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Home , Monoamine oxidase, Norepinephrine, Octopamine

Proc. Nat. Acad. Sci. USA Vol. 68, No. 10, pp. 2370-2373, October 1971

Catecholamines and the Hydroxylation of in Synaptosomes Isolated from Rat (DOPA////) M. KAROBATH Psychiatric Research Laboratories, Massachusetts General Hospital, Boston, Mass. 02114 Communicated by Seymour S. Kety, July 16, 1971

ABSTRACT activity of synapto- removing transmitters from an extraneuronal location at the somes isolated from rat brain was examined. A modified , then incubation of synaptosomes with catechol- tritium-displacement assay was used, which allowed the measurement of tyrosine hydroxylase activity without the should inhibit the formation of DOPA from tyrosine. addition of either inhibitors of the of the The experiments demonstrate that tyrosine hydroxylase hydroxylated products or added exogenous . The activity is affected by uptake. The concentra- activity was strongly inhibited by the addition of tions required to inhibit the synthesis of catechols are in the exogenous and 3,4-dihydroxy-L-phenyl- M. . Aromatic amines other than catechols did not range of 10-7 markedly influence tyrosine hydroxylase activity. These MATERIALS AND METHODS in vitro findings support the hypothesis that synthesis of catecholamines is regulated by a mechanism of end- [3,5-H]HifTyrosine (Tracerlab) was purified by column chro- product inhibition at the tyrosine hydroxylase step. matography. Catechol impurities were absorbed on alumina and tritiated water and anions were removed by Synaptosomes are pinched-off nerve endings with relatively columns (8), by non-neuronal elements (1, 2). They absorption on Dowex-50 resin. Tyrosine was eluted from little contamination Dowex-50 with 25 ml of 4 N HCl; after distillation of the eluate contain the necessary for the synthesis of dopamine (v/v) from tyrosine (3, 4). It has under reduced pressure, tyrosine was dissolved in 5% (3,4-dihydroxyphenethylamine) aqueous . This stock solution was stored at -70°C, not yet been demonstrated that nerve endings of brain contain use to re- that converts dopamine to norepinephrine. and aliquots were lyophilized immediately before the hydroxylase move spontaneously formed tritiated water. Ficoll was ob- Synaptosomes show tyrosine hydroxylase activity, even with- removed from the cofactor by this tained from Pharmacia and salt impurities were out the addition of pteridine required it by dialysis. enzyme (3). This finding suggests that nerve endings contain this cofactor and the reducing system used to regenerate it. Preparation of Synaptosomes. Male rats (170-190 g, Charles There is much evidence that tyrosine hydroxylase is normally River Breeding Co.) were decapitated. were dissected the rate-limiting step in catecholamine (5). The free of the , and homogenized in 0.32 M sucrose in a activity of the enzyme is thought to be regulated by end- glass Elvehjem-type homogenizer with a motor-driven Teflon product inhibition, so that dopamine and norepinephrine may pestle modified to provide 0.25-mm clearance (Kontes Glass regulate their own rates of synthesis by such a mechanism Co.). A crude mitochondrial fraction was prepared (14,000 X g (6, 7). While 3,4-dihydroxyphenylalanine (DOPA) also in- for 15 min, ref. 15), resuspended in isotonic sucrose, and layered hibits tyrosine hydroxylase in vitro (8), the lack of significant over discontinuous Ficoll density gradients (15). The gra- accumulation of DOPA in tissues normally (9) implies that dients were centrifuged in an SW 27 Spinco rotor at 27,000 rpm such a feedback mechanism is not likely to regulate DOPA syn- for 45 min (15). The nerve-ending fraction was removed thesis in vivo. The rate of neuronal catecholamine synthesis by aspiration, washed in isotonic sucrose to remove Ficoll, may be modulated by neuronal activity (10-13). This activity, and resuspended in the incubation medium. The total of by releasing catecholamines at , would lower intra- preparation was 3 hr or less. All preparation were at neuronal catecholamine content and thus decrease the inhibi- 0-4°C. Electron microscopy showed the synaptosome fraction tion of synthesis. Such a system would provide for the re- to contain many structures identified as nerve-ending parti- plenishment of substances as they are being cles, but membrane fragments were also present. inactivated. appears to be a major utilized and of The method means of conserving transmitter catecholamines (14). The Determination Tyrosine Hydroxylase Activity. that could then inhibit further of Nagatsu et at. (16) was used with modifications. In this catecholamines reaccumulate amount amines be an assay, [3,5-3H]Ltyrosine is used as substrate, and the synthesis. Since active uptake of released may is the rate of catecholamine of tritiated water produced during the 3-hydroxylation important factor controlling syn- mM was undertaken to the measured. The incubation medium contained 100 NaCl, thesis, the present study investigate 5 2 mM Na of amines upon mM KCl, 10 mM. , 50 mM sucrose, effects of accumulation exogenous tyrosine was buffered 15 mM in isolated nerve If reuptake of ascorbate, and 2 mM Na EDTA, and by hydroxylase activity endings. at was fresh daily. into terminals an role in Na phosphate pH 6.6. Ascorbate prepared amines presynaptic plays important The final tyrosine concentration was 1.5-2.5 X 10-6 M, with Abbreviation: DOPA, 3,4-dihydroxyphenylalanine. about 1 MCi of [3H]tyrosine present in each tube. The total 2370 Downloaded by guest on September 23, 2021 Proc. Nat. Acad. Sci. USA 68 (1971) Tyrosine Hydroxylation in Synaptosomes 2371

incubation volume was 0.5 ml. Samples were incubated in a aration, the tyrosine hydroxylase assay gave precise results of metabolic shaker-bath at 37°C for 30 min. The reaction was 3.06 + 0.21 (SD) pmol per min per mg of protein (N = 9). stopped by addition of 0.05 ml of 2 M Na acetate buffer (pH Tyrosine hydroxylase in intact synaptosomes incubated 4.5) and the tubes were cooled on ice. After low-speed centrif- as in these experiments was found to be half-saturated by a ugation at 0°C, supernatants were transferred quantitatively substrate concentration in the medium of about 3 X 10- M to a small column of Dowex 50 X 8 resin (200-400 mesh, tyrosine. With a concentration of tyrosine (2 X 10-5 M) H+-form, 0.4 X 2 cm), which was placed on a column of found to be saturating and used in the present experiments, Dowex 1 X 8 resin (200-400 mesh, OH--form, 0.4 X 2 cm). incubation with various amounts of synaptosomes showed Columns were then washed three with 0.85-ml portions that the assay was linear with tissue concentrations from 0.12 of water. Aliquots (2 ml) of the combined effluents were trans- to 0.90 mg of protein per assay tube. The optimum pH for ferred to counting vials, mixed with 13 ml of Bray's solution tyrosine hydroxylase was found to be between 6.0 and 6.7. (17), and counted in a Packard scintillation counter. In order In the present experiments, the reactions were performed at to estimate contaminating neutral metabolites, the remain- pH 6.6 and contained 0.3-0.8 mg of synaptosomal protein per ing effluent material was lyophilized to dryness; the residue assay tube. In confirmation of previous findings (8, 22), 3- was taken up in 1.0 ml of water and counted in 7 ml of Bray's iodotyrosine and a-methyl-p-tyrosine were found to be very solution. Incubation of [3H]tyrosine, either with tissue at strong inhibitors of tyrosine hydroxylase at 10-4 M (Table 1). 0-40C or without tissue at 37°C, permitted estimations of This assay method allows the-measurement of tyrosine hy- the amount of spontaneously formed radioactive water. Both droxylase activity in synaptosomes without interfering with methods indicated that less than 0.1% of the total tritium the further metabolism of the newly synthesized DOPA. present in the incubation mixture was released nonenzymati- The assay was used to study the effect of various compounds cally. Counting efficiency was determined by the internal- on synaptosomal tyrosine hydroxylase activity. The synapto- standard method (18). somes were incubated under conditions known to permit the uptake of exogenous catecholamines (23, 24), and near the op- Estimation of Tyrosine Accumulation by Synaptosomes. A timum pH for tyrosine hydroxylase activity. Since the incuba- Millipore filter technique was used (19). Aliquots of 0.2 ml with were removed from the incubation mixture, pipetted into 5 ml tion mixture was not fortified the pteridine cofactor, tyrosine hydroxylase activity was dependent on the endoge- of ice-cold 0.32 M sucrose, and poured on a moistened 25-mm nous cofactor and its regenerating system. Ascorbic acid and Millipore filter (0.65-,um pore-size), which was washed oxidation of twice with 10-ml portions of 0.32 M sucrose. The filter was EDTA were added to prevent nonenzymatic then dried under an infrared lamp and placed in a counting catecholamines. vial with 10 ml of a toluene solution containing 2,5-diphenyl- In the present experiments, it was found that exogenous oxazole (0.4%) and p-bis- [2-(4-methyl-5-phenyloxazolyl)] ben- norepinephrine, dopamine, or DOPA, in concentrations as low zene (0.01%) (Packard Instrument Co.). as 10-7 M, can inhibit tyrosine hydroxylation (Table 2). Up- take of exogenous amines raises the intrasynaptosomal con- Miscellaneous Procedures. Tyrosine was measured by the centration of the inhibitory amines to concentrations con- fluorometric method of Waalkes et al. (20) after elution from siderably higher than those in the incubation medium (14). Dowex-50 chromatography columns with 1 N NH40H, which The Km values for catecholamine uptake by synaptosomes in was removed by distillation under reduced pressure. Protein homogenates (25) and the concentration of catecholamines in was measured by the method of Lowry et al. (21). the incubation medium required to produce about 50% inhibi- tion of tyrosine hydroxylase (Table 2) are very similar. This RESULTS AND DISCUSSION effect of catecholamine accumulation is probably measured Synaptosomes incubated with [3H]tyrosine form large only in nerve endings that contain tyrosine hydroxylase. amounts of radioactive metabolites, which are mainly acidic Therefore, the measurement of enzyme activity provides a products. These acid metabolites are not retained by Dowex- method to evaluate indirectly the uptake of catecholamines 50 resin, and thus interfere with the measurement of tritiated selectively in and noradrenergic nerve endings, water. While tritiated water was readily removed from the ef- fluent of Dowex-50 columns by lyophilization, up to 80% of TABLE 1. Effect of various compounds on tyrosine hydroxylase the radioactivity could not be removed by this procedure, but activity in synaptosomes prepared from rat brain remained in the residue. In order to make the tritium-dis- placement assay suitable for use with synaptosomes, the Tyrosine hydroxylase method of Nagatsu et al. was modified (16) for the removal Concentration activity of metabolites interfering with the measurement of tritiated Compound (M) (% of control) water. The effluents of the Dowex-50 columns were passed through columns of Dowex-1 resin. Only tritiated water and Di- 10-6 90 neutral metabolites should pass through both columns. When jO-Phenylethylamine 10-6 91 an aliquot of the effluent from Dowex-1 columns was lyophi- 10-6 86 3-Iodotyrosine 10-4 0 lized, less than 5% of the radioactivity in the effluent was DiLa-Methyl-p-tyrosine 10-4 2 found in the residue, indicating that the double-column DiL-c-Methyl-DOPA 10-4 47 method adequately removed the tritiated products interfering with the measurement of labeled water, produced by tyrosine Each value is the mean of two determinations. Incubation hydroxylation. The average activity of tyrosine hydroxylase in time was 30 min. Each preparation was assayed 2-4 times for 8 synaptosomal preparations was 5.19 ± 3.60 (SD) pmol of each compound. A and [3Hvtyrosine were added together, product per min per mg of protein. With a single tissue prep- without prior incubation, to initiate each the incubation. Downloaded by guest on September 23, 2021 2372 Biochemistry: Karobath Proc. Nat. Acad. Sci. USA 68 (1971) TABLE 2. Effect of various agents on the hydroxylation of tyrosine in synaptosomes prepared from rat brain Tyrosine hydroxylase activity (% of control activity i SE) Concentration (M) Agent 1 X 10-7 5X 10-7 1 X 10-6 5X 10-e 1 X 10-5 l-Norepinephrine 77 44 (4) 56 ± 3 (6) 51 i 9 (4) 43 i 4 (4) n.d. Dopamine 76 i 5 (4) 49 ± 9 (4) 43 ±- 94) 37± 4 (4) n.d. i1DOPA 82 (2) n.d. 77 (2) 53 (2) 41 (2) p-Tyramine 90 ± 5 (3) 93 ±- 6 (4) 108 ±- 12 (3) 101 ± 11 (3) n.d. D,i-Octopamine 97 4± 5 (3) 97 ± 8 (3) 93 ± 4 (3) 93 ± 4 (3) n.d.

N, the number of tissue preparations examined, is given in parentheses. The experimental conditions are as in Table 1.; n.d., not done.

even in a heterogenous population of synaptosomes. When cholamine synthesis in intact, sympathetically innervated, tis- synaptosomes from whole brain were examined, dopamine sues have led to similar conclusions. appeared to be more potent then either norepinephrine or Norepinephrine introduced into the synaptosomal incuba- DOPA as an inhibitor of tyrosine hydroxylation. tion, 12 min after the addition of ['Hityrosine led to a rapid It appears that catecholamines are strong inhibitors of tyro- decrease of tyrosine hydroxylase activity, presumably by sine hydroxylation and that aromatic amines, other than cate- enhancing the rate of accumulation of norepinephrine intra- chols are much less active under the experimental conditions. neuronally. Tyramine had little effect on the time course of Thus, only small degrees of inhibition were found with the enzyme activity (Fig. 1). In control samples, the rate of pro- amines: tyramine, DLoctopamine, DL-normetanephrine, ,B- duction of tritiated water by tyrosine hydroxylase was ini- phenylethylamine, and serotonin at 1 MM (Tables 1 and tially rapid and nearly linear for 15-20 min, but became in- 2). The weak inhibitory effect of aromatic amines other creasingly slower thereafter. Whether this spontaneous slow- than catechols on tyrosine hydroxylation may be mediated by ing of tyrosine hydroxylation is mediated by end-product ac- the intraneuronal displacement of catecholamines from storage cumulation or by deterioration of the preparationis not known. sites, or by competition as substrates for monoamine ; The inhibition of tyrosine hydroxylase probably does not the relative potency of the compounds as inhibitors of tyrosine result from a reduction of substrate availability. Thus, the hydroxylation is probably determined by their relative affini- rate of accumulation of [HEItyrosine by isolated nerve endings ties for the catecholamine-uptake system, as well as by their was not altered by adding norepinephrine to the incubation capacity to inhibit the enzyme tryosine hydroxylase. Studies medium at a concentration that strongly inhibited tyrosine of the effect of tyramine (26) and octopamine (27) on cate- hydroxylation (Fig. 2). Tyramine and dopamine (both at 5

700 -

600 -

500 -

t 400 -

K300~~~~~~~~~~0 E300 - / o "

200 -

10 20 30 40 50 MINUTES

FIG. 1. Effect of norepinephrine and tyramine on the time-course of tyrosine hydroxylase activity: Each value is the mean of two -7 determinations. Tyramine (X .... X ) or l-norepinephrine (0-- -0) were introduced (arrow) at a concentration of 5 X 10 M, 12 min after the addition of ['H]tyrosine. Reaction mixtures contained 0.73 mg of protein and 12 nmol of [3,5-3HjLtyrosine (260,000 cpm).

-, control. Downloaded by guest on September 23, 2021 Proc. Nat. Acad. Sci. USA 68 (1971) Tyrosine Hydroxylation in Synaptosomes 2373

accumulate catecholamines from the incubation medium, the intrasynaptosomal concentrations of catecholamines are 4 60 presumably much higher than those initially present in the medium. The finding that tyrosine hydroxylation by synap- 40- tosomes can be inhibited by exogenous catecholamines sug- gests further studies to examine the regulation of catechol- synthesis. 20 I thank Mrs. M. Vogt for preparing the electron micrographs. Discussions and help during the preparation of this manuscript by Drs. R. Baldessarini, M. Huttunen, S. S. Kety, and C. Popper 10 20 30 is gratefully acknowledged. I was a fellow of the Max Kade MINVrES Foundation Inc. New York, N.Y. and supported by U.S. Public Health Service (N.I.M.H.) Grant: MH-16674-2. FIG. 2. Effect of norepinephrine on the accumulation of [3H] tyrosine by synaptosomes: Results are expressed as % of maxi- 1. Gray, E. G., and V. P. Whittaker, J. Anat., 96, 79 (1962). mum uptake of [3,5-'H]ityrosine. Uptake was determined as 2. De-Robertis, E., A. P. DeIraldi, G. R. de L. Arnaiz, and radioactivity associated with particles held back by a 0.65um L. Salganicoff, J. Neurochem., 9, 23 (1962). pore-size Millipore filter. Each value is the mean of two deter- 3. McGeer, E. G., S. P. Bagchi, and E. G. McGeer, Life Sci., 4, minations. Tyrosine concentration was 2 X 10-' M. @-4, con- 1859 (1965). trol; O-- -0, + 5 X 107M l-norepinephrine. 4. Arnaiz, G. R. de L., and E. De Robertis, J. Neurochem., 11, 213 (1964). 5. S. A. and S. X 10-7 M) were also examined under the same Levitt, M., Spector, Sjoerdsma, Udenfriend, incubation J. Pharmacol. Exp. Ther., 148, 1 (1965). conditions, and had no effect on ['Hityrosine accumulation. 6. Udenfriend, S., Pharmacol. Rev., 18, 43 (1966). Therefore, the inhibitory effects appear to depend on intra- 7. Ikeda, M., L. A. Fahien, and S. Udenfriend, J. Biol. Chem., synaptosomal mechanisms. 241, 4452 (1966). The concept that catecholamine synthesis is regulated by 8. Nagatsu, R., M. Levitt, and S. Udenfriend, J. Biol. Chem., end-product 239, 2910 (1964). inhibition of tyrosine hydroxylase has evolved 9. Anton, A. H., and D. F. Sayre, J. Pharmacol. Exp. Ther., from the initial work of Udenfriend and collaborators (6-8). 145, 326 (1964). They demonstrated that a high concentration of norepineph- 10. Oliverio, A., and L. Stjarne, Life Sci., 4, 2339 (1965). rine inhibited a purified tyrosine hydroxylase preparation 11. Alousi, A., and N. Weiner, Proc. Nat. Acad. Sci. USA, 56, that was saturated with exogenous cofactor. Spector et al. 1491 (1966). 12. Roth, R. H., L. Stjarne, and U. S. von Euler, Life Sci., 5, (28) showed that an increase of brain catecholamine concen- 1071 (1966). tration in vivo by inhibition of led to de- 13. Sedval, G. C., V. K. Weise, and I. J. Kopin, J. Pharmacol. creased fromation of norepinephrine from labeled tyrosine, Exp. Ther., 159, 274 (1968). but not from labeled DOPA. Weiner (11) found, by examina- 14. Iversen, L. L., The Uptake and Storage of Noradrenaline in Sympathetic Nerves (Cambridge University Press, London, tion of the isolated of the guinea pig, that release 1967). of norepinephrine by electrical stimulation led to an increased 15. Autilio, L. A., S. H. Appel, P. Pettis, and P. Gambetti, rate of conversion of labeled tyrosine to norepinephrine. Biochemistry, 7, 2615 (1968). Potassium has been used as a depolarizing agent to release 16. Nagatsu, T., M. Levitt, and S. Udenfriend, Anal. Biochem., norepinephrine from tissue slices (29) and excess potassium 9, 122 (1964). 17. Bray, G. A., Anal. Biochem., 1, 279 (1960). increases tyrosine hydroxylase activity in brain slices.* All of 18. Davidson, J. D., and P. Fiegelson, Int. J. Appl. Radiat. these experimental findings are compatible with the concept Isotop., 2, 1 (1957). that alterations of intraneuronal catecholamine concentration 19. Escueta, A. C., and S. H. Appel, Biochemistry, 8 (1969). have an important regulatory influence at the rate-limiting 20. Waalkes, T. P., and S. Udenfriend, J. Lab. Clin. Med., 50, step of catecholamine biosynthesis. 733 (1957). 21. Lowry, 0. H., N. J. Rosenbrough, A. L. Farr, and R. J. Synaptosomes offer several advantages for the study of the Randall, J. Biol. Chem., 193, 265 (1951). synthesis of catecholamines. They allow the direct measure- 22. Weiner, N., and M. Rabadjija, J. Pharmacol. Exp. Ther., ment of tyrosine hydroxylase activity by determination of 164, 103 (1968). the amount of tritiated water formed by substrate hydroxyla- 23. Colburn, R. W., F. K. Goodwin, D. L. Murphy, W. E. Bunney, and J. M. Davis, Biochem. Pharmacol., 17, 957 tion. Furthermore, synaptosomes are a subcellular fraction (1968). that is largely free of contamination by glial and other non- 24. Baldessarini, R. J., and C. Yorke, Nature, 228, 1301 (1970). neuronal tissues. In addition, their metabolism is independent 25. Snyder, S. H., and J. T. Coyle, J. Pharmacol. Exp. Ther., of the influence of neuronal electrical activity. 165, 78 (1969). The present findings 26. Weiner, N., and I. §elvaratam, J. Pharmacol. Exp. Ther., demonstrate that tyrosine hydroxylase 161, 21 (1968). is strongly inhibited by the addition of exogenous catechols, 27. Kopin, I. J., V. K. Weise, and G. C. Sedvall, J. Pharmacol. even in concentrations considerably below those previously Exp. Ther., 170, 246 (1969). required to demonstrate this effect. As nerve-ending particles 28. Spector, S., R. Gordon, A. Sjoerdsma, and S. Udenfriend, Mol. Pharmacol., 3, 549 (1967). 29. Baldessarini, R. J., and I. J. Kopin, J. Pharmacol. Exp. * Harris, J. E., and R. H. Roth, Fed. Proc., 29, 941 Abstr. (1970). Ther., 158, 31 (1967). Downloaded by guest on September 23, 2021