title: Protease-Activated Receptor-2 Up-Regulates Transient Receptor Potential Vanilloid 4 Function in Mouse Esophageal Keratinocyte.
abstract: BACKGROUND
  The reflux of pancreatic-duodenal fluids is implicated in the pathophysiology of proton-pump inhibitor-resistant gastroesophageal reflux disease (GERD). Protease-activated receptor-2 (PAR-2) is activated by proteases, the pancreatic enzyme, trypsin, and the activated PAR-2 enhances transient receptor potential vanilloid 4 (TRPV4) function in neurons. TRPV4 stimulates ATP exocytosis in conjunction with the vesicular nucleotide transporter, which mediates mechano-transduction and vagal stimulation. The aim of the present study was to verify whether the activated PAR-2 up-regulates TRPV4 function in mouse esophageal keratinocytes, which may link to the pathophysiology in PPI-resistant GERD.
  
  
  METHODS
  TRPV4 and PAR-2 expressions were detected by RT-PCR, immunostaining, and western blotting in mouse esophageal keratinocytes. The functional response of TRPV4 to esophageal keratinocytes was analyzed using a Ca(2+) imaging system. Cellular ATP release was examined by luciferase-luciferin reaction. TRPV4 phosphorylation was studied by immunoprecipitation and western blotting.
  
  
  RESULTS
  PAR-2 and TRPV4 mRNAs and proteins were expressed in esophageal keratinocytes. Pre-treatment with trypsin significantly increased the responses to TRPV4 activator in esophageal keratinocytes, probably via the phosphorylation of serine residue of TRPV4 by protein kinase C and resulted in cellular ATP release from the cells.
  
  
  CONCLUSIONS
  Activated PAR-2 with trypsin exposure up-regulated TRPV4 function and increased ATP release in mouse esophageal keratinocytes. This mechanism might be related to the pathophysiology of GERD, especially non-erosive GERD.
authors.affiliation: Department of Gastroenterology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama, Toyama, 930-0194, Japan. nobuhiro@joetsu-hp.jp.
authors.forename: Nobuhiro
authors.initials: N
authors.lastname: Suzuki
authors.affiliation: Department of Gastroenterology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama, Toyama, 930-0194, Japan. m164-tym@umin.net.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Mihara
authors.affiliation: Division of Animal Experimental Laboratory, Life Science Research Center, University of Toyama, 2630 Sugitani, Toyama, Toyama, 930-0194, Japan. hirofumi@cts.u-toyama.ac.jp.
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Nishizono
authors.affiliation: Division of Cell Signaling, Okazaki Institute for Integrative Bioscience (National Institute for Physiological Sciences), National Institutes of Natural Sciences, Nishigonaka 38, Myodaiji, Okazaki, Aichi, 444-8585, Japan. tominaga@nips.ac.jp.
authors.forename: Makoto
authors.initials: M
authors.lastname: Tominaga
authors.affiliation: Department of Gastroenterology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama, Toyama, 930-0194, Japan. tsugi@med.u-toyama.ac.jp.
authors.forename: Toshiro
authors.initials: T
authors.lastname: Sugiyama
mesh_terms: D000818:Animals; D001706:Biopsy; D002478:Cells, Cultured; D004947:Esophagus / Q000166:cytology* / Q000378:metabolism; D005764:Gastroesophageal Reflux / Q000378:metabolism / Q000473:pathology*; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D044464:Receptor, PAR-2 / Q000235:genetics / Q000378:metabolism*; D050916:TRPV Cation Channels / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation
chemical_list: D012333:RNA, Messenger; D044464:Receptor, PAR-2; D050916:TRPV Cation Channels; C486576:Trpv4 protein, mouse
keywords: ATP; GERD; Protease-activated receptor-2; Transient receptor potential vanilloid 4