A novel, inexpensive, and sensitive method for analysis of tyrosine hydroxylase activity in tissue samples.
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Activin co-operates with fibroblast growth factor 2 to regulate tyrosine hydroxylase expression in the basal forebrain ventricular zone progenitors.
Activin and its cognate receptors are expressed during embryogenesis in the rapidly dividing cells of the basal forebrain ventricular zone. This finding prompted us to study the role of activin in regulating neurotransmitter phenotype expression and other aspects of the ventricular zone-derived progenitor cell differentiation. Although virtually ineffective alone, activin co-operated with fibroblast growth factor 2 to induce a rapid tyrosine hydroxylase-immunoreactivity in cultured ventricular zone progenitors. Northern analysis indicated that the increase in tyrosine hydroxylase-immunoreactivity was associated with increased tyrosine hydroxylase gene expression. Activin and fibroblast growth factor 2 action was specific to tyrosine hydroxylase, as it did not induce the expression of choline acetyltransferase, nor enhance the expression of glutamate decarboxylase. Cultures treated with the DNA replication marker bromodeoxyuridine revealed that both proliferating ventricular zone progenitors and their post-mitotic progeny were induced to express tyrosine hydroxylase. In these cultures, activin acted to reduce fibroblast growth factor 2 stimulated mitotic activity. Furthermore, activin permitted neuronal differentiation and survival of the ventricular zone progenitors after three days in vitro. Together these data demonstrate a novel role of activin and fibroblast growth factor 2 in regulating the fate of the embryonic basal forebrain ventricular zone progenitors.
Activin Receptors↗
Formation of amines by intestinal microorganisms and the influence of chlortetracycline.
Although gastric and intestinal contents from rats failed to show amino acid decarboxylase activity when tested against five different amino acids (glutamic acid, arginine, lysine, tyrosine, and histidine), the feces contained at least seven different amines, some known to be pharmacologically active. Putrescine, histamine, and tyramine were identified by means of paper chromatography in both intestinal material and mixed fecal cultures; four other spots were found, three of which had Rf values similar to agmatine, ethanolamine, and ephedrine. The formation of lysine and glutamic acid decarboxylases was not enhanced by an increased acidity during growth while increased oxygen tension was inhibitory to amino acid decarboxylase synthesis in these fecal cultures. The feeding of chlortetracycline to rats, or its presence at a very low concentration in media in which the mixed cultures were grown, reduced the capacity of intestinal microorganisms to produce amines. Cells from mixed fecal cultures grown in the presence of chlortetracycline lacked or contained but weak amino acid decarboxylase activities. The action of the enzymes themselves was unaffected by the presence of the antibiotic in the Warburg cup during assay. The results suggest that amines formed within the intestinal tract might be toxic to the rat, and that chlortetracycline accelerates animal growth by suppressing their production.
Amines↗
The diverse effects of 5'-bromodeoxyuridine on enzyme activities in cultured H35 hepatoma cells.
Reuber (H35) hepatoma cells were grown in medium containing 10(-5)M bromodeoxyuridine (BrdU), which was incorporated into their DNA. Cell growth rate was unaffected by BrdU for the first two generations, after which it was reduced by about 50%. The effect of BrdU incorporation on the activities of several enzymes with rapid turnover rates was examined to test the hypothesis that the synthesis of such enzymes will be preferentially inhibited by BrdU. Tyrosine amino-transferase (TAT) activity decreased by 70% within two generations whereas thymidine kinase activity remained at control values. PEP carboxykinase activity was unchanged during the first generation in BrdU-containing medium but, during the second, its activity increased by at least 30%. Ornithine decarboxylase levels decreased by about 50% only after two generations in the presence of BrdU. There appeared to be no simple relationship between turnover rates and the effect of BrdU on enzyme activity. Incorporation of BrdU was found to inhibit the induction of both TAT and PEP carboxykinase by dexamethasone and to enhance the inhibition of cell growth by this steroid. These results are discussed with respect to possible mechanisms of gene expression and development in both normal and neoplastic cells.
Alanine Transaminase↗
Effects of haloperidol and melatonin on the in situ activity of nigrostriatal tyrosine hydroxylase in male Syrian hamsters.
Haloperidol, an antipsychotic drug, was tested for its effects on the in situ activity of nigrostriatal and hypothalamic tyrosine hydroxylase, in control male Syrian hamsters and in those receiving a high daily dose of melatonin. After receiving daily ip injections (1.25 mg/kg ip) of haloperidol for 21 days, the animals were sacrificed and brain tissue collected for analysis of dopamine and metabolites by HPLC with electrochemical detection. In situ activity of tyrosine hydroyxlase (TH) activity was determined by measuring the accumulation of L-Dopa after administration of the L amino acid decarboxylase inhibitor, mhydroxybenzylhydrazine. Tissue content of dopamine and its metabolites, DOPAC and HVA, was depressed in striatum of animals receiving haloperidol, and tyrosine hydroxylase (TH) activity was significantly decreased 20-24 h after the last injection (from 1823 +/- 63 to 1139 +/- 85 pg l-dopa/mg tissue). The decrease in TH activity in striatum was significantly inhibited by daily injections of a high dose of melatonin (2.5 mg/kg ip) (from 1139 +/- 85 to 1560 +/- 116 pg L-dopa/mg tissue). In the substantia nigra and in the hypothalamus, on the other hand, haloperidol significantly increased the activity of tyrosine hydroxylase. Melatonin administration did not significantly influence TH activity in the substantia nigra, but inhibited TH activity in the hypothalamus and in the pontine brainstem. One explanation for these data is that chronic haloperidol administration in Syrian hamsters increases TH activity in hypothalamus and substantia nigra, but decreases TH activity in striatum by a mechanism involving D2 presynaptic receptors and a melatonin sensitive kinase which regulates TH phosphorylation.
Animals↗
Dopamine synthesis in inbred mouse strains which differ in numbers of dopamine neurons.
BALB/cJ and CBA/J mice have been shown to have different numbers of dopamine (DA) neurons in the central nervous system, with BALB/cJ mice having 20-50% more DA neurons in each dopaminergic cell group which is reflected in a difference in tyrosine hydroxylase activity in these cell groups. The present study compared the levels of DA and the rate of DA synthesis between these two inbred mouse strains. Three measures were used to reflect the rate of DA synthesis: the levels of DA metabolites (DOPAC and HVA) in the striatum, the rate of disappearance of DA following inhibition of tyrosine hydroxylase with alpha-methyl-P-tyrosine, and the rate of accumulation of DOPA following inhibition of aromatic amino acid decarboxylase with NSD-1015. Striatal DA levels were slightly higher in CBA/J mice than BALB/cJ mice. The rate of DA synthesis in the striatum, as estimated from the accumulation of DOPA following NSD-1015 injection or from the decline of DA levels following alpha-methyl-p-tyrosine injection, was from 30-50% greater in the BALB/cJ mice compared to the CBA/J mice. In striatum, DOPAC levels were higher, HVA levels lower, and DOPAC plus HVA levels equal in CBA/J mice compared to BALB/cJ mice. The results show that BALB/cJ mice, with more DA neurons than CBA mice, also synthesize more DA. In addition, the data suggest that DA levels do not necessarily reflect numbers of DA neurons, and that catecholamine metabolite levels are not a good measure for comparing catecholamine synthesis between inbred animal strains.
3,4-Dihydroxyphenylacetic Acid↗
Female lordosis behavior in estrogen-primed male rats treated with para-chlorophenylalanine or alpha-methyl-para-tyrosine.
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Animals↗
The conversion of tyrosine to catecholamines and the biogenesis of N-acetyl-dopamine in isolated epidermis of the fiddler crab, Uca pugilator.
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Acyltransferases↗
Dopamine synthesis by non-dopaminergic neurons in the arcuate nucleus of rat fetuses.
The aim of the present work was to verify the hypothesis that non-dopaminergic neurons expressing individual complementary dopamine synthesis enzymes can perform the co-located synthesis of dopamine. According to this hypothesis, neurons expressing tyrosine hydroxylase use L-tyrosine for the synthesis of L-dihydroxyphenylalanine (L-DOPA), which then enters neurons expressing aromatic amino acid decarboxylase, which converts L-DOPA to dopamine. Experiments were performed using the mediobasal hypothalamus of rat fetuses, which mostly contains single-enzyme neurons (>99%) and occasional double-enzyme neurons (<1%). Controls were obtained from the fetal substantia nigra, which is enriched with dopaminergic neurons. High-performance liquid chromatography was used to measure levels of dopamine and L-DOPA in cell extracts and the incubation medium after incubation in the presence and absence of exogenous L-tyrosine. Addition of L-tyrosine to the medium led to increases in the level of synthesis and release of L-DOPA in the mediobasal hypothalamus and substantia nigra. In addition, L-tyrosine increased dopamine synthesis in the substantia nigra and decreased dopamine synthesis in the mediobasal hypothalamus. This regional difference in levels of dopamine synthesis is probably due to inhibition of the uptake of L-DOPA from the intercellular medium by neurons in the mediobasal hypothalamus containing aromatic amino acid decarboxylase, due to the competitive binding of the L-DOPA transporter by L-tyrosine. Thus, these results provide the first evidence for the co-located synthesis of dopamine by non-dopaminergic neurons expressing single complementary enzymes involved in the synthesis of this neurotransmitter.
Animals↗
Intracranial self-stimulation increases differentially in vivo hydroxylation of tyrosine but similarly in vivo hydroxylation of tryptophan in rat medial prefrontal cortex, nucleus accumbens and striatum.
We have examined using microdialysis the effect of intracranial self-stimulation (ICSS) on the in vivo hydroxylation rate of tyrosine and tryptophan in the medial prefrontal cortex (mPFC), nucleus accumbens (NAC) and striatum (STR). A decarboxylase inhibitor NSD-1015 was included in the perfusate, which enabled the simultaneous measurement of 3,4-dihydroxyphenylalanine (DOPA) and 5-hydroxytryptophan (5-HTP) as an index of the in vivo hydroxylation level of tyrosine and tryptophan. When rats were exposed to 1 h of ICSS at the medial forebrain bundle (MFB), their extracellular levels of DOPA significantly increased in the mPFC, NAC and STR, but with a different magnitude and time course. The same stimulation produced a delayed increase in extracellular 5-HTP, compared to DOPA, in these brain regions. The profile of 5-HTP response demonstrated no apparent difference among the regions. These findings indicate that ICSS of the MFB can increase differentially the in vivo hydroxylation of tyrosine but similarly the in vivo hydroxylation of tryptophan in the mPFC, NAC and STR.
5-Hydroxytryptophan↗
Responsiveness of tuberoinfundibular dopaminergic neurons to 5-hydroxytryptophan: effects of aging.
Serotonin is known to stimulate prolactin secretion by decreasing tyrosine hydroxylase (TH) activity in the tuberoinfundibular dopaminergic (TIDA) neurons. However, the effects of aging on the responsiveness of TIDA neurons to serotonin are not known. An effective way to increase serotonergic activity is to administer 5-hydroxytryptophan (5-HTP), a serotonin precursor. The present study was done to investigate the effects of 5-HTP on TIDA neuronal activity in aging animals. Middle-aged (10-12 mo), old (18-20 mo), and very old (22-24 mo) female Sprague-Dawley rats were bilaterally ovariectomized. Ten days later, they were injected iv with 50 mg/kg body wt of 5-HTP or the vehicle for 5-HTP (PBS-HCI). Twenty minutes later, m-hydroxybenzylhydrazine (NSD), a DOPA decarboxylase inhibitor, was administered. Ten minutes later, the animals were killed, and tyrosine hydroxylase (TH) activity was determined by measuring L-DOPA accumulation in the stalk median eminence by HPLC-EC. In all three groups, administration of 5-HTP increased serum prolactin levels significantly. In control middle-aged rats, TH activity (L-DOPA pg/ microg protein) was 33.0+/-5.6. Treatment with 5-HTP decreased TH activity by 60%. Similarly, 5-HTP treatment decreased TH activity by 52 and 56% in 18- to 20- and 22- to 24-mo-old rats, respectively, compared to the control rats. The magnitudes of the 5-HTP-induced decreases in TH activities in middle-aged, old, and very old rats were not different from each other. These results indicate that TIDA neuronal responsiveness to serotonin does not change with age and that 5-HTP is capable of stimulating PRL release even in very old rats.
5-Hydroxytryptophan↗
GABA axons in synaptic contact with dopamine neurons in the substantia nigra: double immunocytochemistry with biotin-peroxidase and protein A-colloidal gold.
Two different antigens in the same ultrathin section of brain tissue can be revealed by 'double immunocytochemistry' in which one antigen is detected by horseradish peroxidase and the other by silver intensification of colloidal gold (SIG) adsorbed to Protein A. By means of this procedure it has been possible to show that GABAergic axon terminals (containing glutamate decarboxylase) are in synaptic contact with the cell bodies and dendrites of dopaminergic neurons (containing tyrosine hydroxylase) in the substantia nigra of the rat. Thus, several of the physiological and pharmacological effects of GABA and GABAergic drugs in this part of the brain are likely to be mediated by a direct action via postsynaptic GABAergic receptors located on dopaminergic nigrostriatal neurons.
Animals↗
Tyrosine hydroxylase activity in the locus coeruleus of conscious rats: differences with the anesthetized rats.
Microdialysis was used to estimate in vivo tyrosine hydroxylase (TH) activity in locus coeruleus (LC) of conscious rats. An aromatic amino acid decarboxylase inhibitor, difluoromethyl-DOPA (DFMD), was perfused through the dialysis probe and the DOPA accumulating in dialysates was measured. A 3 h perfusion of a 6 x 10(-4) mol/l DFMD solution was needed to obtain measurable levels of DOPA in LC dialysates: a linear increase in DOPA concentrations was first observed and, then, a steady state. DOPA disappeared completely after inhibition of TH by alpha-methyl-p-tyrosine, showing that it reflects TH activity. The DFMD perfusion needed in this study was six times higher in concentration and three times longer in duration than the one required in a previous study we performed in LC of anesthetized rats. Furthermore, TH activity was found lower than the one we previously reported. We conclude that these differences could be explained by the effects of halothane anesthesia on DFMD and DOPA degradation and/or on LC neurons TH activity itself.
Anesthetics↗
Striatonigral GABA, dynorphin, substance P and neurokinin A modulation of nigrostriatal dopamine release: evidence for direct regulatory mechanisms.
The striatonigral pathway contains several neurotransmitters which may regulate the activity of the nigrostriatal dopamine projection in the rat. This was investigated by measuring extracellular dopamine levels in the striatum, using microdialysis, after injections of GABA (300 nmol/0.2 microliters), dynorphin A (0.5 nmol/0.2 microliters), substance P (0.07 mnol/0.2 microliters) or neurokinin A (0.09 nmol/0.2 microliters) into the ipsilateral substantia nigra, pars reticulata (SNR). Intranigral injections of GABA or dynorphin A inhibited, while intranigral injections of substance P or neurokinin A stimulated dopamine levels in the ipsilateral striatum. In rats with ibotenic acid lesions (2.5 micrograms/0.5 microliters) in the SNR, intranigral injections of GABA or dynorphin A inhibited, while intranigral injections of substance P or neurokinin A stimulated dopamine levels in the ipsilateral striatum. These responses were not significantly different than those in unlesioned rats. Analysis of the intranigral lesion with in situ hybridization revealed a heavy loss of glutamic acid decarboxylase mRNA expression in the SNR and a significant loss of tyrosine hydroxylase (TH) mRNA expression in the SNC. Immunohistochemical analysis revealed a disappearance of TH-Like immunoreactivity (LI) im dendrites in the SNR, a considerable loss of TH-LI cell bodies in the SNC and a restricted loss of neuropeptide K-LI in the SNR around the tip of the injection cannula. Furthermore, lesioned rats rotated ipsilateral to the lesion after apomorphine (1 mg/kg, s.c.), indicating that the basal ganglia output mediated via the SNR GABA neurons was impaired on the lesioned side. Analysis of the striatum revealed that a dense TH-LI fiber network could still be seen on the lesioned side. Furthermore, basal and amphetamine stimulated extracellular dopamine levels in the striatum on the lesioned side were not significantly depleted. This indicates that the ascending nigrostriatal dopamine projection was functionally intact on the lesioned side. These findings indicate that intranigral GABA, dynorphin A, substance P and neurokinin A modulation of ipsilateral striatal dopamine release is mediated via direct action on the nigrostriatal projection. Thus, it is suggested that the striatonigral pathway, which contains GABA, dynorphin, substance P and neurokinin A, exerts a direct regulatory effect on the activity of the nigrostriatal dopamine projection.
Animals↗
Effects of imidazoline receptor ligands on monoamine synthesis in the rat brain in vivo.
This study was designed to assess the effects of imidazoline drugs on putative presynaptic imidazoline receptors modulating brain monoamine synthesis in vivo. The accumulation of 3,4-dihydroxyphenylalanine (dopa) and 5-hydroxytryptophan (5-HTP) after decarboxylase inhibition was used as a measure of the rate of tyrosine and tryptophan hydroxylation in various brain regions of naive rats and after irreversible alpha2-adrenoceptor inactivation with EEDQ (1.6 mg/kg, i.p., 6 h). Clonidine (1-3 mg/kg), moxonidine (1-10 mg/kg) and rilmenidine (10 mg/kg) (mixed I1/alpha2 agonists) decreased dopa and 5-HTP synthesis in the cerebral cortex (14%-81%), hippocampus (27%-84%) and/or striatum (29%-56%), but these inhibitory effects were abolished in N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ)-treated rats. Similarly, the stimulatory effect of efaroxan (mixed I1/alpha2 antagonist; 10 mg/kg) on dopa synthesis in the cortex (77%) and hippocampus (57%) was abolished by EEDQ. The selective I1-ligand 2-endo-amino-3-exoisopropylbicyclo-heptane (AGN-192403; 5-10 mg/kg) did not modify dopa or 5-HTP synthesis in any brain region in naive or EEDQ-treated rats. Idazoxan (mixed I2/alpha2 antagonist; 20 mg/kg) increased dopa synthesis in the cortex (111%) and hippocampus (87%), but the stimulatory effects were abolished by EEDQ. Moreover, idazoxan and efaroxan decreased 5-HTP synthesis in the cortex (12%-34%) and hippocampus (30%-34%) in a manner sensitive to blockade by the 5-HT1A receptor antagonist WAY 100135. The selective I2-igands 2-(2-benzofuranyl)-2-imidazoline (2-BFI; 20 mg/kg) and 2-styryl-2-imidazoline (LSL 61122; 10 mg/kg) did not alter the synthesis of dopa or 5-HTP in the cortex or hippocampus. In striatum, 2-BFI (1-20 mg/kg) dose-dependently decreased dopa synthesis (ED50: 5.9 mg/kg), reduced dopamine levels (6%-36%) and increased those of its metabolites DOPAC (15%-95%) and HVA (24%-74%). The inhibitory effect of 2-BFI on dopa/dopamine synthesis in striatum remained unchanged after alkylation of imidazoline receptors with isothiocyanatobenzyl imidazoline (IBI; 60 mg/kg, 6 h) or blockade of these receptors with 2-(2-ethyl 2,3-dihydro-2-benzofuranyl)-2-imidazole (KU-14R; 7-20 mg/kg). Therefore, most imidazoline drugs modulated the synthesis of brain monoamines through interaction with alpha2-adrenoceptors or 5-HT1A receptors. The results do not provide functional evidence for the existence of presynaptic imidazoline receptors regulating the synthesis of monoamines in the rat brain.
5-Hydroxytryptophan↗
Differential effects of gonadal steroids on dopamine metabolism in mesolimbic and nigro-striatal pathways of male rat brain.
Thirty days after castration the concentration of dopamine (DA) was significantly reduced in the septum and n. accumbens septi, but not in the caudate-putamen, of male rat brain. The concentrations of dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), the principle metabolites of DA, also tended to be lower in septum and n. accumbens septi after castration. Chronic s.c. administration of testosterone (T), estradiol (E2), 5 alpha-dihydrotestosterone (DHT), or E2 plus DHT in silastic capsules effectively reversed these effects of castration in septum and n. accumbens septi without affecting concentrations of DA, DOPAC, or HVA in caudate-putamen. The accumulation of DOPA after inhibition of aromatic amino acid decarboxylase activity, which was taken as an in vivo index of tyrosine hydroxylase activity, was not affected in these brain regions by long-term castration or by chronic administration of DHT to castrated males. Acute administration of haloperidol caused equivalent, significant increments in concentrations of DOPAC and HVA in all brain regions studied, regardless of whether castrated rats had been implanted with DHT capsules or no hormone. However, in the absence of haloperidol treatment the concentration of DOPAC in septum and n. accumbens septi, but not in caudate-putamen, was significantly higher in castrated rats implanted with DHT as opposed to no hormone. These results suggest that chronic exposure to T, or to its neural metabolites, E2 and DHT, selectively enhances metabolic activity in mesolimbic DA neurons.
3,4-Dihydroxyphenylacetic Acid↗
Norepinephrine neurons in mouse locus coeruleus express c-fos protein after N-methyl-D,L-aspartic acid (NMDA) treatment: relation to LH release.
In mouse, rat, and monkey, N-methyl-D,L-aspartic acid (NMDA) modulates gonadotropin releasing hormone (GnRH) release by an unknown mechanism. In previous studies we found that normal male mice consistently responded to NMDA administration with increased levels of plasma LH, as did most normal female mice and female hypogonadal mice with fetal preoptic area implants (HPG/POA). To investigate the mechanism of NMDA-induced GnRH release, immunocytochemistry of c-fos protein (FOS) was used for detection of neurons activated by NMDA administration. In both normal male and HPG/POA mice, FOS expression was unchanged in GnRH cells after NMDA administration. That neurosecretory cells can respond to NMDA was shown by the induction of FOS in many CRH (corticotropin-releasing hormone) cells in the paraventricular nucleus. Immunocytochemistry of beta-Endorphin, neuropeptide Y, tyrosine hydroxylase, an enzyme marker for catecholaminergic neurons, and glutamic acid decarboxylase, an enzyme marker for GABA neurons, was combined with that for FOS in normal male mice. Many noradrenergic (NA) neurons in the locus coeruleus (32-61%), and dopaminergic (DA) neurons in the mediobasal hypothalamus (15-31%) expressed FOS after NMDA administration while FOS was only rarely induced in neurons with the other neuromodulators tested. FOS was also induced in the locus coeruleus in male (43, 54%) and female (40, 55, 69%) HPG/POA mice. In contrast, few cells of the locus coeruleus expressed FOS in normal or HPG/POA mice after saline challenge. These results suggested that NMDA did not activate GnRH cells directly, but that NA neurons in the locus coeruleus were activated by NMDA and might be involved in stimulating GnRH release.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Central dopaminergic neurons during development of genetic and DOCA-salt hypertension in the rat.
The in vivo binding of [3H]spiroperidol was measured in discrete areas of the brain in 7-, 9- and 16-week-old spontaneously hypertensive rats (SHR) and age-matched normotensive Wistar-Kyoto (WKY) controls. An increase in the [3H]spiroperidol binding in the striatum, tuberculum olfactorium and frontal cortex but not in the cerebellum was detected at all ages in SHR. The increase was more pronounced in 7- than in 9- or 16-week-old SHR. In vitro data indicated an increase in Bmax but no variation in Kd in the striatum of 7-week-old SHR. Moreover no difference was detectable in the dopaminergic cell bodies (A9, A10). This increase was specific to [3H]spiroperidol binding sites since no difference was observed in the in vivo binding of [3H]QNB and [3H]LSD in the same brain regions. No variation in dopamine level or dopamine utilization, as estimated by measuring the disappearance of the amine induced by alpha-methyl-p-tyrosine, was observed. The DOPA accumulation after injection of the DOPA decarboxylase inhibitor NSD 1015 was greater in the tuberculum olfactorium from 7-week-old SHR. An increase in [3H]spiroperidol binding sites was also observed in the striatum and tuberculum olfactorium after 7 weeks of DOCA-salt treatment. These results suggest that dopaminergic neurons might be implicated in the onset of hypertension in the rat.
Animals↗