In vitro binding and signaling profile of the novel μ opioid receptor agonist endomorphin 2 in rat brain membranes

The recently discovered endogenous mu receptor selective endomorphin 2 was prepared in tritiated form by a catalytic dehalogenation method resulting in a specific radioactivity of 1.98 TBq/mmol (53.4 Ci/mmol), and used for in vitro labelling of rat brain membranes. The binding was saturable, stereospecific and of high affinity (K-d: 0.97 and 1.12 nM based on kinetic and equilibrium binding studies, respectively). The maximal number of binding sites (B-max) was found to be 114.8 fmol/mg protein. [H-3]Endomorphin 2 was displaced by mu-receptor selective specific peptides and heterocyclic compounds with high affinity, whereas kappa and delta receptor specific ligands were much less potent. The K-i values of endomorphin 1 and 2 in inhibiting [H-3]naloxone binding increased by 15-fold in the presence of 100 mM NaCl which indicates the agonist property of these peptides. Endomorphins stimulated [S-35]GTP gamma S binding and inhibited adenylyl cyclase activity which also provides evidence for the agonist character of endomorphins. (C) 1998 Academic Press.