Transporters involved in adult rat cortical astrocyte dopamine uptake: Kinetics, expression and pharmacological modulation.

Astrocytes, glial cells in the central nervous system, perform a multitude of homeostatic functions and are in constant bidirectional communication with neuronal cells, a concept named the tripartite synapse; however, their role in the dopamine homeostasis remains unexplored. The aim of this study was to clarify the pharmacological and molecular characteristics of dopamine transport in cultured cortical astrocytes of adult rats. In addition, we were interested in the expression of mRNA of dopamine transporters as well as dopamine receptors D1 and D2 and in the effect of dopaminergic drugs on the expression of these transporters and receptors. We have found that astrocytes possess both Na+-dependent and Na+-independent transporters. Uptake of radiolabelled dopamine was time-, temperature- and concentration-dependent and was inhibited by decynium-22, a plasma membrane monoamine transporter inhibitor, tricyclic antidepressants desipramine and nortriptyline, both inhibitors of the norepinephrine transporter. Results of transporter mRNA expression indicate that the main transporters involved in cortical astrocyte dopamine uptake are the norepinephrine transporter and plasma membrane monoamine transporter. Both dopamine receptor subtypes were identified in cortical astrocyte cultures. Twenty-four-hour treatment of astrocyte cultures with apomorphine, a D1/D2 agonist, induced upregulation of D1 receptor, norepinephrine transporter and plasma membrane monoamine transporter, whereas the latter was downregulated by haloperidol and L-DOPA. Astrocytes take up dopamine by multiple transporters and express dopamine receptors, which are sensitive to dopaminergic drugs. The findings of this study could open a promising area of research for the fine-tuning of existing therapeutic strategies.

Allosteric Modulator KM822 Attenuates Behavioral Actions of Amphetamine in Caenorhabditis elegans through Interactions with the Dopamine Transporter DAT-1.

Aberrant dopamine (DA) signaling is associated with several psychiatric disorders, such as autism, bipolar disorder, addiction, and Parkinson's disease, and several medications that target the DA transporter (DAT) can induce or treat these disorders. In addition, psychostimulants, such as cocaine and D-amphetamine (AMPH), rely on the competitive interactions with the transporter's substrate binding site to produce their rewarding effects. Agents that exhibit noncompetitive, allosteric modulation of DAT remain an important topic of investigation due to their potential therapeutic applications. We previously identified a novel allosteric modulator of human DAT, KM822, that can decrease the affinity of cocaine for DAT and attenuate cocaine-elicited behaviors; however, whether DAT is the sole mediator of KM822 actions in vivo is unproven given the large number of potential off-target sites. Here, we provide in silico and in vitro evidence that the allosteric site engaged by KM822 is conserved between human DAT and Caenorhabditis elegans DAT-1. KM822 binds to a similar pocket in DAT-1 as previously identified in human DAT. In functional dopamine uptake assays, KM822 affects the interaction between AMPH and DAT-1 by reducing the affinity of AMPH for DAT-1. Finally, through a combination of genetic and pharmacological in vivo approaches we provide evidence that KM822 diminishes the behavioral actions of AMPH on swimming-induced paralysis through a direct allosteric modulation of DAT-1. More broadly, our findings demonstrate allosteric modulation of DAT as a behavior modifying strategy and suggests that Caenorhabditis elegans can be operationalized to identify and investigate the interactions of DAT allosteric modulators. SIGNIFICANCE STATEMENT: We previously demonstrated that the dopamine transporter (DAT) allosteric modulator KM822 decreases cocaine affinity for human DAT. Here, using in silico and in vivo genetic approaches, we extend this finding to interactions with amphetamine, demonstrating evolutionary conservation of the DAT allosteric site. In Caenorhabditis elegans, we report that KM822 suppresses amphetamine behavioral effects via specific interactions with DAT-1. Our findings reveal Caenorhabditis elegans as a new tool to study allosteric modulation of DAT and its behavioral consequences.

Dopamine signaling promotes the xenobiotic stress response and protein homeostasis.

Multicellular organisms encounter environmental conditions that adversely affect protein homeostasis (proteostasis), including extreme temperatures, toxins, and pathogens. It is unclear how they use sensory signaling to detect adverse conditions and then activate stress response pathways so as to offset potential damage. Here, we show that dopaminergic mechanosensory neurons in C. elegans release the neurohormone dopamine to promote proteostasis in epithelia. Signaling through the DA receptor DOP-1 activates the expression of xenobiotic stress response genes involved in pathogenic resistance and toxin removal, and these genes are required for the removal of unstable proteins in epithelia. Exposure to a bacterial pathogen (Pseudomonas aeruginosa) results in elevated removal of unstable proteins in epithelia, and this enhancement requires DA signaling. In the absence of DA signaling, nematodes show increased sensitivity to pathogenic bacteria and heat-shock stress. Our results suggest that dopaminergic sensory neurons, in addition to slowing down locomotion upon sensing a potential bacterial feeding source, also signal to frontline epithelia to activate the xenobiotic stress response so as to maintain proteostasis and prepare for possible infection.

The role of fibroblast growth factor 2 in drug addiction.

Fibroblast growth factor 2 (FGF2) is a member of the FGF-family, which consists of 22 members, with four known FGF receptors (five in humans). Over the last 30 years, FGF2 has been extensively studied for its role in cell proliferation, differentiation, growth, survival and angiogenesis during development, as well as for its role in adult neurogenesis and regenerative plasticity. Over the past decade, FGF2 has been implicated in learning and memory, as well as in several neuropsychiatric disorders, including anxiety, stress, depression and drug addiction. In this review, we present accumulating evidence indicating the involvement of FGF2 in neuroadaptations caused by drugs of abuse, namely, amphetamine, cocaine, nicotine and alcohol. Moreover, evidence suggests that FGF2 is a positive regulator of alcohol and drug-related behaviors. Thus, although additional studies are yet required, we suggest that reducing FGF2 activity may provide a novel therapeutic approach for substance use disorders.

Unique Hydrolysis of an Ester-Type Prodrug of Levodopa in Human Plasma: Relay-Type Role Sharing between Alpha-1 Acid Glycoprotein and Human Serum Albumin.

ONO-2160 is a newly developed oral ester-type prodrug of levodopa for removing the problems in use of levodopa. It has a structure in which two of the same substituents are bound to levodopa. It is important to understand the pharmacokinetics and metabolic pathway for new drug candidate compounds. The aim of this study was to identify the major enzymes that contribute to the metabolism of ONO-2160 in human plasma. ONO-2160 was hydrolyzed by human serum albumin (HSA) and α1-acid glycoprotein (AGP) in human plasma, although the hydrolysis was not inhibited by various reported esterase inhibitors. The value of the intrinsic clearance per milliliter of plasma of ONO-2160 in AGP solution was greater than that in HSA solution and was comparable to that in human plasma. Therefore, AGP is responsible for the hydrolysis of ONO-2160 in human plasma. ONO-M, which is an intermediate metabolite of ONO-2160, has a structure in which one substituent is removed from ONO-2160 and was mainly generated in AGP solution, but not in human plasma or HSA solution. The hydrolysis of ONO-M by HSA was much greater than by AGP. These results indicate that ONO-M, which is mainly generated from ONO-2160 by AGP, is rapidly hydrolyzed by HSA, and that ONO-2160 generates levodopa via ONO-M in a relay-type reaction through AGP and HSA in human plasma. It has not been reported that AGP has esterase-like activity. These findings could be useful information for drug development of the ester-type prodrug.

Dietary supplementation with fish oil reverses high fat diet-induced enhanced sensitivity to the behavioral effects of quinpirole.

Consuming a high fat diet can lead to many negative health consequences, such as obesity, insulin resistance, and enhanced sensitivity to drugs acting on dopamine systems. It has recently been demonstrated that dietary supplementation with fish oil, which is rich in omega-3 fatty acids, can prevent this high fat diet-induced enhanced sensitivity to dopaminergic drugs from developing. However, it is not known whether fish oil supplementation can reverse this effect once it has already developed. To test the hypothesis that dietary supplementation with fish oil will reverse high fat diet-induced enhanced sensitivity to quinpirole, a dopamine D2/D3 receptor agonist, male Sprague-Dawley rats were fed either standard chow (17% kcal from fat), high fat chow (60% kcal from fat), standard chow, or high fat chow supplemented with 20% (w/w) fish oil. Body weight, food consumption, and sensitivity to quinpirole-induced (0.0032-0.32 mg/kg) penile erections were examined throughout the course of the experiment. Eating high fat chow enhanced sensitivity of rats to quinpirole-induced penile erections (i.e. resulted in a leftward shift of the ascending limb of the dose-response curve). Dietary supplementation with fish oil successfully treated this effect, as dose-response curves were not different for rats eating standard chow and rats eating high fat chow with fish oil. These results suggest that in addition to preventing the negative health consequences of eating a high fat diet, fish oil can also reverse some of these consequences once they have developed.

Transcriptome analysis of hemocytes from the white shrimp Litopenaeus vannamei with the injection of dopamine.

As a crucial neuroendocrine-immune factor, dopamine (DA) could regulate the immune system of Litopenaeus vannamei. To understand the immune mechanisms and regulatory pathways of DA in L. vannamei, the transcriptome analysis of hemocytes of L. vannamei with injection of DA (10-6 mol/shrimp) at 3 and 12 h were performed in this study. Moreover, quantitative real-time PCR (qPCR) method was applied to validate the accuracy of transcriptome sequencing and analyze the expression pattern of candidate differentially expressed genes (DEGs) at different time points (0, 3, 6, 12, and 24 h) after DA injection. The results showed that a total of 51382 unigenes with a N50 length of 2341 bp were generated. And 1397 and 457 DEGs were obtained by comparative transcriptome at 3 and 12h respectively. Moreover, the results of functional annotation and enriched pathway showed that the DEGs were involved in phagosome (ko04145), lysosome (ko04142), Endocytosis (ko04144), and NOD-like receptor signaling pathway (ko04621). Besides, the Pearson's correlation coefficient (R) between transcriptome sequencing and qPCR was 0.845, which confirmed the reliability of the transcriptome sequencing results and the accuracy of assembly. Furthermore, the expression pattern of 15 candidate DEGs, containing 9 up-regulated and 6 down-regulated DEGs at 3 h, indicated the regulation of DA in physiological functions especially in the immune system. Therefore, these results revealed that DA induced the expressions of membrane receptors or proteins, activated intracellular signaling pathways, regulated cellular and humoral immune systems, controlled antioxidation and apoptosis, and was involved in the regulation of neuroendocrine system. These findings are helpful to promote the understanding on the effects of biogenic amines on physiological functions and regulatory networks of crustacean, and offer a substantial material and foundation for researching the immune response of crustacean.

[O-methyl-11C]N-(4-(4-(3-Chloro-2-methoxyphenyl)-piperazin-1-yl)butyl)-1H-indole-2-carboxamide ([11C]BAK4-51) Is an Efflux Transporter Substrate and Ineffective for PET Imaging of Brain D3 Receptors in Rodents and Monkey.

Selective high-affinity antagonists for the dopamine D3 receptor (D3R) are sought for treating substance use disorders. Positron emission tomography (PET) with an effective D3R radioligand could be a useful tool for the development of such therapeutics by elucidating pharmacological specificity and target engagement in vivo. Currently, a D3R-selective radioligand does not exist. The D3R ligand, N-(4-(4-(3-chloro-2-methoxyphenyl)piperazin-1-yl)butyl)-1H-indole-2-carboxamide (BAK4-51, 1), has attractive properties for PET radioligand development, including full antagonist activity, very high D3R affinity, D3R selectivity, and moderate lipophilicity. We labeled 1 with the positron-emitter carbon-11 (t1/2 = 20.4 min) in the methoxy group for evaluation as a radioligand in animals with PET. However, [11C]1 was found to be an avid substrate for brain efflux transporters and lacked D3R-specific signal in rodent and monkey brain in vivo.

Regional and source-based patterns of [11C]-(+)-PHNO binding potential reveal concurrent alterations in dopamine D2 and D3 receptor availability in cocaine-use disorder.

Dopamine type 2 and type 3 receptors (D2R/D3R) appear critical to addictive disorders. Cocaine-use disorder (CUD) is associated with lower D2R availability and greater D3R availability in regions primarily expressing D2R or D3R concentrations, respectively. However, these CUD-related alterations in D2R and D3R have not been concurrently detected using available dopaminergic radioligands. Furthermore, receptor availability in regions of mixed D2R/D3R concentration in CUD remains unclear. The current study aimed to extend investigations of CUD-related alterations in D2R and D3R availability using regional and source-based analyses of [11C]-(+)-PHNO positron emission tomography (PET) of 26 individuals with CUD and 26 matched healthy comparison (HC) participants. Regional analysis detected greater binding potential (BPND) in CUD in the midbrain, consistent with prior [11C]-(+)-PHNO research, and lower BPND in CUD in the dorsal striatum, consistent with research using non-selective D2R/D3R radiotracers. Exploratory independent component analysis (ICA) identified three sources of BPND (striatopallidal, pallidonigral, and mesoaccumbens sources) that represent systems of brain regions displaying coherent variation in receptor availability. The striatopallidal source was associated with estimates of regional D2R-related proportions of BPND (calculated using independent reports of [11C]-(+)-PHNO receptor binding fractions), was lower in intensity in CUD and negatively associated with years of cocaine use. By comparison, the pallidonigral source was associated with estimates of regional D3R distribution, was greater in intensity in CUD and positively associated with years of cocaine use. The current study extends previous D2R/D3R research in CUD, demonstrating both lower BPND in the D2R-rich dorsal striatum and greater BPND in the D3R-rich midbrain using a single radiotracer. In addition, exploratory ICA identified sources of [11C]-(+)-PHNO BPND that were correlated with regional estimates of D2R-related and D3R-related proportions of BPND, were consistent with regional differences in CUD, and suggest receptor alterations in CUD may also be present in regions of mixed D2R/D3R concentration.

HIV-1 TAT protein enhances sensitization to methamphetamine by affecting dopaminergic function.

Methamphetamine abuse is common among humans with immunodeficiency virus (HIV). The HIV-1 regulatory protein TAT induces dysfunction of mesolimbic dopaminergic systems which may result in impaired reward processes and contribute to methamphetamine abuse. These studies investigated the impact of TAT expression on methamphetamine-induced locomotor sensitization, underlying changes in dopamine function and adenosine receptors in mesolimbic brain areas and neuroinflammation (microgliosis). Transgenic mice with doxycycline-induced TAT protein expression in the brain were tested for locomotor activity in response to repeated methamphetamine injections and methamphetamine challenge after a 7-day abstinence period. Dopamine function in the nucleus accumbens (Acb) was determined using high performance liquid chromatography. Expression of dopamine and/or adenosine A receptors (ADORA) in the Acb and caudate putamen (CPu) was assessed using RT-PCR and immunohistochemistry analyses. Microarrays with pathway analyses assessed dopamine and adenosine signaling in the CPu. Activity-dependent neurotransmitter switching of a reserve pool of non-dopaminergic neurons to a dopaminergic phenotype in the ventral tegmental area (VTA) was determined by immunohistochemistry and quantified with stereology. TAT expression enhanced methamphetamine-induced sensitization. TAT expression alone decreased striatal dopamine (D1, D2, D4, D5) and ADORA1A receptor expression, while increasing ADORA2A receptors expression. Moreover, TAT expression combined with methamphetamine exposure was associated with increased adenosine A receptors (ADORA1A) expression and increased recruitment of dopamine neurons in the VTA. TAT expression and methamphetamine exposure induced microglia activation with the largest effect after combined exposure. Our findings suggest that dopamine-adenosine receptor interactions and reserve pool neuronal recruitment may represent potential targets to develop new treatments for methamphetamine abuse in individuals with HIV.

Development of CNS multi-receptor ligands: Modification of known D2 pharmacophores.

Several known D2 pharmacophores have been explored as templates for identifying ligands with multiple binding affinities at dopamine and serotonin receptors considered as clinically relevant receptors in the treatment of neuropsychiatric diseases. This approach has resulted in the identification of ligands that target multiple CNS receptors while avoiding others associated with deleterious effects. In particular, compounds 11, 15 and 22 may have potential for further development as antipsychotic agents as they favorably interact with the clinically relevant receptors including D2R, 5-HT1AR, and 5-HT7R. We have also identified the pair of compounds 11 and 10 as high affinity D2R ligands with and without SERT binding affinities, respectively. These differential binding profiles endow the pair with the potential for evaluating SERT contributions to antipsychotic drug activity in animal behavioral models. In addition, compound 11 has no significant affinity for 5-HT2CR and binds only moderately to the H1R, suggesting it may not induce weight gain or sedation when used clinically. Taken together, compound 11 displays an interesting pharmacological profile that necessitates the evaluation of its functional and in vivo effects in animal models which are currently ongoing.

Synthesis, Biological, and Computational Evaluation of Substituted 1-(2-Methoxyphenyl)-4-(1-phenethylpiperidin-4-yl)piperazines and 1-(2-Methoxyphenyl)-4-[(1-phenethylpiperidin-4-yl)methyl]piperazines as Dopaminergic Ligands.

Sixteen new 1-(2-methoxyphenyl)-4-(1-phenethylpiperidin-4-yl)piperazines and 1-(2-methoxyphenyl)-4-[(1-phenethylpiperidin-4-yl)methyl]piperazines were synthesized to be used as probes for mapping the dopamine D2 receptor (D2 DAR) arylpiperazine binding site. All compounds were evaluated for their affinity toward D2 DAR in an in vitro competitive displacement assay. The most active one was 1-(2-methoxyphenyl)-4-{[1-(3-nitrophenethyl)piperidin-4-yl]methyl}piperazine (25) with an affinity of Ki = 54 nM. Docking analysis was conducted on all herein described compounds, whereas molecular dynamic simulation was performed on ligand 25 to establish its mode of interaction with D2 DAR. Two possible docking orientations are proposed; the one with a salt bridge between the piperidine moiety and Asp114 of D2 DAR is more stable.

Brexpiprazole I: in vitro and in vivo characterization of a novel serotonin-dopamine activity modulator.

Brexpiprazole (OPC-34712, 7-{4-[4-(1-benzothiophen-4-yl)piperazin-1-yl]butoxy}quinolin-2(1H)-one) is a novel drug candidate in clinical development for psychiatric disorders with high affinity for serotonin, dopamine, and noradrenaline receptors. In particular, it bound with high affinity (Ki < 1 nM) to human serotonin 1A (h5-HT1A)-, h5-HT2A-, long form of human D2 (hD2L)-, hα1B-, and hα2C-adrenergic receptors. It displayed partial agonism at h5-HT1A and hD2 receptors in cloned receptor systems and potent antagonism of h5-HT2A receptors and hα1B/2C-adrenoceptors. Brexpiprazole also had affinity (Ki < 5 nM) for hD3-, h5-HT2B-, h5-HT7-, hα1A-, and hα1D-adrenergic receptors, moderate affinity for hH1 (Ki = 19 nM), and low affinity for hM1 receptors (Ki > 1000 nM). Brexpiprazole potently bound to rat 5-HT2A and D2 receptors in vivo, and ex vivo binding studies further confirmed high 5-HT1A receptor binding potency. Brexpiprazole inhibited DOI (2,5-dimethoxy-4-iodoamphetamine)-induced head twitches in rats, suggestive of 5-HT2A antagonism. Furthermore, in vivo D2 partial agonist activity of brexpiprazole was confirmed by its inhibitory effect on reserpine-induced DOPA accumulation in rats. In rat microdialysis studies, brexpiprazole slightly reduced extracellular dopamine in nucleus accumbens but not in prefrontal cortex, whereas moderate increases of the dopamine metabolites, homovanillic acid and DOPAC (3,4-dihydroxy-phenyl-acetic acid), in these areas also suggested in vivo D2 partial agonist activity. In particular, based on a lower intrinsic activity at D2 receptors and higher binding affinities for 5-HT1A/2A receptors than aripiprazole, brexpiprazole would have a favorable antipsychotic potential without D2 receptor agonist- and antagonist-related adverse effects. In conclusion, brexpiprazole is a serotonin-dopamine activity modulator with a unique pharmacology, which may offer novel treatment options across a broad spectrum of central nervous system disorders.

Nrf2 deficiency leads to behavioral, neurochemical and transcriptional changes in mice.

Nrf2 is a transcription factor that regulates the antioxidant and detoxification enzyme genes and provides defense against oxidative and electrophilic stresses in various tissues. In brain, while neuroprotective functions of Nrf2 have been well documented, Nrf2 contribution to the brain function remains to be elucidated. To address this issue, we investigated whether Nrf2 deficiency affects psychological behaviors, neurotransmitter systems and gene expressions in mice. We conducted four behavioral tests, social interaction, open-field, rotarod and forced swimming tests and found that Nrf2 knockout mice exhibited reduced immobility in the forced swimming test. Neurochemical analyses revealed that the dopamine and serotonin metabolites increased in the brains of Nrf2 knockout mice. We also present a catalog of genes whose expression is Nrf2-dependent in brain under unstressed conditions, which includes a number of xenobiotic-metabolizing enzyme genes. These results thus support our contention that Nrf2 regulates its target genes in brain under unstressed conditions and loss of Nrf2 affects various brain functions.

Protein kinase C epsilon modulates nicotine consumption and dopamine reward signals in the nucleus accumbens.

Nicotine addiction and alcohol use disorders are very widespread and often occur together. Currently, there is no single drug approved for the simultaneous treatment of both conditions. Although these conditions share common genetic factors, the molecular mechanisms underlying their comorbidity are unknown. We have previously shown that mice lacking protein kinase C epsilon (PKCε) show decreased ethanol self-administration and reward as well as increased aversion to ethanol. Here we find that Prkce(-/-) mice self-administer less nicotine and show decreased conditioned place preference for nicotine compared with wild-type mice. In Prkce(-/-) mice, these behaviors are associated with reduced levels of α(6) and ß(3) nicotinic receptor subunit mRNA in the ventral midbrain and striatum as well as a functional deficit in cholinergic modulation of dopamine release in nucleus accumbens. Our results indicate that PKCε regulates reward signaling through α(6)-containing nicotinic receptors and suggest that PKCε could be a target for the treatment of comorbid nicotine and alcohol addictions.

Dopamine inhibits excitatory neurotransmission in basolateral amygdala during development via pre-synaptic mechanism.

OBJECTIVES: Dopaminergic signaling in the basolateral amygdala (BLA) is important for emotion-related activity. However, little is known about the influence of dopamine (DA) on excitatory synaptic transmission of pyramidal neurons in BLA at early developmental stage. Here in this study, we observed the effect of DA on excitatory neurotransmission in the pyramidal cells of BLA in acute slices. METHODS: Acute slices from amygdala of rats at the age of 14-16 days were prepared and maintained in vitro using standard method. Whole-cell patch clamp recordings were performed to examine the evoked excitatory postsynaptic current (eEPSC), spontaneous excitatory postsynaptic current (sEPSC) and miniature excitatory postsynaptic current (mEPSC). Drugs including DA and synaptic blockers were added in recording solution due to different experimental designs. RESULTS: We found that bath application of DA at a concentration of 100 µM significantly inhibited the amplitude of evoked EPSC. However, the amplitude and frequency of mEPSC were not affected. We also found increased pair pulse facilitation after DA application, indicating DA inhibited excitatory neurotransmission through suppression of release probability at the pre-synaptic terminals. Importantly, DA was also effective in decreasing activity induced upregulation in sEPSCs. Moreover, the DA effects were not affected by either antagonist of dopamine 1 or dopamine 2-like receptors. CONCLUSION: We studied the effects of DA on excitatory neurotransmission and found that DA inhibited glutamatergic synaptic transmission via modulation of pre-synaptic release probability.

Higher binding of the dopamine D3 receptor-preferring ligand [11C]-(+)-propyl-hexahydro-naphtho-oxazin in methamphetamine polydrug users: a positron emission tomography study.

Positron emission tomography (PET) findings suggesting lower D2-type dopamine receptors and dopamine concentration in brains of stimulant users have prompted speculation that increasing dopamine signaling might help in drug treatment. However, this strategy needs to consider the possibility, based on animal and postmortem human data, that dopaminergic activity at the related D3 receptor might, in contrast, be elevated and thereby contribute to drug-taking behavior. We tested the hypothesis that D3 receptor binding is above normal in methamphetamine (MA) polydrug users, using PET and the D3-preferring ligand [11C]-(+)-propyl-hexahydro-naphtho-oxazin ([11C]-(+)-PHNO). Sixteen control subjects and 16 polydrug users reporting MA as their primary drug of abuse underwent PET scanning after [11C]-(+)-PHNO. Compared with control subjects, drug users had higher [11C]-(+)-PHNO binding in the D3-rich midbrain substantia nigra (SN; +46%; p<0.02) and in the globus pallidus (+9%; p=0.06) and ventral pallidum (+11%; p=0.1), whereas binding was slightly lower in the D2-rich dorsal striatum (approximately -4%, NS; -12% in heavy users, p=0.01) and related to drug-use severity. The [11C]-(+)-PHNO binding ratio in D3-rich SN versus D2-rich dorsal striatum was 55% higher in MA users (p=0.004), with heavy but not moderate users having ratios significantly different from controls. [11C]-(+)-PHNO binding in SN was related to self-reported "drug wanting." We conclude that the dopamine D3 receptor, unlike the D2 receptor, might be upregulated in brains of MA polydrug users, although lower dopamine levels in MA users could have contributed to the finding. Pharmacological studies are needed to establish whether normalization of D3 receptor function could reduce vulnerability to relapse in stimulant abuse.

Role of epoxyeicosatrienoic acids (EETs) in mediation of dopamine's effects in the kidney.

We have recently demonstrated that intrarenal dopamine plays an important role in preventing the development of systemic hypertension. Similarly, renal cytochrome P-450 (CYP)-epoxygenase-derived arachidonic acid metabolites, epoxyeicosatrienoic acids (EETs), also are antihypertensive through inhibiting sodium reabsorption and vasodilation. The potential interaction between renal dopamine and epoxygenase systems was investigated. Catechol-O-methyl-transferase (COMT)(-/-) mice with increased intrarenal dopamine levels and proximal tubule deletion of aromatic amino acid decarboxylase (ptAADC(-/-)) mice with renal dopamine deficiency were treated with a low-salt diet or high-salt diet for 2 wk. Wild-type or Cyp2c44(-/-) mice were treated with gludopa, which selectively increased renal dopamine levels. In low salt-treated mice, urinary EET levels were related to renal dopamine levels, being highest in COMT(-/-) mice and lowest in ptAADC(-/-) mice. In high salt-treated mice, total EET and individual EET levels in both the kidney and urine were also highest in COMT(-/-) mice and lowest in ptAADC(-/-) mice. Selective increases in renal dopamine in response to gludopa administration led to marked increases in both total and all individual EET levels in the kidney without any changes in blood levels. qRT-PCR and immunoblotting indicated that gludopa increased renal Cyp2c44 mRNA and protein levels. Gludopa induced marked increases in urine volume and urinary sodium excretion in wild-type mice. In contrast, gludopa did not induce significant increases in urine volume or urinary sodium excretion in Cyp2c44(-/-) mice. These studies demonstrate that renal EET levels are maintained by intrarenal dopamine, and Cyp2c44-derived EETs play an important role in intrarenal dopamine-induced natriuresis and diuresis.

Effects of dopamine D2/D3 receptor ligands on food-cocaine choice in socially housed male cynomolgus monkeys.

Dopamine D2/D3 receptor partial agonists have been suggested as medications for cocaine dependence. The present experiments examined the effect of acute and repeated administration of drugs with varying intrinsic efficacy at D2/D3 receptors on the relative reinforcing strength of cocaine. Use of socially housed cynomolgus monkeys permitted the assessment of whether social status, known to alter D2/D3 receptor availability, influenced the behavioral effects of D2/D3 receptor compounds. The high-efficacy agonist R(-)-norpropylapomorphine [(-)-NPA], low-efficacy agonist aripiprazole (ARI), and antagonist eticlopride (ETIC) were administered acutely to monkeys self-administering cocaine under a food-cocaine choice procedure in which a cocaine self-administration dose-effect curve was determined daily. The effects of 5-day treatment with ARI and (-)-NPA were characterized under conditions in which monkeys did (ARI) or did not [ARI and (-)-NPA] self-administer cocaine during treatment. When administered acutely, ARI and ETIC increased the choice of low cocaine doses, and only (-)-NPA decreased the choice of higher cocaine doses and cocaine intake; effects were similar across social ranks. When administered repeatedly while self administration occurred only on days 1 and 5 of treatment, ARI, but not (-)-NPA, decreased cocaine choice in dominant monkeys, whereas (-)-NPA, but not ARI, did so in subordinates. When dominant monkeys self-administered cocaine on all five days of ARI treatment, however, these effects were not observed. The results indicate that the behavioral effects of D2/D3 receptor agonists can differ according to intrinsic efficacy and subject characteristics. Moreover, these results suggest that exposure to cocaine during treatment can counteract treatment-induced reductions in the reinforcing effects of cocaine.

1H NMR probe for in situ monitoring of dopamine metabolism and its application to inhibitor screening.

Dopamine (DA) is a monoamine neurotransmitter that plays important roles in the brain, and whose levels in the brain are associated with several neurological and psychiatric disorders. Therefore, DA metabolism inhibitors have been used as therapeutic agents. Here, we report a (1)H NMR probe for the in situ analysis of DA metabolism, and its application to DA inhibitor screening. We designed doubly (13)C-labeled DA ((13)C(2)-DA) as the probe. The combination of the (13)C(2)-DA and (1)H-{(13)C-(13)C'} NMR technique allowed the selective and thus in situ monitoring of DA metabolism. Using (13)C(2)-DA, we successfully measured the efficacies of different inhibitors in a tissue sample, allowing us to improve the in situ inhibitory efficacy of the known DA metabolism inhibitor, clorgyline.