Differential expression of tyrosine hydroxylase mRNA in the developing rat mesencephalon

1. With respect to the mesostriatal projection, the mesencephalon is composed of two dopaminergic (DA) cell populations, called dorsal tier and ventral tier. Strong evidence suggests differences in both the spatial and the temporal sequence of the innervation of the striatum between the two groups, with the ventral tier neurons innervating striatal patches prenatally and dorsal tier cells innervating striatal matrix postnatally. 2. Using in situ hybridization, we have examined the expression of the gene coding for tyrosine hydroxylase (TH) in mesencephalic DA neurons with respect to their postnatal development. Two ontogenic patterns of expression were observed: (a) dorsal tier neurons of the medial mesencephalon exhibited a sharp increase in expression beginning after birth, peaking on day 14, then decreasing and, finally, stabilizing; and (b) ventral tier neurons and dorsal tier cells from the lateral and the medial-dorsal mesencephalon showed only a slight increase in TH mRNA, reaching a plateau at P10. 3. The time course of the observed increase in TH gene expression in the first group, generally parallels the innervation of their target cells in the striatal matrix, suggesting that TH gene expression in these cells may be influenced by their postsynaptic cells or by the innervation process.     

The effect of tetrahydrobiopterin on the in situ phosphorylation of tyrosine hydroxylase in rat striatal synaptosomes

Tetrahydrobiopterin (BH₄), the obligatory cofactor of the aromatic amino acid hydroxylases, decreased the in situ³²P-phosphorylation of tyrosine hydroxylase (TH) in rat striatal synaptosomes. Incubation of pre-³²P-labeled synaptosomes with BH₄ in the presence of a permeant analogue of cAMP decreased the cAMP-stimulated level of³²P label incorporation into TH by about 50%, as determined by immunoprecipitation and autoradiography of SDS-polyacrylamide gels. The extent of inhibition mirrored changes in intrasynaptosomal BH₄ levels and varied both as a function of BH₄ concentration and length of incubation. A similar decrease in the amount of TH³²P-labeling was observed with the precursor of BH₄, sepiapterin. This effect, in turn, was reversed by the inhibitor of sepiapterin reductase, N-acetyl-serotonin. Finally, exposure of pre-³²P-labeled synaptosomes to the inhibitor of protein phosphatase 2A, okadaic acid, blocked the response to BH₄. Collectively, the data suggest that BH₄ stimulates the dephosphorylation of TH in situ and thus may play a dual role both as a cofactor for catalysis and a regulator of hydroxylase activity.Special issue dedicated to Dr. Bernard W. Agranoff.     

Paradoxical methylation of the tyrosine hydroxylase gene in mouse preimplantation embryos

The mouse tyrosine hydroxylase (Th) gene is located in an evolutionarily conserved imprinted gene cluster on distal chromosome 7. It is associated with a CpG island that spans the promoter of the gene. Using a bisulfite sequencing method we show that the Th promoter is fully methylated in both male and female mouse germ cells and in human spermatozoa, suggesting that it belongs to the newly identified category of CpG islands, the similarly methylated regions (SMRs). Contrary to other tissue-specific gene sequences, the mouse Th promoter escapes the initial wave of genome demethylation during the first few cell cycles, but becomes demethylated between the morula and the blastocyst stages. This unusual methylation ontogeny may be a characteristic of the SMRs and/or related to the localization of the Th gene in an imprinted gene cluster.     

Expression and characterization of catalytic and regulatory domains of rat tyrosine hydroxylase.

Phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase constitute a family of tetrahydropterin-dependent aromatic amino acid hydroxylases. Comparison of the amino acid sequences of these three proteins shows that the C-terminal two-thirds are homologous, while the N-terminal thirds are not. This is consistent with a model in which the C-terminal two-thirds constitute a conserved catalytic domain to which has been appended discrete regulatory domains. To test such a model, two mutant proteins have been constructed, expressed in Escherichia coli, purified, and characterized. One protein contains the first 158 amino acids of rat tyrosine hydroxylase. The second lacks the first 155 amino acid residues of this enzyme. The spectral properties of the two domains suggest that their three-dimensional structures are changed only slightly from intact tyrosine hydroxylase. The N-terminal domain mutant binds to heparin and is phosphorylated by cAMP-dependent protein kinase at the same rate as the holoenzyme but lacks any catalytic activity. The C-terminal domain mutant is fully active, with Vmax and Km values identical to the holoenzyme; these results establish that all of the catalytic residues of tyrosine hydroxylase are located in the C-terminal 330 amino acids. The results with the two mutant proteins are consistent with these two segments of tyrosine hydroxylase being two separate domains, one regulatory and one catalytic.     

Maternal and embryonic sources of tyrosine hydroxylase during Drosophila embryogenesis

Tyrosine hydroxylase (TH), the enzyme which catalyzes the conversion of tyrosine to L-DOPA and is rate limiting in catecholamine biosynthesis, is biochemically expressed in late stage wild-type Drosophila oocytes as well as in early embryogenesis. Null mutant alleles of TH (pale) are embryonic lethals with death occurring in the late embryonic or early larval periods of development. Staging of embryos demonstrated that inhibition of the enzymatic activity of TH by alpha-methyl-p-tyrosine (alphaMT) retards the progression of embryos primarily during the organogenesis stages of embryonic development, with lesser effects on earlier and later stages. On the other hand, time of gene action studies with a conditional temperature sensitive pale mutant (ple(ts1)) at its restrictive temperature (29 degrees C) indicate an onset of tyrosine hydroxylase gene action beginning in the oocyte stage of development. Thus, maternal as well as embryonic effects on the secretion and/or functionality of this enzyme may play roles in the early developmental program of the organism.     

Localization of tyrosine hydroxylase immunoreactive neurons in the forebrain of the guppy Poecilia reticulata

The current study reports for the first time the distribution of tyrosine hydroxylase immunoreactive (TH-ir) neurons in the forebrain of the guppy Poecilia reticulata . Numerous small TH-ir neurons were observed in the olfactory bulbs, located mainly in the periphery of the bulbs. The TH-ir telencephalic neurons are localized in the ventral telencephalic area where they are grouped in three distinct nuclei (Vv,Vd and Vp) composed of a small number of cells forming a continuous strip. The largest number of forebrain TH-ir neurons was observed in the diencephalon where both small and larger neurons are present. Diencephalic TH-ir neurons are subdivided in large nuclei located in the preoptic region (nSC, nPOp and nPOm), the thalamus (nDM), the pretectal region (nPPv and nAP), the hypothalamus (nPP and nRP) and the posterior tuberculum (nPT). Many diencephalic nuclei are distributed in periventricular regions and no TH-ir cells were observed in the paraventricular organ. A comparative analysis indicates that the present observations are consistent with the general pattern of TH-ir neurons distribution reported for the forebrain of other teleosts, but with some interspecies variability present, mainly in the diencephalon. This paper also provides valuable neuroanatomical information for P. reticulata , a teleost frequently used in toxicological tests, for future studies investigating the effects of environmental pollutants on the catecholaminergic system.     

The effects of enalapril maleate and cold stress exposure on tyrosine hydroxylase activity in some rat tissues

Enalapril is a highly specific and competitive inhibitor of angiotensin-I converting enzyme (ACE) and thus belongs to the category of ACE inhibitors. The beneficial effects of ACE inhibitors appear to result primarily from the suppression of the plasma renin-angiotensin-aldesterone system. This study was designed to detect the effects of enalapril maleate and cold stress on tyrosine hydroxylase (TH) activity in adrenal medulla, heart and hypothalamus in rat. In cold stress treatment (exposed to 8 degrees C cold for 48 h) TH activity was found to be raised significantly (p < 0.05) in adrenal medulla, hypothalamus and heart tissues. In the adrenal medulla, hypothalamus and heart tissues, TH activity of enalapril maleate treated rats (10 mg kg(-1) body weight) group was not raised significantly (p > 0.05). Following intraperitoneal injection of enalapril maleate (10 mg kg(-1) body weight) the rats were exposed to 8 degrees C cold for 48 h. After cold stress and enalapril maleate treatment no statistically significant change in tyrosine hydroxylase activity was detected in adrenal medulla, hypothalamus or heart (p > 0.05). The results of our studies show that enalapril maleate blocks the effect of cold stress on the regulation of TH activity.     

Nicotine induces tyrosine hydroxylase plasticity in the neurodegenerating striatum

It has been shown that nicotine prevents the loss of dopamine (DA) in the corpus striatum (CS) after 6-hydroxydopamine injection in the substantia nigra. To study the role of the enzyme tyrosine hydroxylase (TH; EC 1.14.16.2) in this experimental paradigm, we have examined its activity by assessing the accumulation of l-3,4-dihydroxyphenylalanine after inhibiting the subsequent enzyme in the DA synthetic pathway, aromatic l-amino acid decarboxylase, with 3-hydroxybenzylhydrazine. In addition the amount of TH protein was assessed by western blotting and its distribution in the CS was examined using immunohistochemical methods. 6-hydroxydopamine injection produced a significant decrease in DA levels and l-3,4-dihydroxyphenylalanine accumulation, as well as decreases in TH protein and TH immunoreactive fibres in the CS. After nicotine treatment, the decrease in TH protein in the CS was significantly reduced, with a concomitant preservation of TH activity, but nicotine did not alter the number of TH immunoreactive fibres. The activity and amount of TH did not change in the contralateral (intact) CS. Thus, nicotine induces long lasting TH plasticity in the degenerating CS. A synergistic action of nicotine-activated and lesion-originated signals appears necessary for the expression of this neuronal molecular plasticity.     

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.     

Inhibition of phenylalanine hydroxylase activity by alpha-methyl tyrosine, a potent inhibitor of tyrosine hydroxylase.

Inhibitors of phenylalanine hydroxylase and tyrosine hydroxylase were used in the assay of phenylalanine hydroxylase in liver and kidney of rats and mice. Parachlorophenylalanine (PCPA), methyl tyrosine methyl ester and dimethyl tyrosine methyl ester showed 5–15% inhibition while α-methyl tyrosine seemed to inhibit phenylalanine hydroxylase to the extent of 95–98% at concentrations of 5 × 10 ⁻⁵M –1 × 10 ⁻⁴M. After a phenylketonuric diet (0.12% PCPA + 3% excess phenylalanine), the liver showed 60% phenylalanine hydroxylase activity and kidney 82% that present in pair-fed normals. Hepatic activity was normal after 8 days refeeding normal diet whereas kidney showed 63% of normal activity. The PCPA-fed animals showed 34% in liver and 38% in kidney as compared to normals; in both cases normal activity was noticed after refeeding. The phenylalanine-fed animals showed activity similar to that seen in phenylketonuric animals. The temporary inducement of phenylketonuria in these animals may be due to a slight change in conformation of the phenylalanine hydroxylase molecule; once the normal diet is resumed, the enzyme reverts back to its active form. This paper also suggests that α-methyl tyrosine when fed in conjunction with the phenylketonuric diet may suppress phenylalanine hydroxylase activity completely in the experimental animals thus yielding normal tyrosine levels as seen in human phenylketonurics.     

Galanin inhibits tyrosine hydroxylase expression in midbrain dopaminergic neurons

Galanin (GAL) inhibits midbrain dopamine (DA) activity in several experimental paradigms, yet the mechanism underlying this inhibition is unclear. We examined the effects of GAL on the expression of tyrosine hydroxylase (TH) in primary cultures of rat embryonic (E14) ventral mesencephalon (VM). One micromolar GAL had no effect on the number of TH-immunoreactive (ir) neurons in VM cultures. However, 1 micro m GAL reduced an approximately 100% increase in TH-ir neurons in 1 mm dibutyryl cAMP (dbcAMP)-treated cultures by approximately 50%. TH-ir neuron number in dbcAMP-treated VM cultures was dose-responsive to GAL and the GAL receptor antagonist M40 blocked GAL effects. Semi-quantitative RT-PCR and quantitative immunoblotting experiments revealed that GAL had no effect on TH mRNA levels in VM cultures but reduced TH protein. VM cultures expressed GALR1, GALR2, and GALR3 receptor mRNA. However, dbcAMP treatment resulted in a specific approximately 200% increase in GALR1 mRNA. GALR1 activity is linked to a pertussis toxin (PTX)-sensitive opening of G protein-gated K+ channels (GIRKs). GAL reduction of TH-ir neuron number in dbcAMP + GAL-treated cultures was sensitive to both PTX and tertiapin, a GIRK inhibitor. GAL inhibition of midbrain DA activity may involve a GALR1- mediated reduction of TH in midbrain dopaminergic neurons.     

Tissue-specific developmental requirements of Drosophila tyrosine hydroxylase isoforms

Drosophila tyrosine hydroxylase (DTH) is a key enzyme in dopamine (DA) biosynthesis, which is expressed in neural and hypodermal DA-synthesizing cells. We previously reported that two DTH isoforms are produced in flies through tissue-specific alternative splicing that show distinct regulatory properties. We have now selectively expressed each DTH isoform in vivo in a pale (ple, i.e., DTH-deficient) mutant background. We show that the embryonic lethality of ple can be rescued by expression of the hypodermal, but not the neural, DTH isoform in all DA cells, indicating that the hypoderm- isoform is absolutely required for cuticle biosynthesis and survival in Drosophila. In addition, we report new observations on the consequences of DTH overexpression in the CNS and hypoderm. Our results provide evidence that tissue-specific alternative splicing of the DTH gene is a vital process in Drosophila development.     

Tissue-specific developmental requirements of Drosophila tyrosine hydroxylase isoforms

Drosophila tyrosine hydroxylase (DTH) is a key enzyme in dopamine (DA) biosynthesis, which is expressed in neural and hypodermal DA-synthesizing cells. We previously reported that two DTH isoforms are produced in flies through tissue-specific alternative splicing that show distinct regulatory properties. We have now selectively expressed each DTH isoform in vivo in a pale (ple, i.e., DTH-deficient) mutant background. We show that the embryonic lethality of ple can be rescued by expression of the hypodermal, but not the neural, DTH isoform in all DA cells, indicating that the hypoderm- isoform is absolutely required for cuticle biosynthesis and survival in Drosophila. In addition, we report new observations on the consequences of DTH overexpression in the CNS and hypoderm. Our results provide evidence that tissue-specific alternative splicing of the DTH gene is a vital process in Drosophila development.     

Neuropeptide Y immunoreactivity in the mammalian liver: pattern of innervation and coexistence with tyrosine hydroxylase immunoreactivity

Summary The distribution of nerve fibers displaying neuropeptide Y immunoreactivity in relationship to the catecholaminergic innervation of rat, guinea pig, and rabbit liver was investigated by single- and double-label immunofluorescence methods. In all three species, neuropeptide Y-immunoreactive fibers are prominent in association with the vasculature, biliary pathway, and stromal compartment. The neuropeptide Y innervation of the parenchyma, on the other hand, differs among the three species in term of density. It is quite sparse in the rat and rabbit, particularly in the former species. In the guinea pig liver, numerous single, varicose neuropeptide Y-containing nerve fibers innervate the hepatic parenchyma; often, thin processes surround single hepatocytes and lie close to sinusoids. The immunoreactive pattern of tyrosine hydroxylase, a marker for catecholaminergic neurons and fibers, is comparable to that of neuropeptide Y. Most neuropeptide Y-containing nerve fibers also contain tyrosine hydroxylase immunoreactivity, in all three species, with the exception of the rabbit parenchyma, where a substantial proportion of catecholaminergic fibers lack immunoreactivity for neuropeptide Y. Finally, systemic administration of the sympathetic neurotoxin, 6-hydroxydopamine, in rats and guinea pigs resulted in virtually complete elimination of both neuropeptide Y- and tyrosine hydroxylase-immunoreactive fibers. These findings are consistent with the hypothesis that neuropeptide Y-containing nerve fibers form a subpopulation of the sympathetic innervation of the mammalian liver, which is likely to originate from prevertebral sympathetic ganglia.     

Purification and characterization of the active form of tyrosine hydroxylase from mesangial cells in culture

The capacity of mesangial cells (MC) to produce catecholamines (CAs) has been investigated in our laboratory. To study the CA cascade, it is necessary to examine some steps in their metabolic pathway. Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in the biosynthesis of these biogenic amines (dopamine (DA), norepinephrine (NE), and epinephrine (EPI)). Since the glomerular mesangium is their target in the regulation of renal sodium transport and renin secretion, the aim of the study was to determine the presence of TH in these cells in culture. The CA levels were detected in immortalized MC by high-performance liquid chromatography with electrochemical detection. The following concentrations were found in the intracellular region and in the medium, respectively: NE = 284 +/- 31 and 134 +/- 22, EPI = 75 +/- 14 and 22 +/- 5, and DA = 42 +/- 14, 40 +/- 20 pg/mg cell protein. The enzymatic activity of the cell lysate and medium was measured based on L-dopa formation. In the presence of o-phenanthroline, both samples presented 39% inhibition. The biopterin was detected in the intracellular and in the medium (64.87 and 631.99 pmol/mg protein, respectively) using high-performance liquid chromatography with ultraviolet detection. The cell lysate was submitted to a DEAE-Sephacel column, followed by gel filtration, and Heparin-Sepharose. TH was purified 613.16-fold with a specific activity of 466.0 pg/mg cell protein. Immunoblotting using monoclonal antibody revealed the presence of TH in the different purification steps. Purified TH was sequenced, presenting an alignment with amino-terminal sequence of mouse enzyme. Our results demonstrated the presence of active TH in MC, suggesting that these cells are able to produce CA "in vivo", and establishing a convenient purification method for TH that can be applied to the study of the molecular properties of the enzyme modified "in vivo" by different physiological and pathophysiological stimuli.