Improvement of neurobehavioral disorders in children supplemented with magnesium-vitamin B6. I. Attention deficit hyperactivity disorders.

Some previous studies have reported the involvement of magnesium (Mg) deficiency in children with ADHD syndrome. In this work, 40 children with clinical symptoms of ADHD were followed clinically and biologically during a magnesium-vitamin B6 (Mg-B6) regimen (6 mg/kg/d Mg, 0.6 mg/kg/d vit-B6) which was set up for at least 8 weeks. Symptoms of ADHD (hyperactivity, hyperemotivity/ aggressiveness, lack of attention at school) were scored (0-4) at different times; in parallel, intraerythrocyte Mg2+ (Erc-Mg) and blood ionized Ca2+ (i-Ca) were measured. Children from the ADHD group showed significantly lower Erc-Mg values than control children (n = 36). In almost all cases of ADHD, Mg-B6 regimen for at least two months significantly modified the clinical symptoms of the disease: namely, hyperactivity and hyperemotivity/aggressiveness were reduced, school attention was improved. In parallel, the Mg-B6 regimen led to a significant increase in Erc-Mg values. When the Mg-B6 treatment was stopped, clinical symptoms of the disease reappeared in few weeks together with a decrease in Erc-Mg values. This study brings additional information about the therapeutic role of a Mg-B6 regimen in children with ADHD symptoms.

Improvement of neurobehavioral disorders in children supplemented with magnesium-vitamin B6. II. Pervasive developmental disorder-autism.

Previous studies reported positive results with the use of Mg-vitamin B6 in autism. Despite these reports, this intervention remains controversial. In order to study relationships between changes in clinical symtoms and biological parameters, 33 children (mean age: 4 [1-10] years old) with clinical symptoms of pervasive developmental disorder or autism (PDD, as defined in DSM-IV) were followed for at least 6 months; another group of 36 children (same age) devoided of any known pathology was used as control. All PDD children received a magnesium-vit B6 (Mg-B6) regimen (6 mg/kg/d Mg and 0.6 mg/kg/d vit B6). Intraerythrocyte Mg2+ (Erc-Mg), serum Mg2+ (s-Mg) and blood ionized Ca2+ (i-Ca) were measured before and after treatment. Clinical symptoms of PDD were scored (0 to 4). In contrast to s-Mg or i-Ca, PDD children exhibited significantly lower Erc-Mg values than controls (2.17 +/- 0.4 versus 2.73 +/- 0.23 mmol/L; 16/33). The Mg-B6 regimen led to an increase in Erc-Mg values (2.42 +/- 0.41 (after) versus 2.17 +/- 0.4 mmol/l (before), 11/17) and this supplementation improved PDD symptoms in 23/33 children (p < 0.0001) with no adverse effects: social interactions (23/33), communication (24/33), stereotyped restricted behavior (18/33), and abnormal/delayed functioning (17/33); 15/33 children were improved in the first three groups of symptoms. When the Mg-B6 treatment was stopped, PDD symtoms reappeared in few weeks. A statistically significant relationship was found in Erc-Mg values from children before treatment and their mothers. In conclusion, this study suggests that the behavioral improvement observed with the combination vitamin B6-magnesium in PDD/autism is associated with concomitant modifications of Erc-Mg values.

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.

The COOH-terminal Src kinase Csk is a tumor antigen in human carcinoma.

The cytoplasmic tyrosine kinase cSrc is involved in the regulation of many important cellular functions including cell growth and transformation, and its activity is down-regulated by phosphorylation of the Tyr530 residue by the COOH-terminal Src tyrosine kinase, Csk. Because cSrc was previously found overexpressed, activated, and in some cases mutated in carcinoma, we investigated whether it could act as a tumor antigen. We show that whereas no autoantibodies were found against cSrc or its relative Fyn, up to 20% of patients with carcinoma had high-affinity autoantibodies against Csk. Immunity mainly resulted from a secondary response, as indicated by the presence of IgG1 in the sera. Antibodies were linked to the cancer because they were not detected in healthy subjects nor in patients with unrelated diseases, and their levels decreased in the sera of patients after surgical resection. Furthermore, they behaved as early markers of epithelial transformation because they were present in sera of patients with early-stage tumors and precancerous lesions such as colorectal polyps and in sera of patients that were scored negative for other cancer serological markers (CEA, CA15-3, CA19-9, p53 antibodies). Finally the presence of these antibodies was attributed, at least in part, to a substantial elevation of Csk protein levels in the corresponding tumors. However a strong increase in Src activity was also observed in these tissues, which suggested that Csk cannot regulate Src-like activity in carcinoma. Taken together, these data demonstrate that Csk acts as an autoantigen, and the detection of anti-Csk antibodies may have potential diagnostic usefulness in the early detection and postoperative follow-up of patients with carcinoma.

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.

ACE-like hydrolysis of gastrin analogs and CCK-8 by fundic mucosal cells of different species with release of the amidated C-terminal dipeptide.

Various gastrin analogues and CCK-8 (Asp-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2) are hydrolyzed in vitro by angiotensin-converting enzyme (ACE), the main and initial cleavage occurring at the Met-Asp (or Leu-Asp) bond, releasing the C-terminal dipeptide amide Asp-Phe-NH2. Tetragastrin analogues (e.g., Boc-Trp-Leu-Asp-Phe-NH2) are degraded by a vesicular membrane fraction from rat gastric mucosa, yielding the C-terminal dipeptide Asp-Phe-NH2. We report here on the degradation of gastrin analogues and CCK-8 by a gastric mucosal cell preparation containing specific gastrin receptors. We have shown that gastrin analogues were specifically degraded by gastric mucosal cells from different species (e.g., rabbit and dog) at 37 degrees C (pH 7.4), releasing the C-terminal dipeptide Asp-Phe-NH2, similarly to ACE. This cleavage was found to be temperature and pH sensitive, and was inhibited by metalloproteinase inhibitors and by captopril, strongly suggesting that this enzymatic system closely resembles ACE. We have also demonstrated that a close correlation seems to exist between the apparent affinity of the gastrin analogues for gastrin receptors on gastric mucosal cells, and their ability of being hydrolyzed by this cell preparation. Moreover, all gastrin analogues which have been demonstrated to act as gastrin antagonists remained unaffected in the incubation conditions.

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.

Peptide hormone-membrane interactions. Intervesicular transfer of lipophilic gastrin derivatives to artificial membranes and their bioactivities.

Incorporation of di-fatty acylglycerol moieties at the N-terminus of human little-gastrin-(2-17) leads to self-aggregation of the resulting lipo-gastrins into stable, most probably fluid vesicles. Net intervesicular transfer of the lipo-gastrins to phosphatidyl-choline model bilayers occurs at high rates whereby the chain length of the gastrin lipid moiety was found to affect the transfer rate more decisively than the nature of the acceptor vesicle. Similarly, the bioactivity of the lipo-gastrins is again affected by the nature of the lipid moiety suggesting differentiated interdigitation with the natural bilayer components and thus, different two-dimensional migration rates to the target receptors. Embedment of the lipo-gastrins in phosphatidylcholine bilayers at high lipid/gastrin ratios as mimicry of the cell membrane bound state does not result in onset of ordered structure, but leads to full exposure of the gastrin in essentially randomly coiled form at the water/lipid interface. This may result from the artificial N-terminal anchorage of the gastrin molecules to the bilayers, but also from the relatively tight packing of the phosphatidylcholine vesicles. Nevertheless, this observation might suggest that in the present case membrane-induced conformation and orientation may not represent a pre-requisite for the hormone receptor binding process. However, the results of this study clearly confirm even for the non-amphiphilic hormone gastrin a membrane-bound pathway for receptor recognition and occupancy.

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.

Calcium involvement in the muscarinic response of the gastric parietal cell.

The influence of extracellular Ca2+ on the mediation of carbachol stimulation in isolated rabbit gastric parietal cells was studied. Removing Ca2+ from extracellular medium caused a 42% decrease of the aminopyrine accumulation due to carbachol with the same EC50 value (approximately 5 microM). A short time depletion in extracellular calcium suppressed the carbachol-dependent Ca2+ influx without affecting Ca2+ release from internal stores (fura-2 measurements). Similarly, the production of inositol phosphates under cholinergic stimulation was reduced by 29%. A rapid increase in Ins(1,4,5)P3 was obtained 5 s after carbachol stimulation, and this increase was not changed in Ca2(+)-depleted medium. In contrast, a 20 min incubation with carbachol caused a 50% reduction in both basal and carbachol-stimulated inositol phosphate accumulations. In conclusion, phospholipase C activation, intracellular Ca2+ release and aminopyrine accumulation were sequentially observed following carbachol stimulation of the isolated gastric parietal cell and extracellular calcium contributed to sustain this acid secretory response.

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.

Muscarinic stimulation of inositol phosphate accumulation and acid secretion in gastric fundic mucosal cells.

The muscarinic agonist, carbachol (CCh), was shown to stimulate the production of inositol phosphates (IP) in isolated cells from rabbit fundic mucosa. This stimulatory effect was time- and dose-dependent: EC50 values for IP1, IP2 and IP3 accumulation were not statistically different. The mean value was 30 +/- 8 microM (n = 6). The corresponding maximal stimulation (% of basal value) observed after 20 min incubation in the presence of 100 microM CCh was 160 +/- 15%. CCh-induced IP accumulation was abolished by atropine (Ki = 0.32 +/- 0.18 nM (n = 3)). The CCh concentrations leading to half-maximal inhibition of N-[3H]methylscopolamine binding and half-maximal IP accumulation were similar. The half-maximal value for CCh-induced aminopyrine accumulation was 8-times lower. These results indicate that IP3-mediated mobilization of intracellular Ca2+ might be involved in CCh-induced acid secretion by parietal cells.

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 of human des-tryptophan-1,norleucine-12-minigastrin-II and its biological activities.

A human minigastrin-II analog was prepared by conventional methods in solution using N-benzyloxycarbonyltyrosine O-sulfate as starting material in the synthetic route. Upon removal of the acid-labile protecting groups and purification by preparative reversed-phase chromatography the sulfated gastrin peptide was obtained in satisfactory overall yields as homogeneous material. It was found to be about twice as active as the non-sulfated form in stimulating gastric acid secretion and to exhibit a 10-fold higher affinity to gastrin receptors of purified parietal cells.

Synthesis and biological activity of partially modified retro-inverso pseudopeptide derivatives of the C-terminal tetrapeptide of gastrin.

The effects of partial retro-inverso modifications of selected peptide bonds of the N-terminal tetrapeptide of gastrin have been studied. In some of the synthesized compounds, the phenylalanyl residue has been replaced by the (R,S)-2-benzylmalonyl, 3-phenylpropionyl, benzylcarbamoyl, or benzyloxycarbonyl moieties. All pseudopeptides showed affinity for the gastrin receptor, in vitro, with potencies varying from IC50 = 10(-7) to IC50 = 10(-4) M. These compounds exhibited little or no activity on acid secretion in the anesthetized rat but were able to antagonize the action of gastrin. Among the most potent were Boc-Trp-Leu-gAsp-CO-CH2CH2C6H5 (20) (ED50 = 0.15 microM/kg), Boc-Trp-Leu-gAsp-m(R,S)Phe-NH2 (3) (ED50 = 0.15 microM/kg), and Boc-Trp-gLeu-D-Asp-m(R,S)Phe-NH2 (7) (ED50 = 0.3 microM/kg).

Phenethyl ester derivative analogues of the C-terminal tetrapeptide of gastrin as potent gastrin antagonists.

A series of phenethyl ester derivative analogues of the C-terminal tetrapeptide of gastrin, in which the phenylalanyl residue has been replaced by a phenethyl group and the peptide bond between aspartic acid and phenylalanine by an ester bond, were synthesized. None of these derivatives were able to stimulate gastric acid secretion in the anesthetized rat, whereas they inhibited gastrin-induced acid secretion with ED50 values between 0.02 and 1.5 mg/kg. Among these derivatives, Boc-beta Ala-Trp-Leu-Asp phenethyl ester (9) and Boc-beta Ala-Trp-Leu-Asp p-fluorophenethyl ester (16) were very potent in inhibiting gastrin-induced acid secretion. From these studies, the significant role of the C-terminal dipeptide of gastrin was pointed out. More particularly, the functional role of the phenylalanine through the C-terminal carboxamide and its binding role through its aromatic ring were demonstrated.

Synthesis of gastrin antagonists, analogues of the C-terminal tetrapeptide of gastrin, by introduction of a beta-homo residue.

A series of analogues of Boc-Trp-Leu-Asp-Phe-NH2, a potent gastrin agonist, were synthesized by introducing a beta-homo residue in the sequence. These compounds were tested in vivo on acid secretion, in the anesthetized rat, and for their ability to inhibit binding of labeled gastrin to its receptors on gastric mucosal cells. These analogues behaved as gastrin antagonists. The most potent compounds in this series were Boc-Trp-Leu-beta-homo-Asp-NHCH2C6H5 (10) (IC50 = 1 microM, ED50 = 0.2 mg/kg), Boc-Trp-Leu-beta-homo-Asp-NHCH2CH2C6H5 (11) (IC50 = 0.75 microM, ED50 = 0.5 mg/kg), Boc-Trp-Leu-beta-homo-Asp-Phe-NH2 (12) (IC50 = 1.5 microM, ED50 = 0.1 mg/kg), and Boc-Trp-Leu-beta-homo-Asp-D-Phe-NH2 (13) (IC50 = 2 microM, ED50 = 0.1 mg/kg). We could demonstrate the importance of the region of the peptide bond between leucine and aspartic acid and of the structure of the C-terminal dipeptide Asp-Phe-NH2, for exhibiting biological activity on acid secretion.