Opioid Receptor Blockade Improves Mesenteric Responsiveness in Biliary Cirrhosis

Abstract

Arterial vasodilation with concomitant hyperdynamic circulation is a common finding in cirrhotic subjects. Elevated levels of plasma endogenous opioid peptides have been reported in cholestasis and cirrhosis. Increased opioid peptides contribute to different manifestations of chronic liver disease such as pruritus, ascitis, and hepatic encephalopathy. In this study the potential role of opioid system in cirrhosis-induced vascular hyporesponsiveness was investigated. Bile duct ligated and sham operated animals received daily subcutaneous administration of naltrexone, an opioid receptor antagonist (20 mg/kg/day), or saline for 28 days. After 4 weeks the superior mesenteric artery was cannulated and was perfused according to McGregor method and then phenylephrine vasoconstrictor response of mesenteric vessels (10(-10) to 10(-6 )mol) was examined. In order to evaluate the effects of acute opioid receptor blockade, additional groups of animals were treated by acute single intraperitoneal naltrexone injection (20 mg/kg). Plasma level of nitrite/nitrate as an indicator for nitric oxide production was measured. Biliary cirrhosis was accompanied with a decrease in baseline perfusion pressure in mesenteric vascular bed (P < 0.01). Chronic opioid receptor blockade significantly increased this parameter (P < 0.01). The maximum pressure response to phenylephrine was decreased significantly in cirrhosis while chronic naltrexone treatment completely improved it (P < 0.01). Acute single injection of naltrexone could not influence the understudied homodynamic parameters. Chronic opioid receptor blockade did not modulate the increased nitrite/nitrate levels following cholestasis. This study provided evidence on the contribution of endogenous opioid system to vascular hyporesponsiveness in cirrhosis which is not directly correlated to high plasma NO levels.

Full text

ORIGINAL PAPER
Opioid Receptor Blockade Improves Mesenteric Responsiveness
in Biliary Cirrhosis
Mohammad R. Ebrahimkhani Æ Leila Moezi Æ
Samira Kiani Æ Shahin Merat Æ Ahmad R. Dehpour
Received: 30 December 2007 / Accepted: 26 March 2008
Ó Springer Science+Business Media, LLC 2008
Abstract Arterial vasodilation with concomitant hyper-
dynamic circulation is a common finding in cirrhotic
subjects. Elevated levels of plasma endogenous opioid
peptides have been reported in cholestasis and cirrhosis.
Increased opioid peptides contribute to different manifes-
tations of chronic liver disease such as pruritis, ascitis, and
hepatic encephalopathy. In this study the potential role of
opioid system in cirrhosis-induced vascular hyporespon-
siveness was investigated. Bile duct ligated and sham
operated animals received daily subcutaneous administra-
tion of naltrexone, an opioid receptor antagonist (20 mg/
kg/day), or saline for 28 days. After 4 weeks the superior
mesenteric artery was cannulated and was perfused
according to McGregor method and then phenylephrine
vasoconstrictor response of mesenteric vessels (10
-10
to
10
-6
mol) was examined. In order to evaluate the effects of
acute opioid receptor blockade, additional groups of
animals were treated by acute single intraperitoneal nal-
trexone injection (20 mg/kg). Plasma level of nitrite/nitrate
as an indicator for nitric oxide production was measured.
Biliary cirrhosis was accompanied with a decrease in
baseline perfusion pressure in mesenteric vascular bed
(P \ 0.01). Chronic opioid receptor blockade significantly
increased this parameter (P \ 0.01). The maximum pres-
sure response to phenylephrine was decreased significantly
in cirrhosis while chronic naltrexone treatment completely
improved it (P \ 0.01). Acute single injection of naltrex-
one could not influence the understudied homodynamic
parameters. Chronic opioid receptor blockade did not
modulate the increased nitrite/nitrate levels following
cholestasis. This study provided evidence on the contri-
bution of endogenous opioid system to vascular
hyporesponsiveness in cirrhosis which is not directly cor-
related to high plasma NO levels.
Keywords Mesenteric vascular bed 
Endogenous opioids  Biliary cirrhosis  Bile duct ligated 
Naltrexone  Nitric oxide
Introduction
Cirrhosis of the liver is one of the major causes of mor-
bidity and mortality in the world. It is associated with
hyperdynamic state and arterial vasodilatation, which is
most prominent in mesenteric vascular bed. This phe-
nomenon is responsible for some of the life threatening
events that occur during cirrhosis, such as variceal haem-
orrhage and ascitis. The exact molecular mechanisms are
elusive, but hyporesponsiveness to vasoconstrictors possi-
bly in part due to circulating vasodilators such as nitric
oxide (NO), is thought to play important parts [1].
M. R. Ebrahimkhani  S. Kiani  A. R. Dehpour (&)
Department of Pharmacology, School of Medicine, Tehran
University of Medical Sciences, P.O. Box 13145-784,
Tehran, Iran
e-mail: dehpour@yahoo.com
Present Address:
M. R. Ebrahimkhani
Department of Microbiology and Immunology, David H. Smith
Center for Vaccine Biology and Immunology, Aab Institute
of Biomedical Sciences, University of Rochester Medical
Center, Rochester, NY, USA
L. Moezi
Department of Pharmacology, School of Medicine, Shiraz
University of Medical Sciences, Shiraz, Iran
S. Merat
Digestive Disease Research Centre, Tehran University
of Medical Sciences, Tehran, Iran
123
Dig Dis Sci
DOI 10.1007/s10620-008-0261-7

It has been demonstrated that exogenous opioids can
influence vascular response either by their direct effects on
vascular bed via specific opioid receptors [2] or centrally
via nervous system. Elevated levels of plasma endogenous
opioid peptides mainly methionine enkephaline have been
also reported in patients and animals subjected to chole-
stasis [3]. Opioid peptides contribute partly to the
manifestations of liver disease such as fatigue, pruritis,
ascitis and hepatic encephalopathy. There are also emerg-
ing reports on the role of opioid system in the
pathophysiology of cardiovascular hyporesponsiveness in
short-term cholestasis [4, 5]. However there is not any data
regarding the contribution of endogenous opioid system to
hyperdynamic circulation of cirrhosis which is a more
common clinical situation with worldwide importance.
These observations made us design a study to investi-
gate whether long-term opioid receptor blockade by
naltrexone can improve mesenteric vascular bed response
in secondary biliray cirrhosis. Naltrexone is a non-selec-
tive, long acting opioid receptor blocker which blocks all
three opioid receptors (l, d, j). It is used in clinical
practice for cholestatic pruritis in patients with liver disease
therefore it is readily accessible drug to use in human
studies and future clinical trials.
Materials and Methods
Reagents
All materials were purchased from Sigma (Pool, UK),
otherwise specified in the text.
Animals
Male Sprague Dawley rats (200–230 g) were used in this
study. All animal procedures were in accordance with
‘‘Guide for the Care and Use of Laboratory animals’’ (NIH
US publication no. 85–23 revised 1985) and Pasteur
Institute of Iran.
Study Groups
The rats were randomly divided into two groups of age-
matched rats. One group underwent a sham procedure
(controls) and the other group underwent bile duct ligation
as described [6]. Briefly Laparatomy was performed under
general anesthesia (Ketamine HCl 50 mg/kg and Xylazine
HCl 10 mg/kg i.p.). The bile duct was exposed and triple
ligated, then cut through between the second and third
distal ligations. Sham operation consisted of laparatomy,
bile duct identification and manipulation without ligation.
Mortality rate of procedure is about 5–10% during bile duct
ligation. Each of the two groups was then divided into
saline-treated or naltrexone-treated groups.
Sham-control (n = 8) and cirrhotic-control (n = 8) rats
received daily injection of sterile saline solution (1 ml/kg/
day, s.c.) from the first day of operation. Sham-naltrexone
(n = 8) or cirrhotic-naltrexone (n = 8) rats were injected
with sub-cutaneous naltrexone hydrochloride (20 mg/kg/
day, s.c.) for 28 days post sham procedure or bile duct
ligation [4, 5]. In chronic naltrexone treatment, the last
doses of the drugs were injected 16 h before the experi-
mental protocol. In order to evaluate the effects of acute
opioid receptor blockade, additional groups of animals
were treated by acute single dose intraperitoneal naltrexone
injection 30 min before the experiments (20 mg/kg).
Preparation of Mesenteric Vascular Bed
After 28 days, the rats were anesthetized by sodium pen-
tobarbital (50 mg/kg; i.p.) and the mesenteric vascular bed
was prepared as described in details elsewhere, according
to McGregor method (1965) [7]. Briefly the abdominal
wall was opened and the superior mesenteric artery was
identified, cannulated and gently flushed with modified
Krebs-Henseleit solution that was bubbled with a mixture
of O
2
and CO
2
(pH: 7.4, T = 37°C). A peristaltic pump
(Pump speed control Model 500–1200, Harvard Apparatus,
Dover, MA, USA) provided the constant flow. The perfu-
sion pressure was measured using a pressure transducer
(Pressure Transducer Model P-1000-A, Narco Biosystem,
Houston, TX, USA) placed in the circuit between the outlet
of the pump and the preparation and was recorded on a
Narco physiograph (Desk Model DMP-4B, Narco
Biosystem).
Vasoconstriction Experiment
For measuring the vasoconstriction response of the mes-
enteric vascular bed, phenylephrine, an a
1
-adrenoceptor
agonist, was injected (almost in doses of 0.1 nmol–1 lmol)
into the perfusate before it entered the tissue. The injection
volume was 0.1 ml and injection time was 30 s. The
vasoconstriction, being recorded as an increase in perfusion
pressure, was expressed as mm Hg increase in perfusion
pressure.
Plasma Nitrite/Nitrate Measurement
Plasma nitrate and nitrite levels were measured as indica-
tors of NO production. The measurements were done
according to method by Miranda et al. [8]. Samples were
deproteinized by centrifugation through a 30-kDa molec-
ular weight filter (Centricon Millipore, USA) at
14,000 rpm, for 1.5–3 h at 4°C. After loading the plate
Dig Dis Sci
123

with samples (100 ll), addition of saturated solution of
VCl
3
(100 ll) to each well was rapidly followed by addi-
tion of the Griess reagents (50 ll each). Sulfanilamide and
naphthylethylenediamine dihydrochloride were applied for
preparation of Griess reagents. The plate was incubated at
37°C for 30 min and then absorbance at 540 nm was
measured using standard plate reader. Fresh standard
solutions of nitrate were included in each experiment.
Statistical Analysis
Results were expressed as mean ± SEM. Statistical eval-
uation of data performed by analysis of variances
(ANOVA) followed by Tukey post hoc test. P-values less
than 0.05 were considered statistically significant.
Results
Induction of Cholestasis
One day after laparotomy cirrhotic rats showed manifes-
tation of cholestasis (jaundice, dark urine). Plasma
bilirubin concentration increased in cirrhotic animals con-
sistent with biliary obstruction (10.12 ± 1.05 vs.
0.57 ± 0.09 cirrhotic versus sham animals, P \ 0.05) and
remained significantly elevated all over the study.
Baseline Perfusion Pressure
Perfusion of the mesenteric vascular bed resulted in a
baseline perfusion pressure in the range of 14–24 mmHg.
Bile duct ligation was accompanied by decreased baseline
perfusion pressure (P \0.01). The baseline perfusion
pressure of cirrhotic rats increased significantly by chronic
opioid receptor blockade (17.1 ± 0.7 vs. 21.1 ± 0.7, cir-
rhotic/saline and cirrhotic/naltrexone, respectively;
P \ 0.01). Naltrexone treatment did not influence signifi-
cantly the baseline perfusion pressure in sham group
(Fig. 1).
Phenylephrine-Induced Vasoconstriction
Phenylephrine induced dose-dependent vasoconstriction
that is manifested as an increase in the perfusion pressure
(Fig. 2). The maximum pressure response was higher in
sham-operated group than cirrhotic one (Fig. 2a). The
difference between these two groups became significant in
doses higher than 10
-7
mol and naltrexone treatment could
prevent it significantly (P \ 0.01) (Fig. 2a and b). As it is
clear in Fig. 2b, chronic naltrexone treatment in cirrhotic
rats altered the dose-dependent vasoconstrictor response in
mesenteric vascular bed almost completely toward normal
values. However, acute administration of naltrexone
30 min before experiments could not improve vascular
response (Fig. 3).
0
5
10
15
20
25
Sham
Baseline Perfusion Pressure(mmHg)
Saline
Naltrexone
a
b
Cirrhosis
Fig. 1 The baseline perfusion pressure (mm Hg) of mesenteric
vascular bed of sham operated and 28-day bile duct ligated (cirrhotic)
rats treated 28 days either with saline or naltrexone. (
a
P \ 0.001 vs.
Sham groups;
b
P \ 0.001 vs. cirrhotic/saline group)
Fig. 2 (a and b) The vasoconstriction response curve to phenyleph-
rine for the mesenteric vascular bed of (a) sham operated and bile
duct ligated (cirrhotic) rats treated with saline; (b) sham operated and
bile duct ligated (cirrhotic) rats treated either with saline or naltrexone
for 28 days. Each point represents the mean ± SEM for six to seven
rats. (* P \ 0.01, cirrhotic/saline group in comparison with the
corresponding sham groups or cirrhotic/naltrexone group)
Dig Dis Sci
123

Nitrite and Nitrate Level
Animals undergoing bile duct ligation and treated with
saline had a significantly higher plasma nitrite and nitrate
concentration compared with sham-operated animals
(22.07 ± 2.01 lmol/l vs. 14.2 ± 3.1 lmol/l, cirrhotic/sal-
ine and sham/saline, respectively; P \ 0.05), indicative of
increased NO synthesis following bile duct ligation.
Administration of naltrexone to cirrhotic animals for
28 days could not prevent this increase in plasma nitrite
and nitrate level such that plasma levels were similar
to cirrhotic/saline group (23.1 ± 4.5 lmol/l vs. 22.07 ±
2.01 lmol/l cirrhosis/naltrexone and cirrhosis/saline;
P [ 0.05) (Fig. 4).
Discussion
To our knowledge, the present investigation is the first
study which showes roles for endogenous opioids on vas-
cular hyporesponsiveness in the mesenteric artery vascular
bed of experimental animals with cirrhosis. We showed
that phenylephrine-induced vasoconstriction is impaired in
mesenteric vascular bed of cirrhotic rats and chronic opioid
receptor blockade by naltrexone prevented this hypore-
sponsiveness in cirrhotic animals without modulating
plasma NO levels.
Previously we had shown that 7-day treatment of short-
term cholestatic rats with naltrexone had just partially
restored phenylephrine-induced vasoconstriction [5]. Fur-
thermore, in that study, we could not detect significant
improvement in baseline perfusion pressure of naltrexone
treated cirrhotic rats in comparison to cirrhosis/saline
group [5].
In the present study long-term blockade of opioid
receptors completely normalized those parameters. It is
clear that in the course of progression from cholestasis to
cirrhosis, cardiovascular alterations also progress. Sup-
porting this notion, a dynamic time-dependent decrease in
the vascular response to phenylephrine has been shown
previously during the cholestasis and its subsequent cir-
rhosis [9]. The more pronounced effect of opioid blockade
in cirrhotic animals implies that the interference of
endogenous opioid system with mesenteric contractile
response increases in time and opioid system contributes to
progression of vascular hyporesponsiveness during the
cholestasis to cirrhosis. The difference may be due to the
alteration in density of opioid receptors or their respon-
siveness during the course of disease in vascular system.
Earlier investigations have examined vascular respon-
siveness to different vasoactive agents in experimental
animals and humans with cirrhosis, but with discrepant
proposed mechanisms. While much has been reported
about the role of NO in the genesis of hyperdynamic cir-
culation, it was shown that inhibition of NOS does not
completely abrogate this phenomenon [10]. Clearly, there
are some other undefined mediators involved.
Lee et al. showed that disordered central cardiovascular
regulation contributes to blunted cardiovascular respon-
siveness in cirrhosis and prehepatic portal hypertension
[11]. Opioid receptors behind the blood–brain barrier in
dorsal hippocampal region are involved in the vasorelax-
atory effects of systemically administered opioid agonists
in the hypertensive rats [12]. Therefore; it is probable that
increased opioid transmission in cirrhotic subjects con-
tributes to centrally induced hyporesponsiveness in the
-10 -9
-8
-7 -6 -5 -4
0
10
20
30
40
Cirrhosis+Naltrexon
Cirrhosis+Saline
Phenylephrine log (mol)
Increase in perfusion
pressure (mmHg)
(b)(a)
0
5
10
15
20
25
Cirrhosis+saline
Baseline perfusion pressure (mmHg)
Cirrhosis+naltrexone
Fig. 3 The baseline perfusion
pressure (a) and
vasoconstriction response curve
to phenylephrine (b) for the
mesenteric vascular bed of
cirrhotic rats injected
intraperitoneal, single dose,
with naltrexone 30 min before
assessment of vascular response
0
5
10
15
20
25
30
Sham Cirrhosis
Nitrite+Nitrate (micromolar)
Saline
Naltrexone
*
Fig. 4 The plasma nitrite and nitrate levels of sham operated and 28-
day bile duct ligated (cirrhotic) rats treated either with saline or
naltrexone for 28 days. (* P \ 0.05 cirrhotic/saline group in com-
parison with Sham/saline group)
Dig Dis Sci
123

cardiovascular system. However, arterial nerves of mes-
enteric vascular bed are also known to express d-opioid
receptors [13], activation of which can result in a decrease
neuroeffector release and consequent neurogenic
hyporesponsiveness.
Opioid induced increase in NO production has been well
characterized previously. It has been shown that morphine
can increase intracellular calcium in endothelial cells via
specific l opioid receptors, which then activates NO pro-
duction. In this study we demonstrated that opioid receptor
blockade could not decease plasma nitrite and nitrate lev-
els. This might suggest that, endogenously produced
opioids influence the vascular response in cirrhosis inde-
pendent of NO overproduction. It has been suggested both
increased inducible Nitric oxide synthase (NOS) activation
and impaired constitutive NOS activity can participate in
cholestatic induced vascular changes [14]. However, the
lack of naltrexone effect on plasma NO level does not
completely rule out NO dependent mechanism, since opi-
oid blockade might have prevented the impairment of
constitutive NOS activity. Therefore, further studies are
warranted in this context.
Recently, we have shown that opioid system contributes
to the liver fibrogenesis and naltrexone treatment had
important hepatoprotective effects by modulation of liver
redox sate and hepatic stellate cell activation [15, 16]. The
antifibrogenic roles for opioid antagonist has been also
reported in other models of liver cirrhosis [17]. So, the
observed improvement in mesenteric response after chronic
naltrexone treatment in cirrhotic rats may be due to the
potential hepatoprotective effect of opioid blockade which
lessens the degree of liver injury and its corresponding car-
diovascular alterations. This mechanism is also supported by
our finding that acute administration of naltrexone could not
improve mesenteric vascular response in this study.
In conclusion, there is an impaired vascular contractile
response in cirrhosis. We demonstrated an important role
for endogenous opioid system in the process of hypore-
sponsiveness of mesenteric vascular bed in secondary
biliary cirrhosis.
References
1. Castro A, Jime
´
nez W, Cla
`
ria J, Ros J, Martı
´
nez JM, Bosch M,
Arroyo V, Piulats J, Rivera F, Rode
´
s J (1993) Impaired respon-
siveness to angiotensin II in experimental cirrhosis: role of nitric
oxide. Hepatology 18:367–372
2. Stefano GB, Hartman A, Bilfinger TV, Magazine HI, Liu Y,
Casares F, Goligorsky MS (1995) Presence of the mu3 opiate
receptor in endothelial cells. Coupling to nitric oxide production
and vasodilation. J Biol Chem 270:30290–30293
3. Swain MG, Rothman RB, Xu H, Vergalla J, Bergasa NV, Jones
EA (1992) Endogenous opioids accumulate in plasma in a rat
model of acute cholestasis. Gastroenterology 103:630–635
4. Gaskari SA, Mani AR, Ejtemaei-Mehr S, Namiranian K, Ho-
mayoun H, Ahmadi H, Dehpour AR (2002) Do endogenous
opioids contribute to the bradycardia of rats with obstructive
cholestasis? Fundam Clin Pharmacol 16:273–279
5. Namiranian K, Samini M, Mehr SE, Gaskari SA, Rastegar H,
Homayoun H, Dehpour AR (2001) Mesenteric vascular bed
responsiveness in bile duct-ligated rats: roles of opioid and nitric
oxide systems. Eur J Pharmacol 423:185–193
6. Ebrahimkhani MR, Sadeghipour H, Dehghani M, Kiani S, Pa-
yabvash S, Riazi K, Honar H, Pasalar P, Mirazi N, Amanlou M,
Farsam H, Dehpour AR (2005) Homocysteine alterations in
experimental cholestasis and its subsequent cirrhosis. Life Sci
76:2497–2512
7. Moezi L, Rezayat M, Samini M, Shafaroodi H, Mehr SE, Ebra-
himkhani MR, Dehpour AR (2004) Potentiation of anandamide
effects in mesenteric beds isolated from bile duct-ligated rats:
role of nitric oxide. Eur J Pharmacol 486:53–59
8. Miranda KM, Espey MG, Wink DA (2001) A rapid, simple
spectrophotometric method for simultaneous detection of nitrate
and nitrite. Nitric Oxide 5(1):62–71
9. Kimpel M, Folz IC, Hanisch E (1998) Time course-dependent
evolution of nitric oxide-mediated arterial hyporeactivity to
phenylephrine in rats with ligated bile duct. Scand J Gastroenterol
33:314–318
10. Atucha NM, Ortı
´
z MC, Fortepiani LA, Nadal FJ, Martı
´
nez-Prieto
C, Garcı
´
a-Estan
˜
J (2000) Mesenteric hyporesponsiveness in cir-
rhotic rats with ascitis: role of cGMP and K+ channels. Clin Sci
(Lond) 99:455–460
11. Song D, Sharkey KA, Breitman DR, Zhang Y, Lee SS (2001)
Disordered central cardiovascular regulation in portal hyperten-
sive and cirrhotic rats. Am J Physiol Gastrointest Liver Physiol
280:G420–G430
12. Shen S, Ingenito AJ (1999) Comparison of cardiovascular
responses to intra-hippocampal mu, delta and kappa opioid ago-
nists in spontaneously hypertensive rats and isolation-induced
hypertensive rats. J Hypertens 17:497–505
13. Illes P, Ramme D, Starke K (1986) Presynaptic opioid delta-
receptors in the rabbit mesenteric artery. J Physiol 379:217–228
14. Iwakiri Y, Groszmann RJ (2006) The hyperdynamic circulation
of chronic liver diseases: from the patient to the molecule.
Hepatology 43(2 Suppl 1):S121–S131
15. Kiani S, Ebrahimkhani MR, Shariftabrizi A, Doratotaj B, Paya-
bvash S, Riazi K, Dehghani M, Honar H, Karoon A, Amanlou M,
Tavangar SM, Dehpour AR (2007) Opioid system blockade
decreases collagenase activity and improves liver injury in a rat
model of cholestasis. J Gastroenterol Hepatol 22(3):406–413
16. Ebrahimkhani MR, Kiani S, Oakley F, Kendall T, Shariftabrizi A,
Tavangar SM, Moezi L, Payabvash S, Karoon A, Hoseininik H,
Mani AR, Mann DA, Moore KP, Dehpour AR (2006) Naltrexone,
an opioid receptor antagonist, attenuates liver fibrosis in bile duct
ligated rats. Gut 55(11):1606–1616
17. De Minicis S, Candelaresi C, Marzioni M, Saccomano S, Rosk-
ams T, Casini A, Risaliti A, Salzano R, Cautero N, di Francesco
F, Benedetti A, Svegliate-Baroni G (2008) Role of endogenous
opioids in modulating HSC activity in vitro and liver fibrosis in
vivo. Gut Mar 57(3):352–364
Dig Dis Sci
123