l-3,4-Dihydroxyphenylalanine-Induced Dopamine Release in the Striatum of Intact and 6-Hydroxydopamine-Treated Rats: Differential Effects of Monoamine Oxidase A and B Inhibitors

Abstract: Administration of l -DOPA (50 mg/kg) elicits a significant increase in extracellular dopamine in striata of rats treated with the catecholaminergic neurotoxin 6-hydroxydopamine but not in striata of intact rats. To assess the role of dopaminergic nerve terminals in determining the effects of exogenous l -DOPA on extracellular dopamine levels in striatum, we examined the relative contributions of monoamine oxidase A and monoamine oxidase B to the catabolism of dopamine synthesized from exogenous l -DOPA. Extracellular concentrations of dopamine and its catabolite, 3,4-dihydroxyphenylacetic acid, were monitored with in vivo dialysis in striata of intact rats and of rats with unilateral 6-hydroxydopamine lesions of striatal dopamine. Clorgyline (2 mg/kg), an inhibitor of monoamine oxidase A, significantly increased dopamine and decreased 3,4-dihydroxyphenylacetic acid in intact but not in dopamine-depleted striata. Inhibition of monoamine oxidase B with either l -deprenyl (1 mg/kg) or Ro 19-6327 (1 mg/kg) did not significantly affect dopamine or 3,4-dihydroxyphenylacetic acid in striata of intact or dopamine-depleted rats. In intact rats, administration of clorgyline in conjunction with l -DOPA produced a >20-fold increase in dopamine and prevented the l -DOPA-induced increase in 3,4-dihydroxyphenylacetic acid. Although both l -deprenyl and Ro 19-6327 administered in combination with l -DOPA elicited a small but significant increase in dopamine, levels of 3,4-dihydroxyphenylacetic acid were not affected. In rats pretreated with 6-hydroxydopamine, clorgyline had no significant effect on the increases in dopamine and 3,4-dihydroxyphenylacetic acid elicited by l -DOPA. Furthermore, neither l -deprenyl nor Ro 19-6327 affected l -DOPA-induced increases in dopamine and 3,4-dihydroxyphenylacetic acid in dopamine-depleted striata. The present findings indicate that deamination by monoamine oxidase A is the primary mechanism for catabolism of striatal dopamine, both under basal conditions and after administration of exogenous l -DOPA. Loss of dopaminergic terminals eliminates this action of monoamine oxidase A but does not enhance deamination by monoamine oxidase B. These data support a model in which exogenous l -DOPA elicits enhanced extracellular accumulation of dopamine in the dopamine-depleted striatum because some transmitter synthesis occurs at nondopaminergic sites and the dopamine terminals that normally take up and catabolize this pool of transmitter are absent.     

Biotransformation of l-DOPA to Dopamine in the Substantia Nigra of Freely Moving Rats: Effect of Dopamine Receptor Agonists and Antagonists

Abstract: We investigated the effects of continuous intranigral perfusion of dopamine D1 and D2 receptor agonists and antagonists on the biotransformation of locally applied l -DOPA to dopamine in the substantia nigra of freely moving rats by means of in vivo microdialysis. The "dual-probe" mode was used to monitor simultaneously changes in extracellular dopamine levels in the substantia nigra and the ipsilateral striatum. Intranigral perfusion of 10 µ M l -DOPA for 20 min induced a significant 180-fold increase in extracellular nigral dopamine level. No effect of the intranigral l -DOPA administration was observed on dopamine levels in the ipsilateral striatum, suggesting a tight control of extracellular dopamine in the striatum after enhanced nigral dopamine levels. Continuous nigral infusion with the D1 receptor agonist CY 208243 (10 µ M ) and with the D2 receptor agonist quinpirole at 10 µ M (a nonselective concentration) attenuated the l -DOPA-induced increase in dopamine in the substantia nigra by 85 and 75%, respectively. However, perfusion of the substantia nigra with a lower concentration of quinpirole (1 µ M ) and the D1 antagonist SCH 23390 (10 µ M ) did not affect the nigral l -DOPA biotransformation. The D2 antagonist (−)-sulpiride (10 µ M ) also attenuated the l -DOPA-induced dopamine release in the substantia nigra to ∼10% of that of the control experiments. We confirm that there is an important biotransformation of l -DOPA to dopamine in the substantia nigra. The high concentrations of dopamine formed after l -DOPA administration may be the cause of dyskinesias or further oxidative stress in Parkinson's disease. Simultaneous administration of D1 receptor agonists with l -DOPA attenuates the biotransformation of l -DOPA to dopamine in the substantia nigra. The observed effects could occur via changes in nigral GABA release that in turn influence the firing rate of the nigral dopaminergic neurons.     

l-DOPA Inhibits Complex IV of the Electron Transport Chain in Catecholamine-Rich Human Neuroblastoma NB69 Cells

Abstract: l -3,4-Dihydroxyphenylalanine ( l -DOPA) is toxic for human neuroblastoma cells NB69 and its toxicity is related to several mechanisms including quinone formation and enhanced production of free radicals related to the metabolism of dopamine via monoamine oxidase type B. We studied the effect of l -DOPA on activities of enzyme complexes in the electron transport chain (ETC) in homogenate preparations from the human neuroblastoma cell line NB69. As a preliminary step we compared the activity of ETC in cellular homogenates with that of purified mitochondria from NB69 cells and rat brain. Specific activities for complex I, complex II–III, and complex IV in NB69 cells were, respectively, 65, 96, and 32% of those in brain mitochondria. Complex I activity was inhibited in a dose-dependent way by 1-methyl-4-phenylpyridinium ion with an EC₅₀ of ∼150 µ M . Treatment with 0.25 m M l -DOPA for 5 days reduces complex IV activity to 74% of control values but does not change either complex I or citrate synthase. Ascorbic acid (1 m M ), which protects NB69 cells from l -DOPA-induced neurotoxicity, increases complex IV activity to 133% of the control and does not change other ETC complexes. Ascorbic acid also reverses l -DOPA-induced reduction of complex IV activity in NB69 cells. This observation might indicate that the protection observed with ascorbic acid is related to complex IV activation. In vitro incubation with l -DOPA (0.125–4 m M ) for 2 min produced a dose-dependent reduction of complex IV without change in complex I and II–III activities.     

l-DOPA activates ERK signaling and phosphorylates histone H3 in the striatonigral medium spiny neurons of hemiparkinsonian mice

In the dopamine-depleted striatum, extracellular signal-regulated kinase (ERK) signaling is implicated in the development of l -DOPA-induced dyskinesia. To gain insights on its role in this disorder, we examined the effects of l -DOPA on the state of phosphorylation of ERK and downstream target proteins in striatopallidal and striatonigral medium spiny neurons (MSNs). For this purpose, we employed mice expressing enhanced green fluorescent protein (EGFP) under the control of the promoters for the dopamine D₂ receptor ( Drd2 -EGFP mice) or the dopamine D₁ receptor ( Drd1a -EGFP mice), which are expressed in striatopallidal and striatonigral MSNs, respectively. In 6-hydroxydopamine-lesioned Drd2 -EGFP mice, l -DOPA increased the phosphorylation of ERK, mitogen- and stress-activated kinase 1 and histone H3, selectively in EGFP-negative MSNs. Conversely, a complete co-localization between EGFP and these phosphoproteins was observed in Drd1a -EGFP mice. The effect of l -DOPA was prevented by blockade of dopamine D₁ receptors. The same pattern of activation of ERK signaling was observed in dyskinetic mice, after repeated administration of l -DOPA. Our results demonstrate that in the dopamine-depleted striatum, l -DOPA activates ERK signaling specifically in striatonigral MSNs. This regulation may result in ERK-dependent changes in striatal plasticity leading to dyskinesia.     

Neurotrophic Effects of l-DOPA in Postnatal Midbrain Dopamine Neuron/Cortical Astrocyte Cocultures

Abstract: l -DOPA is toxic to catecholamine neurons in culture, but the toxicity is reduced by exposure to astrocytes. We tested the effect of l -DOPA on dopamine neurons using postnatal ventral midbrain neuron/cortical astrocyte cocultures in serum-free, glia-conditioned medium. l -DOPA (50 µ M ) protected against dopamine neuronal cell death and increased the number and branching of dopamine processes. In contrast to embryonically derived glia-free cultures, where l -DOPA is toxic, postnatal midbrain cultures did not show toxicity at 200 µ M l -DOPA. The stereoisomer d -DOPA (50–400 µ M ) was not neurotrophic. The aromatic amino acid decarboxylase inhibitor carbidopa (25 µ M ) did not block the neurotrophic effect. These data suggest that the neurotrophic effect of l -DOPA is stereospecific but independent of the production of dopamine. However, l -DOPA increased the level of glutathione. Inhibition of glutathione peroxidase by l -buthionine sulfoximine (3 µ M for 24 h) blocked the neurotrophic action of L-DOPA. N -Acetyl- l -cysteine (250 µ M for 48 h), which promotes glutathione synthesis, had a neurotrophic effect similar to that of l -DOPA. These data suggest that the neurotrophic effect of l -DOPA may be mediated, at least in part, by elevation of glutathione content.     

Are cyclooxygenase-2 and nitric oxide involved in the dyskinesia of Parkinson's disease induced by l-DOPA?

Inflammatory mechanisms are proposed to play a role in l-DOPA-induced dyskinesia. Cyclooxygenase-2 (COX2) contributes to inflammation pathways in the periphery and is constitutively expressed in the central nervous system. Considering that inhibition of nitric oxide (NO) formation attenuates l-DOPA-induced dyskinesia, this study aimed at investigating if a NO synthase (NOS) inhibitor would change COX2 brain expression in animals with l-DOPA-induced dyskinesia. To this aim, male Wistar rats received unilateral 6-hydroxydopamine microinjection into the medial forebrain bundle were treated daily with l-DOPA (21 days) combined with 7-nitroindazole or vehicle. All hemi-Parkinsonian rats receiving l-DOPA showed dyskinesia. They also presented increased neuronal COX2 immunoreactivity in the dopamine-depleted dorsal striatum that was directly correlated with dyskinesia severity. Striatal COX2 co-localized with choline-acetyltransferase, calbindin and DARPP-32 (dopamine-cAMP-regulated phosphoprotein-32), neuronal markers of GABAergic neurons. NOS inhibition prevented l-DOPA-induced dyskinesia and COX2 increased expression in the dorsal striatum. These results suggest that increased COX2 expression after l-DOPA long-term treatment in Parkinsonian-like rats could contribute to the development of dyskinesia.     

The Roles of Striatal Serotonin and <Emphasis Type="SmallCaps">l</Emphasis>-Amino-acid Decarboxylase on <Emphasis Type="SmallCaps">l</Emphasis>-DOPA-induced Dyskinesia in a Hemiparkinsonian Rat Model

The administration of l-DOPA is the standard treatment for Parkinson’s disease (PD). However, the symptomatic relief provided by long-term administration may be compromised by l-DOPA-induced dyskinesia (LID) that presents as adverse fluctuations in motor responsiveness and progressive loss of motor control. In the later stages of PD, raphestriatal serotonin neurons compensate for the loss of nigrostriatal dopamine (DA) neurons by converting and releasing DA derived from exogenous l-DOPA. Since the serotonin system does not have an autoregulatory mechanism for DA, raphe-mediated striatal DA release may fluctuate dramatically and precede the development of LID. The 6-hydroxydopamine lesioned rats were treated with l-DOPA (6 mg/kg) and benserazide (15 mg/kg) daily for 3 weeks to allow for the development of abnormal involuntary movement score (AIMs). In rats with LID, chronic treatment with l-DOPA increased striatal DA levels compared with control rats. We also observed a relative increase in the expression of striatal l-amino-acid decarboxylase (AADC) in LID rats, even though tyrosine hydroxylase (TH) expression did not increase. The administration of l-DOPA also increased striatal serotonin immunoreactivity in LID rats compared to control rats. Striatal DA and 5-hydroxytryptamine (5-HT) levels were negatively correlated in l-DOPA-treated rats. These results of this study reveal that 5-HT contributes to LID. Striatal DA positively influences LID, while 5-HT is negatively associated with LID. Finally, we suggest that by strategic modification of the serotonin system it may be possible to attenuate the adverse effects of chronic l-DOPA therapy in PD patients.     

Altered expression and subcellular distribution of GRK subtypes in the dopamine-depleted rat basal ganglia is not normalized by l-DOPA treatment

Dysregulation of dopamine (DA) receptors is believed to underlie Parkinson's disease pathology and l -DOPA-induced motor complications. DA receptors are subject to regulation by G protein-coupled receptor kinases (GRKs) and arrestins. DA lesion with 6-hydroxydopamine caused multiple protein- and brain region-specific changes in the expression of GRKs. In the globus pallidus, all four GRK isoforms (GRK2, 3, 5, 6) were reduced in the lesioned hemisphere. In the caudal caudate-putamen (cCPu) three GRK isoforms (GRK2, 3, 6) were decreased by DA depletion. The decrease in GRK proteins in globus pallidus, but not cCPu, was mirrored by reduction in mRNA. GRK3 protein was reduced in the rostral caudate-putamen (rCPu), whereas other isoforms were either unchanged or up-regulated. GRK6 protein and mRNA were up-regulated in rCPu and nucleus accumbens. l -DOPA (25 mg/kg, twice daily for 10 days) failed to reverse changes caused by DA depletion, whereas D₂/D₃ agonist pergolide (0.25 mg/kg daily for 10 days) restored normal levels of expression of GRK5 and 6. In rCPu, GRK2 protein was increased in most subcellular fractions by l -DOPA but not by DA depletion alone. Similarly, l -DOPA up-regulated arrestin3 in membrane fractions in both regions. GRK5 was down-regulated by l -DOPA in cCPu in the light membrane fraction, where this isoform is the most abundant. The data suggest that alterations in the expression and subcellular distribution of arrestins and GRKs contribute to pathophysiology of Parkinson's disease. Thus, these proteins may be targets for antiparkinsonian therapy.     

Effects of the Serotonin 5-HT<Subscript>1A</Subscript> Receptor Biased Agonists,F13714 and F15599, on Striatal Neurotransmitter Levels Following <Emphasis Type="SmallCaps">l</Emphasis>-DOPA Administration in Hemi-Parkinsonian Rats

Peak-dose dyskinesia is associated with the dramatic increase in striatal dopamine levels that follows l-DOPA administration. The ‘false neurotransmitter’ hypothesis postulates that the latter is likely due to an aberrant processing of l-DOPA by serotonergic neurons. In keeping with this hypothesis, two highly selective ‘biased agonists’ of 5-HT_1A receptors—namely F13714 and F15599 (NLX-101)—were recently shown to exhibit exceptionally potent anti-dyskinetic activity without impairing l-DOPA therapeutic properties despite their differential targeting of 5-HT_1A receptor sub-populations. In this study, we investigated whether these two compounds dampened peak l-DOPA-induced dopamine microdialysate levels in the striatum of hemi-parkinsonian rats. Acute administration of either F13714 (0.04 and 0.16 mg/kg i.p.) or F15599 (0.16 and 0.64 mg/kg, i.p.) blunted l-DOPA (2 mg/kg)-induced increases in dopamine microdialysate levels in the denervated striatum (following unilateral injection of 6-OHDA into the medial forebrain bundle). No significant changes were observed on the intact side of the brain. Concurrently, both drugs profoundly reduced striatal serotonin levels on both sides of the brain. In addition, F13714 and F15599, in the presence of l-DOPA, produced a dose-dependent increase in glutamate levels, but this effect was restricted to later time points. These finding support the interpretation that F13714 and F15599 mediate their anti-dyskinetic effects by blunting of the peak in dopamine levels via activation of somatodendritic serotonin 5-HT_1A receptors and the consequent inhibition of serotonergic neurons. This study adds to the growing body of evidence supporting the development of a potent 5-HT_1A receptor agonist for treatment of peak-dose dyskinesia.     

Chronic intermittent L-DOPA treatment induces changes in dopamine release

3,4-Dihydroxyphenyl- l -alanine (l- DOPA)-induced dyskinesia often develops as a side effect of chronic l -DOPA therapy. This study was undertaken to investigate dopamine (DA) release upon l -DOPA treatment. Chronoamperometric measurements were performed in unilaterally DA-depleted rats, chronically treated with l -DOPA, resulting in dyskinetic and non-dyskinetic animals. Normal and lesioned l -DOPA naïve animals were used as controls. Potassium-evoked DA releases were significantly reduced in intact sides of animals undertaken chronic l -DOPA treatment, independent on dyskinetic behavior. Acute l -DOPA further attenuated the amplitude of the DA release in the control sides. In DA-depleted striata, no difference was found in potassium-evoked DA releases, and acute l -DOPA did not affect the amplitude. While immunoreactivity to serotonin uptake transporter was higher in lesioned striata of animals displaying dyskinetic behavior, no correlation could be documented between serotonin transporter-positive nerve fiber density and the amplitude of released DA. In conclusions, the amplitude of potassium-evoked DA release is attenuated in intact striatum after chronic intermittent l -DOPA treatment. No change in amplitude was found in DA-denervated sides of either dyskinetic or non-dyskinetic animals, while release kinetics were changed. This indicates the importance of studying DA release dynamics for the understanding of both beneficial and adverse effects of l -DOPA replacement therapy.     

Effects of Wild-Type and Mutated Copper/Zinc Superoxide Dismutase on Neuronal Survival and l-DOPA-Induced Toxicity in Postnatal Midbrain Culture

Abstract: Mutations in the free radical-scavenging enzyme copper/zinc superoxide dismutase (Cu/Zn-SOD) are associated with neuronal death in humans and mice. Here, we examine the effects of human wild-type (WT SOD) and mutant (Gly⁹³→ Ala; G93A) Cu/Zn-SOD enzyme on the fate of postnatal midbrain neurons. One-week-old cultures from transgenic mice expressing WT SOD enzyme had significantly more midbrain neurons and fewer necrotic and apoptotic neurons than non-transgenic cultures. In contrast, 1-week-old cultures from transgenic G93A mice expressing mutant SOD enzyme had significantly fewer midbrain neurons and more necrotic and apoptotic neurons than nontransgenic cultures. To subject postnatal midbrain neurons to oxidative stress, cultures were incubated with l -DOPA. l -DOPA at 200 µ M caused ∼50% loss of tyrosine hydroxylase (TH)-positive neurons in nontransgenic cultures and even greater loss in transgenic G93A cultures; no alterations were noted in GABA neuron numbers. In contrast, 200 µ M l -DOPA did not cause any significant reductions in TH-positive or GABA neuron numbers in transgenic WT SOD cultures. l -DOPA at 50 µ M had opposite effects, in that it significantly increased TH-positive, but not GABA neuron numbers in transgenic WT SOD and G93A and in nontransgenic cultures. These results indicate that increased amounts of WT SOD enzyme promote cell survival and protect against l -DOPA-induced dopaminergic neurotoxicity, whereas increased amounts of mutated Cu/Zn-SOD enzyme have inverse effects. As the spontaneous loss and l -DOPA-induced loss of postnatal dopaminergic midbrain neurons appear to be mediated by free radicals, our study supports the view that mutated Cu/Zn-SOD enzyme kills cells by oxidative stress.     

Intrastriatal Grafting of Cos Cells Stably Expressing Human Aromatic l-Amino Acid Decarboxylase: Neurochemical Effects

Abstract: To study the possibility that increasing striatal activity of aromatic l -amino acid decarboxylase (AADC; EC 4.1.1.28) can increase dopamine production in dopamine denervated striatum in response to l -3,4-dihydroxyphenylalanine ( l -DOPA) administration, we grafted Cos cells stably expressing the human AADC gene (Cos- haadc cells) into 6-hydroxydopamine denervated rat striatum. Before grafting, the catalytic activity of the enzyme was assessed in vitro via the generation of ¹⁴CO₂ from l -[¹⁴C]DOPA. The K _m value for l -DOPA in intact and disrupted cells was 0.60 and 0.56 m M , respectively. The cofactor, pyridoxal 5-phosphate, enhanced enzymatic activity with maximal effect at 0.1 m M . The pH optimum for enzyme activity was 6.8. Grafting Cos- haadc cells into denervated rat striatum enhanced striatal dopamine levels measured after systemic administration of l -DOPA. When measured 2 h after l -DOPA administration, the mean dopamine level in the striata of Cos- haadc -grafted animals was 2 µg/g of tissue, representing 31% of normal striatal dopamine concentration. The mean dopamine concentration in the striata grafted with untransfected Cos cells (Cos-ut cells) was 1 µg/g. At 6–8 h after l -DOPA administration, striatal dopamine content in the Cos- haadc -grafted animals was 0.67 µg/g of tissue weight, representing 9% of intact striatum dopamine content. By contrast, the average dopamine content in the Cos-ut-grafted animals was undetectable. These findings demonstrate that enhancing striatal AADC activity can improve dopamine bioformation in response to systemically administered l -DOPA.     

Iron deficiency alters dopamine uptake and response to L-DOPA injection in Sprague-Dawley rats

Iron deficiency (ID) disrupts brain dopamine (DA) and norepinephrine (NE) metabolism including functioning of monoamine transporters and receptors. We employed caudate microdialysis and no net flux (NNF) in post-weaning rats to determine if ID decreased the extraction fraction ( E _d). Five micromolar quinpirole, a dopamine D₂ receptor agonist, resulted in 80% decrease in extracellular DA and 45% higher E _d in control animals. The D₂ agonist had no effect on E _d in ID animals despite a reduction in basal DA. DAT mRNA levels were reduced by 58% with ID, while DAT protein in ventral midbrain and caudate and membrane associated DAT were also reduced by ID. Carbidopa/ l -DOPA was administered to determine if elevated extracellular DA in ID was due to increased release. The DA response to l -DOPA in ID rats was 50% smaller and delayed, whereas the NE response was threefold higher. The caudate concentration of NE was also elevated in ID. Elevated dopamine-β-hydroxylase activity in ID provides a tentative explanation for the increased NE response to l -DOPA. These experiments provide new evidence that ID results in altered synthesis and functioning of DAT and perhaps suggests some compensatory changes in NE metabolism.     

THE PROPORTIONATE LOSS OF l-3,4-DIHYDROXYPHENYLALANINE AND l-5-HYDROXYTRYPTOPHAN DECARBOXYLATING ACTIVITY IN RAT CENTRAL NERVOUS SYSTEM FOLLOWING INTRACISTERNAL ADMINISTRATION OF 5,6-DIHYDROXYTRYPTAMINE OR 6-HYDROXYDOPAMINE

—5,6-Dihydroxytryptamine or 6-hydroxydopamine was administered intracisternally to rats to effect a selective destruction of serotonin or catecholamine-containing neurons. The l -DOPA and l -5-hydroxytryptophan decarboxylating activities of the spinal cord and brain were then determined at several time intervals following this treatment. In both cases the relative loss of l -DOPA decarboxylating activity was the same as the relative loss of l -5-hydroxytryptophan decarboxylating activity. 5,6-Dihydroxytryptamine treatment had little or no effect on catecholamine-containing neurons and 6-hydroxydopamine did not effect serotonin-containing neurons. These data support the idea that only one decarboxylase is involved in the biosynthesis of both serotonin and catecholamines in the rat CNS.     

l-DOPA Cytotoxicity to PC12 Cells in Culture Is via Its Autoxidation

Abstract: The mechanism of cytotoxicity of l -DOPA was studied in the rat pheochromocytoma PC12 cell line. The cytotoxicity of l -DOPA to PC12 cells was time and concentration dependent. Carbidopa, which inhibited the conversion of l -DOPA to dopamine, did not protect against l -DOPA cytotoxicity in PC12 cells. Furthermore, clorgyline, a selective inhibitor of monoamine oxidase type A, and pargyline, an inhibitor of both monoamine oxidase types A and B, both did not have an effect on l -DOPA toxicity. These findings suggest that cytotoxicity was not due to dopamine formed from l -DOPA. Catalase or superoxide dismutase each partially protected against l -DOPA toxicity in PC12 cells. In combination, the effects were synergistic and provided almost total protection against cytotoxicity. 6-Cyano-7-nitroquinoxaline-2,3-dione, an antagonist of non-NMDA receptors, did not protect against l -DOPA toxicity. These data suggest that toxicity of l -DOPA is most likely due to the action of free radicals formed as a result of its autoxidation. Furthermore, these findings suggest that patients on long-term l -DOPA therapy are potentially at risk from the toxic intermediates formed as a result of its autoxidation.     

THE EFFECT OF l-DOPA AND AN INHIBITOR OF PERIPHERAL DECARBOXYLATION ON GLUCOSE METABOLISM IN BRAIN1

Abstract– The effect of the administration of l -DOPA plus an inhibitor of peripheral l -aromatic amino acid decarboxylase (aromatic-l -amino-acid carboxy-lyase; EC 4.1.1.28) on the metabolism of glucose in brain was studied by administering [U-^I4C]glucose (20μCi) to three groups of rats: (1) rats that had been injected with l -DOPA (200mg/kg) 28min earlier; (2) rats that had been similarly injected with l -DOPA and also with N-(d,l -seryl)-N′-(2,3,4-trihydroxybenzyl)hydrazine (50 mg/kg), an inhibitor of l -aromatic amino acid decarboxylase, 30min before the l -DOPA; and (3) appropriate controls. The flux of ¹⁴C from glucose in plasma to those amino acids that are in equilibrium with the tricarboxylic acid cycle intermediates was reduced by treatment with l -DOPA and reduced further by treatment with l -DOPA and the decarboxylase inhibitor. Concentrations of glucose in brain and in plasma were increased after treatment with l -DOPA; these increases were attenuated if the inhibitor was given before the l -DOPA. After treatment with l -DOPA, there were decreases in the concentration of aspartate, tryptophan, and tyrosine in brain. After the administration of l -DOPA and the decarboxylase inhibitor, the concentrations in brain of alanine, glutamate, tyrosine, and phenylalanine were greater, and the concentrations of aspartate, leucine, lysine, histidine, arginine, and tryptophan were less than in control rats.     

The effect of L-3,4-dihydroxyphenylalanine administration on glucose metabolism in brain

The effect of the administration of l -3,4-dihydroxyphenylalanine (l -DOPA) on the metabolism of glucose in brain was studied by administering [U-¹⁴C]glucose to three groups of rats: (1) those injected previously with l -DOPA, 100 mg/kg; (2) those fed 1 % (w/w) l -DOPA in their diet for several months and also injected 15 min before the administration of glucose with l -DOPA, 100 mg/kg; and (3) appropriate controls. Chronic treatment with l -DOPA caused a decrease in the flux of carbon from glucose in plasma to those amino acids in brain that are in equilibrium with the tricarboxylic acid cycle intermediates but not to lactate and alanine. Similar differences from controls, but of smaller magnitude, were observed in rats given a single injection of l -DOPA. Concentrations of glucose in plasma and in brain were increased after acute or chronic treatment with l -DOPA. A single injection of l -DOPA did not cause changes in the levels of the most abundant amino acids in brain, but after chronic treatment with l -DOPA modest changes were noted in the brain levels of some ninhydrin-reacting substances; the contents of taurine and aspartate were lower and those of threonine, serine, glutamine, and glycine were higher.     

l-3,4-Dihydroxyphenylalanine Increases the Quantal Size of Exocytotic Dopamine Release In Vitro

Abstract: The catecholamine precursor l -3,4-dihydroxyphenylalanine ( l -DOPA) is used to augment striatal dopamine (DA), although its mechanism of altering neurotransmission is not well understood. We observed the effects of l -DOPA on catecholamine release in ventral midbrain neuron and PC12 pheochromocytoma cell line cultures. In ventral midbrain neuron cultures exposed to 40 m M potassium-containing media, l -DOPA (100 µ M for 1 h) increased DA release by >10-fold. The elevated extracellular DA levels were not significantly blocked by the DA/norepinephrine transport inhibitor nomifensine, demonstrating that reverse transport through catecholamine-uptake carriers plays little role in this release. In PC12 cells, where DA release from individual secretory vesicles can be observed, l -DOPA (50 µ M for 1 h) elevated DA release in high-potassium media by 370%. Amperometric measurements demonstrated that l -DOPA (50 µ M for 40–70 min) did not raise the frequency of vesicular exocytosis but increased the average size of quantal release to at least 250% of control levels. Together, these findings suggest that l -DOPA can increase stimulation-dependent transmitter release from DA cells by augmenting cytosolic neurotransmitter, leading to increased quantal size.     

Intranasal Administration of the Dopaminergic Agonists l-DOPA, Amphetamine, and Cocaine Increases Dopamine Activity in the Neostriatum: A Microdialysis Study in the Rat

Abstract: The effectiveness of intranasal drug administration to stimulate central neuronal systems is well known from drug addiction and has also been considered as an alternative pharmacokinetic approach to treat brain disorders such as Parkinson's disease. In the present study, the possible neurochemical effects of intranasal administration of the psychostimulants cocaine and amphetamine and of the antiparkinsonian drug l -DOPA were analyzed. By using in vivo microdialysis in the urethane-anesthetized rat, it was found that unilateral intranasal administration of either of the psychostimulants led to huge and rapid increases of extracellular dopamine levels in the neostriatum followed by decreases of its metabolites dihydroxyphenylacetic acid and homovanillic acid. Furthermore, intranasal administration of l -DOPA, but not of the saline vehicle, also led to increased extracellular levels of neostriatal dopamine and to increases of its metabolites. Because the effect of intranasal l -DOPA on neostriatal dopamine was observed only ipsilaterally but not contralaterally to the side of intranasal drug administration, it can be hypothesized that l -DOPA was not effective via passage through the circulation but may have acted through a neuronal or an extraneuronal route. These data provide neurochemical evidence that the intranasal route may not only be efficient in drug abuse, but may also be useful to target the brain therapeutically, as in the case of neurodegenerative brain disorders.     

Role of Aromatic l-Amino Acid Decarboxylase for Dopamine Replacement by Genetically Modified Fibroblasts in a Rat Model of Parkinson's Disease

Abstract: Investigations of gene therapy for Parkinson's disease have focused primarily on strategies that replace tyrosine hydroxylase. In the present study, the role of aromatic l -amino acid decarboxylase in gene therapy with tyrosine hydroxylase was examined by adding the gene for aromatic l -amino acid decarboxylase to our paradigm using primary fibroblasts transduced with both tyrosine hydroxylase and GTP cyclohydrolase I. We compared catecholamine synthesis in vitro in cultures of cells with tyrosine hydroxylase and aromatic l -amino acid decarboxylase together versus cocultures of cells containing these enzymes separately. l -DOPA and dopamine levels were higher in the cocultures that separated the enzymes. To determine the role of aromatic l -amino acid decarboxylase in vivo, cells containing tyrosine hydroxylase and GTP cyclohydrolase I were grafted alone or in combination with cells containing aromatic l -amino acid decarboxylase into the 6-hydroxydopamine-denervated rat striatum. Grafts containing aromatic l -amino acid decarboxylase produced less l -DOPA and dopamine as monitored by microdialysis. These findings indicate that not only is there sufficient aromatic l -amino acid decarboxylase near striatal grafts producing l -DOPA, but also the close proximity of the enzyme to tyrosine hydroxylase is detrimental for optimal dopamine production. This is most likely due to feedback inhibition of tyrosine hydroxylase by dopamine.