Contribution of mu and delta opioid receptors to the pharmacological profile of kappa opioid receptor subtypes

Molecular cloning has identified three opioid receptors: mu (MOR), delta (DOR) and kappa (KOR). Yet, cloning of these receptor types has offered little clarification to the diverse pharmacological profiles seen within the growing number of novel opioid ligands, which has led to the proposal of multiple subtypes. In the present study, utilizing in vitro and in vivo methods including the use of opioid receptor knockout mice, we find that certain antinociceptive effects of the KOR‐1 and KOR‐2 subtype‐selective ligands (+)‐(5α,7α,8β)‐N‐Methyl‐N‐[7‐(1‐pyrrolidinyl)‐1‐oxaspiro[4.5]dec‐8‐yl]‐benzene‐acetamide (U69, 593) and 4‐[(3,4‐Dichlorophenyl)acetyl]‐3‐(1‐pyrrolidinylmethyl)‐1‐piperazine‐carboxylic acid methyl ester fumarate (GR89, 696), respectively, are potentiated by antagonism of MOR and DOR receptors. We believe that our findings can be best explained by the existence of KOR‐DOR and KOR‐MOR heteromers. We only find evidence for the existence of these heteromers in neurons mediating mechanical nociception, but not thermal nociception. These findings have important clinical ramifications as they reveal new drug targets that may provide avenues for more effective pain therapies.     

In vivo characterization of MMP-2200, a mixed δ/μ opioid agonist, in mice

We have previously reported the chemistry and antinociceptive properties of a series of glycosylated enkephalin analogs (glycopeptides) exhibiting approximately equal affinity and efficacy at δ opioid receptors (DORs) and μ opioid receptors (MORs). More detailed pharmacology of the lead glycopeptide MMP-2200 [H?N-Tyr-D-Thr-Gly-Phe-Leu-Ser-(O-β-D-lactose)-CONH?] is presented. MMP-2200 produced dose-related antinociception in the 55°C tail-flick assay after various routes of administration. The antinociceptive effects of MMP-2200 were blocked by pretreatment with the general opioid antagonist naloxone and partially blocked by the MOR-selective antagonist β-funaltrexamine and the DOR-selective antagonist naltrindole. The κ opioid receptor antagonist nor-binaltorphimine and the peripherally active opioid antagonist naloxone-methiodide were ineffective in blocking the antinociceptive effects of MMP-2200. At equi-antinociceptive doses, MMP-2200 produced significantly less stimulation of locomotor activity compared with morphine. Repeated administration of equivalent doses of morphine and MMP-2200 (twice daily for 3 days) produced antinociceptive tolerance (~13- and 5-fold rightward shifts, respectively). In acute and chronic physical dependence assays, naloxone precipitated a more severe withdrawal in mice receiving morphine compared with equivalent doses of the glycopeptide. Both morphine and MMP-2200 inhibited respiration and gastrointestinal transit. In summary, MMP-2200 acts as a mixed DOR/MOR agonist in vivo, which may in part account for its high antinociceptive potency after systemic administration, as well as its decreased propensity to produce locomotor stimulation, tolerance, and physical dependence in mice, compared with the MOR-selective agonist morphine. For other measures (e.g., gastrointestinal transit and respiration), the significant MOR component may not allow differentiation from morphine.     

Comparison of pharmacological activities of three distinct kappa ligands (Salvinorin A, TRK-820 and 3FLB) on kappa opioid receptors in vitro and their antipruritic and antinociceptive activities in vivo

Salvinorin A, TRK-820 (17-cyclopropylmethyl-3,14beta-dihydroxy-4,5alpha-epoxy-6beta-[N-methyl-trans-3-(3-furyl) acrylamido]morphinan hydrochloride), and 3FLB (diethyl 2,4-di-[3-fluorophenyl]-3,7-dimethyl-3,7-diazabicyclo[3.3.1]nonane-9-one-1,5-dicarboxylate) are structurally distinctly different from U50,488H [(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide methanesulfonate], the prototypic selective kappa agonist. Here, we investigated their in vitro pharmacological activities on receptors expressed in Chinese hamster ovary cells and in vivo antiscratch and antinociceptive activities in mice. All three compounds showed high selectivity for the kappa opioid receptor (KOR) over the mu opioid receptor (MOR) and delta opioid receptor (DOR) and nociceptin or orphanin FQ receptors. In the guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding assay, all three were full agonists on the KOR. The rank order of affinity and potency for the KOR was TRK-820 > U50,488H approximately salvinorin A > 3FLB. TRK-820 acted as a partial agonist on MOR and DOR, whereas salvinorin A and 3FLB showed no activities on these receptors. Salvinorin A, TRK-820, and 3FLB caused internalization of the human KOR in a dose-dependent manner. Interestingly, although salvinorin A and U50,488H had similar potencies in stimulating [(35)S]GTPgammaS binding, salvinorin A was about 40-fold less potent than U50,488H in promoting internalization. Following 4-h incubation, all three compounds induced down-regulation of the human KOR, with salvinorin A causing a lower extent of down-regulation. Although TRK-820 was potent and efficacious against compound 48/80-induced scratching, salvinorin A showed low and inconsistent effects, and 3FLB was inactive. In addition, salvinorin A and 3FLB were not active in the acetic acid abdominal constriction test. The discrepancy between in vitro and in vivo results may be due to in vivo metabolism of salvinorin A and 3FLB and possibly to their effects on other pharmacological targets.     

Studies of micro-, kappa-, and delta-opioid receptor density and G protein activation in the cortex and thalamus of monkeys

The aim of this study was to investigate the relative density of micro -, kappa-, and delta-opioid receptors (MOR, KOR, and DOR) and guanosine 5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS) binding stimulated by full agonists in cortical and thalamic membranes of monkeys. The binding parameters [Bmax (femtomoles per milligram)/Kd (nanomolar)] were as follows: [3H][d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO) (MOR; 80/0.7), [3H]U69593 [(5alpha,7alpha,8beta)-(-)-N-methyl-N-(7-(1-pyrrolidinyl)-1-oxaspiro(4,5)dec-8-yl) benzeneacetamide] (KOR; 116/1.3), and [3H][d-Pen2,d-Pen5]-enkephalin (DPDPE) (DOR; 87/1.3) in the cortex; [3H]DAMGO (147/0.9), [3H]U69593 (75/2.5), and [3H]DPDPE (22/2.0) in the thalamus. The relative proportions of MOR, KOR, and DOR in the cortex were 28, 41, and 31% and in the thalamus were 60, 31, and 9%. Full selective opioid agonists, DAMGO (EC50 = 532-565 nM) and U69593 (EC50 = 80-109 nM) stimulated [35S]GTPgammaS binding in membranes of cortex and thalamus, whereas SNC80 [(+)-4-[(alphaR)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethyl-benzamide] (DOR; EC50 = 68 nM) was only active in cortical membranes. The magnitudes of [35S]GTPgammaS binding stimulated by these agonists were similar in the cortex, ranging from 17 to 25% over basal binding. In the thalamus, DAMGO and U69593 increased [35S]GTPgammaS binding by 44 and 23% over basal, respectively. Opioid agonist-stimulated [35S]GTPgammaS binding was blocked selectively by antagonists for MOR, KOR, and DOR. The amount of G protein activated by agonists was highly proportional to the relative receptor densities in both regions. These results distinguish the ability of opioid agonists to activate G proteins and provide a functional correlate of ligand-binding experiments in the monkey brain. In particular, the relative densities of opioid receptor binding sites in the two brain areas reflect their functional roles in the pharmacological actions of opioids in the central nervous system of primates.     

DPDPE-UK14,304 synergy is retained in mu opioid receptor knockout mice

When agonists to alpha(2)adrenergic receptor (AR) and delta opioid receptor (DOR) are co-administered, they act synergistically to inhibit nociceptive elicited behavior. Some previous studies of synergism have used the DOR-selective agonist [D-Pen(2),D-Pen(5)]-enkehphalin (DPDPE), however, DPDPE has been shown to be less potent in mu opioid receptor-knockout (MOR-KO) mice. It is possible, therefore, that MOR contributes to the synergism of DPDPE with the alpha(2)AR agonists. We compared the interactions of spinally administered DPDPE with an alpha(2)AR-adrenergic agonist in MOR-KO and MOR-wildtype (WT) mice. In these mice, morphine is ineffective and the potency of spinally administered DOR agonists, deltorphin II (DELT II) and DPDPE decreased 16- and 250-fold, respectively. Antagonism studies using the MOR-selective antagonist, D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Phe-Thr-NH(2) (CTOP) and the DOR-selective antagonist, naltrindole HCl (naltrindole) demonstrated that while DOR mediates DPDPE-induced antinociception in MOR-KO, both MOR and DOR participate in DPDPE antinociception in WT mice, suggesting that DPDPE is less selective for DOR than previously observed in binding studies when given in vivo. The potency of the alpha(2)AR agonist UK14,304 was equivalent in WT and MOR-KO, demonstrating that the loss of opioid-mediated antinociception in the MOR-KO was not due to generalized impairment of antinociceptive processing. Interestingly, isobolographic analysis showed that, despite substantial loss of DPDPE potency in MOR-KO, DPDPE-UK14,304 synergism is fully retained. Collectively, these experiments demonstrate that although MOR participates in DELT II- and DPDPE-mediated spinal antinociception, DOR independently participates in synergistic antinociception with alpha(2)AR. Resolution of the roles of the opioid receptor subtypes in opioid agonist-induced effects may require comparison of the effects of multiple selective agonists in knockout animals.     

Intramuscular administration of morphine reduces mustard oil‐induced craniofacial muscle pain behavior in lightly anesthetized rats

The present study investigated the role of peripheral opioid receptors in mustard oil‐induced nociceptive behavior and inflammation in the masseter muscles of lightly anesthetized rats. Experiments were carried out on male Sprague–Dawley rats weighing between 300 and 400g. After initial anesthesia with sodium pentobarbital (40mg/kg, i.p.), one femoral vein was cannulated and connected to an infusion pump for the intravenous infusion of sodium pentobarbital. The rate of infusion was adjusted to provide a constant level of anesthesia. Mustard oil (MO, 30μl) was injected into the mid‐region of the left masseter muscle via a 30‐gauge needle. Intramuscularly‐administered morphine significantly reduced shaking behavior but not MO‐induced inflammation. Intramuscular pretreatment with naloxone, an opioid receptor antagonist, reversed antinociception produced by intramuscularly‐administered morphine, while intracisternal administration of naloxone did not affect the antinociception of peripheral morphine. Pretreatment with d ‐Pen‐Cys‐Tyr‐d ‐Trp‐Orn‐Thr‐Pen‐Thr‐NH2 (CTOP), a μ opioid receptor antagonist, but not naltrindole, a δ opioid receptor antagonist, nor norbinaltorphimine (nor‐BNI), a κ opioid receptor antagonist, reversed intramuscularly‐administered morphine‐induced antinociception. These results indicate that intramuscularly‐administered morphine produces antinociception in craniofacial muscle nociception and that this intramuscularly‐administered morphine‐induced antinociception is mediated by a peripheral μ opioid receptor. Our observations further support the clinical approach of administering opioids in the periphery for the treatment of craniofacial muscle nociception.     

Different effects of opioid antagonists on mu-, delta-, and kappa-opioid receptors with and without agonist pretreatment

Opioid receptors display basal signaling (constitutive, agonist-independent activity), which seems to be regulated by agonist exposure. Whereas agonist pretreatment desensitizes receptors to subsequent agonist stimulation, basal signaling of mu-opioid receptor (MOR) was shown to increase. Moreover, agonist pretreatment converts the neutral antagonists naloxone and naltrexone into inverse agonists, suppressing basal signaling, whereas analogs with reduced C6-position, e.g., 6beta-naltrexol, remain neutral antagonists at MOR under any condition. This study compares the regulation of basal signaling of MOR, delta-(DOR), and kappa-(KOR) opioid receptors after pretreatment with morphine or receptor-selective agonists, in transfected human embryonic kidney 293 cell membranes. Moreover, naloxone, naltrexone, and related antagonists were compared for binding potency and effect on basal and agonist-stimulated receptor signaling, measuring guanosine 5'-O-(3-[35S]thio)triphosphate binding. The results demonstrate basal activity for each opioid receptor, which is modulated by pretreatment with agonists. Even closely related opioid antagonists display distinct patterns of neutral and inverse effects before and after agonist pretreatment, including distinct efficacies between naloxone and naltrexone at agonist-pretreated DOR and KOR. Pretreatment with different agonists has varying effects on inverse and neutral activities of some analogs tested. These results demonstrate that antagonist efficacy is context-dependent, possibly accounting for paradoxical pharmacological effects. Activity profiles at the three opioid receptors under different conditions could lead to antagonists with optimal clinical properties in treatment of addiction and adverse opioid effects.     

Endogenous opioid peptides contribute to antinociceptive potency of intrathecal [Dmt1]DALDA

[Dmt(1)]DALDA (H-Dmt-d-Arg-Phe-Lys-NH(2); Dmt = 2',6'-dimethyltyrosine) is a dermorphin analog that shows high affinity and selectivity for the mu opioid receptor. The intrathecal potency of [Dmt(1)]DALDA far exceeded its affinity at mu receptors and suggests that other mechanisms must be involved in its action in the spinal cord. The affinity and selectivity of [Dmt(1)]DALDA was determined using cell membranes expressing cloned human mu, delta, and kappa opioid receptors. Competitive displacement binding with [(3)H][Dmt(1)]DALDA, [(3)H]DPDPE (H-Tyr-d-Pen-Gly-Phe-d-Pen), and [(3)H]U69,593 [(5alpha,7alpha,8beta)-(+)-N-methyl-N-(7-[1-pyrrolidinyl]-1-oxaspiro[4.5]dec-8-yl)-benzeneacetamide] revealed K(i) of 156 +/- 26 pM for mu opioid receptor (MOR), 1.67 +/- 0.04 microM for delta opioid receptor (DOR), and K(i) of 4.4 +/- 1.7 nM for kappa opioid receptor (KOR), respectively. [Dmt(1)]DALDA increased guanosine 5'-O-(3-[(35)S]thiotriphosphate) binding in MOR, DOR, and KOR membranes, with EC(50) being 17 (8.8-33) nM, 2 (1.2-3.2) microM, and 124 (15-1000) nM, respectively. Intrathecal [Dmt(1)]DALDA inhibited the tail-flick response in mice with ED(50) = 1.22 (0.59-2.34) pmol. Intrathecal administration of an antiserum against dynorphin A(1-17) or [Met(5)]enkephalin significantly attenuated the response to i.t. [Dmt(1)]DALDA, resulting in ED(50) of 6.2 (3.6-12.6) pmol and 6.6 (3.5-19.6) pmol, respectively. Neither antisera had any effect on the response to i.t. morphine. Intracerebroventricular (i.c.v.) [Dmt(1)]DALDA was not affected by previous i.c.v. administration of anti-Dyn or anti-ME. Pretreatment with norbinaltorphimine or naltriben also attenuated the antinociceptive response to i.t., but not i.c.v., [Dmt(1)]DALDA. These data suggest that i.t. [Dmt(1)]DALDA causes the release of dynorphin and [Met(5)]enkephalin-like substances that act at kappa and delta receptors, respectively, to contribute to the extraordinary potency of [Dmt(1)]DALDA.     

A role for histamine and H2-receptors in opioid antinociception

To investigate the role of brain H2-receptors in opioid antinociceptive mechanisms, the effects of several antagonists of histamine H2-receptors were determined on morphine (MOR)-induced antinociception, opioid-mediated footshock-induced antinociception (FSIA) and on other opiate effects in rats. Zolantidine dimaleate (ZOL), the first brain-penetrating H2 antagonist (0.03-1.6 mumol/kg s.c.) caused a dose-related inhibition of MOR antinociception in both the tail-flick and hot-plate tests, with no effect on base-line responding. ZOL also inhibited opioid FISA with a similar potency. MOR-induced locomotor activity was also reduced by ZOL, but no effect was seen on MOR-induced hyperthermia, catalepsy or lethality. ZOL (10(-5) M) was inactive at mu, delta or kappa opioid receptors and showed at least 35-fold higher affinity at the H2-receptor than at receptors for serotonin, dopamine, norepinephrine or acetylcholine in brain. To clarify further the role of H2-receptors in ZOL's antiopiate activity, the potencies of seven structural congeners of ZOL were determined on the H2-receptor and on MOR antinociception. Over 3 orders of magnitude, the rank order of potencies of the compounds for inhibiting MOR antinociception was highly correlated with their potencies as H2 antagonists. Cimetidine, unlike other H2 antagonists, potentiated MOR antinociception, potentiated opioid FSIA and increased brain MOR levels, actions that are not likely to be due to blockade of H2-receptors. These findings strongly suggest that stimulation of opioid receptors leads to antinociception by mechanisms that include activation of brain H2-receptors.     

Spinal Endomorphin 2 Antinociception and the Mechanisms That Produce It Are Both Sex- and Stage of Estrus Cycle–Dependent in Rats

Endomorphin 2 (EM2) is the predominant endogenous mu-opioid receptor (MOR) ligand in the spinal cord. Given its endogenous presence, antinociceptive responsiveness to the intrathecal application of EM2 most likely reflects its ability to modulate nociception when released in situ. In order to explore the physiological pliability of sex-dependent differences in spinal MOR-mediated antinociception, we investigated the antinociception produced by intrathecal EM2 in male, proestrus female, and diestrus female rats. Antinociception was reflected by changes in tail flick latency to radiant heat. In females, the spinal EM2 antinociceptive system oscillated between analgesically active and inactive states. During diestrus, when circulating estrogens are low, spinal EM2 antinociceptive responsiveness was minimal. In contrast, during proestrus, when circulating estrogens are high, spinal EM2 antinociception was robust and comparable in magnitude to that manifest by males. Furthermore, in proestrus females, spinal EM2 antinociception required spinal dynorphin and kappa-opioid receptor activation, concomitant with MOR activation. This is required for neither spinal EM2 antinociception in males nor the antinociception elicited in proestrus females by spinal sufentanil or [d-Ala²,N-methyl-Phe⁴,Gly-ol⁵]-enkephalin, which are prototypic MOR-selective nonpeptide and peptide agonists, respectively. These results reveal that spinal EM2 antinociception and the signaling mechanisms used to produce it fundamentally differ in males and females.     

Delta opioid receptor mediated actions in the rostral ventromedial medulla on tail flick latency and nociceptive modulatory neurons

The rostral ventromedial medulla (RVM) is critical for the modulation of dorsal horn nociceptive transmission. Three classes of RVM neurons (ON, OFF, and NEUTRAL) have been described that have distinct responses to noxious stimuli and mu opioid receptor (MOR) agonists. The present study in barbiturate anesthetized rats investigated the effects of the delta 2 opioid receptor (DOR2) agonist, [D-Ala2]deltorphin II (DELT), microinfused into the RVM on the tail flick reflex and activity of RVM neurons. Tail flick latencies increased dose-dependently after administration of DELT (0.6 nmol and 1.2 nmol). Furthermore, DELT inhibited the tail flick related increase in ON cell activity and shortened the tail flick related pause in OFF cell activity. The activity of NEUTRAL cells was not affected. The antinociceptive effects and corresponding changes in ON and OFF cell activity produced by DELT were antagonized by the DOR2 antagonist, naltriben methanesulfonate, administered at the same site. These DOR2 mediated effects on noxious stimulation-evoked changes in RVM neuronal activity are similar to those reported for MOR agonists and suggest that both DOR2 and MOR produce analgesia through activation of OFF cells.     

Heteromerization of the μ- and δ-opioid receptors produces ligand-biased antagonism and alters μ-receptor trafficking

Heteromerization of opioid receptors has been shown to alter opioid receptor pharmacology. However, how receptor heteromerization affects the processes of endocytosis and postendocytic sorting has not been closely examined. This question is of particular relevance for heteromers of the μ-opioid receptor (MOR) and δ-opioid receptor (DOR), because the MOR is recycled primarily after endocytosis and the DOR is degraded in the lysosome. Here, we examined the endocytic and postendocytic fate of MORs, DORs, and DOR/MOR heteromers in human embryonic kidney 293 cells stably expressing each receptor alone or coexpressing both receptors. We found that the clinically relevant MOR agonist methadone promotes endocytosis of MOR but also the DOR/MOR heteromer. Furthermore, we show that DOR/MOR heteromers that are endocytosed in response to methadone are targeted for degradation, whereas MORs in the same cell are significantly more stable. It is noteworthy that we found that the DOR-selective antagonist naltriben mesylate could block both methadone- and [D-Ala2,NMe-Phe4,Gly-ol5]-enkephalin-induced endocytosis of the DOR/MOR heteromers but did not block signaling from this heteromer. Together, our results suggest that the MOR adopts novel trafficking properties in the context of the DOR/MOR heteromer. In addition, they suggest that the heteromer shows "biased antagonism," whereby DOR antagonist can inhibit trafficking but not signaling of the DOR/MOR heteromer.     

Antiexudative effects of opioids and expression of kappa- and delta-opioid receptors during intestinal inflammation in mice: involvement of nitric oxide

The study evaluates the effects of kappa- (KOR), delta- (DOR), and mu-opioid receptor (MOR) agonists on the inhibition of plasma extravasation during acute and chronic intestinal inflammation in mice. The antiexudative effects of KOR and DOR agonists in animals treated with nitric oxide synthase (NOS) inhibitors and their protein levels in the gut (whole jejunum and mucosa) and spinal cord of mice with chronic intestinal inflammation were also measured. Inflammation was induced by the intragastric administration of one (acute) or two (chronic) doses of croton oil. Plasma extravasation was measured using Evans blue and protein levels by Western blot and immunoprecipitation. Plasma extravasation was significantly increased 2.7 times during chronic inflammation. The potency of the KOR agonist trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolydinyl)cyclohexyl]-benzeneazetamine (U50,488H) inhibiting plasma extravasation was enhanced 26.3 times during chronic compared with acute inflammation. [d-Pen(2),d-Pen(5)]-Enkephalin (DPDPE) (a DOR agonist) was also 11.8 times more potent during chronic inflammation, whereas the antiexudative effects of fentanyl (a MOR agonist) were not significantly altered. Receptor-specific antagonists reversed the effects. Protein levels of KOR and DOR in the whole jejunum and mucosa were significantly increased after chronic inflammation. Treatment with NOS inhibitors N(omega)-nitro-l-arginine methyl ester or l-N(6)-(1-iminoethyl)-lysine hydrochloride diminished plasma extravasation and inhibited the increased antiexudative effects of U50,488H and DPDPE during chronic intestinal inflammation. The data show that the enhanced antiexudative effects of KOR and DOR agonists could be related to an increased expression of KOR and DOR in the gut and that the release of nitric oxide may play a role augmenting the effects of opioids during chronic inflammation.     

Involvement of local cholecystokinin in the tolerance induced by morphine microinjections into the periaqueductal gray of rats

The ventrolateral periaqueductal gray (PAG) is a key structure for the development of opioid tolerance. An increased activity of 'anti-opioids' like cholecystokinin (CCK) has been proposed as a possible mechanism for opioid tolerance. The present study evaluates the role of PAG-located CCK in the opioid tolerance induced by repeated microinjections of morphine (MOR) into PAG. Male rats were implanted with chronic guide cannulae aimed at the PAG. Microinjection of MOR (0.5 microg in 0.5 microl) into PAG caused antinociception as quantified with the tail flick and the hot plate tests. When MOR microinjection was repeated twice daily, the antinociceptive effect disappeared within 2 days (tolerance). However, if each MOR microinjection was preceded (within 15 min) by a microinjection of the non-selective CCK receptor antagonist proglumide (PRO), (0.4 microg in 0.5 microl) into the same PAG site, the microinjections of MOR always produced antinociception and did not induce tolerance. If PRO microinjections were suspended, subsequent MOR microinjections induced tolerance. In MOR-tolerant rats, a single PRO microinjection into the same PAG site was enough to restore the antinociceptive effect of MOR. On the other hand, if CCK (1 ng in 0.5 microl) was microinjected into PAG, then MOR microinjection administered 15 min later into the same PAG site did not elicit antinociception. These results show that CCK has anti-opioid activity in PAG and that tolerance to MOR in PAG can be prevented or reversed if CCK receptors are blocked with PRO. Finally, opioid tolerance induced by repeated systemic MOR injections (5mg/kg intraperitoneal ) was reversed by a single microinjection of PRO into PAG. This emphasizes the central importance of PAG in the MOR/CCK interactions that lead to opioid tolerance.     

Regulation of ingestive behaviors in the rat by GSK1521498, a novel micro-opioid receptor-selective inverse agonist

μ-Opioid receptor (MOR) agonism induces palatable food consumption principally through modulation of the rewarding properties of food. N-{[3,5-difluoro-3'-(1H-1,2,4-triazol-3-yl)-4-biphenylyl]methyl}-2,3-dihydro-1H-inden-2-amine (GSK1521498) is a novel opioid receptor inverse agonist that, on the basis of in vitro affinity assays, is greater than 10- or 50-fold selective for human or rat MOR, respectively, compared with κ-opioid receptors (KOR) and δ-opioid receptors (DOR). Likewise, preferential MOR occupancy versus KOR and DOR was observed by autoradiography in brain slices from Long Evans rats dosed orally with the drug. GSK1521498 suppressed nocturnal food consumption of standard or palatable chow in lean and diet-induced obese (DIO) Long Evans rats. Both the dose-response relationship and time course of efficacy in lean rats fed palatable chow correlated with μ receptor occupancy and the plasma concentration profile of the drug. Chronic oral administration of GSK1521498 induced body weight loss in DIO rats, which comprised fat mass reduction. The reduction in body weight was equivalent to the cumulative reduction in food consumption; thus, the effect of GSK1521498 on body weight is related to inhibition of food consumption. GSK1521498 suppressed the preference for sucrose-containing solutions in lean rats. In operant response models also using lean rats, GSK1521498 reduced the reinforcement efficacy of palatable food reward and enhanced satiety. In conclusion, GSK1521498 is a potent, MOR-selective inverse agonist that modulates the hedonic aspects of ingestion and, therefore, could represent a pharmacological treatment for obesity and binge-eating disorders.     

Interaction between medullary and spinal delta1 and delta2 opioid receptors in the production of antinociception in the rat

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.     

Further analysis of acute antinociceptive and anti‐inflammatory actions of 4‐phenylselenyl‐7‐chloroquinoline in mice

A new quinoline containing selenium, 4‐phenylselenyl‐7‐chloroquinoline (4‐PSQ), was described and synthetized by our research group. Recently, we demonstrated the potential antinociceptive and anti‐inflammatory of 4‐PSQ. For this reason, the first objective of this study was to expand our previous findings by investigating the contribution of glutamatergic, serotonergic, and nitrergic systems to the acute antinociceptive action of this compound. Pretreatment with 4‐PSQ (0.01–25 mg/kg, p.o.) reduced the nociception induced by glutamate. MK‐801 (an uncompetitive antagonist of the N‐Methyl‐d ‐aspartate (NMDA) receptor) blocked the antinociceptive effect exerted by 4‐PSQ (25 mg/kg, p.o.) in the acetic acid‐induced abdominal writhing test. The pretreatment with WAY100635 (a selective antagonist of 5‐HT_1A receptor), ketanserin (a selective antagonist of 5‐HT_2A/2C receptor), and pindolol (a nonselective antagonist of 5‐HT_1A/1B receptors) partially blocked the antinociceptive effect caused by 4‐PSQ (25 mg/kg, per oral, p.o.) in the acetic acid‐induced abdominal writhing test. Nitric oxide precursor, l ‐arginine hydrochloride, partially reversed antinociception caused by 4‐PSQ or ω‐nitro‐l ‐arginine (l ‐NOARG). Treatments did not modify the locomotor and exploratory activities of mice. Additionally, the acute anti‐inflammatory effect of 4‐PSQ in a model of pleurisy induced by carrageenan in mice was investigated. 4‐PSQ reduced the cellular migration, pleural exudate accumulation, and myeloperoxidase activity induced by carrageenan exposure. 4‐PSQ protected against the increase in reactive species levels and reduction of nonprotein thiol levels induced by carrageenan. Data presented here showed that the modulation of serotonergic, nitrergic, and glutamatergic systems contributed to the antinociceptive effect of 4‐PSQ and it reinforced the therapeutic potential of this quinolinic compound for acute inflammation.     

Effects of atypical kappa-opioid receptor agonists on intrathecal morphine-induced itch and analgesia in primates

Itch/pruritus is the most common side effect associated with spinal administration of morphine given to humans for analgesia. The aim of this study was to investigate the effectiveness of kappa-opioid receptor (KOR) agonists with diverse chemical structures as antipruritics and to elucidate the receptor mechanism underlying the antipruritic effect in monkeys. In particular, previously proposed non-KOR-1 agonists, including nalfurafine [TRK-820, 17-cyclopropylmethyl-3,14 beta-dihydroxy-4,5 alpha-epoxy-6 beta-[N-methyl-trans-3-(3-furyl)acrylamido]morphinan], bremazocine [(+/-)-6-ethyl-1,2,3,4,5,6-hexahydro-3-[(1-hydroxycyclopropy)-methyl]-11,11-dimethyl-2,6-methano-3-benzazocin-8-ol], and GR 89696 [4-[(3,4-dichlorophenyl)acetyl]-3-(1-pyrrolidinylmethyl)-1-piperazinecarboxylic acid methyl ester] were studied in various behavioral assays for measuring itch/scratching, analgesia, and respiratory depression. Systemic administration of nalfurafine (0.1-1 microg/kg), bremazocine (0.1-1 microg/kg), or GR 89696 (0.01-0.1 microg/kg) dose-dependently attenuated intrathecal morphine (0.03 mg)-induced scratching responses without affecting morphine antinociception. The combination of intrathecal morphine with these KOR agonists did not cause sedation. In addition, pretreatment with effective antiscratching doses of nalfurafine, bremazocine, or GR 89696 did not antagonize systemic morphine-induced antinociception and respiratory depression. The dose-addition analysis revealed that there is no subadditivity for nalfurafine in combination with morphine in the antinociceptive effect. Furthermore, the KOR antagonist study revealed that antiscratching effects of both nalfurafine and a prototypical KOR-1 agonist, U-50488H [trans-(+/-)-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl]-cyclohexyl)-benzeneacetamide], could be blocked completely by a selective KOR antagonist, nor-binaltorphimine (3 mg/kg). These findings suggest that the agonist action on KOR mainly contributes to the effectiveness of these atypical KOR agonists as antipruritics, and there is no evidence for KOR subtypes or mu-opioid antagonist action underlying the effects of these KOR agonists. This mechanism-based study further supports the clinical potential of KOR agonists as antipruritics under the context of spinal opioid analgesia.     

Mechanisms responsible for the enhanced antinociceptive effects of micro-opioid receptor agonists in the rostral ventromedial medulla of male rats with persistent inflammatory pain

This study investigated three possible mechanisms by which the antinociceptive effects of the mu-opioid receptor (MOR) agonist [d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO) and the delta-opioid receptor (DOR) agonist [d-Ala(2),Glu(4)]-deltorphin (deltorphin II) (DELT), microinjected into the rostral ventromedial medulla (RVM), are enhanced in rats with persistent inflammatory injury. Radioligand binding determined that neither the B(max) nor the K(d) values of [(3)H]DAMGO differed in RVM membranes from rats that received an intraplantar injection of saline or complete Freund's adjuvant (CFA) in one hindpaw 4 h, 4 days, or 2 weeks earlier. Likewise, neither the EC(50) nor the E(max) value for DAMGO-induced stimulation of guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding differed in the RVM of saline- or CFA-treated rats at any time point. Microinjection of fixed dose combinations of DAMGO and DELT in the RVM of naive rats indicated that these agonists interact synergistically to produce antinociception when DAMGO is present in equal or greater amounts than DELT and, additively, when DELT is the predominant component. Thus, unlike the periphery or spinal cord, potentiation of MOR-mediated antinociception does not entail an increase in MOR number, affinity, or coupling. Rather, the data are concordant with our proposal that potentiation results from a synergistic interaction of exogenous MOR agonist with DOR-preferring enkephalins whose levels are increased in CFA-treated rats (J Neurosci 21:2536-2545, 2001). Virtually no specific [(3)H]DELT binding nor stimulation of [(35)S]GTPgammaS binding by DELT was obtained in RVM membranes from CFA- or saline-treated rats at any time point. The mechanisms responsible for the potentiation of DELT-mediated antinociception remain to be elucidated.     

Differential antagonism of opioid delta antinociception by [D-Ala2,Leu5,Cys6]enkephalin and naltrindole 5'-isothiocyanate: evidence for delta receptor subtypes.

The present study has investigated the direct opioid delta receptor-mediated antinociception produced by i.c.v. administration of the highly selective delta agonists, [D-Pen2,D-Pen5]enkephalin (DPDPE) and [D-Ala2]deltorphin II, as well as that of the less delta-selective [D-Ser2,Leu5,Thr6]enkephalin (DSLET), by using two novel nonequilibrium opioid antagonists, [D-Ala2,Leu5,Cys6] enkephalin (DALCE) and naltrindole 5'-isothiocyanate (5'-NTII). At times ranging from 8 to 48 hr after a single i.c.v. pretreatment of mice with 5'-NTII, the antinociceptive effects of [D-Ala2] deltorphin II were significantly antagonized. In contrast, 5'-NTII pretreatment at times between 10 min and 24 hr failed to antagonize the antinociceptive effects of DPDPE. Previous studies have shown that pretreatment with i.c.v. DALCE produces a dose- and time-related antagonism of DPDPE, but not morphine, antinociception. However, pretreatment with i.c.v. DALCE failed to antagonize the antinociceptive effects of [D-Ala2]deltorphin II. Similarly, i.c.v. administration of DSLET produced time- and dose-related antinociception which was partially antagonized by either beta-funaltrexamine (beta-FNA) or by ICI 174,864 (N,N-dialyl-Tyr-Aib-Aib-Phe-Leu-OH), suggesting mixed activity at mu and delta receptors. ICI 174,864 produced essentially complete antagonism of DSLET antinociception in beta-FNA-pretreated mice. Pretreatment with 5'-NTII (at -8 to -48 hr), blocked the antinociception produced by DSLET in control or in beta-FNA-pretreated mice. In contrast, pretreatment with DALCE failed to antagonize the antinociception produced by i.c.v. DSLET in either control or in beta-FNA-pretreated mice.(ABSTRACT TRUNCATED AT 250 WORDS)