Superoxide dismutase/catalase mimetics are neuroprotective against selective paraquat-mediated dopaminergic neuron death in the substantial nigra: implications for Parkinson disease

Exposure of mice to the herbicide paraquat has been demonstrated to result in the selective loss of dopaminergic neurons of the substantia nigra, pars compacta (SNpc) akin to what is observed in Parkinson disease (PD). In this study, we investigate the efficacy of two synthetic superoxide dismutase/catalase mimetics (EUK-134 and EUK-189) in protecting against paraquat-induced dopaminergic cell death in both the rat dopaminergic cell line 1RB3AN27 (N27) and primary mesencephalic cultures in vitro and in adult mice in vivo. Our data demonstrate that pretreatment with either EUK-134 or EUK-189 significantly attenuates paraquat-induced neurotoxicity in vitro in a concentration-dependent manner. Furthermore, systemic administration of EUK-189 decreases paraquat-mediated SNpc dopaminergic neuronal cell death in vivo. These findings support a role for oxidative stress in paraquat-induced neurotoxicity and suggest novel therapeutic approaches for neurodegenerative disorders associated with oxidative stress such as PD.     

Role of oxidative stress in paraquat-induced dopaminergic cell degeneration

Systemic treatment of mice with the herbicide paraquat causes the selective loss of nigrostriatal dopaminergic neurons, reproducing the primary neurodegenerative feature of Parkinson's disease. To elucidate the role of oxidative damage in paraquat neurotoxicity, the time-course of neurodegeneration was correlated to changes in 4-hydroxy-2-nonenal (4-HNE), a lipid peroxidation marker. When mice were exposed to three weekly injections of paraquat, no nigral dopaminergic cell loss was observed after the first administration, whereas a significant reduction of neurons followed the second exposure. Changes in the number of nigral 4-HNE-positive neurons suggest a relationship between lipid peroxidation and neuronal death, since a dramatic increase in this number coincided with the onset and development of neurodegeneration after the second toxicant injection. Interestingly, the third paraquat administration did not cause any increase in 4-HNE-immunoreactive cells, nor did it produce any additional dopaminergic cell loss. Further evidence of paraquat-induced oxidative injury derives from the observation of nitrotyrosine immunoreactivity in the substantia nigra of paraquat-treated animals and from experiments with ferritin transgenic mice. These mice, which are characterized by a decreased susceptibility to oxidative stress, were completely resistant to the increase in 4-HNE-positive neurons and the cell death caused by paraquat. Thus, paraquat exposure yields a model that emphasizes the susceptibility of dopaminergic neurons to oxidative damage.     

Na+-dependent transport of large neutral amino acids occurs at the abluminal membrane of the blood-brain barrier

Several Na+-dependent carriers of amino acids exist on the abluminal membrane of the blood-brain barrier (BBB). These Na+-dependent carriers are in a position to transfer amino acids from the extracellular fluid of brain to the endothelial cells and thence to the circulation. To date, carriers have been found that may remove nonessential, nitrogen-rich, or acidic (excitatory) amino acids, all of which may be detrimental to brain function. We describe here Na+-dependent transport of large neutral amino acids across the abluminal membrane of the BBB that cannot be ascribed to currently known systems. Fresh brains, from cows killed for food, were used. Microvessels were isolated, and contaminating fragments of basement membranes, astrocyte fragments, and pericytes were removed. Abluminal-enriched membrane fractions from these microvessels were prepared. Transport was Na+ dependent, voltage sensitive, and inhibited by 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid, a particular inhibitor of the facilitative large neutral amino acid transporter 1 (LAT1) system. The carrier has a high affinity for leucine (Km 21 +/- 7 microM) and is inhibited by other neutral amino acids, including glutamine, histidine, methionine, phenylalanine, serine, threonine, tryptophan, and tyrosine. Other established neutral amino acids may enter the brain by way of LAT1-type facilitative transport. The presence of a Na+-dependent carrier on the abluminal membrane capable of removing large neutral amino acids, most of which are essential, from brain indicates a more complex situation that has implications for the control of essential amino acid content of brain.     

pH Dependence of Histidine Affinity for Blood-Brain Barrier Carrier Transport Systems for Neutral and Cationic Amino Acids

The effects of pH (3.5-7.5) on the brain uptake of histidine by the blood-brain barrier (BBB) carriers for neutral and cationic amino acids were tested, in competition with unlabeled histidine, arginine, or phenylalanine, with the single-pass carotid injection technique. Cationic amino acid ( [14C]arginine) uptake was increasingly inhibited by unlabeled histidine as the pH of the injection solution decreased. In contrast, the inhibitory effect of unlabeled histidine on neutral amino acid ( [14C]phenylalanine) uptake decreased with decreasing pH. Brain uptake indices with varying histidine concentrations indicated that the neutral form of histidine inhibited phenylalanine uptake whereas the cationic form competed with arginine uptake. Since phenylalanine decreased [14C]histidine uptake at all pH values whereas arginine did not, it was concluded that the cationic form of histidine had an affinity for the cationic carrier, but was not transported by it. We propose that the saturable entry of histidine into brain is, under normal physiological circumstances, mediated solely by the carrier for neutral amino acids.     

Amino acid transport by the filamentous fungus Arthrobotrys conoides

Uptake of l-valine by germinated spores of Arthrobotrys conoides has all the characteristics of a system of transport that requires an expenditure of energy by the cells. It is dependent on temperature and has an energy of activation of 16,000 cal/mole. Uptake is optimal at pH 5 to 6. l-Valine accumulated against a concentration gradient and is not lost from the cells by leakage or exchange. The process requires energy supplied by the metabolic reactions that are inhibited by catalytic amounts of 2,4-dinitrophenol and azide. The kinetics of the system are consistent with a mechanism of transport that depends on a limited number of sites on the cell surface, and the Michaelis constant for the system is 1.5 x 10(-5) to 7.5 x 10(-5)m. Modification of the amino or carboxyl group abolishes l-valine uptake. The process is competitively inhibited by d-valine, glycine, and other neutral amino acids (K(i) = 1.5 x 10(-5) to 4.0 x 10(-5)m), indicating a lack of stereospecificity, and also indicating that aliphatic side chain is not required for binding with the carrier. The transport system has less affinity for acidic amino acids (glutamic and aspartic acids) than neutral amino acids, and a greater affinity for basic amino acids (histidine, lysine, and arginine). The range of affinity is in the order of 100, as measured in terms of K(i) values for various compounds. The data presented provide suggestive evidence that the uptake by A. conoides of all amino acids except proline is mediated by a single carrier system that possesses an overall negative charge.     

Phospholipid synthesis in isolated alveolar type II cells exposed in vitro to paraquat and hyperoxia.

Isolated alveolar epithelial type II cells were exposed to paraquat and to hyperoxia by gas diffusion through the thin Teflon bottom of culture dishes. After exposure, type II cells were further incubated in the presence of labelled substrates to assess their capacity to synthesize lipids. Hyperoxia alone (90% O2; 5 h) had minor effects on lipid metabolism in the type II cells. At low paraquat concentrations (5 and 10 microM), hyperoxia enhanced the paraquat-induced decrease of [Me-14C]choline incorporation into phosphatidylcholines. The incorporation rates of [Me-14C]choline, [1-14C]palmitate, [1-14C]glucose and [1,3-3H]glycerol into various phospholipid classes and neutral lipids were decreased by paraquat, depending on the concentration and duration of the exposure. The incorporation of [1-14C]acetate into phosphatidylcholines, phosphatidylglycerols and neutral lipids appeared to be very sensitive to inactivation by paraquat. At 5 microM-paraquat the rate of [1-14C]acetate incorporation was decreased to 50% of the control values. The rate of [1-14C]palmitate incorporation into lipids was much less sensitive; it even increased at low paraquat concentrations. At 10 microM-paraquat both NADPH and ATP were significantly decreased. It is concluded that lipid synthesis in isolated alveolar type II cells is extremely sensitive to paraquat. At low concentrations of this herbicide, lipid synthesis, and particularly fatty acid synthesis, is decreased. The effects on lipid metabolism may be partly related to altered NADPH and ATP concentrations.     

Site-directed mutagenesis of rabbit LAT1 at amino acids 219 and 234

The availability of amino acids in the brain is regulated by the blood-brain barrier (BBB) large neutral amino acid transporter type 1 (LAT1) isoform, which is characterized by a high affinity (low Km) for substrate large neutral amino acids. The hypothesis that brain amino acid transport activity can be altered with single nucleotide polymorphisms was tested in the present studies with site-directed mutagenesis of the BBB LAT1. The rabbit has a high Km LAT1 large neutral amino acid transporter, as compared to the low Km neutral amino acid transporter at the human or rat BBB. The rabbit LAT1 was cloned from a rabbit brain capillary cDNA library. Alignment of the amino acid sequences of rabbit, human, and rat LAT1 revealed two radical amino acid residues that differ in the rabbit relative to the rat or human LAT1. The G219D mutation had a modest effect on the Km and Vmax of tryptophan transport via cloned rabbit LAT1 in frog oocytes, but the W234L variant reduced the Km by 64% and the Vmax by 96%. Conversely, LAT1 transport of either tryptophan or phenylalanine was nearly normalized when the double mutation W234L/G219D variant was produced. These studies show that marked changes in the affinity and capacity of the LAT1 are caused by single nucleotide polymorphisms and that phenotype can be restored with a double mutation.     

Effect of hyperosmolarity on the activity of amino acid transport system L in avian fibroblasts

The transport of selected neutral amino acids known as good substrates of amino acid transport System L has been studied in chick embryo fibroblasts exposed for 4 hours to hyperosmolar culture medium. The activity of the L system, as measured by initial rates of L-phenylalanine uptake, increased in hyperosmolarity treated cells when determined before any cell depletion of intracellular amino acids. This effect was lost after depletion but reappeared after reloading the cells with pertinent substrates of System L. This transport activity appeared to be related to the internal level of amino acids capable of exchange through System L. In hyperosmolarity-treated chick embryo fibroblasts a higher level of System L substrates was obtained during the reloading phase in comparison to control cells. This expanded amino acid pool reflected an increased activity of transport System A, an agency of amino acid mediation known to enlarge its capacity following a hyperosmolar treatment of chick embryo fibroblasts (see Tramacere et al., 1984). L-Methionine, a preferred substrate of both A and L systems, appeared to be involved in the coupling between the activity of amino acid transport Systems A and L in these cells.     

Expression of amino acid transport systems in Xenopus oocytes injected with mRNA of rat small intestine and kidney

Xenopus and Cynops oocytes were injected with exogenous mRNA prepared from rat small intestine and kidney and their electrical responses to amino acids were measured by both the current clamped and the voltage clamped methods. Oocytes injected with mRNA of rat small intestine showed a depolarization response to several neutral and basic amino acids, and almost no response to acidic amino acids. The responses to amino acids increased with incubation time after injection of mRNA, and followed Michaelis-Menten type kinetics. The responses were dependent on both Na+ concentration and membrane potential, and were inactivated by a sulfhydryl reagent, 5,5-dithiobis(2-nitrobenzoate). These results are interpreted as due to the expression of Na+/amino acid cotransporter(s) in oocytes injected with rat small intestine mRNA. On the other hand, the oocyte injected with rat kidney mRNA showed a hyperpolarization response to neutral amino acids, a depolarization response to basic ones, and almost no response to acidic ones in frog Ringer solution. These responses were independent of Na+ concentration and followed Michaelis-Menten type kinetics. These amino acid response characteristics in oocytes injected with rat kidney mRNA are interpreted as due to the expression of facilitated diffusion carrier protein(s) (uniporter) of amino acids in the oocyte.     

Identification of the Cationic Amino Acid Transporter (System y+) of the Rat Blood-Brain Barrier

Abstract: Cationic amino acids are transported from blood into brain by a saturable carrier at the blood-brain barrier (BBB). The transport properties of this carrier were examined in the rat using an in situ brain perfusion technique. Influx into brain via this system was found to be sodium independent and followed Michaelis-Men-ten kinetics with half-saturation constants (K_m) of 50–100 μM and maximal transport rates of 22–26 nmol/min/g for L-lysine, L-arginine, and L-ornithine. The kinetic properties matched that of System y⁺, the sodium-independent cationic amino acid transporter, the cDNA for which has been cloned from the mouse. To determine if the cloned receptor is expressed at the BBB, we assayed RNA from rat cerebral microvessels and choroid plexus for the presence of the cloned transporter mRNA by RNase protection. The mRNA was present in both cerebral microvessels and choroid plexus and was enriched in microvessels 38-fold as compared with whole brain. The results indicate that System y⁺ is present at the BBB and that its mRNA is more densely expressed at cerebral microvessels than in whole brain.     

Kinetics of Neutral Amino Acid Transport Through the Blood-Brain Barrier of the Newborn Rabbit

Abstract: Since protein synthesis in the developing brain may, under certain conditions, be limited by amino acid availability, the present studies were undertaken to characterize the kinetics of large neutral amino acid transport through the blood-brain barrier (BBB) of the newborn rabbit. The K_m, V_max, and K_D of the transport of eight amino acids were determined by a nonlinear regression analysis of data obtained with the carotid injection technique. Compared with kinetic parameters observed for the adult rat, the K_m, V_max, and K_D of amino acid transport were all two- to threefold higher in the newborn. Albumin was found to bind tryptophan actively in vitro , but had no inhibitory effect on tryptophan transport through the newborn BBB. Glutamine was transported through the BBB of the newborn at rates severalfold higher than are seen in the adult rat. However, glutamine transport was not inhibited by high concentrations of N -methylaminoisobutyric acid (NMAIB), a model amino acid that is specific for the alanine-preferring or A-system present in peripheral tissues. In conclusion, these studies show that the BBB neutral amino acid transport system of the newborn rabbit has a lower affinity and higher capacity than does the BBB of the adult rat. Under conditions of high plasma amino acids, the increased capacity of the newborn transport system allows for a higher rate of amino acid transport into brain than would occur via the lower capacity system present in the adult rat brain.     

The Binding Specificity of Amino Acid Transport System y+L in Human Erythrocytes is Altered by Monovalent Cations

System y⁺L is a broad-scope amino acid transporter which binds and translocates cationic and neutral amino acids. Na⁺ replacement with K⁺ does not affect lysine transport, but markedly decreases the affinity of the transporter for l-leucine and l-glutamine. This observation suggests that the specificity of system y⁺L varies depending on the ionic composition of the medium. Here we have studied the interaction of the carrier with various amino acids in the presence of Na⁺, K⁺, Li⁺ and guanidinium ion. In agreement with the prediction, the specificity of system y⁺L was altered by the monovalent cations. In the presence of Na⁺, l-leucine was the neutral amino acid that interacted more powerfully. Elongation of the side chain (glycine - l-norleucine) strengthened binding. In contrast, bulkiness at the level of the β carbon was detrimental. In K⁺, the carrier behaved as a cationic amino acid specific carrier, interacting weakly with neutral amino acids. Li⁺ was found to potentiate neutral amino acid binding and in general the apparent affinities were higher than in Na⁺; elongation of the nonpolar side chain made a more important contribution to binding and the carrier was more tolerant towards β carbon substitution. Guanidinium stimulated the interaction of the carrier with neutral amino acids, but the effect was restricted to certain analogues (e.g., l-leucine, l-glutamine, l-methionine). Thus, in the presence of guanidinium, the carrier discriminates sharply among different neutral amino acids. The results suggest that the monovalent cations stabilize different carrier conformations. Received: 22 January 1996/Revised: 26 April 1996     

G-actin conformational change and polymerization induced by paraquat.

Paraquat (1,1'-dimethyl-4,4'-bipyridilium dichloride) is a broad-spectrum herbicide that is highly toxic to animals (including man), the major lesion being in the lung. In mammalian cells, paraquat causes deep alterations in the organization of the cytoskeleton, marked decreases in cytoskeletal protein synthesis, and alterations in cytoskeletal protein composition; therefore, the involvement of the cytoskeleton in cell injury by paraquat was suggested. We previously demonstrated that monomeric actin binds paraquat; moreover, prolonged actin exposure to paraquat, in depolymerizing medium, induces the formation of actin aggregates, which are built up by F-actin. In this work we have shown that the addition of paraquat to monomeric actin results in a strong quenching of Trp-79 and Trp-86 fluorescence. Trypsin digestion experiments demonstrated that the sequence 61-69 on actin subdomain 2 undergoes paraquat-dependent conformational changes. These paraquat-induced structural changes render actin unable to completely inhibit DNase I. By using intermolecular cross-linking to characterize oligomeric species formed during paraquat-induced actin assembly, we found that the herbicide causes the formation of actin oligomers characterized by subunit-subunit contacts like those occurring in oligomers induced by polymerizing salts (i.e., between subdomain 1 on one actin subunit and subdomain 4 on the adjacent subunit). Furthermore, the oligomerization of G-actin induced by paraquat is paralleled by ATP hydrolysis.     

Investigate the chronic neurotoxic effects of diquat

Chronic exposure to agricultural chemicals (pesticides/herbicides) has been shown to induce neurotoxic effects or results in accumulation of various toxic metabolic by-products. These substances have the relevant ability to cause or increase the risk for neurodegeneration. Diquat is an herbicide that has been extensively used in the United States of America and other parts of the world. Diquat is constantly released into the environment during its use as a contact herbicide. Diquat structurally resembles 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) and paraquat. Rotenone, paraquat, maneb and MPTP reproduce features of movement disorders in experimental animal models. Based on the structural similarity to other neurotoxins, chronic exposure of diquat can induce behavioral and neurochemical alterations associated with dopaminergic neurotoxicity. However, in the present study, diquat unlike other neurotoxins (rotenone, 6-hydroxydopamine, MPTP, paraquat and maneb) did not induce dopamine depletion in the mouse striatum. Although, notable exacerbation in motor impairment (swimming score, akinesia and open field) were evident that may be due to the decreased dopamine turnover and mild nigrostriatal neurodegeneration. These data indicate that, despite the apparent structural similarity to other dopaminergic neurotoxins, diquat did not exert severe deleterious effects on dopamine neurons in a manner that is unique to rotenone and MPTP.     

Blood-brain barrier transport of the alpha-keto acid analogs of amino acids

A number of alpha-keto acid analogs of amino acids have been found to penetrate the blood-brain barrier (BBB). Pyruvate, alpha-ketobutyrate, alpha-ketoisocaproate, and alpha-keto-gamma-methiolbutyrate all cross the BBB by a carrier-mediated process and by simple diffusion. Under normal physiological conditions, diffusion accounts for roughly 15% or less of total transport. Aromatic alpha-keto acids, phenylpyruvate, and p-hydroxyphenylpyruvate do not penetrate the BBB, nor do they inhibit the transport of other alpha-keto acids. Evidence based primarily on inhibition studies indicates that the carrier-mediated transport of alpha-keto acids occurs via the same carrier demonstrated previously for propionate, acetoacetate, and beta-hydroxybutyrate transport, commonly referred to as the monocarboxylate carrier. As a group, the alpha-keto acid analogs of the amino acids have the highest affinity for the carrier, followed by propionate and beta-hydroxybutyrate. Starvation for 4 days induces transport of alpha-keto acids, but transport is suppressed in rats fed commercial laboratory rations and subjected to portacaval shunts. The mitochondrial pyruvate translocator inhibitor alpha-cyanocinnamate has no effect on the BBB transport of alpha-keto acids.     

Emerging roles for sodium dependent amino acid transport in mesenchymal cells

Summary The functional aspects of sodium dependent amino acid transport in mesenchymal cells are the subject of this contribution. In a survey of the cross-talk existing among the various transport mechanisms, particular attention is devoted to the role played by substrates shared by several transport systems, such as L-glutamine. Intracellular levels of glutamine are determined by the activity of System A, the main transducer of ion gradients built on by Na,K-ATPase into neutral amino acid gradients. Changes in the activity of the System are employed to regulate intracellular amino acid pool and, hence, cell volume. System A activity has been found increased in hypertonically shrunken cells and in proliferating cells. Under both these conditions cells have to increase their volume; therefore, System A can be employed as a convenient mechanism to increase cell volume both under hypertonic and isotonic conditions. Although less well characterized, the uptake of anionic amino acids performed by System X^– _AG may be involved in the maintenance of intracellular amino acid pool under conditions of limited availability of neutral amino acids substrates of System A.     

Valine accumulation by alpha-aminobutyric acid-resistant mutants of Serratia marcescens

alpha-Aminobutyric acid, norvaline, and norleucine, which are analogues of branched-chain amino acids, inhibited the growth of Serratia marcescens. The inhibitory effect of these three analogues was counteracted by branched-chain amino acids. A number of mutants resistant to these analogues were isolated. alpha-Aminobutyric acid-resistant (abu-r) mutants markedly accumulated l-valine in the culture medium, but the other analogue-resistant mutants did not. Acetohydroxy acid synthetase, which seems to be rate-limiting for the biosynthesis of l-valine, was derepressed in abu-r mutants. One of the abu-r mutants, no. 140, which accumulated over 8 mg of l-valine per ml, had about a 20-fold increase in the enzyme level. Most of the abu-r mutants had acetohydroxy acid synthetase activity which was sensitive to feedback inhibition by l-valine to the same extent as in the parent strain. However, the enzyme of two of abu-r mutants was less sensitive to l-valine, and one of the two was the best valine accumulator.     

Resveratrol potentiates cytochrome P450 2 d22-mediated neuroprotection in maneb- and paraquat-induced parkinsonism in the mouse

A strong association between polymorphisms of the cytochrome P450 (CYP/Cyp) 2D6 gene and risk to Parkinson's disease (PD) is well established. The present study investigated the neuroprotective potential of Cyp2d22, a mouse ortholog of human CYP2D6, in maneb- and paraquat-induced parkinsonism and the mechanisms involved therein along with the effects of resveratrol on various parameters associated with Cyp2d22-mediated neuroprotection. The animals were treated intraperitoneally with resveratrol (10mg/kg, daily) and paraquat (10mg/kg) alone or in combination with maneb (30 mg/kg), twice a week, for 9 weeks, along with their respective controls. The subsets of animals were also treated intraperitoneally with a Cyp2d22 inhibitor, ketoconazole (100mg/kg, daily). Maneb and paraquat reduced Cyp2d22 and vesicular monoamine transporter type 2 (VMAT-2) expressions, the number of tyrosine hydroxylase-positive cells, and dopamine content and increased paraquat accumulation in the nigrostriatal tissues, oxidative stress, microglial activation, neuroinflammation, and apoptosis. Cyp2d22 inhibitor significantly exacerbated all these neurodegenerative indexes. Resveratrol cotreatment, partially but significantly, ameliorated the neurodegenerative changes by altering Cyp2d22 expression and paraquat accumulation. The results obtained in the study demonstrate that Cyp2d22 offers neuroprotection in maneb- and paraquat-induced dopaminergic neurodegeneration and resveratrol enhances its neuroprotective credentials by influencing Cyp2d22 expression and paraquat accumulation.     

Ameliorative effects of squash (<Emphasis Type="Italic">Cucurbita moschata</Emphasis> Duchesne ex Poiret) leaf extracts on oxidative stress

This study screened paraquat-tolerant plants among 10 plant species, including monocots and dicots angiosperms. Squash (Cucurbita moschata Duchesne ex Poiret) and kidney bean (Phaseolus vulgaris L.) plants exhibited the highest photooxidation-tolerant phenotypes upon a foliar treatment with paraquat. A foliar treatment with paraquat pre-mixed with leaf water extracts from the squash plant significantly alleviated paraquat-induced oxidative damage in maize, but this was not the case after a treatment with the hydrophobic phase of the leaf extracts. In particular, the water extract from young leaves (4th true leaf) of squash plants conferred tenfold higher tolerance to oxidative damage in paraquat-treated leave tissues compared to paraquat-only treatment. This tolerance was tightly linked not only to the increased amounts of ascorbic acid and dehydroascorbate antioxidants in the damaged leaves, but also to the reduced chlorophyll loss, lipid peroxidation, and cellular electrolyte leakage. Moreover, the protective effects of the water extract were apparent when using another bipyridyl herbicide, diquat, but not with a diphenyl-ether herbicide, oxyfluorfen. On the other hand, pre-treatment with the extract prior to the onset of drought or cold stress had no significant antioxidative effect on the treated tissues.