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 hig h-affinity galanin receptor ligand acting as a galanin receptor antago nist in the rat spinal cord, rat hippocampus and isolated mouse pancre atic islets. We have radiolabelled M35 and performed equilibrium bindi ng studies with [I-125]M35 On the rat pancreatic beta-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 d isplaces [I-125]M35 With the same affinity (K-D = 1 nM) as it displace s [I-125]galanin. Displacement of [I-125]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 mu M, respectively. The G TP- and pertussis toxin-sensitivity of M35 binding to Rin m 5F membran es shows that binding of [I-125]M35 is almost completely abolished by the presence of GTP or after pertussis toxin treatment of the cells, i ndicating an agonist-like binding of M35 to the galanin receptors. M35 has a dual effect on the galanin mediated inhibition of forskolin sti mulated cyclic AMP production in Rin m 5F cells: at low concentrations M35 antagonises the effect of galanin, whereas at concentrations abov e 10 nM M35 acts as a galanin receptor agonist. These agonist-like eff ects of galanin and M35 are not additive, thus the mixed agonist/antag onist properties arise from the chimeric nature of M35 [galanin(1-13)- bradykinin(2-9)amide] acting solely at galanin receptors.