Unexpectedly high levels of opioid peptides in rat brain membranes

Abstract

Several considerations led us to directly measure, using radioimmunoassy, the levels of dynorphin-A(1–8), cholecystokinin-8, and Met 5-enkephalin-Arg 6-Gly 7-Leu 8 (MERGL) in lysed-P2 membranes, membranes incubated 60 min at 25°C in 50 mM TRIS-HCl at pH 7.4, and membranes preincubated 60 min at 25°C in TRIS buffer containing 0.4 M NaCl. Lysed-P2 membranes contained neuropeptides at levels between 0.253 and 1.727 pmol/mg protein. Incubating membranes in buffer alone decreased membrane peptide levels by 40 to 60%. Preincubations in the presence of 0.4 M NaCl resulted in even greater reductions in the peptide content of membranes (between 58 and 90%), without changing [ 3H]etorphine binding. Detailed comparisons of the two binding sites labeled by [ 3H]naloxone revealed changes in binding parameters induced by preincubation with NaCl. Taken collectively, these studies demonstrate that lysed-P2 membranes contain sequestered peptides that are not bound to opiate receptors.