Anatomy and Neuroscience - Research Publications
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ItemIdentification of enteroendocrine cells that express TRPA1 channels in the mouse intestine(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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ItemNew Roles of Serotonin and Tachykinins in Intestinal Mucositis?(SPRINGER, 2013-12)
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ItemEffects of Food Components That Activate TRPA1 Receptors on Mucosal Ion Transport in the Mouse Intestine(MDPI AG, 2016-10)TRPA1 is a ligand-activated cation channel found in the intestine and other tissues. Components of food that stimulate TRPA1 receptors (phytonutrients) include allyl isothiocyanate, cinnamaldehyde and linalool, but these may also act at other receptors. Cells lining the intestinal mucosa are immunoreactive for TRPA1 and Trpa1 mRNA occurs in mucosal extracts, suggesting that the TRPA1 receptor is the target for these agonists. However, in situ hybridisation reveals Trpa1 expression in 5-HT containing enteroendocrine cells, not enterocytes. TRPA1 agonists evoke mucosal secretion, which may be indirect (through release of 5-HT) or direct by activation of enterocytes. We investigated effects of the phytonutrients on transmucosal ion currents in mouse duodenum and colon, and the specificity of the phytonutrients in cells transfected with Trpa1, and in Trpa1-deficient mice. The phytonutrients increased currents in the duodenum with the relative potencies: allyl isothiocyanate (AITC) > cinnamaldehyde > linalool (0.1 to 300 μM). The rank order was similar in the colon, but linalool was ineffective. Responses to AITC were reduced by the TRPA1 antagonist HC-030031 (100 μM), and were greatly diminished in Trpa1-/- duodenum and colon. Responses were not reduced by tetrodotoxin, 5-HT receptor antagonists, or atropine, but inhibition of prostaglandin synthesis reduced responses. Thus, functional TRPA1 channels are expressed by enterocytes of the duodenum and colon. Activation of enterocyte TRPA1 by food components has the potential to facilitate nutrient absorption.
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ItemThe gut as a sensory organ(NATURE PUBLISHING GROUP, 2013-12)The gastrointestinal tract presents the largest and most vulnerable surface to the outside world. Simultaneously, it must be accessible and permeable to nutrients and must defend against pathogens and potentially injurious chemicals. Integrated responses to these challenges require the gut to sense its environment, which it does through a range of detection systems for specific chemical entities, pathogenic organisms and their products (including toxins), as well as physicochemical properties of its contents. Sensory information is then communicated to four major effector systems: the enteroendocrine hormonal signalling system; the innervation of the gut, both intrinsic and extrinsic; the gut immune system; and the local tissue defence system. Extensive endocrine-neuro-immune-organ-defence interactions are demonstrable, but under-investigated. A major challenge is to develop a comprehensive understanding of the integrated responses of the gut to the sensory information it receives. A major therapeutic opportunity exists to develop agents that target the receptors facing the gut lumen.
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ItemNovel and Conventional Receptors for Ghrelin, Desacyl-Ghrelin, and Pharmacologically Related Compounds(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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ItemGhrelin and des-acyl ghrelin inhibit aromatase expression and activity in human adipose stromal cells: suppression of cAMP as a possible mechanism(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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ItemAngiotensin II increases nerve-evoked contractions in mouse tail artery by a T-type Ca2+ channel-dependent mechanism(ELSEVIER SCIENCE BV, 2015-08-15)Angiotensin II (Ang II) increases sympathetic nerve-evoked contractions of arterial vessels. Here the mechanisms underlying this effect were investigated in mouse tail artery. Isometrically mounted segments of mouse distal tail artery were used to investigate the effects of endothelium denudation, blocking Ca(2+) channels and inhibiting superoxide signalling on Ang II-induced facilitation of nerve-evoked contractions. In addition, in situ amperometry was used to assess effects of Ang II on noradrenaline release. Ang II (0.1-1nM) increased nerve-evoked contractions but did not change noradrenaline release. Losartan (Ang II type 1 receptor antagonist), but not PD 123319 (Ang II type 2 receptor antagonist), blocked the facilitatory effect of Ang II on nerve-evoked contractions. Ang II increased vascular muscle reactivity to phenylephrine and UK-14304 (α1- and α2-adrenoceptor agonists, respectively). Endothelial denudation increased nerve-evoked contractions and reduced the facilitatory effect of Ang II on these responses. Efonidipine (L- and T-type Ca(2+) channel blocker) and NNC 55-0396 (T-type Ca(2+) channel blocker) also attenuated this effect of Ang II, while nifedipine (L-type Ca(2+) channel blocker) did not. Blockers of superoxide generation/signalling did not change the facilitatory effect of Ang II on nerve-evoked contractions. The findings indicate that Ang II increases the contribution of T-type Ca(2+) channels to neural activation of the vascular muscle. In addition, Ang II appears to reduce the inhibitory influence of the endothelium on nerve-evoked contractions.
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ItemModified Cytoplasmic Ca2+ Sequestration Contributes to Spinal Cord Injury-Induced Augmentation of Nerve-Evoked Contractions in the Rat Tail Artery(PUBLIC LIBRARY SCIENCE, 2014-10-28)In rat tail artery (RTA), spinal cord injury (SCI) increases nerve-evoked contractions and the contribution of L-type Ca2+ channels to these responses. In RTAs from unoperated rats, these channels play a minor role in contractions and Bay K8644 (L-type channel agonist) mimics the effects of SCI. Here we investigated the mechanisms underlying the facilitatory actions of SCI and Bay K8644 on nerve-evoked contractions of RTAs and the hypothesis that Ca2+ entering via L-type Ca2+ channels is rapidly sequestered by the sarcoplasmic reticulum (SR) limiting its role in contraction. In situ electrochemical detection of noradrenaline was used to assess if Bay K8644 increased noradrenaline release. Perforated patch recordings were used to assess if SCI changed the Ca2+ current recorded in RTA myocytes. Wire myography was used to assess if SCI modified the effects of Bay K8644 and of interrupting SR Ca2+ uptake on nerve-evoked contractions. Bay K8644 did not change noradrenaline-induced oxidation currents. Neither the size nor gating of Ca2+ currents differed between myocytes from sham-operated (control) and SCI rats. Bay K8644 increased nerve-evoked contractions in RTAs from both control and SCI rats, but the magnitude of this effect was reduced by SCI. By contrast, depleting SR Ca2+ stores with ryanodine or cyclopiazonic acid selectively increased nerve-evoked contractions in control RTAs. Cyclopiazonic acid also selectively increased the blockade of these responses by nifedipine (L-type channel blocker) in control RTAs, whereas ryanodine increased the blockade produced by nifedipine in both groups of RTAs. These findings suggest that Ca2+ entering via L-type channels is normally rapidly sequestered limiting its access to the contractile mechanism. Furthermore, the findings suggest SCI reduces the role of this mechanism.
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ItemAnalysis of the ghrelin receptor-independent vascular actions of ulimorelin(ELSEVIER SCIENCE BV, 2015-04-05)Ulimorelin (TZP101) is a ghrelin receptor agonist that stimulates intestinal motility, but also reduces blood pressure in rodents and humans and dilates blood vessels. It has been proposed as a treatment for intestinal motility disorders. Here we investigated the mechanisms through which ulimorelin affects vascular diameter. Actions of ulimorelin on wall tension of rodent arteries were investigated and compared with other ghrelin receptor agonists. Saphenous, mesenteric and basilar arteries were obtained from Sprague-Dawley rats (male, 8 weeks) and saphenous arteries were obtained from wild type or ghrelin receptor null mice. These were mounted in myography chambers to record artery wall tension. Ulimorelin (0.03-30µM) inhibited phenylephrine-induced contractions of rat saphenous (IC50=0.6µM; Imax=66±5%; n=3-6) and mesenteric arteries (IC50=5µM, Imax=113±16%; n=3-4), but not those contracted by U46619, ET-1 or 60mM [K(+)]. Relaxation of phenylephrine-constricted arteries was not observed with ghrelin receptor agonists TZP102, capromorelin or AZP-531. In rat saphenous and basilar arteries, ulimorelin (10-100µM) and TZP102 (10-100µM) constricted arteries (EC50=9.9µM; Emax=50±7% and EC50=8µM; Emax=99±16% respectively), an effect not attenuated by the ghrelin receptor antagonist YIL 781 3µM or mimicked by capromorelin or AZP-531. In mesenteric arteries, ulimorelin, 1-10µM, caused a surmountable rightward shift in the response to phenylephrine (0.01-1000µM; pA2=5.7; n=3-4). Ulimorelin had similar actions in mouse saphenous artery from both wild type and ghrelin receptor null mice. We conclude that ulimorelin causes vasorelaxation through competitive antagonist action at α1-adrenoceptors and a constrictor action not mediated via the ghrelin receptor.
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ItemHypotensive effects of ghrelin receptor agonists mediated through a novel receptor(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.