Biomedical subjects
Publications and source records attributed to M A Ghatei.
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Following the discovery of a second gene containing a CGRP-like sequence, we demonstrated that "beta-CGRP" was indeed translated as a 37-amino acid peptide in vivo and was the predominant form of CGRP produced by the enteric nervous system. The presence of CGRP in the islet has been reported by several groups. We now show that beta-CGRP is again the major form. Another 37-amino acid peptide was recently isolated from islet amyloid deposits and found to have approximately 50% amino acid sequence homology with CGRP. Islet amyloid polypeptide, or amylin, is co-localized with insulin to the beta-cell secretory granule and is synthesized and released in parallel with insulin in response to a range of physiological and pharmacological stimuli. IAPP was subsequently shown, like CGRP, to inhibit the release of insulin pharmacologically. Interestingly, it was also shown to decrease the uptake of glucose by striated muscle, though it was considerably less potent than CGRP. This led to the suggestion that IAPP might be a circulating hormone regulating peripheral insulin sensitivity. Infusion of IAPP in human volunteers to produce plasma concentrations more than 100-fold higher than those seen physiologically, however, failed to alter peripheral glucose disposal. We conclude that beta-CGRP and IAPP are likely to play a role in local paracrine control of the islet.
Cholinergic synaptosomes from rat cerebral cortex were isolated by a magnetic immunoaffinity technique, i.e. immunomagnetophoresis. This subpopulation was extracted and subjected to radioimmunoassay for 4 neuropeptides:neuropeptide Y (NPY); vasoactive intestinal peptide (VIP); substance P (SP); and somatostatin (SRIF). Three of the 4 neuropeptides were enriched in the sorted fraction compared with the mother fraction with respect to the cytosolic marker lactate dehydrogenase (LDH). The most enriched neuropeptide was NPY followed by SP and VIP. Somatostatin was not enriched in the cholinergic synaptosome subpopulation. The presence of NPY has not previously been reported in cortical cholinergic neurones.
We have compared the binding of [125I]vasoactive intestinal polypeptide (VIP) to human brain membranes with that of [125I]PACAP27. [125I]VIP was displaced by PACAP27, VIP and two synthetic peptides, peptide-1 (N-terminal PACAP27/C-terminal VIP) and peptide-2 (N-terminal VIP/C-terminal PACAP27), but the IC50 of PACAP27 and peptide-1 were 10-20 times lower than those of VIP and peptide-2. [125I]PACAP27 was readily displaced by PACAP27 and peptide-1, with an IC50 of less than 1 nM, but poorly by VIP and peptide-2. Chemical cross-linking revealed that both labels were bound to polypeptides of Mr 66,000 and Mr 50,000. The results indicate that in human brain membranes both binding sites have a higher affinity to the N-terminal sequence of PACAP27, and VIP binding sites prefer PACAP27 to VIP itself.
Intestinal dysmotility may be an important factor contributing to various gastrointestinal complications associated with cystic fibrosis. Motilin, enteroglucagon, neurotensin, and peptide YY may each play a role as endocrine hormones influencing gastrointestinal motor activity. Fasting children with cystic fibrosis (N = 8) and controls (N = 18) received a liquid nutrient test meal (fat 4 g/100 ml, protein 4 g/100 ml, carbohydrate 20 g/100 ml, 125 kcal/100 ml; 200 ml/m2) containing lactulose (5 g/100 ml), and the plasma concentrations of these peptides were studied. Mouth-to-cecum transit time was simultaneously studied using the breath H2 technique. Fasting levels of peptide YY and the postprandial response of all four peptides were significantly increased in those with cystic fibrosis. In repeat studies on those with cystic fibrosis after a period of altered pancreatic enzyme supplementation, no significant changes in peptide concentrations were observed. A rise in breath H2 permitting estimation of mouth-to-cecum transit time was noted in 17 control subjects (70-220 min, median 140). In contrast, a rise occurred in only two with cystic fibrosis after low-dose enzyme (70 and 180 min), and four after high-dose enzyme replacement (120-230 min, median 155). Altered gut hormone secretion may play a role in the pathophysiology of intestinal dysmotility in patients with cystic fibrosis.
To assess potential mediators of adaptive bowel growth, ileal proglucagon messenger RNA (mRNA) ornithine decarboxylase (ODC) mRNA, plasma enteroglucagons, and plasma glucagonlike peptide I (GLP-I) were analyzed in rats soon after jejunoileal resection or control transection. Analyses were performed before and after refeeding to establish whether responses are nutrient dependent. The elevation of ileal proglucagon and ODC mRNAs within 12 hours after resection and before refeeding shows a nutrient-independent component of the adaptive response. The onset of adaptive growth of the ileum required luminal nutrient but occurred very rapidly, within 4 hours of refeeding. The onset of adaptive growth was accompanied by transient elevation of ileal ODC mRNAs. Ileal proglucagon mRNA and plasma GLP-I levels were also elevated, and these increases were sustained up to 8 days after resection. These early and sustained increases in proglucagon mRNA and plasma GLP-I indicate that in addition to the enteroglucagons, other intestinal proglucagon-derived peptides must be considered as potential mediators of adaptive growth after jejunoileal resection.
GABAergic and cholinergic synaptosomes from rat cerebral cortex were isolated by a magnetic immunoaffinity technique, i.e. immunomagnetophoresis. These subpopulations were extracted and subjected to radioimmunoassay for four neuropeptides: Neuropeptide Y (NPY); vasoactive intestinal peptide (VIP); substance P (SP); and somatostatin (SRIF). In each of the sub-populations three of the four peptides were enriched in the sorted fraction compared with the mother fraction with respect to the cytosolic marker lactate dehydrogenase (LDH). In the GABAergic sub-population the order was SP > SRIF > NPY > or = VIP whilst in the cholinergic sub-population they were enriched in the order VIP > or = NPY > SP > SRIF. The presence of NPY has not previously been reported in cortical cholinergic neurons.
The plasma concentrations of seven gut regulatory peptides were measured in 11 infants suffering from acute gastroenteritis. Samples were taken at the time of the acute illness, upon reintroduction of feeding, and three months after recovery. These results were compared with controls. In the infants with diarrhoea, a massive increase in the fasting plasma mean (SEM) concentrations of enteroglucagon was found at the time of illness (1292 (312) v 79 (27) pmol/l), with concentrations of pancreatic glucagon, peptide tyrosine tyrosine, and motilin also being increased (17.8 (3.1) v 6.3 (1.1) pmol/l, 114.6 (15.2) v 37.0 (11.0) pmol/l, 217.6 (44.1) v 98.5 (18.3 pmol/l) respectively). The preprandial concentrations of motilin were found to be still increased at recovery (183.9 (35.4) pmol/l), but the concentrations of the other three peptides had returned to normal values. No differences in plasma concentrations of vasoactive intestinal polypeptide, neurotensin, or pancreatic polypeptide were found. An increased intestinal permeability was demonstrated at the time of diarrhoea by the urinary ratio of lactulose to mannitol, suggesting simultaneous gut damage. The effects of regulatory peptides may be relevant to the pathophysiology of gastroenteritis in infants.
The distribution of immunoreactivity for the potent vasoconstrictor endothelin-1 was studied in adult and developing human gut using antisera to endothelin-1 (1-21) and the C terminus of big endothelin-1. The coexistence of these peptides with other neuropeptides was investigated using comparative immunocytochemistry. Endothelin-1 like immunoreactivity was detected in extracts of adult (range 20-60 fmol/g wet weight) and fetal (33 fmol/g) gastrointestinal tract and was shown by chromatography to be the predominant isoform of endothelin present in both. It was localised by immunocytochemistry to ganglion cells in the submucous and myenteric plexuses and to scattered nerves, whereas big endothelin-1 like immunoreactivity was found in the submucous plexus only. Colocalisation studies showed immunoreactivity for both endothelin-1 and vasoactive intestinal peptide in the same ganglion cells of the submucous plexus. Although endothelin-1 immunoreactivity was not detected by immunocytochemistry in the fetal human gut until the 32nd week of gestation, big endothelin-1 was found as early as 11 weeks in the developing neural structures and epithelial cells. The latter were shown to be endocrine cells by their immunoreactivity for chromogranin. Our results indicate that endothelin is a neuropeptide found in adult human gut which shows transient expression in endocrine cells during development.
To investigate the enterotrophic effects of bolus doses of long chain triglycerides, two groups of eight female Wistar rats were fed identical diets with 48.2% total calories as the essential fatty acid rich oil Efamol. To one group the oil was given in twice daily bolus doses by gavage, while for the other group the oil was mixed with the remainder of the feed and thus consumed over 24 hours. The animals were killed after 20 to 22 days. Bolus dosing significantly increased parameters of mucosal mass along the length of the small intestine in association with an increase in two hour accumulation of vincristine arrested metaphases in small intestinal crypts. In a second experiment, four replicate studies were carried out, each involving two groups of 12 rats respectively fed as described above. After 21 days one animal from each group was killed every two hours, providing regular plasma samples over 24 hours for measurement of gastrin, cholecystokinin, peptide tyrosine-tyrosine and enteroglucagon. Bolus dosing markedly enhanced release of peptide tyrosine-tyrosine and enteroglucagon, but not of gastrin or cholecystokinin. Thus, the enhanced enterotrophic effects of bolus doses of long chain triglycerides could be mediated by release of a distally located gut peptide, perhaps enteroglucagon.
Peptide YY (PYY) reverses the increased intestinal secretion stimulated by vasoactive intestinal peptide (VIP) in humans. VIP also dilates blood vessels, so we investigated the effect of PYY on the cardiovascular system. Six volunteers received PYY, 0.4 and 1.2 pmol.kg-1 x min-1 i.v. for 2 h, reproducing plasma levels seen postprandially and during a diarrheal illness, respectively. Cardiac function was assessed by echocardiography. PYY infused at 0.4 pmol.kg-1 x min-1 had no effect on cardiovascular parameters. PYY infused at 1.2 pmol.kg-1 x min-1 caused a fall in both stroke volume from 128 +/- 8 to 110 +/- 8 ml/beat (mean +/- 95 confidence interval, P < 0.01) and cardiac output from 7.2 +/- 0.4 to 6.1 +/- 0.4 l/min (P < 0.01). Effects of infusion of PYY into the brachial artery at doses of 0-16 pmol/min were assessed using venous occlusion plethysmography in six subjects. PYY infusion caused a dose-dependent fall in forearm blood flow. Six subjects received VIP, 5 pmol.kg-1 x min-1 i.v., causing a rise in heart rate from 55 +/- 3 to 70 +/- 3 beats/min and increased cardiac output from 7.3 +/- 1.1 to 13.1 +/- 1.1 l/min. The addition of PYY, 0.4 pmol.kg-1 x min-1 i.v., did not affect the heart rate significantly but decreased the cardiac output to 10.4 +/- 1.1 l/min (P < 0.01). Infusions of PYY into the brachial artery at 5 pmol/min decreased local vasodilation induced by VIP infused at 2 pmol/min at the same site by 40% (P < 0.01), even though this dose of PYY had no significant effect on local blood flow when given alone.(ABSTRACT TRUNCATED AT 250 WORDS)
Neuromedin B is a 10-amino-acid mammalian peptide of the bombesin family. We have used a specific radioimmunoassay and Northern blot hybridisation to investigate the possible synthesis of neuromedin-B-like immunoreactivity in the human pituitary gland. The concentration of immunoreactive neuromedin B in whole human pituitary was 15.2 +/- 4.2 pmol/g wet weight in males and 12.8 +/- 2.7 pmol/g wet weight in females (mean +/- SEM, n = 10). In pituitary tumour extracts, neuromedin B immunoreactivity was 9.1 +/- 1.7 pmol/g wet weight (mean +/- SEM, n = 14) in inactive tumours, 18.4 +/- 6.9 pmol/g wet weight (mean +/- SEM, n = 4) in somatotrophs and 10.4 +/- 2.7 pmol/g wet weight (mean +/- SEM, n = 2) in prolactinomas, with no apparent significant difference between the groups. Gel permeation chromatography of pituitary extracts revealed two immunoreactive peaks, the major one of which corresponded in position to that of neuromedin B-32 and a later minor peak to the position of the neuromedin B-10 standard. On fast protein liquid chromatography, neuromedin-B-like immunoreactivity again eluted in two peaks, a minor peak corresponding to the synthetic neuromedin B standard, and a major more hydrophobic peak which was the big neuromedin B form. Northern blot analysis of poly(A)+RNA from human pituitaries revealed the presence of a hybridising band of between 750 and 850 base pairs. These results suggest that neuromedin B is synthesised in the human pituitary gland where it may be of importance in the regulation of pituitary function. Furthermore, the adenomatous condition is not associated with abnormal levels of this peptide.
Dogs were given a prostaglandin analogue, misoprostol, at a dose that significantly increases gastrointestinal epithelial cell proliferation. Both basal and postprandial concentrations of gastrin were significantly higher in the misoprostol-treated dogs and more than doubled after the meal in both the controls and in the test group. Plasma enteroglucagon, cholecystokinin, insulin and glucose-dependent insulinotrophic peptide all increased postprandially, with no effect of misoprostol. Tissue concentrations of bombesin, gastrin and somatostatin were unaffected by misoprostol, but the fundic glucagon-like immunoreactivity was significantly increased. Thus high doses of misoprostol have only minor effects on gastrointestinal regulatory peptides, suggesting that the trophic effect of prostaglandins on the intestinal tract may be direct.
Neuromedin-B shows a widespread distribution throughout mammalian neural and peripheral tissues and may be involved in the modulation of a variety of physiological processes. The highest concentrations of neuromedin-B are found in the anterior pituitary gland, suggesting that it may be of physiological importance within this tissue. To examine this hypothesis, we have used Northern blotting to demonstrate that neuromedin-B is present in the anterior pituitary as a result of local synthesis and have examined the effects of endocrine manipulations on its mRNA and immunoreactive peptide content. In thyroidectomized male rats, neuromedin-B content was decreased (104.1 +/- 5.8 vs. control, 390.9 +/- 23.3 fmol/gland; P less than 0.01) as was its mRNA (7 +/- 1.3% vs. 100%; P less than 0.014), while treatment of intact animals with T4 produced no effect on either peptide content or mRNA. Adrenalectomized male rats showed a significant increase in both neuromedin-B content (313.8 +/- 11.8 vs. control, 233.7 +/- 16.9 fmol/gland; P less than 0.05) and mRNA (377 +/- 27% vs. 100%; P less than 0.014), while dexamethasone treatment increased peptide content (347.8 +/- 32 vs. control, 233.7 +/- 16.9 fmol/gland; P less than 0.01) without any effect on mRNA levels. In female rats, ovariectomy decreased neuromedin-B content (132.4 +/- 13.3 vs. control, 335.0 +/- 37.2 fmol/gland; P less than 0.01) and mRNA levels (6 +/- 2% vs. 100%; P less than 0.014) while estrogen treatment of both ovariectomized and intact rats produced large increases in neuromedin-B (887.8 +/- 114.4 and 1328 +/- 175 fmol/gland, respectively, vs. control, 335.0 +/- 37.2 fmol/gland; both P less than 0.01) and its mRNA (246 +/- 18% and 378 +/- 31%, respectively, vs. 100%; both P less than 0.014). In castrated male rats, no significant alteration in peptide content was observed, and treatment of both castrated and intact male rats with testosterone was similarly without effect. These results demonstrate that 1) neuromedin-B is locally synthesized within the rat anterior pituitary gland; and 2) the local production of neuromedin-B is influenced by endocrine status, which is consistent with an autocrine/paracrine role for this peptide in this tissue.
The abundance of endothelin (ET)-producing endothelial cells in bone marrow and the proximity of these cells to bone-resorbing osteoclasts prompted us to evaluate the action of ET-1 on osteoclast function. Osteoclasts disaggregated from neonatal rat long bones were settled onto devitalized cortical bone substrate, and resorption was quantified by morphometry. The supernatant tartrate-resistant acid phosphatase activity was determined by a spectrophotometric method using paranitrophenol phosphate as substrate. Cell motility was quantified by time lapse video- and computer-assisted image processing using an empirical procedure for morphometric analysis. Cytosolic free calcium levels ([Ca2+]i) were measured in single cells by an indo 1-based microspectrofluorimetric method. Using the area of bone resorbed per slice as response, we found that ET-1 caused a significant (P = 0.011) concentration-dependent inhibition of osteoclastic bone resorption (EC50 = 2.5 nM) without inhibiting acid phosphatase secretion. Exposure of isolated osteoclasts to ET-1 also led to a marked concentration-dependent inhibition of osteoclast motility (EC50 = 7.9 nM; P = 0.013; t1/2 = 18 min) without significant effects on cell spread area. These effects of ET-1 were reversible after removing the peptide, and the cells remained viable during the experiments. In addition, ET-1 did not elevate [Ca2+]i at the concentrations tested. The results suggest that ET-1 specifically interacts with an osteoclast receptor to inhibit osteoclastic bone resorption and cell motility. As the concentration of ET-1 required for osteoclast inhibition was similar to that reported for smooth muscle contraction, it is possible that ET-1, produced locally from the bone marrow endothelial cell, might play a primary role in osteoclast regulation.
Islet amyloid polypeptide (IAPP) is a beta-cell peptide that can oppose insulin action in animal systems, but has not been shown to have any action in man; previously, we failed to show an effect of infused IAPP on iv glucose tolerance in human volunteers. We have reexamined its effects at even higher concentrations in six volunteers who received iv glucose (0.5 g/kg) during infusions of IAPP (25 and 50 pmol/kg.min) or normal saline. IAPP rose from a mean basal of 14.7 +/- 5.3 pmol/L to peak levels of 1,420 +/- 110, 2,240 +/- 140, and 27.7 +/- 9 pmol/L, respectively. IAPP at 25 pmol/kg.min had no effect on the plasma glucose disposal rate or the total incremental insulin response, but, in contrast, at 50 pmol/kg.min decreased the insulin response to glucose compared to the saline infusion (incremental area under the curve, 11,276 +/- 2,353 vs. 17,549 +/- 2,687 U; mean +/- SEM; P less than 0.02). This decrease was observed both during the first phase (0-10 min postglucose) insulin response (3,210 +/- 985 vs. 4,382 +/- 815 U; P less than 0.05) and the second phase response (11-90 min, 8,520 +/- 1,719 vs. 13,679 +/- 2,326 U; P less than 0.03). Glucose disposal rate, however, was unaffected (2.0 +/- 0.2 vs. 1.9 +/- 0.2). Thus, circulating IAPP concentrations greater than 90 times normal postprandial peaks were necessary to affect the insulin response to glucose. IAPP appears unlikely to be a circulating hormone influencing carbohydrate metabolism in man.
The local production of autocrine or paracrine agents in endocrine tissues represents an important level of hormonal regulation. The synthesis of neuropeptide-Y (NPY), substance-P (SP), and vasoactive intestinal peptide (VIP) in the rat anterior pituitary gland has been well demonstrated. We have now studied their expression in human postmortem pituitary tissue. Northern blot analysis of poly(A)+ RNA from whole human pituitaries revealed mRNA encoding the precursors for NPY, SP, and VIP whose hybridization characteristics were indistinguishable from those of the same mRNAs described in previously characterized human tissues. VIP mRNA was detectable in all samples tested, with NPY and preprotachykinin-A mRNA (which encodes SP) detectable in a subset of the pituitaries. The concentration of immunoreactive NPY in whole human pituitary was 3.8 +/- 1.1 pmol/g wet wt in males and 2.9 +/- 0.5 pmol/g wet wt in females (mean +/- SEM; n = 10), that of SP was 3.1 +/- 0.4 pmol/g wet wt in males and 5.2 +/- 1.3 pmol/g wet wt in females (n = 10), and that of VIP was 8.1 +/- 2.9 pmol/g wet wt in males and 5.3 +/- 1.6 pmol/g wet wt in females (n = 10). Size-fractionation of pituitary extracts by gel permeation chromatography revealed single peaks of NPY and VIP-like immunoreactivity in the positions of the standards, while SP-like immunoreactivity mostly eluted in the position of synthetic SP, with two minor immunoreactive peaks eluting earlier. The low levels of NPY, SP, and VIP and their mRNAs in the human pituitary are consistent with peptides having an autocrine/paracrine, rather than endocrine, mode of action.
Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide originally isolated from ovine hypothalami and so called because of its ability to stimulate pituitary adenylate cyclase activity. Alternative amidation and proteolytic processing of prepro-PACAP gives rise to two bioactive-amidated forms, PACAP-NH2(1-38) (PACAP-38) and PACAP-NH2(1-27) (PACAP-27). 7B2 is a polypeptide of 185 amino acids which is predominantly found in secretory granules and is widely distributed in rat and human tissues. We investigated the ability of the two forms of PACAP to stimulate GH, prolactin and 7B2 release by the rat pituitary clonal cell line GH3, and ACTH and 7B2 by the mouse pituitary clonal cell line AtT-20. PACAP-38 and PACAP-27 stimulated 7B2 and GH/prolactin or ACTH secretion with a similar efficacy over the 2-h incubation period from GH3 and AtT-20 cells respectively. 7B2 secretion was also stimulated by corticotrophin-releasing factor (CRF-41) and vasoactive intestinal polypeptide (VIP) in AtT-20 cells, and thyrotrophin-releasing hormone (TRH) and VIP in GH3 cells. Addition of PACAP to CRF-41 resulted in an additive effect on ACTH secretion and a synergistic effect on 7B2 secretion in AtT-20 cells. No synergism was observed when PACAP was added together with TRH, either on GH and prolactin secretion or on 7B2 release from GH3 cells. PACAP-mediated 7B2 secretion from both cell lines and PACAP-stimulated ACTH release from AtT-20 cells were reduced by 5 mg octapeptide synthetic somatostatin analogue/l (5 mg SMS 201-995/l).