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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
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
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
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.
Copyright © 2007 by The American Society for Pharmacology and Experimental Therapeutics 123240/3252691
JPET 323:138–146, 2007 Printed in U.S.A.
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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
. 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
, 1.2 mM KH
, 1.2 mM
, 25 mM NaHCO
, and 11.5 mM D-glucose. This solution had
a final pH of 7.35 after equilibration with 95% O
. The
phosphate buffer used to homogenize the tissue contained 20 mM
NaCl, 2.7 mM KCl, 16 mM Na
, 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
-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,
-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.
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
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
M tetrodotoxin plus 0.1
-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,
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
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
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).
Melatonin in Acute Cholecystitis 141
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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
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).
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
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
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).
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.
Melatonin in Acute Cholecystitis 143
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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
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
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.,
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
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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.
We thank Rosario Moreno for technical assistance.
Alco´n S, Morales S, Camello PJ, Salido GM, Miller SM, and Pozo MJ (2001) Relax-
ation of canine gallbladder to nerve stimulation involves adrenergic and non-
adrenergic non-cholinergic mechanisms. Neurogastroenterol Motil 13:555–566.
Barajas-Lo´pez C, Peres AL, Espinosa-Luna R, Reyes-Vazquez C, and Prieto-Gomez
B (1996) Melatonin modulates cholinergic transmission by blocking nicotinic chan-
nels in the guinea-pig submucous plexus. Eur J Pharmacol 312:319–325.
Beyak MJ, Ramji N, Krol KM, Kawaja MD, and Vanner SJ (2004) Two TTX-resistant
Na currents in mouse colonic dorsal root ganglia neurons and their role in
colitis-induced hyperexcitability. Am J Physiol Gastrointest Liver Physiol287:
Chatterjee PK, Cuzzocrea S, Brown PA, Zacharowski K, Stewart KN, Mota-Filipe H,
and Thiemermann C (2000) Tempol, a membrane-permeable radical scavenger,
reduces oxidant stress-mediated renal dysfunction and injury in the rat. Kidney
Int 58:658673.
Eder MI, Miquel JF, Jongst D, Paumgartner G, and Von Ritter C (1996) Reactive
oxygen metabolites promote cholesterol crystal formation in model bile: role of
lipid peroxidation. Free Radic Biol Med 20:743–749.
Esrefoglu M, Gul M, Emre MH, Polat A, and Selimoglu MA (2005) Protective effect
of low dose of melatonin against cholestatic oxidative stress after common bile duct
ligation in rats. World J Gastroenterol 11:1951–1956.
Gomez-Pinilla PJ, Camello-Almaraz C, Moreno R, Camello PJ, and Pozo MJ (2006a)
Melatonin treatment reverts age-related changes in guinea pig gallbladder neu-
romuscular transmission and contractility. J Pharmacol Exp Ther 319:847– 856.
Gomez-Pinilla PJ, Morales S, Camello-Almaraz C, Moreno R, Pozo MJ, and Camello
PJ (2006b) Changes in guinea pig gallbladder smooth muscle Ca
homeostasis by
acute acalculous cholecystitis. Am J Physiol Gastrointest Liver Physiol 290:G14
Head KA and Jurenka JS (2003) Inflammatory bowel disease Part 1: ulcerative
colitis–pathophysiology and conventional and alternative treatment options. Al-
tern Med Rev 8:247–283.
Hissin PJ and Hilf R (1976) A fluorometric method for determination of oxidized and
reduced glutathione in tissues. Anal Biochem 74:214 –226.
Karasek M (2004) Melatonin, human aging, and age-related diseases. Exp Gerontol
Konturek PC, Konturek SJ, Brzozowski T, Dembinski A, Zembala M, Mytar B, and
Hahn EG (1997) Gastroprotective activity of melatonin and its precursor, L-
tryptophan, against stress-induced and ischaemia-induced lesions is mediated by
scavenge of oxygen radicals. Scand J Gastroenterol 32:433– 438.
Krishna MC, Russo A, Mitchell JB, Goldstein S, Dafni H, and Samuni A (1996) Do
nitroxide antioxidants act as scavengers of O
or as SOD mimics? J Biol Chem
Kvetnoy IM, Ingel IE, Kvetnaia TV, Malinovskaya NK, Rapoport SI, Raikhlin NT,
Trofimov AV, and Yuzhakov VV (2002) Gastrointestinal melatonin: cellular iden-
tification and biological role. Neuro Endocrinol Lett 23:121–132.
Lane EA and Moss HB (1985) Pharmacokinetics of melatonin in man: first pass
hepatic metabolism. J Clin Endocrinol Metab 61:1214 –1216.
Maggi CA, Santicioli P, Renzi D, Patacchini R, Surrenti C, and Meli A (1989) Release
of substance P- and calcitonin gene-related peptide-like immunoreactivity and
motor response of the isolated guinea pig gallbladder to capsaicin. Gastroenterol-
ogy 96:1093–1101.
Mawe GM and Gershon MD (1989) Structure, afferent innervation, and transmitter
content of ganglia of the guinea pig gallbladder: relationship to the enteric nervous
system. J Comp Neurol 283:374 –390.
Mawe GM, Saccone GT, and Pozo MJ (2006) Neural control of the gallbladder and
sphincter of Oddi, in Physiology of the Gastrointestinal Tract (Johnson LR, Barret
KE, Ghishan FK, Merchant JL, Said HM, and Wood JD eds) pp 841–849, Elsevier
Academic Press, San Diego, CA.
McKirdy ML, Johnson CD, and McKirdy HC (1994a) Inflammation impairs neurally
mediated responses to electrical field stimulation in isolated strips of human
gallbladder muscle. Dig Dis Sci 39:2229 –2234.
McKirdy ML, McKirdy HC, and Johnson CD (1994b) Non-adrenergic non-cholinergic
inhibitory innervation shown by electrical field stimulation of isolated strips of
human gall bladder muscle. Gut 35:412– 416.
Mehta SH, Webb RC, Ergul A, Tawfik A, Dorrance AM, and Tawak A (2004)
Neuroprotection by tempol in a model of iron-induced oxidative stress in acute
ischemic stroke. Am J Physiol Regul Integr Comp Physiol 286:R283–R288.
Mourelle M, Guarner F, Molero X, Moncada S, and Malagelada JR (1993) Regulation
of gall bladder motility by the arginine-nitric oxide pathway in guinea pigs. Gut
Ohta Y, Imai Y, Matsura T, Yamada K, and Tokunaga K (2005) Successively
postadministered melatonin prevents disruption of hepatic antioxidant status in
rats with bile duct ligation. J Pineal Res 39:367–374.
Parkman HP, Bogar LJ, Bartula LL, Pagano AP, Thomas RM, and Myers SI (1999a)
Effect of experimental acalculous cholecystitis on gallbladder smooth muscle con-
tractility. Dig Dis Sci 44:2235–2243.
Parkman HP, James AN, Bogar LJ, Bartula LL, Thomas RM, Ryan JP, and Myers
SI (2000) Effect of acalculous cholecystitis on gallbladder neuromuscular trans-
mission and contractility. J Surg Res 88:186 –192.
Parkman HP, Pagano AP, Martin JS, and Ryan JP (1997) Electric field stimulation-
induced guinea pig gallbladder contractions: role of calcium channels in acetylcho-
line release. Dig Dis Sci 42:1919 –1925.
Parkman HP, Pagano AP, and Ryan JP (1999b) Subtypes of muscarinic receptors
regulating gallbladder cholinergic contractions. Am J Physiol 276:G1243–G1250.
Pozo MJ, Camello PJ, and Mawe GM (2004) Chemical mediators of gallbladder
dysmotility. Curr Med Chem 11:1801–1812.
Reiter RJ (1998) Cytoprotective properties of melatonin: presumed association with
oxidative damage and aging. Nutrition 14:691– 696.
Reiter RJ, Tan DX, Mayo JC, Sainz RM, Leon J, and Bandyopadhyay D (2003)
Neurally-mediated and neurally-independent beneficial actions of melatonin in
the gastrointestinal tract. J Physiol Pharmacol 54 (Suppl 4):113–125.
Sann H, Dux M, Schemann M, and Jancso G (1996) Neurogenic inflammation in the
gastrointestinal tract of the rat. Neurosci Lett 219:147–150.
Shiesh SC, Chen CY, Lin XZ, Liu ZA, and Tsao HC (2000) Melatonin prevents
pigment gallstone formation induced by bile duct ligation in guinea pigs. Hepatol-
ogy 32:455–460.
Stewart T, Beyak MJ, and Vanner S (2003) Ileitis modulates potassium and sodium
currents in guinea pig dorsal root ganglia sensory neurons. J Physiol 552:797–807.
Talmage EK, Pouliot WA, Cornbrooks EB, and Mawe GM (1992) Transmitter diver-
sity in ganglion cells of the guinea pig gallbladder: an immunohistochemical study.
J Comp Neurol 317:45–56.
Tan D, Manchester LC, Reiter RJ, Qi W, Hanes MA, and Farley NJ (1999) High
physiological levels of melatonin in the bile of mammals. Life Sci 65:2523–2529.
Tan DX, Reiter RJ, Manchester LC, Yan MT, El Sawi M, Sainz RM, Mayo JC, Kohen
R, Allegra M, and Hardeland R (2002) Chemical and physical properties and
potential mechanisms: melatonin as a broad spectrum antioxidant and free radical
scavenger. Curr Top Med Chem 2:181–197.
Waller RL and Recknagel RO (1977) Determination of lipid conjugated dienes with
tetracyanoethylene-14C: significance for study of the pathology of lipid peroxida-
tion. Lipids 12:914 –921.
Xiao ZL, Chen Q, Biancani P, and Behar J (2001) Abnormalities of gallbladder
muscle associated with acute inflammation in guinea pigs. Am J Physiol Gastroi-
ntest Liver Physiol 281:G490 –G497.
Yau WM and Youther ML (1984) Modulation of gallbladder motility by intrinsic
cholinergic neurons. Am J Physiol Gastrointest Liver Physiol 247:G662–G666.
Yoshimura N, Seki S, Novakovic SD, Tzoumaka E, Erickson VL, Erickson KA,
Chancellor MB, and de Groat WC (2001) The involvement of the tetrodotoxin-
resistant sodium channel Na(v)1.8 (PN3/SNS) in a rat model of visceral pain.
J Neurosci 21:8690 8696.
Address correspondence to: Dr. Marı´a J. Pozo, Department of Physiology,
Nursing School, Avda Universidad s/n, 10071 Ca´ceres, Spain. E-mail:
146 Gomez-Pinilla et al.
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... It has free radical scavenging and antioxidant properties and upon the scavenging generates other compounds that also retain strong reducing potential, which explains the high efficiency of melatonin as an antioxidant compared with other naturally occurring antioxidants [18][19][20]. We have previously shown that melatonin restores smooth muscle function altered by increased oxidative stress-related conditions such as inflammation and aging, including the gastrointestinal smooth muscle [21][22][23][24][25][26][27]. ...
... Beneficial effects of melatonin occur both in proximal and distal colon, although its effects are more prominent in proximal colon, in keeping with our funding of higher oxidative stress and inflammation and apoptosis markers in this colon segment. We have previously reported that melatonin supplementation improves smooth muscle function deteriorated by either aging [21,24,25,27] or inflammation [23,26] through normalization of intracellular calcium homeostasis, mitochondrial membrane potential, Ca 2+ sensitization mechanisms, or neuromuscular function. These effects were also accompanied by restoration of oxidative stress and melatonin secretion by enterochromaffin cells in the case of aged ileum and colon [57]. ...
There is increasing evidence that aging is associated with oxidative damage, inflammation, and apoptosis in different cell types. However, there is limited information regarding aging mechanisms in colon smooth muscle. Old male Wistar rats (22 months) were treated for 10 wks with melatonin or growth hormone (GH). Animals were sacrificed at 24 months of age by decapitation. The colon was dissected and the smooth muscle homogenized. H(2)O(2) and malonyl dialdehyde (MDA) content and catalase and glutathione peroxidase (GPX) activities were determined using colorimetric kits. Expression of nuclear factor kappa B (NF-κB), cyclooxygenase 2 (COX-2), caspase-3, and caspase-9 were determined by Western blot. Aging of colon smooth muscle correlated with an increase in H(2)O(2) and MDA levels when compared with young animals in both proximal and distal segments; these changes were associated with a decrease in the catalase activity in the distal colon. Oxidative stress correlated with an increase in COX-2 and NF-κB expression, which were accompanied by an enhanced expression of the pro-apoptotic enzyme caspase-3 and its upstream enzyme, caspase-9. Melatonin treatment normalized the oxidative, inflammatory, and apoptotic patterns, whereas GH replacement, although effective in reducing oxidative stress in distal colon, did not reverse the age-related inflammation or apoptosis. These results suggest that melatonin should be the treatment of choice to most effectively recover physiological functions in aged colonic smooth muscle.
... Poliovirus, adenoviruses and rabies virus bind to neurons at the neuromuscular junction owing to the neuronal expression of specific receptors. In this context, oxidative stress seems to be implicated in neuromuscular junction impairment, with mitochondrial dysfunction and inflammation being prominent features; related to this, melatonin has been reported to reverse age-related neuromuscular transmission dysfunction and improve muscle physiology [122,123]. ...
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... Thus, melatonin, due to its potent antioxidant activities could be a key player in resolving or preventing this deregulation. In fact, published reports using different animals show that melatonin reverses age-related neuromuscular transmission dysfunction [141] and improves, at the same time, muscle physiology [142]. ...
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... The animals in the study groups were subjected to BDL. The animals in the BDL 24 h group and the BDL 48 h group were subjected to laparotomy and cholecystectomy at 24 and 48 h after surgery, respectively [12][13][14][15]. ...
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Aging is a multifactorial process that involves biochemical, structural and functional changes in mitochondria. The ability of melatonin to palliate the alterations induced by aging is based on its chronobiologic, antioxidant and mitochondrial effects. There is little information about the effects of melatonin on the in situ mitochondrial network of aging cells and its physiological implications. We have studied the ability of melatonin to prevent the functional alterations of in situ mitochondria of smooth muscle cells and its impact on contractility. Mitochondrial membrane potential was recorded in isolated colonic smooth muscle cells from young mice (3 month old), aged mice (22-24 month old) and aged mice treated with melatonin (starting at 14 month age). Aging induced a partial mitochondrial depolarization in resting conditions and reduced the depolarizing response to cellular stimulation. Use of oligomycin indicated that aging enhanced the resting activity of the mitochondrial ATP synthase, whereas in young cells the enzyme operated mainly in reverse mode. Melatonin treatment prevented all these changes. Aging reduced both spontaneous and stimulated contraction of colonic strips and shifted the metabolic dependence of contraction from mitochondria to glycolysis, as indicated the use of mitochondrial and glycolysis inhibitors. These functional alterations were also palliated by melatonin treatment. Aging effects were not related to a decrease in Ca(2+) store mobilization, because this was enhanced in aged cells and restored by melatonin. In conclusion, melatonin prevents the age-induced in situ mitochondrial potential alterations in smooth muscle cells and the associated changes in contractility and metabolism. This article is protected by copyright. All rights reserved.
... In animal models, exogenous melatonin has been shown to reduce the effects of GI inflammation due to acetic acid [56] and dextran sulphate sodium (DSS) [57], common bile duct ligation [58,59] and dinitrobenzene sulfonic acid (DNBS) [60], and reduce gastric damage following stress [61]. Endogenous melatonin can also promote healing in a rat model of gastric ulcer induced by acetic acid [62]. ...
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Neural plasticity is not only the adaptive response of the central nervous system to learning, structural damage or sensory deprivation, but also an increasingly recognized common feature of the gastrointestinal (GI) nervous system during pathological states. Indeed, nearly all chronic GI disorders exhibit a disease-stage-dependent, structural and functional neuroplasticity. At structural level, GI neuroplasticity usually comprises local tissue hyperinnervation (neural sprouting, neural, and ganglionic hypertrophy) next to hypoinnervated areas, a switch in the neurochemical (neurotransmitter/neuropeptide) code toward preferential expression of neuropeptides which are frequently present in nociceptive neurons (e.g., substance P/SP, calcitonin-gene-related-peptide/CGRP) and of ion channels (TRPV1, TRPA1, PAR2), and concomitant activation of peripheral neural glia. The functional counterpart of these structural alterations is altered neuronal electric activity, leading to organ dysfunction (e.g., impaired motility and secretion), together with reduced sensory thresholds, resulting in hypersensitivity and pain. The present review underlines that neural plasticity in all GI organs, starting from esophagus, stomach, small and large intestine to liver, gallbladder, and pancreas, actually exhibits common phenotypes and mechanisms. Careful appraisal of these GI neuroplastic alterations reveals that-no matter which etiology, i.e., inflammatory, infectious, neoplastic/malignant, or degenerative-neural plasticity in the GI tract primarily occurs in the presence of chronic tissue- and neuro-inflammation. It seems that studying the abundant trophic and activating signals which are generated during this neuro-immune-crosstalk represents the key to understand the remarkable neuroplasticity of the GI tract.
Digestive inflammatory processes induce motility alterations associated with an increase in reactive oxygen species production, including monochloramine (NH2Cl). The aim of the study was to characterize the effects of the naturally occurring oxidant monochloramine in the guinea pig gallbladder. We used standard in vitro contractility technique to record guinea pig gallbladder strips contractions. NH2Cl caused a concentration‐dependent contraction which was reduced by inhibition of extracellular Ca²⁺ influx and tyrosine kinase pathways. The PKC antagonist GF109203X also reduced the response but not after previous tyrosine kinase inhibition, suggesting that PKC is activated by tyrosine kinase activity. The NH2Cl contractile effect was also reduced by inhibitors of mitogen‐activated protein kinase (MAPK), nitric oxide synthase, phospholipase A2 and cyclooxygenase. In addition, NH2Cl impaired the responses to CCK, tissue depolarization and electrical field stimulation. In conclusion, we present new evidence that monochloramine impairs not only the gallbladder response to CCK but also to membrane depolarization and nervous plexus stimulation, and that tyrosine kinase, PKC, MAPK and NO pathways are involved in the contractile direct effect of monochloramine.
This chapter provides an overview of the current knowledge of biliary tract motor activity and its regulation. As motility primarily involves the activities of two cell types, neurons and smooth muscle, the chapter also provides a summary of the basic physiology of the nerves and smooth muscle in the gallbladder and sphincter of Oddi (SO) with clinical correlations provided whenever possible. Determination of the cellular mechanisms that are responsible for gallbladder motility in health and disease is difficult in humans; therefore, much of what is known about the structure and function of gallbladder neurons and smooth muscle is derived from animal studies. The chapter describes how the nerves and smooth muscle of these organs function during the bile retention and bile flow phases of the feeding cycle. It examines what is currently known about the roles of nerves and smooth muscle in the biliary tract under pathophysiological conditions.
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A method for measurement of both oxidized (GSSG) and reduced (GSH) glutathione has been developed, with use of o-phthalaldehyde (OPT) as a fluorescent reagent. The method takes advantage of the reaction of GSH with OPT at pH 8 and of GSSG with OPT at pH 12; GSH can be complexed to N-ethylmaleimide to prevent interference of GSH with measurement of GSSG. The method gave “recoveries” of 91 to 110% for both GSH and GSSG and was quite specific for glutathione; and none of the manipulations appeared to influence the amount of glutathione present in the tissue. Results for GSH levels agreed well with earlier reports but levels of GSSG estimated here were higher than earlier reported values. The reasons for the apparently higher levels of GSSG are discussed.
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Hepatic clearance concepts were applied to existing data on iv and oral administration of melatonin to man. A high hepatic extraction ratio was calculated, suggesting prominent first pass hepatic metabolism and reduced bioavailability for orally administered melatonin. Using clearance parameters and previous data, endogenous production rates for melatonin were determined for normal individuals and patients with cirrhosis. Normal melatonin production was 28.8 micrograms/day, while the production rate for cirrhotic patients was 12.3 micrograms/day. Thus, not only do cirrhotic patients have decreased melatonin elimination, as noted in the original report, but they also have decreased daily melatonin production.
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Nitric oxide (NO) synthesised from L-arginine is an intercellular messenger in various biological actions including endothelial dependent relaxation and inhibition of platelet aggregation. This study explored the role of the L-arginine-NO pathway in the regulation of gall bladder motility. Intraluminal gall bladder pressure was recorded in anaesthetised guinea pigs in response to cholecystokinin or bethanechol before and after treatment with specific NO synthase inhibitors (NG-nitro-L-arginine, NG-nitro-L-arginine methyl ester, or NG-monomethyl-L-arginine), or with an NO donor (sodium nitroprusside). Baseline gall bladder pressure significantly increased after treatment with the NO synthase inhibitors. Responses to cholecystokinin (0.025-1.25 nmol/kg) were significantly enhanced after treatment with NG-nitro-L-arginine methyl ester and lasted two to threefold longer than in control experiments. The effect of the inhibitor both on resting pressure and on cholecystokinin induced changes was reversed by L-arginine but not by D-arginine. Pretreatment with the inhibitors also induced a significant enhancement of the response to bethanechol. On the other hand, sodium nitroprusside abolished the response to low dose cholecystokinin and reduced the response to a high dose by about 80%. In vitro experiments with isolated gall bladder strips showed a significant enhancement of the contractile response to cholecystokinin or bethanechol after preincubation with the NO synthase inhibitor. Calcium dependent activity of NO synthase was detected in fresh homogenates from gall bladder tissue and incubation with endotoxin induced considerable calcium independent activity. These findings support the existence of a key L-arginine-nitric oxide pathway regulating gall bladder contraction.
A method for quantitative analysis of conjugated diene unsaturation has been developed utilizing tetracyanoethylene-14C (TCNE-14C) in a Diels-Alder condensation. The amount of C14 found in the Diels-Alder adduct has been shown to be a measure of conjugated diene content. The method has proven successful in analysis of a variety of triglycerides, phospholipids, and peroxidized tissue lipids. In the course of this work, a method for removing the fatty acid substituents from phospholipids using lithium aluminum hydride was developed. TCNE-14C analysis for conjugated dienes in rat liver microsomal lipids after dosing with CCl4 or BrCCl3 has provided conclusive evidence that the increase in ultraviolet absorption at 233 nm of these lipids is due to conjugated dienes.
Several neurotransmitters have been reported to exist in the ganglionated plexus of the guinea pig gallbladder. These include substance P, neuropeptide Y (NPY), calcitonin gene-related peptide, vasoactive intestinal peptide (VIP), acetylcholine, norepinephrine, serotonin, and dopamine. To determine which neuropeptides are intrinsic to gallbladder ganglia, we performed immunohistochemistry on colchicine-treated preparations. In separate, single-labeled preparations, a majority of neurons contained substance P-, NPY-, or somatostatin-like immunoreactivity. In double-labeled preparations, a large majority of the neurons that contained substance P-like immunoreactivity also contained NPY-like immunoreactivity and somatostatin-like immunoreactivity. Immunoreactivity for VIP was present in a small percentage of the gallbladder neurons which did not contain substance P-like immunoreactivity. Additional experiments were done to test for the presence of other compounds, known to exist in the neurons of the gut. Although immunoreactivity was found in control preparations of small intestine, the ganglionated plexus of the gallbladder lacked immunoreactivity for galanin, dynorphin, enkephalin, gastrin-releasing peptide, or gamma-aminobutyric acid. We conclude that ganglia of the guinea pig gallbladder contain at least two populations of neurons, based on transmitter phenotype. One of these populations appears to contain substance P, NPY, and somatostatin. Another population, which represents a small contingent of the total population of neurons, contains VIP.
The aim of this study was to assess whether a local motor response to capsaicin could be observed in the isolated guinea pig gallbladder and to discover the mechanism involved. Capsaicin produced a contraction of this organ that exhibited desensitization, suggesting a specific action on sensory nerves. In preparations preexposed to capsaicin to produce a functional blockade of the capsaicin-sensitive sensory fibers, the contractile response to field stimulation was unaffected as compared with controls. Tachykinins (substance P and neurokinin A) produced a concentration-related contraction of this organ, neurokinin A being more potent than substance P. Spantide, a tachykinin antagonist, markedly inhibited the capsaicin-induced gallbladder contraction, leaving the atropine-sensitive response to field stimulation unaffected. Calcitonin gene-related peptide (CGRP) produced a concentration-related relaxation, which was tetrodotoxin-resistant, suggesting a direct effect on muscle cells. Repeated administration of CGRP produced desensitization. At this stage, application of capsaicin produced a contractile response much larger than in controls. Both substance P- and CGRP-like immunoreactivity were detected in the guinea pig gallbladder by radioimmunoassay and were significantly reduced after systemic capsaicin desensitization. Capsaicin induced the simultaneous release of substance P- and CGRP-like immunoreactivity from superfused isolated gallbladders. These findings indicate that capsaicin-sensitive nerves in the guinea pig gallbladder can produce a local motor response involving the release of multiple neuropeptides. In the guinea pig gallbladder, tachykinins and CGRP might act as "physiologic antagonists," as observed in other viscera from rats and guinea pigs.
Although a well-developed plexus of nerves and ganglia is known to be present in the wall of the gallbladder, little has previously been learned about the function or organization of this innervation. The current study was undertaken in order to evaluate the hypothesis that the ganglionated plexus of the gallbladder is analogous to elements of the enteric nervous system (ENS). The ganglionated plexus of the gallbladder was found to resemble closely the submucosal plexus of the small intestine in its organization into two irregular anastomosing and interwoven networks of ganglia, in the numbers of neurons per ganglion, and in the manifestation of histochemically demonstrable acetylcholinesterase activity in virtually all ganglion cells. In common with enteric ganglia, laminin immunoreactivity was observed to be excluded from the interiors of gallbladder ganglia, which were surrounded by a periganglionic laminin-immunoreactive sheath. As in the submucosal plexus, intrinsic substance P-, vasoactive intestinal polypeptide (VIP)-, and neuropeptide Y (NPY)-immunoreactive neurons were seen in the ganglionated plexus of the gallbladder. Extrinsic nerves in the gallbladder that degenerated following chemical sympathectomy with 6-hydroxydopamine (6-OHDA), and which contained NPY, tyrosine hydroxylase (TH), and dopamine-beta-hydroxylase (DBH) immunoreactivities, formed a perivascular plexus closely associated with blood vessels. Endogenous catecholamines could also be demonstrated in these perivascular nerves by aldehyde-induced histofluorescence. In addition to perivascular nerves, paravascular nerve bundles were observed that were loosely associated with vessels, did not degenerate following administration of 6-OHDA, and contained NPY immunoreactivity. Other paravascular nerves, probably visceral sensory axons, coexpressed substance P and calcitonin-gene-related peptide (CGRP) immunoreactivities. The ganglionated plexus of the gallbladder resembled enteric ganglia in having intrinsic 5-hydroxytryptamine (5-HT)-immunoreactive cells and highly varicose nerve fibers. The 5-HT-immunoreactive gallbladder axons were, like those of the gut, resistant to 6-OHDA, and separate from fibers that expressed TH immunoreactivity. Differences between the ganglionated plexus of the gallbladder and enteric ganglia of the small intestine included in the gallbladder are 1) the presence of TH-immunoreactive cells that contain an endogenous catecholamine, but not DBH; 2) DBH-immunoreactive neurons, some of which coexpress substance P immunoreactivity, but which contain neither a catecholamine nor TH immunoreactivity; 3) an apparent absence of CGRP-immunoreactive cell bodies.(ABSTRACT TRUNCATED AT 400 WORDS)
The mechanical contraction and release of acetylcholine (ACh) were investigated by use of an isolated guinea pig gallbladder preparation subjected to transmural field stimulation. An increase in contraction amplitude was found to be related to the frequency (5-60 Hz) of applied stimulus. Treatment with tetrodotoxin and atropine prevented these electrically induced contractions. A simultaneous increase in the release of ACh was noted with electrical stimulation or depolarization by veratrine hydrochloride, and this increase was susceptible to blockade by tetrodotoxin. Neither contractions nor ACh release could be detected with stimulation at 0.1 Hz. When field stimulation was applied to contracted gallbladder induced by a supramaximal concentration of cholecystokinin octapeptide (CCK-8), a further increase in muscle tone was observed. The magnitude of this increase was frequency dependent. Although the onset of response to field transmural stimulation was relatively more rapid, the maximal response attained was only 70% of that elicited by CCK-8. When tissues were exposed to 2.2 X 10(-6) M CCK-8, the muscle contracted maximally but there was no increase in the efflux of [3H]ACh. These data provide a pharmacological characterization of the intrinsic gallbladder cholinergic neurons and demonstrate a possible role they may play in the control of gallbladder motility.