Isolation, functional characterization, and transcriptome of Mastomys ileal enterochromaffin cells

Abstract

) 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, GABA A, 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 Ͼ70-fold enrichment of EC cells with a serotonin content of 240 Ϯ 22 ng/mg protein. Preparations were 99 Ϯ 0.7% pure by immunostaining for tryptophan hydroxylase. Vasoactive intestinal peptide/ PACAP receptor 1 (VPAC 1) 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 EC 50 values of 5 ϫ 10 Ϫ10 , 4.5 ϫ 10 Ϫ10 , and 1.2 ϫ 10 Ϫ9 M, respectively. Isoproterenol stimulated cAMP levels by ϳ3.5 Ϯ 0.62-fold vs. unstimulated cells (EC50 of ϳ10 Ϫ9 M). Octreotide, acetylcholine, and GABAA inhibited serotonin secretion with IC50 values of 3 ϫ 10 Ϫ11 , 3 ϫ 10 Ϫ10 , and 2.9 ϫ 10 Ϫ10 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 (Ͼ99%) 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.fluorescence-activated cell sorting; serotonin; secretion; small intestine; carcinoid THE ENTEROCHROMAFFIN (EC) cell is distributed throughout the gastrointestinal epithelium and is regarded as the predominant neuroendocrine (NE) cell of the bowel (48). Its location within glands at the base of intestinal crypts and its basal extensions projecting into adjacent glands attest to its local regulatory role (9). Although generally thought of as a specific cell type, EC cells may compr...

Role of CCN2/CTGF in the proliferation of Mastomys enterochromaffin-like cells and gastric carcinoid development

“…Cell contamination by any of these matrix cells could result in the measurement of elevated ccn2 transcript in tumor ECL cells compared with normal cells. This possibility led us to modify methodology initially developed by us for small intestinal EC cells (16) that would facilitate our ability to obtain ECL cell populations of substantially greater purity than the enriched ECL cell populations generated by counterflow elutriation or tumor dissection. Using ECL cellspecific immunostaining by acridine orange, we were able to enrich ECL cells Ͼ98% from both normal mucosa and gastric carcinoids.…”

“…Preparations of normal ECL cells and tumor cells were stained with acridine orange (100 nM) for 30 min (room temperature) (16). The BD FACS Aria Cell-Sorting System (Yale University Department of Immunobiology and Yale Cancer Center) was used to identify and sort cells.…”

Purinergic receptors and gastrointestinal secretomotor function

“…The A 1 receptor also contributes, and an A 1 receptor antagonist (CPT), an A 3 receptor antagonist (MRS1191), or adenosine deaminase could also augment the touch/stretch Ca 2+ response ( [18]. Recent isolation of ileal EC cells [58] may permit further study and comparison of purinergic signaling mechanisms in normal and diseased human ileum. Real-time electrochemical detection of local mucosal 5-HT release is also very promising [5].…”

Role of infection in irritable bowel syndrome

“…Recent studies using isolated enteroendocrine cells indicate the presence of numerous receptors, which allow these cells to integrate many different stimuli. 15 Patients who develop PI-IBS have a 20% increase in EC cells when assessed 3 months after infection 9 and a switch in the ratio of 5-hydroxytryptamine (5HT)/peptide YY (PYY)-containing enteroendocrine cells so that 5HT is the predominant component of enteroendocrine granules after the infection, whereas before PYY predominates. 1…”