glucagon-like-peptide-1-(7-36)amide and adenosine-3--5--cyclic-phosphorothioate

The mechanisms by which glucose-dependent insulinotropic polypeptide (GIP) stimulates insulin secretion were investigated by measurements of whole-cell Ca2+ currents, the cytoplasmic Ca2+ concentration, and cell capacitance as an indicator of exocytosis in individual mouse pancreatic beta-cells maintained in short-term culture. GIP produced a 4.2-fold potentiation of depolarization-induced exocytosis. This stimulation of exocytosis was not associated with a change in the whole-cell Ca2+-current, and there was only a small increase (30%) in the cytoplasmic Ca2+ concentration [intercellular free Ca2+([Ca2+]i)]. The stimulatory effect of GIP on exocytosis was blocked by pretreatment with the specific protein kinase A (PKA) inhibitor Rp-8-Br-cAMPS. Glucagon-like peptide-I(7-36) amide (GLP-I) stimulated exocytosis (90%) in the presence of a maximal GIP concentration (100 nmol/l). Replacement of GLP-I with forskolin produced a similar stimulatory action on exocytosis. These effects of GLP-I and forskolin in the presence of GIP did not involve a change in the whole-cell Ca2+-current or [Ca2+]i. GIP was ineffective in the presence of both forskolin and the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX). Under the same experimental conditions, the protein kinase C (PKC)-activating phorbol ester 4-phorbol 12-myristate 13-acetate (PMA) stimulated exocytosis (60%). Collectively, our data indicate that the insulinotropic hormone GIP stimulates insulin secretion from pancreatic beta-cells, through the cAMP/PKA signaling pathway, by interacting with the secretory machinery at a level distal to an elevation in [Ca2+]i.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Calcium; Cell Membrane; Cells, Cultured; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Electric Conductivity; Enzyme Inhibitors; Exocytosis; Gastric Inhibitory Polypeptide; Gastrointestinal Hormones; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptides; Islets of Langerhans; Membrane Potentials; Mice; Mice, Inbred Strains; Peptide Fragments; Phosphodiesterase Inhibitors; Tetradecanoylphorbol Acetate; Thionucleotides

The actions of glucagon-like peptide-1(7-36)amide (GLP-1(7-36)amide) on cellular signalling were studied in human embryonal kidney 293 (HEK 293) cells stably transfected with the cloned human GLP-1 receptor. The cloned GLP-1 receptor showed a single high-affinity binding site (Kd = 0.76 nM). Binding of GLP-1(7-36)amide stimulated cAMP production in a dose-dependent manner (EC50 = 0.015 nM) and caused an increase in the intracellular free Ca2+ concentration ([Ca2+]i). The latter effect reflected Ca(2+)-induced Ca2+ release and was suppressed by ryanodine. We propose that the ability of GLP-1(7-36)amide to increase [Ca2+]i results from sensitization of the ryanodine receptors by a protein kinase A dependent mechanism.

Topics: Acetylcholine; Calcium; Calcium Channel Blockers; Cell Line; Cloning, Molecular; Cyclic AMP; Embryo, Mammalian; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Glucagon-Like Peptides; Humans; Ionomycin; Kidney; Peptide Fragments; Receptors, Glucagon; Recombinant Proteins; Ryanodine; Thionucleotides