Am J Physiol Regul Integr Comp Physiol 301: R351–R362, 2011.
First published June 1, 2011; doi:10.1152/ajpregu.00656.2010.
Unique properties of muscularis mucosae smooth muscle in guinea pig
urinary bladder
Thomas J. Heppner,1* Jeffrey J. Layne,1* Jessica M. Pearson,1 Hagop Sarkissian,2 and Mark T. Nelson1
1
Department of Pharmacology and 2Department of Surgery, University of Vermont, Burlington, Vermont
Submitted 30 September 2010; accepted in final form 27 May 2011
calcium imaging; large-conductance calcium-activated potassium
channel; potassium channels; calcium flashes; spontaneous phasic
contractions
THE URINARY BLADDER is a hollow, muscular organ that serves
two functions: urine storage and elimination. The bladder wall
is composed of detrusor smooth muscle, which accounts for a
large fraction of the bladder mass, and a layer of transitional
epithelial cells known as the urothelium, which lines the
luminal surface of the urinary bladder. The detrusor smooth
muscle has been extensively characterized and is clearly responsible for the majority of the contractile properties of the
urinary bladder (1, 69). The urothelium, which was originally
thought to simply act as a passive, impermeable barrier that
* T. J. Heppner and J. J. Layne contributed equally to this work.
Address for reprint requests and other correspondence: T. J. Heppner, Dept.
of Pharmacology, Coll. of Medicine, Univ. of Vermont, Burlington, VT 05405
(e-mail: [email protected]).
http://www.ajpregu.org
prevents the movement of solutes from the urine into the
bloodstream, is now recognized to possess a broad range of
sensory and signal transduction functions that greatly influence
bladder physiology (for a review, see Ref. 5).
The detrusor exhibits two primary modes of contractile
behavior: nerve-evoked contractions and spontaneous phasic
contractions (SPCs). Nerve-evoked contractions of the detrusor
are caused by release of ATP and acetylcholine (ACh) from
parasympathetic nerve varicosities onto detrusor myocytes.
ATP and ACh bind to purinergic and muscarinic receptors,
respectively, triggering action potentials that increase intracellular Ca2⫹ and initiate contraction (31, 43, 53). In contrast,
SPCs are induced by spontaneous action potentials in detrusor
myocytes. Ca2⫹ entry through L-type voltage-dependent Ca2⫹
channels (L-VDCCs) mediates the upstroke of the detrusor
smooth muscle action potential and provides Ca2⫹ for SPCs
and nerve-evoked contractions (26, 29, 33, 53, 74). K⫹ efflux
through large-conductance Ca2⫹-activated K⫹ (BK) channels
and voltage-gated K⫹ (KV) channels is responsible for the
repolarization phase of the action potential (29, 63). KV channels contribute to the afterhyperpolarization phase along with
small-conductance Ca2⫹-activated K⫹ (SK) channels (12, 21,
25). Because BK and SK channels play such a central role in
mediating action potential repolarization, blocking them with
toxins such as iberiotoxin (IbTX) or apamin, respectively,
potentiates the detrusor smooth muscle action potential and
greatly increases the force of the resulting contractions (32, 44,
69). Activating BK channels with novel compounds such as
NS11021 (43) or SK channels with NS309 decreases contraction force (unpublished observations).
The region between the basement layer of the urothelium
and the luminal surface of the detrusor contains numerous
sensory and effector nerve fibers (4, 14) and a dense network
of blood vessels and capillaries (39, 40, 50, 51), all of which
are embedded in a connective tissue matrix. A diverse group of
cells reside in this area, including cells described as myofibroblasts, now known as interstitial cells of Cajal (ICCs) (nomenclature designated at the Fifth International Symposium on
Interstitial Cells of Cajal, Ireland, 2007). The physiological
role of these cells is unclear, but they may contribute to sensory
processing and serve a role in mediating communication between the urothelium and detrusor (for review, see Ref. 46). A
type of smooth muscle—the muscularis mucosae— has also
been identified between the basement layer of the urothelium
and the muscularis of the detrusor (10, 42, 54, 59, 67, 68).
Although the existence of the muscularis mucosae is well
established in the medical literature, particularly with respect
to its role in the development of urinary bladder cancer, its
functional role in normal urinary bladder contractility and
physiology has not been characterized. In tissues that possess a
similar layer, such as the gastrointestinal tract (for review, see
0363-6119/11 Copyright © 2011 the American Physiological Society
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Heppner TJ, Layne JJ, Pearson JM, Sarkissian H, Nelson MT.
Unique properties of muscularis mucosae smooth muscle in guinea pig
urinary bladder. Am J Physiol Regul Integr Comp Physiol 301: R351–R362,
2011. First published June 1, 2011; doi:10.1152/ajpregu.00656.2010.—The
muscularis mucosae, a type of smooth muscle located between the
urothelium and the urinary bladder detrusor, has been described,
although its properties and role in bladder function have not been
characterized. Here, using mucosal tissue strips isolated from guinea
pig urinary bladders, we identified spontaneous phasic contractions
(SPCs) that appear to originate in the muscularis mucosae. This
smooth muscle layer exhibited Ca2⫹ waves and flashes, but localized
Ca2⫹ events (Ca2⫹ sparks, purinergic receptor-mediated transients)
were not detected. Ca2⫹ flashes, often in bursts, occurred with a
frequency (⬃5.7/min) similar to that of SPCs (⬃4/min), suggesting
that SPCs are triggered by bursts of Ca2⫹ flashes. The force generated
by a single mucosal SPC represented the maximal force of the strip,
whereas a single detrusor SPC was ⬃3% of maximal force of the
detrusor strip. Electrical field stimulation (0.5–50 Hz) evoked force
transients in isolated detrusor and mucosal strips. Inhibition of cholinergic receptors significantly decreased force in detrusor and mucosal strips (at higher frequencies). Concurrent inhibition of purinergic
and cholinergic receptors nearly abolished evoked responses in detrusor and mucosae. Mucosal SPCs were unaffected by blocking smallconductance Ca2⫹-activated K⫹ (SK) channels with apamin and were
unchanged by blocking large-conductance Ca2⫹-activated K⫹ (BK)
channels with iberiotoxin (IbTX), indicating that SK and BK channels
play a much smaller role in regulating muscularis mucosae SPCs than
they do in regulating detrusor SPCs. Consistent with this, BK channel
current density in myocytes from muscularis mucosae was ⬃20% of
that in detrusor myocytes. These findings indicate that the muscularis
mucosae in guinea pig represents a second smooth muscle compartment that is physiologically and pharmacologically distinct from the
detrusor and may contribute to the overall contractile properties of the
urinary bladder.
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MATERIALS AND METHODS
Animals. Male guinea pigs (250 – 400 g) were euthanized with
isoflurane and exsanguinated according to a protocol approved by the
Institutional Animal Care and Use Committee of the University of
Vermont. The urinary bladders were removed and placed into ice-cold
dissection solution (DS) consisting of (in mM) 80 monosodium
glutamate, 55 NaCl, 6 KCl, 10 glucose, 10 HEPES, and 2 MgCl2,
adjusted to pH 7.3.
Contractility studies. Each bladder was cut open, rinsed with DS to
remove any residual urine, and pinned to a Sylgard-lined dissecting
dish with the serosal surface facing down. The bladders were trimmed
into a rectangle ⬃16 ⫻ 5 mm, and the mucosal layer was separated
from the detrusor by gently lifting up a corner of the mucosae along
a natural plane of division and then snipping away the connective
tissue linking it to the underlying detrusor. The resulting detrusor and
mucosal sheets were then cut into eight equivalent strips, each ⬃5 ⫻
2 mm. Detrusor strips were composed of detrusor smooth muscle,
whereas mucosal strips included urothelium and muscularis mucosae
smooth muscle. Intact bladder strips were prepared as described above
without separation of mucosal and detrusor layers.
The contractile activity of detrusor and mucosal strips was analyzed separately. Each strip was suspended from a force transducer in
a temperature-controlled (37°C) jacketed water bath containing physiological saline solution (PSS) consisting of (in mM) 119 NaCl, 4.7
KCl, 24 NaHCO3, 1.2 KH2PO4, 2.5 CaCl2, 1.2 MgSO4, and 11
glucose. Physiological pH was maintained by bubbling PSS with a gas
mixture of 95% O2-5% CO2. After a brief equilibration period in PSS,
each strip was stretched to ⬃10 mN of tension. Force was measured
and recorded with a MyoMed myograph system (Med Associates,
Georgia, VT). The contractile response to nerve-mediated contractions was characterized by plotting the response to electrical field
stimulation (EFS) of different frequencies (0.5, 2, 3.5, 5, 7.5, 10, 12.5,
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20, 30, 40, and 50 Hz), administered as 20-V, 0.2-ms pulses over a
duration of 2 s. The responses of tissue strips to various agonists and
antagonists, added directly to the bath, were characterized as detailed
below. Contraction amplitudes and force integrals were measured
with Mini Analysis software (Synaptosoft, Fort Lee, NJ). In mucosae,
SPCs often occurred during EFS. At lower stimulation frequencies the
amplitude of SPCs was often much larger than the evoked response,
making it very difficult to accurately measure the evoked response.
For these experiments we used peak amplitudes of SPCs before and
after each stimulus as our baseline and measured the amplitude of the
evoked response that was greater than this value. Evoked responses
less than SPC amplitude were plotted as zero.
Electrophysiology. To compare the electrophysiological properties
of detrusor and mucosal smooth muscle cells, we first separated the
mucosal layer from the detrusor as detailed above. The resulting tissue
sheets were diced into small sections (⬃2 ⫻ 2 mm), which were then
placed into a 1 mg/ml papain solution (Worthington Biochemical,
Freehold, NJ) prepared in DS containing 1 mg/ml dithioerythritol and
incubated for ⬃20 min at 37°C. After a brief rinse in ice-cold DS,
tissue sections were transferred to a vial containing 1 mg/ml collagenase (type II) and 100 ␮M CaCl2 and incubated for ⬃6 min at 37°C.
After rinsing in ice-cold DS, the tissue sections were gently triturated
by using a fire-polished Pasteur pipette to release smooth muscle cells.
The isolated cells in DS were placed in a glass-plated chamber for
⬃20 min and then immersed in a bath solution containing (in mM)
134 NaCl, 6 KCl, 10 glucose, 1 MgCl2, 2 CaCl2, and 10 HEPES,
adjusted to pH 7.3. Whole cell currents were recorded with the
perforated-patch configuration of the patch-clamp technique. Pipettes
with a resistance of 4 –5 M⍀ were filled with a solution consisting of
(in mM) 110 potassium aspartate, 30 KCl, 10 NaCl, 1 MgCl2, 0.05
EGTA, and 10 HEPES with 200 ␮g/ml amphotericin, adjusted to pH
7.3. All reagents were from Sigma-Aldrich (St. Louis, MO) unless
specifically indicated otherwise. Whole cell outward currents were
evoked with 10-mV depolarizing steps (250 ms) from an initial
holding potential of ⫺60 mV up to ⫹60 mV. Where applicable, IbTX
(100 nM; Peptides International, Louisville, KY) was added to block
BK channels, and all currents were normalized to cell capacitance
(pA/pF). All patch-clamp experiments were performed at 22–23°C.
Ca2⫹ imaging. Ca2⫹ events in the guinea pig urinary bladder were
studied in mucosal and detrusor layers, prepared as described above.
The resulting tissues were cut into thin strips (⬃1 ⫻ 3 mm), mounted
to a Sylgard block with small pins, and loaded with the Ca2⫹-sensitive
dye fluo-4 by incubation for 60 –75 min at room temperature with a
solution of fluo-4 AM (10 ␮M) and pluronic acid (2.5 ␮g/ml) in (in
mM) 134 NaCl, 6 KCl, 10 glucose, 1 MgCl2, 2 CaCl2, and 10 HEPES
(pH 7.4). After loading, the tissue was placed in a chamber specialized
for Ca2⫹ imaging and superfused with PSS at a rate of 1–2 ml/min at
37°C. Images were collected with a Noran Oz laser-scanning confocal
microscope and Nikon ⫻20 dry (NA 0.75) or ⫻60 water-immersion
(NA 1.2) fluorescent objectives with the aid of software developed by
Prairie Technologies (Middleton, WI). Fluo-4 was excited with a
krypton-argon laser at 488 nm, and the emitted fluorescence was
collected at ⬎500 nm. Each file was recorded for 30 – 60 s, and images
were collected at a rate of 53 images/s. Imaging fields were 385 ⫻ 385
␮m (256 ⫻ 256 pixels) for the ⫻20 objective and 131 ⫻ 131 ␮m (256 ⫻
256 pixels) for the ⫻60 objective. The raw data files were converted
to TIFF files and analyzed off-line with software developed in our
laboratory by Dr. Adrian Bonev. Fluorescence changes in smooth
muscle were measured by positioning boxes over regions of interest
(ROIs), identified post hoc, and recording changes in fluorescence
within these regions throughout the recording interval. The dimensions of ROIs were 15.2 ⫻ 15.2 ␮m (10 ⫻ 10 pixels) for the ⫻20
objective and 2.5 ⫻ 2.5 ␮m (10 ⫻ 10 pixels) for the ⫻60 objective.
Immunohistochemistry and histology. Tissue strips (intact, detrusor, or mucosal), prepared as detailed above, were fixed in 4%
paraformaldehyde in PSS and then embedded in paraffin blocks. Cross
sections (10 ␮m thick) were cut from the blocks and mounted on glass
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Ref. 65) and esophagus (13, 17, 38), the muscularis mucosae
has been shown to exhibit spontaneous phasic contractility and
also to respond to nerve-induced stimulation. The extent and
nature of spontaneous and nerve-evoked contractions of the
muscularis mucosae in the esophagus exhibit large speciesspecific variability (65), highlighting potential uncertainties
about their functional role in this tissue. In the colon, spontaneous phasic activity of the muscularis mucosae has been
suggested to play a role in modulating mucosal secretion (56,
57, 61), reducing mucosal surface area in response to noxious
stimuli (45, 55), and facilitating the release of acids into the
stomach (62).
The properties of detrusor smooth muscle have often been
investigated in urinary bladder strips with the mucosal or
urothelial layer removed. It has been assumed that the mucosal
layer is devoid of contractile behavior, but cells in this layer
may release factors that could affect detrusor smooth muscle
function. Contrary to this presumption, we found that mucosal
strips exhibited SPCs that can be attributed to a layer of smooth
muscle located just abluminal to the basement layer of the
urothelium—the muscularis mucosae. These SPCs likely result
from bursts of Ca2⫹ (flashes) that elevate Ca2⫹ in the mucosal
layer. The force of SPCs generated by the mucosal layer was
equivalent to that of detrusor SPCs; however, the peak force of
detrusor contractions evoked by 60 mM K⫹ was ⬃40-fold
greater than that of the muscularis mucosae. In contrast to the
detrusor, the mucosal layer exhibited very little SK and BK
channel activity. These findings suggest that the muscularis
mucosae is functionally distinct from the detrusor and may
contribute to bladder physiology.
MUCOSAL SMOOTH MUSCLE PHASIC CONTRACTIONS
RESULTS
Morphology of mucosal strips. Figure 1A shows a fluorescence micrograph of a section of intact urinary bladder (detrusor ⫹ mucosae) immunostained for smooth muscle ␣-actin
(green). Nuclei were stained with DAPI (blue). Smooth muscle
␣-actin was expressed throughout the detrusor smooth muscle
and in the walls of the blood vessels. Notably, between the
detrusor and urothelial layers there were discontinuous sheets
of tissue that stained positive for ␣-actin. In addition to smooth
muscle, some ICCs also express ␣-actin (37). Although this
layer of positive ␣-actin expression may contain both ICCs and
smooth muscle, we have termed this layer the muscularis
mucosae by analogy to similar tissue in human urinary bladder
(10, 42, 54, 59, 67, 68). Mucosal tissue strips, separated from
the detrusor along the natural plane of division (shown in Fig.
1A) and immunostained for ␣-actin, contained the urothelium,
blood vessels, and muscularis mucosae smooth muscle bundles
(Fig. 1B). Located immediately below the basement membrane
was a layer of cells that corresponds to the location of suburothelial ICCs (23). This micrograph has been overexposed to
allow visualization of the urothelium and suburothelial ICC
layer. It is clear that, even with overexposure, there is no
evidence for smooth muscle ␣-actin expression in either the
urothelium or suburothelial ICC layer. Figure 1C is a representative micrograph of a cross section of mucosal tissue
stained with H & E. The suburothelial ICC layer is clearly
visible as a band of cells, approximately three to five cells
thick, lying immediately below the basement layer of the
urothelium. Several mucosal smooth muscle bundles are also
readily apparent. Figure 1D is a schematic of the tissue layers
in the bladder wall showing the location of the muscularis
mucosae. The guinea pig urinary bladder muscularis mucosae
appears to be composed of a discontinuous band of smooth
Fig. 1. General histology of intact bladder and mucosal
strips. A: low-magnification (⫻10) image of an intact
bladder strip showing the detrusor smooth muscle layer
(D) and mucosal smooth muscle layer (MM) immunostained for smooth muscle ␣-actin (green); nuclei were
stained with 4=,6-diamidino-2-phenylindole (DAPI;
blue). The dotted line through the lamina propria indicates the approximate boundary separating the detrusor
and the mucosae. This image has been overexposed to
allow visualization of the urothelial (U) layer. B: lowmagnification image (⫻10) of a mucosal strip immunostained for smooth muscle ␣-actin. Mucosal smooth
muscle bundles (MM) are clearly evident, as are several
blood vessels. The image has been overexposed to allow
visualization of the urothelial lining (U) and the region
where suburothelial ICCs (Su-ICs) are found. C: highpower magnification (⫻40) under oil immersion of a
mucosal strip stained with hematoxylin and eosin (H &
E). Although the Su-IC layer and mucosal smooth muscle bundles show similar H & E staining, the Su-IC layer
was not positive for smooth muscle ␣-actin by immunostaining. D: schematic showing the muscularis mucosae located between the urothelium and the detrusor and
the approximate boundary in the lamina propria separating the detrusor and mucosae. Scale bars, 100 ␮m (A
and B) and 25 ␮m (C).
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slides. Some slides were stained with hematoxylin and eosin (H & E);
others were analyzed by immunohistochemistry.
For immunohistochemistry, tissue cross sections were first permeabilized in 0.2% Triton X-100 in PSS at room temperature for 10 min
and then blocked with 0.1% gelatin in PSS. Slide-mounted sections
were then incubated overnight at 4°C with a mouse anti-smooth
muscle ␣-actin antibody (Sigma-Aldrich; 1:200 dilution in 0.1%
gelatin-PSS). After rinsing in PSS, slides were incubated with fluorescent secondary antibodies (Alexa Fluor 488, 1:500 in gelatin-PSS;
Invitrogen, Carlsbad, CA) for 1 h at room temperature. Nuclei were
stained with 4=,6-diamidino-2-phenylindole (DAPI) before a coverslip
was added.
Statistics. GraphPad Software (San Diego, CA) and SigmaStat,
version 2.0 (SYSTAT Software, Chicago, IL) were used for preparation of graphs and statistical analyses. Data are reported as means ⫾
SE, where n represents the number of tissue strips (contractility
experiments, Ca2⫹ imaging) or isolated myocytes (electrophysiology
experiments). Comparisons between groups were made with a twotailed Student’s t-test; a P value ⬍ 0.05 was considered statistically
significant.
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MUCOSAL SMOOTH MUSCLE PHASIC CONTRACTIONS
similar force integrals and frequencies (Fig. 2, B and C). This
similarity is striking given the much greater smooth muscle mass
in the detrusor strips compared with the sparse distribution of
smooth muscle in the mucosal strips (Fig. 1, A and B).
Mucosal SPCs are attributable to the muscularis mucosae.
One possible origin for the SPCs in mucosal tissue strips is the
suburothelial ICC layer. However, in the guinea pig preparations, the suburothelial ICCs did not stain for smooth muscle
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Fig. 2. Comparison of spontaneous phasic contractions (SPCs) in detrusor
and mucosal strips. A: representative myograph recordings obtained from
detrusor (top) and mucosal (bottom) layers from the same urinary bladder
strip. B: comparison of the force integral of SPCs from mucosal and
detrusor strips. C: comparison of the frequency of SPCs from detrusor and
mucosal strips. Data are expressed as means ⫾ SE. N.S., not significant.
muscle sheets and bundles that are located in close proximity
to the urothelium.
Mucosal tissue strips exhibit phasic contractions. SPCs of
urinary bladder (detrusor) smooth muscle strips from guinea
pig have been extensively studied, but these studies have
typically been performed with the mucosae removed so as to
minimize possible influences from the urothelium (32, 33, 44).
To address the potential functional properties of the mucosae
and relate them to those of the detrusor, we examined the
contractile properties of isolated mucosal and detrusor strips.
Contrary to expectations, mucosal tissue strips exhibited robust
SPCs (Fig. 2A). Interestingly, the SPCs of mucosal and detrusor
strips taken from the same urinary bladder exhibited statistically
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Fig. 3. Ca2⫹ events recorded from the muscularis mucosae and detrusor.
A: bursts of Ca2⫹ flashes at regular intervals were followed by Ca2⫹ waves in
muscularis mucosae (see Supplemental Movies S1 and S2). B: purinergic Ca2⫹
transients recorded from the detrusor. These purinergic Ca2⫹ transients were
absent in recordings from the muscularis mucosae. F/F0 is the fractional
fluorescense. F0 is the baseline fluorescense determinded by averaging 30
images with no Ca2⫹ events.
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MUCOSAL SMOOTH MUSCLE PHASIC CONTRACTIONS
1
Supplemental Material for this article is available online at the Journal
website.
muscle bundles. Periodically, a Ca2⫹ flash (or more often, a burst
of Ca2⫹ flashes) occurs, followed by a quiescent period. The
frequency of Ca2⫹ flashes/bursts was 5.7 ⫾ 0.9/min (n ⫽ 15
mucosal muscle bundles; Fig. 3A), and each Ca2⫹ flash or burst
rapidly elevated global Ca2⫹ levels in the muscle bundles. Importantly, each rise in global Ca2⫹ was followed by an SPC
(compare Ca2⫹ flash/burst frequency in Fig. 3A with SPC frequency in Fig. 2C). Ca2⫹ waves were not observed during Ca2⫹
flash activity; however, as the tissue relaxed, Ca2⫹ levels dropped
and the Ca2⫹ waves again became apparent, until the next burst of
Ca2⫹ flashes. In some experiments (Supplemental Movie S2),
separate muscle bundles bridged by smaller bundles exhibited
concurrent global Ca2⫹ flashes, suggesting a high degree of
coordination between the various sheets and bundles of muscle
that constitute the muscularis mucosae.
It is possible that the bundles of smooth muscle present in
the mucosal region are simply loose bands of detrusor smooth
muscle that have been dissected away with the mucosal strip.
Although we cannot eliminate the possibility that some detrusor tissue might be present in mucosal tissue strips (and vice
versa), our Ca2⫹ signaling data argue against this being a
significant issue. Specifically, detrusor smooth muscle exhibits
characteristic, localized Ca2⫹ transients that reflect activation
of P2X1 receptors (30). In over 40 recordings of mucosal
Fig. 4. Nifedipine, but not cyclopiazonic acid (CPA),
abolished mucosal contractions. Inhibition of voltage-dependent Ca2⫹ channels (VDCCs) with nifedipine (10 ␮M)
eliminated mucosal contractions (A), but inhibition of
sarco(endo)plasmic reticulum Ca2⫹-ATPase (SERCA)
with CPA (10 ␮M) did not inhibit mucosal smooth muscle
SPCs (B). C and D: summary data showing that the force
integral was abolished by nifedipine (C) but was not
significantly altered by CPA (D). Data are expressed as
means ⫾ SE (*P ⬍ 0.05).
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␣-actin (Fig. 1B) and thus seem unlikely to possess contractile
properties. To determine whether SPCs in the guinea pig
mucosae could be specifically linked to the muscularis mucosae, we measured Ca2⫹ activity in muscularis mucosae muscle
bundles with the Ca2⫹ indicator dye fluo-4.
Detrusor smooth muscle cells exhibit four types of Ca2⫹
signals: 1) Ca2⫹ flashes, which are global events that represent
extracellular Ca2⫹ entry during an action potential; 2) Ca2⫹
waves, which have a defined wave front that moves through the
cell, are much slower than Ca2⫹ flashes, and result from inositol
trisphosphate (IP3)-mediated release of Ca2⫹ from the sarcoplasmic reticulum (SR); 3) Ca2⫹ sparks, which are brief, localized
events that represent the release of Ca2⫹ from the SR through
ryanodine receptors; and 4) purinergic Ca2⫹ transients, which are
brief, localized events that represent Ca2⫹ influx into the cell
through P2X1 receptors in response to ATP release from parasympathetic nerve varicosities (30, 74). Representative video recordings of muscularis mucosae Ca2⫹ activity are presented in Supplemental Movies S1 and S2.1 In these recordings, Ca2⫹ waves
(19.2 ⫾ 2.4 Ca2⫹ waves/min; n ⫽ 15 cells) can be seen traveling
along the muscle fibers that compose the muscularis mucosae
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release of ATP or ACh, we exposed mucosal tissue strips to
atropine (20 ␮M) to block muscarinic receptors, ␣,␤-methylene ATP (10 ␮M) to desensitize and block P2X purinergic
receptors, or TTX (10 ␮M) to block neuronal transmission.
None of these treatments significantly affected the frequency of
mucosal SPCs (initial 5.6 ⫾ 0.46/min, atropine 5.9 ⫾ 0.50/
min, ␣,␤-methylene ATP 5.3 ⫾ 0.41/min, TTX 5.8 ⫾ 0.41/
min; n ⫽ 7) or force, measured as peak amplitude (initial 0.88 ⫾
0.21 mN, atropine 0.85 ⫾ 0.23 mN, ␣,␤-methylene ATP 0.86
⫾ 0.16 mN, TTX 0.82 ⫾ 0.23 mN; n ⫽ 7). The persistence of
SPCs in the presence of these inhibitors indicates that they are
most likely nonneurogenic in origin and are not driven by the
release of ACh or ATP from submucosal nerve terminals or the
urothelium.
Ca2⫹ requirements of mucosal SPCs. The Ca2⫹ required for
muscle contractions may be derived from intracellular or extracellular sources, or a combination of the two, depending on
the nature of the muscle (1, 3). In the urinary bladder, treatment
with selective L-VDCC inhibitors (e.g., nifedipine) strongly
inhibits or abolishes spontaneous detrusor activity, indicating
that detrusor contractility depends on the influx of extracellular
Ca2⫹ (26, 29). Although SR Ca2⫹ stores are presumed to
contribute to cholinergic detrusor contractions (3, 20), the role
Fig. 5. Detrusor strips generate significantly greater
force than mucosal strips in response to membrane
depolarization with 60 mM K⫹. A and B: representative
myograph recordings from a detrusor (A) and a mucosal
(B) smooth muscle strip. C and D: comparison of the peak
force of contractions (C) and the phasic amplitude (D)
evoked by 60 mM K⫹. Data are expressed as means ⫾ SE
(*P ⬍ 0.05).
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smooth muscle, such Ca2⫹ transients were never detected.
They were, however, readily apparent in recordings from
detrusor smooth muscle: 8 of 15 detrusor smooth muscle
preparations exhibited spontaneous purinergic Ca2⫹ transients
with a frequency of 3.9 ⫾ 0.9 events·30 s⫺1·field⫺1 (Fig. 3B).
Thus the Ca2⫹ activity of urinary bladder muscularis mucosae
smooth muscle appears to be limited to Ca2⫹ waves and bursts
of Ca2⫹ flashes, typified by the recording shown in Fig. 3A.
The results of our Ca2⫹ imaging indicate that the SPCs of
the guinea pig mucosae most likely arise from bursts of Ca2⫹
flashes in smooth muscle sheets and bundles that compose the
muscularis mucosae. In addition, despite the spatial separation
of mucosal smooth muscle bundles and sheets, mechanisms
that enable a high degree of coordination between the discrete
islands of smooth muscle appear to exist. Finally, the unique
Ca2⫹ signature of the muscularis mucosae indicates that it is
physiologically distinct from the detrusor.
Mucosal phasic contractions are nonneurogenic and spontaneous. The urothelium is known to release both ATP and ACh
(19, 41, 73), and the submucosal space is richly innervated
with nerve fibers that could also potentially release ATP and
ACh (15, 24, 70). To determine whether mucosal phasic
contractions were being driven by urothelial and/or neuronal
MUCOSAL SMOOTH MUSCLE PHASIC CONTRACTIONS
spontaneous contraction of the detrusor only recruits a small
fraction of the total muscle fibers present in the detrusor strip,
whereas each SPC of the mucosal strip recruits a relatively
large proportion of the available smooth muscle. If this is the
case, then the total maximum force-generating capacity of the
detrusor strips should be much greater than that of the mucosal
strips, despite the similarity in spontaneous contraction force.
To investigate this possibility, we contracted mucosal and
detrusor tissue strips by depolarizing with 60 mM K⫹. As
shown in the original recordings (Fig. 5, A and B), depolarization with 60 mM K⫹ had a profoundly greater effect on the
contractile response of detrusor strips, inducing approximately
a 40-fold greater increase in peak force in these strips (36.12 ⫾
7.33 mN) than in mucosal strips (0.88 ⫾ 0.21 mN; P ⬍ 0.05,
n ⫽ 6; Fig. 5C). In addition, the steady-state force (tone)
generated in response to 60 mM K⫹ (baseline in Fig. 5, A and
B) was significantly greater for the detrusor strips (11.22 ⫾
4.53 mN) than the mucosal strips (0.67 ⫾ 44 mN; P ⬍ 0.05,
n ⫽ 6). Finally, the amplitude of the phasic contractions in the
presence of 60 mM K⫹ was also significantly elevated in
detrusor strips (9.15 ⫾ 1.56 mN) compared with mucosal strips
(0.81 ⫾ 0.74 mN; Fig. 5D; P ⬍ 0.05, n ⫽ 8). These findings
indicate that, although the force of mucosal SPCs may be
equivalent to those of the detrusor, the detrusor has the potential to generate much more force than the mucosal smooth
muscle. Interestingly, in the mucosal strips, the peak force
amplitude generated by treatment with 60 mM K⫹ was not
Fig. 6. Electrical field stimulation (EFS; 0.5–50 Hz) evoked frequency-dependent contractions in detrusor and mucosal strips. Aa–Ad: original recordings from
detrusor and mucosae before and after the addition of atropine to block muscarinic receptors, and atropine ⫹ ␣,␤-methylene ATP (␣,␤-meATP) to block
muscarinic receptors and desensitize P2X1 receptors, respectively. Arrows indicate EFS applications in mucosal strips in which the evoked responses were smaller
than the SPCs. Ba–Bd: detrusor (Ba, Bb) and mucosae (Bc, Bd) summary graphs showing amplitudes in control and after muscarinic and purinergic receptor
inhibition. For mucosae, the amplitude of evoked responses was often less than that of SPCs and could not be accurately measured (see MATERIALS AND METHODS).
Responses less than the mean amplitude of SPCs are plotted as zero. Data are expressed as means ⫾ SE (*P ⬍ 0.05).
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of this pathway is still incompletely understood (1, 18, 72).
However, it is well established that SR Ca2⫹ release via
ryanodine receptors in the form of Ca2⫹ sparks plays a role in
opposing detrusor contraction (34, 35, 49).
To determine the relative importance of intracellular and
extracellular Ca2⫹ in mediating mucosal smooth muscle SPCs,
we treated mucosal tissue strips with nifedipine (10 ␮M) to
block L-VDCCs or cyclopiazonic acid (CPA, 10 ␮M) to
deplete SR Ca2⫹ stores. Blocking L-VDCCs rapidly, and
almost completely, eliminated mucosal SPCs (Fig. 4, A and C;
P ⬍ 0.05, n ⫽ 7). In contrast, SR store depletion by CPA
increased baseline tension (control 0.6 ⫾ 0.2 mN, CPA 1.2 ⫾
0.2 mN; P ⬍ 0.05, n ⫽ 7), presumably reflecting elevated
intracellular Ca2⫹ levels caused by blockade of the sarco(endo)plasmic reticulum Ca2⫹-ATPase (SERCA) pump, but did
not significantly elevate the frequency (control 4.6 ⫾ 0.4/min,
CPA 6.3 ⫾ 0.6/min; P ⬎ 0.05; n ⫽ 7) or force integral (P ⬎
0.05; n ⫽ 7) of SPCs (Fig. 4, B and D). These results suggest
that mucosal smooth muscle SPCs are dependent on the influx
of extracellular Ca2⫹ through L-VDCCs but not on SR Ca2⫹
release.
Force generation by mucosal smooth muscle. The force
generated by mucosal SPCs was similar to that of the matched
detrusor strips (Fig. 2). This observation was intriguing because the smooth muscle content in the mucosal strips is
clearly lower than that in the detrusor strips (Fig. 1, A and B).
One possible explanation for this observation is that each
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AJP-Regul Integr Comp Physiol • VOL
Fig. 7. Large-conductance Ca2⫹-activated K⫹ (BK) channel block has a
greater effect on the SPCs of detrusor than mucosal strips. A and B: representative myograph recordings obtained from a detrusor (A) and a mucosal (B)
smooth muscle strip exposed to 100 nM IbTX. C: IbTX significantly increased
the force integral of SPCs in detrusor but not in mucosae. Data are expressed
as means ⫾ SE (*P ⬍ 0.05).
relationship of end-pulse currents (Fig. 8C) shows that the
whole cell IO in mucosal smooth muscle cells was lower at all
positive membrane potentials evaluated (P ⬍ 0.05). At ⫹60
mV, IO was 13.9 ⫾ 2.3 pA/pF (n ⫽ 13) in detrusor myocytes
and 6.9 ⫾ 0.8 pA/pF (n ⫽ 17) in mucosal myocytes—
approximately a twofold difference.
The BK channel blocker IbTX (100 nM) was used to
determine the IBK densities in isolated myocytes. IbTX had a
greater effect on outward currents in detrusor cells than on
those in mucosal smooth muscle cells (Fig. 8, A and B, center);
thus the resulting IbTX difference current (Fig. 8, A and B,
right) was much greater in the detrusor cells. The I-V curves
for end-pulse currents (Fig. 8D) show that IBK was lower in
mucosal smooth muscle cells than in detrusor smooth muscle
cells, a difference that was significant at membrane potentials
of ⫹40 mV and above (P ⬍ 0.05, n ⫽ 13 cells each). At ⫹60
mV, the current density was 5.20 ⫾ 1.15 pA/pF in detrusor
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significantly greater (12.2% ⫾ 2.2%; n ⫽ 6) than the amplitude
of the SPCs that preceded it (Fig. 5B), suggesting that virtually
all contractile cells available within the mucosal strip are
recruited by mucosal SPCs.
EFS evokes contraction of detrusor and mucosal strips. In
addition to SPCs, the urinary bladder also contracts forcefully
to EFS. To study the response of the detrusor and muscularis
mucosae to EFS, we prepared tissue strips as described in
MATERIALS AND METHODS and placed them in tissue baths adjacent to stimulating electrodes. EFS (0.5–50 Hz) was used to
stimulate contractions in detrusor and mucosal strips (Fig. 6).
To measure the contribution of muscarinic and purinergic
pathways to contractions, we applied atropine (10 ␮M) and/or
␣,␤-methylene ATP (10 ␮M) to inhibit muscarinic and purinergic receptors, respectively. In detrusor strips, EFS caused a
frequency-dependent increase in force. Atropine produced a
frequency-dependent reduction in the amplitude of EFS-induced contractions, ranging from 47.4% at 0.5 Hz to 69.6% at
50 Hz (Fig. 6, Aa and Ba; n ⫽ 5 strips/group). In the presence
of both atropine and ␣,␤-methylene ATP, EFS-induced contractions were nearly abolished (mean reduction in amplitude
for all frequencies 96.0%; n ⫽ 5; Fig. 6, Ab and Bb). In
contrast, EFS evoked small increases in mucosal force that
were only evident at higher frequencies. At lower stimulating
frequencies, the evoked responses could not be accurately
measured because of the presence of similar-sized SPCs. Inhibition of mucosal muscarinic receptors with atropine reduced
force amplitude at higher stimulating frequencies (Fig. 6, Ac
and Bc). In the presence of both atropine and the P2X1 receptordesensitizing agent ␣,␤-methylene ATP, evoked responses
were not detected (Fig. 6, Ad and Bd). The maximum amplitude of the evoked contractions in detrusor strips was ⬃10-fold
greater than in mucosae, likely reflecting the much larger
muscle mass of the detrusor.
BK channel expression and activity in mucosal smooth
muscle. Given the prominent role that BK channels play in
regulating detrusor contractility (9, 25, 29, 32, 33, 58, 69), we
sought to determine their role in the regulation of mucosal
smooth muscle contractility. To assess the relative importance
of BK channels in the regulation of mucosal smooth muscle
SPCs, we tested the effects of the BK channel blocker IbTX
(100 nM) on the SPCs of mucosal and detrusor tissue strips. As
expected, block of BK channels with IbTX greatly enhanced
SPCs in detrusor strips (Fig. 7, A and C), increasing the force
integral by almost fivefold (P ⬍ 0.05, n ⫽ 11 strips). In
contrast, block of BK channels did not have a significant effect
on mucosal SPCs (n ⫽ 16 strips; Fig. 7, B and C).
The feeble response of mucosal SPCs to IbTX suggests that
the level of BK channel expression in mucosal smooth muscle
is low relative to that in the detrusor. To investigate this
possibility, we used the patch-clamp technique to compare
IbTX-sensitive BK currents (IBK) in myocytes isolated from
detrusor and mucosal smooth muscle. Whole cell currents were
evoked from freshly isolated myocytes with 10-mV depolarizing steps from a holding potential of ⫺60 mV up to ⫹60 mV.
With a capacitance of 32.3 ⫾ 2.3 pF (n ⫽ 13), detrusor smooth
muscle cells were significantly larger (P ⬍ 0.05) than mucosal
smooth muscle cells (capacitance 25.9 ⫾ 1.6 pF; n ⫽ 17). The
peak outward current density (IO) of mucosal smooth muscle
cells was significantly less than that of detrusor smooth muscle
cells (Fig. 8, A and B, left). The resultant current-voltage (I-V)
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MUCOSAL SMOOTH MUSCLE PHASIC CONTRACTIONS
smooth muscle cells and only 0.92 ⫾ 0.60 pA/pF in mucosal
smooth muscle cells. Collectively, the results of these patchclamp experiments suggest that the diminished contractile
response to IbTX in mucosal tissue strips compared with that
in detrusor strips (Fig. 7) may be due to a reduction in the
density of IBK in mucosal smooth muscle myocytes.
SK channel activity is minimal or absent in mucosal smooth
muscle. Urinary bladder contractility is also modulated by K⫹
efflux through SK channels (27, 33, 35), which contribute to
the afterhyperpolarization of the urinary bladder smooth muscle action potential (12, 21); blocking SK channels results in an
increase in detrusor SPCs (9, 27, 33, 35). To determine whether
SK channel activity, like BK channel activity, was different
between mucosal and detrusor myocytes, we first compared the
effects of the SK channel blocker apamin (300 nM) on mucosal
and detrusor smooth muscle SPCs. Representative myograph
recordings of detrusor (Fig. 9A) and mucosal (Fig. 9B) smooth
muscle strips clearly show a prominent effect of apamin on
detrusor strips and essentially no effect on mucosal strips.
Apamin increased the force integral of detrusor SPCs to 335.5 ⫾
79.7% of the initial force (P ⬍ 0.05, n ⫽ 9) but had no
significant effect on mucosal SPCs (n ⫽ 17; Fig. 9C).
AJP-Regul Integr Comp Physiol • VOL
DISCUSSION
In the present study, we provide the first characterization of
the pharmacology and physiology of urinary bladder muscularis mucosae smooth muscle. Mucosae and detrusor strips
exhibited SPCs of similar amplitudes and frequencies that
depended on functional L-VDCCs. EFS induced a frequencydependent increase in contraction amplitude in detrusor and
mucosal strips that was nearly abolished by inhibition of both
muscarinic and purinergic receptors. However, muscularis mucosae smooth muscle also exhibited distinctly different properties than detrusor smooth muscle. The maximal force-generating capacity (in response to 60 mM K⫹) of detrusor strips
was ⬃40-fold greater than that of mucosal strips. Indeed, the
peak force of mucosal strip SPCs was similar to that induced
by 60 mM K⫹, suggesting that mucosal SPCs engage all
muscle bundles in a strip. The minimal effect of BK and SK
channel blockers on mucosal SPCs is striking compared with
the robust effects of these inhibitors on detrusor contractility.
Contractile properties of the muscularis mucosae. The similarity of SPC amplitude and frequency between mucosal and
detrusor strips from the same bladder is particularly striking.
One explanation for this finding is that similar numbers of
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Fig. 8. BK channel current density is much
larger in detrusor myocytes than in mucosal
myocytes. A and B: representative outward
current density (IO) (Initial; left) and IO after
inhibition of BK channels (⫹IbTX; center);
difference currents (right) obtained from a
detrusor (A) and a mucosal (B) smooth muscle cell represent IbTX-sensitive BK current
(IBK). C: comparison of the current-voltage
(I-V) relationship for IO (Initial) evoked
from ⫺60 to ⫹60 mV for isolated detrusor
and mucosal smooth muscle cells. D: comparison of the I-V relationship for IBK
evoked by 10-mV voltage steps from ⫺60 to
⫹60 mV for isolated detrusor and mucosal
smooth muscle cells. Data are expressed as
means ⫾ SE (*P ⬍ 0.05).
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muscle bundles in the two strips are being driven by the same
type of pacemaker. ICCs have been identified in both the
mucosae and detrusor, although their function is still unclear (47,
71). Indeed, we observed Ca2⫹ signals in structures linking
muscle bundles in the mucosae (see Fig. 3 and Supplemental
Movies S1 and S2). Our data indicate that a SPC engages most of
the smooth muscle in a mucosal strip. In contrast, based on our
measurements, SPCs of the detrusor represent activation of a
small fraction (⬃3%) of the muscle in a strip.
Consistent with this, it has been shown that current injection
into one detrusor myocyte cannot be detected at a distance of
even a few cells from the microelectrode source (8). Similarly,
Gillespie and colleagues (16) have shown that ex vivo whole
bladder preparations exhibit “microcontractions” that are
highly localized to discrete regions of the bladder and do not
propagate across the whole bladder (these bladders also exhibited more complex, integrated contractions as well).
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Fig. 9. Small-conductance Ca2⫹-activated K⫹ (SK) channel block with apamin
had no effect on mucosal strips. A and B: representative myograph recordings
from detrusor (A) and mucosal (B) strips exposed to the SK channel blocker
apamin (300 nM). C: apamin significantly increased the force integral of SPCs
in detrusor but not in mucosae. Data are expressed as means ⫾ SE (*P ⬍ 0.05).
The force of evoked contractile responses in muscularis mucosae, whether induced by EFS or high K⫹, was comparable to that
of SPCs. This was true even at higher stimulating frequencies,
consistent with EFS engaging all of the smooth muscle bundles in
the mucosal strip. There is little information on the innervation of
the muscularis mucosae, although the suburothelial region surrounding the muscularis mucosae is richly innervated (4,
14). Studies of the digestive tract have shown that the
innervation of the muscularis mucosae varies greatly among
different regions and between species and includes cholinergic, peptidergic, and adrenergic nerve fibers (65).
We found that mucosal phasic contractions were absolutely
dependent on Ca2⫹ influx through L-VDCCs, a property that is
shared by detrusor smooth muscle (27, 29). Interestingly, it has
been reported that gastrointestinal muscularis mucosae is insensitive to L-type channel antagonists (64, 66). Like SPCs in the
detrusor (26), the SPCs of bladder mucosal smooth muscle do not
appear to require SR Ca2⫹ stores. The muscularis mucosae exists
in close proximity to the urothelium, nerve fibers, and ICCs, all of
which could be potential sources of contractile modulators (2, 6,
28, 48, 52). However, the phasic contractions exhibited by mucosal strips appear to be nonneurogenic and spontaneous in origin,
as they were not affected by inhibition of muscarinic receptors,
purinergic receptors, or voltage-dependent sodium channels. Recently, Burcher and colleagues (60) reported that mucosal strips of
pig urinary bladder, denuded of the detrusor smooth muscle layer,
contract in response to carbachol and neurokinin A. The authors
attributed this contractile response to the suburothelial ICCs rather
than to the muscularis mucosae. They did, however, note that
sparse smooth muscle was still present in these mucosal strips.
Although we cannot definitively rule out the possibility that
suburothelial ICCs may contribute to the SPCs of mucosal strips,
we found that ␣-actin was only expressed in the discrete bundles
of smooth muscle within the mucosae (Fig. 1). Furthermore, Ca2⫹
flashes, which reflect action potentials, were observed in cells
resembling smooth muscle in the mucosal layer, and these flashes
preceded each spontaneous contraction event (see Fig. 3A and
Supplemental Movie S1). It is possible, however, that ICCs play
some role in coordinating mucosal contractions.
The physiological role of SPCs in urinary bladder has been the
focus of numerous studies; however, the precise role of SPCs
remains unclear. These small, spontaneous nonvoiding contractions, which occur in parts of the bladder wall, have also been
called micromotions (11) or microtransients (16). These events
may be important during the filling phase, playing a role in
adjusting the length of smooth muscle fibers in response to filling
(7) or communicating information on bladder fullness (11, 16,
22). Because the contractile force of muscularis mucosae contractions is ⬃40 times less than that of the detrusor, these events
would be expected to contribute minimally to micturition.
BK and SK channels in muscularis mucosae. The contractility of detrusor smooth muscle is profoundly influenced by
BK and SK channels (27, 32, 33, 69). Given the major role that
these two ion channel types play in modulating the excitability
and contractility of detrusor smooth muscle, it was surprising
that neither seemed to play a role in modulating the SPCs in
mucosal smooth muscle. Neither blocking BK channels nor
blocking SK channels had a significant effect on mucosal SPCs
(Figs. 7 and 9). BK channel current density was also significantly lower in mucosal than in detrusor myocytes (Fig. 8). We
have previously characterized SK currents in detrusor myo-
MUCOSAL SMOOTH MUSCLE PHASIC CONTRACTIONS
cytes (36) and did not explore this issue in mucosal myocytes
because the SK channel blocker apamin did not affect mucosal
contractility (Fig. 9). These results indicate that mucosal smooth
muscle has an ion channel composition distinctly different from
that of detrusor myocytes. Taken together, the results of myography experiments (SK and BK channels) and electrophysiological
data (BK channels) suggest that mucosal smooth muscle has
properties that clearly distinguish it from detrusor smooth muscle.
Perspectives and Significance
ACKNOWLEDGMENTS
We thank Drs. David Hill-Eubanks and Bernhard Nausch for critical
reading of this manuscript.
GRANTS
This work was supported in part by the Totman Trust for Medical Research,
National Institutes of Health (NIH) Grants DK-053832 and DK-065947 to
M. T. Nelson, a Physiology Training grant to J. J. Layne (PHS T32-HL007944), and NIH Grant 2-P20-RR-016435-06 from the COBRE Program of
the National Center for Research Resources.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
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In the present study we describe a separate compartment of
smooth muscle—the muscularis mucosae—located just below
the urothelium in guinea pig urinary bladder that underlies the
SPCs in urothelial strips. The unique Ca2⫹ activity signature
and pharmacological properties of the muscularis mucosae,
such as its relative unresponsiveness to BK and SK channel
blockers, indicate that this tissue layer is functionally distinct
from the detrusor smooth muscle.
SPCs are found in the urinary bladder of many species,
including humans, yet their role is not well understood. They
occur normally during bladder filling and may contribute to
maintaining blood flow during periods of increased intravesicle
pressure as would occur during bladder filling; alternatively,
they may provide information on bladder fullness (22). Histological stains and Ca2⫹ imaging show a thin layer of smooth
muscle—the muscularis mucosae— composed of connecting
sheets and bundles that suggests synchronization over a large
area. The muscularis mucosae is nonneurogenic and contracts
in rhythmic bursts, suggesting local regulation, possibly
through a network of ICC-type cells. Rhythmic bursts or SPCs
with frequency and amplitude similar to those in the mucosae
are also found in isolated strips of detrusor. The muscularis
mucosae may contribute to the increase in nonvoiding contractions found in certain bladder pathologies, such as overactive
bladder, and would provide a novel target for the development
of pharmaceuticals to alleviate this condition.
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