Phillip W. Dickson

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 enzymes 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.

High prealbumin and transferrin mRNA levels in the choroid plexus of rat brain

Expression of plasma protein genes in various parts of the rat brain was studied by hybridizing radioactive cDNA to RNA in cytoplasmic extracts. No mRNA could be detected in brain for the beta subunit of fibrinogen, major acute phase alpha 1-protein, alpha 1-acid glycoprotein and albumin. However, per g tissue, the choroid plexus contained at least 100 times larger amounts of prealbumin mRNA than the liver and about the same amount of transferrin mRNA as liver. No prealbumin mRNA was found in other areas of the brain. The results obtained suggest very active synthesis of prealbumin in choroid plexus, which would be an important link in the transport of thyroid hormones from the blood to the brain via the cerebrospinal fluid.

Methylmercury neurotoxicity is associated with inhibition of the antioxidant enzyme glutathione peroxidase

In this study, we investigated the involvement of glutathione peroxidase-GPx in methylmercury (MeHg)-induced toxicity using three models: (a) in mouse brain after treatment with MeHg (40 mg/L in drinking water), (b) in mouse brain mitochondrial-enriched fractions isolated from MeHg-treated animals, and (c) in cultured human neuroblastoma SH-SY5Y cells. First, adult male Swiss mice exposed to MeHg for 21 days showed a significant decrease in GPx activity in the brain and an increase in poly(ADP-ribose) polymerase cleavage, an index of apoptosis. Second, in mitochondrial-enriched fractions isolated from MeHg-treated mice, there was a significant reduction in GPx activity and a concomitant decrease in mitochondrial activity and increases in ROS formation and lipid peroxidation. Incubation of mitochondrial-enriched fractions with mercaptosuccinic acid, a GPx inhibitor, significantly augmented the toxic effects of MeHg administered in vivo. Incubation of mitochondrial-enriched fractions with exogenous GPx completely blocked MeHg-induced mitochondrial lipid peroxidation. Third, SH-SY5Y cells treated for 24 h with MeHg showed a significant reduction in GPx activity. There was a concomitant significant decrease in cell viability and increase in apoptosis. Inhibition of GPx substantially enhanced MeHg toxicity in the SH-SY5Y cells. These results suggest that GPx is an important target for MeHg-induced neurotoxicity, presumably because this enzyme is essential for counteracting the pro-oxidative effects of MeHg both in vitro and in vivo.

The Acute Phase Response in the Rodenta

In the rodent, the general response to acute inflammation and tissue damage is characterized by a complex rearrangement in the pattern of concentrations of proteins in the plasma leading to an increase in the sedimentation rate of erythrocytes, an increase in leukocyte concentration in the bloodstream, and a decrease in the hematocrit. Body temperature changes only slightly or not at all. The reasons for the change in plasma concentrations of proteins are changes in their rates of synthesis in the liver. Degradation of plasma proteins is not affected. The details of the acute phase response evolved in the interaction of species with their environment. Therefore, it is not surprising to find differences in the details of the acute phase response among species. For example, alpha 2-macroglobulin is a strongly positive acute phase reactant in the rat, but not in the mouse; C-reactive protein is a strongly positive acute phase protein in the mouse, but is not found in the rat. An inducible acute phase cysteine proteinase inhibitor system, which has evolved from a primordial kininogen gene, has been observed so far only in the rat. The changes in the synthesis rates of acute phase proteins during inflammation are closely reflected by corresponding changes in intracellular mRNA levels. In the liver, the capacity to induce the acute phase pattern of synthesis and secretion of plasma proteins probably develops around birth. Changes in mRNA levels are brought about by changes in transcription rates or by changes in mRNA stability. Kinetics of mRNA changes during the acute phase response differ for individual proteins. The main signal compound for eliciting the acute phase response in liver seems to be interleukin-6/interferon-beta 2/hepatocyte stimulating factor, whereas interleukin-1 leads to typical acute phase changes in mRNA levels only for alpha 1-acid glycoprotein, albumin, and transthyretin. Plasma protein genes are expressed in various extrahepatic tissues, such as the choroid plexus, the yolk sac, the placenta, the seminal vesicles, and other sites. All these tissues are involved in maintaining protein homeostasis in associated extracellular compartments by synthesis and secretion of proteins. Synthesis and secretion of plasma proteins in paracompartmental organs other than the liver is not influenced by the acute phase stimuli.

Differential regulation of the human tyrosine hydroxylase isoforms via hierarchical phosphorylation

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the biosynthesis of the catecholamines dopamine, noradrenaline, and adrenaline. In response to short term stimuli TH activity is primarily controlled by phosphorylation of serine 40. We have previously shown that phosphorylation of serine 19 in TH can indirectly activate TH via a hierarchical mechanism by increasing the rate of phosphorylation of serine 40. Here we show that phosphorylation of serine 31 in rat TH increases the rate of serine 40 phosphorylation 9-fold in vitro. Phosphorylation of serine 31 in intact bovine chromaffin cells potentiated the forskolin-induced increase in serine 40 phosphorylation and TH activity more than 2-fold. Humans are unique in that they contain four TH isoforms but to date no significant differences have been shown in the regulation of these isoforms. Phosphorylation of the human TH isoform 1 at serine 31 by extracellular signal-regulated protein kinase (ERK) also produced a 9-fold increase in the rate of phosphorylation of serine 40, whereas little effect was seen in the TH isoforms 3 and 4. ERK did not phosphorylate human TH isoform 2. The effect of serine 19 phosphorylation on serine 40 (44 in TH2) phosphorylation is stronger in TH2 than in TH1. Thus hierarchical phosphorylation provides a mechanism whereby the two major human TH isoforms (1 and 2) can be differentially regulated with only isoform 1 responding to the ERK pathway, whereas isoform 2 is more sensitive to calcium-mediated events.

Sustained phosphorylation of tyrosine hydroxylase at serine 40: a novel mechanism for maintenance of catecholamine synthesis

Tyrosine hydroxylase (TH) is the rate‐limiting enzyme in catecholamine synthesis. Its activity is known to be controlled acutely (minutes) by phosphorylation and chronically (days) by protein synthesis. Using bovine adrenal chromaffin cells we found that nicotine, acting via nicotinic receptors, sustained the phosphorylation of TH at Ser40 for up to 48 h. Nicotine also induced sustained activation of TH, which for the first 24 h was completely independent of TH protein synthesis, and the phosphorylation of TH at Ser31. Imipramine did not inhibit the acute phosphorylation of TH at Ser40 or TH activation induced by nicotine, but did inhibit the sustained responses to nicotine seen at 24 h. The protein kinase(s) responsible for TH phosphorylation at Ser40 switched from being protein kinase C (PKC) independent in the acute phase to PKC dependent in the sustained phase. Sustained phosphorylation and activation of TH were also observed with histamine and angiotensin II. Sustained phosphorylation of TH at Ser40 provides a novel mechanism for increasing TH activity and this leads to increased catecholamine synthesis. Sustained phosphorylation of TH may be a selective target for drugs or pathology in neurons that contain TH and synthesize dopamine, noradrenaline or adrenaline.

Synthesis of Transthyretin (Pre-albumin) mRNA in Choroid Plexus Epithelial Cells, Localized by In Situ Hybridization in Rat Brain

The sites of synthesis of transthyretin in the brain were investigated using in situ hybridization with [35S]-labeled recombinant cDNA probes specific for transthyretin mRNA. Autoradiography of hybridized coronal sections of rat brain revealed specific cellular localization of transthyretin mRNA in choroid plexus epithelial cells of the lateral, third, and fourth ventricles. Transferrin mRNA was also investigated and, in contrast to transthyretin mRNA, was localized mainly in the lateral ventricles. Our results indicate that substantial synthesis of transthyretin and transferrin mRNA may occur in the choroid plexus.

Phosphorylation of Ser19 increases both Ser40 phosphorylation and enzyme activity of tyrosine hydroxylase in intact cells

We have previously shown that the phosphorylation of Ser19 in tyrosine hydroxylase can increase the rate of phosphorylation of Ser40 in tyrosine hydroxylase threefold in vitro. In this report we investigated the role of Ser19 on Ser40 phosphorylation in intact cells. Treatment of bovine chromaffin cells with anisomycin produced a twofold increase in Ser19 phosphorylation with no increase in Ser31 phosphorylation and only a small increase in Ser40 phosphorylation. Treatment of bovine chromaffin cells with forskolin produced a fourfold increase in Ser40 phosphorylation but no significant increase in either Ser19 or Ser31 phosphorylation. When chromaffin cells were first treated with anisomycin, the level of Ser40 phosphorylation after treatment by forskolin was 76% greater than the level of Ser40 phosphorylation in cells treated with forskolin alone. This potentiation of Ser40 phosphorylation by anisomycin could be completely blocked by the p38 MAP (mitogen‐activated protein) kinase inhibitor SB 203580. The potentiation of Ser40 phosphorylation by anisomycin was not due to an increase in Ser40 kinase activity. Anisomycin treatment of chromaffin cells potentiated the forskolin‐induced increase in tyrosine hydroxylase activity by 50%. This potentiation of activity was also blocked by SB 203580. These data provide the first evidence that the phosphorylation of Ser19 can potentiate the phosphorylation of Ser40 and subsequent activation of tyrosine hydroxylase in intact cells.

High levels of messenger RNA for transthyretin (prealbumin) in human choroid plexus

We have investigated the expression of the gene for transthyretin (prealbumin) in the human choroid plexus. RNA was isolated from the human choroid plexus, fractionated by electrophoresis in agarose gel and transferred onto a nitrocellulose filter membrane. Transthyretin messenger RNA (mRNA) was identified by hybridization to radioactive complementary DNA for rat transthyretin. The level of transthyretin mRNA in the human choroid plexus was found to be at least 40 times higher than in human liver, suggesting very active synthesis of transthyretin in the choroid plexus.

Levels of messenger ribonucleic acids for plasma proteins in rat liver during acute experimental inflammation

The levels of mRNA for plasma proteins and for metallothionein in rat liver during the acute-phase response were studied by hybridization to specific cDNA probes. The mRNA forα2-macroglobulin, theβ-chain of fibrinogen, α1,-acid glycoprotein (so-called acute-phase reactants) reached a maximum level between 18 and 36 h after inducing an acute inflammation. The level of mRNA for metallothionein-I peaked earlier, after 12 h. The mRNA for transferrin showed a delayed increase with a broad maximum for its relative level after 36–60 h. The mRNA levels for albumin and α2u-globulin (so-called negative acute-phase reactants) decreased, reaching a minimum of 25 % of the normal level after 36 h (albumin) and after 72 h (α2u-globulin). The ratios of the rates of incorporation of leucine into the proteins over the levels of their mRNA in liver changed only a little, indicating that the rates of synthesis of plasma proteins in the liver are regulated at the mRNA level during the acute-phase response to inflammation.