Neurotrophic Effects of l-DOPA in Postnatal Midbrain Dopamine Neuron/Cortical Astrocyte Cocultures

Abstract: 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 µ M ) 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 µ M l -DOPA. The stereoisomer d -DOPA (50–400 µ M ) was not neurotrophic. The aromatic amino acid decarboxylase inhibitor carbidopa (25 µ M ) 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 µ M for 24 h) blocked the neurotrophic action of L-DOPA. N -Acetyl- l -cysteine (250 µ M 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.     

Lazaroid Treatment Prevents Death of Cultured Rat Embryonic Mesencephalic Neurons Following Glutathione Depletion

Abstract: Reactive oxygen species are believed to play a crucial role in situations where dopamine neurons die, such as in Parkinson's disease or during intracerebral transplantation of embryonic mesencephalic tissue. The present study was designed to address the question whether, and to what extent, the glutathione redox system is important for the viability of rat embryonic dopamine neurons in vitro. Furthermore, we studied whether the lazaroid U-83836E, a 2-methylaminochroman that inhibits lipid peroxidation, affects the survival of cultured mesencephalic neurons subjected to experimentally induced glutathione depletion. Glutathione depletion was achieved by exposing dissociated mesencephalic cell cultures to l -buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis, at four different concentrations (1, 10, 100, and 1,000 µ M ). Dopamine neuron survival was significantly reduced by 65–94% in a concentration-dependent manner by 10–1,000 µ M BSO. The neurotoxic effects of BSO were almost completely prevented by supplementing the culture medium with 0.3 µ M U-83836E. As assessed by HPLC analysis, BSO treatment was associated with a marked reduction of cellular glutathione content, and this depletion was not altered by the presence of U-83836E. We conclude that in the present insult model of severe glutathione depletion, the lazaroid can afford efficient neuroprotection that does not seem to be mediated by a direct interaction with BSO or glutathione, but rather via an independent pathway.     

Energy Stress-Induced Dopamine Loss in Glutathione Peroxidase-Overexpressing Transgenic Mice and in Glutathione-Depleted Mesencephalic Cultures

Abstract: The role of the glutathione system in protecting dopamine neurons from a mild impairment of energy metabolism imposed by the competitive succinate dehydrogenase inhibitor, malonate, was investigated in vitro and in vivo. Treatment of mesencephalic cultures with 10 µ M buthionine sulfoxamine for 24 h reduced total glutathione levels in the cultures by 68%. Reduction of cellular glutathione per se was not toxic to the dopamine population, but potentiated toxicity when the cultures were exposed to malonate. In contrast, transgenic mice overexpressing glutathione peroxidase (hGPE) that received an intrastriatal infusion of malonate (3 µmol) into the left side had significantly less loss of striatal dopamine than their hGPE-negative littermates when assayed 1 week following infusion. These studies demonstrate that manipulation of the glutathione system influences susceptibility of dopamine neurons to damage due to energy impairment. The findings may provide insight into the loss of dopamine neurons in Parkinson's disease in which defects in both energy metabolism and the glutathione system have been identified.     

In Vivo Microdialysis Evidence for Transient Dopamine Release by Benzazepines in Rat Striatum

Abstract: The effects of benzazepine derivatives on extracellular levels of dopamine (DA) and l -3,4-dihydroxyphenylacetic acid (DOPAC) in the dorsal striatum of freely moving rats were studied using in vivo microdialysis. Direct injection of SKF-38393 (0.5 or 1.5 µg/0.5 µl), a selective D₁ receptor agonist, into the striatum through a cannula secured alongside a microdialysis probe produced a rapid dose-dependent transient increase in striatal DA efflux and a more gradual reduction in efflux of DOPAC. The rapid increase in DA efflux was not affected by infusion of tetrodotoxin (TTX; 2 µ M ) or Ca²⁺-free Ringer's solution and occurred after either enantiomer of SKF-38393. A TTX-insensitive increase in DA level similar to that induced by SKF-38393 was also seen after other benzazepines acting as agonists (SKF-75670 and SKF-82958, each 1.5 µg in 0.5 µl) and antagonists (SCH-23390, 1.5 µg in 0.5 µl) at the D₁ receptor and after (+)-amphetamine. These effects were inhibited by infusion of nomifensine (100 µ M ). It is concluded that the transient increases in striatal DA efflux seen after intrastriatal injection of SKF-38393 and other benzazepines are not mediated by presynaptic D₁ receptors but by an amphetamine-like action on the dopamine transporter.     

Novel imine antioxidants at low nanomolar concentrations protect dopaminergic cells from oxidative neurotoxicity

Strong evidence indicates that oxidative stress may be causally involved in the pathogenesis of Parkinson's disease. We have employed human dopaminergic neuroblastoma cells and rat primary mesencephalic neurons to assess the protective potential of three novel bisarylimine antioxidants on dopaminergic cell death induced by complex I inhibition or glutathione depletion. We have found that exceptionally low concentrations (EC₅₀ values ∼20 nM) of these compounds (iminostilbene, phenothiazine, and phenoxazine) exhibited strong protective effects against the toxicities of MPP⁺, rotenone, and l -buthionine sulfoximine. Investigating intracellular glutathione levels, it was found that MPP⁺, l -buthionine sulfoximine, and rotenone disrupted different aspects of the native glutathione equilibrium, while the aromatic imines did not further influence glutathione levels or redox state on any baseline. However, the imines independently reduced protein oxidation and total oxidant flux, saved the mitochondrial membrane potential, and provided full cytoprotection under conditions of complete glutathione depletion. The unusually potent antioxidant effects of the bisarylimines could be reproduced in isolated mitochondria, which were instantly protected from lipid peroxidation and pathological swelling. Aromatic imines may be interesting lead structures for a potential antioxidant therapy of Parkinson's disease and other disorders accompanied by glutathione dysregulation.     

The Effect of Phosphinothricin (Glufosinate) on Glutathione Synthesis in Plants

The inhibitory effect of DL-phosphinothricin (glufosinate) on glutathione synthesis was studied in vivo and in vitro. The influence of phosphinothricin on γ-glutamylcysteine synthetase was compared with the already known effects of l -buthionine sulfoximine and l -methionine sulfoximine. The results showed that phosphinothricin and buthionine sulfoximine are inhibitors of γ-glutamylcysteine synthetase of plants. With both substances the enzyme was inhibited by 50 % at a concentration of 7 . 10^?4M (pI₅₀ = 3.15). Methionine sulfoximine reduced the enzyme activity by 50% at 5 . 10^?2 M (pI₅₀ = 1.30). It is discussed that the target enzyme of phosphinothricin is the glutamine synthetase whereas the γ-glutamylcysteine synthetase is only an accessory target.     

Influence of Cannabinoids on Electrically Evoked Dopamine Release and Cyclic AMP Generation in the Rat Striatum

Abstract: Using the endogenous cannabinoid receptor agonist anandamide, the synthetic agonist CP 55940 {[1α,2β( R )5α]-(−)-5-(1,1-dimethylheptyl)-2-[5-hydroxy-2-(3-hydroxypropyl)cyclohexyl]phenol}, and the specific antagonist SR 141716 [ N -(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1 H -pyrazole-3-carboxamide hydrochloride], second messenger activation of the central cannabinoid receptor (CB1) was examined in rat striatal and cortical slices. The effects of these cannabinoid ligands on electrically evoked dopamine (DA) release from [³H]dopamine-prelabelled striatal slices were also investigated. CP 55940 (1 µ M ) and anandamide (10 µ M ) caused significant reductions in forskolin-stimulated cyclic AMP accumulation in rat striatal slices, which were reversed in the presence of SR 141716 (1 µ M ). CP 55940 (1 µ M ) had no effect on either KCl- or neurotransmitter-stimulated ³H-inositol phosphate accumulation in rat cortical slices. CP 55940 and anandamide caused significant reductions in the release of dopamine after electrical stimulation of [³H]dopamine-prelabelled striatal slices, which were antagonised by SR 141716. SR 141716 alone had no effect on electrically evoked dopamine release from rat striatal slices. These data indicate that the CB1 receptors in rat striatum are negatively linked to adenylyl cyclase and dopamine release. That the CB1 receptor may influence dopamine release in the striatum suggests that cannabinoids play a modulatory role in dopaminergic neuronal pathways.     

Intrastriatal Grafting of Cos Cells Stably Expressing Human Aromatic l-Amino Acid Decarboxylase: Neurochemical Effects

Abstract: To study the possibility that increasing striatal activity of aromatic l -amino acid decarboxylase (AADC; EC 4.1.1.28) can increase dopamine production in dopamine denervated striatum in response to l -3,4-dihydroxyphenylalanine ( l -DOPA) administration, we grafted Cos cells stably expressing the human AADC gene (Cos- haadc cells) into 6-hydroxydopamine denervated rat striatum. Before grafting, the catalytic activity of the enzyme was assessed in vitro via the generation of ¹⁴CO₂ from l -[¹⁴C]DOPA. The K _m value for l -DOPA in intact and disrupted cells was 0.60 and 0.56 m M , respectively. The cofactor, pyridoxal 5-phosphate, enhanced enzymatic activity with maximal effect at 0.1 m M . The pH optimum for enzyme activity was 6.8. Grafting Cos- haadc cells into denervated rat striatum enhanced striatal dopamine levels measured after systemic administration of l -DOPA. When measured 2 h after l -DOPA administration, the mean dopamine level in the striata of Cos- haadc -grafted animals was 2 µg/g of tissue, representing 31% of normal striatal dopamine concentration. The mean dopamine concentration in the striata grafted with untransfected Cos cells (Cos-ut cells) was 1 µg/g. At 6–8 h after l -DOPA administration, striatal dopamine content in the Cos- haadc -grafted animals was 0.67 µg/g of tissue weight, representing 9% of intact striatum dopamine content. By contrast, the average dopamine content in the Cos-ut-grafted animals was undetectable. These findings demonstrate that enhancing striatal AADC activity can improve dopamine bioformation in response to systemically administered l -DOPA.     

Muscarinic Modulation of Striatal Dopamine, Glutamate, and GABA Release, as Measured with In Vivo Microdialysis

Abstract: Intrastriatal microdialysis was used to administer muscarinic drugs in freely moving rats for 40 min at a flow rate of 2 µl/min. Administration of the nonselective agonist pilocarpine at 10 m M increased striatal dopamine release and decreased extracellular GABA and glutamate overflow. Perfusion with the muscarinic M₂ antagonist methoctramine at 75 µ M increased extracellular dopamine and glutamate concentrations but exerted no changes on extracellular GABA levels. Intrastriatal administration of the M₁ antagonist pirenzepine at 0.05 µ M decreased extracellular dopamine overflow. Application of pirenzepine (0.05 and 5 µ M ) exerted no effects on the measured GABA or glutamate levels. There are thus important differences in applied doses of muscarinic drugs needed to obtain modulatory effects. High doses of agonists are probably needed to superimpose on the background of tonic influences of striatal acetylcholine, whereas antagonists can block the receptors in small doses. We further suggest that M₁ receptors might tonically facilitate striatal dopamine release, that M₂ receptors might tonically inhibit striatal glutamate efflux, and that acetylcholine does not exert tonic effects on striatal GABA release. The link with the pilocarpine animal model for temporal lobe epilepsy will be discussed.     

Intrastriatal injection of colchicine induces striatonigral degeneration in mice

Recent studies from environmental toxicology and molecular genetics demonstrate that midbrain dopamine (DA) neurons are particularly vulnerable to microtubule depolymerizing agents, indicating the involvement of microtubule dysfunction in the pathogenesis of Parkinson's disease. Here we show that intrastriatal injection of colchicine (COL), a well-known microtubule disruptor, induced degeneration of striatonigral pathway. Microtubule disruption caused by unilateral injection of COL blocked the retrograde axonal transport of fluorogold previously injected into striatum and induced substantial death of striatal and DA neurons in substantia nigra pars compacta. Furthermore, COL-induced pathologic changes were associated with robust glial reaction, which may be conducive to the degeneration of striatonigral pathway. We also found that intrastriatal injection of COL resulted in side bias in spontaneous turning activities and apomorphine-induced rotational behavior. Together, our results provide in vivo data lending support to the concept that microtubule dysfunction may play a significant role in the death of DA neurons, though glial reaction may be involved and contribute to the degenerative process. Moreover, intrastriatal COL may serve as another experimental model of striatonigral degeneration (Parkinson's variant of multiple system atrophy), given the concurrent loss of both striatal and DA neurons.     

Inhibition of Nitric Oxide Synthase Activity Attenuates Striatal Malonate Lesions in Rats

Abstract: Mitochondrial inhibitors such as malonate are potent neurotoxins in vivo. Intrastriatal injections of malonate result in neuronal damage reminiscent of "excitotoxic" lesions produced by compounds that activate NMDA receptors. Although the mechanism of cell death produced by malonate is uncertain, overactivation of NMDA receptors may be involved; pretreatment of animals with NMDA antagonists provides neuroprotection against malonate lesions. NMDA receptor activation stimulates the enzyme nitric oxide (NO) synthase (NOS). Elevated tissue levels of NO may generate highly reactive intermediates that impair mitochondrial function. We hypothesized that NO may be a mediator of malonate toxicity. We investigated whether in vivo inhibition of NO production by the NOS inhibitor N ^ω-nitro- l -arginine (NLA) would attenuate lesions produced by intrastriatal injections of malonate. We found that systemic injections of 3 mg/kg of NLA significantly reduced the extent of histologic damage elicited by intrastriatal injections of 1.5 µmol of malonate in adult rats.     

Striatal glutamate release evoked in vivo by NMDA is dependent upon ongoing neuronal activity in the substantia nigra, endogenous striatal substance P and dopamine

The aim of the present microdialysis study was to investigate whether the increase in striatal glutamate levels induced by intrastriatal perfusion with NMDA was dependent on the activation of extrastriatal loops and/or endogenous striatal substance P and dopamine. The NMDA-evoked striatal glutamate release was mediated by selective activation of the NMDA receptor-channel complex and action potential propagation, as it was prevented by local perfusion with dizocilpine and tetrodotoxin, respectively. Tetrodotoxin and bicuculline, perfused distally in the substantia nigra reticulata, prevented the NMDA-evoked striatal glutamate release, suggesting its dependence on ongoing neuronal activity and GABA(A) receptor activation, respectively, in the substantia nigra. The NMDA-evoked glutamate release was also dependent on striatal substance P and dopamine, as it was antagonized by intrastriatal perfusion with selective NK(1) (SR140333), D(1)-like (SCH23390) and D(2)-like (raclopride) receptor antagonists, as well as by striatal dopamine depletion. Furthermore, impairment of dopaminergic transmission unmasked a glutamatergic stimulation by submicromolar NMDA concentrations. We conclude that in vivo the NMDA-evoked striatal glutamate release is mediated by activation of striatofugal GABAergic neurons and requires activation of striatal NK(1) and dopamine receptors. Endogenous striatal dopamine inhibits or potentiates the NMDA action depending on the strength of the excitatory stimulus (i.e. the NMDA concentration).     

Changes in the levels of glutathione after cellular and cutaneous damage induced by squalene monohydroperoxide.

Squalene monohydroperoxide (Sq-OOH), the initial product of ultraviolet-peroxidated squalene, was used to investigate the effect of peroxidative challenge upon the glutathione contents in rabbit ear skin and primary-cultured fibroblasts derived from rabbit ear skin. The cellular reduced glutathione (GSH) contents decreased during 30-minute incubations in vitro with Sq-OOH, and oxidized glutathione (GSSG) was formed concomitantly, indicating that Sq-OOH had a potential for GSH-depleting activity in vitro. When Sq-OOH was applied topically to the skin in vivo, only GSSG contents increased significantly within 30 minutes. Moreover, pretreatment with the GSH depletors, DL-buthionine sulfoximine (BSO) and diethyl maleate (DEM), could potentiate the cytotoxicity and comedogenicity induced by Sq-OOH. These findings suggest that the endogenous antioxidant, glutathione, is quite sensitive to Sq-OOH and may be an important material for protecting cells and/or tissues against the oxidative stress induced by Sq-OOH treatment.     

Decarboxylation of Exogenous l-3,4-Dihydroxyphenylalanine in Rat Striatum as Studied by In Vivo Voltammetry

An in vivo voltammetric technique was used to determine whether striatal nondopaminergic neurons take up and decarboxylate exogenous L-3,4-dihydroxyphenylalanine (L-DOPA) and release it as dopamine. After the striatal serotonergic neurons of the rat had been destroyed by intraventricular injection of 5,7-dihydroxytryptamine, L-DOPA was administered intraperitoneally. It was found that changes in the dopamine concentration in the striatal extracellular fluid of the rat were the same as those in the nonlesioned rat. L-DOPA was also administered to the rat after the striatal perikarya had been destroyed by the intrastriatal injection of kainate. The striatal dopamine concentrations of the lesioned rat changed in parallel with 5,7-dihydroxytryptamine-lesioned rats, as well as the nonlesioned rats. Moreover, when normal rats were administered L-DOPA, the dopamine concentration was not increased in the cerebellum, where dopamine neurons do not exist. From these observations, it is concluded that exogenous L-DOPA is taken up, decarboxylated to dopamine, and released only in the striatal dopamine neurons.     

Modulation of Neurosteroid Synthesis/Accumulation by l-Ascorbic Acid in Rat Brain Tissue: Inhibition by Selected Serotonin Antagonists

Abstract: We have investigated the possibility that the synthesis/accumulation of neurosteroids, i.e., brain-produced steroids putatively endowed with modulatory actions in the CNS, is regulated by monoaminergic receptor-mediated mechanisms. In minces of rat brain cortex, l -ascorbic acid concentration-dependently (0.07–1.0 m M ) increases the levels of pregnenolone, allotetrahydrodeoxycorticosterone, and dehydroepiandrosterone. This effect of l -ascorbic acid is region-dependent: in hippocampus, progesterone and allopregnanolone are also increased, whereas dehydroepiandrosterone is unchanged, and in corpus striatum only progesterone is increased significantly. 5-Hydroxytryptamine (10 µ M ), 1-(3-chlorophenyl)piperazine (1.0 µ M ), and 5-methoxytryptamine (0.4 µ M ) mimic the effect of l -ascorbic acid, whereas a pretreatment with p -chlorophenylalanine (400 mg/kg i.p., 2 days) reduces the amplitude of the l -ascorbic acid effect on brain cortical neurosteroids. The effect of l -ascorbic acid is blocked by the nonselective serotonin antagonists methiothepin, clozapine, methysergide, and pizotifen, but not mesulergine, spiperone, MDL 72222, and dl -propranolol, nor by the catecholaminergic receptor antagonists prazosin and S (−)-sulpiride. l -Ascorbic acid is not additive with dibutyryl-cyclic AMP and, furthermore, the inhibition of adenylate cyclase by MDL 12330A, but not of phospholipase C by U-73122, markedly attenuates the l -ascorbic acid-induced increase of pregnenolone in rat brain cortical minces. Together these data suggest that l -ascorbic acid plays a role in the modulation of neurosteroidogenesis, presumably by favoring the activation of the purported serotonin type 6 receptor by endogenous serotonin.     

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.     

Reduction of Brain Glutathione by l-Buthionine Sulfoximine Potentiates the Dopamine-Depleting Action of 6-Hydroxydopamine in Rat Striatum

Sprague-Dawley rats were anesthetized with chloral hydrate, and plastic cannulae were permanently implanted into the lateral ventricles. The animals then were allowed to recover for 1-2 days. L-Buthionine sulfoximine (L-BSO), a selective inhibitor of glutathione (GSH) synthesis, and 6-hydroxydopamine (6-OH-DA), a selective catecholaminergic neurotoxin, were administered intracerebroventricularly. The striatal concentrations of GSH and monoamines were determined by HPLC with electrochemical detection. Two injections of L-BSO (3.2 mg, at a 48-h interval) resulted in a 70% reduction of striatal GSH. 6-OH-DA (150 or 300 micrograms) reduced the concentrations of striatal dopamine and noradrenaline 7 days after the administration, but left the concentrations of 5-hydroxytryptamine unaltered. L-BSO treatment did not produce any changes in the levels of monoamines per se but it potentiated the catecholamine-depleting effect of 6-OH-DA in the striatum. Thus, GSH appears to suppress the toxicity of 6-OH-DA, probably by scavenging the toxic species formed during 6-OH-DA oxidation. In view of these results one may suggest an important role for GSH in catecholaminergic neurons: protecting against the oxidation of endogenous catechols.     

Basic Fibroblast Growth Factor Stimulation of Glial Cells Protects Dopamine Neurons from 6-Hydroxydopamine Toxicity: Involvement of the Glutathione System

Abstract: Neurotrophic factors have been shown to support the survival and promote the recovery of injured neurons both in vivo and in vitro. Here, we investigated whether glial cell line-derived neurotrophic factor (GDNF) and basic fibroblast growth factor (bFGF) could modify the damage to dopamine (DA) neurons in mesencephalic cultures caused by the neurotoxin 6-hydroxydopamine (6-OHDA). The data show that bFGF, but not GDNF, effectively protected DA neurons from 6-OHDA toxicity. Because bFGF is a glial mitogen, whereas GDNF is not, we tested whether glial cells participated in bFGF neuroprotection. Inhibition of glial cell proliferation completely prevented the protective effect of bFGF. Because oxidative events have been associated with 6-OHDA-induced damage, we examined the levels of glutathione (GSH) in control and bFGF-treated cultures. Cultures treated with bFGF had higher levels of GSH, which increased even further in response to 6-OHDA exposure. Control cultures failed to up-regulate GSH levels after 6-OHDA, suggesting a relationship between increased GSH levels and protection from 6-OHDA. Inhibition of glial cell proliferation prevented the rise in GSH in bFGF-treated cultures and abolished the increase after 6-OHDA treatment. Protection from 6-OHDA by bFGF was also diminished when GSH levels were decreased by the GSH synthesis inhibitor l -buthionine sulfoximine. Our study shows that stimulation of glial cells by bFGF allows the up-regulation of antioxidant defenses and supports cell survival during oxidative stress.     

Effect of cellular glutathione depletion on cadmium-induced cytotoxicity in human lung carcinoma cells

The effect of glutathione depletion on cellular toxicity of cadmium was investigated in a subpopulation (T27) of human lung carcinoma A549 cells with coordinately high glutathione levels and Cd⁺⁺-resistance. Cellular glutathione levels were depleted by exposing the cells to diethyl maleate or buthionine sulfoximine. Depletion was dose-dependent. Exposure of the cells to 0.5 mM diethyl maleate for 4 hours or to 10 mM buthionine sulfoximine for 8 hours eliminated the threshold for Cd⁺⁺ cytotoxic effect and deccreased the LD_50S. Cells that were pretreated with 0.5 mM diethyl maleate or 10 mM buthionine sulfoximine and then exposed to these same concentrations of diethyl maleate or buthionine sulfoximine during the subsequent assay for colony forming efficiency produced no colonies, reflecting an enhanced sensitivity to these agents at low cell density. Diethyl maleate was found to be more cytotoxic than buthionine sulfoximine. Synergistic cytotoxic effects were observed in the response of diethyl maleate pretreated cells exposed to Cd⁺⁺. Thus the results demostrated that depletion of most cellular glutathione in A549-T27 cells prior to Cd⁺⁺ exposure sensitizes them to the agent's cytotoxic effects. Glutathione thus may be involved in modulating the early cellular Cd⁺⁺ cytotoxic response. Comparison of reduced glutathione levels and of Cd⁺⁺ cytotoxic responses in buthionine sulfoximine-treated A549-T27 cells with those levels in other, untreated normal and tumor-derived cells suggests that the higher level of glutathione in A549-T27 is not the sole determinant of its higher level of Cd⁺⁺ resistance.Abbreviations BSO DL-buthionine-(R,S)-sulfoximine - DEM diethyl maleate - DMSO dimethyl sulfoxide - GSH reduced glutathione - MT metallothionein     

1, 2, 3, 4-Tetrahydro-2-Methyl-4, 6, 7-Isoquinolinetriol Depletes Catecholamines in Rat Brain

1, 2, 3, 4-Tetrahydro-2-methyl-4, 6, 7-isoquinolinetriol (TMIQ) was synthesised and tested for activity as a dopamine-depleting agent in rat brain. After intracerebroventricular infusion, TMIQ caused reductions in dopamine concentrations in substantia nigra, striatum, hypothalamus, and dorsal raphe, and reduction in noradrenaline concentrations in locus coeruleus. TMIQ also reduced 5-hydroxytryptamine concentrations in dorsal raphe and substantia nigra, although with a lower potency. Comparisons between TMIQ and MPTP showed that they were approximately equipotent in depleting dopamine in the substantia nigra, hypothalamus, and dorsal raphe. Pretreatment of animals with a combination of monoamine oxidase A and B inhibitors completely prevented the TMIQ-induced reductions in dopamine concentrations in substantia nigra and hypothalamus. Direct unilateral intrastriatal injections of TMIQ produced marked ipsilateral reductions in striatal dopamine, correlating with a behavioural response consisting of turning towards the side of injection. The results suggest that TMIQ should be evaluated further as a possible MPTP-like compound, which may derive from endogenous β-hydroxylated catecholamines.