Toll-like receptor 9 expressed in proximal intestinal enteroendocrine cells detects bacteria resulting in secretion of cholecystokinin.

Toll-like receptors (TLRs) play a key role in the recognition of microbes via detection of specific and conserved microbial molecular features. TLRs, mainly expressed in immune cells, interact with intestinal microbiome. Little is known about mechanism(s) of sensing of bacteria by the intestinal surface enteroendocrine cells (EECs). We show here that TLR9 is expressed by the EECs of proximal intestine in a range of species and is co-expressed with the satiety hormone cholecystokinin (CCK). CCK secreted in excess induces emesis (vomiting). Using an EEC model cell line, STC-1, we demonstrate that in response to the TLR9 agonist, DNA containing unmethylated CpG dinucleotide motifs, STC-1 cells secrete CCK and that this secretion is inhibited by specific inhibitors of TLR9. Exposure of STC-1 cells to heat-inactivated pathogenic bacteria, Escherichia coli O55/H7, Shigella flexneri 2457T, Salmonella typhimurium ST4/74, and non-pathogenic Lactobacillus amylovorus GRL1112, results to an increase in CCK secretion compared to untreated control. The magnitudes of CCK release are higher in response to pathogenic bacteria and lowest in response to the non-pathogenic L. amylovorus. The pathogenic strains not only have substantially bigger genomes than L. amylovorus, they also have significantly higher numbers/frequency of RR/CG/YY stimulatory CpG hexamers in their genomic DNA. Pathogen-induced excessive secretion of the gut hormone CCK, provoking emesis can serve as a protective mechanism against development of enteric infections.

High fat diet and body weight have different effects on cannabinoid CB(1) receptor expression in rat nodose ganglia.

Energy balance is regulated, in part, by the orexigenic signaling pathways of the vagus nerve. Fasting-induced modifications in the expression of orexigenic signaling systems have been observed in vagal afferents of lean animals. Altered basal cannabinoid (CB1) receptor expression in the nodose ganglia in obesity has been reported. Whether altered body weight or a high fat diet modifies independent or additive changes in CB1 expression is unknown. We investigated the expression of CB1 and orexin 1 receptor (OX-1R) in the nodose ganglia of rats fed ad libitum or food deprived (24h), maintained on low or high fat diets (HFD), with differing body weights. Male Wistar rats were fed chow or HFD (diet-induced obese: DIO or diet-resistant: DR) or were body weight matched to the DR group but fed chow (wmDR). CB1 and OX-1R immunoreactivity were investigated and CB1 mRNA density was determined using in situ hybridization. CB1 immunoreactivity was measured in fasted rats after sulfated cholecystokinin octapeptide (CCK8s) administration. In chow rats, fasting did not modify the level of CB1 mRNA. More CB1 immunoreactive cells were measured in fed DIO, DR and wmDR rats than chow rats; levels increased after fasting in chow and wmDR rats but not in DIO or DR rats. In HFD fasted rats CCK8s did not reduce CB1 immunoreactivity. OX-1R immunoreactivity was modified by fasting only in DR rats. These data suggest that body weight contributes to the proportion of neurons expressing CB1 immunoreactivity in the nodose ganglion, while HFD blunts fasting-induced increases, and CCK-induced suppression of, CB1-immunoreactivity.

The mechanism of agonist induced Ca2+ signalling in intact endothelial cells studied confocally in in situ arteries.

In endothelial cells there remain uncertainties in the details of how Ca(2+) signals are generated and maintained, especially in intact preparations. In particular the role of the sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA), in contributing to the components of agonist-induced signals is unclear. The aim of this work was to increase understanding of the detailed mechanism of Ca(2+) signalling in endothelial cells using real time confocal imaging of Fluo-4 loaded intact rat tail arteries in response to muscarinic stimulation. In particular we have focused on the role of SERCA, and its interplay with capacitative Ca(2+) entry (CCE) and ER Ca(2+) release and uptake. We have determined its contribution to the Ca(2+) signal and how it varies with different physiological stimuli, including single and repeated carbachol applications and brief and prolonged exposures. In agreement with previous work, carbachol stimulated a rise in intracellular Ca(2+) in the endothelial cells, consisting of a rapid initial phase, then a plateau upon which oscillations of Ca(2+) were superimposed, followed by a decline to basal Ca(2+) levels upon carbachol removal. Our data support the following conclusions: (i) the size (amplitude and duration) of the Ca(2+) spike and early oscillations are limited by SERCA activity, thus both are increased if SERCA is inhibited. (ii) SERCA activity is such that brief applications of carbachol do not trigger CCE, presumably because the fall in luminal Ca(2+) is not sufficient to trigger it. However, longer applications sufficient to deplete the ER or even partial SERCA inhibition stimulate CCE. (iii) Ca(2+) entry occurs via STIM-mediated CCE and SERCA contributes to the cessation of CCE. In conclusion our data show how SERCA function is crucial to shaping endothelial cell Ca signals and its dynamic interplay with both CCE and ER Ca releases.

Plasticity in vagal afferent neurones during feeding and fasting: mechanisms and significance.

The ingestion of food activates mechanisms leading to inhibition of food intake and gastric emptying mediated by the release of regulatory peptides, for example cholecystokinin (CCK), and lipid amides, e.g. oleylethanolamide from the gut. In addition, there are both peptides (e.g. ghrelin) and lipid amides (e.g. anandamide) that appear to signal the absence of food in the gut and that are associated with the stimulation of food intake. Vagal afferent neurones are a common target for both types of signal. Remarkably, the neurochemical phenotype of these neurones itself depends on nutritional status. CCK acting at CCK1 receptors on vagal afferent neurones stimulates expression in these neurones of Y2-receptors and the neuropeptide CART, both of which are associated with the inhibition of food intake. Conversely, in fasted rats when plasma CCK is low, these neurones express cannabinoid (CB)-1 and melanin concentrating hormone (MCH)-1 receptors, and MCH, and this is inhibited by exogenous CCK or endogenous CCK released by refeeding. The stimulation of CART expression by CCK is mediated by the activation of CREB and EGR1; ghrelin inhibits the action of CCK by promoting nuclear exclusion of CREB and leptin potentiates the action of CCK by the stimulation of EGR1 expression. Vagal afferent neurones therefore constitute a level of integration outside the CNS for nutrient-derived signals that control energy intake and that are capable of encoding recent nutrient ingestion.

Feeding-dependent depression of melanin-concentrating hormone and melanin-concentrating hormone receptor-1 expression in vagal afferent neurones.

Food intake is regulated by signals from the gastrointestinal tract. Both stimulation and inhibition of food intake may be mediated by upper gastrointestinal tract hormones, e.g. ghrelin and cholecystokinin that act at least partly via vagal afferent neurones. We now report that vagal afferent neurones in both rat and man express melanin-concentrating hormone and its receptor, melanin-concentrating hormone-1R. In nodose ganglia from rats fasted for 24 h, RT-PCR revealed the expression of both melanin-concentrating hormone and melanin-concentrating hormone-1R, whereas in ganglia from animals fed ad libitum expression was virtually undetectable. Immunohistochemical studies also revealed expression of melanin-concentrating hormone and melanin-concentrating hormone-1R in nodose ganglion neurones of fasted rats, but signals were weak in rats fed ad libitum. Melanin-concentrating hormone and melanin-concentrating hormone-1R were expressed in the same neurones, a high proportion of which also expressed the cholecystokinin-1 receptor. When fasted rats were refed, there was down-regulation of melanin-concentrating hormone and melanin-concentrating hormone-1R expression over a period of 5 h. Similar effects were produced by administration of cholecystokinin to fasted rats. The cholecystokinin-1 receptor antagonist lorglumide inhibited food-induced down-regulation of melanin-concentrating hormone and melanin-concentrating hormone-1R. We conclude that the satiety hormone cholecystokinin acts on vagal afferent neurones to inhibit expression of melanin-concentrating hormone and its receptor. Since the melanin-concentrating hormone system is associated with stimulation of food intake this effect of cholecystokinin may contribute to its action as a satiety hormone.

Expression of the leptin receptor in rat and human nodose ganglion neurones.

There is evidence for interactions between leptin and cholecystokinin in controlling food intake. Since cholecystokinin acts on vagal afferent neurones, we asked whether the leptin receptor was also expressed by these neurones. Primers for different forms of the leptin receptor were used in reverse transcriptase-polymerase chain reaction (RT-PCR) of rat and human nodose ganglia. RT-PCR yielded products corresponding to the long (functional) form as well as short forms of the rat leptin receptor. Moreover, RT-PCR revealed the long form of the leptin receptor in a human nodose ganglion. The identities of RT-PCR products were confirmed by sequencing. Primers corresponding to leptin itself did not give RT-PCR products in nodose ganglia. Immunocytochemical studies revealed leptin-receptor immunoreactivity in neuronal cell bodies. Many neurones co-expressed the leptin and cholecystokinin type A receptors, or leptin receptor and cocaine- and amphetamine-related transcript. We conclude that vagal afferent neurones that express the cholecystokinin type A receptor and cocaine- and amphetamine-related transcript, may also express the long form of the leptin receptor providing a neurochemical basis for observations of interactions between cholecystokinin and leptin.