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NEUROREGULATION AND MOTILITY

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

Gastrointestinal Pathobiology Research Group, Yale University School of Medicine, New Haven, Connecticut

Submitted 6 December 2005 ; accepted in final form 24 January 2006

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, 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 >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 3.5 ± 0.62-fold vs. unstimulated cells (EC₅₀ of 10^–9 M). Octreotide, acetylcholine, and GABA_A 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.

fluorescence-activated cell sorting; serotonin; secretion; small intestine; carcinoid

This article has been cited by other articles:

				M. Kidd, I. M. Modlin, B. I. Gustafsson, I. Drozdov, O. Hauso, and R. Pfragner Luminal regulation of normal and neoplastic human EC cell serotonin release is mediated by bile salts, amines, tastants, and olfactants Am J Physiol Gastrointest Liver Physiol, August 1, 2008; 295(2): G260 - G272. [Abstract] [Full Text] [PDF]

				M. Kidd, G. N Eick, I. M Modlin, R. Pfragner, M. C Champaneria, and J. Murren Further delineation of the continuous human neoplastic enterochromaffin cell line, KRJ-I, and the inhibitory effects of lanreotide and rapamycin J. Mol. Endocrinol., January 1, 2007; 38(1): 181 - 192. [Abstract] [Full Text] [PDF]

				M. Kidd, I. M. Modlin, G. N. Eick, R. L. Camp, and S. M. Mane Role of CCN2/CTGF in the proliferation of Mastomys enterochromaffin-like cells and gastric carcinoid development Am J Physiol Gastrointest Liver Physiol, January 1, 2007; 292(1): G191 - G200. [Abstract] [Full Text] [PDF]