Presence of blood group H antigen on a carcinoembryonic antigen, and its enzymatic modification into blood group A and B specificities.

A carcinoembryonic antigen (CEA-M) was purified from a hepatic metastasis obtained from a blood group O patient with cancer of the rectum. Using 125I-labeled carcinoembryonic antigen (CEA) and blood group antisera, H specificity has been found on the CEA-M. As the addition of anti-H to anti-CEA does not modify the extent of binding of labeled CEA-M to its antibodies (86%), the H and CEA determinants are carried by the same molecule. The affinity chromatography of CEA-M on an immunosorbent "anti-H-Sepharose" demonstrated that a proportion of CEA-M molecules might bear both H and CEA antigenic determinants. In addition, glycosyltransferases were used to modify the blood group H specificity into blood group A or B specificities.

Gastrin and CCK-8 induce inositol 1,4,5-trisphosphate formation in rabbit gastric parietal cells.

The role of phosphoinositide turnover in the mediation of acid secretion was examined in an enriched preparation of isolated rabbit parietal cells (75%). Both gastrin and CCK-8 (octapeptide of cholecystokinin) stimulated [14C]aminopyrine (AP) uptake by cells (EC50 0.07 +/- 0.03 nM (gastrin) and 0.093 +/- 0.065 nM (CCK-8] and increased [3H]inositol phosphates cellular contents (EC50 0.142 +/- 0.016 nM (gastrin) and 0.116 +/- 0.027 nM (CCK-8] in a parallel fashion. In addition, the EC50 values for both phenomenon were quite similar to the Kd values obtained from binding experiments. HPLC analysis of the different [3H]inositol phosphates produced under gastrin or CCK-8 stimulation showed a 2-fold increase in [3H]Ins(1,4,5)P3 levels within 5 s with a concomitant increase in [3H]Ins(1,4)P2 content within 15 s. A low but significant rise in [3H]Ins(1,3,4,5)P4 and [3H]Ins(1,3,4)P3 cellular contents was also observed. No difference between gastrin- and CCK-8-induced inositol phosphates production could be shown. We can conclude that gastrin and CCK-8 display an identical profile of action, suggesting that they stimulate the acid secretory function of parietal cells through the same receptor site coupled to the Ins(1,4,5)P3 production.

Involvement of a pertussis toxin-sensitive G protein in the action of gastrin on gastric parietal cells.

The mechanism whereby gastrin triggers phosphoinositide breakdown was investigated in an enriched preparation of isolated rabbit parietal cells (approx. 75%). In a permeabilized preparation of myo-[3H]inositol-labelled cells, GTP[S], a non-hydrolysable GTP analogue, enhanced [3H]inositol trisphosphate ([3H]InsP3 accumulation in a dose-dependent manner; submaximal concentrations of GTP[S] (less than 10 microM), potentiated gastrin-induced [3H]InsP3 release; preincubation for 5 min with GDP[S], a non-hydrolysable GDP analogue, dose-dependently reduced [3H]InsP3 accumulation stimulated by gastrin even in presence of GTP[S]. Exposure of intact parietal cells for 3 h to pertussis toxin (PTx) (200 ng/ml) led to a 15-50% reduction in gastrin-induced [14C]aminopyrine [(14C]AP) uptake (an index of in vitro acid secretion) and [3H]inositol phosphate ([3H]InsP) accumulation. A decrease in the accumulation of the different [3H]inositol phosphate occurred in gastrin-stimulated parietal cells treated with PTx. A rightward shift of gastrin dose-response curves in the presence of PTx was observed for [14C]AP uptake (EC50 values: 0.125 +/- 0.045 nM without PTx and 1.05 +/- 0.63 nM with PTx), for [3H]InsP accumulation (EC50 values: 0.16 +/- 0.08 nM without PTx and 1.56 +/- 0.58 nM with PTx) and [125I]gastrin binding (IC50 values: 0.247 +/- 0.03 nM without PTx and 2.38 +/- 0.56 nM with PTx). In contrast, cholera toxin (CTx) treatment (100 ng/ml) for 3 h was without effect on gastrin-induced [3H]InsP accumulation. CTx induced a pronounced potentiation of gastrin-stimulated [14C]AP uptake; this effect can be mimicked by IBMX (a phosphodiesterase inhibitor) and by forskolin (an activator of adenylyl cyclase). We conclude that: (i) one or more than one G protein appeared to be involved in gastrin receptor coupling to phospholipase C (PL-C); (ii) these G proteins are not substrates for CTx; (iii) one of these appeared to be a PTx-sensitive 'Gi-like' protein which could be involved in hormone-induced acid secretion, (iiii) the potentiating effect of CTx observed on AP uptake stimulated by gastrin suggests the existence of a cooperative effect between cAMP pathway (CTx) and the gastrin-induced phosphoinositide breakdown in acid secretory activity of parietal cells.

Common or distinct receptors for gastrin and cholecystokinin in gastric mucosa?

The differentiation between gastrin (HG) and cholecystokinin (CCK) receptors in gastric mucosa was examined on isolated parietal (F3) and non-parietal (F1) cells from rabbit fundic mucosa separated by elutriation. Direct binding assays on enriched cell populations were performed using 125I-labeled HG-17, 125I-labeled CCK-8 and 125I-labeled CCK-39 as probes. (1) On F1 cells, the dissociation constants (Kd) for the two labeled CCKs were nearly the same (62 pM for CCK-8 and 74 pM for CCK-39) but the binding capacity for CCK-8 was 2-times higher than for CCK-39. HG-17 also bound to this cell population, but its Kd value as about 2-times higher (110 pM) than that of CCK. The presence of two distinct classes of sites on F1 cells can be suggested from competition studies: one more specific for CCK, which bound CCK-8 and CCK-39 with the same affinity, and another class more specific for gastrin, which bound CCK-8 and HG-17 with the same affinity and CCK-39 with a low affinity. (2) On F3 cells, CCK-8 and HG-17 bound with similar affinities (Kd values 81 pM for CCK-8 and 87 pM for HG-17), but CCK-39 did not specifically bind to this cell population. The presence of a binding site more specific for HG than for CCK on F3 cells was confirmed by competition studies in which CCK-33 competed for binding with labeled HG-17 and labeled CCK-8 with a 50-times lower affinity than the other peptides.

Abilities of some tryptophan and phenylalanine derivatives to inhibit gastric acid secretion.

Benzotript (N-p-chlorobenzoyl-L-tryptophan) has been shown to be a receptor-antagonist in vivo and in vitro for peptides from the gastrin family. In the present study, we examine tryptophan, and some of its N- and C-acylated derivatives, as well as some phenylalanine derivatives, to show their ability to inhibit gastrin-induced acid secretion in the rat in vivo and to compete for the binding of [125I]-(Leu-15)-HG-17 to its cellular receptor on rabbit isolated gastric mucosal cells. N- and C- derivatives of tryptophan and phenylalanine were found to inhibit gastrin-induced acid secretion and binding of [125I]-(Leu-15)-HG-17 to its mucosal cell receptors. By either criterion, the relative antagonistic potencies of the compounds tested were: tert-butyloxycarbonyl-L-tryphophan-p-nitrophenyl ester approximately equal to tert-butyloxycarbonyl-L-tryptophan-carbamoylmethyl ester greater than tert-butyloxycarbonyl-L-tryptophyl-L-methionyl-carbamoylmethyl ester approximately equal to tert-butyloxycarbonyl-L-phenylalanine-carbamoylmethyl ester approximately equal to tert-butyloxycarbonyl-L-tryptophyl-L-methionyl-amide greater than tert-butyloxycarbonyl-L-tryptophan greater than tert-butyloxycarbonyl-L-phenylalanine greater than benzyloxycarbonyl-L-tryptophan approximately equal to benzotript, with minor differences between the in vivo and the in vitro experiments. These results demonstrate that both the nature of the amino acid residue and the N- and C-substitutions are important in determining antagonist activity and affinity for gastrin receptors.

Contraction induced by glicentin on smooth muscle cells from the human colon is abolished by exendin (9-39).

UNLABELLED: Glicentin and glucagon-like peptide-1 (7-36) amide (GLP-1) are gut hormones released during digestion. Glicentin and GLP-1 slow down gastric emptying and glicentin can switch off the duodenojejunal fed motor pattern. The effect of glicentin on the motor activity of colon has never been reported in humans. Our aim was to determine if circular smooth muscle cells (SMC) from the human colon are target cells for glicentin or GLP-1, and if their motility is dependent upon these digestive hormones. METHODS: Twenty-two resections were performed on patients operated for colon adenocarcinoma. The SMC were isolated from colonic circular muscle layer and cell contraction was assessed. RESULTS: Glicentin caused a dose-related contraction of SMC, when GLP-1 determined a contraction of weak amplitude. Exendin-(9-39), described as a GLP-1 receptor antagonist, inhibited contraction due to glicentin or GLP-1. In contrast, on antral SMC from rabbit, GLP-1 exerts neither relaxation nor contraction; however, exendin-(9-39) dose dependently reduced the contractile activity of glicentin [glicentin EC(50) = 5 pM, exendin-(9-39) pA(2) = -9.36]. CONCLUSIONS: The circular muscle from the human colon is a target tissue for glicentin and GLP-1. Whereas glicentin is a long-life digestive hormone which would contribute to segmental contraction, the biological activity of GLP-1 remains unknown on this tissue. On the digestive smooth muscle, exendin-(9-39) behaved as an antagonist for two members of the glucagon-receptor family, GLP-1 and glicentin.

Contraction of human colonic circular smooth muscle cells is inhibited by the calcium channel blocker pinaverium bromide.

UNLABELLED: The effects of L-type calcium channel blockers (CCBs) selective for the gastrointestinal tract (pinaverium) or non-selective (nicardipine and diltiazem), were investigated on CCK-, CCh- or KCl-induced contraction of smooth muscle cells (SMC) isolated from the circular muscle layer of normal or of inflamed human colons. In the normal tissue colon, whatever the contractile agent used, CCK-8 (1nM), CCh (1nM) or KCl (20mM), a micromolar concentration of pinaverium significantly inhibited contraction (88.36%, 93.10%, 93.92% inhibition respectively); this effect was concentration-dependent for CCh (IC50 = 0.73 +/- 0.08nM) and for CCK (IC50 = 0.92 +/- 0.12nM). In parallel, both nicardipine and diltiazem inhibit significantly contraction of isolated SMC. In inflamed colons, pinaverium (1 microM) display a significant higher efficacy than diltiazem or nicardipine to reduce cell contraction induced by CCK-8 or by KCl. In addition, RT-PCR experiments were performed to evidence tissue specificity of the L-type calcium channel. They revealed the expression of the messenger of the a-1 subunit L-type calcium channel (binding site of such CCBs), consistent with the expression of the rbC-2 splice variant of the alpha1-C gene. IN CONCLUSION: (i) the inhibition by calcium channel blockers of agonist-induced contractile activity suggest a modulation of SMC contraction upon extracellular calcium via 'L-type' voltage-dependent calcium channel; (ii) this study provides a rationale for the clinical use of pinaverium in colonic motor disoders affecting the contractility of SMC, since it appeared to decrease the contraction even in pathological situation; and (iii) RT-PCR experiments confirms the presence in human colon SMC of the alpha-1 subunit mRNA of calcium channel.

Short-term inhibitory effect of somatostatin on gastric histamine synthesis.

In this study we investigated the short-term effect of somatostatin on histamine synthesis in a cell population isolated from rabbit gastric mucosa and enriched in enterochromaffin-like cells. Somatostatin inhibited basal and gastrin-stimulated histamine synthesis through a dual mechanism involving a decrease in the affinity of histidine decarboxylase (HDC) for its substrate (L-histidine) and a reduction in the number of functional HDC molecules. H-89 (an inhibitor of cAMP-dependent protein kinase) mimicked somatostatin-induced reduction of HDC affinity, which, on the contrary, was selectively reversed by pertussis toxin (PTX). Furthermore, forskolin was shown to reverse the inhibitory effect of H-89 and to prevent the somatostatin-induced reduction in HDC affinity for L-histidine. Thus, the somatostatin-induced reduction in affinity seems to involve a PTX-sensitive G protein and an inhibition of the cAMP-dependent pathway. On the other hand, the somatostatin-induced decrease in the number of functional HDC molecules seems to be PTX insensitive and independent from a modulation of the cAMP pathway, and does not seem to involve a significant change in HDC messenger RNA expression or a regulation of protein kinase C. The exact nature of this second mechanism will need further studies to be elucidated.

Glicentin and oxyntomodulin modulate both the phosphoinositide and cyclic adenosine monophosphate signaling pathways in gastric myocytes.

We have investigated the transduction pathways mediating the contractile effect of two glucagon-containing peptides, glicentin (GLIC) and oxyntomodulin (OXM), on smooth muscle cells isolated from rabbit antrum. Low concentrations of GLIC induced a biphasic and rapid (first phase at 5-8 sec) Ins(1,4,5)P3 production. By comparison, higher concentrations of OXM or OXM(19-37) were required to obtain biphasic time-courses of Ins(1,4,5)P3 production. In a Ca2+ free medium, the first phase of Ins(1,4,5)P3 production induced by GLIC or OXM was maintained, while the second phase disappeared. In saponin-permeabilized cells, all three peptides induced cell contraction with similar efficacies and potencies. Exogenous Ins(1,4,5)P3 mimicked the contractile effect of the peptides and heparin, which inhibits the Ins(1,4,5)P3 binding to its receptor, prevented contraction stimulated by each effector. We conclude that a Ca2+ mobilization from the intracellular stores is essential in the contractile effects of GLIC and OXM. Using the fluo-3 probe, a [Ca2+]i increase was observed in the presence of GLIC, OXM, or OXM(19-37). The three peptides reduced by 30-40% the cAMP content of cells stimulated by forskolin. This effect was pertussis toxin sensitive as demonstrated with OXM(19-37). Our data constitute important clues for the existence in smooth muscle cells of receptor(s) specific for the GLIC/OXM hormones, coupled via G protein(s) to both Ca2+ and cAMP pathways.

Biphasic kinetics of inositol 1,4,5-trisphosphate accumulation in gastrin-stimulated parietal cells. Effects of pertussis toxin and extracellular calcium.

The effects of Pertussis toxin (PTx) and extracellular Ca2+ were investigated on gastrin-induced Ins(1,4,5)P3 mass level in isolated gastric parietal cells. Basal Ins(1,4,5)P3 content was 5.48 +/- 0.49 pmol/500,000 cells. Gastrin (10 nM) induced a rapid increase in Ins(1,4,5)P3 content which was maximal after 15 s and corresponded to 2-2.5-fold basal level; this Ins(1,4,5)P3 content then decreased within 30 s. After a longer time of gastrin exposure (greater than 1 min), a sustained and unexpected increase in Ins(1,4,5)P3 accumulation was observed which was maximal at 7.5 min (corresponding to 2.3-2.8-fold basal value) and slightly decreased thereafter. PTx treatment of cells (200 ng/ml) for 3 h or removal of extracellular Ca2+ did not affect the rapid rise, but drastically reduced the sustained increase in Ins(1,4,5)P3 content (60-100% inhibition); this inhibition was not evident after 10 min of hormone stimulation. Furthermore, diltiazem, a Ca2+ channel blocker, led to a similar inhibition of the sustained increase. We concluded that: (i) gastrin induced a rapid increase in Ins(1,4,5)P3 content via a mechanism insensitive to PTx and to extracellular Ca2+, and (ii) gastrin induced a sustained increase in Ins(1,4,5)P3 level via a mechanism sensitive to PTx and to extracellular Ca2+. Even though the rapid rise in Ins(1,4,5)P3 content may be involved in the intracellular Ca2+ mobilization occurring after the first seconds of hormone stimulation, the physiological role of the sustained Ins(1,4,5)P3 increased level remains to be elucidated.

Synthesis and biological activities of some pseudo-peptide analogues of tetragastrin: the importance of the peptide backbone.

Pseudo-peptide analogues of the C-terminal tetrapeptide of gastrin, in which a peptide bond has been replaced by a CH2-NH bond, i.e. (tert-butyloxycarbonyl)-L-tryptophyl-psi (CH2-NH)-L-leucyl-L-aspartyl-L-phenylalanine amide (8), (tert-butyloxycarbonyl)-L-tryptophyl-L-leucyl-psi (CH2-NH)-L-aspartyl-L-phenylalanine amide (13), (tert-butyloxycarbonyl)-L-tryptophyl-L-leucyl-L-aspartyl-psi (CH2NH)-L-phenylalanine amide (20), were synthesized. The pseudo-peptides 8 and 13 were shown to have the same affinity as (tert-butyloxycarbonyl)-L-tryptophyl-L-leucyl-L-aspartyl-L-phenylalanine amide (21) for the gastrin receptor on isolated mucosal cells. The pseudo-peptide 20 exhibited lower affinity (IC50 congruent to 10(-5) M). The biological activity of these pseudo-peptides was studied on acid secretion in the anesthetized rat. Compound 8 stimulated acid secretion, identically with that of 21. Compound 13 did not exhibit any agonist activity but was able to antagonize the action of gastrin (ED50 = 0.3 mg/kg). Compound 20 did not show any agonist activity but was able to inhibit gastrin-induced acid secretion, with lower potency (ED50 = 15 mg/kg). The importance of the peptide bonds in the mode of action of gastrin is discussed, and a hypothetical approach of the mechanism of action is presented.

Synthesis and biological activity of new peptide segments of gastrin exhibiting gastrin antagonist property.

A series of C-terminal peptide segments of gastrin, i.e., (tert-butyloxycarbonyl)-L-tryptophyl-L-methionyl-L-aspartic acid amide, (tert-butyloxycarbonyl)-glycyl-L-tryptophyl-L-methionyl-L-aspartic acid amide, (tert-butyloxy-carbonyl)-L-tyrosyl-glycyl-L-tryptophyl-L-methionyl-L-asp artic acid amide, and (benzyloxycarbonyl)-L-glutamyl-L-alanyl-L-tyrosyl-glycyl-L-tryptophyl-L -methionyl-L-aspartic acid amide were prepared and were shown to competitively inhibit the binding of labeled human gastrin to its receptors in an isolated gastric mucosal cell preparation and to antagonize the action of gastrin on gastric acid secretion (ED50 from 1.5 to 7 mg/kg) in vivo in the reperfused rat stomach, determined according to the method of Ghosh and Schild. From these studies, it could be concluded that the C-terminal phenylalanine residue, which is of primary importance for intrinsic biological gastrin-like activity, is not essential for binding to gastrin receptors.

Structure-activity relationships of C-terminal tri- and tetrapeptide fragments that inhibit gastrin activity.

A series of tri- and tetrapeptide derivatives, analogues of the gastrin C-terminal region with no phenylalanine residue, were synthesized. These peptides were tested for their ability to inhibit gastrin-stimulated acid secretion in vivo as well as binding of [125I]-(Nle11)-HG-13 to gastric mucosal cell receptors in vitro. Most of the peptides tested exhibited gastrin antagonist activity in vivo and in vitro. Most active derivatives were 20-30 times more potent than the well-known gastrin antagonist derivatives proglumide and benzotript and had 20-200 times more binding affinity. The smallest fragment exhibiting antagonist activity was the tripeptide Boc-L-tryptophyl-L-methionyl-L-aspartic acid amide.

Muscarinic receptors in isolated gastric fundic mucosal cells. Binding-activity relationships.

Muscarinic receptors are involved in the control of gastric acid secretion. The characteristics of (3H)-N-methyl-scopolamine [(3H)-NMS] binding to isolated cells from rabbit fundic gastric mucosa and inhibition of this binding by muscarinic agonists, antagonists and other pharmacological agents known to regulate acid secretion are reported. Specific binding for (3H)-NMS was described: antagonists interact with high affinity sites (KD = 0.5 nM) whereas binding curves for agonists clearly deviated from the simple mass action isotherm with a flattening of the curve suggesting the presence of more than one class of sites. The low affinity sites for agonists are in the micromolar range. Pirenzine, a gastroselective antimuscarinic compound, known to differentiate between M1 and M2 sites, inhibited (3H)-NMS binding with an IC50 of 0.05 microM. On the same gastric cell population, muscarinic agonist carbachol stimulated (14C)-aminopyrine accumulation in a dose-dependent manner with an ED-50 of 10 microM, value close to that needed to 50% inhibit (3H)-NMS binding. This stimulation was competitively inhibited by muscarinic antagonists and pA2-values for atropine, QNB and pirenzepine, calculated from linear Schild plots, were in the following order: 9.2 for atropine, 8.6 for QNB and 7.0 for pirenzepine. In conclusion, fundic gastric mucosal cells from rabbit, isolated with collagenase and EDTA, contained specific muscarinic receptors coupled to the acid secretory mechanism and pirenzepine interact with these receptors with an intermediate affinity suggesting the presence of functional M2-sites.

Gastrointestinal hormone receptors on isolated smooth muscle cells from gastric antrum of the rabbit.

The regulation by gastrointestinal polypeptide hormones of contraction and relaxation of functionally isolated smooth muscle cells from gastric antrum of the rabbit has been investigated. Gastrin, cholecystokinin (CCK-8) and motilin induced a rapid contraction of isolated cells: significant response occurred within a 5-sec incubation with these peptides and maximal response (40% decrease in cell length) after 30 sec. A higher sensitivity of smooth muscle cells to gastrin and CCK-8 than to motilin stimulations was demonstrated (EC50 = 10 pM for both gastrin and CCK-8 and EC50 = 1 nM for motilin). The minimal gastrin fragment required to get full contraction was the C-terminal pentapeptide amide common to gastrin and CCK. Proglumide inhibited gastrin- or CCK-8- but not motilin-induced contractions with an IC50 of 50 microM. contraction induced by gastrin and motilin required normal levels of extracellular calcium, whereas that due to CCK-8 seemed to be independent of extracellular calcium. Vasoactive intestinal polypeptide (VIP) caused a relaxation of smooth muscle cells maximally contracted by carbachol or CCK-8 or gastrin (EC50 = 2.2 nM) with a parallel increase in intracellular cAMP content.

M3-subtype muscarinic receptor that controls intracellular calcium release and inositol phosphate accumulation in gastric parietal cells.

The muscarinic receptor subtype which triggers acid secretion was investigated in isolated rabbit gastric parietal cells. Cytosolic free Ca2+ concentration ([Ca2+]i), measured with the fluorescent indicator FURA-2, increased rapidly after full agonist (carbachol) stimulation (6-8 sec), then returned to an intermediate sustained value. Other M2-agonists, oxotremorine and arecoline, produced a partial [Ca2+]i increase, whereas M1-agonists, pilocarpine and [4-m-chlorophenylcarbamoyloxyl]-2-butynyl-trimethylammonium, were without any significant effect. [Ca2+]i rise was inhibited by selective muscarinic antagonists: atropine greater than 4-diphenylacetoxy-N-methyl-piperidine methbromide greater than quinuclidinylbenzilate (QNB) greater than pirenzepine greater than 11-[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H- pyrido[2,3-b][1,4]benzodiazepine-6-one, this sequence being characteristic of the involvement of an M3-subtype. This inhibition was shown to be stereoselective; dexetimide and (-)QNB were more potent than levetimide and (+)QNB. The IC50 values for inhibition of [Ca2+]i increase by muscarinic antagonists were in good agreement with those obtained for inhibition of phospholipase C activation. In conclusion, the muscarinic receptor that controls acid secretion appears to be of the M3-subtype and the biochemical events coupled to the activation of this receptor system are also controlled through the same subtype.

"Gastrin" and "CCK" receptors on histamine- and somatostatin-containing cells from rabbit fundic mucosa--I. Characterization by means of agonists.

A previous study has suggested the presence of two distinct binding sites for gasrin and cholecystokinin (CCK) in isolated non-parietal cells from rabbit gastric mucosa: a receptor which binds CCK-8 and CCK-39 with a high affinity and a receptor which binds gastrin and CCK-8 with the same high affinity and CCK-39 with a lower affinity. To characterize these receptors, their ability to induce phosphoinositide breakdown was investigated. Gastrin (HG-17), CCK-39 and CCK-8 induced [3H]-inositol phosphate ([3H]InsP) accumulation from [3H]inositol prelabelled cells with a high potency (EC50: 0.3-2.7 nM) but CCK-8 exhibited a higher efficacy than HG-17 or CCK-39. HG-17, CCK-8 and CCK-39 induced a rapid accumulation of [3H]inositol monophosphate ([3H]InsP1), [3H]inositol bisphosphate ([3H]InsP2) and [3H]inositol trisphosphate ([3H]InsP3) but CCK-8 caused a two times higher accumulation than HG-17 or CCK-39. Histamine- and somatostatin-containing cells appeared to be located in this non-parietal cells population. HG-17, CCK-8 and CCK-39 dose-dependently induced histamine release with the following order of potency: HG-17 = CCK-8 (EC50 approximately 0.2 nM) greater than CCK-39 (EC50 approximately 4 nM). In addition, HG-17 exhibited the highest efficacy. HG-17, CCK-8 and CCK-39 enhanced somatostatin-like immunoreactivity (SLI) release with the following order of potency: CCK-8 (EC50 approximately 0.1 nM) = CCK-39 greater than HG-17 (EC50 approximately 10 nM); CCK-8 and CCK-39 exhibited the highest efficacy. These results led us to the following conclusions: (i) existence of a "gastrin-type" and of a "CCK-type" receptor mediating phosphoinositide breakdown in these gastric non-parietal cells. CCK-8 interacts with both receptor-types with the same affinity; (ii) the release of histamine from histamine-containing cells could be induced following "gastrin-type" receptors activation; (iii) somatostatin release from D-cells present in this non-parietal cells population could be induced following "CCK-type" receptors activation.

"Gastrin" and "CCK" receptors on histamine- and somatostatin-containing cells from rabbit fundic mucosa-II. Characterization by means of selective antagonists (L-364,718 and L-365,260).

In the preceding paper, by means of selective agonists to gastrin (HG-17) and cholecystokinin (CCK-39), we evidenced the existence of "gastrin-type" receptors that could regulate histamine release and "CCK-type" receptors that could stimulate somatostatin release in isolated rabbit fundic non-parietal cells (F1 cells). Furthermore, these receptors could induce phosphoinositide breakdown. To confirm the involvement of these receptor types in these biological and biochemical processes, we used selective antagonists, L-364,718 (3-(benzoylamino)-benzodiazepine) specific to "CCK-A-type" receptor and L-365,260 (3-(acylamino)-benzodiazepine) specific to "gastrin/CCK-B-type" receptor. Neither L-364,718 nor L-365,260 alone caused any significant stimulation of [3H]inositol phosphate ([3H]InsP) production and release of histamine or somatostatin-like immunoreactivity (SLI). Each analogue inhibited in a dose-dependent manner [125I]HG-17 or [125I]CCK-39 binding to F1 cells, [3H]InsP accumulation and histamine and SLI release stimulated by HG-17 or CCK-39. L-365,260 appeared to be 30-70 times more potent than L-364,718 in inhibiting [125I]HG-17 binding to F1 cells, as well as HG-17-induced [3H]InsP accumulation and HG-17-or CCK-39-enhanced histamine release (IC50 values: approximately 5-20 nM for L-365,260 and approximately 200-1500 nM for L-364,718). In contrast, L-364,718 was 200 to 400 times more potent than L-365,260 in inhibiting [125I]CCK-39 binding to F1 cells, CCK-39-induced [3H]-InsP accumulation and SLI release stimulated by CCK-39 or HG-17 (IC50 values: approximately 0.3-1 nM for L-364,718 and 100-200 nM for L-365,260). These results led to conclude: (i) the existence of a "gastrin-type" receptor related to histamine release: (ii) the existence of a "CCK-A-type" receptor related to somatostatin release; (iii) the existence of "gastrin type" and "CCK-A-type" receptors linked to the phosphoinositide breakdown pathway.

Muscarinic receptors in isolated smooth muscle cells from gastric antrum.

Smooth muscle cells from the gastric antrum of the rabbit were isolated using collagenase and pronase. We examined the characteristics of muscarinic receptors that control contraction of the muscle cell: kinetics, stoichiometry and specificity of both contractile response to muscarinic agents and binding of labeled N-methyl-scopolamine. Cells contracted in the presence of muscarinic agents after a short time (30 sec) while binding of (3H)-NMS reached a plateau after 10 min exposure. Specific binding was saturable and Scatchard analysis revealed a single class of high-affinity binding sites (Kd: 0.5 nM). Oxotremorine was the most potent agonist with an ED50 of 0.6 pM; acetylcholine and carbachol were 10 times less potent. Muscarinic antagonists competed with (3H)-NMS for binding with IC50 values in the same range (nanomolar or less) than those obtained for inhibition of acetylcholine-induced contractions. Pirenzepine antagonized contractile effect of muscarinic agonists with EC50 in a micromolar range. Intracellular levels of cyclic AMP were lowered by muscarinic agonists. Monoclonal anti-muscarinic receptor antibodies M-35 displayed agonist-like activities triggering contraction and lowering cyclic AMP levels of the cells. However, although the antagonist inhibits M-35-induced contractions and cAMP decrease, M-35 had no effect on binding of the antagonist to the muscarinic receptor. These data revealed the presence of an M2-muscarinic receptor subtype involved in the contractile response of the isolated smooth muscle cell.

Intracellular coupling of prostaglandin inhibition of acid secretion in isolated rabbit gastric parietal cells.

Acid secretion from isolated rabbit gastric parietal cells can be stimulated by gastric secretagogues, histamine (cyclic-AMP pathway) and carbachol (inositol phosphate pathway). Prostaglandins (PG) from E series are potent inhibitors of acid secretion. The intracellular mechanism of this inhibition was examined by using a stable PGE1-analogue, misoprostol. Aminopyrine (AP) accumulations due to histamine, IBMX and forskolin were dose-dependently inhibited by misoprostol, whereas a weak but significant biphasic effect on carbachol-induced AP accumulation was observed. The cyclic-AMP formation induced by histamine and IBMX were also inhibited by misoprostol in a non-competitive way. The potent effect of forskolin on cyclic-AMP levels was not modified by misoprostol in parietal cells, whereas it was potentiated in non-parietal cells. The inhibitory effect of misoprostol on AP accumulation was reduced by incubation of parietal cells with Bordetella pertussis toxin (IAP) but not with Cholera toxin (CT). Pretreatment of the cells with IAP did not alter cyclic-AMP levels of resting and histamine-stimulated parietal cells but abolished the inhibitory effect of misoprostol. Treatment with CT increased basal and histamine-stimulated cyclic-AMP levels and masked the inhibitory effect of misoprostol. The biphasic effect of misoprostol on carbachol-stimulated AP accumulation in parietal cells was confirmed on carbachol-stimulated phospholipase C activity and on [Ca2+]i stimulated by carbachol. These data confirm a direct and specific effect of the prostanoid on the Gi-subunit of the adenylate cyclase coupled to the histamine H2-receptor, and a biphasic effect on the phospholipase C pathway of the parietal cells.