Studies on submaxillary gland immunoreactive glucagon

Bhathena, Sam J.; Smith, S. S.; Voyles, Nancy R.; Penhos, J. C.; Recant, Lillian

doi:10.1016/0006-291x(77)90622-2

Cited by 32 publications

(26 citation statements)

References 12 publications

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“…By previous studies we know that the rat salivary gland has significant amounts of a polypeptide with the same physicochemical, immunological and biological properties as pancreatic glucagon [3,5,12,15]. In addition, when isolated cells of this gland are incubated with 3H-L-tryptophan, a radioactive polypeptide of 3.5 Kdaltons is immunoprecipitated with a C-terminal glucagon antiserum (30 K); suggesting that the salivary gland represents a source for synthesis of extrapancreatic glucagon rather than an organ that stores this hormone.…”

Section: Discussionmentioning

confidence: 99%

Evidence of glucagon biosynthesis involving protein intermediates in rat salivary glands

Pérez-Castillo

Blázquez

1984

Diabetologia

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Summary.In an attempt to determine the ability of rat submaxillary glands to synthesise glucagon via protein intermediates, isolated cells from these glands were incubated in vitro with 3H-L-tryptophan and the acid-ethanol extracts of the cells were purified on Bio-Gel P-30 columns. Aliquots of the eluates were incubated with a C-terminal glucagon antiserum (30K) and the radioactivity bound to the glucagon antibody appeared to be distributed among proteins of Molecular weight > 40.14 and 3.5 Kdaltons. A similar elution pattern was obtained in the presence of urea (7 mol/1) and guanidine hydrochloride (6 tool/l). To determine the molecular weight of the immunoreactive material eluting before the 3.5 Kdalton polypeptide, aliquots of the cell extracts were immunoprecipitated and analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Polypeptides of 125.8, 63.1, 42.6 and 14.4 Kdaltons were obtained. These polypeptides incorporate more radioactive tryptophan with increase in the time of incubation. Pulse-chase experiments with unlabelled tryptophan, cycloheximide-treatment of isolated cells and limited tryptic digestion of the larger glucagon immunoreactive component, transform it into a 3.5 Kdalton polypeptide with immunological characteristics indistinguishable from pancreatic glucagon. These results suggest that the larger molecule contains glucagon and thus may serve as a precursor or an intermediate of extrapancreatic glucagon biosynthesis.Key words: Glucagon biosynthesis, protein intermediates, rat submaxillary glands.Our knowledge concerning the location of cells that produce and secrete polypeptide hormones has changed rapidly during the last few years. Gastrointestinal hormones have been detected in unforeseen locations [1] and peptides of neural origin have been found in pancreatic and gastrointestinal endocrine cells [2]. Glucagon, a hormone previously considered to be exclusively a product of pancreatic islets, has recently been detected in several extrapancreatic locations [3][4][5][6], where it could represent synthesised hormone, trapped or stored as an aggregate. However, cells similar to pancreatic A cells have been identified in the stomach [7,8], where large amounts of immunoreactive glucagon is released in response to specific stimuli [9][10][11]. Demonstration of active hormonal biosynthesis could offer conclusive evidence for the existence of extrapancreatic glucagon. In previous reports we have described that isolated cells of human [12] and rat submaxillary glands [5] incorporate 3H-L-tryptophan into a 3,500dalton polypeptide with specific immune reaction with a C-terminal glucagon antiserum, suggesting that salivary glands actively synthesise glucagon rather than selectively trap or store this hormone. Therefore, the present study was designed in order to determine the ability of isolated rat submaxillary gland cells to synthesise glucagon, with special regard to defining the different forms of immunoreactive glucagon and the probable precursor-product relationship. Material ...

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Section: Discussionmentioning

confidence: 99%

Evidence of glucagon biosynthesis involving protein intermediates in rat salivary glands

Pérez-Castillo

Blázquez

1984

Diabetologia

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“…The existence of glucagon-like substances in the rat salivary gland has been reported in recent years [1][2][3][4][5][6][7]. It has been postulated that salivary gland may be one of the candidates for extrapancreatic sources of circulating glucagon, which occurs even in the pancreatectomized or eviscerated rat [1][2][3]7].…”

Section: Glucagon-degrading Activity Affinity Chromatographymentioning

confidence: 99%

“…However, the question whether or not the glucagonlike substance in acid saline extract of the submaxillary gland is identical to pancreatic glucagon and/or its precursor has been controversial because glucagon-like substances in the salivary gland have been found only in high molecular forms [2][3][4][5], and because of the difficulty in demonstrating it successfully by the immunohistochemical method.…”

Section: Glucagon-degrading Activity Affinity Chromatographymentioning

confidence: 99%

“…(5) 2N acetic acid method: the material was homogenized in 2N acetic acid, centrifuged at 2,000 g for 30 min and the supernatant fluid was collected as extract. (6) Acid saline method: after homogenization in saline (0.154mol/1) acidified with HCI to pH2.8, the sample was centrifuged at 10,000 g for 30 min and the supernatant was used as the extract [2][3][4][5][6][7].…”

Section: Extraction Proceduresmentioning

confidence: 99%

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125I-glucagon-degrading activity in acid-saline extracts of rat salivary gland

et al. 1984

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Summary. The antibody-binding ability of the glucagon-like substance in rat submaxillary gland acid saline extract was examined by affinity chromatography, and the biological activity studied using the isolated liver perfusion method. We found that the glucagon-like substances in acid saline extract could not be bound to anti-glucagon antibody and that the gel-filtration peak on ultrogel AcA 54 could increase neither glucose nor cyclic AMP output from isolated perfused rat liver. Furthermore, the radioactivity peak of 125I-glucagon on Bio Gel P-6 column chromatography moved from its original position and eluted in later fractions after incubation with an acid saline extract of the submaxillary gland. In consequence, there was 125I-glucagon degrading activity in the submaxillary gland, but no glucagon-related peptide. Therefore, it is suggested that the glucagon-like substance, which has been reported in acid saline extract of the rat salivary gland, may be an artifact due to tracer degrading activity.

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“…Large amounts of glucagon-like substances have been reported in acid saline extracts of the submaxillary gland of the rat, mouse and rabbit (Bhathena et al, 1974;Silverman and Dunbar, 1974;Lawrence et al, 1976;Dunbar et al, 1976;Paulo et al, 1986). In addition, immunocytochemical studies have shown glucagonlike peptides in the rat salivary gland (Smith and Tomo, 1986).…”

mentioning

confidence: 99%

Degradation of 125I-Glucagon, -Pancreatic Polypeptide and -Insulin by Acid Saline Extract of Rat Submaxillary Gland and Their Protection by Proteinase Inhibitors.

Tasaka¹,

Marumo²,

Inoue³

et al. 1989

Endocrinol Japon

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The acid saline extract (ASE) of rat submaxillary gland exerts a powerful degrading effect on 125I-glucagon. In order to study the degradation of other 125I-peptides by ASE and the effects of their inhibitors , 125I-pancreatic polypeptide (PP) and 125I-insulin were used together with 125I-glucagon. The degradation studies were done by the trichloroacetic acid (TCA) method or gel in the ordinary immunoassay system using the TCA method, but 125I-insulin was intact in the presence of ASE. Leupeptin, and to a lesser extent pchloromercuriphenyl-sulfonic acid (PCMS) and N-ethylmaleimide, inhibited the destruction of 125I-glucagon or -PP under the TCA method. PCMS was especially protective at high concentrations, for example 16 mM. These findings were confirmed by gel filtration of the assay mixture. In the presence of leupeptin (0.4mM) and PCMS (16mM), no shift in the peak of labelled glucagon or PP occurred. Thus ASE degrades not only 125I-glucagon but -PP, and thiol proteinase inhibitors have a strong inhibitory action on them.

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Studies on submaxillary gland immunoreactive glucagon

Cited by 32 publications

References 12 publications

Evidence of glucagon biosynthesis involving protein intermediates in rat salivary glands

Evidence of glucagon biosynthesis involving protein intermediates in rat salivary glands

125I-glucagon-degrading activity in acid-saline extracts of rat salivary gland

Degradation of 125I-Glucagon, -Pancreatic Polypeptide and -Insulin by Acid Saline Extract of Rat Submaxillary Gland and Their Protection by Proteinase Inhibitors.

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