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Query: UNIPROT:P01275 (glucagon)
 26,492 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Fetal bovine pancreas was extracted for glucagon using (A) ethanol-Hcl after trichloroacetic acid (TCA) treatment of the pancreas, (B) ethanol-HCl and (C) urea-acetic acid. Fractionation of the acetic acid soluble proteins vi Sephadex G-50 columns yielded glucagon immunoreactivity in the void volume, high molecular weight glucagon immunoreactivities (HMW-IRGs), "proglucagon" (approximately equal to 9 K delta), and true glucagon (3.5 K delta) regions. HMW-IRGs were obtainable using all three methods of extraction. The material obtained from the ethanol-HCl-TCA method appeared stable on Sephadex G-100 (1 M acetic acid) rechromatography. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis analysis showed immunoreactive species corresponding to approximately 40 K delta and approximately 12 K delta. HMW-IRGs did not bind to concanavalin A (Con A)-agarose. SDS-polyacrylamide gel electrophoresis of the Con A-agarose filtered IRG again showed a major immunoreactive peak of approximately 40 K delta. Dose-response RIA studies indicated that the HMW-IRGs from both the gel filtration and SDS-polyacrylamide gel experiments were immunochemically indistinguishable from glucagon. HMW-IRGs bind to antiglucagon antibody agarose, further indicating their reactivity towards glucagon antibodies. When HMW-IRGs are incubated with guanidinium hydrochloride and gel filtered in the same system, a significant fraction of HMW-IRG (representing up to 25% of the total IRG analysed) was found to resist disruption. Our data support the contention that a significant portion of the HMW-IRGs (molecular weight greater than 20 K delta) extracted from fetal bovine pancreas are composed of glucagon covalently linked to larger protein unit(s).
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PMID:Glucagon from the bovine fetal pancreas: chromatographic and electrophoretic characterizations of high molecular weight immunoreactive species. 700 59

The effects of varying concentrations of ethanol (1, 10, and 30 mM) and its metabolites (1 mM acetate and 1 and 10 mM acetaldehyde) on insulin and glucagon secretion induced by glucose (11.1 mM) and arginine (20 mM) were studied in isolated perfused pancreas of Sprague-Dawley rats. Ethanol and its metabolites did not significantly modify basal secretion of the two hormones. Ethanol reduced glucose-induced insulin secretion by means of a dose-related effect. Arginine-induced insulin output did not seem to be influenced to any significant degree. Acetate and acetaldehyde significantly inhibited glucose and arginine-induced insulin secretion. While ethanol (10 and 30 mM ) did not modify glucagon output during arginine perfusion, acetate and acetaldehyde markedly enhanced it. The block of insulin secretion and the increased secretion of glucagon could explain the diabetogenic effect of ethanol demonstrated in vivo. The mechanism by which ethanol acts on the pancreatic beta- and alpha-cells is discussed.
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PMID:Effect of ethanol, acetaldehyde, and acetate on insulin and glucagon secretion in the perfused rat pancreas. 702 Dec 70

The conversion of proglucagon and proinsulin by secretory granules isolated from both prelabeled and unlabeled anglerfish islets was investigated. Either granules isolated from tissue labeled with [3H]tryptophan and [14C]isoleucine or [35S]cysteine, or lysed granules from unlabeled tissue to which exogenously labeled prohormones had been added were incubated under various conditions. Acetic acid extracts of these granule preparations were analyzed for prohormone and hormone content by gel filtration. Both prelabeled and lysed, unlabeled secretory granules converted radiolabeled precursor peptides (Mr 8,000-15,000) to labeled insulin and glucagon. The accuracy of the cleavage process was established by demonstrating comigration of products obtained from in vitro cleavage with insulin and glucagon extracted from intact islets using electrophoresis and high-pressure liquid chromatography (HPLC). The pH optimum for granule-mediated conversion was found to be in the range of pH 4.5-5.5. Conversion of both proglucagon and proinsulin by secretory granules was significantly inhibited in the presence of antipain, leupeptin, p-chloromercuribenzoate (PCMB) or dithiodipyridine (DDP) but not chloroquine, diisopropyl fluorophosphate, EDTA, p-nitrophenyl guanidinobenzoate, soybean trypsin inhibitor, or N-p-tosyl-L-lysine chloromethyl ketone HCl. The inhibitory action of PCMB and DDP was reversed in the presence of dithiothreitol. Both membranous and soluble components of the secretory granules possessed significant converting activity. HPLC and electrophoretic analysis of cleavage products demonstrated that the converting activities of the membranous and soluble components were indistinguishable. The amount of inhibition of proinsulin and proglucagon conversion caused by 600 micrograms/ml porcine proinsulin was significantly lower than that caused by the same concentration of unlabeled anglerfish precursor peptides. These results indicate that the proinsulin and proglucagon converting enzyme(s) in the anglerfish pancreatic islet is a unique intracellular thiol proteinase(s) that may be granule membrane-associated and may require the presence of prohormone sequences in addition to the dibasic residues at cleavage sites for substrate recognition and/or binding.
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PMID:Characterization of proinsulin- and proglucagon-converting activities in isolated islet secretory granules. 702 70

The synthesis of the heterobifunctional cross-linking reagent 2-nitro-4-azidophenylsulfenyl chloride (NAPSCl) is described. This reagent can be used to specifically attach a photoactivatable nitrophenyl azide to tryptophan-containing polypeptides and proteins lacking sulfhydryl groups. The sulfenyl chloride group of NAPSCl reacts with the indole ring of tryptophan following second-order reaction kinetics in 50-100% acetic acid. The labeled product can be effectively photolyzed at wavelengths above 300 nm. The reaction of glucagon, a peptide hormone containing a single tryptophan residue at position 25 and no cysteine, with NAPSCl gave one major product, the photosensitive derivative glucagon-NAPS. The structure and properties of the purified derivative were established by amino acid analysis, absorption spectroscopy, and photolysis. Only the tryptophan residue of this derivative was modified. The photosensitive glucagon was shown to activate the adenylate cyclase of hepatocyte plasma membranes to the same extent as the native hormone at equimolar concentrations. Glucagon-NAPS could be radiolabeled by the lactoperoxidase-catalyzed iodination of the peptide. A glucagon-specific antibody bound both radiolabeled glucagon and glucagon-NAPS peptides. The covalent labeling of protein molecules with radiolabeled glucagon-NAPS peptide upon photolysis was demonstrated. Glucagon-NAPS can be used as an effective photoaffinity probe for labeling the glucagon receptor site in plasma membranes of target cells.
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PMID:Synthesis and characterization of a heterobifunctional photoaffinity reagent for modification of tryptophan residues and its application to the preparation of a photoreactive glucagon derivative. 742 13

We synthesized and iodinated an exendin-4 analogue, [Y39]exendin-4 (700 Ci/mmol), for use as a radioligand to characterize exendin receptors on dispersed pancreatic acini and gastric chief cells from guinea pig. Binding of this bioactive radioligand was rapid, temperature-dependent and specific (not inhibited by other pancreatic or gastric secretagogues). Measurement of the ability of exendin-4 to inhibit the binding of 125I-[Y39]exendin-4 indicated the presence of two classes of receptors. Pancreatic acini had 12.5.10(10) binding sites/mg acinar protein of which 6% were high affinity (Kd = 0.5 nM) and 94% were low affinity (Kd = 0.1 microM). Chief cells had 3370 binding sites/cell of which 9% were high affinity (Kd = 0.3 nM) and 91% were low affinity (Kd = 0.2 microM). Washing with 0.2 M acetic acid (pH 2.5), 0.2 M glycine (pH 10.5), or trypsin (100 micrograms/ml) after 30 min incubation at 37 degrees C, indicated that 63 and 49% of radioligand was internalized in acini and chief cells, respectively. Truncated glucagon-like peptide-1 (tGLP-1), a mammalian peptide sharing 53% homology with exendin-4, inhibited radioligand binding at the same concentrations that altered secretion from acini and chief cells. Glucagon, GLP-1 and GLP-2 inhibited 125I-[Y39]exendin-4 binding only at concentrations > or = 100 nM. Exendin(9-39)NH2, a specific exendin-receptor antagonist, potently inhibited 125I-[Y39] exendin-4 binding (IC50 = 6.1 and 3.5 nM in acini and chief cells, respectively). In pancreatic acini and gastric chief cells from guinea pig, exendin-3, exendin-4 and tGLP-1 increase cellular cAMP and modulate enzyme secretion by interacting with high-affinity exendin receptors. 125I-[Y39] exendin-4 is a useful radioligand for studying exendin receptors.
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PMID:Use of 125I-[Y39]exendin-4 to characterize exendin receptors on dispersed pancreatic acini and gastric chief cells from guinea pig. 780 Aug 58

Glucagon injected in the lateral hypothalamus stimulates sympathetic activity and suppresses monamine metabolism. The central hypothesis underlying this study is that there is a reciprocal relationship between food intake and sympathetic activity to IBAT. This hypothesis was tested by using intrahypothalamic microinjections of glucagon, a peptide that has been reported to decrease food intake. Sympathetic nerve activity to interscapular brown adipose tissue (IBAT) was measured as electrophysiological discharges of sympathetic nerves to IBAT. The microinjection of glucagon into the lateral hypothalamus (LH) increased sympathetic nerve activity by +103.8 +/- 35.0% (mean +/- S.E.M.) from pre-injection basal level by 30 min after injection. There was a gradual return to baseline. Micro-injection of glucagon into the LH depressed food intake. Monoamine metabolism was measured by using a microdialysis probe attached to a guide cannula for microinjection of glucagon into the LH. After microinjection of glucagon, the dialysates were collected over 30 min intervals and assayed for norepinephrine (NE), serotonin (5-HT), dopamine (DA) and their metabolites (3-methoxy-4-hydroxyphenylglycol (MHPG); 5-hydroxyindole-3-acetic acid (5-HIAA); and 3,4-dihydroxyphenylacetic acid (DOPAC). Glucagon suppressed both NE and MHPG concentrations in the lateral hypothalamus (LH), and the concentration of DOPAC was also decreased. There was no change of 5-HT concentration but 5-HIAA levels were reduced by glucagon treatment. These data show that glucagon injected in the LH stimulates sympathetic activity and suggest that this may have occurred by suppression of norepinephrine, dopamine and serotonin turnover in the LH of freely moving rats. These data support the hypothesis of a reciprocal relationship between food intake and sympathetic activity.
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PMID:Glucagon injected in the lateral hypothalamus stimulates sympathetic activity and suppresses monoamine metabolism. 811 8

Cholecystokinin (CCK) produced in the mucosa of the upper small intestine exerts several biological functions. Its secretion in physiological amounts is modulated by the interaction of extracellular regulators and by binding to intracellular receptors of the target cells. The relative affinity of CCK to its receptor has been characterized in various biological and pharmacological studies and it is now well established that CCK has a higher affinity to the CCKA than to the CCKB receptor. Furthermore CCK influences the secretion of pancreatic enzymes in several species but very little is known about the relationship between CCK and the islet hormone-producing cells in the pig pancreas. The localization of this receptor at the cellular level showed conflicting results in animal studies and has not been described in pigs. The aim of the present study was to characterize the precise cellular location of the CCKA receptor in the porcine pancreas. Polyclonal antiserum was raised against the N-terminal epitope of the CCKA receptor molecule and used for localization studies. Using immunohistochemistry on methanol/acetic acid-fixed, paraffin-embedded pancreas, the CCKA receptor could successfully be localized in islet cells. Parallel staining of serial sections with antibodies directed against insulin and glucagon revealed colocalization with glucagon in alpha cells. No immunoreaction was found in the exocrine pancreas. Our results support the concept that in the porcine species the stimulation of the exocrine pancreas is mediated by the CCKB rather than the CCKA receptor, as it is known for the rat species.
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PMID:Cell-specific localization of the cholecystokininA receptor in the porcine pancreas. 1119 80

Endocrine cells containing gastrin/cholecystokinin (CCK)-like immunoreactivity were localized to the islet tissue in the pancreas of the spiny dogfish. Most of these cells were located in the 'intestinal' lobe of the pancreas; only occasional cells were observed in the 'splenic' lobe. The gastrin/CCK-like immunoreactive cells were often co-localized with the 'classical' pancreas hormones (insulin, glucagon and somatostatin). Radioimmunoassay of water extracts with a C-terminally directed antiserum revealed high levels of immunoreactive material in the intestinal part (48.6 +/- 19.9 pmol/g) and lower levels (4.5 +/- 0.6 pmol/g) in the splenic part. Acetic acid extracts of the intestinal lobe contained low levels (6.8 +/- 3.3 pmol/g) of gastrin/CCK-like immunoreactivity, whereas corresponding extracts of the splenic part showed no immunoreactivity. When the extracts were subjected to DEAE ion-exchange chromatography the gastrin/CCK-like peptides eluted as a major peak. After Sephadex gel filtration, pooled immunoreactive material from the main DEAE chromatographic peak eluted at a position close to that of CCK4. Further characterization by ion-exchange and reversed-phase HPLC showed that, in general, the immunoreactive material behaved like the shorter forms of the gastrin/CCK family (CCK4/G5 and CCK8/Cae 3-10).
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PMID:Endocrine cells with gastrin/cholecystokinin-like immunoreactivity in the pancreas of the spiny dogfish, Squalus acanthias. 1250 16

We present a method for the quantitation of glucagon from rat plasma by protein precipitation and LC/MS. No internal standard was used, as a labeled standard was not available and similar peptides did not show comparable extraction characteristics to glucagon. The LC system included a Keystone C18, 300 A pore size column; a linear gradient was used with a mobile phase consisting of water and acetonitrile, each with 0.2% acetic acid and 0.02% trifluoroacetic acid. Glucagon was detected with the mass spectrometer in positive ion mode monitoring the 4+ charge state at m/z 871.7. The method had an approximated limit of detection of 1 ng/mL. The lower limit of quantitation (LLOQ) was 25 ng/mL (7.2 fmol/mL), which could be reduced with an appropriate internal standard. External calibration was used and calibration curves were found to be linear over the range from 25 to 1000 ng/mL (7.2 to 290 fmol/mL). The method showed a high degree of precision and accuracy both within and between runs at four validation points, including the LLOQ.
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PMID:Quantitation of the large polypeptide glucagon by protein precipitation and LC/MS. 1538 87

The cholecystokinin (CCK) family of peptides and receptors is present throughout the brain and gastrointestinal tract including pancreatic tissue. The expression of these molecules in the gut and pancreas is species-specifically regulated and the role of CCK in porcine pancreatic islet hormone secretion is still a matter of discussion. Therefore, in this study we have determined the cell-type specific localization of and its high affinity CCKA-receptor in islet cells using immunohistochemical techniques. Serial sectioning followed by double-immunostaining of methanol/acetic acid-fixed, paraffin-embedded pancreatic tissues were performed with antibodies against CCK, CCKA-receptor, glucagon and somatostatin. To determine whether CCK specific mRNA is locally expressed, total RNA was isolated, transcribed to cDNA and analysed with specific primer for CCK gene expression. Our results clearly show that CCK and the CCKA-receptor coexist in glucagon--but not in somatostatin-producing cells. Moreover our RT-PCR experiments demonstrate that there is no local gene expression of CCK in the porcine pancreas. Our results provide evidence that, in the porcine species, blood-borne CCK binds specifically to the CCKA-receptor and may thereby modulate the glucose homeostasis via a direct action on A-cells.
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PMID:Determination of target cells for cholecystokinin in the porcine pancreas. 1613 Aug 20


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