adiponectin enhances calcium-dependency of mouse...

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Adiponectin enhances calcium-dependency of

mouse bladder contraction mediated by protein

kinase Cα expression

Koji Nobe, Akiko Fujii, Kiyomi Saito, Takaharu Negoro, Yoshio

Ogawa, Yasuko Nakano, Terumasa Hashimoto and Kazuo Honda

Departments of (K.N., A.F., T.H., K.H.) Pharmacology, (K.S.) Clinical and Molecular

Pharmacokinetics/Pharmacodynamics, and (T.N., Y.N.) Pharmacogenomics, School of

Pharmacy, Showa University; (Y.O.) Department of Urology, Showa University

Hospital, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan

JPET Fast Forward. Published on January 30, 2013 as DOI:10.1124/jpet.112.202028

Copyright 2013 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on January 30, 2013 as DOI: 10.1124/jpet.112.202028

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Running title page

Running Title: Association of adiponectin with bladder contraction

Address correspondence to:

Koji Nobe, Ph.D., Department of Pharmacology, School of Pharmacy, Showa

University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan

Tel: +81-3-3784-8212

Fax: +81-3-3784-3232

E-mail: [email protected]

Counts

Number of text pages: 27

Number of tables: 1

Number of figures: 5

Number of references: 30

Number of words in the Abstract: 249

Number of words in the Introduction: 282

Number of words in the Discussion: 1168

List of non-standard abbreviations

Adip-R, adiponectin-specific receptor; A-kinase, cAMP dependent protein

kinase; [Ca2+]i, intracellular calcium concentration; CCh, carbachol; CREB,

cAMP-response element-binding protein; FFA, free fatty acids; MLC, myosin


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light chain; MLCP, myosin light chain phosphatase; PL, phospholipids; PKC,

protein kinase C; p-PKCα, phosphorylated-PKCα; PSS, physiological salt

solution; total-Cho, total cholesterol.

Recommended section

Endocrine and Diabetes


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ABSTRACT Adiponectin is an adipose tissue-secreted protein and is a

multi-functional adipocytokine. However, the association of adiponectin with bladder

contraction has not been investigated. In this study, the adiponectin-sense transgenic

mouse (Adip-Sen mouse; 16–24 weeks, male) and age-matched controls (C57Bl mouse)

were studied. The Adip-Sen mouse showed a significant increase in plasma adiponectin

levels (56.2%; p<0.01) compared with those in the C57Bl mouse, without affecting

other lipid parameters. Isometric force development in bladder smooth muscle tissues

were detected using an organ-bath system. Although carbachol (CCh; 0.1-100

µM)-induced time- and dose-dependent contractions in Adip-Sen mouse bladder were

slightly enhanced compared with those in the C57Bl mouse during a low range (0.3-1.0

µM) of CCh, differences could not be detected with other CCh concentrations. However,

the reduction in contraction under Ca2+-replaced conditions was significantly different

between Adip-Sen and C57Bl mice (94.1% and 66.3% of normal contraction,

respectively; n= 5). A parameter of Ca2+ sensitivity, the relation between intracellular

Ca2+ concentration and contraction, was increased in the Adip-Sen mouse compared

with that in the C57B1 mouse. This Ca2+ dependency in the Adip-Sen mouse was

reduced by a protein kinase C (PKC) inhibitor, but not by a Rho kinase inhibitor.

Expression of the calcium-dependent isoform of PKC, PKCα, was increased in the

Adip-Sen mouse bladder and CCh-induced phosphorylation of PKCα was also

enhanced compared with those in the C57Bl mouse. In conclusion, adiponectin is

associated with bladder smooth muscle contraction, which involves an increase in Ca2+

dependency of contraction mediated by PKCα expression.


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Introduction

Adiponectin is an adipocyte-secreted hormone and is present in the circulation of

healthy humans at high concentrations (Goldstein and Scalia, 2004). Unlike most other

adipocytokines, adiponectin levels decrease in individuals with obesity (Stumvoll et al.,

2002), and adiponectin levels are further reduced in type-II diabetes (Hotta et al., 2000).

Because adiponectin plays a role in increases in glucose incorporation and insulin

sensitization, it is thought that adiponectin is an endogenous anti-diabetic factor

(Kadowaki and Yamauchi, 2005). Moreover, it has been suggested that adiponectin is

associated with coronary artery disease, stroke, non-alcoholic steatohepatitis, and

several types of cancers (Lam and Xu, 2005; Trujillo and Scherer, 2005; Wang et al.,

2007). In some of these adiponectin-related diseases, dysfunction of the urinary system

is recognized as a complication. For example, alterations of urinary bladder smooth

muscle tissue are found in diabetes, hypertension, and hyperlipidemia. It has also been

reported that adiponectin affects vascular smooth muscle contraction (Ding et al., 2012).

Therefore, we hypothesized that blood adiponectin levels are associated with bladder

smooth muscle contractions. However, the association of adiponectin with urinary

systems has not been investigated. To understand this association, we hypothesized that

the role of adiponectin can be clearly defined under adiponectin-enhanced conditions. In

2006, we established an adiponectin-sense-transgenic (Adip-Sen) mouse (Saito et al.,

2006). We found that plasma adiponectin levels were significantly increased in the

Adip-Sen mouse (56.2%) compared with those of the wild type mouse, suggesting a

role of adiponectin in regulation of energy homeostasis.

This study aimed to determine if there is an association of adiponectin with bladder

smooth muscle contraction, which is an important function of the urinary system, using


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the Adip-Sen mouse. Mechanisms governing this association were also considered.


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Materials and Methods

Generation and maintenance of the transgenic mice. Male Adip-Sen mice

(16–24 weeks) and age-matched control (C57Bl/6J) mice were prepared and maintained

as described previously (Saito et al., 2006). Mice were housed at constant room

temperature (20 ± 2°C) with 12-h light and dark cycles. Mice were fed standard mouse

chow, which included 4.5% fat (Oriental Yeast Corp., Tokyo, Japan). Food and water

were available ad libitum and mice grew satisfactorily. Animals were used for

experiments at 16–24 weeks of age. This study was approved by the care and use of

laboratory animals of the Japanese Pharmacological Society.

Blood collection and plasma biochemical assays. Blood samples were obtained

from the inferior vena cava under ether anesthesia. The plasma supernatant was used for

the detection of plasma glucose, phospholipids (PL), free fatty acids (FFA),

triacylglycerol, and total cholesterol (total-Cho) levels in clinical laboratory tests

conducted by SRL Inc. (Tokyo, Japan).

Bladder smooth muscle tissue preparation. Mice were sacrificed by

over-treatment of ether, and decapitation and bloodletting were then performed. The

urinary bladder was isolated and the tissue was rinsed in physiological salt solution

(PSS). Subsequently, fat and connective tissues were removed from bladder tissue strips

using cotton and micro-scissors under stereoscopic microscopy. Urothelium was also

removed. PSS, which was supplemented with 118 mM NaCl, 5.8 mM KCl, 2.5 mM

CaCl2, 1.2 mM MgCl2, 1.4 mM NaH2PO4, 21.4 mM NaHCO3, and 11.1 mM glucose,

was aerated with 95% O2 and 5% CO2 at 37°C. Prior to measurements, the wet weight

of each tissue was determined.

Measurement of isometric force development and intracellular calcium


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concentration ([Ca2+]i). Each tissue was positioned vertically in a

temperature-controlled 5-mL organ bath. One end of the tissue was connected to a strain

gauge transducer (Type T-7-8-240, Orientec, Tokyo, Japan) to monitor contractile

responses. Measurements were made under normal PSS and calcium-free (no CaCl2

added to PSS: Ca2+-free PSS) conditions. Bladder contractions were normalized to the

cross-sectional area, as described previously (Nobe et al., 2009). Isometric force and

[Ca2+]i were simultaneously measured using

4-[3-[3-[2-[2-[[5-[(Acetoxymethoxy)carbonyl]oxazole]-2-yl]-6-[bis[2-(acetoxymethoxy

)-2-oxoethyl]amino]benzofuran-5-yloxy]ethoxy]-4-[bis[2-(acetoxymethoxy)-2-oxoethyl

]amino]phenyl]-1-oxopropyl]piperazine-1-acetic acid (Fura-PE3) acetoxymethyl ester

(fura-PE3/AM; TEF Lab, Inc., Austin, TX) as reported in our previous study (Nobe et

al., 2001).

Western blot analysis. Isolated fresh tissues were treated under various conditions

and then reactions were terminated by liquid nitrogen. Plasma membrane fractions for

western blot analysis were prepared as described previously (Nobe et al., 2008). Sodium

dodecyl sulfate polyacrylamide gel electrophoresis was performed according to the

method of Laemmli (Laemmli, 1970), using a 12% polyacrylamide gel. Detection of

each protein was performed in a similar manner as in our previous study (Nobe et al.,

2010) using primary antibodies at 1:1000 dilution (polyclonal, Abcam Co., Cambridge,

UK) followed by a horseradish peroxidase-conjugated secondary antibody (polyclonal

IgG, Santa Cruz Biotechnology Inc., CA).

Measurement of mRNA levels. Messenger RNA levels in each bladder tissue were

measured as reported in our previous study (Saito et al., 2006).

Data analysis. Values are presented as the mean ± SEM obtained from at least five


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animals. Statistical differences (p<0.01) for multiple comparisons were assessed with

one-way analysis of variance for repeated measurements followed by the

Student-Newman-Keuls test (Y-Stat Program; Igaku Tosyo Shuppan Co., Ltd., Tokyo,

Japan).


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Results

Basic characteristics of Adip-Sen mice. Plasma adiponectin levels were

significantly increased (56.2%) in Adip-Sen mice compared with those in C57Bl mice at

the age of 16–24 weeks, but body weight and the wet weight of bladder tissue were

similar between the groups (Table 1). Moreover, plasma glucose, total-Cho, PL, and

FFA levels were also similar between the groups.

Changes in carbachol-induced bladder force development in Ca2+-free PSS.

Resting levels of isometric force in C57Bl and Adip-Sen mice bladders were 0.96 ±

0.05 and 1.11 ± 0.04 mN/mm2 (n = 5), respectively. KCl (50 mM)-induced sustained

force development was also similar between the groups (data not shown). Cumulative

addition of 2-[(aminocarbonyl)oxy]-N,N,N-trimethylethanaminium chloride (carbachol;

CCh, Sigma-Aldrich, St. Louis, MO) induced significant increases in isometric force

under normal calcium conditions. During 0.3-1 µM CCh stimulation, force responses in

the Adip-Sen mouse were enhanced compared with those in the C57Bl mouse, but

increases in maximal response were not evident. Maximal force levels in C57Bl and

Adip-Sen mice in the presence of 30 µM CCh were 4.37 ± 0.27 and 4.35 ± 0.14

mN/mm2 (n = 5), respectively.

To demonstrate an association between extracellular Ca2+ concentration and bladder

contractility, force development was measured under Ca2+-free conditions (Fig. 1A and

1B). Pretreatment of C57Bl mice with Ca2+-free PSS for 10 min significantly reduced

the CCh-induced force development compared with the response in normal PSS, and

33.8% of the normal response remained. However, the CCh-induced response in

Ca2+-free PSS in Adip-Sen mice resulted in a suppression of this response. In the


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presence of 30 µM CCh, only 5.9% of the normal response was detected.

The relation between [Ca2+]i and force development was examined (Fig. 2). In

Fura-PE3-loaded tissue, changes in [Ca2+]i were expressed as R340/380. Resting and 50

mM KCl-treated R340/380 values were similar in C57Bl and Adip-Sen mice (data not

shown). Isometric force development elicited by CCh was simultaneously measured

(Fig. 1). In the C57Bl mouse, a relationship was evident between R340/380 and force

development. The slope of this relation was 1.04. In the Adip-Sen mouse, the

CCh-induced increase in force development was similar to the response in the C57Bl

mouse, but it was detected at a lower R340/380 compared with that in the C57Bl mouse.

Therefore, the slope of this relation in the Adip-Sen mouse was increased (2.57).

Effects of Rho kinase and protein kinase C (PKC) inhibitors on CCh-induced

force development. To determine the associations of Rho kinase and PKC with

CCh-induced bladder force development, we used (R)-(＋ )- trans-N-(4-pyridyl)-4

-(1-aminoethyl)-cyclohexanecarboxamide・2HCl (Y27632; Wako Pure Chemical Co.,

Osaka, Japan) (Uehata et al., 1997), an inhibitor of Rho kinase, and

12-(2-Cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo[2,3-a]pyrrolo[3,4-

c]carbazole (Gö6976; Sigma-Aldrich, St. Louis, MO) (Martiny-Baron et al., 1993), an

inhibitor of calcium-dependent PKC (Fig. 3). Pretreatment of the tissues with 1 µM

Y27632 slightly reduced CCh-induced responses without affecting resting levels (Fig.

3A), but significant differences (p<0.01) between Adip-Sen and C57Bl mice remained,

which were similar to the responses in the absence of Y27632. Pretreatment with 1 µM

Gö6976 also slightly reduced force development in the C57Bl mouse (Fig. 3B).

However, the inhibitory effect of Gö6976 in Adip-Sen mice was significantly increased

(p<0.01). In the presence of 1 µM Gö6976, 30 µM CCh-induced responses in C57Bl


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and Adip-Sen mice remained at 85.2% and 53.1% of the control response, respectively.

Changes in PKC isoforms in the Adip-Sen mouse bladder. To identify the

distribution of PKC isoforms in mouse bladder smooth muscle, the expression of each

isoform was assessed in C57Bl mice in the non-stimulated resting state (Fig. 4A). The

calcium-dependent PKC isoforms PKCα and PKCβ were detected, and

calcium-independent PKC isoforms, PKCμ and PKCθ were also observed. The effect of

PKCα and PKCβ expression on enhancement of adiponectin levels was assessed (Fig.

4B). In the Adip-Sen mouse, we observed a significant increase (119%; p<0.01) in

PKCα levels compared with those in the C57Bl mouse, but they were not affected by

addition of 30 µM CCh at 37°C for 5 min. Adiponectin levels and CCh treatment did

not alter PKCβ, PKCμ, and PKCθ levels in C57Bl and Adip-Sen mice (data not shown).

To confirm an increase in protein levels in the Adip-Sen mouse, PKCαmRNA levels

were also measured (Fig. 4C). Relative PKCα mRNA levels in C57Bl and Adip-Sen

mice were 0.011 ± 0.001 (n = 5) and 0.045 ± 0.003 (n = 5), respectively (p<0.01).

Significant increases in both PKCα protein and mRNA expression were confirmed in

the Adip-Sen mouse.

Activation of PKC involves auto-phosphorylation of PKC in many types of cells

(Stempka et al., 1999; Bayer et al., 2003). In the non-stimulated resting state,

phosphorylated-PKCα (p-PKCα) levels in C57Bl and Adip-Sen mice were not observed.

However, a 30 µM CCh-induced increase in p-PKCα levels was detected only in the

Adip-Sen mouse (Fig. 4D). This CCh-induced increase was 90.5% of resting levels (n =

5).


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Discussion

The current study found an association of adiponectin with bladder smooth muscle

contraction. This finding suggested that this association was involved in activation of

PKCα-mediated calcium dependency of bladder smooth muscle contraction.

To investigate the association between adiponectin and bladder smooth muscle

contraction, we developed the Adip-Sen mouse, because changes in bladder function(s)

by adiponectin might be detectable under conditions of chronically increased

adiponectin levels. In the Adip-Sen mouse, increases in adiponectin levels were

confirmed without affecting body weight and other blood parameters (Table 1). These

results are similar to our previous report (Saito et al., 2006). Based on these results, we

consider that changes in the Adip-Sen mouse were caused by chronically increased

adiponectin levels, and these changes were not due to secondary effects in these

transgenic mice.

Dose-response curves of CCh stimulation in Adip-Sen and C57Bl mice were

similar without 0.3-1 µM CCh stimulation (Fig. 1), but extracellular calcium

dependency was significantly enhanced only in the Adip-Sen mouse. This result

suggests that adiponectin affects bladder smooth muscle contraction, which is mediated

by an increase in calcium dependency. A relation between [Ca2+]i and isometric force

also supported this possibility (Fig. 2), because developed force levels in the Adip-Sen

mouse were significantly increased compared with those in the C57Bl mouse, when

CCh-induced [Ca2+]i levels were similar. This indicates an increase in

“calcium-sensitivity” of the Adip-Sen mouse bladder contraction. Interestingly, 50 mM

KCl induced h [Ca2+]i and force development, which were similar among C57Bl and

Adip-Sen mice (data not shown). These results indicate that enhancement of calcium


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dependency in Adip-Sen mice is involved in receptor-mediated signaling systems,

without involving enhancement of affinity between calcium and contractile proteins.

Intracellular calcium dependency of bladder contraction has been previously reported

(Ekman et al., 2009; Nobe et al., 2009). However, changes in calcium dependency and

its regulatory mechanism are not clearly understood. Therefore, we hypothesize that

adiponectin is a regulatory factor of calcium sensitivity of bladder contraction.

In smooth muscle contraction, the Rho-Rho kinase pathway is one of the major

signaling pathways. This pathway enhances calcium dependency mediated by inhibition

of myosin light chain (MLC) phosphatase and it induces accumulation of

phosphorylated MLC (Hirano et al., 2004). Moreover, a role for PKC has also been

proposed in smooth muscle contraction (Salamanca and Khalil, 2005). PKC enhances

MLC kinase and other intracellular contractile factors. Associations of these pathways

in bladder contraction have been previously reported (Yamaguchi, 2004; Durlu-Kandilci

and Brading, 2006), and our results are consistent with these previous reports (Fig. 3).

However, we found that inhibitory effects of Gö6976 were significantly enhanced only

in the Adip-Sen mouse (Fig. 3B). These results indicate that the contribution of the PKC

pathway in bladder contraction is enhanced in the Adip-Sen mouse. Therefore, we

speculate that adiponectin regulates calcium dependency, which is mediated by

activation of the PKC pathway.

We evaluated protein levels of PKC to examine the change in PKC with

adiponectin-mediated increases in calcium dependency. It is generally accepted that

PKC involves 10 or more isoforms, which involve calcium-dependent PKC (PKCα, β

and γ), calcium-independent PKC (PKCδ, μ and θ), and atypical PKC isoforms

(Salamanca and Khalil, 2005). In the current study, in bladder smooth muscle tissue,


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PKCα, β, μ and θ isoforms were detected (Fig. 4A). Among these isoforms, only PKCα

was significantly enhanced, which depended on plasma adiponectin levels (Fig. 4B).

These results indicate that calcium-dependent PKCα is chronically enhanced in the

Adip-Sen mouse. Our results suggested that adiponectin enhanced PKCα expression

and it increased calcium sensitivity of bladder contraction. Our findings of increased

PKCα mRNA levels support this suggestion (Fig. 4C). To confirm that an increase in

PKCα expression is associated with calcium dependency of Adip-Sen mouse bladder

contraction, an important step of PKCα phosphorylation was investigated. The

CCh-induced increase in p-PKCα levels was enhanced in the Adip-Sen mouse. Because

phosphorylation is essential for PKCα activation (Stempka et al., 1999), this suggests

that PKCα activity is enhanced in the Adip-Sen mouse. Both enhancement of PKCα

expression and over-activation of PKCα might contribute to the increase in calcium

sensitivity of bladder contraction. Myosin light chain phosphatase (MLCP) inhibitory

protein, CPI-17, which is a downstream signaling pathway of PKCα activation, might

play a role in bladder contraction. In many types of smooth muscle tissue, CPI-17 acts

as an effector of PKC, and phosphorylation of CPI-17 contributes to an enhancement of

the contraction mediated by inactivation of MLCP (Hirano, 2007). In our preliminary

trials, phosphorylated-CPI-17 levels in the Adip-Sen mouse bladder were enhanced

compared with those in the C57Bl mouse (unpublished data). Therefore, we considered

that adiponectin-mediated alteration of bladder contraction might involve the pathway

of PKCα and CPI-17.

The mechanisms involved in adiponectin-induced PKCα expression are not clearly

understood. The relation between adiponectin and PKCα is unknown. However, it has


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been reported that adiponectin stimulates the adiponectin-specific receptor (Adip-R)

(Yamauchi and Kadowaki, 2008). This receptor is distributed in many types of tissue

(Trujillo and Scherer, 2005) and we have also detected Adip-R mRNA in C57Bl mouse

bladder (data not shown). Stimulation of Adip-R increases cAMP and activates

cAMP-dependent protein kinase (A-kinase) (Ouchi et al., 2000). Activation of A-kinase

contributes to both glucose-incorporation and sensitization of insulin receptors

(Ouedraogo et al., 2006; Wu et al., 2007). Moreover, cAMP regulates some gene

expressions, which are mediated by cAMP-response element-binding protein (CREB)

(Paolillo et al., 1999; Cypess et al., 2011). An association of CREB with PKCα activity

has been suggested. Therefore, we speculate that adiponectin-induced PKCα expression

in bladder smooth muscle contraction is also mediated by cAMP and/or A-kinase

activation.

Based on our results, enhancement of force reduction in Ca2+-free PSS in Adip-Sen

mouse bladder can be interpreted as follows (Fig. 5). (1) Both Ca2+-dependent

(involving PKCα) and Ca2+-independent (nPKC and/or rho/ROCK) pathways were

involved in C57Bl mouse bladder contraction in normal PSS. These pathways

contribute to CCh-induced force development as a normal contractile response. (2) In

Ca2+-free PSS, part of the contraction mediated by the Ca2+-dependent pathway was

replaced in the C57Bl mouse bladder. Therefore, Ca2+-independent pathway-associated

contraction remained. (3) In Adip-Sen mouse bladder contraction, the developed force

level was similar to the response in the C57Bl mouse. However, the contribution ratio of

Ca2+-dependent/Ca2+-independent pathways in the Adip-Sen mouse was different from

the ratio in the C57Bl mouse. In Adip-Sen mouse bladder contraction, contribution of

the Ca2+-dependent pathway was significantly enhanced by enhancement of PKCα


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expression. (4) Similar to the response in the C57Bl mouse bladder, part of the

Ca2+-dependent contraction in the Adip-Sen mouse bladder was suppressed in Ca2+-free

PSS. Because a major part of the contraction was suppressed, the total force level in the

Adip-Sen mouse was significantly reduced compared with that in the C57Bl mouse. We

consider that the PKCα-mediated Ca2+-dependent pathway plays a major role in

changes in Adip-Sen mouse bladder contraction, but the association of the

Ca2+-independent pathway with these changes is unknown.

Adiponectin plays a role as a regulatory factor of bladder contraction. This involves

enhancement of calcium sensitivity of the contraction, mediated by both expression and

activation of PKCα.


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Acknowledgment

We thank Mr. S. Imawaka for technical support.

Authorship Contributions

Participated in research design: Nobe, Ogawa, and Honda.

Conducted experiments: Nobe and Negoro.

Contributed new reagents or analytic tools: Fujii and Saito.

Performed data analysis: Fujii and Hashimoto.

Wrote or contributed to the writing of the manuscript: Nobe, Nakano, and Honda.

Conflict of interest statement: None.


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Footnotes

This study was supported by a Grant-in-Aid for Encouragement of Young Scientists

from the Ministry of Education, Culture, Sports, Science and Technology (MEXT;

21590290) in Japan and a Private University High Technology Research Center Project

matching fund subsidy from MEXT (NEXT; S1001011).


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Figure Legends

Fig. 1. Effects of carbachol (CCh) on isometric force under normal and Ca2+-free

conditions in the bladder of C57Bl and Adip-Sen mice. CCh-induced changes in

isometric force (mN/mm2) were measured as described in the Methods. Typical changes

observed in bladder preparations isolated from C57Bl (A) and Adip-Sen (B) mice.

Bladder tissues were pre-incubated in normal PSS (left panel) and Ca2+-free PSS (right

panel) for 10 min, and then the indicated contractions of CCh were introduced.

Concentration-response relationships for CCh-induced isometric force responses in the

bladder of C57Bl (open symbols) and Adip-Sen (closed symbols) mice under normal

(circles) and Ca2+-free (squares) conditions are indicated (C). Each value represents the

mean ± SEM of at least five independent determinations. *p<0.01 and #p<0.01

compared with the values in the C57Bl mouse and responses in normal PSS,

respectively.

Fig. 2. Changes in calcium sensitivity in CCh-induced bladder contraction in C57Bl and

Adip-Sen mice bladders. Isolated bladder tissues were pre-incubated with

Fura-PE3/AM (5 µM), containing PSS at room temperature for 90 min. Relative

fluorescence intensities (R340/380) were measured as a parameter of intracellular calcium

concentration ([Ca2+]i). Isometric force development was simultaneously measured. The

relation between R340/380 and isometric force development in C57Bl (open circles) and

Adip-Sen (closed circles) mice were plotted as % maximal response in the C57Bl

mouse.


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Fig. 3. Effect of Rho kinase (A) and PKC (B) inhibitors on CCh-induced isometric force

in the bladder of C57Bl (open symbols) and Adip-Sen (closed symbols) mice.

CCh-induced changes in isometric force (mN/mm2) were measured as described in Fig.

1. Bladder tissues were pre-incubated in the presence (squares) or absence (circles) of 1

µM Y27632 or 1 µM Gö6976 for 10 min, and subsequently, the indicated contractions

of CCh were introduced. Each value represents the mean ± SEM of at least five

independent determinations. *p<0.01 and #p<0.01 compared with values in the C57Bl

mouse and the response in the absence of inhibitors, respectively.

Fig. 4. Change in PKC expression in C57Bl and Adip-Sen mice bladders. Distribution

of PKC isoforms (αβ��γ δμ and θ) in non-stimulated C57Bl mouse bladders were

measured as described in the Methods (A). The rat brain was used as a positive control

(PC). Expression levels of PKCα and PKCβII in C57Bl and Adip-Sen mice were

assessed in the presence (+) and absence (-) of 30 µM CCh at 37°C for 5 min. Western

blot images (B-upper panels) and the ratio of PKC/β-actin levels are shown (B-lower

panel). Relative expression levels of PKCα mRNA (C) and phosphorylated-PKCα

(p-PKCα�D) levels were measured as described in the Methods. A typical image of

p-PKCα levels is shown (inset; 1: un-stimulated C57Bl mouse, 2: CCh-treated C57Bl

mouse, 3: un-stimulated Adip-Sen mouse, 4: CCh-treated Adip-Sen mouse). Each value

represents the mean ± SEM of at least five independent determinations. *p<0.01 and

#p<0.01 compared with values in the C57Bl mouse and PKCα levels, respectively.

Fig. 5. Adip-dependent alteration of CCh-induced bladder contraction in Ca2+-free PSS


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in the Adip-Sen mouse.


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Table 1. Basic characteristics of C57Bl and Adip-Sen mice

Mice n Body weight Adiponectin Glucose t-Cho PL FFA Bladder Wt

(g) (µg/dL) (mg/dL) (mg/dL) (mg/dL) (µEQ/L) (mg)

C57Bl 5 28.5 ± 1.04 20.1 ± 0.55 86.7 ± 2.76 104.7 ± 5.63 206.2 ± 9.47 1005.4 ± 61.4 19.7 ± 1.03

Adip-Sen 5 27.6 ± 2.15 31.4 ± 0.61* 89.4 ± 3.07 94.2 ± 5.97 205.8 ± 7.30 1041.6 ± 70.1 20.2 ± 1.46

* p<0.01 vs. values in C57Bl mice.


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0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

0.0 0.1 1 10 100

0.1 0.3 1 3 10 30 100

CCh (µM)

1 min

2 m

N/m

m2

0.1 0.3 1 3 10 30 100

CCh (µM)

Ca2+-free PSS

B Adip-Sen mouse

C Dose-response curves

CCh (µM)

Isom

etri

c F

orce

(m

N/m

m2 )

＊#＊#

##

#

##

## #

＊#

＊

＊

A C57Bl mouse

0.1 0.3 1 3 10 30 100

CCh (µM)

1 min2

mN

/mm

2

0.1 0.3 1 3 10 30 100

CCh (µM)

Ca2+-free PSS

Figure 1This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on January 30, 2013 as DOI: 10.1124/jpet.112.202028

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c F

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R340/380 (% of control)

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120

0 20 40 60 80 100

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0.5

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＊

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Y27632

Gö6976


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PKCα

PKC

PKCβII PKCγ

PC C57Bl PC C57Bl PC C57Bl

PKCδ PKCμ PKCθ

PC C57Bl PC C57Bl PC C57Bl

A

B C57Bl Adip-Sen

+-30 µMCCh +-

0

1

2

3

4

5

6

30 µM CCh - +

PKCαPKCβ

Rat

io (P

KC

/β-a

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) **

##

C57Bl Adip-Sen- +

C

D

0

2

4

6

8

10

C57Bl Adip-Sen

Rat

io (p

-PK

Cα/

β-ac

tin)

Resting30 µM CCh

0

0.01

0.02

0.03

0.04

0.05

0.06

C57Bl Adip-Sen

Rel

ativ

e E

xpre

ssio

ns

＊

＊

p<0.011 2 3 4

PKC

PKCα

PKCβΙΙ


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Ca2+-independent Ca2+-dependentC57Bl

Adip-Sen

Ca2+-free +

Adip-Sen

CCh-induced force developments

Ca2+-independent Ca2+-dependent

Ca2+-independent

Ca2+-independentCa2+-free

+C57Bl

(2)

(1)

(3)

(4)


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