Tyrosine Hydroxylase RNA Research Articles

Abstract TP277: CD4 T Cells Increase Tyrosine Hydroxylase Expression With Aging and Stroke in Mice

Introduction: Activated T lymphocytes are present in the brain and play a detrimental role in aging and potentiate inflammation after ischemic injury. Previous studies have shown that T lymphocytes (T cells) synthesize and secrete catecholamines that modulate their activation. Tyrosine hydroxylase (TH), is a rate-limiting enzyme in catecholamine synthesis. Little is known about the role and contribution of TH synthesis specifically in T lymphocytes in aging and ischemic stroke injury. We hypothesized that aging will lead to increase TH + T cells in the brain that will have an activated phenotype and contribute to the progression of ischemic injury. Methods: Young (8-12 weeks) and old (22-24 months) C57BL/6 male mice were used. Spleen, bone marrow and brain were harvested and flow cytometry was used to immunophenotype the TH + T cells. Expression of TH on sorted CD4 + and CD8 + T cells was assessed by a real-time polymerase chain reaction. Splenic lymphocytes were stimulated with PMA/ionomycin and different T cell subsets were analyzed. The CD4 + T cells were purified from young and old mouse spleens and ex vivo stimulated to measure cytokine production. Finally, young and old mice underwent 60 minutes of middle cerebral artery occlusion (MCAo) and were euthanized 4 days later to determine the role of TH + T lymphocytes in stroke injury. Results: The percentage and MFI of TH was increased on CD4 + T cells with aging however, no change in TH percentage or MFI on CD8 + T cells was observed. TH RNA was increased in CD4 + T cells derived from old animals. The TH + CD4 + T cells were activated and had an effector memory phenotype in aging. On acute stimulation, old TH + CD4 + T cells increased the production of INF-γ, IL-4, and Il-17. After MCAo the percentage TH + CD4 + T cells increased in both young and old mice in the brain. The MFI IL-4/INF-γ ratio was higher in the young MCAo versus old MCAo reflecting a Th2 related response. Conclusion: TH + CD4 + T cells increase with age. These cells have an activated and effector memory phenotype and secrete inflammatory cytokines on acute stimulation. After MCAo, TH + CD4 + T cells demonstrated Th2 related protective responses in only in young mice.

Angiotensin II mediates the axonal trafficking of tyrosine hydroxylase and dopamine β-hydroxylase mRNAs and enhances norepinephrine synthesis in primary sympathetic neurons.

In the sympatho-adrenal system, angiotensin II (Ang II) acts as a key neuromodulatory component. At sympathetic nerve terminals, Ang II influences sympathetic transmission by enhancing norepinephrine (NE) synthesis, facilitating NE release and inhibiting NE uptake. Previously, it was demonstrated that tyrosine hydroxylase (TH) mRNA is trafficked to the distal axons of primary superior cervical ganglia (SCG) neurons, directed by a cis-acting regulatory element (i.e. zipcode) located in the 3'UTR of the transcript. Results of metabolic labeling studies established that the mRNA is locally translated. It was further shown that the axonal trafficking of the mRNA encoding the enzyme plays an important role in mediating dopamine (DA) and NE synthesis and may facilitate the maintenance of axonal catecholamine levels. In the present study, the hypothesis was tested that Ang II induces NE synthesis in rat primary SCG neurons via the modulation of the trafficking of the mRNAs encoding the catecholamine synthesizing enzymes TH and dopamine β-hydroxylase (DBH). Treatment of SCG neurons with the Ang II receptor type 1 (AT1R) agonist, L-162,313, increases the axonal levels of TH and DBH mRNA and protein and results in elevated NE levels. Conversely, treatment of rat SCG neurons with the AT1R antagonist, Eprosartan, abolished the L-162,313-mediated increase in axonal levels of TH and DBH mRNA and protein. In a first attempt to identify the proteins involved in the Ang II-mediated axonal transport of TH mRNA, we used a biotinylated 50-nucleotide TH RNA zipcode as bait in the affinity purification of TH zipcode-associated proteins. Mass spectrometric analysis of the TH zipcode ribonucleoprotein (RNP) complex immune-purified from SCG neurons led to the identification of 163 somal and 127 axonal proteins functionally involved in binding nucleic acids, the translational machinery or acting as subunits of cytoskeletal and motor proteins. Surprisingly, immune-purification of the TH axonal trafficking complex, results in the acquisition of DBH mRNA, suggesting that these mRNAs maybe transported to the axon together, possibly in the same RNP complex. Taken together, our results point to a novel mechanism by which Ang II participates in the regulation of axonal synthesis of NE by modulating the local trafficking and expression of TH and DBH, two key enzymes involved in the catecholamine biosynthetic pathway.

Role of Propolis on Tyrosine Hydroxylase Activity and Blood Pressure in Nitric Oxide Synthase-Inhibited Hypertensive Rats

Reduction in the synthesis or bioavailability of nitric oxide plays a significant role in the development of hypertension. Propolis is a resinous product collected by honeybees from various plant sources. Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the biosynthesis of catecholamines. The aim of this study was to examine the effect of propolis on blood pressure (BP), TH, and total RNA levels in the adrenal medulla, heart, and hypothalamus tissues in chronic nitric oxide synthase (NOS)-inhibited rats by Nw-nitro-l-arginine methyl ester (L-NAME). Rats received NOS inhibitor (L-NAME) for 15 days to produce hypertension and propolis for the last 5 days. TH activity and total RNA levels significantly increased in adrenal medulla, heart, and hypothalamus tissues in L-NAME-treated groups (P < .05). TH activity and total RNA levels of L-NAME+propolis-treated rats reduced (P < .05) compared with L-NAME-treated groups. TH activity in propolis-treated rats was reduced to the control values. L-NAME led to a significant increase in BP compared with the control group. Propolis administration to L-NAME-treated rats reduced BP but this was not statistically significant compared to L-NAME-treated groups. These results suggest that propolis decreases TH activity in NOS-inhibited hypertensive rats and thereby may modulate the synthesis of catecholamine and BP.

Chronic nicotine treatment leads to induction of tyrosine hydroxylase in locus ceruleus neurons: the role of transcriptional activation.

Chronic nicotine treatment (two daily subcutaneous injections administered approximately 12 h apart for 14 days) is associated with long-term inductions of tyrosine hydroxylase (TH) protein and TH mRNA in locus ceruleus (LC) neurons. These increases persist for at least 3 days after the final nicotine injection in LC cell bodies and for at least 7 to 10 days in LC nerve terminal regions. We tested whether this long-term response is due to sustained stimulation of TH gene transcription rate. A semiquantitative reverse transcription-polymerase chain reaction assay was developed to assess changes in the levels of TH RNA primary transcripts; these changes are an indirect measurement of changes in TH gene transcription rate. TH RNA primary transcript levels increase rapidly in the LC after a single nicotine administration and return to basal levels by 24 h. A similar rapid and transient induction of LC TH RNA primary transcripts occurs after chronic nicotine administration. In contrast, TH RNA primary transcript levels remain elevated for a sustained period of time (at least 1 day) in the adrenal medulla after chronic nicotine administration. Similar rapid, but transient changes in LC TH RNA primary transcript levels are observed after repeated immobilization stress. These results suggest that TH gene transcription rate in the LC is stimulated rapidly after each nicotine injection; however, in contrast to the adrenal medulla, there is no sustained transcriptional response elicited by chronic nicotine treatment or repeated immobilization stress in the LC, suggesting that post-transcriptional mechanisms may also play a role in these long-term responses.

Common Proteins Bind mRNAs Encoding Erythropoietin, Tyrosine Hydroxylase, and Vascular Endothelial Growth Factor

The hypoxia-inducible genes erythropoietin (Epo), tyrosine hydroxylase (TH), and vascular endothelial growth factor (VEGF) are regulated post-transcriptionally by proteins binding to specific regions located in the 3′ untranslated region (UTR) of their mRNAs. To determine whethertrans-factors binding to this region in all three of these RNAs are similar, we generated riboprobes containing the 3′ UTR of erythropoietin, tyrosine hydroxylase, and vascular endothelial growth factor mRNA and assayed them by electrophoretic mobility shift assay (EMSA) and UV cross-linking experiments. Each riboprobe formed similar shifted protein complexes using human hepatoma cell (Hep3B) cytoplasmic lysates in the EMSA. Hep3B proteins bound to each probe could be cross-competed by the specific unlabeled Epo, TH, or VEGF riboprobes. By contrast, a non-specific 3′ UTR riboprobe did not compete for binding with the Epo, TH, or VEGF RNA shifted protein complexes. UV cross-linking studies revealed proteins of similar molecular weights for the Epo, TH, and VEGF RNA shifted protein complexes. Taken together, these results suggest a common post-transcriptional regulatory mechanism for hypoxia-inducible genes.

Subcellular localization of tyrosine hydroxylase (TH) gene transcripts: New insights into the pattern of TH gene expression in the locus coeruleus under pharmacological stimulation

To document the subcellular compartmentalization of the tyrosine hydroxylase (TH) primary (heterogeneous nuclear, hnRNA) and processed mRNA transcripts in vivo, we have studied the expression of the TH gene in the locus coeruleus of the rat brain in basal conditions and under pharmacological stimulation by reserpine. Using exon-specific probes and electron microscopic in situ hybridization in reserpine-treated animals, we have demonstrated that the TH mRNA is localized both in the perikaryal cytoplasm (in association with domains of the endoplasmic reticulum) and in the nucleus (in large and smaller aggregates). By comparing hybridization patterns with intron- and exon-specific probes, we have shown that the two main foci of nuclear TH RNA labeling correspond to the Primary (hnRNA) transcript, while accessory secondary tracks or dots represent mature transcript devoid of introns. Finally, our data indicate that the pattern of expression of the TH gene is heterogeneous within the locus coeruleus neuron population. Under basal conditions, many neurons exhibit no detectable intro signal, although all locus coeruleus neurons express the TH gene. Other neurons display high intron labeling, and obviously express the gene at a much higher level. Upon reserpine stimulation, the number of neurons displaying detectable intro signals, as well as the intensity of the intron signal per neuron progressively increases during the first 24 h, suggesting that TH expressing neurons are progressively recruited for higher expression of this gene as the stimulation progresses.

Neonatal stress: effects of hypoglycemia and hypoxia on adrenal tyrosine hydroxylase gene expression.

Catecholamines (CA) are released from and resynthesized in the adrenal medulla in response to stress. In the mature animal, stimulus-secretion-synthesis coupling occurs through transsynaptic (neuronal) activity. In contrast, in the immature animal, before functional adrenal innervation, certain stressors (hypoglycemia and glycopenia) do not result in CA release. Additionally, it is not known whether release and biosynthesis remain coupled in the neonate as they are in the adult. Therefore, to evaluate whether neonatal stressors can induce CA biosynthesis at the genomic level "directly" before function adrenal innervation, we studied the expression of the tyrosine hydroxylase (TH) gene, the rate-limiting enzyme in CA biosynthesis. Newborn rat pups were made either hypoxic, hypoglycemic, or cellularly glycopenic (2-deoxyglucose). Neither hypoxic stress nor insulin-induced hypoglycemic stress altered steady state levels of TH mRNA in the neonate. However, cellular glycopenia resulted in a significant 2-fold rise in TH mRNA levels (p < 0.05). As expected, each of these stressors increased TH mRNA levels in the mature adult rat. Thus, neonatal hypoxia and hypoglycemia appear to require intact neurogenic impulse activity, whereas cellular glycopenia may "directly" induce TH RNA, perhaps through hormonal mechanisms. This developmental model allows for the analysis of mechanisms governing adrenal CA release separate from those governing biosynthesis at the level of TH RNA. Acute neonatal hypoxic stress results in adrenal CA release without increasing TH RNA. Intrauterine growth retardation from chronic prenatal hypoxemia results in neonatal CA depletion and decreased CA responsiveness. We speculate that chronic hypoxia alters CA pathways, increasing the susceptibility of these infants to later stressors.

Long term alteration in tyrosine hydroxylase mRNA levels in rat locus coeruleus after intraventricular injection of 5,6-dihydroxytryptamine

The time course variations in tyrosine hydroxylase (TH) activity and specific mRNA were measured in the rat locus coeruleus (LC) and substantia nigra after an intracerebroventricular (i.c.v.) injection of 5,6-dihydroxytryptamine (5,6-DHT), a neurotoxin known to selectively destroy serotoninergic neurons. In this study, the TH activity and TH mRNA were both analyzed from homogenates of single tissue samples (micropunches). TH mRNA was extracted and quantified by densitometry using a northern blot method and an artificial TH RNA as an external standard. 5,6-DHT injection led to a long-lasting increase in TH activity and TH mRNA in LC but not in substantia nigra. The elevation in LC was progressive and reached its maximum value (+75%) at day 4 and day 8 after 5,6-DHT. This effect on TH activity was accompanied by a parallel change in TH mRNA whose amplitude was +57%, +81% and +45% at day 2, 4, and 8 respectively after the neurotoxin injection. Return to normal values was observed at day 16. Variations in TH activity and TH mRNA in LC were of similar amplitude. These results suggest that serotonin could be a potent modulator of TH gene expression within noradrenergic LC neurons.

Interaction of cyclic AMP and cell-cell contact in the control of tyrosine hydroxylase RNA

The interaction between cell-cell contact and cyclic AMP-mediated control of the rat tyrosine hydroxylase (TH) gene was investigated in subclones of the PC12 rat pheochromocytoma cell line. Increasing cell culture density and elevation of intracellular cyclic AMP levels with forskolin both cause augmentation of TH RNA levels. However, the extent of increase in TH RNA following forskolin treatment is less in cultures grown at high density than those at low density, suggesting that there may be an interaction in the mechanism by which these two treatments modulate TH RNA levels. The role of cis-acting sequences in the TH gene in the induction of TH RNA by cyclic AMP and cell density was determined by the use of plasmid constructs containing the 5′-flanking sequences of the TH gene directing the transcription of the reporter gene, chloroamphenicol acetyltransferase (CAT). Using transient transfection assays in PC12 cells, we have mapped the site of cyclic AMP regulation of the TH gene to a region between −60 and −41. Stable transformants of PC12 cells which express p5TH CAT ( −773 +27 ) were isolated and the activity of CAT following treatment of cells with forskolin and growth at different cell densities was evaluated. CAT activity does not differ between cells grown at low or high density. Forskolin induces CAT activity 2–4 fold, but the extent of induction does not vary with changes in cell culture density. We conclude from these experiments that the intracellular mechanism by which increased cell-cell contact modulates TH RNA levels is not through interaction with the same genomic elements as those which regulate gene expression by cyclic AMP.

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

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

Identification and cell type specificity of the tyrosine hydroxylase gene promoter.

Genomic DNA encoding the rat tyrosine hydroxylase (TH) gene was isolated from a lambda phage library using a nick-translated fragment from a cDNA clone for rat TH. We have determined the initiation site for TH RNA synthesis and have sequenced 1100 bases of the primary transcript and 5' flanking region. The 5' end of the transcript is the same in several rat tissues in which TH is expressed as well as in rat pheochromocytoma cells (PC). RNA prepared from PC cells that had been stimulated with dexamethasone also mapped to the same transcription start site. Sequence upstream from the initiation site contains the canonical TATA box, but no apparent CAAT box. When a portion of the 5' flanking region of the TH gene (-773 to + 27) is fused to the chloramphenicol acetyltransferase (CAT) gene, it promotes expression of CAT in pheochromocytoma cells and GH4 cells, but not in two neural tumour lines, RT4-D and B103, nor in several non neural cell lines. This suggests that this region of the TH gene has features that confer tissue-restricted expression on the TH promoter.