Identification of progastrin in gastrinomas, antrum, and duodenum by a novel radioimmunoassay.

Recent studies on the gene sequence encoding the human pyloric antral hormone, gastrin, indicate a precursor of 101 residues. We have now raised antibodies to a synthetic analogue corresponding to (Tyr)-human progastrin COOH-terminal pentapeptide. The antibodies could be used in radioimmunoassay to measure this peptide, but they did not react with corresponding fragments of procholecystokinin, porcine progastrin, or other human progastrin-derived peptides, notably heptadecapeptide gastrin (G17), and 34-residue gastrin (G34). Radioimmunoassay of human antral and duodenal extracts revealed a major peak of activity that corresponded to the native COOH-terminal fragment of progastrin, and occurred in approximately equimolar amounts with COOH-terminal G17 immunoreactivity. In addition, there was a minor peak of apparently higher molecular weight material. In some gastrinomas the latter material was the predominant immunoreactive form, and it occurred in higher molar concentrations than any other form of gastrin. Digestion of this material with trypsin liberated peptides that reacted with antibodies specific for the NH2-terminus of G34, and G17. On this basis the high molecular weight component was identified as a form of gastrin that extended from the COOH-terminus of the precursor to a point at least beyond the NH2-terminus of G34, and probably included the entire progastrin sequence. The results suggest differences in posttranslational processing pathways of progastrin in antrum, duodenum, and gastrinomas. They also indicate that the present experimental approach allows the identification of progastrin-like substances, which should open the way to studying the mechanisms of gastrin biosynthesis.

Differential control of somatostatin messenger RNA in rat gastric corpus and antrum. Role of acid, food, and capsaicin-sensitive afferent neurons.

Somatostatin messenger RNA in the antrum and corpus of rat stomach was quantified by Northern and slot blotting using a probe generated by the polymerase chain reaction. Fasting for 48 h enhanced the abundance of somatostatin mRNA in the pyloric antral region, but not in the acid-secreting region of the stomach. In fasted rats, somatostatin mRNA in antrum, but not corpus, was decreased by inhibition of acid secretion with omeprazole. In contrast, in rats treated with capsaicin to lesion small diameter afferents there was a significant decrease in somatostatin mRNA abundance in the corpus but not antrum. The effects of capsaicin cannot be attributed to nonspecific changes in gastric endocrine cell gene expression, since the abundance of histidine decarboxylase mRNA (which is a functionally regulated marker for a different gastric endocrine cell type) did not change with capsaicin. Gastric capsaicin-sensitive afferents are rich in calcitonin gene-related peptide, and in rats with antibodies to this peptide there was reduced corpus somatostatin mRNA. Moreover, infusion of calcitonin gene-related peptide in control rats produced a significant increase in somatostatin mRNA in the gastric corpus. The results indicate that somatostatin mRNA abundance is controlled by the gastric luminal contents and the extrinsic afferent innervation, but the relative importance of these factors differs in antrum and corpus: luminal contents are relatively more important in antrum and primary afferents using calcitonin gene-related peptide in the corpus.

Feeding-dependent depression of melanin-concentrating hormone and melanin-concentrating hormone receptor-1 expression in vagal afferent neurones.

Food intake is regulated by signals from the gastrointestinal tract. Both stimulation and inhibition of food intake may be mediated by upper gastrointestinal tract hormones, e.g. ghrelin and cholecystokinin that act at least partly via vagal afferent neurones. We now report that vagal afferent neurones in both rat and man express melanin-concentrating hormone and its receptor, melanin-concentrating hormone-1R. In nodose ganglia from rats fasted for 24 h, RT-PCR revealed the expression of both melanin-concentrating hormone and melanin-concentrating hormone-1R, whereas in ganglia from animals fed ad libitum expression was virtually undetectable. Immunohistochemical studies also revealed expression of melanin-concentrating hormone and melanin-concentrating hormone-1R in nodose ganglion neurones of fasted rats, but signals were weak in rats fed ad libitum. Melanin-concentrating hormone and melanin-concentrating hormone-1R were expressed in the same neurones, a high proportion of which also expressed the cholecystokinin-1 receptor. When fasted rats were refed, there was down-regulation of melanin-concentrating hormone and melanin-concentrating hormone-1R expression over a period of 5 h. Similar effects were produced by administration of cholecystokinin to fasted rats. The cholecystokinin-1 receptor antagonist lorglumide inhibited food-induced down-regulation of melanin-concentrating hormone and melanin-concentrating hormone-1R. We conclude that the satiety hormone cholecystokinin acts on vagal afferent neurones to inhibit expression of melanin-concentrating hormone and its receptor. Since the melanin-concentrating hormone system is associated with stimulation of food intake this effect of cholecystokinin may contribute to its action as a satiety hormone.

Degradation of human gastrin and CCK by endopeptidase 24.11: differential behaviour of the sulphated and unsulphated peptides.

The degradation of human sulphated heptadecapeptide gastrin (G17s) by human endopeptidase 24.11 was studied in vitro. The products of degradation were characterized by HPLC, region-specific gastrin radioimmunoassay and amino acid analysis. The enzyme cleaved G17s at four sites, Trp4-Leu5, Ala11-Tyr12, Gly13-Trp14 and Asp16-Phe17. The patterns of fragments produced when sulphated and unsulphated G17s are hydrolysed by endopeptidase 24.11 indicate that the enzyme cleaves both substrates at the same four bonds. However, the sulphated G17 was 3-times less rapidly degraded than the unsulphated G17 (G17ns). In contrast, the rate of cleavage of the octapeptide cholecystokinin (CCK8) was faster when the peptide was sulphated. The kinetic data of endopeptidase 24.11 indicated similar Km values for sulphated or unsulphated gastrin and CCK; sulphated CCK8 exhibited a 2-fold higher kcat/Km value compared to unsulphated CCK8, whereas G17s exhibited a 2-fold lower kcat/Km value compared to G17ns. The results indicate that the presence of a sulphate group causes a marked reduction in the rate of hydrolysis of gastrin by endopeptidase 24.11, whereas sulphation enhances cholecystokinin degradation by the same enzyme. They also suggest that endopeptidase 24.11 may be responsible for the difference in metabolism of sulphated and unsulphated G17, previously observed in human circulation.

A novel gastrin-processing pathway in mammalian antrum.

An antiserum, L221, has been developed that is specific for the C-terminal region of the N-terminal tridecapeptide (i.e., 1-13) fragment of the acid-stimulating hormone, G17. In contrast to N-terminal G17 antisera previously used to estimate 1-13 G17, L221 does not cross-react with other N-terminal gastrin fragments or with C-terminal extensions of G17. Using L221 in conjunction with conventional gastrin antisera, and reversed-phase HPLC, it has been possible to identify in addition to 1-13 G17 a further, formerly unrecognised gastrin fragment, 1-11 G17, in stomach extracts. The production of 1-13 G17, 1-11 G17 and other gastrin forms such as the biologically active hexapeptide G6 which is known to occur naturally cannot be explained by tryptic cleavage of progastrin. Instead, their biosynthesis could be explained by the actions of an enzyme with an endopeptidase 24.11-like specificity. In porcine antrum, unsulphated and sulphated G17 are present in similar amounts, but unsulphated 1-13 G17 was about twice as abundant as sulphate 1-13 G17. This is consistent with previous in vitro findings that endopeptidase 24.11 has a higher affinity for the Ala-11-Tyr-12 and Gly-13-Trp-14 bonds in unsulphated G17, than in sulphated G17. The results suggest a novel albeit minor, processing pathway for gastrin biosynthesis in pig antrum involving an enzyme resembling endopeptidase 24.11.

Isolation, sequence and biosynthetic significance of a novel fragment of gastrin-releasing peptide from chicken proventriculus.

The isolation of bombesin-related peptides in chicken proventriculus was monitored by radioimmunoassay using a C-terminal specific bombesin antibody. Two peptides were identified, one corresponded to the 27-residue, chicken gastrin-releasing peptide (GRP-27) previously identified; the other corresponded to its C-terminal hexapeptide. Chicken GRP-27 stimulated pancreatic and gastric acid secretion in anaesthetized turkeys, but the hexapeptide was inactive. No evidence could be found to suggest that the hexapeptide was an artifact of degradation generated during extraction or isolation. It is proposed that the hexapeptide is produced either by chymotryptic-like cleavage of GRP-27 or by trypsin-like cleavage followed by two cycles of dipeptidylaminopeptidase cleavage. This type of biosynthetic processing may be more common than formerly supposed.

The isolation and chemical characterization of a novel vasoactive intestinal peptide-related peptide from a teleost fish, the cod, Gadus morhua.

An octacosapeptide that shows sequence homology to porcine vasoactive intestinal peptide (VIP) has been isolated from a teleost fish, the cod, Gadus morhua. The full primary sequence is His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Ser-Arg-Phe-Arg-Lys-Gln-Met-Ala-Ala- Lys-Lys-Tyr-Leu-Asn-Ser-Val-Leu-Ala. This peptide contains four, or five substitutions, compared with dogfish and porcine VIP, respectively. The residues in positions 13, 26 and 28 are different in all three species. These substitutions seem to have little effect on bioactivity, since cod VIP was virtually equipotent with porcine VIP in stimulating amylase release from guinea-pig pancreatic acini. During the isolation procedure an N-terminally modified form of VIP (Des-His, or 2-28 cod VIP) was also isolated. The available data suggest the sequence of VIP is well conserved in vertebrates which is consistent with an important biological role.

A novel vasoactive intestinal peptide (VIP) from elasmobranch intestine has full affinity for mammalian pancreatic VIP receptors.

A peptide that cross reacted with N-terminal, but not C-terminal, antisera to vasoactive intestinal peptide (VIP) was isolated from extracts of intestine from the dogfish Scyliorhinus canicula. Microsequence analysis gave the structure His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Ser-Arg-Ile-Arg-Lys-Gln-Met-Ala-Val-Lys - Lys-Tyr-Ile-Asn-Ser-Leu-Leu-Ala-NH2. C-terminal amidation was determined by HPLC analysis of phenylthiocarbamyl amino acid derivatives after carboxypeptidase Y digestion. The peptide differs at five positions from the porcine octacosapeptide. Dogfish VIP was equipotent with its porcine counterpart in inhibiting binding of 125I-labelled VIP to guinea pig dispersed pancreatic acini, and in stimulating amylase secretion by the same preparation. The data indicate a strong conservation of VIP during evolution and permit identification of residues crucial for bioactivity.

The isolation and sequence analysis of vasoactive intestinal peptide from a ganglioneuroblastoma.

A ganglioneuroblastoma was excised at surgery from a 1-yr-old girl with severe watery diarrhea. The tumor, weighing 1 g, was extracted in trifluoracetic acid and contained 8.3 nmol immunoreactive vasoactive intestinal peptide. The peptide was isolated by affinity chromatography and high pressure liquid chromatography and was found to be identical to porcine vasoactive intestinal peptide by amino acid analysis and microsequence analysis.

Chromogranin A and pancreastatin-like immunoreactivity in normal pregnancies.

Placenta is a neuroendocrine organ, and we therefore wanted to study the occurrence of the general neuroendocrine marker chromogranin A (CgA) and its split product pancreastatin. CgA and pancreastatin-like immunoreactivity (PST-LI) were determined by ELISA and RIA methods, respectively, in homogenates from term placentas, sera from pregnant women, nonpregnant women, umbilical cords, and in amniotic fluids. In placental homogenates, the mean level of CgA was 7.1 +/- 8.6 pmol/g wet wt (mean +/- SD), whereas PST-LI was not detectable. CgA immunoreactivity was demonstrated by immunofluorescence studies of isolated trophoblasts and decidual cells from term placentas. In trophoblasts, CgA was colocalized with human chorionic gonadotropin (hCG) and human placental lactogen. By Northern blotting, a distinct band corresponding to CgA messenger RNA (mRNA) was demonstrated in the placental cell line, whereas, in placental homogenates, a mRNA band of a slightly larger size was found. Median CgA level in maternal sera at term tended to be higher (median: 469 pmol/L, range 61-980 pmol/L, P < 0.1) than at 6-11 weeks (286 pmol/L, 61-653 pmol/L) or in sera from nonpregnant women (306 pmol/L, 204-469 pmol/L). In umbilical cord sera, median CgA level was significantly higher (898 pmol/L, 102-2245 pmol/L, P < 0.05) than in term sera. Median serum level of PST-LI was significantly higher at term (38 pmol/L, 0-131 pmol/L) than at 6-11 weeks (9 pmol/L (0-85 pmol/L, P < 0.05), than in nonpregnant women (6 pmol/L, 0-52 pmol/L, P < 0.05), and in umbilical cord sera (12 pmol/L, 0-76 pmol/L, P < 0.05). In amniotic fluid, median CgA value was significantly higher at term (1163 pmol/L, 714-1673 pmol/L) than at 14-17 weeks (551 pmol/L, 82-980 pmol/L, P < 0.01), whereas median level of PST-LI was significantly higher at 14-17 weeks (32 pmol/L, 6-97 pmol/L) than at term (0 pmol/L, 0-15 pmol/L, P < 0.01). To our knowledge, this is the first report describing the presence of CgA and PST-LI in placenta and amniotic fluid and the occurrence CgA mRNA in placental tissue and in a placental cell line. The presence of CgA in placenta may indicate a physiological role in pregnancy.

Neurochemical activities in human temporal lobe related to aging and Alzheimer-type changes.

Activities relating to 3 neurotransmitter and 4 neuropeptide systems have been examined in human temporal lobe (post mortem) for their relationships with age and Alzheimer-type changes (senile plaques and cognitive function). Significant alterations with increasing age (from 61 to 92 years) in a series of non-demented cases included a reduction of the cholinergic enzyme, choline acetyltransferase, and an increase in vasoactive intestinal peptide immunoreactivity. In cases of alzheimer's disease the only neurochemical activity investigated which correlated significantly with cognitive impairment (assessed from a Mental Test Score obtained shortly before death) and with the severity of Alzheimer-type abnormalities (senile plaques density) was choline acetyltransferase. Further analyses of the data in relation to the severity of plaque formation suggest that alterations in other neurochemical activities including reductions in dopamine-beta-hydroxylase activity, cholecystokinin octapeptide (aqueous extracted) and somatostatin immunoreactivities and an increase in substance P immunoreactivity, may occur at later stages of the disease process. These comparative data suggest that biochemical changes in this brain area associated with age and earlier stages of Alzheimer's disease may be relatively selective.

Histidine decarboxylase gene expression in rat fundus is regulated by gastrin.

The conversion of histidine to histamine by histidine decarboxylase (HDC) is of central importance in the control of vertebrate acid secretion. We have used PCR-generated probes to study the regulation of HDC gene expression in rat fundic mucosa. When circulating gastrin levels were lowered by fasting or elevated by treatment with omeprazole, there were parallel changes in HDC mRNA abundance. However, when animals with elevated gastrin levels were concurrently treated with the gastrin/CCK-B receptor antagonist PD 134308, HDC mRNA levels were not increased. These data are consistent with the hypothesis that HDC gene expression is regulated by gastrin, over the physiological range of circulating hormone concentrations.

Isolation from chicken antrum, and primary amino acid sequence of a novel 36-residue peptide of the gastrin/CCK family.

A peptide that cross-reacted with C-terminal gastrin/CCK antisera was isolated from chicken antral extracts by a combination of gel filtration and reversed-phase HPLC. The sequence was: Phe-Leu-Pro-His- Val-Phe-Ala-Glu-Leu-Ser-Asp-Arg-Lys-Gly-Phe-Val-Gln-Gly-Asn-Gly-Ala- Val-Glu-Ala-Leu-His-Asp-His-Phe-Tyr-Pro-Asp-Trp-Met-Asp-Phe(NH2). Aside from the C-terminal tetrapeptide and the Tyr residue, the molecule does not resemble other known forms of gastrin or CCK. The peptide was a potent stimulus of avian gastric acid but not pancreatic secretion. The results have important implications for the structure-activity and evolutionary relationships of the gastrin/CCK family.

Functional control of gastrin releasing peptide (GRP) mRNA in rat stomach.

The gastric factors controlling abundance of mRNA encoding the important neuropeptide, gastrin releasing peptide (GRP) in rat stomach, were examined by Northern and slot blot analysis. Withdrawal of food increased antral GRP mRNA, as did treatment of fed rats with the acid inhibitory drug, omeprazole. There was no change in GRP mRNA abundance in gastric corpus. The data indicate functionally distinct populations of GRP neurons in different regions of the stomach, and control of antral neuropeptide biosynthesis by the gastric luminal contents.

Antibodies to fragments of provasoactive intestinal peptide reveal subpopulations of vasoactive intestinal peptide containing neurons in the rat gut.

The cellular origin of peptides derived from preprovasoactive intestinal peptide has been studied in rat stomach and ileum. Antisera specific for the C-terminal regions of the N-terminal flanking peptide (preprovasoactive intestinal peptide 22-80), bridging peptide (preprovasoactive intestinal peptide 111-124), C-terminal flanking peptide (preprovasoactive intestinal peptide 156-170) and vasoactive intestinal peptide were used in immunohistochemical studies on sections and whole mounts. All four antisera stained nerve fibres and cell bodies in the stomach and intestine. However, there were distinct differences in the pattern of colocalization of peptides derived from provasoactive intestinal peptide. In the sub-mucous plexus of the ileum virtually 100% of neurons reacting with vasoactive intestinal peptide antibodies also reacted with antibodies to the other three peptides. In contrast, in the stomach, while all vasoactive intestinal peptide-immunoreactive neurons of the myenteric plexus contained C-terminal flanking peptide- and bridging peptide-like immunoreactivity, only 50% of these cells reacted with the antiserum to N-terminal flanking peptide. The data indicate that in a population of neurons in the myenteric plexus of the rat stomach, preprovasoactive intestinal peptide is processed in such a way that the antigenic determinant of the N-terminal flanking peptide is not produced. In a second population of enteric neurons in the stomach, and in the intestine, it appears that processing of preprovasoactive intestinal peptide results in the production of peptides reacting with antibodies to vasoactive intestinal peptide, the flanking and bridging peptides.

Characterization of cholecystokininA and cholecystokininB receptors expressed by vagal afferent neurons.

The cholecystokinin receptors expressed by vagal afferent neurons mediate the effect of cholecystokinin in inhibiting food intake and gastric emptying. We have determined the relative abundance of cholecystokininA, gastrin-cholecystokininB and gastrin-cholecystokininC receptor populations in the rat vagus by autoradiography using [125I]Bolton Hunter-cholecystokinin-8, [125I]Bolton Hunter-heptadecapeptide gastrin and [125I]Leu(15)2-17Glycine-extended heptadecapeptide gastrin, together with the selective antagonists devazepide and L-740093. The results indicate approximately three-fold higher abundance of cholecystokininA compared with gastrin-cholecystokininB receptors, and no significant representation of gastrin-cholecystokininC receptors. Topical capsaicin applied to the vagal nerve trunk abolished the accumulation of sites binding both [125I]Bolton Hunter-labelled cholecystokinin-8 and heptadecapeptide gastrin indicating that both cholecystokininA and gastrin-cholecystokininB receptor populations were present on afferent fibres. The molecular identity of the receptors expressed by rat and human nodose ganglia was examined using the reverse transcription polymerase chain reaction. Products of the predicted size for the cholecystokininA and gastrin-cholecystokininB receptors were identified. The human and rat cholecystokininA receptor products were cloned and the sequences were found to be 99% homologous to those published for receptors expressed by rat pancreas and human gall bladder. We conclude that cholecystokininA and gastrin-colecystokininB receptors are synthesized by nodose ganglion cells, and that the receptor proteins are transported to the periphery along afferent fibres. While there is a clear role for vagal cholecystokininA receptors, the function of vagal afferent gastrin-cholecystokininB receptors remains to be determined.

Expression of the leptin receptor in rat and human nodose ganglion neurones.

There is evidence for interactions between leptin and cholecystokinin in controlling food intake. Since cholecystokinin acts on vagal afferent neurones, we asked whether the leptin receptor was also expressed by these neurones. Primers for different forms of the leptin receptor were used in reverse transcriptase-polymerase chain reaction (RT-PCR) of rat and human nodose ganglia. RT-PCR yielded products corresponding to the long (functional) form as well as short forms of the rat leptin receptor. Moreover, RT-PCR revealed the long form of the leptin receptor in a human nodose ganglion. The identities of RT-PCR products were confirmed by sequencing. Primers corresponding to leptin itself did not give RT-PCR products in nodose ganglia. Immunocytochemical studies revealed leptin-receptor immunoreactivity in neuronal cell bodies. Many neurones co-expressed the leptin and cholecystokinin type A receptors, or leptin receptor and cocaine- and amphetamine-related transcript. We conclude that vagal afferent neurones that express the cholecystokinin type A receptor and cocaine- and amphetamine-related transcript, may also express the long form of the leptin receptor providing a neurochemical basis for observations of interactions between cholecystokinin and leptin.

Carbachol stimulation of gastric acid secretion and its effects on the parietal cell.

1. The acid secretagogue effect of gastrin is mainly mediated by the release of enterochromaffin-like (ECL) cell histamine, but the mechanism of muscarinic stimulation of acid secretion remains unclear. The results of studying aminopyrine uptake in isolated parietal cells, and histamine release in isolated ECL cells suggest that muscarinic agents may act both directly on the parietal cell and indirectly via histamine release from ECL cells. 2. We examined parietal and ECL cell responses to the muscarinic agent carbamylcholine (carbachol) in conscious rats and in rat isolated vascularly perfused stomachs. 3. Intravenous carbachol stimulated acid secretion in conscious gastric fistula rats and increased H+K+ ATPase mRNA abundance, indicating activation of parietal cells. In these experiments there was no increase in portal venous histamine, or in oxyntic mucosal histidine decarboxylase (HDC) enzyme activity and HDC mRNA abundance. 4. In rat isolated stomachs stimulated with carbachol in the dose range 10 nM(-1) mM only the 1 microM concentration increased venous histamine significantly. 5. We concluded that the muscarinic agent carbachol stimulates acid secretion and H+K+ ATPase mRNA in vivo by a direct effect on the parietal cell, that does not depend on the release of ECL cell histamine.

Studies on the renin response to vasoactive intestinal polypeptide (VIP) in the conscious rabbit.

Vasoactive intestinal polypeptide (VIP) has been found within the renal cortex and is believed to be a neurotransmitter. Although it produces systemic vasodilatation and renin release, the exact mechanism of the latter response is uncertain. When infused into conscious rabbits, VIP elicits a rise in plasma renin activity (PRA) and an increase in heart rate. The rise in PRA is unaltered by pretreatment with propranolol, but is attenuated by indomethacin. The tachycardia is inhibited by propranolol, but unaffected by indomethacin. We conclude that the renin response to VIP is in part prostaglandin-dependent and that the heart rate response is due to direct or reflex beta-adrenoceptor stimulation.

Cholecystokinin octapeptide in rat central nervous system: Immunocytochemical studies using a monoclonal antibody that does not react with CGRP.

The biological activity of the important neuropeptide cholecystokinin octapeptide (CCK8) resides at the C-terminus. Antibodies with C-terminal specificity have been reported to cross-react with a different neuropeptide, calcitonin gene related peptide (CGRP) and this has frustrated the interpretation of immunohistochemical studies. We describe here the properties of a monoclonal antibody to the CCK-related peptide, caerulein, that reacts with the C-terminal region of CCK8, but does not react with CGRP in radioimmunoassay or immunohistochemistry. The distribution of CCK-like activity revealed by immunohistochemistry using this antibody broadly resembles that described previously with a single major exception: in the dorsal horn of the spinal cord. The results support the suggestion that apparent CCK activity in the terminals of rat primary sensory neurones is due to cross-reactivity with CGRP.