1-methyl-4-phenylpyridinium (MPP+) decreases mitochondrial oxidation-reduction (REDOX) activity and membrane potential (Deltapsi(m)) in rat striatum

Mitochondrial dysfunction has long been implicated in the death of nigrostriatal dopaminergic neurons in Parkinson's disease (PD) and its experimental models. Here we further analyzed changes in the mitochondrial oxidation-reduction (REDOX) activity and membrane potential (Deltapsi(m)) of striatal synaptosomes after the infusion of 1-methyl-4-phenylpyridinium (MPP+) into rat striatum. MPP+ (40 nmol) treatment produced decreases in mitochondrial REDOX activity and Deltapsi(m) at 18 h, as measured by fluorometric analysis with both Alamar blue and JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide) dyes. At this time point, tyrosine hydroxylase (TH) and dopamine transporter (DAT) protein levels were not altered, but both decreased at 7 days after MPP+ (40 nmol) infusion. Both measures of mitochondrial dysfunction induced by MPP+ (40 nmol) at 18 h were attenuated, at least in part, by pretreatment with a selective dopamine uptake inhibitor GBR-12909 (1-(2-(bis(4-fluorophenyl)methoxy)ethyl)-4-(3-phenylpropyl) piperazine). In addition, GBR-12909 partially attenuated MPP+ (40 nmol)-caused a loss of striatal nerve terminal as indicated by decreases in TH and DAT immunoreactivities as well as dopamine and its metabolites levels. The present study indicates that decreases in mitochondrial REDOX activity and Deltapsi(m) may play a role in MPP+ -induced dopaminergic neurotoxicity, and further provides that improvement of mitochondrial dysfunction may be a better way to slow progressive dopaminergic neurodegeneration commonly associated with PD.     

Terminally differentiated SH-SY5Y cells provide a model system for studying neuroprotective effects of dopamine agonists

We characterized undifferentiated (UN) and three differentiation conditions of the SH-SY5Y neuroblastoma cell line for phenotypic markers of dopaminergic cells, sensitivity to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridinium ion (MPP+), the requirement to utilize the dopamine (DA) transporter (DAT) for MPP+ toxicity, and the neuroprotective effects of pramipexole. Cells were differentiated with retinoic acid (RA), 12-O-tetradecanoyl-phorbol-13-acetate (TPA), and RA followed by TPA (RA/TPA). RA/TPA treated cells exhibited the highest levels of tyrosine hydroxylase and DAT but lower levels of vesicular monoamine transporter. The kinetics of [3H]DA uptake and [3H]MPP+ uptake to DAT in RA/TPA differentiated cells were similar to that of rat and mouse caudate-putamen synaptosomes. RA/TPA differentiated cells evidenced high sensitivity to the neurotoxic effects of MPP+ (0.03 to 3.0 mM), and the neurotoxic effects of MPP+ were blocked with the DAT inhibitor 1-(2-[bis(4-fluorophenyl)methoxy]ethyl)-4-(3-phenylpropyl)piperazine (GBR 12909). DA-induced cell death was not more sensitive in RA vs RA/TPA differentiated cells and was not inhibited by transporter inhibitors. RA/TPA differentiated cells exhibited 3-fold and 6-fold higher levels, respectively, of DA D2 and D3 receptors than UN or RA differentiated cells. Pretreatment with pramipexole was protective against MPP+ in the RA/TPA differentiated cells but not in undifferentiated or RA differentiated cells. The neuroprotective effect of pramipexole was concentration-dependent and dopamine D2/D3 receptor dependent. In contrast, protection by pramipexole against DA was not DA receptor dependent. Further characterization of the neuroprotective effects of DA agonists in this model system can provide unique information about DA receptor dependent and independent mechanisms of neuroprotection.     

Terminally differentiated SH-SY5Y cells provide a model system for studying neuroprotective effects of dopamine agonists

We characterized undifferentiated (UN) and three differentiation conditions of the SH-SY5Y neuroblastoma cell line for phenotypic markers of dopaminergic cells, sensitivity to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridinium ion (MPP), the requirement to utilize the dopamine (DA) transporter (DAT) for MPP toxicity, and the neuroprotective effects of pramipexole. Cells were differentiated with retinoic acid (RA), 12-O-tetradecanoly-phorbol-13-acetate (TPA), and RA followed by TPA (RA/TPA). RA/TPA treated cells exhibited the highest levels of tyrosine hydroxylase and DAT but lower levels of vesicular monoamine transporter. The kinetics of [³H]DA uptake and [³H]MPP uptake to DAT in RA/TPA differentiated cells were similar to that of rat and mouse caudate-putamen synaptosomes. RA/TPA differentiated cells evidenced high sensitivity to the neurotoxic effects of MPP (0.03 to 3.0 mM), and the neurotoxic effects of MPP were blocked with the DAT inhibitor 1-(2-[bis(4-fluorophenyl)methoxy]ethyl)-4-(3-phenylpropyl)piperazine (GBR 12909). DA-induced cell death was not more sensitive in RA vs RA/TPA differentiated cells and was not inhibited by transporter inhibitors. RA/TPA differentiated cells exhibited 3- fold and 6-fold higher levels, respectively, of DA D₂ and D₃ receptors than UN or RA differentiated cells. Pretreatment with pramipexole was protective against MPP in the RA/TPA differentiated cells but not in undifferentiated or RA differentiated cells. The neuroprotective effect of pramipexole was concentration-dependent and dopamine D₂/D₃ receptor dependent. In contrast, protection by pramipexole against DA was not DA receptor dependent. Further characterization of the neuroprotective effects of DA agonists in this model system can provide unique information about DA receptor dependent and independent mechanisms of neuroprotection.     

Role of dopamine tone in the pursuit of brain stimulation reward

Dopaminergic neurons contribute to intracranial self-stimulation (ICSS) and other reward-seeking behaviors, but it is not yet known where dopaminergic neurons intervene in the neural circuitry underlying reward pursuit or which psychological processes are involved. In rats working for electrical stimulation of the medial forebrain bundle, we assessed the effect of GBR-12909 (1-[2-[bis(4-fluorophenyl)-methoxy]ethyl]-4-[3- phenylpropyl]piperazine), a specific blocker of the dopamine transporter. Operant performance was measured as a function of the strength and cost of electrical stimulation. GBR-12909 increased the opportunity cost most subjects were willing to pay for a reward of a given intensity. However, this effect was smaller than that produced by a regimen of cocaine administration that drove similar increases in nucleus accumbens (NAc) dopamine levels in unstimulated rats. Delivery of rewarding stimulation to drug-treated rats caused an additional increase in dopamine concentration in the NAc shell in cocaine-treated, but not GBR-12909-treated, rats. These behavioral and neurochemical differences may reflect blockade of the norepinephrine transporter by cocaine but not by GBR-12909. Whereas the effect of psychomotor stimulants on ICSS has long been attributed to dopaminergic action at early stages of the reward pathway, the results reported here imply that increased dopamine tone boosts reward pursuit by acting at or beyond the output of the circuitry that temporally and spatially summates the output of the directly stimulated neurons underlying ICSS. The observed enhancement of reward seeking could be attributable to a decrease in the value of competing behaviors, a decrease in subjective effort costs, or an increase in reward-system gain.     

(-)-Epigallocatechin gallate regulates dopamine transporter internalization via protein kinase C-dependent pathway

Dopamine transporter (DAT) provides not only an integral component of dopaminergic neurotransmission but also a molecular gateway for the accumulation of some neurotoxins such as 1-methyl-4-phenylpyridinium (MPP(+)), a metabolite of 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP). Previous study reported that the neuroprotective effects of green tea polyphenols against MPP(+)-induced neurotoxicity were related to its inhibitory effect on MPP(+) uptake via DAT in dopaminergic cells. To extend the study, we investigated (-)-epigallocatechin gallate (EGCG), a monomer of green tea polyphenols, on DAT internalization in DAT-overexpressed PC12 cells. We found that EGCG (1-100 microM) can induce a dose-dependent inhibition of dopamine uptake in DAT-PC12 cells. In parallel, treatment of EGCG decreased membrane-bound DAT by 15% to 60%. Furthermore, protein kinase C (PKC) inhibitor GF109203X at 2 microM can markedly diminish the inhibitory effects of EGCG on dopamine uptake and reverse the EGCG-induced internalization of DAT. In addition, semiquantitative RT-PCR analysis indicated that EGCG did not affect DAT mRNA expression in the PC12 cells. These data suggest that EGCG exerts its inhibitory effect on DAT by modulating DAT internalization, in which PKC activation may be involved.     

Dopamine transport sites selectively labeled by a novel photoaffinity probe: 125I-DEEP

The dopamine transporter was labeled using a photosensitive compound related to GBR-12909, 125I-1-[2-(diphenylmethoxy)ethyl]-4-[2- (4-azido-3-iodophenyl)ethyl]piperazine (125I-DEEP). 125I-DEEP bound reversibly and with high affinity to the dopamine transport protein in the absence of light and could be covalently attached to the protein following exposure to UV light. In rat striatal homogenates, 125I-DEEP was found to incorporate covalently into a protein with apparent molecular weight of 58,000 Da. The properties of this binding protein were characteristic of the dopamine transporter since covalent attachment could be inhibited by dopamine-uptake blockers with the proper pharmacological rank order of potencies. Covalent binding was also inhibited in a stereospecific manner by (+) and (-) cocaine, as well as other cocaine analogs. The protein was not found in the cerebellum. The dopamine transporter appears to exist in a glycosylated form since photoaffinity-labeled transport sites could adsorb to wheat germ-agglutinin and could be specifically eluted from the column by beta-N-acetylglucosamine.     

Dose-response and duration effects of acute administrations of cocaine and GBR12909 on dopamine synthesis and transporter in the conscious monkey brain: PET studies combined with microdialysis

The dose-response and duration effects of acute administration of the dopamine transporter (DAT) blocker cocaine and GBR12909 on dopamine synthesis and transporter availability were evaluated in the brains of conscious monkeys using high-resolution positron emission tomography (PET) in combination with microdialysis. Rate of dopamine synthesis and DAT availability were evaluated using L-[beta-11C]DOPA and [11C]beta-CFT (WIN35,428), respectively. Administration of cocaine (0.5, 2 and 5 mg/kg) resulted in dose-dependent elevation of dopamine level in the striatal extracellular fluid (ECF) at 0.5 h after injection, and returned to the baseline level within 1.5 h post-injection. At 0.5 post-injection, cocaine reduced dopamine synthesis rate and DAT availability in a dose-dependent manner. The reduction of DAT availability by cocaine (2 mg/kg) returned to baseline level at 3 h post-injection and thereafter. Interestingly, dopamine synthesis rate was significantly higher at 3 h than baseline level and returned to baseline level 5.5 h post-injection. When GBR12909 (0.5, 2 and 5 mg/kg) was administered 0.5 h before tracer injection, dopamine synthesis rate and DAT availability were significantly decreased in a dose-dependent manner. These reductions induced by GBR12909 (2 mg/kg) lasted at least until 5.5 h post-injection. GBR12909 induced dose-dependent elevation of dopamine level in ECF, and the elevation lasted up to 7 h. The present results indicated that cocaine and GBR12909 affect dopamine synthesis rate and DAT availability in the striatum with difference time courses as measured by PET in the conscious monkey brains.     

GBR-12909 effect on dopamine outflow depends on phosphorylation in the caudate nucleus of the rat

Modulation of the dopamine (DA) transporter inhibitor GBR-12909 effect on DA release by protein kinases and protein phosphatases was studied in slices of the rat caudate nucleus measuring DA outflow in the superfusate of static chambers. Activation of protein kinase A and C markedly enhanced the effect of GBR-12909, whereas protein kinase inhibition by H7 reduced the GBR-12909 effect. Inhibition of protein phosphatases (PPP) 1 and 2A by okadaic acid did not modify basal outflow of DA. However, after the addition of okadaic acid a dramatic and biphasic effect was found when DA outflow was enhanced by GBR-12909. Inhibition of PPP 2A enhanced extracellular DA levels, while inhibition of PPP 1 and 2A completely abolished the effect of GBR-12909. In contrast to tetrodotoxin, the voltage-activated calcium channel blocker omega-conotoxin MVIIC inhibited GBR-12909 effects on DA outflow. Additionally, in aCSF devoid of calcium GBR-12909 did not increase DA liberation. These results suggest a complex and strong influence of phosphorylation on GBR-12909 effects on calcium channel-dependent DA outflow at low-affinity piperazine binding sites in slices of the rat caudate nucleus in vitro.     

Effects of endothelin B receptor agonists on amyloid beta protein (25-35)-induced neuronal cell death

MPTP or its metabolite MPP+ are used to produce a Parkinsonism syndrome in a variety of animal species. The present study describes the effects of intranigral MPP+ administration either at 10 or 40 microg on the neuronal dopamine transporter (DAT) activity measured in rat striatal synaptosomes at different times after lesion. The 40 microg MPP+ injection induced a maximal toxic effect on day 7. However, 10 microg MPP+ progressively inhibited DA uptake on the injured side. V(max) decreased in a time-dependent manner and the lowest value was observed on day 21 after lesion. At this time, the K(m) value began to increase and was continuously accentuated until day 45 as compared to the contralateral side. Treatments either with the antioxidant alpha-tocopherol acetate or the MAO inhibitor pargyline, given daily for 7 days after lesion, partially prevented the 40 microg MPP(+)-induced inhibition of DA uptake. Conversely, both treatments given daily for 21 days after lesion completely prevented the alteration of DAT activity in the ipsilateral striatum induced by 10 microg MPP+. The absence of protection when both treatments were stopped 2 weeks before DA uptake measurements indicated that free radicals and DA oxidized products were continuously accumulated and gradually affected the functionality of the DAT. These results demonstrate that a rat intranigral lesion with 10 microg MPP+ led to a progressive impairment of DAT activity.     

Dopaminergic therapy improves upper limb motor performance in aged rhesus monkeys

The potential of dopaminergic treatments to improve upper limb motor movements was tested in 7 aged rhesus monkeys using L-3,4-dihydroxyphenylalanine (L-dopa) or the selective dopamine uptake inhibitor 1-2(bis[4-fluorophenyl] methoxy]ethyl)-4-(3-phenylpropyl) piperazine hydrochloride (GBR-12909). Six young monkeys were studied for comparison. L-Dopa or GBR-12909 improved upper limb motor performance by up to 40% in the aged animals. At this point their performance was comparable to that of young adults. Dopaminergic therapy could be useful in elderly humans experiencing declines in upper limb motor functions.     

Synthesis and preliminary characterization of a high-affinity novel radioligand for the dopamine transporter

In our effort to develop a novel radioligand selective for the dopamine transporter, compound 1b (O-972) was designed and characterized. The compound 1b was characterized for its binding both in monkey and rat striatum tissue, which demonstrated its high selectivity for the dopamine transporter (DAT) when its binding was compared with that at the serotonin transporter (SERT). The compound 5, which is a precursor for the tritiated radiolabel ligand [3H]O-972, was synthesized and biologically characterized. The preliminary characterization of this novel radioligand revealed its strong binding affinity for the DAT. Thus, the pharmacological profile of [3H]O-972 indicated that DAT inhibitors, which include GBR 12909, mazindol, CFT, and cocaine, could potently displace this novel radioligand from monkey brain striatum tissue. On the other hand, compounds known to be not selective for and potent at the DAT were very weak to do so. Initial binding results also indicate that [3H]O-972 may interact with the DAT in a manner that is not identical to that for GBR 12909 and tropane analogs.     

A rapid binding assay for solubilized dopamine transporters using [3H]WIN 35,428.

The cocaine analog [3H]WIN 35,428 was used to label digitonin-solubilized dopamine transporters from dog caudate nucleus. The assay consists of incubation of extracts with the ligand followed by separation of free from bound ligand by centrifugation after adding activated charcoal. Specific binding was observed in dog caudate but was absent in dog cerebellar extracts. Binding was linear with tissue, saturable, and of high affinity (Kd = 16 nM). In competition studies, soluble [3H]WIN 35,428 binding was inhibited strongly by mazindol, GBR 12909, and (-)-cocaine but only weakly by citalopram, desipramine, and (+)-cocaine; this is typical of binding to the dopamine transporter. Compared to assays using [3H]GBR 12935, (-)-cocaine was relatively more potent, suggesting that the cocaine and GBR 12935 binding sites are somewhat different. When soluble extract was chromatographed on a wheat germ agglutinin-Sepharose column, [3H]WIN 35,428 binding activity was eluted with N-acetylglucosamine in a manner similar to photoaffinity-labeled dopamine transporters.     

Dopamine transporter: involvement in selective dopaminergic neurotoxicity and degeneration

The carrier molecule that transports dopamine (DA) into dopamine neurons by an electrogenic, Na(+)- and Cl(-)-transport-coupled mechanism is known as the dopamine transporter (DAT). This uptake system is exclusively expressed in DA neurons with significantly higher levels of DAT expression in cells of the substantia nigra pars compacta than those of the ventral tegmental area and arcuate hypothalamic neurons. The expression density of DAT strongly correlates with the extent of DA cell loss in Parkinson's disease (PD). There are also DAT gene polymorphisms associated with PD. These data suggest a role of the DAT in the pathogenesis of PD. Though selective for its respective neurotransmitter, the DAT can also transport synthetic/natural analogues of the transmitter. Should such compounds interact with vital intracellular structures, their penetration into the neuron might have significant consequences. This sequence of toxic events could indeed demonstrated for the synthetic toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which produces selective degeneration of DA neurons characteristic of PD. Dopaminergic toxicity of its active metabolite 1-methyl-4-pyridinium (MPP(+)) is mediated by the DAT through accumulation into DA neurons, where it inhibits mitochondrial complex I activity. Various endogenous and exogenous heterocyclic molecules, which are structurally related to MPTP/MPP(+), such as isoquinolines and beta-carbolines, have been reported to exhibit similar toxic properties on DA cells, which are conferred by their uptake by the DAT. Taken together, there is large body of evidence from morphological, molecular biological and toxicological studies indicating that the DAT might be responsible for the selectivity of DA cell death in PD.     

Acute mitochondrial and chronic toxicological effects of 1-methyl-4-phenylpyridinium in human neuroblastoma cells

At low micromolar concentrations, 1-methyl-4-phenylpyridinium (MPP+), the toxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) selectively kills nigrostriatal dopaminergic neurons by mechanisms believed to involve impairment of mitochondrial complex I. A human neuroblastoma cell line expressing the dopamine transporter (DAT) was utilized to examine the effects of MPP+ on acute physiologic responses and subsequent cell death. Acute responses were measured by microphysiometry and by monitoring mitochondrial membrane potential with [3H]tetraphenylphosphonium (TPP+) uptake. MPP+ (10 microM) increased extracellular proton excretion in DAT-expressing cells within 2-3 min, but had no effect in untransfected cells. The lipophilic complex I inhibitor, rotenone, increased proton excretion in both cell lines. In DAT-expressing cells, mitochondrial membrane potential was reduced within I h of 10 microM MPP+ exposure. Rotenone reduced mitochondrial membrane potential in both cell lines. MPP+ caused apoptotic death of DAT-transfected cells 2-3 days after drug application, but did not kill untransfected cells. Thus, MPP+ produces immediate mitochondrial impairment only in cells that express DAT, and these changes occur days before overt cellular toxicity. The magnitude, time course and nature of these changes were similar to those produced by rotenone, confirming the site of action of MPP+ as mitochondrial complex I. These immediate mitochondrial effects appear to be an accurate predictor of subsequent cell death.     

Different effects of selective dopamine uptake inhibitors, GBR 12909 and WIN 35428, on HIV-1 Tat toxicity in rat fetal midbrain neurons

Drug abuse is a risk factor for neurological complications in HIV infection. Cocaine has been shown to exacerbate HIV-associated brain pathology and enhance neurotoxicity of HIV-1 Tat and gp120 proteins. In this study, we found that the selective inhibitor of dopamine transporter (DAT) function, 1-[2-[bis(4-fluorophenyl) methoxy]ethyl]-4-(3-phenylpropyl) piperazine (GBR 12909, vanoxerine), but not the selective inhibitors of serotonin and norepinephrine (SERT and NET) transporters, sertraline and nizoxetine, emulated cocaine-mediated enhancement of Tat neurotoxicity in rat fetal midbrain primary cell cultures. Similar to cocaine, the significant increase of Tat toxicity in midbrain cell cultures was observed at micromolar dose (5 μM) of GBR 12909. However, different doses of another selective dopamine uptake inhibitor, WIN 35428 did not affect Tat neurotoxicity. The study supports the hypothesis that changes in control of dopamine (DA) homeostasis are important for the cocaine-mediated enhancement of HIV-1 Tat neurotoxicity. Our results also demonstrate that inhibitors of DA uptake, which can bind to different domains of DAT, differ in their ability to mimic synergistic toxicity of cocaine and HIV-1 Tat in the midbrain cell culture.     

Mice lacking alpha-synuclein are resistant to mitochondrial toxins

Abnormalities in the function of alpha-synuclein are implicated in the pathogenesis of Parkinson's disease (PD). We found that alpha-synuclein-deficient mice are resistant to MPTP-induced degeneration of dopaminergic neurons. There was dose-dependent protection against loss of both dopamine in the striatum and dopamine transporter (DAT) immunoreactive neurons in the substantia nigra. These effects were not due to alterations in MPTP processing. We found that alpha-synuclein-deficient mice are also resistant to both malonate and 3-nitropropionic acid (3-NP) neurotoxicity. There was reduced generation of reactive oxygen species in alpha-synuclein-deficient mice following administration of 3-NP. These findings implicate alpha-synuclein as a modulator of oxidative damage, which has been implicated in neuronal death produced by MPTP and other mitochondrial toxins.     

Rapid reduction of ATP synthesis and lack of free radical formation by MPP+ in rat brain synaptosomes and mitochondria

MPTP is a neurotoxin thought to damage dopaminergic neurons through free radical formation. MPTP is metabolized in the brain to MPP(+), which is taken up into dopaminergic neurons via the dopamine transporter and assumed to impair mitochondrial function. We used striatal synaptosomes and telencephalic mitochondria to further investigate MPP(+) mechanism of action. For comparison, the respiratory toxins FCCP, a cyanide analog that uncouples mitochondrial ATP production, and rotenone, a NADH dehydrogenase inhibitor, were also tested. FCCP, MPP(+) and rotenone caused a rapid but stable decrease in [3H]dopamine (DA) uptake by striatal synaptosomes. Two free radical scavengers, the salen-manganese complex EUK-134, and the spin trap s-PBN, did not prevent MPP(+)-induced decrease in DA uptake. However, addition of ATP during synaptosome preparation resulted in partial recovery of MPP(+)-induced [3H]DA uptake decrease. Generation of oxygen free radicals by treatment of telencephalic mitochondria with MPP(+), FCCP, or rotenone, was evaluated by measuring DCF fluorescence, while light emission by the luciferin-luciferase complex was used to determine ATP levels. MPP(+), unlike rotenone, did not produce oxygen free radicals, but rather blocked ATP production in mitochondria, as did FCCP and rotenone. Taken together, these results suggest that MPP(+) toxicity, at least during its initial stages, is primarily due to a decrease in ATP synthesis by mitochondria and not to free radical formation.     

Vasoactive intestinal peptide (VIP) prevents neurotoxicity in neuronal cultures: relevance to neuroprotection in Parkinson's disease

Vasoactive intestinal peptide (VIP) provides neuroprotection against beta-amyloid toxicity in models of Alzheimer's disease. A superactive analogue, stearyl-Nle17-VIP (SNV) is a 100-fold more potent than VIP. In primary neuronal cultures, VIP protective activity may be mediated by femtomolar-acting glial proteins such as activity-dependent neurotrophic factor (ADNF), activity-dependent neuroprotective protein (ADNP), peptide derivatives ADNF-9 (9aa) and NAP (8aa), respectively. It has been hypothesized that beta-amyloid induces oxidative stress leading to neuronal cell death. Similarly, dopamine and its oxidation products were suggested to trigger dopaminergic nigral cell death in Parkinson's disease. We now examined the possible protective effects of VIP against toxicity of dopamine, 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenylpyridinium ion (MPP+) in neuronal cultures [rat pheochromocytoma (PC12), human neuroblastoma (SH-SY5Y) and rat cerebellar granular cells]. Remarkably low concentrations of VIP (10(-16)-10(-8) M), ADNF-9 and NAP (10(-18)-10(-10) M) protected against dopamine and 6-OHDA toxicity in PC12 and neuroblastoma cells. VIP (10(-11)-10(-9) M) and SNV (10(-13)-10(-11) M), protected cerebellar granule neurons against 6-OHDA. In contrast, VIP did not rescue neurons from death associated with MPP+. Since dopamine toxicity is linked to the red/ ox state of the cellular glutathione, we investigated neuroprotection in cells depleted of reduced glutathione (GSH). Buthionine sulfoximine (BSO), a selective inhibitor of glutathione synthesis, caused a marked reduction in GSH in neuroblastoma cells and their viability decreased by 70-90%. VIP, SNV or NAP (over a wide concentration range) provided significant neuroprotection against BSO toxicity. These results show that the mechanism of neuroprotection by VIP/SNV/NAP may be mediated through raising cellular resistance against oxidative stress. Our data suggest these compounds as potential lead compounds for protective therapies against Parkinson's disease.     

Subtle differences in the discriminative stimulus effects of cocaine and GBR-12909

1. In addition to inhibiting the dopamine transporter, cocaine affects a variety of other neurotransmitter systems. In the present study, the involvement of both dopaminergic and the nondopaminergic systems in the behavioral effects of cocaine was studied using an intravenous drug discrimination procedure. 2. One group (Group 1) of rats were trained to discriminate cocaine (1 mg/kg, i.v.) from saline, while a second group (Group 2) of rats were trained to discriminate the same dose of cocaine from both GBR-12909 (1 mg/kg i.v.), a dopamine-selective uptake inhibitor, and saline. 3. Following training, substitution tests with different doses of cocaine and several drugs pharmacologically related to cocaine were conducted. When cocaine dose was varied, there was a dose-dependent generalization to the cocaine-training stimulus in both groups of rats. Conversely, GBR-12909 and GBR-12935, another dopamine-selective uptake inhibitor, generalized to the cocaine-training stimulus in Group 1, but there was minimal or no generalization in Group 2 4. The norepinephrine-selective uptake inhibitors, desipramine and nisoxetine, and the serotonin-selective uptake inhibitor, zimeldine, produced little or no generalization to the cocaine-training stimulus in either group of rats. The sodium channel blocker, dimethocaine which has a relatively high affinity for the dopamine transporter fully generalized to the cocaine stimulus in both groups of rats, while procaine which has a low affinity for the dopamine transporters only partially generalized to the cocaine-training stimulus in both groups of rats 5. Finally, lidocaine, which has negligible affinity for the dopamine transporter, did not generalize to the cocaine-training stimulus in either group of rats. The findings suggest similarities as well as subtle, but important, differences between the discriminative stimulus effects of cocaine and the dopamine uptake inhibitors, GBR-12909 and GBR12935.