Dataset Information

Involvement of VNUT-exocytosis in transient receptor potential vanilloid 4-dependent ATP release from gastrointestinal epithelium.

ABSTRACT: 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.

SUBMITTER: Mihara H

PROVIDER: S-EPMC6203352 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Involvement of VNUT-exocytosis in transient receptor potential vanilloid 4-dependent ATP release from gastrointestinal epithelium.

Mihara Hiroshi H Uchida Kunitoshi K Koizumi Schuichi S Moriyama Yoshinori Y

PloS one 20181026 10

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 pr ...[more]

PMID: 30365528

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Project description:PurposeHydration and ionic composition of the subretinal space (SRS) is modulated by the retinal pigment epithelium (RPE). In particular calcium concentration (Ca(2+)) in the SRS varies with light exposure, and although this change is regulated by RPE transport activity, the specific transport proteins involved have yet to be defined. Two members of the transient receptor potential vanilloid family, TRPV5 and TRPV6, are calcium selective ion channels and are known to be expressed in calcium-transporting epithelial tissues. The present work characterizes of TRPV5 and TRPV6 in RPE.MethodsReverse transcriptase PCR was used to examine the presence of TRPV5 and TRPV6 mRNA in cultured human RPE. Protein expression was assessed by western blotting using TRPV5- and TRPV6-specific antibodies. Immunocytochemistry was employed to examine subcellular localization of TRPV5 and TRPV6 in frozen, formaldehyde-fixed sections of native RPE-choroid tissue and in cultured human RPE monolayers. Finally, TRPV5/TRPV6 activity was assessed in cultured RPE, using Ca(2+) indicator dyes to follow [Ca(2+)](i) as a function of changes in [Ca(2+)](o) with and without addition of the TRPV5/TRPV6 inhibitor ruthenium red.ResultsDirect sequencing of PCR DNAs documented the presence of TRPV5 and TRPV6 transcripts in human RPE. Immunocytochemistry showed that TRPV5 and TRPV6 are expressed in native RPE-choroid tissue with strong immunoreactivity for both channels on the apical as well as the basal plasma membranes. Immunostaining for both channels was also positive in monolayers of cultured RPE cells. In cultured cells subcellular localization was variable with immunoreactivity present in the cytoplasmic domain as well as on the plasma membrane. Plasma membrane staining was increased with phagocytosis. The reported molecular weight of the core protein for both TRPV5 and TRPV6 is about 75 kDa, with the expected size of the glycosylated proteins in the range of 85-100 kDa. Western blot analysis of TRPV6 in RPE detected a distinct band at approximately 85 kDa, with another strong band at approximately 60 kDa. A similar pattern was seen for TRPV5, with strong bands at 82 kDa and 71 kDa. In live-cell imaging experiments, [Ca(2+)](i) was lower in the presence of the TRPV5/TRPV6 inhibitor ruthenium red.ConclusionsRPE expresses the epithelial calcium channels TRPV5 and TRPV6, the most calcium-selective channels of the TRP superfamily. Present findings suggest that these channels could function in RPE to mediate calcium influx from SRS and thus regulate changes in SRS calcium composition that accompany light/dark transitions.

Dataset Information

Involvement of VNUT-exocytosis in transient receptor potential vanilloid 4-dependent ATP release from gastrointestinal epithelium.

Publications

Involvement of VNUT-exocytosis in transient receptor potential vanilloid 4-dependent ATP release from gastrointestinal epithelium.

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