Submitted on December 6, 2005
Accepted on January 24, 2006
Isolation, Functional Characterization and Transcriptome of Mastomys Ileal Enterochromaffin Cells
Mark Kidd1, Irvin M. Modlin1*, Geeta N. Eick1, and Manish C. Champaneria1
1 Gastrointestinal Pathobiology Research Group, Yale University School of Medicine, New Haven, CT, USA
* To whom correspondence should be addressed. E-mail: imodlin{at}optonline.net.
Although the enterochromaffin (EC) cell is one of the primary neuroendocrine (NE) 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 have 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, Nycodenz gradient centrifuged, and EC cells collected by FACS sorting 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. 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 measured by ELISA. Gene Chip Affymetrix profiling of FACS sorted cells was undertaken to obtain the EC cell transcriptome. FACS sorting produced a >70-fold enrichment of EC cells with a serotonin content of 240±+22ng/mg protein. Preparations were 99±0.7% pure by immunostaining for tryptophan hydroxylase. VPAC1 and SSTR2 receptors were present whereas PAC1 and CCK2 receptors were undetectable. Forskolin, isoproterenol and PACAP-38 stimulated serotonin secretion at EC50 values of 5x10-10M, 4.5x10-10M and 1.2x10-9M, respectively. Isoproterenol stimulated cAMP levels ~3.5±0.62-fold versus unstimulated cells (EC50 of ~10-9M). Octreotide, acetylcholine and GABAA inhibited serotonin secretion with IC50 values of 3x10-11M, 3x10-10M, 2.9x10-10M, 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 have been able to isolate homogeneous preparations (>99%) of live ileal EC cells and demonstrated regulation of serotonin secretion as well as establish 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.
