Conformational analysis in solution of gastrin releasing peptide

Gastrin releasing peptide (GRP) is the first peptide isolated from porcine gastric and intestinal tissues and is homologous to the carboxyl terminus of bombesin (Bn) isolated from the skin of the frog Bombina bombina. It is a member of the Bn-like peptides, which are important in numerous biological and pathological processes. The Bn-like peptides show high sequence homology in their C-terminal regions, but they have different selectivity for their receptors. In particular, GRP selectively binds to the GRP receptor (GRPR). However, the molecular basis for this selectivity remains largely unknown. Here, we report the three-dimensional structure of GRP. Hopefully, it could be helpful in a better understanding of the binding selectivity between GRP and GRPR.     

Capsaicin-sensitive vagal afferents and CCK in inhibition of gastric motor function induced by intestinal nutrients

The role of vagal afferent pathways and cholecystokinin (CCK) in mediating changes in gastric motor function after a meal was investigated in urethane-anesthetized rats. Proximal gastric motor function was measured manometrically, and nutrients were infused into an isolated segment of duodenum. Inhibition of gastric motility in response to duodenal infusion of protein (peptone or casein), but not carbohydrate (glucose), was significantly attenuated by administration of the CCK antagonist, L364,718. Selective ablation of vagal afferents by perineural treatment with the sensory neurotoxin, capsaicin, significantly reduced responses to both duodenal protein and glucose. These results suggest that protein in the duodenum decreases proximal gastric motor function via release of CCK and a vagal capsaicin-sensitive afferent pathway. In contrast, glucose acts via a capsaicin-sensitive vagal pathway not involving CCK. Thus separate neural and hormonal mechanisms mediate the effects of different nutrients in the duodenal feedback regulation of gastric motor function.     

Localisation of somatostatin- and gastrin-like immunoreactivity in the gastrointestinal tract of Ciona intestinalis L.

Summary Somatostatin- and gastrin-like immunoreactivity has been found by immunofluorescence in cells of the stomach and intestinal epithelia of Ciona intestinalis L. The cells containing the peptide immunoreactive to mammalian anti-gastrin can be restained with the Grimelius' technique for argyrophilia.     

Cultured gastrointestinal smooth muscle cells: cell response to contractile agonists depends on their phenotypic state

In the digestive tract, the transit of ingested food induces a local contraction-relaxation reflex of which the smooth muscle cell (SMC) represents the functional unit. Although freshly isolated SMCs have been extensively used for in vitro studies, in specific cases cultured cells appear necessary. Because conventionally cultured SMCs lose their contractile properties, we have developed: (1) differentiated, contractile rabbit gastric SMCs (D-stim cells), cultured in a medium supplemented with insulin, and (2) proliferative, dedifferentiated rabbit gastric SMCs (P-stim cells), cultured in a medium supplemented with insulin, fetal serum, EGF and b-FGF. The proliferative index was 5±4% and 82±10%, respectively, for D-stim and P-stim cells. Expression of SM-myosin heavy chain was observed in 90% of D-stim cells, whereas it was progressively lost in P-stim cells. Carbachol (1–100 nM), glicentin (2 nM) and gastrin-17 (100 nM) induced contraction of D-stim cells cultured for 3 or 6 days, whereas they did not induce the contraction of P-stim cells; in contrast, gastrin-17 (10 nM) was able to stimulate DNA synthesis (1.86±0.09-fold increase) in P-stim cells. The coupling of muscarinic receptors to intracellular transduction pathways was evaluated in D-stim cells: at day 3, carbachol (100 nM) induced a twofold increase in the production of inositol tri-tetra-phosphates; in parallel, a phosphorylation of ERK MAP kinases occurred within 1 min of carbachol stimulation. In conclusion, cultured functional myocytes derived from mature tissue may be used for long-term studies concerning the events coupled either to proliferation or to motility regulation of differentiated SMCs due to the activation of G-protein-coupled receptors.This study was supported in part by grants from the AFM (Association Française contre les Myopathies).     

The role of calcium in agonist-stimulated hydrolysis of phosphatidylinositol in mouse pancreas

The effects of Ca²⁺ on agonist-stimulated hydrolysis of myo-[2-³H]inostol-labelled phosphatidylinositol in mouse pancreas in vitro, were studied. The increase in cytosol Ca²⁺ concentration produced by the ionophore A23187 did not stimulate the breakdown of phosphatidylinositol. Cholecystokinin-octapeptide (CCK-8) stimulated the hydrolysis of phosphatidylinositol under conditions in which intracellular calcium stores were depleted. The breakdown of phosphatidylinositol was stimulated by bethanechol and CCK-8 in Ca²⁺-free Krebs solution, and the addition of Ca²⁺ to the medium potentiated the effects of these agonists. Lanthanum significantly reduced bethanechol and CCK-8 stimulated hydrolysis of phosphatidylinositol in Krebs solution, but was without effect in Ca²⁺-free Krebs solution. The results of this study support the proposal that PI hydrolysis does not occur as a result of Ca²⁺ mobilization and may be involved in Ca²⁺ gating in the pancreas.     

Ileal interposition attenuates the satiety responses evoked by cholecystokinin-8 and -33

One of the possible mechanisms by which the weight-reducing surgical procedure ileal interposition (II) works is by increasing circulating levels of lower gut peptides that reduce food intake, such as glucagon like peptide-1 and peptide YY. However, since this surgery involves both lower and upper gut segments, we tested the hypothesis that II alters the satiety responses evoked by the classic upper gut peptide cholecystokinin (CCK). To test this hypothesis, we determined meal size (MS), intermeal interval (IMI) and satiety ratio (SR) evoked by CCK-8 and -33 (0, 1, 3, 5 nmol/kg, i.p.) in two groups of rats, II and sham-operated. CCK-8 and -33 reduced MS more in the sham group than in the II group; CCK-33 prolonged IMI in the sham group and increased SR in both groups. Reduction of cumulative food intake by CCK-8 in II rats was blocked by devazepide, a CCK₁ receptor antagonist. In addition, as previously reported, we found that II resulted in a slight reduction in body weight compared to sham-operated rats. Based on these observations, we conclude that ileal interposition attenuates the satiety responses of CCK. Therefore, it is unlikely that this peptide plays a significant role in reduction of body weight by this surgery.     

Recombinant prohormone convertase 1 and 2 cleave purified pro cholecystokinin (CCK) and a synthetic peptide containing CCK 8 Gly Arg Arg and the carboxyl-terminal flanking peptide

Purified recombinant prohormone convertase 1 and 2 (PC1 and PC2) cleave a peptide containing cholecystokinin (CCK) 8 Gly Arg Arg and the carboxyl-terminal peptide liberating CCK 8 Gly Arg Arg. PC1 and PC2 also cleave purified pro CCK, liberating the amino terminal pro-peptide while no carboxyl-terminal cleavage was detected. Under the conditions of the in vitro cleavage assay, it appears that the carboxyl-terminal cleavage site of pro CCK is not accessible to the enzymes while this site is readily cleaved in a synthetic peptide. Additional cellular proteins that unfold the prohormone may be required to expose the carboxyl-terminal site for cleavage.     

Effect of H2O2 on CCK-8-evoked changes in mitochondrial activity in isolated mouse pancreatic acinar cells

BACKGROUND INFORMATION: This paper studies the effect of H(2)O(2) on mitochondrial responses evoked by CCK-8 (cholecystokinin 8) in mouse pancreatic acinar cells. Cytosolic ([Ca(2+)](c)) and mitochondrial ([Ca(2+)](m)) free-calcium concentrations, mitochondrial inner membrane potential (psi(m)) and FAD autofluorescence were monitored using confocal laser scanning microscopy. RESULTS: CCK-8 induced an increase in [Ca(2+)](m) that slowly declined towards the prestimulation level. Depolarization of psi(m) that partially recovered, as well as increases in FAD autofluorescence, could also be observed in response to the hormone. Pretreatment of cells with 1 mM H(2)O(2) alone resulted in marked changes in mitochondrial parameters and, moreover, H(2)O(2) inhibited the CCK-8-evoked changes in [Ca(2+)](m), psi(m) and FAD autofluorescence. The results of the present study have demonstrated that CCK-8 can evoke marked changes in pancreatic acinar cell mitochondrial activity and that CCK-8-evoked responses are blocked by H(2)O(2). Additionally, H(2)O(2) releases Ca(2+) from intracellular stores and inhibits pancreatic acinar cell responses to CCK-8. CONCLUSION: The effects observed reflect an impairment of mitochondrial activity in the presence of H(2)O(2) that could represent some of its mechanisms of action to induce cellular damage leading to cell dysfunction and generation of pathologies.     

Effects of cholecystokinin octapeptide (CCK-8) on food intake in adult and aged rats under different feeding conditions

The effects of CCK on food intake were investigated under fixed feeding conditions in comparison to a test meal taken after 16 h of food deprivation. The experiments were performed on young adult rats (8 weeks old) as well on aged rats (23 months old). Intraperitoneal CCK-8 (8 and 40 μg/kg) significantly reduced the size of a test meal following 16-h food deprivation. This effect was independent of the age of the rats. However, under fixed feeding conditions neither of the doses used in this study reduced food intake in the young adult rats, whereas the highest dose of 40 μg/kg did so in the aged rats. These results suggest that the hypophagic effect of exogenous CCK-8 depends on experimental conditions, food intake being reduced after a period of food deprivation but not under a fixed feeding regimen in adult animals. Furthermore, the data suggest that age is a factor contributing to the complex behavioral actions of CCK, because only old animals were more susceptible to an anorectic action of CCK under the fixed feeding schedule. An explanation may lie in an interaction of other known behavioral effects of CCK (e.g., anxiogenic, mnemonic action) with its effects under the different feeding schedules.     

EVI-1 modulates arsenic trioxide induced apoptosis through JNK signalling pathway in leukemia cells

High expression of the oncogene ecotropic viral integration site-1 (EVI-1) is an independent negative prognostic indicator of survival in leukemia patients. This study aimed to examine the effects of arsenic trioxide (ATO) on EVI-1 in acute myeloid leukemia (AML). Mononuclear cells were isolated from the bone marrow and peripheral blood of AML patients and healthy donors. EVI-1 expression in hematopoietic cells was evaluated by RT-qPCR and Western blot analysis. EVI-1 was highly expressed in both primary AML and leukemia cell lines (THP-1 and K562). ATO down-regulated EVI-1 mRNA in zebrafish in vivo as well as in primary leukemia cells and THP-1 and K562 cells in vitro. Additionally, ATO treatment induced apoptosis, down-regulated both EVI-1 mRNA and oncoprotein expression, increased the expression of pro-apoptosis proteins, and decreased the expression of anti-apoptotic proteins in leukemia cells in vitro. EVI-1 expression in leukemia cells (THP-1 and K562) transduced with EVI-1 siRNA was significantly reduced. Silencing EVI-1 had a significant effect on the activation of the JNK pathway and the induction of leukemia cell apoptosis. ATO may downregulate EVI-1 mRNA and oncoprotein levels and block the inhibitory effects of EVI-1 on the JNK pathway, which activates the JNK apoptotic pathway, thereby leading to the apoptosis of EVI-1 in AML patients.