Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.14.16.2 (tyrosine hydroxylase)
 14,760 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Defined regions (septum, substantia nigra) of the embryonic central nervous system (CNS) were transplanted into the sciatic nerves of young adult rats. Immunocytochemical techniques were used to examine the expression of neurotransmitter related enzymes and neuronal cytoskeletal proteins in the grafts. The origin of the septal grafts was confirmed by immunoreactivity in neurons to choline acetyltransferase and the beta-nerve growth factor receptor (192-IgG). In substantia nigra grafts, neuronal perikarya and processes were identified with an antibody directed against tyrosine hydroxylase. Typical spatial distributions of phosphorylated (Mr 200,000) and non-phosphorylated (Mr 168,000 & 200,000) neurofilaments were observed in the short term (1-2 months) grafts with the monoclonal antibodies RT97 and SMI-32 respectively. Dense dendrite arbors and neuronal cell bodies were immunostained with an antibody that recognizes a high molecular weight microtubule associated protein (MAP2). In the long term (1 year) transplants, prominent cytoskeletal changes in the somata, axons and dendrites of neurons were evident. The cells showed a shift in phosphorylated neurofilament staining from the axon to the soma accompanied by a reduction in axonal immunoreactivity in the adjacent neuropil. Other abnormal features included swollen perikarya, hypertrophied axonal segments and short segments of kinked axons. Regression of the dendrite trees in the long standing grafts was also apparent when sections were reacted with the MAP2 antibody. These experiments indicate that grafted fetal neurons, isolated in the peripheral nervous system, differentiate and express markers like their counterparts in situ. After extended time periods under these circumstances, cytoskeletal modifications become apparent in the neurons. These aberrant changes are similar to morphological characteristics associated with aging and neurodegenerative disorders. This experimental paradigm offers a new approach to study cytoskeletal disturbances in neurons and provides a unique opportunity to examine conditions that may modulate the abnormal changes.
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PMID:Transplantation of fetal CNS tissue into the peripheral nervous system: a model to study aberrant changes in the neuronal cytoskeleton. 166 43

In the presence of nerve growth factor (NGF) PC12 pheochromocytoma cells develop properties of sympathetic neurons and extend long microtubule-containing neurites. PC12 cell microtubule protein was isolated using an assembly and disassembly procedure, either directly from an unlabeled cell extract or by copolymerization of a [35S]-methionine-labeled cell extract with rat brain microtubule proteins. Microtubule proteins of PC12 cells treated and untreated with NGF did not reveal any differences as analyzed by SDS-PAGE. The major constituents were the tubulin dimer and microtubule-associated proteins, mainly one high molecular weight component (HMW2) (Mr = 290,000), which resembles by several criteria the high molecular weight protein MAP2 of rat brain microtubule protein. PC12 cell microtubule protein contained intrinsic cAMP-dependent and cAMP-independent protein kinase activity. In vitro phosphorylation experiments indicated that, unlike in rat brain microtubule preparations, both subunits of the tubulin dimer were substrate for the endogenous kinase activity. The addition of cAMP to the incubation medium exerted a slight increase in phosphorylation of the HMW2 protein and an 80,000 polypeptide, and most effectively the phosphorylation of a Mr = 62,000 peptide was increased. This component could be identified as tyrosine hydroxylase by indirect immunoprecipitation procedures. Thus, the in vitro phosphorylation pattern of PC12 cell microtubule protein differed substantially from the one obtained from rat brain microtubule preparations.
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PMID:Microtubule proteins in PC12 pheochromocytoma cells. Isolation, composition and in vitro phosphorylation. 632 Nov 30

The neuropeptides galanin (GAL) and vasoactive intestinal polypeptide (VIP) are upregulated in spinal and vagal sensory as well as in cranial motor neurons after axonal transection. In this study an increase of both peptides is demonstrated in axotomized principal ganglionic neurons (PGN) of the rat sympathetic superior cervical ganglion by use of double-labeling immunofluorescence. Compared to control ganglia that do not contain more than 1% GAL- or VIP-positive cells, about 26% of all PGN exhibit GAL immunoreactivity by day 1 after transection of the major postganglionic branches. The proportion of immunoreactive neurons reaches its maximum after 30 days (40%) and decreases to about 27% within the second month after axotomy. The percentage of VIP-positive neurons is much lower than for GAL: 2% of the PGN exhibit VIP immunoreactivity at day 1 and about 7% are observed 30 and 60 days after axotomy. In order to further characterize newly GAL- and VIP-positive PGN, their cell diameters were determined 12 days after axotomy. Compared to the mean overall neuron diameter of 24.8 microns, GAL-immunoreactive neurons are predominantly of small and intermediate size (22.2 microns), whereas VIP occurs mainly in larger neurons (26.1 microns). Besides cell bodies, many intraganglionic nerve fibers stain positive for GAL or VIP, particularly at day 6. Most likely, these fibers represent axons, as indicated by the absence of MAP2, a cytoskeletal protein found in neuronal somata and dendrites. They establish direct membrane contacts with postganglionic perikarya, as revealed by pre-embedding immuno-electron microscopy. Some cell bodies and fibers contain both peptides. Colocalization of GAL or VIP with tyrosine hydroxylase (TH), the rate-limiting enzyme of catecholamine synthesis, reveals a reduced immunoreactivity for TH in intensely GAL- or VIP-positive cells, and vice versa at day 6. However, no difference in staining intensity for VIP or GAL, and TH, is observed after 30 and 60 days. Possible implications of GAL and VIP for peripheral nerve regeneration and their regulation by target-derived factors are discussed.
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PMID:Plasticity of postganglionic sympathetic neurons in the rat superior cervical ganglion after axotomy. 752 68

Double-labelling immunofluorescence was applied on single sections of the rat superior cervical ganglion to evaluate neurochemistry and connectivity of intraganglionic SIF cells. The synaptic vesicle membrane protein synaptophysin and secretoneurin, a newly discovered neuropeptide derived from secretogranin II, proved reliable molecular markers of this cell type, whereas serotonin and tyrosine hydroxylase immunoreactivities were observed in slightly incongruent SIF cell subpopulations. Immunolabelling for vasoactive intestinal polypeptide and neuropeptide Y occurred in few SIF cells. None of the above immunoreactivities were visibly altered by preganglionic or postganglionic denervation, while some SIF cells were immunolabelled for galanin or for the neuronal microtubule-associated protein MAP2 after postganglionic denervation. SIF cells were nonreactive for the pan-neuronal marker protein gene product (PGP) 9.5 or neurofilament 160 kD. Intense staining of NADPH-diaphorase in some SIF cells, suggesting catalytic activity of nitric oxide synthase, could not be substantiated by immunoreactivity for this enzyme. SIF cells were approached by nonidentical fiber populations immunoreactive for PGP 9.5, neurofilament, or neuropeptide Y, whereas immunoreactivities for galanin and vasoactive intestinal polypeptide were colocalized in fiber meshes around SIF cells. The findings indicate (1) neurochemical SIF cell heterogeneity, (2) SIF cell plasticity in response to ganglionic perturbation, and (3) a differentiated innervation of SIF cells in the rat superior cervical ganglion.
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PMID:Immunohistochemistry of small intensely fluorescent (SIF) cells and of SIF cell-associated nerve fibers in the rat superior cervical ganglion. 781 35

Intact bovine adrenal medullary chromaffin cells were preincubated with 32PO4, and the multiple-site phosphorylation of tyrosine hydroxylase (TH) was studied. Up to eight 32P-labeled peptides were produced by tryptic hydrolysis of TH; however, all of the tryptic phosphopeptides were derived from four phosphorylation sites--Ser8, Ser19, Ser31 and Ser40. In situ regulation of 32P incorporation into the latter three sites was demonstrated with a diverse set of pharmacological agents. 32P incorporation into Ser19 was preferentially increased by brief exposures to depolarizing secretagogues. Longer treatments also increased Ser31 and Ser40 phosphorylation. Nicotine, muscarine and vasoactive intestinal polypeptide--reflecting cholinergic and non-cholinergic components of sympatho-adrenal transmission--each produced different patterns of multiple-site phosphorylation of TH. Nicotine, bradykinin and histamine increased 32P incorporation at each of the three sites whereas muscarine, angiotensin II, endothelin III, prostaglandin E1, GABA and ATP selectively increased Ser31 phosphorylation. Nerve growth factor did not influence TH phosphorylation in chromaffin cells from adult adrenal glands but selectively increased Ser31 phosphorylation in chromaffin cells isolated from calf adrenal glands. 32P incorporation into Ser40 was selectively increased by forskolin and other cAMP-acting agents whereas vasoactive intestinal polypeptide increased Ser31 and Ser40 phosphorylation. Thus, the phosphorylation of TH in bovine chromaffin cells appears to be regulated at three sites by three separate intracellular signaling pathways--Ser19 via Ca2+/calmodulin-dependent protein kinase II; Ser31 via ERK (MAP2 kinases); and Ser40 via cAMP-dependent protein kinase. These signaling pathways, as well as the extracellular signals that were effective in stimulating them, are similar to those previously described for TH in rat pheochromocytoma cells. However, several of the pharmacological agents produced different patterns of multiple-site TH phosphorylation in the bovine chromaffin cells. These differences between tissues could be accounted for by differences in the coupling/access between the extracellular signal transduction systems and the intracellular signaling pathways as opposed to differences in the intracellular signaling pathways per se.
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PMID:Multiple signaling pathways in bovine chromaffin cells regulate tyrosine hydroxylase phosphorylation at Ser19, Ser31, and Ser40. 809 28

The search for specific neurotrophic factors that will eventually be used to reduce or arrest the rate of degeneration of dopaminergic neurons in Parkinson's disease is being pursued by first testing the ability of putative compounds to increase the survival of dopaminergic neurons in primary cultures of the fetal, ventral mesencephalon. This research has intensified in recent years. The experimental procedures used by different laboratories in these studies differ widely, and meaningful comparisons of the results obtained are accordingly difficult to make. Some important experimental variables include the age of the fetal tissue used; the dissection technique used to isolate the ventral mesencephalon; the percentage of dopaminergic neurons present in the culture initially; handling of the tissue during dissection; the technique used to disperse the cells; the use of serum; the technique of plating the cells; the attachment factors used; detachment and loss of cells during the staining procedure; the age of the cultures at the time of analysis; the uneven distribution of cells at the time of analysis and the use of imaging techniques in the analysis. We show that when the E14 rat embryo is used, it is possible to consistently obtain a culture with 20% of tyrosine hydroxylase-positive neurons. Neither the plating density in the range of 7.8 x 10(3) to 1.25 x 10(5) cells/cm2, nor the percentage of serum in the growth medium affected the percentage of cells that expressed TH initially, at 4 or 12 h after plating. When the cells were plated as 25 microliters droplets, called microislands (area approximately 12.5 mm2), and allowed to attach before additional growth medium was added, cell density remained uniform at the center of the microisland for the duration of the culture. Restriction of the analysis of cell survival to the center of the microisland therefore helped to decrease the variability in counting that could occur when cells are dispersed over a larger area. In contrast, in an 8-well chamber slide or 35 mm petri dish, in which the whole area is plated, cell density was consistently higher at the edge (edge effect), versus the centre, by a factor of about three. The use of microisland cultures also has the additional benefit of increasing by a factor of about five the number of individual cultures that can be set up per liter, and a proportionate reduction in the number of animals used per experiment. When the percentage of serum in the growth medium was 0% always, or 10% for the first 12 h, and 0% thereafter, or 10% always, the number of TH-pos neurons per field (using a x 20 objective, column factor 1.25; area 320 microns2) after 5 days in culture (DIV5) was < 1,3-8 and 14-22, respectively. Under the same experimental conditions, the number of neurons (MAP2-positive) per field was 5-8, 18-30 and 45-65 (N = 10 in all cases), respectively. Serum deprivation therefore has a highly deleterious effect on neuronal survival in culture. We suggest that cultures that were exposed to serum at any stage of the experiment, should not be referred to as "serum-free', since even a brief exposure to serum exerts a protective effect on neurons, and especially on dopaminergic neurons. Instead, the percentage and kind of serum used, the exact usage, and the duration of exposure of the cells to serum should be stated. Finally, it is suggested that where possible, an imaging system with manual count and journaling capabilities be used in the analysis. The methods described are illustrated by dose-response curves of the neurotrophic effects of BDNF, NGF-beta and IL-6 versus percentage survival on dopaminergic neurons, when grown in serum-free medium throughout.
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PMID:Standardized methods to bioassay neurotrophic factors for dopaminergic neurons. 884 22

An adenovirus encoding tyrosine hydroxylase (TH) activity was inserted in neuronal and glial cultured cells obtained from human fetal central nervous system (CNS) tissue. Using a double fluorescence immunostaining, we characterized inoculated CNS cells, with a TH antiserum and one of the following antibodies: microtubule-associated protein (MAP2) and GABA for neuronal cells, vimentin (Vim) for glial cells and glial fibrillary acidic protein (GFAP) for astrocytes. The characterization of inoculated neuronal cells was established by the detection of TH-MAP2-stained neurons in cultures obtained from the thoracic and lumbar parts of the spinal cord where no intrinsic TH cells are described. Inoculated glial cells were characterized by the detection of TH-Vim and TH-GFAP-stained CNS cultured cells. We also observed GABA neurons expressing TH immunoreactivity which could be considered as inoculated neurons expressing the GABA phenotype. Whatever the time of inoculation, transfection was observed in both neuronal and glial cells, after up to 4 months of culture. Although no precise quantitation was performed, the percentage of inoculation was found on microscopic inspection to be greater in glia than in neurons, as previously reported. We concluded that a gene coding for a key neuronal enzyme can be incorporated in embryonic human glial and neuronal cells through the use of a recombinant adenovirus.
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PMID:An adenovirus vector encoding tyrosine hydroxylase activity may enter human CNS cells in primary dissociated cultures. 893 Jun 92

The effects of striatal target cells on the morphological development of dopaminergic neurons were studied in dissociated cultures of embryonic rat mesencephalon. Mesencephalic neurons were cultured for four days in presence of target striatal cells or non target cerebellar ones. The outgrowth of dopaminergic neurons, visualized after tyrosine hydroxylase immunohistochemistry, was examined by quantitative morphometry. In cocultures, the increased complexity of dopaminergic neurites (branching) was the most striking pattern. It was dependent on the presence of target striatal cells as compared to non target ones. Cultures raised in presence or absence of serum lead to suggest the implication of striatal neurons rather than glia. Using MAP2 and phosphorylated neurofilaments immunohistochemistry in combination with tyrosine hydroxylase immunolabelling, it could be shown that the target-induced branching effect concerned only axonal and not dendritic processes. To further define whether diffusible factors from the striatal target would participate in the axonal branching effect, mesencephalic cells were cultured in conditioned medium from striatal neurons. Striatal conditioned medium enhanced dopamine uptake and dopamine neuron branching to the same extent as that observed in striatal cocultures. These findings demonstrate that soluble factors secreted by striatal neurons themselves selectively influence the branching of dopaminergic axons in vitro.
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PMID:Striatal target-induced axonal branching of dopaminergic mesencephalic neurons in culture via diffusible factors. 916 62

Metabotropic glutamate receptors (mGluRs), which couple glutamate to second messengers, have important roles in the regulation of movement by the basal ganglia. We used two polyclonal antisera to mGluR1a and mGluR2/3 and confocal laser microscopy to investigate the localization of these receptors in the basal ganglia of the rat. The mGluRs were visualized in combination with an antibody to tyrosine hydroxylase (TH), an antibody to microtubule-associated protein 2 (MAP2, a dendritic marker), or SV2 (an antibody to a protein associated with presynaptic terminals). In the neostriatum, punctate mGluR1a immunoreactivity (ir) was present in the neuropil. This staining did not colocalize with MAP2-ir or SV2-ir and was not altered by decortication or unilateral 6-hydroxydopamine (6-OHDA) lesions. In the globus pallidus and substantia nigra pars reticulata, however, mGluR1a-ir was tightly clustered along large MAP2-ir dendrites. In contrast to the variations in mGluR1a-ir staining, similar punctate neuropil mGluR2/3-ir staining was observed within all basal ganglia structures. In the neostriatum, these puncta were abundant; unlike mGluR1a, many mGluR2/3-ir puncta colocalized with SV2-ir, and striatal mGluR2/3-ir puncta were markedly reduced in number after decortication. Neither mGluR1a-ir nor mGluR2/3-ir could be detected in TH-ir soma within substantia nigra pars compacta, or in TH-ir striatal terminals. Overall, our observations suggest that mGluR1a and mGluR2/3 receptors have distinct cellular localizations in different components of the basal ganglia circuitry and are likely to subserve distinct functions. Our data support the presence of mGluR2/3 on the terminals of corticostriatal afferents, where they may regulate glutamate release. In contrast, mGluR1a appears to be a postsynaptic receptor of neurons in the neostriatum, globus pallidus, and substantia nigra pars reticulata.
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PMID:Immunohistochemical localization of metabotropic glutamate receptors mGluR1a and mGluR2/3 in the rat basal ganglia. 945 72

Previous work has demonstrated that the bone morphogenetic proteins (BMP)-2, BMP-4, and BMP-7 can promote the development of tyrosine hydroxylase (TH)-positive and catecholamine-positive cells in quail trunk neural crest cultures. In the present work, we showed that mRNA for the type I bone morphogenetic protein receptor IA (BMPR-IA) was present in neural crest cells grown in the absence or presence of BMP-4. We have used a replication-competent avian retrovirus to express a constitutively active form of BMPR-IA in neural crest cells in culture. Cultures grown in the absence of BMP-4 and infected with retrovirus containing a construct encoding this activated BMPR-IA developed five times more TH-immunoreactive and catecholamine-positive cells than uninfected control cultures or cultures infected with virus bearing the wild-type BMPR-IA cDNA. The number of TH-positive cells which developed was dependent on the concentration of virus bearing the activated receptor cDNA used in the experiments. Most TH-positive cells which developed also contained viral p19 protein. Total cell number was not affected by infection with the virus containing the activated receptor construct. The effect of the activated receptor was phenotype-specific since infection with the virus bearing the activated receptor cDNA did not alter the number or morphology of microtubule-associated protein (MAP)2-immunoreactive cells, which are distinct from the TH-positive cell population. These findings are consistent with the observation that MAP2-positive cells are not affected by the presence of BMP-4. Taken together, these results suggest that activity of BMPR-IA is an important element in promoting the development of the adrenergic phenotype in neural crest cultures.
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PMID:Expression of a constitutively active type I BMP receptor using a retroviral vector promotes the development of adrenergic cells in neural crest cultures. 952 84


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