Article

Aging impairs Ca2+ sensitization pathways in gallbladder smooth muscle

Department of Physiology, Nursing School, University of Extremadura, Avda. Universidad s/n, Caceres, Spain.
Age (Impact Factor: 3.45). 07/2011; 34(4):881-93. DOI: 10.1007/s11357-011-9285-6
Source: PubMed

ABSTRACT

Calcium sensitization is an important physiological process in agonist-induced contraction of smooth muscle. In brief, calcium sensitization is a pathway that leads to smooth muscle contraction independently of changes in [Ca(2+)](i) by mean of inhibition of myosin light chain phosphatase. Aging has negative impacts on gallbladder contractile response due to partial impairment in calcium signaling and alterations in the contractile machinery. However, information regarding aging-induced alterations in calcium sensitization is scanty. We hypothesized that the calcium sensitization system is negatively affected by age. To investigate this, gallbladders were collected from adult (4 months old) and aged (22-24 months old) guinea pigs. To evaluate the contribution of calcium sensitization pathways we assayed the effect of the specific inhibitors Y-27632 and GF109203X on the "in vitro" isometric gallbladder contractions induced by agonist challenges. In addition, expression and phosphorylation (as activation index) of proteins participating in the calcium sensitization pathways were quantified by Western blotting. Aging reduced bethanechol- and cholecystokinin-evoked contractions, an effect associated with a reduction in MLC20 phosphorylation and in the effects of both Y-27632 and GF109203X. In addition, there was a drop in ROCK I, ROCK II, MYPT-1 and PKC expression and in the activation/phosphorylation of MYPT-1, PKC and CPI-17 in response to agonists. Interestingly, melatonin treatment for 4 weeks restored gallbladder contractile responses due to re-establishment of calcium sensitization pathways. These results demonstrate that age-related gallbladder hypocontractility is associated to alterations of calcium sensitization pathways and that melatonin treatment exerts beneficial effects in the recovery of gallbladder contractility.

Full-text

Available from: Pedro J Camello, Jun 12, 2015
Aging impairs Ca
2+
sensitization pathways in gallbladder
smooth muscle
Beatriz Macias & Pedro J. Gomez-Pinilla & Cristina Camello-Almaraz &
Patricia Pascua & Jesus Af. Tresguerres & Pedro J. Camello & Maria J. Pozo
Received: 20 January 2011 / Accepted: 19 June 2011 /Published online: 12 July 2011
#
American Aging Association 2011
Abstract Calcium sensitization is an important physi-
ological process in agonist-induced contraction of
smooth muscle. In brief, calcium sensitization is a
pathway that leads to smooth m uscle contraction
independently of changes in [Ca
2+
]
i
by mean of
inhibition of myosin light chain phosphatase. Aging
has negative impacts on gallbladder contractile response
due to partial impairment in calcium signaling and
alterations in the contractile machinery. However,
information regarding aging-induced alterations in
calcium sensitization is scanty. We hypothesized that
the calcium sensitization system is negatively affected
by age. To investigate this, gallbladders were collected
from adult (4 months old) and aged (2224 months old)
guinea pigs. To evaluate the contribution of calcium
sensitization pathways we assayed the effect of the
specific inhibitors Y-27632 and GF109203X on the in
vitro isometric gallbladder contractions induced by
agonist challenges. In addition, expression and phos-
phorylation (as activation index) of proteins participat-
ing in the calcium sensitization pathways were
quantified by Western blotting. Aging reduced
bethanechol- and cholecystokinin-evoked contractions,
an effect associated with a reduction in MLC20
phosphorylation and in the effects of both Y-27632
and GF109203X. In addition, there was a drop in
ROCK I, ROCK II, MYPT-1 and PKC expression and
in the activation/phosphorylation of MYPT-1, PKC and
CPI-17 in response to agonists. Interestingly, melatonin
treatment for 4 weeks restored gallbladder contractile
responses due to re-establishment of calcium sensitiza-
tion pathways. These results demonstrate that age-
related gallbladder hypocontractility is associated to
alterations of calcium sensitization pathways and that
melatonin treatment exerts beneficial effects in the
recovery of gallbladder contractility.
Keywords Gallbladder
.
Bethanechol
.
CCK
.
Calcium
sensitization
.
Aging
.
Oxidative stress
.
Melatonin
Introduction
Gallbladder functions (bile storage and release) are
controlled by the contractile state of the gallbladder
smooth muscle. This comprises periods of active and
passive relaxations (during which hepatic bile is
stored and concentrated) followed by contr actions
that release the bile in response to the lipids present in
the food (Pozo et al. 2004). Smooth muscle contrac-
tion is mainly achieved by increases in cytoplasmatic
AGE (2012) 34:881893
DOI 10.1007/s11357-011-9285-6
B. Macias and P.J. Gomez-Pinilla have contributed equally to
this work
B. Macias
:
P. J. Gomez-Pinilla
:
C. Camello-Almaraz
:
P. Pascua
:
P. J. Camello
:
M. J. Pozo (*)
Department of Physiology, Nursing School,
University of Extremadura,
Avda. Universidad s/n,
10003 Caceres, Spain
e-mail: mjpozo@unex.es
J. A. Tresguerres
Department of Physiology, Medical School, University
Complutense of Madrid,
28040 Madrid, Spain
Page 1
calcium concentration ([Ca
2+
]
i
) that activate myosin
light chain kinase (MLCK) to catalyze the phosphory-
lation of myosin regulatory light chain (MLC
20
)causing
force development (Karaki et al. 1997). An additional
signaling pathway involved in force maintenance,
termed calcium sensitization pathway, evokes smooth
muscle contraction independently of changes in [Ca
2+
]
i
by inhibition of myosin light chain phosphatase
(MLCP) and the consequent dephosphorylation of
MLC
20
in the presence of basal (Ca
2+
-dependent or
Ca
2+
-independen t) or incr eased MLCK a ctivity
(Somlyo and Somlyo 2003). MLCP inhibition occurs
mainly by two pathways: Rho A/ROCK/MYPT-1 and
PKC/CPI-17. Activation of some G-protein coupled
receptors by their agonists induce an increase in
membrane-bound Rho A-GTP necessary for the
activation of Rho kinases (ROCK I and ROCK II),
that phosphorylate, amongst other proteins, the myosin
phosphatase targeting 1 (MYPT-1), a subunit of MLCP.
p-MYPT-1 inhibits MLCP activity, thus favouring
contraction. G proteins c ause also activation of
phospholipase C (PLC), diacylglycerol (DAG) output
and activation of protein kinase-C (PKC). PKC
phosphorylates C-kinase-dependent phosphatase inhib-
itor of 17 kDa (CPI-17), an inhibitor of the MLCP
activity via its PP1c subunit, resulting in smooth
muscle contraction (Eto et al. 1995; Somlyo and
Somlyo 2003). The activity of MLCP can also be
regulated by altering its heterotrimeric conformation
(Hirano et al. 2004) and by the protein scaffold-myosin
phosphatase-Rho interacting protein (M-RIP) (reviewed
by Kim et al. 2008). In addition, in the absence of
changes in [Ca
2+
]i, MLC
20
can be phosphorylated
driving to contraction by Ca
2+
-independent MLCKs
such as ZIP kinase and integrin-linked kinase (Murthy
2006). The impact of aging on calcium sensitization
pathways has been poorly explored.
It has been described that aging compromises
gallbladder function and drives to an elevated incidence
of cholesterol gallstones in old individuals (Chen et al.
1999; van de Heijning et al. 1999; Xu and Shaffer
1996). We found that aging modifies neurological
control of guinea pig gallbladder and impairs contractile
response as the result of alterations in the contractile
machinery without affecting calcium release from
intracellular stores (Gomez-Pinilla et al. 2006). The
fact that impairment in the contractile capacity does not
run in parallel with alterations in calcium signals
suggests that calcium-independent mechanisms, such
as calcium sensitization pathways, would be additional
targets for the harmful effects of age. Herein we explore
this hypothesis to give some insights into the intracel-
lular mechanisms affected. In order to test whether age-
related effects in Ca
2+
-independent contractility could
be ameliorated by melatonin we treated a group of aged
guinea pig with the indoleamine for 28 days.
Our data indicate that aging decreases the partic-
ipation of calcium sensitization pathways in guinea
pig gallbladder, which is translated into impaired
contractile responses to agonists. Melatonin treatment
normalizes calcium sensitization pathways and the
contractile responses, indicating that both processes
run in parallel in the gallbladder.
Material and methods
Animals
Female guinea pigs (DunkinHartley) were divided
into two groups according to age: young adults
(4 months old, n=12) and aged (2224 months old,
n=10). Animals were housed in light (12 h lightdark
cycles) and temperature (20°C) controlled rooms and
had free access to water and food. A group of aged
guinea pigs (n=10) was treated orally with melatonin
(2.5 mg kg
1
day
1
) every day 1 h before the dark
phase for 28 days. All experi ments were performed
according to European guidelines for animal research
and approved by the Animal Ethics Committees of
the University of Extremadura.
Contraction recording of guinea pig gallbladder strips
Deep halothane anesthetized guinea pigs were killed
by cervical dislocation, the gallbladder was removed
and placed in KrebsHenseleit solution. The gallblad-
der was cut in longitudinal full-thickness strips (3×
8 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 measured using force transducers
and digitized using a MacLab syst em (ADInstru-
ments, Colorado Springs, CO, USA). Gallbladder
strips were mounted under an initial resting tension
equivalent to 1.5 g load and allowed to equilibrate for
60 min, with solution changes every 20 min. Previous
tension recordings, in response to 100 μM bethane-
882 AGE (2012) 34:881893
Page 2
chol, at different loads showed that 1.5 g load was the
optimal stretch for recording the maximal tension in
both age groups. Every strip coming from the same
animal was used in a different experimental protocol.
Force was normalized with respect to the cross
section area (CSA) of each muscle strip.
In order to find the contribution of calcium
sensitization pathways in the agonist-induced gallblad-
der smooth muscle contraction a protocol of cumula-
tive doseresponse curves for bethanechol (BE) and
cholecystokinin (CCK) was designed. The cumulative
doseresponse curve commenced with the lowest
concentration of the agonist and the subsequent doses
were not added until the contractile response became
constant. After the maximal response was reached, 1 h
was required for washing out and returning to the
resting tension. Then, the muscle strips were incubated
for 30 min with the antagonists (5 μM Y-27632 or
5 μM GF109203X, inhibitors of ROCK- and PKC-
dependent pathways, respectively) and the cumulative
concentrationresponse curve was repeated.
Analysis of protein expression and phosphorylation
by Western blot
Guinea pig gallbladder smooth muscle was cut in three
pieces, one was not stimulated (control), one was
challenged with BE (0.1 μM) and the last one with
10 nM of CCK. The stimuli were applied for 1 min.
After challenges tissues were quickly frozen, pulverized
in liquid nitrogen, extracted in lysis buffer and then
sonicated for 5 s. Lysates were centrifuged at 10,000×g
for 15 min at 4°C to remove nuclei and intact cells and
the protein concentration was measured.
Protein extracts (30 μg) were heat-denatured at 95°C
for5minwithDTT,electrophoresedon7.5%or15%
polyacrylamide-SDS gels and then transferred to nitro-
cellulose membranes. Membranes were blocked for 1 h at
room temperature using 10% bovine serum albumin
(BSA) and incubated overnight at 4°C with affinity-
purified polyclonal antibodies against ROCK I and II and
COX-2 (1:500; Becton-Dickinson Bioscience, Erembo-
deguen, Belgium), Rho A, p-CPI-17, CPI-17, p-MYPT-1,
MYPT-1, pMLC20 and MLC20 (1:500; 1:750 for
pMLC20 and 1:7,000 for MLC20; Santa Cruz Biotech-
nology, Santa Cruz, CA, USA), p-PKC (pan antibody,
1:5,000; Cell Signaling Technology, Danvers, MA,
USA). A rabbit anti-actin polyclonal antibody (1:5,000;
Sigma, St. Louis, MO, USA) was used as control for
protein loading. After washing, the membranes were
incubated for 1 h at room temperature with anti-mouse
(1:10,000; Amersham Biosciences, Bucks, UK) or anti-
rabbit (1:7,000; Santa Cruz Biotechnology, Santa Cruz,
CA, USA) IgG-horseradish peroxidase conjugated sec-
ondary antibodies. Bands were visualized using the
supersignal west pico chemiluminescent substrate
(Pierce, Rockford, IL, USA) and quantified using the
software gel-pro analyzer (4.0, Media Cybernetics,
Bethesda, MD, USA) and normalized to actin content.
In protein phosphorylation assays, two similar gels
were run and one membrane was incubated with the
primary antibody against the tota l protein and the
other with an anti-phosphoprotein of interest.
Solutions and drugs
KrebsHenseleit solution (in mM): 113 NaCl, 4.7 KCl,
2.5 CaCl
2
,1.2KH
2
PO
4
,1.2MgSO
4
,25NaHCO
3
and
11.5
D-glucose (pH=7.35). Lysis buffer (in mM): 50
TrisHCl pH 7.4, 150 NaCl, 1% Triton X-100,1%
deoxycholate, 0.5% (w/v)NaN
3
,0.4EDTA,supple-
mented with a cocktail of protease and phosphatase
inhibitors (Complete, EDTA-free and PhosSTOP;
Roche Molecular Biochemicals, Mannheim, Germany).
Drug concentrations are expressed as final bath concen-
trations of active species. Drugs and chemicals were
obtained from the following sources: BE, CCK and
melatonin are from Sigma and Y-27632 and
GF109203X from Calbiochem (La Jolla, CA, USA).
Quantification and statistics
Results are expressed as means±SEM of n gallbladder
strips or blots. Gallbladder tension is given in milli-
newtons per square millimeter of tissue since data were
normalized respect to CSA. Overall comparison between
concentrationresponse curves was performed by a
mixed two-way analysis of variance (ANOVA) model
using age/treatment × concentration as fixed and
repeated factors, respectively. To compare maximum
effects (E
max
), we used a priori planned contrast.
Statistical differences between means were determined
by Studentst-test. All data were inspected to exclude
gross departures from normality or homoskedascity and
correlation between mean and variance sizes between
groups. The statistical power was adjusted to be at least
0.8 (0.2 type II error). Differences were considered
significant at P<0.05.
AGE (2012) 34:881893 883
Page 3
Results
Effects of aging on gallbladder contractile response
In a previous report (Gomez-Pinilla et al. 2006)we
showed that aging impaired contraction of the guinea
pig gallbladder in response to agonists. In the current
study, we give some insights on the age induced
alterations of stimuluscontraction coupling mecha-
nisms. To evaluate the effects of aging we exposed
guinea pig gallbladder strips to increasing levels of
BE, a musc arinic receptor agonist, or CCK octapep-
tide, a pro-contractile hormone secreted in response to
lipid content of food.
Bethanechol (10 nM to 10 mM) and CCK (10 pM
to 1 μM) produced a dose-dependent contraction with
a maximal effect at 1 mM and 1 μM, respectively
(Fig. 1a, b). Analysis by two-w ay ANOVA showed
that age causes a significant decrease in gallbladder
contractile responses to both agonists (BE: F=191.2
and P<0.0001; CCK: F=58.87 and P<0.0001).
Thus, the E
max
to BE and CCK were lower in aged
0.0
0.5
1.0
1.5
2.0
2.5
*
MLC-20
phosphorylation
-9
-8 -7 -6 -5 -4 -3 -2 -1
0
10
20
30
40
50
***
Log [BE] (M)
Tension (mN/mm
2
)
Tension (mN/mm
2
)
-12
-11 -10
-9
-8
-7
-6 -5
0
10
20
30
40
50
Adult
Senescent
***
Log [CCK] (M)
BA
Bethanechol Cholecistokinin
0.0
0.5
1.0
1.5
2.0
2.5
*
MLC-20
phosphorylation
p-MLC20
MLC20
Actin
42 KDa
18 KDa
20 KDa
C
p-MLC20
MLC20
Actin
C CCK
42 KDa
18 KDa
20 KDa
BE
Adult Aged
Adult Aged
Ad Ag
Ad Ag
CCK
CBE
C
C
Fig. 1 Aging reduces guinea pig gallbladder myogenic
response. Strips were exposed to increasing concentrations of
a bethanechol (BE, 10 nM to 10 mM) and b cholecystokinin
(CCK, 10 pM to 1 μM). Note a significant reduction in the
contractile response in gallbladders from aged animals. In (a)
and (b), contractile responses are expressed as mean±SEM. n=
1927 strips (***P<0.0001, adult vs. aged). c Original Western
blots on gallbladder smooth muscle from adult (Ad) and aged
(Ag) guinea pigs for phospho-MLC20 and total MLC20 in
unstimulated (control) or agonist-treated gallbladder samples.
After normalization to actin content, the optic density of
phosphorylated protein vs. total protein was calculated for
control and treated samples in both age groups. Then, the
resulting ratio for treated samples was expressed as fold
increase respect to unstimulated conditions. Note that protein
phosphorylations were decreased in the aged group The
histograms (mean±SEM) show summarized data of changes
in MLC20 phosphorylation for 810 blots (*P<0.05, adult vs.
aged)
884 AGE (2012) 34:881893
Page 4
muscles with a 45.62% (BE) and 34.44% (CCK) of
reduction associated to aging (BE E
max
: 4.91±0.28
vs. 2.67±0.25 mN/mg, n=22 and 19, P<0.001; CCK
E
max
: 4.21±0.28 vs. 2.76±0.27 mN/mg, n=27 and
19, P<0.001; Fig. 1a, b).
The age-induced reduction in the contractile force
was associated to a decrease in the ratio of MLC20
phosphorylation as illustrates Fig. 1c. The extent of
MLC20 phosphorylation induced by BE and CCK in
aged strips was 30% and 36% smaller than in adult
strips (P<0.05, n=8 and 10). Age-related alterations
could be explained by alterations in calcium handling,
as calcium is the main second messenger controlling
contraction in smooth muscle. However, we have
previously shown that age-related decreases in gall-
bladdercontractiondonotruninparallelwith
impairment in calcium-dependent signaling (Gomez-
Pinilla et al. 2006). Thus, we explored the status of
calcium-independent mechanism leading to contrac-
tion or calcium sensitization during aging.
Effects of aging on contractile response sensitivity
to Y-27632 and GF109203X
In order to investigate the contribution of calcium
sensitization pathways in the contractile response to
BE and CCK, concentrationresponse curves were
performed in the absence and presence of Y-27632
(5 μM) and GF109203X (5 μM), specific inhibitors
of ROCK and PKC, respectively (Toullec et al. 1991;
Uehata et al. 1997). The pre-treatment with Y-27632
reduced significantly the doseresponse curve to BE
in adult and aged strips, (adult: F=95.90, P<0.001;
aged: F =50.53, P <0.001; Fig. 2a, b)butthe
reduction in the maximal response to BE was lower
in aged tissues (39.1±4.6 vs. 23.9±5.74% of inhibi-
-9 -8
-7 -6 -5 -4 -3
-2
-1
0
25
50
75
100
125
***
Log [BE] (M)
Maxime response (%)
Control
Y-27632
A
Adult Aged
-9 -8
-7
-6 -5 -4 -3
-2 -1
0
25
50
75
100
125
***
Log [BE] (M)
Control
Y-27632
B
Adult Aged
Control
GFX
Control
GFX
-9 -8
-7 -6
-5
-4 -3
-2
-1
0
25
50
75
100
125
***
Log [BE] (M)
Maxime response (%)
Maxime response (%)
Maxime response (%)
-9 -8 -7 -6 -5 -4 -3
-2
-1
0
25
50
75
100
125
**
Log [BE] (M)
Fig. 2 Aging reduces the sensitivity of bethanechol-induced
gallbladder contractions to Y-27632 and GF109203X, inhibitors
of ROCK and PKC, respectively. Effects of 5 μM Y-27632 (a
and b) and 5 μM GF109203X ( c and d) on bethanechol (BE)
evoked contractions in gallbladder strips from adult and aged
guinea pigs. Note that the effects of the inhibitors are lower in
aged than adult animals indicative of a lower participation of
calcium sensitization pathways. Data are expressed as mean±
SEM. n=1015 strips (**P<0.01 and ***P<0.001, adult vs.
aged)
AGE (2012) 34:881893 885
Page 5
tion for adult and aged strips, respectively, P<0.05).
Similar results were found in the presence of
GF109203X (adult: F=97.21, P<0.001; aged: F=
32.90, P<0.01; Fig. 2c, d) although the inhibition of
maximal response to BE in old animals was even
lower (42.8±6.8 vs. 16.9±8.4% of inhibition for adult
and aged strips, respectively, P<0.01). When CCK
was used as agonists we found a similar picture
(Fig. 3), the antagonists reduced the dose response
curves of both agonists in both age groups but their
effects wer e less marked in old tissue, especially those
of GF109203X.
This pharmacological approach points out that the
contribution of calcium sensitization pathways are
less prominent in aged animal, which could be
responsible for the age-i nduced impairment of gall-
bladder contractile response. To characterize the age-
induced impairment, we assessed by Western blot the
expression and activation of the more relevant
proteins involved in calcium sensitization pathways.
Effects of aging on expression of members of calcium
sensitization pathways
Consistent with the reduced inhibition evoked by Y-
27632 and GF109203X on the contractile agonist
response in old animals, aging reduced the expression
of some proteins participating in calcium sensitization
mechanisms. In this regard, aging induced a signi fi-
cant reduction of some components of Rho A/ROCK
pathway without affecting the expression of Rho A
(ROCK I 50% of adult, ROCK II 33% of adult and
A
-12
-11
-10 -9
-8 -7
-6
-5
0
25
50
75
100
125
***
Log [CCK] (M)
Maxime response (%)
-12 -11
-10 -9
-8 -7 -6 -5
0
25
50
75
100
125
***
Log [CCK] (M)
Adult
Adult
Aged
Aged
Control
Y-27632
Control
Y-27632
Control
GFX
Control
GFX
-12 -11 -10 -9 -8 -7 -6 -5
0
25
50
75
100
125
***
Log [CCK] (M)
Maxime response (%)
Maxime response (%)
Maxime response (%)
-12 -11 -10 -9 -8 -7 -6 -5
0
25
50
75
100
125
**
Log [CCK] (M)
Fig. 3 Aging reduces the sensitivity of CCK-induced contrac-
tile response to Y-27632 and GF109203X, inhibitors of ROCK
and PKC, respectively. Effects of 5 μM Y-27632 (a and b) and
5 μM GF109203X (c and d) on CCK-evoked contractions in
gallbladder strips from adult and aged guinea pigs. Note that
the effects of the inhibitors are smaller in aged than adult
animals indicative of a lower participation of calcium sensiti-
zation pathways. Data are expressed as mean±SEM. n=69
strips (**P <0.01 and ***P<0.001, adult vs. aged)
886 AGE (2012) 34:881893
Page 6
Rho A
A
Actin
ROCK I
B
Actin
ROCK II
C
Actin
MYPT-1
D
Ad Ag Ad Ag
Ad Ag Ad Ag
Actin
42 KDa
22 KDa
42 KDa
130 KDa
42 KDa
150 KDa
42 KDa
140 KDa
*
*
**
Adult Aged
A
g
ed A
g
ed
Aged
0.0
0.5
1.0
1.5
2.0
Expression
0.0
0.5
1.0
1.5
2.0
Expression
Adult
0.0
0.5
1.0
1.5
2.0
Expression
0.0
0.5
1.0
1.5
2.0
Expression
Adult
Adult
Fig. 4 Aging decreases the
expression of proteins
involved in Rho A/ROCK
pathway. Original Western
blots on gallbladder smooth
muscle from adult (Ad) and
aged (Ag) guinea pigs for a
Rho A, b ROCK I, c ROCK
II and d MYPT-1. There are
not differences in the
expression of Rho A while
aging reduces the expres-
sion of ROCK I, ROCK II
and MYPT-1. Actin was
used as loading control and
proteins expression was
calculated respect to actin
expression. Histograms
summarize proteins expres-
sion (mean±SEM) for
68 blots (*P<0.05 and
** P<0.01, adult vs. aged)
p-PKC
A
Actin
CPI 17
B
Ad Ag Ad Ag
Actin
42 KDa
75 KDa
42 KDa
17 KDa
**
Adult Aged AgedAdult
0.0
0.5
1.0
1.5
2.0
0.0
0.5
1.0
1.5
2.0
Expression
Expression
Fig. 5 Aging decreases the expression of proteins involved in
PKC/CPI-17 pathway. Original Western blots on gallbladder
smooth muscle from adult (Ad) and aged ( Ag) guinea pigs for a
phospho-PKC and b CPI-17. Note that aging reduces the
expression of phospho-PKC. Actin was used as loading control
and proteins expr ession was c alcula ted respect t o actin
expression. Histograms summarize protein expression (mean±
SEM) for 68 blots (** P<0.01, adult vs. aged)
AGE (2012) 34:881893 887
Page 7
MYPT-1 40% of adult, Fig. 4). In addition, the
expression of phospho-PKC was reduced by 40% in
aged gallbladder while the expression of its effector
CPI-17 was similar in adult and aged tissues (Fig. 5).
Effects of aging on activation of calcium sensitization
pathways
Activation of calcium sensitization was explored
using phosphoprotein-specific antibodies agains t the
key components of the pathways. Phosphorylation of
MYPT-1 (at Thr-853), PKC (at Ser-660) and CPI-17
(at Thr-38) in response to BE and CCK is shown in
Fig. 6. In adult gallbladders, the ratios phosphorylated/
total proteins after agonist challenge were higher than 1
(the ratios for unstimulated tissues are referred as 1),
indicating an increase in phosphorylation. By the
contrary, in aged animals phosphorylation in response
to BE and CCK was reduced for the three regulatory
proteins tested (Fig. 6). The decreased phosphorylation
found in aged animals together with the reduction in
protein expression can explain the reduced effects of
the inhibitors tested in contractility, and altogether
evidence that aging is associated to a partial loss of
calcium sensitization which impairs gallbladder con-
tractile response.
Effects of melatonin treatment on gallbladder
contractility and calcium sensitization pathways
The beneficial effects of melatonin treatment against
age-associated gallbladder hypocontractility has been
Adult Aged
p-MYPT-1
t-MYPT-1
Actin
42 KDa
130 KDa
130 KDa
BA
DC
Adult Aged
Adult Aged
Control CCK Control CCK
Control CCK Control CCK
Control CCK Control CCK
FE
p-PKC
Actin
42 KDa
75 KDa
p-CPI -17
t-CPI -17
Actin
42 KDa
17 KDa
17 KDa
*
**
*
**
**
*
Ad Ag
Bethanechol Cholecistokinin
Ad Ag
0
1
2
Phosphorylation
0
1
2
MYPT-1
Phosphorylation
Ad Ag
0
1
2
Ad
Ag
0
1
2
Phosphorylation
Ad
Ag Ad Ag
0
1
2
0
1
2
Phosphorylation
PKC
Phosphorylation
CPI-17
Phosphorylation
Fig. 6 Aging impairs activation of Ca
2+
sensitization path-
ways. a Western blots using anti-p-Thr853-MYPT-1 and anti-
MYPT-1 in unstimulated (control) or CCK-treated gallbladder
samples from adult (Ad) and aged (Ag) guinea pigs. c Changes
in phospho-PKC expression in response to CCK for both
groups of animals. e Representative Western blots using anti-p-
Thr38-CPI-17 and anti-CPI-17 in unstimulated (control) or
CCK-treated tissues. The histograms show summarized data of
changes in b MYPT-1, d PKC and f CPI-17 phosphorylation.
After normalization to actin content, the optic density of
phosphorylated protein vs. total protein was calculated for
control and treated samples in both age groups. Then, the
resulting ratio fo r tr eated samples was exp ressed as fold
increase respect to unstimulated conditions. Note that protein
phosphorylations were decreased in the aged group for all the
proteins studied and for both challenges. n=68 experiments.
(*P<0.05, ** P<0.01, adult vs. aged)
888 AGE (2012) 34:881893
Page 8
previously described (Gomez-Pinilla et al. 2006), and
here we confirm previous result showing that in
vivo melatonin treatment for 28 days almost normal-
izes BE and CCK concentrationresponse curves
(Fig. 7, left bars). Given that melatonin exerts potent
antioxidant effects at multiple stages of the signal
pathways (Pozo et al. 2010), we treated aged animals
with melatonin to find if aged-induced alterations in
the effects of Y-27632 and GF109203X and in the
level of expression and activation of calcium
sensitizing proteins coul d be prevented. In line
with the recovery of the contractile capacity, Fig. 7
shows that melatonin treatment normalizes the
effects of the inhibitors (Fig. 7, middle and right
bars). Melatonin treatment also increases ROCK I,
ROCK II, MYPT-1 (Fig. 8a) and phospho-PKC
(Fig. 8b) expression and enhances the activation/
phosphorylation of MYPT-1, PKC and CPI-17
(Fig. 8c, d) in response to BE and CCK challenge.
These results indicate that melatonin-associated
recovery of calcium sensitization pathways could
be responsible for normalization of gallbladder
contractile capacity.
The melatonin-induced normalization of gallblad-
der contractile capacity correlated with the reestab-
lishment of COX-2 expression that was enhanced in
aged gallbladder (Fig. 9).
Discussion
Data presen ted here support the hypothesis that
calcium sensitization signaling is negatively affected
by aging in the guinea pig gallbladder. The main
finding of this report is the reduction in the
contribution of calcium sensiti zation mechanisms to
the contractility of aged guinea pig gallbladder. Here,
we describe that the targets for aging are the proteins
participating in both Rho A/ROCK/MYPT-1 and
PKC/CPI-17 pathways at expression and activation
levels. The fact that melatonin treatment restored
calcium sensitization contribution to the contractile
response and normalized COX-2 expression could
point out to an oxidative stress imbalance as cause of
the impairment in calcium sensitization participation,
although we cannot rule out other mechanisms of
action of melatonin.
Aging-associated changes have been described
in the gastrointestinal tract, such as reduction in
the ability to e at large boluses of food (Rayner et
al. 2000), as well as alterations in gastrointestinal
function and motility disorders affecting gastric
empty and/or colonic transit (Camilleri et al.
2000). However, molecular or cellular mechanisms
responsible for these alterations have been poorly
explored.
BA
BE-induced response CCK-induced response
**
*
**
**
**
**
Ag Mel Ag
Mel
Ag Mel
0
25
50
75
100
125
150
Tension
Inhibition
by Y27632
% of Adult
Ag
Mel Ag Mel
Ag
Mel
0
25
50
75
100
125
150
% of Adult
Inhibition
by GFX
Tension
Inhibition
by Y27632
Inhibition
by GFX
Fig. 7 Melatonin normalizes contractile responses and calcium
sensitization contribution to contraction in aged gallbladders.
Histograms represent maximal contractile response to a
bethanechol and b cholecystokinin in aged animals (gray bars)
and aged animals treated with melatonin (2.5 mg kg
1
day
1
)
for 28 days (empty bars). Gallbladder contractility and the
inhibitory effects of Y-27632 and GF109203X were increased
after melatonin treatment. Data are expressed as percentage of
adult values. n=815 strips (*P<0.05 and **P<0.01, aged vs.
aged treated with melatonin)
AGE (2012) 34:881893 889
Page 9
The main contracting signals for gallbladder
muscle are acetylcholine and CCK (Pozo et al.
2004). Here, we confirm previous findings (Gomez-
Pinilla et al. 2006) about the impairment in myogenic
gallbladder responses associated to aging and we further
show that a decrease in the phosphorylation of MLC20
is associated to this hypocontractility. In addition, we
also unveil the effects of aging in calcium-independent
pathways for gallbladder contraction.
Intracellular Ca
2+
levels play an important role in
the modulation of contractionrelaxation cycle of
smooth muscle cells. So, increases in [Ca
2+
]
i
lead to
contraction in a calmodulin- and MLCK-dependent
way (Horowitz et al. 1996; Karaki et al. 1997).
Classically, disturbances of Ca
2+
homeostasis and/or
signaling are potential targets for intervention in aged
tissues. It is well established that aging evokes tissue-
specific changes in Ca
2+
homeostasis in nerves and in
skeletal, cardiac and smooth muscles (Gomez-Pinilla
et al. 2007; Gomez-Pinilla et al. 2011; Puzianowska-
Kuznicka and Kuznicki 2009). In the guinea pig
gallbladder, aging reduced calcium influx but not
calcium mobilization from internal stores even if
contractile impairment is present for both maneuvers
(Gomez-Pinilla et al. 2006). These are clear evidences
that changes in Ca
2+
homeostasis can neither fully
explain the impairment in gallbladder contractility
during aging nor the normalization in contractility
Ag
Mel
Ag
M
e
l
Ag
Mel
Ag
Mel
0
25
50
75
100
125
150
Ag
M
el
Ag
M
el
Ag
Mel
Ag
Mel
0
25
50
75
100
125
150
Ag Mel
Ag
Mel Ag Mel Ag Mel
0
25
50
75
100
125
150
% of Adult
Rho A
ROCK I ROCK II MYPT-1
BA
Ag Mel Ag Mel
0
25
50
75
100
125
150
% of Adult
% of Adult
% of Adult
**
*
****
p-PKC CPI 17
DC
*
*
**
**
**
**
MYPT-1 PKC
CPI 17
CCK-induced phosphorylationBE-induced phosphorylation
Protein expression Protein expression
MYPT-1
PKC
CPI 17MLC20 MLC20
*
**
Fig. 8 Melatonin restores the expression and activation of
proteins involved in calcium sensitization pathways in aged
gallbladders. Histograms represent protein expression of pro-
teins involved in the a Rho A/ROCK and b PKC/CPI-17
calcium sensitization pathways in aged animals (gray bars) and
aged animals treated with melatonin (empty bars). c BE-
induced and d CCK-induced phosphorylation of MYPT-1 (left),
PKC (middle) and CPI-17 (right) in aged animals (gray bars)
and aged animals treated with melatonin (empty bars). Note the
significant increase in protein expression and activation after
melatonin treatment. Data are expressed as percentage of adult
values. n=68 blots (*P<0.05 and **P<0.01, aged vs. aged
treated with melatonin)
890 AGE (2012) 34:881893
Page 10
induced by melatonin treatment even without the
reestablishment of calcium influx (Gomez-Pinilla et
al. 2006). In this report, we show that impaired
calcium sensitization pathways run in parallel with
gallbladder hypomotility and decreased MLC20
phosphorylation. The impairment in calcium sensiti-
zation contribution to reduced gallbladde r contractil-
ity associated to aging is driven by a reduction in the
protein expression of ROCK I, ROCK II, MYPT-1
and PKC together with a reduction in the activation of
MYPT-1, PKC and CPI-17 in response to agonist
challenges. These changes in protein expression and
activation were accompanied by an expected reduc-
tion of the impact of the specific inhibitors Y-27632 or
GF109203X on the contractile responses to agonists.
Molecular and pharmacological findings fully explain
the reduction in gallbladder contractile capacity
associated to aging.
According to our results , calcium sensit ization
pathways are not static and their contribution can
change depending of the environment, indicative of
plasticity in those pathways. It has been shown that
increases in calcium-independent contractions play an
important r ole in d iseases such as hypertension
(Uehata et al. 1997), bronchial asthma (Chiba et al.
1999), erectile dysfunction (Wang et al. 2002) and
hyperactive bladder (Persson et al. 1998). Here, we
show that aging displays opposite effects since it
leads to an impairment of calcium sensitization
pathways that is translated into hypocontractility of
the gallbladder. These findings, together with results
obtained in gallbladders from newborn guinea pigs
(Camello-Almaraz et al. 2009), draw the whole
picture about the state of calcium sensitization along
life. In guinea pig gallbladder, calcium sensitization
pathway increases from birth to adult ages (Camello-
Almaraz et al. 2009) and after that it suffers a decline.
However, calcium handling runs in the opposite
direction, with a reduction after birth (Camello-
Almaraz et al. 2009) and partial maintenance with
aging (Gomez-Pinilla et al. 2006). In the three age
points, the state of calcium sensitization matches
better with the contractile capacity than calcium
signaling does. The effects of aging on calcium
sensitization in guinea pig gallbladder described here
are similar to those found in aged urinary bladder
(Gomez-Pinilla et al. 2008), suggesting that in
different contra ctile organs aging initiat es similar
harmful effects, which seem to be related to the
imbalance in oxidative stress. However, it is likely the
presence of species-related differences, sinc e in
mouse urinary bladder calcium handling and contrac-
tile activity run in parallel during aging (Gomez-
Pinilla et al. 2011) in spite that calcium sensitization
was not explored.
Melatonin (the main pineal gland hormone but
with an important presence also in the gastrointestinal
tract) has a substantial direct and indirect antioxidant
activity and it can counteract the exacerbated free
radicals damage accumulated during aging (Cuesta et
al. 2010; Forman et al. 2010; Kireev et al. 2007a, b,
2008; Reiter et al. 1999a, b). Age-related decline in
both circulating levels and the amplitude of the
rhythm of melatonin are a well documented phenom-
ena in a variety of species (Pang et al. 1990) and leads
to an increase in oxidative stress due to an imbalance
between pro-oxidants and antioxidant (Reiter et al.
1999b). In fact, we have found th at melatonin
preserves the membrane potential of in situ mito-
chondria, the main source of intracellular oxidants, in
several models (Camello-Almaraz et al. 2008
;
Cox2
Ad Ag
Actin
42 KDa
70 KDa
Adult Aged Melatonin
0.0
0.5
1.0
1.5
*
Expression
Mel
Fig. 9 Aging decreases the expression of COX-2. Original
Western blots on gallbladder smooth muscle from adult (Ad),
aged (Ag) and aged animals treated with melatonin (Mel)
guinea pigs for COX-2. Note that aging increases the
expression of COX-2 and that melatonin normalizes this
expression pattern. Actin was used as loading control and
protein expression was calculated respect to actin expression.
Histograms summarize protein expression (mean± SEM) for
1012 blots (* P<0.05, aged vs. adult and aged treated with
melatonin)
AGE (2012) 34:881893 891
Page 11
Gomez-Pinilla et al. 2008). In addition to its relevant
antioxidant activity, melatonin exerts many of its
physiological actions by binding to nuclear receptors
of the orphan family and by interacting with mem-
brane MT
1
and MT
2
receptors and intracellular
proteins such as quinone reductase 2, calmodulin,
calreticulin and tubulin (Pandi-Perumal et al. 2008;
Reiter et al. 2010), which could underline the
beneficial effects reported for melatonin.
Exogenous administration of melatonin to aged
guinea pigs restored gallbladder contractility indepen-
dently of changes in calcium handling (Gomez-Pinilla
et al. 2006). In this report, we confirm the beneficial
effects of melatonin on gallbladder contractile capac-
ity and describe that it is mediated by an increase in
calcium sensitization contribution to the contractile
response. Melatonin treatment reestablishes the expres-
sion of proteins involved in these pathways and their
activation in response to agonist challenges. Accumu-
lated data strongly suggest that continuous (chronic)
upregulation of pro-inflammatory mediators are induced
during the aging process due to an age-related redox
imbalance that activates many pro-inflammatory signal-
ing pathways (reviewed by Chung et al. 2009). We have
just reported that aging of gastrointestinal smooth
muscle is associated to an increase in oxidative stress
that correlates with increased NF-κB and COX-2
expression indicative of a subtle inflammation pattern
in aging (Pascua et al. 2011). Here we show that
increased expression of COX-2 in aged gallbladder is
normalized by melatonin treatment, which would
suggest that the improvement in Ca
2+
sensitization-
mediated contractility could be related to the amelio-
ration of the oxidative insult and subtle inflammation.
However, other actions of melatonin can not been ruled
out.
These observations are important in several respects.
First, they strengthen the parallelism between calcium
sensitization pathways and contractility. Second, they
suggest that the impairment in calcium sensitization
contribution is due to the increase in oxidative stress and
subtle inflammation. Finally, this report reinforces the
beneficial effects of melatonin treatment on smooth
muscle pathological conditions related to oxidative
stress imbalance (Pozo et al. 2010).
In summary, we found an age-related decline in the
gallbladder contractility due to the reduction of the
contribution of calcium sensitization mechanisms,
shown at the molecular and pharmacological level.
The administration of exogenous melatonin normal-
ized calcium sensitization participation and gallblad-
der contractile response and COX expression,
suggesting that calcium sensitization pathways could
be sensitive to oxidative stress.
Acknowledgements This work was supported by Spanish
Ministery of Science and Education (BFU 200760563), Junta
de Extremadura (PRI07A069) and Instituto de Salud Carlos III
(RETICEF RD06/0013/1012 and RD06/0013/0002).
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Page 13
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