title: Intravital imaging of mouse urothelium reveals activation of extracellular signal-regulated kinase by stretch-induced intravesical release of ATP.
abstract: To better understand the roles played by signaling molecules in the bladder, we established a protocol of intravital imaging of the bladder of mice expressing a Förster/fluorescence resonance energy transfer (FRET) biosensor for extracellular signal-regulated kinase (ERK), which plays critical roles not only in cell growth but also stress responses. With an upright two-photon excitation microscope and a vacuum-stabilized imaging window, cellular ERK activity was visualized in the whole bladder wall, from adventitia to urothelium. We found that bladder distention caused by elevated intravesical pressure (IVP) activated ERK in the urothelium, but not in the detrusor smooth muscle. When bladder distension was prevented, high IVP failed to activate ERK, suggesting that mechanical stretch, but not the high IVP, caused ERK activation. To delineate its molecular mechanism, the stretch-induced ERK activation was reproduced in an hTERT-immortalized human urothelial cell line (TRT-HU1) in vitro. We found that uniaxial stretch raised the ATP concentration in the culture medium and that inhibition of ATP signaling by apyrase or suramin suppressed the stretch-induced ERK activation in TRT-HU1 cells. In agreement with this in vitro observation, pretreatment with apyrase or suramin suppressed the high IVP-induced urothelial ERK activation in vivo. Thus, we propose that mechanical stretch induces intravesical secretion of ATP and thereby activates ERK in the urothelium. Our method of intravital imaging of the bladder of FRET biosensor-expressing mice should open a pathway for the future association of physiological stimuli with the activities of intracellular signaling networks.
authors.affiliation: Department of Pathology and Biology of Diseases, Graduate School of Medicine, Kyoto University, Kyoto, Japan.|Department of Urology, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Sano
authors.affiliation: Department of Urology, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Kobayashi
authors.affiliation: Department of Urology, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
authors.forename: Hiromitsu
authors.initials: H
authors.lastname: Negoro
authors.affiliation: Department of Urology, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Sengiku
authors.affiliation: Department of Pathology and Biology of Diseases, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
authors.forename: Takuya
authors.initials: T
authors.lastname: Hiratsuka
authors.affiliation: Department of Pathology and Biology of Diseases, Graduate School of Medicine, Kyoto University, Kyoto, Japan.|Department of Molecular Genetics, Institute of Biomedical Science, Kansai Medical University, Osaka, Japan.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Kamioka
authors.affiliation: Department of Urology, Cambridge Health Alliance, Cambridge, Massachusetts.
authors.forename: Louis S
authors.initials: LS
authors.lastname: Liou
authors.affiliation: Department of Urology, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
authors.forename: Osamu
authors.initials: O
authors.lastname: Ogawa
authors.affiliation: Department of Pathology and Biology of Diseases, Graduate School of Medicine, Kyoto University, Kyoto, Japan matsuda.michiyuki.2c@kyoto-u.ac.jp.
authors.forename: Michiyuki
authors.initials: M
authors.lastname: Matsuda
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001081:Apyrase / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism* / Q000502:physiology; D005260:Female; D000069416:Intravital Microscopy / Q000379:methods*; D051379:Mice; D008810:Mice, Inbred C57BL; D009130:Muscle, Smooth / Q000378:metabolism / Q000502:physiology; D015398:Signal Transduction / Q000235:genetics*; D000090324:Tumor Protein, Translationally-Controlled 1; D001743:Urinary Bladder / Q000000981:diagnostic imaging* / Q000502:physiology; D019459:Urothelium / Q000166:cytology / Q000000981:diagnostic imaging* / Q000502:physiology / Q000648:ultrastructure
chemical_list: C000715648:Tpt1 protein, mouse; D000090324:Tumor Protein, Translationally-Controlled 1; D000255:Adenosine Triphosphate; D048049:Extracellular Signal-Regulated MAP Kinases; D001081:Apyrase
keywords: Extracellular signal‐regulated kinase; in vivo imaging; urothelium