Effects of L-dopa treatment on methylation in mouse brain: implications for the side effects of L-dopa

The effects of L-dopa on methylation process in the mouse brain were investigated. The study is based on recent findings that methylation may play an important role in Parkinson's disease (PD) and in the actions of L-dopa. The methyl donor, S-adenosylmethionine (SAM) and a product of SAM, methyl beta-carboline, were shown to cause PD-like symptoms, when injected into the brain of animals. Furthermore, large amounts of 3-O-methyl dopa, the methyl product of L-dopa, are produced in PD patients receiving L-dopa treatment, and L-dopa induces methionine adenosyl transferase, the enzyme that produces SAM. The results show that, at 0.5 hr, L-dopa (100 mg/kg) decreased the methyl donor, S-adenosylmethionine (SAM) by 36%, increased its metabolite S-adenosylhomocysteine (SAH) by 89% and increased methylation (SAH/SAM) by about 200%. All parameters returned to control values within 4 hr. But 2, 3 and 4 consecutive injections of L-dopa, given at 45 min intervals, depleted SAM by 60, 64 and 76% and increased SAM/SAH to 818, 896, and 1524%. L-dopa (50, 100 and 200 mg/kg) dose-dependently depleted SAM from 24.9 +/- 1.7 nmol/g to 13.0 +/- 0.8, 14.7 +/- 0.8 and 7.7 +/- 0.7 nmol/g, and increased SAH from 1.88 +/- 0.14 to 3.43 +/- 0.26, 4.22 +/- 0.32 and 6.21 +/- 0.40 nmol/g. Brain L-dopa was increased to 326, 335 and 779%, dopamine to 138, 116 and 217% and SAH/SAM to 354, 392 and 1101%. The data show that L-dopa depletes SAM, and increases methylation 4-5 times more than dopamine, therefore, methylation may play a role in the actions of L-dopa. This and other studies suggest that the high level of utilization of methyl group by L-dopa leads to the induction of enzymes to replenish SAM and to increase the methylation of L-dopa as well as DA. These changes may be involved in the side effects of L-dopa.     

Regional distribution of catecholamines in rat brain after L-3-O-methyldopa and L-dopa

The distribution of labelled dopamine (DA) and noradrenaline (NA) in the various brain regions of the rat was similar after administration of L-¹⁴C-3-0-methyldopa (OMD) or L-¹⁴C-dopa, DA showing the greatest accumulation in the striatum and NA in the hypothalamus. The concentration of catecholamines 2 hours after OMD amounted to 2–15 % of those found after L-dopa. In the whole brain, the cerebral catecholamines formed from dopa decreased more rapidly than those originating from OMD. In conclusion, OMD is a precursor of cerebral catecholamines; however, it is less effective than dopa.     

Effects of Catechol-O-Methyltransferase Inhibitors and L-3,4-Dihydroxyphenylalanine With or Without Carbidopa on Extracellular Dopamine in Rat Striatum

Abstract: The effects of two new catechol- O -methyltransferase (COMT) inhibitors, OR-611 and Ro 40-7592, in combination with L-3,4-dihydroxyphenylalanine (L-dopa) with or without carbidopa on extracellular levels of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 3- O -methyldopa (3-OMD), and 5-hydroxyindoleacetic acid in rat striatum were studied. A dose of 10 mg/kg i.p. of Ro 40-7592 alone, in contrast to the same dose of OR-611, decreased the dialysate level of HVA and increased that of DOPAC; this dose was thus used to differentiate between the effects of central and peripheral COMT inhibition. L-Dopa (50 mg/kg i.p.) alone slightly increased extracellular levels of DA, DOPAC, and HVA. The effects of L-dopa were potentiated by carbidopa (50 mg/kg i.p.), and even 3-OMD levels in dialysate samples became detectable. Both OR-611 and Ro 40-7592 significantly further increased the DA and DOPAC efflux from striatum produced by L-dopa. This increase was more pronounced when carbidopa was added to the treatment. OR-611 did not modify the effect of L-dopa or carbidopa/L-dopa on dialysate HVA levels, whereas Ro 40-7592 markedly reduced those levels. Both OR-611 and Ro 40-7592 very clearly suppressed dialysate 3-OMD levels produced by carbidopa/L-dopa. Ro 40-7592 was more effective than OR-611 in potentiating the effects of L-dopa or carbidopa/L-dopa. These in vivo data show that the new COMT inhibitors markedly inhibit the O -methylation of L-dopa and increase its availability to brain, which is reflected as increased DA formation. A significant effect can be achieved even by inhibiting only the peripheral COMT activity. The data suggest that COMT inhibitors may be of clinical importance as adjuncts in the treatment of Parkinson's disease.     

Effects of aging on LH and prolactin after LHRL L-dopa, methyl-dopa, and stress in male rat.

Hypothalamic-pituitary control of prolactin and LH secretion was tested in young (4-6 months) and aged (22-30 months) male Long-Evans rats given L-dopa, methyl dopa, LHRH, or stress treatments. Pretreatment serum LH levels were consistently higher in young than in the aged groups. The increase in serum LH after LHRH injection was only about half as much in aged as compared to young control males. Although acute stress caused a prompt increase in serum LH in young male rats, this treatment was without effect in the aged group. Methyl dopa treatment stimulated serum prolactin secretion in both young and old rats. Although L-dopa treatment caused a reduction in serum prolactin in both age groups, the sensitivity, magnitude, and duration of the reduction was smaller in the aged rats.     

Dopaminergic modulation of footshock induced aggression in paired rats.

Footshock induced aggression (FIA) was induced in weight matched paired rats and three paradigms of aggressive behaviour was recorded, namely, the latency to fight (LF), total period of physical contact (TPP) and cumulative aggression scores (CAS). Dopamine (DA), administered centrally, and peripherally administered L-dopa (with benserazide, a peripheral decarboxylase inhibitor), a DA precursor, and the postsynaptic D2 receptor agonists, apomorphine, N-n-propyl-norapomorphine (PNA), bromocriptine, lisuride and pergolide, induced a dose-related facilitation of FIA characterized by decrease in LF and increase in TPP and CAS. However, the DA presynaptic receptor agonist, BHT-920, induced a biphasic effect with inhibition of FIA being induced by a lower dose and facilitation of the aggressive behaviour produced by a higher dose. The postsynaptic D2 receptor antagonists, haloperidol, spiperone and pimozide, induced a dose-related attenuation of FIA, an effect not seen with domperidone, a peripheral DA receptor antagonist. The results indicate that central dopaminergic postsynaptic D2 receptors have a modulatory facilitative effect on FIA, while the presynaptic DA autoreceptors mitigate aggressive behaviour. However, the presynaptic DA receptor agonist, BHT-920, appears to lose its receptor specificity on dose increment. Long term administration of haloperidol, followed by withdrawal, or desipramine, induced per se augmentation of FIA and potentiated the aggression-facilitative effects of L-dopa, apomorphine and PNA. Since both these treatments are known to induce supersensitivity of central postsynaptic dopamine D2 receptors, the effects are likely to be related to augmented function of dopamine neurones. The findings, in conjunction with a recent report from this laboratory indicating an increase in rat brain DA levels in FIA, support the contention that the central DA system has a facilitative effect on FIA.     

Evidence for L-dopa incorporation into cell proteins in patients treated with levodopa

Levodopa (L-dopa) is the most widely used agent for the symptomatic relief of Parkinson's disease. There is concern that chronic L-dopa treatment may be detrimental, with some studies suggesting that L-dopa may be neurotoxic. A potentially important mechanism whereby L-dopa may exert neurotoxic effects has been overlooked: that of the incorporation of L-dopa into proteins by protein synthesis. L-Dopa competes with tyrosine as a substrate in protein synthesis in vitro. We provide evidence that L-dopa can also be incorporated into proteins in vivo. Blood from L-dopa-treated and -non-treated patients was separated into protein, erythrocyte and lymphocyte fractions and levels of protein-incorporated dopa quantified by HPLC. Levels of protein-incorporated dopa were significantly increased in lymphocyte cell proteins from L-dopa-treated patients. This has not arisen from oxidative pathways as there was no evidence of oxidative damage to proteins. In addition, there was no increase in protein-incorporated dopa in erythrocytes, which are not actively synthesizing proteins. We suggest that protein-incorporated dopa could also be generated in the CNS. The accumulation of protein-incorporated dopa in cells is associated with oxidative stress and impaired function and could contribute to some of the problems associated with long-term L-dopa treatment.     

Aromatic-L-amino-acid decarboxylase activity in mouse pancreatic islets

Aromatic-L-amino-acid decarboxylase activity has been measured in intact or homogenised pancreatic islets of ob/ob mice (Ume? ob/ob). The method used involves the trapping and measuring of the 14CO2 released from L-[1-14C]dihydroxyphenylalanine (L-dopa). Islets showed a decarboxylase activity which was dependent on pyridoxal phosphate and inhibitable by 0.1 mM benserazide or 0.1 mM alpha-monofluoromethyldopa. Maximum activity in intact islets was about 330 mmol/kg dry islet per h with an apparent Km of 3.3 mM. Islet homogenates had a Vmax of about 120 mmol/kg per h with a Km of 0.3 mM. L-5-Hydroxytryptophan, m-tyrosine and o-tyrosine interfered with the decarboxylation of L-dopa in a way that suggested a high activity also towards those substrates. L-Phenylalanine, L-tyrosine and D-glucose had no effect. At 0.05 mM L-dopa islet homogenates showed a much higher activity than homogenates of liver, kidney, or spleen. Islet uptake of L-[3H]dopa was well in excess of the decarboxylation rate and thus probably not rate-limiting. It is concluded that mouse pancreatic islets have a high activity of aromatic-L-amino-acid decarboxylase. This is in accordance with previous suggestions of a stimulatory effect of this enzyme on insulin secretion.     

Phosphorylated mixed isomers of L-dopa increase melanin content in skins of Skh-2 pigmented hairless mice

Dopa phosphates, a new class of compounds, contain phosphate-ester linkages at the 3- and/or 4- positions of the phenylalanine ring of L-dopa. Dopa phosphates have been shown to increase pigment production in the epidermis of hairless mice. Groups of Skh-2 pigmented hairless mice were treated topically with various concentrations of dopa phosphates daily for five weeks. Half of each group received suberythemal UVB radiation three times weekly for four weeks from a bank of filtered FS20 lamps. UVB and dopa phosphates alone each caused a modest increase in epidermal pigmentation. However, treatment of mice with dopa phosphates plus UVB radiation resulted in a marked increase in pigmentation, greater than with either treatment alone. The optimal concentration of dopa phosphates was 0.01% (100 micrograms/ml Tris-glycerol buffer) whether or not they were applied in conjunction with UVB radiation. Histological analyses revealed that dopa phosphates and UVB radiation each caused an increase in the number of pigmented melanocytes in the epidermis. Control groups treated with Tris-glycerol buffer alone, or buffer containing L-phenylalanine or L-dopa showed no significant changes in pigmentation. Our results indicate that dopa phosphates stimulate the production of melanin and affect the development and distribution of melanocytes in the skin of Skh-2 mice. By these criteria, dopa phosphates and UVB act in a similar manner to increase melanin content in the skin. The processes may be related to those recently observed in cultured mouse melanoma cells where dopa phosphates are incorporated into melanin, presumably following enzymatic hydrolysis by cellular phosphatases with the resultant production of L-dopa and inorganic phosphate.     

Neuroendocrine changes in male hamsters following photostimulation

Transfer of gonadally regressed male golden hamsters from a short (5 L:19 D) to a stimulatory (14 L:10 D) photoperiod elicits, within 24 hr, significant changes in hypothalamic dopamine, serotonin, and possibly norepinephrine metabolism. Hypothalamic LHRH content was significantly elevated in short-photoperiod animals, but within 24 hr of transfer to a 14:10 photoperiod, LHRH declined to levels not different from those in hamsters maintained continuously in a long photoperiod. Plasma FSH levels were also significantly elevated within 24 hr of transfer, but increases in plasma LH were somewhat slower. Chronic treatment with the tyrosine hydroxylase inhibitor, alpha-methyl tyrosine (alpha MPT), which inhibits catecholamine synthesis, blocked the effect of a stimulatory photoperiod on plasma FSH levels, while treatment of 5:19 hamsters with the catecholamine precursor, L-dopa, mimicked the effects of photostimulation on plasma FSH levels. Testicular weights were not affected by alpha MPT or L-dopa treatment for 1 week. From these data, it appears that endocrine events associated with photoperiod-induced testicular recrudescence are under the control of hypothalamic neurotransmitters.     

L-DOPA: from a biologically inactive amino acid to a successful therapeutic agent

Summary.  The article traces the development of research on the naturally occurring amino acid L-3,4-dihydroxyphenylalanine (L-dopa), from the first synthesis of its D,L racemate in 1911, and the isolation of its L-isomer from seedling of Vicia faba beans to the amino acid's successful application, from 1961 onward, as the most efficacious drug treatment of Parkinson's disease (PD). Upon its isolation from legumes in 1913, L-dopa was declared to be biologically inactive. However, two early pharmacological studies, published in 1927 and 1930 respectively, proved (in the rabbit) that D,L-dopa exerted significant effects on glucose metabolism (causing marked hyperglycemia) and on arterial blood pressure. Interest in L-dopa's biological activity increased considerably following the discovery, in 1938, of the enzyme L-dopa decarboxylase and the demonstration that in the animal and human body L-dopa was enzymatically converted to dopamine (DA), the first biologically active amine in the biosynthetic chain of tissue catecholamines. This prompted, in the 1940s, many studies, both in animals and in humans, especially concerned with the vasopressor potential of L-dopa/DA. In the 1950s, the focus of L-dopa research shifted to its potential for replenishing the experimentally depleted (by insulin or reserpine) peripheral and brain catecholamine stores and the concomitant restoration of normal function. During that period, of special interest were the observations that L-dopa reversed the reserpine-induced state of “tranquilisation” and that its decarboxylation product DA occurred in high amounts in animal and human brain, with a preferential localization in the basal ganglia. These observations set the stage for the beginning of DA studies in PD brain. In 1960, the severe brain DA deficit, confined to patients with PD was discovered, and a year later L-dopa's strong therapeutic effect in patients with PD was demonstrated. In 1967, the chronic high-dose oral L-dopa regimen was successfully introduced into clinical practice. Despite some initial doubts about L-dopa's mechanism of action in PD, it is now generally recognized that L-dopa use in PD is a classic example of a brain neurotransmitter replacement therapy. However, the DA replacement potential of L-dopa may not be its sole action of interest, as suggested by recent evidence that L-dopa may also have its own biological activity in the CNS, independent of DA. Received June 29, 2001 Accepted August 6, 2001 Published online June 17, 2002     

Molecular Effects of L-dopa Therapy in Parkinson’s Disease

Parkinson’s disease (PD) is one of the most common neurological diseases in elderly people. The mean age of onset is 55 years of age, and the risk for developing PD increases 5-fold by the age of 70. In PD, there is impairment in both motor and nonmotor (NMS) functions. The strategy of PD motor dysfunction treatment is simple and generally based on the enhancement of dopaminergic transmission by means of the L-dihydroxyphenylalanine (L-dopa) and dopamine (DA) agonists. L-dopa was discovered in the early -60''s of the last century by Hornykiewicz and used for the treatment of patients with PD. L-dopa treatment in PD is related to decreased levels of the neurotransmitter (DA) in striatum and ab-sence of DA transporters on the nerve terminals in the brain. L-dopa may also indirectly stimulate the receptors of the D1 and D2 families. Administration of L-dopa to PD patients, especially long-time therapy, may cause side effects in the form of increased toxicity and inflammatory response, as well as disturbances in biothiols metabolism. Therefore, in PD pa-tients treated with L-dopa, monitoring of oxidative stress markers (8-oxo-2’-deoxyguanosine, apoptotic proteins) and in-flammatory factors (high-sensitivity C-reactive protein, soluble intracellular adhesion molecule), as well as biothiol com-pounds (homocysteine, cysteine, glutathione) is recommended. Administration of vitamins B6, B12, and folates along with an effective therapy with antioxidants and/or anti-inflammatory drugs at an early stage of PD might contribute to improvement in the quality of the life of patients with PD and to slowing down or stopping the progression of the disease.     

Phosphorylated isomers of L-dopa stimulate MSH binding capacity and responsiveness to MSH in cultured melanoma cells

L-dopa is a key metabolite in the process of melanogenesis. However, it is difficult to use in biological experiments because it is subject to auto-oxidation and relatively insoluble at neutral pH. Dopa phosphates contain phosphate ester linkages at positions 3 and/or 4 of the phenylalanine ring of L-dopa, rendering them highly soluble and stable to auto-oxidation when compared to L-dopa. Dopa phosphates are readily taken up by melanoma cells in culture and converted to L-dopa and inorganic phosphate by cellular phosphatases, making them useful for studying L-dopa effects in vivo. Here we investigated the effects of dopa phosphates on receptors for MSH in cultured melanoma cells. We found that dopa phosphates caused a 3-fold stimulation of MSH binding capacity by the cells which probably occurred through an increase in the number of receptors for MSH with no apparent change in affinity of the receptors. The increased binding capacity for MSH was followed by increased cellular tyrosinase activity and melanogenesis. Thus dopa phosphates and/or L-dopa can act as regulators of the MSH receptor system. The observations suggest a novel mechanism for regulation of hormonal responsiveness: hormonal signal amplification by a metabolite in the target pathway.     

垂体在刺激脑内渗透压感受器引起的肾脏反应中的作用

实验在α氯醛糖和氨基甲酸乙酯混合麻醉的大鼠中进行。脑室内注射高张盐水(icv.HS)后,肾血浆流量、肾小球滤过率、尿量、尿钠排出量、尿钾排出量和渗透物质清除率均增加,游离水清除率下降。去除垂体后,icv.HS不再能引起上述肾脏反应。另外给大鼠静脉注射血管升压素(VP)拮抗剂(V_1和V_2受体拮抗剂),并不能削弱上述icv.HS引起的肾脏反应。脑室内注射高张盐水后,尿中多巴胺(DA)排出量无显著增多;给予多巴脱羧酶抑制剂苄丝肼也不能削弱icv.HS引起的肾脏反应。上述实验结果表明,在本实验条件下刺激脑内渗透压感受器引起的肾脏反应依赖于垂体的完整性,但看来并不依赖于外周的VP和DA,故垂体通过何种机制介导icv.HS引起上述肾脏反应,有待于进一步的研究。     

Age-related motor and catecholomine alterations in mice on levodopa supplemented diet

Old mice reared on regular diet show reduced motor activity, decreased basal adenylate cyclase, and increased MAO activities compared to adults. Brain DDC and COMT activities, DA, NE levels and DA-stimulated adenylate cyclase remained unchanged. By contrast, mice fed levodopa for life did not develop decreased motor activity with aging, lived about 50% longer, had slightly elevated whole brain DA and NE levels and failed to develop the expected rise in MAO activity with aging. Levodopa did not alter the number of dopaminergic and muscarinic cholinergic receptors or the adenylate cyclase activity in the striatum during aging. On levodopa, hepatic and renal DA, dopa, and HVA increased but the latter two returned to basal levels by mid life. In liver, DDC was unchanged but MAO tended to be higher in levodopa-fed mice. Thus, motor impairment is an age-related phenomenon in mice associated with selective alterations in brain dopaminergic systems, which may be prevented by dietary levodopa. Extracerebral tissues, through possibly adaptive metabolic mechanisms, play a significant role in regulating brain catecholamines during chronic administration of large doses of levodopa.     

Effects of dopamine metabolites on locomotor activities and on the binding of dopamine: relevance to the side effects of L-dopa

L-dopa is the major treatment for Parkinson's disease (PD), but its efficacy is limited by the presence of dyskinesia. The dyskinesia develops over a period of exposure to L-dopa and is related to the dosage, therefore, the cause may involve inductive changes that produce toxic levels of metabolites, interfering with dopamine (DA) neurotransmission. Chronic L-dopa induces catechol-O-methyltransferase (COMT) and methionine adenosyl transferase (MAT), enzymes involved in the methylation of catecholamines (CA). In addition, high levels of 3-O-methyl-dopa have been reported in the plasma of dyskinetic PD patients, treated with L-dopa, as compared to non-dyskinetic patients, therefore, the methyl metabolites of CA may be increased during L-dopa therapy and may be involved in the dyskinesia. Since large amounts of DA are produced from L-dopa, and DA is extensively methylated, the methyl metabolites of DA, 3-methoxytyramine (3-MT) and 3,4-dimethoxyphenylethylamine (DIMPEA), may be also involved. The first step in knowing this, is to assess the behavioral and DA-receptor activities of 3-MT and DIMPEA. In the rat, the intraventricular injection of 0.5 micromol of DIMPEA increased the total distance traveled (TD) by over 100%, the number of movement (NM) made by 40% and the time spent moving (MT) by about 36%. Identical doses of 3-MT decreased the TD by 42%, NM by 22% and MT by 39%. DIMPEA (1 mM) increased the binding of DA with brain membranes by 44.7%, whereas 3-MT decreased it by 15.8%. The results show that 3-MT and DIMPEA are behaviorally active, and in parallel, they interact with the binding sites for DA, consequently, they may contribute to the side effects of L-dopa. L-dopa produces high levels of DA and induces MAT and COMT. It is proposed, therefore, that DA will be methylated to 3-MT and 3-MT to DIMPEA. At threshold level each product will inhibit, allosterically, its enzyme of methylation, causing sequential and rhythmic up and down regulation of its concentration. At peak levels these hydrophobic metabolites will modulate the actions of DA on synaptic membranes, causing abnormal movements, at times, resembling the "on-off effects".     

Catecholamines in larvae and juveniles of the prosobranch gastropod, Crepidula fornicata

We investigated roles of catecholamines in metamorphosis of the prosobranch gastropod, Crepidula fornicata. Levels of DOPA, norepinephrine (NE) and dopamine (DA) were measured by high-pressure liquid chromatography (HPLC) in competent larvae and juvenile siblings that metamorphosed in response to the natural adult-derived cue or to elevated K+. Competent larvae contained 1.58 +/- 0.26 (S.E.M.) x 10(-2) pmol DOPA, 0.91 +/- 0.45 x 10(-2) pmol NE, and 0.290 +/- 0.087 pmol DA (mean values per microg total protein, n = 4 batches of larvae). Levels of DA per individual were not different between larvae and juvenile siblings; levels of NE were higher in juveniles. The tyrosine hydroxylase (TH) inhibitor alpha-methyl-DL-m-tyrosine (alpha-MMT) depleted DOPA and DA to approximately half of control values without affecting levels of NE. Depletion of DOPA and DA was accompanied by inhibition of metamorphosis in response to the natural cue but not to elevated K+. The dopamine-beta-hydroxylase inhibitor diethyldithiocarbamate (DDTC) induced high frequencies of metamorphosis at concentrations of 0.1-10 microM. In juveniles induced by 10 microM DDTC, levels of both NE and DA averaged approximately 80% of those in control larvae. Catecholamines may function as endogenous regulators of metamorphosis in C. fornicata.     

Protein Phosphatases but not Reactive Oxygen Species Play Functional Role in Acute Amphetamine-Mediated Dopamine Release

Drug abuse-induced neurodegeneration can be triggered by elevated production of reactive oxygen species (ROS). Involvement of oxidative stress in acute amphetamine (AMPH)-mediated dopamine (DA) release, however, has not been completely understood yet. In order to elucidate the dopaminergic response of PC12 cells to a single dose of 10 μM AMPH, ROS production was measured as related to the extracellular DA level. Due to the spontaneous oxidation of peroxide-sensitive fluorophore 2′,7′-dichlorofluorescin diacetate (DCFH-DA) to 2′,7′-dichlorofluorescein (DCF), the increase in fluorescence could not be unambiguously attributed to AMPH-triggered ROS production. Based on Amplex Red fluorescence, no ROS production was detected after acute AMPH application. Our data strongly suggest that ROS development was not the main triggering factor for immediate DA release after acute AMPH treatment. On the other hand, AMPH-induced elevation of DA levels in rat brain striatal slices was quenched by the water soluble antioxidant, N-acetylcysteine (NAC) at 10 mM. In this study, we also investigated the contribution of protein phosphatases to the AMPH-induced rat brain striatal dopaminergic response. The experimental protocol, double AMPH challenge was applied for screening the effect of NAC and cantharidin on AMPH-mediated DA release. Here we show that AMPH-mediated DA release increased nearly twofold in striatal rat brain slices pretreated for 30 min with 1000 μM cantharidin, a selective PP1 and PP2A inhibitor. These findings prove the lack of ROS inhibitory action on protein phosphatase activity in acute AMPH-mediated DA efflux.     

The reciprocal exclusion by L-dopa (L-3,4-dihydroxyphenylalanine) and L-tyrosine of their incorporation as single units into a soluble rat brain protein.

Several compounds, structurally and metabolically related to phenylalanine and tyrosine, were tested for their effects on the incorporations of phenylalanine and tyrosine as single units into a protein of the soluble subcellular fraction of rat brain. Of the compounds tested, only L-dopa (L-3,4-dihydroxyphenylalanine) inhibited these incorporations. Further, L-dopa was incorporated into a protein of the same fraction in such a way that it excluded the incorporation of tyrosine as a single unit. Conversely, tyrosine inhibited and excluded the incorporation of L-dopa. The incorporation of L-dopa required ATP (apparent Km = 0.23mM), KCl (apparent Km = 20mM) and MgCl2 (optimal concentration range, 5-16mM). These requirements were similar to those previously determined for the incorporation of tyrosine and phenylalanine. The inactivation rate of the enzymic systems for L-tyrosine and L-dopa incorporations, when kept at 37 degrees C, was the same for both amino acids (half-life = 80 min). It is suggested that the acceptor for the incorporation of dopa is the same as that for the incorporation of tyrosine.     

Role of Serotonin2A and Serotonin2B/2C Receptor Subtypes in the Control of Accumbal and Striatal Dopamine Release Elicited In Vivo by Dorsal Raphe Nucleus Electrical Stimulation

This study investigates, using in vivo microdialysis, the role of serotonin2A (5-HT2A) and 5-HT(2B/2C) receptors in the effect of dorsal raphe nucleus (DRN) electrical stimulation on dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), and 5-hydroxyindoleacetic acid (5-HIAA) extracellular levels monitored in the nucleus accumbens (NAC) and the striatum of halothane-anesthetized rats. Following DRN stimulation (300 microA, 1 ms, 20 Hz, 15 min) DA release was enhanced in the NAC and reduced in the striatum. The 5-HT2A antagonist SR 46349B (0.5 mg/kg) and the mixed 5-HT(2A/2B/2C) antagonist ritanserin (0.63 mg/kg) significantly reduced the effect of DRN stimulation on DA release in the NAC but not in the striatum. DA responses to DRN stimulation were not affected by the 5-HT(2B/2C) antagonist SB 206553 (5 mg/kg) in either region. None of these compounds was able to modify the enhancement of DOPAC and 5-HIAA outflow induced by DRN stimulation in either the NAC or the striatum. Finally, in both brain regions basal DA release was significantly increased only by SB 206553. These results indicate that 5-HT2A but not 5-HT(2B/2C) receptors participate in the facilitatory control exerted by endogenous 5-HT on accumbal DA release. Conversely, 5-HT(2B/2C) receptors tonically inhibit basal DA release in both brain regions.