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

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.