Palten Knut
Formation of S – Nitrosothiols as intermediates in the bioactivation of nitroglycerin
Diplomarbeit
zur Erlangung des akademischen Grades eines Magisters an der Naturwissenschaftlichen Fakultät der Karl-Franzens-Universität Graz
O.Univ.-Prof. Dr. Bernhard-Michael Mayer Institut für Pharmakologie and Toxikologie
2010
Acknowledgment
This work was supported by grants of the “Fonds zur Föderung der Wissenschaftlichen Forschung” in Austria to B. Mayer.
This thesis is dedicated to my parents Roland and Johanna, who made my studies possible in the first place.
I would like to thank my supervisor O.Univ.-Prof. Dr. Bernhard-Michael Mayer and all members of the department of pharmacology and toxicology, especially the “NO-group”, for their support and for accepting me as a full-fledged team member.
Special thanks are due to Dr. Antonius Gorren who advised me on all my questions and gave me a lot of feedback, Mag. Verena Wenzl who introduced me into all methods and Dr. Matteo Beretta who kindly proofread this work.
Last but not least I want to thank Gwendolyn for helping me with this work and just being part of my life.
II
Table of contents
1. Introduction...... 1
1.1. Nitric Oxide ...... 1 1.1.1. Pharmacology of nitric oxide...... 2 1.1.2. Organic nitrates – exogenous NO...... 4 1.1.3. Biotransformation of organic nitrates by ALDH2...... 4 1.1.4. Nitrate tolerance...... 6 1.1.4.1. Pseudotolerance...... 6 1.1.4.2. Classical tolerance...... 7
1.2. Low Molecular Weight S-Nitrosothiols...... 9 1.2.1. Stability...... 9 1.2.2. Transport...... 10 1.2.3. Detection...... 10 1.2.4. Biological actions...... 10 1.2.4.1. Via NO release ...... 11 1.2.4.2. Via S–transnitrosation...... 11 1.2.4.3. As direct effector...... 12 1.2.5. Therapeutic use ...... 12 1.2.6. Formation of S-nitrosothiols...... 13 1.2.7. Decomposition of S-nitrosothiols ...... 15 1.2.7.1. Homolytic and heterolytic decomposition...... 15 1.2.7.2. Reductive decomposition...... 16 1.2.7.3. Enzymatic decomposition...... 17
2. Methods...... 18
2.1. Materials ...... 18
2.2. Nitroglycerin Metabolism...... 21 2.2.1. Procedure...... 21 2.2.2. Calculation...... 22
2.3. Soluble Guanylate Cyclase Activity...... 23 2.3.1. Procedure...... 23 2.3.2. Calculation...... 24
2.4. NO Release...... 25 2.4.1. Procedure...... 25
3. Results...... 26
3.1. Nitroglycerin Metabolism...... 26 3.1.1. Effect of DTT and GSH on GTN metabolism ...... 26 3.1.2. Effect of the DTT concentration on GTN metabolism in the presence of GSH...... 27
3.2. Soluble Guanylate Cyclase Activity...... 28 3.2.1. Effect of DTT and GSH on sGC activity (50 ng sGC)...... 28 3.2.2. Effect of DTT and GSH on sGC activity (25 ng sGC)...... 29 3.2.3. Effect on sGC activition of substituting NAD+ by NADH ...... 30 3.2.4. Effect of the DEA/NO concentration on sGC activation ...... 31 3.2.5. Effect of the GSH concentration on the activation of sGC by GTN/ALDH-2 ...... 32 3.2.6. Effect of the ALDH2 concentration on the activation of sGC by GTN...... 33
III
3.2.7. Effect of DTT on the inhibition by GSH of GTN/ALDH2-mediated sGC activition ...... 34
3.3. NO Release...... 35 3.3.1. Effect of DTT and GSH on ALDH2-mediated NO release by GTN ...... 35 3.3.2. Effect of DTT and GSH on NO release by DEA/NO...... 37 3.3.3. Effect of GSH on NO release by DEA/NO in the absence of oxygen ...... 40 3.3.4. Effect of GSH on NO release by DEA/NO at varying concentrations...... 41 3.3.5. Effect of various cations on formation and decomposition of GSNO...... 44
4. Discussion...... 46
4.1. Effect of DTT and GSH on GTN metabolism...... 46
4.2. Effect of DTT and GSH on sGC activity...... 46
4.3. Effect of DTT and GSH on ALDH2-mediated NO release by GTN...... 46
4.4. Effect of DTT and GSH on NO release by DEA/NO ...... 47 4.4.1. Comparison of the reactions of NO with GSH and DTT...... 48 + 2+ 4.4.2. Effects of NAD , SOD, O2 and Mg on GSH nitrosation by NO ...... 49
5. Conclusions...... 50
6. References...... 51
7. List of Figures...... 63
8. List of Tables ...... 64
9. Abstract...... 65
10. Abbreviations...... 66
IV INTRODUCTION
1. Introduction For over a century organic nitrates, especially the most prominent representative nitroglycerin (GTN), have been used for the symptomatic therapy of coronary heart diseases angina pectoris, myocardial infarction, and heart failure and still remain a mainstay of therapy (2). The beneficial effect of these NO releasing pro-drugs, a dilatation of large coronary vessels and thus increased coronary blood supply, is limited by reduction of efficacy during long-term application. A complete loss of anti-anginous effects can be observed after 24 – 48h of continuous application (30). This so-called “nitrate tolerance” necessitates an increase of dosage or a 6 – 8h interval of non-application and is therefore of great clinical relevance. Since the mitochondrial aldehyde dehydrogenase (ALDH2) has been shown to be responsible for the bioactivation of GTN (1), an ALDH2 dependent mechanism has been discussed to cause nitrate tolerance. S-Nitrosothiols (RSNO), also known as thionitrites, are reaction products of nitric oxide (NO) or its metabolites with sulfhydryl groups of proteins (31, 32), glutathione (GSH) or cysteine (Cys). S-Nitrosothiols have been shown to occur in biological systems (10,13) and to cause physiological responses like vasodilatation or inhibition of platelet aggregation, which are strongly reminiscent of NO. Therefore these compounds have been suggested by Ignarro et al. to act as intermediates in the activation of soluble guanylate cyclase (sGC) by GTN (2,33).
1.1. Nitric Oxide In the year 1980 Furchgott et al. made an important discovery: Acetylcholine (ACh) was renowned for its vasorelaxant effects, but if the endothelium was removed from the blood vessels, ACh caused a vasoconstriction instead of a vasorelaxation (34). Thus, an endothelium-derived intermediate was postulated, which was simply called “endothelium derived relaxing factor” (EDRF). Based on the similarities of EDRF to NO, it was suggested in 1986 by Furchgott et al. that they might be identical (34,35). This suggestion was supported by various studies (36,37). Nitric oxide (NO) is one of the smallest known biologically active messengers. Although NO is a radical its reactivity with other molecules is surprisingly small. Important reactions of NO are its interaction with oxyhaemoglobin to form methaemoglobin and nitrate as well as with •! ! superoxide (O2 ) to form peroxynitrite (ONOO ), which is a potent nitrating and oxidizing agent in vivo. NO can react with thiols to form S-nitrosothiols in vivo, such as S- nitrosocysteine (CysNO) and S-nitrosoglutathione (GSNO), although the mechanism remains unclear at present (30).
In the presence of oxygen NO• is oxidized to NO2•. The precise mechanism has not been clarified yet and two pathways have been proposed (40): • The first pathway is based on dimerization of NO to dinitrogen dioxide (N2O2)
2 NO• ! N2O2
N2O2 + O2 " N2O4
N2O4 ! 2 NO2• The second pathway involves the formation of an ONOO• radical
NO• + O2 ! ONOO• ONOO• + NO•" ONOONO
ONOONO " 2 NO2•
1 INTRODUCTION
The autoxidation of NO follows second order kinetics with respect to NO and first order with respect to O2 (26). Consequently the reaction proceeds fast in high concentrations, but slowly in low concentrations of NO.
Further reactions of NO2• can yield potent nitrosating species such N2O4 and N2O3, which will be hydrolyzed to nitrite and nitrate or alternatively transform thiol moieties RS-H to nitrosothiols RS-NO (41).
- - + 2 NO2• ! N2O4 N2O4 + H2O " NO2 + NO3 + 2 H - + NO2• + NO• ! N2O3 N2O3 + H2O " 2 NO2 + 2 H - + N2O4 + RS-H " NO3 + RS-NO + H - + N2O3 + RS-H " NO2 + RS-NO + H
Another important reaction is the oxidation of NO by oxyhemoglobin (oxy-Hb), the major sink - for nitric oxide in plasma. The products are nitrate (NO3 ) and methemoglobin (metHb) (105).
- oxyHb + NO• " metHb + NO3
1.1.1. Pharmacology of nitric oxide Today it is well established that NO plays an important role as a second messenger in many biological processes. NO can be formed endogenously or applied exogenously as organic nitrates or other NO donors. Humoral transmitter substances such as Acetylcholine (ACh), Bradykinin (BK), Adenosinetriphosphate (ATP), Endothelin (ET) and Histamine (H) bind Gq coupled receptors on endothelial cells and activate the phospholipase C (PLC). This enzyme catalyzes the hydrolysis of phosphatidylinositol-biphosphate (PIP2) to inositoltriphosphate (IP3) and diacylglycerol (DAG). IP3 binds IP3-receptors on the surface of the sarcoplasmic reticulum (SR), which activates the unselective calcium channel and causes release of calcium into the cytosol. Binding of Ca2+ to calmodulin (CaM) stimulates its association to the endothelial nitric oxide synthase (eNOS) resulting in enzyme activation. Besides the endothelial NOS, inducable and neuronal isoforms exist. Also shear stress activates eNOS through phosphorylation by protein kinase B (PKB). eNOS catalyzes the conversion of L- arginine to L-citrulline and NO (46). NO can diffuse through the membrane of the smooth muscle cell and activate the NO sensitive soluble guanylate cyclase (sGC). sGC is a heterodimeric hemoprotein that is activated by the binding of NO to the heme. This results in a conformational change at the catalytic site, which increases the maximal rate of of formation of cyclic guanosine monophosphate (cGMP) 100 – 200-fold. Finally, the second messenger cGMP activates the protein kinase G (PKG), causing phosphorylation of myosin light chain phosphatase (MLCP), IP3-receptor associated cGMP-kinase substrate (IRAG), potassium channels, vasodilator stimulated posphoprotein (VASP), and other targets (47). As a result the cytosolic calcium concentration decreases (48) and Ca2+ dissociates from CaM, which causes vasorelaxation and inhibition of platelet aggregation and adhesion (34,46,50,51,52,53). These pathways are summarized in Figure 1.
2 INTRODUCTION
The sGC activity depends on its redox status: If the heme is oxidized, the activity decreases (5), and the enzyme becomes insensitive to NO. On the other hand, if the heme is reduced, sGC activity is stimulated strongly by NO (49). The decomposition of the second messenger cGMP to the non-cyclical, inactive guanosine monophosphate (GMP) is catalyzed by phosphodiesterases (PDE) (54). Therefore, selective inhibition of PDE type 5 (PDE-5), resulting in stimulation of NO-mediated vasorelaxation, is used for the treatment of erectile dysfunction or pulmonary hypertension (55).
Figure 1: NO-mediated smooth muscle relaxation. Acetylcholine (ACh), bradykinin (BK), adenosine triphosphate (ATP), endothelin (ET), histamine (H), guanine nucleotide-binding protein q (Gq), phospholipase C (PLC), phosphatidylinositolbiphosphate (PIP2), inositoltriphosphate (IP3), diacylglycerol (DAG), calmodulin (CaM), endothelial nitric oxide synthase (eNOS), nitric oxide (NO), glyceroltrinitrate (GTN), mitochondrial aldehyde-dehydrogenase (ALDH2), glyceroldinitrate (GDN), nitric oxide species (NOx), soluble guanylate cyclase (sGC), cyclic guanosine monophosphate (cGMP), phosphodiesterase (PDE), non-cyclical guanosine monophosphate (GMP), proteine kinase C (PKC), IP3-receptor associated cGMP - kinase substrate (IRAG), myosin light chain phosphatase (MLCP)
3 INTRODUCTION
1.1.2. Organic nitrates – exogenous NO For over 150 years scientists have been studying organic nitrates and their properties. The most prominent representative of these drugs is glyceroltrinitrate (GTN), which is also known as nitroglycerin. GTN was synthesized by the Italian chemist Ascanio Sobrero in order to produce a potent explosive agent. As Sobrero analyzed the new substance with the tip of his tongue, he observed that small amounts of GTN cause strong headaches. Workers in dynamite factories in the late nineteenth century also suffered from the same symptom, when they returned to work after the weekend. This so-called nitrate-headache, is caused by vasodilatation of cerebral blood vessels. After the investigation of the vasorelaxant effects of organic nitrates, Wiliam Murrell was the first to use GTN for the treatment of angina pectoris in the year 1876 (42). However, the mode of action of GTN still remained a mystery for a long time, until in 1977 it was hypothesised that the active principle could be NO (43). Today, drugs acting through release of NO are summarized under the name nitrovasodilators. All organic nitrates are esters of nitric acid and are typical pro-drugs, which means that they are administered in an inactive form and are transformed in vivo to the active metabolite, NO. Therapeutic concentrations of organic nitrates need to be bioactivated by the mitochondrial enzyme ALDH2 (1,2). Although the therapeutic use is limited by nitrate tolerance, organic nitrates are still the drugs of choice for the treatment of coronary heart disease. Furthermore, they are used for the treatment of myocardial infarct, cardiac insufficiency, osteoporosis, anal fissure and as tocolytic agent (45). The structure of GTN is shown in Figure 2. Other nitrovasodilators are isosorbide dinitrate, 5-isosorbide mononitrate, sodium nitroprusside, pentaerithrityltetranitrate and molsidomine.
Figure 2: GTN formula The relaxation induced by GTN results primarily in the dilatation of large venous blood vessels causing a reduction in preload by “venous pooling” and secondarily in a slight dilatation of large arterial blood vessels causing a reduction in afterloa. Both mechanisms reduce myocardial oxygen consumption. In addition, organic nitrates dilate the epicardial coronary arteries especially at fractional stenosis, which increases the oxygen availability (46). The effect of “coronary stealing” does not occur, because the tone of the arterial resistance vessels is sustained. Overall, the imbalance between the heart's oxygen demand and supply can be corrected, rendering these drugs the medication of choice for the treatment of angina pectoris. The exact mechanism of action of organic nitrates is still unclear. In 1977 Katsuki et al. discovered NO as the effective agent (43). In 1978 Murad et al. discovered that, just like NO, nitrovasodilators such as GTN activate sGC and thus convert GTP to cGMP (6,7).
1.1.3. Biotransformation of organic nitrates by ALDH2 In contrast to direct NO donors (for example DEA/NO), nitrates (oxidation number +V) must be bioactivated to NO (oxidation number + II) (34). Various studies reported the transformation of GTN to NO in-vitro and in-vivo (56,66,67), but only at unphysiological high doses of GTN. Elucidation of the mechanism of GTN bioconversion at physiologically significant concentrations is hampered by the difficulty to detect small amounts of NO and NO-related species, which are highly reactive radicals and have short life-times in the range of seconds or less.
4 INTRODUCTION
Needleman et al. observed a correlation between the availability of thiol containing compounds (R-SH) like GSH and Cys and the vasorelaxant effect of GTN (58). Today this phenomenon is known as “non-enzymatic bioactivation” by thiols, especially cysteine (59,30). It was postulated that a depletion of such “R-SH pools” causes nitrate tolerance (58,60). This hypothesis was disproved when it was shown, that GTN-tolerant tissues still contain free thiols (64,72). The first enzyme that was suggested to metabolize GTN to NO was the GSH- dependent glutathione-S-transferase (GST) (57). Other enzymes proposed as GTN bioactivators: are cytochrome reductase (61,62) and xanthine-oxidoreductase (63). In 2002 Chen et al. identified the mitochondrial isoform of aldehyde dehydrogenase (ALDH2) as the key enzyme in GTN bioactivation. For the first time the predominant formation of 1,2- glyceroldinitrate (1,2-GDN) and the bioactivation of GTN at therapeutic doses could be explained. Unselective ALDH2 inhibitors like disulfiram and chloral hydrate blocked 1,2-GDN formation (1,2). Presently, two distinct pathways of GTN biotransformation are discerned: an ALDH2-independent “low-potency” (high Michaelis constant Km) pathway, that metabolizes pharmacological high concentrations (> 1"M) of GTN predominantly to 1,3-GDN and a “high- potency” (low Michaelis constant Km) pathway, that metabolizes physiological low concentrations (< 1"M) of GTN predominantly to 1,2-GDN (2). Within the “low-potency” pathway, cytochrome P450 (Cyp450) metabolizes high doses of GTN in the endoplasmatic reticulum (ER) to NO. Within the “high-potency” pathway, ALDH2 metabolizes GTN in the - mitochondria to nitrite (NO2 ) and NO or NO-related species (NOX) (60,30). ALDH2 is an important enzyme for ethanol metabolism. In addition to the dehydrogenase activity, which depends on the cofactor nicotinamide adenine dinucleotide (NAD+), ALDH2 shows NAD+-independent esterase activity (65). Although, in contrast to aldehyde metabolism, GTN bioactivation is a reductive reaction, NAD+ accelerates the reaction rate 10-fold. The catalytic site of ALDH2 contains three redox-sensitive cysteines: Cys301, Cys303 and Cys302 acting as the main binding site for all substrates. It has been proposed that GTN binds to the thiol of Cys302 via nucleophilic attack leading to a nitric thioester. The free thiol of Cys301 or Cys303 is thought to reduce the nitric thioester to nitrite and to form a disulfide bridge, resulting in inactivation of ALDH2 by GTN after one turnover (1,30).
Cys301 – SH Cys301 – SH Cys301 – S
- Cys302 – SH + GTN " Cys302 – S – NO2 + 1,2-GDN " Cys302 – S + NO2 Cys303 – SH Cys303 – SH Cys303 – SH active ALDH2 RED inactive ALDH2 OX
Activity can be partially restored by reductants like the thiol containing molecules dithiothreitol (DTT) and 2-mercaptoethanol (2-ME) (1). Dihydrolipoic acid (LPA-H2) is less effective than DTT, while the most abundant intracellular low-molecular weight thiols glutathione (GSH) and cysteine (Cys), as well as ascorbate, show no effect on ALDH2 activity (3,73). These compounds reduce the disulfide-bridge at the cysteine site of ALDH2 and thus enable multiple turnovers of the enzyme (1).
Cys301 – S 2-MERED Cys301 – SH 2-MEOX
Cys302 – S + DTTRED " Cys302 – SH + DTTOX
Cys303 – SH LPA-H2 RED Cys303 – SH LPA-H2 OX
inactive ALDH2 OX Reductants active ALDH2 RED
5 INTRODUCTION
However, reactivation of ALDH2 requires relatively high concentrations ("M - mM) of these reducing agents. Furthermore, ALDH2 is only partially restored by reductants, suggesting an irreversible component to ALDH2 inactivation (73). In addition, none of the reducing agents occurs in effective concentrations in biological systems. Additional evidence for the involvement of ALDH2 in GTN bioactivation is the significantly lower GTN affinity of the East-Asian variant (ALDH2*2) of the enzyme (75). About 35-57% of the Asian population suffer from a markedly reduced alcohol tolerance, which is caused by a mutation of the enzyme. At position 487 a lysine is changed for glutamate and thus these patients show a decreased affinity for GTN (76). Significant inhibition of GTN-induced cGMP formation and vasorelaxation can be observed in ALDH2 “knock-out” mice (1,77,78,68). GTN yields stoichiometric amounts of nitrite and 1,2-GDN, but the link between the formation of inorganic nitrite and sGC activation is still unclear. Chen et al. suggested a reduction of nitrite to NO or a NO-related species by a respiratory chain-associated enzyme like cytochrome c oxidase (2,72). Accordingly, nitrite could serve as a vascular storage pool for NO in vivo (70). Other studies did not support the theory that GTN bioactivation is coupled to mitochondrial respiration, and inorganic nitrite could be excluded as an intermediate (68,69). Alternatively, GTN metabolism by ALDH2 may yield NO directly via a three-electron reduction (68,71).
1.1.4. Nitrate tolerance In 1888, Stewart described a case of GTN tolerance in a patient who required 1.3 g of pure GTN to accomplish the same hypotensive effect as the initial dose of 13 mg (44). The mechanisms behind this tolerance are still poorly understood. Nitrate tolerance is a complex phenomenon resulting in reduced responsiveness of blood vessels to GTN and other organic nitrates. A distinction can be made between nitrate-unspecific “pseudotolerance”, which is caused by a neurohormonal counter-regulation against vasodilatation, and nitrate-specific “classical tolerance”, which is associated with diminished responsiveness to GTN. Desensitization of vasodilator response to NO donors or endothelium-derived NO caused by another nitrate is known as “cross-tolerance” (60,79). One has to differentiate between in vivo tolerance, which is caused by chronic application of therapeutic doses of GTN, and in vitro tolerance, which is caused by high pharmacological doses of GTN in isolated cells (49).
1.1.4.1. Pseudotolerance Neurohormonal counter-regulation is generally caused by nitrate-induced, dose-dependent hypotension, which activates a baroreceptor-mediated cascade of regulatory mechanisms. The activation of the renin-angiotensin-aldosterone system (RAAS) results in increased blood concentrations of catecholamines, antidiuretic hormone (ADH), angiotensin II, and aldosterone (Figure 3). As a result, the blood volume increases and thus weakens the preload reducing effect of GTN. Nitrate headache is potentially caused by this rebound-effect (46,49).
6 INTRODUCTION
Figure 3: GTN-induced rebound effect by RAAS regulatory mechanisms. Noradrenalin (NA), angiotensin I-converting enzyme (ACE), phospholipase C (PLC),
inositol triphosphate (IP3), protein kinase C (PKC), phosphorylation (+Pi), antidiuretic hormone (ADH), increased expression of aldosterone-induced proteins (expr.), basolateral Na+/K+ ATPase (ATPase), adenylate cyclase (AC), cyclic adenosine monophosphate (cAMP)
1.1.4.2. Classical tolerance Many mechanisms have been suggested to be responsible for nitrate-dependent tolerance: desensitization of sGC, decreased bioactivation, inactivation of PKG, stimulation of PDE, endothelial dysfunction and enhanced sensitivity against vasoconstrictors. Nitrate tolerance seems to be a multifactorial phenomenon. Therefore several proposed mechanisms may act in concert (60). Desensitization of sGC could be due to oxidation of the ferrous heme iron at the NO binding site (56). Oxidation can be caused by high concentrations of GTN, NO, oxygen (5), or other oxidants. However, desensitization of sGC is only significant after exposure to pharmacological high GTN concentrations in vitro (30). Needleman et al. suggested the involvement of intracellular thiol-pools (GSH and Cys) in the bioactivation of GTN and proposed that vascular thiol depletion provoked by GTN results in reduced GTN bioactivation (58,60). However, several other studies found no correlation between thiol levels, GTN bioactivity, or nitrate tolerance (64,72). .- In 1995 Münzel et al. proposed reactive oxygen species (ROS) such as superoxide (O2 ) as the primary cause for nitrate tolerance (80). Uncoupled endothelial NOS, generated by a controversially discussed (30) lack of tetrahydrobiopterin (BH4) (81), and NADPH oxidase (82) are considered as the main sources of superoxide. Cardiovascular diseases like diabetes, hypertension, arteriosclerosis, hyperlipidemia, and smoking are all associated with increased vascular superoxide production. Superoxide reacts with NO to peroxynitrite (see 1.1), which is a potent oxidant and causes endothelial dysfunction (83).
7 INTRODUCTION
Antioxidants like ascorbate, resveratrol, vitamin E, folate, and others potentially prevent oxidation by superoxide and/or peroxynitrite and protect against endothelial dysfunction (30). Finally, Chen et al. identified ALDH2 as the key enzyme of GTN metabolism and suggested that the enzyme undergoes oxidative inactivation (see 1.1.3). In line with those results, Mayer et al. reported strong evidence for superoxide-mediated oxidative inactivation of ALDH2 as a major cause of nitrate tolerance (30), whereas DiFabio et al. argued that tolerance cannot result from ALDH2 inactivation (77). Increased amounts of ROS were reported in GTN-tolerant tissues, but it his still unclear, how GTN induces ROS formation in mitochondria. Dysfunction of manganese superoxide dismutase (Mn-SOD) due to GTN- dependent oxidation could be responsible for ROS formation (84). Additionally, inactivation of ALDH2 could also occur as a consequence of the depletion of an essential reductant (see 1.1.3), which might be exacerbated by GTN-induced formation of superoxide and/or peroxynitrite (30,81,85,86).
8 INTRODUCTION
1.2. Low Molecular Weight S-Nitrosothiols Because of the short life-time of NO, its beneficial effects appeared to be limited to the immediate vicinity of its formation for a long time. In 1987 Ignarro et al. suggested S- Nitrosothiols (RSNO) as intermediates in the activation of sGC and in 1992 Stamler et al. postulated that NO forms RSNO in vivo (90, 91, 92), which plays an important role in the endogenous storage and transport of NO. Subsequent studies support this theory (88,89). According to Stamler et al. RSNO acts as storage pool of NO, which can be released when required and influence several physiological and pathophysiological processes. Decreased blood levels of RSNO are associated with cardiovascular and respiratory diseases (93). However, there is still no clear picture how S-nitrosothiols are formed endogenously. The nitrosation of Cys containing proteins leads to more stable high molecular weight (HMW) RSNO, while the nitrosation of small thiol containing peptides such as GSH and Cys leads to less stable low molecular weight (LMW) RSNO. Between HMW- and LMW-RSNO exists an equilibrium, which is regulated by S-transnitrosation through nucleophilic attack by the thiolate (109,110). The proportion of these pools depends on the cell type, because GSNO is metabolized faster in some cells than others (111). According to Stamler et al the main part of RSNO (about 90%) occurs in vivo as HMW-RSNO (94,95), principally as the RSNO of the plasma protein albumin at a concentration of approx. 7 µM (90,91,92,112,113). LMW-RSNO such as S-nitrosocysteine (CysNO) and S-nitrosoglutathione (GSNO) (Figure 4) occur in lower concentrations. Because of their absorption of visible light, RSNO are strongly coloured. GSNO is pink and exhibits maximal absorption at 336 nm (! = 778 M x cm-1). GSNO is found in all tissues with concentrations in the range of 10 nM up to 250 nM in erythrocytes and 100 nM in aortic rings (93). Considering that mM amounts of GSH are available in vivo (96), the degree of GSNO is only 1 % or less. The levels of free GSNO and NO (5 nM) in vivo (28) seem to be comparable, so GSNO could be an important factor for NO regulation. A concentration of 1 mM GSH in vivo increases the life-time of NO to ca. 2.75 h (94). Even if the exact mechanism remains unclear, the physiological significance of S-nitrosation is beyond any doubt. Figure 4: GSNO formula
1.2.1. Stability The stability of GSNO is restricted by concentration-dependent thermal decomposition, photolysis, and catalytic decomposition by trace metals like iron and copper. Because of its instability in aqueous solutions, GSNO-stock solutions should be prepared freshly and not preserved more than a few hours. For long time storage GSNO should be kept in a crystalline, frozen state (93). Decomposition by trace metals can be prevented by addition of the unselective metal ion chelator ethylenediaminetetraacetate (EDTA) or the copper (I) selective metal ion chelator neocupreine (10,11,12,14,193). thermal and catalytic decomposition 2 RS-NO ! RS – SR + 2 NO• The reported decomposition reaction (87) releases NO and disulfides. Besides copper, ferrous (Fe2+) ions also catalyze RSNO decomposition, but iron-catalyzed decomposition occurs via a nitrosyl-iron complex (93). In vitro experiments showed that NO and GSH form - GSNO in the presence of oxygen, which is reduced to superoxide (O2• ). The formation of superoxide can be prevented by addition of superoxide dismutase (SOD) (97).
9 INTRODUCTION
The mechanisms proposed for this reaction are summarized in chapter 1.2.6. Reversible transnitrosation to other thiol containing compounds may also play an important role in the life-time of RSNO. Transnitrosation rates from GSH to other thiols at around 80 M-1 x s-1 (37 °C, pH 7.4) and lower rates from S-nitrosoalbumin to GSH at around 3-9 M-1 x s-1 were reported (98). The life-time of GSNO (hours) is reduced to minutes in the presence of other thiols (99,100). This can occur by transnitrosation or by formation of a disulfide bridge (101): - - Transnitrosation R1 – S – NO + S – R2 !R1 – S + NO – S – R2 Disulfide bridge RSNO + GS- "RS – SG + NO- In vivo albumin was shown to be the main target of transnitrosation by GSNO in blood. Oxyhemoglobin (oxy-Hb), a potent NO scavenger, on the other hand, shows only low affinity to transnitrosation agents. The degree of hemoglobin nitrosation is only about a few percent (102). In contrast to NO, small amounts of GSNO or other LMW RSNOs can exist several hours in the presence of oxy-Hb (106) and thus the NO moiety in GSNO is prevented from oxidation to nitrate. RSNOs induce S-nitrosation of the "93 residue of oxy-Hb without formation of nitrate. Transnitrosation is inhibited in the presence of the metal chelators neocupreine and DTPA, implicating the need of catalytic copper for this reaction (106).
1.2.2. Transport Another important point is the permeability of RSNO in biological systems. The cell membrane is impermeable to hydrophilic molecules like GSNO and GSH. Thus, S-nitrosation occurs preferentially on the cell surface or in the extracellular space (103). Intracellular GSH levels cannot be increased by exogenous application (104). However, this fact is discussed controversially. Two mechanisms for transmembrane transport of RSNOs have been suggested: active transport by L-Cys selective amino acid transporters (107) and transnitrosation of intracellular thiols by protein disulfide isomerases (PDI) (108). Furthermore, a coupled mechanism has been suggested, in which PDI catalyzes the decomposition from LMW RSNOs like GSNO to the nitrosation agent N2O3, which in turn transnitrosates extracellular L-Cys. The L-Cys-NO thus generated passes through the membrane via the amino acid transporter, and, consequently, the intracellular S-nitrosothiol level increases.
1.2.3. Detection The detection and quantification of GSNO occurs via decomposition of the S-NO bond by reduced copper, mercury, or iodine ions, and electrochemical detection of the released NO using a NO Clark-type electrode. Alternatively, chemoluminescence with an ozone analyzer, spin-trapping with electron paramagnetic resonance, or fluorescence methods can be used for detection. Pre-separation, using high performance liquid chromatography (HPLC) or ultrafiltration membranes (93) with selectivity for NMW or LMW RSNO, may be required.
1.2.4. Biological actions There is a large body of evidence for GSNO exhibiting biological activities such as vasorelaxation (114), bronchodilatation (115), and inhibition of platelet aggregation (116,117), which are not exclusively due to release of NO (118). RSNOs can act as a buffer pool of oxidation-resistant NO, but additionally affect cell communication by reversible post- translational modification of signal proteins like receptors, enzymes and transcription factors (111).
10 INTRODUCTION
S-Nitrosothiols can exert biological actions in three different ways: a) Via NO release. LMW RSNOs can act as NO donors, with NO acting as the effective agent. b) Via S-transnitrosation of other thiol moieties on LMW thiols or cysteine containing proteins by nitrosation agents such as N2O3. c) Directly. GSNO can itself, NO-independently, activate enzymes like sGC, for example by causing S-glutathiolation.
1.2.4.1. Via NO release Both LMW and HMW RSNO release NO by decomposition of the S-N bond, which is mainly responsible for the sGC-mediated physiological effects. A suggested decomposing agent is copper (119) (for details see 1.2.7). Previous studies proposed a NO-independent mechanism (120), which led to the hypothesis that RSNOs are more than simple NO donors (101). However, the vasodilating effect of GSNO is comparable to ACh and is decreased in the presence of superoxide (121). The beneficial effects like relaxation of blood vessels, relaxation of bronchial smooth muscle and the inhibition of platelet aggregation are strongly reminiscent of NO donors. High dosage of GSNO can also produce adverse effects by activating the apoptotic pathway (122,123,124).
1.2.4.2. Via S–transnitrosation In addition to the NO-mediated pathway, RSNOs possibly affect biological processes by reversible S-transnitrosation of Cys of regulator proteins. Interestingly, S-transnitrosation, for example between extracellular and intracellular RSNOs (107) or neuronal stimulation regulating proteins (125), are L-stereoselective. As noted before, the nitrosation is reversible by adding excess thiol-containing compounds like GSH or DTT and by high intensity light (161). Nitrosation does not occur exclusively at one binding site of the protein. Hb for example can be nitrosylated either at the Cys"93 residue or at the heme site.
Some observed effects of S-transnitrosation on several proteins are: • Inactivation of glutathione reductase (126) • Inactivation of caspase 3 (127) • Inactivation of creatine kinase (128) • Inactivation of glyceraldehydes-3-phosphate dehydrogenase (129) • Inactivation of alcohol dehydrogenase (130) • Inactivation of protein tyrosine phosphatase (131) • Inactivation of aconitase (132) • Inactivation of adenyl cyclase (133) • Decrease of ligand-binding affinity of the glucocorticoid receptor (134) • Decrease of DNA-binding ability of NF-#B (135) • Decrease of DNA-binding ability of estrogen receptor (162) • Inactivation of Complex I (136) • Inactivation of ornithine decarboxylase (137) • Inactivation of cathepsin K (138)
11 INTRODUCTION
• Inactivation of adenosyl transferase (139) • Regulation of thioredoxin (141) • Inactivation of NMDA-channel (163) • Inactivation of Na+ channels in C-type neurons (164) • Activation of ryanodine receptor (140) • Activation of Ca2+ activated K+ channels (165) • Activation of ATP sensitive K+ channels (166)
The described post-translational modification results in: • Decreased energy metabolism (complex I, cytochrome c oxidase, hemoglobin, etc.) • Prevention from protein degradation and apoptosis (caspase family) • Inhibition of DNA binding of transcription factors via S-nitrosylation of the zinc-fingers (estrogen receptor, glucocorticoid receptor, NF-#B) • Inactivation (NMDA receptor, Na+ channels in C-type neurons) or activation (Ryanodine receptor, Ca2+-activated K+ channels, ATP-sensitive K+ channels) of membrane ion channels • Inactivation of kinases and phosphatases (Protein tyrosine phosphatase, glyceraldehyde-3-phosphate dehydrogenase, etc.)
1.2.4.3. As direct effector RSNOs can produce an effect in biological systems without the need for NO release or S- transnitrosation. One established direct effect of RSNOs like GSNO is the interruption of the replication cycle of viruses like human immunodeficiency virus 1 (142), perhaps by inhibition of the protease (145), herpes simplex (143), Epstein-Barr (144), and coronavirus (146). The exact mode of action is still not clear, but an effect on the virion maturation (143,146) by S- glutathiolation of Cys residues is supposed. Another important point is a neuroprotective effect in general (148) or against amyloid "-peptides (147) in particular. RSNOs also inhibit bacteriological $1-protease and thus cause a bacteriostatic effect (149), induce increased expression of 5-lipoxygenase (150) in human bronchial cells, or promote the release of noradrenalin in neuronal cells (133). If S-glutathiolation by GSNO is discussed as a possible mode of action, it is important to mention that S-glutathiolation only occurs in the presence of oxygen. GSNO and oxygen produce GS-O-SG or GS-(O2)-SG intermediates, which in turn induce S-glutathiolation (17). The process is reversible by the disulfide reductant DTT (151). However, the nitrosation of a protein does not necessarily result in a functional effect.
1.2.5. Therapeutic use In general, the therapeutic use of LMW RSNOs like GSNO is problematic, because of the unpredictability of NO release, nitrosation, and glutathiolation (152). In addition, the strong and acute biological response to RSNOs reduces their practical utilization. For example, the strong vasodilatation caused by GSNO provokes the risk of excessive hypotension (92), rebound phenomena, and the occurrence of side effects such as headache. Nevertheless, GSNO is known to have several beneficial effects: • Arterio-selective vasodilatation and improved cerebral blood flow (114) • Bronchodilatation (more potent than theophylline) (115) • Decrease of inflammation during stroke (114) 12 INTRODUCTION
• Protection of endothelial cells from oxidized low-density lipoproteins (101) • Decrease of blood pressure of pregnant women with severe praeclampsia (153) • Inhibition of ADP-induced platelet aggregation and prevention from (154,155) thrombosis after post-coronary angioplasty • Improved wound healing by external use of GSNO containing hydrogels (156) • Inhibition of the growth of the malaria parasite Plasmodium falciparum (194)
The enhanced healing can be explained by increased collagen formation (157), sterilizing effect of NO, or a better blood supply to the wound area through vasodilatation (92). The external application of RSNOs could be an interesting possibility to overcome the strong physiological response. RSNOs also seem to play a role in respiratory diseases. Asthmatic patients show depleted levels of GSNO in their airways, while pneumonia patients show inflammation induced by high levels of RSNO, which may provide new therapeutic approaches to asthma (158,159,160). Interestingly, GSNO does not induce nitrate tolerance or exhibit cross-tolerance in GTN-tolerant blood vessels (190). Selectivity in the therapeutic use of RSNOs can be achieved by coupling to selective, monoclonal antibodies or simply through enhancing the lipophilicity by increasing the length of the associated alkyl group (39).
1.2.6. Formation of S-nitrosothiols To understand the physiological effects of NO it is necessary to know the mechanisms of formation of RSNO in vivo. On the one hand, enzymatic mechanisms have been suggested. Various enzymes such as ceruloplasmin, Hb, or NOS (167) have been proposed to catalyze RSNO formation, but their roles in vivo have not yet been corroborated (32). On the other hand, non-enzymatic pathways were suggested to be responsible for the formation of RSNOs. Three mechanisms have been proposed (Figure 5), all of which were reported to occur in vitro in the presence of NO, oxygen and GSH as the thiol. Pathway 1, first suggested by Czapski et al. (169), is based on the formation of the nitrosating intermediate N2O3 from NO in the presence of oxygen, and was confirmed in subsequent studies (21,22). The supposed reactions in detail are:
NO• + O2 ! ONOO•
ONOO• + NO• " 2 •NO2
•NO2 + NO• ! N2O3
N2O3 + GSH " GSNO + HNO2 7 -1 -1 The reaction between N2O3 and GSH is very fast with k = 6.6 x 10 M x s (175), while the formation of N2O3 is much slower and therefore rate-determining. Alternatively, pathway 2 involves the direct one-electron oxidation of GSH to a thiyl radical by •NO2 followed by a combination of the thiyl and NO radicals to GSNO (13,173). The formation mechanism of •NO2 is identical to pathway 1. The supposed reactions in detail are: - + GSH + •NO2 " NO2 + H GS• + NO• " GSNO
13 INTRODUCTION
The third pathway was suggested by Gow et al. (25) and involves the direct reaction between NO and GSH to produce the radical intermediate GSN•OH, which is further oxidized to GSNO by oxygen or another electron acceptor like NAD+. The proposed reactions in detail are: GSH + NO• ! GSN•OH • - GSN OH + O2 " GSNO + O2 - - NO• + O2 " ONOO
Figure 5: RSNO formation mechanisms. On the basis of Keszler et al. (168) According to Keszler et al. (168) S-nitrosation occurs simultaneously through a radical (pathway 1) and non-radical (pathway 2) mechanism, while a direct addition of NO to GSH in the presence of an electron acceptor (pathway 3) is insignificant for two reasons: first, the reaction of thiols with NO to RSNO does not occur in the absence of oxygen (10,11,12,13,24,168) and thus nitrosating intermediates have to be formed by aerobic oxidation of NO. Second, the absence or presence of NAD+ as electron acceptor instead of oxygen does not show any difference (168). Other suggested mechanisms of RSNO formation in vivo involve trace metal ions (15) and dinitrosyl iron complexes (176). Thiols are possibly oxidized to thiyl radicals by passing one electron to trace metals like copper and further react with NO to GSNO (15,92,176,177). Alternatively, a nitrosating intermediate NO+ is formed in the presence of transition metals (178).
14 INTRODUCTION
Another interesting point is that S-nitrosated proteins mainly occur in the mitochondria, which is the primary intracellular superoxide source (179). Further, formation of RSNO also depends on the availability of GSH- and Cys-containing proteins. Depletion of intracellular GSH significantly increases the S-nitrosation of proteins (179). In general, the transformation + of thiols to S-nitrosthiols requires an S-nitrosating intermediate like NO , •NO2, or N2O4, because NO itself is not a nitrosating agent (170,171,172). Although the reactions are well studied in vitro, the in vivo mechanism is still unclear (22). S- Nitrosation in a complex biological system might be significantly different from the reaction in buffered in vitro solutions. In biological systems the neutral nitrosating intermediates can accumulate in hydrophobic compartments like the membrane (167,174) or hydrophobic pockets of proteins (24). This accumulation is potentially responsible for the predominant S- nitrosation of the "93-Cys of hemoglobin, which is close to hydrophobic regions of the protein (24). However, whereas some studies have shown that hydrophobic compartments stimulate the oxidation of NO by oxygen (174), others do not show any correlation between hydrophobic subenvironments and S-nitrosation rates (168).
1.2.7. Decomposition of S-nitrosothiols The general stability of RSNOs was discussed in chapter 1.2.1. In this chapter the possible decomposing mechanisms in vitro but also in vivo are discussed in detail. The mechanism of NO release from RSNO is complex, especially in biological systems. Decomposition rates at room temperature range from seconds to years, depending on suggested dissociation energies between 20 kcal x mol-1 (27) and 30 kcal x mol-1 (181). The S-N bond is weak, sterically unencumbered and strongly polarized (180). In line with these properties, RSNOs are fairly resistant to thermolysis (182). However, decomposition can occur, photolytically, reductively, or enzymatically, yielding the corresponding disulfide and NO. Depending on the conditions, the cleavage can occur homolytically or heterolytically, generating NO, NO+, or NO-. It follows first-order kinetics, as long as the presence of UV light, metal ions, enzymes, and reducing agents is excluded (180).
1.2.7.1. Homolytic and heterolytic decomposition Barrett et al. observed homolytic cleavage by light in 1966 (183). Furchgott et al. showed light-induced relaxation of blood vessels (184) by releasing NO from a light-sensitive (185) glutathione pool (186,187,188). Grossi et al. also observed homolytic cleavage by thermal decomposition (189). However, according to Bartberger et al. the homolytic cleavage of S-N bonds is irrelevant under physiological conditions (181) and biological effects are regulated by heterolytic NO+ transfer, rather than by direct, homolytic NO release (190). On the other hand, Stamler et al. argued that homolytic cleavage is the biological relevant low-energy mechanism, while the heterolytic cleavage is insignificant under physiological conditions (180). The supposed reactions (180) in detail are:
Homolytic 2 R – S – NO " NO• + 2 R – S• " R – S – S – R Heterolytic RSNO + H+ " RSH + NO+ RSH " RS- + H+ RS- " RS• + e- (oxidation) NO+ + e- " NO• (reduction)
15 INTRODUCTION
1.2.7.2. Reductive decomposition The decomposition of RSNOs is highly influenced by the presence of transition metal ions like iron(II) (15,22,87) and especially copper(I) (10,39,178). In contrast to copper, iron forms 2+( dinitrosyl iron complexes {Fe NO)2} in the presence of NO. Thus, in the case of iron, decomposition of RSNOs is not exclusively due to redox activity, but involves the formation of an catalytic complex without changing its redox state. Copper, on the other hand, shows a 2+ + low redox potential of Eo=+0.15 V between its redox states Cu and Cu (192) and therefore is easily able to catalyze the decomposition of RSNO by reduction (87): RS-NO + Cu+ " RS- + NO• + Cu2+ Cu2+ + RS- " Cu+ + RS• 2 RS• " RS – SR One electron reduction of RSNO by monovalent copper(I) decomposes the molecule and releases NO. The thiolate anion reduces copper(II) to its original redox state and is itself oxidized to a thiyl radical. Thiyl radicals easily react with each other to produce disulfides. The reduction does not depend on the presence of oxygen or other oxidants and works well under anoxic conditions (192). The decomposition by trace metals can be prevented by addition of the unselective metal ion chelator EDTA or the copper (I)-selective metal ion chelator neocupreine, but not by the Cu2+-selective metal ion chelator cuprizone (10,11,12,14,193). Gorren et al. argued that if the cleavage of S-N bonds is caused by a simple reduction, Cu2+-selective chelators should also be less effective inhibitors of RSNO decomposition. Furthermore, Cu+ should only decompose stoichiometric amounts of RSNO, contrary to observations (10). In addition, the susceptibility of the various RSNOs to copper- catalyzed decomposition does not correlate with their particular reduction potentials. Thus, the decomposition by copper cannot exclusively involve a reductive mechanism, but may involve metal ion chelation and inner-sphere electron transfer (180). Copper(II) can be reduced to the active decomposing agent copper (I) in the presence of reducing agents like GSH via the formation of a complex between copper and GSH (10,195): GSH ! GS- + H+ GS- + Cu2+ " GS-%Cu2+ GS-%Cu2+ " GS• + Cu+ GS- + Cu+ " GS-%Cu+ Silver (I) and mercury (II) ions show catalytic activity towards hydrolysis of the S-N bond (196), while Zn2+, Ca2+, Mg2+, Ni2+, Co2+, Mn2+, and Cr3+ show no catalytic activity (87). However, all these observations were based on in vitro experiments and as the concentrations of free copper ions under physiological conditions are extremely low, the involvement of free copper in GSNO breakdown in vivo is questionable. Release of NO from RSNO can be promoted by exogenous and endogenous reductants. A reductant with potential biological relevance is ascorbic acid (197,198), which occurs in vivo at relatively high concentrations. Ascorbate-stimulated nitrosothiol decomposition increases with pH, indicating that the dissociated anion is the more active agent. The vasodilator activity of GSNO is increased in the presence of ascorbate in vivo (199). Interestingly, EDTA inhibits the decomposition by ascorbate, so that the stimulation by ascorbate may originate from the formation and/or regeneration of low levels of cuprous ion by reducing Cu2+ to Cu+ (180). Superoxide has also been shown to decompose GSNO (20), but is far less effective than copper ions (93).
16 INTRODUCTION 1.2.7.3. Enzymatic decomposition In biological systems, additional mechanisms exist for the decomposition of RSNOs. Although tissue homogenates retain the ability to decompose GSNO to NO after removal of all HMW compounds, including all proteins (200), several authors suggested an enzymatic pathway as the primary in vivo mechanism of GSNO decomposition (108,109,110). Some candidate enzymes are GSNO-lyase (201), &-glutamyltranspeptidase (202), thioredoxin/ thioredoxin reductase (203), anaerobic xanthine oxidase (205), protein disulfide dismutase (206) and the GSH-dependent formaldehyde dehydrogenase (GSNO reductase), also referred to as alcohol dehydrogenase 3 (ADH3) (110). With exception of GSNO reductase, which can consume large quantities of GSNO (109,110,159), all suggested enzymes only use small amounts of RSNO and do not seem to play a major role in vivo (93). GSNO reductase occurs in high amounts in liver (207). In 2004 Liu et al. observed increased levels of RSNO in GSNO reductase knock-out mice, which also became hypotensive under anaesthesia (109). As described in chapter 1.2.5, human asthmatics show decreased levels of GSNO and increased levels of GSNO reductase in bronchial fluids. Possibly high levels of GSNO reductase increase the decomposition rate of GSNO and thus deplete endogenous NO storage pools. Hausladen et al. showed copper-independent decomposition of GSNOs in bacterial extracts (204). However, it is still unclear if there is any enzyme that can metabolize RSNO directly to NO.
17 METHODS
2. Methods
2.1. Materials All solutions prepared in nano-pure water, if no specific solvent is declared (Barnstead ultrafiltered type I, resistence > 18 M! x cm-1) [14C]GTN [2-14C]Glycerol-1,2,3- American Radiolabeled trinitrate Chemicals 50 mCi/mmol [32P]GTP Guanosine-5’-[$32P]- Amersham Bioscience triphosphate 10 mCi/ml aqueous solution 1,2-GDN 1,2-dinitroglycerin Cerilliant 100"g/ml acetonitrile 1,3-GDN 1,3-dinitroglycerin Cerilliant 100"g/ml acetonitrile
Al2O3 Aluminium oxide (crystalline) MERCK ALDH2 Human mitochondrial Expressed in and purified dehydrogenase from Escherichia coli in accordance to (208) In MES buffer
CaCl2 Calcium chloride MERCK cGMP Cyclic guanosine SIGMA monophosphate
CuSO4 Copper sulfate SIGMA DEA/NO 2,2-diethyl-1-nitroso- ALEXIS oxyhydrazin in 10 mM NaOH
(C2O5)2O Diethylether MERCK DTPA Diethylenetriamine- MERCK pentaacetic acid 10 mM in 50 mM NaOH DTT 1,4-dithio-DL-threitol SIGMA EDTA Ethylene-1,2-diamine- SIGMA N,N,N’,N’-tetraacetic acid
CH3COOC2H5 Ethyl acetate MERCK
EtOH Ethanol C2H6O MERCK
18 METHODS
GSH Glutathione SIGMA #-L-Glutamyl-L- cysteinylglycine 100 mM in 100 mM NaOH GSNO S – Nitroso glutathione ALEXIS In 50 mM TEA.HCl buffer GTN Glyceroltrinitrate Sanova Pharma 1 mg/ml isotonic glucose From a local pharmacy solution Nitro Pohl Ampullen " GTN (TLC-Standart) Glyceroltrinitrate Cerilliant 1000"g/ml acetonitrile GTP Guanosine-5’-triphosphate SIGMA
HClO4 Perchloric acid (60%) MERCK Hydralazine 1-hydrazinylphthalazine KCl Potassium chloride MERCK
KH2PO4 Potassium dihydrogen MERCK phosphate KI (calibration solution) Potassium iodide SIGMA
0.1 M KI in 0.1 H2SO4 MES 2-(N-morpholino) SIGMA ethanesulfonic acid
MgCl2 Magnesium chloride MERCK
MgSO4 Magnesium sulfate MERCK (anhydrous)
MnCl2 Manganese chloride MERCK
Na2CO3 Sodium carbonate MERCK NaCl Sodium chloride MERCK NAD+ Nicotinamide adenine SIGMA dinucleotide NADH Nicotinamide adenine SIGMA dinucleotide (reduced) NADPH Nicotinamide adenine SIGMA dinucleotide phosphate
NaNO2 (calibrating solution) Sodium nitrite MERCK Neocuproine 2,9-dimethyl-1,10- SIGMA phenanthroline 10 mM neocupreine in 20 mM HCl 19 METHODS sGC Soluble guanylate cyclase Generously provided by Prof. Doris Koesling, Institute of
Pharmacol. and Toxicology, Ruhr-Universität Bochum SOD Superoxide dismutase SIGMA 10kU/ml in 50 mM TEA .HCl buffer
Na(CH3COO). 3 H2O Sodium acetate trihydrate ROTH TEA Triethanolamine SIGMA
Toluene C6H5CH3 MERCK TRX Thioredoxin SIGMA TRX-R Thioredoxin reductase SIGMA
Zn(CH3COO)2.2H2O Zinc acetate MERCK
ZnCl2 Zinc chloride MERCK
20 METHODS
2.2. Nitroglycerin Metabolism
2.2.1. Procedure (210) The rates of 1,2- and 1,3- GDN formation by purified recombinant human ALDH2 were determined by radio thin layer chromatography using a modified method on the basis of (209). All solutions were prepared fresh and all steps were performed on ice. The respective basic reaction mixtures contained (unless indicated otherwise):
Chapter 3.1.1 Chapter 3.1.2
3 mM MgCl2 5 mM MgCl2 1 mM EDTA 100 "M DTPA 1 mM NAD+ 1 mM NAD+ 2 mM GSH 1 kU/ml SOD 2 mM DTT 2 mM GSH 2 "M [14C]GTN 2 mM DTT 10 "M [14C]GTN
in 50 mM K2HPO4 buffer (pH 7.4) in 50 mM TEA-HCl buffer (pH 7.4) Reaction volume = 200 "l Reaction volume = 200 "l Incubation time = 10 min Incubation time = 2 min
Table 1: Basic reaction mixtures of GTN denitration assays
In these mixtures, purified ALDH2 (4 "g), dissolved in MES buffer, was incubated in the absence or presence of thiols at 37 °C for 10 or 2 minutes. For blank measurements the same amount of MES buffer was added to the reaction mixture and treated in the same way. The reaction was stopped by cooling on ice and addition of 1ml of diethyl ether. The metabolites 1,2-GDN and 1,3-GDN as well as the remaining GTN were extracted twice with 1 ml of diethyl ether. The ether phase was dried with about 10 mg MgSO4 and samples were centrifuged at 14000 rpm and 4 °C for five minutes (Eppendorf centrifuge 5417R). The supernatant was transferred into new 2 ml tubes and evaporated. After evaporation 40 "l of ethanol were added to each residue of the samples and vortexed. 8 "l of this solution were then transferred into Packard vials with 5 ml liquid scintillator (PerkinElmer' Ultima Gold XR) to determine the amount of radioactivity before the separation by thin layer chromatography (TLC). The remaining sample was applied to a 20 x 20 cm silica gel plate (Silica gel 60 F254, MERCK), next to a standard mixture containing 10 "l of GTN, 25 "l 1,2- GDN and 25 "l 1,3- GDN standards each. The TLC plate was placed inside a TLC chamber with mobile phase (225ml toluene + 25ml ethyl acetate two times saturated with H2O) for about 60 minutes runtime. After the run, the plate was taken out and, after drying, the standard lane was sprayed with diphenylamine (1 g/100 ml methanol). The resulting brown spots were visualized by exposing the plate to UV light (366 nm) for 5 minutes. According to the location of the standard spots on the plate the appropriate areas of the samples were scraped off, put into a 15 ml Packard vial containing 5 ml liquid scintillator and vortexed before measuring the radioactivity by liquid scintillation counting (Packard Tri-Carb 2100TR).
21 METHODS
2.2.2. Calculation The disintegrations per minute (dpm) measured by the liquid scintillation counter were transformed into the rates of GTN metabolism as follows:
dpm[sample] radioactivity of the sample dpm[blank] radioactivity of the blank nmol[GTN] amount of [14C]GTN dpm[100%] radioactivity of the whole sample (dpm[1,2-GDN+1,3-GDN+GTN]) min incubation time mg[protein] amount of used protein
22 METHODS
2.3. Soluble Guanylate Cyclase Activity
2.3.1. Procedure (210) The activity of soluble guanylate cyclase (isolated from bovine lung) was determined by measuring the amount of [32P]cGMP formed from [32P]GTP using a modified method that was first described by Schultz et al. (211). All solutions were prepared fresh and all steps were performed on ice. Unless indicated otherwise, sGC activity was tested in the presence of 10 "M GTN and 4 "g ALDH2. As standards 10 nM and 1 "M DEA/NO were used. The respective basic reaction mixtures contained (unless indicated otherwise):
Chapter 3.2.1 Chapters 3.2.2 - 3.2.7 Effect of DTT and GSH on sGC activity (50 Effect of DTT and GSH on sGC activity (25 ng sGC) ng sGC)
5 mM MgCl2 5 mM MgCl2 1 mM cGMP 1 mM cGMP 0.5 mM GTP 0.5 mM GTP [32P]GTP (about 250,000 cpm) [32P]GTP (about 250,000 cpm) 1 mM NAD+ or NADH 1 mM NAD+ or NADH 1 kU/ml SOD 1 kU/ml SOD 100 "M DTPA 100 "M DTPA 2 mM DTT 2 mM DTT 2 mM GSH 2 mM GSH 4 "g ALDH2 4 "g ALDH2 50 ng sGC 25 ng sGC In 50 mM TEA-HCl buffer (pH 7.4) in 50 mM TEA-HCl buffer (pH 7.4) Reaction volume = 100 "l Reaction volume = 200 "l Incubation time = 2 min Incubation time = 2 min
Table 2: Basic reaction mixtures of sGC activity assays
First, ALDH2 and water were introduced into the reaction tubes. Next the reaction mixture was prepared and added for blank and 100 % values before addition of sGC. Then the final mixture (including sGC) was divided into the sample tubes before starting the reaction by adding the NO-donor (GTN or DEA/NO) and incubating at 37 °C for 2 minutes. The reaction was stopped by adding 450 "l of zinc acetate (120 mM) and 450 "l of sodium carbonate (120 mM) to each sample, leading to a zinc carbonate co-precipitation of the side products. Then the samples were centrifuged for 5 minutes at 14000 rpm and 4 °C. The separation of cGMP and GTP occurred via adsorption chromatography: separation columns were filled with alumina (Al2O3) and eluted with 5 ml of HClO4 (100 mM) each before addition of the samples. After draining, the columns were washed with 10 ml aqua-dest. before eluting cGMP with 5 ml of sodium acetate (250 mM) into Packard vials. As 32P is a high-energy beta emitter, the Cerenkov radiation was measured without using liquid scintillator.
23 METHODS
2.3.2. Calculation The amount of the product cGMP was calculated as follows:
nmol[cGMP] amount of formed cGMP cpm[sample] radioactivity (counts per minute) of each sample cpm[blank] radioactivity without adding sGC, mitochondria, and NO-donor nmol[GTP] amount of GTP cpm[100%] radioactivity of total GTP added per tube recovery 50 % (due to aliquoting and partial absorption of cGMP by zinc carbonate)
Calculation of the sGC activity:
nmol[cGMP] amount of formed cGMP mg[protein] amount of used sGC 2min incubation time
24 METHODS
2.4. NO Release
2.4.1. Procedure The determination of the released amounts of NO was performed with a commercially available Clark-type electrode (Iso-NO, World Precision Instrument, Berlin, Germany) in accordance with an established method (8). The NO meter was connected to an Apple Macintosh via an analog to digital (A/D) converter (MacLab, World Precision Instruments). The output current was recorded at 0.6 Hz under constant stirring at 37°C. The data were recorded with the CHART( program (V6.4, AD Instrument Ldt., Hasting, Britain). Three-point calibration of the electrode was performed daily by determination and standardization of the current [mV] that is produced by the reduction of 0.5 "M NaNO2 to NO by 100 "M KI diluted in 100 "M H2SO4. All solutions were prepared fresh and kept on ice. If not indicated otherwise, the NO release by 100 "M GTN and 125 "g ALDH2 was measured. As standard 1 "M DEA/NO was used. The respective basic reaction mixtures contained (unless indicated otherwise):
Chapter 3.3 NO Release
5 mM MgCl2 1 kU/ml SOD 100 "M DTPA 1 mM NAD+ or NADH 2 mM GSH 2 mM DTT 125 "g ALDH2 + 100 "M GTN or 1 "M DEA/NO In 50 mM TEA.HCl buffer (pH 7.4) Reaction volume = 500 "l
Table 3: Basic reaction mixtures for NO determination
Under aerobic conditions, the basic reaction solution was incubated in water-jacketed open plastic vials and the electrode was put into the solution. After a stable baseline was reached, 100 "M GTN or 1 "M DEA/NO was injected. Unless indicated otherwise, 4 mM of an aqueous CuSO4 solution was injected until the baseline was reached again. Under anaerobic conditions, all solutions were saturated with argon and stored in closed vessels on ice. The basic reaction solution was incubated in water-jacketed vials closed by a membrane, which was penetrated by the electrode and a gas supply line. The solution was aerated with argon for 15 min. After removal of the gas supply line, the measurement was started. For quantification of the amounts of NO that are released from GSNO, concentrations between 100 "M and 2 mM were applied. The concentration of the GSNO used for calibration was checked spectroscopically ()=340 nm, !=750 M-1 x cm-1). Cleavage of the N- S bond occurred in the presence of 2 mM GSH and was initiated by the injection of 4 mM CuSO4. Finally, the quantification was performed using the maximal NO concentration obtained. 25 RESULTS
3. Results
3.1. Nitroglycerin Metabolism
3.1.1. Effect of DTT and GSH on GTN metabolism Since the identification of mitochondrial ALDH2 as the major GTN metabolizing enzyme and the putative oxidative formation of a disulfide bond at the active site, that inactivates the enzyme after one turnover (1), many potential regenerating agents were tested. The unphysiological compound dithiothreitol (DTT) is the most efficient reductant reported to date (2). Glutathione (GSH), the most abundant intracellular thiol, on the other hand shows no significant effect on ALDH2 activity. In fact, GSH seems to slightly decrease the dehydrogenase-activity of the enzyme and to cause reversible glutathionylation of proteins (3). To corroborate those observations ALDH2-dependent 1,2-GDN formation from GTN was assayed (Figure 6).
Figure 6: GTN metabolism by 4 "g ALDH2 ± 2 mM GSH/DTT. Incubation with 2 "M 14C-GTN for 10 min at 37°C. As expected, DTT enabled several turnovers, while GSH showed no effect. To investigate a potential effect of other antioxidants, this experiment was repeated with the vasodilator hydralazine (2mM ± 1mM NAD+) and the enzyme combination thioredoxin-2/ thioredoxin-reductase (2mM + 1 mM NAD+ + 1mM NADPH). None of these increased 1,2- GDN formation and thioredoxin-2/thioredoxin-reductase also had no effect on ALDH2- mediated NO release from GTN, as measured with a NO-selective Clark-type electrode (data not shown).
26 RESULTS
3.1.2. Effect of the DTT concentration on GTN metabolism in the presence of GSH For better comparison of the data and to exclude direct inhibition of ALDH2-mediated denitration by GSH under different conditions, the GTN denitration assay was repeated under the same conditions that were applied in the cyclase assay. To investigate a possible protective effect of DTT on the denitration activity of ALDH2 in the presence of GSH, increasing concentrations of DTT were added (Figure 7).
Figure 7: GTN metabolism by 4 "g ALDH ± 2 mM GSH ± 0-2 mM DTT. Incubation with 10 "M 14C-GTN for 2 min at 37°C; V = 200 "l.
As a consequence of the changed conditions denitratation activity of ALDH2 was higher, but, like in Figure 6, GSH had no effect. DTT had no effect at concentrations $ 1 "M, but approximately doubled the activity at 10 "M. The stimulatory effect of 10 "M DTT seemed to be slightly smaller in the presence of 2 mM GSH, but the difference did not reach statistical significance. Additional studies with DTT concentrations between 10 "M and 1000 "M in the presence and absence of GSH need to be done.
27 RESULTS
3.2. Soluble Guanylate Cyclase Activity
3.2.1. Effect of DTT and GSH on sGC activity (50 ng sGC) The NO-mediated activation of soluble guanylate cyclase (sGC) by the NO-donor DEA/NO and by GTN with ALDH2 is shown in Figure 8.
Figure 8: Activition of sGC by DEA/NO (10 nM or 1 "M) or by GTN (10 "M) and ALDH2 (2 "g) in the presence and absence of 2 mM DTT and/or GSH. Incubation with 50 ng sGC for 10 min. at 37°C; V = 100 "l.
DEA/NO-mediated cyclase activity decreased up to 50 % in the presence of DTT and GSH, especially at low NO concentrations. With GTN/ALDH2, GSH also seemed to cause a reduction of activity, perhaps by scavenging the produced NO.
28 RESULTS
3.2.2. Effect of DTT and GSH on sGC activity (25 ng sGC) The sGC stock solutions contain DTT for a higher stability, which may increase basal activity. To minimize that effect, we added half the amount of cyclase and doubled the reaction volume, resulting in 4-fold lower final cyclase and basal DTT concentrations. In addition, the incubation time was reduced from 10 min. to 2 min.
Figure 9: Activition of sGC by DEA/NO (10 nM or 1 "M) or by GTN (10 "M) and ALDH2 (4 "g) in the presence and absence of 2 mM DTT and/or GSH. Incubation with 25 ng sGC for 2 min. at 37°C; V = 200 "l.
Under these conditions (Figure 9), a two-fold higher cyclase activity was observed, except in the presence of 1 "M DEA/NO. With 10 nM DEA/NO the activity was still decreased by thiols, in particular by GSH. Inhibition was no longer observed when the cyclase was maximally activated in the presence of 1 "M DEA/NO. Close to maximal activation was also achieved with GTN/ALDH2; interestingly, this activity was substantially lowered in the presence of 2 mM GSH, but not in the presence of 2 mM DTT or of both GSH and DTT.
29 RESULTS
3.2.3. Effect on sGC activition of substituting NAD+ by NADH Since NAD+ has a profound stimulatory effect on ALDH2-mediated GTN bioactivation, omission of NAD+ greatly diminishes activation of sGC by GTN/ALDH-2. However, NADH can partially substitute NAD+ in the activation of GTN metabolism by ALDH-2. Therefore, by addition of NADH instead of NAD+, a potential role of NAD+ as a redox-active agent in the cyclase assay was investigated.
Figure 10: Activation of sGC by DEA/NO (10 nM or 1 "M) or by GTN (10 "M) and ALDH2 (4 "g) in the presence and absence of 2 mM DTT and/or GSH. Incubation with 25 ng sGC for 2 min. at 37°C; V = 200 "l.
In the presence of 1 mM NADH cyclase activity was about 40% lower than with NAD+. Activity was lower under all conditions (10 nM DEA/NO, 1 "M DEANO, and 10"M GTN/4"g ALDH-2) suggesting that it is the maximal activity of the cyclase that is affected. Whether the phenomenon is due to stimulation by NAD+ or inhibition by NADH is not clear yet. Surprisingly, no inhibition by GSH occurred in the presence of 1 mM NADH. The reason is unclear and requires further investigation.
30 RESULTS
3.2.4. Effect of the DEA/NO concentration on sGC activation Since the inhibitory effect of DTT and GSH seemed to depend on the NO concentration, we determined the effect of both thiols on the activation of sGC by DEA/NO in the concentration range from 10 pM to 10 "M. The results are shown in Figure 11.
Figure 11: Activation of sGC by DEA/NO in the presence and absence of 2 mM DTT and GSH. Incubation with 25 ng sGC for 2 min. at 37°C; V = 200 "l.
The curves show a 50 % decrease of cyclase activity at low DEA/NO concentrations for both thiols. In all cases maximal activity was reached at 1 mM DEA/NO. Maximal activity was not affected by the thiols (19.8 ±0.3, 19.7±0.5, and 20.8+0.7 "M x min–1 x mg–1 for control, DTT, and GSH, respectively), while basal activity was slightly lower (1.8 ±0.3, 0.9±0.4, and 0.4±0.5 "M x min–1 x mg–1 for control, DTT, and GSH, respectively). The main effect of the thiols was an increase of the EC50 (6.6 ±0.9, 15±3, and 17±4 nM for control, DTT, and GSH, respectively). Although these results confirm an inhibitory effect of GSH on sGC activation by NO, the effect was rather small and the same effect was observed with DTT. Consequently, these observations do not explain the low GTN/ALDH2-mediated cyclase activity in the presence of GSH (Figure 9).
31 RESULTS
3.2.5. Effect of the GSH concentration on the activation of sGC by GTN/ALDH-2 The inhibition of GTN/ALDH-2-induced sGC activation by GSH was further explored by variation of the concentration of GSH at fixed concentrations of GTN and ALDH2.
Figure 12: Activation of sGC by 10 "M GTN and 4 "g ALDH2 in the presence of GSH. Incubation with 25 ng sGC for 2 min. at 37°C; V = 200 "l.
At a concentration of 100 "M, GSH did not affect sGC activity. At higher concentrations, GSH inhibited sGC activation by 54±2% with an IC50 of 0.25±0.03 mM.
32 RESULTS
3.2.6. Effect of the ALDH2 concentration on the activation of sGC by GTN In principle, the inhibitory effect of GSH could be due to a decrease of the ALDH-2 activity, a decrease of the cyclase activity, or an interaction with the activating/inhibiting species that are generated from GTN. To further explore the mechanism of inhibition, we studied the stimulation at varying ALDH-2 concentrations.
Figure 13: Activation of sGC by 10 "M GTN and 0 – 50 "g ALDH2 in the presence and absence of 2 mM GSH. Incubation with 25 ng sGC for 2 min. at 37°C; V = 200 "l.
In the absence of GSH, ALDH-2 mediated the activation of sGC by GTN with an EC50 of 5±2 mg/ml. Addition of GSH did not affect maximal activity, but caused a ~10-fold increase of the EC50 to 59±13 mg/ml.
33 RESULTS
3.2.7. Effect of DTT on the inhibition by GSH of GTN/ALDH2- mediated sGC activition
As previously shown (Figure 9), DTT prevented GSH from lowering the cyclase activity. To further investigate this phenomenon, we determined the effect of varying concentrations of DTT at a fixed GSH concentration of 2 mM (Figure 14).
Figure 14: Activation of sGC by DEA/NO (10 nM or 1 "M) or by 10 "M GTN and 4 "g ALDH2 in the presence of 2 mM GSH and varying concentrations of DTT. Incubation with 25 ng sGC for 2 min. at 37°C; V = 200 "l.
Under these conditions DTT increased activity from 8.1±0.3 to 16.1±0.4 "M x min–1 x mg–1 with an EC50 of 3.5±1.2 "M. Consequently, the potency of DTT in preventing inhibition seems to exceed that of GSH in causing inhibition by at least two orders of magnitude. In the presence of a saturating DEA/NO concentration (1 "M) sGC activity was hardly affected, ruling out an effect of DTT on maximal sGC activity. At a concentration of 10 nM DEA/NO DTT caused a 60 % activity increase in the presence of 2 mM GSH, which is not much smaller than the effect observed with GTN/ALDH-2 (~100 %). However, since this effect appeared to occur at an approx. 10-fold higher concentration of DTT, it is probably not responsible for the activation observed with GTN/ALDH-2, although it may have contributed at concentrations of 10 "M DTT and higher. The control experiments in the presence of 1 "M DEA/NO suggest that reactivation by DTT may not be complete, since the maximal activity observed with GTN/ALDH-2 was about 17 % lower.
34 RESULTS
3.3. NO Release The results presented in 3.1 and 3.2 prompted us to look into the effects of GSH and DTT on NO released by GTN/ALDH and by DEA/NO. To determine the NO concentration we used a NO-sensitive Clark-type electrode (8).
3.3.1. Effect of DTT and GSH on ALDH2-mediated NO release by GTN For a direct comparison with the data of the enzymatic assays (chapters 3.1 and 3.2), it would be desirable to use the exact same reaction conditions. Although the detection limit of the electrode is 1 nM (8), the signal produced by 10 "M GTN and 20 "g/ml ALDH2 is not detectable. Because of that, concentrations were increased to 100 "M GTN and 250 "g/ml ALDH2. Pilot studies (not shown) indicated that in the presence of GSH smaller amounts of NO might be released. According to previous reports (10,11,12,13) GSH could act as an effective scavenger for NO under aerobic conditions. The product of NO and GSH was identified as S-nitrosoglutathione (GSNO), which is stable in aqueous solutions but can be decomposed by trace metals such as copper (14) and iron (15) in the presence of reductants like GSH and ascorbate (10). To check for the formation of GSNO, 4 mM Cu(II)SO4 were injected, when the signal reached the baseline (Figure 15).
Figure 15: NO release by 125 "g ALDH2 in the presence and absence of 2 mM GSH and DTT. Injection of 100 "M GTN (at time zero) and 4 mM CuSO4 (indicated by arrows) at 37 °C; V = 500 "l. Injection of 100 "M GTN in the presence of 250 "g/ml ALDH2 caused only a minor NO signal that was greatly enhanced (>10-fold) in the presence of DTT. This agrees with the observation that DTT enabled multiple turnovers of ALDH2 (Figure 6 and Figure 7). Also in line with those results, no stimulation was observed with GSH, which instead caused a decrease of the signal. Similarly, stimulation of NO release by DTT was somewhat decreased by GSH. These observations suggest that GSH either scavenged NO or inhibited ALDH2.
35 RESULTS
Previous results (Figure 6 and Figure 7) showed a small decrease of ALDH2 activity in the presence of DTT and GSH in comparison to DTT alone, but the difference was statistically not significant. Therefore, the lack of NO is probably caused by GSH acting as NO scavenger. After injection of copper (II) ions (black arrows in Figure 15), small quantities of NO (0.05 – 0.1 "M) were released exclusively in the presence of GSH, suggesting GSNO formation. The combination of DTT and GSH produced 50 % higher amounts of NO than GSH alone, which may be explained by the greater NO production in the presence of DTT. However, the stimulation of NO formation by DTT far exceeded the increase in copper-induced NO release, indicating that in the presence of DTT a far smaller fraction of the NO formed is converted into a nitrosothiol. To investigate the effect of NAD+, the experiment was repeated in its absence (Figure 16).
Figure 16: NO release (peak height) by 125 "g ALDH2 in the presence and absence of 2 mM GSH and DTT. Injection of 100 "M GTN and 4 mM CuSO4 (GSNO) at 37 °C; V = 500 "l.
In the absence of NAD+ all peak levels were about 70 % lower, in agreement with the stimulatory effect of NAD+ on denitration (4). The amounts of NO released from GSNO under these conditions were below the detection limit of the electrode.
36 RESULTS
3.3.2. Effect of DTT and GSH on NO release by DEA/NO As a control experiment we also determined the effect of DTT and GSH on NO release by DEA/NO under the same conditions. Copper was injected after 720 seconds.
Figure 17: NO release from 1 "M DEA/NO in the presence and absence of 2 mM DTT and GSH. Injection of DEA/NO at zero time and of 4 mM CuSO4 after 720s (arrow) at 37 °C in the presence of SOD and NAD+; V = 500 "l.
With DEA/NO as the NO source, GSH decreased the peak height by 70 % in comparison to the control curve (Figure 17). Addition of copper ions after return to baseline of the signal resulted in a large NO signal, indicating that a sizeable fraction had been transformed into GSNO. By contrast, DTT had only a minor effect on the NO peak height, and hardly any NO was released after copper ion injection. In the combined presence of DTT and GSH the NO peak height was almost indiscernable from that observed with DTT alone, and copper-induced NO release was only slightly higher than with DTT alone. To investigate the role of NAD+ the experiments were repeated in its absence and in the presence of NADH (Figure 18).
37 RESULTS
Figure 18: Effect of NAD+ and NADH on NO release from 1 "M DEA/NO in the presence and absence of 2 mM DTT and GSH. Conditions as for Fig. 17; V = 500 "l.
In the absence of NAD+ peak heights were decreased by about 6 – 8 % (with exception of GSH alone), while GSNO formation was not affected. NADH did not affect NO release; GSNO formation appeared to be somewhat smaller in the presence of NADH. However, control experiments with authentic GSNO suggested that this may be due to an effect of NADH on copper-mediated NO release rather than on GSNO formation (not shown). .- Several reports have suggested a superoxide (O2 )-dependent mechanism for GSNO formation (11) or decomposition (20). To investigate the effect of superoxide on the formation or decomposition of GSNO, the experiments were repeated in the absence of superoxide– dismutase (SOD) (Figure 19).
38 RESULTS
Figure 19: NO release from 1 "M DEA/NO before and after addition of 4 mM CuSO4 in the absence and presence of 2 mM GSH at 37 °C in the absence of SOD; V = 500 "l.
In comparison to the results presented in Figure 18, omission of SOD showed no statistically significant difference in peak heights (except for a slight increase in the GSNO control), but decay of the NO signal was two times faster (Figure 20 vs. Figure 17).
Figure 20: NO release from 1 "M DEA/NO before and after addition of 4 mM CuSO4 (arrows) in the absence and presence of 2 mM GSH at 37 °C in the absence of SOD; V = 500 "l.
39 RESULTS 3.3.3. Effect of GSH on NO release by DEA/NO in the absence of oxygen
Oxygen (O2) was proposed in several studies to be involved in GSNO formation (21,22,11,23,24), while on the other hand an oxygen-independent pathway was suggested in the presence of an electron acceptor (25). Furthermore, oxygen has been suggested to be involved in decomposition as well (27). To study the effect of oxygen on the formation and decomposition of GSNO, NO release was measured in the absence of oxygen by bubbling the reaction solution and all stock solutions with argon (Figure 21).
Figure 21: NO release from 1 "M DEA/NO before and after addition of 4 mM CuSO4 (arrows) in the presence of 2 mM GSH at 37 °C; V = 500 "l.
NO peak height in the absence of oxygen was about 4 times higher than in its presence. Residual oxygen, not removed from the solution, and slow readmission during the incubation may be responsible for the slow decay of the signal. After 270s a fairly stable NO concentration was achieved. Injection of copper (II) at this time resulted in release of about 0.3 "M NO, which is much less than the amount produced in the presence of oxygen (0.9 "M). 140s after copper (II) injection a stable NO concentration was observed. These results suggest that GSNO formation is oxygen-dependent, whereas GSNO decomposition is not (For details see discussion).
40 RESULTS
3.3.4. Effect of GSH on NO release by DEA/NO at varying concentrations To extrapolate the effects of GSH on NO release from DEA/NO to physiologically more relevant concentrations (28), we repeated the experiments with DEA/NO concentrations between 10 and 1,000 nM (Figure 22).
Figure 22: NO release from 10 – 1,000 nM DEA/NO in the presence and absence of 2 mM GSH before and after addition of 4 mM CuSO4 (GSNO) at 37 °C in the presence of SOD and NAD+. To ease interpretation of the results the DEA/NO concentrations 0 – 1,000 nM (Figure 23), 0 – 400 nM (Figure 24) and 0 – 50 nM (Figure 25) were separately drawn non-logarithmical. Representative curves in the presence of 2 mM GSH with DEA/NO concentrations between 50 and 1,000 nM are shown in Figure 26. In the absence of GSH, the peak height increased linearly with the DEA/NO concentration, with an apparent yield of 0.897±0.006 mol/mol DEA/NO. In the presence of 2 mM GSH, linearity was lost, with high DEA/NO concentrations exhibiting higher NO yields. Below 50 nM DEA/NO, the NO peak was no longer detectable. These observations suggest more efficient scavenging on NO by GSH at low NO concentrations, which agrees with previous results (3.2.2, 3.2.6). The copper-induced, GSNO-derived peak height in the presence of GSH increased linearly with the DEA/NO concentrations (Figure 23), with an apparent yield of 1.061±0.011 mol/mol DEA/NO. The apparent discrepancy between the total yields of NO (i.e before and after copper injection) observed in the absence and presence of GSH is explained by the different rates of NO release from DEA/NO and GSNO. Whereas GSNO releases NO within seconds, with peak heights approximating the released amounts of NO fairly accurately, the much slower release of NO from DEA/NO results in peak height that are strongly affected by the simultaneous disappearance of NO.
41 RESULTS
Figure 23: Linear representation of the semi-logarithmic plots in Fig. 22.
Figure 24: Linear representation of the semi-logarithmic plots in Fig. 22 in the concentration range from 0 to 400 nM DEA/NO.
42 RESULTS
Figure 25: Linear representation of the semi-logarithmic plots in Fig. 22 in the concentration range from 0 to 50 nM DEA/NO.
Figure 26: Representative curves of the NO release from DEA/NO in the concentration range between 50 nM and 1 "M in the presence of 2 mM GSH before and after addition of 4 mM CuSO4.
43 RESULTS
3.3.5. Effect of various cations on formation and decomposition of GSNO All reaction discussed so far were performed in the presence of 5 mM Mg2+, which is an important ALDH2 activating cofactor (29). In the absence of Mg2+ the ability of GSH to scavenge NO was decreased and less NO was released after CuSO4 addition (Figure 27). To investigate the effect of cations on formation and decomposition of GSNO, the experiment was repeated in the presence of various salts (Figure 27).
Figure 27: NO release from 1 "M DEA/NO in the presence and absence of 2 mM GSH and the cations Mg2+, Ca2+ Na+, Mn2+, and Zn2+ (5 mM each), before and after addition of + 4 mM CuSO4 at 37 °C in the absence of SOD and NAD ; V = 500 "l.
Mg2+ and Ca2+ both did not affect NO release from DEA/NO in the absence of GSH, but markedly reduced peak heights in its presence, and vastly increased NO release after + 2+ addition of CuSO4. By contrast, Na had no effect. With Mn the peak height after DEA/NO addition in the absence of GSH was somewhat reduced, whereas that in the presence of GSH was little affected; however, NO release after CuSO4 addition was equally high as or slightly higher than that observed for the other bivalent cations. Zn2+ drastically reduced the peak height in the absence of GSH but appeared to have little effect in its presence. The results with Zn2+ and to a lesser extent with Mn2+ are difficult to interpret, since both ions decreased the rate of NO release by DEA/NO, resulting in lower initial rates of NO formation, lower peak heights, and slower apparent decay rates (Figure 28).
44 RESULTS
Figure 28: NO release from 1 "M DEA/NO in the absence of GSH and in the presence of the 2+ 2+ 2+ Mg , Mn , and Zn (5 mM each), before and after addition of 4 mM CuSO4 at 37 °C in the absence of SOD and NAD+; V = 500 "l. Curves in the presence of Ca2+ and Na+ (not shown) were all but identical to the one shown for Mg2+.
45 DISCUSSION
4. Discussion
4.1. Effect of DTT and GSH on GTN metabolism Only DTT enables more turnovers of ALDH2 DTT but not GSH stimulated GTN metabolism (3.1) and NO release (3.3.1), because only DTT could reduce the disulfide of oxidized ALDH2 (1). Therefore, this effect was specific for GTN/ALDH reactions.
4.2. Effect of DTT and GSH on sGC activity GSH decreases ALDH2-mediated sGC activity The presence of DTT had no effect on the cyclase activity under the presented conditions obviously, since the maximum activity was already achieved without DTT (3.2.2.). However, GSH showed a pronounced restraining effect. If the inhibition by GSH was caused by the shortened incubation time (2min), limited to the presence of NAD+ (or absence of NADH) (3.2.3) or just occurred by the use of specific ALDH2 concentrations (3.2.6), is still not clear and needs further investigations. But, this GSH-effect must not be equated with the not particularly large and not GSH-specific right shift of sGC-activity curves, which could be observed by the use of DEA/NO instead of ALDH2 (3.2.4). GSH, as well as DTT, showed a 50 % decrease of cyclase activity at low DEA/NO concentrations (* 10 nM), but not at high concentrations (+ 100 nM). Clear is only that GSH affected the ALDH2-mediated sGC activity in a concentration-depending way: high GSH concentrations (+ 300 "M) produced inhibition of sGC activity (up to 54±2%), while low concentrations (* 100 "M) did not. The maximum rate vmax of sGC was not affected (Figure 13), although an effect on NO-sensitivity cannot be excluded yet. DTT prevents inhibition by GSH at low concentrations The potency of DTT in preventing inhibition (+10 "M) seems to exceed that of GSH in causing inhibition (+ 300 "M) by at least two orders of magnitude (3.2.7). At a saturating DEA/NO concentration (1 "M) sGC activity was nearly unaffected, ruling out an effect of DTT on maximal sGC activity. But, at low DEA/NO concentrations (10 nM), DTT caused a 60 % activity increase in the presence of 2 mM GSH, which is not much smaller than the effect observed with GTN/ALDH-2 (~100 %).
4.3. Effect of DTT and GSH on ALDH2-mediated NO release by GTN 10x higher amounts of NO with DTT, but no signal with GSH alone In the presence of DTT the NO-signal that was greatly enhanced (>10-fold) (3.3.1), because DTT enabled multiple turnovers of ALDH2 (1). No stimulation was observed with GSH, which instead caused a decrease of the signal. These observations suggest that GSH either scavenged NO or inhibited ALDH2. Copper (II) ions released small quantities of NO (0.05 – 0.1 "M) exclusively in the presence of GSH, suggesting GSNO formation. The combination of DTT and GSH produced 50 % higher amounts of NO than GSH alone, which may be explained by the greater NO production in the presence of DTT. However, the stimulation of NO formation by DTT far exceeded the increase in copper-induced NO release, indicating that in the presence of DTT a far smaller fraction of the NO formed is converted into a nitrosothiol.
46 DISCUSSION
4.4. Effect of DTT and GSH on NO release by DEA/NO Very efficient GSNO formation from submicromolar DEA/NO in the presence of GSH By the use of DEA/NO instead of ALDH/GTN as the source of NO (Figure 17 and Figure 18) a slight decrease (14±3 %) of the maximal NO concentration could be observed with DTT. In the presence of GSH the decrease was much greater (67±2 %), but this effect was completely abolished in the combined presence of DTT. Since it was suspected that the decrease in peak height might be due to nitrosothiol formation, CuSO4 was added at the end of the reaction (Figure 17). Unsurprisingly, no nitrosothiols were detected in the absence of thiols, but a large signal was evident in the presence of GSH, indicating that the lower NO peak in the presence of that thiol can be attributed to GSNO formation. Much smaller, but detectable amounts of nitrosothiols were observed in the presence of DTT or DTT + GSH. NO autoxidation is a minor process under the experimental conditions There are several surprising aspects to the curves in Figure 17 that need to be addressed. The decay phases of the curves show no evidence for second order behavior, as would be expected if NO autoxidation were the dominating process (26). For the curves generated by DEA/NO the second-order nature may be partially masked by the simultaneous first-order NO release from DEA/NO, but the decay curves were also perfectly first-order after CuSO4 addition, which results in instantaneous NO release, and should not affect the subsequent decay curve. Furthermore, a nearly perfect linear relationship between the height of the NO peak and the DEA/NO concentration could be observed (Figure 22), which also suggests that the decay phase is caused by a first-order process. Indeed, observed decay rates were considerably faster than might be explained by autoxidation (26). The cause of this set of observations lies in the experimental conditions applied in these studies. These experiments were performed in open, stirred vessels with DEA/NO concentrations of 1 "M and lower. Under such conditions most NO disappears by diffusion out of the reaction vessel. NO autoxidation is not involved in GSNO formation under the conditions of the present study A second set of observations, however, cannot be explained in this way. The observation that first suggested to us the involvement of GSNO formation was the pronounced decrease of the NO peaks in the presence of GSH. Yet, it is exactly this observation that cannot be accommodated by the presently leading hypotheses. NO autoxidation results in NO2 formation in a reaction that is second-order in NO and first-order in O2 (Eq. I). According to one hypothesis (22, 175), NO2 reacts with another molecule of NO to the strong nitrosating agent N2O3, which reacts with GSH to GSNO and nitrite (Eqs. II and III). According to another hypothesis (13, 173, 212) NO2 reacts directly with GSH to produce nitrite and a glutathiyl radical (GS•) that will instantaneously combine with NO to GSNO (Eqs. IV and V). What both hypotheses have in common, is rate-limiting autoxidation of NO, preceding the reaction with GSH. Consequently, the presence of GSH should not affect the NO formation/consumption curve in agreement with observations (212). It might be argued that GSNO formation consumes one extra NO molecule (Eq. V), which might change the stoichiometry of the reaction, and hence the NO peak values, but the same NO molecule is also consumed during autoxidation (Eqs. 2 and 5).) Hence, the striking decrease of the NO peak, observed here, is inconsistent with the leading proposals for nitrosation by NO/O2. The involvement of NO autoxidation is also unlikely in the light of the observation that, under the conditions applied here, most NO will diffuse out of the solution before autoxidation can occur (see above). Instead, the data strongly suggest a direct reaction between NO and GSH. Such a reaction is also suggested by the linear relationship between the observed GSNO concentration, as revealed by the CuSO4–induced NO peak, and the DEA/NO concentration (Figure 22), because the height of this peak should reflect the order (in NO) of the GSNO-producing reaction.
47 DISCUSSION
2 NO !! 2 NO2 (I)
NO2 + NO " N2O3 (II) + – N2O3 + GSH ! GSNO + H + NO2 (III) • + – NO2 + GSH ! GS + H + NO2 (IV) • NO + GS ! GSNO (V) A direct reaction between NO and GSH has been previously proposed by Gow et al. (25). According to their hypothesis, binding of NO to GSH results in a radical intermediate GSN•OH, which in the presence of suitable oxidizing agents will be oxidized to GSNO (Eqs. – VI and VII). Under aerobic conditions, O2 will be reduced to O2 (Eq. VIII). Evidence was presented that anaerobically NAD+ can substitute for O2 (25). Importantly, it was suggested that this mechanism accounts for GSNO formation at low ($ 1"M) concentrations of NO, with the conventional autoxidation-based mechanism predominating at higher concentrations (% 50 "M). • NO + GSH " GSN OH (VI) GSN•OH + A ! GSNO + H+ + A– (VII) • + – GSN OH + O2 ! GSNO + H + O2 (VIII) Despite the potential physiological implications, the hypothesis met with a fairly cool reception for several reasons. The data presented by Gow et al. were mostly qualitative, and the evidence for the proposed reaction rather indirect and sometimes ambiguous. Moreover, a couple of inconsistencies, mainly involving the order of the reaction in NO, may have dampened enthusiasm for the hypothesis. Gow et al. explained the observation that at higher NO concentrations autoxidation-mediated nitrosation predominated by the second-order nature in NO of autoxidation (25), which makes little sense since they reported their mechanism to be second-order in NO as well. Moreover, to explain the second-order nature – of the reaction, they suggested that the O2 formed in the aerobic reaction reacts rapidly with another NO molecule to produce peroxynitrite (Eq. IX). However, such a fast reaction following a slow initial step would change the net stoichiometry of the reaction (from 1 NO/GSNO to 2 NO/GSNO) but not the order of the reaction. The present data, however, do support a mechanism along the lines proposed by Gow et al. The simplest way out of the conundrum is to assume that the reaction is actually first-order in NO, as is also suggested by the presented data. The second-order nature of the reaction in Gow et al. was inferred from quite noisy data, and hinged critically on one specific data point (see Fig. 2A in ref. 25). – – O2 + NO ! ONOO (IX)
4.4.1. Comparison of the reactions of NO with GSH and DTT Contrasting with the pronounced effects of GSH, DTT caused a much smaller decrease of the NO peak and only a small amount of GSNO could be detected by addition of CuSO4 (Figure 17 and Figure 18). The explanation for the difference may lie in the lower stability of nitrosated DTT (DTT-SNO) (214). Additionally, it cannot be ruled out that the reaction between DTT and NO is slower than that between GSH and NO. DTT-SNO is known to decompose along different pathways depending on the DTT/NO ratio. At low relative DTT concentrations the dinitrosated compound (DTT(SNO)2) will be formed, which decomposes to oxidized DTT and NO (214).
48 DISCUSSION
At higher relative DTT concentrations, such as applied in this study, the mononitrosated compound will predominate, which decomposes to oxidized DTT and HNO (214). The small effect of DTT on the NO peak together with the small Cu-catalyzed NO release suggest that rather little DTT-SNO is formed. When added together, the combination of DTT and GSH behaved as DTT alone. Rapid transnitrosation is expected to occur between GSH and DTT, but this reaction is expected to favor GSNO formation. Moreover, the combination would be expected to remove at least as much NO from solution as GSH alone, particularly if DTT-SNO decomposition played any part, since that reaction does not yield NO and would therefore remove even more NO from solution. The best explanation for the observations is that DTT reduces GSNO effectively to GSH and NO, thereby neutralizing the effects of GSH on the NO curves and leaving only the small effects of DTT itself.
+ 2+ 4.4.2. Effects of NAD , SOD, O2 and Mg on GSH nitrosation by NO Experiments under quasi-anaerobic conditions yielded much higher NO peaks and considerably diminished copper-catalyzed GSNO decomposition (Figure 21), indicating that the formation of GSNO from DEA/NO in the presence of GSH is O2-dependent, as previously observed. Consequently, a mechanism along the lines proposed by Gow et al. is favored (apart from the fact that the reaction is first-order in NO). I would also like to point out that the data cannot discriminate the order in which NO and O2 react, which implies that it also conceivable that NO reacts with a GSOOH/GSOO –-complex. Experiments in the absence of NAD+ or in the presence of NADH indicated that these compounds did not significantly affect NO or RSNO formation (Figure 18). These observations enhance the physiological relevance of the results. Also, although they do not + invalidate the observation by Gow et al. that NAD can substitute for O2 under anerobic + + conditions (25), they do demonstrate that NAD is a poor substitute for O2, since 1 mM NAD was unable to compete with ~0.2 mM O2, suggesting a rate constant at least two orders of magnitude smaller. Omission of SOD from the reaction mixture had little effect (Figure 18). GSH lowered the NO peak to about the same extent with and without SOD. The most prominent effect appeared to be an approx. 2-fold increase in the apparent decay rate constant of NO both in the presence and absence of GSH (Figure 20). Finally, the potential effect of Mg2+ on NO and GSNO formation was investigated. Somewhat surprisingly, it could be observed that omission of Mg2+ yielded a considerably higher NO peak (1.8±0.3-fold) and a much lower GSNO peak (0.33±0.06-fold), indicating that Mg2+ stimulates GSNO formation (Figure 27). Additional experiments demonstrated that Ca2+ has an equally stimulating effect on GSNO formation. A third divalent cation, Mn2+, also clearly stimulated GSNO formation, although in this case the interpretation is complicated by the fact that Mn2+ slowed down NO release from DEA/NO in the absence of GSH (Figure 27). An even stronger inhibition of DEA/NO decomposition precluded determination of the effect of Zn2+ (Figure 27). The monovalent cation Na+ had no effect on GSNO formation (Figure 27). Typical divalent cations like Mg2+ and Ca2+ have been reported to form weak complexes with the carboxylates of GSH (213). It is conceivable that coordination of the negative carboxylate groups increases the reactivity of the thiol group by lowering its pKa value. Alternatively, the complexes may present a more favorable conformation.
49 CONCLUSIONS
5. Conclusions Allowing for the results of the present study as a whole it can be inferred that GSH was somehow affecting ALDH2, sGC or was scavenging the mediating NO. As a result decreased sGC activities (3.2) or NO peaks (3.3) could be observed. DTT showed the ability to enable multiple more turnovers of ALDH2 and to prevent from decreased sGC activation in the presence of GSH. Two reasons could account for the inhibition by GSH and its prevention by DTT: a) GSH could restrain the ALDH2, for example by causing S-glutathiolation of an important side group. The prevention by DTT could be based on reduction of this modified group. b) In addition, it is still possible that the effect of GSH is caused by formation of GSNO, whereby GSNO would be far less effective to activate sGC under presented conditions than NO. The effect of DTT would correspond to the decomposition of GSNO, which is compatible with the remaining data. A key observation of the present study is that GSH can be directly nitrosated by DEA/NO- derived NO with high efficiency. The CuSO4-liberated NO peak from 1 "M DEA/NO amounted to approx. 1 "M. Assuming a NO over DEA/NO stoichiometry of ~1.5 (19), this corresponds to a level of nitrosation of ~2/3. This fits nicely with the observed decrease of the NO peak height to ~1/3 of that in the absence of GSH. An even greater nitrosated fraction would have been found under conditions where autoxidation rather than diffusion were the main process accounting for NO disappearance in the absence of NO. The mechanism of endogenous cellular nitrosothiol formation is a matter of much contention, but most parties agree that direct nitrosation by NO is physiologically irrelevant. From the present results, however, it can be estimated that under physiological conditions (50 "M O2, 2 mM GSH), a steady-state concentration of 1 nM NO will give rise to a rate of GSNO formation of ~1 pM/s or 3.6 "M/hr. It is difficult to assess the physiological relevance of that rate, in view of present uncertainties regarding nitrosothiol concentrations in vivo, but it would definitely fit reported values at the lower end of the spectrum (107). The present observations, therefore, put direct nitrosation by NO back in contention as a major candidate for biological nitrosothiol formation.
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62 LIST OF FIGURES / TABLES
7. List of Figures Figure 1: NO-mediated smooth muscle relaxation...... 3 Figure 2: GTN formula...... 4 Figure 3: GTN-induced rebound effect by RAAS regulatory mechanisms...... 7 Figure 4: GSNO formula...... 9 Figure 5: RSNO formation mechanisms...... 14 Figure 6: GTN metabolism by 4 "g ALDH2 ± 2 mM GSH/DTT. Incubation with 2 "M 14C-GTN for 10 min at 37°C...... 26 Figure 7: GTN metabolism by 4 "g ALDH ± 2 mM GSH ± 0-2 mM DTT. Incubation with 10 "M 14C-GTN for 2 min at 37°C; V = 200 "l...... 27 Figure 8: Activition of sGC by DEA/NO (10 nM or 1 "M) or by GTN (10 "M) and ALDH2 (2 "g) in the presence and absence of 2 mM DTT and/or GSH. Incubation with 50 ng sGC for 10 min. at 37°C; V = 100 "l...... 28 Figure 9: Activition of sGC by DEA/NO (10 nM or 1 "M) or by GTN (10 "M) and ALDH2 (4 "g) in the presence and absence of 2 mM DTT and/or GSH. Incubation with 25 ng sGC for 2 min. at 37°C; V = 200 "l...... 29 Figure 10: Activation of sGC by DEA/NO (10 nM or 1 "M) or by GTN (10 "M) and ALDH2 (4 "g) in the presence and absence of 2 mM DTT and/or GSH. Incubation with 25 ng sGC for 2 min. at 37°C; V = 200 "l...... 30 Figure 11: Activation of sGC by DEA/NO in the presence and absence of 2 mM DTT and GSH. Incubation with 25 ng sGC for 2 min. at 37°C; V = 200 "l...... 31 Figure 12: Activation of sGC by 10 "M GTN and 4 "g ALDH2 in the presence of GSH. Incubation with 25 ng sGC for 2 min. at 37°C; V = 200 "l...... 32 Figure 13: Activation of sGC by 10 "M GTN and 0 – 50 "g ALDH2 in the presence and absence of 2 mM GSH. Incubation with 25 ng sGC for 2 min. at 37°C; V = 200 "l...... 33 Figure 14: Activation of sGC by DEA/NO (10 nM or 1 "M) or by 10 "M GTN and 4 "g ALDH2 in the presence of 2 mM GSH and varying concentrations of DTT. Incubation with 25 ng sGC for 2 min. at 37°C; V = 200 "l...... 34 Figure 15: NO release by 125 "g ALDH2 in the presence and absence of 2 mM GSH and DTT. Injection of 100 "M GTN (at time zero) and 4 mM CuSO4 (indicated by arrows) at 37 °C; V = 500 "l...... 35 Figure 16: NO release (peak height) by 125 "g ALDH2 in the presence and absence of 2 mM GSH and DTT. Injection of 100 "M GTN and 4 mM CuSO4 (GSNO) at 37 °C; V = 500 "l...... 36 Figure 17: NO release from 1 "M DEA/NO in the presence and absence of 2 mM DTT and GSH. Injection of DEA/NO at zero time and of 4 mM CuSO4 after 720s (arrow) at 37 °C in the presence of SOD and NAD+; V = 500 "l...... 37 Figure 18: Effect of NAD+ and NADH on NO release from 1 "M DEA/NO in the presence and absence of 2 mM DTT and GSH. Conditions as for Fig. 17; V = 500 "l...... 38
Figure 19: NO release from 1 "M DEA/NO before and after addition of 4 mM CuSO4 in the absence and presence of 2 mM GSH at 37 °C in the absence of SOD; V = 500 "l...... 39
63 LIST OF FIGURES / TABLES
Figure 20: NO release from 1 "M DEA/NO before and after addition of 4 mM CuSO4 (arrows) in the absence and presence of 2 mM GSH at 37 °C in the absence of SOD; V = 500 "l...... 39
Figure 21: NO release from 1 "M DEA/NO before and after addition of 4 mM CuSO4 (arrows) in the presence of 2 mM GSH at 37 °C; V = 500 "l...... 40 Figure 22: NO release from 10 – 1,000 nM DEA/NO in the presence and absence of 2 mM GSH before and after addition of 4 mM CuSO4 (GSNO) at 37 °C in the presence of SOD and NAD+...... 41 Figure 23: Linear representation of the semi-logarithmic plots in Fig. 22...... 42 Figure 24: Linear representation of the semi-logarithmic plots in Fig. 22 in the concentration range from 0 to 400 nM DEA/NO...... 42 Figure 25: Linear representation of the semi-logarithmic plots in Fig. 22 in the concentration range from 0 to 50 nM DEA/NO...... 43 Figure 26: Representative curves of the NO release from DEA/NO in the concentration range between 50 nM and 1 "M in the presence of 2 mM GSH before and after addition of 4 mM CuSO4...... 43 Figure 27: NO release from 1 "M DEA/NO in the presence and absence of 2 mM GSH and the cations Mg2+, Ca2+ Na+, Mn2+, and Zn2+ (5 mM each), before and after + addition of 4 mM CuSO4 at 37 °C in the absence of SOD and NAD ; V = 500 "l...... 44 Figure 28: NO release from 1 "M DEA/NO in the absence of GSH and in the presence of the 2+ 2+ 2+ Mg , Mn , and Zn (5 mM each), before and after addition of 4 mM CuSO4 at 37 °C in the absence of SOD and NAD+; V = 500 "l. Curves in the presence of Ca2+ and Na+ (not shown) were all but identical to the one shown for Mg2+...... 45
8. List of Tables Table 1: Basic reaction mixtures of GTN denitration assays...... 21 Table 2: Basic reaction mixtures of sGC activity assays...... 23 Table 3: Basic reaction mixtures for NO determination...... 25
64 ABSTRACT
9. Abstract Nitric oxide (NO) is an important biomolecule with a vast range of functions in signal transduction and the immune response. The majority of its functions in signal transduction are mediated by its most important target, the NO-sensitive (soluble) isoform of guanylate cyclase (sGC). However, under some conditions NO will be converted to metabolites with distinct properties, that may alter its (patho)physiological impact. Nitrosothiols are endogenously occurring formal adducts of protein- or low molecular weight thiols with the 1- electron oxidized form of nitric oxide, NO+. There is growing awareness in the scientific community that nitrosothiols may perform distinct functions in biology. Since nitrosothiols will release NO under certain conditions and are generally more stable than NO, they may function as a storage and transport form of NO. However, nitrosothiols also exhibit biological actions completely different from NO, since the nitrosation of specific cysteine residues on proteins will alter their function. Hence, S-nitrosation is increasingly regarded as a post- translational modification akin to phosphorylation. Despite these potential (patho)physiological ramifications, however, there is still no consensus about the way in which nitrosothiols may be generated cellularly. Probably the best-studied pathway of nitrosothiol formation is the aerobic reaction of NO with thiols. Anaerobically, NO does not form nitrosothiols, but in the presence of oxygen, nitrosothiols are formed in a reaction that is first order in O2 and second order in NO. Because of the low (submicromolar) concentrations of NO and the second-order nature in NO of the reaction, this pathway is expected to be too slow to make a significant contribution under physiological conditions. The present study demonstrates that in the submicromolar domain, the aerobic nitrosation of glutathione by NO is first order in NO and far more efficient than previously thought, making it a serious candidate as the origin of nitrosothiol formation in vivo. Furthermore, a decreasing effect of glutathione on the NO-release from glycerol trinitrate (GTN) by aldehyde dehydrogenase 2 (ALDH2) and the enzymatic activity of sGC, probably by formation of nitrosothiols, was shown under specific conditions. Dithiothreitol (DTT) was found partially to prevent decrease.
65 ABBREVIATIONS
10. Abbreviations
[14C]GTN [2-14C]Glycerol-1,2,3-trinitrate [32P]GTP Guanosine-5’-[&32P]-triphosphate AC Adenylate cyclase ACE Angiotensin I – converting enzyme ACh Acetylcholine ADH Antidiuretic hormone
Al2O3 Aluminium oxide ALDH Aldehyde dehydrogenase ATP Adenosine triphosphate ATPase basolateral Na+/K+ ATPase BK Bradykinin
CaCl2 Calcium chloride CaM Calmodulin cAMP cyclic adenosine monophosphate cGMP cyclic guanosine monophosphate cpm Counts per minute
CuSO4 Copper sulfate Cyp450 Cytochrome P450 DAG Diacyl glycerol DEA/NO 2,2-diethyl-1-nitroso-oxyhydrazine dpm Disintegrations per minute DTPA Diethylene triamine pentaacetic acid DTT 1,4-dithio-DL-threitol EDTA Ethylene diamine tetraacetic acid eNOS endothelial nitric oxide synthase ER endoplasmatic reticulum ET Endothelin EtOH Ethanol GDN Glycerol dinitrate GSH Glutathione, #-L-Glutamyl-L-cysteinylglycine GSNO S – Nitrosoglutathione GST Glutathione-S-transferase GTN Glycerol trinitrate GTP Guanosine triphosphate 66 ABBREVIATIONS
H Histamine iNOS inducable nitric oxide synthase
IP3 Inositol triphosphate
IRAG IP3 – receptor associated cGMP – kinase substrate
K2HPO4 Potassium hydrogen phosphate KCl Potassium chloride KI Potassium iodide MES 2-(N-morpholino)ethanesulfonic acid
MgCl2 Magnesium chloride
MgSO4 Magnesium sulfate (anhydrous) MLCP Myosin light chain phosphatase
MnCl2 Manganese chloride NA Noradrenalin
Na2CO3 Sodium carbonate NaCl Sodium chloride NAD+ Nicotinamide adenine dinucleotide NADH Nicotinamide adenine dinucleotide (reduced) NADPH Nicotinamide adenine dinucleotide phosphate
NaNO2 Sodium nitrite Neocuproine 2,9-dimethyl-1,10-phenanthroline nNOS neuronal nitric oxide synthase NO Nitric oxide
PIP3 Phospatidyl inositol biphosphate PKB Protein kinase B PKG Protein kinase G PLC Phospholipase C RSNO S - Nitrosothiol sGC Soluble guanylate cyclase SOD Superoxide dismutase TEA Triethanolamine TRX Thioredoxin TRX-R Thioredoxin reductase VASP Vasodilator stimulated phosphoprotein
ZnCl2 Zinc chloride
67