Karl Franzens University Graz Faculty of Natural Sciences Institute of Pharmaceutical Sciences

Department of Pharmacology and Toxicology Chair: O. Univ.-Prof. Dr. Bernd Mayer

ROLE OF ALDEHYDE DEHYDROGENASE-2 LOCALIZATION IN NITROGLYCERIN-INDUCED VASODILATION

Diploma thesis submitted by

Corina Theresia Madreiter

For the award of the academic degree

Mag.a pharm.

July, 2012

Diese Diplomarbeit ist meinem wissenschaftsbegeisterten Großvater Johann Nitzinger gewidmet.

CONTENTS

Contents ...... 1 Introduction...... 2 Clinical use of glyceryl trinitrate ...... 2 Role of NO in endothelium-dependent vasodilation ...... 4 Bioactivation of GTN ...... 6 Nitrate tolerance ...... 8 Working hypothesis and aims...... 10 Materials and methods ...... 11 Chemicals ...... 11 Rodents ...... 14 Porcine and bovine coronary arteries ...... 15 Human coronary arteries ...... 15 Organ bath experiments ...... 16 Sample preparation and protein determination ...... 19 Gel electrophoresis (SDS-PAGE) ...... 20 Western blot ...... 22 Statistical analysis ...... 24 Results ...... 25 Vasodilation in aortas and coronary arteries ...... 25 Subcellular localization of ALDH2 ...... 28 Discussion ...... 31 Abstract ...... 34 Zusammenfassung ...... 35 Acknowledgment ...... 36 References ...... 37 Abbreviations ...... 41

1 INTRODUCTION

Clinical use of glyceryl trinitrate

Due to its vasodilating effect nitroglycerin (glycerol trinitrate; GTN) has been used as anti-ischemic drug since the late nineteenth century. The beneficial clinical effect is caused by dilation of large coronary arteries, resulting in improved blood supply to the heart, and venodilation, which increases venous pooling and consequently reduces cardiac preload [1].

Besides GTN other organic esters, like isosorbide mononitrate, isosorbide dinitrate or pentaerythritol tetranitrate (Figure 1) are used for the treatment of coronary artery disease. Because of their rapid action organic nitrates - especially GTN - are the first choice in the treatment of acute angina pectoris. However, the clinical use of organic nitrates is limited by the development of tolerance after long-term continuous application [2]. To take advantage of its rapid action, GTN is administered as a spray or sublingual tablet (single doses 0.4-2.4 mg) in situations of acute stenocardia. Since GTN shows a high first pass-effect in the liver after oral administration, transdermal therapeutic systems are frequently used for chronic treatment [3]. To avoid loss of efficacy after 24-48 h of continuous application (nitrate tolerance), nitrate-free intervals – usually overnight - are necessary [4]. However, intermitted application may lead to anginal episodes [5].

Other common side effects include hypotension, which is often asymptomatic, but may be accompanied by headache, slower heart rate, and syncopes. Furthermore coronary steal and myocardial ischemia are sometimes reported [5].

Since phosphodiesterase 5 inhibitors used for the treatment of erectile dysfunction can potentiate the hypotensive effect of nitrovasodilators, even resulting in death, their co-administration is strictly contraindicated in nitrate therapy [5].

2

Glycerol trinitrate (GTN) Isosorbide dinitrate (ISDN)

Isosorbide mononitrate (ISMN) Pentaerythritol tetranitrate (PETN) Figure 1: Organic nitrates

3 Role of NO in endothelium-dependent vasodilation

The wall of blood vessels is composed of three layers. • The inner coat, tunica intima , consists of flattened endothelial cells, which are resting on a thin layer of connective tissue. • Tunica media , containing spindle-shaped, smooth muscle cells as well as elastin and collagen fibers, is responsible for mechanical strength and contractile power. • Tunica adventitia provides a loose connection to the surrounding tissue. Moreover, the adventitia of most vessels contains sympathetic fiber terminals to regulate local resistance and blood flow [6].

In 1980 it was reported that acetylcholine (ACh) requires the presence of endothelial cells to promote vasodilation through the release of a substance, named endothelium-derived relaxing factor (EDRF) [7]. Seven years later EDRF was identified as nitric oxide (NO) [8] and NO-mediated vasodilation found its way into standard textbooks of pharmacology.

As shown in Figure 2, ACh binds to the muscarinic receptor M3 [9], a G-protein coupled receptor that is expressed on endothelial cells, resulting in activation of phospholipase C. This enzyme catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP 2) into inositol 1,4,5-trisphosphate (IP 3) and diacyl glycerol (DAG). IP 3 binds to the IP 3-receptor of the endoplasmatic reticulum and causes the release of Ca 2+ into the cytoplasm. The binding of Ca 2+ to calmodulin (CaM) triggers high affinity binding of Ca 2+ /CaM to endothelial NO synthase (eNOS) [3]. Another mechanism responsible for up-regulation of eNOS activity is the phosphatidylinositol 3 kinase/Akt signaling pathway, which is activated by mechanical stress and various agonists, including vascular endothelial growth factor (VEGF) [10]. Furthermore, shear stress and growth factors lead to activation of AMP-activated protein kinase (AMPK), resulting in phosporylation of eNOS at Ser633 and Ser1177 [11]. Endothelial NOS catalyzes the oxidation of L-arginine to L-citrulline and NO, which diffuses into smooth muscle cells and activates soluble guanylate cyclase (sGC) through binding to a

4 regulatory heme group. As a result GTP is converted to cyclic GMP (cGMP) [3]. The cGMP signaling pathway affects three downstream targets: Protein kinase G (PKG), cGMP-regulated phosphodiesterases, and cyclic-nucleotide-gated channels [12]. PKG reduces intracellular Ca 2+ and the function of contractile 2+ elements through several mechanisms, including inhibition of IP 3-dependent Ca release from the sarcoplasmatic reticulum [13], increased Ca 2+ uptake into the sarcoplasmatic reticulum [14] and decreased extracellular Ca 2+ influx [15]. The lower intracellular Ca 2+ concentration reduces myosin light chain (MLC) phosphorylation by MLC kinase (MLCK), which is essential for contraction of muscle cells [16]. Moreover, PKG activation results in inhibition of the Rho- associated coiled-coil containing protein kinase (ROCK) pathway [17]. This leads to activation of MLC phosphatase (MLCP) and, therefore, reduced phosphorylation of MLC [18].

Figure 2: The role of NO in endothelium-dependent vasodilation.

5 Bioactivation of GTN

Although the physiology of NO in vasorelaxation has been clarified, the biochemical mechanisms underlying the pharmacological action of organic nitrates are still elusive. In contrast to the NO ● donor diethylamine/NONOate (DEA/NO), the prodrug GTN has to be bioactivated to release NO or a related species (NOx) and causes nitrate tolerance [19]. There has been extensive research to identify molecules or enzymes that are involved in the GTN metabolism, which can be clearance-based, resulting in nitrite formation, or mechanism-based, yielding NO or NOx [20].

Reduced thiols react with GTN in a non-enzymatic manner, yielding 1,2-glycerol dinitrate (1,2-GDN) and 1,3-glycerol dinitrate (1,3-GDN) together with inorganic nitrite. This reaction seems to be clearance-based without concomitant formation of NOx [21]. A notable exception is the reaction with cysteine and some cysteine derivates, which does indeed lead to NO-mediated activation of sGC in vitro [22]. Since high concentrations of GTN and cysteine are needed, this reaction may not be significant in vivo [23]. Therefore enzymatic metabolism of GTN appears to be more relevant. Several enzymes have been proposed to metabolize GTN, including GSH-S-transferase, cytochrome P450, cytochrome P450 reductase and xanthine oxidase [4]. In 2002 mitochondrial aldehyde dehydrogenase-2 (ALDH2) was identified as the key enzyme catalyzing GTN-bioactivation.

ALDH2 is one of the 19 members of the aldehyde dehydrogenase superfamily, consisting of enzymes responsible for the conversion of aldehydes into the corresponding carboxylic acids. ALDH2 is a homotetramer with subunits of approximately 54 kDa. Its main function is the detoxification of ethanol-derived acetaldehyde in the liver [24]. Besides its classical dehydrogenase activity, ALDH2 catalyzes hydrolysis of ester substrates [24] and in 2002, ALDH2 was identified as a major GTN-metabolizing enzyme in cultured RAW 267.4 macrophages [25]. Based on the mitochondrial localization of liver ALDH2 [26], ALDH2 is commonly designated as mitochondrial aldehyde dehydrogenase, to distinguish it from other

6 isoforms, like ALDH1, an exclusively cytosolic protein (25). However, recent data from our group demonstrated that ALDH2 is mainly (85-95 %) cytosolic in mouse aortas and human coronary arteries [27]. Although the biochemical mechanism of GTN bioactivation by ALDH2 is still elusive, the initial step seems to be nucleophilic attack on GTN by Cys302 of ALDH2, forming a thionitrate intermediate and releasing 1,2-GDN. Cys302 and one of the adjacent cysteine residues (Cys301 or Cys303) appear to be involved in the main pathway of GTN bioconversion, yielding nitrite and a protein disulfide [20]. Reducing agents like dithiothreitol (DTT) and dihydrolipoic acid (LPA-H2) restore ALDH2 activity by reduction of the disulfide intermediate [28]. In contrast, the reaction with Glu268, which is not essential for GTN denitration but may be involved in structural organization of the NAD-binding pocket [29], might result in heterolysis of the sulfenyl nitrite, which possibly yields a protein-sulfinate and nitroxyl. Formation of a sulfinate, which is not reducible by DTT, would explain irreversible inactivation of the enzyme upon exposure to GTN [20]. Combined lack of the cysteines and Glu268 shifts the reaction towards formation of free NO radicals, most probably through homolytic cleavage of the sulfenyl nitrite intermediate. This third pathway seems to be essential in GTN bioactivation, although it accounts just for 5 to 10 % of total GTN turnover [20].

7 Nitrate tolerance

Continuous application (24-48 h) of organic nitrates leads to decreased responsiveness of blood vessels to organic nitrates and loss of efficacy of GTN [30]. This phenomenon of nitrate tolerance caused the so-called “Monday disease” of workers in dynamite factories. These workers were exposed to high levels of organic nitrates during the week and suffered from headache, due to vasodilation, only after weekends, when they had lost nitrate tolerance [31].

In addition to pseudo-tolerance, which is caused by a neurohormonal response to nitrate therapy, possibly resulting in reflectory release of endogenous vasoconstrictors [32], several intrinsic processes appear to be involved in the development of vascular nitrate tolerance.

It has been demonstrated that continuous GTN treatment is associated with increased vascular superoxide production [33]. Superoxide reacts with NO radicals ● ●- - to form peroxynitrite ( NO + O 2 -> ONOO ) and therefore limits NO bioavailability [34]. Observations indicate that most of the cytotoxicity attributed to NO is rather due to peroxynitrite. Peroxynitrite interacts with lipids, DNA, and proteins via direct oxidative reactions or via indirect, radical-mediated mechanisms. These reactions trigger cellular responses ranging from subtle modulations of cell signaling to overwhelming oxidative injury [35]. The hypothesis of vascular superoxide production is also supported by the observation that supplementation of the antioxidant vitamin C partially prevents nitrate tolerance [36].

Another possible explanation for nitrate tolerance is the inactivation of ALDH2. As mentioned before, GTN seems to be metabolized through three distinct pathways, one of which (5-10 % total GTN metabolism) accounts for NO formation, whereas the other two pathways appear to be clearance-based and unrelated to GTN bioactivation. These clearance-based pathways could contribute to nitrate tolerance through partially irreversible enzyme oxidation [20]. The hypothesis of three distinct pathways contributing to a different extent to the development of nitrate tolerance fits well to a study on PETN [37]. Like GTN,

8 PETN is bioactivated by ALDH2, but, in contrast to GTN, PETN does not cause nitrate tolerance. This observation may be explained by a very low rate of ALDH2 inactivation, most likely as a result of the low affinity of PETN for the clearance-based denitration pathway [37]. Since oxidized ALDH2 needs a reductant to restore its activity, reductant depletion after long term GTN application may contribute to the development of nitrate tolerance [38]. Reducing agents like DTT and ascorbate, which prevent nitrate tolerance, are able to partly restore ADLH2 activity, possibly by reduction of oxidized thiols [28].

Besides a decrease in ALDH2 activity, changes in ALDH2 protein expression during continuous GTN exposure were also observed. However, after a drug-free period, vasodilator responses to GTN and the rates of GTN denitration had returned to control values, whereas vascular ALDH2 expression and aortic ALDH activity were still significantly depressed [39]. These observations indicate that other factors than ALDH2-mediated GTN bioactivation contribute to nitrate tolerance.

9 WORKING HYPOTHESIS AND AIMS

Recent data from our group demonstrated that ALDH2 is mainly (85-95 %) cytosolic in mouse aortas and human coronary arteries. Together with the observation that GTN-induced relaxation of ALDH2-deficient aortic rings was restored by overexpression of ALDH2 in cytosol, whereas targeting of the protein to mitochondria had no effect, these findings challenge the general view according to which ALDH2-catalyzed GTN bioactivation takes place in vascular smooth muscle mitochondria. Interestingly, the response to GTN of mouse aortas with exclusively cytosolic expression of ALDH2 was monophasic, whereas relaxation of wildtype vessels (expressing approximately 15 % of ALDH2 in the mitochondria) was biphasic, suggesting that ALDH2 in mitochondria might counteract vascular GTN bioactivation, possibly through superoxide-mediated inactivation of GTN- derived NO.

It was the aim of my diploma work to test this hypothesis by comparing the characteristics of GTN-induced vasodilation with the subcellular distribution of ALDH2 in the following blood vessels: murine, rat, guinea pig and rabbit aorta and bovine, porcine and human coronary arteries. I carefully analyzed the data for a potential correlation of the relative amount of mitochondrial ALDH2 with GTN potency or biphasic response to the organic nitrate. In addition, I compared the expression levels of cytosolic and mitochondrial ALDH in arteries and veins to see whether the claimed venoselectivity of organic nitrates is due to differential subcellular localization of ALDH2.

10 MATERIALS AND METHODS

Chemicals

Acrylamide 30 % Electrophoresis Reagent Sigma-Aldrich

Ammonium persulfate (APS) Sigma-Aldrich

Calcium chloride CaCl 2 Fluka

Roche Complete Protease and Phosphatase Inhibitor Cocktail Diagnostics GmbH

D(+)-Glucose-Monohydrate Fluka

Diethyl ether Merck

Disodium hydrogen phosphate Na 2HPO 4 Sigma-Aldrich

ECL TM Prime Western Blotting Detection Reagent GE Healthcare

Ethylenediaminetetraacetic acid Sigma-Aldrich (EDTA)

11

Glycine Sigma-Aldrich

Hydrochloric acid HCl Merck

Ketamine Graeub

Magnesium chloride MgCl 2 Sigma-Aldrich

2-Mercaptoethanol Sigma-Aldrich

Medetomidine Orion Pharma

Methanol CH 3OH Merck

Milk powder Non-fat dry milk BioRAD

2-(N-Morpholino) ethanesulfonic acid Sigma-Aldrich (MES) N,N,N’,N’– Tetramethylethylenediamine Sigma-Aldrich

(TEMED)

Pohl Boskamp (Nitro POHL ® Nitroglycerin ampoules )

Thermo PageRuler TM Prestained Protein Ladder Scientific

12 Phenylmethylsulfonyl fluoride Sigma-Aldrich (PMSF)

Thermo Pierce® BCA Protein Assay Kit Bicinchoninic acid Scientific

Ponceau S Staining Solution Sigma-Aldrich

Potassium chloride KCl Fluka

Potassium dihydrogen phosphate KH 2PO 4 Merck

Potassium hydrogen phosphate K2HPO 4 Merck

Thermo Restore TM Western Blot Stripping Buffer Scientific

Sodium cloride NaCl Fluka

Sodium hydrogen carbonate NaHCO 3 Fluka

Sodium hydroxide NaOH Sigma-Aldrich

Sodium lauryl sulfate (SDS) Sigma-Aldrich

Sucrose Sigma-Aldrich

13 Trans-Blot Paper BioRAD

Trizma® base Tris(hydroxymethyl) Sigma-Aldrich aminomethane

Tween® 20 Sigma-Aldrich

9,11-dideoxy-11 α,9 α∝- U-46619 epoxymethanoprostaglandin Sigma-Aldrich thromboxane A2 receptor agonist F2 α

Rodents

All animals (Table 1) received care in accordance with the Austrian law on experimentation with laboratory animals (last amendment, 2005), which is based on the U. S. National Institutes of Health guidelines. Studies were performed with wild type animals of either sex.

Animals Weight Strain Guinea pigs 400-500 g Dunkin -Hartley Mice 25-35 g C57BL/6 Rabbits 4,000-5,000 g New Zealand Rats 250-350 g Sprague Dawley Table 1: Rodents used in the present work .

14 Porcine and bovine coronary arteries

Porcine and bovine right coronary arteries were freshly obtained from a local slaughter house and cleaned from the surrounding adipose tissue before experiments.

Human coronary arteries

After approval by the local ethical committee, pieces of the arteria coronaria dextra and vena cordis magna (underlined in Figure 3) were explanted from six non- failing hearts not suitable for transplantation. Vessels were cleaned from the surrounding adipose tissue before experiments.

Figure 3: Anatomy of the heart; Hafferl, A., Lehrbuch der topographischen Anatomie , Springer-Verlag, 1957

15 Organ bath experiments

Mice, rats and guinea pigs were anaesthetized with diethyl ether. Afterwards mice got a lethal dose of urethane (1 g/kg), rats and guinea pigs were decapitated. Rabbits were narcotized with ketamine (10 mg/kg) and medetomidine (1 mg/kg) before exsanguination. Aortas (from mice, rats, guinea pig and rabbits) and right coronary arteries (from pig, cow and human) were cut into pieces of approximately 3-4 mm length. Aortic and coronary rings were suspended in 5 ml organ baths for isometric tension measurements, containing oxygenated Krebs-Henseleit buffer (Figure 4).

force transducer

tissue holder

tissue holder

outlet of prewarmed water prewarmed buffer

vessel inlet of

prewarmed water inlet of oxymix (95% O 2/5% CO 2) outlet of buffer Figure 4: Organ bath

To prepare 1 liter of Krebs-Henseleit buffer 2 g glucose monohydrate (final concentration: 10 mM) and 2.1 g NaHCO 3 (final concentration: 25 mM) were

16 added to 50 mL of stock solution 1, 2 and 3 (all stored at 4 °C) and filled up with distilled water (Table 2).

Solution 1 for Krebs-Henseleit buffer Volume: 1 l

NaCl 138.40 g KCl 7.46 g

KH 2PO 4 3.24 g

Distilled H 2O Ad 1,000 ml Solution 2 for Krebs-Henseleit buffer Volume: 1 l

CaCl 2.2H 2O 7.35 g

Distilled H 2O Ad 1,000 ml Solution 3 for Krebs-Henseleit buffer Volume: 1 l

MgCl 2.6H 2O 4.88 g

Distilled H 2O Ad 1,000 ml Table 2: Krebs-Henseleit buffer

Vessels were equilibrated for 60 minutes at basal tension of 0.5-2.0 g (Table 3).

Animal Vessel Basal tension Cow Coronary Artery 2.0 g Guinea Pig Aorta 1.0 g Human Coronary Artery 1.0 g Mouse Aorta 0.5 g Pig Coronary Artery 2.0 g Rabbit Aorta 2.0 g Rat Aorta 1.0 g Table 3: Basal tension

After setting the basal tension, vessels were contracted three times with 60 mM KCl-solution (Table 4) to test for appropriate contractile function.

17 60 mM KCl-solution Volume: 1 l

NaCl 3.56 g KCl 4.47 g

KH 2PO 4 0.16 g

CaCl 2.2H 2O 0.37 g

MgCl 2.6H 2O 0.24 g Glucose monohydrate 2.00 g

NaHCO 3 2.10 g

Distilled H 2O Ad 1,000 ml Table 4: 60 mM KCl-solution

After a wash out step, rings were contracted with thromboxane A2 receptor agonist 9,11-dideoxy-11 α,9 α-epoxymethanoprostaglandin F2 α (U-46619; 50 nM). As contraction had reached a stable plateau, cumulative concentration-response curves for GTN (0.1 nM-100 µM) were established, as reported in Table 5.

Initial GTN concentration Pipetted volume Final GTN concentration 50 nM 10 µl 0.1 nM 50 nM 20 µl 0.3 nM 500 nM 7 µl 1 nM 500 nM 20 µl 3 nM 5 µM 7 µl 10 nM 5 µM 20 µl 30 nM 50 µM 7 µl 100 nM 50 µM 20 µl 300 nM 500 µM 7 µl 1 µM 500 µM 20 µl 3 µM 4,4 mM 8 µl 10 µM 4,4 mM 22,7 µl 30 µM 4,4 mM 80 µl 100 µM Table 5: Pipetting scheme applied to establish cumulative GTN concentration- response curves.

18 Contractile force corresponding to each agonist concentration was recorded and expressed as percent of precontraction [40]. Rings were snap-frozen in liquid nitrogen and stored at –80 °C for western blot anal ysis.

Sample preparation and protein determination

20-100 µg of blood vessels were homogenized with a rough glass potter in 200-400 µl TES+ buffer (Table 6, Table 7). Volume of TES+ and number of strokes depended on stiffness and tissue size.

TES buffer Volume: 1 l

Trizma 6.06 g Sucrose 85.58 g EDTA 1 M 500 µl HCl -> pH 7.2

Distilled H 2O Ad 1,000 ml Table 6: TES buffer

TES+ buffer Volume: 1,040 µl

TES buffer 1,000 µl PMSF 100 mM 20 µl Proteinase inhibitor solution (complete stock) 20 µl Table 7: TES+ buffer

Homogenates were centrifuged at 510 g for five minutes at 4 °C to remove large pieces of tissue. To separate cytosolic and crude mitochondrial fractions, homogenates were centrifuged at 20,800 g for ten minutes at 4 °C. Supernatants (cytosolic fractions) were transferred to fresh tubes, while pellets (crude mitochondrial fractions) were re-suspended in half of the volume of TES+ that was used to homogenize the tissues. For determination of the protein concentration with BCA protein assay kit, 1:5 and 1:10 dilutions of each fraction were prepared in MES buffer (Table 8).

19 MES buffer for BCA Volume: 1 l

MES 19.52 g

MgCl 2 (2 M solution) 100 µl

Distilled H 2O Ad 1,000 ml Table 8: MES buffer for BCA

25 µl of standard-solution (albumin-solution at different concentrations) or samples as well as 200 µl of bicinchoninic acid assay (BCA) protein assay kit reagent was added into each well of the 96-well plate. After 30 minutes at 37 °C the color change from green to violet – depending on the protein concentration – was measured at 550 nm on a Titertek photometer. The method is based on the reduction of Cu 2+ by peptide bonds to Cu +, which forms a purple chelate-complex with two bicinchoninic acid molecules.

Gel electrophoresis (SDS-PAGE)

60 µl of each homogenate was added to 15 µl of Laemmli buffer 5 × and then boiled for 5 minutes at 95 °C to avoid protein aggr egation.

Laemmli buffer stock Volume: 200 ml

Trizma 7.55 g Glycerol 100 ml SDS 20 g Bromophenol blue 100 mg HCl -> pH 6.8

Distilled H2O Ad 200 ml Table 9: Laemmli buffer stock To obtain 1 ml of Laemmli buffer 5 ×, 750 µl of stock solution (Table 9) was mixed with 250 µl of 2-mercaptoethanol.

20 Separating gels were prepared by casting 12 % running gels (Table 10) and 5 % stacking gel (Table 11) as described in the following tables.

Running Gel 12 % For one gel

Distilled water 2,550 µl

1.5 M Tris-HCl, pH 8.8 18.18 g Tris / 100 ml H 2O 1,950 µl With HCl -> pH 8.8 Polyacrylamide 30 % 3,000 µl SDS 10 % 75 µl

APS 10 % 1 g APS / 10 ml H 2O 75 µl TEMED 20 µl Table 10: Running Gel 12 %

Stacking Gel For two gels

Distilled water 3,500 µl

1.5 M Tris-HCl, pH 6.8 12.12 g Tris / 100 ml H 2O 625 µl With HCl -> pH 6.8 Polyacrylamide 30 % 825 µl SDS 10 % 50 µl

APS 10 % 1 g APS / 10 ml H 2O 50 µl TEMED 20 µl Table 11: Stacking Gel

Loading slots were washed with distilled water before loading the samples. Gels were then placed into the running chamber and the inner chamber was filled with electrode buffer, which was prepared from a 10-fold concentrated stock (Table 12).

21 Electrode buffer (10-fold concentrated) Volume: 1 l

Glycine 160 g Trizma 30.2 g SDS 10 % 100 ml Distilled water Ad 1,000 ml Table 12: Electrode buffer (10-fold concentrated) Glycine and Trizma were dissolved in about 500 ml pre-warmed distilled water, before bringing the volume to 900 ml with distilled water and 100 ml SDS 10 %. To obtain electrode buffer , 900 ml of distilled H 2O was mixed with 100 ml of a 10-fold concentrated stock (Table 12).

Samples were loaded onto the gels, which were run at 250 V for approximately 30 minutes (sufficient separation of the colored standard proteins).

Western blot

After gel electrophoresis, the stacking gel was removed and the running gel was washed with cooled transfer buffer to remove traces from SDS, which could reduce the efficiency of transfer on nitrocellulose.

Transfer buffer (10-fold concentrated) Volume: 1 l

Glycine 160 g Trizma 30.2 g

Distilled H 2O Ad 1,000 ml Table 13: Transfer buffer (10-fold concentrated) Glycine and Trizma were dissolved in about 500 ml pre-warmed distilled water, before bringing the volume to 1000 ml with distilled water. To obtain transfer buffer, 700 ml of distilled H 2O was mixed with 200 ml of methanol and 100 ml of a 10-fold concentrated stock (Table 13).

22 The Western blot sandwich was built by putting a piece of nitrocellulose over the running gel between two pieces of filter paper and two sponges. The sandwich was then inserted in the blotting chamber containing transfer buffer, and proteins were electrophoretically transferred on nitrocellulose for 50-55 minutes at a constant current of 320 mA. After electroblotting, membranes were collected, incubated in Ponceau S for 3-5 minutes, and washed with distilled water. Paper copies were made for documentation. Depending on the target size of the proteins, visualized after Ponceau S staining, membranes were cut into horizontal stripes. After wash out of the Ponceau S staining with PBST, non-specific sites were blocked by shaking at room-temperature with PBSTM (5 %) for one hour.

PBS (10-fold concentrated) Volume: 1 l

NaCl 80 g KCl 2 g

Na 2HPO 4.2H2O 14.4 g

KH 2PO 4 2 g

Distilled H 2O Ad 1,000 ml Table 14: PBS (10-fold concentrated) 1 liter PBST was prepared by dilution of a 10-fold concentrated stock (Table 14) and addition of 500 µl Tween-20. PBSTM was prepared dissolving 2.5 g of milk powder in 50 ml PBST.

After 1 hour the milk solution was replaced by 10 ml of fresh PBSTM and specific antibodies (Table 15) were added. For detection of the proteins of mice, rats, guinea pigs, and humans the ALDH2 antibody of Dr. Weiner was used [27]. Since porcine and bovine proteins did not react with this antibody, the antibody from Acris Antibodies was used. Membranes were incubated with primary antibodies by shaking at 4 °C overnight.

23 Primary Traget Amount of Secondary Dilution Supplier antibody Size antibody antibody 10 µl Dr. Henry ALDH 2 54 kDa 1:5,000 Rabbit 1:20 dil. Weiner Acris ALDH 2 54 kDa 1:5,000 2 µl Goat Antibodies 5 µl β-actin 43 kDa 1:100,000 Mouse Sigma-Aldrich 1:100 dil. Table 15: Primary Antibodies

On the following day the membrane stripes were washed three times with PBST for 5 minutes and afterwards secondary antibodies (Table 16) were added in 10 ml PBSTM. Incubation lasted one hour.

Secondary Dilution Amount of antibody Supplier antibody Goat 1:100,000 1 µl; 1:100 dil. Sigma-Aldrich Mouse 1:5,000 2 µl Sigma-Aldrich Rabbit 1:5,000 2 µl Sigma-Aldrich Table 16: Secondary Antibodies

Membranes were washed three times with PBST for 5 minutes and afterwards incubated with ECL reagent for 5 minutes. The membrane stripes were collected, put into a transparent envelope, and band intensity was recorded using a densitometer [27].

Statistical analysis

One-way unpaired analysis of variance (ANOVA) with post hoc Student-Newman- Keuls test was used for comparison between groups. P values <0.05 were considered as statistically significant.

24 RESULTS

Vasodilation in aortas and coronary arteries

The relaxation of aortic rings induced by increasing concentrations of GTN was determined in organ baths by isometric tension measurements. Individual concentration-response curves, obtained from different vessel segments of a species, were averaged and counted as n=1.

Figure 5: Nitroglycerin (GTN)-induced aortic relaxation in rodents. Contractile force of wild type aortas of mice, rats, guinea pigs and rabbits was measured in response to GTN (10 -10 -10 -4 mol/L) and is expressed as percent of pre-contraction produced by U-46619 (50 nM). Data are mean values ± standard error; 30 rings from 9 mice; 40 rings from 5 rats; 46 rings from 6 guinea pigs; 38 rings from 5 rabbits. ANOVA analysis of concentration-response curves revealed significant differences between all species (P<0.05).

25 As shown in Figure 5, GTN had a noteworthy biphasic effect in aortic rings of mice and rabbits, while aortic rings of rats and guinea pigs exhibited a monophasic response to GTN. Relaxation curves of rabbit aortas were considerably different from those obtained with blood vessels from other species. Even concentrations higher than 100 µM GTN did not improve relaxation. Notably, several laboratories reported monophasic curves, with virtually complete relaxation of rabbit aortas [25, 41, 42]. Since the method is well established in our laboratory and rabbits got no special diet, it is hard to find an explanation for the different reaction compared to what was reported previously. Differences in the set up – different basal tension or pre-contraction with phenylephrine [25, 41, 42] – could be possible reasons.

Figure 6: Nitroglycerin (GTN)-induced relaxation of coronary arteries. Contractile force of bovine, porcine and human right coronary arteries was measured in response to GTN (10 -10 -10 -4 mol/L) and is expressed as percent of pre-contraction produced by U-46619 (50 nM). Data are mean values ± standard error; 40 rings from 5 pigs; 27 rings from 5 cows; 27 rings from 5 humans. ANOVA analysis of concentration- response curves revealed significant differences between all species (P<0.05).

As shown in Figure 6, GTN had a monophasic effect in porcine, bovine, and human coronary arteries.

26 Since aortas of mice, rabbits, and rats, though to a much lower extent, exhibited biphasic responses to GTN, EC 50 values (Table 17) were calculated for the high affinity pathway (mouse, rat: 0.1 nM-1 µM; rabbit: 0.1 nM-10 µM). To make comparison between aortas possible, EC 50 values for relaxation of guinea pig aortas were estimated for the range of the high affinity pathway (0.1 nM-1 µM).

EC 50 of Species high affinity pathway Range [mol/L] Guinea Pig 2.60*10 -8 ± 1.44*10 -9 0.1 nM-1 µM Rat 3.05*10 -8 ± 3.02*10 -9 0.1 nM-1 µM Mouse 5.00*10 -8 ± 6.10*10 -9 0.1 nM-1 µM Rabbit 9.18*10 -8 ± 1.84*10 -8 0.1 nM-10 µM

Table 17: EC 50 values for GTN-induced relaxation of aortas (high affinity pathway).

As shown in Figure 6, GTN had monophasic effects in coronary arteries of pigs, cows, and humans. Therefore, EC 50 values (Table 18) were calculated for complete curves (0.1 nM-100 µM).

EC Species 50 Range [mol/L] Cow 1.38*10 -8 ± 6.03*10 -9 0.1 nM-100 µM Human 3.48*10 -8 ± 4.10*10 -8 0,1 nM-100 µM Pig 1.91*10 -7 ± 3.26*10 -8 0.1 nM-100 µM

Table 18: EC 50 values for GTN-induced relaxation of coronary arteries.

27 Subcellular localization of ALDH2

Subcellular distribution of ALDH2 was studied in aortas of mice, rats, guinea pigs and rabbits as well as in porcine, bovine and human coronary arteries. To quantify the total amount of enzyme, purified human ALDH2 was used as standard, assuming the same affinity of proteins from different species to the antibody. For quantification of the subcellular distribution of ALDH2 in rodent aortas and in human vessels, the ALDH2 antibody of Dr. Weiner was used, while in porcine and bovine coronary arteries ALDH2 was detected with a polyclonal antibody purchased from Acris Antibodies. β-actin was used as loading control.

Figure 7 shows the mitochondrial (M; 10 µg of protein) and cytosolic fractions (C; 10 µg of protein) of mouse (m), rat (r), guinea pig (gp) and rabbit (rb) aorta, as well as of human coronary artery (ha) and human coronary vein (hv). S = 25 ng purified human ALDH2 in 25 µg ALDH2 KO mouse aorta lysate. The ALDH2 antibody kindly provided by Dr. Henry Weiner was used and the exposure time was 1 minute.

In Figure 8 mitochondrial (M; 25 µg for pig, 10 µg for cow) and cytosolic fractions (C; 25 µg for pig, 10 µg for cow) of porcine (p) and bovine (c) coronary arteries are shown. Polyclonal ALDH2 antibody from Acris antibodies was used. The exposure time was 10 minutes for ALDH2 and 1 minute for β-actin.

Figure 7 and Figure 8 show representative blots of cytosolic and mitochondrial fractions of aortas and coronary arteries. Note that the higher loading volume of mitochondrial fractions resulted in broadening of the bands.

28 Aortas

Coronary Arteries

Vein

Figure 9: Amount of ALDH2 in mitochondrial and cytosolic fractions of blood vessels from different species. Total amount of ALDH2 in vessels was quantified with a human standard, assuming same affinity for proteins of different species, and expressed as ng ALDH2/mg tissue.

Figure 9 shows the amount of ALDH2 in cytosolic and mitochondrial fractions of the blood vessels analyzed in the present study. The total amount of ALDH2 in murine aortas is significantly higher (P<0.05) than in vessels of all other species.

29 ALDH2 in ALDH2 in

Species Vessel cytosolic mitochondrial EC 50 fraction [%] fraction [%] Pig Coronary artery 98.04 ± 1.08 1.96 ± 1.08 1.91*10 -7 ± 3.26*10 -8 Rat Aorta 96.23 ± 1.98 3.77 ± 1.98 3.05*10 -8 ± 3.02*10 -9 Guinea Pig Aorta 96.00 ± 1.08 4.00 ± 1.08 2.60*10 -8 ± 1.44*10 -9 Human Vein 95.24 ± 2.62 4.76 ± 2.62 n.d. Human Coronary artery 94.34 ± 1.74 5.66 ± 1.74 3.48*10 -8 ± 4.10*10 -8 Mouse Aorta 89.26 ± 2.75 10.74 ± 2.75 5.00*10 -8 ± 6.10*10 -9 Rabbit Aorta 87.62 ± 5.36 12.38 ± 5.36 9.18*10 -8 ± 1.84*10 -8 Cow Coronary artery 86.50 ± 1.88 13.50 ± 1.88 1.38*10 -8 ± 6.03*10 -9 Table 19: Distribution of vascular ALDH2 in cytosolic and mitochondrial fractions.

Table 19 shows a summary of the data sorted by increasing relative amounts of ALDH2 in mitochondrial fractions. According to these results, ALDH2 is mainly cytosolic in all blood vessels tested, including human veins. However, the fraction of ALDH2 present in mitochondria increased from approximately 2 to 14 % (found in porcine and bovine coronary arteries, respectively).

30 DISCUSSION

Recent data from our group demonstrated that ALDH2 is mainly (85-95 %) cytosolic in mouse aortas and human coronary arteries, a finding that is in striking contrast to the almost exclusive mitochondrial localization of the protein in liver [27]. A possible explanation for the different localization of vascular ALDH2 could be the loss of the signal peptide for translocation into mitochondria by alternative splicing [27]. Furthermore, it is possible that ALDH2 follows a translocation pathway from mitochondria to cytosol, as it has been reported for fumarase [43]. My diploma thesis confirmed the recent findings on the subcellular distribution of ALDH2 in vascular smooth muscle. In aortas of rodents, as reported in 2003 [44], as well as in bovine, human and porcine coronary arteries, ALDH2 is primarily cytosolic. Like in coronary arteries (94 %), ALDH2 is mainly present in the cytosol of coronary veins (95 %). This observation indicates that other factors than subcellular ALDH2 distribution - such as thickness of the smooth muscle layer or expression levels of sGC - cause venoselectivity of GTN [45]. However, the results of subcellular distribution of ALDH2 in veins provide strong support that cytosolic localization of ALDH2 is essential for GTN bioactivation in any blood vessel type. Surprisingly coronary veins were considerably less sensitive to GTN than coronary arteries in relaxation studies in vitro (data not shown), although GTN is known to be a venoselective nitrovasodilator [46]. But the functional results obtained with veins in the current study could be affected by the preparation and the relaxation protocol, which were designed for arteries. Consequently, the method would need optimization before drawing conclusions on the vessel specificity of GTN. Another possible reason for the lower GTN sensitivity of coronary veins could be the lower total amount of ALDH2 (5.5 ng/mg tissue) in comparison to coronary arteries (12.5 ng/mg tissue). Moreover, it is likely that large venous capacity vessels are more sensitive to GTN than coronary veins. Our scientific group made the observation that relaxation response to GTN of ALDH2 knockout aortas was restored by cytosolic but not by mitochondrial overexpression of ALDH2. This led to the hypothesis that GTN is mainly metabolized by ALDH2 in the cytosol [27]. In the same study it has been reported

31 that murine aortas expressing ALDH2 exclusively in the cytosol showed a monophasic response to GTN, in contrast to wild type vessels, which reacted in a biphasic manner. This indicates that relaxation is antagonized at GTN concentrations ≥ 1 µM by a mechanism absent in vessels containing only cytosolic ALDH2 [27]. However, this conclusion is challenged by completely monophasic relaxation of bovine coronary arteries, in which a relatively large amount (10-15 %) of ALDH2 was found in mitochondria. Since complete relaxation of bovine coronary arteries was already obtained at GTN concentrations of 1 µM, a possibly existing low affinity pathway (range in aortic relaxation curves: 1 µM-100 µM) could remain hidden. Moreover, it is possible that coronary vessels react in a different manner than aortas. However, results of GTN relaxation in aortas agree well with observations suggesting a possible adverse effect of mitochondria on GTN metabolism. In aortas appears to be indeed a correlation between the subcellular distribution of ALDH2 and the profile of relaxation curves. Aortas of mice and rabbits, in which more than 10 % of ALDH2 was found in mitochondria, showed biphasic response, while aortas of guinea pigs and rats (5 % ALDH2 in mitochondria) reacted in a monophasic manner. The suggestion that mitochondrial GTN metabolism may counteract cytosolic GTN bioactivation was also supported by the observation of our group that cGMP accumulation in response to GTN was increased after the knock-down of mitochondrial function with ethidium bromide [27]. The mechanism of this supposed mitochondrial interference on GTN bioactivation is still unknown. A possible explanation for a counteracting mitochondrial mechanism could be superoxide production by mitochondrial ALDH2 [47]. Superoxide reacts with NO radicals to form ● ●- - peroxynitrite ( NO + O 2 -> ONOO ), which may cause reduction of vascular NO production, and therefore nitrate tolerance [34]. Observations that mice lacking mitochondrial superoxide dismutase got nitrate-tolerant agree well with this hypothesis [48]. The proposed mechanism of nitrate tolerance development is illustrated in Figure 10.

32 Figure 10: Proposed mechanism of nitrate tolerance development.

In summary, the data obtained in the current study confirm the recent findings that vascular ALDH2 is - against prevailing view - mainly cytosolic. Observations relating to a possible metabolism of mitochondrial ALDH2, which is counteracting GTN bioactivation in the cytosol, are still controversial, because a clear correlation between subcellular ALDH2 distribution and GTN vasorelaxation has not been found. Therefore, further work is required to clarify this issue.

33 ABSTRACT

Due to the vasodilating effect of nitric oxide (NO), the prodrug nitroglycerin (glycerol trinitrate; GTN) is used in the treatment of ischemic heart disease. In 2002 mitochondrial aldehyde dehydrogenase-2 (ALDH2) was identified as the key enzyme in GTN metabolism. Since ALDH2 was well characterized as mitochondrial protein catalyzing oxidation of ethanol in hepatocytes, the enzyme was commonly designated as mitochondrial aldehyde dehydrogenase. However, recent data showed that ALDH2 is mainly cytosolic in the vasculature of mice and humans, and that denitration rates of murine smooth muscle cells were higher than those of cells without functional mitochondria. This observation suggested possible adverse effects of mitochondria. It was the aim of my diploma work to test this hypothesis. Therefore the subcellular distribution of ALDH2 in aortas (of mice, rats, guinea pigs, and rabbits) and coronary arteries (of pigs, cows, and humans) was analyzed by western blotting and compared with the potency of GTN determined by recording cumulative concentration-response curves. In the vasculature of all species ALDH2 was mainly cytosolic, with only about 2-15 % of the protein found in mitochondrial fractions. GTN had a biphasic effect in aortas expressing more than 10 % of ALDH2 in mitochondria (mice and rabbit aortas). In contrast, all coronary arteries exhibited monophasic response to GTN, although about 13 % of bovine ALDH2 was found in mitochondria. The results obtained with aortas support the hypothesis that mitochondria may counteract GTN metabolism. It is conceivable that mitochondrial GTN metabolism yields superoxide, which ● ●- - reacts with NO radicals to form peroxynitrite ( NO + O 2 -> ONOO ), a possible cause for nitrate tolerance and endothelial dysfunction.

34 ZUSAMMENFASSUNG

Nitroglycerin (Glyceroltrinitrat, GTN) wird aufgrund der gefäßerweiternden Wirkung von freigesetztem Stickstoffmonoxid (NO) bei koronaren Herzkrankheiten eingesetzt. Im Jahr 2002 wurde Aldehyddehydrogenase-2 (ALDH2) als wesentliches Enzym der GTN-Bioaktivierung identifiziert. Da ALDH2, welche eine Schlüsselrolle im Alkohol-Metabolismus einnimmt, in Hepatozyten nahezu ausschließlich in Mitochondrien vorkommt, wird dieses Enzym auch als mitochondriale ALDH bezeichnet. Kürzliche Arbeiten zeigten aber, dass ALDH2 in Blutgefäßen von Mäusen und Menschen überwiegend im Zytosol exprimiert wird und Zellen mit funktionslosen Mitochondrien sogar höhere Denitrierungsraten aufweisen als gewöhnliche Muskelzellen. Dies lässt nachteilige Effekte von Mitochondrien auf die GTN-Bioaktivierung vermuten. Ziel meiner Arbeit war, diese Hypothese zu überprüfen. Hierfür wurde die subzelluläre ALDH2-Verteilung in Aorten von Mäusen, Ratten, Meerschweinchen sowie Hasen und in Koronararterien von Schweinen, Rindern und Menschen mithilfe von Western Blots ermittelt. Organbadversuche sollten zudem Auskunft über den Verlauf der GTN-Relaxationskurven dieser Gefäße geben. In den untersuchten Gefäßen wurde ALDH2 hauptsächlich (85-98 %) im Zytosol gefunden. Relaxationskurven von Aorten mit mehr als 10 % mitochondrialer ALDH2 (Maus, Hase) zeigten einen biphasischen Verlauf. Die Relaxationskurven von Koronargefäßen waren hingegen auch bei höherem Anteil an mitochondrialer ALDH2 (Rind: 13 %) monophasisch. Ergebnisse mit Aorten unterstützen, im Gegensatz zu jenen mit Koronarterien, die Hypothese, dass Mitochondrien einen nachteiligen Effekt auf die GTN-Bioaktivierung haben. Möglicherweise führt der mitochondriale GTN- Metabolismus zur Bildung von Superoxid, welches mit NO Peroxynitrit bildet ● ●- - ( NO + O 2 -> ONOO ). Peroxynitrit wiederum kann zur Entstehung von Nitrattoleranz und endothelialer Dysfunktion beitragen.

35 ACKNOWLEDGMENT

Mein größter Dank gebührt Gerald Wölkart , welcher mir in allen Arbeitsphasen mit seiner Kompetenz und angenehm ruhigen Art zur Seite gestanden ist. Zudem möchte ich mich herzlich bei Matteo Beretta bedanken, welcher keine Zeit und Mühe geschont hat, um unzählige Fragen zu beantworten, und mit seinem Enthusiasmus und Fleiß ein Vorbild für mich darstellt. Weiters möchte ich mich bei meiner ALDH2-Gruppenkollegin Regina Neubauer , welche mir in den letzten Monaten zu einer lieben Freundin geworden ist, für ihre Hilfsbereitschaft und stets aufmunternden Worte bedanken. Vor allem danke ich natürlich Prof. Bernd Mayer , welcher mir die tolle Möglichkeit gegeben hat, den Forschungsalltag kennen zu lernen.

Darüber hinaus gebührt mein besonderer Dank meiner lieben Mutter, welche mich beim Verfolgen meiner Ziele immerzu tatkräftig unterstützt hat!

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40 ABBREVIATIONS

ACh acetylcholine ALDH2 aldehyde dehydrogenase-2 AMPK AMP-activated protein kinase CaM calmodulin cGMP cyclic GMP DAG diacyl glycerol DTT dithiothreitol EDRF endothelium-derived relaxing factor eNOS endothelial NO synthase 1,2-GDN 1,2-glycerol dinitrate 1,3-GDN 1,3-glycerol dinitrate GTN glycerol trinitrate

IP 3 inositol 1,4,5-trisphosphate ISDN isosorbide dinitrate ISMN isosorbide mononitrate

LPA-H2 dihydrolipoic acid MLC myosin light chain MLCK myosin light chain kinase MLCP myosin light chain phosphatase NO nitric oxide - NO 2 nitrite NOx related NO species PETN pentaerythritol tetranitrate

PIP 2 phosphatidylinositol 4,5-bisphosphate PKG protein kinase G Rho0 deficient of mitochondrial DNA SEM standard error of the mean sGC soluble guanylate cyclase

41