2010 THE AUTHORS. JOURNAL COMPILATION
Investigative Urology
2010 BJU INTERNATIONAL
c-KIT-POSITIVE ICCS AND BLADDER CONTRACTIONS IN DIABETIC RATS
VAHABI
ET AL.
BJUI
The role of c-kit-positive interstitial cells in
mediating phasic contractions of bladder strips
from streptozotocin-induced diabetic rats
BJU INTERNATIONAL
Bahareh Vahabi, Neil G. McKay, Kim Lawson and Donna J. Sellers*
Biomedical Sciences, Owen Building, Sheffield Hallam University, Sheffield, UK, and *Health Sciences & Medicine,
Bond University, Queensland, Australia
Accepted for publication 1 April 2010
OBJECTIVE
• To investigate the role of c-kit-positive
interstitial cells (ICCs) in mediating
muscarinic receptor-induced phasic
contractions of isolated bladder strips from
streptozotocin(STZ)-induced diabetic rats
and to confirm the expression and location
of ICCs in the rat bladder.
MATERIALS AND METHODS
• Bladders were removed from STZ-induced
diabetic rats at 1, 4 and 12 weeks after
induction of diabetes and from age-matched
controls.
• To investigate the functional role of ICCs
in mediating phasic contractions, bladder
strips were isolated from control and
diabetic rats and mounted in tissue baths.
• Strips were stimulated with low
concentrations of the muscarinic receptor
agonist carbachol (CCH; 0.1 μM) to induce
phasic contractions and the effect of
increasing concentrations (1–50 μM) of
imatinib (Glivec® or Gleevec®, formerly
STI571), a c-kit tyrosine kinase inhibitor, was
then investigated.
• For molecular studies, to detect expression
of the c-kit tyrosine kinase receptor (c-kit),
total cellular RNA was extracted from rat
bladders and reverse-transcribed to obtain
complementary DNA (cDNA).
• Reverse transcription-polymerase chain
reaction (RT-PCR) was then performed using
primers specific to the c-kit sequence and
amplified products separated by agarose gel
electrophoresis.
INTRODUCTION
In recent years, it has become apparent
that bladder function is much more complex
1480
What’s known on the subject? and What does the study add?
Although the functional significance of ICCs in the bladder is still not clear, they have been
linked with detrusor overactivity. In this study we demonstrate the presence of c-kit
positive interstitial cells in rat urinary bladder for the first time and their importance in
mediating muscarinic receptor-induced phasic contractions of bladder strips from control
and diabetic rats. The role of these cells does not seem to be significantly altered by the
diabetic state.
• Amplified PCR products were excised from
the gel, sequenced and compared with the
known c-kit sequence to confirm their
identity.
• For immunohistochemical detection,
whole mount preparations of control rat
bladders were fixed in acetone and labelled
using antibodies directed to the ICC marker
c-kit.
RESULTS
• In functional studies, CCH induced phasic
contractions in bladder strips from control
and diabetic rats. Bladder strips from 1-week
diabetic rats showed CCH-induced phasic
contractions, which were greater in
amplitude, but had lower frequency, than the
controls, whilst no such differences were
apparent at later time points of diabetes.
• Imatinib decreased the amplitude and the
frequency of the CCH-induced phasic
contractions in both control and diabetic
tissues in a concentration-dependent
manner, although in diabetic tissues this
effect was only seen at the higher
concentrations of imatinib. RT-PCR of
bladder cDNA yielded a single amplicon of
480 bp.
than originally thought. During filling, the
bladder develops phasic or autonomous
activity consisting of rhythmical transient
contractions, similar to peristaltic movements
• The sequence of this amplicon showed a
98% homology with the published c-kit
sequence, thus confirming c-kit mRNA
expression in both control and 1-week
diabetic rat bladder.
• Expression of c-kit protein was also
detected in a network of cells on the edge of
and between smooth muscle bundles of
control rat bladders by positive
immunoreactivity to c-kit specific
antibodies.
CONCLUSION
• These data show the presence of
c-kit-positive ICCs in rat urinary bladder
and their importance in mediating
muscarinic receptor-induced phasic
contractions of bladder strips from control
and diabetic rats. The role of these ICCs does
not seem to be significantly altered by the
diabetic state.
KEYWORDS
interstitial cells, c-kit, rat bladder,
streptozotocin, diabetes, imatinib
of the gastrointestinal tract [1]. This
phasic activity occurs in the absence
of neural stimulation, but can be
modulated by activation of various
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2 0 1 0 B J U I N T E R N A T I O N A L | 1 0 7 , 1 4 8 0 – 1 4 8 7 | doi:10.1111/j.1464-410X.2010.09507.x
c-KIT-POSITIVE ICCS AND BLADDER CONTRACTIONS IN DIABETIC RATS
receptors including muscarinic and purinergic
receptors [2,3].
The physiological relevance of this phasic
activity is not yet clear, but phasic
contractions may play a key role in bladder
function, mediating bladder tone and relaying
sensory afferent information from the
detrusor. Recently, a novel population of cells
has been found in the bladder wall and linked
to phasic activity [4,5]. These cells resemble
the interstitial cells (ICCs) of the
gastrointestinal tract, which are known to act
as pacemakers and drive peristalsis, as well as
play a role in processing sensory information
[6]. Various terms have been used to describe
these cells, including myofibroblasts,
interstitial cells (ICs), interstitial cells of
Cajal-like cells and pacemaking cells, but a
recent consensus was reached to term them
‘ICCs’ (Fifth International symposium on ICC,
2007).
ICCs were first reported in the bladder
by Smet et al. [7] who showed cGMPimmunopositive cells in both guinea-pig
and human bladder, which had similar
morphology to ICCs in the gastrointestinal
tract. Further studies showed these cells to be
vimentin-positive and positive for the protooncogene that encodes the tyrosine kinase
receptor Kit, which is expressed by ICCs but
not by smooth muscle or fibroblast cells. Thus,
c-Kit has become an established ICC marker
and ICCs have been observed in the
interstitium, amongst smooth muscle cells
and between smooth muscle bundles of the
detrusor [4,5,8,9]. Although ICCs have been
demonstrated in the human, mouse and
guinea-pig bladder, no studies have so far
reported these cells in rat urinary bladder.
The functional significance of ICCs in the
bladder is not clear. The observation that
these cells are immunopositive for connexion
43 and vanilloid receptor-like protein 1
(VRL1), along with the previously reported
immunopositivity for cGMP and their close
proximity to nitrergic and peptidergic nerve
fibres, suggests a functional role for these
cells [7,10,11]. It has been suggested that ICCs
may function as a sensing network, receiving/
sending signals from/to the urothelium,
modulating afferent bladder innervation and/
or activating a spinal or intramural reflex arc
[12]. It has also been shown that ICCs in the
bladder are capable of generating Ca2+
transients [13], but do not have an obvious
role in pacemaking. Instead, these cells might
©
mediate the communication between
subgroups of smooth-muscle cells in the
bladder and thus might modulate phasic
activity and micromotions of the bladder wall
[13]. Imatinib mesylate (Glivec® or Gleevec®,
formerly STI571), normally used in the
treatment of Philadelphia chromosomepositive chronic myeloid leukaemia and c-kitpositive gastrointestinal stromal tumours,
pharmacologically inhibits the tyrosine kinase
activity of c-kit [14] and has been used
extensively for studying the role of ICCs in
generation of spontaneous electrical activity
of various smooth muscle organs such as the
uterus, intestine, myometrium and bladder
[15–18].
As it has been speculated that ICCs are
involved in generation of phasic contractions,
changes in the properties of ICCs may
therefore be important in mediating the
increased phasic contractions seen in
detrusor overactivity (DO) and animal models
of bladder dysfunction [19–21]. This is
supported by studies showing an increased
number of ICCs in the bladder of patients with
idiopathic and neurogenic DO [15], while, in a
BOO guinea-pig model of DO, it has been
shown that ICCs are more widely distributed
in the suburothelial layers than in normal
guinea-pigs [22]. In addition, De Jongh et al.
[23] have shown that cGMP-positive cells on
the outer surface of the bladder were
increased in the obstructed guinea-pig
bladder compared with the normal bladder,
and suggested that these changes in the
network of ICCs may play a role in the altered
functioning and remodelling of the
obstructed bladder, rendering the underlying
mechanisms more sensitive to excitatory
inputs and subsequently increasing the
afferent discharge and phasic contractions. In
contrast, Roosen et al. [24] have shown that
such an up-regulation of ICC expression or cKit immunoreactivity is not seen in
neurogenic or idiopathic DO in humans. Thus,
it is still unclear whether changes in ICCs are
linked to bladder dysfunction.
Since in the streptozotocin (STZ)-induced
diabetic rat, which has commonly been used
as a model of bladder dysfunction, we have
previously shown enhanced phasic activity of
the bladder [21], it was postulated that ICCs
may play a role in regulation of bladder
contractility in this model, and that their
function may be changed by the diabetic
state. Thus, the aim of the present study was
to investigate the role of c-kit-positive ICCs in
mediating muscarinic receptor-induced
phasic contractions of isolated bladder strips
from STZ-induced diabetic rats and confirm
the expression and location of these cells in
the rat bladder.
MATERIALS AND METHODS
Male Wistar-Hannover rats (Charles River,
UK), ≈ 250 g body weight, were used in this
study. Diabetes was induced by a single i.p.
injection of STZ (65 mg/kg body weight;
dissolved in 0.01 M citrate buffer, pH 4.5).
After induction of diabetes, the rats were kept
for a period of 1, 4 or 12 weeks, with free
access to food and water. At the end of the
experimental period, the rats were killed
humanely and rats with blood glucose levels
of >15 mM were considered diabetic. Control
rats were age-matched. All procedures were
performed in accordance with UK Home
Office Project and Personal licences.
FUNCTIONAL STUDIES
For investigation of the functional role of ICCs
in mediating the phasic activity of bladder, the
bladder was removed from 1, 4 and 12-week
diabetic rats and their age-matched controls.
A longitudinal incision was made through the
bladder from the base to the dome and the
bladder was opened up to form a flat sheet.
Using a razor blade, the base and the top of
the dome were removed and 3–4 longitudinal
strips (depending on bladder size) measuring
2–4 × 6–12 mm were then cut from the
bladder body. Tissues were suspended in
15 mL organ baths containing Krebsbicarbonate solution (in mM: 118.3 NaCl, 11.7
D-Glucose, 24.9 NaHCO3, 4.7 KCl, 1.15
MgSO4, 1.15 KH2PO4 and 1.9 CaCl2)
including 5 μM indomethacin (a
cyclooxygenase inhibitor), maintained at
37 °C and gassed with 95% O2 and 5% CO2.
The tension developed by the tissues was
measured using isometric force transducers
(Pioden Controls Ltd, UK) connected to a
Powerlab data acquisition system using
‘Chart’ software (AD Instruments, UK). At the
start of the experiments, the strips were
placed under 2 g tension and left to
equilibrate for 60 min. After equilibration,
tissues were stimulated with 0.1 μM carbachol
(CCH) to induce phasic activity, as previously
described by Ng et al. [2]. In the continuous
presence of CCH, increasing cumulative
concentrations of imatinib (1–50 μM; stock
solution dissolved in water, with subsequent
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V A H A B I ET AL.
dilutions in Krebs–bicarbonate buffer) were
added to the tissues and the effects of this
drug on the amplitude and the frequency of
phasic activity was assessed. The tissues were
incubated with each concentration of
imatinib for 10–15 min.
To calculate the amplitude and the frequency
of phasic activity, a slightly modified method
to that proposed by Imai et al. [25] was used
to define a single phasic contraction event.
Firstly, the maximum amplitude of contractile
activity over a 5-min period was calculated in
control tissues. Any contractions over and
above 30% of this mean were considered as
single phasic contractions and counted for
calculation of the frequency. The same 30%
threshold was also used to determine the
frequency of phasic activity in diabetic
tissues. The mean contraction amplitude/mg
tissue and the frequency during a 5-min
period within a 10–15 min interval were
calculated and data expressed as the mean ±
SEM. The percentage change in the amplitude
and the frequency of phasic activity at each
concentration of imatinib relative to that in
the absence of imatinib, i.e. in the presence of
0.1 μM CCH only, was calculated for both
control and diabetic tissues.
For statistical analyses repeated measures
ANOVA followed by Dunnett’s post hoc test for
intra-tissue variations and unpaired Student’s
t-test for inter-tissue variations were used; P
< 0.05 was considered to indicate statistical
significance.
mixture was incubated at 50 °C for 60 min,
followed by 10 min at 70 °C to inactivate the
reaction. PCR reactions were performed in a
volume of 25 μL with 22.5 μL of PCR master
mix (1.1x; Abgene, UK), 0.2 μM of primers and
0.01 μg of cDNA. Each control and diabetic
cDNA sample was first amplified using
glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) forward (5′ ACCCAGAAGACTGTG
GATGG 3′) and reverse (5′ CACATTGGGGG
TAGGAACAC 3′) primers to confirm successful
reverse transcription (positive control). Sterile
water was included instead of the cDNA
template as a negative control reaction. After
confirmation of the presence of amplifiable
cDNA, samples were amplified using primers
specific to c-kit, forward (5′ GGCCTAGCCAG
AGACATCAG 3′) and reverse (5′ GAGAGGCTGT
GTGGAAGAGG 3′). All primers were supplied
by Invitrogen, UK.
The amplification conditions consisted of
denaturation at 95 °C for 5 min, followed by
35 cycles of 95 °C for 15 s, 59 °C for 1 min
and polymerization at 72 °C for 1 min. The
PCR products were then separated by agarose
gel electrophoresis to confirm successful
amplification and then purified from the
gel using a QIAquick Gel (QIAGEN, Crawley,
UK) extraction kit and sent to GATC-Biotech
(Konstanz) for direct sequencing to confirm
the identity of the amplified products. The
sequenced data was then verified by
comparison with the genome database
using BLAST from the NCBI website (http://
blast.ncbi.nlm.nih.gov/blast.cgi).
MOLECULAR STUDIES
IMMUNOHISTOCHEMISTRY
For investigation of c-kit tyrosine kinase
receptor mRNA expression, urinary bladders
were removed from control (n = 5) and 1week diabetic (n = 5) rats. The bladders were
opened longitudinally and the mucosa was
carefully dissected away from the underlying
tissue. Subsequently total cytoplasmic RNA
was isolated using a Ribo PureTM kit (Ambion,
Warrington, UK) according to the protocol
provided by the manufacturer, including the
removal of trace genomic DNA. RNA integrity
and concentration were analysed using an
Experion automated electrophoresis system
(Bio-Rad laboratories, Hemel Hempstead, UK).
First-strand complementary DNA (cDNA) was
prepared from RNA by reverse transcription
(RT). RT reactions contained; RNA (325 ng/μL),
oligo dT18 primers (0.04 mg/mL), dNTPs
(0.5 mM) and superscript III enzyme (4 units/
μL) (Invitrogen, UK) in a volume of 100 μL. The
1482
Immunohistochemistry to identify c-kit
protein expression in the rat urinary bladder
was performed using a modified version of
the method described by McCloskey and
Gurney [8]. Whole mount preparations of
bladders from control rats were prepared. The
bladders were cut into two longitudinal strips.
The top and the bottom of each longitudinal
strip were removed and the mucosa was
carefully dissected away using curved
scissors. Bladder preparations were fixed in
ice-cold acetone for 10 min and then washed
three times for 5 min at room temperature in
PBS (0.1 M). Tissues were then blocked for 1 h
in 0.1 M PBS with 1% (v/v) donkey serum
(Sigma, Dorset, UK) before incubation with
1 : 200 anti c-kit goat polyclonal IgG primary
antibody (C-19; Santa-Cruz, Heidelberg,
Germany) diluted in PBS containing 1%
donkey serum for 1 h at room temperature.
For negative controls, tissues were incubated
in PBS containing 1% donkey serum only.
After incubation with the primary antibody or
under control conditions, tissues were washed
three times for 10 min with PBS, and then
incubated with the secondary antibody,
donkey anti-goat IgG fluorescent antibody
(Alexa 488; Invitrogen, Paisely, UK) diluted
(1 μg/mL) in PBS containing 5% donkey
serum and 0.05% (v/v) Tween, for 1.5 h at
room temperature. After three washes for
5 min in PBS, tissues were mounted on Lpolylysine-coated slides using aqueous
mounting media (70% glycerol/30% PBS).
Images were captured using a Zeiss 510
confocal scanning laser microscope equipped
with krypton/argon laser as the source of the
ion beam. Alexa 488-labelled antibodies were
visualized by excitation at 488 nm with a
506–538 band-pass emission filter. Acquiring
10–20 optical sections in the Z-plane and
running the series-scanning mode from the
deepest focus point to the highest focus
point, allowed a projected image and data set
to be generated from which composite
projected images were saved. Images were
analysed using Zeiss LSM5 software.
RESULTS
FUNCTIONAL INVESTIGATION OF THE ROLE OF
ICCs IN MEDIATING MUSCARINIC RECEPTORINDUCED PHASIC ACTIVITY OF DETRUSOR
STRIPS FROM CONTROL AND DIABETIC RATS
CCH (0.1 μM) induced phasic contractions in
bladder strips from control and diabetic rats
(Fig. 1). The amplitude of phasic contractions
in 1-week diabetic tissues was significantly
(P < 0.05) greater than in the aged-matched
control tissues (Fig. 2A). However, there was
no significant difference in the amplitude
of CCH-stimulated phasic activity of the
detrusor strips from 4-week and 12-week
diabetic rats vs their aged-matched control
tissues (Fig. 2A). In contrast to amplitude, the
frequency of phasic activity in 1-week
diabetic tissues was significantly (P < 0.001)
less than in the age-matched control tissues
(Fig. 2B). However, there was no significant
difference between 4-week and 12-week
diabetic tissues vs their aged-matched control
tissues (Fig. 2B).
Imatinib inhibited the amplitude and
frequency of CCH-induced phasic activity in a
concentration-dependent manner in both
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c-KIT-POSITIVE ICCS AND BLADDER CONTRACTIONS IN DIABETIC RATS
FIG. 1. Typical tension recordings showing the effect of increasing concentrations of imatinib, and vehicle
(water), on CCH-induced phasic activity in detrusor strips from representative control (C) and diabetic (D)
rats.
4
1μM Imatinib 5μM Imatinib
3
g
10μM Imatinib
50μM Imatinib
0.1μM CCH
2
1
C
0
0.1μM CCH
1μM Imatinib
5μM Imatinib
10μM Imatinib
50μM Imatinib
4
3
g2
1
1-week D
0
0.1μM CCH 1μM Imatinib
4
g
5μM Imatinib
10μM Imatinib
3
50μM Imatinib
2
1
4-week D
0
1μM Imatinib
0.1μM CCH
4
5μM Imatinib
10μM Imatinib
IDENTIFICATION OF C-KIT GENE EXPRESSION
IN CONTROL AND DIABETIC RAT BLADDERS
50μM Imatinib
3
g
2
1
12-week D
0
H2O
4 0.1μM CCH
g
H2O
H2O
H2O
3
2
1
0
10 min
Vehicle control
FIG. 2. The amplitude and frequency of phasic activity in detrusor strips from diabetic (D) and age-matched
control (C) rats upon stimulation with 0.1 μM CCH. *P < 0.05, ***P < 0.001 vs the relevant age-matched control
groups (unpaired t-test). Data is presented as the mean ± SEM (n = 8–17).
A
Frequency, events in 5min
0.10
0.05
70
60
50
40
***
30
20
10
12-week D
12-week C
4-week D
4-week C
1-week D
1-week C
12-week D
12-week C
4-week D
4-week C
0
1-week D
0.00
1-week C
Amplitude, g/mg tissue
*
The presence of cDNA in control and diabetic
bladders was initially confirmed by
amplification of GAPDH and observation of a
band at the expected product size of ≈ 170 bp
(Fig. 4). Subsequently, RT-PCR showed the
expression of c-kit in cDNA samples from
both control and 1-week diabetic rat bladders,
at the expected product size of 480 bp (Fig. 4).
Direct sequencing of the c-kit amplicons
yielded partial sequences, 272 bp for 1-week
diabetic and 113 bp for control samples. These
sequences showed 98% homology with
R.rattus mRNA for c-kit receptor tyrosine
kinase isoform (accession no: D12524.1)
confirming the expression of c-kit tyrosine
receptor kinase in normal and diabetic rat
urinary bladder.
IDENTIFICATION OF C-KIT PROTEIN
EXPRESSION IN RAT URINARY BLADDER
B
0.15
©
control and diabetic tissues (Fig. 3). The effect
of vehicle (time control) was not significant
for any tissue group (Fig. 1). Imatinib
significantly inhibited the amplitude of phasic
contractions in detrusor strips from 1-week
diabetic and aged-matched control rats
at all concentrations (Fig. 3). Imatinib also
significantly inhibited the frequency of phasic
activity in control tissues at all concentrations
(Fig. 3). However, in 1-week diabetic rats the
frequency of phasic activity was significantly
inhibited only at higher concentrations of
imatinib (10 μM and 50 μM; Fig. 3B). The
inhibitory effect of imatinib on the amplitude
and the frequency of phasic contractions also
differed between 4-week diabetic tissues and
their age-matched controls (Fig. 3C,D) and
12-week diabetic tissues and their agematched controls (Fig. 3E,F). The amplitude
and frequency of phasic activity in the 4- and
12-week diabetic rats were significantly
decreased only at the higher concentrations
of imatinib as compared with the respective
age-matched controls (Fig. 3C–F).
Positive immunoreactivity to an anti-c-kit
polyclonal antibody showed a network of ckit-positive cells in the rat urinary bladder.
Figure 5 shows this immunostaining for c-kit
on a network of connected cells on the edge
of, as well as between smooth muscle bundles
in the rat urinary bladder. These c-kit-positive
cells had elongated cell bodies and extended
fine processes consistent with the
morphology reported for c-kit-positive ICCs in
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FIG. 3. Effect of increasing concentrations of imatinib on the amplitude and frequency of phasic activity
induced by 0.1 μM CCH in 1-week (A,B), 4-week (C,D) and 12-week (E,F) control (C) and diabetic (D) tissues.
*P < 0.05, **P < 0.01 vs 0 μM imatinib. Data is presented as the mean ± SEM (n = 8–17).
DISCUSSION
A
The presence of ICCs in bladder is well
documented. In the guinea-pig, ICCs are
found throughout the muscle bundles and
within the interstitium of the bladder, and
particularly in the outer fibromuscular coat
[7]. In addition, ICCs have been located on the
boundary of smooth muscle bundles in the
guinea-pig bladder, running axial with
smooth muscle cells and positioned in close
proximity to intramural nerves, particularly to
neuronal nitric oxide synthase-containing
neurones [8]. Hashitani et al. [13] reported
three subpopulations of these cells in the
guinea-pig urinary bladder: adjacent to the
boundary of muscle bundles, scattered among
smooth muscle cells within muscle bundles
and in connective tissues between smooth
muscle bundles; whilst Gillespie et al.
[4,5] also reported cGMP-positive ICCs in
guinea-pig urinary bladder immediately
1484
0.050
**
**
**
**
**
0.025
0
1
5x 0 –6
10 –
6
1
5x 0 –5
10 –
5
0
1
5x 0 –6
10 –
6
10 –
5x 5
10 –
5
0.000
** **
**
**
*
**
0
1
5x 0 –6
10 –
6
10 –
5x 5
10 –
5
* **
1-Week D
5
*
0.075
1-Week C
70
60
50
40
30
20
10
0
0
1
5x 0 –6
10 –
6
10
5x –5
10 –
0.100
Frequency, events in 5 min
1-Week D
Concentration of Imatinib
D
0.075
0.050
** **
**
**
0.025
**
5
0
1
5x 0 –6
10 –
6
10 –
5x 5
10 –
0
1
5x 0 –6
10 –
6
10 –
5x 5
10 –
5
0.000
**
**
4-Week D
**
**
0
1
5x 0 –6
10 –
6
10 –
5
5x
10 –
5
0.100
4-Week C
70
60
50
40
30
20
10
0
5
0.125
4-Week D
0
1
5x 0 –6
10 –
6
10 –
5x 5
10 –
4-Week C
Frequency, events in 5 min
C
Concentration of Imatinib
Concentration of Imatinib
F
12-Week D
0.100
0.075
0.050
** **
0.025
**
**
0
1
5x 0 –6
10 –
6
10 –
5x 5
10 –
5
5
0
1
5x 0 –6
10 –
6
10 –
5x 5
10 –
0.000
12-Week C
70
60
50
40
30
20
10
0
12-Week D
**
**
**
0
1
5x 0 –6
10 –
6
10 –
5x 5
10 –
5
0.125
12-Week C
Frequency, events in 5 min
E
0
1
5x 0 –6
10 –
6
10 –
5x 5
10 –
5
Amplitude, g/mg tissue
In the present study, we investigated CCHinduced phasic contractions of the rat bladder
and the role of ICCs. Phasic contractions were
induced by low concentrations (0.1 μM) of the
muscarinic agonist CCH in strips of bladder
from control and diabetic rats. As both
development and maintenance of ICCs
require the tyrosine kinase receptor c-kit,
imatinib, a c-kit tyrosine kinase inhibitor, was
used to investigate a role for these cells in
mediating this phasic activity, as shown
previously in guinea-pig and human bladders
[15]. In the present study, imatinib reduced
both the amplitude and the frequency of
CCH-induced phasic contractions in control
and diabetic tissues in a concentrationdependent manner, confirming a role for ckit-positive ICCs in mediating phasic activity
in rat bladder.
B
1-Week C
0.125
Concentration of Imatinib
Amplitude, g/mg tissue
The role of ICCs in the bladder is not clear,
although they may play a significant role in
the generation and modulation of bladder
sensation and contractile function [1,26].
Recently ICCs have been linked to the
cholinergic regulation of phasic activity and it
has been postulated that in overactive bladder
enhanced spontaneous contractions may be
caused by leakage of small amounts of
acetylcholine from intramural nerves acting
partly on muscarinic receptors expressed on
ICCs [2,27].
Amplitude, g/mg tissue
other tissues (Fig. 5A–C). There was no
immunostaining for c-kit in the negative
control (minus the primary antibody) (Fig. 5D).
Concentration of Imatinib
Concentration of Imatinib
FIG. 4. c-DNA samples from typical control and 1-week diabetic rat bladders amplified with primers specific
to c-kit. The gel shows amplified products of c-kit and GAPDH. Lane 1: 100 bp ladder. Lane 2: Positive control
amplified with primers to GAPDH (≈ 170 bp). Lane 3: negative control (water). Lane 4: 1-week diabetic rat
bladder sample amplified with primers to c-kit (≈ 480 bp). Lane 5: Control rat bladder sample amplified with
primers to c-kit.
1
500 bp
400 bp
200 bp
2
3
4
5
GAPDH
c-kit
100 bp
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FIG. 5. Detection of c-kit positive cells in rat bladder. Panel A: morphology of c-kit positive cells with elongated
cell bodies and several slender processes. Panels B, C: c-kit positive cells can be seen in a network on the edge
of (#) and between smooth muscle bundles (*). D: negative control (minus primary antibody).
B
A
B
#
10 μm
C
20 μm
D
*
*
20 μm
20 μm
beneath the urothelium and on the outer
muscle layers. In human bladder, a network
of ICCs is localized beneath the urothelium
and between smooth muscle cells of the
bladder [10], whilst Sui et al. [28] reported
a population of these cells beneath the
urothelium forming a functional syncytium.
ICCs have also been reported in mouse
bladder, in the outer part of the detrusor
muscle layer and between the muscle
bundles but not in the suburothelium or the
urothelium [11]. However, no studies have so
far reported the location of these cells in rat
urinary bladder.
on a network of cells between detrusor
smooth muscle bundles and at the edge of the
bundles, with morphology consistent with
that seen in other tissues. Morphological
studies on guinea-pig and human bladders
have similarly identified ICCs on the boundary
of smooth muscle bundles and in the spaces
between the bundles [8,10]. Previous studies
have also confirmed the presence of ICCs in
the suburothelium/lamina propria region. We
were unable to confirm the presence of ICCs
in the suburothelial region of the rat bladder,
similar to previous immunological studies on
the mouse bladder [11].
In the present study we were able to confirm
expression of c-kit-positive ICCs in the rat
bladder using RT-PCR and sequencing of the
resulting amplicons. The results confirmed the
expression of c-kit tyrosine kinase mRNA at
the expected product size of 480 bp. In
addition, we detected c-kit immunoreactivity
There has been some controversy regarding
the use of c-kit antibodies to detect ICCs.
Some of this may be due to the heterogeneity
and diversity of this cell population. ICCs were
first described in the guinea-pig and human
bladder as target cells for nitric oxide, and
responded to this stimulus with an increase in
©
immunoreactivity for cGMP [7]. However, ICCs
in human and guinea-pig bladders also show
immunoreactivity for vimentin [9,10] and in
human bladder the vanilloid receptor VR1/
TRPV1 and the vanilloid receptor-like protein
VRL1/TRPV2 are expressed on ICCs, indicating
a possible role in noxious stimuli transduction
[10,29]. Sui et al. [28] also reported a network
of ICCs beneath the urothelium in human
bladders that were immunoreactive to
connexin 43. However, it was found that this
suburothelial cell layer stained poorly for the
c-kit receptor. Sui et al. [28] also showed that
c-kit and a coincidental staining for desmin
were apparent in the basal layer and even in
the intermediate regions of the urothelial
layer rather than in the connexion 43/
vimentin-positive cell layer in the
suburothelial space and thus concluded that
c-kit staining could not be used as a marker to
distinguish suburothelial ICCs. However,
whilst many c-kit antibodies fail to detect
ICCs in positive-control tissues, ICCs have
been identified in the guinea-pig bladder by
McCloskey and Gurney [8] using antibodies
directed to the c-kit receptor and ICCs with
immunoreactivity to c-kit, and a similar
morphology to those described by McCloskey
and Gurney, have also been reported by
Hashitani et al. [13] in guinea-pig bladder.
In the present study, bladder strips from the
1-week diabetic rat model had significantly
increased amplitude of CCH-induced phasic
contractions compared with age-matched
control tissues. However, in contrast there
was a decrease in the frequency of phasic
contractions in these diabetic tissues when
compared with their age-matched controls.
This difference was not observed at later time
points in the diabetic model. We have also
previously observed that basal phasic activity
is increased in a similar diabetic rat model of
diabetes [21]. The reason for the increase in
amplitude of CCH-induced phasic activity in
1-week diabetic tissues is unknown, although
it was postulated that changes in the number
or function of ICCs could be an important
factor. Increased numbers of ICCs have been
shown in the bladders of patients with
idiopathic and neurogenic DO [15], and in the
BOO guinea-pig model of DO [22]. However, in
the present study the data from functional
inhibition of phasic activity with imatinib
does not support this idea. Imatinib inhibited
CCH-induced phasic activity in bladder
strips from control and diabetic rats in a
similar manner. Indeed, in general, higher
concentrations of imatinib were required to
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1485
V A H A B I ET AL.
inhibit phasic activity in bladder strips from
diabetic rat compared with in control tissues,
although this trend towards a reduced
sensitivity was not statistically significant.
This is in contrast to a previous study that
showed imatinib to have a more profound
effect in reducing phasic activity in human DO
than in normal tissues [15]. Imatinib also
reduced spontaneous contractions in bladder
sheets from spinal cord-transected rats, but
not in those from normal rats [28]. The reason
for these differences is not clear, although
they may be due to variations in the species
and model used, as well as diversity in
the experimental procedures and tissue
preparation techniques. The present findings
are however, in agreement with a recent
report showing no up-regulation of ICC
expression or c-kit immunoreactivity in
neurogenic and idiopathic DO in humans [24].
As we did not investigate the distribution and
density of the ICCs in diabetic rat bladders
and their age-matched controls in the present
study, we cannot comment on whether there
are changes at this level in the diabetic
bladders, although we can conclude that the
functional role of these cells in phasic activity
does not seem to be significantly altered by
the diabetic state.
frequency phasic contractions during
postnatal development. This is in agreement
with more controlled micturition in these
postnatal rats [2].
In conclusion, c-kit-positive ICCs are present
in the rat urinary bladder and play a role in
mediating muscarinic receptor-induced
phasic contractions in bladders from control
and diabetic rats. Although diabetic tissues
showed a trend towards reduced sensitivity to
the inhibitory effect of imatinib on phasic
contractions, the role of these cells does not
seem to be significantly altered by the diabetic
state.
1486
8
9
ACKNOWLEDGEMENTS
10
We would like to thank Novartis (Basel,
Switzerland) for providing the imatinib.
CONFLICT OF INTEREST
11
None declared.
REFERENCES
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Alternatively, explanations for the increased
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Correspondence: Donna J. Sellers, Faculty
of Health Sciences & Medicine, Bond
University, Robina, Queensland 4220,
Australia.
e-mail: [email protected]
Abbreviations: ICC, interstitial cell; STZ,
streptozotocin; CCH, carbachol; cDNA,
complementary DNA; VRL1, vanilloid
receptor-like protein 1; DO, detrusor
overactivity; RT, reverse transcription;
GAPDH, glyceraldehyde-3-phosphate
dehydrogenase.
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