Activation of Tyrosine Hydroxylase in PC12 Cells by the Cyclic GMP and Cyclic AMP Second Messenger Systems

Tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, is subject to regulation by a variety of agents. Previous workers have found that cyclic AMP-dependent protein kinase and calcium-stimulated protein kinases activate tyrosine hydroxylase. We wanted to determine whether cyclic GMP might also be involved in the regulation of tyrosine hydroxylase activity. We found that treatment of rat PC12 cells with sodium nitroprusside (an activator of guanylate cyclase), 8-bromocyclic GMP, forskolin (an activator of adenylate cyclase), and 8-bromocyclic AMP all produced an increase in tyrosine hydroxylase activity measured in vitro or an increased conversion of [14C]tyrosine to labeled catecholamine in situ. Sodium nitroprusside also increased the relative synthesis of cyclic GMP in these cells. In the presence of MgATP, both cyclic GMP and cyclic AMP increased tyrosine hydroxylase activity in PC12 cell extracts. The heat-stable cyclic AMP-dependent protein kinase inhibitor failed to attenuate the activation produced in the presence of cyclic GMP. It eliminated the activation produced in the presence of cyclic AMP. Sodium nitroprusside also increased tyrosine hydroxylase activity in vitro in rat corpus striatal synaptosomes and bovine adrenal chromaffin cells. In all cases, the cyclic AMP-dependent activation of tyrosine hydroxylase was greater than that of the cyclic GMP-dependent second messenger system. These results indicate that both cyclic GMP and cyclic AMP and their cognate protein kinases activate tyrosine hydroxylase activity in PC12 cells.     

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.     

Regulation of GTP cyclohydrolase I and dihydropteridine reductase in rat pheochromocytoma PC 12 cells

The addition of 8-bromo cyclic AMP, forskolin, theophylline, and 3-isobutyl-1-methylxanthine to the medium of PC 12 cells resulted in an increase in GTP cyclohydrolase I activity, but had no effect on dihydropteridine reductase activity, except theophylline which caused a decrease in dihydropteridine reductase activity at 96 h. GTP cyclohydrolase I activity peaked at 24 h and returned to normal 96 h after drug treatment. Cycloheximide decreased GTP cyclohydrolase I activity at 48 and 96 h, but had little effect on dihydropteridine reductase activity. The addition of reserpine selectively increased only GTP cyclohydrolase I activity. The addition of tetrahydrobiopterin and sepiapterin, however, coordinately inhibited both GTP cyclohydrolase I and dihydropteridine reductase activities. It appears that GTP cyclohydrolase I activity in PC 12 cells is regulated by cyclic AMP stimulation and by end-product inhibition, whereas dihydropteridine reductase activity is only subject to pterin inhibition.     

Activation of Tyrosine Hydroxylase in the Superior Cervical Ganglion by Nicotinic and Muscarinic Agonists

Both dimethylphenylpiperazinium (DMPP), a nicotinic agonist, and bethanechol, a muscarinic agonist, increase 3,4-dihydroxyphenylalanine (DOPA) synthesis in the superior cervical ganglion of the rat. DMPP causes approximately a fivefold increase in DOPA accumulation in intact ganglia whereas bethanechol causes about a two-fold increase in DOPA accumulation. These effects are additive with each other and with the increase in DOPA accumulation produced by 8-bromo cyclic AMP. The action of DMPP is dependent on extracellular Ca2+ while the actions of bethanechol and 8-bromo cyclic AMP are not dependent on extracellular Ca2+. Cholinergic agonists and cyclic nucleotides produce a stable activation of tyrosine hydroxylase (TH) in the ganglion. The activation of TH by nicotinic and muscarinic agonists can be detected after 5 min of incubation of the ganglia with these agents. The nicotinic response disappears after 30 min of incubation, whereas the muscarinic response persists for at least 30 min. The Ca2+ dependence of the TH activation produced by these agents is similar to the Ca2+ dependence of their effects on DOPA accumulation in intact ganglia. These data are consistent with the hypothesis that nicotinic agonists, muscarinic agonists, and cyclic AMP analogues increase TH activity by three distinct mechanisms. The activation of TH presumably underlies the increase in DOPA synthesis produced by these agents.     

Insulin-Like Growth Factor-I Enhances Tyrosine Hydroxylase Activation in Bovine Chromaffin Cells

Previous studies have shown that insulin-like growth factor-I (IGF-I) enhances secretagogue-stimulated Ca2+ uptake and catecholamine release in bovine chromaffin cells. This report describes the effect of IGF-I on the activity of tyrosine hydroxylase (tyrosine 3-monooxygenase, EC 1.14.16.2), the major regulatory enzyme in the pathway of catecholamine biosynthesis. Tyrosine hydroxylase activity was assayed by measuring 3,4-dihydroxyphenylalanine (Dopa) accumulation in the presence of brocresine, an inhibitor of Dopa decarboxylase. Chromaffin cells cultured in serum-free medium produced approximately 40% less Dopa when stimulated by 55 mM K+ than did cells that had been cultured in the presence of serum. Incubation of cells for 3 days in serum-free medium containing 10 nM IGF-I restored high K(+)-stimulated Dopa accumulation to a level comparable to that seen in cells cultured continuously in serum-containing medium. In eight experiments, IGF-I increased high K(+)-stimulated Dopa accumulation (expressed as picomoles per minute per milligram of protein) by 96 +/- 13%. IGF-I increased the protein content of chromaffin cells by approximately 30%; consequently, its effect on tyrosine hydroxylase activity was even greater when Dopa synthesis was expressed as picomoles per minute per 10(7) cells. IGF-I also enhanced the rate of Dopa accumulation in cells stimulated by dimethylphenylpiperazinium, 8-bromo-cyclic AMP, phorbol 12,13-dibutyrate, or Ba2+. The effect of IGF-I on high K(+)-stimulated tyrosine hydroxylase activity was measurable when enzyme activity was assayed in vitro, suggesting that this effect was due to a stable modification of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulation of DOPA Synthesis in the Superior Cervical Ganglion by Veratridine

We have investigated the effect of veratridine on DOPA (3,4-dihydroxyphenylalanine) accumulation by the superior cervical ganglion of the rat. Incubation of the ganglion with veratridine (50 microM) causes a 10-fold increase in the rate of DOPA accumulation. Veratridine-stimulated DOPA accumulation is blocked by tetrodotoxin, but not by cholinergic or adrenergic antagonists or by decentralization of the ganglion. The cyclic nucleotide 8-bromo cyclic GMP does not increase DOPA accumulation, and 8-bromo cyclic AMP causes only a 2-fold increase in DOPA accumulation, which is additive with the effect of veratridine. Thus, the action of veratridine appears to be independent of these cyclic nucleotides. The effect of veratridine on DOPA accumulation is probably due to a stable modification of tyrosine hydroxylase, since an increase in tyrosine hydroxylase activity can be measured in cell-free extracts of veratridine-treated ganglia. Both the increase in DOPA accumulation and the stable activation of tyrosine hydroxylase are dependent upon extracellular Ca2+. The activation of tyrosine hydroxylase by veratridine may be mediated by the depolarization of, and the subsequent entry of Ca2+ into, ganglionic neurons.     

Adenosine Receptor Activation and the Regulation of Tyrosine Hydroxylase Activity in PC 12 and PC 18 Cells

We compared the response of rat PC12 cells and a derivative PC18 cell line to the effects of adenosine receptor agonists, antagonists, and adenine nucleotide metabolizing enzymes. We found that theophylline (an adenosine receptor antagonist), adenosine deaminase, and AMP deaminase all decreased basal cyclic AMP content and tyrosine hydroxylase activity in the PC12 cells, but not in PC18 cells. Both cell lines responded to the addition of 2-chloroadenosine and 5'-N-ethylcarboxamidoadenosine, adenosine receptor agonists, by exhibiting an increase in tyrosine hydroxylase activity and cyclic AMP content. The latter finding indicates that both cell lines contained an adenosine receptor linked to adenylate cyclase. We found that the addition of dipyridamole, an inhibitor of adenosine uptake, produced an elevation of cyclic AMP and tyrosine hydroxylase activity in both cell lines. Deoxycoformycin, an inhibitor of adenosine deaminase, failed to alter the levels of cyclic AMP or tyrosine hydroxylase activity. This suggests that uptake was the primary inactivating mechanism of adenosine action in these cells. We conclude that both cell types generated adenine nucleotides which activate the adenosine receptor in an autocrine or paracrine fashion. We found that PC12 cells released ATP in a calcium-dependent process in response to activation of the nicotinic receptor. We also measured the rates of degradation of exogenous ATP, ADP, and AMP by PC12 cells. We found that the rates of metabolism of the former two were at least an order of magnitude greater than that of AMP. Any released ATP would be rapidly metabolized to AMP and then more slowly degraded to adenosine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tyrosine Hydroxylase Is Activated and Phosphorylated on Different Sites in Rat Pheochromocytoma PC12 Cells Treated with Phorbol Ester and Forskolin

Abstract: Incubation of rat pheochromocytoma PC12 cells with 4β-phorbol-12β-myristate-13α-acetate (PMA), an activator of Ca²⁺/phospholipid-dependent protein kinase (protein kinase C), or forskolin, an activator of adenylate cyclase, is associated with increased activity and enhanced phosphorylation of tyrosine hydroxylase. Neither the activation nor increased phosphorylation of tyrosine hydroxylase produced by PMA is dependent on extracellular Ca²⁺. Both activation and phosphorylation of the enzyme by PMA are inhibited by pretreatment of the cells with trifluo-perazine (TFP). Treatment of PC 12 cells with l-oleoyl-2-acetylglycerol also leads to increases in the phosphorylation and enzymatic activity of tyrosine hydroxylase; 1, 2-diolein and 1, 3-diolein are ineffective. The effects of forskolin on the activation and phosphorylation of the enzyme are independent of Ca²⁺ and are not inhibited by TIT⁵. Forskolin elicits an increase in cyclic AMP levels in PC 12 cells. The increases in both cyclic AMP content and the enzymatic activity and phosphorylation of tyrosine hydroxylase following exposure of PC 12 cells to different concentrations of forskolin are closely correlated. In contrast, cyclic AMP levels do not increase in cells treated with PMA. Tryptic digestion of the phosphorylated enzyme isolated from untreated cells yields four phosphopeptides separable by HPLC. Incubation of the cells in the presence of the Ca²⁺ ionophore ionomycin increases the phosphorylation of three of these tryptic peptides. However, in cells treated with either PMA or forskolin, there is an increase in the phosphorylation of only one of these peptides derived from tyrosine hydroxylase. The peptide phosphorylated in PMA-treated cells is different from that phosphorylated in forskolin-treated cells. The latter peptide is identical to the peptide phosphorylated in dibutyryl cyclic AMP-treated cells. These results indicate that tyrosine hydroxylase is activated and phosphorylated on different sites in PC 12 cells exposed to PMA and forskolin and that phosphorylation of either of these sites is associated with activation of tyrosine hydroxylase. The results further suggest that cyclic AMP-dependent and Ca²⁺/ phospholipid-dependent protein kinases may play a role in the regulation of tyrosine hydroxylase in PC 12 cells.     

Vasoactive Intestinal Peptide Stimulates Catecholamine Biosynthesis in Isolated Adrenal Chromaffin Cells: Evidence for a Cyclic AMP-Dependent Phosphorylation and Activation of Tyrosine Hydroxylase

Vasoactive intestinal peptide (VIP) increased catecholamine biosynthesis in bovine adrenal chromaffin cells by 50–200%. Six related peptides produced no effects. In addition, VIP increased tyrosine hydroxylase (TH) activity measured in gel-filtered supernatants prepared from homogenates of treated cells. The hypothesis that cyclic AMP is the second messenger involved in these effects of VIP was also evaluated. VIP led to an elevation of cyclic AMP levels, and this increase occurred over a similar concentration range and time course as the activation of TH and the increase in catecholamine biosynthesis. Each measure reached maximal levels at 10–20 γM VIP within 1 min and remained elevated for at least 16 min. These changes produced by VIP were paralleled by enhanced phosphorylation of TH, and this phosphorylation occurred on a single tryptic peptide that was the same peptide whose phosphorylation has been previously shown to be stimulated by forskolin. In contrast to VIP and forskolin, 12-O-tetradecanoylphorbol 13-acetate, a phorbol ester known to activate protein kinase C, increased the phosphorylation on a total of three tryptic peptides of TH. Our results indicate that VIP stimulates catecholamine biosynthesis in chromaffin cells through the phosphorylation and activation of TH and support the conclusion that a cyclic AMP-dependent phosphorylation of TH is responsible for these effects.     

Tetrahydrobiopterin biosynthetic activities in human macrophages, fibroblasts, THP-1, and T 24 cells. GTP-cyclohydrolase I is stimulated by interferon-gamma, and 6-pyruvoyl tetrahydropterin synthase and sepiapterin reductase are constitutively present

Interferon-gamma induces tetrahydrobiopterin biosynthesis in human cells and cell lines. Macrophages are peculiar in the formation of large amounts of neopterin derivatives as compared to tetrahydrobiopterin (Werner, E. R., Werner-Felmayer, G., Fuchs, D., Hausen, A., Reibnegger, G., and Wachter, H. (1989) Biochem J. 262, 861-866). Here we compare the impact of interferon-gamma treatment on activities of GTP-cyclohydrolase I (EC 3.5.4.16), 6-pyruvoyl tetrahydropterin synthase, and sepiapterin reductase (EC 1.1.1.153) in human peripheral blood-derived macrophages, normal dermal fibroblasts, THP-1 myelomonocytic cells, and the T 24 bladder transitional-cell carcinoma line. Upon interferon-gamma treatment, GTP-cyclohydrolase I activity is increased 7- to 40-fold, whereas 6-pyruvoyl tetrahydropterin synthase and sepiapterin reductase activities, which are constitutively present in all four investigated cells, remain unchanged. In fibroblasts and T 24 cells GTP cyclohydrolase I activity is the rate-limiting step of tetrahydrobiopterin biosynthesis. In macrophages and in THP-1 cells, however, the induced GTP cyclohydrolase I activity is higher than the 6-pyruvoyl tetrahydropterin synthase activity, leading to the accumulation of neopterin and neopterin phosphates.     

In situ phosphorylation of tyrosine hydroxylase in chromaffin cells: Localization to soluble compartments

The present studies investigated the subcellular distribution of acetylcholine's effects upon the phosphorylation of tyrosine hydroxylase in isolated purified bovine adrenal chromaffin cells. After labeling the intact chromaffin cells with ³²P_i, over 90% of the [³²P]tyrosine hydroxylase was found in soluble fractions. Stimulation of the cells with acetylcholine, the natural secretagogue of chromaffin cells, increased the phosphorylation of tyrosine hydroxylase and over 90% of the increase was associated with soluble tyrosine hydroxylase. Homogenates and subcellular fractions from chromaffin cells were also prepared and phosphorylated in vitro in an attempt to optimize detection of tyrosine hydroxylase phosphorylation. In chromaffin cell homogenates, both 8-bromo-cyclic AMP and calcium increased ³²P incorporation into tyrosine hydroxylase, and again over 90% of the increase was observed in soluble fractions. In the particulate fraction, phosphorylation of a band which comigrated with tyrosine hydroxylase in electrophoresis was occasionally detected but only with very long autoradiographic exposures.Tyrosine hydroxylase enzymatic activity in the isolated purified chromaffin cells was also found to be associated predominantly (approx 90%) with soluble fractions. In contrast, a large portion (40–50%) of the tyrosine hydroxylase activity from crude bovine adrenal medullae was associated with the particulate fraction.The data indicate that although tyrosine hydroxylase (and possibly kinases) can associate with particulate fractions when isolated from crude bovine adrenal medullae, the enzyme is predominantly soluble when isolated from the isolated cells. Further, the effects of acetylcholine on the isolated chromaffin cells are predominantly associated with this soluble tyrosine hydroxylase and its attendant kinases.     

Relationship Between the Regulation of Enkephalin-Containing Peptide and Dopamine β-Hydroxylase Levels in Cultured Adrenal Chromaffin Cells

Adrenal medullary chromaffin cells were maintained under conditions known to increase their cellular levels of enkephalin-containing peptides and the effects of these treatments on another chromaffin vesicle component, dopamine beta-hydroxylase, were examined. Catecholamine-depleting drugs, such as tetrabenazine, and cyclic nucleotide-elevating drugs, including forskolin, 8-bromo-cyclic AMP, and theophylline, increase chromaffin cell enkephalin-containing peptide levels but fail to increase dopamine beta-hydroxylase. In contrast, insulin treatment increases the levels of both enkephalin-containing peptides and dopamine beta-hydroxylase. The increased amounts of enkephalin-containing peptides produced by tetrabenazine and by insulin are stored in subcellular particles with properties identical to chromaffin vesicles on density-gradient centrifugation. These results suggest that following insulin treatment chromaffin cells synthesize new chromaffin vesicles with a full complement of enkephalin-containing peptides, but that after treatment with catecholamine-depleting or cyclic nucleotide-related agents enkephalin-containing peptides can be inserted into preexisting vesicles or that new vesicles, made as a part of the normal turnover of cellular components, contain elevated peptide levels.     

Enhanced Phosphorylation of Tyrosine Hydroxylase at More Than One Site Is Induced by 56 mM K+ in Rat Pheochromocytoma PC 12 Cells in Culture

Incubation of rat pheochromocytoma PC12 cells with dibutyryl cyclic AMP or 56 mM K+ is associated with increased activity and enhanced phosphorylation of tyrosine hydroxylase in situ. Following incubation of the PC12 cells with 32Pi, rapid isolation of the tyrosine hydroxylase, and tryptic digestion of the enzyme, two distinct 32P-peptides can be identified after paper electrophoresis. 56 mM K+ increases 32Pi incorporation into both of these peptides, whereas dibutyryl cyclic AMP increases 32Pi incorporation into only one of these peptides. The rate of increase in the incorporation of 32Pi into these two peptides in cells treated with 56 mM K+ is similar. The phosphorylation of tyrosine hydroxylase in PC12 cells occurs exclusively on serine residues. These results suggest that tyrosine hydroxylase in PC12 cells is phosphorylated on serine residues at two or more distinct sites after 56 mM K+ -induced depolarization. Since only one of these sites is phosphorylated by cyclic AMP-dependent protein kinase, activation of tyrosine hydroxylase by 56 mM K+ may involve phosphorylation by multiple protein kinases in rat pheochromocytoma PC12 cells.     

Effect of Carbachol and 56 mM-Potassium Chloride on the Cyclic AMP-Mediated Induction of Tyrosine Hydroxylase in Neuroblastoma Cells in Culture

Abstract: Tyrosine hydroxylase (TH) activity is increased two- to threefold in neuroblastoma cell line NBP² maintained in culture for 48 h in the presence of either the inhibitor of cyclic AMP-phosphodiesterase (PDE), 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (RO 20- 1724), or the activator of adenylate cyclase, prostaglandin E₁ (PGE₁). Cyclic AMP levels are elevated 70–80% and 30–40% throughout the 48-h treatment with RO 20-1724 and PGE₁, respectively. Carbachol does not affect either basal TH activity or cyclic AMP levels in the cells. However, the cholinergic agonist delays the induction of TH elicited by either RO 20-1724 or PGE₁. This delay is prevented by atropine. The elevation in cyclic AMP levels elicited by either RO 20-1724 or PGE₁ is blocked for 1 h or 15 min. respectively, after treatment with carbachol. Cyclic AMP levels then begin to rise until they reach those levels observed in the presence of RO 20-1724 or PGE₁ alone by 12 h or 1 h of treatment, respectively. Time course studies demonstrate that this transient inhibition of the elevation of cyclic AMP is associated with a 48-h delay in the induction of TH elicited by either RO 20-1724 or PGE₁. In contrast, the induction elicited by 8-bromo cyclic AMP is unaffected by carbachol. A depolarizing concentration (56 mM) of KCl produces a 24-h delay in the induction of TH elicited by RO 20-1724, without affecting the concomitant elevation of cyclic AMP produced by the PDE inhibitor. Furthermore, 56 mM-KCl inhibits the induction of TH elicited by 8-bromo cyclic AMP. It thus appears that carbachol delays the induction of TH by transiently inhibiting the elevation of cyclic AMP, whereas potassium depolarization delays the induction of TH by inhibiting a process with a site of action that is distal to the elevation of cyclic AMP.     

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.     

Dopamine Autoreceptors Modulate the Phosphorylation of Tyrosine Hydroxylase in Rat Striatal Slices

The hypothesis that dopamine (DA) autoreceptors modulate the phosphorylation of tyrosine hydroxylase (TH; EC 1.14.16.2) was investigated in rat striatal slices. Tissue was prelabeled with 32P inorganic phosphate, and TH recovered by immunoprecipitation with anti-TH rabbit serum. The TH monomer was resolved on sodium dodecyl sulfate polyacrylamide gels, and the extent of phosphorylation was determined by scanning densitometry of autoradiographs. Depolarization of striatal slices with 55 mM K+ markedly increased the incorporation of 32P into several proteins, including the TH monomer (Mr = 60,000). A similar increase in TH phosphorylation occurred in response to the adenylate cyclase activator forskolin and the cyclic AMP analog dibutyryl cyclic AMP. An increase in TH phosphorylation was not observed in response to the D1-selective agonist SKF 38393. The D2-selective DA autoreceptor agonist pergolide decreased the phosphorylation of TH below basal levels and blocked the increase in phosphorylation elicited by 55 mM K+. The inhibitory effect of pergolide was antagonized by the D2-selective antagonist eticlopride. Changes observed in the phosphorylation of TH were mirrored by changes in tyrosine hydroxylation in situ. These observations support the hypothesis that a reduction in TH phosphorylation is the mechanism by which DA autoreceptors modulate tyrosine hydroxylation in nigrostriatal nerve terminals.     

Sepiapterin reductase in cultured human cells

Sepiapterin reductase, an enzyme involved in the synthesis of tetrahydrobiopterin (the natural cofactor for phenylalanine, tyrosine and tryptophan hydroxylases), has been assayed in cultured human amniotic fibroblasts and in cultured mononuclear blood cells. In both cases, the Michaelis constants for sepiapterin and NADPH were essentially equal; 20 microM and 6 microM respectively for stimulated mononuclear blood cells and 22 microM and 5 microM respectively for amniotic fibroblasts. The inhibition by N-acetylserotonin was also similar in both cases. The concentration that produced 50% inhibition in stimulated mononuclear blood cells and in amniotic fibroblasts was 2 microM. The results strongly suggest that the same enzyme is acting in both types of cells, at least when grown in culture.     

In Vitro Phosphorylation of Bovine Adrenal Chromaffin Cell Tyrosine Hydroxylase by Endogenous Protein Kinases

Under phosphorylating conditions, addition of Ca2+ or cyclic AMP to the 100,000 g supernatant of purified bovine adrenal chromaffin cells increases both the incorporation of 32P into tyrosine hydroxylase and the activity of the enzyme. Combining maximally effective concentrations of each of these stimulating agents produces an additive increase in both the level of 32P incorporation into tyrosine hydroxylase and the degree of activation of the enzyme. The increased phosphorylation by Ca2+ is due to stimulation of endogenous Ca2+-dependent protein kinase activity and not inhibition of phosphoprotein phosphatases. When the chromaffin cell supernatant is subjected to diethylaminoethyl (DEAE) chromatography to remove calmodulin and phospholipids, tyrosine hydroxylase is no longer phosphorylated or activated by Ca2+; on the other hand, phosphorylation and activation of tyrosine hydroxylase by cyclic AMP are not affected. Subsequent replacement of either Ca2+ plus calmodulin or Ca2+ plus phosphatidylserine to the DEAE-fractionated cell supernatant restores the phosphorylation, but not activation of the enzyme. Reverse-phase HPLC peptide mapping of tryptic digests of tyrosine hydroxylase from the 100,000 g supernatant shows that the Ca2+-dependent phosphorylation occurs on three phosphopeptides, whereas the cyclic AMP-dependent phosphorylation occurs on one of these peptides. In the DEAE preparation, either cyclic AMP alone or Ca2+ in the presence of phosphatidylserine stimulates the phosphorylation of only a single phosphopeptide peak, the same peptide phosphorylated by cyclic AMP in the crude supernatant. In contrast, Ca2+ in the presence of calmodulin stimulates the phosphorylation of three peptides having reverse-phase HPLC retention times that are identical to peptides phosphorylated by Ca2+ addition to the crude unfractionated 100,000 g supernatant. Rechromatography of the peaks from each of the in vitro phosphorylations, either in combination with each other or in combination with each of the seven peaks generated from phosphorylation of tyrosine hydroxylase in situ, established that cyclic AMP, Ca2+/phosphatidylserine, and Ca2+/calmodulin all stimulate the phosphorylation of the same reverse-phase HPLC peptide: in situ peptide 6. Ca2+/calmodulin stimulates the phosphorylation of in situ peptides 3 and 5 as well. Thus, tyrosine hydroxylase can be phosphorylated in vitro by protein kinases endogenous to the chromaffin cell. Phosphorylation occurs on a maximum of three of the seven in situ phosphorylated sites, and all three of these sites can be phosphorylated by a Ca2+/calmodulin-dependent protein kinase.     

Expression of Tyrosine Hydroxylase Gene in Cultured Hypothalamic Cells: Roles of Protein Kinase A and C

Abstract: In hypothalamic cells cultured in serum-free medium, the quantity of tyrosine hydroxylase mRNA increases after treatment with an activator of the protein kinase A pathway (8-bromoadenosine cyclic AMP, 3-isobutyl-1-methylxanthine, or forskolin) or an activator of protein kinase C (12- O -tetradecanoylphorbol 13-acetate or sn -1,2-diacylglycerol). The tyrosine hydroxylase mRNA level decreases in the cells after inhibition of protein kinase C with calphostin C or after depletion of protein kinase C by extended phorbol ester treatment. These data suggest that both protein kinase pathways regulate tyrosine hydroxylase gene expression in hypothalamic cells. As simultaneous activation of both pathways has less than an additive effect on the tyrosine hydroxylase mRNA level, they appear to be interrelated. Compared with the rapid and dramatic increase of the tyrosine hydroxylase mRNA level in pheochromocytoma cells, activation of the protein kinase A or protein kinase C pathway in the cultured hypothalamic cells induces slow changes of a small magnitude in the amount of tyrosine hydroxylase mRNA. The slow regulation of tyrosine hydroxylase gene expression in hypothalamic dopaminergic neurons corresponds to the relatively high stability of tyrosine hydroxylase mRNA (half-life = 14 ± 1 h) in these cells.