Wojciech J. Rossowski

Adrenomedullin, amylin, calcitonin gene-related peptide and their fragments are potent inhibitors of gastric acid secretion in rats.

Adrenomedullin, amylin and calcitonin gene-related peptides (CGRP) share close sequence homology and have overlapping spectra of biological activities, particularly with respect to cardiovascular and gastrointestinal functions. Comparisons of the effects of these three peptides on gastric acid release have been made by i.v. infusions in conscious rats equipped with gastric fistulae. All peptides were extremely potent inhibitors of basal, pentagastrin- and 2-deoxy-D-glucose-stimulated gastric acid secretion with IC50 values in the subnanomolar to nanomolar range. These effects were not inhibited by C-terminal extra-cyclic fragments of the peptides which often act as competitive receptor antagonists in other biological systems. At high concentrations C-terminal fragments of human adrenomedullin and rat alpha-calcitonin gene-related peptide displayed some receptor agonist activity. Furthermore, the N-terminally situated disulfide-bridged ring fragments, human adrenomedullin-(15-22), rat amylin-(1-8) and rat alpha-calcitonin gene-related peptide-(1-8), retained significant gastric acid inhibitory potencies thus suggesting involvement of receptor(s) with significantly differing ligand binding profiles than those characterized previously.

Galanin binding sites in rat gastric and jejunal smooth muscle membrane preparations

Receptors for galanin in membranes from the rat gastric and jejunal smooth muscle were studied using [125I] radioiodinated synthetic porcine galanin. Specific binding was time and temperature dependent. At 32 degrees C radioligand was degraded in the presence of smooth muscle membranes in a time-dependent manner. At optimal experimental conditions, the equilibrium binding analyses showed the presence of a single population of high affinity binding sites in both the rat stomach and jejunum (Kd value of 2.77 +/- 0.78 nM and 4.93 +/- 1.74 nM for stomach and jejunal smooth muscle membranes, respectively). The concentration of the high affinity binding sites was 58.19 +/- 11.04 and 32.36 +/- 5.68 fmol/mg protein, for gastric and jejunal preparations, respectively. Specific binding was completely inhibited by 10(-6) M of nonradioactive galanin; was 75% blocked by 1 microM of galanin(9-29); it was 10% blocked by 1 microM of galanin(15-29). Galanin(1-15) at a concentration of 1 microM was ineffective for inhibiting [125I]galanin binding. Deletion of four C-terminal amino acid residues from galanin(9-29) to give galanin(9-25) also resulted in almost complete loss of affinity. Radioiodinated galanin and N-terminally deleted fragments had receptor binding potency in the following order: galanin(1-29) greater than galanin(9-29) greater than galanin(15-29). We conclude that the C-terminal part of the galanin chain is important for the rat gastric and jejunal smooth muscle membrane receptor recognition and binding and that N-terminal amino acid sequences are probably not so important, since galanin(1-15) was not active but galanin(9-29) retained most of the receptor binding activity.

Characterization of somatostatin receptor subtypes controlling rat gastric acid and pancreatic amylase release

An examination of the binding characteristics of a large number of somatostatin analogues with respect to the five known somatostatin receptor subtypes has recently resulted in the discovery of several peptides with some selectivity for types 2, 3, and 4 and little affinity for type 1 or 5 receptor. A panel of these peptides has thus far implicated type 2 receptors in the inhibition of release of pituitary growth hormone and type 4 receptors in inhibiting pancreatic insulin release. In the present article, we have examined the inhibitory effects of the same group of peptides on in vivo rat gastric acid and pancreatic amylase release and binding to rat pancreatic acinar cells. The type 2-selective ligand NC-8-12 was a potent inhibitor of gastric acid release (EC50s in the 1.5 nM region) whereas the type 4-selective ligand, DC-23-99, elicited little response. However, some involvement of type 3 receptors could not be ruled out because the type 3-selective analogue, DC-25-20, exhibited inhibitory effects at higher dose levels (EC50 > 10 nM). Conversely, the type 4 analogue was a potent inhibitor of amylase release (EC50 1.1 nM) whereas the type 3 analogue had no significant effects at doses tested. DC-23-99 also bound with high affinity to rat acinar cells (EC50 3.8 nM), whereas DC-25-20 exhibited more than 10-fold less affinity. Thus, these two major biological functions of somatostatin appear to be controlled by different receptors and, furthermore, effects on both endocrine and exocrine pancreas appear to be type 4 receptor mediated.

Pituitary adenylate cyclase-activating polypeptide relaxes rat gastrointestinal smooth muscle

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a new member of the secretin/glucagon peptides family, being most homologous to vasoactive intestinal peptide (VIP). The present study was designed to investigate a possible effect of PACAP on the rat gastrointestinal smooth muscle in vitro. We demonstrated that 1) PACAP reduced basal smooth-muscle contractions in all portions of the gastrointestinal tract, but the effect of VIP was region-specific. The inhibitory effect of PACAP in midcolon was approximately 100 times greater than that of VIP. 2) PACAP significantly inhibited smooth-muscle contractions induced by acetylcholine or carbachol. The inhibitory effect of PACAP was not affected by hexamethonium and was additive to the inhibitory effect of atropine and pirenzepine. 3) PACAP inhibited smooth-muscle contractions induced by substance P, cholecystokinin, and galanin, even after atropine treatment. Although the exact mechanism of the inhibitory action of PACAP remains to be clarified, PACAP appears to exert its effect in the rat at a site other than muscarinic receptors, probably through a direct effect on gastrointestinal smooth muscle in vitro.

Examination of somatostatin involvement in the inhibitory action of GIP, GLP-1, amylin and adrenomedullin on gastric acid release using a new SRIF antagonist analogue.

1 The effect of a new type 2 selective somatostatin (SRIF) receptor antagonist (DC‐41‐33) on somatostatin‐induced inhibition of pentagastrin‐stimulated gastric acid secretion in conscious, chronic gastric fistula equipped rats was studied. 2 Infused intravenously, DC‐41‐33 dose‐dependently inhibits SRIF‐induced inhibition of pentagastrin‐stimulated gastric acid secretion with an IC50 of 31.6±1.2 nmol kg−1 versus 10 nmol kg−1 SRIF and blocks the inhibitory effects of SRIF when simultaneously co‐infused. Its effectiveness provides additional evidence that SRIF‐inhibition of gastric acid release is a SRIF type 2 receptor‐mediated process. 3 DC‐41‐33 is able to completely reverse the inhibitory effect of glucose‐dependent insulinotropic polypeptides, GIP and GIP‐(1–30)NH2, and glucagon‐like polypeptide, GLP‐1(7‐36)NH2, on pentagastrin‐stimulated gastric acid secretion thus confirming that they exert these effects through stimulation of endogenous SRIF release. 4 DC‐41‐33 only partially blocks potent amylin and adrenomedullin‐induced inhibition of gastric acid secretion, therefore suggesting that somatostatin may not function as a primary mediator in the action of these peptides. 5 Our results indicate that DC‐41‐33, is a potent in vivo inhibitor of exogenous and endogenous SRIF in rats. It represents a new class of SRIF analogues which should eventually provide excellent tools for further evaluating the many physiological roles of SRIF and its five receptor subtypes.

Inhibitory action of galanin on gastric acid secretion in pentobarbital-anesthetized rats

The effects of galanin and two galanin fragments, GAL(9-29) and GAL(15-29), were studied for potential effects on pentagastrin- and bethanechol-stimulated gastric acid secretion in a pentobarbital-anesthetized rat experimental model. At a dose of 10 micrograms/kg/h, galanin potently inhibited pentagastrin-stimulated gastric acid secretion whereas inhibition of bethanechol-stimulated gastric acid secretion was not statistically significant. Simultaneous iv infusion of galanin and atropine did not affect the inhibitory action of the former. In similar experiments, a GAL(15-29) fragment was completely inactive whilst GAL(9-29) retained only about 5% potency. These results indicate that galanin probably induces its inhibitory effects by acting directly on the parietal cells rather than through a cholinergic pathway. They also demonstrate that the rat gastric acid inhibitory activity of galanin depends critically on the integrity of the first fourteen N-terminal amino acids.

Structural requirements at the N-terminus of urotensin II octapeptides

Urotensin II is the latest of a growing list of peptides exhibiting potent cardiovascular effects. It is an extremely potent vasoconstrictor in primates; its excretion is elevated in hypertensive patients thus suggesting therapeutic potential for urotensin II analogues, particularly receptor antagonists. In the present study, a number of interesting structural features pertaining to the N-terminus of urotensin II have been evaluated for binding to cloned human and rat urotensin II receptors and functional effects on rat upper thoracic aorta smooth muscle preparations. Shortened octapeptides retained full binding affinities and functional activities, did not require a free N-terminal amino group, and could tolerate an amidated C-terminus. The N-terminal Asp residue present in the octapeptides did not require a negatively charged side chain, merely one which contained a hydrogen bond acceptor CO group which could be present at a variety of positions on the side chain. The side chain could be constrained into a trans-olefinic configuration with full retention of potency, but potency was lost in the cis configuration. N-terminal aromatic amino substituted with polar groups such as OH and NO(2) also resulted in high affinity analogues. Overall, the correlation between binding affinities for the human and rat receptors was quite good. These findings could be of value in the development of more potent urotensin II receptor antagonists based on the previously identified somatostatin antagonist octapeptides which we have recently found, function as relatively weak urotensin II antagonists.

Galanin: structure-dependent effect on pancreatic amylase secretion and jejunal strip contraction

Rat and porcine galanin and their fragments inhibited cholecystokinin-8 (CCK-8)-stimulated amylase secretion with the following activities: rat galanin-(1-29) = porcine galanin-(1-29) = galanin-(1-15) = rat galanin-(3-29) > rat galanin-(2-29) = porcine galanin-(2-29) > galanin-(1-10). Fragments of rat galanin-(9-29) and N alpha-acetyl-galanin-(9-29) were able to inhibit CCK-8-stimulated pancreatic amylase secretion but only at higher dose levels. Porcine galanin-(15-29) and rat galanin-(21-29) were unable to produce significant inhibition. Rat and porcine galanin-(1-29), galanin-(1-15) and rat N alpha-acetyl-galanin-(9-29) also inhibited basal pancreatic amylase secretion. In the rat jejunal strip contraction model, rat galanin-(1-29) and porcine galanin-(1-29) have similar potencies. Galanin-(1-15) and galanin-(1-10) stimulate rat jejunal strip contraction with decreasing potencies. Elimination of Gly1 from the N-terminus of both rat and porcine galanin had no significant effect either on pancreatic amylase secretion or on jejunal strip contraction. The rat galanin-(3-29) and (9-29) are not active in the stimulation of rat jejunal strip contraction. Acetylation of porcine galanin-(9-29) created a peptide that was a powerful stimulator of rat jejunal strip contraction. The present data indicate that N-terminal rat galanin amino acid residues are crucial for rat jejunal strip contraction but are not required for inhibition of pancreatic amylase. These results suggest that the galanin amino acid sequence contains several specific domains, which can be recognized by specific galanin receptor subsets.

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.

Inhibitory effect of galanin on basal and pentagastrin-stimulated gastric acid secretion in rats

The dose-dependent effects of galanin on basal and pentagastrin-stimulated gastric acid secretion in pentobarbital-anesthetized rats were examined. Intravenous infusion of galanin at doses of 0.31, 0.62, 1.25, 1.87, 3.11, and 6.22 nmol/kg-1/h-1 into pentagastrin-stimulated rats produced a diminution in gastric acid secretion which was maximal (54.7%) at the level of the 1.87 nmol/kg-1/h-1 dose. Furthermore, the effect was biphasic, since both lower and higher doses of peptide were less effective. At the optimum concentration of 1.87 nmol/kg-1/h-1 galanin also inhibited basal gastric acid secretion. We conclude that endogenous galanin might be involved in the physiologic regulation of gastric acid secretion.