dynorphins and preproenkephalin

The dynorphin/κ-opioid receptor system has been implicated in the pathogenesis and pathophysiology of several psychiatric disorders. In the present review, we present evidence indicating a key role for this system in modulating neurotransmission in brain circuits that subserve mood, motivation, and cognitive function. We overview the pharmacology, signaling, post-translational, post-transcriptional, transcriptional, epigenetic and cis regulation of the dynorphin/κ-opioid receptor system, and critically review functional neuroanatomical, neurochemical, and pharmacological evidence, suggesting that alterations in this system may contribute to affective disorders, drug addiction, and schizophrenia. We also overview the dynorphin/κ-opioid receptor system in the genetics of psychiatric disorders and discuss implications of the reviewed material for therapeutics development.

Topics: Brain; Central Nervous System Stimulants; Cyclic AMP Response Element-Binding Protein; Dynorphins; Enkephalins; Epigenesis, Genetic; Gene Expression Regulation; Humans; Mental Disorders; Models, Animal; Protein Precursors; Receptors, Opioid, kappa; Self Stimulation; Signal Transduction

Addictions to cocaine or heroin/prescription opioids [short-acting μ-opioid receptor (MOPr) agonists] involve relapsing cycles, with experimentation/escalating use, withdrawal/abstinence, and relapse/re-escalation. κ-Opioid receptors (KOPr; encoded by OPRK1), and their endogenous agonists, the dynorphins (encoded by PDYN), have counter-modulatory effects on reward caused by cocaine or MOPr agonist exposure, and exhibit plasticity in addictive-like states. KOPr/dynorphin activation is implicated in depression/anxiety, often comorbid with addictions. In this opinion article we propose that particular stages of the addiction cycle are differentially affected by KOPr/dynorphin systems. Vulnerability and resilience can be due to pre-existing (e.g., genetic) factors, or epigenetic modifications of the OPRK1 or PDYN genes during the addiction cycle. Pharmacotherapeutic approaches limiting changes in KOPr/dynorphin tone, especially with KOPr partial agonists, may hold potential for the treatment of specific drug addictions and psychiatric comorbidity.

Topics: Adaptation, Biological; Animals; Behavior, Addictive; Disease Models, Animal; Drug Discovery; Dynorphins; Enkephalins; Genetic Predisposition to Disease; Humans; Illicit Drugs; Narcotic Antagonists; Polymorphism, Genetic; Protein Precursors; Receptors, Opioid, kappa; Recurrence

1. The working hypothesis that neuropeptide gene expression in a neuron is an indicator of that neuron's physiological activity is discussed. 2. Representative examples from the literature are presented to support the hypothesis. 3. Further, we discuss the regulation of expression of two opioid peptides, preproenkephalin and preprodynorphin, in laminae I and II of the spinal cord and in nucleus caudalis of the trigeminal nuclear complex, where they may play a role in pain modulation. 4. The expression of the opioid peptide genes can be induced by both painful and nonnoxious stimuli in neurons in time-dependent and sensory-specific fashions.

Topics: Animals; Dynorphins; Enkephalins; Gene Expression Regulation; Nucleic Acid Hybridization; Protein Precursors; RNA, Messenger; Spinal Cord; Trigeminal Nuclei

Topics: Animals; Cholecystokinin; Dynorphins; Endorphins; Enkephalins; Hippocampus; Neuropeptides; Protein Precursors; RNA, Messenger; Seizures

Topics: Amino Acid Sequence; Animals; Dynorphins; Endorphins; Enkephalins; Humans; Neurosecretory Systems; Nociceptors; Placenta; Pro-Opiomelanocortin; Protein Precursors; Receptors, Opioid; Reproduction

Of the many factors that influence food intake, there is strong evidence that opioid and CCK peptides, which stimulate feeding and elicit satiety, respectively, are important components that may act in concert to regulate energy balance. Cholecystokinin peptides have been isolated in both the brain and gastrointestinal tract, and changes in concentration in the brain and in plasma have been shown to vary with feeding. Peripherally injected CCK has been shown to elicit satiety in many species, including humans, an effect that may be mediated in the CNS via the vagus. In several species, most notably the sheep, direct injection into the CSF potently decreases food intake. Questions remaining regarding the role of CCK peptides in eliciting satiety include the sites and mechanisms of action. It is unknown whether CCK acts directly on receptors, indirectly on some other parameter, or as a neurotransmitter. Although opioid peptides have also been localized in portions of both the periphery and brain, a specific physiological role for their presence has not yet been determined. Opioid peptides from three families--endorphins, enkephalins, and dynorphins--have been shown to stimulate feeding in various species. They have been active at several opioid receptor types in the CNS, but there is limited evidence to suggest they affect food intake when administered peripherally. In contrast, peripheral injection of opiate antagonists has effectively decreased food intake, an observation that led to the original hypothesis that opioids were involved in the hunger component in the control of food intake and that excess concentrations might be involved in the development of obesity. An increasing body of evidence supports the concept that opioid and CCK peptides may interact to control food intake, but the evidence is more suggestive than conclusive.

Topics: Amino Acid Sequence; Animals; Behavior, Animal; beta-Endorphin; beta-Lipotropin; Brain; Ceruletide; Cholecystokinin; Digestive System Physiological Phenomena; Dynorphins; Eating; Endorphins; Enkephalins; Fasting; Food; Humans; Immunologic Techniques; Kinetics; Morphine; Nervous System; Neurons; Obesity; Peptide Fragments; Protein Precursors; Receptors, Cell Surface; Receptors, Cholecystokinin; Satiation; Sincalide; Species Specificity; Structure-Activity Relationship; Tissue Distribution

Peptides derived from prodynorphin and provasopressin precursors coexist within neurosecretory vesicles of magnocellular neurons of the rat hypothalamus projecting to the posterior pituitary. The secretory activity of these neurons can be stimulated with physiological manipulations that elevate plasma levels of vasopressin (VP), such as dehydration and salt-loading. Evidence indicates that both VP- and prodynorphin-derived peptides are secreted under such conditions. With chronic osmotic challenge, the mRNAs for both prodynorphin and provasopressin increase in parallel in the supraoptic and paraventricular nuclei of the hypothalamus, and not within nonmagnocellular cell groups projecting elsewhere in the brain. The results indicate an example of coordinate regulation of mRNA expression for coexisting peptides within the brain. These results from microdissected tissues have been coupled with the more anatomically precise method of in situ hybridization histochemistry. Using 35S-radiolabeled synthetic oligonucleotides complementary to VP and dynorphin mRNAs, these mRNAs have been autoradiographically localized to magnocellular parikarya in the rat hypothalamus. Results also indicate that this technology can be used for regulatory studies, as evidenced by the increased hybridization of VP oligonucleotide to hypothalamic nuclei from salt-loaded rats.

Topics: Animals; Dynorphins; Enkephalins; Histocytochemistry; Hypothalamus; Nucleic Acid Hybridization; Protein Precursors; Rats; Rats, Brattleboro; RNA, Messenger; Vasopressins

Topics: Animals; Dynorphins; Endorphins; Enkephalins; Genes; Neurons; Neurotransmitter Agents; Pro-Opiomelanocortin; Protein Precursors; Protein Processing, Post-Translational

Topics: Acetylcholine; Afferent Pathways; Analgesics; Animals; beta-Endorphin; Brain Stem; Dynorphins; Endorphins; Enkephalins; Medulla Oblongata; Models, Biological; Neural Pathways; Neurons; Neurotensin; Norepinephrine; Pain; Peptide Fragments; Periaqueductal Gray; Pituitary Hormones, Anterior; Pro-Opiomelanocortin; Protein Precursors; Spinal Cord; Stress, Physiological

Topics: Amino Acid Sequence; Animals; Brain; Dynorphins; Endorphins; Enkephalins; Guinea Pigs; Mice; Pain; Protein Precursors; Rats; Stress, Physiological

The olfactory tubercle (OT) is a striatal region that receives olfactory inputs. mRNAs of prodynorphin (Pdyn) and preproenkephalin (Penk), precursors of dynorphins and enkephalins, respectively, are strongly expressed in the striatum. Both produce opioid peptides with various physiological effects such as pain relief and euphoria. Recent studies have revealed that OT has anatomical and cytoarchitectonic domains that play different roles in odor-induced motivated behavior. Neuronal subtypes of the OT can be distinguished by their expression of the dopamine receptors D1 (Drd1) and D2 (Drd2). Here, we addressed whether and which type of opioid peptide precursors the D1- and D2-expressing neurons in the OT express. We used multiple fluorescence

Topics: Animals; Corpus Striatum; Dynorphins; Enkephalins; In Situ Hybridization, Fluorescence; Mice; Neurons; Olfactory Tubercle; Protein Precursors; Receptors, Dopamine D1; RNA, Messenger

Maintaining stable body temperature through environmental thermal stressors requires detection of temperature changes, relay of information, and coordination of physiological and behavioral responses. Studies have implicated areas in the preoptic area of the hypothalamus (POA) and the parabrachial nucleus (PBN) as nodes in the thermosensory neural circuitry and indicate that the opioid system within the POA is vital in regulating body temperature. In the present study we identify neurons projecting to the POA from PBN expressing the opioid peptides dynorphin and enkephalin. Using mouse models, we determine that warm-activated PBN neuronal populations overlap with both prodynorphin (Pdyn) and proenkephalin (Penk) expressing PBN populations. Here we report that in the PBN

Topics: Animals; Dynorphins; Enkephalins; Female; Hot Temperature; Male; Mice; Mice, Transgenic; Optogenetics; Parabrachial Nucleus; Preoptic Area; Protein Precursors

Early-life stressful events affect the neurobiological maturation of cerebral circuitries including the endogenous opioid system and the effects elicited by adolescent cocaine exposure on this system have been poorly investigated. Here, we evaluated whether cocaine exposure during adolescence causes short- or long-term alterations in mRNAs codifying for selected elements belonging to the opioid system. Moreover, since brain-derived neurotrophic factor (BDNF) may undergo simultaneous alterations with the opioid peptide dynorphin, we also evaluated its signaling pathway as well.. Adolescent male rats were exposed to cocaine (20 mg/kg/day) from post-natal day (PND) 28 to PND42, approximately corresponding to human adolescence. After short- (PND45) or long-term (PND90) abstinence, prodynorphin-κ-opioid receptor (pDYN-KOP) and pronociceptin-nociceptin receptor (pN/OFQ-NOP) gene expression were evaluated in the nucleus accumbens (NAc) and hippocampus (Hip) together with the analysis of BDNF signaling pathways.. In the NAc of PND45 rats, pDYN mRNA levels were up-regulated, an effect paralled by increased BDNF signaling. Differently from NAc, pDYN mRNA levels were down-regulated in the Hip of PND45 rats without significant changes of BDNF pathway. At variance from PND45 rats, we did not find any significant alteration of the investigated parameters either in NAc and Hip of PND90 rats.. Our results indicate that the short-term withdrawal from adolescent cocaine exposure is characterized by a parallel pDYN mRNA and BDNF signaling increase in the NAc. Given the depressive-like state experienced during short abstinence in humans, we hypothesize that such changes may contribute to promote the risk of cocaine abuse escalation and relapse.

Topics: Animals; Brain-Derived Neurotrophic Factor; Cocaine-Related Disorders; Dynorphins; Enkephalins; Gene Expression; Male; Nociceptin Receptor; Nucleus Accumbens; Opioid Peptides; Protein Precursors; Rats; Rats, Sprague-Dawley; Receptors, Opioid; Receptors, Opioid, kappa; RNA, Messenger; Signal Transduction; Substance Withdrawal Syndrome

The serotonergic system is a well-established modulator of l-dopa-induced dyskinesia. To date, targeting serotonin transporters or serotonin receptor subtype 1A (5-HT. The goal of the present study was to characterize Vilazodone's effects on l-dopa-induced behaviors, neurochemistry and gene expression in unilateral 6-hydroxydopamine-lesioned hemi-parkinsonian rats.. In experiments 1 and 2, l-dopa-naïve and l-dopa-primed animals were coadministered Vilazodone and l-dopa daily for 3 weeks to model subchronic use, and behavioral, neurochemical, and messenger RNA (mRNA) expression changes were measured. In experiment 3, dyskinetic behavior was assessed following 5-HT. Vilazodone significantly suppressed developing and established l-dopa-induced dyskinesia without compromising the promotor effects of l-dopa therapy. In the dopamine-depleted striatum, Vilazodone-l-dopa cotreatment increased dopamine content, suggesting a normalization of dopamine kinetics in dyskinetic brain, and reduced l-dopa-induced c-Fos and preprodynorphin mRNA overexpression, indicative of attenuated dopamine D. Our findings show Vilazodone has a serotonin-dependent effect on rodent l-dopa-induced dyskinesia and implicate the potential for repositioning Vilazodone against l-dopa-induced dyskinesia development and expression in Parkinson's disease patients. © 2018 International Parkinson and Movement Disorder Society.

Topics: Animals; Antiparkinson Agents; Disease Models, Animal; Dynorphins; Dyskinesia, Drug-Induced; Enkephalins; Gene Expression Regulation; Levodopa; Male; Oxidopamine; Parkinsonian Disorders; Piperazines; Protein Precursors; Proto-Oncogene Proteins c-fos; Pyridines; Rats; Rats, Sprague-Dawley; Receptors, Serotonin; RNA, Messenger; Selective Serotonin Reuptake Inhibitors; Serotonin; Serotonin Antagonists; Time Factors; Vilazodone Hydrochloride

Opiates act through opioid receptors to diminish pain. Here, we investigated whether mu (MOR) and delta (DOR) receptor endogenous activity assessed in the whole mouse body or in particular at peripheral receptors on primary nociceptive neurons, control colonic pain.. We compared global MOR and DOR receptor knockout (KO) mice, mice with a conditional deletion of MOR and DOR in Nav1.8-positive nociceptive primary afferent neurons (cKO), and control floxed mice of both genders for visceral sensitivity. Visceromotor responses to colorectal distension (CRD) and macroscopic colon scores were recorded on naïve mice and mice with acute colitis induced by 3% dextran sodium sulphate (DSS) for 5 days. Transcript expression for opioid genes and cytokines was measured by quantitative RT-PCR.. Naïve MOR and DOR global KO mice show increased visceral sensitivity that was not observed in cKO mice. MOR and preproenkephalin (Penk) were the most expressed opioid genes in colon. MOR KO mice had augmented kappa opioid receptor and Tumour-Necrosis-Factor-α and diminished Penk transcript levels while DOR, preprodynorphin and Interleukin-1β were unchanged. Global MOR KO females had a thicker colon than floxed females. No alteration was detected in DOR mutant animals. A 5-day DSS treatment led to comparable hypersensitivity in the different mouse lines.. Our results suggest that mu and delta opioid receptor global endogenous activity but not activity at the peripheral Nav1.8 neurons contribute to visceral sensitivity in naïve mice, and that endogenous MOR and DOR tones were insufficient to elicit analgesia after 5-day DSS-induced colitis.. Knockout mice for mu and delta opioid receptor have augmented colon sensitivity in the CRD assay. It shows endogenous mu and delta opioid analgesia that may be explored as potential targets for alleviating chronic intestinal pain.

Topics: Analgesics, Opioid; Animals; Colitis; Dextran Sulfate; Dynorphins; Enkephalins; Female; Interleukin-1beta; Male; Mice; Mice, Knockout; Pain; Pain Management; Protein Precursors; Receptors, Opioid, delta; Receptors, Opioid, mu; Tumor Necrosis Factor-alpha

Neuropeptide precursors are traditionally viewed as proteins giving rise to small neuropeptide molecules. Prodynorphin (PDYN) is the precursor protein to dynorphins, endogenous ligands for the κ-opioid receptor. Alternative mRNA splicing of neuropeptide genes may regulate cell- and tissue-specific neuropeptide expression and produce novel protein isoforms. We here searched for novel PDYN mRNA and their protein product in the human brain.. Novel PDYN transcripts were identified using nested PCR amplification of oligo(dT) selected full-length capped mRNA. Gene expression was analyzed by qRT-PCR, PDYN protein by western blotting and confocal imaging, dynorphin peptides by radioimmunoassay. Neuronal nuclei were isolated using fluorescence-activated nuclei sorting (FANS) from postmortem human striatal tissue. Immunofluorescence staining and confocal microscopy was performed for human caudate nucleus.. Two novel human PDYN mRNA splicing variants were identified. Expression of one of them was confined to the striatum where its levels constituted up to 30% of total PDYN mRNA. This transcript may be translated into ∆SP-PDYN protein lacking 13 N-terminal amino acids, a fragment of signal peptide (SP). ∆SP-PDYN was not processed to mature dynorphins and surprisingly, was targeted to the cell nuclei in a model cellular system. The endogenous PDYN protein was identified in the cell nuclei in human striatum by western blotting of isolated neuronal nuclei, and by confocal imaging.. High levels of alternatively spliced ∆SP-PDYN mRNA and nuclear localization of PDYN protein suggests a nuclear function for this isoform of the opioid peptide precursor in human striatum.

Topics: Adult; Aged; Aged, 80 and over; Amino Acids; Animals; Caudate Nucleus; Cell Line, Tumor; Cell Nucleus; Dynorphins; Enkephalins; Female; Gene Expression Regulation; Gene Silencing; Humans; Male; Middle Aged; Opioid Peptides; Protein Isoforms; Protein Precursors; Rats; RNA, Messenger; Young Adult

The opioidergic neuropeptides dynorphin (DYN) and enkephalin (ENK) and the D1 and D2 dopaminergic receptors (D1R, D2R) are involved in the striatal control of motor and behavioral function. In Parkinson's disease, motor disturbances such as "on-off" motor fluctuations and involuntary movements (dyskinesia) are severe complications that often arise after chronic l-dihydroxyphenylalanine (l-DOPA) treatment. Changes in the striatal expression of preproENK (PPENK), proDYN (PDYN), D1R, and D2R mRNA have been observed in parkinsonian animals treated with l-DOPA. Enhanced opioidergic transmission has been found in association with l-DOPA-induced dyskinesia, but the connection of PPENK, PDYN, D1R, and D2R mRNA expression with locomotor activity remains unclear. In this study, we measured PPENK, PDYN, D1R and D2R mRNA levels by in situ hybridization in the striatum of 6-OHDA hemi-parkinsonian rats treated with l-DOPA (PD+l-DOPA group), along with two control groups (PD+saline and naive+l-DOPA). We found different levels of expression of PPENK, PDYN, D1R and D2R mRNA across the experimental groups and correlated the changes in mRNA expression with dyskinesia and locomotor variables assessed by open field test during several phases of l-DOPA treatment. Both PDYN and PPENK mRNA levels were correlated with the severity of dyskinesia, while PPENK mRNA levels were also correlated with the frequency of contralateral rotational movements and with locomotor variables. Moreover, a strong correlation was found between D1R mRNA expression and D2R mRNA expression in the PD+l-DOPA group. These findings suggest that, in parkinsonian animals treated with l-DOPA, high levels of PPENK are a prerequisite for a locomotor sensitization to l-DOPA treatment, while PDYN overexpression is responsible only for the development of dyskinesia.

Topics: Analysis of Variance; Animals; Antiparkinson Agents; Disease Models, Animal; Dynorphins; Dyskinesia, Drug-Induced; Enkephalins; Female; Gene Expression Regulation; Levodopa; Oxidopamine; Parkinson Disease; Protein Precursors; Rats; Rats, Sprague-Dawley; Receptors, Dopamine; RNA, Messenger; Statistics as Topic; Sympatholytics; Tyrosine 3-Monooxygenase

Spinocerebellar ataxia type 23 (SCA23) is caused by missense mutations in prodynorphin, encoding the precursor protein for the opioid neuropeptides α-neoendorphin, Dynorphin (Dyn) A and Dyn B, leading to neurotoxic elevated mutant Dyn A levels. Dyn A acts on opioid receptors to reduce pain in the spinal cord, but its cerebellar function remains largely unknown. Increased concentration of or prolonged exposure to Dyn A is neurotoxic and these deleterious effects are very likely caused by an N-methyl-d-aspartate-mediated non-opioid mechanism as Dyn A peptides were shown to bind NMDA receptors and potentiate their glutamate-evoked currents. In the present study, we investigated the cellular mechanisms underlying SCA23-mutant Dyn A neurotoxicity. We show that SCA23 mutations in the Dyn A-coding region disrupted peptide secondary structure leading to a loss of the N-terminal α-helix associated with decreased κ-opioid receptor affinity. Additionally, the altered secondary structure led to increased peptide stability of R6W and R9C Dyn A, as these peptides showed marked degradation resistance, which coincided with decreased peptide solubility. Notably, L5S Dyn A displayed increased degradation and no aggregation. R6W and wt Dyn A peptides were most toxic to primary cerebellar neurons. For R6W Dyn A, this is likely because of a switch from opioid to NMDA- receptor signalling, while for wt Dyn A, this switch was not observed. We propose that the pathology of SCA23 results from converging mechanisms of loss of opioid-mediated neuroprotection and NMDA-mediated excitotoxicity.

Topics: Amino Acid Sequence; Animals; Cell Culture Techniques; Computer Simulation; Dynorphins; Endorphins; Enkephalins; Mice; Mice, Inbred C57BL; N-Methylaspartate; Neurons; Neurotoxins; Protein Precursors; Protein Structure, Secondary; Receptors, N-Methyl-D-Aspartate; Signal Transduction; Spinal Cord; Spinocerebellar Degenerations

Neuropeptide Y (NPY) is a hypothalamic neuropeptide that plays a prominent role in feeding and energy homeostasis. Expression of the NPY Y1 receptor (Y1R) is highly concentrated in the nucleus accumbens (Acb), a region important in the regulation of palatable feeding. In this study, we performed a number of experiments to investigate the actions of NPY in the Acb.. First, we determined caloric intake and food choice after bilateral administration of NPY in the Acb in rats on a free-choice diet of saturated fat, 30% sucrose solution, and standard chow and whether this was mediated by the Y1R. Second, we measured the effect of intra-Acb NPY on neuronal activity using in vivo electrophysiology. Third, we examined co-localization of Y1R with enkephalin and dynorphin neurons and the effect of NPY on preproenkephalin messenger RNA levels in the striatum using fluorescent and radioactive in situ hybridization. Finally, using retrograde tracing, we examined whether NPY neurons in the arcuate nucleus projected to the Acb.. In rats on the free-choice, high-fat, high-sugar diet, intra-Acb NPY increased intake of fat, but not sugar or chow, and this was mediated by the Y1R. Intra-Acb NPY reduced neuronal firing, as well as preproenkephalin messenger RNA expression in the striatum. Moreover, Acb enkephalin neurons expressed Y1R and arcuate nucleus NPY neurons projected to the Acb.. NPY reduces neuronal firing in the Acb resulting in increased palatable food intake. Together, our neuroanatomical, pharmacologic, and neuronal activity data support a role and mechanism for intra-Acb NPY-induced fat intake.

Topics: Action Potentials; Animals; Arcuate Nucleus of Hypothalamus; Choice Behavior; Corpus Striatum; Dietary Fats; Dietary Sucrose; Dynorphins; Eating; Enkephalins; Feeding Behavior; Male; Mice, Inbred C57BL; Neurons; Neuropeptide Y; Nucleus Accumbens; Protein Precursors; Rats, Wistar; Receptors, Neuropeptide Y; RNA, Messenger

Behavioral flexibility is known to be mediated by corticostriatal systems and to involve several major neurotransmitter signaling pathways. The current study investigated the effects of inactivation of glutamatergic N-methyl-D-aspartate-(NMDA) receptor signaling in the dorsal striatum on behavioral flexibility in mice. NMDA-receptor inactivation was achieved by virus-mediated inactivation of the Grin1 gene, which encodes the essential NR1 subunit of NMDA receptors. To assess behavioral flexibility, we used a water U-maze paradigm in which mice had to shift from an initially acquired rule to a new rule (strategy shifting) or had to reverse an initially learned rule (reversal learning). Inactivation of NMDA-receptors in all neurons of the dorsal striatum did not affect learning of the initial rule or reversal learning, but impaired shifting from one strategy to another. Strategy shifting was also compromised when NMDA-receptors were inactivated only in dynorphin-expressing neurons in the dorsal striatum, which represent the direct pathway. These data suggest that NMDA-receptor-mediated synaptic plasticity in the dorsal striatum contributes to strategy shifting and that striatal projection neurons of the direct pathway are particularly relevant for this process.

Topics: Animals; Cognition; Corpus Striatum; Dynorphins; Enkephalins; Executive Function; Exploratory Behavior; HEK293 Cells; Humans; Maze Learning; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Nerve Tissue Proteins; Neuronal Plasticity; Protein Precursors; Psychomotor Performance; Receptors, N-Methyl-D-Aspartate; Reversal Learning

Kisspeptin/neurokinin B/dynorphin (KNDy) neurons, which coexpress kisspeptins (Kps), neurokinin B (NKB), and dynorphin (Dyn), regulate gonadotropin secretion. The KNDy model proposes that NKB (a stimulator, through NK3R) and Dyn (an inhibitor, through κ-opioid receptor) shape Kp secretion onto GnRH neurons. However, some aspects of this paradigm remain ill defined. Here we aimed to characterize the following: 1) the effects of NKB signaling on FSH secretion and 2) the role of Dyn in gonadotropin secretion after NK3R activation; 3) additionally, we explored the roles of other tachykinin receptors, NK1R and NK2R, on gonadotropin release. Thus, the effects of the NK3R agonist, senktide, on FSH release were explored across postnatal development in male and female rats; gonadotropin responses to agonists of NK1R substance P and NK2R [neurokinin A (NKA)] were also monitored. Moreover, the effects of senktide on gonadotropin secretion were assessed after antagonizing Dyn actions by nor-binaltorphimine didydrochloride. Before puberty, rats of both sexes showed increased FSH secretion to senktide (and Kp-10). Conversely, adult female rats were irresponsive to senktide in terms of FSH, despite proven LH responses, whereas the adult males did not display FSH or LH responses to senktide, even at high doses. In turn, substance P and NKA stimulated gonadotropin secretion in prepubertal rats, whereas in adults modest gonadotropin responses to NKA were detected. By pretreatment with a Dyn antagonist, adult males became responsive to senktide in terms of LH secretion and displayed elevated basal LH and FSH levels; nor-binaltorphimine didydrochloride treatment uncovered FSH responses to senktide in adult females. Furthermore, the expression of Pdyn and Opkr1 (encoding Dyn and κ-opioid receptor, respectively) in the mediobasal hypothalamus was greater in males than in females at prepubertal ages. Overall, our data contribute to refining our understanding on how the elements of the KNDy node and related factors (ie, other tachykinins) differentially participate in the control of gonadotropins at different stages of rat postnatal maturation.

Topics: Aging; Animals; Dynorphins; Enkephalins; Female; Follicle Stimulating Hormone; Hypothalamus; Kisspeptins; Luteinizing Hormone; Male; Neurokinin B; Peptide Fragments; Protein Precursors; Rats, Wistar; Receptors, Neurokinin-1; Receptors, Neurokinin-2; Substance P

Despite considerable knowledge that prenatal ethanol exposure can lead to devastating effects on the developing fetus, alcohol consumption by pregnant women remains strikingly prevalent. Both clinical and basic research has suggested that, in addition to possible physical, behavioral, and cognitive deficits, gestational exposure to alcohol may lead to an increased risk for the development of later alcohol-related use and abuse disorders. The current work sought to characterize alterations in endogenous opioid signaling peptides and gene expression produced by ethanol exposure during the last days of gestation.. Experimental subjects were 4-, 8-, and 12-day old infant rats obtained from pregnant females that were given daily intubations of 0, 1, or 2g/kg ethanol during the last few days of gestation (GDs 17-20). Using real-time RT-PCR, western blotting analysis, and enzyme immunoassays, we examined mRNA and protein for three opioid receptors and ligands in the nucleus accumbens, ventral tegmental area, and hypothalamus.. Three main trends emerged - (1) mRNA for the majority of factors was found to upregulate across each of the three postnatal ages assessed, indicative of escalating ontogenetic expression of opioid-related genes; (2) prenatal ethanol significantly reduced many opioid peptides, suggesting a possible mechanism by which prenatal exposure can affect future responsiveness towards ethanol; and (3) the nucleus accumbens emerged as a key site for ethanol-dependent effects, suggesting a potential target for additional assessment and intervention towards understanding the ethanol's ability to program the developing brain.. We provide a global assessment of relatively long-term changes in both opioid gene expression and protein following exposure to only moderate amounts of ethanol during a relatively short window in the prenatal period. These results suggest that, while continuing to undergo ontogenetic changes, the infant brain is sensitive to prenatal ethanol exposure and that such exposure may lead to relatively long-lasting changes in the endogenous opioid system within the reward circuitry. These data indicate a potential mechanism and target for additional assessments of ethanol's ability to program the brain, affecting later responsiveness towards the drug.

Topics: Age Factors; Alcohol Drinking; Analysis of Variance; Animals; Animals, Newborn; Brain; Central Nervous System Depressants; Dynorphins; Enkephalins; Ethanol; Female; Gene Expression Regulation, Developmental; Gestational Age; Male; Pregnancy; Prenatal Exposure Delayed Effects; Pro-Opiomelanocortin; Protein Precursors; Rats; Rats, Sprague-Dawley; Receptors, Opioid; RNA, Messenger

Neuropeptides induce signal transduction across the plasma membrane by acting through cell-surface receptors. The dynorphins, endogenous ligands for opioid receptors, are an exception; they also produce non-receptor-mediated effects causing pain and neurodegeneration. To understand non-receptor mechanism(s), we examined interactions of dynorphins with plasma membrane. Using fluorescence correlation spectroscopy and patch-clamp electrophysiology, we demonstrate that dynorphins accumulate in the membrane and induce a continuum of transient increases in ionic conductance. This phenomenon is consistent with stochastic formation of giant (~2.7 nm estimated diameter) unstructured non-ion-selective membrane pores. The potency of dynorphins to porate the plasma membrane correlates with their pathogenic effects in cellular and animal models. Membrane poration by dynorphins may represent a mechanism of pathological signal transduction. Persistent neuronal excitation by this mechanism may lead to profound neuropathological alterations, including neurodegeneration and cell death.

Topics: Analgesics, Opioid; Animals; Cell Membrane; Dynorphins; Endorphins; Enkephalins; Humans; Ligands; Microscopy, Confocal; Neuropeptides; Opioid Peptides; PC12 Cells; Protein Precursors; Rats; Signal Transduction

Medium spiny neurons (MSNs) constitute 95% of neurons in the dorsal striatum subdivided into direct (striatonigral) and indirect (striatopallidal) pathways. Whereas D1 and D2 receptors and several neuropeptides, including dynorphin and enkephalin, are differentially expressed in these neurons, 5-hydroxytryptamine 6 receptors (5-HT6) are expressed in both pathways. Previous results demonstrate that concurrent 5-HT6 receptor overexpression in MSNs of both pathways in the dorsomedial striatum (DMS) interferes with instrumental learning and that 5-HT6 overexpression in the dorsolateral striatum (DLS) relieves rats from inflexible habitual behaviors. We hypothesized that 5-HT6 receptor-mediated co-activation of both pathways interferes with the differential activation/inhibition of direct/indirect pathways by dopamine. To test this idea, we cloned novel viral vectors to selectively overexpress 5-HT6 receptors in direct or indirect pathway MSNs to deconstruct their role in modulating instrumental learning and habitual responding. We found that increasing 5-HT6 receptor expression in either direct or indirect pathway MSNs of the posterior DMS selectively enhanced or impaired initial acquisition of a discrete instrumental learning task respectively, though all rats were ultimately able to learn the task. In a separate set of experiments, 5-HT6 receptor overexpression in indirect pathway MSNs of the DLS facilitated behavioral flexibility in rats overtrained on a repetitive pressing task using a variable interval schedule of reinforcement, during an omission contingency training session and subsequent probe testing. Together these findings further the notion that 5-HT6 signaling causes balanced activation of opposing MSN pathways by serotonin in sub-regions of the dorsal striatum allowing for more reflective modalities of behavior.

Topics: Animals; Conditioning, Operant; Dynorphins; Enkephalins; Genetic Vectors; Male; Neostriatum; Neurons; Protein Precursors; Rats; Rats, Long-Evans; Receptors, Serotonin

The mechanisms underlying lateralization and progression of motor symptoms from unilateral to bilateral in Parkinson's disease (PD) remain to be elucidated. In addition, the molecular mechanisms involved in levodopa-induced dyskinesias (LIDs) depending on lateralization and disease progression from unilaterally to bilateral have not been described yet. We investigated motor symptoms, LIDs and associated striatal molecular markers expression after unilateral left or right, and after a sequential bilateral 6-hydroxydopamine (6-OHDA)-induced nigrostriatal lesions in rats. Sequentially bilateral lesioned animals showed a bilateral increase in striatal preproenkephalin (PPE) mRNA without changes in pre-prodynorphin (PDyn) mRNA expression. The increase in dyskinesias when parkinsonism becomes bilateral was mostly due to an increase in orolingual dyskinesias associated to a increase in PDyn mRNA expression. Right lesion induces, or facilitates when first-done, a greater level of LIDs and an increase in striatal PPE and PDyn mRNAs in the second lesioned side. We describe a new striatal molecular pattern that appears when parkinsonism becomes bilateral and the relevance of the lateralization for the development of LIDs.

Topics: Animals; Antiparkinson Agents; Corpus Striatum; Dynorphins; Dyskinesia, Drug-Induced; Enkephalins; Functional Laterality; Levodopa; Male; Motor Activity; Oxidopamine; Parkinsonian Disorders; Protein Precursors; Rats, Sprague-Dawley; RNA, Messenger

Cocaine induces neurochemical changes of endogenous prodynorphin-kappa opioid receptor (pDYN-KOP) and pronociceptin/orphaninFQ-nociceptin receptor (pN/OFQ-NOP) systems. Both systems play an important role in rewarding mechanisms and addictive stimulus processing by modulating drug-induced dopaminergic activation in the mesocortico-limbic brain areas. They are also involved in regulating stress mechanisms related to addiction. The aim of this study was to investigate possible changes of gene expression of the dynorphinergic and nociceptinergic system components in the nucleus accumbens (NA) and in medial and lateral caudate putamen (mCPu and lCPu, respectively) of rats, following chronic subcutaneous infusion of cocaine. In addition, the epigenetic histone modifications H3K4me3 and H3K27me3 (an activating and a repressive marker, respectively) at the promoter level of the pDYN, KOP, pN/OFQ and NOP genes were investigated. Results showed that cocaine induced pDYN gene expression up-regulation in the NA and lCPu, and its down-regulation in the mCPu, whereas KOP mRNA levels were unchanged. Moreover, cocaine exposure decreased pN/OFQ gene expression in the NA and lCPu, while NOP mRNA levels appeared significantly increased in the NA and decreased in the lCPu. Specific changes of the H3K4me3 and H3K27me3 levels were found at pDYN, pN/OFQ, and NOP gene promoter, consistent with the observed gene expression alterations. The present findings contribute to better define the role of endogenous pDYN-KOP and pN/OFQ-NOP systems in neuroplasticity mechanisms following chronic cocaine treatment. The epigenetic histone modifications underlying the gene expression changes likely mediate the effects of cocaine on transcriptional regulation of specific gene promoters that result in long-lasting drug-induced plasticity.

Topics: Animals; Cocaine; Corpus Striatum; Dynorphins; Enkephalins; Epigenesis, Genetic; Gene Expression Regulation; Histones; Male; Nociceptin; Nociceptin Receptor; Nucleus Accumbens; Opioid Peptides; Protein Precursors; Rats; Receptors, Opioid; Receptors, Opioid, kappa

Brain glutamate overactivity is well documented in Parkinson's disease (PD) and antiglutamatergic drugs decrease L-3,4-dihydroxyphenylalanine (l-DOPA)-induced dyskinesias (LID); the implication of dopamine neurotransmission is not documented in this anti-LID activity. Therefore, we evaluated changes of dopamine receptors, their associated signaling proteins and neuropeptides mRNA, in normal control monkeys, in saline-treated 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned monkeys and in L-DOPA-treated MPTP monkeys, without or with an adjunct treatment to reduce the development of LID: 2-methyl-6-(phenylethynyl)pyridine (MPEP), the prototypal metabotropic glutamate 5 (mGlu5) receptor antagonist. All de novo treatments were administered for 1 month and the animals were sacrificed thereafter. MPTP monkeys treated with l-DOPA + MPEP developed significantly less LID than MPTP monkeys treated with l-DOPA alone. [(3)H]SCH-23390 specific binding to D1 receptors of all MPTP monkeys was decreased as compared to controls in the basal ganglia and no difference was observed between all MPTP groups, while striatal D1 receptor mRNA levels remained unchanged. [(3)H]raclopride specific binding to striatal D2 receptors and mRNA levels of D2 receptors were increased in MPTP monkeys compared to controls; l-DOPA treatment reduced this binding in MPTP monkeys while it remained elevated with the l-DOPA + MPEP treatment. Striatal [(3)H]raclopride specific binding correlated positively with D2 receptor mRNA levels of all MPTP-lesioned monkeys. Striatal preproenkephalin/preprodynorphin mRNA levels and phosphorylated ERK1/2 and Akt/GSK3β levels increased only in L-DOPA-treated MPTP monkeys as compared to controls, saline treated-MPTP and l-DOPA + MPEP treated MPTP monkeys. Hence, reduction of development of LID with MPEP was associated with changes in D2 receptors, their associated signaling proteins and neuropeptides.

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Animals; Antiparkinson Agents; Basal Ganglia; Corpus Striatum; Dopamine Antagonists; Dopamine D2 Receptor Antagonists; Dynorphins; Dyskinesia, Drug-Induced; Enkephalins; Excitatory Amino Acid Antagonists; Female; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Levodopa; Macaca fascicularis; MAP Kinase Signaling System; Parkinsonian Disorders; Protein Precursors; Proto-Oncogene Proteins c-akt; Pyridines; Receptor, Metabotropic Glutamate 5; Receptors, Dopamine D1; Receptors, Dopamine D2; RNA, Messenger

To measure the plasma concentrations of three endogenous opioid peptides and the levels of preproenkephalin (PPE) and preprodynorphin (PPD) mRNA in peripheral blood lymphocytes of patients during scheduled surgery performed under intravenous general anaesthesia combined with an epidural block.. Patients were anaesthetized and arterial blood was collected at 0 (baseline), 20, 40, 60, and 80 min during surgery. The plasma concentrations of β-endorphin, leucine-enkephalin and dynorphin A were measured using radioimmunoassay. Reverse transcription-polymerase chain reaction was used to measure the levels of PPD and PPE mRNA in peripheral blood lymphocytes collected during surgery.. Fifteen patients participated in this prospective study. The plasma concentrations of β-endorphin were significantly lower at all time-points compared with the baseline value. The plasma concentrations of leucine-enkephalin and dynorphin A were significantly lower at 40, 60, and 80 min compared with baseline. The PPD/β-actin ratio was significantly lower at 80 min compared with baseline, while the PPE/β-actin ratio showed no significant change.. The level of mRNA from two pre-endogenous opioid peptide genes either decreased or remained unchanged during surgery under intravenous general anaesthesia with epidural block, suggesting that patients remained pain free during surgery.

Topics: Abdomen; Adult; Anesthesia, Epidural; Anesthesia, General; Anesthetics, Intravenous; beta-Endorphin; Bupivacaine; Dynorphins; Enkephalin, Leucine; Enkephalins; Female; Fentanyl; Gene Expression; Humans; Male; Midazolam; Middle Aged; Pain; Prospective Studies; Protein Precursors; Radioimmunoassay; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Vecuronium Bromide

Molecular mechanisms of adaptive transformations caused by alcohol exposure on opioid dynorphin and nociceptin systems have been investigated in the rat brain. Alcohol was intragastrically administered to rats to resemble human drinking with several hours of exposure: water or alcohol (20% in water) at a dose of 1.5 g/kg three times daily for 1 or 5 days. The development of tolerance and dependence were recorded daily. Brains were dissected 30 minutes (1- and 5-day groups) or 1, 3 or 7 days after the last administration for the three other 5-day groups (groups under withdrawal). Specific alterations in opioid genes expression were ascertained. In the amygdala, an up-regulation of prodynorphin and pronociceptin was observed in the 1-day group; moreover, pronociceptin and the kappa opioid receptor mRNAs in the 5-day group and both peptide precursors in the 1-day withdrawal group were also up-regulated. In the prefrontal cortex, an increase in prodynorhin expression in the 1-day group was detected. These data indicate a relevant role of the dynorphinergic system in the negative hedonic states associated with multiple alcohol exposure. The pattern of alterations observed for the nociceptin system appears to be consistent with its role of functional antagonism towards the actions of ethanol associated with other opioid peptides. Our findings could help to the understanding of how alcohol differentially affects the opioid systems in the brain and also suggest the dynorphin and nociceptin systems as possible targets for the treatment and/or prevention of alcohol dependence.

Topics: Alcohol Drinking; Alcoholic Intoxication; Amygdala; Animals; Brain; Central Nervous System Depressants; Dose-Response Relationship, Drug; Dynorphins; Enkephalins; Ethanol; Gene Expression; Male; Nociceptin; Opioid Peptides; Protein Precursors; Rats; Rats, Sprague-Dawley; Reflex, Righting; Substance Withdrawal Syndrome

The endogenous opioid system (EOS) plays a critical role in addictive processes. Molecular dysregulations in this system may be specific for different stages of addiction cycle and neurocircuitries involved and therefore may differentially contribute to the initiation and maintenance of addiction. Here we evaluated whether the EOS is altered in brain areas involved in cognitive control of addiction including the dorsolateral prefrontal cortex (dl-PFC), orbitofrontal cortex (OFC) and hippocampus in human alcohol-dependent subjects. Levels of EOS mRNAs were measured by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and levels of dynorphins by radioimmunoassay (RIA) in post-mortem specimens obtained from 14 alcoholics and 14 controls. Prodynorphin mRNA and dynorphins in dl-PFC, κ-opioid receptor mRNA in OFC and dynorphins in hippocampus were up-regulated in alcoholics. No significant changes in expression of proenkephalin, and µ- and δ-opioid receptors were evident; pro-opiomelanocortin mRNA levels were below the detection limit. Activation of the κ-opioid receptor by up-regulated dynorphins in alcoholics may underlie in part neurocognitive dysfunctions relevant for addiction and disrupted inhibitory control.

Topics: Adaptation, Physiological; Adult; Alcoholism; Analysis of Variance; Animals; Behavior, Addictive; Case-Control Studies; Dynorphins; Enkephalins; Hippocampus; Humans; Male; Opioid Peptides; Prefrontal Cortex; Protein Precursors; Radioimmunoassay; Receptors, Opioid; Reverse Transcriptase Polymerase Chain Reaction; Reward; RNA, Messenger; Statistics, Nonparametric; Up-Regulation

The aim of this study was to determine the role of the endogenous dynorphin/kappa opioid receptor (DYN/KOP) system in ethanol-induced state-dependent conditioned place preference (CPP). To this end, mice lacking the pro-DYN gene and their wild-type littermates/controls were tested for baseline place preference on day 1, received 15-min morning and afternoon conditionings with saline or ethanol (2g/kg) each day for three consecutive days and were then tested for CPP under a drug-free state on day 5 and following a saline or ethanol (1 or 2g/kg) challenge on day 8. Given that compensatory developmental changes may occur in knockout mice, the effect of nor-binaltorphimine (nor-BNI), a KOP antagonist, on state-dependent CPP induced by ethanol was also studied in wild-type mice. On day 1, mice were tested for baseline place preference and, 4h later, treated with saline or nor-BNI (10mg/kg). On days 2-4, mice received 15-min morning and afternoon conditionings and were tested for CPP under a drug-free state on day 5 and following an ethanol (1g/kg) challenge on day 8. A comparable CPP was observed in mice lacking the pro-DYN gene and their wild-type littermates/controls as well as in wild-type mice treated with nor-BNI and their saline-treated controls. However, these mice compared to their respective controls exhibited a greater CPP response following an ethanol (1g/kg) challenge, suggesting that the endogenous DYN/KOP system may negatively regulate ethanol-induced state-dependent CPP.

Topics: Animals; Central Nervous System Depressants; Conditioning, Operant; Drug Administration Schedule; Dynorphins; Enkephalins; Ethanol; Female; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Naltrexone; Narcotic Antagonists; Protein Precursors; Sex Factors; Time Factors

The transcription factor cAMP response element binding (CREB) protein plays a pivotal role in drug-dependent neuronal plasticity. CREB phosphorylation at Ser133 is enhanced by drugs of abuse, including nicotine. Dynorphin (Dyn) contributes to the addictive process and its precursor gene prodynorphin (PD) is regulated by CREB. PD mRNA and Dyn synthesis were enhanced in the striatum following acute nicotine, suggesting genomic regulation.. These studies investigated PD transcription in mice acutely treated with nicotine, determined the role of CREB, and characterized the receptors involved.. Acute nicotine increased adenylyl cyclase activity, cAMP, and pCREB Ser133 levels in striatum and enhanced CREB binding to CRE elements (DynCREs) of the PD promoter, preferentially DynCRE3. DynCRE3 binding was dose dependent with 1 mg of nicotine giving a maximal response. Additionally, DynCRE binding was time dependent, rising by 15 min, reaching a maximum at 1 h, and returning to control by 3 h, a temporal pattern similar to that of cAMP and pCREB. Supershift experiments showed that CREB and pCREB Ser133 were the major contributors to DynCRE3 binding complex. The nAChR antagonist mecamylamine and the dopamine D1-like receptor antagonist SCH 23390 prevented the nicotine-induced increase of pCREB and nuclear protein binding to DynCRE3.. Our findings suggest that nicotine regulates PD expression in striatum at the transcriptional level and CREB is involved. Dopamine D1 receptor stimulation by nAChR-released dopamine appears to be an underlying mechanism. Altered Dyn synthesis might be relevant for the behavioral actions of nicotine and especially its aversive properties.

Topics: Adenylyl Cyclases; Animals; Benzazepines; Corpus Striatum; Cyclic AMP; Cyclic AMP Response Element-Binding Protein; Dose-Response Relationship, Drug; Dynorphins; Enkephalins; Male; Mecamylamine; Mice; Nicotine; Phosphorylation; Protein Binding; Protein Precursors; Response Elements; Time Factors; Transcription, Genetic

Tissue injury and/or opioids induce plastic changes in the spinal cord resulting in pain hypersensitivity; the contribution of the dorsal root ganglia (DRG) is poorly understood. We evaluated DRG phenotypic changes induced by surgery and/or remifentanil in a mice model of postoperative pain using as neuronal markers ERK1/2 and c-Fos; prodynorphin mRNA and dynorphin levels were also determined. We hypothesized that a correlation between nociception and DRG reactivity would occur. Surgery and/or remifentanil induced mechanical hypersensitivity, correlated with ERK1/2 phosphorylation and c-Fos expression in the DRG; changes were greater in the remifentanil + incision group and still present on day 14 (p < 0.01 vs. control). Intrathecal PD98059 (ERK1/2 inhibitor) partially reversed the mechanical hypersensitivity (44%, p < 0.05) observed in the remifentanil + incision group. In this group, significant increases in prodynorphin mRNA (at 2, 7, and 14 days, p < 0.01) roughly coincided with increases in dynorphin (days 2 and 14, p < 0.001) in the DRG. Remifentanil or incision (alone) also induced an up-regulation in prodynorphin mRNA expression on days 7 and 14 (p < 0.01, p < 0.05, respectively), partially correlating with dynorphin levels. On day 21, all molecular changes returned to control levels in all experimental conditions, concurring with the complete recovery of nociceptive thresholds. Surgery and/or remifentanil induce up-regulation of c-Fos and pERK in the DRG, approximately correlating with nociceptive behavior, also associated with an increased expression of prodynorphin/dynorphin. These changes support the role of the DRG in the development and maintenance of pain hypersensitivity after surgery. The findings could contribute to the development of new therapeutic agents focused on peripheral targets.

Topics: Analgesics, Opioid; Animals; Behavior, Animal; Dynorphins; Enkephalins; Ganglia, Spinal; Hyperalgesia; Male; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Pain, Postoperative; Piperidines; Protein Precursors; Proto-Oncogene Proteins c-fos; Remifentanil; RNA, Messenger

Naltrexone is not recommended during pregnancy. However, sustained-release naltrexone implant use in humans has resulted in cases of inadvertent foetal exposure. Here, we used clinically relevant dosing to examine the effects of maternally administered sustained-release naltrexone on the rat brain by examining offspring at birth and in adulthood. Maternal treatment (naltrexone or placebo implant) started before conception and ceased during gestation, birth or weaning. Morphometry was assessed in offspring at birth and adulthood. Adult offspring were evaluated for differences in locomotor behaviour (basal and morphine-induced, 10 mg/kg, s.c.) and opioid neurochemistry, propensity to self-administer morphine and cue-induced drug-seeking after abstinence. Blood analysis confirmed offspring exposure to naltrexone during gestation, birth and weaning. Naltrexone exposure increased litter size and reduced offspring birth-weight but did not alter brain morphometry. Compared to placebo, basal motor activity of naltrexone-exposed adult offspring was lower, yet they showed enhanced development of psychomotor sensitization to morphine. Developmental naltrexone exposure was associated with resistance to morphine-induced down-regulation of striatal preproenkephalin mRNA expression in adulthood. Adult offspring also exhibited greater operant responding for morphine and, in addition, cue-induced drug-seeking was enhanced. Collectively, these data show pronounced effects of developmental naltrexone exposure, some of which persist into adulthood, highlighting the need for follow up of humans that were exposed to naltrexone in utero.

Topics: Animals; Behavior, Animal; Brain; Delayed-Action Preparations; Dynorphins; Enkephalins; Female; Gene Expression; Male; Morphine; Motor Activity; Naltrexone; Narcotic Antagonists; Narcotics; Neostriatum; Pregnancy; Prenatal Exposure Delayed Effects; Protein Precursors; Rats; Rats, Sprague-Dawley; Receptors, Opioid, mu; Self Administration

Adenosine A(₂A) receptor antagonism provides a promising approach to developing nondopaminergic therapy for Parkinson's disease (PD). Clinical trials of A(₂A) antagonists have targeted PD patients with L-3,4-dihydroxyphenylalanine (L-DOPA)-induced dyskinesia (LID) in an effort to improve parkinsonian symptoms. The role of adenosine in the development of LID is little known, especially regarding its actions via A₁ receptors. We aimed to examine the effects of genetic deletion and pharmacological blockade of A₁ and/or A(₂A) receptors on the development of LID, on the induction of molecular markers of LID including striatal preprodynorphin and preproenkephalin (PPE), and on the integrity of dopaminergic nigrostriatal neurons in hemiparkinsonian mice. Following a unilateral 6-hydroxydopamine lesion A₁, A(₂A) and double A₁-A(₂A) knockout (KO) and wild-type littermate mice, and mice pretreated with caffeine (an antagonist of both A₁ and A(₂A) receptors) or saline were treated daily for 18-21 days with a low dose of L-DOPA. Total abnormal involuntary movements (AIMs, a measure of LID) were significantly attenuated (p<0.05) in A₁ and A(₂A) KOs, but not in A₁-A(₂A) KOs and caffeine-pretreated mice. An elevation of PPE mRNA ipsilateral to the lesion in WT mice was reduced in all KO mice. In addition, neuronal integrity assessed by striatal dopamine content was similar in all KOs and caffeine-pretreated mice following 6-hydroxydopamine lesioning. Our findings raise the possibility that A₁ or A(₂A) receptors blockade might also confer a disease-modifying benefit of reduced risk of disabling LID, whereas the effect of their combined inactivation is less clear.

Topics: Adrenergic Agents; Animals; Antiparkinson Agents; Caffeine; Corpus Striatum; Disease Models, Animal; Dynorphins; Dyskinesia, Drug-Induced; Enkephalins; Gene Expression Regulation; Levodopa; Mice; Mice, Inbred C57BL; Mice, Knockout; Oxidopamine; Parkinson Disease; Protein Precursors; Purinergic P1 Receptor Antagonists; Receptor, Adenosine A1; Receptor, Adenosine A2A; RNA, Messenger; Statistics, Nonparametric; Time Factors

Opioid peptides are involved in various pathophysiological processes, including algesia, epilepsy, and drug dependence. A strong association between L-DOPA-induced dyskinesia (LID) and elevated prodynorphin mRNA levels has been established in both patients and in animal models of Parkinson's disease, but to date the endogenous prodynorphin peptide products have not been determined. Here, matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS) was used for characterization, localization, and relative quantification of striatal neuropeptides in a rat model of LID in Parkinson's disease. MALDI IMS has the unique advantage of high sensitivity and high molecular specificity, allowing comprehensive detection of multiple molecular species in a single tissue section. Indeed, several dynorphins and enkephalins could be detected in the present study, including dynorphin A(1-8), dynorphin B, α-neoendorphin, MetEnkRF, MetEnkRGL, PEnk (198-209, 219-229). IMS analysis revealed elevated levels of dynorphin B, α-neoendorphin, substance P, and PEnk (220-229) in the dorsolateral striatum of high-dyskinetic animals compared with low-dyskinetic and lesion-only control rats. Furthermore, the peak-intensities of the prodynorphin derived peptides, dynorphin B and α-neoendorphin, were strongly and positively correlated with LID severity. Interestingly, these LID associated dynorphin peptides are not those with high affinity to κ opioid receptors, but are known to bind and activate also μ- and Δ-opioid receptors. In addition, the peak intensities of a novel endogenous metabolite of α-neoendorphin lacking the N-terminal tyrosine correlated positively with dyskinesia severity. MALDI IMS of striatal sections from Pdyn knockout mice verified the identity of fully processed dynorphin peptides and the presence of endogenous des-tyrosine α-neoendorphin. Des-tyrosine dynorphins display reduced opioid receptor binding and this points to possible novel nonopioid receptor mediated changes in the striatum of dyskinetic rats. Because des-tyrosine dynorphins can only be detected by mass spectrometry, as no antibodies are available, these findings highlight the importance of MALDI IMS analysis for the study of molecular dynamics in neurological diseases.

Topics: Animals; Antiparkinson Agents; Disease Models, Animal; Dynorphins; Dyskinesia, Drug-Induced; Enkephalins; Female; Humans; Levodopa; Mice; Neostriatum; Parkinson Disease; Protein Precursors; Rats; Rats, Sprague-Dawley; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

L-DOPA-induced dyskinesia is a troublesome complication of L-DOPA pharmacotherapy of Parkinson's disease and has been associated with disturbed brain opioid transmission. However, so far the results of clinical and preclinical studies on the effects of opioids agonists and antagonists have been contradictory at best. Prodynorphin mRNA levels correlate well with the severity of dyskinesia in animal models of Parkinson's disease; however the identities of the actual neuroactive opioid effectors in their target basal ganglia output structures have not yet been determined. For the first time MALDI-TOF imaging mass spectrometry (IMS) was used for unbiased assessment and topographical elucidation of prodynorphin-derived peptides in the substantia nigra of a unilateral rat model of Parkinson's disease and L-DOPA induced dyskinesia. Nigral levels of dynorphin B and alpha-neoendorphin strongly correlated with the severity of dyskinesia. Even if dynorphin peptide levels were elevated in both the medial and lateral part of the substantia nigra, MALDI IMS analysis revealed that the most prominent changes were localized to the lateral part of the substantia nigra. MALDI IMS is advantageous compared with traditional molecular methods, such as radioimmunoassay, in that neither the molecular identity analyzed, nor the specific localization needs to be predetermined. Indeed, MALDI IMS revealed that the bioconverted metabolite leu-enkephalin-arg also correlated positively with severity of dyskinesia. Multiplexing DynB and leu-enkephalin-arg ion images revealed small (0.25 by 0.5 mm) nigral subregions with complementing ion intensities, indicating localized peptide release followed by bioconversion. The nigral dynorphins associated with L-DOPA-induced dyskinesia were not those with high affinity to kappa opioid receptors, but consisted of shorter peptides, mainly dynorphin B and alpha-neoendorphin that are known to bind and activate mu and delta opioid receptors. This suggests that mu and/or delta subtype-selective opioid receptor antagonists may be clinically relevant for reducing L-DOPA-induced dyskinesia in Parkinson's disease.

Topics: Animals; Dynorphins; Dyskinesias; Enkephalins; Female; Immunohistochemistry; Levodopa; Male; Mass Spectrometry; Mice; Mice, Knockout; Parkinson Disease; Protein Precursors; Rats; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Dopamine denervation in Parkinson's disease and repeated Levodopa (L-DOPA) administration that induces dyskinesias are associated with an enhancement of basal ganglia neuropeptide transmission. Various adjunct non-dopaminergic treatments to Levodopa were shown to reduce and/or prevent dyskinesias. The aim of this study was to seek if non-dopaminergic drug treatments to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) lesioned monkeys combined with L-DOPA to prevent dyskinesia were associated with changes of striatal neuropeptides. Chronic treatment with Ro 61-8048 a kynurenine hydroxylase inhibitor, docosahexaenoic acid (DHA) a polyunsaturated fatty acid (omega-3), naltrexone an opioidergic antagonist and CI-1041 an N-methyl-D-aspartate (NMDA) glutamate receptor antagonist with L-DOPA prevented dyskinesias to various extents except naltrexone whereas all MPTP monkeys treated with L-DOPA alone developed dyskinesias. Striatal preproenkephalin (PPE), preprodynorphin (PPD) and preprotachykinin A (PPT-A) mRNA levels were measured by in situ hybridization. An increase of PPE and PPD mRNA levels was observed in anterior caudate nucleus of L-DOPA treated MPTP monkeys compared to controls and to Saline-treated MPTP monkeys whereas PPT-A mRNA levels were unchanged. Striatal PPE and PPD mRNA levels remained elevated in L-DOPA plus naltrexone-treated MPTP monkeys, while co-treatment with DHA, CI-1041 or Ro 61-8048 prevented their increase to various extents. Maximal dyskinesias scores of MPTP monkeys correlated significantly with striatal PPE and PPD mRNA levels but not with PPT-A mRNA levels. These results show that drugs displaying a wide range of pharmacological activities can modulate L-DOPA induced dyskinesias and this activity is correlated with striatal PPD and PPE mRNA levels suggesting a convergent mechanism.

Topics: Animals; Antiparkinson Agents; Benzoxazoles; Cocaine; Corpus Striatum; Disease Models, Animal; Docosahexaenoic Acids; Dopamine; Dopamine Uptake Inhibitors; Dynorphins; Dyskinesia, Drug-Induced; Enkephalins; Female; Iodine Isotopes; Levodopa; Macaca fascicularis; Naltrexone; Neuropeptides; Ovariectomy; Parkinsonian Disorders; Piperidines; Protein Precursors; RNA, Messenger; Sulfonamides; Tachykinins; Thiazoles; Time Factors

Dynorphin A in the spinal cord is considered to contribute to nociceptive stimuli. However, it has not yet been determined whether activation of the spinal dynorphinergic system under nociceptive stimuli plays a role in direct acceleration of the ascending nociceptive pathway. In this study, the authors investigated the role of spinal dynorphinergic transmission in ongoing brain activation under noxious stimuli in mice.. The changes in prodynorphin messenger RNA expression and dynorphin A (1-17)-like immunoreactivity in the mouse spinal cord were determined after the intraplantar injection of complete Freund's adjuvant in mice. The signal intensity in different brain regions after the intraplantar injection of complete Freund's adjuvant or intrathecal injection of dynorphin A (1-17) was measured by a pharmacological functional magnetic resonance imaging analysis.. Complete Freund's adjuvant injection produced pain-associated behaviors and induced a dramatic increase in signal intensity in the mouse cingulate cortex, somatosensory cortex, insular cortex, and thalamic nuclei. These effects were not seen in prodynorphin knockout mice. Prodynorphin messenger RNA expression and dynorphin A (1-17)-like immunoreactivity on the ipsilateral side of the spinal cord were markedly increased in complete Freund's adjuvant-injected mice. Furthermore, intrathecal injection of dynorphin A (1-17) at relatively high doses caused pain-associated behaviors and a remarkable increase in the activities of the cingulate cortex, somatosensory cortex, insular cortex, and medial and lateral thalamic nuclei in mice.. These findings indicate that spinally released dynorphin A (1-17) by noxious stimuli leads to the direct activation of ascending pain transmission.

Topics: Animals; Behavior, Animal; Brain; Brain Chemistry; Dynorphins; Enkephalins; Freund's Adjuvant; Immunohistochemistry; Inflammation; Injections, Spinal; Magnetic Resonance Imaging; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Pain; Protein Precursors; Receptors, N-Methyl-D-Aspartate; Reverse Transcriptase Polymerase Chain Reaction; RNA; Spinal Cord; Synaptic Transmission

Functional evidence suggests that opioid peptides such as dynorphin are involved in the regulation of airway macrophage functions and of human cancer growth. However, anatomical evidence for components of a putative dynorphin network within lung cancer patients is scarce. Tissue from lung cancer patients was examined immunohistochemically for all components of a local dynorphin (DYN) network. Double immunofluorescence microscopy analysis revealed colocalization of the opioid precursor PDYN with its end-product DYN, and key processing enzymes prohormone convertases 1 and 2 and carboxypeptidase E, as well as the kappa-opioid receptor (KOR) within alveolar macrophages and cancerous cells in varying degrees among patients. Moreover, chromograninA-immunoreactive pulmonary neuroendocrine cells expressing DYN were close to substance P- and KOR-immunoreactive sensory nerves. Our findings give a first hint of a neuroanatomical basis for a peripheral DYN network, conceivably regulating pulmonary, immune and cell-proliferative functions within the human lung, most likely in a paracrine/autocrine fashion.

Topics: Aged; Biomarkers, Tumor; Bronchi; Carboxypeptidase H; Carcinoma; Dynorphins; Enkephalins; Female; Fluorescent Antibody Technique, Indirect; Humans; Immunoenzyme Techniques; Lung Neoplasms; Macrophages, Alveolar; Male; Microscopy, Fluorescence; Middle Aged; Neurotransmitter Agents; Proprotein Convertase 1; Proprotein Convertase 2; Protein Precursors; Receptors, Opioid, kappa; Respiratory Mucosa

The transcription factor DeltaFosB is induced in the nucleus accumbens (NAc) by drugs of abuse. This study was designed to evaluate the possible modifications in FosB/DeltaFosB expression in both hypothalamic and extrahypothalamic brain stress system during morphine dependence and withdrawal. Rats were made dependent on morphine and, on day 8, were injected with saline or naloxone. Using immunohistochemistry and western blot, the expression of FosB/DeltaFosB, tyrosine hydroxylase (TH), corticotropin-releasing factor (CRF) and pro-dynorphin (DYN) was measured in different nuclei from the brain stress system in morphine-dependent rats and after morphine withdrawal. Additionally, we studied the expression of FosB/DeltaFosB in CRF-, TH- and DYN-positive neurons. FosB/DeltaFosB was induced after chronic morphine administration in the parvocellular part of the hypothalamic paraventricular nucleus (PVN), NAc-shell, bed nucleus of the stria terminalis, central amygdala and A(2) noradrenergic part of the nucleus tractus solitarius (NTS-A(2)). Morphine dependence and withdrawal evoked an increase in FosB/DeltaFosB-TH and FosB/DeltaFosB-CRF double labelling in NTS-A(2) and PVN, respectively, besides an increase in TH levels in NTS-A(2) and CRF expression in PVN. These data indicate that neuroadaptation to addictive substances, observed as accumulation of FosB/DeltaFosB, is not limited to the reward circuits but may also manifest in other brain regions, such as the brain stress system, which have been proposed to be directly related to addiction.

Topics: Animals; Brain; Corticotropin-Releasing Hormone; Dynorphins; Enkephalins; Hypothalamo-Hypophyseal System; Male; Morphine; Morphine Dependence; Pituitary-Adrenal System; Protein Precursors; Proto-Oncogene Proteins c-fos; Rats; Rats, Wistar; Stress, Physiological; Substance Withdrawal Syndrome; Tyrosine 3-Monooxygenase

Spinocerebellar ataxias (SCAs) are dominantly inherited neurodegenerative disorders characterized by progressive cerebellar ataxia and dysarthria. We have identified missense mutations in prodynorphin (PDYN) that cause SCA23 in four Dutch families displaying progressive gait and limb ataxia. PDYN is the precursor protein for the opioid neuropeptides, α-neoendorphin, and dynorphins A and B (Dyn A and B). Dynorphins regulate pain processing and modulate the rewarding effects of addictive substances. Three mutations were located in Dyn A, a peptide with both opioid activities and nonopioid neurodegenerative actions. Two of these mutations resulted in excessive generation of Dyn A in a cellular model system. In addition, two of the mutant Dyn A peptides induced toxicity above that of wild-type Dyn A in cultured striatal neurons. The fourth mutation was located in the nonopioid PDYN domain and was associated with altered expression of components of the opioid and glutamate system, as evident from analysis of SCA23 autopsy tissue. Thus, alterations in Dyn A activities and/or impairment of secretory pathways by mutant PDYN may lead to glutamate neurotoxicity, which underlies Purkinje cell degeneration and ataxia. PDYN mutations are identified in a small subset of ataxia families, indicating that SCA23 is an infrequent SCA type (∼0.5%) in the Netherlands and suggesting further genetic SCA heterogeneity.

Topics: Cerebellum; Dynorphins; Enkephalins; Female; Glutamate Plasma Membrane Transport Proteins; Humans; Male; Mutation, Missense; Pedigree; Protein Precursors; Purkinje Cells; Spinocerebellar Degenerations

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

Nicotine displays rewarding and aversive effects, and while dopamine has been linked with nicotine's reward, the neurotransmitter(s) involved with aversion remains speculative. The kappa-dynorphinergic system has been associated with negative motivational and affective states, and whether dynorphin (Dyn) contributes to the behavioral pharmacology of nicotine is a pertinent question.. We determined whether administration of a single dose of nicotine alters the biosynthesis of Dyn in the striatum of mice.. Nicotine free base, 1 mg/kg, sc, induced a biphasic, protracted increase of striatal Dyn, an initial rise by 1 h, which declined to control levels by 2 h, and a subsequent increase, between 6 and 12 h, lasting over 24 h. At 1 h, the nicotine effect was dose dependent, with doses>or=0.5 mg/kg inducing a response. Prodynorphin mRNA increased by 30 min for over 24 h, and in situ hybridization demonstrated elevated signal in caudate/putamen and nucleus accumbens. The nicotinic antagonist mecamylamine prevented the Dyn response, and a similar effect was observed with D1- and D2-like dopamine receptor antagonists, SCH 23390, sulpiride, and haloperidol. The glutamate NMDA receptor antagonist MK-801 reversed the nicotine-induced increase of Dyn, while the AMPA antagonist NBQX had a marginal effect.. We interpret our findings to indicate that acute nicotine enhances the synthesis and release of striatal Dyn. We propose that nicotine influences Dyn primarily through dopamine release and that glutamate plays a modulatory role. A heightened dynorphinergic tone may contribute to the aversive effects of nicotine in naive animals and first-time tobacco smokers.

Topics: Animals; Caudate Nucleus; Corpus Striatum; Dopamine; Dose-Response Relationship, Drug; Dynorphins; Enkephalins; Male; Mice; Nicotine; Nicotinic Agonists; Nucleus Accumbens; Protein Precursors; Putamen; Receptors, N-Methyl-D-Aspartate; RNA, Messenger; Time Factors

We investigated the effect of early vs. late initiation of levodopa treatment on dyskinetic movements, rotational behavior and molecular markers in hemiparkinsonian rats. Male Sprague-Dawley rats received a unilateral 6-hydroxydopamine (6-OHDA) administration in the nigrostriatal pathway. Rats were divided into three groups treated with: (i) levodopa (6 mg/kg) twice daily for 22 days starting at 4 weeks after 6-OHDA (Early group); (ii) levodopa at the same dose, regimen and duration but starting at 12 weeks after 6-OHDA (Late group), and (iii) saline starting at 4 weeks after 6-OHDA and continuing until the Late group finished treatment. Dyskinesias were quantified on days 1 and 22 of levodopa treatment. Striatal expression of preproenkephalin and preprodynorphin mRNAs, subthalamic cytochrome oxidase mRNA, and glutamate decarboxylase 67 mRNA in the pars reticulata of the substantia nigra was measured by in-situ hybridization. After 22 days of levodopa treatment, the percentage of rats showing dyskinesia was lower in the Early group than in the Late group (60% vs. 100%, respectively). No significant differences in total dyskinesia score were observed between both groups with the exception of the orolingual dyskinesias that were significantly less frequent in the Late group (P < 0.01). No significant differences were observed in the molecular markers between the Early and Late groups. Prompt initiation of levodopa treatment might be able to delay some of the basal ganglia molecular and circuitry changes underlying the development of dyskinesia but, once developed, they are behaviorally and molecularly similar to those appearing after late initiation of levodopa.

Topics: Animals; Corpus Striatum; Dopamine Plasma Membrane Transport Proteins; Dynorphins; Dyskinesias; Electron Transport Complex IV; Enkephalins; Glutamate Decarboxylase; Immunohistochemistry; In Situ Hybridization; Levodopa; Male; Neurons; Oxidopamine; Parkinson Disease, Secondary; Protein Precursors; Rats; Rats, Sprague-Dawley; RNA, Messenger; Substantia Nigra; Subthalamic Nucleus

Coadministration of entacapone with levodopa attenuates motor complications in experimental models of Parkinson's disease. The mechanisms underlying entacapone effects are unknown. We investigated the effect of entacapone, on: long-duration response (LDR) to levodopa, levodopa-induced postsynaptic pharmacodynamic mechanisms and molecular changes in hemiparkinsonian rats. 6-Hydroxydopamine-unilaterally lesioned rats were treated with levodopa (25 mg/kg)+vehicle; levodopa+entacapone (30 mg/kg) or saline, twice daily for 22 days. The LDR and the apomorphine-induced rotations were measured. In situ hybridization was performed measuring the expression of striatal preproenkephalin, preprodynorphin and dopamine D-3 receptor mRNAs, subthalamic cytochrome oxidase mRNA and nigral glutamic acid decarboxylase mRNA. Entacapone potentiated the LDR but did not modify either the apomorphine-induced rotational behavior or the molecular changes. Our results suggest that the effects of entacapone on levodopa-induced motor response are not mediated by postsynaptic mechanisms and that administration of entacapone is not able to normalize the molecular alterations induced by levodopa in the basal ganglia.

Topics: Animals; Antiparkinson Agents; Apomorphine; Brain; Catechols; Corpus Striatum; Dynorphins; Electron Transport Complex IV; Enkephalins; Gene Expression; Glutamate Decarboxylase; Immunohistochemistry; In Situ Hybridization; Levodopa; Male; Motor Activity; Nitriles; Parkinsonian Disorders; Protein Precursors; Rats; Rats, Sprague-Dawley; Receptors, Dopamine D3; RNA, Messenger; Substantia Nigra; Subthalamus

Nicotine is neuronal stimulating drug in the central nervous system and elicits various effects through nicotinic acetylcholine receptors. As previously reported, nicotine has an antinociceptive effect through activation of endogenous opioid neurons. However, detailed mechanisms of nicotine-induced antinociception are uncertain. In this study, we focused on spinal cord and investigated the involvement of endogenous opioidergic neurons in nicotine-induced antinociception in mice. In the tail-pinch test, subcutaneously administered nicotine (5 mg/kg) produced maximal antinociception 0.5 h after nicotine administration; this was attenuated by mecamylamine (MEC, 3 mg/kg, s.c.) or naloxone (NLX, 1 mg/kg, s.c.) administration. Intrathecal nicotine (10 mug) produced maximal antinociception at 2 min and this was also attenuated by MEC (3 mg/kg, s.c.) or NLX (1 mg/kg, s.c.) administration. The preproenkephalin (ppENK) mRNA level in spinal cord, but not dorsal root ganglion, was significantly increased 2 h following nicotine administration and recovered to control level 4 h after nicotine (5 mg/kg, s.c.) administration. This increase in ppENK mRNA level was inhibited by MEC (3 mg/kg, s.c.). The mRNA levels of preprodynorphin and preproopiomelanocortin were not increased by nicotine (5 mg/kg, s.c.). In the dorsal horn of the lumbar spinal cord, methionine-enkephalin (Met-ENK)-like IR was remarkably reduced at 0.5 h following nicotine administration and recovered to control levels by 2 h after nicotine (3 mg/kg, s.c.) administration. These results suggest that nicotine has an antinociceptive effect by promoting the release of Met-ENK, but not dynorphins and endorphins, from activated opioidergic neurons in spinal cord.

Topics: Analgesics; Animals; Dose-Response Relationship, Drug; Down-Regulation; Dynorphins; Enkephalin, Methionine; Enkephalins; Male; Mecamylamine; Mice; Mice, Inbred ICR; Naloxone; Narcotic Antagonists; Nicotine; Nicotinic Agonists; Nicotinic Antagonists; Nociceptors; Pain; Pain Measurement; Pain Threshold; Posterior Horn Cells; Pro-Opiomelanocortin; Protein Precursors; RNA, Messenger

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

Regulations of hormonal stress responses entail the initiation, amplitude and termination of the reaction, as well as its integration with other stress response systems. This study investigates the role of endogenous opioids in the regulation and integration of behavioral, thermal and hormonal stress responses, as these neuromodulators and their receptors are expressed in limbic structures responsible for stress responses. For this purpose, we subjected mice with selective deletion of beta-endorphin, enkephalin or dynorphin to the zero-maze test, a mildly stressful situation, and registered behaviors and stress hormone levels. Behavioral stress reactivity was assessed using zero-maze, light-dark and startle-reactivity paradigms. Animals lacking enkephalin displayed increased anxiety-related behavioral responses in each three, dynorphin knockouts in two models, whereas the responses of beta-endorphin knockouts indicated lower anxiety level in the zero-maze test. All knockout strains showed marked changes in hormonal stress reactivity. Increase in ACTH level after zero-maze test situation, unlike in wild type animals, failed to reach the level of significance in Penk1(-/-) and Pdyn(-/-) mice. Corticosterone plasma levels rapidly increased in all strains, with a lower peak response in knockouts. In wild-type and beta-endorphin-deficient mice, corticosterone levels returned to baseline within 60min after stress exposure. In contrast, mice lacking dynorphin and enkephalin showed longer-lasting elevated corticosterone levels, indicating a delayed termination of the stress reaction. Importantly, the behavioral and hormonal responses correlated in wild-type but not in knockout mice. Hyperthermia elicited by stress was reduced in animals lacking dynorphin and absent in Penk1(-/-) mice, despite of the heightened behavioral anxiety level of these strains. These results demonstrate an important role on the endogenous opioid system in the integration of behavioral and hormonal stress responses.

Topics: Adrenocorticotropic Hormone; Amygdala; Analysis of Variance; Animals; Anxiety; beta-Endorphin; Corticosterone; Dynorphins; Enkephalins; Exploratory Behavior; Hypothermia; Limbic System; Male; Maze Learning; Mice; Mice, Inbred C57BL; Mice, Knockout; Opioid Peptides; Paraventricular Hypothalamic Nucleus; Protein Precursors; Proto-Oncogene Proteins c-fos; Reflex, Startle; Stress, Psychological; Time Factors

Nicotine withdrawal causes somatic and negative affective symptoms that contribute to relapse and continued tobacco smoking. So far, the neuronal substrates involved are not fully understood, and an opioid role has been suggested. In this regard, the opioid dynorphin (Dyn) is of interest as it produces aversive states and has been speculated to play a role in the nicotine behavioral syndrome. These studies explore whether Dyn metabolism is altered during withdrawal following chronic administration of nicotine. Mice were administered nicotine, 2 mg/kg, s.c., four times daily for 14 days, and Dyn and prodynorphin (PD) mRNA estimated in selective brain regions at various times (30 min to 96 h) following drug discontinuation. The content of Dyn, estimated by RIA, was decreased in the striatum for a protracted time, from 30 min to over 72 h. In contrast, the mRNA for PD, evaluated by Northern blot, was elevated, appearing by 8 h and lasting over 96 h. Dyn was decreased in both ventral and dorsal striatum, and PD mRNA was differentially increased in the two striatal compartments as demonstrated by in situ hybridization. PD message was predominantly augmented in the nucleus accumbens, rostral pole, core, and shell, and the medial aspects of caudate/putamen. We interpret these data to indicate increased activity of striatal, particularly accumbal, dynorphinergic neurons during nicotine withdrawal resulting in enhanced peptide release and compensatory synthesis. Heightened dynorphinergic tone might be responsible, in part, for the emergence of the negative affective states observed during nicotine withdrawal.

Topics: Analysis of Variance; Animals; Behavior, Animal; Brain Mapping; Corpus Striatum; Drug Administration Schedule; Dynorphins; Enkephalins; Gene Expression Regulation; Male; Mice; Nicotine; Protein Precursors; Radioimmunoassay; RNA, Messenger; Substance Withdrawal Syndrome; Time Factors

The Fischer 344 (F344) rat strain differs from the Lewis strain in the response to neuropathic pain. Recently, we found that F344 rats totally recover from mechanical allodynia induced by chronic constriction injury (CCI) of the sciatic nerve 28 days after surgery whereas Lewis rats are initiating their recovery at this time point. Thus, the use of this neuropathic pain model in these different rat strains constitutes a good strategy to identify possible target genes involved in the development of neuropathic pain. Since differences between Lewis and F344 rats in their response to pain stimuli in acute pain models have been related to differences in the endogenous opioid and noradrenergic systems, we aimed to determine the levels of expression of key genes of both systems in the spinal cord and dorsal root ganglia (DRG) of both strains 28 days after CCI surgery. Real time RT-PCR revealed minimal changes in gene expression in the spinal cord after CCI despite the strain considered, but marked changes in DRG were observed. A significant upregulation of prodynorphin gene expression occurred only in injured DRG of F344 rats, the most resistant strain to neuropathic pain. In addition, we found a significant downregulation of tyrosine hydroxylase and proenkephalin gene expression levels in both strains whereas delta-opioid receptor was found to be significantly downregulated only in injured DRG of Lewis rats although the same trend was observed in F344 rats. The data strongly suggest that dynorphins could be involved in strain differences concerning CCI resistance.

Topics: Animals; Chronic Disease; Denervation; Disease Models, Animal; Down-Regulation; Dynorphins; Enkephalins; Ganglia, Spinal; Gene Expression Regulation; Hyperalgesia; Ligation; Male; Neurons, Afferent; Norepinephrine; Peripheral Nerve Injuries; Peripheral Nerves; Peripheral Nervous System Diseases; Protein Precursors; Rats; Rats, Inbred F344; Rats, Inbred Lew; Receptors, Opioid, delta; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Species Specificity; Spinal Cord; Tyrosine 3-Monooxygenase

It has been shown that chronic cocaine increases prodynorphin mRNA in the caudate putamen and decreases it in the hypothalamus. In addition, treatment with a kappa-opioid receptor agonist produced the opposite effect on prodynorphin gene expression in these brain regions and also evoked a decrease in the hippocampus. It is already known that kappa-opioid receptor agonists decrease the development of sensitization to some of the behavioral effects of cocaine. The serotonin system has also been shown to regulate dynorphin gene expression and a continuous infusion of fluoxetine induced prodynorphin gene expression in the same pattern as the kappa-opioid agonist (+)(5a,7a,8b)-N-methyl-N-[7-(1-pyrrolidinyl)-1 oxaspiro[4.5]dec-8-yl]-benzeneacetamide (U-69593) in the brain regions investigated. It is interesting to note that treatment with a continuous infusion of cocaine produced different effects on this parameter. To determine whether serotonin plays a role in the regulation of prodynorphin mRNA by kappa-opioid agonists or cocaine, rats were treated with the serotonin depleter parachloroamphetamine (PCA). Beginning 24 h later, rats were treated with the selective kappa-opioid agonist U-69593 for 5 days or continuously with cocaine for 7 days and prodynorphin mRNA was measured. Prodynorphin mRNA was decreased significantly in the hypothalamus, caudate putamen, and hippocampus of rats treated with a single injection of PCA. Subsequent to PCA administration the effects of U-69593 or cocaine on prodynorphin mRNA were differentially affected across brain regions. Prodynorphin gene expression was still increased by U-69593 treatment in the hypothalamus and decreased in the caudate putamen. Cocaine treatment still produced a decrease in this parameter in the hypothalamus and an increase in the caudate putamen. In contrast, in the hippocampus, the decrease in prodynorphin mRNA produced by U-69593 was no longer evident after PCA and cocaine, which previously had no effect, now increased it in the serotonin-depleted group. These findings suggest that serotonin is necessary to maintain normal levels of dynorphin mRNA in all of the investigated brain areas and that the regulation of prodynorphin mRNA expression by chronic treatment with a kappa-opioid receptor agonist or cocaine requires serotonin in the hippocampus, but not in the hypothalamus or caudate putamen.

Topics: Animals; Benzeneacetamides; Blotting, Northern; Central Nervous System; Cocaine; Dynorphins; Enkephalins; Fluoxetine; Hippocampus; Hypothalamus; Male; Neostriatum; Protein Precursors; Pyrrolidines; Rats; Rats, Sprague-Dawley; Receptors, Opioid, kappa; RNA Probes; RNA, Messenger; Selective Serotonin Reuptake Inhibitors; Serotonin

Dynorphins, endogenous peptides for the kappa opioid receptor, play important roles in many physiological and pathological functions. Here, we examined how prolonged treatment with three major prodynorphin peptides, dynorphin A (1-17) (Dyn A), dynorphin B (1-13) (Dyn B) and alpha-neoendorphin (alpha-Neo), regulated the human kappa opioid receptor (hKOR) stably expressed in Chinese hamster ovary (CHO) cells. Results from receptor binding and [(35)S]GTPgammaS binding assays showed that these peptides were potent full agonists of the hKOR with comparable receptor reserve and intrinsic efficacy to stimulate G proteins. A 4-h incubation with alpha-Neo at a concentration of approximately 600xEC(50) value (from [(35)S]GTPgammaS binding) resulted in receptor down-regulation to a much lower extent than the incubation with Dyn A and Dyn B at comparable concentrations ( approximately 10% vs. approximately 65%). Extending incubation period and increasing concentrations did not significantly affect the difference. The plateau level of alpha-Neo-mediated receptor internalization (30 min) was significantly less than those of Dyn A and Dyn B. Omission of the serum from the incubation medium or addition of peptidase inhibitors into the serum-containing medium enhanced alpha-Neo-, but not Dyn A- or Dyn B-, mediated receptor down-regulation and internalization; however, the degrees of alpha-Neo-induced adaptations were still significantly less than those of Dyn A and Dyn B. Thus, these endogenous peptides differentially regulate KOR after activating the receptor with similar receptor occupancy and intrinsic efficacy. Both stability in the presence of serum and intrinsic capacity to promote receptor adaptation play roles in the observed discrepancy among the dynorphin peptides.

Topics: Animals; Binding, Competitive; Blotting, Western; CHO Cells; Cricetinae; Cricetulus; Down-Regulation; Dynorphins; Electrophoresis, Polyacrylamide Gel; Endorphins; Enkephalins; Flow Cytometry; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Ligands; Peptides; Protease Inhibitors; Protein Precursors; Receptors, Opioid, kappa

L-DOPA-induced dyskinesia (LID) is one of the main limitations of long term L-DOPA use in Parkinson's disease (PD) patients. We show that chronic L-DOPA treatment induces novel dyskinetic behaviors in aphakia mouse with selective nigrostriatal deficit mimicking PD. The stereotypical abnormal involuntary movements were induced by dopamine receptor agonists and attenuated by antidyskinetic agents. The development of LID was accompanied by preprodynorphin and preproenkephalin expression changes in the denervated dorsal striatum. Increased FosB-expression was also noted in the dorsal striatum. In addition, FosB expression was noted in the pedunculopontine nucleus and the zona incerta, structures previously not examined in the setting of LID. The aphakia mouse is a novel genetic model with behavioral and biochemical characteristics consistent with those of PD dyskinesia and provides a more consistent, convenient, and physiologic model than toxic lesion models to study the mechanism of LID and to test therapeutic approaches for LID.

Topics: Afferent Pathways; Animals; Antiparkinson Agents; Aphakia; Corpus Striatum; Disease Models, Animal; Dynorphins; Dyskinesia, Drug-Induced; Enkephalins; Levodopa; Mice; Mice, Inbred C57BL; Mice, Neurologic Mutants; Parkinsonian Disorders; Protein Precursors; Proto-Oncogene Proteins c-fos; Substantia Nigra

Chronic nicotine administration has been shown previously to produce mechanical hypersensitivity in the rat although the mechanism of this effect is unknown. Rats treated with chronic systemic nicotine 3.6 or 8.6 mg/(kg day) for 14-21 days displayed mechanical hypersensitivity coincident with an increase of prodynorphin immunoreactivity and dynorphin content within the spinal cord. The administration of dynorphin antiserum intrathecally significantly attenuated chronic nicotine-induced mechanical hypersensitivity. Our results suggest that chronic nicotine administration produces an increase in spinal dynorphin content and release that contributes to mechanical hypersensitivity.

Topics: Animals; Cold Temperature; Dose-Response Relationship, Drug; Dynorphins; Enkephalins; Immunochemistry; Immunohistochemistry; Injections, Spinal; Male; Nicotine; Nicotinic Agonists; Pain Measurement; Pain Threshold; Physical Stimulation; Pressure; Protein Precursors; Rats; Rats, Sprague-Dawley; Spinal Cord

The prodynorphin system is implicated in the neurochemical mechanism of psychostimulants. Exposure to different drugs of abuse can induce neuroadaptations in the brain and affect opioid gene expression. The present study aims to examine the possibility of a common neurobiological substrate in drug addiction processes. We studied the effects of single and repeated 3,4-methylenedioxy-N-methylamphetamine ('Ecstasy') on the gene expression of the opioid precursor prodynorphin, and on the levels of peptide dynorphin A in the rat brain. Acute (8 mg/kg, intraperitoneally) 3,4-methylenedioxy-N-methylamphetamine markedly raised, two hours later, prodynorphin mRNA levels in the prefrontal cortex, and in the caudate putamen, whereas it decreased gene expression in the ventral tegmental area. Chronic (8 mg/kg, intraperitoneally, twice a day for 7 days) 3,4-methylenedioxy-N-methylamphetamine increased prodynorphin mRNA in the nucleus accumbens, hypothalamus and caudate putamen and decreased it in the ventral tegmental area. Dynorphin A levels increased after chronic treatment in the ventral tegmental area and decreased after acute treatment in the nucleus accumbens, prefrontal cortex and hypothalamus. These findings confirm the role of the dynorphinergic system in mediating the effects of drugs of abuse, such as 3,4-methylenedioxy-N-methylamphetamine, in various regions of the rat brain, which may be important sites for the opioidergic mechanisms activated by addictive drugs.

Topics: Animals; Blotting, Northern; Brain; Dynorphins; Enkephalins; Gene Expression; Hallucinogens; Male; N-Methyl-3,4-methylenedioxyamphetamine; Protein Precursors; Radioimmunoassay; Rats; Rats, Sprague-Dawley; RNA, Messenger; Time Factors

Effects of big dynorphin (Big Dyn), a prodynorphin-derived peptide consisting of dynorphin A (Dyn A) and dynorphin B (Dyn B) on memory function, anxiety, and locomotor activity were studied in mice and compared to those of Dyn A and Dyn B. All peptides administered i.c.v. increased step-through latency in the passive avoidance test with the maximum effective doses of 2.5, 0.005, and 0.7 nmol/animal, respectively. Effects of Big Dyn were inhibited by MK 801 (0.1 mg/kg), an NMDA ion-channel blocker whereas those of dynorphins A and B were blocked by the kappa-opioid antagonist nor-binaltorphimine (6 mg/kg). Big Dyn (2.5 nmol) enhanced locomotor activity in the open field test and induced anxiolytic-like behavior both effects blocked by MK 801. No changes in locomotor activity and no signs of anxiolytic-like behavior were produced by dynorphins A and B. Big Dyn (2.5 nmol) increased time spent in the open branches of the elevated plus maze apparatus with no changes in general locomotion. Whereas dynorphins A and B (i.c.v., 0.05 and 7 nmol/animal, respectively) produced analgesia in the hot-plate test Big Dyn did not. Thus, Big Dyn differs from its fragments dynorphins A and B in its unique pattern of memory enhancing, locomotor- and anxiolytic-like effects that are sensitive to the NMDA receptor blockade. The findings suggest that Big Dyn has its own function in the brain different from those of the prodynorphin-derived peptides acting through kappa-opioid receptors.

Topics: Amino Acid Sequence; Animals; Anti-Anxiety Agents; Anxiety; Behavior, Animal; Dose-Response Relationship, Drug; Dynorphins; Enkephalins; Hot Temperature; Male; Memory; Mice; Molecular Sequence Data; Motor Activity; Naltrexone; Pain Measurement; Protein Precursors; Reaction Time; Receptors, N-Methyl-D-Aspartate

Self-administration procedures have not yet provided evidence that freely moving mice can reliably acquire and maintain an operant behavior to self-administer cannabinoid agonists. The aim of the present work was to establish a model of cannabinoid operant intravenous self-administration in freely moving mice given the relevance of this species for the use of genetically modified animals. In addition, the possible involvement of the kappa/dynorphin system in cannabinoid self-administration was evaluated by using pro-dynorphin knockout mice. Outbred CD1 wild-type mice as well as pro-dynorphin knockout and wild-type mice were trained to self-administer the cannabinoid receptor agonist WIN 55,212-2 under an FR1 schedule of reinforcement. Two cannabinoid training doses (6.25 and 12.5 microg/kg/infusion) were used in the acquisition studies in outbred mice. Animals acquired a reliable operant responding to self-administer WIN 55,212-2 (12.5 microg/kg/infusion), but required as many as 15 sessions to attain this behavior. Interestingly, when a previous injection of WIN 55,212-2 (0.1 mg/kg, i.p.) was administered in the home-cage 24 h before the first session, mice acquired operant responding for cannabinoid self-administration by the fourth session. When the kappa-opioid agonist antagonist nor-binaltorphimine (5 mg/kg s.c.) was administered 4 h before the first session, the time required to acquire a reliable cannabinoid self-administration was also significantly reduced. Finally, a shift to the left in the dose-intake curve to self-administer WIN 55,212-2 was observed in pro-dynorphin knockout mice when compared to wild-type mice. These results indicate that the activation of the kappa/dynorphin opioid system after WIN 55,212-2 administration could counteract cannabinoid rewarding effects.

Topics: Animals; Benzoxazines; Cannabinoids; Conditioning, Operant; Dose-Response Relationship, Drug; Dynorphins; Enkephalins; Male; Mice; Mice, Knockout; Morpholines; Naltrexone; Naphthalenes; Protein Precursors; Receptor, Cannabinoid, CB1; Receptors, Opioid, kappa; Self Administration

The aim of this study was to determine whether inhibition of the extracellular-regulated kinase signaling pathway decreases acute amphetamine-induced behavioral activity and neuropeptide gene expression in the rat striatum. Western blotting revealed that extracellular-regulated kinase1/2 phosphorylation was highly induced in the rat striatum 15 min after an acute amphetamine (2.5 mg/kg, i.p.) injection without altering the total amount of extracellular-regulated kinase protein. In a separate experiment, the systemic injection of SL327, a selective inhibitor of extracellular regulated kinase kinase that crosses the blood-brain barrier, 1 h prior to amphetamine administration decreased amphetamine-induced vertical and horizontal activity. Quantitative in situ hybridization histochemistry showed that SL327 abolished the high levels of preproenkephalin and preprodynorphin mRNA induced by amphetamine in the striatum with no alteration of their basal levels. In another set of experiments, the hyperlocomotor activity induced by amphetamine was reduced by pretreatment with intra-striatal infusion of U0126. U0126 also blocked the amphetamine-induced increases in phospho-extracellular-regulated kinase and preproenkephalin and preprodynorphin gene expression in the striatum. These data indicate that activation of the extracellular-regulated kinase cascade contributes to the behavioral effects and changes in striatal neuropeptide gene expression induced by acute amphetamine.

Topics: Aminoacetonitrile; Amphetamine; Amphetamine-Related Disorders; Animals; Butadienes; Corpus Striatum; Disease Models, Animal; Dynorphins; Enkephalins; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation; Male; MAP Kinase Signaling System; Motor Activity; Neuropeptides; Nitriles; Phosphorylation; Protein Precursors; Rats; Rats, Sprague-Dawley; RNA, Messenger; Up-Regulation

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

Activation of dopamine (DA) D1 receptors (D1Rs) in the nucleus accumbens (Acb) markedly affects the levels of prodynorphin, the precursor of aversion-associated dynorphin peptides. The location of prodynorphin, specifically as related to the dopaminergic inputs and D1Rs in the Acb, is fundamental for establishing the physiologically relevant sites. To determine these sites, we examined the electron microscopic dual-immunolabeling of prodynorphin and D1R or tyrosine hydroxylase (TH), a marker of catecholamine terminals in the rat Acb shell. This subregion is targeted by mesolimbic dopaminergic inputs affecting reward-aversion responses and locomotor activity. Prodynorphin was prominently localized to large (100-200 nm) granular aggregates in somatodendritic and axonal profiles, some of which expressed dynorphin A/B. In somata and dendrites, prodynorphin was often found in punctate clusters in the cytoplasm. Of the total prodynorphin-labeled dendrites, approximately 63% expressed D1Rs, which were largely located on the plasma membranes. In comparison with dendrites, many more axon terminals contained prodynorphin, although only 15% of these terminals contained D1R-labeling. Prodynorphin terminals formed symmetric synapses with D1R-labeled or unlabeled dendrites, and also apposed TH-containing axon terminals. Our results provide ultrastructural evidence that in the Acb shell, the prodynorphin is available for cleavage to physiologically active peptides in both dendrites and terminals of neurons that express D1Rs. They also indicate that dynorphin peptides have distributions that would enable their participation in modulation of DA release or D1R-mediated postsynaptic responses in Acb shell neurons.

Topics: Animals; Axons; Blotting, Western; Dendrites; Dynorphins; Enkephalins; Immunohistochemistry; Male; Microscopy, Electron, Transmission; Nucleus Accumbens; Protein Precursors; Rats; Rats, Sprague-Dawley; Receptors, Dopamine D1; Transfection

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

Antagonism of the CB(1) cannabinoid receptor (CB(1) receptor) by rimonabant (SR141716) reduces self-administration of alcohol and other drugs of abuse in animal models. These findings suggest that the CB(1) receptor may be a target for genetic differences that modify the salient features of rewarding drugs. In the present study, wild-type (CB(1) (+/+)) are compared to transgenic mice deficient in CB(1) receptors (CB(1) (-/-)). The goal was to investigate the influences of the cannabinoid receptor system on opioid peptide gene expression and on dopamine receptor gene expression which is commonly influenced by substances of abuse. We demonstrate using reverse transcription and real-time polymerase chain reaction (PCR) that striatal mRNA for preproenkephalin (PPENK) and preprodynorphin (PPDYN) in the CB(1) (-/-) striatum increases when compared to CB(1) (+/+). Real-time PCR analyses to evaluate D(2) and D(4) dopamine receptor gene expression in striatum isolated from CB(1) (+/+) and CB(1) (-/-) revealed a nearly 2-fold increase in D(4) receptor mRNA in the striatum from CB(1) (-/-) mice and no significant change in D(2) expression. In contrast, treatment of C57BL/6 mice with the CB(1) receptor antagonist, rimonabant, produced a reduction of both D(2) and D(4) dopamine receptor expression in the striatum. These data suggest that genetic differences in CB(1) receptor may exert a modulatory effect on D(4) dopamine receptor and opioid peptide gene expression.

Topics: Animals; Corpus Striatum; Dynorphins; Enkephalins; Gene Expression; Mice; Mice, Knockout; Opioid Peptides; Piperidines; Protein Precursors; Pyrazoles; Receptor, Cannabinoid, CB1; Receptors, Dopamine D2; Receptors, Dopamine D4; Reverse Transcriptase Polymerase Chain Reaction; Rimonabant; RNA, Messenger

Previous studies showed that opioid drugs-oxycodone-6-oxime and 14-methoxy-5-methyl-dihydromorphinone (14-methoxymetopon)-produced less respiratory depressive effect and slower rate of tolerance and dependence, respectively. It was also reported that morphine decreased the prodynorphin gene expression in the rat hippocampus, striatum and hypothalamus. In this study, we determined the prodynorphin gene expression and dynorphin levels in selected brain regions of opioid tolerant rats. We found that in the striatum morphine decreased, while oxycodone-6-oxime increased and 14-methoxymetopon did not alter the prodynorphin gene expression. In the nucleus accumbens, morphine and oxycodone-6-oxime did not change, while 14-methoxymetopon increased the prodynorphin gene expression. In the hippocampus both oxycodone-6-oxime and 14-methoxymetopon enhanced, whereas morphine did not alter the prodynorphin gene expression. In the rat striatum only oxycodone-6-oxime increased dynorphin levels significantly in accordance with the prodynorphin mRNA changes. In the hippocampus both opioid agonists increased the dynorphin levels significantly similarly to the augmented prodynorphin gene expression. In ventral tegmental area only 14-methoxymetopon increased dynorphin levels significantly. In nucleus accumbens and the temporal-parietal cortex the changes in the prodynorphin gene expression and the dynorphin levels did not correlate. Since the endogenous prodynorphin system may play a modulatory role in the development of opioid tolerance, the elevated supraspinal dynorphin levels appear to be partly responsible for the reduced degree of tolerance induced by the investigated opioids.

Topics: Animals; Blotting, Northern; Brain; Drug Tolerance; Dynorphins; Enkephalins; Gene Expression; Male; Morphine Derivatives; Narcotics; Oxycodone; Protein Precursors; Radioimmunoassay; Rats; Rats, Sprague-Dawley; RNA, Messenger; Time Factors

Intrathecal (i.t.) administration into mice of N-ethylmaleimide (NEM), a cysteine protease inhibitor, produced a characteristic behavioral response, the biting and/or licking of the hindpaw and the tail along with slight hindlimb scratching directed toward the flank. The behavior induced by NEM was inhibited by the intraperitoneal injection of morphine. We have recently reported that dynorphin A and, more potently big dynorphin, consisting of dynorphins A and B, produce the same type of nociceptive response whereas dynorphin B does not [Tan-No K, Esashi A, Nakagawasai O, Niijima F, Tadano T, Sakurada C, Sakurada T, Bakalkin G, Terenius L, Kisara K. Intrathecally administered big dynorphin, a prodynorphin-derived peptide, produces nociceptive behavior through an N-methyl-d-aspartate receptor mechanism. Brain Res 2002;952:7-14]. The NEM-induced nociceptive behavior was inhibited by pretreatment with dynorphin A- or dynorphin B-antiserum and each antiserum also reduced the nociceptive effects of i.t.-injected synthetic big dynorphin. The characteristic NEM-evoked response was not observed in prodynorphin knockout mice. Naloxone, an opioid receptor antagonist, had no effects on the NEM-induced behavior. Ifenprodil, arcaine and agmatine, antagonists at the polyamine recognition site on the N-methyl-D-aspartate (NMDA) receptor ion-channel complex, and MK-801, an NMDA ion-channel blocker inhibited the NEM-induced effects. Ro25-6981, an antagonist of the NMDA receptor subtype containing NR2B subunit was not active. NEM completely inhibited degradation of dynorphin A by soluble and particulate fractions of mouse spinal cord. Collectively, the results demonstrate that endogenous prodynorphin-derived peptides are pronociceptive in uninjured animals, and required for the NEM-induced behavior. The NEM effects may be mediated through inhibition of the degradation of endogenous dynorphins, presumably big dynorphin that in turn activates the NMDA receptor ion-channel complex by acting on the polyamine recognition site.

Topics: Agmatine; Analysis of Variance; Animals; Behavior, Animal; Biguanides; Dizocilpine Maleate; Dose-Response Relationship, Drug; Drug Interactions; Dynorphins; Enkephalins; Enzyme Inhibitors; Ethylmaleimide; Excitatory Amino Acid Antagonists; Immune Sera; Injections, Spinal; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Morphine; Narcotics; Nociceptin Receptor; Piperidines; Protein Precursors; Receptors, Opioid; Spinal Cord; Time Factors

Dynorphins, endogenous kappa-opioid agonists widely expressed in the central nervous system, have been reported to increase following diverse pathophysiological processes, including excitotoxicity, chronic inflammation, and traumatic injury. These peptides have been implicated in cognitive impairment, especially that associated with aging. To determine whether absence of dynorphin confers any beneficial effect on spatial learning and memory, knockout mice lacking the coding exons of the gene encoding its precursor prodynorphin (Pdyn) were tested in a water maze task. Learning and memory assessment using a 3-day water maze protocol demonstrated that aged Pdyn knockout mice (13-17 months) perform comparatively better than similarly aged wild-type (WT) mice, based on acquisition and retention probe trial indices. There was no genotype effect on performance in the cued version of the swim task nor on average swim speed, suggesting the observed genotype effects are likely attributable to differences in cognitive rather than motor function. Young (3-6 months) mice performed significantly better than aged mice, but in young mice, no genotype difference was observed. To investigate the relationship between aging and brain dynorphin expression in mice, we examined dynorphin peptide levels at varying ages in hippocampus and frontal cortex of WT 129SvEv mice. Quantitative radioimmunoassay demonstrated that dynorphin A levels in frontal cortex, but not hippocampus, of 12- and 24-month mice were significantly elevated compared to 3-month mice. Although the underlying mechanisms have yet to be elucidated, the results suggest that chronic increases in endogenous dynorphin expression with age, especially in frontal cortex, may adversely affect learning and memory.

Topics: Age Factors; Aging; Analysis of Variance; Animals; Behavior, Animal; Brain Chemistry; Dynorphins; Enkephalins; Frontal Lobe; Gene Expression Regulation; Genotype; Hippocampus; Maze Learning; Mice; Mice, Knockout; Protein Precursors; Radioimmunoassay; Reaction Time; Retention, Psychology; Space Perception; Spatial Behavior; Swimming; Time Factors

In Parkinson's disease (PD), the striatal dopamine depletion and the following overactivation of the indirect pathway of the basal ganglia leads to very early disinhibition of the subthalamic nucleus (STN) that may contribute to the progression of PD by glutamatergic overstimulation of the dopaminergic neurons in the substantia nigra. Adenosine A2A antagonism has been demonstrated to attenuate the overactivity of the striatopallidal pathway. To investigate whether neuroprotection exerted by the A2A antagonist 8-(3-chlorostyryl)caffeine (CSC) correlates with a diminution of the striatopallidal pathway activity, we have examined the changes in the mRNA encoding for enkephalin, dynorphin, and adenosine A2A receptors by in situ hybridization induced by subacute systemic pretreatment with CSC in rats with striatal 6-hydroxydopamine(6-OHDA) administration. Animals received CSC for 7 days until 30 min before 6-OHDA intrastriatal administration. Vehicle-treated group received a solution of dimethyl sulfoxide. CSC pretreatment partially attenuated the decrease in nigral tyrosine hydroxylase immunoreactivity induced by 6-OHDA, whereas no modification of the increase in preproenkephalin mRNA expression in the dorsolateral striatum was observed. The neuroprotective effect of the adenosine A2A antagonist CSC in striatal 6-OHDA-lesioned rats does not result from a normalization of the increase in striatal PPE mRNA expression in the DL striatum, suggesting that other different mechanisms may be involved.

Topics: Adenosine A2 Receptor Antagonists; Animals; Caffeine; Cell Count; Dynorphins; Enkephalins; Globus Pallidus; Immunohistochemistry; In Situ Hybridization; Male; Microinjections; Neostriatum; Neural Pathways; Neuroprotective Agents; Oxidopamine; Parkinson Disease, Secondary; Protein Precursors; Rats; Rats, Sprague-Dawley; Receptor, Adenosine A2A; RNA, Messenger; Stereotyped Behavior; Substantia Nigra; Sympatholytics; Tyrosine 3-Monooxygenase

We elaborate herein a novel theory of basal ganglia function that accounts for why palatable, energy-dense foods retain high incentive value even when immediate physiological energy requirements have been met. Basal ganglia function has been studied from the perspective of topographical segregation of processing within parallel circuits, with primary focus on motor control and cognition. Recent findings suggest, however, that the striatum can act as an integrated unit to modulate motivational state. We describe evidence that the striatal enkephalin system, which regulates the hedonic impact of preferred foods, undergoes coordinated gene expression changes that track current motivational state with regard to food intake. Striatal enkephalin gene expression is also downregulated by an intrastriatal infusion of a cholinergic muscarinic antagonist, a manipulation that greatly suppresses food intake. To account for these findings, we propose that signaling through a hypothalamic-midline thalamic-striatal axis impinges on the cholinergic interneurons of the striatum, which via their large, overlapping axonal fields act as a network to modulate enkephalin-containing striatal output neurons. A key relay in this circuit is the paraventricular thalamic nucleus, which receives convergent input from orexin-coded hypothalamic energy-sensing and behavioral state-regulating neurons, as well as from circadian oscillators, and projects to cholinergic interneurons throughout the striatal complex. We hypothesize that this system evolved to coordinate feeding and arousal, and to prolong the feeding central motivational state beyond the fulfillment of acute energy needs, thereby promoting "overeating" and the consequent development of an energy reserve for potential future food shortages.

Topics: Acetylcholine; Animals; Arousal; Dopamine; Dynorphins; Energy Metabolism; Enkephalins; Food; Hypothalamus; Models, Biological; Narcotics; Nucleus Accumbens; Protein Precursors; Rats; Receptors, Opioid, kappa; Reward; RNA, Messenger; Thalamus

Behavioral studies have demonstrated that chronic food restriction augments the rewarding and motor-activating effects of centrally injected psychostimulants and direct dopamine (DA) receptor agonists. Recently, it has been shown that intracerebroventricular (i.c.v.) injection of the D-1 DA receptor agonist, SKF-82958, produces an enhanced locomotor-activating effect as well as increased activation of striatal ERK 1/2 MAP kinase, CaM kinase II, CREB, and c-fos in food-restricted (FR) relative to ad libitum fed (AL) rats. Striatal neurons that express the D-1 DA receptor coexpress dynorphin and substance P, and CREB is known to couple D-1 DA receptor stimulation to preprodynorphin (ppD) gene expression. The purpose of the present study was to examine possible genomic consequences of FR using real-time quantitative RT-PCR to measure striatal neuropeptide gene expression 3 h after i.c.v. injection of SKF-82958 (20 microg). Results indicate that, in nucleus accumbens (NAc), basal levels of ppD and preprotachykinin (ppT) mRNA are lower in FR than AL rats. This may reflect a decrease in tonic DA transmission during FR which precedes the compensatory upregulation of postsynaptic D-1 DA receptor-mediated cell signaling. In response to SKF-82958 challenge, however, FR subjects displayed greater levels of ppD and ppT mRNA in NAc than did AL subjects. A similar trend was seen in caudate-putamen (CPu). SKF-82958 also increased preproenkephalin (ppE) mRNA in Nac, but not CPu, with no difference between feeding groups. The present findings regarding ppD and ppT are consistent with prior findings of increased behavioral and cellular responses to acute D-1 DA agonist challenge in FR rats. The functional consequences of increased neuropeptide gene expression in response to acute drug challenge remain to be investigated but may include modulation of behavioral effects that emerge with repeated drug exposure, including sensitization, tolerance, and addiction.

Topics: Animals; Benzazepines; Caudate Nucleus; Dopamine Agonists; Dynorphins; Eating; Enkephalins; Food Deprivation; Gene Expression Regulation; Genes, fos; Injections, Intraventricular; Male; Neuropeptides; Nucleus Accumbens; Protein Precursors; Proto-Oncogene Proteins c-fos; Putamen; Rats; Rats, Sprague-Dawley; Receptors, Dopamine D1; Reward; RNA, Messenger; Signal Transduction; Stimulation, Chemical; Tachykinins

The investigation of rodent cocaine self-administration (SA) under conditions that promote escalating patterns of intake may provide insight into the loss of control over drug use that is central to human addiction.. This study examines the effects of daily long-access (LgA) SA of high or low cocaine doses on drug intake, extinction, reinstatement, and brain mRNA levels.. Three groups of male Sprague-Dawley rats were trained to self-administer cocaine during multiple-dose sessions. Short-access (ShA) rats were tested daily for multi-dose SA then remained in the chambers for 7 h with no cocaine available. LgA rats had access to low (0.5 mg/kg per infusion; LgA-LD) or high (2.0 mg/kg per infusion; LgA-HD) cocaine doses for 7 h after multi-dose SA. After 14 days, responding was extinguished, cocaine-induced reinstatement was determined, and preproenkephalin (ppENK), preprodynorphin (ppDYN), corticotropin releasing factor (CRF) and dopamine D(2) receptor (D(2)R) mRNA levels were measured in various brain regions using a quantitative solution hybridization RNase protection assay.. Whereas SA was not altered in ShA rats and only increased during the "loading phase" in LgA-LD rats, a general escalation of intake was found in LgA-HD rats. LgA, particularly LgA-HD, rats were more susceptible to reinstatement than ShA rats. Caudate-putamen ppENK and nucleus accumbens D(2)R mRNA levels were elevated in LgA-HD rats. Overall, D(2)R mRNA levels were positively correlated with reinstatement.. The escalation of cocaine SA under LgA conditions is dose-dependent and is associated with heightened susceptibility to drug-induced relapse. The characterization of neurobiological alterations that accompany escalated SA should facilitate the identification of mechanisms underlying the onset of human addiction.

Topics: Animals; Behavior, Addictive; Brain; Cocaine; Cocaine-Related Disorders; Corticotropin-Releasing Hormone; Dose-Response Relationship, Drug; Dynorphins; Enkephalins; Extinction, Psychological; Male; Opioid Peptides; Protein Precursors; Rats; Rats, Sprague-Dawley; Receptors, Dopamine D2; Recurrence; RNA, Messenger; Self Administration; Time Factors

Preprodynorphin and preproenkephalin are protein precursors from which are derived two classes of opioid neurotransmitter peptides. Dynorphin A((1-17)) is produced by proteolytic processing of prodynorphin, and processing of proenkephalin yields the enkephalin peptides. We report here on the isolation and sequencing of multiple clones for these two mRNAs from a cDNA library. Two cDNA clones of preprodynorphin contained the full-length sequence (2.35 kb) with the primary structure predicted from the guinea pig gene sequence. In contrast, one clone encoded the full-length sequence but also an additional 192 nt at the 5' end. This sequence has high homology to the 5' flanking region of the human preprodynorphin gene, and RNase protection assays demonstrated that in addition to a primary initiation site, transcription of this mRNA is initiated at several sites 160-190 nt 5' with respect to the primary site. This difference may alter translational efficiency or mRNA stability. The sequence of preproenkephalin cDNA clones confirmed the structure predicted from the gene sequence. One clone, however, contained sequences encoded by exons 2 and 3, and initiated within the first intron (intron A) of the gene. We used RNase protection mapping to assess the abundance in the brain and pituitary of preproenkephalin transcripts that initiate within intron A. These studies confirmed that the primary transcription start site is 28 nucleotides downstream from the TATAA site, and that intron A sequences are not present in significant amounts in these tissues.

Topics: Animals; Base Sequence; Brain; Dynorphins; Enkephalins; Guinea Pigs; Male; Molecular Sequence Data; Pituitary Gland; Protein Precursors; RNA Processing, Post-Transcriptional; RNA, Messenger; Transcription Initiation Site

Release of endogenous dynorphin opioids within the spinal cord after partial sciatic nerve ligation (pSNL) is known to contribute to the neuropathic pain processes. Using a phosphoselective antibody [kappa opioid receptor (KOR-P)] able to detect the serine 369 phosphorylated form of the KOR, we determined possible sites of dynorphin action within the spinal cord after pSNL. KOR-P immunoreactivity (IR) was markedly increased in the L4-L5 spinal dorsal horn of wild-type C57BL/6 mice (7-21 d) after lesion, but not in mice pretreated with the KOR antagonist nor-binaltorphimine (norBNI). In addition, knock-out mice lacking prodynorphin, KOR, or G-protein receptor kinase 3 (GRK3) did not show significant increases in KOR-P IR after pSNL. KOR-P IR was colocalized in both GABAergic neurons and GFAP-positive astrocytes in both ipsilateral and contralateral spinal dorsal horn. Consistent with sustained opioid release, KOR knock-out mice developed significantly increased tactile allodynia and thermal hyperalgesia in both the early (first week) and late (third week) interval after lesion. Similarly, mice pretreated with norBNI showed enhanced hyperalgesia and allodynia during the 3 weeks after pSNL. Because sustained activation of opioid receptors might induce tolerance, we measured the antinociceptive effect of the kappa agonist U50,488 using radiant heat applied to the ipsilateral hindpaw, and we found that agonist potency was significantly decreased 7 d after pSNL. In contrast, neither prodynorphin nor GRK3 knock-out mice showed U50,488 tolerance after pSNL. These findings suggest that pSNL induced a sustained release of endogenous prodynorphin-derived opioid peptides that activated an anti-nociceptive KOR system in mouse spinal cord. Thus, endogenous dynorphin had both pronociceptive and antinociceptive actions after nerve injury and induced GRK3-mediated opioid tolerance.

Topics: Animals; Astrocytes; Disease Models, Animal; Disease Progression; Drug Tolerance; Dynorphins; Enkephalins; G-Protein-Coupled Receptor Kinase 3; Hyperalgesia; Lumbosacral Region; Mice; Mice, Inbred C57BL; Mice, Knockout; Narcotic Antagonists; Narcotics; Neuralgia; Neurons; Protein Precursors; Protein Serine-Threonine Kinases; Receptors, Opioid; Receptors, Opioid, kappa; Sciatic Neuropathy; Spinal Cord

Previous studies have shown that peripheral electrical stimulation (PES) can suppress morphine-induced conditioned place preference (CPP) and the reinstatement of extinguished CPP in the rat. The present study was performed to elucidate if preproenkephalin (PPE) and preprodynorphin (PPD) mRNAs in the nucleus accumbens (NAc) play a role in this event. Rats were trained with morphine for 4 days to establish CPP paradigm. They were then given 15-min test once a day for eight consecutive days for extinction trial. Twenty-four hours after the 8th session of extinction trials, rats were given peripheral electrical stimulation (PES) at 2 or 100 Hz once a day for 3 days, then a morphine-priming injection at a dose of 1, 2, or 4 mg/kg to reinstate the extinguished CPP. At the end of the experiment, PPE and PPD mRNA levels in the nucleus acccumbens (NAc) were determined by the semiquantitative RT-PCR technique. The results showed that PES at 2- and 100-Hz administered 30 min a day for 3 days suppressed both the expression of morphine-induced CPP and the reinstatement of extinguished CPP. PES at 2 Hz increased preproenkephalin (PPE) mRNA levels, whereas PES of 100 Hz that of preprodynorphin (PPD) mRNA levels in the NAc. These findings suggest that enkephalin and dynorphin in NAc may play important roles in the mechanisms underlying the inhibitory effect of PES on the expression and reinstatement of morphine-induced CPP in rats.

Topics: Analysis of Variance; Animals; Behavior, Animal; Conditioning, Operant; Dose-Response Relationship, Drug; Dose-Response Relationship, Radiation; Dynorphins; Electric Stimulation; Enkephalins; Extinction, Psychological; Gene Expression Regulation; Hindlimb; Male; Morphine; Motor Activity; Narcotics; Nucleus Accumbens; Protein Precursors; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Time Factors

Topics: Animals; Antisense Elements (Genetics); Cocaine; Corpus Striatum; Dopamine Uptake Inhibitors; Dynorphins; Enkephalins; Humans; In Situ Hybridization; Protein Precursors; Rats; RNA Probes; RNA, Messenger

The prodynorphin gene and its product, dynorphin B, have been found to promote cardiogenesis in embryonic cells by inducing the expression of GATA-4 and Nkx-2.5, two transcription factor-encoding genes essential for cardiogenesis. The molecular mechanism(s) underlying endorphin-induced cardiogenesis remain unknown. In the present study, we found that GTR1 embryonic stem (ES) cells expressed cell surface kappa opioid receptors, as well as protein kinase C (PKC)-alpha, -beta1, -beta2, -delta, -epsilon, and -zeta. Cardiac differentiation was associated with a marked increase in the Bmax value for a selective opioid receptor ligand and complex subcellular redistribution of selected PKC isozymes. PKC-alpha, -beta1, -beta2, -delta, and -epsilon all increased in the nucleus of ES-derived cardiac myocytes, compared with nuclei from undifferentiated cells. In both groups of cells, PKC-delta and -epsilon were mainly expressed at the nuclear level. The nuclear increase of PKC-alpha, -beta1, and -beta2 was due to a translocation from the cytosolic compartment. In contrast, the increase of both PKC-delta and PKC-epsilon in the nucleus of ES-derived cardiomyocytes occurred independently of enzyme translocation, suggesting changes in isozyme turnover and/or gene expression during cardiogenesis. No change in PKC-zeta expression was observed during cardiac differentiation. Opioid receptor antagonists prevented the nuclear increase of PKC-alpha, PKC-beta1, and PKC-beta2 and reduced cardiomyocyte yield but failed to affect the nuclear increase in PKC-delta and -epsilon. PKC inhibitors prevented the expression of cardiogenic genes and dynorphin B in ES cells and abolished their development into beating cardiomyocytes.

Topics: Animals; Cell Differentiation; Cell Line; Cell Nucleus; Dynorphins; Embryo, Mammalian; Embryo, Nonmammalian; Endorphins; Enkephalins; Enzyme Inhibitors; Gene Expression Regulation, Developmental; Heart; Isoenzymes; Myocytes, Cardiac; Myosin Heavy Chains; Narcotic Antagonists; Protein Kinase C; Protein Precursors; Receptors, Opioid, kappa; RNA, Messenger; Signal Transduction; Stem Cells

The cardiac differentiation of embryonic stem (ES) cells was found to involve prodynorphin gene and dynorphin B expression and was associated with the interaction of secreted dynorphin B with cell surface opioid receptors coupled with protein kinase C (PKC) signaling and complex subcellular redistribution patterning of selected PKC isozymes. Here, confocal microscopy revealed the presence of immunoreactive dynorphin B-like material in GTR1 ES cells, suggesting that dynorphin peptides may also act intracellularly. Opioid binding sites were identified in ES cell nuclei, with a single dissociation constant in the low nanomolar range. A significant increase in Bmax for a kappa opioid receptor ligand was observed in nuclei isolated from ES-derived cardiomyocytes compared with nuclei from undifferentiated cells. Direct exposure of nuclei isolated from undifferentiated ES cells to dynorphin B or U-50,488H, a synthetic kappa opioid receptor agonist, time- and dose-dependently activated the transcription of GATA-4 and Nkx-2.5, 2 cardiac lineage-promoting genes. Nuclear exposure to dynorphin B also enhanced the rate of prodynorphin gene transcription. These responses were abolished in a stereospecific fashion by the incubation of isolated nuclei with selective opioid receptor antagonists. Nuclei isolated from undifferentiated cells were able to phosphorylate the acrylodan-labeled MARCKS peptide, a high-affinity fluorescent PKC substrate. Exposure of isolated nuclei to dynorphin B induced a remarkable increase in nuclear PKC activity, which was suppressed by opioid receptor antagonists. Nuclear treatment with PKC inhibitors abolished the capability of dynorphin B to prime the transcription of cardiogenic genes.

Topics: Animals; Cell Differentiation; Cell Line; Cell Lineage; Cell Nucleus; DNA-Binding Proteins; Dynorphins; Embryo, Mammalian; Embryo, Nonmammalian; Endorphins; Enkephalins; Enzyme Activation; GATA4 Transcription Factor; Gene Expression Regulation, Developmental; Heart; Homeobox Protein Nkx-2.5; Homeodomain Proteins; Myocytes, Cardiac; Protein Kinase C; Protein Precursors; Receptors, Opioid; RNA, Messenger; Signal Transduction; Stem Cells; Transcription Factors; Transcriptional Activation; Xenopus Proteins

Various hypothalamic neuropeptides are involved in central regulation of food intake and expression of genes encoding these peptides changes with alterations in the bodyweight/metabolic status/nutritional status. Orexin(s) and dynorphin have been implicated in the regulation of appetite and neuroendocrine systems, but the function of these peptides is not well understood. We have employed in situ hybridization to examine the effects of long-term alterations in the bodyweight on expression of mRNA for preproorexin and prodynorphin in the putative feeding centers of the ovine hypothalamus. Expression of preproorexin was localized to the dorsomedial hypothalamic nucleus, perifornical area and lateral hypothalamic area. Cells expressing prodynorphin were localized to the periventricular, supraoptic, paraventricular, ventromedial hypothalamic nuclei and the thalamus. Small numbers of single scattered cells were seen in other brain areas. A few scattered prodynorphin-expressing cells were found in the lateral hypothalamic area but, in contrast to observations in the rat, there was no colocalization with preproorexin. Long-term alterations in the bodyweight did not influence the level of expression of preproorexin or prodynorphin. These findings suggest that orexin and dynorphin may not play a direct role in appetite regulation in sheep, although regulation at the level of the receptors for these peptides remains a possibility.

Topics: Animals; Appetite Regulation; Body Weight; Carrier Proteins; Dynorphins; Enkephalins; Feeding Behavior; Female; Food Deprivation; Gene Expression Regulation; Hypothalamus; Intracellular Signaling Peptides and Proteins; Neurons; Neuropeptides; Neurosecretory Systems; Orexins; Ovariectomy; Protein Precursors; RNA, Messenger; Sheep; Time Factors

Environmental manipulations early in life may induce persistent alterations in adult behaviour and physiology. The underlying neural mechanisms of these responses are not yet clear. We have previously reported long-term changes in brain opioid peptide levels in male and female Sprague-Dawley rats after short periods (15 min, known as neonatal handling) of maternal separation (MS) until weaning. To study this further, we investigated behavioural and neurochemical effects of repeated MS in male Wistar rats. The rat pups were separated from their dams in litters for either 360 min (MS360) or 15 min (MS15) daily from postnatal day 1 to 21 or exposed to normal animal facility rearing. Behavioural analysis showed that MS360 rats had increased ultrasonic calls on postnatal day 5 compared to MS15 rats, but not on postnatal day 6. Moreover, the MS360 rats had more animals with higher frequency of calls at day 5 than 6 than the MS15 rats. Analysis of the opioid peptides dynorphin B and Met-enkephalin-Arg(6)Phe(7) with radioimmunoassay 7 weeks after the MS procedure, revealed long-term neurochemical changes in several brain areas and in the pituitary gland. Immunoreactive dynorphin B and Met-enkephalin-Arg(6)Phe(7) levels were affected in the hypothalamus and dynorphin B levels in the neurointermediate pituitary lobe, amygdala, substantia nigra and the periaqueductal gray. Together, these findings show that repeated periods of MS early in life in male Wistar rats affect the development of the ultrasonic call response and induce long-lasting and possibly permanent alterations in the opioid peptide systems.

Topics: Animals; Animals, Newborn; Behavior, Animal; Body Weight; Brain; Corticosterone; Dynorphins; Endorphins; Enkephalin, Methionine; Enkephalins; Female; Male; Maternal Deprivation; Opioid Peptides; Pituitary Gland; Protein Precursors; Radioimmunoassay; Rats; Rats, Wistar; Time Factors; Ultrasonics

Projection neurons in the ventral striatum, the accumbens nucleus and olfactory tubercle, were examined by combining the retrograde tracing method and immunocytochemistry with antibodies against C-terminals of the preprodynorphin (PPD), preproenkephalin (PPE), preprotachykinin A (PPTA) and preprotachykinin B (PPTB). When the retrograde tracer was injected into the ventral pallidum, about 60% and 40% of retrogradely labeled neurons in the accumbens nucleus were immunoreactive for PPD and PPE, respectively. In contrast, all accumbens nucleus neurons projecting to the ventral mesencephalic regions including the substantia nigra and ventral tegmental area were immunopositive for PPD but not for PPE. Although no olfactory tubercle neurons projected fibers to the mesencephalic regions, 60% and 40% of olfactory tubercle neurons projecting to the ventrolateral portion of the ventral pallidum were immunoreactive for PPD and PPE, respectively, as were the accumbens nucleus neurons. About 70% of accumbens nucleus and olfactory tubercle neurons projecting to the ventral pallidum and all accumbens nucleus neurons projecting to the ventral mesencephalic regions showed PPTA immunoreactivity. A small population (2-12%) of accumbens neurons projecting to the ventral pallidum and mesencephalic regions displayed immunoreactivity for PPTB. Compared with the dorsal striatopallidal projection neurons that were reported to mostly express PPE, it was characteristic of the ventral striatum that only the smaller population (about 40%) of ventral striatopallidal projection neurons expressed PPE. This suggests that the ventral striatopallidal projection system is less specialized than the dorsal striatopallidal system in terms of peptide production, or that the ventral pallidum should be compared with a combined region of the globus pallidus and entopeduncular nucleus in the dorsal system.

Topics: Animals; Basal Ganglia; Dynorphins; Enkephalins; Fluorescent Antibody Technique; Immunoenzyme Techniques; Immunohistochemistry; Male; Neural Pathways; Neurokinin B; Neurons; Nucleus Accumbens; Olfactory Pathways; Peptide Fragments; Protein Precursors; Rats; Rats, Wistar; Tachykinins

Effects of c-fos antisense oligodeoxynucleotide (ASO) on serotonin (5-HT)-induced upregulation of preprodynorphin (ppDyn), preproenkephalin (ppEnk), and glutamic acid decarboxylase (GAD), a special chemical marker for gamma-aminobutyric acid (GABA) neurons, mRNAs in cultured spinal dorsal horn neurons were investigated in order to extend our understanding of expressions of opioid peptides and GABA in spinal cord regulated by the descending serotonergic efferents. Reverse transcription-polymerase chain reaction analysis revealed a time-course increase in the expression of mRNAs encoding c-fos, ppDyn, ppEnk, and GAD after administration of 5-HT (100 nM). Administration of c-fos ASO (0.02 nM) 30 min prior to 5-HT application markedly blocked the expression of c-fos gene. Moreover, c-fos ASO pretreatment significantly decreased the 5-HT-induced upregulation of ppDyn and ppEnk mRNAs, but failed to affect the expression level of GAD mRNA. These results suggest that the serotoningic raphe-spinal efferents might play an important role in regulating the synthesis of enkephalin, dynorphin, and GABA in the spinal dorsal horn neurons. The immediate early oncogene c-fos might be involved in the 5-HT-induced increase in ppDyn and ppEnk expression. However, under the present experimental conditions, c-fos does not seem to be associated with the upregulation of GAD mRNA induced by 5-HT.

Topics: Animals; Cells, Cultured; Dynorphins; Enkephalins; Gene Expression Regulation; Glutamate Decarboxylase; Oligodeoxyribonucleotides, Antisense; Posterior Horn Cells; Protein Precursors; Proto-Oncogene Proteins c-fos; Rats; Rats, Wistar; RNA, Messenger; Serotonin; Serotonin Antagonists; Up-Regulation

Nerve injury-induced afferent discharge is thought to elicit spinal sensitization and consequent abnormal pain. Experimental neuropathic pain, however, also depends on central changes, including descending facilitation arising from the rostral ventromedial medulla (RVM) and upregulation of spinal dynorphin. A possible intersection of these influences at the spinal level was explored by measuring evoked, excitatory transmitter release in tissues taken from nerve-injured animals with or without previous manipulation of descending modulatory systems. Spinal nerve ligation (SNL) produced expected tactile and thermal hyperesthesias. Capsaicin-evoked calcitonin gene-related peptide (CGRP) release was markedly enhanced in lumbar spinal tissue from SNL rats when compared with sham-operated controls. Enhanced, evoked CGRP release from SNL rats was blocked by anti-dynorphin A(1-13) antiserum; this treatment did not alter evoked release in tissues from sham-operated rats. Dorsolateral funiculus lesion (DLF) or destruction of RVM neurons expressing mu-opioid receptors with dermorphin-saporin, blocked tactile and thermal hypersensitivity, as well as SNL-induced upregulation of spinal dynorphin. Spinal tissues from these DLF-lesioned or dermorphin-saporin-treated SNL rats did not exhibit enhanced capsaicin-evoked CGRP-IR release. These data demonstrate exaggerated release of excitatory transmitter from primary afferents after injury to peripheral nerves, supporting the likely importance of increased afferent input as a driving force of neuropathic pain. The data also show that modulatory influences of descending facilitation are required for enhanced evoked transmitter release after nerve injury. Thus, convergence of descending modulation, spinal plasticity, and afferent drive in the nerve-injured state reveals a mechanism by which some aspects of nerve injury-induced hyperesthesias may occur.

Topics: Afferent Pathways; Analgesics, Opioid; Animals; Calcitonin Gene-Related Peptide; Capsaicin; Disease Models, Animal; Dynorphins; Enkephalins; Hyperesthesia; Ligation; Lumbosacral Region; Male; Medulla Oblongata; Microinjections; N-Glycosyl Hydrolases; Nerve Compression Syndromes; Neuralgia; Neuronal Plasticity; Neurotransmitter Agents; Oligopeptides; Opioid Peptides; Pain Measurement; Protein Precursors; Rats; Rats, Sprague-Dawley; Receptors, Opioid, mu; Recombinant Fusion Proteins; Ribosome Inactivating Proteins, Type 1; Saporins; Sensory Thresholds; Spinal Cord; Spinal Nerves

Cocaine administration increases activity at dopamine receptors, increases preprodynorphin (ppDyn) gene expression in the caudate-putamen (CPu), and activates the stress responsive hypothalamic-pituitary-adrenal (HPA) axis. To examine the hypothesis that mu-opioid receptors (MOR) may play roles in these cocaine effects, we tested the effects of acute "binge" pattern cocaine administration in mice with targeted disruption of the MOR gene. Wild-type (+/+) and homozygous MOR-deficient (-/-) mice received three injections of 15 mg/kg cocaine at 1-h intervals. Mice were sacrificed 30 min after the last injection and mRNAs for ppDyn and preproenkephalin (ppEnk) in the CPu and nucleus accumbens (NAc), and for type I corticotropin-releasing hormone receptor (CRH(1) receptor) and pro-opiomelanocortin (POMC) in the hypothalamus and pituitary, were measured by solution hybridization RNase protection assays. Cocaine elevated ppDyn mRNA in the CPu, but not NAc, of both the MOR -/- and wild-type mice. ppEnk mRNA in the CPu, but not NAc, was lower in MOR -/- mice than in wild-type mice following cocaine administration. Hypothalamic CRH(1) receptor and POMC mRNAs were expressed at similar levels in untreated and in cocaine-treated mice of each genotype. However, there were lower basal levels of CRH(1) receptor mRNA in the anterior pituitary of the MOR -/- mice than in wild-type mice and the MOR -/- mice failed to show the cocaine-induced decreases in CRH(1) receptor mRNA found in the wild-type mice. Cocaine activated the HPA axis similarly in MOR -/- and wild-type mice, as reflected in similar increases in plasma corticosterone levels in both genotypes. These results support a specific role for MORs in acute cocaine effects on striatal ppEnk gene expression and fail to support critical roles for these receptors in acute cocaine's effects on either ppDyn gene expression or HPA activation. MOR -/- mice are useful models for studying cocaine effects on ppEnk gene expression that could aid interpretation of the similar postmortem phenomena found in human cocaine addicts.

Topics: Animals; Cocaine; Corpus Striatum; Corticotropin-Releasing Hormone; Dopamine Uptake Inhibitors; Dynorphins; Enkephalins; Frontal Lobe; Gene Expression; Hypothalamo-Hypophyseal System; Male; Medulla Oblongata; Mice; Mice, Knockout; Pituitary-Adrenal System; Pons; Pro-Opiomelanocortin; Protein Precursors; Receptors, Opioid, mu; 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

Preprodynorphin (PPD), preproenkephalin (PPE) and preprotachykinins A (PPTA) and B (PPTB) are known to be expressed by neostriatal projection neurons. In the present study, we investigated the distributions and colocalizations of immunoreactivities for those prepropeptides in the ventral striatum, such as the accumbens nucleus (Acb) and olfactory tubercle (OT). Antibodies raised against C-terminal portions of the prepropeptides labeled cell bodies of neurons with diameters of 8-15 microm. PPD-, PPE- and PPTA-immunoreactive neurons were distributed throughout the Acb and concentrated in the dense cell layer of the OT. PPTB-immunoreactive neurons were observed to form cell clusters, which were localized in mu-opioid receptor-immunoreactive patchy regions in the Acb, but were very rarely found in the dense cell layer of the OT. Double-immunofluorescence analysis revealed that PPD, PPE and PPTB immunoreactivities were shown in 69%, 19% and 14% of PPTA-immunoreactive neurons, respectively, in the Acb core region, and in 92%, 7% and 25% of PPTA-immunoreactive neurons, respectively, in the Acb shell region. In the olfactory bulb, 51%, 19% and 3% of PPTA-immunoreactive neurons showed PPD, PPE and PPTB immunoreactivities, respectively. PPD and PPE immunoreactivities were rarely coexpressed in single neurons of all striatal regions. The present results indicated that, although PPTA and PPE were occasionally coexpressed in single neurons of the ventral striatum, the segregated expression of PPD and PPE in the ventral striatum was similar to that in the dorsal striatum. The clustered localization of PPTB-expressing neurons in the Acb and near absence of PPTB-expressing neurons in the dense cell layer of the OT suggests that neurokinin B is a key substance in differentiating between the ventral and dorsal striatal regions.

Topics: Animals; Dynorphins; Enkephalins; Fluorescent Antibody Technique; Immunohistochemistry; Neurokinin B; Neurons; Nucleus Accumbens; Olfactory Pathways; Peptide Fragments; Protein Precursors; Rats; Rats, Wistar; Tachykinins

Intrathecal (i.t.) administration of big dynorphin (1-10 fmol), a prodynorphin-derived peptide consisting of dynorphin A and dynorphin B, to mice produced a characteristic behavioral response, the biting and/or licking of the hindpaw and the tail along with slight hindlimb scratching directed toward the flank, which peaked at 5-15 min after an injection. Dynorphin A produced a similar response, though the doses required were higher (0.1-30 pmol) whereas dynorphin B was practically inactive even at 1000 pmol. The behavior induced by big dynorphin (3 fmol) was dose-dependently inhibited by intraperitoneal injection of morphine (0.125-2 mg/kg) and also dose-dependently, by i.t. co-administration of D(-)-2-amino-5-phosphonovaleric acid (D-APV) (1-4 nmol), a competitive N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801 (0.25-4 nmol), an NMDA ion-channel blocker, and ifenprodil (2-8 pmol), an inhibitor of the NMDA receptor ion-channel complex interacting with the NR2B subunit and the polyamine recognition site. On the other hand, naloxone, an opioid receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a non-NMDA glutamate receptor antagonist, 7-chlorokynurenic acid, a competitive antagonist of the glycine recognition site on the NMDA receptor ion-channel complex, [D-Phe(7),D-His(9)]-substance P(6-11), a specific antagonist for substance P (NK1) receptors, and MEN-10376, a tachykinin NK2 receptor antagonist, had no effect. These results suggest that big dynorphin-induced nociceptive behavior is mediated through the activation of the NMDA receptor ion-channel complex by acting on the NR2B subunit and/or the polyamine recognition site but not on the glycine recognition site, and does not involve opioid, non-NMDA glutamate receptor mechanisms or tachykinin receptors in the mouse spinal cord.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Analgesics, Opioid; Animals; Behavior, Animal; Dizocilpine Maleate; Dynorphins; Endorphins; Enkephalins; Excitatory Amino Acid Antagonists; Injections, Spinal; Kynurenic Acid; Male; Mice; Morphine; Neurokinin A; Nociceptors; Peptide Fragments; Piperidines; Protein Precursors; Receptors, N-Methyl-D-Aspartate; Receptors, Tachykinin; Substance P

Whereas the role of dopaminergic tone in the cortico-striatal-thalamic system is well-established, the role of endogenous opioids in the function of this system is less understood. We show that Borna disease virus infection of adult rats results in an increase in preproenkephalin transcripts in the striatum of Borna-infected rats, a region important for forming coordinated sequential motor actions and in developing programmes of thought and motivation. Stereotypic behaviours and dyskinesias, the clinical hallmarks of infection in adult Lewis rats (BD rats), are accompanied by a disrupted pattern of immediate early gene c-fos activation in the motor thalamus, with significance for the breakdown in coordinated sequential motor actions. We also find increased preproenkephalin in infected cultured neuroblastoma and rat foetal glial cells. The expression pattern of enkephalin mRNA in vivo and in vitro suggest that increased enkephalin function is one of the neuropharmacological means by which Borna disease virus causes motor disease of animals and possibly cognitive and affective disease in man, and further suggest that enkephalins play a critical role in the maintenance of a balanced tone of activity in the cortico-basal ganglia-thalamo-cortical loops.

Topics: Animals; Astrocytes; Borna Disease; Borna disease virus; Cells, Cultured; Dynorphins; Enkephalins; Gene Expression; Genes, fos; Humans; Male; Neuroblastoma; Protein Precursors; Putamen; Rats; Rats, Inbred Lew; Receptors, Dopamine D2; Tumor Cells, Cultured

A decrease in inhibitory tone of endogenous opioid peptide on the afternoon of proestrus is one event underlying generation of the ovulatory luteinizing hormone (LH) surge, since premature removal of this inhibitory tone (i.e., disinhibition) results in an early onset of the surge. Our laboratory demonstrated that blockade of kappa-opioid receptors in the medial preoptic area (MPOA) advanced the onset of the LH surge on proestrus. Since dynorphin is the endogenous ligand for the kappa-opioid receptor, the present studies examined the possible role of dynorphin in this disinhibition response. 1) Neutralization of endogenous dynorphin peptides, by push-pull perfusion of the MPOA with antibodies specific for dynorphin A1-17 or A1-8 from 1030-1355 h on proestrus, tended to prematurely advance the increase in plasma LH levels normally occurring on this day of the estrous cycle. Although this increase was not statistically significant when compared with controls, plasma LH levels in two antibody-treated rats were sufficiently elevated to cause full ovulation, a response that did not occur in controls. These data suggest that dynorphin A1-7 and A1-8 might have a role in the MPOA, although a minor one, in suppressing LH secretion early on proestrus. MPOA levels of prodynorphin mRNA decreased at 1700-1800 h on proestrus when plasma LH levels were high, compared with values at 1300-1400 h when plasma LH levels were low. This change did not occur on diestrous d 1 when there was no LH surge. 2) MPOA levels of kappa-opioid receptor mRNA did not change on proestrus or diestrous d 1. These results suggest that a reduction in prodynorphin gene expression on the afternoon of proestrus may be one event involved in a possible decrease in dynorphin inhibitory tone on the ovulatory LH surge-generating signal.

Topics: Animals; Antibodies; Dynorphins; Enkephalins; Female; Luteinizing Hormone; Ovulation; Peptide Fragments; Perfusion; Preoptic Area; Proestrus; Protein Precursors; Rats; Rats, Sprague-Dawley; Receptors, Opioid, kappa; RNA, Messenger

The cAMP response element-binding protein (CREB) is a critical integrator of neural plasticity that is responsive in a brain region-specific manner to a variety of environmental and pharmacological stimuli, including widely prescribed antidepressant medications. We developed inducible transgenic lines of mice that express either CREB or a dominant-negative mutant of CREB (mCREB) in forebrain regions and used these mice to determine the functional significance of this transcription factor in the learned helplessness paradigm, a behavioral model of depression. We also use a complementary viral-mediated gene transfer approach to directly test the effect of mCREB in the nucleus accumbens, a brain region important for motivation and reward. The results demonstrate that blockade of CREB by overexpression of mCREB in transgenic mice or by viral expression of mCREB in the nucleus accumbens produces an antidepressant-like effect, whereas overexpression of CREB in transgenic mice results in the opposite phenotype. In addition, mCREB expression was colocalized with and decreased the expression of prodynorphin in nucleus accumbens medium spiny neurons, and antagonism of dynorphin in the nucleus accumbens was sufficient to produce an antidepressant-like effect similar to that observed after blockade of CREB. Together, the results demonstrate that nucleus accumbens CREB-dynorphin influence behavior in the learned helplessness model and suggest that this signaling cascade may contribute to symptoms of depression.

Topics: Animals; Antidepressive Agents; Behavior, Animal; Cyclic AMP Response Element-Binding Protein; Depression; Dynorphins; Enkephalins; Helplessness, Learned; Immunohistochemistry; Male; Mice; Mice, Transgenic; Mutation; Naltrexone; Nucleus Accumbens; Prosencephalon; Protein Precursors; Rats; Rats, Sprague-Dawley; Receptors, Opioid, kappa

In a previous study, we have shown in unilaterally dopamine-depleted rats that increased behavioral responsiveness to the dopamine D1-receptor agonist SKF-38393, which was induced by pretreatment with L-DOPA, is paralleled by specific alterations in striatal neuropeptide mRNA levels. The behavioral 'priming' effect of L-DOPA is prevented if L-DOPA is preceded by the NMDA-receptor antagonist MK-801. In the present study, the question is addressed whether blockade of the increased behavioral responsiveness with MK-801 also prevents the observed changes in striatal neuropeptide mRNA levels. After a challenge with SKF-38393 (3 mg/kg, s.c.), the striatal levels of preprodynorphin, preprotachykinin, and preproenkephalin mRNA were compared between unilaterally dopamine-depleted rats that were either primed with a single administration of L-DOPA (50 mg/kg, i.p.) or with L-DOPA preceded by MK-801 (0.1 mg/kg, i.p.). Priming with L-DOPA enhanced the increase in dynorphin mRNA levels in the dorsolateral part of the dopamine-depleted striatum that occurred after SKF-38393. On the other hand, it had no significant effect on substance P or enkephalin mRNA levels. MK-801 prior to L-DOPA prevented the increased responsiveness of dynorphin regulation. However, it induced a decreased response to dopamine D1-receptor stimulation in the substance P mRNA levels in dorsal regions of the dopamine-depleted striatum. The levels of enkephalin mRNA after challenge with SKF-38393 were not affected by the MK-801 administration. These results demonstrate that the increased behavioral responsiveness to the D1-receptor agonist SKF-38393 after priming with L-DOPA is primarily related to the upregulation of dynorphin mRNA levels in the dopamine-depleted striatum.

Topics: 2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine; Animals; Disease Models, Animal; Dizocilpine Maleate; Dopamine Agents; Dopamine Agonists; Drug Interactions; Dynorphins; Dyskinesia, Drug-Induced; Enkephalins; Excitatory Amino Acid Antagonists; Immunohistochemistry; Levodopa; Male; Motor Activity; Neostriatum; Neurons; Neuropeptides; Oxidopamine; Parkinsonian Disorders; Protein Precursors; Rats; Rats, Wistar; Receptors, Dopamine D1; Receptors, N-Methyl-D-Aspartate; RNA, Messenger; Tachykinins; Tyrosine 3-Monooxygenase

Metabotropic glutamate receptor 1 (mGluR1) is highly expressed in striatonigral projection neurons of rat striatum. To define the role of mGluR1 in the regulation of striatal gene expression, the responsiveness of the three neuropeptide gene expression to a single injection of the dopamine D(1) agonist SKF-82958 was compared between mGluR1 mutant and wild-type control mice. We found that acute injection of SKF-82958 increased preprodynorphin (PPD), substance P (SP), and preproenkephalin (PPE) mRNAs in the dorsal and ventral striatum of mutant and wild-type mice in a dose-dependent manner (0.125, 0.5, and 2 mg/kg, i.p.) as revealed by quantitative in situ hybridization. However, the induction of PPD mRNA in both the dorsal and ventral striatum of mGluR1 minus sign/minus sign mice was significantly less than that of wild-type +/+ mice in response to the two higher doses of SKF-82958. In contrast to PPD, SP and PPE in the dorsal and ventral striatum of mGluR1 mutant mice were elevated to a similar level as that of wild-type mice. There were no differences in basal levels and distribution patterns of all three mRNAs between the two genotypes of mice treated with saline. These results indicate that mGluR1 selectively participates in striatonigral PPD induction in response to D(1) receptor stimulation.

Topics: Animals; Benzazepines; Dose-Response Relationship, Drug; Dynorphins; Enkephalins; Gene Expression Regulation; Injections, Intraperitoneal; Male; Mice; Mice, Knockout; Nucleus Accumbens; Protein Precursors; Putamen; Receptors, Dopamine D1; Receptors, Metabotropic Glutamate; RNA, Messenger; Up-Regulation

Group I metabotropic glutamate receptors (mGluRs) are positively coupled to phosphoinositide hydrolysis through G-proteins and are densely expressed in the medium-sized spiny neurons of striatum. Activation of this group of mGluRs in the striatum produces long-lasting stimulation of behavioral activity. In this study, the role of group I mGluRs in the modulation of neuropeptide mRNA expression in striatal neurons was investigated using a Group I-selective agonist, 3,5-dihydroxyphenylglycine (DHPG) in chronically cannulated rats. Unilateral injections of DHPG into the dorsal striatum (caudoputamen) at behaviorally active doses of 20, 40, and 80 nmol elevated basal levels of preprodynorphin (PPD), substance P (SP), and preproenkephalin (PPE) mRNAs in the injected dorsal striatum as revealed by quantitative in situ hybridization. The elevation of all three mRNAs was dose-dependent and the responsiveness of opioid peptide mRNAs (PPD and PPE) to acute injection of DHPG at each dose surveyed was greater than that of SP mRNA. Induction of the mRNAs was delayed and prolonged as increases in hybridization signal became evident at 2 (SP and PPE) or 3 (PPD) h, reached a peak between 3 and 6 h, and returned to normal levels 24 h after DHPG injection. Coadministration of a Group I-selective antagonist, n-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carbo xamide (PHCCC, 10 nmol), with DHPG markedly attenuated DHPG-stimulated PPD, PPE, and, to a lesser extent, SP expression. Administration of PHCCC alone had no significant effect on basal levels of three mRNA expression in the striatum. This study provides a detailed description of the dose- and time-related alterations in striatonigral PPD/SP and striatopallidal PPE mRNA expression in response to a single injection of the Group I agonist DHPG. Data obtained demonstrate a facilitatory, dynamic regulation of constitutive expression of PPD, SP, and PPE mRNAs by local enhancement of glutamatergic tone on DHPG- and PHCCC-sensitive Group I mGluRs.

Topics: Animals; Behavior, Animal; Dose-Response Relationship, Drug; Dynorphins; Enkephalins; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glycine; Male; Neostriatum; Neurons; Nucleus Accumbens; Protein Precursors; Rats; Rats, Wistar; Receptors, Metabotropic Glutamate; Resorcinols; RNA, Messenger; Substance P; Time Factors

Group I metabotropic glutamate receptors (mGluRs) are positively coupled to phosphoinositide hydrolysis, and are expressed in medium spiny neurons of rat striatum in vivo. By modifying intracellular activities, this group of mGluRs is involved in the regulation of gene expression important for neuroplasticity. To characterize the regulatory role of group I receptors in opioid peptide mRNA expression in vitro, primary cultures of striatal cells were prepared from neonatal day-1 rat pups. Cells were cultured in the presence of a mitotic inhibitor, cytosine arabinoside, which generated predominant neuronal cell cultures after 12-14 days in culture as demonstrated by dense immunostaining of more than 90% of cultured cells to a specific marker for neurons (microtubule-associated protein) but not for astroglial cells (glial fibrillary acidic protein). The vast majority of neurons (>90%) were also verified as GABAergic neurons according to their positive immunoreactivity to GABA and glutamic acid decarboxylase-65/67 antibodies. A few large neurons (<5%) showed high levels of choline acetyltransferase immunoreactivity, presumably cholinergic neurons. To confirm group I mGluR expression in cultured neurons, both in situ hybridization and immunocytochemistry were performed, which detected moderate levels of mGluR1 and mGluR5 mRNAs and protein products in most neurons (>70%), respectively. On this culture system, quantitative in situ hybridization was then performed to quantify changes in preprodynorphin (PPD) and preproenkephalin (PPE) mRNA levels in response to mGluR stimulation. Acute incubation of a non-subgroup selective agonist, 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD), increased PPD and PPE mRNA levels in a concentration-dependent manner (176 and 189% over control for PPD and PPE after 100 microM ACPD incubation, respectively). Application of a selective group I agonist, 3,5-dihydroxyphenylglycine (DHPG), produced much greater induction of either mRNA (285 and 289% over control for PPD and PPE after 100 microM DHPG incubation, respectively). Co-incubation of a selective group I antagonist, n-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxamide (PHCCC), blocked both ACPD- and DHPG-induced PPD/PPE expression. These data demonstrate the validity of a neuronal cell culture model for studying the molecular regulation of opioid gene expression in vitro. Selective activation of identified group I mGluRs facilitates constitutive expression of PPD

Topics: Animals; Animals, Newborn; Cells, Cultured; Corpus Striatum; Cycloleucine; Dynorphins; Enkephalins; Gene Expression; Immunohistochemistry; In Situ Hybridization; Methoxyhydroxyphenylglycol; Neurons; Neuroprotective Agents; Nucleus Accumbens; Phenotype; Protein Precursors; Rats; Receptor, Metabotropic Glutamate 5; Receptors, Metabotropic Glutamate; RNA, Messenger

The purpose of this study was to investigate the effects of intrastriatal blockade of GABA(A) receptors on dopamine D(1)/D(2) receptor interactions in the intact rat striatum. Muscarinic receptors mediate the ability of the D(2) receptor antagonist, eticlopride, to block an increase in striatonigral neuropeptide messenger RNA stimulated by the full D(1) agonist, SKF-82958. However, because D(2) receptor antagonists activate striatopallidal neurons, it is possible that increased GABA release from local medium spiny axon collaterals also contributes to the ability of eticlopride to block the effects of SKF-82958. This hypothesis was addressed by infusing the GABA(A) receptor antagonist, bicuculline, into the dorsal striatum in rats treated with eticlopride and SKF-82958. In contrast to the actions of the muscarinic antagonist, scopolamine, bicuculline did not affect the increase in behaviors induced by SKF-82958 or the ability of eticlopride to block them. Quantitative in situ hybridization demonstrated that bicuculline did not significantly affect basal preprodynorphin messenger RNA, nor did it affect the ability of eticlopride to decrease SKF-82958-induced preprodynorphin messenger RNA. However, the level of the preprodynorphin hybridization signal in bicuculline plus SKF-82958-treated rats was significantly lower than in saline plus SKF-82958-treated rats. In contrast, bicuculline, eticlopride or SKF-82958 by themselves increased basal preproenkephalin messenger RNA. However, there was no significant interaction among bicuculline, eticlopride and SKF-82958 on preproenkephalin messenger RNA levels.These data indicate that blockade of striatal GABA(A) receptors has only a subtle effect on acute dopamine agonist-induced changes in gene expression. These results are discussed in the context of local intrastriatal interactions.

Topics: Animals; Behavior, Animal; Benzazepines; Bicuculline; Corpus Striatum; Dopamine Agonists; Dopamine Antagonists; Drug Administration Schedule; Dynorphins; Enkephalins; GABA Antagonists; GABA-A Receptor Antagonists; Gene Expression; In Situ Hybridization; Injections, Subcutaneous; Male; Microinjections; Protein Precursors; Rats; Rats, Wistar; Receptors, Dopamine D1; Receptors, Dopamine D2; Receptors, GABA-A; RNA, Messenger; Salicylamides

Excitotoxic spinal cord injury (SCI) causes anatomic, physiologic and molecular changes within the spinal cord and brain. Intraspinal injection of quisqualic acid (QUIS) produces an excitotoxic injury that leads to the onset of behavioral syndromes, believed to be related to the clinical condition of chronic pain. The opioid system, classically involved in the suppression of pain transmission, has been associated with the onset of pain-related behaviors and changes in spinal opioid peptide expression have been demonstrated in various models of SCI and chronic pain. Recently, changes in opioid peptide expression have been demonstrated in both spinal and supraspinal areas following excitotoxic SCI. Therefore, the purpose of this study was to examine changes in opioid peptide gene expression as they relate to the onset of pain behaviors following excitotoxic SCI. Male, Long-Evans rats were given an intraspinal injection of 1.2 microl of 125 mM QUIS and allowed to survive for 10 days, a duration sufficient for the development of pain-related behaviors. Animals were assessed daily for the presence of excessive grooming behavior, i.e. self-directed biting and scratching resulting in damage to superficial and deeper layers of the skin. Animals were also tested for thermal hypersensitivity using a cold plate apparatus on days 5, 7, and 10 following QUIS injection. After sacrifice, quantitative in situ hybridization was performed on regions of the spinal cord surrounding the lesion site as well as whole brain sections through various levels of the thalamus and cortex. Spinal preproenkephalin (PPE) and preprodynorphin (PPD) expression was significantly increased in animals that developed excessive grooming behaviors vs. those that did not. For PPE, this difference was seen bilaterally, in areas of cord caudal to the site of injury. For PPD, this difference was seen only ipsilateral to the site of injection, rostral to the site of injury. In addition, PPE expression in the anterior cingulate cortex and PPD expression in the contralateral parietal cortex were significantly higher in grooming vs. non-grooming animals. These results support previous conclusions that both spinal and supraspinal regulation of endogenous opioid peptide expression plays a role in the response to or onset of post-SCI pain. These results also suggest that the opioid peptides are regulated independently and serve different functions in response to SCI.

Topics: Animals; Brain; Dynorphins; Enkephalins; Excitatory Amino Acid Agonists; Gene Expression; Grooming; Male; Opioid Peptides; Pain; Protein Precursors; Quisqualic Acid; Rats; Rats, Long-Evans; RNA, Messenger; Spinal Cord; Spinal Cord Injuries

After repeated administration of cocaine at intervals, sensitization phenomena can be observed, so that its behavioural effects are enhanced. Since this phenomenon is long-lasting, it was of interest to study which persistent alterations in the activity of dopaminergic neurones or of endogenous opioid systems downstream of dopaminergic synapses in the basal ganglia are involved in the sensitization. Cocaine (10 mg/kg i.p.) was administered to rats on days 1, 3, 5 and 7 and saline on days 2, 4 and 6 ("repeated cocaine"), or saline was injected on days 1-6 and cocaine on day 7 ("acute cocaine"), or saline was injected on days 1-7 ("saline group"). The "repeated cocaine" schedule led to a significant sensitization to the locomotor activation produced by cocaine on day 7 or on day 17, 10 days after the end of sensitization protocol. Microdialysis in the nucleus accumbens which was performed after administration of cocaine (10 mg/kg i.p.) on day 7, or after an administration of the same dose 10 days after the last administration of cocaine, respectively, revealed significant acute increases of extracellular dopamine to about 200% of basal values. These increases were similar in "acute cocaine" and in "repeated cocaine" animals both after 7 days and after 17 days. For in situ hybridization studies, rats were sacrificed on day 7, 4.5 h after the last cocaine or saline administration. The mRNA for tyrosine hydroxylase (TH) in substantia nigra + ventral tegmental area was significantly elevated to about 140% of saline controls both in the "repeated cocaine" and the "acute cocaine" group as compared with the "saline group". In contrast, there were no differences between the three groups in the mRNAs of preprodynorphin or preproenkephalin levels measured in the nucleus accumbens (core and shell). These results suggest that sensitization phenomena to cocaine are not necessarily connected with alterations in the dopaminergic activity in the mesolimbic system or in the transcription of precursors of endogenous opioid peptides which are located downstream of the dopaminergic synapses.

Topics: Animals; Behavior, Animal; Cocaine; Dopamine; Dynorphins; Enkephalins; In Situ Hybridization; Locomotion; Male; Microdialysis; Neurons; Nucleus Accumbens; Protein Precursors; Rats; Rats, Wistar; RNA, Messenger; Tyrosine Transaminase

Intraspinal injection of quisqualic acid, a mixed kainic acid/2-amino-3(3-hydroxy-5-methylisoxazol-4-yl)propionic acid and metabotropic glutamate receptor agonist, produces an excitotoxic injury that leads to the onset of both spontaneous and evoked pain behavior as well as changes in spinal and cortical expression of opioid peptide mRNA, preprodynorphin and preproenkephalin. What characteristics of the quisqualic acid-induced injury are attributable to activation of each receptor subtype is unknown. This study attempted to define the role of activation of the kainic acid/2-amino-3(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) and metabotropic glutamate receptor subtypes in the regulation of opioid peptide expression and the onset of spontaneous and evoked pain-related behavior following excitotoxic spinal cord injury by comparing quisqualic acid-induced changes with those created by co-injection of quisqualic acid and the kainic acid/AMPA antagonist, 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo[f]quinoxaline, (NBQX) or the metabotropic antagonist, (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA). Therefore, 42 male Long-Evans adult rats were divided into seven treatment groups and received intraspinal microinjections of saline (sham), 0.5% dimethylsulphoxide (sham), quisqualic acid (1.2 microl, 125 mM), NBQX (1.2 microl, 60 microM), AIDA (1.2 microl, 250 microM), quisqualic acid/NBQX (1.2 microl, 125 mM/60 microM), or quisqualic acid/AIDA (1.2 microl, 125 mM/250 microM) directed at spinal levels thoracic 12-lumbar 2. Behavioral observations of spontaneous and evoked pain responses were completed following surgery. After a 10-day survival period, animals were killed and brain and spinal cord tissues were removed and processed for histologic analysis and in situ hybridization. Both AIDA and NBQX affected the quisqualic acid-induced total lesion volume but only AIDA caused a decrease in the percent tissue damage at the lesion epicenter. Preprodynorphin and preproenkephalin expression is increased in both spinal and cortical areas in quisqualic acid-injected animals versus sham-, NBQX or AIDA-injected animals. NBQX did not affect quisqualic acid-induced spinal or cortical expression of preprodynorphin or preproenkephalin except for a significant decrease in preproenkephalin expression in the spinal cord. In contrast, AIDA significantly decreases quisqualic acid-induced preprodynorphin and preproenkephalin expression within the spinal cord and cortex. AIDA, b

Topics: Animals; Behavior, Animal; Dynorphins; Enkephalins; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Grooming; Indans; Male; Nerve Degeneration; Neurons; Neurotoxins; Opioid Peptides; Pain; Pain Measurement; Protein Precursors; Quinoxalines; Rats; Rats, Long-Evans; Receptors, AMPA; Receptors, Glutamate; Receptors, Kainic Acid; Receptors, Metabotropic Glutamate; RNA, Messenger; Spinal Cord; Spinal Cord Injuries

Previous work has shown that oligodendrocytes (OLs) express both micro- and kappa-opioid receptors. In developing OLs, micro receptor activation increases OL proliferation, while the kappa-antagonist nor-binaltorphimine (NorBNI) affects OL differentiation. Because exogenous opioids were not present in our defined culture medium, we hypothesized that NorBNI blocked endogenous opioids produced by the OLs themselves. To test this, intact and partially processed proenkephalin and prodynorphin-derived peptides were assessed in OLs using immunocytochemistry or Western blot analysis, or both. Immature OLs possessed large amounts of intact and partially processed proenkephalin precursors, as well as posttranslational products of prodynorphin including dynorphin A (1-17). With maturation, however, intact or partially processed proenkephalin was expressed by only about 50% of OLs, while dynorphin A (1-17) was undetectable. To assess the function of OL-derived opioids, the effect of kappa-agonists/antagonists on OL differentiation and death was explored. kappa-Agonists alone had no effect. In contrast, NorBNI significantly increased OL death. Additive OL losses were evident when NorBNI was paired with toxic levels of glutamate, suggesting that kappa-receptor blockade alone is sufficient to induce OL death. Thus, the results indicate that OLs express proenkephalin and prodynorphin peptides in a developmentally regulated manner, and further suggest that opioids produced by OLs modulate OL maturation and survival through local (i.e., autocrine and/or paracrine) mechanisms.

Topics: Aging; Animals; Animals, Newborn; Autocrine Communication; Brain; Cell Differentiation; Cell Survival; Cells, Cultured; Dynorphins; Enkephalins; Immunohistochemistry; Mice; Narcotic Antagonists; Narcotics; Oligodendroglia; Opioid Peptides; Paracrine Communication; Protein Precursors; Receptors, Opioid, kappa; Stem Cells

Neurotransmission mediated via opioid and dopamine receptors is believed to be involved in the reinforcing and/or rewarding effects of ethanol consumption. We previously examined the effect of ethanol consumption (and naltrexone treatment, used clinically to treat alcoholism) on micro-opioid receptor density. We describe here the effect of free-choice ethanol consumption and naltrexone treatment on preproenkephalin, preprodynorphin, and dopamine D1 and D2 receptor mRNA expression in the central nervous system.. Fawn-hooded rats were given continual free-choice access to a 5% ethanol solution or water (4 weeks) followed by 2 weeks of water alone. At the end of this abstinence period, osmotic minipumps were implanted subcutaneously to deliver saline (n = 4) or naltrexone (n = 4; 8.4 mg/kg/day for 4 weeks). After recovery from surgery, the rats again were given access to 5% ethanol under the same free-choice conditions (4 weeks). A third group of age-matched controls drank only water during the behavioral trial. At the end of the behavioral trial, the rats were decapitated, and a quantitative examination of peptide precursor mRNAs was made by using in situ hybridization histochemistry.. Naltrexone treatment significantly decreased preprodynorphin expression in the nucleus accumbens, but neither naltrexone treatment nor ethanol consumption significantly affected dopamine D1 and D2 receptor mRNA expression. In contrast, ethanol consumption increased preproenkephalin mRNA in the central and intercalated nuclei of the amygdala but decreased preproenkephalin mRNA in the nucleus accumbens and olfactory tubercle. The decreased level of preproenkephalin mRNA in the nucleus accumbens may reflect a neuroadaptive response to increased release of dopamine, whereas the increased level of preproenkephalin mRNA in the central nucleus of the amygdala may be associated with an anxiolytic effect of ethanol consumption.. The data support the putative role of opioid peptides in the effects of ethanol and suggest that the nucleus accumbens and central nucleus of the amygdala are loci for the reinforcing effects of ethanol.

Topics: Amygdala; Animals; Brain; Dynorphins; Enkephalins; Ethanol; Gene Expression; Male; Naltrexone; Narcotic Antagonists; Nucleus Accumbens; Olfactory Pathways; Protein Precursors; Rats; Receptors, Dopamine D1; Receptors, Dopamine D2; RNA, Messenger; Self Administration

Dynorphin A, a prodynorphin-derived peptide, is able to induce neurological dysfunction and neuronal death. To study dynorphin cytotoxicity in vitro, prodynorphin-derived peptides were added into the culture medium of nonneuronal and neuronal cells or delivered into these cells by lipofection or electroporation. Cells were unaffected by extracellular exposure when peptides were added to the medium. In contrast, the number of viable cells was significantly reduced when dynorphin A or "big dynorphin," consisting of dynorphins A and B, was transfected into cells. Big dynorphin was more potent than dynorphin A, whereas dynorphin B; dynorphin B-29; [Arg(11,13)]-dynorphin A(-13)-Gly-NH-(CH(2))(5)-NH(2), a selective kappa-opioid receptor agonist; and poly-l-lysine, a basic peptide more positively charged than big dynorphin, failed to affect cell viability. The opioid antagonist naloxone did not prevent big dynorphin cytotoxicity. Thus, the toxic effects were structure selective but not mediated through opioid receptors. When big dynorphin was delivered into cells by lipofection, it became localized predominantly in the cytoplasm and not in the nuclei. Big dynorphin appeared to induce toxicity through an apoptotic mechanism that may involve synergistic interactions with the p53 tumor-suppressor protein. It is proposed that big dynorphin induces cell death by virtue of its net positive charge and clusters of basic amino acids that mimic (and thereby perhaps interfere with) basic domains involved in protein-protein interactions. These effects may be relevant for a pathophysiological role of dynorphins in the brain and spinal cord and for control of death of tumor cells, which express prodynorphin at high levels.

Topics: Apoptosis; Cation Exchange Resins; Cell Compartmentation; Cell Survival; Central Nervous System; Cytoplasm; Cytotoxins; Dynorphins; Enkephalins; Immunohistochemistry; Lipids; Naloxone; Narcotic Antagonists; Nerve Degeneration; Peptide Fragments; Protein Precursors; Protein Structure, Tertiary; Receptors, Opioid; Receptors, Opioid, kappa; Transcription, Genetic; Tumor Cells, Cultured; Tumor Suppressor Protein p53

The purpose of this study was to investigate the role that kappa opioid receptor stimulation has upon stimulant-induced behavior and neuropeptide gene expression in the striatum. Acute administration of amphetamine (2.5 mg/kg i.p.) caused an increase in behavioral activity and preprodynorphin, substance P, and preproenkephalin mRNA expression. When amphetamine-treated rats were pretreated with U69593, a kappa agonist (0.16 or 0.32 mg/kg s.c.), there was a significant decrease in behavioral activity. Quantitative in situ hybridization histochemistry revealed that 0.32 mg/kg U69593 significantly decreased amphetamine-induced mRNA expression of all three neuropeptides; however, only the induction of preproenkephalin mRNA was decreased by 0.16 mg/kg. These data suggest that stimulation of kappa receptors decreases acute amphetamine-induced behavior and mRNA expression of neuropeptides in the rat striatum.

Topics: Amphetamine; Analgesics; Animals; Behavior, Animal; Benzeneacetamides; Central Nervous System Stimulants; Corpus Striatum; Dynorphins; Enkephalins; Gene Expression; In Situ Hybridization; Male; Neuropeptides; Protein Precursors; Pyrrolidines; Rats; Rats, Sprague-Dawley; Receptors, Opioid, kappa; RNA, Messenger; Stimulation, Chemical; Substance P

The involvement of dynorphin on Delta-9-tetrahydrocannabinol (THC) and morphine responses has been investigated by using mice with a targeted inactivation of the prodynorphin (Pdyn) gene. Dynorphin-deficient mice show specific changes in the behavioral effects of THC, including a reduction of spinal THC analgesia and the absence of THC-induced conditioned place aversion. In contrast, acute and chronic opioid effects were normal. The lack of negative motivational effects of THC in the absence of dynorphin demonstrates that this endogenous opioid peptide mediates the dysphoric effects of marijuana.

Topics: Analgesia; Analgesics, Opioid; Animals; Avoidance Learning; Behavior, Animal; Brain Chemistry; Dronabinol; Dynorphins; Enkephalins; Female; Gene Targeting; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Mutant Strains; Morphine; Motivation; Motor Activity; Narcotics; Pain Measurement; Protein Precursors; Receptors, Opioid, kappa; Spatial Behavior; Substance-Related Disorders

In the present study, we investigated the selectivity and specificity associated with continuous intrastriatal treatment with antisense oligonucleotides. Rats were given intrastriatal infusions for 72 h with phosphodiester, and fully and endcap phosphorothioated oligonucleotide probes complementary to prodynorphin mRNA. Dynorphin (Dyn) peptide levels were measured by radioimmunoassay. The integrity of three other striatal transmitter systems, the neuropeptide Y (NPY)-ergic interneurons, the cholinergic interneurons and the dopaminergic afferent innervation, was assessed histochemically. The gross morphology of the striatum and the distribution of fluorescently labelled antisense probes were also investigated. Brains infused with phosphodiester probes had tissue Dyn levels not different from control. They also showed little or no change in staining for NPY, acetylcholinesterase (AChE) and tyrosine hydroxylase (TH) and essentially normal striatal gross morphology. In contrast, brains treated with fully phosphorothioated oligonucleotides showed significant decreases in striatal Dyn levels but also severe tissue damage accompanied by massive cell infiltration and decreases in immunoreactivities for the striatal neurochemical markers. Fluorescently labelled phosphorothioate probes were observed widely in the striatum and adjacent structures and, presumably retrogradely transported, in the dopamine cell bodies in the substantia nigra, also revealing the presence of abnormal cellular structures within the striatum. By comparison, endcap probes significantly reduced striatal Dyn levels and showed good tissue penetration without inducing major changes in tissue morphology or histochemistry of non-dynorphinergic systems, except for cell infiltration. The deleterious tissue effects of fully phosphorothioated oligonucleotides and the ineffectiveness of phosphodiester oligonucleotides in inhibiting protein synthesis suggest that, of the probes examined in this study, endcap oligonucleotides are the most useful for in vivo studies in the central nervous system.

Topics: Acetylcholinesterase; Animals; Brain; Corpus Striatum; Dynorphins; Enkephalins; Gene Expression Regulation; Infusions, Parenteral; Neurons; Neuropeptide Y; Oligodeoxyribonucleotides, Antisense; Protein Precursors; Rats; RNA, Messenger; Thionucleotides; Transcription, Genetic; Tyrosine 3-Monooxygenase

Effects of obesity on gene expression for opioid peptides and neuropeptide-Y (NPY) in the arcuate nucleus (ARC), and on opioid peptides and alpha-melanocyte stimulating hormone (alpha-MSH) in the paraventricular nucleus (PVN) were examined in obese Zucker rats (18 weeks old). Obese Zucker rats are insulin-resistant, diabetic and hyperleptinemic as indicated by high serum glucose, insulin and leptin levels. ARC proOpiomelanocortin (POMC) mRNA levels were significantly lower in the obese relative to lean Zucker rats and ARC proNeuropeptide Y (proNPY) mRNA levels were higher (P<0.05). There were no differences in proDynorphin and proEnkephalin mRNA levels in the ARC (0.05). Obese Zucker rats had lower alpha-MSH and dynorphin A(1-17) peptide levels in the paraventricular nucleus (PVN) (P<0.05), but did not have lower PVN beta-endorphin peptide levels (0.05). The decrease in POMC in the ARC and decrease in alpha-MSH in the PVN seen in the obese Zucker rat in the present study suggest that reduced activity of the melanocortin system in the ARC to PVN pathway may contribute to the related hyperphagia. Reduced activity of the melanocortin system in the ARC to PVN pathway may be due to a disturbance of leptin signaling coupling to POMC.

Topics: alpha-MSH; Animals; Arcuate Nucleus of Hypothalamus; beta-Endorphin; Blood Glucose; Dynorphins; Energy Metabolism; Enkephalins; Feeding Behavior; Gene Expression; Insulin; Leptin; Male; Melanocytes; Neuropeptide Y; Obesity; Paraventricular Hypothalamic Nucleus; Pro-Opiomelanocortin; Protein Precursors; Rats; Rats, Zucker; RNA, Messenger

mu-Opioid receptor-expressing neurons in the rat cerebral neocortex were characterized by an immunolabeling method with an antibody to a carboxyl terminal portion of the receptor. They were small, bipolar, vertically elongated, non-pyramidal neurons, and scattered mainly in layers II-IV. We examined chemical characteristics of mu-opioid receptor-expressing neocortical neurons by the double immunofluorescence method. Almost all neuronal cell bodies expressing mu-opioid receptor-like immunoreactivity showed immunoreactivity for GABA, suggesting that they were cortical inhibitory interneurons. mu-Opioid receptor-immunoreactive neurons were further studied by the double staining method with markers for the subgroups of cortical GABAergic neurons. Immunoreactivities for vasoactive intestinal polypeptide, corticotropin releasing factor, choline acetyltransferase, calretinin and cholecystokinin were found in 92, 79, 67, 35 and 35% of mu-opioid receptor-immunoreactive cortical neurons, respectively. In contrast, less than 10% of mu-opioid receptor-immunoreactive neurons showed immunoreactivity for parvalbumin, calbindin, somatostatin, neuropeptide Y or nitric oxide synthase. Moreover, mu-opioid receptor-immunoreactive neurons very frequently exhibited preproenkephalin immunoreactivity, but not preprodynorphin immunoreactivity. The present results indicate that mu-opioid receptor-expressing neurons belong to a distinct subgroup of neocortical GABAergic neurons, because vasoactive intestinal polypeptide, corticotropin releasing factor, choline acetyltransferase, calretinin and cholecystokinin have often been reported to coexist with one another in single neocortical neurons. Methionine-enkephalin, which is a major product of the preproenkephalin gene, is known to be one of the most potent endogenous ligands for mu-opioid receptor. Thus, the expression of mu-opioid receptor in preproenkephalin-producing neurons suggested that mu-opioid receptor serves as an autoreceptor for the subpopulation of GABAergic interneurons at a single-neuron or population level.

Topics: Animals; Calbindin 2; Calbindins; Cerebral Cortex; Cholecystokinin; Choline O-Acetyltransferase; Corticotropin-Releasing Hormone; Dynorphins; Enkephalins; gamma-Aminobutyric Acid; Interneurons; Neuropeptide Y; Nitric Oxide Synthase; Parvalbumins; Protein Precursors; Rats; Rats, Wistar; Receptors, Opioid, mu; S100 Calcium Binding Protein G; Somatostatin; Vasoactive Intestinal Peptide

Spinal cord injury in rats is known to cause anatomical, physiological and molecular changes within the spinal cord. These changes may account for behavioral syndromes that appear following spinal cord injury, syndromes believed to be related to the clinical condition of chronic pain. Intraspinal injection of quisqualic acid produces an excitotoxic injury with pathological characteristics similar to those associated with ischemic and traumatic spinal cord injury. In addition, recent studies have demonstrated changes in blood flow, neuronal excitability and gene expression in the brain following excitotoxic injury, indicating that behavioral changes may result from modification of neuronal substrates at supraspinal levels of the neuraxis. Because changes in spinal opioid peptide expression have been demonstrated in models of traumatic spinal cord injury and chronic pain, the present study investigated messenger RNA expression of the opioid peptides, preproenkephalin and preprodynorphin, at spinal and supraspinal levels following excitotoxic spinal cord injury. Male, Long-Evans rats were given three intraspinal injections of quisqualic acid (total 1.2 microl, 125mM). After one, three, five, seven or 10days, animals were killed and quantitative in situ hybridization performed on regions of the spinal cord surrounding the lesion site, as well as whole-brain sections through various levels of the thalamus. Preproenkephalin and preprodynorphin expression was increased in spinal cord areas adjacent to the site of quisqualic injection and in cortical regions associated with nociceptive function, preproenkephalin in the cingulate cortex and preprodynorphin in the parietal cortex, both ipsilaterally and contralaterally at various time-points following injury. These results further our knowledge of the secondary events that occur following spinal cord injury, specifically implicating supraspinal opioid systems in the CNS response to spinal cord injury.

Topics: Animals; Dynorphins; Enkephalins; Excitatory Amino Acid Agonists; Lumbar Vertebrae; Male; Opioid Peptides; Protein Precursors; Quisqualic Acid; Rats; Rats, Long-Evans; RNA, Messenger; Spinal Cord Injuries; Thoracic Vertebrae

Zinc finger-containing transcription factor GATA-4 and homeodomain Nkx-2.5 govern crucial developmental fates and have been found to promote cardiogenesis in embryonic cells exposed to the differentiating agent DMSO. Nevertheless, intracellular activators of these transcription factors are largely unknown. In this study, pluripotent P19 cells expressed the prodynorphin gene, an opioid gene encoding for the dynorphin family of opioid peptides. P19 cells were also able to synthesize and secrete dynorphin B, a biologically active end product of the prodynorphin gene. DMSO-primed GATA-4 and Nkx-2.5 gene expression was preceded by a marked increase in prodynorphin gene expression and dynorphin B synthesis and secretion. The DMSO effect occurred at the transcriptional level. In the absence of DMSO, dynorphin B triggered GATA-4 and Nkx-2.5 gene expression and led to the appearance of both alpha-myosin heavy chain and myosin light chain-2V transcripts, two markers of cardiac differentiation. Moreover, dynorphin B-exposed cells were positively stained in the presence of MF 20, a mouse monoclonal antibody raised against the alpha-myosin heavy chain. Opioid receptor antagonism and inhibition of opioid gene expression by a prodynorphin antisense phosphorothioate oligonucleotide blocked DMSO-induced cardiogenesis, suggesting an autocrine role of an opioid gene in developmental decisions.

Topics: Animals; Autocrine Communication; Cell Differentiation; Dimethyl Sulfoxide; DNA-Binding Proteins; Dynorphins; Endorphins; Enkephalins; Fetal Heart; Fetal Proteins; GATA4 Transcription Factor; Gene Expression Regulation, Developmental; Homeobox Protein Nkx-2.5; Homeodomain Proteins; Mice; Oligonucleotides, Antisense; Opioid Peptides; Protein Precursors; Recombinant Fusion Proteins; RNA, Messenger; Stem Cells; Thionucleotides; Transcription Factors; Transfection; Xenopus Proteins; Zinc Fingers

The aim of this study was to determine at which developmental stage and how dopamine regulates the expression of striatal dopamine receptor and neuropeptide mRNAs. For this, we studied the expression of these mRNAs, in relation to dopamine innervation, in normal mice from gestational day 13 (G13) to adult. Particularly, we investigated the adaptive changes in the expression of these markers in mice lacking the dopamine transporter during development. We detected tyrosine hydroxylase, by immunohistochemistry, in the ventral mesencephalon and the striatal anlage in both genotypes at G13, whereas the dopamine transporter appeared in the striatum of normal mice at G14. By in situ hybridization, we detected striatal dopamine D1, D2, D3 receptor, and substance P mRNAs at G13, preproenkephalin A mRNA at G14 and dynorphin mRNA at G17 in normal mice. Although the time of initial detection and the distribution were not affected in mutant mice, quantitative changes were observed. Indeed, D1 and D2 receptor as well as preproenkephalin A mRNA levels were decreased from G14 on, and dynorphin mRNA level was increased from G17 on. In contrast, substance P mRNA level was unaffected. Our data demonstrate that the influence of dopamine on striatal neurons occurs early during the development of the mesostriatal system as quantitative changes appeared in mutant mice as soon as G14. These findings bring new insights to the critical influence of dopamine on the expression of striatal dopamine receptor and neuropeptide mRNAs during development, and suggest that mesostriatal dopamine transmission functions from G14 on.

Topics: Animals; Carrier Proteins; Corpus Striatum; Dopamine; Dopamine Plasma Membrane Transport Proteins; Dynorphins; Enkephalins; Female; Gene Expression Regulation, Developmental; Gestational Age; In Situ Hybridization; Male; Membrane Glycoproteins; Membrane Transport Proteins; Mesencephalon; Mice; Mice, Knockout; Nerve Tissue Proteins; Neurons; Pregnancy; Protein Precursors; Receptors, Dopamine D1; Receptors, Dopamine D2; Receptors, Dopamine D3; RNA, Messenger; Substance P; Tyrosine 3-Monooxygenase

In general, administration of methamphetamine and cocaine alters preprodynorphin and preproenkephalin mRNA levels in striatum. However, no study has directly compared the effects of these stimulants on opioid peptides in striatum. This study used in situ hybridization to compare directly the effects of cocaine and methamphetamine on preprodynorphin and preproenkephalin mRNAs in distinct striatal regions. Male Sprague-Dawley rats received a single administration of 15 mg/kg methamphetamine or 30 mg/kg cocaine and were killed 30 min or 3 h later. Methamphetamine and cocaine differentially affected preprodynorphin mRNA in striatum after 3 h. Densitometric analysis of film autoradiograms revealed that cocaine, but not methamphetamine, significantly increased preprodynorphin. This effect was seen throughout rostral striatum and dorsally in caudal striatum. However, specific analysis of "patches" in which preprodynorphin expression is high revealed a significantly greater effect of methamphetamine versus cocaine. In contrast, both cocaine and methamphetamine had similar effects on preproenkephalin mRNA, decreasing levels after 30 min in rostral striatum and in the core of nucleus accumbens. These data suggest that methamphetamine and cocaine have distinct postsynaptic consequences on striatal neurons.

Topics: Animals; Autoradiography; Cocaine; Corpus Striatum; Dynorphins; Enkephalins; In Situ Hybridization; Male; Methamphetamine; Nucleus Accumbens; Opioid Peptides; Protein Precursors; Rats; Rats, Sprague-Dawley; RNA, Messenger

There is growing evidence suggesting that neurotrophins have modulating effects on the pain signaling system at spinal levels. In order to determine whether neurotransmitter expression is modulated in response to the elevation of neurotrophins, the changes in c-fos, neuropeptide and glutamic acid decarboxylase (GAD) mRNAs expression was evaluated after BDNF or NT-3 was applied to cultured spinal neurons. Reverse transcription polymerase chain reaction analysis revealed that BDNF induced a significant increase in the expression of preprodynorphin (pDYN), preproenkephalin (pENK), neuropeptide Y (NPY) and GAD mRNAs. In contrast, the pENK, not the pDYN, NPY and GAD, mRNA levels increased after the treatment of NT-3. Both BDNF and NT-3 produced a rapid increase in c-fos mRNA. These results suggest that BDNF and NT-3 have differential neuronal effects on the synthesis of spinal cord neurotransmitters that are involved in the modulation of nociceptive information.

Topics: Animals; Brain-Derived Neurotrophic Factor; Cells, Cultured; Dynorphins; Enkephalins; Gene Expression Regulation; Genes, fos; Glutamate Decarboxylase; Neurons; Neuropeptide Y; Neuropeptides; Neurotrophin 3; Protein Precursors; Proto-Oncogene Proteins c-fos; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Spinal Cord

Chronic administration of morphine or cocaine affects opioid gene expression. To better understand the possible existence of common neuronal pathways shared by different classes of drugs of abuse, we studied the effects of methamphetamine on the gene expression of the opioid precursor prodynorphin and on the levels of peptide dynorphin A in the rat brain. Acute (6 mg/kg, intraperitoneally, i.p.) and chronic (6 mg/kg, i.p. for 15 days) methamphetamine markedly raised prodynorphin mRNA levels in the hypothalamus, whereas no effect was observed in the hippocampus. Dynorphin A levels increased after chronic treatment in the hypothalamus and in the striatum, whereas no significant changes were detected after acute treatment. These results indicate that methamphetamine affects prodynorphin gene expression in the hypothalamus, which may be an important site (also for its relevant neuroendocrine correlates) for opioidergic mechanisms activated by addictive drugs.

Topics: Animals; Brain; Dynorphins; Enkephalins; Gene Expression Regulation; Hippocampus; Hypothalamus; Male; Methamphetamine; Protein Precursors; Rats; Rats, Sprague-Dawley; Substance-Related Disorders; Time Factors; Visual Cortex

Limited proteolysis of the dynorphin precursor (prodynorphin) at dibasic and monobasic processing sites results in the generation of bioactive dynorphins. In the brain and neurointermediate lobe of the pituitary, prodynorphin is processed to produce alpha and beta neo endorphins, dynorphins (Dyn) A-17 and Dyn A-8, Dyn B-13, and leucine-enkephalin. The formation of Dyn A-8 from Dyn A-17 requires a monobasic cleavage between Ile and Arg. We have identified an enzymatic activity capable of processing at this monobasic site in the rat brain and neurointermediate lobe of the bovine pituitary; this enzyme is designated "dynorphin A-17 processing enzyme." In the rat brain and neurointermediate lobe, a majority of the Dyn A processing enzyme activity is membrane-associated and can be released by treatment with 1% Triton X-100. This enzyme has been purified to apparent homogeneity from the membrane extract of the neurointermediate lobe using preparative iso-electrofocussing in a granulated gel pH 3.5 to 10, FPLC using anion exchange chromatography, and non-denaturing electrophoresis. The Dyn A processing enzyme exhibits a pI of about 5.8 and a molecular mass of about 65 kDa under reducing conditions. The Dyn A processing enzyme is a metalloprotease and has a neutral pH optimum. It exhibits substantial sensitivity to metal chelating agents and thiol agents suggesting that this enzyme is a thiol-sensitive metalloprotease. Specific inhibitors of other metallopeptidases such as enkephalinase [EC 3.4.24.11], the enkephalin generating neutral endopeptidase [EC 3.4.24.15], or NRD convertase do not inhibit the Dyn A processing enzyme activity. In contrast, specific inhibitors of angiotensin converting enzyme inhibit the activity. The purified enzyme is able to process a number of neuropeptides at both monobasic and dibasic sites. These characteristics are consistent with a role for the Dyn A processing enzyme in the processing of Dyn A-17 and other neuropeptides in the brain.

Topics: Amino Acid Sequence; Animals; Cattle; Dynorphins; Enkephalins; Metalloendopeptidases; Molecular Sequence Data; Neprilysin; Organ Specificity; Protein Precursors; Rats; Rats, Sprague-Dawley

We determined the effects of morphine on mRNA levels for the opioid ligands preprodynorphin (PPD) and preproenkephalin (PPE) and the kappa opioid receptor (KOR). Rats received six injections of morphine (6.25 mg/kg/injection) every 2 h, and were sacrificed 30 min later. mRNA levels were measured in brain tissue after removal of the cortex, cerebellum and brainstem. There were increases in PPD and KOR mRNA levels (P<0.05 and P<0.005, respectively), with no alteration of PPE. These alterations in the kappa/dynorphin system may counter morphine-induced effects on the brain.

Topics: Animals; Brain Chemistry; Dynorphins; Enkephalins; Gene Expression; Male; Morphine; Narcotics; Protein Precursors; Rats; Rats, Wistar; Receptors, Opioid, kappa; RNA, Messenger

Preprodynorphin (PPD) and preproenkephalin (PPE) gene expression in a rat model of orofacial inflammation were examined in order to further characterize the neurochemical mechanisms underlying orofacial inflammation and hyperalgesia. Deep and cutaneous orofacial inflammation was produced by a unilateral injection of complete Freund's adjuvant (CFA) into the rat temporomandibular joint (TMJ) or perioral skin (PO), respectively. RNA blot analysis of the tissues including the spinal trigeminal complex revealed that the PPD mRNA level ipsilateral to TMJ inflammation was increased by 56.5+/-14.7% (n=4) when compared to the Naive group, and was significantly greater than the contralateral PPD mRNA level (p<0.05). The distribution of neurons that exhibited PPD mRNA after inflammation was localized by in situ hybridization (naive approximately 0). In TMJ-inflamed rats (n=6) PPD mRNA-positive neurons were found ipsilaterally in the medial portion of laminae I-II of the upper cervical dorsal horn (4.5+/-0.3), the dorsal portion of the subnucleus caudalis and caudal subnucleus interpolaris (5.2+/-0.3), and the paratrigeminal nucleus (6.4+/-1.2). A very localized induction of PPD mRNA was also identified in a group of neurons in the intermediate portion of the subnucleus caudalis (2.4+/-0.4) in PO-inflamed rats (n=6). The distribution of these PPD mRNA-positive neurons was somatotopically relevant to the site of injury. There were no significant changes in PPE mRNA expression in both TMJ- and PO-inflamed rats. These results indicate that TMJ inflammation resulted in a more intense and widespread increase in PPD mRNA expression when compared to PO inflammation. These changes may contribute to persistent central hyperexcitability and pain associated with temporomandibular disorders.

Topics: Animals; Dynorphins; Enkephalins; Freund's Adjuvant; Gene Expression; Hyperalgesia; In Situ Hybridization; Male; Neurogenic Inflammation; Neurons, Afferent; Protein Precursors; Rats; Rats, Sprague-Dawley; RNA, Messenger; Skin; Temporomandibular Joint; Temporomandibular Joint Disorders; Trigeminal Nuclei

Neostriatal GABAergic neurons projecting to the globus pallidus synthesize the opioid peptide enkephalin, while those innervating the substantia nigra pars reticulata and the entopeduncular nucleus synthesize dynorphin. The differential control exerted by dopamine on the activity of these two efferent projections concerns also the biosynthesis of these opioid peptides. Using in situ hybridization histochemistry, we investigated the role of opioid co-transmission in the regulation of neostriatal and pallidal activity. The expression of the messenger RNAs encoding glutamate decarboxylase-the biosynthetic enzyme of GABA-and the precursor peptides of enkephalin (preproenkephalin) and dynorphin (preprodynorphin) were measured in rats after a sustained blockade of opioid receptors by naloxone (s.c. implanted osmotic minipump, eight days, 3 mg/kg per h), and/or a subchronic blockade of D2 dopamine receptors by haloperidol (one week, 1.25 mg/kg s.c. twice a day). The density of mu opioid receptors in the neostriatum and globus pallidus was determined by autoradiography. Naloxone treatment resulted in a strong up-regulation of neostriatal and pallidal mu opioid receptors that was not affected by the concurrent administration of haloperidol. Haloperidol alone produced a moderate down-regulation of neostriatal and pallidal micro opioid receptors. Haloperidol strongly stimulated the expression of neostriatal preproenkephalin and preprodynorphin messenger RNAs. This effect was partially attenuated by naloxone, which alone produced moderate increases in preproenkephalin and preprodynorphin messenger RNA levels. In the neostriatum, naloxone did not affect either basal or haloperidol-stimulated glutamate decarboxylase messenger RNA expression. A strong reduction of glutamate decarboxylase messenger RNA expression was detected over pallidal neurons following either naloxone or haloperidol treatment, but concurrent administration of the two antagonists did not result in a further decrease. The amplitude of the variations of mu opioid receptor density and of preproenkephalin and preprodynorphin messenger RNA levels suggests that the regulation of neostriatal and pallidal micro opioid receptors is more susceptible to a direct opioid antagonism, while the biosynthesis of opioid peptides in the neostriatum is more dependent on the dopaminergic transmission. The down-regulation of mu opioid receptors following haloperidol represents probably an adaptive change to increased enke

Topics: Animals; Dopamine; Dopamine Antagonists; Dynorphins; Enkephalins; Globus Pallidus; Glutamate Decarboxylase; Haloperidol; Male; Naloxone; Narcotic Antagonists; Neostriatum; Neurons; Opioid Peptides; Protein Precursors; Rats; Rats, Wistar; Receptors, Opioid, mu; RNA, Messenger

Sixty days following neonatal dopamine depletion (>98%) with 6-hydroxydopamine, preprotachykinin and preprodynorphin mRNA levels were significantly reduced (67 and 78% of vehicle controls, respectively) in the anterior striatum as determined by in situ hybridization while preproenkephalin mRNA expression was elevated (133% of vehicle controls). Suppression of the serotonin hyperinnervation phenomenon in the dopamine-depleted rat with 5,7-dihydroxytryptamine yielded no significant alterations in reduced striatal preprotachykinin (66%) or preprodynorphin (64%) mRNA levels, while preproenkephalin mRNA expression remained significantly elevated (140%). These data suggest that striatal serotonin hyperinnervation does not contribute to the development of dysregulated striatal neuropeptide transmission in either direct or indirect striatal output pathways following neonatal dopamine depletion.

Topics: 5,7-Dihydroxytryptamine; Animals; Animals, Newborn; Chromatography, High Pressure Liquid; Corpus Striatum; Denervation; Dopamine; Dynorphins; Enkephalins; Gene Expression Regulation; In Situ Hybridization; Neuropeptides; Nucleic Acid Hybridization; Oxidopamine; Protein Precursors; Rats; Rats, Sprague-Dawley; RNA, Messenger; Serotonin; Tachykinins

Topics: Animals; Appetite; Basal Ganglia; Carrier Proteins; Cocaine; Desoxycorticosterone; Dopamine Plasma Membrane Transport Proteins; Dynorphins; Enkephalins; Gene Expression Regulation; Male; Membrane Glycoproteins; Membrane Transport Proteins; Nerve Tissue Proteins; Nucleus Accumbens; Protein Precursors; Rats; Rats, Sprague-Dawley; RNA, Messenger; Sodium, Dietary; Transcription, Genetic; Tyrosine 3-Monooxygenase

Topics: Animals; Central Nervous System Stimulants; Corpus Striatum; Dopamine Agonists; Dopamine Antagonists; Dynorphins; Enkephalins; Excitatory Amino Acid Antagonists; Gene Expression Regulation; Genes, Immediate-Early; Glutamic Acid; Neurons; Neuropeptides; Protein Precursors; Receptors, Dopamine D1; Receptors, Dopamine D2; Substance-Related Disorders; Transcription, Genetic

Opioid peptides are known to play a role in the function and growth of the mammalian heart. Although some information about gene expression of opioids in the heart is available, there is no data on the cellular location of opioid gene expression during development or in the adult. Using in situ hybridization and rat heart ranging from embryonic day 14 (E14) to adulthood, we have evaluated the distribution of gene expression for proenkephalin, proopiomelanocortin, and prodynorphin. With respect to preproenkephalin mRNA (PPE mRNA), message in the ventricle was abundant from E14 (the first time point examined) until shortly after birth, with a marked reduction noted on postnatal days 5, 10, and 21. Adults displayed considerable message, though less than in preparations of embryonic and neonatal heart. PPE mRNA was detected in epicardial, myocardial, and endocardial cells, as well as the walls of blood vessels, capillaries, and fibroblasts. Preproopiomelanocortin (POMC) mRNA was only found in adults, and was localized to the myocardium. Message for preprodynorphin could not be observed in the ventricles of developing or adult rats. These results are the first to define the temporal and spatial ontogeny of opioid gene expression with regard to the emergence of cardiac architecture. The data suggest that gene expression for proenkephalin is especially prevalent in embryonic and neonatal rats and may be related to the modulatory activity of the opioid growth factor, [Met5]-enkephalin, on cell proliferation and differentiation. The role of PPE and POMC mRNA in adult rat heart requires elucidation.

Topics: Animals; Animals, Newborn; Dynorphins; Enkephalins; Female; Gene Expression Regulation, Developmental; Heart; In Situ Hybridization; Male; Myocardium; Opioid Peptides; Pericardium; Pregnancy; Pro-Opiomelanocortin; Protein Precursors; Rats; Rats, Sprague-Dawley; RNA, Messenger

It is established that dopamine (DA) controls the expression of preprodynorphin (PPDYN), preprotachykinin A (PPT-A) and preproenkephalin (PPE) mRNAs in striatal structures. Since cocaine, nicotine and ethanol enhance extracellular DA concentration, we have examined whether their repeated administration produced common changes in the expression of these mRNAs. Quantitative in situ hybridization histochemistry was performed in rats 2 h after a final challenge subsequent to repeated subcutaneous injections (3 X a day) of cocaine (12.5 mg/kg), nicotine (0.4 mg/kg) for 14 days and ethanol (160 mg/kg) for 7 days. In the dorsal striatum, cocaine produced simultaneous PPDYN and PPT-A mRNA increases without PPE mRNA change whereas nicotine and ethanol produced no modification. After cocaine, PPDYN mRNA was preferentially increased in striatal patch compartment. In the nucleus accumbens, the effects were more complex. In cocaine-treated rats, we measured concomitant increases of PPDYN and PPE mRNA in the rostral pole, an isolated induction of PPT-A mRNA signals in the core without any change in the two shell subregions: the cone and the ventral shell. In contrast, after nicotine and ethanol, the ventral shell was the only accumbal subregion which showed a neuropeptide mRNA alteration, nicotine leading to decreased PPDYN mRNA and ethanol to increased PPT-A mRNA contents. The neuropeptide regulation after chronic treatment with these psychostimulant drugs does not strictly conform to a general DA control scheme in the dorsal and the ventral striatum. The cocaine effects can be clearly distinguished from those of nicotine and ethanol in terms of neuropeptide regulation and striatal subregions affected.

Topics: Animals; Body Weight; Cocaine; Corpus Striatum; Drug Administration Schedule; Dynorphins; Enkephalins; Ethanol; In Situ Hybridization; Injections, Subcutaneous; Male; Nicotine; Nucleus Accumbens; Protein Precursors; Rats; Rats, Sprague-Dawley; RNA, Messenger; Tachykinins

Metabotropic glutamate receptors (mGluR) are coupled to multiple intracellular second messenger systems through G-proteins and densely expressed by medium spiny projection neurons in the rat striatum. Unlike ionotropic glutamate receptors which mediate rapid synaptic transmission, mGluRs are important for relatively long-lasting modulation of neuronal metabotropic activity, possibly including gene expression, in response to cellular stimulation. In this study, the effects of acute injection of the selective mGluR agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) on behavior and striatal neuropeptide mRNA expression were evaluated in chronically-cannulated rats. Unilateral injection of ACPD into the dorsal striatum at doses of 0.8, 4, 20, 100, 500 and 1000 nmol had no significant effect on spontaneous behavioral activity. However, intrastriatal ACPD (0.8, 4, 20 and 100 nmol) dose-dependently elevated preprodynorphin (PPD), substance P (SP) and preproenkephalin (PPE) mRNA expression in the dorsal striatum as revealed by quantitative in situ hybridization. PPD/SP mRNAs showed a biphasic response to a single injection of ACPD as the expression of these two mRNAs was increased at 3 and 6 h, decreased at 11 h, and returned to normal 24 h after ACPD administration. PPE induction in the dorsal striatum was significantly elevated as early as 2 h and remained even 24 h after ACPD was injected. In addition, the PPD and PPE mRNA induction by ACPD was blocked by intrastriatal pretreatment with the selective mGluR antagonist, (+)-alpha-methyl-4-carboxyphenyl-glycine. These data demonstrate a facilitatory regulation of constitutive expression of striatonigral PPD/SP, and striatopallidal PPE, mRNAs by local mGluR-mediated glutamatergic transmission.

Topics: Animals; Corpus Striatum; Cycloleucine; Dynorphins; Enkephalins; Globus Pallidus; In Situ Hybridization; Male; Microinjections; Neurons; Neuropeptides; Protein Precursors; Rats; Rats, Wistar; Receptors, Metabotropic Glutamate; RNA, Messenger; Substance P; Substantia Nigra

To investigate the effects of type I (mineralocorticoid) and type II (glucocorticoid) receptor activation on striatal neuropeptide [preproenkephalin (PPE), preprotachykinin (PPT), and preprodynorphin (DYN)] mRNA and midbrain cholecystokinin (CCK) mRNA as well as striatal tyrosine hydroxylase radioimmunoreactivity (TH-RIC) levels, we administered either replacement levels of corticosterone (CORT; 0.5 mg/kg/day, s.c.) or pharmacological levels of deoxycorticosterone acetate (DOCA; a mineralocorticoid steroid with ability to bind to type I and type II receptors; 5 mg/kg, s.c.) to adrenalectomized adult male rats. After 1 week of recovery from adrenalectomy surgery, animals were injected daily with sesame oil or CORT for 1, 3, or 7 days or DOCA for 3 or 7 days and killed 16 h after the last injection. Adrenalectomy resulted in a decrease in all three striatal neuropeptide mRNA levels, compared with sham-operated rats. CORT replacement resulted in recovered PPE and PPT mRNA levels after 1 day and elevated PPE mRNA levels over those in sham-operated controls after 3 days. In contrast, DYN mRNA levels showed recovery after 7 days of CORT replacement. Results after DOCA treatment largely paralleled those after CORT replacement. There were no significant treatment effects on indirect markers of midbrain dopaminergic activity, i.e., CCK mRNA and TH-RIC. From these results we conclude that compared with striatal tachykinin and dynorphinergic neurons, enkephalinergic cells show greater sensitivity, whereas the dopaminergic system, including mesencephalic CCK, demonstrates an insensitivity to physiological CORT and to pharmacological DOCA treatment.

Topics: Adrenalectomy; Animals; Anti-Inflammatory Agents; Basal Ganglia; Cholecystokinin; Corticosterone; Desoxycorticosterone; Dynorphins; Enkephalins; Gene Expression; Male; Neuropeptides; Protein Precursors; Radioimmunoassay; Rats; Rats, Sprague-Dawley; RNA, Messenger; Tachykinins; Tyrosine 3-Monooxygenase

Preprodynorphin and preproenkephalin mRNA expression was examined in the CNS of two rat strains, the alcohol-preferring Fawn-Hooded (FH) and the alcohol-non-preferring Wistar-Kyoto (WKY), using in situ hybridisation histochemistry. Relative to the WKY, the FH showed significantly lower levels of preproenkephalin mRNA in the striatum and nucleus accumbens (-24% and -17% respectively), but a higher level of preprodynorphin mRNA in the hippocampus (+33%). The depressed level of preproenkephalin mRNA in the nucleus accumbens may be implicated in alcohol-seeking behaviour.

Topics: Alcohol Drinking; Animals; Dynorphins; Enkephalins; Gene Expression; Histocytochemistry; In Situ Hybridization; Limbic System; Protein Precursors; Rats; Rats, Inbred Strains; Rats, Inbred WKY; RNA, Messenger; Tissue Distribution

To investigate adaptive responses of pancreatic beta-cells to hyperglycemia, genes induced by glucose stimulation were identified by subtraction cloning. Among 53 clones representing differentially expressed genes, 20 encoded the endogenous opioid precursor, prodynorphin. The amino acid sequence of murine prodynorphin is identical to the rat protein in sequences comprising the opioid peptides and 86% identical in the remainder of the molecule. Stimulation of MIN6 cells increased prodynorphin RNA levels to more than 20-fold in proportion to physiological glucose concentrations. Similar induction levels were observed in murine betaTC3 and rat Rinm5F beta-cell lines. Prodynorphin RNA expression increased within 1 h of glucose stimulation, achieved maximal levels by 4 h, and remained elevated for at least 24 h. By using RIA, MIN6 cells were shown to contain and secrete increased amounts of dynorphin-A following glucose stimulation. Treatment of MIN6 cells with KCl, forskolin, or isobutyl-methyl-xanthine strongly induced prodynorphin RNA expression, suggesting that induction may be related to secretion-coupled signaling pathways. The induction of prodynorphin in several beta-cell lines is consistent with previous demonstrations of beta-cell synthesis of other endogenous opioids, including beta-endorphin, and suggests that opioids may have a potentially significant role in regulating beta-cell secretion.

Topics: Amino Acid Sequence; Animals; Base Sequence; Cell Line; Dynorphins; Enkephalins; Glucose; Islets of Langerhans; Male; Molecular Sequence Data; Protein Precursors; Rats; Rats, Inbred Lew; RNA, Messenger

Protracted changes in basal "steady-state" opioid peptide gene expression in the brain may represent adaptations underlying the behavioral effects of drugs of abuse, observed long after drug exposure. Here, we have studied the long-term effects of two distinct regimens of morphine administration ("intermittent" vs "chronic" morphine treatment) on behavioral sensitization and "steady-state" striatal preprodynorphin and preproenkephalin gene expression in rats. Opioid peptide gene expression was investigated using in situ hybridization at three rostrocaudal levels (rostral, intermediate and caudal) of the caudate-putamen and the nucleus accumbens. Behavioral studies showed that the intermittent morphine treatment resulted in a significantly greater enhancement of morphine-induced locomotion than the chronic morphine treatment three weeks after cessation of opiate exposure. The intermittent morphine treatment resulted in an initial decrease of preprodynorphin gene expression of about 5-10% in the caudate-putamen and the nucleus accumbens at the rostral and intermediate levels one day after the last morphine administration. In contrast, a protracted increase of preprodynorphin gene expression of about 20% throughout the caudate-putamen and of about 6% in intermediate sections of the nucleus accumbens was observed 21 days after cessation of intermittent morphine treatment. Although the chronic morphine treatment induced a decrease of preprodynorphin messenger RNA levels one day after the last administration, no significant changes were observed three weeks after cessation of chronic morphine treatment. No long-term changes were observed in preproenkephalin gene expression after either morphine treatment. Since the intermittent morphine administration induced long-term behavioral sensitization much more effectively than the chronic morphine treatment, we tentatively suggest that the protracted increase of preprodynorphin gene expression may play a facilitative role in the long-term character of opiate-induced behavioral sensitization.

Topics: Animals; Autoradiography; Behavior, Animal; Corpus Striatum; Dynorphins; Enkephalins; Gene Expression; Male; Morphine; Motor Activity; Narcotics; Protein Precursors; Rats; Rats, Wistar; Time Factors; Tissue Distribution

Cocaine exposure in utero is known to cause a variety of behavioral and motor deficits that may be attributable to alterations in the dopamine neurocircuitry. To ascertain cocaine effects in the fetus, we developed a nonhuman primate model in which pregnant monkeys were administered cocaine from day 20 through day 60 or 70 of gestation. Fetuses from these pregnancies develop a repertoire of neural deficiencies, including decreased mRNA expression of tyrosine hydroxylase in the midbrain and increased mRNA expression of dopamine receptor subtypes in the rostral forebrain. Presently, we studied the effects of maternal cocaine treatment on the mRNA expression of the endogenous opioids preprodynorphin (PPD) and preproenkephalin (PPE) in fetal monkey brains. Fetuses exposed to saline (0.9%) or cocaine (3 mg/kg) were delivered by Caesarean section, the fetal brains were dissected, and tissue RNA was extracted and quantified using ribonuclease protection assay analysis. The opioid peptides PPD and PPE were expressed in the fetal monkey brain by day 60, and even higher levels were found in day 70 fetuses. Maternal exposure to cocaine increased gene expression of PPD and PPE in the fetus at both day 60 and day 70 of gestation. Dynorphin mRNA levels were significantly elevated in the striatum, whereas enkephalin mRNA was elevated in both the frontal cortex and the striatal area of fetuses whose mothers received cocaine. Changes in the expression of these opioid peptides in presumed dopamine target neurons, which mediate motivation and reward, as well as motor control, provide further evidence for profound consequences of in utero cocaine exposure on the developing dopamine neurocircuitry.

Topics: Animals; Brain; Cocaine; Dynorphins; Enkephalins; Female; Gene Expression Regulation; Macaca mulatta; Narcotics; Pregnancy; Protein Precursors; RNA, Messenger

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

Systemic administration of the muscarinic receptor antagonist, scopolamine, augments, whereas the muscarinic receptor agonist, oxotremorine, attenuates behaviors (locomotion and stereotypies) and preprodynorphin (PPD) and substance P (SP) gene expression in striatonigral neurons induced by the indirect dopamine receptor agonist, amphetamine (AMPH). In contrast, systemic scopolamine blocks, whereas oxotremorine augments, AMPH-stimulated preproenkephalin (PPE) gene expression in striatopallidal neurons. This study investigated the site of action of these effects by administering scopolamine and oxotremorine directly into the striatum and assessing the expression of neuropeptide mRNAs with quantitative in situ hybridization. Unilateral injection of scopolamine into the dorsal striatum augmented, and oxotremorine attenuated, AMPH (2.5 mg/kg, i.p.)-stimulated behaviors. Intrastriatal scopolamine at a concentration of 62 mM, but not 6.2 mM, increased basal levels of PPD and SP mRNAs in the dorsal striatum. In addition, both 6.2 and 62 mM scopolamine significantly augmented AMPH-stimulated PPD and SP mRNA levels. Intrastriatal infusion of 1.6 or 8.1 mM oxotremorine did not alter basal levels of striatal PPD and SP mRNAs. However, both concentrations of oxotremorine completely blocked AMPH-stimulated SP mRNA and oxotremorine at 8.1 mM blocked AMPH-stimulated PPD mRNA. In contrast, PPE induction by AMPH was blocked by 62, but not 6.2, mM scopolamine. Both concentrations of oxotremorine tended to augment basal and AMPH-stimulated PPE mRNA in the dorsal striatum but the trend was not significant. These data demonstrate an inhibition of striatonigral, and facilitation of striatopallidal, gene expression through activation of local striatal muscarinic receptors, which is consistent with the changes seen after systemic administration of muscarinic agents. Therefore, muscarinic cholinergic regulation of basal and stimulated expression of neuropeptide mRNA is processed within the striatum.

Topics: Amphetamine; Animals; Behavior, Animal; Corpus Striatum; Dynorphins; Enkephalins; Injections; Male; Muscarinic Agonists; Muscarinic Antagonists; Neuropeptides; Oxotremorine; Protein Precursors; Rats; Rats, Wistar; Reference Values; RNA, Messenger; Scopolamine; Substance P

We previously identified a DNA binding element termed the upstream regulatory element (URE) that contains the consensus initiator sequence (Inr) in the upstream promoter of the rat prodynorphin gene. The URE displays specific binding to the upstream regulatory element binding protein (UREB1), a novel transcription regulator. Here, we report that the URE functions as a suppressor element. A series of chloramphenicol acetyltransferase reporters (pCAT) were constructed by subcloning either wild-type or mutated URE sequences into a pCAT reporter plasmid 5' of bases -135 to +58 of the prodynorphin gene. The basal -135 to +58 dynorphin promoter (pCAT 0.2) has robust transcriptional activity in Chinese hamster ovary (CHO) cells but not in rat pheochromocytoma PC12 cells. This robust transcriptional activity was completely inhibited in the presence of wildtype URE, whereas the mutations of the URE had no effect. Gel mobility shift assays showed that the complex formed by the URE and nuclear protein extracts can be competed by addition of wild-type URE oligonucleotide but not by specific mutations of the URE, defining particular bases required for protein interaction with the URE. The identical URE sequence is also found upstream in the promoter of human macrophage inflammatory protein 1 beta (hMIP 1 beta). The suppressive activity of the rat dynorphin URE can be replaced by the hMIP 1 beta URE. These data suggest that the URE may serve as a suppressor element in the regulation of dynorphin and hMIP 1 beta gene transcription.

Topics: Animals; CHO Cells; Cricetinae; DNA-Binding Proteins; Dynorphins; Enkephalins; Mutation; Oligodeoxyribonucleotides; Phosphoproteins; Promoter Regions, Genetic; Protein Precursors; Rats; Regulatory Sequences, Nucleic Acid; Suppression, Genetic; Transcriptional Activation; Transfection; Tumor Suppressor Proteins; Ubiquitin-Protein Ligases

Complex and contradictory data have been reported regarding the changes in spinal opioidergic systems associated with chronic inflammatory pain in the rat. In an attempt to solve these discrepancies, the in vivo release of met-enkephalin and dynorphin and the expression of the corresponding propeptide genes were investigated at the spinal level in arthritic rats and paired controls. A dramatic increase in the concentration of prodynorphin mRNA (+300-550%) and a less pronounced elevation of that of dynorphin-like material (+40-50%) were found in the dorsal part of cervical and lumbar segments of the spinal cord in rats rendered arthritic by an intradermal injection of Freund's adjuvant four weeks prior to these measurements. In addition, the spinal release of dynorphin-like material (assessed through an intrathecal perfusion procedure in halothane-anaesthetized animals) was approximately twice as high in arthritic rats as in controls. In spite of significant elevations in the levels of both met-enkephalin (+30-70%) and proenkephalin A mRNA (+40-50%) in the dorsal part of cervical and lumbar segments, the spinal release of met-enkephalin-like material was decreased (-50%) in arthritic rats as compared to paired controls. Proenkephalin A mRNA (but not prodynorphin mRNA) could be measured in dorsal root ganglia, and its levels were dramatically reduced in ganglia at the lumbar segments in arthritic rats. Such parallel reductions in the spinal release of met-enkephalin-like material and the levels of proenkephalin A mRNA in dorsal root ganglia of arthritic rats support the idea that the activity of primary afferent enkephalinergic fibres decreases markedly during chronic inflammatory pain.

Topics: Animals; Arthritis, Experimental; Blotting, Northern; DNA, Complementary; Dynorphins; Enkephalins; Ganglia, Spinal; In Situ Hybridization; Male; Neurons; Protein Precursors; Radioimmunoassay; Rats; Rats, Sprague-Dawley; RNA, Messenger; Spinal Cord

The neurosteroid tetrahydrodeoxycorticosterone (THDOC) interacts with gamma-aminobutyric acid (GABA)/ benzodiazepine (BZ) receptors. To test the hypothesis that THDOC works partially through mechanisms associated with GABAA/BZ receptor function, deoxycorticosterone acetate (DOCA) and the benzodiazepine, diazepam (DZ), were administered short- (1 day) and long-term (11 days). Levels of mRNA for dynorphin, preprotachykinin and preproenkephalin in the striatum of adult male Sprague-Dawley rats were measured by in situ hybridization. Acute DOCA and DZ treatment produced parallel neuropeptide mRNA profiles, whereas chronic DOCA and DZ treatment yielded different patterns of neuropeptide gene expression. Chronic DZ treatment resulted in no significant increase in salt intake whereas chronic DOCA activated salt appetite. We suggest that acute DZ and DOCA interact with GABAA/BZ receptors; however, the results of chronic treatment suggest that DZ and DOCA operate through dissimilar mechanisms.

Topics: Analysis of Variance; Animals; Appetite; Corpus Striatum; Desoxycorticosterone; Diazepam; Dynorphins; Enkephalins; Male; Neurons; Protein Precursors; Rats; Rats, Sprague-Dawley; RNA, Messenger; Sodium, Dietary; Tachykinins; Transcription, Genetic

The levels of dynorphin A-like immunoreactivity (Dyn A-LI) and dynorphin B-like immunoreactivity (Dyn B-LI) were determined in various regions of brain, spinal cord and pituitary gland in spontaneously hypertensive rats (SHRs) as compared with the normotensive Wistar-Kyoto rats (WKYs). SHRs had significantly lower levels of Dyn A-LI and Dyn B-LI in the neurointermediate pituitary lobe and in the hippocampus. Conversely, the levels of Dyn A-LI and Dyn B-LI were higher in the hypothalamus, striatum and periaqueductal gray of the SHRs.

Topics: Animals; Biomarkers; Brain; Dynorphins; Endorphins; Enkephalins; Hypertension; Immunohistochemistry; Lumbosacral Region; Male; Pituitary Gland; Protein Precursors; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Spinal Cord

Caffeine is a widely consumed substance that elicits psychomotor stimulant effects and also displays addictive properties. In order to assess the effect of caffeine on striatal neuropeptide mRNA expression, male rats were injected (i.p.) with caffeine at 20, 40 or 80 mg/kg of body weight twice daily for 9 consecutive days. Preproenkephalin (PPE), preprotachykinin A (PPT-A) and preprodynorphin (PPD) mRNA levels were determined in coronal sections of brain tissue by in situ hybridization histochemistry. PPE mRNA levels were increased by chronic caffeine in all subdivisions of the striatum at 80 mg/kg (dorsolateral caudate-putamen (dlCPu), +139%; dorsomedial CPu (dmCPu), +42%; ventrolateral CPu (vlCPu), +102%; ventromedial CPu (vmCPu), +20%; and anterior CPu (aCPu), +75% relative to vehicle-injected controls that were normalized to 0% change). Similarly, PPD mRNA expression was increased in all aspects of the striatum at 80 mg/kg (dlCPu, dmCPu, vlCPu, vmCPu and aCPu, +98%, +25%, +104%, +9% and +85%, respectively). In contrast to PPE mRNA, PPD mRNA was increased +117% above control in the nucleus accumbens (NAc) at 20 mg/kg of caffeine. PPT-A mRNA expression was not significantly affected by caffeine treatment in the CPu or NAc. The data demonstrate that repeated exposure to caffeine selectively increases opioid neuropeptide mRNA expression in the striatum and the NAc of the rat brain by a dopamine-independent mechanism.

Topics: Animals; Autoradiography; Caffeine; Central Nervous System Stimulants; Corpus Striatum; DNA Probes; Dynorphins; Enkephalins; Gene Expression; Male; Nucleus Accumbens; Protein Precursors; Rats; Rats, Sprague-Dawley; RNA, Messenger

Specific antibodies were produced against C-terminal portions of rat preprodynorphin (PPD), preproenkephalin (PPE), and preprotachykinin A (PPT). PPD, PPE, and PPT C-terminal immunoreactivity was observed in many cell bodies of medium-sized neurons in the rat neostriatum (caudate-putamen). Intense PPE immunoreactivity was found in neuropil of the globus pallidus, whereas intense to moderate PPD and PPT immunoreactivity was distributed in neuropil of the substantia nigra and the entopeduncular nucleus. A double-immunofluorescence analysis revealed that PPE-immunoreactive neostriatal neurons rarely showed immunoreactivity for PPD (<1%) or PPT (<2%). In contrast, more than 95% of PPD-immunoreactive neostriatal neurons showed PPT immunoreactivity, and vice versa. No PPD-, PPE-, or PPT-immunoreactive neostriatal neurons showed immunoreactivity for the markers of neostriatal intrinsic neurons, such as calretinin, choline acetyltransferase, parvalbumin, or somatostatin. When tetramethylrhodamine-dextran amine (TMR-DA) was injected into the substantia nigra, almost all neurons that were labeled retrogradely with TMR-DA showed immunoreactivity for PPD (98%) or PPT (99%), but very few of them exhibited PPE immunoreactivity (1%). After injection of TMR-DA into the globus pallidus, 86%, 17%, and 10% of the retrogradely labeled neurons showed immunoreactivity for PPE, PPD, and PPT, respectively. These results support the notion that the neostriatal projection neurons are divided into at least two groups: The projection neurons of one group contain enkephalins and send projection fibers almost exclusively to the globus pallidus, and the others contain tachykinins and dynorphins/Leu-enkephalin and send projection fibers mainly to the substantia nigra.

Topics: Amino Acid Sequence; Animals; Dynorphins; Enkephalins; Fluorescent Antibody Technique, Direct; Guinea Pigs; Immunoblotting; Immunoenzyme Techniques; Immunohistochemistry; Male; Molecular Sequence Data; Neostriatum; Neurons; Protein Precursors; Rabbits; Rats; Rats, Wistar; Tachykinins

The selective M4 muscarinic receptor toxin, MT3, was used in vivo to evaluate the role of M4 receptors in cholinergic inhibition of neuropeptide mRNA expression in striatonigral neurons. Unilateral injection of the muscarinic toxin 3 (0.04-4 nmol) into the dorsal striatum of chronically-cannulated rats elevated basal levels of preprodynorphin, substance P and preproenkephalin mRNAs in the ipsilateral dorsal striatum as revealed by quantitative in situ hybridization. Pretreatment with muscarinic toxin 3 also augmented amphetamine (2.5 mg/kg, i.p.)-stimulated preprodynorphin and substance P expression in the dorsal striatum in a manner similar to that observed after the muscarinic antagonist, scopolamine. Since muscarinic toxin 3 has a much greater affinity for muscarinic M4 receptors than for other subtypes, it is possible that muscarinic toxin 3, by interacting with the muscarinic M4 subtype, regulates basal and/or dopamine-stimulated striatal neuropeptide gene expression.

Topics: Amphetamine; Animals; Corpus Striatum; Dopamine; Dopamine Uptake Inhibitors; Dynorphins; Enkephalins; In Situ Hybridization, Fluorescence; Intercellular Signaling Peptides and Proteins; Male; Muscarinic Antagonists; Neurotoxins; Peptides; Protein Precursors; Radionuclide Imaging; Rats; Rats, Wistar; RNA, Messenger; Substance P

The effects of the acute administration of the serotonin-selective tropane analog, [2beta-propanoyl-3beta-(4-isopropylphenyl)-tropane, WF-31, on spontaneous locomotor activity were measured and compared to those of the highly selective serotonin uptake inhibitor, fluoxetine and cocaine, a non-selective re-uptake inhibitor of dopamine and serotonin. WF-31 (1, 10 and 30 mg/kg)-elicited increases in locomotor behaviors when compared to vehicle-treated rats. This increased activity was blocked by pre-treatment with the dopaminergic antagonist, flupenthixol, suggesting that these effects may be mediated by dopaminergic mechanisms. Cocaine, but not fluoxetine, also elicited increases in behaviors. In addition, the effects of these three compounds on opioid peptide gene expression were also assessed using in situ hybridization histochemistry in the same animals. The acute administration of both WF-31 and cocaine increased the expression of preprodynorphin mRNA in the dorsal striatum whereas fluoxetine had no effect. Expression of striatal preproenkephalin mRNA was augmented by all three compounds. Within the nucleus accumbens, PPD mRNA levels were affected only by treatment with WF-31, an effect that was blocked by pre-treatment with flupenthixol. In contrast, the acute administration of both WF-31 and fluoxetine, but not cocaine, increased the expression of preproenkephalin mRNA. These increases, however, were not reversed by pre-treatment with flupenthixol. Despite its profile in vitro as a relatively selective serotonin re-uptake inhibitor, some of the in vivo actions of WF-31 appear to be mediated by dopaminergic mechanisms. These data further suggest that the mechanisms underlying expression of the opioid peptides in the nucleus accumbens may vary from those in the dorsal striatum.

Topics: Animals; Behavior, Animal; Cocaine; Corpus Striatum; Dopamine Uptake Inhibitors; Dynorphins; Enkephalins; Fluoxetine; Gene Expression; Genome; Male; Motor Activity; Protein Precursors; Rats; Rats, Sprague-Dawley; Selective Serotonin Reuptake Inhibitors; Tropanes

Previous findings from this laboratory have shown that low (2 Hz) and high (100 Hz)-frequency electroacupuncture (EA) accelerated the release of different kinds of opioid peptides in the CNS. In the present study, we tried to elucidate whether EA of different frequencies would affect the transcription of genes encoding different opioid peptides. Digoxin-labeled antisense cRNA probes were used for in situ hybridization to detect the mRNA encoding preproenkephalin (PPE), preprodynorphin (PPD) and proopiomelanocortin (POMC) in the rat brain. The results showed that: (1) Neither 2 Hz nor 100 Hz EA altered the POMC mRNA level in the rat brain. (2) EA of the two frequencies induced a similar degree of increase of PPE mRNA in rostromedial reticular formation (gigantocellular, paragigantocellular and lateral reticular nucleus); whereas in supraoptic nucleus, suprachiasmatic nucleus, arcuate nucleus, paraventricular hypothalamic nucleus, ventromedial nucleus and the nucleus of lateral lemniscus, 2 Hz EA induced a higher PPE mRNA expression than 100 Hz EA. (3) 100 Hz EA markedly increased the PPD mRNA levels in supraoptic nucleus, paraventricular hypothalamic nucleus, ventromedial nucleus and parabrachial nucleus, while 2 Hz was without effect. Since de novo peptide synthesis is regarded as a natural outcome following accelerated peptide release, the present results substantiate our previous observation that EA of different frequencies exert different acceleratory effects on the release and synthesis of different opioid peptides in the central nervous system.

Topics: Acupuncture Analgesia; Animals; Brain; Dynorphins; Electroacupuncture; Enkephalins; Female; Gene Expression; Pro-Opiomelanocortin; Protein Precursors; Rats; Rats, Wistar

Research has suggested that exogenous opioid substances can have direct effects on cardiac muscle or influence neurotransmitter release via presynaptic modulation of neuronal inputs to the heart. In the present study, multiple-labelling immunohistochemistry was employed to determine the distribution of endogenous opioid peptides within the guinea-pig heart. Approximately 40% of cardiac ganglion cells contained immunoreactivity for dynorphin A (1-8), dynorphin A (1-17) and dynorphin B whilst 20% displayed leu-enkephalin immunoreactivity. Different populations of opioid-containing ganglion cells were identified according to the co-existence of opioid immunoreactivity with immunoreactivity for somatostatin and neuropeptide Y. Immunoreactivity for prodynorphin-derived peptides was observed in many sympathetic axons in the heart and was also observed, though to a lesser extent, in sensory axons. Leu-enkephalin immunoreactivity was observed in occasional sympathetic and sensory axons. No immunoreactivity was observed for met-enkephalin-arg-gly-leu or for beta-endorphin. These results demonstrate that prodynorphin-derived peptides are present in parasympathetic, sympathetic and sensory nerves within the heart, but suggest that only the prodynorphin gene is expressed in guinea-pig cardiac nerves. This study has shown that endogenous opioid peptides are well placed to regulate cardiac function via both autonomic and sensory pathways.

Topics: Adrenergic Fibers; Animals; Antibody Specificity; Axons; beta-Endorphin; Dynorphins; Endorphins; Enkephalin, Leucine; Enkephalin, Methionine; Enkephalins; Female; Ganglia, Autonomic; Guinea Pigs; Heart; Immunohistochemistry; Male; Neurons, Afferent; Opioid Peptides; Parasympathetic Fibers, Postganglionic; Protein Precursors

Disruption of the mouse dopamine transporter gene results in spontaneous hyperlocomotion despite major adaptive changes, such as decreases in neurotransmitter and receptor levels. In homozygote mice, dopamine persists at least 100 times longer in the extracellular space, explaining the biochemical basis of the hyperdopaminergic phenotype and demonstrating the critical role of the transporter in regulating neurotransmission. The dopamine transporter is an obligatory target of cocaine and amphetamine, as these psychostimulants have no effect on locomotor activity or dopamine release and uptake in mice lacking the transporter.

Topics: Amphetamine; Animals; Carrier Proteins; Cell Line; Cocaine; Dopamine; Dopamine Plasma Membrane Transport Proteins; Dynorphins; Enkephalins; Female; Gene Expression Regulation; Gene Targeting; Locomotion; Male; Membrane Glycoproteins; Membrane Transport Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Nerve Tissue Proteins; Protein Precursors; Rats

The present study examined the modulatory role of dopamine (DA) on striatonigral preprotachykinin (PPT) and prodynorphin (PD) gene expression, employing the DA uptake inhibitor, GBR-12909 (GBR), as a tool. The striatal and nigral levels of tachykinin (substance P (SP), neurokinin A (NKA)) and dynorphin (dynorphin A(1-8) (DYN)) peptides were determined by radioimmunoassays. The abundance of mRNAs in the striatum was quantified by Northern blot analysis. The rate of transcription of PPT and PD genes in the striatum was measured by transcription run-on assays. A regimen of repeated administration of GBR (20 mg/kg/day, i.p., for 1-4 days) to female Sprague-Dawley rats increased striatal and nigral SP, NKA, and DYN peptide levels. The increased peptide levels were associated with increases in the abundance of PD mRNA and PPT mRNA and increases in the rate of transcription of PD and PPT genes in the striatum, suggesting a GBR-induced activation of the striatonigral tachykinin and dynorphin neurons. Dopaminergic denervation with 6-hydroxydopamine (6OHDA) blocked the GBR-induced increases in SP and DYN and PPT and PD mRNAs. The concurrent administration of the D1 DA antagonist, SCH-23390, blocked the GBR-induced increases in SP, NKA and PPT mRNA but failed to affect DYN or PD mRNA levels; the concurrent administration of the D2 DA antagonist, spiperone, blocked the GBR-induced increases in SP, NKA and PPT mRNA and also DYN and PD mRNA. The study reveals that repeated administration of GBR enhances the levels of tachykinin and dynorphin peptides in striatonigral neurons by a stimulus-transcription-biosynthesis coupling mechanism. The GBR-induced effects are dependent on the integrity of nigrostriatal dopaminergic neurons and the presence of D1 and/or D2 DA receptors.

Topics: Animals; Cell Nucleus; Corpus Striatum; Denervation; Dopamine; Dopamine Uptake Inhibitors; Dose-Response Relationship, Drug; Dynorphins; Enkephalins; Female; Gene Expression Regulation; Kinetics; Organ Specificity; Piperazines; Protein Precursors; Rats; Rats, Sprague-Dawley; RNA, Messenger; Substantia Nigra; Tachykinins; Transcription, Genetic

The effects of a repeated treatment with nicotine on the expression of mRNAs encoding preproenkephalin (PPE), preprotachykinin-A (PPT-A), and preprodynorphin (PPDYN) were examined by in situ hybridization histochemistry in various subregions of the nucleus accumbens (Acb). In saline-treated rats, optical density measurements on autoradiographic films showed marked anteroposterior decreasing gradients for PPE and PPT-A mRNAs in the rostral pole and the core, in the cone, and in the ventral shell of the Acb, whereas a lower anteroposterior gradient was observed for PPDYN mRNA signals. The intensity of the three mRNA signals also varied according to Acb subregion. However, analysis of percentages of prepropeptide mRNA-containing neurons as compared to total neurons showed, in the rostral pole, the core, and the cone, a similar percentage of PPE mRNA (around 45%)- and PPT-A mRNA (around 40%)-expressing neurons. The ventral shell can be distinguished from the other subregions by a lower percentage of PPE mRNA (35.8%)- and PPT-A mRNA (30.6%)-expressing neurons. The percentage of PPDYN mRNA-containing neurons, by contrast, was similar (around 37%) in the core, the cone, and the ventral shell. Repeated nicotine administration increases the PPE mRNA level in the rostral pole and the anterior third of the core without any change in PPT-A and PPDYN mRNA levels in the various Acb subregions examined. The PPE mRNA increase does not support an effect mediated through an interaction of nicotine with DA neurons. The effect could be linked to a nicotine activation of other afferents to the anterior Acb and/or to a direct nicotine stimulation of PPE mRNA neurons.

Topics: Animals; Dynorphins; Enkephalins; In Situ Hybridization; Male; Nicotine; Nucleus Accumbens; Protein Precursors; Rats; Rats, Sprague-Dawley; RNA, Messenger; Tachykinins

Our previous work has demonstrated a dose-dependent induction of striatal preprodynorphin (PPD) in response to a single injection of the psychostimulants amphetamine (AMPH) or methamphetamine (METH). In the present study, dose-response effects of acute administration of these stimulants on preproenkephalin (PPE) mRNA expression in the rat striatum were investigated with quantitative in situ hybridization histochemistry 3 h after injection. Acute AMPH or METH at equimolar doses (3.75, 7.5, 15, and 30 mumol/kg) significantly increased PPE mRNA expression in dorsal (caudoputamen), but not ventral (nucleus accumbens), striatum in a dose-dependent fashion. In addition, the role of D1 and D2 dopamine receptors in mediating AMPH- and METH-stimulated PPE and PPD expression was also evaluated by using subtype-specific antagonists. Pretreatment of rats with SCH 23390 (0.1 mg/kg, i.p.), a selective D1 receptor antagonist, completely blocked acute AMPH (21 mumol/kg, i.p.)- or METH (21 mumol/kg, i.p.)-induced PPE as well as PPD mRNA expression in the caudoputamen. Pretreatment with eticlopride (0.5 mg/kg, i.p.), a selective D2 receptor antagonist, also blocked PPD induction by the two stimulants, but PPE induction was unaffected. Furthermore, SCH 23390 decreased, and eticlopride elevated, constitutive PPE mRNA levels in the caudoputamen. Neither antagonist had a significant effect on the basal level of PPE or PPD mRNA in the nucleus accumbens. These results demonstrate a clear dose-related responsiveness of PPE gene expression in striatal neurons in response to acute administration of amphetamines, although the intensity of the response is far less than that for striatal PPD. Furthermore, both D1 and D2 subtypes of dopamine receptors mediate AMPH- and METH-stimulated striatal PPD mRNA expression, whereas D1 receptor activation alone mediates amphetamine-stimulated PPE mRNA expression in the rat striatum.

Topics: Amphetamine; Analysis of Variance; Animals; Corpus Striatum; Dynorphins; Enkephalins; In Situ Hybridization; Male; Methamphetamine; Protein Precursors; Rats; Rats, Wistar; Receptors, Dopamine; RNA, Messenger

The main purpose of this study was to investigate differences regarding endogenous opioids in post-mortem frontal cortex of adult patients with Down syndrome (DS), patients with Alzheimer disease (AD) and neurologically healthy persons, respectively, using specific radioimmunoassays. The results of this study show that there is an increase in the levels of leu-enkephalin and dynorphin A in the frontal cortex of patients with DS as compared to the control group. An almost identical increase was also observed when comparing patients with AD to controls. In conclusion, the results of this study suggest a relationship between elevated tissue levels of leuenkephalin and dynorphin A in cerebral cortex and cognitive impairments in patients with DS and AD.

Topics: Aged; Alzheimer Disease; Down Syndrome; Dynorphins; Endorphins; Enkephalin, Leucine; Enkephalin, Methionine; Enkephalins; Female; Frontal Lobe; Gene Expression Regulation; Humans; Male; Middle Aged; Protein Precursors; Radioimmunoassay

Most adrenal chromaffin cells synthesize opioids derived from proenkephalin but not from prodynorphin. However, human pheochromocytomas and the PC12 rat pheochromocytoma cell line synthesize dynorphins. The aim of this study was to confirm the presence of the authentic prodynorphin transcript and its dynorphin product in PC12 cells. We have found that the sequence of a 458 bp cDNA fragment derived from RT-PCR amplification of total PC12 RNA was in complete accordance with the published sequence of the equivalent region of the prodynorphin gene. It encodes the potent endogenous kappa opioid agonists alpha-neo-endorphin, dynorphin A and dynorphin B. Furthermore, immunoaffinity-purified PC12 cell extracts were subjected to RP-HPLC. Most of its IR-dynorphin eluted on a peak exhibiting the retention time of similarly treated rat anterior pituitary. The expression of the prodynorphin gene in pheochromocytomas can be explained as either the result of (a) the process of dedifferentiation of chromaffin cells to pheochromocytoma which may thus cause the expression of a previously unexpressed prodynorphin or that (b) those pheochromocytomas expressing the prodynorphin gene derive from the few, centrally located chromaffin cells, which express this gene even under normal conditions.

Topics: Adrenal Medulla; Animals; Chromatography, Affinity; Chromatography, High Pressure Liquid; Dynorphins; Endorphins; Enkephalins; Gene Expression; PC12 Cells; Pheochromocytoma; Pituitary Gland; Polymerase Chain Reaction; Protein Precursors; Rats; Sequence Analysis

This study investigated the effects of pharmacological blockade or stimulation of muscarinic receptors on constitutive and amphetamine-stimulated preprodynorphin, substance P and pre-proenkephalin gene expression in rat striatum. Acute administration of the non-selective muscarinic antagonist, scopolamine (2.5, 5 and 10 mg/kg, s.c.), caused a dose-dependent increase in preprodynorphin and substance P, but not preproenkephalin, messenger RNA expression in the dorsal and ventral striatum as revealed by quantitative in situ hybridization. In contrast, acute injection of the non-selective muscarinic receptor agonist, oxotremorine (0.125, 0.25 and 0.5 mg/kg, s.c.), caused a dose-dependent increase in basal levels of preproenkephalin messenger RNA in the dorsal striatum, without causing a significant effect on constitutive striatal preprodynorphin and substance P expression. Pretreatment with scopolamine (2.5 mg/kg, s.c.) significantly augmented striatal induction of preprodynorphin and substance P messenger RNA induced by acute injection of amphetamine (1.25 and 2.5 mg/kg, i.p.), whereas scopolamine blocked amphetamine-stimulated striatal preproenkephalin expression. Pretreatment with oxotremorine (0.25 mg/kg, s.c.) significantly attenuated amphetamine (1.25 and 2.5 mg/kg, i.p.)-stimulated striatal preprodynorphin and, to a lesser degree, substance P messenger RNA expression. Oxotremorine tended to increase amphetamine-stimulated preproenkephalin messenger RNA expression, but the effect did not reach statistical significance. In addition, scopolamine increased spontaneous, and enhanced amphetamine-stimulated, behavioral activity, whereas oxotremorine attenuated amphetamine-stimulated behaviors. These data support the concept that cholinergic transmission, via interaction with muscarinic receptors, inhibits basal and D1 receptor-stimulated striatonigral dynorphin/substance P gene expression and facilitates striatopallidal enkephalin gene expression.

Topics: Acetylcholine; Amphetamine; Animals; Corpus Striatum; Dynorphins; Enkephalins; Gene Expression Regulation; Male; Muscarinic Agonists; Muscarinic Antagonists; Nerve Tissue Proteins; Neuropeptides; Oxotremorine; Protein Precursors; Rats; Rats, Wistar; Receptors, Dopamine D1; Receptors, Muscarinic; Scopolamine; Substance P

The modulation by selective dopamine receptor antagonists of the effects of "binge' cocaine (3 x 15 mg kg-1 day-1, i.p., for 3 days after 11 days of adaptation to saline injections) on preproenkephalin, preprodynorphin, kappa opioid receptor and dopamine transporter mRNAs was determined. Administration of cocaine was preceded by daily single injections of a D1 (SCH 23390; 2 mg kg-1) or the D2 (sulpiride; 50 mg kg-1) dopamine receptor antagonist. The D1, and not the D2, antagonist blocked cocaine-induced preprodynorphine and preproenkephalin increases in the caudate-putamen. Sulpiride alone, and sulpiride plus cocaine, increased preproenkephalin mRNA. Dopamine transporter mRNA levels showed a cocaine treatment-antagonist interaction. These data indicate that this administration paradigm elevates both preprodynorphin and preproenkephalin mRNAs by a D1-dependent mechanism not requiring D2 activation.

Topics: Analysis of Variance; Animals; Benzazepines; Brain; Carrier Proteins; Cocaine; DNA Primers; Dopamine Antagonists; Dopamine Plasma Membrane Transport Proteins; Drug Administration Schedule; Dynorphins; Enkephalins; Male; Membrane Glycoproteins; Membrane Transport Proteins; Nerve Tissue Proteins; Polymerase Chain Reaction; Protein Precursors; Rats; Rats, Inbred F344; Receptors, Opioid, kappa; RNA, Messenger; Substance-Related Disorders; Sulpiride; Transcription, Genetic

Antisense oligodeoxynucleotides (ODNs) of c-fos and/or c-jun were used in this study to investigate the role of Fos and Jun proteins in electroacupuncture (EA)-induced transcription of the opioid genes, preproenkephalin (PPE), preprodynorphin (PPD) and proopiomelanocortin (POMC). As the results showed, EA-induced Fos and fun expression was blocked efficiently and specifically by e-fos and c-jun antisense ODNs, respectively. This treatment significantly prevented EA-induced PPD, but not PPE, mRNA expression. These results suggest that Fos and Jun proteins are involved in PPD rather than PPE gene transcription activated by EA stimulation.

Topics: Animals; Brain; Dynorphins; Electroacupuncture; Enkephalins; Female; In Situ Hybridization; Protein Precursors; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Rats; Rats, Wistar; Transcription, Genetic

This study tested the role of N-methyl-D-aspartate and kainate/AMPA receptors in mediating mRNA expression of the immediate early gene zif/268 and the opioid peptide genes preprodynorphin and preproenkephalin in rat forebrain following a single injection of methamphetamine. At 3 h after acute methamphetamine [4 mg/kg, intraperitoneally (IP)], quantitative in situ hybridization histochemistry revealed that zif/268 mRNA expression was increased in the dorsal striatum (caudoputamen) and in the sensory cortex. Preprodynorphin was increased in both dorsal and ventral striatum (nucleus accumbens) and preproenkephalin was increased in the dorsal striatum. Pretreatment with (+ or -)-3-(2-carboxypiperazin-4-yl)-propyl-1 -phosphonic acid (CPP) (10 mg/kg, IP), an N-methyl-D-aspartate receptor antagonist, blocked the methamphetamine-induced zif/268 mRNA expression in the striatum and in the region of sensory cortex representing the upper limb and nose. 6,7-Dinitro-quinoxaline-2,3-dione (DNQX) (100 mg/kg, IP), a kainate/AMPA receptor antagonist, did not reduce the ability of methamphetamine to induce zif/268 mRNA in striatal and cortical neurons. Furthermore, both antagonists caused a parallel blockade of methamphetamine-stimulated preprodynorphin mRNA expression in the dorsal and ventral striatum but did not significantly affect methamphetamine-stimulated preproenkephalin mRNA expression. CPP and DNQX reduced basal levels of zif/268 mRNA in cortical and striatal neurons but did not affect the constitutive expression of the two opioid mRNAs in the striatum. Neither antagonist had a significant effect on methamphetamine-induced hyperlocomotion and stereotypies. These results demonstrate that both N-methyl-D-aspartate and kainate/AMPA receptor-mediated glutamatergic transmission is linked to modulation of the methamphetamine-stimulated oploid peptide gene expression in rat forebrain. Furthermore, N-methyl-D-aspartate receptors participate in methamphetamine-stimulated zif/268 expression.

Topics: Animals; Corpus Striatum; DNA-Binding Proteins; Dynorphins; Early Growth Response Protein 1; Enkephalins; Excitatory Amino Acid Agonists; Immediate-Early Proteins; Male; Methamphetamine; Nerve Tissue Proteins; Protein Precursors; Rats; Rats, Wistar; Receptors, AMPA; Receptors, Kainic Acid; Receptors, N-Methyl-D-Aspartate; RNA, Messenger; Stimulation, Chemical; Time Factors; Transcription Factors

The present work was designed (a) to study comparatively the effect of 2Hz and 100Hz electroacupuncture (EA) on the expression of oncogene c-fos/c-jun and three opioid (preproenkephalin-PPE; preprodynorphin-PPD; proopiomelanocortin-POMC) genes in the rat brain; (b) to clarify the role of Fos/Jun (AP-1) on opioid genes expression induced by EA stimulation through specific blockade of EA-induced Fos/Jun expression using antisense oligodeoxynucleotides (ODNs) of c-fos/c-jun. The results were: (a) 2Hz and 100Hz EA induced differential Fos expression in different brain areas; (b) EA of both frequencies accelerated PPE gene transcription, but 2Hz EA was more effective than 100Hz EA; (c) PPD expression was accelerated by 100Hz EA, but not by 2Hz EA; (d) the blockade of Fos/Jun expression by c-fos/c-jun antisense ODNs prevented EA from accelerating PPD but not PPE mRNA expression.

Topics: Animals; Dynorphins; Electroacupuncture; Enkephalins; Protein Precursors; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Rats; Rats, Wistar; RNA, Messenger

Dynorphin B (Dyn B-13, also known as rimorphin) is generated from Dyn B-29 (leumorphin) by the cleavage at a single Arg residue. An enzymatic activity capable of processing at this monobasic site has been previously reported in neurosecretory vesicles of the bovine pituitary and pituitary-derived cell lines. This enzyme termed "the dynorphin-converting enzyme" (DCE) has been purified to apparent homogeneity from the neurointermediate lobe of the bovine pituitary using hydrophobic chromatography on phenyl-Sepharose, preparative isoelectrofocusing in a granulated gel between pH 4 to 6.5, and non-denaturing electrophoresis on 5% polyacrylamide gel. DCE exhibits a pI of about 5.1 and a molecular mass of about 54 kDa under reducing conditions. DCE is a metallopeptidase and exhibits a neutral pH optimum. Specific Inhibitors of soluble metallopeptidases such as enkephalinase (EC 3.4.24.11) or enkephalin generating neutral endopeptidase (EC 3.4.24.15) do not inhibit DCE activity indicating that DCE is distinct from these two enzymes. Cleavage site determination with matrix-assisted laser desorption ionization time of flight (MALDITOF) mass spectrometry shows that DCE cleaves the Dyn B-29 N terminus to the Arg14 generating Dyn B-13 and Dyn B-(14-29). Among other peptides derived from Dyn B-29, DCE cleaves only those peptides that fit the predicted "consensus motif" for monobasic processing. These data are consistent with a broader role for the dynorphin converting enzyme in the biosynthesis of many peptide hormones and neuropeptides by processing at monobasic sites.

Topics: Amino Acid Sequence; Animals; Binding Sites; Cattle; Dynorphins; Endorphins; Enkephalins; Isoelectric Point; Kinetics; Molecular Sequence Data; Molecular Weight; Peptide Fragments; Pituitary Gland; Protein Precursors; Protein Processing, Post-Translational; Serine Endopeptidases; Substrate Specificity

Pharmacological studies suggest that diabetes produces changes in the brain opioid system, affecting several behavioral functions including analgesia, feeding and self-stimulation. Previous investigations of opioid receptor binding have failed to explain the unusual opioid pharmacology of the diabetic animal. In the present study, the effects of streptozotocin-induced diabetes on levels of three immunoreactive (ir)-prodynorphin-derived peptides, ir-dynorphin A1-17 (A1-17), ir-dynorphin A1-8 (A1-8) and ir-dynorphin B1-13 (B1-13), were determined in eleven brain regions known to be involved in appetite, taste and reward. Diabetes was found to increase levels of A1-17 in the ventromedial and dorsomedial hypothalamic nuclei (+60% and +25%, respectively) and levels of A1-8 in the dorsomedial and lateral hypothalamus (+45% and +35%, respectively). The possible significance of these results is discussed in relation to (i) diabetic hyperphagia, (ii) medial hypothalamic transduction of circulating insulin levels, and (iii) the potentiation of reward by metabolic need states.

Topics: Animals; Brain; Diabetes Mellitus, Experimental; Dynorphins; Enkephalins; Hypothalamic Hormones; Male; Peptide Fragments; Protein Precursors; Rats; Rats, Sprague-Dawley

Proteolytic processing of prodynorphin-derived peptides in rat brain was studied with the help of high performance size exclusion chromatography (SEC) connected to electrospray ionization mass spectrometry. Extracts from rat striatum were incubated with individual synthetic dynorphin peptides. Dynorphin A was the most resistant to proteolytic cleavage, converting slowly to Leu-enkephalin (0.3 pmol/min), whereas dynorphin B was processed to this pentapeptide at a 10(4)-fold higher rate. Minor cleavage was also observed between Arg6-Arg7. Alphaneoendorphin was also rapidly metabolized to Leu-enkephalin (6 nmol/min) and, to a lesser extent, to Leu-enkephalinArg6. This new strategy for studying peptidases can easily be adapted to identification of components present in body fluids.

Topics: Animals; Anti-Bacterial Agents; Chromatography, Gel; Corpus Striatum; Dynorphins; Endorphins; Enkephalin, Leucine; Enkephalins; Male; Mass Spectrometry; Peptide Hydrolases; Peptides; Protein Precursors; Rats; Sensitivity and Specificity; Tissue Extracts

Several studies have demonstrated that cocaine increases preprodynorphin, c-fos, and zif/268 mRNAs in rat dorsal striatum. Multiple, closely spaced exposures to cocaine appear to elicit the greatest increases in dynorphin. However, the response of preproenkephalin, c-fos and zif/268 mRNAs to such a dosing regimen is unknown. Therefore, we used a 'binge' paradigm to evaluate changes in mRNA for preprodynorphin, preproenkephalin, c-fos and zif/268. Male Wistar rats received three hourly i.p. injections of saline or 10 or 20 mg/kg cocaine for 1, 5, or 10 days. Although cocaine-induced locomotor and stereotypical behaviors were significantly increased as compared to saline on days 1, 5 and 10, these behaviors were significantly less on day 10 than on days 1 and 5. One hour after the last injection on days 1, 5, or 10, the rats were anesthetized and decapitated for quantitative in situ hybridization histochemistry. C-fos mRNA was undetectable in all treatment groups whereas zif/268 mRNA in the dorsal striatum was increased in a dose-dependent manner (20 mg/kg > 10 mg/kg) but the intensity of hybridization signal decreased over time (1 day >> 5 days > 10 days) as compared to that in saline-treated controls. In contrast, 10 mg/kg cocaine binges caused an increase in preprodynorphin, but not preproenkephalin, mRNA in the dorsal, but not ventral, striatum in a time-dependent manner (day 10 >> day 5 > day 1) whereas 20 mg/kg cocaine binges caused an increase in striatal preprodynorphin that was greater on day 1 and day 5 than on day 10. These data indicate that (1) c-fos, zif/268 and preprodynorphin mRNAs are differentially regulated in dorsal striatum, (2) behavioral tolerance results from chronic binges with 10 and 20 mg/kg cocaine and (3) the preprodynorphin genomic response exhibits tolerance to chronic high dose, but not low dose, cocaine binges.

Topics: Analysis of Variance; Animals; Behavior, Animal; Cocaine; Corpus Striatum; Disease Models, Animal; DNA-Binding Proteins; Drug Administration Schedule; Dynorphins; Early Growth Response Protein 1; Enkephalins; Immediate-Early Proteins; Male; Nerve Tissue Proteins; Protein Precursors; Proto-Oncogene Proteins c-fos; Rats; Rats, Wistar; RNA, Messenger; Transcription Factors

Rats self-administered cocaine or received saline during 3 daily 5 h sessions and were euthanized 1 h after the final session. Quantitative in situ hybridization revealed that cocaine self-administration increased levels of preprodynorphin, but not preproenkephalin, c-fos, or zif/268 mRNAs in a patchy pattern in the dorsal striatum. These data demonstrate that the regulation of preprodynorphin gene expression is dissociable from that of c-fos and zif/268 in dorsal striatum following short-term cocaine self-administration.

Topics: Animals; Cocaine; Dynorphins; Enkephalins; Gene Expression; Image Processing, Computer-Assisted; In Situ Hybridization; Male; Neostriatum; Nucleus Accumbens; Protein Precursors; Rats; Rats, Wistar; RNA, Messenger; Self Administration

A striatal dopaminergic denervation leads to changes in the expression of messenger RNA encoding prepropeptides contained in striatal efferent neurons. Such a dopaminergic lesion also abolishes a functional equilibrium between dopaminergic and cholinergic transmissions, generally believed to operate within the neostriatum, which constitutes the theoretical basis for the clinical use of antimuscarinic drugs in extrapyramidal diseases. It is possible, therefore, that changes in prepropeptide messenger RNA expression are mediated by an alteration in cholinergic transmission. To test this hypothesis, we have examined in rats whether trihexyphenidyl, an antimuscarinic drug of wide clinical use, can counteract the changes in preproenkephalin, preprotachykinin and preprodynorphin messenger RNA expression produced by a unilateral 6-hydroxydopamine lesion of substantia nigra dopaminergic neurons. Two weeks after the lesion, trihexyphenidyl was continuously administered through an osmotic minipump (5 mg/day for 15 days) to half of the lesioned and sham-operated rats, the other half receiving the vehicle. Using quantitative in situ hybridization histochemistry, messenger RNAs were analysed at two rostrocaudal levels (anterior and central) of the neostriatum. In parallel, M1 muscarinic receptors were measured by autoradiography of [3H]pirenzepine binding sites. In sham-operated rats, trihexyphenidyl administration produced a significant increase (17-27%) in M1 binding sites. In addition, preproenkephalin messenger RNA levels were decreased (-38%) in the central part, while preprodynorphin messenger RNA levels were significantly increased (+22%) at both striatal levels. In 6-hydroxydopamine-lesioned rats, the expected changes in messenger RNAs were observed when ipsi- versus contralateral side values were compared, but changes were not always detected when comparison was established between values from the dopamine-denervated neostriatum and those from sham-operated rats. The trihexyphenidyl administration in 6-hydroxydopamine-lesioned animals was unable to reproduce the up-regulation of M1 receptors, even in the intact neostriatum. This antimuscarinic treatment further increased preproenkephalin messenger RNA levels in the denervated anterior neostriatum, amplifying the ipsi- versus contralateral difference. It also potentiated the imbalance in preprotachykinin messenger RNA expression, mainly as a result of an increase of preprotachykinin messenger RNA levels in the

Topics: Animals; Autoradiography; Dopamine; Dynorphins; Enkephalins; In Situ Hybridization; Male; Muscarinic Antagonists; Neostriatum; Oxidopamine; Pirenzepine; Protein Precursors; Rats; Rats, Wistar; RNA, Messenger; Substantia Nigra; Sympathectomy, Chemical; Tachykinins; Trihexyphenidyl

Using in situ hybridization histochemistry, we studied the distribution of neurons that express preproopiomelanocortin (pre-POMC), preprodynorphin (pre-PDYN), and preproenkephalin (pre-PENK) gene transcripts within the human hypothalamus and surrounding structures. Of the three opioid systems, pre-POMC neurons have the most restricted distribution. Pre-POMC cells are most numerous in the infundibular nucleus and retrochiasmatic area of the mediobasal hypothalamus; a few labeled cells are present within the boundaries of the ventromedial nucleus and infundibular stalk. Pre-POMC message was not found in the limited samples of structures adjacent to the hypothalamus. In contrast to neurons that express pre-POMC, neurons expressing pre-PDYN and pre-PENK are more widely represented throughout the hypothalamus and extrahypothalamic structures. However, pre-PDYN and pre-PENK cells differ from one another in distribution. Pre-PDYN message is especially abundant in neurons of the tuberal and mammillary regions, with a distinct population of labeled cells in the premammillary nucleus and dorsal posterior hypothalamus. Pre-PDYN gene expression also is found in neurons of the dorsomedial nucleus, ventromedial nucleus, caudal magnocellular portion of the paraventricular nucleus, dorsolateral supraoptic nucleus, tuberomammillary nucleus, caudal lateral hypothalamus, and retrochiasmatic area. In structures immediately adjacent to the hypothalamus, pre-PDYN neurons were observed in the caudate nucleus, putamen, cortical nucleus of the amygdala, and bed nucleus of the stria terminalis. Pre-PENK neurons occur in varying numbers in all hypothalamic nuclei except the mammillary bodies. The chiasmatic region is particularly rich in pre-PENK neurons, with the highest packing density in the intermediate nucleus [the intermediate nucleus (Braak and Braak [1987] Anat. Embryol. 176:315-330) has also been termed the sexually dimorphic nucleus of the preoptic area (SDA-POA; Swaab and Fliers [1985] Science 228:1112-1115) or the interstitial nucleus of the anterior hypothalamus 1 (Allen et al. [1989] J. Neurosci. 9:497-506)], dorsal suprachiasmatic nucleus, medial preoptic area, and rostral lateral hypothalamic area. Pre-PENK neurons are numerous in the infundibular nucleus, ventromedial nucleus, dorsomedial nucleus, caudal parvicellular portion of the paraventricular nucleus, tuberomammillary nucleus, lateral hypothalamus, and retrochiasmatic area. Only a few lightly labeled cells we

Topics: Adolescent; Adult; Dynorphins; Enkephalins; Gene Expression; Histocytochemistry; Humans; Hypothalamus; In Situ Hybridization; Male; Middle Aged; Neurons; Pro-Opiomelanocortin; Protein Precursors; Sensitivity and Specificity

The effect of N-methyl-D-aspartate (NMDA) receptor blockade on the expression of preproenkephalin (PPE), preprotachykinin (PPT) and preprodynorphin (PPD) mRNAs in the caudate-putamen and nucleus accumbens was assessed with the non-competitive NMDA receptor antagonist MK-801. Administration of MK-801 once daily for 7 consecutive days increased the abundance of all three neuropeptide mRNAs in the caudate-putamen (CPU) and nucleus accumbens (NAc). (1) PPE mRNA abundance was increased in the anterior CPU (26%) as well as dorsal and ventral CPU (46% and 39%, respectively) but was unaffected in the NAc. (2) PPT mRNA was increased in the NAc (33%), anterior CPU (27%), dorsal CPU (43%) and ventral CPU (67%). In the ventral CPU, PPT mRNA abundance doubled when the dose of MK-801 increased two-fold (from 67% to 119% above control). (3) PPD mRNA was elevated in dorsal and ventral regions of the CPU (49% and 24%, respectively) and in anterior CPU (50%). In the NAc PPD mRNA was increased only at the higher dose (0.1 mg/kg) of MK-801. Cellular analysis of the distribution of grains per cell shows that increases are due to increased accumulation of mRNA by previously expressing cells of the CPU and NAc. These observations demonstrate that NMDA receptor activity plays a significant role in the regulation of neuropeptide expression in the caudate-putamen and accumbens of the rat brain.

Topics: Animals; Base Sequence; Corpus Striatum; Dizocilpine Maleate; Dynorphins; Enkephalins; Male; Molecular Probes; Molecular Sequence Data; Neuropeptides; Nucleus Accumbens; Protein Precursors; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; RNA, Messenger; Tachykinins

Cyclase response elements (CREs) are located in the promoter regions of several neuropeptide and immediate early genes. Activation of the adenylate cylase/cAMP second messenger cascade leads to phosphorylation of CRE-binding proteins (P-CREBs) which bind to CREs in the promoter regions of these genes and alter their rate of transcription. We have previously reported an increase in striatal immunoreactivity for P-CREB (phosphorylated on Ser-133) and Fos following intracerebroventricular (ICV) injection of H2O-soluble forskolin, a direct activator of adenylate cyclase. Because CREs are located in the promoter regions of the opioid peptide genes, preproenkephalin (PPE) and preprodynorphin (PPD), we investigated what effect continuous ICV infusion of H2O-soluble forskolin has on striatal PPE and PPD mRNA levels. Quantitative in situ hybridization histochemistry demonstrated that continuous activation of the adenylate cyclase/cAMP second messenger cascade results in a significant induction of striatal PPE and PPD mRNA at 6, 24, and 72 h. The sustained induction of striatal PPE and PPD mRNA indicates that pro-opioid gene transcription is not desensitized following 72 h of continuous adenylate cyclase activation. Continuous ICV infusion of 1, 9-dideoxyforskolin, a forskolin analog which does not activate adenylate cyclase, did not induce striatal PPE and PPD mRNA. These data are consistent with cAMP-dependent protein kinase-induced phosphorylation and binding of CREBs to CREs in the promoter regions of pro-opioid genes during sustained activation of adenylate cyclase.

Topics: Animals; Colforsin; Corpus Striatum; Dynorphins; Enkephalins; Histocytochemistry; In Situ Hybridization; Injections, Intraventricular; Male; Protein Precursors; Rats; Rats, Sprague-Dawley; RNA, Messenger

Cholestatic liver disease is associated with clinical and experimental findings consistent with increased opioidergic neuromodulation, increased plasma total opioid activity, and elevated plasma enkephalin concentrations. In contrast to the normal adult rat liver, preproenkephalin mRNA was detected by Northern blotting in livers of adult rats with cholestasis due to bile duct resection and not in the sham-resected controls. Preprodynorphin mRNA was not detected in livers of either group, while preproopiomelanocortin mRNA was found in very low levels in both groups. Preproenkephalin mRNA was not expressed in the livers of rats with acute hepatocellular necrosis induced by thioacetamide. Hybridization histochemistry of cholestatic livers demonstrated the presence of preproenkephalin mRNA primarily over cells in the periportal areas, some of which appeared to be proliferating bile ductular cells. Immunohistochemical staining of cholestatic liver indicated the production of at least Met-enkephalin in association with preproenkephalin gene expression. These findings suggest that the liver itself, by synthesizing enkephalins, contributes directly to the abnormalities of the opioid system reported in cholestasis.

Topics: Animals; Cholestasis; Dynorphins; Enkephalins; Histocytochemistry; Immunohistochemistry; Liver; Male; Necrosis; Nucleic Acid Hybridization; Pro-Opiomelanocortin; Protein Precursors; Rats; Rats, Sprague-Dawley; RNA, Messenger; Tissue Distribution

Prodynorphin (Prodyn)-derived peptides are synthesized in a subset of gonadotrope cells and released concomitantly with LH and FSH, and their levels in the rat adenohypophysis are influenced by the gonadal steroid environment. In several hormonal systems, factors that affect peptide levels may modulate the transcription of messenger RNA (mRNA) encoding for the target gene. Therefore, the present study was designed to investigate the effects of gonadal ablation and estrogen replacement on changes in steady state levels of anterior pituitary Prodyn mRNA and on the transcription rate in the adult female rat. The antiestrogen tamoxifen was employed for further exploring the relationships between estrogens and dynorphin (dyn)-related peptides. Adopting a solution hybridization-ribonuclease protection assay, steady state levels of Prodyn mRNA doubled in 2-week ovariectomized (OVX) rats, in parallel with a 3-fold increase in immunoreactive dyn-A-(1-17)-like material (irdyn-A). Estradiol (E2) replacement through sc SILASTIC implants for 1, 3, 7, and 14 days, which produces serum E2 levels between 25-35 pg/ml, restored in a time-dependent manner mRNA and peptide concentrations to values in sham-OVX rats. A significant decrease was observed after 3 days, and after 7 days, the effect was maximal. Tamoxifen (250 micrograms/kg.day, sc) administered simultaneously antagonized the action of E2 on Prodyn gene expression. Tamoxifen administered without E2 for 7 or 14 days significantly raised anterior pituitary levels of Prodyn mRNA and ir-dyn-A. To establish whether E2 and tamoxifen exert their effects on adenohypophyseal Prodyn mRNA by influencing the transcriptional activity of this gene, an in vitro transcriptional elongation assay was performed on nuclei from the anterior pituitary. The transcriptional rate of the Prodyn gene was significantly increased in 2-week OVX rats. Prodyn mRNA synthesis was suppressed in OVX rats exposed to E2, an effect antagonized by tamoxifen administered concomitantly. The antiestrogen administered alone for 14 days further elevated the transcriptional rate of Prodyn mRNA induced by gonadal ablation. In conclusion, E2 down-regulated the synthesis of Prodyn-derived peptides in adenohypophyseal cells. The antiestrogen tamoxifen antagonized the effect of E2 and, when chronically administered to OVX rats, further elevated the postcastrational rise in Prodyn gene expression.

Topics: Animals; Dynorphins; Enkephalins; Estradiol; Female; Gene Expression; Ovariectomy; Pituitary Gland, Anterior; Protein Precursors; Rats; Rats, Sprague-Dawley; RNA, Messenger; Tamoxifen; Transcription, Genetic

Prodynorphin mRNA and immunoreactive dynorphin A (ir-dynorphin A) levels were measured in different brain areas at various time points after amygdala kindled seizures. In the hippocampus, striatum and hypothalamus, prodynorphin mRNA levels were not significantly changed in kindled rats (killed 1 week after the last stimulus-evoked seizure), but they were significantly increased 1 h after seizures. The relative increase was the highest in the hippocampus (approximately 3-fold). In the brainstem, midbrain and cerebral cortex no changes in prodynorphin mRNA were detected in kindled rats, 1 h or 1 week after a kindled seizure. ir-Dynorphin A levels were significantly reduced in the hippocampus and in the striatum of kindled rats, as well as 5 and 60 min after kindled seizures, but they were increased back to control levels after 120 min. In the hypothalamus, ir-dynorphin A levels were significantly increased 120 min after a kindled seizure. ir-Dynorphin A levels were also significantly reduced in the brainstem and in the frontal, parietal and temporal cortex 120 min, but not 5 or 60 min, after a kindled seizure. Taken together, these data support the hypothesis that the dynorphinergic system is activated after amygdala kindled seizures, with different kinetics in different brain areas.

Topics: Amygdala; Animals; Blotting, Northern; Dynorphins; Enkephalins; Kindling, Neurologic; Male; Protein Precursors; Radioimmunoassay; Rats; Rats, Sprague-Dawley; RNA Probes; RNA, Messenger; Seizures

Opioid peptide gene expression was characterized in adult rat ventricular cardiac myocytes that had been cultured in the absence or the presence of phorbol 12-myristate 13-acetate. The phorbol ester induced a concentration- and time-dependent increase of prodynorphin mRNA, the maximal effect being reached after 4 h of treatment. The increase in mRNA expression was suppressed by incubation of cardiomyocytes with staurosporine, a putative protein kinase C inhibitor, and was not observed when the cells were cultured in the presence of the inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate. Incubation of cardiac myocytes with phorbol 12-myristate 13-acetate also elicited a specific and staurosporine-sensitive increase in immunoreactive dynorphin B, a biologically active end product of the precursor, both in the myocardial cells and in the culture medium. In vitro run-off transcription assays indicated that transcription of the prodynorphin gene was increased both in nuclei isolated from phorbol ester-treated myocytes and in nuclei isolated from control cells and then exposed to phorbol 12-myristate 13-acetate. No transcriptional effect was observed when cardiac myocytes or isolated nuclei where exposed to 4 alpha-phorbol 12,13-didecanoate. The phorbol ester-induced increase in prodynorphin gene transcription was prevented by pretreatment of myocytes or isolated nuclei with staurosporine, suggesting that myocardial opioid gene expression may be regulated by nuclear protein kinase C. In this regard, cardiac myocytes expressed protein kinase C-alpha, -delta, -epsilon, and -zeta, as shown by immunoblotting. Only protein kinase C-delta and protein kinase C-epsilon were expressed in nuclei that have been isolated from control myocytes, suggesting that these two isotypes of the enzyme may be part of the signal transduction pathway involved in the effect elicited by the phorbol ester on opioid gene transcription in isolated nuclei. The incubation of myocardial nuclei isolated from control cells in the presence of a protein kinase C activator induced the phosphorylation of the myristoylated alanine-rich protein kinase C substrate peptide, a specific fluorescent substrate of the enzyme. The possibility that prodynorphin gene expression may control the heart function through autocrine or paracrine mechanisms is discussed.

Topics: Alkaloids; Animals; Cell Nucleus; Cells, Cultured; Dose-Response Relationship, Drug; Dynorphins; Endorphins; Enkephalins; Enzyme Inhibitors; Gene Expression; Heart Ventricles; Isoenzymes; Kinetics; Male; Myocardium; Phorbol Esters; Protein Kinase C; Protein Precursors; Rats; Rats, Wistar; Staurosporine; Tetradecanoylphorbol Acetate; Transcription, Genetic

Colocalization of proenkephalin and prodynorphin mRNAs with each other as well as with D1, D2, and D3 dopamine receptor mRNAs was analyzed in the nucleus accumbens of the rat. Distinct combinations were detected in the rostral pole, core, and shell subdivisions of the nucleus accumbens. Proenkephalin and prodynorphin mRNAs were principally localized in separate cells in the core. All detectable prodynorphin cells in the core expressed D1 mRNA but not D2 mRNA. Conversely, approximately 95% of the proenkephalin-positive cells in this region expressed D2 mRNA but not D1 mRNA. This pattern was identical to that observed in the caudate putamen. In the rostral pole and the shell, embedded in a background of this "typical" colocalization pattern, clusters of cells expressing a distinct configuration were found. In these clusters, proenkephalin-positive cells expressed both prodynorphin and D1 mRNAs, but they did not express D2 mRNA. D3 and prodynorphin mRNAs were colocalized in "limbic" striatal areas, including the ventromedial caudate putamen, the rostral pole, and the medial shell. In contrast, D3 mRNA was not detected in any proenkephalin-positive cells. Together with the prodynorphin/D1 data, this suggests that a subset of prodynorphin cells expresses both D1 and D3 mRNAs. It is concluded that 1) clusters of cells that coexpress proenkephalin, prodynorphin, and D1 mRNAs overlap extensively with previously defined cytoarchitectural cell clusters in the nucleus accumbens and 2) a subset of the prodynorphin cells in the ventromedial caudate putamen and the nucleus accumbens contains both D1 and D3 mRNAs.

Topics: Animals; Caudate Nucleus; Dynorphins; Enkephalins; In Situ Hybridization; Male; Neurons; Nucleus Accumbens; Protein Precursors; Putamen; Rats; Rats, Sprague-Dawley; Receptors, Dopamine; RNA, Messenger

The biochemical and cellular mechanisms involved in the development and/or maintenance of morphine tolerance remain unclear. In the adult central nervous system (CNS) results are contradictory. For the neonate, a variety of drug induced deficits have been observed following prenatal addiction to opioids, although very little work on the biochemical and molecular level has been done. Therefore, the present study was carried out to investigate the effects of prenatal morphine treatment on the levels and expression of endogenous opioid peptides in brain regions of newborns. Dams were implanted with one morphine pellet (75 mg each) 1 week prior to the birth of pups. Changes in mRNA levels for the opioid peptides were determined by Northern blot analysis. Alterations in opioid peptide levels were determined by radioimmunoassays. Prenatal morphine treatment significantly increased proenkephalin mRNA levels and decreased met-enkephalin levels in striatum of newborns. These data are in contrast to what is observed in the adult CNS. These data indicate that prenatal morphine treatment may increase met-enkephalin release and/or cause inhibition at the level of translation. In addition, increased transcription may be necessary to maintain equilibrium in the system when there is an increase in met-enkephalin release.

Topics: Aging; Animals; Animals, Newborn; Blotting, Northern; Brain; Corpus Striatum; Dynorphins; Enkephalin, Methionine; Enkephalins; Female; Frontal Lobe; Gene Expression; Hippocampus; Hypothalamus; Morphine; Multivariate Analysis; Organ Specificity; Pregnancy; Prenatal Exposure Delayed Effects; Protein Biosynthesis; Protein Precursors; Rats; Rats, Sprague-Dawley; RNA, Messenger

Quantitative in situ hybridization histochemistry was used to examine the effects of unilateral 6-hydroxydopamine lesions of the ascending dopaminergic fibres on levels of mRNA encoding the neuropeptides enkephalin, dynorphin and substance P in subregions of the nucleus accumbens. The nucleus accumbens was divided into quadrants and changes in mRNA were measured along the rostrocaudal extent of the nucleus. Two weeks after the lesion an increase was found in enkephalin mRNA in the lesioned side compared to the non-lesioned side, whereas a decrease was observed for dynorphin and substance P mRNA. The changes in mRNA levels differed from quadrant to quadrant and were not uniformly distributed along the rostrocaudal axis. Both types of changes, i.e. increase and decrease, were much higher in rostral parts of the nucleus than in caudal parts, indicating regional differences in the effects of blockade of the dopaminergic neurotransmission. The lesion-induced increases and decreases in mRNA levels occurred in both the shell and the core subregions of the nucleus accumbens and were not specifically related to either of these areas. Factors are discussed that may contribute to the rostrocaudal gradient in the changes of enkephalin, substance P and dynorphin mRNA levels. On the basis of their afferent and efferent connections, the rostral and caudal parts of the nucleus accumbens are considered to be involved in different functions. The present results suggest that dopamine depletion may affect these functions in a differential manner.

Topics: Animals; Dopamine; Dynorphins; Enkephalins; Male; Neural Pathways; Nucleus Accumbens; Oxidopamine; Protein Precursors; Rats; Rats, Wistar; Receptors, Dopamine; RNA, Messenger; Substance P; Tachykinins

The effects of short- and long-term cocaine exposure on the expression of the nuclear transcription factor genes, c-fos and zif/268, as well as the opioid peptides, preprodynorphin (PPD) and preproenkephalin (PPE), in various regions of rat brain were evaluated by injecting i.p. saline or 10, 20, or 30 mg/kg cocaine HCl once daily for 1 or 10 days. In situ hybridization histochemistry was performed using 40mer oligonucleotides coding for c-fos and zif/268, or 48mers coding for PPD and PPE, followed by quantitative image analysis. Behavioral ratings demonstrated that on day 1 cocaine-induced activity increased in a dose-dependent manner. Statistical analysis revealed that repeated administration of cocaine for 10 days resulted in an augmentation of the behavioral response elicited by acute exposure to cocaine. Image analysis of film autoradiograms demonstrated that 1 h after a single injection of cocaine, the expression of c-fos and zif/268, but not PPD or PPE in the dorsal striatum and cortex, was enhanced in a dose-dependent manner as compared to that in saline controls. In contrast, administration of 30, but not 10 or 20, mg/kg cocaine for 10 days increased the expression of PPD but not PPE, mRNA and downregulated that of c-fos and zif/268 as compared to acute cocaine. These data indicate that repeated, high dose cocaine administration induces an increased PPD but not PPE genomic response and that the expression of c-fos and zif/268 is dissociable from that of PPD.

Topics: Animals; Cell Nucleus; Cocaine; Dynorphins; Endorphins; Enkephalins; Gene Expression Regulation; Image Processing, Computer-Assisted; In Situ Hybridization; Male; Neostriatum; Protein Precursors; Rats; Rats, Wistar; RNA, Messenger; Transcription Factors

The distributions of four prodynorphin-derived peptides, dynorphin A (1-17), dynorphin A (1-8), dynorphin B, and alpha-neo-endorphin were determined in 10 cortical regions and the striatum of the old world monkey (Macaca nemestrina). alpha-neo-endorphin was the most abundant peptide in both cortex and striatum. The concentrations of all four peptides were significantly greater in the striatum compared to the cortex. In general, concentrations of each peptide tended to be higher in allocortex than in neocortex. Possible inter- and intradomain processing differences, as estimated by ratios of these peptides, did not vary within cortex, but the intradomain peptide ratio, dyn A (1-17)/dyn A (1-8), was significantly greater in cortex than in striatum. These results indicate that prodynorphin is, in some ways, uniquely processed in the primate. Particularly unusual is the relatively low abundance of prodynorphin-derived products in the cortex, in the face of moderately high levels of kappa opiate receptor expression.

Topics: Animals; Cerebral Cortex; Corpus Striatum; Dynorphins; Endorphins; Enkephalins; Macaca nemestrina; Peptide Fragments; Protein Precursors; Tissue Distribution

Differences in behavioral and neurochemical responses to drugs of abuse and environmental stress have been observed between rats that have a greater locomotor response in a novel environment (high responders: HR) compared to those that have a low response to novelty (low responders: LR). This study examined nuclei associated with the nigrostriatal and mesolimbic systems for differences in mRNA content between HR and LR using Northern blot analysis. These brain regions were chosen because of their role in both drug abuse and stress responses. The mRNAs examined code for either peptide transmitters that interact with the dopaminergic system or components of the dopaminergic system that have not been previously examined for differences between HR and LR. HR rats had approximately 50% lower levels of mRNA for beta-preprotachykinin (PPT) in the core of the nucleus accumbens (NACC) compared to LR. No differences between HR and LR in mRNA levels for dynorphin (DYN), preproenkephalin (PPE), glutamic acid decarboxylase (GAD) or neurotensin (NT) were observed in the core of the NACC. In the shell region of the NACC, HR exhibited a 25% reduction in the level of mRNA for NT compared to LR. No differences between HR and LR in mRNA levels for PPT, DYN, PPE or GAD were observed in the shell of the NACC. In the medial frontal cortex and the dorsal striatum, no differences between HR and LR in mRNA levels for PPT, DYN, PPE, GAD or NT were found. In the substantia nigra and ventral tegmental area no differences between HR and LR in mRNA levels for tyrosine hydroxylase, GAD, cholecystokinin, or NT were noted.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Blotting, Northern; Cholecystokinin; Dopamine; Dynorphins; Enkephalins; Exploratory Behavior; Genetic Code; Glutamate Decarboxylase; Male; Motor Activity; Neurotensin; Protein Precursors; Rats; Rats, Sprague-Dawley; RNA, Messenger; Tachykinins; Tyrosine 3-Monooxygenase

Chronic food restriction produces a variety of physiological and behavioral adaptations including a potentiation of the reinforcing effect of food, drugs and lateral hypothalamic electrical stimulation. Previous work in this laboratory has revealed that the lowering of self-stimulation threshold by food restriction is reduced by mu- and kappa-selective opioid antagonists. In the present study, the effect of chronic food restriction on levels of three prodynorphin-derived peptides, namely dynorphin A1-17 (A1-17), dynorphin A1-8 (A1-8) and dynorphin B1-13 (B1-13) were measured in eleven brain regions known to be involved in appetite, taste and reward. Food restriction increased levels of A1-17 in dorsal medial (+19.6%), ventral medial (+24.2%) and medial preoptic (+82.9%) hypothalamic areas. Levels of A1-17 decreased in the central nucleus of the amygdala (-35.1%). Food restriction increased levels of A1-8 in nucleus accumbens (+34.4%), bed nucleus of the stria terminalis (+24.5%) and lateral hypothalamus (+41.9%). Food restriction had no effect on levels of B1-13. A1-17 is highly kappa-preferring and the brain regions in which levels increased all have a high ratio of kappa: mu and delta receptors. A1-8 is less discriminating among opioid receptor types and the brain regions in which levels increased have a low ratio of kappa: mu and delta receptors. The present results suggest that food restriction alters posttranslational processing within the dynorphin A domain of the prodynorphin precursor, possibly leading to a change in the balance between kappa and non-kappa opioid receptor stimulation in specific brain regions.

Topics: Animals; Diet; Dynorphins; Enkephalins; Hypothalamic Hormones; Male; Neuropeptides; Peptide Fragments; Protein Precursors; Radioimmunoassay; Rats; Rats, Sprague-Dawley; Time Factors

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

The effects of a single dose of amphetamine or methamphetamine on opioid peptide gene expression in the dorsal and ventral striatum of rats were investigated by quantitative in situ hybridization 3, 6, or 18 h after injection. Although amphetamine-treated rats exhibited significantly different behaviors than those treated with methamphetamine, both drugs caused a patchy increase in preprodynorphin, but not preproenkephalin, mRNA in the caudate at all 3 time points. No changes were detected in the nucleus accumbens. These data indicate that prolonged elevation of preprodynorphin expression may alter the responsiveness of striatonigral neurons to subsequent amphetamine exposure.

Topics: Amphetamine; Animals; Corpus Striatum; Dynorphins; Enkephalins; Male; Methamphetamine; Protein Precursors; Rats; Rats, Wistar; RNA, Messenger

Rats were treated with escalating doses of cocaine until they experienced a convulsion and were euthanized 1 or 3 h after the last injection. Quantitative in situ hybridization histochemistry revealed that c-fos and zif/268 mRNAs were induced at 1 h in many limbic structures and declined 3 h after cocaine-induced convulsions. Preprodynorphin and preproenkephalin signals increased in many of the same structures 3 h, but not 1 h, after cocaine-induced convulsions.

Topics: Animals; Cocaine; Convulsants; DNA-Binding Proteins; Dynorphins; Early Growth Response Protein 1; Enkephalins; Gene Expression Regulation; Immediate-Early Proteins; In Situ Hybridization; Kindling, Neurologic; Limbic System; Male; Nerve Tissue Proteins; Protein Precursors; Proto-Oncogene Proteins c-fos; Rats; Seizures; Time Factors; Transcription Factors

Neurons expressing messenger RNA encoding the opioid peptide precursors, preproenkephalin and preprodynorphin were localized in the medulla oblongata of the rat by in situ hybridization of specific DNA oligonucleotide probes. Neurons containing preproenkephalin messenger RNA were found throughout the medullary reticular formation in the gigantocellular and paragigantocellular reticular nuclei, the parvicellular and lateral reticular nuclei; commissural, medial and ventrolateral subnuclei in the nucleus tractus solitarius and the nucleus of the spinal trigeminal tract. Labelled cells were also concentrated in the more medial regions of the area postrema. In contrast, neurons containing preprodynorphin messenger RNA had a more restricted distribution and were detected in the commissural and ventrolateral nucleus tractus solitarius and nucleus of the spinal trigeminal tract, especially in the more dorsal regions. Expression of preproenkephalin and preprodynorphin messenger RNA was also examined in the dorsal vagal complex of rats that had undergone a unilateral nodose ganglionectomy or cervical vagotomy. Twenty-four hours after both cervical vagotomy and nodose ganglionectomy, there was a specific 1.5-2-fold elevation in preproenkephalin and preprodynorphin messenger RNA levels in the ventrolateral subnucleus of the contralateral nucleus tractus solitarius relative to levels in the ipsilateral nucleus tractus solitarius and in the nucleus tractus solitarius of sham-operated animals. Previous immunohistochemical studies demonstrating the co-localization of enkephalin and dynorphin in the ventrolateral nucleus tractus solitarius suggest that these changes occurred in the same population of neurons. In light of the suggested role of the ventrolateral nucleus tractus solitarius as a central respiratory centre and the activation of the intact pulmonary afferents that innervate this area following a unilateral vagotomy (which increases inspiration volume and expiratory time by affecting the Hering-Breuer reflex), our results suggest a specific involvement of enkephalin- and dynorphin-containing neurons in the ventrolateral nucleus tractus solitarius in central respiratory control mechanisms.

Topics: Animals; Dynorphins; Endorphins; Enkephalins; Ganglionectomy; Gene Expression; In Situ Hybridization; Male; Medulla Oblongata; Neck; Neurons; Nodose Ganglion; Protein Precursors; Rats; Rats, Sprague-Dawley; Respiratory System; RNA, Messenger; Vagotomy

The mechanisms involved in the development of morphine tolerance and dependence are still unknown. Recently much attention has been directed toward the changes in post receptor events. Opiate receptors, like other hormone and neurotransmitter receptors, have been shown to mediate their effects through guanine nucleotide binding proteins (G-proteins). This, in turn, may cause alterations in intracellular events, one of which is transcription of specific genes. We investigated the changes in the levels of mRNA of proenkephalin (PPE) and prodynorphin (DYN) and the stimulatory G protein alpha subunit (G alpha s) in adult morphine tolerant rats. Chronic morphine treatment induced reciprocal alterations in the levels of opioid peptide mRNA and G alpha s mRNA in discrete brain regions. In striatum, PPE mRNA decreased by 49% (P < .01) and in hypothalamus, DYN mRNA showed a decrease of 21% (P < .01). In contrast, G alpha s mRNA increased 20% (P < .01) in striatum and 97% (P < .01), in hypothalamus. In hippocampus the changes were reversed: PPE mRNA increased (55%, P < .05) and G alpha s mRNA decreased (33%, P < .01). Frontal cortex exhibited a small decrease in PPE (11.5%, P < .05) without any change on G alpha s or DYN mRNA levels. These reciprocal alterations suggest an opposing mode of regulation of G alpha s and PPE/DYN gene expression in morphine tolerant animals.

Topics: Analysis of Variance; Animals; Blotting, Northern; Brain Chemistry; Drug Tolerance; Dynorphins; Enkephalins; Gene Expression; GTP-Binding Proteins; Hypothalamus; Male; Morphine; Protein Precursors; Rats; Rats, Sprague-Dawley; RNA, Messenger

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

Big dynorphin (prodynorphin 209-240), dynorphin A (prodynorphin 209-225), dynorphin B (prodynorphin 228-240), beta-endorphin (beta-lipotrophin 61-90), or Met-enkephalin, each infused into the third ventricle, were tested for their effect on PRL release in the anesthetized turkey hen. Laying hens that received big dynorphin at the rate of 0.35 nmol/min showed a 5.1-fold increase in serum PRL at the end of a 30-min infusion period. In a second experiment, the big dynorphin-induced PRL increase was 2.6-fold. Nest-deprived, previously incubating hens that received big dynorphin displayed an 8.2-fold increase in serum PRL. Laying and nest-deprived incubating control birds infused with saline displayed no PRL increases. Laying hens that received dynorphin A (0.35 nmol/min) showed a 1.5-fold increase in serum PRL after 30 min of infusion; after 40 min of infusion, this increase rose to 2.7-fold. Infusions of beta-endorphin (0.35 nmol/min), or Met-enkephalin (0.35 nmol/min) failed to evoke PRL increases in either laying or nest-deprived incubating turkeys. Infusion of big dynorphin or dynorphin A for 120 min maintained an elevated PRL level across the period, a level equal to that evoked by electrical stimulation of the medial preoptic nucleus (ES/POM). Infusion of dynorphin B (0.48 nmol/min) or a reduced dose of dynorphin A (0.09 nmol/min) augmented the PRL response evoked by ES/POM. No augmentation was noted for beta-endorphin or Met-enkephalin, nor for saline-infused controls. The dynorphin-induced PRL response appeared to be dose-dependent.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; beta-Endorphin; Dose-Response Relationship, Drug; Dynorphins; Electric Stimulation; Enkephalin, Methionine; Enkephalins; Female; Kinetics; Prolactin; Protein Precursors; Turkeys

The distributions and extent of processing of four prodynorphin-derived peptides (dynorphin A (1-17), dynorphin A (1-8), dynorphin B, and alpha-neoendorphin) were determined in ten regions of the cortex as well as in the striatum of the guinea-pig. There were significant differences between concentrations of these peptides in most cortical regions, with alpha-neoendorphin being several times more abundant than the other peptides, and dynorphin A (1-17) being present in the least amount. There were significant between-region differences in concentration for each peptide, although most regions had concentrations similar to those seen in the striatum. Concentrations of each peptide tended to be higher in piriform, entorhinal, motor, and auditory cortex than in other cortical regions. The extent of processing of prodynorphin varied across cortical regions as well, primarily due to the extent of processing to alpha-neoendorphin. Prodynorphin mRNA levels were not significantly different between cortical regions or from the amount observed in the striatum. Although specific regional variation exists, it appears that in general prodynorphin is expressed and processed in a similar manner in the cortex as in the striatum.

Topics: Animals; Blotting, Northern; Cerebral Cortex; Corpus Striatum; Dynorphins; Endorphins; Enkephalins; Guinea Pigs; Male; Organ Specificity; Peptide Fragments; Protein Precursors; Radioimmunoassay; RNA, Messenger

The PC12 is a cloned rat pheochromocytoma cell line that retains a number of chromaffin cell characteristics, such as the presence of nicotinic cholinergic receptors, the synthesis and secretion of catecholamines, and the expression of a number of neuropeptide genes. The PC12 cell line is a useful model for the study of neuronal development, since PC12 cells can be induced to differentiate toward sympathetic neurons after exposure to nerve growth factor (NGF). PC12 cells can also be induced to differentiate toward the opposite direction, i.e. toward mature chromaffin cells. Morphological and biochemical changes mark the differentiation of PC12 cells toward either direction. Among the substances proposed as biochemical markers of PC12 cell differentiation toward chromaffin cells is the endogenous opioid precursor proenkephalin and its posttranslational peptide products. Indeed, the proenkephalin gene is expressed in both adrenal chromaffin and PC12 cells. The secretion of enkephalins from PC12 cells increases by several-fold after differentiation toward chromaffin cells. On the other hand, prodynorphin (another endogenous opioid precursor) is not present in normal adrenal chromaffin cells, but it is synthesized by human pheochromocytomas. It, thus, appears that dedifferentiation of chromaffin cells induces expression of the prodynorphin gene. Consequently, the aim of this study was to determine whether the rat pheochromocytoma-derived PC12 cell line expresses the prodynorphin gene, if it secretes dynorphins, and if the NGF-induced differentiation of PC12 cells toward sympathetic neurons affects the secretion of the latter. We found the following. 1) PC12 cells synthesize prodynorphin and secrete its peptide products. 2) The size of the prodynorphin transcript and the mol wt of its dominant form of dynorphin appear to be similar or identical to those of prodynorphin in rat anterior pituitary. 3) PC12 dynorphin secretion is increased, in a dose-dependent manner, after nicotinic cholinergic stimulation, an effect blocked by the specific nicotine antagonist hexamethonium. Thus, it appears that after cholinergic stimulation, PC12 dynorphin is cosecreted with catecholamines, a phenomenon described for a number of neuropeptides, including the proenkephalin-derived opioids. 4) The NGF-mediated differentiation of PC12 cells into sympathetic neurons exerted a stimulatory effect on basal, nicotine-induced, and depolarization-dependent dynorphin secretion. However, NG

Topics: Animals; Blotting, Northern; Cell Differentiation; Chromatography, Gel; Dynorphins; Enkephalins; Gene Expression; Hexamethonium; Hexamethonium Compounds; Molecular Weight; Nerve Growth Factors; Nicotine; PC12 Cells; Potassium Chloride; Protein Precursors; Rats; RNA, Messenger

An animal model of nociception involving unilateral hindpaw inflammation has been used to examine behavioral, molecular, and biochemical aspects of well-characterized spinal cord neural circuits involved in pain transmission. The neurotoxin capsaicin administered neonatally was used to modify this neuronal system by producing a selective destruction of most small, unmyelinated primary afferent axons. Capsaicin had minimal effects on the behavioral hyperalgesia and edema associated with the hindpaw inflammation and on the constitutive expression of preprodynorphin (PPD) mRNA and preproenkephalin mRNA in the spinal cord. However, the inflammation-induced increases in Fos-like immunoreactivity (Fos-LI) and in PPD mRNA were greatly attenuated by neonatal capsaicin treatment. The data indicate that input from small-diameter unmyelinated primary afferents is important for the stimulus-induced increase in Fos-LI and PPD mRNA. Our finding that neonatal capsaicin reduces the levels of Fos-LI and PPD mRNA in a related fashion in the spinal dorsal horn provides further evidence for a relationship between the protein product of the c-fos protooncogene and regulation of dynorphin gene transcription.

Topics: Animals; Animals, Newborn; Capsaicin; Dynorphins; Enkephalins; Female; Foot Diseases; Gene Expression; Hyperalgesia; Inflammation; Male; Protein Precursors; Proto-Oncogene Proteins c-fos; Rats; Rats, Inbred Strains; Spinal Cord; Substance P

The opioid peptide precursors, preprodynorphin and preproenkephalin show structure similarity with a transcription factor, hunchback and the putative helix-loop-helix DNA-binding proteins, lil-1, tal and twist. Segments with similarity contain the three enkephalin sequences in preprodynorphin and one in preproenkephalin which are present within heptapeptide repeats characteristic of an alpha-helical coiled-coil structure distinctive of an amphipathic helix-loop-helix DNA-binding motif. Hunchback and the opioid prohormones also have cystein-rich regions characteristic of zinc-finger domains in common.

Topics: Amino Acid Sequence; Animals; Base Sequence; Cattle; DNA; DNA-Binding Proteins; Dynorphins; Enkephalins; Humans; Molecular Sequence Data; Protein Precursors; Sequence Homology, Nucleic Acid; Transcription Factors

The expression of preprodynorphin has been studied using the Northern blot technique. Ten human cell lines, six small cell lung carcinoma (SCLC), one large cell carcinoma (LCC), two neuroblastoma and one lymphoblast-like cell line, were screened with a preprodynorphin cRNA-probe. Tryptic digestion followed by radioimmunoassay for Leu-enkephalin-Arg6 was used to detect possible translation of the preprodynorphin transcript. Of the ten cell lines investigated we found that all expressed preprodynorphin-mRNA to various degrees, and that this transcript is also translated. Two of the cell lines, neuroblastoma SK-N-MC and SCLC H69, also expressed preproenkephalin-mRNA. This set of cell lines provides a useful model of human origin in which the regulation of the preprodynorphin gene and the posttranslational processing of its products can be studied and compared.

Topics: Blotting, Northern; Carcinoma, Small Cell; Dynorphins; Enkephalins; Humans; Protein Precursors; RNA, Messenger; Tumor Cells, Cultured

Stimulation of the perforant path elicits a behavioral response, wet dog shakes (WDS), and reduction in hippocampal dynorphin A(1-8) immunoreactivity (DYN-IR) and prodynorphin mRNA (DYN mRNA) in rats. This study examined whether glutamate, the proposed endogenous transmitter released by perforant fibers, mediated the above responses. A glutamate antagonist, gamma-D-glutamylglycine (DGG, 25 micrograms/0.5 microliters), or artificial cerebrospinal fluid (ACSF, 0.5 microliters) was injected into the ventral hippocampus 10-20 min prior to acute or daily stimulation of the left perforant path in rats. In acute stimulation experiments, 4 consecutive stimulation trials elicited a total of 73 +/- 4 WDS at an average threshold intensity of 0.46 +/- 0.03 mA in ACSF-treated rats. The hippocampal DYN-IR in these animals decreased by more than 40% in both dorsal and ventral hippocampus relative to sham-stimulated rats. DGG injections significantly elevated the threshold for WDS (0.78 +/- 0.05 mA, P less than 0.01), reduced the number of WDS (45 +/- 6, P less than 0.01), and partially antagonized stimulation-induced reduction of DYN-IR in the ventral, but not dorsal, hippocampus. In daily stimulation experiments, rats received a single trial of stimulation once per day for 6 days. Daily DGG pretreatment almost completely abolished WDS at control threshold intensities, and significantly inhibited stimulation-induced decrease of DYN-IR in both dorsal and ventral hippocampus. In situ hybridization using a 35S-labeled oligodeoxyribonucleotide probe demonstrated a clear depletion of DYN mRNA signal in the dentate granule cell layer of ACSF-treated animals. This depletion was completely prevented in DGG-treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Dipeptides; Dynorphins; Enkephalins; Excitatory Amino Acid Antagonists; Glutamic Acid; Hippocampus; Histocytochemistry; Male; Neural Pathways; Peptide Fragments; Protein Precursors; Rats; Rats, Inbred F344; RNA, Messenger; Tremor

This study describes three different endopeptidases in human spinal cord with high affinity for the dynorphins. The enzymes, with strict specificity towards paired basic residues, release Leu-enkephalin and its C-terminal extensions - Arg6 and -Arg6 -Arg7 from the substrate peptides. They differ with regard to molecular size, charge properties, and inhibitory profile. Two of the enzymes show optimal activity at neutral pH, whereas the third enzyme has its activity optimum at pH 5.6. They are all suggested to be of importance for the formation of Leu-enkephalin from prodynorphin.

Topics: Amino Acid Sequence; Dynorphins; Enkephalin, Leucine; Enkephalins; Humans; Hydrogen-Ion Concentration; In Vitro Techniques; Male; Molecular Sequence Data; Molecular Weight; Protein Precursors; Serine Endopeptidases; Serine Proteinase Inhibitors; Spinal Cord

Experiments were conducted (i) to determine the hemodynamic (blood pressure and heart rate) responses of conscious rats following intrathecal (IT) administration of endogenous prodynorphin-derived opioids into the lower thoracic space, (ii) to identify the receptors involved in mediating their cardiovascular responses, and (iii) to reveal any possible hemodynamic interactions with the neuropeptide arginine vasopressin. Male Sprague-Dawley rats were surgically prepared with femoral arterial and venous catheters as well as a spinal catheter (into lower thoracic region, T9-T12). After recovery, hemodynamic responses were observed in conscious rats for 5-10 min after IT injections of artificial cerebrospinal fluid (CSF) solution, prodynorphin-derived opioids (dynorphin A, dynorphin B, dynorphin A (1-13), dynorphin A (1-10), alpha- and beta-neoendorphin, leucine enkephalin (LE), methionine enkephalin (ME), arginine vasopressin (AVP), or norepinephrine (NE)). IT injections of AVP (10 or 20 pmol), dynorphin A (1-13), or dynorphin A (10-20 nmol) caused pressor effects associated with a prolonged and significant bradycardia. Equimolar (20 nmol) concentrations of LE, ME, alpha- and beta-neoendorphin, and dynorphin A (1-10) caused no significant blood pressure or heart rate changes. Combined IT injections of dynorphin A (1-13) and AVP caused apparent additive pressor effects when compared with the same dose of either peptide given alone. IT infusion of the specific AVP-V1 antagonist d(CH2)5Tyr(Me)AVP before subsequent IT AVP, dynorphin A (1-13), or NE administration inhibited only the subsequent pressor responses to AVP. The kappa-opioid antagonist (Mr2266) infused IT blocked the pressor actions of subsequent dynorphin A administration and not AVP or NE.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Arginine Vasopressin; beta-Endorphin; Blood Pressure; Dynorphins; Endorphins; Enkephalin, Leucine; Enkephalin, Methionine; Enkephalins; Heart Rate; Hemodynamics; Injections, Spinal; Male; Narcotics; Norepinephrine; Peptide Fragments; Protein Precursors; Rats; Rats, Inbred Strains

The regulatory effect of the perforant path on opioid gene expression in the entorhinal cortex-hippocampal region was investigated. The left perforant path was electrically stimulated at the angular bundle under conditions which elicit wet dog shakes but no motor seizures in rats. Animals were given either an acute stimulation composed of several consecutive stimulation trials, or daily stimulations with a single trial every day for 6 days. Rats were then sacrificed at 24 h or 6 days after the last trial. The amounts of prodynorphin mRNA (DYN mRNA) and proenkephalin A mRNA (EK mRNA) in the hippocampus and entorhinal cortex were measured by RNA blot analysis. Dynorphin A(1-8) and [Met5]enkephalin immunoreactivities were determined by radioimmunoassay. A decrease in DYN mRNA level of approximately 50-80% was found on both sides of the hippocampus 24 h after both acute and daily stimulation. Hippocampal dynorphin A(1-8) immunoreactivity was also reduced at 24 h, and persisted for at least 6 days. In contrast, bilateral increases in EK mRNA level were observed in the hippocampus (54-101%) and entorhinal cortex (97-165%) 24 h after the acute stimulation. Also, [Met5]enkephalin immunoreactivity in the hippocampus tended to be increased at this time. These results indicate that activation of the perforant path inhibits the gene expression of prodynorphin, but enhances that of proenkephalin in the entorhinal cortex-hippocampal region.

Topics: Animals; Cerebral Cortex; Dynorphins; Electric Stimulation; Enkephalins; Gene Expression Regulation; Hippocampus; Male; Nucleic Acid Hybridization; Peptide Fragments; Protein Precursors; Rats; Rats, Inbred F344; RNA, Messenger

Young pigs, which are useful experimental animals for biomedical research, were prepared with lateral intracerebroventricular (ICV) cannulae and housed individually in cages fitted with operant panels, with food and water ad lib. ICV injection of 200 micrograms of dynorphin A 1-17 or 1-13 resulted in a significant meal commencing within 2-5 min. Shorter fragments of dynorphin (1-10, 1-9, 1-8) were ineffective at inducing feeding as was dynorphin B (rimorphin). In the same situation, leumorphin and alpha neo-endorphin (200 micrograms) elicited significant feeding but beta neo-endorphin did not. Dynorphin 1-17 or 1-13, administered 5 min before feeding started, increased meal size when pigs were fed after 4-h deprivation. Naloxone ICV (0.4 mg) significantly reduced food intake in pigs feeding after 4-h deprivation and its main effect was in the second half of the meal. Naloxone also abolished the effect of ICV dynorphin. It is concluded that dynorphin and related endogenous opioids are involved in the regulation of food intake in pigs.

Topics: Animals; Appetitive Behavior; Brain; Conditioning, Operant; Dynorphins; Endorphins; Enkephalins; Feeding Behavior; Female; Injections, Intraventricular; Male; Motivation; Naloxone; Peptide Fragments; Protein Precursors; Receptors, Opioid; Swine

We report here that prodynorphin mRNA and prodynorphin-derived peptides are synthesized in the R2C rat Leydig tumor cell line. The size of the prodynorphin transcript found in these cells (approximately 2200 nucleotides) is identical to that found in the intact testis. R2C cells also contain proteolytically processed prodynorphin-derived peptides. In R2C cells, the endogenous prodynorphin gene and cellular levels of prodynophin-derived peptides are positively regulated by cAMP analogs, while phorbol esters exert a slight negative regulation of the prodynorphin mRNA. Using gene transfer techniques, we have identified a 210-basepair fragment of the rat prodynorphin gene which initiates the transcription of the bacterial reporter molecule, chloramphenicol acetyl transferase. The chimeric fusion gene, when transfected into R2C cells, exhibited the same positive response to cAMP analogs as the endogenous gene. The results suggest that a cAMP regulatory element resides within the cloned rat prodynorphin fragment, and that the element is functionally active in R2C cells.

Topics: 1-Methyl-3-isobutylxanthine; 8-Bromo Cyclic Adenosine Monophosphate; Animals; Blotting, Northern; Cyclic AMP; Dynorphins; Enkephalin, Leucine; Enkephalins; Gene Expression Regulation; Leydig Cell Tumor; Male; Mice; Nucleic Acid Hybridization; Pro-Opiomelanocortin; Protein Precursors; Rats; RNA, Messenger; Testicular Neoplasms; Testis; Tetradecanoylphorbol Acetate; Transcription, Genetic; Tumor Cells, Cultured

Steady-state analyses of prodynorphin-derived opioid peptides were conducted on acid extracts of the brain of the frog. Xenopus laevis. Radioimmunoassays specific for dynorphin A(1-17), dynorphin A(1-8), alpha-neoendorphin and dynorphin B coupled with gel filtration chromatography and reverse phase high performance liquid chromatography were used. The major prodynorphin-related end-product detected was alpha-neoendorphin. Interestingly, Leu-enkephalin was also detected. Since the Xenopus proenkephalin precursor does not contain the Leu-enkephalin sequence, these data suggest that some of the prodynorphin-related end-products had been cleaved to yield Leu-enkephalin.

Topics: Animals; Brain; Dynorphins; Endorphins; Enkephalins; Protein Precursors; Xenopus laevis

Radioimmunoassay revealed increased dynorphin A(1-8)-like immunoreactivity [dynA(1-8)LI] in the aged rat brain. Among a number of brain regions examined, an age-related dynA(1-8)LI elevation was found only in the hippocampal formation and frontal cortex. Moreover, the increase in dynA(1-8)LI in the aged hippocampus was associated with a decline in spatial learning ability: dynA(1-8)LI distinguished aged rats that were behaviorally impaired from aged cohorts that learned the spatial task as rapidly as younger animals. Northern blot hybridization using a 32P-labeled complementary RNA probe encoding rat prodynorphin indicated that the abundance of prodynorphin mRNA was also significantly increased in the hippocampal formation of aged rats with identified spatial learning impairments.

Topics: Aging; Animals; Blotting, Northern; Cognition; Dynorphins; Enkephalins; Hippocampus; Learning; Protein Precursors; Rats; RNA, Messenger

Guinea-pig ileum was dissected and the mucosa, submucosa and external musculature extracted with aqueous acetic acid for measurement of four prodynorphin-derived peptides, namely dynorphin A 1-8, dynorphin A 1-17, dynorphin B, and alpha-neoendorphin. The peptide-like immunoreactive material extracted from the external musculature was characterized by multi-dimensional chromatographic analysis and compared to synthetic porcine standards. The chromatographic methods utilized were: reversed-phase high performance liquid chromatography (RP-HPLC), using two different eluants; cation exchange high performance liquid chromatography (CE-HPLC) and gel filtration chromatography. The dynorphin A 1-8-like immunoreactive material was homogeneous and coeluted with the standard in all chromatographic modes. The dynorphin A 1-17-like and dynorphin B-like immunoreactive material was heterogeneous but showed a peak that coeluted with synthetic standard in all chromatographic modes. The alpha-neoendorphin-like immunoreactive material also appeared to be heterogeneous with the major component on CE-HPLC coeluting with the synthetic peptide standard while the major component on RP-HPLC eluted differently. It was concluded that the guinea-pig ileum contains immunoreactivity for peptides derived from all coding regions of the prodynorphin gene and that these peptides may be present in multiple immunoreactive forms.

Topics: Animals; Chromatography, Gel; Chromatography, High Pressure Liquid; Dynorphins; Endorphins; Enkephalins; Guinea Pigs; Ileum; Peptide Fragments; Protein Precursors; Radioimmunoassay

Peripheral inflammation produces a rapid elevation (within 4 h) in preprodynorphin mRNA in neurons of the dorsal spinal cord and an even more rapid elevation in c-fos proto-oncogene mRNA (within 30 min). During this period a relatively modest increase is also observed in spinal cord preproenkephalin mRNA. Previous anatomical studies have shown that the neurons in which these transcripts increase have overlapping distributions. Assuming that these events occur in the same cells, it suggests the possibility that newly synthesized c-fos protein may participate in transcriptional regulation of opioid genes in spinal cord.

Topics: Animals; Dynorphins; Enkephalins; Gene Expression Regulation; Hyperalgesia; Inflammation; Male; Protein Precursors; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-fos; Rats; Rats, Inbred Strains; RNA, Messenger; Spinal Cord

The response of dynorphinergic neurons in the lumbosacral spinal cord of the rat to chronic arthritic inflammation was studied by the combined use of biochemical and immunohistochemical procedures. In polyarthritic rats, in which all four limbs showed a swelling, inflammation and hyperalgesia, a pronounced elevation was seen in the level of messenger ribonucleic acid encoding prodynorphin (pro-enkephalin B) in the lumbosacral spinal cord. In addition, the levels of immunoreactive dynorphin A1-17, a primary gene product of this precursor, were greatly increased. This activation was reflected in a striking intensification of the immunohistochemical staining of both dynorphin and alpha/beta-neo-endorphin, a further major product of pro-dynorphin. In control animals perikarya were stained exceedingly rarely and encountered only in laminae I and II. Stained fibres and varicosities were seen throughout the dorsal and ventral gray matter, being most concentrated in laminae I, II, IV and V of the dorsal horn and dorsolateral to the central canal. In polyarthritic rats, fibres and varicosities were much more intensely stained throughout the cord, particularly in laminae I/II, IV and V and dorsolateral to the central canal. Many strongly-stained perikarya could be seen: these comprised many small diameter cells in laminae I and II, and some large diameter marginal neurons and large diameter cells, heterogenous in appearance, in the deeper laminae IV and V. Monolaterally inflamed rats injected in the right hind-paw showed pathological changes only in this limb. Correspondingly, in unilateral inflammation, an elevation in immunoreactive dynorphin was seen exclusively in the right dorsal horn and the above-described intensification of staining for dynorphin and neo-endorphin was seen only in this quadrant. This reveals the neuroanatomical specificity of the response. Thus, in the lumbosacral cord of the rat, pro-dynorphin neurons are most preponderant in laminae I, II, IV and V. A pronounced intensification of the immunohistochemical staining of these neurons is seen in chronic arthritis. Furthermore, there is a parallel elevation in the levels of messenger ribonucleic acid encoding pro-dynorphin and of its primary products dynorphin and neo-endorphin. These findings demonstrate an enhancement in the functional activity of spinal cord localized dynorphin neurons in the response to chronic arthritic inflammation.

Topics: Animals; Arthritis; Dynorphins; Enkephalins; Gene Expression Regulation; Immunohistochemistry; Male; Pain; Protein Precursors; Rats; Rats, Inbred Strains; RNA, Messenger; Spinal Cord

The neuroanatomical distribution of the prodynorphin precursor molecule in the forebrain of the male Syrian hamster (Mesocricetus auratus) has been studied with a novel antiserum directed against the C-terminus of the leumorphin [dynorphin B (1-29)] peptide product. C-peptide staining in sections from colchicine-treated hamsters is compared to staining in sections from untreated animals. In addition, the pattern of C-peptide immunostaining in hamster brain is compared to that in the rat brain. Finally, the C-peptide immunolabeling patterns in hamsters and rats are compared to those obtained with antisera to dynorphin A (1-17) and dynorphin B (1-13). Areas of heaviest prodynorphin immunoreactivity in the hamster include the hippocampal formation, lateral septum, bed nucleus of the stria terminalis, medial preoptic area, medial and central amygdaloid nuclei, ventral pallidum, substantia nigra, and numerous hypothalamic nuclei. Although this C-peptide staining pattern is similar to dynorphin staining reported previously in the rat, several species differences are apparent. Whereas moderate dentate gyrus granule cell staining and no CA4 cell staining have been reported in the rat hippocampal formation, intense immunostaining in the dentate gyrus and CA4 cell labeling are observed in the hamster. In addition, the medial preoptic area, bed nucleus of the stria terminalis, and medial nucleus of the amygdala stain lightly for prodynorphin-containing fibers and cells in the rat, compared to heavy cell and fiber staining in the hamster in all three of these regions. In the rat there is no differential staining between tissues processed with the C-peptide, dynorphin A, and dynorphin B antisera, but numerous areas of the hamster brain show striking differences. In most hamster brain areas containing prodynorphin peptides, the C-peptide antiserum immunolabels more cells and fibers than the dynorphin B antiserum, which in turn labels more cells and fibers than dynorphin A antiserum. However, exceptions to this hierarchy of staining intensity are found in the lateral hypothalamus, substantia nigra, arcuate nucleus, and habenula. The differences in staining patterns between rat and hamster are greatest when C-peptide antiserum is used; apparent species differences are present, though less pronounced, in dynorphin B- and dynorphin A-immunostained material.

Topics: Animals; Brain Chemistry; Colchicine; Cricetinae; Dynorphins; Endorphins; Enkephalins; Immune Sera; Immunoenzyme Techniques; Male; Mesocricetus; Peptide Fragments; Perfusion; Protein Precursors; Rats; Rats, Inbred Strains; Species Specificity

Levels of mRNAs coding for prodynorphin (Pro-Dyn) and proenkephalin (Pro-Enk) as well as the levels of immunoreactive (ir)-dynorphin (Dyn) and (ir)-Met-enkephalin (Met-Enk) were measured in the spinal cord of rats, 65 h following transection or injury of the spinal cord at the T6 spinal segment. Levels of both Pro-Dyn mRNA and of ir-Dyn were significantly increased between 60 and 150%, above control levels in the whole spinal cord, whereas those of Pro-Enk mRNA and of ir-Met-Enk remained unchanged. The increase in spinal levels of Pro-Dyn mRNA were highest in the areas close to the side of transection and indicate an involvement of the Pro-Dyn opioid system in the response to spinal injury and transection.

Topics: Animals; Dynorphins; Enkephalin, Methionine; Enkephalins; Gene Expression Regulation; Male; Nucleic Acid Hybridization; Protein Precursors; Rats; Rats, Inbred Strains; RNA; RNA, Complementary; RNA, Messenger; Spinal Cord Injuries

By the use of highly selective antisera and an immunohistochemical technique the possible coexistence of proenkephalin- (PRO-ENK)- and prodynorphin (PRO-DYN)-derived peptides was examined in 4- to 6-micron thick serial sections of the L4-L5 segments of the spinal cord of non-colchicine-treated polyarthritic rats. In control, non-colchicine treated animals, virtually no cell bodies stained for the PRO-ENK-derived peptides, heptapeptide (MRF) and octapeptide (MRGL), nor for the PRO-DYN-derived peptides, dynorphin A (DYN) and alpha-neoendorphin (NEO). In contrast, in polyarthritic rats, numerous large (15-30 micron) multipolar neurons could be visualized with each antiserum in laminae IV/V. Alternate staining of adjacent sections with either anti-MRF or anti-MRGL antisera, followed by either anti-DYN or anti-NEO antisera, revealed a clear coexistence of PRO-ENK and PRO-DYN peptides. It was possible to demonstrate co-localization of all 4 opioids in a single cell. It appeared that all cells staining for PRO-ENK peptides in laminae IV/V also stained for PRO-DYN peptides.

Topics: Animals; Arthritis, Rheumatoid; Dynorphins; Endorphins; Enkephalins; Male; Oligopeptides; Protein Precursors; Rats; Rats, Inbred Strains; Spinal Cord

Cerebrospinal fluid activity of a dynorphin-converting enzyme transforming prodynorphin-derived peptides to [Leu]enkephalin-Arg6 was measured in 12 women at term pregnancy before cesarean section and in eight nonpregnant, nonpuerperal controls. In pregnant women, the dynorphin-converting enzyme activity was significantly lower (mean +/- SD 6.8 +/- 3.8 U/L) than in nonpregnant controls (11.7 +/- 2.6 U/L; P less than .01). Furthermore, prodynorphin-derived [Leu]enkephalin-Arg6-containing polypeptides were significantly increased in samples from pregnant women (P less than .05). This indicates that a reduced activity of opioid peptide-degrading enzymes might contribute to an increased resistance to pain at term pregnancy.

Topics: Cesarean Section; Chromatography, High Pressure Liquid; Dynorphins; Enkephalin, Leucine; Enkephalins; Female; Humans; Luteal Phase; Pregnancy; Protein Precursors; Radioimmunoassay; Serine Endopeptidases

The aim of this study was to understand the possible influence of the antimanic drug, lithium, and the neuroleptic, haloperidol, alone or in combination, on the regulation of dynorphin biosynthesis in the striatum. The study was done using male Fisher-344 rats subjected to a regimen of subchronic administration of lithium chloride (4 mEq/kg/day for 1,2,4 or 6 days, i.p.) or a regimen of chronic oral administration of a diet containing lithium carbonate (1.5 g/kg of the diet). Subchronic administration of lithium increased striatal dynorphin A(1-8)-like immunoreactivity (DN-LI) in a time-related fashion. Immunocytochemistry revealed an increase in DN-LI in fibers and cells clustered in 'patches' throughout striatum. The increase in DN-LI was reversible on cessation of lithium administration. Concurrent administration of lithium and an opiate antagonist, naltrexone, or a dopamine receptor antagonist, haloperidol, did not influence the changes induced by lithium. Chronic oral administration of lithium for 21 days led to an increase in DN-LI in the striatum. Co-administration of haloperidol with the 21 day regimen of lithium administration failed to affect the increase in DN-LI. The prodynorphin mRNA abundance in the striatum was quantitated by a molecular hybridization procedure using a prodynorphin 32P-cRNA probe generated from the Riboprobe system. Evidence from the Northern blot analysis reveals that lithium increases the prodynorphin mRNA abundance in the striatum. These results indicate that lithium affects the dynamics of prodynorphin biosynthesis in the striatum, presumably increasing transcription and/or translational processes.

Topics: Animals; Basal Ganglia; Chlorides; Corpus Striatum; Dose-Response Relationship, Drug; Dynorphins; Enkephalins; Gene Expression Regulation; Genes; Haloperidol; Immunoenzyme Techniques; Lithium; Lithium Carbonate; Lithium Chloride; Male; Peptide Fragments; Protein Precursors; Rats; Rats, Inbred F344; RNA, Messenger; Transcription, Genetic

A unilateral experimental inflammation of the hindlimb produces hyperalgesia to both mechanical and radiant thermal stimuli that is rapid in onset. During this period, parameters of dynorphin biosynthesis are elevated to a much greater degree than those of the enkephalin system. An increase in the content of the peptide dynorphin A(1-8) occurs in the spinal cord segments that receive sensory input from the affected limb. This is accompanied by a rapid (within 24 h) and pronounced increase in the levels of mRNA coding for the dynorphin protein precursor. Maximum elevations (6- to 8-fold) of preprodynorphin mRNA are observed between days 2 and 5 subsequent to the induction of inflammation. Compared to the increase in mRNA, the increase in dynorphin A(1-8) peptide was appreciably delayed and proportionately less; maximal increases in peptide (3-fold) were seen at day 5 of inflammation. Dorsal spinal cord preproenkephalin mRNA is elevated to a lesser degree (50-80%). However, the increase in preproenkephalin mRNA is apparently not enough to yield a measurable increase in the proenkephalin-derived peptide met5-enkephalin-Arg6-Gly7-Leu8, the levels of which showed no significant change during the 14-day inflammatory period. These data suggest the active participation of opioid neurons, especially those containing dynorphin, at the spinal level, in the modulation of sensory afferent input during peripheral inflammatory pain states.

Topics: Animals; DNA; Dynorphins; Enkephalin, Methionine; Enkephalins; Hyperalgesia; Hyperesthesia; Male; Nucleic Acid Hybridization; Peptide Fragments; Protein Precursors; Rats; Rats, Inbred Strains; RNA, Messenger

It has long been disputed whether mammalian enterochromaffin (EC-) cells contain a peptide in addition to serotonin. Previous immunohistochemical studies have provided evidence for the presence of enkephalins in EC-cells. These findings, however, are equivocal. Therefore, the problem of opioid peptides in EC-cells has been re-examined in the gastro-intestinal mucosa of dog, guinea-pig and man. A battery of antisera against derivatives of pro-opiomelanocortin, pro-enkephalin and pro-dynorphin have been applied to semithin serial sections of the tissues, in combination with fluorescence histochemistry and serotonin immunocytochemistry. Our findings indicate that EC-cells of the investigated species contain pro-dynorphin-related peptides, i.e. dynorphin A and alpha-neo-endorphin, but no derivatives from pro-opiomelanocortin or pro-enkephalin. Since remarkable interspecies variations occur with respect to the number and staining characteristics of opioid immunoreactive EC-cells, it is concluded that pro-dynorphin shows specific routes of post-translational processing depending upon the species and the gastro-intestinal segment investigated. Future studies should focus on the mutual relationships between serotonin and dynorphins and on the physiological significance of these peptides in the gastrointestinal tract.

Topics: Adrenal Medulla; Animals; Cattle; Chromaffin System; Digestive System; Dogs; Dynorphins; Endorphins; Enkephalins; Enterochromaffin Cells; Guinea Pigs; Humans; Immunohistochemistry; Pancreas; Pituitary Gland; Pro-Opiomelanocortin; Protein Precursors

Preproenkephalin and preprodynorphin mRNAs can be detected by in situ hybridization in medium-sized striatal neurons in normal rats and in rats with unilateral cerebral cortical lesions. Hybridization of 35S-labeled oligonucleotide cDNAs complementary to specific regions of each mRNA reveals that preproenkephalin-expressing neurons are more numerous than cells expressing preprodynorphin. Hybridization densities above enkephalin-positive neurons are also more than twice those noted above preprodynorphin-expressing cells. Northern analyses of mRNA extracted from the striatum are consistent with these relationships. The striatal preproenkephalin hybridization densities are decreased ipsilateral to cerebral cortical lesions; this change evolves largely between 1 and 5 d following the lesion. Striatal preproenkephalin mRNA is thus more prominent than preprodynorphin mRNA and depends on cerebral cortical inputs for its full expression.

Topics: Animals; Blotting, Northern; Cerebral Cortex; Corpus Striatum; DNA; Dynorphins; Enkephalins; Male; Neurons; Neurons, Afferent; Nucleic Acid Hybridization; Protein Precursors; Rats; RNA, Messenger

A procedure is described for the isolation of intact hippocampal mossy fiber synaptosomes. Electron microscopic examination revealed numerous synaptosomal profiles which are clearly of mossy fiber origin, indicated by their large size (2-6 micron diameter) and characteristic morphology. Furthermore, this fraction is enriched in zinc and dynorphin B which appear to be concentrated in mossy fiber terminals in vivo. Synaptosomes isolated by this procedure accumulated 2-deoxyglucose and retained 88% of total lactate dehydrogenase activity after incubation at 30 degrees C for 60 minutes, indicating a high degree of membrane integrity. Oxygen consumption was stimulated 4-fold by veratridine (0.1 mM) and inhibited 90% by ouabain (1 mM), suggesting that synaptosomal metabolism remained tightly coupled to ouabain-sensitive ATPase activity. Potassium-stimulated (45 mM) release of dynorphin B was completely dependent upon the presence of extrasynaptosomal calcium, while only 30% of the evoked release of glutamate was calcium-dependent. D-aspartate, which exchanges glutamate out of the cytoplasmic pool, virtually eliminated the calcium-independent component of glutamate release. This synaptosomal preparation will be useful in identifying the factors that modulate the release of amino acid and opioid neurotransmitters from hippocampal nerve terminals and in the investigation of their presynaptic mechanisms of action.

Topics: Animals; Aspartic Acid; Deoxyglucose; Dynorphins; Enkephalins; Glutamates; Glutamic Acid; Hippocampus; Male; Microscopy, Electron; Protein Precursors; Rats; Rats, Inbred Strains; Synaptosomes

We demonstrated the presence and the secretion in vivo and in vitro of immunoreactive preproenkephalin B-derived opioid peptides (alpha-neoendorphin, dynorphin and leumorphin) in human phaeochromocytomas. In seventeen human phaeochromocytomas and two human adrenal medullas, the tissue contents of immunoreactive preproenkephalin B-derived opioid peptides (alpha-neoendorphin, dynorphin and leumorphin) and leu-enkephalin were studied by specific RIAs. Compared with a remarkable wide distribution in amounts of immunoreactive leu-enkephalin (1063 +/- 437 pg/mg, mean +/- SE), small amounts of immunoreactive alpha-neoendorphin (22.6 +/- 6.4 pg/mg) and dynorphin (8.5 +/- 1.2 pg/mg) were detected in all seventeen human phaeochromocytomas and the two human adrenal medullas. Leumorphin-like immunoreactivity was detected in only four tumours. Gel chromatographic studies revealed the presence of preproenkephalin B-derived peptides and their high molecular forms. A significant positive correlation between the tumour tissue contents of immunoreactive alpha-neoendorphin and of dynorphin was observed. Nicotine (10(-5), 10(-4) mol/l) significantly stimulated the secretion of immunoreactive alpha-neoendorphin and dynorphin as well as leu-enkephalin and catecholamines from cultured human phaeochromocytoma cells. Administration of 1 mg of glucagon to a patient with medullary phaeochromocytoma induced a rapid increase in the plasma concentration of immunoreactive alpha-neoendorphin with a concomitant increase in plasma catecholamines. These results indicate the presence of preproenkephalin B-derived opioid peptides in human phaeochromocytomas and human adrenal medullas and their secretion in human phaeochromocytomas.

Topics: Adolescent; Adrenal Gland Neoplasms; Adult; Cells, Cultured; Dynorphins; Endorphins; Enkephalins; Epinephrine; Female; Glucagon; Humans; Male; Middle Aged; Nicotine; Norepinephrine; Pheochromocytoma; Protein Precursors; Radioimmunoassay

The present investigation examined the effects of neonatal and adult 6-hydroxydopamine (6-OHDA)-induced lesions of dopaminergic neurons on opioid and tachykinin peptides and their gene expression in the rat basal ganglia. This work was undertaken to determine if changes in these neuropeptide systems were contributing to the differing behavioral responses observed between neonatally and adult-lesioned rats after dopamine agonist administration. [Met5]Enkephalin (ME) content was increased in striatal tissue from both 6-OHDA-lesioned groups when compared with unlesioned controls. Dynorphin-A (1-8) content was not altered by the 6-OHDA lesions. The tachykinin peptides substance P and neurokinin A were significantly decreased in level in the striatum and substantia nigra of neonatally lesioned rats, but not in the adult-lesioned rats, when compared with unlesioned controls. Proenkephalin mRNA abundance (quantified by an RNA-cDNA hybridization technique) and precursor level (as reflected by cryptic ME content) were increased in the striatum of both neonatally and adult-lesioned rats. The abundance of preprotachykinin mRNA coding for the tachykinin peptides was markedly decreased in the neonatally lesioned rats, whereas only a small reduction was observed in the adult-lesioned rats. These results suggest that destruction of dopamine-containing terminals with 6-OHDA elevates the level of ME by accelerating transcriptional and/or translational processes; conversely, the reduced content of tachykinins in neonatally lesioned rats may be due to a reduction in such processes. Thus, preproenkephalin-A and preprotachykinin gene expression are differentially regulated after lesioning of catecholamine-containing neurons, an observation suggesting a close functional relationship among these neurotransmitter systems. Furthermore, of the peptides studied, only levels of the tachykinin peptides were differentially altered in the striatum and substantia nigra of the neonatally lesioned rats compared with adult-lesioned rats; therefore, these peptides may be associated with the distinctive behavioral differences between neonatally and adult 6-OHDA-lesioned rats given dopamine agonists.

Topics: 2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine; Aging; Animals; Animals, Newborn; Benzazepines; Corpus Striatum; Dopamine; Dynorphins; Enkephalin, Methionine; Enkephalins; Ergolines; Female; Gene Expression Regulation; Hippocampus; Hydroxydopamines; Male; Motor Activity; Neuropeptides; Oxidopamine; Peptide Fragments; Protein Precursors; Quinpirole; Rats; Rats, Inbred Strains; RNA, Messenger; Substantia Nigra; Tachykinins

The contents of methionine-enkephalin-Arg-Gly-Leu, dynorphin A, dynorphin B and alpha-neoendorphin have been measured in both control and Huntington's disease brains obtained postmortem. All 4 peptides were significantly reduced in the caudate nucleus and putamen of Huntington's disease compared with the control group. No differences were observed in frontal cortex or hypothalamus. Immunocytochemistry showed a marked depletion of dynorphin-like immunoreactivity in Huntington's disease substantia nigra.

Topics: Basal Ganglia; Caudate Nucleus; Dynorphins; Endorphins; Enkephalins; Globus Pallidus; Humans; Huntington Disease; Protein Precursors; Putamen; Substantia Nigra

Dynorphin B (rimorphin) is formed from leumorphin (dynorphin B-29) by the action of a thiol protease from rat brain membranes, in a single step. This represents a "single-arginine cleavage" between threonine-13 and arginine-14 of the substrate. We have observed that in addition to dynorphin B, dynorphin B-14 is formed from dynorphin B-29. Among the various protease inhibitors tested, none except p-chloromercuribenzensulfonic acid inhibited the formation of the two products. Both temperature and pH had similar effects on the formation of dynorphin B-14 and dynorphin B. The inhibitory potencies of adrenocorticotropic hormone, peptide E, and dynorphin A were virtually identical for the formation of the two products. These results suggest that the same enzyme may be responsible for the formation of dynorphin B-14 and dynorphin B.

Topics: Adrenocorticotropic Hormone; Cysteine Endopeptidases; Dynorphins; Endopeptidases; Endorphins; Enkephalins; Hydrogen-Ion Concentration; Peptides; Protease Inhibitors; Protein Precursors; Temperature

Leumorphin, an opioid peptide whose functions are unknown, is found in mammalian brain and pituitary and stimulates lordosis behavior in estrogen-treated female rats. To elucidate the role of leumorphin in the physiological control of female sexual behavior, the levels of immunoreactive (ir) leumorphin as well as ir dynorphin (dynorphin A) were measured in the rat brain and pituitary during the estrous cycle. There was a clear variation of ir leumorphin in the hypothalamus and anterior pituitary during the estrous cycle. The levels of ir leumorphin in the hypothalamus and anterior pituitary on the afternoon of proestrus were significantly higher (P less than 0.01) than those on the afternoons of estrus and metestrus. The rise in the hypothalamic levels of ir leumorphin on the afternoon of proestrus was correlated with the receptivity of lordosis during the estrous cycle. Furthermore, there was a close correlation with ir dynorphin levels. These findings are in agreement with studies demonstrating a common precursor for leumorphin and dynorphin. Ir leumorphin in the hippocampus and neurointermediate pituitary did not change significantly during the estrous cycle. Because the leumorphin antiserum used recognizes rimorphin (dynorphin B) 1.78 times more than porcine leumorphin on a molar basis, high performance-gel permeation chromatography was done on pooled extracts of hypothalamus taken at proestrus and estrus. The peak in the leumorphin-like substance in the activation of sexual behavior is discussed.

Topics: Animals; Antigen-Antibody Reactions; Dynorphins; Enkephalins; Estrus; Female; Hypothalamus; Pituitary Gland, Anterior; Protein Precursors; Rats; Rats, Inbred Strains; Sexual Behavior, Animal

Male Fischer-344 rats were given a single intrastriatal injection of kainic acid (KA; 1 microgram/rat), which caused recurrent motor seizures lasting 3-6 hr. During the convulsive period, native Met5-enkephalin-like (ME-LI) and dynorphin A (1-8)-like (DYN-LI) immunoreactivities in hippocampus decreased by 31 and 63%, respectively. By 24 hr after dosing, the hippocampal opioid peptides had returned to control levels, and by 48 hr ME-LI had increased 270% and DYN-LI 150%. Immunocytochemical analysis revealed that ME-LI and Leu5-enkephalin-like (LE-LI) immunostaining in the mossy fibers of dentate granule cells and the perforant-temporoammonic pathway had decreased visibly by 6 hr and had increased markedly by 48 hr following KA. A visible decrease in DYN-LI in mossy fiber axons within 6 hr was followed by a substantial increase by 48 hr. To determine whether the increases in hippocampal ME-LI reflected changes in ME biosynthesis, levels of mRNA coding for preproenkephalin (mRNAenk) and cryptic ME-LI cleaved by enzyme digestion from preproenkephalin were measured. Following the convulsive period (6 hr), mRNAenk was 400% of control, and by 24 hr, cryptic ME-LI was 300% of control. Increases in native and cryptic ME-LI and in mRNAenk were also noted in entorhinal cortex, but not in hypothalamus or uninjected striatum. Our data suggest that KA-induced seizures cause an increase in ME release, followed by a compensatory increase in ME biosynthesis in the hippocampus and entorhinal cortex.

Topics: Animals; Dynorphins; Enkephalin, Methionine; Enkephalins; Hippocampus; Histocytochemistry; Immunochemistry; Kainic Acid; Protein Precursors; Radioimmunoassay; Rats; Rats, Inbred F344; RNA, Messenger; Seizures

The total content of rat pituitary anterior lobe (AL) immunoreactive (ir) dynorphin A (ir-Dyn A) and ir-dynorphin B (Dyn B) increased in male rats between 15 and 58 days of age, but there was little alteration in the concentration of ir-Dyn A or B expressed relative to protein content. Adult rats (90 days of age) had lower concentrations of these peptide immunoreactivities in the AL. Castration of 58-day-old male rats produced a testosterone-reversible loss of ir-Dyn A and B by 50-60% 3 days after surgery. Thereafter, the levels of these peptides gradually increased to 2.5 times the levels found in control animals at 1 month after castration. These effects of castration on AL dynorphin were not seen in 15-day-old rats and were much less marked in adults. Similar changes were seen in the levels of other prodynorphin products, alpha- and beta-neo-endorphin (ir-alpha-nEnd and ir-beta-nEnd), and ir-[Leu5]enkephalin (ir-LE). Administration of testosterone (100 micrograms/100 g BW) to castrated rats for 2 days largely prevented the drop in the levels of AL ir-Dyn A and B. Ovariectomy produced an increase in the levels of ir-Dyn A, Dyn B, alpha-nEnd, beta-nEnd, and LE 2 weeks after surgery, but, in contrast to castration, no significant decrease was seen 3 days after ovariectomy. These changes in AL content of dynorphin-related peptides after castration or ovariectomy directly reflect those previously reported for AL content of LH. The mechanisms regulating storage (and perhaps secretion) of AL peptides derived from prodynorphin may be similar to those regulating storage and secretion of LH and FSH in rat AL. AL ir-LE could potentially arise from proenkephalin A or prodynorphin (proenkephalin B). Ir-LE levels in AL were approximately 10 times higher than the levels of ir-[Met5]-enkephalinyl-Arg-Gly-Leu (ME-RGL) in male rat AL, and changes in ir-LE content after castration were very similar to those observed in other prodynorphin-derived peptides, but different from the effects of castration on ir-ME-RGL. It is possible that prodynorphin is a major source of AL ir-LE.

Topics: Animals; Dynorphins; Endorphins; Enkephalin, Leucine; Enkephalins; Female; Gonadal Steroid Hormones; Male; Orchiectomy; Ovariectomy; Pituitary Gland, Anterior; Protein Precursors; Rats; Testosterone

Bridge peptide, the sequence of amino acids which joins alpha-neo-endorphin and dynorphin A 1-17, was determined to exist as a free molecule in the rat anterior pituitary. Gel filtration chromatography revealed the presence of the free peptide as well as a high molecular weight intermediate of 8-9 kilodaltons.

Topics: Animals; Dynorphins; Enkephalins; Male; Molecular Weight; Pituitary Gland, Anterior; Protein Precursors; Protein Processing, Post-Translational; Rats; Rats, Inbred Strains

Serial sections from araldite-embedded rat and man pancreata were investigated immunohistochemically for the presence of prodynorphin-related peptides and alpha-endorphin. Immunoreactivities were visualized by the avidin/biotin-peroxidase complex (ABC) technique. In the human pancreas, none of the endocrine cells could be immunostained for prodynorphin-, proopiomelanocortin-related peptides and enkephalins. In the rat pancreas, however, all glucagon cells exhibited immunoreactivities for both beta-neoendorphin and dynorphin A. In addition, these cells contain alpha-endorphin-like immunoreactivity but no immunoreactivities for corticotropin, melanotropin, 16 K-fragment, alpha-N-acetyl-alpha-endorphin and enkephalins. All specificity controls confirmed that the rat endocrine pancreas might be an other source of dynorphin and endorphin with a biosynthetic pathway different from that in the pituitary or in other locations. However, concerning synthesis or degradation of peptide precursor substances interspecies differences may exist.

Topics: Adrenocorticotropic Hormone; Animals; Dynorphins; Enkephalins; Frozen Sections; Humans; Immune Sera; Immunoenzyme Techniques; Islets of Langerhans; Peptide Fragments; Protein Precursors; Rats; Species Specificity; Staining and Labeling

Dynorphin B (rimorphin), a proenkephalin B (prodynorphin)-derived peptide, and met-enkephalin-Arg6, Gly7, Leu8 (met-enkephalin octapeptide), a proenkephalin A-derived peptide, were identified in the mammalian cochlea by specific radioimmunoassays. The antisera are directed against unique sequences in the peptides, and this immunoreactivity cannot be ascribed to cross-reaction with any other known opioid peptides. Met-enkephalin octapeptide and dynorphin B can for this reason serve as reliable markers for the proenkephalin A- and proenkephalin B-derived peptides, respectively. Lesion studies in the cochlea indicate that dynorphin B is confined to olivocochlear efferents. It has not been determined if the dynorphin-containing neurons are the same as those known to contain enkephalin-related peptides, or if they may be cholinergic. Different, presumably inhibitory, neurotransmitters or modulators in the olivocochlear fibers create the possibility of separately modulating the effects of inner or outer hair cells on auditory nerve activity, and so becoming able to study their individual actions in audition. The olivocochlear fiber-hair cell-eighth nerve interaction may provide a valuable model for a complex multi-transmitter synaptic junction.

Topics: Animals; Cochlea; Dynorphins; Endorphins; Enkephalin, Methionine; Enkephalins; Female; Guinea Pigs; Hair Cells, Auditory; Nerve Tissue Proteins; Protein Precursors

Leu-Enkephalin (LE) is an endogenous opioid peptide that can arise from two distinct precursors: proenkephalin and prodynorphin. Experiments were designed to differentiate LE derived from proenkephalin versus that derived from prodynorphin. The most dense collections of dynorphin-positive fibers and terminals are in the substantia nigra and posterior pituitary, areas rich in dynorphin-related peptides. The concentration of LE in these regions is significantly higher than that of ME-Arg-Gly-Leu; the ratio of LE to ME-Arg-Gly-Leu is therefore greater than that found in the proenkephalin precursor, which is unity. Globus pallidus deafferentation resulted in a significant decrease of dynorphin B and LE, but not ME-Arg-Gly-Leu, in the substantia nigra. Mild intermittent foot shock (0.2 mA, 20 min) causes a significant increase of dynorphin B and LE in the substantia nigra, but has no effect on ME-Arg-Gly-Leu concentrations. Thus, in the substantia nigra LE may be derived primarily from prodynorphin. Likewise, in the posterior pituitary, osmotic stimulus (e.g., 2% NaCl as drinking fluid) causes marked depletion in dynorphin and LE but has no effect on ME levels suggesting that in the posterior pituitary LE is derived primarily from dynorphin.

Topics: Animals; Corpus Striatum; Dynorphins; Enkephalin, Leucine; Enkephalins; Male; Neural Pathways; Pituitary Gland, Posterior; Protein Precursors; Rats; Rats, Inbred Strains; Substantia Nigra

The effect of leumorphin (LM), one of big leu-enkephalins derived from preproenkephalin B, on PRL secretion was studied in the rat in vivo and in vitro. Intracerebroventricular injection of synthetic porcine LM (0.06-6 nmol/rat) caused a dose-related increase in plasma PRL levels in urethane-anesthetized male rats and in conscious freely moving rats. Intravenous injection of LM (3 nmol/100 g BW) also raised plasma PRL levels in these animals. The plasma PRL response to intracerebroventricular LM (0.6 nmol/rat) was blunted by naloxone (125 micrograms/100 g BW, iv). The stimulating effect of LM on PRL release was the most potent among the peptides derived from preproenkephalin B. In in vitro studies, PRL release from superfused anterior pituitary cells was stimulated in a dose-related manner by LM (10(-9)-10(-6) M), and the effect was blunted by naloxone (10(-5) M). These results suggest that LM has a potent stimulating effect on PRL secretion from the pituitary in the rat by acting, at least in part, directly at the pituitary through an opiate receptor.

Topics: Animals; beta-Endorphin; Dynorphins; Endorphins; Enkephalins; Injections, Intraventricular; Male; Naloxone; Pituitary Gland, Anterior; Prolactin; Protein Precursors; Rats; Swine; Thyrotropin-Releasing Hormone

Antibodies have been raised to a synthetic peptide corresponding to the C-terminal 15-amino acid residues of prodynorphin, the common precursor to the neo-endorphins and dynorphins. The amino acid sequence of the antigen was based on the sequence deduced from mRNA isolated and cloned from porcine hypothalamus (Kakidani, H., Y. Furutani, H. Takahashi, M. Noda, Y. Morimoto, T. Hirose, M. Asai, S. Inayama, S. Nakanishi, and S. Numa (1982) Nature 298: 245-248). Using a radioimmunoassay developed from these antibodies we have isolated an endogenous prodynorphin C-fragment from bovine caudate nucleus. The isolated peptide displayed characteristics on gel filtration similar to those of synthetic prodynorphin C-fragment predicted from the porcine mRNA sequence but had low cross-reactivity in the radioimmunoassay. Sequencing and amino acid analysis showed a substitution of serine for asparagine at position 6 in the porcine sequence. Dynorphin B (rimorphin), which is adjacent to prodynorphin C-fragment in the precursor, was isolated from the same extract. Amino acid analysis and elution position on a gel filtration column confirmed its structure as that previously characterized from bovine pituitary extracts. The release of prodynorphin C-fragment and the C-terminus of dynorphin B from the porcine precursor would require cleavage at a single arginine residue. However, a terminal arginine was not present on either of these prodynorphin peptides isolated from bovine caudate. The data would suggest that processing at a single arginine residue results in elimination of the arginine, a feature in common with processing at paired basic residues.

Topics: Animals; Cattle; Caudate Nucleus; Dynorphins; Endorphins; Enkephalins; Protein Precursors; Rabbits; Radioimmunoassay

Dynorphin B (rimorphin) is formed from dynorphin B-29 (leumorphin) by the action of a thiol protease from rat brain membranes. This represents a "single-arginine cleavage" between threonine-13 and arginine-14 of the substrate. In isotope dilution experiments we find that the radioactivity from radiolabelled dynorphin B-29, which appears in dynorphin B during incubation with the enzyme preparation, is not diminished by addition of a high concentration of dynorphin B-Arg14. Moreover, in pulse-chase experiments, radioactivity that appeared in dynorphin B-Arg14 did not decrease, nor did the radioactivity in dynorphin B increase, after chasing with a high concentration of non-radioactive dynorphin B-29. These results indicate that although some dynorphin B-Arg14 is formed by the impure enzyme preparation, it is not an intermediate in the conversion of dynorphin B-29 to dynorphin B. Thus the formation of dynorphin B does not involve the action of a trypsin-like enzyme followed by removal of arginine-14 by a carboxypeptidase B-like enzyme. It appears that a single enzyme converts dynorphin B-29 to dynorphin B in a single step.

Topics: Animals; Brain; Cell Membrane; Chromatography, High Pressure Liquid; Cysteine Endopeptidases; Dynorphins; Endopeptidases; Endorphins; Enkephalins; Iodine Radioisotopes; Kinetics; Protein Precursors; Rats

When measured in postmortem parkinsonian brains, dynorphin levels were unchanged, as compared to control brains, in mesencephalic, striatal and corticolimbic areas. A significant reduction in Leu5-enkephalin and Met5-enkephalin levels had been previously observed in the pallidum and putamen whereas only Met5-enkephalin concentrations were decreased in the substantia nigra of parkinsonian brains. These data suggest that L-Enk could be generated either from proenkephalin A in the striatal areas or from prodynorphin in the nigral areas.

Topics: Aged; Brain; Brain Chemistry; Dynorphins; Enkephalin, Leucine; Enkephalin, Methionine; Enkephalins; Humans; Parkinson Disease; Protein Precursors

Dehydration significantly reduced the concentration of immunoreactive dynorphin A(1-17), dynorphin A(1-8), alpha-neo-endorphin, beta-neo-endorphin, and leu-enkephalin in the rat pituitary posterior-intermediate lobe. A statistically significant increase in immunoreactive dynorphin A(1-8), alpha-neo-endorphin and leu-enkephalin was observed in the hypothalamus. Comparison of the molar ratios of dynorphin A(1-17): dynorphin A(1-8) and alpha-neo-endorphin: beta-neo-endorphin showed an altered profile of stored pro-dynorphin cleavage products in the posterior-intermediate lobe of the pituitary of dehydrated rats.

Topics: Animals; beta-Endorphin; Dehydration; Dynorphins; Endorphins; Enkephalin, Leucine; Enkephalins; Food Deprivation; Hypothalamus; Male; Peptide Fragments; Pituitary Gland, Posterior; Protein Precursors; Rats; Rats, Inbred Strains

Analysis of an acid extract of the striatum of the rhesus monkey revealed that the molar ratio of dynorphin A(1-8)-sized material and dynorphin (A(1-17)-sized material is approximately 1:1. In addition, the molar ratios of the dynorphin A-related end products to both dynorphin B(1-13)-sized material and alpha-neo-endorphin-sized material were approximately 1:1. Fractionation of an acid extract of the substantia nigra by gel filtration and reverse phase HPLC revealed the following molar ratios for pro-dynorphin-related end products. The molar ratio of dynorphin A(1-8) to dynorphin A(1-17) is approximately 6:1. The molar ratios of dynorphin A-related end products to dynorphin B(1-13) and alpha-neo-endorphin were approximately 0.5 and 0.8, respectively. Comparisons between proteolytic processing patterns of pro-dynorphin in the striatum and the substantia nigra of the rhesus monkey are considered. In addition, comparisons between pro-dynorphin processing in the substantia nigra of the rhesus monkey and the substantia nigra of the rat are discussed.

Topics: Animals; Chromatography, Gel; Chromatography, High Pressure Liquid; Corpus Striatum; Dynorphins; Endorphins; Enkephalins; Female; Macaca mulatta; Peptide Fragments; Protein Precursors; Radioimmunoassay; Substantia Nigra

A rat brain membrane extract was shown to convert synthetic dynorphin B-29 ("leumorphin") to dynorphin B [dynorphin B-29-(1-13), "rimorphin"]. This represents a "single arginine cleavage" at Thr-Arg at positions 13 and 14 of the substrate. The product was identified by immunoprecipitation with a highly specific dynorphin B antiserum and by coelution with radiolabeled dynorphin B on reversed-phase high-performance liquid chromatography. The converting activity exhibits a pH optimum of 8. It is inhibited by a thiol protease inhibitor but not by inhibitors of cathepsin B or of serine proteases. It is inhibited by dynorphin A but not by various dynorphin A fragments. These results suggest that the converting activity is due to a novel thiol protease distinct from any known protease believed to function in the processing of biologically active peptides.

Topics: Animals; Brain; Cysteine Endopeptidases; Dynorphins; Endopeptidases; Endorphins; Enkephalins; Hydrogen-Ion Concentration; Kinetics; Male; Peptide Fragments; Protease Inhibitors; Protein Precursors; Rats; Substrate Specificity

The distribution of peptides derived from the novel opioid peptide precursor proenkephalin B (prodynorphin) was studied in lobes of the pituitary with antibodies against alpha-neoendorphin (alpha-neo-E) beta-neoE, dynorphin (DYN)-(1-17), DYN-(1-8), and DYN B in combination with gel filtration and high pressure liquid chromatography. In the posterior pituitary, all five opioid peptides occurred in high and about equimolar concentrations, whereas putative precursor peptides were found in only minor quantities. In contrast, in the anterior pituitary immunoreactive (ir-) DYN-(1-17) and ir-DYN B consisted exclusively of a common precursor species with a mol wt of about 6000. Six thousand-dalton DYN may be comprised of the C-terminal portion of proenkephalin B, with the sequence of DYN-(1-17) at its N-terminus. Moreover, the major portions of ir-alpha-neo-E and ir-beta-neoE in the anterior pituitary were found to be of an apparent mol wt of 8000. These findings indicate a differential processing of the opioid peptide precursor proenkephalin B in the two lobes of the pituitary. The anterior pituitary seems to process proenkephalin B predominantly into high mol wt forms of neo-E and DYNs, whereas in the posterior pituitary proenkephalin B undergoes further proteolytic processing to the smaller opioid peptides alpha-neo-E, beta-neo-E, DYN-(1-17), DYN-(1-8), and DYN B. Thus, processing differences may enable the selective liberation of different (opioid) peptides with distinct biological properties from one precursor within different tissues.

Topics: Animals; beta-Endorphin; Chromatography, High Pressure Liquid; Dynorphins; Endorphins; Enkephalin, Leucine; Enkephalins; Male; Molecular Weight; Peptide Fragments; Pituitary Gland; Pituitary Gland, Anterior; Pituitary Gland, Posterior; Protein Precursors; Radioimmunoassay; Rats; Rats, Inbred Strains; Tissue Distribution

The opioid activity and opioid receptor type specificity of synthetic human leumorphin were studied in vitro. Human leumorphin inhibited the contraction of the myenteric plexus-longitudinal muscle preparation of the guinea pig ileum and was similar in opioid potency to porcine leumorphin. This action of human leumorphin was antagonized less effectively by naloxone than by the opiate antagonist Mr 2266. Human leumorphin also inhibited the contraction of the rabbit vas deferens which has only the kappa-type opioid receptor. It is concluded that human leumorphin has potent opioid activity and acts as an agonist at the kappa-type opioid receptor, like porcine leumorphin and other peptides derived from preproenkephalin B.

Topics: Animals; Benzomorphans; Depression, Chemical; Dynorphins; Endorphins; Enkephalins; Guinea Pigs; Humans; Ileum; Male; Naloxone; Protein Precursors; Rabbits; Receptors, Opioid; Receptors, Opioid, kappa; Swine; Vas Deferens

The postnatal development of several pro-enkephalin-B-derived opioid peptides - dynorphin 1-17, dynorphin 1-8, dynorphin B, alpha-neo-endorphin and beta-neo-endorphin - was examined in rat pituitary lobes. The concentrations of pro-enkephalin-B-derived peptides from the anterior pituitary were between 4- and 12-fold and those from the neurointermediate pituitary between 17- and 122-fold lower in newborn as compared to adult rats. Similarly, the concentrations of vasopressin in the neurointermediate pituitary increased 50-fold between birth and adulthood; those of oxytocin, however, increased more than 540-fold over this period. The molecular weight pattern of dynorphin 1-17, dynorphin 1-8, dynorphin B, alpha- and beta-neo-endorphin-immunoreactive peptides in the anterior and neurointermediate pituitary did not differ between 3-day-old pups and adult rats. In the neurointermediate pituitary, the major immunoreactive components had the same chromatographic properties as synthetic dynorphin 1-17, dynorphin 1-8, dynorphin B, alpha- and beta-neo-endorphin, respectively, on gel filtration and high-performance liquid chromatography (HPLC). This indicates that neonatal rats were already capable of processing the precursor pro-enkephalin B into these various opioid peptides. In newborn rats, however, the amount of dynorphin 1-8 in the neurointermediate pituitary was three times lower than that of its putative intermediate precursor peptide dynorphin 1-17. Similarly, the amount of beta-neo-endorphin was almost four times lower than that of its putative precursor alpha-neo-endorphin. In contrast, in the neurointermediate pituitary of adult rats, dynorphin 1-17 and dynorphin 1-8, in addition to a alpha- and beta-neo-endorphin, occurred in equimolar amounts.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Animals, Newborn; Cell Differentiation; Chromatography, High Pressure Liquid; Dynorphins; Endorphins; Enkephalin, Leucine; Enkephalins; Female; Male; Molecular Weight; Oxytocin; Peptide Fragments; Peptides; Pituitary Gland, Anterior; Pituitary Gland, Posterior; Pregnancy; Protein Precursors; Rats; Rats, Inbred Strains; Vasopressins

The primary structure of the common precursor of porcine beta-neo-endorphin and dynorphin (preproenkephalin B) has shown the existence of a third leucine-enkephalin (leu-enkephalin) sequence with a C-terminal extension of 24 amino acids. This nonacosapeptide, named leumorphin, was approximately 70 times more potent than leu-enkephalin in inhibiting the contraction of the myenteric plexus-longitudinal muscle preparation of the guinea pig ileum. This action of leumorphin, like those of beta-neo-endorphin and dynorphin, was antagonized less effectively by naloxone than that of leu-enkephalin, but more effectively by Mr2266, an antagonist relatively specific for the kappa type opiate receptor. The inhibitory action of leumorphin or beta-neo-endorphin on the contraction of the guinea pig ileum muscle strip was reduced in a dose-dependent manner by pretreatment with dynorphin and vice versa. Leumorphin as well as beta-neo-endorphin and dynorphin inhibits the contraction of the rabbit vas deferens which is known to have only the kappa type opiate receptor. This action was also effectively antagonized by Mr2266. It is concluded that leumorphin has potent opioid activity and acts at the kappa receptor, like other opioid peptides derived from preproenkephalin B.

Topics: Animals; Dynorphins; Endorphins; Enkephalins; Guinea Pigs; Ileum; Male; Muscle Contraction; Naloxone; Neuromuscular Junction; Protein Precursors; Receptors, Opioid; Receptors, Opioid, kappa; Structure-Activity Relationship; Swine; Vas Deferens

The contents and molecular forms of five different prodynorphin-derived opioid peptides were compared in extracts of rat hippocampus by radioimmunoassay after C18-HPLC resolution. Dynorphin (Dyn) A(1-17) immunoreactivity (ir) and Dyn B-ir were heterogeneous in form; Dyn A(1-8)-ir, alpha-neoendorphin (alpha neo)-ir and beta-neoendorphin (beta neo)-ir each eluted as single homogeneous peaks of immunoreactivity. The fraction of immunoreactivity having the same retention as the appropriate synthetic standard was used to estimate the actual hippocampal content of each peptide. Comparison of these values showed that the concentrations of Dyn B, alpha neo, and Dyn A(1-8) were nearly equal, whereas both Dyn A(1-17) and beta neo were 1/5th to 1/10th the value of the other three. Calcium-dependent K+-stimulated release of these prodynorphin-derived opioids from hippocampal slices was detected. The stimulated rates of release were highest for Dyn B-ir followed by alpha neo-ir, then beta neo-ir and Dyn A(1-8)-ir with Dyn A(1-17)-ir lowest. The relative rates of stimulated release were in agreement with the relative proportions of peptide present within the tissue. This evidence of the presence and release of these opioid peptides considerably strengthens the hypothesis that this family of endogenous opioids plays a neurotransmitter role in the hippocampus.

Topics: Amino Acid Sequence; Animals; Chromatography, High Pressure Liquid; Dynorphins; Endorphins; Enkephalins; Hippocampus; Kinetics; Male; Peptide Fragments; Protein Precursors; Radioimmunoassay; Rats; Rats, Inbred Strains

The carboxy-terminal nonacosapeptide sequence of porcine preproenkephalin B contains the sequence of Leu-enkephalin at its amino terminus. The endogenous existence of this peptide, leumorphin, has not yet been proved. Synthesis of leumorphin was carried out by a solid-phase technique and the purity and structure of the synthetic peptide were confirmed. Synthetic porcine leumorphin exhibited a dose-dependent opiate effect (ED50 4.70 X 10(-9) M) on electrically stimulated contraction of the guinea pig ileum preparation. The potency was about 100 times as high as that of Leu-enkephalin. Leumorphin was less potent than dynorphin(1-13) (ED50 0.38 X 10(-9) M) but it was more active than beta h-endorphin (ED50 18 X 10(-9) M). The opiate activity was only partially reversed by naloxone. Intracisternal injection of synthetic leumorphin caused significant analgesia in mice (ED50 7.31 nmol/mouse). The potency was lower than that of beta h-endorphin (ED50 0.60 nmol/mouse) but higher than that of dynorphin(1-13) (ED50 16.10 nmol/mouse). Intracisternally injected leumorphin did not produce such a violent behavioral effect as did dynorphin(1-13), and it exhibited a mild sedative effect. The data supports the concept that leumorphin is a new type of opioid peptide and that the synthetic preparation will be useful for further biological and immunological studies on this peptide.

Topics: Analgesia; Animals; beta-Endorphin; Biological Assay; Dynorphins; Electric Stimulation; Endorphins; Enkephalins; Guinea Pigs; Ileum; Indicators and Reagents; Mice; Muscle Contraction; Naloxone; Narcotics; Peptide Fragments; Protein Precursors; Swine

Leumorphin and rimorphin (dynorphin B) were nearly equipotent in inhibiting the contraction of the myenteric plexus-longitudinal muscle preparation of the guinea pig ileum and the rabbit vas deferens. Leumorphin had long duration of action in vitro after wash-out, whereas the duration of action of rimorphin was relatively shorter. These action of leumorphin and rimophin were antagonized less effectively by naloxone than by Mr2266, an antagonist relatively specific for the kappa-receptor. Rimorphin as well as leumorphin inhibits the contraction of the rabbit vas deferens which contains solely kappa-receptors. It is concluded that leumorphin and rimorphin have equipotent opioid activity and act at the kappa-receptor, like other opioid peptides derived from preproenkephalin B.

Topics: Animals; Biological Assay; Dynorphins; Electric Stimulation; Endorphins; Enkephalin, Leucine; Enkephalins; Guinea Pigs; Ileum; Male; Muscle Contraction; Muscle, Smooth; Naloxone; Protein Precursors; Rabbits; Receptors, Opioid; Receptors, Opioid, kappa; Vas Deferens

Using synthetic leumorphin, we obtained antisera for leumorphin and set up two radioimmunoassays (RIAs) with different specificities. Gel exclusion chromatography coupled with the two RIAs showed the existence of a considerable amount of leumorphin-like peptide in water extracts from porcine neuro-intermediate pituitaries. Reverse phase high performance liquid chromatography revealed that leumorphin-like peptide in the water extracts was indistinguishable from synthetic leumorphin. These results along with potent opioid activity of leumorphin indicate that leumorphin is a novel endogenous opioid peptide derived from preproenkephalin B.

Topics: Animals; Chemical Phenomena; Chemistry; Chromatography, Gel; Chromatography, High Pressure Liquid; Dynorphins; Electrophoresis, Polyacrylamide Gel; Endorphins; Enkephalin, Leucine; Enkephalins; Pituitary Gland; Protein Precursors; Radioimmunoassay; Swine