J.M. Conlon

Measurement and partial characterization of the multiple forms of neurokinin A-like immunoreactivity in carcinoid tumours

An antiserum raised against neurokinin A has been used to demonstrate storage and release of neurokinin A-like immunoreactivity by carcinoid tumours. The antiserum showed reactivity towards members of the tachykinin family of polypeptides in the order: neurokinin A greater than eledoisin greater than neurokinin B greater than kassinin greater than substance P greater than physalaemin but the magnitude of the cross-reactivity with substance P and physalaemin was less than 1% of that of neurokinin A. A sensitive (IC50 238 fmol/ml; minimum detectable concentration, 9 fmol/ml) radioimmunoassay was set up using this antiserum. Extracts of metastatic tumour tissue from four patients with a primary carcinoid tumour in the midgut contained both neurokinin A-like immunoreactivity (NKA-LI) and substance P-like immunoreactivity (SP-LI). The concentrations (pmol/g wet weight) of NKA-LI and SP-LI in the tumours were: patient A 210, 201; patient B 2276, 6849; patient C 1198, 834 and patient D 424, 379. Analysis of the tumour extracts by reverse phase HPLC indicated that the NKA-LI was heterogeneous. Under two different conditions of chromatography, one component was eluted with the same retention time as neurokinin A. Two further components were more hydrophobic than neurokinin A but were not eluted with the retention time of neurokinin B. Analysis of these components by gel filtration indicated a molecular weight in the 3000-4000 range suggesting that they may be related to neuropeptide K, an N-terminally extended form of neurokinin A. NKA-LI and SP-LI were undetectable in the plasma of patients A and D but were elevated in patient B (NKA-LI 1005 +/- 114; SP-LI 345 +/- 85 fmol/ml) and patient C (NKA-LI 80 +/- 31; SP-LI 21 +/- 13 fmol/ml).

Characterization of an amidated form of pancreatic polypeptide from the daddy sculpin (Cottus scorpius)

The primary structure of pancreatic polypeptide from the teleostean fish, Cottus scorpius (daddy sculpin) was established as: YPPQPESPGGNASPEDWAKYHAAVRHYVNLITRQRYNH2 The presence of a COOH-terminally alpha-amidated amino acid was established using an HPLC method of general applicability. Although the peptide shows strong homology towards anglerfish pancreatic polypeptide (86%), homology towards porcine peptide YY (PYY) (61%) and porcine neuropeptide Y (NPY) (61%) was greater than towards porcine pancreatic polypeptide (PP) (47%). This result supports suggestions that the gene duplication events which led to PP, NPY and PYY formation took place after the time of divergence of fish and mammals.

Circulating Tachykinins (Substance P, Neurokinin A, Neuropeptide K) and the Carcinoid Flush

Antisera of defined regional specificity have been used to measure the concentration of substance P-like immunoreactivity (SP-LI) and neurokinin A-like immunoreactivity (NKA-LI) during a meal-induced flush in 10 patients with metastatic carcinoid tumours. Although all patients flushed, NKA-LI levels in five patients and SP-LI in six patients were not elevated relative to healthy subjects (NKA-LI, less than 3 pg/ml; SP-LI, less than 10 pg/ml) both in the fasted state and after food. In the patients with elevated basal plasma tachykinin levels, increases in NKA-LI and SP-LI after food were erratic and did not correspond to a defined digestive phase or the occurrence of the flush. Chromatographic analysis of plasma demonstrated the presence of neuropeptide K and neurokinin A, and the detection of COOH-terminal fragments of substance P is consistent with the higher levels of circulating SP-LI measured with a COOH-terminally directed antiserum compared with an NH2-terminally directed antiserum. Subcutaneous injection of the somatostatin analogue SMS 201-995 (50 micrograms) alleviated symptoms of flush in two of three patients but only partially suppressed NKA-LI and SP-LI concentrations. It is concluded that circulating tachykinins cannot be solely responsible for the meal-induced carcinoid flush.

Proglucagon-derived peptides: nomenclature, biosynthetic relationships and physiological roles

The decade of the 1980s has seen enormous advances in the field of molecular endocrinology brought about in large measure by application of the methods of recombinant DNA technology. The increase in our knowledge and understanding of the glucagon-related peptides provides an excellent illustration of this point. Cloning and sequence analysis of cDNAs and DNA fragments from genomic libraries has led to the elucidation of the primary structure of the biosynthetic precursor of glucagon (pre-proglucagon) from a variety of species including man [1-6]. With this information, it has been possible to interpret the complex structural relationships between the multiple forms of the glucagon-like peptides in tissues and in the circulation. Probably the most exciting development in the field is the realisation that the pre-proglucagon gene in mammals encodes, in addition to glucagon, two additional peptides with structural similarity to glucagon, termed glucagon-like peptide-I (GLP-1) and glucagon-like peptide-2 (GLP-2). This review attempts to summarise recent advances with a view to clarifying previous ambiguities regarding the nomenclature and biosynthetic relationships between the peptides and speculates as to the physiological role of the peptides.

Primary structure of glucagon and a partial sequence of oxyntomodulin (glucagon=37) from the guinea pig

Two glucagon-like peptides have been isolated from guinea pig pancreas and their primary structures determined. A 29-residue peptide is identical to the glucagons from all other mammals yet studied in the N-terminal region (residues 1-20) but the C-terminal region [Gln-Phe-Leu-Lys-Trp-Leu-Leu-Asn-Val] contains five substitutions. A 37-residue peptide probably represents guinea pig glucagon extended from the C-terminus by [Lys-Arg-Asn-Arg-Asn-Asn-Ile-Ala] and is analogous to human oxyntomodulin (glucagon-37). The structures suggest evolutionary pressure to conserve the N-terminal region of glucagon and the C-terminal region of oxyntomodulin. The biological activity of guinea pig glucagon has not yet been determined but it is speculated that changes in the C-terminal region of glucagon may have produced a molecule with reduced biological potency that is appropriate to the reduced potency of guinea pig insulin.

CCK-antagonist L-364,718: influence on rat pancreatic growth induced by caerulein and bombesin-like peptides

The present study investigates the inhibitory effect of the novel potent benzodiazepine-related CCK-antagonist L-364,718 on pancreatic growth in the rat induced by chronic administration of caerulein and bombesin-like peptides. Caerulein, injected s.c. twice daily at a dose of 1 microgram/kg body weight, and bombesin (10 micrograms/kg) induced a similar increase (1.5-3-fold) in pancreatic wet weight, total protein, amylase, trypsin, putrescine and spermidine content after 14 days of treatment. Growth induced by caerulein showed a significant increase in total DNA content suggesting cellular hyperplasia, whereas bombesin-like peptides led to cellular hypertrophy. In comparison to bombesin the decapeptide neuromedin C (10 micrograms/kg) was found to be 30-50% less potent. In the same dose range, neuromedin B and the tachykinins neurokinin A and B, all structurally related to bombesin, had no significant trophic effect on the rat pancreas. Administration of the CCK-antagonist L-364,718 twice daily at a dose of 0.1 mg/kg or at 1.0 mg/kg, either s.c. or orally, led dose-dependently to a near-complete inhibition of the caerulein-induced trophic effect. In contrast, L-364,718 administered at identical dosages, did not affect pancreatic hypertrophy induced by bombesin and neuromedin C. It is concluded that both peptides mediate their effect on the rat pancreas directly and not via release of endogenous cholecystokinin. Tachykinins are not involved in the regulation of pancreatic growth. Caerulein- and bombesin-like peptides have comparable effects on the stimulation of protein and polyamine synthesis.

Substance P, neurokinin A, vasoactive intestinal polypeptide and gastrin releasing peptide in the intestine and pancreas of spontaneously obese-diabetic mice

The concentrations and contents of immunoreactive substance P (SP), neurokinin A (NKA), vasoactive intestinal polypeptide (VIP) and gastrin releasing peptide (GRP) were measured in acid-ethanol extracts of intestine (duodenum-jejunum-ileum) and pancreas of C57BL/KsJ diabetes-obese (db/db) mice, Aston obese-hyperglycaemic (ob/ob) mice, and their respective lean controls. The intestinal concentration of GRP and pancreatic concentrations of VIP and GRP were 36-57% lower in lean Aston mice than lean C57BL/KsJ mice, indicating the influence of genetic background in control mice. Intestinal concentrations of SP and NKA were reduced by 19-33% in the db/db and ob/ob mutants compared with their lean controls, but the intestinal contents of these peptides were normal or greater than normal due to intestinal hypertrophy of the mutant mice. The intestinal VIP concentration was not altered, but the content was increased by 87% and 25% respectively in db/db and ob/ob mice, whereas the intestinal GRP concentration was reduced by 51% in ob/ob mice. Pancreatic concentrations and contents of NKA, VIP and GRP were similar in lean and db/db C57BL/KsJ mice. However, pancreatic concentrations and contents of VIP and GRP were reduced by 51-55% in ob/ob mice compared with their lean controls. The sensitivity of the present assay did not permit accurate determination of the low pancreatic concentrations of SP. The results suggest that the spontaneous ob/ob and db/db syndromes of obesity and diabetes in mice are associated with reduced intestinal concentrations of SP and NKA. The ob/ob mouse also exhibited reductions of intestinal GRP and pancreatic GRP and VIP concentrations. These changes in regulatory peptides may relate to abnormalities of intestinal and possibly pancreatic function in obese and diabetic mutant mice.

Sulphated CCK-8-like peptides in the neural ganglion of the protochordate Ciona intestinalis.

Immunochemical studies were carried out on extracts of the neural ganglion from the ascidian Ciona intestinalis in order to the characterize the peptide(s), which react with antibodies against the C-terminal sequence common for the mammalian hormones, cholecystokinin (CCK) and gastrin. Radioimmunoassays specific for the sulphotyrosyl-containing N-terminus of CCK-8, for the common alpha-carboxyamidated C-terminus and for gastrin were used to monitor gel chromatography and reverse-phase HPLC of the extracts. Only neutral extracts contained immunoreactive material (634 (524-785) pmol eqv.CCK-8/g) (mean and range, n = 4)). HPLC revealed a small peak eluting almost like CCK-8 and a larger peak eluting earlier. By subsequent gel chromatography the larger peak eluted in the same position as sulphated CCK-8. The material was recognized almost equally by the N- and C-terminal CCK radioimmunoassays, whereas the specific C-terminal gastrin radioimmunoassay did not measure the peptides. Treatment with arylsulphatase removed the binding to the antiserum specific for the sulphotyrosyl-containing sequence of CCK. The results indicate that the ganglion of Ciona intestinalis contains a tyrosyl-sulphated peptide resembling mammalian CCK-8.

Effects of a transplantable insulinoma upon regulatory peptide concentrations in the gastrointestinal tract of the rat

SummaryThe rapid growth (0.8±0.3 g/day) of a transplantable insulinoma, which also contained substance P (2.9±2.3 pmol/g) and gastrin-releasing peptide (3.2± 2.1 pmoll/g), resulted in the development of hyperphagia, hyperinsulinaeinia and hypoglycaemia in rats (n=8). After a 14-day growth period, the insulinoma-bearing rats showed an increase (49%; p<0.01) in the weight of the small intestine but no significant change in stomach weight compared with control animals. The content (pmol/organ) of somatostatin, substance P, neurokinin A and vasoactive intestinal peptide in the stomachs of the tumour rats was unchanged. A depletion in the content (53% p<0.01) and concentration (57%; p<0.01) of gastrin-releasing peptide, however, suggested either hypersecretion, possibly mediated through hypoglycaemia-induced vagal stimulation, or inhibition of synthesis. The concentration and content of glucagon-like immunoreactivity (enteroglucagon) in the small intestine of the insulinoma rats increased markedly (47%; p<0.01 and 120%; p<0.01). This increase is consistent with a proposed role of this peptide as a factor trophic to the intestinal mucosa. No significant changes in the concentrations of somatostatin, substance P, neurokinin A, vasoactive intestinal peptide and gastrin-releasing peptide in the small intestine were observed. However, the increase in gut weight resulted in a greater content of vasoactive intestinal peptide (40%; p<0.01) and substance P (37%; p<0.05) in the insulinoma rats.