Targeted antioxidative and neuroprotective properties of the dopamine agonist pramipexole and its nondopaminergic enantiomer SND919CL2x [(+)2-amino-4,5,6,7-tetrahydro-6-Lpropylamino-benzathiazole dihydrochloride].

Pramipexole has been shown to possess neuroprotective properties in vitro that are partly independent of its dopaminergic agonism. The site of neuroprotective action is still unknown. Using [(3)H]pramipexole, we show that the drug enters and accumulates in cells and mitochondria. Detoxification of reactive oxygen species (ROS) by pramipexole is shown in vitro and in vivo by evaluating mitochondrial ROS release and aconitase-2 activity, respectively. Pramipexole and its (+)-enantiomer SND919CL2X [low-affinity dopamine agonist; (+)2-amino-4,5,6,7-tetrahydro-6-l-propylamino-benzathiazole dihydrochloride] possess equipotent efficacy toward hydrogen peroxide and nitric oxide generated in vitro and inhibit cell death in glutathione-depleted neuroblastoma cells. IC(50) values ranged from 15 to 1000 microM, consistent with the reactivity of the respective radical and the compartmentalization of ROS generation and ROS detoxification. Finally, both compounds were tested in superoxide dismutase 1-G93A mice, a model of familial amyotrophic lateral sclerosis. SND919CL2X (100 mg/kg) prolongs survival time and preserves motor function in contrast to pramipexole (3 mg/kg), which shows an increase in running wheel activity before disease onset, presumably caused by the dopaminergic agonism. We conclude that both enantiomers, in addition to their dopaminergic activity, are able to confer neuroprotective effects by their ability to accumulate in brain, cells, and mitochondria where they detoxify ROS. However, a clinical use of pramipexole as a mitochondria-targeted antioxidant is unlikely, because the high doses needed for antioxidative action in vitro are not accessible in vivo due to dopaminergic side effects. In contrast, SND919CL2X may represent the prototype of a mitochondria-targeted neuroprotectant because it has the same antioxidative properties without causing adverse effects.

Microglial cells in culture express a prominent glutathione system for the defense against reactive oxygen species.

To obtain information on the glutathione metabolism of microglial cells, the content of glutathione and activities of enzymes involved in the defense against peroxides were determined for microglia-rich cultures from rat brain. These cultures contain approximately 90% microglia cells as determined by immunocytochemical staining for glial markers, by the phagocytosis activity of the cells and by the production of superoxide after stimulation of the cells with phorbolester. For these cultures, a glutathione content of 41.2 +/- 11.2 nmol/mg protein and a specific activity of glutathione reductase of 15.2 +/- 3.2 nmol/(min x mg protein) were determined. These values are significantly higher than those found for astroglial or neuronal cultures. In addition, with 68.7 +/- 23.5 nmol/(min x mg protein), the specific activity of glutathione peroxidase in microglial cultures was 78% higher than in cultured neurons. The specific catalase activity of microglial cultures was less than 40% that of astroglial or neuronal cultures. Microglial cultures contain only marginal amounts of oxidized glutathione. However, on application of oxidative stress by incubation of microglial cultures with hydrogen peroxide or with the superoxide-producing hypoxanthine/xanthine oxidase system, cellular glutathione was rapidly oxidized. These results demonstrate that microglial cells have a prominent glutathione system, which is likely to reflect the necessity for self-protection against reactive oxygen species when produced by these or surrounding brain cells.

Glycogen is mobilized during the disposal of peroxides by cultured astroglial cells from rat brain.

Regeneration of reduced nicotinamide adenine dinucleotide phosphate (NADPH) is essential for the activity of glutathione redox cycling during cellular peroxide detoxification. In order to test for a function of astroglial glycogen to serve as endogenous precursor for glucose-6-phosphate, the substrate for the regeneration of NADPH by the pentose phosphate pathway, the content of glycogen in astroglia-rich primary cultures derived from the brains of newborn rats was determined after application of peroxides. In the presence of hydrogen peroxide or cumene hydroperoxide in concentrations of 200 microM glycogen was mobilized with a half-life of 16 min in incubation medium containing 20 mM glucose, whereas in the absence of peroxides the glycogen content decreased more slowly with a half-life of 42 min. After 30 min of incubation with or without peroxides 30 and 73%, respectively, of the initial glycogen content was found. The degree of glycogen mobilization was reduced by lowering the initial concentration of the peroxides. These results demonstrate that in astroglial cells (i) glucosyl residues of glycogen are mobilized after application of peroxides despite the presence of exogenous glucose, and (ii) that the demand for glucose-6-phosphate as substrate for NADPH regeneration via the pentose phosphate pathway can, at least partially, be met by mobilization of glycogen.

The detoxification of cumene hydroperoxide by the glutathione system of cultured astroglial cells hinges on hexose availability for the regeneration of NADPH.

The ability of astroglia-rich primary cultures derived from the brains of newborn rats to detoxify exogenously applied cumene hydroperoxide (CHP) was analyzed as a model to study glutathione-mediated peroxide detoxification by astrocytes. Under the conditions used, 200 microM CHP disappeared from the incubation buffer with a half-time of approximately 10 min. The half-time of CHP in the incubation buffer was found strongly elevated (a) in cultures depleted of glutathione by a preincubation with buthionine sulfoximine, an inhibitor of glutathione synthesis, (b) in the presence of mercaptosuccinate, an inhibitor of glutathione peroxidase, and (c) in the absence of glucose, a precursor for the regeneration of NADPH. The involvement of glutathione peroxidase in the clearance of CHP was confirmed by the rapid increase in the level of GSSG after application of CHP. The restoration of the initial high ratio of GSH to GSSG depended on the presence of glucose during the incubation. The high capacity of astroglial cells to clear CHP and to restore the initial ratio of GSH to GSSG was fully maintained when glucose was replaced by mannose. In addition, fructose and galactose at least partially substituted for glucose, whereas exogenous isocitrate and malate were at best marginally able to replace glucose during peroxide detoxification and regeneration of GSH. These results demonstrate that CHP is detoxified rapidly by astroglial cells via the glutathione system. This metabolic process strongly depends on the availability of glucose or mannose as hydride donors for the regeneration of the NADPH that is required for the reduction of GSSG by glutathione reductase.

The glutathione system of peroxide detoxification is less efficient in neurons than in astroglial cells.

The ability of neurons to detoxify exogenously applied peroxides was analyzed using neuron-rich primary cultures derived from embryonic rat brain. Incubation of neurons with H2O2 at an initial concentration of 100 microM (300 nmol/3 ml) led to a decrease in the concentration of the peroxide, which depended strongly on the seeding density of the neurons. When 3 x 10(6) viable cells were seeded per dish, the half-time for the clearance by neurons of H2O2 from the incubation buffer was 15.1 min. Immediately after application of 100 microM H2O2 to neurons, glutathione was quickly oxidized. After incubation for 2.5 min, GSSG accounted for 48% of the total glutathione. Subsequent removal of H2O2 caused an almost complete regeneration of the original ratio of GSH to GSSG within 2.5 min. Compared with confluent astroglial cultures, neuron-rich cultures cleared H2O2 more slowly from the incubation buffer. However, if the differences in protein content were taken into consideration, the ability of the cells to dispose of H2O2 was identical in the two culture types. The clearance rate by neurons for H2O2 was strongly reduced in the presence of the catalase inhibitor 3-aminotriazol, a situation contrasting with that in astroglial cultures. This indicates that for the rapid clearance of H2O2 by neurons, both glutathione peroxidase and catalase are essential and that the glutathione system cannot functionally compensate for the loss of the catalase reaction. In addition, the protein-normalized ability of neuronal cultures to detoxify exogenous cumene hydroperoxide, an alkyl hydroperoxide that is reduced exclusively via the glutathione system, was lower than that of astroglial cells by a factor of 3. These results demonstrate that the glutathione system of peroxide detoxification in neurons is less efficient than that of astroglial cells.

Detoxification of exogenous hydrogen peroxide and organic hydroperoxides by cultured astroglial cells assessed by microtiter plate assay.

Peroxides are often applied to cultured brain cells to investigate functions of these cells under oxidative stress. However, little is known about the ability of brain cells to detoxify peroxides. In order to investigate peroxide clearance of adherent cultured cells, the peroxide assay originally described for the determination of hydrogen peroxide production during experimental protein glycation by Jiang et al. [Z.-Y. Jiang, A.C.S. Woollard, S.P Wolff, Hydrogen peroxide production during experimental protein glycation, FEBS Lett. , 268 (1990) 69-71.] was adapted to microtiter plates. Besides hydrogen peroxide, with this assay organic hydroperoxides such as tertiary butylhydroperoxide (tBHP), and cumene hydroperoxide (CHP) can also be quantified. Up to an amount of 2.5 nmol of each peroxide per well of a plate the absorption measured was proportional to the concentration of the peroxide. Using the assay described the ability of astroglia-rich primary cultures to detoxify peroxides was monitored by measuring the peroxide content in 10 microliter samples collected at several time points from the peroxide-containing incubation buffer of one dish. If peroxides were applied at a concentration of 100 muM, hydrogen peroxide, tBHP, and CHP disappeared from the incubation buffer in reactions following first order kinetics with apparent half-times of 3.1 min, 2.9 min, and 4.2 min, respectively. In the absence of cells H2O2 and CHP were stable in the incubation buffer for at least 30 min, whereas tBHP decayed slowly in a spontaneous reaction. In conclusion, the method presented allows the determination of the rapid detoxification of various peroxides by cultured cells.

Rapid clearance of tertiary butyl hydroperoxide by cultured astroglial cells via oxidation of glutathione.

The ability of astroglial cells to detoxify exogenously applied tertiary butyl hydroperoxide (tBHP) was tested using astroglia-rich primary cultures derived from the brains of newborn rats. If 200 microM tBHP was applied, this compound disappeared from the incubation buffer with an apparent half-life of about 5 min. After 20 min incubation tBHP was not detectable any more. A decay of tBHP was found even in the absence of cells. Therefore, half-times for the cell-dependent tBHP clearance were corrected for the cell-independent decay of tBHP. The cell-dependent half-time of tBHP in the incubation buffer was found strongly elevated i) with increasing concentration of tBHP, ii) after decrease of the glutathione content of the cells by a preincubation with buthionine sulfoximine, an inhibitor of glutathione synthesis, iii) in the presence of mercaptosuccinate, an inhibitor of glutathione peroxidase, and iv) in the absence of glucose, the precursor for the generation of NADPH. Incubation of astroglial cells with 200 microM tBHP in the absence of glucose led to a 46% oxidation of the cellular glutathione within 30 s. Under these conditions the cells were unable to restore the original high ratio of the concentrations of GSH to GSSG within 30 min of incubation. In contrast, if glucose was present the level of GSSG encountered on incubation with tBHP was lower (32% of total glutathione after 30 s) and the original ratio of the levels of GSH to GSSG was essentially reestablished within 10 min. In the presence of 3 mM mercaptosuccinate oxidation of glutathione was almost completely inhibited. These results demonstrate that an exogenous hydroperoxide is detoxified rapidly by astroglial cells via the glutathione system.