Tyrosine Hydroxylase Messenger RNA Research Articles

Tyrosine hydroxylase messenger RNA corroborates protein localization in the nervous system of the pond snail, Lymnaea stagnalis

AbstractCatecholaminergic neurons are abundant in molluscs and are involved in a variety of behaviors such as feeding, respiration, learning, and locomotion. However, previous identification of these neurons has relied almost exclusively on immunohistochemistry using antibodies, which have not been fully validated for use in molluscs. We employed tissue‐specific quantitative PCR in adults of Lymnaea stagnalis (a pulmonate gastropod) and whole‐mount in situ hybridization in larvae to both quantify and visualize messenger RNA of the catecholamine synthesis enzyme, tyrosine hydroxylase (TH). TH messenger RNA was found to localize primarily in the foot and the central nervous system, with smaller quantities present in the cephalic sensory organs. Additionally, we performed western blots that validated a popular antibody used as a marker for catecholaminergic neurons in molluscs. Taken together, these data indicate that TH messenger RNA is present in the central and peripheral nervous system of L. stagnalis and support the specificity of past immunohistochemical labeling of the TH protein. These findings have potentially broad implications, given the wide range of biological processes that have been studied in L. stagnalis.

Increased expression of dopamine receptors in synovial fibroblasts from patients with rheumatoid arthritis: inhibitory effects of dopamine on interleukin-8 and interleukin-6.

Observations in both animal models of arthritis and patients with rheumatoid arthritis (RA) suggest a role for dopamine and its receptors in RA. Because synovial fibroblasts (SFs) contribute to inflammation and joint destruction in RA, the aim of this study was to investigate dopaminergic pathways in SFs obtained from patients with RA and, for comparison, in SFs from patients with osteoarthritis (OA) undergoing knee joint replacement surgery. The expression of all dopamine receptors (D1 -D5 ) and dopamine transporter was assessed by immunofluorescence and immunohistochemical staining. The levels of dopamine receptor and tyrosine hydroxylase messenger RNA were measured by real-time polymerase chain reaction. The intracellular content of dopamine, its precursor, and its main metabolites was assayed by high-performance liquid chromatography. The influence of dopamine on proinflammatory interleukin-6 (IL-6) and IL-8, matrix metalloproteinase 3, and tissue inhibitor of metalloproteinases 1 (TIMP-1) and TIMP-2 was studied in SFs. SFs possess an intrinsic dopaminergic system, including dopamine receptors, dopamine transporter, and tyrosine hydroxylase, and contain dopamine, its precursor, and its main metabolites. SFs from patients with RA, in comparison with those from patients with OA, showed increased expression of dopamine receptors D1 and D5 , and exogenous dopamine strongly inhibited the production of IL-8 in patients with RA. SFs from patients with RA and patients with OA show a dopaminergic phenotype. The expression of D1-like dopamine receptors was higher in RASFs, and this increased expression may lead to antiinflammatory effects, as demonstrated by the expression of IL-8. Studies in animal models and patients with RA are needed to assess the therapeutic potential of endogenous, local production of dopamine in synoviocytes.

Methylphenidate and cocaine self‐administration produce distinct dopamine terminal alterations

Methylphenidate (MPH) is a commonly abused psychostimulant prescribed for the treatment of attention deficit hyperactivity disorder. MPH has a mechanism of action similar to cocaine (COC) and is commonly characterized as a dopamine transporter (DAT) blocker. While there has been extensive work aimed at understanding dopamine (DA) nerve terminal changes following COC self-administration, very little is known about the effects of MPH self-administration on the DA system. We used fast scan cyclic voltammetry in nucleus accumbens core slices from animals with a 5-day self-administration history of 40 injections/day of either MPH (0.56 mg/kg) or COC (1.5 mg/kg) to explore alterations in baseline DA release and uptake kinetics as well as alterations in the interaction of each compound with the DAT. Although MPH and COC have similar behavioral effects, the consequences of self-administration on DA system parameters were found to be divergent. We show that COC self-administration reduced DAT levels and maximal rates of DA uptake, as well as reducing electrically stimulated release, suggesting decreased DA terminal function. In contrast, MPH self-administration increased DAT levels, DA uptake rates and DA release, suggesting enhanced terminal function, which was supported by findings of increased metabolite/DA tissue content ratios. Tyrosine hydroxylase messenger RNA, protein and phosphorylation levels were also assessed in both groups. Additionally, COC self-administration reduced COC-induced DAT inhibition, while MPH self-administration increased MPH-induced DAT inhibition, suggesting opposite pharmacodynamic effects of these two drugs. These findings suggest that the factors governing DA system adaptations are more complicated than simple DA uptake blockade.

Chronic corticotropin‐releasing factor type 1 receptor antagonism with antalarmin regulates the dopaminergic system of Fawn‐Hooded rats

Corticotropin-releasing factor is a neuropeptide associated with the integration of physiological and behavioural responses to stress and also in the modulation of affective state and drug reward. The selective, centrally acting corticotropin-releasing factor type 1 receptor antagonist, antalarmin, is a potent anxiolytic and reduces volitional ethanol consumption in Fawn-Hooded rats. The efficacy of antalarmin to reduce ethanol consumption increased with time, suggestive of adaptation to reinforcement processes and goal-directed behaviour. The aim of the present study was to examine the effects of chronic antalarmin treatment on reward-related regions of Fawn-Hooded rat brain. Bi-daily antalarmin treatment (20 mg/kg, i.p.) for 10 days increased tyrosine hydroxylase messenger RNA expression throughout the ventral mesencephalon. Following chronic antalarmin the density of dopaminergic terminals within the basal ganglia and amygdaloid complex were reduced, as was dopamine transporter binding within the striatum. Receptor autoradiography indicated an up-regulation of dopamine D2, but no change in D1, binding in striatum, and Golgi-Cox analysis of striatal medium spiny neurones indicated that chronic antalarmin treatment increased spine density. Thus, chronic antalarmin treatment modulates dopaminergic pathways and implies that chronic treatment with drugs of this class may ultimately alter postsynaptic signaling mechanisms within the basal ganglia.

Characterization of five evolutionary conserved regions of the human tyrosine hydroxylase (TH) promoter: Implications for the engineering of a human TH minimal promoter assembled in a self‐inactivating lentiviral vector system

A DNA fragment of about 13 kb containing the human tyrosine hydroxylase (TH) promoter was previously isolated from a genomic DNA library and sequenced. The 11 kb from the transcription start of the human TH promoter was successively joined to the green fluorescent protein (GFP) to generate a transgenic mouse model. High levels of GFP expression could be observed in TH-positive cells of the Substantia nigra of embryonic and adult mice. Intriguingly, the sequence of the human TH promoter showed a low degree of homology with the mouse and rat TH promoters. In fact, comparative analysis of the sequences of human, rat, and mouse TH promoters revealed only five small regions of high homology. These five evolutionarily conserved regions were numbered in numeric progression from the 5' end of human TH promoter. In the present study, a panel of minimal human TH promoters was generated to analyze the transcriptional activity and specificity of gene expression conferred by the five conserved regions (CRs). The series of constructs was termed 250 bp and contained the first -194 bp of the human TH promoter immediately upstream of the transcription start, the first 35 bp the human TH messenger RNA leader, plus one or more of the five CRs. All the constructs were assembled in a self-inactivating form of the latest series of lentiviral vector system based on the human immunodeficiency virus type 1 (HIV-1). Lentiviral-mediated gene transfer was highly efficient for the in vitro transduction of human neuronal progenitor cells (hNPCs). Since a subset of hNPCs express TH following in vitro treatment with a mixture of differentiating agents, it was possible to assess specificity of expression for all the minimal human TH promoters. Overall, the successive addition of the five conserved regions produced a greater degree of specificity in induced TH-positive hNPCs, in particular after the addition of CRI (-8,917, -8,876). However, the human TH minimal promoters did not show any specificity for TH-positive differentiated mouse primary striatal and S. nigra cells, indicating a difference of TH gene regulation between the human and mouse systems. The human TH minimal promoters may provide the opportunity for the selection of TH-positive human embryonic and adult stem cells for brain transplantation experiments in animal models for Parkinson's disease.

Role of ROS and NO in hypoxia-induced increase in tyrosine hydroxylase-messenger RNA in PC12 cells.

Paraganglia such as the carotid body and retroperitoneal paraganglia constitute a protective system against the adverse effects of hypoxia by measuring arterial p02 and initiating appropriate reflexes. One of these protective reflexes is release of catecholamines into the blood stream by retroperitoneal paraganglia and the fetal adrenal medulla. Concomitant to this release, catecholamine synthesis is enhanced by hypoxia-induced increase in transcription of the rate-limiting enzyme of catecholamine synthesis, i.e. tyrosine hydroxylase (TH). A tumor cell line derived from a rat adrenal medullary tumor, PC 12 cells, is a suitable model to study this hypoxia-induced increased TH gene transcription (Czyzyk-Krzeska et al. 1994; Kroll and Czyzyk-Krzeska, 1998).

Transcriptional and post-transcriptional regulation of Tyrosine Hydroxylase messenger RNA in PC12 cells during persistent stimulation by VIP and PACAP38: differential regulation by protein kinase A and protein kinase C -dependent pathways

VIP and PACAP38 are closely related peptides that are released in the adrenal gland and sympathetic ganglia and regulate catecholamine synthesis and release. We used PC12 cells as a model system to examine receptor and second messenger pathways by which each peptide stimulates transcriptional and post-transcriptional mechanisms that regulate the level of the mRNA for tyrosine hydroxylase (TH), the rate-limiting enzymatic step in catecholamine synthesis. Concentration–response studies revealed that PACAP38 had both greater efficacy and potency than VIP. The specific PAC1 receptor antagonist PACAP[6–38] blocked the effects of each peptide on THmRNA content while the PACAP/VIP type II receptor antagonist (N-AC-Tyr1-D-Phe2)-GRF-(1–29)-NH2 was without effect. At equipotent concentrations, each peptide stimulated a transient increase in TH gene transcription lasting less than 3h. Continuous VIP treatment stimulated a transient increase in TH mRNA lasting less than 24h. In contrast, continuous exposure to PACAP38 stimulated a stable increase in TH mRNA that persisted for 2 days in the absenceofelevated transcription, pointing to different post-transcriptional effects of the two peptides. PACAP38 alone had no effect on the magnitude of TH gene transcription or TH mRNA in A126-1B2 PKA-deficient PC12 cells. However, when combined with dexamethasone, PACAP38 produced a synergistic increase in TH mRNA in the absence of PACAP38-stimulated TH gene transcription. In contrast, VIP had no effect on either TH mRNA content or TH gene transcription in this model. PACAP38, but not VIP, stimulated PKC activity. Calphostin C antagonized the effect of PACAP38 on the persistent post-transcriptional elevation in TH mRNA. Thus, the results support the conclusion thatVIP and PACAP38 each stimulate PAC1 receptors to increase TH gene transcription through a PKA-controlled pathway, but their divergent post-transcriptional effects result at least partly from differing abilities to stimulate PKC.

Gene expression of catecholamine biosynthetic enzymes following exercise: modulation by age.

Both age and exercise training are associated with tissue specific alterations in the catecholaminergic system. We examined the effect of short-term exercise training on tyrosine hydroxylase and dopamine beta-hydroxylase gene expression in adrenals and specific brain regions with aging. In addition, we examined activator protein-1 and cyclic AMP response element transcription factor binding activity in the adrenal medulla. Male, six- and 24-month-old F-344 rats were exercised by treadmill running for five consecutive days. One group was killed immediately and a second group was killed 2h after the last training session. Exercise significantly elevated tyrosine hydroxylase messenger RNA equally in adrenals of both young and old rats. Training had no effect on dopamine beta-hydroxylase messenger RNA in adrenals of young, but levels were elevated in old rats. Binding activities of both activator protein-1 and cyclic AMP response element binding protein were diminished with age in the adrenal medulla. Exercise training had no significant effect on the binding activity of cyclic AMP response element binding protein in either young or old animals, whereas activator protein-1 binding activity increased equally in young and old animals. Exercise training revealed divergent changes in tyrosine hydroxylase messenger RNA in brain catecholaminergic neurons. In the locus coeruleus and the ventral tegmental areas, training elevated tyrosine hydroxylase messenger RNA levels only in young rats. In the substantia nigra, there was no change in young, but a 45% increase in tyrosine hydroxylase messenger RNA in old rats. In the ventral tegmental area, training increased tyrosine hydroxylase gene expression 80% in young but not in old rats. These results indicate that short-term exercise training increases tyrosine hydroxylase messenger RNA levels in young animals in the adrenals, the locus coeruleus and the ventral tegmental area. The responses for exercise training of aged animals differed from the young in brain noradrenergic and dopaminergic nuclei, especially in the substantia nigra, and to some extent in the locus coeruleus and the ventral tegmental area.

Prepro-galanin messenger RNA levels are increased in rat locus coeruleus after treadmill exercise training

The effects of treadmill exercise training on prepro-galanin (GAL) and tyrosine hydroxylase (TH) gene expression in the locus coeruleus (LC) were examined. Male Fischer-344 rats ( n=9) were assigned to 6 weeks of treadmill running. An additional group of animals comprised the sedentary home cage control group ( n=9). Levels of GAL and TH messenger RNA (mRNA) in the LC were measured using in situ hybridization histochemistry with autoradiography. Levels of GAL mRNA were higher in treadmill trained animals compared to sedentary animals, but there was no effect of treadmill running on TH mRNA. These results suggest that gene expression for galanin is responsive to repeated exercise stress and may have a neuromodulatory role in LC-noradrenergic adaptation to treadmill exercise training.

Expression of peptides and other neurochemical markers in hypothalamus and olfactory bulb of mice devoid of all known thyroid hormone receptors

We have investigated with histochemical techniques the expression of peptides and other neurochemical markers in the hypothalamus and olfactory bulb of male mice, in which the genes encoding the α and β thyroid hormone receptors (TRα1, TRβ1 and TRβ2) have been deleted. Thyrotropin-releasing hormone messenger RNA levels were increased in the hypothalamic paraventricular nucleus and in the medullary raphe nuclei of mutant mice lacking the thyroid hormone receptors α1 and β (α1 -/-β -/-), as compared to wild-type mice. In contrast, galanin messenger RNA levels were lower in the hypothalamic paraventricular nucleus of mutant animals, as was galanin-like immunoreactivity in the internal layer of the median eminence. Substance P messenger RNA levels were unchanged in the medullary raphe nuclei. Thyrotropin-releasing hormone receptor messenger RNA levels were increased in motoneurons, unchanged in the subiculum, and lower in the amygdala of mutant animals. Galanin messenger RNA levels were unchanged in the hypothalamic dorsomedial and arcuate nuclei of the thyroid hormone receptor α1 -/-β -/- mice, as was the immunocytochemistry for oxytocin and for vasopressin in the hypothalamic paraventricular nucleus. A reduction in tyrosine hydroxylase messenger RNA levels was found in the arcuate nucleus of mutant mice. In the olfactory bulb, immunohistochemistry for calbindin and for tyrosine hydroxylase revealed a reduction in the intensity of labeling of nerve processes in the glomerular layer of thyroid hormone receptor α1 -/-β -/- mice. The tyrosine hydroxylase messenger RNA levels were also slightly reduced. In contrast, the levels of galanin and neuropeptide Y messenger RNA in this region were unchanged in thyroid hormone receptor α1 -/-β -/- mice as compared to wild-type mice. Together these studies reveal many regional and neurochemically selective alterations in neuronal phenotype of mice devoid of all known thyroid hormone receptors.

Increased transcription of the tyrosine hydroxylase gene in individual locus coeruleus neurons following footshock stress

Footshock-evoked change in transcriptional activity of tyrosine hydroxylase in neurons of the locus coeruleus was examined using an intron-specific in situ hybridization histochemical technique. A significant increase in the cellular concentration of tyrosine hydroxylase primary transcripts was found in locus coeruleus neurons 3 h following 30 min of intermittent footshock. However, the footshock-induced increase in tyrosine hydroxylase transcription was not homogeneously expressed in locus coeruleus neurons. Similarly, administration of the α 2-adrenergic receptor antagonist idazoxan produced a significant increase in the cellular concentration of tyrosine hydroxylase primary transcripts that was heterogeneously distributed among locus coeruleus neurons. Both footshock and idazoxan significantly increased the regional levels of tyrosine hydroxylase messenger RNA in the locus coeruleus. The time-course of changes in tyrosine hydroxylase transcription rate and messenger RNA levels in the locus coeruleus was examined after a 15 min exposure to footshock. A robust increase in tyrosine hydroxylase transcription rate was found at the end of 15 min of footshock, which remained elevated for 6 h and was back to the control levels by 24 h. In contrast, in response to a 15 min period of footshock tyrosine hydroxylase messenger RNA concentrations in the locus coeruleus did not increase until 6 h and remained elevated at 24 h. These findings demonstrate that transcription of the tyrosine hydroxylase gene in locus coeruleus neurons in response to footshock stress occurs rapidly, is sustained for many hours and is heterogeneously distributed. These data also suggest that the increase in tyrosine hydroxylase messenger RNA following footshock is mediated, at least in part, by an increase in tyrosine hydroxylase gene transcription.

Central corticotropin-releasing hormone receptors modulate hypothalamic–pituitary–adrenocortical and sympathoadrenal activity during stress

The role of brain corticotropin-releasing hormone receptors in modulating hypothalamic–pituitary–adrenal and sympathoadrenal responses to acute immobilization stress was studied in conscious rats under central corticotropin-releasing hormone receptor blockade by intracerebroventicular injection of a peptide corticotropin-releasing hormone receptor antagonist. Blood for catecholamines, adrenocorticotropic hormone and corticosterone levels was collected through vascular catheters, and brains were removed at 3 h for in situ hybridization for tyrosine hydroxylase messenger RNA in the locus coeruleus, and corticotropin-releasing hormone and corticotropin-releasing hormone receptor messenger RNA in the hypothalamic paraventricular nucleus. Central corticotropin-releasing hormone receptor blockade reduced the early increases in plasma epinephrine and dopamine, but not norepinephrine, during stress. Immobilization stress increased tyrosine hydroxylase messenger RNA levels in the locus coeruleus by 36% in controls, but not in corticotropin-releasing hormone antagonist-injected rats. In control rats, corticotropin-releasing hormone messenger RNA and type 1 corticotropin-releasing hormone receptor messenger RNA in the paraventricular nucleus increased after stress ( P<0.01), and these responses were attenuated by central corticotropin-releasing hormone receptor blockade. In contrast, central corticotropin-releasing hormone antagonist potentiated plasma adrenocorticotropic hormone responses, but slightly attenuated plasma corticosterone responses to stress. The inhibition of plasma catecholamine and locus coeruleus tyrosine hydroxylase messenger RNA responses to stress by central corticotropin-releasing hormone receptor blockade supports the notion that central corticotropin-releasing hormone regulates sympathoadrenal responses during stress. The attenuation of stress-induced corticotropin-releasing hormone and corticotropin-releasing hormone receptor messenger RNA responses by central corticotropin-releasing hormone receptor blockade suggests direct or indirect positive feedback effects of corticotropin-releasing hormone receptor ligands on corticotropin-releasing hormone expression, whereas additional mechanisms potentiate adrenocorticotropic hormone responses at the pituitary level. In addition, changes in neural activity by central corticotropin-releasing hormone are likely to modulate adrenocortical responsiveness during stress.

A Human Tyrosine Hydroxylase Isoform Associated with Progressive Supranuclear Palsy Shows Altered Enzymatic Activity

A novel human tyrosine hydroxylase (HTH) messenger RNA subgroup generated by alternative splicing and characterized by the absence of the third exon was recently identified. The corresponding putative protein lacks 74 amino acids including Ser31 and Ser40, two major phosphorylation sites implicated in the regulation of HTH activity. These mRNA species are detected in adrenal medulla and are overexpressed in patients suffering from progressive supranuclear palsy, a neurodegenerative disease mostly affecting catecholaminergic neurons of the basal ganglia. In the present work, an HTH protein isoform lacking exon 3 was identified in human adrenal medulla. For this purpose, an antibody was raised against the HTH exon 3. The effect of the removal of exon 3 on the enzymatic activity of HTH was studied in vitro by comparing a purified recombinant fusion protein without exon 3 (glutathione S-transferase (GST)-HTHDelta3) to the equivalent protein containing exon 3 (GST-HTH3). In initial velocity conditions, GST-HTHDelta3 has 30% of the maximal velocity of GST-HTH3. Moreover, the skipping of exon 3 results in the absence of activation of GST-HTH by heparin and increases by 10-fold the retroinhibition constant for dopamine, demonstrating the involvement of exon 3 in the regulation of HTH enzymatic activity. The identification of a variably expressed HTH isoform that lacks an exon implicated in activity regulation supports the view that HTH alternative splicing contributes to the functional diversity within the catecholaminergic system and may be implicated in some neurological diseases.

Endogenous opioid peptides contribute to suckling-induced prolactin release by suppressing tyrosine hydroxylase activity and messenger ribonucleic acid levels in tuberoinfundibular dopaminergic neurons.

The endogenous opioid peptides have been implicated in the control of the suckling-induced PRL rise during lactation. This study examined the role of the endogenous opioid peptides in suppressing tuberoinfundibular dopaminergic neuronal activity during lactation. In the first experiment, lactating rats were constantly exposed to pups. Naloxone (NAL; 60 mg/kg x h; i.v.), an opioid receptor antagonist, or saline was infused for 12 h. Blood was collected before and at 2-h intervals during the infusion. NAL suppressed circulating PRL levels to less than 36% of control values at 4, 6, 8, and 12 h after the onset of the infusion. Tyrosine hydroxylase (TH) activity in the stalk-median eminence and TH messenger RNA signal levels in the arcuate nucleus were determined at the end of the NAL infusion. TH activity and TH messenger RNA signal levels were increased 2.5- and 2.7-fold, respectively, after the 12-h NAL infusion. Even though the time spent with their pups was similar between the two groups, the pups in the NAL-treated group failed to gain weight during the 12-h NAL infusion period, whereas the control litters (8 pups) gained 5 g. In a second experiment, pups were removed from the dams before the 12-h NAL infusion and were returned after 11 h. Blood was collected before the infusion, at 3-h intervals during the pup separation period, and at 15-min intervals after reunion with the pups. Plasma PRL in control and NAL-treated rats was low (1-15 ng/ml) and similar during the separation period. The suckling-induced PRL surge in NAL-treated rats was markedly attenuated to 9-25% of control levels (350-650 ng/ml). After a 1-h suckling episode, TH activity in the stalk-median eminence of NAL-treated rats was 4.5-fold greater than controls. Litter weight gains were significantly less in NAL-treated rats during the 1-h suckling episode. These data indicate that the endogenous opioid peptides are an integral component for increasing PRL release in response to suckling and they act to decrease tuberoinfundibular dopaminergic neuronal activity during lactation, in part, by suppressing TH gene expression.

Angiotensin II-induced nuclear targeting of the angiotensin type 1 (AT1) receptor in brain neurons.

Angiotensin II (Ang II) interaction with the neuronal AT1 receptor results in a chronic stimulation of neuromodulation that involves the expression of norepinephrine transporter (NET) and tyrosine hydroxylase (TH). In view of this unique property and the presence of putative nuclear localization signal (NLS) consensus sequence in the AT1 receptor, this study was conducted to investigate the hypothesis that Ang II would induce nuclear sequestration of this G protein-coupled receptor and that the sequestration may have implications on Ang II-induced expression of NET and TH genes. Incubation of neuronal cultures with Ang II caused a time- and dose-dependent increase in the levels of AT1 receptor immunoreactivity in the nucleus. A 6.7-fold increase was observed with 100 nM Ang II, in 15 min, that was blocked by losartan, an AT1 receptor-specific antagonist. Ang II-induced nuclear sequestration was specific for AT1 receptor, because Ang II failed to produce a similar effect on neuronal AT2 receptors. The presence of the putative NLS sequence in the cytoplasmic tail of the AT1 receptor seems to be the key in nuclear targeting because: 1) nuclear targeting was attenuated by a peptide of the AT1 receptor that contained the putative NLS sequence; and 2) Ang II failed to cause nuclear translocation of the AT2 receptor, which does not contain the putative NLS. Ang II also caused a time- and dose-dependent stimulation of P62 phosphorylation, a glycoprotein of the nuclear pore complex. A 6-fold stimulation of phosphorylation was observed with 100 nM Ang II, in 15 min, that was completely blocked by losartan and not by PD123,319, an AT2 receptor specific antagonist. Preloading of neurons with p62-pep (a peptide containing consenses of mitogen-activated protein kinase in p62) resulted in a loss of Ang II-induced p62 phosphorylation and stimulation of NET and TH messenger RNA levels. In conclusion, these data demonstrate that Ang II induces nuclear sequestration of AT1 receptor involving NLS in the AT1 receptor and p62 of the nuclear pore complex in brain neurons. A possible role of such a nuclear targeting of the AT1 receptor on chronic neuromodulatory actions of Ang II has been discussed.

Induction of tyrosine hydroxylase by forskolin: modulation with age

With aging, circulating catecholamines are elevated in both humans and animals. This may be related to the increased basal levels of tyrosine hydroxylase messenger RNA (mRNA) levels and tyrosine hydroxylase enzyme activity in the adrenal medulla of senescent compared with younger animals. In addition, tyrosine hydroxylase gene expression in the senescent rat is resistant to further stimulation by cold exposure as compared with younger animals. Collectively, these observations suggest either that tyrosine hydroxylase expression is already maximally stimulated in senescent rats or that tyrosine hydroxylase gene induction pathways are impaired with senescence. To help distinguish between these possibilities, we examined the induction of tyrosine hydroxylase mRNA, tyrosine hydroxylase immunoreactivity and tyrosine hydroxylase enzyme activity in the adrenal medulla following forskolin administration to young and old F-344 rats. Forskolin at doses of 1.8 and 3.5 mg/kg increased tyrosine hydroxylase mRNA levels 2.5-fold in adrenal medulla from young rats but did not increase either tyrosine hydroxylase immunoreactivity or tyrosine hydroxylase enzyme activity 5 h after administration. Prolonged treatment with forskolin (3 doses, 12 h apart) increased tyrosine hydroxylase mRNA levels and tyrosine hydroxylase immunoreactivity and tyrosine hydroxylase enzyme activity. In senescent rats, the baseline level of tyrosine hydroxylase mRNA was more than 2-fold higher compared with young rats. A single injection of the lower dose of forskolin increased tyrosine hydroxylase mRNA levels by the same increment in senescent as compared with young rats. These data indicate that the tyrosine hydroxylase gene in the adrenal medulla from senescent rats is still capable of further stimulation. © 1997 Elsevier Science B.V. All rights reserved.

Developmental expression of tyrosine hydroxylase, D2-dopamine receptor and substance P genes in the carotid body of the rat.

Alterations in the level of putative neurotransmitters/neuromodulators and corresponding receptors may be a possible mechanism involved in changes in chemosensitivity of peripheral chemoreceptors in the carotid body during development. Using quantitative in situ hybridization histochemistry, levels of messenger RNAs encoding tyrosine hydroxylase, the rate-limiting enzyme for dopamine synthesis, the D2-dopamine receptor and substance P of newborn rats at postnatal days 0, 2, 14 and 21 were determined. For comparison, during the same time points during development, we also determined the level of expression of these messenger RNAs in the cells of the superior cervical ganglion which are not chemosensitive. Tyrosine hydroxylase and D2-dopamine receptor messenger RNAs were co-localized in many of the cells in both the carotid body and the superior cervical ganglion. In the carotid body, the level of tyrosine hydroxylase messenger RNA expression was greatest at birth, significantly decreased by 48 h postnatal age and remained decreased at 14 and 21 postnatal days. In contrast, D2-dopamine receptor messenger RNA levels significantly increased with postnatal age in the carotid body. This profile of an D2-dopamine receptor was not observed in the superior cervical ganglion where tyrosine hydroxylase and D2-dopamine receptor messenger RNAs levels did not significantly change from postnatal days 0 to 21. Lastly, in the rat carotid body, substance P messenger RNA was not detected. However, substance P messenger RNA was abundant in the nodose and petrosal ganglion. The increasing contribution of carotid body on ventilation with increasing postnatal age is associated with changes in levels of gene expression for tyrosine hydroxylase and D2-dopamine receptor in the carotid body.

Accumulation of tyrosine hydroxylase messenger RNA molecules in the rat mesencephalon by chronic caffeine treatment

In the present study we assessed the effect of chronic treatment with caffeine on the levels of the messenger RNA molecule encoding the enzyme tyrosine hydroxylase (TH) by in situ hybridization histochemistry in the ventral tegmental area (VTA) and the substantia nigra compacta (SNc) of the rat brain. Animals that received caffeine for nine consecutive days at doses of 20, 40 and 80 mg/kg of body weight displayed increased TH mRNA levels in the SNc (up to 64% above vehicle-injected controls) and the VTA (33% above controls). Moreover, the increases observed at 80 mg/kg of caffeine were prevented by concurrent administration of the non-competitive N-methyl- d-aspartate (NMDA) receptor antagonist MK-801 (0.25 mg/kg). These results demonstrate that chronic exposure to caffeine, an adenosine A2 receptor antagonist, alters the levels of expression of the mRNA encoding the rate limiting enzyme in catecholamine biosynthesis.

Contrasting effects of repeated treatment vs. withdrawal of methamphetamine on tyrosine hydroxylase messenger RNA levels in the ventral tegmental area and substantia nigra zona compacta of the rat brain.

We have assessed the effect of repeated treatment with methamphetamine (METH) on the abundance of the messenger ribonucleic acid molecules encoding the enzyme tyrosine hydroxylase (TH) and preprocholecystokinin (PPCCK) in the substantia nigra zona compacta (SNc) and the ventral tegmental area (VTA) by in situ hybridization histochemistry. Rats were injected twice daily with METH (4 mg/kg of body weight) for 6 consecutive days and sacrificed either 5 h or 15 days after the last injection. TH mRNA in the VTA was unaffected by repeated METH treatment but was decreased 25% relative to controls in the SNc. Concurrent administration of METH and MK-801 decreased TH mRNA levels in the SNc to 47% relative to controls. In contrast, TH mRNA levels were found increased in the VTA (42%) but not SNc 15 days post-METH treatment. Coadministration of MK-801 with METH prevented the increase in TH mRNA in the VTA. PPCCK mRNA levels were not significantly affected by METH treatment in VTA or SNc either 5 h or 15 days posttreatment. The results demonstrate that exposure to repeated methamphetamine elicits changes of TH mRNA levels in the VTA that become manifest 2 weeks after withdrawal from this psychostimulant drug.

Angiotensin II regulation of tyrosine hydroxylase gene expression in the neuronal cultures of normotensive and spontaneously hypertensive rats.

In the present study we investigated the regulation of tyrosine hydroxylase (TH) by angiotensin II (Ang II) in an attempt to provide cellular and molecular evidence that this hormone has increased neuromodulatory actions in the spontaneously hypertensive (SH) rat brain. Neuronal cells in primary culture from the hypothalamus-brain stem of both normotensive [Wistar-Kyoto (WKY)] and SH rats have been used. These cultures mimic in vivo situations. Ang II caused a time-dependent increase in TH activity in WKY rat brain neurons. A maximal increase of 2.5-fold was observed with 100 nM Ang II in an actinomycin- and cycloheximide-dependent process. In addition, Ang II caused a parallel increase in TH messenger RNA (mRNA) levels, with a maximal stimulation of 5-fold in 4 h by 100 nM Ang II in WKY rat brain neurons. The stimulation of TH mRNA was mediated by the AT1 receptor subtype, resulted from an increase in its transcription, and involved activation of phospholipase C and protein kinase C. Antisense oligonucleotide for c-fos attenuated Ang II stimulation of TH mRNA in a time- and dose-dependent fashion, indicating an involvement of c-fos as a putative third messenger in Ang II stimulation of TH. Ang II also caused stimulation of TH activity and its mRNA levels in neuronal cultures of SH rat brain by a mechanism similar to that observed for neuronal cultures of WKY rat brain, involving AT1 receptors, protein kinase C, and c-fos. However, the stimulation of TH activity and that of TH mRNA were approximately 30% and 80% higher, respectively, in the SH rat brain neurons than those in the WKY rat brain neurons. In vivo experiments have been carried out to validate the elevated response of TH gene expression to Ang II in SH rat brain neuronal cultures. Ang II stimulated both TH activity and TH mRNA levels in the hypothalami and brain stems of adult WKY and SH rats. The level of stimulation in the brain of the SH rat was significantly higher than that in the WKY rat. These observations are consistent with an increase in AT1, receptor gene expression and suggest that increased TH gene expression could be the cellular/molecular basis for the greater neuromodulatory action of Ang II in the SH rat brain.