D-Ala2, MePhe4, Gly-ol5]enkephalin Research Articles

Gender differences in opioid-mediated analgesia: animal and human studies.

Gender differences in opioid-mediated analgesia: animal and human studies.

Opioids modulate cell division in the germinal zone of the late embryonic neocortex.

Opioid effects on cell division in the embryonic cerebral cortex were examined using two experimental approaches: (i) the presence of opioid receptors in the embryonic day 16 mouse neocortex was tested using immunohistochemical techniques; (ii) the values of the indices of [3H]thymidine pulse labelled cells and mitotic indices were estimated in the ventricular zone of the embryonic day 16 mouse neocortex 2.5, 4.5 and 8.5 h after administration to pregnant females of selected opioid receptor agonists or the opioid antagonist naloxone. The immunohistochemical study demonstrated that distinct subpopulations of the ventricular zone cells express mu, delta or kappa opioid receptors. Acute exposure of mouse embryos to mu, delta and kappa opioid receptor agonists or naloxone differentially affects the indices of [3H] thymidine pulse labelled cells and mitotic indices indicating changes in the cell cycle composition. Treatment with the mu opioid receptor agonist D-Ala2-MePhe4, Gly-ol5-enkephalin (DAGO), or the partially selective kappa opioid receptor agonist bremazocine, increased the [3H]thymidine labelling and mitotic indices. In contrast, the delta receptor agonist (D-Ser8)-leucine enkephalin-Thr (DSLET) produced a decrease in the labelled cell indices and mitotic indices. Naloxone provided a biphasic effect: a decrease in the values of labelled cell indices 2.5 h after naloxone administration, followed by an increase in the values of the indices at 4.5 and 8.5 h. These results suggest that the endogenous embryonic/maternal opioid systems are involved in the regulation of cell division in the ventricular zone of the late embryonic cortex.

Evidence for the implication of phosphoinositol signal transduction in mu-opioid inhibition of DNA synthesis.

An opioid receptor agonist, [D-Ala2,Me-Phe4,Glyol5]enkephalin (DAMGE), decreased [3H]thymidine incorporation into DNA of fetal rat brain cell aggregates. This action proved to depend on the dose of this enkephalin analog and the interval the aggregates were maintained in culture. The opioid antagonist naltrexone and the mu-specific antagonist cyclic D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr amide (CTOP) reversed the DAMGE effect, arguing for a receptor-mediated mechanism. The mu-opioid nature of this receptor was further established by inhibiting DNA synthesis with the highly mu-selective agonist morphiceptin and blocking its action with CTOP. Several other opioids, pertussis toxin, and LiCl also diminished DNA synthesis, whereas cholera toxin elicited a modest increase. Naltrexone completely reversed the inhibition elicited by the combination of DAMGE and low doses of LiCl but not by that of high levels of LiCl alone. The enkephalin analog also reduced basal [3H]inositol trisphosphate and glutamate-stimulated [3H]inositol monophosphate and [3H]inositol bisphosphate accumulation in the aggregates. These DAMGE effects were reversed by naltrexone and were temporally correlated with the inhibition of DNA synthesis. A selective protein kinase C inhibitor, chelerythrine, also inhibited thymidine incorporation dose-dependently. The effect of DAMGE was not additive in the presence of chelerythrine but appeared to be consistent with their actions being mediated via a common signaling pathway. These results suggest the involvement of the phosphoinositol signal transduction system in the modulation of thymidine incorporation into DNA by DAMGE.

Dog cerebral cortex contains μ-, δ- and κ-opioid receptors at different densities: apparent lack of evidence for subtypes of the κ-receptor using selective radioligands

The biochemical and pharmacological properties of mu (μ), kappa (κ) and delta (δ) opioid receptors were ascertained in dog cerebral cortex homogenates. The selective peptides, [ 3H] d-Pen 2- d-Pen 5enkephalin ([ 3H]DPDPE) and [ 3H] d-Ala 2-MePhe 4-Glyol 5-enkephalin ([ 3H]Glyol; [ 3H]DAMGO), bound to δ- and μ-opioid receptors with high affinity (dissociation constants, K dvalues= 4.7and1.6nM) but to different densities of binding sites (B max values of 49.2 and 6.6 fmol/mg protein, respectively) in washed homogenates of dog cerebral cortex. In contrast, the non-peptides, [ 3H]U69593 ([ 3H]U69) and [ 3H]etorphine ([ 3H]ET), labeled a high concentration of κ-opioid receptors (respectiveB max values of 67.2 and 76.6 fmol/mg protein) of high affinity (respectiveK ds of 1.4 and 0.47 nM) in the same tissue homogenates. Thus, the relative rank order of opioid receptor densities was:κ > δ ≫ μ. The selective labeling of the κ-receptors with two different drugs ([ 3H]U69 and [ 3H]ET) failed to reveal the possible existence of multiple κ-sites based on the relativeB max values of the two radioglands. This conclusion was further supported by the similarity of pharmacological specificity of both [ 3H]U69 and [ 3H]ET binding, where all the opioids tested produced 100% inhibition of these labels and where the rank order of potency of opioids at inhibiting the binding of these probes was:U50488 to >U69593 >dynophin-(1–8) >naloxone≫morphine≫Glyol (DAMGO)>DPDPE. The fact that [ 3H]Glyol selectively bound μ-receptors was borne-out by the relative rank order of inhibitory potency of different compounds against [ 3H]Glyol binding to dog cortex membranes, thus:Glyol (DAMGO)>morphine>naloxone>dynorphin-(1–8) ≫U50488 >U69593. The pharmacological selectivity of [ 3H]DPDPE binding to δ-receptors was different to the other receptor subtypes, being:DPDPE>dynorphin-(1–8) >naloxone>morphine⩾glyol≫U69 >U50488. These data have therefore helped identify the presence of μ-, δ and κ-opioid receptors in the dog cerebral cortex, and have shown the lack of existence of subtypes of the κ-receptor in this tissue.

Estimation of opioid receptor agonist dissociation constants with beta-chlornaltrexamine, an irreversible ligand which also displays agonism.

1. The irreversible opioid receptor antagonist beta-chlornaltrexamine (beta-CNA) has been shown previously to have agonist activity in the guinea-pig ileum preparation. However, the receptor type or types mediating this effect have not been established. 2. In this study, the agonism of beta-CNA was investigated by use of the competitive antagonist 16-methylcyprenorphine (RX8008M). Non-cumulative concentration-effect curves for beta-CNA were displaced in a non-parallel fashion indicating that the agonism was mediated by both mu- and kappa-receptors. 3. In principle, expression of agonism by an irreversible receptor antagonist could compromise its use in estimating agonist dissociation constants (pKAs) due to desensitization operating in addition to receptor inactivation. For kappa-receptors, this possibility was checked by use of ethylketocyclazocine (EKC) to mimic the agonist effects of beta-CNA and test whether subsequent EKC concentration-effect curves were displaced. For mu-receptors it was necessary to perform more involved experiments in which [D-Ala2, MePhe4, Gly-ol5]enkephalin (DAGOL) was used as a standard agonist and its pKA was estimated under different conditions of beta-CNA incubation. 4. These analyses indicated that neither the mu- nor the kappa-receptor-mediated agonism of beta-CNA was associated with appreciable receptor desensitization. In turn it was concluded that the usefulness of beta-CNA as a pharmacological tool for the estimation of mu- and kappa-opioid receptor agonist dissociation constants is not compromised by the agonist effects that the compound demonstrates at these receptors.

Pharmacological analysis of the calcium-dependence of mu-receptor agonism.

1 A number of studies in isolated tissues have shown that mu-opioid-receptor-mediated agonism can be augmented or potentiated with reduction in extracellular calcium concentration [( Ca2+]o). These effects have been ascribed to alterations in post-receptor coupling but the nature of the changes involved have not been quantitatively elucidated. 2 In this paper, logistic curve-fitting and operational model-fitting (Black & Leff, 1983) were used to analyse the effects of variations in [Ca2+]o on the mu-receptor-mediated effects of [D-Ala2, MePhe4, Gly-ol5]enkephalin (DAGOL) in the isolated, coaxially-stimulated ileum of the guinea-pig. At each value of [Ca2+]o, the effects of irreversible receptor alkylation by beta-chlornaltrexamine (beta-CNA) were also investigated. 3 From these analyses it is concluded that with reduction in [Ca2+]o the efficacy of DAGOL in this system is increased and the sensitivity of the transducer relation is also enhanced, the latter indicating a trend towards positive co-operativity. Reduction of [Ca2+]o also appeared to produce a reduction in agonist affinity. 4 Experimental manipulation of [Ca2+]o may provide a useful means of enhancing mu-agonist efficacy, allowing detection of agonism in compounds with low intrinsic efficacies. However, the accompanying change in co-operativity of the transducer relation must be considered when quantifying agonism under these conditions.

Differential ontogeny of divalent cation effects on rat brain δ-, μ-, and χ-opioid receptor binding

The effect of the divalent cations, Mn 2+, Mg 2+ and Ca 2+ on rat forebrain δ-, μ- and χ-receptor binding was examined during postnatal development. It was found that δ-receptor binding, assessed with [ 3H] d-Ala 2- d-Leu 5-enkephalin ([ 3H]DADLE) (+10 nM d-Ala 2-MePhe 4-Gly-ol 5-enkephalin (DAMGE)), was stimulated by the 3 cations in a dose- and developmental time-dependent manner. δ-Binding was most sensitive to the cations during the first week postnatal, prior to the appearance of high-affinity δ-binding. In contrast, inhibition of μ-receptor binding ([ 3H]DAMGE) by divalent cations appeared early in development and remained constant throughout the postnatal period. Divalent cation inhibition of χ-binding ([ 3H]ethylketocyclazocine ([ 3H]EKC) +100 nM DAMGE and 100 nM DADLE) appeared after the second week postnatal. These results demonstrate that the characteristics and postnatal development of divalent cation modulation of μ-, δ- and χ-binding is distinctly different. Thus, the neonate may be a good model system to examine the binding properties and functions of δ- and χ-receptor subtypes.