Manganese induces sustained Ser40 phosphorylation and activation of tyrosine hydroxylase in PC12 cells

Manganese (Mn²⁺) is an essential metal involved in normal functioning of a range of physiological processes. However, occupational overexposure to Mn²⁺ causes neurotoxicity. The dopaminergic system is a particular target for Mn²⁺ neurotoxicity. Tyrosine hydroxylase (TH) is the rate limiting enzyme for dopamine synthesis and is regulated acutely by phosphorylation at Ser40 and chronically by protein synthesis. In this study we used pheochromocytoma 12 cells to investigate the effects of Mn²⁺ exposure on the phosphorylation and activity of TH. Mn²⁺ treatment for 24 h caused a sustained increase in Ser40 phosphorylation and TH activity at a concentration of 100 μM, without altering the level of TH protein or PC12 cell viability. Inhibition of protein kinase A and protein kinase C and protein kinases known to be involved in sustained phosphorylation of TH in response to other stimuli did not block the effects of Mn²⁺ on Ser40 phosphorylation. A substantial increase in H₂O₂ production occurred in response to 100 μM Mn²⁺. The antioxidant Trolox^TM completely inhibited H₂O₂ production but did not block TH phosphorylation at Ser40, indicating that oxidative stress was not involved. Sustained TH phosphorylation at Ser40 and the consequent activation of TH both occurred at low concentrations of Mn²⁺ and this provides a potential new mechanism for Mn²⁺-induced neuronal action that does not involve H₂O₂-mediated cell death.     

Angiotensin-(1–7) through AT2 receptors mediates tyrosine hydroxylase degradation via the ubiquitin–proteasome pathway

Hypothalamic norepinephrine (NE) release regulates arterial pressure by altering sympathetic nervous system activity. Because angiotensin (Ang) (1–7) decreases hypothalamic NE release and this effect may be correlated with a diminished NE synthesis, we hypothesize that Ang-(1–7) down-regulates tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamines biosynthesis. We investigated the effect of Ang-(1–7) on centrally TH activity and expression. TH activity was evaluated by the release of tritiated water from ³H- l -tyrosine. TH expression and phosphorylation were determined by western blot. Hypothalami from normotensive or spontaneously hypertensive rats pre-incubated with Ang-(1–7) showed a significant decrease in TH specific activity. Ang-(1–7) caused a decrease in TH phosphorylation at Ser19 and Ser40 residues. The heptapeptide induced a decrease in TH expression that was blocked by an AT₂ receptor antagonist and not by an AT₁ or Mas receptor antagonist, suggesting the involvement of AT₂ receptors. The proteasome inhibitor MG132 blocked the Ang-(1–7)-mediated TH reduction. In addition, Ang-(1–7) increased the amount of TH–ubiquitin complexes, indicating that the Ang-(1–7)-mediated TH degradation involves ubiquitin conjugation prior to proteasome degradation. We conclude that Ang-(1–7) down-regulates TH activity and expression centrally leading to a decrease in the central NE system activity.     

Evidence for Protein Kinase C Involvement in the Short-Term Activation by Prolactin of Tyrosine Hydroxylase in Tuberoinfundibular Dopaminergic Neurons

Abstract: The mechanism of the short-term activation by prolactin (PRL) of tyrosine hydroxylase (TH) in tuberoinfundibular dopaminergic neurons was examined in vitro on hypothalamic slices from ovariectomized rats. TH activity (determined by 3,4-dihydroxyphenylalanine accumulation in the median eminence after blockade of decarboxylase with NSD 1055) showed a dose-dependent increase within 2 h of incubation of the hypothalamic slices with PRL. To determine whether a phosphorylation process was involved in this increase in TH activity, we studied the sensitivity of the enzyme to dopamine (DA) feedback inhibition. In control median eminences, two kinetically different forms of TH coexisted, one exhibiting a K _1(DA) value of 29.92 ± 0.49 μ M , the other being × 15-fold more sensitive to DA inhibition with a K _1(DA) of 1.96 ± 0.09 μ M , likely corresponding to a phosphorylated and active form and to a nonphosphorylated and less active form, respectively. After PRL treatment, the TH form of low K _1(DA) remained unaffected, whereas the K _1(DA) of the purported active form of TH increased to 62.6 ± 0.8 μ M , suggesting an increase in the enzyme phosphorylation. This increase in the K _I(DA) of TH was selectively prevented by GF 109203X, a potent and selective inhibitor of protein kinase C, but not by a specific inhibitor of protein kinase A or calmodulin. Finally, this action of PRL could be mimicked by 12- O -tetradecan-oylphorbol 13-acetate (a direct activator of protein kinase C). These results suggest that PRL, at the median eminence level, activates TH by increasing the enzyme phosphorylation and that this action may involve an activation of protein kinase C.     

Tyrosine Hydroxylase from Bovine Striatum: Catalytic Properties of the Phosphorylated and Nonphosphorylated Forms of the Purified Enzyme

Abstract: The properties of purified tyrosine hydroxylase (TH) from bovine corpus striatum, both native and phosphorylated forms of the enzyme, were studied. TH had a tendency toward greater affinity for tetrahydrobiopterin (BH₄) than for the synthetic cofactor 6-methyltetrahydropterin (6-MPH₄), although the maximal velocity of the TH-catalyzed reaction was greater with 6-MPH4. Phosphorylation increased the affinity of TH for cofactor at pH 6.0, with little change in V _max. At pH 7.0, phosphorylation caused increased activation of TH by increasing V _max as well as reducing the K _m for cofactor. The K _m for dopamine was increased twofold by phosphorylation at pH 6.0, but eightfold at pH 7.0. Phosphorylation was not associated with a change in K _m for tyrosine at any pH or with any cofactor studied, although the K _m for tyrosine of TH was cofactor-dependent and seven to eight times greater with 6-MPH₄ than with BH₄ as cofactor. Heparin and NaCl activated native TH at pH 6.0, but not at pH 7.0. Phosphorylated TH was unaffected by heparin or salt at pH 6.0, but was relatively inhibited at pH 7.0. The data are presented in the context of the physiological environment of TH.     

Short-Term Inhibitory Effect of Estradiol on Tyrosine Hydroxylase Activity in Tuberoinfundibular Dopaminergic Neurons In Vitro

Abstract: The short-term inhibition by estradiol of tyrosine hydroxylase (TH) in tuberoinfundibular dopaminergic neurons was examined in vitro on hypothalamic slices from ovariectomized rats. TH activity (determined by L-3,4-di-hydroxyphenylalanine accumulation in the median eminence after blockade of decarboxylase with NSD 1055) showed a 30–40% decrease within 1 h of incubation with estradiol. To determine whether a dephosphorylation process was involved in this decline in TH activity, we studied the sensitivity of the enzyme to dopamine (DA) feedback inhibition: In controls, we observed that two kinetically different forms of TH coexisted, with one exhibiting a K_l(DA) of 26.4 ± 2 μM the other being ∼ 10-fold more sensitive to DA inhibition, with a [k_1{DA)] of 2.56 ± 0.17 μM. likely corresponding to a phosphorylated and active form and to a non-phosphorylated and poorly active form, respectively. Conversely. after estradiol treatment all TH molecules exhibited the same K_1(DA) of 2.5 ± 0.3 μM. This effect was stereospecific, because 17α-estradiol could not promote it. whereas with 17β-estradiol. it could be observed at only 10⁻¹¹M and after a short delay (30 min). Finally, this decrease in the K_1(DA) of the purported active form of TH could be prevented by okadaic acid (an inhibitor of protein phosphatases). These results suggest that estradiol can act directly on the mediobasal hypothalamus to trigger a rapid decline in TH activity and that this action may involve a decrease in TH phosphorylation.     

Tyrosine Hydroxylase Phosphorylation in Digitonin-Permeabilized Bovine Adrenal Chromaffin Cells: The Effect of Protein Kinase and Phosphatase Inhibitors on Ser19 and Ser40 Phosphorylation

Abstract: The protein kinases and protein phosphatases that act on tyrosine hydroxylase in vivo have not been established. Bovine adrenal chromaffin cells were permeabilized with digitonin and incubated with [γ-³²P]ATP, in the presence or absence of 10 µ M Ca²⁺, 1 µ M cyclic AMP, 1 µ M phorbol dibutyrate, or various kinase or phosphatase inhibitors. Ca²⁺ increased the phosphorylation of Ser¹⁹ and Ser⁴⁰. Cyclic AMP, and phorbol dibutyrate in the presence of Ca²⁺, increased the phosphorylation of only Ser⁴⁰. Ser³¹ and Ser⁸ were not phosphorylated. The Ca²⁺-stimulated phosphorylation of Ser¹⁹ was incompletely reduced by inhibitors of calcium/calmodulin-stimulated protein kinase II (46% with KN93 and 68% with CaM-PKII 273–302), suggesting that another protein kinase(s) was contributing to the phosphorylation of this site. The Ca²⁺-stimulated phosphorylation of Ser⁴⁰ was reduced by specific inhibitors of protein kinase A (56% with H89 and 38% with PKAi 5–22 amide) and protein kinase C (70% with Ro 31-8220 and 54% with PKCi 19–31), suggesting that protein kinases A and C contributed to most of the phosphorylation of this site. Results with okadaic acid and microcystin suggested that Ser¹⁹ and Ser⁴⁰ were dephosphorylated by PP2A.     

Chronic Ethanol Reduces Immunologically Detectable Gqα/11α in NG108–15 Cells

Abstract: The amyloid protein (βA₄) is found in the CNS of patients with Alzheimer's disease; however, the pathogenic role of this protein is not known. In the present study, a peptide fragment of βA₄βA₄ 25–35; Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-NH₂), which contains the conserved C-terminal sequence of substance P (X-Gly-Leu-Met-NH₂), and the neuropeptide substance P (SP) were examined for their ability to modulate nicotine-evoked secretion from cultured bovine adrenal chromaffin cells. Secretion of the released endogenous catecholamines was monitored by electrochemical detection after separation by HPLC. Secretion induced by 10⁻⁵ M nicotine was inhibited by SP and βA₄ 25–35. The IC₅₀ of SP and βA₄ 25–35 was 3 × 10⁻⁶ and 3 × 10⁻⁵ M , respectively. SP and βA₄ 25–35 both protected against nicotinic receptor desensitization. However, βA₄ 25–35 was ∼ 10-fold less effective than SP in its protective effect. The present work shows that βA₄ 25–35 can mimic the modulatory actions of SP on the nicotinic response of cultured bovine chromaffin cells, i.e., inhibition of the nicotinic response and protection against nicotinic desensitization. These modulatory actions may be associated with changes in nicotinic receptor levels reported to occur in Alzheimer's disease.     

cDNAs induced by ozone from Atriplex canescens (saltbush) and their response to sulfur dioxide and water-deficit

Ozone effects on plant water relations have been reported to be similar to those of water-deficit. The objective was to identify ozone-inducible (OI) clones from Atriplex canescens (saltbush) and determine if they were also responsive to water-deficit as well as SO₂. cDNA clones derived from four different polyA RNAs which accumulate in 8-month-old shrub leaves exposed to ozone (0.2 μl I⁻¹, 6 h day⁻¹, 7 days) were isolated by differential screening, analyzed by northern blots, sequenced, and gene product homologies with other plant genes were determined. Clone OI12A-3 has homology with wound-inducible proteinase inhibitors, whereas clone OI8–3 protein is homologous to thiol proteases. Clones OI2–2 and OI14–3 putatively code for glycine-rich proteins with repeated motifs (Gly-Gly-Gly-Tyr-Gly-His)_n and putative cell-wall-targeting signal peptides. Clone OI2–2 and particularly clone OI14–3 were also induced by both SO₂ and water-deficit. These data indicate that woody plant genes associated with cell wall protein production and whose expression is induced by several stress factors may be responding to common oxidative stress pathways.     

Evidence for the Existence of Differential O-Glycosylated α5-Subunits of the γ-Aminobutyric AcidA Receptor in the Rat Brain

Abstract: Polyclonal antibodies were raised to synthetic peptides having amino acid sequences corresponding with the N- or C-terminal part of the γ-aminobutyric acid_A (GABA_A) receptor α₅-subunit. These anti-peptide α₅(2–10) or anti-peptide α₅(427–433) antibodies reacted specifically with GABA_A receptors purified from the brains of 5–10-day-old rats in an enzyme-linked immunosorbent assay and were able to dose-dependently immunoprecipitate up to 6.3 or 13.1% of the GABA_A receptors present in the incubation, respectively. In immunoblots, each of these antibodies reacted with the same two protein bands with apparent molecular mass of 53 or 57 kDa. After exhaustive treatment of purified GABA_A receptors with N -Glycanase, each of these antibodies identified two proteins with apparent molecular masses of 46 and 48 kDa. Additional treatment of GABA_A receptors with neuraminidase and O -Glycanase resulted in an apparently single protein with molecular mass of 47 kDa, which again was identified by both the anti-peptide α₅(2–10) and the anti-peptide α₅(427–433) antibody. These results indicate the existence of at least two different α₅-sub-units of the GABA_A receptor that differ in their carbohydrate content. In contrast to other α- or β-subunits of GABA_A receptors so far investigated, at least one of these two α₅-subunits contains O-linked carbohydrates.     

Myelin P0 Glycoprotein: Identification of the Site Phosphorylated In Vitro and In Vivo by Endogenous Protein Kinases

Abstract: Myelin membrane prepared from mouse sciatic nerve possesses both kinase and substrates to incorporate [³²P]PO₄³⁻ from [γ-³²P]ATP into protein constituents. Among these, P₀ glycoprotein is the major phosphorylated species. To identify the phosphorylated sites, P₀ protein was in vitro phosphorylated, purified, and cleaved by CNBr. Two ³²P-phosphopeptides were isolated by HPLC. The exact localization of the sequences around the phosphorylated sites was determined. The comparison with rat P₀ sequence revealed, besides a Lys¹⁷² to Arg substitution, that in the first peptide, two serine residues (Ser¹⁷⁶ and Ser¹⁸¹) were phosphorylated, Ser¹⁷⁶ appearing to be modified subsequently to Ser¹⁸¹. In the second peptide, Ser¹⁹⁷, Ser¹⁹⁹, and Ser²⁰⁴ were phosphorylated. All these serines are clustered in the C-terminal region of P₀ protein. This in vitro study served as the basis for the identification of the in vivo phosphorylation sites of the C terminal region of P₀. We found that, in vivo, Ser¹⁸¹ and Ser¹⁷⁶ are not phosphorylated, whereas Ser¹⁹⁷, Ser¹⁹⁹, Ser²⁰⁴, Ser²⁰⁸, and Ser²¹⁴ are modified to various extents. Our results strongly suggest that the phosphorylation of these serine residues alters the secondary structure of this domain. Such a structural perturbation could play an important role in myelin compaction at the dense line level.     

Brain β-Spectrin Phosphorylation: Phosphate Analysis and Identification of Threonine-347 as a Heparin-Sensitive Protein Kinase Phosphorylation Site

Abstract: Phosphorylation of brain spectrin was studied by a combination of in vivo and in vitro approaches. Chemical analysis of phosphate groups on electrophoretically purified mouse brain β-spectrin yielded a stoichiometry of 3.2 ± 0.18 mol of PO₄/mol of β-spectrin. The spectrin isolated by chromatographic methods from mouse brain, pig brain, and human erythrocytes yielded 4.1, 5.6, and 3.2 mol of PO₄/mol of spectrin heterodimer, respectively. The ³²P labeling of spectrin in retinal ganglion cell neurons or NB 2a/d1 neuroblastoma cells with [³²P]orthophosphate showed phosphorylation of only β-spectrin in vivo. Two-dimensional phosphopeptide map analyses showed that most of the in vivo sites on β-spectrin were phosphorylated by either a heparin-sensitive endogenous cytoskeleton-associated protein kinase or protein kinase A. Phosphoamino acid analysis of in vivo and in vitro phosphorylated β-spectrin showed that [³²P]phosphate groups were incorporated into both serine (>90%) and threonine residues. In vitro, phosphate groups were incorporated into threonine residues by the heparin-sensitive endogenous protein kinase. The amino acid sequence VQQQLQAFNTY of an α-chymotryptic ³²P-labeled peptide phosphorylated by the heparin-sensitive cytoskeleton-associated endogenous protein kinase corresponded to amino acid residues 338–348 on the β1 repeat of β-spectrin_G (βSPIIa) gene. These data suggest that phosphorylation of Thr³⁴⁷, which is localized on the presumptive synapsin I binding domain of β-spectrin_G, may play a role in synaptic function by regulating the binding of spectrin to synaptic vesicles.     

β-Amyloid-Induced Neurotoxicity of a Hybrid Septal Cell Line Associated with Increased Tau Phosphorylation and Expression of β-Amyloid Precursor Protein

Abstract: Recent evidence suggests that β-amyloid peptide (β-AP) may induce tau protein phosphorylation, resulting in loss of microtubule binding capacity and formation of paired helical filaments. The mechanism by which β-AP increases tau phosphorylation, however, is unclear. Using a hybrid septal cell line, SN56, we demonstrate that aggregated β-AP_1–40 treatment caused cell injury. Accompanying the cell injury, the levels of phosphorylated tau as well as total tau were enhanced as detected immunochemically by AT8, PHF-1, Tau-1, and Tau-5 antibodies. Alkaline phosphatase treatment abolished AT8 and PHF-1 immunoreactivity, confirming that the tau phosphorylation sites were at least at Ser^199/202 and Ser³⁹⁶. In association with the increase in tau phosphorylation, the immunoreactivity of cell-associated and secreted β-amyloid precursor protein (β-APP) was markedly elevated. Application of antisense oligonucleotide to β-APP reduced expression of β-APP and immunoreactivity of phosphorylated tau. Control peptide β-AP_1–28 did not produce significant effects on tau phosphorylation, although it slightly increased cell-associated β-APP. These results suggest that βAP_1–40-induced tau phosphorylation may be associated with increased β-APP expression in degenerated neurons.     

Tyrosine hydroxylase activity is regulated by two distinct dopamine-binding sites

Tyrosine hydroxylase (TH), the rate-limiting enzyme in the biosynthesis of the catecholamines dopamine, noradrenaline and adrenaline, is regulated acutely by feedback inhibition by the catecholamines and relief of this inhibition by phosphorylation of serine 40 (Ser40). Phosphorylation of serine 40 abolishes the binding of dopamine to a high affinity ( K _D < 4 nM) site on TH, thereby increasing the activity of the enzyme. We have found that TH also contains a second low affinity ( K _D = 90 nM) dopamine-binding site, which is present in both the non-phosphorylated and the Ser40-phosphorylated forms of the enzyme. Binding of dopamine to the high-affinity site decreases V _max and increases the K _m for the cofactor tetrahydrobiopterin, while binding of dopamine to the low-affinity site regulates TH activity by increasing the K _m for tetrahydrobiopterin. Kinetic analysis indicates that both sites are present in each of the four human TH isoforms. Dissociation of dopamine from the low-affinity site increases TH activity 12-fold for the non-phosphorylated enzyme and 9-fold for the Ser40-phosphorylated enzyme. The low-affinity dopamine-binding site has the potential to be the primary mechanism responsible for the regulation of catecholamine synthesis under most conditions.     

Monoclonal Antibodies to the Human γ2 Subunit of the GABAA/Benzodiazepine Receptors

Abstract: The large intracellular loop (IL) of the γ₂ subunit of the cloned human γ-aminobutyric acid_A (GABA_A) receptor (γ₂IL) was expressed in bacteria as glutathione- S -transferase and staphylococcal protein A fusion proteins. Mice were immunized with the fusion proteins (one protein per animal), and monoclonal antibodies were obtained. Six monoclonal antibodies reacted with the γ₂IL moiety of the fusion proteins. Three of these monoclonal antibodies also immunoprecipitated a high proportion of the GABA_A/benzodiazepine receptors from bovine and rat brain and reacted with a wide 44,000–49,000-M_r peptide band in immunoblots of affinity-purified GABA_A receptors. These monoclonal antibodies are valuable reagents for the molecular characterization of the GABA_A receptors in various brain regions.     

Presence of Pituitary Adenylate Cyclase-Activating Polypeptide Receptors in Y-79 Human Retinoblastoma Cells

Abstract: Cytochemical analysis demonstrated that a high percentage of human Y-79 retinoblastoma cells displayed a specific labeling by the biotinyl derivative of pituitary adenylate cyclase-activating polypeptide (PACAP), a novel neuropeptide of the secretin-vasoactive intestinal peptide (VIP) family of peptides. In cell membranes, the two molecular forms of PACAP, the one with 38 (PACAP 38) and the other with 27 (PACAP 27) amino acids, displaced the binding of ¹²⁵I-PACAP 27 with IC₅₀ values in the picomolar range and increased adenylyl cyclase activity by 100-fold with EC₅₀ values of 27 and 180 p M , respectively. VIP, human peptide histidine-isoleucine, glucagon, and secretin were much less effective and potent in both receptor assays. The PACAP receptor antagonists PACAP 6–27 and PACAP 6–38 and an antiserum directed against the stimulatory G protein G_s inhibited the PACAP stimulation of adenylyl cyclase. In intact cells, both PACAPs and VIP failed to stimulate the phosphoinositide hydrolysis, whereas in cell membranes PACAP 38, but not the other peptides, produced a modest increase (40%) of inositol phosphate formation with an EC₅₀ value of 22 n M . However, this effect was not antagonized by either PACAP 6–38 or PACAP 6–27. These data demonstrate the presence in human Y-79 retinoblastoma cells of specific PACAP receptors and provide further evidence that PACAP may act as a neurotransmitter/neuromodulator in mammalian retina.     

Pharmacological Characterization of Tachykinin Receptors Controlling Acetylcholine Release from Rat Striatum: An In Vivo Microdialysis Study

Abstract: The regulation of striatal cholinergic function by tachykinins was examined in urethane-anesthetized rats by using microdialysis. Substance P (0.01–1 µ M ), [Sar⁹,Met(O₂)¹¹]substance P (1–10 µ M ), septide (0.1–3 µ M ), neurokinin (NK) A (0.1–10 µ M ), and senktide (0.1–10 µ M ) produced concentration-dependent increases in striatal acetylcholine (ACh) release. Septide was the most potent agonist for inducing release of ACh, whereas the stimulating effect of senktide was less pronounced and more progressive in onset. The response to septide was prevented by intraperitoneal administration of the nonpeptide NK₁ antagonist SR 140333 (1–3 mg/kg) but not by the nonpeptide NK₂ receptor antagonist SR 48968, indicating that the effect was mediated specifically by NK₁ receptors. ACh release caused by NKA was reduced by SR 48968 (1–3 mg/kg) and slightly affected by SR 140333, indicating a principal role for NK₂ receptors in the peptide response. The similar efficacy of SR 140333 and SR 48968 in blocking substance P-induced ACh release suggested that the effect of this peptide involves the stimulation of both NK₁ and NK₂ receptors. Finally, our results indicate that the increase in striatal ACh release induced by the D₁ agonist (+)-SKF-38393 (3 µ M ) may be mediated indirectly through local release of NKA or substance P acting at NK₂ receptors.