u-50488 and preproenkephalin

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; Corpus Striatum; Dopamine; Enkephalins; Hydroxydopamines; Locomotion; Motor Neurons; Movement; Naloxone; Neural Pathways; Oxidopamine; Protein Precursors; Pyrrolidines; Receptors, Opioid; Receptors, Opioid, kappa; Substantia Nigra

Emotionally arousing events like encounter with an unfamiliar con-species produce strong and vivid memories, whereby the hippocampus and amygdala play a crucial role. It is less understood, however, which neurotransmitter systems regulate the strength of social memories, which have a strong emotional component. It was shown previously that dynorphin signalling is involved in the formation and extinction of fear memories, therefore we asked if it influences social memories as well. Mice with a genetic deletion of the prodynorphin gene Pdyn (Pdyn(-/-)) showed a superior partner recognition ability, whereas their performance in the object recognition test was identical as in wild-type mice. Pharmacological blockade of kappa opioid receptors (KORs) led to an enhanced social memory in wild-type animals, whereas activation of KORs reduced the recognition ability of Pdyn(-/-) mice. Partner recognition test situation induced higher elevation in dynorphin A levels in the central and basolateral amygdala as well as in the hippocampus, and also higher dynorphin B levels in the hippocampus than the object recognition test situation. Our result suggests that dynorphin system activity is increased in emotionally arousing situation and it decreases the formation of social memories. Thus, dynorphin signalling is involved in the formation of social memories by diminishing the emotional component of the experience.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Amygdala; Animals; Enkephalins; Hippocampus; Memory; Mice; Mice, Knockout; Naltrexone; Narcotic Antagonists; Protein Precursors; Receptors, Opioid, kappa; Recognition, Psychology; Social Behavior

We recently demonstrated that endogenous prodynorphin-derived peptides mediate anticonvulsant, antiepileptogenic and neuroprotective effects via kappa opioid receptors (KOP). Here we show acute and delayed neurodegeneration and its pharmacology after local kainic acid injection in prodynorphin knockout and wild-type mice and neuroprotective effect(s) of KOP activation in wild-type mice. Prodynorphin knockout and wild-type mice were injected with kainic acid (3 nmoles in 50 nl saline) into the stratum radiatum of CA1 of the right dorsal hippocampus. Knockout mice displayed significantly more neurodegeneration of pyramidal cells and interneurons than wild-type mice 2 days after treatment. This phenotype could be mimicked in wild-type animals by treatment with the KOP antagonist GNTI and rescued in knockout animals by the KOP agonist U-50488. Minor differences in neurodegeneration remained 3 weeks after treatment, mostly because of higher progressive neurodegeneration in wild-type mice compared with prodynorphin-deficient animals. In wild-type mice progressive neurodegeneration, but not acute neuronal loss, could be mostly blocked by U-50488 treatment. Our data suggest that endogenous prodynorphin-derived peptides sufficiently activate KOP receptors during acute seizures, and importantly in situations of reduced dynorphinergic signaling-like in epilepsy-the exogenous activation of KOP receptors might also have strong neuroprotective effects during excitotoxic events.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; CA1 Region, Hippocampal; Enkephalins; Guanidines; Humans; Interneurons; Kainic Acid; Mice; Mice, Inbred C57BL; Mice, Knockout; Morphinans; Neurodegenerative Diseases; Protein Precursors; Pyramidal Cells; Receptors, Opioid, kappa; Seizures

Exposure to inescapable stressors increases both the rewarding properties and self-administration of cocaine through the signaling of the kappa-opioid receptor (KOR), but the effect of this signaling on other reinforcing agents remains unclear.. The objective of this study is to test the hypothesis that signaling of the KOR mediates the forced swim stress (FSS)-induced potentiation of ethanol reward and self-administration.. Male C57Bl/6J mice were tested in a biased ethanol-conditioned place preference (CPP) procedure, and both C57Bl/6J and prodynorphin gene-disrupted (Dyn -/-) mice were used in two-bottle free choice (TBC) assays, with or without exposure to FSS. To determine the role of the KOR in the resulting behaviors, the KOR agonist U50,488 (10 mg/kg) and antagonist nor-binaltorphimine (nor-BNI, 10 mg/kg) were administered prior to parallel testing.. C57Bl/6J mice exposed to repeated FSS 5 min prior to daily place conditioning with ethanol (0.8 g/kg) demonstrated a 4.4-fold potentiation of ethanol-CPP compared to unstressed mice that was prevented by nor-BNI pretreatment. Likewise, pretreatment with U50,488 90 min prior to daily ethanol place conditioning resulted in a 2.8-fold potentiation of ethanol-CPP. In the TBC assay, exposure to FSS significantly increased the consumption of 10% (v/v) ethanol by 19.3% in a nor-BNI-sensitive manner. Notably, Dyn -/- mice consumed a similar volume of ethanol as wild-type littermates and C57Bl/6J mice, but did not demonstrate significant stress-induced increases in consumption.. These data demonstrated a stress-induced potentiation of the rewarding effects and self-administration of ethanol mediated by KOR signaling.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; Conditioning, Psychological; Enkephalins; Ethanol; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Naltrexone; Protein Precursors; Receptors, Opioid, kappa; Reward; Self Administration; Signal Transduction; Stress, Physiological

Kappa-opioid receptors (κOR) are positioned to modulate pre- and post-synaptic responses of norepinephrine-containing neurons in the rat locus coeruleus (LC). The ability of an acute systemic injection of a long acting κOR agonist, U50,488, to induce trafficking of κOR was assessed in the LC using immunogold-silver detection in male Sprague-Dawley rats. U50,488 administration shifted immunogold-silver labeling indicative of κOR from primarily plasmalemmal sites to intracellular sites when compared to vehicle-treated subjects. This translocation from the plasma membrane to the cytoplasmic compartment was prevented by pre-treatment with the κOR antagonist, norbinaltorphimine (norBNI). To determine whether agonist stimulation could induce adaptations in the expression of the noradrenergic synthesizing enzyme, dopamine beta hydroxylase (DβH), and κOR expression, Western blot analysis was used to compare expression levels of DβH and κOR following U50,488 administration. Expression levels for DβH and κOR were significantly increased following U50,488 administration when compared to controls. These data indicate that a systemic injection of a κOR agonist stimulates internalization of κORs in noradrenergic neurons and can impact κOR and DβH expression levels in this stress-sensitive brain region.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; Dopamine beta-Hydroxylase; Endocytosis; Enkephalins; Locus Coeruleus; Male; Microscopy, Immunoelectron; Neurons; Norepinephrine; Protein Precursors; Rats; Rats, Sprague-Dawley; Receptors, Opioid, kappa

Stress and anxiety are mainly regulated by amygdala and hypothalamic circuitries involving several neurotransmitter systems and providing physiological responses to peripheral organs via the hypothalamic-pituitary-adrenal axis and other pathways. The role of endogenous opioid peptides in this process is largely unknown. Here we show for the first time that anxiolytic parameters of explorative behavior in mice lacking prodynorphin were increased 2-4-fold in the open field, the elevated plus maze and the light-dark test. Consistent with this, treatment of wild-type mice with selective kappa-opioid receptor antagonists GNTI or norbinaltorphimine showed the same effects. Furthermore, treatment of prodynorphin knockout animals with U-50488H, a selective kappa-opioid receptor agonist, fully reversed their anxiolytic phenotype. These behavioral data are supported by an approximal 30% reduction in corticotropin-releasing hormone (CRH) mRNA expression in the hypothalamic paraventricular nucleus and central amygdala and an accompanying 30-40% decrease in corticosterone serum levels in prodynorphin knockout mice. Although stress-induced increases in corticosterone levels were attenuated in prodynorphin knockout mice, they were associated with minor increases in depression-like behavior in the tail suspension and forced swim tests. Taken together, our data suggest a pronounced impact of endogenous prodynorphin-derived peptides on anxiety, but not stress coping ability and that these effects are mediated via kappa-opioid receptors. The delay in the behavioral response to kappa-opioid receptor agonists and antagonist treatment suggests an indirect control level for the action of dynorphin, probably by modulating the expression of CRH or neuropeptide Y, and subsequently influencing behavior.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Amygdala; Animals; Anxiety; Brain Stem; Corticosterone; Corticotropin-Releasing Hormone; Dynorphins; Enkephalins; Exploratory Behavior; Female; Guanidines; Hypothalamus; Male; Maze Learning; Mesencephalon; Mice; Mice, Inbred C57BL; Mice, Knockout; Morphinans; Naltrexone; Neuropeptide Y; Neuropeptides; Protein Precursors; Raphe Nuclei; Receptors, Opioid, kappa; Stress, Psychological

We hypothesized that mice subjected to prolonged stress would demonstrate decreased performance in a learning and memory task attributable to the endogenous activation of the kappa opioid receptor (KOR). C57BL/6J mice were tested using the novel object recognition (NOR) assay at various time points after exposure to repeated forced swim stress (FSS). Unstressed mice demonstrated recognition of the novel object at the end of a procedure using three 10-min object interaction phases, with a recognition index (RI) for the novel object of 71.7+/-3.4%. However, 1 h after exposure to FSS, vehicle-pretreated mice displayed a significant deficit in performance (RI=58.2+/-4.1%) compared with unstressed animals. NOR was still significantly reduced 4 but not 24 h after FSS. Treatment with the KOR-selective antagonist norbinaltorphimine (10 mg/kg, i.p.) prevented the decline in learning and memory performance. Moreover, direct activation of the KOR induced performance deficits in NOR, as exogenous administration of the KOR agonist U50,488 [(+/-)-trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzeneacetamide] (0.3 mg/kg, i.p.) suppressed NOR (RI=56.0+/-3.9%). The effect of FSS on NOR performance was further examined in mice lacking the gene for the endogenous KOR agonist dynorphin (Dyn). Dyn gene-disrupted mice exposed to FSS did not show the subsequent learning and memory deficits (RI=66.8+/-3.8%) demonstrated by their wild-type littermates (RI=49.7+/-2.9%). Overall, these results suggest that stress-induced activation of the KOR may be both necessary and sufficient to produce subsequent deficits in novel object recognition.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics, Non-Narcotic; Analysis of Variance; Animals; Behavior, Animal; Enkephalins; Gene Expression Regulation; Immobility Response, Tonic; Learning Disabilities; Male; Memory Disorders; Mice; Mice, Inbred C57BL; Mice, Knockout; Motor Activity; Naltrexone; Narcotic Antagonists; Protein Precursors; Receptors, Opioid, kappa; Recognition, Psychology; Stress, Psychological; Swimming; Time Factors

Dynorphin is the presumed endogenous ligand for the kappa-opioid receptor. The dynorphin gene may play a role in psychotropic agent-mediated behavioral changes via dopaminergic modulation. Therefore, in this study, possible involvement of the dynorphin gene in nalbuphine-mediated behavioral responses was examined using prodynorphin (Pdyn) gene knock-out (-/-) mice. Pdyn gene deficiency potentiates nalbuphine-induced behavioral sensitization of locomotor activity and accumbal c-Fos expression. Administration of nalbuphine induced a significant increase in the dialysate dopamine level in the nucleus accumbens. This increase was more pronounced in the Pdyn (-/-) mice than in the wild-type (WT) mice. In addition, Pdyn (-/-) mice were more vulnerable to the naloxone-precipitated withdrawal syndrome (i.e., teeth chattering, wet dog shakes, forepaw tremors, jumping, weight loss, and global withdrawal score) after repeated treatment with nalbuphine than the WT mice. Consistently, nor-binaltorphimine, a kappa-opioid receptor antagonist, significantly potentiated nalbuphine-induced behavioral effects in WT mice, whereas U-50488H, a kappa-opioid receptor agonist, significantly attenuated these changes in Pdyn (-/-) mice in a dose-dependent manner. Our data suggest that the kappa-opioid receptor/dynorphin system is specifically modulated in response to behavioral sensitization and withdrawal signs induced by nalbuphine.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics, Non-Narcotic; Analgesics, Opioid; Animals; Area Under Curve; Blotting, Western; Dopamine; Enkephalins; Gene Expression; Genes, fos; Mice; Mice, Knockout; Microdialysis; Motor Activity; Nalbuphine; Naloxone; Naltrexone; Narcotic Antagonists; Nucleus Accumbens; Protein Precursors; Receptors, Opioid, kappa; Reverse Transcriptase Polymerase Chain Reaction; Substance Withdrawal Syndrome

Prior activation of the kappa opioid system by repeated stress or agonist administration has been previously shown to potentiate the rewarding properties of subsequently administered cocaine. In the present study, intermittent and uncontrollable footshock, a single session of forced swim, or acute administration of the kappa agonist U50,488 (5 mg/kg) were found to reinstate place preference in mice previously conditioned with cocaine (15 mg/kg) and subsequently extinguished by repeated training sessions without drug.. Stress-induced reinstatement did not occur for mice pretreated with the kappa opioid receptor antagonist norbinaltorphimine (10 mg/kg) and did not occur in mice lacking either kappa opioid receptors (KOR -/-) or prodynorphin (Dyn -/-). In contrast, the initial cocaine conditioning and extinction rates were not significantly affected by disruption of the kappa opioid system. Cocaine-injection also reinstated conditioned place preference in extinguished mice; however, cocaine-primed reinstatement was not blocked by kappa opioid system disruption.. The results suggest that stress-induced drug craving in mice may require activation of the dynorphin/kappa opioid system.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; Behavior, Animal; Cocaine-Related Disorders; Conditioning, Operant; Disease Models, Animal; Enkephalins; Extinction, Psychological; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Protein Precursors; Receptors, Opioid, kappa; Stress, Psychological; Swimming

Stress is a complex human experience having both positive and negative motivational properties. When chronic and uncontrollable, the adverse effects of stress on human health are considerable and yet poorly understood. Here, we report that the dysphoric properties of chronic stress are encoded by the endogenous opioid peptide dynorphin acting on specific stress-related neuronal circuits. Using different forms of stress presumed to evoke dysphoria in mice, we found that repeated forced swim and inescapable footshock both produced aversive behaviors that were blocked by a kappa-opioid receptor (KOR) antagonist and absent in mice lacking dynorphin. Injection of corticotropin-releasing factor (CRF) or urocortin III, key mediators of the stress response, produced place aversion that was also blocked by dynorphin gene deletion or KOR antagonism. CRF-induced place aversion was blocked by the CRF2 receptor antagonist antisauvigine-30, but not by the CRF1 receptor antagonist antalarmin. In contrast, place aversion induced by the KOR agonist U50,488 was not blocked by antisauvigine-30. These results suggest that the aversive effects of stress were mediated by CRF2 receptor stimulation of dynorphin release and subsequent KOR activation. Using a phospho-selective antibody directed against the activated KOR to image sites of dynorphin action in the brain, we found that stress and CRF each caused dynorphin-dependent KOR activation in the basolateral amygdala, nucleus accumbens, dorsal raphe, and hippocampus. The convergence of stress-induced aversive inputs on the dynorphin system was unexpected, implicates dynorphin as a key mediator of dysphoria, and emphasizes kappa-receptor antagonists as promising therapeutics.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics, Non-Narcotic; Animals; Behavior, Animal; Conditioning, Operant; Corticotropin-Releasing Hormone; Dose-Response Relationship, Drug; Drug Interactions; Dynorphins; Enkephalins; Mice; Mice, Inbred C57BL; Mice, Knockout; Naltrexone; Narcotic Antagonists; Odorants; Phosphorylation; Protein Precursors; Receptors, Opioid, kappa; Stress, Physiological; Swimming; Urocortins

Neuropsychiatric disorders are one of the main challenges of human medicine with epilepsy being one of the most common serious disorders of the brain. Increasing evidence suggest neuropeptides, particularly the opioids, play an important role in epilepsy. However, little is known about the mechanisms of the endogenous opioid system in epileptogenesis and epilepsy. Therefore, we investigated the role of endogenous prodynorphin-derived peptides in epileptogenesis, acute seizure behaviour and epilepsy in prodynorphin-deficient mice. Compared with wild-type littermates, prodynorphin knockout mice displayed a significantly reduced seizure threshold as assessed by tail-vein infusion of the GABA(A) antagonist pentylenetetrazole. This phenotype could be entirely rescued by the kappa receptor-specific agonist U-50488, but not by the mu receptor-specific agonist DAMGO. The delta-specific agonist SNC80 decreased seizure threshold in both genotypes, wild-type and knockout. Pre-treatment with the kappa selective antagonist GNTI completely blocked the rescue effect of U-50488. Consistent with the reduced seizure threshold, prodynorphin knockout mice showed faster seizure onset and a prolonged time of seizure activity after intracisternal injection of kainic acid. Three weeks after local injection of kainic acid into the stratum radiatum CA1 of the dorsal hippocampus, prodynorphin knockout mice displayed an increased extent of granule cell layer dispersion and neuronal loss along the rostrocaudal axis of the ipsi- and partially also of the contralateral hippocampus. In the classical pentylenetetrazole kindling model, dynorphin-deficient mice showed significantly faster kindling progression with six out of eight animals displaying clonic seizures, while none of the nine wild-types exceeded rating 3 (forelimb clonus). Taken together, our data strongly support a critical role for dynorphin in the regulation of hippocampal excitability, indicating an anticonvulsant role of kappa opioid receptors, thereby providing a potential target for antiepileptic drugs.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics; Animals; Benzamides; Cell Count; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Epilepsy, Temporal Lobe; Guanidines; Hippocampus; Kindling, Neurologic; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Morphinans; Naltrexone; Nerve Degeneration; Piperazines; Protein Precursors; Receptors, Opioid, kappa; Synaptic Transmission; Time Factors

Repeated forced-swim stress (FSS) produced analgesia, immobility and potentiation of cocaine-conditioned place preference (CPP) in wild-type C57Bl/6 mice, but not in littermates lacking the kappa opioid receptor (KOR) gene. These results were surprising because kappa agonists are known to produce conditioned place aversion and to suppress cocaine-CPP when coadministered with cocaine. The possibility that disruption of the kappa system blocked the stress response by adversely affecting the hypothalamic-pituitary axis was examined by measuring plasma corticosterone levels. However, disruption of the dynorphin/kappa system by gene deletion or receptor antagonism did not reduce the FSS-induced elevation of plasma corticosterone levels. A second explanation for the difference is that kappa receptor activation caused by FSS occurred prior to cocaine conditioning rather than contemporaneously. To test this hypothesis, we measured the effects of the kappa agonist (trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide (U50,488) administered to mice at various intervals preceding cocaine conditioning. The results showed that the interaction between the kappa system and cocaine reinforcement depended on the timing of the drug pairing. Mice given U50,488 60 min prior to cocaine showed a robust, nor-BNI-sensitive potentiation of cocaine-CPP, whereas administration 15 min before cocaine significantly suppressed cocaine-CPP. In the absence of cocaine, U50,488 given 60 min prior to saline conditioning produced no place preference, whereas administration 15 min before saline conditioning produced significant place aversion. The results of this study suggest that kappa receptor activation induced by FSS prior to the cocaine-conditioning session may be both necessary and sufficient for potentiation of the reinforcing actions of cocaine.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics, Non-Narcotic; Analysis of Variance; Animals; Behavior, Animal; Cocaine; Conditioning, Operant; Drug Interactions; Enkephalins; Enzyme Activation; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Motor Activity; Pain Measurement; Protein Precursors; Reaction Time; Receptors, Opioid, kappa; Stress, Physiological; Swimming; Time Factors

The molecular mechanisms involved in the reversion of levodopa-induced motor fluctuations by the adenosine A2A antagonist 8-(3-chlorostryryl) caffeine (CSC) were investigated in rats with a 6-hydroxydopamine (6-OHDA)-induced lesion and compared with the ones achieved by the kappa-opioid agonist, U50,488. Animals were treated with levodopa (50 mg/kg/day) for 22 days and for one additional week with levodopa + CSC (5 mg/kg/day), levodopa + U50,488 (1 mg/kg/day), or levodopa + vehicle. The reversion of the decrease in the duration of levodopa-induced rotations by CSC, but not by U50,488, was maintained until the end of the treatment and was associated with a further increase in levodopa-induced preprodynorphin mRNA in the lesioned striatum, being higher in the ventromedial striatum. The increase in striatal preprodynorphin expression, particularly in the ventromedial striatum, may be related to the reversion of levodopa-induced motor fluctuations in the CSC-treated animals, suggesting a role of the direct striatal output pathway activity in the ventromedial striatum in the pathophysiology of motor fluctuations.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Adenosine; Adrenergic Agents; Animals; Caffeine; Corpus Striatum; Dynorphins; Dyskinesias; Enkephalins; Immunohistochemistry; In Situ Hybridization; Levodopa; Male; Oxidopamine; Parkinsonian Disorders; Protein Precursors; Rats; Rats, Sprague-Dawley; Receptors, Opioid, kappa; RNA, Messenger

Calcium-binding proteins regulate transcription and secretion of pancreatic islet hormones. Here, we demonstrate neuroendocrine expression of the calcium-binding downstream regulatory element antagonistic modulator (DREAM) and its role in glucose-dependent regulation of prodynorphin (PDN) expression. DREAM is distributed throughout beta- and alpha-cells in both the nucleus and cytoplasm. As DREAM regulates neuronal dynorphin expression, we determined whether this pathway is affected in DREAM(-/-) islets. Under low glucose conditions, with intracellular calcium concentrations of <100 nM, DREAM(-/-) islets had an 80% increase in PDN message compared with controls. Accordingly, DREAM interacts with the PDN promoter downstream regulatory element (DRE) under low calcium (<100 nM) conditions, inhibiting PDN transcription in beta-cells. Furthermore, beta-cells treated with high glucose (20 mM) show increased cytoplasmic calcium (approximately 200 nM), which eliminates DREAM's interaction with the DRE, causing increased PDN promoter activity. As PDN is cleaved into dynorphin peptides, which stimulate kappa-opioid receptors expressed predominantly in alpha-cells of the islet, we determined the role of dynorphin A-(1-17) in glucagon secretion from the alpha-cell. Stimulation with dynorphin A-(1-17) caused alpha-cell calcium fluctuations and a significant increase in glucagon release. DREAM(-/-) islets also show elevated glucagon secretion in low glucose compared with controls. These results demonstrate that PDN transcription is regulated by DREAM in a calcium-dependent manner and suggest a role for dynorphin regulation of alpha-cell glucagon secretion. The data provide a molecular basis for opiate stimulation of glucagon secretion first observed over 25 years ago.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; Calcium; Cell Line; Cell Nucleus; DNA; Dynorphins; Electrophoretic Mobility Shift Assay; Enkephalins; Gene Expression Regulation; Glucagon; Glucagon-Secreting Cells; Glucose; Humans; Insulin-Secreting Cells; Islets of Langerhans; Kv Channel-Interacting Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Naltrexone; Protein Binding; Protein Precursors; Receptors, Opioid, kappa; Repressor Proteins

We previously reported that (-)-nicotine and kappa-opioid receptor agonists lessened impairment of learning and/or memory in several animal models. Furthermore, these drugs prevented neurodegenerative damage induced by ischemia or beta-amyloid peptide (25-35). In the present study, we tested whether (-)-nicotine and U-50,488H prevent delayed-memory impairment induced by beta-amyloid peptide (25-35), and changes of expression of alpha7-type nicotinic acetylcholine receptor mRNA and prodynorphin mRNA. Seven days after treatment with beta-amyloid peptide (25-35) (9 nmol/mouse, i.c.v.), memory impairment was observed in the Y-maze test. Memory impairment was prevented when (-)-nicotine (6.16 micromol/kg, s.c.) or U-50,488H (21 micromol/kg, s.c.) was administered 1 h before, but not 1 h after, beta-amyloid peptide (25-35) treatment. There was no change in prodynorphin mRNA or alpha7-type nicotinic acetylcholine receptor mRNA expression in the hippocampus 10 days after beta-amyloid peptide (25-35) treatment alone. Of interest, mRNA expression of not only prodynorphin, but also the alpha7-type nicotinic acetylcholine receptor, was significantly decreased when U-50,488H was administered 1 h before, but not 1 h after, treatment with beta-amyloid peptide (25-35). However, these changes were not observed after the administration of (-)-nicotine. These results suggest that activation of the kappa-opioid system, but not beta7-type nicotinic receptors has a neuroprotective effect on beta-amyloid peptide (25-35)-induced memory impairment, and may be involved in the long-lasting changes in the expression of these mRNAs.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; alpha7 Nicotinic Acetylcholine Receptor; Amyloid beta-Peptides; Animals; Enkephalins; Gene Expression Regulation; Male; Mice; Nicotine; Peptide Fragments; Protein Precursors; Receptors, Nicotinic; RNA, Messenger

The objective of this study was to determine whether prodynorphin-derived opioid peptides could block the spontaneous luteinizing hormone (LH) surge and ovulation, and if so, whether this inhibitory action was mediated through kappa-opioid receptors. Various doses of dynorphin peptides (dynorphin A(1-17), dynorphin A(1-8), dynorphin B, alpha- and beta-neoendorphin) were infused into the brain through third-ventricle cannulae in rats between 1330-1800 h on proestrus. Each dynorphin peptide blocked the LH surge and ovulation in a dose-dependent manner. Dynorphin A(1-17) and A(1-8) were equally effective in producing these actions, and more potent than either dynorphin B or alpha- or beta-neoendorphin. U50,488H, a specific kappa-opioid receptor agonist, also blocked the LH surge and ovulation. When a mixture of five dynorphin peptides was infused intraventricularly, each at a dose that inhibited the LH surge, both the surge and ovulation were blocked. However, when norbinaltorphimine, a specific kappa-opioid receptor antagonist, was coinfused with the mixture of dynorphin peptides, the LH surge and ovulation were fully restored. These results demonstrate that prodynorphin-derived opioid peptides, acting through kappa-opioid receptors, can block the LH surge and ovulation. Dynorphin A(1-17) and A(1-8) are the most potent in this regard.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; Brain; Dynorphins; Enkephalins; Female; Luteinizing Hormone; Naltrexone; Ovulation; Peptide Fragments; Proestrus; Protein Precursors; Rats; Rats, Sprague-Dawley; Receptors, Opioid, kappa

***micro***-, delta- and kappa-opioid receptors are widely expressed in the central nervous system where they mediate the strong analgesic and mood-altering actions of opioids, and modulate numerous endogenous functions. To investigate the contribution of the kappa-opioid receptor (KOR) to opioid function in vivo, we have generated KOR-deficient mice by gene targeting. We show that absence of KOR does not modify expression of the other components of the opioid system, and behavioural tests indicate that spontaneous activity is not altered in mutant mice. The analysis of responses to various nociceptive stimuli suggests that the KOR gene product is implicated in the perception of visceral chemical pain. We further demonstrate that KOR is critical to mediate the hypolocomotor, analgesic and aversive actions of the prototypic kappa-agonist U-50, 488H. Finally, our results indicate that this receptor does not contribute to morphine analgesia and reward, but participates in the expression of morphine abstinence. Together, our data demonstrate that the KOR-encoded receptor plays a modulatory role in specific aspects of opioid function.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; Behavior, Animal; Enkephalins; Female; Gene Expression Regulation; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Morphine; Pain; Pro-Opiomelanocortin; Protein Precursors; Receptors, Opioid, kappa; Substance Withdrawal Syndrome; Viscera

Prodynorphin mRNA and dynorphin B expression have been previously shown to be greatly increased in cardiac myocytes of BIO 14.6 cardiomyopathic hamsters. Here we report that exogenous dynorphin B induced a dose-dependent increase in prodynorphin mRNA levels and stimulated prodynorphin gene transcription in normal hamster myocytes. Similar responses were elicited by the synthetic selective kappa opioid receptor agonist U-50,488H. These effects were counteracted by the kappa opioid receptor antagonist Mr-1452 and were not observed in the presence of chelerythrine or calphostin C, two specific protein kinase C (PKC) inhibitors. Treatment of cardiomyopathic cells with Mr-1452 significantly decreased both prodynorphin mRNA levels and prodynorphin gene transcription. In control myocytes, dynorphin B induced the translocation of PKC-alpha to the nucleus and increased nuclear PKC activity without affecting the expression of PKC-delta, -epsilon, or -zeta. Acute release of either U-50,488H or dyn B over single normal or cardiomyopathic cells transiently increased the cytosolic Ca2+ concentration. A sustained treatment with each opioid agonist increased the cytosolic Ca2+ level for a more prolonged period in cardiomyopathic than in control myocytes and led to a depletion of Ca2+ from the sarcoplasmic reticulum in both groups of cells. The possibility that prodynorphin gene expression may affect the function of the cardiomyopathic cell through an autocrine mechanism is discussed.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; Calcium; Cardiomyopathy, Hypertrophic; Cell Compartmentation; Cell Nucleus; Cricetinae; Cytosol; Dynorphins; Endorphins; Enkephalins; Enzyme Inhibitors; Gene Expression; Mesocricetus; Myocardium; Protein Kinase C; Protein Precursors; Pyrrolidines; Receptors, Opioid, kappa; Ryanodine; Sarcoplasmic Reticulum

The expression of the prodynorphin gene was investigated in adult cultured rat ventricular cardiac myocytes by using a sensitive solution hybridization RNase protection assay for the quantitative analysis of prodynorphin mRNA. Myocyte culture in high KCl resulted, after 4 h, in a marked increase in cellular prodynorphin mRNA, while a KCl treatment for 6, 12, or 24 h progressively down-regulated the levels of prodynorphin mRNA below the control value. Immunoreactive dynorphin B, a biologically active end product of the precursor, was found to be present in the culture medium in significantly higher amounts than in the cardiac myocytes. The levels of this biologically active K opioid receptor agonist significantly increased after 4 h of KCl treatment and were markedly reduced following a 24-h exposure of the cardiac myocytes to KCl. These KCl-induced effects were all abolished by cell incubation in the presence of the calcium channel blocker verapamil. In single cardiac myocytes, acute stimulation of K opioid receptors with dynorphin B or with the selective agonist U-50,488H increased the level of cytosolic calcium. This effect was abolished by the specific K opioid receptor antagonist (Mr-1452) and was not affected by the removal of calcium from the bathing medium. These results suggest that an opioid gene may influence the myocardial function in an autocrine or paracrine fashion.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics; Animals; Calcium; Cells, Cultured; Cytosol; Dynorphins; Enkephalins; Gene Expression; Heart; Heart Ventricles; Male; Myocardium; Potassium Chloride; Protein Precursors; Pyrrolidines; Rats; Rats, Wistar; Receptors, Opioid, kappa; RNA, Messenger

To investigate the effects of the endogenous kappa-receptor agonists dynorphin and leumorphin on neurons of the supraoptic nucleus in the rat hypothalamus, intracellular recordings were made from 62 supraoptic neurons in slice preparations. Bath application of dynorphin and leumorphin at 10(-7) M to 3 x 10(-6) M decreased the spontaneous firing rate with slight hyperpolarization of the membrane potential (-3.8 +/- 0.5 mV, mean +/- S.E.M.) but did not detectably change input resistance. The inhibitory effects were blocked by the relatively selective kappa-antagonist MR-2266. The synthetic kappa-receptor agonist U-50,488H had similar inhibitory effects on supraoptic neurons. Postsynaptic potentials evoked by electrical stimulation dorsal or dorsolateral to the supraoptic nucleus were suppressed by dynorphin and leumorphin. Morphine and [D-Ala, D-Leu]enkephalin, which are relatively selective to mu- and delta-receptors, respectively, influenced the postsynaptic potentials less. Dynorphin and leumorphin also decreased the duration of action potentials that were prolonged by either bath application of tetraethylammonium chloride at 5-10 mM or intracellular injection of Cs ions from the recording electrodes which were filled with 3 M cesium citrate. The prolongation was blocked by 1 mM MnCl2 and 2 mM CoCl2, which suggested that the components were due to voltage-dependent Ca2+ influx. The results suggest that endogenous kappa-receptor agonists inhibit neurosecretory cells of the supraoptic nucleus to suppress synaptic events and Ca2+ components of action potentials.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Action Potentials; Analgesics; Animals; Benzomorphans; Calcium; Cesium; Dynorphins; Electric Stimulation; Electrophysiology; Enkephalins; Humans; In Vitro Techniques; Male; Membrane Potentials; Narcotic Antagonists; Neurosecretory Systems; Protein Precursors; Pyrrolidines; Rats; Rats, Wistar; Receptors, Opioid, kappa; Supraoptic Nucleus; Synapses; Tetraethylammonium Compounds

We have investigated the influence of endogenous opioids on oxytocin secretion during pregnancy. In blood-sampled conscious rats on days 18 and 21 of pregnancy plasma oxytocin concentration, measured by radioimmunoassay, was significantly increased compared to non-pregnant or post-partum rats. On days 15, 18 and 21 of pregnancy but not in non-pregnant, early pregnant or post-partum rats, the opioid antagonist naloxone caused a significant increase in plasma oxytocin compared to vehicle injection, indicating activation of an endogenous opioid restraint over oxytocin secretion. Electrically stimulated neural lobes isolated from 16- and 21-day pregnant rats released more oxytocin than those from non-pregnant rats. However, naloxone (10(-5) M) was less effective at potentiating, and the kappa-opioid agonist U50,488 (10(-5)M) was less effective at inhibiting, stimulated release at the end of pregnancy than in non-pregnant rats suggesting desensitization of oxytocin nerve terminals to actions of endogenous opioids. Neural lobes from male rats drinking 2% saline for 4 days also showed desensitization of oxytocin nerve endings to naloxone. Neither neural lobe content of dynorphin A(1-8), an endogenous kappa-opioid, nor prodynorphin mRNA expression, measured by in situ hybridization histochemistry in the supraoptic nucleus altered during pregnancy. However, neural lobe content of Met5-enkephalin significantly decreased by day 21 of gestation suggesting enhanced release.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; Dynorphins; Electric Stimulation; Endorphins; Enkephalin, Methionine; Enkephalins; Female; Male; Naloxone; Oxytocin; Peptide Fragments; Pituitary Gland, Posterior; Pregnancy; Pregnancy, Animal; Protein Precursors; Pyrrolidines; Rats; Rats, Wistar; RNA, Messenger; Vasopressins

The effects of long-term administration of opioid agonists on the regulation of prodynorphin gene expression in rat brain were investigated. Chronic intracerebroventricular treatment with the synthetic opioid agonist acting on the kappa receptor, U-50,488H, and the classic mu agonist morphine markedly decreased prodynorphin mRNA levels in hypothalamus, hippocampus and striatum of tolerant rats. Levels of ir-Dynorphin A remained unchanged except in two cases. Chronic exposure to opiates thus appears to induce modifications of the endogenous opioid system, as regards gene expression regulation.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics; Animals; Blotting, Northern; Brain Chemistry; Corpus Striatum; DNA Probes; Down-Regulation; Enkephalins; Gene Expression Regulation; Hippocampus; Hypothalamus; Male; Morphine; Narcotics; Protein Precursors; Pyrrolidines; Rats; Rats, Inbred Strains; RNA, Messenger

The three endogenous opioid precursors of almost 30000 Da are pro-opiocortin, proenkephalin and prodynorphin. Pro-opiocortin contains beta-endorphin, melanotropins and ACTH. Proenkephalin yields one [Leu5]enkephalin, three [Met5]enkephalins, one [Met5] enkephalyl-Arg-Arg-Val-NH2 (metorphamide or adrenorphin), one [Met5]enkephalyl-Arg-Gly-Leu and one [Met5]enkephalyl-Arg-Phe. [Leu5]enkephalin is common to all fragments of prodynorphin; its carboxyl extension by Arg-Lys leads to alpha- and beta-neo-endorphin and its carboxyl extension by Arg-Arg gives two dynorphins A and B of 17 and 13 amino acids, respectively. Another endogenous peptide is dynorphin A (1-8). The three main opioid binding sites are mu, delta and kappa. Their analysis has been facilitated by the synthesis of analogues of peptides and non-peptide compounds, which have selective agonist or antagonist action at only one site. The various physiological roles of the three types of the opiate receptor have so far not been sufficiently investigated.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; Benzomorphans; Binding Sites; Brain; Cyclazocine; Diprenorphine; Enkephalin, Leucine; Enkephalins; Ethylketocyclazocine; Guinea Pigs; Ileum; In Vitro Techniques; Male; Mice; Muscle Contraction; Muscle, Smooth; Myenteric Plexus; Naloxone; Pro-Opiomelanocortin; Protein Precursors; Pyrrolidines; Radioligand Assay; Rats; Receptors, Opioid; Vas Deferens