Caerulein secretion by dermal glands in Xenopus laevis.

Since gastrin and its related peptides are secreted by a minority population of widely dispersed cells in mamamalian tissues it has, in the past, been difficult to study the subcellular aspects of their secretion. From published reports (1, 2) it seemed possible that a satisfactory system for such studies might be provided by the skin of certain amphibians such as Xenopus laevis since in these tissues high concentrations of peptides such as caerulein exist, and there is some indication (3) that this, or a similar gastrin-like peptide, may be a dermal gland secretory product. We have therefore explored this possibility by studying the structure, secretory process, and secretory product of the most prominent non mucous type of gland in the skin of X. laevis. These studies clearly demonstrate that most, if not all, of the caerulein in which the skin is contained in secretion granules within the dermal glands and that its release can be specifically evoked by adrenergic stimulation. The release process by a holocrine mechanism expels all of the stored secretion onto the skin surface and thus for biosynthetic studies it should now be possible to synchronize the processes which lead to the replenishment of the peptide.

Substance P and cholecystokinin-like peptides in Helix neurons and cholecystokinin and serotonin in a giant neuron.

Immunohistochemical procedures revealed that the serotonin-containing cell situated in each metacerebral ganglion of the snail Helix also contains a cholecystokinin-like peptide, but is devoid of substance P. In radioimmunoassays the cholecystokinin-like material showed similarities to the carboxy terminal regions of mammalian cholecystokinin and gastrin. Since much is known about the morphology and synaptic connections of this serotonin neuron, the role of the cholecystokinin-like peptides can now be investigated by neurophysiological methods, thus opening the way to discovering whether a neuron can use more than one transmitter.

Hypergastrinemia increases gastric epithelial susceptibility to apoptosis.

Plasma concentrations of the hormone gastrin are elevated by Helicobacter pylori infection and by gastric atrophy. It has previously been proposed that gastrin acts as a cofactor during gastric carcinogenesis and hypergastrinemic transgenic INS-GAS mice are prone to developing gastric adenocarcinoma, particularly following H. pylori infection. We hypothesised that the increased risk of carcinogenesis in these animals may partly result from altered susceptibility of gastric epithelial cells to undergo apoptosis. Gastric corpus apoptosis was significantly increased 48 h after 12Gy gamma-radiation in mice rendered hypergastrinemic by transgenic (INS-GAS) or pharmacological (omeprazole treatment of FVB/N mice) methods and in both cases the effects were inhibited by the CCK-2 receptor antagonist YM022. However, no alteration in susceptibility to gamma-radiation-induced gastric epithelial apoptosis was observed in mice overexpressing progastrin or glycine-extended gastrin. Apoptosis was also significantly increased in gastric corpus biopsies obtained from H. pylori-infected humans with moderate degrees of hypergastrinemia. We conclude that hypergastrinemia specifically renders cells within the gastric corpus epithelium more susceptible to induction of apoptosis by radiation or H. pylori. Altered susceptibility to apoptosis may therefore be one factor predisposing to gastric carcinogenesis in INS-GAS mice and similar mechanisms may also be involved in humans.

Pathways of processing of the gastrin precursor in rat antral mucosa.

The precursor of the acid-stimulating hormone gastrin gives rise to multiple peptides differing markedly in biological activity, but the relevant biosynthetic pathways are poorly understood. We have used antibodies to amidated gastrins, gastrins with COOH-terminal glycine (Gly) gastrins with COOH-terminal hydroxyglycine (GlyOH) and to the COOH terminus of progastrin, to immunoprecipitate peptides labeled with [35S]sulfate or [3H]tyrosine during incubation of rat antral mucosa in vitro. Labeled progastrin was detectable after 30 min of continuous incubation with isotopic precursors, G34 and G34-Gly after 60 min, and G17 and G17-Gly after 120 min. Pulse chase experiments indicated that progastrin is converted to G34-Gly which then follows one of two pathways: (a) hydroxylation of COOH-terminal Gly and conversion to G34 followed by cleavage yielding G17, or (b) cleavage to G17-Gly. The kinetics of G17-Gly and G17 labeling were similar, suggesting that G17-Gly is a product in its own right, and not simply an intermediate in G17 synthesis. Since the two peptides are reported to have distinct biological activities, they appear to be alternative mature products of progastrin processing.

Identification and measurement of molecular variants of cholecystokinin in duodenal mucosa and plasma. Diminished concentrations in patients with celiac disease.

The amount and type of cholecystokinin (CCK) in duodenal extracts and plasma of celiac patients and normal subjects was studied by radioimmunoassay and gel filtration. In both groups there were similar patterns of molecular forms in extracts of duodenal biopsies, but concentrations in celiac disease were significantly depressed. In boiling water extracts of duodenal mucosa from both groups a factor with the properties of the COOH-terminal octapeptide of cholecystokinin predominated, but there were also significant amounts of a larger molecular weight form. In acid extracts of mucosa a factor with the properties of the 33 or 39 residue form was identified in amounts that were approximately 25% those of CCK8; there were also similar amounts of an acid-soluble form that had an apparent molecular weight higher than CCK39. Plasma immunoreactive cholecystokinin was studied after concentration by immunoaffinity adsorption and fractionation by gel filtration. In normal subjects fasting CCK-like immunoreactivity was less than 0.8 pmol/liter, and after a light breakfast increased to 2.0 +/- 0.7 (range 1.0 to 4.8) pmol/liter; CCK8-like activity accounted for all the increased immunoreactivity. In five of six celiac patients the concentrations of both fasting and postprandial CCK-like immunoreactivity in plasma were undetectable (less than 0.8 pmol/liter). We conclude that diminished production and release of CCK could account for the impaired pancreatic and gall bladder responses to intraluminal stimuli in celiac disease.

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.

Metabolism of heptadecapeptide gastrin in humans studied by region-specific antisera.

The metabolism of synthetic human heptadecapeptide gastrin (G17) in vivo, and in serum in vitro, was studied by radioimmunoassay using region specific antisera, gel filtration, ion exchange chromatography, and high performance liquid chromatography. After infusion of G17 intravenously in normal human volunteers, COOH-terminal and NH2-terminal immunoreactive G17 fragments were generated. At a steady state, approximately 15% of COOH-terminal immunoreactivity was attributable to G14-like material and up to 25% of total NH2-terminal immunoreactivity was attributable to two NH2-terminal fragments; one had the chromatographic properties of 1-13 G17, and the other was less acidic and less hydrophobic. After stopping the infusion of G17, the latter fragments accounted for progressively greater proportions of total gastrin activity. When G17 was incubated in serum in vitro, there was time-dependent and temperature-dependent loss of immunoreactivity, and again COOH-terminal and NH2-terminal immunoreactive fragments were formed. Removal of the NH2-terminal pyroglutamic acid was probably the rate limiting step because synthetic 2-17 G17 was degraded more rapidly in serum (t1/2, 2-3 h) than G17 (t1/2, 3-5 h). EDTA blocked degradation at the COOH-terminus of both 2-17 G17 and G17 but cleavage at the NH2-terminus still occurred, giving rise to a G14-like peptide. The rate of conversion of G17 in serum was not enough to account for the production of fragments in vivo, and it is proposed that these are formed when G17 encounters enzymes on cell surfaces, perhaps during passage through the capillary circulation. The production of these fragments needs to be considered in interpreting studies of the identity, metabolism, and release of gastrin in health and disease.

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.

Gastrin is a target of the beta-catenin/TCF-4 growth-signaling pathway in a model of intestinal polyposis.

Mutations in the adenomatous polyposis coli (APC) tumor suppressor gene occur in most colorectal cancers and lead to activation of beta-catenin. Whereas several downstream targets of beta-catenin have been identified (c-myc, cyclin D1, PPARdelta), the precise functional significance of many of these targets has not been examined directly using genetic approaches. Previous studies have shown that the gene encoding the hormone gastrin is activated during colon cancer progression and the less-processed forms of gastrin are important colonic trophic factors. We show here that the gastrin gene is a downstream target of the beta-catenin/TCF-4 signaling pathway and that cotransfection of a constitutively active beta-catenin expression construct causes a threefold increase in gastrin promoter activity. APC(min-/+) mice overexpressing one of the alternatively processed forms of gastrin, glycine-extended gastrin, show a significant increase in polyp number. Gastrin-deficient APC(min-/+) mice, conversely, showed a marked decrease in polyp number and a significantly decreased polyp proliferation rate. Activation of gastrin by beta-catenin may therefore represent an early event in colorectal tumorigenesis and may contribute significantly toward neoplastic progression. The identification of gastrin as a functionally relevant downstream target of the beta-catenin signaling pathway provides a new target for therapeutic modalities in the treatment of colorectal cancer.

Processing and proliferative effects of human progastrin in transgenic mice.

Incompletely processed gastrins have been postulated to play a role in growth of the gastrointestinal tract, but few studies have examined the effects of progastrin on mucosal proliferation in vivo. Human gastrin gene expression and progastrin processing were therefore studied in transgenic mice containing a human gastrin (hGAS) minigene, and compared to processing in mice bearing an insulin gastrin (INS-GAS) transgene that overexpresses amidated gastrin. Progastrin processing was studied using region-specific antisera and radioimmunoassays, biosynthetic labeling, immunoprecipitation, and HPLC. Proliferative effects due to overexpression of processed and unprocessed gastrin in INS-GAS and hGAS mice, respectively, were determined using routine histology and BrdU incorporation. The pancreatic islets of INS-GAS mice were able to produce carboxyamidated G-17, resulting in a twofold elevation of serum amidated gastrin, marked thickening of the oxyntic mucosa, and an increased BrdU labeling index (LI) of the gastric body. In contrast, livers of adult hGAS mice expressed abundant human gastrin mRNA and human progastrin but were unable to process this peptide to the mature amidated form, resulting in markedly elevated serum progastrin levels and normal amidated gastrin levels. Nevertheless, there was a marked increase in the BrdU labeling index of the colon in hGAS mice (LI 7.46+/-1.90%), as well as in INS-GAS mice (LI 6.16+/-1.17%), compared to age-matched, wild type control mice (LI 4.01+/-0.98%, P < 0.05). These studies suggest that incompletely processed gastrin precursors may contribute to colonic mucosal proliferation in vivo.

Overexpression of glycine-extended gastrin in transgenic mice results in increased colonic proliferation.

Gastrin is a peptide hormone involved in the growth of both normal and malignant gastrointestinal tissue. Recent studies suggest that the glycine-extended biosynthetic intermediates mediate many of these trophic effects, but the in vivo relevance of glycine-extended gastrin (G-Gly) has not been tested. We have generated mice (MTI/G-GLY) that overexpress progastrin truncated at glycine-72 to evaluate the trophic effects of G-Gly in an in vivo model. MTI/G-GLY mice have elevated serum and colonic mucosal levels of G-Gly compared with wild-type mice. MTI/G-GLY mice had a 43% increase in colonic mucosal thickness and a 41% increase in the percentage of goblet cells per crypt. MTI/G-GLY mice exhibited increased colonic proliferation compared with wild-type controls, with an expansion of the proliferative zone into the upper third of the colonic crypts. Continuous infusion of G-Gly into gastrin-deficient mice for two weeks also resulted in elevated G-Gly levels, a 10% increase in colonic mucosal thickness, and an 81% increase in colonic proliferation when compared with gastrin-deficient mice that received saline alone. To our knowledge, these studies demonstrate for the first time that G-Gly's contribute to colonic mucosal proliferation in vivo.

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.

Substitution of phosphotyrosine for sulphotyrosine in biologically active peptides. Enzymatic phosphorylation of a progastrin peptide confers immunoreactivity reminiscent of the sulphated derivative.

The peptide SAEEEDQYN, corresponding to the carboxyl-terminal tryptic fragment of rat progastrin, whose penultimate tyrosyl residue is sulphated in the native peptide, is phosphorylated with Km values of 120 and 180 microM by two spleen tyrosine protein kinases, termed TPK-IIB and TPK-III, respectively. Another spleen tyrosine protein kinase related to the src family (TPK-I/lyn) is poorly active toward this peptide, displaying a Km 6.5 mM. The Tyr-phosphorylated peptide is recognized by an antibody (L304), which reacts with both sulphated and unmodified peptides, while it is not recognized by a second antibody (L303), which reacts with unmodified peptide yet not with the sulphated derivative. These data, in conjunction with previous observations (Hofsteenge, J., Stone, S.R., Donella-Deana, A. and Pinna, L.A. (1990) Eur. J. Biochem. 188, 55-59) support the view that phosphotyrosine is an effective surrogate for sulphotyrosine in a wide spectrum of biological activities.

Discrimination between constitutive secretion and basal secretion from the regulated secretory pathway in GH3 cells.

The present experiments were undertaken to characterize basal release from vesicles of the regulated secretory pathway. In transfected GH3 cells, progastrin was released by the constitutive route, and mature, bioactive, amidated gastrin by the regulated secretory pathway. Studies using brefeldin A and bafilomycin A1 which inhibit progression through the Golgi complex suggested that both basal and stimulated release of amidated gastrin originated from mature secretory granules. Basal, but not stimulated, secretion of amidated gastrin was strongly inhibited at 22 degrees C. Mature secretory vesicles therefore support both basal and evoked secretion although the mechanisms underlying the two processes differ in their temperature sensitivity.

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.

Characterisation of N-terminally extended met-enkephalin Arg6Gly7Leu8 variants in the porcine upper digestive tract.

Proenkephalin A-derived peptides are known to occur in the gut, but their precise identity is uncertain. We report here the isolation of N-terminally extended forms of Met-enkephalin Arg6Gly7Leu8 from porcine upper digestive tract monitored by radioimmunoassay. A single major form was identified in pyloric antral muscle and mucosa, but in the duodenum two major forms were detected. Microsequence analysis together with immunological data revealed that the antral mucosal peptide and the most acidic duodenal peptide had identical amino-acid sequences, corresponding to a 5.3 kDa peptide terminating in Met-enkephalin Arg6Gly7Leu8. The data indicate that high-molecular-weight peptides may constitute a major proportion of gut opioid peptide immunoactivity.

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.

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.

Isolation, structure and properties of the C-terminal flanking peptide of preprocholecystokinin from rat brain.

The C-terminal flanking peptide of preprocholecystokinin has been isolated from rat brain. Micro-sequence analysis revealed the primary structure: Ser-Ala-Glu-Asp-Tyr-Glu-Tyr-Pro-Ser. Arylsulphatase and mild acid hydrolysis suggested that both tyrosine residues are sulphated. The peptide was not active in bioassay systems that respond to CCK8; the significance of the conserved tripeptide Ser-Ala-Glu is discussed.

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.