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Blackwell Munksgaard
Copyright
#
Blackwell Munksgaard 2004
doi: 10.1046/j.1600-0854.2003.00156.x
Review
The Uroepithelium: Not Just a Passive Barrier
Gerard Apodaca
Renal-Electrolyte Division of the Department of Medicine,
Laboratory of Epithelial Cell Biology, and Department of
Cell Biology and Physiology, University of Pittsburgh,
Pittsburgh, PA 15261, USA, [email protected]
The uroepithelium lines the inner surface of the renal
pelvis, the ureters, and the urinary bladder, where it
forms a tight barrier that allows for retention of urine,
while preventing the unregulated movement of ions,
solutes, and toxic metabolites across the epithelial barrier. In the case of the bladder, the permeability barrier
must be maintained even as the organ undergoes cyclical
changes in pressure as it fills and empties. Beyond
furthering our understanding of barrier function, new
analysis of the uroepithelium is providing information
about how detergent-insoluble membrane/protein
domains called plaques are formed at the apical plasma
membrane of the surface umbrella cells, how mechanical
stimuli such as pressure alter exocytic and endocytic
traffic in epithelial cells such as umbrella cells, and how
changes in pressure are communicated to the underlying
nervous system.
Key words: bladder, endocytosis, epithelium, exocytosis,
mechanical stimuli, neural–epithelial interactions, uroepithelium, uroplakin, pressure
Received and accepted for publication 3 November 2003
Epithelial cells cover external surfaces or line the inner
surfaces of organ systems such as the gut, blood vessels,
and urogenital tract. In the case of the lower urinary tract
(including the renal pelvis, ureters, and bladder), the surface is coated by a specialized epithelium called the uroepithelium. The uroepithelium is stratified and is comprised
of three cell types including basal cells, intermediate cells,
and umbrella cells. Basal cells are small ( 10 mm in diameter), form a single layer that contacts the underlying
connective tissue and capillary bed, and serve as precursors
for the other cell layers. Their estimated half-life is
3–6 months, although estimates are hard to make because
their mitotic index is very low (on the order of 0.1–0.5%)
(1,2). Intermediate cells are pyriform in shape (10–25 mm in
diameter), sit on top of the underlying basal cells, and form a
layer that appears in cross-section anywhere from one to
several cell layers thick. In some species the intermediate
cells have long, thin cytoplasmic processes that connect
them to the basement membrane (1–3). The outermost
umbrella cell layer is comprised of very large polyhedral cells
with diameters of 25–250 mm (Figure 1). In some species,
such as rat and guinea pig, multinucleate cells are common, and like intermediate cells they can also have thin
projections that connect them to the basement membrane
(3). Although umbrella cells are long lived, they are rapidly
regenerated when the uroepithelium is damaged. This
regeneration can result from cell division within any of
the three cell layers, and generation of the multinucleate
umbrella cells is likely the result of intermediate cell–cell
fusion (2,3).
A primary function of epithelia, including the uroepithelium, is to form a barrier that prevents entry of pathogens
and selectively controls the passage of water, ions,
solutes, and large macromolecules across the mucosal
surface of the cell into the underlying tissue. Barrier function depends, in part, on the presence of specialized membrane domains that form a seal between the plasma
membranes of adjacent epithelial cells. In the case of the
uroepithelium, high resistance tight junctions are found in
the umbrella cell layer that effectively divide the cell surface of these cells into apical and basolateral membrane
domains (4). In addition, the apical membrane of umbrella
cells has a unique lipid and protein composition that also
contributes to the low permeability of this membrane
domain to water and solutes (4–6).
The uroepithelium was long treated as an impermeable
cellular plastic coating that allowed for urine storage, and
the majority of analyses of the lower urinary tract have
focused on the bladder musculature and innervation. A
renewed interest in the uroepithelium indicates that it
can alter the ion and protein composition of the urine
(4,7,8), and study of the uroepithelium is providing new
insight into how specialized membrane domains are
assembled, how epithelial cells sense and respond to
mechanical stimuli such as pressure, and how epithelial
cells may communicate mechanical stimuli to the nervous
system. This review will summarize new information pertinent to each of these areas of inquiry.
Specialized Membrane Domains at the Apical
Surface of the Umbrella Cell
The apical surface of umbrella cells contains unique structural and biochemical features. When examined by scanning electron microscopy the surface of the umbrella cells,
such as those from rabbit bladders, appears pleated and
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Apodaca
A
UC
UC
IC
10 µm
IC
IC
B
IC
IC
IC
20 µm
Figure 1: The umbrella and intermediate cell layers. A
transmission electron micrograph of rabbit uroepithelium shows a
bi-nucleate umbrella cell and underlying intermediate cells. The location
of the tight junction between adjacent umbrella cells is marked with
an arrow. Legend: UC, umbrella cell; IC, intermediate cell.
each cell is surrounded by a tight junctional ring (Figure 2A).
Higher magnification views show that the surface is covered by raised ridges, also called hinges or microplicae,
and intervening areas called plaques (Figure 2B). The
arrangement of hinges and plaques give the apical surface
its characteristic scalloped appearance, which is apparent
when the apical surface of cross-sectioned umbrella cells
is viewed by transmission electron microscopy (Figure 3A).
The hinge areas are not well understood, but contain at
least one unique protein called urohingin (9), and presumably all other non-plaque proteins. Plaques are thought to
occupy approximately 70–90% of the surface of the
umbrella cell (2,10,11).
The membrane associated with the hinge and plaque
regions is highly detergent insoluble, even in relatively
harsh detergents like sarkosyl (12). The detergent insolubility, described some 30 years ago, may reflect the
unusual lipid composition of this membrane, which is rich
in cholesterol, phosphatidyl choline, phosphatidyl ethanolamine, and cerebroside – a lipid profile similar to myelin
(13). Cholesterol-rich and detergent-insoluble membranes
such as ‘rafts’ and caveolae have recently received
considerable attention (14). Essentially, the entire apical
surface of the umbrella cell is composed of two lipid raft
subdomains: plaques and hinges, and study of these
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H
5 µm
Figure 2: Ultrastructure of umbrella cell apical membrane. (A)
Scanning electron micrograph of mucosal surface of rabbit
umbrella cell layer. The tight junction ring of an individual
umbrella cell is demarcated by arrows. (B) High magnification
view of apical surface of umbrella cell. Examples of hinges (‘H’)
are marked with arrows.
subdomains may provide important clues to how
detergent-insoluble rafts are formed and how they function.
An additional characteristic of the membrane associated
with the plaque regions is that the outer leaflet appears to
be twice as thick as the inner leaflet, thus forming an
asymmetric unit membrane (AUM; Figure 3B) (10,15,16).
The AUM is composed of a paracrystalline array of 16-nm
diameter AUM particles that are apparent when detergentsolubilized membranes are negatively stained, or when the
apical membrane is examined using high-resolution, quickfreeze, deep-etch techniques (Figure 4). AUM particles
exhibit six-fold symmetry, are composed of an inner ring
containing six large particles and an outer ring containing
six small particles, and each subunit forms a twisted ribbon
structure (16). A plaque is comprised of approximately
1000–3000 AUM particles.
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The Uroepithelium
A
P
50 nm
H
P
100 nm
Figure 4: Asymmetric unit membrane (AUM) particles at the
apical surface of the umbrella cell. A rapid-freeze, deep-etch
micrograph shows hinge areas (‘H’) and plaques (‘P’). Inset: higher
magnification view of AUM particles found in plaque region.
1 µm
B
0.5 mm
Figure 3: Surface specializations at the apical surface of the
umbrella cell. (A) Transmission electron micrograph of crosssectioned rat umbrella cell apical membrane demonstrating hinge
and plaque regions. Hinge areas are marked with arrows. The
intervening areas of plasma membrane are plaques. The boxed area
is magnified in panel B. (B) High magnification view of asymmetric
unit membrane (AUM). Hinges are marked with arrows, and a
plaque with readily identifiable AUM is marked with an arrowhead.
Potential constituents of the AUM particles include the
uroplakins (UPs), a family of at least five proteins including
the tetraspan family members UPIa and UPIb, and the type
I single-span proteins UPII, UPIIIa, and UPIIIb (Figure 5)
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(17,18). The latter was only recently described (18). UPIa,
UPII, UPIIIa, and UPIIIb are only expressed in the uroepithelium and are concentrated in the umbrella cell layer.
UPIb is also expressed in the cornea and conjunctiva (19).
UPIa serves as a receptor for uropathogenic Escherichia
coli (20,21). How UPs combine to generate the six-fold
symmetry of the AUM particles is currently unknown.
Furthermore, the currently described UPs may not be the
sole constituents of AUM particles, as there are other
proteins associated with plaques that have not been
characterized. These include the antigen recognized by
the AE-31 monoclonal antibody, an uncharacterized 27 kDa
protein localized to plaques and originally thought to be
UPI (22).
Assembly of plaque proteins
The membrane trafficking and assembly of UPs into AUM
particles is only now being analyzed. In general, this analysis has been hampered by the lack of a polarized uroepithelial cell line that forms AUM particles, although a
recently described primary uroepithelial culture model
that is highly polarized, expresses UPs, and forms AUMs
may be useful in this regard (23). Biochemical experiments
using purified proteins established that UPIa pairs with
UPII and UPIb pairs with UPIIIa or UPIIIb (18,24). The
physiological significance of these paired interactions
was recently elucidated when UPs were ectopically
expressed alone or in combination in human embryonic
kidney 293T cells (18,25). The only singly expressed UP
that can exit the endoplasmic reticulum (ER) and be delivered to the cell surface is UPIb. Exit from the ER and
surface delivery of UPII is dependent on coexpression
with UPIa, and exit and delivery of UPIIIa or UPIIIb is
dependent on coexpression of UPIb. These results indicate that the initial steps in AUM particle assembly involve
heterodimer formation within the ER. It is unknown if
heterodimer formation occurs in umbrella cells or if ectopic
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Apodaca
UP Ia
UP II
UP IIIa
UP Ib
UP IIIb
Figure 5: The uroplakins. UPIa and UPIb are members of the tetraspanning family of proteins that cross the plasma membrane four times.
UPII, UPIIIa, and UPIIIb are single-pass type I membrane proteins. They share a conserved domain (shown in red) at the start of the
transmembrane region that in UPIIIa and UPIIIb extends toward the N-terminus. The region of the extracellular domain of UPIIIb colored in
yellow is >90% identical to a portion of the human DNA mismatch repair enzyme-related PMSR6 protein. The small green circles represent
potential sites for N-linked glycan addition. The brackets denote known pairing interactions between UP isoforms. Figure adapted from
reference (18).
expression of all five known UPs leads to AUM particle
formation. Also unknown is if AUM assembly occurs solely
in the ER or whether assembly and maturation occur in
other organelles as well. Early studies by Hicks indicated
that packaging of AUM particles into vesicles might occur
in the Golgi apparatus of umbrella cells (26).
An additional system that may provide information concerning plaque assembly is the recent generation of UPIIIa
knockout mice (27). The umbrella cells of these mice contain few plaques, and those plaques remaining are small in
size. The ability to form any plaques may reflect the ability
of UPIIIb to substitute, albeit poorly, for UPIIIa, although
this remains to be established. Alternatively, the UPIa–
UPII complex may be able to form AUM particles inefficiently in the absence of UPIII. In fact, UPIa and UPII are
detected at the apical surface of the UPIII-deficient
umbrella cells. In contrast, UPIb is not found to accumulate
at the surface of these cells. This latter observation is
somewhat inconsistent with the cell surface delivery of
UPIb observed in HEK293T cells (25), and may indicate
that traffic of UPs in umbrella cells may be different than
that observed in heterologous non-polarized cell systems.
It will be intriguing to determine which steps in plaque
assembly can occur in the absence of UPIIIa, and whether
plaque formation occurs in animals lacking expression of
both UPII and UPIII.
Function of plaques
Although plaques and attendant AUM particles have been
known for decades, the role of these structures is not well
understood. However, the UPIIIa knockout mice described
above are providing some intriguing and somewhat
unexpected roles for UPIIIa and plaque function.
A previously ascribed function to plaques is that they may
help maintain the permeability barrier associated with the
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apical membrane of umbrella cells. Consistent with such a
role, the apical membranes of umbrella cells from UPIIIa
knockout animals show increased permeability to the normally membrane impermeant dye methylene blue (27).
Recent biophysical analyses confirm these findings and
demonstrate that bladders from knockout animals have a
significant increase in water and urea permeability across
the umbrella cell layer; however, junctional permeability
appeared to be unchanged (6). These results are the first
indication that integral membrane proteins may contribute
to the apical membrane permeability barrier of the uroepithelium. Because UPIIIa deficiency is associated with
several growth and developmental defects that are
described below, it is important to confirm that the lipid
composition of the apical membrane is not affected by
UPIIIa depletion, and that UPIIIa alone or in combination
with other plaque proteins can change the permeability
across other membranes including reconstituted liposomes of a defined lipid composition.
Lack of UPIIIa expression has other dramatic consequences, including formation of a hyperplastic uroepithelium with small umbrella cells (consistent with increased
turnover of the epithelium), enlarged ureters, and vesicoureteral reflux (leakage of urine from the bladder back to the
kidneys) (27). Although vesicoureteral reflux is a hereditary
disease that affects 0.5–1.0% of the population, the
genetics of this condition are not well understood (28).
The knockout animals indicate that deficiencies in plaque
subunits or plaque formation may account for some of
these genetic abnormalities. Why UPIIIa deficiency leads
to these growth and developmental defects is unknown. It
could simply reflect the altered permeability barrier, or
there may be other causes. For example, the dystrophin
complex links plasma membrane proteins to the underlying cytoskeleton of muscle cells, and disruption of this
complex leads to muscular dystrophy (29). This disease is
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The Uroepithelium
characterized by growth defects in muscle fibers including
small size, hypertrophy, and leaky plasma membranes.
Compared to the other UPs, UPIIIa has a relatively long
cytoplasmic domain that has been proposed to interact
with the cytoskeleton (27). Although this interaction may
be indirect, and remains to be demonstrated, UPIIIa interactions with the cytoskeleton could stabilize membrane
domains important for normal plasma membrane function
in umbrella cells.
Another function ascribed to plaques is that they may
modulate the apical surface area of the umbrella cell by
regulating insertion (during filling) and recovery (during
voiding) of plaque membrane (15). In addition to the apical
plasma membrane, UPs are also found in a population of
vesicles that occupy the cytoplasm of the umbrella cell and
have, depending on species, either a fusiform or discoidal
appearance in cross-section. In mice, the mature vesicles
have a fusiform appearance, while in rabbits the vesicles
are discoidal in shape (Figure 6). Fusiform/discoidal vesicles are thought to be formed in the Golgi apparatus (26),
and as will be discussed below are likely involved in
transport of UPs and other secretory cargo to the apical
surface of the umbrella cells (30,31). Whether UPs are
required for fusiform/discoidal vesicle formation or fusion
with the apical plasma membrane is an open question.
Although beyond the scope of this review, there is
emerging evidence that cargo proteins may modulate
their intracellular transport (32). The cytoplasm of umbrella
cells from UPIIIa knockout mice is replete with ‘immature’
vesicles that have a discoidal instead of fusiform appearance (27), indicating that significant vesicle formation
is occurring even in the absence of UPIIIa expression.
However, it is unknown if these vesicles contain other UP
cargo and whether they undergo exocytosis in a normal
manner. These questions can be readily answered using
the morphological and electrical techniques described below.
Response of the Uroepithelium to Bladder
Filling/Voiding
An area of cell biology that is again receiving attention is
the response of the uroepithelium to cyclical changes in
hydrostatic pressure as the bladder fills and empties. In the
mammalian bladder, pressure rises in a tri-phasic manner
as the organ fills with urine. The first rise occurs rapidly and
then pressure remains relatively constant for an extended
period of time called the storage phase. In rabbits, for
example, this phase lasts 4–5 h on average but can extend
to 12–15 h (33,34). The storage phase is followed by the
micturition phase, which is characterized by a rapid rise in
bladder pressure, punctuated by large spikes in pressure
as the smooth muscle contracts. Upon voiding, the pressure returns to baseline and the process begins anew. A
crucial aspect of the barrier function of the uroepithelium is
that it must be maintained in the face of these changes in
hydrostatic pressure.
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A
DV
0.5 µm
B
FV
0.5 µm
Figure 6: Discoidal and fusiform vesicles in the apical
cytoplasm of rabbit and mouse umbrella cell. (A) Discoidal
vesicles (DV) are found in rabbit umbrella cells. (B) Fusiform
vesicles (FV) are more common in mouse umbrella cells.
The increased urine volume is accommodated by the uroepithelium in at least two ways. The chief mechanism is
likely to be unfolding of the mucosal surface, which is
highly wrinkled in the empty bladder (Figure 7). The other
mechanism occurs at the cellular level and involves transitions in the morphology and function of the uroepithelium.
As the bladder fills, the uroepithelium becomes thinner,
apparently the result of intermediate and basal cells being
pushed laterally to accommodate the increased urine
volume (2,3). The umbrella cells undergo a large shape
change that involves progression from a roughly cuboidal
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Apodaca
with a significant decrease in vesicle surface area (30).
This change occurs gradually (over a 5-h time period),
indicating that exocytosis is a graded process. It was also
noted that little change in surface area or secretion of
35
S-labeled proteins occurred in the absence of pressure,
and that pressure-induced exocytosis of 35S-labeled
proteins was sensitive to brefeldin A (30). No significant
changes were observed in basolateral surface area.
Fourth, as described above, UPs are major constituents
of discoidal/fusiform vesicles (30,38) and, as predicted,
increased amounts of UPIII are found at the cell surface
of umbrella cells exposed to hydrostatic pressure (30).
100 µm
Figure 7: Scanning electron micrograph of mucosal surface
of the rabbit bladder. Note the large folds.
morphology in the empty bladder to one that is flat and
squamous in the filled bladder (2,3). In the classical model
for vesicle dynamics, the umbrella cell shape transformation is hypothesized to be accompanied by discoidal/
fusiform vesicle exocytosis (Figure 8A). This would
increase the apical surface area of the umbrella cell and
the overall surface area of the bladder, allowing the
bladder to accommodate additional urine volume (30,35).
Upon voiding, it is hypothesized that apical membrane
added during filling is rapidly internalized, replenishing
the pool of discoidal vesicles. An alternative model proposes that there are no changes in umbrella cell surface
area and, instead, changes in umbrella cell shape are
accomplished by folding/unfolding of the apical plasma
membrane (36).
Exocytosis in response to increased hydrostatic
pressure
The current data are most consistent with the classical
model’s tenet that pressure induces exocytosis of
discoidal/fusiform vesicles, resulting in increased umbrella
cell apical surface area. First, serial sectioning and electron
microscopic analysis demonstrates that discoidal/fusiform
vesicles are in fact distinct entities that are not connected
to one another or to the cell surface (30). Second,
discoidal/fusiform vesicles move from a scattered distribution in the cell cytoplasm to a position just underneath the
apical plasma membrane of umbrella cells exposed to
hydrostatic pressure (2,26,30). Third, early studies demonstrated that the volume fraction and numerical density of
discoidal/fusiform vesicles are significantly decreased in
filled vs. voided bladders (37). However, this analysis did
not take into account changes in cell volume that might
occur as the bladder fills. Stereology and estimates
of apical membrane capacitance (where 1 mF 1 cm2 of
surface area) were recently used to demonstrate in
isolated tissue that increased hydrostatic pressure stimulates a 50% increase in apical surface area that is coupled
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It remains to be formally shown in a rigorous pulse-chase
analysis that discoidal/fusiform vesicle membrane proteins
(e.g. UPs) are indeed fusing with the plasma membrane in
response to increased pressure. It is also possible that
other membrane-bound organelles are fusing in response
to pressure. Also unknown is whether intermediate and
basal cells undergo adjustments in cell volume or surface
area to accommodate increased urine volume. Intriguingly,
the 1 or 2 layers of intermediate cells closest to the
umbrella cell layer also possess small vesicles that have
a discoidal-like appearance (Figure 9). Whether these vesicles contain UPs and whether they turnover during bladder
filling have not been determined.
Pressure-induced endocytosis in umbrella cells
Two pieces of data are inconsistent with the classical
model described above (30). First, the amount of membrane added to the apical membrane of umbrella cells
when the epithelium is exposed to pressure
( 1500 mm2) is significantly less than the amount of
membrane present in the steady-state pool of discoidal
vesicles ( 7000 mm2). Second, the total amount of cellassociated UPIII decreases significantly after exposing
the uroepithelium to pressure for 5 h. This conundrum
was solved when biotin protection assays and lectin internalization assays were used to demonstrate that
increased hydrostatic pressure not only stimulates exocytosis, but also stimulates rapid endocytosis (30). The
endocytosed membrane components including UPs are
likely delivered to lysosomes, where they are degraded
(30), although this has not been formally proven. Whether
recycling of internalized membrane is occurring is also
unknown. Apparently, the rates of endocytosis and exocytosis are such that the net effect is to add membrane to
the apical surface of the cell. These data lead to a refinement of the classical model for vesicle transport to
include an endocytic pathway that operates simultaneously with the exocytic pathway (Figure 8B) (30).
At first glance, it seems counterintuitive that hydrostatic
pressure would simultaneously induce exocytosis and
endocytosis; however, hydrostatic pressure-induced
endocytosis would modulate the increase in apical surface area brought about by exocytosis, and it would
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A
Classical model
Exocytosis
of vesicles
Increase in apical surface
area accompanied by
change in cell shape
Filling
Voiding
Endocytosis of
membrane
Re-establishment of
vesicle population
B Revised model
Exocytosis of
vesicles coupled
with endocytosis
Filling
Increase in apical surface
area coupled with
delivery of endocytosed
vesicles to lysosomes
lysosome
Voiding
Re-establishment of
vesicle population
ensure turnover of membrane components such as AUM
particles. As described earlier, AUM particles may play
important roles in barrier function and plasma membrane
events. Furthermore, endocytosis and exocytosis are
coupled in other cell types, such as neurons, where
these two processes maintain the unique composition
of the presynaptic membrane (39). Intriguingly, neither
clathrin-coated pits nor caveolae are detected at the
apical surface of the umbrella cells, indicating that internalization may be via a nonclathrin-dependent pathway.
Because so little is understood about these pathways for
endocytosis (40), umbrella cells may provide a model system to define the machinery that drives them and study
how they are regulated.
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Endocytosis of added
membrane coupled
with vesicle formation
in the Golgi
Figure 8: Models for vesicle
dynamics in umbrella cells. (A)
In the classical model bladder filling
is accompanied by exocytosis of
discoidal/fusiform vesicle, thereby
increasing the apical surface of the
umbrella cells. Upon voiding, the
added membrane is endocytosed,
thus re-establishing the population
of vesicles. (B) In the revised model
filling stimulates both exocytosis
and endocytosis. The net rates of
these processes are such that
membrane is added to the apical
surface. Endocytosed membrane
is delivered to lysosomes where
contents are degraded. Upon
voiding the added membrane is
internalized and reestablishment of
the vesicle pool may result from
both endocytosis and de novo
synthesis along the biosynthetic
pathway.
There is limited evidence that mechanical stimuli can
alter endocytosis in other cell types, including endothelial cells (41,42); however, the underlying machinery
that transduces mechanical stimuli to changes in endocytosis is completely unexplored, and almost nothing is
known in the umbrella cell system. Recent evidence
indicates that exposing the apical surface of the epithelium to hypertonic solutions stimulates apical endocytosis (43), but the mechanism is unknown, and endocytosis
that accompanies return from hypotonic medium (which
causes cell swelling) to isotonic medium is blocked in
cells treated with the actin disrupting agent cytochalasin
B (44), indicating a role for actin in umbrella cell apical
endocytosis.
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Apodaca
inner ear (42). How mechanical stimuli are sensed and how
they are transduced into downstream changes in vesicular
traffic is not well understood, although the cytoskeleton,
stretch-activated channels, increased cytoplasmic Ca2þ,
integrins, phospholipases, tyrosine kinases, ATP, and
cAMP have been implicated in these events (42).
UC
IC
DV
N
1 µm
Figure 9: Presence of discoidal-like vesicles in intermediate
cells of rabbit bladder. Legend: DV, examples of discoidal-like
vesicles; IC, intermediate cell, N, nucleus; UC, umbrella cell.
Events following voiding
The classical model proposes that, upon voiding, apical
membrane added during filling is rapidly endocytosed
(Figure 8A). Although highly likely, the current evidence
is scant. Early experiments to demonstrate post-voiding
endocytosis involved instilling fluid-phase endocytic
markers into bladder just after urination (15,26). However, filling the bladder increases hydrostatic pressure
and induces the hydrostatic pressure-stimulated endocytosis described above (30). Other evidence comes
from studies in which endocytosis was studied in tissue
placed in hypotonic, then isotonic buffers (45); however,
the physiological significance of this experimental
manipulation is unclear. Finally, there is evidence that
short-term application of hydrostatic pressure (for 5 min)
across isolated uroepithelium increases surface area,
and this increase returns to baseline when the pressure
is released, presumably the result of endocytosis (44).
The intracellular fate of membrane internalized after
voiding is an open question. The classical model proposes that it serves to reestablish the population of
discoidal vesicles (Figure 8A) (15,26). However, there
are few data that support this conclusion. In fact, endocytosed marker proteins (including fluid-phase and
membrane-bound lectins) only label a small fraction of
the total discoidal vesicle pool (15,26,46), indicating that
the majority of discoidal vesicles may be formed de
novo along the biosynthetic pathway (Figure 8B).
Regulation of Discoidal Vesicle Exocytosis
Mechanical stimuli alter exocytic traffic in many cell types,
including endothelial cells, myocytes, kidney epithelia,
type II alveolar cells, osteocytes, and the hair cells in the
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Secondary messenger cascades and other regulatory
pathways involved in pressure-induced exocytosis
In the umbrella cell, we are only just beginning to understand the mechano-transduction pathways involved in
discoidal/fusiform vesicle exocytosis, although a requirement
for metabolic energy (in the form of ATP) and the actin,
intermediate filament, and microtubule cytoskeleton was
proposed some years ago (44,47,48). Like other regulated
secretory events, Ca2þ signaling is likely to play an important role in modulating exocytosis in umbrella cells (35).
Raising cytoplasmic Ca2þ, by treating the epithelium either
with the Ca2þ ionophore A23187 or with the ER Ca2þ uptake
inhibitor thapsigargin, stimulates exocytosis in the umbrella
cell layer. As expected, pressure-induced exocytosis in
umbrella cells is blocked when tissue is bathed in a nominally
Ca2þ-free solution or if cells are treated with inhibitors of
inositol 1,4,5-trisphosphate (IP3)-inducible Ca2þ release pathways from the ER (35). The latter results indicate that both
extracellular and intracellular Ca2þ may play a role in this
process. One possible upstream signal for Ca2þ release (purinergic signaling) is discussed below. The downstream target
of Ca2þ is unknown, although possible effectors include
Ca2þ- sensitive isoforms of adenylyl cyclase, as well as phospholipase Cd, and other channels and pumps (49). Because
the epithelium is multilayered, Ca2þ imaging techniques
will need to be used to confirm that pressure increases
cytoplasmic Ca2þ in the umbrella cell layer. An alternative is
that Ca2þ induces the release of a secretagogue from intermediate/basal cells, which then stimulates umbrella cell
exocytosis.
An additional secondary messenger that is produced when
the uroepithelium is exposed to hydrostatic pressure is
cAMP (30). Artificially raising cAMP in umbrella cells, by
treating tissue in the absence of pressure with forskolin,
causes a dramatic increase in exocytic activity, but not
endocytic activity (30). Unopposed by endocytosis, forskolin induces change in the apical surface area of umbrella
cells of greater than 120%. Forskolin also stimulates exocytosis in isolated uroepithelial cells (7). Treatment with
H-89, an inhibitor of the downstream cAMP effector
protein kinase A (PKA), partially inhibits (by 50%) hydrostatic pressure-induced changes in the apical surface area
of umbrella cells (30). These data indicate that cAMP,
acting through PKA, modulates hydrostatic pressureinduced exocytic traffic in umbrella cells. The isoform of
PKA involved and the targets of this kinase are presently
unknown. Again, it is possible that cAMP stimulates secretagogue release from basal/intermediate cells, which then
acts upon umbrella cells to induce exocytosis.
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Obviously a great deal of work is necessary to understand
the regulation of exocytosis in the uroepithelium. For
example, the role of specific Rab GTPases in this process
is an open question. Rab GTPases are important in regulating tethering/docking of vesicles to their target compartment leading to membrane fusion, and have also been
implicated in cargo selection, vesicle budding, and organelle motility (50). Preliminary data indicate that Rab27b is
expressed in the bladder (51), and we find that it is
expressed in the umbrella cell layer (G. Apodaca, D. Barral,
and M. Seabra, unpublished observations). The other isoform of Rab27, Rab27a, has been implicated in melanosome traffic and exocytosis of lytic granules in cytotoxic
T-cells (52). Rab27a links melanosomes, through melanophilin, to the unconventional myosin Va, which interacts with
the actin cytoskeleton and serves to retain melanosomes
at the cell periphery (52). As described earlier, discoidal
vesicles move from a random position in the cytoplasm to
below the apical membrane as the bladder fills, and actin
depolymerization inhibits exocytosis (10,30,53). In a manner analogous to melanocytes, Rab27b may interact with a
myosin motor to tether discoidal/fusiform vesicles to the
actin cytoskeleton, ultimately promoting vesicle exocytosis. Localizing Rab27b to discoidal/fusiform vesicles, and
generation of Rab27b knockout mice would be important
first steps in understanding any potential role for this
GTPase in vesicle exocytosis.
Upstream signals governing discoidal vesicle
exocytosis: role of ATP
Most cells release ATP when exposed to mechanical stimuli, and ATP is known to modulate exocytosis in several
cell systems (54). Mechanisms of release can include ATP
transporters, exocytosis, hemi-gap junctions, ATPconducting ion channels, and cell damage or death (54).
Released ATP can bind to one of two cell-associated purinergic receptors: P2X and P2Y (55). At least six P2Y
receptors (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, and P2Y12)
and eight P2X receptors (P2X1-7 and P2XM) have been
described (55). P2Y receptors are seven-transmembrane
domain receptors that upon ATP binding couple through
heterotrimeric G-proteins to modulate the activity of
adenylyl cyclases, which generate cAMP, and phospholipases, leading to generation of diacylglycerol and
IP3. In turn, IP3 binds to IP3 receptors that stimulate
increased cytosolic Ca2þ. P2X receptors are similar in
structure to the subunits of the epithelial sodium channel
and act as ligand-gated channels that directly stimulate
Ca2þ entry into the cell (55). As described above, Ca2þ
and cAMP are secondary messengers that stimulate exocytosis in umbrella cells.
The uroepithelium releases ATP when exposed to hydrostatic pressure (56–58), and P2X2, P2X3, P2X4, and P2X5
receptors have been localized to the serosal surface of
uroepithelial cells (59–61). ATP release may depend on
Traffic 2004; 5: 117–128
the activity of the amiloride-sensitive epithelial sodium
channel, which is proposed to act as a mechanosensor in
this system (57). ATP release may also require expression
of the transient receptor potential channel, vanilloid
subfamily member (TRPV1), an ion channel expressed by
nociceptive (pain sensing) afferent neurons and the uroepithelium (62). Isolated bladders from TRPV1 knockout
animals fail to release ATP in response to pressure and do
not increase umbrella cell apical surface area in response
to pressure (62). The link between TRPV1 and ATP release
is unknown, but could reflect TRPV1 conductance of extracellular Ca2þ into the cell.
Intriguingly, addition of apyrase (a membrane impermeant
exonucleotidase) to the serosal, but not mucosal, side of
isolated uroepithelial tissue prevents pressure-induced
exocytosis in umbrella cells (E. Wang, L. Birder, and
G. Apodaca, unpublished observations), indicating that
extracellular ATP release acts as an upstream signal for
exocytosis (Figure 10). Furthermore, we observe that
general inhibitors of P2 receptors inhibit pressure-induced
exocytosis, pointing to P2 receptor involvement in this
process. P2X2 and P2X3 knockout mice were recently
generated (63) (D. Cockayne, Roche Palo Alto, unpublished observations) and pressure-induced exocytosis
is blocked in bladder tissue taken from these animals
(E. Wang, L. Birder, and G. Apodaca, unpublished observations), implicating these particular purinergic receptors in
discoidal vesicle exocytosis. ATP-stimulated exocytosis is
blocked by treatments that remove extracellular Ca2þ or
prevent release of Ca2þ from intracellular stores, indicating
that Ca2þ is an important secondary messenger in this
process. Any role for P2Y receptors is unknown at present.
Again, it is unknown whether ATP directly acts on umbrella
cells, or if the effect is indirect.
Sensing Bladder Fullness: Cross-talk Between
the Uroepithelium and the Nervous System
A growing body of evidence indicates that epithelia
exposed to mechanical stimuli, such as those lining the
gut, blood vessels, airways of the lung, and lower urinary
tract, receive and transmit signals to submucosal neurons
(64,65). In the case of the bladder, there is evidence that
the uroepithelium may communicate bladder fullness to
the underlying nervous system through a paracrine signaling pathway involving ATP release (63,65,66).
Transmission of signals between uroepithelial cells
and afferent nerves
Afferent nerve processes (axons) have an intriguing distribution in the bladder. In addition to abutting blood vessels
and surrounding muscle bundles, these axonal processes
are found within the uroepithelium and in a nerve plexus
just below the basal cell layer (63,67). These nerve processes express P2X3 purinergic receptors (60), however
125
Apodaca
infrequently, have increased bladder capacity, and their
bladders fail to undergo contractions when experimentally
filled. We have noted that the bladders are enlarged in
these animals (E. Wang, L. Birder, and G. Apodaca, unpublished observations). Bladder pressures are normal in P2X3
knockout animals (63). The enlargement of the bladder
may be an adaptation to maintain low pressures, in part
because the umbrella cells of P2X3-deficient animals are
unable to undergo pressure-induced exocytosis. The
above data indicate that ATP, possibly released from the
uroepithelium, binds in a paracrine fashion to P2X3 receptors on afferent nerve processes, signaling bladder fullness, and also binds in an ‘autocrine’ fashion to P2X2 and
P2X3 receptors on the umbrella cell to stimulate exocytosis (Figure 10). Thus, ATP has a dual function, allowing for
the epithelium to accommodate more urine and also communicating filling to the nervous system.
Figure 10: Role of ATP in signaling exocytosis and bladder
fullness. Bladder filling increases hydrostatic pressure,
stimulating release of ATP from the uroepithelium (steps 1 & 2)
through an unknown mechanism. The released ATP can bind to
P2X3 receptors present on afferent nerve processes (step 3),
increasing nerve firing and relaying bladder filling to the central
nervous system (CNS) (step 4). Afferents can also release ATP
through a mechanism likely involving exocytosis of synaptic
vesicle (SV) content (step 5). ATP released from either the
afferents or the uroepithelium can bind to P2X2 and/or P2X3
receptors, and perhaps P2Y receptors (dashed line in step 6), on
the umbrella cells (step 6). Ligand binding causes increased
cytoplasmic Ca2þ (a result of Ca2þ influx from outside the cell and
efflux from intracellular stores), which signals discoidal/fusiform
vesicle exocytosis (step 7). ATP may also bind to purinergic
receptors present on basal/intermediate cells (step 8), which
stimulate release of ‘secretagogues’ that act upon umbrella cells
to stimulate vesicle exocytosis (step 9).
see (59) for an alternative view, and have recently been
implicated in some forms of nociception (pain sensation)
and warmth perception (63,66,68). It has been proposed
that ATP released from the uroepithelium during bladder
filling binds to P2X receptors on afferent nerve processes
signaling bladder fullness (Figure 10) (63,65).
Consistent with this model, the uroepithelium releases
ATP in response to pressure (56–58); however, ATP
release from other cell types including endothelial cells
and smooth muscle cells may also contribute to this signaling. Also consistent with the model, blocking P2X
receptors significantly decreases nerve firing when bladders are filled in a urinary bladder/pelvic nerve preparation
(69). Even stronger evidence comes from studies in which
bladder function was examined in the P2X3 knockout mice
described above (63). The P2X3-deficient mice urinate
126
Communication between the uroepithelium and the
nerves that innervate the bladder is likely to be bidirectional. In addition to releasing neurotransmitters such as
ATP and NO (56–58,70,71), uroepithelial cells express ion
channels and receptors characteristic of sensory neurons.
These include the P2X receptors described above (59–61),
as well as TRPV1 and the b-adrenoceptor (62,71). Because
bladder afferents can release both ATP as well as peptide
neurotransmitters, binding of these neurotransmitters to
the uroepithelium could act in a paracrine fashion to stimulate changes in umbrella cells, including enhanced exocytosis. There is some evidence that neurotransmitters
released from efferent nerves can alter barrier function in
the uroepithelium (72), and it was recently observed that
blocking nerve transmission prevents the acute disruption
of the uroepithelium observed after spinal cord injury (73).
Summary and Perspectives
The composition of urine is markedly different from
plasma, with urine osmolality ranging from 50 to 1200
Osmol, a pH ranging between 4.5 and 10, and containing
high concentrations of ammonia, urea, as well as other
toxins. In mammals, the bladder must store this urine for
prolonged periods of time without permitting the passage
of highly permeable molecules such as ammonia into the
bloodstream. The barrier to ion, solute, and toxin flux is
formed by the uroepithelium, which lines the inner surface
of the bladder and must also adapt to large variations
in pressure as the bladder fills and empties. In addition,
to providing important information on how barriers
are formed and maintained, recent analysis of the uroepithelium is providing insight into the assembly of protein
particles into specialized membrane domains called plaques. Defects in plaque assembly increase membrane
permeability and may lead to diseases such as vesicoureteral reflux (6,27). In addition, study of the uroepithelium
is providing clues to how epithelial cells sense mechanical
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The Uroepithelium
stimuli such as pressure, and transduce changes in these
stimuli into cellular events such as membrane traffic.
Increased pressure, for example, stimulates exocytosis
and endocytosis in umbrella cells (30). Exocytosis is modulated by purinergic signaling cascades, Ca2þ, and cAMP
(30,35). Nothing is known about regulation of pressureinduced endocytosis, but it may not involve clathrin. Future
analysis of this pathway may lead to a better understanding of clathrin-independent pathways of internalization.
Finally, the uroepithelium interfaces with an underlying
nervous system, and bidirectional signaling between
these two systems may communicate the degree of bladder filling, and may allow the nervous system to modulate
uroepithelial barrier function.
Acknowledgments
I thank Dr Lori Birder, Chris Guerriero, Asli Oztan, Raul Rojas, Dr. Debbie
Cockayne and Edward Wang for their constructive and helpful comments
during preparation of this manuscript. The transmission and scanning electron micrographs were prepared by W. Giovanni Ruiz, and the rapid-freeze,
deep-etch micrograph was provided by Dr John Heuser. This work was
supported by a grant to GA from the NIH (RO1DK54425).
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