delta-Opioid receptor agonists produce antinociception and [35S]GTPgammaS binding in mu receptor knockout mice

We examined the effects of [D-Pen(2),D-Pen(5)]enkephalin (DPDPE), [D-Ala(2),Glu(4)]deltorphin (DELT), and (+)-4-[(alphaR)-alpha((2S, 5R)-4-Allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N, N-diethylbenzamide (SNC80) on [35S]GTPgammaS binding in brain membranes prepared from micro-opioid receptor knockout (-/-) mice. The potency and maximal response (E(max)) of these agonists were unchanged compared to control mice. In contrast, while the potency of [D-Pen(2),pCl-Phe(4),D-Pen(5)]enkephalin (pCl-DPDPE) was not significantly different, the E(max) was reduced as compared to controls. In the tail-flick test, intracerebroventricular (i.c.v.) or intrathecal (i.th.) DELT produced antinociceptive effects in -/- mice with potency that did not differ significantly from controls. In contrast, the antinociceptive potency of i.c.v. and i.th. DPDPE was displaced to the right by 4- and 9-fold in -/- compared to control mice, respectively. Reduced DPDPE antinociceptive potency in -/- mice, taken together with reduced DPDPE- and pCl-DPDPE- stimulated G protein activity in membranes prepared from -/- mice, demonstrate that these agonists require mu-opioid receptors for full activity. However, because DELT mediated G protein activation and antinociception were both comparable between -/- and wild type mice, we conclude that the mu-opioid receptor is not a critical component of delta-opioid receptor function.     

Functional interaction among opioid receptor types: up-regulation of mu- and delta-opioid receptor functions after repeated stimulation of kappa-opioid receptors

It has been widely accepted that repeated administration of kappa-opioid receptor agonists leads to the development of antinociceptive tolerance. The present study was designed to investigate the effect of repeated administration of a selective kappa-opioid receptor agonist (1S-trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzeneacetamide hydrochloride ((-)U-50,488H) on the mu- and delta-opioid receptor agonist-induced antinociception and G-protein activation in mice. The mice were injected either subcutaneously (s.c.) or intracerebroventricularly (i.c.v.) pretreated with saline or (-)U-50,488H once a day for seven consecutive days. Two hours after the last injection, the mice were challenged by either mu- or delta-opioid receptor agonist for the antinociceptive assay. Repeated treatment with (-)U-50,488H (s.c. or i.c.v.) significantly enhanced antinociceptive effect of both mu-opioid receptor agonist (morphine) and delta-opioid receptor agonists ([d-Ala2]deltorphin (DELT) and (+)-4-[(alphaR)-alpha-((2S,5R)-4-allyl-2,5-dime thyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC-80) compared to saline-treated groups. Under these conditions, repeated s.c. injection of (-)U-50,488H significantly enhanced both mu- and delta-opioid receptor agonist-stimulated [35S]GTPgammaS binding in the membrane of the thalamus. On the contrary, either repeated administration of morphine (s.c. or i.c.v.) or SNC-80 failed to affect the kappa-opioid receptor agonist-induced antinociception and G-protein activation. Taken together, these results suggest that repeated stimulation of kappa-opioid receptor markedly increases the functional mu- and delta-opioid receptors, whereas repeated stimulation of either mu- or delta-opioid receptor had no direct effect on kappa-opioidergic function in mice.     

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.     

Central delta-opioid receptor interactions and the inhibition of reflex urinary bladder contractions in the rat.

The in vivo effects of a number of opioid agonists and antagonists were studied on the spontaneous reflex contractions of the urinary bladder recorded isometrically in the rat anesthetized with urethane. All substances were administered into the central nervous system by the intracereboventricular (i.c.v.) or spinal intrathecal (i.t.) route. The conformationally restricted enkephalin analogues [2-D-penicillamine, 5-L-cysteine] enkephalin (DPLCE), [2-D-penicillamine, 5-L-penicillamine] enkephalin (DPLPE) and [2-D-penicillamine, 5-D-penicillamine] enkephalin (DPDPE) produced dose-related inhibition of reflex bladder contractions when administered by the i.c.v. or i.t. route. Both the novel delta-opioid receptor antagonist ICI 154,129 (200-600 micrograms) [N,N-bisallyl-Tyr-Gly-Gly-Psi-(CH2S)-Phe-Leu-OH) and ICI 174,864 (1-3 micrograms) [N,N-dially-Tyr-Aib-Aib-Phe-Leu-OH: Aib = alpha-aminoisobutyric acid] attenuated or abolished the effects of DPLCE, DPLPE and DPDPE when administered by the i.c.v. or i.t. route. The antagonism observed was selective since the equipotent inhibition produced by the mu-opioid receptor agonist [D-Ala2, Me-Phe4, Gly(ol)5] enkephalin (DAGO) was unaffected. Overall, ICI 154,129 was considerably weaker than ICI 174,864 and both antagonists inhibited bladder activity at doses higher than those required to demonstrate delta-receptor antagonism. Further studies of the agonistic effect of ICI 174,864 showed that it was insensitive to low doses of naloxone (2 micrograms, i.c.v. or i.t.) but could be abolished by higher (10-15 micrograms) doses of naloxone. These observations suggested that the agonistic effect of ICI 174,864 was not mediated by mu-opioid receptor. beta-Endorphin (0.2-1.0 micrograms, i.c.v.) inhibited bladder contractions but following recovery from this effect, appeared to prevent the expression of delta-receptor antagonism by ICI 174,864. In addition a previously subthreshold dose of ICI 174,864 now exhibited marked agonistic activity. The inhibitory effect of a submaximal dose of DPDPE was also potentiated by beta-endorphin under these circumstances. These observations suggest that supra-spinal and spinal delta-opioid receptors are involved in the opioid-mediated inhibition of reflex bladder contractions in the rat. Moreover beta-endorphin may be important in regulating central delta-opioid receptors.     

The antinociceptive properties of endomorphin-1 and endomorphin-2 in the mouse

Two highly selective mu-opioid receptor agonists, endomorphin-1 (EM-1) and endomorphin-2 (EM-2), have been identified and postulated to be endogenous mu-opioid receptor ligands. The present minireview describes the antinociceptive properties with the tail-flick test of these two ligands given intracerebroventricularly (i.c.v.) and intrathecally (i.t.) in ICR mice. EM-1 or EM-2 given i.c.v. or i.t. dose-dependently produce antinociception. These antinociceptive effects induced by EM-1 and EM-2 given i.c.v. or i.t. are selectively mediated by the stimulation of mu-, but not delta- or kappa-opioid receptors. Like other mu-opioid agonists morphine and DAMGO ([D-Ala2,NMePhe4,Gly5-ol]enkephalin), EM-1 and EM-2 given i.c.v. activate descending pain controls by the releases of noradrenaline and 5-HT and subsequently act on alpha2-adrenoceptors and 5-HT receptors, respectively, in the spinal cord to produce antinociception. However, the antinociception induced by EM-2 given i.c.v. or i.t. also contain an additional component, which is mediated by the release of dynorphin A(1-17) acting on kappa-opioid receptors at the supraspinal and spinal sites. In addition, the antinociception induced by EM-2 given i.c.v. contains another component, which is mediated by the release of Met-enkephalin acting on delta2-opioid receptors in the spinal cord. It is proposed that there are two subtypes of mu-opioid receptors,which are involved in EM-1- and EM-2-induced antinociception. One subtype of mu-opioid receptors is stimulated by EM-1, EM-2 and other mu-opioid agonists morphine and DAMGO; and another subtype of mu-opioid is sorely stimulated by EM-2 and is involved in the releases of dynorphin A(1-17) and Met-enkephalin for the production of antinociception.     

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.     

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

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

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.     

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.     

Contribution of supraspinal mu- and delta-opioid receptors to antinociception in the rat.

This study evaluated the contribution of supraspinal opioid receptors to the production of antinociception, in the rat. I.c.v. administration of a selective mu- (DAMGO) and a selective delta- (DPDPE), but not a selective kappa- (U50,488H) opioid receptor agonist, produced significant dose-dependent increase in mechanical nociceptive thresholds. ICI 174,864, a delta-opioid receptor antagonist, completely blocked the antinociceptive effects produced by DPDPE ([D-Pen2,D-Pen5]enkephalin) at a dose that had no effect on the increases in nociceptive thresholds produced by DAMGO ([D-Ala2,N-MePhe4,Gly5-ol]enkephalin). The simultaneous i.c.v. administration of a low-antinociceptive dose of DAMGO or DPDPE given in combination with sequentially increasing doses of the other opioid agonist, produced synergy (i.e., a more than additive antinociceptive effect), at the lower doses tested. The results of these experiments provide evidence to support the suggestion that both supraspinal mu- and delta-opioid receptors contribute to the production of antinociception, in the rat.     

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.     

Blockade of mu-opioid receptor-mediated G-protein activation and antinociception by TRK-820 in mice

The effects of kappa-opioid receptor agonists trans-3,4-dichloro-N-(2-(1-pyrollidinyl)-cyclohexyl) benzeneacetamide ((-)-U50,488H) and 17-cyclopropylmethyl-3,14beta-dihydroxy-4,5alpha-epoxy-6beta-[N-methyl-trans-3-(3-furyl)acrylamido]morphinan hydrochloride (TRK-820) on the G-protein activation and antinociception induced by the selective mu-opioid receptor agonist, [D-Ala(2),N-MePhe(4),Gly-ol(5)]enkephalin (DAMGO), were determined in mice. G-protein activation was measured by monitoring the guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS) binding in the mouse pons/medulla. DAMGO (10 microM) produced a marked increase of [35S]GTPgammaS binding to the mouse pons/medulla membrane. On the other hand, both TRK-820 and (-)-U50,488H produced small but significant increases of [35S]GTPgammaS binding to the mouse pons/medulla membrane. These increases by both TRK-820 and (-)-U50,488H were completely reversed by the selective kappa-opioid receptor antagonist, norbinaltorphimine. Under these same conditions, the DAMGO-induced increase of [35S]GTPgammaS binding was significantly attenuated by TRK-820 in a concentration-dependent manner, but not by (-)-U50,488H. In the tail-flick test, DAMGO (16 ng) given intracerebroventricularly (i.c.v.), produced a marked antinociception. The antinociception induced by DAMGO was dose-dependently blocked by co-treatment with TRK-820, but not (-)-U50,488H, in mice pretreated with norbinaltorphimine (5 microg, i.c.v.). The present results provide direct evidence for the antagonistic property of TRK-820 for mu-opioid receptors, in addition to the full agonistic property for kappa-opioid receptors.     

The antianxiety-like effects of antagonists of group I and agonists of group II and III metabotropic glutamate receptors after intrahippocampal administration

RATIONALE: Substances acting as agonists of group II mGlu receptors with joint group I mGlu receptor antagonist effects, or group II mGlu receptors agonists, were shown to induce antianxiety-like effect in rats after intrahippocampal administration. OBJECTIVE: The present study was undertaken to establish whether a more selective group I, II, III mGlu receptors agonists/antagonists induce anxiolytic-like effects after injection to the hippocampus. METHODS: (S)-4-Carboxyphenylglycine [(S)-4CPG] and 7-(hydroxyimino)cyclopropan[b]chromen-1alpha-carboxylic ethyl ester (CPCCOEt), selective antagonists at group I mGlu receptors, or (+)1S, 2S, 5R, 6S-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid (LY354740) and (2S, 1'S, 2'S)-2-(carboxycyclopropyl)glycine (L-CCG-I), two selective agonists of group II mGlu receptors, as well as (1S, 2S, 4S, 5S)-2-aminobicyclo[2.1.1]hexane-2,5-dicarboxylic acid-I (ABHxD-I), an agonist at all three groups of mGlu receptors and L-serine-O-phosphate (L-SOP), an agonist at group III mGlu receptors, were used. All compounds were administered into the CA1 region of the dorsal hippocampus. The conflict drinking Vogel test in rats was used to estimate the anxiolytic-like effects of all the compounds. RESULTS: After intrahippocampal administration, both selective group I mGlu receptors antagonists (S)-4CPG and CPCCOEt, as well as the selective agonists of group II mGlu receptors LY 354740 and L-CCG-I, and an agonist of group III mGlu receptors, L-SOP, induced anticonflict effects. CONCLUSION: Selective antagonists of group I mGlu receptors and agonists of group II and group III mGlu receptors exhibit anxiolytic-like activity in the conflict drinking test. It seems that the hippocampus may be one of the brain structures involved in the anticonflict effect of mGlu receptor agonists/antagonists.     

Subgroups among mu-opioid receptor agonists distinguished by ATP-sensitive K+ channel-acting drugs.

1. We evaluated the effects of the i.c.v. administration of different K+ channel blockers (gliquidone, 4-aminopyridine and tetraethylammonium) and an opener of K+ channels (cromakalim) on the antinociception induced by several mu-opioid receptor agonists in a tail flick test in mice. 2. The s.c. administration of all agonists of mu-opioid receptors tested (morphine, 1-16 mg kg-1; metadone, 1-6 mg kg-1; buprenorphine, 0.04-0.64 mg kg-1; fentanyl, 0.02-0.32 mg kg-1 and levorphanol, 0.2-3.2 mg kg-1) elicited a dose-dependent antinociceptive effect. 3. The ATP-sensitive K+ channel blocker, gliquidone (0.06-16 micrograms per mouse, i.c.v.) antagonized the antinociception induced by buprenorphine, morphine and metadone. In contrast, gliquidone (0.25-160 micrograms per mouse) did not modify the antinociceptive effects of fentanyl and levorphanol. 4. Cromakalim (4-64 micrograms per mouse, i.c.v.), an opener of ATP-sensitive K+ channels, enhanced the antinociception produced by buprenorphine, morphine, and methadone, and did not significantly modify the antinociceptive effects of fentanyl and levorphanol. 5. The i.c.v. administration of the K+ channel blockers tetraethylammonium (10 micrograms per mouse) or 4-aminopyridine (25 ng per mouse) did not significantly modify the antinociception induced by any mu-opioid receptor agonist tested. 6. These results suggest that the opening of ATP-sensitive K+ channels is involved in the antinociceptive effect of morphine, buprenorphine and methadone, but not in that of fentanyl or levorphanol. Consequently, we suggest that at least two subgroups can be distinguished among mu-opioid receptor agonists, each inducing antinociception through different effector mechanisms.     

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).     

Determination of the receptor selectivity of opioid agonists in the guinea-pig ileum and mouse vas deferens by use of beta-funaltrexamine

The irreversible inhibitor of mu-opioid receptor-mediated effects, beta-funaltrexamine (beta-FNA), was used to investigate the selectivity of various opioid agonists at mu-opioid receptors in the electrically stimulated guinea-pig ileum and mouse vas deferens preparations in vitro. In the guinea-pig ileum, pretreatment with beta-FNA (3 X 10(-8) - 3 X 10(-6)M) produced a concentration-dependent antagonism of the inhibitory effect produced by the mu-opioid receptor agonist [D-Ala2, MePhe4, Gly(ol)5]enkephalin (DAGO). High concentrations of beta-FNA (3 X 10(-6) - 1 X 10(-5)M) also antagonized the inhibitory effects of the kappa-opioid agonist U50488. Pretreatment of guinea-pig ileum with beta-FNA at 1 X 10(-6)M resulted in blockade of the effect of some opioid agonists. The compounds which showed the largest rightward shifts in their concentration-response curves, and hence the greatest mu/kappa opioid receptor selectivity, were nalbuphine, [D-Ser2, Leu5]enkephalinyl-Thr6(DSLET), morphine, DAGO and normorphine. Responses to tifluadom, Mr 2034, ethylketocyclazocine, butorphanol, nalorphine, proxorphan and U50488 were not inhibited by beta-FNA. In the mouse vas deferens, pre-treatment with beta-FNA (1 X 10(-6)M) produced a similar shift in the dose-response curves for normorphine as in the guinea-pig ileum. The concentration-response curves for the delta-receptor agonists [D-Ala2, D-Leu5] enkephalin (DADLE) and DSLET were, however, also shifted, indicating that beta-FNA will also block delta-opioid receptors. Since beta-FNA does not block kappa-opioid receptor-mediated effects, it can be used in the guinea-pig ileum preparation as a selective mu-receptor inhibitor. However, its lack of selectivity between mu- and delta-opioid receptors should be taken into account in many other isolated tissues and experiments in vivo.     

Activation of spinal metabotropic glutamate receptors elicits cardiovascular responses in pentobarbital anesthetized rats

The aim of this study was to examine whether intrathecal (i.t.) injection of metabotropic glutamate (mGlu) receptor agonists at the thoracolumbar level of the spinal cord causes changes either in the blood pressure or in the heart rate of pentobarbital anesthetized rats. The broad spectrum mGlu receptor agonist (+/-)-1-aminocyclopentane- trans-1,3-dicarboxylic acid ( trans-ACPD) and the Group III mGluR agonist L-(+)-2-amino-4-phosphonobutyric acid ( L-AP4) induced pressor effects at doses of 300 nmol and 600 nmol (i.t.) but did not induce changes at a lower dose (150 nmol, i.t.). The specific Group I mGlu receptor agonist ( RS)-3,5-dihydroxyphenylglycine (3,5-DHPG), as well as the highly selective Group II mGlu receptor agonist 2 R,4 R-4-aminopyrrolidine-2,4-dicarboxilate (2 R,4 R-APDC), induced pressor effects at a dose of 300 nmol only. The compounds (150-600 nmol) did not modify the heart rate in these experiments. On the other hand, low doses of Group II mGlu receptor agonists (75 nmol 2 R,4 R-APDC; 1.5 nmol 2 S,2' R,3' R)-2-(2',3'-dicarboxychloropropyl)glycine; DCG IV) induced hypotension and bradycardia when spinal N-methyl- D-aspartate (NMDA) receptors were previously blocked by 2-amino-5-phosphonovaleric acid (APV; 30 nmol; i.t.). The pressor response to trans-ACPD was probably mediated by activation of both Group I and Group II mGluRs because i.t. injection of either the selective Group I mGlu receptor antagonist ( S)-4-carboxyphenylglycine (4CPG) or the selective Group II mGlu receptor antagonist (2 S,3 S,4 S)-2-methyl-2-(carboxycyclopropyl)glycine (MCCG) antagonized the increases in the blood pressure produced by the agonist. Moreover, 4CPG and MCCG antagonized the pressor effects of 3,5-DHPG and 2 R,4 R-APDC, respectively. Blockade of spinal Group II mGlu receptors by MCCG also prevented the hypotensive and bradycardic effects of 2 R,4 R-APDC and DCG IV in rats pretreated with APV. On the other hand, the pressor response to L-AP4 (300 nmol) was prevented by the selective antagonist ( S)-2-amino-2-methyl-4-phosphonobutanoic acid (MAP4).These results suggest that activation of spinal Group I, II and III mGlu receptors increases the mean blood pressure in pentobarbital anesthetized rats and that, after blockade of NMDA receptors, low doses of Group II mGlu receptor agonists induce hypotension and bradycardia.     

Role of metabotropic glutamate receptors in the hypersusceptibility to pentylenetetrazole-induced seizure during diazepam withdrawal

To clarify the contribution of metabotropic glutamate (mGlu) receptors in brain to benzodiazepine withdrawal signs, we now examine the effects in mice of selective ligands for three subgroups of mGlu receptor on the hypersusceptibility to pentylenetetrazole-induced seizure during diazepam withdrawal. The seizure threshold for pentylenetetrazole was significantly decreased by the discontinuation of chronic diazepam treatment. The decrease in the seizure threshold for pentylenetetrazole during diazepam withdrawal was significantly suppressed by intracerebroventricular (i.c.v.) pretreatment with the group 1 mGlu receptor antagonist, (S)-4-carboxyphenylglycine ((S)-4CPG: 56 and 100 nmol). These doses of (S)-4CPG did not alter the seizure threshold in chronically vehicle-treated (control) mice. Pretreatment i.c.v. with a presynaptic mGlu receptor agonist (the group 2 mGlu receptor agonist (2S,1'S,2'S)-2-(carboxycyclopropyl)-glycine (L-CCG-I: 3.0 and 5.6 nmol) and the group 3 mGlu receptor agonist, L-amino-4-phosphonobutyric acid (L-AP4: 3.0 and 5.6 nmol)) failed to suppress the decrease in seizure threshold in diazepam-withdrawn mice, but increased the seizure threshold in control mice. Pretreatment i.c.v. with the group 1 mGlu receptor antagonist/group 2 mGlu receptor agonist, (S)-4-carboxy-3-hydroxyphenylglycine ((S)-4C3HPG: 56 and 100 nmol), significantly increased the seizure threshold in control mice and suppressed the decrease in seizure threshold in diazepam-withdrawn mice. These findings suggest that enhancement of group 1 mGlu receptor function and a decline in both group 2 and group 3 mGlu receptor functions may play an important role in the hypersusceptibility to pentylenetetrazole-induced seizure during diazepam withdrawal.     

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.     

Face-washing behavior induced by the group I metabotropic glutamate receptor agonist (S)-3,5-DHPG in mice is mediated by mGlu1 receptor

It is known for the non-selective group I metabotropic glutamate (mGlu) receptors agonist (S)-3,5-dihydroxyphenylglycine (S-3,5-DHPG) to cause convulsions, which are mediated by mGlu1 receptor. However, the behavioral changes other than convulsions caused by (S)-3,5-DHPG have not been well studied. The purpose of the present study was to explore the behavioral changes elicited by activation of group I mGlu receptors with (S)-3,5-DHPG and to clarify which, mGlu1 receptor or mGlu5 receptor, is responsible for such behavior. (S)-3,5-DHPG at doses of 3-30 nmol caused characteristic face-washing behavior. This behavioral change was inhibited by both the competitive mGlu1 receptor antagonists (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA) and (S)-4-carboxyphenylglycine (S-4CPG) and the non-competitive mGlu1 receptor antagonist, 4-[1-(2-fluoropyridin-3-yl)-5-methyl-1H-1,2,3-triazol-4-yl]-N-isopropyl-N-methyl-3,6-dihydropyridine-1(2H)-carboxamide (FTIDC), but not by the mGlu5 receptor antagonist 2-Methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP), the mGlu2/3 receptor agonist (-)-2-oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate (LY379268), the mGlu2/3 receptor antagonist (2S)-2-amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid (LY341495), the N-methyl-d-asparate (NMDA) receptor antagonist 5R,10S-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801), or the competitive non-NMDA receptor antagonist 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX). These findings indicate that face-washing behavior is due to selective activation of mGlu1 receptor by (S)-3,5-DHPG, and that the face-washing behavior induced by (S)-3,5-DHPG in mice can be used for in vivo testing of the antagonistic potency of both competitive and non-competitive mGlu1 receptor antagonists.