RESUMEN
Pancreastatin (PST), a chromogranin A-derived peptide, is a potent physiological inhibitor of glucose-induced insulin secretion. PST also triggers glycogenolysis in liver and reduces glucose uptake in adipocytes and hepatocytes. Here, we probed for genetic variations in PST sequence and identified two variants within its functionally important carboxyl terminus domain: E287K and G297S. To understand functional implications of these amino acid substitutions, we tested the effects of wild-type (PST-WT), PST-287K, and PST-297S peptides on various cellular processes/events. The rank order of efficacy to inhibit insulin-stimulated glucose uptake was: PST-297S > PST-287K > PST-WT. The PST peptides also displayed the same order of efficacy for enhancing intracellular nitric oxide and Ca(2+) levels in various cell types. In addition, PST peptides activated gluconeogenic genes in the following order: PST-297S ≈ PST-287K > PST-WT. Consistent with these in vitro results, the common PST variant allele Ser-297 was associated with significantly higher (by â¼17 mg/dl, as compared with the wild-type Gly-297 allele) plasma glucose level in our study population (n = 410). Molecular modeling and molecular dynamics simulations predicted the following rank order of α-helical content: PST-297S > PST-287K > PST-WT. Corroboratively, circular dichroism analysis of PST peptides revealed significant differences in global structures (e.g. the order of propensity to form α-helix was: PST-297S ≈ PST-287K > PST-WT). This study provides a molecular basis for enhanced potencies/efficacies of human PST variants (likely to occur in â¼300 million people worldwide) and has quantitative implications for inter-individual variations in glucose/insulin homeostasis.
Asunto(s)
Variación Genética , Mutación Missense , Hormonas Pancreáticas , Células 3T3-L1 , Adulto , Sustitución de Aminoácidos , Animales , Glucemia/metabolismo , Dicroismo Circular , Femenino , Células Hep G2 , Humanos , Insulina/sangre , Masculino , Ratones , Hormonas Pancreáticas/sangre , Hormonas Pancreáticas/química , Hormonas Pancreáticas/genética , Hormonas Pancreáticas/farmacología , Estructura Terciaria de Proteína , Relación Estructura-ActividadRESUMEN
We describe a new, simple, robust and efficient method based on direct-tissue matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry that enables consistent semi-quantitation of peptide hormones in isolated pancreatic islets from normal and diabetic rodents. Prominent signals were measured that corresponded to all the main peptide hormones present in islet-endocrine cells: (α-cells) glucagon, glicentin-related polypeptide/GRPP; (ß-cells) insulin I, insulin II, C-peptide I, C-peptide II, amylin; (δ-cells) somatostatin-14; and (PP-cells), and pancreatic polypeptide. The signal ratios coincided with known relative hormone abundances. The method demonstrated that severe insulin deficiency is accompanied by elevated levels of all non-ß-cell-hormones in diabetic rat islets, consistent with alleviation of paracrine suppression of hormone production by non-ß-cells. It was also effective in characterizing hormonal phenotype in hemizygous human-amylin transgenic mice that express human and mouse amylin in approx. equimolar quantities. Finally, the method demonstrated utility in basic peptide-hormone discovery by identifying a prominent new Gcg-gene-derived peptide (theoretical monoisotopic molecular weight 3263.5 Da), closely related to but distinct from GRPP, in diabetic islets. This peptide, whose sequence is HAPQDTEENARSFPASQTEPLEDPNQINE in Rattus norvegicus, could be a peptide hormone whose roles in physiology and metabolic disease warrant further investigation. This method provides a powerful new approach that could provide important new insights into the physiology and regulation of peptide hormones in islets and other endocrine tissues. It has potentially wide-ranging applications that encompass endocrinology, pharmacology, phenotypic analysis in genetic models of metabolic disease, and hormone discovery, and could also effectively limit the numbers of animals required for such studies.
Asunto(s)
Islotes Pancreáticos/química , Hormonas Pancreáticas/análisis , Análisis de Secuencia de Proteína/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Secuencia de Aminoácidos , Animales , Diabetes Mellitus Experimental , Glicentina/análisis , Glicentina/química , Histocitoquímica , Humanos , Polipéptido Amiloide de los Islotes Pancreáticos/análisis , Polipéptido Amiloide de los Islotes Pancreáticos/química , Polipéptido Amiloide de los Islotes Pancreáticos/genética , Islotes Pancreáticos/metabolismo , Masculino , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Hormonas Pancreáticas/química , Fragmentos de Péptidos/análisis , Fragmentos de Péptidos/química , Ratas , Ratas Wistar , Reproducibilidad de los Resultados , Alineación de SecuenciaRESUMEN
Chromogranin A (CgA), chromogranin B (CgB), and secretogranin II (SgII) belong to a family of uniquely acidic secretory proteins in elements of the diffuse neuroendocrine system. These "granins" are characterized by numerous pairs of basic amino acids as potential sites for intra- and extragranular processing. In response to adequate stimuli, the granins are coreleased with neurotransmitters and hormones and appear in the circulation as potential modulators of homeostatic processes. This review is directed towards functional aspects of the secreted CgA, CgB, and SgII and their biologically active sequences. Widely different effects and targets have been reported for granin-derived peptides. So far, the CgA peptides vasostatin-I, pancreastatin, and catestatin, the CgB peptides CgB(1-41) and secretolytin, and the SgII peptide secretoneurin are the most likely candidates for granin-derived regulatory peptides. Most of their effects fit into patterns of direct or indirect modulations of major functions, in particular associated with inflammatory conditions.
Asunto(s)
Cromogranina A/química , Cromogranina B/química , Hormonas/metabolismo , Sistemas Neurosecretores/fisiología , Secretogranina II/química , Animales , Diferenciación Celular , Homeostasis , Humanos , Modelos Biológicos , Neuropéptidos/química , Hormonas Pancreáticas/química , Fragmentos de Péptidos/química , Péptidos/químicaRESUMEN
Chromogranin A (CgA) belongs to the granin family of uniquely acidic secretory proteins co-stored and co-secreted with other hormones and peptides in elements of the diffuse neuroendocrine system. The granins arise from different genes and are characterized by numerous sites for post-translational cleavage into shorter peptides with postulated regulatory properties. This review is directed towards endocrine aspects of CgA and its biologically active peptides. There is ample evidence from in vitro studies of distinct effects and targets for three CgA-derived peptides, vasostatin-I, pancreastatin and catestatin. Endocrine regulations are indicated from in vivo studies, consistent with the postulated prohormone function of CgA for peptides with regulatory properties. Most of the effects fit into patterns of direct or indirect, inhibitory modulations of major functions, implicating CgA peptides in regulation of calcium and glucose metabolism, cardiovascular functions, gastrointestinal motility and nociception, tissue repair, inflammatory responses and as host defense peptides in the first phase of microbial invasions.
Asunto(s)
Cromogranina A/fisiología , Glándulas Endocrinas/fisiología , Animales , Calcio/metabolismo , Metabolismo de los Hidratos de Carbono , Fenómenos Fisiológicos Cardiovasculares , Cromogranina A/química , Humanos , Inmunidad Innata , Inflamación/fisiopatología , Modelos Biológicos , Modelos Moleculares , Neoplasias/patología , Neoplasias/fisiopatología , Tumores Neuroendocrinos/fisiopatología , Sistemas Neurosecretores/fisiología , Hormonas Pancreáticas/química , Hormonas Pancreáticas/fisiología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/fisiología , Relación Estructura-ActividadRESUMEN
The physiologic effects of the chromogranin A peptide fragment, pancreastatin, were studied in six healthy Caucasian men, ages 25-46 years. Synthetic pancreastatin (human chromogranin A(273-301)-amide) was infused into the brachial artery of each subject to achieve a local concentration of approximately 200 nM over 15 minutes. Forearm blood flow was measured by strain-gauge plethysmography while (A-V)(glucose) was monitored by arterial and venous sampling. Pancreastatin infusion significantly reduced forearm glucose uptake (mean reduction +/- 1 SEM, 54 +/- 15%; P = 0.028) but did not alter forearm blood flow-indicating a metabolic, rather than hemodynamic, effect. Simultaneous infusion of pancreastatin with insulin (0.1 mU/kg/min) did not diminish insulin-induced forearm glucose uptake, suggesting pancreastatin is not simply a negative insulin modulator. The results of this study suggest that pancreastatin may contribute to the dysglycemia associated with type 2 diabetes and essential hypertension, two common human disease states in which plasma pancreastatin levels are elevated.
Asunto(s)
Brazo/irrigación sanguínea , Hormonas Pancreáticas/química , Hormonas Pancreáticas/fisiología , Adulto , Velocidad del Flujo Sanguíneo/efectos de los fármacos , Glucemia/metabolismo , Cromogranina A , Diabetes Mellitus Tipo 2/fisiopatología , Glucosa/farmacocinética , Hemodinámica , Humanos , Masculino , Persona de Mediana Edad , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismoRESUMEN
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 alpha(q/11)-PLC-beta pathway leads to an increase in [Ca2+]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.
Asunto(s)
Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , ADN/antagonistas & inhibidores , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Óxido Nítrico/biosíntesis , Hormonas Pancreáticas/farmacología , Inhibidores de la Síntesis de la Proteína/farmacología , Animales , Carcinoma Hepatocelular/patología , División Celular/efectos de los fármacos , Cromogranina A , Cromograninas , GMP Cíclico/biosíntesis , ADN/biosíntesis , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Neoplasias Hepáticas/patología , Óxido Nítrico/antagonistas & inhibidores , Hormonas Pancreáticas/química , Ratas , Células Tumorales Cultivadas , omega-N-Metilarginina/farmacologíaRESUMEN
The African bullfrog Pyxicephalus adspersus is generally classified along with frogs of the genus Rana in the subfamily Raninae of the family Ranidae but precise phylogenetic relationships between species are unclear. Pancreatic polypeptide (PP), insulin, and glucagon-like peptide (GLP-1) were isolated from an extract of P. adspersus pancreas and characterized structurally. A comparison of the amino acid sequence of Pyxicephalus PP (APSEPQHPGG(10)QATPEQLAQY(20)YSDLYQYITF(30)ITRPRF++ +. NH(2)) with those of the known amphibian PP molecules in a maximum parsimony analysis generates a single phylogenetic tree in which Pyxicephalus is the sister to the clade comprising the members of the genus Rana. The three orders of living amphibians form discrete clades with the representative of the Gymnophiona appearing as sister to the Caudata-Anura. In contrast, Pyxicephalus insulin (A chain, GIVEQCCHSA(10)CSLYDLENYC(20)N; B-chain, LANQHLCGSH(10)LVEALYMVCG(20)ERGFFYYPKS(30)) and and GLP-1 (HAEGTFTSDM(10)TSYLEEKAAK(20)EFVDWLIKGR(30)PK) resemble more closely the corresponding peptides from the cane toad Bufo marinus than the peptides from any species of Rana. Cladistic analysis based upon the amino acid sequences of insulin produced a polyphyletic assemblage with the Gymnophiona nesting within an unresolved clade containing the non-ranid frogs. The data support the assertion that the amino acid sequence of PP, but not those of the other islet hormones, is of value as a molecular marker for inferring phylogenetic relationships between early tetrapod species.
Asunto(s)
Hormonas Pancreáticas/química , Filogenia , Ranidae/clasificación , Secuencia de Aminoácidos , Animales , Cromatografía Líquida de Alta Presión , Glucagón/química , Glucagón/aislamiento & purificación , Péptido 1 Similar al Glucagón , Insulina/química , Insulina/aislamiento & purificación , Datos de Secuencia Molecular , Polipéptido Pancreático/química , Polipéptido Pancreático/aislamiento & purificación , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Precursores de Proteínas/química , Precursores de Proteínas/aislamiento & purificación , Homología de Secuencia , Especificidad de la EspecieAsunto(s)
Hormonas Pancreáticas/fisiología , Adipocitos/metabolismo , Animales , Cromogranina A , Humanos , Islotes Pancreáticos/metabolismo , Glucógeno Hepático/metabolismo , Modelos Biológicos , Páncreas/metabolismo , Hormonas Pancreáticas/química , Hormonas Pancreáticas/genética , Glándulas Paratiroides/metabolismo , Células Parietales Gástricas/metabolismo , Receptores de la Hormona Gastrointestinal/fisiología , Estrés Fisiológico/fisiopatologíaRESUMEN
Studies of pancreatic development have suggested that the islet cells develop through multihormonal stages. Abundant data have confirmed that multihormonal cells are common during pancreatic development. A number of transcription factors and homeotic proteins have also been found to be important to pancreatic and islet cell development. While one of these factors (Isl1) is important for the development of the dorsal pancreatic bud and mesenchyme, another factor (Pdx1) is needed for growth and branching of both pancreatic buds. Studies of the expression patterns of pancreatic hormones and transcription factors and other marker proteins seem at present to be most compatible with the view that early glucagon and glucagon + insulin expressing cells are precursors to the glucagon cells of the islets while mature B cells arise through differentiation from glucagon-negative precursor cells. Recent data also point to possibilities of local paracrine interactions between islet cell types and the parenchymal tissue during development.
Asunto(s)
Islotes Pancreáticos/embriología , Páncreas/embriología , Animales , Diferenciación Celular , Desarrollo Embrionario y Fetal , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Islotes Pancreáticos/citología , Islotes Pancreáticos/metabolismo , Páncreas/metabolismo , Hormonas Pancreáticas/química , Hormonas Pancreáticas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The ECL cells constitute the predominant endocrine cell population in the mucosa of the acid-secreting part of the stomach (fundus). They are rich in chromogranin A (CGA), histamine and histidine decarboxylase (HDC). They secrete CGA-derived peptides and histamine in response to gastrin. The objective of this investigation was to examine the expression of pancreastatin (rat CGA266-314) and WE14 (rat CGA343-356) in rat stomach ECL cells. The distribution and cellular localisation of pancreastatin- and WE14-like immunoreactivities (LI) were analysed by radioimmunoassay and immunohistochemistry with antibodies against pancreastatin, WE14 and HDC. The effect of food deprivation on circulating pancreastatin-LI was examined in intact rats and after gastrectomy or fundectomy. Rats received gastrin-17 (5 nmol/kg/h) by continuous intravenous infusion or omeprazole (400 micromol/kg) once daily by the oral route, to induce hypergastrinemia. CGA-derived peptides in the ECL cells were characterised by gel permeation chromatography. The expression of CGA mRNA was examined by Northern blot analysis. Among all of the endocrine cells in the body, the ECL cell population was the richest in pancreastatin-LI, containing 20-25% of the total body content. Food deprivation and/or surgical removal of the ECL cells lowered the level of pancreastatin-LI in serum by about 80%. Activation of the ECL cells by gastrin infusion or omeprazole treatment raised the serum level of pancreastatin-LI, lowered the concentrations of pancreastatin- and WE14-LI in the ECL cells and increased the CGA mRNA concentration. Chromatographic analysis of the various CGA immunoreactive components in the ECL cells of normal and hypergastrinemic rats suggested that these cells respond to gastrin with a preferential release of the low-molecular-mass forms.
Asunto(s)
Cromograninas/metabolismo , Mucosa Gástrica/metabolismo , Proteínas de Neoplasias/biosíntesis , Hormonas Pancreáticas/biosíntesis , Animales , Cromogranina A , Cromograninas/química , Cromograninas/genética , Células Enterocromafines , Mucosa Gástrica/química , Mucosa Gástrica/citología , Gastrinas/sangre , Inmunohistoquímica , Masculino , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Hormonas Pancreáticas/sangre , Hormonas Pancreáticas/química , Hormonas Pancreáticas/genética , ARN Mensajero/química , Ratas , Ratas Sprague-DawleyRESUMEN
Histological studies were performed on 24 pancreases of normal human embryos and fetuses aged 7 to 38 weeks. For immunocytochemistry, the avidin-biotin-peroxidase method was used to identify and localize insulin, glucagon, somatostatin, and pancreatic polypeptide (PP) cells. In 7 wk old embryos, cells containing somatostatin and PP are observed. One week later appear single glucagon-positive cells. In the 9th wk, insulin producing cells are visible. During the fetal period two populations of the investigated cells are found: Langerhans islets and dispersed cells. The latter cells containing insulin, glucagon or somatostatin are localized in the walls of pancreatic ducts throughout the whole gland, while PP-positive cells are seen mainly in the part of the pancreas, which develops from the ventral anlage (anteroinferior part of the head and adjacent part of the main pancreatic duct). During the development of islets we have observed four stages: (1) scattered cells (7 to 10 weeks); (2) grouping cells (11 to 15 weeks); (3) mantle and zonular islets (10 to 29 weeks), in which B cells located inside are surrounded by a thick zone of A, PP and somatostatin-producing cells; (4) mixed islets (from 30 weeks on) - all cells are scattered over the whole transverse section of the islet. In the developing pancreas, the glucagon- and somatostatin-containing cells are the most numerous, while the insulin and PP-containing cells occur in lesser quantities.
Asunto(s)
Desarrollo Embrionario y Fetal , Islotes Pancreáticos/química , Islotes Pancreáticos/embriología , Hormonas Pancreáticas/química , Embrión de Mamíferos , Femenino , Glucagón/química , Glucagón/inmunología , Humanos , Inmunohistoquímica , Insulina/química , Insulina/inmunología , Islotes Pancreáticos/citología , Masculino , Hormonas Pancreáticas/inmunología , Polipéptido Pancreático/química , Polipéptido Pancreático/inmunología , Somatostatina/química , Somatostatina/inmunologíaRESUMEN
Using a specific antiserum for the C-terminal glycine amide region of human pancreastatin (PST), pancreastatin-like immunoreactivity (PST-LI) was measured in cerebrospinal fluid (CSF) from 447 subjects (368 +/- 10.8 pmol/l, mean +/- S.E.M.) free from endocrine diseases. The CSF contents of PST-LI showed a mountain-shape type change which peaked at 40 years of age. The highest concentration was found in the group of ages 40-49 years old (412 +/- 22.9 pmol/l) and the lowest concentration was found in the group of ages 80-89 years old (293.2 +/- 45.2 pmol/l) among various age groups. Gel chromatographic examination revealed the presence of two major forms (MW 13,500 and 5,400) of PST-LI in CSF. Because of the character of this antibody, the large molecular form is possibly an N-terminally elongated PST and the other may be PST-52. This may be the first report on the unique age-related change of PST concentration in CSF.
Asunto(s)
Envejecimiento/líquido cefalorraquídeo , Hormonas Pancreáticas/líquido cefalorraquídeo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Cromatografía en Gel , Cromogranina A , Femenino , Humanos , Masculino , Persona de Mediana Edad , Peso Molecular , Hormonas Pancreáticas/química , Hormonas Pancreáticas/inmunología , RadioinmunoensayoRESUMEN
A radioimmunoassay of human pancreastatin was developed using a rabbit antiserum that selectively recognized the C-terminal amidated end of the peptide, and it was used for the identification of the molecular forms of pancreastatin in human gut (stomach, duodenum, small intestine, colon) and endocrine tumor extracts (liver metastasis of a gastrinoma and a medullary carcinoma of thyroid, one nonsecreting pancreatic tumor, one recurrence of a gut carcinoid, one vipoma and one insulinoma). In all gut extracts, a gel filtration chromatography revealed the presence of three peaks of pancreastatin-like immunoreactivity. The predominant form eluted with an apparent molecular weight higher than that of pancreastatin. This form was also predominant in the endocrine tumors analyzed, except in the insulinoma, where a lower molecular weight form predominated. The high molecular form was further purified from a liver metastasis of a gastrinoma. The pancreastatin-like immunoreactivity eluted in all the chromatographical systems (reverse-phase, ion exchange) as a single peak that was finally purified to homogeneity and sequenced. The sequence of the first 29 N-terminal amino acids was obtained unambiguously and corresponded to the sequence 210-238 of chromogranin A. Considering the selectivity of the assay used for peptide identification, this major form was identified as the fragment 210-301 of chromogranin A. It is likely that the predominant form of pancreastatin in human gut extracts and noninsular tumors is a 92 amino acid peptide.
Asunto(s)
Cromograninas/química , Neoplasias de las Glándulas Endocrinas/química , Hormonas Gastrointestinales/química , Hormonas Pancreáticas/química , Fragmentos de Péptidos/química , Extractos de Tejidos/química , Secuencia de Aminoácidos , Animales , Cromatografía en Gel , Cromogranina A , Cromograninas/genética , ADN Complementario/genética , Datos de Secuencia Molecular , Peso Molecular , Especificidad de la Especie , PorcinosRESUMEN
Glucagon-like peptide-I (GLP-I) is encoded together with glucagon by the glucagon gene and is related in its structure to the glucagon-secretin family of peptides. Three of the predicted forms of the peptide, a 37-residue long GLP-I(1-37), a 31-residue GLP-I(7-37) and a 30-residue GLP-I(7-36)amide as well as three analogs des [Gly37, Arg36] GLP-I(7-37), des [Gly37, Arg36, Gly35] GLP-I(7-37) and des [His7] GLP-I(7-37) were synthesized by the stepwise solid phase method. These synthetic peptides were used to define the structural domains required for the binding of GLP-I to the pancreatic beta cell. The competitive binding experiments showed that both the amino and carboxyl terminal domains of the molecule contribute to GLP-I binding. In these experiments glucagon, another peptide that stimulates insulin secretion, was a weak full agonist of GLP-I binding. Results from these studies provide further characterization of the physiological role of this new peptide.
Asunto(s)
Hormonas Pancreáticas/química , Péptidos/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Unión Competitiva/fisiología , Línea Celular , Código Genético/genética , Péptidos Similares al Glucagón , Humanos , Insulina/metabolismo , Secreción de Insulina , Hígado/fisiología , Datos de Secuencia Molecular , Hormonas Pancreáticas/genética , Hormonas Pancreáticas/fisiología , Péptidos/genética , Péptidos/fisiología , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Relación Estructura-ActividadRESUMEN
Pancreastatin is a 49 amino acid peptide with a C-terminal glycine amide originally isolated from porcine pancreas. There are strong indications that pancreastatin is derived from chromogranin A, since the amino acid sequence 240-288 in porcine chromogranin A contains pancreastatin flanked by typical signals for proteolytic processing. In the present study the distribution and molecular nature of immunoreactive pancreastatin were examined in selected porcine tissues. For this purpose a radioimmunoassay specific for the C-terminal sequence of porcine pancreastatin, that did not cross-react with porcine chromogranin A was used in combination with gel permeation chromatography and reverse-phase high-pressure liquid chromatography (HPLC). We demonstrated the presence of pancreastatin, a C-terminal pancreastatin fragment and N-terminally extended molecular forms in the examined tissues. Pancreastatin predominated in the pancreas and stomach antrum, while N-terminally extended molecular forms were mainly present in the stomach body, jejunum and adrenal gland. The specific distribution pattern of the molecular forms probably reflects a tissue-specific processing of chromogranin A.
Asunto(s)
Hormonas Pancreáticas/análisis , Glándulas Suprarrenales/química , Animales , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Cromogranina A , Cromograninas/metabolismo , Yeyuno/química , Páncreas/química , Hormonas Pancreáticas/química , Fragmentos de Péptidos/análisis , Antro Pilórico/química , Radioinmunoensayo/normas , Radioinmunoensayo/estadística & datos numéricos , Estómago/química , Porcinos , Distribución TisularRESUMEN
It is known that pancreastatin-like immunoreactivity (PLI) occurs in the secretory granules of the islet B- and D-cells in the pig pancreas, and that porcine pancreastatin inhibits insulin secretion in rats and mice. In this study, we characterized the porcine plasma PLI and examined whether PLI is released from the pig pancreas in vivo. We found that PLI in unextracted pig plasma largely consists of two high-molecular fractions, with Mr values of 80-85,000 and 300-350,000, respectively. In addition, a small peak of PLI eluted after gel filtration at the position of synthetic porcine pancreastatin. After extraction on octadecylsilyl silica, virtually all PLI disappeared except in the fraction co-eluting with porcine pancreastatin. In thiopenthal-anesthetized pigs, plasma samples were obtained from the carotid artery and the superior pancreaticoduodenal vein. By multiplying the venous-arterial concentration difference by the pancreatic venous plasma flow, a net pancreatic output of PLI of 420 +/- 120 pmol/min was found. This pancreatic PLI output was significantly reduced by electrical stimulation of the local autonomic nerves along the superior artery during atropine administration (p less than 0.001). Furthermore, the pancreatic venous PLI levels were elevated during intravenous infusion of glucose (p less than 0.01). We conclude that pig plasma PLI levels can be measured by radioimmunoassay after extraction on octadecylsilyl silica and that there is a net pancreatic output of PLI, which is reduced by sympathetic stimulation and enhanced during hyperglycemia.
Asunto(s)
Páncreas/metabolismo , Hormonas Pancreáticas/metabolismo , Animales , Sistema Nervioso Autónomo/fisiología , Cromatografía en Gel , Cromogranina A , Estimulación Eléctrica , Glucosa/farmacología , Insulina/sangre , Peso Molecular , Páncreas/inervación , Hormonas Pancreáticas/química , PorcinosRESUMEN
Pancreastatin is a 49 amino acid peptide originally isolated from porcine pancreas on the basis of its C-terminal glycinamide as isolation criterion. It is derived by proteolytic processing from chromogranin A, an acidic protein component of secretory granules in endocrine and neuronal cells. The primary structures of human, porcine, bovine and rat pancreastatin have been determined on the protein or cDNA level and show 70% sequence homology. By immunocytochemistry, pancreastatin has been detected in the pituitary, adrenal gland, pancreas, CNS and throughout the gastrointestinal tract. In pancreatic islets, pancreastatin is co-localized with insulin, glucagon and somatostatin. The principle biological activities of this peptide are: inhibition of insulin release and of exocrine pancreatic secretion. These effects which can be assigned to the amidated C-terminal part of the molecule have been demonstrated in several species. Whether or not pancreastatin can be classified as a novel peptide hormone that under physiological conditions plays a role in the regulation of the endocrine and exocrine pancreas, is still a matter of controversy.