Effects of [D-Ala2, D-Leu5]enkephalin and [D-Pen2, L-Pen5]enkephalin on apomorphine-induced motor activity in the mouse.

The effects of intracerebroventricular injections of opioid peptides such as DADL [( D-Ala2, D-Leu5]enkephalin) and DPLPE [( D-Pen2, L-Pen5]enkephalin) with different degrees of selectivity for delta- over mu-receptor on apomorphine (0.1, 0.3, 1.0 and/or 3.0 mg/kg)-induced motor activity were investigated in the mouse using multi-dimensional behavioral analyses. Lower doses (0.1 and 0.3 mg/kg) of apomorphine failed to affect significantly motor activity, whilst higher doses (1.0 and 3.0 mg/kg) of the drug produced a marked increase in linear locomotion, circling, rearing, and/or grooming behaviors. DADL (0.03, 0.1 or 0.3 microgram) by itself did not influence behaviors, while the peptide (0.1 and 0.3 microgram) produced a marked inhibition on apomorphine (1.0 but not 3.0 mg/kg)-induced increase in rearing behaviors. Furthermore, the inhibitory effect of DADL (0.3 micrograms) on the apomorphine (1.0 mg/kg)-induced increase in rearing was reversed by treatment with the alkylating agent beta-FNA (beta-funaltrexamine) (5.0 micrograms). In contrast to the effects of DADL, the much more delta-selective opioid agonist DPLPE (0.3, 1.0 or 1.75 micrograms) had no marked effects on apomorphine (1.0 mg/kg)-induced behaviors. These results suggest that delta opioid receptors do not play a principal role in the apomorphine-induced increase in circling, rearing or grooming behaviors.     

D-Ala2,NMePhe4,Gly-ol5]enkephalin, but not [D-Pen2,L-Pen5]enkephalin, specifically inhibits behaviors induced by the dopamine D2 agonist RU 24213.

The effects of intracerebroventricular injection (10 microliters) of mu- and delta-selective opioid peptides on behaviors induced by the dopamine D2-selective agonist RU 24213 were investigated in the mouse, using multi-dimensional behavioral analyses. Fifteen to 30 min after the start of behavioral measurements, a 3.0 mg/kg dose of RU 24213 produced a marked increase in linear locomotion, circling, rearing and grooming behaviors. Although the mu-selective opioid peptide [D-Ala2,NMePhe4,Gly-ol5]enkephalin (DAGO) (0.003 and 0.01 microgram) itself did not significantly affect behaviors, DAGO (0.01 microgram) antagonized the RU 24213 (3.0 mg/kg)-induced increase in behaviors such as linear locomotion, circling, rearing, and grooming. Additionally, the effects of DAGO on RU 24213-induced behaviors were fully reversed by treatment with the mu-selective alkylating agent beta-funaltrexamine (beta-FNA) (5.0 micrograms). In contrast, the delta-selective opioid peptide [D-Pen2,L-Pen5]enkephalin (0.3 or 1.0 micrograms) had no marked effects on RU 24213 (3.0 mg/kg)-induced behaviors. These results suggest that mu- but not delta-opioid receptors play an inhibitory role in the behaviors induced by the selective activation of dopamine D2 receptors.     

Topographically designed analogues of [D-Pen,D-Pen5]enkephalin.

The conformationally restricted, cyclic disulfide-containing delta opioid receptor selective enkephalin analogue [D-Pen2,D-Pen5]enkephalin (1, DPDPE) was systematically modified topographically by addition of a methyl group at either the pro-S or pro-R position of the beta carbon of an L-Phe4 or D-Phe4 residue to give [(2S,3S)-beta-MePhe4]DPDPE (2), [(2R,3R)-beta-MePhe4]DPDPE (3), [(2S,3R)-beta-MePhe4]DPDPE (4), and [(2R,3S)-beta-MePhe4]DPDPE (5). The four corresponding isomers were prepared in which the beta-methylphenylalanine residue was p-nitro substituted, that is with a beta-methyl-p-nitrophenylalanine (beta-Me-p-NO2Phe) residue, to give [(2S,3S)-beta-Me-p-NO2Phe4]DPDPE (6), [(2R,3R)-beta-Me-p-NO2Phe4]DPDPE (7), [(2S,3R)-beta-Me-p-NO2Phe4] DPDPE (8), and [(2R,3S)-beta-Me-p-NO2Phe4]DPDPE (9), respectively. The potency and selectivity (delta vs mu opioid receptor) were evaluated by radioreceptor binding assays in the rat brain using [3H]CTOP (mu ligand) and [3H]DPDPE (delta ligand) and by bioassay with mouse vas deferens (MVD, delta receptor assay) and guinea pig ileum (GPI, mu receptor assay). The eight analogues of DPDPE showed highly variable binding and bioassay activities particularly at the delta opioid receptor (4 orders of magnitude), but also at the mu opioid receptor, which led to large differences (3 orders of magnitude) in receptor selectivity. For example, [(2S,3S)-beta-MePhe4]DPDPE (2) is 1800-fold selective in binding to the delta vs mu receptor, making it one of the most selective delta opioid receptor ligands in the enkephalin series as assessed by the rat brain binding assay, whereas the corresponding (2R,3R)-beta-Me-p-NO2Phe-containing analogue 9 is only 4.5-fold selective (nonselective) in this same assay. On the other hand, in the bioassay systems, [(2S,3S)-beta-Me-p-NO2Phe4]DPDPE (5) is more potent than DPDPE and 8800-fold selective for the MVD (delta receptor) vs the GPI (mu receptor), making it the most highly selective ligand in this series for the delta opioid receptor on the basis of these bioassays. In these assay systems, the (2R,3S)-beta-MePhe4-containing analogue 5 had very weak potency and virtually no receptor selectivity (4.4-fold). These results demonstrate that topographical modification alone in a conformationally restricted peptide ligand can significantly modulate both potency and receptor selectivity of peptide ligands that have multiple sites of biological activity and suggest that this approach may have general application to peptide ligand design.     

Theoretical study of the flexibility and solution conformation of the cyclic opioid peptides [D-Pen2,D-Pen5]enkephalin and [D-Pen2,L-Pen5]enkephalin

An investigation of the conformational profiles of two cyclic delta-selective opioid peptides, [D-Pen2,D-Pen5]-enkephalin and [D-Pen2,L-Pen5]-enkephalin, has been made. The methods and procedures used are more extensive and systematic than those previously reported, involving a combination of nested grid rotations, cyclic ring-closing algorithms, molecular dynamic simulations at high and low temperature, and total geometry optimizations. The reexamination is a necessary first step in further characterization of the bioactive form of delta-selective peptides. This study also addresses the question of how rigid such cyclic analogs actually are. Finally, the effect of solvent environment on the low energy conformers obtained from the extensive search strategy has been determined. Simulation of the effect of water as a solvent by a continuum dielectric constant of 80 results in the breaking of internal hydrogen bonds and rearrangement of the rank order of energy of the conformers. The lowest energy solution conformation for [D-Pen2,D-Pen5]-enkephalin, obtained without utilizing any experimental data, is in excellent agreement with the geometric properties deduced from its solution NMR spectra.     

Radioiodinated [D-Ala2, Met 5] enkephalin

¹²⁵I[D-Ala², Met⁵] enkephalin with high specific activity (122–185 Ci/mmol) was prepared and purified by Sep-Pak C₁₈ reverse phase cartridge followed by high performance liquid chromatography (HPLC). HPLC at pH 3.0 resolved ¹²⁵I[D-Ala², Met⁵] enkephalin into two fractions, which ran as a single spot in thin-layer chromatography with the same R_f values. Alkaline hydrolysates of the HPLC-purified fractions showed a single spot corresponding to monoiodotyrosine standard when analysed by thin-layer chromatography. Binding kinetics of the tracer was found to approach equilibrium after 30 min at 24d?. Scatchard analysis of the saturation equilibrium binding studies gave an equilibrium dissociation constant of 3.58 nM and the number of binding site of 30 fmol/mg protein. Enkephalin analogs were capable of displacing ¹²⁵I[D-Ala², Met⁵] enkephalin binding from the rat brain plasma membrane. The effective concentration of [D-Ala², Met⁵] enkephalin and [D-Ala², Leu⁵] enkephalin for 50% inhibition of ¹²⁵I[D-Ala², Met⁵] enkephalin binding was estimated to be 79 nM and 23 nM, respectively. Both substance P and gastrin tetrapeptide failed to displace the ¹²⁵I[D-Ala², Met⁵] enkephalin binding to any significant extent. The ¹²⁵I[d-Ala², Met⁵] enkephalin prepared by the present procedure is therefore a useful tracer. This method of preparing radioiodinated peptide may be applicable to other enkephalin analogs or neuropeptides in general.     

Interaction between the mu-agonist dermorphin and the delta-agonist [D-Ala2, Glu4]deltorphin in supraspinal antinociception and delta-opioid receptor binding.

1. In rats, the interaction between the mu-opioid agonist dermorphin and the delta-opioid agonist [D-Ala2, Glu4]deltorphin was studied in binding experiments to delta-opioid receptors and in the antinociceptive test to radiant heat. 2. When injected i.c.v., doses of [D-Ala2, Glu4]deltorphin higher than 20 nmol produced antinociception in the rat tail-flick test to radiant heat. Lower doses were inactive. None of the doses tested elicited the maximum achievable response. This partial antinociception was accomplished with an in vivo occupancy of more than 97% of brain delta-opioid receptors and of 17% of mu-opioid receptors. Naloxone (0.1 mg kg-1, s.c.), and naloxonazine (10 mg kg-1, i.v., 24 h before), but not the selective delta-opioid antagonist naltrindole, antagonized the antinociception. 3. In vitro competitive inhibition studies in rat brain membranes showed that [D-Ala2, Glu4]deltorphin displaced [3H]-naltrindole from two delta-binding sites of high and low affinity. The addition of 100 microM Gpp[NH]p produced a three fold increase in the [D-Ala2, Glu4]deltorphin Ki value for both binding sites. The addition of 10 nM dermorphin increased the Ki value of the delta-agonist for the high affinity site five times. When Gpp[NH]p was added to the incubation medium together with 10 nM dermorphin, the high affinity Ki of the delta-agonist increased 15 times. 4. Co-administration into the rat brain ventricles of subanalgesic doses of dermorphin and [D-Ala2, Glu4]deltorphin resulted in synergistic antinociceptive responses. 5. Pretreatment with naloxone or with the non-equilibrium mu-antagonists naloxonazine and beta-funaltrexamine completely abolished the antinociceptive response of the mu-delta agonist combinations. 6. Pretreatment with the delta-opioid antagonists naltrindole and DALCE reduced the antinociceptive response of the dermorphin-[D-Ala2, Glu4]deltorphin combinations to a value near that observed after the mu-agonist alone. At the dosage used, naltrindole occupied more than 98% of brain delta-opioid receptors without affecting mu-opioid-receptors. 7. These data suggest that in the rat tail-flick test to radiant heat, mu- and delta-opioid agonists co-operate positively in evoking an antinociceptive response. Although interactions between different opioid pathways cannot be excluded, in vitro binding results indicate that this co-operative antinociception is probably mediated by co-activation of the delta-opioid receptors at the cellular level by the mu- and delta-agonist.     

Photoreactive enkephalin analogue: [D-Ala2, p-N3-Phe 4-Met 5] enkephalin

A photoreactive [D-Ala², p-N₃-Phe⁴-Met⁵]enkephalin was synthesized by classical solution peptide synthetic methods. The hydroxysuccinimide ester was used in all the coupling steps in the presence of a weak base, triethylamine. The deprotected enkephalin analogue was purified on high performance liquid chromatography using a Waters, C₁₈μBondapak reverse phase column and its purity was assessed by thin-layer chromatography. The composition of the analogue was determined and confirmed by elemental analysis and amino acid analysis. Its photoreactivity was demonstrated by the time dependent ultraviolet spectral changes on exposure to light. Competition receptor binding showed that [D-Ala², p-N₃-Phe⁴-Met⁵]enkephalin was 4-fold more potent than [D-Ala², Met⁵]-enkephalin in competing for binding to the enkephalin binding site. The data presented suggest that this photoreactive enkephalin analogue may be suitable for use in the in situ photoaffinity labeling of the enkephalin receptor.     

Chronic effect of [D-Pen2,D-Pen5]enkephalin on rat brain opioid receptors.

In previous studies, we have demonstrated that chronic etorphine or [D-Ala2,D-Leu5]enkephalin (DADLE) treatment of rats results in the reduction of mu- and delta-opioid receptor binding activities as tolerance develops. As both etorphine and DADLE are relatively non-specific opioid ligands, interacting with both mu- and delta-receptors, these studies could not determine whether down-regulation of a specific receptor type occurs. Therefore, in the present studies, animals were rendered tolerant to the delta-opioid receptor-selective agonist [D-Pen2,D-Pen5]enkephalin (DPDPE), and receptor binding activities were measured. Treating Sprague-Dawley rats with increasing doses of DPDPE (80-160-240-320 micrograms/kg) i.c.v. for 1 to 4 days resulted in a time-dependent increase in the AD50 of DPDPE to elicit an antinociceptive response. When delta-receptor binding was determined by using [3H]DPDPE, a 40-50% decrease in binding in the midbrain and cortex, and 25-35% decrease in binding in the striatum were observed after 3 or 4 days of DPDPE treatment. Scatchard analysis of the [3H]DPDPE saturation binding data revealed a decrease in Bmax values and no significant change in Kd values. To our surprise, when mu-receptor binding was determined by using [3H]Tyr-D-Ala-Gly-MePhe-Gly-ol (DAMGO), a 10-15% decrease in binding was also observed in the midbrain and cortex after 4 days of DPDPE treatment. Our conclusion is that chronic DPDPE treatment preferentially reduces delta-opioid receptor binding activity. Its minor effect on the mu-opioid receptor maybe due to an interaction between delta cx and mu cx binding sites.     

Effect of [D-Ala2,MePhe4,Gly-ol5]enkephalin and [D-Ala2,D-Leu5]enkephalin on ion and amino acid transport in rabbit ileum

The selective mu-opioid agonist [D-Ala2,MePhe4,Gly-ol5]enkephalin (DAGO) (10 microM) reduced the short circuit (Isc) and the L-valine induced increase of the transepithelial potential difference and Isc(delta Vms and delta Isc) measured in-vitro in rabbit ileum, with a mechanism antagonized by naloxone (1 microM). [D-Ala2,D-Leu5]enkephalin (DADLE) (10 microM) had no significant effect on the transepithelial potential difference (Vms), Isc, delta Vms and delta Isc. In the ileum deprived of the serosa and muscolaris, DAGO reduced the delta Vms and delta Isc, but not the Vms and Isc, suggesting localization of the receptors responsible for this latter effect in the myenteric plexus and/or the muscularis mucosae. These preliminary results suggest that in the rabbit ileum opioids influence electrolyte and amino acid transport and these effects may be at least partly mediated by mu-receptors.     

Pharmacological characterization of [D-Ala2,Leu5,Ser6]enkephalin (DALES): antinociceptive actions at the delta non-complexed-opioid receptor.

Substantial evidence has been accumulated which suggests that opioid delta receptors may be distinguished on the basis of their involvement in the modulation (i.e., increase or decrease in potency) of mu-mediated antinociception. On this basis, it has been hypothesized that some opioid delta receptors exist within a functional complex with mu receptors (delta complexed (delta cx) receptors) while other delta sites do not (delta non-complexed (delta ncx) receptors). Recent work with [D-Ala2,Leu5,Cys6]enkephalin (DALCE) has demonstrated that this compound produces initial antinociceptive actions, does not modulate morphine antinociception and appears to bind irreversibly to the delta ncx site, presumably by means of thiol-disulfide exchange between the receptor and the cysteine sulfhydryl group. To determine if a structural basis exists for actions at the hypothesized delta ncx receptor, in the present study we report the synthesis and pharmacological characterization of [D-Ala2,Leu5,Ser6] enkephalin (DALES), a close structural analogue of DALCE. If a structural basis for action at the delta ncx site exists, then DALES would be predicted to produce antinociception, fail to modulate morphine antinociception and, since it lacks the free sulfhydryl group present in DALCE, fail to exhibit irreversible antagonistic actions; these predictions were supported. Additionally, pretreatment with DALCE at -24 h, but not with DALES, blocked DALES-induced antinociception. These observations in vivo support the concept of a structural basis for activity at the hypothesized delta ncx site and suggest that DALES, like DALCE, may be a useful probe for pharmacological characterization of putative delta receptor subtypes.     

Ring substituted and other conformationally constrained tyrosine analogues of [D-Pen2,D-Pen5]enkephalin with delta opioid receptor selectivity.

The conformationally restricted, cyclic disulfide-containing delta opioid receptor selective enkephalin analogue [D-Pen2,D-Pen5] enkephalin (DPDPE) was modified by 2' (CH3) and 3' (I, OCH3, NO2, NH2) ring substitutions and by beta-methyl conformationally constrained beta-methyltyrosine derivatives in the 1 position. The potency and selectivity of these analogues were evaluated by bioassay in the mouse vas deference (MVD, delta receptor assay) and guinea pig ileum (GPI, mu receptor assay) assays and by radioreceptor binding assays in the rat brain using [3H]CTOP (mu ligand) and [3H][p-ClPhe4]DPDPE (delta ligand). The analogues showed highly variable potencies in the binding assays and in the bioassays. Aromatic ring substituents with positive Hammett constants had decreased potency, while substituents with negative Hammett constraints has increased potency for the opioid receptor. The most potent and most selective compound based on the binding was [2'-MeTyr1]DPDPE (IC50 = 0.89 nM and selectivity ratio 1310 in the binding assays). The 6-hydroxy-2-aminotetralin-2-carboxylic acid-containing analogue, [Hat1]DPDPE, also was highly potent and selective in both assays, demonstrating that significant modifications of tyrosine in enkephalins are possible with maintenance of high potency and delta opioid receptor selectivity. Of the beta-methyl-substituted Tyr1 analogues, [(2S,3R)-beta-MeTyr1]DPDPE was the most potent and the delta receptor selective. The results with substitution of beta-MeTyr or Hat instead of Tyr also demonstrate that topographical modification in a conformationally restricted ligand can significantly modulate both potency and receptor selectivity of peptide ligands that have multiple sites of biological activity.     

Orphanin FQ/nociceptin potentiates [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin-Induced mu-opioid receptor phosphorylation

In this study, we investigate the molecular mechanisms by which acute orphanin FQ/nociceptin (OFQ/N), acting through the nociceptin opioid peptide (NOP) receptor, desensitizes the mu-opioid receptor. We described previously the involvement of protein kinase C and G-protein-coupled receptor kinases (GRK) 2 and 3 in OFQ/N-induced mu receptor desensitization. Because phosphorylation of the mu receptor triggers the successive regulatory mechanisms responsible for desensitization, such as receptor uncoupling, internalization, and down-regulation, we investigated the ability of OFQ/N to modulate [d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO)-induced mu receptor phosphorylation in BE(2)-C human neuroblastoma cells transfected with epitope-tagged mu receptors. OFQ/N treatment (100 nM, 60 min) potentiated DAMGO-induced mu receptor phosphorylation; inhibition of GRK2 or protein kinase C concomitant with OFQ/N treatment blocked the OFQ/N-mediated increase in DAMGO-induced phosphorylation. Inclusion of the NOP antagonist peptide III-BTD during OFQ/N pretreatment blocked the potentiation of DAMGO-induced phosphorylation by OFQ/N, which is consistent with the potentiation being mediated via actions of the NOP receptor. In addition, in cells expressing mu receptors in which the GRK-mediated phosphorylation site Ser(375) was mutated to alanine, OFQ/N treatment failed to potentiate DAMGO-induced mu receptor phosphorylation and failed to desensitize the mu receptor. However, DAMGO-induced mu receptor phosphorylation and OFQ/N-induced mu receptor desensitization occurred in cells expressing mu receptors lacking non-GRK phosphorylation sites. These data suggest that OFQ/N binds to NOP receptors and activates protein kinase C, which then increases the ability of GRK2 to phosphorylate the agonist-occupied mu receptor, heterologously regulating homologous mu receptor desensitization.     

Systemic analgesic activity and delta-opioid selectivity in [2,6-dimethyl-Tyr1,D-Pen2,D-Pen5]enkephalin.

The cyclic peptide [2,6-dimethyl-Tyr1,D-Pen2,D-Pen5]enkephalin (2) was synthesized by solid-phase techniques and contains the optically pure unnatural amino acid 2,6-dimethyltyrosine (DMT) as a replacement for the Tyr1 residue of [D-Pen2,D-Pen5]enkephalin (DPDPE, 1). This structural modification resulted in a 10-fold increase in the potency of 2 at the delta opioid receptor and a 35-fold increase in potency at the mu receptor while substantial delta receptor selectivity was maintained. In addition, 2 was 86-fold more effective than 1 at inhibiting electrically stimulated contractions of the mouse vas deferens. In the hot plate test, 2 was 7-fold more potent than 1 after intracerebroventricular administration in the mouse. While 1 was inactive following systemic administration of doses as high as 30 mg/kg, subcutaneous administration of 2 significantly inhibited writhing with an ED50 of 2.6 mg/kg. These results demonstrate that the potency and systemic activity of DPDPE are significantly increased by replacement of Tyr1 with DMT.     

The use of [3H]-[D-Pen2,D-Pen5]enkephalin as a highly selective ligand for the delta-binding site.

The characteristics of the binding of [3H]-[D-Pen2,D-Pen5]enkephalin were determined in homogenates of guinea-pig and rat brain. In the guinea-pig, the maximum binding capacity for [3H]-[D-Pen2,D-Pen5]enkephalin was 4.19 pmol g-1 and the KD 1.61 nM. In the rat, the corresponding values were 2.47 pmol g-1 and 5.42 nM. In both species, the maximum binding capacity and the affinity were not altered when mu-binding was suppressed with [D-Ala2,MePhe4,Gly-ol5]enkephalin. The mu-agonists, [D-Ala2,MePhe4,Gly-ol5]enkephalin and morphine, displaced a small portion of the binding of [3H]-[D-Pen2,D-Pen5]enkephalin with high affinities.     

Lipid membrane permeability of modified c[D-Pen2, D-pen5]enkephalin peptides

Permeability coefficients of a series of analogues of a potent opioid peptide, c[D-Pen², D-Pen⁵]enkephalin, were measured in a model membrane system. The analogues included hydrophobic amino acid substitutions on position 3. Liposomes of a mixed composition consisting of zwitterionic lipids and cholesterol served as the model membranes. The obtained permeability coefficients range between 0.38 × 10^?12 and 2.9 × l0^?12 cm/s. These data were correlated with the hydrophobicity scale of Nozaki and Tanford (J. Biol. Chem. 246, 1971, 2211-2217) (correlation coefficient = 0.9933) and with determinations of lipid order perturbation by differential scanning calorimetry (correlation coefficient = -0.9779). The reasonably good correlation obtained within the family of analogues substituted on position 3 (Gly, Ala, Leu, Phe) indicates that changes in permeabilities are primarily related to increases in the partition coefficient of the peptide. However, Phe residue added on the N-terminal end of the peptide (position 0) does not appear to follow the observed trend, showing stronger lipid perturbation and lower permeability compared to the Phe³ analog. This observation demonstrates that each class of peptide modifications requires a new basis of permeability analysis and predictions. © Munksgaard 1996.     

Analgesic and tolerance-inducing effects of the highly selective delta opioid agonist [D-Pen2,D-Pen5]enkephalin in mice

The novel and highly selective, conformationally restricted enkephalin analogue for delta-opioid receptors, [D-Pen2,D-Pen5]enkephalin (DPDPE; Pen = penicillamine), was studied in various in vivo tests for analgesia, tolerance and physical dependence. Intracerebroventricular (i.c.v.) administration of DPDPE caused a dose-dependent, naloxone-reversible antinociception, measured with the heat-irradiant (tail-flick) method. Acute tolerance developed to the antinociceptive effect of DPDPE. DPDPE also caused mild signs of physical dependence (withdrawal hypothermia and body weight loss) after repeated peptide treatment. Severe signs of morphine withdrawal (e.g. withdrawal jumping) on the other hand, could not be reversed by the administration of DPDPE. It is concluded that the activation of central delta-opioid receptors may play a role in controlling pain mechanisms, and that this activation is followed by the rapid development of a tolerance to this action.     

Chronic [D-Ala2, D-Leu5]enkephalin treatment increases the nerve growth factor in adult mouse brain

The delta opioid peptide [D-Ala(2), D-Leu(5)]enkephalin (DADLE) has been shown to enhance the survival of dopaminergic neurons. Here, we found that chronic treatment with DADLE caused a significant increase in nerve growth factor (NGF) in the hippocampus and the midbrain of adult albino Swiss (CD-1) mice, but not in the striatum or frontal cortex. Glia-derived neurotrophic factor (GDNF) was not significantly affected. Thus, the neuroprotective action of DADLE may be mediated in part by NGF.     

Effect of [D-Ala2,Met5]enkephalinamide and [D-Ala2,D-Leu5]enkephalin on cholinergic and noradrenergic neurotransmission in isolated atria

In rabbit isolated atria, [D-Ala2,Met5]enkephalinamide and [D-Ala2,D-Leu5]enkephalin (0.1-3 microM) inhibited responses to cholinergic nerve stimulation in a concentration-dependent manner without affecting responses to exogenous acetylcholine. The inhibitory effect was blocked by the opiate receptor antagonist naloxone (1 microM). In rabbit atria in which the transmitter acetylcholine stores had been radioactively labelled by preincubating the tissue in [3H]choline, tetrodotoxin (100 ng/ml) significantly (P less than 0.001) blocked the stimulation-induced (2 Hz for 3 min) release of radioactivity. Both [D-Ala2,Met5]enkephalinamide and [D-Ala2,D-Leu5]enkephalin (0.3 and 1 microM) significantly decreased stimulation-induced radioactivity release and their effects were blocked by naloxone (1 microM). In rat isolated atria, [D-Ala2,Met5]enkephalinamide and [D-Ala2,D-Leu5]enkephalin (0.3-3 microM) inhibited responses to cholinergic nerve stimulation without affecting responses to exogenous acetylcholine. The inhibitory effect was blocked by naloxone (1 microM). In guinea-pig isolated atria, responses to cholinergic nerve stimulation were unaffected by the enkephalin analogues. In rabbit, rat and guinea-pig isolated atria, responses to noradrenergic nerve stimulation and exogenous noradrenaline were unaffected by the enkephalin analogues.     

Modulation of mu-mediated antinociception by delta agonists in the mouse: selective potentiation of morphine and normorphine by [D-Pen2,D-Pen5]enkephalin

The effect of the delta-selective agonist [D-Pen2,D-Pen5]enkephalin (DPDPE) on the antinociception produced by intracerebroventricular (i.c.v.) administration of the mu agonists morphine, [D-Ala2,NMePhe4,Gly-ol5]enkephalin (DAGO), [NMePhe3,D-Pro4]morphiceptin (PLO17), beta-endorphin, phenazocine, etorphine and sufentanil was studied in mice. Only the antinociceptive effects of morphine and normorphine were modulated by i.c.v. coadministration of a dose of DPDPE which did not produce any significant antinociception alone. Both the morphine and normorphine dose-response lines were displaced to the left in the presence of DPDPE. The delta-selective antagonist ICI174,864 (N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH) (where Aib is alpha-aminoisobutyric acid) blocked the modulation of morphine antinociception by DPDPE. ICI 174,864 alone failed to produce either a significant increase or decrease of morphine, phenazocine, etorphine or beta-endorphin antinociception. The results of the present study provide support for the hypothesis that the enkephalins may function to modulate antinociception produced at the mu receptor; such modulation may come about via the existence of an opioid mu-delta receptor complex. The mu receptors existing in such a complex may be selectively activated by morphine and normorphine, but not the other mu agonists studied here. Thus, the enkephalins may function both to directly initiate, as well as to modulate, some forms of supraspinal mu receptor-mediated antinociception.     

Delta-opioid-receptor activation by [D-Pen2,D-Pen5]enkephalin and morphine inhibits substance P release from trigeminal nucleus slices.

The release of substance P (SP) from spinal dorsal horn slices is partially inhibited by micromolar concentrations of selective delta-opioid receptor agonists. In the present study, we have examined the effect of nanomolar concentrations of [D-Pen2,D-Pen5]enkephalin (DPDPE, delta-opioid receptor agonist) and low micromolar of concentrations morphine on K(+)-evoked SP release from rat trigeminal nucleus caudalis (TNC) slices. DPDPE and morphine inhibited SP release with an apparent maximal effect at 3 nM and at 3 microM, respectively. DPDPE and morphine produced U-shaped concentration-response curves that were completely autoinhibited at 100 nM DPDPE and 1 microM morphine. The inhibition of SP release produced by 3 nM DPDPE and 3 microM morphine was blocked by the opioid receptor antagonists naloxone (30 nM; non-selective) and ICI 174,864 (0.3 microM; delta-selective) but not by nor-binaltorphimine (3 nM n-BNI; kappa-selective), naloxonazine (1 nM; micro 1-selective) or beta-funaltrexamine (20 nM beta-FNA; mu-selective). These findings indicate that delta-opioid receptor-mediated inhibition of SP release from TNC can be achieved by nanomolar concentrations of selective delta-opioid receptor agonists. Activation of delta-opioid receptors by morphine might be involved in the residual analgesia observed after mu 1-opioid receptor blockade and in the analgesia produced by high doses of morphine.