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.

RCAN1 is a chromosome 21 gene that controls secretion in endocrine cells, regulates mitochondrial function, and is sensitive to oxidative stress. Regulator of calcineurin 1 (RCAN1) is also an endogenous inhibitor of the protein phosphatase calcineurin, the inhibition of which leads to hypoinsulinemia and diabetes in humans and mice. However, the presence or the role of RCAN1 in insulin-secreting β-cells and its potential role in the pathogenesis of diabetes is unknown. Hence, the aim of this study is to investigate the presence of RCAN1 in β-cells and identify its role in β-cell function. RCAN1 is expressed in mouse islets and in the cytosol of pancreatic β-cells. We find RCAN1 is a glucose-responsive gene with a 1.5-fold increase in expression observed in pancreatic islets in response to chronic hyperglycemia. The overexpression of the human RCAN1.1 isoform in mice under the regulation of its endogenous promoter causes diabetes, age-associated hyperglycemia, reduced glucose tolerance, hypoinsulinemia, loss of β-cells, reduced β-cell insulin secretion, aberrant mitochondrial reactive oxygen species production, and the down-regulation of key β-cell genes. Our data therefore identifies a novel molecular link between the overexpression of RCAN1 and β-cell dysfunction. The glucose-responsive nature of RCAN1 provides a potential mechanism of action associated with the β-cell dysfunction observed in diabetes.

Mitochondria are the primary site of cellular energy generation and reactive oxygen species (ROS) accumulation. Elevated ROS levels are detrimental to normal cell function and have been linked to the pathogenesis of neurodegenerative disorders such as Down's syndrome (DS) and Alzheimer's disease (AD). RCAN1 is abundantly expressed in the brain and overexpressed in brain of DS and AD patients. Data from nonmammalian species indicates that increased RCAN1 expression results in altered mitochondrial function and that RCAN1 may itself regulate neuronal ROS production. In this study, we have utilized mice overexpressing RCAN1 (RCAN1ox) and demonstrate an increased susceptibility of neurons from these mice to oxidative stress. Mitochondria from these mice are more numerous and smaller, indicative of mitochondrial dysfunction, and mitochondrial membrane potential is altered under conditions of oxidative stress. We also generated a PC12 cell line overexpressing RCAN1 (PC12RCAN1). Similar to RCAN1ox neurons, PC12RCAN1 cells have an increased susceptibility to oxidative stress and produce more mitochondrial ROS. This study demonstrates that increasing RCAN1 expression alters mitochondrial function and increases the susceptibility of neurons to oxidative stress in mammalian cells. These findings further contribute to our understanding of RCAN1 and its potential role in the pathogenesis of neurodegenerative disorders such as AD and DS.

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Enterochromaffin cells are the major site of serotonin (5-HT) synthesis and secretion providing ∼95% of the body's total 5-HT. 5-HT can act as a neurotransmitter or hormone and has several important endocrine and paracrine roles. We have previously demonstrated that EC cells release small amounts of 5-HT per exocytosis event compared to other endocrine cells. We utilized a recently developed method to purify EC cells to demonstrate the mechanisms underlying 5-HT packaging and release. Using the fluorescent probe FFN511, we demonstrate that EC cells express VMAT and that VMAT plays a functional role in 5-HT loading into vesicles. Carbon fiber amperometry studies illustrate that the amount of 5-HT released per exocytosis event from EC cells is dependent on both VMAT and the H(+)-ATPase pump, as demonstrated with reserpine or bafilomycin, respectively. We also demonstrate that increasing the amount of 5-HT loaded into EC cell vesicles does not result in an increase in quantal release. As this indicates that fusion pore size may be a limiting factor involved, we compared pore diameter in EC and chromaffin cells by assessing the vesicle capture of different-sized fluorescent probes to measure the extent of fusion pore dilation. This identified that EC cells have a reduced fusion pore expansion that does not exceed 9 nm in diameter. These results demonstrate that the small amounts of 5-HT released per fusion event in EC cells can be explained by a smaller fusion pore that limits 5-HT release capacity from individual vesicles.