Neurobiology 7. (1999)
1. szám - Preliminary notes
Neurobiology; 7 (I) pp. 83-84 (1999) Preliminary note AFFINITY LABELING OF OPIOID RECEPTORS IN VIVO AND IN VITRO KICSI, E., BOZÓ, B., FARKAS J., MÁCSAI M.,1 Szabó, G.' and Szűcs, M. Institute of Biochemistry, Biological Research Center, Szeged; and Institute of Pathophysiology, SZAOTE Medical University, Szeged, Hungary Affinity labels are useful probes to study receptor mechanisms both in vivo and in vitro. Previously, we have been successful in preparing opioid affinity labels by furnishing enkephalin molecules with chemically reactive chloromethyl ketone (-CH2CI) or melphalan groups which ligands were able to alkylate nucleophilic side chains of the opioid receptors under proper experimental conditions (1-3). These ligands, however, labelled not only the p opioid receptors, but displayed some crossreactivity to other opioid sites. Another disadvantage that restrained their use was that their analgesic effect in vivo did not seem to be irreversible (1). When DAMGO, the highly pselective opioid agonist was equipped with a chloromethyl ketone group at the C-terminal, the resulting compound DAMCK, Tyr-D-Ala-Gly-MePhe- CH2CI retained the p-receptor specificity of the parent compound (4). The present work has revealed that 0.01-100 ng/kg DAMCK administered either centrally (icv), or systemically (iv) produced a dose-dependent, opioid receptor mediated, profound antinociception measured with the tail-flick assay in male rats. At 100 ng/kg icv dose, its analgesic effect persisted up to 240 min, the longest time tested. Conversely, the effect of identical dose of DAMGO peaked at 30 min, and gradually deceased afterwards. The effect of DAMCK in vivo was not only long-lasting, but the opioid antagonist naloxone, given 45 min after the peptide, only partially reversed its effect. This result suggested that 5070% of the DAMCK effect in vivo was due to irreversible binding. [3H]DAMCK specifically and covalently labelled p opioid binding proteins of about 54, 40 and 32 kDa in synaptic plasmamembranes (SPM) of rat brains prepared as published (5). Very similar labelling patterns were observed in SPMs purified from morphine-tolerant rat brains. The finding 1218-8068/99 $ 5.00 © 1999 Akadémiai Kiadó. Budapest