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Effects of L-dopa treatment on methylation in mouse brain: Implications for the side effects of L-dopa
Life Sciences. Vol. 66. No. 23. m. 2277-2288.2000 &yri& 0 Zoo0 tisevier Scieke Inc. F’rinted in the USA. All rights mewed 0024-3205/00&s& bent matter
PII SO0243205(00)00557-9
ELSEVIER
EFFECTS
OF GDOPA TREATMENT ON METRYLATION IN MOUSE IMPLICATIONS FOR THE SIDE EFFECTS OF L-DOPA
BRAIN:
X.X. Liu, K. Wilson and C.G. Charlton College of Pharmacy and Pharmaceutical Florida A&M University Tallahassee, FL 3 23 07
Sciences
(Received in final form January 7,200O)
Summary 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, Sadenosylmethionine (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 (SAWSAM) 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 8 18, 896, and 1524%. L-dopa (50, 100 and 200 mg/kg) dose-dependently depleted SAM from 24.9 311.7nnioVg to 13.0 + 0.8, 14.7 f 0.8 and 7.7 k 0.7 nmol/g, and increased SAHfrom 1.88 f0.14 to 3.43 kO.26, 4.22H.32and6.21 f0,40mnol/g. BrainLdopa was increased to 326, 335 and 779%, dopamine to 138, 116 and 217% and SAH&4M 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. Key Words:
S-adenosylmethionine, S-adenosylhomocysteine, L-dihydroxyphenylalanine, methylation, Parkinson’s
disease
Correspondences should be addressed to: Clivel G. Charlton, Ph.D., College of Pharmacy and Pharm&euti~ Sciences, Florida A&M University, Tallahassee, FL. 32307, USA. Tel.#: (850) 5993716, Fax.#: (850) 599-8444.
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L-dopa, alone or in combination with a peripheral decarboxylase inhibitor, remains the most efficacious drug for the treatment of Parkinson’s disease (PD) (1). The L-dopa that reaches the brain is decarboxylated and replenishes the neurotransmitter, dopamine (DA), which is depleted in the nigrostriatal system of PD patients. A significant amount of L-dopa is also 0-methylated by catechol0-methyltransferase (COMT) in a reaction using S-adenosylmethionine (SAM) as the methyl donor, and SAM is utilized during the process. Since, relatively large doses of L-dopa are administered, on a stoichiometric basis, the depletion of SAM is marked, and the rate of methylation is correspondingly significant. This is highlighted by the ftnding that the cerebrospinal fluid (CSF) concentrations of 3-0methyldopa, a methyl metabolite of SAM and L-dopa, were about 5 to 10 times higher than the levels of L-dopa, itself, in PD patients treated with L-dopa (2). It was also shown that more than half of the total urinary metabolites of L-dopa in men are 0-methylated (3). The direct analysis of the levels of SAM tinther suggests a possible role of transmethylation in L-dopa treated patients, because decreased concentrations of SAM occur in the blood (4 ) and CSF of Ldopa treated PD patients (5). SAM was also decreased in the brain and liver of L-dopa treated animals (6,7,8). These studies were interpreted in terms of the metabolic significance of methylation, but a less restricted analysis suggests that the reduction of SAM may contribute to the success of Ldopa as an anti-Parkinson’s agent. Interestingly, the combination of L-dopa with carbidopa, a peripheral decaboxylase inhibitor, increases the effectiveness of L-dopa therapy and correspondingly, increases the levels of 3-O-methyl dopa, a product of the reaction of L-dopa with SAM (2). Although, it is well understood that the effectiveness of carbidopa reflects the level of L-dopa that reaches the brain and is converted to DA, more recent findings suggest, also, that the reaction of Ldopa with SAM and the depletion of SAM may be of importance in the actions of L-dopa and the reactions to L-dopa. It was reported that PD patients methylate nicotinamide at a rate 90 times higher than controls (9) and when injected into the brain of animals, both SAM, the methyl donor (10, 11, 12) and N-methyl beta-carboline, a SAM-dependent metabolite (13) caused reproducible PD-like symptoms. Those studies indicate that methylation may be increased in PD, therefore, the ability of L-dopa to interact with SAM and to deplete SAM may represent a mode-of-action for L-dopa (14). There are ancillary reasons to support this idea, because methylation reactions are increased in the aged population; the group that is most susceptible to develop PD. So, it can be reasoned that as people age, essential constituents, such as protein lipids, nucleic acids and biogenicamines are increasingly methylated. The methylation of DA norepinephrine and serotonin can deplete the levels of these neurotransmitters, changes that occur in PD. Methylation can increase N-methyl beta-carboline, that has been reported to cause PD-types of effects (13) and methylation produces surface active phospholipids, which decreases the viscosity of cell membranes (15). It also increases the hydrophobicity of protein by neutralizing negative charges (16) and silent gene transcription (17). The expression of tyrosine hydroxylase (TH)-mRNA for example, is suppressed by DNA methylation of the TH gene (18). Furthermore, N-methylation has been shown to increase the cytotoxicity of the monoamine derivative, isoquinoline, that is proposed to serve as pathogenic agents in PD (19). It is likely, therefore, that an increase in methylation may induce the wear and tear that lead to PD-types of changes in the susceptible individual, such as a person endowed with low number of nigrostriatal DA cells, or having nigrostriatal dopamine cells with poor metabolic qualities. There is a putative role of methylation in the actions of, and the reactions to, L-dopa because chronic L-dopa has been shown to increase the activities of methionine adenosyl transferase (MAT) (20) and COMT (21) as well as the protein and nucleic acids for those enzymes (22). Once induced, MAT will increase the synthesis of SAM, and COMT will transfer the methyl group of SAM to L-dopa and DA and will decrease their levels, More L-dopa will be required to maintain the levels of DA so, patients
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with high capacity for the induction of MAT and COMT may experience short-lived beneficial effects from L-dopa. Moreover, toxic metabolites and metabolites that interfere with L-dopa, may be produced. An example is 3-O-methyl dopa. It has been shown that when 3-0-methyldopa was given with L-dopa to PD patients the clinical responses to L-dopa was reduced (23, 24, 25) and 3-0methyldopa also decreased the accumulation of dopamine in rats treated with MPTP (26) as well as in normal rats (27,28). In addition, evidence shows that the mono and di-methyl metabolites of DA, 3-methoxytyramine (3-MT) (29) and 3,4-dimethoxy-phenylethylamine (DIMPEA) (29, 30) are behaviorally active, and interact with DA receptors (unpublished data). Most of the studies concerning the interaction of L-dopa with the methylation process were performed in the seventies, and focused on the metabolism of L-dopa. Since methylation may play important roles in the actions of L-dopa, the effects of L-dopa on transmethylation were reexamined. These studies are necessary, also, in light of the availability of more sensitive and reliable analytical techniques, utilizing HPLC-combined electrochemical and spectrophotometric analytical procedures. The studies make it possible to compare the present data with earlier data, to perform the investigation under acute, subchronic and time-dependent conditions and to examine more parameters, such as the values for SAM, its demethylated product, S-adenosylhomocysteine, brain L-dopa and dopamine, as well as changes in the rate of methylation (SAWSAM). Mouse, which seems to be a more sensitive model in certain PD studies, and which we have used to investigate the chronic effects of L-dopa on MAT and COMT, are used instead of the rat, on which most methylation data have been collected. The results show that L-dopa depletes SAM and increases SAH. It increases methylation much more than it increases DA. The effects were dose-dependent and accumulative, The data help us to understand why L-dopa induces the enzymes, MAT and COMT and it may help us to know more about the side effects of L-dopa.
Materials and Methods Animals In this study, Swiss Webster, albino male mice (25-35g), were purchased from Harlan Sprague Dawley. They were kept under a 12 hr light-12 hr dark cycle at a constant temperature (22 “C), with access to food and water. The animals were allowed at least 5 days acclimation before use in any experiment. For the time course study, both single and multiple injections were performed. Groups of 4 mice, intraperitoneally, received a single dose of L-dopa (100 mg/kg) suspended in phosphate buffered saline (PBS). They were decapitated at 0.5, 1, 2, 4 and 8 hr(s) after injection. For the study that evaluates the accumulative effects of L-dopa, groups of 7 mice each received one, two, three or four injections ofL-dopa (100 mg/kg) at 45-min intervals and were decapitated 30 min after the last dose. In the dose-effect study, mice received 0,50, 100,200 or 300 mg/kg of L-dopa and were decapitated 30 min after the injections. In all the experiments, matching control groups received the vehicle alone and were sacrificed with each experimental group to control for changes due to the daily rhythm in SAM concentrations in the brain. Chemicals S-adenosylmethionine (SAM) (p-toluene sulfonate salt), S-adenosylhomocysteine (SAH), L-dopa, dopamine and l-octane sulfonic acid sodium salt (HPLC grade) were purchased from Sigma Chemical Company, St. Louis, MO. Triethylamine (TEA) was obtained from Aldrich, Milwaukee, WI. HPLC grade acetonitrile and phosphoric acid were purchased from Fisher Scientific, Fair Lawn, NJ. All other chemicals used were obtained from Fisher Scientific and were of analytical grade.
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Preparation of brain homogenates The brains were removed immediately after sacrificing the animals. Brain tissues were homogenized in 2 volumes (w/v) of ice-cold 0.4 M perchloric acid using a PolyScience (model X520) Polytron. The homogenates were centrifuged at 9,000 x g for 20 min at 4 “C. The supematants were filtered (0.45 urn) and stored at -70 “C until analyzed by high-pressure liquid chromatography (HPLC). HPLC Assay Fifty microliters of the brain supematants were injected onto an HPLC system. The HPLC system consisted of a Thermal Separation Products (TSP) Solvent Delivery ConstaMetric 4100 pump with an online membrane degasser, a TSP Spectra System AS3500 autosampler with a 100 up1 sample loop and a 25 cm x 4.6 mm id., 5~ Cl8 Hypersil column (Whatman EQC). The detection system consisted of a TSP SpectroMonitor 3200 UV detector and an ESA Coulochem II electrochemical detector (5200 A model) with a high sensitivity analytical cell ( Model 5011). The UV detector was operated at 260.2 nm and the electrochemical detection was performed with guard cell set at 3 50 mV and analytical electrodes set at 300 mV for the second electrode and -50 mV for the first one. The sensitivity of the second analytical cell was set at 5 PA. The mobile phase consisted of 75 mM NaH,PO,.HO, 5.1 mM l-octane sulfonic acid sodium salt, 100 uV1 TEA and 10% acetronitrile. The final pH was adjusted to 3.0 with phosphoric acid. The flow rate was 1.5 mI/min and a column pressure of 1938-2023 psi was maintained. Concentrations were calculated by reference to peak area of external standards. All standards were freshly prepared in 0.4 M perchloric acid immediately before analysis. Recovery Experiments Brain tissues were homogenized in 2 volume of 0.4 M perchloric acid containing known amounts of SAM, SAH, L-dopa and dopamine. Standard peak areas then corrected by subtracting the contributions from the endogenous origin which was obtained from another set of tissue preparations without added standards. Seven replicates were prepared and analyzed as described above. Statistical Analysis Values for the brain concentrations of SAM, SAH, L-dopa and dopamine, and the calculated SAIWAM were given as the mean + the standard error of the mean (SEM). Two tailed paired t-test were used to compare the matched controls and the tested values. All the statistical analysis was performed using GraphPad Prism software.
Results HPLC-combined electrochemical andspectrophotometric detection of SAM, SAH, L-dopa and DA. Both SAM and SAH, as well as L-dopa and DA were extracted and determined from the same tissue samples into 0.4 M perchloric acid. Using an HPLC system with online W and electrochemical detectors enabled us to detect these four compounds simultaneously. The levels of SAM and SAH were determined by the W detector. The levels of L-dopa and DA were detected by the electrochemical detector. These four compounds were well separated (Fig. 1) using the described method. It was determined that the recovery of SAM, SAH, L-dopa and dopamine from brain extracts was 62.5f4.3%, 73.9+6.0%, 65.3+5.0% and 59.8+5.0% (n=7), respectively. Although the recovery was appreciable less than 100°/o, the one cycle low volume extraction procedure optimized the concentration of the metabolites and speeded up the extraction and detection procedures. The calibration curves, based.on area-under-the-peak from standards of 1.00-10.0 uM SAM, 0.200-1.50 uM SAH, 0.200-7.50 @l L-dopa and 0.500-10.0 p.M DA showed a good linearity, with correlation coefficients of 0.998, 0.994, 0.990 and 0.997 for each curve, respectively.
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(A)
Time (tin) Fig, 1 HPLC Chromatograms of SAM, SAH, L-dopa and dopamine from 50 l.tl of mouse brain extracts. SAM and SAH were analyzed using UV detector. The retention times for SAM and SAH were 19.5 min and 14.2 min respectively (A). L-dopa and dopamine were recorded from electrochemical detector. The retention times for Ldopa and dopamine were 4.1 min and 11.4 min, respectively (B). The HPLC conditions were as described in the Materials and Methods. Time-course eSfect of a single dose of L-dopa. The effects of a single injection of L-dopa on the levels of SAM and SAH were determined. At 0.5 hr after the injection of 100 mg/kg of L-dopa the levels of SAM were markedly decreased to 64% of control value. The concentration of SAM for the L-dopa treated animals was 21.4 +l 1 nmol/g of wet tissue, as compared to the value of 33.5 + 0.3 nmol/g for the control animals. The concentration of SAH, the demethylated metabolite of SAM, was increased to 2.38 f 0.21 nmolfg, as compared to the control value of 1.28 f 0.16 nmol/g. This represents an increase of 86% (Fig. 2). At 2 hr post-injection the concentration of SAM was still depressed to 80% of controls and that of SAH returned to normal. At the 4 and 8 hr post-injection periods the levels of SAM and SAH both returned to control values (Fig. 2). The ratio of the value of SAH to that of SAM (SAH/SAM) is used to indicate the extent of transmethylation. It is therefore an index of methylation, and the value is proportionate to the levels of methylation. The results in Fig.2 show that L-dopa increases methylation in the brain of mice, noted by a dramatic 200% increase in SAHISAM at the 0.5 hr postinjection period. At 4 and 8 hr post-injection the methylation rates were statistically the same for both groups.
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q SAHfSApvr
2 3002 g z 200-
0
sfw
0
SAM
0 s
loo-
o!
I
-1
1
5
t L-dopa
I
5
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Time (h)
Fig.2 The time course of the effect of a single dose of L-dopa (100 mg/kg) on methylation in the mouse brain. Each point represents the mean and standard error of the mean for data from four animals. Data are expressed as a percentage of the levels found in the corresponding control group. Statistically significant changes are indicated by an asterisk (*PC 0.05). Effects of repeated injection ofL&pa on methylation. L-dopa has a sustained depleting effects on SAM when given to groups of mice for 1 time or for 2, 3 or 4 consecutive times at 45611 intenals and determined 30 min after the last dose (Table I). Mice that received one injection of L-dopa showed a decrease in the concentration of SAM to 77% of control value. Two consecutive injections of L-dopa further depleted the levels of SAM to 40% of control value. Three consecutive injections reduced SAM to 37% of control level and four infections Table I. l%e sustained efsects ofl-dopa on SAM, SAH and SAH/SAkf values in the mouse brain. Four groups of mice (N=7) received 1 dose or 2, 3 or 4 doses of L-dopa (100 mg/kg, ip) at 45 min intervals and were sacrificed 30 min after the last injection. Concentrations are expressed in nmoYg of fresh tissue and all values represent the mean + SEM P
1 2
s) Control 38.9H.7
SAM L-dopa 29.6_+3.3*
SAH Control L-dooa 2.24f0.04 4.95+0.68*
&4H/SAM(x 102) Control L-dooa 5.8kO.2 19.3M.5*
41.1M.8
16.3f1.5*
2.45kO.12
7.24,0.67*
6.OkO.3
49.1f9.7*
3
34.9k2.1
12.4+1.4*
2.61f0.18
7.89f0.53*
7.5kO.3
67.2f6.4*
4
36.4k1.8
8.8M.8*
2.29f0.32
8.27f1.02*
6.3M.9
96.0+1.1*
decreased the concentration of SAM to 25% of controls. After the same series of L-dopa injections, SAH was increased to 223% after the first, and plateau at over 300% of controls after the 2”d , 3” and 4” injections (Table I). The SAWSAM kept increasing with the repeated doses of
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L-dopa and reached the highest value of 1702% of controls after the fourth dose. Again, this is indicative of an L-dopa induced increase in methylation. Dose-e#ect ofL&pa on methylation. The dose-effect of exogenous L-dopa on the concentrations of SAM, SAH, the levels of methylation (SAH/SAM) and on brain L-dopa and dopamine levels were examined (Fig.3). The
SAHISAM
Brain
E-dopa
DA
L-dopa
dose (mg/kg) Fig. 3
The dose-effects of L-dopa on methylation in the mouse brain. Each point shows the mean of data from seven animals. All data are expressed as percentages of the levels found in the control group. Opened circles represent the values for SAH, filled circles represent the values for SAM (A). In B, SAH/SAM is represented by squares, L-dopa by filled triangles and DA by opened triangles.
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L-dopa concentration detected in the control animals was 1.13H.09 mnol/g. Treating the mice with L-dopa of 50, 100 and 200 mglkg increased brain L-dopa to 334, 340 and 796% of control value, respectively. For the same L-dopa doses, the mean DA levels increased by 138, 126 and 232%, respectively. L-dopa and DA react avidly with SAM and this was evident by the dosedependent L-dopa-induced reduction of SAM to 5 1, 58 and 28% of controls, SAH, on the other hand, was increased to 201, 219 and 336% of control values for the respective doses of L-dopa. The rate of methylation was dose-dependently increased to 369, 402 and 1135% of control by Ldopa. Discussion The data show that exogenous L-dopa administration has significant effects on the biological methylation process in the mouse brain. The depletion of SAM by L-dopa is consistent with the results of previous studies (3 l), but the analysis of SAH, brain L-dopa and DA in this study, help to evaluate the comparative effects of L-dopa on DA and the methylation process. L-dopa increases transmethylation to a greater extent than it increases the levels of brain L-dopa and DA; a proportional increase of about 4:3: 1 occurred, respectively. So, the increased brain levels of L-dopa, from the injected L-dopa, corresponds more closely with the methylation process than with the increment in dopamine. The data suggest that biological methylation may play a role in the actions of L-dopa, in addition to the well established role for L-dopa, of increasing the levels of DA. The results are relevant to previous findings. Some of the findings, include the report that the injection of the methyl donor, SAM, causes PD-like changes in animals. The changes include tremor, rigidity and hypokinesia, the depletion of tyrosine hydroxylase and dopamine, and nigrostriatal neuronal degeneration in rodents. The behavioral effects were inhibited by L-dopa and other agents that seem to compete for the methyl donor (29,11,12). Collins et al. ( 13) also, found that N-methyl beta-carboline, a methyl metabolite of SAM, caused PD-like symptoms in animals. Furthermore, it was observed that the methylation of nicotinamide, used as a measure of methylation, was increased more than ninety times in PD patients as compared to age-matched controls (9). These findings, when taken together, suggest that an increase of methylation may occur in PD. Accordingly, as a therapy for PD, L-dopa may express part of its actions through the reaction with SAM and the depletion of SAM. The depletion of SAM would have a sparing effect on DA metabolism, while L-dopa replenishes DA. The interaction of L-dopa with the methylation process, though, will change over a period of time during L-dopa administration. During the early stages, L-dopa will compete with other catecholamine for the methyl donor, SAM. This has been corroborated in studies showing that the methylation of norepinephrine was decreased in the brain of L-dopa treated rats (7). Similarly, a decrease in the methylation of DA and a corresponding increase in its availability should occur following the acute administration of L-dopa. The acute or subacute effects of L-dopa, however, will be different from the chronic effects, The continuous utilization of SAM by L-dopa will lead to an L-dopa-induced increase in the activities of the enzyme that catalyzes the production of SAM (MAT) (20) and the enzyme that transfers the methyl group of SAM to the catecholamines (COMT) (2 1). The induction of MAT by L-dopa has been reported (20). It requires relatively high doses of L-dopa, administered at frequent intervals (3-4 times/day) over several days (20). The induction of MAT may be viewed as a counter-response measure to replenish the depletion of SAM caused by L-dopa (20). The increase in MAT, as a counter-response
to the depletion of SAM by L-dopa will produce more
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SAM. The rebound increase in SAM will increase the methylation and destruction of L-dopa and DA. Therefore, the levels of brain L-dopa and DA usually achieved during the early periods of L-dopa therapy will no longer be attainable. More L-dopa will be required to sustain the DA level required for the control of the symptoms of PD, so the effects of L-dopa will wear 0% and a form of tolerance to L-dopa will develop. Thus, the interaction of L-dopa with the methylation process may create a vicious cycle in which by reacting with, and utilizing SAM, L-dopa will eventually induce methylation enzymes that lead to the conversion of dopamine as well as the administered L-dopa. High rates of the methylation of L-dopa and DA will deplete these biogenic amines, causing the loss of the effects of L-dopa and the production of their methylated metabolites (Fig. 4). High levels of 3-O-methyl dopa have been well documented following L-dopa therapy (23,2). At present, however, there is a lack of information regarding the accumulation of the methyl metabolites of dopamine during L-dopa therapy. It is well accepted, though, that increases in the methylation of dopamine should parallel the increases in the methylation of L-dopa. The accumulation of methylated metabolites of L-dopa and DA may help to explain the adverse effects of L-dopa seen following long-term therapy, because 3-O-methyl dopa, for example, has been shown to antagonize the effects of L-dopa (23,24,25,26,27,28). Such an effect may be compared with, and contributed to, the “wearing-off-effects”. The effects of the methyl metabolites of dopamine, 3-MT and DIMPEA, which, respectively, decreases and increases locomotor activities (29) and DA binding in rats may be compared to the “on-off effects” seen following chronic L-dopa administration to PD patients and to MPTP treated monkeys. The “on-off’ types of effects may be explained by the scenario of a sequential methylation of DA, to produce 3- MT and DIMPEA. Initially, DA is monomethylated at the 3-OH position, producing toxic levels of 3-MT, and causing hypokinesia. The accumulation of 3-MT, however, will inhibit further mono-methylation of DA (end-product inhibition), at the same time 3-MT will serve as substrate for the production of DIMPEA via methylation at the 4-OH position. The accumulation of toxic levels of DIMPEA will cause hyperkinesia. However, the accumulation of DIMPEA will inhibit its own synthesis, and the levels of 3-MT will again increase to cause hypokinesia. Thus, this important phasic or rhythmic event may be caused by a relatively simple biochemical process, as illustrated in Fig. 4. In summarizing the results, the data and the corroborative evidence present an interesting scenario. On the basis that excess methylation may be involved in PD-like changes (29,l 3, 9), the depletion of SAM by L-dopa may help in alleviating of the symptoms of PD. The depletion of SAM, however, creates great demands for the already limiting methyl donor, SAM. Such a biochemical stress on the methylation process activates and/or induces MAT to replenish SAM. Evidence also showed that Ldopa induces the enzyme COMT (21). The induction of MAT causes a rebound elevation in the production of SAM, and the induction of COMT will promote aggressive transmethylation reactions, leading to the methylation and depletion of L-dopa and dopamine. It means that higher doses of Ldopa will be required to maintain the therapeutic DA levels, a state of condition that is remindful of the “wearing-off effect” or a form of tolerance. The methyl metabolites of L-dopa and dopamine will also be produced. These metabolites are more hydrophobic than their parents, and will interact more readily with cell membranes, and may, through their mass actions, interfere with the availability of Ldopa and the receptor actions of DA. Thus, the interaction of L-dopa with SAM may underlie the actions and/or the toxic side-effects of L-dopa. As illustrated in Fig.4, the effects of L-dopa can also decrease the levels of ATP, because, every mole of L-dopa has the potential of utilizing at least two moles of ATP, via the utilization of SAM and the replenishment of SAM by the reaction of ATP with methionine. The production of secondary metabolites, such as homocysteine and adenosine from SAH should also be noteworthy.
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the Methylation
SAM ATP
+Met=
Process
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Pool SAM
Ldopa+ A
Fig. 4 The illustration highlights the production of dopamine from L-dopa and the known and putative transmethylation reactions that occur. Vertical short arrows (” or ‘) indicate decrease or increase in activities/concentrations. Solid arrowed-lines (+) indicate the direction of the reaction, and broken arrowed-lines represent feed back inhibition of reactions. The question mark (?) represents unknown pathway. Abbreviations: BAMT (biogenicamine methyl transferase); COMT (catecholamine methyl transferase); MAT (methinonine-adenosyltransferase); SAM (Sadenosyhnetbionine); Met (methionine); SAH (S-adenosylhomocysteine; L-dopa (L3,4-dihydroxyphenylalanine); 3-OMD (3-methoxy-4-hydroxyphenylalanine); 3,4OMD (3,4dimethoxyphenylalanine); 3-MT (3-methoxytyramine); DIMPEA (3,4-dimethoxyphenylethylamine).
Acknowledgments Support for this research was provided by the National Institutes of Health grants GO8 111 , RR03020 and NS 28432.
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R. SURTEES and K. HYLAND, J. Neurol. Neurosurg. Psychiatry, 53 569-572 (1990). R.J. WURTMAN, A& Biochem. Psychophurmacol , E. COSTA, L.L. IVERSEN and R. PAOLETTI (Eds), Vo16 24 l-246, Raven Press, New York (1972). J.P. CHALMERS, R.J. BALDESSARINI, R. J. WURTMAN, Proc. Natl. Acad. Sci. USA, 68 662-666 (1971). D. DALY, J.W. MILLER M.R. NADEAU and J. SELHUB, [Abstract], FASEB. J 8 A920 (1994). A.C. WILLIAMS, H.S. PALL, G.B. STEVENTON, S. GREEN, S. BUTTRUM, H. MOLLOY and R.H. WARING, Adv. in Neural., 60 194-196 (1993). C.G. CHARLTON and B. CROWELL, JR., Pharmacol. Biochem. Behav., 43 423-431 (1992). C.G. CHARLTON, and B. CROWELL, JR., Mol. Chem. Neuropathol., 26 269-284 (1995). B. CROWELL. JR BENSON, D. SHOCKLEY and C.G. CHARLTON., Behav. and Neural Biol., 59 186-193 (1993). M.A. COLLINS, E.J. NEAFSEY, K. MASTSUBARA, R.J. COBUZZI and H. ROLLEMA, Brain. Res. 570 154-160 (1992). C.G. CHARLTON, Basic, Clinical, and Therapeutic Aspects of Alzheimer ‘s and Purkkzson’s Disease ,T. NAGATSU et al. (Eds), Vol 1 333-339, Plenum Press, New York (1990). F. I-IIRATA, J. WARREN, S. MATTER and J. AXELROD, Proc. Natl. Acad. Sci. USA, 76 368-372 (1979). C. GAGNON, J. AXELROD and,M.J. BROWNSTEIN, Life Sci., 22 2155-2164 (1978). L.L. MAYS-HOOPES, Molecular Biology of Aging: Gene Stability and Gene Expression, R.S. SOHAL (Eds), 49-65, Raven Press, New York (1985). K. OKUSE, I. MATSUOKA, K. KURMARA, Mol. Brain. Res., 46 197-207 (1997). M. NAOI, W. MARUYAMA, T. NIWA, and T. NAGATSU, J. Neural. Transm. (Suppl.) 41 197-205 (1994). R. BENSON, B. CROWELL, JR., B. HILL, K. DOONQUAH and C. CI-IARLTON, 1993. Neurochem. Res., 18 325-330 (1993). R. BENSON. Studies of biochemical processes that may affect the efSacy of L-dopa therapy in Parkinson ‘s disease, Ph.D. Thesis, School of Graduate Studies, Meharty Medical College, Nashville, Tennessee (1992). W.Q. ZHAO, D. OLSEN, X.X. LIU and C. CHARLTON, [Abstr], c33 5 1, The 1998 National Minority Research Symposium, New York, (1998). D.B. CALNE, F. KAROUM, C.R.J. RUTHVEN, M. SANDER Brit. J. Pharmacol., 37 57-63 (1969). M.D. MUENTER R.P. DINAPOLI, N.S. SHARPLESS and G.M. TYCE, Mayo Clin. Proc 48 173-183 (1973). J.G. NUTT, W.R. WOODWARD, S.T. GANCHER and D. MERRICK. Ann. Neurol. 2L 584-588 (1987). J. FUJITAKE, S. KUNO and E. MIZUTA, Advances in Neurology, Parkinson ‘s Disease: Anatomy, Pathology, and Therapy, eds., M.B. STRElFLER, A.D. KOREZYN, E. MELAMED and M.B.H. YOUDIM, Vol53 225-229, Raven Press, New York (1990). A. RECHES and S. FAHN, Ann. Neurol., 12 267-271 (1982). J.J. GERVAS, V. MURADAS, E. BAZAN, E.G. AGUADO and J.G. YEBENESM, Neurol, 33 278-282 (1983). C.G. CHARLTON, R. BENSON and B. CROWELL, [Abstr.], 231dAnnual Meeting, Sot for Neurosci. (1993). A. BARBEAU, P. SINGI-I, P. GAUDREAU and M. JOUBERT, [Abstr.], Medical Meetings: Can. Sot. for Clin. Invest., Can. Med. Ass. J., 94 659 (1966).
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R.J. WURTMAN, C.M. ROSE, S. MATOTHYSSE, BALDESSARINI, Sci., 169 395-397 (1970).
J.
STEPENSON
and
R.
PII SO0243205(00)00557-9
ELSEVIER
EFFECTS
OF GDOPA TREATMENT ON METRYLATION IN MOUSE IMPLICATIONS FOR THE SIDE EFFECTS OF L-DOPA
BRAIN:
X.X. Liu, K. Wilson and C.G. Charlton College of Pharmacy and Pharmaceutical Florida A&M University Tallahassee, FL 3 23 07
Sciences
(Received in final form January 7,200O)
Summary 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, Sadenosylmethionine (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 (SAWSAM) 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 8 18, 896, and 1524%. L-dopa (50, 100 and 200 mg/kg) dose-dependently depleted SAM from 24.9 311.7nnioVg to 13.0 + 0.8, 14.7 f 0.8 and 7.7 k 0.7 nmol/g, and increased SAHfrom 1.88 f0.14 to 3.43 kO.26, 4.22H.32and6.21 f0,40mnol/g. BrainLdopa was increased to 326, 335 and 779%, dopamine to 138, 116 and 217% and SAH&4M 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. Key Words:
S-adenosylmethionine, S-adenosylhomocysteine, L-dihydroxyphenylalanine, methylation, Parkinson’s
disease
Correspondences should be addressed to: Clivel G. Charlton, Ph.D., College of Pharmacy and Pharm&euti~ Sciences, Florida A&M University, Tallahassee, FL. 32307, USA. Tel.#: (850) 5993716, Fax.#: (850) 599-8444.
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L-dopa, alone or in combination with a peripheral decarboxylase inhibitor, remains the most efficacious drug for the treatment of Parkinson’s disease (PD) (1). The L-dopa that reaches the brain is decarboxylated and replenishes the neurotransmitter, dopamine (DA), which is depleted in the nigrostriatal system of PD patients. A significant amount of L-dopa is also 0-methylated by catechol0-methyltransferase (COMT) in a reaction using S-adenosylmethionine (SAM) as the methyl donor, and SAM is utilized during the process. Since, relatively large doses of L-dopa are administered, on a stoichiometric basis, the depletion of SAM is marked, and the rate of methylation is correspondingly significant. This is highlighted by the ftnding that the cerebrospinal fluid (CSF) concentrations of 3-0methyldopa, a methyl metabolite of SAM and L-dopa, were about 5 to 10 times higher than the levels of L-dopa, itself, in PD patients treated with L-dopa (2). It was also shown that more than half of the total urinary metabolites of L-dopa in men are 0-methylated (3). The direct analysis of the levels of SAM tinther suggests a possible role of transmethylation in L-dopa treated patients, because decreased concentrations of SAM occur in the blood (4 ) and CSF of Ldopa treated PD patients (5). SAM was also decreased in the brain and liver of L-dopa treated animals (6,7,8). These studies were interpreted in terms of the metabolic significance of methylation, but a less restricted analysis suggests that the reduction of SAM may contribute to the success of Ldopa as an anti-Parkinson’s agent. Interestingly, the combination of L-dopa with carbidopa, a peripheral decaboxylase inhibitor, increases the effectiveness of L-dopa therapy and correspondingly, increases the levels of 3-O-methyl dopa, a product of the reaction of L-dopa with SAM (2). Although, it is well understood that the effectiveness of carbidopa reflects the level of L-dopa that reaches the brain and is converted to DA, more recent findings suggest, also, that the reaction of Ldopa with SAM and the depletion of SAM may be of importance in the actions of L-dopa and the reactions to L-dopa. It was reported that PD patients methylate nicotinamide at a rate 90 times higher than controls (9) and when injected into the brain of animals, both SAM, the methyl donor (10, 11, 12) and N-methyl beta-carboline, a SAM-dependent metabolite (13) caused reproducible PD-like symptoms. Those studies indicate that methylation may be increased in PD, therefore, the ability of L-dopa to interact with SAM and to deplete SAM may represent a mode-of-action for L-dopa (14). There are ancillary reasons to support this idea, because methylation reactions are increased in the aged population; the group that is most susceptible to develop PD. So, it can be reasoned that as people age, essential constituents, such as protein lipids, nucleic acids and biogenicamines are increasingly methylated. The methylation of DA norepinephrine and serotonin can deplete the levels of these neurotransmitters, changes that occur in PD. Methylation can increase N-methyl beta-carboline, that has been reported to cause PD-types of effects (13) and methylation produces surface active phospholipids, which decreases the viscosity of cell membranes (15). It also increases the hydrophobicity of protein by neutralizing negative charges (16) and silent gene transcription (17). The expression of tyrosine hydroxylase (TH)-mRNA for example, is suppressed by DNA methylation of the TH gene (18). Furthermore, N-methylation has been shown to increase the cytotoxicity of the monoamine derivative, isoquinoline, that is proposed to serve as pathogenic agents in PD (19). It is likely, therefore, that an increase in methylation may induce the wear and tear that lead to PD-types of changes in the susceptible individual, such as a person endowed with low number of nigrostriatal DA cells, or having nigrostriatal dopamine cells with poor metabolic qualities. There is a putative role of methylation in the actions of, and the reactions to, L-dopa because chronic L-dopa has been shown to increase the activities of methionine adenosyl transferase (MAT) (20) and COMT (21) as well as the protein and nucleic acids for those enzymes (22). Once induced, MAT will increase the synthesis of SAM, and COMT will transfer the methyl group of SAM to L-dopa and DA and will decrease their levels, More L-dopa will be required to maintain the levels of DA so, patients
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with high capacity for the induction of MAT and COMT may experience short-lived beneficial effects from L-dopa. Moreover, toxic metabolites and metabolites that interfere with L-dopa, may be produced. An example is 3-O-methyl dopa. It has been shown that when 3-0-methyldopa was given with L-dopa to PD patients the clinical responses to L-dopa was reduced (23, 24, 25) and 3-0methyldopa also decreased the accumulation of dopamine in rats treated with MPTP (26) as well as in normal rats (27,28). In addition, evidence shows that the mono and di-methyl metabolites of DA, 3-methoxytyramine (3-MT) (29) and 3,4-dimethoxy-phenylethylamine (DIMPEA) (29, 30) are behaviorally active, and interact with DA receptors (unpublished data). Most of the studies concerning the interaction of L-dopa with the methylation process were performed in the seventies, and focused on the metabolism of L-dopa. Since methylation may play important roles in the actions of L-dopa, the effects of L-dopa on transmethylation were reexamined. These studies are necessary, also, in light of the availability of more sensitive and reliable analytical techniques, utilizing HPLC-combined electrochemical and spectrophotometric analytical procedures. The studies make it possible to compare the present data with earlier data, to perform the investigation under acute, subchronic and time-dependent conditions and to examine more parameters, such as the values for SAM, its demethylated product, S-adenosylhomocysteine, brain L-dopa and dopamine, as well as changes in the rate of methylation (SAWSAM). Mouse, which seems to be a more sensitive model in certain PD studies, and which we have used to investigate the chronic effects of L-dopa on MAT and COMT, are used instead of the rat, on which most methylation data have been collected. The results show that L-dopa depletes SAM and increases SAH. It increases methylation much more than it increases DA. The effects were dose-dependent and accumulative, The data help us to understand why L-dopa induces the enzymes, MAT and COMT and it may help us to know more about the side effects of L-dopa.
Materials and Methods Animals In this study, Swiss Webster, albino male mice (25-35g), were purchased from Harlan Sprague Dawley. They were kept under a 12 hr light-12 hr dark cycle at a constant temperature (22 “C), with access to food and water. The animals were allowed at least 5 days acclimation before use in any experiment. For the time course study, both single and multiple injections were performed. Groups of 4 mice, intraperitoneally, received a single dose of L-dopa (100 mg/kg) suspended in phosphate buffered saline (PBS). They were decapitated at 0.5, 1, 2, 4 and 8 hr(s) after injection. For the study that evaluates the accumulative effects of L-dopa, groups of 7 mice each received one, two, three or four injections ofL-dopa (100 mg/kg) at 45-min intervals and were decapitated 30 min after the last dose. In the dose-effect study, mice received 0,50, 100,200 or 300 mg/kg of L-dopa and were decapitated 30 min after the injections. In all the experiments, matching control groups received the vehicle alone and were sacrificed with each experimental group to control for changes due to the daily rhythm in SAM concentrations in the brain. Chemicals S-adenosylmethionine (SAM) (p-toluene sulfonate salt), S-adenosylhomocysteine (SAH), L-dopa, dopamine and l-octane sulfonic acid sodium salt (HPLC grade) were purchased from Sigma Chemical Company, St. Louis, MO. Triethylamine (TEA) was obtained from Aldrich, Milwaukee, WI. HPLC grade acetonitrile and phosphoric acid were purchased from Fisher Scientific, Fair Lawn, NJ. All other chemicals used were obtained from Fisher Scientific and were of analytical grade.
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Preparation of brain homogenates The brains were removed immediately after sacrificing the animals. Brain tissues were homogenized in 2 volumes (w/v) of ice-cold 0.4 M perchloric acid using a PolyScience (model X520) Polytron. The homogenates were centrifuged at 9,000 x g for 20 min at 4 “C. The supematants were filtered (0.45 urn) and stored at -70 “C until analyzed by high-pressure liquid chromatography (HPLC). HPLC Assay Fifty microliters of the brain supematants were injected onto an HPLC system. The HPLC system consisted of a Thermal Separation Products (TSP) Solvent Delivery ConstaMetric 4100 pump with an online membrane degasser, a TSP Spectra System AS3500 autosampler with a 100 up1 sample loop and a 25 cm x 4.6 mm id., 5~ Cl8 Hypersil column (Whatman EQC). The detection system consisted of a TSP SpectroMonitor 3200 UV detector and an ESA Coulochem II electrochemical detector (5200 A model) with a high sensitivity analytical cell ( Model 5011). The UV detector was operated at 260.2 nm and the electrochemical detection was performed with guard cell set at 3 50 mV and analytical electrodes set at 300 mV for the second electrode and -50 mV for the first one. The sensitivity of the second analytical cell was set at 5 PA. The mobile phase consisted of 75 mM NaH,PO,.HO, 5.1 mM l-octane sulfonic acid sodium salt, 100 uV1 TEA and 10% acetronitrile. The final pH was adjusted to 3.0 with phosphoric acid. The flow rate was 1.5 mI/min and a column pressure of 1938-2023 psi was maintained. Concentrations were calculated by reference to peak area of external standards. All standards were freshly prepared in 0.4 M perchloric acid immediately before analysis. Recovery Experiments Brain tissues were homogenized in 2 volume of 0.4 M perchloric acid containing known amounts of SAM, SAH, L-dopa and dopamine. Standard peak areas then corrected by subtracting the contributions from the endogenous origin which was obtained from another set of tissue preparations without added standards. Seven replicates were prepared and analyzed as described above. Statistical Analysis Values for the brain concentrations of SAM, SAH, L-dopa and dopamine, and the calculated SAIWAM were given as the mean + the standard error of the mean (SEM). Two tailed paired t-test were used to compare the matched controls and the tested values. All the statistical analysis was performed using GraphPad Prism software.
Results HPLC-combined electrochemical andspectrophotometric detection of SAM, SAH, L-dopa and DA. Both SAM and SAH, as well as L-dopa and DA were extracted and determined from the same tissue samples into 0.4 M perchloric acid. Using an HPLC system with online W and electrochemical detectors enabled us to detect these four compounds simultaneously. The levels of SAM and SAH were determined by the W detector. The levels of L-dopa and DA were detected by the electrochemical detector. These four compounds were well separated (Fig. 1) using the described method. It was determined that the recovery of SAM, SAH, L-dopa and dopamine from brain extracts was 62.5f4.3%, 73.9+6.0%, 65.3+5.0% and 59.8+5.0% (n=7), respectively. Although the recovery was appreciable less than 100°/o, the one cycle low volume extraction procedure optimized the concentration of the metabolites and speeded up the extraction and detection procedures. The calibration curves, based.on area-under-the-peak from standards of 1.00-10.0 uM SAM, 0.200-1.50 uM SAH, 0.200-7.50 @l L-dopa and 0.500-10.0 p.M DA showed a good linearity, with correlation coefficients of 0.998, 0.994, 0.990 and 0.997 for each curve, respectively.
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(A)
Time (tin) Fig, 1 HPLC Chromatograms of SAM, SAH, L-dopa and dopamine from 50 l.tl of mouse brain extracts. SAM and SAH were analyzed using UV detector. The retention times for SAM and SAH were 19.5 min and 14.2 min respectively (A). L-dopa and dopamine were recorded from electrochemical detector. The retention times for Ldopa and dopamine were 4.1 min and 11.4 min, respectively (B). The HPLC conditions were as described in the Materials and Methods. Time-course eSfect of a single dose of L-dopa. The effects of a single injection of L-dopa on the levels of SAM and SAH were determined. At 0.5 hr after the injection of 100 mg/kg of L-dopa the levels of SAM were markedly decreased to 64% of control value. The concentration of SAM for the L-dopa treated animals was 21.4 +l 1 nmol/g of wet tissue, as compared to the value of 33.5 + 0.3 nmol/g for the control animals. The concentration of SAH, the demethylated metabolite of SAM, was increased to 2.38 f 0.21 nmolfg, as compared to the control value of 1.28 f 0.16 nmol/g. This represents an increase of 86% (Fig. 2). At 2 hr post-injection the concentration of SAM was still depressed to 80% of controls and that of SAH returned to normal. At the 4 and 8 hr post-injection periods the levels of SAM and SAH both returned to control values (Fig. 2). The ratio of the value of SAH to that of SAM (SAH/SAM) is used to indicate the extent of transmethylation. It is therefore an index of methylation, and the value is proportionate to the levels of methylation. The results in Fig.2 show that L-dopa increases methylation in the brain of mice, noted by a dramatic 200% increase in SAHISAM at the 0.5 hr postinjection period. At 4 and 8 hr post-injection the methylation rates were statistically the same for both groups.
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q SAHfSApvr
2 3002 g z 200-
0
sfw
0
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0 s
loo-
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Time (h)
Fig.2 The time course of the effect of a single dose of L-dopa (100 mg/kg) on methylation in the mouse brain. Each point represents the mean and standard error of the mean for data from four animals. Data are expressed as a percentage of the levels found in the corresponding control group. Statistically significant changes are indicated by an asterisk (*PC 0.05). Effects of repeated injection ofL&pa on methylation. L-dopa has a sustained depleting effects on SAM when given to groups of mice for 1 time or for 2, 3 or 4 consecutive times at 45611 intenals and determined 30 min after the last dose (Table I). Mice that received one injection of L-dopa showed a decrease in the concentration of SAM to 77% of control value. Two consecutive injections of L-dopa further depleted the levels of SAM to 40% of control value. Three consecutive injections reduced SAM to 37% of control level and four infections Table I. l%e sustained efsects ofl-dopa on SAM, SAH and SAH/SAkf values in the mouse brain. Four groups of mice (N=7) received 1 dose or 2, 3 or 4 doses of L-dopa (100 mg/kg, ip) at 45 min intervals and were sacrificed 30 min after the last injection. Concentrations are expressed in nmoYg of fresh tissue and all values represent the mean + SEM P
1 2
s) Control 38.9H.7
SAM L-dopa 29.6_+3.3*
SAH Control L-dooa 2.24f0.04 4.95+0.68*
&4H/SAM(x 102) Control L-dooa 5.8kO.2 19.3M.5*
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decreased the concentration of SAM to 25% of controls. After the same series of L-dopa injections, SAH was increased to 223% after the first, and plateau at over 300% of controls after the 2”d , 3” and 4” injections (Table I). The SAWSAM kept increasing with the repeated doses of
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L-dopa and reached the highest value of 1702% of controls after the fourth dose. Again, this is indicative of an L-dopa induced increase in methylation. Dose-e#ect ofL&pa on methylation. The dose-effect of exogenous L-dopa on the concentrations of SAM, SAH, the levels of methylation (SAH/SAM) and on brain L-dopa and dopamine levels were examined (Fig.3). The
SAHISAM
Brain
E-dopa
DA
L-dopa
dose (mg/kg) Fig. 3
The dose-effects of L-dopa on methylation in the mouse brain. Each point shows the mean of data from seven animals. All data are expressed as percentages of the levels found in the control group. Opened circles represent the values for SAH, filled circles represent the values for SAM (A). In B, SAH/SAM is represented by squares, L-dopa by filled triangles and DA by opened triangles.
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L-dopa concentration detected in the control animals was 1.13H.09 mnol/g. Treating the mice with L-dopa of 50, 100 and 200 mglkg increased brain L-dopa to 334, 340 and 796% of control value, respectively. For the same L-dopa doses, the mean DA levels increased by 138, 126 and 232%, respectively. L-dopa and DA react avidly with SAM and this was evident by the dosedependent L-dopa-induced reduction of SAM to 5 1, 58 and 28% of controls, SAH, on the other hand, was increased to 201, 219 and 336% of control values for the respective doses of L-dopa. The rate of methylation was dose-dependently increased to 369, 402 and 1135% of control by Ldopa. Discussion The data show that exogenous L-dopa administration has significant effects on the biological methylation process in the mouse brain. The depletion of SAM by L-dopa is consistent with the results of previous studies (3 l), but the analysis of SAH, brain L-dopa and DA in this study, help to evaluate the comparative effects of L-dopa on DA and the methylation process. L-dopa increases transmethylation to a greater extent than it increases the levels of brain L-dopa and DA; a proportional increase of about 4:3: 1 occurred, respectively. So, the increased brain levels of L-dopa, from the injected L-dopa, corresponds more closely with the methylation process than with the increment in dopamine. The data suggest that biological methylation may play a role in the actions of L-dopa, in addition to the well established role for L-dopa, of increasing the levels of DA. The results are relevant to previous findings. Some of the findings, include the report that the injection of the methyl donor, SAM, causes PD-like changes in animals. The changes include tremor, rigidity and hypokinesia, the depletion of tyrosine hydroxylase and dopamine, and nigrostriatal neuronal degeneration in rodents. The behavioral effects were inhibited by L-dopa and other agents that seem to compete for the methyl donor (29,11,12). Collins et al. ( 13) also, found that N-methyl beta-carboline, a methyl metabolite of SAM, caused PD-like symptoms in animals. Furthermore, it was observed that the methylation of nicotinamide, used as a measure of methylation, was increased more than ninety times in PD patients as compared to age-matched controls (9). These findings, when taken together, suggest that an increase of methylation may occur in PD. Accordingly, as a therapy for PD, L-dopa may express part of its actions through the reaction with SAM and the depletion of SAM. The depletion of SAM would have a sparing effect on DA metabolism, while L-dopa replenishes DA. The interaction of L-dopa with the methylation process, though, will change over a period of time during L-dopa administration. During the early stages, L-dopa will compete with other catecholamine for the methyl donor, SAM. This has been corroborated in studies showing that the methylation of norepinephrine was decreased in the brain of L-dopa treated rats (7). Similarly, a decrease in the methylation of DA and a corresponding increase in its availability should occur following the acute administration of L-dopa. The acute or subacute effects of L-dopa, however, will be different from the chronic effects, The continuous utilization of SAM by L-dopa will lead to an L-dopa-induced increase in the activities of the enzyme that catalyzes the production of SAM (MAT) (20) and the enzyme that transfers the methyl group of SAM to the catecholamines (COMT) (2 1). The induction of MAT by L-dopa has been reported (20). It requires relatively high doses of L-dopa, administered at frequent intervals (3-4 times/day) over several days (20). The induction of MAT may be viewed as a counter-response measure to replenish the depletion of SAM caused by L-dopa (20). The increase in MAT, as a counter-response
to the depletion of SAM by L-dopa will produce more
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SAM. The rebound increase in SAM will increase the methylation and destruction of L-dopa and DA. Therefore, the levels of brain L-dopa and DA usually achieved during the early periods of L-dopa therapy will no longer be attainable. More L-dopa will be required to sustain the DA level required for the control of the symptoms of PD, so the effects of L-dopa will wear 0% and a form of tolerance to L-dopa will develop. Thus, the interaction of L-dopa with the methylation process may create a vicious cycle in which by reacting with, and utilizing SAM, L-dopa will eventually induce methylation enzymes that lead to the conversion of dopamine as well as the administered L-dopa. High rates of the methylation of L-dopa and DA will deplete these biogenic amines, causing the loss of the effects of L-dopa and the production of their methylated metabolites (Fig. 4). High levels of 3-O-methyl dopa have been well documented following L-dopa therapy (23,2). At present, however, there is a lack of information regarding the accumulation of the methyl metabolites of dopamine during L-dopa therapy. It is well accepted, though, that increases in the methylation of dopamine should parallel the increases in the methylation of L-dopa. The accumulation of methylated metabolites of L-dopa and DA may help to explain the adverse effects of L-dopa seen following long-term therapy, because 3-O-methyl dopa, for example, has been shown to antagonize the effects of L-dopa (23,24,25,26,27,28). Such an effect may be compared with, and contributed to, the “wearing-off-effects”. The effects of the methyl metabolites of dopamine, 3-MT and DIMPEA, which, respectively, decreases and increases locomotor activities (29) and DA binding in rats may be compared to the “on-off effects” seen following chronic L-dopa administration to PD patients and to MPTP treated monkeys. The “on-off’ types of effects may be explained by the scenario of a sequential methylation of DA, to produce 3- MT and DIMPEA. Initially, DA is monomethylated at the 3-OH position, producing toxic levels of 3-MT, and causing hypokinesia. The accumulation of 3-MT, however, will inhibit further mono-methylation of DA (end-product inhibition), at the same time 3-MT will serve as substrate for the production of DIMPEA via methylation at the 4-OH position. The accumulation of toxic levels of DIMPEA will cause hyperkinesia. However, the accumulation of DIMPEA will inhibit its own synthesis, and the levels of 3-MT will again increase to cause hypokinesia. Thus, this important phasic or rhythmic event may be caused by a relatively simple biochemical process, as illustrated in Fig. 4. In summarizing the results, the data and the corroborative evidence present an interesting scenario. On the basis that excess methylation may be involved in PD-like changes (29,l 3, 9), the depletion of SAM by L-dopa may help in alleviating of the symptoms of PD. The depletion of SAM, however, creates great demands for the already limiting methyl donor, SAM. Such a biochemical stress on the methylation process activates and/or induces MAT to replenish SAM. Evidence also showed that Ldopa induces the enzyme COMT (21). The induction of MAT causes a rebound elevation in the production of SAM, and the induction of COMT will promote aggressive transmethylation reactions, leading to the methylation and depletion of L-dopa and dopamine. It means that higher doses of Ldopa will be required to maintain the therapeutic DA levels, a state of condition that is remindful of the “wearing-off effect” or a form of tolerance. The methyl metabolites of L-dopa and dopamine will also be produced. These metabolites are more hydrophobic than their parents, and will interact more readily with cell membranes, and may, through their mass actions, interfere with the availability of Ldopa and the receptor actions of DA. Thus, the interaction of L-dopa with SAM may underlie the actions and/or the toxic side-effects of L-dopa. As illustrated in Fig.4, the effects of L-dopa can also decrease the levels of ATP, because, every mole of L-dopa has the potential of utilizing at least two moles of ATP, via the utilization of SAM and the replenishment of SAM by the reaction of ATP with methionine. The production of secondary metabolites, such as homocysteine and adenosine from SAH should also be noteworthy.
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SAM ATP
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Pool SAM
Ldopa+ A
Fig. 4 The illustration highlights the production of dopamine from L-dopa and the known and putative transmethylation reactions that occur. Vertical short arrows (” or ‘) indicate decrease or increase in activities/concentrations. Solid arrowed-lines (+) indicate the direction of the reaction, and broken arrowed-lines represent feed back inhibition of reactions. The question mark (?) represents unknown pathway. Abbreviations: BAMT (biogenicamine methyl transferase); COMT (catecholamine methyl transferase); MAT (methinonine-adenosyltransferase); SAM (Sadenosyhnetbionine); Met (methionine); SAH (S-adenosylhomocysteine; L-dopa (L3,4-dihydroxyphenylalanine); 3-OMD (3-methoxy-4-hydroxyphenylalanine); 3,4OMD (3,4dimethoxyphenylalanine); 3-MT (3-methoxytyramine); DIMPEA (3,4-dimethoxyphenylethylamine).
Acknowledgments Support for this research was provided by the National Institutes of Health grants GO8 111 , RR03020 and NS 28432.
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