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Context: The global prevalence of diabetes mellitus, and in particular type 2 diabetes mellitus is increasing at an alarming rate. Risk factors for development of diabetes include obesity and advancing age., Objectives: The distribution of insulin, glucagon, somatostatin and pancreatic polypeptide in the pancreatic islets has been investigated in young and elderly type 2 Zucker diabetic fatty (ZDF) rats and age-matched Zucker lean (ZL) controls., Methods: Experiments were performed in male animals aged either 9-13 weeks or 30-34 weeks. Immunohistochemistry was used to label insulin, glucagon, somatostatin and pancreatic polypeptide in islet cells., Results: The percentage of insulin-positive cells was unaltered in young but decreased in elderly ZDF (35.5 ± 2.5%) rats compared to ZL controls (57.9 ± 1.8%). The percentage of glucagon-positive cells was increased in young ZDF (58.7 ± 3.4%) compared to ZL controls (23.4 ± 2.1%). However, in elderly rats the percentage of glucagon-positive cells declined in ZDF rats and was no longer different from ZL controls. The percentage of somatostatin-positive cells was unaltered in young and elderly ZDF rats compared to ZL controls. The percentage of pancreatic polypeptide-positive cells was unaltered in young but increased in elderly ZDF (22.0 ± 2.5%) rats compared to ZL controls (13.8 ± 1.8%)., Conclusions: The distribution of pancreatic hormones is altered to varying extents in the ZDF rat and during the normal aging process.

Peptide libraries have proven to be useful in applications such as substrate profiling, drug candidate screening and identifying protein-protein interaction partners. However, issues of fidelity, peptide length, and purity have been encountered when peptide libraries are chemically synthesized. Biochemically produced libraries, on the other hand, circumvent many of these issues due to the fidelity of the protein synthesis machinery. Using thioredoxin as an expression partner, a stably folded peptide scaffold (avian pancreatic polypeptide) and a compatible cleavage site for human rhinovirus 3C protease, we report a method that allows robust expression of a genetically encoded peptide library, which yields peptides of high purity. In addition, we report the use of methodological synchronization, an experimental design created for the production of a library, from initial cloning to peptide characterization, within a 5-week period of time. Total peptide yields ranged from 0.8% to 16%, which corresponds to 2-70 mg of pure peptide. Additionally, no correlation was observed between the ability to be expressed or overall yield of peptide-fusions and the intrinsic chemical characteristics of the peptides, indicating that this system can be used for a wide variety of peptide sequences with a range of chemical characteristics.

Short-term regulation of food intake controls what, when and how much we eat within a single day or a meal. This regulation results from an integrated response to neural and humoral signals that originate from the brain, gastrointestinal (GI) tract and adipose tissue. In the GI tract, multiple sites including the stomach, duodenum, distal small intestine, colon, and pancreas are involved in this process. Ingested food evokes satiety by mechanical stimulation and by release of peptides in the GI tract. The intestine in particular plays a key role in satiety through various peptides secreted in response to food. Many of the intestinal peptides inhibit also gastric emptying thus enhancing gastric mechanoreceptor stimulation. In this review, the current knowledge about the effects of different macronutrients and fibre on the release of GI satiety-related peptides in humans is discussed.

Normal circadian variations in vasoactive intestinal polypeptide, somatostatin, cholecystokinin and pancreatic polypeptide were measured to determine if these alter with aging and to identify gastrointestinal regulatory hormones that might control the dramatic diurnal variation in the gastric cytoprotective trefoil protein TFF2. Plasma pancreatic polypeptide concentrations showed a marked diurnal rhythm (p < 0.0001). Basal and postprandial pancreatic polypeptide concentrations increased with age (p < 0.01). The timing of the diurnal rhythm was consistent with pancreatic polypeptide inhibiting TFF2 secretion and there was a negative association between pancreatic polypeptide and TFF2 concentrations (p < 0.002). The much higher pancreatic polypeptide concentrations in older people will induce increased satiety that may contribute to 'anorexia of ageing'. These results identify potential therapies for treatment of gastric mucosal morbidity and age-associated loss of appetite.

Potent and specific inhibitors of protein.protein interactions have potential both as therapeutic compounds and biological tools, yet discovery of such molecules remains a challenge. Our laboratory has recently described a strategy, called protein grafting, for the identification of miniature proteins that bind protein surfaces with high affinity and specificity and inhibit the formation of protein.protein complexes. In protein grafting, those residues that comprise a functional alpha-helical binding epitope are stabilized on the solvent-exposed alpha-helical face of the small yet stable protein avian pancreatic polypeptide (aPP). Here we use protein grafting in combination with molecular evolution by phage display to identify phosphorylated peptide ligands that recognize the shallow surface of CBP KIX with high nanomolar to low micromolar affinity. Furthermore, we show that grafting of the CBP KIX-binding epitope of CREB KID onto the aPP scaffold yields molecules capable of high affinity recognition of CBP KIX even in the absence of phosphorylation. Importantly, both classes of designed ligands exhibit high specificity for the target CBP KIX domain over carbonic anhydrase and calmodulin, two unrelated proteins that bind hydrophobic or alpha-helical molecules that might be encountered in vivo.

The purpose of this study was to compare the effects of pancreastatin (PST) (400 pmol/kg/hr) and somatostatin (SRIF) (400 pmol/kg/hr) on food-induced release of gastrin, pancreatic polypeptide (PP), and peptide YY (PYY) in conscious dogs. The present findings indicate that SRIF is more potent than PST on the inhibition of food-induced release of PP; that SRIF and PST do not influence food-induced release of gastrin; and that PST cannot inhibit food-induced release of PYY, whereas SRIF inhibits PYY release in a potent fashion.

Xenin is a 25 amino acid peptide detected in the gastric mucosa of various mammals. It has since been found in low concentrations in other tissues. Xenin plasma concentrations increase after meals. The present study reports some gastroenteropancreatic effects of this peptide in the dog. Intravenous infusion of 64 pmol/kg min synthetic xenin significantly inhibited pentagastrin-stimulated gastric acid secretion and stimulated exocrine pancreatic secretion of volume and protein. Further, intravenous infusion of xenin in a dose of 1.0 pmol/kg min stimulated jejunal motility in the anaesthetized dog. An intravenous infusion of 32 pmol/kg min xenin raised plasma concentrations of pancreatic polypeptide, vasoactive intestinal polypeptide, insulin and glucagon. The present experiments therefore indicate manifold bioactive properties of intravenously infused xenin in the dog, with jejunal motility the most sensitive target. Conclusions as to the physiological role of xenin cannot be drawn from the present experiments. The release of other hormonal peptides indicates a complex action of xenin.

Hepatic porphyrias are characterized by neurological symptoms manifested by abdominal pain, neuropathies and mental aberrations. Porphyrins are ubiquitous and essential biochemical constituents of living beings acting as mediators of oxidation reaction in the metabolism of the steroid, drugs, environmental chemicals or as a mean of exchanging gases, such as oxygen and carbon dioxide between the environment and the tissue of the body using endogenous polypeptide properties. The different porphyrins arising from the arrangement of normal heme synthesis are characterized by an accumulation and excretion of specific intermediate porphyrins and/or of precursors exerting toxic effect, initiating cascades of generations of polypeptides, neurotransmitters and gut-brain axis peptide responsible for the symptoms of clinical status. We studied polypeptide levels in 27 patients (19 females, 8 males) presenting acute attack of hepatic porphyria: 2 with ALA dehydratase-deficient porphyria; 9 with acute intermittent porphyria; 12 with porphyria cutanea tarda and 4 with variegate porphyria. During acute attacks of porphyria, polypeptides were found to be constantly increased: vasoactive intestinal polypeptide (VIP); neurotensin (NT); substance P; pancreatic polypeptide; gastrin-releasing peptide; gastrin and motilin. Administration of the somatostatin (antagonizing polypeptide), which was undetectable or low before treatment, apparently alleviated the acute symptomatology. Elevated levels of polypeptides, at least partly, contribute to appearance of acute symptoms in porphyria patients.

Traditionally, neuropeptide Y (NPY) receptors have been divided into Y1 and Y2 subtypes based on peptide pharmacology and synaptic localization. Other receptor subtypes have been proposed based on preferences for NPY, peptide YY (PYY), or pancreatic polypeptide (PP). Recently, we discovered a novel human member of this receptor family exhibiting high affinity for PP and PYY. In the current study, we expressed a DNA clone encoding this human PP-preferring receptor [hPP1 (or Y4)] in Chinese hamster ovary cells and performed a peptide structure-activity study. [125I]pPYY bound to homogenates of hPP1-Chinese hamster ovary cells with a Kd of 0.064 +/- 0.006 nM and a Bmax of 244 +/- 12 fmol/mg protein. Human PP inhibited binding with a Ki of 0.023 nM, whereas human PYY (Ki = 0.31 nM) and human NPY Ki = 12 nM) were significantly less potent. Rat, porcine, and bovine PP inhibited binding with similar affinities to human PP, whereas avian PP was substantially less potent (Ki = 1 nM). Deletion of the first four amino acids reduced the affinity of bovine PP to 1 nM. Carboxyl-terminal fragments of NPY and PYY also had reduced potency compared with the native peptides. In addition, deletion of Tyr36-amide produced a substantial reduction in affinity. Pro34-substituted NPY and PYY had modestly increased affinity compared with the native peptides, although Gln34-bPP had similar affinity compared with bovine PP. The carboxyl-terminally derived Y1 antagonist 1229U91 was a very potent (Ki = 0.042 nM) inhibitor of binding to hPP1. Thus, the carboxyl-terminal region of PP seems to be the most important part of the peptide for high affinity binding to hPP1. A few key residues (amino acids 2 and 3) in the amino-terminal region of PP contribute to the high affinity of the native peptide. Thus, features required for peptide recognition by the hPP1 receptor seem to be distinct from the Y1 and Y2 receptor.

The neuropeptide Y family of peptides consists of neuropeptide Y (NPY), which is expressed in the central and peripheral nervous systems, and peptide YY (PYY) and pancreatic polypeptide (PP) which are gut endocrine peptides. All three peptides are 36 amino acids long and act on G-protein-coupled receptors. NPY and PYY are present in all vertebrates, whereas PP probably arose as a copy of PYY in an early tetrapod ancestor. NPY is one of the most conserved peptides during evolution and no gnathostome (jawed) species differs from the ancestral gnathostome sequence at more than five positions. PYY is more variable, particularly in mammals which have nine differences to the gnathostome ancestor. PP may be the most rapidly evolving neuroendocrine peptide among tetrapods with only 50% identity between mammals, birds, and amphibians. Ancestral gnathostome NPY and PYY seem to have differed at only four positions, suggesting that the gene duplication occurred shortly before the appearance of the gnathostomes. The two peptides differ from one another at 9-12 positions in tetrapod species and share at least two receptor subtypes in mammals. In bony and cartilaginous fishes, NPY and PYY have only 5-6 differences which, together with more extensive neuronal localization of PYY, indicate an even greater functional overlap between the two peptides in these animal groups. The emergence of sequence information for several receptor subtypes from various species will shed additional light on the evolution of the functions of the NPY-family peptides.

Neuropeptide Y (NPY), peptide YY (PYY), and pancreatic polypeptide (PP) are structurally related peptides found in all higher vertebrates. NPY is expressed exclusively in neurons, whereas PYY and PP are produced primarily in gut endocrine cells. Several receptor subtypes have been identified pharmacologically, but only the NPY/PYY receptor of subtype Y1 has been cloned. This is a heptahelix receptor that couples to G proteins. We utilized Y1 sequence information from several species to clone a novel human receptor with 43% amino acid sequence identity to human Y1 and 53% identity in the transmembrane regions. The novel receptor displays a pharmacological profile that distinguishes it from all previously described NPY family receptors. It binds PP with an affinity (Ki) of 13.8 pM, PYY with 1.44 nM, and NPY with 9.9 nM. Because these data may identify the receptor as primarily a PP receptor, we have named it PP1. In stably transfected Chinese hamster ovary cells the PP1 receptor inhibits forskolin-stimulated cAMP synthesis. Northern hybridization detected mRNA in colon, small intestine, pancreas, and prostate. As all three peptides are present in the gut through either endocrine release or innervation, all three peptides may be physiological ligands to the novel NPY family receptor PP1.

1. Confluent epithelial layers of a human adenocarcinoma cell line called Colony-6 have been shown to respond to nanomolar concentrations of vasoactive intestinal polypeptide (VIP), peptide YY (PYY), neuropeptide Y (NPY) and somatostatin (Som). 2. The VIP-induced increase in basal short-circuit current (SCC) was attenuated by basolateral application of Som, PYY or NPY, and also by the Y1-receptor agonist [Leu31,Pro34]NPY, as well as pancreatic polypeptide (PP). High concentrations (0.1-3.0 microM) of NPY(2-36) were effective but the C-terminal fragment NPY(13-36) (0.1-1.0 microM) and desamidoNPY (0.6 microM) were not active. A rank order of agonist EC50 values was: PYY > NPY > [Leu31,Pro34]NPY > PP > NPY(2-36) >> NPY (13-36). 3. Receptors for all these peptides were preferentially located within the basolateral domain. Apical addition of PP (1 microM) and Som (100 nM) had no effect upon basal SCC while apical VIP (10 nM) responses were 18%, and apical PYY (100 nM) were 27% the size of respective basolateral controls (100%). 4. Cross-desensitization was observed between [Leu31,Pro34]NPY (1 microM) and both PYY (100 nM) and PP (1 microM) and between PYY and NPY(2-36) (1 microM), but was not significant between PYY (100 nM) and PP (1 microM). We suggest that either these cells express a single new Y-receptor with an unusual phenotype or that two Y-receptor populations exist in Colony-6 cells.

The purpose of these experiments was to investigate the neural control of peptide YY (PYY) secretion. The effects of various pharmacological manipulations and vagotomy on peptide YY (PYY) secretion was examined in dogs. Atropine, hexamethonium and atropine plus hexamethonium treatment blocked food-induced release of PYY significantly. Integrated release of PYY in response to food alone and in combination with atropine, hexamethonium and atropine plus hexamethonium were 8.8 +/- 2.2, -1.1 +/- 2.3, -2.7 +/- 2.2 and -3.2 +/- 3.1 (ng (0-150) min/ml), respectively. beta-Adrenergic blockade with propranolol or depletion of nerve terminal stores of catecholamines with reserpine did not affect food-stimulated release of PYY. Truncal vagotomy resulted in significant elevations of basal and food-induced release of PYY. IV administration of bethanechol, a cholinergic agonist, and electrical stimulation of the vagus nerve resulted in release of PYY. Together, these data suggest that food-stimulated PYY secretion is dependent on ganglionic transmission and an atropine-blockable postganglionic parasympathetic pathway; and that PYY release is inhibited tonically, probably through a vagal cholinergic mechanism. Adrenergic pathways do not participate in food-stimulated PYY release; however, electrical stimulation of the splanchnic nerves increased basal levels of PYY, suggesting that the sympathetic nervous system affects release of PYY.

The coexistence of immunoreactivities to cholecystokinin, glucagon, glucagon-like peptide 1, salmon pancreatic polypeptide, neuropeptide tyrosine, and peptide tyrosine tyrosine was studied immunocytochemically, revealing for the first time in fish intestine the existence in the same cell of immunoreactivities to cholecystokinin-glucagon/glucagon-like peptide 1, cholecystokinin-salmon pancreatic polypeptide, glucagon/glucagon-like peptide 1-salmon pancreatic polypeptide, glucagon/glucagon-like peptide 1-neuropeptide tyrosine, salmon pancreatic polypeptide tyrosine tyrosine, and glucagon/glucagon-like peptide 1-peptide tyrosine tyrosine. Colocalization of cholecystokinin-salmon pancreatic polypeptide was observed only in the pyloric caeca of the rainbow trout Oncorhynchus mykiss, while the other colocalizations also occurred in proximal and middle intestinal segments. In all cases, endocrine cells immunoreactive to only one of the paired antisera were detected except for anti-glucagon and anti-glucagon-like peptide 1, which always immunostained the same cells.

Peptide YY (PYY), a 36 amino acid peptide, is a member of the structurally and functionally related pancreatic polypeptide (PP) family of gastrointestinal and neurally active peptides. Peptide YY is released postprandially from the distal small intestine and colon and has been shown to inhibit many physiologic actions of cholecystokinin (CCK), an integral foregut hormonal stimulant of pancreatic and gastric secretion. The specific signals for the release of PYY have not been ascertained, although foregut signals, both neural and hormonal, are likely. In this study, we evaluated the possible role for CCK in postprandial PYY release in eight conscious dogs. Conscious dogs were given a fat meal or a one hour intravenous infusion of CCK-8. On separate days, the dogs were pretreated with the specific CCK receptor antagonist L-364,718. Peripheral blood samples were collected for radioimmunoassay for PYY, PP and neuropeptide Y. The fat meal and exogenous CCK stimulated PYY and PP release, effects that were abolished by pretreatment with the CCK receptor antagonist. The results provide support for a physiologic role of CCK in the mediation of postprandial PYY and PP release. Furthermore, an inhibitory feedback loop is suggested between the hindgut (PYY) and the foregut (CCK).

The objectives of this study were to characterize the distribution of immunoreactive peptide-YY (PYY) in the dog pancreas and to examine whether vagal stimulation can release PYY from the pancreas. Levels of pancreatic polypeptide (PP) were also measured. PYY levels in extracts of the right lobe (0.43 +/- 0.03 ng/mg tissue) and head (0.35 +/- 0.12 ng/mg tissue) of the pancreas were approximately 10- to 20-fold higher than those in extracts of the stomach (0.03 +/- 0.01 ng/mg tissue; P less than 0.05) and left lobe of the pancreas (0.02 +/- 0.01 ng/mg tissue). However, PYY levels in extracts of the pancreas and stomach were significantly lower than PYY levels in extracts of the colonic mucosa (7.63 +/- 0.71 ng/mg tissue). PP levels were significantly (300-fold) higher in pancreatic extracts than in stomach and colonic extracts. Immunoreactive PYY and PP in pancreatic and colonic mucosal extracts coeluted with synthetic PYY and PP standards on HPLC. Electrical vagal stimulation of dogs resulted in a significant (P less than 0.05) release of PYY and PP, which was abolished by atropine treatment (2 mg/kg, iv). PYY levels in the pancreatic veins increased more quickly than in the peripheral veins. The integrated levels of PYY in the pancreatic veins [1.63 +/- 0.30 ng (0-30 min)/ml] were significantly (P less than 0.05) higher than those in peripheral veins [0.86 +/- 0.16 ng (0-30 min)/ml], but lower than those in colonic veins [3.14 +/- 0.22 ng (0-30 min)/ml]. Our results indicate that PYY is primarily produced in the colon; however, the pancreas contains a measurable amount of PYY, which is mainly distributed in the right lobe and head of the pancreas. In addition, PYY can be released from the pancreas in response to vagal stimulation. These data suggest that pancreatic PYY may participate in the regulation of pancreatic and gastrointestinal functions through endocrine and paracrine actions.

Gastric acidity is influenced by systemic and local peptide effects. Previous work by others has shown that intraluminally secreted peptides may have a role in local control of gastric acidity; however, the response of these peptides to acute changes in gastric pH is unknown. To determine the effects of acute changes in pH on systemic and intraluminal peptide levels, 14 normal volunteers underwent placement of a nasogastric tube after an overnight fast. Blood and gastric fluid were analyzed on a control day, 2 hours after completion of 24 hours of aluminum-magnesium antacid therapy and after 24 hours of H2 blockade. Plasma and acid-alcohol-extracted gastric peptide levels were measured with specific radioimmunoassays. Specimens were subdivided into two groups: 28 gastric fluid specimens with a pH less than 4 and 10 specimens with a pH greater than 4. In the patients with a pH greater than 4, the luminal peptides, motilin, neurotensin, pancreatic polypeptide, somatostatin, substance P, and gastrin, were decreased by 50% to 90% and gastrin-releasing peptide was decreased by 36% compared with specimens with a pH less than 4. Conversely, intraluminal vasoactive intestinal polypeptide and calcitonin levels were elevated by 60% and 27%, respectively, in the samples with a pH greater than 4. Intraluminal peptide concentrations are responsive to changes in intragastric pH; however, this response was not seen in plasma peptide levels.

Peptide YY (PYY) and pancreatic polypeptide (PP) are related hormones released systemically after a meal. The effects of glucose stimulation and vagal involvement on circulating and ileal luminal PYY and PP concentrations were evaluated in awake dogs. An oral glucose tolerance test (OGTT), intravenous glucose tolerance test (IVGT), 2-deoxyglucose bolus (2-DG), or atropine pretreatment prior to OGTT were administered to awake dogs with 25-cm ileal Thiry-Vella fistulas. Circulating and ileal intraluminal PYY and PP levels were measured by radioimmunoassay. No changes were noted in circulating PYY, and circulating PP increased (p less than 0.05) only after administration of 2-DG. Ileal luminal PP recovery was minimal (less than 60 pg/mL) and was unchanged after all tests. Ileal luminal PYY recovery increased significantly after both OGTT and IVGT. Pretreatment with atropine abolished the luminal PYY response to OGTT, and 2-DG did not affect luminal PYY recovery. Blood glucose and insulin levels were similar in all groups. Peripheral cholinergic control of luminal PYY release is suggested by our findings, whereas a central mediation of circulatory PP release is supported by 2-DG stimulation.

By affinity cross-linking and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, we identified a novel cell surface receptor on intact rat cells, which bound, with similar dissociation constants, pancreatic polypeptide (PP), neuropeptide Y (NPY) and peptide YY (PYY), the members of the PP family. The receptor was detected on pancreatic islet and acinar cells, hepatocytes and epithelial cells of the stomach, duodenum and small intestine. Its molecular weight was estimated to be 65,000, and the cross-linking of [125I] labeled ligands was inhibited by an excess of unlabeled PP, NPY or PYY. The results suggest that the 65-kDa molecule is a common receptor for PP family peptides.

The release of insulin, glucagon, somatostatin and pancreatic polypeptide (PP) by isolated mouse pancreatic islets was determined during 30-min incubations at 5.6 and 16.7 mmol glucose/l in the absence and presence of gastric inhibitory polypeptide (GIP), vasoactive intestinal polypeptide (VIP) and peptide histidine isoleucine (PHI) at concentrations of 1-1000 nmol/l. Insulin release was enhanced (greater than 50%) by GIP (100-1000 nmol/l) and VIP (1 mumol/l) at 5.6 mmol glucose/l, but not at 16.7 mmol glucose/l. Glucagon release was increased by GIP (100-1000 nmol/l), and by VIP and PHI (1-1000 nmol/l) at both glucose concentrations in a dose-related manner (maximum increases greater than tenfold). Somatostatin release was similarly increased by GIP (10-1000 nmol/l) at both glucose concentrations. Only the highest concentration (1 mumol/l) of PHI tested increased somatostatin release (twofold) at 5.6 mmol glucose/l, whereas PHI and VIP (1-1000 nmol/l) reduced (greater than 37%) somatostatin release at 16.7 mmol glucose/l. PP release was increased (49-58%) by 100-1000 nmol GIP/l, but was not significantly altered by VIP, and was reduced (39-56%) by PHI. The results indicate that GIP, VIP and PHI each stimulate glucagon release in a dose-related manner, but they exert discretely different effects on other islet hormones depending upon the dose and the prevailing glucose concentration.

Four dogs with chronic gastric fistulas were give intravenous bombesin nonapeptide (B9), ranatensin, and litorin by constant infusion for 90 min at 1.2 micrograms x kg-1 on separate days. A dose response study with substance P (1.5, 3.0, 60, 18 and 54 micrograms x kg-1 x h-1) was also carried out and all tests compared to a standard protein meal (10g x kg-1). Plasma gastrin and PP were measured by radioimmunoassay and gastric acid by autobiuret titration. Substance P failed to stimulate gastric acid secretion or release either pancreatic polypeptide (PP) or gastrin. Basal gastrin levels were 8 +/-2 fmol/ml. The peak increment of gastrin released by bombesin was 95 +/- 16, ranatensin 22 +/- 6, litorin 18 +/- 4, and meal 39 +/- 5 fmol/ml. Bombesin caused significantly greater release of gastrin than a meal, litorin or ranatensin (P less than 0.01). Basal gastric secretion was 23 +/- 4 microequiv./min. B9 produced a peak acid secretion of 356 +/- 124 muequiv./min. There was no significant difference between the bombesin-like peptides (P less than 0.01). Basal plasma PP was 38 +/- 12 fmol/ml. B9 produced a peak PP increment of 600 +/- 50, litorin 137 +/- 36, ranatensin 98 +/- 11, and a meal 305 +/- 58 fmol/ml. B9 released significantly more PP than either litorin of ranatensin (P less than 0.01). The different amino acid sequences of the peptides are probably responsible for their potency. The substitution of a penultimate phenylalanine residue in litorin and ranatensin for leucine in bombesin does not prevent PP or gastrin release by bombesin-like peptides. Since bombesin-like peptides are widely distributed in the gastrointestinal tract of man and stimulate both acid and gut hormone secretion, it is possible that they might play a physiological role in the modulation of gastrointestinal function.

The short-term effects of truncal vagotomy and antrectomy on bombesin-stimulated pancreatic secretion and release of gastrin and pancreatic polypeptide (PP) were studied in 18 anesthetized dogs. Together with an intravenous infusion of secretin (250 ng/kg/hr) bombesin (500 ng/kg/hr) was given before and after truncal vagotomy, antrectomy, and sham operation (N = 6 dogs per group). Peak incremental pancreatic protein output in response to bombesin was about 2-3 times higher before than after the different surgical procedures (tachyphylaxis). Neither truncal vagotomy nor antrectomy significantly altered the pancreatic protein response to bombesin when compared with sham operation. Bombesin produced a mean 1-hr increase over basal of 196 pM for gastrin, which was abolished by antrectomy but not appreciably affected by truncal vagotomy and sham operation. The mean 1-hr increment (207 pM) for PP in response to bombesin was not changed by truncal vagotomy, antrectomy, and sham operation. This study shows in the anesthetized dog that exogenous bombesin stimulates release of PP as well as gastrin; that the release of gastrin by bombesin is not vagally dependent; that neither truncal vagotomy nor antrectomy alter the release of PP by bombesin; and that the action of bombesin on pancreatic protein secretion does not depend on release of gastrin or on intact vagal nerves.

Phylogeny of biogenic peptides and their source cells was studied by immunohistochemistry and electron microscopy. The distribution of the peptide containing neurons and paraneurons in the brain and in the gastroenteropancreatic endocrine system was depicted, especially in the bullfrog as the representative of deuterostomia and in the cockroach and some other insects as the representatives of protostomia. Stress was given to: (1) calcitonin-immunoreactive neurons in bullfrog hypothalamus and PP-reactive neurons in the cockroach protocerebrum as instances of transmissional-hormonal partition of a neuropeptide, (2) open-type endocrine cells in the gut structurally and functionally common to the protostomia and deuterostomia, and (3) phylogeny of the prohormones with special reference to big gastrin and proglucagon (glicentin).

The sixth lumbar and first sacral spinal cord segments in the rat contain parasympathetic preganglionic neurons which innervate the pelvic viscera. There have been few studies, however, which have specifically considered the distribution of putative peptide neurotransmitters in these cord segments. The present paper describes and compares the immunohistochemical distribution of dynorphin (1-8)-, enkephalin-, somatostatin-, cholecystokinin octapeptide-, avian pancreatic polypeptide-, FMRF-NH2-, neurotensin-, and vasoactive intestinal polypeptide-like immunoreactivities in the dorsal gray commissure and sacral parasympathetic nucleus of the sixth lumbar and first sacral spinal cord segments in colchicine-treated rats. Antisera against all of the peptides, except avian pancreatic polypeptide, stained cells in the sacral parasympathetic nucleus. Dynorphin (1-8-), enkephalin-, and substance P-like immunoreactive cells were present in significantly greater numbers than somatostatin-, neurotensin-, cholecystokinin-, FMRF-NH2-, and vasoactive intestinal polypeptide-like immunoreactive cells. All of the antisera also stained fibers in the sacral parasympathetic nucleus in varying densities, and a fiber bundle which extended between the dorsal gray commissure and the sacral parasympathetic nucleus. Antisera against substance P and cholecystokinin stained a bundle of fibers that extended between the dorsal horn and the sacral parasympathetic nucleus. Antisera against somatostatin, cholecystokinin octapeptide, substance P and FMRF-NH2 stained an additional fiber bundle which extended between the lateral edge of the dorsal horn and the dorsal gray commissure. All the remaining antisera, except neurotensin, also stained fibers that extended between the sacral parasympathetic nucleus and the dorsal gray commissure, but in a sparser distribution. Immunoreactive cells were localized to the dorsal gray commissure in sections stained with each of the antisera. Dynorphin (1-8) and enkephalin antisera stained the greatest number of cells, followed by FMRF-NH2, neurotensin, somatostatin and avian pancreatic polypeptide. The smallest number of immunoreactive cells was present in substance P, cholecystokinin and vasoactive intestinal polypeptide immunostained sections. A significant difference was noted between the number of dynorphin, enkephalin, somatostatin, cholecystokinin, avian pancreatic polypeptide, FMRF-NH2, neurotensin and vasoactive intestinal polypeptide immunoreactive cells in the sacral parasympathetic nucleus and dorsal gray commissure.(ABSTRACT TRUNCATED AT 400 WORDS)

Peptide-YY (PYY) is a novel enteric peptide that is structurally related to pancreatic polypeptide and neuropeptide-Y. The objectives of the present experiments were to characterize the following aspects of PYY metabolism: the distribution of PYY in the canine gastrointestinal tract, the release of PYY in response to oral ingestion of a mixed meal or intraduodenal (ID) administration of oleic acid, the effect of ileocolectomy on the release of PYY in response to ID administration of oleic acid when transit of chyme to the distal ileum and colon is prevented, the effect of interruption of intramural neural pathways of the small bowel on the release of PYY, and the effect of iv cholecystokinin on the release of PYY. The results of these experiments demonstrate that PYY immunoreactivity is distributed primarily in the terminal ileum, colon, and rectum. Circulating levels of PYY increase significantly (P less than 0.05) within 10-30 min after ingestion of a meal or to ID administration of a fatty acid. Complete interruption of the flow of chyme to the site of PYY-containing cells (i.e. ileum-colon) did not block the release of PYY; however, ileocolectomy abolished the release of PYY in response to ID administration of oleic acid. Severance of intramural neural pathways along the small bowel did not alter the release of PYY in response to an oral meal. Intravenous administration of graded doses of cholecystokinin stimulated the release of PYY in a dose-related manner. The results of these experiments indicate that the release of PYY from the distal ileum and colon is controlled, at least in part, by an extramural neural, endocrine, or a combination of both types of mechanisms which originate in the foregut.

The effects of a newly synthesized peptide, human gastrin-releasing peptide (hGRP), on the pancreatic exocrine secretion and the release of pancreatic polypeptide (PP) were examined in the conscious rat. Plasma PP concentrations were determined by a recently established specific radioimmunoassay for rat PP. Amounts of 0.18, 0.35, and 3.5 nmol/kg/h hGRP significantly stimulated both pancreatic exocrine secretion and 0.35 nmol/kg/h of hGRP increased PP release. Simultaneously infused proglumide (300 mg/kg/h) did not affect either pancreatic exocrine secretion or PP release. However, simultaneous infusion of atropine (100 micrograms/kg/h) slightly inhibited PP release, but did not restrict the incremental response of pancreatic protein secretion to hGRP. These results suggest that hGRP directly stimulates pancreatic exocrine secretion and PP release.

The 27-amino acid peptide gastrin releasing peptide (GRP-(1-27] was infused at 4 dose levels (0.01, 0.1, 1.0, and 10 nM) into the arterial line of the isolated perfused porcine pancreas. Infusions were performed at 3 different perfusate glucose levels (3.5, 5.0, and 8.0 mM) and at two levels of amino acids (5 and 15 mM). GRP-(1-27) stimulated insulin and pancreatic polypeptide secretion and inhibited somatostatin secretion in a dose-dependent manner. Glucagon secretion was unaffected by infusion of GRP under all circumstances. The effect of GRP-(1-27) on insulin secretion was enhanced with increasing perfusate glucose levels, whereas the effects upon somatostatin and pancreatic polypeptide secretion were independent of perfusate glucose levels. The responses to GRP were unaffected by elevation of the concentration of amino acids in the perfusate. The effects of GRP were unaffected by atropine at 10(-6) M. The localization of GRP within the porcine pancreas, its release during electrical stimulation of the vagus nerve, and its potent effects upon pancreatic endocrine secretion make it conceivable that the peptide participates in parasympathetic regulation of pancreatic endocrine secretion.

Three dimensional analysis of retinal neuropeptides and monoamine-containing amacrine cells were performed on flat-mount preparations of the chick retina by using indirect immunofluorescence method. somatostatin (SOM), neurotensin (NT), leu-enkephalin (ENK), vasoactive intestinal polypeptide (VIP), substance P (SP), corticotropin releasing factor (CRF), avian pancreatic polypeptide (APP), glucagon (GLC), 5-hydroxytryptamine (5HT) and tyrosine hydroxylase (TH) were examined with specific antisera. To localize these substances in the amacrine cells, and to see in which layers their processes arborize, frozen sections were examined. There were four patterns of distribution. (1) Substances with more immunoreactive cells in the central than in the peripheral portions (SOM, NT, VIP, SP, GLC, 5HT), (2) Substances with more immunoreactive cells in the peripheral portion than in the central portion (APP), (3) Substances for which such cells were evenly distributed (TH), and (4) Substances with more immunoreactive cells in the inferior than in the superior portion (CRF). Subtypes were identified among the amacrine cells containing single peptides or monoamine.

The distribution and ontogeny of polypeptide YY (PYY)- and pancreatic polypeptide (PP)-immunoreactive cells in the gastrointestinal tract of rat were investigated. PYY-immunoreactive cells were numerous in the pylorus, ileum and colon and only a few cells were observed in the corpus, duodenum, jejunum and rectum. On the other hand, a few PP-immunoreactive cells were seen in the colon only. Both PYY- and PP-immunoreactive cells were of the open type, i.e., they extended from the basal lamina to the gut lumen. PYY-immunoreactive cells were observed first in the lower half of the stomach and in the intestine of 19 day-old embryo. The localization of the cells seemed to move along towards the pylorus and the lower part of the intestine. PP-immunoreactive cells could only be detected for the first time in the colon of 2 day-old rat. These cells appeared temporarily in the pylorus and rectum during the period 7 to 21 days after birth. It was concluded that the difference between PYY- and PP-immunoreactive cells in the distribution, frequency and ontogeny provide further evidence that PYY and PP occur in two independent cell types.

We studied the effect of the intravenous infusion of 16,16-dimethylprostaglandin E2 methyl ester (di-M-PGE2) and somatostatin on bombesin-stimulated gastric acid secretion, plasma gastrin and plasma pancreatic polypeptide in four chronic gastric fistula dogs. Bombesin-stimulated gastric acid secretion was significantly inhibited by somatostatin and virtually abolished by di-M-PGE2. Both agents caused significant, but indistinguishable inhibition of gastrin release (P less than 0.05). Bombesin-stimulated pancreatic polypeptide release was also significantly inhibited by both somatostatin and di-M-PGE2; the inhibitory effect of somatostatin was significantly greater than that of di-M-PGE2 (P less than 0.05). This study provides further evidence in support of the complex interrelationships between agents responsible for the modulation of gastrointestinal physiology.

Substance P (SP)-, leucine-enkephalin (LENK)-, cholecystokinin octapeptide (CCK8)-, bombesin (BOM)-, and avian pancreatic polypeptide (APP)-like immunoreactivities were analyzed in the optic tectum of Rana pipiens 5-99 days after unilateral eye enucleation, or optic nerve ligation, by means of the peroxidase-antiperoxidase and indirect fluorescence single and double labeling methods. The normal pattern (Kuljis and Karten, '81, '82c) of peptide-like immunoreactivity was observed ipsilaterally to the operation. Contralateral deafferented tectae displayed conspicuous modifications in the normal pattern of peptide-like immunoreactivity unique to each of the substances studied. The modifications of peptide-like immunoreactivity observed varied depending both on the survival time and on the particular peptide analyzed, with either increment or diminution or disappearance--or combinations thereof--in the staining intensity of some of the peptide-positive bands in the superficial one-third (superficial neuropil) of the tectum. The onset of these changes is detectable immunocytochemically between the sixth and tenth day following deafferentation. By 2-4 weeks full expression of the long-term changes is reached with no apparent further modification up to the 99th postoperative day. The rapid onset of these phenomena suggests the existence of a previously unrecognized retinal ganglion cell terminal peptidergic contribution to the tectum and/or rapid transsynaptic effects. The former possibility is suggested by recent observations demonstrating peptide-like immunoreactivity for SP, LENK, CCK8 and BOM in the retinal stump of ligated optic nerves (Kuljis and Karten, '82b). The fact that no vertebrate retinal ganglion cells have been shown to contain any of these peptides (Brecha et al., '79; Famiglietti et al., '80; Eldred and Karten, '81; Karten et al., '82), however, argues against the possibility of a retinal terminal contribution to peptide-like immunoreactivity in the tectum and suggests that transsynaptic phenomena are involved.

The effects of 1-h infusions of bombesin and gastrin releasing peptide (GRP) at 50 pmol/kg per h and neurotensin at 100 pmol/kg per h on gastrin, pancreatic polypeptide (PP) and neurotensin release in man were determined following either saline or atropine infusion (20 micrograms/kg). Bombesin produced a rise in plasma neurotensin from 32 +/- 6 to 61 +/- 19 pmol/l and of PP from 26 +/- 8 to 36 +/- 7 pmol/l. There was a further rise of plasma PP to 50 +/- 13 pmol/l after cessation of the infusion. GRP had no significant effect on plasma neurotensin, but compared to bombesin, produced a significantly greater rise in plasma PP from 34 +/- 6 to 66 +/- 19 pmol/l during infusion. There was no post-infusional increase. At this dose, GRP was as effective as bombesin in releasing gastrin, although unlike bombesin its effect was enhanced by atropine. Neurotensin produced a rise in plasma PP from 17 +/- 4 to 38 +/- 8 pmol/l. Atropine blocked the release of PP during GRP and neurotensin infusion. Atropine had no effect on neurotensin or PP release during bombesin infusion, but did block the rise in plasma PP following bombesin infusion. We conclude that, in contrast to meal-stimulated neurotensin release, bombesin-stimulated neurotensin release is cholinergic independent. Despite structural homology, bombesin and GRP at the dose used are dissimilar in man in their actions and sensitivity to cholinergic blockade.

A radioimmunoassay has been developed for the new intestinal hormonal peptide tyrosine tyrosine [peptide YY (PYY)]. Peptide YY concentrations were measured in separated layers of the human gastrointestinal tract, where PYY was found exclusively in the mucosal epithelium which contained the endocrine cells. Peptide YY was found throughout the small intestine, in very low concentrations (5 pmol/g) in duodenum (6 pmol/g) and jejunum (5 pmol/g), but in higher concentrations in the terminal ileum (84 pmol/g). High concentrations were found throughout the colon (ascending 82 pmol/g, sigmoid 196 pmol/g), being maximum in the rectum (480 pmol/g). The major molecular form of PYY-like immunoreactivity in human intestine appeared to be identical to pure porcine hormone, both as judged by gel permeation chromatography and by reverse-phase high-pressure liquid chromatography. Basal plasma concentrations of PYY were low but rose in response to food, remaining elevated for several hours postprandially. The known potent biologic actions of PYY, its high concentrations in gut endocrine cells, and its release into the circulation after a normal meal suggest that this peptide may function physiologically as a circulating gut hormone.

Galanin is a 29 amino acid peptide, initially isolated from the porcine small intestine. The peptide has been shown to occur in intrapancreatic nerves in close association to the islets. Its effects on islet hormone secretion and its possible mechanisms behind these effects are reviewed. Galanin has been shown to inhibit basal and stimulated insulin secretion both in vivo and in vitro under a variety of experimental conditions. The peptide has also been shown to inhibit somatostatin secretion and the secretion of pancreatic polypeptide (PP). With regard to glucagon secretion, however, results in the literature are not consistent since both stimulatory and inhibitory effects have been reported. A direct interaction with the pancreatic beta-cells has been proposed behind its inhibitory action on insulin secretion, since galanin inhibits insulin secretion from isolated beta-cells from obese, hyperglycaemic, mice. Galanin has thereby also been shown to induce repolarization and to reduce the free Ca2+ concentration, [Ca2+]i. The reduction in [Ca2+]i is probably not due to a direct interference with the voltage-activated Ca2+ channels, since there is no effect of galanin when these channels are opened by depolarization induced by high concentrations of K+. Instead, preliminary studies indicate that galanin activates the K+ channels that are regulated by ATP, in turn inducing a repolarization-induced reduction in [Ca2+]i resulting in reduced insulin secretion. However, the possibility that galanin inhibits the insulin secretory mechanism at a step distal to the regulation of cytoplasmic free Ca2+ concentration should not be overlooked.

Valosin, a novel 25-amino acid gastrointestinal peptide with N-terminal valine and C-terminal tyrosine, has recently been isolated from porcine upper gut extracts. Its physiologic role is unknown and it does not belong to one of the structurally related gut peptide families. Assuming that valosin may influence gastrointestinal functions, we investigated the effect of high-performance liquid chromatography-pure valosin on gastric and exocrine pancreatic secretion and on the intestinal myoelectric activity in conscious dogs. Intravenous injection of valosin (0.125-1 microgram/kg) dose-dependently increased gastric acid secretion 80-fold over basal, corresponding to 18% of the maximal pentagastrin-induced effect. Pepsin output increased 10-fold over basal (30% of the pentagastrin-stimulated secretion). Half-maximal stimulation by pentagastrin could be further increased dose-dependently by simultaneous administration of valosin. Pancreatic bicarbonate secretion was stimulated 11-fold over basal at 1.0 microgram/kg, reaching about 6% of the secretin-induced maximal output, whereas protein secretion increased 12-fold over basal, corresponding to about 55% of the cholecystokinin-induced maximal output. In fasted dogs, spontaneously occurring migrating myoelectric complexes were substantially delayed during infusion of valosin at a dose of 0.2 microgram/kg. These experiments indicate that valosin may represent a novel member of the regulatory gastrointestinal peptides.

We have investigated the effect of galanin infusion on unstimulated pancreatic polypeptide (PP) release as well as on the PP response to arginine by the perfused rat pancreas. Galanin significantly reduced unstimulated PP output. Addition of arginine to the perfusate evoked a biphasic pattern of PP release; the second phase of this PP response was delayed when galanin was simultaneously infused. These findings point to a regulatory role of galanin in the control of PP secretion.

The influence of gastric emptying of nutrients on plasma cholecystokinin and pancreatobiliary functions is poorly understood. We therefore temporally related the emptying of fat, protein, and glucose of a mixed meal to release of the gut hormones cholecystokinin, pancreatic polypeptide, and peptide YY and outputs of trypsin, lipase, bilirubin, and bile salts. Five healthy volunteers with a multilumen duodenal tube ingested a mixed meal with phase-specific markers for the aqueous phase, liquid fat, solid fat, and solid protein phases. Duodenal passage was determined by intraduodenal infusion of a second set of phase-specific nonabsorbable markers. Plasma cholecystokinin levels and pancreatobiliary secretions rose to a maximum at 30-60 min and then gradually declined (p less than 0.01) despite continued entry of protein and fat into the duodenum throughout the whole 4-h experimental period. High levels of both pancreatic polypeptide and peptide YY were observed in the last 2 h of the experiment. Release of factors capable of inhibiting cholecystokinin release and subsequently pancreatobiliary secretion may be responsible for the observed time-course.

Intact vagi after ulcer operations are often implicated in the cause of recurrent ulcer. The stimulation of gastric acid by insulin hypoglycemia is dangerous and the measurement of acid secretion after gastrectomy unreliable. This study was undertaken to assess and compare PP release by bombesin or insulin as an indicator of vagal integrity. Eight dogs with a chronic gastric fistula were tested with bombesin (100 nanograms per kilogram) and insulin (0.1 unit per kilogram) intravenous bolus after unilateral and, then, bilateral truncal vagotomy. Each study was 120 minutes, and blood was taken at one, three, five, seven and then ten minute intervals. Gastric acid was measured by autobiuret titration. Plasma was stored at minus 20 degrees C. until assayed for PP by radioimmunoassay. Bombesin-stimulated gastric acid secretion was not significantly altered by vagotomy (p greater than 0.05), whereas that stimulated by insulin was significantly inhibited by bilateral truncal vagotomy (p less than 0.05). Bilateral and right hemivagotomy significantly inhibited PP release by bombesin (p less than 0.05), however, only bilateral truncal vagotomy significantly inhibited PP release by insulin (p less than 0.05). These results suggest that the measurement of PP release by insulin or bombesin is a sensitive index of vagal integrity and that bombesin-released PP may specifically delineate the integrity of the right vagus. Since the measurement of gastric acid secretion after operation is both uncomfortable and often difficult to interpret, the value of a simple blood test to determine vagal integrity may be of considerable clinical relevance.

Peptide YY (PYY), neuropeptide Y, and pancreatic polypeptide (PP) are the third family of structurally related peptides to be extracted from the brain-gut axis. In the present study we have validated a sensitive and specific assay for PYY and used it to measure the distribution of PYY in the canine gastrointestinal tract. In addition we have compared the PYY and PP responses to both an intragastric meal and to insulin hypoglycemia. The highest concentration of PYY was present in the canine ileum (1610 +/- 123 ng/g) and colon (1607 +/- 194 ng/g). Significant (p less than 0.05) increases in both circulating PP and PYY concentrations were observed in response to the meal (mean delta PP = 180 +/- 43 pg/ml; mean delta PYY = 205 +/- 61 pg/ml). In contrast, only serum PP concentrations (mean delta PP = 294 +/- 36 pg/ml) increased significantly (p less than 0.01) in response to insulin hypoglycemia, demonstrating differences in the sensitivity of the PP and PYY cells to vagal stimulation. The present demonstration of PYY release in response to a physiological stimulus fulfills one of the prerequisites for hormonal status.