Pretreatment with adenosine and adenosine A1 receptor agonist protects against intestinal ischemia-reperfusion injury in rat.

Abstract

To examine the effects of adenosine and A1 receptor activation on reperfusion-induced small intestinal injury.
Rats were randomized into groups with sham operation, ischemia and reperfusion, and systemic treatments with either adenosine or 2-chloro-N(6)-cyclopentyladenosine, A1 receptor agonist or 8-cyclopentyl-1,3-dipropylxanthine, A1 receptor antagonist, plus adenosine before ischemia. Following reperfusion, contractions of ileum segments in response to KCl, carbachol and substance P were recorded. Tissue myeloperoxidase, malondialdehyde, and reduced glutathione levels were measured.
Ischemia significantly decreased both contraction and reduced glutathione level which were ameliorated by adenosine and agonist administration. Treatment also decreased neutrophil infiltration and membrane lipid peroxidation. Beneficial effects of adenosine were abolished by pretreatment with A1 receptor antagonist.
The data suggest that adenosine and A1 receptor stimulation attenuate ischemic intestinal injury via decreasing oxidative stress, lowering neutrophil infiltration, and increasing reduced glutathione content.

Full text

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PO Box 2345, Beijing 100023, China World J Gastroenterol 2007 January 28; 13(4): 538-547
www.wjgnet.com World Journal of Gastroenterology ISSN 1007-9327
wjg@wjgnet.com © 2007 The WJG Press. All rights reserved.
Pretreatment with adenosine and adenosine A1 receptor
agonist protects against intestinal ischemia-reperfusion
injury in rat
V Haktan Ozacmak, Hale Sayan
www.wjgnet.com
BASIC RESEARCH
V Haktan Ozacmak, Hale Sayan, Department of Physiology,
School of Medicine, Zonguldak Karaelmas University, Kozlu
67600, Zonguldak, Turkey
Supported by Zonguldak Karaelmas University Research Proje-
cts Fund, No. 2003-01-09
Correspondence to: Dr. VH Ozacmak, Department of Physio-
logy, School of Medicine, Zonguldak Karaelmas University, Koz-
lu 67600, Zonguldak, Turkey. vhaktan@yahoo.com
Telephone: +90-372-2610243 Fax: +90-372-2610264
Received: 2006-07-07 Accepted: 2006-11-03
Abstract
AIM: To examine the effects of adenosine and A1 re-
ceptor activation on reperfusion-induced small intestinal
injury.

METHODS: Rats were randomized into groups with
sham operation, ischemia and reperfusion, and systemic
treatments with either adenosine or 2-chloro-N6-cyclo-
pentyladenosine, A1 receptor agonist or 8-cyclopentyl-
1,3-dipropylxanthine, A1 receptor antagonist, plus ade-
nosine before ischemia. Following reperfusion, contra-
ctions of ileum segments in response to KCl, carbachol
and substance P were recorded. Tissue myeloperoxidase,
malondialdehyde, and reduced glutathione levels were
measured.

RESULTS: Ischemia significantly decreased both contra-
ction and reduced glutathione level which were ameliora-
ted by adenosine and agonist administration. Treatment
also decreased neutrophil infiltration and membrane lipid
peroxidation. Beneficial effects of adenosine were abolis-
hed by pretreatment with A1 receptor antagonist.

CONCLUSION: The data suggest that adenosine and
A1 receptor stimulation attenuate ischemic intestinal in-
jury via decreasing oxidative stress, lowering neutrophil
infiltration, and increasing reduced glutathione content.

© 2007 The WJG Press. All rights reserved.
Key words: Adenosine; Adenosine A1 receptor; Intestinal
ischemia; Pharmacological preconditioning
Ozacmak VH, Sayan H. Pretreatment with adenosine and
adenosine A1 receptor agonist protects against intestinal
ischemia-reperfusion in rat. World J Gastroenterol 2007;
13(4): 538-547
http://www.wjgnet.com/1007-9327/13/538.asp
INTRODUCTION
Ischemia-reperfusion (I/R) injury of the intestine is
a significant problem in a numerous situations such
as abdominal aortic aneurysm surgery, small bowel
transplantation, cardiopulmonary bypass, strangulated
hernias, and neonatal necrotizing enterocolitis[1]. Decreased
contractile activity, increased microvascular permeability,
and dysfunction of mucosal barrier are all associated with
intestinal I/R[2,3]. I/R injury of the intestine is an intricate
and multifactorial pathophysiological process that involves
the formation and action of oxygen free radicals (OFRs)[3-9],
inflammatory cytokines, the complement system[1,3] and
neutrophil infiltration[1-3,6,7,10] at the site of damage.
The purine nucleoside adenosine is one of the major
local regulators of normal tissue function, acting in
both an autocrine and paracrine fashion. Its regulatory
function becomes pronounced especially when energy
supply ceases abruptly as in the case of ischemia, and
fails to meet cellular energy demand. Adenosine exerts
its effects by interacting with its receptors, four of which
have been cloned and characterized as adenosine A1, A2a,
A2b, and A3[11-13]. The physiological role of adenosine in the
gastrointestinal tract is still poorly understood, particularly
with regard to colonic and ileal motor functions. It
has been reported that A1 adenosine receptor (A1AR)
antagonists increase defecation in rats[13] and that A1AR
agonists can inhibit intestinal fluid secretion and peristalsis
via adenosine A2B and A1 receptors, respectively[14].
One of the cellular events observed during ischemia
is the increased consumption of ATP, leading to
accumulation of adenosine with thereby elevating
extracellular adenosine. The accumulation of adenosine
is believed to contribute to cytoprotection in the ischemic
tissue[11,12]. Furthermore, adenosine which is released
during short periods of ischemia followed by reperfusion,
provides cytoprotection against a subsequent sustained
ischemia in heart, resulting in reduced infarct size[15-20].
This is known as the preconditioning effect of adenosine,
which is mediated mostly through the activation of cardiac
A1ARs before ischemia[11,12,18]. It is well documented
that the early[19,20] and late[11,15,18] phases of ischemic
tolerance are mediated by adenosine in myocardium. That
adenosine exerts anti-ischemic actions is indicated by

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Ozacmak VH et al. Adenosine and A1 receptor agonist on intestinal injury 539
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a number of studies using adenosine receptor agonists
and antagonists[15-18] as well as animals overexpressing or
lacking A1AR[21,22]. Administration of adenosine either
prior to ischemia or during reperfusion has been shown
to attenuate myocardial injury[23,24]. Treatment with
adenosine A1AR agonist initiates preconditioning not only
in heart[15-18,25] but also in tissues such as kidney[26,27] and
brain[28,29], resulting in attenuation of ischemic injury. One
of the underlying mechanisms suggested for adenosine
receptor-mediated preconditioning in the heard is through
involvement of protein kinase C (PKC) in heart[11,12,30].
Activation of PKC induces opening of ATP-sensitive K+
channels[11,12,31]. Among other effectors that most likely
contribute to the cytoprotection by adenosine are mitogen
activated protein (MAP) kinases[25,31-33], heat shock proteins
(HSPs)[11-15,34], antioxidant enzymes[15,34] and inducible
nitric oxide synthase (iNOS)[11,12]. Moreover, induction and
activation of manganese superoxide dismutase (Mn-SOD)
is also believed to be a significant factor in mediating
myocardial adaptation in response to activation of A1AR[15].
In the phenomenon of ischemic preconditioning
(IPC), a short period of ischemia protects the organs (e.g.
heart) against a subsequent more substantial ischemic
injury[35]. In fact, IPC has been one of the most promising
strategies against reperfusion injury during the last few
years. It appears to elevate the tolerance of the intestine
to I/R injury. A number of experimental studies have
shown that reperfusion injury in small intestine is
prevented by IPC[36-40]. IPC conducted in small intestine
reduces postischemic leukocyte adhesion by maintaining
the bioavailability of nitric oxide[41]. Moreover, it lowers
the expression of P-selectin[38], which is a downstream
effector target of the adenosine-initiated, PKC dependent,
signalling pathway in intestine. Although activation of
PKC triggered by adenosine has been a crucial factor for
initiating the beneficial actions of IPC in most tissues,
the effector of the preconditioning phenomenon appears
to vary among tissues. Activated-K+ channels[42], nitric
oxide[39,41] and endogenous opioid peptides[43] have reported
to be the other downstream effectors of IPC in intestine.
Based primarily on animal experiments, the identification
of the molecular mechanisms that are responsible for
protection by IPC, has provided opportunities to consider
several rational targets for pharmacological intervention.
Consequently, a variety of drugs have been demonstrated
to be able to mimic IPC when applied instead of ischemia.
This is known as pharmacological preconditioning (PPC).
Recently, various studies carried out in rat small intestine
have demonstrated that establishing PPC by administration
of either adenosine[37] or A1AR agonist[38] mimics the
protective effects of IPC. Intensive investigation has been
focused on explaining how adenosine accomplishes the
beneficial effect of preconditioning. For instance, currently
published studies suggest important anti-ischemic roles
of the A1[18,25,30], A3[17,21] or A2a[44,45] adenosine receptors
in heart. On the other hand, relatively little data are
available on the role of the different adenosine receptors
in mediating cytoprotection in intestinal tissue which is
exposed to I/R. The majority of studies strongly suggest
that adenosine can promote protection against I/R injury
via activation of different adenosine receptors in various
tissues. A substantial number of studies report that IPC
has been beneficial in human heart and the liver. However,
both prospective controlled studies in human and
experimental studies in animals are lacking[1]. Furthermore,
research based on administration of drugs that can mimic
the effects of IPC is required further to explore the
cellular events during I/R injury of the intestine. To date,
there is no direct evidence showing possible effects of
adenosine and A1AR activation on reduced contractility
of intestinal smooth muscle due to I/R injury. Therefore,
the present study was constructed to explore the possible
effects of adenosine and A1AR activation on reperfusion
injury of small intestinal tissue by evaluating contractile
response and levels of thiobarbituric acid-reactive
substances (TBARS, a marker of lipid peroxidation),
reduced glutathione (GSH, an endogenous antioxidant),
and myeloperoxidase (MPO an index of neutrophil
infiltration), in terminal ileum subjected to I/R.
MATERIALS AND METHODS
Animals
Following Ethical Committee approval, forty adult
male Wistar rats, weighing 200-230 g, were obtained
from the Experimental Research Section of Zonguldak
Karaelmas University, where animals have been reared and
maintained under standard conditions, such as stable room
temperature (23 ± 2℃), a 12 h light: 12 h dark cycle, and
feeding with commercial rat chow and tap water ad libitum.
Experimental manipulations and surgical operations
were approved by the Animal Ethical Committee of the
University. Maximum care and a humane approach to use
of animals was of primary consideration.
Experimental groups and operative procedures
The surgical goal was to induce mesenteric ischemia in rats
for 30 min followed by a 180 min reperfusion period. On
the day before surgery, each animal was fasted overnight
with unlimited access to water. Briefly, each animal was
anesthetized by an intraperitoneal injection of 50 mg/kg
sodium thiopenthal followed by a midline incision made
into the peritoneal cavity. The small bowel was exteriorized
gently to the left onto moist gauze, and then the superior
mesenteric artery (SMA) was carefully exposed, isolated,
and clamped using a microvascular clamp. Intestinal isch-
emia was confirmed by obvious lack of pulse in the SMA
and paleness of the jejunum and ileum. The intestines
were then meticulously placed back into the abdomen
which was closed with two small clamps. Following 30
min of occlusion time, the clamping was gently released
and the intestine inspected for proper reperfusion char-
acterized by regular pulsation. Throughout the surgical
procedure, each animal was placed under a heating lamp
to maintain constant body temperature (e.g. 37℃). For
the purpose of assessing the roles of adenosine and A A1AR
agonist, animals were randomly divided into five groups��nimals were randomly divided into five groups��
(1) Sham-operated group, subjected to laparatomy without
performing the occlusion of the SMA; (2) I/R group, sub-
jected to the occlusion of SMA followed by reperfusion; (3)
CPA-treated group (0.1 mg/kg, 5 min prior to ischemia) +
I/R; (4) Adenosine-treated group (10 mg/kg, 5 min prior

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540 ISSN 1007-9327 CN 14-1219/R World J Gastroenterol January 28, 2007 Volume 13 Number 4
to ischemia) + I/R; (5) DPCPX pretreatment (1 mg/kg,
15 min prior to adenosine administration) + adenosine
treatment (10 mg/kg, 5 min prior to ischemia) + I/R. In
the last group, confirming the possible effect of selective
A1AR agonist CPA on reperfusion injury, the selective
A1AR antagonist DPCPX was administered 15 min prior
to adenosine treatment. The route and volume for drug
administration were the tail vein and 200 μL, respectively.
To animals in both sham-control and I/R-control groups
were given sterile serum physiological solution in the same
volume instead. Choice of dose regimen for the drugs was
based on published studies in the literature[22,37,38].
Preparation of terminal ileum
Upon completion of the I/R period, and whilst still unc-
oncious, the animals were sacrificed by exsanguination of
the abdominal aorta. Strips of terminal ileum of 10 mm
length were immediately removed 10 cm oral to the ileoce-
cal junction and transferred into a Petri dish containing
Krebs solution (in mmol/L: NaCl 118, NaHCO3 24.88,
KH2PO4 1.18, KCl 4.7, MgSO4 1.16, CaCl2 2.52 and glu-
cose 11.1). Then, tissue was longitudinally suspended in a
standard organ chamber, and continuously perfused with
20 mL of preoxygenated Krebs solution (pH 7.4), which
was bubbled constantly with a mixture of 950 mL/L O2
and 50 mL/L CO2 gas and maintained at a temperature
of 37℃. One end of the tissue strip was tied to a fixed
post and the other attached to an isometric force trans-
ducer under a resting tension of 2 g. Isometric responses
were monitored by external force displacement transducer
(FDA-10A, Commat Iletisim Co., Ankara, Turkey) and
recorded on the computer using MP 30 software (Biopac
Systems Inc., Santa Barbara, CA, USA). In the organ bath,
each strip was allowed to equilibrate for 1 h with interven-
ing washes every 15 min before adding any compound.
Tissue samples also obtained from small intestine approxi-
mately 10 cm proximal to the ileocecal area were frozen
immediately and stored at -40℃ for biochemical measure-
ments.
Concentration-response curves
At the beginning of each experiment to observe dose-
contractile response relationship, KCl was added to the or-
gan chamber to a final concentration of 30 mmol/L. For
the preparation of high K+ solutions, NaCl was exchanged
for an equimolar amount of KCl to maintain the physi-
ological osmolarity of the Krebs solution. The contraction
recorded in response to KCl was considered as a referen-
ce response. Afterwards, the contractions in response to
carbachol and substance P at various final concentrations
ranging from 10-9 mol/L to 10-2 mol/L were recorded by
pipetting these compounds into the organ bath in a cu-
mulative fashion at equal intervals. At the end of the ex-
periment, the response to 30 mmol/L KCl was measured
again to confirm and evaluate the degree of tissue viability.
The amplitude of all contractions was then normalized for
each g of tissue and expressed as percentage of the initi-
al KCl-reference response. The number of experiments,
represented as “n”, indicates that each experiment was
performed with a tissue sample taken from one animal.
as a referen-
All experiments were conducted in a paired way. For the
purpose of evaluating the effects of ligand, agonist, and
antagonist, the maximum response (Emax) and pD2 values
(e.g. the negative logarithm of the concentration for the
half-maximal response, ED50) were computed by using
GraphPad Prism Software 3.02 (GraphPad Prism Inc., San
Diego, CA, USA)[46]. The pD2 values (apparent agonist affi-
nity constants) were calculated from each agonist concen-
tration–response curve by linear regression of the linear
median part of the sigmoid curve and taken as a measure
of the sensitivity of the tissues to each agonist.
Drugs
Adenosine, CPA, DPCPX, carbachol and substance P were
purchased from Sigma (Sigma Chemical Co., St. Louis,
MO, USA). They were dissolved in double distilled water,
except for CPA and DPCPX which were initially prepared
in dimethyl sulphoxide and then diluted in physiological
saline. Adenosine, CPA, and DPCPX were prepared fresh
just before usage. Carbachol and substance P were made
up at different concentrations and kept frozen in aliquots.
Compounds, which were used for preparing Krebs soluti-
on, were purchased from Merck (Merck KGaA, Darmsta-
dt, Germany). All other reagents, including trichloroacetic
acid (TCA), thiobarbituric acid (TBA), butylated hydroxy
toluene (BHT), and dithiobisnitrobenzoate (DTNB) were
obtained from Sigma.
Determination of tissue TBARS and GSH
Tissue TBARS content was measured in order to estimate
the extent of lipid peroxidation in the injured terminal
ileum. Samples obtained from each group were stored at
-40℃ until assayed. Tissue samples were washed in ice-
cold Krebs solution, blotted on absorbent paper and
weighed. Afterwards, each sample was minced followed
by homogenization with 10 mL of 100 g/L TCA per g of
tissue, using a motor-driven homogenizer (Heidolph Diax
900, Heidolph Elektro GmbH&Co.KG, Kelheim, Germa-
ny). Then, the tissue TBARS levels were measured spec-
trophotometrically based on a method described by Casini
et al[47] and expressed as nmol/g of tissue weight. Briefly,
following two consecutive centrifugations at 3000 g for 15 centrifugations at 3000 g for 15
min, 750 μL supernatant was added to equal volume of 6.7
g/L TBA and heated to 100℃ for 15 min. The absorbance
of the samples was then measured spectrophotometrically
at 535 nm (Smart Spectro, LaMotte Co., Chestertown, MD,
USA).
The GSH content of the samples were measured by
applying a modified Ellman method[48]. In brief, 2 mL of
0.3 mol/L Na2HPO4 solution was mixed with 0.5 mL of
supernatant obtained by employing the homogenization
procedure described above. Into the mixture, 0.2 mL of
DTNB solution was added followed by reading absor-
bance at 412 nm. The tissue GSH levels were expressed as
μmol/g of tissue weight.
Casini
Measurement of tissue MPO activity
The degree of neutrophil accumulation in the intestinal
tissue samples was measured by assaying MPO activity as
described by Bradley et al[49]. Briefly, upon thawing, each
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sample was very finely minced with surgical blade in a petri
dish containing 50 mmol/L potassium phosphate buffer
(PB, pH 6.0) at a volume 20 times the tissue weight (e.g. 1
mL) followed by homogenization for 5 min in ice-cold PB
by means of motor driven homogenizer. The homogenate
was centrifuged at 40 000 g for 15 min at 4℃. The ho-
mogenized tissue pellet was suspended in 50 mmol/L PB
containing 5 g/L hexadecyltrimethylammonium bromide
(HETAB) and then homogenized again. Following three
freeze and thaw cycles with sonication (Bandelin Sonopuls
HD2070, Bandelin Electronic GmbH&CO.KG, Berlin,
Germany) between cycles, the samples were centrifuged
at 40 000 g for 10 min. Aliquots of supernatant (0.1 mL)
were added to 2.9 mL of reaction mixture containing 0.167
mg/mL of o-dianisidine, and 20 mmol/L H2O2 solution,
which were prepared in 50 mmol/L of PB. Immediately
after adding the aliquot to the mixture, the change in ab-
sorbance at 460 nm was measured for 5 min. One unit of
MPO activity was defined as that degrading 1 μmol of
peroxide per min at 25℃. The activity was then normal-
ized as unit per mg of tissue (U/mg).
Statistical analysis
Values for the experiments dealing with contractility were
normalized for per g of tissue followed by expressing them
as percentage of KCl response. Each data point represents
mean ± SE. For statistical evaluation, SPSS 11.0 statistical
software package programme was used (SPSS Inc., Chi-
cago, IL, USA). One-way analysis of variance (ANOVA)
was applied for statistical comparison of groups, followed
by analysis with Tukey-Kramer test so as to determine dif-
ferences between the groups. Probability value (P) of 0.05
or less was considered statistically meaningful.
Each data point represents
RESULTS
Ileal longitudinal muscle contractility
For longitudinal ileum muscle collected from sham
control, CPA-treated, adenosine-treated, and DPCPX-
adenosine-treated animals, mean contraction responses
to 30 mmol/L KCl were measured as 0.59 ± 0.01 g; 0.40
± 0.09 g; 0.50 ± 0.22 g; and 0.44 ± 0.21 g, respectively,
which were statistically indistinguishable (Figure 1). In I/R
control group however, the contractile response (0.26 ±
0.08 g) was significantly reduced when compared to that in
sham-operated control group (P = 0.012).
The addition of carbachol at concentrations from
10-9 mol/L to 10-2 mol/L into the organ bath resulted
in a dose-dependent contractile effect on the terminal
ileum segments from all groups (Figure 2), providing
sigmoid curves with Emax and pD2 values (Figure 3). Emax
value for carbachol was significantly lower in the I/R
control group than in the sham-operated control group
(157.04% ± 41.35% vs 488.66% ± 47.01%, respectively).
In other words, contraction in response to carbachol
was significantly reduced by induction of I/R. Statistical
difference between the groups appeared to be meaningful
at 10-6 mol/L (P = 0.02), reaching a maximal level at
10-3 mol/L of carbachol (P = 0.0001). The I/R-induced
reduction in contractility was significantly restored by
treatments with both CPA and adenosine but not by
pretreatment with DPCPX. Amelioration of reduced
contractions with CPA and adenosine therapies became
statistically significant at millimolar doses of carbachol (P
= 0.03 at 10-3 mol/L and 10-2 mol/L).
Comparison of the Emax values showed that average
contraction of ileum samples in I/R group was just
32% of that in sham-operated control group, while
those in CPA- and adenosine-treated groups were
approximately 81% and 76%, respectively (Table 1). In
the group pretreated with DPCPX, Emax was found to be
approximately 60% of that in sham control group, which
was statistically significant (P < 0.05). On the other hand,
no statistically significant change was detected in the
corresponding pD2 values in any group (Table 1).
In response to various concentrations of substance
Table 1 E max and pD 2 values of carbachol and substance P (n = 8, means ± SE)
Sham ControlI/R ControlCPA-I/RADO-I/RDPCPX + ADO-I/R
Carbachol
Emax
pD2
Substance P
Emax
pD2
488.67 ± 47.01
6.30 ± 0.19
157.05 ± 41.35a
7.02 ± 0.31
395.05 ± 32.62c
6.24 ± 0.40
372.21 ± 54.68c
6.94 ± 0.13
212.35 ± 40.09a
5.81 ± 0.18
255.94 ± 31.17
6.51 ± 0.05
115.00 ± 13.36a
8.29 ± 1.08
148.19 ± 13.80a
7.00 ± 0.22
242.93 ± 46.55c
6.69 ± 0.28
259.61 ± 24.62c
6.51 ± 0.07
aP < 0.05 vs sham-operated control group, cP < 0.05 vs I/R control group.
0.75
0.50
0.25
0.00
Contraction (g)
Sham control
I/R control
CPA
Adenosine
DPCPX-Adenosine
a
KCL (30 mmol/L)
Figure 1 Mean contraction of longitudinal ileum muscle isolated from sham-
operated control, I/R control, CPA-I/R, adenosine-I/R, and DPCPX-adenosine-I/R
rats in response to 30 mmol/L KCl. Data are expressed as means ± SE (n = 8). aP
< 0.05 vs sham-operated control group.
Ozacmak VH et al. Adenosine and A1 receptor agonist on intestinal 541
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P ranging from 10-9 mol/L to 10-2 mol/L, terminal ileum
samples contracted in a dose-dependent fashion in all
groups (Figure 4), rendering sigmoid curves with Emax and
pD2 values (Figure 5). The contractile response induced
by substance P was significantly and dose-dependently
inhibited by induction of I/R. Statistical difference
between sham-operated control rats and I/R control
animals was significant at 10-6 mol/L and over doses of
substance P (P < 0.05). Reduced contractility due to I/R
was alleviated significantly by adenosine treatment (P <
0.05). This effect of adenosine was completely lost once
DPCPX was given prior to adenosine administration.
However, the exacerbating effect of DPCPX was
significantly evident in response to substance P at doses
lower than 10-5 mol/L (Figure 5). Above this concentration,
as shown in the ascending part of the curve, responses in
both adenosine- and DPCPX-adenosine-treated groups
were statistically indistinguishable. Accordingly, there was
a statistically significant difference between I/R control
group and DPCPX-adenosine-treated group in response
to 10-4 mol/L (P = 0.022), 10-3 mol/L (P = 0.004), and
10-2 mol/L (P = 0.011) of substance P. Regarding the
corresponding pD2 values, no statistically significant
change was detected in any group (Table 1).
TBARS level
Average TBARS content of intestinal samples from sham-
operated animals was 54.18 ± 3.26 nmol/g tissue, while
that from I/R control rats was 78.27 ± 7.60 nmol/g tissue
(Figure 6). I/R caused approximately 1.45 fold increase
in TBARS content of the tissue, which was significantly
different from that measured in samples from sham-
operated animals (P = 0.002). Administration of either
CPA or adenosine prior to the induction of ischemia
significantly reduced the elevated TBARS content to the
levels observed in sham control rats. Mean values of the
both groups (39.87 ± 11.02 nmol/g tissue and 56.49 ± 7.03
nmol/g tissue, respectively) were significantly different
from that of the I/R control group (P = 0.001). On the
other hand, in the case of DPCPX pretreatment before
Sham control
I/R control
CPA
Adenosine
DPCPX-Adenosine
Contraction (% of KCL)
600
500
400
300
200
100
0
10-9 10-8 10-7 10-6 10-5 10-4 10-3 10-2
Concentration of carbachol (mol/L)
a
a
a
a
a
a
a
c
c
Figure 3 Dose-response curves of carbachol in longitudinal ileum muscle isolated
from sham-operated control, I/R control, CPA-I/R, adenosine-I/R, and DPCPX-
adenosine-I/R rats. Data are expressed as means ± SE (n = 8). aP < 0.05 vs
sham-operated control, cP < 0.05 vs I/R control groups.
Figure 2 Representative traces showing responses generated by various
concentrations of carbachol in longitudinal ileum muscle isolated from sham-
operated control (A), I/R control (B), CPA–I/R (C), adenosine-I/R (D), and DPCPX-
adenosine-I/R (E) rats.
4 3 2
5
6
7
8
9
4
5
6
7
8
9
8
9
7
6
5
4 3 2
4
3
2
5
6
7
8
9
9
8
76543
2
1 min
0.5 g
A B C D E
Figure 4 Representative traces showing responses generated by various
concentrations of substance P in longitudinal ileum muscle isolated from sham-
operated control (A), I/R control (B), CPA-I/R (C), adenosine-I/R (D), and DPCPX-
adenosine-I/R (E) rats.
3 2
4 3 2
4
5
6
7
8
9
987
6
54
32
5
6
7
8
9
987
6
5
4
3
2
2
3
4
5
6
7
8
9
1 min
0.5 g
A B C D E
Figure 5 Dose-response curves of substance P in longitudinal ileum muscle
isolated from sham-operated control, I/R control, CPA-I/R, adenosine-I/R, and
DPCPX-adenosine-I/R rats. Data are expressed as means ± SE (n = 8). aP < 0.05
vs sham-operated control, cP < 0.05 vs I/R control groups.
Sham control
I/R control
CPA
Adenosine
DPCPX-Adenosine
Contraction (% of KCL)
400
300
200
100
0
10-9 10-8 10-7 10-6 10-5 10-4 10-3 10-2
Concentration of substance P (mol/L)
a
a
a
a
a
a
c
c
c
c
c
a
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542 ISSN 1007-9327 CN 14-1219/R World J Gastroenterol January 28, 2007 Volume 13 Number 4