Jean-Marie Zajac

Pre- and postsynaptic distribution of μ, δ and κ opioid receptors in the superficial layers of the cervical dorsal horn of the rat spinal cord

Highly selective tritiated ligands and quantitative autoradiography have been used to study μ, δ and κ binding sites in the dorsal horn of the rat spinal cord. We have measured the proportions of the 3 main types of opioid binding sites in the superficial layers of the cervical dorsal horn (laminae I and II). The proportions of μ, δ and κ sites were70 ± 4%, 23 ± 2%and7 ± 1%, respectively, over the whole C4-T2 extent. Similar percentages were encountered at the level of each individual segment from C4 to T2. Eight days after a unilateral dorsal rhizotomy C4-T2, dramatic decreases were seen on the ipsilateral side to the lesion by comparison to the intact side. In the C7 segment, these decreases were76 ± 1%, 61 ± 1%and53 ± 3% for μ, δ and κ binding sites, respectively. The C7 segment can be considered as completely deafferented, so we attribute the residual values to postsynaptic binding whereas the decrease can be attributed to a loss of the presynaptic sites. These results are discussed with respect to the contribution of pre- and postsynaptic depressive effects of opiates on the transmission of noxious messages at the level of the dorsal horn.

Deltakephalin, Tyr-D-Thr-Gly-Phe-Leu-Thr: A new highly potent and fully specific agonist for opiate δ-receptors

Deltakephalin, Tyr-D-Thr-Gly-Phe-Leu-Thr (DTLET) was rationally designed as pure delta-probe from proposed models of mu and delta opiate receptors. On peripheral organs, deltakephalin displays a 3000 times higher inhibitory potency on the electrically stimulated mouse vas deferens (IC50 = 0.15 nM) as on the guinea pig ileum (IC50 = 460 nM). As expected [3H]deltakephalin interacts at 35 degrees C in rat brain tissue to a single class of binding sites (delta) (Bmax = 0.115 pmole/mg protein) with a high affinity: KD = 1.35 nM from equilibrium measurements and KD = 0.43 nM from kinetic determinations. Deltakephalin occurs as the most specific ligand for delta-binding sites as shown by the following discrimination ratios KI(mu)/KI(delta): 0.31 for D-Ala2-D-Leu5-enkephalin; 0.15 for D-Ser2-Thr6-Leu-enkephalin and 0.05 for deltakephalin.

Opioid receptor subtypes in the rat spinal cord: electrophysiological studies with μ- and δ-opioid receptor agonists in the control of nociception

We have compared the ability of selective mu- and delta-opiate agonists to modulate nociceptive transmission at the level of the rat dorsal horn using electrophysiological approaches. Single-unit extracellular recordings were made from neurones in the lumbar dorsal horn of the intact rat under halothane anaesthesia. Neurones could be activated by both A- and C-fibre electrical stimulation (and by natural innocuous and noxious stimuli). Agonists were applied directly onto the cord in a volume of 50 microliters. The intrathecal administration of 3 agonists, Tyr-D-Ala-Gly-MePhe-Gly-ol (DAGO) (mu-selective) (2 X 10(-3)-10 nmol) Tyr-D-Thr-Gly-Phe-Leu-Thr (DTLET) (mu/delta) (7 X 10(-4)-70 nmol), and cyclic Tyr-D-Pen-Gly-Phe-D-Pen (DPDPE) (delta) (2 X 10(-2)-100 nmol) produced dose-dependent inhibitions of C-fibre-evoked neuronal activity whilst A-fibre activity was relatively unchanged. DAGO produced near-maximal inhibitions which could be completely reversed by naloxone (1.5 nmol) whilst DPDPE causes less marked inhibitions which could only be partially reversed by naloxone (1.5-13.5 nmol). DTLET produced effects intermediate to those of DAGO and DPDPE. The results suggest that both mu- and delta-opioid receptors can modulate the transmission of nociceptive information in the rat spinal cord.

The μ rather than the δ subtype of opioid receptors appears to be involved in enkephalin-induced analgesia

The analgesic activity of some opioid peptides which display a relative selectivity for either the μ-receptor subtype, [D-Ala2,MePhe4,Gly-ol5]enkephalin (DAGO) or the δ-receptor subtype, [D-Ala2,D-Leu5]enkephalin (DADLE), [D-Ser2, Leu5]enkephalyl-Thr (DSLET) and [D-Thr2,Leu5]enkephalyl-Thr (DTLET) is highly correlated with their affinity for central or peripheral μ- but not δ-receptors. Moreover their analgesic effects as well as those elicited by degrading enzyme inhibitors (bestatin + thiorphan) of endogenous enkephalins were easily antagonized by naloxone with similar pA2 values but not by the δ-antagonist ICI 154,129. Therefore the analgesia produced by opioid peptides including endogenous enkephalins is likely connected to μ-receptor stimulation. Finally, there was no obvious potentiation by δ-agonists of the analgesia resulting from either administration of the μ-agonist morphine or endogenous enkephalins. This suggested that in the hot plate test, there is no modulation of the effect resulting from μ-receptor stimulation by a δ-receptor interaction. Likewise, enkephalinergic activity such as that due to thiorphan + bestatin does not appear to be regulated through μ- or δ-receptor stimulation.

Autoradiographic study of μ and δ opioid binding sites and neutral endopeptidase-24.11 in rat after dorsal root rhizotomy

Highly selective tritiated ligands and autoradiography have been used to study μ and δ binding sites as well as neutral endopeptidase-24.11 in the dorsal horn of the rat spinal cord. Two weeks and 4 months after dorsal root rhizotomy, both opioid binding sites were dramatically decreased (≅60%). In contrast, the level of neutral endopeptidase-24.11 remained unaltered.

Differences in Binding Properties of μ and δ Opioid Receptor Subtypes from Rat Brain: Kinetic Analysis and Effects of Ions and Nucleotides

Abstract: Differences in binding properties of μ and δ opioid receptors were investigated using DAGO (Tyr‐d‐Ala‐Gly‐MePhe‐Gly‐ol) and DTLET (Tyr‐d‐Thr‐Gly‐Phe‐Leu‐Thr), which occur, respectively, as the most selective μ and δ radioligands available. At high concentration, each agonist is able to interact with its nonspecific sites. Competition experiments indicated that a two‐site competitive model was adequate to explain the interactions of DAGO and DTLET with [3H]DTLET and [3H]DAGO binding sites, respectively. The weak cross‐reactivity (≃10%) of DTLET for μ sites was taken into account in these experiments. On the other hand, DAGO and DTLET exhibit differential binding kinetics. Thus, at 35°C, the lifetime of DTLET within its receptor site about 14 times longer than that of the μ agonist. Sodium and manganese ions decrease the maximal number of high affinity μ and δ sites, but the sensitivity of μ receptors is three times higher towards Na+ and 20‐fold higher towards Mn2+ than that of δ receptors. GTP reduces similarly the μ and δ binding whereas only the DAGO binding was modified by the nonhydrolyzable analogue guanylylimidodiphosphate [GMP‐P(NH)P]. However, in the presence of Na+ ions, GMP‐P(NH)P inhibits the DTLET binding in a concentration‐dependent manner. The effects of Na+ and GMP‐P(NH)P could be explained by a sequential transformation of δ receptors to low‐affinity states. This model predicts that Na+, by lowering the affinity of a fraction of sites, produces a decrease in the maximal number of high‐affinity δ receptors and that GMP‐P(NH)P enhances the Na+, effect. Moreover, the binding kinetic to this high‐affinity state was also modified by Na+ and nucleotides. All of these data support the existence of two independent μ and δ binding sites, the properties of which are differentially regulated by these endogenous effectors.

Occurrence of two cholecystokinin binding sites in guinea-pig brain cortex.

Saturation experiments of the highly potent cholecystokinin analogue [3H]Boc(diNle28,31)CCK27-33 ([3H]BNDL-CCK7, 100 Ci/mmol) with guinea pig brain cortex in a large concentration range (0.05 nM to 30 nM) show the presence of two different binding sites (A site: KD = 0.13 nM, Bmax = 35 fmol/mg; B site: KD = 6.4 nM, Bmax = 92 fmol/mg). Both sites exhibit different sensitivity to sodium ions and therefore can be selectively investigated at [3H]BDNL-CCK7 concentration lower than 1 nM for the A site in Tris buffer and in Krebs buffer for the B site. The selectivity factors KIB/KIA of various CCK related peptides vary from 58 for CCK4 to 26 for CCK8 and 4 for the antagonist (Nle28,31) CCK27-32-NH2. The occurrence of two different CCK binding sites in the brain could explain biphasic pharmacological effects of CCK8.

Regional distribution of μ, δ and κ opioid receptors in human brains from controls and parkinsonian subjects

The binding properties of μ and δ opioid receptors were investigated in several areas of human brain by using [3H]Tyr-d-Ala-Gly-(Me)Phe-Gly-ol and [3H]Tyr-d-Thr-Gly-Phe-Leu-Thr as respective selective ligands, while the totality of opioid receptors was measured by using [3H]etorphine as a non-selective agonist. Receptor densities were highest in cerebral cortex, amygdala and striatum, and lowest in the substantia nigra (pars compacta). In the different brain areas of patients with Parkinsons disease, the density and the proportion of the various opioid receptors were not significantly different from control subjects.

Binding in vivo of selective μ and δ opioid receptor agonists: opioid receptor occupancy by endogenous enkephalins

Abstract The in vivo binding properties of cerebral μ and δ opioid receptors were investigated in mice after the intrastriatal injection of [ 3 H][D-Ala 2 , MePhe 4 , Gly-ol 5 ]enkephalin (DAGO) or [ 3 H][D-Thr 2 , Leu 5 ]enkephalyl-Thr (DTLET). Both peptides exhibited similar diffusion kinetics in the brain and 30–40% of [ 3 H]DAGO or [ 3 H]DTLET was shown to be present in the tissue 15 min after injection when maximal binding was observed. The specific binding of both agonists, defined as the fraction of the radioactivity bound to brain membranes which was displaced by 10 nmol of cold ligand, was reversible, saturable and displayed a pharmacological profile similar to that found in in vitro experiments. At doses producing a similar analgesic effect in the hot-plate test in mice, DTLET occupied 64% of δ sites and DAGO 15% of μ sites. However, because of the residual cross-reactivity of DTLET for μ sites, it appeared that both ligands occupied a similar number of μ receptors at their ED 50 values, thus supporting a preferential involvement of μ opioid binding sites in the supraspinal pain control. [Met 5 ]enkephalin inhibited the in vivo binding of both agonists only when the peptide was protected from degradation by the co-administration of a mixed inhibitor of enkephalin degrading enzymes RB38A (N[3(R)(hydroxyaminocarbonyl)-2-benzyl-1-oxopropyl]-L-phenylalanine). Unlike thiorphan, 5 nmol RB38A alone was able to inhibit [ 3 H]DAGO binding by 60%. This result is the first direct demonstration of the existence of an in vivo tonic control of μ opioid receptor occupation by endogenous opioid peptides.

[3H] BOC [NLE28,31]CCK27−33, a new highly labelle ligand for CCK receptors: Binding on brain and on pancreas

Preliminary results on the binding of [3H]Boc[Nle28, 31]CCK27-33, designated [3H]Boc[diNle]CCK7, on mouse brain and rat pancreas membranes are presented. This new ligand for CCK receptors possesses a high specific activity (144 Ci/mmole), and binds in a saturable manner to mouse brain (Kd = 0.49 nM, Bmax = 49 fmoles/mg protein) and rat pancreas (Kd = 4.4 nM, Bmax = 696 fmoles/mg protein). Unlabelled Boc[diNle]CCK7 displaces [125I]CCK8 from its binding sites on mouse brain membranes with a high affinity, slightly superior to that of CCK8. The order of potencies to displace [3H]Boc[diNle]CCK7 from its binding sites was the same in mouse brain and rat pancreas: [3H]Boc[diNle]CCK7 greater than CCK8, Boc-CCK7 greater than non-sulfated CCK8, the pancreas binding sites being more discriminative than the brain binding sites. Thus, [3H]Boc[diNle]CCK7 is a very promising new probe for the characterization of CCK receptors and their interaction with different CCK fragments.