Key points• Enterochromaffin (EC) cells are enteroendocrine cells that synthesise ∼95% of the body's total serotonin (5-HT).• Although 5-HT release from EC cells plays a number of important physiological roles, primary EC cells have not been studied at the single cell level.• This study provides the first functional characterisation of single primary guinea-pig and human EC cells.• EC cells release 5-HT from large dense core vesicles in a calcium-dependent manner with kinetics surprisingly resembling release from synaptic vesicles.• 3D modelling indicates that the quantity of 5-HT released per vesicle fusion event is physiologically relevant to GI tract function in terms of the concentrations needed to activate local 5-HT receptors.• These findings represent significant advances in our understanding of EC cell function and will be of broad interest to researchers in endocrine cell biology, gastroenterology, neuroscience, exocytosis and glucose control.Abstract The major source of serotonin (5-HT) in the body is the enterochromaffin (EC) cells lining the intestinal mucosa of the gastrointestinal tract. Despite the fact that EC cells synthesise ∼95% of total body 5-HT, and that this 5-HT has important paracrine and endocrine roles, no studies have investigated the mechanisms of 5-HT release from single primary EC cells. We have developed a rapid primary culture of guinea-pig and human EC cells, allowing analysis of single EC cell function using electrophysiology, electrochemistry, Ca 2+ imaging, immunocytochemistry and 3D modelling. Ca 2+ enters EC cells upon stimulation and triggers quantal 5-HT release via L-type Ca 2+ channels. Real time amperometric techniques reveal that EC cells release 5-HT at rest and this release increases upon stimulation. Surprisingly for an endocrine cell storing 5-HT in large dense core vesicles (LDCVs), EC cells release 70 times less 5-HT per fusion event than catecholamine released from similarly sized LDCVs in endocrine chromaffin cells, and the vesicle release kinetics instead resembles that observed in mammalian synapses. Furthermore, we measured EC cell density along the gastrointestinal tract to create three-dimensional (3D) simulations of 5-HT diffusion using the minimal number of variables required to understand the physiological relevance of single cell 5-HT release in the whole-tissue milieu. These models indicate that local 5-HT levels are likely to be maintained around the activation threshold for mucosal 5-HT receptors and that this is dependent upon stimulation and location within the gastrointestinal tract. This is the first study demonstrating single cell 5-HT release in primary EC cells. The mode of 5-HT release may represent a unique mode of exocytosis amongst endocrine cells and is functionally relevant to gastrointestinal sensory and motor function.

BackgroundEnteroendocrine cells collectively constitute our largest endocrine tissue, with serotonin (5-HT) secreting enterochromaffin (EC) cells being the largest component (~50 %). This gut-derived 5-HT has multiple paracrine and endocrine roles. EC cells are thought to act as nutrient sensors and luminal glucose is the major absorbed form of carbohydrate in the gut and activates secretion in an array of cell types. It is unknown whether EC cells release 5-HT in response to glucose in primary EC cells. Furthermore, fasting augments 5-HT synthesis and release into the circulation. However, which nutrients cause fasting-induced synthesis of EC cell 5-HT is unknown. Here we examine the effects of acute and chronic changes in glucose availability on 5-HT release from intact tissue and single EC cells.MethodsWe utilised established approaches in our laboratories measuring 5-HT release in intact mouse colon with amperometry. We then examined single EC cells function using our published protocol in guinea-pig colon. Single cell Ca2+ imaging and amperometry were used with these cells. Real-time PCR was used along with amperometry, on primary EC cells cultured for 24 h in 5 or 25 mM glucose.ResultsWe demonstrate that acute increases in glucose, at levels found in the gut lumen rather than in plasma, trigger 5-HT release from intact colon, and cause Ca2+ entry and 5-HT release in primary EC cells. Single cell amperometry demonstrates that high glucose increases the amount of 5-HT released from individual vesicles as they undergo exocytosis. Finally, 24 h incubation of EC cells in low glucose causes an increase in the transcription of the 5-HT synthesising enzyme Tph1 as well as increasing in 5-HT secretion in EC cells.ConclusionsWe demonstrate that primary EC cells respond to acute changes in glucose availability through increases in intracellular Ca2+ the activation of 5-HT secretion, but respond to chronic changes in glucose levels through the transcriptional regulation of Tph1 to alter 5-HT synthesis.