Urotelio

Traffic 2004; 5: 117–128 Blackwell Munksgaard

Copyright

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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 117

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UC UC

IC 10 µm

IC IC

B IC

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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 118

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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. Traffic 2004; 5: 117–128

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P 50 nm

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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

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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) Traffic 2004; 5: 117–128

(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 119

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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 120

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 Traffic 2004; 5: 117–128

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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. Traffic 2004; 5: 117–128

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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 121

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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 122

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 Traffic 2004; 5: 117–128

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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. Traffic 2004; 5: 117–128

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. 123

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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 124

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. Traffic 2004; 5: 117–128

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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

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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 Traffic 2004; 5: 117–128

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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