The dopamine transporter (DAT) terminates dopamine (DA) neurotransmission by reuptake of DA into presynaptic neurons. -(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059) prevented the quinpirole-evoked increase in ASPϩ accumulation, whereas inhibition of PI3K was without effect. Fluorescence flow cytometry and biotinylation studies revealed a rapid increase in DAT cell-surface expression in response to D 2 R stimulation. These experiments demonstrate that D 2S R stimulation increases DAT cell surface expression and therefore enhances substrate clearance. Furthermore, they show that the increase in DAT function is ERK1/2-dependent but PI3K-independent. Our data also suggest the possibility of a direct physical interaction between DAT and D 2 R. Together, these results suggest a novel mechanism by which D 2S R autoreceptors may regulate DAT in the central nervous system. Dopamine (DA) is the predominant catecholamine neurotransmitter in the central nervous system. Dysregulation of DA neurons has been implicated in the pathogenesis of Parkinson's disease, schizophrenia, and drug addiction (Sotnikova et al., 2006). Extracellular DA levels are primarily regulated by the DA transporter (DAT), an integral membrane protein that is a member of the Na ϩ /Cl Ϫ -dependent J.J., A.Z., and T.S.S. contributed equally to this work.

Morphine produces analgesia by activating mu opioid receptors encoded by the MOR-1 gene. Although morphine-6 beta-glucuronide (M6G), heroin and 6-acetylmorphine also are considered mu opioids, recent evidence suggests that they act through a distinct receptor mechanism. We examined this question in knockout mice containing disruptions of either the first or second coding exon of MOR-1. Mice homozygous for either MOR-1 mutation were insensitive to morphine. Heroin, 6-acetylmorphine and M6G still elicited analgesia in the exon-1 MOR-1 mutant, which also showed specific M6G binding, whereas M6G and 6-acetylmorphine were inactive in the exon-2 MOR-1 mutant. These results provide genetic evidence for a unique receptor site for M6G and heroin analgesia.

We have identified four new mu-opiod receptor (MOR)-1 exons, indicating that the gene now contains at least nine exons spanning more than 200 kilobases. Replacement of exon 4 by combinations of the new exons yields three new receptors. When expressed in Chinese hamster ovary cells, all three variants displayed high affinity for mu-opioid ligands, but kappa and delta drugs were inactive. However, there were subtle, but significant, differences in the binding profiles of the three variants among themselves and from MOR-1. Immunohistochemically, the major variant, MOR-1C, displayed a regional distribution quite distinct from that of MOR-1. Region-specific processing also was seen at the mRNA level. Antisense mapping revealed that the four new exons were all involved in morphine analgesia. Together with two other variants generated from alternative splicing of exon 4, there are now six distinct MOR-1 receptors.

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Genetic and pharmacological approaches were used to examine -opioid receptor (KOR-1) regulation of dopamine (DA) dynamics in the nucleus accumbens and vulnerability to cocaine. Microdialysis revealed that basal DA release and DA extraction fraction (E d ), an indirect measure of DA uptake, are enhanced in KOR-1 knock-out mice. Analysis of DA uptake revealed a decreased K m but unchanged V max in knock-outs. Knock-out mice exhibited an augmented locomotor response to cocaine, which did not differ from that of wild-types administered a behavioral sensitizing cocaine treatment. The ability of cocaine to increase DA was enhanced in knock-outs, whereas c-fos induction was decreased. Although repeated cocaine administration to wild types produced behavioral sensitization, knock-outs exhibited no additional enhancement of behavior. Administration of the long-acting KOR antagonist nor-binaltorphimine to wild-type mice increased DA dynamics. However, the effects varied with the duration of KOR-1 blockade. Basal DA release was increased whereas E d was unaltered after 1 h blockade. After 24 h, release and E d were increased. The behavioral and neurochemical effects of cocaine were enhanced at both time points.These data demonstrate the existence of an endogenous KOR-1 system that tonically inhibits mesoaccumbal DA neurotransmission. Its loss induces neuroadaptations characteristic of "cocaine-sensitized" animals, indicating a critical role of KOR-1 in attenuating responsiveness to cocaine. The increased DA uptake after pharmacological inactivation or gene deletion highlights the plasticity of mesoaccumbal DA neurons and suggests that loss of KOR-1 and the resultant disinhibition of DA neurons trigger short-and long-term DA transporter adaptations that maintain normal DA levels, despite enhanced release.

Salvinorin A (SalA), a selective κ-opioid receptor (KOR) agonist, produces dysphoria and pro-depressant like effects. These actions have been attributed to inhibition of striatal dopamine release. The dopamine transporter (DAT) regulates dopamine transmission via uptake of released neurotransmitter. KORs are apposed to DAT in dopamine nerve terminals suggesting an additional target by which SalA modulates dopamine transmission. SalA produced a concentration-dependent, nor-binaltorphimine (BNI)- and pertussis toxin-sensitive increase of ASP+ accumulation in EM4 cells coexpressing myc-KOR and YFP-DAT, using live cell imaging and the fluorescent monoamine transporter substrate, trans 4-(4-(dimethylamino)-styryl)-N-methylpyridinium) (ASP+). Other KOR agonists also increased DAT activity that was abolished by BNI pretreatment. While SalA increased DAT activity, SalA treatment decreased serotonin transporter (SERT) activity and had no effect on norepinephrine transporter (NET) activity. In striatum, SalA increased the Vmax for DAT mediated DA transport and DAT surface expression. SalA up-regulation of DAT function is mediated by KOR activation and the KOR-linked extracellular signal regulated kinase-½ (ERK1/2) pathway. Co-immunoprecipitation and BRET studies revealed that DAT and KOR exist in a complex. In live cells, DAT and KOR exhibited robust FRET signals under basal conditions. SalA exposure caused a rapid and significant increase of the FRET signal. This suggests that the formation of KOR and DAT complexes is promoted in response to KOR activation. Together, these data suggest that enhanced DA transport and decreased DA release resulting in decreased dopamine signaling may contribute to the dysphoric and pro-depressant like effects of SalA and other KOR agonists.

The D 3 dopamine (DA) 4 receptor, a member of the D2-like family of DA receptors, is expressed in limbic brain regions, both presynaptically on DA neurons as well as postsynaptically. The D3 receptor has gained increasing attention as a target for the treatment of schizophrenia, psycho-stimulant abuse, and DA cell neurodegeneration (1-4). Its restricted central nervous system distribution, relative to D2 receptors, suggests that D3 receptor ligands may have fewer side effects than currently available therapeutic agents.Studies using D3 receptor knock-out mice (5-7) or D3 antisense (8, 9) revealed that D3 receptors regulate extracellular DA in ventral striatum. This effect was attributed to D3 regulation of a long negative feedback loop in which postsynaptic D3 receptors on medium spiny neurons modulate the activity of accumbens output neurons projecting to DA cell bodies in mid-brain (5). However, this hypothesis is incompatible with the effects of DA receptor ligands in tissue preparations in which efferent projections to midbrain DA nuclei are disrupted; modulation of extracellular DA by D3 receptors has been demonstrated in striatal slices (7) and tissue suspensions (10). Pharmacological studies examining the mechanism of such regulation have been precluded by the lack of selective ligands that discriminate between D2 and D3 receptors in vivo (5,(11)(12)(13).DA signaling is terminated by the DA transporter (DAT), an integral membrane protein that re-uptakes DA released into the extracellular space (14). Receptor and second messengerlinked kinase cascades regulate DAT function and cell surface

The rat -opioid receptor clone in which novel exon 5 was found in the place of exon 4 (MOR-1B) was one of the first MOR-1 variants described. We now have identified the mouse homolog of the rat MOR-1B as well as four additional variants derived from splicing from exon 3 into different sites within exon 5. The sequences of all of the variants were identical except for the intracellular tip of the C terminus encoded by exon 5, where each variant predicted a unique amino acid sequence ranging from 2 to 39 amino acids. All of the mMOR-1B variants were selective for -opioids in receptorbinding assays, as anticipated, because they all have identical binding pockets defined by the transmembrane domains. However, the relative potency and efficacy of -agonists to each other varied from variant to variant in guanosine 5Ј-O-(3-[ 35 S]thio)triphosphate-binding studies, as shown by morphine-6␤-glucuronide, which was the most efficacious agent against mouse MOR-1B1 (mMOR-1B1) and the least efficacious agent against mMOR-1B2. mMOR-1B4 was quite unusual. Although mMOR-1B4 was -selective in receptor-binding studies and antagonists labeled mMOR-1B4 well, the binding affinities of most of the -agonists were far lower than those seen with mMOR-1, suggesting that the 39 amino acids at the C terminus of mMOR-1B4 influences the conformation of the receptor and its ligand recognition site itself either directly or through its interactions with other proteins. In conclusion, alterations in the amino acid sequence of the C terminus do not alter the -specificity of the receptor but they can influence the binding characteristics, efficacy, and potency of -opioids.After the classification of the -, -, and ␦-opioid receptors (Martin et al., 1976;Lord et al., 1977;Kosterlitz and Leslie, 1978), drugs were classified by their selectivity profiles in receptor-binding assays. Most clinical analgesics are -selective, having little affinity for either -or ␦-receptors, yet clinicians have long known that patient responses to -opioids can vary widely, both in terms of their relative analgesic activity and their side effects (Payne and Pasternak, 1992). Clinicians also have used incomplete cross-tolerance among -opioids to regain analgesic activity in patients highly tolerant to one -drug by switching them to a different -opioid, a technique termed opioid rotation (Cherny et al., 2001). These and other clinical observations raised the possibility that all of the -opioids might not be acting through identical receptor mechanisms.The concept of multiple -opioid receptors was formally proposed almost 25 years ago (Wolozin and Pasternak, 1981) based on identification of a novel receptor-binding site (Pasternak and Snyder, 1975a;Lutz et al., 1984) and the actions of the novel antagonists naloxonazone and naloxonazine, agents that dissociated opioid analgesia from other opioid actions, including respiratory depression, the inhibition of gastrointestinal transit, and most signs of physical dependence (Pasternak et al., 1980;Ling et al., 1984Ling et a...