O gastrópode terrestre Helix aspersa (Müller) é um herbívoro generalista, que habita a região mediterrânea. Os gastrópodes terrestres em geral entram em estados dormentes durante o seu ciclo de vida. A dormência é uma forma de inatividade associada a uma redução na taxa metabólica, sem grandes alterações no estado hídrico do animal (Withers & Cooper, 2010). Os gastrópodes terrestres quando saem de um estado dormente podem apresentar um aumento na produção de espécies reativas de oxigênio (ROS) nas mitocôndrias (Turrens et al., 1982) levando a um quadro de possível estresse oxidativo (Hermes-Lima & Zenteno-Savin, 2002). Cerca de 0,1% a 2% da respiração normal celular in vitro resulta em formação de ânion superóxido (Fridovich, 2004; Murphy, 2009; Hamanaka & Chandel, 2010). Muitos estudos apontam para um aumento na produção de ROS (Duranteau et al., 1998; Chandel et al., 1998; Wood et al., 1999; Killilea et al., 2000) durante a hipóxia. O estresse oxidativo é definido como o desequilíbrio no balanço entre agentes pró-oxidantes e agentes antioxidantes, em favor dos pró-oxidantes, levando a uma perturbação na sinalização e no controle redox e/ou dano molecular (Sies & Jones, 2007). A GSH é o principal grupo sulfidrila não proteico encontrado em células de mamíferos. Esta normalmente em uma concentração de 1 a 10 mM, enquanto a GSSG é encontrada em uma concentração de 10 a 100 vezes menor (Rossi et al., 1995; Griffith, 1999). A GSH atua desativando radicais livres, preservando o status redox celular e defendendo o organismo contra xenobióticos (Meister, 1995a). A ativação do sistema de defesa antioxidante, incluindo aumento da atividade de enzimas antioxidantes, durante situações de depressão metabólica foi chamada de preparo para o estresse oxidativo (Hermes-Lima et al., 1998). Esta ativação protege o organismo durante o hipometabolismo e durante a reoxigenação/despertar de um possível estresse oxidativo. Os objetivos deste estudo foram: analisar as possíveis respostas durante um ciclo de anoxia e reoxigenação do sistema de defesa antioxidante de Helix aspersa com níveis reduzidos de glutationa total (eq-GSH); e examinar a liberação de ROS em mitocôndrias isoladas de Helix aspersa em estivação. O metabolismo de GSH mostrou-se em nosso estudo como importante fator na manutenção do equilíbrio redox de Helix aspersa durante a anoxia e reoxigenação, lidando com um provável aumento de produção de ROS durante a reoxigenação. E durante a estivação, foi demonstrado que as mitocôndrias de glândula digestiva de Helix aspersa liberam mais H2O2 in vitro. Este aumento na liberação de ROS na mitocôndria pode estar relacionado com a indução de respostas antioxidantes, que ocorrem durante a estivação em gastrópodes terrestres em diversos estudos (Hermes-Lima & Storey, 1995; Ramos-Vasconcelos & Hermes-Lima, 2003; Ramos-Vasconcelos et al., 2005) / The gastropod Helix aspersa (Müller) is a generalist herbivore that inhabits the Mediterranean region. The terrestrial gastropods generally go into dormant states during their life cycle. Dormancy is a form of inactivity associated with a reduction in metabolic rate, without major changes in the water status of the animal (Withers & Cooper, 2010). The terrestrial gastropods when they leave a dormant state may experience an increased production of reactive oxygen species (ROS) in mitochondria (Turrens et al., 1982) leading to a potential oxidative stress (Hermes-Lima & Zenteno-Savin, 2002). About 0.1% to 2% of the normal cellular respiration in vitro results in formation of superoxide anion (Fridovich, 2004; Murphy, 2009; Hamanaka & Chandel, 2010). Many studies point to an increased production of ROS (Duranteau et al. 1998; Chandel et al., 1998, Wood et al. 1999; Killilea et al., 2000) during hypoxia. Oxidative stress is defined as the imbalance between pro-oxidant agents and antioxidants in favor of pro-oxidants, leading to a disruption of redox signaling and redox control and/or molecular damages (Sies & Jones, 2007). GSH is the main non-protein sulfhydryl group found in mammalian cells. It´s usually in a concentration of 1 to 10 mM, whereas GSSG is found at a concentration of 10 to 100 times lower (Rossi et al. 1995; Griffith, 1999). GSH acts by disabling free radicals, maintaining the cellular redox status and defending the body against xenobiotics (Meister, 1995a). The activation of the antioxidant defense system, including increased activity of antioxidant enzymes, during situations of metabolic depression is called \"preparation for oxidative stress (Hermes-Lima et al., 1998). This activation protects the body during hypometabolism and during recovery of a possible situation of oxidative stress. The objectives of this study were: to analyze the possible response during a cycle of anoxia and reoxygenation of the antioxidant defense system of Helix aspersa with reduced levels of total glutathione (GSH-eq) and to examine the release of ROS in isolated mitochondria from Helix aspersa in aestivation. The metabolism of GSH presented itself in our study as an important factor in maintaining the redox balance of Helix aspersa during anoxia and reoxygenation, dealing with a probable increase in ROS production during reoxygenation. And during aestivation, it was demonstrated that the digestive gland mitochondria of Helix aspersa released more H2O2 in vitro. This increased release of ROS in mitochondria may be related to induction of antioxidant responses that occur during aestivation in terrestrial gastropods in several studies (Hermes-Lima & Storey, 1995; Ramos-Vasconcelos & Hermes-Lima, 2003, Ramos- Vasconcelos et al., 2005)

Salinity stress is one of the main challenges for crop growth and production. The estimated loss of crop yield due to salinity stress is up to 20% worldwide each year. Plants have evolved an array of mechanisms to defend themselves against salinity stress. A key aspect of plant responses to salinity stress is the engagement of a nitrosative burst that results in nitric oxide (NO) accumulation. A major mechanism for the transfer of NO bioactivity is S-nitrosylation which is a modification of the reactive thiol group of a rare but highly active cysteine residue within a protein through the addition of a NO moiety to generate an S-nitrosothiol (SNO). S-nitrosylation can result in altered structure, function and cellular localisation of a protein. Our findings suggest that S-nitrosylation is a key regulator of plant responses to salinity stress. Glutathione (GSH), a tripeptide cellular antioxidant, is S-nitrosylated to form S-nitrosoglutathione (GSNO), which functions as a stable store of NO bioactivity. Cellular GSNO levels are directly controlled by S-nitrosoglutathione reductase (GSNOR), thereby, regulating global SNO levels indirectly. The absence of this gene results in high levels of SNOs. In Arabidopsis, previous research has shown that loss-of-function mutation in GSNOR1 results in pathogen susceptibility (Feechan et al., 2005). In our study, we investigated salt tolerance in gsnor1-3 plants. We have found that this line is salt sensitive at various stages of their life cycle. Interestingly, classical salt stress signalling pathways are fully functional in gsnor1-3 plants. We have also explored non-classical pathways involved in salt tolerance. Autophagy is a cellular catabolic process which is involved in the recycling and degradation of unwanted cellular materials under stressed and non-stressed conditions. We have demonstrated that gsnor1-3 plants have impaired autophagy during salt stress. An accumulation of the autophagy marker NBR1 supports the lack of autophagosome formation. We hypothesised that S-nitrosylation might regulate upstream nodes of autophagosome formation. Our study demonstrated that at least one key player involved in autophagosome biogenesis is regulated by S-nitrosylation. ATG7, an E1-like activating enzyme, which regulates ATG8-PE and ATG12-ATG5 ubiquitin like conjugation systems, is S-nitrosylated in vitro and in vivo. S-nitrosylation of ATG7 impairs its function in vitro. We showed that S-nitrosylation of ATG7 is mediated by GSNO. Interestingly, ATG7 is also transnitrosylated by thioredoxin (TRX), another important redox regulatory enzyme. We suggest that similar mechanisms might exist in planta. Finally, work in this study revealed that S-nitrosylation of Cys558 and Cys637 cause the inhibition of ATG7 function. In aggregate, this study revealed a novel mechanism for the redox-based regulation of autophagy during salt stress.

Several lines of evidence implicate oxidative stress in the pathophysiology of bipolar disorder (BPD). The mood stabilizers lithium and valproate have been shown to protect against oxidative stress-induced cell death. This study examined whether an increase in cellular reductive potential due to glutathione (GSH) synthesis up-regulation underlies this neuroprotective effect. Using primary rat cortical neurons as a model, this study demonstrated that unlike lithium and valproate, carbamazepine and lamotrigine do not exert neuroprotective effects against H2O2-induced cell death. Moreover, the level of GSH and the GSH:GSSG ratio in neurons and in rat brain remained unchanged following chronic treatment with either lithium or valproate. Similarly, this study did not find a significant effect of treatment on the expression of genes encoding γ-glutamylcysteine ligase sub-units, Gclc and Gclm, in both neurons and the rat brain. These findings suggest that other molecular targets of lithium and valproate likely mediate the observed neuroprotective effects.

Several lines of evidence implicate oxidative stress in the pathophysiology of bipolar disorder (BPD). The mood stabilizers lithium and valproate have been shown to protect against oxidative stress-induced cell death. This study examined whether an increase in cellular reductive potential due to glutathione (GSH) synthesis up-regulation underlies this neuroprotective effect. Using primary rat cortical neurons as a model, this study demonstrated that unlike lithium and valproate, carbamazepine and lamotrigine do not exert neuroprotective effects against H2O2-induced cell death. Moreover, the level of GSH and the GSH:GSSG ratio in neurons and in rat brain remained unchanged following chronic treatment with either lithium or valproate. Similarly, this study did not find a significant effect of treatment on the expression of genes encoding γ-glutamylcysteine ligase sub-units, Gclc and Gclm, in both neurons and the rat brain. These findings suggest that other molecular targets of lithium and valproate likely mediate the observed neuroprotective effects.

Primary brain injury in stroke is followed by oxidative stress and further neural damage. Glutathione (GSH) is critical in antioxidant defense. Since cysteine is limiting in GSH synthesis, Phase 1 of this study investigated the effect of a dietary sulphur amino acid deficiency (-SAA) on neural damage in global hemispheric hypoxia-ischemia (GHHI). Rats were fed a -SAA or control diet for 6 days, and subjected to GHHI after 3 days. Histologically evaluated neural damage at 7 days post hypoxia-ischemia was greater in -SAA rats. Brain GSH concentration was decreased in -SAA rats 3 days after ischemia. A cysteine precursor, L-2-oxothiazolidine-4-carboxylic acid (OTC) administered to -SAA rats did not ameliorate neural damage. GSH is decreased by protein-energy malnutrition (PEM) in some tissues. Phase 2 investigated the effect of PEM on brain oxidative stress, neural damage and behaviour after global ischemia in adult male gerbils. In a 2x2 factorial design, gerbils were fed an adequate protein (12%; C) or low protein (2%; PEM) diet for 4 weeks, then subjected to transient ischemia (I) or sham surgery (S). After 12 hours of reperfusion, brain from half the gerbils was collected for biochemical analyses. Remaining gerbils were fed pre-surgery diets for 10 more days. To assess functional consequences of ischemia, gerbils were placed in an open field on Days 3, 7 and 10 after surgery. On Day 10, viable hippocampal CA1 neurons were counted. C-I gerbils did not habituate as readily in the open field on day 3 as C-S, but normalized by day 7. PEM-I gerbils failed to habituate by day 10, traveled greater distance than other gerbils and 7 of 12 displayed thigmotaxis, a <i>wall-hugging</i> preference for the outer perimeter of the open field. CA1 neuron loss in I was 61.5% of S, but unaffected by PEM. Four of 12 PEM-I gerbils had marked increases in hippocampal glia. Hippocampus protein thiols were reduced by PEM and by ischemia, consistent with oxidative stress. GSH concentration, glutathione reductase activity and thiobarbituric acid reactive substances were not significantly affected by PEM or ischemia. Findings from these two studies suggest well-nourished but not nutritionally-deficient rodents tolerate a mild brain insult. This is clinically relevant because many elderly stroke victims suffer from PEM at the time of ischemia, which may compromise recovery.

Primary brain injury in stroke is followed by oxidative stress and further neural damage. Glutathione (GSH) is critical in antioxidant defense. Since cysteine is limiting in GSH synthesis, Phase 1 of this study investigated the effect of a dietary sulphur amino acid deficiency (-SAA) on neural damage in global hemispheric hypoxia-ischemia (GHHI). Rats were fed a -SAA or control diet for 6 days, and subjected to GHHI after 3 days. Histologically evaluated neural damage at 7 days post hypoxia-ischemia was greater in -SAA rats. Brain GSH concentration was decreased in -SAA rats 3 days after ischemia. A cysteine precursor, L-2-oxothiazolidine-4-carboxylic acid (OTC) administered to -SAA rats did not ameliorate neural damage. GSH is decreased by protein-energy malnutrition (PEM) in some tissues. Phase 2 investigated the effect of PEM on brain oxidative stress, neural damage and behaviour after global ischemia in adult male gerbils. In a 2x2 factorial design, gerbils were fed an adequate protein (12%; C) or low protein (2%; PEM) diet for 4 weeks, then subjected to transient ischemia (I) or sham surgery (S). After 12 hours of reperfusion, brain from half the gerbils was collected for biochemical analyses. Remaining gerbils were fed pre-surgery diets for 10 more days. To assess functional consequences of ischemia, gerbils were placed in an open field on Days 3, 7 and 10 after surgery. On Day 10, viable hippocampal CA1 neurons were counted. C-I gerbils did not habituate as readily in the open field on day 3 as C-S, but normalized by day 7. PEM-I gerbils failed to habituate by day 10, traveled greater distance than other gerbils and 7 of 12 displayed thigmotaxis, a <i>wall-hugging</i> preference for the outer perimeter of the open field. CA1 neuron loss in I was 61.5% of S, but unaffected by PEM. Four of 12 PEM-I gerbils had marked increases in hippocampal glia. Hippocampus protein thiols were reduced by PEM and by ischemia, consistent with oxidative stress. GSH concentration, glutathione reductase activity and thiobarbituric acid reactive substances were not significantly affected by PEM or ischemia. Findings from these two studies suggest well-nourished but not nutritionally-deficient rodents tolerate a mild brain insult. This is clinically relevant because many elderly stroke victims suffer from PEM at the time of ischemia, which may compromise recovery.

The glyoxalase pathway is widespread in both prokaryotic and eukaryotic organisms. This system utilizes two enzymes (glyoxalase I (GlxI) and glyoxalase II (GlxII)) to catalyze the formation of D-lactate from the substrates glutathione (GSH) and methylglyoxal (MG). The latter chemical is a harmful byproduct of glycolysis. This thesis gives detailed studies of the behavior of the GlxI enzyme as it pertains to its thiol co-substrate specificity, its structural similarity among its superfamily members (most particularly with the fosfomycin resistance protein (FosA)) and residue identification that would alter its metal selectivity. The thiol co-substrate GSH was thought to be the only thiol utilizied by the glyoxalase system. However, reports identified organisms that utilized the thiols trypanothione (T(SH)2) and glutahionylspermidine (GspdSH) as co-substrates. These organisms, known as the trypanosomes, are very well known in tropical environments to cause diseases. E. coli does not contain T(SH)2 but does contain GspdSH and manufactures the latter in increasing amounts under conditions of cell duress. Substrate specificity studies were conducted replacing GSH with GspdSH and T(SH)2. In addition to this, to ensure the thiols reacted in a true glyoxalase system, substrate specificity studies were also conducted on the second enzyme GlxII and verification of the product D-lactate was performed. To continue, structurally, the enzyme GlxI belongs to the βαβββ superfamily of proteins that are known to have very similar structure but to catalyze very different reactions. Comparing the active site of E. coli GlxI and FosA, there is one significant difference at one residue. Therefore an E56A mutation was performed on GlxI and the mutant bacterium were subjected to growth analysis in the presence of fosfomycin and MG. The mutant enzyme was also tested for its performance in the presence of MG and various divalent metals. Further, the Glx I enzyme from E. coli is known to be active in the presence of non-zinc bivalent metals, while the human counterpart is active in the presence of Zn2+. When one compares GlxI from E. coli with the human GlxI, there are many differences in the primary structure that could be viable areas that determine the metal specificity of the enzyme. Mutation analysis was performed on these areas to determine catalytic performance as well as metal specificity. These studies display how versatile the glyoxalase system is with regard to the use of its thiol co-substrates. These thiols participate in the detoxification pathway for MG in the cell especially under late log phase conditions. Structural studies can give some knowledge concerning the possible evolution of the enzyme among its family members, and is of monumental significance to the scientific community as it relates to enzyme metal selectivity and the development of enzymes over time.

The glyoxalase pathway is widespread in both prokaryotic and eukaryotic organisms. This system utilizes two enzymes (glyoxalase I (GlxI) and glyoxalase II (GlxII)) to catalyze the formation of D-lactate from the substrates glutathione (GSH) and methylglyoxal (MG). The latter chemical is a harmful byproduct of glycolysis. This thesis gives detailed studies of the behavior of the GlxI enzyme as it pertains to its thiol co-substrate specificity, its structural similarity among its superfamily members (most particularly with the fosfomycin resistance protein (FosA)) and residue identification that would alter its metal selectivity. The thiol co-substrate GSH was thought to be the only thiol utilizied by the glyoxalase system. However, reports identified organisms that utilized the thiols trypanothione (T(SH)2) and glutahionylspermidine (GspdSH) as co-substrates. These organisms, known as the trypanosomes, are very well known in tropical environments to cause diseases. E. coli does not contain T(SH)2 but does contain GspdSH and manufactures the latter in increasing amounts under conditions of cell duress. Substrate specificity studies were conducted replacing GSH with GspdSH and T(SH)2. In addition to this, to ensure the thiols reacted in a true glyoxalase system, substrate specificity studies were also conducted on the second enzyme GlxII and verification of the product D-lactate was performed. To continue, structurally, the enzyme GlxI belongs to the βαβββ superfamily of proteins that are known to have very similar structure but to catalyze very different reactions. Comparing the active site of E. coli GlxI and FosA, there is one significant difference at one residue. Therefore an E56A mutation was performed on GlxI and the mutant bacterium were subjected to growth analysis in the presence of fosfomycin and MG. The mutant enzyme was also tested for its performance in the presence of MG and various divalent metals. Further, the Glx I enzyme from E. coli is known to be active in the presence of non-zinc bivalent metals, while the human counterpart is active in the presence of Zn2+. When one compares GlxI from E. coli with the human GlxI, there are many differences in the primary structure that could be viable areas that determine the metal specificity of the enzyme. Mutation analysis was performed on these areas to determine catalytic performance as well as metal specificity. These studies display how versatile the glyoxalase system is with regard to the use of its thiol co-substrates. These thiols participate in the detoxification pathway for MG in the cell especially under late log phase conditions. Structural studies can give some knowledge concerning the possible evolution of the enzyme among its family members, and is of monumental significance to the scientific community as it relates to enzyme metal selectivity and the development of enzymes over time.

Glutathione (GSH), a 3-amino acid compound is ubiquitously expressed in eukaryotic cells and is the most abundant low molecular weight thiol. The importance of GSH is highlighted by its multitude of effects. Within the vascular wall GSH plays a crucial role as an intracellular antioxidant and it possess the ability to act as a signalling intermediate and store for nitric oxide (NO). The importance of NO and its role in vascular wall homeostasis is well recognized. Within the coronary circulation, NO is the primary dilator of many of the large arteries and the smaller arterioles. In addition to controlling coronary vascular tone, the importance of NO is highlighted by its antithrombotic, antihypertrophic, and antriproliferative effects. During instances of cardiovascular disease and normal aging, increases in the production of reactive oxygen species occur. A portion of the deleterious vascular effects of reactive oxygen species are believed to be due to reduction in NO bioavailability as a result of increased ROS-mediated destruction of NO. Altered GSH production in humans has been demonstrated to reduce endothelial function. Conversely, supplementation with GSH augments endothelium-dependent dilation. The mechanisms by which these alterations in GSH influence vasomotor function have not been resolved. The purpose of the studies within this thesis was to examine the impact of chronic and acute GSH modulations on coronary vascular resistance (CVR) and endothelium dependent dilation. In all experiments vascular reactivity was assessed in the isolated perfused rat heart. The advantage of this technique is that it allows the global coronary vasomotor functioning to be examined. Hearts were allowed to stabilize for 30 minutes to allow for the development of spontaneous coronary vascular resistance, followed by a bradykinin (BK) dose-response curve to assess endothelium-dependent dilation. The coronary circulation was then maximally dilated using an endothelium-independent agonist. In all cases BK-mediated dilation is expressed as a percentage of the endothelium-independent dilation. Chapter 2 of this document examines the chronic nature of GSH depletion and examines whether GSH depletion augments the influence of natural aging. Animals (mean age 33 and 65 weeks) were randomized to receive L-Buthionine-(S,R)-sulphoximine (BSO) in the tap water in order to inhibit GSH synthesis, or regular tap water (normal controls). Following 10 days of BSO treatment, ventricular GSH content was reduced in the BSO group compared to the control (0.182±0.021 vs 2.022±0.084 nmol/mg wet weight, p<0.05) and there was increased ventricular H2O2 content (1.345±0.176 vs 0.877±0.123 pmol/µg PRO, p<0.05). Baseline CVR was significantly reduced in the older animals compared to the adult animals (3.92±0.34 vs 4.76±0.20 and 3.67±0.24 vs 5.12±0.37 mmHg/ml×min-1 in the control and BSO treated groups, p<0.05). Conversely, in the presence of LNAME there was a significant increase in CVR in the adult BSO group (14.15±0.99, p<0.05) compared to all other groups. In the absence of LNAME, maximal dilation (percent endothelium-independent response) was reduced in the older animals compared to the adult animals (77±10.3% vs 95.0±1.0% for older and adult control and 92.7±4.5% vs 98.6±0.6% for the older and adult BSO, main effect of age). In the presence of LNAME the adult BSO group had a significantly reduced sensitivity (EC50) compared to all other groups (-7.39±0.09 Log M, p<0.05). Additionally, adult BSO treated animals had an increase in eNOS protein content. These results demonstrate that chronic thiol depletion resulted in an increased reliance on NO in the adult BSO group only. In chapter 3 the beneficial effects of GSH supplementation on BK mediated dilation were examined. Acute GSH was administered in the perfusate at either 0 (control) or with 10 µM for 2 reasons, 1) this concentration does not reduce basal coronary vascular resistance, allowing for a similar baseline CVR across conditions and 2) the 10 µM concentration is a physiologically relevant concentration of plasma/extracellular fluid GSH. The sensitivity to the endothelial agonist bradykinin was enhanced in the presence of GSH (-8.70±0.16 vs -7.94±0.06 LogM, p<0.01). The GSH effect was not dependent on NO production or utilization by soluble guanylate cyclase (sGC) as the enhanced dilation in the GSH group was maintained despite NOS (LNAME) and/or sGC inhibition. When the hearts were supplemented with a ROS scavenger TEMPOL, enhanced dilation was seen in the control group, but was not further enhanced in the GSH group. The requirement for ROS was best demonstrated when both the CON and GSH groups were supplemented with both TEMPOL and LNAME. This condition resulted in similar sensitivity (-7.76±0.19 vs -7.75±0.17 LogM, p>0.05) and area under the curve (182.33±12.70 vs 170±13.86, p>0.05) between GSH and CON. Thus, it was concluded that the effects of GSH administration requires the presence of ROS and exerts its effect in the microvasculature. The study presented in chapter 4 examined the effects of acute thiol modulation (depletion) on CVR and endothelium-dependent dilation. Previous reports have suggested that a reduction in intracellular GSH causes impaired NO production, and functional data support this contention. However, a majority of the data regarding the effects of thiol manipulation are from endothelial-removed vessels. The following agents were used to reduce GSH: the glutathione reductase inhibitor, BCNU; the thiol oxidizing agent, diamide; the thiol conjugating agent, ethacrynic acid (EA); and a thioredoxin inhibitor (CDNB). Preliminary data revealed that only CDNB (11.46±0.71 mmHg/ml×min-1) and EA (8.61±0.36 mmHg/ml×min-1) caused an elevation in CVR compared to the control (6.73±0.24 mmHg/ml×min-1). Conversely, Diamide and BCNU did not significantly affect baseline CVR, or the BK mediated responses. In the presence of EA, there was an overall blunting of the BK-response curve as observed by reduced EC50 (-7.85±0.07 Log M) and maximal dilation (90.8±1.8 %, percent endothelium-independent dilation) compared to the control group (-8.42±0.08 Log M and 97.7±1.6%). In the presence of CDNB the maximal dilation was 74.4±1.9% and the EC50 was -8.83±0.28 Log M. In addition to altering BK mediated responses, acute thiol depletion with all agents resulted in an increased minimal CVR with significant increases observed in the presence of CDNB and EA. There was a significant correlation with GSH:GSSG ratio and baseline (-0.547, p<0.05) and minimal CVR (r=-0.581, p<0.05). This study demonstrates that modulation of the GSH:GSSG ratio using a variety of agents with diverse mechanisms elicits differential responses within the vasculature. Specifically conjugation of GSH and inhibition of thioredoxin significantly alters BK mediated response, where as BCNU and dimaide did not. These results suggest that a modulation in the GSH:GSSG ratio impairs endothelium-dependent dilation and alters total dilatory capacity (baseline-minimal CVR) and thus may have implications for adequate tissue perfusion. Across all studies there was significant correlation between GSH and GSSG with both baseline and minimal CVR. Therefore it is likely that changes in overall glutathione content plays a role in determining baseline and minimal coronary vascular resistance. These results demonstrate the complexity that manipulations of GSH have on both CVR and endothelium-dependent dilation, and provide mechanistic insight into how changes in GSH alter coronary vascular resistance and endothelium-dependent dilation.

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