Rab11a and Rab8 work in conjunction with myosin5B to promote discoidal/fusiform vesicle exocytosis at the apical surface of umbrella cells. It is predicted that similar Rab cascades will be common to other regulated secretory pathways.

Epithelial cells are continuously exposed to mechanical forces including shear stress and stretch, although the effect these forces have on tight junction (TJ) organization and function are poorly understood. Umbrella cells form the outermost layer of the stratified uroepithelium and undergo large cell shape and surface area changes during the bladder cycle. Here we investigated the effects of bladder filling and voiding on the umbrella cell TJ. We found that bladder filling promoted a significant increase in the length of the TJ ring, which was quickly reversed within 5 min of voiding. Interestingly, when isolated uroepithelial tissue was mounted in Ussing chambers and exposed to physiological stretch, we observed a 10-fold drop in both transepithelial electrical resistance (TER) and the umbrella cell junctional resistance. The effects of stretch on TER were reversible and dependent on the applied force. Furthermore, the integrity of the umbrella cell TJ was maintained in the stretched uroepithelium, as suggested by the limited permeability of biotin, fluorescein, and ruthenium red. Finally, we found that depletion of extracellular Ca(2+) by EGTA completely disrupted the TER of unstretched, but not of stretched uroepithelium. Taken together, our studies indicate that the umbrella cell TJ undergoes major structural and functional reorganization during the bladder cycle. The impact of these changes on bladder function is discussed.

Changes in the urothelial barrier are observed in patients with cystitis, but whether this leads to inflammation or occurs in response to it is currently unknown. To determine whether urothelial barrier dysfunction is sufficient to promote cystitis, we employed in situ adenoviral transduction to selectively overexpress the pore-forming tight junction-associated protein claudin-2 (CLDN-2). As expected, the expression of CLDN-2 in the umbrella cells increased the permeability of the paracellular route toward ions, but not to large organic molecules. In vivo studies of bladder function revealed higher intravesical basal pressures, reduced compliance, and increased voiding frequency in rats transduced with CLDN-2 vs. controls transduced with green fluorescent protein. While the integrity of the urothelial barrier was preserved in the rats transduced with CLDN-2, we found that the expression of this protein in the umbrella cells initiated an inflammatory process in the urinary bladder characterized by edema and the presence of a lymphocytic infiltrate. Taken together, these results are consistent with the notion that urothelial barrier dysfunction may be sufficient to trigger bladder inflammation and to alter bladder function.

The bladder uroepithelium transmits information to the underlying nervous and musculature systems, is under constant cyclical strain, expresses all four adenosine receptors (A 1, A2A, A2B, and A3), and is a site of adenosine production. Although adenosine has a welldescribed protective effect in several organs, there is a lack of information about adenosine turnover in the uroepithelium or whether altering luminal adenosine concentrations impacts bladder function or overactivity. We observed that the concentration of extracellular adenosine at the mucosal surface of the uroepithelium was regulated by ecto-adenosine deaminase and by equilibrative nucleoside transporters, whereas adenosine kinase and equilibrative nucleoside transporters modulated serosal levels. We further observed that enriching endogenous adenosine by blocking its routes of metabolism or direct activation of mucosal A 1 receptors with 2-chloro-N 6 -cyclopentyladenosine (CCPA), a selective agonist, stimulated bladder activity by lowering the threshold pressure for voiding. Finally, CCPA did not quell bladder hyperactivity in animals with acute cyclophosphamideinduced cystitis but instead exacerbated their irritated bladder phenotype. In conclusion, we find that adenosine levels at both surfaces of the uroepithelium are modulated by turnover, that blocking these pathways or stimulating A1 receptors directly at the luminal surface promotes bladder contractions, and that adenosine further stimulates voiding in animals with cyclophosphamide-induced cystitis.cyclophosphamide-induced cystitis; voiding ADENOSINE IS A UBIQUITOUSLY occurring nucleoside that is important for the homeostasis of diverse organ systems including the kidneys, heart, lungs, and brain (20,46,55). It is found both intracellularly and in the extracellular space, and its concentration in these compartments is dependent on its biogenesis and turnover (43,44). The intracellular pool of adenosine is synthesized de novo by the hydrolysis of S-adenosylmethionine or is generated from the nucleotidase-dependent breakdown of ATP to ADP to AMP to adenosine. The extracellular pool of adenosine is also formed from the hydrolysis of ATP by ecto-nucleotidases or alkaline phosphatases or by metabolism of cAMPs to AMPs to adenosine. In addition, sodiumcoupled concentrative or equilibrative nucleoside transporters shuttle adenosine in a concentration-dependent manner between the intracellular and extracellular compartments (23). The turnover of extracellular and intracellular adenosine is mediated by two enzymes: adenosine deaminase and adenosine kinase, which decrease adenosine by converting it to inosine or AMP, respectively (13,30,32,52).

The role of phosphorylation in ADAM17-dependent shedding is controversial. We show that the A1 adenosine receptor stimulates exocytosis in umbrella cells by a pathway that requires phosphorylation of ADAM17–Ser-811, followed by HB-EGF shedding and EGF receptor transactivation. Preventing ADAM17 phosphorylation blocks these downstream events.

In response to bladder filling and voiding, umbrella cells undergo changes in cell‐shape and vesicular trafficking. However, it is not known how filling/voiding impact paracellular transport in these cells. To investigate how changes in umbrella cell stretch affect paracellular permeability, bladder mucosa was mounted in Ussing chambers and studied under current clamp conditions. In this system the transepithelial resistance (TER) was dependent on whether the tissue was bowed outwards, simulating filling, or bowed inwards as observed during voiding when the epithelium is refolded. Outward bowing provoked by an increase in mucosal hydrostatic pressure of ~1 cm of water caused a decrease in junctional resistance from 21.1 ± 8.5 to 1.8 ± 0.4 kΩ.cm2. These effects were reversible and dependent on the applied force. The integrity of the tight junctions (TJ) was assessed by examining the distribution of ruthenium red (RR) added to the mucosal surface of the tissue and examined by electron microscopy. The TJ appeared intact and no RR was found along the basolateral surface of the cells during experimental filling. Finally, depletion of extracellular Ca2+ by EGTA completely disrupted the TER of control, but not of stretched uroepithelium, indicating a change in the junctional barrier. In summary, our results indicate that filling and voiding impacts the junctional complex of umbrella cells.

Bladder filling promotes a reorganization of the umbrella cell tight junctions (TJ) ring, which is accompanied by a large drop in junctional resistance. The goal of this study was to assess the impact of increased paracellular permeability on bladder function. Claudins are structural components of the TJ that define the permeability of the paracellular barrier present in epithelia. To increase the paracellular permeability of the rat uroepithelium we expressed claudin‐2 using in situ adenoviral transduction. Fluorescent microscopy showed prominent expression of claudin‐2 in the umbrella cells of the transduced bladders. The electrical resistance and potential difference of the urinary bladders mounted in Ussing chambers were significantly lower in rats transduced with claudin‐2, 0.5 ± 0.1 kΩ.cm2 and ‐1.3 ± 0.2 mV, than in rats transduced with GFP, 4.6 ± 0.3 kΩ.cm2 and ‐12 ± 2 mV. Conversely, the permeability of the uroepithelium to large molecules (MW >350), fluorescein and NHS‐biotin, was similar in rats transduced with GFP and claudin‐2. In vivo studies of bladder function using continuous cystometry revealed higher intravesical basal pressures and shorter intervoiding intervals in rats transduced with claudin‐2 than in rats transduced with GFP. In conclusion, the expression of claudin‐2 in the uroepithelium increases its permeability to ions and augments voiding frequency, consistent with the notion that paracellular ion transport modulates bladder function.Support: NIH R01 DK099196 and P30 DK079307 Pittsburgh Center for Kidney Research ‐ O'Brien Pilot.