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Gallbladder stasis is associated to experimental acute cholecystitis. Impaired contractility could be, at least in part, the result of inflammation-induced alterations in the neuromuscular function. This study was designed to determine the changes in gallbladder neurotransmission evoked by acute inflammation and to evaluate the protective and therapeutic effects of melatonin. Experimental acute cholecystitis was induced in guinea pigs by common bile duct ligation for 2 days, and then the neuromuscular function was evaluated using electrical field stimulation (EFS; 5-40 Hz). In a group of animals with the bile duct ligated for 2 days, a deligation of the duct was performed, and after 2 days, the neuromuscular function was studied. The EFS-evoked isometric gallbladder contraction was significantly lower in cholecystitic tissue. In addition, inflammation changed the pharmacological profile of these contractions that were insensitive to tetrodotoxin but sensitive to atropine and omega-conotoxin, indicating that acute cholecystitis affects action potential propagation in the intrinsic nerves. Nitric oxide (NO)-mediated neurotransmission was reduced by inflammation, which also increased the reactivity of sensitive fibers. Melatonin treatment prevented qualitative changes in gallbladder neurotransmission, but it did not improve EFS-induced contractility. The hormone recovered gallbladder neuromuscular function once the biliary obstruction was resolved, even when the treatment was started after the onset of gallbladder inflammation. These findings show for the first time the therapeutic potential of melatonin in the recovery of gallbladder neuromuscular function during acute cholecystitis.
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Effects of Melatonin on Gallbladder Neuromuscular Function in
Acute Cholecystitis
Pedro J. Gomez-Pinilla, Pedro J. Camello, and Marı´a J. Pozo
Department of Physiology, Nursing School, University of Extremadura, Caceres, Spain
Received March 22, 2007; accepted July 3, 2007
ABSTRACT
Gallbladder stasis is associated to experimental acute chole-
cystitis. Impaired contractility could be, at least in part, the
result of inflammation-induced alterations in the neuromuscular
function. This study was designed to determine the changes in
gallbladder neurotransmission evoked by acute inflammation
and to evaluate the protective and therapeutic effects of mel-
atonin. Experimental acute cholecystitis was induced in guinea
pigs by common bile duct ligation for 2 days, and then the
neuromuscular function was evaluated using electrical field
stimulation (EFS; 5–40 Hz). In a group of animals with the bile
duct ligated for 2 days, a deligation of the duct was performed,
and after 2 days, the neuromuscular function was studied. The
EFS-evoked isometric gallbladder contraction was significantly
lower in cholecystitic tissue. In addition, inflammation changed
the pharmacological profile of these contractions that were
insensitive to tetrodotoxin but sensitive to atropine and
-conotoxin, indicating that acute cholecystitis affects action
potential propagation in the intrinsic nerves. Nitric oxide (NO)-
mediated neurotransmission was reduced by inflammation,
which also increased the reactivity of sensitive fibers. Melatonin
treatment prevented qualitative changes in gallbladder neuro-
transmission, but it did not improve EFS-induced contractility.
The hormone recovered gallbladder neuromuscular function
once the biliary obstruction was resolved, even when the treat-
ment was started after the onset of gallbladder inflammation.
These findings show for the first time the therapeutic potential
of melatonin in the recovery of gallbladder neuromuscular func-
tion during acute cholecystitis.
Gallbladder tone is mainly regulated by both myogenic
mechanisms and neurohormonal inputs. The neural control
of gallbladder motility involves reflexes that include both
efferent and afferent nerve fibers as well as the intrinsic
plexus in the gallbladder wall (Mawe et al., 2006). Acetylcho-
line (ACh) released from cholinergic neurons induces con-
traction of the gallbladder smooth muscle through musca-
rinic receptors (Parkman et al., 1999b), and it has
neuromodulatory functions, promoting or inhibiting the re-
lease of other neurotransmitters (Parkman et al., 1999b).
Cholinergic neurons coexpress other neurotransmitters such
as NO and several neuropeptides (Talmage et al., 1992).
Afferent nerve fibers containing calcitonin gene-related pep-
tide and tachykinins have also been described in the gangli-
onated plexus of the gallbladder (Mawe and Gershon, 1989).
Acute acalculous cholecystitis (AC) is characterized by gall-
bladder inflammation in the absence of gallstones. Although
its pathogenesis is unknown, gallbladder stasis is always
present, probably as the result of the deleterious neural and
muscular actions of inflammatory mediators such as reactive
oxygen species and prostaglandins (Pozo et al., 2004). In
animal models, it has been described that cholecystitis re-
duces gallbladder contractile responses to agonists that act
directly on smooth muscle cells (Parkman et al., 1999a; Xiao
et al., 2001) and that it also causes alterations in calcium
signaling and contractile machinery (Gomez-Pinilla et al.,
2006b). In addition, EFS-induced contractions are also im-
paired in inflamed gallbladder, mainly due to the reduction
in the function of cholinergic nerves and the up-regulation of
the inhibitory nitrergic component (Parkman et al., 2000).
The effect of cholecystitis on afferent fibers has not yet been
explored.
Melatonin (MEL), the main product of pineal gland, is a
potent free radical scavenger, and it activates a broad group
of antioxidant cellular mechanisms (Tan et al., 2002). These
properties made melatonin efficacious against different dis-
eases where oxidative stress is the main cause (Karasek,
2004). The gastrointestinal tract is an important source of
melatonin (Kvetnoy et al., 2002). The liver and the gallblad-
This work was supported by Ministerio de Educacion y Ciencia BFU 2004-
0637 and Junta de Extremadura 2PR03A020.
P.J.G.-P. is recipient of a doctoral fellowship from Junta de Extremadura.
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
doi:10.1124/jpet.107.123240.
ABBREVIATIONS: ACh, acetylcholine; AC, acute acalculous cholecystitis; MEL, melatonin; CBDL, common bile duct ligation; DL, deligation; EFS,
electrical field stimulation; MDA, malondialdehyde; GSH, glutathione; L-NAME, N
-nitro-L-arginine methyl ester; DMSO, dimethyl sulfoxide;
ANOVA, analysis of variance; TTX, tetrodotoxin.
0022-3565/07/3231-138–146$20.00
T
HE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 323, No. 1
Copyright © 2007 by The American Society for Pharmacology and Experimental Therapeutics 123240/3252691
JPET 323:138–146, 2007 Printed in U.S.A.
138
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der are especially exposed to high levels of the hormone,
because hepatic metabolism is the major pathway for deac-
tivation of melatonin (Lane and Moss, 1985), which is also
present in active form in bile and concentrated in the gall-
bladder (Tan et al., 1999). In the gastrointestinal tract, mel-
atonin has a gastroprotective function (Konturek et al., 1997)
and therapeutic effects against malignancy associated with
irritable bowel syndrome (Head and Jurenka, 2003).
The aims of this study were to investigate the effects of
acute cholecystitis in the neuromuscular transmission and to
evaluate the impact of melatonin treatment. Our results
indicate that melatonin restores neuromuscular function in
inflamed gallbladder, which can be of importance to recover
gallbladder contractility in this pathological condition.
Materials and Methods
Design: Animal Preparations. Male guinea pigs, weighing 400
to 600 g, were used in the study. AC was induced to animals by
common bile duct ligation (CBDL) for 2 days, as described previously
(Gomez-Pinilla et al., 2006b). This method was approved by the
Animal Care and Ethical Committees of the University of Extrema-
dura (Caceres, Spain). In brief, after anesthesia with 20 mg/kg i.p.
ketamine hydrochloride and 5 mg/kg i.p. xylacine, a laparotomy was
performed, and the distal end of the common bile duct was ligated.
Two days after, the animals were sacrificed for tissue harvest (n
28). In the model used in this study, the gallbladder is stretched as
the result of bile duct ligation and the continuous bile output. Taking
this into account, it would be difficult to see any improvement in the
neuromuscular function by keeping the bile duct ligated, which rep-
resents a remarkably extreme pathological condition. To solve this
dilemma, in a group of animals 2 days later, CBDL, the common bile
duct, was deligated (DL) under anesthesia with microsurgical scis-
sors, and 2 days later, the animals were sacrificed (n 28). For both
experimental models, a group of guinea pigs were sham-operated
(n 4), which included all of the surgical steps, with the exception of
common bile duct ligation.
Melatonin and Tempol Administration. Guinea pigs were
treated orally with melatonin (2.5 or 30 mg/kg/day). Melatonin was
dissolved in glucose solution (1.5%), and it was placed in the oro-
pharynx by using a syringe. This treatment was applied daily at the
same time, just before the light in the animal house was switched off
(7:00 PM). Melatonin was administered 14 days before the sacrifice
of the animals in both experimental groups, AC and DL. In a group
of animals subjected to DL, melatonin treatment (30 mg/kg) started
12 h after CBDL was performed, and it continued until the sacrifice
of the animal. Tempol was administered in the drinking water at 1
mM for 14 days before the animal was sacrificed.
Functional Studies. At the appropriated time, the animals were
killed with deep halothane anesthesia and cervical dislocation. Gall-
bladders were removed, and they were immediately placed in ice-cold
Krebs-Henseleit solution (for composition, see “Solutions and
Drugs”) at pH 7.35. The gallbladder was cut in longitudinal full-
thickness strips (3 10 mm) that were placed vertically in a 10-ml
organ bath filled with Krebs-Henseleit solution maintained at 37°C
and gassed with 95% O
2
,5%CO
2
. Isometric contractions were mea
-
sured using force displacement transducers that were interfaced
with a Macintosh computer using a MacLab hardware unit and
software (ADInstruments, Colorado Springs, CO). The muscle strips
were placed under an initial resting tension equivalent to a 1.5-g
load. Intrinsic nerves were activated by EFS with a pair of external
platinum ring electrodes connected to a square-wave stimulator
(CS9/3BO; Cibertec, Madrid, Spain). Trains of stimuli (0.3-ms dura-
tion; 5– 40 Hz; 350-mA current strength) were delivered for 10 s at
3-min intervals. After construction of a frequency-response curve
and to pharmacologically characterize the neurogenic responses, an-
tagonists/inhibitors were added to the organ bath for 20 min, and
then the EFS protocols were repeated.
Malondialdehyde and Reduced Glutathione Assays. Gall-
bladder fragments of approximately 10 mg were placed in an ice-cold
phosphate buffer at a proportion of 1:5 (w/v), homogenized with an
homogenizer (Ika-Werke, Staufen, Germany) for 2 min, and centri-
fuged at 10,000 rpm for 15 min at 4°C. The protein concentration was
then quantified with a commercial kit (TPRO-562; Sigma-Aldrich,
St. Louis, MO), and the rest of homogenate was treated with ice-cold
perchloric acid (7%, v/v) to eliminate proteins, and it was kept at
80°C until analysis. Malondialdehyde (MDA) level, an index of
lipidic peroxidation, was determined based on colorimetric Reckna-
gel’s method (Waller and Recknagel, 1977). In brief, the samples
were incubated with 0.4% thiobarbituric acid at 80°C for 20 min, and
then the sample absorbance at 550 nm was measured. Reduced
glutathione determination was carried out following the Hissin and
Hilf (1976) method. Samples were incubated with 0.005% orthoph-
thaldehyde in darkness at room temperature for 45 min, and the
fluorescent complex that was formed, indicative of reduced glutathi-
one (GSH) level, was measured with a fluorimeter (excitation, 350
nm; emission, 425 nm).
Solutions and Drugs. The Krebs-Henseleit solution contained
113 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl
2
, 1.2 mM KH
2
PO
4
, 1.2 mM
MgSO
4
, 25 mM NaHCO
3
, and 11.5 mM D-glucose. This solution had
a final pH of 7.35 after equilibration with 95% O
2
,5%CO
2
. The
phosphate buffer used to homogenize the tissue contained 20 mM
NaCl, 2.7 mM KCl, 16 mM Na
2
HPO
4
,and4mMNaH
2
PO
4
, pH 7.4.
Drug concentrations are expressed as final bath concentrations of
active species. Drugs and chemicals were obtained from the following
sources: atropine,
L-NAME, melatonin, and tempol (4-hydroxy-
2,2,6,6-tetramethylpiperidine-N-oxyl or 4-hydroxy-tempo) were from
Sigma-Aldrich.
-Conotoxin GVIA, (E)-capsaicin, and tetrodotoxin
citrate were from Tocris Cookson Inc. (Bristol, UK). Ketamine was
from Merial (Lyon, France). Xylacine was supplied by Bayer AG
(Kiel, Germany). Other chemicals used were of analytical grade from
Panreac (Barcelona, Spain). Stock solutions of atropine, capsaicin,
and
-conotoxin GVIA were prepared in DMSO. The solutions were
diluted such that the final concentration of DMSO was 0.1% (v/v).
This concentration of DMSO did not have effects on gallbladder tone.
Data Analysis. Results are expressed as means S.E.M. of n
gallbladder strips from at least five different animals. Gallbladder
tension is given in millinewtons per milligram of tissue. Statistical
differences between multiple groups or the effects of inhibitor treat-
ments were tested using appropriate analysis of variance (ANOVA).
Differences were considered significant at P 0.05.
Results
Effects of Acute Cholecystitis on Gallbladder Neuro-
muscular Function. EFS was used to stimulate the neuro-
nal network in the gallbladder wall, and the recording of
isometric tension allowed us to evaluate the neuromuscular
function. EFS evoked a frequency-dependent contraction in
control strips that was significantly decreased in animals
subjected to CBDL compared with sham controls (Fig. 1, A
and B). The diminished response was reflected by reductions
in both the amplitude and the duration of the contractions
(P 0.01 and P 0.001, two-way ANOVA for both; Fig. 1, C
and D).
To determine the neural and myogenic components of the
EFS-evoked contractions, the nerve Na
channel inhibitor
tetrodotoxin (TTX) was used. In control strips, 1
M TTX
abolished EFS-elicited responses (Fig. 2A). In inflamed
strips, tetrodotoxin was not effective (3.6% enhancement at
25 Hz) (Fig. 2B), but when the strips were coincubated with
1
M tetrodotoxin plus 0.1
M
-conotoxin GVIA, an N-type
Melatonin in Acute Cholecystitis 139
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calcium channel blocker, there was a reduction (63% inhibi-
tion at 25 Hz) in the contractile response evoked by EFS (Fig.
2B). These results indicate that in inflamed gallbladder, the
transmission of the action potential along neural fibers is
impaired and that EFS stimulates neurotransmitter release
directly from nervous terminals.
To elucidate the neurotransmitters involved in the EFS-
induced contraction, we tested several antagonists/inhibi-
tors on this neural response. In control animals, we found
that 1
M atropine reduced the EFS-elicited contractile
response (82% inhibition at 25 Hz; Fig. 3A), but the strips
from cholecystitic animals were less sensitive to atropine
blockade (30% inhibition at 25 Hz; Fig. 3D). The impact of
inflammation on the contribution of NO was tested by
using the inhibitor of the nitric-oxide synthase,
L-NAME,
at 100
M. This inhibitor enhanced EFS-induced contrac-
tion in strips from control guinea pigs, especially at the
lowest frequencies assayed (90% enhancement at 5 Hz;
Fig. 3B), but it had little effect in inflamed strips (17%
enhancement at 5 Hz; Fig. 3E).
Small-diameter sensory neurons that are sensitive to cap-
saicin play a major role in the generation of neurogenic
inflammation (Sann et al., 1996). When we induced sensory
nerve desensitization by treatment with a high concentration
of capsaicin (10
M), we found no effect in control strips (Fig.
3C), but this treatment induced an inhibition of EFS-elicited
contractile responses in cholecystitic strips (56% inhibition;
Fig. 3F).
Effects of Melatonin on Neuromuscular Function in
Acute Cholecystitis. We have reported previously that mel-
atonin treatment was able to restore gallbladder neuromus-
cular function in aging (Gomez-Pinilla et al., 2006a). To de-
termine whether this hormone had beneficial effects in the
alterations described above, we treated the animals with 2.5
and 30 mg/kg melatonin (MEL 2.5; MEL 30) as described
under Materials and Methods. Under these conditions, none
of melatonin doses used enhanced the amplitude of the con-
tractile responses evoked by EFS (Fig. 4A), but the contrac-
tions partially recovered the sensitivity to TTX (85 and 77%
inhibition for MEL 2.5 and MEL 30, respectively, at 25 Hz;
Fig. 4B). Although the treatment dose-dependently increased
the inhibitory effects of atropine and it decreased the inhib-
itory effects of capsaicin significantly at some frequencies
(Fig. 4, C and E), these changes were small. However, mela-
tonin was able to protect nitrergic nerves, because when
L-NAME was added to the organ bath, the EFS-evoked con-
Fig. 2. AC induces a TTX-resistant
gallbladder response to EFS. Effects
of 1
M TTX on EFS-elicited contrac-
tile response in control (A) and AC
gallbladder strips (B). After EFS was
performed in control conditions (solid
lines) strips were incubated for 20
min with the antagonist, and EFS
was repeated (dotted line). Note the
lack of effects of TTX on inflamed tis-
sue and the reduction in the response
after incubation with 0.1
M
-cono-
toxin. Data are mean S.E.M. n 7
and 6 strips for control and AC strips,
respectively (ⴱⴱ, P 0.01; ANOVA).
Fig. 1. Inflammation impairs EFS-
elicited contractile responses in
guinea pig gallbladder. A, original re-
cordings showing guinea pig gallblad-
der contractile responses elicited by
EFS (0.3-ms duration, 5– 40 Hz, 350
mA, for 10 s every 3 min) applied to
control and AC strips. Traces are typ-
ical of 26 to 18 strips for control and
AC strips, respectively. B, superim-
posed recordings of the EFS-induced
response to 25 Hz showing in more
details the inflammation-related re-
duction in the peak amplitude and du-
ration of the response. C and D, sum-
mary data of EFS induced-responses
(peak amplitude in C and duration in
D) in both experimental groups (,
P 0.01; ⴱⴱ, P 0.001; ANOVA).
140 Gomez-Pinilla et al.
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tractile responses were enhanced in similar proportions to
those found in control strips (91 and 85% enhancement for
MEL 2.5 and MEL 30, respectively, at 5 Hz; Fig. 4D). These
results indicate that melatonin had some effects on inflam-
mation of the gallbladder, but there are some contractile
disabilities related to the experimental method used in this
study, as indicated under Materials and Methods, that mel-
atonin treatment cannot restore.
Thus, we tested melatonin effects in animals that under-
went the deligation protocol. When deligation was per-
formed in animals that were not treated with melatonin,
the neuromuscular function worsened, as indicated by the
reduction in the EFS-induced contraction (Fig. 5, A and B).
Taking into account the small amplitude of these contrac-
tions, we did not apply antagonists/inhibitors of the neu-
rotransmitters to determine the nature of this response.
However, when the animals were treated with melatonin
10 days before performing the surgical procedures and
until the animal was sacrificed, there was a very notice-
able improvement in gallbladder neuromuscular function.
As shown in Fig. 5C, melatonin treatment increased the
gallbladder neurogenic responses in a dose-dependent
way. In the strips from animals treated with melatonin,
the EFS-elicited responses recovered the sensitiveness to
TTX (70 and 73% inhibition for MEL 2.5 and MEL 30,
respectively, at 25 Hz; Fig. 6A) and atropine (73 and 76%
inhibition for MEL 2.5 and MEL 30, respectively, at 25 Hz;
Fig. 6B) to a level comparable with that seen in control
tissue. Although capsaicin still induced a small inhibition
of EFS-induced responses (5 and 10% inhibition for MEL
2.5 and MEL 30, respectively, at 25 Hz %; Fig. 6D), the
reduction was significantly smaller than that found in
inflamed tissue; at the highest frequencies, this effect was
not different from that registered in control tissue. In this
experimental group, 30 mg/kg melatonin also re-estab-
lished the sensitivity to
L-NAME (Fig. 6C), but this was not
the case for 2.5 mg/kg melatonin, suggesting that the ef-
fects of CBDL in the nitrergic function are exacerbated by
the deligation procedure.
These results suggest that melatonin has prophylactic ef-
fects preventing neuromuscular damage during cholecystitis.
To check whether melatonin also has a therapeutic role in
the management of acute cholecystitis, melatonin treatment
(30 mg/kg) was initiated after the onset of gallbladder inflam-
mation. As represented in Figs. 5C and 6, melatonin recov-
ered gallbladder contractility in response to EFS and the
pharmacological profile of the neurotransmission, indicating
that indoleamine can ameliorate the neuromuscular damage
induced by acute cholecystitis.
These effects of melatonin could be related to its antioxi-
dant and scavenger properties as indicated by the reduction
in the lipidic peroxidation and the increase in the levels of
GSH induced by melatonin treatment (Table 1).
Fig. 3. AC impairs the efferent innervation and increases the excitability of sensory contractile fibers. Effects of 1
M atropine, 100
M L-NAME, and
10
M capsaicin on EFS-elicited contractile response in control (A–C) and AC gallbladder strips (D–F). After EFS was performed under control
conditions (solid lines), strips were incubated for 20 min with the antagonist/inhibitor, and EFS was repeated (dotted line). Note the lack of effectsof
L-NAME on inflamed tissue and the reduction in the response after incubation with capsaicin. Data are mean S.E.M. n 8 to 21 strips (, P 0.05;
ⴱⴱ, P 0.01; ANOVA).
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Effects of Tempol on Neuromuscular Function in
Acute Cholecystitis. To test whether other antioxidants/
scavengers also have beneficial effects on the impaired neu-
romuscular transmission in acute cholecystitis, we also
tested the effects of tempol, a membrane-permeable superox-
ide dismutase mimetic (Krishna et al., 1996). Administration
Fig. 5. Melatonin treatment improves
the neurogenic damage exacerbated
by the deligation procedure. A, origi-
nal recordings showing guinea pig
gallbladder contractile responses elic-
ited by EFS (0.3-ms duration, 5–40
Hz, 350 mA, for 10 s every 3 min)
applied to AC and DL strips. Traces
are typical of 16 and 17 strips for AC
and DL strips, respectively. B, sum-
mary data of EFS induced-responses
(peak amplitude) in both experimen-
tal groups (ⴱⴱⴱ, P 0.001; ANOVA).
C, effects of melatonin treatment (2.5
and 30 mg/kg, DL MEL 2.5 and DL
MEL 30, respectively; 30 mg/kg for
4 days after the onset of AC, DL
MEL 4 days) on EFS-elicited contrac-
tile response gallbladder strips from
animals that underwent the deliga-
tion procedure. Data are mean
S.E.M. (n 12–28 strips; , P 0.05;
ⴱⴱ, P 0.01; ⴱⴱⴱ, P 0.001 versus
AC; ANOVA).
Fig. 4. Melatonin treatment protects intrinsic neurons, but it does not improve EFS-induced contraction. A, effects of melatonin treatment (2.5 and
30 mg/kg) on EFS-induced gallbladder contractions in acute cholecystitic animals. Histograms represent the effects of 1
M TTX (B), 1
M atropine
(C), 100
M L-NAME (D), and 10
M capsaicin (E) on EFS-elicited contractile response in control, AC, and AC melatonin-treated gallbladder strips.
Data are mean S.E.M. n 5 to 18 strips. Note that EFS-induced responses recover TTX and
L-NAME sensitivity, whereas melatonin has less effect
on cholinergic and sensory fibers (, P 0.01 AC versus control; †, P 0.05 MEL30 versus AC; and
, P 0.01 MEL 30 versus AC; ANOVA).
142 Gomez-Pinilla et al.
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of tempol in the drinking water for 14 days to guinea pigs
that underwent the protocol of deligation prevented the func-
tional impairment of EFS-induced contraction, although to a
lesser extent than 30 mg/kg melatonin (63 and 26% recovery
for MEL 30 and tempol, respectively; Fig. 7, A and B; P
0.01). The recovery was accompanied by the normalization of
the neurotransmission, because TTX, atropine,
L-NAME, and
capsaicin had similar effects in tempol-treated animals as in
control animals (Fig. 7, C–F). These prophylactic effects also
correlated with a decrease in the MDA levels and an increase
in the GSH content that were altered by the AC and DL
protocols (Table 1).
Discussion
The current report shows that the impairment in guinea
pig gallbladder neurotransmission evoked by inflamma-
tion was associated with a decrease in the contribution of
the efferent plexus and the up-regulation of sensory affer-
ent fibers. In addition, melatonin treatment caused an
improvement in the neurogenic contractile response and
the normalization of the different neural components that
were probably related to its antioxidant and scavenger
properties
Our results indicate that EFS evokes a gallbladder re-
Fig. 6. Melatonin treatment normalizes the different neural components stimulated by EFS. Effects of 100
M TTX (A), 1
M atropine (B), 100
M
L-NAME (C), and 10
M capsaicin (D) on EFS-elicited contractile responses in control, AC, and melatonin-treated gallbladder strips. Melatonin was
administered to animals that underwent the deligation protocol (2.5 and 30 mg/kg, DL MEL 2.5 and DL MEL 30, respectively; 30 mg/kg for 4 days
after the onset of AC, DL MEL 4 days). After EFS was performed under control conditions, strips were incubated for 20 min with the
antagonist/inhibitor, and EFS was repeated. Data are mean S.E.M. (n 7–9 strips; , P 0.01 versus control;
, P 0.01 versus AC; ANOVA).
TABLE 1
Effect of acute cholecystitis, melatonin, and tempol treatment on oxidative stress markers
Data are expressed as mean S.E.M. of -fold increase respect to levels found in control tissue. n 5 to 6 animals.
AC DL DL MEL 2.5 DL MEL 30 DL MEL 4 Days DL Tempol (1 mM)
MDA level 4.82 0.43 4.32 0.41 1.90 0.37**
,
␦␦
1.44 0.26**
,
␦␦
1.20 0.26**
,
␦␦
1.20 0.21**
,
␦␦
GSH level 0.31 0.05 0.35 0.05 0.76 0.07*
,
0.91 0.06*
,
0.89 0.06*
,
0.96 0.04*
,
* P 0.05; ** P 0.01 vs. AC.
P 0.05;
␦␦
P 0.01 vs. DL.
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Fig. 7. Tempol treatment improves the neuromuscular damage exacerbated by the deligation procedure, and it normalizes the contribution of the
different neural components in the inflamed gallbladder. A, original recordings showing guinea pig gallbladder contractile responses elicited by EFS
(0.3-ms duration, 5– 40 Hz, 350 mA, for 10 s every 3 min) applied to DL strips from animals treated with 1 mM tempol (DL tempol 1 mM). Traces
are typical of 17 and 20 strips for DL and DL tempol 1 mM, respectively. B, summary data of EFS induced-responses (peak amplitude) in those
experimental groups (ⴱⴱⴱ, P 0.001 and ⴱⴱ, P 0.01 versus DL; ANOVA). C to F, histograms showing the effects of 100
M TTX, 1
M atropine, 100
M L-NAME, and 10
M capsaicin on EFS-elicited contractile responses in control, AC, and DL tempol groups. Data are mean S.E.M. (n 10 –21
strips; , P 0.01 versus control;
, P 0.01 versus AC; ANOVA).
144 Gomez-Pinilla et al.
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sponse by the release of different inhibitory and excitatory
neurotransmitters. The smaller contractile responses to EFS
in cholecystitic strips suggest the existence of an inflamma-
tion-induced impairment in the gallbladder intrinsic nerves,
in agreement with previous results in human and animal
models (McKirdy et al., 1994a; Parkman et al., 2000). How-
ever, the reduced gallbladder smooth muscle contractility to
ACh found in cholecystitis (Parkman et al., 1999a, 2000;
Gomez-Pinilla et al., 2006b) could also contribute to the im-
paired neuromuscular function in inflamed tissue. The most
striking finding in our study was the lack of sensitivity
shown by cholecystitic strips to TTX, which could be ex-
plained by a direct release of neurotransmitter from nervous
terminal. This was confirmed by the sensitivity of the EFS-
induced responses to
-conotoxin GVIA, a blocker of N-type
calcium channel located in the presynaptic membrane whose
activation is necessary for neurotransmitter release. These
results suggest that inflammation evokes a functional dener-
vation in the gallbladder that avoids the genesis or propaga-
tion of action potential through efferent fibers. Alterations in
the properties and/or expression levels of voltage-dependent
Na
channels have been implicated in a variety of patholog
-
ical states, including inflammation of the viscera (Yoshimura
et al., 2001; Stewart et al., 2003; Beyak et al., 2004). In this
regard, the more common effect of inflammation on Na
channels is the up-regulation of TTX-resistant slow (Nav1.8)
type (Yoshimura et al., 2001; Beyak et al., 2004). Alterations
in the pharmacological profile of Na
channels could also
explain the TTX-resistant contractions reported in this
study.
Classically, ACh released in response to EFS is the main
excitatory component of the gallbladder contraction (Yau and
Youther, 1984; Parkman et al., 1997). Here, we show that in
control conditions, atropine abolished EFS-induced contrac-
tion, whereas in inflamed tissue, it just reduced EFS-elicited
contraction approximately 50%, indicative of a functional
denervation of the cholinergic component, similar to results
described previously in inflamed gallbladder (Parkman et al.,
2000).
Nonadrenergic noncholinergic neurotransmission in
guinea pig gallbladder was described more than a decade ago
(Mourelle et al., 1993), and NO is the main nonadrenergic
noncholinergic neurotransmitter involved (McKirdy et al.,
1994b; Alco´n et al., 2001). Inflammation evokes a functional
impairment in gallbladder nitrergic innervation as demon-
strated by the lack of effects of
L-NAME in cholecystitic strips
compared with control tissue. This result does not support
the study from Parkman et al. (2000), where
L-NAME only
had an effect in inflamed tissue, indicating that normal gall-
bladder does not release NO from the intrinsic plexus. This is
in conflict with the presence of nitrergic nerves described in
guinea pig gallbladder (Mawe et al., 2006) and with the
functional data reported above.
Neurotransmitters released from sensory nerves evoked
contraction or relaxation of the gallbladder (Maggi et al.,
1989). In our study, sensory denervation with capsaicin had
no effect in control conditions, whereas it reduced EFS-elic-
ited contractile response in inflammation, suggesting excita-
tory neurotransmitter release from sensory nerves in in-
flamed gallbladder. The major participation of the sensory
innervation is a common finding in neurogenic inflammation
(Sann et al., 1996). In the gallbladder, we have shown that
aging, which is also related to increased oxidative stress, is
associated with over-reactivity of sensory fibers (Gomez-Pi-
nilla et al., 2006a).
The most important finding of our study is that melatonin
has prophylactic and therapeutic effects on inflammation-
induced impairment in gallbladder neuromuscular function.
Thus, with 14-day melatonin treatment, the EFS-induced
contractile response recovered the sensitiveness to TTX, in-
dicating that melatonin protects the voltage-dependent Na
channels involved in the neural transmission of the action
potential. Furthermore, the nitrergic innervation recovered
its functionality and sensory fibers became less sensitive to
EFS. However, melatonin itself did not improve the contrac-
tile response to EFS unless the obstruction of the bile duct
was relieved. Under these conditions, melatonin reversed the
impairment in contractility in a dose-dependent manner, and
it fully recovered the different neural components stimulated
by EFS. It must be pointed out that 2.5 mg/kg melatonin had
no effects on the nitrergic innervation after deligation, al-
though this treatment was efficacious in increasing the par-
ticipation of these inhibitory nerves with the bile duct-ligated
animals. Deligation itself worsened gallbladder contractility,
as consequence probably of an increase in oxidative stress
insult due to reperfusion of the organ once the mechanical
stretch was alleviated. This is supported by the increase in
the MDA levels indicative of lipidic peroxidation and oxida-
tive stress injury found after deligation. On this basis, it
seems that nitrergic innervation is especially sensitive to the
enhanced oxidative stress after deligation. In agreement
with this, we have recently reported a minor participation of
nitrergic nerves in neuromuscular transmission in aging and
its recovery after melatonin treatment (Gomez-Pinilla et al.,
2006a). Furthermore, melatonin has been shown to have
neurally mediated actions in the gut, regulating either cho-
linergic, nitrergic, and/or sensory innervation (Barajas-Lo´pez
et al., 1996; Reiter et al., 2003).
According to our results, melatonin not only protects
against inflammation but also resolves the inflammation-
induced impairment of neuromuscular function. Thus, when
melatonin treatment started after the onset of gallbladder
inflammation, there was an enhancement of the contractile
response to EFS that also recovered the neurotransmission
pattern. However, the prophylactic administration of mela-
tonin was more effective than the therapeutic administra-
tion, which could be related to the increase in the antioxidant
defenses induced by the administration of melatonin before
the oxidative insult.
It is well accepted that melatonin administration at phar-
macological doses decreases free radical formation and leads
to a substantial recovery of the major antioxidant enzymes
(Reiter, 1998). Recent evidence has shown that melatonin
has protective effects on liver and hepatic injury after extra-
hepatic bile duct ligation in rats (Shiesh et al., 2000; Esrefo-
glu et al., 2005; Ohta et al., 2005). In addition to liver and
hepatic damage, free radical accumulation associated with
bile duct ligation has been implicated in the genesis of gall-
stone (Eder et al., 1996). In this regard, antioxidant treat-
ment with melatonin not only reversed the increased oxida-
tive stress but also prevented gallstone formation (Shiesh et
al., 2000). In our preparation, either prophylactic or thera-
peutic melatonin treatments were effective in reducing MDA
levels and in increasing the endogenous antioxidant defense
Melatonin in Acute Cholecystitis 145
by guest on December 27, 2011jpet.aspetjournals.orgDownloaded from
GSH, indicating that melatonin antioxidant effects can be
responsible for the improvement in the neuromuscular func-
tion. In fact, the treatment of the animals with the mem-
brane-permeant superoxide dismutase mimetic tempol also
induced a significant improvement in the neuromuscular
function of inflamed gallbladder, which is in agreement with
other reports showing that tempol reduces the dysfunctions
associated to oxidative stress insult (Chatterjee et al., 2000;
Mehta et al., 2004). Collectively, our data suggest a prophy-
lactic and therapeutic role of melatonin in experimental
acute cholecystitis, a remarkable finding due to the lack of an
effective pharmacological treatment for acute cholecystitis.
In conclusion, the results obtained in the present study
indicate that inflammation impairs gallbladder neuromuscu-
lar function as the result of changes in the neural inputs to
smooth muscle. These changes can be summarized as a de-
nervation of efferent nerves together with a hyperactivity of
afferent fibers. Melatonin significantly ameliorated the in-
flammation-related changes in gallbladder neuromuscular
transmission, indicating its potential to combat inflamma-
tion-induced gallbladder damage.
Acknowledgments
We thank Rosario Moreno for technical assistance.
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