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14 beta-(Bromoacetamido)morphine irreversibly labels mu opioid receptors in rat brain membranes
J M Bidlack1, D K Frey, A Seyed-Mozaffari
1Department of Pharmacology, University of Rochester School of Medicine, New York 14642.
Abstract:
The binding properties of 14 beta-(bromoacetamido)morphine (BAM) and the ability of BAM to irreversibly inhibit opioid binding to rat brain membranes were examined to characterize the affinity and selectivity of BAM as an irreversible affinity ligand for opioid receptors. BAM had the same receptor selectivity as morphine, with a 3-5-fold decrease in affinity for the different types of opioid receptors. When brain membranes were incubated with BAM, followed by extensive washing, opioid binding was restored to control levels. However, when membranes were incubated with dithiothreitol (DTT), followed by BAM, and subsequently washed, 90% of the 0.25 nM [3H] [D-Ala2,(Me)Phe4,Gly(ol)5]enkephalin (DAGO) binding was irreversibly inhibited as a result of the specific alkylation of a sulfhydryl group at the mu binding site. This inhibition was dependent on the concentrations of both DTT and BAM. The mu receptor specificity of BAM alkylation was demonstrated by the ability of BAM alkylated membranes to still bind the delta-selective peptide [3H] [D-penicillamine2,D-penicillamine5]enkephalin (DPDPE) and (-)-[3H]bremazocine in the presence of mu and delta blockers, selective for kappa binding sites. Under conditions where 90% of the 0.25 nM [3H]DAGO binding sites were blocked, 80% of the 0.8 nM [3H]naloxone binding and 50% of the 0.25 nM 125I-labeled beta h-endorphin binding were inhibited by BAM alkylation. Morphine and naloxone partially protected the binding site from alkylation with BAM, while ligands that did not bind to the mu site did not afford protection.2+hese studies have demonstrated that when a disulfide bond
Insights
14 beta-(bromoacetamido)morphine (BAM) irreversibly inhibits opioid binding by alkylating sulfhydryl groups at mu opioid receptors. This selective inhibition is concentration-dependent and demonstrates BAM
Area of Science:
- Pharmacology
- Neuroscience
- Biochemistry
Background:
- Opioid receptors are crucial targets for pain management and addiction therapies.
- Understanding the specific binding properties of opioid ligands is essential for developing selective therapeutics.
- Irreversible ligands offer potential for prolonged receptor blockade.
Purpose of the Study:
- To characterize 14 beta-(bromoacetamido)morphine (BAM) as an irreversible affinity ligand for opioid receptors.
- To determine the selectivity and mechanism of BAM's inhibition of opioid binding.
- To investigate the role of sulfhydryl groups in opioid receptor binding.
Main Methods:
- Binding assays using rat brain membranes and radiolabeled opioid ligands.
- Incubation of membranes with BAM, dithiothreitol (DTT), and various opioid ligands.
- Assessment of irreversible inhibition of [3H]DAGO, [3H]naloxone, and 125I-labeled beta h-endorphin binding.
- Evaluation of receptor selectivity using selective blockers for mu, delta, and kappa opioid receptors.
Main Results:
- BAM exhibits similar receptor selectivity to morphine but with reduced affinity.
- Pre-incubation with DTT followed by BAM resulted in irreversible inhibition of 90% of [3H]DAGO binding.
- Inhibition was attributed to specific alkylation of a sulfhydryl group at the mu binding site.
- BAM alkylation selectively inhibited mu receptor binding while sparing delta and kappa receptors.
- Morphine and naloxone partially protected the binding site from BAM alkylation.
Conclusions:
- BAM functions as a selective, irreversible affinity ligand for mu opioid receptors.
- The mu opioid receptor binding site contains a critical sulfhydryl group susceptible to alkylation by BAM.
- BAM's properties make it a valuable tool for studying opioid receptor structure and function.