Binding and agonist/antagonist actions of M35, galanin(1-13)-bradykinin(2-9) amide chimeric peptide, in Rin m 5F insulinoma cells

• Published in: Regulatory peptides 1995-11, Vol.59 (3), p.341-348
• Main Authors: Kask, Kalev, Berthold, Malin, Bourne, James, Andell, Siv, Langel, Ülo, Bartfai, Tamas
• Format: Article
• Language: English
• Subjects: Animals; Binding Sites; Binding, Competitive; Biological and medical sciences; Bradykinin - analogs & derivatives; Bradykinin - metabolism; Bradykinin - pharmacology; Cell Membrane - metabolism; Cell receptors; Cell structures and functions; Chimeric peptide; Colforsin - antagonists & inhibitors; Colforsin - pharmacology; Cyclic AMP - metabolism; Cyclic AMP production; Fundamental and applied biological sciences. Psychology; Galanin - agonists; Galanin - antagonists & inhibitors; Galanin - metabolism; Galanin - pharmacology; Galanin receptor; Guanosine Triphosphate - pharmacology; Insulinoma; Ligands; Molecular and cellular biology; Neuropeptide receptors; Peptide antagonist; Peptide Fragments - metabolism; Peptide Fragments - pharmacology; Pertussis Toxin; Protein Binding; Rats; Receptors, Galanin; Receptors, Gastrointestinal Hormone - agonists; Receptors, Gastrointestinal Hormone - antagonists & inhibitors; Receptors, Gastrointestinal Hormone - metabolism; Recombinant Fusion Proteins - metabolism; Recombinant Fusion Proteins - pharmacology; Rin m 5F insulinoma cell line; Tumor Cells, Cultured; Virulence Factors, Bordetella - pharmacology
• ISSN: 0167-0115; 1873-1686
• DOI: 10.1016/0167-0115(95)00089-T

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 β-cell line Rin m 5F, whereby we show the existence of a high-affinity binding site ( K D = 0.9 ± 0.1 nM) with a B max of 72 ± 3 fmol/mg protein. Galanin displaces [ 125I]M35 with the same affinity ( K D = 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 K D-values of 0.3 ± 0.1 nM and 0.52 ± 0.03 μM, 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.