title: Binding and agonist/antagonist actions of M35, galanin(1-13)-bradykinin(2-9)amide chimeric peptide, in Rin m 5F insulinoma cells.
abstract: The chimeric peptide M35 [galanin(1-13)-bradykinin(2-9) amide] is a high-affinity galanin receptor ligand acting as a galanin receptor antagonist in the rat spinal cord, rat hippocampus and isolated mouse pancreatic islets. We have radiolabelled M35 and performed equilibrium binding studies with [125I]M35 on the rat pancreatic beta-cell line Rin m 5F, whereby we show the existence of high-affinity binding site (KD = 0.9 +/- 0.1 nM) with a Bmax of 72 +/- 3 fmol/mg protein. Galanin displaces [125I]M35 with the same affinity (KD = 1 nM) as it displaces [125I]galanin. Displacement of [125I]galanin by M35 from Rin m 5F cell membranes shows the presence of two binding sites for M35 with KD-values of 0.3 +/- 0.1 nM and 0.52 +/- 0.03 microM, respectively. The GTP- and pertussis toxin-sensitivity of M35 binding to Rin m 5F membranes shows that binding of [125I]M35 is almost completely abolished by the presence of GTP or after pertussis toxin treatment of the cells, indicating an agonist-like binding of M35 to the galanin receptors. M35 has a dual effect on the galanin mediated inhibition of forskolin stimulated cyclic AMP production in Rin m 5F cells: at low concentrations M35 antagonises the effect of galanin, whereas at concentrations above 10 nM M35 acts as a galanin receptor agonist. These agonist-like effects of galanin and M35 are not additive, thus the mixed agonist/antagonist properties arise from the chimeric nature of M35[galanin(1-13)-bradykinin(2-9)amide] acting solely at galanin receptors.
authors.affiliation: Department of Neurochemistry and Neurotoxicology, Arrhenius Laboratories, Stockholm University, Sweden.
authors.forename: K
authors.initials: K
authors.lastname: Kask
authors.forename: M
authors.initials: M
authors.lastname: Berthold
authors.forename: J
authors.initials: J
authors.lastname: Bourne
authors.forename: S
authors.initials: S
authors.lastname: Andell
authors.forename: U
authors.initials: U
authors.lastname: Langel
authors.forename: T
authors.initials: T
authors.lastname: Bartfai
mesh_terms: D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D001920:Bradykinin / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D005576:Colforsin / Q000037:antagonists & inhibitors / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D019004:Galanin / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000494:pharmacology*; D006160:Guanosine Triphosphate / Q000494:pharmacology; D007340:Insulinoma; D008024:Ligands; D010446:Peptide Fragments / Q000378:metabolism* / Q000494:pharmacology; D037342:Pertussis Toxin; D011485:Protein Binding; D051381:Rats; D044088:Receptors, Galanin; D011964:Receptors, Gastrointestinal Hormone / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
chemical_list: D008024:Ligands; D010446:Peptide Fragments; D044088:Receptors, Galanin; D011964:Receptors, Gastrointestinal Hormone; D011993:Recombinant Fusion Proteins; D010566:Virulence Factors, Bordetella; C074069:galanin-(1-13)-bradykinin-(2-9)-amide; D005576:Colforsin; D006160:Guanosine Triphosphate; D019004:Galanin; D000242:Cyclic AMP; D037342:Pertussis Toxin; D001920:Bradykinin