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Published on: July 4, 2018
Isolation, functional characterization, and transcriptome of Mastomys ileal enterochromaffin cells
1Gastrointestinal Pathobiology Research Group, Yale University School of Medicine, CT, USA.
Insights
Researchers developed a novel method to isolate pure enterochromaffin (EC) cells from the ileum. This breakthrough enables detailed study of EC cell function and the gut
Area of Science:
- Gastroenterology and Hepatology
- Neuroendocrinology
- Cell Biology
Background:
- Enterochromaffin (EC) cells are crucial neuroendocrine regulators in the small intestine.
- Previous limitations in isolating pure EC cells hindered comprehensive physiological and molecular characterization.
- Understanding EC cell function is vital for elucidating gastrointestinal regulatory mechanisms.
Purpose of the Study:
- To develop a robust methodology for isolating pure populations of Mastomys ileal EC cells.
- To evaluate the functional regulation of isolated EC cells, specifically serotonin secretion.
- To define the complete transcriptome of EC cells for molecular insights.
Main Methods:
- Isolation of EC cells using enzymatic digestion, Nycodenz gradient centrifugation, and fluorescence-activated cell sorting (FACS).
- Confirmation of purity and identity via immunostaining (tryptophan hydroxylase, chromogranin A), serotonin content analysis, and electron microscopy.
- Assessment of receptor expression (RT-PCR) and functional responses (ELISA, cAMP assays) to various stimuli; transcriptome analysis using GeneChip Affymetrix profiling.
Main Results:
- Achieved >70-fold enrichment and >99% purity of ileal EC cells.
- Demonstrated functional regulation of serotonin secretion by various agonists and antagonists, including PACAP-38, forskolin, isoproterenol, octreotide, acetylcholine, and GABAA.
- Established the EC cell transcriptome, revealing expression of key marker genes and a broad range of neurotransmitter and hormone receptors.
Conclusions:
- Successfully developed and validated a method for isolating highly pure, viable ileal EC cells.
- Characterized the functional responses and molecular profile of EC cells, providing a foundation for further research.
- This methodology is applicable to human ileum, paving the way for understanding EC cell pathophysiology in disease states.
Abstract:
Although the enterochromaffin (EC) cell is one of the primary neuroendocrine regulatory cells of the small intestine, the lack of a purified cell system has precluded characterization of the cell and limited precise physiological evaluation. We developed methodology to obtain a pure population of Mastomys ileal EC cells, evaluated their functional regulation, and defined the transcriptome. Mastomys ilea were everted, end ligated, pronase-collagenase digested, and Nycodenz gradient centrifuged, and EC cells were collected by fluorescence-activated cell sorting (FACS) of acridine orange-labeled cells. Enrichment was confirmed by immunostaining of tryptophan hydroxylase and chromogranin A, specific EC cell markers, serotonin content, EC cell marker gene expression, and electron microscopy. Pituitary adenylate cyclase-activating polypeptide (PACAP), somatostatin, and gastrin receptor expression was determined by real-time RT-PCR. Live post-FACS-sorted cells were cultured, and the effects of forskolin, isoproterenol, acetylcholine, GABAA, PACAP-38, and gastrin on serotonin secretion were measured by ELISA. GeneChip Affymetrix profiling of FACS-sorted cells was undertaken to obtain the EC cell transcriptome. FACS produced a >70-fold enrichment of EC cells with a serotonin content of 240 +/- 22 ng/mg protein. Preparations were 99 +/- 0.7% pure by immunostaining for tryptophan hydroxylase. Vasoactive intestinal peptide/PACAP receptor 1 (VPAC1) and somatostatin receptor 2 were present, whereas PACAP receptor 1 (PAC1) and CCK2 receptors were undetectable. Forskolin, isoproterenol, and PACAP-38 stimulated serotonin secretion at EC50 values of 5 x 10(-10), 4.5 x 10(-10), and 1.2 x 10(-9) M, respectively. Isoproterenol stimulated cAMP levels by approximately 3.5 +/- 0.62-fold vs. unstimulated cells (EC50 of approximately 10(-9) M). Octreotide, acetylcholine, and GABAA inhibited serotonin secretion with IC50 values of 3 x 10(-11), 3 x 10(-10), and 2.9 x 10(-10) M, respectively. Gastrin had no effect on serotonin secretion. The naive EC cell transcriptome revealed highly expressed EC cell marker genes, the absence of marker genes for other small intestinal cell types, and a receptor profile that included cholinergic, adrenergic, dopaminergic, serotoninergic, GABAergic, and prostaglandin receptors. We were able to isolate homogeneous preparations (>99%) of live ileal EC cells and demonstrated regulation of serotonin secretion as well as established the normal EC cell transcriptome. Application of this methodology to normal and diseased human ileum will facilitate the elucidation of the pathophysiology of EC cells.
