Calcium signalling in Cajal-like interstitial cells of the lower urinary tract

Abstract

Interstitial cells of Cajal (ICC) serve several critical physiological roles in visceral smooth muscle organs, including acting as electrical pacemakers to modulate phasic contractile activity and as intermediaries in motor neurotransmission. The major roles of ICC have been described in the gastrointestinal tract, however, ICC-like cells (ICC-LC) can also be found in other visceral organs, including those of the lower urinary tract (LUT), where they provide similar functions, acting as electrical pacemakers and as intermediary cells involved in the modulation of neurotransmission to adjacent smooth muscle cells. The physiological functions of ICC-LC, in particular their role as pacemakers, relies on their ability to generate transient and propagating intracellular Ca(2+) events. The role of ICC-LC as pacemakers and neuromodulators in the LUT is increasingly apparent and the study of their intracellular Ca(2+) dynamics will provide a better understanding of their role in LUT excitability.

Full text

NATURE REVIEWS
|
UROLOGY ADVANCE ONLINE PUBLICATION
|
1
Department of
Physiology & Cell
Biology, University
ofNevada School of
Medicine, Reno,
NV89557, USA (B.T.D.,
S.D.K., S.M.W). Wake
Forest Institute for
Regenerative Medicine,
Wake Forest University
School of Medicine,
Winston-Salem,
NC27106, USA
(K.‑E.A.).
Correspondence to:
B.T.D.
bdrumm@
medicine.nevada.edu
Calcium signalling in Cajal‑like interstitial
cells of the lower urinary tract
Bernard T. Drumm, Sang Don Koh, Karl-Erik Andersson and Sean M. Ward
Abstract | Interstitial cells of Cajal (ICC) serve several critical physiological roles in visceral smooth muscle
organs, including acting as electrical pacemakers to modulate phasic contractile activity and as intermediaries
in motor neurotransmission. The major roles of ICC have been described in the gastrointestinal tract, however,
ICC-like cells (ICC-LC) can also be found in other visceral organs, including those of the lower urinary tract
(LUT), where they provide similar functions, acting as electrical pacemakers and as intermediary cells involved
in the modulation of neurotransmission to adjacent smooth muscle cells. The physiological functions of
ICC-LC, in particular their role as pacemakers, relies on their ability to generate transient and propagating
intracellular Ca2+ events. The role of ICC-LC as pacemakers and neuromodulators in the LUT is increasingly
apparent and the study of their intracellular Ca2+ dynamics will provide a better understanding of their role in
LUT excitability.
Drumm, B. T. et al. Nat. Rev. Urol. advance online publication 16 September 2014; doi:10.1038/nrurol.2014.241
Introduction
Interstitial cells of Cajal (ICC) are a specialized popu-
lation of cells involved in smooth muscle excitability
that are mesenchymal in origin.1–3 ICC possess few
contractile elements but contain large numbers of mito-
chondria, an abundance of endoplasmic reticulum (ER)
and caveolae—organelles essential for intracellular and
extracellular calcium handling in these cells—and dis-
tinct membrane channels for their specialized functions
as pacemakers and neuromodulators.4 The discovery
that ICC in the gastrointestinal tract (GIT) express
Kit, a tyrosine kinase receptor, enabled investigators to
label these cells using antibodies against the receptor
and study their distribution and function in a variety of
v isceral organs.
Manipulation of the Kit receptor and mutant animal
models with defects in the Kit signalling pathway (and
greatly reduced numbers of ICC) has enabled the study
of the physiological roles of these cells in visceral-
smooth-muscle organs. A number of roles have been
suggested for these cells in the GIT. ICC are thought to
act as pacemakers by generating electrical slow waves
that organize phasic contractile behaviour of neighbour-
ing smooth muscle cells.5–7 They also provide a propaga-
tion pathway for the regeneration of slow waves so that
large areas of visceral organs can be entrained to con-
tract synchronously.8 ICC mediate excitatory (choliner-
gic) and inhibitory (nitrergic) motor inputs into visceral
muscles,9–11 and serve as stretch receptors to regu late
electrical excitability of the smooth-muscle–ICC syn-
cytium and pacemaker frequency.12 Finally, ICC are also
thought to have a role in vagal afferent signalling.13
Since the function of ICC was initially elucidated in the
GIT, many groups have reported the discovery of cells
with similar morphology in tissues of the urogenital tract.
These cells—termed ICC-like cells (ICC-LC)—have been
identified in the urethra,14 vas deferens,15 prostate,16
bladder,17 corpus cavernosum,18,19 ureter,20 fallopian
tube,21 oviduct22 and uterus.23 Physiological experi-
ments have revealed that ICC-LC might act as electrical
pacemakers in some urogenital tissues (including the
urethra,14 ureter24,25 and prostate16,26) in a similar manner
to ICC in the GIT. However, the roles of ICC-LC in the
bladder, renal pelvis and penis (in which smooth muscle
or atypical smooth muscle cells can generate pacemaking
activity) have not been fully explored.27 In the bladder,
the close association (20–40 nm as demonstrated with
electron microscopy) between nerve fibres and ICC-LC
suggests that they might act as intermediary cells, trans-
ducing nerve signals to detrusor smooth muscle cells.28–31
ICC-LC responded to electrical field stimulation of nerve
elements by firing coordinated Ca2+ transients insitu,
demonstrating functional innervation.32 Recent studies
on the corpus cavernosum and the prostate suggest that
ICC-LC might also act as intermediary cells between
nerves and smooth muscle in these tissues.33,34
In 2012, a new class of interstitial cell was identified in
the bladder that expresses platelet-derived growth recep-
tor alpha (PDGFRα) but not Kit.35,36 Similar to ICC-LC,
PDGFRα+ cells have morphological and functional
character isticsincluding close apposition to motor
nerves, expression of neural receptors and ionic conduc-
tances to mediate postjunctional responses to smooth
muscle cellsthat make them probable intermediary
cells involved in neuromodulation or in paracrine or
hormonal signalling.35–38 Thus, it seems that ICC-LC and
Competing interests
The authors declare no competing interests.
REVIEWS
© 2014 Macmillan Publishers Limited. All rights reserved

2
|
ADVANCE ONLINE PUBLICATION www.nature.com/nrurol
PDGFRα+ cells in the bladder might affect the contractility
of smooth muscle cells either by acting as primary or sup-
portive electrical pacemakers, or by acting as intermediary
n euromodulators (Figure1).
The stochastic release of calcium from intracellular
stores that lead to the generation of intracellular transient
Ca2+ waves and propagating Ca2+ waves seems to be funda-
mental to the physiological functions of ICC-LC in visceral
organs, especially in their role as pacemakers and inter-
mediaries in motor neurotransmission. In the past 15years,
studies investigating the role of Ca2+ signalling in these cells
have been performed in organs of the upper and lower
urinary tract. These include several excellent morphologi-
cal, Ca2+ imaging and electrical characterisation studies
in the renal pelvis and ureter and there is some evidence
that ICC-LC might act as pacemakers or neuromodula-
tors in these organs24,25,39–47 However, for the scope of this
Review we will focus on studies from the lower urinary
tract (LUT), namely the urethra, bladder and prostate. In
this Review, the generation and propagation of intracellular
Ca2+ events and the physiological effects of this activity in
the urethra, bladder and prostate will be discussed.
Ca2+ signalling in ICC-LC
At the single cell level, the spontaneous electrical activity
of ICC-LC in the LUT is thought to be due to their ability
to mobilize intracellular Ca2+. Ca2+ mobilization tends to
manifest as spontaneous oscillations and propagating
waves. Spontaneous intracellular Ca2+ oscillations have
been observed in ICC-LC from both the upper and lower
urinary tract of several animal models including rabbit
urethra,48 mouse renal pelvis,39,40,42 guinea pig prostate49
and bladders of guinea pig17,50,51 and rat.52 These Ca2+
signals typically exhibit similar temporal characteristics
among species and tissues, and are of greater amplitude
but occur at a lower frequency than Ca2+ transients found
in adjacent smooth muscle bundles (Figure2).51
Ca2+ mobilization involves a combination of influx from
the extracellular space and release of Ca2+ from intracellu-
lar ER stores and mitochondria.53 Mitochondria can act as
a reversible Ca2+ storage organelle, with Ca2+ transport in
and out of the mitochondria affecting cytosolic Ca2+ con-
centrations and whole-cell Ca2+ signalling;54 ICC-LC are
known to contain a dense distribution of mitochondria.14,55
Ca2+ release from the ER typically involves the activation
of inositol 1,4,5-triphosphate (IP3)-sensitive receptors
(IP3Rs), ryanodine receptors (RyRs) or a combination of
the two.54 Ca2+ influx can have a major effect on the fre-
quency and amplitude of Ca2+ oscillations by refilling the
ER Ca2+ stores, increasing cytosolic Ca2+ to sensitize IP3Rs
and RyRs or by activating the receptors directly.56,57
Berridge58 described a mechanism for the generation
of spontaneous Ca2+ oscillations in ICC and ICC-LC,
whereby the oscillations originate as random Ca2+ release
events of limited spread. These release events could be
either a Ca2+ spark—a localised release of Ca2+ originat-
ing from the ER via RyRs—or a Ca2+ puff, which is also a
localised release of Ca2+, but originates from IP3Rs. This
initial trigger initiates a Ca2+ transient or wave that is
amplified by a positive feedback loop of calcium-induced
calcium release along the ER. The oscillations terminate
when the ER stores are depleted and Ca2+ is extruded
from the cytosol via the sarcoplasmic/endoplasmic reticu-
lumCa2+-ATPase (SERCA) pump, the plasma membrane
Ca2+-ATPase and uptake into the mitochondria. The ER
store refills for the next transient via Ca2+ influx from
the extracellular space, uptake via the SERCA pump and
p ossibly Ca2+ shuttling from the mitochondria (Figure3).
Key points
Interstitial cell of Cajal-like cells (ICC-LC) are found throughout the lower urinary
tract (LUT), where they form a functional syncytium with nerves, smooth muscle
cells and recently identified PDGFRα+ cells
ICC-LC are believed to function as electrical pacemakers in the urethra and
prostate, and in the bladder they might act as intermediary cells to transduce
nerve signals to smooth muscle cells
The generation of intracellular Ca2+ transients originating from the release of
Ca2+ from the endoplasmic reticulum (ER) seems to be fundamental to the
physiological functions of ICC-LC
Ca2+ release from the ER involves the activation of receptors sensitive to inositol
1,4,5-triphosphate and ryanodine. Propagation of this Ca2+ signal is subsequently
supported by Ca2+ influx and mitochondrial Ca2+ handling of cytosolic Ca2+ levels
Several translational studies have revealed that ICC-LC might serve as
valuable targets for LUT dysfunctions including overactive bladder and benign
prostatichyperplasia
The modulation of muscle excitability in the LUT depends on Ca2+ signalling
originating in ICC-LC. Further examination of these events provides an approach
to evaluate therapeutic agents on LUT function
Figure 1 | Syncytium of nerves, ICC-LC, PDGFRα+ cells and smooth muscle cells in the
bladder detrusor. ICC-LC and PDGFRα+ cells are likely to mediate neural input inthe
bladder detrusor. Innervation of these interstitial cells by various neural pathways can
modulate their activity, including their intracellular Ca2+ dynamics. This activity can
then affect the excitability of adjacent smooth-muscle cells, which are electrically
connected to interstitial cells via low-resistance gap junctions. Thus, excitatory and
inhibitory nerves, innervating interstitial cells (ICC-LC) and recently identified PDGFRα+
cells are coupled to smooth-muscle cells to form a functional syncytium, which
enables them to act as neuromodulators to the bulk smooth muscle in some organs
(bladder, penis) and also act as electrical pacemakers inothers (urethra, prostate).
Abbreviations: Ach, acetylcholine; ICC-LC, interstitial cells of Cajal-like cells;
P2Yreceptor, purine receptor; PDGFRα, platelet-derived-growth-factor receptor α.
PDGFRα+ cell
ICC-LC
Gap junction
Smooth muscle cell
Excitatory motor neuron (ACh)
Inhibitory motor neuron (nitric oxide/ATP)
NO NO
ACh
Muscarinic
receptor
P2Y receptor
ATP
REVIEWS
© 2014 Macmillan Publishers Limited. All rights reserved

NATURE REVIEWS
|
UROLOGY ADVANCE ONLINE PUBLICATION
|
3
Urethra
A role for intracellular Ca2+ signalling in the genera-
tion of ICC-LC pacemaker activity outside of the GIT
was first proposed based on experiments in the rabbit
urethra.14 A number of early studies found that pace-
maker electrical activity in rabbit urethral cells was
inhibi ted by agents that disrupted the intracellular Ca2+
stores andCa2+ influx.14,59–61 A rhythmical release of intra-
cellular Ca2+, possibly in the form of a propagating Ca2+
wave, was proposed by Segeant etal.60 and Hollywood
etal.61 to be required for generating pacemaking electri-
cal activity. This hypothesis was later c onfirmed with Ca2+
imagingstudies.48
Ca2+ waves were first imaged in enzymatically dis-
persed rabbit urethral ICC-LC using confocal micro-
scopy and the Ca2+ indicator fluo-4 acetoxymethyl ester.48
The waves were inhibited by the SERCA pump inhibi-
tor cyclopiazonic acid (CPA), the RyR blockers ryano-
dine and tetracaine, and the phospholipase C inhibitors
2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate and
U73122.48 Inhibiting IP3Rs with 2-aminoethoxy diphenyl
borate (2-APB) reduced the amplitude and spatial spread
of the waves without affecting their frequency, whereas
tetra caine abolished all Ca2+ activity.48,62 Similar results
were reported for insitu recordings of spontaneous Ca2+
transients from rabbit urethral ICC-LC, where waves were
also blocked by ryanodine, 2-APB, caffeine and CPA.63
Taken together these findings imply that Ca2+ release
from the ER via RyRs, in the form of Ca2+ sparks, acts as
the ‘prime oscillator’ in generating Ca2+ oscillations, and
further Ca2+ release from IP3Rs is required to amplify this
initial signal into a propagating wave. An article published
in 2014 provides evidence that the Ca2+ release mediated
by IP3Rs in rabbit urethral ICC-LC might be modulatedby
protein kinase A.64 However, the exact mechanism is
c urrently unclear and warrants further study.
Ca2+ influx from the extracellular space is an essen-
tial component of spontaneous intracellular Ca2+ waves
in ICC-LC of the rabbit urethra, and the influx pathway
has been studied extensively. The frequency of Ca2+
waves depends on the external Ca2+ concentration.48
Voltage-dependent L-type Ca2+ channels (Cav 1.2) do
not appear to have a role in the influx pathway, as block-
ing these channels with the dihydropyridines nifedipine
and nicar dipine failed to block Ca2+ transients at both the
cellular and tissue level.48,63 Initial results suggested that
Ca2+ influx was dependent on capacitive calcium entry,48
but later studies revealed that this was not the case.65 It
is now thought that Ca2+ transients in urethral ICC-LC
rely on Ca2+ influx from reverse-mode sodium/calcium
exchange (NCX). The Na+/Ca2+ exchanger is a bidirec-
tional ion transport protein that can exchange three Na+
ions for one Ca2+ ion and mediates Ca2+ influx or efflux
depending on the net electrochemical driving force.66
Bradley etal.67 demonstrated that rabbit urethral ICC-LC
expressed the NCX3 isoform and that decreasing [Na+]o
a
c
b7
20 s
2.0 (F/F0) 80 μm
(F/F0)
0
20 s
30 μm
0 s
1.2 s
2.4 s
3.6 s
0.4 s
1.6 s
2.8 s
4.0 s
0.8 s
2.0 s
3.2 s
4.4 s
Figure 2 | Spontaneous Ca2+ waves in urethral ICC-LC. a | Montage of an isolated rabbit urethral ICC-LC, recorded at 37oC
firing a propagating Ca2+ wave. Acolour-coded system has been imported into the image to show low Ca2+ fluorescence
intensity as cold colours (blue/green) and high Ca2+ fluorescence intensity as warmer colours (yellow/red). The images in
this montage were taken at 400 ms intervals. The wave initiated at the top right region of the cell then propagated along
the cell length. b | x, t linescan plot of spontaneous Ca2+ waves. This linescan was acquired from recordings of Ca2+ waves
using an Andor camera and iQ software (Andor Technology, Belfast, UK). Movie files recorded in iQ were converted to a
stack of TIFF (tagged image file format) images and imported into Image J software (version 1.40, National Institutes of
Health, MD, USA) for posthoc analysis. Prior to analysis, background fluorescence was subtracted from the stack. A single
pixel line was drawn along the mid axis of the cell and, using the ‘reslice’ function in Image J, a pseudo-linescan image was
produced with distance along the cell (m) on the vertical axis and time (sec) on the horizontal axis. Basal fluorescence
was obtained from areas of the cell displaying the most uniform and least intense fluorescence (F0). A colour-coded
systemwas then imported into the TIFF to show low fluorescence intensity as cold colours (blue/green) and high
fluorescence intensity as warmer colours (yellow/red). c | Plot profile showing a trace of the Ca2+ oscillations from
thelinescan discussed above.
REVIEWS
© 2014 Macmillan Publishers Limited. All rights reserved

4
|
ADVANCE ONLINE PUBLICATION www.nature.com/nrurol
from 130 mM to 13 mM (to increase the driving force for
reverse-mode NCX) increased the frequency of Ca2+ waves
and spontaneous transient inward currents in ICC-LC.
Specific reverse-mode NCX inhibitors blocked this effect.
Similar results were achieved by raising the external K+
concentration to increase the driving force for reverse-
mode NCX.68 However, there is not widespread agreement
onthe i dentity of the urethral ICC-LC influx pathway,
with thecGMP-gated Ca2+ channel also suggested to be a
c andidate influx channel.69
It should be noted that the concept of Ca2+ influx being
required to refill a depleted ER store between oscilla-
tions might not occur in urethral ICC-LC as the Berridge
model suggests.58 Ca2+-free solutions abolished Ca2+ waves
in u rethral ICC-LC and under these conditions, 10 mM
caffeine responses remained intact, indicating that influx
might not be required to refill the Ca2+ stores during oscil-
lations.48 Perhaps Ca2+ sparks from RyRs cannot initiate a
propagating Ca2+ wave unless neighbour ing IP3Rs on the
ER membrane are sufficiently sensitized to be activated
by Ca2+. This sensitization might occur by raising the
intracellular [Ca2+]i through Ca2+ influx.70 Disrupting
the Ca2+ handling of mito chondria with electron trans-
port chain inhibitors and proto nophores, such as carbonyl
cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP)
and carbonyl cyanide m- chlorophenylhydrazone (CCCP)
inhibited spontaneous Ca2+ waves.71 Furthermore, activat-
ing mitochondrial Ca2+ uptake with kaempferol induced
a Ca2+ transient, followed by a burst of higher frequency
Ca2+ oscillations with progressively smaller ampli-
tudes.71 This effect was blocked by removing Ca2+ from
the bathing solution but not by 2-APB, demonstrating
that it was dependent on Ca2+ influx and did not cause
direct release from intracellular stores.71 Co nv er sely,
when Ca2+ efflux via the mitochondrial sodium/calcium
exchanger was inhibited with CGP 37517, wave fre-
quency was decreased.72 This finding suggests that Ca2+
efflux from the mitochondria might refill Ca2+ stores or,
more likely, prevent adequate sensitization of Ca2+ recep-
tors on the ER so they are not activated in response to
an initial Ca2+ release signal. Taken together, we observe
intra cellular Ca2+ transients in urethral ICC-LC that give
rise to propagating Ca2+ waves that subsequently activate
calcium-activated chlor ide channels (CaCC) in the plasma
membrane leading to depolarization. This depolariza-
tion is f undamental to the pacemaker function of these
cells(Figure4).
Urethral ICC-LC are noted to be in close a ssociation
with nerves and might act as intermediary cells to trans-
duce neural inputs to the bulk smooth muscle.73,74 Several
studies have attempted to evaluate whether neuro-
transmitters can affect the intracellular Ca2+ signal ling
of urethral cells. The frequency of Ca2+ waves in rabbit
urethral ICC-LC is increased by exogenous application
of ATP and this effect is mimicked by purine receptor
(P2Y) agonists and blocked by P2Y inhibitors but not
P2X purine receptor inhibitors.75,76 This finding led to the
suggestion that contractions of urethral tissue evoked by
activation of P2Y receptors by purinergic nerves could be
mediated via Ca2+ waves in ICC-LC, but further experi-
ments revealed that these urethral contractions are caused
by activation of P2X receptors on smooth muscle cells.77
The velocity and frequency of Ca2+ waves in ICC-LC can
also be increased by activating α1-adrenoceptors with
noradrenaline or phenylephrine; these receptors probably
sensitize IP3Rs to respond to an initial Ca2+ signal from
RyRs.63,70,76 Conversely, the amplitude and spread of Ca2+
waves is inhibited by nitric oxide donors and activators of
the cGMP/PKG pathway,63,78 which is also attributed to an
effect on IP3Rs.78
ICC-LC in the LUT lack the well-defined anasto mosing
networks of ICC in the gut,4 which seems contradictory
to a pacemaker system. Ca2+ transients in rabbit urethra
show little temporal correlation with Ca2+ transients in
the adjacent smooth muscle, leading to the suggestion
that a ‘loose pacemaker’ system is involved, in which the
Mitochondrion
ER
1
3
4
6
2
Ca2+
Ca2+
Ca2+
Plasma
membrane
Ca2+
Ca CC
Cl–
RyR
IP
3
R
5
NCX
Na+
Ca2+
Ca2+
ATPase
Ca2+
Ca2+
SERCA
5
6
L-type
Ca2+
T-type
Ca2+
Ca2+
Ca2+
Ca2+
Spark
Puff Puff
STD
Ca2+
STIC
+
++
+
Ca2+
wave
Figure 3 | ICC-LC mechanism of Ca2+ wave propagation. Stochastic release of Ca2+
from the ER via RyRs generates a Ca2+ spark or a Ca2+ puff from IP3Rs (1), which
diffuses to a neighbouring cluster of IP3Rs and subsequently activates them.
Theinitial trigger initiates a Ca2+ transient or wave that is amplified by a positive
feedback loop of calcium-induced calcium release along the ER (2). An increase
incytosolic calcium in ICC-LC that act as pacemakers activates CaCCs (3), which
generates STICs, leading to STDs (4). STDs manifest as pacemaker
depolarizations. Membrane depolarization causes activation of T-type and L-type
voltage-dependent Ca2+ channels (dependent upon cell type and organ). The
increase in cytosolic Ca2+ is balanced by Ca2+ extrusion via the plasma membrane
Ca2+ATPase (5) and uptake by the SERCA pump. The ER store refills for the
nextCa2+ transient via Ca2+ influx from the extracellular space, uptake via the
SERCA pump and possibly Ca2+ shuttling from the mitochondria (6). Depending
upon the cell type, this mechanism can occur via voltage-dependent calcium
channels or the NCX (6). Abbreviations: CaCC, calcium-activated chloride channels;
ER, endoplasmic reticulum; ICC-LC, interstitial cells of Cajal-like cells; IP3R, inositol
1,4,5-triphosphate receptor; NCX, sodium/calcium exchanger; RyR, ryanodine
receptor; SERCA, sarcoplasmic/endoplasmic reticulum Ca2+-ATPase; STD,
spontaneous transient depolarization; STIC, spontaneous transient inward current.
REVIEWS
© 2014 Macmillan Publishers Limited. All rights reserved

NATURE REVIEWS
|
UROLOGY ADVANCE ONLINE PUBLICATION
|
5
majority of Ca2+ activity in ICC-LC occurs independently
of the smooth muscle.76 Ca2+ transients in ICC-LC seldom
initiate intercellular Ca2+ waves within smooth muscle
bundles. Instead, the summation of the signals from
ICC-LC to smooth muscle cells might increase the overall
excitability of the urethra. However, it should be noted that
even the concept of ICC-LC Ca2+ waves mediating ure-
thral contractions in electrically coupled smooth muscle
cells is not completely accepted. Sancho etal.79 found that
inhibiting IP3Rs with 2-APB led to a decrease in electrical-
field-stimulated contractions in sheep urethra but not rat
urethra, suggesting that the role of Ca2+ release in ICC-LC
pacemaker activity might be species dependent.
Bladder
ICC-LC in the bladder detrusor muscle were initially
believed to act as pacemakers,17 but Ca2+ transients in blad-
der ICC-LC occur asynchronously with adjacent smooth
muscle, which deems a pacemaking role for these transi-
ents unlikely.51 Little evidence is available to currently
support the ICC-LC pacemaker theory in bladder, and
the exact role of the intracellular Ca2+ signals in these cells
remains unknown, although it has been noted that ICC-LC
Ca2+ signalling can be upregulated or d ownregulated in
patients with sacral spinal cordinjuries.80
ICC-LC are found in different anatomical locations
within the bladder wall and are therefore likely to have
different physiological functions. ICC-LC found closely
apposed to detrusor smooth muscle cells (Figure1) do
not appear to be directly involved in pacemaker activity,
but might be involved in regulating smooth muscle excit-
ability or tone during, for example, the bladder filling-
phase when stretch is imposed on detrusor muscle.28,32,81
ICC-LC are also found in the lamina propria where they
might act as neuromodulators.28,32 A second population
of ICC-LC is found within the suburothelium, the exact
function of these cells is currently unknown although
pacemaking functions and stromal signalling have been
suggested.82 Ca2+ and membrane potential transients,
produced by stretch or the cholinergic agonist carbachol
(CCh) have been recorded in the suburothelial region
near the dome of the neonatal rat bladder wall and then
spread to the detrusor muscle. However, these activi-
ties are lost in adult tissues where Ca2+ and electrical
activity becomes less coordinated.83 ICC-LC might be
responsible for this activity, but this proposal has not yet
been verified. ICC-LC are also found in close apposition
toafferent nerve fibres and may be involved in urothelial
afferent nerve signalling.28 These different populations
of ICC-LC in the bladder might provide investiga-
tors with possible cellular targets for the t reatment of
bladderdysfunction.
Some groups have suggested that the Ca2+ activity of
bladder ICC-LC could be utilized to transmit signals to
nearby nerves or the underlying smooth muscle cells.27,28
Guinea pig detrusor ICC-LC are in close apposition to
cholinergic nerves terminals, suggesting that these cells
might modulate neural input to the detrusor smooth
muscle.84 This observation was supported by the find-
ings that bladder ICC-LC from rat express the M2 and
M3 muscarinic receptors and that CCh-induced Ca2+
currents in these cells were blocked by the muscarinic
antagonist atropine.85 CCh also induced Ca2+ oscilla-
tions in guinea-pig ICC-LC , which were blocked by the
muscari nic M3 blocker 4-DAMP.86 Similar results were
also found in cultured mouse ICC-LC; CCh increased
the amplitude of intracellular Ca2+ transients and this
effect was blocked by atropine.87 In the absence of neural
input, electrical field stimulation of ICC-LC in the detru-
sor and lamina propria of the guinea pig bladder evoked
insitu asynchronous spontaneous Ca2+ oscillations,32
demonstrating that Ca2+ activity in ICC-LC could be
modulated by neurotransmission. Taken together, these
findings suggest that innervation of ICC-LC by neural
networks might affect smooth muscle contraction in the
bladder (and urethra) by modulation of the frequency,
amplitude or the spatial spread of Ca2+ waves.
Similarly to the rabbit urethra, Ca2+ transients in
guinea-pig-bladder ICC-LC seem to rely on Ca2+ release
from ER stores via IP3Rs and RyRs, as demonstrated
by the inhibition of Ca2+ waves using U73122, xesto-
spongin C, ryanodine and tetracaine.86 Also similar to
the urethra, bladder ICC-LC have L-type Ca2+ channels,88
and like the urethra these are not essential for sponta-
neous Ca2+ activity as inhibiting L-type Ca2+ channels
Mitochondrion
ER
1
3
4
5
6
2
7
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
Plasma
membrane
Ca2+
NCX
Ca2+
3Na+
IP3
PLC
Cl Ca
Cl–
Cl–
RyR
IP
3
R
Figure 4 | Urethral ICC-LC pacemaking mechanism. Stochastic release of Ca2+
from the ER via RyRs generates a Ca2+ spark (1), which diffuses to a neighbouring
cluster of IP3Rs and subsequently activates them (2). Activation of the IP3Rs
clusters leads to regenerative release from the ER and results in a propagating
Ca2+ wave (3). TheCa2+ wave activates chloride channels on the plasma
membrane, leading to the generation of pacemaker STICs (4). The regenerative
release of Ca2+ from IP3Rs requires that the receptors be adequately sensitized to
activate. This sensitization occurs through an increased [Ca2+]i via Ca2+ influx
through reverse mode NCX (5). IP3Rs are also sensitized by a continuous
production of IP3 resulting from the hydrolysis of membrane bound PLC (6).
Mitochondria can contribute to this mechanism by removing excess Ca2+ from
RyRs, thus preventing their inactivation athigh Ca2+ levels. The extrusion of Ca2+
from the mitochondria via an NCX protein might also increase Ca2+ near the mouth
of RyRs, increasing their sensitivity to activate (7). Abbreviations: ER, endoplasmic
reticulum; ICC-LC, interstitial cells of Cajal-like cells; IP3, inositol
1,4,5-triphosphate; IP3R, inositol 1,4,5-triphosphate receptor; NCX, sodium/
calcium exchanger; PLC, phospholipaseC; RyR, ryanodine receptor; STIC,
spontaneous transient inward current.
REVIEWS
© 2014 Macmillan Publishers Limited. All rights reserved

6
|
ADVANCE ONLINE PUBLICATION www.nature.com/nrurol
does not affect spontaneous Ca2+ activity in ICC-LC in
the guinea pig bladder.50 In the rat, there is some evi-
dence to suggest that influx might occur via T-type Ca2+
c hannels or reverse-mode NCX. Three T-type Ca2+ chan-
nels subtypes have been identified in ICC-LC isolated
from rat bladder. The exact role of each subtype has
not been clarified.52 Application of the selective T-type
Ca2+ channel blocker mibefradil significantly decreased
the spontaneous intracellular Ca2+ waves of rat bladder
ICC-LC loaded with fluo-3 AM. Rat bladder ICC-LC
express the NCX3 isoform (there are three isoforms of
the NCX protein overall) but smooth muscle cells do
not.89 Reducing extracellular [Na+] to induce reverse
mode NCX led to an increase in [Ca2+]i, which could be
blocked by the NCX inhibitor KB-R 7943.89
PDGFRα+ cells have been implicated in puriner-
gic neurotransmission in the colon90–93 and there is a
growing body of evidence that they also act as neuro-
modulators or as intermediaries between purinergic
nerves and smooth muscle cells in the bladder.37,38 Inthe
colon, PDGFRα+ cells are thought to generate Ca2+
transients in response to purinergic agonists through a
P2Y1 receptor and IP3-dependent pathway.91,92 Although
an in-depth study of Ca2+ dynamics in PDGFRα+ cells
of the bladder has not yet been performed, it is likely
that intracellular Ca2+ release events or an increased
[Ca2+]i due to Ca2+ influx enable PDGFRα+ cells to carry
out their physiological function as n euromodulators in
the bladder.
Prostate
ICC-LC in the prostate are amongst the most stud-
iedICC-LC outside of the GIT, in terms of their Ca2+
dynamics and pacemaking mechanisms at the cellu-
lar level. The guinea-pig prostate fires pacemaker slow
waves, which are believed to originate in ICC-LC.94
These slow waves can be blocked with the SERCA pump
inhibitor CPA and are decreased in frequency by 2-APB,
xestospongin C, U73122 and neomycin.95 Conv ersel y,
application of RyR-activating concentrations of ryano-
dine increased slow wave frequency. The underlying Ca2+
events were later visualized as spontaneous Ca2+ waves
and Ca2+ sparks that were inhibited by CPA and caffeine.49
Slow waves that occur in the prostate gland are voltage
dependent,96 which is reflected in the Ca2+ activity of
the pacemaker ICC-LC in this tissue. Unlike ICC-LC
in other regions of the LUT, Ca2+ transients in prostate
ICC-LC seem to rely on Ca2+ influx via several types of
voltage-gated Ca2+ channels. Patch-clamp experiments
have revealed that isolated ICC-LC in the prostate possess
both L-type and T-type Ca2+ channels, both of which con-
tribute to the generation of pacemaker currents resulting
from the activation of CaCC in the plasma membrane.97
An initial release of Ca2+ from the ER is thought to trigger
CaCC, resulting in a small membrane depolarization and
the opening of low-voltage-activated T-type Ca2+ chan-
nels. The resulting Ca2+ influx can trigger regenerative
release from the ER leading to a propagating Ca2+ wave
that can activate sufficient numbers of CaCC to trigger
spontaneous transient depolarization, which is crucial for
prostate ICC-LC to coordinate contraction in electrically
coupled smooth muscle cells. This finding suggests that
although Ca2+ release from ER stores is crucial for the
generation of intracellular Ca2+ transients, the mainten-
ance of these events and the subsequent electrical pace-
maker activity also relies heavily on Ca2+ influx via T-type
Ca2+ channels.97
The role for L-type Ca2+ channels in this pacemaking
mechanism is not clear. Both spontaneous Ca2+ sparks
and waves are abolished by the L-type Ca2+ channel
blocker nicardipine,49 suggesting that Ca2+ influx from
L-type Ca2+ channels is required to initially activate RyRs
and for the propagation of Ca2+ waves.
Recent electrophysiological studies have revealed that
Ca2+ influx through T-type Ca2+ channels is more impor-
tant for the maintenance of pacemaker activity than Ca2+
influx via L-type Ca2+ channels.96,97 The CaCC in pros-
tate ICC-LC is activated upon Ca2+ entry via T-type Ca2+
channels, suggesting a close association between the
two channel populations.97 Notably, 1 M of the L-type
calcium channel blocker nicardipine was sufficient to
abolish all Ca2+ activity in isolated ICC-LC from guinea-
pig prostate,49 whereas high concentrations of another
L-type Ca2+ channel blocker (10 M nifedipine) often
failed to fully inhibit slow waves and contractions in
tissue recordings. Residual slow waves and contractions
can be blocked with inhibitors of T-type Ca2+ channels,
further supporting a close association between T-type
Ca2+ channels and CaCC.96 It is possible that, invivo,
ICC-LC rely more heavily on Ca2+ influx via T-type
Ca2+ channels to generate the initial ER release events
that lead to propagating intracellular Ca2+ waves, and
that Ca2+ influx through L-type Ca2+ channels might be
involved in maintaining the Ca2+ waves—this mechanism
would explain why L-type Ca2+ channel inhibition only
partially suppresses slow waves and contractions in tissue
experiments, whereas inhibition of T-type Ca2+ channels
can block the activity entirely. Isolated prostate ICC-LC
might have a more depolarized membrane poten-
tial, owing to the loss of electrical coupling with adja-
cent smooth muscle cells.96 This depolarization could
lead to a full inactivation of the T-type Ca2+ channels
and, therefore, Ca2+ influx through L-type Ca2+ chan-
nels might become crucial to initiate the i ntracellular
pacemakermechanism.96
It has also been hypothesized that the voltage depen-
dency of Ca2+ signalling in prostate ICC-LC could be
due to modulation of G-protein-coupled IP3 production,
owing to changes in membrane potential.96 Investigators
suggest that the Ca2+ storage capacity of the ER in pros-
tate ICC-LC might be significantly lower than that of
ICC-LC in other LUT organs.97 As mentioned previously,
depleting the ER stores with CPA or caffeine abolished
all Ca2+ transients in prostate ICC-LC.49 However, this
effect was not associated with a rise in basal Ca2+ as
observed in the urethra,48 suggesting that the ER load
in prostate ICC-LC is relatively low, thus making Ca2+
influx via voltage gated Ca2+ channels more important
for regenerative Ca2+ release from the ER and Cl– channel
activation than in the urethra.
REVIEWS
© 2014 Macmillan Publishers Limited. All rights reserved

NATURE REVIEWS
|
UROLOGY ADVANCE ONLINE PUBLICATION
|
7
In 2009, two reports of studies using guinea-pig pros-
tate implicated mitochondrial Ca2+ handling in modu-
lating ICC-LC Ca2+ activity. FCCP, CCCP and rotenone
abolished slow waves observed in intracellular mem-
brane recordings, with slow waves believed to be gener-
ated by ICC-LC.95 Similarly, Nguyen etal.98 showed that
pacemaker potentials could be blocked by CCCP. These
results suggest a role for mitochondrial Ca2+ handling in
generating ICC-LC Ca2+ signals; however, a mechanism
for this effect has not yet been determined, although Ca2+
shuttling between the ER and mitochondria has been
proposed (Figure5).98
ICC-LC in the prostate might also be affected by
neurotransmission. Work by Dey etal.99 showed that
8-bromo-GMP inhibited spontaneous contractions of
the guinea-pig prostate, potentially owing to a similar
inhibitory mechanism on Ca2+ transients via IP3Rs in
ICC-LC as is observed in the urethra.78 The prostate
can also be innervated by adrenergic neurotransmission
through activation of α1-adrenoceptors.100 Studies have
shown that an increase in the frequency of pacemaker dis-
charges (believed to originate in ICC-LC) can be induced
by α1-adrenoceptor agonists such as phenylephrine.98
ICC-LC as therapeutic targets in the LUT
The specialized functions that interstitial cells perform
in the LUT, including the generation of pacemaker
activity, mediation in motor neurotransmission, and
afferent nerve signalling, would lead to significant
pathological changes in the various organs of the LUT
if the cellular numbers, interactions with each other and
other cells, phenotype or cellular functions of ICC-LC
were impaired. A growing body of evidence suggests that
the numbers of ICC-LC are disrupted in several LUT
disorders and in spinal-cord injury.31 Kit+ ICC-LC were
shown to be present in significantly greater numbers in
human overactive bladder than normal specimens.29
Although this study was limited in the number of
samples tested, warranting further studies, disease-
associated disruption in specific cellular functions in
these cells provides an opportunity for cellular targeting
for pharmacologicalinterventions.
Studies have also shown that the Kit inhibitor, imati-
nib, reduced the contractile activity of detrusor smooth
muscle in overactive bladder.101 Imatinib improved
bladder capacity, compliance, voided volumes, urinary
frequency, and reduced contraction thresholds and
spontaneous activity during cystometry in a guinea-
pig model.101 A similar study also showed that imati-
nib inhibited phasic contractions of bladder muscle
in the pig.102 More recently, an invivo study examined
the effects of persistent neonatal inhibition of Kit using
imatinib on the development of ICC-LC in bladder and
the functional consequences of the loss of these cells.
Imatinib caused a significant decrease in the number of
ICC-LC in neonatal tissues and also reduced the phasic
contractile frequency and muscarinic-induced contrac-
tions in bladder.103 These data support the hypothesis that
poor development of ICC-LC in the bladder has signifi-
cant repercussions on bladder function. In a rat animal
model of suprasacral cord injury (SSCI), numbers of Kit-
expressing ICC-LC and associated contractile activity of
detrusor muscle were increased, whereas in a model of
sacral cord injury (SCI) ICC-LC numbers and contrac-
tions were decreased.80 Furthermore, in a model of mod-
erate spinal cord contusion injury, animals that received
imatinib displayed enhanced bladder function.104
In the human prostate, increased numbers of ICC-LCs
are found in tissues affected by BPH,105 suggesting a role
for ICC-LC in the pathophysiology of this condition.
BPH is associated with an increase in α1-adrenoceptor
expression, which has led to the use of α1-antagonists as
clinical treatments for this condition.100 Tamsulosin—a
clinically used α1-adrenoceptor antagonist—reduces the
spontaneous electrical activity of the guinea-pig pros-
tate.106 This effect suggests that α1-adrenoceptors might
be located on ICC-LCs and that their activation might
contribute to Ca2+ mobilization that leads to the genera-
tion of pacemaker activity in this tissue.106 Studies have
also shown that inhibition of Kit with imatinib decreased
spontaneous contractile activity in both young and older
guinea-pig models.107 A greater effect of the Kit inhibi-
tor was observed in the younger age group, suggesting
that the ageing guinea-pig prostate is less reliant on
pacemaker activity generated by ICC-LC. However, it
should be noted that, in this study, imatinib decreased
contractile activity to a greater degree than the under-
lying pacemaker activity. The concentrations of imati-
nib that were used and its acute administration in the
studies on bladder and prostate might have side effects
Mitochondrion
ER
1
3
4
5
6
7
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
Plasma
membrane
Ca2+
Ca2+ IP3
PLC
CC
Ca
Cl–
Cl–
RyR
IP
3
R
L-type Ca CC
Cl–
Cl–
2
Ca2+
T-type
Figure 5 | Schematic of prostate ICC-LC pacemaking mechanism. Release of Ca2+
from the RyRs occurs as a Ca2+ spark (1). The Ca2+ spark activates a small number
of Ca2+ activated Cl– channels (CaCC) on the plasma membrane (2), which causes
membrane depolarisation, leading to the activation of T-type Ca2+ channels,
resulting in Ca2+ influx and an increased cytosolic [Ca2+] (3). The increased [Ca2+]i
leads to the activation of IP3Rs and regenerative Ca2+ release from the ER (4).
Thesummation of this release is sufficient to activate adequate numbers of Cl–
channels to generate STDs (5). IP3Rs are sensitized by a continuous production of
IP3 resulting from the hydrolysis of membrane bound PLC (6). Ca2+ shuttling
between the mitochondria andthe ER might also contribute to this mechanism (7).
Abbreviations: CaCC, calcium-activated chloride channels; ER, endoplasmic
reticulum; ICC-LC, interstitial cells of Cajal-like cells; IP3, inositol 1,4,5-triphosphate;
IP3R, inositol 1,4,5-triphosphate receptor; PLC, phospholipaseC; RyR,ryanodine
receptor; STD, spontaneous transient depolarization.
REVIEWS
© 2014 Macmillan Publishers Limited. All rights reserved

8
|
ADVANCE ONLINE PUBLICATION www.nature.com/nrurol
on L-type calcium channels on smooth muscle cells.108
These data suggest that imatinib might affect Ca2+ mobi-
lization in smooth muscle rather than ICC-LCs. It is also
worth noting that imatinib has also been reported to
inhibit PDGFRα signalling and these studies should be
viewed in the light that cellular signalling in PDGFRα+
i nterstitial cells might also be inhibited by imatinib.
To date, translational studies have not been performed
to determine the functional role of ICC-LC in the urethra.
Such studies are critical to evaluate whether ICC-LC are
therapeutic targets in pathophysiological conditions. As
intracellular calcium waves are essential for the genera-
tion of pacemaker activity or regulation of neuroeffec-
tor signalling that modulates the excitability of the NIPS
(Nerves, ICC-LC, PDGFRα+ cells, Smooth muscle cells)
syncytium in various tissues of the LUT, an examination
of calcium waves in ICC-LC would provide a suitable
approach to evaluate therapeutic agents on LUT function.
Conclusions
Spontaneous Ca2+ events in ICC-LC can affect smooth
muscle contraction by leading to the generation of pace-
maker electrical activity or might enable ICC-LC to act
as intermediary cells in neurotransmission. The roles of
various components of the intracellular Ca2+ machinery
have been investigated extensively. Results from these
studies combine to form a general picture throughout the
LUT, that ICC-LC fire spontaneous Ca2+ events and these
require release from the ER. In all tissues discussed, store
release via both RyRs and IP3Rs is essential, with one
receptor possibly acting as an ‘amplifier’ for the ‘initia-
tor’. These initiator events, Ca2+ sparks, have already been
observed in the prostate49 and urethra.70 In most cases,
Ca2+ influx is required to modulate the intracellular Ca2+
signal and, with the exception of the prostate, this does
not occur via voltage-gated Ca2+ channels. In the urethra,
influx occurs via reverse-mode NCX and there is some
evidence to suggest that this might also be the case in
the bladder. However, a satisfactory model of how Ca2+
oscillations are generated and propagated at the single
cell level is currently lacking. Although a defined role for
RyR-mediated and IP3R-mediated store release is appar-
ent, controversy still exists regarding which receptor has
the initial or amplification role in signal generation. The
role of mitochondria in modulating the intracellular Ca2+
signal has been outlined in the urethra and prostate, but
no such work has yet been carried out in the bladder.
Similarly, more study is required to identify the Ca2+
influx pathway, as well as to fully understand the role of
neural innervation on intracellular Ca2+ mobilization in
ICC-LC of all tissues in the LUT. This point is particu-
larly prudent in the case of the bladder, where ICC-LC
are likely candidates to act as neurotransductors to the
smooth muscle. The recent identification of a second
population of interstitial cells expressing PDGFRα in the
LUT system, which are closely apposed to nerve fibres
and respond to neurotransmitters, suggests that neuro-
modulation and hormonal modulation of motor func-
tion in these visceral tissues is more complicated than
previously thought. Further analysis of the physiologi-
cal roles of these cells in the LUT is required before we
can reach a comprehensive understanding of the normal
function and changes that occur in the activity of these
cells in disease states of these organs.
Review criteria
Literature for this Review was selected by searching for
articles using the PubMed database. Search terms used
included “Cajal”, “ICC”, “calcium”, “Ca2+”, “interstitial”,
“pacemaker” in conjunction with the terms “urethra”,
“bladder”, “prostate”, “urology”, “LUT”, “continence”.
Only full text articles were used. The reference lists of
identified papers were then checked for other papers
toinvestigate.
1. Lecoin, L., Lecoin, G. & Le Douarin, N. Origin of
the c-kit-positive interstitial cells in the avian
bowel. Development 122, 725–733 (1996).
2. Young, H.M. Embryological origin of interstitial
cells of Cajal. Microsc. Res. Tech. 47, 303–308
(1999).
3. Young, H.M., Ciampoli, D., Southwell, B.R. &
Newgreen, D.F. Origin of interstitial cells of Cajal
in the mouse intestine. Dev. Biol. 180, 97–107
(1996).
4. Sanders, K.M. A case for interstitial cells
ofCajal as pacemakers and mediators of
neurotransmission in the gastrointestinal tract.
Gastroenterology 111, 492–515 (1996).
5. Huizinga, J.D. etal. W/kit gene required for
interstitial cells of Cajal and for intestinal
pacemaker activity. Nature 373, 347–349
(1995).
6. Torihashi, S. etal. c-kit-dependent development
of interstitial cells and electrical activity in the
murine gastrointestinal tract. Cell. Tiss. Res. 280,
97–111 (1995).
7. Ward, S.M., Burns, A.J., Torihashi, S. &
Sanders, K.M. Mutation of the proto-oncogene
c-kit blocks development of interstitial cells of
Cajal and electrical rhythmicity in murine
intestine. J.Physiol. 480, 91–97 (1994).
8. Horiguchi, K., Semple, G.S., Sanders, K.M. &
Ward, S.M. Distribution of pacemaker function
through the tunica muscularis of the canine
gastric antrum. J.Physiol. 537, 237–250
(2001).
9. Beckett, E.A., Horiguchi, K., Khoyi, M.,
Sanders,K.M. & Ward, S.M. Loss of enteric
motor neurotransmission in the gastric fundus
of Sl/Sld mice. J.Physiol. 543, 871–887 (2002).
10. Burns, A.J., Lomax, A.E., Torihashi, S.,
Sanders,K.M. & Ward, S.M. Interstitial cells
ofCajal mediate inhibitory neurotransmission
inthe stomach. Proc. Natl Acad. Sci. USA 93,
12008–12013 (1996).
11. Ward, S.M. etal. Interstitial cells of Cajal
mediate cholinergic neurotransmission from
enteric motor neurons. J.Neurosci. 20,
1393–1403 (2000).
12. Won, K.J., Sanders, K.M. & Ward, S.M.
Interstitial cells of Cajal mediate
mechanosensitive responses in the stomach.
Proc. Natl Acad. Sci. USA 102, 14913–14918
(2005).
13. Fox, E.A., Phillips, R.J., Martinson, F.A.,
Baronowsky, E.A. & Powley, T.L. C-Kit mutant
mice have a selective loss of vagal
intramuscular mechanoreceptors in the
forestomach. Anat. Embryol. 204, 11–26
(2001).
14. Sergeant, G.P., Hollywood, M.A.,
McCloskey,K.D., Thornbury, K.D. &
McHale,N.G. Specialised pacemaking cells in
the rabbit urethra. J.Physiol. 526, 359–366
(2000).
15. Burton, L.D. etal. P2X2 receptor expression by
interstitial cells of Cajal in vas deferens
implicated in semen emission. Auton. Neurosci.
84, 147–161 (2000).
16. Exintaris, B., Klemm, M.F. & Lang , R.J.
Spontaneous slow wave and contractile activity
of the guinea pig prostate. J.Urol. 168, 315–322
(2002).
17. McCloskey, K.D. & Gurney, A.M. Kit positive cells
in the guinea pig bladder. J.Urol. 168,
832–836 (2002).
18. Hashitani, H. & Suzuki, H. Identification
ofinterstitial cells of Cajal in corporal tissues of
the guinea-pig penis. Br. J.Pharmacol. 141,
199–204 (2004).
19. Uckert, S. et al. C-kit-positive multipolar cells
inhuman penile erectile tissue: expression of
connexin 43 and relation to trabecular smooth
muscle cells. Georgian Med. News 180, 9–13
(2010).
REVIEWS
© 2014 Macmillan Publishers Limited. All rights reserved

NATURE REVIEWS
|
UROLOGY ADVANCE ONLINE PUBLICATION
|
9
20. Pezzone, M.A. etal. Identification of c-kit
positive cells in the mouse ureter: the interstitial
cells of Cajal of the urinary tract. Am. J.Physiol.
Cell. Physiol. 284, F925–F929 (2003).
21. Popescu, L.M. etal. Novel type of interstitial cell
(Cajal-like) in human fallopian tube. J.Cell. Mol.
Med. 9,479–523 (2005).
22. Dixon, R.E. etal. Chlamydia infection causes
loss of pacemaker cells and inhibits oocyte
transport in the mouse oviduct. Biol. Reprod. 80,
665–673 (2009).
23. Duquette, R.A. etal. Vimentin-positive,
c-kit-negative interstitial cells in human and rat
uterus: a role in pacemaking. Biol. Reprod. 72,
276–283 (2005).
24. Eken, A. etal. Immunohistochemical and
electron microscopic examination of Cajal cells
in ureteropelvic junction obstruction. Can. Urol.
Assoc. 7, E311–E316 (2013).
25. Metzger, R., Neugebauer, A., Rolle, U., Böhlig, L.
& Till, H. C-kit receptor (CD117) in the porcine
urinary tract. Pediatr. Surg. Int. 24, 67–76
(2008).
26. Lam, M., Dey, A., Lang, R.J. & Exintaris, B.
Effects of imatinib mesylate on the spontaneous
activity generated by the guinea-pig prostate.
BJU Int. 112, E398–E405 (2013).
27. Hashitani, H. Interaction between interstitial
cells and smooth muscles in the lower urinary
tract and penis. J.Physiol. 576, 707–714
(2006).
28. McCloskey, K.D. Interstitial cells in the urinary
bladder—localisation and function.
Neurourol.Urodyn. 29, 82–87 (2010).
29. Kubota, Y. etal. Role of KIT-positive interstitial
cells of Cajal in the urinary bladder and possible
therapeutic targets for overactive bladder.
Adv.Urol. 2011, 816342 (2011).
30. Wiseman, O.B., Fowler, C.J. & Landon, D.N.
Therole of the human bladder lamina propia
myofibroblast. BJUInt. 91, 89–93 (2003).
31. Johnston, L. etal. Altered distribution of
intersitial cells and innervation in the rat urinary
bladder following spinal cord injury. J.Cell. Mol.
Med. 16, 1533–1543 (2012).
32. Gray, S.M., McGeown, J.G., McMurray, G. &
McClosky, K.D. Functional innervation of guinea-
pig bladder interstitial cells of Cajal subtypes:
neurogenic stimulation evokes insitu calcium
transients. PLoSONE 8, e53423 (2013).
33. Wang, J.P., Ding, G.F. & Wang, Q.Z. Interstitial
cells of Cajal mediate excitatory sympathetic
neurotransmission in guinea pig prostate.
Cell.Tissue Res. 352, 479–486 (2013).
34. Fu, W. etal. Ultrastructural features and possible
functional role of kit-positive interstitial cells in
the guinea pig corpus cavernosum. Int. J.Impot.
Res. 23, 173–179 (2011).
35. Monaghan, K.P., Johnston, L. & McCloskey, K.D.
Identification of PDGFRα positive population
ofinterstitial cells in human and guinea-pig
bladders. J.Urol. 188, 639–647 (2012).
36. Koh, B.H. etal. Platelet-derived growth factor
receptor-α cells in mouse urinary bladder: a new
class of interstitial cells. J.Cell. Mol. Med. 16,
691–700 (2012).
37. Lee, H., Koh, B.H., Peri, L.E., Sanders, K.M. &
Koh, S.D. Functional expression of SK channels
in murine detrusor PDGFRα+ cells. J.Physiol.
591, 503–513 (2013).
38. Lee, H., Koh, B.H., Peri, L.E., Sanders, K.M.
&Koh, S.D. Purinergic inhibitory regulation of
murine detrusor muscles mediated by PDGFRα+
interstitial cells. J.Physiol. 592, 1283–1293
(2014).
39. Lang, R.J., Hashitani, H., Tonta, M.A.,
Parkington, H.C. & Suzuki, H. Spontaneous
electrical and Ca2+ signals in typical and atypical
smooth muscle cells and interstitial cell of Cajal-
like cells of mouse renal pelvis. J.Physiol. 583,
1049–1068 (2007).
40. Lang, R.J., Hashitani, H., Tonta, M.A., Suzuki, H.
& Parkington, H.C. Role of Ca2+ entry and Ca2+
stores in atypical smooth muscle cell
autorhythmicity in the mouse renal pelvis.
Br.J.Pharmacol. 152. 1248–1259 (2007).
41. Lang, R.J., Zoltkowski, B.Z., Hammer, J.M.,
Meeker, W.F. & Wendt, I. Electrical
characterisation of interstitial cells of Cajal-like
cells and smooth muscle cells isolated from the
mouse ureteropelvic junction. J.Urol. 177,
1573–1580 (2007).
42. Lang, R.J. etal. Spontaneous electrical and Ca2+
signals in the mouse renal pelvis that drive
pyeloureteric peristalsis. Clin. Exp. Pharmacol.
Physiol. 37, 509–515 (2010).
43. Lee, H.W., Baak, C.H., Lee, M.Y. & Kim, Y.C.
Spontaneous contractions augmented by
cholinergic and adrenergic systems in the
human ureter. Korean J.Physiol. Pharmacol. 15,
37–41 (2011).
44. Metzger, R., Schuster, T., Till, H., Franke, F.E.
&Dietz, H.G. Cajal-like cells in the upper urinary
tract: comparative study in various species.
Pediatr. Surg. Int. 21, 169–174 (2005).
45. Lang, R.J. & Klemm, M.F. Interstitial cells of
Cajal-like cells in the upper urinary tract. J.Cell.
Mol. Med. 9, 543–556 (2005).
46. Lang, R.J. etal. Pyeloureteric peristalsis: role
ofatypical smooth muscle cells and interstitial
cells of Cajal-like cells as pacemakers. J.Physiol.
576, 695–705 (2006).
47. Di Benedetto, A. etal. Pacemakers in the upper
urinary tract. Neurourol. Urodyn. 32, 349–353
(2013).
48. Johnston, L., Sergeant, G.P., Hollywood, M.A.,
Thornbury, K.D. & McHale, N.G. Calcium
oscillations in interstitial cells of the rabbit
urethra. J.Physiol. 565, 449–461 (2005).
49. Lam, H., Shigemasa, Y., Exintaris, B., Lang, R.J.
& Hashitani, H. Spontaneous Ca2+ signalling
ofinterstitial cells in the guinea pig prostate.
J.Urol. 186, 2478–2486 (2011).
50. Hashitani, H., Yani, Y. & Suzuki, H. Role of
interstitial cells and gap junctions in the
transmission of spontaneous Ca2+ signals in
detrusor smooth muscles of the guinea-pig
urinary bladder. J.Physiol. 559, 567–581
(2004).
51. Hashitani, H. & Lang, R.J. Functions of ICC-like
cells in the urinary tract and male genital
organs. J.Cell. Mol. Med. 14. 1199–1211
(2010).
52. Deng, J. etal. Identification of T-type calcium
channels in the interstitial cells of Cajal in rat
bladder. Urology 80, 1389.e1–e7 (2012).
53. Berridge, M. J. & Dupont, G. Spatial and
temporal signalling by calcium. Curr. Biol. 6,
267–274 (1994).
54. Berridge, M. J., Lipp, P. & Bootman, M.D. The
versatility and universality of calcium signalling.
Nat. Rev. Mol. Cell. Biol. 4, 517–529 (2000).
55. Cunningham, R.M., Larkin, P. & McCloskey, K.D.
Ultrastructural properties of interstitial cells
ofCajal in the guinea pig bladder. J.Urol. 185,
1123–1131 (2011).
56. Berridge, M.J. & Galione, A. Cytosolic calcium
oscillators. FASEBJ. 2, 3074–3082 (1988).
57. Sneyd, J., Tsaneava-Atanasova, K., Yule, D.I.,
Thompson, J.L. & Shuttleworth, T.J. Control
ofcalcium oscillations by membrane fluxes.
Proc.Natl Acad. Sci. USA 101, 1392–1396
(2004).
58. Berridge, M.J. Smooth muscle cell calcium
activation mechanisms. J.Physiol. 586,
5047–5061 (2008).
59. Sergeant, G.P., Hollywood, M.A.,
McCloskey,K.D., McHale, N.G. &
Thornbury,K.D. Role of IP3 in modulation
ofspontaneous activity in pacemaker cells of
rabbit urethra. Am.J.Physiol. Cell. Physiol. 280,
C1349–C1356 (2001).
60. Sergeant, G.P., Thornbury, K.D., McHale, N.G.
& Hollywood, M.A. Characterisation of
norepinephrine-evoked currents in interstitial
cells isolated from the rabbit urethra.
Am.J.Physiol. Cell. Physiol. 283, C885–C984
(2002).
61. Hollywood, M.A., Sergeant, G.P., McHale, N.G.
& Thornbury, K.D. Activation of Ca2+ -activated
Cl– current by depolarizing steps in rabbit
urethral interstitial cells. Am. J.Physiol. Cell.
Physiol. 285, C327–C333 (2003).
62. McHale, N.G., Hollywood, M.A., Sergeant, G.P.
& Thornbury, K.D. Origin of spontaneous
rhythmicity in smooth muscle. J.Physiol. 570,
23–28 (2006).
63. Hashitani, H. & Suzuki, H. Properties of
spontaneous Ca2+ transients recorded from
interstitial cells of Cajal-like cells of the rabbit
urethra insitu. J.Physiol. 583, 505–519
(2007).
64. Drumm, B.T. etal. The role of cAMP
dependentprotein kinase in modulating
spontaneous intracellular Ca2+ waves in
interstitial cells ofCajal from the rabbit urethra.
Cell. Calcium http://dx.doi.org/10.1016/
j.ceca.2014.07.002.
65. Bradley, J.E., Hollywood, M.A., McHale, N.G.,
Thornbury, K.D. & Sergeant, G.P. Pacemaker
activity in urethral interstitial cells is not
dependent on capacitive calcium entry.
Am.J.Physiol. Cell. Physiol. 289, C625–C632
(2005).
66. Blaustein, M.P. & Lederer, W.J. Sodium /
calcium exchange: Its physiological implications.
Physiol. Rev. 79 763–854 (1999).
67. Bradley, J.E. etal. Contribution of reverse
Na+-Ca2+ exchange to spontaneous activity in
interstitial cells of Cajal in the rabbit urethra.
J.Physiol. 574, 651–661 (2006).
68. Drumm, B.T. etal. The effect of high [K+]o on
spontaneous Ca2+ waves in freshly isolated
interstitial cells of Cajal from the rabbit urethra.
Physiol. Rep. 2, e00203 (2014).
69. Triguero, D., Sancho, M., Garcia-Flores, M. &
Garcia-Pascual, A. Presence of cyclic nucleotide-
gated channels in the rat urethra and their
involvement in nerve-mediated nitrergic
relaxation. Am. J.Physiol. Renal Physiol. 297,
F1353–F1360 (2009).
70. Drumm, B.T. etal. Role of Ca2+ influx and
diffusion in the initiation and propagation of
calcium waves in ICC freshly isolated from the
rabbit urethra. Proc. Physiol. Soc. 27, PC353
(2012).
71. Sergeant, G.P., Bradley, J.E., Thornbury, K.D.,
McHale, N.G. & Hollywood, M.A. Role of
mitochondria in modulation of spontaneous Ca2+
waves in freshly dispersed interstitial cells of
Cajal from the rabbit urethra. J.Physiol. 586,
4631–4642 (2008).
72. Hashitani, H., Lang, R.J. & Suzuki, H. Role of
perinuclear mitochondria in the spatiotemporal
dynamics of spontaneous Ca2+ waves in
interstitial cells of Cajal-like cells of the rabbit
urethra. Br. J.Pharmacol. 161, 680–694 (2010).
73. Smet, P.J., Jonavicius, J., Marshall, V.R. &
DeVente, J. Distribution of nitric oxide synthase-
immunoreactive nerves & identification of the
cellular targets of nitric oxide in guinea-pig
andhuman urinary bladder by cGMP
immunohistochemistry. J.Neurosci. 71,
337–348 (1996).
REVIEWS
© 2014 Macmillan Publishers Limited. All rights reserved

10
|
ADVANCE ONLINE PUBLICATION www.nature.com/nrurol
74. Lyons, A.D., Gardiner, T.A. & McCloskey, K.D.
Kit-positive interstitial cells in the rabbit urethra:
Structural relationships with nerves and smooth
muscle. Br. J.Urol. 99, 687–694 (2007).
75. Bradley, J.E. etal. Novel excitatory effects
ofadenosine triphosphate on contractile and
pacemaker activity in rabbit urethral smooth
muscle. J.Urol. 183, 801–811 (2010).
76. Thornbury, K.D., Hollywood, M.A., McHale, N.G.
& Sergeant, G.P. Cajal beyond the gut:
interstitial cells in the urinary system—towards
general regulatory mechanisms of smooth
muscle contractility? Acta Gastro-Enterol. Bel.
74, 536–542 (2011).
77. Bradley, J.E. etal. P2X receptor currents in
smooth muscle cells contribute to nerve
mediated contractions of rabbit urethral smooth
muscle. J.Urol. 186, 745–752 (2011).
78. Sergeant, G.P., Johnston, L., McHale, N.G.,
Thornbury, K.D. & Hollywood, M.A. Activation
ofcGMP/PKG pathway inhibits electrical activity
in rabbit urethral interstitial cells of Cajal by
reducing the spread of Ca2+ waves. J.Physiol.
574, 167–181 (2006).
79. Sancho, M., Garcia-Pascual, A. & Triguero, D.
Presence of the Ca2+-activated chloride channel
anoctamin 1 in the urethra and its role in
excitatory neurotransmission. Am. J.Physiol.
Renal Physiol. 302, F390–-F400 (2012).
80. Deng, J. etal. The effects of Glivec on the urinary
bladder excitation of rats with supra sacral or
sacral spinal cord transection. J.Surg. Res. 183,
598–605 (2013).
81. Johnston, L. etal. Morphological expression of
KIT positive interstitial cells of Cajal in human
bladder. J.Urol. 184, 370–377 (2010).
82. Rusu, M.C., Folescu, R., Manoiu, V. S. &
Didilescu, A.C. Suburothelial interstitial cells.
Cells Tissues Organs http://dx.doi.org/
10.1159/000360816.
83. Kanai, A. etal. Origin of spontaneous activity
inneonatal and adult rat bladders and its
enhancement by stretch and muscarinic
agonists. Am. J.Physiol. Renal Physiol. 292,
F1065–F1072 (2007).
84. Davidson, R.A. & McCloskey, K.D. Morphology
and localization of interstitial cells on the guinea-
pig bladder: structural relationships with smooth
muscle and neurons. J.Urol. 173, 1385–1390
(2005).
85. Wu, Y. etal. Expression and function of
muscarinic subtype receptors in bladder
interstitial cells of Cajal in rats. Urol. J.11,
1642–1647 (2014).
86. Johnston, L., Carson, C., Lyons, A.D.,
Davidson,R.A. & McCloskey, K.D.
Cholinergic-induced Ca2+ signalling in interstitial
cells of Cajal from the guinea pig bladder. Am.
J.Physiol. Renal Physiol. 294, F645–F655 (2008).
87. Kim, S.O. etal. Spontaneous electrical activity
of cultured interstitial cells of Cajal from mouse
urinary bladder. Korean J.Physiol. Pharmacol. 17,
531–536 (2013).
88. McCloskey, K.D. Calcium currents in interstitial
cells from the guinea-pig bladder. BJUInt. 97,
1383–1443 (2006).
89. Zhong, X. etal. Reverse mode of the sodium/
calcium exchanger subtype 3 in interstitial cells
of Cajal from rat bladder. Urology 82,
254.e7–e12 (2013).
90. Iino, S., Horiguchi, K., Horiguchi, S. & Nojyo, Y.
c-Kit-negative fibroblast-like cells express
platelet-derived growth factor alpha in the
murine gastrointestinal musculature.
Histochem.Cell. Biol. 131, 691–702 (2009).
91. Kurahashi, M. etal. A functional role of
fibroblast-like cells in gastrointestinal smooth
muscles. J.Physiol. 589, 697–710 (2011).
92. Baker, S.A. etal. Distribution and Ca2+
signaling of fibroblast-like cells in the murine
gastric fundus. J.Physiol. 591, 6193–6208
(2013).
93. Kurahashi, M., Mutafova-Yambolieva, V.,
Koh,S.D. & Sanders, K.M. Platelet-derived
growth factor receptor α positive cells and not
smooth muscle cells mediate purinergic
hyperpolarization in murine colonic muscles.
Am.J.Physiol. Cell. Physiol. http://dx.doi.org/
ajpcell.00080.2014.
94. Nguyen, D.T., Dey, A., Lang, R.J., Ventura, S.
&Exintaris, B. Contractility and pacemaker cells
in the prostate gland. J.Urol. 185, 347–351
(2011).
95. Exintaris, B., Nguyen, D.T., Lam, M. & Lang, R.J.
Inositol triphosphate-dependent Ca2+ stores and
mitochondria modulate slow wave activity arising
from the smooth muscle cells of the guinea pig
prostate gland. Br. J.Pharmacol. 156,
1098–1106 (2009).
96. Shigemasa, Y., Lam, M., Mitsui, R. &
Hashitani,H. Voltage-dependency of slow wave
frequency in the guinea pig prostate. J.Urol.
http://dx.doi.org/10.1016/j.juro.2014.03.034.
97. Lang, R.J., Tonta, M.A., Takano, H. &
Hashitani,H. Voltage-operated Ca2+ currents and
Ca2+-activated Cl- currents in single interstitial
cells of the guinea pig prostate. BJUInt. http://
dx.doi.org/10.111/bju.12656.
98. Nguyen, D.T., Lang, R.J. & Exintaris, B.
α1-adrenoceptor modulation of spontaneous
electrical waveforms in the guinea-pig prostate.
Eur. J.Pharmacol. 608, 62–70 (2009).
99. Dey, A., Lang, R.J. & Exintaris, B. Nitric oxide
signaling pathways involved in the inhibition of
spontaneous activity in the guinea pig prostate.
J.Urol. 187, 2254–2260 (2012).
100. Michel, M.C. & Vryday, W. α1, α2 and
β-adrenoceptors in the urinary bladder, urethra
and prostate. Br. J.Pharmacol. 147, S88–S119
(2006).
101. Biers, S.M., Reynard, J.M., Doore, T. &
Brading,A.F. The functional effects of a c-kit
tyrosine inhibitor on guinea-pig and human
detrusor. BJUInt. 97, 612–616 (2006).
102. Vahabi, B., Sellers, D.J., Bijos, D.A. &
Drake,M.J. Phasic contractions un urinary
bladder from juvenile versus adult pigs.
PLoSONE 8, e58611 (2013).
103. Gevaert, T. etal. Administration of imatinib
mesylate in rats impairs the neonatal development
of intramuscular interstitial cells of Cajal in bladder
and results in altered contractile properties.
Neurourol. Urodyn. 33, 461–468 (2013).
104. Sharp, K. G., Yee, K.M. & Steward, O.
Are-assessment of treatment with a tyrosine
kinase inhibitor (imatinib) on tissue sparing
andfunctional recovery after spinal cord injury.
Exp.Neurol. 254, 1–11 (2014).
105. Imura, M. etal. Regulation of cell proliferation
through a KIT-mediated mechanism in benign
prostatic hyperplasia. Prostate 72, 1506–1513
(2012).
106. Chakrabarty, B., Dey, A., Lam, M., Ventura, S. &
Exintaris, B. Tamsulosin modulates, but does not
abolish the spontaneous activity in the guinea
pig prostate gland. Neurourol. Urodyn. http://
dx.doi.org/10.1002/nau.22557.
107. Lam, M., Dey, A., Lang, R.J. & Exintaris, B.
Effects of imatinib mesylate on the spontaneous
activity generated by the guinea-pig prostate.
BJUInt. 112, E398–-E405 (2013).
108. Hashitani, H., Hayase, M. & Suzuki, H. Effects of
imatinib mesylate on spontaneous electrical and
mechanical activity in smooth muscle of the
guinea-pig stomach. Br. J.Pharmacol. 154,
451–4459 (2008).
Acknowledgements
This work was supported by grant support: R01
DK098388 to S.D.K. and R01 DK-57236 & P01
DK41315 to S.M.W.
Author contributions
B.T.D. and S.M.W. researched data for the article
andwrote the manuscript. B.T.D., K.-E.A. and S.M.W.
made substantial contributions to discussion of
content. All authors reviewed and edited the
manuscript before submission.
REVIEWS
© 2014 Macmillan Publishers Limited. All rights reserved