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Recent studies reveal complex patterns of hormone coexpression within enteroendocrine cells (EECs), contrary to the traditional view that gut hormones are expressed individually in EECs. Moreover, different hormones have been found in separate subcellular vesicles. However, detailed analysis of relative expression of multiple hormones has not been made. Subcellular studies have been confined to peptide hormones, and have not included the indolamine 5-hydroxytryptamine (5-HT) or the neuroendocrine protein chromogranin A (CgA). In the present work, coexpression of 5-HT, CgA, secretin, cholecystokinin (CCK), ghrelin, and glucagonlike peptide (GLP)-1 in mouse duodenum was quantified at a cellular and subcellular level by semiautomated cell counting and quantitative vesicle measurements. We investigated whether relative numbers of cells with colocalized hormones analyzed at a cell level matched the numbers revealed by examination of individual storage vesicles within cells. CgA and 5-HT were frequently expressed in EECs that contained combinations of GLP-1, ghrelin, secretin, and CCK. Separate subcellular stores of 5-HT, CgA, secretin, CCK, ghrelin, and GLP-1 were identified. In some cases, high-resolution analysis revealed small numbers of immunoreactive vesicles in cells dominated by a different hormone. Thus the observed incidence of cells with colocalized hormones is greater when analyzed at a subcellular, compared with a cellular, level. Subcellular analysis also showed that relative numbers of vesicles differ considerably between cells. Thus separate packaging of hormones that are colocalized is a general feature of EECs, and EECs exhibit substantial heterogeneity, including the colocalization of hormones that were formerly thought to be in cells of different lineages.

Patients with Hirschsprung disease lack enteric ganglia in the distal colon and propulsion of colorectal content is substantially impaired. Proposed stem cell therapies to replace neurons require surgical bypass of the aganglionic bowel during re-colonization, but there is inadequate knowledge of the consequences of bypass. We performed bypass surgery in Ednrb−/− Hirschsprung rat pups. Surgically rescued rats failed to thrive, an outcome reversed by supplying electrolyte- and glucose-enriched drinking water. Histologically, the bypassed colon had normal structure, but grew substantially less in diameter than the functional region proximal to the bypass. Extrinsic sympathetic and spinal afferent neurons projected to their normal targets, including arteries and the circular muscle, in aganglionic regions. However, although axons of intrinsic excitatory and inhibitory neurons grew into the aganglionic region, their normally dense innervation of circular muscle was not restored. Large nerve trunks that contained tyrosine hydroxylase (TH)-, calcitonin gene-related peptide (CGRP, encoded by Calca or Calcb)-, neuronal nitric oxide synthase (nNOS or NOS1)-, vasoactive intestinal peptide (VIP)- and tachykinin (encoded by Tac1)-immunoreactive axons occurred in the distal aganglionic region. We conclude that the rescued Ednrb−/− rat provides a good model for the development of cell therapies for the treatment of Hirschsprung disease.

Oligophrenin-1 is a RhoGTPase-activating protein (RhoGAP) that is involved in the regulation of shape changes in dendritic spines, and outgrowth of axons and dendrites in the brain. These changes in neuronal morphology are central to the mechanisms of plasticity, learning, and memory. Although the enteric nervous system also exhibits long-term changes in neuronal function, the expression and involvement of oligophrenin-1 has not previously been investigated. We show by RT-PCR analysis that oligophrenin-1 mRNA is expressed in the myenteric plexus (MP) of the guinea pig ileum. Sequencing of RT-PCR products showed that guinea pig oligophrenin-1 mRNA is 98% and 87% homologous to human and mouse oligophrenin-1, respectively, except that a 42 bp sequence is absent from the guinea pig mRNA. This 42 bp sequence codes for a sequence of 14 amino acids located near the carboxy-terminal end of the RhoGAP domain in the human sequence. An antibody that recognizes human oligophrenin-1 identified a 91 kDa protein band in rat and mouse brain lysates and in guinea pig sciatic nerve, and a 36 kDa protein band in both purified enteric ganglion cell and brain lysate from guinea pig. Oligophrenin-1 is localized specifically to neurons and varicose axons in the MPs and submucosal plexuses (SMPs) of the guinea pig and rat, but is not detectable in glial cells, smooth muscle, or other cell types. These findings indicate that oligophrenin-1 is expressed in the enteric nervous system, where it may regulate morphological changes in axons and dendrites, and thus modulate neuronal connectivity. Anat Rec Part A 273A:671–676, 2003. © 2003 Wiley-Liss, Inc.

Oligophrenin-1 is a RhoGTPase-activating protein (RhoGAP) that is involved in the regulation of shape changes in dendritic spines, and outgrowth of axons and dendrites in the brain. These changes in neuronal morphology are central to the mechanisms of plasticity, learning, and memory. Although the enteric nervous system also exhibits long-term changes in neuronal function, the expression and involvement of oligophrenin-1 has not previously been investigated. We show by RT-PCR analysis that oligophrenin-1 mRNA is expressed in the myenteric plexus (MP) of the guinea pig ileum. Sequencing of RT-PCR products showed that guinea pig oligophrenin-1 mRNA is 98% and 87% homologous to human and mouse oligophrenin-1, respectively, except that a 42 bp sequence is absent from the guinea pig mRNA. This 42 bp sequence codes for a sequence of 14 amino acids located near the carboxy-terminal end of the RhoGAP domain in the human sequence. An antibody that recognizes human oligophrenin-1 identified a 91 kDa protein band in rat and mouse brain lysates and in guinea pig sciatic nerve, and a 36 kDa protein band in both purified enteric ganglion cell and brain lysate from guinea pig. Oligophrenin-1 is localized specifically to neurons and varicose axons in the MPs and submucosal plexuses (SMPs) of the guinea pig and rat, but is not detectable in glial cells, smooth muscle, or other cell types. These findings indicate that oligophrenin-1 is expressed in the enteric nervous system, where it may regulate morphological changes in axons and dendrites, and thus modulate neuronal connectivity. Anat Rec Part A 273A:671–676, 2003. © 2003 Wiley-Liss, Inc.