Harald Reither

Carrier-mediated release, transport rates, and charge transfer induced by amphetamine, tyramine, and dopamine in mammalian cells transfected with the human dopamine transporter.

Abstract: Amphetamine and related substances induce dopamine release. According to a traditional explanation, this dopamine release occurs in exchange for amphetamine by means of the dopamine transporter (DAT). We tested this hypothesis in human embryonic kidney 293 cells stably transfected with the human DAT by measuring the uptake of dopamine, tyramine, and d‐ and l‐amphetamine as well as substrate‐induced release of preloaded N‐methyl‐4‐[3H]phenylpyridinium ([3H]MPP+). The uptake of substrates was sodium‐dependent and was inhibited by ouabain and cocaine, which also prevented substrate‐induced release of MPP+. Patch‐clamp recordings revealed that all four substrates elicited voltage‐dependent inward currents (on top of constitutive leak currents) that were prevented by cocaine. Whereas individual substrates had similar affinities in release, uptake, and patch‐clamp experiments, maximal effects displayed remarkable differences. Hence, maximal effects in release and current induction were ∼25% higher for d‐amphetamine as compared with the other substrates. By contrast, dopamine was the most efficacious substrate in uptake experiments, with its maximal initial uptake rate exceeding those of amphetamine and tyramine by factors of 20 and 4, respectively. Our experiments indicate a poor correlation between substrate‐induced release and the transport of substrates, whereas the ability of substrates to induce currents correlates well with their releasing action.

Is Parkinson's Disease a Vesicular Dopamine Storage Disorder? Evidence from a Study in Isolated Synaptic Vesicles of Human and Nonhuman Primate Striatum

The cause of degeneration of nigrostriatal dopamine (DA) neurons in idiopathic Parkinsons disease (PD) is still unknown. Intraneuronally, DA is largely confined to synaptic vesicles where it is protected from metabolic breakdown. In the cytoplasm, however, free DA can give rise to formation of cytotoxic free radicals. Normally, the concentration of cytoplasmic DA is kept at a minimum by continuous pumping activity of the vesicular monoamine transporter (VMAT)2. Defects in handling of cytosolic DA by VMAT2 increase levels of DA-generated oxy radicals ultimately resulting in degeneration of DAergic neurons. Here, we isolated for the first time, DA storage vesicles from the striatum of six autopsied brains of PD patients and four controls and measured several indices of vesicular DA storage mechanisms. We found that (1) vesicular uptake of DA and binding of the VMAT2-selective label [3H]dihydrotetrabenazine were profoundly reduced in PD by 87–90% and 71–80%, respectively; (2) after correcting for DA nerve terminal loss, DA uptake per VMAT2 transport site was significantly reduced in PD caudate and putamen by 53 and 55%, respectively; (3) the VMAT2 transport defect appeared specific for PD as it was not present in Macaca fascicularis (7 MPTP and 8 controls) with similar degree of MPTP-induced nigrostriatal neurodegeneration; and (4) DA efflux studies and measurements of acidification in the vesicular preparations suggest that the DA storage impairment was localized at the VMAT2 protein itself. We propose that this VMAT2 defect may be an early abnormality promoting mechanisms leading to nigrostriatal DA neuron death in PD.

Lower efficacy of the dopamine D1 agonist, SKF 38393, to stimulate adenylyl cyclase activity in primate than in rodent striatum

The selective D1 agonist, SKF 38393, stimulated adenylyl cyclase by about 40% of basal activity in rat striatum but by only about 10% in the striatum of rhesus monkeys. In contrast, dopamine stimulated striatal adenylyl cyclase in both species with equal efficiency (70-80%). SKF 38393 30 microM inhibited the effect of 30 microM dopamine by about 45% in rat and by about 75% in primate tissue. This difference may be due to a lower D1 receptor reserve in primate than in rodent tissue and suggests that only selective D1 agonists with full efficacy at D1 receptors can be expected to have beneficial effects in patients with Parkinsons disease.

Induction by low Na+ or Cl− of cocaine sensitive carrier‐mediated efflux of amines from cells transfected with the cloned human catecholamine transporters†

COS‐7 cells transfected with the cDNA of the human dopamine transporter (DAT cells) or the human noradrenaline transporter (NAT cells) were loaded with [3H]‐dopamine or [3H]‐noradrenaline and superfused with buffers of different ionic composition. In DAT cells lowering the Na+ concentration to 0, 5 or 10 mM caused an increase in 3H‐efflux. Cocaine (10 μM) or mazindol (0.3 μM) blocked the efflux at low Na+, but not at 0 Na+. Lowering the Cl− concentration to 0, 5 or 10 mM resulted in an increased efflux, which was blocked by cocaine or mazindol. Desipramine (0.1 μM) was without effect in all the conditions tested. In NAT cells, lowering the Na+ concentration to 0, 5 or 10 mM caused an increase in 3H‐efflux, which was blocked by cocaine or mazindol. Desipramine produced a partial block, its action being stronger at 5 or 10 mM Na+ than at 0 mM Na+. Efflux induced by 0, 5 or 10 mM Cl− was completely blocked by all three uptake inhibitors. In cross‐loading experiments, 5 mM Na+‐ or 0 Cl−‐induced efflux was much lower from [3H]‐noradrenaline‐loaded DAT, than NAT cells and was sensitive to mazindol, but not to desipramine. Efflux from [3H]‐dopamine‐loaded NAT cells elicited by 5 mM Na+ or 0 Cl− was blocked by mazindol, as well as by desipramine. Thus, cloned catecholamine transporters display carrier‐mediated efflux of amines if challenged by lowering the extracellular Na+ or Cl−, whilst retaining their pharmacological profile. The transporters differ with regard to the ion dependence of the blockade of reverse transport by uptake inhibitors.

Chronic exposure to manganese decreases striatal dopamine turnover in human alpha-synuclein transgenic mice

Interaction of genetic and environmental factors is likely involved in Parkinsons disease (PD). Mutations and multiplications of alpha-synuclein (α-syn) cause familial PD, and chronic manganese (Mn) exposure can produce an encephalopathy with signs of parkinsonism. We exposed male transgenic C57BL/6J mice expressing human α-syn or the A53T/A30P doubly mutated human α-syn under the tyrosine hydroxylase promoter and non-transgenic littermates to MnCl₂-enriched (1%) or control food, starting at the age of 4 months. Locomotor activity was increased by Mn without significant effect of the transgenes. Mice were sacrificed at the age of 7 or 20 months. Striatal Mn was significantly increased about three-fold in those exposed to MnCl₂. The number of tyrosine hydroxylase positive substantia nigra compacta neurons was significantly reduced in 20 months old mice (-10%), but Mn or transgenes were ineffective (three-way ANOVA with the factors gene, Mn and age). In 7 months old mice, striatal homovanillic acid (HVA)/dopamine (DA) ratios and aspartate levels were significantly increased in control mice with human α-syn as compared to non-transgenic controls (+17 and +11%, respectively); after Mn exposure both parameters were significantly reduced (-16 and -13%, respectively) in human α-syn mice, but unchanged in non-transgenic animals and mice with mutated α-syn (two-way ANOVA with factors gene and Mn). None of the parameters were changed in the 20 months old mice. Single HVA/DA ratios and single aspartate levels significantly correlated across all treatment groups suggesting a causal relationship between the rate of striatal DA metabolism and aspartate release. In conclusion, under our experimental conditions, Mn and human α-syn, wild-type and doubly mutated, did not interact to induce PD-like neurodegenerative changes. However, Mn significantly and selectively interacted with human wild-type α-syn on indices of striatal DA neurotransmission, the neurotransmitter most relevant to PD.

α-Synuclein selectively increases manganese-induced viability loss in SK-N-MC neuroblastoma cells expressing the human dopamine transporter

The established or potentially toxic agents implicated in the nigral cell death in Parkinsons disease, dopamine, 1-methyl-4-phenylpyridinium (MPP(+)), iron, and manganese, were examined as to their effects on the viability of cells overexpressing alpha-synuclein. SK-N-MC neuroblastoma cells stably expressing the human dopamine transporter were transfected with human alpha-synuclein and cell clones with and without alpha-synuclein immunoreactivity were obtained. Cells were exposed for 24-72 h to 1-10 microM dopamine, 0.1-3 microM MPP(+), 0.1-1 mM FeCl(2) or 30-300 microM MnCl(2) added to the culture medium. There was no difference between cells expressing alpha-synuclein and control cells after exposure to dopamine, MPP(+) or FeCl(2). However, MnCl(2) resulted in a significantly stronger decreased viability of cells overexpressing alpha-synuclein after 72 h. These findings suggest that manganese may co-operate with alpha-synuclein in triggering neuronal cell death such as seen in manganese parkinsonism. The relevance of our observations for the pathoetiology of Parkinsons disease proper remains to be determined.

Zinc regulates the dopamine transporter in a membrane potential and chloride dependent manner

The dopamine transporter (DAT), a membrane protein specifically expressed by dopaminergic neurons and mediating the action of psychostimulants and dopaminergic neurotoxins, is regulated by Zn(2+) which directly interacts with the protein. Herein, we report a host-cell-specific direction of the Zn(2+) effect on wild type DAT. Whereas low mumolar Zn(2+) decreased dopamine uptake by DAT expressing HEK293 cells, it stimulated uptake by DAT expressing SK-N-MC cells. Inhibition or stimulation was lost in a DAT construct without the binding site for Zn(2+). Also reverse transport was differentially affected by Zn(2+), dependent on whether the DAT was expressed in HEK293 or SK-N-MC cells. Pre-treatment of DAT expressing cells with phorbol-12-myristate-13-acetate, an activator of protein kinase C, attenuated the inhibitory effect of Zn(2+) on uptake in HEK293 cells and increased the stimulatory effect in SK-N-MC cells. Patch-clamp experiments under non-voltage-clamped conditions revealed a significantly higher membrane potential of HEK293 than SK-N-MC cells and a reduced membrane potential after phorbol ester treatment. Lowering chloride in the uptake buffer switched the stimulatory effect of Zn(2+) in SK-N-MC cells to an inhibitory, whereas high potassium depolarization of HEK293 cells switched the inhibitory effect of Zn(2+) to a stimulatory one. This study represents the first evidence that DAT regulation by Zn(2+) is profoundly modulated by the membrane potential and chloride.

Functional sensitization of striatal dopamine D1 receptors in the 6-hydroxydopamine-lesioned rat

Dopamine-stimulated adenylyl cyclase activity was measured in striatal homogenates of rats in which the nigrostriatal pathway was lesioned by 6-hydroxydopamine 20-24 months before the experiments. In the intact (contralateral) striatum the potency and the efficacy of dopamine in stimulating adenylyl cyclase was lower in the presence of high NaCl concentrations (120 mM) compared with the effects of dopamine in an NaCl-poor assay medium (20 mM). The same effect of NaCl was observed in the striatum on the side of a weak, behaviourally ineffective 6-hydroxydopamine lesion resulting in a loss of 57% of striatal dopamine. This effect of NaCl was absent in the strongly denervated striatum, i.e. in rats having a 99.8% dopamine loss and rotating when challenged with a low dose of apomorphine. Thus, in denervated vs intact striatum, in the presence of a physiological concentration of NaCl, dopamine-stimulated adenylyl cyclase showed a sensitization which was absent in assays with 20 mM NaCl. The inhibition of adenylyl cyclase by dopamine via D2 receptors, which was seen in the presence of 120 mM NaCl and the D1 antagonist SCH 23390, was not affected by denervation. We suggest that chronic dopaminergic denervation of the striatum results in a stabilized, i.e. NaCl-insensitive, high affinity state of D1 receptors. This may be the basis for a sensitization of the coupling mechanism of the denervated D1 receptors to adenylyl cyclase.

Zn2+ modulates currents generated by the dopamine transporter: parallel effects on amphetamine-induced charge transfer and release

The psychostimulant drug amphetamine increases extracellular monamines in the brain acting on neurotransmitter transporters, especially the dopamine transporter. Mediated by this plasmalemmal pump, amphetamine does not only induce release but also charge transfer which might be involved in the release mechanism. To study a potential link between the two phenomena, we used Zn(2+) as an acute regulatory agent which modulates dopamine uptake by a direct interaction with the transporter protein. Charge transfer was investigated in patch-clamp experiments on HEK 293 cells stably expressing the human dopamine transporter, release was studied in superfusion experiments on cells preloaded with the metabolically inert transporter substrate [(3)H]1-methyl-4-phenylpyridinium. Ten micromoles of Zn(2+) had only minor effects in the absence of amphetamine but stimulated release and inward currents induced by amphetamine depending on the concentration of the psychostimulant: the effect of 0.2 microM was not significantly modulated, whereas the effect of 1 and 10 microM amphetamine was stimulated, and the stimulation by Zn(2+) was significantly stronger at 10 microM than at 1 microM amphetamine. The stimulatory action of Zn(2+) on release and inward current was in contrast to its inhibitory action on dopamine uptake. This supports a release mechanism of amphetamine different from facilitated exchange diffusion but involving ion fluxes through the dopamine transporter.

Antiproliferative action of dopamine and norepinephrine in neuroblastoma cells expressing the human dopamine transporter

The classical catecholamine transmitters dopamine and norepinephrine are also involved in neurodevelopment and neuron cell death. These effects are thought to be mediated by receptors coupled to intracellular second messengers and by oxidative stress, respectively. Using human SK‐N‐MC neuroblastoma cells stably transfected with the dopamine or norepinephrine transporter, we show that catecholamines have profound effects on cell viability not mediated by any of the above mechanisms. Low micromolar concentrations of dopamine or norepinephrine (1–10 μM) affected cell growth depending on the expression level of plasmalemmal transport. The growth inhibition was accompanied by a profound effect on the cell cycle profile with an arrest in G1 as determined by flow cytometry measuring DNA content. In a proportion of cells, apoptosis was present. Various antioxidants did not prevent the catecholamine effect, and there was no indication of accumulation of reactive oxygen species (as determined by hydroethidine, dichlorofluorescein, and the inactivation‐reactivation pattern of aconitase), ruling out involvement of oxidative stress. Reversal by FeCl3 and parallel effects of the iron‐chelator deferoxamine suggest that the catecholamine action was related to intracellular iron chelation. This novel biological action of dopamine and norepinephrine unrelated to neurotransmitter receptors, second messengers, or oxidative stress may be important for cell differentiation during neurodevelopment and survival of differentiated neurons.