Chronic Cocaine Administration Increases CNS Tyrosine Hydroxylase Enzyme Activity and mRNA Levels and Tryptophan Hydroxylase Enzyme Activity Levels

Cocaine is an inhibitor of dopamine and serotonin reuptake by synaptic terminals and has potent reinforcing effects that lead to its abuse. Tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH) catalyze the rate-limiting steps in dopamine and serotonin biosynthesis, respectively, and are the subject of dynamic regulatory mechanisms that could be sensitive to the actions of cocaine. This study assessed the effects of chronic cocaine on brain TH and TPH activities. Cocaine was administered (0.33 mg/infusion, i.v.) to rats for 7 days every 8 min for 6 h per day. This administration schedule is similar to patterns of self-administration by rats when given ad libitum access to this dose. This chronic, response-independent administration increased TH enzyme activity in the substantia nigra (30%) and ventral tegmental area (43%). Moreover, TH mRNA levels were also increased (45 and 50%, respectively). In contrast to the enzymatic and molecular biological changes in the cell bodies, TH activity was unchanged in the terminal fields (corpus striaturn and nucleus accumbens). Similarly, TPH activity was increased by 50% in the raphe nucleus (serotonergic cell bodies). In summary, the chronic response-independent administration of cocaine produces increases in the expression of TH mRNA and activity in both the cell bodies of motor (nigrostriatal) and reinforcement (mesolimbic) dopamine pathways. These increases are not manifested in the terminal fields of these pathways.     

Regulation of Tyrosine Hydroxylase Gene Expression in the Rat Carotid Body by Hypoxia

The activity (Vmax) of tyrosine hydroxylase (TH; EC 1.14.16.2), the rate limiting enzyme in the synthesis of catecholamines, is increased in carotid body, superior cervical ganglion, and the adrenal medulla during hypoxia (i.e., reduced PaO2). The present study was undertaken to determine if the increase in TH activity in these tissues during hypoxia is regulated at the level of TH mRNA. Adult rats were exposed to hypoxia (10% O2) or room air for periods lasting from 1 to 48 h. The carotid bodies, superior cervical ganglia, and adrenals were removed and processed for in situ hybridization using 35S-labeled oligonucleotide probes. The concentration of TH mRNA was increased by hypoxia at all time points in carotid body type I cells, but not in cells of either superior cervical ganglion or adrenal medulla. The increase in TH mRNA in carotid body during hypoxia did not require innervation of the carotid body or intact adrenal glands. In addition, hypercapnia, another physiological stimulus of carotid body activity, failed to induce an increase in TH mRNA in type I cells. Our findings suggest that hypoxia stimulates TH gene expression in the carotid body by a mechanism that is intrinsic to type I cells.     

Long-Term Induction of Tyrosine Hydroxylase Expression: Compensatory Response to Partial Degeneration of the Dopaminergic Nigrostriatal System in the Rat Brain

Abstract: The present study was undertaken to examine the adaptive changes occurring 1 and 6 months after moderate or severe unilateral 6-hydroxydopamine-induced lesions confined to the lateral part of the rat substantia nigra pars compacta (SNC). The expression of tyrosine hydroxylase (TH) enzyme was analyzed in the remaining dopaminergic nigral cell bodies and in the corresponding striatal nerve endings. In the cell bodies of the lesioned SNC, TH mRNA content was increased (+20 to +30%) 6 months after the lesion without changes in cellular TH protein amounts. The depletion of TH protein in the nerve terminal area was less severe than the percentage of cell loss observed in the SNC at 1- and 6-month postlesion intervals. Moreover, the decrease in TH protein in the ipsilateral striatum was less pronounced 6 months after lesion than 1 month after. That no corresponding change in TH protein content was observed in the cell bodies at a time when TH increased in nerve terminals suggests that the newly synthesized protein is probably rapidly transported to the striatal fibers. These results suggest the existence of a sequence of changes in TH expression between cell bodies and fibers, occurring spontaneously after partial denervation of the nigrostriatal pathway.     

Induction of Gene Expression of the Catecholamine-Synthesizing Enzymes by Insulin-Like Growth Factor-I

Abstract: The effects of insulin-like growth factor-I (IGF-I) on gene expression and the activities of the three enzymes specific for catecholamine biosynthesis, tyrosine hydroxylase (TH), dopamine β-hydroxylase (DBH), and phenylethanolamine N -methyltransferase (PNMT), were determined in bovine adrenomedullary chromaffin cells primary cultured in serum-free medium. The mRNA level of TH was maximally elevated in the presence of IGF-I by 3.1 ± 0.4-fold after 48 h, DBH by 5.1 ± 0.3-fold in 24 h, and PNMT by 2.8 ± 0.5-fold in 72 h. In addition, the activity of TH was increased by 77%, DBH by 70%, and PNMT by 23% in IGF-I-exposed cultures. In the absence of the growth factor, the mRNA levels of TH and DBH were decreased to 45 ± 10% and 35 ± 12% of the time-zero control within 48 h while PNMT mRNA was decreased to 82 ± 5% only after 72 h. When the cells were cotreated with the protein tyrosine kinase inhibitor genistein, DBH induction by IGF-I was suppressed, confirming that the effect is mediated by tyrosine kinase. Cotreatment with the protein kinase A (PKA) inhibitor H89 caused complete reversal of the IGF-I-induced DBH increase and the effects of IGF-I treatment and PKA activation by forskolin were not additive, suggesting that PKA is involved in the signaling initiated by IGF-I in these cells.     

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.     

The Roles of CRE, TRE, and TRE-Adjacent S1 Nuclease Sensitive Element in the Regulation of Tyrosine Hydroxylase Gene Promoter Activity by Angiotensin II

Abstract: The cis elements mediating activation of the tyrosine hydroxylase gene by angiotensin II were examined by transfecting tyrosine hydroxylase promoter-luciferase constructs into cultured bovine adrenal medullary cells. Angiotensin II-responsive elements are located within −54/+25-bp and −269/−55-bp promoter regions and were identified, respectively, as cyclic AMP (CRE)- and 12- O -tetradecanoylphorbol 13-acetate responsive element (TRE)-like sequences. Unlike CRE, TRE also supports basal promoter activity. Mutations of TRE or CRE that reduced angiotensin II stimulation abolished in vitro binding of nuclear proteins to those elements, suggesting that proteins forming CRE- and TRE-inducible complexes may mediate angiotensin II stimulation. The TRE is adjacent to a dyad symmetry element. Those two sites form a common regulatory unit in which the dyad symmetry element acts as a repressor of the TRE site. Isolated dyad symmetry element did not bind nuclear proteins in vitro. In supercoiled DNA it exhibited S1 nuclease sensitivity and was recognized by a DNA cruciform-specific antibody consistent with the extrusion of a cruciform structure that overlaps with the TRE. A mutation that abolished formation of the cruciform correlated with a loss of repressor activity. We propose a novel model of tyrosine hydroxylase gene regulation in which functions of the TRE are modulated via structural transition in the adjacent DNA.     

Short-term Effects of Endothelins on Tyrosine Hydroxylase Activity and Expression in the Olfactory Bulb of Normotensive Rats

The olfactory system in rats is part of the limbic region with extensive afferent connections with brain areas involved in the regulation of behaviour and autonomic responses. The existence of the endothelin system and catecholaminergic neurons in the olfactory bulb suggests that endothelins may modulate noradrenergic transmission and diverse olfactory mediated processes. In the present work we studied the effect of endothelin-1 and -3 on neuronal norepinephrine release and the short-term regulation of tyrosine hydroxylase in the olfactory bulb. Results showed that both endothelins increased tyrosine hydroxylase activity through the activation of a non-conventional endothelin G-protein coupled receptor, coupled to the stimulation of protein kinase A and C, as well as Ca²⁺/calmodulin-dependent protein kinase II. On the other hand, neither endothelin-1 nor endothelin-3 modified tyrosine hydroxylase total protein levels, but both peptides increased the phosphorylation of serine residues of the enzyme at sites 19 and 40. Furthermore, endothelins enhanced norepinephrine release in olfactory neurons suggesting that this event may contribute to increased tyrosine hydroxylase activity by reducing the feedback inhibition. Taken together present findings show a clear interaction between the endothelin system, and the catecholaminergic transmission in the olfactory bulb. Additional studies are required to evaluate the physiological functions regulated by endothelins at this brain level.     

Elevation of RNA Coding for Tyrosine Hydroxylase in Rat Adrenal Gland by Reserpine Treatment and Exposure to Cold

When rats are treated daily with reserpine or maintained at 4 degrees C, the level of a specific RNA coding for tyrosine hydroxylase is elevated in the adrenal gland. The increase in this specific RNA temporally precedes and is quantitatively equal to the increase in adrenal tyrosine hydroxylase enzyme activity elicited by these treatments. These results suggest that prolonged stress may lead to changes in the levels of specific RNA species in the adrenal gland.     

An HSV-1 Vector Containing the Rat Tyrosine Hydroxylase Promoter Enhances Both Long-Term and Cell Type-Specific Expression in the Midbrain

Abstract: A defective herpes simplex virus type one (HSV-1) vector that contains a 6.8-kb fragment of the rat tyrosine hydroxylase promoter (pTHlac-7kb) was examined for its capability to target catecholaminergic cell type-specific expression in the CNS. Cell type-specific expression was assessed by comparison with a control vector (pHSVlac) that uses the HSV-1 immediate early 4/5 promoter to support expression in multiple cell types. In initial experiments comparing expression in catecholaminergic and noncatecholaminergic cell lines, pTHlac-7kb supported a seven- to 20-fold increase in reporter gene expression in catecholaminergic cell lines. Four days after stereotactic injection into the midbrain of adult rats, pTHlac-7kb supported a 10-fold targeting of β-galactosidase expression to tyrosine hydroxylase-expressing neurons in the substantia nigra pars compacta compared with pHSVlac. Expression from pTHlac-7kb was stably maintained for 6 weeks with no significant changes in the pattern of expression. Long-term expression from pTHlac-7kb was confirmed by RNA and DNA analysis. In contrast, reporter gene expression in the midbrain from pHSVlac decreased ∼30-fold between 4 days and 6 weeks after gene transfer. Thus, within the context of this HSV-1 vector system, the tyrosine hydroxylase promoter enhanced cell type-specific expression and contributed to stable, long-term expression of a recombinant gene product in neurons. The capability to target recombinant gene expression to catecholaminergic neurons in specific brain areas may be useful for studies on the roles of these neurons in brain physiology and behavior.     

Chronic Electroconvulsive Seizures Increase the Expression of Serotonin2 Receptor mRNA in Rat Frontal Cortex

Abstract: The present study examines the influence of electroconvulsive seizure (ECS), as well as antidepressant drugs, on levels of serotonin₂ (5-HT₂) receptor mRNA in rat frontal cortex. Using a sensitive RNase protective assay, preliminary studies demonstrated the predicted regional distribution for the 5-HT₂ receptor mRNA: levels of 5-HT₂ mRNA were highest in frontal cortex (2.58 amol/μg of total RNA), intermediate in neostriatum, thalamus, and midbrain, and lowest in hippocampus, cerebellum, and choroid plexus. Chronic (10 or 14 days), but not acute (1 or 3 days), ECS treatment significantly increased levels of 5-HT₂ receptor mRNA. ECS treatment resulted in a similar time-dependent up-regulation of 5-HT₂ receptor ligand binding; chronic, but not acute, ECS treatment significantly increased levels of [³H]ketanserin ligand binding, confirming previous reports. Northern blot analysis demonstrated that 5-HT₂ receptor mRNA occurs as two bands (～5 and 6 kb in size), both of which were increased by chronic ECS treatment. The influence of antidepressant drug treatments on 5-HT₂ receptor mRNA was also examined. Chronic fluoxetine treatment increased levels of 5-HT₂ receptor mRNA, although levels of [³H]ketanserin ligand binding were not altered. In contrast, chronic administration of imipramine, mianserin, and tranylcypromine, treatments that decreased ligand binding, did not decrease levels of 5-HT₂ receptor mRNA. In fact, mianserin treatment caused a small, but significant, increase in levels of receptor mRNA. The results suggest that ECS up-regulation of 5-HT₂ receptor mRNA could underlie the increased density of 5-HT₂ receptor binding sites in response to this treatment, but that other mechanisms likely operate in the down-regulation of 5-HT₂ receptor ligand binding by antidepressant drug treatments.     

基因表达与mRNA结构的关系

基因的转录调控和转录后水平的调控在基因表达过程中起着重要作用。mRNA的结构与基因表达调控的关系非常密切。目前对于mRNA结构对表达的影响因素,主要集中于起始密码子和S-D序列的结构和间隔长度、基因和基因间的间隔区序列和长度,5’末端与3’末端非翻译区、多聚（A）尾、内含子序列对翻译起始效率、发夹结构对mRNA的稳定性的影响和mRNA翻译起始区等对基因表达影响。     

Effects of Electroconvulsive Shock on Tetrahydrobiopterin and GTP-Cyclohydrolase Activity in the Brain and Adrenal Gland of the Rat

The effects of a single and repeated electroconvulsive shock (ECS) (300 mA, 0.2 s) on tetrahydrobiopterin (BH4) levels and GTP-cyclohydrolase activity in the brain and adrenal glands of rats were examined. Twenty-four hours after the last ECS treatment (one/day for 7 days), biopterin levels were significantly elevated in the locus coeruleus, hippocampus, frontal cortex, hypothalamus, ventral tegmental area, and adrenal gland. There were no changes in biopterin levels after a single application of ECS. GTP-cyclohydrolase activity was significantly increased in the locus coeruleus, frontal cortex, hippocampus, hypothalamus, and adrenal gland 24 h after repeated ECS and remained elevated in certain tissues up to 8 days after the last treatment. Kinetic analysis of adrenal and locus coeruleus GTP-cyclohydrolase 1 day after 7 days of ECS showed significant changes in both Km and Vmax values. These data suggest that the long-term increases in BH4 levels and GTP-cyclohydrolase activity after repeated ECS may play a part in the mediation of the antidepressant effects of ECS.     

Induction of Adrenal Tyrosine Hydroxylase mRNA by Single Immobilization Stress Occurs Even After Splanchnic Transection and in the Presence of Cholinergic Antagonists

Abstract: Immobilization (IMO) stress elevates plasma catecholamines and increases tyrosine hydroxylase (TH) gene expression in rat adrenals. This study examined the mechanism(s) of IMO-induced changes in adrenal TH mRNA levels. Innervation of the adrenal medulla is predominantly cholinergic and splanchnicotomy as well as nicotinic receptor antagonists prevent the cold-induced rise in TH mRNA levels. In this study, the IMO-induced rise in plasma catecholamines, but not TH mRNA levels, was reduced by the antagonist chlorisondamine. Muscarinic antagonist atropine also did not prevent the IMO stress-elicited rise in TH mRNA. Furthermore, denervation of the adrenals by unilateral splanchnicotomy did not block the IMO-induced rise in TH mRNA but completely prevented the induction of neuropeptide Y mRNA. These results suggest that (1) the large increase in adrenal TH gene expression elicited by a single IMO stress is not regulated via cholinergic receptors or splanchnic innervation, and (2) there is a dissociation between regulatory mechanisms of catecholamine secretion and elevation of TH gene expression in the adrenal medulla of rats during IMO stress.