Calcium, ATP, and Magnesium Activate Soluble Tyrosine Hydroxylase from Rat Striatum

Soluble tyrosine hydroxylase (TH) prepared from rat striatum by sonication, centrifugation, and gel filtration on Sephadex G-25 was activated by preincubation with Ca2+, ATP, and Mg2+. Activation occurred with micromolar concentrations of Ca2+ and required the presence of both ATP and Mg2+. The activation was reversible and was characterized by a large decrease of apparent Km for the pteridine cofactor and a small increase of Vmax. Ca2+-induced activation was small when TH activity was assayed at pH values near the optimum, but the magnitude of the activation increased with increasing assay pH. The activation apparently did not involve Ca2+-activated protease because it was not affected by the protease inhibitor leupeptin. Nor did it involve cyclic AMP-dependent protein kinase, as evidenced by the failure of the heat-stable inhibitor of this kinase to decrease Ca2+-induced TH activation. Furthermore, the activation of TH evoked by Ca2+ and that produced by cyclic AMP was additive. These experiments indicate that striatal TH can be activated in vitro by an endogenous Ca2+-dependent mechanism. The similarity of the Ca2+-induced activation of TH to that elicited by increased neuronal activity and terminal depolarization is discussed.     

Activation and Multiple-Site Phosphorylation of Tyrosine Hydroxylase in Perfused Rat Adrenal Glands

Tryptic digestion of tyrosine hydroxylase (TH) isolated from rat adrenal glands labeled with 32Pi produced five phosphopeptides. Based on the correspondence of these phosphopeptides with those identified in TH from rat pheochromocytoma cells, four phosphorylation sites (Ser8, Ser19, Ser31, and Ser40) were inferred. Field stimulation of the splanchnic nerves at either 1 or 10 Hz (300 pulses) increased 32P incorporation into TH. At 10 Hz, the phosphorylation of Ser19 and Ser40 was increased, whereas at 1 Hz, Ser19, Ser31, and Ser40 phosphorylation was increased. Stimulation at either 1 or 10 Hz also increased the catalytic activity of TH, as measured in vitro (pH 7.2) at either 30 or 300 microM tetrahydrobiopterin. Nicotine (3 microM, 3 min) increased Ser19 phosphorylation, vasoactive intestinal polypeptide (10 microM, 3 min) increased Ser40 phosphorylation, and muscarine (100 microM, 3 min) increased TH phosphorylation primarily at Ser19 and Ser31. Vasoactive intestinal polypeptide, but not nicotine or muscarine, mimicked the effects of field stimulation on TH activity. Thus, the regulation of rat adrenal medullary TH phosphorylation by nerve impulses is mediated by multiple first and second messenger systems, as previously shown for catecholamine secretion. However, different sets of second messengers are involved in the two processes. The action of vasoactive intestinal polypeptide as a secretagogue involves the mobilization of intracellular calcium, whereas its effects on TH phosphorylation are mediated by cyclic AMP. This latter effect of vasoactive intestinal polypeptide and the consequent increase in Ser40 phosphorylation appear to be responsible for the rapid activation of TH by splanchnic nerve stimulation.     

Activation of Tyrosine Hydroxylase by Nicotine in Rat Adrenal Gland

The administration of nicotine activates tyrosine hydroxylase in the rat adrenal gland. This activation is apparently maximal 25 min after a single subcutaneous injection of nicotine at 2.3 mg/kg. Repeated injections of nicotine (seven injections once every 30 min) are associated with a persistent activation of adrenal tyrosine hydroxylase for at least 3 h. The nicotinic receptor antagonist hexamethonium does not significantly inhibit the nicotine-mediated activation of tyrosine hydroxylase in innervated adrenal glands. However, hexamethonium completely blocks the activation of adrenal tyrosine hydroxylase by nicotine in denervated adrenal glands. Furthermore, even though a single injection of nicotine activates tyrosine hydroxylase in both innervated and denervated adrenal glands, repeated injections of nicotine do not activate tyrosine hydroxylase in denervated adrenal glands. Our results suggest that the systemic administration of nicotine activates adrenal tyrosine hydroxylase by two mechanisms: (1) via direct interaction with adrenal chromaffin cell nicotinic receptors; and (2) via stimulation of the CNS leading to the release from the splanchnic nerve of substances that interact with adrenal chromaffin cell receptors other than the nicotinic receptor.     

Four Forms of Tyrosine Hydroxylase Are Present in Human Adrenal Medulla

Alternative splicing can result in up to four forms of human tyrosine hydroxylase (HTH) mRNA (mRNAHTH). In the adrenal gland, a major site of catecholamine biosynthesis, the presence of all four mRNAHTH forms is controversial. In the present study, postmortem human adrenal medullary tissue was analyzed for the presence of multiple forms of HTH protein by blot immunolabeling. Electrophoretic transfers were developed with affinity-purified rabbit anti-HTH antibodies raised against peptides that reproduced the unique amino acid sequences predicted by the four mRNAHTH forms. All four of the predicted HTH protein forms were present in the adrenal glands from a diverse sample population.     

Presynaptic Dopamine Autoreceptors Control Tyrosine Hydroxylase Activation in Depolarized Striatal Dopaminergic Terminals

The possible control of tyrosine hydroxylase (TH) activity by dopaminergic receptor-dependent mechanisms was investigated using rat striatal slices or synaptosomes incubated in the presence of various 3,4-dihydroxyphenylethylamine (dopamine or DA) agonists and antagonists. Under "normal" conditions (4.8 mM K+ in the incubating medium), the DA agonists apomorphine, 6,7-dihydroxy-N,N-dimethyl-2-aminotetralin (TL-99), 7-hydroxy-N,N-dipropyl-2-aminotetralin (7-OH-DPAT), Trans-(-)-4,4a,5,6,7,8,8a,9-octahydro-5-propyl-2H-pyrazolo-3,4- quinoline, and 3-(3-hydroxyphenyl)-N-n-propylpiperidine decreased TH activity in soluble extracts of incubated tissues. In the case of the catechol-containing drugs apomorphine and TL-99, this effect was partly due to a direct inhibition of the enzyme, but in all other cases it appeared to depend on the stimulation of presynaptic DA autoreceptors. No effect of DA antagonists was detected on TH activity under "normal" conditions. In contrast, when tissues were incubated in a K+ -enriched (60 mM) medium, (-)-sulpiride and other DA antagonists enhanced TH activation due to depolarization whereas DA agonists were ineffective. Because (-)-sulpiride also increased the enzyme activity in striatal slices exposed to drugs inducing release of DA, such as veratridine and d-amphetamine, it is concluded that the stimulating effect of the DA antagonist resulted in fact from the blockade of the negative control of TH normally triggered by endogenous DA acting on presynaptic autoreceptors. In contrast to TH activation due to K+ -induced depolarization, the activation evoked by tissue incubation with dibutyryl cyclic AMP was unaffected by the typical agonist 7-OH-DPAT or the antagonist (-)-sulpiride. This would suggest that TH control via presynaptic DA autoreceptors normally concerns possible modulations of the cyclic AMP-dependent phosphorylation of the enzyme.     

Effects of Cold Exposure on Rat Adrenal Tyrosine Hydroxylase: An Analysis of RNA, Protein, Enzyme Activity, and Cofactor Levels

Long-term cold exposure (5-7 days) is known to induce concomitant increases in the levels of adrenomedullary tyrosine hydroxylase (TH) RNA, protein, and enzyme activity. In this report, we compare the time courses of these changes and investigate the effects of cold exposure on the levels of biopterin, the cofactor required for tyrosine hydroxylation. After only 1 h of cold exposure, TH mRNA abundance increased 71% compared with nonstressed controls. Increases in total cellular TH RNA levels were maximal (threefold over control values) within 3-6 h of cold exposure and remained elevated throughout the duration of the experiment (72 h). TH protein levels increased rapidly after 24 h of cold exposure and reached a maximal value threefold above that of controls at 48-72 h. Despite the relatively rapid and large elevations in TH RNA and protein content, only modest increases in TH activity were detected during the initial 48 h of cold exposure. Adrenomedullary biopterin increased rapidly after the onset of cold exposure, rising to a level approximately twofold that of the nonstressed controls at 24 h, and remained at this level throughout the duration of the stress period. Taken together, the results of this time course study indicate that cold-induced alterations in adrenal TH activity are mediated by multiple cellular control mechanisms, which may include pre- and posttranslational regulation. Our findings also suggest that cold stress-induced increases in the levels of the TH cofactor may represent another key event in the sympathoadrenal system's response to cold stress.     

Influence of Calcium on Dopamine Synthesis and Tyrosine Hydroxylase Activity in Rat Striatum

Abstract: We have investigated three aspects of the relationship between calcium and tyrosine hydroxylase activity in rat striatum. In the first series of experiments, we examined the hypothesis that the rise in dopamine synthesis during increased impulse flow results from a calcium-induced activation of tyrosine hydroxylase. Calcium (12.5–200 μ M ) had no effect when added to crude enzyme or enzyme partially purified by gel filtration. Moreover, incubation of synaptosomes with excess calcium (up to 3.5 m M ) had little or no effect on dopamine synthesis. Incubation with the depolarizing alkaloid veratridine (75 μ M ) did increase dopamine synthesis, but did not alter the activity of tyrosine hydroxylase subsequently prepared from the synaptosomes, despite the presumed rise in intracellular calcium. In the second series we examined the hypothesis that increased dopamine synthesis after axotomy results from activation of tyrosine hydroxylase owing to a decrease in intracellular calcium. Addition of the calcium chelator EGTA (100 μ M ) to crude or partially purified enzyme was without effect, whereas incubation of synaptosomes with EGTA (500 μM ) decreased cell-free enzyme activity. In the third experimental series we examined the relationship between calcium and activation of tyrosine hydroxylase by dibutyryl cyclic AMP. EGTA failed to alter the increase in the activity of tyrosine hydroxylase prepared from synaptosomes incubated with dibutyryl cyclic AMP. However, it blocked the increase in synaptosomal dopamine synthesis and dopamine content normally produced by the cyclic AMP analogue. Thus, tyrosine hydroxylase does not appear to be activated by either increases or decreases in calcium availability. However, calcium may be important for the maintenance of basal tyrosine hydroxylase activity, and may play an indirect role in the expression of tyrosine hydroxylase activation produced by other means.     

Elevation of RNA Coding for Tyrosine Hydroxylase in Rat Adrenal Gland by Reserpine Treatment and Exposure to Cold

When rats are treated daily with reserpine or maintained at 4 degrees C, the level of a specific RNA coding for tyrosine hydroxylase is elevated in the adrenal gland. The increase in this specific RNA temporally precedes and is quantitatively equal to the increase in adrenal tyrosine hydroxylase enzyme activity elicited by these treatments. These results suggest that prolonged stress may lead to changes in the levels of specific RNA species in the adrenal gland.     

Inactivation of Tyrosine Hydroxylase Activity by Ascorbate In Vitro and in Rat PC12 Cells

Abstract: Tyrosine hydroxylase activity is reversibly modulated by the actions of a number of protein kinases and phosphoprotein phosphatases. A previous report from this laboratory showed that low-molecular-weight substances present in striatal extracts lead to an irreversible loss of tyrosine hydroxylase activity under cyclic AMP-dependent phosphorylation conditions. We report here that ascorbate is one agent that inactivates striatal tyrosine hydroxylase activity with an EC₅₀ of 5.9 μM under phosphorylating conditions. Much higher concentrations (100 mM) fail to inactivate the enzyme under nonphosphorylating conditions. Isoascorbate (EC₅₀, 11 μM) and dehydroascorbate (EC₅₀, 970 μM) also inactivated tyrosine hydroxylase under phosphorylating but not under nonphosphorylating conditions. In contrast, ascorbate sulfate was inactive under phosphorylating conditions at concentrations up to 100 mM. Since the reduced compounds generate several reactive species in the presence of oxygen, the possible protecting effects of catalase, peroxidase, and superoxide dismutase were examined. None of these three enzymes, however, afforded any protection against inactivation. We also examined the effects of ascorbate and its congeners on the activity of tyrosine hydroxylase purified to near homogeneity from a rat pheochromocytoma. This purified enzyme was also inactivated by the same agents that inactivated the impure corpus striatal enzyme. Under conditions in which ascorbate almost completely abolished enzyme activity, we found no indication for significant prote-olysis of the purified enzyme as determined by sodium do-decyl sulfate-polyacrylamide gel electrophoresis. We also found that pretreatment of PC12 cells in culture for 4 h with 1 mM ascorbate, dehydroascorbate, or isoascorbate (but not ascorbate sulfate) also decreased tyrosine hydroxylase activity 25–50%. The inactivation seen under in vitro conditions appears to have a counterpart under more physiological conditions.     

Cholinergic Receptor-Mediated Phosphorylation and Activation of Tyrosine Hydroxylase in Cultured Bovine Adrenal Chromaffin Cells

We have identified a 56-kilodalton protein in cultured bovine adrenal chromaffin cells that is phosphorylated when catecholamine secretion is stimulated. Immunodetection on Western blots from both one- and two-dimensional polyacrylamide gels indicated that this protein was tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis. Two-dimensional polyacrylamide gel electrophoresis of proteins from unstimulated cells revealed small amounts of phosphorylated protein with a molecular weight of 56K and pI values of 6.37 and 6.27 which were subunits of tyrosine hydroxylase. Nicotinic stimulation of chromaffin cells caused the phosphorylation of three proteins of 56 kilodaltons with pI values of approximately 6.37, 6.27, and 6.15 which were tyrosine hydroxylase. The immunochemical analysis also revealed that there was unphosphorylated tyrosine hydroxylase 56 kilodaltons with a pI of 6.5 which may have decreased on nicotinic stimulation. The phosphorylation of tyrosine hydroxylase was associated with an increase in in situ conversion of [3H]tyrosine to [3H]dihydroxyphenylalanine ([3H]DOPA). Muscarinic stimulation also caused phosphorylation of tyrosine hydroxylase, but to a smaller extent than did nicotinic stimulation. The secretagogues, elevated K+ and Ba2+, stimulated phosphorylation of tyrosine hydroxylase and [3H]DOPA production. The effects of nicotinic stimulation and elevated K+ on tyrosine hydroxylase phosphorylation and [3H]DOPA production were Ca2+-dependent. Nicotinic agonists also raised cyclic AMP levels in chromaffin cells after 2 min. Dibutyryl cyclic AMP and forskolin, which have little effect on catecholamine secretion, also caused phosphorylation of tyrosine hydroxylase. These stimulators of cyclic AMP-dependent processes caused the appearance of two phosphorylated subunits of tyrosine hydroxylase with pI values of 6.37 and 6.27. There was also a small amount of phosphorylated subunit with a pI of 6.15. Both agents stimulated [3H]DOPA production. The experiments indicate that tyrosine hydroxylase is phosphorylated and activated when chromaffin cells are stimulated to secrete. The data suggest that the earliest phosphorylation of tyrosine hydroxylase induced by a nicotinic agonist occurs through stimulation of a Ca2+-dependent protein kinase. After 2 min phosphorylation by a cyclic AMP-dependent protein kinase may also occur. Phosphorylation of tyrosine hydroxylase is associated with an increase in in situ tyrosine hydroxylase activity.     

Effects of Phospholipases on the Kinetic Properties of Rat Striatal Membrane-Bound Tyrosine Hydroxylase

Abstract: Rat striatal tyrosine hydroxylase can be isolated in both a soluble and a synaptic membrane-bound form. The membrane-bound enzyme, which exhibits lower K _ms for both tyrosine (7 μ M ) and reduced pterin cofactor (110 μ M ) relative to the soluble enzyme (47 μ M and 940 μ M , respectively), can be released from the membrane fraction with mild detergent, and concomitantly its kinetic properties revert to those of the soluble enzyme. Treatment of membrane-bound tyrosine hydroxylase with C. perfringens phospholipase C increased the K _m of the enzyme for tyrosine to 27 μ M and the V _max by 60% without changing the K _m for cofactor. In contrast, treatment of membrane-bound tyrosine hydroxylase with V. russelli phospholipase A₂ increased the K _m for tyrosine to 48 μ M increased the V _max and increased the K _m for cofactor to 560 μ M . The enzyme remained bound to the membrane fraction following both phospholipase treatments. Addition of phospholipids to treated enzyme could partially reverse the effects of phospholipase A₂ treatment, but not the effects of phospholipase C treatment. The kinetic properties of phospholipase-treated, detergent-solubilized tyrosine hydroxylase were identical to those of the control solubilized enzyme. Tyrosine hydroxylase appears to interact with synaptic membrane components to produce at least two separately determined consequences for the kinetic properties of the enzyme.     

Subcellular Distribution of Tyrosine Hydroxylase in Some Catecholaminergic Rat Brain Areas Determined by a Quantitative Immunoblot Assay

The subcellular distribution of the protein tyrosine hydroxylase (TH) after fractionation of rat brain tissue was studied by a sensitive technique of immunoblot quantification in the dopaminergic nigrostriatal and the dorsal noradrenergic pathways and in the ventrolateral medulla. This repartition indicates that in all catecholaminergic regions of the cell bodies studied, the contribution of the nerve endings to the total TH amount is very low (less than 7%), in contrast to that observed in the terminal fields. The correlative subcellular determination of the TH amount and activity in the same tissue could be a useful approach for studying experimentally induced mechanisms of catecholamine synthesis modulation in different brain catecholaminergic pathways.     

Circadian Variations in the Activity of Tyrosine Hydroxylase, Tyrosine Aminotransferase, and Tryptophan Hydroxylase: Relationship to Catecholamine Metabolism

Abstract— Circadian variations in the activity of tyrosine hydroxylase, tyrosine aminotransferase, and tryptophan hydroxylase were observed in the rat brain stem. Tyrosine hydroxylase exhibited a bimodal pattern with peaks occurring during both the light and dark phases of the circadian cycle. Tyrosine aminotransferase had one daily peak of activity occurring late in the light phase, whereas tryptophan hydroxylase activity was maximal late in the dark phase. Circadian fluctuations in tyrosine hydroxylase activity did not correlate well with circadian variations in the turnover rates of norepinephrine or dopamine nor with levels of these catecholamines. This supports the idea that although tyrosine hydroxylase is the rate-limiting enzyme in the synthesis of catecholamines, other factors must also be involved in the in vivo regulation of this process. Administration of α -methyl- p -tyrosine (AMT) methyl ester HC1 (100 mg/kg) had no effect on the activity of tryptophan hydroxylase, but effectively eliminated the peak of tyrosine hydroxylase activity that occurred during the light phase. AMT also lowered levels of tyrosine aminotransferase, but only at times near the daily light to dark transition. These chronotypic effects of AMT emphasize the importance of "time of day" as a factor that must be taken into account in evaluating the biochemical as well as the pharmacological and toxicological effects of drugs.     

Bovine Adrenal Tyrosine Hydroxylase: Comparative Study of Native and Proteolyzed Enzyme, and Their Interaction with Anions

Abstract: The pH optimum of native adrenal medulla tyrosine hydroxylase activity is shifted from 5.8 to 6.4 by polyanions (heparin, dextran sulphate), salts (NaCl, Na₂SO₄) and the anionic buffer 2-( N -morpholino)ethanesulphonic acid (MES). Simultaneously, the activity at the optimal pH is increased. Kinetic studies have shown that this activation is associated with a decrease of the apparent K _m of the enzyme for the cofactor 6,7-dimethyltetrahydropterin (DMPH₄) and an increase in the V _max for tyrosine and DMPH₄. The K _m for the tyrosine remained unchanged. These data have been interpreted in terms of the polyelectrolyte theory. The adsorption of tyrosine hydroxylase on various affinity gels containing heparin, dextran sulphate or unsulphated polymer dextran as ligands indicate that the activation of the enzyme is mediated by electrostatic interactions with the anionic species. The site of electrostatic interaction possesses some specificity since the binding constants are higher for heparin or dextran sulphate than for NaCl or MES buffer. Moreover, 3-( N -morpholino)propanesulphonic acid (MOPS) a slightly structurally different buffer inhibits the enzyme activity whereas N -(2-acetamido)-2-amino-ethanesulphonic acid (ACES) has no effect. A limited proteolytic digestion which preserves the enzymatic activity, destroys the effects of the anions. The isoelectric point and the molecular parameters of tyrosine hydroxylase are markedly altered after limited digestion. It is therefore suggested that the interaction between the hydroxylase and anionic compounds occurs on a part of the protein which is different from the active site and which is lost by proteolysis. This portion of the protein might be involved in regulation of native tyrosine hydroxylase.     

Short-Term Regulation of Hydroxylase Cofactor Activity in Rat Brain

Differential drug effects on hydroxylase cofactor activities were observed in the corpus striatum and the locus coeruleus when conditions of sacrifice were controlled. A conformational stability-dependent variable degree of stoichiometric coupling between quinonoid dihydropteridine reductase and tyrosine hydroxylase is proposed as a short-latency influence on hydroxylase cofactor levels.