ABSTRACT
The vasopressin V2 receptor (V2R) is a validated therapeutic target for autosomal dominant polycystic kidney disease (ADPKD), with tolvaptan being the first FDA-approved antagonist. Herein, we used Gaussian accelerated molecular dynamics simulations to investigate the spontaneous binding of tolvaptan to both active and inactive V2R conformations at the atomic-level. Overall, the binding process consists of two stages. Tolvaptan binds initially to extracellular loops 2 and 3 (ECL2/3) before overcoming an energy barrier to enter the pocket. Our simulations result highlighted key residues (e.g., R181, Y205, F287, F178) involved in this process, which were experimentally confirmed by site-directed mutagenesis. This work provides structural insights into tolvaptan-V2R interactions, potentially aiding the design of novel antagonists for V2R and other G protein-coupled receptors.
ABSTRACT
Autosomal dominant polycystic kidney disease (ADPKD) is a common monogenic kidney disease. Emerging research indicates that the Notch signaling pathway plays an indispensable role in the pathogenesis of numerous kidney diseases, including ADPKD. Herein, we identified that Notch3 but not other Notch receptors was overexpressed in renal tissues from mice with ADPKD and ADPKD patients. Inhibiting Notch3 with γ-secretase inhibitors, which block a proteolytic cleavage required for Notch3 activation, or shRNA knockdown of Notch3 significantly delayed renal cyst growth in vitro and in vivo. Subsequent mechanistic study elucidated that the cleaved intracellular domain of Notch3 (N3ICD) and Hes1 could bind to the PTEN promoter, leading to transcriptional inhibition of PTEN. This further activated the downstream PI3K-AKT-mTOR pathway and promoted renal epithelial cell proliferation. Overall, Notch3 was identified as a novel contributor to renal epithelial cell proliferation and cystogenesis in ADPKD. We envision that Notch3 represents a promising target for ADPKD treatment.
Subject(s)
Cell Proliferation , Polycystic Kidney, Autosomal Dominant , Receptor, Notch3 , Animals , Receptor, Notch3/metabolism , Receptor, Notch3/genetics , Cell Proliferation/drug effects , Cell Proliferation/physiology , Polycystic Kidney, Autosomal Dominant/metabolism , Polycystic Kidney, Autosomal Dominant/drug therapy , Polycystic Kidney, Autosomal Dominant/pathology , Polycystic Kidney, Autosomal Dominant/genetics , Mice , Humans , Mice, Inbred C57BL , Male , Kidney/metabolism , Kidney/pathology , Kidney/drug effectsABSTRACT
The dysregulated intracellular cAMP in the kidneys drives cystogenesis and progression in autosomal dominant polycystic kidney disease (ADPKD). Mounting evidence supports that vasopressin V2 receptor (V2R) antagonism effectively reduces cAMP levels, validating this receptor as a therapeutic target. Tolvaptan, an FDA-approved V2R antagonist, shows limitations in its clinical efficacy for ADPKD treatment. Therefore, the pursuit of better-in-class V2R antagonists with an improved efficacy remains pressing. Herein, we synthesized a set of peptide V2R antagonists. Peptide 33 exhibited a high binding affinity for the V2R (Ki = 6.1 ± 1.5 nM) and an extended residence time of 20 ± 1 min, 2-fold that of tolvaptan. This prolonged interaction translated into sustained suppression of cAMP production in washout experiments. Furthermore, peptide 33 exhibited improved efficacies over tolvaptan in both ex vivo and in vivo models of ADPKD, underscoring its potential as a promising lead compound for the treatment of ADPKD.
Subject(s)
Polycystic Kidney, Autosomal Dominant , Humans , Tolvaptan/therapeutic use , Tolvaptan/metabolism , Polycystic Kidney, Autosomal Dominant/drug therapy , Polycystic Kidney, Autosomal Dominant/metabolism , Antidiuretic Hormone Receptor Antagonists/pharmacology , Antidiuretic Hormone Receptor Antagonists/therapeutic use , Kidney/metabolism , Vasopressins/metabolism , Receptors, Vasopressin/metabolismABSTRACT
Bifunctional ligands possessing both µOR agonism and σ1R antagonism have shown promise in producing strong analgesic effects with reduced opioid-related side effects. However, the µOR agonism activity of most dual ligands diminishes compared with classical opioids, raising concern about their effectiveness in managing nociceptive pain. In this study, a new class of dual µOR agonist/σ1R antagonist was reported. Through structure-activity relationship analyses, we identified the optimal compound, 4x, which displayed picomolar µOR agonism activity (EC50: 0.6 ± 0.2 nM) and good σ1R inhibitory activity (Ki: 363.7 ± 5.6 nM) with excellent selectivity. Compound 4x exhibited robust analgesic effects in various pain models, with significantly reduced side effects. Importantly, compound 4x also possessed good safety profiles and no abnormalities were observed in biological parameters even under a high dosage. Our findings suggest that 4x may be a promising lead compound for developing safer opioids and warrants further in-depth studies.
Subject(s)
Analgesics , Receptors, Opioid, mu , Humans , Analgesics/adverse effects , Pain/drug therapy , Pain/chemically induced , Analgesics, Opioid/adverse effects , Structure-Activity Relationship , LigandsABSTRACT
The adenosine A1 receptor plays important roles in tuning free fatty acid (FFA) levels and represents an attractive target for metabolic disorders. Though remarkable progress has been achieved in the exploitation of effective (orthosteric) A1 receptor agonists in modulating aberrant FFA levels, the effect of A1 receptor allosteric modulation on lipid homeostasis is less investigated. Herein we sought to explore the effect of an allosteric modulator on the action of an A1 receptor orthosteric agonist in regulating the lipolytic process in vitro and in vivo. We examined the binding kinetics of a selective A1 receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA) in the absence or presence of an allosteric modulator (2-amino-4,5-dimethyl-3-thienyl)-[3-(trifluoromethyl)-phenyl]methanone (PD81,723) on rat adipocyte membranes. We also examined the allosteric effects of PD81,723 on mediating the CCPA-induced inhibition of cAMP accumulation, HSL (hormone-sensitive lipase) phosphorylation and FFA production in in vitro and in vivo models. Our results demonstrated that PD81,723 slowed down the dissociation of CCPA from the A1 receptor, which, consequently, potentiated the antilipolytic action of CCPA through downregulating the cAMP/HSL pathway. Our study exemplified the application of A1 receptor allosteric modulators as an alternative for metabolic disease treatments.
Subject(s)
Adipose Tissue , Receptors, Purinergic P1 , Rats , Animals , Receptors, Purinergic P1/metabolism , Adipose Tissue/metabolism , Adipocytes , Lipolysis , Adenosine/metabolism , Receptor, Adenosine A1/metabolism , Allosteric RegulationABSTRACT
Vasopressin V2 receptors (V2R) are a promising drug target for autosomal dominant polycystic kidney disease (ADPKD). As previous research demonstrated that the residence time of V2R antagonists is critical to their efficacy in both ex vivo and in vivo models of ADPKD, we performed extensive structure-kinetic relationship (SKR) analyses on a series of benzodiazepine derivatives. We found that subtle structural modifications of the benzodiazepine derivatives dramatically changed their binding kinetics but not their affinity. Compound 18 exhibited a residence time of 77 min, which was 7.7-fold longer than that of the reference compound tolvaptan (TVP). Accordingly, compound 18 exhibited higher efficacy compared to TVP in an in vivo model of ADPKD. Overall, our study exemplifies a kinetics-directed medicinal chemistry effort for the development of efficacious V2R antagonists. We envision that this strategy may also have general applicability in other therapeutic areas.
Subject(s)
Anti-Anxiety Agents , Polycystic Kidney, Autosomal Dominant , Humans , Antidiuretic Hormone Receptor Antagonists/pharmacology , Antidiuretic Hormone Receptor Antagonists/therapeutic use , Polycystic Kidney, Autosomal Dominant/drug therapy , Tolvaptan/pharmacology , Tolvaptan/therapeutic use , Vasopressins/pharmacology , Vasopressins/metabolism , Hypnotics and Sedatives , Anticonvulsants/therapeutic use , Benzodiazepines/pharmacology , Benzodiazepines/therapeutic use , Receptors, Vasopressin/metabolismABSTRACT
Opioids are efficacious analgesics for pain treatments. However, their repeated use in large doses often leads to analgesic tolerance, which limits the clinical application. Sigma-1 receptor (σ1R) antagonists were reported to synergistically enhance the analgesic effect of mu opioid receptor (MOR) agonists without amplifying the adverse effects. Therefore, the σ1R is considered a promising drug target for pain management. Based on the recently elucidated co-crystal structure of σ1R with 4-IBP, we designed and developed a series of σ1R antagonists harboring the 2,6-diazaspiro[3.4]octan-7-one scaffold. Through a detailed structure-activity relationship study, we identified compound 32 as a potent σ1R antagonist, which significantly enhanced the antinociceptive effect of morphine and rescued morphine-induced analgesic tolerance. Our results support σ1R antagonism as a promising strategy to develop novel analgesics and highlight the therapeutic potential of compound 32 to prevent morphine tolerance.
Subject(s)
Morphine , Receptors, sigma , Morphine/pharmacology , Morphine/therapeutic use , Analgesics/pharmacology , Analgesics/therapeutic use , Analgesics, Opioid/pharmacology , Analgesics, Opioid/therapeutic use , Receptors, Opioid, mu , Dose-Response Relationship, Drug , Sigma-1 ReceptorABSTRACT
Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease, which is characterized by progressive growth of multiple renal cysts in bilateral kidneys. In the past decades, mechanistic studies have entailed many essential signalling pathways that were regulated through post-translational modifications (PTMs) during cystogenesis. Among the numerous PTMs involved, the effect of ubiquitination and deubiquitination remains largely unknown. Herein, we identified that USP28, a deubiquitinase aberrantly upregulated in patients with ADPKD, selectively removed K48-linked polyubiquitination and reversed protein degradation of signal transducer and activator of transcription 3 (STAT3). We also observed that USP28 could directly interact with and stabilize c-Myc, a transcriptional target of STAT3. Both processes synergistically enhanced renal cystogenesis. Furthermore, pharmacological inhibition of USP28 attenuated the cyst formation both in vivo and in vitro. Collectively, USP28 regulates STAT3 turnover and its transcriptional target c-Myc in ADPKD. USP28 inhibition could be a novel therapeutic strategy against ADPKD.
Subject(s)
Cysts , Polycystic Kidney, Autosomal Dominant , Ubiquitin Thiolesterase , Humans , Cysts/metabolism , Deubiquitinating Enzymes , Kidney/metabolism , Polycystic Kidney, Autosomal Dominant/drug therapy , Polycystic Kidney, Autosomal Dominant/genetics , Polycystic Kidney, Autosomal Dominant/metabolism , Signal Transduction , Ubiquitin Thiolesterase/antagonists & inhibitors , Ubiquitin Thiolesterase/metabolism , Animals , Mice , STAT3 Transcription Factor/metabolism , Proto-Oncogene Proteins c-myc/metabolismABSTRACT
Renal cyst development and expansion in autosomal dominant polycystic kidney disease (ADPKD) involves over-proliferation of cyst-lining epithelial cells and excessive cystic fluid secretion. While metformin effectively inhibits renal cyst growth in mouse models of ADPKD it exhibits low potency, and thus an adenosine monophosphate-activated protein kinase (AMPK) activator with higher potency is required. Herein, we adopted a drug repurposing strategy to explore the potential of PF-06409577, an AMPK activator for diabetic nephropathy, in cellular, ex vivo and in vivo models of ADPKD. Our results demonstrated that PF-06409577 effectively down-regulated mammalian target of rapamycin pathway-mediated proliferation of cyst-lining epithelial cells and reduced cystic fibrosis transmembrane conductance regulator-regulated cystic fluid secretion. Overall, our data suggest that PF-06409577 holds therapeutic potential for ADPKD treatment.
Subject(s)
Cysts , Metformin , Polycystic Kidney, Autosomal Dominant , AMP-Activated Protein Kinases/metabolism , Adenosine Monophosphate , Animals , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Indoles , Mammals , Metformin/pharmacology , Mice , Polycystic Kidney, Autosomal Dominant/drug therapy , Polycystic Kidney, Autosomal Dominant/metabolism , TOR Serine-Threonine Kinases/metabolismABSTRACT
Cyst formation and enlargement in autosomal dominant kidney disease (ADPKD) is mainly driven by aberrantly increased cytosolic cAMP in renal tubule epithelial cells. Because the vasopressin V2 receptor (V2R) regulates intracellular cAMP levels in kidneys, a series of benzodiazepine derivatives were developed targeting the V2R. Among these derivatives, compound 25 exhibited potent binding affinity to the V2R (Ki = 9.0 ± 1.5 nM) and efficacious cAMP inhibition (IC50 = 9.2 ± 3.0 nM). This led to the suppression of cyst formation and growth in both an MDCK cell model and an embryonic kidney cyst model. Further advancing compound 25 in a murine model of ADPKD demonstrated a significantly improved in vivo efficacy compared with the reference compound tolvaptan. Overall, compound 25 holds therapeutic potential for the treatment of ADPKD.
Subject(s)
Cysts , Polycystic Kidney, Autosomal Dominant , Animals , Antidiuretic Hormone Receptor Antagonists/pharmacology , Antidiuretic Hormone Receptor Antagonists/therapeutic use , Benzodiazepines/metabolism , Benzodiazepines/pharmacology , Benzodiazepines/therapeutic use , Cyclic AMP/metabolism , Cysts/metabolism , Humans , Kidney/metabolism , Mice , Polycystic Kidney, Autosomal Dominant/drug therapy , Polycystic Kidney, Autosomal Dominant/metabolism , Receptors, Vasopressin/metabolism , Vasopressins/metabolismABSTRACT
Prevailing strategies directing early-phase drug discovery heavily rely on equilibrium-based metrics such as affinity, which overlooks the kinetic process of a drug molecule interacting with its target. Herein, we developed a number of vasopressin V2 receptor (V2R) antagonists with divergent binding affinities and kinetics for autosomal dominant polycystic kidney disease (ADPKD). Surprisingly, the residence time of the V2R antagonists, but not their affinity, was correlated with the efficacy in both ex vivo and in vivo models of ADPKD. We envision that the kinetics-directed drug candidate selection and development may have general applicability for ADPKD and other therapeutic areas as well.
Subject(s)
Antidiuretic Hormone Receptor Antagonists , Polycystic Kidney, Autosomal Dominant , Receptors, Vasopressin , Antidiuretic Hormone Receptor Antagonists/chemistry , Antidiuretic Hormone Receptor Antagonists/pharmacology , Drug Design , Humans , Polycystic Kidney, Autosomal Dominant/drug therapy , Polycystic Kidney, Autosomal Dominant/metabolism , Receptors, Vasopressin/metabolismABSTRACT
Piccolo, a presynaptic cytomatrix protein, plays a role in synaptic vesicle trafficking in the presynaptic active zone. Certain single-nucleotide polymorphisms of the Piccolo-encoding gene PCLO are reported to be associated with mental disorders. However, a few studies have evaluated the relationship between Piccolo dysfunction and psychotic symptoms. Therefore, we investigated the neurophysiological and behavioral phenotypes in mice with Piccolo suppression in the medial prefrontal cortex (mPFC). Downregulation of Piccolo in the mPFC reduced regional synaptic proteins, accompanied with electrophysiological impairments. The Piccolo-suppressed mice showed an enhanced locomotor activity, impaired auditory prepulse inhibition, and cognitive dysfunction. These abnormal behaviors were partially ameliorated by the antipsychotic drug risperidone. Piccolo-suppressed mice received mild social defeat stress showed additional behavioral despair. Furthermore, the responses of these mice to extracellular glutamate and dopamine levels induced by the optical activation of mPFC projection in the dorsal striatum (dSTR) were inhibited. Similarly, the Piccolo-suppressed mice showed decreased depolarization-evoked glutamate and -aminobutyric acid elevations and increased depolarization-evoked dopamine elevation in the dSTR. These suggest that Piccolo regulates neurotransmission at the synaptic terminal of the projection site. Reduced neuronal connectivity in the mPFC-dSTR pathway via suppression of Piccolo in the mPFC may induce behavioral impairments observed in schizophrenia.
ABSTRACT
The vasopressin V2 receptor belongs to the superfamily of G protein-coupled receptors (GPCRs) and is a potential drug target for water balance disorders such as polycystic kidney disease. Traditionally, the discovery of novel agents for the vasopressin V2 receptor has been guided by evaluating their receptor affinity, largely ignoring the binding kinetics. However, the latter is receiving increasing attention in the drug research community and has been proved to be a more complete descriptor of the dynamic process of ligand-receptor interaction. Herein we aim to revisit the molecular basis of ligand-vasopressin V2 receptor interaction from the less-investigated kinetic perspective. A homogenous time-resolved fluorescence resonance energy transfer (TR-FRET) assay was set up and optimized, which enabled accurate kinetic profiling of unlabeled vasopressin V2 receptor ligands. Receptor occupancy profiles of two representative antagonists with distinct target residence time were simulated. Their functional effects were further explored in cAMP assays. Our results showed that the antagonist with longer receptor residence time (lixivaptan) displayed sustained target occupancy than the antagonist with shorter receptor residence time (mozavaptan). In accordance, lixivaptan displayed insurmountable antagonism and wash-resistant inhibitory effect on the cellular cAMP level, while not so for mozavaptan. Together, our data provide evidence that binding kinetics, next to their affinity, offers additional information for the dynamic process of ligand-receptor interaction. Hopefully, this study may lead to more kinetics-directed medicinal chemistry efforts and aid the design and discovery of different-in-class of vasopressin V2 receptor ligands for clinical applications.
Subject(s)
Receptors, Vasopressin/metabolism , Animals , Antidiuretic Hormone Receptor Antagonists/pharmacology , CHO Cells , Cricetulus , Cyclic AMP/metabolism , HEK293 Cells , Humans , Kinetics , Ligands , Protein Binding , Receptors, Vasopressin/geneticsABSTRACT
Transmembrane protein 168 (TMEM168) comprises 697 amino acid residues, including some putative transmembrane domains. It is reported that TMEM168 controls methamphetamine (METH) dependence in the nucleus accumbens (NAc) of mice. Moreover, a strong link between METH dependence-induced adaptive changes in the brain and mood disorders has been evaluated. In the present study, we investigated the effects of accumbal TMEM168 in a battery of behavioral paradigms. The adeno-associated virus (AAV) Tmem168 vector was injected into the NAc of C57BL/6J mice (NAc-TMEM mice). Subsequently, the accumbal TMEM168 mRNA was increased approximately by seven-fold when compared with the NAc-Mock mice (controls). The NAc-TMEM mice reported no change in the locomotor activity, cognitive ability, social interaction, and depression-like behaviors; however, TMEM168 overexpression enhanced anxiety in the elevated-plus maze and light/dark box test. The increased anxiety was reversed by pretreatment with the antianxiety drug diazepam (0.3 mg/kg i.p.). Moreover, the NAc-TMEM mice exhibited decreased prepulse inhibition (PPI) in the startle response test, and the induced schizophrenia-like behavior was reversed by pretreatment with the antipsychotic drug risperidone (0.01 mg/kg i.p.). Furthermore, accumbal TMEM168 overexpression decreased the basal levels of extracellular GABA in the NAc and the high K+ (100 mM)-stimulated GABA elevation; however, the total contents of GABA in the NAc remained unaffected. These results suggest that the TMEM168-regulated GABAergic neuronal system in the NAc might become a novel target while studying the etiology of anxiety and sensorimotor gating deficits.
Subject(s)
Membrane Proteins/metabolism , Nucleus Accumbens/metabolism , Sensory Gating , Animals , Behavior, Animal , Diazepam/pharmacology , Dopamine/metabolism , Glutamic Acid/metabolism , Male , Membrane Proteins/genetics , Mice , Mice, Inbred C57BL , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Risperidone/pharmacology , Sensory Gating/drug effects , Serotonin/metabolism , gamma-Aminobutyric Acid/metabolismABSTRACT
Chronic exposure to methamphetamine causes adaptive changes in brain, which underlie dependence symptoms. We have found that the transmembrane protein 168 (TMEM168) is overexpressed in the nucleus accumbens of mice upon repeated methamphetamine administration. Here, we firstly demonstrate the inhibitory effect of TMEM168 on methamphetamine-induced behavioral changes in mice, and attempt to elucidate the mechanism of this inhibition. We overexpressed TMEM168 in the nucleus accumbens of mice by using an adeno-associated virus vector (NAc-TMEM mice). Methamphetamine-induced hyperlocomotion and conditioned place preference were attenuated in NAc-TMEM mice. Additionally, methamphetamine-induced extracellular dopamine elevation was suppressed in the nucleus accumbens of NAc-TMEM mice. Next, we identified extracellular matrix protein osteopontin as an interacting partner of TMEM168, by conducting immunoprecipitation in cultured COS-7 cells. TMEM168 overexpression in COS-7 cells induced the enhancement of extracellular and intracellular osteopontin. Similarly, osteopontin enhancement was also observed in the nucleus accumbens of NAc-TMEM mice, in in vivo studies. Furthermore, the infusion of osteopontin proteins into the nucleus accumbens of mice was found to inhibit methamphetamine-induced hyperlocomotion and conditioned place preference. Our studies suggest that the TMEM168-regulated osteopontin system is a novel target pathway for the therapy of methamphetamine dependence, via regulating the dopaminergic function in the nucleus accumbens.
Subject(s)
Locomotion/drug effects , Membrane Proteins/metabolism , Methamphetamine/pharmacology , Osteopontin/metabolism , Animals , COS Cells , Chlorocebus aethiops , In Situ Hybridization , Male , Mice , Mice, Inbred C57BL , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Background: Several clinical studies have suggested that N-acetylaspartate and N-acetylaspartylglutamate levels in the human brain are associated with various psychiatric disorders, including major depressive disorder. We have previously identified Shati/Nat8l, an N-acetyltransferase, in the brain using an animal model of psychosis. Shati/Nat8l synthesizes N-acetylaspartate from L-aspartate and acetyl-coenzyme A. Further, N-acetylaspartate is converted into N-acetylaspartylglutamate, a neurotransmitter for metabotropic glutamate receptor 3. Methods: Because Shati/Nat8l mRNA levels were increased in the dorsal striatum of mice following the exposure to forced swimming stress, Shati/Nat8l was overexpressed in mice by the microinjection of adeno-associated virus vectors containing Shati/Nat8l gene into the dorsal striatum (dS-Shati/Nat8l mice). The dS-Shati/Nat8l mice were further assessed using behavioral and neurochemical tests. Results: The dS-Shati/Nat8l mice exhibited behavioral despair in the forced swimming and tail suspension tests and social withdrawal in the 3-chamber social interaction test. These depression-like behaviors were attenuated by the administration of a metabotropic glutamate receptor 2/3 antagonist and a selective serotonin reuptake inhibitor. Furthermore, the metabolism of N-acetylaspartate to N-acetylaspartylglutamate was decreased in the dorsal striatum of the dS-Shati/Nat8l mice. This finding corresponded with the increased expression of glutamate carboxypeptidase II, an enzyme that metabolizes N-acetylaspartylglutamate present in the extracellular space. Extracellular serotonin levels were lower in the dorsal striatum of the dS-Shati/Nat8l and normal mice that were repeatedly administered a selective glutamate carboxypeptidase II inhibitor. Conclusions: Our findings indicate that the striatal expression of N-acetylaspartate synthetase Shati/Nat8l plays a role in major depressive disorder via the metabotropic glutamate receptor 3-mediated functional control of the serotonergic neuronal system.
Subject(s)
Corpus Striatum/metabolism , Depression/genetics , Depression/pathology , Gene Expression Regulation/genetics , Receptors, Metabotropic Glutamate/metabolism , Serotonin/metabolism , Acetyltransferases/genetics , Acetyltransferases/metabolism , Animals , Aspartic Acid/analogs & derivatives , Aspartic Acid/metabolism , Depression/metabolism , Dipeptides/metabolism , Disease Models, Animal , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HEK293 Cells , Hindlimb Suspension , Humans , Interpersonal Relations , Male , Membrane Proteins/metabolism , Mice , Mice, Inbred C57BL , Microdialysis , Microinjections , Swimming/psychology , Transduction, GeneticABSTRACT
RATIONALE: Although dependence to methamphetamine (METH) is associated with serious psychiatric symptoms and is a global health and social problem, no effective therapeutic approaches have been identified. Pseudoginsenoside-F11 (PF11) is an ocotillol-type saponin that is isolated from Panax quinquefolius (American ginseng) and was shown to have neuroprotective effects to promote learning and memory and to antagonize the pharmacological effects of morphine. Furthermore, PF11 also shows protective effects against METH-induced neurotoxicity in mice. However, the effects of PF11 on METH-induced preference and dopamine (DA) release have not been defined. OBJECTIVES: We investigated the effects of PF11 administration on METH-induced hyperlocomotion and conditioned place preference (CPP) in mice. Subsequently, extracellular DA and gamma-aminobutyric acid (GABA) levels were determined in the nucleus accumbens (NAc) of mice after co-administration of PF11 and METH using in vivo microdialysis analyses. Moreover, the effects of PF11 administration on the µ-opioid neuronal responses, DAMGO (µ-opioid receptor agonist; [D-Ala(2), N-MePhe(4), Gly-ol]-enkephalin)-induced hyperlocomotion and accumbal extracellular DA increase were investigated to elucidate how PF11 inhibits METH-induced dependence by dopaminergic neuronal hyperfunction. RESULTS: Co-administration of PF11 and METH for 6 days attenuated METH-induced locomotor sensitization compared with treatment with METH alone. In the CPP test, PF11 administration also inhibited METH-induced place preference. In vivo microdialysis analyses indicated that co-administration of PF11 and METH for 7 days prevented METH-induced extracellular DA increase in the NAc and repeated PF11 administration with or without METH for 7 days increased extracellular GABA levels in the NAc, whereas single administration of PF11 did not. Furthermore, DAMGO-induced hyperlocomotion and accumbal extracellular DA increase were significantly inhibited by acute PF11 administration. CONCLUSIONS: The present data suggest that PF11 inhibits METH-induced hyperlocomotion, preference, and accumbal extracellular DA increase by regulating GABAergic neurons and µ-opioid receptors.
Subject(s)
Behavior, Animal/drug effects , Central Nervous System Stimulants/antagonists & inhibitors , Dopamine Uptake Inhibitors/antagonists & inhibitors , Dopaminergic Neurons/drug effects , Ginsenosides/pharmacology , Methamphetamine/antagonists & inhibitors , Neurons/drug effects , Nucleus Accumbens/drug effects , gamma-Aminobutyric Acid/physiology , Analgesics, Opioid/pharmacology , Animals , Conditioning, Operant/drug effects , Dopamine/metabolism , Dopamine Uptake Inhibitors/pharmacology , Enkephalin, Ala(2)-MePhe(4)-Gly(5)-/pharmacology , Male , Methamphetamine/pharmacology , Mice , Mice, Inbred ICR , Nucleus Accumbens/cytology , Receptors, Opioid, mu/drug effectsABSTRACT
A novel N-acetyltransferase, Shati/Nat8l, was identified in the nucleus accumbens (NAc) of mice with methamphetamine (METH) treatment. Previously we reported that suppression of Shati/Nat8l enhanced METH-induced behavioral alterations via dopaminergic neuronal regulation. However, the physiological mechanisms of Shati/Nat8l on the dopaminergic system in the brain are unclear. In this study, we injected adeno-associated virus (AAV) vector containing Shati/Nat8l into the NAc or dorsal striatum (dS) of mice, to increase Shati/Nat8l expression. Overexpression of Shati/Nat8l in the NAc, but not in the dS, attenuated METH-induced hyperlocomotion, locomotor sensitization, and conditioned place preference in mice. Moreover, the Shati/Nat8l overexpression in the NAc attenuated the elevation of extracellular dopamine levels induced by METH in in vivo microdialysis experiments. These behavioral and neurochemical alterations due to Shati/Nat8l overexpression in the NAc were inhibited by treatment with selective group II metabotropic glutamate receptor type 2 and 3 (mGluR2/3) antagonist LY341495. In the AAV vector-injected NAc, the tissue contents of both N-acetylaspartate and N-acetylaspartylglutamate (NAAG), endogenous mGluR3 agonist, were elevated. The injection of peptidase inhibitor of NAAG or the perfusion of NAAG itself reduced the basal levels of extracellular dopamine in the NAc of naive mice. These results indicate that Shati/Nat8l in the NAc, but not in the dS, plays an important suppressive role in the behavioral responses to METH by controlling the dopaminergic system via activation of group II mGluRs.