Tissue-specific and high-level expression of the human tyrosine hydroxylase gene in transgenic mice.

Transgenic mice carrying multiple copies of the human tyrosine hydroxylase (TH) gene have been produced. The transgenes were transcribed correctly and expressed specifically in brain and adrenal gland. The level of human TH mRNA in brain was about 50-fold higher than that of endogenous mouse TH mRNA. In situ hybridization demonstrated an enormous region-specific expression of the transgene in substantia nigra and ventral tegmental area. TH immunoreactivity in these regions, though not comparable to the increment of the mRNA, was definitely increased in transgenic mice. This observation was also supported by Western blot analysis and TH activity measurements. However, catecholamine levels in transgenics were not significantly different from those in nontransgenics. These results suggest unknown regulatory mechanisms for human TH gene expression and for the catecholamine levels in transgenic mice.     

Effects of haloperidol and melatonin on the in situ activity of nigrostriatal tyrosine hydroxylase in male Syrian hamsters

Haloperidol, an antipsychotic drug, was tested for its effects on the in situ activity of nigrostriatal and hypothalamic tyrosine hydroxylase, in control male Syrian hamsters and in those receiving a high daily dose of melatonin. After receiving daily ip injections (1.25 mg/kg ip) of haloperidol for 21 days, the animals were sacrificed and brain tissue collected for analysis of dopamine and metabolites by HPLC with electrochemical detection. In situ activity of tyrosine hydroyxlase (TH) activity was determined by measuring the accumulation of L-Dopa after administration of the L amino acid decarboxylase inhibitor, mhydroxybenzylhydrazine. Tissue content of dopamine and its metabolites, DOPAC and HVA, was depressed in striatum of animals receiving haloperidol, and tyrosine hydroxylase (TH) activity was significantly decreased 20-24 h after the last injection (from 1823 +/- 63 to 1139 +/- 85 pg l-dopa/mg tissue). The decrease in TH activity in striatum was significantly inhibited by daily injections of a high dose of melatonin (2.5 mg/kg ip) (from 1139 +/- 85 to 1560 +/- 116 pg L-dopa/mg tissue). In the substantia nigra and in the hypothalamus, on the other hand, haloperidol significantly increased the activity of tyrosine hydroxylase. Melatonin administration did not significantly influence TH activity in the substantia nigra, but inhibited TH activity in the hypothalamus and in the pontine brainstem. One explanation for these data is that chronic haloperidol administration in Syrian hamsters increases TH activity in hypothalamus and substantia nigra, but decreases TH activity in striatum by a mechanism involving D2 presynaptic receptors and a melatonin sensitive kinase which regulates TH phosphorylation.     

MOUSE BRAIN TYROSINE HYDROXYLASE AND GLUTAMIC ACID DECARBOXYLASE FOLLOWING TREATMENT WITH ADRENOCORTICOTROPHIC HORMONE, VASOPRESSIN OR CORTICOSTERONE

The activities of tyrosine hydroxylase (TH) and glutamic acid decarboxylase (GAD) from several mouse brain regions were assayed following repeated administration of adrenocorticotrophic hormone (ACTH), lysine vasopressin (LVP) or corticosterone. Although similar treatments with ACTH have been shown to result in changes of catecholamine turnover and GABA content, no changes in the activity of either TH or GAD were observed in any brain region. Likewise LVP had no effect on either enzyme. Since the assays for TH were performed with concentrations of tyrosine and tetrahydrobiopterin cofactor below their respective Michaelis constants, this suggests that the changes of catecholamine turnover are not mediated by changes of TH activity. Twice daily corticosterone adrninistration for four days increased TH activity in the hypothalamus but not in any other brain region.     

Cretinism: influence on rate-limiting enzymes of amine synthesis in rat brain.

The effect of neonatal hypothyroidism on tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH) activity, as well as on water content, was studied in different regions of the developing rat brain. Neonatal hypothyroidism, induced by daily treatment with propylthiouracil starting at birth, led to a cretinoid syndrome with a marked impairment of body and brain growth. Compared to control littermates, 30- and 45-day-old cretinous rats had elevated levels of water in the brain stem. The activities of TH and TPH were increased in a time-dependent manner in the brain stem, basal ganglia and hypothalamus of maturing cretinous animals. The increased activity of these rate-limiting enzymes of mono-amine synthesis may account for the elevated levels of brain norepi-nephrine and serotonin in rats subjected to neonatal hypothyroidism.     

Change of tyrosine hydroxylase in the parkinsonian brain and in the brain of MPTP-treated mice as revealed by homospecific activity

Changes in homospecific activity (unit of enzyme activity per unit of enzyme protein; Rush, Kindler and Udenfriend, 1974. Biochem. Biophys. Res. Commun., 61, 38) of tyrosine hydroxylase (TH) in the striatum of the brain were examined in MPTP-treated mice and parkinsonian patients. After a single injection of MPTP to mice, TH activity was acutely inhibited onlyin situ without changes in in vitro TH activity (Vmax) and TH protein; TH homospecific activity (TH Vmax/TH protein) did not change. After repeated injection of MPTP to mice for 8 days, in situ TH activity, in vitro TH Vmax, and TH protein were decreased in parallel, and TH homospecific activity did not change The result indicates that the decreases in in situ TH activity and in TH Vmax are due to the decrease in TH protein by nerve degeneration of dopaminergic neurons in MPTP treated mice. However, when MPP⁺ was infused in the striatum of rats for 3 hours, in vitro TH activity (Vmax) was decreased without changes in TH protein. Thus, TH homospecific activity was decreased. The results indicate that MPP⁺ inactivates TH protein in the striatum after continued infusion. In contrast, the homospecific activity of TH in post-mortem parkinsonian striatum was increased 3-fold. The increase in homospecific activity of residual TH in parkinsonian brain suggests such molecular changes in TH molecules as result in a compensatory increase in TH activity.Special issue dedicated to Dr. Sidney Udenfriend.     

Substrate-mediated enhancement of phosphorylated tyrosine hydroxylase in nigrostriatal dopamine neurons: evidence for a role of alpha-synuclein

Tyrosine hydroxylase (TH) protein, phosphorylated at serine-40, serine-31 and serine-19, and enzyme catalytic activity were compared under basal conditions and in activated nigrostriatal dopamine (NSDA) neurons of wild-type and homozygous alpha-synuclein knockout mice. Mice were injected with the D2 antagonist raclopride to stimulate NSDA neuronal activity in the presence or absence of supplemental l-tyrosine. There was no difference in phosphorylated TH levels or TH catalytic activity between wild-type and alpha-synuclein knockout mice under basal conditions or following raclopride-induced acceleration of NSDA activity. In wild-type animals, tyrosine administration potentiated the raclopride-induced increase in phosphorylated TH and enzyme activity. However, tyrosine administration did not enhance phosphorylated TH levels or enzyme catalytic activity in raclopride-stimulated NSDA neurons in alpha-synuclein knockout mice. These findings suggest that alpha-synuclein plays a role in the ability of tyrosine to either enhance TH phosphorylation or hinder TH inactivation during accelerated neuronal activity. The present study supports the hypothesis that alpha-synuclein functions as a molecular chaperone protein that regulates the phosphorylation state of TH in a substrate and activity-dependent manner.     

Ontogenesis of Monoamine-Synthesizing Enzyme Activities and Biopterin Levels in Rat Brain or Salivary Glands, and the Effect of Thyroxine Administration

Neonatal changes in the activities of tyrosine hydroxylase (TH) and tryptophan hydroxylase (TrpH) and in the content of the co-factor, biopterin, were studied in rat midbrain for the first 20 days after birth. Changes in TH activity in the parotid and submandibular glands were also examined. Changes in TH activity per unit weight in the developing rat brain were briefly similar to those in the salivary glands; the activity increased from day 2 or 4 to day 9 after birth, and remained constant or slightly decreased at day 12, then rapidly increased on day 16. TrpH activity in the midbrain increased about twofold up to day 16. The biopterin concentration in the brain increased, reached a maximum level on day 12 after birth, and thereafter decreased. The effect of hyperthyroidism in rats given 0.2 mg/kg i.p. of thyroxine every 2 days postnatally was studied on the activity of TH in rat salivary glands at 12-day-old rats. In parotid or submandibular gland of hyperthyroid rats, TH activity increased at day 12 postnatally. In comparison with the effect on TH activity in the salivary glands, TH activity in the midbrain on day 20 postnatally was not induced by hyperthyroidism. Furthermore, increase of the TrpH activity and biopterin and catecholamine levels in the midbrain of hyperthyroid rats was not found on day 20 after birth in comparison with the corresponding controls. From these data, we suppose that postnatal hyperthyroidism may cause precocious induction of TH in rat salivary gland, but may not increase the activity of TH or TrpH, and the level of their co-factor, biopterin, in rat midbrain.     

Region - specific alterations in tyrosine hydroxylase activity in rats exposed to lead

Previous studies from our laboratory showed that subchronic exposure to low levels of Pb resulted in significant decrease in dopamine (DA) content, attenuation of stimulus-induced release of DA in the dopaminergic projection area of nucleus accumbens (NA), and alterations in tyrosine hydroxylase (TH) activity in rat whole brain homogenates. The present study reported here was conducted to assess the functional integrity of DA synthesis in different brain regions of rats subchronically (90-days) exposed to 50 ppm Pb by measuring the activity of the rate limiting enzyme, tyrosine hydroxylase, in seven brain regions. In Pb-exposed rats, TH activity was reduced in two of the seven brain regions investigated, i.e., nucleus accumbens (42% reduction) and frontal cortex (61% reduction) when compared to controls. In contrast, Pb exposure did not affect the TH activity in cerebellum, brainstem, hippocampus, hypothalamus and striatum. The changes in TH activity in nucleus accumbens (NA) and frontal cortex (FC) in Pb-exposed rats were further confirmed by Western blot analysis using TH polyclonal antibody. Collectively, these results indicate that low level subchronic Pb exposure may affect TH protein in these brain regions.     

Coordinate Regulation of the Cyclic AMP System with Firing Rate and Expression of Tyrosine Hydroxylase in the Rat Locus Coeruleus: Effects of Chronic Stress and Drug Treatments

Recent studies have demonstrated that chronic stress increases the firing rate and expression of tyrosine hydroxylase (TH) in neurons of the locus coeruleus (LC), the major noradrenergic nucleus in brain. The present study was undertaken to examine the influence of chronic stress and other treatments known to influence the activity of LC neurons on the cyclic AMP (cAMP) second messenger system in these neurons. Chronic (5 days) cold exposure significantly increased levels of TH immunoreactivity in the LC, as previously reported, but not in substantia nigra (SN) or ventral tegmentum (VT), two dopaminergic nuclei studied for comparison. Chronic cold exposure increased levels of cAMP-dependent protein kinase activity in soluble, but not particulate, fractions of the LC, and increased basal and GTP- and forskolin-stimulated adenylate cyclase activity in this brain region. In contrast, levels of the protein kinase and adenylate cyclase in VT, SN, and frontal cortex were not significantly influenced by cold exposure. To study further the relationship between regulation of LC firing rate, TH expression, and the cAMP system in the LC, other treatments known to influence TH were examined. Reserpine treatment, shown previously to increase levels of TH, was found to increase both LC firing rate and levels of soluble cAMP-dependent protein kinase activity in the LC. 6-Hydroxydopamine, shown previously to increase levels of TH and firing rate of LC neurons, also increased soluble levels of protein kinase activity. Other treatments known to either increase (adrenalectomy) or decrease (chronic imipramine) levels of TH in the LC were also found to increase or decrease, respectively, levels of cAMP-dependent protein kinase activity in this brain region.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solitary hypothalamic neurons inherently express vasopressin and tyrosine hydroxylase

Hypothalamic neurons were grown as single cells in three-dimensional culture. Solitary neurons lacking cell contacts were immunocytochemically examined for inherent expression of vasopressin (VP), tyrosine hydroxylase (TH), and luteinizing hormone releasing hormone (LHRH). Immunoreactive VP and TH were detected within a day. Sixty to eighty-five percent of neurons displayed homogeneously distributed reaction product for VP or TH. One percent exhibited intense punctate staining of somas and varicosities. Few neurons stained for LHRH. Results indicate that hypothalamic neurons can express appropriate neuropeptides and transmitter-specific products without contacting other neurons or nonneuronal cells. Thus, this culture system may provide a useful model to study intrinsic neuronal processes.     

Effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration in prenatal stage on the dopamine system in the postnatal mouse brain.

A dopaminergic neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine (MPTP), administered to a pregnant female was found to affect postnatally the catecholamine metabolism of the pups. MPTP (5 mg/kg body weight/day) was administered to pregnant C57 Black BYA mice daily for 7 days between the 12th and 18th day of gestation. Dopamine levels and tyrosine hydroxylase (TH) activity were measured in the whole brain from the pups sacrificed after birth. In MPTP-treated pups at 7 days of age, TH total activity (TH activity/brain) did not change (92% of the control value), while TH specific activity (TH activity/mg protein) was increased to 163% of that in control mice. Thus, TH homospecific activity (TH activity/mg TH protein) doubled compared to the control mice. At 28 days of age, both the total activity and the specific activity of TH in the brains of postnatal mice were reduced to 50% and 78% of the control, respectively. Dopamine concentration in the striatum was also reduced significantly. Reduction in the TH activity and dopamine concentration were also observed at the age of 12 weeks. These data suggest that the prenatal exposure to MPTP induced a prolonged reduction of TH activity in the brains of mice with a transient increase of TH homospecific activity during the postnatal period.     

Cyclin-dependent kinase 5 phosphorylates serine 31 of tyrosine hydroxylase and regulates its stability

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine biosynthesis, and its activity is regulated by phosphorylation in the N-terminal regulatory domain. The proline-directed serine/threonine kinase cyclin-dependent kinase 5 (cdk5) plays an important role in diverse neuronal processes. In the present study, we identify TH as a novel substrate of cdk5. We show that cdk5 phosphorylates TH at serine 31 and that this phosphorylation is associated with an increase in total TH activity. In transgenic mice with increased cdk5 activity, the immunoreactivity for phosphorylated TH at Ser-31 is enhanced in neurons of the substantia nigra, a brain region enriched with TH-positive neurons. In addition, we demonstrate that co-expression of cdk5 and its regulatory activator p35 with TH increases the stability of TH. Consistent with these findings, TH protein levels are reduced in cdk5 knock-out mice. Importantly, the TH activity and protein turnover of the phosphorylation-defective mutant TH S31A was not altered by cdk5 activity. Taken together, these data suggest that cdk5 phosphorylation of TH is an important regulator of TH activity through stabilization of TH protein levels.     

Elevated renal norepinephrine in proANP gene-disrupted mice is associated with increased tyrosine hydroxylase expression in sympathetic ganglia

The sympatholytic properties of atrial natriuretic peptide (ANP) contribute to its vasodilatory and natriuretic effects. High circulating catecholamine levels, along with renal dysfunction, present in proANP gene-disrupted (-/-) mice are thought to contribute to the hypertension characteristic of this model. To further understand the mechanism by which the absence of ANP leads to stimulation of sympathetic activity we measured tyrosine hydroxylase expression in mice with and without ANP. The adrenal and prevertebral ganglionic expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine production, was significantly increased in ANP -/- mice. ANP's sympatholytic properties include the depression of ganglionic and adrenal TH expression and catecholamine production. Thus, these results suggest that the absence of ANP's sympatholytic effects is not completely compensated for in ANP -/- mice. In addition, mice devoid of ANP display an increase in renal sympathetic nerve activity from birth through to adulthood which may lead to structural and/or functional changes in the early postnatal kidney that contribute to the hypertensive phenotype of ANP -/- mice. The over-activation of the sympathetic nervous system in mice lacking ANP confirms the important role of this peptide in the modulation of sympathetic nerve activity and its contribution to blood pressure homeostasis.     

Short-term effect of stress on tyrosine hydroxylase activity

The short-term influences of stress on the activities of tyrosine hydroxylase in vivo and in vitro were examined in mice. The in vivo tyrosine hydroxylase activity was estimated by the rate of dopa accumulation which was measured at 30 min after the injection of NSD-1015 (100 mg kg), an aromatic l-amino acid decarboxylase inhibitor, intraperitoneally and was compared with tyrosine hydroxylase activity measured in vitro. For the in vivo assay, both the accumulation of dopa (tyrosine hydroxylase activity) and that of 5-hydroxytryptophan (tryptophan hydroxylase activity) and the levels of monoamines and the metabolites (noradrenalin, adrenalin, dopamine, normetanephrine, 3-methoxytyramine and serotonin) and those of precursor amino acids, tyrosine and tryptophan, were investigated in ten different brain regions and in adrenals. The amount of dopa accumulation in the brain as a consequence of decarboxylase inhibition, in vivo tyrosine hydroxylase activity, was significantly increased by stress, in nerve terminals (striatum, limbic brain, hypothalamus, cerebral cortex and cerebellum) and also in adrenals. The effect of stress on tyrosine hydroxylase activity in vitro at a subsaturating concentration of 6-methyltetrahydropterin cofactor was also observed in nerve terminals (striatum, limbic brain, hypothalamus, and cerebral cortex). The amount of 5-hydroxytryptophan accumulation, the in vivo tryptophan hydroxylase activity, was also significantly increased in bulbus olfactorius, limbic brain, cerebral cortex, septum and lower brain stem. The influence of stress was also observed on the levels of precursor amino acids, tyrosine and tryptophan and monoamines in specific brain parts. These results suggest that the stress influences both catecholaminergic neurons and serotonergic neurons in nerve terminals in the brain. This effect was also observed on tyrosine hydroxylase activity in vitro in nerve terminals. However, in adrenals, the influence by stress was not observed on the in vitro activity, although dopa accumulation was increased.     

The catecholamine system in rat fetal brain with a disrupted corticosteroid balance]

The disturbance of corticosteroids balance of female rats on the 16 and 18 days of pregnancy by injections of exogenous corticosterone or methopyrone--blocker of endogenous hormone formation--decreased both body weight and activity of the rate-limiting catecholamine synthesising enzyme--tyrosine hydroxylase (TH) in the stem half of the 21 day fetal brain. Concomitantly with inhibitory action, which may be caused by general retardation of the organism development, corticosteroids stimulated TH activity during prenatal ontogenesis. Fetuses developed under elevated corticosteroid level had lower body weight but higher TH activity in comparison with fetuses endured the deficit of these hormones. Besides, corticosterone injection to the females on the 20th day of gestation increased in 6 hours TH activity in stem half of their fetus brain. The data obtained suggested the prominent role of corticosteroids in the prenatal development of brain catecholaminergic system.     

Effect of LPS treatment on tyrosine hydroxylase expression and Parkinson-like behaviors

Puberty is a critical period of development during which the brain undergoes reorganizing and remodeling. Exposure to stress during this period is thought to interfere with normal brain development and increase susceptibility to mental illnesses. In female mice, pubertal exposure to lipopolysaccharide (LPS), a bacterial endotoxin, has been shown to alter sexual, anxiety-like, and depression-like behaviors and cognition in an enduring manner. However, the mechanisms underlying these effects remain unknown. The present study examined age and sex difference in tyrosine hydroxylase (TH) expression and dopamine-dependent and Parkinson-like behaviors following LPS treatment. The results show that LPS treatment during adulthood causes an enduring increase in TH expression in many of the brain regions examined. In contrast, there is no change in TH expression following LPS treatment during puberty. However, pubertal LPS treatment induces enduring behavioral deficits in tests of Parkinson-like behaviors, more so in male than in female mice. These results suggest that the low levels of TH following exposure to pubertal immune challenge may predispose mice to Parkinson-like behavior. These findings add to our understanding of stress and immune responses during puberty and their impact on mental health later in life.     

Effects of nitric oxide synthase inhibitor on tyrosine hydroxylase mRNA in the adrenal medulla of spontaneously hypertensive and Wistar Kyoto rats.

We studied the effects of N(G)-nitro-l-arginine methyl ester (L-NAME) on catecholamine levels, tyrosine hydroxylase (TH) activity, and TH mRNA levels in the adrenal medulla of spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY). L-NAME (100 mg/L in drinking water) and atropine (10 mg/L in drinking water) were administered for 2 weeks. Epinephrine and norepinephrine levels, TH activity, and TH mRNA levels in the adrenal medulla of L-NAME-treated WKY were significantly decreased. These parameters were not significantly altered in the adrenal medulla of L-NAME-treated SHR. Nitrite/nitrate levels in the adrenal medulla of L-NAME-treated WKY were significantly decreased; however, no significant change in L-NAME-treated SHR was observed. Ca(2+)-dependent nitric oxide synthase (NOS) activity in the adrenal medulla of SHR was significantly decreased compared to that of WKY. TH mRNA levels in L-NAME + atropine-treated and L-NAME-treated WKY were significantly lower than TH mRNA levels in control WKY. These results suggest that nitric oxide in the adrenal medulla may enhance the catecholamine biosynthetic pathway via increased TH mRNA expression. Our results also suggest that this effect is suppressed in SHR due to decreased NOS activity in the adrenal medulla.