QUANTITATIVE HISTOCHEMICAL STUDIES OF FORMALDEHYDE-INDUCED PARENCHYMAL FLUORESCENCE FOLLOWING L-DOPA ADMINISTRATION

A microfluorimetric method is described for quantitative histochemical study of diffuse formaldehyde-induced fluorescence in the neuropil of various brain regions and was used to investigate the effects of L-DOPA in the rat and cat. Regional and species differences in the effects of L-DOPA on the CNS are reported and discussed. In the rat, suitable doses of L-DOPA led to increases in the intensity of fluorescence in the neuropil of the samples of caudate nucleus, which were greater than the increases in the neuropil of the other regions studied. Marked increases in the intensity of fluorescence in the neuropil of the cat brain samples were produced by doses of L-DOPA/kg body wt. which were too low to produce detectable increases in the fluorescence of the rat brain samples.     

3-METHOXY-4-HYDROXYPHENYLETHANOL IN THE RAT BRAIN

Abstract— The neutral dopamine metabolite, 3-methoxy4hydroxypenylethanol (MOPET) can be measured in the rat brain by a GLC method using a pentaflouropropionic derivative and electron capture detector. The identity of MOPET is verified by mass spectrographic analyses.
The endogenous level of MOPET of 16.6 ng/g whole rat brain can be raised more than four-fold by intraperitoneal injection of L-DOPA, dopamine or MOPET. In contrast intraventricular injection of dopamine or intraperitoneal injection of L-DOPA plus a peripheral decarboxylase inhibitor (Ro 4-4602)., results in small and insignificant increase bf MOPET in the CNS.
It is concluded that MOPET is probably of low significance to central dopamine metabolism and that MOPET found in the rat brain is predominantly of peripheral origin.     

The effect of intrastriatal injection of liposome-entrapped tyrosinase on the dopamine levels in the rat brain.

Parkinson's disease is a neurodegenerative disorder which is mainly characterized by degeneration of the dopaminergic cells in the nigro-striatal system. Due to a lowered L-tyrosine 3-monooxygenase activity, L-tyrosine is not sufficiently transformed to L-DOPA. To date the most common therapy is the administration of the dopamine precursor L-DOPA, with severe collateral effects. Therefore, the substitution of the lacking tyrosine hydroxylase with tyrosinase might be a novel therapeutical approach that would generate specifically L-DOPA from L-tyrosine. We present here evidence that stereotaxic injection of liposome-entrapped tyrosinase is able to significatively increase the levels of dopamine in the rat brain. The catecholamines L-DOPA, dopamine, L-epinephrine, L-norepinephrine were extracted by acid treatment from the brains and detected by HPLC.     

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.     

Effect of catecholamines and serotonin on RNA-synthesizing activity in isolated nuclei and chromatin of the rat brain and liver

It has been shown that intraperitoneal injections of L-DOPA cause an increase in the matrix activity of chromatin and stimulate the incorporation of [3H]uridine into the nuclear fraction of rat brain cells by 35%. In vitro studies have shown that preincubation of brain chromatin with L-DOPA diminishes the inhibiting effect of actinomycin D on RNA synthesis. It has been found that the rate of RNA synthesis in vitro depends on concentrations of catecholamines (L-DOPA, dopamine, norepinephrine) and serotonin.     

Striatal L-DOPA Decarboxylase Activity in Parkinson's Disease In Vivo: Implications for the Regulation of Dopamine Synthesis

Abstract: L-DOPA is a large neutral amino acid subject to transport out of, as well as into, brain tissue. Competition between dopamine synthesis and L-DOPA egress from striatum must favor L-DOPA egress if decarboxylation declines relatively more than transport in Parkinson's disease. To test this hypothesis, we injected patients with Parkinson's disease with a radidabeled analogue of L-DOPA and recorded regional brain radioactivity as a function of time by means of positron emission tomography. We simultaneously estimated the activity of the decarboxylating enzyme and the amino acid transport. In the striatum of patients, we found the L-DOPA decarboxylase activity to be reduced in the head of the caudate nucleus and the putamen. However, the rate of egress of the DOPA analogue was unaffected by the disease and thus inhibited dopamine synthesis more than predicted in the absence of L-DOPA egress.     

L-DOPA interaction with the brain tissue DNA of rats

The possibility of direct interaction of L-DOPA with DNA isolated from rat brain tissue has been considered. The spectral and viscosity data suggest conformational changes in L-DOPA-treated DNA in conditions excluding the accumulation of L-DOPA oxidation products.     

Regulatory pathways and uptake of L-DOPA by capillary cerebral endothelial cells, astrocytes, and neuronal cells

We examined the nature and regulation of the inward L-3,4-dihydroxyphenylalanine (L-DOPA) transporter in rat capillary cerebral endothelial (RBE4) cells, type 1 astrocytes (DI TNC1), and Neuro-2a neuroblastoma cells. In all three cell types, the inward transfer of L-DOPA was largely promoted through the 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid-sensitive and sodium-independent L-type amino acid transporter. Only in DI TNC1 cells was the effect of maneuvers that increase intracellular cAMP levels accompanied by increases in L-DOPA uptake. Also, only in DI TNC1 cells was the effect of the guanylyl cyclase inhibitor LY-83583 accompanied by a 65% increase in L-DOPA accumulation, whereas the nitric oxide donor sodium nitroprusside produced a 25% decrease in L-DOPA accumulation. In all three cell types, the Ca2+/calmodulin inhibitors calmidazolium and trifluoperazine inhibited L-DOPA uptake in a noncompetitive manner. Thapsigargin (1 and 3 microM) and A-23187 (1 and 3 microM) failed to alter L-DOPA accumulation in RBE4 and Neuro-2a cells but markedly increased L-DOPA uptake in DI TNC1 cells. We concluded that L-DOPA in RBE4, DI TNC1, and Neuro-2a cells is transported through the L-type amino acid transporter and appears to be under the control of Ca2+/calmodulin-mediated pathways. Astrocytes, however, are endowed with other processes that appear to regulate the accumulation of L-DOPA, responding positively to increases in intracellular Ca2+ and cAMP and to decreases in cGMP.     

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.     

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.     

The cytotoxicity of dopamine may be an artefact of cell culture

Administration of L-DOPA is commonly used to treat Parkinson's disease, yet controversy continues as to whether the dopamine arising from it aggravates neuronal loss. Several authors have reported cytotoxic effects of L-DOPA and dopamine on cultured cells, but others have not. In this report using the rat pheochromocytoma cell line PC12 and the M14 human melanoma cell line we show that dopamine-mediated cell death is not specific for neuronal cells. Moreover, our data show that both L-DOPA and dopamine interact with commonly used cell culture media, undergoing oxidation to generate hydrogen peroxide and dopamine semiquinones/quinones. Catalase and reduced glutathione could protect against cytotoxicity. These results suggest that caution needs to be employed when using cell culture studies to predict effects of L-DOPA and/or dopamine in vivo because of the extracellular generation of reactive species in the culture media.     

Missing proof of cooperative synthesis of dopamine by non-dopaminergic neurons

L-DOPA accumulation in the extracellular medium was detected when the transfer of L-DOPA from the neurons containing tyrosine hydroxylase to the neurons containing aromatic L-amino acid decarboxylase was blocked, under conditions of inhibition of the L-DOPA degradation enzyme. Thus, the missing proof confirming the existence of cooperative synthesis of dopamine by neurons non-dopaminergic was obtained.     

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.     

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.     

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.     

Dopamine antibodies in the pathogenesis of parkinsonism]

L-DOPA and dopamine (DA) binding antibodies were found in the blood serum of Parkinsonian patients and middle-aged and elderly normal persons. DA-binding serum gamma-globulins of parkinsonian patients injected into rat caudate nuclei induced the pathogenetic mechanism of Parkinson's syndrome (generator of pathologically enhanced excitation) in these brain part and evoked main parkinsonian symptoms (oligokinesia, rigidity, tremor). The serum gamma-globulins of Parkinsonian patients without Da-antibodies caused less pronounced EEG disturbances. Parkinsonian symptoms developed rarely and were shorter and less pronounced compared with the DA-antibody effect. The DA binding antibodies role in Parkinson's syndrome pathogenesis and is L-DOPA therapeutic tolerance formation was discussed.     

Nigrostriatal Dopaminergic Neurons Remain Undamaged in Rats Given High Doses of l-DOPA and Carbidopa Chronically

Rats were fed maximally tolerated doses of L-3,4-Dihydroxyphenylalanine (L-DOPA) and carbidopa daily for 120 days in order to achieve a sustained elevation in brain dopamine levels. Some animals were also given buthionine sulfoximine, a gamma-glutamylcysteine synthetase inhibitor, in an unsuccessful effort to reduce brain glutathione contents. L-DOPA- and carbidopa-treated animals displayed no behavioral changes suggestive of nigrostriatal dopaminergic neuronal loss. When sacrificed 60 days after L-DOPA treatment ended, all rats had normal tyrosine hydroxylase activities and dopamine contents in their striata, and cell counts were normal in the substantia nigra. It therefore seems unlikely that a model of Parkinson's disease, suitable for exploring the etiological importance of glutathione deficiency, can be produced in rats merely by administering the largest tolerable doses of L-DOPA.     

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.     

Inhibition of calcium-independent luminal uptake of L-dopa by calmodulin antagonists in immortalized rat capillary cerebral endothelial cells

The present study examined the involvement of protein kinase A (PKA), protein kinase G (PKG), protein kinase C (PKC), protein tyrosine kinase (PTK) and Ca(2+)/calmodulin mediated pathways on the luminal uptake of L-DOPA through the L-type amino acid transporter in immortalized rat capillary cerebral endothelial (REB-4) cells. Non-linear analysis of the saturation curve for L-DOPA revealed a K(m)value (in microM) of 71+/-9 and a V(max)value of 17+/-1 (in nmol mg protein/6 min). L-DOPA uptake at the luminal cell border was a sodium-independent process and insensitive to N-(methylamino)-isobutyric acid (MeAIB, 1 m m), but sensitive to 2-aminobicyclo(2,2,1)-heptane-2-carboxylic acid (BHC, IC(50)=140 microM). The Ca(2+)/calmodulin inhibitors calmidazolium and trifluoperazine inhibited L-DOPA (2.5 microM) uptake with IC(50)s of 23 and 33 microM, respectively. The inhibitory effect of BHC on the accumulation of L-DOPA was of the competitive type, whereas that of calmidazolium and trifluoperazine was of the non-competitive type. Modulators of PKA (cyclic AMP, forskolin, isobutylmethylxanthine and cholera toxin), PKG (cyclic GMP, zaprinast, LY 83583 and sodium nitroprusside), PKC (phorbol 12,13-dibutyrate, staurosporine and chelerythrine) and PTK (genistein and tyrphostin 25) failed to affect the accumulation of a non-saturating (2.5 microM) concentration of L-DOPA. It is concluded that L-DOPA uptake in RBE-4 cells is promoted through the L-type amino acid transporter and appears to be under the control of calmodulin mediated pathways.     

Immunohistochemistry of endogenous L-DOPA in the rat posterior hypothalamus

The aim of this work was to study L-DOPA-containing neuronal structures of the rat posterior and dorsal hypothalamus by means of immunohistochemistry using antiserum against glutaraldehyde conjugated L-DOPA. Aspects and distribution of L-DOPA immunoreaction among cells of the supramammillary nucleus and the A11, A13c and A13 cell groups are described and compared to dopamine immunoreactivity, mainly through a double colored labelling procedure employing a color modification of the DAB reaction by metallic ions. Differences between L-DOPA and dopamine stainings within cell groups as the presence of cells with predominant or exclusive L-DOPA coloration are tentatively explained under the light of previous findings using immunohistochemistry of catecholamines synthesizing enzymes and catecholamines histofluorescence.