Nodose immunostaining: Similar methods as above were utilised except the following primary antibodies were used: P2X3 (Alomone, APR-016, 1:200 or Neuromics, GP10108, 1:200), GFP (Abcam, ab13970, 1:2000) and GLP1R antibody51 (0.1?mg?ml?1). epithelium and generate signals in response to food ingestion. Whilst traditionally considered hormone-producing cells, there is evidence that they also initiate activity in the afferent vagus nerve and thereby signal directly to the brainstem. We investigate whether enteroendocrine L-cells, well known for their production of the incretin hormone glucagon-like peptide-1 (GLP-1), also release other neuro-transmitters/modulators. We demonstrate regulated ATP release by ATP measurements in cell supernatants and by using sniffer patches that generate electrical currents upon ATP exposure. Employing purinergic receptor antagonists, we demonstrate that evoked ATP release from L-cells triggers electrical responses in neighbouring enterocytes through P2Y2 and nodose ganglion neurones in co-cultures through P2X2/3-receptors. We conclude that L-cells co-secrete ATP together with GLP-1 and PYY, and that ATP acts as an additional signal triggering vagal activation and potentially synergising with the actions of locally elevated peptide hormone concentrations. Introduction Enteroendocrine cells (EECs) are specialized hormone-releasing cells scattered along the gastrointestinal epithelium. In response to various stimuli following food ingestion, they release a host of gut peptide hormones, including glucagon-like peptide 1 (GLP-1), which is secreted from a subpopulation of EECs traditionally called L-cells, that at least in the distal intestine often co-secrete peptide YY (PYY)1. GLP-1 acts as an incretin hormone, boosting glucose dependent insulin release from pancreatic -cells and both GLP-1 and PYY suppress food intake1. The anorexic action of these hormones is thought at least in part to be mediated through activation of their cognate G-protein coupled receptors (GLP1R and NPY2R, respectively) located on vagal afferent nerve terminals, originating from neurons with somata in the nodose ganglia2. We showed previously that GLP-1 application in isolation did (±)-BAY-1251152 little to cytosolic Ca2+-concentrations in subunit expression levels (2?Ct values) of ND neurons from intact ganglia (black circles), acutely dissociated neurons (black squares), and after 3 days in vitro cultures (black triangles). Samples for each type of preparation were prepared from ND ganglia pooled from 2 to 3 3 mice, repeated three independent times. Individual data points represent independent preparations and lines represent mean??SEM (subunit expression from individually picked ND neurons. Each column represents a single ND neuron. Range indicator for heat map on left. Sample GLP1R negative (c) and GLP1R-positive (d) NeuroD1-EYFP neuron immunostained for P2X3 (Alomone P2X3 antibody APR-016 in c, Neuromics P2X3 antibody GP10108 in d) and GLP1R. Scale bars represent 20?m. e Scatterplot of % block of exogenous ATP (100?M) application by 100?M PPADs (grey filled circles, and subunits (Fig.?6a). Heterogeneity of subunit expression in ND neurons was evident from single-cell expression LHR2A antibody analysis (Fig.?6b); however, expression was present in all ND neurons examined and its levels were the highest compared with all other subunits. Immunostaining for P2X3 in dissociated ND cultures confirmed protein expression in GLP1R negative (Fig.?6c) and positive (Fig.?6d) neurons. To examine the functional contribution of P2X3 in signalling between L-cells and vagal afferents, the more selective P2X2/P2X3 blocker Ro51 was tested on co-cultures of Gq-DREADD transfected GLUTag cells and ND neurons (Fig.?6f). GLP1R-positive ND neurons were also examined using the GLP1R-Cre mouse line3 to identify GLP1R-expressing ND neurons. Ro51 reduced the peak amplitude of CNO-induced Ca2+ responses in most ND neurons (Fig.?6g) and overall inhibited CNO-triggered Ca2+ elevations by 54% (Fig.?6h), (±)-BAY-1251152 thus supporting the role of P2X3 in ATP signalling between L-cells and vagal afferent neurons. Signalling from L-cells to sensory neurones in intact colon To examine whether L-cell-released ATP triggers afferent nerve signalling within the intact gut, we measured changes in mesenteric nerve activity from the proximal colon following AngII mediated L-cell activation. Reproducible biphasic increases in nerve discharges were elicited by bath application of AngII (1?M) following pretreatment with IBMX (100?M; Supplementary Figure?5a, b, f). This consisted of a rapid transient increase in nerve firing followed by a sustained plateau of activity lasting more than 10?min. Repetitive AngII responses could be obtained from the same sample with similar response profiles and minimal desensitization (Supplementary Figure?5c, d, e). No significant change was observed in the transient response in the presence of a purinergic antagonist, whilst the plateau phase of AngII responses was largely attenuated following pre-treatment with PPADS (Supplementary Figure?5e, g, h). Discussion Beyond its roles as an energy source for numerous biochemical processes and a stabilizer of catecholamine loading in secretory vesicles20, ATP has been widely regarded as a signalling molecule in its own right21. In this study, we provide evidence for (±)-BAY-1251152 regulated ATP release from enteroendocrine L-cells and demonstrate functional purinergic signalling from L-cells to enterocyte and neuronal targets within.