Alterations in dopamine release but not dopamine autoreceptor function in dopamine D3 receptor mutant mice

Dopamine (DA) autoreceptors expressed along the somatodendritic extent of midbrain DA neurons modulate impulse activity, whereas those expressed at DA nerve terminals regulate both DA synthesis and release. Considerable evidence has indicated that these DA autoreceptors are of the D2 subtype of DA receptors. However, many pharmacological studies have suggested an autoreceptor role for the DA D3 receptor. This possibility was tested with mice lacking the D3 receptor as a result of gene targeting. The basal firing rates of DA neurons within both the substantia nigra and ventral tegmental area were not different in D3 receptor mutant and wild-type mice. The putative D3 receptor-selective agonist R(+)-trans-3,4,4a, 10b-tetrahydro-4-propyl-2H,5H-(1)benzopyrano(4,3-b)-1,4-oxazin+ ++-9-ol (PD 128907) was equipotent at inhibiting the activity of both populations of midbrain DA neurons in the two groups of mice. In the gamma-butyrolactone (GBL) model of DA autoreceptor function, mutant and wild-type mice were identical with respect to striatal DA synthesis and its suppression by PD 128907. In vivo microdialysis studies of DA release in ventral striatum revealed higher basal levels of extracellular DA in mutant mice but similar inhibitory effects of PD 128907 in mutant and wild-type mice. These results suggest that the effects of PD 128907 on dopamine cell function reflect stimulation of D2 as opposed to D3 receptors. Although D3 receptors do not seem to be significantly involved in DA autoreceptor function, they may participate in postsynaptically activated short-loop feedback modulation of DA release.     

PET measurement of dopamine D2 receptor-mediated changes in striatopallidal function

This study was designed to validate an in vivo measurement of the functional sensitivity of basal ganglia neuronal circuits containing dopamine D2 receptors. We hypothesized that a D2 agonist would decrease striatopallidal neuronal activity, and hence regional cerebral blood flow (rCBF) over the axon terminals in the globus pallidus. Quantitative pallidal blood flow was measured using positron emission tomography (PET) with bolus injections of H215O and arterial sampling in six baboons before and after intravenous administration of the selective D2 agonist U91356a. We also tested whether the response to U91356a was modified by previous acute administration of various antagonists. Another baboon had serial measurements of blood flow under identical conditions, but received no dopaminergic drugs. In all animals that received U91356a, pallidal flow decreased in a dose-related manner. Global CBF had a similar response, but the decline in pallidal flow was greater in magnitude and remained significant after accounting for the global effect. A D2 antagonist, but not antagonists of D1, serotonin-2, or peripheral D2 receptors, prevented this decrease. This work demonstrates and validates an in vivo measure of the sensitivity of D2-mediated basal ganglia pathways. It also supports the hypothesis that activation of the indirect striatopallidal pathway, previously demonstrated using nonselective D2-like agonists, can be mediated specifically by D2 receptors. We speculate that the U91356a-PET technique may prove useful in detecting functional abnormalities of D2-mediated dopaminergic function in diseases such as parkinsonism, dystonia, Tourette syndrome, or schizophrenia.     

Re-evaluation of the role of the dopamine transporter in dopamine system homeostasis

Mice with a genetic deletion of the dopamine transporter (DAT) were used to assess its role in the function of dopamine (DA) neurons. Profound alterations in the homeostasis of the nigrostriatal DA system were induced by the absence of the DAT. Extracellular levels of DA were elevated and clearance of released DA was 300-times slower than in control mice. This was accompanied by a 20-fold decrease in tissue DA levels and a paradoxical doubling of the rate of DA synthesis. A crucial role is indicated for the DAT in maintenance of DA neuron presynaptic function, particularly in the control of storage mechanisms.     

Synthesis and dopamine transporter affinity of 2-(methoxycarbonyl)-9-methyl-3-phenyl-9-azabicyclo[3.3.1]nonane derivatives

A series of 9-methyl-3 beta-phenyl-2-substituted-9-azabicyclo[3.3.1]nonane derivatives were synthesized and evaluated as cocaine-binding site ligands at the dopamine transporter (DAT). The conformation of the bicyclic structures and the stereochemistry of the substituents were determined by NMR and X-ray crystallography. The in vitro binding affinity (Ki) of the 9-azabicyclo[3.3.1]nonane derivatives was measured in rat caudate-putamen tissue, and they were found to be 100-fold (Ki = 2-14 microM) less potent than cocaine and other tropane analogs. From these results it is evident that the cocaine-binding site at the DAT is very sensitive to structural modifications of the unsubstituted methylene bridge [C(6)-C(7)] of cocaine and cocaine-like compounds.     

Synthesis, structure, dopamine transporter affinity, and dopamine uptake inhibition of 6-alkyl-3-benzyl-2-[(methoxycarbonyl)methyl]tropane derivatives

A series of 6-alkyl-3 beta-benzyl-2-[(methoxycarbonyl)methyl]tropane analogues were synthesized and evaluated as cocaine binding site ligands at the dopamine transporter (DAT). The in vitro affinity (Ki) for the DAT of the 6-alkyl-3 beta-benzyl-2-[(methoxycarbonyl) methyl]tropane analogues was determined by inhibition of [3H]WIN 35,428 in rat caudate putamen tissue. The inhibition of dopamine uptake (IC50) was also measured for selected compounds which demonstrated moderate affinity for the dopamine transporter. The unsubstituted enantiopure analogues (-)-19a (Ki = 33 nM) and surprisingly (+)-20a (Ki = 60 nM) were found to be almost equipotent with the high-affinity binding components of cocaine and WIN 35,065-2 and exhibited slightly more potent dopamine uptake inhibition than both cocaine and WIN 35,065-2. In general, substitution at the 6-position of racemic 19a and 20a with alkyl groups was found to result in decreased activity relative to increased chain length of the substituent. The 3 beta-benzyl-2 beta-[(methoxycarbonyl)methyl]-6 beta-methyltropane (21b; Ki = 57 nM) was the only 6-alkyl derivative to exhibit moderately potent activity. The 6 beta-isomer 21b was 4-fold more potent than the 6 alpha-isomer 19b (Ki = 211 nM) and was nearly equipotent with (-)-19a and (+)-20a as well as with cocaine and WIN 35,065-2. The results of this study further demonstrate the steric constraints associated with the C(6)-C(7) methylene bridge of the tropane ring system for molecular recognition of cocaine analogues at the cocaine binding site(s) on the DAT.     

Implication of dopamine transporter system on 1-methyl-4-phenylpyridinium and rotenone effect in striatal synaptosomes

Absorption and endogenous excretion of P by male broiler chicks (14-29 d old) were quantitatively evaluated at different Ca:P ratios (1, 1:1; 2, 1.5:1; 3, 2:1; 4, 2.5:1) in four groups given experimental diets ad lib. The P content was the same in all diets. An isotope-dilution technique was used to determine endogenous faecal and renal excretion. Ca and P retentions in the whole body were estimated according to the comparative slaughter technique. P absorption was calculated from retention and endogenous excretion. Absorption and endogenous excretion of P amounted to (mg P/d per chick): 304, 270, 160 and 158; and 135, 109, 31 and 30 in groups 1, 2, 3 and 4 respectively. Widening of the Ca:P ratio in the feed limited the P absorption. Availability of feed P amounted to (%): (1) 66, (2) 57, (3) 32 and (4) 30, and the amounts of absorbed P retained were (%): (1) 56, (2) 60, (3 and 4) 81. The increasing Ca concentration in the feed showed a greater effect on P absorption than on P retention. The ratios of relative retention to relative endogenous excretion of absorbed P were: (1) 1.27, (2) 1.50, (3 and 4) 4.26.     

Novel N-substituted 3 alpha-[bis(4'-fluorophenyl)methoxy]tropane analogues: selective ligands for the dopamine transporter

A series of N-substituted 3 alpha-[bis(4'-fluorophenyl)methoxy]tropane analogues has been prepared that function as dopamine uptake inhibitors. The N-methylated analogue of this series had a significantly higher affinity for the dopamine transporter than the parent compound, N-methyl-3 alpha- (diphenylmethoxy)tropane (benztropine, Cogentin). Yet like the parent compound, it retained high affinity for muscarinic receptors. A series of N-substituted compounds were prepared from nor-3 alpha-[bis(4'-fluorophenyl)methoxy]tropane via acylation followed by hydride reduction of the amide or by direct alkylation. All compounds containing a basic tropane nitrogen displaced [3H]WIN 35,428 at the dopamine transporter (Ki range = 8.5-634 nM) and blocked dopamine uptake (IC50 range = 10-371 nM) in rat caudate putamen, whereas ligands with a nonbasic nitrogen were virtually inactive. None of the compounds demonstrated high binding affinity at norepinephrine or serotonin transporters. Importantly, a separation of binding affinities for the dopamine transporter versus muscarinic m1 receptors was achieved by substitution of the N-methyl group with other N-alkyl or arylalkyl substituents (eg. n-butyl, allyl, benzyl, 3-phenylpropyl, etc.). Additionally, the most potent and selective analogue in this series at the dopamine transporter, N-(4"-phenyl-n-butyl)-3 alpha-[bis(4'-fluorophenyl)methoxy]tropane analogue failed to substitute for cocaine in rats trained to discriminate cocaine from saline. Potentially, new leads toward the development of a pharmacotherapeutic for cocaine abuse and other disorders affecting the dopamine transporter may be discovered.     

Immunochemical analysis of dopamine transporter protein in Parkinson's disease

The plasma membrane dopamine transporter (DAT) is considered to be a reliable marker of presynaptic dopaminergic terminal loss. Previous in vivo imaging and postmortem binding studies have detected a loss in striatal DAT binding in Parkinson's diseased (PD) brain; however, these techniques have poor spatial resolution and may suffer from nonspecific binding of some ligands. In this study, we use novel highly specific monoclonal antibodies to distinct epitopes of human DAT to quantify and localize the protein. Western blot analysis revealed marked reductions in DAT immunoreactivity in putamen, caudate, and nucleus accumbens of PD brain compared with control cases, and the reductions were significantly correlated to disease duration. Immunohistochemistry revealed DAT-immunoreactive fibers and puncta that were dense throughout the striatum of control brains but that were drastically reduced in putamen of PD brains. Caudate from PD brains showed a significant degree of sparing along the border of the ventricle, and the nucleus accumbens was relatively preserved. An unexpected finding was that discrete islands of DAT immunoreactivity were preserved within the matrix of PD putamen. Thus, immunological analysis of DAT protein provides novel and sensitive means for localizing and quantifying DAT protein in PD and other neurological disorders involving dopaminergic systems.     

N-[2-[4-(4-Chlorophenyl)piperazin-1-yl]ethyl]-3-methoxybenzamide: a potent and selective dopamine D4 ligand

A series of new 1-aryl-4-alkylpiperazines containing a terminal benzamide fragment or a tetralin-1-yl nucleus on the alkyl chain were synthesized and tested for binding at cloned human dopamine D4 and D2 receptor subtypes. A SAFIR (structure-affinity relationship) study on this series is herein discussed. The most relevant D4 receptor affinities were displayed by N-[omega-[4-arylpiperazin-1-yl]alkyl]-methoxybenzamides (compounds 5, 16-20), their IC50 values ranging between 0.057 and 7.8 nM. Among these, N-[2-[4-(4-chlorophenyl)piperazin-1-yl]ethyl]-3-methoxybenzamide (17) emerged since it exhibited very high affinity for dopamine D4 receptor (IC50 = 0.057 nM) with selectivity of >10 000 for the D4 versus the D2 receptor; compound 17 was also selective versus serotonin 5-HT1A and adrenergic alpha1 receptors.     

14-3-3 proteins in neuronal development and function

The 14-3-3 proteins are small, cytosolic, evolutionarily conserved proteins expressed abundantly in the nervous system. Although they were discovered more than 30 yr ago, their function in the nervous system has remained enigmatic. Several recent studies have helped to clarify their biological function. Crystallographic investigations have revealed that 14-3-3 proteins exist as dimers and that they contain a specific region for binding to other proteins. The interacting proteins, in turn, contain a 14-3-3 binding motif; proteins that interact with 14-3-3 dimers include PKC and Raf, protein kinases with critical roles in neuronal signaling. These proteins are capable of activating Raf in vitro, and this role has been verified by in vivo studies in Drosophila. Most interestingly, mutations in the Drosophila 14-3-3 genes disrupt neuronal differentiation, synaptic plasticity, and behavioral plasticity, establishing a role for these proteins in the development and function of the nervous system.     

SH3 binding domains in the dopamine D4 receptor

The dopamine D4 receptor is a G protein-coupled receptor (GPCR) that belongs to the dopamine D2-like receptor family. Functionally, the D2-like receptors are characterized by their ability to inhibit adenylyl cyclase. The dopamine D4 receptor as well as many other catecholaminergic receptors contain several putative SH3 binding domains. Most of these sites in the D4 receptor are located in a polymorphic repeat sequence and flanking sequences in the third intracellular loop. Here we demonstrate that this region of the D4 receptor can interact with a large variety of SH3 domains of different origin. The strongest interactions were seen with the SH2-SH3 adapter proteins Grb2 and Nck. The repeat sequence itself is not essential in this interaction. The data presented indicate that the different SH3 domains in the adapter proteins interact in a cooperative fashion with two distinct sites immediately upstream and downstream from the repeat sequence. Removal of all the putative SH3 binding domains in the third intracellular loop of the dopamine D4 receptor resulted in a receptor that could still bind spiperone and dopamine. Dopamine could not modulate the coupling of these mutant receptors to adenylyl cyclase and MAPK, although dopamine modulated receptor-G protein interaction appeared normal. The receptor deletion mutants show strong constitutive internalization that may account for the deficiency in functional activation of second messengers. The data indicates that the D4 receptor contains SH3 binding sites and that these sites fall within a region involved in the control of receptor internalization.     

Tyrosine-533 of rat dopamine transporter: involvement in interactions with 1-methyl-4-phenylpyridinium and cocaine

To improve our understanding of structure-function relationships for neurotransmitter transporters, we performed site-directed mutagenesis of the rat dopamine transporter (DAT) and assessed the functions of the mutants in transiently-expressing COS cells. Tyrosine-533 of rat DAT lies in the 11th transmembrane region, where the corresponding amino acid of human DAT is phenylalanine. Alanine substitution of tyrosine-533 (Y533A) conferred an increased affinity for 1-methyl-4-phenylpyridinium (MPP+). Phenylalanine substitution of tyrosine-533 (Y533F) increased the velocity of MPP+ uptake but decreased DAT's affinity for MPP+. Cocaine's potency in inhibiting dopamine uptake was unchanged with Y533A, but increased with Y533F. Differences in the uptake kinetics and inhibitory potency of cocaine between rat and human DATs were similar to the differences observed between the wild-type and Y533F mutants DATs. Tyrosine-533 may be important for the DAT function and for species differences in transporter functions, including differential sensitivities to cocaine and 1-methyl-1,2,3,6-tetrahydropyridine (MPTP) in humans and rats.     

Genetic polymorphism of the 3' VNTR region of the human dopaminergic function gene DAT1 (human dopamine transporter gene) in the Mongolian population

The hypervariable region of the dopamine transporter gene (DAT1) was amplified from samples in the Mongolian population. This region includes a variable number of tandem repeats of a 40-bp core unit in the 3' untranslated region of DAT1. Vandenbergh et al. (1992) reported variability in the number of repeats of this 3' flanking region ranging from 3 to 11 times in white and black populations. We examined polymorphism at the DAT1 locus in 78 native Mongolian subjects. We found alleles with 7 to 13 repeats, which is different from the findings of Vandenbergh et al. (1992). The allele distribution of the Mongolian population is similar to that in the Japanese population, reported previously by Nakatome et al. (1995). Chi-square analysis showed a significant lack of homogeneity between our findings in Mongolian subjects and those reported previously in white and black populations. The DAT1 locus was estimated to have a heterozygosity index of 14.1%, and the polymorphic information content was calculated to be 0.16.     

Substrate transport and cocaine binding of human dopamine transporter is reduced by substitution of carboxyl tail with that of bovine dopamine transporter

A chimeric dopamine transporter (DAT) cDNA encoding mutant human DAT (hDAT) protein in which the intracellular carboxyl-terminal tail is replaced by that of the bovine dopamine transporter (bDAT) was constructed. The chimeric hDAT cDNA was expressed in COS-7 cells, and [3H]dopamine and [3H]MPP+ uptake and [3H]CFT binding capacities were assessed. Substrate transport and ligand binding of bDAT were reduced by 32-43% as a result of substitution of the carboxyl tail in hDAT, suggesting that the functional characteristics of bDAT arise from differences in the carboxyl tail between human and bovine DAT. Thus, it appears that the sequences encoded within the carboxyl terminal of DAT would be one of the important determinants for its functions.     

Evidence for the sequential formation of two complexes between an uptake inhibitor, GBR 12783 [1-[2-(diphenylmethoxy)ethyl]-4-(3-phenyl-2-propenyl)piperazine], and the neuronal transporter of dopamine

Incubation of a crude synaptosomal fraction from rat striatum with GBR 12783 at 37 degrees C produced an inhibition of the specific uptake of [3H]dopamine that increased with time. The inhibition increased when GBR 12783 was present during preincubation and incubation (IC50 = 1.85+/-0.1 nM) instead of incubation alone (IC50 = 25+/-3.5 nM). Time-course studies of uptake inhibition demonstrated that a first collision transporter-inhibitor complex (TI) was formed immediately after addition of GBR 12783 so that the initial uptake velocity (V0) decreased for increasing concentrations of inhibitor (Ki > or = 20 nM). TI slowly isomerized to a more stable complex TI* (Ki* < or = 5 nM) with a value of t1/2 = 20-270 s. Fits of data to model 2 in which the steady-state uptake (VS) is set to zero were generally preferred, suggesting that formation of TI* could tend to irreversibility, as a consequence of a very low reverse isomerization. As expected, k, V0, and VS tended to steady-state values in an asymptotic manner for high concentrations of GBR 12783. GBR 12783 at 2.5 nM produced a mixed inhibition of the uptake, with an increase in KM and a decrease in Vmax; these effects were improved for 10 nM GBR 12783 and at 20 degrees C. These results are discussed in relation to previous data concerning [3H]GBR 12783 binding. The present work gives the first experimental demonstration that dopamine uptake blockers can act according to a two-step mechanism of inhibition; this is of great interest, because these inhibitors can oppose the effects of cocaine or amphetamine on the transporter according to a reaction that is partly nondependent on the concentration of the abused agent.     

Synthesis and ligand binding studies of 4'-iodobenzoyl esters of tropanes and piperidines at the dopamine transporter

Four analogs and two homologs of cocaine, designed as potent cocaine antagonists, were synthesized. The SN2 reaction between ecgonine methyl ester (13) or appropriately substituted piperidinol (19, 21) and appropriately substituted 4-iodobenzoyl chloride gave 4-iodobenzoyl esters of tropanes and piperidines (5-8). 2'-Hydroxycocaine (9) was obtained from 2'-acetoxycocaine (12) by selective transesterification with MeOH saturated with dry HCl gas. 2'-Acetoxycocaine (12) was synthesized from acetylsalicyloyl chloride (23) and ecgonine methyl ester (13). The binding affinities of these compounds were determined at the dopamine transporter for the displacement of [3H]WIN-35428. An iodo group substitution at the 4'-position of cocaine decreased dopamine transporter binding potency, while a hydroxy or acetoxy group at the 2'-position exhibited increased binding potency for the dopamine transporter compared to cocaine (10- and 3.58-fold, respectively). 2'-Hydroxylation also enhanced the bidning potency of 4'-iodococaine (5) by 10-fold. Replacement of the tropane ring with piperidine led to poor binding affinities.     

Mechanisms of amphetamine action revealed in mice lacking the dopamine transporter

Amphetamine (AMPH) inhibits uptake and causes release of dopamine (DA) from presynaptic terminals. AMPH can act on both vesicular storage of DA and directly on the dopamine transporter (DAT). To assess the relative importance of these two processes, we have examined the releasing actions of AMPH in mice with a genetic deletion of the DAT. The sequence of actions of AMPH has been determined by following the real time changes of DA in the extracellular fluid of intact tissue with fast scan cyclic voltammetry. In striatal slices from wild-type mice, AMPH causes a gradual (approximately 30 min) increase in extracellular DA, with a concomitant disappearance of the pool of DA available for depolarization-evoked release. Conversely, in slices from mice lacking the DAT, although a similar disappearance of electrically stimulated DA release occurs, extracellular DA does not increase. Similarly, microdialysis measurements of DA after AMPH in freely moving animals show no change in mice lacking the DAT, whereas it increases 10-fold in wild-type mice. In contrast, redistribution of DA from vesicles to the cytoplasm by the use of a reserpine-like compound, Ro4-1284, does not increase extracellular DA in slices from wild-type animals; however, subsequent addition of AMPH induces rapid (<5 min) release of DA. Thus, the DAT is required for the releasing action, but not the vesicle-depleting action, of AMPH on DA neurons, and the latter represents the rate-limiting step in the effects of AMPH. Furthermore, these findings suggest that in the absence of pharmacological manipulation, such as the use of amphetamine, endogenous cytoplasmic DA normally does not reach sufficient concentrations to reverse the DAT.     

Profound neuronal plasticity in response to inactivation of the dopamine transporter

The dopamine transporter (DAT) plays an important role in calibrating the duration and intensity of dopamine neurotransmission in the central nervous system. We have used a strain of mice in which the gene for the DAT has been genetically deleted to identify the DAT's homeostatic role. We find that removal of the DAT dramatically prolongs the lifetime (300 times) of extracellular dopamine. Within the time frame of neurotransmission, no other processes besides diffusion can compensate for the lack of the DAT, and the absence of the DAT produces extensive adaptive changes to control dopamine neurotransmission. Despite the absence of a clearance mechanism, dopamine extracellular levels were only 5 times greater than control animals due to a 95% reduction in content and a 75% reduction in release. Paradoxically, dopamine synthesis rates are doubled despite a decrease of 90% in the levels of tyrosine hydroxylase and degradation is markedly enhanced. Thus, the DAT not only controls the duration of extracellular dopamine signals but also plays a critical role in regulating presynaptic dopamine homeostasis. It is interesting to consider that the switch to a dopamine-deficient, but functionally hyperactive, mode of neurotransmission observed in mice lacking the DAT may represent an extreme example of neuronal plasticity resulting from long-term psychostimulant abuse.