Rod Dimaline

Expression of cannabinoid CB1 receptors by vagal afferent neurons is inhibited by cholecystokinin

Both inhibitory (satiety) and stimulatory (orexigenic) factors from the gastrointestinal tract regulate food intake. In the case of the satiety hormone cholecystokinin (CCK), these effects are mediated via vagal afferent neurons. We now report that vagal afferent neurons expressing the CCK-1 receptor also express cannabinoid CB1 receptors. Retrograde tracing established that these neurons project to the stomach and duodenum. The expression of CB1 receptors determined by RT-PCR, immunohistochemistry and in situ hybridization in rat nodose ganglia was increased by withdrawal of food for ≥12 hr. After refeeding of fasted rats there was a rapid loss of CB1 receptor expression identified by immunohistochemistry and in situ hybridization. These effects were blocked by administration of the CCK-1 receptor antagonist lorglumide and mimicked by administration of CCK to fasted rats. Because CCK is a satiety factor that acts via the vagus nerve and CB1 agonists stimulate food intake, the data suggest a new mechanism modulating the effect on food intake of satiety signals from the gastrointestinal tract.

Regulation by gastric acid of the processing of progastrin‐derived peptides in rat antral mucosa

1 Inhibition of gastric acid secretion by proton pump inhibitors like omeprazole increases the synthesis and secretion of the pyloric antral hormone gastrin. We report here how omeprazole influences the conversion of the gastrin precursor to its final products, and the abundance of mRNAs encoding proteins associated with progastrin processing in rat antral mucosa. 2 Progastrin processing was studied using a pulse–chase protocol in antral mucosa, incubated in vitro, from rats treated with omeprazole for up to 5 days. Labelled peptides were detected by on–line scintillation counting after immunoprecipitation and HPLC. The mRNAs encoding prohormone‐processing enzymes were identified by Northern blot, polymerase chain reaction or ribonuclease protection assay, and their cellular origins identified by immunocytochemistry. 3 Cleavage of [3H]‐ and [35S]‐labelled progastrins at Arg‐94–95 or Arg‐57–58, and amidation at Phe‐92 were not influenced by pretreatment with omeprazole. In contrast, cleavage of G34 (the thirty‐four amino acid amidated gastrin) at Lys‐74–75 to give G17 (the seventeen amino acid amidated gastrin), and of G34–Gly to Gl7–Gly (G34 and G17 with COOH‐terminal glycine), was increased 3‐fold after treatment with omeprazole for either 1 or 5 days. 4 Approximately 20% of newly synthesized amidated and Gly‐extended gastrins were secreted within 240 min of the labelling period in omeprazole‐treated samples, but secretion of labelled gastrins from control tissue was undetectable over a comparable period. 5 The amidating enzyme, peptidylglycine α‐amidating mono‐oxygenase (PAM), the prohormone convertases PC1/3, PC2, PC5 and the PC2 chaperone 7B2 were localized to rat antral gastrin cells by immunocytochemistry. The relative abundance of mRNA species encoding 7B2, PC5 and PAM were unchanged after treatment with omeprazole for 5 days, whereas gastrin, PC1/3 and PC2 mRNAs are known to increase at this time. 6 The main consequence of increased cleavage at Lys‐74–75 is the production of G17 and G17–Gly at the expense of G34 and G34–Gly, respectively. The latter have longer plasma half‐lives, and so their increased cleavage may serve to limit the rise in plasma gastrin concentration after inhibition of acid secretion. Changes in the abundance of mRNAs encoding prohormone‐processing enzymes cannot account for the rapidity of the changes in cleavage of progastrin at Lys residues after omeprazole.

Neurochemical activities in human temporal lobe related to aging and Alzheimer-type changes

Activities relating to 3 neurotransmitter and 4 neuropeptide systems have been examined in human temporal lobe (post mortem) for their relationships with age and Alzheimer-type changes (senile plaques and cognitive function). Significant alterations with increasing age (from 61 to 92 years) in a series of non-demented cases included a reduction of the cholinergic enzyme, choline acetyltransferase, and an increase in vasoactive intestinal peptide immunoreactivity. In cases of alzheimers disease the only neurochemical activity investigated which correlated significantly with cognitive impairment (assessed from a Mental Test Score obtained shortly before death) and with the severity of Alzheimer-type abnormalities (senile plaques density) was choline acetyltransferase. Further analyses of the data in relation to the severity of plaque formation suggest that alterations in other neurochemical activities including reductions in dopamine-beta-hydroxylase activity, cholecystokinin octapeptide (aqueous extracted) and somatostatin immunoreactivities and an increase in substance P immunoreactivity, may occur at later stages of the disease process. These comparative data suggest that biochemical changes in this brain area associated with age and earlier stages of Alzheimers disease may be relatively selective.

VIP-, substance P-, gastrin/CCK-, bombesin-, somatostatin- and glucagon-like immunoreactivities in the gut of the rainbow trout, Salmo gairdneri

SummaryThe presence of peptides in the gastrointestinal tract of the rainbow trout, Salmo gairdneri, was investigated immunocytochemically. VIP-like immunoreactivity was demonstrated in nerves in all layers of the stomach and the intestine, whereas substance P-like immunoreactivity was localized to endocrine cells, predominantly in the mucosa of the stomach, and to nerves mainly concentrated in the myenteric plexus throughout the gut. Endocrine cells reactive to gastrin/CCK antiserum were demonstrated in the intestinal mucosa, while no immunoreactivity was found in the stomach. Bombesin-immunoreactive and somatostatin-immunoreactive cells were localized in the stomach mucosa, and cells reactive to glucagon antiserum in the intestinal mucosa. Radioimmunoassay of stomach mucosa and muscle confirmed the presence of VIP-like and substance P-like immunoreactivity in these tissues, while gastrin/CCK-like immunoreactivity was low and bombesin-like immuno-reactivity was insignificant. In conclusion, molecules resembling the mammalian brain-gut peptides may be involved in the neuronal and hormonal control of gut function in fish.

Characterization of cholecystokininA and cholecystokininB receptors expressed by vagal afferent neurons.

The cholecystokinin receptors expressed by vagal afferent neurons mediate the effect of cholecystokinin in inhibiting food intake and gastric emptying. We have determined the relative abundance of cholecystokininA, gastrin-cholecystokininB and gastrin-cholecystokininC receptor populations in the rat vagus by autoradiography using [125I]Bolton Hunter-cholecystokinin-8, [125I]Bolton Hunter-heptadecapeptide gastrin and [125I]Leu(15)2-17Glycine-extended heptadecapeptide gastrin, together with the selective antagonists devazepide and L-740093. The results indicate approximately three-fold higher abundance of cholecystokininA compared with gastrin-cholecystokininB receptors, and no significant representation of gastrin-cholecystokininC receptors. Topical capsaicin applied to the vagal nerve trunk abolished the accumulation of sites binding both [125I]Bolton Hunter-labelled cholecystokinin-8 and heptadecapeptide gastrin indicating that both cholecystokininA and gastrin-cholecystokininB receptor populations were present on afferent fibres. The molecular identity of the receptors expressed by rat and human nodose ganglia was examined using the reverse transcription polymerase chain reaction. Products of the predicted size for the cholecystokininA and gastrin-cholecystokininB receptors were identified. The human and rat cholecystokininA receptor products were cloned and the sequences were found to be 99% homologous to those published for receptors expressed by rat pancreas and human gall bladder. We conclude that cholecystokininA and gastrin-colecystokininB receptors are synthesized by nodose ganglion cells, and that the receptor proteins are transported to the periphery along afferent fibres. While there is a clear role for vagal cholecystokininA receptors, the function of vagal afferent gastrin-cholecystokininB receptors remains to be determined.

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.

Gastrin: old hormone, new functions

It is exactly a century since the gastric hormone gastrin was first described as a blood-borne regulator of gastric acid secretion. The identities of the main active forms of the hormone (the “classical gastrins”) and their cellular and molecular sites of action in regulating acid secretion have all attracted sustained attention. However, recent work on peptides derived from the gastrin precursor that do not stimulate acid secretion (“non-classical gastrins”), together with studies on mice over-expressing the gene, or in which the gastrin gene has been deleted, suggest hitherto unsuspected roles in regulating cell proliferation, migration, and differentiation. Moreover, microarray and proteomic studies have identified previously unsuspected target genes of the classical gastrins. Some of the newer actions have implications for our understanding of the progression to cancer in oesophagus, stomach, pancreas and colon, all of which have recently been linked in one way or another to dysfunctional signalling involving products of the gastrin gene. The present review focuses on recent progress in understanding the biology of both classical and non-classical gastrins.

Cocaine- and Amphetamine-Regulated Transcript: Stimulation of Expression in Rat Vagal Afferent Neurons by Cholecystokinin and Suppression by Ghrelin

The neuropeptide transmitter cocaine- and amphetamine-regulated transcript (CART) inhibits food intake and is expressed by both vagal afferent and hypothalamic neurons. Here we report that cholecystokinin (CCK) regulates CART expression in rat vagal afferent neurons. Thus, CART was virtually undetectable after energy restriction for 24 h, but administration of CCK to fasted rats increased CART immunoreactivity, and refeeding of fasted animals promptly increased CART by a mechanism sensitive to a CCK-1 receptor antagonist. In vagal afferent neurons incubated in serum-free medium, CART was virtually undetectable, whereas the orexigenic peptide melanin-concentrating hormone (MCH) was readily detected. The addition of CCK rapidly induced CART expression and downregulated MCH. Using a CART promoter–luciferase reporter vector transfected into cultured vagal afferent neurons, we showed that CCK stimulation of CART transcription was mediated by activation of protein kinase C and cAMP response element-binding protein (CREB). The action of CCK on CART expression was inhibited by the orexigenic peptide ghrelin, through a mechanism that involved exclusion of phosphorylated CREB from the nucleus. Thus, CCK reciprocally regulates expression of CART and MCH within the same vagal afferent neuron; ghrelin inhibits the effect of CCK at least in part through control of the nuclear localization of phosphoCREB, revealing previously unsuspected modulation of gut–brain signals implicated in control of food intake.

Mutations of RegIα are associated with enterochromaffin-like cell tumor development in patients with hypergastrinemia

BACKGROUND & AIMS The RegIalpha gene (Reg) encodes a secretory protein proposed to regulate islet beta-cell and gastric mucous cell growth. Reg is expressed in rat gastric enterochromaffin-like (ECL) cells. The aim of this study was to examine Reg expression in human corpus and to determine the identity of Reg in ECL cell carcinoid tumors in hypergastrinemic patients. METHODS Reg messenger RNA (mRNA) abundance was quantified by Northern blot in extracts of gastric corpus from patients with and without ECL cell tumors and in AR4-2J cells stimulated by gastrin; cellular origins were determined by immunocytochemistry. Mutations of Reg were determined by reverse-transcription polymerase chain reaction, cloning, and sequencing, and the mutated protein was expressed in HIT-T15 cells. RESULTS Reg mRNA abundance was increased approximately threefold in the corpus of hypergastrinemic patients compared with controls, and was enriched in 3 of 7 ECL cell carcinoid tumors but not in non-endocrine cell gastric polyps. In AR4-2J cells, gastrin stimulated Reg mRNA abundance; this was eliminated by the gastrin/cholecystokinin B antagonist L-740,093 (10(-9) mol/L). Immunocytochemistry indicated that Reg was located in both chief cells and ECL cells in human corpus. Mutations of Reg were identified in 3 of 5 patients with ECL cell carcinoid tumors; in 2 cases, mutation of the initiator methionine residue led to exclusion of the protein from the secretory pathway. CONCLUSIONS Gastrin regulates Reg mRNA abundance in human corpus. Mutations of Reg that prevent secretion are associated with ECL cell carcinoids, suggesting a function as an autocrine or paracrine tumor suppressor.

Histidine decarboxylase gene expression in rat fundus is regulated by gastrin

The conversion of histidine to histamine by histidine decarboxylase (HDC) is of central importance in the control of vertebrate acid secretion. We have used PCR‐generated probes to study the regulation of HDC gene expression in rat fundic mucosa. When circulating gastrin levels were lowered by fasting or elevated by treatment with omeprazole, there were parallel changes in HDC mRNA abundance. However, when animals with elevated gastrin levels were concurrently treated with the gastrin/CCK‐B receptor antagonist PD 134308, HDC mRNA levels were not increased. These data are consistent with the hypothesis that HDC gene expression is regulated by gastrin, over the physiological range of circulating hormone concentrations.