Direct esterification of amides by the dimethylsulfate-mediated activation of amide C-N bonds.

Amides are important intermediates in organic chemistry and the pharmaceutical industry, but their low reactivity requires catalysts and/or severe reaction conditions for esterification. Here, a novel approach was devised to convert amides into esters without the use of transition metals. The method effectively overcomes the inherent low reactivity of amides by employing dimethylsulfate-mediated reaction to activate the C-N bonds. To confirm the proposed reaction mechanism, control experiments and density functional theory (DFT) calculations were conducted. The method demonstrates a wide array of substrates, including amides with typical H/alkyl/aryl substitutions, N,N-disubstituted amides, amides derived from alkyl, aryl, or vinyl carboxylic acids, and even amino acid substrates with stereocentres. Furthermore, we have shown the effectiveness of dimethylsulfate in removing acyl protective groups in amino derivatives. This study presents a method that offers efficiency and cost-effectiveness in broadening the esterification capabilities of amides, thereby facilitating their increased utilization as synthetic compounds in diverse transformations.

Design and synthesis of cantharidin derivative DCZ5418 as a TRIP13 inhibitor with anti-multiple myeloma activity in vitro and in vivo.

Natural product cantharidin can inhibit multiple myeloma cell growth in vitro, while serious adverse effects limited its clinical application. Therefore, the structural modification of cantharidin is needed. Herein, inspired by the structural similarity of the aliphatic endocyclic moiety in cantharidin and TRIP13 inhibitor DCZ0415, we designed and synthesized DCZ5418 and its nineteen derivatives. The molecular docking study indicated that DCZ5418 had a similar binding mode to TRIP13 protein as DCZ0415 while with a stronger docking score. Moreover, the bioassay studies of the MM-cells viability inhibition, TRIP13 protein binding affinity and enzyme inhibiting activity showed that DCZ5418 had good anti-MM activity in vitro and definite interaction with TRIP13 protein. The acute toxicity test of DCZ5418 showed less toxicity in vivo than cantharidin. Furthermore, DCZ5418 showed good anti-MM effects in vivo with a lower dose administration than DCZ0415 (15 mg/kg vs 25 mg/kg) on the tumor xenograft models. Thus, we obtained a new TRIP13 inhibitor DCZ5418 with improved safety and good activity in vivo, which provides a new example of lead optimization by using the structural fragments of natural products.

Discovery of a CB2 and 5-HT1A receptor dual agonist for the treatment of depression and anxiety.

Cannabinoid CB2R agonists have gained considerable attention as potential novel therapies for psychiatric disorders due to their non-psychoactive nature, in contrast to CB1R agonists. In this study, we employed molecular docking to design and synthesize 23 derivatives of cannabidiol (CBD) with the aim of discovering potent CB2R agonists rather than CB2R antagonists or inverse agonists. Structure-activity relationship (SAR) investigations highlighted the critical importance of the amide group at the C-3' site and the cycloalkyl group at the C-4' site for CB2R activation. Interestingly, three CBD derivatives, namely 2o, 6g, and 6h, exhibited substantial partial agonistic activity towards the CB2 receptor, in contrast to the inverse agonistic property of CBD. Among these, 2o acted as a CB2R and 5-HT1AR dual agonist, albeit with some undesired antagonist activity for CB1R. It demonstrated significant CB2R partial agonism while maintaining a level of 5-HT1AR agonistic and CB1R antagonistic activity similar to CBD. Pharmacokinetic experiments confirmed that 2o possesses favorable pharmacokinetic properties. Behavioral studies further revealed that 2o elicits significant antidepressant-like and anxiolytic-like effects while maintaining a good safety profile.

A Cre Driver Line for Genetic Targeting of Kappa Opioid Receptor Expressing Cells.

Here we describe the generation and characterization of a Cre knock-in mouse line that harbors a Cre insertion in the 3'UTR of the κ opioid receptor gene (Oprk1) locus and provides genetic access to populations of κ opioid receptor (KOR)-expressing neurons throughout the brain. Using a combination of techniques including RNA in situ hybridization and immunohistochemistry, we report that Cre is expressed with high fidelity in KOR-expressing cells throughout the brain in this mouse line. We also provide evidence that Cre insertion does not alter basal KOR function. Baseline anxiety-like behaviors and nociceptive thresholds are unaltered in Oprk1-Cre mice. Chemogenetic activation of KOR-expressing cells in the basolateral amygdala (BLAKOR cells) resulted in several sex-specific effects on anxiety-like and aversive behaviors. Activation led to decreased anxiety-like behavior on the elevated plus maze and increased sociability in female but not in male Oprk1-Cre mice. Activation of BLAKOR cells also attenuated KOR agonist-induced conditioned place aversion (CPA) in male Oprk1-Cre mice. Overall, these results suggest a potential role for BLAKOR cells in regulating anxiety-like behaviors and KOR-agonist mediated CPA. In summary, these results provide evidence for the utility of the newly generated Oprk1-Cre mice in assessing localization, anatomy, and function of KOR circuits throughout the brain.

Cocaine self-administration augments kappa opioid receptor system-mediated inhibition of dopamine activity in the mesolimbic dopamine system.

Prior studies examining the effects of cocaine on the dynorphin/kappa opioid receptor (Dyn/KOR) system primarily focus on non-contingent cocaine exposure, but the effects of self-administration, which more closely reflects human drug-taking behaviors, are not well studied. In this study we characterized the effects of escalated intravenous cocaine self-administration on the functional state of the Dyn/KOR system and its interaction with mesolimbic dopamine signaling. Rats self-administered cocaine in an extended access, limited intake cocaine procedure, in which animals obtained 40 infusions per day (1.5 mg/kg/inf) for 5 consecutive days to ensure comparable consumption levels. Following single day tests of cue reactivity and progressive ratio responding, quantitative real-time polymerase chain reaction was used to measure levels of Oprk and Pdyn transcripts in the ventral tegmental area and nucleus accumbens. Additionally, after self-administration, ex vivo fast-scan cyclic voltammetry in the NAc was used to examine the ability of the KOR agonist U50,488 to inhibit dopamine release. We found that KOR-induced inhibition of dopamine release was enhanced in animals that self-administered cocaine compared to controls, suggesting upregulated Dyn/KOR activity after cocaine self-administration. Furthermore, expression levels of Pdyn in the nucleus accumbens and ventral tegmental area, and Oprk in the nucleus accumbens, were elevated in cocaine animals compared to controls. Additionally, Pdyn expression in the nucleus accumbens was negatively correlated with progressive ratio breakpoints, a measure of motivation to self-administer cocaine. Overall, these data suggest that cocaine self-administration elevates KOR/Dyn system activity in the mesolimbic dopamine pathway.

Atorvastatin differentially regulates the interactions of cocaine and amphetamine with dopamine transporters.

The function of the dopamine transporter (DAT) is regulated by membrane cholesterol content. A direct, acute removal of membrane cholesterol by methyl-ß-cyclodextrin (MßCD) has been shown to reduce dopamine (DA) uptake and release mediated by the DAT. This is of particular interest because a few widely prescribed statins that lower peripheral cholesterol levels are blood-brain barrier (BBB) penetrants, and therefore could alter DAT function through brain cholesterol modulation. The goal of this study was to investigate the effects of prolonged atorvastatin treatment (24 h) on DAT function in neuroblastoma 2A cells stably expressing DAT. We found that atorvastatin treatment effectively lowered membrane cholesterol content in a concentration-dependent manner. Moreover, atorvastatin treatment markedly reduced DA uptake and abolished cocaine inhibition of DA uptake, independent of surface DAT levels. These deficits induced by atorvastatin treatment were reversed by cholesterol replenishment. However, atorvastatin treatment did not change amphetamine (AMPH)-induced DA efflux. This is in contrast to a small but significant reduction in DA efflux induced by acute depletion of membrane cholesterol using MßCD. This discrepancy may involve differential changes in membrane lipid composition resulting from chronic and acute cholesterol depletion. Our data suggest that the outward-facing conformation of DAT, which favors the binding of DAT blockers such as cocaine, is more sensitive to atorvastatin-induced cholesterol depletion than the inward-facing conformation, which favors the binding of DAT substrates such as AMPH. Our study on statin-DAT interactions may have clinical implications in our understanding of neurological side effects associated with chronic use of BBB penetrant statins.

Chemo- and Site-Selective Lysine Modification of Peptides and Proteins under Native Conditions Using the Water-Soluble Zolinium.

Site-selective lysine modification of peptides and proteins in aqueous solutions or in living cells is still a big challenge today. Here, we report a novel strategy to selectively quinolylate lysine residues of peptides and proteins under native conditions without any catalysts using our newly developed water-soluble zoliniums. The zoliniums could site-selectively quinolylate K350 of bovine serum albumin and inactivate SARS-CoV-2 3CLpro via covalently modifying two highly conserved lysine residues (K5 and K61). In living HepG2 cells, it was demonstrated that the simple zoliniums (5b and 5B) could quinolylate protein lysine residues mainly in the nucleus, cytosol, and cytoplasm, while the zolinium-fluorophore hybrid (8) showed specific lysosome-imaging ability. The specific chemoselectivity of the zoliniums for lysine was validated by a mixture of eight different amino acids, different peptides bearing potential reactive residues, and quantum chemistry calculations. This study offers a new way to design and develop lysine-targeted covalent ligands for specific application.

Prolonged ethanol exposure modulates constitutive internalization and recycling of 5-HT1A receptors.

Alcohol exposure alters the signaling of the serotoninergic system, which is involved in alcohol consumption, reward, and dependence. In particular, dysregulation of serotonin receptor type 1A (5-HT1AR) is associated with alcohol intake and withdrawal-induced anxiety-like behavior in rodents. However, how ethanol regulates 5-HT1AR activity and cell surface availability remains elusive. Using neuroblastoma 2a cells stably expressing human 5-HT1ARs tagged with hemagglutinin at the N-terminus, we found that prolonged ethanol exposure (18 h) reduced the basal surface levels of 5-HT1ARs in a concentration-dependent manner. This reduction is attributed to both enhanced receptor internalization and attenuated receptor recycling. Moreover, constitutive 5-HT1AR internalization in ethanol naïve cells was blocked by concanavalin A (ConA) but not nystatin, suggesting clathrin-dependent 5-HT1AR internalization. In contrast, constitutive 5-HT1AR internalization in ethanol-treated cells was blocked by nystatin but not by ConA, indicating that constitutive 5-HT1AR internalization switched from a clathrin- to a caveolin-dependent pathway. Dynasore, an inhibitor of dynamin, blocked 5-HT1AR internalization in both vehicle- and ethanol-treated cells. Furthermore, ethanol exposure enhanced the activity of dynamin I via dephosphorylation and reduced myosin Va levels, which may contribute to increased internalization and reduced recycling of 5-HT1ARs, respectively. Our findings suggest that prolonged ethanol exposure not only alters the endocytic trafficking of 5-HT1ARs but also the mechanism by which constitutive 5-HT1AR internalization occurs.

Discovery of chiral N-2'-aryletheryl-1'-alkoxy-ethyl substituted arylisoquinolones with anti-inflammatory activity from the nucleophilic addition reactions of the thiophenols and oxazolinium.

Herein we disclosed the novel nucleophilic addition reactions of the thiophenols and oxazolinium (DCZ0358) to produce N-2'-aryletheryl-1'-alkoxy-ethyl substituted arylisoquinolones. After evaluating the anti-inflammatory activity in vitro, 2d was found having significant anti-TNFα activity. Through the amplified synthesis of 2d, four monomers (3a-b and 4a-d) were obtained by chiral separation of the product. The reaction mechanism was proposed and explored by the control experiments. However, only the R-stereoisomers 3b and 4b have significant anti-TNFα activity in vitro (IC50 = 56 and 14 nM, respectively). Moreover, 4b exerts potent therapeutic effects on ulcerative colitis in vivo (30 mg/kg bw, qd, i. g.). The subsequent bio-target exploration of compound 4bvia molecular docking and the experimental validation disclosed that 4b has 3-fold selectivity of binding activity on estrogen receptor (ER) beta (ß) (Ki = 760.86 nM) vs. alpha (α) (Ki = 2320.58 nM). Thus, it provides a novel type of non-steroidal leads for developing anti-inflammatory drugs.

Metabotropic glutamate 2,3 receptor stimulation desensitizes agonist activation of G-protein signaling and alters transcription regulators in mesocorticolimbic brain regions.

Metabotropic glutamate (mGlu) receptors are regulators of glutamate release and targets for development of therapies for hyperactive glutamatergic signaling. However, the effects of long-term stimulation of mGlu receptors on cellular signaling in the brain have not been described. This study investigated the effects of 2-day and 14-day osmotic mini-pump administration of the mGlu2,3 agonist LY379268 (3.0 mg kg-1  day-1 ) to rats on receptor-mediated G-protein activation and signaling in mesocorticolimbic regions in rat brain sections. A significant reduction in LY379268-stimulated [35 S]GTPγS binding was observed in the 14-day group in some cortical regions, prefrontal cortex, nucleus accumbens, and ventral pallidum. The 14-day LY379268 treatment group exhibited mGlu2 mRNA levels significantly lower in hippocampus, nucleus accumbens, caudate, and ventral pallidum. In both 2-day and 14-day treatment groups immunodetectable phosphorylated cAMP Response Element-Binding protein (CREB) was significantly reduced across all brain regions. In the 2-day group, we observed significantly lower immunodetectable CREB protein across all brain regions, which was subsequently increased in the 14-day group but failed to achieve control values. Neither immunodetectable extracellular signal-regulated kinase (ERK) protein nor phosphorylated ERK from 2-day or 14-day treatment groups differed significantly from control across all brain regions. However, the ratio of phosphorylated ERK to total ERK protein was significantly greater in the 14-day treatment group compared with the control. These results identify compensatory changes to mGlu2,3 signal transduction in rat brains after chronic systemic administration of agonist, which could be predictive of the mechanism of action in human pharmacotherapies.

Cocaine Self-administration Regulates Transcription of Opioid Peptide Precursors and Opioid Receptors in Rat Caudate Putamen and Prefrontal Cortex.

The brain opioid system plays an important role in cocaine reward. Altered signaling in the opioid system by chronic cocaine exposure contributes to cocaine-seeking and taking behavior. The current study investigated concurrent changes in the gene expression of multiple components in rat brain opioid system following cocaine self-administration. Animals were limited to 40 infusions (1.5 mg/kg/infusion) within 6 h per day for five consecutive days. We then examined the mRNA levels of opioid receptors including mu (Oprm), delta (Oprd), and kappa (Oprk), and their endogenous opioid peptide precursors including proopiomelanocortin (Pomc), proenkephalin (Penk), prodynorphin (Pdyn) in the dorsal striatum (CPu) and the prefrontal cortex (PFC) 18 h after the last cocaine infusion. We found that cocaine self-administration significantly increased the mRNA levels of Oprm and Oprd in both the CPu and PFC, but had no effect on Oprk mRNA levels in either brain region. Moreover, cocaine had a greater influence on the mRNA levels of opioid peptide precursors in rat CPu than in the PFC. In the CPu, cocaine self-administration significantly increased the mRNA levels of Penk and Pdyn and abolished the mRNA levels of Pomc. In the PFC, cocaine self-administration only increased Pdyn mRNA levels without changing the mRNA levels of Pomc and Penk. These data suggest that cocaine self-administration influences the expression of multiple genes in the brain opioid system, and the concurrent changes in these targets may underlie cocaine-induced reward and habitual drug-seeking behavior.

A novel silicone derivative of natural osalmid (DCZ0858) induces apoptosis and cell cycle arrest in diffuse large B-cell lymphoma via the JAK2/STAT3 pathway.

Diffuse large B-cell lymphoma (DLBCL) is a highly heterogeneous malignant tumor characterized by diffuse growth. DCZ0858 is a novel small molecule with excellent antitumor effects in DLBCL. This study explored in depth the inhibitory effect of DCZ0858 on DLBCL cell lines. Cell Counting Kit-8 (CCK-8) and plate colony formation assays were used to evaluate cell proliferation levels. Flow cytometry was employed to analyze apoptosis and the cell cycle, and western blotting was used to quantify the expression of cell cycle regulators. The results indicated that DCZ0858 inhibited cell growth in a concentration-dependent and time-dependent manner while inducing no significant toxicity in normal cells. Moreover, DCZ0858 initiated cell apoptosis via both internal and external apoptotic pathways. DCZ0858 also induced cell cycle arrest in the G0/G1 phase, thereby controlling cell proliferation. Further investigation of the molecular mechanism showed that the JAK2/STAT3 pathway was involved in the DCZ0858-mediated antitumor effects and that JAK2 was the key target for DCZ0858 treatment. Knockdown of JAK2 partly weakened the DCZ0858-mediated antitumor effect in DLBCL cells, while JAK2 overexpression strengthened the effect of DCZ0858 in DLBCL cells. Moreover, a similar antitumor effect was observed for DCZ0858 and the JAK2 inhibitor ruxolitinib, and combining the two could significantly enhance cancer-suppressive signaling. Tumor xenograft models showed that DCZ0858 inhibited tumor growth in vivo and had low toxicity in important organs, findings that were consistent with the in vitro data. In summary, DCZ0858 is a promising drug for the treatment of DLBCL.

A novel phosphoramide compound, DCZ0847, displays in vitro and in vivo anti-myeloma activity, alone or in combination with bortezomib.

Multiple myeloma (MM) is an incurable hematological malignancy, for which novel effective therapies are urgently needed. We synthesized a novel phosphoramide compound, DCZ0847, showing a potent anti-myeloma activity both in vitro and in vivo. DCZ0847 showed high cytotoxicity towards primary MM cells but had no effect on normal cells and was well tolerated in vivo. The anti-myeloma activity of DCZ0847 was associated with inhibition of cell proliferation; promotion of cell apoptosis via mitochondrial transmembrane potential collapse and caspase-mediated extrinsic or intrinsic apoptotic pathways; and the induction of G2/M phase arrest via downregulation of CDC25C, CDK1, and cyclin B1. In particular, DCZ0847 induced DNA damage and triggered a DNA-damage response by enhancing the levels of γ-H2A.X, phosphorylated (p)-ATM, p-ATR, p-Chk1, and p-Chk2. Additionally, DCZ0847 was able to overcome the bone marrow stromal cells-induced proliferation of MM cells and blocked JAK2/STAT3 signaling. Importantly, DCZ0847 acted synergistically with bortezomib, with the combination exerting greater cytotoxic effects in vitro and in vivo. Together, our results indicate that DCZ0847, alone or in combination with bortezomib, may represent a potential new therapy for patients with MM.

Acute ethanol exposure reduces serotonin receptor 1A internalization by increasing ubiquitination and degradation of ß-arrestin2.

Acute alcohol exposure alters the trafficking and function of many G-protein-coupled receptors (GPCRs) that are associated with aberrant behavioral responses to alcohol. However, the molecular mechanisms underlying alcohol-induced changes in GPCR function remain unclear. ß-Arrestin is a key player involved in the regulation of GPCR internalization and thus controls the magnitude and duration of GPCR signaling. Although ß-arrestin levels are influenced by various drugs of abuse, the effect of alcohol exposure on ß-arrestin expression and ß-arrestin-mediated GPCR trafficking is poorly understood. Here, we found that acute ethanol exposure increases ß-arrestin2 degradation via its increased ubiquitination in neuroblastoma-2a (N2A) cells and rat prefrontal cortex (PFC). ß-Arrestin2 ubiquitination was likely mediated by the E3 ligase MDM2 homolog (MDM2), indicated by an increased coupling between ß-arrestin2 and MDM2 in response to acute ethanol exposure in both N2A cells and rat PFC homogenates. Importantly, ethanol-induced ß-arrestin2 reduction was reversed by siRNA-mediated MDM2 knockdown or proteasome inhibition in N2A cells, suggesting ß-arrestin2 degradation is mediated by MDM2 through the proteasomal pathway. Using serotonin 5-HT1A receptors (5-HT1ARs) as a model receptor system, we found that ethanol dose-dependently inhibits 5-HT1AR internalization and that MDM2 knockdown reverses this effect. Moreover, ethanol both reduced ß-arrestin2 levels and delayed agonist-induced ß-arrestin2 recruitment to the membrane. We conclude that ß-arrestin2 dysregulation by ethanol impairs 5-HT1AR trafficking. Our findings reveal a critical molecular mechanism underlying ethanol-induced alterations in GPCR internalization and implicate ß-arrestin as a potential player mediating behavioral responses to acute alcohol exposure.

Design, Synthesis and Pharmacological Evaluation of Novel Hsp90N-terminal Inhibitors Without Induction of Heat Shock Response.

Heat shock protein 90 (Hsp90) is a potential oncogenic target. However, Hsp90 inhibitors in clinical trial induce heat shock response, resulting in drug resistance and inefficiency. In this study, we designed and synthesized a series of novel triazine derivatives (A1-26, B1-13, C1-23) as Hsp90 inhibitors. Compound A14 directly bound to Hsp90 in a different manner from traditional Hsp90 inhibitors, and degraded client proteins, but did not induce the concomitant activation of Hsp72. Importantly, A14 exhibited the most potent anti-proliferation ability by inducing autophagy, with the IC50 values of 0.1 µM and 0.4 µM in A549 and SK-BR-3 cell lines, respectively. The in  vivo study demonstrated that A14 could induce autophagy and degrade Hsp90 client proteins in tumor tissues, and exhibit anti-tumor activity in A549 lung cancer xenografts. Therefore, the compound A14 with potent antitumor activity and unique pharmacological characteristics is a novel Hsp90 inhibitor for developing anticancer agent without heat shock response.

Plasticity and Function of Spinal Oxytocin and Vasopressin Signaling during Recovery from Surgery with Nerve Injury.

WHAT WE ALREADY KNOW ABOUT THIS TOPIC: WHAT THIS ARTICLE TELLS US THAT IS NEW: BACKGROUND:: Recovery from pain after surgery is faster after cesarean delivery than after other abdominal procedures. The authors hypothesized that recovery in rats after surgery could be reversed by antagonism of spinal oxytocin or vasopressin receptors, that there may be a sex difference, and that spinal oxytocin innervation could change after surgery. METHODS: Male and female rats underwent partial spinal nerve ligation surgery. Effects of nonselective and selective oxytocin and vasopressin 1A receptor antagonists on mechanical hypersensitivity during partial recovery were assessed (n = 8 to 14/group). Oxytocin immunoreactivity in the dorsal horn of the spinal cord (n = 7 to 8/group) and messenger RNA (mRNA) expression for oxytocin-binding receptors in dorsal root ganglia and spinal cord (n = 8/group) were measured. RESULTS: Intrathecal injection of oxytocin and vasopressin receptor antagonists were similarly effective at reducing withdrawal threshold (in all experiments from 22 [19, 26] median [first quartile, third quartile]) g to 8.3 [6.4, 12] g after injection) in both sexes, while having no or minimal effects in animals without surgery. Oxytocin fiber immunoreactivity was 3- to 5-fold greater in lumbar than other regions of the spinal cord and was increased more than 2-fold in lumbar cord ipsilateral to surgery. Injury was also associated with a 6.5-fold increase in oxytocin receptor and a 2-fold increase in vasopressin 1A receptor messenger RNA expression in the L4 dorsal root ganglion ipsilateral to surgery. CONCLUSIONS: These findings suggest that the capacity for oxytocin signaling in the spinal cord increases after surgery and that spinal oxytocin signaling plays ongoing roles in both sexes in recovery from mechanical hypersensitivity after surgery with known nerve injury.

RGS2 modulates the activity and internalization of dopamine D2 receptors in neuroblastoma N2A cells.

Dysregulated expression and function of dopamine D2 receptors (D2Rs) are implicated in drug addiction, Parkinson's disease and schizophrenia. In the current study, we examined whether D2Rs are modulated by regulator of G protein signaling 2 (RGS2), a member of the RGS family that regulates G protein signaling via acceleration of GTPase activity. Using neuroblastoma 2a (N2A) cells, we found that RGS2 was immunoprecipitated by aluminum fluoride-activated Gαi2 proteins. RGS2 siRNA knockdown enhanced membrane [(35)S] GTPγS binding to activated Gαi/o proteins, augmented inhibition of cAMP accumulation and increased ERK phosphorylation in the presence of a D2/D3R agonist quinpirole when compared to scrambled siRNA treatment. These data suggest that RGS2 is a negative modulator of D2R-mediated Gαi/o signaling. Moreover, RGS2 knockdown slightly increased constitutive D2R internalization and markedly abolished quinpirole-induced D2R internalization assessed by immunocytochemistry. RGS2 knockdown did not compromise agonist-induced ß-arrestin membrane recruitment; however, it prevents ß-arrestin dissociation from the membrane after prolonged quinpirole treatment during which time ß-arrestin moved away from the membrane in control cells. Additionally, confocal microscopy analysis of ß-arrestin post-endocytic fate revealed that quinpirole treatment caused ß-arrestin to translocate to the early and the recycling endosome in a time-dependent manner in control cells whereas translocation of ß-arrestin to these endosomes did not occur in RGS2 knockdown cells. The impaired ß-arrestin translocation likely contributed to the abolishment of quinpirole-stimulated D2R internalization in RGS2 knockdown cells. Thus, RGS2 is integral for ß-arrestin-mediated D2R internalization. The current study revealed a novel regulation of D2R signaling and internalization by RGS2 proteins.

A Single Amphetamine Infusion Reverses Deficits in Dopamine Nerve-Terminal Function Caused by a History of Cocaine Self-Administration.

There are ∼ 1.6 million people who meet the criteria for cocaine addiction in the United States, and there are currently no FDA-approved pharmacotherapies. Amphetamine-based dopamine-releasing drugs have shown efficacy in reducing the motivation to self-administer cocaine and reducing intake in animals and humans. It is hypothesized that amphetamine acts as a replacement therapy for cocaine through elevation of extracellular dopamine levels. Using voltammetry in brain slices, we tested the ability of a single amphetamine infusion in vivo to modulate dopamine release, uptake kinetics, and cocaine potency in cocaine-naive animals and after a history of cocaine self-administration (1.5 mg/kg/infusion, fixed-ratio 1, 40 injections/day × 5 days). Dopamine kinetics were measured 1 and 24 h after amphetamine infusion (0.56 mg/kg, i.v.). Following cocaine self-administration, dopamine release, maximal rate of uptake (Vmax), and membrane-associated dopamine transporter (DAT) levels were reduced, and the DAT was less sensitive to cocaine. A single amphetamine infusion reduced Vmax and membrane DAT levels in cocaine-naive animals, but fully restored all aspects of dopamine terminal function in cocaine self-administering animals. Here, for the first time, we demonstrate pharmacologically induced, immediate rescue of deficits in dopamine nerve-terminal function in animals with a history of high-dose cocaine self-administration. This observation supports the notion that the DAT expression and function can be modulated on a rapid timescale and also suggests that the pharmacotherapeutic actions of amphetamine for cocaine addiction go beyond that of replacement therapy.

Amphetamine self-administration attenuates dopamine D2 autoreceptor function.

Dopamine D2 autoreceptors located on the midbrain dopaminergic neurons modulate dopamine (DA) neuron firing, DA release, and DA synthesis through a negative-feedback mechanism. Dysfunctional D2 autoreceptors following repeated drug exposure could lead to aberrant DA activity in the ventral tegmental area (VTA) and projection areas such as nucleus accumbens (NAcc), promoting drug-seeking and -taking behavior. Therefore, it is important to understand molecular mechanisms underlying drug-induced changes in D2 autoreceptors. Here, we reported that 5 days of amphetamine (AMPH) self-administration reduced the ability of D2 autoreceptors to inhibit DA release in the NAcc as determined by voltammetry. Using the antibody-capture [(35)S]GTPγS scintillation proximity assay, we demonstrated for the first time that midbrain D2/D3 receptors were preferentially coupled to Gαi2, whereas striatal D2/D3 receptors were coupled equally to Gαi2 and Gαo for signaling. Importantly, AMPH abolished the interaction between Gαi2 and D2/D3 receptors in the midbrain while leaving striatal D2/D3 receptors unchanged. The disruption of the coupling between D2/D3 receptors and Gαi2 by AMPH is at least partially explained by the enhanced RGS2 (regulator of G-protein signaling 2) activity resulting from an increased RGS2 trafficking to the membrane. AMPH had no effects on the midbrain expression and trafficking of other RGS proteins such as RGS4 and RGS8. Our data suggest that midbrain D2/D3 receptors are more susceptible to AMPH-induced alterations. Reduced D2 autoreceptor function could lead to enhanced DA signaling and ultimately addiction-related behavior. RGS2 may be a potential non-dopaminergic target for pharmacological intervention of dysfunctional DA transmission and drug addiction.

Protein kinase Cß is a modulator of the dopamine D2 autoreceptor-activated trafficking of the dopamine transporter.

The strength and duration of extracellular dopamine concentrations are regulated by the presynaptic dopamine transporter (DAT) and dopamine D2 autoreceptors (D2autoRs). There is a functional interaction between these two proteins. Activation of D2autoRs increases DAT trafficking to the surface whereas disruption of this interaction compromises activities of both proteins and alters dopaminergic transmission. Previously we reported that DAT expression and activity are subject to modulation by protein kinase Cß (PKCß). Here, we further demonstrate that PKCß is integral for the interaction between DAT and D2autoR. Inhibition or absence of PKCß abolished the communication between DAT and D2autoR. In mouse striatal synaptosomes and transfected N2A cells, the D2autoR-stimulated membrane insertion of DAT was abolished by PKCß inhibition. Moreover, D2autoR-stimulated DAT trafficking is mediated by a PKCß-extracellular signal-regulated kinase signaling cascade where PKCß is upstream of extracellular signal-regulated kinase. The increased surface DAT expression upon D2autoR activation resulted from enhanced DAT recycling as opposed to reduced internalization. Further, PKCß promoted accelerated DAT recycling. Our study demonstrates that PKCß critically regulates D2autoR-activated DAT trafficking and dopaminergic signaling. PKCß is a potential drug target for correcting abnormal extracellular dopamine levels in diseases such as drug addiction and schizophrenia.