[3H]Naltrindole: a potent and selective ligand for labeling δ-opioid receptors

doi:10.1016/0006-8993(93)90363-R

Brain Research

Volume 604, Issues 1–2, 26 February 1993, Pages 160-164

https://doi.org/10.1016/0006-8993(93)90363-R Get rights and content

Abstract

Naltrindole (NTI) is a selective and potent δ-opioid antagonist which preferentially antagonizes a subset of selective δ-opioid agonists. The purpose of this study was to evaluate whether [³H]NTI, the first radiolabeled δ-opioid antagonist, could selectively label δ-opioid receptors in a synaptosomal preparation. Increasing temperature and protein concentration (0.1–1.6 mg protein) increased the specific binding of [³H]NTI. Monovalent and divalent cations (0.01–100 mM) had minimal effects on the binding properties of [³H]NTI, in contrast to their effects on binding of the delta agonists [³H]DPDPE and [³H]DSLET. Subfractionation of rat brain homogenates revealed that [³H]NTI and [³H]DSLET primarily labeled binding sites in synaptosomal and microsomal fractions, whereas [³H]DPDPE labelled half as many sites in synaptosomal fraction. The B_max determined for [³H]NTI in crude synaptosomal fraction was 95 ± 12 fmol/mg. The dissociation constant (K_d) was determined from three different methods to be 0.08 ± 0.02 nM (Scatchard analysis), 0.07 ± 0.02 nM (competition study) and 0.03 ± 0.005 nM (kinetic analysis). [³H]NTI binding was not significantly inhibited by μ- or κ-opioid ligands or by nonopioid compounds. These results demonstrate that [³H]NTI is a potent and selective radioligand for δ-opioid receptors in rat brain preparations.

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To completely rule out crosstalk between the fluorescent detection channels, the multitracking configuration was used. To determine if RTX treatment would alter the binding number and affinity of the δ-opioid receptors in the spinal cord, saturation binding of [3H]-naltrindole, a specific radioligand for δ-opioid receptors (Contreras et al., 1993), was carried out using the dorsal spinal cord tissue membranes. Four RTX- and four vehicle-treated rats were decapitated after being anesthetized with 2–3% isoflurane.
Most δ-opioid receptors are located on the presynaptic terminals of primary afferent neurons in the spinal cord. However, their presence in different phenotypes of primary afferent neurons and their contribution to the analgesic effect of δ-opioid agonists are not fully known. Resiniferatoxin (RTX) is an ultra-potent transient receptor potential vanilloid type 1 channel (TRPV1) agonist and can selectively remove TRPV1-expressing primary afferent neurons. In this study, we determined the role of δ-opioid receptors expressed on TRPV1 sensory neurons in the antinociceptive effect of the δ-opioid receptor agonists [d-Pen²,d-Pen⁵]-enkephalin and [d-Ala²,Glu⁴]-deltorphin. Nociception was measured by testing the mechanical withdrawal threshold in the hindpaw of rats. Changes in the δ-opioid receptors were assessed using immunocytochemistry and the [³H]-naltrindole radioligand binding. In RTX-treated rats, the δ-opioid receptor on TRPV1-immunoreactive dorsal root ganglion neurons and afferent terminals in the spinal cord was diminished. RTX treatment also significantly reduced the maximal specific binding sites (31%) of the δ-opioid receptors in the dorsal spinal cord. Interestingly, intrathecal injection of [d-Pen²,d-Pen⁵]-enkephalin or [d-Ala²,Glu⁴]-deltorphin produced a large and prolonged increase in the nociceptive threshold in RTX-treated rats. These findings indicate that loss of TRPV1-expressing afferent neurons leads to a substantial reduction in presynaptic δ-opioid receptors in the spinal dorsal horn. However, the effect of δ-opioid agonists on mechano-nociception is paradoxically potentiated in the absence of TRPV1-expressing sensory neurons. This information is important to our understanding of the cellular sites and mechanisms underlying the spinal analgesic effect of δ-opioid agonists.
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CTOP and nor-BNI also prevent stress-induced analgesia at doses that are substantially lower than used in this study [34,53]. The Kd for [3H]-naltrindole binding to delta opioid receptors (0.08 nM) [14] is also considerably lower than the naltrindole concentration used here (2 mM) and previous studies have shown that naltrindole injection into the vlPAG inhibits analgesia evoked from the amygdala at doses (0.3–8 nmol) [43,48] similar to the dose used in the present study. These considerations support the conclusion that delta, but not mu or kappa, receptors mediate the fall in arterial pressure and heart rate evoked by visceral nociception.
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[3H]Naltrindole: a potent and selective ligand for labeling δ-opioid receptors

Abstract

Eur. J. Pharmacol.

Peptides

J. Biol. Chem.

Eur. J. Pharmacol.

Eur. J. Pharmacol.

Characterization of [3H]2-d-penicillamine, 5-d-penicillamine]enkephalin binding to δ-opioid receptors in the rat brain and neuroblastoma-glioma hybrid cell line (NG-108-15)

Delta opioid antagonist, naltrindole, selectively blocks analgesia induced by DPDPE but not DAGO or morphine

Pharmacol Biochem. Behav.

Synthesis of [3H]naltrindole

J. Labelled Compounds

[³H]Naltrindole: a potent and selective ligand for labeling δ-opioid receptors

Characterization of [³H]2-d-penicillamine, 5-d-penicillamine]enkephalin binding to δ-opioid receptors in the rat brain and neuroblastoma-glioma hybrid cell line (NG-108-15)

Synthesis of [³H]naltrindole