Differential agonist regulation of the human kappa-opioid receptor

Opiates are potent analgesics used clinically in the treatment of pain. A significant drawback to the chronic use and clinical effectiveness of opiates is the development of tolerance. To investigate the cellular mechanisms of tolerance, the cloned human kappa-opioid receptor was stably expressed in human embryonic kidney (HEK 293) cells, and the effects of opioid agonist treatment were examined. The receptor-expressing cells showed specific high-affinity membrane binding for a kappa-selective opioid, 3H-labeled (+)-(5alpha,7alpha,8beta)-N-methyl-N-[7-(1-pyrrolidiny l)-1-oxaspiro [4,5] dec-8-yl] benzeneacetamide ([3H]U69,593), and a nonselective opioid antagonist, [3H]diprenorphine. Pretreatment with pertussis toxin or guanosine 5'-O-(3-thiotriphosphate) reduced [3H]69,593 binding, indicating that the human K receptor coupled to G proteins of the Gi or Go families in HEK 293 cells. The receptor-mediated inhibition of adenylyl cyclase was abolished by pertussis toxin pretreatment and was blocked by a kappa-selective antagonist, norbinaltorphimine. A 3-h pretreatment with a kappa-selective agonist, (+/-)-trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl] benzeneacetamide (U50,488), caused receptor down-regulation, whereas no receptor down-regulation was found after levorphanol pretreatment. U50,488 or dynorphin A(1-17) pretreatments (3 h) desensitized the ability of U50,488 or dynorphin A(1-17) to inhibit cyclic AMP accumulation, as evidenced by a decrease in functional potency. Also, U50,488 pretreatment desensitized the ability of levorphanol to inhibit forskolin-stimulated cyclic AMP accumulation. In contrast, pretreatment of cells with either levorphanol or a potent nonselective opioid, etorphine, resulted in no apparent receptor desensitization. Taken together, these results demonstrate that the human kappa receptor is differentially regulated by selective and nonselective opioid agonists, with selective agonists able to desensitize the receptor.     

Activation of the cloned human kappa opioid receptor by agonists enhances [35S]GTPgammaS binding to membranes: determination of potencies and efficacies of ligands

Activation of kappa receptors inhibits adenylate cyclase, enhances K+ conductance and reduces Ca++ conductance via pertussis toxin-sensitive G proteins. We recently cloned a human kappa opioid receptor and stably expressed it in Chinese hamster ovary (CHO) cells. In this study, the effects of activation of the human kappa receptor by agonists on [35S]GTPgammaS binding to CHO cell membranes were examined. The presence of GDP and Mg++ was essential for the kappa agonist (-)-U50,488H-induced increase in [35S]GTPgammaS binding to be observed and the optimal concentration was 3 microM and 5 mM, respectively. The presence of 100 mM Na+ was necessary to produce the maximal signal-to-background ratio. (-)U50,488H-induced increase in [35S]GTPgammaS binding was time- and tissue concentration-dependent. (-)U50,488H increased [35S]GTPgammaS binding in a dose-dependent manner with an EC50 of 3.1 nM. (+)-U50,488H had no effect, which indicates that this effect is stereospecific. Naloxone (1 microM) or norbinaltorphimine (10 nM) shifted the dose-response curve of (-)-U50,488H to the right by 100-fold. These results indicate that enhancement of [35S]GTPgammaS binding by (-)-U50,488H is a kappa receptor-mediated event. Pretreatment of the cells with pertussis toxin, but not cholera toxin, abolished the (-)-U50,488H-induced increase in [35S]GTPgammaS binding, which indicates the involvement of Gi and/or Go proteins. [35S]GTPgammaS binding induced by (-)-U50,488H had a Kd value of 0.34 +/- 0.08 nM and a Bmax value of 431 +/- 29 fmol/mg protein. The rank order of potencies of opioid ligands tested in stimulating [35S]GTPgammaS binding was dynorphin A 1-17 > (+/-)-ethylketocyclazocine > beta-funaltrexamine, (-)-U50,488H, tifluadom > nalorphine > pentazocine, nalbuphine > buprenorphine. Dynorphin A 1-17, (+/-)-ethylketocyclazocine, (-)-U50,488H, tifluadom and beta-funaltrexamine were full agonists, but nalorphine and pentazocine were partial agonists producing maximal responses of 68% and 23% of those of full agonists, respectively. Nalbuphine and buprenorphine had low levels of agonist activities. Norbinaltorphimine and naloxone were antagonists devoid of activities. Enhancement of [35S]GTPgammaS binding by kappa agonists provides a simple functional measure for receptor activation and can be used for determination of potencies and efficacies of opioid ligands at the kappa receptor.     

Effects of chronic U50,488H treatment on binding and mechanical responses of the rat hearts

The effects of chronic injection of U50,488H (trans-3,4-dichloro-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeacetamidel++ +), a selective kappa opioid agonist, on the properties of the binding sites of tritiated U69593 [(5 alpha,7 alpha,8 beta)-(-)-N-methyl-N-(7-(1-pyrrolidinyl)-1-oxaspiro (4,5)dec-8-yl)benzeneacetamide], another selective kappa opioid agonist, and mechanical responses to U50,488H of the heart were studied. Rats received injection twice a day with U50,488H for 4 days. Binding studies on the crude membrane homogenates revealed that there was no change in maximum binding, but a significant increase in Kd after the treatment, indicating that the number of kappa binding sites remained unchanged whereas the affinity of the binding sites to kappa-agonist decreased. The study on the mechanical responses to U50,488H in the isolated perfused heart preparation showed that although the agonist at 10(-6) M caused MR2266 reversible reductions in heart rate and force of contraction as well as ventricular ectopic beat in the heart of rats in the control group, its effects were absent in the U50,488H-treated group, indicating the development of tolerance to the mechanical effects of U50,488H on the heart. The results indicate that the development of tolerance to the mechanical effects of a kappa-agonist after chronic treatment with the agonist was not accompanied by down-regulation, but only a slight and significant reduction in affinity of kappa binding sites in the rat heart.     

Ligand binding profiles of U-69, 593-sensitive and-insensitive sites in human cerebral cortex membranes: evidence of kappa opioid receptors heterogeneity

Receptor binding studies were performed to characterize the properties of subtypes of kappa opioid receptors in membrane preparations of human cerebral cortex. [3H]U69,593 ([3H]U69), a selective kappa 1-agonist, and [3H]diprenorphine ([3H]DIP), a non-selective opioid antagonist, in the presence of 1 microM each of DAMGO, DPDPE and U-69 to block mu-, delta-, and kappa 1-sites, labeled single population of binding sites, respectively. [3H]U-69 binding sites (KD = 3.8 +/- 0.2 nM, Bmax = 6.3 +/- 0.2 fmol/mg protein) had a binding profile that correspond to kappa 1-receptor. That is, dynorphin A (1-13) (Dyn A), bremazocine (BZC), U50,488H (U50), (-)ethylketocyclazocine (EKC) and nor-binaltorphimine (nor-BNI) bound to this site with high affinities. [3H]DIP labeled binding sites (Kd = 7.3 +/- 0.2 nM, Bmax = 102 +/- 9 fmol/mg protein) that were not sensitive to U-50, but to BZC, EKC and nor-BNI. These results indicate that kappa 1 and Kappa 2 opioid receptors exist in human cerebral cortex with different ligand binding profiles.     

Identification of an opioid kappa receptor subtype-selective N-substituent for (+)-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine

A three-component library of compounds was prepared in parallel using multiple simultaneous solution-phase synthetic methodology. The compounds were biased toward opioid receptor antagonist activity by incorporating (+)-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine (a potent, nonselective opioid pure antagonist) as one of the monomers. The other two monomers, which included N-substituted or unsubstituted Boc-protected amino acids and a range of substituted aryl carboxylic acids, were selected to add chemical diversity. Screening of these compounds in competitive binding experiments with the kappa opioid receptor selective ligand [3H]U69,593 led to the discovery of a novel kappa opioid receptor selective ligand, N-?(2'S)-[3-(4-hydroxyphenyl)propanamido]-3'-methylbutyl?-(3R, 4R)-dimethyl-4-(3-hydroxyphenyl)piperidine (8, RTI-5989-29). Additional structure-activity relationship studies suggested that 8 possesses lipophilic and hydrogen-bonding sites that are important to its opioid receptor potency and selectivity. These sites appear to exist predominantly within the kappa receptor since the selectivity arises from a 530-fold loss of affinity of 8 for the mu receptor and an 18-fold increase in affinity for the kappa receptor relative to the mu-selective ligand, (+)-N-[trans-4-phenyl-2-butenyl]-(3R, 4R)-dimethyl-4-(3-hydroxyphenyl)piperidine (5a). The degree of selectivity observed in the radioligand binding experiments was not observed in the functional assay. According to its ability to inhibit agonist stimulated binding of [35S]GTPgammaS at all three opioid receptors, compound 8 behaves as a mu/kappa opioid receptor pure antagonist with negligible affinity for the delta receptor.     

Dietary isoflavones reduce plasma cholesterol and atherosclerosis in C57BL/6 mice but not LDL receptor-deficient mice

To assess the cardiovascular effects of systemically administered opioid agonists, changes in blood pressure and heart rate were observed after intravenous (i.v.) administration of U50,488H (trans-3,4-dichloro-N-[2-(1-pyrrolidinyl) cyclohexyl]benzeneacetamide), a selective kappa-opioid receptor agonist, and DAMGO (D-Ala2, N-Me-Phe4, Gly5-ol), a selective mu-opioid-receptor agonist. Intravenous administration of U50,488H (1.2 mg/kg) and DAMGO (0.3 mg/kg) to the awake sheep resulted in an immediate increase in blood pressure. The pressor response to U50,488H was accompanied by an increase in heart rate. In contrast, there was no accompanying change in heart rate in response to DAMGO. We hypothesized that the lack of a reflex bradycardia to the pressor responses of both the mu- and kappa-opioid-receptor agonists was due to a blunting of baroreflex-mediated bradycardia. The reflex bradycardia to norepinephrine (0.6 microg/kg/min) was significantly reduced in the presence of DAMGO but not U50,488H. In view of the lack of effect of U50,488H on the baroreflex, we further hypothesized that the tachycardia it elicited was due to an increase in sympathetic activity. Pretreatment with propranolol (0.1 mg/kg) completely blocked the tachycardia elicited by U50,488H. These data suggest that the lack of a reflex bradycardia to the pressor response of DAMGO is due to a blunting of baroreflex-mediated bradycardia. In contrast, the increase in heart rate caused by U50,488H is mediated by sympathetic activation of the heart.     

Anti asialoglycoprotein receptor antibodies and soluble interleukin-2 receptor levels as marker for inflammation in autoimmune hepatitis

Previous results using an amphibian model showed that systemic and spinal administration of opioids selective for mu, delta and kappa-opioid receptors produce analgesia. It is not known whether non-mammalian vertebrates also contain supraspinal sites mediating opioid analgesia. Thus, opioid agonists selective for mu (morphine; fentanyl), delta (DADLE, [D-Ala2, D-Leu5]-enkephalin; DPDPE, [D-Pen2, D-Pen5]-enkephalin) and kappa (U50488, trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl] benzeneacetamide methanesulfonate; CI977, (5R)-(544alpha,744alpha,845beta)-N-methyl-N-[7-(1-p yrr olidinyl)-1-oxaspiro[4,5]dec-8yl]-4-benzofuranaceta mide++ + monohydrochloride) opioid receptors were tested for analgesia following i.c.v. administration in the Northern grass frog, Rana pipiens. Morphine, administered at 0.3, 1, 3 and 10 nmol/frog, produced a dose-dependent and long-lasting analgesic effect. Concurrent naltrexone (10 nmol) significantly blocked analgesia produced by i.c.v. morphine (10 nmol). ED50 values for the six opioids ranged from 2.0 for morphine to 63.9 nmol for U50488. The rank order of analgesic potency was morphine > DADLE > DPDPE > CI977 > fentanyl > U50488. These results show that supraspinal sites mediate opioid analgesia in amphibians and suggest that mechanisms of supraspinal opioid analgesia may be common to all vertebrates.     

No evidence for presynaptic opioid receptors on cholinergic, but presence of kappa-receptors on dopaminergic neurons in the rabbit caudate nucleus: involvement of endogenous opioids

The effects of various opioid receptor agonists and antagonists were studied in rabbit caudate nucleus slices preincubated with either [3H]dopamine or [3H]choline, superfused with medium (containing in most experiments the D2 receptor antagonist domperidone) and subjected to electrical field stimulation. The stimulation-evoked [3H]overflow from slices prelabeled with [3H]dopamine (evoked [3H]dopamine release) was significantly reduced by preferential kappa-opioid receptor agonists, like U-50,488 H, but not by mu- or delta-opioid receptor selective drugs. Opioid receptor antagonists shifted the concentration/response curve of U-50,488 H to the right (apparent pA2-value of the kappa-selective antagonist nor-binaltorphimine: 10.1) and enhanced the evoked dopamine release in the presence of a mixture of peptidase inhibitors. On the other hand, the [3H]overflow from rabbit caudate nucleus slices prelabeled with [3H]choline (evoked acetylcholine release) remained almost unaffected by any opioid receptor agonist, as long as the presynaptic D2 heteroreceptor was blocked with domperidone: in the absence of domperidone, U-50,488 H exhibited facilitatory effects. For comparison, the effects of the preferential delta-opioid receptor agonist DPDPE was also studied in slices of the rat striatum, where it clearly inhibited the evoked acetylcholine release. From our data we conclude that in the rabbit caudate nucleus the evoked dopamine release is inhibited by both exogenous and endogenous opioids via presynaptic kappa-opioid receptors, whereas the evoked release of acetylcholine is not, or only indirectly (via released dopamine) affected by opioids.     

Differential antagonism by MK-801 against antinociception induced by opioid receptor agonists administered supraspinally in mice

Various doses of MK-801 ((+/-)-5-methyl-10,11-dihydro-5H-dibenzo(a,d) cyclohepten-5, 10-imine maleate), a non-competitive N-methyl-D-aspartic acid (NMDA) receptor antagonist (0.001-1 microgram) injected intracerebroventricularly (i.c.v.) alone did not show any antinociceptive effect. MK-801 (0.001-1 microgram i.c.v.) dose dependently attenuated the inhibition of the tail-flick and hot plate responses induced by i.c.v. administered morphine (1 microgram), [D-Pen2, D-Pen5]enkephalin (DPDPE; 10 micrograms), and U50,488H (trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeoce tamide ) 60 micrograms). However, the inhibition of the tail-flick and hot plate responses induced by i.c.v. administered beta-endorphin (1 microgram) was not changed by i.c.v. administered MK-801. Our results indicate that, at the supraspinal level, NMDA receptors are involved in the production of antinociception induced by supraspinally administered morphine, DPDPE, and U50,488H but not beta-endorphin.     

Regulation of opioid receptors in rat sensory neurons in culture

To determine whether opioid receptors in sensory neurons are regulated by chronic exposure to opioids, we assessed the binding of various opioid ligands to membranes derived from isolated rat dorsal root ganglia neurons grown in culture. Equilibrium binding of [3H]diprenorphine onto membranes from cells grown for 13-15 days revealed a saturable binding site with a Kd value of 0.3 +/- 0.2 nM and an approximate Bmax value of 1300 +/- 200 fmol/mg of protein. [3H]Diprenorphine binding increased 3-fold from 1-15 days in culture. The mu receptors represent approximately 70 +/- 11% of the [3H]diprenorphine binding sites, as indicated by saturation binding of [3H]DAMGO. The kappa and delta receptors represent approximately 10 +/- 3% and approximately 5 +/- 2% of the [3H]diprenorphine binding sites, respectively. Preexposure of neurons to 10 microM naloxone for 48 hr up-regulated the receptors by 40%, whereas incubation with 100 nM to 10 microM DAMGO for 48 hr resulted in a significant decrease in the Bmax value of opioid receptors, with a maximum reduction of 70%. The identification of a high level of opioid receptors expressed in isolated sensory neurons and their modulation by opioids demonstrates that cultured sensory neurons are an excellent model with which to study opioid receptor regulation.     

Enhanced kappa-opioid receptor-mediated analgesia by antisense targeting the sigma1 receptor

In the current study, we used an antisense oligodeoxynucleotide targeting the recently cloned sigma1 receptor to assess its functions within the nervous system. Sigma1 antagonists potentiate the analgesic actions of opioids. Similarly, the antisense probe targeting the sigma1 receptor enhanced the analgesic activity of the kappa1-opioid receptor agonist U50,488H (trans-3,4-dichloro-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeacetamidel++ +) and the kappa3-opioid receptor agonist naloxone benzoylhydrazone. A mismatch control was inactive. These results confirm the role of sigma1 receptors in an anti-opioid analgesic system and illustrate the utility of antisense approaches towards the elucidation of sigma receptor functions.     

Suppression of phospholipase C blocks Gi-mediated inhibition of adenylyl cyclase activity

The potential effect of inhibition of phospholipase C on the response of Gi-coupled receptors was investigated in neuroblastoma x glioma hybrid (NG108-15) cells. The phospholipase C specific inhibitor 1-[6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H -pyrrole-2,5-dione (U73122), which did not affect basal and forskolin-stimulated adenylyl cyclase activities, time- and dose-dependently blocked delta-opioid receptor-mediated inhibition of adenylyl cyclase activity, the EC50 (0.5 microM) of which was consistent with that for inhibition of bradykinin-dependent phospholipase C activation (EC50 = 1 microM). U73122 treatment also blocked functional responses of m4 muscarinic receptor and alpha2-adrenoceptor in NG108-15 cells and three opioid receptors (mu, delta and opioid receptor-like receptor (ORL1)) in human neuroblastoma SK-N-SH cells. 1-[6-((17Beta-3-Methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-2, 5-pyrrolidinedione (U73343), an inactive analog of U73122, did not show any effect, which suggests that the blockade by U73122 of Gi-coupled receptor-mediated signaling is probably mediated through inhibition of phospholipase C, although a possible direct modification of G proteins can not be excluded. Furthermore, treatment with U73122 but not U73343 blocked the GTP-induced inhibition of adenylyl cyclase, indicating blockade at the level of Gi proteins.     

Characterization, expression, and immunohistochemical localization of 5 alpha-reductase in human skin

The effect of the mu opioid agonist DAGO, delta opioid agonist DPDPE and kappa opioid agonist U50,488H on 3H-dopamine (3H-DA) uptake was studied in synaptosomes prepared from rat striatum and nucleus accumbens. Over the range of concentrations tested (1 nM-10 microM) DAGO and DPDPE were devoid of effects on 3H-DA uptake in the striatum and the nucleus acumbens. In contrast, U50,488H significantly decreased 3H-DA uptake in both structures. The inhibition of uptake induced by the kappa agonist was not reversed in the presence of the opiate antagonists naloxone (10 microM) or nor-binaltorphimine (0.1 microM). Dynorphin A (1-13) also induced a significant reduction in 3H-DA uptake in both structures at the concentrations of 10 and 30 microM. This inhibitory effect was not reversed by naloxone (10 microM). These data suggest that kappa opioid agonists modulate dopamine uptake in the striatum and the nucleus accumbens and their effects may not be due to an activation of opioid receptors.     

Inverse agonism at adrenergic and opioid receptors: studies with wild type and constitutively active mutant receptors

Ligands which display inverse agonism at G protein-coupled receptors do so by decreasing the intrinsic ability of a receptor to active the cellular G protein population in the absence of an agonist ligand. Expression of the murine delta opioid receptor in Rat-1 fibroblasts resulted in the inverse agonist ICI174864 being able to cause inhibition of basal high affinity GTPase activity and of the binding of [35S]GTP gamma S in membranes of a clone (D2) of these cells which expresses high levels of the receptor. These effects were blocked by co-addition of the neutral antagonist TIPP[psi], demonstrating a requirement for the delta opioid receptor, and by pertussis toxin pretreatment of the cells, showing them to be produced via a Gi-like G protein. The inverse agonist properties of ICI174864 could also be demonstrated in whole cells. Stimulation of forskolin-amplified adenylyl cyclase activity was produced by ICI174864 following [3H]adenine prelabelling of the cells. Constitutively activated mutants of receptors should provide a convenient means to detect inverse agonists. Incubation of cells either transiently or stably transfected with a constitutively activated mutant of the human beta 2-adrenoceptor with the beta 2-inverse agonists betaxolol or sotalol, which are both able to inhibit CAM beta 2-adrenoceptor-mediated basal adenylyl cyclase activity, resulted in a strong upregulation of levels of the receptor. In the stable cells lines this effect was prevented by co-incubation with neutral antagonists but could not be reproduced by an adenylyl cyclase P-site ligand which also inhibited basal adenylyl cyclase levels.     

Regulation of hsp expression during rodent spermatogenesis

As part of an effort to develop peptides with selective kappa-opioid antagonist activity, a series of N-alkylated [D-Pro10]dynorphin A-(1-11) derivatives were made through solid-phase peptide synthesis: R-Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-D-Pro-LysOH, where R = N-benzyl, N-cyclopropylmethyl, N,N-dicyclopropylmethyl, or N,N-diallyl. These derivatives and dynorphin A-(1-13)NH2 were evaluated for kappa-opioid receptor binding affinity and potency as inhibitors of adenylyl cyclase. Equilibrium competition binding experiments using [3H]diprenorphine (approximately 600 pM) were performed on membranes prepared from cultured Chinese hamster ovary (CHO) cells stably expressing the rat kappa-opioid receptor. Tissue prepared from this cell line was used to evaluate opioid peptide inhibition of forskolin-stimulated (50 microM) adenylyl cyclase activity. Displacement of [3H]diprenorphine specific binding by these peptides was observed with a rank order of affinity (Ki, nM) = [D-Pro10]dynorphin A-(1-11) (0.13) > dynorphin A-(1-13)NH2 (0.34) > N-cyclopropylmethyl- (1.4) > N,N-dicyclopropylmethyl- (12.6) approximately N-benzyl- (18.3) approximately N,N-diallyl-[D-Pro10]dynorphin A-(1-11) (26.0). A similar rank order was observed for potency of adenylyl cyclase inhibition (IC50, nM): [D-Pro10]dynorphin A-(1-11) (0.12) approximately dynorphin A-(1-13)NH2 (0.19) > N-cyclopropylmethyl- (2.7) > N,N-dicyclopropylmethyl- (13.2) approximately N,N-diallyl- (18.0) approximately N-benzyl-[D-Pro10]dynorphin A-(1-11) (36.4). The peptides differed in their percent maximal inhibition of adenylyl cyclase activity: dynorphin A-(1-13)NH2 (100%) approximately N-cyclopropylmethyl- (94.3%) approximately [D-Pro10]dynorphin A-(1-11) (87.9%) > N-benzyl- (71.4%) > N,N-dicyclopropylmethyl- (23.6%) approximately N,N-diallyl-[D-Pro10]dynorphin A-(1-11)(18.9%). As the N,N-dicyclopropylmethyl- and N,N-diallyl-[D-Pro10]dynorphin A-(1-11) derivatives were found to have only weak partial agonist activity with respect to adenylyl cyclase inhibition, they were evaluated for their ability to reverse dynorphin A-(1-13)NH2 (10 nM) inhibition of adenylyl cyclase activity. N,N-dicyclopropylmethyl- and N,N-diallyl-[D-Pro10]dynorphin A-(1-11) reversed dynorphin A-(1-13)NH2 inhibition to levels equal to the maximal inhibition produced by N,N-dicyclopropylmethyl- and N,N-diallyl-[D-Pro10]dynorphin A-(1-11) alone. This weak partial agonism combined with nanomolar potency render the N,N-dicyclopropylmethyl- and N,N-diallyl-[D-Pro10]dynorphin A-(1-11) compounds promising leads for further attempts to synthesize peptide kappa-opioid receptor antagonists.     

Selective inhibition of [D-Ala2, Glu4]deltorphin antinociception by supraspinal, but not spinal, administration of an antisense oligodeoxynucleotide to an opioid delta receptor

Evidence in vivo has suggested the existence of subtypes of the delta opioid receptor (DOR), which have been termed delta 1 and delta 2. These proposed DOR subtypes are thought to be activated by [D-Pen2, D-Pen5]enkephalin (DPDPE, delta 1) and [D-Ala2, Glu4]deltorphin (delta 2). Recent work in which an antisense oligodeoxynucleotide (oligo) to a cloned DOR was administered by the intrathecal (i.th.) route has demonstrated a reduction in the antinociceptive actions of both i.th. DPDPE and [D-Ala2, Glu4]deltorphin, but not of [D-Ala2, NMPhe4, Gly-ol]enkephalin (DAMGO, mu agonist) in mice. The present investigation has extended these observations by administering the same DOR antisense oligo sequence by the intracerebroventricular (i.c.v.) route and evaluating the antinociceptive actions of i.c.v. agonists selective for delta, mu and kappa receptors. I.th. treatment with DOR antisense oligo, but not mismatch oligo, significantly inhibited the antinociceptive actions of both i.th. DPDPE and [D-Ala2, Glu4]deltorphin but not of i.th. DAMGO or U69,593 (kappa agonist), confirming previous data. In contrast, i.c.v. DOR antisense oligo, but not mismatch oligo, selectively inhibited the antinociceptive response to i.c.v. [D-Ala2, Glu4]deltorphin without altering the antinociceptive actions of i.c.v. DPDPE, DAMGO or U69,593. The data suggest that the cloned DOR corresponds to that pharmacologically classified as delta 2 and further, suggest that this delta receptor subtype may play a major role in eliciting spinal delta-mediated antinociception.     

Mu/kappa opioid interactions in rhesus monkeys: Implications for analgesia and abuse liability.

Mu opioid receptor agonists are clinically valuable as analgesics; however, their use is limited by high abuse liability. Kappa opioid agonists also produce antinociception, but they do not produce mu agonist-like abuse-related effects, suggesting that they may enhance the antinociceptive effects and/or attenuate the abuse-related effects of mu agonists. To evaluate this hypothesis, the present study examined interactions between the mu agonist fentanyl and the kappa agonist U69,593 in three behavioral assays in rhesus monkeys. In an assay of schedule-controlled responding, monkeys responded under a fixed-ratio 30 (FR 30) schedule of food presentation. Fentanyl and U69,593 each produced rate-decreasing effects when administered alone, and mixtures of 0.22:1, 0.65:1, and 1.96:1 U69,593/fentanyl usually produced subadditive effects. In an assay of thermal nociception, tail withdrawal latencies were measured from water heated to 50 °C. Fentanyl and U69,593 each produced dose-dependent antinociception, and effects were additive for all mixtures. In an assay of drug self-administration, rhesus monkeys responded for intravenous drug injection, and both dose and FR values were manipulated. Fentanyl maintained self-administration, whereas U69,593 did not. Addition of U69,593 to fentanyl produced a proportion-dependent decrease in rates of fentanyl self-administration. Moreover, addition of U69,593 increased the sensitivity of fentanyl self-administration to increases in the FR value. Taken together, these results suggest that simultaneous activation of mu and kappa receptors, either with a mixture of selective drugs or with a single drug that targets both receptors, may reduce abuse liability without reducing analgesic effects relative to selective mu agonists administered alone. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Cyclic AMP protects against staurosporine and wortmannin-induced apoptosis and opioid-enhanced apoptosis in both embryonic and immortalized (F-11kappa7) neurons

The mechanism by which opiates affect fetal development is unknown, but one potential target is the programmed cell death (apoptosis) pathway of neurons. Apoptosis was induced in both primary neuronal cultures from embryonic day 7 cerebral hemispheres of chick brain (E7CH) and the F-11kappa7 cell line (an immortalized mouse neuroblastoma x dorsal root ganglion hybrid stably transfected to overexpress kappa-opioid receptors) by either staurosporine or the phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002. Cells pretreated with either the mu-specific opioid agonist morphiceptin (E7CH) or the kappa-specific opioid agonist U69,593 (F-11kappa7) for 24 h showed increased apoptosis in response to staurosporine or wortmannin when compared with non-pretreated cells. The effects of morphiceptin and U69,593 were time- and dose-dependent and antagonist-reversible, suggesting that they were receptor-mediated. Neither morphiceptin nor U69,593 by themselves had any measurable effect on cell viability or DNA fragmentation, and coaddition of opiates at the same time as staurosporine, wortmannin, or LY294002 did not enhance apoptosis. Time course studies indicated a maximal opioid effect at a time (16-24 h) when inhibition of adenylate cyclase had been maximal for many hours. Addition of dibutyryl cyclic AMP either before or at the time of opioid addition protected against apoptosis and reduced fragmentation to levels seen for staurosporine plus dibutyryl cyclic AMP alone. The specificity for cyclic AMP was confirmed by showing protection with the specific agonist Sp-adenosine 3',5'-cyclic monophosphothioate and increased killing with the antagonist Rp-adenosine 3',5'-cyclic monophosphothioate. We conclude that the opioid enhancement of apoptosis is based on the inhibition of adenylate cyclase and that the effect is time-dependent.