PubMed Health⌕ Search

Biomedical subjects

B Göke

Publications and source records attributed to B Göke.

At least 19 recordsLinked to original sources
PubMed1992

Expression of the ras-related rab3a gene in insulinoma-derived cell lines.

This study was designed to search for the expression of the small-molecular-weight GTP-binding protein rab3a in endocrine pancreatic cell lines. Total RNA was isolated from five different cell lines (RINm5F, RIN 104836, beta-TC1, HIT-15, and INRI-G9) and from whole rat brain. The expression of rab3a was analyzed by Northern blots. Similar as in brain two transcripts of 1300 and 1800 bp were detected in RIN-cells at low stringency conditions with the predominant signal at 1300 bp. At high stringency the stronger signal was at 1800 bp. When a 300 bp PstI fragment derived from the coding region of rab3a was utilized as probe the 1800 bp signal was predominant under each condition. Only a faint band at 1800 bp occurred in preparations from beta TC1-cells and no signal at all was found in HIT-15 and INRI-G9-cells. In conclusion, rab3a is expressed in rat insulin-releasing insulinoma-derived RIN-cells with a specific 1800 bp transcription product.

Animals↗
PubMed1992

Characterisation of the expression and post-translational processing of the preprotachykinin-I gene and the regulated release of tachykinins by the RINm5F cell-line.

Normal transcription and postranslational processing of the preprotachykinin (PPT)-I gene and regulated release of substance P and neurokinin A by the rat pancreatic endocrine cell-line, RINm5F, has been demonstrated, using radioimmunoassays (RIAs), reversed-phase (rp)HPLC and Northern blot analysis. This is the first stable cell-line found to express the PPT-I gene and provides an opportunity for investigating PPT-I gene expression and tachykinin biosynthesis. RIN5mF cells are a model for the pancreatic beta-cell, which is not known to exhibit PPT-I gene expression which may, therefore, be a feature of the transformed state of these cells. These data may imply that the tachykinins are important in pancreatic islet embryogenesis.

Animals↗
PubMed1992

Solubilization of active GLP-1 (7-36)amide receptors from RINm5F plasma membranes.

Glucagon-like peptide-1 (7-36)amide (GLP-1 (7-36)amide) represents a physiologically important incretin in mammals including man. Receptors for GLP-1 (7-36)amide have been described in RINm5F cells. We have solubilized active GLP-1(7-36)amide receptors from RINm5F cell membranes utilizing the detergents octyl-beta-glucoside and CHAPS; Triton X-100 and Lubrol PX were ineffective. Binding of radiolabeled GLP-1(7-36)amide to the solubilized receptor was inhibited concentration-dependently by addition of unlabeled peptide. Scatchard analysis of binding data revealed a single class of binding sites with Kd = 0.26 +/- 0.03 nM and Bmax = 65.4 +/- 21.24 fmol/mg of protein for the membrane-bound receptor and Kd = 22.54 +/- 4.42 microM and Bmax = 3.9 +/- 0.79 pmol/mg of protein for the solubilized receptor. The binding of the radiolabel to the solubilized receptor was dependent both on the concentrations of mono- and divalent cations and the protein/detergent ratio in the incubation buffer. The membrane bound receptor is sensitive to guanine-nucleotides, however neither GTP-gamma-S nor GDP-beta-S affected binding of labeled peptide to solubilized receptor. These data indicate that the solubilized receptor may have lost association with its G-protein. In conclusion, the here presented protocol allows solubilization of the GLP-1(7-36)amide receptor in a functional state, and opens up the possibility for further molecular characterization of the receptor protein.

Animals↗
PubMed1992

Cardiac natriuretic peptide hormones during artificial cardiac pacemaker stimulation and left heart catheterization.

Brain natriuretic peptide (BNP) is synthesized and released predominantly in the ventricular myocardium whereas atrial natriuretic peptide (ANP) is produced mainly in the atria. This study evaluated whether artificial pacemaker stimulation or left heart catheterization results in specific changes in BNP and ANP plasma levels. Both BNP and ANP responded sensitively to changes in pacemaker stimulation (single-chamber pacemakers; pacing rates of 72 and 92/min) and during the left heart catheterization procedure. However, whereas higher pacing resulted in a more pronounced increase in plasma BNP levels, a stronger ANP release followed catheterization. This incongruous rise in ANP and BNP plasma concentrations points to at least partly independent mechanisms governing the release of BNP and ANP.

Atrial Natriuretic Factor↗
PubMed1992

Glucagon-like peptide-1(7-37)/(7-36)amide is a new incretin.

Glucagon-like peptide-1 (GLP-1) is the main product of the intestinal processing of proglucagon. It is released from the intestinal K-cells into the circulation in response to the oral ingestion of food. At the pancreatic beta cell GLP-1 is a potent insulin secretagogue in the presence of elevated glucose levels, defining glucagon-like peptide-1 as a new incretin. Its action is mediated by specific receptors coupled to the adenylate cyclase system by a stimulatory G-protein. Finally, glucagon-like peptide-1 stimulates proinsulin gene expression and it is thus involved at several levels in the regulation of insulin synthesis and secretion.

Adenylyl Cyclases↗
PubMed1992

Glucagon-like peptide-1 cells in the gastrointestinal tract and pancreas of rat, pig and man.

A highly specific monoclonal antibody directed against the C-terminal part of glucagon-like peptide-1 (GLP-1) was raised to immunohistochemically evaluate the distribution of GLP-1 containing cells in the entire gastrointestinal tract including pancreas of rat, pig and man. In the pancreas GLP-1-immunoreactive cells were found variously shaped and predominantly located in the periphery of the islets. Ultrastructurally, GLP-1 was co-localized with glucagon in the alpha-granula of A-cells and was mainly restricted to the electrondense core. In the intestine open type cells reaching the lumen via a slender apical process were stained with the GLP-1 antibody. They occurred in all parts of the crypts but predominantly in the basal portion. The density of GLP-1 immunoreactive cells varied between species in a characteristic order: rat greater than pig greater than man. In pig and human gut a large number of cells occurred in the distal jejunum and ileum. A continuous increase of cell densities was found from the proximal to the distal colon resulting in highest numbers in the rectum. In rats the highest cell density occurred in the ileum. Again, a continuous increase of GLP-1-positive cell numbers was evident from the proximal to the distal portion of small and large bowel. GLP-1 was partly co-localized with PYY. The GLP-1 positive cells appeared electronmicroscopically as L-cells with the typical large granula. This morphological data indicates that GLP-1-releasing cells in the small intestine are appropriately positioned in the distal part to sense and respond to the presence of nutrients that have escaped the absorptive surface of the upper small intestine.

Animals↗
PubMed1992

Low molecular mass GTP-binding proteins in subcellular fractions of the pancreas: regulated phosphoryl G proteins.

Low molecular mass guanine nucleotide-binding proteins [small guanosine 5'-triphosphate (GTP)-binding proteins] and phosphoproteins of the pancreatic acinar cell were compared by two-dimensional gel electrophoresis. [35S]GTP alpha S blotting analysis of the total cell protein revealed 20 GTP-binding proteins ranging in molecular mass from 20 to 28 kDa and pI of 4.8-6.4. Analysis of 32P-labeled total cell protein revealed over 300 phosphoproteins. The subcellular distribution of the small GTP-binding proteins was examined: 17 were located in the rough endoplasmic reticulum (RER) fraction, 19 in the smooth microsome fraction, 14 in the zymogen granule membrane fraction, and 11 in the cytosolic fraction, with overlap between fractions. Of the GTP-binding proteins, two were also found to be phosphoproteins, one located on the RER and one on the zymogen granule membrane. The phosphorylation of both small GTP-binding proteins was increased by secretagogue stimulation of the cells but with different time courses. The RER small GTP-binding protein demonstrated a rapid and transient increase in 32P labeling, whereas the granule membrane small GTP-binding protein showed an increase at longer times (30 min). Two of the cytosolic small GTP-binding proteins were also seen in particulate fractions, especially in the zymogen granule membrane fraction, suggesting the possibility of cycling between cytosolic and membrane-associated forms.

Animals↗
PubMed1992

HPLC and FPLC. Recent progress in the use of automated chromatography systems for resolution of pancreatic secretory proteins.

Automated chromatography techniques such as high-performance (high-pressure) liquid chromatography (HPLC) or fast protein liquid chromatography (FPLC) represent a promising, new approach toward resolving the complex protein mixture of pancreatic juice. To achieve highly reproducible and rapid resolution of pancreatic protein profiles, reversed-phase, hydrophobic interaction, and ion-exchange techniques are employed. Until now, none of the respectively reported methods has provided a convenient, specific procedure for diagnostic purposes. However, experimental data prove that these methods are very sensitive and furthermore, easy to handle. Therefore, it seems possible to utilize such methods for diagnostics in future.

Animals↗
PubMed1991

Characterization of glucagon-like peptide-I(7-36)amide receptors of rat lung membranes by covalent cross-linking.

125I-labelled GLP-I(7-36)amide was cross-liked to a specific binding protein in rat lung membranes using disuccinimidyl suberate. A single radio-labelled band at Mr 66,000 was identified by SDS-PAGE after solubilization of the ligand-binding protein complex which is consistent with the presence of a single class of binding sites on rat lung membranes. The band was undetectable when 1 mumol/l GLP-I(7-36)amide was included in the binding assay. No change in the mobility of the band was observed under reducing conditions suggesting that the binding protein in the receptor is not part of a larger disulphide-linked protein. The intensity of the radiolabelled protein band was reduced when the incubation with 125I-labelled GLP-I(7-36)amide was carried out in the presence of guanine nucleotides suggesting that the GLP-I(7-36)amide receptor is coupled to the adenylate cyclase system.

Animals↗
PubMed1991

Priming effect of glucagon-like peptide-1 (7-36) amide, glucose-dependent insulinotropic polypeptide and cholecystokinin-8 at the isolated perfused rat pancreas.

The priming effect of glucagon-like peptide-1 (7-36) amide (GLP-1 (7-36) amide), glucose-dependent insulin-releasing polypeptide (GIP) and cholecystokinin-8 (CCK-8) on glucose-induced insulin secretion from rat pancreas was investigated. The isolated pancreas was perfused in vitro with Krebs-Ringer bicarbonate buffer containing 2.8 mmol/l glucose. After 10 min this medium was supplemented with GLP-1 (7-36) amide, GIP or CCK-8 (10, 100, 1000 pmol/l) for 10 min. After an additional 10 min period with 2.8 mmol/l glucose alone, insulin secretion was stimulated with buffer containing 10 mmol/l glucose for 44 min. In control experiments the typical biphasic insulin response to 10 mmol/l glucose occurred. Pretreatment of the pancreas with GIP augmented insulin secretion: 10 pmol/l GIP enhanced only the first phase of the secretory response to 10 mmol/l glucose; 100 and 1000 pmol/l GIP stimulated both phases of hormone secretion. After exposure to CCK-8, enhanced insulin release during the first (at 10 and 1000 pmol/l CCK-8) and the second phase (at 1000 pmol/l) was observed. Priming with 100 pmol/l GLP-1 (7-36) amide significantly amplified the first and 1000 pmol/l GLP-1 (7-36) amide both secretion periods, 10 pmol/l GLP-1 (7-36) amide had no significant effect. All three peptide hormones influenced the first, quickly arising secretory response more than the second phase. Priming with forskolin (30 mM) enhanced the secretory response to 10 mM glucose plus 0.5 nM GLP-1 (7-36) amide 4-fold. With a glucose-responsive B-cell line (HIT cells), we investigated the hypothesis that the priming effect of GLP-1 (7-36) amide is mediated by the adenylate cyclase system. Priming with either IBMX (0.1 mM) or forskolin (2.5 microM) enhanced the insulin release after a consecutive glucose stimulation (5 mM). This effect was pronounced when GLP-1 (7-36) amide (100 pM) was added during glucose stimulation. Priming capacities of intestinal peptide hormones may be involved in the regulation of postprandial insulin release. The incretin action of these hormones can probably, at least in part, be explained by these effects. The priming effect of GLP-1 (7-36) amide is most likely mediated by the adenylate cyclase system.

1-Methyl-3-isobutylxanthine↗
PubMed1991

Helodermin and islet hormone release in isolated rat pancreas.

The effect of helodermin (1,10,100 pmol/L), a new member of the VIP/secretin/glucagon peptide family, was investigated in glucose- (10 mmol/L) and arginine- (10 mmol/L) induced insulin, glucagon, and somatostatin secretion from the isolated perfused rat pancreas. Helodermin stimulated the somatostatin release in a concentration-dependent manner during the first (controls, 100%; 1 pmol/L, 113%, ns; 10 pmol/L, 175%, p less than 0.05; 100 pmol/L, 233%, p less than 0.05) and the second secretion phases (controls, 100%; 1 pmol/L, 117%, ns; 10 pmol/L, 210%, p less than 0.05; 100 pmol/L, 362%, p less than 0.05). There was a biphasic glucagon response with an inhibition during the first phase (controls, 100%; 1 pmol/L, 92%; 10 pmol/L, 63%, p less than 0.05; 100 pmol/L, 52%, p less than 0.05%) and a stimulation during the second phase (controls, 100%; 1 pmol/L, 117%, ns; 10 pmol/L, 210%, p less than 0.05; 100 pmol/L, 362%, p less than 0.05). Helodermin had weak stimulatory effects on pancreatic beta cells with a maximum at 10 pmol/L (first phase: controls, 100%; 1 pmol/L, 96%, ns; 10 pmol/L, 148%, p less than 0.05; 100 pmol/L, 136%, ns; second phase: controls, 100%; 1 pmol/L, 104%, ns; 10 pmol/L, 170%, p less than 0.05; 100 pmol/L, 136%, ns).

Animals↗
PubMed1991

Intracellular mediators of bombesin action on rat pancreatic acinar cells.

The effects of bombesin on physiological responses (amylase secretion, protein synthesis) and intracellular mediators [inositol 1,4,5-trisphosphate (1,4,5-IP3), [Ca2+]i, and diacylglycerol] were studied in isolated rat pancreatic acini and compared with the actions of cholecystokinin (CCK). Bombesin stimulated amylase secretion to the same extent as CCK. However, it failed to reproduce the inhibition of amylase secretion by high concentrations of CCK and likewise did not inhibit incorporation of [3H]leucine into protein in contrast to high concentrations of CCK. Low concentrations of bombesin (1-100 pM) induced repetitive oscillations in [Ca2+]i, whereas higher concentrations of bombesin (1-10 nM) induced a large transient increase in [Ca2+]i followed by a small sustained plateau. Bombesin (1-100 nM) induced an early peak of 1,4,5-IP3 at 5-15 s but was without measurable effect at lower concentrations. These effects on [Ca2+]i and 1,4,5-IP3 were similar to those seen with CCK except that bombesin was approximately 10-fold less potent than CCK. Bombesin induced an increase in acinar 1,2-diacylglycerol with a biphasic time course similar to CCK. However, the magnitude of the response to bombesin was much smaller than the response to CCK. The results suggest that bombesin receptors initiate similar intracellular messengers as does CCK. However, CCK induces a larger increase of diacylglycerol and probably an as yet unidentified messenger responsible for its inhibitory effects.

Amylases↗
PubMed1991

Binding, internalization, and processing of bombesin by rat pancreatic acini.

The binding and subsequent fate of 125I-labeled bombesin was studied in rat pancreatic acini. At both 37 degrees C and 4 degrees C binding occurred to a single saturable site, with Kd equal to 1.7 nM. The amount of steady-state tracer binding was reduced at 4 degrees C (5.7%/mg protein) compared with 37 degrees C (8.7%/mg), with a similar relative change in calculated binding capacity. With the use of an acid wash procedure to remove surface bound ligand, 55% of cell-associated 125I-bombesin was internalized in the steady state at 37 degrees C but only 5% at 4 degrees C. Preincubation at 4 degrees C followed by an increase to 37 degrees C led to rapid internalization of bound bombesin, which was blocked by the metabolic inhibitor antimycin. 125I-bombesin was found to be degraded by two acinar systems. One was not related to receptor binding but could be inhibited with bacitracin. The other occurred after internalization and was partially blocked with chloroquine. Thus, after binding, bombesin is internalized, and the degradation products are released from the cell. Exposure to bombesin is also accompanied by a subsequent decrease in cell surface binding (54% after 1 h exposure to 100 nM bombesin), suggesting that the bombesin receptor may also be internalized in a ligand-dependent manner.

Animals↗