Effects of l -DOPA and l -3-o-methyl-DOPA on uptake of [3H]l -methionine by synaptosomes

Neuro~harmacok%?~, 1972.11.715-720 PergmonPress. Printedin Gt. Britain.

EFFECTS UPTAKE

Psychiatric

OF

L-DOPA

AND

L-3-0-METHYL-DOPA

OF [3H]~-METHIONINE

ON

BY SYNAPTOSOMES*

R. J. BALDEssARINIt and M. KAROBATH$ Research Laboratories, Department of Psychiatry, Massachusetts Harvard Medical School, Boston, Massachusetts

General Hospital and

(Accepted 4 February 1972)

Summary-Uptake of [aH]L-methionine by nerve endings in homogenates of rat brain occurred by a rapid, temperature-dependent process, saturable with increasing substrate concentrations. The apparent Michaelis constant (Km) for methionine was 0.21 mM. Transport was inhibited by dinitrophenol, but was not dependent on Na+, nor inhibited by ouabain. L-DOPA or L-3-0-methyl-dopa inhibited this transport process if incubated with [SH]r,-methionine. L-DOPA acted as a competitive inhibitor of methionine uptake (Ki about 0.15 mM). When nerve endings were preloaded with either aromatic amino acid, initial uptake of methionine was enhanced, probably by a countertransport process. We suggest that presence of L-DOPA or of 3-0-methyl-dopa, which accumulates in the brain after L-DOPA administration, could alter the availability of methionine for synthesis of the methyl donor S-adenosylmethionine. THE SUCCESSFUL use of L-dihydroxyphenylalanine (L-DOPA) in the treatment of Parkinson’s disease has involved the unprecedented administration of very large doses of an aromatic amino acid to man for extended periods. A number of toxic side effects of this treatment have been reported (BARBEAUand MCDOWELL, 1970), although their mechanisms remain obscure. Several biochemical effects of L-DOPA are also known. For example, L-DOPA as a substrate of catechol-0-methyl-transferase (COMT, EC 2.1.1.6) utilizes methyl groups of S-adenosyl-methionine (SAMe) and leads to depletion of this important methyl donor in animal tissues including brain (CHALMERS,BALDESSARINI and WURTMAN,1971) and in human blood (MATTHYSSE, LIPINSKIand SHIH, 1971). Alterations of SAMe in the rat brain following chronic administration of L-DOPA follow a bi-phasic course (CHALMERSet al., 1971). Thus, there is an initial depletion (90 %) of SAMe during the hour following a final dose of L-DOPA (100 mg/kg i.p.) followed by a rise to levels up to 304O’A above control for several hours. The initial depletion of SAMe is presumably the result of utilization of the methyl donor at rates in excess of synthesis. The “rebound” increase of SAMe concentrations in brain is not readily explained. However, SAMe levels are also elevated in leukocytes of patients with chronic myelocytic leukemia (BALDESSARINI and CARBONE,1965). Increased methionine availability can increase the rate of synthesis of SAMe (BALDESSARINI, 1966) and the uptake of methionine by leukemic leukocytes is reported to be higher than normal (SLOANE and

BRIDGES, 1968). Furthermore,

L-DOPA

can affect

the uptake

of other

amino

*Supported by U.S. Public Health Service (N.I.M.H.) Grant MH-16674. fRecipient of Research Scientist Development Award, Type II, National Institute of Mental Health K02MH-74370. $Fellow of the Max Kade Foundation, Inc., New York, N.Y. Present Address: Department of Psychiatry, University of Vienna (Psychiatrische Universitatsklinik, Allgemeines Krankenhaus der Stadt Wien, A-1090, Wien, Austria). 715

116

R. J. BALDESSARINI and M. KAROBATH

acids into isolated nerve endings and can alter levels of endogenous aromatic amino acids in the rat brain (KAROBATH,DIAZ and HUTTUNEN,1971, 1972). Thus, we have studied the effects of L-DOPA and L-3-0-methyl-dopa upon the uptake of methionine into isolated nerve endings.

METHODS Amino acid uptake was studied, using a partially purified preparation of synaptosomes from whole brains of male Sprague-Dawley rats (180-210 g). Tissue preparation, incubation and radioactivity counting methods were those of GRAHAME-SMITH and PARFITT(1970) with only minor modifications. Thus, “Pi’ (crude mitochondrial-synaptosomal) pellets (17,000 g) were prepared and then incubated in a simplified Krebs-Ringer-PO, buffered medium containing (mM): NaCl, 120; KCl, 5; Sucrose, 40; D-glucose, 10; and Na-phosphate, 20 at pH 7.4. Most incubations were carried out at 37°C with tissue (0.3-0.5 mg protein) in the presence of O-5 &i of chromatographically pure [3H-methyl]L-methionine (New England Nuclear, 3.3 Ci/m mol) with 0.1 mM or 1.0 mM L-methionine (Calbiochem) in 500 ~1 total volume. The [3H]L-methionine (chromatographically found to be more than 95% pure) was either used within a month of manufacture, or for kinetic experiments, repurified by ascending paper chromatography in n-butanol: acetic acid: water (25 : 4 : 10, ~01s) followed by descending elution with 70 ‘A (vol) ethanol to remove oxidation products which form at the rate of about 5% a month (personal communication, New England Nuclear Corp.). In some experiments methionine concentration was varied. In others, tissues were either pre-incubated (5 min) or simultaneously incubated in normal medium or with various concentrations of L-DOPA (donated by Hoffman-LaRoche) or L-3-0-methyl-dopa (L-3methoxytyrosine, donated by Sterling-Winthrop). When pre-incubation was used, tissue was recovered by centrifugation and then resuspended in medium with or without L-DOPA or L-3-0-methyl-dopa. Reactions were stopped with 5 ml of iced medium and synaptosomes containing [3H]-methionine were recovered (BALDESSARINI and VOGT, 1971) from the incubating medium by ultrafiltration (25 mm Millipore filters, 0.8 t.~pores) and washed with 5 ml of 0.32 M sucrose. The 3H on the filters was counted by scintillation spectrometry. “Blanks” were determined as uptake at 4°C and subtracted. Kinetic data were analysed by plots of l/velocity vs l/substrate concentration or l/velocity vs inhibitor concentration. In order to identify the labelled material taken up, [3H]L-methionine was incubated for 5 min at 37°C with preparations of “Pz)’ pellet as described. The tissue was recovered by centrifugation at 17,000 g, washed with fresh incubation medium and extracted by homogenization into 70 % (~01s) ethanol. The extracted material was chromatographed as already described, along with authentic labelled and unlabelled L-methionine. Similarly, “P,” tissue was incubated for 2 min, centrifuged, washed with 0.32 M sucrose, taken up in the sucrose and layered onto sucrose density gradients prepared as described by GRAHAMESMITHand PARFITT(1970) and centrifuged at 100,000 g for 85 min. The various fractions were carefully removed by aspiration, and aliquots were prepared for scintillation spectrometry by solubilization with 4N NH,OH and “N.C.S.” (Amersham/Searle Corp.). In other experiments, the “P2” tissue preparation was exposed to sonic or osmotic stress and recovered by centrifugation as described by GRAHAME-SMITH and PARFITT(1970), following incubation with [3H]L-methionine for 5 min, or exposed to a various drugs or modified media for 5 min and then incubated with [3H]L-methionine for 1 min.

L-DOPA, 3-0-methyl-dopa

and methionine uptake

717

RESULTS

When [3H]L-methionine was incubated with the tissue preparation containing synaptosomes and mitochondria, there was a very rapid uptake of the amino acid. The process increased linearly with time for not more than 1-2 min (Fig. 1) and from 2 to 5 min little EFFECTS OF L-DOPA ON UPTAKE OF [3H]L-METHIONINE A. PRE-INCUBATED L- DOPA

WITH

B.

CO - INCUBATED L- DOPA

WITH

FIG. 1. Altered uptake of [3H]L-methionine in the presence of L-DOPA.

(A) Partially isolated nerve endings (resuspended

crude mitochondrial pellet) were preincubated with L-DOPA ( 0), 1 mM, or in normal medium ( l) for 5 min, centrifuged and transferred to fresh medium and incubated with [SH]L-methionine (1 mM). (5) Similar nerve endings were co-incubated with [SH]L-methionine (1 mM) and with LDOPA (ImM) ( 0) or in medium without L-DOPA (0). In both experiments, nerve endings were recovered by ultrafiltration, and their 3H was counted. Data are mean % of maximum uptake (N = 3-6); 100% f S.E.M. = 7.12 h 0.43 nmol [3H]L-methionine/2 min/mg protein. The differences at 1 min are highly significant (P-z OWS) when values of “t” are calculated from the raw data (nmol/min/mg).

further increase in uptake occurred. Incubation for times longer than 10 min resulted in a gradual loss of accumulated radioactivity. The radioactive material accumulated in 5 min was found by chromatographic analysis to migrate in a single peak of radioactivity (more than 97% of total 3H), identical in Rf to authentic L-methionine. When the tissue was exposed to hypoosmotic stress, 75-80 % of the accumulated [3H]L-methionine escaped into the medium and more than 60% escaped during sonic stress. When the tissue was ultracentrifuged on discontinuous sucrose gradients, about 90 % of the 3H could be accounted for and the mitochondrial fraction contained only 2.950.4 % of the total 3H (mean f S.E.M. for 8 gradients). The uptake process was found to be dependant on temperature as the 1 min uptake was decreased about 50 % at 25°C and more than 90% decreased at 4°C. Dinitrophenol(l0 mM) completely inhibited uptake and desmethylimipramine (1 mM) had a weak inhibitory effect, whereas ouabain or cocaine (1 mM) had no inhibitory effect. When Na+ was replaced by choline or Li+ in a sodium-free TRIS (tris-[hydroxymethyllamino-methane, Sigma) buffered

718

R. J. BALDESSARINIand

M. KAROBATH

medium, uptake was not inhibited, while substitution of K+ for Na+ decreased uptake by 43 %. The uptake of [3H]L-methionine by isolated nerve endings appeared to follow saturable Michaelis-Menten kinetics (Fig. 2). The mean apparent Michaelis constant (Km) was 0.21 mM and the apparent Vmax was 5.2 n mol/min/mg protein, which is equivalent to about 97 nmol/min/g of intact whole brain tissue. When L-DOPA was added to the incubating medium (0.4 or 1*OmM), inhibition of the initial phase of uptake of [3H]L-methionine occurred (Fig. 1, Table 1). However, when TABLE 1. ALTERED UPTAKE OF [‘WJL-METHIONINEIN THE PRESENCEOF L-DOPA OR L-3-0-METHYL-DOPA Condition 0-methyl-dopa, preincubated L-DOPA, preincubated 0-methyl-dopa, co-incubated L-DOPA, co-incubated

% of control 188.3 238.4 19.1 19.9

Partially isolated nerve endings were pre-incubated for 5 min with L-DOPA or 3-0-methyl-dopa or in normal medium, separated by centrifugation, transferred to normal medium or to medium containing L-DOPA or 3-0-methyl-dopa (0.4 mM), and incubated for 1 min with [3H]L-methionine (0.1 mM). Nerve endings were then recovered by ultrafiltration and their radioactivity was counted. The differences are all highly significantly (P < 0.001) different from control (100% f S.E.M. = 1.1 nmol/min/mg protein & 0.04) for N = 6-12 when values of “t” were calculated from the raw data (nmoI/min/mg).

tissues had been pre-incubated with L-DOPA or in normal medium for 5 min, recovered by centrifugation, and then incubated with radioactive methionine, there was an acceleration of initial uptake by tissue previously exposed to L-DOPA (Fig. 1, Table 1). When the tissue was pre-incubated with L-DOPA and then also incubated with L-DOPA during uptake of [3H]L-methionine, there was no difference from uptake by tissues not exposed to L-DOPA. The 3-0-methylated analog of L-DOPA had similar effects (Table 1). L-DOPA appeared to interact competitively with the uptake of methionine (Fig. 2) and values for its inhibitory constant (Ki) were about 0.15 mM (range, 0.13-0.17 mM).

DISCUSSION The present findings demonstrate that pharmacologic aspects of the uptake of amino acids can be studied using preparations of isolated nerve endings. The uptake of methionine appeared to follow Michaelis-Menten kinetics. A striking feature of the uptake of amino acids by synaptosomes as compared with brain slices is the extreme rapidity of the former process, in which maximal accumulation occurs within 5 min as noted previously by GRAHAME-SMITH and PARFITT(1970), although uptake was linear with time for less than 2-3 min. Nevertheless, ultrafiltration permits the recovery of synaptosomes within a few seconds and thus allows studies of the kinetic properties of initial uptake of amino acids or amines (BALDESSARINI and VOGT, 1971). The uptake process appeared to involve entry mainly into isolated nerve endings because stresses which are known to disrupt the integrity of cell

L-DOPA, 3-0-methyl-dopa

and methionine uptake

719

INHIBITION OF UPTAKE OF PHIL-METHIONINE BY L-DOPA A.

i/V

vs

I/[!31

II-0,1

(f/ thErtffohwE]j

x to-3_M

0

O,l

L -DOPA

0.‘2

0.3

0.4

0,5

fm,M,!

FIG. 2. Kinetic analysis of the inhibition of uptake of [3H]L-methionine by L-DOPA. (A) Resuspended crude mitochondrial pellets were incubated for 1 min with [3H]t-methionine (0.05-0.80 mM) and with ( 0) or without ( l) added L-DOPA (0.25-0.5 mM). (B) Similar incubations were done with [aH]L-methionine at 0~0.5,0~10,0~20 or 0.40 mM, with L-DOPA present (up to 0.5 mM). In both experiments, nerve endings were recovered by ultrafiltration and their 3H was counted. Data were calculated as l/velocity (V=nmoi [aH]L-methionine/min/mg protein) vs l/methionine concentration (A) or vs L-DOPA concentration (B) and plotted by linear regression analysis. Values for Ki ranged from 0.133 to 0.174 mM.

membranes (GRAHAME-SMITH and PARFITT, 1970) released most of the accumulated radioactivity identified chromatographically as unmetabolized [3H]L-methionine and because uptake into mitochondria, the other membrane enclosed structures likely to occur in the tissue preparation, accounted for less than 3 % of the total. Unlike the transport of amines at nerve endings, the uptake of methionine was apparently not absolutely dependant on sodium, as Lif or choline could replace Naf adequately, although K-b could not; the process was not inhibited by ouabain or by other drugs known to block the transport of amines, except that desmethylimipramine had a small inhibitory effect at high concentrations (mM). Thus, the lack of sodium-dependency is similar to that reported for tryptophan at isolated nerve endings (GRAHAME-SMITH and PARFITT, 1970), or for the transport of leucine by alveolar macrophages (TSAN and BERLIN, 1971), and for the relative lack of dependence on sodium of the transport of leucine by neuronal cells in contrast to glia (HAMBERGER, 1971). L-methionine has been described as having a transport mechanism in brain tissues similar to that of the aromatic amino acids, and to be capable of competing for their uptake, or of facilitating their uptake by a process of counter-transport, both in vitro and in vivo (e.g. GRAHAME-SMITH and PARFITT, 1970). Whether inhibition or facilitation of transport will occur apparently depends on the relative concentrations of the competing amino acids

720

R. J. BALDESSARINI and M. KAROBATH

inside and outside of the cell (Fig. 1, Table 1). The implications of the present findings for amino acid metabolism in the intact nervous system subjected to large doses of L-DOPA include the possibility that L-DOPA may initially compete for uptake with a variety of amino acids including aromatic amino acids and methionine. However, since L-DOPA is so rapidly metabolized, this effect may not last for more than 2-3 hr, after an acute dose, by which time L-DOPA is no longer detectable in the brain (CHALMERSet al., 1971). At later times, however, the 3-0-methylated amino acid analog of L-DOPA is known to accumulate in the brain and other tissues and to undergo a relatively slow clearance, with a half-life of about 12 hr, partly because it is a relatively poor substrate for L-aromatic amino acid decarboxylase (EC 4.1.1.26; BARTHOLINI and PLETSCHER, 1970). Thus it is possible that at such later times, the efflux of 3-0-methyl-dopa could facilitate the transport of other amino acids, including methionine, into brain cells. The time course of the fall and then rise in methyl donor levels in the brain is very similar to the time course of the rise and fall of tissue concentrations of 3-0-methyl-dopa. Therefore, the present findings offer an hypothesis to explain the observed “rebound” increases in accumulation of SAMe in the brain several hours after administration of L-DOPA to rats (CHALMERSet al., 1971). Acknowledgements-The excellent technical assistance of Mrs. E. GREINERand Mrs. J. STEPHENSON and the gifts of drugs by Hoffman-LaRoche Co. and the Sterling-Winthrop Research Labs are gratefully acknowledged.

REFERENCES BALDESSARINI, R. J. (1966). Alterations in tissue levels of S-adenosyl-methionine. Biochem. Pharmac. 15: 741-74s. BALDESSARINI, R. J. and CARBONE,P. (1965). Adenosylmethionine elevation in leukemic white blood cells. Science, N. Y. 149: 644-645. BALDESSARINI, R. J. and VOG~, M. (1971). The uptake and release of norepinephrine by rat brain tissue fractions prepared by ultrafiltration. J. Neurochem. 18: 951-963. BARBEAU,A. and MCDOWELL, F. H. (1970). L-DOPA andParkinsonism, F. A. Davis, Philadelphia. BARTHOLINI,G. and PLETSCHER,A. (1970). Distribution and metabolism of L-3-0-methyl-dopa in rats. Br. J. Pharmac. Chemother. 40: 461467. CHALMERS,J. P., BALDESSARINI, R. J. and WURTMAN, R. J. (1971). Effects of L-DOPA on norepinephrine metabolism in the rat brain. Proc. Nutn. Acad. Sci. (U.S.A.) 68: 662-666. GRAHAME-SMITH,D. G. and PARFITT, A. G. (1970). Tryptophan transport across the synaptosomal membrane. J. Neurochem. 17: 1339-1353. HAMBERGER,A. (1971). Amino acid uptake in neuronal and glial cell fractions from rabbit cerebral cortex. Brain Res. 31: 169-178. KAROBATH, M., DIAZ, J.-L. and HUTTUNEN,M. 0. (1971). The effect of L-DOPA on the concentrations of tryptophan, tyrosine and serotonin in rat brain. Eur. J. Pharmac. 14: 393-396. KAROBATH,M., DIAZ, J.-L. and HUTTUNEN,M. 0. (1972). Serotonin synthesis with rat brain synaptosomes: Effects of L-DOPA, L-3-methoxytyrosine and catecholamines. Biochem. Pharmac. (in press.) MATTHYSSE,S., LIPINSKI,J. F. and SHIH,V. (1971). L-DOPA and S-adenosyl-methionine. Clinica. chim. Acta 3.5: 253-254. SLOANE,K. M. and BRIDGES, J. M. (1968). The in vitro uptake of 35S-L-methionine by normal and leukaemic leukocytes. Acta Haemat. 40: 18-27. TSAN, M. F. and BERLIN,R. D. (1971). Membrane transport in the rabbit alveolar macrophage. The specificity and characteristics of amino acid transport systems. Biochim. biophys. Acta. 241:155-169.