L-Dopa decarboxylase interaction with the major signaling regulator ΡΙ3Κ in tissues and cells of neural and peripheral origin.

L-Dopa decarboxylase (DDC) catalyzes the decarboxylation of L-Dopa to dopamine and 5-hydroxytryptophan (5-HTP) to serotonin. Although DDC has been purified from a variety of peripheral organs, including the liver, kidney and pancreas, the physiological significance of the peripherally expressed enzyme is not yet fully understood. DDC has been considered as a potential novel biomarker for various types of cancer, however, the role of DDC in the development of hepatocellular carcinoma (HCC) remains to be evaluated. Phosphatidylinositol 3-kinase (PI3K), on the other hand, has been shown to play a key role in the tumorigenesis, proliferation, metastasis, apoptosis, and angiogenesis of HCC by regulating gene expression. We initially identified the interaction of DDC with PI3K by means of the phage display methodology. This association was further confirmed in human hepatocellular carcinoma cell lines, human embryonic kidney cells, human neuroblastoma cells, as well as mouse brain, by the use of specific antibodies raised against DDC and PI3K. Functional aspects of the above interaction were studied upon treatment with the DDC inhibitor carbidopa and the PI3K inhibitor LY294002. Interestingly, our data demonstrate the expression of the neuronal type DDC mRNA in HCC cells. The present investigation provides new evidence on the possible link of DDC with the PI3K pathway, underlining the biological significance of this complex enzyme.

Clinical Value of 18F-FDOPA PET/CT With Contrast Enhancement and Without Carbidopa Premedication in Patients with Insulinoma.

AIM: We evaluated the clinical usefulness of 6-[18F]fluoro-3,4-dihydroxy-L-phenylalanine(18F-FDOPA)-positron-emission tomography (PET)/computed tomography (CT) in insulinoma detection with contrast enhancement, early acquisition time, and no carbidopa premedication. PATIENTS AND METHODS: Twenty-six patients diagnosed with hyperinsulinemic hypoglycemia underwent an 18F-FDOPA PET/CT examination. Patients without carbidopa premedication and contrast-enhanced CT were included. Imaging findings were compared to the overall final diagnosis (histological findings). RESULTS: In 10 of 26 patients (eight women, two men; mean age=53 years; age range=30-94 years), a detected lesion was confirmed histologically as an insulinoma. 18F-FDOPA PET detected the tumor in five out of ten patients. Contrast-enhanced CT also detected the tumor in five out of ten. Overall, 18F-FDOPA PET/CT, with contrast enhancement and without carbidopa premedication, was able to detect the insulinoma in seven out of ten patients (70%). CONCLUSION: Based on our data, 18F-DOPA PET/CT, with contrast enhancement and without carbidopa premedication, as a 'one-stop' diagnostic modality is a viable option for insulinoma detection.

A multi-biomarker analysis of the antioxidant efficacy of Parkinson's disease therapy.

Substantial evidences suggest that reactive oxygen species participate in the normal aging process and in cancer and neurodegenerative age-related diseases. Parkinson's disease (PD), one of the most common oxidative stress-associated pathology in aging people, is treated with a standard pharmacological protocol consisting in a combined therapy l-dopa plus an inhibitor of dopa-decarboxylase, such as carbidopa. The therapy is well validated for the ability to restoring dopaminergic neurotransmission in PD patients, while l-dopa and carbidopa ability in modulating oxidative stress is currently under discussion. Our aim was to evaluate the impact of l-dopa and carbidopa on several biomarkers of exogenously-induced oxidative stress to validate the overall antioxidant effectiveness of the therapy. For this purpose we used peripheral blood lymphocytes from healthy donors treated in vitro with l-dopa and carbidopa and then challenged by different concentrations of H2O2. Glutathione (GSH, GSSG, GSH/GSSG), malondialdehyde (TBARs), protein carbonyls as well as DNA damage (8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and micronuclei (MN)), modulation was evaluated. Our results show that l-dopa, but not carbidopa, decreases the markers of lipid and protein oxidation and increases the total content of glutathione. Both l-dopa and carbidopa (alone or in combination) are able to counteract the formation of 8-oxodG and to reduce H2O2-induced micronuclei.

Suppression of camptothecin biosynthetic genes results in metabolic modification of secondary products in hairy roots of Ophiorrhiza pumila.

Camptothecin is a monoterpenoid indole alkaloid that exhibits anti-tumor activity. In Ophiorrhiza pumila, production of camptothecin and its related alkaloids was high in the hairy roots, but not in the cell suspension culture derived from hairy roots. To identify the intermediates in camptothecin biosynthesis, expression of genes encoding tryptophan decarboxylase (TDC) and secologanin synthase (SLS), the two enzymes catalyzing the early steps in camptothecin biosynthesis, were suppressed in the hairy roots of O. pumila by RNA interference (RNAi), and metabolite changes were investigated. In most TDC- and SLS-suppressed lines, accumulation of camptothecin and related alkaloids, strictosidine, strictosamide, pumiloside, and deoxypumiloside was reduced. The accumulation levels of secologanin exhibited a strong negative correlation with the expression level of TDC, and that of loganin exhibited a negative correlation with the expression level of SLS. Some hairy root-specific chromatographic peaks detected by liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR-MS) exhibited positive or negative correlation with TDC expression, suggesting their possible involvement in camptothecin biosynthesis.

Protective effects of L-dopa and carbidopa combined treatments on human catecholaminergic cells.

Parkinson's disease (PD) is one of the most common neurodegenerative disorders characterized by decreased levels of the neurotransmitter dopamine (DA) in the striatum of the brain, as a result of degeneration of DA neurons. Levodopa (L-Dopa) crosses the blood-brain barrier and its administration replenishes the loss of DA in dopaminergic neurons in PD patients. Despite the evident beneficial effects, L-Dopa use may cause side effects and its toxicity found in in vitro assays has been attributed to the generation of reactive oxygen species (ROS): L-Dopa is converted to DA and its metabolism and autoxidation gives rise to quinones, semiquinones, and hydrogen peroxide. Despite this evidence, L-Dopa in some in vivo and in vitro experiments showed no toxic effects, or even antioxidant effects. Two major peripheral L-Dopa metabolic pathways, driven by the enzymes Aromatic L-amino acid decarboxylase (AADC) and catechol-O-methyl transferase (COMT), significantly deplete the amount of L-Dopa reaching the brain. The low bioavailability of L-Dopa may cause a wide variation in clinical response between patients. Strategies addressing to improve the bioavailability of L-Dopa include coadministering L-Dopa with carbidopa, a decarboxylase inhibitor, as multiple daily doses. We utilized catecholaminergic human neuroblastoma cells to study DNA damage and ROS production after L-Dopa and carbidopa treatments. Our data lead us to confirm that L-Dopa may have a protective effect on dopaminergic cells especially at certain concentrations, in particular, toward the production of ROS and their toxic effects on DNA. Furthermore in the combined treatment, with induction of ROS following administration of H(2)O(2), carbidopa is effective in reducing the damage caused by reactive oxygen intermediates both alone and in combination with L-Dopa.

Cysteine decrease following acute Levodopa intake in patients with Parkinson's disease.

The thiol homeostasis determines the redox milieu and thus scavenging of free radicals by antioxidants like glutathione (GSH). GSH is formed out of cysteine in combination with l-glycine and glutamine acid. An up regulation of free radical occurrence is looked upon as one key feature of chronic neurodegeneration. Levodopa (LD) is under suspicion to support synthesis of free radicals via the degradation of its derivative dopamine in abundant mitochondria. Objectives were to investigate the impact of LD on free cysteine turnover in plasma. 200mg LD/50mg carbidopa (CD) were administered to 13 patients with Parkinson's disease under standardised conditions. Plasma levels of LD and free cysteine were measured before, 60- and 80-min after the LD/CD application. Cysteine concentrations decayed, expectedly LD levels increased. Cysteine decrease may result from an up regulation of GSH synthesis to encounter augmented appearance of free radicals associated with LD turnover via mitochondrial monoaminooxidase.

5-(2-18F-fluoroethoxy)-L-tryptophan as a substrate of system L transport for tumor imaging by PET.

UNLABELLED: Large neutral l-amino acids are substrates of system L amino acid transporters. The level of one of these, LAT1, is increased in many tumors. Aromatic l-amino acids may also be substrates of aromatic l-amino acid decarboxylase (AADC), the level of which is enhanced in endocrine tumors. Increased amino acid uptake and subsequent decarboxylation result in the intracellular accumulation of the amino acid and its decarboxylation product. (18)F- and (11)C-labeled neutral aromatic amino acids, such as l-3,4-dihydroxy-6-(18)F-fluorophenylalanine ((18)F-FDOPA) and 5-hydroxy-l-[ß-(11)C]tryptophan, are thus successfully used in PET to image endocrine tumors. However, 5-hydroxy-l-[ß-(11)C]tryptophan has a relatively short physical half-life (20 min). In this work, we evaluated the in vitro and in vivo characteristics of the (18)F-labeled tryptophan analog 5-(2-(18)F-fluoroethoxy)-l-tryptophan ((18)F-l-FEHTP) as a PET probe for tumor imaging. METHODS: (18)F-l-FEHTP was synthesized by no-carrier-added (18)F fluorination of 5-hydroxy-l-tryptophan. In vitro cell uptake and efflux of (18)F-l-FEHTP and (18)F-FDOPA were studied with NCI-H69 endocrine small cell lung cancer cells, PC-3 pseudoendocrine prostate cancer cells, and MDA-MB-231 exocrine breast cancer cells. Small-animal PET was performed with the respective xenograft-bearing mice. Tissues were analyzed for potential metabolites. RESULTS: (18)F-l-FEHTP specific activity and radiochemical purity were 50-150 GBq/µmol and greater than 95%, respectively. In vitro cell uptake of (18)F-l-FEHTP was between 48% and 113% of added radioactivity per milligram of protein within 60 min at 37°C and was blocked by greater than 95% in all tested cell lines by the LAT1/2 inhibitor 2-amino-2-norboranecarboxylic acid. (18)F-FDOPA uptake ranged from 26% to 53%/mg. PET studies revealed similar xenograft-to-reference tissue ratios for (18)F-l-FEHTP and (18)F-FDOPA at 30-45 min after injection. In contrast to the (18)F-FDOPA PET results, pretreatment with the AADC inhibitor S-carbidopa did not affect the (18)F-l-FEHTP PET results. No decarboxylation products of (18)F-l-FEHTP were detected in the xenograft homogenates. CONCLUSION: (18)F-l-FEHTP accumulates in endocrine and nonendocrine tumor models via LAT1 transport but is not decarboxylated by AADC. (18)F-l-FEHTP may thus serve as a PET probe for tumor imaging and quantification of tumor LAT1 activity. These findings are of interest in view of the ongoing evaluation of LAT1 substrates and inhibitors for cancer therapy.

Structural perspective on the direct inhibition mechanism of EGCG on mammalian histidine decarboxylase and DOPA decarboxylase.

Histidine decarboxylase (HDC) and l-aromatic amino acid decarboxylase (DDC) are homologous enzymes that are responsible for the synthesis of important neuroactive amines related to inflammatory, neurodegenerative, and neoplastic diseases. Epigallocatechin-3-gallate (EGCG), the most abundant catechin in green tea, has been shown to target histamine-producing cells and to promote anti-inflammatory, antitumor, and antiangiogenic effects. Previous experimental work has demonstrated that EGCG has a direct inhibitory effect on both HDC and DDC. In this study, we investigated the binding modes of EGCG to HDC and DDC as a first step for designing new polyphenol-based HDC/DDC-specific inhibitors.

5-Hydroxytryptophan attenuates somatic signs of nicotine withdrawal.

Abrupt nicotine cessation after chronic use disrupts monoaminergic systems and causes withdrawal signs/symptoms. In this study, the precursor of serotonin 5-hydroxytryptophan (5-HTP) relieved nicotine withdrawal signs. (-)-Nicotine bitartrate or equimolar sodium tartrate was infused into each rat via a s.c. osmotic minipump for 7 days. Somatic abstinence signs (teeth-chattering/chews and shakes, etc.) were counted one day after pump removal. Somatic signs were attenuated by the i.p. injection of 5-HTP, but not by NSD-1015, a centrally-acting L-aromatic amino acid decarboxylase inhibitor, indicating that 5-HTP mitigates somatic signs mainly through its conversion to 5-HT.

Increased renal dopamine and acute renal adaptation to a high-phosphate diet.

The current experiments explore the role of dopamine in facilitating the acute increase in renal phosphate excretion in response to a high-phosphate diet. Compared with a low-phosphate (0.1%) diet for 24 h, mice fed a high-phosphate (1.2%) diet had significantly higher rates of phosphate excretion in the urine associated with a two- to threefold increase in the dopamine content of the kidney and in the urinary excretion of dopamine. Animals fed a high-phosphate diet had a significant increase in the abundance and activity of renal DOPA (l-dihydroxyphenylalanine) decarboxylase and significant reductions in renalase, monoamine oxidase A, and monoamine oxidase B. The activity of protein kinase A and protein kinase C, markers of activation of renal dopamine receptors, were significantly higher in animals fed a high-phosphate vs. a low-phosphate diet. Treatment of rats with carbidopa, an inhibitor of DOPA decarboxylase, impaired adaptation to a high-phosphate diet. These experiments indicate that the rapid adaptation to a high-phosphate diet involves alterations in key enzymes involved in dopamine synthesis and degradation, resulting in increased renal dopamine content and activation of the signaling cascade used by dopamine to inhibit the renal tubular reabsorption of phosphate.

Expression of functional dopaminergic phenotype in purified cultured Müller cells from vertebrate retina.

Purified retina glial Müller cells can express the machinery for dopamine synthesis and release when maintained in culture. Dopamine is detected in cell extracts of cultures exposed to its precursor, L-DOPA. A large portion of synthesized dopamine is recovered in the superfusing medium showing the tendency of the accumulated dopamine to be released. Müller cells purified from developing chick and mouse retinas express L-DOPA decarboxylase (DDC; aromatic-L-amino-acid decarboxylase; EC 4.1.1.28) and the dopamine transporter DAT. The synthesis of dopamine from L-DOPA supplied to Müller cultures is inhibited by m-hydroxybenzylhydrazine, a DDC inhibitor. Dopamine release occurs via a transporter-mediated process and can activate dopaminergic D(1) receptors expressed by the glia population. The synthesis and release of dopamine were also observed in Müller cell cultures from mouse retina. Finally, cultured avian Müller cells display increased expression of tyrosine hydroxylase, under the influence of agents that increase cAMP levels, which results in higher levels of dopamine synthesized from tyrosine. A large proportion of glial cells in culture do express Nurr1 transcription factor, consistent with the dopaminergic characteristics displayed by these cells in culture. The results show that Müller cells, deprived of neuron influence, differentiate dopaminergic properties thought to be exclusive to neurons.

Community and long-term care management of Parkinson's disease in the elderly: focus on monoamine oxidase type B inhibitors.

Parkinson's disease affects up to 1 million people in the US, most of them elderly. Motor and non-motor symptoms can be significantly disabling to the point of necessitating institutionalisation. Age-related changes in drug absorption, distribution, metabolism and excretion complicate the treatment of elderly patients with Parkinson's disease. General management principles include initiation of medication at low doses with gradual titration based on clinical effects, avoidance of certain classes of drugs (e.g. anticholinergics), and attention to polypharmacy and its risk for potentially toxic drug interactions. Levodopa remains the most efficacious anti-Parkinson's disease medication and should be the cornerstone of therapy in the elderly Parkinson's disease patient. Use of dopamine receptor agonists, amantadine and anticholinergic drugs in the elderly is limited by high risk for psychotoxicity. Catechol-O-methyltransferase inhibitors may be used to augment levodopa in the setting of 'wearing off' (i.e. motor fluctuations). Monoamine oxidase type B (MAO-B) inhibitors can be used across the spectrum of disease severity, but selegiline (deprenyl), the prototype in this class, is characterised by low and erratic bioavailability of the parent drug and conversion to amphetamine metabolites that may increase the risk of adverse events. A new orally disintegrating tablet formulation overcomes some of these limitations. Rasagiline is a new, selective, second-generation MAO-B inhibitor that is chemically and metabolically distinct from selegiline. The favourable safety profile of rasagiline in the elderly and its once-daily formulation may maximise drug adherence and improve outcomes.

Nicotine induces tyrosine hydroxylase plasticity in the neurodegenerating striatum.

It has been shown that nicotine prevents the loss of dopamine (DA) in the corpus striatum (CS) after 6-hydroxydopamine injection in the substantia nigra. To study the role of the enzyme tyrosine hydroxylase (TH; EC 1.14.16.2) in this experimental paradigm, we have examined its activity by assessing the accumulation of l-3,4-dihydroxyphenylalanine after inhibiting the subsequent enzyme in the DA synthetic pathway, aromatic l-amino acid decarboxylase, with 3-hydroxybenzylhydrazine. In addition the amount of TH protein was assessed by western blotting and its distribution in the CS was examined using immunohistochemical methods. 6-hydroxydopamine injection produced a significant decrease in DA levels and l-3,4-dihydroxyphenylalanine accumulation, as well as decreases in TH protein and TH immunoreactive fibres in the CS. After nicotine treatment, the decrease in TH protein in the CS was significantly reduced, with a concomitant preservation of TH activity, but nicotine did not alter the number of TH immunoreactive fibres. The activity and amount of TH did not change in the contralateral (intact) CS. Thus, nicotine induces long lasting TH plasticity in the degenerating CS. A synergistic action of nicotine-activated and lesion-originated signals appears necessary for the expression of this neuronal molecular plasticity.

Neuropeptide Y induced attenuation of catecholamine synthesis in the rat mesenteric arterial bed.

The effect of neuropeptide Y (NPY) on the basal and nerve stimulation-induced increase in norepinephrine synthesis was studied in the isolated and perfused mesenteric arterial bed of the rat. Tyrosine hydroxylation, the rate-limiting step in catecholamine (CA) biosynthesis, was assessed by measuring the accumulation of DOPA in the perfusate/superfusate overflow after perfusion of the mesenteric arterial bed with the decarboxylase inhibitor m-hydroxybenzyl hydralazine (NSD-1015). Treatment with NDS-1015 resulted in a time-dependent increase in DOPA production and nerve stimulation (8 Hz, supramaximal voltage, 2 ms duration) increased DOPA production even further. NPY 1 to 100 nM was observed to produce a concentration-dependent attenuation in both the basal and nerve stimulation-induced increase in DOPA formation. To come to an understanding of the NPY receptor subtype mediating the inhibition of CA synthesis, the rank order of potency of a series of NPY analogs with varying selectivity for NPY receptor subtypes including intestinal polypeptide (PYY), PYY 13-36, Leu36 Pro34 NPY, human pancreatic polypeptide (h-PP), and rat pancreatic polypeptide (r-PP) were determined. In addition, the effect of various selective NPY antagonists on the inhibitory effect of NPY was also examined. These included the Y1 antagonist BIB03304, the Y2 antagonist BIIE0246, and the Y5 antagonist CGP71683. The IC50's for NPY, PYY, PYY13-36, Leu31 Pro34 NPY, and hPP in inhibiting CA synthesis were 5, 7, 15, 30, and 33 nM respectively. rPP failed to inhibit CA synthesis. All 3 of the NPY antagonists produced attenuation of the NPY-induced inhibition of CA synthesis, but it took a combination of all 3 to completely block the effect of a maximal inhibitory concentration of NPY. These results demonstrate that NPY inhibits CA synthesis in the perfused mesenteric arterial bed and can do so by activation of a variety of receptors including the Y1, Y2, and Y5.

Tolcapone decreases plasma levels of S-adenosyl-L-homocysteine and homocysteine in treated Parkinson's disease patients.

BACKGROUND: Elevated plasma total homocysteine (tHcy) appeared in levodopa/dopadecarcoxylase inhibitor (DDI) treated patients with Parkinson's disease (PD). One therapeutic approach for tHcy reduction is vitamine supplementation, since folic acid and cobalamine catalyse and enhance metabolism of tHcy to methionine. A further therapeutic alternative is inhibition of catechol-O-methyltransfrase (COMT) on a regular basis, when levodopa/DDI treatment is performed. METHODS: We measured the concentrations of S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), tHcy, levodopa and 3-O-methyldopa in plasma of 13 levodopa treated PD patients before first drug intake at 0600 hours. Blood samples were taken before and after 2 days of additional application of the centrally acting catechol-O-methyltransferase inhibitor tolcapone 100 mg t.i.d. RESULTS: Plasma levels of SAH [day 1: 48.32+/-22.52, 23.92-98.25 (mean+/-SD, range; micromol/l); day 3: 37.72+/-15.84, 23.4-61.89; p = 0.01] and tHcy (day 1: 13.88+/-5.62, 7.63-24.81; day 3: 11.38+/-4.44, 5.98-20.45; p = 0.04) significantly reduced. Plasma levels of levodopa did not significantly (p = 0.17) increase, whereas 3-OMD concentrations significantly (p = 0.0002) reduced after additional tolcapone intake. There was no significant change of SAM plasma levels (p = 0.22). CONCLUSION: Our prospective trial shows, that COMT inhibition with tolcapone lowers tHcy synthesis. Tolcapone may also possess beside its proven, occasional, hepatotoxic potency also beneficial effects via decrease of SAH and tHcy. This may hypothetically reduce homocysteine mediated progress of neuronal degeneration and the risk for onset of dementia, vascular disease and polyneuropathy in levodopa treated PD patients in the long term.

Possibility of autocrine beta-adrenergic signaling in C2C12 myotubes.

Levodopa reportedly inhibits insulin action in skeletal muscle. Here we show that C2C12 myotubes produce levodopa and that insulin-stimulated glucose transport is enhanced when endogenous levodopa is depleted. Exogenous levodopa prevented the stimulation of glucose transport by insulin (P < 0.05) and increased cAMP concentrations (P < 0.05). The decrease in insulin-stimulated glucose transport caused by levodopa was attenuated by propranolol (a beta-adrenergic antagonist) and prevented by NSD-1015 (NSD), an inhibitor of DOPA decarboxylase (DDC; converts levodopa to dopamine). Propranolol and NSD both prevented levodopa-related increases in [cAMP]. However, the effects of levodopa were unlikely to be dependent on the conversion of levodopa to catecholamines because we could detect neither DDC in myotubes nor catecholamines in media after incubation of myotubes with levodopa. The data suggest the possibility of novel autocrine beta-adrenergic action in C2C12 myotubes in which levodopa, produced by myotubes, could have hormone-like effects that impinge on glucose metabolism.

Hydrogen peroxide degradation and selective carbidopa-induced cytotoxicity against human tumor lines.

The carcinoid tumor, an uncommon neuroendocrine neoplasm, is associated with serotonin overproduction as is more common small cell lung carcinoma (SCLC). alpha-Methyl-dopahydrazine (carbidopa), an inhibitor of the serotonin synthetic enzyme aromatic-L-amino acid decarboxylase, proved lethal to NCI-H727 lung carcinoid cells as well as NCI-H146 and NCI-H209 SCLC cells, but not to five other human tumor cell lines of differing origins [Gilbert JA, Frederick LM, Ames MM. The aromatic-L-amino acid decarboxylase inhibitor carbidopa is selectively cytotoxic to human pulmonary carcinoid and small cell lung carcinoma cells. Clin. Cancer Res. 2000;6:4365-72]. The mechanism of carbidopa cytotoxicity remained an unanswered question. We present data here that incubation of the catechol carbidopa (100 microM) in RPMI and DMEM culture media yielded molar equivalents of hydrogen peroxide (H2O2) within 2-4 h. Alkaline elution studies revealed carbidopa-dependent single-strand DNA breaks in sensitive carcinoid cells comparable to those induced by similar concentrations of H2O2. Neither compound induced significant DNA damage in carbidopa-resistant NCI-H460 large cell lung carcinoma cells. Furthermore, when carbidopa was incubated with a variety of tumor cell types, not only were decreased media H2O2 concentrations detected in the presence of cells, but cell lines least sensitive to carbidopa degraded exogenous H2O2 more rapidly than did sensitive cells. Implicated in these studies, pyruvate degraded H2O2 in RPMI in a dose- and time-dependent manner and reversed carbidopa-induced cytotoxicity to carcinoid cells. Extracellular pyruvate levels produced per h by resistant large cell lung carcinoma cells averaged four-fold that of sensitive carcinoid cells plated at equal density (24 h time course). Finally, carbidopa exposure (100 microM, 24 h) depleted extracellular pyruvate from sensitive carcinoid cells, but reduced pyruvate levels from resistant NCI-H460 cells less than 17%.

Transport of dopamine and levodopa and their interaction in COS-7 cells heterologously expressing monoamine neurotransmitter transporters and in monoaminergic cell lines PC12 and SK-N-SH.

The present study investigated the effects of levodopa, a precursor of dopamine (DA) therapeutically used for the treatment of Parkinson's disease, on DA transport in the two different systems, COS-7 cells heterologously expressing rat monoamine transporter cDNA and in monoaminergic cell lines PC12 and SK-N-SH. Levodopa enhanced uptake of [3H]DA and [3H]norepinephrine (NE) but not [3H]serotonin in the transfected COS-7 cells in a concentration-dependent manner. On the other hand, in PC12 and SK-N-SH cells where NET is functionally expressed, levodopa enhanced [3H]DA and [3H]NE uptake at low concentrations and inhibited the uptake at higher concentrations. The effects of levodopa on catecholamine transporters in the opposite direction suggest a different mechanism at the intra- and extracellular sites in a levodopa transport-dependent and independent manner.

Aromatic L-amino acid decarboxylase (AAAD) inhibitors as carcinoid tumor-imaging agents: synthesis of 18F-labeled alpha-fluoromethyl-6-fluoro-m-tyrosine (FM-6-FmT).

The aromatic L-amino acid decarboxylase (AAAD) enzyme is significantly upregulated in neuroendocrine tumors and, thus, would be a good target for PET imaging agents. Alpha-fluoromethyl-DOPA (FMDOPA) is one of the most potent irreversible AAAD inhibitor and its non-catechol derivative, alpha-fluoromethyl-m-tyrosine (FMmT), is a promising AAAD imaging agent. We synthesized FMmT and its direct electrophilic fluorination provided a mixture of products identified by NMR analysis after HPLC purification as 6-fluoro-, 2-fluoro- and 2,6-difluoro-derivatives of FMmT. Using rat striatal homogenates, alpha-fluoromethyl-6-fluoro-m-tyrosine (FM-6-FmT) was found to have AAAD inhibitory activity comparable to that of FMDOPA. Electrophilic radiofluorination of FMmT using [18F]AcOF gave 18F labeled 6-fluoro-, 2-fluoro- and 2,6-difluoro-FMmT derivatives in 22.0%, 21.9% and 8.5% radiochemical yields, respectively. Based on its proposed mechanism of inhibition, FM-6-[18F]FmT is expected to irreversibly bind to AAAD and, hence, could be used as a PET agent to image tumors of endocrine origin containing high concentrations of AAAD. Since FM-6-FmT lacks the catechol moiety, it is expected to be better than FMDOPA since it is not a substrate for catechol-O-methyltransferase.

The parkinsonian neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)) mediates release of l-3,4-dihydroxyphenylalanine (l-DOPA) and inhibition of l-DOPA decarboxylase in the rat striatum: a microdialysis study.

Reactive oxygen species (ROS) and reactive nitrogen species (RNS), particularly peroxynitrite, have been implicated as key participants in the dopaminergic neurotoxicity of 1-methyl-4-phenylpyridinium (MPP(+)). However, on the basis of available information, it is not clear whether the MPP(+)-induced overproduction of ROS and RNS occurs in the intraneuronal and/or extracellular compartment. Early steps in the neurotoxic mechanism evoked by MPP(+) include a profound dopaminergic energy impairment, which mediates a massive release of dopamine (DA), glutathione (GSH), and cysteine (CySH). In the event that MPP(+) mediates extracellular generation of ROS (such as superoxide and/or hydroxyl radicals) and/or peroxynitrite, released DA, GSH, and CySH should be oxidized forming thioethers of DA and disulfides. Using microdialysis experiments in which MPP(+) was perfused into the striatum of awake rats, the present study was unable to detect the presence of such biomarkers of extracellular ROS and/or RNS generation. However, MPP(+) induced a transient, concentration-dependent rise of extracellular l-3,4-dihydroxyphenylalanine (l-DOPA), identified on the basis of dialysate analysis using several HPLC methods and its conversion to DA by purified l-DOPA decarboxylase (DDC). Methamphetamine (30 mg/kg, i.p.) similarly caused a significant but transient rise of l-DOPA in the rat striatum. Antioxidants such as salicylate and mannitol had no effect on the MPP(+)-mediated elevation of extracellular l-DOPA, suggesting that it is not formed by nonenzymatic hydroxylation of l-tyrosine by ROS or RNS. Rather, in vivo, but not in vitro, MPP(+) caused rapid inhibition of DDC, which appears to result in intraneuronal accumulation and subsequent release of l-DOPA. Because l-DOPA can mediate l-glutamate release, as well as be an excitotoxin, the possibility is raised that l-DOPA may play a role in the dopaminergic neurotoxicity of MPP(+).