MP28-08 INTRAVITAL TWO-PHOTON IMAGING OF BLADDER REVEALED THE ACTIVATION OF UROTHELIAL ERK BY STRETCH-INDUCED INTRAVESICAL RELEASE OF ADENOSINE TRIPHOSPHATE

INTRODUCTION AND OBJECTIVE: Although imaging of live animals at microscopic resolution (intravital imaging) represents a powerful tool to observe the behavior of cells and tissues over time under physiological conditions, there is no report that describes any dynamic phenomenon in the bladder as investigated by this technique. METHODS: To better understand the molecular signaling in the bladder, we have established a method for intravital imaging of the mouse bladder expressing a Förster/fluorescence resonance energy transfer (FRET) based biosensor for extracellular signal-regulated kinase (ERK), which plays critical roles not only in cell growth but also in stress responses. Using an upright two-photon excitation microscope and a vacuum-stabilized organ fixation device, cellular ERK activity was visualized in the whole bladder wall, extending from adventitia to urothelium. RESULTS: We found that distention of the bladder to 60-100 cm H2O caused by intravesical pressure (IVP) activated ERK in the urothelium, but not in the detrusor smooth muscle. Although we confirmed that the blood stream was decreased or stopped when IVP was elevated to 60-100 cm H2O, hypoxia in the bladder caused by aortic cross-clamping did not activate ERK in the urothelium. When bladder distension was prevented, elevated IVP failed to activate ERK; thus, suggesting that mechanical stretch, but not hypoxia or hydrostatic pressure in the bladder caused ERK activation. Similar stretch-induced ERK activation was observed in an hTERT-immortalized human urothelial cell line (TRT-HU1) in vitro. Uniaxial stretch increased the ATP concentration in the culture media; and inhibition of ATP signaling by apyrase or suramin suppressed the stretch-induced ERK activation in TRT-HU1 cells. In line with these in vitro observations, bladder distension by saline injection or injecting their own urine increased the concentration of ATP in the bladder; and pretreatment of the bladder with apyrase or suramin suppressed the bladder distension-induced urothelial ERK activation in vivo. CONCLUSIONS: We have shown here that mechanical stretch of the urothelium induces ATP release into the bladder, and thereby activates urothelial ERK in an autocrine/paracrine manner. Our protocol for intravital imaging of the bladder of FRET biosensor-expressing mice should open a new pathway for studying the association of physiological stimuli with the activities of intracellular signaling networks in future. Source of Funding: The Platform for Dynamic Approaches to Living Systems from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. A Grant-in-Aid for Scientific Research on Innovative Areas (no. 22113002)A Grant-in-Aid for Young Scientists A (no. 15H05682) . A Grant-in-Aid for Young Scientists A (no. 25713055)