Amino Acid Decarboxylase Inhibitor Research Articles

Increased neostriatal dopamine activity after intraperitoneal or intranasal administration of L-DOPA: on the role of benserazide pretreatment.

L-DOPA provides the most potent medication to treat Parkinson's disease, and such systemic treatment is usually combined with a peripheral amino acid decarboxylase inhibitor to amplify its central effectiveness. Since L-DOPA can lose its efficacy or can lead to adverse effects with prolonged application, current pharmacokinetic and dynamic research is aimed at improving the drug's applicability. In a previous study, performed with in vivo microdialysis in the anesthetized rat, we have shown that intranasal L-DOPA administration (without prior decarboxylase inhibition) can increase extracellular dopamine levels in the neostriatum. Using similar experimental conditions in the present experiment, we tested the neurochemical effects of L-DOPA treatment in combination with the peripheral amino acid decarboxylase inhibitor benserazide. In accordance with other data, it was found that the combination of i.p. benserazide and i.p. L-DOPA led to pronounced increases of extracellular levels of dopamine, dihydroxyplenylacetic acid and homovanillic acid in the neostriatum, whereas i.p. L-DOPA alone only moderately increased dopamine, but strongly increased the metabolite levels. Furthermore, increased dopamine levels, and weaker increases of dihydroxyplenylacetic acid and homovanillic acid were observed after i.p. benserazide followed by intranasal L-DOPA. Finally, we found that i.p. benserazide alone can lead to pronounced increases in neostriatal dopamine and moderate increases of dihydroxyplenylacetic acid levels, whereas it did not affect homovanillic acid. Thus, not only the combination of L-DOPA (i.p. or intranasal) with the presumed peripheral L-DOPA decarboxylase inhibitor benserazide, but also each component alone can affect dopamine activity in the brain. Especially the findings with benserazide treatment might be of relevance for understanding the mechanisms of current L-DOPA therapy, since they indicate that part of the treatment's actions may possibly be determined by central dopaminergic effects of the accompanying amino acid decarboxylase inhibitor.

Neurotrophic effects of L-DOPA in postnatal midbrain dopamine neuron/cortical astrocyte cocultures.

L-DOPA is toxic to catecholamine neurons in culture, but the toxicity is reduced by exposure to astrocytes. We tested the effect of L-DOPA on dopamine neurons using postnatal ventral midbrain neuron/cortical astrocyte cocultures in serum-free, glia-conditioned medium. L-DOPA (50 microM) protected against dopamine neuronal cell death and increased the number and branching of dopamine processes. In contrast to embryonically derived glia-free cultures, where L-DOPA is toxic, postnatal midbrain cultures did not show toxicity at 200 microM L-DOPA. The stereoisomer D-DOPA (50-400 microM) was not neurotrophic. The aromatic amino acid decarboxylase inhibitor carbidopa (25 microM) did not block the neurotrophic effect. These data suggest that the neurotrophic effect of L-DOPA is stereospecific but independent of the production of dopamine. However, L-DOPA increased the level of glutathione. Inhibition of glutathione peroxidase by L-buthionine sulfoximine (3 microM for 24 h) blocked the neurotrophic action of L-DOPA. N-Acetyl-L-cysteine (250 microM for 48 h), which promotes glutathione synthesis, had a neurotrophic effect similar to that of L-DOPA. These data suggest that the neurotrophic effect of L-DOPA may be mediated, at least in part, by elevation of glutathione content.

Reduced striatal tyrosine hydroxylase activity is not accompanied by change in responsiveness of dopaminergic receptors following chronic treatment with deprenyl

Deprenyl is the only selective monoamine oxidase B (MAO-B) inhibitor that is in clinical use for the treatment of Parkinson's disease. Our previous studies showed that chronic treatment of rats with low (MAO-B selective) doses of deprenyl inhibited dopamine (DA) re-uptake and enhanced DA release in the striatum. These changes could affect DA synthesis rate by activation of negative feedback loops. Chronic deprenyl treatment has also been suggested to cause down-regulation of release-modulating DA receptors. The effects of chronic and acute treatment with deprenyl on ex vivo striatal tyrosine hydroxylase activity were therefore studied, by determination of steady-state tissue level of DOPA following administration of NSD-1015 (100 mg/kg i.p.). In addition, we assessed changes in the in vivo sensitivity of dopaminergic receptors from the reduction in DOPA extracellular level after systemic apomorphine administration (2.5 mg/kg s.c.), following elevation of microdialysate DOPA by systemic or local aromatic amino acid decarboxylase inhibition with NSD-1015. Chronic treatment with deprenyl (0.25 mg/kg s.c. daily for 21 days) caused a significant reduction in tyrosine hydroxylase activity to 60% of control, with no change in the apomorphine-induced reduction of microdialysate DOPA and DOP AC. The reduction in tyrosine hydroxylase activity is compatible with our previous results showing an increase in striatal DA extracellular level following chronic treatment with deprenyl. The increased extracellular striatal DA level could reduce tyrosine hydroxylase activity through activation of a negative feedback loop, by activation of either presynaptic or postsynaptic DA receptors.

Influence of an Aromatic Amino Acid Decarboxylase Inhibitor on GTP Cyclohydrolase I Activity in the Rat Brain

Summary Based on the hypothesis that catecholamine decreases may cause increases in biopterin, we measured the GTP cyclohydrolase I(GTPCH-I) activity and biopterin content in the cerebral cortex, midbrain/diencephalon, cerebeilum, pons/medulla oblongata, striatum , and hippocampus of brains from rats given NSDlOIS, an inhibitor for aromatic amino acid decarboxylase . The catecholamine contents decreased in all the regions tested. The changes in the GTPCH -I activity and bioptcrin content were different among the regions; they decreased in some regions contrary to expectations and increased in other regions. However, either the enzyme activity or biopterin content increased in the midbrain/ diencephalon and pons/ medulla oblongata, in which similar levels of catecholamines, GTPCH -I activity and biopterin were detected. These results suggest that for the midbrain/diencephalon and pons/medulla oblongata the surmise that a decrease in catecholamines causes an increase in the biopterin synthesis may be correct.

Cerebral 6-[ 18F]fluoro- l-DOPA uptake in rhesus monkey: pharmacological influence of aromatic amino acid decarboxylase (AAAD) and catechol- O-methyltransferase (COMT) inhibition

FDOPA/PET scans were performed in one rhesus monkey to study the influence of three catechol- O-methyltransferase (COMT) inhibitors (CGP 28014, OR-611 and Ro 40-7592) on FDOPA pharmacokinetics. COMT inhibitors were administered in combination with carbidopa, a peripherally acting inhibitor of the aromatic amino acid decarboxylase (AAAD). FDOPA was administered intravenously and its metabolic fate in plasma was determined using an HPLC system with an on-line γ- γ coincidence detector. Cerebral tracer uptake was assessed in the striatum and in a non-dopaminergic brain region (occipital cortex). In the periphery, the pharmacokinetic efficiency of FDOPA was increased due to the combined inhibition of COMT and AAAD activity. All three COMT inhibitors reduced the FDOPA methylation rate constant in plasma, with complete suppression obtained in the case of Ro 40-7592. In the brain, specific 18F radioactivity (striatal minus brain reference radioactivity) increased as a result of the increase in FDOPA plasma availability following the administration of COMT and AAAD inhibitors. We established a significant linear correlation between striatal radioactivity and FDOPA plasma levels ( r=0.924±0.048, P<0.0001 for total striatal and r=0.948±0.054, P<0.0001 for specific striatal radioactivity). Using plasma FDOPA radioactivity as input, we found that the striatal FDOPA uptake rate constant K i FD was not changed by any of the inhibitors. Thus, the enhancement of striatal radioactivity after application of enzyme inhibitors is a consequence of the increase in plasma FDOPA that becomes available for conversion to fluorodopamine in the striatal dopaminergic nerve terminals. By contrast, using the radioactivity in a non-dopaminergic region (cortex) as input, we found that the striatal FDOPA uptake rate constant K i ref was significantly ( P<0.0001) increased following pretreatment with COMT inhibitors. Our analysis demonstrated that K i ref and the 3-OMFD contribution to the cerebral radioactivity were inversely correlated.

Ex vivo inhibitory effect of the 5-HT uptake blocker citalopram on 5-HT synthesis.

Serotonin (5-hydroxytryptamine, 5-HT) synthesis was determined in vivo by measuring the accumulation of 5-hydroxytryptophan (5-HTP) in rat frontal cortex after inhibition of aromatic amino acid decarboxylase by administrative of m-hydroxybenzylhydrazine (NSD 1015) (100 mg/kg, i.p.). The selective 5-HT reuptake inhibitor, citalopram, the 5-HT1A agonists, (+/-) 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT), ipsapirone, gepirone and the 5-HT1A/B agonist, 7-trifluoromethyl-4(4-methyl-1-piperazinyl-pyrolo[1,2-a]-quinox ali ne (CGS 12066B), the 5-HT1A/B ligands and beta-adrenoceptor antagonists, (+/-) pindolol and (+/-) alprenolol, and the non-selective 5-HT ligands, m-chlorophenylpiperazine (mCPP) and metergoline, all inhibited the synthesis of 5-HT. The 5-HT1A/5-HT2 antagonist, spiperone, alone, had no effect on basal 5-HT synthesis, however it attenuated the effect of 8-OH-DPAT by 56% and CGS 12066B by 39% but only barely that of citalopram by 17%. The selective 5-HT1A antagonist, WAY 100635, which did not modify by itself 5-HT synthesis, had no effect on citalopram-induced reduction of 5-HT synthesis. Neither the 5-HT2 agonist, (+/-)1-(2,5-dimethoxy-4-indophenyl)-2-aminopropane (DOI) nor the 5-HT2 antagonist, ritanserin, had any effect on the synthesis of 5-HT. In addition, ritanserin did not modify the inhibitory effect of citalopram. Methiothepin was the only compound to increase 5-HT synthesis. These results suggest that the effect of citalopram on the synthesis of 5-HT is not mediated by 5-HT1A or 5-HT2 receptors and that other receptors may be involved.

Histamine in the chick pineal gland: origin, metabolism, and effects on the pineal function.

The chick pineal gland contains histamine and tele-methylhistamine. The levels of both substances are elevated after treatment of chicks with the amino acid precursor of histamine, L-histidine (1 g/kg, ip). In control and L-histidine-loaded animals the pineal levels of histamine and tele-methylhistamine are higher in light-exposed than in dark-adapted animals (measured at the end of the light phase and in the middle of the dark phase of 12 hr light, 12 hr dark illumination cycle, respectively). The chick pineal gland contains histamine-immunofluorescent cells displaying mast cell morphology; they are seen in the vicinity of the capsule and in the parenchyma. Enzymatic studies showed the presence of the activity of histamine synthesizing and inactivating enzyme, i.e., L-histidine decarboxylase (HDC) and histamine-methyltransferase (HMT). The detected enzyme activities were sensitive to specific inhibitors of HDC (alpha-fluoromethylhistidine and alpha-hydrazinohistidine) and HMT (quinacrine and metoprine); inhibitors of aromatic amino acid decarboxylase alpha-methyl-DOPA and NSD-1015 were inactive on HDC. Exogenous histamine added to organ-cultured chick pineals strongly stimulated endogenous cyclic AMP accumulation and moderately increased melatonin secretion. The data, considered collectively, suggest that in avians histamine, probably originating from the pineal mast cell compartment, may function as a regulator of pineal gland activity.

Catechol O-Methyltransferase Inhibition and the Treatment of Parkinson's Disease

Publisher Summary Catechol O-methyltransferase (COMT) plays an important role in the metabolism of the dopamine precursor, l -dopa, and of dopamine. Inhibitors of COMT could alter the metabolism of L-dopa and dopamine to therapeutic advantage by a number of mechanisms in L-dopa-treated patients with Parkinson's disease (PD). The introduction of entacapone and tolcapone, reversible inhibitors of COMT that are well absorbed and well tolerated, has permitted investigation of the clinical importance of COMT in Parkinson's disease. Entacapone is primarily a peripheral inhibitor of COMT; tolcapone penetrates the blood brain barrier and inhibits brain COMT as well as peripheral COMT. This chapter discusses l -dopa absorption. COMT is in high concentration in the gut and liver and may contribute to first-pass metabolism of levodopa. Inhibition of gut and liver COMT would then be expected to increase the bioavailability of orally administered l -dopa. Pharmacokinetically, this should be manifest by an increase in peak concentration of plasma l -dopa and a decrease in the time-to-peak concentration. Neither entacapone nor tolcapone increases the peak concentration of levodopa or shortens the time-to-peak concentration in subjects with PD. These results are in contrast to the effect of inhibition of first-pass decarboxylation of l -dopa; the aromatic amino acid decarboxylase inhibitors, carbidopa and benserazide, markedly increase the peak plasma L-dopa concentrations. These observations argue that first-pass O-methylation of l -dopa is not quantitatively important. To the extent that dyskinesia and, perhaps, some mental effects are related to peak concentrations of l -dopa, this lack of effect on peak concentrations by COMT inhibitors may be beneficial. The chapter also discusses L-dopa elimination, Antiparkinsonian effects of COMT inhibition, l -dopa transport into brain and inhibition of brain COMT. Potential benefit of inhibition of brain COMT is sparing of S- adenosylmethionine, the methyl donor for O-methylation reactions. Low cerebrospinal fluid S-adenosylmethionine concentrations have been linked to depression and dementia.

Neurotensin inhibits the activation of midbrain serotonergic neurons produced by random inescapable sound

Previous studies indicate that exposure of rats to randomly presented, inescapable loud sound, referred to as sound stress, increases central serotonin turnover as well as the ex vivo activity of tryptophan hydroxylase (EC 1.14.16.4), the rate-limiting enzyme in serotonin biosynthesis. The purpose of this investigation was to determine whether intracerebroventricular (i.c.v.) administration of neurotensin (NT), a tridecapeptide found within the midbrain raphe, influences the activation of the midbrain serotonergic neurons by sound stress. Accumulation of 5-hydroxytryptophan (5-HTP) in vivo, in the presence of the aromatic amino acid decarboxylase inhibitor, NSD 1015 (m-hydroxybenzylhydrazine, 100 mg/kg i.p.) given immediately before a 30 min sound stress, was used as an index of in vivo tryptophan hydroxylase activity. Sound-stressed rats had significantly higher levels of 5-HTP in cortex and midbrain compared to sham-stressed controls. NT (0.01–3.3 nmol total), given i.c.v., 5 min prior to 30 min sound stress, completely blocked the enhanced accumulation of 5-HTP, but had no effect on basal accumulation of 5-HTP, except at the highest doses of 1.0 or 3.3 nmol, which others have previously shown to inhibit basal serotonergic metabolism. NT (0.3 and 3.3 nmol) blocked the increase in cortical tryptophan hydroxylase activity, ex vivo, in response to 30 min sound stress, without affecting basal enzyme activity. These and other recent data suggest a possible role for endogenous NT in the regulation of serotonergic neuronal activity within the midbrain raphe.

An aromatic L-amino acid decarboxylase inhibitor blocks L-dopa-induced air-stepping in neonatal rats.

Rat pups suspended in air and administered L-DOPA engage in a locomotor behavior termed air-stepping. The role of L-DOPA itself was investigated by administering several doses of an aromatic L-amino acid decarboxylase inhibitor, NSD 1015, prior to 100 mg/kg L-DOPA to 5-day-old rats. NSD 1015 dose-dependently increased the latency to onset and decreased the duration of L-DOPA-induced air-stepping. Thus L-DOPA induces air-stepping only after its conversion to dopamine and/or noradrenaline.

Depressor action of l- threo-dihydroxyphenylserine in the rat nucleus tractus solitarii

Microinjections of l- threo-dihydroxyphenylserine ( l- threo-DOPS, 0.1–3 ng), a synthetic precursor amino acid of noradrenaline, into the medial area of the nucleus tractus solitarii produced dose-dependent depressor and bradycardic responses in anesthetized rats treated with or without i.p. 3-hydroxybenzylhydrazine, a central inhibitor of l-aromatic amino acid decarboxylase. d- threo-DOPS (3 ng) produced no effect. l-Dihydroxyphenylalanine ( l-DOPA) methyl ester (1 μg), a competitive antagonist of l-DOPA, microinjected into the nucleus tractus solitarii, blocked the depressor and bradycardic responses to l- threo-DOPS. l- threo-DOPS itself produces vasodepressor actions without its conversion to noradrenaline, probably via a recognition site for l-DOPA in the rat nucleus tractus solitarii.

Synthesis and evaluation of an 18F-labeled dopa prodrug as a PET tracer for studying brain dopamine metabolism

In the quantitative studies of presynaptic dopamine metabolism by PET with 6-[ 18F]fluoro- l-dopa (6-[ 18F]FDOPA), metabolic analysis in the plasma is required to determine the precise input function because of susceptibility of the compound to peripheral metabolism. In this study, we prepared 6-[ 18F]-fluoro-O-pivaloyl- l-dopa (6-[ 18F]FPDOPA) as a prodrug of 6-[ 18F]FDOPA, and evaluated its potential as a PET tracer in mice. If the 6-[ 18F]FPDOPA is stable peripherally and is hydrolyzed to 6-[ 18F]FDOPA in the brain tissues, disadvantage of the 6-[ 18F]FDOPA will be overcome. Compared with the 6-[ 18F]FDOPA, the initial brain uptake of the 6-[ 18F]FPDOPA was lower; however, the uptake in the latter become comparable, and the uptake ratios of striatum to other reference regions were larger. Medication of mice with inhibitors of aromatic amino acid decarboxylase and catechol-O-methyl transferase greatly enhanced the striatal uptake of the two compounds. The reduced brain uptake of the compounds by l-phenylalanine-loading suggested transport through the blood-brain barrier by the neutral amino acid transporter. HPLC analysis showed the presence of 6-[ 18F]FPDOPA, 6-[ 18F]FDOPA and 6-[ 18F]fluorodopamine in the striatum; however, 6-[ 18F]fluoro-3-O-methyl- l-dopa was a predominate metabolite in the brain and plasma as in the case of [ 18F]FDOPA. Results suggested that 6-[ 18F]FPDOPA had characteristics as a prodrug of 6-[ 18F]FDOPA; however, the compound was also labile to metabolic alteration in vivo.

Enzymic sulphation of dopa and tyrosine isomers by HepG2 human hepatoma cells: stereoselectivity and stimulation by Mn2+.

HepG2 human hepatoma cells, labelled with [35S]sulphate in media containing L-3,4-dihydroxyphenylalanine (L-dopa), (D-dopa), DL-m-tyrosine or D-p-tyrosine, were found to produce the [35S]sulphated forms of these compounds. Addition to the labelling media of m-hydroxybenzylhydrazine, an aromatic amino acid decarboxylase inhibitor, greatly enhanced the production of L-dopa O-[35S]sulphate and DL-m-tyrosine O-[35S]sulphate, with a concomitant decrease in the formation of dopamine O-[35S]sulphate and m-tyramine O-[35S]sulphate. With 3'-phosphoadenosine 5'-phospho[35S]sulphate as the sulphate donor., HepG2-cell cytosol was shown to contain enzymic activity catalysing the sulphation of L-dopa, D-dopa, L-m-tyrosine, D-m-tyrosine, L-p-tyrosine and D-p-tyrosine. The pH optimum of the enzyme, designated dopa/tyrosine sulphotransferase, was determined to be 8.75 with D-m-tyrosine as the substrate. The enzyme exhibited stereoselectivity for the D-form of dopa or tyrosine isomers. Addition of 10mM MnCl2 to the reaction mixture resulted in a remarkable stimulation of dopa/tyrosine sulphotransferase activity, being as high as 267.8 times with D-p-tyrosine as the substrate. Quantitative assays revealed L-dopa, D-dopa and D-m-tyrosine to be better substrates than L-p-tyrosine. When the HepG2-cell cytosol was subjected to DEAE Bio-Gel and hydroxyapatite column chromatography, dopa/tyrosine sulphotransferase was co-eluted with the thermolabile 'M-form' phenol sulphotransferase. Furthermore dopa/tyrosine sulphotransferase displayed properties similar to that of the M-form phenol sulphotransferase with respect to thermostability and sensitivity to 2,6-dichloro-4-nitrophenol. Whether the M-form phenol sulphotransferase is truly (solely) responsible for the dopa/tyrosine sulphotransferase activity present in HepG2 cells remains to be clarified.

Increased neostriatal tyrosine hydroxylation during stress: role of extracellular dopamine and excitatory amino acids.

We examined the regulation of neostriatal tyrosine hydroxylation during acute stress, testing the hypothesis that excitatory amino acids (EAAs) contribute to the stress-evoked increase in dopamine (DA) synthesis. Dialysis probes implanted into neostriatum permitted delivery of drugs and sampling of extracellular fluid. Rats were exposed to 30 min of intermittent tail shock during infusion of an inhibitor of aromatic amino acid decarboxylase (AAAD), NSD-1015 (100 microM), and DOPA was measured in the dialysate. Tail shock was applied beginning either 15 min after the onset of NSD-1015 treatment (the initial rate of DOPA accumulation) or 75 min after the onset of treatment (when DOPA had approached steady state). Tail shock increased the steady-state levels of extracellular DOPA in neostriatum (+40%). However, there was no change in the initial rate of DOPA accumulation unless animals also received the D2 receptor antagonist eticlopride (50 nM), in which case an increase was observed (+228%). The impact of tail shock on the steady-state level of DOPA was attenuated by the D2 agonist quinpirole (100 microM), or by 2-amino-5-phosphonovalerate (APV) (100 microM) or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (100 microM), EAA antagonists acting at NMDA or D,L-alpha-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionate (AMPA) receptors, respectively. These data suggest that acute stress normally has little effect on tyrosine hydroxylation in neostriatum due to the inhibitory influence of DA in the extracellular fluid. However, when that influence is absent (e.g., during extended inhibition of DOPA decarboxylation or blockade of DA receptors), stress increases tyrosine hydroxylation via EAAs acting on NMDA and AMPA receptors. Thus, EAAs released from corticostriatal projections may stimulate DA synthesis and thereby restore dopaminergic activity under conditions in which the availability of DA for release has been compromised.

Sanguinarine and chelerythrine as inhibitors of aromatic amino acid decarboxylase.

Quaternary benzo[c]phenanthridine alkaloids sanguinarine, chelerythrine and their dihydroderivatives were tested as inhibitors of aromatic amino acid decarboxylase (EC 4.1, 1.28, AAD) from rat liver. Sanguinarine and chelerythrine exhibited strong inhibition of AAD with Ki 1.2 x 10(-4) M and 5.8 x 10(-4) M, respectively, while no inhibitory effect was observed for their dihydroderivatives. The inhibition was found to be irreversible. The enzyme-inhibitor interaction apparently stabilized AAD against thermal inactivation. Pyridoxal-5'-phosphate partially decreased but did not reverse the inhibition. Dithiothreitol prevented the inhibitory effect of sanguinarine and chelerythrine which indicates that the interaction with thiol groups essential for AAD activity is included in the inhibition mechanism.

Psychotic complications of long term levodopa treatment of Parkinson's disease

Psychotic symptoms such as hallucinations, delusions and confusion are well known side-effects of levodopa (LD) therapy of Parkinson's disease (PD), even if there is much confusion regarding the variability of the psychotic manifestations. We studied 18 patients clinically treated with LD associated with an inhibitor of peripheral aminoacid decarboxylase (DI). Daily dosage of LD ranged between 250 and 1750 mg; the mean age of patients was 72.7 years; the age at the onset of the disease was 63.3 and duration of the disease 8.4 years. In all patients any possible etiology of Parkinsonism were excluded. Brain CT scan excluded focal lesions in all cases. Psychotic complications were seen in 8 patients: using DSM-III-R-criteria, the various LD-induced psychotic states can be classified in two groups: simple (including or hallucinations with preserved insight) and complex ones (including chronic confusion without preserved insight). Patients with complex symptoms were younger at the onset of the disease and they developed these symptoms later, and these patients were also more susceptible to dyskinesias developed before psychotic complications.

In vivo microdialysis to determine the relative pharmacokinetics of drugs.

The purpose of this study was to evaluate a simultaneous microdialysis method in blood and brain striatum to determine the relative pharmacokinetics and metabolism of L-3,4-dihydroxypenylalanine (L-dopa). L-Dopa (250 mumol/kg) was administered to rats with or without the aromatic amino acid decarboxylase (AADC) inhibitor carbidopa (25 mumol/kg) or benserazide (25 or 62.5 mumol/kg). L-Dopa, its metabolites, and AADC inhibitors in dialysates were analyzed by high performance liquid chromatography with an electrochemical detector. A moment analysis was also made to obtain pharmacokinetic parameters. After administration of L-dopa alone, it and its related metabolites were detected in both dialysates of blood and brain striatum. Coadministration of carbidopa (25 mumol/kg) or benserazide (62.5 mumol/kg) significantly enhanced the striatal amount of L-dopa by 8.0 and 6.1 times, respectively. Carbidopa and benserazide also increased striatal amounts of L-dopa metabolites, such as 3,4-dihydroxyphenylacetic acid, homovanillic acid, and 3-methoxy-4-hydroxyphenylethyleneglycol. Inhibition effect of benserazide on an extracerebral decarboxylation of L-dopa to dopamine (DA) was stronger than that of carbidopa. Carbidopa showed a higher striatal level of DA than benserazide. These results suggest a different effect of the two inhibitors on the DA formations in blood and brain striatum, and on the L-dopa transport through the blood-brain barrier (BBB). Thus, microdialysis is an easy and available method for simultaneously assessing the in vivo relative pharmacokinetics and metabolism of drugs in systemic circulation and a target organ.

Hypoprolactinemia decreases tyrosine hydroxylase activity in the tuberoinfundibular dopaminergic neurons acutely by protein dephosphorylation and chronically by changes in gene expression

This study evaluated the roles of protein dephosphorylation or suppressed gene expression in reducing tyrosine hydroxylase activity in tuberoinfundibular dopaminergic neurons after acute or chronic bromocriptine (BROMO) administration. Diestrous or ovariectomized rats were injected with BROMO (3 mg/kg, s.c.) at 1000 h and were sacrificed 4 h later or were injected with BROMO at 12 h intervals for 3 days.In vitro tyrosine hydroxylase activity was assessed by incubating hypothalamic explants with brocresine, an L-aromatic amino acid decarboxylase inhibitor, and measuring 3,4-dihydroxyphenylalanine (DOPA) accumulation in the stalk-median eminence (SME). The incubation medium also contained either 2 μM okadaic acid, a protein phosphatase 1 and 2A inhibitor, or its vehicle (0.25% dimethylsulfoxide). Acute (4 h) and chronic (3 days) BROMO treatment suppressed circulating PRL levels from 10-12 ng/ml to<1 ng/ml and reduced tyrosine hydroxylase activity in the SME by 60% or 40% in diestrous or ovariectomized rats, respectively. Okadaic acid increased tyrosine hydroxylase activity in the SME 2-fold in control diestrous or ovariectomized rats. The reduced tyrosine hydroxylase activity in the SME after acute BROMO treatment was increased by okadaic acid 5-or 3-fold in diestrous or ovariectomized rats respectively, to a value similar to the controls. In sharp contrast, after chronic BROMO treatment, the response to okadaic acid was blunted. As assessed byin situ hybridization, tyrosine hydroxylase mRNA signal levels in the arcuate nucleus of diestrous rats were not altered after acute BROMO treatment, but were reduced by 70% after chronic BROMO treatment. The acute BROMO-induced decrease in tyrosine hydroxylase activity was reversed by co-administration of oPRL or rPRL, indicating that the action of BROMO is via a reduction in PRL. The data suggest that protein dephosphorylation may be a primary mechanism for the rapid BROMO-dependent suppression of tyrosine hydroxylase activity, whereas suppression of tyrosine hydroxylase gene expression may contribute to the lower tyrosine hydroxylase activity after chronic BROMO treatment.

Differential activation of the 5-hydroxytryptamine-containing neurons of the midbrain raphe of the rat in response to randomly presented inescapable sound

Estimates of 5-hydroxytryptamine (5-HT) turnover in response to 30 min of inescapable, randomly presented, loud sound (sound stress) were obtained for regions of rat brain containing 5-HT perikarya by means of 5-hydroxytryptophan (5-HTP) accumulation after administration of an inhibitor of aromatic amino acid decarboxylase (100 mg/kg i.p., m-hydroxybenzylhydrazine, NSD 1015). Sound stress increased 5-HTP accumulation in the median raphe nucleus (MRN) twofold over that from sham-stressed controls, but did not change 5-HTP accumulation significantly in dorsal raphe nucleus (DRN) or hindbrain. These findings indicate that the 5-HT perikarya of the MRN but not those of the DRN or hindbrain are activated by sound stress, and thus, provide further evidence for a functional distinction between the 5-HT neurons of these two midbrain nuclei.

The yohimbine-induced anticonflict effect in the rat, Part II. Neurochemical findings.

In a companion paper the alpha 2-adrenoceptor antagonist yohimbine was found to produce a dose-dependent anticonflict effect in a modified Vogel's conflict test. The behavioral data further indicated that noradrenergic and serotonergic neurons as well as the benzodiazepine (BDZ) receptor may be involved in the anticonflict effect of yohimbine. In the present study the effects on rat brain monoamine neurochemistry and GABAA/BDZ receptor function (36Cl-uptake in corticohippocampal synaptoneurosomes) of a maximally anticonflict producing dose of yohimbine (4.0 mg/kg, i.p.) were studied. The levels of rat brain catecholamines and indoleamines were measured ex vivo using high performance liquid chromatography with electrochemical detection (HPLC-ED). Yohimbine decreased noradrenaline levels both in the hippocampus and the hemispheres but instead increased DOPAC levels in these brain regions as well as in the limbic forebrain. Yohimbine also markedly enhanced DOPA accumulation in the hippocampus and the hemispheres after inhibition of 1-aromatic amino acid decarboxylase by means of NSD 1015, whereas in the limbic system only a modest increase was obtained. The yohimbine-induced effects on the catecholamine synthesis rate were largely abolished in animals severely depleted of NA by means of 6-hydroxy-dopamine (6-OH-DA) pretreatment. Yohimbine decreased both the 5-HIAA/5-HT quotient (an indicator of 5-HT turnover) and 5-HTP accumulation after NSD 1015 in the hemispheres, whereas in the hippocampus and the limbic system only 5-HTP accumulation was decreased. The yohimbine-induced effect on the indoleamine synthesis rate was not influenced by 6-OH-DA pretreatment, whereas this effect and that on the catecholamine synthesis rate were both abolished by reserpine pretreatment. Neither in vivo nor in vitro administration of yohimbine significantly altered baseline or GABA-induced accumulation of 36Cl- in corticohippocampal synaptoneurosomes. In conclusion, the present study provides neurochemical support for the suggestion that yohimbine may exert its anticonflict effect in a modified Vogel's conflict test by increasing and decreasing NA and 5-HT neurotransmission, respectively, whereas no evidence was obtained for a direct interaction of yohimbine with GABAA/BDZ receptor function.