Different effects of NMDA/group I metabotropic glutamate receptor agents in delta- and mu-opioid receptor agonist-induced supraspinal antinociception

The N-methyl-D-aspartate (NMDA) and metabotropic glutamate (mGlu) receptors are involved in nociceptive transmission in the central nervous system. The present study was designed to study the effects of NMDA and group I mGlu receptor agents on delta- and mu-opioid receptor agonist-induced antinociception in the mouse brain. Intracerebroventricular (i.c.v.) treatment with the non-competitive NMDA receptor antagonist dizocilpine and the group I mGlu receptor antagonist (S)-4-carboxyphenylglycine ((S)-4CPG) significantly attenuated the antinociception induced by the delta-opioid receptor agonists [D-Pen(2), Pen(5)]enkephalin (DPDPE), (-)-TAN 67 and [D-Ala(2)]deltorphin II. On the contrary, i.c.v. administration of dizocilpine and (S)-4CPG slightly but significantly enhanced the antinociception induced by the mu-opioid receptor agonist [D-Ala(2), N-Me-Phe(4), Gly(5)-ol]enkephalin (DAMGO). Under these conditions, i.c.v. administration of NMDA and the group I mGlu receptor agonist 3,5-dihydrophenylglycine (DHPG) significantly enhanced the antinociception induced by delta-opioid receptor agonists, whereas both reduced DAMGO-induced antinociception. These findings suggest that the supraspinal antinociceptive actions of mu- and delta-opioid receptor agonists appear to be modulated differently by NMDA and group I mGlu receptors in the mouse.     

Effects of the delta-opioid receptor antagonist naltrindole on antinociceptive responses to selective delta-agonists in post-weanling rats.

1. Antagonism, by the selective delta-opioid receptor antagonist naltrindole, of the antinociceptive effects of [D-Pen2, D-Pen5] enkephalin (DPDPE), [D-Ser2, Leu5, Thr6] enkephalin (DSLET) and D-Ala2 deltorphin I (DELT I) has been studied in 25 day old rats. 2. Antinociception was measured by the 50 degrees C tail immersion test following i.p. administration of agonists and/or antagonists. 3. Dose-related antinociception was observed with DPDPE, DSLET and DELT I and ED75 doses were computed (0.66 mg kg-1, 0.65 mg kg-1, 0.032 mg kg-1 respectively) and used for antagonism studies. 4. Naltrindole (0.01 mg kg-1) significantly attenuated the antinociceptive effects of DPDPE and DSLET with 0.1 mg kg-1 producing complete reversal of the effects of the ED75 dose. In contrast, naltrindole at 0.01 and 0.1 mg kg-1 did not alter antinociceptive responses to DELT I. Naltrindole at 1 mg kg-1 significantly attenuated DELT I antinociception. 5. Naloxone (1 mg kg-1) produced equivalent degrees of antagonism of the antinociceptive effects of DPDPE, DSLET and DELT I. ICI 174,864 (1 mg kg-1) also antagonized antinociception with a differential degree of attenuation (DSLET > DPDPE > DELT I). 6. Naltrindole (1 mg kg-1) had no effect on the antinociception induced by the selective mu-agonist alfentanil (60 micrograms kg-1). Naltrindole, naloxone or ICI 174,864 had no effect on nociceptive latencies. 7. The differential antagonism by naltrindole of the effects of three selective delta-agonists suggests delta-receptor heterogeneity.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of delta-opioid receptor subtypes in neuropathic pain

A large body of evidence suggests an important role of delta-opioid receptor agonists in antinociception at the level of the spinal cord. Our study was undertaken to analyse the spinal antinociceptive and antiallodynic effects of delta(1)- and delta(2)-opioid receptor agonists and antagonist after their acute and chronic intrathecal administration in a neuropathic pain model in the rat. In rats with a crushed sciatic nerve, the delta(1)-opioid receptor agonist [D-Pen(2), D-Pen(5)]enkephalin (DPDPE, 5-25 microg i.t.) and the delta(2)-opioid receptor agonist deltorphin II (1.5-25 microg i.t.) dose dependently antagonized the cold-water allodynia which developed after sciatic nerve injury. These effects of DPDPE were antagonized by 7-benzylidenenaltrexon (BNTX, 1 microg i.t.) while the effects of deltorphin II were antagonized by 5'naltrindole izotiocyanate (5'NTII, 25 microg i.t.). Both agonists had a dose-dependent, statistically significant effect on the tail-flick latency in two tests, with focused light and cold water. Chronic administration of DPDPE (25 microg i.t.) and deltorphin II (15 microg i.t.) resulted in significant prolongation of the reaction time determined on days 2, 4 and 6 post-injury. In conclusion, our results show an antiallodynic and antinociceptive action of DPDPE and deltorphin II at the spinal cord level, which suggests that both delta-opioid receptor subtypes play a similar role in neuropathic pain. This indicates that not only delta(1)- but also delta(2)-opioid receptor agonists can be regarded as potential drugs for the therapy of neuropathic pain.     

Mu-opioid receptor specific antagonist cyprodime: characterization by in vitro radioligand and [35S]GTPgammaS binding assays

The use of compounds with high selectivity for each opioid receptor (mu, delta and kappa) is crucial for understanding the mechanisms of opioid actions. Until recently non-peptide mu-opioid receptor selective antagonists were not available. However, N-cyclopropylmethyl-4,14-dimethoxy-morphinan-6-one (cyprodime) has shown a very high selectivity for mu-opioid receptor in in vivo bioassays. This compound also exhibited a higher affinity for mu-opioid receptor than for delta- and kappa-opioid receptors in binding assays in brain membranes, although the degree of selectivity was lower than in in vitro bioassays. Cyprodime has recently been radiolabelled with tritium resulting in high specific radioactivity (36.1 Ci/mmol). We found in in vitro binding experiments that this radioligand bound with high affinity (K(d) 3. 8+/-0.18 nM) to membranes of rat brain affording a B(max) of 87. 1+/-4.83 fmol/mg. Competition studies using mu, delta and kappa tritiated specific ligands confirmed the selective labelling of cyprodime to a mu-opioid receptor population. The mu-opioid receptor selective agonist [D-Ala(2),N-MePhe(4),Gly(5)-ol]enkephalin (DAMGO) was readily displaced by cyprodime (K(i) values in the low nanomolar range) while the competition for delta- ([D-Pen(2), D-Pen(5)]enkephalin (DPDPE)) and kappa- (5alpha,7alpha, 8beta-(-)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro(4, 5)dec-8-yl]-benzene-acetamide (U69,593)) opioid receptor selective compounds was several orders of magnitude less. We also found that cyprodime inhibits morphine-stimulated [35S]GTPgammaS binding. The EC(50) value of morphine increased about 500-fold in the presence of 10 microM cyprodime. These findings clearly indicate that cyprodime is a useful selective antagonist for mu-opioid receptor characterization.     

Cross-tolerance between analgesia produced by xylazine and selective opioid receptor subtype treatments

Opioid receptor agonists produce analgesia through multiple systems activated by stimulation of mu(1), mu(2), delta(1), delta(2) and kappa(1) opioid receptors. Morphine analgesia is modulated by stimulation of alpha(2) adrenoceptors. To understand how multiple opioid analgesic systems interact with alpha(2)-adrenoceptor systems, analgesic cross-tolerance between the alpha(2) adrenoceptor agonist xylazine and opioid receptor agonists was studied using the mouse tail-flick assay. Mice received either xylazine (20 mg/kg, s.c.) or saline (1 ml/kg) for five days. On day six, mice received a dose of s.c. xylazine, i.c.v. [D-Ala(2),MePhe(4),Gly(ol)(5)]enkephalin (DAMGO), i.t. Tyr-Pro-Trp-Gly-NH(2) (Tyr-W-MIF-1), i.c.v. or i.t. [D-Pen(2),D-Pen(5)]enkephalin (DPDPE), i.t. [D-Ala(2)]deltorphin II (deltorphin II), or s.c. trans-(+/-)-3, 4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl-cyclohexyl] benzeneacetamide (U50,488). Xylazine tolerant mice required 4. 57-fold more xylazine to elicit the same response as saline treated animals and showed a 2.55-fold shift in i.c.v. DAMGO and a 3.37-fold shift in i.c.v. DPDPE antinociception. No cross-tolerance was seen with i.c.v. deltorphin II, i.t.Tyr-W-MIF-1, i.t. DPDPE, i.t. Tyr-W-MIF-1 or s.c. U50,488. These results implicate alpha(2) adrenoceptor systems in the modulation of supraspinal mu(1), and delta(1) opioid analgesic circuitry and raise the possibility that mu(2), delta(2) or kappa(1) opioid receptor agonists may be alternated with alpha(2) adrenoceptor agonists to minimize tolerance or treat opioid-tolerant patients.     

Pertussis toxin abolishes mu- and delta-opioid agonist-induced place preference.

The effects of i.c.v. treatment with pertussis toxin (PTX) on the motivational effect of opioid agonists were examined in mice. Morphine (0.1-10 nmol, i.c.v.), [D-Ala2, N-MePhe4, Gly-ol5]enkephalin (DAGO, 0.001-0.1 nmol, i.c.v.), a selective mu-opioid receptor agonist, and [D-Pen2, D-Pen5]enkephalin (DPDPE, 1-15 nmol, i.c.v.), a selective delta-opioid receptor agonist, produced a dose-related place preference in mice. Administration of PTX (0.5 micrograms, i.c.v.) to mice resulted in no preference for either the drug- or vehicle-associated place. Pretreatment with PTX abolished the place preferences induced by DAGO (0.1 nmol), morphine (10 nmol) and DPDPE (15 nmol). These findings demonstrate that the appetitive effects of opioids result from the activation of central mu- and delta-receptors, and suggest that PTX-sensitive GTP-binding proteins in the central nervous system may be involved in the motivational effects of mu- and delta-opioid agonists.     

Several delta-opioid receptor ligands display no subtype selectivity to the human delta-opioid receptor

Pharmacological studies performed in vivo suggested that the delta-opioid receptor could exist as two distinct subtypes, delta(1) and delta(2), while in vitro studies are inconclusive. Therefore, we measured the binding and functional selectivity of several putative delta(1)- and delta(2)-opioid receptor-selective compounds in membranes from Chinese hamster ovary cells stably expressing the human delta-opioid receptor. The compounds characterized were the agonists [D-Pen(2),D-Pen(5)]enkephalin (DPDPE, delta(1)) and deltorphin II (delta(2)), and the antagonists 7-benzylidenenaltrexone (BNTX, delta(1)), naltriben (delta(2)), naltrindole 5'-isothiocyanate (delta(2)), and naltrindole (delta(1) and delta(2)). In competition binding assays, all compounds tested showed no preference for the [3H]DPDPE, [3H]deltorphin II, or [3H]naltrindole binding sites. BNTX also showed no selectivity for the delta-opioid receptor over the mu-opioid receptor. In functional assays, the stimulation of [35S]GTPgammaS binding induced by either DPDPE or deltorphin II was potently inhibited by both delta(1)- and delta(2)-opioid receptor-selective antagonists. Together, these results indicate that these compounds are not selective for either the delta(1)- or delta(2)-opioid receptor binding sites in binding or functional assays.     

Delta-opioid receptor binding sites in rodent spinal cord.

1. The delta-opioid receptor agonist [D-Pen2,D-Pen5]enkephalin showed an antinociceptive effect in the mouse tail-flick test, following intrathecal administration. This action was reversed by naloxone and by the selective delta-opioid receptor antagonist ICI 174864. 2. High affinity, saturable binding of [3H]-[D-Pen2,D-Pen5]enkephalin has been demonstrated in spinal cord homogenates from guinea-pig, hamster, rat and both adult and young (18-20 g) mice. The binding was shown by autoradiography to be concentrated in the superficial laminae of the dorsal horn. 3. Competition studies confirmed that the binding of [3H]-[D-Pen2,D-Pen5]enkephalin was to the delta-opioid site. However, anomalies were seen with displacement assays using mu-ligands, which may suggest some common high affinity site for delta- and mu-opioid receptor agonists in the spinal cord. 4. The results add further evidence for a role of the delta-opioid receptor in spinally-mediated antinociception.     

Lack of antinociceptive efficacy of intracerebroventricular [D-Ala2,Glu4]deltorphin, but not [D-Pen2,D-Pen5]enkephalin, in the mu-opioid receptor deficient CXBK mouse.

The antinociceptive efficacy of [D-Pen2,D-Pen5]enkephalin (DPDPE) (delta 1 agonist) and [D-Ala2,Glu4]deltorphin (delta 2 agonist) was evaluated following intracerebroventricular (i.c.v.) or intrathecal (i.t.) administration in CD-1 and CXBK strains of mice using the radiant heat tail-flick test. Following i.c.v. administration, [D-Ala2,Glu4]deltorphin was effective in CD-1, but not CXBK, mice; DPDPE was approximately equiactive in both strains. While i.c.v. [D-Ala2,Glu4]deltorphin did not produce antinociception in the CXBK mouse, it effectively antagonized the antinociceptive actions of i.c.v. DPDPE. [D-Ala2,Glu4]deltorphin was effective following i.t. administration in both strains. These data suggest possible differences in the supraspinal populations of opioid delta receptor subtypes in the CXBK strain. On the basis of previously established selectivity of these agonists, the CXBK mouse may have a predominate population of supraspinal opioid delta 1, rather than delta 2, receptors.     

Lack of mu-opioid receptor-mediated G-protein activation in the spinal cord of mice lacking Exon 1 or Exons 2 and 3 of the MOR-1 gene

The G-protein activation induced by mu-opioid receptor agonists was determined in spinal cord membranes from two types of mu-opioid receptor knockout mice: mice with a disruption of exon 1 (MOR (Exon 1)-KO) or exons 2 and 3 (MOR (Exons 2 and 3)-KO) of the mu-opioid receptor gene. The G-protein activation induced by the opioid agonists was measured by monitoring the increases of guanosine-5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTP gamma S) binding. The mu-opioid receptor agonists (D-Ala(2),N-MePhe (4),Gly-ol(5)]enkephalin, endomorphin-1, endomorphin-2, morphine, morphine-6 beta-glucuronide, and fentanyl produced concentration-dependent increases of [(35)S]GTP gamma S binding to spinal cord membranes in wild-type mice, but not in MOR (Exon 1)-KO mice or MOR (Exons 2 and 3)-KO mice. On the other hand, the delta-opioid receptor agonist [D-Pen (2,5)]enkephalin, the kappa-opioid receptor agonist (-)U50,488H, or the ORL1-receptor agonist nociception increased [(35)S]GTP gamma S binding in the spinal cord membranes from both MOR (Exon 1)-KO mice and MOR (Exons 2 and 3)-KO mice to the same extent as in the corresponding wild-type mice. The results provide further information about the important roles of the sequences encoded within exon 1 and exons 2 and 3 of mu-opioid receptor gene for the activation of G-proteins by mu-opioid receptor agonists in the mouse spinal cord.     

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.     

Absence of G-protein activation by mu-opioid receptor agonists in the spinal cord of mu-opioid receptor knockout mice

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.     

Effects of competitive and noncompetitive antagonists of the N-methyl-D-aspartate receptor on the analgesic action of delta 1- and delta 2-opioid receptor agonists in mice.

1. The effects of MK-801, a noncompetitive antagonist of the N-methyl-D-aspartate (NMDA) receptor and LY 235959, a competitive antagonists of the NMDA receptor on the analgesic actions of [D-Pen2,D-Pen5]enkephalin (DPDPE) and [D-Ala2, Glu4] deltorphin II (deltorphin II), the putative delta 1- and delta 2-opioid receptor agonists, respectively, were determined in the male Swiss-Webster mice. 2. Intracerebroventricular administration of DPDPE or deltorphin II produced analgesia. MK-801 administered intraperitoneally 10 min before the injection of DPDPE or deltorphin II, dose-dependently antagonized the analgesic actions of both drugs. 3. LY 235959 also dose-dependently antagonized the analgesic actions of DPDPE and deltorphin II. 4. The effects of MK-801 and LY 235959 on the binding of [3H]-DPDPE to mouse brain membranes were also determined. Neither of the NMDA receptor antagonists had any effect on the binding of [3H]-DPDPE. 5. It is concluded that competitive and noncompetitive antagonists of the NMDA receptor antagonize the analgesic action of delta 1- and delta 2-opioid receptor agonists and that such effects are not mediated via a direct interaction with brain delta-opioid receptors.     

Analgesic activity and opioid receptor selectivity of stereoisomers of ohmefentanyl isothiocyanate.

Ohmefentanyl is a very potent and highly selective agonist for mu-opioid receptors. We now study analgesia, in vitro activity and opioid receptor affinity of the stereoisomers of ohmefentanyl isothiocyanate. We found that some isomers of ohmefentanyl isothiocyanate had a potent analgesic effect and that all isomers except (3R,4S,2'S)-ohmefentanyl isothiocyanate had a more potent inhibitory action on the electrically evoked contractions of mouse vas deferens than of guinea pig ileum. The inhibitory actions could be antagonized by naloxone. However, compared with the activity of the corresponding stereoisomers of ohmefentanyl, these ohmefentanyl isothiocyanates had significantly reduced analgesia and in vitro activity. They also inhibited the binding of [3H]DPDPE ([D-Pen(2),D-Pen(5)]enkephalin) and [3H]DAGO ([D-Ala(2),Mephe(4),Gly-ol(5)]enkephalin) to opioid receptors in mouse brain membranes. The inhibitory effect of stereoisomers of ohmefentanyl isothiocyanate at mu-opioid receptors was markedly lower than that of their parent compounds. The affinity of stereoisomers of ohmefentanyl isothiocyanate for delta-opioid receptors was, however, greater than or equal to that of their corresponding stereoisomers of ohmefentanyl. The results showed that the introduction of an isothiocyanato group into the phenyl ring in position-1 of ohmefentanyl reduced bioactivity and affinity to mu-opioid receptors but that the selectivity of these compounds for delta-opioid receptors was enhanced. Isomer (3R,4S,2'R)-ohmefentanyl isothiocyanate showed highest selectivity for delta-opioid receptors (K(i)(mu)/K(i)(delta)=13.6) and potent analgesic activity (ED(50)=0.25 mg/kg).     

Evidence for lack of modulation of mu-opioid agonist action by delta-opioid agonists in the mouse vas deferens and guinea-pig ileum.

1. There is evidence from in vivo studies for an interaction of mu- and delta-opioid ligands. In the present work this concept has been investigated using the mouse vas deferens and guinea-pig ileum myenteric plexus-longitudinal preparations. 2. In field stimulated vasa deferentia of the mouse, co-administration of sub-effective concentrations of the delta-opioid agonist [D-Pen2,D-Pen5]enkephalin (DPDPE) and [Met5]- or [Leu5]enkephalin had no effect on the dose-response curves of the mu-agonists [D-Ala2,MePhe4, Gly-ol5]enkephalin (DAMGO) and morphine. Similarly, the delta-opioid agonists did not alter the potency of morphine and DAMGO when added at different times prior to the mu-opioid agonists, or when EC50 concentrations of delta-opioid ligands were co-administered. Compounds with preferred activity for the putative delta 1-(DPDPE) or delta 2-([D-Ala2,Glu4]deltorphin II (Delt II)) opioid receptors were ineffective in this respect. 3. The guinea-pig ileum contains delta-opioid receptors. No function of these receptors in mediating blockage of field-stimulated contractions was observed with ligands having affinity for the putative delta 1 or delta 2 subtypes nor were the agonists able to modulate responses to mu-opioid ligands in this tissue. 4. The results demonstrate the modulation of mu-opioid agonists by delta-opioid agonists does not occur in the isolated peripheral tissues examined. Thus the findings do not support the concept of a functional coupling of opioid receptors, though the results may be explained by differences between opioid systems in the brain and peripheral tissues examined.     

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.     

Modulation of mu-mediated antitussive activity in rats by a delta agonist.

Effects of selective mu and delta receptor agonists on capsaicin-induced cough reflex in rats were studied. Intracisternal injection (i.cist.) of a selective mu receptor agonist [D-Ala2,Mephe4,Gly-ol5]enkephalin (DAMGO) produced dose-related depression of coughs over the 0.003-0.03 nmol dose range. The antitussive potency of DAMGO was 100-fold more potent than morphine. The antitussive effects of DAMGO and morphine were significantly reduced by naloxone (1 nmol i.cist.). The selective delta receptor agonist, [D-Pen2,D-Pen5]enkephalin (DPDPE), at a dose of 10 nmol (i.cist.), had no significant effect on the number of coughs. When co-administered i.cist., DPDPE (10 nmol) consistently and significantly decreased the antitussive potencies of DAMGO and morphine. The decrease in the antitussive effects of DAMGO and morphine caused by DPDPE were prevented by selective delta receptor antagonist, naltrindole (3 nmol). These results suggest that the antitussive effects of opioids are mediated predominantly by mu receptors, and delta receptors may play an inhibitory role in antitussive processes that are mediated by the mu receptors.     

Mutation S363A in the human delta-opioid receptor selectively reduces down-regulation by a peptide agonist

Chemically distinct opioid agonists have different abilities to down-regulate opioid receptors. The present study investigated the role of Ser(363) in human delta-opioid receptor down-regulation by a delta-selective peptide- and non-peptide agonist. Cyclic[D-Pen(2),D-Pen(5)]enkephalin (DPDPE)-mediated down-regulation was significantly attenuated by a S363A mutation. In contrast, this mutation had no effect on down-regulation by (+)-4-[(alpha R)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]N,N-diethylbenzamide (SNC80). These results demonstrate that the molecular mechanism of the human delta-opioid receptor down-regulation is agonist-specific.     

Anti-exudative effects of opioid receptor agonists in a rat model of carrageenan-induced acute inflammation of the paw

We evaluated the anti-exudative effects (Evan's blue) of mu-, delta- and kappa-opioid receptor agonists in a rat model of carrageenan-induced acute inflammation. The contribution of different components was assessed after the administration of: cyclosporine A, capsaicin, 6-hydroxydopamine, compound 48/80, and specific histamine-receptor antagonists. The results show that the mu-opioid receptor agonists morphine and fentanyl and the delta-opioid receptor agonists DPDPE (enkephalin, [D-Pen(2,5)]) and SNC 80 ((+)-4-[(alpha R)-alpha((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N diethylbenzamide) decrease plasma extravasation in a dose-dependent manner, with a biphasic response. The effects were reversed by specific antagonists, and are predominantly mediated by peripheral opioid receptors. The integrity of sensory and sympathetic fibres is essential for the anti-exudative effects of fentanyl and DPDPE. Histamine and functional histamine H(2) and H(3) receptors are required for morphine and fentanyl (but not DPDPE) inhibition of plasma extravasation, suggesting different mechanism for mu- and delta-opioid receptor agonists. The present findings implicate multiple sites and mechanisms in the anti-exudative effects of exogenous opioids.     

The R7 subfamily of RGS proteins assists tachyphylaxis and acute tolerance at mu-opioid receptors.

Members of the R7 subfamily of regulators of G-protein signaling (RGS) proteins (RGS6, RGS7, RGS9-2, and RGS11) are found in the mouse CNS. The expression of these proteins was effectively reduced in different neural structures by blocking their mRNA with antisense oligodeoxynucleotides (ODNs). This was achieved without noticeable changes in the binding characteristics of labeled beta-endorphin to opioid receptors. Knockdown of R7 proteins enhanced the potency of antinociception promoted by morphine and [D-Ala(2), N-MePhe(4), Gly-ol(5)]-enkephalin (DAMGO)-both agonists at mu-opioid receptors. The duration of morphine analgesia was greatly increased in RGS9-2 and in RGS11 knockdown mice. The impairment of R7 proteins brought about different changes in the analgesic activity of selective delta agonists. Knockdown of RGS11 reduced [D-Ala(2)]deltorphin II analgesic effects. Those of RGS6 and RGS9-2 proteins caused [D-Ala(2)]deltorphin II to produce a smoothened time-course curve-the peak effect blunted and analgesia extended during the declining phase. RGS9-2 impairment also promoted a similar pattern of change for [D-Pen(2,5)]-enkephalin (DPDPE). RGS7-deficient mice showed an increased response to both [D-Ala(2)]deltorphin II and DPDPE analgesic effects. A single intracerebroventricular (i.c.v.) ED(80) analgesic dose of morphine gave rise to acute tolerance in control mice, but did not promote tolerance in RGS6, RGS7, RGS9-2, or RGS11 knockdown animals. Thus, R7 proteins play a critical role in agonist tachyphylaxis and acute tolerance at mu-opioid receptors, and show differences in their modulation of delta-opioid receptors.