Open Journal of Gastroenterology, 2011, 1, 13-22
doi:10.4236/ojgas.2011.12003 Published Online November 2011 (http://www.SciRP.org/journal/ojgas/ OJGas
).
Published Online November 2011 in SciRes. http://www.scirp.org/journal/OJGas
Role of constitutive nitric oxide synthase in regulation of
Helicobacter pylori-induced gastric mucosal cyclooxygenase-2
activation through S-nitrosylation: mechanism
of ghrelin action
Bronislaw L. Slomiany, Amalia Slomiany
University of Medicine and Dentistry of New Jersey, Newark, USA.
Email: slomiabr@umdnj.edu
Received 18 July 2011; revised 21 September 2011; accepted 3 October 2011.
ABSTRACT
Gastric mucosal inflammatory responses to H. pylori
lipopolysaccharide (LPS), are characterized by the
excessive NO and prostaglandin (PGE2) generation
due to the disturbances in nitric oxide synthase (NOS)
and cyclooxygenase (COX) systems. Here, we report
that the LPS-induced enhancement in gastric muco-
sal inducible (i) iNOS) activity and up-regulation in
PGE2 production was associated with the suppression
in Akt kinase activity and the impairment in consti-
tutive (c) cNOS activation. The stimulatory effect of
the LPS on PGE2 production, furthermore, was sus-
ceptible to suppression by COX-2 inhibitor, NS-398,
and iNOS inhibitor, 1400 W. Further, we show that
the countering effect of peptide hormone, ghrelin, on
the LPS-induced changes was reflected in up-regu-
lation in Akt activity and the increase in cNOS acti-
vation through phosphorylation, and accompanied by
the suppression in iNOS expression and the reduction
in COX-2 activity associated with the loss in COX-2
protein S-nitrosylation. Moreover, the effect of ghre-
lin on the LPS-induced COX-2 S-nitrosylation was
subject to repression by Akt inhibition. Our findings
demonstrate that induction in iNOS with H. pylori in-
fection leads to COX-2 activation through S-nitro-
sylation and up-regulation in PGE2 generation, and
that ghrelin counters these untoward consequences of
the LPS through Akt-mediated up-regulation in cNO-
S activation required for the iNOS gene repression.
Keywords: H. pylori; Gastric Mucosa; iNOS Induction;
COX-2 Activation; S-Nitrosylation; Ghrelin
1. INTRODUCTION
Helicobacter pylori, a Gram-negative bacterium colo-
nizing the gastric mucosa, is recognized as a primary
cause of gastric disease, and its cell-wall lipopolysac-
charide (LPS) has been identified as a potent endotoxin
capable of eliciting mucosal inflammatory changes that
characterize gastritis and duodenal ulcer [1-3]. Indeed,
the gastric mucosal inflammatory responses to H. pylori
infection in humans as well as well as those characteriz-
ing mucosal inflammatory changes in the animal model
of H. pylori LPS-induced gastritis are manifested by a
marked increase in epithelial cell apoptosis and proin-
flammatory cytokine production, and the excessive nitric
oxide (NO) and prostaglandin (PGE2) generation [4-7].
A growing body of evidence, moreover, points to the
disturbances in NO generated by nitric oxide synthase
(NOS) isozyme system, and the formation of PGE2 syn-
thesized from arachidonic acid (AA) by the action of
cyclooxygenase (COX) system, as the events defining
the extent of gastric mucosal inflammatory involvement
in response to H. pylori colonization [8-12].
The physiological and pathophysiological implica-
tions of NO and PGE2 depend on the type of isozyme
system involved in their generation, their subcellular
targeting and the local concentrations [12-16]. Of the
three NOS isozymes responsible for NO generation, the
two expressed constitutively (cNOS) are calcium/calmo-
dulindependent and provide precise pulses of NO for a
fine modulation of the cellular processes [14,16]. The
third, inducible isoform (iNOS) is calcium/calmodu-
lin-independent, and its activation by proinflammatory
cytokines and bacterial LPS provides the high output of
NO that is importance to host defense against microbial
invasion. However, its massive and sustained production
is also associated with transcriptional disturbances and
the induction of apoptosis [14-18]. The cyclooxygenase
system consists of two COX isozymes, the constitutive
isoform or COX-1, responsible for maintaining normal
B. L. Slomiany et al. / Open Journal of Gastroenterology 1 (2011) 13-22
14
physiological prostaglandin production, and the induc-
ible form or COX-2, which accounts for up-regulation in
PGE2 production in response to inflammatory stimulus
[8,9,12]. Moreover, a large volume of data supports the
existence of functional and signaling cross-talk between
the products of NOS and COX systems [13,14,19-22].
Indeed, the stimulation of NO production through
iNOS induction or the exogenous NO donors leads to
COX enzymes activation and the increase in PGE2 gen-
eration, while a decrease in PGE2 formation has been
observed in the presence of pharmacological inhibitors
of NOS system [20-23]. Furthermore, the COX-2 activa-
tion for the increase in PGE2 synthesis has been linked to
the enzyme protein S-nitrosylation via NO derived
through LPS-elicited induction in iNOS [20]. There are
also indications for the role of cNOS in the iNOS-de-
pendent COX-2 activation [21,24]. Interestingly, the
disturbances in NO and PGE2 production associated with
H. pylori colonization are reflected in the massive up-
regulation in iNOS activity and the suppression of cNOS
activation [4,5,25,26]. Moreover, we have recently sh-
own that Akt-mediated cNOS activation through phos-
phorylation at Ser1179 plays an essential role in the mod-
ulatory influence of peptide hormone, ghrelin, on the
extent of gastric mucosal inflammatory responses to H.
pylori LPS [26].
As gastric ghrelin is recognized as an important regu-
lator of NOS and COX enzyme systems, and implicated
in the control of local inflammations, gastroprotection,
and modulation of the mucosal inflammatory responses
to bacterial infection [18,27-31], in this study we inves-
tigated the nature of inflammatory changes induced in
gastric mucosal cells by H. pylori LPS and the mecha-
nism of ghrelin modulatory influence on the cross-talk
between the NOS and COX systems. Our data revealed
that the LPS-elicited induction in iNOS leads to COX-2
activation through S-nitrosylation and up-regulation in
PGE2 generation, and that ghrelin counters these unto-
ward consequences of the LPS through up-regulation in
cNOS phosphorylation and the suppression of iNOS
gene induction.
2. MATERIALS AND METHODS
2.1. Gastric Mucosal Cell Incubation
The mucosal cells, collected by scraping the mucosa of
freshly dissected rat stomachs with a blunt spatula, were
suspended in five volumes of ice-cold Dulbecco’s modi-
fied (Gibco) Eagle’s minimal essential medium (DM-
EM), supplemented with fungizone (50 µg/ml), penicil-
lin (50 U/ml), streptomycin (50 µg/ml), and 10% fetal
calf serum, and gently dispersed by trituration with a
syringe, and settled by centrifugation [5]. Following
rinsing, the cells were resuspended in the medium to a
concentration of 2 × 107 cell/ml, transferred in 1 ml ali-
quots to DMEM in culture dishes and incubated under
95% O2 - 5% CO2 atmosphere at 37˚C for 16 h in the
presence of 0 ng/ml - 200 ng/ml of H. pylori LPS [18].
In the experiments evaluating the effect of ghrelin (rat,
Sigma), cNOS inhibitor, L-NAME, iNOS inhibitor, 1400
W, Akt inhibitor, SH-5 (Calbiochem), COX-1 inhibitor,
SC-560, COX-2 inhibitor, NS-398 and ascorbate (Sig-
ma), the cells were first preincubated for 30 min with the
indicated dose of the agent or vehicle before the addition
of the LPS. The viability of cell preparations before and
during the experimentation, assessed by Trypan blue dye
exclusion assay [11], was greater than 97%.
2.2. Helicobacter pylori Lipopolysaccharide
H. pylori used for LPS preparation was cultured from
clinical isolates obtained from ATCC No. 4350 [2,5].
The bacterium was homogenized with liquid phenol-
chloroform-petroleum ether, centrifuged, and the LPS
contained in the sup ernatant was precipitated with water,
washed with 80% phenol solution and dried with ether.
The dry residue was dissolved in a small volume of wa-
ter at 45˚C, centrifuged at 100,000 × g for 4 h, and the
resulting LPS sediment subjected to lyophilization.
2.3. PGE2 and NO Quantification
The aliquots of cell suspension from the control and
various experimental conditions were centrifuged at
1500 × g for 5 min and the conditioned medium super-
natant collected. PGE2 assays were carried out using an
enzyme-linked immunoassay (Cayman) and 100 µl
aliquots of the spent medium supernatant, according to
the manufacturer’s instructions. The amount of PGE2
released into culture medium was determined by meas-
uring the absorbance at 405 nm [30]. To assess NO
production in gastric mucosal cells, we measured the
stable NO metabolite, nitrite, accumulation in the cul-
ture medium using Griess reaction [32]. A 100 µl of
spent culture medium was incubated for 10 min at
room temperature with 0.1 ml of Griess reagent and the
absorbance was measured at 570 nm.
2.4. COX-2 Activity Assay
For measurements of COX-2 activity, the gastric mu-
cosal cells from the control and various experimental
conditions were settled by centrifugation, rinsed with
phosphate-buffered saline, and homogenized in 0.3 ml
cold sample buffer containing 0.1 M Tris-HCl, pH 7.8,
and 1 mM EDTA, centrifuged at 12,000 × g for 10 min
at 4˚C, and the supernatant collected. The COX-2 ac-
tivity in 40 µl aliquots of the resulting supernatant was
measured using COX activity assay kit in the absence
and the presence of COX-1 inhibition (SC-560), ac-
cording to the manufacturer’s (Cayman) instruction.
The absorbance was read at 590 nm.
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2.5. cNOS and iNOS Activity Assay
Nitric oxide synthase activities of cNOS and iNOS
enzymes in the gastric mucosa l cells were measured by
monitoring the conversion of L-[3H] arginine to L-[3H]
citrulline using NOS-detect kit (Stratagene). The cells
from the control and experimental treatments were
homogenized in a sample buffer containing either 10
mM EDTA (for Ca2+-independent iNOS) or 6 mM
CaCl2 (for Ca2+-depenedent cNOS), and centrifuged.
The aliquots of the resulting supernatant were incu-
bated for 30 min at 25˚C in the presence of 50 µCi/ml
of L-[3H] arginine, 10 mM NAPDH, 5 µM tetrahydro-
biopterin, and 50 mM Tis-HCl buffer, pH 7.4, in a final
volume of 250 µl. Following addition of stop buffer
and Dowex-50 W (Na+) resin, the mixtures were tran-
sferred to spin cups, centrifuged and the formed L-[3H]
citrulline contained in the flow through was quantified
by scintillation counting.
2.6. Akt Activity Assay
The kinase activity of Akt in gastric mucosal cells was
measured with the Akt Activity Kit (Calbiochem) by
quantifying phosphorylation of a biotinylated peptide
substrate (GRPRTSSFAEG). The cells were lysed in
lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl,
10% glycerol, 1% Triton X-100, 1% deoxycholate, 2
mM EDTA, 1 mM sodium orthovanadate, 1 mM PAF,
and 1 mM NaF), containing protease inhibitor cocktail
(Sigma), at 4˚C for 30 min, centrifuged at 14,000 × g for
15 min, and immunoprecipitated with anti-Akt antibody
for 1 h at 4˚C. Protein A/G agarose beads were then
added for an additional 1 h, and the immune complex
was recovered by centrifugation and thoroughly washed
with lysis buffer [26]. The agarose beads were then sus-
pended for 30 min at room temperature in the kinase
assay buffer, centrifuged, and the supernatants used for
the Akt activity assay by following the manufacturer’s
instruction.
2.7. COX-2 Protein S-Nitrosylation Assay
A biotin switch procedure was employed to assess
COX-2 protein S-nitrosylation [33,34]. The gastric mu-
cosal cells were treated with iNOS inhibitor, 1400 W (40
µM) or ghrelin (0.5 µg/ml), or Akt inhibitor, SH-5 (30
µM) + ghrelin (0.5 µg/ml), and incubated for 16 h in the
presence of 100 ng/ml of H. pylori LPS. Following cen-
trifugation at 500 × g for 5 min, the recovered cells were
lysed in 0.2 ml of HEN lysis buffer (250 mM HEPES, 1
mM EDTA, 0.1 mM neocuprin, pH 7.7), and the unni-
trosylated thiol groups were blocked with S-methyl me-
thanethiosulfonate reagent at 50˚C for 20 min [34]. The
proteins were precipitated with acetone, resuspended in
0.2 ml of HEN buffer containing 1% SDS, and subjected
to targeted nitrothiol group reduction with sodium as-
corbate (100 mM). The free thiols were then labeled
with biotin and the biotinylated proteins were recovered
on streptavidin beads. The formed streptavidin bead-
protein complex was washed with neutralization buffer,
and the bound proteins were dissociated from strepta-
vidin beads with 50 µl of elution buffer (20 mM HEPES,
100 mM NaCl, 1 mM EDTA, pH 7.7) containing 1%
2-mercaptoethanol [34]. The obtained proteins were then
analyzed by Western blotting.
2.8. Immunoblotting Analysis
The mucosal cells from the control and experimental
treatments were collected by centrifugation and resus-
pended for 30 min in ice-cold lysis buffer (20 mM
Tris-HCl, pH 7.4, 150 mM NaCl, 10 % glycerol, 1% Tri-
ton X-100, 2 mM EDTA, 1 mM sodium orthovanadate, 4
mM sodium pyrophosphate, 1 mM PMSF, and 1 mM
NaF), containing 1 µg/ml leupeptin and 1 µg/ml pep-
statin [18]. Following brief sonication, the lysates were
centrifuged at 12,000 g for 10 min, and the supernatants
were normalized with respect to protein concentration
using BCA protein assay kit (Pierce). The samples, in-
cluding those subjected to biotin switch procedure, were
then resusp ended in load ing buffer, boiled for 5 min, and
subjected to SDS-PAGE using 40 µg protein/lane. The
separated proteins were transferred onto nitrocellulose
membranes, blocked for 1 h with 5% skim milk in
Tris-buffered Tween (20 mM Tris-HCl, pH 7.4, 150 mM
NaCl, 0.1% Tween-20), and probed with specific poly-
clonal rabbit antibodies directed against COX-1, COX-2,
and iNOS (Calbiochem). The phosphorylated cNOS
(pcNOS) was analyzed using specific antibody (Calbio-
chem) directed against phospho-cNOS (mouse anti-
eNOS, pSer11 79), and following stripping, probed with
antibody against to tal cNOS.
2.9. Data Analysis
All experiments were carried out using duplicate sam-
pling, and the results are expressed as means ± SD.
Analysis of variance (ANOVA) and nonparametric
Kruskal-Wallis tests were u sed to determine significance.
Any difference detected was evaluated by means of post
hoc Bonferroni test, and the significance level was set at
P < 0.05.
3. RESULTS
To further ascertain the nature of gastric mucosal in-
flammatory responses to H. pylori and to reveal the
modulatory role ghrelin, we investigated the interaction
between the systems responsible for NO generation and
prostaglandin production. Using rat gastric mucosal cells
exposed to H. pylori key virulence factor, LPS, we
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16
demonstrated that the LPS caused a dose-dependent in-
crease in the mucosal cell PGE2 and NO production,
which at 100 ng/ml LPS reached the respective values of
12.7-fold and 14.5-fold over that of controls (Figure 1).
Moreover, we also established that the LPS effect on NO
production was reflected in a 20.5-fold up-regulation in
the mucosal cell iNOS activity (0.42 cpm x 105/mg pro-
tein for the control vs. 8.67 cpm × 105/mg protein for
100 ng/ml LPS), and a 76.8% decrease (2.80 cpm ×
105/mg protein for the control vs. 0.65 cpm × 105/mg
protein for 100 ng/ml LPS) in the activity of cNOS
(Figure 2).
To gain additional insights into the nature of the
LPS-induced changes, we examined NO production and
PGE2 generation by gastric mucosal cell in the presence
of NOS and COX systems inhibition. For this, the mu-
cosal cells prior to incubation with the LPS were pre-
treated with cNOS inhibitor, L-NAME and iNOS in-
hibitor, 1400 W (Figure 3), or COX-1 inhibitor, SC-560
and COX-2 inhibitor, NS-398 (Figure 4), and assayed
for PGE2 generation and NO production. As shown in
Figure 3, the effect of cNOS inhibition was reflected in
a moderate up-regulation in the LPS-induced NO and
PGE2 production, while the inhibition of iNOS lead to a
profound reduction in the LPS effect on the mucosal cell
NO and PGE2 production. Furthermore, the effect of the
LPS on the mucosal cell capacity for NO and PGE2
production was not discernibly affected by COX-1 inhi-
bition (Figure 4), whereas preincubation with COX-2
inhibitor, NS-398, produced a marked reduction in PGE2
generation but had no effect on the LPS-induced NO
generation (Figur e 4).
Further, we found that preincubation of gastric muco-
sal cells with ghrelin led to a concentration-dependent
suppression of the LPS-induced effect on PGE2 genera-
tion and the activity o f iNOS (Figure 5), while the activ-
ity of cNOS showed an increase (Figure 6). Indeed, we
Figure 1. Effect of H. pylori LPS on PGE2 and nitrite produc-
tion in rat gastric mucosal cells. The cells were treated with the
indicated concentrations of the LPS and incubated for 16 h.
Values represent the means ± SD of five experiments. *P <
0.05 compared with that of control (LPS – 0).
Figure 2. Effect of H. pylori LPS on the expression of cNOS
and iNOS activities in rat gastric mucosal cells. The cells were
treated with the indicated concentrations of the LPS and incu-
bated for 16 h. Values represent the means ± SD of five ex-
periments. *P < 0.05 compared with that of control (LPS - 0).
Figure 3. Effect of nitric oxide synthase inhibitors on H. pylori
LPS-induced changes in the production of PGE2 and nitrite by
gastric mucosal cells. The cells, preincubated with the indi-
cated concentrations of cNOS inhibitor, L-NAME (LN), or
iNOS inhibitor, 1400 W, were treated with the LPS at 100 ng/
ml and incubated for 16 h. Values represent the means ± SD of
five experiments. *P < 0.05 compared with that of control. **P
< 0.05 compared with that of LPS alone.
Figure 4. Effect of cyclooxygenase inhibitors on H. pylori
LPS-induced changes in the production of PGE2 and nitrite by
gastric mucosal cells. The cells, preincubated with the indi-
cated concentrations of COX-1 inhibitor, SC-560 (SC), or
COX-2 inhibitor, NS-398 (NS), were treated with the LPS at
100 ng/ml and incubated for 16 h. Values represent the means
± SD of five experiments. *P < 0.05 compared with that of
control. **P < 0.05 compared with that of LPS alone.
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observed that ghrelin at 0.5 µg/ml, elicited an 84.1%
drop in the LPS-induced mucosal cell PGE2 generation
and a 90.2% decrease in the activity of iNOS (Figure 5),
whereas the activity of cNOS showed a 4.4-f old incr ease
(Figure 6). Moreover, the increase in cNOS activation in
the presence of ghrelin was associated with a concentra-
tion-dependent up -regulation in gastric mucosal cell Akt
kinase activity, which at 0.5 µg/ml of ghrelin increased
2-folds over that of the LPS (Figure 6).
As cNOS is known to undergo a rapid posttransla-
tional activation through phosphorylation at Ser1179 [18,
29,30], we have also examined the effect of the LPS and
ghrelin on gastric mucosal cell cNOS phosphorylation.
For this, the cells prior to incubation with ghrelin were
pretreated with Akt kinase inhibitor, SH-5, and the lys-
ates were probed with antibodies directed against cNOS
and phosphor ylated cNOS at Ser1179 (Figure 7). We obse-
rved that the LPS-induced suppression in cNOS activity
Figure 5. Effect of ghrelin on H. pylori LPS-induced changes
in the production of PGE2 and the expression iNOS activity in
gastric mucosal cells. The cells, preincubated with the indi-
cated concentrations of ghrelin, were treated with the LPS at
100 ng/ml and incubated for 16 h. Values represent the means
± SD of five experiments. *P < 0.05 compared with that of
control. **P < 0.05 compared with that of LPS alone.
Figure 6. Effect of ghrelin on H. pylori LPS-induced changes
in the expression of Akt kinase and cNOS activities in gastric
mucosal cells. The cells, preincubated with the indicated con-
centrations of ghrelin, were treated with the LPS at 100 ng/ml
and incubated for 16 h. Values represent the means ± SD of
five experiments. *P < 0.05 compared with that of control.
**P < 0.05 compared with that of LPS alone.
was associated with the inhibition of the enzyme phos-
phorylation, while the up-regulation in cNOS activation
by ghrelin was reflected in a marked increase in the en-
zyme protein phosphorylation at Ser1179. Moreover, the
suppression of ghrelin effect on cNOS phosphoryla- tion
was attained in the presence of Akt inhibitor, SH-5
(Figure 7).
To gain further understanding of the events resulting
in the suppression of H. pylori LPS-induced up-regu-
lation in PGE2 generation and iNOS activation by ghre-
lin, we examined the influence of the LPS and ghrelin on
the gastric mucosal cell expression of iNOS, and COX-1
and COX-2 prot ei ns (Figure 8). The results revealed that
the LPS-induced up-regulation in iNOS activity and
PGE2 production was associated with the induction in
iNOS and COX-2 proteins expression, while the sup-
pression of the LPS effect by ghrelin was reflected in a
marked inhibition of the iNOS protein expression, but no
Figure 7. Effect of ghrelin (Gh) and Akt kinase
inhibitor, SH-5 (SH), on H. pylori LPS-induced
changes in cNOS phosphorylation in gastric
mucosal cells. The cells were treated with ghre-
lin at 0.5 µg/ml, or Akt inhibitor, SH-5 (30 µM)
+ Gh, and incubated for 16 h in the presence of
100 ng/ml LPS. Cell lysates were resolved on
SDS-PAGE, transferred to nitrocellulose, and
probed with phosphorylation-specific cNOS
(pcNOS) antibody, and after stripping reprobed
with anti-cNOS antibody. The immunoblots
shown are representative of three experiments.
Figure 8. Effect of ghrelin on H. pylori
LPS-induced expression of COX-1, COX-2, and
iNOS proteins in gastric mucosal cells. The cells
were treated with the LPS at 100 ng/ml or ghre-
lin (Gh) at 0.5 ng/ml + LPS and incubated for 16
h. Cell lysates were resolved on SDS-PAGE, tr-
ansferred to nitrocellulose, and probed with
anti-COX-1, anti-COX-2, or anti-iNOS antibody.
The immunoblots shown are representative of
three experiments.
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18
COX-2 protein expression was discerned. We have also
apparent diminution in observed that the COX-1 protein
expression remained essentially unaffected by the inclu-
sion of the LPS or ghrelin (Figure 8). Thus, the enzy-
matic activity of the LPS-induced COX-2 protein for
up-regulation in PGE2 production shows an apparent
dependence on NO generated by the iNOS system.
Therefore, to ascertain the requirement of H. pylori
LPS-induced up-regulation in COX-2 activ ation for NO,
the mucosal cells prior to incubation with ghrelin were
pretreated with iNOS inhib itor, 1400 W, or Akt inhibito r,
SH-5, or nitrosothiol reducing agent, ascorbate, and as-
sayed for COX-2 activity. We found that the LPS-in-
duced up-regulation in COX-2 activity was subject to
suppression not only by the pretreatment with ghrelin,
but also sho wed su scep tibility to iNOS inh ibito r, 1400 W,
whereas Akt inhibitor, SH-5, had no effect on the extent
of the LPS-induced COX-2 activation (Figure 9).
Moreover, while the iNOS inhibitor, 1400 W, produced
amplification in the inhibitory effect of ghrelin on
COX-2 activity, the Ak t inhibitor, SH-5, caused the sup-
pression in ghrelin effectiveness. Further, we found that
the LPS-induced COX-2 activation displayed suscepti-
bility to suppression by nitrosothiol reducing agent, as-
corbate, which also produced enhancement in the effect
of ghrelin on COX-2 activity (Figure 9).
Finally, we also examined the dependence of COX-2
S-nitrosylation on the LPS-induced iNOS activation by
the biotin switch method [33,34]. The gastric mucosal
cells were incubated with H. pylori LPS or ghrelin +
LPS in the absence and presence of Akt inhibitor, SH-5,
or iNOS inhibitor, 1400 W + LPS, and t he lysates following
the biotin switch procedure were examined for COX-2
protein S-nitrosylation (Figure 10). Western blot analy-
sis revealed that COX-2 in the cells exposed to the LPS
alone showed a marked increase in the protein S-nitro-
sylation, whereas the prein cubation with iNOS inhibitor,
1400 W, resulted in the loss of the LPS-induced COX-2
S-nitrosylation. A pronounced decrease in the LPS-in-
duced COX-2 S-nitrosylation was also attained in the
presence of ghrelin. Moreover, this effect of ghrelin on
COX-2 S-nitrosylation was subject to suppression by the
inclusion of Akt inhibitor, SH-5. These data suggest that
induction in iNOS elicited by H. pylori LPS leads to
COX-2 activation through S-nitrosylation that results in
an excessive PGE2 generation, and that ghrelin counters
the detrimental consequences of iNOS induction by way
of cNOS-dependent suppression of iNOS gene induc-
tion.
4. DISCUSSION
H. pylori lipopolysaccharide is recognized as a potent
endotoxin capable of eliciting mucosal inflammatory
Figure 9. Effect of iNOS inhibitor, 1400 W, Akt inhibitor,
SH-5, and ascorbate on the ghrelin (Gh)-induced changes in
COX-2 activity in gastric mucosal cell exposed to H. pylori
LPS. The cells, preincubated with 40 µM 1400 W (14 W), 30
µM SH-5 (SH), or 300 µM ascorbate (As), were treated with
Gh at 0.5 µg/ml and incubated for 16h in the presence of 100
ng/ml LPS. Values represent the means ± SD of five experi-
ments. *P < 0.05 compared with t hat of control. **P < 0.05 com-
pared with that of LPS alone. ***P < 0.05 compared with that of
Gh LPS .
Figure 10. Effect of ghrelin (Gh) on H. pylori
LPS-induced COX-2 S-nitrosylation. The gastric
mucosal cells were treated with 40 µM of iNOS
inhibitor, 1400 W (14 W), or Gh (0.5 µg/ml), or
30 µM Akt inhibitor, SH-5 (SH) + Gh, and incu-
bated for 16 h in the presence of 100 ng/ml L PS.
A portion of the cell lysates was processed by
biotin switch procedure for protein S-nitrosylation
and, along with the reminder of the lysates, re-
solved on SDS-PAGE, transferred to nitrocellu-
lose and probed with anti-COX-2 antibody. The
immunoblots shown are representative of three
experiments.
responses akin to those that characterize gastritis and
duodenal u lcer [1-3]. Indeed, H. pylori LPS, like LPS of
other Gram-negative bacteria, is known to trigger a wide
variety of transcriptional factors, including NF-B and
AP-1, that exert transcrip tional control over such impor-
tant mediators of inflammation as iNOS and COX-2
systems, which along with the constitutively expressed
isozyme forms, cNOS and COX-1, are responsible for
NO and PGE2 production [4-6,20-22]. Moreover, a
growing volume of data consistently points towards the
existence of a functional and signaling relationship be-
tween the products of NOS and COX enzyme systems
[13,14,19,20]. In particular, there are strong indications
for the involvement of iNOS-derived NO in COX-2 ac-
tivation through S-nitrosylation for the increase in PGE2
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generation [20-22]. Furthermore, the gastric mucosal
inflammatory responses to H. pylori as well as its LPS
are reflected in the suppression in cNOS activation
[5,7,25,26]. Hence, in this study presented herein, we
investigated the nature of interaction between the systems
responsible for NO generation and PGE2 production.
Using rat gastric mucosal cells exposed to H. pylori
LPS, we demonstrated that the LPS-induced enhance-
ment in iNOS activity and up-regulation in PGE2 pro-
duction was accompanied by the suppression in Akt ki-
nase activity and the impairment in cNOS activation
through phosphorylation. The stimulatory effect of the
LPS on PGE2 production was susceptible to suppression
by COX-2 inhibitor, NS-398, as well as the inhibitor of
iNOS, 1400 W. However, the LPS-induced up-regulatio n
in the mucosal cell NO generation was not affected by
the inhibitors of COX-1 and COX-2 systems. These lat-
ter findings are thus in keeping with the literature data as
to the role of iNOS in the regulation of COX-2 activa-
tion for the increase in PGE2 production [2 0 -23].
Further, we found that preincubation of the mucosal
cells with gastric hormone, ghrelin, recognized for its
modulatory influence on the inflammatory responses to
bacterial infection [18,27,29-31,35], exerted countering
effect on the LPS-induced suppression in Akt activity
and lead to the increase in cNOS activation through
phosphorylation at Ser1179. We also observed that these
effects of ghrelin were accompanied by the suppression
in iNOS protein expression and the reduction in COX-2
activity. Moreover, ghrelin-induced up-regulation in
cNOS activation was susceptible to suppression by Akt
inhibitor, SH-5, which also caused the abrogation in
ghrelin-induced reduction in COX-2 activity. From this,
we concluded that ghrelin countering effect on H. pylori
LPS-induced inflammatory changes occurs with the in-
volvement of Akt-mediated cNOS activation through
phosphorylation, and that Akt kinase plays an essential
role in the action of ghrelin on COX-2 activity. Indeed,
Akt kinase occupies a central role in the receptor (GHS-
R)-mediated responses to ghrelin stimulation [26,36],
and the involvement of signaling through Akt for a rapid
up-regulation in cNOS activity through the enzyme pro-
tein phosphorylation at Ser1179 is well documented [29,
30,37].
Our further examination of the influence of ghrelin on
H. pylori LPS-induced up-regulation in iNOS activity
and PGE2 generation revealed that while the expression
of COX-1 protein remained essentially unaffected by the
LPS or ghrelin, the effect of the LPS was associated with
the induction in iNOS and COX-2 proteins. The coun-
tering effect of ghrelin, moreover, was reflected in a
marked inhibition of the iNOS protein expression, but no
apparent diminution in COX-2 protein was discerned.
Thus, in concordance with our earlier results, it is ap-
parent that whilst the countering effect of ghrelin on the
LPS-induced enhancement in iNOS activity is associated
with the inhibition of iNOS gene expression at the tran-
scriptional level [38], the suppression of the LPS-in-
duced COX-2 activity by ghrelin does not involve the
inhibition in the enzyme protein expression. However,
the enzymatic activity of the LPS-induced COX-2 pro-
tein for up-regulation in PGE2 production shows an ap-
parent dependence on NO generated by the iNOS system.
Indeed, the stimulation of NO production through iNOS
induction has been reported to lead to COX-2 protein
modification through S-nitrosylation that results in the
enzyme activation and the increase in PGE2 generation
[20-22].
Hence, to reveal further insights into the mechanism
of ghrelin suppression of the LPS-induced gastric mu-
cosal inflammatory disturbances, we examined the
COX-2 activity and its protein S-nitrosylation in the
presence of the inhibitors of iNOS and Akt, as well as
nitrosothiols reducing agent, ascorbate. We found that
the countering effect of ghrelin on the LPS-induced
up-regulation in COX-2 activity was further amplified in
the presence of iNOS inhibitor, 1400 W, while the Akt
inhibitor, SH-5, caused the suppression in ghrelin effec-
tiveness. Moreover, the LPS-induced COX-2 activation
displayed susceptibility to suppression by ascorbate,
which also produced an amplification in ghrelin effect
on the mucosal COX-2 activity. These data, together
with the demonstrated susceptibility of the LPS-induced
COX-2 activation to inhibition by iNOS inhibitor, 1400
W, as well as known vulnerability of S-nitrosylated pro-
teins to reduction by ascorbate [20,21,26,33,34], sug-
gest that the countering effect of ghrelin on H. pylori
LPS-induced changes in COX-2 activation are intimately
linked to the events of Akt activation and a rapid up-
regulation in cNOS activity through Akt-med iated cNOS
protein phosphorylation. Consequently, our results point
to the involvement of cNOS in controlling the extent of
iNOS-dependent COX-2 activation.
The supporting evidence as to the involvement of
Akt-mediated cNOS activation in the modulatory action
of ghrelin against H. pylori LPS-induced gastric mucosal
consequences of iNOS induction and up-regulation in
COX-2 activation comes from the results of biotin
switch assay. We found that the mucosal cells exposed to
incubation with H. pylori LPS showed a marked incr e as e
in COX-2 protein S-nitrosylation, while the suppression
of the LPS-induced iNOS activity with a specific inhibi-
tor, 1400 W, caused the loss in COX-2 S-nitrosylation.
Further, we observed a pronounced drop in the LPS-
induced COX-2 S-nitrosylation in the presence of ghre-
lin, the effect of which was subject to the repression by
Akt inhibitor, SH-5. These findings, together with the
results of COX-2 activity assays, suggest that induction
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20
in gastric mucosal iNOS expression elicited by H. pylori
LPS leads to COX-2 activation through S-nitrosylation
that results in an excessive PGE2 generation. It is also
apparent that ghrelin counters these untoward conse-
quences of H. pylori via Akt-mediated up-regulation in
cNOS activation that is required for the suppression of
iNOS gene induction.
While the intricate details of the role of cNOS in the
regulation of iNOS g ene induction remain ob scure, there
are indications that cNOS is capable of affecting tran-
scriptional factor NF-B activation and therefore to in-
fluence the extent of promoter activity and iNOS gene
transcription [12,21,24]. More interestingly, the litera-
ture data suggest that S-n itrosylation of an inhibitor pro-
tein, IB kinase complex (IKK) interferes with ubiquit-
inylation and proteasomal degradation of IB, thus pre-
venting the nuclear translocation of NF-B, and result-
ing in its inability to promote target gene transcription
[39-41]. Furthermore, we have shown that the LPS-in-
duced suppression in cNOS activity was associated with
the induction in iNOS protein expression, while the
countering effect of ghrelin, like that NF-B inhibitor,
PPM-18, was reflected in a marked inhibition of the
iNOS protein expression [38]. Moreover, the effect of
ghrelin was intimately linked to Akt-mediated cNOS
activation through phosphorylation [26].
Taken togeth er, these observations lead us to postulate
that the initial phase gastric mucosal inflammatory re-
sponses elicited by H. pylori involves the suppression in
Akt kinase-dependent cNOS activation that leads to ab-
rogation of cNOS-mediated IKK S-nitrosylation and the
increase in proteasomal degradation of IB, thus allow-
ing NF-B translocation to the nucleus to promote iNOS
gene transcription. The induction in iNOS expression, in
turn, leads to iNOS-mediated COX-2 activation through
S-nitrosylation that results in an excessive PGE2 genera-
tion. We also postulate that ghrelin counters these unto-
ward consequences of H. pylori LPS via up-regulation in
Akt-dependent cNOS activation through phosphoryla-
tion that results in up-regulation in cNOS-mediated IKK
coplex S-nitrosylation which interferes with proteasomal
degradation of IB, thereby preventing the nuclear
translocation of NF-B and the induction in iNOS gene
transcription. Hence, the repression of iNOS expression
by ghrelin triggers the loss in COX-2 activation through
iNOS-dependent S-nitrosylation and leads to a reduction
in PGE2 generati o n.
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