Peptide YY kinetics and effects on blood pressure and circulating pancreatic and gastrointestinal hormones and metabolites in man.

Peptide YY (PYY) is a 36 amino acid peptide produced by mucosal endocrine cells of the ileum and colon which inhibits acid secretion and intestinal transit in man. To assess its effects on metabolites and digestive hormones PYY was infused into 18 fasting normal subjects at three dose levels (0.06, 0.19, and 0.57 pmol kg-1 min-1), each for a period of 1 h. During the infusions mean plasma PYY levels increased by 8, 25, and 73 pmol/liter, respectively. The mean disappearance half-time on stopping the infusions was 9.2 +/- 0.4 (SEM) min. The mean MCR was 7.3 +/- 0.7 ml kg-1 min-1 and the apparent volume of distribution was calculated to be 94 +/- 9 ml kg-1. During the highest dose infusion there was a significant increase in both systolic and diastolic blood pressure, of 8.6 +/- 3.7 mmHg (P less than 0.05) and 10.9 +/- 3.0 mmHg (P less than 0.01), respectively. PYY caused a significant 50% reduction in plasma pancreatic polypeptide concentrations (P less than 0.05) and a 55% reduction in circulating motilin levels (P less than 0.05). PYY had no significant effect on circulating concentrations of insulin, glucagon, gastrin, gastric inhibitory peptide, neurotensin, enteroglucagon, or vasoactive intestinal peptide. PYY also had no significant effect on circulating concentrations of glucose, lactate, glycerol, or nonesterified fatty acids. This recently discovered human intestinal hormonal peptide thus has significant effects both on gastrointestinal hormones (motilin and pancreatic polypeptide) and blood pressure in man, but appears not to influence glucose or lipid metabolism.

Human and porcine immunoreactive gastric inhibitory polypeptides (IR-GIP) are not identical.

Immunoreactive gastric inhibitory polypeptide (IR-GIP) from human and porcine intestine was quantified by radioimmunoassay and the molecular forms characterised by gel permeation and reverse-phase high pressure liquid chromatography (HPLC). Gel filtration revealed two major immunoreactive peaks corresponding to the previously described 5-kDa and 8-kDa molecular forms, which appeared similar in both species. Isocratic reverse-phase HPLC revealed that the major immunoreactive GIP peak (5-kDa) in the human tissue eluted earlier than the corresponding porcine molecular form, indicating the latter to be less hydrophobic. These findings suggest significant species differences between human and porcine GIP.

Is the C-terminal flanking peptide of rat cholecystokinin double sulphated?

A specific radioimmunoassay was developed to the predicted nine amino acid C-terminal flanking peptide of cholecystokinin (peptide serine serine, PSS). In aqueous extracts of rat brain, PSS was undetectable unless the extracts were first treated with arylsulphatase, which also resulted in desulphation of cholecystokinin. The reverse-phase HPLC analysis of partially desulphated extracts showed the presence of two peaks intermediate to the naturally occurring and the completely desulphated forms. It is therefore proposed that the CCK-flanking peptide PSS has both tyrosine residues sulphated.

Distribution and postprandial release of porcine peptide YY.

Peptide YY (PYY), a thirty-six amino acid intestinal hormonal peptide with a tyrosine residue at each end (hence YY as Y represents tyrosine in the new peptide nomenclature), was found throughout the gastrointestinal tract of the pig. Concentrations were very low in the foregut (antrum, 3.4 +/- 0.3 pmol/g; duodenum, 1.1 +/- 1.5 pmol/g), higher in the distal small intestine (ileum, 100 +/- 13 pmol/g) and very high in the large bowel (descending colon, 270 +/- 45 pmol/g). Peptide YY was found to circulate in plasma and concentrations rose substantially in response to eating (fasting, 138 +/- 15 pmol/l; postprandial, 263 +/- 21 pmol/l; P less than 0.001). There was a small but significant portal/arterial gradient in postprandial PYY levels. More than 90% of the immunoreactive PYY in gut extracts eluted, on gel permeation chromatography, in an identical position to pure PYY standard, but small amounts of higher molecular weight material, possibly precursors, were detected. In contrast, plasma from fasting pigs contained a large proportion (60-70%) of these large molecular forms. These findings suggest that the putative pro-PYY may be cleared more slowly from the circulation than the 36 amino acid hormonal peptide. The high concentrations of immunoreactive PYY in the circulation of the young pig may reflect a species difference between pig and man or may indicate an important role for PYY in the developing animal.

Measurement of cholecystokinin octapeptide using a new specific radioimmunoassay.

A peptide analogue of CCK-8 (Tyroc) which has a tyrosine in place of the amide group in the C-terminal end, has been used both for raising antisera and for iodination. The antisera produced by immunisation with Tyroc are directed towards the N-terminal end of the CCK-8 molecule. The assay system appears totally specific for the CCK-8 sulphated molecule and shows no significant cross-reaction with other molecular forms of CCK, or with the gastrins. The assay can detect changes between adjacent tubes of 0.25 fmol/tube CCK-8 with 95% confidence. The assay is robust, reliable and reproducible and can be used to measure tissue and plasma levels of CCK-8.

Oxidation/reduction of methionine residues in CCK: a study by radioimmunoassay and isocratic reverse phase high pressure liquid chromatography.

The study was undertaken to investigate the oxidation and reduction of cholecystokinin (CCK) both as pure standards and as endogenous porcine peptides. Furthermore an attempt was made to prevent oxidation of the endogenous porcine peptides in the extraction procedure. CCK-8 and CCK-33 standards were always oxidized in weak solutions, CCK-8 varying from 26% to 67% oxidized and CCK-33 from 18% to 70%. Similarly, tissue extracts of porcine brain and duodenum contained oxidized forms of the peptide. CCK standards were readily oxidized in the presence of hydrogen peroxide. Oxidized CCK-8 standard and CCK-8 in porcine brain was 90% reduced and oxidized CCK-33 standard and in duodenal extracts was reduced by 70% by a 40 hour incubation with 0.725 mol/l dithiothreitol at 37 degrees C. Extraction of CCK peptides in the presence of 65 mmol/l dithiothreitol resulted in almost complete prevention of oxidation with over 95% of the peptides being obtained in the reduced state. This additive is therefore recommended for all tissue quantitation studies.

Distribution and heterogeneity of immunoreactive cholecystokinin (CCK) in the mucosa of the porcine gastrointestinal tract.

The concentration and molecular nature of cholecystokinin-like immunoreactivity (CCK-LI) in extracts of porcine intestinal mucosa were determined using sequence-specific radioimmunoassays. Highest CCK concentrations were measured in duodenal mucosa (258 +/- 60 pmol/g in the distal duodenum) followed by jejunal mucosa (204 +/- 36 pmol/g in the proximal jejunum) and pylorus (51 +/- 9 pmol/g). All other gastrointestinal regions proximal to the pylorus and distal to the jejunum contained less than 20 pmol/g. Pancreas contained less than 1 pmol/g. Gel chromatography in 6 M urea revealed four immunoreactive forms and this was confirmed by reverse-phase high-pressure liquid chromatography (HPLC). The predominant molecular form in acid extracts of duodenal mucosa resembled CCK-33 although high concentrations of the larger CCK form ('CCK-58') and of the form intermediate in size between CCK-33 and CCK-8 were measured. A molecular form resembling CCK-8 was the principal form in neutral extracts of the duodenum.

Rat immunoreactive cholecystokinin (CCK): characterization using two chromatographic techniques.

Acid and neutral extracts of rat cerebral cortex and upper small intestine were prepared and the endogenous concentrations of cholecystokinin-like immunoreactivity (CCK-LI) measured by three new CCK-specific radioimmunoassays. The characterization of the immunoreactive CCK molecular forms was undertaken using gel permeation chromatography in the presence of 6 M urea to minimise problems relating to peptide adsorption or aggregation. Reverse-phase high-performance liquid chromatography (HPLC) was also performed on the rat tissue extracts. Rat cortex contained 268 +/- 12 pmol/g CCK-LI, and over 90% resembled the sulphated CCK-8, which was preferentially extracted at neutral pH. In contrast, the rat upper small intestine (97 +/- 8 pmol/g of CCK-LI) contained less than 20% CCK-8, the majority of immunoreactive CCK being of larger molecular size and being preferentially extracted at acid pH. In the small intestine the predominant molecular form(s) was intermediate in size between CCK-33 and CCK-8. Large amounts of CCK-33 and of a molecular form larger than CCK-33 were also detected. It is concluded that post-translational cleavage of CCK differs in rat brain and gut.

24-hour variation in content and release of hypothalamic neuropeptides in the rat.

The tissue content of up to eight neuropeptides, viz bombesin (BOM), cholecystokinin (CCK-8), neurotensin (NT), neuropeptide Y (NPY), peptide histidine isoleucine amide (PHI), somatostatin (SRIF), substance P (SP) and vasoactive intestinal polypeptide (VIP), in rat hypothalami removed at various times of the day, was measured using specific radioimmunoassays. There was significant variation in the content of BOM, CCK-8, NT, PHI, SP and VIP across a 24-h period. The levels of BOM, CCK-8 and NT were lowest around the onset of darkness (1900 h) and rose throughout the night to reach a peak around the time of lights on. Hypothalamic content of all eight peptides fell between 0700 h and 1300 h by an average of 45 +/- 4%. Basal release of these peptides, as well as that in the presence of 48 mM potassium (K+), was measured from hypothalami removed between 0700 and 1900 h and incubated in vitro in a CSF-like medium. Basal secretion of NT significantly increased, whilst that of CCK-8 significantly decreased over the same period. There was no significant change in the basal release of the other neuropeptides. The release in the presence of 48 mM K+ of SP decreased significantly during the day, whilst that of VIP significantly increased. There was also a significant change in the stimulated release of BOM, levels falling during the morning and rising again at 1900 h. 48 mM K+ caused a significant increase in the release of SRIF and SP at all times tested. Whilst 48 mM K+ induced a significantly higher release of CCK-8 and NT in the morning, this stimulus was ineffective in the evening. The contrary was true in the case of BOM, NPY and VIP, where a significant stimulation was induced only at 1900 h. The possible implications of these findings are discussed.

Peptides, the limbic lobe and schizophrenia.

The human brain contains several peptides with probable synaptic actions, some of which form complex neuronal networks in the limbic lobe (amygdala, hippocampus and temporal cortex). A limbic lobe abnormality has been postulated in schizophrenia on the basis of similarities between schizophrenic symptoms and symptoms in cases of known limbic pathology. Cholecystokinin (CCK), somatostatin (SRIF), neurotensin (NT), vasoactive intestinal polypeptide (VIP) and substance P (SP)-like immunoreactivities were measured by radioimmunoassay in 10 brain areas of 14 schizophrenics and 12 controls. In the schizophrenic group symptoms had been rated in life and the group was divided into Type I (n = 7) and Type II (n = 7) subgroups on the basis of the absence or presence of morbid negative symptoms. In control brains each peptide showed a characteristic distribution with high levels in cortex (CCK), limbic lobe (SOM, NT, VIP) or striatal areas (SP) and low levels of each of the peptides in thalamus. Significant (P less than 0.05) differences between groups were: reductions of CCK and SOM in hippocampus and CCK in amygdala in Type II schizophrenics, and CCK in the temporal cortex of the total schizophrenic group; and elevations of VIP in amygdala in Type I schizophrenics and of SP in the hippocampus in the total schizophrenic group. The findings could not be explained by variables such as age, delay between death and necropsy or to neuroleptic medication. These clinical-state related alterations in the peptide content of the limbic system in schizophrenia may illuminate the pathophysiological basis of the disease, particularly the distinction between Type I and II syndromes.

Measurement and characterisation of human cholecystokinin-like immunoreactivity (CCK-LI) in tissues by radioimmunoassay.

Two radioimmunoassays specific for cholecystokinin-like immunoreactivity (CCK-LI) in human tissue are described. The first assay employed an antiserum (Z-69) directed to the sulphated tyrosine at the C-terminal end of CCK-33 and measured all biologically active molecular forms of CCK except the controversial C-terminal tetrapeptide amide (CCK4). The sensitivity of this assay was 0.6 pmol/g. A second assay (employing antiserum Z-91) measured CCK-LI forms larger than the octapeptide and had a sensitivity of 0.2 pmol/g. Both assays were characterised with endogenous human peptides. Acid (pH 2.5) and neutral extracts (pH 6.5) of human intestine and brain were assessed for CCK-LI concentrations and gel chromatography performed in the presence of 6 mol/l urea to elucidate the various molecular forms. Human cerebral cortex CCK-LI was almost all sulphated CCK-8, but large molecular mass forms were present, particularly in acid extracts, forming about 10% of the whole. Human duodenum and jejunum contained approximately equal amounts of large CCK, CCK 33/39 and of CCK-8. Both intestine and brain possess not yet isolated sulphated molecular forms which eluted between the pure CCK-8 and CCK-33/39 standards. The results obtained from this study indicate that the biosynthesis of CCK in human brain and gut is quantitatively different.

Chromatographic characterisation of neurotensin-like immunoreactivity in plasma and tissue extracts from hepatoma patients.

To determine the nature of the neurotensin-like immunoreactivity produced by some cases of primary liver carcinoma, plasma and tumour neurotensin-like immunoreactivities were characterised in patients with neurotensin-producing hepatoma by chromatographic analysis in conjunction with radioimmunoassay with C- and N-terminally directed antisera. Gel filtration revealed that the majority of neurotensin-like immunoreactivity extracted from the tumour was co-eluted with synthetic neurotensin, whereas a substantial amount of the plasma immunoreactive neurotensin emerged at the void volume. Tumour neurotensin-like immunoreactivity showed a degree of heterogeneity on gel filtration and high pressure liquid chromatography that contrasted with neurotensin-like immunoreactivity extracted from ileal mucosa, suggesting a different post-translational processing of neurotensin within tumour tissue. The ratio of neurotensin 1-8 to neurotensin 1-13 in the patients' plasma suggested that neurotensin was metabolised in a manner similar to that in healthy subjects. The clinical and biological significance of the ectopic production of neurotensin in some hepatomas remains unclear.

Nippostrongylus brasiliensis: changes in plasma levels of gastrointestinal hormones in the infected rat.

Plasma concentrations of gastrointestinal hormones were measured by radioimmunoassay in fasted rats 9 days after infection with a range of doses of Nippostrongylus brasiliensis. Values for infected rats fed ad libitum were compared with those of weight matched, pair fed, uninfected rats to control for the possible effects of dose-dependent reductions in food intake associated with infection. The plasma concentrations of some of the gastrointestinal hormones in infected rats were very different from those of their pair fed partners. The magnitude and direction of the changes varied according to the hormone being examined. Plasma concentrations of gastrin and pancreatic polypeptide were similar in pair fed and infected rats at all doses used. For the other hormones assayed, infection was associated with dose-related changes. The plasma concentrations of cholecystokinin and insulin were slightly but significantly reduced in infected rats. In contrast, secretin, enteroglucagon, and pancreatic glucagon concentrations were markedly increased. At the highest dose given (52 larvae/g body wt), the plasma levels of secretin and enteroglucagon in infected rats were elevated 9 X and 15 X, respectively. A comparison of the changes seen in N. brasiliensis-infected rats with those reported for other helminth infections revealed striking differences. The possible etiology of alterations in plasma gastrointestinal hormone concentrations and their contribution to the pathological changes seen in animals infected with helminths are discussed.

Pentagastrin-stimulated gastric acid output and plasma enteroglucagon in acarbose-treated rats.

Although the purified porcine enteroglucagons glicentin and oxyntomodulin inhibit pentagastrin-stimulated gastrin acid secretion when given parenterally to rats, it is not known whether the postprandial rise in endogenous enteroglucagons is capable of exerting a similar effect. We have used the alpha-glucosidase inhibitor acarbose in combination with a sucrose- and starch-rich semisynthetic diet over 8 days to bring about a mean increase of 89 pmol/l in the fasting plasma enteroglucagon concentration in rats, without significantly affecting plasma gastrin concentrations. There was no significant suppression of pentagastrin-stimulated gastric acid secretion in the acarbose-treated rats, suggesting that endogenous enteroglucagons do not act as physiological inhibitors of gastric acid secretion.

Peptide YY abnormalities in gastrointestinal diseases.

Plasma concentrations of peptide YY (PYY), a newly isolated peptide produced by ileal and colonic endocrine cells, were measured in several groups of patients with digestive disorders after a standardized normal breakfast. Peptide YY levels were found to be grossly elevated in patients with steatorrhea due to small intestinal mucosal atrophy (tropical sprue). Basal levels in these patients were 79 +/- 18 pM, which was nearly 10-fold higher than those seen in healthy controls (8.5 +/- 0.8 pM). Patients with steatorrhea due to chronic destructive pancreatitis also had substantially increased basal PYY levels (47.5 +/- 6.3 pM), and their postprandial response was also greater than that of normal subjects. Moderately elevated plasma PYY concentrations were seen in patients with inflammatory bowel disease and patients recovering from acute infective diarrhea. In contrast, patients with diverticular disease, duodenal ulcer, and functional bowel disease had normal PYY responses. These changes in the secretion of PYY responses. These changes in the secretion, may shed light on the physiologic role of this newly discovered peptide and on intestinal adaptation to common digestive disorders.

Human distribution and release of a putative new gut hormone, peptide YY.

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.

Effect of peptide YY on gastric, pancreatic, and biliary function in humans.

The effect of peptide YY (PYY) on gastric and pancreatico-biliary secretion was studied in humans. Peptide YY was infused into groups of 6 healthy volunteers at doses of 0.59, 0.20, and 0.064 pmol X kg-1 X min-1. The two higher doses caused a significant suppression of gastric acid and pepsin output during background stimulation with pentagastrin. The middle dose of PYY (0.20 pmol X kg-1 X min-1) that increased plasma PYY levels by 27 +/- 2 pM caused a 90% +/- 18% (mean +/- SEM; p less than 0.001) reduction in the incremental gastric volume response to pentagastrin. Similarly this dose of PYY caused a substantial inhibition of the acid (77% +/- 14%; p less than 0.005) and pepsin (96% +/- 22%; p less than 0.01) response to pentagastrin; in 2 subjects, pepsin output fell to below basal levels. In contrast, the highest dose of PYY (0.62 pmol X kg-1 X min-1) had no significant influence on duodenal juice volume, output of bicarbonate, trypsin, or bilirubin during low dose stimulation with secretin (0.25 pmol X kg-1 X min-1) and cholecystokinin-8 (0.15 pmol X kg-1 X min-1). Thus PYY concentrations in the circulation similar to those seen after the ingestion of food cause a marked reduction in gastric secretion. This peptide should therefore be considered as one of the possible candidates for the classical enterogastrone.

Effect of food on porcine gut regulatory peptides in arterial and portal venous blood.

The concentrations of gut regulatory peptides were monitored simultaneously in the portal and arterial circulations of 8 healthy conscious swine in the fasted state and after a standard mixed meal. The sampling was achieved via chronic cannulations of these two vessels. Portal/arterial differences of insulin and glucagon were similar to those previously described, indicating the importance of hepatic extraction of these two hormones. Portal/arterial differences for the gastrointestinal regulatory peptides were relatively small, however, and the liver is unlikely to be of major importance in the metabolism of these peptides.

Lymph, pancreatic and gastrointestinal hormones in response to feeding in the conscious pig.

Concentrations of several gastrointestinal hormonal peptides were measured in lymph from the cisterna chyli and in arterial plasma; in healthy, conscious pigs during ingestion of a meal. Lymph concentrations of the pancreatic hormones insulin, glucagon and pancreatic polypeptide were small compared with plasma concentrations, although postprandial increments were significant. In contrast lymph concentrations of gastrin, cholecystokinin, motilin and gastric inhibitory peptide (GIP) from the foregut showed a more marked postprandial rise than the pancreatic hormones. Indeed the total integrated responses of these peptides in lymph reached about 50% of those seen in arterial plasma. It would appear unlikely that the lymphatics constitute an important transport mechanisms for these regulatory peptides. However, lymph concentrations of hormones may reflect levels in interstitial fluid better than plasma and may be of value in assessing putative physiological actions within a target tissue.

A VIP-containing system concentrated in the lumbosacral region of human spinal cord.

Neuropeptides were first localized in the human spinal cord by immunocytochemistry and substance P has been shown, by the same method, to be reduced ipsilaterally in the dorsal horn after limb amputation and bilaterally in the Riley-Day syndrome. Several neuropeptides increasingly fulfil the criteria to establish them as neurotransmitters or neuromodulators, and they may also have trophic actions in the spinal cord. Using radioimmunoassay and immunocytochemistry, we present here for the first time a quantitative regional distribution and localization of vasoactive intestinal polypeptide (VIP), substance P, somatostatin, bombesin and cholecystokinin (CCK-8) in normal postmortem human spinal cord. A comparison of the distribution of these peptides reveals an exceptional pattern for VIP, with relatively much higher levels in the lumbosacral region. Immunocytochemical analysis shows a distinctive distribution of VIP-containing fibres and terminals at the lumbosacral segments. This VIP-containing system may have an important role in the spinal control of urogenital function in man.