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A comparative analysis of the infarct-limiting activity of δ- and κ-opioid receptors (OR) agonists was carried out on a model of coronary occlusion (45 min) and reperfusion (120 min) in male Wistar rats. We used selective δ 2 -OR agonist deltorphin II (0.12 mg/kg), δ-OR agonists BW373U86 and p-Cl-Phe DPDPE (0.1 and 1 mg/kg), selective agonists of δ 1 -OR DPDPE (0.1 and 0.969 mg/kg), κ 1 -OR U-50,488 (0.1 and 1 mg/kg), κ 2 -OR GR-89696 (0.1 mg/kg), and κ-OR ICI-199,441 (0.1 mg/kg). All drugs were administered intravenously 5 min before reperfusion. Deltorphin II, BW373U86 (1 mg/kg), p-Cl-Phe DPDPE (1 mg/kg), U-50,488 (1 mg/kg), and ICI-199,441 had a cardioprotective effect. The most promising compounds for drug development are ICI-199,441 and deltorphin II., (© 2024. Springer Science+Business Media, LLC, part of Springer Nature.)

Signaling bias refers to G protein-coupled receptor ligand ability to preferentially activate one type of signal over another. Bias to evoke signaling as opposed to sequestration has been proposed as a predictor of opioid ligand potential for generating tolerance. Here we measured whether delta opioid receptor agonists preferentially inhibited cyclase activity over internalization in HEK cells. Efficacy (τ) and affinity (KA) values were estimated from functional data and bias was calculated from efficiency coefficients (log τ/KA). This approach better represented the data as compared to alternative methods that estimate bias exclusively from τ values. Log (τ/KA) coefficients indicated that SNC-80 and UFP-512 promoted cyclase inhibition more efficiently than DOR internalization as compared to DPDPE (bias factor for SNC-80: 50 and for UFP-512: 132). Molecular determinants of internalization were different in HEK293 cells and neurons with βarrs contributing to internalization in both cell types, while PKC and GRK2 activities were only involved in neurons. Rank orders of ligand ability to engage different internalization mechanisms in neurons were compared to rank order of E max values for cyclase assays in HEK cells. Comparison revealed a significant reversal in rank order for cyclase E max values and βarr-dependent internalization in neurons, indicating that these responses were ligand-specific. Despite this evidence, and because kinases involved in internalization were not the same across cellular backgrounds, it is not possible to assert if the magnitude and nature of bias revealed by rank orders of maximal responses is the same as the one measured in HEK cells.

Delta-opioid (DOP) receptors are members of the G protein-coupled receptor (GPCR) sub-family of opioid receptors, and are evolutionarily related, with homology exceeding 70%, to cognate mu-opioid (MOP), kappa-opioid (KOP), and nociceptin opioid (NOP) receptors. DOP receptors are considered attractive drug targets for pain management because agonists at these receptors are reported to exhibit strong antinociceptive activity with relatively few side effects. Among the most potent analgesics targeting the DOP receptor are the linear and cyclic enkephalin analogs known as DADLE (Tyr-D-Ala-Gly-Phe-D-Leu) and DPDPE (Tyr-D-Pen-Gly-Phe-D-Pen), respectively. Several computational and experimental studies have been carried out over the years to characterize the conformational profile of these penta-peptides with the ultimate goal of designing potent peptidomimetic agonists for the DOP receptor. The computational studies published to date, however, have investigated only a limited range of timescales and used over-simplified representations of the solvent environment. We provide here a thorough exploration of the conformational space of DADLE and DPDPE in an explicit solvent, using microsecond-scale molecular dynamics and bias-exchange metadynamics simulations. Free-energy profiles derived from these simulations point to a small number of DADLE and DPDPE conformational minima in solution, which are separated by relatively small energy barriers. Candidate bioactive forms of these peptides are selected from identified common spatial arrangements of key pharmacophoric points within all sampled conformations., (Copyright © 2013 Wiley Periodicals, Inc.)

In some regions of the developing rat brain such as the nucleus accumbens (Acb), mu opioid (MOP) receptor specific binding in the perinatal period exceeds that in the adult. To investigate the significance of these developmental changes, MOP and nociceptin/orphanin FQ (NOP) receptor binding and G protein coupling as determined by GTPgammaS binding experiments were examined in mesolimbic regions of postnatal day 2 (P2) pups and compared to those of their dams. Acb of the P2 pup exhibited 2-fold greater MOP receptor specific binding than that of the dam. In the ventral tegmental area (VTA), NOP specific binding was about 2-fold higher in the P2 pup. A correlation was found between MOP and NOP binding and their coupling to G protein on dam and P2 pup brain sections. However, the magnitude of increases in MOP and NOP receptor G protein coupling to G protein in P2 pups exceeded the 2-fold differences in binding between pups and dams. Furthermore, the amplitude of the MOP receptor G protein coupling in female P2 Acb was greater than increases in male P2 pup Acb. Differences in MOP and NOP binding and G protein coupling in other mesolimbic regions between P2 pups and dams were rarely observed. The data indicate that greater binding and G protein coupling of MOP and NOP receptors occur in discrete, mesolimbic regions of P2 pups when compared to their dams. It may be of significance that these brain regions, Acb and VTA, are undergoing maturation on P2.

Study Objectives: To trace the entire oxyhemoglobin dissociation curve (ODC) in a cohort of cirrhotic patients in stable condition who were candidates for orthotopic liver transplantation (OLT)., Design: Prospective cohort study., Setting: A large academic hospital., Patients and Methods: We traced the entire ODC in whole blood in standard conditions (pH 7.4; PCO2, 40 mm Hg; temperature, 37 degrees C) for 50 cirrhotic candidates for OLT (27 men and 23 women) and 50 age- and height-matched healthy subjects (27 men and 23 women). All subjects were nonsmokers or ex-smokers for at least 5 years. We also measured 2,3 diphosphoglycerate (2,3 DPG) in RBCs, plasma ions, and arterial blood gases in all subjects according to standard methods. Mixed venous blood was also obtained from the 50 cirrhotic patients., Results: Mean ODC was the same in the two groups. However, for the cirrhotic patients, the dispersion of the PO2 values of oxygen saturation percentage (SO2%) from 20 to 80% was significantly larger (p < 0.01 to p < 0.0001). In the cirrhotic patients, the mean PO2 for half-saturation of hemoglobin (P50) was 7.11 + 0.14 mEq/L chloride (p < 0.001) plus 0.36 mEq/L inorganic phosphate (p < 0.05) plus 0.25 micromol/gram of hemoglobin (gHb) 2,3 DPG (p < 0.00002) in absolute numerical values. Sodium, potassium, and calcium, three plasma ions disturbed in cirrhotic patients, did not contribute to determine the mean P50., Discussion: In patients with cirrhosis, increased dispersion of PO2 values for a given level of SO2% may be related to four factors: (1) an observed alteration of the enzymes controlling the phosphoglycerate shunt; (2) hypothyroidism, which may affect 7 to 20% of patients with primary biliary cirrhosis; (3) the type of ongoing treatment, eg, diuretics and/or propranolol; and (4) plasma ion disturbances., Conclusions: We describe the ODC by three indexes: shape, position, and an index of dispersion of the PO2 values for a given level of SO2%. In addition, when the latter is increased, we suggest that other factors than pH, temperature, carbon dioxide, and inorganic phosphates are acting on the position of the ODC.

The p38 mitogen-activated protein kinase (MAPK) cascade transduces multiple extracellular signals from cell surface to nucleus and is employed in cellular responses to cellular stresses and apoptotic regulation. The involvement of the p38 MAPK cascade in opioid- and opioid receptor-like receptor-1 (ORL1) receptor-mediated signal transduction was examined in NG108-15 neuroblastoma x glioma hybrid cells. Stimulation of endogenous delta-opioid receptor (DOR) or ORL1 resulted in activation of p38 MAPK. It also induced the activation of extracellular signal-regulated kinases (ERKs), another member of the MAPK family, with slower kinetics. Activation of p38 MAPK was abolished by selective antagonists of DOR or ORL1, pretreatment with pertussis toxin, or SB203580, a specific inhibitor of p38 MAPK. Inhibition of p38 MAPK had no significant effect on opioid-induced ERK activation, indicating that p38 MAPK activity was not required for ERK activation, though its stimulation preceded ERK activation. Inhibition of protein kinase A (PKA) strongly diminished p38 activation mediated by DOR or ORL1 but had no significant effect on ERK activation, and protein kinase C (PKC) inhibitors potentiated stimulation of p38 while inhibiting activation of ERKs. Taken together, our results provide the first evidence for coupling of DOR and ORL1 to the p38 MAPK cascade and clearly demonstrate that receptor-mediated activation of p38 MAPK both involves PKA and is negatively regulated by PKC.

Endogenous enkephalins and delta opiates affect sensory function and pain sensation by inhibiting synaptic transmission in sensory circuits via delta opioid receptors (DORs). DORs have long been suspected of mediating these effects by modulating voltage-dependent Ca(2+) entry in primary sensory neurons. However, not only has this hypothesis never been validated in these cells, but in fact several previous studies have only turned up negative results. By using whole-cell current recordings, we show that the delta enkephalin analog [D-Ala(2), D-Leu(5)]-enkephalin (DADLE) inhibits, via DORs, L-, N-, P-, and Q-high voltage-activated Ca(2+) channel currents in cultured rat dorsal root ganglion (DRG) neurons. The percentage of responding cells was remarkably high (75%) within a novel subpopulation of substance P-containing neurons compared with the other cells (18-35%). DADLE (1 microM) inhibited 32% of the total barium current through calcium channels (I(Ba)). A delta (naltrindole, 1 microM), but not a mu (beta-funaltrexamine, 5 microM), antagonist prevented the DADLE response, whereas a DOR-2 subtype (deltorphin-II, 100 nM), but not a DOR-1 (DPDPE, 1 microM), agonist mimicked the response. L-, N-, P-, and Q-type currents contributed, on average, 18, 48, 14, and 16% to the total I(Ba) and 19, 50, 26, and 20% to the DADLE-sensitive current, respectively. The drug-insensitive R-type current component was not affected by the agonist. This work represents the first demonstration that DORs modulate Ca(2+) entry in sensory neurons and suggests that delta opioids could affect diverse Ca(2+)-dependent processes linked to Ca(2+) influx through different high-voltage-activated channel types.

In addition to cytosolic efflux, reversal of excitatory amino acid (EAA) transporters evokes glutamate exocytosis from the striatum in vivo. Both kappa-opioid and muscarinic receptor agonists suppress this calcium-dependent response. These data led to the hypothesis that the calcium-independent efflux of striatal glutamate evoked by transporter reversal may activate a transsynaptic feedback loop that promotes glutamate exocytosis from thalamo- and/or corticostriatal terminals in vivo and that this activation is inhibited by presynaptic kappa and muscarinic receptors. Corollaries to this hypothesis are the predictions that agonists for these putative presynaptic receptors will selectively inhibit the calcium-dependent component of glutamate released from striatal synaptosomes, whereas the calcium-independent efflux evoked by an EAA transporter blocker, L-trans-pyrrolidine-2,4-dicarboxylic acid (L-trans-PDC), will be insensitive to such receptor ligands. Here we report that a muscarinic agonist, oxotremorine (0.01-10 microM), and a kappa-opioid agonist, U-69593 (0.1-100 microM), suppressed the calcium-dependent release of glutamate that was evoked by exposing striatal synaptosomes to the potassium channel blocker 4-aminopyridine. The presynaptic inhibition produced by these ligands was concentration dependent, blocked by appropriate receptor antagonists, and not mimicked by the delta-opioid agonist [D-Pen2,5]-enkephalin. The finding that glutamate efflux evoked by L-trans-PDC from isolated striatal nerve endings was entirely calcium independent supports the notion that intact basal ganglia circuitry mediates the calcium-dependent effects of this agent on glutamate efflux in vivo. Furthermore, because muscarinic or kappa-opioid receptor activation inhibits calcium-dependent striatal glutamate release in vitro as it does in vivo, it is likely that both muscarinic and kappa receptors are inhibitory presynaptic heteroceptors expressed by striatal glutamatergic terminals.

We used the Xenopus oocyte expression system to examine the regulation of rat kappa opioid receptor (rKOR) function by G protein receptor kinases (GRKs). kappa agonists increased the conductance of G protein-activated inwardly rectifying potassium channels in oocytes co-expressing KOR with Kir3.1 and Kir3.4. In the absence of added GRK and beta-arrestin 2, desensitization of the kappa agonist-induced potassium current was modest. Co-expression of either GRK3 or GRK5 along with beta-arrestin 2 significantly increased the rate of desensitization, whereas addition of either beta-arrestin 2, GRK3, or GRK5 alone had no effect on the KOR desensitization rate. The desensitization was homologous as co-expressed delta opioid receptor-evoked responses were not affected by KOR desensitization. The rate of GRK3/beta-arrestin 2-dependent desensitization was reduced by truncation of the C-terminal 26 amino acids, KOR(Q355Delta). In contrast, substitution of Ala for Ser within the third intracellular loop [KOR(S255A,S260A, S262A)] did not reduce the desensitization rate. Within the C-terminal region, KOR(S369A) substitution significantly attenuated desensitization, whereas the KOR(T363A) and KOR(S356A,T357A) point mutations did not. These results suggest that co-expression of GRK3 or GRK5 and beta-arrestin 2 produced homologous, agonist-induced desensitization of the kappa opioid receptor by a mechanism requiring the phosphorylation of the serine 369 of rKOR.

Differences in the specificity of coupling of delta-opioid receptor with G-protein have been reported in the literature. We have observed a differential desensitization of delta-opioid receptors, endogenously expressed in the neuroblastoma cell line SK-N-BE, induced by peptide and alkaloid agonists. By combining photoaffinity labelling of receptor-activated G-proteins with [alpha-(32)P]azidoanilide-GTP and an anti-sense oligodeoxynucleotide strategy, we examined whether the chemical nature of opioid agonists, alkaloid or peptide, has a critical role in determining a G(i)alpha/G(o)alpha-protein-selective activation by the human delta-opioid receptors. Etorphine, a non-selective alkaloid agonist, was shown to stimulate the incorporation of [alpha-(32)P]azidoanilide-GTP into G(i)alpha1, G(i)alpha2, G(i)alpha3 and pertussis-toxin-insensitive Galpha subunits. In contrast, [d-Pen(2),d-Pen(5)]enkephalin (DPDPE; Pen is penicillamine) and Tyr-d-Ala-Phe-Asp-Val-Val-Gly-NH(2) (deltorphin I), selective peptide agonists, mainly activated G(i)alpha2 and G(o)alpha2 subunits. The 'knock-down' of G(o)alpha2 subunits by anti-sense oligodeoxynucleotides selectively decreased the inhibition of adenylate cyclase induced by DPDPE and deltorphin I, whereas anti-sense oligodeoxynucleotides directed against G(i)alpha2 subunits only decreased the potency of etorphine in inhibiting cAMP accumulation. These results suggest that the nature of the agonist, peptide or alkaloid is critical in determining the interaction between human delta-opioid receptors and Galpha subunits.

1. The actions of opioid receptor agonists on synaptic transmission in substantia gelatinosa (SG) neurones in adult (6- to 10-week-old) rat spinal cord slices were examined by use of the blind whole-cell patch-clamp technique. 2. Both the mu-receptor agonist DAMGO (1 microM) and the delta-receptor agonist DPDPE (1 microM) reduced the amplitude of glutamatergic excitatory postsynaptic currents (EPSCs) which were monosynaptically evoked by stimulating Adelta afferent fibres. Both also decreased the frequency of miniature EPSCs without affecting their amplitude. 3. In contrast, the kappa-receptor agonist U-69593 (1 microM) had little effect on the evoked and miniature EPSCs. 4. The effects of DAMGO and DPDPE were not seen in the presence of the mu-receptor antagonist CTAP (1 microM) and the delta-receptor antagonist naltrindole (1 microM), respectively. 5. Neither DAMGO nor DPDPE at 1 microM affected the responses of SG neurones to bath-applied AMPA (10 microM). 6. Evoked and miniature inhibitory postsynaptic currents (IPSCs), mediated by either the GABAA or the glycine receptor, were unaffected by the mu-, delta- and kappa-receptor agonists. Similar results were also obtained in SG neurones in young adult (3- to 4-week-old) rat spinal cord slices. 7. These results indicate that opioids suppress excitatory but not inhibitory synaptic transmission, possibly through the activation of mu- and delta- but not kappa-receptors in adult rat spinal cord SG neurones; these actions are presynaptic in origin. Such an action of opioids may be a possible mechanism for the antinociception produced by their intrathecal administration.

Monoclonal antibodies generated against multiple antigenic peptides of the N-terminal sequence (3LVPSARAELQSSPLV17) of the cloned delta opioid receptor immunoprecipitated a 58 kDa protein from CHAPS-solubilized NG108-15 membranes. The immunoprecipitates bound [3H]DPDPE--but not [3H]DAMGO--with a Kd of 6.4 nM and a Bmax of 75 pM. Western blot analysis revealed a distinct band of 58 kDa. The antibodies inhibited basal and PGE1-stimulated cAMP levels, and mimicked the effect of agonists manifest in a compensatory increase in cAMP formation. The antibody will be potentially useful in the analysis of functional epitopes on the delta opioid receptor.

The aim of this study was to investigate the analgesic effects of epidural opioids upon persistent pain sensitivity in neonatal rat pups. Two models of persistent pain were used, subcutaneous injection of carrageenan, and topical application of capsaicin cream, both to the hind paw. The contribution of individual opioid receptor subtypes in the spinal cord to analgesia were tested at different developmental stages using epidural mu (morphine sulphate), delta (DPDPE) and kappa (U69593) opioid receptor agonists in neonatal rats aged P (postnatal day) 3, 10 and 21. Rat pups at all three ages displayed a reduction in mechanical (von Frey hair) threshold following carrageenan-induced inflammation of the hind paw that was evident at 3 h and was still present 5 h after application. This effect was greatest in magnitude at P21. This response was blocked by low doses of all three agonists at all ages, relative effectiveness varying with age. Comparison with potencies in acute tests (Marsh, D., Dickenson, A., Hatch, D. and Fitzgerald, M., Epidural opioid analgesia in infant rats I: mechanical and heat responses, Pain 82 (1999) 23-32) show that opioid potency is significantly greater in the presence of carrageenan inflammation at all ages. Topical capsaicin application to the hind paw produced a significant fall in withdrawal latencies to noxious heat. Generally, epidural opioid agonists did not block this C-fibre induced sensitization except at P3, when morphine and DPDPE did prevent the fall in threshold in a dose dependent manner. The results show that newborn rat pups are capable of displaying both allodynia and hyperalgesia following experimental inflammation that is blocked by epidural mu, delta and kappa opioids. The opioid potency is enhanced compared with antinociception in acute tests. This is not observed following capsaicin hyperalgesia and is therefore not a general consequence of C fibre induced increases in central excitability but relies upon mechanisms special to inflammatory pain.

Previous studies have shown that stressors modify endogenous opioid systems. However, the consequences of social stress on the function of endogenous opioid systems is not well documented. The present studies investigated the effect of rank and housing condition on response to SNC-80, a delta receptor agonist. Triad-housed rats were assessed for dominance status by their behavior and alteration in body weights. At 3 and 50 days, triad- and individually housed rats were injected with SNC-80 (35 mg/kg i.p.) or saline, and evaluated using a test battery consisting of open field behaviors, rectal temperature, analgesia, and air-puff-induced ultrasonic vocalizations. After 50 days of housing, plasma corticosterone, adrenal catecholamines, and the density of cyclic[D-penicillamine2-D-penicillamine2]enkephalin-stimu lat ed guanylyl 5'-[gamma[35S]thio]-triphosphate binding in the prefrontal cortex, the amygdala, nucleus accumbens, thalamus, arcuate, and median eminence were also determined. The first 24 h of triad housing resulted in loss of body weight in subdominant (betas and gammas) but not dominant alpha rats. SCN-80-induced hypothermia was smaller, and there was no depression of headpoke and locomotor behavior in the periphery and the center of the field of alpha rats, in contrast to subdominant and singly housed rats. Rank status did not influence SNC-80's analgesic effect or its inhibition of air-puff-induced ultrasonic vocalizations. Plasma corticosterone levels of alphas and gammas were lower compared with betas and singly housed rats. Agonist stimulation of delta receptor guanylyl 5'-[gamma[35S]thio]-triphosphate binding was lateralized in prefrontal cortex and amygdala, but not nucleus accumbens. Binding was highest in all brain areas of singly housed rats and lowest in the thalamus of beta and of gamma rats. Lateralized binding in amygdala, high locomotor activity, and sensory sensitivity correlated positively with greater sensitivity to SNC-80-induced depression in these measures. Higher binding in the right amygdala correlated with higher plasma corticosterone levels. These findings indicate that dominant rats displayed stimulant rather than depressant responses to delta-opioid activation. Therefore in rodents rank-related stress can alter responsiveness of the endogenous opioid system, and dominance can increase the excitatory effects of delta agonists.

The aim of this study was to investigate the analgesic effects of epidural opioids in neonatal rat pups. The contribution of individual opioid receptor subtypes in the spinal cord to analgesia at different developmental stages was investigated using epidural mu (morphine sulphate), delta (DPDPE) and kappa (U69593) opioid receptor agonists in neonatal rats aged postnatal day (P) 3, 10 and 21. Thresholds for flexion withdrawal reflexes to mechanical stimuli (von Frey hairs) and to noxious heating of the hind paw were low in neonates and increased with postnatal age. The analgesic action of each opioid receptor agonist followed an individual developmental pattern. In mechanical tests, all three opioid agonists were considerably more efficacious analgesics in younger animals and ED50s at P3 were always lower than at P21. In heat tests, the pattern differed. The efficacy of the kappa opioid agonist decreased with postnatal age, morphine efficacy increased over the same period and the effects of the delta agonist remained relatively unchanged. The distribution and concentration of tritiated morphine in the spinal cord following epidural administration did not alter significantly with postnatal age, suggesting that opioid access is not a major determinant of the effects reported here. It is concluded that whereas heat pain is particularly sensitive to spinal kappa opioids in neonates, mechanical sensory thresholds are generally sensitive to all spinal opioids in the newborn. The differing epidural opioid requirements compared to older subjects is likely to be due to developmental changes in spinal cord opioid receptor distribution or pharmacology.

The potential modulation of opioid receptor signaling by kainic acid (KA) has been investigated in neuroblastoma x glioma NG 108-15 hybrid cells and neuroblastoma SK-N-SH cells. Acute incubation of KA significantly attenuated delta opioid receptor (DOR) signaling induced by the DOR agonist [D-Pen2, D-Pen5]-enkephalin (DPDPE), as measured by activation of G proteins and inhibition of cAMP accumulation. The attenuation by KA was time- and dose-dependent and could be blocked by antagonists of kainate/AMPA receptors, suggesting possible mediation through kainate/AMPA receptors. KA attenuation of DPDPE-stimulated G protein activation was reversed by inhibitors of protein kinase C or by removal of both extracellular Ca2+ and intracellular Ca2+. In contrast, NMDA attenuation of DPDPE-stimulated G protein activation was independent of intracellular Ca2+, indicating that different mechanism(s) may underlie the modulation effect of KA and NMDA. This notion was further supported by the results that KA did not alter nociceptin/orphanin FQ-stimulated G protein activation in NG 108-15 cells but NMDA did. In addition, pretreatment of NG 108-15 cells with antagonists of kainate/AMPA receptors blocked the acute desensitization of DOR signaling. These data provide evidence that KA may be involved in the modulation of opioid receptor signal transduction.

The role of enkephalin and the opioid receptors in modulating GABA release within the rat globus pallidus (GP) was investigated using whole-cell patch recordings made from visually identified neurons. Two major GP neuronal subtypes were classified on the basis of intrinsic membrane properties, action potential characteristics, the presence of the anomalous inward rectifier (Ih), and anode break depolarizations. The mu opioid receptor agonist [D-Ala2-N-Me-Phe4-Glycol5]-enkephalin (DAMGO) (1 microM) reduced GABAA receptor-mediated IPSCs evoked by stimulation within the striatum. DAMGO also increased paired-pulse facilitation, indicative of presynaptic mu opioid receptor modulation of striatopallidal input. In contrast, the delta opioid agonist D-Pen-[D-Pen2, 5]-enkephalin (DPDPE) (1 microM) was without effect. IPSCs evoked by stimulation within the GP were depressed by application of [methionine 5']-enkephalin (met-enkephalin) (30 microM). Met-enkephalin also reduced the frequency, but not the amplitude, of miniature IPSCs (mIPSCs) and increased paired-pulse facilitation of evoked IPSCs, indicative of a presynaptic action. Both DAMGO and DPDPE reduced evoked IPSCs and the frequency, but not amplitude, of mIPSCs. However, spontaneous action potential-driven IPSCs were reduced in frequency by met-enkephalin and DAMGO, whereas DPDPE was without effect. Overall, these results indicate that presynaptic mu opioid receptors are located on striatopallidal terminals and pallidopallidal terminals of spontaneously firing GP neurons, whereas presynaptic delta opioid receptors are preferentially located on terminals of quiescent GP cells. Enkephalin, acting at both of these receptor subtypes, serves to reduce GABA release in the GP and may therefore act as an adaptive mechanism, maintaining the inhibitory function of the GP in basal ganglia circuitry.

In the striatum, DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of 32 kDa) is highly expressed by virtually all projection medium-sized spiny neurons. cAMP-dependent phosphorylation of DARPP-32 is stimulated via activation of dopamine D1 receptors in striatonigral neurons, and via activation of adenosine A2A receptors in striatopallidal neurons. In this study, we have examined the contribution of mu-, delta- and kappa-opioid receptors to the regulation of DARPP-32 phosphorylation, in rat striatal slices. The results show that, at low concentrations (100 pm-1 nm), the mu-opioid agonist, Tyr-D-Ala-Gly-N-Me-Phe-glycinol (DAMGO), inhibits the increase in DARPP-32 phosphorylation induced by activation of D1, but not by activation of A2A receptors. Conversely, the delta-receptor agonist, Tyr-D-Pen-Gly-Phe-D-Pen (DPDPE), inhibits DARPP-32 phosphorylation induced by activation of A2A, but not by activation of D1 receptors. The kappa-receptor agonist, U50488, does not affect DARPP-32 phosphorylation induced by either D1 or A2A agonists. Thus, mu-opioid receptors interact with dopamine D1 receptors on striatonigral neurons, whereas delta-opioid receptors interact with adenosine A2A receptors on striatopallidal neurons. These results suggest that regulation of DARPP-32 phosphorylation is involved in mediating some of the effects exerted by enkephalin on striatal medium-sized spiny neurons.

The effect of opioid peptides on cultured, oestradiol-stimulated human myometrial cells was examined. Oestradiol increased cell densities in mixed-cell (smooth muscle cells + stromal fibroblasts) cultures by 40%. This oestradiol-induced stimulation of cell proliferation was decreased to control values by D-met2-pro5-enkephalinamide. The half-effective inhibitory concentration of enkephalinamide was 0.3 nmol/l. The opioid-induced inhibition of cell proliferation was blocked completely by the specific opiate receptor antagonist naloxone, while naloxone did not have any effect on its own. This opioid effect was mediated dominantly by the mu opiate receptor. The optimal concentration for oestradiol to stimulate uterine cell proliferation was 2.2 nM. The basal rate of cell proliferation was not affected by enkephalinamide. In saturation experiments, the parameters of specific [3H]-naloxone binding were: dissociation constant = 1.02 nM, maximal binding capacity = 2910 binding sites/cell, Hill coefficient = 1.029. In human myometrial pure smooth muscle cell cultures, oestradiol decreased the proliferation of cells. Progesterone potentiated these oestradiol effects, but had no effect on its own. Enkephalinamide was also able to block the effects of oestradiol, but naloxone did not antagonize it. In summary, here we present a novel inhibitory role of endogenous opioid peptides in the regulation of cell growth and proliferation in the human uterus.

Previous work supports the existence of two types of delta opioid receptor (delta1 and delta2) and a role of both subtypes in the spinal cord and the ventromedial medulla (VMM) in the production of antinociception. Although it is well established that spinal and supraspinal mu opioid receptors interact in a synergistic manner to produce antinociception, little is known about the interaction of delta opioid receptors. This study used isobolographic analysis to determine how delta1 and delta2 opioid receptors in the VMM interact with their respective receptors in the spinal cord to produce antinociception. Concurrent administration of the delta1 opioid receptor agonist [D-Pen2,D-Pen5]enkephalin at spinal and supraspinal sites in a fixed-dose ratio produced antinociception in an additive manner in the tail-flick test. In contrast, concurrent administration of very low doses of the delta2 opioid receptor agonist [D-Ala2,Glu4]deltorphin at spinal and medullary sites produced antinociception in a synergistic manner. However, as the total dose of [D-Ala2,Glu4]deltorphin increased, this interaction converted to additivity. These observations suggest that different mechanisms mediate the antinociceptive effects of different doses of delta2 opioid receptor agonists. The difference in the nature of the interaction produced by delta1 and delta2 opioid receptor agonists provides additional evidence for the existence of different subtypes of the delta opioid receptor. These results also suggest that delta2 opioid receptor agonists capable of crossing the blood-brain barrier will be more potent or efficacious analgesics than delta1 opioid receptor agonists after systemic administration.

The voltage-dependent inhibition of N-type Ca2+ channel current by the delta-opioid agonist [D-pen2, D-pen5]-enkephalin (DPDPE) was investigated in the mammalian cell line NG108-15 with 10 microM nifedipine to block L-type channels, with whole-cell voltage clamp methods. In in vitro differentiated NG108-15 cells DPDPE reversibly decreased omega-conotoxin GVIA-sensitive Ba2+ currents in a concentration-dependent way. Inhibition was maximal with 1 microM DPDPE (66% at 0 mV) and was characterized by a slowing of Ba2+ current activation at low test potentials. Both inhibition and kinetic slowing were attenuated at more positive potentials and could be relieved up to 90% by strong conditioning depolarizations. The kinetics of removal of inhibition (de-inhibition) and of its retrieval (re-inhibition) were also voltage dependent. Both de-inhibition and re-inhibition were single exponentials and, in the voltage range from -20 to +10 mV, had significantly different time constants at a given membrane potential, the time course of re-inhibition being faster than that of de-inhibition. The kinetics of de-inhibition at -20 mV and of reinhibition at -40 mV were also concentration dependent, both processes becoming slower at lower agonist concentrations. The rate of de-inhibition at +80/+120 mV was similar to that of Ca2+ channel activation at the same potentials measured during application of DPDPE (approximately 7 ms), both processes being much slower than channel activation in controls (<1 ms). Moreover, the amplitude but not the time course of tail currents changed as the depolarization to +80/+120 mV was made longer. The state-dependent properties of DPDPE Ca2+ channel inhibition could be simulated by a model that assumes that inhibition by DPDPE results from voltage- and concentration-dependent binding of an inhibitory molecule to the N-type channel.

Increased cerebrovascular permeability is an important factor in the development of cerebral edema after stroke, implicating the blood-brain barrier (BBB) in the pathology of stroke. Present investigations modeled stroke at the level of the cerebral capillary endothelium by analyzing BBB permeability changes to the membrane-impermeant marker [14C]sucrose after hypoxia/aglycemia. Under hypoxia alone, long exposures (48 h) were necessary to result in a significant increase in permeability of bovine brain microvessel endothelial cells to [14C]sucrose. Hypoxia/aglycemia exposures resulted in a much shorter time (i.e., 1-3 h) required for a corresponding increase in permeability to [14C]sucrose. Statistically significant changes in basal permeability were observed between 3 and 6 h of hypoxia/aglycemia; however, 6 h of aglycemia alone had no significant effect on BBB permeability. Both rat astroglioma (C6) cells and C6 conditioned medium showed no improvements in barrier function measured by transendothelial cell resistance or permeability to [14C]sucrose. Changes in endothelial cell calcium flux may be responsible for the permeability change observed after both 48 h of hypoxia and 6 h of hypoxia/aglycemia because nifedipine (10 and 100 nM) and SKF 96365 (100 nM) decreased the permeability change. Immunocytochemical studies also revealed a change in the distribution of endothelial cell F-actin. This study provides evidence that the BBB is sensitive to short exposures of hypoxia/aglycemia and that changes in endothelial cell calcium flux may be responsible for structural and functional variations in the BBB during ischemic stress.

1. We have studied the effects of mu and delta opioids on intestinal function (permeability, PER; gastrointestinal transit, GIT), and their antagonism after the intracerebroventricular (i.c.v.) administration of specific antibodies (ABs) or antisense oligodeoxynucleotides (ODN) to mu-receptors (OR). Central versus peripheral site/s of action of subcutaneous (s.c.) mu-opioids, were also assessed. 2. Male Swiss CD-1 mice were used. GIT was measured with charcoal and PER by the passage of 51Cr-EDTA from blood to lumen. 3. Morphine and fentanyl (i.c.v. and s.c.) inhibited GIT and PER in a dose-related manner; they were more potent by i.c.v. route, both on GIT and PER (70 and 17 times for morphine and fentanyl). They also had a greater effect on GIT than PER (4.3 and 1.6 times). DPDPE had a lower potency than mu-agonists in all experiments, and no dose-response could be obtained after s.c. administration on GIT. 4. Pretreatment with i.c.v. ABs (24 h) or antisense ODN (5 days), decreased the effects (GIT and PER) of i.c.v. morphine and fentanyl, while those of DPDPE remained unchanged. The ABs did not alter the peripheral effects of mu-opioids. 5. The results show that (i.c.v. or s.c.) mu opioids produce dose-related inhibitions of PER and GIT, being more potent by the i.c.v. route. Delta-opioids had a greater effect on PER than GIT, while the opposite occurred for mu-agonists. Pretreatment with ABs or ODN to mu-OR, blocked the central effects of mu (but not delta) agonists on GIT and PER.

The efficacy of different opioid agonists to induce acute desensitization of the human delta-opioid receptor-mediated inhibition of cAMP accumulation was investigated in the neuroblastoma cell line SK-N-BE, which endogenously expresses these receptors. While etorphine, a non-selective alkaloid agonist, caused 50% desensitization after a 30-min incubation, the same treatment in the presence of the selective peptide agonists, DPDPE ([D-Pen2,D-Pen5]enkephalin) and deltorphin I (Tyr-D-Ala-Phe-Asp-Val-Val-Gly), almost totally desensitized the delta-opioid receptor-mediated inhibition of adenylyl cyclase. When SK-N-BE cells were prechallenged either with alkaloid or peptide agonist, we observed a cross-desensitization that was less marked when cells were pretreated with peptide agonists and then challenged with etorphine. Taken together, these results demonstrate that human delta-opioid receptors are differentially desensitized by alkaloid and peptide agonists.

Chronic activation of the mu-opioid receptor (MOR1TAG) results in the loss of agonist response that has been attributed to desensitization and down-regulation of the receptor. It has been suggested that opioid receptor phosphorylation is the mechanism by which this desensitization and down-regulation occurs. When MOR1TAG was stably expressed in both neuroblastoma neuro2A and human embryonic kidney HEK293 cells, the opioid agonist [D-Ala2,MePhe4, Gly5-ol]enkephalin (DAMGO) induced a time- and concentration-dependent phosphorylation of the receptor, in both cell lines, that could be reversed by the antagonist naloxone. Protein kinase C can phosphorylate the receptor, but is not involved in DAMGO-induced MOR1TAG phosphorylation. The rapid rate of receptor phosphorylation, occurring within minutes, did not correlate with the rate of the loss of agonist-mediated inhibition of adenylyl cyclase, which occurs in hours. This lack of correlation between receptor phosphorylation and the loss of response was further demonstrated when receptor phosphorylation was increased by either calyculin A or overexpression of the G-protein receptor kinases. Calyculin A increased the magnitude of MOR1TAG phosphorylation without altering the DAMGO-induced loss of the adenylyl cyclase response. Similarly, when mu- and delta-opioid (DOR1TAG) receptors were expressed in the same system, overexpression of beta-adrenergic receptor kinase 2 elevated agonist-induced phosphorylation for both receptors. However, in the same cell lines under the same conditions, overexpression of beta-adrenergic receptor kinase 2 and beta-arrestin 2 accelerated the rate of DPDPE- but not DAMGO-induced receptor desensitization. Thus, these data show that phosphorylation of MOR1TAG is not an obligatory event for the DAMGO-induced loss in the adenylyl cyclase regulation by the receptor.

The delta opioid receptor antagonist [D-penicillamine2,5]enkephalin (DPDPE) is an enzymatically stable peptide analogue of Met-enkephalin. DPDPE uses a saturable transport mechanism to cross the blood-brain barrier (BBB), though the exact mechanism is not fully understood. The aim of the present study was to identify the mechanism by which DPDPE enters the brain. The effect of phenylarsine oxide (PAO), an endocytosis inhibitor, on the transport of [3H]DPDPE was investigated using both in vitro and in situ transport studies. Two in vitro models of the BBB utilizing primary bovine brain microvascular endothelial cells (BBMEC) were studied. [3H]DPDPE permeability across monolayers of BBMEC grown on polycarbonate filters was studied. PAO significantly reduced the permeability of [3H]DPDPE across the monolayer. PAO also reduced the uptake of [3H]DPDPE into BBMEC cells, without affecting binding to the cells. The in situ perfusion model of the BBB was also studied, PAO reduced DPDPE uptake by the brain in a dose-dependent manner. These studies indicate that DPDPE enters the brain via an energy-dependent transcytotic mechanism.

In addition to effects in the periphery through inhibition of prostaglandin synthesis, several lines of evidence suggest that nonsteroidal anti-inflammatory drugs (NSAIDs) act in the central nervous system. The possibility that the central action of NSAIDs involves regulation of opioid receptors was investigated by quantitative autoradiography of mu, delta, and kappa sites in rat brain slices. Increased (p < 0.05) labeling of mu receptors was observed in thalamic nuclei, gyrus dentate, and layers of the parietal cortex of rats treated for 10 days with lysine clonixinate. Labeling of delta receptors was lower in the lateral septum, and kappa sites decreased in thalamic nuclei. These effects were not mediated through direct interaction with opioid-binding sites, since receptor-binding assays using rat brain membranes confirmed that clonixinate up to 1 x 10(-4) mol/l does not inhibit mu, delta, and kappa receptor specific binding. Central effects of NSAIDs might, therefore, involve interaction with the opioid receptor system through indirect mechanisms.

Phosducin (Phd) regulates the function of G proteins by its ability to tightly bind Gbetagamma subunits. Because the internalization of opioid receptors as well as the activity of adenylyl cyclase (AC) activity depends on G proteins, we tested Phd on these parameters. NG 108-15 hybrid cells stably expressing the phosphoprotein were challenged with [D-penicillamine2,D-penicillamine5]enkephalin to inhibit cAMP generation, demonstrating an increased efficacy of the opioid on AC. Studying the binding of [35S]guanosine-5'-O-(gamma-thio)-triphosphate to membranes from Phd overexpressing cells, we found that [D-penicillamine2, D-penicillamine5 ]enkephalin failed, in the presence of Phd (0.1 nM), to elevate incorporation of the nucleotide. Phd also strongly inhibited opioid-stimulated GTPase activity. NG 108-15 cells were also employed to investigate the effect of Phd on opioid receptor internalization. Control cells and cells overexpressing Phd were transiently transfected to express mu-opioid receptors fused to green fluorescence protein. In controls and in Phd overexpressing cells confocal microscopy identified fluorescence associated with the membrane. Time-lapse series microscopy of living control cells challenged with etorphine (1 microM) revealed receptor internalization within 30 min. In contrast, Phd overexpressing cells largely failed to respond to the opioid. Thus, in Phd overexpressing cells, opioids exhibit an increased efficacy despite the inhibitory action of the phosphoprotein on opioid-stimulated incorporation of [35S]guanosine-5'-O-(gamma-thio)-triphosphate. We suggest that inhibition of GTPase stabilizes the opioid-induced G protein Gi-GTP complex, which is believed to enhance AC inhibition. Finally, scavenging of Gbetagamma by Phd attenuates internalization of opioid receptors, which may contribute to the efficacy of opioids.

Objective: To investigate the cardiovascular effects of microinjection into the hippocampus of selective mu, delta and kappa opioid receptor agonists in anesthetized spontaneously hypertensive rats, isolation-induced hypertensive rats and their normotensive Wistar-Kyoto and group-housed Sprague-Dawley controls., Methods and Results: The microinjection of a selective kappa agonist, spiradoline mesylate, (+/-)-(5alpha, 7alpha, 8beta)-3,4-dichloro-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro++ +[4.5]dec-8-yl]-benzeneacetamide mesylate) (5 nmol) into the dorsal region of hippocampus, where injection of control saline failed to affect cardiovascular activities, induced centrally mediated decreases in mean blood pressure and heart rate in both hypertensive and normotensive rats. The effects were blocked by prior treatment of the hippocampus with nor-binaltorphimine dihydrochloride, a selective kappa opioid receptor antagonist The hypotensive and bradycardic effects were quantitatively similar between spontaneously hypertensive rats and Wistar-Kyoto rats and between isolated hypertensive rats and normotensive group-housed rats. The sequential administration of increasing doses (5, 10, 50 nmol) of the selective mu agonist [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin and delta agonists [D-Ala2, D-Leu5]-enkephalin or [D-Pen2, D-Pen5]-enkephalin into the same areas of the hippocampus as used for the kappa agonist had no significant effects on mean blood pressure and heart rate in either hypertensive or normotensive rats., Conclusion: The present results extend our previous findings of a hippocampally mediated hypotensive effect of kappa agonists in the spontaneously hypertensive rat to the isolated rat model of hypertension and they establish that mu and delta opioid receptor agonists similarly applied are ineffective. Hippocampal kappa receptors may have a greater role in cardiovascular control than mu and delta receptors.

1. The actions of opioid receptor agonists on the calcium channel currents (IBa) of acutely dissociated periaqueductal grey (PAG) neurons from C57B16/J mice and mutant mice lacking the first exon of the mu-opioid receptor (MOR-1) were examined using whole cell patch clamp techniques. These effects were compared with the GABA(B)-receptor agonist baclofen. 2. The endogenous opioid agonist methionine-enkephalin (met-enkephalin, pEC50 6.8, maximum inhibition 40%), the putative endogenous mu-opioid agonist endomorphin-1 (pEC50 6.2, maximum inhibition 35%) and the mu-opioid selective agonist DAMGO (Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol enkephalin, pEC50 6.9, maximum inhibition 40%) inhibited IBa in 70% of mouse PAG neurons. The inhibition of IBa by each agonist was completely prevented by the mu-receptor antagonist CTAP (D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2). The delta-opioid receptor agonists DPDPE ([D-Pen2,5]enkephalin, 1 microM) and deltorphin II (1 microM), and the kappa-opioid receptor agonist U-69593 (1-10 microM), did not affect IBa in any cell tested. 3. The GABA(B) agonist baclofen inhibited IBa in all neurons (pEC50 5.9, maximum inhibition 42%). 4. In neurons from the MOR-1 deficient mice, the mu-opioid agonists met-enkephalin, DAMGO and endomorphin-1 did not inhibit IBa, whilst baclofen inhibited IBa in a manner indistinguishable from wild type mice. 5. A maximally effective concentration of endomorphin-1 (30 microM) partially (19%), but significantly (P<0.005), occluded the inhibition of IBa normally elicited by a maximally effective concentration of met-enkephalin (10 microM). 6. This study indicates that mu-opioid receptors, but not delta- or kappa-opioid receptors, modulate somatic calcium channel currents in mouse PAG neurons. The putative endogenous mu-agonist, endomorphin-1, was a partial agonist in mouse PAG neurons.

Clinical and experimental observations suggest that opiates can exert different influences on the perception of stimuli from distinct sensory modalities. Thermally-induced nociception is classically responsive to opiate agonists. mu-Opioid receptor-deficient transgenic mice are more sensitive to thermal nociceptive stimuli and morphine fails to attenuate the nociceptive responses to thermal stimuli in these animals. To enhance our understanding of opiate influences on mechanical sensitivity, we have examined withdrawal responses to a sequence of ascending forces of mechanical stimuli in mice with normal (wild type), half-normal (heterozygous) and absent (homozygous) mu-opioid receptor levels. We report data from mice examined without drug pretreatment or following pretreatment with morphine, the selective kappa-opioid agonist, U50488H, and the selective delta-opioid agonist, DPDPE. Saline-pretreated mice of each genotype displayed similar, monotonically increasing frequency of withdrawal responses to the graded stimuli. Subcutaneously administered morphine produced a dose-dependent reduction in withdrawal responses in wild type and heterozygous mice, but had no significant effect in homozygous mice. Intraventricular administration of DPDPE also reduced the frequency of paw withdrawal (FPW) in wild type mice, but not in homozygous mice. In contrast, systemic U50488H produced a dose-dependent attenuation of paw withdrawal in both wild type and homozygous mice. These findings suggest that (1) interactions of endogenous peptides with mu-opioid receptors may not play a significant role in the response to mechanical stimuli in drug-free animals, and (2) deficiency of mu-opioid receptors has no functional consequence on the response to the prototypical kappa-opioid receptor agonist, but decreases responses to the prototypical mu- and delta-opioid receptor agonists., (Copyright 1999 Elsevier Science B.V.)

In the light of functional studies, it has been suggested that antibodies directed to alpha subunits of G-proteins delivered into cerebrospinal fluid (CSF) reached and blocked the function of neural transducer proteins. Current understanding indicates that IgGs do not move freely across plasma membranes. Therefore, to characterize the uptake of these antibodies by neural cells, anti-Gi2alpha IgGs were labeled with 125I, fluorescein or with gold particles. The expression of Galpha subunits was also reduced by blocking their mRNA with antisense oligodeoxynucleotides (ODN). Following intracerebroventricular (icv) injection of gold-conjugated anti-Gi2alpha IgGs, electrondense particles entered and became distributed in the cytoplasm and plasma membranes of neural cells. Scattered particles were also found in dendrites and nuclei. Unlabeled IgGs diminished cerebral signals of fluorescein-labeled anti-Galpha IgGs, indicating that this uptake can be saturated. Cerebral radiostaining promoted by in vivo anti-Gi2alpha 125I-IgGs was almost absent in Gi2alpha knocked-down mice, but not after decreasing the quantity of Gzalpha subunits. The immunosignals of CSF anti-Galpha 125I-IgGs, as well as the impairment of opioid-evoked antinociception, were increased by agonist-induced activation of G protein-coupled receptors. The impairing effect of the antibodies on opioid-evoked antinociception was prevented by agents blocking the cellular uptake of proteins, i.e., cytochalasin B, BSA, DMSO, H7, and by down regulation of protein kinase Cbeta1 (PKCbeta1). In mice treated with an ODN to PKCbeta1 mRNA, 125I-IgGs to Gi2alpha subunits remained bound to periventricular structures and did not spread to deeper areas of the CNS. These results indicate that IgGs delivered into the CSF show a saturable binding to Galpha subunits that translocate to the external side of the neural membrane before being internalized by a PKCbeta1-dependent mechanism., (Copyright 1999 Elsevier Science B.V.)

We report here on the binding affinity and bioassay results of cyclic enkephalin analogs comprising a cyclic moiety and C-terminal fragment of MERGL, where ME denotes methionine enkephalin. MERGL (YGGFMRGL) has been suggested to be cleaved enzymatically by membrane-bound enkephalinase 24.11 to leave ME and the tripeptide RGL. In our study we have synthesized hybrids of DPDPE or DPLCE and the C-terminal tripeptide RGL in order to mimic a prohormone able to cross the blood-brain barrier. The study has shown that of the homologs presented here, analogs of DPLCE often are more potent at delta opioid receptors both in binding affinity and in bioactivity at the MVD, than DPDPE. Our hypothesis that hybrids (consisting of the drug and the spacer for the carrier) could be designed which would either have no opioid activity or, alternatively, be by themselves very active, has been verified.

In this study, we evaluated the gastric effects of methylnaltrexone, an opioid receptor antagonist that does not cross the blood-brain barrier in vivo, on mu, kappa and delta opioid agonists induced brainstem unitary responses in an in vitro neonatal rat brainstem-gastric preparation. Single units in the medial subnucleus of the nucleus tractus solitarius (NTS), responding to electrical stimulation of subdiaphragmatic vagal fibers, were recorded. Selective opioid receptor agonists and antagonists were applied only to the gastric compartment of the bath chamber and thus, the brainstem functions of the preparation were not affected by the drugs. The peripheral gastric effects of a mu opioid receptor agonist, DAMGO, and a kappa opioid receptor agonist, U-50,488H, were evaluated on 58 tonic units that received the subdiaphragmatic vagal inputs. For approximately 78% of the units observed, DAMGO (1.0 microM) and U-50,488H (1.0 microM) induced a concentration-dependent inhibition of 62.1+/-9.3% (mean +/- SE) and 49.2+/-6.5% of the control level of the NTS neuronal activity, respectively. Methylnaltrexone competitively antagonized the DAMGO-induced brainstem neuronal effects. Methylnaltrexone at an 18.8-fold higher concentration also reversed U-50,488H-induced NTS neuronal responses. Naloxone, a non-selective opioid receptor antagonist, reversed the inhibitory effects of DAMGO and U-50,488H at much lower concentrations (3.8% and 0.5%, respectively) compared to methylnaltrexone. Only 18% of the NTS neurons evaluated showed inhibitory responses to a delta receptor agonist, DPDPE, (19.7+/-5.0% at 10 microM), and this inhibition could not be reversed by methylnaltrexone in the concentration range we tested. In addition, when methylnaltrexone (1.0 microM) alone was applied to the gastric compartment, there was an activation (8.5+/-2.1%) of the NTS neurons receiving subdiaphragmatic vagal inputs, suggesting an endogenous gastric opioid action in the modulation of brainstem neuronal activities.

1. The antinociceptive effects of the delta opioid receptor selective agonist, DPDPE [(D-Pen2,D-Pen5)-enkephalin] was studied in rats aged postnatal day (P) 14, P21, P28 and P56. 2. Antinociceptive effects of DPDPE were measured as percentage inhibition of the C-fibre evoked response and post-discharge of dorsal horn neurones evoked by peripheral electrical stimulation. DPDPE was administered by topical application, akin to intrathecal injection. 3. DPDPE (0.1-100 microg) produced dose-related inhibitions at all ages; these inhibitions were reversed by 5 microg of the opioid antagonist naloxone. 4. The dose-response curves for C-fibre evoked response and post-discharge of the neurones were not different in rats aged P14 and P21. DPDPE was significantly more potent at P14 and P21 compared with its inhibitory effects on these responses at P28 and P56. 5. DPDPE produced minor inhibitions of the A-fibre evoked response of the neurones at P14, P21, P28 and P56, suggesting that the inhibitory effects of DPDPE are mediated via presynaptic receptors on the terminals of C-fibre afferents. 6. Since spinal delta opioid receptor density changes little over this period, the increased antinociceptive potency of DPDPE in the rat pups compared with the adult is likely to be due to post-receptor events, or in developmental changes in the actions of other transmitter/receptor systems within the spinal cord.

Previous work indicates that the antianalgesic action of pentobarbital and neurotensin administered intracerebroventricularly in mice arises from activation of a descending system to release cholecystokinin (CCK) in the spinal cord where CCK is known to antagonize morphine analgesia. Spinal dynorphin, like CCK, has an antianalgesic action against intrathecally administered morphine. This dynorphin action is indirect; even though it is initiated in the spinal cord, it requires the involvement of an ascending pathway to the brain and a descending pathway to the spinal cord where an antianalgesic mediator works. The aim of the present investigation was to determine if the antianalgesic action of intrathecal dynorphin A involved spinal CCK. All drugs were administered intrathecally to mice in the tail flick test. Morphine analgesia was inhibited by dynorphin as shown by a rightward shift of the morphine dose-response curve. The effect of dynorphin was eliminated by administration of the CCK receptor antagonists lorglumide and PD135 158. One hour pretreatment with CCK antiserum also eliminated the action of dynorphin. On the other hand, the antianalgesic action of CCK was not affected by dynorphin antiserum. Thus, CCK did not release dynorphin. Both CCK and dynorphin were antianalgesic against DSLET but not DPDPE, delta 2 and delta 1 opioid receptor peptide agonists, respectively. The results suggest that the antianalgesic action of dynorphin occurred through an indirect mechanism ultimately dependent on the action of spinal CCK.

The analgesic effect of three different opioid agonists, DAMGO ([D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin), U-50,488H (trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidynyl)cyclohexyl] benzene-aceramide methane sulphonate), and [D,Pen2-D,Pen5]-enkephalin, which act upon mu, delta and kappa opioid receptors, respectively, was compared in the presence and absence of salmon-calcitonin (s-CT). The analgesic test used was the writhing test in mice. The analgesic effect of the opioids was significantly enhanced by pretreatment of the animals with s-CT intraperitoneally (i.p.) administered. This effect was more evident for the delta and kappa-agonists. The present result suggests that the joint administration of s-CT and opioids may be a useful and interesting alternative in the treatment of painful diseases resistant to other treatments.

Avian vocal control regions of adult male songbirds contain opioid peptides and receptors, suggesting that opioids play a role in avian vocal behavior control. In a previous study, we found no difference in opioid receptor densities in singing versus nonsinging adult male dark-eyed juncos (Junco hyemalis), leading us to hypothesize that opioids are not involved in controlling song production. To assess whether opioids may be involved in other aspects of vocal behavior, we used quantitative in vitro autoradiography to compare mu and delta opioid receptor densities in vocal control regions of singing adult males with those of adult females and adolescent (about 3 months old) males and females. We found mu and delta receptors in all vocal control regions measured. Adolescents had significantly higher opioid receptor densities than did adults in area X (delta), robust n. of the archistriatum (delta and mu), and n. intercollicularis (mu), suggesting a developmental role for opioids in the vocal control system. Based on opioid roles in other animal models, we propose that opioids may be involved in song learning, auditory processing, and/or vocal control system development.

Endogenous opioid peptides and opioid receptors are expressed by brain cells early during normal development, and exogenous opiate exposure in this period is known to affect brain cell proliferation and maturation. Despite the abundant evidence that opioids affect brain development, little is known about the mechanisms involved. In this study cortical astrocytes in primary culture were examined immunohistochemically by using antibodies against the opioid receptors. The immunoreactivity for delta-opioid receptors was strongly upregulated during mitosis with an increase in immunostaining that started in early prophase and lasted through the M-phase to cytokinesis. Similar effects could not be observed when antibodies against the mu- or kappa-opioid receptor subtypes were used. Cultured neurons and microglia presented a strong and homogenous immunostaining for the delta-opioid receptor and no further upregulation of immunoreactivity could be detected in these cells. The presence of functional delta-opioid receptors on the mitotic astrocytes was verified by using microspectrofluorometry for detection of delta-opioid agonist induced changes in intracellular free calcium concentrations ([Ca2+]i). In these experiments fluo-3/AM incubated cells showed a rapidly induced delta-opioid agonist (DPDPE, 10(-6) M) evoked increase in [Ca2+]i. These results suggest an upregulation of the delta-opioid receptors that could represent a mechanism involved in the response to opioids in the developing brain.

Purpose: This study was conducted to examine the influence of P-glycoprotein (P-gp) modulation on the pharmacodynamics of the model opioid peptide DPDPE., Methods: Mice (n = 5-7/group) were pretreated with a single oral dose of the P-gp inhibitor GF120918 (25 or 250 mg/kg) or vehicle. 3H-DPDPE (10 mg/kg) or saline was administered 2.5 hr after pretreatment. Antinociception was determined, and blood and brain tissue were obtained, 10 min after DPDPE administration., Results: A significant difference (p < 0.001) in DPDPE-associated antinociception was observed among mice pretreated with a 25- (83 +/- 16% MPR) or 250- (95 +/- 5% MPR) mg/kg dose of GF120918 in comparison to mice pretreated with vehicle (24 +/- 14% MPR) or mice receiving GF120918 without DPDPE (12 +/- 8% MPR). A significant difference (p < 0.01) in brain tissue DPDPE concentration also was observed among treatment groups [25 +/- 6 ng/g (vehicle), 37 +/- 11 ng/g (25 mg/kg GF120918), 70 +/- 8 ng/g (250 mg/kg GF120918)]. In contrast, blood DPDPE concentrations were not statistically different between groups (678 +/- 66, 677 +/- 130, and 818 +/- 236 ng/ml for vehicle, GF120918 [25 mg/kg], and GF120918 [250 mg/kg], respectively). A single 100-mg/kg i.p. dose of (+)verapamil increased the brain:blood DPDPE concentration ratio by approximately 70% relative to saline-treated control mice (0.139 +/- 0.021 vs. 0.0814 +/- 0.0130, p < 0.01), a change in partitioning similar to that observed with the low dose of GF120918. These data provide further support for a P-gp-based mechanism of brain:blood DPDPE distribution., Conclusions: The present study demonstrates that GF120918 modulates blood-brain disposition and antinociception of DPDPE. Coadministration of a P-gp inhibitor with DPDPE may improve the pharmacologic activity of this opioid peptide.

Cocaine can produce antinociception in a number of animal models. The present experiments were designed to determine if opioid receptor agonists modulate cocaine-induced antinociception in rats. Cocaine produced a dose-dependent increase in antinociception in the hot-plate, but not paw-pressure, test. The combination of cocaine and morphine or [D-Pen2, D-Pen5]enkephalin (DPDPE) produced results no greater than simple additivity in the hot-plate test. However, the combination of cocaine and morphine produced greater antinociception than morphine alone in the paw-pressure test. A low dose of U69,593 potentiated the effects of cocaine in the hot-plate test. In contrast, cocaine attenuated the effect of U69,593 in the paw-pressure test. Both naltrexone and the selective kappa-opioid receptor antagonist nor-binaltorphamine (nor-BNI) blocked the potentiation of cocaine-induced antinociception by U69,593. The combination of U69,593 and cocaine can produce superadditive or subadditive effects, depending upon the doses and antinociceptive assay used.

Opioid receptors (mu, delta and kappa) are known to regulate diverse physiological functions and yet, at the molecular level, they are coupled to a seemingly identical set of G proteins. A recent study has discerned subtle differences between the opioid receptors in their ability to activate the pertussis toxin-insensitive G16. Differences in microarchitecture might be magnified when these receptors are provided with 'non-native' partners. Here, we examined whether the opioid receptors can interact productively with a set of chimeric Galphaq subunits which are known to link many Gi-coupled receptors to phosphoinositide-specific phospholipase C (PI-PLC). The qi5, qo5 and qz5 chimeras have the last five residues of Galphaq replaced by those of Galphai, Galphao and Galphaz, respectively. Except for mu-receptor and qo5, each pair of opioid receptor and Galphaq chimera allowed opioid agonists to stimulate PI-PLC in transfected COS-7 cells. The Galphaq chimera-mediated responses were ligand selective, agonist dose dependent and saturable. The most robust responses were obtained with kappa-receptor and qi5 or qz5, whereas the coupling of delta- and mu-receptors to Galphaq chimeras produced much weaker responses. Among the Galphaq chimeras, qo5 was less efficiently coupled to the opioid receptors. As revealed by radioligand binding assays and immunoblot analysis, differences in the efficiency of coupling were not due to variations in the expression of receptors and Galphaq chimeras. Differences in the magnitude of PI-PLC responses are thus likely to represent structural incompatibility between opioid receptors and Galphaq chimeras, suggesting that each opioid receptor possesses unique structural surfaces for the binding of G proteins.

For the further elucidation of the central functions of nociceptin/orphanin FQ (noc/OFQ), the endogenous ligand of the G protein-coupled opioid receptor-like receptor ORL1, centrally acting specific antagonists will be most helpful. In this study it was found that the hexapeptide acetyl-RYYRIK-NH2 (Ac-RYYRIK-NH2), described in literature as partial agonist on ORL1 transfected in CHO cells, antagonizes the stimulation of [35S]-GTPgammaS binding to G proteins by noc/OFQ in membranes and sections of rat brain. The antagonism of the peptide was competitive, of high affinity (Schild constant 6.58 nM), and specific for noc/OFQ in that the stimulation of GTP binding by agonists for the mu-, delta-, and kappa-opioid receptor was not inhibited. The hexapeptide also fully inhibited the chronotropic effect of noc/OFQ on neonatal rat cardiomyocytes. It is suggested that Ac-RYYRIK-NH2 may provide a promising starting point for in vivo tests for antagonism of the action of noc/OFQ and for the further development of highly active and specific antagonists.

The piglet displays similar postnatal development in respiration and sleep-wake behavior to the human. To shed light on the possible influence of opioid systems on these functions, this study assessed the density of mu- and delta-opioid receptors in brainstems of 2-3 and 5-7 (young), 14-17 (intermediate) and 20-21 (older) day-old piglets, using quantitative autoradiography. Serial 10 microns sections from fresh-frozen brains were incubated with either mu-(125I-DAGO) or delta-(125I-DPDPE) opioid ligands. The binding characteristics of each receptor remained unchanged over the age-range studied. delta-opioid receptor density was minimal in the young piglets, and increased over the age-range studied in all brainstem regions. mu-opioid receptor density exceeded delta-opioid density in all brainstem regions in young and older piglets, and remained unchanged with age. We conclude that, as in other species, the development of delta-opioid receptors in swine lags behind that of mu-opioid receptors, and that the distribution of each in the piglet's brainstem is distinct. The present findings help explain the changing influence of the mu- and delta-opioid systems on breating and state during postnatal development.

Enkephalins are opioid peptides that bridge the neuroendocrine and immune systems. Using flow cytometry and a fluorescein conjugate of the endogenous pentapeptide methionine-enkephalin (ME), we have identified enkephalin receptors on cultured human lymphocytes. Cell surface recognition sites that bound ME with high affinity and specificity were detected for NALM 6 (pre-B acute lymphoblastic leukemia) and Jurkat (T lymphoma) cells. Brain-like enkephalin receptors were measured for these lymphocytes using conventional radioligand-receptor assays and the highly delta opioid receptor-selective enkephalin analog [3H]DPDPE. Upon activation, the lymphocyte enkephalin receptors transmitted signals that enhanced the accumulation of intracellular cAMP. These studies provide evidence that cultured human lymphocytes of the B (NALM 6 cells) and T (Jurkat cells) lineages express functional enkephalin receptors and suggest that such receptors may be instrumental in the lymphocyte response to opioid peptides and alkaloids., (Copyright 1999 Academic Press.)

The adrenal medulla produces opioids which exert paracrine effects on adrenal cortical and chromaffin cells and on adrenal splanchnic nerves, via specific binding sites. The opioid binding sites in the adrenals are detectable mainly in the medullary part of it and differ in type between species. Thus, the bovine adrenal medulla contains mostly kappa-opioid binding sites and fewer delta- and mu-opioid binding sites while primate adrenals contain mainly delta sites and few kappa-opioid binding sites. Most chromaffin cell tumors, the pheochromocytomas, produce opioids which suppress catecholamine production by the tumor. The aim of the present work was to identify the types of opioid binding sites in human pheochromocytomas. For this purpose, we characterized the opioid binding sites on crude membrane fractions prepared from 14 surgically excised pheohromocytomas and on whole KAT45 cells, a recently characterized human pheochromocytoma cell line. Our data showed that human pheohromocytomas are heterogeneous, as expected, with regard to the production of catecholamines and the distribution and profile of their opioid binding sites. Indeed, only one out of the 14 pheochromocytomas expressed exclusively delta and mu opioid sites, while in the remaining 13 tumors kappa-type binding sites were dominant. The KAT45 cell line possessed a significant number of kappa1 binding sites, fewer kappa2-opioid binding sites and kappa3-opioid binding sites, and minimal binding capacity for delta- and mu-opioid receptor agonists sites. More specifically, the kappa1 sites/cell were approximately 18,000, the kappa2 4500/cell and the kappa3 sites 2000/cell. Our findings for the surgical specimens and the cell line combined with previously published pharmacological data obtained from KAT45 cells suggest that kappa sites appear to be the most prevalent opioid binding sites in pheochromocytomas. Finally, in normal bovine adrenals the profile of opioid binding sites differs in adrenaline and noradrenaline producing chromaffin cells. To test the hypothesis that the type of catecholamine produced by a pheochromocytoma depends on its cell of origin, we compared our binding data with the catecholamine content of each pheochromocytoma examined. We found no correlation between the type of the predominant catecholamine produced and the opioid binding profile of each tumor suggesting that this hypothesis may not be valid.

We have previously shown that methionine-enkephalin (MENK) alters in dose-dependent fashion the capacity of human neutrophils to produce superoxide anion. The response of neutrophils from different donors was diverse and this effect could be due to variable activity of proteolytic enzymes involved in the degradation of the neuropeptide. In this study, we have demonstrated a highly individual aminopeptidase N (APN) activity of neutrophils from different donors. Preincubation of neutrophils with MENK, but not with the synthetic agonist of the mu (DAGO) or the delta (DPDPE) opioid receptor, down-regulated the APN activity. This was paralleled by a loss in cell surface expression of APN at physiological (10(-10) M) concentrations of MENK. The level of APN activity from different donors correlated with the effect of MENK on superoxide anion release. Neutrophils with low APN activity, if preincubated with MENK, released reduced amounts of superoxide anion. In contrast, neutrophils with high APN activity released increased amounts of superoxide anion after preincubation with MENK. Thus, the highly individual APN activity on the surface of neutrophils from different donors seems to be altered by MENK and to be related to the respiratory burst.

1. The ability of mu-opioid receptor agonists to activate G-proteins in the spinal cord of mu-opioid receptor knockout mice was examined by monitoring the binding to membranes of the non-hydrolyzable analogue of GTP, guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS). 2. In the receptor binding study, Scatchard analysis of [3H][D-Ala2,NHPhe4,Gly-ol]enkephalin ([3H]DAMGO; mu-opioid receptor ligand) binding revealed that the heterozygous mu-knockout mice displayed approximately 40% reduction in the number of mu-receptors as compared to the wild-type mice. The homozygous mu-knockout mice showed no detectable mu-binding sites. 3. The newly isolated mu-opioid peptides endomorphin-1 and -2, the synthetic selective mu-opioid receptor agonist DAMGO and the prototype of mu-opioid receptor agonist morphine each produced concentration-dependent increases in [35S]GTPgammaS binding in wild-type mice. This stimulation was reduced by 55-70% of the wild-type level in heterozygous, and virtually eliminated in homozygous knockout mice. 4. No differences in the [35S]GTPgammaS binding stimulated by specific delta1- ([D-Pen2,5]enkephalin), delta2-([D-Ala2]deltorphin II) or kappa1-(U50,488H) opioid receptor agonists were noted in mice of any of the three genotypes. 5. The data clearly indicate that mu-opioid receptor gene products play a key role in G-protein activation by endomorphins, DAMGO and morphine in the mouse spinal cord. They support the idea that mu-opioid receptor densities could be rate-limiting steps in the G-protein activation by mu-opioid receptor agonists in the spinal cord. These thus indicate a limited physiological mu-receptor reserve. Furthermore, little change in delta1-, delta2- or kappa1-opioid receptor-G-protein complex appears to accompany mu-opioid receptor gene deletions in this region.

Chronic in vivo treatment with opioid antagonists increases opioid receptor density and the potency of opioid agonists without altering receptor mRNA levels. To determine if basal receptor density affects opioid receptor upregulation, we examined the effect of chronic naltrexone treatment on mu-opioid receptor density and mRNA in two mice strains that differ in mu-opioid receptor density. CXBK mice (mu-opioid receptor deficient) and outbred Swiss Webster mice were implanted s.c. with a placebo or 15 mg naltrexone pellet for 8 days, the pellets removed and 24 hr later opioid receptor density (mu, delta) and receptor mRNA level (mu) determined in whole brain; or morphine dose-response studies conducted. In placebo-treated CXBK mice, mu-opioid receptor density was approximately 40% less than in Swiss Webster mice, although mu-opioid receptor mRNA abundance was similar in both strains. In placebo-treated CXBK mice, morphine potency was approximately 6-fold less than Swiss Webster mice. Naltrexone treatment increased morphine potency (1.7-fold) and mu- (approximately 90%) and delta- (approximately 20-40%) opioid receptor density in CXBK and Swiss Webster mouse brain similarly. Mu-opioid receptor mRNA was unchanged by naltrexone treatment in either strain. There was no difference in the basal or naltrexone-treated whole brain G(i alpha2) protein levels in CXBK or Swiss Webster mouse. These data indicate that a deficiency in mu-opioid receptors does not alter the regulation of opioid receptors by opioid antagonists in vivo, and suggest that adaptive responses to chronic opioid antagonist treatment are independent of opioid receptor density.

We compared the effects of three micro-(DAMGO, DALDA, TNPO) and three delta-(DPDPE, DELT, SNC-80) opioid agonists on arterial blood gas after IV administration in awake sheep. None of the mu agonists altered pO2, pCO2 or pH. All three mu agonists decreased pO2 increased pCO2 and decreased pO2, and this effect was not sensitive to naloxone or TIPPpsi, a delta-antagonist, suggesting that it is not mediated by beta-opioid receptors. When administered to pregnant animals, there were significant changes in fetal pCO2 and pH. It may be possible to develop delta-selective opioid agonists which do not produce respiratory depression.