Phyllis G. Paterson^(1)
(1)University of Saskatchewan - Saskatoon. Canada

CENTRAL ROLE OF GLUTATHIONE IN STROKE

The cascade of events responsible for the death of neural cells following a stroke include depletion of ATP, glutamate excitotoxicity, calcium overload, and production of strong oxidants that can overwhelm antioxidant defense. These events have been extensively reviewed ^(1-4). A variety of mechanisms contribute to the increase in reactive species including mitochondrial leakage, increased intracellular calcium, increased xanthine oxidase activity, altered arachidonic acid metabolism and activated neutrophils, catecholamine oxidation, and the development of lactacidosis ^(4-9). Restoration of blood flow to the ischemic tissue amplifies the production of free radicals ⁽⁹⁾. While the increased superoxide anion produced can adversely affect cell function, more importantly it can interact to produce powerful oxidizing agents such as the hydroxyl radical. The superoxide dismutase family can dismutate the superoxide anion into hydrogen peroxide, but the latter must be removed or it can be reduced to the hydroxyl radical by reduced transition metals such as ferrous iron. The hydroxyl radical is damaging to cells by causing DNA strand breaks, protein oxidation, and lipid peroxidation. The latter is particularly damaging since once initiated, this starts a chain of peroxidations, ultimately resulting in the formation of lipid peroxyl radicals and lipid hydroperoxides that disturb cell membrane function. The lipid hydroperoxides in the presence of iron can be converted to alkoxy and peroxy radicals resulting in new chains of lipid peroxidation ^(4-10). An upregulation of pro-inflammatory genes occurs as a result of the oxidative insult with upregulation of cell adhesion molecules on the endothelium and leukocyte infiltration, resulting in an inflammatory state that also makes an important contribution to cell damage ^(11,12).

Glutathione (GSH) has a central role within the finely tuned network of antioxidant systems that can respond to the oxidative insult. The key importance of GSH and glutathione peroxidase to efficient peroxide scavenging in neural cells has been reviewed ^(3,6,13). This is carried out by enzymes that dismutate hydrogen peroxide into water and molecular oxygen. Catalase can remove only hydrogen peroxide, and is effective only at high concentrations of this peroxide. Of more importance is the family of glutathione peroxidases that remove not only hydrogen peroxide but also lipid peroxides ^(8,10,14). Glutathione peroxidase activity is dependent upon the presence of GSH which is oxidized in the process. As the efficiency of glutathione peroxidase for scavenging peroxides increases as a function of GSH concentration, small changes in GSH can have a large influence on the ability of the cell to scavenge peroxides ⁽⁷⁾. GSH is also used by glutathione-S-transferases to detoxify the aldehyde breakdown products of lipid hydroperoxides ⁽¹⁵⁾. GSH functions to regenerate vitamin E that is important for scavenging lipid peroxyl radicals in membranes; GSH reduces oxidized ascorbate, which directly reduces the alpha-tocopherol radical ^{(5,7,8,10,14)}. GSH exerts direct antioxidant effects and influences other cell processes important in determining the extent of cell injury -reviewed in ^(1)(16)-. These include prevention of the formation of advanced glycation products and inhibition of the transcription factor, NFkB that is required for the expression of pro-inflammatory genes.

MODULATION OF BRAIN GLUTATHIONE IN STROKE

Brain GSH under conditions of cerebral oxidative insult is determined by the balance among its utilization, de novo synthesis, and reduction of oxidized-glutathione by glutathione reductase. Severe oxidative stress depletes cellular GSH ^(17,18). Brain GSH is decreased during ischemia and reperfusion in some rat models of stroke ^(19-23). In some cases, an increase in oxidized glutathione accounts for only a small portion of depleted GSH (<1%), and the rest can be recovered as protein-GSH mixed disulfide with accompanying loss of protein thiols ⁽²⁰⁾. Active transport of GSSG out of the cell is an additional mechanism to protect the cell from a shift in redox equilibrium ⁽¹⁸⁾.

In the synthesis of GSH (L-gamma-glutamyl-L-cysteinylglycine), gamma-glutamylcysteine is formed in the initial rate-limiting step catalyzed by gamma-glutamylcysteine synthetase. This step is important for regulating maximum tissue GSH concentration in vivo. GSH synthetase then catalyzes the reaction between glycine and gamma-glutamylcysteine to form GSH ^(24-26). As plasma cysteine concentrations are low, additional sources are supplied by reduction of cystine and synthesis from methionine via the transsulfuration pathway in liver. In many extrahepatic tissues, the amino acid substrates for GSH synthesis are also provided by efflux of hepatic GSH into plasma and the uptake of plasma GSH via gamma-glutamyltranspeptidase located on the external surface of cell membranes ^(27,28); this reaction also provides a salvage pathway for GSH synthesis ⁽²⁷⁾. Some uptake of intact GSH by a Na-dependent GSH uptake system may also occur in tissues such as brain capillaries ⁽²⁹⁾. While intraorgan and interorgan cycles of GSH are well described for liver and kidney, information about their relative importance in brain is quite limited ⁽³⁰⁾.

A second major determinant of the rate of GSH synthesis is the cellular level of the limiting substrate, cysteine. Previous studies have documented the depressing effects of fasting, low-protein diets, or diets limiting in sulfur amino acids on the GSH concentration of nonneural tissues ^(31-33). Our laboratory has therefore used a nutritional approach to study the effects of GSH depletion in a rat model of stroke. A previous study ⁽³⁴⁾ showed that an acute, severe deficiency of sulfur amino acids used as a model of reduced cysteine supply causes a dramatic reduction in the GSH and cysteine concentrations of liver, an important site of GSH synthesis. This was accompanied by a decline of 10-14% in the GSH concentration of a number of brain regions; this was statistically significant in the neocortex and thalamus, but the other regions showed a similar trend. We also measured protein carbonyl content of the tissues by a previously published method ⁽³⁵⁾ as a marker of oxidative modification of proteins. Brain and liver protein carbonyls tended to be increased in the deficient group, suggesting an increase in oxidative stress. The lack of statistical significance is possibly related to the small sample size in this initial experiment because the trends were very strong.

We hypothesized that this decline in brain GSH, although modest compared to liver, would be critical when the brain was subjected to oxidative stress. Our second study ⁽³⁶⁾ investigated whether acute sulfur amino acid deficiency exacerbates brain damage in a previously described rat model of global hemispheric hypoxic ischemia ⁽³⁷⁾. After feeding adult Long-Evans rats either the sulfur amino acid - deficient or a control diet for 3 days, the right common carotid artery was severed followed by 35 minutes of hypoxia in 12% oxygen. The rats were continued on the same diets for 3 days following the insult, at which time the deficient group was returned to the control diet. At 7 days following the insult, brains were perfusion-fixed, embedded, sectioned at coordinates of ~0.4 mm and ~3.1 mm from bregma, and stained with hematoxylin and eosin. Neural damage to the neocortex, hippocampus, striatum, and thalamus was assessed in the hemisphere ipsilateral to the ligated artery in the anterior and posterior sections. The H&E-stained sections were scored for damage using a semi-quantitative approach ⁽³⁸⁾. Adjacent sections were stained with a mouse monoclonal anti-microtubule-associated protein 2 (MAP2) IgG, using the avidin-biotin method of immunocytochemistry with 3,3´-diaminobenzidine tetrachloride as the chromagen ⁽³⁹⁾. MAP2 provides information on dendritic function as it is located in the dendritic processes of neurons, and its absence has been used as an early marker of ischemic damage ^(40,41). The extent of total neural damage was dramatically greater in the sulfur amino acid-deficient group. The accompanying figure, which focuses on the hippocampus, illustrates the range of severity of neural damage (Fig 1).

These findings are supported by a previous report that GSH depletion by buthionine sulfoximine, an inhibitor of gamma-glutamylcysteine synthetase, exacerbated ischemic injury in a rat focal cerebral ischemia model ⁽⁴²⁾. A number of strategies have also been developed to enhance cellular GSH ⁽⁴³⁾. Gotoh et al. ⁽⁴⁴⁾ and Noguchi et al. ⁽²³⁾ reported that administering a GSH ester immediately after an ischemic insult increases brain GSH and offers neuroprotection. An alternative approach is to administer GSH delivery agents such as N-acetylcysteine (NAC), the antidote for acetaminophen overdose ⁽⁴⁵⁾, which promote GSH synthesis by acting as a cysteine precursor ⁽⁴³⁾. N-acetylcysteine enhanced hippocampal neuronal survival in a transient forebrain ischemia model in the rat ⁽⁴⁶⁾. Another compound of current interest is alpha-lipoic acid, long known for its role in oxidative metabolism, and its corresponding dithiol, dihydrolipoate (reviewed in (47, 48)). Among other antioxidant effects, these compounds increase intracellular GSH ^(49-51). Since cystine uptake is a limiting factor in GSH synthesis, the alpha-lipoic acid-mediated increase in intracellular GSH is proposed to be the result of reduction to dihydrolipoate and its release, followed by the extracellular reduction of cystine by dihydrolipoate, and uptake of cysteine by the ASC transporter more efficiently than cystine can be taken up by the xc^- system ⁽⁵¹⁾. Thus the mechanism would be to increase intracellular availability of cysteine for GSH synthesis by bypassing the glutamate-sensitive xc^- transport system. This mechanism may also explain how alpha-lipoic acid protects against glutamate-induced cytotoxicity ^(52,53), since alpha-lipoic acid could circumvent the glutamate-induced downregulation of cellular GSH which occurs because glutamate is a competitive inhibitor of the xc^- transporter ^(50,51). Alpha-lipoic acid was protective in some but not all studies of stroke in animal models, and discrepancies may be related to differences in route of administration, enantiomeric form, or conversion to the dithiol form. Panigrahi et al. ⁽⁵⁴⁾ reported dramatic results by administering alpha-lipoic acid intravenously in the rat model of bilateral carotid artery occlusion combined with hypotension and reperfusion. GSH losses in cortex, striatum, and hippocampus were 50-57%, and alpha-lipoic acid returned these to about 90% of normal; mortality was reduced from 79% to 25%. Alpha-lipoic acid given intravenously crosses the blood-brain barrier in rats not exposed to ischemia ⁽⁵⁴⁾. Prehn et al. ⁽⁵⁵⁾ earlier showed in middle cerebral artery occlusion in mice and rats that dihydrolipoate (but not alpha-lipoic acid) given intraperitoneally reduced the size of the infarct. Wolz and Krieglstein ⁽⁵⁶⁾ reported in mouse and rat models of focal ischemia that alpha-lipoic acid was protective when given subcutaneously but not when administered intraperitoneally, suggesting that liver alpha-lipoic acid metabolism can keep brain levels low. Unfortunately, these compounds have only been administered prior to induction of the ischemia.

CONCLUSIONS

These studies suggest that the maintenance of brain GSH is an important determinant of the extent of secondary tissue damage in stroke. Strategies to enhance GSH in brain should be tested for their therapeutic efficacy in the human condition of stroke. Our studies of depletion of brain GSH by acute sulfur amino acid deficiency may have practical significance. Ongoing studies will determine whether more clinically relevant states such as fasting and chronic protein-energy malnutrition have similar effects that can be reversed by the administration of GSH delivery agents. Protein-energy depletion, which is known to decrease GSH in other tissues ^(31-33), has been documented in 16% or more of elderly patients at the time of admission for acute stroke -reviewed in ⁽⁵⁷⁾-.

ACKNOWLEDGEMENTS

I thank the Heart and Stroke Foundation of Saskatchewan for funding my research.

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Discussion Board

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[ABSTRACT] [CENTRAL ROLE OF GLUTATHIONE IN STROKE] [MODULATION OF BRAIN GLUTATHIONE IN STROKE] [IMAGES] [CONCLUSIONS] [ACKNOWLEDGEMENTS] [BIBLIOGRAPHY] [Discussion Board]

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Phyllis G. Paterson
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Last update: 13/01/00