Dopamine synthesis by non-dopaminergic neurons of the rat fetus arquate nucleus

Our hypothesis was tested in respect to dopamine synthesis by non-dopaminergic neurons expressing individual complementary enzymes of the DA synthetic pathway. According to the hypothesis, L-dihydroxyphenylalanine (L-DOPA) synthesised in tyrosine hydroxylase(TH)-expressing neurons for conversion to dopamine. The mediobasal hypothalamus of rats on the 21st embryonic day was used as an experimental model. The fetal substantia nigra containing dopaminergic neurons served as control. Dopamine and L-DOPA were measured by high performance liquid chromatography in cell extracts and incubation medium in presence or absence of L-tyrosine. L-tyrosine administration increased L-DOPA synthesis in the mediobasal hypothalamus and substantia nigra. Moreover, L-tyrosine provoked an increase of dopamine synthesis in substantia nigra and a decrease in the mediobasal hypothalamus. This is, probably, due to an L-tyrosine-induced competitive inhibition of the L-DOPA transport to monoenzymatic AADC neurons after its release from the monoenzymatic TH neurons. This study provides a convincing evidence of dopamine synthesis by non-dopaminergic neurons expressing TH or AADC, in cooperation.     

Cooperative synthesis of dopamine in rat mediobasal hypothalamus as a compensatory mechanism in hyperprolactinemia

Dopamine (DA), synthesized in the mediobasal hypothalamus by dopaminergic neurons containing two enzymes of DA synthesis–tyrosine hydroxylase and decarboxylase of aromatic L-amino acids, or by monoenzymatic non-dopaminergic neurons containing one DA synthesis enzyme in cooperation, is known to have an inhibitory effect on prolactin secretion. Deterioration of this inhibitory control leads to an increase in prolactin concentration in the blood and to the development of hyperprolactinemia syndrome. In a rat model of hyperprolactinemia induced by administration of a neurotoxin causing degeneration of dopaminergic and noradrenergic neurons, the level of DA first decreases, leading to an increase in prolactin level (decompensation stage), while later both levels are restored to normal (compensation stage). However, the mechanism of such compensation is still not clear. The aim of the present study was to analyze whether the increase in cooperative synthesis of DA by monoenzymatic neurons during hyperprolactinemia is a manifestation of a compensatory mechanism representing a particular case of neuroplasticity. The level of cooperative synthesis in the hyperprolactinemia model and in the control was estimated as the level of synthesis of DA and L-dihydroxyphenylalanine (L-DOPA)–an intermediate product of DA synthesis, when L-DOPA transfer from neurons containing tyrosine hydroxylase into neurons containing aromatic L-amino acid decarboxylase is inhibited. The level of DA synthesis during the decompensation stage was not changed, while during the compensation stage it was lower than the control. Along with a reduction in DA level, during the compensation stage an increase in the extracellular L-DOPA level in the medium was detected. Thus, the compensation of DA deficiency after degeneration of dopaminergic neurons in the mediobasal hypothalamus is due to the increase in cooperative synthesis of DA by monoenzymatic neurons containing one of the complementary enzymes of the DA synthesis pathway.     

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.     

Expression of the enzymes of dopamine synthesis in non-dopaminergic neurons: functional significance and regulation

Dopamine(DA), the most widely distributed in the nervous system and functionally important chemical signal, is synthesized in DA-ergic neurons from L-tyrosine by means of two enzymes, tyrosine hydroxylase (TH) and aromatic L-amino acid decarboxylase (AADC). Apart from the enzymes, specific DA transporter is an attribute of DA-ergic neurons. In the mid eighties of the last century, in addition to DA-ergic neurons, those expressing only one enzyme, TH or AADC, have been discovered. These "monoenzymatic" neurons occurred to be more numerous and more widely distributed in the brain compared to DA-ergic neurons that manifests their wide involvement to the brain functioning. It has been demonstrated that the monoenzymatic neurons expressing complementary enzymes of DA synthesis produce this neurotransmitter in cooperation. In this case, L-tyrosine is transformed to L-DOPA in TH containing neurons that is followed by L-DOPA release and uptake from the intercellular space to AADC containing neurons for DA synthesis. Moreover, the L-DOPA uptake to DA-ergic or serotoninergic neurons results either in the increase or the onset of DA synthesis in addition to serotonin, respectively. The expression of the enzymes of DA synthesis in non-dopaminergic neurons is one of the adaptive reactions serving to compensate the functional insufficiency of DA-ergic neurons. For instance, hyperprolactinemia and the deficiency of DA, prolactin-inhibiting hormone, which is developed under degeneration of DA-ergic neurons of the arcuate nucleus, are compensated with time due to the increase of the number of monoenzymatic neurons and cooperative synthesis of DA in the nucleus. It is supposed that the same compensatory cooperative synthesis of DA is turned on under the degeneration of DA-ergic neurons of the nigrostriatal system that is manifested by the appearance of non-dopaminergic neurons expressing enzymes of DA synthesis in the deafferentated striatum. The expression of the enzymes of DA synthesis in non-dopaminergic neurons is under the control by intercellular signals, catecholamines, neurotrophic (growth) factors and, perhaps, hormones. Thus, non-dopaminergic monoenzymatic neurons expressing enzymes of DA synthesis produce this neurotransmitter in cooperation that is a compensatory reaction under functional insufficiency of DA-ergic neurons, in neurodegenerative diseases, hyperprolactinemia and Parkinson's disease, in particular.     

L-3,4-Dihydroxyphenylalanine Synthesis by Genetically Modified Schwann Cells

We have investigated whether Schwann cells can be modified by gene transfer to synthesize L-3,4-dihydroxyphenylalanine (L-DOPA), the immediate precursor in the formation of dopamine. By using a retrovirus containing a rat tyrosine hydroxylase (TH) cDNA, we established an immortalized rodent Schwann cell line that stably expressed high levels of TH and secreted L-DOPA in vitro when supplied with tyrosine and the essential cofactor biopterin. We also infected primary Schwann cells and demonstrated that cells expressing TH secreted L-DOPA while maintaining their capacity to myelinate neurons in vitro. This study indicate that it may be feasible to utilize autotransplantation of genetically modified Schwann cells to alleviate the movement disorders in Parkinson's disease.     

Ontogeny of cholinergic ganglionic cells which take up L-3,4-dihydroxyphenylalanine and contain vasoactive intestinal polypeptide in the mouse tongue

During the prenatal development of the mouse tongue, the intralingual ganglion was investigated histochemically and immunohistochemically for acetylcholinesterase (AChE) activity, the capacity to take up L-3,4-dihydroxyphenylalanine (L-DOPA) and vasoactive intestinal polypeptide (VIP)-like immunoreactivity. AChE-positive cells were first observed at gestational day 12, and AChE-positive neurons which take up L-DOPA were found at gestational day 14, while VIP-like immunoreactive neurons were not seen until gestational day 16. The present results suggested that the cholinergic neurons within the mouse tongue can take up L-DOPA from gestational day 14 and synthesize VIP from gestational day 16.     

Catechol-O-methyltransferase inhibition protects against 3,4-dihydroxyphenylalanine (DOPA) toxicity in primary mesencephalic cultures: new insights into levodopa toxicity

Inhibition of catechol-O-methyltransferase (COMT) has protective effects on levodopa (L-DOPA), but not D-DOPA toxicity towards dopamine (DA) neurons in rat primary mesencephalic cultures [Mol. Pharmacol. 57 (2000) 589]. Here, we extend our recent studies to elucidate the mechanisms of these protective effects. Thus, we investigated the effects of all main L-DOPA/DA metabolites on survival of tyrosine hydroxylase immunoreactive (THir) neurons in primary rat mesencephalic cultures. 3-O-Methyldopa, homovanillic acid, dihydroxyphenyl acetate and 3-methoxytyramine had no effects at concentrations up to 300 micro M after 24h, whereas DA was more toxic than L-DOPA with toxicity at concentrations of >or=1 micro M. The coenzyme of COMT, S-adenosyl-L-methionine (SAM), and its demethylated product S-adenosylhomocystein caused no relevant alteration of THir neuron survival or L-DOPA toxicity. In contrast, inhibition of SAM synthesis by selenomethionine showed time- and dose-dependent increase of THir neuron survival, but did not affect L-DOPA toxicity. L-DOPA-induced lipid peroxidation in mesencephalic cultures was not modified by the COMT inhibitor Ro 41-0960 (1 micro M). Increased contamination of the cultures with glial cells attenuated L- and D-DOPA toxicity, but caused significant enhancement of protection by COMT inhibitors against L-DOPA toxicity only. Investigations of L-DOPA uptake in rat striatal cultures using HPLC revealed a significant reduction of extracellular L-DOPA concentrations by Ro 41-0960. Our data confirm that L-DOPA toxicity towards DA neurons is mediated by an autooxidative process, which is attenuated by glial cells. In addition, we demonstrate a second mechanism of L-DOPA toxicity in vitro mediated by a COMT- and glia-dependent pathway, which is blocked by COMT inhibitors, most likely due to enhanced glial uptake of L-DOPA.     

L-DOPA Neurotoxicity Is Mediated by Up-Regulation of DMT1?IRE Expression

Background

The mechanisms underlying neurotoxicity caused by L-DOPA are not yet completely known. Based on recent findings, we speculated that the increased expression of divalent metal transporter 1 without iron-response element (DMT1−IRE) induced by L-DOPA might play a critical role in the development of L-DOPA neurotoxicity. To test this hypothesis, we investigated the effects of astrocyte-conditioned medium (ACM) and siRNA DMT-IRE on L-DOPA neurotoxicity in cortical neurons.

Methods and Findings

We demonstrated that neurons treated with L-DOPA have a significant dose-dependent decrease in neuronal viability (MTT Assay) and increase in iron content (using a graphite furnace atomic absorption spectrophotometer), DMT1−IRE expression (Western blot analysis) and ferrous iron (55Fe(II)) uptake. Neurons incubated in ACM with or without L-DOPA had no significant differences in their morphology, Hoechst-33342 staining or viability. Also, ACM significantly inhibited the effects of L-DOPA on neuronal iron content as well as DMT1−IRE expression. In addition, we demonstrated that infection of neurons with siRNA DMT-IRE led to a significant decrease in DMT1−IRE expression as well as L-DOPA neurotoxicity.

Conclusion

The up-regulation of DMT1−IRE and the increase in DMT1−IRE-mediated iron influx play a key role in L-DOPA neurotoxicity in cortical neurons.     

Striatal Astrocytes Act as a Reservoir for L-DOPA

L-DOPA is therapeutically efficacious in patients with Parkinson’s disease (PD), although dopamine (DA) neurons are severely degenerated. Since cortical astrocytes express neutral amino acid transporter (LAT) and DA transporter (DAT), the uptake and metabolism of L-DOPA and DA in striatal astrocytes may influence their availability in the dopaminergic system of PD. To assess possible L-DOPA- and DA-uptake and metabolic properties of striatal astrocytes, we examined the expression of L-DOPA, DA and DAT in striatal astrocytes of hemi-parkinsonian model rats after repeated L-DOPA administration, and measured the contents of L-DOPA, DA and their metabolite in primary cultured striatal astrocytes after L-DOPA/DA treatment. Repeated injections of L-DOPA induced apparent L-DOPA- and DA-immunoreactivities and marked expression of DAT in reactive astrocytes on the lesioned side of the striatum in hemi-parkinsonian rats. Exposure to DA for 4h significantly increased the levels of DA and its metabolite DOPAC in cultured striatal astrocytes. L-DOPA was also markedly increased in cultured striatal astrocytes after 4-h L-DOPA exposure, but DA was not detected 4 or 8h after L-DOPA treatment, despite the expression of aromatic amino acid decarboxylase in astrocytes. Furthermore, the intracellular level of L-DOPA in cultured striatal astrocytes decreased rapidly after removal of extracellular L-DOPA. The results suggest that DA uptaken into striatal astrocytes is rapidly metabolized and that striatal astrocytes act as a reservoir of L-DOPA that govern the uptake or release of L-DOPA depending on extracellular L-DOPA concentration, but are less capable of converting L-DOPA to DA.     

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.     

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.     

Production of L-DOPA by cell suspension cultures of Mucuna pruriens

The endogenous synthesis of 3-(3,4-dihydroxyphenyl)-L-alanine (L-DOPA) by cell suspension cultures of Mucuna pruriens was found to be influenced by several environmental parameters. The nature of the nitrogen source as well as the concentration of nitrogen containing salts, sucrose and phosphate in the culture medium were found to affect the biosynthesis of L-DOPA. Addition of 2, 4-dichlorophenoxyacetic acid to the medium suppressed L-DOPA production; continuous illumination of the cultures had a strong beneficial effect on L-DOPA production. L-DOPA was accumulated intracellularly by the cell suspension cultures. These observations further demonstrate that for certain products of plant cell suspensions product synthesis can be manipulated by a proper selection of specified nutrients.     

The brain is one of the sources of L-dihydroxyphenylalanine in systemic circulation in fetuses and neonatal rats

The performed study was aimed at checking our hypothesis that the developing brain is a source of L-dihydroxyphenylalanine (L-DOPA), a precursor of dopamine in the total circulation system. At the initial stage, the L-DOPA concentration in peripheral blood was analyzed at the 18th and 21st embryonal days (E18 and E21), at the 3rd postnatal day (P3), and at the prepubertal period (P30). The highest L-DOPA concentration was revealed at the perinatal period, while decreased 4–12 times for the first month of life. The subsequent analysis of dynamics of the total blood L-DOPA content showed that maintenance of the constant L-DOPA concentration at the perinatal period on the background of a gradual increase of the blood serum volume is due to a rise of its secretion. At the postnatal period (P3–P30), the blood L-DOPA content increased twice in males, whereas it decreased to the same extent in females. Analysis of the L-DOPA concentration in two most important brain centers, hypothalamus and mesencephalon-rhombencephalon, showed its twofold decrease in hypothalamus during E18–E21 of development; then it slightly increased from E21 to P3 and fell 4–5 times by P30. In mesencephalon-rhombencephalon, the L-DOPA concentration was slightly reduced from E18 to E21 (only in females), while on P3 it returned to the E18 level and decreased 7–9 times by P30. The direct proof for the L-DOPA release from the developing brain into the systemic circulation follows from comparison of the blood L-DOPA concentration in shamoperated and encephalectomized rat fetuses after mechanical destruction of neurons of the two abovementioned most important dopaminergic centers. Thus, encephalectomy led to a twofold reduction of the blood L-DOPA concentration (statistically significant differences were observed only in females). Thus, the work presents evidence that the developing brain is one of L-DOPA sources in the total circulation system in rats during prenatal and early postnatal periods of ontogenesis.     

Pharmacologic study of adrenergic mechanisms of compensatory hypertrophy of the ovary]

Treatment of hemicastrated adult female rats with adrenoblockers, chlorpromazine and alpha-methyl-DOPA decreased the ovarian compensatory hypertrophy (OCH) and prevented the stilbestrol suppression of the OCH. Disulfiram (dophamine-beta-hydroxylase inhibitor) potentiated the stilbestrol suppression of the OCH. Small doses of L-DOPA stimulated the OCH, and high doses of L-DOPA and dilantin failed to act on the ACH, but potentiated the estrogeninduced OCH inhibition. It is suggested that the FSH secretion was mediated by the release of norepinephrine in the central adrenergic neurons and that the estrogen action inhibiting the FSH secretion was mediated through the stimulation of dophamine release.     

Ethanol triggers sphingosine 1-phosphate elevation along with neuroapoptosis in the developing mouse brain

Recent studies suggest that l-3,4 dihydroxyphenylalanine (L-DOPA)-induced dyskinesia (LID), a severe complication of conventional L-DOPA therapy of Parkinson's disease, may be caused by dopamine (DA) release originating in serotonergic neurons. To evaluate the in vivo effect of a 5-HT(1A) agonist [(±)-8-hydroxy-2-(dipropylamino) tetralin hydrobromide, 8-OHDPAT] on the L-DOPA-induced increase in extracellular DA and decrease in [(11) C]raclopride binding in an animal model of advanced Parkinson's disease and LID, we measured extracellular DA in response to L-DOPA or a combination of L-DOPA and the 5-HT(1A) agonist, 8-OHDPAT, with microdialysis, and determined [(11) C]raclopride binding to DA receptors, with micro-positron emission tomography, as the surrogate marker of DA release. Rats with unilateral 6-hydroxydopamine lesions had micro-positron emission tomography scans with [(11) C]raclopride at baseline and after two pharmacological challenges with L-DOPA?+?benserazide with or without 8-OHDPAT co-treatment. Identical challenge regimens were used with the subsequent microdialysis concomitant with ratings of LID severity. The baseline increase of [(11) C]raclopride-binding potential (BP(ND) ) in lesioned striatum was eliminated by the L-DOPA challenge, while the concurrent administration of 8-OHDPAT prevented this L-DOPA-induced displacement of [(11) C]raclopride significantly in lesioned ventral striatum and near significantly in the dorsal striatum. With microdialysis, the L-DOPA challenge raised the extracellular DA in parallel with the emergence of strong LID. Co-treatment with 8-OHDPAT significantly attenuated the release of extracellular DA and LID. The 8-OHDPAT co-treatment reversed the L-DOPA-induced decrease of [(11) C]raclopride binding and increase of extracellular DA and reduced the severity of LID. The reversal of the effect of L-DOPA on [(11) C]raclopride binding, extracellular DA and LID by 5-HT agonist administration is consistent with the notion that part of the DA increase associated with LID originates in serotonergic neurons.     

Some parasympathetic neurons in the guinea-pig heart express aspects of the catecholaminergic phenotype in vivo

Summary In a histochemical study of intrinsic cardiac ganglia of the guinea-pig in whole-mount preparations, it was found that some 70–80% of the neurons express aspects of the catecholaminergic phenotype. These neurons have an uptake mechanism for L-DOPA, and contain the enzymes for converting L-DOPA, (but not D-DOPA) to dopamine and noradrenaline, i.e. aromatic L-aminoacid decarboxylase and dopamine -hydroxylase. Monoamine oxidase is also present within some of the neurons. In these respects, the neurons resemble noradrenergic neurons of sympathetic ganglia, so we refer to them as intrinsic cardiac amine-handling neurons. However, these neurons do not contain tyrosine hydroxylase and show little or no histochemically detectable uptake of -methyldopa, dopamine or noradrenaline, even after depletion of endogenous stores of amines by pre-treatment with reserpine. Noradrenergic fibres from the sympathetic chain form pericellular baskets around nerve cell bodies. The uptake of L-DOPA into nerve cell bodies is not prevented by treatment with 6-hydroxydopamine sufficient to cause transmitter-depletion or degeneration of the extrinsic noradrenergic fibres. Such degeneration experiments suggest that axons of the amine-handling neurons project to cardiac muscle, blood vessels and other intrinsic neurons. The cardiac neurons do not show any immunohistochemically detectable serotonergic characteristics; there is no evidence for uptake of the precursors L-tryptophan and 5-hydroxytryptophan or 5-HT itself, whereas the extrinsic noradrer ergic nerve fibres within the ganglia can take up 5-HT when it is applied in high concentrations.Abbreviations AChE acetylcholinesterase - DBH-IR dopamine -hydroxylase-like immunoreactivity - L-DOPA L-dihydroxyphenylalamine - 5-HT-IR 5-hydroxytryptamine-like immunoreactivity - 6-OHDA 6-hydroxydopamine - methyldopa L--methyl-dihydroxyphenylalanine - MAO monoamine oxidase - NPY neuropeptide Y - SIF small intensely fluorescent cells - TH-IR tyrosine hydroxylase-like immunoreactivity - VIP vasoactive intestinal polypeptide     

Production of L-DOPA from cell suspension culture of Mucuna pruriens f. pruriens

Summary Production of L-DOPA was studied in cell suspension culture of Mucuna pruriens f. pruriens. Suspension culture was established in MSI medium composed of half concentration of Murashige and Skoog's salts and 2% sucrose. A two-stage cell suspension culture was developed for enhanced accumulation of L-DOPA. In the first stage, the culture system was composed of MSI medium without CaCl, which was suitable for cell growth and in the second stage MSI medium containing 42.5 mg.l^–1 KH₂PO₄ and 4% sucrose favoured L-DOPA production. A discernible higher production of L-DOPA was obtained in this two-stage cell suspension culture in comparison to single stage culture.     

Toxic and Protective Effects of l-DOPA on Mesencephalic Cell Cultures

Abstract: The autoxidation of L-DOPA or dopamine (DA) and the metabolism of DA by monoamine oxidase generate a spectrum of toxic species, namely, hydrogen peroxide, oxy radicals, semiquinones, and quinones. When primary dissociated cultures of rat mesencephalon were incubated with L-DOPA (200 μ M ) for 48 h, the number of tyrosine hydroxylase-positive neurons (DA neurons) was reduced to 69.7% of control values, accompanied by a decrease in [³H]DA uptake to 42.3% of control values; the remaining DA neurons exhibited reduced neurite length and overall deterioration. Lack of simultaneous change in the number of neurons stained with neuron-specific enolase indicated that toxicity was relatively specific for DA neurons. At the same time, the level of GSH, a major cellular antioxidant, rose to 125.2% of control values. Thus, exposure of mesencephalic cultures to L-DOPA results in both damaging and antioxidant actions. Ascorbate (200 μ M ), an antioxidant, prevented the rise in GSH. The effect of ascorbate on GSH points to an oxidative signal to initiate the rise in GSH content. On the other hand, neither inhibition of monoamine oxidase with pargyline nor addition of superoxide dismutase or catalase to the culture medium prevented the rise in GSH level or the loss in [³H]DA uptake. The latter results tend to exclude the products of monoamine oxidase activity or the presence of hydrogen peroxide or superoxide in the medium as responsible agents for the rise in GSH or neuronal toxicity. In cultures treated with L-buthionine sulfoximine (L-BSO), an inhibitor of GSH synthesis, l-DOPA prevented cell death by L-BSO.     

The hypothermic effect of L-dopa in the rat

L-DOPA given to rats together with a peripheral decarboxylase inhibitor Ro 4-4602, caused a fall in rectal temperature, a decrease of the brain 5-HT level and an increase of the 5-HIAA level. These effects were counteracted by pretreatment with p-chlorophenylalanine. The dopamine receptor blocking agents, pimozide and spiroperidol, did not influence the rectal temperature in normal rats and did not antagonize the hypothermia induced by L-DOPA. It was suggested that the L-DOPA-hypothermia does not involve central dopaminergic neurons. It may result from the release of 5-hydroxytryptamine from its storage sites.     

5-s-Cysteinyl-conjugates of catecholamines induce cell damage,extensive DNA base modification and increases in caspase-3 activity in neurons

A decrease in reduced glutathione levels in dopamine containing nigral cells in Parkinson's disease may result from the formation of cysteinyl-adducts of catecholamines, which in turn exert toxicity on nigral cells. We show that exposure of neurons (CSM 14.1) to 5-S-cysteinyl conjugates of dopamine, L-DOPA, DOPAC or DHMA causes neuronal damage, increases in oxidative DNA base modification and an elevation of caspase-3 activity in cells. Damage to neurons was apparent 12-48 h of post-exposure and there were increases in caspase-3 activity in neurons after 6 h. These changes were paralleled by large increases in pyrimidine and purine base oxidation products, such as 8-OH-guanine suggesting that 5-S-cysteinyl conjugates of catecholamines are capable of diffusing into cells and stimulating the formation of reactive oxygen species (ROS), which may then lead to a mechanism of cell damage involving caspase-3. Indeed, intracellular ROS were observed to rise sharply on exposure to the conjugates. These results suggest one mechanism by which oxidative stress may occur in the substantia nigra in Parkinson's disease.