Anatomy and Neuroscience - Research Publications

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    Identification of enteroendocrine cells that express TRPA1 channels in the mouse intestine
    Cho, H-J ; Callaghan, B ; Bron, R ; Bravo, DM ; Furness, JB (SPRINGER, 2014-04)
    TRPA1 is an ion channel that detects specific chemicals in food and also transduces mechanical, cold and chemical stimulation. Its presence in sensory nerve endings is well known and recent evidence indicates that it is expressed by some gastrointestinal enteroendocrine cells (EEC). The purpose of the present work is to identify and quantify EEC that express TRPA1 in the mouse gastrointestinal tract. Combined in situ hybridisation histochemistry for TRPA1 and immunofluorescence for EEC hormones was used. TRPA1 expressing EEC were common in the duodenum and jejunum, were rare in the distal small intestine and were absent from the stomach and large intestine. In the duodenum and jejunum, TRPA1 occurred in EEC that contained both cholecystokinin (CCK) and 5-hydroxytryptamine (5HT) and in a small number of cells expressing 5HT but not CCK. TRPA1 was absent from CCK cells that did not express 5HT and from EEC containing glucagon-like insulinotropic peptide. Thus TRPA1 is contained in very specific EEC populations. It is suggested that foods such as garlic and cinnamon that contain TRPA1 stimulants may aid digestion by facilitating the release of CCK.
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    Glucagon-like peptide 1 and peptide YY are in separate storage organelles in enteroendocrine cells
    Cho, H-J ; Robinson, ES ; Rivera, LR ; McMillan, PJ ; Testro, A ; Nikfarjam, M ; Bravo, DM ; Furness, JB (SPRINGER, 2014-07)
    A sub-group of enteroendocrine cells (L cells) release gastrointestinal hormones, GLP-1 and PYY, which have different but overlapping physiological effects, in response to intraluminal nutrients. Whilst their release profiles are not identical, how the plasma levels of these two hormones are differentially regulated is not well understood. We investigate the possibility that GLP-1 and PYY are in separate storage vesicles. In this study, the subcellular location of GLP-1 and PYY storage organelles is investigated using double-labelling immunohistochemistry, super resolution microscopy and high-resolution confocal microscopy. In all species tested, human, pig, rat and mouse, most cytoplasmic stores that exhibited GLP-1 or PYY immunofluorescence were distinct from each other. The volume occupancy, determined by 3D analysis, overlapped by only about 10∼20 %. At the lower resolution achieved by conventional confocal microscopy, there was also evidence of GLP-1 and PYY being in separate storage compartments but, in subcellular regions where there were many storage vesicles, separate storage could not be resolved. The results indicate that different storage vesicles in L cells contain predominantly GLP-1 or predominantly PYY. Whether GLP-1 and PYY storage vesicles are selectively mobilised and their products are selectively released needs to be determined.
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    Both exogenous 5-HT and endogenous 5-HT, released by flu oxetine, enhance distension evoked propulsion in guinea-pig ileum in vitro
    Gwynne, RM ; Clarke, AJ ; Furness, JB ; Bornstein, JC (FRONTIERS MEDIA SA, 2014-09-19)
    The roles of 5-HT3 and 5-HT4 receptors in the modulation of intestinal propulsion by luminal application of 5-HT and augmentation of endogenous 5-HT effects were studied in segments of guinea-pig ileum in vitro. Persistent propulsive contractions evoked by saline distension were examined using a modified Trendelenburg method. When 5-HT (30 nM), fluoxetine (selective serotonin reuptake inhibitor; 1 nM), 2-methyl-5-HT (5-HT3 receptor agonist; 1 mM), or RS 67506 (5-HT4 receptor agonist, 1 μM) was infused into the lumen, the pressure needed to initiate persistent propulsive activity fell significantly. A specific 5-HT4 receptor antagonist, SB 207266 (10 nM in lumen), abolished the effects of 5-HT, fluoxetine, and RS 67506, but not those of 2-methyl-5-HT. Granisetron (5-HT3 receptor antagonist; 1 μM in lumen) abolished the effect of 5-HT, fluoxetine, RS 67506, and 2-methyl-5-HT. The NK3 receptor antagonist SR 142801 (100 nM in lumen) blocked the effects of 5-HT, fluoxetine, and 2-methyl-5-HT. SB 207266, granisetron, and SR 142801 had no effect by themselves. Higher concentrations of fluoxetine (100 and 300 nM) and RS 67506 (3 and 10 μM) had no effect on the distension threshold for propulsive contractions. These results indicate that luminal application of exogenous 5-HT, or increased release of endogenous mucosal 5-HT above basal levels, acts to lower the threshold for propulsive contractions in the guinea-pig ileum via activation of 5-HT3 and 5-HT4 receptors and the release of tachykinins. The results further indicate that basal release of 5-HT is insufficient to alter the threshold for propulsive motor activity.
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    Selenium-Enriched Agaricus bisporus Mushroom Protects against Increase in Gut Permeability ex vivo and Up-Regulates Glutathione Peroxidase 1 and 2 in Hyperthermally-Induced Oxidative Stress in Rats
    Maseko, T ; Dunshea, FR ; Howell, K ; Cho, H-J ; Rivera, LR ; Furness, JB ; Ng, K (MDPI AG, 2014-06)
    Dietary effects of organic Se supplementation in the form of Se-enriched Agaricus bisporus mushroom on ileal mucosal permeability and antioxidant selenoenzymes status in heat induced oxidative stress in rats were evaluated. Acute heat stress (40 °C, 21% relative humidity, 90 min exposure) increased ileum baseline short circuit current (Isc; 2.40-fold) and epithelial conductance (Ge; 2.74-fold). Dietary supplementation with Se-enriched A. bisporus (1 µg Se/g feed) reduced (p < 0.05) ileum Isc and Ge during heat stress to 1.74 and 1.91 fold, respectively, indicating protection from heat stress-induced mucosal permeability increase. The expression of ileum glutathione peroxidase (GPx-) 1 and 2 mRNAs were up-regulated (p < 0.05) by 1.90 and 1.87-fold, respectively, for non-heat stress rats on the Se-enriched diet relative to the control. The interplay between heat stress and dietary Se is complex. For rats on the control diet, heat stress alone increased ileum expression of GPx-1 (2.33-fold) and GPx-2 (2.23-fold) relative to thermoneutral conditions. For rats on the Se-enriched diet, heat stress increased (p < 0.05) GPx-1 expression only. Rats on Se-enriched + α-tocopherol diet exhibited increased expression of both genes (p < 0.05). Thus, dietary Se-enriched A. bisporus protected against increase in ileum permeability and up-regulated GPx-1 and GPx-2 expression, selenoenzymes relevant to mitigating oxidative stress.
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    Novel and Conventional Receptors for Ghrelin, Desacyl-Ghrelin, and Pharmacologically Related Compounds
    Callaghan, B ; Furness, JB ; Christopoulos, A (AMER SOC PHARMACOLOGY EXPERIMENTAL THERAPEUTICS, 2014-10)
    The only molecularly identified ghrelin receptor is the growth hormone secretagogue receptor GHSR1a. Its natural ligand, ghrelin, is an acylated peptide whose unacylated counterpart (UAG) is almost inactive at GHSR1a. A truncated, nonfunctional receptor, GHSR1b, derives from the same gene. We have critically evaluated evidence for effects of ghrelin receptor ligands that are not consistent with actions at GHSR1a. Effects of ghrelin are observed in cells or tissues where the expression of GHSR1a is not detectable or after the Ghsr gene has been inactivated. In several, effects of ghrelin are mimicked by UAG, and ghrelin binding is competitively reduced by UAG. Effects in the absence of GHSR1a and sites at which ghrelin and UAG have similar potency suggest the presence of novel nonspecific ghrelin receptors (ghrelin receptor-like receptors [GRLRs]). A third class of receptor, the UAG receptors, at which UAG, but not ghrelin, is an agonist has been proposed. None of the novel receptors, with the exception of the glycoprotein CD36, which accounts for ghrelin action at a limited number of sites, have been identified. GHSR1a and GHSR1b combine with other G protein-coupled receptors to form heterodimers, whose pharmacologies differ from their components. Thus, it is feasible some GRLRs and some UAG receptors are heterodimers. Effects mediated through GRLRs or UAG receptors include adipocyte lipid accumulation, myoblast differentiation, osteoblast proliferation, insulin release, cardioprotection, coronary artery constriction, vascular endothelial cell proliferation, and tumor cell proliferation. The molecular identification and pharmacologic characterization of novel ghrelin receptors are thus important objectives.
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    Ghrelin and des-acyl ghrelin inhibit aromatase expression and activity in human adipose stromal cells: suppression of cAMP as a possible mechanism
    Docanto, MM ; Yang, F ; Callaghan, B ; Au, CC ; Ragavan, R ; Wang, X ; Furness, JB ; Andrews, ZB ; Brown, KA (SPRINGER, 2014-08)
    Aromatase converts androgens into estrogens and its expression within adipose stromal cells (ASCs) is believed to be the major driver of estrogen-dependent cancers in older women. Ghrelin is a gut-hormone that is involved in the regulation of appetite and known to bind to and activate the cognate ghrelin receptor, GHSR1a. The unacylated form of ghrelin, des-acyl ghrelin, binds weakly to GHSR1a but has been shown to play an important role in regulating a number of physiological processes. The aim of this study was to determine the effect of ghrelin and des-acyl ghrelin on aromatase in primary human ASCs. Primary human ASCs were isolated from adipose tissue of women undergoing cosmetic surgery. Real-time PCR and tritiated water-release assays were performed to examine the effect of treatment on aromatase transcript expression and aromatase activity, respectively. Treatments included ghrelin, des-acyl ghrelin, obestatin, and capromorelin (GHSR1a agonist). GHSR1a protein expression was assessed by Western blot and effects of treatment on Ca(2+) and cAMP second messenger systems were examined using the Flexstation assay and the Lance Ultra cAMP kit, respectively. Results demonstrate that pM concentrations of ghrelin and des-acyl ghrelin inhibit aromatase transcript expression and activity in ASCs under basal conditions and in PGE2-stimulated cells. Moreover, the effects of ghrelin and des-acyl ghrelin are mediated via effects on aromatase promoter PII-specific transcripts. Neither the GHSR1a-specific agonist capromorelin nor obestatin had any effect on aromatase transcript expression or activity. Moreover, GHSR1a protein was undetectable by Western blot and neither ghrelin nor capromorelin elicited a calcium response in ASCs. Finally, ghrelin caused a significant decrease in basal and forskolin-stimulated cAMP in ASC. These findings suggest that ghrelin acts at alternate receptors in ASCs by decreasing intracellular cAMP levels. Ghrelin mimetics may be useful in the treatment of estrogen-dependent breast cancer.
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    Hypotensive effects of ghrelin receptor agonists mediated through a novel receptor
    Callaghan, B ; Kosari, S ; Pustovit, RV ; Sartor, DM ; Ferens, D ; Ban, K ; Baell, J ; Nguyen, TV ; Rivera, LR ; Brock, JA ; Furness, JB (WILEY, 2014-03)
    BACKGROUND AND PURPOSE: Some agonists of ghrelin receptors cause rapid decreases in BP. The mechanisms by which they cause hypotension and the pharmacology of the receptors are unknown. EXPERIMENTAL APPROACH: The effects of ligands of ghrelin receptors were investigated in rats in vivo, on isolated blood vessels and on cells transfected with the only molecularly defined ghrelin receptor, growth hormone secretagogue receptor 1a (GHSR1a). KEY RESULTS: Three agonists of GHSR1a receptors, ulimorelin, capromorelin and CP464709, caused a rapid decrease in BP in the anaesthetized rat. The effect was not reduced by either of two GHSR1a antagonists, JMV2959 or YIL781, at doses that blocked effects on colorectal motility, in vivo. The rapid hypotension was not mimicked by ghrelin, unacylated ghrelin or the unacylated ghrelin receptor agonist, AZP531. The early hypotension preceded a decrease in sympathetic nerve activity. Early hypotension was not reduced by hexamethonium or by baroreceptor (sino-aortic) denervation. Ulimorelin also relaxed isolated segments of rat mesenteric artery, and, less potently, relaxed aorta segments. The vascular relaxation was not reduced by JMV2959 or YIL781. Ulimorelin, capromorelin and CP464709 activated GHSR1a in transfected HEK293 cells at nanomolar concentrations. JMV2959 and YIL781 both antagonized effects in these cells, with their pA2 values at the GHSR1a receptor being 6.55 and 7.84. CONCLUSIONS AND IMPLICATIONS: Our results indicate a novel vascular receptor or receptors whose activation by ulimorelin, capromorelin and CP464709 lowered BP. This receptor is activated by low MW GHSR1a agonists, but is not activated by ghrelin.