DOPA Levels Research Articles

Structure-activity relationships in the 8-amino-6,7,8,9-tetrahydro-3H-benz[e]indole ring system. 2. Effects of 8-amino nitrogen substitution on serotonin receptor binding and pharmacology.

A series of analogs of the potent and selective 5-HT1A agonist 8-(di-n-propylamino)-6,7,8,9-tetrahydro-3H-benz[e]indole-1-carbaldehyde (2b) (OSU191) was prepared in which the dipropylamino group was modified to bear a variety of substituents. These compounds were evaluated for both in vitro and in vivo effects, including the establishment of a receptor binding profile for these analogs at the 5-HT1A, dopamine D-2, dopamine D-3, 5-HT1D alpha, and 5-HT1D beta sites. Several of the analogs were evaluated for their biochemical effects in reserpinized rats, specifically with regard to in vivo changes in brain levels of 5-HTP and DOPA. Nearly all of the compounds prepared for this study were exceedingly potent at the 5-HT1A receptor, although most also displayed significant affinity for the dopamine D-2 receptor. A strong preference for the 5-HT1D alpha over the 5-HT1D beta receptor was also apparent. An analog bearing a butylglutarimide side chain, S-7k, was extremely selective for the 5-HT1A receptor. Although this compound possessed a Ki of 0.6 nM, it elicited only modest changes in 5-HTP brain levels. However, this compound did not appear as an antagonist when tested in a cyclic-AMP-based intrinsic activity assay.

Biochemical pathway of melanotic encapsulation of Brugia malayi in the mosquito, Armigeres subalbatus

The mosquito, Armigeres subalbatus, is naturally resistant to the filarial worm, Brugia malayi, and microfilariae (mf) penetrating the midgut are killed by melanotic encapsulation reactions in the hemocoel within 48 h following ingestion. This vector-parasite system was used to assess changes in hemolymph tyrosine, tyrosine derivatives, and catecholamine-metabolizing enzyme activities using high pressure liquid chromatography with electrochemical detection (HPLC-ED) during melanotic encapsulation reactions against mf. Tyrosine and dopa were detected in the hemolymph of both control and immune-activated (mf-exposed) mosquitoes, but not dopamine or N-acetyl dopamine (NADA). Tyrosine was significantly increased in immune-activated mosquitoes at 6 and 12 h post blood feeding, but was depleted following intrathoracic inoculation of mf in the absence of a blood meal. Dopa also was elevated in immune-activated mosquitoes at 6, 12, and 24 h post-exposure to mf. There were significant increases in phenol oxidase (PO) and dopa decarboxylase (DDC) activities in immune-activated mosquitoes as compared to controls, and these elevated activities were correlated with changes in tyrosine and dopa levels in the hemolymph. No significant differences in N-acetyl transferase (NAT) and dopachrome conversion enzyme (DCE) activities between control and immune-activated mosquitoes were observed. The possible roles these molecules play in melanotic encapsulation reactions of A. subalbatus against mf are discussed.

Drug delivery to the brain. DOPA prodrugs based on a ring-closure reaction to quaternary thiazolium compounds

In order to explore the possibility of an alternative redox chemical delivery system to a dihydropyridine-pyrid́inium interconversion system, we prepared DOPA prodrugs coupled with thiazolium precursors, like thiamine disulfides, by forming an ester bond with the amino acid carboxylic moiety while protecting the catechol function with pivalyl groups. The disposition of the prodrugs was evaluated by measuring the concentrations of DOPA regenerated after intravenous administration of the prodrugs and the results were compared with those for DOPA itself. The plasma levels of DOPA demonstrated no significant differences between DOPA and the prodrugs. In contrast, however, brain levels of DOPA were remarkably elevated following administration of the prodrugs. Among the prodrugs examined, ZiPr-DOPA(P) 2 was found to most efficiently facilitate delivery of DOPA to brain and this compound showed 30- and 3.7-fold greater increases in the AUC and MRT of DOPA in brain, respectively, than did DOPA itself. These findings suggest that a redox ring-closure system to a quaternary thiazolium can be used as an alternative chemical delivery system to the brain.

6R-L-erythro-5,6,7,8-Tetrahydrobiopterin and Dopamine Release

We previously reported that intracerebroventricular administration of 6R-L-erythro-5,6,7,8-tetrahydrobiopterin (6R-BJL), a cofactor for aromatic amino acid hydroxylases and nitric oxide synthase, induces an increase in DOPA levels (as an index of DA biosynthesis) and in levels of DA metabolites (as an index of DA release) in the rat brain: the increase in DOPA levels was larger than that of DA metabolites, indicating that 6R-BH4enhances DA release. In the present study, we examined the effects of 6R-BH4on DA release in vivo from the striatum of male Wistar rats and its mechanism of action using brain microdialysis. When various concentrations of 6R-BJL, 6S-BH4 (a diastereoisomer of 6R-BH4 ) or 6-methyltetrahydropterin (6-MPH4) were added to the perfusion fluid of microdialysis probes, DA levels collected in dialysates increased in a concentration-dependent manner: the 6R-BJL-induced increase was far greater than the increase induced by 6S-BH4 or 6-MPH4. Biopterin had little effect on the DA levels. After i.p. injection of 250 mg/kg of α-methyl-p-tyrosine (α-MT; an inhibitor of tyrosine hydroxylase), most of the 6RBH4- induced increase persisted but the increase induced by 6S-BJL or 6-MPH4 was abolished. The 6R-BH4 -induced increase in DA levels in dialysates was abolished after addition of tetrodotoxin (50 μM) to the perfusion fluid, but it persisted after i.p. injection of 100 mg/kg of nomifensine which completely inhibited DA reuptake. The increase in DA levels also persisted after administration of nitric oxide synthase inhibitors. The 6R-BJL-induced increase in DA levels was inhibited by co-administration of 6S-BJL or 6-MPH4. Administration of sepiapterin (an immediate precursor of 6R-BJL) increased tissue levels of reduced biopterin (mainly consisting of 6R-BH4) but not its extracellular levels monitored by brain microdialysis, indicating that it selectively increases the intracellular 6R-BJL levels. In contrast, administration of 6R-BH4increased both tissue levels of reduced biopterin and its extracellular levels, indicating that it increases both the intra- and extra-cellular 6R-BH4leveles. DA levels in dialysates was elevated following administration of sepiaterin (an immediate precursor of 6R-BH4), but the elevation was abolished after pretreatment with α-MT. Furthermore, 6R-BH4 increased firing rates of neurons in the dorsal motor nucleus of vagus monitored by a slice patch method. These results suggest that 6R-BH4 has a DA releasing action independent of its cofactor for tyrosine hydroxylase and nitric oxide synthase, which is mediated by a specific recognition site for 6R-BH4 on DA neurons or related cells.

3,4-dihydroxyphenylalanine (DOPA) decarboxylase deficiency and resultant high levels of plasma DOPA and dopamine in unfavorable neuroblastoma.

Neuroblastoma (NB) is a tumor which arises from neural crest cells. In the developing neural crest cells, the induction of 3,4-dihydroxyphenylalanine (DOPA) decarboxylase is more delayed than that of tyrosine hydroxylase and dopamine-beta-hydroxylase. If NB cells are arrested in an early stage of neural crest development, the induction of DOPA decarboxylase is insufficient and the accumulation and secretion of DOPA can be caused. The biochemically immature phenotype is thought to represent the undifferentiated characteristics of the cells and might correlate with the grade of malignancy. To investigate whether the hypothesis is clinically applicable or not, we have measured plasma DOPA, dopamine and urinary catecholamine metabolites in NB patients. The levels of plasma DOPA, dopamine, urinary homovanillic acid (HVA) and vanillactic acid (VLA) were significantly higher in patients with unfavorable NBs and the higher plasma DOPA level was significantly associated with the patients' age (> 1 year old), tumor stage (III, IV) and DNA diploidy. Serial determination of plasma DOPA was a good monitor of the disease course. These results are compatible with the hypothesis on DOPA decarboxylase deficiency and DOPA secretion in undifferentiated, unfavorable NBs. In conclusion, the plasma DOPA can be used to predict patients' prognosis as well as to follow up patients with NB.

The effects of cisapride on plasma L-dopa levels and clinical response in Parkinson's disease.

Cisapride (CIS) is a prokinetic agent that increases gastrointestinal motility in normal individuals and improves constipation in Parkinson's disease (PD). We studied the effects of CIS on the clinical response and the peripheral pharmacokinetics of orally administered L-dopa given to patients with PD. Twenty patients with idiopathic PD and chronic constipation, whose response to L-dopa was suboptimal or characterized by fluctuations, agreed to participate in an open study that lasted for 2 weeks. Fourteen patients completed the study (mean age 65 +/- 9.3 years, mean duration of treatment 5.7 +/- 4.2 years, mean L-dopa daily doses 658.9 +/- 269.9 mg); six patients were excluded due to lack of compliance or changes in medication during the study. The end points of the study included the mean levels of L-dopa, the height of the peak of L-dopa in plasma, mean plasma levels of 3-OM-dopa, and the speed and quality of gait and visuomanual coordination before and during treatment with CIS. CIS increased peak plasma levels of L-dopa by 37% and the mean plasma levels of L-dopa by 13% with respect to those obtained with the same dose of L-dopa before the addition of CIS. Therefore, CIS appears to increase early absorption of L-dopa through acceleration of gastric emptying. CIS also increased plasma 3-OM-dopa levels, improved visuomanual coordination, and reduced gait disability. CIS improves gastrointestinal function and response to L-dopa in patients with PD and could be a helpful add-on medication in these patients.

Nuclear DNA ploidy pattern and tissue levels of dihydroxyphenylalanine and catecholamines in pheochromocytoma.

Flow-cytometric analysis of nuclear DNA and measurements of tissue levels of dihydroxyphenylalanine (dopa) and catecholamines were performed using fresh samples of tissue taken from 17 patients with pheochromocytoma. Nine (53%) tumors had a diploid pattern of DNA, and 8 (47%) had an aneuploid pattern. The tissue concentrations of dopa, dopamine, norepinephrine, and epinephrine did not differ significantly between pheochromocytomas with a diploid and those with an anoeuploid pattern. These results suggest that there is no correlation between nuclear DNA ploidy and tissue levels of dopa and catecholamines in pheochromocytomas.

New approaches to evaluate sympathoadrenal system activity in experiments on Earth and in space

In previous studies the activity of the sympathoadrenal system (SAS) in cosmonauts during space flights was evaluated by measuring plasma catecholamines (CA) levels and urinary CA and their metabolites concentrations. Plasma CA levels are accepted indicators of SAS activity, however, they are determined by the plasma clearances as well as the rates of CA release (spillover-SO) into the bloodstream. Nowadays methods are available which evaluate not only plasma levels of CA but also their release, spillover, uptake, reuptake, degradation and also CA synthesis in vivo measured by plasma levels of dihydroxyphenylalanine (DOPA). Plasma concentrations of DOPA, the CA noradrenaline (NE), adrenaline (ADR), and dopamine (DA), the deaminated catechol metabolites dihydroxyphenylglycol (DHPG) and dihydroxyphenylacetic acid (DOPAC), and the O-methylated metabolites methoxyhydroxyphenylglycol (MHPG) and homovanillic acid (HVA) were measured during immobilization stress (IMO) in conscious rats. Radiotracer methods were used to measure NE SO. IMO markedly increased arterial NE levels but NE SO was less elevated bacause the NE clearance was slightly reduced in IMO rats. Simultaneous measurements of plasma CA and their metabolites provide another means to obtain information about SAS function. For instance, dissociation between changes of plasma DHPG and NE levels can indicate changes in neuronal reuptake of NE. We found marked parallel increases in plasma NE and DHPG levels during acute IMO; however after repeated IMO, plasma NE levels were increased but DHPG responses were less pronounced suggesting a reduced NE reuptake. DOPA, the CA precursor, circulates in plasma at a concentration higher than NE. During stress, increased sympathoneural outflow stimulates DOPA synthesis and release into the circulation supporting the view that changes in plasma DOPA levels during stress reflect in vivo changes in the rate of CA synthesis. We propose to measure the new plasma indicators of SAS activity in cosmonauts and/or in animals before, during and after space flights.

Dopamine-releasing action of 6R-L-erythro-tetrahydrobiopterin: analysis of its action site using sepiapterin.

Recently, we reported that 6R-L-erythro-tetrahydrobiopterin (6R-BH4), a natural cofactor for hydroxylases of tyrosine and tryptophan, has a monoamine-releasing action independent of its cofactor activity. Here we attempted to determine whether 6R-BH4 acts inside the cell or from the outside of the cell by using brain microdialysis in the rat striatum. For this purpose, sepiapterin, and immediate precursor of 6R-BH4 in the salvage pathway, was used to selectively increase the intracellular 6R-BH4 levels. Dialytic perfusion of sepiapterin increased tissue levels of reduced biopterin (mainly 6R-BH4) but not the extracellular levels. Administration of sepiapterin increased the extracellular levels of 3,4-dihydroxyphenylalanine (DOPA) (an index of in vivo tyrosine hydroxylase activity) and of dopamine (DA) (an index of in vivo DA release). Either of the increases was eliminated after pretreatment with a tyrosine hydroxylase inhibitor alpha-methyl-p-tyrosine. Administration of 6R-BH4 increased extracellular levels of reduced biopterin. DOPA, and DA. After pretreatment with alpha-methyl-p-tyrosine, the increase in DOPA levels was abolished, but most of the increase in DA levels persisted. The increase in DA levels also persisted after pretreatment with nitric oxide synthase inhibitors. These data demonstrate that 6R-BH4 stimulates DA release directly, independent of its cofactor action for tyrosine hydroxylase and nitric oxide synthase, by acting from the outside of neurons.

The Effect of Clozapine on Plasma Norepinephrine: Relationship to Clinical Efficacy

Clozapine is an atypical neuroleptic medication with superior efficacy to conventional antipsychotic agents for patients with chronic, symptomatic schizophrenia. Neurochemical characteristics that distinguish clozapine from other neuroleptics and contribute to its differential efficacy are not known. We assessed the effects of clozapine on plasma levels of norepinephrine (NE) in a double-blind, parallel groups comparison of clozapine (n = 11) and haloperidol (n = 15) in chronic schizophrenic outpatients who had been previously treated with fluphenazine. Simultaneous measurements were obtained for plasma levels of the catecholamine precursor dopa, the dopamine metabolite dihydroxyphenylacetic acid (DOPAC), the NE metabolite 3,4-dihydroxyphenylglycol (DHPG), adrenocorticotropin (ACTH), cortisol, and hemodynamic parameters. Clozapine produced marked increases (471%) in plasma NE levels, whereas haloperidol had no significant effects on plasma NE levels. Clozapine also increased dopa and tended to increase DOPAC levels, without effects on DHPG, ACTH, or cortisol levels and without consistent changes in blood pressure. Across patients, the magnitude of clozapine-induced increments in plasma NE levels was positively related to improvement in positive symptoms and global symptomatology and was unrelated to the occurrence of extrapyramidal symptoms. The results suggest that clozapine differs importantly from other neuroleptics in increasing plasma NE levels, with the peripheral noradrenergic stimulation related to its superior efficacy profile. The unchanged DHPG levels and absence of hypertension suggest a more complex mechanism of action of clozapine than heightened NE release alone.

Increased Urinary Dopamine Excretion in Young Patients with Essential Hypertension

The evidence that some older patients with essential hypertension have low urinary dopamine excretion has brought into question the levels of urinary dopamine and plasma dopa, the major source of urinary dopamine, in young patients with essential hypertension. Twenty-four-hour urine sodium, creatinine, dopamine and noradrenaline and plasma dopa were evaluated in 48 patients with essential hypertension aged 18 to 27 years and 25 normotensive subjects. In comparison with age-matched normotensive subjects, the hypertensive patients had higher urinary dopamine (1920 +/- 80 vs 1520 +/- 130 nmol/day, p < 0.01) and noradrenaline (216 +/- 11 vs 179 +/- 12 nmol/day, p < 0.05) excretion. There was a significant correlation between urinary dopamine and noradrenaline excretion. There was no difference in plasma dopa levels between normotensive and hypertensive subjects. These results suggest that the elevated conversion of dopa to dopamine in the kidney is leading to increased urinary dopamine excretion in young patients with essential hypertension.

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 × 10 −4 mol/ l DFMD solution was needed to obtain measurable levels of DOPA in LC dialy sates: a linear increase in DOPA concentrations was first observed and, then, a steady state. DOPA disappeared completely after inhibition of TH by α-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.

Effects of 5‐HT1A Receptor Agonists on Patterns of Rat Motor Activity in Relation to Effects on Forebrain Monoamine Synthesis

The effects of the 5-HT1A receptor agonists 8-OH-DPAT (0.15-2.5 mumol kg-1 subcutaneously), flesinoxan (0.6-10.0 mumol kg-1 subcutaneously) and buspirone (1.9-30.0 mumol kg-1 subcutaneously) on spontaneous motor activity in male Sprague-Dawley rats was examined in a photocell-equipped open-field arena. Following motor activity observations, the cerebral aromatic L-amino acid decarboxylase inhibitor NSD-1015 (100 mg kg-1 intraperitoneally) was administered and 30 min. later the animals were decapitated for subsequent analysis of the accumulated forebrain DOPA and 5-HTP levels, as an estimate of the rate of monoamine synthesis. 8-OH-DPAT and flesinoxan produced a similar and characteristic pattern of changes of the spontaneous motor activity in normal animals i.e. a moderate decrease in locomotor activity, a marked suppression of rearing and an increase in the relative amount of forward locomotion and of activity in the periphery of the open-field arena. This behavioural profile was closely related to a decrease in forebrain 5-HTP accumulation, indicating 5-HT receptor stimulation. In agreement with these observations buspirone also produced an increase in peripheral activity and a suppression of rearing. In contrast to effects by 8-OH-DPAT and flesinoxan, however, buspirone produced a further reduction of locomotor activity and reduced the forward locomotion. This difference in behavioural profile between buspirone and the other two compounds is probably explained by its DA receptor blocking properties, as indicated by an increased DOPA accumulation in the neostriatum. At least partially, 8-OH-DPAT, flesinoxan and buspirone, all antagonized reserpine-induced (5 mg kg-1 subcutaneously--16 hr) suppression of locomotor activity. This stimulation of locomotor activity in reserpine-treated rats is in all probability related to 5-HT1A receptor stimulation since concomitant DA D2 receptor blockade, in the case of buspirone, did not markedly affect this behavioural response.

Protein-bound 3,4-dihydroxyphenylalanine is a major reductant formed during hydroxyl radical damage to proteins.

Proteins and aromatic amino acids previously exposed to hydroxyl radicals reduced cytochrome c, free iron, and copper ions. A major product of hydroxyl radical addition to tyrosine is 3,4-dihydroxyphenylalanine (DOPA), which has these reducing properties. The reduction of nitro blue tetrazolium by radical-damaged protein was consistent with the generation of quinones in the protein. By acid hydrolysis followed by high-performance C18 reversed-phase liquid chromatography we have shown that hydroxyl radical-damaged proteins contain significant amounts of protein-bound DOPA (PB-DOPA). The authenticity of the DOPA measured was confirmed by gas chromatography-mass spectrometry. PB-DOPA was also generated enzymatically using mushroom tyrosinase, which catalyzes the hydroxylation of tyrosine residues. By comparing the levels of DOPA in radical-damaged or enzyme-treated protein with that of cytochrome c reduction, we show that PB-DOPA is a major source of the observed reducing activity. PB-DOPA may have a role in the replenishment of reduced transition metal ions involved in free radical generating systems in vivo.

Biosynthesis of dopamine and serotonin in the rat brain after repeated cocaine injections: a microdissection mapping study.

The purpose of the present study was to examine the effects of chronic cocaine on dopamine (DA) and serotonin (5-HT) synthesis in several rat brain regions implicated in drug reinforcement. Male rats were treated twice daily with cocaine (15 mg/kg, ip) or saline for 1 week. After 42 hr of abstinence, rats were challenged with either cocaine (15 mg/kg, ip) or saline, followed by the aromatic L-amino acid decarboxylase inhibitor 3-hydroxybenzylhydrazine (NSD-1015; 100 mg/kg, ip). Animals were decapitated 30 min after NSD-1015 and discrete brain regions were microdisected from 300 microns frozen sections. Postmortem tissue levels of 3,4-dihydroxyphenylalanine (DOPA) and 5-hyroxytryptophan (5-HTP) were quantified by HPLC with electrochemical detection and used to estimate biosynthesis of DA and 5-HT, respectively. In chronic saline-treated rats, cocaine dramatically suppressed DA and 5-HT synthesis in all forebrain regions examined, including: medial prefrontal cortex, nucleus accumbens, caudate nucleus, olfactory tubercle, and basolateral amygdala. The degree of inhibition ranged from 35-65% and was more pronounced in 5-HT neurons compared to DA neurons in the same tissue sample. In general, chronic cocaine did not significantly alter basal levels of DOPA or 5-HTP; a notable exception was lateral hypothalamus, where chronic cocaine reduced basal DA synthesis to 75% of control. After repeated cocaine injections, the synthesis-inhibiting effect of a challenge injection of cocaine was attenuated in many brain areas. These data suggest that whereas acute cocaine decreases DA and 5-HT synthesis in forebrain, chronic cocaine is not neurotoxic to DA and 5-HT neurons. In addition, the mechanism(s) mediating cocaine-induced suppression of monoamine synthesis may become desensitized by chronic exposure to the drug.

Activity of Tyrosine Hydroxylase in the Striatum of Newborn Piglets in Response to Hypocapnic Hypoxia

The effect of graded hypoxia induced by hyperventilation on the activity of tyrosine hydroxylase was measured in vivo by microdialysis. Microdialysis probes were inserted into the striatum of newborn piglets and perfused with medium containing 3-hydroxybenzylhydrazine, an inhibitor of L-aromatic amino acid decarboxylase. The level of 3,4-dihydroxyphenylalanine (DOPA) measured in the effluent dialysate was then an index of tyrosine hydroxylase activity. The oxygen pressure in the veins and capillaries of the cortex was measured, through a cranial window placed over the parietal cortex, by the phosphorescence lifetime of palladium-meso-tetra(4-carboxyphenyl)porphine added to the blood. After baseline measurements, PaCO2 was decreased from 38 torr (control value) to 19, 13, and 11 torr resulting in decreases in the cortical oxygen pressure from 40 +/- 6 torr to 26 +/- 3, 23 +/- 4, and 20 +/- 4 torr, respectively. Decrease in the oxygen pressure to 26 +/- 3 torr caused a statistically significant increase of 25-30% in the level of DOPA in the effluent perfusate. During the next step of increase in ventilator rate, when oxygen decreased only slightly, the level of DOPA remained at the higher level. Ventilation rates that lowered the oxygen pressure to below 20 torr, however, caused a progressive decrease in the level of DOPA. During recovery, the level of DOPA steadily increased, attaining 160% of control value after 1.5 h. When the oxygen pressure was decreased to 16 +/- 2 torr by a single increase in ventilator rate, the DOPA level decreased in the effluent to 15-20% below control.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of acute and chronic cocaine on the activity of tuberoinfundibular dopamine neurons in the rat

We investigated the effects of acute and chronic cocaine on dopamine (DA) synthesis in tuberoinfundibular dopamine (TIDA) neurons by measuring the accumulation of 3,4-dihydroxyphenylalaine (DOPA) in the median eminence (ME). Cocaine-induced prolactin (PRL). Chronic cocaine did not alter were evaluated for comparison. Acute cocaine inhibited DA synthesis in the ME and decreased circulating PRL. Chronic cocaine did not alter basal DOPA levels in TIDA nerve terminals or PRL levels in plasma. After repeated cocaine injections, cocaine's inhibitory effect on DA synthesis was abolished in the ME while the PRL response to drug was unchanged. These results suggest that TIDA neurons become tolerant to cocaine-induced suppression of DA synthesis after chronic cocaine exposure, but the altered sensitivity of TIDA neurons is not revealed by plasma PRL measures.

Inhibition of aromatic l-amino acid decarboxylase under physiological conditions: optimization of 3-hydroxybenzylhydrazine concentration to prevent concurrent inhibition of monoamine oxidase

The activity of the enzyme tyrosine hydroxylase (TH; EC 1.14.16.2) is commonly studied indirectly by quantifying the formation of the product, 3,4-dihydroxyphenylalanine (DOPA), after inhibition of aromatic l-amino acid decarboxylase (AAAD; EC 4.1.1.28), the enzyme which metabolizes DOPA. This study was done to determine if the concentration of the hydrazine derivative 3-hydroxybenzylhydrazine (NSD-1015), a drug frequently used in vitro to inhibit AAAD, could be adjusted such that it would inhibit that enzyme, but would not simultaneously inhibit a second, potentially important enzyme, monoamine oxidase (MAO; EC 1.4.3.4). MAO catalyzes the formation of 3,4-dihydroxyphenylacetic acid (DOPAC) and 3,4-dihydroxyphenylglycol (DOPEG) from dopamine (DA) and norepinephrine (NE), respectively. Five concentrations of NSD-1015 in superfusate (0.01 to 20 μM) were tested in strips of canine portal vein superfused and stimulated in vitro. DOPA, DA, NE, and DOPEG in superfusate and in the veins after superfusion were quantified by HPLC with electrochemical detection. The efficacy of NSD-1015 in inhibiting AAAD and MAO was determined by examining the levels of DOPA and DOPEG, respectively. NSD-1015, only when applied at 0.1 μM, resulted in the marked augmentation of total DOPA levels, but did not affect levels of DOPEG, which suggests that this concentration of the drug inhibits AAAD, but does not inhibit MAO. Therefore, it is concluded that, of the concentrations of NSD-1015 tested, 0.1 μM is the optimum concentration to use in this preparation for studies designed to examine TH activity by measuring DOPA after the inhibition of AAAD.

Serotonin-induced decrease in hypothalamic tyrosine hydroxylase activity and corresponding increase in prolactin release are abolished at midpregnancy and by transplants of rat choriocarcinoma cells.

We investigated the effect of central serotonin (5-hydroxytryptamine, 5-HT) administration on hypothalamic tuberoinfundibular dopamine neurons and related changes in neuronal activity to circulating PRL levels in two physiological models: 1) pregnant rats expressing (day 8) or not expressing (days 11 and 16) PRL surges, and 2) ovariectomized rats transplanted with rat choriocarcinoma cells, which secrete functional placental lactogen-I. Over a 4-min period between 0900 and 1400 h, rats were administered either vehicle or 5-HT (20 micrograms/6 microliters) through lateral ventricular cannulae. Plasma PRL levels were determined by RIA. NSD 1015 (25 mg/kg intraarterial), a dihydroxyphenylalanine (DOPA) decarboxylase inhibitor, was injected 20 min after initiation of ventricular infusion. Ten min later, the stalk-median eminence (SME) was dissected. The rate of DOPA accumulation, determined by measuring DOPA levels in the SME by HPLC, was used as an index of tyrosine hydroxylase catalytic activity, indicating tubero infundibular dopamine neuronal activity. In day-8 pregnant rats 5-HT reduced DOPA accumulation to 57% of vehicle-injected controls and increased circulating PRL levels 13-fold. In contrast, on days 11 and 16 of pregnancy 5-HT did not alter DOPA accumulation in the SME or plasma PRL levels. In nonpregnant rats ovariectomized for 24 h, 5-HT decreased DOPA accumulation in the SME to 43% of vehicle-infused controls and increased PRL levels approximately 26-fold. However, in nonpregnant rats with rat choriocarcinoma cells, 5-HT produced no changes in either DOPA accumulation in the SME or in circulating PRL levels. The inability of 5-HT to reduce tyrosine hydroxylase activity after mid-pregnancy may account for the lack of a PRL response. Placental lactogens secreted at midpregnancy, particularly placental lactogen-1, may induce this loss of 5-HT effect.

Central Administration of Serotonin Decreases Tyrosine Hydroxylase Catalytic Activity and Messenger Ribonucleic Acid Signal Levels in the Hypothalamus of Female Rats

We investigated the effect of central serotonin (5-hydroxytryptamine, 5-HT) administration on hypothalamic tuberoinfundibular dopamine neurons and related changes in neuronal activity to circulating prolactin (PRL) levels. Ovariectomized rats were treated with either vehicle or 5-HT through a lateral ventricular cannula in one of two dose paradigms: 1) a bolus of 20 μg, with tissues taken at 30 min, or 2) the same bolus immediately followed by 20 μg/30 min via a syringe pump for 120 min, and tissues taken at 120 min. Blood samples were taken throughout experiments and plasma PRL determined by radioimmunoassay. Under both paradigms, NSD 1015, a dihydroxyphenylalanine (DOPA) decarboxylase inhibitor (25 mg/kg intraarterially) was injected 10 min before decapitation and brain excision followed by stalk-median eminence dissection. The rate of DOPA accumulation, determined by measuring DOPA levels in the stalk-median eminence by high-performance liquid chromatography with electrochemical detection was used as a measure of tyrosine hydroxylase (TH) catalytic activity. Stalk-median eminence DOPA accumulation in control rats was 29.9 ± 4.2 and 28.8 ± 4,4 ng/mg protein (30 and 120 min experiments, respectively). DOPA accumulation in 5-HT-treated rats was significantly reduced (P<0.05) after 30 min to 17.8 ± 1.2 ng/mg protein, but it was similar (21.7 ± 3.9) to controls after 120 min of 5-HT infusion. 5-HT levels in the stalk-median eminence of rats treated with 5-HT were 13- to 17-fold greater than controls (16.9 to 18.5 ng/mg protein). Plasma PRL levels in both groups increased 10-fold after 5-HT treatment with a peak at 5 min, returning to baseline by 120 min. TH mRNA levels were determined by in situ hybridization in a second group of rats which were treated with the 20μg bolus and subsequent 120 min infusion of 5-HT. TH mRNA signal levels in the arcuate nucleus of control rats averaged 144 ± 21 grains/cell. After treatment with 5-HT, TH mRNA levels in the arcuate nucleus were significantly lower (P<0.0001) with 69±14 grains/cell. In a third group of rats, the effects of the 30 min 5-HT treatment on TH catalytic activity and circulating PRL levels was challenged with two 5-HT(2) receptor antagonists, LY53857 (5 mg/kg intraperitoneally) or ketanserin (10 mg/kg intraperitoneally). Neither the 5-HT-induced decrease in TH catalytic activity nor the increase in PRL was altered by pretreatment (120 min) with 5-HT(2) antagonists. These data suggest that central 5-HT is capable of decreasing TH activity and TH mRNA levels in the tuberoinfundibular dopamine neurons and that the decrease in dopaminergic neuronal activity may contribute to the 5-HT-induced PRL rise. The changes in TH catalytic activity and PRL after intracerebroventricular administration of 5-HT do not appear to be mediated by 5-HT(2) receptors.