Mesencephalic THmRNA-reduced expression by blocking axonal transport with colchicine.

Colchicine, an axonal transport blocking agent, was unilaterally injected in the medial forebrain bundle of rats. As early as 18 h after the injection a rapid decrease in TH-mRNA level was observed in the substantia nigra and the ventral tegmental area (SN/VTA) on the injected side. In contrast, TH protein levels remained stable for 48 h, and decreased later in both cells bodies and terminals (caudate/putamen). The number of TH-immunopositive cells in SN/VTA increased after colchicine equally in both sides, excluding a neurotoxic effect. These results suggest that TH gene expression is controlled by a retrogradely transported activating factor rather than by feedback inhibition by the end product, i.e. TH protein.

Acute stimulatory effect of estradiol on striatal dopamine synthesis.

The acute effect of physiological doses of estradiol (E2) on the dopaminergic activity in the striatum was studied. In a first series of experiments, ovariectomized rats were injected with 17 alpha or 17 beta E2 (125, 250, or 500 ng/kg of body weight, s.c.), and in situ tyrosine hydroxylase (TH) activity (determined by DOPA accumulation in the striatum after intraperitoneal administration of NSD 1015) was quantified. A dose-dependent increase in striatal TH activity was observed within minutes after 17 beta (but not 17 alpha) E2 treatment. To examine whether E2 acts directly on the striatum, in a second series of experiments, anesthetized rats were implanted in the striatum with a push-pull cannula supplied with an artificial CSF containing [3H]tyrosine. The extracellular concentrations of total and tritiated dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) were measured at 20-min intervals. Addition of 10(-9) M 17 beta (but not 17 alpha) E2 to the superfusing fluid immediately evoked an approximately 50% increase in [3H]DA and [3H]DOPAC extracellular concentrations, but total DA and DOPAC concentrations remained constant. This selective increase in the newly synthesized DA and DOPAC release suggested that E2 affects DA synthesis rather than DA release. Finally, to determine whether this rapid E2-induced stimulation of DA synthesis was a consequence of an increase in TH level of phosphorylation, the enzyme constant of inhibition by DA (Ki(DA)) was calculated. Incubation of striatal slices in the presence of 10(-9) M 17 beta (but not 17 alpha) E2 indeed evoked an approximate twofold increase in the Ki(DA) of one form of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of tyrosine hydroxylase gene expression in mesencephalic dopamine neurons: effect of imipramine treatment.

The effects of a chronic imipramine treatment on the mesoamygdaloid pathway of rats were examined. Using semiquantitative immunocytochemical techniques, it was observed that the level of TH mRNA was decreased in the ventral tegmental area (VTA). In contrast, the TH protein was increased in both the VTA and amygdala. The TH activity was decreased in the amygdala when assessed under normal conditions but increased after a preincubation to phosphorylate the enzyme, suggesting a lowering of the protein-specific activity in the terminals. These results show that TH protein turnover in the mesoamygdaloid neurons can be reduced by chronic imipramine treatments, thereby producing an accumulation of inactive TH protein in the neurons while also decreasing TH gene activity in the cell bodies.

Evidence for protein kinase C involvement in the short-term activation by prolactin of tyrosine hydroxylase in tuberoinfundibular dopaminergic neurons.

The mechanism of the short-term activation by prolactin (PRL) of tyrosine hydroxylase (TH) in tuberoinfundibular dopaminergic neurons was examined in vitro on hypothalamic slices from ovariectomized rats. TH activity (determined by 3,4-dihydroxyphenylalanine accumulation in the median eminence after blockade of decarboxylase with NSD 1055) showed a dose-dependent increase within 2 h of incubation of the hypothalamic slices with PRL. To determine whether a phosphorylation process was involved in this increase in TH activity, we studied the sensitivity of the enzyme to dopamine (DA) feedback inhibition. In control median eminences, two kinetically different forms of TH coexisted, one exhibiting a Ki(DA) value of 29.92 +/- 0.49 microM, the other being approximately 15-fold more sensitive to DA inhibition with a Ki(DA) of 1.96 +/- 0.09 microM, likely corresponding to a phosphorylated and active form and to a nonphosphorylated and less active form, respectively. After PRL treatment, the TH form of low Ki(DA) remained unaffected, whereas the Ki(DA) of the purported active form of TH increased to 62.6 +/- 0.8 microM, suggesting an increase in the enzyme phosphorylation. This increase in the Ki(DA) of TH was selectively prevented by GF 109203X, a potent and selective inhibitor of protein kinase C, but not by a specific inhibitor of protein kinase A or calmodulin. Finally, this action of PRL could be mimicked by 12-O-tetradecanoylphorbol 13-acetate (a direct activator of protein kinase C). These results suggest that PRL, at the median eminence level, activates TH by increasing the enzyme phosphorylation and that this action may involve an activation of protein kinase C.

Evidence of tyrosine hydroxylase mRNA in the anterior and neurointermediate lobes of female rat pituitary.

The anterior pituitary is thought to be unable to synthesize dopamine (DA) except under experimental conditions where a tyrosine hydroxylase (TH) activity, the rate-limiting step of its synthesis, has been demonstrated. In this work, we tested whether the enzyme described as active under particular conditions comes from de novo TH gene transcription or from a pre-existing TH mRNA poorly translated or untranslated under physiological conditions. Therefore, we searched for the presence of TH mRNA in normal female rat pituitary using the polymerase chain reaction following reverse transcription (RT/PCR) and in situ hybridization (ISH). The neurointermediate lobe (NIL) of the hypophysis was used as negative tissue, since it is thought to be unable to synthesize TH. As expected, no ISH labelling could be seen in the neural lobe (NL). However, scarce labelled cells were found in the intermediate lobe (IL) confirming the positive results observed in the NIL by RT/PCR. The anterior lobe (AL) also presented TH mRNA by PCR and ISH. The TH gene expression in sparse cells of the AL is discussed in regard to the ability of the AL to synthesize DA under particular conditions from a pre-existing mRNA.