Redox-based therapeutics in neurodegenerative disease.

This review describes recent developments in the search for effective therapeutic agents that target redox homeostasis in neurodegenerative disease. The disruption to thiol redox homeostasis in Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis and multiple sclerosis is discussed, together with the experimental strategies that are aimed at preventing, or at least minimizing, oxidative damage in these diseases. Particular attention is given to the potential of increasing antioxidant capacity by targeting the Nrf2 pathway, the development of inhibitors of NADPH oxidases that are likely candidates for clinical use, together with strategies to reduce nitrosative stress and mitochondrial dysfunction. We describe the shortcomings of compounds that hinder their progression to the clinic and evaluate likely avenues for future research. LINKED ARTICLES: This article is part of a themed section on Redox Biology and Oxidative Stress in Health and Disease. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.12/issuetoc.

Comparative potencies of 3,4-methylenedioxymethamphetamine (MDMA) analogues as inhibitors of [3H]noradrenaline and [3H]5-HT transport in mammalian cell lines.

BACKGROUND AND PURPOSE: Illegal 'ecstasy' tablets frequently contain 3,4-methylenedioxymethamphetamine (MDMA)-like compounds of unknown pharmacological activity. Since monoamine transporters are one of the primary targets of MDMA action in the brain, a number of MDMA analogues have been tested for their ability to inhibit [3H]noradrenaline uptake into rat PC12 cells expressing the noradrenaline transporter (NET) and [3H]5-HT uptake into HEK293 cells stably transfected with the 5-HT transporter (SERT). EXPERIMENTAL APPROACH: Concentration-response curves for the following compounds at both NET and SERT were determined under saturating substrate conditions: 4-hydroxy-3-methoxyamphetamine (HMA), 4-hydroxy-3-methoxymethamphetamine (HMMA), 3,4-methylenedioxy-N-hydroxyamphetamine (MDOH), 2,5-dimethoxy-4-bromophenylethylamine (2CB), 3,4-dimethoxymethamphetamine (DMMA), 3,4-methylenedioxyphenyl-2-butanamine (BDB), 3,4-methylenedioxyphenyl-N-methyl-2-butanamine (MBDB) and 2,3-methylenedioxymethamphetamine (2,3-MDMA). KEY RESULTS: 2,3-MDMA was significantly less potent than MDMA at SERT, but equipotent with MDMA at NET. 2CB and BDB were both significantly less potent than MDMA at NET, but equipotent with MDMA at SERT. MBDB, DMMA, MDOH and the MDMA metabolites HMA and HMMA, were all significantly less potent than MDMA at both NET and SERT. CONCLUSIONS AND IMPLICATIONS: This study provides an important insight into the structural requirements of MDMA analogue affinity at both NET and SERT. It is anticipated that these results will facilitate understanding of the likely pharmacological actions of structural analogues of MDMA.

Blockade of noradrenaline transport abolishes 4-methylthioamphetamine-induced contraction of the rat aorta in vitro.

The aim of this study was to characterize the effects of 4-methylthioamphetamine (4-MTA) on contractility and noradrenaline (NA) transport and release in the isolated rat aorta. Descending thoracic aortic rings were isolated from male Wistar rats (220-240 g) and the effect of 4-MTA on contractility was measured by isometric force displacement. 4-MTA (0.1 microm-1 mm) induced a concentration-dependent contraction of aortic rings, with a pD(2) of 4.40 +/- 0.38, and an E(max) of 0.80 +/- 0.05 g tension. The alpha(1)-adrenoceptor antagonist, prazosin (1 microm) and alpha(2) antagonist, yohimbine (1 microm) inhibited maximal contraction to 100 microm 4-MTA by 45.0 +/- 6.7% and 53.5 +/- 7.1% of control values respectively, whereas the 5-hydroxytryptamine (5-HT) antagonist, ketanserin (100 nm) had no effect on the 4-MTA-mediated contraction. The specific NA transport inhibitor, nisoxetine (1 microm) abolished contraction of the aorta by 4-MTA. 4 Nisoxetine-sensitive [(3)H]-NA transport in aortic rings was measured over a concentration range of 0-5 microm [(3)H]-NA, and had a maximal rate of transport (V(max)) of 0.77 +/- 0.07 pmol [(3)H]-NA min(-1) mg(-1) protein and a Michaelis affinity constant (K(M)) of 2.3 +/- 0.5 microm. 4-MTA inhibited nisoxetine-sensitive [(3)H]-NA transport with a pIC(50) of 6.16 +/- 0.18 and the pIC(50) for inhibition of nisoxetine-sensitive [(3)H]-NA transport by 3,4-methylenedioxymethamphetamine (MDMA) was 6.83 +/- 0.13. 4-MTA (1-100 microm) significantly stimulated release of pre-loaded [(3)H]-NA from aortic rings and 4-MTA-induced [(3)H]-NA release was inhibited by 1 microm nisoxetine. These data suggest that 4-MTA causes contraction of the rat aorta in vitro by a mechanism that is consistent with an ability to cause release of NA at the level of the NA transporter. It is concluded that 4-MTA has the potential to increase the extracellular concentration of NA peripherally as well as centrally, and that this may cause adverse cardiovascular effects in its users.

In vitro neuronal and vascular responses to 5-HT in rats chronically exposed to MDMA.

1. This study examined the effects of chronic exposure of rats to 3,4-methylenedioxymethamphetamine (MDMA) on [(3)H]5-hydroxytryptamine ([(3)H]5-HT) re-uptake into purified rat brain synaptosomes, 5-HT-induced isometric contraction of aortic rings and [(3)H]5-HT re-uptake into rat aorta. 2. Rats were administered MDMA (20 mg kg(-1) i.p.) twice daily over 4 days. One, 7, 14 or 21 days post treatment, whole brain synaptosomes and descending thoracic aortic rings were prepared for investigation. 3. Chronic MDMA treatment significantly reduced the maximum rate (V(max)) of specific high-affinity [(3)H]5-HT re-uptake 1 day after treatment and for up to 21 days post-final administration of MDMA. Direct application of MDMA (100 microM) abolished synaptosomal re-uptake of [(3)H]5-HT in vitro. 4. Chronic MDMA administration significantly reduced the maximum contraction (E(max)) to 5-HT at 1 and 7 days after treatment, but not at 14 or 21 days. 5. Chronic MDMA administration had no effect on sodium-dependent [(3)H]5-HT re-uptake into aorta 1 day after treatment, nor did 100 microM MDMA have any direct effect on [(3)H]5-HT uptake into aortic rings in vitro. 6. These results show, for the first time, an altered responsiveness of vascular tissue to MDMA after chronic administration. In addition, they demonstrate a difference in the sensitivity of central and peripheral 5-HT uptake systems to chronic MDMA exposure, and suggest that the action of MDMA in the cardiovascular system does not arise from a direct effect of MDMA on peripheral 5-HT transport.

Molecular mechanisms of cystine transport.

The transport of L-cystine into cells of the mammalian brain is an essential step in the supply of cysteine for synthesis of the antioxidant glutathione. Uptake of L-cystine in rat brain synaptosomes occurs by three mechanisms that are distinguishable on the basis of their ionic dependence, kinetics of transport and specificity of inhibitors. Almost 90% of L-cystine transport is by a low-affinity, sodium-dependent mechanism (K(m)=473+/-146 microM), that is mediated by the X(AG)- family of glutamate transporters. Both L-glutamate (IC(50)=9.1+/-0.4 microM) and L-cysteine sulphinate (IC(50)=16.4+/-3.6 microM) are non-competitive inhibitors of sodium-dependent L-[(14)C]cystine transport, whereas L-trans-pyrrolidine-2,4-dicarboxylic acid (IC(50)=5.6+/-2.0 microM), L-serine-O-sulphate (IC(50)=13.2+/-5.4 microM), kainate (IC(50)=215+/-78 microM) and L-cysteine (IC(50)=363+/-63 microM) are competitive inhibitors. L-Cystine has no effect on the sodium-dependent uptake of D-[(3)H]aspartate. These results suggest that L-cystine binds to a site that is different from the L-glutamate recognition site on X(AG)- glutamate transporters. In rat brain slices, sodium-dependent transport of both L-glutamate and L-cystine is necessary for maintaining glutathione levels. Uptake of L-cystine is sensitive to inhibition by an increased extracellular concentration of L-glutamate, which has important implications for understanding conditions that may initiate oxidative stress.

Stimulation of synaptosomal D-[(3)H]aspartate transport by substance P in rat brain.

The effect of the neuropeptide, substance P, on the transport of D-[(3)H]aspartate into rat striatal synaptosomes was studied. Almost 90% of the total transport of D-[(3)H]aspartate was sodium-dependent and the maximum rate (V(max)) of this transport was increased by 34% of control by 2.5 nM substance P (EC(50)=0.52 nM). In contrast, sodium-independent transport was inhibited by substance P. The NK(1) antagonist, L706303 (500 nM) blocked the stimulation of D-[(3)H]aspartate transport by 2.5 nM substance P, but did not alter D-aspartate uptake in the absence of substance P. These results indicate that high affinity glutamate transporters in the brain may be under positive regulation by substance P, and suggest a previously-unidentified mechanism of control of glutamate transport.

Kinetic and pharmacological analysis of L-[35S]cystine transport into rat brain synaptosomes.

The synaptosomal transport of L-[35S]cystine occurs by three mechanisms that are distinguishable on the basis of their ionic dependence, kinetics of transport and the specificity of inhibitors. They are (a) low affinity sodium-dependent transport (Km 463 +/- 86 microM, Vmax 185 +/- 20 nmol mg protein-1 min-1), (b) high affinity sodium-independent transport (Km 6.90 +/- 2.1 microM, Vmax 0.485 +/- 0.060 nmol mg protein(-1) min(-1)) and (c) low affinity sodium-independent transport (Km 327 +/- 29 microM, Vmax 4.18 +/- 0.25 nmol mg protein(-1) min(-1)). The sodium-dependent transport of L-cystine was mediated by the X(AG)- family of glutamate transporters, and accounted for almost 90% of the total quantity of L-[35S]cystine accumulated into synaptosomes. L-glutamate (Ki 11.2 +/- 1.3 microM) was a non-competitive inhibitor of this transporter, and at 100 microM L-glutamate, the Vmax for L-[35S]cystine transport was reduced to 10% of control. L-cystine did not inhibit the high-affinity sodium-dependent transport of D-[3H]aspartate into synaptosomes. L-histidine and glutathione were the most potent inhibitors of the low affinity sodium-independent transport of L-[35S]cystine. L-homocysteate, L-cysteine sulphinate and L-homocysteine sulphinate were also effective inhibitors. 1 mM L-glutamate reduced the sodium-independent transport of L-cystine to 63% of control. These results suggest that the vast majority of the L-cystine transported into synaptosomes occurs by the high-affinity glutamate transporters, but that L-cystine may bind to a site that is distinct from that to which L-glutamate binds. The uptake of L-cystine by this mechanism is sensitive to inhibition by increased extracellular concentrations of L-glutamate. The importance of these results for understanding the mechanism of glutamate-mediated neurotoxicity is discussed.

Chronic haloperidol treatment impairs glutamate transport in the rat striatum.

High-affinity, Na(+)-dependent transport of glutamate into neurons and glial cells maintains the extracellular concentration of this neurotransmitter at a sub-toxic level. Chronic blockade of dopamine D(2) receptors with haloperidol elevates extracellular glutamate levels in the striatum. The present study examines the effect of long-term haloperidol treatment on glutamate transporter activity using an assay based on measuring the uptake of D-[3H]aspartate in striatal synaptosomes prepared from male Wistar rats. The maximal rate of glutamate transport in the striatum is reduced by 63% following 27 weeks of haloperidol treatment. This impairment of glutamate transport may be important in chronic neuroleptic drug action.

Inhibition of the high-affinity uptake of D-[3H]aspartate in rate by L-alpha-aminoadipate and arachidonic acid.

The mechanism of inhibition of the high-affinity sodium-dependent transport of D-[3H]aspartate by the gliotoxin, L-alpha-aminoadipate, and also by the endogenous fatty acid, arachidonic acid (cis-5,8,11,14 eicosatetraenoic acid), into rat brain synaptosomes has been investigated. L-alpha-Aminoadipate competitively inhibited the transport of D-[3H]aspartate with a K1 value of 192 microM. Superfusion of coronal slices of rat brain for 40 min with 1 mM L-alpha-aminoadipate reduced the glutathione concentration of the tissue by 20%. Neither glutamate nor kainate depleted the glutathione level of the slices. Pre-incubation of synaptosomes with arachidonic acid (10 microM) for 10-60 min produced a marked potentiation of the inhibition of D-[3H]aspartate transport, compared to experiments in which the acid was added concurrently with the D-[3H]aspartate ('co-incubation' experiments). Inhibition of D-[3H]aspartate transport by arachidonic acid was not blocked by addition of nordihydroguaretic acid to the pre-incubation medium. Staurosporine (50 nM) reduced the inhibition of transport occurring during pre-incubation with 10 microM arachidonic acid, and there was no longer any significant difference from the level of inhibition obtained in co-incubation experiments. Phorbol, 12-myristate, 13-acetate (1 microM) reduced the transport of D-[3H]aspartate to 73% of control after 20 min pre-incubation of the synaptosomes. This study highlights the fact that inhibition of glutamate transport may affect brain function in a number of different ways. Competitive inhibition by a structural analogue of glutamate, such as L-alpha-aminoadipate, leads to a reduction in the glutathione level, which may be an important factor in L-alpha-aminoadipate-mediated toxicity. On the other hand, the more long-term effects of non-competitive inhibition of glutamate transport by arachidonic acid, in a mechanism involving protein kinase C, may represent a physiological means for regulation of transporter activity in the brain.