Cytosolic phospholipase A2 S-nitrosylation in ghrelin protection against detrimental effect of ethanol cytotoxicity on gastric mucin synthesis ——Ghrelin in gastric mucosal protection

doi:10.4236/health.2010.29152

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Vol.2, No.9, 1033-1039 (2010) Health

doi:10.4236/health.2010.29152

Cytosolic phospholipase A2 S-nitrosylation in ghrelin

protection against detrimental effect of ethanol

cytotoxicity on gastric mucin synthesis

——Ghrelin in gastric mucosal protection

Bronislaw L. Slomiany, Amalia Slomiany

Research Center University of Medicine and Dentistry of New Jersey Newark, Newark, USA; Corresponding Author: slomiabr@umdnj.edu

Received 12 March 2010; revised 8 April 2010; accepted 11 April 2010.

ABSTRACT

Ghrelin, a peptide hormone produced mainly in

the stomach, has emerged recently as an im-

portant regulator of nitric oxide synthase (NOS)

and cyclooxygenase (COX) enzyme systems,

the products of which play direct cytoprotective

function in the maintenance of gastric mucosal

integrity. In this study, using gastric mucosal

cells, we report on the role of ghrelin in coun-

tering the cytotoxic effect of ethanol on mucin

synthesis. We show that the countering effect of

ghrelin on mucin synthesis was associated with

the increase in NO and PGE2 production, and

characterized by a marked up-regulation in cy-

tosolic phospholipase A2 (cPLA2) activity. The

ghrelin-induced up-regulation in mucin synthe-

sis, like that of cPLA2 activity, was subject to

suppression by Src inhibitor, PP2 and ERK in-

hibitor, PD98059, as well as ascorbate. Moreover,

the loss in countering effect of ghrelin on the

ethanol cytotoxicity and mucin synthesis was

attained with cNOS inhibitor, L-NAME as well as

COX-1 inhibitor SC-560. The effect of L-NAME

was reflected in the inhibition of ghrelin-induced

mucosal cell capacity for NO production, cPLA2

S-nitrosylation and PGE2 generation, whereas

COX-1 inhibitor caused only the inhibition in

PGE2 generation. Our findings suggest that the

activation of gastric mucosal cPLA2 through

cNOS-induced S-nitrosylation plays an essen-

tial role in the countering effect of ghrelin on the

disturbances in gastric mucin synthesis caused

by ethanol cytotoxicity.

Keywords: Ghrelin; Ethanol Cytotoxicity; Gastric

Mucin; CNOS; CPLA2; S-Nitrosylation

1. INTRODUCTION

Acute gastric mucosal lesions, mucosal inflammatory

changes, and gastro-duodenal ulceration are well-recogni-

zed consequences of alcohol abuse on the health of gas-

trointestinal tract [1-3]. The gastric mucosal responses to

ethanol cytotoxicity are manifested by microcirculatory

changes, elevation in proinflammatory cytokine produc-

tion, enhancement in epithelial cell apoptosis, and the

impairment in prostaglandin and nitric oxide signaling

pathways [2,4,5]. Furthermore, the disturbances in mu-

cus producing gastric mucosal cells by ethanol affect the

synthesis of mucins, the glycoproteins that maintain the

integrity and strength of the protective mucus layer which

constitutes the pre-epithelial element of gastric mucosal

defense [6,7]. Hence, the alterations in the synthesis and

processing of gastric mucins induced by ethanol are di-

rectly reflected in the impairment of mucus coat function

that weakens the inherent resistance of the mucosa to

injury, and facilitates the onset of gastric disease [3,6,7].

Recent advances in understanding the nature of fac-

tors involved in the maintenance gastric mucosal integ-

rity revealed that the extent of mucosal protection

against ethanol cytotoxicity is influenced by ghrelin, a

28-amino acid peptide hormone produced mainly in the

stomach [4,8,9]. This endogenous ligand for the growth

hormone secretagogue receptor, has been implicated in

the control of local inflammations, healing experimen-

tally induced gastric ulcers, and the protection of gastric

mucosa against acute injury by ethanol [4,9,10]. More-

over, ghrelin emerged as an important regulator of the

cross-talk between NOS and COX enzyme systems [9],

the products of which, NO and PGE2, play direct cyto-

protective role in maintaining the gastric mucosal integ-

rity under normal physiological conditions [11,12].

Indeed, the literature data support the existence of a

functional relationship between the products of NOS and

B. L. Slomiany et al. / HEALTH 2 (2010) 1033-1039

1034

COX systems, and there are strong indications that the

enzyme compartmentalization and substrate availability

determines the segregated utilization of the respective

products in physiological and pathophysiological proc-

esses [13-15]. The stimulation of NO production through

NOS induction or the exogenous NO donors leads to up-

regulation in COX enzymes activation and the increase

in prostaglandin generation [14-17], while the inhibition

of NOS decreases prostaglandin formation. Moreover, it

has been reported that the NO-induced enzyme protein

S-nitrosylation impacts the events of cytosolic phos-

pholipase (cPLA2) activation, and hence influence the

processes associated with arachidonic acid release for

prostaglandin synthesis [18].

In our previous report, using rat gastric mucosal cells,

we have shown that ghrelin protection against ethanol

cytotoxicity involves cNOS-mediated cPLA2 activation

for the increase in PGE2 production [19]. Here, we ex-

amined the influence of ghrelin on the disturbances in

gastric mucin synthesis caused by ethanol.

2. MATERIALS AND METHODS

2.1. Cell Preparation and Mucin Synthesis

The gastric mucosal cells, collected from freshly dis-

sected rat stomachs, were suspended 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 serum, and gently dispersed by tritu-

ration with a syringe, and settled by centrifugation [5].

Following rinsing, the cells were resuspended in the me-

dium to a concentration of 2 × 107 cell/ml, transferred in

1 ml aliquots to DMEM in culture dishes containing

[3H]glucosamine (110 µCi), used as a marker of mucin

synthesis, and incubated under 95% O2-5% CO2 atmos-

phere at 37˚C for 16 h [20]. After washing with DMEM

containing 5% albumin to remove free radiolabel, the

cells were resuspended in fresh DMEM and incubated

for 2 h in the presence of 3% ethanol [5]. In the experi-

ments evaluating the effect of ghrelin (rat, Sigma), cNOS

inhibitor, L-NAME, iNOS inhibitor, 1400 W, Src inhibi-

tor, PP2, ERK1/2 inhibitor, PD98059 (Calbiochem),

COX-1 inhibitor, SC-560, COX-2 inhibitor, NS398, and

ascorbate (Sigma), the cells were first preincubated for

30 min with the indicated dose of the agent or vehicle

followed by incubation with ethanol. The viability of

cell preparations before and during the experimentation,

assessed by Trypan blue dye exclusion assay, was

greater than 97%. At the end of the specified incubation

period, the cells were centrifuged, washed with phos-

phate-buffered saline, and the combined supernatants used

for mucin assay.

2.2. Mucin Analysis and Ethanol

Cytotoxicity Assay

The combined cell wash and incubation medium con-

taining 3H-labeled mucin were treated at 4˚C with 10

volumes of 2% phosphotungstic acid in 20% trichloro-

acetic acid for 4 h and the formed precipitates were col-

lected by centrifugation. The glycoprotein precipitates

were dissolved in 6 M urea and chromatographed on a

Bio-Gel A-1.5 column, and the mucin fractions eluted in

the excluded volume were subjected to analysis for in-

corporation of radiolabel and protein content [20]. For

the measurement of ethanol-induced cytotoxicity, the

aliquots of cell suspension from the control and various

experimental conditions were centrifuged at 300 × g for

5 min and the supernatants used for the assay of cyto-

toxicity using TOX-7 lactate dehydrogenase assay kit

(Sigma).

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 a

PGE2 EIA kit (Cayman) and 100 µl aliquots of the spent

medium supernatant [5]. To assess nitric oxide produc-

tion in rat gastric mucosal cells, we measured the stable

NO metabolite, nitrite, accumulation in the culture me-

dium using Griess reaction [21].

2.4. CPLA2 Activity Assay

The measurement of cPLA2 activity in the gastric mu-

cosal cells following various experimental conditions

was carried out using cPLA2 assay kit (Cayman). The

cells were homogenized in 1 ml of 50 mM Hepes buffer,

pH 7.4, containing 1 mM EDTA, and centrifuged at

10,000 × g for 15 min at 4˚C. The supernatants were

filtered through an Amicon YM30 filter concentrators,

followed by 15 min incubation with 5 µM of calcium-

independent PLA2 inhibitor, bromoenol lactone, and the

aliquots (10 µl) of such prepared cell lysates were sub-

jected to cPLA2 assay [19].

2.5. CPLA2 S-Nitrosylation

A biotin switch procedure was employed to assess the

cPLA2 protein S-nitrosylation [22,23]. The gastric mu-

cosal cells, treated with ghrelin (0.8 µg/ml) or L-NAME

(300 µM)+ghrelin and incubated for 2 h in the presence

of 3% ethanol, were lysed in 0.2 ml of HEN lysis buffer,

pH 7.7, and the unnitrosylated thiol groups were blocked

with S-methyl methanethiosulfonate reagent [23]. The

proteins were precipitated with acetone, resuspended in

B. L. Slomiany et al. / HEALTH 2 (2010) 1033-1039

1035

0.2 ml of HEN buffer containing 1% SDS, and subjected

to targeted nitrothiol group reduction with sodium ascor-

bate (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) containing 1% 2-mercapto-

ethanol [22]. The obtained proteins were then analyzed

by Western blotting.

2.6. Western Blot Analysis

The mucosal cells, collected by centrifugation, were re-

suspended for 30 min in ice-cold lysis buffer [19], and

following brief sonication the lysates were centrifuged at

12,000 g for 10 min and the supernatants subjected to

protein determination using BCA protein assay kit

(Pierce). The samples, including those subjected to bio-

tin switch procedure, were then resuspended in loading

buffer, boiled for 5 min, and subjected to SDS-PAGE

using 50 µg protein/lane. The separated proteins were

transferred onto nitrocellulose membranes and probed

with the antibody (Cell Signaling) against the cPLA2,

and after incubation with the horseradish peroxidase-

conjugated secondary antibody, the protein bands were

revealed using an enhanced chemiluminescence detec-

tion.

2.7. Data Analysis

All experiments were carried out using duplicate sam-

pling, and the results are expressed as means ± SD.

Analysis of variance (ANOVA) was used to determine

significance and the significance level was set at P <

0.05.

3. RESULTS

To assess the influence of ghrelin on the disturbances in

gastric mucin synthesis caused by alcohol cytotoxicity,

we employed rat gastric mucosal cells exposed to etha-

nol at the dose range (3%) that impairs the mucosal ca-

pacity for mucin synthesis and prostaglandin generation

[1-3]. We determined that preincubation of the mucosal

cells with ghrelin led to a concentration-dependent pre-

vention of ethanol cytotoxicity, and afforded nearly com-

plete protection at 0.8 µg/ml of ghrelin (Figure 1). Fur-

ther, our results revealed that cytotoxicity induced in

gastric mucosal cells by 3% ethanol was reflected in a

32.6% decrease in mucin synthesis (Figure 1), as well as

a 29.8% reduction in PGE2 generation and a 51.7% drop

in NO production (Figure 2). Ghrelin at its optimal con-

centration (0.8 µg/ml) for the protection against ethanol

Figure 1. Effect of ghrelin on ethanol-induced cytotoxicity and

the synthesis of mucin in rat gastric mucosal cells. The cells,

labeled with [3H]glucosamine as a marker of mucin synthesis,

were treated with the indicated concentrations of ghrelin and

incubated for 2 h in the presence of 3% ethanol (Et). Values

represent the means ± SD of five experiments. *P < 0.05 com-

pared with that of control. **P < 0.05 compared with that of Et

alone.

Figure 2. Effect of ghrelin on ethanol-induced changes in the

production of PGE2 and nitrite by gastric mucosal cells. The

cells were treated with the indicated concentrations of ghrelin

and incubated for 2 h in the presence of 3% ethanol (Et). Val-

ues represent the means ± SD of five experiments. *P < 0.05

compared with that of control. **P < 0.05 compared with that

of Et alone.

cytotoxicity evoked a 55.4% increase in the mucosal cell

capacity for mucin synthesis (Figure 1), while NO pro-

duction increased 2.1-folds and PGE2 generation by

53.1% (Figure 2).

Moreover, we found that significant loss in the pre-

ventive effect of ghrelin on the ethanol-induced toxicity

and the cell capacity for mucin synthesis was attained

with cNOS inhibitor, L-NAME as well as specific COX-1

inhibitor, SC-560, while selective iNOS inhibitor, 1400W

and a specific COX-2 inhibitor, NS-398 had no effect

(Figure 3). The effect of L-NAME, furthermore, was

reflected in the inhibition of ghrelin-induced mucosal

cell capacity for NO production as well as PGE2 genera-

tion, whereas the pretreatment with COX-1 inhibitor,

B. L. Slomiany et al. / HEALTH 2 (2010) 1033-1039

1036

Figure 3. Effect of nitric oxide synthase and cyclooxygenase

inhibitors on the ghrelin (Gh)-induced protection against etha-

nol (Et) cytotoxicity, and the synthesis of mucin in gastric mu-

cosal cells. The cells, labeled with [3H] glucosamine, and pre-

incubated with 300 µM L-NAME (LN), 20 µM 1400 W (14

W), 30 µM PP2, 30 µM NS-398 (NS), 20 µM SC-560 (SC) or

30 µM PD98059 (PD), were treated with Gh at 0.8 µg/ml and

incubated for 2h in the presence of 3% Et. Values represent the

means ± SD of five experiments. *P < 0.05 compared with that

of control. **P < 0.05 compared with that of Et alone. ***P <

0.05 compared with that of Gh + Et.

SC560, led only to the inhibition in ghrelin-induced

PGE2 generation (Figure 4). The stimulatory effect of

ghrelin on the mucosal cell capacity for NO and PGE2

production, however, was not affected by the inclusion

of iNOS inhibitor 1400W and COX-2 inhibitor, NS-398

(Figure 4). Further, we found that the countering effect

of ghrelin on the ethanol-induced changes in gastric mu-

cosal cell capacity for mucin synthesis, and NO and

PGE2 production, were subject to suppression by Src

kinase inhibitor, PP2, while the inhibitor of MAPK/ERK1/2,

PD98059, caused the suppression in mucin synthesis and

PGE2 generation, but had no effect on NO production

(Figures 3 and 4).

As the initial and rate limiting step in prostaglandin

production is the liberation of arachidonic acid from me-

mbrane phospholipids by highly selective cPLA2 [5,24],

we next analyzed the effect of ghrelin on the mucosal

cell cPLA2 enzymatic activity. We found that ghrelin

countering effect on the ethanol-induced cytotoxicity and

the mucosal cell capacity for mucin synthesis was re-

flected in a 56.5% increase in cPLA2 activity (Figure 5).

Furthermore, the ghrelin-induced up-regulation in cPLA2

activity, like that of mucin synthesis, was subject to sup-

pression by Src inhibitor, PP2 and ERK1/2 inhibitor,

PD98059, as well as the inhibitor of cNOS, L-NAME

(Figure 5). The pretreatment with COX-1 inhibitor, SC-

560, however, while not causing any discernible altera-

tion in the cPLA2 activity, exerted the inhibitory effect

on mucin synthesis (Figure 5).

Figure 4. Effect of nitric oxide synthase and cyclooxygenase

inhibitors on the ghrelin (Gh)-induced changes in the produc-

tion of PGE2 and nitrite by gastric mucosal cells in the pres-

ence of ethanol (Et). The cells, preincubated with 300 µM

L-NAME (LN), 20 µM 1400 W (14 W), 30 µM PP2, 30 µM

NS-398 (NS), 20 µM SC-560 (SC) or 30 µM PD98059 (PD),

were treated with 0.8 µg/ml Gh and incubated for 2 h in the

presence of 3% Et. Values represent the means ± SD of five

experiments. *P < 0.05 compared with that of control. **P <

0.05 compared with that of Et alone. ***P < 0.05 compared

with that of Gh + Et.

Figure 5. Effect of nitric oxide synthase and cyclooxygenase

inhibitors on the ghrelin (Gh)-induced changes in mucin syn-

thesis and cPLA2 activity in gastric mucosal cells in the pres-

ence of ethanol (Et). The cells, labeled with [3H] glucosamine,

and preincubated with 300 µM L-NAME (LN), 30 µM PP2, 30

µM PD98059 (PD), 300 µM ascorbate (As) or 20 µM SC-560

(SC), were treated with Gh at 0.8 µg/ml and incubated for 2 h

in the presence of 3% Et. Values represent the means ± SD of

five experiments. *P < 0.05 compared with that of control. **P

< 0.05 compared with that of Et alone. ***P < 0.05 compared

with that of Gh  Et.

Since in addition to phosphorylation, the activation of

cPLA2 involves the NO-dependent enzyme protein S-

nitrosylation [18,19], we further analyzed the influence

of S-nitrosylation on gastric mucosal cPLA2 activity.

The results revealed that ghrelin-induced up-regulation

in cPLA2 activity and the cell capacity for mucin synthe-

sis was susceptible to suppression not only by the inhi-

B. L. Slomiany et al. / HEALTH 2 (2010) 1033-1039

1037

bitor of cNOS, L-NAME, but also by ascorbate (Figure

5), which is in keeping with known susceptibility of

S-nitrosylated proteins to this reducing agent [14,23].

Moreover, Western blot analysis of the mucosal cell lys-

ates subjected to biotin switch procedure, with antibody

against cPLA2, revealed that ghrelin prevention of the

ethanol-induced cytotoxicity was manifested in the in-

crease in cPLA2 protein S-nitrosylation, whereas prein-

cubation with L-NAME blocked the ghrelin-induced

cPLA2 nitrosylation (Figure 6).

4. DISCUSSION

Acute gastric mucosal injury and gastro-duodenal ulcera-

tions are the most common consequences of alcohol

abuse on the health of gastrointestinal tract [1-3]. More-

over, the disturbances in gastric mucosal cells caused by

ethanol cytotoxicity affect the synthesis of mucins, the

glycoproteins that maintain the integrity and strength of

the protective mucus layer, and hence weaken the inher-

ent resistance of the mucosa to injury and facilitate the

onset of gastric disease [6,7]. Therefore, considering the

recent evidence for the involvement of ghrelin in gastric

mucosal protection against injury by ethanol [4,9], in the

study presented herein we examined the influence of this

28-amino acid peptide hormone on the synthesis of gas-

tric mucin.

Using rat gastric mucosal cells exposed to ethanol at

the concentration range that impairs mucosal cell capac-

ity for mucin synthesis and prostaglandin generation

[1-3], we demonstrated that the protective effect of ghre-

lin against ethanol cytotoxicity was associated with up-

regulation in mucin synthesis, and accompanied by the

increase in NO and PGE2 production, and the enhance-

Figure 6. Effect of cNOS inhibitor, L-NAME (LN) on ghrelin

(Gh)-induced cPLA2 S-nitrosylation in gastric mucosal cells

exposed to ethanol (Et). The cells were treated with Gh or

L-NAME + Gh and incubated for 2 h in the presence of Et. A

portion of the cell lysate was processed by biotin switch pro-

cedure for protein S-nitrosylation and, along with the reminder

of the lysates, subjected to SDS-PAGE, transferred to nitrocel-

lulose and probed with anti-cPLA2 antibody. The immunoblots

shown are representative of three experiments.

ment in cPLA2 activity. A significant loss in the coun-

tering effect of ghrelin on the ethanol-induced cytotoxic-

ity and the cell capacity for mucin synthesis was attained

with cNOS inhibitor, L-NAME as well as specific COX-1

inhibitor, SC-560, whereas COX-2 inhibitor, NS-398,

and iNOS inhibitor, 1400W, had no effect. These find-

ings are thus in keeping with the results of earlier studies

suggesting that the detrimental effect of ethanol on gas-

tric mucin synthesis are associated with the impairment

in NO and PGE2 generation [1-3,6,25], and lend further

credence as to the role of ghrelin in regulation of the

cross-talk between NOS and COX enzyme systems [9].

Further, we found that the countering effect of ghrelin

on the ethanol-induced changes in mucin synthesis, and

NO and PGE2 production, were subject to suppression

by Src kinase inhibitor, PP2, whereas the inhibitor of

MAPK/ERK, PD98059, elicited suppression in mucin

synthesis and PGE2 generation, but had no effect on NO

production. Thus the activation of Src appears to be a

triggering event whereby ghrelin is capable of affecting

the mucosal cell capacity for mucin synthesis as well as

NO and PGE2 generation. Moreover, our data on the

inhibition of ghrelin-induced mucosal cell capacity for

mucin synthesis, and NO and PGE2 generation by L-

NAME, and only that of PGE2 and mucin synthesis by

COX-1 inhibitor, SC-560, suggest that ghrelin-induced

up-regulation in mucin synthesis and PGE2 generation

occurs with the involvement of cNOS-derived NO, and

requires COX-1 participation. Indeed, the role of NO in

modulation of COX enzymes activity in a variety of dif-

ferent systems is well documented [15-17].

As the initial and rate limiting event in prostaglandin

production is the release of AA from membrane phos-

pholipids by highly selective cPLA2 [5,24], we further

assessed the influence of ghrelin on the processes of

cPLA2 activation. We found that ghrelin elicited up-regu-

lation in the mucosal cell cPLA2 activation, which like

that of mucin synthesis was subject to suppression by

Src inhibitor, PP2, and ERK1/2 inhibitor, PD98059, as

well as the inhibitor of cNOS, L-NAME. However,

COX-1 inhibitor, SC-560, while not causing discernible

alteration in the cPLA2 activity, exerted the inhibitory

effect on mucin synthesis. Hence, we concluded that the

activation of gastric mucosal cPLA2 by ghrelin for the

increase in mucin synthesis to counter ethanol cytotoxic-

ity occurs with the involvement of cNOS and requires

Src kinase-dependent MAPK/ERK participation. Indeed,

the literature data indicate that MAPK/ERK-dependent

cPLA2 phosphorylation on the critical Ser505 residue

plays a crucial role in Ca2+-dependent translocation of

cPLA2 from cytosol to membrane to gain access to AA-

rich phospholipid substrates [5,26].

Whilst regulation of the key enzymes for prostaglandin

B. L. Slomiany et al. / HEALTH 2 (2010) 1033-1039

1038

production through phosphorylation is well recognized,

recent evidence indicates that the increase in prostaglan-

din formation may also result from NO-induced enzyme

protein S-nitrosylation [14,17-19]. Indeed, the post-tran-

slational modification through S-nitrosylation at the

critical Cys526 residue has been linked to the NO-induced

enhancement in catalytic activity of COX-2 [14], and

cPLA2 activation through S-nitrosylation at Cys152 was

reported to be responsible for up-regulation in arachi-

donic acid release in human epithelial cells [18]. There-

fore, to assess the role of ghrelin in cPLA2 activation, we

further analyzed the influence of S-nitrosylation on gas-

tric mucosal cPLA2 activity and the capacity for mucin

synthesis. We found that ghrelin-induced up-regulation

in cPLA2 activity and the cell capacity for mucin synthe-

sis was susceptible to suppression not only by the in-

hibitor of cNOS, L-NAME, but also by ascorbic acid,

which is keeping with well known susceptibility of S-

nitrosylated proteins to this reducing agent [14,23].

Moreover, Western blot analysis of the mucosal cell lys-

ates subjected to biotin switch procedure with antibody

against cPLA2 revealed that ghrelin prevention of the

ethanol-induced cytotoxicity was manifested in the in-

creased cPLA2 protein S-nitrosylation, whereas prein-

cubation with cNOS inhibitor, L-NAME resulted in the

blockage of the ghrelin-induced cPLA2 protein S-nitro-

sylation. Therefore, consistent with our results, the cPLA2

activation by ghrelin through S-nitrosylation is of direct

relevance to the mucosal capacity for mucin synthesis

and PGE2 generation, and hence the protection of gastric

mucosal cells against ethanol cytotoxicity.

In summary, our study demonstrates that the activa-

tion of gastric mucosal cPLA2 through S-nitrosylation

plays an essential role in the countering effect of ghrelin

on the ethanol-induced disturbances in gastric mucin sy-

nthesis.

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