Open Journal of Cell Biology, 2011, 1, 1-10
doi:10.4236/ojcb.2011.11001 Published Online December 2011 (http://www.SciRP.org/journal/ojcb)
Ghrelin Suppression of Helicobacter pylori-Induced Gastric
Mucosal Expression of iNOS is Mediated through the
Inhibition of IKK-β Activation by cNOS-Dependent
S-Nitrosylation
Bronislaw L. Slomiany*, Amalia Slomiany
University of Medicine and Dentistry of New Jersey, Newark, USA
Received November 7, 2011; revised December 3, 2011; accepted December 12, 2011
Abstract
Excessive nitric oxide generation, caused by the disturbances in nitric oxide synthase (NOS) isozyme system,
plays a key role in defining the extent of gastric mucosal inflammatory response to H. pylori infection. Here,
we report that H. pylori LPS-induced enhancement in gastric mucosal inducible (i) iNOS expression and the
impairment in constitutive (c) cNOS activity was associated with up-regulation in the inhibitory kB kinase-β
(IKKβ) activation through phosphorylation, rise in IκB-α degradation, and the increase in the transcriptional
factor, NF-κB, nuclear translocation. Further, we show that the countering effect of peptide hormone, ghrelin,
on the LPS-induced disturbances in NOS isozyme system was reflected in the increase in Src/Akt-dependent
cNOS activation through phosphorylation and the suppression of IKK-β activity through cNOSmediated
IKK-β protein S-nitrosylation. As a consequence, ghrelin exerted the inhibitory effect on the LPS-induced
rise in IκB-α degradation and NF-κB nuclear translocation, thus leading to iNOS gene suppression and the
repression of iNOS induction. These results point to a central role of cNOS activation in controlling the sig-
naling pathways of the LPS-triggered iNOS gene induction. Moreover, our findings suggest a molecular
mechanism by which ghrelin suppresses the gastric mucosal proinflammatory consequences of H. pylori in-
fection.
Keywords: H. Pylori, Gastric Mucosa, Ghrelin, cNOS Activation, IKK-β S-Nitrosylation
1. Introduction
Cell-wall lipopolysaccharide (LPS) of H. pylori, a Gram-
negative bacterium colonizing the gastric mucosa of over
50% of the human population, is recognized as a potent
endotoxin capable of eliciting mucosal inflammatory
changes that characterize gastritis and duodenal ulcer
[1-3]. The stimulation of gastric mucosa with H. pylori
LPS is known to provoke a marked up-regulation in pro-
inflammatory cytokine production, massive rise in epi-
thelial cell apoptosis, and the excessive nitric oxide (NO)
generation caused by the disturbances in nitric oxide
synthase (NOS) system [4-6]. A growing body of evi-
dence, moreover, points to the disturbances in NO pro-
duction associated with H. pylori colonization, and re-
flected in continual activation of iNOS and the suppres-
sion of cNOS isozyme system, as the event defining the
extent of gastric mucosal inflammatory involvement. [4,
7-9]. Moreover, studies indicate that the expression of
proinflammatory mediators induced by H. pylori infec-
tion is regulated by the activity of the nuclear transcrip-
tion factor, NF-κB [6,10-12].
Indeed, NF-κB is ubiquitously present in all types of
mammalian cells, where it controls a variety of genes
involved in immune, inflammatory, and proliferative res-
ponses, including those of the NOS isozyme system that
are responsible for NO production [12-14]. While the
factor is known to consist of five homo- and heter-
odimers of the Rel protein family, the most characterized
NF-κB complex is a p50/p65 hetrodimer, which is se-
questered in the cytoplasm of resting cells bound to a
family of inhibitory IκB proteins [14-16]. Activation of
NF-κB in response to such stimuli as TNF-α or LPS oc-
curs through a rapid IκB-α phosphorylation by an IκB

B. L. SLOMIANY ET AL. 2
kinase (IKK) complex, that targets IκB-α for degradation
through the ubiquitin-proteasomal pathway, thus allow-
ing nuclear translocation of NF-κB, its binding to pro-
moter response elements, and activation of the transcrip-
tion of genes implicated in immunoregulation, inflam-
mation and cellular proliferation [14-17].
The IKK complex consists of two catalytic subunits,
IKK-α and IKK-β, and one regulatory subunit, IKK-γ,
also known as NEMO (NF-κB essential modulator) [12,
14,15,17]. While the activation of IKK complex requires
phosphorylation of IKK-α and IKK-β on the specific
conserved serine residues in the activation loops, the
IKK-γ regulatory subunit appears to be responsible for
IKK complex stabilization and the interaction with up-
stream regulatory proteins [15,17]. Furthermore, al-
though IKK-α and IKK-β show a high degree of homol-
ogy in their structural domains, the available literature
indicates that IKK-β displays up to 50 fold higher level
of kinase activity towards IκB proteins, and the gene
deletion studies demonstrated that it is IKK-β and not
IKK-α which is responsible for NF-κB activation in re-
sponse to stimulation by TNF-α or bacterial LPS [18- 20].
In addition, recent reports indicate that the activity of
IKK complex may also be regulated through S-nitrosy-
lation of a specific cysteine residue within the activation
loop of IKK-β, by endogenous NO donors, leading to
suppression of IκB-α degradation and the inhibition of
NF-κB nuclear translocation, thus blocking the transcrip-
tion of NF-κB-regulated gene products [17,21,22].
As peptide hormone, ghrelin, is recognized as an im-
portant modulator of gastric mucosal inflammatory re-
sponses to H. pylori through the regulation of NOS sys-
tem [4,9,23-26], in this study we investigated the effect
of H. pylori LPS on the activation of NF-κB signaling
pathway in gastric mucosal cells, and the mechanism of
ghrelin modulatory action. Our results demonstrate that
the LPS elicit induction in iNOS expression via up-
regulation in IKK-β activation through phosphorylation.
We further show that ghrelin inhibits the LPS-induced
activation of IKK-β through cNOS-mediated IKK-β pro-
tein S-nitrosylation, thus leading to suppression of IκB-α
phosphorylation and degradation, inhibition of NF-κB
nuclear translocation, and the repression of iNOS gene
induction.
2. Materials and Methods
2.1 Gastric Mucosal Cell Incubation
The cells were collected by scraping the mucosa of
freshly dissected rat stomachs with a blunt spatula, and
suspended in five volumes of ice-cold Dulbecco’s modi-
fied (Gibco) Eagle’s minimal essential medium (DMEM),
supplemented with fungizone (50 µg/ml), penicillin (50
U/ml), streptomycin (50 µg/ml), and 10% fetal calf se-
rum. The cells were then gently dispersed by trituration
with a syringe, settled by centrifugation, and following
rinsing resuspended in the medium to a concentration of
2 × 107 cell/ml [9]. Cell aliquots (1 ml) were then trans-
ferred to DMEM in culture dishes and incubated under
95% O2 - 5% CO2 atmosphere at 37˚C for 16 h in the
presence of 0 - 100 ng/ml of H. pylori LPS [25]. H. py-
lori used for LPS preparation was cultured from clinical
isolates obtained from ATCC No. 4350 [5]. In the ex-
periments evaluating the effect of ghrelin (rat, Sigma),
cNOS inhibitor, L-NAME, iNOS inhibitor, 1400W, Akt
inhibitor, SH-5, Src inhibitor, PP2 (Calbiochem), and
ascorbate (Sigma), 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 pre-
parations before and during the experimentation, asse-
ssed by Trypan blue dye exclusion assay [9], was greater
than 97%.
2.2. cNOS and iNOS Activity Assay
Nitric oxide synthase activities of cNOS and iNOS en-
zymes in the gastric mucosal 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 ho-
mogenized in a sample buffer containing either 10 mM
EDTA (for Ca2+-independent iNOS) or 6 mM CaCl2
(for Ca2+-depenedent cNOS), and centrifuged. The ali-
quots of the resulting supernatant were incubated for 30
min at 25˚C in the presence of 50 µCi/ml of L-[3H] ar-
ginine, 10 mM NAPDH, 5 µM tetrahydrobiopterin, 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 transferred to spin cups,
centrifuged and the formed L-[3H] citrulline contained in
the flow through was quantified by scintillation counting.
2.3. Nuclear Protein Extraction
The aliquots of gastric mucosal cell suspension from the
control and various experimental conditions were settled
by centrifugation at 1500 xg for 5 min, rinsed with phos-
phate-buffered saline, and lysed by incubation for 10 min
on ice in the lysis buffer, containing 10 mM HEPES, pH
7.9, 10 mM KCl, 0.1 mM EDTA, 0.5% Nonidet P-40, 1
mM dithiothreitol, and 0.5 mM PMSF [13]. Following
centrifugation at 12,000 xg for 10 min at 4˚C, the super-
natant was subjected to centrifugation at 100,000 xg for
1h and the obtained soluble fraction was used as source
of cytosolic extract [27]. The pellets, from 12,000 xg

B. L. SLOMIANY ET AL.3
centrifugation, containing crude nuclei were suspended
for 20 min at 4˚C in the extraction buffer, containing 20
mM HEPES, pH 7.9, 25% glycerol, 400 mM NaCl, 1.5
mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, and 1
mM PMSF. The samples were centrifuged at 15,000 xg
for 10 min at 4˚C, and the supernatants containing nu-
clear extracts were collected and stored at −70˚C until
use.
2.4. IκB Kinase Activity Assay
To measure the IKK-β activity we utilized the ELISA-
based detection kit, K-LISATM (Calbiochem). The GST-
IkB-a 50-amino acid peptide that includes the Ser32 and
Ser36 of IkB-α phosphorylation sites was used as a sub-
strate [28]. The gastric mucosal cell cytosolic extracts
were incubated a glutathione-coated 96-well plate with
GST-tagged IκB-α at room temperature for 30 min, and
the phosphorylated GST-IκB-α substrate was detected
using anti-phospho IkB-α (Ser32/Ser36) as first antibody,
followed by horseradish peroxidase-conjugated secon-
dary antibody. Following washing the retained complex
was probed TMB reagent for spectrophotometric quanti-
fication at 450 nm.
2.5. IKK-β Protein S-Nitrosylation Assay
A biotin switch procedure was employed to assess IKK-β
protein S-nitrosylation [29,30]. The gastric mucosal cells
were treated with ghrelin (0.5 µg/ml), or Akt inhibitor,
SH-5 (20 µM) + ghrelin (0.5 µg/ml), and incubated for
1h in the presence of 100ng/ml of H. pylori LPS. Fol-
lowing centrifugation at 500 xg 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 unnitrosylated thiol groups were blocked with S-
methyl methanethiosulfonate reagent at 50˚C for 20 min
[30]. The proteins were precipitated with acetone, resus-
pended in 0.2 ml of HEN buffer containing 1% SDS, and
subjected to targeted nitrothiol group reduction with so-
dium ascorbate (100 mM). The free thiols were then la-
beled with biotin and the biotinylated proteins were re-
covered on streptavidin beads. The formed streptavidin
bead-protein complex was washed with neutralization
buffer, and the bound proteins were dissociated from
streptavidin beads with 50 µl of elution buffer (20 mM
HEPES, 100 mM NaCl, 1 mM EDTA, pH 7.7) contain-
ing 1% 2-mercaptoethanol [30]. The obtained proteins
were then analyzed by Western blotting.
2.6. 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% Triton 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 pepstatin [25].
Following brief sonication, the lysates were centrifuged
at 12,000 g for 10 min, and the supernatants were col-
lected and normalized with respect to protein concentra-
tion using BCA protein assay kit (Pierce). The samples,
including those subjected to biotin switch procedure,
were then resuspended in loading buffer, boiled for 5
min, and subjected to SDS-PAGE using 40 µg protein/
lane. The separated proteins were transferred onto nitro-
cellulose 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
polyclonal rabbit antibodies directed against IκB-α and
cNOS (Calbiochem), phospho-IκB-α and phospho-IKK-
β (Cell Signaling), NF-κB p65 and IKK-β (EMD Milli-
pore). The phosphorylated cNOS (pcNOS) was analyzed
using specific antibody (Calbiochem) directed against
phospho-cNOS (mouse anti-eNOS, pSer1179).
2.7. Data Analysis
All experiments were carried out using duplicate samp-
ling, and the results are expressed as means ± SD. Ana-
lysis of variance (ANOVA) and nonparametric Kruskal-
Wallis tests were used 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
We have reported that the modulatory effect of ghrelin
on gastric mucosal inflammatory responses to H. pylori
LPS, characterized by the disturbances in NO production,
was reflected in the cNOS activation through phos-
phorylation and the suppression of iNOS protein expres-
sion [9,25]. Indeed, as illustrated in Figure 1, the LPS at
100 ng/ml elicited a 19.8-flold increase in the mucosal
cell iNOS activity, whereas the cNOS activity showed a
4.3-fold decrease. Moreover, preincubation with ghrelin
at 0.5 µg/ml lead to a 90.2% reduction in the LPS-in-
duced iNOS activity, while the activity of cNOS in-
creased by a 77%. Since iNOS expression is controlled
primarily by the transcription factor, NF-κB [10-12], we
further investigated the effect on ghrelin on the LPS-
induced NF-κB activation.
As NF-κB activation requires phosphorylation and
degradation of the inhibitory protein IκB-α, we exposed

B. L. SLOMIANY ET AL. 4
Figure 1. Effect of ghrelin on H. pylori LPS-induced expres-
sion of cNOS and iNOS activities in gastric mucosal cells.
The cells, in the absence and presence of preincubation with
ghrelin (0.5 µg/ml), were treated with H. pylori LPS (100
ng/ml) and incubated for 16 h. Values are presented as
percent of control, and represent the means ± SD of five
experiments. *P < 0.05 compared with that of control. *P <
0.05 compared with that of the LPS.
the gastric mucosal cells for different time periods to H.
pylori LPS in the absence or presence of ghrelin, and the
cell lysates were analyzed for IκB- α phosphorylation and
degradation. As shown in Figure 2(a), the LPS induced a
marked phosphorylation of IκB-α at 10 min, while a dis-
tinct reduction in IκB-α protein level occurred at 30 min
(Figure 2(b)). Moreover, we found that in the presence
of ghrelin, the LPS-induced phosphorylation of IκB-α
was significantly inhibited (Figure 2(a)), and the extent
of the induced IκB-α protein degradation showed a sig-
nificant decrease (Figure 2(b)).
Further, as IκB-α degradation leads to translocation of
p65 NF-κB from the cytoplasm to nucleus [14-17], we
assessed the nuclear content of p65 NF-κB in response to
H. pylori LPS alone and in the presence of preincubation
with ghrelin. The results of immunoblots analysis re-
vealed that incubation of the mucosal cells with the LPS
lead to an increase of p65 NF-κB in the nuclear extracts
(Figure 3), while In the presence of preincubation with
ghrelin, the LPS-induced nuclear translocation of p65
NF-κB was significantly diminished (Figure 3).
As the extent of IκB-α phosphorylation and its pro-
teasomal degradation is mediated through IKK-β of the
IKK complex [12,14,17], we next analyzed the influence
of ghrelin on changes in IKK-β activity in gastric muco-
sal cells exposed to H. pylori LPS. The results revealed
that the LPS (at 100 ng/ml) induced a 8.4-fold increase in
the mucosal cell IKK-β activity, and that preincubation
with ghrelin lead to a concentration-dependent suppres-
sion of the LPS effect (Figure 4). As a result the LPS-
induced activity of IKK-β, in the presence 0.5 µg/ml of
ghrelin, showed an 83% decrease. Moreover, as the sup-
pression of the LPS-induced iNOS protein expression by
ghrelin is associated with rapid cNOS activation through
phosphorylation [24,25], we have also examined the
Figure 2. Effect of ghrelin on H. pylori LPS-induced IκB-α
serine phosphorylation (a) and IκB-α protein degradation
(b) in rat gastric mucosal cells. The cells were preincubated
for 30 min with 0 or 0.5 µg/ml of ghrelin and incubated for
the indicated time periods with the LPS at 100 ng/ml. Cell
lysates were analyzed by Western blotting with anti-phos-
pho-IκB-α or anti-IκB-α antibody. Relative densities are
expressed as fold of control (time, 0). Values represent the
means ±SD of four separate experiments. *P < 0.05 com-
pared with that of LPS.
mucosal cell IKK-β activity in the presence of the in-
hibitors of cNOS phosphorylation. For this, the gastric
mucosal cells prior to incubation with the LPS were pre-

B. L. SLOMIANY ET AL.5
Figure 3. Effect of ghrelin on H. pylori LPS-induced nuclear
translocation of p65 NF-κB in rat gastric mucosal cells. The
cells were preincubated for 30 min with 0 or 0.5 µg/ml of
ghrelin and incubated for 30 min with the LPS at 100 ng/ml.
Cells were lysed and the level of p65 NF-κB protein in the
nuclear fraction was analyzed immunochemically. Relative
densities are expressed as fold of control. The data repre-
sent the means ± SD of four separate experiments. *P < 0.05
compared with that of LPS.
Figure 4. Effect of ghrelin on H. pylori LPS-induced changes
in IKK-β activity in gastric mucosal cells. The cells, prein-
cubated for 30 min with the indicated concentrations of
ghrelin, were treated with the LPS at 100 ng/ml and incu-
bated for 30 min. 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.
treated with the inhibitors of Src/Akt pathway in the ab-
sence or presence of ghrelin, and assayed for IKK-β ac-
tivity. We found that the countering effect of ghrelin on
H. pylori LPS-induced up-regulation in the mucosal cell
IKK-β activity was subject to suppression by both, the
inhibitor of Src, PP2, as well as Akt inhibitor, SH-5
(Figure 5). A significant decrease in the effectiveness of
ghrelin on the LPS-induced IKK-β activation, further-
more, was observed in the presence of cNOS inhibitor,
L-NAME, whereas the inhibitor of iNOS, 1400 W, had
no effect (Figure 5). Further, examination of the effect
of H. pylori LPS and ghrelin on IKK-β and cNOS phos-
Figure 5. Effect of Src inhibitor, PP2, Akt inhibitor, SH-5,
cNOS inhibitor, L-NAME, and iNOS inhibitor, 1400 W, on
the ghrelin (Gh)-induced changes in IKK-β activity in gas-
tric mucosal cells exposed to H. pylori LPS. The cells, pre-
incubated with 30 µM PP2, 20 µM SH-5 (SH), 200 µM
L-NAME (LN), or 40 µM 1400W (14W), were treated with
Gh at 0.5 µg/ml and incubated for 30 min in the presence of
100 ng/ml of LPS. 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. ***P < 0.05 com-
pared with that of Gh  LPS.
phorylation revealed that the LPS-induced up-regulation
in IKK-β activity was associated with a marked increase
in IKK-β phosphorylation and the suppression in cNOS
protein phosphorylation (Figure 6). Moreover, while
preincubation with ghrelin lead to the increase in cNOS
phosphorylation that was susceptible to suppression by
Akt inhibitor, SH-5, ghrelin produced no apparent effect
of the extent of the LPS-induced IKK-β phosphorylation
(Figure 6). Thus, the countering effect of ghrelin on H.
pylori LPS-induced up-regulation in gastric mucosal
IKK-β activation shows an apparent dependence on NO
generated by the cNOS system.
To ascertain further the role of NO generated by cNOS
system in the modulatory influence of ghrelin on H. py-
lori LPS-induced up-regulation in gastric mucosal IKK-β
activation, we assessed the effect of nitrosothiols reduc-
ing agent, ascorbate. As show in Figure 7, preincubation
with ascorbate lead to a significant relieve in the inhibi-
tory effect of ghrelin on the LPS-induced IKK-β activity.
However, ascorbate produced no discernible effect on
the extent of the LPS-induced IKK-β activation. More-
over, we also examined the dependence of IKK-β S-ni-
trosylation on the ghrelin-induced cNOS activation
through Akt-mediated phosphorylation by the biotin
switch method [29,30]. The gastric mucosal cells were
incubated with H. pylori LPS or ghrelin + LPS in the
absence and presence of Akt inhibitor, SH-5, and the
lysates following the biotin switch procedure were ex-
amined for IKK-β protein S-nitrosylation (Figure 8).
Western blot analysis revealed that ghrelin countering
effect on the LPS-induced up-regulation in IKK-β active-

B. L. SLOMIANY ET AL. 6
Figure 6. Effect of H. pylori LPS and ghrelin (Gh) on cNOS
and IKK-β phosphorylation in gastric mucosal cells ex-
posed to Akt inhibitor, SH-5 (SH). The cells were preincu-
bated for 30 min with Gh at 0.5 µg/ml, or SH (20 µM) + Gh,
and incubated for 1 h in the presence of 100 ng/ml of LPS.
Cell lysates were resolved on SDS-PAGE, transferred to
nitrocellulose and probed with phosphorylation specific
cNOS (pcNOS) and pIKKβ antibody, and after stripping
reprobed with anti-cNOS and anti-IKKβ antibody. The
immunoblots shown are representative of three experi-
ments.
Figure 7. Effect of ascorbate on the ghrelin (Gh)-induced
changes in the expression of IKK-β activity in gastric mu-
cosal cells exposed to H. pylori LPS. The cells, preincubated
with 300 µM ascorbate (As), were treated with Gh at 0.5
µg/ml and incubated for 30 min in the presence of 100
ng/ml LPS. Values represent the means ± SD of five ex-
periments. *P < 0.05 compared with that of control. **P <
0.05 compared with that of LPS alone. ***P < 0.05 com-
pared with that of Gh  LPS.
ity was manifested in a marked increase in the enzyme
protein S-nitrosylation, and that this effect of ghrelin on
IKK-β S-nitrosylation was subject to suppression by Akt
inhibitor, SH-5. These data suggest that ghrelin counter-
ing effect of the detrimental consequences of up-regula-
tion in IKK-β activity by H. pylori LPS is mediated
through cNOS-dependent IKK-β protein S-nitrosylation.
4. Discussion
Infection with gastric pathogen, H. pylori, initiates the
course of mucosal inflammatory events that increases the
risk of chronic gastritis, peptic ulcer disease, and gastric
Figure 8. Effect of H. pylori LPS and ghrelin (Gh) on IKK-β
S-nitrosylation in gastric mucosal cells exposed to Akt in-
hibitor, SH-5 (SH). The cells, were preincubated for 30 min
with Gh at 0.5 µg/ml, or SH at (20 µM) + Gh, and incubated
for 1 h in the presence of 100 ng/ml LPS. A portion of the
cell lysates was processed by biotin switch procedure for
protein S-nitrosylation and, along with the reminder of the
lysates, resolved on SDS-PAGE, transferred to nitrocellulose
probed with anti-IKKbantibody. The immunoblots shown
are representative of three experiments.
adenocarcinoma [1-3,12]. Indeed, colonization of gastric
mucosa by H. pylori in humans or stimulation of gastric
mucosal cells with H. pylori LPS is known to induce
up-regulation in proinflammatory cytokine release, en-
hances the epithelial cell apoptosis, and causes the exces-
sive NO generation due to disturbances in NOS isozyme
system [4-7,9]. These events define the extent of gastric
mucosal inflammatory involvement, and are carefully
regulated at the early response stage by a family of tran-
scriptional factors, known as NF-κB, which transduce the
inflammatory stimulus into the activation of a variety
genes involved in ant-microbial responses, including those
accountable for NO generation [6, 10-12, 31,32]. As gas-
tric mucosal inflammatory responses to H. pylori as well
as its LPS are reflected in continual induction of iNOS
and the suppression of cNOS activation, and a peptide
hormone, ghrelin, is recognized as an important regulator
of NOS system [4,5,7,9,23-26], the objective of this stu-
dy was to ascertain the influence of ghrelin on the proc-
esses associated with NF-κB activation by H. pylori LPS.
The data obtained with rat gastric mucosal cells re-
vealed that H. pylori LPS-induced enhancement in the
expression of iNOS and the impairment in cNOS activity
was associated with up-regulation in IKK-β activation
through phosphorylation, rise in IκB-α degradation, and
the increase in NF-κB nuclear translocation. These find-
ings are thus in keeping with the preponderant literature
evidence as to the involvement of bacterial LPS in the
classical signaling pathway of NF-κB activation [14-17].
In this pathway, NF-κB activity is regulated by cytosolic
sequestration through interaction with inhibitory IκB
proteins. Activation of NF-κB by proinflammatory stim-
uli, such as LPS or TNF-α, involves phosphorylation of
the inhibitory IκB-α at two conserved N-terminal serine
residues (Ser32 and Ser36) by IκB-kinase (IKK) complex,
a process that signals IκB-α for degradation by the ubiq-
uitin-proteasomal pathway [15-18]. Then, the released

B. L. SLOMIANY ET AL. 7
activated NF-κB, usually consisting of p50 and p65 sub-
units, translocates to the nucleus, and activates the tran-
scription of various proinflammatory target genes, in-
cluding those coding for cytokines, apoptotic factors, and
iNOS [10-12,31,32]. Indeed, we have found earlier that
the induction in gastric mucosal iNOS enzyme protein
expression in response to H. pylori LPS was susceptible
to suppression by the inhibitor of NF-κB activation,
PM-18 as well as peptide hormone, ghrelin [9,33]. Fur-
thermore, as shown herein, ghrelin exerted the inhibitory
effect on H. pylori LPS-induced rise in gastric mucosal
IkB-β degradation and NF-κB nuclear translocation.
Hence, to reveal further insights into the mechanism
by which ghrelin exerts modulatory control over gastric
mucosal inflammatory disturbances caused by H. pylori
infection, we examined the influence of ghrelin on H.
pylori LPS-induced changes in the activity of IKK-β a
key enzyme of NF-κB activation pathway that controls
the extent of IκB-α phosphorylation and its proteasomal
degradation [12,14,17]. We found that countering effect
of ghrelin on the LPS-induced up-regulation in gastric
mucosal cell IKK-β activity was subject to suppression
by Src inhibitor, PP2 as well as Akt inhibitor, SH-5. A
significant decrease in the effectiveness of ghrelin to
counter the LPS-induced IKK-β activation was also ob-
served in the presence of cNOS inhibitor, L-NAME,
while the inhibitor of iNOS, 1400 W, had no effect. Fur-
thermore, in consonance with the documented involve-
ment of Akt in rapid cNOS activation through phos-
phorylation at Ser1179 [9,24,33], ghrelin-induced up-
regulation in cNOS activity was reflected in the increase
of enzyme phosphorylation that was susceptible to sup-
Figure 9. Schematic diagram illustrating modulatory mechanism of ghrelin action in countering the gastric mucosal proin-
flammatory events triggered by H. pylori LPS-induced NF-κB activation. Binding of the LPS to Toll-like receptor 4 (TLR4)/
MD2 leads to IKK complex activation by phosphorylation at its IKK-βcatalytic subunit, thus triggering up-regulation in IkB-α
phosphorylation and its proteasomal degradation. This causes the increase in NF-κB translocation to the nucleus to promote
iNOS gene transcription. Ghrelin, through binding to growth hormone secretagogue receptor (GHSR), inhibits the LPS-in-
duced IKK activation via up-regulation in Src/Akt-dependent cNOS activation that results in cNOS-mediated IKK-β S-nitro-
sylation, thus leading to suppression of IκB-α phosphorylation and degradation, inhibition of NF-κB nuclear translocation, and
the repression of iNOS gene transcription.

B. L. SLOMIANY ET AL. 8
pression by Akt inhibitor, SH-5. However, the hormone
produced no apparent effect on the extent of the LPS-
induced phosphorylation of IKK-β. From this, we con-
cluded that the countering effect of ghrelin on H. pylori
LPS-induced up-regulation in gastric mucosal IKK-β
activation and iNOS induction occurs with the involve-
ment of Akt-mediated cNOS activation. Indeed, signal-
ing through Src/Akt pathway is known to occupy a cen-
tral stage in the receptor (GHS-R)-mediated responses to
ghrelin stimulation [26,34], and the recent reports sug-
gest that the activity of IKK complex may also be subject
to inhibition through S-nitrosylation of a specific cys-
teine residue within the activation loop of IKK-β by the
endogenous NO donors [21,22,31].
Therefore, to address further the role of cNOS in the
regulation of IKK-β activation by ghrelin, we assessed
the effect of nitrosothiol reducing agent, ascorbate. We
found that while ascorbate produced no discernible effect
on the extent of the LPS-induced IKK-β activation, the
agent elicited a marked relieve in the inhibitory effect of
ghrelin on the LPS-induced IKK-β activity. Hence, tak-
ing into consideration known susceptibility of S-nitro-
sylated proteins to reduction by ascorbic acid [26,29,30,
33], we surmised that the countering effect of ghrelin on
H. pylori LPS-induced IKK-β activation are intimately
linked to the events of IKK-β protein S-nitrosylation by
NO generated by the cNOS system. Indeed, examination
of IKK-β S-nitrosylation by the biotin switch method,
revealed that ghrelin countering effect on the LPS-in-
duced up-regulation in IKK-β activity was manifested by
a marked increase in the kinase protein S-nitrosylation,
and subject to suppression by Akt inhibitor, SH-5. These
findings, together with the results of IKK-β activity as-
says, suggest that proinflammatory events elicited in
gastric mucosa by H. pylori and manifested by the induc-
tion in iNOS expression are the result of the LPS-in-
duced up-regulation in IKK-β activation through phos-
phorylation. Further, it is also evident that ghrelin exerts
the inhibitory effect on the LPS-induced activation
IKK-β via cNOS-mediated IKK-β S-nitrosylation, thus
leading to suppression in IκB-α phosphorylation and
degradation, inhibition in of NF-κB nuclear translocation,
and the repression of iNOS gene transcription. Conse-
quently, the role of ghrelin in modulation of the extent of
gastric mucosal inflammatory involvement appears to be
intimately linked to signaling events associated with
Src/Akt-mediated cNOS activation through phosphoryla-
tion.
Taken together, our results suggest that the initial phase
gastric mucosal proinflammatory events triggered by H.
pylori involves the disturbances in Src/Akt kinase-de-
pendent cNOS phosphorylation that leads to abrogation
of cNOS-mediated control over IKKβ activity through
S-nitrosylation and the increase in proteasomal degrada-
tion of IκB-α, thus allowing NF-κB translocation to the
nucleus to promote iNOS gene transcription. We also re-
port that ghrelin counters these untoward consequences
of H. pylori LPS via up-regulation in Src/Akt-dependent
cNOS activation through phosphorylation that results in
up-regulation in cNOS-mediated IKK-β S-nitrosylation
which interferes with IκB-α phosphorylation and protea-
somal degradation, thereby preventing the nuclear trans-
location of NF-κB and the activation of iNOS gene tran-
scription. A schematic diagram illustrating the proposed
mechanism of anti-H. pylori action of ghrelin is depicted
in Figure 9.
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