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The short-term inhibition by estradiol of tyrosine hydroxylase (TH) in tuberoinfundibular dopaminergic neurons was examined in vitro on hypothalamic slices from ovariectomized rats. TH activity (determined by L-3,4-dihydroxyphenylalanine accumulation in the median eminence after blockade of decarboxylase with NSD 1055) showed a 30-40% decrease within 1 h of incubation with estradiol. To determine whether a dephosphorylation process was involved in this decline in TH activity, we studied the sensitivity of the enzyme to dopamine (DA) feedback inhibition: In controls, we observed that two kinetically different forms of TH coexisted, with one exhibiting a Ki(DA) of 26.4 +/- 2 microM and the other being approximately 10-fold more sensitive to DA inhibition, with a Ki(DA) of 2.56 +/- 0.17 microM, likely corresponding to a phosphorylated and active form and to a nonphosphorylated and poorly active form, respectively. Conversely, after estradiol treatment all TH molecules exhibited the same Ki(DA) of 2.5 +/- 0.3 microM. This effect was stereospecific, because 17 alpha-estradiol could not promote it, whereas with 17 beta-estradiol, it could be observed at only 10(-11) M and after a short delay (30 min). Finally, this decrease in the Ki(DA) of the purported active form of TH could be prevented by okadaic acid (an inhibitor of protein phosphatases). These results suggest that estradiol can act directly on the mediobasal hypothalamus to trigger a rapid decline in TH activity and that this action may involve a decrease in TH phosphorylation.

We have analyzed some functional aspects of the promoter of the human dopamine beta-hydroxylase (DBH) gene. A fragment of 1,247 bp directly 5' to the transcriptional start was progressively shortened, placed in front of a reporter gene, and tested in a human neuroblastoma cell line expressing DBH (SK-N-SH-TFM) and in a monkey kidney cell line (CV-1). A remarkably short region (267 bp), directly upstream from the transcription start, was sufficient to confer activity and tissue-specific expression. Furthermore, the expression of the DBH gene was shown to be inducible by cyclic AMP in SK-N-SH-TFM cells. This effect was demonstrated to occur at the transcriptional level, as shown by run-on assays, and was due to the presence of a near-consensus cyclic AMP-responsive element located in the untranscribed 5' regulatory region of the gene.

A possible interference of the GnRH-associated peptide (GAP) with the metabolism of dopamine in the rat striatum was investigated. The presence of the precursor of the peptide in this central region of dopaminergic terminals was first evidenced using specific RIA. The action of GAP on dopamine release was investigated in the caudate nucleus using the local superfusion with a push-pull cannula supplied with an artificial CSF containing the tritiated precursor of dopamine [( 3H]tyrosine). Addition of GAP (1 microM) to the superfusing fluid resulted in an increase of the release of the newly synthesized dopamine without a significant modification of the total amine release. In situ neutralization of GAP by addition in the CSF of a rabbit serum containing antibodies directed against the GAP produced opposite effects evidencing a tonic function for this peptide. In addition to the increased specific activity of the dopamine released during GAP treatment, the alterations observed in the efflux (and the specific activity) of dihydroxyphenyl acetic acid and the activation of dopamine synthesis obtained in vitro in striatal slices in the presence of GAP led us to conclude that the GAP system could be considered as a positive control of dopamine synthesis.

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.

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.

Rats were submitted to a series of 10 daily electroconvulsive shocks (ECS). A first group of animals was killed 1 day after the last seizure and a second group 30 days later. Tyrosine hydroxylase (TH) activity was measured using an in vitro assay in the nucleus caudatus, anterior cortex, amygdala, substantia nigra, ventral tegmental area, and locus ceruleus. The mRNA corresponding to this enzyme (TH-mRNA) was evaluated using a cDNA probe at the cellular level in the ventral tegmental area, substantia nigra, and locus ceruleus. Met-enkephalin (MET)-immunoreactivity and the mRNA coding for the preproenkephalin (PPE-mRNA) were assayed in striatum and the central nucleus of the amygdala. The day after the last ECS an increase of TH activity was observed in the ventral tegmental area, locus ceruleus, and substantia nigra in parallel with a similar increase in the amygdala and striatum; in the anterior cortex TH activity remained unchanged. TH-mRNA was increased in the locus ceruleus, evidencing the presence in this structure of a genomic activation. The amounts of MET and PPE-mRNA were unaffected in the striatum but increased in the amygdala. Thirty days after the last ECS we observed a decrease of TH activity in the amygdala and of TH-mRNA amount in the ventral tegmental area. In the locus ceruleus TH-mRNA remained higher in treated animals than in controls whereas TH activity returned to control levels. These results demonstrate that a series of ECS induces an initial increase of the activity of mesoamygdaloid catecholaminergic neurons followed by a sustained decrease through alterations of TH gene expression which could mediate the clinical effect of the treatment.

Two full-length cDNA clones that encode functional rat tryptophan hydroxylase (EC 1.14.16.4), the key enzyme in serotonin synthesis, have been isolated from a rat pineal gland library. These two clones correspond to the 1.8- and 4-kilobase mRNA species, respectively. They contain the same coding sequence corresponding to a 51,010-dalton protein and differ in the length of their 3' untranslated regions.