CCK and gastrin inhibit adenylate cyclase activity through a pertussis toxin-sensitive mechanism in the tumoral rat pancreatic acinar cell line AR 4-2J (original) (raw)
Related papers
British Journal of Pharmacology, 1997
1In the pig, the secretory response of the pancreas is not inhibited by the antagonist MK329 suggesting that cholecystokininA (CCKA) receptors are not involved.2Membranes were isolated from the pancreas of 6 Large White pigs to characterize their CCK receptors.3The binding of [M125I]-BH-[Thr, Nle]CCK-9 was dependent on pH, maximal after a 90 min incubation period, saturable and reversible. Saturation analysis of the binding demonstrated a single class of high affinity sites (Kd = 0.22 ± 0.02 nM) and a binding capacity, Bmax= 110.64±12.50 fmol mg−1 protein.4Competition binding by agonists and antagonists of CCKA and CCKB/gastrin receptors demonstrated the presence of two distinct binding components, sites presenting a high affinity for [Thr, Nle]CCK-9, gastrin, PD 135158, L-365,260 and a low affinity for MK329, SR 27897, and sites presenting a high affinity for [Thr, Nle]CCK-9, MK329, SR 27897 and a low affinity for gastrin, PD 135158, L-365,260.5These pharmacological data demonstrate the presence of both CCKA and CCKB/gastrin receptors in the pig pancreas, the latter being predominant.6Two distinct membrane proteins (50 and 85–100 kDa, respectively) display pharmacological features of CCKB/gastrin and CCKA receptors.7In pigs, as in calves and humans, CCKB/gastrin receptors are predominant in the pancreas.In the pig, the secretory response of the pancreas is not inhibited by the antagonist MK329 suggesting that cholecystokininA (CCKA) receptors are not involved.Membranes were isolated from the pancreas of 6 Large White pigs to characterize their CCK receptors.The binding of [M125I]-BH-[Thr, Nle]CCK-9 was dependent on pH, maximal after a 90 min incubation period, saturable and reversible. Saturation analysis of the binding demonstrated a single class of high affinity sites (Kd = 0.22 ± 0.02 nM) and a binding capacity, Bmax= 110.64±12.50 fmol mg−1 protein.Competition binding by agonists and antagonists of CCKA and CCKB/gastrin receptors demonstrated the presence of two distinct binding components, sites presenting a high affinity for [Thr, Nle]CCK-9, gastrin, PD 135158, L-365,260 and a low affinity for MK329, SR 27897, and sites presenting a high affinity for [Thr, Nle]CCK-9, MK329, SR 27897 and a low affinity for gastrin, PD 135158, L-365,260.These pharmacological data demonstrate the presence of both CCKA and CCKB/gastrin receptors in the pig pancreas, the latter being predominant.Two distinct membrane proteins (50 and 85–100 kDa, respectively) display pharmacological features of CCKB/gastrin and CCKA receptors.In pigs, as in calves and humans, CCKB/gastrin receptors are predominant in the pancreas.
Gut, 1987
Trophic changes of the exocrine pancreas after in vivo gastrin (G)/CCK treatment are well documented but up to now the study of the mechanisms involved is restricted by the lack of a suitable in vitro model. Nevertheless the in vivo trophic effect induced by gastrin/CCK peptides has been associated with an increase of ornithine decarboxylase (ODC) activity. In the present work, using the AR42J cell line in which CCK receptors and stimulation of amylase release by CCK peptides has already been demonstrated, we investigated the presence of gastrin binding sites and the possible modulation of proliferation by an inhibitor of ODC activity. '25I-BH-Gl7ns binding is saturable, reversible and specific. Potencies of the different analogues tested are G17ns > CCK8 > CCK8ns > G6s > G/CCK4. Furthermore dBt cGMP, a non-peptide antagonist for CCK recep-
Regulatory Peptides, 1999
The CCK-B / gastrin receptor has been characterised in both normal and tumour tissues. Endocytosis of the CCK-B / gastrin receptor has recently been demonstrated and this has similarly been described for other peptide receptors. In addition, ligand and ligand-receptor translocation to the nucleus has been demonstrated for other peptides. The aim of this study was to identify the sites of expression of the CCK-B / gastrin receptor in the known CCK-B / gastrin receptor bearing pancreatic acinar AR42J cells. The specificity of the CCK-B / gastrin receptor antibody (a-CCKBR-Ser antibody) was demonstrated by inhibition ELISA studies, radioligand inhibition studies and immunofluorescence binding studies on AR42J cells. Western blotting and immunogold electron microscopy techniques were used to identify the receptor in AR42J cell preparations. The affinity purified a-CCKBR-Ser antibody was shown to be specific for the CCK-B / gastrin receptor. The receptor was expressed on the cell membrane, in the cytoplasm and within the nucleus. Isoforms of the receptor protein identified in extra-nuclear and nuclear extracts ranged in molecular weight from 58 to 66 kDa. We conclude that the CCK-B / gastrin receptor is not only expressed on the cell membrane, but also in the cytoplasm and nucleus of AR42J pancreatic acinar cells.
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1997
Recent studies suggest that in some tissues GRP receptor activation can both stimulate phospholipase C and the adenylate cyclase pathway and that activation of the latter pathway may be important in mediating some of its well-described growth effects. However, other studies suggest GRP-R may not be coupled to adenylate cyclase. To investigate this possibility, in the present study we determined the coupling of the GRP receptors to each pathway in mouse, rat, and guinea pig pancreatic acini and compared it to that in mouse Swiss 3T3 cells and human SCLC cells, all of which possess well-characterized GRP receptors. Moreover, we tested the effect of PKC activation on the ability of GRP-related peptides to increase cAMP accumulation in these tissues. Changes in cAMP levels were determined with or without IBMX present, with or without forskolin, or both to amplify small increases in cAMP. In mouse, rat and guinea pig pancreatic acini, murine Swiss 3T3 cells and human SCLC cells, GRP-related peptides caused a 600%, 500%, 250%, 300% and 60% w 3 x increase, respectively, in H IP with 1-3 nM causing a half-maximal effect. In murine Swiss 3T3 cells, IBMX, forskolin, and IBMX plus forskolin caused a 300%, 3500% and 10 500% increase in cAMP, respectively. GRP-related peptides and Ž. VIP caused an additional 70% increase in cAMP with GRP causing a half-maximal EC increase in cAMP at 2.1 " 0.5 50 w 3 x nM, which was not significantly different from the EC of 3.1 " 0.9 nM for increasing H IP in these cells. GRP-related 50 peptides did not stimulate increases in cAMP in mouse, rat or guinea pig pancreatic acini or in SCLC cells either alone, with IBMX or forskolin or both. However, in pancreatic acini IBMX, forskolin or both increased cAMP 3 to 8-, 10 to 500-, and 100 to 1000-fold increase and the addition of VIP caused an additional 20-, 2-, and 3-fold increase in cAMP in the different species. In mouse pancreatic acini with TPA alone or IBMX plus TPA, neither bombesin nor GRP increased cAMP. Furthermore, in mouse pancreatic acini, neither TPA nor TPA plus IBMX altered basal or VIP-stimulated increases in cAMP. In mouse Swiss 3T3 cells TPA significantly increased cAMP stimulated by Bn, GRP or VIP. These results demonstrated that GRP receptor activation in normal tissues from three different species and a human tumoral cell line do not result in adenylate cyclase activation, whereas in Swiss 3T3 cells it causes such activation. The results suggest that the difference in coupling to adenylate cyclase is likely at least partially due to a difference in coupling to an adenylate cyclase
Regulatory Peptides, 1996
The novel radioligand [3H]PD140376 was used to label receptors that bind cholecystokinin (CCK) and related peptides in membranes prepared from guinea-pig brain and gastric glands. Under control conditions, measurements of the apparent affinity of 11 agonist and 16 antagonist ligands in both tis,;ues revealed a strong positive relationship between the affinity of a compound in either tissue (slope of the regression line = 0.89, r 2 = 0.908). Agonists consistently showed higher affinity for sites in gastric glands compared to brain. If agonists were excluded from the analysis, the degree of correspondence between affinities measured in each tissue was almost perfect (slope = 0.93, r 2 = 0.986). I:a the presence of the guanyl nucleotide 5'-guanylimidodiphosphate (GppNHp), agonist affinity in gastric glands, but not brain, was reduced such that there was a direct relationship between binding affinity in each tissue. These data are consistent with the notion that the receptor sites in brain and gastric glands, which recognise CCK and gastrin related compounds, are the same and of the CCK-B/gastrin subtype. The receptors in the two respective tissues, however, do appear to differ in the degree of post-receptor coupling. These findings may explain previously reported differences between gastrin and CCK-B receptors that were based upon binding studies using agonist ligands. 8s, which are thought to represent the receptors through which these peptides exert their influence, are distributed throughout the brain and the gastrointestinal tract . These receptor binding sites have been differentiated into subtypes on the basis of their affinity for a range of related peptide agonists and non-peptide antagonists. In the case of receptor binding sites for which the endogenous ligand can be considered as being CCK-8s, there are two main subtypes; CCK-A and CCK-B sites . CCK-A receptors discriminate well between sulphated and unsulphated forms of CCK octapeptide and bind the non-peptide antagonist devazepide (L-364,718, MK 329) with picomolar affinity, whilst displaying much lower binding affinity for the peptoid antagonist CI-988 and the benzodiazepine L-365,260. CCK-A receptors are found in high concentrations in the pancreas, gall bladder and also in discrete regions of the brain . CCK-B receptors are ubiquitous throughout the brain and may be defined by 0167-0115/96/$15.00
European Journal of Pharmacology: Molecular Pharmacology, 1995
We have shown that gastrin and cholecystokinin octapeptide (CCK-8) are differently coupled to G protein (GTP-binding protein) through type B cholecystokinin receptors in guinea-pig brain membranes and Jurkat cells. Indeed, the gastrin-13 binding affinity is strongly reduced by stable guanyl nucleotides, whereas CCK-8 binding is only slightly affected. In order to determine the structural requirements regulating such coupling, we have synthesized several gastrin and choleeystokinin fragments (sulphated or unsulphated) elongated at the N-terminus of the common C-terminal tetrapeptide. We investigated their interaction with CCK s receptors in guinea pig brain membranes and Jurkat cells and their involvement in the G protein coupling. Their apparent binding affinities to CCK u receptors were measured by inhibition of [l~I]Bolton Hunter-CCK-8 (3-[125I]iodo-4-hydroxyphenyl)propionyl-CCK-8) binding in the presence or absence of GTPyS (guanosine 5'-O-(3-thio)triphosphate) or aluminium tetrafluoride (AIF~). Activation of the G proteins by GTPyS or AIF 4-led to a decrease in binding affinity for the gastrin related peptides, the common CCK-gastrin C-terminal forms, the cholecystokinin hexapeptide and the unsulphated cholecystokinin heptapeptide. Sulphated CCK-7, CCK-8, and cionin apparent binding affinities were not affected. These finding indicated that the sulphated tyrosine in position 7 in CCK (as counted from the C-terminus), provides the cholecystokinin selectivity for the CCK B receptor compared to gastrin. The results are discussed with the aim to better clarify the physiological relevance of gastrin and cholecystokinin toward CCK B receptors and their related intracellular events.
The Cellular Localization of the Cholecystokinin 2 (Gastrin) Receptor in the Stomach
Pharmacology and Toxicology, 2002
The role of the gastric acid secretagogues acetylcholine, gastrin and histamine has been debated for decades. Initially, the mast cell was considered the source of acid stimulatory histamine. Later, showed that the entero-chromaffinlike (ECL) cell produces and stores histamine in several species, including rat and man. showed that food and gastrin stimulated oxyntic mucosal histamine synthesis and release, that histamine and cholinergics but not gastrin induced acid secretion in isolated oxyntic glands and parietal cells, and Rangachari (1995) that acetylcholine or gastrin released histamine in isolated mucosa. These findings suggested that gastrin stimulates acid secretion through release of ECL cell histamine. Studying simultaneous histamine release and acid secretion in isolated oxyntic mucosal cells, we found that gastrin stimulated acid secretion only in preparations releasing histamine. Moreover, in the isolated rat stomach, gastrin stimulated both histamine release and acid secretion. Maximal acid output was higher with histamine than with gastrin, and augmented by acetylcholine but not by gastrin. These findings strongly suggested that gastrin acts by releasing histamine. Finally, a fluorescein-labelled gastrin analogue bound to the ECL cell, not to the parietal or stem cell regions. This is interesting, recalling that gastrin has a potent and specific trophic effect on the ECL cell and only a general effect on all other oxyntic cell types. In conclusion, physiological observations are best explained by localising the CCK2 receptor only to the ECL cell, the other effects of gastrin on the gastric mucosa being secondary to the release of mediators from the ECL cell.