Addition of the calcium-chelating agent ethylene glycol bis(β-aminoethyl ether)-N,N’-tetraacetic acid (EGTA) to a high-speed supernatant preparation obtained from rat striatum produced a dramatic increase in the activity of tyrosine hydroxylase assayed in the presence of subsaturating concentrations of tyrosine and reduced pterin cofactor. This activation appeared to be mediated by changes in the kinetic properties of tyrosine hydroxylase. In the presence of EGTA (50 µM) the Km of the enzyme for tyrosine decreased nearly 8-fold (from 54 µM to 9 µM), the Km for 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine decreased 7-fold (from 0.89 mM to 0.13 mM), and the Ki for dopamine increased 700-fold (from 0.11 mM to 74 mM). No significant change in Vmax was observed. All these kinetic alterations could be reversed by the addition of calcium to the assay medium, while magnesium, even in high concentrations (1 mM), was ineffective. A similar activation was observed with tyrosine hydroxylase isolated from other dopamine-containing regions of rat brain (median eminence, olfactory tubercle) and in the dopamine-rich pedal ganglion of Mercenaria mercenaria (Mollusca). EGTA produced no significant change in the activity of tyrosine hydroxylase prepared from central (medulla-pons) or peripheral noradrenergic neurons. These results suggest that tyrosine hydroxylase associated with dopaminergic neurons may differ from the enzyme found in noradrenergic neurons. This unique response of the tyrosine hydroxylase present in dopaminergic neurons may serve as a useful biochemical marker for identifying these neurons.

The formation of [ 14 C]‐3,4‐dihydroxyphenylalanine (DOPA) from [ 14 C]‐tyrosine, in the presence of the amino acid decarboxylase inhibitor, brocresine (3‐hydroxy‐4‐bromobenzyloxy‐amine dihydrogen phosphate), was greatly enhanced in rat vasa deferentia depolarized by a KCl‐enriched Krebs‐Henseleit solution (52 mM KCl) compared with tissues maintained in unmodified Krebs‐Henseleit solution. When the conversion of tyrosine was allowed to proceed as far as catecholamine (brocresine absent) no significant difference was observed between the accumulation of [ 14 C]‐catecholamines (CA) in depolarized rat vasa deferentia and the accumulation in control (non‐depolarized) tissues. Endogenous CA levels in the depolarized rat vasa deferentia fell to 67% of the controls after a 1 h incubation period and to 53% at the end of 2 hours. Chromatographic separation on Amberlite CG‐120 columns of the newly synthesized CA and catechol metabolites from the rat vas deferens revealed that a very high proportion was present as dopamine. The percentage distribution after 1 h incubation in control Krebs‐Henseleit was: noradrenaline (NA): 30.6 ± 5.2; dopamine 56.9 ± 5.9; acid metabolites: 12.8 ± 1.1; and in KCl‐rich Krebs‐Henseleit, NA: 32; dopamine: 44.7 and acid metabolites 23.3. In contrast to the newly synthesized ( 14 C‐labelled) CA, endogenous dopamine comprises only 10% of the endogenous CA stores in rat vas deferens. The distribution of newly synthesized NA and dopamine in rat vas deferens is strikingly different from that of guinea‐pig vas deferens where more than 80% of newly formed amine is present as NA. In the latter tissue depolarization with K + causes a striking increase in CA biosynthesis.

Addition of CaCl2 to soluble preparations of tyrosine hydroxylase from rat medulla pons produces a marked activation of the enzyme assayed with subsaturating concentrations of tyrosine (10 µM) and pteridine cofactor (2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine, 100 µM). While some increase in activity occurs with Ca++ concentrations as low as 10 µM, activation is maximal at 50 µM Ca++ and remains unchanged up to 1.0 mM. BaC12 produces similar although less pronounced effects. MgCl2 prevents the activation of the enzyme if added to the reaction mixture before CaCl2. Alone MgCl2 has no effect in concentrations up to 1 mM. Ethylene glycol bis(β-aminoethyl ether)- N,N '-tetraacetic acid has no direct effects on the enzyme but completely antagonizes the activation produced by Ca++. The activation of tyrosine hydroxylase by Ca++ is reflected in changes in the kinetic properties of the enzyme. The Km for tyrosine decreases from 58.1 to 10.3 µM, the Km for pteridine cofactor decreases from 673 to 125 µM, and the Ki for norepinephrine increases almost 20-fold, from 0.34 to 6.27 mM, in the presence of Ca++. Thus norepinephrine is a much less effective inhibitor of the Ca++-activated enzyme. The proposal is made that Ca++ which enters the nerve terminal during nerve stimulation may enhance norepinephrine synthesis by activating tyrosine hydroxylase in a manner similar to the activation observed in vitro with CaCl2. Similar findings are reported for tyrosine hydroxylase isolated from rat cerebral cortex. ACKNOWLEDGMENTS Thanks are due to Ms. Ilona Decerbo and Ms. Anne Morrison for their excellent technical assistance.

1.1. The dopamine (DA) and 5-hydroxytryptamine (5-HT) contents of the ganglia of Mercenaria mercenaria expressed as ng. per ganglion were as follows: Pedal ganglion:236±16 ng. DA101± NG. 5-HT.Visceral ganglion:76±3 ng. DA116±7 NG. 5-HT.Cerebral ganglion:37±5 ng. DA107±15 ng. 5-HT.1.2. Pedal ganglia synthesize [14C]DA from [14C]tyrosine but do not form significant amounts of [14C]noradrenaline (NA). [14C]5-HT is also formed from [14C]tryptophan in pedal ganglia.2.3. Incubation of pedal ganglia in KCl-rich sea water accelerates synthesis of [14C]DA from [14C]tyrosine two- to threefold compared with controls. The specific activity of DA isolated from the K+-stimulation ganglia is corresponding increased.3.4. The acceleration of DA synthesis appears to take place at the tyrosine hydroxylation step.4.5. Synthesis of DA from tyrosine in pedal ganglia is inhibited up to 60 per cent by 10−4M DA in the medium.

1. Reserpine in vitro (10(-5)M) caused a profound inhibition (>85%) of the formation of both (14)C-catecholamine ((14)C-CA) and (14)C-dihydroxyphenylalanine ((14)C-DOPA) (in the presence of the amino acid decarboxylase inhibitor brocresine) from (14)C-tyrosine in guinea-pig vas deferens. The magnitude of the inhibition was similar for both (14)C-CA and (14)C-DOPA suggesting that the inhibition occurred primarily at the tyrosine hydroxylase step.2. One hour after in vivo treatment with reserpine (1 mg/kg) when tissue stores of noradrenaline (NA) were depleted by 50%, there was a significant inhibition of the formation of (14)C-DOPA. Twenty-four hours after such treatment, when endogenous NA could no longer be detected, synthesis of (14)C-DOPA was indistinguishable from untreated controls. However a 45% inhibition of (14)C-DOPA synthesis from (14)C-tyrosine could be produced in tissues which had been depleted of NA for 24 h or 48 h by the addition of reserpine, 10(-5)M, to the incubation medium.3. Addition of pteridine cofactor, 2-amino-6,7,-dimethyl-4-hydroxy-5,6,7,8-tetrahydropteridine, to the incubation medium in a concentration of 5 x 10(-3)M enhanced the formation of both (14)C-CA and (14)C-DOPA from (14)C-tyrosine in guinea-pig vas deferens. In 52 mM KCl Krebs-Henseleit medium (14)C-CA formation increased from 2.58+/-0.20 (nmol/g)/h to 6.35+/-0.47 (nmol/g)/h whilst (14)C-DOPA formation increased from 5.04+/-0.88 (nmol/g)/h to 11.29+/-0.59 (nmol/g)/h.4. Pteridine cofactor (5 x 10(-3)M) did not reverse the inhibition of (14)C-DOPA formation seen with reserpine (10(-5)M) in previously untreated tissues or in vasa deferentia from animals pretreated with reserpine 1 mg/kg for 24 hours. However, the inhibition did disappear in the presence of pteridine cofactor when treatment with reserpine was prolonged to 48 h and included two doses of reserpine of 2 mg/kg.5. Tyramine (5.8 x 10(-5)M) and bretylium (10(-5)M) in vitro inhibited the formation of (14)C-CA and (14)C-DOPA from (14)C-tyrosine to the same extent in guinea-pig vas deferens again indicating that their major site of action is on tyrosine hydroxylase. The inhibitory effects were reversed by pteridine cofactor.6. Synthesis of (14)C-NA from (14)C-tyrosine in calf splenic nerve was not increased by incubating the tissue in 52 mM KCl-Krebs-Henseleit solution.

Summary . Increasing the concentration of KC1 in Krebs‐Henseleit bicarbonate solution enhanced the formation of 14C‐noradrenaline (14C‐NA) from 14C‐tyrosine in the guinea‐pig vas deferens. In 52 mm KC1 Krebs‐Henseleit solution the specific activity of the newly formed 14C‐NA was double that of controls. . The rate of synthesis of 14C‐NA from 14C‐tyrosine was constant for up to 2 h in 52 mm KC1 Krebs‐Henseleit solution and for 4 h in unmodified Krebs‐Henseleit solution. . There was no increase in NA formation in the presence of KC1 rich Krebs‐Henseleit solution if 14C‐DOPA was used as the starting substrate instead of 14C‐tyrosine. . The specific activity of 14C‐tyrosine in the high KC1 treated vas deferens was 80% of that of control tissues. Thus the enhanced synthesis of 14C‐NA in high KC1 Krebs‐Henseleit solution did not arise from an increase in the specific activity of precursor. . The effect of K+ on NA synthesis was not mimicked by ganglionic stimulants nor blocked by tetrodotoxin. . Removal of Ca2+ ions or increasing the concentration of Mg2+ ions abolished the increase in synthesis of NA seen in high KC1 Krebs‐Henseleit solution but left the basal rate of NA synthesis in unmodified Krebs‐Henseleit solution unaltered. . The spontaneous release of newly synthesized catecholamines (14C‐labelled) or tritiated noradrenaline (3H‐NA) from vasa deferentia was increased in 52 mm KC1 Krebs‐Henseleit solution. Removal of Ca2+ ions reduced the increased efflux of newly synthesized amine in high KC1 media to that seen in unmodified Krebs‐Henseleit solution. The efflux of 3H‐NA was reduced to one‐third of its former rate in the absence of Ca2+. . High KC1 Krebs‐Henseleit solution caused a substantial contraction of the vas deferens which was not abolished by tetrodotoxin. Release of 3H‐NA paralleled the contractile response, and was likewise unaffected by tetrodotoxin. . No evidence was obtained for any alterations in the activity of tyrosine hydroxylase, the rate limiting enzyme in the formation of NA from tyrosine, in homogenates of vas deferens which had been treated with 52 mm KC1 Krebs‐Henseleit solution. . These results support the hypothesis that acceleration of NA synthesis occurs when tyrosine hydroxylase is freed from end‐product inhibition by the release of noradrenaline, brought about in this case, by high concentrations of KC1.