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Query: UNIPROT:P06889 (Mol)
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The addition of epidermal growth factor (EGF) to cultures of the rat PCG2 pheochromocytoma cell line increased the level of RNA coding for tyrosine hydroxylase (TH). A region of DNA containing 5'-flanking sequences of the TH gene was fused to a heterologous gene and transfected into a rat anterior pituitary cell line, GH4. The TH gene sequences from +27 to -272 contained information sufficient for the induction of TH by EGF. Two regions within this TH DNA were extensively homologous to the EGF regulatory element of the rat prolactin gene.
Mol Cell Biol 1987 Sep
PMID:Regulated expression of the tyrosine hydroxylase gene by epidermal growth factor. 289 99

PC12 cells on extracellular matrix (ECM) or plastic were incubated with 3H-tyrosine (3H-TY) in the presence and absence of serum or cold tyrosine. 3H-Dopamine (3H-DA) was determined in medium and cells from 1 to 48 h later with Dowex cation exchange chromatography. In serum-free and tyrosine-free medium, PC12 cells on ECM released significantly more 3H-DA, whereas cells on plastic had a significantly higher cellular content of 3H-DA, but total 3H-DA (medium plus cells) was equal in ECM and plastic cultures. When 3H-TY was added to tyrosine-containing medium, there was a significant decrease in the levels of 3H-DA detected and the differences between ECM and plastic cultures were attenuated, but the patterns of secretion and storage were similar to those observed with tyrosine-free medium and total synthesis did not decline at 48 h. Serum decreased the efficiency of the resin to retain 3H-DA from culture medium, attenuated the difference in dopamine release between ECM and plastic cultures, and contributed to variations in 3H-TY uptake. The morphometric relationship between the cell membrane and the internal compartment in PC12 cells of different shapes was also characterized. The perimeter length and area of the midsection of cells were determined with a modular system for quantitative digital analysis. The perimeter length of cells on ECM was significantly greater than cells on plastic, whereas the internal areas were similar. The ratio of perimeter length to area (P/A) for all cells on ECM was 30% higher than the P/A ratio for cells on plastic. The ratio of P/A for a subpopulation of very flat cells on ECM was 70% higher than the ratio for round cells on plastic. Immunocytochemistry for tyrosine hydroxylase revealed a more diffuse distribution of this enzyme in cells on ECM. These data suggest that there is an increase in the ratio of cell surface area to cell volume as PC12 cells spread on ECM which could facilitate secretory vesicle fusion with the cell membrane, and hence, exocytosis. Although there is a concomitant increase in the secretion of dopamine and a decrease in the storage of dopamine, the change in cell shape does not appear to immediately alter the synthesis of dopamine.
Mol Cell Endocrinol 1987 Nov
PMID:Extracellular matrix changes PC12 cell shape and processing of newly synthesized dopamine. 289 May 44

Activation of neurotransmitter receptors can regulate transcription in postsynaptic cells through the actions of second messengers. Trans-synaptic regulation of transcription appears to be an important mechanism controlling the synthesis of molecules involved in neuronal signaling, especially neuropeptides. Proenkephalin, vasoactive intestinal polypeptide, and somatostatin have been shown to be transcriptionally regulated by the second messenger, cyclic AMP (cAMP), as has the catecholamine synthesizing enzyme tryosine hydroxylase. cAMP-inducible elements have been mapped within these genes, and trans-acting factors which bind to several such elements have been identified. With the discovery that individual neurons generally contain multiple transmitters within their synaptic terminals, it has become important to understand in detail the mechanisms by which the synthesis of transmitters can be coregulated. Here we compare the structure and function of the proenkephalin cAMP-inducible enhancer with the mapped cAMP-inducible elements of the vasoactive intestinal polypeptide, somatostatin, and tyrosine hydroxylase genes and a putative cAMP-inducible element in the proto-oncogene c-fos. We have previously shown that the proenkephalin enhancer is composed of two different elements, ENKCRE-1 and ENKCRE-2. We show here that one of these, ENKCRE-2, is structurally similar to elements found within the vasoactive intestinal polypeptide, somatostatin, and tyrosine hydroxylase genes and binds a trans-acting factor that is competed for both in cotransfection experiments (in vivo) and in DNase I footprint assays (in vitro) by these other elements. The c-fos element has similar structural requirements to confer transcriptional induction by cAMP but competes less strongly. Protein purified by affinity chromatography with the ENKCRE-2 sequence binds to each of these elements. A second element within the proenkephalin cAMP-inducible enhancer, ENKCRE-1, binds a factor that is not competed for by these other genes and is therefore distinct. This analysis suggests a potential mechanism of transcriptional coregulation of the neuronally expressed genes investigated in this study and also demonstrates that multiple factors are involved in transcriptional activation by cAMP.
Mol Cell Biol 1988 Oct
PMID:A common trans-acting factor is involved in transcriptional regulation of neurotransmitter genes by cyclic AMP. 290 36

Steroid hormones modify several brain functions, at least in part by altering expression of particular genes. Of interest are those genes that are involved in cell-cell communication in the brain, for instance neuropeptide genes and genes that code for enzymes involved in synthesis of neurotransmitters. Steroid regulation of mRNA levels for several genes has been reported, including the genes coding for the neuropeptides vasopressin, corticotropin releasing factor, luteinizing hormone-releasing factor, pro-opiomelanocortin; somatostatin, preproenkephalin, and the enzyme tyrosine hydroxylase. Steroid control of releasing factor genes is consistent with classical neuroendocrine concepts of negative feedback. Steroid-induced plasticity of gene expression is sometimes in evidence, with the presence or absence of a particular steroid inducing expression of a neuropeptide gene in neurons that under other conditions do not express the gene. As a means of gaining some insight into the mechanism of action of steroid hormones, several groups have determined some of the neuropeptide profiles of neurons that contain receptors for steroid hormones. Marked heterogeneity is found, in that often only a subpopulation of phenotypically-similar neurons, even within a single brain area, contains receptors for a given steroid.
Mol Neurobiol 1988
PMID:Regulation of neuropeptide gene expression by steroid hormones. 307 66

1. We have used horseradish peroxidase-conjugated protein A- and 125I-protein A to develop immunohistochemical and radioimmunohistochemical methods for the localization of antigens in brain and other tissues of the rat. 2. We visualized methionine-enkephalin fibers in the rat brain by incubating tissue sections with a specific polyclonal antibody and peroxidase-conjugated protein A. The method is simple, fast, and less expensive and more sensitive than classical immunohistochemical techniques and the principle could be used to visualize many other tissue antigens. 3. Incubation of tissue samples with specific polyclonal antibodies and 125I-protein A, followed by autoradiography, allows the permanent recording of the radioimmunohistochemical localization of brain methionine-enkephalin, tyrosine hydroxylase, and angiotensin-converting enzyme and of pituitary vasopressin and could be applied to the localization of many other tissue antigens. 4. A new quantitative radioimmunohistochemical technique for methionine-enkephalin allows the determination of the endogenous peptide content in discrete brain nuclei from 16-microns-thick sections. The method is based on the quantitative determination of the amount of 125I-protein A bound to specific tissue areas after incubation with a specific polyclonal antibody, followed by autoradiography and computerized microdensitometry. To quantify the endogenous peptide content, the values obtained are interpolated into a methionine-enkephalin internal standard curve. This standard curve was constructed by measuring endogenous concentrations of methionine-enkephalin by radioimmunoassay in specific brain regions and correlating these values with quantitative autoradiographic determinations in homologous areas of adjacent sections. Similar methods can be developed for other tissue antigens. 5. These new methods allow for the localization and quantification of tissue antigens in very discrete areas of the brain and other tissues and have a wide application in neurobiology and pathology.
Cell Mol Neurobiol 1988 Mar
PMID:Radioimmunochemical methods for the quantitative autoradiographic determination of antigens in brain and other tissues. 340

PC12 cells are spherical when cultured on plastic, but flatten and spread extensively when cultured on extracellular matrix (ECM) produced by bovine corneal endothelial cells. We previously demonstrated that cells which have spread on ECM release more dopamine and contain less intracellular dopamine than cells which are rounded on plastic cultureware. Glucocorticoids increase dopamine production in PC12 cells presumably via an increase in tyrosine hydroxylase gene transcription. We questioned whether cell shape as determined by ECM would change the response of PC12 cells to glucocorticoids. Dopamine release and cell content were measured by a radioenzymatic assay in serum-free cultures of PC12 cells on ECM and plastic treated with various glucocorticoids in a dose-response fashion for 24 or 48 h. In addition, the response of cells on both substrata to one dose of dexamethasone was examined from 3-48 h. PC12 cells on ECM and plastic exhibited a dose-related increase in dopamine release and content when treated for 24 h with corticosterone or for 48 h with dexamethasone, corticosterone or cortisol. The ED50s for dexamethasone- and corticosterone-stimulated release and content were similar for cells on ECM and plastic as were the doses at which the first significant increases were detected. The average times at which the first significant increase in release and content occurred were also similar for cells on ECM and plastic. PC12 cells on ECM continue to release more dopamine and store less dopamine than cells on plastic with glucocorticoid treatment. However, glucocorticoid-treated cells on ECM showed a greater percent increase over ECM controls in the cell content of dopamine, whereas glucocorticoid-treated cells on plastic showed a greater percent increase over controls in the release of dopamine. It is hypothesized that glucocorticoids increase dopamine production to a similar extent in cells on ECM and plastic, but that storage is facilitated in cells on ECM.
Mol Cell Endocrinol 1987 Apr
PMID:Glucocorticoid stimulation of dopamine production in PC12 cells on extracellular matrix and plastic. 356 53

Differential screening of cDNA libraries was used to detect and prepare probes for mRNAs that are regulated in PC12 rat pheochromocytoma cells by long-term (2-week) treatment with nerve growth factor (NGF). In response to NGF, PC12 cells change from a chromaffin cell-like to a sympathetic-neuron-like phenotype. Thus, one aim of this study was to identify NGF-regulated mRNAs that may be associated with the attainment of neuronal properties. Eight NGF-regulated mRNAs are described. Five of these increase 3- to 10-fold and three decrease 2- to 10-fold after long-term NGF exposure. Each mRNA was characterized with respect to the time course of the NGF response, regulation by agents other than NGF, and rat tissue distribution. Partial sequences of the cDNAs were used to search for homologies to known sequences. Homology analysis revealed that one mRNA (increased 10-fold) encodes the peptide thymosin beta 4 and a second mRNA (decreased 2-fold) encodes tyrosine hydroxylase. Another of the increased mRNAs was very abundant in sympathetic ganglia, barely detectable in brain and adrenals, and undetectable in all other tissues surveyed. One of the decreased mRNAs, by contrast, was very abundant in the adrenals and nearly absent in the sympathetic ganglia. With the exception of fibroblast growth factor, which is the only other agent known to mimic the differentiating effects of NGF on PC12 cells, none of the treatments tested (epidermal growth factor, insulin, dibutyryl cyclic AMP, dexamethasone, phorbol ester, and depolarization) reproduced the regulation observed with NGF. These and additional findings suggest that the NGF-regulated mRNAs may play roles in the establishment of the neuronal phenotype and that the probes described here will be useful to study the mechanism of action of NGF and the development and differentiation of neurons.
Mol Cell Biol 1987 Sep
PMID:Identification and characterization of mRNAs regulated by nerve growth factor in PC12 cells. 367 Mar 9

The effects of lisuride and of the R(-)- and S(+)-enantiomers of apomorphine were examined on 3,4-dihydroxyphenylalanine (DOPA) production by striatal synaptosomes and by crude, soluble striatal tyrosine hydroxylase. Due to their catechol structure, the enantiomers were almost equally effective in blocking soluble tyrosine hydroxylase (EC 1.14.16.2) (IC50 = 470 and 890 nM for R(-)- and S(+)-apomorphine, respectively), provided incubations were performed at pH 7.2 with 1 mM tetrahydrobiopterin as cofactor. The enantiomers were similarly effective in blocking synaptosomal DOPA production (IC50 = 410 and 970 nM for R(-)- and S(+)-apomorphine, respectively). As S(+)-apomorphine but not R(-)-apomorphine is considered to be a dopamine antagonist, these results support the assumption that the block of synaptosomal DOPA production by both apomorphine enantiomers is due to a direct inhibition of tyrosine hydroxylase. Lisuride at high concentrations (10-100 microM) blocked DOPA production in striatal synaptosomes; simultaneously, intrasynaptosomal dopamine was depleted. These data support the assumption that lisuride inhibits DOPA production indirectly, similar to reserpine. In accordance with this assumption, lisuride was without effect on DOPA production in dopamine-depleted synaptosomes. These results demonstrate that inhibition of synaptosomal DOPA production by at least some dopamine agonists may be explained by direct inhibitory effects on tyrosine hydroxylase.
Mol Pharmacol 1985 Dec
PMID:Effects of apomorphine enantiomers and of lisuride on 3,4-dihydroxyphenylalanine production in striatal synaptosomes. 393 8

Incubation of pheochromocytoma cells with 56 mM K+ or with cholera toxin increases the conversion of [14C]tyrosine to [14C]catecholamines (Chalfie, M., Settipani, L., and Perlman, R. L. (1979) Mol. Pharmacol. 15, 263-270). We have now measured the tyrosine content and the rate of dihydroxyphenylalanine production in these cells. Incubation with 56 mM K+ or with cholera toxin increases the rate of dihydroxyphenylalanine production but decreases the tyrosine content of the cells. We have also measured the uptake of tyrosine into pheochromocytoma cells. The rate of tyrosine uptake is more than 1 order of magnitude greater than the rate of dihydroxyphenylalanine production. Moreover, tyrosine uptake is not affected by cholera toxin and is decreased by approximately 30% in media that contain 56 mM K+. These results provide direct evidence that tyrosine 3-monooxygenase regulates catecholamine synthesis in pheochromocytoma cells and that incubation with 56 mM K+ or with cholera toxin causes the activation of this enzyme in these cells.
 ...
PMID:Tyrosine 3-monooxygenase regulates catecholamine synthesis in pheochromocytoma cells. 610 63

The steady-state kinetics of tyrosine hydroxylase [L-tyrosine, tetrahydropteridine: oxygen oxidoreductase (3-hydroxylating); EC 1.14.16.2] frequently exhibits complex features which confound interpretation of the results. Using an assay-enzyme system which is essentially devoid of the major mitigating kinetic features, a comprehensive kinetics data base has been compiled. The studies employed L-tyrosine, 5,6,7,8-tetrahydrobiopterin, and oxygen as substrates, and 3-(3',4'-dihydroxyphenyl)L-alanine, a deazapterin, 3-iodo-L-tyrosine, and dopamine as product, substrate analogue, and product analogue inhibitors, respectively. All three reactants were varied pairwise, and all inhibitors (except dopamine) were tested with each of the three substrates as variable substrate. The entire data base was interpreted exclusively in terms of models for classic saturation kinetics of enzyme catalysis, providing an internally consistent kinetic model and evidence for a sequential mechanism with partially ordered sequences for substrate addition and product release. Some possible mechanisms and experimental variables relating these results to more complex kinetics of tyrosine hydroxylase are considered briefly.
Mol Pharmacol 1983 Jan
PMID:Steady-state kinetics of bovine striatal tyrosine hydroxylase. 613 41


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