Distinct apical and basolateral membrane requirements for stretch-induced membrane traffic at the apical surface of bladder umbrella cells

Abstract

Epithelial cells respond to mechanical stimuli by increasing exocytosis, endocytosis, and ion transport, but how these processes are initiated and coordinated and the mechanotransduction pathways involved are not well understood. We observed that in response to a dynamic mechanical environment, increased apical membrane tension, but not pressure, stimulated apical membrane exocytosis and ion transport in bladder umbrella cells. The exocytic response was independent of temperature but required the cytoskeleton and the activity of a nonselective cation channel and the epithelial sodium channel. The subsequent increase in basolateral membrane tension had the opposite effect and triggered the compensatory endocytosis of added apical membrane, which was modulated by opening of basolateral K(+) channels. Our results indicate that during the dynamic processes of bladder filling and voiding apical membrane dynamics depend on sequential and coordinated mechanotransduction events at both membrane domains of the umbrella cell.

Figure 1.

System for stretching the uroepithelium. Uroepithelial tissue was mounted between two closed Ussing stretch chambers, the chambers were filled with Krebs buffer, and the tissue was equilibrated for 30–45 min. (A) Control tissue was not exposed to mechanical stimuli. (B) Tissue was bowed outward in response to stepwise increases in the hydrostatic pressure head. (C) Hydrostatic pressure was simultaneously raised stepwise in both mucosal and serosal hemichambers. (D) Both the mucosal and serosal hemichambers were maintained at 1 cm H₂O pressure, and the tissue was pulled by a filament toward the serosal hemichamber. (E) Tissue was bowed outward in response to raising hydrostatic pressure in the mucosal hemichamber. (F) Tissue was bowed inward in response to raising hydrostatic pressure in the serosal hemichamber. (G) Tissue was bowed outward as the mucosal hemichamber was filled at different rates. M, mucosal hemichamber; S, serosal hemichamber. The green line between the chambers shows the initial position of uroepithelium and the red curve shows the position of the tissue upon bowing.

Figure 2.

Modulation of electrophysiological parameters in response to mechanical stimuli. (A) Responses in tissue exposed to stepwise increases in pressure. (B) Responses to pulling the tissue toward the serosal hemichamber under constant pressure. The experiments were repeated ≥3 times, and representative results are shown.

Figure 3.

Mechanical and morphological features of distended umbrella cells. (A) In response to filling the mucosal hemichamber, tension initially develops in the apical membrane, which causes the tissue to bow outward. The apical membrane tension can be dissipated in part by increased exocytosis. Subsequently, tension increases in the basolateral membrane, which unlike the apical membrane does not accommodate increased tension by modulating surface area. (B) The apical membrane acts like a spring that can accommodate heightened tension by increasing apical surface area, but the spring is limited by the basolateral membrane, which may act like a rope to further constrain tension release (in the case of the umbrella cell by promoting endocytosis of apical membrane). (C) In response to filling the serosal hemichamber, tension is increased in the basolateral membrane, which causes the tissue to bow inward. As the tissue bows further, tension increases in the apical membrane.

Figure 4.

Modulation of umbrella cell electrophysiological parameters by changes in the direction of the mechanical force. (A) Responses in tissue bowed outward by a 2-cm H₂O pressure head in the mucosal hemichamber. (B) Responses in tissue bowed inward by a 1-cm H₂O pressure head in the serosal hemichamber. The experiments were repeated ≥3 times, and representative results are shown.

Figure 5.

Electrophysiological responses to different pressure heads and rates of chamber filling. (A) Effect of different pressure heads on electrophysiological parameters as the tissue was bowed outward. Insets show responses observed when the hydrostatic pressure was raised to 8 cm H₂O. (B) Responses to different rates of filling when the pressure head was maintained at 2 cm H₂O in the mucosal hemichamber. The legend shows the gauge of needle and approximate rate of filling. The insets show data for the 25-gauge needle. (C) Effect of different pressure heads on electrophysiological parameters as the tissue was bowed inward. The experiments were repeated ≥3 times and representative results are shown. Data for the responses to 2 cm H₂O (outward bowing) and 1 cm H₂O (inward bowing) are reproduced from Figure 4.

Figure 6.

Increased membrane turnover in response to acute changes in hydrostatic pressure. (A) Tissue was mounted, equilibrated, and then cooled to 4°C for 30 min. The apical surface of the cells was treated with the indicated concentration of sulfo-NHS acetate for 60 min, washed, and then incubated with 0.5 mg/ml sulfo-NHS–SS-biotin for 15 min to biotinylate unblocked apical membrane proteins. The percent change in biotinylated proteins way quantified by densitometry and is plotted in the graph to the right. (B and C) The apical surface of the umbrella cells was incubated with 1 mg/ml sulfo-NHS acetate for 60 min at 4°C, and then the tissue was bowed outward (2 cm H₂O pressure using a 20-gauge needle) for 5 min at 37°C (B) or 10 min at 4°C (C). The apical surface of tissue was then incubated with 0.5 mg/ml sulfo-NHS–SS-biotin for 15 min to biotinylate newly inserted apical membrane proteins. The percent change in biotinylated proteins is shown in the panel to the right. (D) The tissue was incubated with 1 mg/ml sulfo-NHS acetate, treated with cytochalasin D for 60 min at 37°C, stretched 10 min at 4°C, and the apical surface biotinylated. The percent change in biotinylated proteins is shown in the panel to the right. (E) Percent change in biotinylated proteins for tissue bowed outward (2 cm H₂O pressure using a 20-gauge needle) at 37°C in the presence of 75 μM 2-APB. (A–E) Data are expressed as mean ± SEM (n ≥ 3). Representative lysates from control (C) or stretched (Str) samples are shown in B–E. Statistically significant difference (* p < 0.05) relative to control. (F) Tissue was mounted, equilibrated, and then cooled to 4°C for 30 min. WGA was added to the mucosal hemichamber and incubated for 30 min, and the tissue was bowed outward for 10 min at 4°C by filling the chamber using a 20-gauge needle at a 2 cm H₂O pressure head. Unstretched tissue served as control. Shown are individual optical sections taken from the apical, middle, and basal regions of stretched umbrella cells. Rhodamine phalloidin was used to label the actin cytoskeleton (red), and Topro-3 was used to label the nuclei (blue). A three-dimensional reconstruction of the cell is shown at the right. Bar, 20 μm.

Figure 7.

Role of the cytoskeleton in the electrophysiological responses to outward bowing. The tissue was pretreated with the indicated blocker for 30 min at 37° C. The tissue was then bowed outward using a 20-gauge needle to fill the mucosal hemichamber at a 2 cm H₂O pressure head. Untreated tissue served as a control. The experiments were repeated ≥3 times, and representative results are shown.

Figure 8.

Requirement for apical membrane cation channels and intracellular Ca²⁺ release in the phase 1 response to outward bowing. (A and B) The indicated channel blockers were added to the mucosal (A) or serosal (B) hemichamber and preincubated for 30 min at 37°C. The tissue was then bowed outward using a 20-gauge needle to fill the mucosal hemichamber at a 2 cm H₂O pressure head. (C) Tissue was pretreated with 2-APB or ryanodine for 1 h and then stretched as described in A and B. Alternatively, the apical solution was exchanged for one lacking Ca²⁺, and the tissue was stretched. In A-C untreated tissue served as a control and is reproduced from Figure 7. The experiments were repeated ≥3 times, and representative results are shown.

Figure 9.

K_ATP is expressed in umbrella cells and modulates the stretch response. (A) Expression of the indicated K⁺ channels was detected by RT-PCR of total RNA isolated from mouse bladder uroepithelium. Numbers above the DNA species are the expected product sizes of the RT-PCR reaction (in base pairs). (B) A lysate of rat uroepithelium was resolved by SDS-PAGE and a Western blot probed with antibody specific for the Kir6.1 subunit of K_ATP. The nominal mass of the major protein detected in the lysate is indicated to the right of the panel. (C) Frozen thin sections of rat bladder tissue were labeled with an antibody to the Kir6.1 subunit of K_ATP (green), claudin-4 (red) to label the basolateral membranes of the umbrella cells and plasma membranes of the underling intermediate and basal cells and Topro-3 to label the cell nuclei (blue). A merged panel is shown at the right. Bar, 10 μm. (D) The indicated K⁺ channel openers were added to the serosal hemichamber and incubated with tissue for 30 min. The tissue was then bowed outward by filling the chamber with a 20-gauge needle at a 2 cm H₂O pressure head. The experiments were repeated ≥3 times, and representative results are shown.

Figure 10.

Model for stretch-induced umbrella cell responses. As the bladder fills, the folded mucosal surface of the bladder is pushed outward, and increased tension at the apical membrane causes opening of an NSCC, stimulating influx of extracellular Ca²⁺. The increased [Ca²⁺]_i triggers Ca²⁺-dependent Ca²⁺ release from IP₃ dependent stores, which stimulates exocytosis and may modulate the activity of other channels. Exocytosis may amplify the initial response by stimulating delivery of additional stretch sensing channels to the apical surface of the cell. ENaC, perhaps acting downstream of the NSCC, is also opened and may modulate the exocytic response by changing the membrane potential or the driving force for the entry of other ions (e.g., by modulating the activity of the Na⁺,K⁺ ATPase). As the epithelium bows further outward, tension in the basolateral membrane would further increase. This would cause an increase in apical membrane endocytosis, which would modulate the exocytic response by stimulating internalization of apical membrane channels and other sensory molecules (not shown). The mechanosensor at this membrane is unknown but may include the cytoskeleton and associated integrins (not shown) or stretch-modulated K⁺ channel(s), which may close in response to the increased tension or other intracellular mediators such as ATP. During voiding, tension would increase in the basolateral membrane as the mucosa refolds. The increased tension would further stimulate endocytosis of apical membrane and its constituents, readying the umbrella cell for the next cycle of filling. For clarity we do not include the underlying cell layers or connective tissue; however, they may contribute to the events in the overlying umbrella cell layer by release of mediators and effects on umbrella cell tension.