American Journal of Molecular Biology, 2011, 1, 43-51
doi:10.4236/ajmb.2011.12006 Published Online July 2011 (http://www.SciRP.org/journal/ajmb/ AJMB ).
Published Online July 2011 in SciRes. http://www.scirp.org/journal/AJMB
Ghrelin-induced cSrc activation through constitutive nitric
oxide synthase-dependent S-nitrosylation in modulation of
salivary gland acinar cell inflammatory responses to
Porphyromonas gingivalis
Bronislaw L. Slomiany, Amalia Slomiany
Research Center, University of Medicine and Dentistry of New Jersey, Newark, USA.
Received 9 March 2011; revised 20 April 2011; accepted 28 April 2011.
ABSTRACT
A peptide hormone, ghrelin, recognized for its role in
the regulation of nitric oxide production has emerged
as an important modulator of oral mucosal inflam-
matory responses to periodontopathic bacterium, P.
gingivalis. As cSrc kinase plays a major role in con-
trolling the activity of nitric oxide synthase (NOS)
system, in this study we investigated the influence of
P. gingivalis LPS on the processes of Src activation in
rat sublingual gland acinar cells. The LPS-induced
enhancement in the activity of inducible (i) iNOS and
the impairment in constitutive (c) cNOS were re-
flected in the suppression in cSrc activity and the ex-
tent of its phosphorylation at Tyr416. Further, we show
that the countering effect of ghrelin on the LPS-
induced changes in cSrc activity and the extent of its
phosphorylation was accompanied by a marked re-
duction in iNOS and the increase in cNOS activation
through phosphorylation at Ser1179. Moreover, the
effect of ghrelin on cSrc activation was associated
with the kinase S-nitrosylation that was susceptible to
the blockage by cNOS inhibition. Our findings sug-
gest that P. gingivalis-induced up-regulation in iNOS
leads to disturbances in cNOS phosphorylation that
exerts the detrimental effect on the processes of cSrc
activation through cNOS mediated S-nitrosylation.
We also show that the effect of ghrelin on P. gin-
givalis-induced inflammatory changes are manifested
in the enhancement in cSrc activation through
S-nitro- sylation and the increase in its phosphoryla-
tion at Tyr416.
Keywords: Ghrelin; P. Gingivalis; Salivary Acinar
Cells; cNOS; cSrc Activation; S-Nitrosylation
1. INTRODUCTION
Ghrelin, a 28-amino acid peptide hormone, initially iso-
lated from the stomach [1,2], and more recently identi-
fied in oral mucosa, saliva and the acinar cells of sali-
vary glands [3], is recognized as an important modulator
of oral mucosal inflammatory responses to periodonto-
pathic bacterium, P. gingivalis through the regulation of
nitric oxide synthase (NOS) isozyme system [4-6]. The
signaling mechanism that underlies the regu lation of NO
by ghrelin involves the stimulation of growth-hormone
secretagogue receptor type 1a (GHSR1a), a seven-trans-
membrane G-protein coupled receptor, that leads to ac-
tivation of heterotrimeric G protein-dependent network
of protein kinases, including that of cellular Src (cSrc), a
member of the non-receptor protein tyrosine kinase Src
family [7-10]. All currently known eleven members of
the Src family kinases share similar structural organiza-
tion, consisting of N-terminal myristoylation motif that
facilitates membrane attachment, followed by th e protein
binding SH3 and SH2 homology domains, a protein -
tyrosine kinase domain, and a C-terminal regulatory tail
[8,10].
The 60 kDa phosphoprotein (pp60c-src) Src is encoded
by a physiological c-src gene, which is cellular homo-
logue of the highly transforming v-src gene of Rouse
sarcoma virus [8,11,12]. The activity of cSrc is tightly
regulated by reversible phosphorylation on Tyr527 and
Tyr416 amino acid residues, which either inactivates or
activates the kinase. The inhibitory phosphorylation at
Tyr527 of C-terminal tail locks the kinase in an inactive
dormant conformation through the interaction with its
SH2 domain [9]. The dormant form of the enzyme is
destabilized by dephos ph or y l ation or displ a cement of the
inhibitory Tyr527 from the SH2 binding pocket, thus ex-

B. L. Slomiany et al. / American Journal of Molecular Biology 1 (2011) 43-51 44
posing the activation loop to autophosphorylation at
Tyr416 which stabilizes the enzyme in its active state [9,
11]. Furthermore, there are indications that in addition to
phosphorylation/dephosphorylation-based circuitry of Src
activation, the activity of cSrc may be also regulated
through S-nitrosylation at the kinase cysteine residues
located within the C-terminal region of its catalytic do-
main [11,13,14].
Indeed, protein modification through targeted S-nitro-
sylation at the critical cysteine residues, with the invol-
vement of both constitu tive and inducible forms of NOS
system, is gaining recognition as an important post-
translational event of significance to a variety of bio-
logical processes affected by NO [4-6,13,14]. Moreo ver,
the NO-induced Src protein S-nitrosylation has been
shown to promote the kinase activation through auto-
phosphorylation at Tyr416 that appears to be independent
of the phosphorylation status of C-terminal Tyr527
autoinhibitory site [14-16].
As oral mucosal inflammatory responses to periodon-
topathic bacterium, P. gingivalis, are characterized by
the disturbances in NO production , and since cSrc kinase
plays a central role in transduction of signals that regu-
late the activity of NOS isozyme system [4,5,13,14], in
this study we investigated th e impact of P. gingivalis key
virulence factor, LPS, on the cSrc kinase activity in sub-
lingual salivary gland acinar cells. Moreover, consider-
ing the demonstrated role of ghrelin in the regulation of
NOS system [4,5,17], we examined the influence of this
peptide hormone on processes of cSrc activation through
S-nitrosylation.
2. MATERIALS AND METHODS
2.1. Sublingual Salivary Gland Cell Incubation
The acinar cells of sublingual salivary gland, collected
from freshly dissected rat salivary glands, were sus-
pended in five volumes of ice-cold Dulbecco’s modified
(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, and gently dispersed by trituration with a syringe,
and settled by centrifugation [18]. After rinsing, the cells
were resuspended in the medium to a concentration of 2
× 107 cell/ml, transferred in 1 ml aliquots to DMEM in
culture dishes and incubated under 95% O2 - 5% CO2
atmosphere at 37˚C for 16h in the presence of P. gin-
givalis LPS [5]. In the experiments evaluating the effect
of ghrelin (rat, Sigma), cNOS inhibitor, L-NAME, iNOS
inhibitor, 1400 W, 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
preparations before and during the experimentation, as-
sessed by Trypan blue dye exclusion assay [18], was
greater than 98%.
2.2. Porphyromonas Gingivalis
Lipopolysaccharide
P. gingivalis used for LPS preparation was cultured from
clinical isolates obtained from ATCC No. 33277 [19].
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.
Analyses indicated that such obtained LPS preparation
was essentially free of nucleic acids as determined by
absorption at 260 nm, and its protein content, measured
by BCA assay kit, was less than 0.2%.
2.3. NO Production, and cNOS and iNOS
Activity Assay
NO production in the acinar cells of sublingual salivary
gland was determined by measuring the stable NO me-
tabolite, nitrite, accumulation in the culture medium us-
ing Griess reaction [20]. A 100 µl of spent culture me-
dium was incubated for 10 min with 0.1 ml of Griess
reagent (Sigma) and the absorbance was measured at
570 nm. The activity of cNOS and iNOS enzymes was
measured by monitoring the conversion of L-[3H] argin-
ine to L-[3H] citrulline u sing NOS-detect k it (Stratagene).
The acinar cells from the control and experimental treat-
ments were homogenized in a sample buffer containing
either 10 mM EDTA (for Ca2+-independent iNOS) or 6
mM CaCl2 (for Ca2+-dependent cNOS), and centrifuged
[5,18]. The aliquots of the resulting supernatant were
incubated for 30 min at 25˚C in the presence of 50
µCi/ml of L-[3H] arginine, 10 mM NAPDH, 5 µM tet-
rahydrobiopterin, 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 quanti-
fied by scintillation counting.
2.4. Src Kinase Activity Assay
Tyrosine kinase activity of cSrc in sublingual salivary
gland acinar cells was measured by using polyE4Y
(Sigma) and [

-32P] ATP (Amersham) as the substrates
[21]. The cells were lysed in lysis buffer (20 mM Tris-
HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.5%
sodium deoxycholate, 2 mM EDTA, 1 mM sodium or-
thovanadate, 1 mM PAF, and 1 mM NaF), containing
protease inhibitor cocktail (Sigma), at 4˚C for 30 min,
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centrifuged at 12,000 × g for 10 min, and the super-
natants were subjected to protein determination using
BCA protein assay kit (Pierce). The supernatant samples
containing equal total protein content were then im-
munoprecipitated with anti-Src antibody (Sigma) for 2 h
at 4˚C. Protein A/G agarose beads were added for an
additional 1 h, and the immune complex was recovered
by centrifugation and thoroughly washed with lysis
buffer. The agarose beads were then suspended for 30
min at room temperature in the kinase assay buffer (10
mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM MgCl2, 5
mM MnCl2, 2 mM EDTA, and 2 mM dithithreitol), cen-
trifuged, and the supernatants used for the Src activity
assay. For this, the samples containing 25 µg of the cell
homogenate protein in 50 µl of Src kinase assay buffer
were incubated with 1 mg/ml of polyE4Y and 100 mM
ATP containing 10 µCi of [

-32P] ATP, at 30˚C for 20
min. The reaction was terminated by adding 25 µl of
40% trichloroacetic, the samples were spotted onto phos-
phorcellulose paper, and following thorough washing
with 1% phosphoric acid and then acetone [22], the ra-
dioactivity incorpo r ated into polyE4Y was determined by
liquid scintillation counting.
2.5. Src Kinase Phosphorylation Assay
Measurements of the phosphorylation status of cSrc at
Tyr416 in sublingual salivary gland acinar cells were
conducted using Phospho-Src ( Tyr416) ELISA kits (Cell
Signaling). The acinar cells were lysed on ice for 30 min
in lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl,
1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM
sodium pyrophosphate,1 mM sodium orthovanadate, 1
mM β-glycerophosphate, 1 mM PMSF, 1 µg/ml leu-
peptin), and centrifuged at 12,000 × g for 10 min. The
supernatants diluted (1:1) in stan dard diluent buffer were
pipetted in 100 µl aliquots into wells containing immo-
bilized capture antibody specific for Src (pTyr416), and
following washing the complex was reacted with Src
detection antibody. After washing, the retained complex
was labeled with horseradish peroxidase and probed
with TMB reagent for spectrophotometric quantification
at 450 nm.
2.6. Src Protein S-Nitrosylation Assay
Assessment of cSrc kinase S-nitrosylation in the acinar
cells of sublingual salivary gland was conducted using a
biotin switch procedure for protein S-nitrosylation [23,
24]. The cells were treated with iNOS inhibitor, 1400W
(30 µM) or cNOS inhibitor, L-NAME (200 µM), or
ghrelin (0.6 µg/ml), and incubated for 16 h in the pres-
ence of 100 ng/ml of P. gingivalis 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-
thane-thiosu lfonate reagent at 50˚C for 20 min [24]. 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
streptavidin beads with 50 µl of elution buffer (20 mM
HEPES, 100 mM NaCl, 1 mM EDTA, pH 7.7) contain-
ing 1% 2-mercap-toethanol [24]. The obtained proteins
were then analyzed b y Western blotting.
2.7. Immunoblotting Analysis
The acinar cells of sublingual salivary gland from the
control and experimental treatments were collected by
centrifugation and resuspended 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 leu-
peptin and 1 µg/ml p epstatin [5]. Follo wing brief sonica-
tion, the lysates were centrifuged at 10,000 × g for 10
min, and the supernatants were subjected to protein de-
termination using BCA protein assay kit (Pierce). The
samples, including those subjected to biotin switch pro-
cedure, were then resuspended in loading buffer, boiled
for 5 min, and subjected to SDS-PAGE using 40 µg pro-
tein/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 the
antibody against phosphorylated protein at 4˚C for 16 h.
After 1 h incubation with the horseradish peroxidase-
conjugated secondary antibody, the phosphorylated pro-
teins were revealed using an enhanced chemilumines-
cence. Membranes were stripped by incubation in 1M
Tris-HCl (pH 6.8), 10% SDS, and 10 mM dithiotreitol
for 30 min at 55˚C, and probed with antibody against
total cNOS, iNOS or Src. Immunoblotting was perform-
ed using specific antibodies directed against iNOS,
cNOS and phospho-cNOS (Ser1179), (Calbiochem), and
cSrc (monoclonal 327, Sigma) and phospho-Src (Tyr416),
(Cell Signaling).
2.8. 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 Kru-
skal-Wallis tests were used to determine significance.
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Figure 1. Effect of P. gingivalis LPS on the expression of Src
kinase activity and nitrite production in rat sublingual salivary
gland acinar cells. The acinar cells were treated with the indi-
cated concentrations of the LPS and incubated for 16 h. Values
represent the means ±SD of five experiments. *p < 0.05 com-
pared with that of control (LPS - 0).
Figure 2. Effect of P. gingivalis LPS on Src kinase tyrosine
(Ty r416) phosphorylation and the expression of inducible nitric
oxide synthase (iNOS) activity in rat sublingual salivary gland
acinar cells. The cells were treated with the indicated concen-
trations of the LPS and incubated for 16 h. *p < 0.05 compared
with that of control (LPS - 0).
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 ex amine the influence of periodontopathic bacterium,
P. gingivalis, on the relationship between cSrc kinase
activation and the disturbances in NO production, we
employed rat sublingual salivary gland acinar cells ex-
posed to P. gingivalis key virulence factor, LPS. We
found that the LPS-induced massive in crease in NO pro-
duction was associated with a dose-dependent decrease
in the acinar cell Src activity, which at 100 ng/ml LPS
showed a 28.6% decrease (Figure 1). Moreover, the
inhibitory effect of the LPS, at 100 ng/ml, on cSrc activ-
ity was reflected in a 25% drop in the enzyme Tyr416
phosphorylation, and a 30% drop in the enzyme Tyr416
Figure 3. Effect of ghrelin on P. gingivalis LPS-induced
changes in sublingual salivary gland acinar cell expression of
Src kinase and cNOS activities. The cells, preincubated with
the indicated concentrations of ghrelin, were treated with the
LPS at 100 ng/ml and incubated for 16 h. *p < 0.05 compared
with that of control. **p < 0.05 compared with that of LPS
alone.
phosphorylation occurred at 200 ng/ml LPS (Figure 2).
We also established that the disturbances in NO produc-
tion elicited in the acinar by P. gingivalis LPS at 100
ng/ml were manifested by a 26.4-fold up-regulation in
iNOS activity (Figure 2), while the activity of cNOS
showed a 5.2-fold decrease (Figure 3).
Our further results revealed that preincubation of the
acinar cells with ghrelin led to a concentration-depend-
ent reversal in the LPS-induced suppression of cSrc ac-
tivity and the extent of its protein phosphorylation on
Tyr416. As a result the activity of cSrc in the presence of
0.6 µg/ml ghrelin increased 2.2-fold over that of the LPS
(Figure 3), while the Src protein phosphorylation at
Tyr416 showed a 2.3-fold increase (Figure 4). Further-
Figure 4. Effect of ghrelin on P. gingivalis LPS-induced
changes in Src kinase tyrosine (Tyr416) phosphorylation and the
expression of iNOS activity in sublingual salivary gland acinar
cells. The cells, preincubated with the indicated concentrations
of ghrelin, were treated with the LPS at 100ng/ml and incu-
bated for 16 h. 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.
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Figure 5. Effect of iNOS inhibitor, 1400W, and cNOS inhibitor,
L-NAME, on the ghrelin (Gh)-induced changes in Src kinase
activity and its phosphorylation at tyrosine (Tyr416) in the sali-
vary gland acinar cell exposed to P. gingivalis LPS. The cells,
preincubated with 30 µM 1400 W (14 W), or 200 µM
L-NAME (LN), were treated with Gh at 0.6 µg/ml and incu-
bated for 16 h in the presence of 100 ng/ml LPS. Values repre-
sent 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 compared with that of Gh  LPS.
Figure 6. Effect of ghrelin (Gh) on P. gingivalis LPS-induced
changes in the expression of iNOS protein and cNOS phos-
phorylation in the salivary gland acinar cells. The cells were
treated with Gh at 0.6 µg/ml or Src inhibitor, PP2 at 20 µM +
Gh, and incubated for 16 h in the presence of 100 ng/ml LPS.
Cell lysates were resolved on SDS-PAGE, transferred to nitro-
cellulose and probed with phosphorylation specific cNOS
(pcNOS) antibody, and after stripping the membranes were
probed with anti-cNOS and anti-iNOS antibody. The immuno-
blots shown are representative of three experiments.
more, we observed that ghrelin at 0.6 µg/ml evoked a
5.3-fold increase in the acinar cell cNOS activity (Fig-
ure 3), and produced a 13.9-fold reduction in the LPS-
induced iNOS activity (Figure 4).
To reveal further insight into the relationship between
ghrelin-induced changes in the activity cNOS and iNOS
enzymes and the acinar cell cSrc activation, we exam-
ined the role of NO generated by the NOS isozyme sys-
tem. For this, the cells prior to incubation with ghrelin
were pretreated with cNOS inhibitor, L-NAME, or iNOS
inhibitor, 1400 W, and assayed for cSrc activity as w ells
as the extent of its pro tein phosphorylation at Tyr 416. The
results revealed that ghrelin-induced up-regulation in
cSrc activity and Tyr416 phosphorylation was subject to
inhibition by cNOS inhibitor, L-NAME, whereas prein-
cubation with iNOS inhibitor, 1400 W, produced an am-
plification in the effect of ghrelin on Src activity and the
extent of its protein phosphorylation at Tyr416 (Figure 5).
These data, thus, indicate that ghrelin-induced Src acti-
vation throu gh its Tyr416 phosphorylation occurs with the
involvement of cNOS.
As NO generated by iNOS plays a role in P. gingivalis
LPS-induced cNOS S-nitrosylation that interferes with
the enzyme activation throu gh phosphorylation at Ser1179
[5], we next examined the influence of ghrelin on the
expression of iNOS protein and cNOS phosphorylation.
The acinar cells prior to incubation with the LPS were
pretreated with gh relin or g hre lin p lus Src inhib itor, PP2,
and the lysates were probed with antibodies directed
against iNOS, cNOS and phosphorylated cNOS (Ser1179).
As shown in Figure 6, the effect of the LPS was mani-
fested in the induction of iNOS protein expression and
the inhibition in cNOS phosphorylatio n, while the coun-
tering effect of ghrelin was reflected in a marked reduc-
tion in the iNOS protein expression and the increase in
cNOS phosphorylation at Ser1179. Moreover, the sup-
pression of ghrelin effect on cNOS phosphorylation as
well as further reduction in iNOS protein was observed
in the presence of Src kinase inhibitor, PP2 (Figure 6),
thus suggesting th e involv ement of cSrc in the regulatio n
of NOS isozyme system at both translational and post-
translational levels.
Figure 7. Effect of ascorbate on the ghrelin (Gh)-induced
changes in the expression of Src kinase and cNOS activities in
sublingual salivary gland acinar cells exposed to P. gingivalis
LPS. The cells, preincubated with 300 µM ascorbate (As),
were treated with Gh at 0.6 µg/ml and incubated for 16h in the
presence of 100 ng/ml 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
compared with that of Gh  LPS.
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Figure 8. Effect of ghrelin (Gh) on P. gingivalis LPS-induced
Src kinase S-nitrosylation. The salivary gland acinar cells were
treated with Gh (0.6 µg/ml), or cNOS inhibitor, L-NAME (200
µM) + Gh, and incubated for 16 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, trans-
ferred to nitrocellulose and probed with phospho-Src (Tyr416)
antibody, and after stripping reprobed with anti-Src antibody.
The immunoblots shown are representative of three experi-
ments.
Further, we found that ghrelin countering effect on the
LPS-induced Src activation in the acin ar cells of salivary
gland displayed susceptibility to suppression by nitro-
sothiols reducing agent, ascorbate, which also produced
enhancement in the effect of ghrelin on cNOS activity
(Figure 7). Hence, to ascertain the relationship between
Src kinase S-nitrosylation and its activation through
phosphorylation, we examined the dependence of cSrc
S-nitrosylation on the ghrelin-induced cNOS activation
by the biotin switch method [23,24]. The acinar cells
were incubated with P. gingivalis LPS or ghrelin + LPS
or cNOS inhibitor, L-NAME, + ghrelin + LPS, and the
lysates following the biotin switch procedure were
probed with antibodies directed against phospho-cSrc
(Tyr416) and total Src. Western blot analysis revealed that
ghrelin countering effect on the LPS-induced suppres-
sion in cSrc activity was manifested by a marked in-
crease in both the Src kinase S-nitrosylation as well as
its protein phosphorylation. Moreover, the blockage of
cNOS activity with L-NAME, not only caused the loss
in cSrc S-nitrosylation but was also accompanied by a
substantial decrease in Src protein phosphorylation at
Tyr416 (Figure 8). Thus, ghrelin countering effects on P.
gingivalis-induced inflammatory changes are manifested
in the enhancement in Src activation through cNOS-
dependent S-nitrosylation and the increase in its phos-
phorylation.
4. DISCUSSION
P. gingivalis is a Gram-negative bacterium found in pe-
riodontal packets of patien ts with periodon titis, a chronic
inflammatory disease that affects 10% - 15% of adult
population and is a major cause of adult tooth loss [25,
26]. The oral mucosal responses to P. gingivalis and its
key virulence factor, cell wall LPS, are characterized by
a massive rise in epithelial cell apoptosis and proin-
flammatory cytokine production, and the disturbances in
NOS isozyme system responsible for NO generation [4-
6,19,27]. Investigations into the endogenous factors ca-
pable of influencing the extent of mucosal inflammatory
responses along the alimentary tract, including that of
oral cavity, have brought to focus the importance of a
peptide hormone, ghrelin [1-4,28]. A growing body of
evidence, furthermore, points to ghrelin as a principal
modulator of the mucosal NOS [4-6,17]. The mechanism
that underlies the regulation of NO signaling by ghrelin
relies on the receptor (GHS-R1a)-mediated activation of
G protein-dependent network of protein kinases, includ-
ing that of membrane-associated non-receptor tyrosine
kinase, cSrc [7-10].
As Src kinase plays a pivotal role in the transduction
of signals that regulate the activity of NOS isozyme sys-
tem [8,13,14], in this study we investigated the influence
of P. gingivalis LPS on the processes associated with Src
activation. Our findings revealed that the LPS-induced
drop in sublingual salivary gland acinar cells activity of
cNOS and up-regulation in iNOS was associated with
the suppression in the activity of cSrc. Moreover, the
suppression in cSrc activity was reflected in a decrease
in the kinase phosphorylation at Tyr416. Furthermore,
preincubation of the acinar cells with ghrelin exerted
countering effect on the LPS-induced impairment in
cSrc activity and the extent of its phosphorylation on
Tyr416, and was accompanied by an increase in the cNOS
activity and a marked reduction in the activity of iNOS.
These findings are thus in keeping with the literature
data suggesting the involvement of cSrc kinase in the
regulation of NOS isozyme system at both transcrip-
tional and post-transcriptional levels [13,14].
Indeed, in concordance with the documented in-
volvement of cSrc in post-translational cNOS activation
through phosphorylation [5,18,29], we found that the
induced up-regulation in cNOS activity by ghrelin was
reflected in the increase of enzyme protein phosphoryla-
tion at Ser1179. Also, as up-regulation in iNOS activity in
response to LPS involves transcriptional factor NF-B
transactivation of iNOS gene for the induction in the
enzyme protein [30-32], we analyzed the influence of
ghrelin on the acinar cell iNOS protein expression. We
found that P. gingivalis LPS induction in iNOS activity
was associated with the increase in the enzyme protein
expression, while the countering effect of ghrelin, was
reflected in a marked inhibition of the iNOS protein ex-
pression that was further suppressed in the presence of
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Src kinase inhibitor, PP2.
Next we addressed the relationship between the ghre-
lin-induced changes in activity of cNOS and iNOS en-
zymes, and the Src kinase activation. As up-regulation in
cSrc activation through autophosphorylation on Tyr416
has been reported in the presence of the exogenous NO
donors as well as the NO produced by endothelial NOS
[13-15], we examined the effect of NOS inhibitors on
cSrc activity and the extent of its protein phosphoryla-
tion at Tyr416. We found that ghrelin-induced up-regula-
tion in the acinar cell cSrc activity and Tyr416 phos-
phorylation displayed susceptibility to cNOS inhibitor,
L-NAME, while an amplification in the ghrelin effect on
cSrc activation was attained with the inhibitor of iNOS,
1400 W. From this, we concluded that cNOS plays an
essential role in ghrelin-induced activation of cSrc in th e
acinar cells. Furthermore, the countering effect of ghre-
lin on the LPS-induced changes in cSrc activation was
susceptible to the suppression by nitrosothiols reducing
agent, ascorbate, which also produced an enhancement
in the effect of ghrelin on cNOS activity. These data,
together with the known susceptibility of S-nitrosylated
proteins to reduction by ascorbic acid [4,5,14,15,23,24],
demonstrate that P. gingivalis LPS-induced disturbances
in the acinar cell cNOS and cSrc protein S-nitrosylation
patterns interfere with the activation of both cNOS and
cSrc. Moreover, our results suggest that ghrelin-induced
up-regulation in cSrc activity th rough phosph orylation at
Tyr416 is intimately linked to the events of the kinase
protein S-nitrosylation by NO generated by the cNOS.
Our assertion is further supported by the literature
evidence indicating that ascorbate treatment both in-
creases cNOS activity and reduces the enzyme protein S-
nitrosylation, and th at the countering effect of ghrelin on
the LPS-induced impairment in cNOS activity is associ-
ated with the loss of the enzyme protein S-nitrosylation
and the increase in its phosphorylation at Ser1179 [5,13,
32]. Indeed, the accumulating evidence demonstrates
that protein modification through targeted S-nitrosyla-
tion at the critical cysteine, with the participation of bo th
constitutive and inducible forms of NOS system, is a
post-translational event of significance to the regulation
of signal transduction pathways by NO [4-6,13-15,17,32,
33]. Moreover, NO-induced Src S-nitrosylation at the
critical cysteine residues located within the C-terminal
region of its catalytic domain has been reported to pro-
mote cSrc activation through autophosphorylation at
Tyr416 [13-15].
Hence, to assess the role of ghrelin in countering P.
gingivalis LPS-induced interference with cSrc activation
in the acinar cells, we examined the dependence of Src
S-nitrosylation on the ghrelin-induced cNOS activation
by the biotin switch assay. Western blot analysis of the
acinar cell lysates revealed that ghrelin countering effect
on the LPS-induced suppression in cSrc activity was
manifested by a marked increase in the kinase protein S-
nitrosylation as well as phosphorylation at Tyr416. Fur-
thermore, the suppression of ghrelin effect on NO pro-
duction with cNOS inhibitor, L-NAME, caused the loss
in cSrc S-nitrosylation and a decrease in the kinase
phosphorylation. These findings thus imply that the
changes evoked by P. gingivalis in the activity of NOS
isozyme system involved in NO generation are of direct
relevance to cSrc kinase activation through autophos-
phorylation at Tyr416, and point to the role of cSrc
S-nitrosylation in the protective mechanism of ghrelin
action.
In conclusion, our findings demonstrate that P. gin-
givalis-induced up-regulation in the acinar cell iNOS
leads to disturbances in cNOS phosphorylation that ex-
erts the detrimental effect on the processes of cSrc
kinase activation through cNOS mediated S-nitrosyla-
tion. We also show that the effects of ghrelin on P. gin-
givalis-induced inflammatory disturbances are mani-
fested in the enhancement in Src activation through S-
nitrosylation and the increase in its phosphorylation at
Tyr416.
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