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Adenosine triphosphate (ATP) modulates mechanosensitive vagal afferent nerves in the gastrointestinal tract. ATP is stored in secretory vesicles via the ATP transporter VNUT. Recently, the bisphosphate clodronate was reported to inhibit VNUT and was suggested to be a safe potent therapeutic option for chronic pain. Transient receptor potential vanilloid 4 (TRPV4) is activated by mechanical stimuli and some epoxyeicosatrienoic acids and becomes sensitized under inflammatory conditions. We have previously reported that TRPV4 and VNUT are expressed in mouse esophageal keratinocytes and that TRPV4 activation induces ATP release in gastric epithelial cells. Here we show the expression of TRPV4 and VNUT in normal human gastrointestinal cell derived cell lines (GES-1 and CCD 841) and in tissues from normal and VNUT-KO mice. TRPV4 agonists (GSK101 or 8,9-EET) induced an increase in cytosolic Ca2+ and/or current responses in mouse primary colonic epithelial cells and CCD 841 cells, but not in cells isolated from TRPV4-KO mice. TRPV4 agonists (GSK101 or 5.6-EET) also induced ATP release in GES-1 and CCD 841 cells, which could be blocked by the VNUT inhibitor, clodronate. Thus, VNUT inhibition with clodronate could represent a novel therapeutic option for visceral pain., Competing Interests: The authors have declared that no competing interests exist.

Background: Transient receptor potential vanilloid 4 (TRPV4) is activated by stretch (mechanical), warm temperature, some epoxyeicosatrienoic acids, and lipopolysaccharide. TRPV4 is expressed throughout the gastrointestinal epithelia and its activation induces adenosine triphosphate (ATP) exocytosis that is involved in visceral hypersensitivity. As an ATP transporter, vesicular nucleotide transporter (VNUT) mediates ATP storage in secretory vesicles and ATP release via exocytosis upon stimulation. Summary: TRPV4 is sensitized under inflammatory conditions by a variety of factors, including proteases and serotonin, whereas methylation-dependent silencing of TRPV4 expression is associated with various pathophysiological conditions. Gastrointestinal epithelia also release ATP in response to hypo-osmolality or acid through molecular mechanisms that remain unclear. These synergistically released ATP could be involved in visceral hypersensitivity. Low concentrations of the first generation bisphosphate, clodronate, were recently reported to inhibit VNUT activity and thus clodronate may be a safe and potent therapeutic option to treat visceral pain. Key Messages: This review focuses on: (1) ATP and TRPV4 activities in gastrointestinal epithelia; (2) factors that could modulate TRPV4 activity in gastrointestinal epithelia; and (3) the inhibition of VNUT as a potential novel therapeutic strategy for functional gastrointestinal disorders. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]