Long-term alteration in tyrosine hydroxylase mRNA levels in rat locus coeruleus after intraventricular injection of 5,6-dihydroxytryptamine.

The time course variations in tyrosine hydroxylase (TH) activity and specific mRNA were measured in the rat locus coeruleus (LC) and substantia nigra after an intracerebroventricular (i.c.v.) injection of 5,6-dihydroxytryptamine (5,6-DHT), a neurotoxin known to selectively destroy serotoninergic neurons. In this study, the TH activity and TH mRNA were both analyzed from homogenates of single tissue samples (micropunches). TH mRNA was extracted and quantified by densitometry using a northern blot method and an artificial TH RNA as an external standard. 5,6-DHT injection led to a long-lasting increase in TH activity and TH mRNA in LC but not in substantia nigra. The elevation in LC was progressive and reached its maximum value (+75%) at day 4 and day 8 after 5,6-DHT. This effect on TH activity was accompanied by a parallel change in TH mRNA whose amplitude was +57%, +81% and +45% at day 2, 4, and 8 respectively after the neurotoxin injection. Return to normal values was observed at day 16. Variations in TH activity and TH mRNA in LC were of similar amplitude. These results suggest that serotonin could be a potent modulator of TH gene expression within noradrenergic LC neurons.

Retinoic acid induces cholinergic differentiation of cultured newborn rat sympathetic neurons.

Many studies provide evidence that retinoic acid (RA), an endogenous derivative of vitamin A, plays a role in the development of the nervous system. We now report that RA controls the neurotransmitter phenotype of post-mitotic rat sympathetic neurons in cell culture. RA added to the culture medium increased the specific activity of choline acetyltransferase (ChAT) and the level of acetylcholine (ACh). Concomitantly, RA reduced the specific activities of two catecholamine synthetic enzymes, tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) and the level of norepinephrine (NE). After a 2 week treatment with 5 microM RA, ChAT was increased by 5-10 fold, whereas TH and DBH were decreased by 10-15 fold and 2-3 fold, respectively, as compared to sympathetic neurons grown in the absence of RA. The modulation of the activity of the three enzymes was dose-dependent and followed a similar time course. The decrease of TH expression was demonstrated to be due to a decreased number of TH molecules.

Tyrosine hydroxylase regulation in neurotransmission and neuroplasticity.

The modulation of neurotransmitter synthesis is a fundamental mechanism influencing neurotransmission and neuronal plasticity during development. The regulation of the tyrosine hydroxylase (TH) has been used to elucidate specific adaptative responses in neurons. Trans-synaptic impulse activity elicits sort- and long-term changes in the activity of TH. Acute regulation involves the activation of preexisting TH molecules via phosphorylation and possibly through alternative splicing events in humans, whereas long-term regulation results from an increased synthesis of the enzyme due in part to the transcriptional stimulation of the TH gene. The long-term increase of TH activity was addressed using the drug reserpine known to modify the secretion of neurotransmitters and the tetradecanoyl phorbol acetate (TPA). Inductions of TH expression by reserpine in vivo as well as by TPA in vitro seem to be mediated by an AP-1 complex acting on a TPA responsive element (TRE) of the rat TH promoter indicating that the TRE-TH site plays a critical role in trans-synaptic induction. Our results also demonstrate a degree of adaption by sympathetic neurons to their environment by conversion from adrenergic to cholinergic phenotype.

Induction of tyrosine hydroxylase in the rat substantia nigra by local injection of forskolin.

Forskolin (FSK) was locally injected into the substantia nigra (SN) of anesthetised rats. The day after injection (24 and 36 hr), tyrosine hydroxylase (TH) activity increased locally in this structure but remained unmodified in the ipsilateral caudate nucleus (CN). The amount of messenger RNA for TH (TH-mRNA) was also increased in the SN 24 hr after the injection. However, TH protein content was modified neither locally in the SN nor in the ipsilateral CN. In addition, the decrease of the ratio between dopamine and its first metabolite in the CN and the SN suggested a decreased activity of the dopaminergic nigral cells. The absence of increase of the protein synthesis in spite of the fact that TH-gene transcription was initiated could be the consequence of the inhibition of dopaminergic cells by the drug. These results confirm that, in vivo, TH induction is cAMP-dependent and demonstrate that the TH-gene activity is not strictly coupled to the activity of dopaminergic cells in the SN.

In vitro and in vivo regulation of the expression of the tyrosine hydroxylase gene.

Preganglionic nerve stimulation has been shown to induce delayed or long term changes in the neuron. Different models were used to study the trans-synaptic regulation of the tyrosine hydroxylase (TH): the rat adrenal medulla (AM) and the superior cervical ganglia (SCG). Northern blot analysis, western blot and enzymatic assays demonstrated that the TH mRNA paralleled both an increase in the protein amount and its enzymatic activity. Results of in vitro transcription on nuclei isolated from AM or from SCG after treatment with reserpine suggest that this increase in TH expression is due to an effect on the transcriptional activity of the TH gene. Other gene, cfos, is also induced by reserpine indicating that the TH transcription in these neurons may be mediated by "third messengers". Several putative regulatory elements, in particular an octamer sequence AP1 has been localized in the promoter of TH gene. Gel shift assays with nuclear extracts from untreated and phorbol ester treated cells strongly suggest that a protein complex binds to this AP1 like sequence. Comparative analysis of gel shift assays with AM nuclear extracts exhibit a similar pattern suggesting that this AP1 site could be involved in the trans-synaptic regulation of TH.

Inhibitory actions of acute estradiol treatment on the activity and quantity of tyrosine hydroxylase in the median eminence of ovariectomized rats.

Abstract The effects of acute estradiol (E(2)) treatment on both the activity of tyrosine hydroxylase (TH) in the median eminence and the serum level of prolactin (PRL) were investigated. Twelve-day-ovariectomized rats were injected with 17beta-E(2) (25mug sc) at 1100 h and sacrificed hourly from 1200 to 2300 h. TH activity was quantified by measuring the amount of exogenous tyrosine converted to L-DOPA in vitro by aliquots of median eminence homogenates. Serum PRL levels were evaluated by radioimmunoassay. A biphasic response of TH activity to treatment was observed: an immediate decrease occurred-preceding and accompanying a rise in serum PRL-followed by an increase beyond control levels 2 h after the maximal release of PRL. The increase in TH activity could be prevented by the pretreatment of rats with a specific rat PRL antiserum, suggesting it was not due to E(2) per se but rather mediated by the E(2)-induced PRL elevation. To pin-point the process underlying the E(2)-induced decrease in TH activity, we evaluated the kinetic parameters of TH in the median eminence as well as its quantity (by Western blot analysis) in the median eminence and arcuate nucleus. Finally, we used a sensitive dot-blot assay to quantify specific TH messenger ribonucleic acid in the arcuate nucleus. The decrease in TH activity after E(2) treatment paralleled an immediate decrease in the affinity of TH for its pterin cofactor (6-MPH4), while V(max) remained unchanged. A decrease in the amount of TH protein in the arcuate nucleus and median eminence as well as in the TH messenger ribonucleic acid level in the arcuate nucleus was also observed, but the latency of these effects precluded a major involvement in the immediate decline of TH activity. Therefore, when observed separately from those of PRL, E(2) effects on TH in tuberoinfundibular dopaminergic neurons are clearly inhibitory consisting of a 'deactivation' of the enzyme together with a reduction of its synthesis.

Transcriptional and post-transcriptional regulation of tyrosine hydroxylase gene by protein kinase C.

The role played by protein kinase C (PKC) in TH gene regulation was investigated at transcriptional and post-transcriptional levels using PC12 cells. The cells were treated with the phorbol ester TPA, which not only activates PKC but also causes down-regulation. PKC levels were monitored by [3H]PDBU binding assay and by using an anti-PKC antibody that detected intact PKC (79 kd) as well as its catalytic and regulatory domains. The [3H]PDBU binding to the membrane-associated PKC increased within 15-30 min of TPA treatment; thereafter total cellular [3H]PDBU binding decreased to a minimum of 20% of the control at 8 h. The rate of decrease in binding was greater than the decrease in the intensity of the staining of PKC holo enzyme visualized by anti-PKC antibody. TH mRNA levels, measured over the same time period, rose within 15 min of TPA treatment to peak at 4 h and subsequently declined below control level, paralleling the depletion of PKC. If cells depleted of PKC were reincubated in the normal medium, a recovery in PKC level was seen and, in parallel, TH mRNA levels increased to above control level. Furthermore, if down-regulation of PKC was prevented by incubating the cells with the protease inhibitor leupeptin, a decrease beyond control level in TH mRNA was not observed. TPA rapidly induced TH gene transcription; a maximal increase of two-fold was observed at 15 min, but the transcriptional rate then declined although it did not decrease beyond control values after 8 and 24 h of TPA treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of tyrosine hydroxylase gene expression in rat brain and adrenals by exposure to cold.

The long-term changes in tyrosine hydroxylase (TH) activity induced by chronic exposure to cold in brain noradrenergic neurons of the locus coeruleus (LC) were analyzed and compared to those measured in a peripheral tissue such as adrenals. This analysis was made possible at the level of one single tissue corresponding to one animal by the use of sensitive methods that allow assay of TH activity, protein, and mRNA levels in parallel from the same homogenate. The three parameters were measured in brain structures and adrenals of rats maintained at 4 degrees C during 4 days and were compared to those of control animals kept at normal housing temperature (22 degrees C). LC of rats exposed to cold contained 200% more TH mRNA than controls. The amount of TH protein in this area rose to as much as 164% that of controls. Similarly, the activity of the enzyme increased to 140% of the normal value. Thus, these observations show that 1) the increase in TH mRNA was much higher than the increase in protein levels, and that 2) the newly synthesized molecules have about the same activity as that present under normal conditions. In contrast to the LC, no variation of these parameters was observed in the substantia nigra. In the adrenals, the variations in the different parameters were qualitatively similar to that observed in the LC, although they were quantitatively higher: TH mRNA, TH protein, and TH activity levels were respectively 330%, 182%, and 167% that of control adrenals. Altogether, these results demonstrate that exposure to cold induces an alteration in TH synthesis in brain noradrenergic neurons as well as in adrenals.

Regulation of neurotransmitter metabolic enzymes and tyrosine hydroxylase mRNA level by nerve growth factor in cultured sympathetic neurones.

The survival of new-born rat sympathetic neurones in culture was increased in a dose-dependent manner by 7S nerve growth factor (NGF). NGF also increased, in a parallel manner, the specific activities of tyrosine hydroxylase (TOH) and choline acetyltransferase (CAT). Total acetylcholinesterase (AcChE) activity increased with NGF concentration, although less distinctly than TOH and CAT. However, NGF caused a large induction of the asymmetric A12 form of AcChE, and to a lesser extent of the globular G1 and G2 forms, whereas the globular G4 form was little affected. This suggests that NGF differentially regulates the synthesis and/or assembly of the various AcChE molecular forms. The levels of TOH mRNA in neurone cultures grown with increasing NGF concentrations were measured by Northern blot analysis with a rat cDNA probe. To correct for variations in the total mass of RNA per neurone, the filters were rehybridized with an 18S rRNA probe. The level of TOH mRNA, measured by the ratio (TOH:18S) of the hybridization signals increased 3.4-fold between 92 and 740 ng ml-1 7S NGF. Increases of TOH specific activity of the same order of magnitude were observed in sister cultures. The deficit in the level of mature TOH mRNA at low NGF concentration was not accompanied by a compensatory accumulation in unprocessed TOH transcripts. As TOH induction is insensitive to RNA polymerase inhibitors, we suggest that NGF regulates the maturation of TOH pre-mRNAs, and that the unprocessed transcripts are rapidly degraded. The long-term regulation of TOH by NGF may thus constitute a case of process-versus-discard control, as defined by J.E. Darnell.

Identification of a human glial fibrillary acidic protein cDNA: a tool for the molecular analysis of reactive gliosis in the mammalian central nervous system.

Two clones encoding human glial fibrillary acidic protein (GFAP) were isolated from a human astrocytoma cDNA library. The clones pHGFAP1 and pHGFAP2 were selected by the combined use of differential colony hybridization and hybridization-selection technique with polyclonal anti GFAP antiserum. The longer one, pHGFAP1, encompasses 3.0 kb and includes the 1.8 kb long 3' untranslated region specific to the human mRNA. Sequence data disclosed an extensive homology within the coding region of human and mouse GFAP cDNAs even in the end domains. Blot hybridization analysis of RNAs from human, rat and mouse brain revealed a single GFAP mRNA species of 3.1, 2.8 and 2.7 kb respectively and Southern blot experiments indicated that this mRNA is most probably transcribed from a unique gene. In situ hybridization performed with biotinylated probes on cultured mouse brain cells suggests both the sorting and the transport of GFAP mRNA throughout the cytoplasm and processes of the astrocytes. As a model of reactive gliosis secondary to degenerative disorders, 6-hydroxydopamine (6-OHDA) lesion of the substantia nigra in the rat was performed. GFAP mRNA increased 1.4 fold in the ipsilateral striatum on day 10 after the lesion. It then declined to the control level 4 months later contrasting with the lower and more sustained increase in preproenkephalin (PPE) mRNA. The interspecies cross-reactivity of the HGFAP probes make them useful as a tool for the molecular analysis of reactive gliosis in various experimental models.

The primary structure of human dopamine-beta-hydroxylase: insights into the relationship between the soluble and the membrane-bound forms of the enzyme.

A full length dopamine-beta-hydroxylase (DBH) cDNA clone was isolated from a human pheochromocytoma lambda gt11 library. Both structural and functional evidence confirms the authenticity of the clone: (i) antibodies selected with fusion proteins generated by positive clones precipitate DBH activity, (ii) the sequence of three internal DBH tryptic peptides are included in the deduced DBH sequence, (iii) the previously reported N-terminal 15 amino acids of bovine DBH exhibits a nearly complete identity with that predicted for human DBH. The polypeptide chain of DBH comprises 578 amino acids corresponding to an unmodified protein of 64 862 daltons and is preceded by a cleaved signal peptide of 25 residues. DBH exists in both membrane-bound and soluble forms. The hydropathy plot reveals no obvious hydrophobic segment, except the signal peptide. S1 mapping analysis indicates no diversity in the 5' and 3' extremities of the DBH mRNA. Taken together with available biochemical data, these observations suggest that the membrane attachment of DBH probably results from a post-translational modification, glypiation being the most likely candidate. Comparative amino acid sequence analysis establishes that DBH shares no homology with the other catecholamine synthesizing enzymes, tyrosine hydroxylase and phenylethanolamine-N-methyl transferase.

The use of a tyrosine-hydroxylase cDNA probe to study the neurotransmitter plasticity of rat sympathetic neurons in culture.

We have compared quantitatively the effects of muscle-conditioned medium (CM) and elevated K+ concentration (40 mM) on the enzymatic activity of tyrosine hydroxylase (TH) and on TH-mRNA levels in primary cultures of rat sympathetic neurons. Northern blot analysis of RNA from cultured neurons with a 32P-labeled rat TH-cDNA probe was performed. The probe hybridized strongly with a single RNA species of 1.9 kb, similar in size to the TH-mRNA from PC12 pheochromocytoma cells. In agreement with earlier data both CM and a partially purified factor from CM increased choline acetyltransferase activity up to 200-fold and depressed TH activity by 2- to 7-fold in cultured sympathetic neurons. These effects were accompanied by a decrease in TH-mRNA level, which correlated with the decrease in TH activity. On the other hand, a culture medium supplemented with 40 mM KCl caused a 1.5- to 5-fold increase in TH activity, which was accompanied by an increase in TH-mRNA level of the same order of magnitude. As a working hypothesis, we suggest that CM and neuronal depolarization control the transcription of the TH gene in an antagonistic manner.

Molecular genetics of catecholamines as an approach to the biochemistry of manic-depression.

Manic depressive illness has been clearly established to exhibit a strong genetic component and is therefore amenable to linkage analysis using random DNA markers. In view of the catecholamine hypothesis of this disorder, the gene encoding tyrosine hydroxylase (TH) the limiting enzyme in catecholamines is a good candidate to investigate. This gene has been localized to chromosome 11 in close linkage with Harvey-ras-1. The various transcriptional and post-transcriptional mechanisms that modulate short and long-term TH activity are discussed. Human tyrosine hydroxylase is coded by at least three distinct mRNAs derived from a single gene. This variation has clear functional consequences and could represent a novel mode of regulating catecholamines levels in normal and pathological neurons.

Sequential changes in cytomegalovirus antigenic pattern during infection of renal transplant patients.

Changes in the antigenic pattern of cytomegalovirus occurring during the course of infection were studied in two renal transplant patients. The virus was isolated several times from each patient. After two to five passages in vitro, the immune precipitate formed either with guinea-pig antiserum raised to Davis reference strain, or with human serum taken from the same patient during the acute phase of the infection, was analysed by SDS-polyacrylamide gel electrophoresis. Isolates from each patient showed a slightly different antigenic pattern with both human and experimental antisera. These differences possibly reflect modulation of the expression of proteins during the course of viral infection in the patients and during adaptation to cell culture in vitro.

[Histochemical localization of messenger RNA coding for tyrosine hydroxylase: in situ hybridization with a single-stranded cDNA probe]. / Localisation histochimique des ARN messagers codant pour la tyrosine hydroxylase: hybridation in situ à l'aide d'une sonde ADNc monocaténaire.

The expression of tyrosine-hydroxylase (TH) gene was analysed in tissue sections of bovine adrenal glands, by in situ hybridization using a single-stranded cDNA probe. Tissue fixation and hybridization conditions were found that led to a specific and sensitive detection of TH.

Identification of cDNA clones coding for rat tyrosine hydroxylase antigen.

Five recombinant DNA plasmids have been constructed that contain structural gene sequences for rat tyrosine hydroxylase [TyrOHase; tyrosine 3-monooxygenase; L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2]. Rat pheochromocytoma PC 12 cell line, which contains relatively high levels of catecholamine-synthesizing enzymes, was used to purify RNA. TyrOHase cDNA clones were identified by screening 350 cDNA clones constructed from partially purified TyrOHase mRNA. A rapid and powerful screening of the recombinant clones by differential colony hybridization was possible because TyrOHase is a tissue-specific protein. The final selection relied on the ability of cDNA inserts to hybridize specifically to TyrOHase mRNA as judged by cell-free translation and immunoprecipitation. Blot hybridization analysis of polyadenylylated RNA from PC 12 cells indicated a major mRNA species of 1.9 kilobases. A species of the same size was identified from a human pheochromocytoma tumor, indicating a crossreactivity between rat TyrOHase cDNA and human TyrOHase mRNA.

Modification of steroidogenesis in a mouse adrenal cell line (Y-1) transformed by simian adenovirus SA-7.

Transformation of a steroidogenic mouse adrenal cell line (Y-1) by simian adenovirus SA7 produced a cell line with low apparent steroidogenic activity. The effect of ACTH and cholera toxin on cyclic AMP production was similar in both not transformed and virus-transformed cells and activity of cyclic AMP-dependent protein kinase was also similar in both cells. In transformed cells, cholesterol was metabolized to delta 5-3 beta-hydroxysteroids, mainly 20 alpha-dihydropregnenolone while in not transformed cells, the major metabolites were delta 4-3 ketosteroids (20 alpha-dihydro- and 11 beta-hydroxy-20 alpha-dihydroprogesterone). In both cell lines ACTH increased the metabolism of cholesterol. Further studies with labelled pregnenolone and progesterone revealed a loss of delta 5-3 beta-hydroxysteroid dehydrogenase/isomerase and 11 beta-hydroxylase activity in the transformed cells.