Microdialysis Monitoring of 3,4-Dihydroxyphenylalanine Accumulation After Decarboxylase Inhibition: A Means to Estimate In Vivo Changes in Tyrosine Hydroxylase Activity of the Rat Locus Ceruleus

Abstract: An on-line microdialysis approach was developed to estimate changes in tyrosine hydroxylase activity in the locus ceruleus noradrenergic neurons of anesthetized rats by measuring the 3,4-dihydroxyphenylalanine (DOPA) acumulation in the extracellular fluid during perfusion of an aromatic amino acid decarboxylase inhibitor through a dialysis probe. The aromatic amino acid decarboxylase inhibitor used was difluoromethyl-DOPA, which was shown to be more stable than NSD 1015 or Ro 4-4602 in the perfusion fluid. A 1-h perfusion of a 10⁻⁴ mol/L of difluoromethyl-DOPA solution induced a linear increase in DOPA concentration in the locus ceruleus dialysates that achieved a steady state within 1 h. The identity of DOPA accumulated in dialysates during aromatic amino acid decarboxylase inhibition was confirmed by the disappearance of the chromatographic peak when DOPA formation was blocked by the administration of α-methyl- p -tyrosine. Systemic administration of the α₂-antagonist piperoxane before difluoromethyl-DOPA perfusion markedly increased the DOPA concentration during both the accumulation and the steady-state periods, showing that the present technique is a suitable in vivo approach to monitor changes in tyrosine hydroxylase activity occurring in the locus ceruleus neurons.     

Effect of Precursor Loading on the Synthesis Rate and Release of Dopamine and Serotonin in the Striatum: A Microdialysis Study in Conscious Rats

The effects of systemic administration of tyrosine and phenylalanine on the extracellular levels of tyrosine and dopamine were determined by microdialysis in the striatum of awake rats. In addition, the effects of these precursors on in vivo 3,4-dihydroxyphenylalanine (DOPA) formation were determined during continuous infusion of a decarboxylase inhibitor. Both precursors increased the dialysate levels of tyrosine sixfold, but only phenylalanine administration stimulated DOPA formation. However, neither precursor affected the release of dopamine. When the precursor administration was repeated in rats in which the release of dopamine was stimulated by haloperidol pretreatment, again no effect was seen on the release of dopamine. Systemic administration of tryptophan (100 mg/kg, i.p.) during continuous infusion of a decarboxylase inhibitor induced a threefold increase in the formation of 5-hydroxytryptophan and caused an increase in the release of serotonin during infusion of an uptake inhibitor to about 150% of controls. Finally, we investigated whether dietary precursors were able to influence neurotransmitter formation and release. Rats trained to consume their daily food in a period of 2 h were implanted with microdialysis probes. Scheduled eating induced a small increase in the extracellular levels of tyrosine (135% of controls), but the release of dopamine and the formation of 5-hydroxytryptophan during continuous infusion of a decarboxylase inhibitor were not affected.     

Dopamine Release in Rat Striatum: Physiological Coupling to Tyrosine Supply

Intracerebral microdialysis was used to monitor dopamine release in rat striatal extracellular fluid following the intraperitoneal administration of dopamine's precursor amino acid, L-tyrosine. Dopamine concentrations in dialysates increased transiently after tyrosine (50-100 mg/kg) administration. Pretreatment with haloperidol or the partial lesioning of nigrostriatal neurons enhanced the effect of tyrosine on dopamine release, and haloperidol also prolonged this effect. These data suggest that nigrostriatal dopaminergic neurons are responsive to changes in precursor availability under basal conditions, but that receptor-mediated feedback mechanisms limit the magnitude and duration of this effect.     

Enhancement of Dopamine Release In Vivo from the Rat Striatum by Dialytic Perfusion of 6R-l-erythro-5,6,7,8-Tetrahydrobiopterin

We have previously reported that intracerebroventricular administration of 6R-L-erythro-5,6,7,8-tetrahydrobiopterin (6R-BH4), a cofactor for tyrosine hydroxylase, enhances biosynthesis of 3,4-dihydroxyphenylethylamine (dopamine) in the rat brain. In the present study, we have more precisely examined the effects of 6R-BH4 on dopamine release in vivo from the rat striatum using brain microdialysis. The amount of dopamine collected in striatal dialysates was determined using HPLC with electrochemical detection after purification with an alumina batch method. When the striatum was dialyzed with Ringer solution containing various concentrations of 6R-BH4 (0.25, 0.5, and 1.0 mM), dopamine levels in striatal dialysates increased in a concentration-dependent manner. Biopterin had little effect on dopamine levels in dialysates. The 6R-BH4-induced increase in dopamine levels in dialysates was abolished after pretreatment with tetrodotoxin (50 microM) added to the perfusion fluid, but after pretreatment with nomifensine (100 mg/kg, intraperitoneal injection), an inhibitor of dopamine uptake mechanism, a larger increase was observed. After inhibition of tyrosine hydroxylase by pretreatment with alpha-methyl-p-tyrosine (250 mg/kg, intraperitoneal injection), most of the increase persisted. These results suggest that 6R-BH4 has a dopamine-releasing action, which is not dependent on biosynthesis of dopamine.     

ALTERATIONS IN RECEPTORS CONTROLLING DOPAMINE SYNTHESIS AFTER CHRONIC ETHANOL INGESTION

The influence of acute and chronic ethanol treatment and withdrawal on regulation of dopamine synthesis in striatal and mesolimbic areas of mouse brain was evaluated. Tyrosine hydroxylase activity was estimated by measuring in vivo DOPA accumulation after inhibition of aromatic amino acid decarboxylase. Eight hours after a single (3 g/kg) dose of ethanol, DOPA synthesis was increased and pimozide, a dopamine receptor antagonist, stimulated DOPA synthesis to the same degree in ethanol-treated and control animals. On the other hand, 8 h after withdrawal of animals from chronic ethanol treatment, endogenous dopamine synthesis was the same in ethanol-withdrawn and control animals, but the stimulation of dopamine synthesis produced by low doses of pimozide or haloperidol was significantly less in the animals that had consumed ethanol. This effect was even more apparent at 24h after withdrawal; by 3 days after withdrawal the decreased response of ethanol-withdrawn animals to the administration of dopamine receptor blockers was no longer statistically significant. At all time points tested, high doses of pimozide or haloperidol stimulated DOPA synthesis equally in control and ethanol-withdrawn animals. Chronic ethanol treatment and withdrawal may alter the coupling between dopamine receptors which regulate dopamine synthesis and tyrosine hydroxylase.     

Extracellular Striatal Concentrations of Endogenous 3,4-Dihydroxyphenylalanine in the Absence of a Decarboxylase Inhibitor: A Dynamic Index of Dopamine Synthesis In Vivo

Abstract: Basal levels of endogenous 3,4-dihydroxyphenylalanine (DOPA) were detected by HPLC coupled with coulometric detection in dialysates from freely moving rats implanted 48–72 h earlier with transversal dialysis fibers in the dorsal caudate. Because decarboxylase inhibitor is absent in the Ringer's solution, this method allows monitoring of basal output of dopamine (DA) and 3,4-dihydroxyphenylacetic acid, as well as DOPA. Extracellular DOPA concentrations were reduced by the tyrosine hydroxylase inhibitor α-methylparatyrosine (200 mg/kg, i.p.) and by the dopaminergic agonist apomorphine (0.25 mg/kg, s.c.). The dopaminergic antagonist haloperidol (0.2 mg/kg, s.c.) stimulated DOPA output by about 60% over basal values. γ-Butyrolactone, at doses of 700 mg/kg, i.p., which are known to block dopaminergic neuronal firing and which reduce DA release, stimulated DOPA output maximally by 130% over basal values. Tetrodotoxin, which blocks DA release by blocking voltage-dependent Na⁺ channels, increased DOPA output maximally by 100% over basal values. The results indicate that basal DOPA can be detected and monitored in the extracellular fluid of the caudate of freely moving rats by transcerebral dialysis and can be taken as a dynamic index of DA synthesis in pharmacological conditions.     

Studies on Tyrosine Hydroxylase System in Rat Brain Slices Using High-Performance Liquid Chromatography with Electrochemical Detection

A new method for the measurement of tyrosine hydroxylase (TH; EC 1.14.16.2) activity in brain slices was developed by using high-performance liquid chromatography (HPLC) with electrochemical detection (ED). To estimate TH activity in brain slices containing all of the components of the enzyme system, tetrahydrobiopterin, dihydropteridine reductase, and TH itself, slices were incubated with NSD-1055, an inhibitor of aromatic L-amino acid decarboxylase, and 3,4-dihydroxyphenylalanine (DOPA) formed from endogenous tyrosine was measured using HPLC-ED. Hydroxylation of endogenous tyrosine to DOPA in striatal slices was linear up to 90 min at 37 degrees C, and increased by incubation with 20 mM K+ to depolarize the nerve cells. Furthermore, the formation of DOPA could be detected in all parts of brain regions examined, and the activity in this slice system was nearly parallel to the maximal velocity of the homogenate from the slices as enzyme in the presence of saturating concentrations of tyrosine and 6-methyltetrahydropterin as cofactor. This assay system should be useful to study the regulatory mechanisms of TH in relatively intact tissue preparations.     

Enhanced Tyrosine Hydroxylation in Hippocampus of Chronically Stressed Rats upon Exposure to a Novel Stressor

We have used microdialysis to measure the in vivo level of tyrosine hydroxylation in hippocampus of the freely moving rat. An inhibitor of aromatic amino acid decarboxylase, NSD-1015, was administered through the dialysis probe and the resulting accumulation of 3,4-dihydroxyphenylalanine (DOPA) in extracellular fluid of hippocampus was quantified. Administration of the tyrosine hydroxylase inhibitor, alpha-methyl-p-tyrosine, decreased extracellular DOPA to undetectable level. In addition, both systemic and local application of clonidine, an alpha 2-adrenergic agonist, produced a decrease in extracellular DOPA. In response to acute tail shock, a significant increase in extracellular DOPA was observed. Thus, it appears that in vivo accumulation of DOPA after local administration of NSD-1015 provides a reliable index of hippocampal tyrosine hydroxylation. We have used this technique to investigate whether prior exposure to chronic stress alters the in vivo level of tyrosine hydroxylation in hippocampus under basal conditions as well as in response to a novel stressor. In rats previously exposed to chronic cold stress, the basal accumulation of extracellular DOPA did not differ from naive controls. Acute tail shock, however, produced a significantly greater and more prolonged elevation in extracellular DOPA of chronically stressed rats. These data suggest that enhanced biosynthetic capacity of noradrenergic terminals may be one mechanism underlying adaptation to chronic stress.     

Effect of dopamine agonists and antagonists on DOPA formation in the substantia nigra

Abstract: The effect of different psychotropic drugs on the rate of DOPA accumulation after administration of a decarboxylase inhibitor (NSD 1015) was compared in the substantia nigra (SN) and caudate nucleus (CN) by a new radioenzymatic method. Inhibition of monoamine oxidase with pargyline or stimulation of dopamine (DA) receptors with apomorphine, N -n-propyl-norapomorphine or d -amphetamine reduced DOPA formation in the CN and SN to the same extent. Vice versa, both inhibition of DA receptors with haloperidol or (-)sulpiride and depletion of DA concentration with reserpine enhanced DOPA formation to a greater extent in the CN than in the SN. Apomorphine antagonized not only the effect of haloperidol and (-)sulpiride, but also, and even more effectively, that of reserpine. The results indicate that DA synthesis in the SN is controlled by both end-product inhibition and DA receptor-mediated mechanisms.     

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

Abstract: 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, P_aCO₂ 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. With return of the ventilator rate to control values, the DOPA levels again increased to well above control and stayed higher even after 1.5 h. The slow return of tyrosine hydroxylase activity to control indicates relatively long-term modification of the enzyme activity. Activation of tyrosine hydroxylase occurs when the oxygen pressure is decreased, but at <16 torr the reaction rate becomes limited by the availability of oxygen and decreases with further decrease in oxygen pressure. Our results showed that even small changes in cortical oxygen pressure modulate the activity of tyrosine hydroxylase. This alteration in the metabolism of catecholamines in newborn brain may have significant impact on later development of the organism.     

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.     

Role of tyrosine, DOPA and decarboxylase enzymes in the synthesis of monoamines in the brain of the locust

The metabolic transformation of tyrosine (TYR) by the decarboxylase and hydroxylase enzymes was investigated in the central nervous system of the locust, Locusta migratoria. It has been demonstrated that the key amino acids, 3,4-dihydroxyphenylalanine (DOPA), 5-hydroxytryptophan (5HTP) and tyrosine are decarboxylated in all part of central nervous system. DOPA and 5HTP decarboxylase activities show parallel changes in the different ganglia, but the rank order of the activity of TYR decarboxylase is different. Enzyme purification has revealed that the molecular weights of TYR decarboxylase and DOPA/5HTP decarboxylase are 370,000 and 112,000, respectively. The decarboxylation of DOPA by DOPA/5HTP decarboxylase is stimulated, whereas the decarboxylation of DOPA by TYR decarboxylase is inhibited in the presence of the cofactor pyridoxal-5'-phosphate. TYR hydroxylase could not be detected and 3H-TYR is found to be metabolised to tyramine (TA), but not to DOPA. The haemolymph contains a significant concentration of DOPA (120 pmol/100 microl haemolymph), and the ganglia incorporates DOPA from the haemolymph by a high affinity uptake process (K(M)=12 microM and V(max)=24 pmol per ganglion/10 min). Our results suggest that no tyrosine hydroxylase is present in the locust CNS and the DOPA uptake into the ganglia by a high affinity uptake process as well as the DOPA decarboxylase enzyme may be responsible for the regulation of the ganglionic dopamine (DA) level. Two types of decarboxylases exist, one of them decarboxylating DOPA and 5HTP (DOPA/5HTP decarboxylase), other decarboxylating TYR (TYR decarboxylase). The DOPA/5HTP decarboxylase enzyme present in the insect brain may correspond to the 5HTP/DOPA decarboxylase in vertebrate brain, whereas TYR decarboxylase is characteristic only for the insect brain.     

Reduction in brain tyrosine hydroxylase activity following acetylcholinesterase blockade in rats.

Activation of cholinergic neurons in the brain is produced by administration of the acetylcholinesterase inhibitors physostigmine and diisopropylfluorophosphate (DFP). This activation has a biphasic effect on tyrosine hydroxylase (EC 4.14.3-) activity. The acute effect of DFP, 1 mg/kg, intraperitoneally, or physostigmine, 0.2 mg/kg, intravenously, or 10 mug, intraventricularly, was a rapid reduction in tyrosine hydroxylase activity in the hypothalamus. The activities of DOPA decarboxylase (EC 4.1.1.28) and dopamine-beta-hydroxylase (EC 1.14.17.1) were not changed. In contrast to the acute effect, chronic administration of physostigmine, 0.2 mg/kg, intravenously, twice daily for 7 days produced an increase in tyrosine hydroxylase activity in the hypothalamus. The rapid acute effects may be due to an allosteric inactivation of tyrosine hydroxylase, while the chronic effects may reflect enzyme induction.     

Tyrosine availability and dopamine synthesis in the striatum: studies with gamma-butyrolactone

The administration of tyrosine (200 mg/kg) to adult male rats significantly enhanced the increase in striatal dopamine (DA) levels that followed gamma-butyrolactone (GBL) injection. Tyrosine injection also stimulated the rise in striatal dihydroxyphenylalanine (DOPA) accumulation after injection of m-hydroxybenzylhydrazine dihydrochloride (NSD-1015) that resulted from GBL administration. These results identify a new paradigm in which an increase in the brain levels of tyrosine enhances the rate of formation of dopamine. In addition, They support the notion that tyrosine hydroxylase must be “activated” in order for tyrosine availability to influence DA synthesis.     

Regulation of tyrosine hydroxylase activity and phosphorylation at Ser(19) and Ser(40) via activation of glutamate NMDA receptors in rat striatum

The activity of tyrosine hydroxylase, the rate-limiting enzyme in the biosynthesis of dopamine, is stimulated by phosphorylation. In this study, we examined the effects of activation of NMDA receptors on the state of phosphorylation and activity of tyrosine hydroxylase in rat striatal slices. NMDA produced a time-and concentration-dependent increase in the levels of phospho-Ser(19)-tyrosine hydroxylase in nigrostriatal nerve terminals. This increase was not associated with any changes in the basal activity of tyrosine hydroxylase, measured as DOPA accumulation. Forskolin, an activator of adenylyl cyclase, stimulated tyrosine hydroxylase phosphorylation at Ser(40) and caused a significant increase in DOPA accumulation. NMDA reduced forskolin-mediated increases in both Ser(40) phosphorylation and DOPA accumulation. In addition, NMDA reduced the increase in phospho-Ser(40)-tyrosine hydroxylase produced by okadaic acid, an inhibitor of protein phosphatase 1 and 2A, but not by a cyclic AMP analogue, 8-bromo-cyclic AMP. These results indicate that, in the striatum, glutamate decreases tyrosine hydroxylase phosphorylation at Ser(40) via activation of NMDA receptors by reducing cyclic AMP production. They also provide a mechanism for the demonstrated ability of NMDA to decrease tyrosine hydroxylase activity and dopamine synthesis.     

Biochemical Evidence for Alteration of Neostriatal Dopaminergic Function by 5,7-Dihydroxytryptamine

Unilateral injection of 5,7-dihydroxytryptamine (DHT) into the rat neostriatum markedly reduced not only striatal tryptophan hydroxylase (TPH) activity but also striatal tyrosine hydroxylase (TH) activity and dopamine (DA) concentration measured 10--15 days later. The decrease in striatal TH activity was dose related over the range of 8--32 micrograms of DHT; a dose of 16 micrograms reduced striatal TH activity to 40--50% of control, DA concentration to 38% of control, and TPH activity to 5--20% of control. Intrastriatal injection of 16 micrograms of DHT reduced TH activity in the ipsilateral substantia nigra to 51% of control. Pretreatment with amfonelic acid, a potent DA uptake inhibitor, significantly reduced the effect of DHT on striatal and nigral TH activity and striatal DA concentration without affecting the DHT-induced decrease in striatal TPH activity. Desmethylimipramine (5 and 25 mg/kg) had no effect on the DHT-induced decrease in striatal TH activity. Striatal choline acetyltransferase and glutamic acid decarboxylase activities were not decreased by 16 micrograms of DHT. The results indicate that DHT can alter dopaminergic function in the rat neostriatum through a direct effect of the drug on DA neurons.     

Acute administration of cocaine regulates the phosphorylation of serine-19, -31 and -40 in tyrosine hydroxylase

Acute cocaine can inhibit catecholamine biosynthesis by regulating the enzymatic activity of tyrosine hydroxylase via alterations in the phosphorylation state of the enzyme. The mechanisms underlying acute cocaine-dependent regulation of tyrosine hydroxylase phosphorylation have not been determined. In this study, 0, 15 or 30 mg/kg cocaine was administered intraperitoneally to rats and the phosphorylation state of tyrosine hydroxylase in the brain was examined using antibodies specific for the phosphorylated forms of serine-19, -31 and -40 in tyrosine hydroxylase. In the caudate and nucleus accumbens, cocaine dose-dependently decreased the levels of phosphorylated serine-19, -31 and -40. In the ventral tegmental area, the levels of phosphorylated serine-19, but not serine-31 and -40, were decreased by 15 and 30 mg/kg cocaine. In the amygdala, the levels of phosphorylated serine-19, but not serine-31 or -40, were decreased. The functional effects of these alterations in phosphorylation state were assessed by measuring tyrosine hydroxylase activity in vivo (accumulation of DOPA after administration of the decarboxylase inhibitor NSD-1015). Acute administration of 30 mg/kg cocaine significantly decreased l-DOPA production in caudate and accumbens but not in amygdala. These data suggest that the phosphorylation of serine-31 or -40, but not serine-19, is involved in the regulation of tyrosine hydroxylase activity by acute cocaine.     

Extracellular Concentrations of Dopamine and Metabolites in the Rat Caudate After Oral Administration of a Novel Catechol-O-Methyltransferase Inhibitor Ro 40–7592

The effect of the systemic administration of a novel, orally active, catechol-O-methyltransferase (COMT) inhibitor, Ro 40-7592, on the in vivo extracellular concentrations of dopamine (DA) and its metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), was studied by transcerebral microdialysis in the dorsal caudate of freely moving rats. Ro 40-7592 (at doses of 3.0, 7.5, and 30 mg/kg p.o.) elicited a marked and long-lasting reduction of HVA, and at doses of 7.5 and 30 mg/kg, an increase of DOPAC output, but it failed to increase DA output. The administration of L-beta-3,4-dihydroxyphenylalanine (L-DOPA, 20 and 50 mg/kg p.o.) with a DOPA decarboxylase inhibitor (benserazide) increased both HVA and DOPAC output, but failed to modify significantly extracellular DA concentrations in dialysates; in contrast, combined administration of L-DOPA+benserazide with Ro 40-7592 (30 mg/kg p.o.) resulted in a significant increase in DA output. Ro 40-7592 prevented the L-DOPA-induced increase in HVA output and markedly potentiated the increase in DOPAC output. To investigate to what extent the increase in extracellular DA concentrations was related to an exocitotic release, tetrodotoxin (TTX) sensitivity was tested. Addition of TTX to Ringer, although abolishing DA output in the absence of L-DOPA, partially reduced it in the presence of L-DOPA+Ro 40-7592 and even more so after L-DOPA without the COMT inhibitor. The results of the present study suggest that metabolism through COMT regulates extracellular concentrations of DA formed from exogenously administered L-DOPA but not of endogenous DA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of Aromatic L-Amino Acid Decarboxylase and Tyrosine Aminotransferase by the Monoamine Oxidase Inhibitor Phenelzine

The concentration of p-tyramine in the rat striatum was increased significantly by intraperitoneal injection of phenelzine (5 or 100 mg/kg). Unlike other monoamine oxidase (MAO) inhibitors, phenelzine had no effect on p-tyramine levels in the first 1-2 h following injection. The high dose of phenelzine increased the p-tyramine levels much more than the low dose. In addition, the high dose of phenelzine increased striatal p-tyrosine levels significantly 12 h after injection. Further studies showed that phenelzine inhibited the tyrosine aminotransferase activity of rat liver homogenates; the IC50 was 50 microM. Phenelzine also inhibited the aromatic L-amino acid decarboxylase activity of rat brain homogenate with an IC50 of 25 microM. Following intraperitoneal injection of 100 mg/kg phenelzine, the initial concentration of phenelzine in the striatum appears to be high enough to inhibit aromatic L-amino acid decarboxylase. It is suggested that the multiple enzyme inhibition caused by administration of high doses of phenelzine accounts for its unusual effects on striatal p-tyramine levels compared with other MAO inhibitors, i.e., its initial lack of effect on p-tyramine levels followed later by very large increases in p-tyramine levels.     

Role of Aromatic l-Amino Acid Decarboxylase for Dopamine Replacement by Genetically Modified Fibroblasts in a Rat Model of Parkinson's Disease

Abstract: Investigations of gene therapy for Parkinson's disease have focused primarily on strategies that replace tyrosine hydroxylase. In the present study, the role of aromatic l -amino acid decarboxylase in gene therapy with tyrosine hydroxylase was examined by adding the gene for aromatic l -amino acid decarboxylase to our paradigm using primary fibroblasts transduced with both tyrosine hydroxylase and GTP cyclohydrolase I. We compared catecholamine synthesis in vitro in cultures of cells with tyrosine hydroxylase and aromatic l -amino acid decarboxylase together versus cocultures of cells containing these enzymes separately. l -DOPA and dopamine levels were higher in the cocultures that separated the enzymes. To determine the role of aromatic l -amino acid decarboxylase in vivo, cells containing tyrosine hydroxylase and GTP cyclohydrolase I were grafted alone or in combination with cells containing aromatic l -amino acid decarboxylase into the 6-hydroxydopamine-denervated rat striatum. Grafts containing aromatic l -amino acid decarboxylase produced less l -DOPA and dopamine as monitored by microdialysis. These findings indicate that not only is there sufficient aromatic l -amino acid decarboxylase near striatal grafts producing l -DOPA, but also the close proximity of the enzyme to tyrosine hydroxylase is detrimental for optimal dopamine production. This is most likely due to feedback inhibition of tyrosine hydroxylase by dopamine.