Chromogranin A-derived Peptide Research Articles

Inhibition of Insulin Secretion by Betagranin, an N-terminal Chromogranin A Fragment

Betagranin, an N-terminal fragment of chromogranin A, results from a proteolytic processing, and is co-secreted with insulin. While other chromogranin A-derived peptides negatively modulate hormone secretion, the role of betagranin in pancreatic beta-cells is so far unknown. We have recently shown that pancreatic islet betagranin levels are down-regulated in obese, leptin-deficient mice. In the present study, we have investigated the distribution of betagranin in primary mouse islets and cells of the MIN6 line and have evaluated its effects on insulin secretion. We showed that betagranin co-localizes with insulin within secretory granules and strongly inhibited insulin secretion in response to both glucose and potassium, by blocking the influx of calcium. The data demonstrated a hitherto unknown inhibitory effect of betagranin on insulin secretion.

Recombinant preparation and characterization of interactions for a calmodulin‐binding chromogranin A peptide and calmodulin

Chromogranin-derived peptides have important and varied biological activities. They affect a wide spectrum of targets such as fungal membranes, blood vessels, myocardial cells, and pancreatic cells. Despite the biological significance and the diverse activities, the molecular mechanisms of the interactions between the peptides and the target proteins have not been well understood. Here, we studied the interaction between a chromogranin A-derived peptide (CGA40-65) and its target protein, calmodulin, with NMR spectroscopy. Calmodulin was easily prepared with standard recombinant technology, but CGA40-65 posed challenges requiring multistep procedures. The recombinantly produced peptide retained the calmodulin-binding property of the full-length CGA, as shown by the HSQC binding experiment. By applying resonance assignments, we identified the residues in calmodulin involved in the CGA40-65 binding. We also found that the peak changes are close to those exhibited by the peptides having the wrap-around binding mechanism. Further analysis revealed that the CGA40-65-induced changes are more similar to those by CaMKIp peptide than those by smMLCKp peptide among the wrap-around binding peptides, suggesting that CGA40-65 can be categorized as a CaMKIp-like peptide. Our report is the first residue-resolution mechanistic study involving chromogranin peptides and their target proteins. Our approaches should be applicable to interaction studies involving other chromogranin-derived peptides and their cellular target proteins.

New biological aspects of Chromogranin A-derived peptides: Focus on vasostatins

Chromogranin A (CgA), one component of the granin family, represents the major soluble protein co-stored and co-released with catecholamines, within chromaffin cells secretory granules. It is considered a diagnostic and prognostic marker of several diseases, including a variety of tumours and cardiac heart failure. It also represents a precursor of biologically active fragments, generated after proteolytic cleavage at the level of the multiple pairs of dibasic sites which enrich its sequence. CgA, and its derived fragments show an old evolutionary history being ubiquitously present throughout the animal word, from mammals to invertebrates. Their biological functions include control of hormone production, and several paracrine and autocrine actions mainly attributed to its derived peptides. Two N-terminal fragments, named vasostatins 1 (VS-1: CgA(1-76)) and vasostatin 2 (VS-2: CgA(1-113)) due to their ability to dilate pre-constricted vessels, exert a large spectrum of homeostatic actions, including antifungal and antimicrobial effect, modulation of cell adhesion, and inhibition of parathyroid hormone secretion. Recently, on isolated heart preparations from eel, frog and rat they were shown to act as negative inotropic agents able to counteract the effects of beta-adrenergic stimulation. This short note introduces the abstracts of the contributions at the "International Workshop on Vasostatins and Chromogranin A-derived peptides" (Island of Capri, Italy; September 2005). The Workshop was focused on recent findings on the role of vasostatins (VSs) in cardiovascular and gastrointestinal systems, extracellular fluids composition, and innate immunity. Particular attention has been given to the still elusive mechanism of action of these peptides.

Crucial role of cytoskeleton reorganization in the negative inotropic effect of chromogranin A-derived peptides in eel and frog hearts

Vasostatins (VSs), i.e. the main biologically active peptides generated by the proteolytic processing of chromogranin A (CGA) N-terminus, exert negative inotropism in vertebrate hearts. Here, using isolated working eel ( Anguilla anguilla) and frog ( Rana esculenta) heart preparations, we have studied the role of the cytoskeleton in the VSs-mediated inotropic response. In both eel and frog hearts, VSs-mediated-negative inotropy was abolished by treatment with inhibitors of cytoskeleton reorganization, such as cytochalasin-D (eel: 10 nM; frog: 1 nM), an inhibitor of actin polymerisation, wortmannin (0.01 nM), an inhibitor of PI3-kinase (PI3-K)/protein kinase B (Akt) signal-transduction cascade, butanedione 2-monoxime (BDM) (eel: 100 nM; frog: 10 nM), an antagonist of myosin ATPase, and N-(6-aminohexil)-5-chloro-1-naphthalenesulfonamide (W7) (eel: 100 nM; frog: 1 nM), a calcium-calmodulin antagonist. These results demonstrate that changes in cytoskeletal dynamics play a crucial role in the negative inotropic influence of VSs on eel and frog hearts.

Antifungal coating by biofunctionalized polyelectrolyte multilayered films

The surface of medical devices is a common site of bacterial and fungal adhesion, first step to the constitution of a resistant biofilm leading frequently to chronic infections. In order to prevent such complications, several physical and chemical modifications of the device surface have been proposed. Here, we experiment a new type of topical antifungal coating using the layer-by-layer technique. The nanometric multilayer film obtained by this technique is functionalized by the insertion of a chromogranin A-derived antifungal peptide (CGA 47-66, chromofungin). We show that the embedded peptide keeps its antifungal activity by interacting with the fungal membrane and penetrating into the cell. In vitro studies demonstrate that such an antifungal coating is able to inhibit the growth of yeast Candida albicans by 65% and completely stop the proliferation of filamentous fungus Neurospora crassa. The cytotoxicity of such a coating was also assessed by growing human gingival fibroblasts at its surface. Finally, the antifungal coating of poly(methylmethacrylate), a widely used material for biomedical devices, is successfully tested in an in vivo oral candidiasis rat model. Taken together, these results assessed the functionalized multilayer films containing a new potent antifungal non-toxic peptide, as a novel and promising technique for local antifungal protection.

Somatostatin, misoprostol and galanin inhibit gastrin- and PACAP-stimulated secretion of histamine and pancreastatin from ECL cells by blocking specific Ca 2+ channels

The oxyntic mucosa is rich in ECL cells. They secrete histamine and chromogranin A-derived peptides, such as pancreastatin, in response to gastrin and pituitary adenylate cyclase-activating peptide (PACAP). Secretion is initiated by Ca 2+ entry. While gastrin stimulates secretion by opening L-type and N-type Ca 2+ channels, PACAP stimulates secretion by activating L-type and receptor-operated Ca 2+ channels. Somatostatin, galanin and prostaglandin E 2 (PGE 2) inhibit gastrin- and PACAP-stimulated secretion from the ECL cells. In the present study, somatostatin and the PGE 2 congener misoprostol inhibited gastrin- and PACAP-stimulated secretion 100%, while galanin inhibited at most 60–65%. Bay K 8644, a specific activator of L-type Ca 2+ channels, stimulated ECL-cell secretion, an effect that was inhibited equally effectively by somatostatin, misoprostol and galanin (75–80% inhibition). Pretreatment with pertussis toxin, that inactivates inhibitory G-proteins, prevented all three agents from inhibiting stimulated secretion (regardless of the stimulus). Pretreatment with nifedipine (10 μM), an L-type Ca 2+ channel blocker, reduced PACAP-evoked pancreastatin secretion by 50–60%, gastrin-evoked secretion by ∼ 80% and abolished the response to Bay K 8644. The nifedipine-resistant response to PACAP was abolished by somatostatin and misoprostol but not by galanin. Gastrin and PACAP raised the intracellular Ca 2+ concentration in a biphasic manner, believed to reflect mobilization of internal Ca 2+ followed by Ca 2+ entry. Somatostatin and misoprostol blocked Ca 2+ entry (and histamine and pancreastatin secretion) but not mobilization of internal Ca 2+. The present observations on isolated ECL cells suggest that Ca 2+ entry rather than mobilization of internal Ca 2+ triggers exocytosis, that gastrin and PACAP activate different (but over-lapping) Ca 2+ channels, that somatostatin, misoprostol and galanin interact with inhibitory G-proteins to block Ca 2+ entry via L-type Ca 2+ channels, and that somatostatin and misoprostol (but not galanin) in addition block N-type and/or receptor-operated Ca 2+ channels.

ENOS, nNOS, cGMP and protein kinase G mediate the inhibitory effect of pancreastatin, a chromogranin A-derived peptide, on growth and proliferation of hepatoma cells

Pancreastatin (PST), a chromogranin A-derived peptide, has an anti-insulin metabolic effect and inhibits growth and proliferation by producing nitric oxide (NO) in HTC rat hepatoma cells. When NO production is blocked, a proliferative effect prevails due to the activation a Gα q/11-phospholipase C-β (PLC-β) pathway, which leads to an increase in [Ca 2+] i, protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) activation. The aim of the present study was to investigate the NO synthase (NOS) isoform that mediates these effects of PST on HTC hepatoma cells and the possible roles of cyclic GMP (cGMP) and cGMP-dependent protein kinase. DNA and protein synthesis in response to PST were measured as [ 3H]-thymidine and [ 3H]-leucine incorporation in the presence of various pharmacological inhibitors: N-monomethyl- l-arginine (NMLA, nonspecific NOS inhibitor), l-NIO (endothelial nitric oxide synthase (eNOS) inhibitor), espermidine (neuronal nitric oxide synthase (nNOS) inhibitor), LY83583 (guanylyl cyclase inhibitor), and KT5823 (protein kinase G inhibitor, (PKG)). l-NIO, similarly to NMLA, reverted the inhibitory effect of PST on hepatoma cell into a stimulatory effect on growth and proliferation. Nevertheless, espermidine also prevented the inhibitory effect of PST, but there was no stimulation of growth and proliferation. When guanylyl cyclase activity was blocked, there was again a reversion of the inhibitory effect into a stimulatory action, suggesting that the effect of NO was mediated by the production of cGMP. PKG inhibition prevented the inhibitory effect of PST, but there was no stimulatory effect. Therefore, the inhibitory effect of PST on growth and proliferation of hepatoma cells may be mainly mediated by eNOS activation. In turn, the effect of NO may be mediated by cGMP, whereas other pathways in addition to PKG activation seem to mediate the inhibition of DNA and protein synthesis by PST in HTC hepatoma cells.

Chromogranin A-derived peptides: interaction with the rat posterior cerebral artery

Chromogranin A (CgA), an acidic granule protein of the regulated secretory pathway in the diffuse neuroendocrine system, is postulated to serve as a prohormone for regulatory peptides. Betagranin (rCgA 1–128), the first N-terminal cleavage product of rat CgA, is 87% homologous to the bovine vasostatin I (bCgA 1–76), previously shown to be vasoinhibitory in bovine resistance arteries. In this study the vasoactivity of homologous rat and bovine peptides was investigated in the rat posterior cerebral artery. Firstly, we examined the interaction of rhodamine (Rh)-labelled bCgA 7–40 and bCgA 47–70 with elements of the arterial wall by fluorescence microscopy. Secondly, rCgA 7–57, bCgA 1–40, bCgA 7–40 and bCgA 47–66 (chromofungin) were studied for effects on arterial tone and intracellular calcium as function of pressure in an arteriograph. Although without dilator or constrictor responses at 60–150 mm Hg, the rat peptide (rCgA 7–57) evoked a significant delay in the onset of forced dilatation at 170 mm Hg, in contrast to the bovine peptides bCgA 1–40, bCgA 7–40 and bCgA 47–66 (chromofungin). Neither Rh-bCgA 7–40 nor Rh-bCgA 47–70 stained the endothelial layer, while Rh-bCgA 47–70 but not Rh-bCgA 7–40 stained the smooth muscle compartment. Analogously, bCgA 47–66 but not bCgA 7–40 reduced intracellular calcium, however without modifying the myogenic response. Thus, the betagranin peptide rCgA 7–57 and the two bovine chromofungin-containing peptides, highly homologous to the corresponding sequence (rCgA 47–66), affected the rat cerebral artery without vasodilator effects, indicating significant species differences in vasoactivity of the N-terminal domain of CgA.

The effect of a chromogranin A-derived peptide (CgA4-16) in the writhing nociceptive response induced by acetic acid in rats

The nociceptive effects of i.p administration of a synthetic peptide (CgA4-16) derived from chromogranin A (CgA) were studied on a model of inflammatory (somato-visceral) pain. Inflammatory mediators participate in controlling the activity of enterochromaffin cells that store and release chromogranins. Adult male Wistar rats were injected i.p with diluted acetic acid (AA) to induce abdominal writhes. Pharmacological agents were injected prior to CgA4-16 and/or AA together. While i.p CgA4-16 alone did not produce any effect, the peptide increased the number of abdominal constrictions induced by i.p AA administration in a dose-related manner. To determine the possible mechanisms involved in CgA4-16 produced pronociceptive effect, i.p diltiazem or indomethacin were tested. The pronociceptive effect induced by CgA4-16 was blocked by pretreatment of either substance. I.p administration of CGRP, substance P (SP) or capsaicin evoked dose-related abdominal writhing. CgA4-16, 20 min prior to CGRP or capsaicin, potentiated the nociceptive effects induced by CGRP or capsaicin, but not those induced by SP. Taken together, these data suggest for the first time that a CgA-derived peptide may modulate inflammatory pain.

Pancreastatin, a chromogranin A-derived peptide, inhibits leptin and enhances UCP-2 expression in isolated rat adipocytes.

Leptin, the ob gene product, is an adipocyte-secreted hormone that centrally regulates weight by decreasing caloric intake and increasing energy expenditure. Expression of leptin is regulated by dietary status, insulin, glucocorticoids and catecholamines. Pancreastatin (PST), a chromogranin A-derived peptide, correlates with catecholamine levels, and may play a role in the physiology of stress, modulating endocrine secretion and metabolism. Thus, PST has been found to exert a lipolytic and anti-insulin effect in white adipocytes. The aim of the present work was to investigate a possible role of PST modulating the expression of key genes involved in lipid storage and metabolism: leptin, PPAR-gamma2, UCP-1 and UCP-2. We incubated isolated rat epididymal adipocytes with 100 nM PST for 16 and 24 h. Leptin, UCP-2 and UCP-1 mRNA levels were assessed by RT-PCR, followed by Southern blot. Leptin secretion was also measured by ELISA. PST inhibited leptin expression and secretion at 16-h incubation, but this effect was no longer observed after 24 h. On the other hand, PST stimulated the expression of UCP-2 after 16 h. However, the effect was still significant after 24 h. The inhibitory effect of PST on leptin expression and secretion and the stimulation of UCP-2 expression were prevented by blocking PKC. UCP-1 and PPR-gamma2 expression did not change after PST stimulation. Leptin differentially regulates the expression of key genes in the rat adipocyte, upregulating the expression of UCP-2 and inhibiting the expression and secretion of leptin by a mechanism that involves PKC activity. These effects may contribute to the metabolic action of catecholamines in physiological and pathophysiological conditions with increased sympathetic activity.

Pancreastatin, a chromogranin A-derived peptide, activates protein synthesis signaling cascade in rat adipocytes

Pancreastatin (PST), a chromogranin A-derived peptide, has been found to modulate glucose, lipid, and protein metabolism in rat adipocytes. PST has an overall counterregulatory effect on insulin action by activating a specific receptor–effector system (Gα q/11 protein-PLC-β-PKC classical). However, PST stimulates both basal and insulin-mediated protein synthesis in rat adipocytes. In order to further investigate the mechanisms underlying the effect of PST stimulating protein synthesis, we sought to study the regulation of different components of the core translational machinery by the signaling triggered by PST. Thus, we studied ribosomal p70 S6 kinase, phosphorylation of the cap-binding protein (initiation factor) eIF4E, and phosphorylation of the eIF4E-binding protein 4E-BP1 (PHAS-I). We have found that PST stimulates the S6 kinase activity, as assessed by kinase assay using specific immunoprecipitates and substrate. This effect was checked by Western blot with specific antibodies against the phosphorylated S6 kinase. Thus, PST dose-dependently stimulates Thr421/Ser424 phosphorylation of S6 kinase. Moreover, PST promotes phosphorylation of regulatory sites in 4E-BP1 (PHAS-I) (Thr37, Thr46). The initiation factor eIF4E itself, whose activity is also increased upon phosphorylation, is phosphorylated in Ser209 by PST stimulation. Finally, we have found that these effects of PST on S6 kinase and the translation machinery can be blocked by preventing the activation of PKC. These results indicate that PST stimulates protein synthesis machinery by activating PKC and provides some evidence of the molecular mechanisms involved, i.e., the activation of S6K and the phosphorylation of 4E-BP1 (PHAS-I) and the initiation factor eIF4E.

The spectrum of endogenous human chromogranin A-derived peptides identified using a modified proteomic strategy.

The hypothesis that chromogranin A (CgA), a protein of neuroendocrine cell secretory granules, may be a precursor of biologically active peptides, rests on observed activities of peptide fragments largely produced by exogenous protease digestion of the bovine protein. Here we have adopted a modified proteomic strategy to isolate and characterise human CgA-derived peptides produced by endogenous prohormone convertases. Initial focus was on an insulinoma as previous studies have shown that CgA is rapidly processed in pancreatic beta cells and that tumours arising from these express appropriate prohormone convertases. Eleven novel peptides were identified arising from processing at both monobasic and dibasic sites and processing was most evident in the C-terminal domain of the protein. Some of these peptides were identified in endocrine tumours, such as mid-gut carcinoid and phaeochromocytoma, which arise from endocrine cells of different phenotype and in different anatomical sites. Two of the most interesting peptides, GR-44 and ER-37, representing the C-terminal region of CgA, were found to be amidated. These data would imply that the intact protein is C-terminally amidated and that these peptides are probably biologically active. The spectrum of novel CgA-derived peptides, described in the present study, should provide a basis for biological evaluation of authentic entities.

Localization and characterization of evolutionarily conserved chromogranin A-derived peptides in the rat and human pituitary and adrenal glands.

Chromogranin A (CgA) is a neuroendocrine protein that undergoes proteolytic cleavage in secretory granules. The aim of the present study was to characterize the peptides WE14 and EL35 that are derived from evolutionarily conserved regions of CgA in rat and human endocrine tissues. In the rat pituitary, HPLC analysis revealed that WE14 is present as a single immunoreactive peak, whereas EL35 elutes in two molecular forms. Authentic WE14 is also produced in both rat and human adrenal glands, while EL35 displays a variable elution profile depending on the tissue extract, indicating the existence of different forms of EL35 in these tissues. Immunohistochemical labeling of the rat pituitary showed that WE14 and EL35 occur in gonadotropes and melanotropes, but not in corticotropes. A strong immunoreaction for both peptides was also observed in rat adrenochromaffin cells. In the human adrenal gland, the WE14 and EL35 antisera revealed intense labeling of adrenomedullary cells in adult and nests of chromaffin progenitor cells in fetal adrenal. Finally, WE14 and EL35 immunoreactivity was detected in pheochromocytoma tissue where WE14 occurred as a single immunoreactive form, while EL35 displayed different forms. The observations that WE14 and EL35: (1). have been preserved during vertebrate evolution, (2). are processed in a cell-specific manner, and (3). occur during ontogenesis of the adrenal gland strongly suggest that these peptides play a role in endocrine tissues. In addition, the existence of differentially processed CgA-derived peptides in normal and tumorous tissues may provide new tools for the diagnosis and prognosis of neuroendocrine tumors.

Pancreastatin, a chromogranin A-derived peptide, inhibits DNA and protein synthesis by producing nitric oxide in HTC rat hepatoma cells

Background/Aims : Pancreastatin, a chromogranin A-derived peptide, has a counter-regulatory effect on insulin action. We have previously characterized pancreastatin receptor and signalling in rat liver and HTC hepatoma cells. A G α q/11 -PLC- β pathway leads to an increase in [Ca 2+] i, PKC and mitogen activated protein kinase (MAPK) activation. These data suggested that pancreastatin might have a role in growth and proliferation, similar to other calcium-mobilizing hormones. Methods : DNA and protein synthesis were measured as [ 3H]-thymidine and [ 3H]-leucine incorporation. Nitric oxide (NO) was determined by the Griess method and cGMP production was quantified by enzyme-linked immunoassay. Results : Contrary to the expected results, we have found that pancreastatin inhibits protein and DNA synthesis in HTC hepatoma cells. On the other hand, when the activity of NO synthase was inhibited by N -monomethyl- l -arginine (NMLA), the inhibitory effect of pancreastatin on DNA and protein synthesis was not only reverted, but a dose-dependent stimulatory effect was observed, probably due to MAPK activation, since it was prevented by PD98059. These data strongly suggested the role of NO in the inhibitory effect of pancreastatin on protein and DNA synthesis, which is overcoming the effect on MAPK activation. Moreover, pancreastatin dose-dependently increased NO production in parallel to cyclic guanosine monophosphate (cGMP). Both effects were prevented by NMLA. Finally, an indirect effect of pancreastatin through the induction of apoptosis was ruled out. Conclusions : Therefore, the NO and the cGMP produced by the NO-activated guanylate cyclase may mediate the dose-dependent inhibitory effect of pancreastatin on growth and proliferation in HTC hepatoma cells.

Neurohormonal regulation of secretion from isolated rat stomach ECL cells: a critical reappraisal

ECL cells are endocrine/paracrine cells in the oxyntic mucosa. They produce, store and secrete histamine and chromogranin A-derived peptides such as pancreastatin. The regulation of ECL-cell secretion has been studied by several groups using purified ECL cells, isolated from rat stomachs. Reports from different laboratories often disagree. The purpose of the present study was to re-evaluate the discrepancies by studying histamine (or pancreastatin) secretion from standardized preparations of pure, well-functioning ECL cells. Cells from rat oxyntic mucosa were dispersed by pronase digestion, purified by repeated counter-flow elutriation and subjected to density gradient centrifugation. The final preparation consisted of more than 90% ECL cells (verified by histamine and/or histidine decarboxylase immunocytochemistry). They were maintained in primary culture for 48 h before they were exposed to candidate stimulants and inhibitors for 30 min after which the medium was collected for determination of mobilized histamine (or pancreastatin). Gastrin-17 and sulphated cholecystokinin octapeptide (CCK-8s) raised histamine secretion 4-fold, the EC 50 for both peptides being around 100 pM. The neuropeptide pituitary adenylate cyclase activating peptide (PACAP-27) (5-fold increase) and the related neuropeptides vasoactive intestinal peptide (VIP) and peptide histidine isoleucine (PHI) (3-fold increase) mobilized histamine with similar potency (EC 50 ranging from 80 to 140 pM). Adrenaline, isoprenaline and terbutaline stimulated secretion by activating a β 2 receptor subtype, while acetylcholine and carbachol were without effect. Secretion experiments were invariably run in parallel with a gastrin standard curve. Somatostatin, prostaglandin E 2 (PGE 2) and the PGE 1 congener misoprostol inhibited PACAP- and gastrin-stimulated secretion by more than 90%, with IC 50 values ranging from 90–720 (somatostatin) to 40–200 (misoprostol) pM. The neuropeptide galanin inhibited secretion by 60–70% with a potency similar to that of somatostatin. Proposed inhibitors such as peptide YY, neuropeptide Y and the cytokines interleukin 1-β and tumor necrosis factor α induced at best a moderate inhibition of gastrin- or PACAP-stimulated secretion at high concentrations, while calcitonin gene-related peptide, pancreatic polypeptide and histamine itself were without effect. Inhibition of gastrin- or PACAP-stimulated secretion was routinely compared to a somatostatin standard curve. In conclusion, gastrin, PACAP, VIP/PHI and adrenaline stimulated secretion. Somatostatin and PGE 2 were powerful inhibitors of both gastrin- and PACAP-stimulated secretion; although equally potent, galanin was less effective than somatostatin and PGE 2.

Pancreastatin, a chromogranin A-derived peptide, activates Gα 16 and phospholipase C-β 2 by interacting with specific receptors in rat heart membranes

Pancreastatin (PST) is one of the chromogranin A (CGA)-derived peptides with known biological activity. It has a general inhibitory effect on secretion in many exocrine and endocrine systems including the heart atrium. Besides, a role of PST as a counter-regulatory peptide of insulin action has been proposed in the light of its effects on glucose and lipid metabolism in the liver and adipose tissue, where receptors and signaling have been described. Gα q/11 pathway seems to mediate PST action. Since PST has been shown to function as a typical calcium-dependent hormone, and increased plasma levels have been found in essential hypertension correlating with catecholamines, we sought to study its possible interaction and signaling in heart membranes. Here, we are characterizing specific PST binding sites and signaling in rat heart membranes. We have found that PST receptor has a K d of 0.5 nM and a B max of 34 fmol/mg of protein. The PST binding is inhibited by guanine nucleotides, suggesting the functional coupling of the receptor with GTP binding proteins (G proteins). Moreover, PST dose-dependently increases GTP binding to rat heart membranes. Finally, we have studied PST signaling-effector system by measuring phospholipase C (PLC) activity using blocking antibodies against different G proteins and PLC isoforms. We have found that PST stimulates PLCβ 2>PLCβ 1>PLCβ 3 by activating Gα 16 in rat heart membranes. These data suggest that PST may modulate the cardiac function.

Pancreastatin modulates insulin signaling in rat adipocytes: mechanisms of cross-talk.

Pancreastatin (PST), a chromogranin A-derived peptide, has counterregulatory effects on insulin in the hepatocyte and the adipocyte, suggesting a possible role in insulin resistance. The mechanism of PST action on glucose and lipid metabolism is typical of a calcium-mobilizing hormone and involves a receptor Gq/11 protein-phospholipase C (PLC)-beta pathway. In the rat adipocyte, PST inhibits insulin-mediated glucose transport, glucose utilization, and lipid synthesis, and it has a lipolytic effect but stimulates basal and insulin-stimulated protein synthesis. We have also recently studied the PST receptor-effector system in adipocyte membranes. To further investigate the mechanisms of PST effect on insulin action, we studied the cross-talk of PST with insulin signaling in the rat adipocyte. We found that PST inhibits insulin-stimulated GLUT4 translocation to the membrane, which may explain the reported inhibition of glucose transport. Tyrosine phosphorylation of the activated insulin receptor, insulin receptor substrate (IRS)-1, and p60-70 was also blunted, preventing their association with p85 phosphatidylinositol 3-kinase (PI3K) and their activity. The mechanism of this inhibition involves the activation of the "classical" protein kinase C isoforms and the serine phosphorylation of insulin receptor and IRS-1. On the other hand, PST activates the mitogen-activated protein kinase (MAPK) signaling module and enhances the effect of insulin. This pathway may account for the described effect of PST on protein synthesis. In conclusion, PST seems to inhibit the insulin-stimulated PI3K pathway in the adipocyte, whereas it activates the MAPK pathway. These data provide some clues to the PST cross-talk with insulin signaling that may explain the PST effects on glucose metabolism and protein synthesis.

Characterization of pancreastatin receptor and signaling in rat HTC hepatoma cells

Pancreastatin, a chromogranin A-derived peptide widely distributed throughout the neuroendocrine system, has a general inhibitory effect on endocrine secretion and a counterregulatory effect on insulin action. We have recently described the cross-talk of pancreastatin with insulin signaling in rat hepatoma cells (HTC), where it inhibits insulin action and signaling through the serine phosphorylation of the insulin receptor, thereby impairing tyrosine kinase activity. Here, we have characterized pancreastatin receptors and signaling in HTC cells. The pancreastatin effector systems were studied by determining phospholipase C activity in HTC membranes and mitogen-activated protein kinase (MAPK) phosphorylation activity in HTC cells. Binding studies with radiolabeled pancreastatin showed a population of high affinity binding sites, with a B max of 8 fmol/mg protein and a K d of 0.6 nM. Moreover, we assessed the coupling of the receptor with a G protein system by inhibiting the binding with guanine nucleotide and by measuring the GTP binding to HTC membranes. We found that pancreastatin receptor was coupled with a Gα q/11 protein which activates phospholipase C-β 1 and phospholipase C-β 3, in addition to MAPK via both βγ and α q/11.

Histamine depletion does not affect pancreastatin secretion from isolated rat stomach ECL cells

ECL cells co-secrete histamine and pancreastatin, a chromogranin A-derived peptide, in response to gastrin. The aim of the study was to explore possible ways to deplete ECL cells of histamine without affecting pancreastatin and to examine how histamine depletion affects pancreastatin secretion. Isolated rat stomach ECL cells (80–85% purity), prepared by counter-flow elutriation, were cultured for 48 h in the presence of α-fluoromethylhistidine (histidine decarboxylase inhibitor), bafilomycin A 1 (inhibitor of vacuolar-type proton-translocating ATPase) or reserpine (inhibitor of vesicular monoamine transporter). At this stage, the cells were challenged with 10 nM (EC 100) gastrin-17 for 30 min. Histamine and pancreastatin were determined by radioimmunoassay. Maximally effective concentrations of α-fluoromethylhistidine, bafilomycin A 1 and reserpine were found to lower ECL-cell histamine (by 60%, 78% and 80%, respectively) without affecting pancreastatin. Basal histamine secretion was reduced in a dose-dependent manner by all three drugs. Gastrin-evoked histamine secretion was reduced greatly by the three agents, while pancreastatin secretion was unaffected. The results show that histamine can be depleted not only by inhibiting its formation (α-fluoromethylhistidine), but also (and more effectively) by inhibiting histamine vesicular uptake, directly (reserpine) or indirectly (bafilomycin A 1). The results also indicate that although histamine is co-stored with pancreastatin, it is not required for either storage or secretion of pancreastatin.

Gastric submucosal microdialysis: a method to study gastrin- and food-evoked mobilization of ECL-cell histamine in conscious rats

Rat stomach ECL cells are rich in histamine and chromogranin A-derived peptides, such as pancreastatin. Gastrin causes the parietal cells to secrete acid by flooding them with histamine from the ECL cells. In the past, gastric histamine release has been studied using anaesthetized, surgically manipulated animals or isolated gastric mucosa, glands or ECL cells. We monitored gastric histamine mobilization in intact conscious rats by subjecting them to gastric submucosal microdialysis. A microdialysis probe was implanted into the submucosa of the acid-producing part of the stomach (day 1). The rats had access to food and water or were deprived of food (48 h), starting on day 2 after implantation of the probe. On day 4, the rats received food or gastrin (intravenous infusion), and sampling of microdialysate commenced. Samples (flow rate 1.2 μl min −1) were collected every 20 or 60 min, and the histamine and pancreastatin concentrations were determined. The serum gastrin concentration was determined in tail vein blood. Exogenous gastrin (4-h infusion) raised microdialysate histamine and pancreastatin dose-dependently. This effect was prevented by gastrin receptor blockade (YM022). Depletion of ECL-cell histamine by α-fluoromethylhistidine, an irreversible inhibitor of the histamine-forming enzyme, suppressed the gastrin-evoked release of histamine but not that of pancreastatin. Fasting lowered serum gastrin and microdialysate histamine by 50%, while refeeding raised serum gastrin and microdialysate histamine and pancreastatin 3-fold. We conclude that histamine mobilized by gastrin and food intake derives from ECL cells because: 1) Histamine and pancreastatin were released concomitantly, 2) histamine mobilization following gastrin or food intake was prevented by gastrin receptor blockade, and 3) mobilization of histamine (but not pancreastatin) was abolished by α-fluoromethylhistidine. Hence, gastric submucosal microdialysis allows us to monitor the mobilization of ECL-cell histamine in intact conscious rats under various experimental conditions not previously accessible to study. While gastrin receptor blockade lowered post-prandial release of ECL-cell histamine by about 80%, unilateral vagotomy reduced post-prandial mobilization of ECL-cell histamine by about 50%. Hence, both gastrin and vagal excitation contribute to the post-prandial release of ECL-cell histamine.