Tyrosine Hydroxylase: Activation by Nerve Stimulation (vas deferens/calcium/potassium depolarization/regulation of norepinephrine biosynthesis/

Transcription

1 Proc. Nat. Acad. Sci. USA Vol. 71, No. 11, pp , November 1974 Tyrosine Hydroxylase: Activation by Nerve Stimulation (vas deferens/calcium/potassium depolarization/regulation of norepinephrine biosynthesis/ tyrosine 3-monooxygenase) VICTOR H. MORGENROTH, III, MARGARET BOADLE-BIBER, AND ROBERT H. ROTH Departments of Pharmacology and Psychiatry, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 651 Communicated by Alfred Gilman, August 14, 1974 ABSTRACT The synthesis of the sympathetic neurotransmitter, norepinephrine, is accelerated by electrical stimulation of the guinea pig vas deferens. The molecular mechanism responsible for this enhanced formation of transmitter is unknown but has been attributed to an increase in the activity of tyrosine hydroxylase (EC ; tyrosine 3-monooxygenase) during nerve stimulation. In the present experiments, we found that crude preparations of tyrosine hydroxylase isolated from guinea pig vasa deferentia that were electrically stimulated or depolarized by potassium show an increase in activity compared with enzyme obtained from untreated paired control tissues. This increase in activity is partially antagonized by addition of the Ca + + chelator, ethylene glycol bis(8-aminoethyl etlier)-n,n'-tetraacetic acid (EGTA), to the assay medium, and can be completely blocked if Ca++ is removed from the potassium-rich medium used todepolarizetheintact tissue, before preparation of the enzyme. A similar increase in enzyme activity occurs when Ca++ ions are added directly to enzyme prepared from untreated vasa deferentia. In this instance, the activation is completely reversed by EGTA. The increase in activity produced by addition of Ca++ to the isolated enzyme or by electrical stimulation or potassium depolarization of the tissue before isolation of the enzyme appears to be mediated by changes in the kinetic properties of tyrosine hydroxylase. All treatments appear to activate tyrosine hydroxylase by causing an increase in its affinity for substrate and pteridine cofactor and by decreasing its affinity for the end-product inhibitor, norepinephrine. These results provide direct evidence that the enhanced formation of norepinephrine seen during stimulation of sympathetically innervated tissues arises from an activation of tyrosine hydroxylase. The fact that the activation produced by nerve stimulation is mimicked by Ca++ ions raises the intriguing possibility that the influx or mobilization of Ca++ that accompanies nerve stimulation and that is intimately involved in release of transmitter may also participate in the activation of tyrosine hydroxylase. Abbreviations: EGTA, ethylene glycol bis(,8-aminoethyl ether)- N,N'-tetraacetic acid; Me2H4pterin, 6,7-dimethyl-,5,6,7,8-tetrahydropterin Tyrosine hydroxylase [EC ; tyrosine 3-monooxygenase; I-tyrosine, tetrahydropteridine: oxygen oxidoreductase (3-hydroxylating) ] is the rate-limiting enzyme in the biosynthesis of norepinephrine and, as such, sets the pace for the overall synthesis of the sympathetic neurotransmitter. Thus, studies on the properties of this key enzyme are of importance for understanding the mechanisms by which the sympathetic neuron regulates its transmitter supply to meet altered conditions of demand. Numerous experiments in the past decade have demonstrated that an increase in sympathetic nerve activity is associated with an acceleration of norepinephrine biosynthesis in the tissue containing the terminals of the sympathetic nerves under study (1-5). This increase in synthesis is believed to occur as a result of an increase in the activity of tyrosine hydroxylase. Since tyrosine hydroxylase is inhibited by catechols and catecholamines, presumably due to their ability to competitively antagonize the binding of the pteridine cofactor to the apoenzyme (6, 7), it has been proposed that free intraneuronal norepinephrine may control its own synthesis by negative feedback inhibition of tyrosine hydroxylase (8). In support of this concept is the observation that an increase in the endogenous norepinephrine content of sympathetically innervated tissues by prior treatment with monoamine oxidase inhibitors results in a marked reduction of norepinephrine biosynthesis (9). In view of these as well as other observations, it has been suggested that the acceleration of norepinephrine biosynthesis that occurs during increased sympathetic activity results from the depletion of a small strategic pool of norepinephrine which releases tyrosine hydroxylase from negative feedback inhibition, thus causing an apparent activation of the enzyme (2, 4, 1). Consistent with the above hypothesis is the observation that synthesis and release appear to be intimately coupled. Thus, the acceleration of norepinephrine biosynthesis that occurs during potassium-induced depolarization of sympathetic nerves is blocked when the depolarization-induced release is prevented by removal of Ca++ from the medium (11, 12). However, some recent indirect evidence indicates that the increase in the conversion of tyrosine to norepinephrine which occurs during depolarization cannot be completely explained by a simple reduction in the size of a regulatory pool of norepinephrine and, more likely, involves some other activation process (13). Gutman and Segal (14) have indicated that sodium and calcium ions can affect adrenal tyrosine hydroxylase activity, and we have recently demonstrated that addition of calcium to high-speed supernatants obtained from brain areas rich in noradrenergic neurons results in an activation of tyrosine hydroxylase (15). Thus, it seems possible that an influx of calcium, which occurs during nerve stimulation and depolarization of the sympathetic nerve terminals, may be responsible for activating tyrosine hydroxylase. Ini order to test this possibility, we have examined the activity and kinetic properties of tyrosine hydroxylase isolated from guinea pig vasa deferentia after electrical stimulation or potassium depolarization of the sympathetic nerves in this tissue. Furthermore, we have compared the changes produced by such treatments to those which occur when Ca++ is added to the isolated tyrosine hydroxylase. The following is an account of such a study. METHODS MAale Hartley guinea pigs (3-4 g) were killed by cervical dislocation. Vasa deferentia were dissected, frozen on dry ice,

2 4284 Biochemistry: Morgenroth et al. Proc. Nat. Acad. Sci. USA 71 (1974) and stored at -7. At the time of assay, frozen vasa deferentia were pooled, homogenized in 1 volumes of ice-cold 5 mm Tris-acetate buffer (ph 6.), and centrifuged at 14, X g for 9 min at 4. The supernatant served as the source of soluble tyrosine hydroxylase. In most stimulation experiments, vasa deferentia were set up for field stimulation essentially as described by Birmingham and Wilson (16). One vas deferens was field stimulated for 1 sec at 2 Hz every 3 sec for periods of up to 3 min, while the vas deferens from the contralateral side was incubated under identical conditions without stimulation and served as a control. The response of the tissue was recorded on a Grass Polygraph by means of a Grass FT- 13 Force-displacement Transducer. In one set of experiments, the organs were prepared according to the method of Hukovic (17) and the hypogastric nerve was stimulated at a distance of about 4 cm from the vas deferens. The same stimulus parameters were used as described above. In all experiments the bath was maintained oxygenated and at 37. Upon the conclusion of the stimulation period both vasa deferentia were blotted dry and rapidly frozen on dry ice. The supernatant was prepared as described above. In other experiments freshly dissected vasa deferentia were incubated for 15 min at 37 in Krebs-Henseleit medium, Ca++-free Krebs-Henseleit medium, a K+-enriched Krebs-Henseleit medium in which 4% of the NaCl had been replaced by KCl, giving a final concentration of KCl of 52 mm, and K+-enriched Krebs- Henseleit medium from which calcium had been removed (11). At the end of the incubation period each vas deferens was blotted dry, frozen on dry ice, and stored at -7 until assayed. The supernatant was prepared for assay as before. The supernatants obtained from the vasa deferentia were assayed for tyrosine hydroxylase activity by a modification of the assay of Shiman et al. (18) and Coyle (19). The reaction was carried out in a total volume of 1. ml; 1 Al of supernatant was added to a reaction mixture containing.2 M acetate buffer ph 6. (2 Al),.1 mm 4-bromo-3-hydroxybenzyloxyamine NSD 155 (5 Ml), 33 units of catalase in glass-distilled water (2 Ml), sheep liver dihydropteridine reductase (EC ) in 25 mm Tris HCl buffer (ph 7.4) (1 Al, 83 Ag of protein), 1 mm NADPH (1 Al), and 6,7- dimethyl-5,6,7,8-tetrahydropterin. HCl (Me2H4pterin) (1 Al). Solutions under study or H2 were added in a volume of 1 Al. Blanks consisted of either boiled enzyme (2 min) or complete incubation mixtures to which 5 Ml of glacial acetic acid had been added. After a 5-min preincubation, the reaction was initiated by the addition of substrate, [3-5-3H ]tyrosine (1 MCi/Mmole). Protein was determined according to the method of Lowry et al. (2). All calculations were performed on a Hewlett Packard Programmable Calculator model 981 A. Statistics were determined by use of paired tests. Statistical estimations of Km were performed according to the method of Wilkinson (21). The Ki of norepinephrine was determined by the method of Dixon (22) at three concentrations of Me2H4 pterin. Kinetics were determined on the linear portion of the time course and protein concentration curves. RESULTS Effect of Electrical Stimulation and Depolarization by Potassium on Tyrosine Hydroxylase Activity. Electrical stimulation of the vas deferens produced a marked increase in the activity of tyrosine hydroxylase measured in vitro when compared with Effect of electrical stimulation on the activity TABLE 1. of tyrosine hydroxylase from guinea pig vas deferens Tyrosine hydroxylase activity (pmoles Assay of dopa/mg of pro- Pretreatment conditionst tein per min) Unstimulated control Standard 58.8 ± 4.5 Electrical stimulation* Standard i 18.2 Unstimulated control +1 /M Ca i 22.6 Electrical stimulation* +1iAM Ca ± 15.9 Electrical stimulation* +5 MM EGTA ± 8. 1 * Each vas deferens was field stimulated at 2 Hz for 1 sec every 3 sec for 3 min. t Tyrosine hydroxylase was assayed in supernatant fractions as described in Methods. I Each value is the mean i SEM of 12 determinations carried out on four separate vasa deferentia. the unstimulated paired control vas deferens (Table 1). A similar increase in tyrosine hydroxylase activity was seen in the enzyme isolated from vasa deferentia incubated for 15 min in high potassium (52 mm) Krebs-Henseleit medium (Table 2). This increase was dependent upon the presence of calcium in the incubation medium, since the activity of tyrosine hydroxylase was unchanged when calcium was omitted from the potassium-enriched incubation medium. The activation of tyrosine hydroxylase produced by electrical stimulation or incubation in potassium-enriched Krebs medium could be partially reversed by addition of the calcium-chelating agent, ethylene glycol bis(f3-aminoethyl ether)-nn'-tetraacetic acid (EGTA). Effect of Calcium on Tyrosine Hydroxylase Actiity. Addition of calcium in concentrations as low as.5,av to the reaction mixture resulted in a significant activation of tyrosine hydroxylase of vas deferens. Further studies indicated that maximal activation was achieved at 6. MM calcium chloride when the reaction was carried out in the presence of 5 AMM tyrosine and.1 mm Me2H4pterin. In subsequent experiments a calcium concentration of 1 MAM was used to ensure maximal activation. The addition of Ca++ ions (1 1AM) to the reaction mixture containing enzyme prepared from tissues that had been stimulated electrically or depolarized with a K+-rich incubation medium produced no further increase in the activity of tyrosine hydroxylase (Tables 1 and 2). However, the activity of the enzyme isolated from untreated tissues could be increased to that of enzyme from stimulated tissues by addition of Ca++ to the reaction medium. Effect of Various Ions on Tyrosine Hydroxylase Activity. Barium chloride (5 MM) produced a 32% increase in tyrosine hydroxylase activity at 1 AMM tyrosine and.1 mm Me2H4- pterin, while calcium chloride (1 MM) produced an even greater activation of 198% at the same tyrosine and Me2H4- pterin concentrations. In contrast, magnesium chloride had no effect on tyrosine hydroxylase activity at concentrations as high as 1 mm. The addition of the calcium chelator, EGTA (5 MM), completely reversed the effect of calcium. EGTA alone at concentrations as high as 1. mm (Table 3) had no effect on tyrosine hydroxylase activity of vas deferens. In order to elucidate further the mechanisms that might be involved in the increase in tyrosine hydroxylase activity which

3 Proc. Nat. Acad. Sci. USA 71 (1974) TABLE 2. Effect of depolarization of the guinea pig vas deferew', with potassium-rich Krebs-Henseleit medium on tyrosine hydroxylase activity Tyrosine hydroxylaset (pmoles of dopa/ Assay mg of protein Pretreatment medium* conditionst per min) Krebs-Henseleit Standard 63.5 == 4.7 Ca+ +-free Krebs-Henseleit Standard KCI Standard It 16.2 Ca++-4ree Krebs-Henseleit + 52 mm KCl Standard KCI + 5 AM EGTA 89.3 It 9. KCl + 1 IM Ca t 14.8 * Vasa deferentia were incubated for 15 min at 37 in the various media, blotted, frozen on dry ice, and subsequently homogenized and assayed for tyrosine hydroxylase. t Tyrosine hydroxylase was assayed in supernatant fractions as described in Methods. t Each value is the mean SEM of 12 determinations carried out on four separate vasa deferentia, except in the experiment where 1MIM Ca++ was added to the assay medium, when duplicate determinations were made on three vasa deferentia. results from stimulating the sympathetic nerves before enzyme isolation (or addition of calcium ions directly to the isolated enzyme), the kinetics of tyrosine hydroxylase were determined after each treatment. Effect of Calcium and Nerve Stimulation on the Km for Tyrosine. Addition of calcium chloride (1 MM) to the untreated isolated enzyme caused a 5-fold decrease in the am parent Km for tyrosine from 47.9 to 9.3 MAM. Electrical stimulation of the guinea pig vas deferens for 3 min before isolation of the enzyme caused a 5-fold decrease in the Km for tyrosine from 47.9 to 8.1 MMA. There was no change in the Vmax for tyrosine (Table 4) in either instance. Effect of Calcium and Nerve Stimulation on the Km for AMe2H4- pterin. Calcium chloride (1 MAIM) produced a 3-fold decrease in the apparent Km of the enzyme for Me2H4pterin TABLE 3. Effect of divalent cations on the activity of tyrosine hydroxylase from guinea pig vas deferens Tyrosine hydroxylaset (pmoles of dopa/mg Assay conditions n* of protein per min) Standard Calcium (1OuM) Barium (5 MM) Magnesium (1. mm) i+5. 1 EGTA (1.OmM) Calcium (1 AM) + EGTA (5MIAM) ±6.6 * Number of determinations. t Values are expressed as the mean SEM of assays conducted at a tyrosine concentration of 1 MM and a Me2H4pterin concentration of.1 mm. Activation of Tyrosine Hydroxylase 4285 TABLE 4. Effects of electrical stimulation and calcium addition on the kinetic properties of tyrosine hydroxylase Km Km Me2H4- Vmax Tyrosinet Va,,x ty- pterint Me2H4- Treatment (X 1-6) rosinet (X 1-s) pterint Unstimulated control Unstimulated control + Ca++* Electrical stimulation * Ca++ (1MM) was added to the assay medium. t Km and Van for tyrosine were determined by the method of Lineweaver and Burke at a Me2H4pterin concentration of 1 mm and seven tyrosine concentrations ranging from 1 X 1-4 to 1-7 M. Each value is the mean of the intercepts generated from six separate lines. t Km and Vinax for Me2H4pterin were determined by the method of Lineweaver and Burke at a tyrosine concentration of.1 mm and six Me2H4pterin concentrations ranging from 1-3 to 1-5 M. Each value is the mean of the intercepts generated from six separate lines. Vmax results are expressed in terms of pmoles of dopa per mg of protein per min. from.28 mm to.9 mm, with a significant change in the Vmax from 238 to 37 pmoles of dopa formed per mg of protein per min. Electrical stimulation caused a 5-fold decrease in the apparent Km for Me21H4pterin, from.28 to.5 mm again with a change in Vmax from 233 to 361 pmoles of dopa formed per mg of protein per min (Table 4). Effect of Calcium and Nerve Stimulation on the Ki of Norepinephrine. Calcium chloride (1,MM) caused a 4-fold increase in the Ki for norepinephrine from.24 to 1.1 mma. Intermittent electrical stimulation at 2 Hz for 3 min caused an even greater increase, from.24 to 3.8 mm (Fig. 1). This change in the inhibitory activity for the natural endproduct inhibitor, norepinephrine, observed in the activated enzyme is also reflected by a change of the inhibitory potency of another catechol compound, a-propyldopacetamide. Whereas, 2 MM a-propyldopacetamide causes a 54% inhibition of control enzyme, the same concentration causes only a 12% inhibition of the enzyme isolated from electrically stimulated vas deferens, a 15% inhibition of the calcium-activated tyrosine hydroxylase isolated from control tissue, and an 11% inhibition of the enzyme obtained from potassium-stimulated vasa deferentia (Table 5). DISCUSSION Since the discovery that tyrosine hydroxylases of both peripheral and central origin are inhibited by catecholamines (6, 8), it has been speculated that noradrenergic neurons regulate the synthesis of their transmitter by means of end-product feedback inhibition of tyrosine hydroxylase (1). Thus, it has been argued that in the resting state tyrosine hydroxylase is partially inhibited in the nerve terminal by a small "strategic" pool of norepinephrine and that during periods of increased nerve activity this end-product inhibition is decreased, presumably as a consequence of the release of norepinephrine (2, 4). This regulatory pool of norepinephrine was always envisioned as regulating tyrosine hydroxylase by means of its ability to competitively antagonize the binding of the pteridine cofactor to the apoenzyme (7, 23). However, some recent

4 4286 Biochemistry: Morgenroth et al..c E )., cl EX. E. cl ) E oo co C', a) co E )i ~ ~ ~ ~ --- I-- o Ki Control Colcium 1.1mM Electricol Ki 3.8mM Stimulotion ID QS [Norepinephrine] mm FIG. 1. Ki was determined by the method of Dixon (2) at three Me2H4pterin concentrations of 1-4, 1C5, and 1Ce M, a constant ityrosine concentration of 1-4 M, and seven norepinephrine concentrations. Each point is the mean of three determinations. Stimulation parameters are the same as those indicated in Table 2. indirect evidence indicates that the regulatory process may be somewhat more complex. The argument is as follows: If the activity of tyrosine hydroxylase is determined only by the extent of competition between end-product inhibitor and pteridine cofactor, then it should be possible to bring control rates of norepinephrine synthesis (i.e., unstimulated) up to stimulated rates simply by providing a source of excess exogenous pteridine cofactor. This has never proved possible to do experimentally (12, 13). By the same token, addition of exogenous norepinephrine to the incubation medium should bring the tyrosine hydroxylase of both control and stimulated tissues to the same baseline level. Here again this has never been observed experimentally, since the synthesis rate in stimulated tissues is always higher than that of the controls regardless of the manipulations that have been carried out (2, 13, 24). Observations of this kind recently led Cloutier and Weiner (13) to conclude that the activation of tyrosine hydroxylase that occurs during nerve stimulation is not simply a result of a decrease in end-product inhibition, but may also involve some as yet unknown positive allosteric activator. In this report we have presented direct evidence that tyrosine hydroxylase is activated during nerve stimulation and that this activation may in part be linked to the influx or mobilization of calcium ions that occurs during depolarization of sympathetic nerve terminals. As illustrated in Table 3, addition of calcium (1,M) to the high-speed supernatants obtained from the guinea pig vas deferens causes a marked increase (198%) in tyrosine hy- 15 TABLE 5. Alteration of inhibitory potency of ca-propyldopacetamide on the activated form of tyrosine hydroxylase Tyrosine hydroxylase$ (pmoles of dopa/mg of protein per min) a-propyl- % In- Treatment Control dopacetamide hibition None 56.2 i ± 6 1 um Ca++ in assay medium Electrical stimulation* i i ± 1 Krebs-Henseleit medium i i i 2 mm KCIt * Electrical stimulation of hypogastric nerve at 2 Hz for 1 sec every 3 sec for 15 min. t Incubation for 15 min at 37 in Krebs-Henseleit medium + 52 mm KCL. $ Each value is the mean + SEM of six determinations. 2,uM a-propyldopacetamide. droxylase activity. This effect could be readily reversed by removal of calcium with the calcium chelator, EGTA, while EGTA alone had no significant effect. This effect of calcium on the tyrosine hydroxylase obtained from the vas deferens is similar in extent to that observed on tyrosine hydroxylase prepared from other peripheral and central tissues rich in norepinephrine-containing neurons (15) Proc, Nat. Acad. Sci. USA 71 (1974) (Roth, Morgenroth, and Hughes, unpublished data). Previous experiments conducted in our laboratory have demonstrated that depolarization of the guinea pig vas deferens prel)aration by incubation in p)otassium-enriched Krebs-Henseleit medium increases the conversion of [3H]- tyrosine to norepinephrine and to dopa under conditions in which the tissue is treated with a decarboxylase inhibitor (l l). In the present experiments (Table 2), potassium depolarization of the vas deferens preparation for periods of 15 mil caused an increase in the activity of tyrosine hydroxylase isolated from the vas deferens. The presence of calcium in the potassium-enriched nmedium is essential for this activation to be observed. The activation of tyrosine hydroxylase p)roduced by preincubation in potassium-enriched medium is similar in extent to that observed upon addition of calcium to tyrosine hydroxylase isolated from control tissue (198% versus 195%). Furthermore, the addition of EGTA just prior to assay of the tyrosine hydroxylase prepared from the potassium-depolarized tissue partially reverses the increase in tyrosine hydroxylase activity observed. The addition of calcium to the reaction mixture containing the enzyme isolated from the potassium-depolarized tissue produced no further increase in tyrosine hydroxylase activity. However, addition of calcium to the reaction mixture containing the tyrosine hydroxylase isolated from tissues incubated in normal Krebs-Henseleit medium increased the activity to a level similar to that observed for the enzyme isolated from potassium-depolarized tissue. Nearly identical results were obtained on the tyrosine hydroxylase isolated from electrically stimulated vasa deferentia. The tyrosine hydroxylase activity was increased to approximately the same extent as that observed when calcium was added to the tyrosine hydroxylase prepared from the unstimulated contralateral control tissue. However, as observed in the potassium-

5 Proc. Nat. Acad. Sci. USA 71 (1974) stimulated tissue, addition of calcium to the tyrosine hydroxylase obtained from the stimulated tissue produced no further increase in activity. Addition of a calcium chelator to the tyrosine hydroxylase obtained from the electrically stimulated tissue again partially reversed the increase in tyrosine hydroxylase activity, although the reversal was not as great as that observed in the enzyme activated by incubation in high potassium (Tables 1 and 2). In an attempt to further elucidate the mechanisms involved in the activation of tyrosine hydroxylase produced by nerve stimulation, we determined the effects of electrical stimulation and calcium addition to the tyrosine hydroxylase obtained from the paired control tissue on the kinetic properties of tyrosine hydroxylase. Both treatments appeared to produce similar kinetic alterations in tyrosine hydroxylase (Table 4). The addition of calcium and electrical stimulation caused a 5-fold decrease in the Km for substrate tyrosine and a 4-fold decrease in the Km for artificial cofactor, Me2H4pterin. Calcium addition also caused a 4-fold increase in the K, for norepinephrine, whereas electrical stimulation caused about a 16-fold increase in the Kj for norepinephrine (Fig. 1). The above results provide direct evidence that alterations in tyrosine hydroxylase activity occur in peripheral sympathetic neurons during periods of increased nerve activity and indicate that they may be mediated by an increase in the affinity of the enzyme for substrate and cofactor and a decrease in affinity for the endogenous end-product inhibitor norepinephrine. The question as to which kinetic alteration is most important in producing the activation of tyrosine hydroxylase in vivo remains to be determined. However, since tissue levels of tyrosine are quite high (probably saturating), it is unlikely that an increase in the affinity of the enzyme for substrate would influence tyrosine hydroxylase activity in tivo. On the other hand, it is quite likely that an increase in the affinity of the enzyme for pteridine cofactor or a decrease in the affinity of the enzyme for end-product inhibitor norepinephrine could effectively activate tyrosine hydroxylase in vivo. The similarity of the changes produced by nerve stimulation and those observed UpOfl calcium addition to the tyrosine hydroxylase isolated from paired control tissue suggests that calcium may play an important role in activating tyrosine hydroxylase. Calcium could produce this activation directly. Alternatively, calcium could mediate this activation by, for example, changing the affinity of the enzyme for some other as yet unknown allosteric effector, or by initiating an increase in the formation of a positive allosteric effector or by removing an allosteric inhibitor. In view of recent findings in our laboratory (Morgenroth and Roth, unpublished data) it seems quite possible that cyclic AMP may be involved in this activation process. Thanks are due to Ms. Anne Morrison for expert technical assistance. Supported in part by grants from USPHS, MH-1492, NS-9389, and NS Roth, R. H., Stjarne, L. & von Euler, U. S. (1966) "Acceleration of noradrenaline biosynthesis by nerve stimulation," Life Sci. 5, Alousi, A. & Weiner, N. (1966) "The regulation of norepinephrine synthesis in sympathetic nerves. Effects of nerve stimulation cocaine and catecholamine releasing agents," Proc. Nat. Acad. Sci. USA 56, Activation of Tyrosine Hydroxylase Gordon, R., Reid, J. V. 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