Immunocytochemical localisation of pancreastatin and chromogranin A in porcine neuroendocrine tissues.

Pancreastatin is a 49 amino acid peptide with a C-terminal glycine amide originally isolated from porcine pancreas. In the present study the cellular localisation of pancreastatin in porcine neuroendocrine tissue was examined immunocytochemically using an antiserum raised against porcine pancreastatin (33-49) that does not cross-react with porcine chromogranin A. In order to study the possible precursor-product relationship between chromogranin A and pancreastatin the cellular localisation of both peptides was examined in peripheral tissues using simultaneous double immunostaining. The pancreastatin antiserum immunostained cells and nerve fibers throughout the neuroendocrine system. In most of the examined tissues we found colocalisation of pancreastatin and chromogranin A immunostaining. These results support the precursor-product concept for chromogranin A and pancreastatin. However, in the gastrointestinal tract and the adenohypophysis a minor population of the endocrine cells exhibited immunostaining with only one of the two antibodies. This discrepancy between immunostaining with pancreastatin antiserum and monoclonal chromogranin A antibody could be due to absence of, or extensive, processing of chromogranin A in certain cell populations.     

Distribution and partial characterisation of immunoreactivity to the putative C-terminus of rat pancreastatin

The presumptive C-terminal nonapeptide of rat pancreastatin was synthesised based upon the sequence of rat chromogranin A (CGA) analogous to that of porcine pancreastatin as contained within porcine CGA. Antisera were produced which were used to determine the qualitative and quantitative distribution of pancreastatin-like immunoreactivity in rat tissues by immunocytochemistry and radioimmunoassay respectively. Pancreastatin-like immunoreactivity was most abundant in pituitary, adrenal, gastric corpus and thyroid with considerably lower levels detected in the remainder of the gastroentero-pancreatic system and brain. Immunoreactivity was localised exclusively in endocrine cells and the relative abundance of immunoreactive cells paralleled the levels obtained radioimmunometrically. Chromatographic characterisation of pancreastatin-like immunoreactivity revealed molecular heterogeneity. Immunoreactive peptides of similar size to synthetic rat pancreastatin were present in gastrointestinal tissues and thyroid. These data indicate a tissue specific processing of CGA in the rat.     

Isolation and characterization of a tumor-derived human pancreastatin-related protein

A protein with pancreastatin-like immunoreactivity has been isolated and purified from liver metastasis of a patient with insulinoma. NH2-terminal sequence analysis in conjunction with the use of antibodies specific for the C-terminal structure of pancreastatin identified this protein as a 186-amino acid residue protein corresponding to human chromogranin A-116-301. Using a sensitive radioimmunoassay it was found that serum from the patient with insulinoma contains two peptide species; one comigrates with the 186-amino acid residue pancreastatin and the other the 48-residue pancreastatin.     

Isolation and characterization of a tumor-derived human protein related to chromogranin A and its in vitro conversion to human pancreastatin-48

A protein with pancreastatin-like immunoreactivity has been isolated and purified from liver metastasis of a patient with insulinoma. NH2-terminal residue analysis, in conjunction with the use of antibodies that are specific for the C-terminal amide peptide of porcine pancreastatin, identified this protein as a 186-amino-acid protein corresponding to human chromogranin A-116-301 (the fragment corresponding to the positions from 116 to 301 of human chromogranin A). Digestion of this protein with trypsin yielded a 48-amino-acid peptide with the retention of full pancreastatin activity. Serum from patient with insulinoma contains a peptide specie(s) that comigrates with the 48-amino-acid pancreastatin, suggesting that this peptide might be a physiologically important circulation form of pancreastatin in humans. A sensitive radioimmunoassay was established using antibody developed against a synthetic 29-amino-acid peptide amide of pancreastatin. Immunocytochemical staining revealed that a major population of human pancreatic islet cells were immunoreactive to the antiserum but with varying intensity of staining. Pancreastatin-like immunoreactivity was not observed in exocrine acinar cells.     

Rat pancreastatin inhibits both pancreatic exocrine and endocrine secretions in rats.

Effects of synthetic rat pancreastatin C-terminal fragment on both exocrine and endocrine pancreatic functions were examined in rats, in vivo and in vitro. Pancreastatin (20, 100 pmol, 1 nmol/kg/h) significantly inhibited CCK-8-stimulated pancreatic juice flow and protein output in a dose-related manner, in vivo. The inhibitory effect on bicarbonate output was not statistically significant. Pancreastatin did not significantly inhibit basal pancreatic secretions in vivo, and did not inhibit amylase release from the dispersed acini, in vitro. Insulin release stimulated by intragastric administration of glucose (5 g/kg) was significantly inhibited by pancreastatin (1 nmol/kg/h), in vivo. Plasma glucose concentrations were increased by pancreastatin infusion, but the increase was not statistically significant. Furthermore, pancreastatin inhibited insulin release from isolated islets, in vitro. Synthetic rat C-terminal pancreastatin fragment has bioactivities on both exocrine and endocrine pancreatic functions in rats.     

Effects of pancreastatin and chromogranin A on insulin release stimulated by various insulinotropic agents

The effects of porcine pancreastatin on insulin release stimulated by insulinotropic agents, glucagon, cholecystokinin-octapeptide (CCK-8), gastric inhibitory polypeptide (GIP) and L-arginine, were compared to those of bovine chromogranin A (CGA) using the isolated perfused rat pancreas. Pancreastatin significantly potentiated glucagon-stimulated insulin release (first phase: 12.5 +/- 0.9 ng/8 min; second phase: 34.5 +/- 1.6 ng/25 min in controls; 16.5 +/- 1.1 ng/8 min and 44.0 +/- 2.2 ng/25 min in pancreastatin group), whereas CGA was ineffective. The first phase of L-arginine-stimulated insulin release was also potentiated by pancreastatin (6.9 +/- 0.5 ng/5 min in controls, 8.4 +/- 0.6 ng/5 min in pancreastatin group), but not by CGA. Pancreastatin did not affect CCK-8 or GIP-stimulated insulin release. Similarly, CGA did not affect insulin release stimulated by CCK-8 or GIP. These findings suggest that pancreastatin stimulates insulin release in the presence of glucagon. Because pancreastatin can have multiple effects on insulin release, which are dependent upon the local concentration of insulin effectors, pancreastatin may participate in the fine tuning of insulin release from B cells.     

Pancreastatin producing cell line from human pancreatic islet cell tumor

It has been characterized that cell line QGP-1 derived from human non-functioning pancreatic islet cell tumor produces human pancreastatin. Exponentially growing cultures produced 5.7 fmol of pancreastatin/10(6) cells/hr. Human pancreastatin immunoreactivities in plasma and tumor after xenografting with QGP-1 into nude mouse were 92.7 fmol/ml and 160.2 pmol/g wet weight, respectively. Immunocytochemical study revealed both chromogranin A and pancreastatin immunoreactive cells in the tumor. Gel filtrations of culture medium and tumor extract identified heterogenous molecular forms of PST-LI which eluted as large and smaller molecular species. These results suggest that plasma pancreastatin levels may be useful as a tumor marker of endocrine tumor of the pancreas, and the pancreastatin producing cell line may be useful for studies of the mechanism of secretions and processing of chromogranin A and pancreastatin.     

Effects of systemic pancreastatin on memory retention

Pancreastatin, a peptide isolated from the pancreas, was shown to enhance memory retention after peripheral administration in mice when administration following T-maze footshock avoidance training. The effect of pancreastatin on memory retention, one week after training, was time dependent showing enhancement of retention when pancreastatin was administered 0 and 30 min but not 60 min after training. Pancreastatin reversed the amnesia produced by scopolamine. The pancreastatin fragment (33-49) also enhanced memory. Pancreastatin did not increase glucose in vivo. We conclude that peripherally administered pancreastatin modulates memory processing.     

Inhibitory effect of pancreastatin on pancreatic exocrine secretion in the conscious rat

The effect of newly discovered pancreastatin on pancreatic secretion stimulated by a diversion of bile-pancreatic juice (BPJ) from the intestine was examined in the conscious rat. Exogenous pancreastatin infusion (20, 100 and 200 pmol/kg.h) inhibited pancreatic protein and fluid outputs during BPJ diversion in a dose-dependent manner. Pancreastatin did not affect plasma cholecystokinin (CCK) concentrations. Pancreastatin (100 pmol/kg.h) inhibited CCK-stimulated pancreatic secretion, but did not inhibit secretin-stimulated pancreatic secretion. Pancreastatin alone, however, did not affect basal pancreatic secretion. In contrast, pancreastatin (10(-10)-10(-7)M) did not suppress CCK-stimulated amylase release from isolated rat pancreatic acini. These results indicate that pancreastatin has an inhibitory action on exocrine function of the pancreas. This action may not be mediated by direct mechanisms and nor via an inhibition of CCK release. It is suggested that pancreastatin may play a role in the regulation of the intestinal phase of exocrine pancreatic secretion.     

Light and electron microscopical immunocytochemical localization of pancreastatin-like immunoreactivity in porcine tissues

Pancreastatin is a 49 amino acid comprising peptide isolated from porcine pancreas that is derived by proteolytic processing from chromogranin A. Using an antibody against the synthetic C-terminal fragment pancreastatin (33-49), we examined the light and electron microscopical immunocytochemical localization of this peptide in porcine tissues. Pancreastatin-like immunoreactivity (PLI) was found in pancreatic somatostatin-, insulin- and glucagon cells in varying intensities; pancreatic polypeptide cells were always negative. At the electron microscopical (EM) level the immunoreactivity was confined to the electron dense core of the secretory granules in the case of somatostatin and insulin cells or to the less electron dense "halo" of the glucagon granules. In the antrum PLI positive cells represented gastrin (G), somatostatin (D) and enterochromaffin (EC) cells, in the duodenum in addition to EC- and G-cells a small number of PLI positive cells showed a positive immunoreaction for glucagon-like peptide (GLP) I and secretin in serial sections. Both norepinephrine and epinephrine containing cells of the adrenal medulla exhibited a strong reaction for PLI. In the pituitary several cell populations stained with varying intensities, including gonadotrophs and thyrotrophys. PLI is present in a distinct and characteristic subpopulation of neuroendocrine cells in various organs. The subcellular localization may indicate a function in the granular concentration, packaging and storage of peptides and amines in the brain-gut endocrine system.     

Glycogenolytic effect of pancreastatin in the rat

Pancreastatin is a novel 49-amino acid peptide with a C-terminal glycine amide. The peptide was isolated from porcine pancreatic extracts and shows a structural similarity to chromogranin A. The effect of synthetic porcine pancreastatin on blood glucose levels and hepatic glycogen content was investigated in ratsin vivo. Pancreastatin (300 pmol/kg) produced a time-dependent decrease in glycogen content of liver and a slight hyperglycemia. Basal plasma insulin and glucagon levels were not modified by pancreastatin. We suggest that pancreastatin could play a biological role in the glucose metabolism through a glycogenolytic effect.     

Effects of pancreastatin on insulin, glucagon and somatostatin secretion by the perfused rat pancreas

Pancreastatin is a novel peptide, isolated from porcine pancreatic extracts, which has been shown to inhibit glucose-induced insulin release "in vitro". To achieve further insight into the influence of pancreastatin on pancreatic hormone secretion, we have studied the effects of this peptide on unstimulated insulin, glucagon and somatostatin output, as well as on the responses of these hormones to glucose and to tolbutamide in the perfused rat pancreas. Pancreastatin strongly inhibited unstimulated insulin release as well as the insulin responses to glucose and to tolbutamide. It did not significantly affect glucagon or somatostatin output under any of the above-mentioned conditions. These findings suggest that pancreastatin inhibits B-cell secretory activity directly, and not through an A-cell or D-cell paracrine effect.     

Reprint of: Metabolic effects and mechanism of action of the chromogranin A-derived peptide pancreastatin

Pancreastatin is one of the regulatory peptides derived from intracellular and/or extracellular processing of chromogranin A, the soluble acidic protein present in the secretory granules of the neuroendocrine system. While the intracellular functions of chromogranin A include formation and maturation of the secretory granule, the major extracellular functions are generation of biologically active peptides with demonstrated autocrine, paracrine or endocrine activities. In this review, we will focus on the metabolic function of one of these peptides, pancreastatin, and the mechanisms underlying its effects. Many different reported effects have implicated PST in the modulation of energy metabolism, with a general counterregulatory effect to that of insulin. Pancreastatin induces glycogenolysis in liver and lipolysis in adipocytes. Metabolic effects have been confirmed in humans. Moreover, naturally occurring human variants have been found, one of which (Gly297Ser) occurs in the functionally important carboxy-terminus of the peptide, and substantially increases the peptide's potency to inhibit cellular glucose uptake. Thus, qualitative hereditary alterations in pancreastatin's primary structure may give rise to interindividual differences in glucose and lipid metabolism. Pancreastatin activates a receptor signaling system that belongs to the seven-spanning transmembrane receptor coupled to a Gq-PLCβ-calcium-PKC signaling pathway. Increased pancreastatin plasma levels, correlating with catecholamines levels, have been found in insulin resistance states, such as gestational diabetes or essential hypertension. Pancreastatin plays important physiological role in potentiating the metabolic effects of catecholamines, and may also play a pathophysiological role in insulin resistance states with increased sympathetic activity.     

Light and electron microscopical immunocytochemical localization of pancreastatin-like immunoreactivity in porcine tissues

Summary Pancreastatin is a 49 amino acid comprising peptide isolated from porcine pancreas that is derived by proteolytic processing from chromogranin A. Using an antibody against the synthetic C-terminal fragment pancreastatin (33–49), we examined the light and electron microscopical immunocytochemical localization of this peptide in porcine tissues. Pancreastatin-like immunoreactivity (PLI) was found in pancreatic somatostatin-, insulin- and glucagon cells in varying intensities; pancreatic polypeptide cells were always negative. At the electron microscopical (EM) level the immunoreactivity was confined to the electron dense core of the secretory granules in the case of somatostatin and insulin cells or to the less electron dense halo of the glucagon granules. In the antrum PLI positive cells represented gastrin (G), somatostatin (D) and enterochromaffin (EC) cells, in the duodenum in addition to EC- and G-cells a small number of PLI positive cells showed a positive immunoreaction for glucagon-like peptide (GLP) I and secretin in serial sections. Both norepinephrine and epinephrine containing cells of the adrenal medulla exhibited a strong reaction for PLI. In the pituitary several cell populations stained with varying intensities, including gonadotrophs and thyrotrophs. PLI is present in a distinct and characteristic subpopulation of neuroendocrine cells in various organs. The subcellular localization may indicate a function in the granular concentration, packaging and storage of peptides and amines in the brain-gut endocrine system.     

Pancreastatin-like immunoreactivity in human carcinoid disease

Pancreastatin-like immunoreactivity has been demonstrated in human carcinoid tumors by immunohistochemistry and radioimmunoassay, employing antisera raised to a synthetic C-terminal fragment of porcine pancreastatin. Immunohistochemistry revealed intense immunoreactivity in all tumors. By radioimmunoassay, high concentrations of pancreastatin-like immunoreactivity were measured in carcinoid tumors arising from the fore-gut (mean +/- S.D. and range: 369 +/- 955 and 9.4-3670 pmol g-1, respectively, n = 14), mid-gut (mean +/- S.D. and range: 1354 +/- 1538 and 337-3978 pmol g-1, respectively, n = 5) and in metastases associated with mid-gut tumors (mean +/- S.D. and range: 684 +/- 739 and 31-2255 pmol g-1, respectively, n = 7), compared to corresponding normal tissues (less than 1.4 pmol g-1). Individuals with hepatic metastases and carcinoid syndrome had elevated circulating levels of pancreastatin-like immunoreactivity (mean +/- S.D. and range: 770 +/- 1249 and 42-4120 pmol l-1; n = 12), significantly above the normal, fasting range (mean +/- S.D. and range: 14.9 +/- 7.5 and 4-37.5 pmol l-1, respectively, n = 42). However, patients with non-metastatic carcinoid tumors (n = 4), who had been clinically cured after primary tumor resection, had plasma levels within the normal range. Chromatographic analysis of extracts of primary lung and ileal tumors, hepatic metastases from ileal tumors and plasma from individuals with carcinoid syndrome revealed molecular heterogeneity of pancreastatin-like immunoreactivity.     

Ontogenetic expression of chromogranin A and its derived peptides, WE-14 and pancreastatin, in the rat neuroendocrine system

 The ontogenetic expression of chromogranin A (CgA) and its derived peptides, WE-14 and pancreastatin (PST), was studied in the rat neuroendocrine system employing immunohistochemical analysis of fetal and neonatal specimens from 12.5-day embryos (E12.5), to 42-day postnatal (P42) rats. CgA immunostaining was first detected in endocrine cells of the pancreas, stomach, intestine, adrenal gland and thyroid at E13.5, E14.5, E15.5, E15.5 and E18.5, respectively. PST-like immunoreactivity was detected in endocrine cells of the pancreas at E13.5, stomach, intestine at E15.5, adrenal gland at E17.5 and thyroid at E18.5. WE-14 immunoreactivity was first observed in the immature pancreas at E15.5, mucosal cells of the stomach at E15.5, scattered chromaffin cells in the immature adrenal gland and mucosal cells of the intestine at E17.5 and thyroid parafollicular cells at E18.5. These data confirm that the translation of the CgA gene is regulated differentially in various neuroendocrine tissues and, moreover, suggests that the posttranslational processing of the molecule is developmentally controlled. Accepted: 18 October 1996     

The distribution of GAWK-like immunoreactivity in neuroendocrine cells of the human gut, pancreas, adrenal and pituitary glands and its co-localisation with chromogranin B

GAWK is a recently discovered peptide isolated from extracts of human pituitary gland and subsequently shown to be identical to sequence 420-493 of human chromogranin B. The distribution of this peptide was studied in human gut, pancreas, adrenal and pituitary glands using antisera to two portions of the 74 amino acid peptide (sequences 1-17 and 20-38). In addition, the co-existence of GAWK immunoreactivity with other peptides and chromogranin B was investigated using comparative immunocytochemistry. In the gut, GAWK was localised mainly to serotonin-containing cells of the mucosal epithelium, where electron microscopy showed it to be stored in typical electron-dense (250 nm diameter) granules, and to a moderate population of nerve fibres in the gut wall. Considerable quantities of GAWK-like immunoreactivity were measured in the gut, up to 36.3 +/- 18 pmol GAWK 1-17/g wet weight of tissue (mean +/- SEM) and 12.4 +/- 2.9 pmol GAWK 20-38/g. Chromatography of gut extracts revealed several GAWK-like immunoreactive peaks. GAWK-like immunoreactivity was also detected in endocrine cells of pancreas, pituitary gland and adrenal medulla, where the highest concentrations of GAWK-like immunoreactivity were measured (GAWK 1-17 2071.8 +/- 873.2 and GAWK 20-38 1292.7 +/- 542.7 pmol/g). Endocrine cells containing GAWK-like immunoreactivity were found also to be immunoreactive for chromogranin B. Our results define a discrete distribution of GAWK immunoreactivity in human endocrine cells and nerves and provide morphological support for the postulated precursor-product relationship between chromogranin B and GAWK. Details of the functions of this peptide are awaited.     

Effects of pancreastatin on insulin and pancreatic polypeptide secretion in the dog

Porcine pancreastatin (1.19 nmol) was administered into the peripheral vein (i.v.) or the third cerebral ventricle (i.t.v.) of dogs and its effect on the secretion of insulin and pancreatic polypeptide (PP) studied. Neither means of administration had any effect on basal and glucose-induced insulin or PP secretion. However, i.v. pancreastatin did inhibit the i.v. CCK-8-induced insulin but not PP release. Pancreastatin may thus play a role in the regulation of insulin secretion in the canine pancreas.     

Isolation and characterization of bovine pancreastatin

Bovine pancreastatin, a 47 amino acid residue peptide, was isolated from the pancreas and the pituitary gland using a chemical method which detects its C-terminal glycine amide structure. The complete amino acid sequence of the pancreatic peptide is 74% homologous to that of porcine pancreastatin and is identical to bovine chromogranin A-(248-294), as deduced from its cDNA sequence. The sequence of the first 28 amino-terminal residues of the pituitary peptide was determined to be identical to the corresponding sequence of the pancreatic peptide. Since the pituitary peptide also contains the C-terminal glycine amide, it is therefore likely to be identical in structure to the pancreatic peptide. Thus, we conclude that bovine chromogranin A is the precursor of bovine pancreastatin. Synthetic bovine pancreastatin inhibited pancreatic exocrine secretion in a similar manner to porcine pancreastatin.