Laura Anselmi

Enteroendocrine cells: a site of ‘taste’ in gastrointestinal chemosensing

Purpose of reviewThis review discusses the role of enteroendocrine cells of the gastrointestinal tract as chemoreceptors that sense lumen contents and induce changes in gastrointestinal function and food intake through the release of signaling substances acting on a variety of targets locally or at a distance. Recent findingsRecent evidence supports the concept that chemosensing in the gut involves G protein-coupled receptors and effectors that are known to mediate gustatory signals in the oral cavity. These include sweet-taste and bitter-taste receptors, and their associated G proteins, which are expressed in the gastrointestinal mucosa, including selected populations of enteroendocrine cells. In addition, taste receptor agonists elicit a secretory response in enteroendocrine cells in vitro and in animals in vivo, and induce neuronal activation. SummaryTaste-signaling molecules expressed in the gastrointestinal mucosa might participate in the functional detection of nutrients and harmful substances in the lumen and prepare the gut to absorb them or initiate a protective response. They might also participate in the control of food intake through the activation of gut–brain neural pathways. These findings provide a new dimension to unraveling the regulatory circuits initiated by luminal contents of the gastrointestinal tract.

Galanin receptors in the rat gastrointestinal tract

Galanin functions are mediated by three distinct G-protein-coupled receptors, galanin receptor 1 (GalR1), GalR2 and GalR3, which activate different intracellular signaling pathways. Here, we quantified mRNA levels of GalR1, GalR2 and GalR3 in the gastrointestinal tract using real time RT-PCR. GalR1 and GalR2 mRNAs were detected in all segments with the highest levels in the large intestine and stomach, respectively. GalR3 mRNA levels were quite low and mostly confined to the colon. We also investigated the effect of galanin 1-16, which has high affinity for GalR1 and GalR2 and low affinity for GalR3 on depolarization-evoked Ca2+ increases in rat cultured myenteric neurons using Ca2+-imaging. Intracellular Ca2+ changes in myenteric neurons were monitored using the Ca2+ sensitive dye, fluo-4, and confocal microscopy. Galanin 1-16 (1 microM) markedly inhibited the K+-evoked Ca2+ increases in myenteric neurons. In summary, the differential distribution of GalRs supports the hypothesis that the complex effects of galanin in the gastrointestinal tract result from the activation of multiple receptor subtypes. Furthermore, this study confirms the presence of functional GalRs and suggests that galanin modulates transmitter release from myenteric neurons through inhibition of voltage-dependent calcium channels involving a G(i/o)-coupled GalR.

Morphine Induces μ Opioid Receptor Endocytosis in Guinea Pig Enteric Neurons Following Prolonged Receptor Activation

BACKGROUND & AIMS The μ opioid receptor (μOR) undergoes rapid endocytosis after acute stimulation with opioids and most opiates, but not with morphine. We investigated whether prolonged activation of μOR affects morphines ability to induce receptor endocytosis in enteric neurons. METHODS We compared the effects of morphine, a poor μOR-internalizing opiate, and (D-Ala2,MePhe4,Gly-ol5) enkephalin (DAMGO), a potent μOR-internalizing agonist, on μOR trafficking in enteric neurons and on the expression of dynamin and β-arrestin immunoreactivity in the ileum of guinea pigs rendered tolerant by chronic administration of morphine. RESULTS Morphine (100 μmol/L) strongly induced endocytosis of μOR in tolerant but not naive neurons (55.7% ± 9.3% vs 24.2% ± 7.3%; P < .001) whereas DAMGO (10 μmol/L) strongly induced internalization of μOR in neurons from tolerant and naive animals (63.6% ± 8.4% and 66.5% ± 3.6%). Morphine- or DAMGO-induced μOR endocytosis resulted from direct interactions between the ligand and the μOR because endocytosis was not affected by tetrodotoxin, a blocker of endogenous neurotransmitter release. Ligand-induced μOR internalization was inhibited by pretreatment with the dynamin inhibitor, dynasore. Chronic morphine administration resulted in a significant increase and translocation of dynamin immunoreactivity from the intracellular pool to the plasma membrane, but did not affect β-arrestin immunoreactivity. CONCLUSIONS Chronic activation of μORs increases the ability of morphine to induce μOR endocytosis in enteric neurons, which depends on the level and cellular localization of dynamin, a regulatory protein that has an important role in receptor-mediated signal transduction in cells.

Diet-Induced Regulation of Bitter Taste Receptor Subtypes in the Mouse Gastrointestinal Tract

Bitter taste receptors and signaling molecules, which detect bitter taste in the mouth, are expressed in the gut mucosa. In this study, we tested whether two distinct bitter taste receptors, the bitter taste receptor 138 (T2R138), selectively activated by isothiocyanates, and the broadly tuned bitter taste receptor 108 (T2R108) are regulated by luminal content. Quantitative RT-PCR analysis showed that T2R138 transcript is more abundant in the colon than the small intestine and lowest in the stomach, whereas T2R108 mRNA is more abundant in the stomach compared to the intestine. Both transcripts in the stomach were markedly reduced by fasting and restored to normal levels after 4 hours re-feeding. A cholesterol-lowering diet, mimicking a diet naturally low in cholesterol and rich in bitter substances, increased T2R138 transcript, but not T2R108, in duodenum and jejunum, and not in ileum and colon. Long-term ingestion of high-fat diet increased T2R138 RNA, but not T2R108, in the colon. Similarly, α-gustducin, a bitter taste receptor signaling molecule, was reduced by fasting in the stomach and increased by lowering cholesterol in the small intestine and by high-fat diet in the colon. These data show that both short and long term changes in the luminal contents alter expression of bitter taste receptors and associated signaling molecules in the mucosa, supporting the proposed role of bitter taste receptors in luminal chemosensing in the gastrointestinal tract. Bitter taste receptors might serve as regulatory and defensive mechanism to control gut function and food intake and protect the body from the luminal environment.

Expression of galanin receptor messenger RNAs in different regions of the rat gastrointestinal tract.

Galanin effects are mediated by three G-protein-coupled receptors: galanin receptor 1 (GalR1), GalR2 and GalR3. We quantified mRNA levels of GalR1, GalR2 and GalR3 in the rat stomach, small and large intestine using real-time RT-PCR. All three GalR mRNAs were detected throughout the gut at different levels. GalR1 and GalR2 mRNA levels were higher in the large than in the small intestine. GalR2 mRNA was most abundant in the stomach. GalR3 mRNA levels were generally quite low. The differential regional distribution of GalRs suggests that the complex effects of galanin in the gut are the result of activating multiple receptor subtypes, whose density, subtype and signaling vary along the gastrointestinal tract.

Protective role of μ opioid receptor activation in intestinal inflammation induced by mesenteric ischemia/reperfusion in mice.

Intestinal ischemia is a clinical emergency with high morbidity and mortality. We investigated whether activation of μ opioid receptor (μOR) protects from the inflammation induced by intestinal ischemia and reperfusion (I/R) in mice. Ischemia was induced by occlusion of the superior mesenteric artery (45 min), followed by reperfusion (5 hr). Sham‐operated (SO) and normal (N) mice served as controls. Each group received subcutaneously 1) saline solution, 2) the μOR selective agonist [D‐Ala2, N‐Me‐Phe4, Gly5‐ol]‐enkephalin (DAMGO; 0.01 mg kg−1), 3) DAMGO and the selective μOR antagonist [H‐D‐Phe‐Cys‐Tyr‐D‐Trp‐Arg‐Thr‐Pen‐Thr‐NH2] (CTAP; 0.1 mg kg−1), or 4) CTAP alone. I/R induced intestinal inflammation as indicated by histological damage and the significant increase in myeloperoxidase (MPO) activity, an index of tissue neutrophil accumulation. Tumor necrosis factor‐α (TNF‐α) and interleukin‐10 (IL‐10) mRNA levels were also increased in I/R mice compared with SO. DAMGO significantly reduced tissue damage, MPO activity, and TNF‐α mRNA levels in I/R, and these effects were reversed by CTAP. By contrast, DAMGO did not modify IL‐10 mRNA levels or gastrointestinal transit. DAMGOs effects are receptor mediated and likely are due to activation of peripheral μORs, because it does not readily cross the blood–brain barrier. These findings suggest that activation of peripheral μOR protects from the inflammatory response induced by I/R through a pathway involving the proinflammatory cytokine TNF‐α. Reduction of acute inflammation might prevent I/R complications, including motility impairment, which develop at a later stage of reperfusion and likely are due to inflammatory cell infiltrates.

Role of galanin receptor 1 in peristaltic activity in the guinea pig ileum

Galanin effects are mediated by distinct receptors, galanin receptor 1 (GAL-R1), GAL-R2 and GAL-R3. Here, we analyzed 1) the role of GAL-R1 in cholinergic transmission and peristalsis in the guinea-pig ileum using longitudinal muscle-myenteric plexus preparations and intact segments of the ileum in organ bath, and 2) the distribution of GAL-R1 immunoreactivity in the myenteric plexus with immunohistochemistry and confocal microscopy. Galanin inhibited electrically stimulated contractions of longitudinal muscle-myenteric plexus preparations with a biphasic curve. Desensitization with 1 microM galanin suppressed the high potency phase of the curve, whereas the GAL-R1 antagonist, RWJ-57408 (1 microM), inhibited the low potency phase. Galanin (3 microM) reduced the longitudinal muscle contraction and the peak pressure, and decreased the compliance of the circular muscle. All these effects were antagonized by RWJ-57408 (1 or 10 microM). RWJ-57408 (10 microM) per se did not affect peristalsis parameters in normal conditions, nor when peristalsis efficiency was reduced by partial nicotinic transmission blockade with hexamethonium. In the myenteric plexus, GAL-R1 immunoreactivity was localized to neurons and to fibers projecting within the plexus and to the muscle. GAL-R1 was expressed mostly by cholinergic neurons and by some neurons containing vasoactive intestinal polypeptide or nitric oxide synthase. This study indicates that galanin inhibits cholinergic transmission to the longitudinal muscle via two separate receptors; GAL-R1 mediates the low potency phase. The reduced peristalsis efficiency could be explained by inhibition of the cholinergic drive, whereas the decreased compliance is probably due to inhibition of descending neurons and/or to the activation of an excitatory muscular receptor. Endogenous galanin does not appear to affect neuronal pathways subserving peristalsis in physiologic conditions via GAL-R1.

Activation of μ opioid receptors modulates inflammation in acute experimental colitis

μ opioid receptors (μORs) are expressed by neurons and inflammatory cells, and mediate immune response. We tested whether activation of peripheral μORs ameliorates the acute and delayed phase of colitis.

Opioid-Induced Mitogen-Activated Protein Kinase Signaling in Rat Enteric Neurons following Chronic Morphine Treatment

Opioids, acting at μ opioid receptors, are commonly used for pain management. Chronic opioid treatment induces cellular adaptations, which trigger long-term side effects, including constipation mediated by enteric neurons. We tested the hypothesis that chronic opioid treatment induces alterations of μ opioid receptor signaling in enteric neurons, which are likely to serve as mechanisms underlying opioid-induced constipation. In cultured rat enteric neurons, either untreated (naïve) or exposed to morphine for 4 days (chronic), we compared the effect of morphine and DAMGO (D-Ala2,MePhe4,Gly-ol5 enkephalin) on μ opioid receptor internalization and downstream signaling by examining the activation of the mitogen-activated protein kinase/extracellular signal-regulated kinases 1 and 2 (MAPK/ERK) pathway, cAMP accumulation and transcription factor cAMP Response Element-Binding protein (CREB) expression. μ opioid receptor internalization and MAPK/ERK phosphorylation were induced by DAMGO, but not morphine in naïve neurons, and by both opioids in chronic neurons. MAPK/ERK activation was prevented by the receptor antagonist naloxone, by blocking receptor trafficking with hypertonic sucrose, dynamin inhibitor, or neuronal transfection with mutated dynamin, and by MAPK inhibitor. Morphine and DAMGO inhibited cAMP in naïve and chronic enteric neurons, and induced desensitization of cAMP signaling. Chronic morphine treatment suppressed desensitization of cAMP and MAPK signaling, increased CREB phosphorylation through a MAPK/ERK pathway and induced delays of gastrointestinal transit, which was prevented by MAPK/ERK blockade. This study showed that opioids induce endocytosis- and dynamin-dependent MAPK/ERK activation in enteric neurons and that chronic morphine treatment triggers changes at the receptor level and downstream signaling resulting in MAPK/ERK-dependent CREB activation. Blockade of this signaling pathway prevents the development of gastrointestinal motility impairment induced by chronic morphine treatment. These findings suggest that alterations in μ opioid receptor downstream signaling including MAPK/ERK pathway in enteric neurons chronically treated with morphine contribute to the development of opioid-induced constipation.

Galanin Inhibition Of Voltage Dependent Ca2+ Influx In Rat Cultured Myenteric Neurons Is Mediated By Galanin Receptor 1

Galanin activates three receptors, the galanin receptor 1 (GalR1), GalR2, and GalR3. In the gastrointestinal tract, GalR1 mediates the galanin inhibition of cholinergic transmission to the longitudinal muscle and reduction of peristalsis efficiency in the small intestine. Galanin has also been shown to inhibit depolarization‐evoked Ca2+ increases in cultured myenteric neurons. Because GalR1 immunoreactivity is localized to cholinergic myenteric neurons, we hypothesized that this inhibitory action of galanin on myenteric neurons is mediated by GalR1. We investigated the effect of galanin 1‐16, which has high affinity for GalR1 and GalR2, in the presence or absence of the selective GalR1 antagonist, RWJ‐57408, and of galanin 2‐11, which has high affinity for GalR2 and GalR3, on Ca2+ influx through voltage‐dependent Ca2+ channels in cultured myenteric neurons. Myenteric neurons were loaded with fluo‐4 and depolarized by high K+ concentration to activate voltage‐dependent Ca2+ channels. Intracellular Ca2+ levels were quantified with confocal microscopy. Galanin 1‐16 (0.01–1 μM) inhibited the depolarization‐evoked Ca2+ increase in a dose‐dependent manner with an EC50 of 0.172 μM. The selective GalR1 antagonist, RWJ‐57408 (10 μM), blocked the galanin 1‐16 (1 μM)‐mediated inhibition of voltage‐dependent Ca2+ channel. By contrast, the GalR2/GalR3 agonist, galanin 2‐11 did not affect the K+‐evoked Ca2+ influx in myenteric neurons. GalR1 immunoreactivity was localized solely to myenteric neurons in culture, as previously observed in intact tissue. These findings indicate that the inhibition of depolarization‐evoked Ca2+ influx in myenteric neurons in culture is mediated by GalR1 and confirm the presence of functional GalR1 in the myenteric plexus. This is consonant with the hypothesis that GalR1 mediates galanin inhibition of transmitter release from myenteric neurons.