Expansion and contraction of the umbrella cell apical junctional ring in response to bladder filling and voiding

The epithelial junctional complex, composed of tight junctions, adherens junctions, desmosomes, and an associated actomyosin cytoskeleton, forms the apical junctional ring (AJR), which must maintain its continuity in the face of external mechanical forces that accompany normal physiological functions. The AJR of umbrella cells, which line the luminal surface of the bladder, expands during bladder filling and contracts upon voiding; however, the mechanisms that drive these events are unknown. Using native umbrella cells as a model, we observed that the umbrella cell’s AJR assumed a nonsarcomeric organization in which filamentous actin and ACTN4 formed unbroken continuous rings, while nonmuscle myosin II (NMMII) formed linear tracts along the actin ring. Expansion of the umbrella cell AJR required formin-dependent actin assembly, but was independent of NMMII ATPase function. AJR expansion also required membrane traffic, RAB13-dependent exocytosis, specifically, but not trafficking events regulated by RAB8A or RAB11A. In contrast, the voiding-induced contraction of the AJR depended on NMMII and actin dynamics, RHOA, and dynamin-dependent endocytosis. Taken together, our studies indicate that a mechanism by which the umbrella cells retain continuity during cyclical changes in volume is the expansion and contraction of their AJR, processes regulated by the actomyosin cytoskeleton and membrane trafficking events.

Bladder filling and voiding affect umbrella cell tight junction organization and function

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 Ca2+ 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.

Adenosine receptor expression and function in bladder uroepithelium

The uroepithelium of the bladder forms an impermeable barrier that is maintained in part by regulated membrane turnover in the outermost umbrella cell layer. Other than bladder filling, few physiological regulators of this process are known. Western blot analysis established that all four adenosine receptors (A1, A2a, A2b, and A3) are expressed in the uroepithelium. A1 receptors were prominently localized to the apical membrane of the umbrella cell layer, whereas A2a, A2b, and A3 receptors were localized intracellularly or on the basolateral membrane of umbrella cells and the plasma membrane of the underlying cell layers. Adenosine was released from the uroepithelium, which was potentiated 10-fold by stretching the tissue. Administration of adenosine to the serosal or mucosal surface of the uroepithelium led to increases in membrane capacitance (where 1 μF ≈ 1 cm2 tissue area) of ∼30% or ∼24%, respectively, after 5 h. Although A1, A2a, and A3 selective agonists all stimulated membrane capacitance after being administrated serosally, only the A1 agonist caused large increases in capacitance after being administered mucosally. Adenosine receptor antagonists as well as adenosine deaminase had no effect on stretch-induced capacitance increases, but adenosine potentiated the effects of stretch. Treatment with U-73122, 2-aminoethoxydiphenylborate, or xestospongin C or incubation in calcium-free Krebs solution inhibited adenosine-induced increases in capacitance. These data indicate that the uroepithelium is a site of adenosine biosynthesis, that adenosine receptors are expressed in the uroepithelium, and that one function of these receptors may be to modulate exocytosis in umbrella cells.