Pha maceu ical Sciences 1997,
3:
609-612
Recei ed July 21, 1997
Accep ed Oc obe 1, 1997
0
1997 Pha maceu ical Sciences
P o ec i e E ec o L-A ginine Agains Ibup o en-induced
Gas ic Inju y in Ra s
M.
J.
MARTIN
CALERO,
M. D.
JIMENEZ,
c.
ALARCON
DE
LA
LASTRA,
c.
LA
CASA,
J.
M. HERRERIAS*, L.BRUSEGHINI ,
A.
ESTERAS? AND V. MOTILVA
Depa men
o
Pha macology, Facul y
o
Pha macy, Uni e si y
o
Se ille,
*Depa men
o
Gas oen e ology, Vi gen Maca ena Hospi al, Uni e si y
o
Se ille, and
TZambon G oup, Ba celona, Spain
Abs ac
This s udy has been designed o con i m he p o ec i e e ec o di e en single o al doses
o L-a ginine in he p esence o equimola doses o ibup o en, and o compa e he esul s
wi h hose ob ained a e ea men wi h ibup o en alone. Di e en pa ame e s we e
assessed in a s: gas ic damage (mm2 and sco e), a io o lesiona ed s omachs/ o al
s omachs e alua ed, and p esence o haemo hage.
Six hou s a e dosing, o al adminis a ion o ibup o en (0.3,
0.6
and 1.2 mmol kg-')
p oduced a p og essi e dose-dependen inc ease in damage o he gas ic mucosa. All
ea men s wi h equimola doses o L-a ginine conside ably educed lesions (mm2 and
sco e) and he same endency was obse ed wi h he o he pa ame e s examined. We also
e alua ed he gas op o ec i e e ec o L-a ginine agains an i-ulce e e ence d ugs,
ani idine and oxa idine ( wo an isec e o y agen s) and misop os ol (a cy op o ec i e
d ug). The deg ee o inhibi ion o damage p o ided by L-a ginine was simila o hose
ob ained wi h he o he d ugs.
Thus, we conclude ha he simul aneous adminis a ion o equimola doses o ibup o en
and L-a ginine o e s signi ican p o ec ion compa ed wi h gas olesi e doses o ibup o en
alone, wi h an impo an dec ease in he lesiona ed a eas and imp o emen o he ascula
s a e. The ex en o his p o ec i e ac ion is compa able wi h ha obse ed wi h an i-ulce
e e ence d ugs.
Non-s e oidal an i-in lamma o y d ugs (NSAIDs)
a e
among he mos commonly used d ugs in he
wo ld, bu hei use in he ea men o pain, e e
and in lamma ion, and in pa icula , in he ea -
men o a h i is, is associa ed wi h signi ican
un owa d e ec s on he gas oin es inal ac . The
bleeding and ulce a ion induced in he s omach by
NSAIDs is he mos common ad e se eac ion o
medica ion and has a signi ican inancial impac on
heal h ca e. Thei gas ic oxici y is ela ed o
supp ession o p os aglandin syn hesis, h ough
inhibi ion o cyclooxygenase ac i i y, and in addi-
ion, se e al NSAIDs ha e opical i i an p ope -
ies on gas ic mucosa.
In he las ew yea s, a numbe o di e en s a-
egies o de eloping NSAlDs wi h educed gas ic
Co espondence:
M.
J.
Ma in Cale o, Labo a o io de Fa m-
acologia, Facul ad de Fa macia,
P o .
Ga cia Gonzilez
s/n,
41012-Se illa, Spain.
oxici y ha e been used. These include o mula ing
he agen s wi h an en e ic coa ing o p e en
abso p ion in he s omach, he de elopmen o p o-
d ugs, which
a e
no ac i e un il hey ha e unde -
gone me abolism by he li e , and co-adminis a-
ion o ei he supp esso s o acid (an i-H2 o p o on
pump inhibi o s) o exogenous p os aglandins.
Recen ly, a new s a egy ela ed o he ole
o
ni ic oxide (NO) in he gas oin es inal ac has
been p oposed. NO is a memb ane-pe meable gas
ha se es as a media o o many physiological
e en s (Moncada e a1 1991).
I
is p oduced om
L-
a ginine by a calcium-dependen NO syn hase.
Nume ous s udies ha e implica ed
NO
as a c i ical
media o o mucosal de ence, simila o p os-
aglandins (Whi le e a1 1990). NO, o d ugs ha
gene a e NO (e.g. sodium ni op usside, glyce yl
ini a e) ha e been shown o educe he se e i y
o gas ic mucosal inju y in expe imen al models
(Whi le e a1 1990; B own e a1 1993) and some
610
M.
J.
MART~N
ET
AL
au ho s ha e sugges ed ha he gas op o ec i e
e ec o L-a ginine could be h ough an NO-pa h-
way (Ki agawa e a1 1990; Kon u ek
e
a1 1993)
Ibup o en is a po en NSAID ha ecen ly has been
o mula ed wi h equimola doses o L-a ginine in
o de o imp o e pha macokine ic pa ame e s
(highe and mo e apid abso p ion) wi h quicke
and mo e po en analgesic e ec . In a p e ious
epo we ound ha adminis a ion o L-a ginine
be o e ibup o en ea men induced signi ican
inhibi ion o gas ic lesions (Mo il a
e
a1 1996).
Thus, we designed his s udy o con i m he p o-
ec i e e ec o he ibup o en and L-a ginine o -
mula ion s ibup o en alone, and o compa e his
gas op o ec ion wi h o he adi ional he apies
such as misop os ol
(PGE1
analogue), ani idine (a
classic an isec e o y agen ) o oxa idine (a new
an i-H2 d ug).
Ma e ial and Me hods
Animal g oups and compounds
Male Wis a a s (supplied by Animal Se ices,
Facul y o Medicine, Se ille), 180-250 g, we e
used. The animals we e ed a no mal labo a o y
die in a con olled oom ( empe a u e 22-24°C
and humidi y a 70-75%). They we e placed in
single cages wi h wi e-ne loo s o p e en
cop ophagy, and we e dep i ed o ood o
18
h
be o e he expe imen s bu had ee access o wa e .
G oups o 9-10 a s we e ea ed wi h di e en
doses o ibup o en (Sigma Chemical Co., MO),
ibup o en and L-a ginine (Zambon S.A., Ba celona,
Spain), ani idine (Sigma Chemical Co., MO),
oxa idine (Zambon S.A., Ba celona, Spain) and
misop os ol (CECOFAR, Se ille, Spain). The
d ugs we e suspended in dis illed wa e and we e
adminis e ed by he in agas ic ou e, in a dose o
1
mL/lOO
g body weigh . Con ol g oups ecei ed
ehicle in compa able olume. Each expe imen
was pe o med a leas wice and esul s we e
pooled.
Expe imen al p o ocol
Di e en g oups o animals we e ea ed wi h
ibup o en (0.3, 0.6 and 1-20 mmol kg-') and
equimola doses o ibup o en and L-a ginine
(0.3/0-3, 0.6/0-6 and 1-20/1.20 mmol kg-'). Six
hou s a e ea men he animals we e killed using
an o e dose o e hyl e he and he s omachs we e
emo ed and cu along he smalle cu a u e. The
gas ic lesions we e immedia ely e alua ed and he
ollowing pa ame e s ela ed o damage we e
assessed. Gas ic damage (sco e):
0,
absence o
lesions; 1, haemo hagic o nec o ic mucosa; 2,
poin ed lesions; 3, poin ed lesions wi h lesions
<
3 mm; 4, mainly lesions
>
3 mm. A ea o gas-
ic damage (mm2): he p oduc o ulce leng h and
wid h was calcula ed. Mucosal damage
(%):
educ ion o damaged a ea o he di e en g oups
compa ed wi h he espec i e equimola dose o
ibup o en alone (100%). Ra io lesioned s o-
machs/ o al s omachs e alua ed. P esence o hae-
mo hage (sco e):
0,
absence; 1, sligh
haemo hage;
2,
ma ked haemo hage.
The in e media e gas olesi e dose o ibup o en
(0.6 mmol kg-') no ed in hese expe imen s was
selec ed o examine he p o ec i e e ec o mis-
op os ol, ani idine and oxa idine. New g oups o
animals ecei ed he ollowing ea men s: ibup o-
en alone; equimola dose o ibup o en and
L-
a ginine (0-6/0.6 mmol kg-'); ibup o en and
misop os ol (0-6/0.05
x
mmol kg-l); ibu-
p o en and ani idine (0.6/0.05 mmol kg-'); and
ibup o en and oxa idine (0-6/0.05 mmol kg-
').
A e he expe imen al pe iod, he animals we e
killed and he s omachs emo ed, opened and
assessed as in he p e ious expe imen s. The same
pa ame e s we e e alua ed.
Analysis
o
esul s
S a is ical analysis was pe o med using con en-
ional me hods (a i hme ic mean
s.e.). One-way
analysis o a iance was used o es o signi ican
di e ences o means o ea men g oups. Mul iple
compa ison be ween means was pe o med using
he Mann-Whi ney U- es and X2- es a a p o ec-
ion le el o
0.05.
Resul s
Six hou s a e dosing, ibup o en gi en o ally in
a ious doses
(0.3,
0-6 and 1.20 mmol kg-') p o-
duced a p og essi e dose-dependen inc ease o
damage o he gas ic mucosa (4.12
z
1.01,
17.88
2.74 and 66.41
9.17 mm2, espec i ely).
O al ea men o he animals wi h equimola doses
o L-a ginine conside ably educed he gas ic
lesions (0.6 and 1.20 mmol kg-',
P
<
0.001
agains ibup o en alone, mm2 and sco e). The same
endency was e alua ed wi h he o he pa ame e s,
deg ee o haemo hage and pe cen age o mucosal
damage (Table 1).
Signi ican dec eases in he gas ic lesion pa a-
me e s we e ound wi h all he ea men s shown in
Table
2.
The mos ma ked educ ion o he lesion
(mm2 and sco e), as well as haemo hagic sco e
and pe cen age o mucosal damage, was obse ed
wi h ani idine, oxa idine and misop os ol
(P
<
0.001). Howe e , L-a ginine ea men also
L-ARGININE PROTECTS AGAINST IBUPROFEN-INDUCED GASTRIC INJURY IN RATS
61
1
Table
1.
The an i-ulce p o ec ion o di e en doses o L-a ginine in he p esence o equimola doses o ibup o en in a s.
T ea men Haemo hage Gas ic damage Mucosal Lesioned s omachs Gas ic damage
(sco e) (mm2) damage
(%)
s omachs e alua ed (sco e)
None
0.00
0.00
0.00
0.00
0.0
0120
0.00
0.00
Ibup o en
0.20
0.06 4.12
1.01
100 19/20 2.15 0.20
Ibup o en and
0.08
0.04 2.47
0.68 59.8 19/20 1.95
0.15
(0.3
mmol kg-I)
L-a ginine
(0.3
mmol kg-I)
(0.6
mmol kg-
I)
L-a ginine
(0.6
mmol kg-I)
Ibup o en
1.03 0.12 17.88
2.74 100 19/19 3.21 0.10
Ibup o en and
0.32
0.1
1
*
6.29
1.33* 35.2 19/19 2.31 0.11
Ibuu o en
1.82
0.10 66.41
9.17 100 19/19 4.0
0.01
(1.20
mmol kg-')
Ibup o en and
0.23 0.09* 17.97 4.15* 27.1 19/20 2.45
0.18*
L-a ginine
(1.20
mmol kg-
I)
Resul s a e means
*
s.e. (n
=
19
o
20).
*P
<
0.001, ibup o en and L-a ginine s ibup o en alone (Mann-Whi ney U- es ).
Table
2.
The e ec s o di e en ea men s in he p esence o ibup o en
(0.06
mmol kg-I): L-a ginine
(0.6
mmol kg-I),
ani idine
(0.05
mmol kg-'), oxa idine
(0.05
mmol kg-') and misop os ol
(0.05
x
mmol kg-')
on
pa ame e s o gas ic
damage in a s.
T ea men Haemo hage Gas ic damage Mucosal Lesioned s omachs Gas ic damage
(sco e) (mm2) damage(%) s omachs e alua ed
(%)
(sco e)
Ibup o en alone
1.03
0.12
17.88
2.74 100 100 3.21 0.10
+
L-A ginine
0.32
*
0.11
*
6.29
1.33* 35.2
100
2.31
0.11*
+
Misop os ol
0.13
0.06* 2.25
*
0.76* 12.6 89.5 1.95
0.21
*
+
Rani idine
0.10
0.05* 1.43
0.61
*
8.0
68.41-
1.50
0.29*
+
Roxa idine
0.10
0.02*
0.08
0.07
0.5
66.7 1
.OO
0.25
Resul s a e means
s.e. (n
=
19
o
20).
*P
<
0,001,
ea men g oups s ibup o en alone, Mann-Whi ney
U
es ;
P
<
0.01,
x2
es .
induced e y ma ked dec eases in all pa ame e s
e alua ed
(P
<
0.001).
Discussion
The e is o e whelming e idence ha NO is one o
he mos impo an media o s o mucosal de ence,
a ec ing ac o s such as mucus sec e ion, mucosal
blood low, ulce epai and he ac i i y o a a ie y
o mucosal immunocy es. Endogenous NO has he
capaci y o down- egula e in lamma o y esponses
in he gas oin es inal ac (Kubes
&
Wallace
1995), and i has been shown o be in ol ed in
ce ain ypes o gas op o ec ion such as ha
induced by capsaicin, papa e ine (B zozowski e a1
1993), suc al a e and mild i i an s (Kon u ek e a1
1994). In his way, he abili y o
NO
o educe he
se e i y o damage induced by NSAIDs has
ecen ly been exploi ed in an a emp o p oduce
d ugs which
a e
no ulce ogenic. By a aching an
NO- eleasing moie y o s anda d NSAIDs (by
inco po a ion o a ni oxybu yl moie y (e.g. lu bi-
p o en, ke op o en and diclo enac ni oxy-bu yl-
es e ), he gas oin es inal oxic e ec s o hese
compounds was g ea ly educed bu did no in e -
e e wi h hei abili y o supp ess in lamma o y
p ocesses, inhibi p os aglandin syn hesis o inhibi
pla ele agg ega ion (Wallace
&
Ci ino 1994).
O he ecen s a egies such as p ophylac ic he apy
wi h he
PGEl
analogue misop os ol ha e been
shown o educe he incidence
o
NSAID-induced
ulce s signi ican ly, bu he use o his d ug is
limi ed by i s ad e se e ec s in a conside able
numbe o pa ien s.
Howe e , he e ec o L-a ginine, he p ecu so
o NO, on he gas ic mucosal in eg i y has no
612
M.
J.
MART~N
ET
AL
been much s udied. Ou da a show ha simul a-
neous ea men wi h L-a ginine signi ican ly
dec eases ibup o en-induced gas ic damage in a
dose-dependen way. This esul is in ag eemen
wi h Ki agawa e a1 (1990) who epo ed ha
L-
a ginine gi en in a enously p e en ed gas ic
lesions caused by
0-6
M
HC1 o wa e -imme sion
s ess. Simila esul s we e ob ained by Takeuchi e
a1 (1993) who ound ha o al ea men wi h
L-
a ginine exhibi s gas ic cy op o ec ion agains
0.6
M
HC1-induced lesions in a dose dependen
manne . This e ec was mimicked by he enan-
iome D-a ginine and was no a ec ed by p e ious
adminis a ion o
N
G-ni o-L-a ginine me hyl es e
(L-NAME), a po en inhibi o o NO biosyn hesis.
Thus hey sugges ed ha he mucosal p o ec i e
ac ion o L-a ginine (p.0.) is un ela ed o he NO
pa hway, and i s mechanism may appea h ough
adap a i e cy op o ec ion media ed by endogenous
p os aglandins. In con as , in a p e ious s udy, we
did no obse e any p o ec i e e ec agains
diclo enac-induced gas ic inju y a e D-a ginine
ea men . Howe e , he p o ec ion induced by
L-
a ginine was main ained a e adminis a ion o
L-NAME sugges ing, a leas in pa , he in ol e-
men o NO in he p o ec i e mechanism o o al
L-
a ginine (Mo il a e a1 1997).
Fe az e a1 (1994) ound ha in a-a e ial
adminis a ion o L-a ginine be o e he applica ion
o
20%
e hanol p oduced a dose-dependen
inc ease in he ex en o damage. L-A ginine sig-
ni ican ly educed gas ic blood low ela i e o
con ols, and he au ho s sugges ed ha he inc ease
o gas ic inju y was pa ly NO dependen and
pa ly NO independen . They sugges ed a pa a-
doxical e ec
o
L-a ginine on gas ic mucosal
in eg i y. While small amoun s o NO p oduced
cons i u i ely by endo helial cells appea o be
c i ical o main enance o mucosal pe usion, la ge
amoun s o NO can exace ba e mucosal inju y,
p obably as a consequence o he cy o oxic p op-
e ies o he NO adical o he pe oxyni i e adical
o med h ough he in e ac ion o NO wi h supe -
oxide.
We conclude ha , unde ou expe imen al con-
di ions, in agas ic adminis a ion o L-a ginine
p o ec s he gas ic mucosa agains ibup o en-
induced inju y, and ha his p o ec i e e ec is
compa able wi h hose o an isec e o y agen s o
misop os ol. Howe e he mechanism in ol ed in
his bene icial ac ion ha e no been s udied. I is
possible ha L-a ginine achie es di ec cy op o-
ec i e e ec on mucosal issue and ha his
p ope y could be ela ed o he NO-pa hway, bu
hese supposi ions equi e u he explo a ion.
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