Effects of Catechol Estrogens1 and Catecholamines on Hypothalamic and Corpus Striatal Tyrosine Hydroxylase Activity

Abstract: The effects of catechol estrogens on tyrosine hydroxylase activity in hypothalamic and corpus striatal extracts were evaluated. When assayed in the presence of subsaturating concentrations of pterin cofactor, tyrosine hydroxylase activity was depressed by 2-hydroxyestrone, 2-hydroxyestradiol, Lnorepinephrine, or dopamine. However, estrone, 17β-estradiol, 2- methoxyestrone, or 2-methoxyestradiol had no consistent inhibitory effect on tyrosine hydroxylase activity under in vitro conditions. Moreover, a decrease in pterin binding affinity (elevated K_m) in the presence of either catecholamines or 2-hydroxyestrogens was found. These findings were suggestive of a competitive interaction between catechols and pterin. Catechol estrogens and catecholamines were shown to inhibit both membrane-bound and soluble forms of tyrosine hydroxylase. The membrane-bound form of tyrosine hydroxylase, however, was found to have a greater binding affinity (lower K_l) for 2hydroxyestradiol and norepinephrine than did the soluble form. The results of the present study are suggestive of a cytoplasmic effect of estrogen that may be mediated by 2-hydroxyestrogen and terminated by O-methylation.     

A comparison of the long-term and short-term regulations of tyrosine hydroxylase activity

The activity of tyrosine hydroxylase, the enzyme that catalyzes the rate-limiting step in catecholamine biosynthesis, increases in postganglionic sympathetic neurons following electrical stimulation of their afferent preganglionic input. Two types of changes in enzyme activity occur: an acute increase and a delayed and long-lasting increase. The pharmacological mechanisms involved in these transsynaptic effects and the biochemical mechanisms underlying the changes in tyrosine hydroxylase activity are discussed.     

MINIMUM DURATION OF TRANS-SYNAPTIC STIMULATION REQUIRED FOR THE INDUCTION OF TYROSINE HYDROXYLASE BY RESERPINE IN THE RAT SUPERIOR CERVICAL GANGLION

—The period during which trans-synaptic stimulation is required by the rat superior cervical ganglion for induction of tyrosine hydroxylase by reserpine has been studied. Ganglia were decentralized on one side at various times before or after an injection of reserpine. The tyrosine hydroxylase activity of the denervated and control ganglia was assayed 72 h after drug treatment. When decentralization was performed 8 h after an injection of reserpine the increase in tyrosine hydroxylase activity was blocked in the denervated ganglia. Decentralization 12 h after reserpine treatment or later had no effect on the enzyme induction. The actual increase in tyrosine hydroxylase activity occurred between 24 and 48 h after injection of reserpine.     

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.     

Preganglionic stimulation increases the phosphorylation of tyrosine hydroxylase in the superior cervical ganglion by both cAMP-dependent and Ca2+-dependent protein kinases

Electrical stimulation of the preganglionic cervical sympathetic trunk increases the phosphorylation of tyrosine hydroxylase in the superior cervical ganglion of the rat by a nicotinic mechanism and by a noncholinergic mechanism. We have measured the incorporation of [32P]Pi into specific tryptic phosphopeptides in tyrosine hydroxylase in order to identify the protein kinases that phosphorylate this enzyme in electrically stimulated ganglia. 32P-labeled tyrosine hydroxylase was isolated from the ganglion by immunoprecipitation and polyacrylamide gel electrophoresis and was subjected to tryptic hydrolysis. Seven tryptic peptides were resolved from these hydrolysates by two-dimensional thin-layer electrophoresis and chromatography. Preganglionic stimulation (20 Hz, 5 min) increased the incorporation of 32P into four of these peptides. In the presence of cholinergic antagonists, however, electrical stimulation increased the labeling of only one phosphopeptide. From a comparison of the effects of preganglionic stimulation with the effects of agonists that activate specific protein kinases, we conclude that electrical stimulation increases the phosphorylation of tyrosine hydroxylase by both a cAMP-dependent protein kinase and a Ca2+/calmodulin-dependent protein kinase. The nicotinic component of preganglionic stimulation appears to be mediated by a Ca2+/calmodulin-dependent protein kinase, while the noncholinergic component appears to be mediated by cAMP-dependent protein kinase. Although protein kinase C can phosphorylate tyrosine hydroxylase, this kinase does not appear to participate in the stimulation-induced phosphorylation of tyrosine hydroxylase in the superior cervical ganglion.     

Effects of substance P on the long-term regulation of tyrosine hydroxylase activity and catecholamine levels in cultured adrenal chromaffin cells

Acetylcholine, released from splanchnic nerve terminals innervating adrenal chromaffin cells, is known to increase synthesis of adrenal tyrosine hydroxylase, the rate-limiting enzyme in catecholamine synthesis. The neuropeptide substance P is also present in the splanchnic nerve innervating the adrenal medulla, and this study examined whether substance P has any long-term effects on tyrosine hydroxylase activity and catecholamine levels in cultures of adult bovine adrenal chromaffin cells. When cultures were incubated for 3 days with substance P and carbachol, a cholinergic agonist, substance P (10(-6) M, and greater) completely inhibited the increase in tyrosine hydroxylase activity normally induced by carbachol. Long-term stimulation with carbachol also depleted endogenous catecholamines from the cells and substance P prevented this carbachol-induced depletion of catecholamine content. Substance P by itself, in the absence of carbachol, had only a slight effect on tyrosine hydroxylase activity. 8-Bromoadenosine 3':5'-cyclic monophosphate, an analogue of adenosine 3':5'-cyclic monophosphate, also increases tyrosine hydroxylase activity in chromaffin cells; however, substance P had no effect on the increase in tyrosine hydroxylase activity induced by this analogue. These results indicate that substance P's effects are relatively specific for the carbachol-induced increased in tyrosine hydroxylase activity and that the primary site of action of substance P is not a site common to the mechanism of tyrosine hydroxylase induction by carbachol and 8-bromoadenosine 3':5'-cyclic monophosphate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrical Stimulation of the Substantia Nigra and Changes of 2-Phenylethylamine Synthesis in the Rat Striatum

In rats pretreated with deprenyl (2 mg/kg), electrical stimulation of the left substantia nigra produced an increase in the concentrations of 3,4-dihydroxyphenylacetic acid and homovanillic acid in the left striatum by 57 and 45%, but the levels of 2-phenylethylamine and p-tyramine decreased by 22 and 41%, respectively, as compared with those in the right striatum. The administration of alpha-methyl-p-tyrosine (1.25 mg/kg, i.p.), a tyrosine hydroxylase inhibitor, 1 h before nigral stimulation, did not affect the concentration of 2-phenylethylamine in unstimulated striata but prevented the stimulation-induced decrease in the concentration of 2-phenylethylamine. Neither stimulation nor alpha-methyl-p-tyrosine affected the activity of monoamine oxidase A or B, and stimulation did not produce any change in striatal blood flow, a finding demonstrating that the changes in the rate of accumulation of 2-phenylethylamine were not due to changes in catabolism or removal of 2-phenylethylamine from the brain. These experiments demonstrate that the rate of synthesis of striatal 2-phenylethylamine is decreased following nigral stimulation and that this effect is blocked after partial inhibition of tyrosine hydroxylase. This suggests that 2-phenylethylamine is present in tyrosine hydroxylase-containing neurons and therefore supports the coexistence of 2-phenylethylamine and dopamine in the nigrostriatal pathway.     

Central effects of the tetrapeptide tuftsin

Under systemic administration tuftsin produces a marked influence on behavior and emotional reactivity in rats: enhances motor (vertical) activity, perception of pain stimulation and related aggressiveness and residual excitation. Activating effect is accompanied by an aggravation of the acquisition of passive avoidance reaction during single reinforcement. In vitro experiments revealed a direct inhibitory effect of tuftsin on the reaction rate of brain tyrosine hydroxylase activity. In vivo there was shown an increase in hypothalamic and especially striatal tyrosine hydroxylase activity. The data obtained indicate direct relationships between tuftsin central effects and the changes in brain catecholaminergic processes participating in the regulation of emotional-motivational and motor reactions.     

Effects of Peptides of the Secretin-Glucagon Family and Cyclic Nucleotides on Tyrosine Hydroxylase Activity in Sympathetic Nerve Endings

Previous studies have shown that certain peptides of the secretin-glucagon family stimulate tyrosine hydroxylase activity in sympathetic neurons of the superior cervical ganglion and three of its end organs, i.e., the iris, pineal gland, and submaxillary gland. To determine whether a similar regulation occurs in other sympathetic neurons, the effects of two of these peptides, secretin and vasoactive intestinal peptide, were examined in the right cardiac ventricle of the rat, a tissue innervated primarily by the middle and inferior cervical ganglia. Both peptides stimulated tyrosine hydroxylase activity, measured in situ, in this tissue. In addition, several second messenger systems were investigated as possible mediators of this peptidergic stimulation of tyrosine hydroxylase activity in autonomic end organs. 8-Bromoadenosine 3',5'-cyclic monophosphate and forskolin elevated tyrosine hydroxylase activity in slices of both the right ventricle and the submaxillary gland. 8-Bromoguanosine 3',5'-cyclic monophosphate also stimulated tyrosine hydroxylase activity in both tissues, whereas nitroprusside stimulated activity only in the submaxillary slices. Furthermore, the phosphodiesterase inhibitors 3-isobutyl-1-methylxanthine and/or Ro 20-1724 potentiated the stimulation by secretin, as well as the stimulations by forskolin and nitroprusside. Phorbol 12,13-dibutyrate also stimulated tyrosine hydroxylase activity in cardiac and submaxillary slices; however, no potentiation of these effects was seen following addition of either phosphodiesterase inhibitor. These data, taken together with those of previous studies, suggest a role for a cyclic nucleotide, probably adenosine 3',5'-cyclic monophosphate, in the peptidergic stimulation of tyrosine hydroxylase activity in sympathetic nerve terminals.     

Activation of Retinal Tyrosine Hydroxylase In Vitro by Cyclic AMP-Dependent Protein Kinase: Characterization and Comparison to Activation In Vivo by Photic Stimulation

Abstract: Tyrosine hydroxylase in rat retina is activated in vivo as a consequence of photic stimulation. Tyrosine hydroxylase in crude extracts of dark-adapted retinas is activated in vitro by incubation under conditions that stimulate protein phosphorylation by cyclic AMP-dependent protein kinase. Comparison of the activations of the enzyme by photic stimulation in vivo and protein phosphorylation in vitro demonstrated several similarities. Both treatments decreased the apparent K _m of the enzyme for the synthetic pterin cofactor 6MPH₄. Both treatments also produced the same change in the relationships of tyrosine hydroxylase activity to assay pH. When retinal extracts containing tyrosine hydroxylase activated either in vivo by photic stimulation or in vitro by protein phosphorylation were incubated at 25°C, the enzyme was inactivated in a time-dependent manner. The inactivation of the enzyme following both activation in vivo and activation in vitro was partially inhibited by sodium pyrophosphate, an inhibitor of phosphoprotein phosphatase. In addition to these similarities, the activation of tyrosine hydroxylase in vivo by photic stimulation was not additive to the activation in vitro by protein phosphorylation. These data indicate that the mechanism for the activation of tyrosine hydroxylase that occurs as a consequence of light-induced increases of neuronal activity is similar to the mechanism for activation of the enzyme in vitro by protein phosphorylation. This observation suggests that the activation of retinal tyrosine hydroxylase in vivo may be mediated by phosphorylation of tyrosine hydroxylase or some effector molecule associated with the enzyme.     

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.     

Plasma gonadotropin,prolactin levels and hypothalamic tyrosine hydroxylase activity in rats during estrous cycle,after overiectomy and after blockade of catecholamine biosynthesis

Plasma gonadotropin, prolactin levels and hypothalamic tyrosine hydroxylase activity were evaluated at 0900, 1200 and 1700 h during diestrus, proestrus and estrus, ovariectomized and after systemic administration of reserpine or α-methyl p-tyrosine, which interfere with catecholamine biosynthesis, in rats. Gonadotropin and prolactin levels showed peak values during the afternoon of proestrus, while hypothalamic tyrosine hydroxylase activity was markedly lowered at 1200 on proestrus. Gonadotropin levels were slightly lowered whereas prolactin concentrations and hypothalamic tyrosine hydroxylase activity were significantly increased by reserpine. Depletion of hypothalamic dopamine by reserpine apparently resulted in significant elevation of prolactin levels which inturn induce tyrosine hydroxylase. Gonadotropin levels and hypothalamic tyrosine hydroxylase activity were significantly suppressed after the administration of α-methyl p-tyrosine. Prolactin levels, however, were elevated significantly. These results indicate that catecholamines are involved in the control of gonadotropin and prolactin release during estrous cycle and inhibition of catecholamines biosynthesis by α-methyl p-tyrosine could result in suppression of gonadotropin levels, whereas removal of tonic inhibition of hypothalamic dopamine by α-methyl-p-tyrosine elevate prolactin levels.     

Tyrosine hydroxylase activity in rabbit autonomic nervous system ganglia in acute experimental emotional stress

The Zigmond method was made use of to study tyrosine hydroxylase activity in cervical, stellate and nodosal ganglia of rabbits predisposed and resistant to stress. Experimental acute emotional stress was induced by concurrent stimulation of the ventromedial nuclei of the hypothalamus and different skin areas. Experiments have shown that changes in metabolic activity of the sympathetic and parasympathetic systems were phasic in nature throughout 3 hours of the experiment. The changes in tyrosine hydroxylase activity in nodosal and stellate ganglia of rabbits predisposed to stress differs from those in stress-resistant animals. Stress-predisposed rabbits manifested a decrease in the activity of tyrosine hydroxylase in stellate and nodosal ganglia.     

Mechanisms involved in the long-term modulation of tyrosine hydroxylase by endothelins in the olfactory bulb of normotensive rats

The olfactory bulbs play a relevant role in the interaction between the animal and its environment. The existence of endothelin-1 and -3 in the rat olfactory bulbs suggests their role in the control of diverse functions regulated at this level. Tyrosine hydroxylase, a crucial enzyme in catecholamine biosynthesis, is tightly regulated by short- and long-term mechanisms. We have previously reported that in the olfactory bulbs endothelins participate in the short-term tyrosine hydroxylase regulation involving complex mechanisms. In the present work we studied the effect of long-term stimulation by endothelins on tyrosine hydroxylase in the rat olfactory bulbs. Our findings show that endothelin-1 and -3 modulated catecholaminergic transmission by increasing enzymatic activity. However, these peptides acted through different receptors and intracellular pathways. Endothelin-1 enhanced tyrosine hydroxylase activity through a super high affinity ET(A) receptor and cAMP/PKA and CaMK-II pathways, whereas, endothelin-3 through a super high affinity atypical receptor coupled to cAMP/PKA, PLC/PKC and CaMK-II pathways. Endothelins also increased tyrosine hydroxylase mRNA and the enzyme total level as well as the phosphorylation of Ser 19, 31 and 40 sites. Furthermore, both peptides stimulated dopamine turnover and reduced its endogenous content. These findings support that endothelins are involved in the long-term regulation of tyrosine hydroxylase, leading to an increase in the catecholaminergic activity which might be implicated in the development and/or maintenance of diverse pathologies involving the olfactory bulbs.     

Low-Na+ Medium Increases the Activity and the Phosphorylation of Tyrosine Hydroxylase in the Superior Cervical Ganglion of the Rat

Incubation of the rat superior cervical ganglion in Na+-free or low-Na+ medium increased the rate of synthesis of 3,4-dihydroxyphenylalanine (DOPA) in the ganglion fourfold and caused a concomitant stable activation of tyrosine hydroxylase. DOPA synthesis was half-maximal in medium containing about 20 mM Na+. Low-Na+ medium also increased the incorporation of 32Pi into tyrosine hydroxylase; the dependence of tyrosine hydroxylase phosphorylation on the Na+ concentration resembled that of DOPA synthesis. The stimulatory effects of low-Na+ medium on DOPA production and on tyrosine hydroxylase activity in vitro were dependent on extra-cellular Ca2+. The stimulation of DOPA synthesis in low-Na+ medium was inhibited by methoxyverapamil, an inhibitor of Ca2+ uptake, and was partially blocked by tetrodotoxin, but it was not affected by the cholinergic antagonists hexamethonium and atropine. Ionomycin, a calcium ionophore, stimulated DOPA synthesis to about the same extent as low-Na+ medium and also increased the incorporation of 32Pi into tyrosine hydroxylase. 8-Bromo cyclic AMP (1 mM) also stimulated DOPA production in the ganglion, and this stimulation was more than additive with that produced by low-Na+ medium. These data support the hypothesis that low-Na+ medium stimulates DOPA synthesis by raising intracellular Ca2+, which then promotes the phosphorylation of tyrosine hydroxylase.     

Bovine Tyrosine Hydroxylase Gene-Promoter Regions Involved in Basal and Angiotensin II-Stimulated Expression in Nontransformed Adrenal Medullary Cells

Abstract: The tyrosine hydroxylase gene is expressed specifically in catecholaminergic cells, and its activity is regulated by afferent stimuli. To characterize molecular mechanisms underlying those regulations, we have constructed chimeric genes consisting of bovine tyrosine hydroxylase gene promoters (wild-type or deletion mutants) and a luciferase reporter gene. The basal expression of these genes and their regulation by angiotensin II were examined in cultured bovine adrenal medullary cells. Luciferase activity was normalized to the amount of transfected plasmid DNA. A pTHgoodLUC plasmid containing the -428/+21-bp fragment of the tyrosine hydroxylase gene promoter expressed luciferase activity at severalfold higher levels than the promoterless pOLUC plasmid. Deletion of the -194/-54-bp promoter fragment containing POU/Oct, SP1, and other putative regulatory elements increased luciferase expression fivefold. An additional deletion further upstream (-269/-194 bp), including a 12-O-tetradecanoylphorbol 13-acetate (TPA)-responsive element (TRE)-like site, reduced promoter activity. These results indicate the presence of negatively and positively acting regions in the bovine tyrosine hydroxylase gene promoter controlling basal promoter activity in adrenal medullary cells. Angiotensin II stimulated the expression of endogenous tyrosine hydroxylase gene and pTHgood-LUC approximately threefold without affecting the expression of pOLUC. A comparable threefold stimulation was observed following the deletion of the -194/-54-bp promoter region, despite the increase in basal promoter activity. Additional deletion of the -269/-194-bp promoter fragment reduced stimulation by angiotensin II to 1.5-fold. These results indicate that the angiotensin II receptor-responsive element is located in the -269/-194-bp promoter region containing the TRE-like site. Additional angiotensin II-responsive site(s) may be present outside this region. Gel mobility shift assays demonstrated constitutive and angiotensin II-induced protein binding to the tyrosine hydroxylase gene promoter. Some DNA-protein complexes were displaced with c-Fos antibodies. The results suggest that c-Fos-related antigens support basal promoter activity and mediate activation of tyrosine hydroxylase by angiotensin II receptor.     

Effect of tuftsin and hydroxymethacil on tyrosine hydrolase of macrophages in chronic stress

It has been established that rat peritoneal macrophages possess tyrosine hydroxylase activity which has been characterized by KM4, 2 microM, Vmax 30 nmole/min per mg of protein. After 15 days of electronociseptive stimulation almost all tyrosine hydroxylase activity has been established in the form which has tyrosine KM47.0 M and Vmax 18.6 nmole/min. per mg of protein. Immunostimulator hydroxymethacil++, at its intraperitoneal injections to stressed rats during 7 days induced the appearance of low-affinity form of tyrosine hydroxylase with KM270 microM and Vmax 27.8 nmole/min.per mg of protein. The same low affinity form of the enzyme has been established after injections of tuftsin which possesses immunostimulating properties.     

Tyrosine hydroxylase phosphorylation: regulation and consequences

The rate-limiting enzyme in catecholamine synthesis is tyrosine hydroxylase. It is phosphorylated at serine (Ser) residues Ser8, Ser19, Ser31 and Ser40 in vitro, in situ and in vivo. A range of protein kinases and protein phosphatases are able to phosphorylate or dephosphorylate these sites in vitro. Some of these enzymes are able to regulate tyrosine hydroxylase phosphorylation in situ and in vivo but the identity of the kinases and phosphatases is incomplete, especially for physiologically relevant stimuli. The stoichiometry of tyrosine hydroxylase phosphorylation in situ and in vivo is low. The phosphorylation of tyrosine hydroxylase at Ser40 increases the enzyme's activity in vitro, in situ and in vivo. Phosphorylation at Ser31 also increases the activity but to a much lesser extent than for Ser40 phosphorylation. The phosphorylation of tyrosine hydroxylase at Ser19 or Ser8 has no direct effect on tyrosine hydroxylase activity. Hierarchical phosphorylation of tyrosine hydroxylase occurs both in vitro and in situ, whereby the phosphorylation at Ser19 increases the rate of Ser40 phosphorylation leading to an increase in enzyme activity. Hierarchical phosphorylation depends on the state of the substrate providing a novel form of control of tyrosine hydroxylase activation.     

Increased cardiac production of dihydroxyphenylalanine (DOPA) during sympathetic stimulation in anaesthetized dogs.

Entry of dihydroxyphenylalanine (DOPA) into plasma from specific organs may reflect regional activity of tyrosine hydroxylase, the enzyme responsible for the immediate synthesis of DOPA and rate-limiting for subsequent formation of catecholamines. Therefore, cardiac spillovers of DOPA, noradrenaline and the intraneuronal metabolite of noradrenaline, dihydroxyphenylglycol (DHPG), were examined during two periods of graded electrical stimulation of the sympathetic nerves to the heart in anesthetized dogs. Responses were examined before and after neuronal uptake blockade with desipramine. Cardiac spillover of DOPA increased by 1.8- and 4.4-fold during sympathetic stimulation before desipramine and by 1.6- and 3.3-fold after desipramine. Fold increases in cardiac spillover of DOPA were much lower than but positively related with fold increases in noradrenaline spillover (5.9- and 13.8-fold increases before and 9.0- and 15.8-fold increases after desipramine). Increases in cardiac spillover of DHPG (1.5- and 2.3-fold increases) were blocked by desipramine so that fold changes in spillover of DOPA were greater than and poorly related to changes in spillover of DHPG. Fold increases in cardiac spillover of DOPA showed a close one-to-one positive relationship with fold increases in the sum of cardiac spillovers of noradrenaline and dihydroxyphenylglycol before and after desipramine. For a given fold increase in noradrenaline release, transmitter turnover is increased fractionally and noradrenaline synthesis need also only increase fractionally to maintain transmitter stores constant. The close relationship between fold increases in cardiac spillover of DOPA and combined spillovers of noradrenaline and DHPG is consistent with regulation of tyrosine hydroxylase activity to match changes in noradrenaline synthesis with changes in noradrenaline turnover. Changes in cardiac spillover of DOPA appear to reflect local changes in tyrosine hydroxylase activity.     

Similar Time Course Changes in Striatal Levels of Glutamic Acid Decarboxylase and Proenkephalin mRNA Following Dopaminergic Deafferentation in the Rat

An immunoblot procedure was developed to quantify the amount of tyrosine hydroxylase protein in homogenate of small brain regions. With the use of this method we have studied the variations in tyrosine hydroxylase activity and protein levels in some catecholaminergic neurons at different times following a single reserpine injection (10 mg/kg s.c.) and reevaluated the anatomical specificity of tyrosine hydroxylase induction by this drug. Reserpine administration provoked a long-lasting increase in both tyrosine hydroxylase activity and protein levels within locus ceruleus neurons. This effect culminated at day 4 after injection. At this time, the enzyme activity and protein levels in treated animals were respectively 2.7 and 2.6 times that measured in vehicle-treated animals. Both parameters varied in parallel so that tyrosine hydroxylase specific activity did not change over time. In contrast, reserpine did not cause any changes in tyrosine hydroxylase activity in the dopaminergic neurons of the substantia nigra, but provoked a moderate increase in tyrosine hydroxylase protein level. This latter effect was maximal (1.5 times) 4 days after treatment. In the adjacent dopaminergic area, i.e., the ventral tegmental area, a small decrease in the enzyme activity was recorded at day 2 without any significant change in the level of the protein. In conclusion, first, our data show the capacity of our method to assay tyrosine hydroxylase protein amounts in small brain catecholaminergic nuclei. Second, our results confirm and extend previous studies on the effect of reserpine on the regulation of tyrosine hydroxylase level within brain noradrenergic neurons.(ABSTRACT TRUNCATED AT 250 WORDS)