guanosine-5--o-(3-thiotriphosphate) and Insulinoma

Hypoglycemic sulfonylureas stimulate insulin release by binding to a regulatory subunit of plasma membrane ATP-sensitive K+ (K(ATP)) channels. The consequent closure of K(ATP) channels leads to depolarization, opening of voltage-dependent Ca2+ channels, Ca2+ influx, and a rise in intracellular [Ca2+]. Recently, however, it has been suggested that sulfonylureas may have an additional action on secretion, independent of changes in intracellular [Ca2+] but dependent on the activity of protein kinase C (PKC). We have investigated the mechanisms involved in the PKC-dependent effect of sulfonylureas on the secretion machinery in beta-cells. In MIN6 beta-cells permeabilized by streptolysin O, insulin release was stimulated by elevation of [Ca2+] from 10(-8) to 10(-5) mol/l. At a [Ca2+] of 10(-8) mol/l, insulin release from permeabilized beta-cells was stimulated by addition of GTP-gamma-S, or by addition of a phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA). TPA, but not GTP-gamma-S, also increased insulin release when [Ca2+] was 10(-5) mol/l. Insulin release from permeabilized beta-cells was stimulated by tolbutamide (0.1-1 mmol/l) at 10(-8) but not at 10(-5) mol/l Ca2+. The effect of tolbutamide was blocked either by inhibition of PKC or when phorbol ester-sensitive PKC isoforms were maximally stimulated by TPA. Meglitinide and glibenclamide also stimulated insulin release from permeabilized beta-cells. To assess the possibility that direct activation of PKC mediates the exocytotic response to sulfonylureas, we studied the effect of tolbutamide and glibenclamide on PKC activity. Purified brain PKC was not activated by tolbutamide or glibenclamide, whether tested in the absence or presence of phosphatidylserine or TPA, or at low or high [Ca2+]; nor was the total PKC activity in extracts of MIN6 beta-cells affected by tolbutamide. Neither tolbutamide nor glibenclamide elicited translocation of any isoform of PKC in intact or permeabilized beta-cells under conditions in which TPA evoked a marked redistribution of PKC alpha- and epsilon-isoforms. We conclude that although the plasma membrane K(ATP) channel-independent stimulation of exocytosis by sulfonylureas may require functional PKC, the mechanism does not involve a direct activation of the enzyme.

Topics: Animals; Calcium; Enzyme Activation; Exocytosis; Glyburide; Guanosine 5'-O-(3-Thiotriphosphate); Hypoglycemic Agents; Insulinoma; Islets of Langerhans; Isoenzymes; Mice; Pancreatic Neoplasms; Phosphatidylserines; Potassium Channels; Protein Kinase C; Sulfonylurea Compounds; Tetradecanoylphorbol Acetate; Tolbutamide; Tumor Cells, Cultured

Several GTP-binding proteins (G-proteins) undergo post-translational modifications (isoprenylation and carboxyl methylation) in pancreatic beta cells. Herein, two of these were identified as CDC42 and rap 1, using Western blotting and immunoprecipitation. Confocal microscopic data indicated that CDC42 is localized only in islet endocrine cells but not in acinar cells of the pancreas. CDC42 undergoes a guanine nucleotide-specific membrane association and carboxyl methylation in normal rat islets, human islets, and pure beta (HIT or INS-1) cells. GTPgammaS-dependent carboxyl methylation of a 23-kD protein was also demonstrable in secretory granule fractions from normal islets or beta cells. AFC (a specific inhibitor of prenyl-cysteine carboxyl methyl transferases) blocked the carboxyl methylation of CDC42 in five types of insulin-secreting cells, without blocking GTPgammaS-induced translocation, implying that methylation is a consequence (not a cause) of transfer to membrane sites. High glucose (but not a depolarizing concentration of K+) induced the carboxyl methylation of CDC42 in intact cells, as assessed after specific immunoprecipitation. This effect was abrogated by GTP depletion using mycophenolic acid and was restored upon GTP repletion by coprovision of guanosine. In contrast, although rap 1 was also carboxyl methylated, it was not translocated to the particulate fraction by GTPgammaS; furthermore, its methylation was also stimulated by 40 mM K+ (suggesting a role which is not specific to nutrient stimulation). AFC also impeded nutrient-induced (but not K+-induced) insulin secretion from islets and beta cells under static or perifusion conditions, whereas an inactive structural analogue of AFC failed to inhibit insulin release. These effects were reproduced not only by S-adenosylhomocysteine (another methylation inhibitor), but also by GTP depletion. Thus, the glucose- and GTP-dependent carboxyl methylation of G-proteins such as CDC42 is an obligate step in the stimulus-secretion coupling of nutrient-induced insulin secretion, but not in the exocytotic event itself. Furthermore, AFC blocked glucose-activated phosphoinositide turnover, which may provide a partial biochemical explanation for its effect on secretion, and implies that certain G-proteins must be carboxyl methylated for their interaction with signaling effector molecules, a step which can be regulated by intracellular availability of GTP.

Topics: Acetylcysteine; Animals; Blotting, Western; cdc42 GTP-Binding Protein; Cell Cycle Proteins; Cell Line; Cells, Cultured; Enzyme Inhibitors; Glucose; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Humans; Insulin; Insulin Secretion; Insulinoma; Islets of Langerhans; Kinetics; Male; Methylation; Pancreatic Neoplasms; Potassium; Protein Methyltransferases; Rats; Rats, Sprague-Dawley

Glucagon-like peptide-I (GLP-I) is a potent incretin hormone and mediates its actions via the cyclic AMP (cAMP) pathway. The GLP-I receptor belongs to the family of seven-transmembrane domain receptors coupled to G proteins. We have analyzed the regulation of GLP-I receptor function and expression by its own ligand and the cAMP-dependent pathway in rat insulinoma-derived beta cells (RINm5F). The GLP-I receptor underwent rapid homologous desensitization, which occurred at the receptor level. This was characterized by a reduced binding capacity not mediated by protein kinase A (PKA). GLP-I receptor mRNA levels were down-regulated during incubation of cells by agents increasing cAMP levels including GLP-I itself. This effect was dependent upon time and concentration. Forskolin, the PKA activator 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole-3, 5-monophosphorothiotate, and GLP-I stabilized the GLP-I receptor mRNA. All induced down-regulation of the GLP-I receptor number within 3 h, a time point at which GLP-I receptor mRNA levels were not decreased. This effect was not influenced by cycloheximide. Therefore, in addition to transcriptional effects, posttranslational mechanisms exist to regulate GLP-I receptor numbers in insulin-secreting cells.

Topics: Animals; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cycloheximide; Enzyme Activation; Gene Expression; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Guanosine 5'-O-(3-Thiotriphosphate); Insulin; Insulin Secretion; Insulinoma; Islets of Langerhans; Pancreatic Neoplasms; Peptide Fragments; Protein Precursors; Protein Synthesis Inhibitors; Rats; Receptors, Glucagon; RNA, Messenger; Tumor Cells, Cultured

Calcitonin gene-related peptide (CGRP) shares about 46% and 20% amino acid sequence homology with islet amyloid polypeptide (IAPP) and salmon calcitonin (sCT). We investigated whether these related peptides could cross-react with the specific binding of 125I-[His]hCGRP I to the CGRP receptor in hamster insulinoma cell membranes. A rapid dissociation of membrane bound 125I-[His]hCGRP I could be induced in the presence of 1 microM chicken CGRP (cCGRP). The specific 125I-[His]hCGRP I binding was inhibited by the related peptides and their half-maximal inhibitory concentrations (IC50) were: cCGRP (0.1 nM), rat CGRP I and human CGRP I and II (1.0-2.0 nM), fragment of hCGRP I (8-37) (150 nM), human IAPP (440 nM). The non-amidated form of hIAPP; human diabetes-associated peptide (hDAP) did not inhibit the binding of 125I-[His]hCGRP I and sCT was only effective at a high concentration (1 microM). Binding of 125I-[His]hCGRP I was dose dependently inhibited by guanosine-5'-O-(3-thiotriphosphate) or (GTP gamma S) and a 70% reduction of binding was obtained with 0.1 mM GTP gamma S. The IC50 value of cCGRP (0.1 nM) was increased 100-fold in the presence of 0.1 mM GTP gamma S. Human CGRP I and cCGRP at 2.5 microM did not stimulate the activity of hamster insulinoma cell membranes adenylate cyclase, while glucagon (1 microM) induced a 2-fold increase. Thus, specific CGRP receptors present in hamster beta cells are associated with G protein (s) and IAPP can interact with these receptors. These results and the observation that cCGRP and hCGRP I did not influence adenylate cyclase activity provide further evidence for CGRP receptor subtypes.

Topics: Adenosine Triphosphate; Amyloid; Animals; Binding, Competitive; Calcitonin; Calcitonin Gene-Related Peptide; Cell Membrane; Chickens; Cricetinae; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Humans; Insulinoma; Islet Amyloid Polypeptide; Islets of Langerhans; Kinetics; Pancreatic Neoplasms; Rats; Receptors, Calcitonin Gene-Related Peptide

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.

Topics: Animals; Cations; Detergents; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Glucagon-Like Peptides; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Insulinoma; Pancreatic Neoplasms; Peptide Fragments; Peptides; Protein Binding; Rats; Receptors, Cell Surface; Receptors, Glucagon; Solubility; Thionucleotides; Tumor Cells, Cultured

Galanin, an inhibitor of insulin secretion in pancreatic beta-cells, exerts its multiple effects through mechanisms that are sensitive to pertussis toxin (PTX). G proteins have been characterized in RINm5F cells. By ADP ribosylation and immunoblotting, the alpha-subunits of Gi1, Gi2, Gi3, and two forms of Go were identified, Gi alpha 2 being predominant. As expected from a G protein-linked receptor, GTP and its nonhydrolyzable analogue GTP-gamma-S decreased tracer galanin binding to cell membranes. This resulted from a change in receptor affinity without any modification in the number of sites. Selective antibodies against the COOH-terminal decapeptide of the alpha-subunits of the Gi and Go proteins were used to block G protein interaction before we studied galanin binding. Antibody AS, which selectively recognizes Gi alpha 1 and Gi alpha 2, decreased tracer galanin binding to membranes at concentrations where there were no effects of other antibodies specifically directed against Gi alpha 3 or G alpha o. These data suggest that Gi1 and/or Gi2 interact with the galanin receptor and probably mediate the effects of galanin in pancreatic beta-cells.

Topics: Amino Acid Sequence; Animals; Antibodies; Brain; Cell Line; Cell Membrane; Galanin; GTP-Binding Proteins; Guanine Nucleotides; Guanosine 5'-O-(3-Thiotriphosphate); Immunoassay; Insulinoma; Kidney; Macromolecular Substances; Molecular Sequence Data; NAD; Pancreatic Neoplasms; Peptides; Pertussis Toxin; Rats; Receptors, Galanin; Receptors, Gastrointestinal Hormone; Virulence Factors, Bordetella

We found that, besides dihydropyridine-sensitive Ca channels, insulin-secreting RINm5F cells also contain a minority (15-25%) of omega-conotoxin (omega-CgTx)-sensitive channels that show a high-affinity binding to [125I] omega-CgTx (Kd 51 pM). Noradrenaline (NA, 10 microM) slows down Ca-channel activation in these cells and produces a sizeable reduction of Ca currents that is relieved by strong pre-conditioning depolarizations (facilitation). The action of NA is mimicked by intracellular application of GTP-gamma-S and is prevented by pertussis toxin (PTX) or by cell pre-incubation with omega-CgTx. This suggests specific noradrenergic inhibition of omega-CgTx-sensitive Ca channels that is modulated by membrane potentials and PTX-sensitive G-protein activation.

Topics: Animals; Calcium Channel Blockers; Calcium Channels; Cell Line; Guanosine 5'-O-(3-Thiotriphosphate); Insulin; Insulin Secretion; Insulinoma; Kinetics; Norepinephrine; omega-Conotoxin GVIA; Pancreatic Neoplasms; Peptides, Cyclic; Rats

The existence of a single or of multiple populations of glibenclamide binding sites is a subject of controversy. In the present study, radioligand binding techniques were employed to determine whether multiple populations of [3H]glibenclamide binding sites exist in pancreatic tumor (insulinoma) cells. Additional studies were performed to further characterize the binding of [3H]glibenclamide to insulinoma and cardiac membranes. [3H]Glibenclamide bound to high (0.1 nM) and low (240 nM) affinity binding sites in insulinoma membranes. The physiological relevance of multiple populations of sites is unknown. The binding of glibenclamide to insulinoma and cardiac membranes was altered by guanine nucleotides and not adenine nucleotides. This suggests glibenclamide binding can be modulated by G-proteins. Glibenclamide binding was also modulated by divalent cations. The divalent cations, Ca2+ and Zn2+, stimulated specific glibenclamide binding to cardiac and insulinoma membranes, while Mg2+ and Mn2+ enhanced cardiac binding only. Moreover, the lowering of pH from 7.4 to 6.5 was found to enhance specific glibenclamide binding. Interestingly, the magnitude of this effect was much larger in cardiac membranes. The specific nature of the regulation of glibenclamide binding by guanine nucleotides, divalent cations and pH remains to be explored.

Topics: Animals; Cations, Divalent; Dogs; Female; Glyburide; Guanosine 5'-O-(3-Thiotriphosphate); Guanylyl Imidodiphosphate; Hydrogen-Ion Concentration; In Vitro Techniques; Insulinoma; Male; Membranes; Myocardium; Nucleotides; Pancreatic Neoplasms; Potassium Channels; Rats; Receptors, Drug; Trypsin; Tumor Cells, Cultured

Guanosine triphosphate (GTP) can release Ca2+ and enhance responses to D-myo-inositol 1,4,5-trisphosphate (IP3) in crude liver microsomes in the presence of polyethylene glycol (PEG) (Dawson et al. (1986) Biochem. J. 234, 311-315). The mechanism of these responses has been further investigated. GTP gamma S which antagonizes the actions of GTP on microsomes, does not promote Ca2+ re-uptake when added after the completion of GTP-mediated Ca2+ release. However, the effects of GTP could be reversed by washing or dilution of the microsomes. Addition of PEG to the incubation medium promoted the aggregation of microsomes. Electron microscopy provided no evidence for the fusion of microsomal vesicles in the presence or absence of GTP. In the presence of PEG, GTP produced an alteration of the permeability properties of the microsomal membrane as indicated by increased leakage of an intraluminal esterase, a reduction in the mean buoyant density of the vesicles, and a decrease in the latency of mannose 6-phosphate hydrolysis. All three effects developed relatively slowly, whereas the effects of GTP on Ca2+ fluxes occurred more rapidly (complete within 15 min). A low permeability to mannose 6-phosphate was restored upon washing away the GTP. These results suggest that non-specific permeability changes may underly the effects of GTP on Ca2+ release and that, under certain conditions, GTP can reversibly modulate the permeability of a transmembrane 'pore' in microsomal membranes that can pass ions and macromolecules. The possibility that such a pore serves to link IP3-sensitive vesicles with other Ca2+-containing compartments is discussed.

Topics: Animals; Brain; Calcium; Centrifugation, Density Gradient; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Insulinoma; Microsomes, Liver; Permeability; Polyethylene Glycols; Rats; Thionucleotides