Occurrence of L-DOPA and dopamine in plants and cell cultures ofMucuna pruriens and effects of 2,4-d and NaCl on these compounds

The development of the L-DOPA-content of roots, stems and leaves ofMucuna pruriens during growth of the plants is described. Besides L-DOPA, the leaves, but not the stems and the roots, also contain the related catechol dopamine. The time course of dopamine accumulation is compared to that of L-DOPA. In cell suspension cultures ofM. pruriens dopamine can be detected as well. Its level is strongly increased by addition of the growth regulator 2,4-d to the medium, a condition that suppresses cell growth and L-DOPA-accumulation. Dopamine induction appears to be a specific metabolic effect of 2,4-d. Salt stress, as caused by the addition of NaCl, gives no induction of dopamine formation, whereas L-DOPA is released into the medium.     

Further studies of dopamine metabolism and function in Tetrahymena

The large amounts of dopamine accumulated by cells of Tetrahymena pyriformis strain NT-1 and secreted into their growth medium were found to depend primarily upon an extracellular, non-enzymatic conversion of tyrosine to L-dihydroxyphenylalanine (L-DOPA); L-DOPA was then rapidly taken into the cells and transformed into dopamine enzymatically. Efforts to find physiologically significant dopamine binding sites on the cell surface or dopamine-sensitive adenylate cyclase activity were unsuccessful, suggesting that the catecholamine does not function in Tetrahymena as it does in higher animals.     

Neuroprotective Mechanisms of Antiparkinsonian Dopamine D2-Receptor Subfamily Agonists

Numerous studies have shown that endogenous and/or environmental neurotoxins and oxidative stress may participate in the pathogenesis of Parkinson's disease (PD), but the detailed mechanisms are still unclear. While dopamine (DA) replacement therapy with L-DOPA (levodopa) improves PD symptoms, it does not inhibit the degeneration of DA neurons in the substantia nigra. Recently, bromocriptine, pramipexole and several other agonists of the dopamine D₂-receptor subfamily (including D₂, D₃ and D₄-subtypes) have been shown to have neuroprotective effects in parkinsonian models in vitro and in vivo. Their neuroprotective effects may be mediated directly and/or indirectly by antioxidant effects, mitochondrial stabilization or induction of the antiapoptotic Bcl-2 family.     

Dopamine transporter binding is unaffected by L-DOPA administration in normal and MPTP-treated monkeys

Background

Radiotracer imaging of the presynaptic nigrostriatal dopaminergic system is used to assess disease progression in Parkinson''s disease (PD) and may provide a useful adjunct to clinical assessment during therapeutic trials of potential neuroprotective agents. Several clinical trials comparing dopamine agonists to L-DOPA or early vs. late L-DOPA have revealed differences between clinical assessment and imaging of the presynaptic dopaminergic system, hence questioning the comparability of these measures as neuroprotection outcome variables. Thus, results of these studies may have been affected by factors other than the primary biological process investigated.

Methodology/Principal Findings

We tested the possibility that L-DOPA might interfere with DAT binding. Post-mortem DAT binding was conducted in normal and MPTP-treated macaque monkeys that were administered L-DOPA, acutely or chronically. In parallel, DAT SPECT was conducted in MPTP-treated animals that were administered chronic L-DOPA. [99mTc]TRODAT-1 SPECT binding was similarly reduced in all MPTP monkeys regardless of L-DOPA treatment. L-DOPA had no significant effect on post-mortem DAT binding either in saline or in MPTP-lesioned animals.

Conclusions/Significance

These data indicate that L-DOPA does not induce modifications of DAT expression detectable by SPECT of by DAT binding autoradiography, suggesting that differences between clinical assessment and radiotracer imaging in clinical trials may not be specifically related to L-DOPA treatment.     

Antiparkinsonian Effects of Aqueous Methanolic Extract of Hyoscyamus niger Seeds Result From its Monoamine Oxidase Inhibitory and Hydroxyl Radical Scavenging Potency

Hyoscyamus species is one of the four plants used in Ayurveda for the treatment of Parkinson’s disease (PD). Since Hyoscyamus niger was found to contain negligible levels of L-DOPA, we evaluated neuroprotective potential, if any, of characterized petroleum ether and aqueous methanol extracts of its seeds in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of PD in mice. Air dried authenticated H. niger seeds were sequentially extracted using petroleum ether and aqueous methanol and were characterized employing HPLC-electrochemistry and LCMS. Parkinsonian mice were treated daily twice with the extracts (125–500 mg/kg, p.o.) for two days and motor functions and striatal dopamine levels were assayed. Administration of the aqueous methanol extract (containing 0.03% w/w of L-DOPA), but not petroleum ether extract, significantly attenuated motor disabilities (akinesia, catalepsy and reduced swim score) and striatal dopamine loss in MPTP treated mice. Since the extract caused significant inhibition of monoamine oxidase activity and attenuated 1-methyl-4-phenyl pyridinium (MPP⁺)-induced hydroxyl radical (·OH) generation in isolated mitochondria, it is possible that the methanolic extract of Hyoscyamus niger seeds protects against parkinsonism in mice by means of its ability to inhibit increased ·OH generated in the mitochondria.     

Chemical lesion and drug induced supersentivity and subsensitivity of caudate dopamine receptors

In order to elucidate the mechanism of denervation supersensitivity, the effects of 6-hydroxydopamine lesion, placed in the substantia nigra, were examined on rat brain caudate adenylate cyclase and ³H-haloperidol binding to membrane dopamine receptors. In addition, the effects of chronic administration of L-DOPA, bromocriptine and piribedil were also investigated on ³H-haloperidol binding and dopamine, K⁺ isoproterenol (IPNE) and 2-Cl-adenosine stimulated formation of cyclic AMP in caudate slices. 6-Hydroxydopamine lesions resulted in significantly greater stimulation of adenylate cyclase by dopamine at various concentrations tested. The haloperidol binding sites were increased by 28% on lesioned side caudate without changes in dissociation constants (K_D). Three weeks after treatment with L-DOPA, bromocriptine or piribedil, the ³H-haloperidol binding sites were decreased by 40% with no change in K_D. The stimulatory effect of dopamine on cyclic AMP formation was also abolished, although there was no change in IPNE, K⁺, or 2-Cl-adenosine stimulated cyclic AMP formation in caudate slices, suggesting a specific effect of dopamine agonists on dopamine receptors. The results of these studies suggest a close relationship between at least some populations of dopamine receptors and adenylate cyclase in the caudate nucleus.     

A site-specific mutation of tyrosine hydroxylase reduces feedback inhibition by dopamine in genetically modified cells grafted in parkinsonian rats

Aromatic L-amino acid decarboxylase (AADC) is necessary for conversion of L-DOPA to dopamine. Therefore, AADC gene therapy has been proposed to enhance pharmacological or gene therapies delivering L-DOPA. However, addition of AADC to the grafts of genetically modified cells expressing tyrosine hydroxylase (TH) and GTP cyclohydrolase 1 (GCH1), which produce L-DOPA in parkinsonian rats, resulted in decreased production of L-DOPA and dopamine owing to feedback inhibition of TH by dopamine. End-product feedback inhibition has been shown to be mediated by the regulatory domain of TH, and site-specific mutation of serine 40 makes TH less susceptible to dopamine inhibition. Therefore, we investigated the efficacy of using TH with serine 40 mutated to leucine (mTH) in an ex vivo gene-therapy paradigm. Primary fibroblasts (PF) from Fischer 344 rats were transduced with retrovirus to express mTH or wild-type rat TH cDNA (wtTH). Both cell types were also transduced with GCH1 to provide the obligate TH cofactor, tetrahydrobiopterin. PF transfected with AADC were used as coculture and cografting partners. TH activities and L-DOPA production in culture were comparable between PFwtTHGC and PFmTHGC cells. In cocultures with PFAADC cells, PFmTHGC cells showed significant reduction in the inhibitory effect of dopamine compared with PFwtTHGC cells. In vivo microdialysis measurement showed that cografting PFAADC cells with PFmTHGC cells resulted in smaller decreases in L-DOPA and no reduction in dopamine levels compared with cografts of PFAADC cells with PFwtTHGC cells, which decreased both L-DOPA and dopamine levels. Maintenance of dopamine levels with lower levels of L-DOPA would result in more focused local delivery of dopamine and less potential side-effects arising from L-DOPA diffusion into other structures. These data support the hypothesis that mutation of serine 40 attenuates TH end-product inhibition in vivo and illustrates the importance of careful consideration of biochemical pathways and interactions between multiple genes in gene therapy.     

Adeno-associated virus-mediated gene transfer of human aromatic L-amino acid decarboxylase protects mixed striatal primary cultures from L-DOPA toxicity

Although L-DOPA is the drug of choice for Parkinson's disease, prolonged L-DOPA therapy results in decreased drug effectiveness and the appearance of motor complications. This may be due in part to the progressive loss of the enzyme, aromatic L-amino acid decarboxylase (AADC). We have developed an adeno-associated virus vector (AAV-hAADC) that contains human AADC cDNA under the control of the cytomegalovirus promoter. Infusion of this vector into the striatum of parkinsonian rats and monkeys improves L-DOPA responsiveness by improving AADC-mediated conversion of L-DOPA to dopamine. This is now the basis of a proposed therapy for advanced Parkinson's disease. A key concern has been that over-production of dopamine in striatal neurons could cause dopamine toxicity. To investigate this possibility in a controlled system, mixed striatal primary rat neuronal cultures were prepared. Exposure of cultures to high concentrations of L-DOPA induced the following changes: cell death in nigral and striatal neurons, aggregation of neurofilaments and focal axonal swellings, abnormal expression of DARPP-32, and activation of astroglia and microglial cells. Transduction of cultures with AAV-hAADC resulted in efficient and sustained neuronal expression of the AADC protein and prevented all the L-DOPA-induced toxicities. The protective effects were due primarily to AADC-dependent conversion of L-DOPA to dopamine and an increase in induction of vesicular monoamine transporter resulting in dopamine storage in cultured cells. These results suggest a neuroprotective role for AADC gene transfer against L-DOPA toxicity.     

L-DOPA does not enhance hydroxyl radical formation in the nigrostriatal dopamine system of rats with a unilateral 6-hydroxydopamine lesion

The debate about the toxicity of L-DOPA to dopaminergic neurons has not been resolved. Even though enzymatic and nonenzymatic metabolism of L-DOPA can produce hydrogen peroxide and oxygen free radicals, there has been controversy as to whether L-DOPA generates an oxidant stress in vivo. This study determined whether acute or repeated administration of L-DOPA caused in vivo production of hydroxyl radicals in striatum and other brain regions in rats with a unilateral 6-hydroxydopamine lesion of the dopaminergic nigrostriatal projections. Salicylate trapping combined with in vivo microdialysis provided measurements of extracellular 2,3-dihydroxybenzoic acid (2,3-DHBA) in striatum following L-DOPA administration systemically (100 mg/kg, i.p.) or by intrastriatal perfusion (1 mM, via the microdialysis probe). Tissue concentrations of 2,3-DHBA and salicylate were also measured in striatum, ventral midbrain, and cerebellum following repeated administration of L-DOPA (50 mg/kg, i.p., once daily for 16 days). In each instance, treatment with L-DOPA did not increase 2,3-DHBA concentrations, regardless of the nigrostriatal dopamine system's integrity. When added to the microdialysis perfusion medium, L-DOPA resulted in a significant decrease in the striatal extracellular concentration of 2,3-DHBA. These results suggest that administration of L-DOPA, even at high doses, does not induce hydroxyl radical formation in vivo and under some conditions may actually diminish hydroxyl radical activity. Furthermore, prior damage to the nigrostriatal dopamine system does not appear to predispose surviving dopaminergic neurons to increased hydroxyl radical formation following L-DOPA administration. Unlike L-DOPA, systemic administration of methamphetamine (10 mg/kg, s.c.) produced a significant increase in the concentration of 2,3-DHBA in striatal dialysate, suggesting that increased formation of hydroxyl radicals may contribute to methamphetamine neurotoxicity.     

Phenoloxidase in the scallop Chlamys farreri: purification and antibacterial activity of its reaction products generated in vitro

Phenoloxidase (PO) was purified from hemocytes of the scallop Chlamys farreri using native-PAGE and gel permeation column chromatography, and then substrate specificity and antibacterial activity generated from reaction products of purified PO were analyzed. The results showed purified PO had a molecular mass of 576 kDa in native-PAGE and 53 kDa in denatured PAGE, and could catalyze the substrates L-3,4-dihydroxyphenylalanine (L-DOPA), dopamine, catechol and hydroquinone suggesting it is a type of p-diphenoloxidase. Using dopamine as a substrate, PO reaction products significantly inhibited the growth of Vibrio alginolyticus, Vibrio parahaemolyticus and Aeromonas salmonicida. No significant inhibition was found in Streptococcus dysgalactiae, Streptococcus iniae, Micrococcus lysodeikticus and Edwardsiella tarda. When L-DOPA was used as a substrate, significant inhibition occurred in A. salmonicida only.     

Apical and basolateral 4F2hc and the amino acid exchange of L-DOPA in renal LLC-PK1 cells

Summary. The present study aimed to examine the presence and define the role of 4F2hc, a glycoprotein associated with the LAT2 amino acid transporter, in L-DOPA handling by LLC-PK₁ cells. For this purpose we have measured the activity of the apical and basolateral inward and outward transport of [¹⁴C] L-DOPA in cell monolayers and examined the influence of 4F2hc antisense oligonucleotides on [¹⁴C] L-DOPA handling. The basal-to-apical transepithelial flux of [¹⁴C] L-DOPA progressively increased with incubation time and was similar to the apical-to-basal transepithelial flux. The spontaneous and the L-DOPA-stimulated apical fractional outflow of [¹⁴C] L-DOPA were identical to that through the basal cell side. The L-DOPA-induced fractional outflow of [¹⁴C] L-DOPA through the apical or basal cell side was accompanied by marked decreases in intracellular levels of [¹⁴C] L-DOPA. In cells treated with an antisense oligonucleotide complementary to 4F2hc mRNA for 72 h, [¹⁴C] L-DOPA inward transport and 4F2hc expression were markedly reduced. Treatment with the 4F2hc antisense oligonucleotide markedly decreased the spontaneous fractional outflow of [¹⁴C] L-DOPA through the apical or the basal cell side. It is likely that the Na⁺-independent and pH-sensitive uptake of L-DOPA include the hetero amino acid exchanger LAT2/4F2hc, which facilitates the trans-stimulation of L-DOPA and its outward transfer at both the apical and basal cell sides.     

The determination of hydroxydopamines and other trace amines in the urine of Parkinsonian patients and normal controls

5- and 6-Hydroxydopamine, which we had earlier identified as naturally occurring amines in human urine, were quantified in Parkinson's patients treated with L-DOPA, Parkinson's patients whose treatment did not include L-DOPA and in age matched controls. Analysis was carried out by GCMS of the ditrifluoromethylbenzoyl-trimethylsilyl (DTFMB-TMS) derivatives of the compounds. The concentrations of 5- and 6-hydroxydopamine in the urine of DOPA treated Parkinson's patients were significantly higher than the concentrations from patients not treated and from normal controls. Urinary dopamine levels were greatly elevated in DOPA treated Parkinson's patients whilep-tyramine levels were suppressed. No marked differences were seen between the three groups in terms of the urinary concentrations of any of the other amines measured.     

Decarboxylation of Exogenous l-3,4-Dihydroxyphenylalanine in Rat Striatum as Studied by In Vivo Voltammetry

An in vivo voltammetric technique was used to determine whether striatal nondopaminergic neurons take up and decarboxylate exogenous L-3,4-dihydroxyphenylalanine (L-DOPA) and release it as dopamine. After the striatal serotonergic neurons of the rat had been destroyed by intraventricular injection of 5,7-dihydroxytryptamine, L-DOPA was administered intraperitoneally. It was found that changes in the dopamine concentration in the striatal extracellular fluid of the rat were the same as those in the nonlesioned rat. L-DOPA was also administered to the rat after the striatal perikarya had been destroyed by the intrastriatal injection of kainate. The striatal dopamine concentrations of the lesioned rat changed in parallel with 5,7-dihydroxytryptamine-lesioned rats, as well as the nonlesioned rats. Moreover, when normal rats were administered L-DOPA, the dopamine concentration was not increased in the cerebellum, where dopamine neurons do not exist. From these observations, it is concluded that exogenous L-DOPA is taken up, decarboxylated to dopamine, and released only in the striatal dopamine neurons.     

Germination growth response of different plant species to the allelochemical L-3,4-dihydroxyphenylalanine (L-DOPA)

The aim of this study was to investigate the seed germination response of different plant families to L-3,4-dihydroxyphenylalanine (L-DOPA), one of the strongest allelochemicals in nature. Three types of responses in terms of colouration changes on filter paper were obtained; black and gray (Gramineae and Compositae), no change (Leguminosae, Brassicaceae, and Cucurbitaceae) and an obstructed-circle around the seeds with black colouration on the outer side of the circle (Hydrophyllaceae) when L-DOPA solution was applied during seed germination. Radicle growth in the Gramineae and Leguminosae families was inhibited less by a single treatment of L-DOPA solution (250 g/ml) than in the other families. However, continuous treatment with L-DOPA demonstrated that the Gramineae family was less affected in terms of the inhibition of radicle growth than the Leguminosae family. When more seeds were added to the L-DOPA solution less inhibition of radicle growth was observed in all plants tested. The EC₅₀ of L-DOPA for bluebell (Hydrophyllaceae), white clover (Leguminosae), and lettuce (Compositae) was approximately 200, 100, and 50 g/ml, respectively. However, in perennial ryegrass (Gramineae) no EC₅₀ was observed even at 250 g/ml L-DOPA. In the Gramineae family, addition of more seeds into the L-DOPA solution increased the colouration on the filter paper. These results demonstrated that each seed functions to oxidize or dissolve L-DOPA. In the Gramineae, Leguminosae, Compositae, and Hydrophyllaceae, increasing the number of seeds imbibed in the L-DOPA solution increased the rate of L-DOPA disappearance from the petri-dish. Of the Grammaceous plants tested, only perennial ryegrass, which showed fairly weak allelopathic activity, metabolised L-DOPA to dopamine. Although the relationships between the changes in colouration of the filter paper and the inhibition of radicle growth in these experiments are still unknown, there appears to be a strong response in each species to protect the cell from L-DOPA damage.     

Uptake and intracellular fate of L-DOPA in a human intestinal epithelial cell line: Caco-2

The aim of the presentstudy was to examine the kinetic characteristics of theL-3,4-dihydroxyphenylalanine (L-DOPA)transporter and the fate of newly formed dopamine in Caco-2 cells. Inthe presence of 50 µM benserazide (an inhibitor of aromaticL-amino acid decarboxylase), L-DOPA was rapidlyaccumulated in Caco-2 cells. At equilibrium (30 min of incubation) theintracellular L-DOPA concentration was 10.2 ± 0.1 µM ata medium concentration of 0.5 µM. In saturation experiments theaccumulation of L-DOPA was saturable with aMichaelis-Menten constant (K_m) of 60 ± 10 µMand a maximal reaction velocity (V_max) of 6.6 ± 0.3 nmol · mg protein¹ · 6 min¹; at 4°C the amount of L-DOPAaccumulated in the cells was nonsaturable. When cells were incubatedwith increasing concentrations of L-DOPA (10-100 µM)in the absence of benserazide, a substantial amount of theL-DOPA that was taken up was decarboxylated to dopamine, with an apparent K_m of 27.2 µM. In experimentsperformed in cells cultured in polycarbonate filters, theaccumulation of L-DOPA in the presence of benserazide wasgreater when the substrate was applied from the basolateral cell borderthan when it was applied from the apical cell border. In the absence ofbenserazide, L-DOPA applied from the basolateral cellborder resulted in a nonlinear formation of dopamine(K_m = 43 ± 7 µM,V_max = 23.7 ± 1.2 nmol · mgprotein¹ · 6 min¹). Theamount of dopamine leaving the cell through the apical cell border waslower than the amount that escaped through the basolateral cell border,and the process was saturable (K_m = 623 ± 238 µM, V_max = 0.19 ± 0.02 nmol · mgprotein¹ · 6 min¹). Inconclusion, the data presented here show that Caco-2 cells are endowedwith an efficient L-DOPA uptake system, and intracellular L-DOPA was found to be rapidly converted to dopamine, someof which diffuses out of the cell. The utilization of Caco-2 cells cultured on polycarbonate filters probably provides a better way tolook at processes such as the outward transfer of intracellular molecules, namely, the outward transfer of newly formed dopamine.

     

Innovative Effect of Illite on Improved Microbiological Conversion of L-Tyrosine to 3,4 Dihydroxy Phenyl L-Alanine (L-DOPA) by Aspergillus oryzae ME2 Under Acidic Reaction Conditions

In the present investigation, the previous ultraviolet irradiated mutant strain of Aspergillus oryzae UV-7 was further improved in terms of 3,4 dihydroxy phenyl L-alanine (L-DOPA) activity after chemical mutagenesis through 1-methyl 3-nitro 1-nitroso guanidine (MNNG = 250–1500 μg/ml) treatment (0–30 min). Among several mutant variants, the one that produced a larger amount of L-DOPA from L-tyrosine was designated to as ME₂ and it was made 2-deoxy-D-glucose-resistant by growing it at various concentrations of 2 dg (0.01–0.025 %, w/v) in Vogel’s agar medium. Relatively better production of L-DOPA (> 0.60 mg/ml) was obtained when 2.0% (w/v) glucose was used as a carbon source in the mycelium production medium and the tyrosinase activity increased constitutively (1.08 mg/ml), which resulted in a greater production of L-DOPA. At optimum pH₀ (pH 6.0) and reaction time (60 min), more than 65% sugar was utilized for cell mass formation. The maximum conversion of L-tyrosine to L-DOPA (0.428 mg/ml) was achieved 60 min after the biochemical reaction. Mould mycelium was used for microbiological conversion of L-tyrosine to L-DOPA because tyrosinases, β-carboxylases, and tyrosine hydroxylases are intracellular enzymes. The effect of illite (1.0 × 10⁶–6.0 × 10⁶ M) on biochemical conversion of L-tyrosine to L-DOPA by Aspergillus oryzae ME₂ was also carried out. Best results of L-DOPA biosynthesis were observed when the concentration of illite was 3.5 × 10⁻⁶ M (1.686 mg/ml L-DOPA produced with 1.525 mg/ml consumption of L-tyrosine). It was noted that the addition of illite not only increased enzyme activity but also enhanced the permeability of cell membrane to facilitate the secretion of enzymes into the reaction broth. The comparison of kinetic parameters showed the ability of mutant to yield L-DOPA (i.e., Y_p/x 7.360 ± 0.04 mg/mg). When the culture grown on various illite concentrations was monitored for Q_p, Q_s, and q_p, there was significant enhancement (p < 0.025) in these variables over the control, which indicate that the study can be commercially applicable on stirred and magnetic rotary drums. Overall, there was up to 3-fold (Q_p = 0.290 mg/L-DOPA produced/ml/h) enhancement in the product formation rate, which is highly encouraging (HS, LSD 0.456).     

Dopamine Transporter Loss in 6-OHDA Parkinson’s Model Is Unmet by Parallel Reduction in Dopamine Uptake

The dopamine transporter (DAT) regulates synaptic dopamine (DA) in striatum and modulation of DAT can affect locomotor activity. Thus, in Parkinson’s disease (PD), DAT loss could affect DA clearance and locomotor activity. The locomotor benefits of L-DOPA may be mediated by transport through monoamine transporters and conversion to DA. However, its impact upon DA reuptake is unknown and may modulate synaptic DA. Using the unilateral 6-OHDA rat PD model, we examined [³H]DA uptake dynamics in relation to striatal DAT and tyrosine hydroxylase (TH) protein loss compared with contralateral intact striatum. Despite >70% striatal DAT loss, DA uptake decreased only ∼25% and increased as DAT loss approached 99%. As other monoamine transporters can transport DA, we determined if norepinephrine (NE) and serotonin (5-HT) differentially modulated DA uptake in lesioned striatum. Unlabeled DA, NE, and 5-HT were used, at a concentration that differentially inhibited DA uptake in intact striatum, to compete against [³H]DA uptake. In 6-OHDA lesioned striatum, DA was less effective, whereas NE was more effective, at inhibiting [³H]DA uptake. Furthermore, norepinephrine transporter (NET) protein levels increased and desipramine was ∼two-fold more effective at inhibiting NE uptake. Serotonin inhibited [³H]DA uptake, but without significant difference between lesioned and contralateral striatum. L-DOPA inhibited [³H]DA uptake two-fold more in lesioned striatum and inhibited NE uptake ∼five-fold more than DA uptake in naïve striatum. Consequently, DA uptake may be mediated by NET when DAT loss is at PD levels. Increased inhibition of DA uptake by L-DOPA and its preferential inhibition of NE over DA uptake, indicates that NET-mediated DA uptake may be modulated by L-DOPA when DAT loss exceeds 70%. These results indicate a novel mechanism for DA uptake during PD progression and provide new insight into how L-DOPA affects DA uptake, revealing possible mechanisms of its therapeutic and side effect potential.     

The effect of chronic L-DOPA administration on supersensitive pre- and postsynaptic dopaminergic receptors in rat brain

Chronic administration of haloperidol induced supersensitivity of the pre- and postsynaptic dopaminergic receptors in rat brain. The response of the presynaptic receptors was determined by an enhanced inhibitory effect of apomorphine on dopamine synthesis after gamma-butyrolactone injection. This change in the receptor function was detected both in the nigrostriatal and mesolimbic pathways. Haloperidol also increased the ³H-spiperone binding sites in striatal membranes, indicating supersensitivity of the postsynaptic receptors. Subsequent prolonged treatment with high doses of L-DOPA/carbidopa resulted in a decrease in ³H-spiperone binding sites, but had no effect on the supersensitive presynaptic receptors. It is suggested that tardive dyskinesia may be a state of both pre- and postsynaptic dopamine receptor supersensitivity and that chronic L-DOPA treatment may have a differential effect on these sites.     

Long-Term L-DOPA Treatment Causes Indiscriminate Increase in Dopamine Levels at the Cost of Serotonin Synthesis in Discrete Brain Regions of Rats

(1) The treatment of choice for Parkinson’s disease (PD) is 3,4-dihydroxyphenylalanine (L-DOPA) with peripheral decarboxylase inhibitor, but long-term therapy leads to motor and psychiatric complications. In the present study we investigated 5-hydroxytryptamine (5-HT) and dopamine concentrations in serotonergic and dopaminergic nuclei following chronic administration of L-DOPA to find whether the neurotransmitter synthesis in these brain areas are compensated. (2) Rats were administered L-DOPA (250 mg/kg) and carbidopa (25 mg/kg) daily for 59 and 60 days, and killed on the 60th day, respectively at 24 h and 30 min after the last dose. L-DOPA, norepinephrine, 5-HT, 5-hydroxyindoleacetic acid (5-HIAA), dopamine, homovanillic acid (HVA), and 3,4-dihydroxyphenylacetic acid (DOPAC) were measured in striatum, nucleus raphe dorsalis (NRD), nucleus accumbens (NAc), substantia nigra, cerebellum, and cortex employing HPLC-electrochemical procedure. (3) Prolonged treatment of L-DOPA caused depression in the animals as revealed in a forced swim test. Serotonin content was significantly decreased in all brain regions studied 30 min after long-term L-DOPA, except in NAc. The cortex and striatum showed lowered levels of this indoleamine 24 h after 59 doses of L-DOPA. Dopamine, HVA, and DOPAC concentrations were significantly higher in all the regions studied after 30 min, and in the cerebellum after 24 h of L-DOPA. The levels of DOPAC were elevated in all the brain areas studied 24 h after prolonged L-DOPA treatment. (4) The present results suggest that long-term L-DOPA treatment results in significant loss of 5-HT in serotonergic and dopaminergic regions of the brain. Furthermore, while L-DOPA metabolism per se was uninfluenced, dopamine synthesis was severely impaired in all the regions. The imbalance of serotonin and dopamine formation may be the cause of overt cognitive, motor, and psychological functional aberrations seen in parkinsonian patients following prolonged L-DOPA treatment.     

Dopamine agonists reverse the elevated 3H-neuroleptic binding in neuroleptic-pretreated rats.

Chronic administration of large doses of haloperidol (10 mg/kg/day for 21 days) resulted in a 37–45% increase in the specific binding of ³H-spiperone and a 28% increase in the specific binding of ³H-apomorphine in rat striatal homogenates. The increase in ³H-spiperone binding in neuroleptic-pretreated rats could be reversed significantly by a five day administration of either bromocryptine (35 mg/kg p.o.) or L-DOPA (200 mg/kg p.o.)+ Carbidopa (20 mg/kg p.o.). Treatment of normal rats for 5 days with either L-DOPA or bromocryptine alone had no effect on ³H-spiperone binding.These results indicate that dopamine agonists can reverse the neuroleptic-induced elevation of brain neuroleptic binding, suggesting that short-term high-dose therapy with dopamine agonists might be of some value in alleviating neuroleptic-induced tardive dyskinesia clinically.