Anatomy and Neuroscience - Research Publications

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    Distribution and characterisation of CCK containing enteroendocrine cells of the mouse small and large intestine
    Fakhry, J ; Wang, J ; Martins, P ; Fothergill, LJ ; Hunne, B ; Prieur, P ; Shulkes, A ; Rehfeld, JF ; Callaghan, B ; Furness, JB (SPRINGER, 2017-08)
    There is general consensus that enteroendocrine cells, EEC, containing the enteric hormone cholecystokinin (CCK) are confined to the small intestine and predominate in the duodenum and jejunum. Contrary to this, EEC that express the gene for CCK have been isolated from the large intestine of the mouse and there is evidence for EEC that contain CCK-like immunoreactivity in the mouse colon. However, the human and rat colons do not contain CCK cells. In the current study, we use immunohistochemistry to investigate CCK peptide presence in endocrine cells, PCR to identify cck transcripts and chromatography to identify CCK peptide forms in the mouse small and large intestine. The colocalisation of CCK and 5-HT, hormones that have been hypothesised to derive from cells of different lineages, was also investigated. CCK immunoreactivity was found in EEC throughout the mouse small and large intestine but positive cells were rare in the rectum. Immunoreactive EEC were as common in the caecum and proximal colon as they were in the duodenum and jejunum. CCK gene transcripts were found in the mucosa throughout the intestine but mRNA for gastrin, a hormone that can bind some anti-CCK antibodies, was only found in the stomach and duodenum. Characterisation of CCK peptides of the colon by extraction, chromatographic separation and radioimmunoassay revealed bioactive amidated and sulphated forms, including CCK-8 and CCK-33. Moreover, CCK-containing EEC in the large intestine bound antibodies that target the biologically active sulfated form. Colocalisation of CCK and 5-HT occurred in a proportion of EEC throughout the small intestine and in the caecum but these hormones were not colocalised in the colon, where there was CCK and PYY colocalisation. It is concluded that authentic, biologically active, CCK occurs in EEC of the mouse large intestine.
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    Quantitation and chemical coding of enteroendocrine cell populations in the human jejunum
    Coles, TEF ; Fothergill, LJ ; Hunne, B ; Nikfarjam, M ; Testro, A ; Callaghan, B ; McQuade, RM ; Furness, JB (SPRINGER, 2020-01)
    Recent studies reveal substantial species and regional differences in enteroendocrine cell (EEC) populations, including differences in patterns of hormone coexpression, which limit extrapolation between animal models and human. In this study, jejunal samples, with no histologically identifiable pathology, from patients undergoing Whipple's procedure were investigated for the presence of gastrointestinal hormones using double- and triple-labelling immunohistochemistry and high-resolution confocal microscopy. Ten hormones (5-HT, CCK, secretin, proglucagon-derived peptides, PYY, GIP, somatostatin, neurotensin, ghrelin and motilin) were localised in EEC of the human jejunum. If only single staining is considered, the most numerous EEC were those containing 5-HT, CCK, ghrelin, GIP, motilin, secretin and proglucagon-derived peptides. All hormones had some degree of colocalisation with other hormones. This included a population of EEC in which GIP, CCK and proglucagon-derived peptides are costored, and four 5-HT cell populations, 5-HT/GIP, 5-HT/ghrelin, 5-HT/PYY, and 5-HT/secretin cell groups, and a high degree of overlap between motilin and ghrelin. The presence of 5-HT in many secretin cells is consistent across species, whereas lack of 5-HT and CCK colocalisation distinguishes human from mouse. It seems likely that the different subclasses of 5-HT cells subserve different roles. At a subcellular level, we examined the vesicular localisation of secretin and 5-HT, and found these to be separately stored. We conclude that hormone-containing cells in the human jejunum do not comply with a one-cell, one-hormone classification and that colocalisations of hormones are likely to define subtypes of EEC that have different roles.
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    Analysis of enteroendocrine cell populations in the human colon
    Martins, P ; Fakhry, J ; de Oliveira, EC ; Hunne, B ; Fothergill, LJ ; Ringuet, M ; Reis, DD ; Rehfeld, JF ; Callaghan, B ; Furness, JB (SPRINGER, 2017-02)
    Recent studies have shown that patterns of colocalisation of hormones in enteroendocrine cells are more complex than previously appreciated and that the patterns differ substantially between species. In this study, the human sigmoid colon is investigated by immunohistochemistry for the presence of gastrointestinal hormones and their colocalisation. The segments of colon were distant from the pathology that led to colectomy and appeared structurally normal. Only four hormones, 5-hydroxytryptamine (5-HT), glucagon-like peptide 1 (GLP-1), peptide YY (PYY) and somatostatin, were common in enteroendocrine cells of the human colon. Cholecystokinin, present in the colon of some species, was absent, as were glucose-dependent insulinotropic peptide, ghrelin and motilin. Neurotensin cells were extremely rare. The most numerous cells were 5-HT cells, some of which also contained PYY or somatostatin and very rarely GLP-1. Almost all GLP-1 cells contained PYY. It is concluded that enteroendocrine cells of the human colon, like those of other regions and species, exhibit overlapping patterns of hormone colocalisation and that the hormones and their patterns of expression differ between human and other species.
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    Evidence that central pathways that mediate defecation utilize ghrelin receptors but do not require endogenous ghrelin
    Pustovit, RV ; Callaghan, B ; Ringuet, MT ; Kerr, NF ; Hunne, B ; Smyth, IM ; Pietra, C ; Furness, JB (WILEY, 2017-08)
    In laboratory animals and in human, centrally penetrant ghrelin receptor agonists, given systemically or orally, cause defecation. Animal studies show that the effect is due to activation of ghrelin receptors in the spinal lumbosacral defecation centers. However, it is not known whether there is a physiological role of ghrelin or the ghrelin receptor in the control of defecation. Using immunohistochemistry and immunoassay, we detected and measured ghrelin in the stomach, but were unable to detect ghrelin by either method in the lumbosacral spinal cord, or other regions of the CNS In rats in which the thoracic spinal cord was transected 5 weeks before, the effects of a ghrelin agonist on colorectal propulsion were significantly enhanced, but defecation caused by water avoidance stress (WAS) was reduced. In knockout rats that expressed no ghrelin and in wild-type rats, WAS-induced defecation was reduced by a ghrelin receptor antagonist, to similar extents. We conclude that the ghrelin receptors of the lumbosacral defecation centers have a physiological role in the control of defecation, but that their role is not dependent on ghrelin. This implies that a transmitter other than ghrelin engages the ghrelin receptor or a ghrelin receptor complex.
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    Sites of action of ghrelin receptor ligands in cardiovascular control
    Callaghan, B ; Hunne, B ; Hirayama, H ; Sartor, DM ; Nguyen, TV ; Abogadie, FC ; Ferens, D ; McIntyre, P ; Ban, K ; Baell, J ; Furness, JB ; Brock, JA (AMER PHYSIOLOGICAL SOC, 2012-10)
    Circulating ghrelin reduces blood pressure, but the mechanism for this action is unknown. This study investigated whether ghrelin has direct vasodilator effects mediated through the growth hormone secretagogue receptor 1a (GHSR1a) and whether ghrelin reduces sympathetic nerve activity. Mice expressing enhanced green fluorescent protein under control of the promoter for growth hormone secretagogue receptor (GHSR) and RT-PCR were used to locate sites of receptor expression. Effects of ghrelin and the nonpeptide GHSR1a agonist capromorelin on rat arteries and on transmission in sympathetic ganglia were measured in vitro. In addition, rat blood pressure and sympathetic nerve activity responses to ghrelin were determined in vivo. In reporter mice, expression of GHSR was revealed at sites where it has been previously demonstrated (hypothalamic neurons, renal tubules, sympathetic preganglionic neurons) but not in any artery studied, including mesenteric, cerebral, and coronary arteries. In rat, RT-PCR detected GHSR1a mRNA expression in spinal cord and kidney but not in the aorta or in mesenteric arteries. Moreover, the aorta and mesenteric arteries from rats were not dilated by ghrelin or capromorelin at concentrations >100 times their EC(50) determined in cells transfected with human or rat GHSR1a. These agonists did not affect transmission from preganglionic sympathetic neurons that express GHSR1a. Intravenous application of ghrelin lowered blood pressure and decreased splanchnic nerve activity. It is concluded that the blood pressure reduction to ghrelin occurs concomitantly with a decrease in sympathetic nerve activity and is not caused by direct actions on blood vessels or by inhibition of transmission in sympathetic ganglia.