Distribution and Ca2+ signalling of fibroblast-like (PDGFRα+) cells in the murine gastric fundus

Platelet-derived growth factor receptor positive (PDGFR(+)) 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 PDGFR(+) cells in murine gastric fundus, load cells with Ca2+ indicators, and follow their activity via digital imaging. Immunolabelling demonstrated a high density of PDGFR(+) 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 PDGFR(+) cells. Ca2+ imaging was used to characterize spontaneous Ca2+ transients and responses to purines in PDGFR(+) cells in situ. ATP, ADP, UTP and -NAD elicited robust Ca2+ transients in PDGFR(+) cells. Ca2+ 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 -NAD were absent in PDGFR(+) cells from P2ry1((-/-)) mice, but responses to ATP were retained. Purine-evoked Ca2+ transients were mediated through Ca2+ release mechanisms. Inhibitors of phospholipase C (U-73122), IP3 (2-APB), ryanodine receptors (Ryanodine) and SERCA pump (cyclopiazonic acid and thapsigargin) abolished Ca2+ transients elicited by purines. This study provides a link between purine binding to P2Y1 receptors and activation of SK3 channels in PDGFR(+) cells. Activation of Ca2+ release is likely to be the signalling mechanism in PDGFR(+) cells responsible for the transduction of purinergic enteric inhibitory input in gastric fundus muscles.