Distribution and Ca(2+) signalling of fibroblast-like (PDGFR(+)) cells in the murine gastric fundus

Salah A Baker; Grant W Hennig; Anna K Salter; Masaki Kurahashi; Sean M Ward; Kenton M Sanders

doi:10.1113/jphysiol.2013.264747

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Distribution and Ca(2+) signalling of fibroblast-like (PDGFR(+)) cells in the murine gastric fundus

Journal article

Open access

Peer reviewed

Distribution and Ca(2+) signalling of fibroblast-like (PDGFR(+)) cells in the murine gastric fundus

Salah A Baker, Grant W Hennig, Anna K Salter, Masaki Kurahashi, Sean M Ward and Kenton M Sanders

The Journal of physiology, Vol.591(24), pp.6193-6208

12/15/2013

DOI: 10.1113/jphysiol.2013.264747

PMCID: PMC3892471

PMID: 24144881

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Abstract

Platelet-derived growth factor receptor α positive (PDGFRα(+)) cells are suggested to mediate purinergic inputs in GI muscles, but the responsiveness of these cells to purines in situ has not been evaluated. We developed techniques to label and visualize PDGFRα(+) cells in murine gastric fundus, load cells with Ca(2+) indicators, and follow their activity via digital imaging. Immunolabelling demonstrated a high density of PDGFRα(+) cells in the fundus. Cells were isolated and purified by fluorescence-activated cell sorting (FACS) using endogenous expression of enhanced green fluorescent protein (eGFP) driven off the Pdgfra promoter. Quantitative PCR showed high levels of expression of purinergic P2Y1 receptors and SK3 K(+) channels in PDGFRα(+) cells. Ca(2+) imaging was used to characterize spontaneous Ca(2+) transients and responses to purines in PDGFRα(+) cells in situ. ATP, ADP, UTP and β-NAD elicited robust Ca(2+) transients in PDGFRα(+) cells. Ca(2+) transients were also elicited by the P2Y1-specific agonist (N)-methanocarba-2MeSADP (MRS-2365), and inhibited by MRS-2500, a P2Y1-specific antagonist. Responses to ADP, MRS-2365 and β-NAD were absent in PDGFRα(+) cells from P2ry1((-/-)) mice, but responses to ATP were retained. Purine-evoked Ca(2+) transients were mediated through Ca(2+) release mechanisms. Inhibitors of phospholipase C (U-73122), IP3 (2-APB), ryanodine receptors (Ryanodine) and SERCA pump (cyclopiazonic acid and thapsigargin) abolished Ca(2+) transients elicited by purines. This study provides a link between purine binding to P2Y1 receptors and activation of SK3 channels in PDGFRα(+) cells. Activation of Ca(2+) release is likely to be the signalling mechanism in PDGFRα(+) cells responsible for the transduction of purinergic enteric inhibitory input in gastric fundus muscles.

Adenosine Diphosphate - pharmacology

Animals

Calcium - metabolism

Calcium Channel Blockers - pharmacology

Calcium Signaling

Fibroblasts - drug effects

Fibroblasts - metabolism

Flow Cytometry - methods

Gastric Fundus - cytology

Gastric Fundus - metabolism

Mice

Mice, Inbred C57BL

NAD - pharmacology

Purinergic Agents - pharmacology

Receptor, Platelet-Derived Growth Factor alpha - genetics

Receptor, Platelet-Derived Growth Factor alpha - metabolism

Type C Phospholipases - antagonists & inhibitors

Details

Title: Subtitle: Distribution and Ca(2+) signalling of fibroblast-like (PDGFR(+)) cells in the murine gastric fundus
Creators: Salah A Baker - University of Nevada, Reno
Grant W Hennig - University of Nevada, Reno
Anna K Salter - University of Nevada, Reno
Masaki Kurahashi - University of Nevada, Reno
Sean M Ward - University of Nevada, Reno
Kenton M Sanders - University of Nevada, Reno
Resource Type: Journal article
Publication Details: The Journal of physiology, Vol.591(24), pp.6193-6208
DOI: 10.1113/jphysiol.2013.264747
PMID: 24144881
PMCID: PMC3892471
NLM abbreviation: J Physiol
eISSN: 1469-7793
Grant note: DK57236 / NIDDK NIH HHS P01-DK41315 / NIDDK NIH HHS R01 DK091336 / NIDDK NIH HHS
Language: English
Date published: 12/15/2013
Academic Unit: Internal Medicine
Record Identifier: 9984359795502771

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