Cyclic hexa- and pentapeptide somatostatin analogues with reduced gastric inhibitory activity

The gastric inhibitory activity of cyclic hexa- and pentapeptide analogues of somatostatin was investigated in conscious cats with gastric fistulae. Gastric acid and pepsin secretions were stimulated by pentagastrin. Cyclo(Phe-Phe-D-Trp-Lys-Thr-Phe) showed no inhibition of acid secretion at molar doses up to 50-times the ID50 for somatostatin. This peptide inhibited pepsin secretion at the highest dose (50 micrograms kg-1 hr-1), and its potency is approximately 0.005 compared with somatostatin (1.0). Cyclo(Pro-Phe-D-Trp-Lys-Thr-Phe) inhibited acid (approximately 50%) and pepsin (approximately 85%) secretions, but the inhibition was not dose-related being similar with doses of 10 to 50 micrograms kg-1 hr-1. The cyclic pentapeptide, cyclo(7-aminoheptanoyl-Phe-D-Trp-Lys-Thr), was inactive in the dose range studied, with a potency less than 0.01. Cyclo[7-aminoheptanoyl-Phe-D-Trp-Lys-Thr(Bzl)] has been described as a somatostatin antagonist with respect to inhibition of growth hormone, insulin and glucagon release in rats [2]. Up to 60-fold molar excesses of this peptide failed to antagonise the inhibitory activity of somatostatin in the stomach. The results demonstrate that residues outside the central 6-11 region of somatostatin are very important for its gastric activity. The lack of gastric antagonistic activity of the pentapeptide antagonist indicates that these residues are likely to be involved in receptor recognition/binding.     

The primary structure of human urogastrone.

Urogastrone is a potent inhibitor of gastric acid secretion which is present in human urine. Its existence has been known for over 30 years but it has only recently been isolated in a sufficiently pure form for detailed structural studies to be undertaken. Two separate polypeptides beta- and gamma-urogastrone were isolated. The structures were established by carrying out enzymic degradations of S-carboxymethyl and S-carboxamidomethyl derivatives with trypsin, chymotrypsin, thermolysin and a protease derived from the fungus Armillaria mellea. Sequences of the smaller peptides thus obtained were determined by the dansyl Edman method. Partial acid hydrolysis of urogastrone itself gave fragments containing single intact disulphide bonds, and oxidation then allowed the direction of individual bonds to be established. Beta-Urogastrone was shown to be a 53-amino acid residue polypeptide containing three disulphide bonds, and gamma-urogastrone had an identical sequence but lacked the C-terminal arginine residue. Urogastrone was subsequently found to be structurally related to mouse epidermal growth factor in that 37 of the 53 residues were commonly located in each polypeptide. Furthermore, as both peptides has similar effects upon gastric acid secretion and upon epidermal growth, urogastrone was also a human epidermal growth factor. The 16 variable residues were spread across the molecule, all apart from two were compatible with single base changes in the triplet condons, and the overall effect was to make uorgastrone more acidic than EGF. The smallest biologically active unit has not been defined but at least six residues can be removed from the C-terminus without causing a reduction in potency.     

Reduced gastric acid inhibitory effect of a pGIP(1-30)NH2 fragment with potent pancreatic amylase inhibitory activity.

Gastric inhibitory polypeptide (GIP) strongly stimulates insulin secretion in the presence of glucose and also stimulates somatostatin release from gastric mucosa. It was reported recently that both stimulatory activities can be dissociated by removing the C-terminal 12 amino acid residues. Since insulin and somatostatin are involved in regulation of exocrine pancreatic and gastric secretion in rats, we compared the inhibitory effects of pGIP and the pGIP(1-30)NH2 fragment on pancreatic amylase and gastric acid secretion. pGIP(1-30)NH2 displayed full activity on inhibition of bombesin (BN)-stimulated amylase release relative to GIP itself, but was about 10-fold less potent in inhibiting gastric acid secretion. These results suggest that the receptors involved in these two events have quite different ligand binding requirements and that more specific analogues of GIP can be designed which should be of value in elucidating the physiological roles of this hormone.     

Prostaglandins may not mediate inhibition of gastric acid secretion by somatostatin in the rat

The role of prostaglandins as mediators of the inhibitory effect of somatostatin on gastric acid secretion has been evaluated in conscious and anesthetized rats. The effect of somatostatin on bethanechol-stimulated gastric acid secretion was determined with or without indomethacin pretreatment. Prostaglandin synthesis inhibition (less than 90%) by indomethacin was verified with PGE2-generation assay on gastric mucosal tissue. In both conscious and anesthetized rats somatostatin significantly inhibited the stimulated acid output in the control and indomethacin pretreated groups. The present findings do not support a role for prostaglandins in the inhibition of gastric acid secretion by somatostatin in the rat.     

Inhibition of gastric acid secretion in dogs by neurotensin

Neurotensin is a tridacapeptide which has been isolated from bovine hypothalamus. The action of synthetic neurotensin was studied on gastric acid secretion in dogs provided with gastric pouches. Intravenously infused neurotensin, 50 ng × kg^?1 × min^?1, was found to produce a considerable inhibition of pentagastrin stimulated gastric acid secretion. On the other hand, there was no sign of inhibition of histamine induced gastric acid secretion. The experiments show that neurotensin, isolated from the central nervous system is a potent gastric secretory inhibitor and that it has a selective action in inhibiting gastric acid responses to pentagastrin but not to histamine.     

Stereoisomeric relationships among anti-inflammatory activity, inhibition of platelet aggregation, and inhibition of prostaglandin synthetase

This study compares the affects of a new non-steroidal anti-inflammatory drug, d,1-6-chloro-alpha-methyl-carbazole-2-acetic acid, its enantiomers, and indomethacin on platelet aggregation, prostaglandin synthetase, adjuvant arthritis, gastric ulceration and arachidonic acid induced diarrhea. In the adjuvant arthritic rat, doses producing anti-inflammatory activity were similar for all compounds with the exception of the I-isomer which was much less active. On the other hand, indomethacin was 10 to 25 times more potent with regard to inhibition of platelet aggregation, inhibition of prostaglandin synthetase, inhibition of arachidonic acid induced diarrhea, and induction of gastric ulceration than the racemate and its isomers. Such divergence of potencies suggests that the racemate, unlike indomethacin, would have no affect on platelet aggregation and, hence, produce no prolongation of bleeding time at doses possessing anti-inflammatory activity. The data also suggest that the racemate and d-isomer have greater specificity toward anti-arthritic activity and are less ulcerogenic than indomethacin. The d-isomer apparently is the more active component of the racemate in all the systems tested since: (a) the d-isomer has 2 to 3 times the inhibitory potency of the racemate and (b) the I-isomer, at high dosages or high concentrations had considerably less affect. Comparison of potencies relative to inhibition of platelet aggregation and of prostaglandin synthetase, are quite close; therefore, mechanistically, the anti-aggregatory affects of these drugs, or lack thereof, may be related to inhibition of prostaglandin synthetase.     

Biochemical studies of toxic agents. The metabolism of 1- and 2-bromopropane in rats

1. (+)-n-Propylmercapturic acid sulphoxide, i.e. (+)-N-acetyl-S-n-propyl-l-cysteine S-oxide, was prepared as the dicyclohexylammonium salt, (-)-n-propyl-mercapturic acid sulphoxide was prepared as the free acid, and S-isopropyl-l-cysteine and isopropylmercapturic acid were also prepared. 2. The metabolism of 1- and 2-bromopropane was studied by radiochromatographic examination of the urine excreted by rats that had been fed with a diet containing (35)S-labelled yeast and then injected subcutaneously with these compounds. In addition to n-propyl-mercapturic acid and 2-hydroxypropylmercapturic acid, the excretion of which has already been reported, n-propylmercapturic acid sulphoxide was shown to be a metabolite of 1-bromopropane. Sulphur-containing metabolites of 2-bromopropane, if present in the urine at all, were there in very small amounts. 3. n-Propylmercapturic acid and isopropylmercapturic acid were isolated from the urine of rats that had been injected subcutaneously with S-n-propyl-l-cysteine and S-isopropyl-l-cysteine respectively.     

Peptide YY interacts with secretin and duodenal acidification to inhibit gastric acid secretion

Previous studies have indicated that plasma levels of peptide YY (PYY) increase significantly after a meal. The purpose of this study was to characterize the interaction of PYY and secretin in the inhibition of gastric acid secretion, and to determine whether PYY can influence acid-induced inhibition of gastric acid secretion in conscious dogs. I.v. administration of PYY at 200 pmol/kg/h inhibited pentagastrin (1 microgram/kg/h)-stimulated gastric acid output (P less than 0.05). PYY further augmented i.v. secretin-induced inhibition of pentagastrin-stimulated gastric acid output by 32 +/- 7%, and intraduodenal hydrochloric acid-induced inhibition of pentagastrin-stimulated gastric acid output by 40 +/- 12%. The mean integrated release of secretin response to duodenal acidification (3.9 +/- 1.0 ng-[0-60] min/ml) was not affected by PYY (3.3 +/- 0.9 ng-[0-60] min/ml). The present study demonstrates that PYY can interact with secretin and duodenal acidification in an additive fashion to inhibit pentagastrin-stimulated gastric acid secretion. Our results suggest that several hormones that are released postprandially can interact with each other to inhibit gastric acid secretion.     

Peripheral adrenomedullin inhibits gastric emptying through CGRP8-37 -sensitive receptors and prostaglandins pathways in rats

The effects of intravenous (iv) adrenomedullin (AM) on gastric emptying were investigated in conscious rats. AM induced a maximal 50% inhibition of gastric emptying at a dose of 1.2 nmol/kg. AM was about two-fold less potent than alpha-calcitonin gene-related peptide (alpha-CGRP), which induced a similar 50% maximal inhibition of gastric emptying at 0.6 nmol/kg. Delayed gastric emptying induced by i.v. AM and alpha-CGRP was prevented by peripheral injection of the selective CGRP1 antagonist, CGRP8-37, and by pretreatment with indomethacin, while not altered by blockade of the sympathetic nervous system with propranolol. These data indicate that peripheral AM inhibits gastric emptying through the interaction with CGRP8-37 -sensitive receptors, likely CGRP1 receptors, and the recruitment of prostaglandin-dependent mechanisms.     

Small doses of capsaicin given intragastrically inhibit gastric basal acid secretion in healthy human subjects.

Although the direct inhibitory effect of small dose of capsaicin on gastric secretory responses was proved in animal observations, the role of capsaicin-sensitive afferent nerves (CSAN) and the effect of capsaicin applied in small and high doses on gastric secretion in human has not been clarified yet. In this study we investigated the influence of different small doses (100-800 microg) of capsaicin given intragastrically through an orogastric tube on gastric basal secretory responses in 10 healthy human subjects. Gastric basal secretory responses (volume, H+-concentration, H+-output) were measured from the suctions of gastric juice for a 1-h period. It has been found that: a) capsaicin dose-dependently inhibited the volume and H+-output of gastric juice; b) ID50 was found to be about 400 microg for capsaicin on gastric acid secretion; c) the time interval for capsaicin-induced gastric inhibition existed for about 1 h indifferently from the higher dose (800 microg) of capsaicin given after. It has been concluded that the capsaicin (given in small doses) inhibits the gastric basal acid output via stimulation of the inhibition of capsaicin sensitive afferent nerves.     

Comparison of gastric and intestinal antisecretory and protective effects of prostacyclin and its stable thia-imino-analogue (Hoe 892) in conscious rats

The effects of prostacyclin (PGI2) and its stable thia-thimo-analogue (Hoe 892) on gastric and intestinal secretions and gastric mucosal lesions have been determined in conscious rats. Both PGI2 and Hoe 892 given subcutaneously (s.c.) reduced dose-dependent gastric acid secretion, the ID50 (dose producing 50% inhibition) being about 48.6 and 11.8 micrograms/kg, respectively. In contrast, intragastric (i.g.) PGI2 and Hoe 892 did not cause any change in gastric acid secretion at doses ranging from 1 to 100 micrograms/kg. Both PGI2 and Hoe 892 reduced significantly intestinal fluid secretion (antienteropooling activity). PGI2 and Hoe 892 given i.g. or s.c. reduced dose-dependent gastric ulcer formation induced by acidified aspirin (ASA), Hoe 892 being somewhat less potent than PGI2. Both PGI2 and Hoe 892 were equally effective against gastric mucosal necrosis induced by absolute ethanol and this effect was observed both after i.g. and s.c. administration of these agents. We conclude that stable thia-imino-PGI2 analogue, Hoe 892, has similar gastric and intestinal antisecretory and protective activity as PGI2 and may be useful in the prevention of gastric damage by various noxious agents.     

The effects of intravenously administered bombesin on pentagastrin-stimulated acid secretion in cats

The effects of bombesin (BBS) infusion or BBS injection on the plateau gastric secretion stimulated by pentagastrin (Pg) were compared in cats fitted with gastric fistula (GF) and Heidenhain pouch (HP). Injection of 81 pmol/kg of BBS inhibited Pg-stimulated acid secretion in both GF and HP by 47 +/- 5% and 37 +/- 5% (P less than 0.01), respectively. Infusion of 324 pmol/kg.h of BBS did not significantly modify acid secretion, but as soon as the infusion stopped, an inhibition appeared which lasted 1 h (37 +/- 5% in GF and 53 +/- 4% in HP P less than 0.01). The inhibition was reversed in GF by infusion of BBS 324 pmol/kg.h. In HP, reversion of inhibition required the addition in the Pg infusion of subthreshold dose of carbachol. We suggest that under non-steady state conditions (i.e. injection or after the end of the infusion) a concentration gradient of BBS is created which favors the response of D-cells over that of G-cells, whereas under steady-state conditions (i.e. during infusion) the effects of BBS on G- and D-cells are balanced. This finding argues for a physiological role of BBS in the regulation of gastric acid secretion.     

Enkephalinergic control of somatostatin secretion from the perfused rat stomach

A role for the enkephalins in the regulation of gastric somatostatin (SLI) secretion has been investigated in an isolated perfused rat stomach model. Both methionine- and leucine-enkephalins caused a dose-dependent inhibition of gastric inhibitory polypeptide (GIP) stimulated SLI secretion. Leu-enkephalin was one order of magnitude less potent than met-enkephalin: 50% inhibition by met-enkephalin was at 4 X 10(-9) M and with leu-enkephalin 3.5 X 10(-8) M. Naloxone (100 nM) had no effect on basal secretion but blocked the inhibitory action of met-enkephalin (1 nM or 1 microM). Vagal stimulation (7 V, 10 Hz, 5 ms) inhibited GIP-stimulated SLI release. Administration of naloxone partially reversed this inhibition, suggesting that endogenous opioids were at least partially responsible for vagally induced inhibition. A number of possible pathways by which endogenous enkephalins may modulate SLI release have been proposed.     

N-acetyl-GRP(20-26)-O-CH3 reverses intracisternal bombesin-induced inhibition of gastric acid secretion in rats

Intracisternal injection of 19 pmoles of bombesin in light-ether-anesthetized rats, five minutes after intracisternal vehicle, produced a 75% and 63% inhibition in gastric acid output and concentration, respectively, in 2-hour pylorus-ligated rats. Pretreatment of rats with the characterized peripheral bombesin antagonist N-acetyl-GRP(20-26)-O-CH3 (1 nmole) reversed the inhibitory effect of bombesin on gastric acid output and concentration. In contrast, the related bombesin antagonist N-acetyl-GRP-O-CH2-CH3 (1 nmole) was ineffective in this system. In urethane-anesthetized, acute gastric fistula rats infused with pentagastrin, intracisternal N-acetyl-GRP(20-26)-O-CH3 protected against the inhibition in gastric acid output produced by intracisternal bombesin (19 pmoles). Thus the recently characterized peripheral bombesin antagonist N-acetyl-GRP(20-26)-O-CH3 also appears to be effective in antagonizing central bombesin-induced inhibition in gastric acid secretion in two models. This represents a first report of a synthetic bombesin antagonist effective in reversing central bombesin-induced effects on gastric function.     

Stereoisomeric relationships among anti-inflammatory activity, inhibition of platelet aggregation, and inhibition of prostaglandin synthetase

This study compares the affects of a new non-steroidal anti-inflammatory drug, d,l-6-chloro-alpha-methyl-carbazole-2-acetic acid, its enantiomers, and indomethacin on platelet aggregation, prostaglandin synthetase, adjuvant arthritis, gastric ulceration and arachidonic acid induced diarrhea. In the adjuvant arthritic rat, doses producing anti-inflammatory activity were similar for all compounds with the exception of the l-isomer which was much less active. On the other hand, indomethacin was 10 to 25 times more potent with regard to inhibition of platelet aggregation, inhibition of prostaglandin synthetase, inhibition of arachidonic acid induced diarrhea, and induction of gastric ulceration than the racemate and its isomers. Such divergence of potencies suggests that the racemate, unlike indomethacin, would have no affect on platelet aggregation and, hence, produce no prolongation of bleeding time at doses possessing anti-inflammatory activity. The data also suggest that the racemate and d-isomer have greater specificity toward anti-arthritic activity and are less ulcerogenic than indomethacin. The d-isomer apparently is the more active component of the racemate in all the systems tested since: (a) the d-isomer has 2 to 3 times the inhibitory potency of the racemate and (b) the l-isomer, at high dosages or high concentrations had considerably less affect. Comparison of potencies relative to inhibition of platelet aggregation and of prostaglandin synthetase, are quite close; therefore, mechanistically, the anti-aggregatory affects of these drugs, or lack thereof, may be related to inhibition of prostaglandin synthetase.     

Cyclopamine inhibition of the sonic hedgehog pathway in the stomach requires concomitant acid inhibition

The Shh pathway has been implicated in gastric carcinogenesis, and inhibition of this pathway has been shown to inhibit tumour growth in gastric cell lines. Assessing the in vivo efficacy of Shh pathway antagonists in blocking Shh signaling in the stomach is important for clinical trial design, but has not been previously investigated. We investigated the in vivo efficacy of a Shh antagonist, cyclopamine, in correlation to the secondary effects induced by this treatment on gastrin levels and acid secretion. Gastrin has been shown to induce Shh production, processing and activity, which is believed to be mediated by acid secretion. We tested this hypothesis and showed that hypergastrinaemia induces Shh production in vivo, and confirmed that this effect on Shh is mediated by acid secretion. We showed that cyclopamine treatment induces both hypergastrinaemia and Shh, and does not inhibit Gli-1. Inhibition of the effect of hypergastrinaemia on the Shh pathway, in cyclopamine-treated mice, was demonstrated by use of lansoprazole which concomitantly inhibited Gli-1, and did not increase Shh production. Therefore, this evidence suggests that hypergastrinaemia, via increased acid secretion, may increase expression of Shh and that Shh antagonists may require concomitant acid inhibition to successfully inhibit a pathway known to be up-regulated in gastric carcinogenesis.     

Demonstration of glycoprotein antigens associated with stomach tumors

After precipitation of the glycoproteins from gastric tumor tissues by caprylic acid, immunochemical methods were applied to the research of specific gastric tumor antigens; this precipitation process by caprylic acid was less aggressive than the use of urea or proteases. The purification of a fraction associated with tumor tissue has been carried out by affinity chromatography with a specific antiserum immobilized on Sepharose. The obtained fraction contains 4 proteic antigens. One of them is common with all the gastric extracts. Neither CEA, nor alpha-foetoprotein has been detected in this fraction. The corresponding antiserum seems to show a tumor specificity, whereas tissue specificity has to be demonstrated.     

Lack of association between pro-inflammatory genotypes of the interleukin-1 (IL-1B -31 C/+ and IL-1RN *2/*2) and gastric cancer/duodenal ulcer in Korean population

IL-1beta is a pro-inflammatory cytokine with multiple biological effects and is a potent inhibitor of gastric acid secretion, and IL-1RN has been shown to be associated with enhanced IL-1beta production in vitro. Recently, it was reported that the pro-inflammatory genotypes, IL-1B -31 C/+ and IL-1RN *2/*2, were associated with an increased risk of gastric cancer in a Caucasian population. We tested the association between the polymorphisms and 190 gastric cancer, 117 duodenal ulcer, and 172 healthy subjects as controls in the Korean population. The allele frequency of IL-1B -31 C was more prevalent in Korean (51%) than in Caucasian (30%), while the frequency of IL-1RN *2 allele was less in Korean (6%) than in Caucasian (27%). Using the IL-1B TT genotype as a reference group, the CC genotype was not associated with an increased risk of gastric cancer or duodenal ulcer in the Korean population (odds ratios (OR)=0.90, 95% confidence interval (CI)=0.50-1.64; OR=0.72, 95% CI=0.36-1.46, respectively). Similarly, IL-1RN*2 was not a risk genotype for either gastric cancer or duodenal ulcer. No association was recognized on the haplotype analysis of the two genes, either. Our results did not support the previous report that IL-1B -31 C/IL-1RN*2 polymorphisms were associated with an increased risk of gastric cancer. The lack of association with duodenal ulcer also suggested that the polymorphisms were not directly related to the acid-secreting capability.     

The biological importance of each amino acid residue of the troponin I inhibitory sequence 104-115 in the interaction with troponin C and tropomyosin-actin

To systematically evaluate the contribution of each amino acid residue of the troponin I (TnI) inhibitory region (104-115), 14 synthetic analogs were synthesized by the solid-phase method. The analogs consisted of either single glycine or multiglycine replacements. The importance of the substituted amino acid(s) was determined from the extent of inhibition of the acto-S1 ATPase activity and the strength of binding to a troponin C (TnC) high pressure liquid chromatography affinity column of each synthetic analog. Every residue of the TnI sequence (104-115) is necessary to achieve maximum inhibition of the ATPase activity. However, the analogs quantitatively differed in the amount of inhibition induced. The TnI analogs bound less tightly to the TnC affinity column than the native synthetic peptide indicating that all residues in the TnI sequence contribute to the binding of TnC in the presence of Mg2+ or Ca2+. In the presence of Ca2+, there is a definite increase in the strength of the interaction between most analogs and TnC. This is accompanied with a shift toward a more specific interaction with the C terminus of the TnI inhibitory sequence.     

Effects of the synthetic PGE1 derivative misoprostol on basal, stimulated and nocturnal acid secretion in duodenal ulcer cases. Mechanism of action

We studied the antisecretory effect of the synthetic PGE1 derivative misoprostol. Basal, histamine-stimulated and nocturnal acid secretion were determined on separate days in 3 groups of endoscopically confirmed duodenal ulcer patients. Misoprostol or placebo were given in random order in doses of 200, 400 and 800 mcg. H+ concentration, acid output and pH of gastric content were determined. Additionally, the effect of misoprostol on purified and gastric mucosa carbonic anhydrase was determined according to Maren's micromethod. Misoprostol reduced dose-dependently basal acid secretion (by 54% after 200 mcg, by 82% after 400 mcg and, respectively, by 94% after 800 mcg) as well as stimulated secretion which was reduced after the same doses by 22% (p less than 0.05), 48% and 64% (p less than 0.001), respectively. Nocturnal secretion was significantly reduced for a period of 4 h, with a consecutive rise of gastric pH. In vitro, misoprostol inhibited dose-dependently purified and gastric mucosa carbonic anhydrase basal activity. In conclusion, misoprostol inhibits basal, nocturnal and histamine-stimulated secretion in duodenal ulcer patients, an effect which could be mediated by inhibition of carbonic anhydrase.