Quantitation and chemical coding of enteroendocrine cell populations in the human jejunum.

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.

Relationships of endocrine cells to each other and to other cell types in the human gastric fundus and corpus.

Gastric endocrine cell hormones contribute to the control of the stomach and to signalling to the brain. In other gut regions, enteroendocrine cells (EECs) exhibit extensive patterns of colocalisation of hormones. In the current study, we characterise EECs in the human gastric fundus and corpus. We utilise immunohistochemistry to investigate EECs with antibodies to ghrelin, serotonin (5-HT), somatostatin, peptide YY (PYY), glucagon-like peptide 1, calbindin, gastrin and pancreastatin, the latter as a marker of enterochromaffin-like (ECL) cells. EECs were mainly located in regions of the gastric glands populated by parietal cells. Gastrin cells were absent and PYY cells were very rare. Except for about 25% of 5-HT cells being a subpopulation of ECL cells marked by pancreastatin, colocalisation of hormones in gastric EECs was infrequent. Ghrelin cells were distributed throughout the fundus and corpus; most were basally located in the glands, often very close to parietal cells and were closed cells i.e., not in contact with the lumen. A small proportion had long processes located close to the base of the mucosal epithelium. The 5-HT cells were of at least three types: small, round, closed cells; cells with multiple, often very long, processes; and a subgroup of ECL cells. Processes were in contact with their surrounding cells, including parietal cells. Mast cells had very weak or no 5-HT immunoreactivity. Somatostatin cells were a closed type with long processes. In conclusion, four major chemically defined EEC types occurred in the human oxyntic mucosa. Within each group were cells with distinct morphologies and relationships to other mucosal cells.

Neural pathways for colorectal control, relevance to spinal cord injury and treatment: a narrative review.

STUDY DESIGN: Narrative review. OBJECTIVES: The purpose is to review the organisation of the nerve pathways that control defecation and to relate this knowledge to the deficits in colorectal function after SCI. METHODS: A literature review was conducted to identify salient features of defecation control pathways and the functional consequences of damage to these pathways in SCI. RESULTS: The control pathways for defecation have separate pontine centres under cortical control that influence defecation. The pontine centres connect, separately, with autonomic preganglionic neurons of the spinal defecation centres and somatic motor neurons of Onuf's nucleus in the sacral spinal cord. Organised propulsive motor patterns can be generated by stimulation of the spinal defecation centres. Activation of the somatic neurons contracts the external sphincter. The analysis aids in interpreting the consequences of SCI and predicts therapeutic strategies. CONCLUSIONS: Analysis of the bowel control circuits identifies sites at which bowel function may be modulated after SCI. Colokinetic drugs that elicit propulsive contractions of the colorectum may provide valuable augmentation of non-pharmacological bowel management procedures.

Analysis of enteroendocrine cell populations in the human colon.

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.

Distribution and characterisation of CCK containing enteroendocrine cells of the mouse small and large intestine.

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.

Ghrelin and Motilin Control Systems in GI Physiology and Therapeutics.

Ghrelin and motilin are released from gastrointestinal endocrine cells during hunger, to act through G protein-coupled receptors that have closely related amino acid sequences. The actions of ghrelin are more complex than motilin because ghrelin also exists outside the GI tract, it is processed to des-acyl ghrelin which has activity, ghrelin can exist in truncated forms and retain activity, the ghrelin receptor can have constitutive activity and is subject to biased agonism and finally additional ghrelin-like and des-acyl ghrelin receptors are proposed. Both ghrelin and motilin can stimulate gastric emptying, acting via different pathways, perhaps influenced by biased agonism at the receptors, but research is revealing additional pathways of activity. For example, it is becoming apparent that reduction of nausea may be a key therapeutic target for ghrelin receptor agonists and perhaps for compounds that modulate the constitutive activity of the ghrelin receptor. Reduction of nausea may be the mechanism through which gastroparesis symptoms are reduced. Intriguingly, a potential ability of motilin to influence nausea is also becoming apparent. Ghrelin interacts with digestive function through its effects on appetite, and ghrelin antagonists may have a place in treating Prader-Willi syndrome. Unlike motilin, ghrelin receptor agonists also have the potential to treat constipation by acting at the lumbosacral defecation centres. In conclusion, agonists of both ghrelin and motilin receptors hold potential as treatments for specific subsets of digestive system disorders.

Novel and conventional receptors for ghrelin, desacyl-ghrelin, and pharmacologically related compounds.

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.

The chemical coding of 5-hydroxytryptamine containing enteroendocrine cells in the mouse gastrointestinal tract.

The majority of 5-HT (serotonin) in the body is contained in enteroendocrine cells of the gastrointestinal mucosa. From the time of their discovery over 80 years ago, the 5-HT-containing cells have been regarded as a class of cell that is distinct from enteroendocrine cells that contain peptide hormones. However, recent studies have cast doubt on the concept of there being distinct classes of enteroendocrine cells, each containing a single hormone or occasionally more than one hormone. Instead, data are rapidly accumulating that there are complex patterns of colocalisation of hormones that identify multiple subclasses of enteroendocrine cells. In the present work, multiple labelling immunohistochemistry is used to investigate patterns of colocalisation of 5-HT with enteric peptide hormones. Over 95 % of 5-HT cells in the duodenum also contained cholecystokinin and about 40 % of them also contained secretin. In the jejunum, about 75 % of 5-HT cells contained cholecystokinin but not secretin and 25 % contained 5-HT plus both cholecystokinin and secretin. Small proportions of 5-HT cells contained gastrin or somatostatin in the stomach, PYY or GLP-1 in the small intestine and GLP-1 or somatostatin in the large intestine. Rare or very rare 5-HT cells contained ghrelin (stomach), neurotensin (small and large intestines), somatostatin (small intestine) and PYY (in the large intestine). It is concluded that 5-HT-containing enteroendocrine cells are heterogeneous in their chemical coding and by implication in their functions.

Differences in hormone localisation patterns of K and L type enteroendocrine cells in the mouse and pig small intestine and colon.

This study has investigated the patterns of colocalisation of the conventional K cell marker, glucagon-like insulinotropic peptide (GIP), and the L cell markers, glucagon like peptide-1 (GLP-1) and peptide YY (PYY), in enteroendocrine cells (EEC) of the small intestine and colon of mouse and pig. All combinations of the hormones, 3 in a cell, 2 in a cell and 1 at a time, were encountered. In both species, the three most common EEC types contained (1) both GLP-1 and PYY but not GIP, (2) GLP-1 alone or (3) GIP plus GLP-1 without PYY. Few GIP plus PYY cells and rare cells containing all 3 hormones were encountered. Gradients of cell types occurred along the intestine. For example, in mouse, there were no PYY cells in the duodenum and few in the jejunum, but >50% of labelled EEC in the distal ileum and colon were PYY immunoreactive. By contrast, over 40% of EEC in the pig duodenum contained PYY, and most also contained either GLP-1 or GIP. The gradient in pig was less pronounced. It is concluded that the traditional classification of K and L cells requires revision, and that there are major inter-species differences in the patterns of colocalisation of hormones that have been used to characterise K and L cells.

Ghrelin and des-acyl ghrelin inhibit aromatase expression and activity in human adipose stromal cells: suppression of cAMP as a possible mechanism.

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.

Identification of enteroendocrine cells that express TRPA1 channels in the mouse intestine.

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.

The enteric nervous system and gastrointestinal innervation: integrated local and central control.

The digestive system is innervated through its connections with the central nervous system (CNS) and by the enteric nervous system (ENS) within the wall of the gastrointestinal tract. The ENS works in concert with CNS reflex and command centers and with neural pathways that pass through sympathetic ganglia to control digestive function. There is bidirectional information flow between the ENS and CNS and between the ENS and sympathetic prevertebral ganglia.The ENS in human contains 200-600 million neurons, distributed in many thousands of small ganglia, the great majority of which are found in two plexuses, the myenteric and submucosal plexuses. The myenteric plexus forms a continuous network that extends from the upper esophagus to the internal anal sphincter. Submucosal ganglia and connecting fiber bundles form plexuses in the small and large intestines, but not in the stomach and esophagus. The connections between the ENS and CNS are carried by the vagus and pelvic nerves and sympathetic pathways. Neurons also project from the ENS to prevertebral ganglia, the gallbladder, pancreas and trachea.The relative roles of the ENS and CNS differ considerably along the digestive tract. Movements of the striated muscle esophagus are determined by neural pattern generators in the CNS. Likewise the CNS has a major role in monitoring the state of the stomach and, in turn, controlling its contractile activity and acid secretion, through vago-vagal reflexes. In contrast, the ENS in the small intestine and colon contains full reflex circuits, including sensory neurons, interneurons and several classes of motor neuron, through which muscle activity, transmucosal fluid fluxes, local blood flow and other functions are controlled. The CNS has control of defecation, via the defecation centers in the lumbosacral spinal cord. The importance of the ENS is emphasized by the life-threatening effects of some ENS neuropathies. By contrast, removal of vagal or sympathetic connections with the gastrointestinal tract has minor effects on GI function. Voluntary control of defecation is exerted through pelvic connections, but cutting these connections is not life-threatening and other functions are little affected.

Stratification of enterochromaffin cells by single-cell expression analysis.

Dynamic interactions between gut mucosal cells and the external environment are essential to maintain gut homeostasis. Enterochromaffin (EC) cells transduce both chemical and mechanical signals and produce 5-hydroxytryptamine (5-HT) to mediate disparate physiological responses. However, the molecular and cellular basis for functional diversity of ECs remains to be adequately defined. Here, we integrated single-cell transcriptomics with spatial image analysis to identify fourteen EC clusters that are topographically organized along the gut. Subtypes predicted to be sensitive to the chemical environment and mechanical forces were identified that express distinct transcription factors and hormones. A Piezo2+ population in the distal colon was endowed with a distinctive neuronal signature. Using a combination of genetic, chemogenetic and pharmacological approaches, we demonstrated Piezo2+ ECs are required for normal colon motility. Our study constructs a molecular map for ECs and offers a framework for deconvoluting EC cells with pleiotropic functions.

Structure and activity of alpha-conotoxin PeIA at nicotinic acetylcholine receptor subtypes and GABA(B) receptor-coupled N-type calcium channels.

α-Conotoxins are peptides from cone snails that target the nicotinic acetylcholine receptor (nAChR). RgIA and Vc1.1 have analgesic activity in animal pain models. Both peptides target the α9α10 nAChR and inhibit N-type calcium channels via GABA(B) receptor activation, but the mechanism of action of analgesic activity is unknown. PeIA has previously been shown to inhibit the α9α10 and α3ß2 nAChRs. In this study, we have determined the structure of PeIA and shown that it is also a potent inhibitor of N-type calcium channels via GABA(B) receptor activation. The characteristic α-conotoxin fold is present in PeIA, but it has a different distribution of surface-exposed hydrophobic and charged residues compared with Vc1.1. Thus, the surface residue distribution, rather than the overall fold, appears to be responsible for the 50-fold increase in selectivity at the α3ß2 nAChR by PeIA relative to Vc1.1. In contrast to their difference in potency at the nAChR, the equipotent activity of PeIA and Vc1.1 at the GABA(B) receptor suggests that the GABA(B) receptor is more tolerant to changes in surface residues than is the nAChR. The conserved Asp-Pro-Arg motif of Vc1.1 and RgIA, which is crucial for potency at the α9α10 nAChR, is not required for activity at GABA(B) receptor/N-type calcium channels because PeIA has a His-Pro-Ala motif in the equivalent position. This study shows that different structure-activity relationships are associated with the targeting of the GABA(B) receptor versus nAChRs. Furthermore, there is probably a much more diverse range of conotoxins that target the GABA(B) receptor than currently realized.

Embryonic toxin expression in the cone snail Conus victoriae: primed to kill or divergent function?

Predatory marine cone snails (genus Conus) utilize complex venoms mainly composed of small peptide toxins that target voltage- and ligand-gated ion channels in their prey. Although the venoms of a number of cone snail species have been intensively profiled and functionally characterized, nothing is known about the initiation of venom expression at an early developmental stage. Here, we report on the expression of venom mRNA in embryos of Conus victoriae and the identification of novel α- and O-conotoxin sequences. Embryonic toxin mRNA expression is initiated well before differentiation of the venom gland, the organ of venom biosynthesis. Structural and functional studies revealed that the embryonic α-conotoxins exhibit the same basic three-dimensional structure as the most abundant adult toxin but significantly differ in their neurological targets. Based on these findings, we postulate that the venom repertoire of cone snails undergoes ontogenetic changes most likely reflecting differences in the biotic interactions of these animals with their prey, predators, or competitors. To our knowledge, this is the first study to show toxin mRNA transcripts in embryos, a finding that extends our understanding of the early onset of venom expression in animals and may suggest alternative functions of peptide toxins during development.

Sites of action of ghrelin receptor ligands in cardiovascular control.

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.

Identification of neurons that express ghrelin receptors in autonomic pathways originating from the spinal cord.

Functional studies have shown that subsets of autonomic preganglionic neurons respond to ghrelin and ghrelin mimetics and in situ hybridisation has revealed receptor gene expression in the cell bodies of some preganglionic neurons. Our present goal has been to determine which preganglionic neurons express ghrelin receptors by using mice expressing enhanced green fluorescent protein (EGFP) under the control of the promoter for the ghrelin receptor (also called growth hormone secretagogue receptor). The retrograde tracer Fast Blue was injected into target organs of reporter mice under anaesthesia to identify specific functional subsets of postganglionic sympathetic neurons. Cryo-sections were immunohistochemically stained by using anti-EGFP and antibodies to neuronal markers. EGFP was detected in nerve terminal varicosities in all sympathetic chain, prevertebral and pelvic ganglia and in the adrenal medulla. Non-varicose fibres associated with the ganglia were also immunoreactive. No postganglionic cell bodies contained EGFP. In sympathetic chain ganglia, most neurons were surrounded by EGFP-positive terminals. In the stellate ganglion, neurons with choline acetyltransferase immunoreactivity, some being sudomotor neurons, lacked surrounding ghrelin-receptor-expressing terminals, although these terminals were found around other neurons. In the superior cervical ganglion, the ghrelin receptor terminals innervated subgroups of neurons including neuropeptide Y (NPY)-immunoreactive neurons that projected to the anterior chamber of the eye. However, large NPY-negative neurons projecting to the acini of the submaxillary gland were not innervated by EGFP-positive varicosities. In the celiaco-superior mesenteric ganglion, almost all neurons were surrounded by positive terminals but the VIP-immunoreactive terminals of intestinofugal neurons were EGFP-negative. The pelvic ganglia contained groups of neurons without ghrelin receptor terminal innervation and other groups with positive terminals around them. Ghrelin receptors are therefore expressed by subgroups of preganglionic neurons, including those of vasoconstrictor pathways and of pathways controlling gut function, but are absent from some other neurons, including those innervating sweat glands and the secretomotor neurons that supply the submaxillary salivary glands.

Three-dimensional growth of breast cancer cells potentiates the anti-tumor effects of unacylated ghrelin and AZP-531.

Breast cancer is the most common type of cancer in women and notwithstanding important therapeutic advances, remains the second leading cause of cancer-related death. Despite extensive research relating to the hormone ghrelin, responsible for the stimulation of growth hormone release and appetite, little is known of the effects of its unacylated form, especially in cancer. The present study aimed to characterize effects of unacylated ghrelin on breast cancer cells, define its mechanism of action, and explore the therapeutic potential of unacylated ghrelin or analog AZP-531. We report potent anti-tumor effects of unacylated ghrelin, dependent on cells being cultured in 3D in a biologically-relevant extracellular matrix. The mechanism of unacylated ghrelin-mediated growth inhibition involves activation of Gαi and suppression of MAPK signaling. AZP-531 also suppresses the growth of breast cancer cells in vitro and in xenografts, and may be a novel approach for the safe and effective treatment of breast cancer.

Analgesic alpha-conotoxins Vc1.1 and Rg1A inhibit N-type calcium channels in rat sensory neurons via GABAB receptor activation.

alpha-Conotoxins Vc1.1 and Rg1A are peptides from the venom of marine Conus snails that are currently in development as a treatment for neuropathic pain. Here we report that the alpha9alpha10 nicotinic acetylcholine receptor-selective conotoxins Vc1.1 and Rg1A potently and selectively inhibit high-voltage-activated (HVA) calcium channel currents in dissociated DRG neurons in a concentration-dependent manner. The post-translationally modified peptides vc1a and [P6O]Vc1.1 were inactive, as were all other alpha-conotoxins tested. Vc1.1 inhibited the omega-conotoxin-sensitive HVA currents in DRG neurons but not those recorded from Xenopus oocytes expressing Ca(V)2.2, Ca(V)2.1, Ca(V)2.3, or Ca(V)1.2 channels. Inhibition of HVA currents by Vc1.1 was not reversed by depolarizing prepulses but was abolished by pertussis toxin (PTX), intracellular GDPbetaS, or a selective inhibitor of pp60c-src tyrosine kinase. These data indicate that Vc1.1 does not interact with N-type calcium channels directly but inhibits them via a voltage-independent mechanism involving a PTX-sensitive, G-protein-coupled receptor. Preincubation with a variety of selective receptor antagonists demonstrated that only the GABA(B) receptor antagonists, [S-(R*,R*)][-3-[[1-(3,4-dichlorophenyl)ethyl]amino]-2-hydroxy propyl]([3,4]-cyclohexylmethyl) phosphinic acid hydrochloride (2S)-3[[(1S)-1-(3,4-dichlorophenyl)-ethyl]amino-2-hydroxypropyl](phenylmethyl) phosphinic acid and phaclofen, blocked the effect of Vc1.1 and Rg1A on Ca2+ channel currents. Together, the results identify Ca(V)2.2 as a target of Vc1.1 and Rg1A, potentially mediating their analgesic actions. We propose a novel mechanism by which alpha-conotoxins Vc1.1 and Rg1A modulate native N-type (Ca(V)2.2) Ca2+ channel currents, namely acting as agonists via G-protein-coupled GABA(B) receptors.