Peptide Dynorphin Research Articles

Dynorphin modulates motivation through a pallido-amygdala cholinergic circuit.

The endogenous opioid peptide dynorphin and its receptor κ-opioid receptor (KOR) have been implicated in divergent behaviors, but the underlying mechanisms remain elusive. Here we show that dynorphin released from nucleus accumbens dynorphinergic neurons exerts powerful modulation over a ventral pallidum (VP) disinhibitory circuit, thereby controlling cholinergic transmission to the amygdala and motivational drive in mice. On one hand, dynorphin acts postsynaptically via KORs on local GABAergic neurons in the VP to promote disinhibition of cholinergic neurons, which release acetylcholine into the amygdala to invigorate reward-seeking behaviors. On the other hand, dynorphin also acts presynaptically via KORs on dynorphinergic terminals to limit its own release. Such autoinhibition keeps cholinergic neurons from prolonged activation and release of acetylcholine, and prevents perseverant reward seeking. Our study reveals how dynorphin exquisitely modulate motivation through cholinergic system, and provides an explanation for why these neuromodulators are involved in motivational disorders, including depression and addiction.

Control of motivation for sucrose in the paraventricular hypothalamic nucleus by dynorphin peptides and the kappa opioid receptor

The dynorphin peptides are the endogenous ligands for the kappa opioid receptor (KOR) and regulate food intake. Administration of dynorphin-A1-13 (DYN) in the paraventricular hypothalamic nucleus (PVN) increases palatable food intake, and this effect is blocked by co-administration of the orexin-A neuropeptide, which is co-released with DYN in PVN from neurons located in the lateral hypothalamus. While PVN administration of DYN increases palatable food intake, whether it increases food-seeking behaviors has yet to be examined. We tested the effects of DYN and norBNI (a KOR antagonist) on the seeking and consumption of sucrose using a progressive ratio (PR) and demand curve (DC) tasks. In PVN, DYN did not alter the sucrose breaking point (BP) in the PR task nor the elasticity or intensity of demand for sucrose in the DC task. Still, DYN reduced the delay in obtaining sucrose and increased licks during sucrose intake in the PR task, irrespective of the co-administration of orexin-A. In PVN, norBNI increased the delay in obtaining sucrose and reduced licks during sucrose intake in the PR task while increasing elasticity without altering intensity of demand in the DC task. However, subcutaneous norBNI reduced the BP for sucrose and increased the delay in obtaining sucrose in the PR task while reducing the elasticity of demand. Together, these data show different effects of systemic and PVN blockade of KOR on food-seeking, consummatory behaviors, and incentive motivation for sucrose and suggest that KOR activity in PVN is necessary but not sufficient to drive seeking behaviors for palatable food.

The opioid peptide dynorphin interferes with testicular activity in Mozambique tilapia

The opioid peptide dynorphin interferes with testicular activity in Mozambique tilapia

Antagonism of kappa opioid receptors accelerates the development of L-DOPA-induced dyskinesia in a preclinical model of moderate dopamine depletion

Levels of the opioid peptide dynorphin, an endogenous ligand selective for kappa-opioid receptors (KORs), its mRNA and pro-peptide precursors are differentially dysregulated in Parkinson’s disease (PD) and following the development of l-DOPA-induced dyskinesia (LID). It remains unclear whether these alterations contribute to the pathophysiological mechanisms underlying PD motor impairment and the subsequent development of LID, or whether they are part of compensatory mechanisms. We sought to investigate nor-BNI, a KOR antagonist, 1) in the dopamine (DA)-depleted PD state, 2) during the development phase of LID, and 3) via measuring of tonic levels of striatal DA. While nor-BNI (3 mg/kg; s.c.) did not lead to functional restoration in the DA-depleted state, it affected the dose-dependent development of abnormal voluntary movements (AIMs) in response to escalating doses of l-DOPA in a rat PD model with a moderate striatal 6-hydroxdopamine (6-OHDA) lesion. We tested five escalating doses of l-DOPA (6, 12, 24, 48, 72 mg/kg; i.p.), and nor-BNI significantly increased the development of AIMs at the 12 and 24 mg/kg l-DOPA doses. However, after reaching the 72 mg/kg l-DOPA, AIMs were not significantly different between control and nor-BNI groups. In summary, while blocking KORs significantly increased the rate of development of LID induced by chronic, escalating doses of l-DOPA in a moderate-lesioned rat PD model, it did not contribute further once the overall severity of LID was established. While we observed an increase of tonic DA levels in the moderately lesioned dorsolateral striatum, there was no tonic DA change following administration of nor-BNI.

Design and development of novel, short, stable dynorphin-based opioid agonists for safer analgesic therapy.

Kappa opioid receptors have exceptional potential as an analgesic target, seemingly devoid of many problematic Mu receptor side-effects. Kappa-selective, small molecule pharmaceutical agents have been developed, but centrally mediated side-effects limit clinical translation. We modify endogenous dynorphin peptides to improve drug-likeness and develop safer KOP receptor agonists for clinical use. Using rational, iterative design, we developed a series of potent, selective, and metabolically stable peptides from dynorphin 1-7. Peptides were assessed for in vitro cAMP-modulation against three opioid receptors, metabolic stability, KOP receptor selectivity, desensitisation and pERK-signalling capability. Lead peptides were evaluated for in vivo efficacy in a rat model of inflammatory nociception. A library of peptides was synthesised and assessed for pharmacological and metabolic stability. Promising peptide candidates showed low nanomolar KOP receptor selectivity in cAMP assay, and improved plasma and trypsin stability. Selected peptides showed bias towards cAMP signalling over pERK activity, also demonstrating reduced desensitisation. In vivo, two peptides showed significant opioid-like antinociception comparable to morphine and U50844H. These highly potent and metabolically stable peptides are promising opioid analgesic leads for clinical translation. Since they are somewhat biased peptide Kappa agonists they may lack many significant side-effects, such as tolerance, addiction, sedation, and euphoria/dysphoria, common to opioid analgesics.

Withdrawal: Rink-Jan Lohman, Karnaker Reddy Tupally, Ajit Kandale, Peter J. Cabot, Harendra S. Parekh (2022) Design and development of novel, short, stable dynorphin-based opioid agonists for safer analgesic therapy. Br J Pharmacol; https://doi.org/10.1111/bph.15862.

The kappa opioid receptor has exceptional potential as an analgesic target, seemingly devoid of the many peripheral side-effects of Mu opioid receptors. Kappa-selective, small molecule pharmaceutical agents have been developed, but centrally-mediated side effects have limited their clinical translation. Here, we modify an active endogenous dynorphin peptide to improve drug-likeness and develop safer KOPr agonists for clinical use. Using rational, iterative design and modern peptide chemistry, we developed a series of potent, selective and metabolically stable peptides from dynorphin 1-7. Peptides were assessed for cAMP-modulation against Kappa, Mu and Delta opioid receptors, metabolic stability, Kappa receptor specificity and binding, and interrogated for in vitro desensitisation and pERK signalling capability. Finally, lead peptides were evaluated for efficacy in the Freund's complete adjuvant rat model of inflammatory nociception. A library of 70 peptides was synthesised and assessed for pharmacological and metabolic stability factors. At least 10 peptide candidates showed low nanomolar activity (˂50 nM) in a cAMP assay, specificity for Kappa opioid receptor, and plasma half-life >60 min, with 6 candidates also stable in trypsin. None of the selected peptides showed pERK activity, with a bias towards cAMP signalling, and reduced desensitisation, In vivo, KA305 and KA311 showed antinociception opioid receptor-specific activity comparable to morphine and U50844H. These highly potent and metabolically stable peptides are promising opioid analgesic leads for clinical translation. Since they are biased peptide Kappa agonists, it is plausible they lack many of the most significant side effects, such as tolerance, addiction, sedation and euphoria/dysphoria, common to opioid analgesics.

Dynorphin Neuropeptides Decrease Apparent Proton Affinity of ASIC1a by Occluding the Acidic Pocket.

Prolonged acidosis, as it occurs during ischemic stroke, induces neuronal death via acid-sensing ion channel 1a (ASIC1a). Concomitantly, it desensitizes ASIC1a, highlighting the pathophysiological significance of modulators of ASIC1a acid sensitivity. One such modulator is the opioid neuropeptide big dynorphin (Big Dyn) which binds to ASIC1a and enhances its activity during prolonged acidosis. The molecular determinants and dynamics of this interaction remain unclear, however. Here, we present a molecular interaction model showing a dynorphin peptide inserting deep into the acidic pocket of ASIC1a. We confirmed experimentally that the interaction is predominantly driven by electrostatic forces, and using noncanonical amino acids as photo-cross-linkers, we identified 16 residues in ASIC1a contributing to Big Dyn binding. Covalently tethering Big Dyn to its ASIC1a binding site dramatically decreased the proton sensitivity of channel activation, suggesting that Big Dyn stabilizes a resting conformation of ASIC1a and dissociates from its binding site during channel opening.

Improving the Utility of a Dynorphin Peptide Analogue Using Mannosylated Glycoliposomes.

Peptide therapeutics offer numerous advantages in the treatment of diseases and disorders of the central nervous system (CNS). However, they are not without limitations, especially in terms of their pharmacokinetics where their metabolic lability and low blood–brain barrier penetration hinder their application. Targeted nanoparticle delivery systems are being tapped for their ability to improve the delivery of therapeutics into the brain non-invasively. We have developed a family of mannosylated glycoliposome delivery systems for targeted drug delivery applications. Herein, we demonstrate via in vivo distribution studies the potential of these glycoliposomes to improve the utility of CNS active therapeutics using dynantin, a potent and selective dynorphin peptide analogue antagonist of the kappa opioid receptor (KOR). Glycoliposomal entrapment protected dynantin against known rapid metabolic degradation and ultimately improved brain levels of the peptide by approximately 3–3.5-fold. Moreover, we linked this improved brain delivery with improved KOR antagonist activity by way of an approximately 30–40% positive modulation of striatal dopamine levels 20 min after intranasal administration. Overall, the results clearly highlight the potential of our glycoliposomes as a targeted delivery system for therapeutic agents of the CNS.

Compartment-specific opioid receptor signaling is selectively modulated by different dynorphin peptides.

Many signal transduction systems have an apparent redundancy built into them, where multiple physiological agonists activate the same receptors. Whether this is true redundancy, or whether this provides an as-yet unrecognized specificity in downstream signaling, is not well understood. We address this question using the kappa opioid receptor (KOR), a physiologically relevant G protein-coupled receptor (GPCR) that is activated by multiple members of the Dynorphin family of opioid peptides. We show that two related peptides, Dynorphin A and Dynorphin B, bind and activate KOR to similar extents in mammalian neuroendocrine cells and rat striatal neurons, but localize KOR to distinct intracellular compartments and drive different post-endocytic fates of the receptor. Strikingly, localization of KOR to the degradative pathway by Dynorphin A induces sustained KOR signaling from these compartments. Our results suggest that seemingly redundant endogenous peptides can fine-tune signaling by regulating the spatiotemporal profile of KOR signaling.

Parabrachial opioidergic projections to preoptic hypothalamus mediate behavioral and physiological thermal defenses.

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 Prodynorphin (Pdyn) and Proenkephalin (Penk) mRNA expressing neurons are partially overlapping subsets of a glutamatergic population expressing Solute carrier family 17 (Slc17a6) (VGLUT2). Using optogenetic approaches we selectively activate projections in the POA from PBN Pdyn, Penk, and VGLUT2 expressing neurons. Our findings demonstrate that Pdyn, Penk, and VGLUT2 expressing PBN neurons are critical for physiological and behavioral heat defense.

Brain site-specific regulation of hedonic intake by orexin and DYN peptides: role of the PVN and obesity

ABSTRACT The orexin peptides promote hedonic intake and other reward behaviors through different brain sites. The opioid dynorphin peptides are co-released with orexin peptides but block their effects on reward in the ventral tegmental area (VTA). We previously showed that in the paraventricular hypothalamic nucleus (PVN), dynorphin and not orexin peptides enhance hedonic intake, suggesting they have brain-site-specific effects. Obesity alters the expression of orexin and dynorphin receptors, but whether their expression across different brain sites is important to hedonic intake is unclear. We hypothesized that hedonic intake is regulated by orexin and dynorphin peptides in PVN and that hedonic intake in obesity correlates with expression of their receptors. Here we show that in mice, injection of DYN-A1–13 (an opioid dynorphin peptide) in the PVN enhanced hedonic intake, whereas in the VTA, injection of OXA (orexin-A, an orexin peptide) enhanced hedonic intake. In PVN, OXA blunted the increase in hedonic intake caused by DYN-A1–13. In PVN, injection of norBNI (opioid receptor antagonist) reduced hedonic intake but a subsequent OXA injection failed to increase hedonic intake, suggesting that OXA activity in PVN is not influenced by endogenous opioid activity. In the PVN, DYN-A1–13 increased the intake of the less-preferred food in a two-food choice task. In obese mice fed a cafeteria diet, orexin 1 receptor mRNA across brain sites involved in hedonic intake correlated with fat preference but not caloric intake. Together, these data support that orexin and dynorphin peptides regulate hedonic intake in an opposing manner with brain-site-specific effects.

Unique Pharmacological Properties of the Kappa Opioid Receptor Signaling Through Gαz as Shown with Bioluminescence Resonance Energy Tranfer.

Opioid receptors (ORs) convert extracellular messages to signaling events by coupling to the heterotrimeric G proteins, Gα•βγ Classic pharmacological methods, such as [35S]GTPγS binding and inhibition of cyclic AMP production, allow for general opioid characterization, but they are subject to the varying endogenous Gα proteins in a given cell type. Bioluminescence resonance energy transfer (BRET) technology offers new insight by allowing the direct observation of Gα subunit-specific effects on opioid pharmacology. Using a Venus-tagged Gβγ and nanoluciferase-tagged truncated G protein receptor kinase 3, an increase in BRET signal correlated with OR activation mediated by a specific Gα protein. The magnitude of the BRET signal was normalized to the maximum response obtained with 10 µM 2-(3,4-dichlorophenyl)-N-methyl-N-[(1R,2R)-2-pyrrolidin-1-ylcyclohexyl]acetamide (U50,488) for the kappa OR (KOR). Opioids reached equilibrium with the KOR, and concentration-response curves were generated. Although the full agonists U50,488, salvinorin A, nalfurafine, and dynorphin peptides were equally efficacious regardless of the Gα subunit present, the concentration-response curves were leftward shifted when the KOR was signaling through Gαz compared with other Gαi/o subunits. In contrast, the Gα subunit distinctly affected both the efficacy and potency of partial kappa agonists, such as the benzomorphans, and the classic mu opioid antagonists, naloxone, naltrexone, and nalmefene. For example, (-)pentazocine had EC50 values of 7.3 and 110 nM and maximal stimulation values of 79% and 35% when the KOR signaled through Gαz and Gαi1, respectively. Together, these observations suggest KOR pharmacology varies based on the specific Gα subunit coupled to the KOR. SIGNIFICANCE STATEMENT: Opioid receptors couple to various heterotrimeric Gαβγ proteins to convert extracellular cues to precise intracellular events. This paper focuses on how the various inhibitory Gα subunits influence the pharmacology of full and partial agonists at the kappa opioid receptor. Using a bioluminescent assay, the efficacy and potency of kappa opioids was determined. Opioid signaling was more potent through Gαz compared with other Gα proteins. These observations suggest that Gαz may impact opioid pharmacology and cellular physiology more than previously thought.

Effects of beta-endorphin and dynorphin A on in vitro apoptosis in human peripheral blood lymphocytes

Along with functioning in the nerve system, the endogenous opioid peptides as the ligands of mu-, delta-, and kappa-opioid receptors exert multiple effects on immune cells. Moreover, experimental evidence showed that morphine as an exogenous agonist of mu-opioid receptors affects immune cell viability. Such effects were discovered in experiments with cultured cells and laboratory animals. Hence, we studied effects of endogenous opioid peptides dynorphin A and beta-endorphin on viability of human peripheral blood mononuclear cells in vitro. For this, we used samples of peripheral blood cells collected from the fourteen healthy volunteers, who provided with signed informed consent and might request any information regarding the research. Mononuclear cells were collected from the heparinized blood samples according to standard protocol and cultivated in the humid atmosphere for 72 hours. Two μCi 3H-Methyl-thymidine was added into each test tube at 18 hours before the end of the cultivation period. Scintillation counting was performed by using Guardian liquid scintillation analyzer (Wallac, Finland) expressing the data as count per minute. To assess apoptosis, the cells were cultured for 24 hours in similar conditions except for adding radioactive probe. Next, the cells were stained with Annexin V-FITC/7-AAD kit (Beckman Coulter, USA) according to the manufacturer’s instructions for further recording apoptotic cells in flow cytometers BD FACSCalibur (Becton Dickinson, USA) or CytoFLEX S (Beckman Coulter, USA). The lymphocyte gate set by light scatter parameters was shown in typical Annexin V-FITC vs 7-AAD plot followed by counting Annexin V+/7-AADcells. All data were expressed as means ± S.E. Statistical significance was assessed by using Student’s t-test. It was found that physiologic concentrations of mu- and kappa-opioid receptor agonists beta-endorphin and dynorphin A exerted multidirectional effects on proliferation of human peripheral blood lymphocytes. In particular, dynorphin A increased basal proliferation and proliferation in response to suboptimal mitogen stimulation. Moreover, beta-endorphin enhanced effects of mitogen stimulation at suboptimal concentration but profoundly suppressed proliferation in maximally activated cells. The modulating effects of beta-endorphin and dynorphin A on in vitro proliferation are not associated with augmented cell apoptosis.

Modulation of feeding behavior and metabolism by dynorphin

The neuronal regulation of metabolic and behavioral responses to different diets and feeding regimens is an important research area. Herein, we investigated if the opioid peptide dynorphin modulates feeding behavior and metabolism. Mice lacking dynorphin peptides (KO) were exposed to either a normal diet (ND) or a high-fat diet (HFD) for a period of 12 weeks. Additionally, mice had either time-restricted (TR) or ad libitum (AL) access to food. Body weight, food intake and blood glucose levels were monitored throughout the 12-week feeding schedule. Brain samples were analyzed by immunohistochemistry to detect changes in the expression levels of hypothalamic peptides. As expected, animals on HFD or having AL access to food gained more weight than mice on ND or having TR access. Unexpectedly, KO females on TR HFD as well as KO males on AL ND or AL HFD demonstrated a significantly increased body weight gain compared to the respective WT groups. The calorie intake differed only marginally between the genotypes: a significant difference was present in the female ND AL group, where dynorphin KO mice ate more than WT mice. Although female KO mice on a TR feeding regimen consumed a similar amount of food as WT controls, they displayed significantly higher levels of blood glucose. We observed significantly reduced levels of hypothalamic orexigenic peptides neuropeptide Y (NPY) and orexin-A in KO mice. This decrease became particularly pronounced in the HFD groups and under AL condition. The kappa opiod receptor (KOR) levels were higher after HFD compared to ND feeding in the ventral pallidum of WT mice. We hypothesize that HFD enhances dynorphin signaling in this hedonic center to maintain energy homeostasis, therefore KO mice have a more pronounced phenotype in the HFD condition due to the lack of it. Our data suggest that dynorphin modulates metabolic changes associated with TR feeding regimen and HFD consumption. We conclude that the lack of dynorphin causes uncoupling between energy intake and body weight gain in mice; KO mice maintained on HFD become overweight despite their normal food intake. Thus, using kappa opioid receptor agonists against obesity could be considered as a potential treatment strategy.

Kappa Opioid Receptor Antagonists as Potential Therapeutics for Mood and Substance Use Disorders.

The kappa opioid receptor (KOR) and its primary cognate ligands, the dynorphin peptides, are involved in diverse physiological processes. Disruptions to the KOR/dynorphin system have been found to likely play a role in multiple neuropsychological disorders, and hence KOR has emerged as a potential therapeutic target. Targeting KOR is complicated by close homology to the mu and delta opioid receptors (MOR and DOR), and many KOR ligands have at least moderate affinity to MOR and/or DOR. Animal models utilizing primarily very long-lasting selective KOR antagonists (>3weeks following a single dose) have demonstrated that KOR antagonism attenuates certain anxiety-like and depression-like behaviors and blocks stress- and cue-induced reinstatement to drug seeking. Recently, relatively selective KOR antagonists with medication-like pharmacokinetic and pharmacodynamic properties and durations of action have been developed. One of these, JNJ-67953964 (also referred to as CERC-501, LY2456302, OpraKappa or Aticaprant) has been studied in humans, and shown to be safe, relatively KOR selective, and able to substantially attenuate binding of a KOR PET tracer to CNS localized KOR for greater than 24h. While animal studies have indicated that compounds of this structural class are capable of normalizing withdrawal signs in animal models of cocaine and alcohol dependence and reducing cocaine and alcohol intake/seeking, additional studies are needed to determine the value of these second generation KOR antagonists in treating mood disorders and substance use disorders in humans.

Thymosin Beta 4 Is Involved in the Development of Electroacupuncture Tolerance.

Background: Electroacupuncture (EA) tolerance, a negative therapeutic effect, is a gradual decline in antinociception because of its repeated or prolonged use. This study aims to explore the role of thymosin beta 4 (Tβ4), having neuro-protection properties, in EA tolerance (EAT).Methods: Rats were treated with EA once daily for eight consecutive days to establish EAT, effect of Tβ4 on the development of EAT was determined through microinjection of Tβ4 antibody and siRNA into the cerebroventricle. The mRNA and protein expression profiles of Tβ4, opioid peptides (enkephalin, dynorphin and endorphin), and anti-opioid peptides (cholecystokinin octapeptide, CCK-8 and orphanin FQ, OFQ), and mu opioid receptor (MOR) and CCK B receptor (CCKBR) in the brain areas (hypothalamus, thalamus, cortex, midbrain and medulla) were characterized after Tβ4 siRNA was administered.Results: Tβ4 levels were increased at day 1, 4, and 8 and negatively correlated with the changes of tail flick latency in all areas. Tβ4 antibody and siRNA postponed EAT. Tβ4 siRNA caused decreased Tβ4 levels in all areas, which resulted in increased enkephalin, dynorphin, endorphin and MOR levels in most measured areas during repeated EA, but unchanged OFQ, CCK-8, and CCKBR levels in most measured areas. Tβ4 levels were negatively correlated with enkephalin, dynorphin, endorphin, or MOR levels in all areas except medulla, while positively correlated with OFQ and CCK-8 levels in some areas.Conclusion: These results confirmed Tβ4 facilitates EAT probably through negatively changing endogenous opioid peptides and their receptors and positively influencing anti-opioid peptides in the central nervous system.

(350) The Persistent Pain Transcriptome: Distinct Transcriptional Signatures of Post-Surgical and Inflammatory Models Define Convergent Pathways to the Transition to Hyperalgesia

Most models of induced hyperalgesia display stereotyped behavioral responses to modalities such as heat and low threshold mechanical stimuli. Despite this, the nociceptive apparatus and the subsequent subjective experiences that define hyperalgesia differ greatly, as do accompanying symptoms of the associated pain state. Based on several recent in-depth studies of the activation patterns of primary afferents, we predicted that divergent activation paradigms could trigger distinct plastic events at second-order neurons that converge to sensitize the spinal circuits to evoked stimuli. The spinal circuits engaged by inflammatory and post-surgical pain states can be elucidated based on multiplex labeling and time course evaluations of the transcriptional signatures using deep sequencing. These experiments revealed a population of neurons apparently undetected by single-cell and single-nucleus sequencing methods because of its sparse representation in the superficial laminae of the dorsal horn. Investigations into the character and connectivity of these second order spinal neurons revealed that they participate similarly in at least two models of hyperalgesia. These models are driven by different transcriptional machinery at the level of the dorsal spinal cord, and the most transcriptionally evident neurons express very high levels of both the opioid peptide dynorphin and receptors that may be involved in phenotypic switching during hyperalgesia. We identified multiple cell populations, representing opponent processes acting to promote and abrogate hyperalgesia. Surprisingly, both neural and glial substrates of plasticity were divergent between the models, but converged at the pathway level. Examples of convergent pathways included immune-like activation and upregulation of the dynorphin precursor gene, whereas the activation of neuronal genes was divergent. These investigations outline a multidisciplinary strategy for identification of targets for analgesia and hyperalgesia research, and identify a set of neuronal receptors for future analgesic target development.

Short-term withdrawal from repeated exposure to cocaine during adolescence modulates dynorphin mRNA levels and BDNF signaling in the rat nucleus accumbens

BackgroundEarly-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. MethodsAdolescent 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. ResultsIn 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. ConclusionsOur 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.

Structure and dynamics of dynorphin peptide and its receptor.

Dynorphin is a neuropeptide involved in pain, addiction and mood regulation. It exerts its activity by binding to the kappa opioid receptor (KOP) which belongs to the large family of G protein-coupled receptors. The dynorphin peptide was discovered in 1975, while its receptor was cloned in 1993. This review will describe: (a) the activities and physiological functions of dynorphin and its receptor, (b) early structure-activity relationship studies performed before cloning of the receptor (mostly pharmacological and biophysical studies of peptide analogues), (c) structure-activity relationship studies performed after cloning of the receptor via receptor mutagenesis and the development of recombinant receptor expression systems, (d) structural biology of the opiate receptors culminating in X-ray structures of the four opioid receptors in their inactive state and structures of MOP and KOP receptors in their active state. X-ray and EM structures are combined with NMR data, which gives complementary insight into receptor and peptide dynamics. Molecular modeling greatly benefited from the availability of atomic resolution 3D structures of receptor-ligand complexes and an example of the strategy used to model a dynorphin-KOP receptor complex using NMR data will be described. These achievements have led to a better understanding of the complex dynamics of KOP receptor activation and to the development of new ligands and drugs.

Ginsenoside Re protects methamphetamine-induced dopaminergic neurotoxicity in mice via upregulation of dynorphin-mediated \u03ba-opioid receptor and downregulation of substance P-mediated neurokinin 1 receptor

BackgroundWe previously reported that ginsenoside Re (GRe) attenuated against methamphetamine (MA)-induced neurotoxicity via anti-inflammatory and antioxidant potentials. We also demonstrated that dynorphin possesses anti-inflammatory and antioxidant potentials against dopaminergic loss, and that balance between dynorphin and substance P is important for dopaminergic neuroprotection. Thus, we examined whether GRe positively affects interactive modulation between dynorphin and substance P against MA neurotoxicity in mice.MethodsWe examined changes in dynorphin peptide level, prodynorphin mRNA, and substance P mRNA, substance P-immunoreactivity, homeostasis in enzymatic antioxidant system, oxidative parameter, microglial activation, and pro-apoptotic parameter after a neurotoxic dose of MA to clarify the effects of GRe, prodynorphin knockout, pharmacological inhibition of κ-opioid receptor (i.e., nor-binaltorphimine), or neurokinin 1 (NK1) receptor (i.e., L-733,060) against MA insult in mice.ResultsGRe attenuated MA-induced decreases in dynorphin level, prodynorphin mRNA expression in the striatum of wild-type (WT) mice. Prodynorphin knockout potentiated MA-induced dopaminergic toxicity in mice. The imbalance of enzymatic antioxidant system, oxidative burdens, microgliosis, and pro-apoptotic changes led to the dopaminergic neurotoxicity. Neuroprotective effects of GRe were more pronounced in prodynorphin knockout than in WT mice. Nor-binaltorphimine, a κ-opioid receptor antagonist, counteracted against protective effects of GRe. In addition, we found that GRe significantly attenuated MA-induced increases in substance P-immunoreactivity and substance P mRNA expression in the substantia nigra. These increases were more evident in prodynorphin knockout than in WT mice. Although, we observed that substance P-immunoreactivity was co-localized in NeuN-immunreactive neurons, GFAP-immunoreactive astrocytes, and Iba-1-immunoreactive microglia. NK1 receptor antagonist L-733,060 or GRe selectively inhibited microgliosis induced by MA. Furthermore, L-733,060 did not show any additive effects against GRe-mediated protective activity (i.e., antioxidant, antimicroglial, and antiapoptotic effects), indicating that NK1 receptor is one of the molecular targets of GRe.ConclusionsOur results suggest that GRe protects MA-induced dopaminergic neurotoxicity via upregulatgion of dynorphin-mediated κ-opioid receptor and downregulation of substance P-mediated NK1 R.