the stimulation of
pulmonary artery fibroblasts, with both enzymes able to
induce DNA synthesis over a similar concentration range.
As soybean t rypsin inhibitor was able to completely inhibit
the duodenase effect, this demonstrates that the catalytic
activity is essential for its action. However, only a short
exposure to duodenase is required to induce maximal DNA
synthesis suggesting a rapid activation p rofile. Conditioned

PAR3 and PAR4, whereas trypsin cleaves and activates
PAR2. As duodenase is capable of cleaving certain
substrates with trypsin-like primary specificity, we initially
hypothesized that induction of DNA synthesis by duoden-
ase is mediated through a PAR2 mechanism.
Surprisingly, we could find no evidence to support the
involvement of a classic PAR2 in mediating the mitogenic
effects of duodenase, specifically: (a) the synthetic peptide
Ser-Leu-Ile-Gly-Arg-Leu, which is specific for PAR2, was
unable to i nduce [
3
H]thymidine incorporation in fibroblasts,
and a similar lack of mimickery was evident for peptides
specific for PAR1 and PAR4; and (b) duodenase cleave d t he
model PAR2 substrate more slowly than either trypsin or
tryptase, and generated a very different array of peptides,
suggesting that duodenase may cleave PAR2 at different
sites. Activation of PAR3 by duodenase seems unlikely, as
this receptor has limited intrinsic signalling capacity [27] and
so far has only b een found to be activated by thrombin [12].
Schechter et al. [28] have described the action of mast cell
tryptase on keratinocytes, as acting through a subpopula-
tion of PAR2 receptors, suggesting the existence o f subtypes
Table 1. Effect of cytokines on duodenase-induced DNA synthesis.
Bovine p ulmonary artery fibroblasts were assessed for [
3
H]thymidine
incorporation induced by d u odenase (30 n
M
), following pretreatment

n ¼ 12, over three separate experiments.
b
n ¼ 8, over two sepa-
rate experiments. * p > 0.001.
1178 A. D. Pemberton et al. (Eur. J. Biochem. 269) Ó FEBS 2002
of this receptor. In addition, it has been demonstrated that
regulation of intestinal ion transport in rat jejenum is
mediated by a P AR that, a lthough similar in many r espects
to PAR2, showed distinct and atypical orders of potency
when a range of peptide agonists were assessed [29]. These
reports and the data from this study, in particular the
pertussis toxin-sensitivity of DNA synthesis induction and
the ability o f duodenase to stimulate [
35
S]GTPcS binding to
pulmonary artery fibroblast membranes, would suggest that
the m itogenic action of duodenase is mediated via direct
interaction with a proteolytically activated G
i/o
-coupled
receptor. While the precise PAR subtype remains to be fully
identified, it may be an atypical P AR2 that is not activated
by existing classic PAR2 peptides. To date, no bo vine PAR
sequences have been published and analysis o f cleavage sites
on these receptors may reveal species-specific activation
motifs that are distinct from those in mouse, rat and
humans an d explain the lack of efficacy of current PAR2-
activating peptides in our model system.
A number of s ignalling pathways and intermediates such
as Ca

canine tracheal smooth muscle through an ERK1/2-depen-
dent mechanism, proliferation being inhibited completely by
PD98059 [31]. Moreover, in pulmonary artery fibroblasts,
inhibition of PtdIns 3-kinase by wortmannin or LY294002
inhibited completely duodenase-induced [
3
H]thymidine in-
corporation. This would suggest that activation of P tdIns 3-
kinase is the key regulatory step in the proliferative p athway
and that each of the other pathways interacts with this
pathway with the magnitude of the cellular response
determined by the integrated sum of each of these
components. Our data is supported by previous reports
demonstrating t hat thrombin a cts i n a PtdIns 3-kinase- a nd
p70
s6k
-dependent manner to induce DNA synthesis in
pulmonary artery fibroblasts [32]. In addition, this report
noted that downregulation of protein kinase C partially
attenuated thrombin-induced p70
s6k
activation, which
would concur with our findings that inhibition of protein
kinase C results in partial inhibition of DNA synthesis.
To date, identification of downstream signalling path-
ways for PARs have principally concentrated on PAR1 and
PAR2. P AR1 c ouples to members of the G
12/13
,G
q

cytokines cause the fibroblasts either to become refractory
to mitogens or to enter into S-phase more slowly over the
time period examined. It remains to be established whether
chronic exposure to TNFa and IL-1b would result in a
sensitization of these cells to mitogenic stimuli. These
results support further our hypothesis that duodenase is
not acting via a classical PAR2.
In summary, this study has demonstrated that duoden-
ase induces DNA synthesis in pulmonary artery fibro-
blasts and that this response may be mediated by an
atypical PAR, either an i soform of PAR2 or an uniden-
tified receptor. It is important to recognize that the
current study was undertaken in a fully homologous
system, using a bovine serine protease a nd bovine
pulmonary fibroblasts. This would indicate that the
proteolytic event and subsequent downstream signalling
and functional responses we have described m ay be an
important consequence o f duodenase release from Brun-
ner’s glands, or of mast cell activation in vivo. Indeed,
mast cell hyperplasia is known to be a prominent event in
many forms of chronic inflammation in the lung such as
cryptogenic fibrosing alveolitis, and fibroblast proliferation
is the most significant feature in the pathology of these
clinical conditions [9]. The precise nature and character-
ization of the receptor that mediates the effects of
duodenase requires further investigation.
ACKNOWLEDGEMENTS
This work was funded by the Norman Salvesen Emphysema Research
Trust, the Wellcome Trust, and the National Asthma Campaign (UK).
We thank Dr Joh n Huntley and Ms Anne Mackellar for providing the

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