INCREASED S-NITROSYLATION IMPAIRS CONTRACTION AND

RELAXATION IN MOUSE AORTA

By

Hyehun ·Choi

Submitted to the Faculty of the College of Graduate Studies

of the Georgia Health Sciences University in partial fulfillment

of the Requirements of the Degree of

Doctor of Philosophy

June

2011 INCREASED S-NITROSYLATION IMPAIRS CONTRACTION AND

RELAXATION IN MOUSE AORTA

This dissertation is submitted by Hyehun Choi and has been examined and approved by an appointed committee of the faculty of the College of

Graduate Studies of the Georgia Health Sciences University.

The signatures which appear below verify the fact that all required changes have been incorporated and that the dissertation has received final approval with reference to content, form and accuracy of presentation.

This dissertation is therefore in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

Jk(.t., 1. "b> ,, Date HYEHUN CHOI Increased S-nitrosylation Impairs Contraction and Relaxation in Mouse Aorta (Under the direction of R. CLINTON WEBB, Ph.D.)

S-Nitrosylation is a ubiquitous protein modification in redox-based signaling. This modification uses nitric oxide (NO) to forms S-nitrosothiol (SNO) on cysteine residues. Thioredoxin (Trx) and Trx reductase (TrxR) play a role in limiting S- nitrosylation. We hypothesized overall that S-nitrosylation of intracellular signaling molecules impairs contraction and relaxation of vascular smooth muscle cells. Aortic rings from C57BL/6 mice were used to measure vascular contraction and relaxation. The rings were treated with TrxR inhibitors, auranofin or 1-chloro-2,4-dinitrobenzene (DNCB), and/or NO donors, propylamine propylamine NONOate (PANOate) or S-nitrosocysteine (CysNO), to increase S- nitrosylation. Contractile responses of aortic rings to phorbol-12,13-dibutyrate

(PDBu), a PKC activator, were attenuated by auranofin, DNCB, PANOate, and

CysNO. PKCa S-nitrosylation was increased by a TrxR inhibitor and CysNO; concomitantly, PKCa activity and downstream signaling were inhibited as compared to control protein. Vascular relaxation in aortic rings from normotensive (Sham) and angiotensin II (Angll)-induced hypertensive mice was measured after contraction with phenylephrine in the presence or absence of

DNCB. DNCB reduced relaxation to acetylcholine (ACh) compared to vehicle, but the antioxidants, apocynin and tempol, normalized DNCB-induced impaired relaxation to ACh in sham aorta. Soluble guanylyl cyclase (sGC) S-nitrosylation was increased by DNCB, and sGC activity (cyclic GMP assay) was reduced in sham aorta. In aortic rings from Angll-treated mice, DNCB did not change relaxation to ACh compared to vehicle. DNCB decreased relaxation to s.odium nitroprusside (SNP) in aortic rings from both sham and Angll mice. Total protein

S-nitrosylation was enhanced in Angll aorta compared to sham, and TrxR activity was inhibited in Angll aorta compared to sham. These data suggest that PKC is inactivated by S-nitrosylation and this modification inhibits contractile responses to PDBu. TrxR inhibition reduces vascular relaxation via increasing oxidative stress and sGC S-nitrosylation. In Angll-induced hypertensive mice, augmented

S-nitrosylation is associated with impaired vasodilation. Thus, TrxR and S- nitrosylation may provide a critical mechanism in hypertension associated with abnormal vascular reactivity.

INDEX WORDS: S-nitrosylation, protein kinase C, thioredoxin reductase, oxidative stress, vascular contraction, vascular relaxation, angiotensin II, hypertension ACKNOWLEDGEMENTS

I would like to thank everyone who helps and supports me, because this dissertation would have not been possible without the support of a number of individuals. Since I have met very kind and nice people in our laboratory, department, and school, I think that I can finish my Ph.D. work. In the future, I would like to support people wherever as I got help from people and also, I would like to do my best like what I learned during my Ph.D. I specially thank my mentor, Dr. Clinton Webb, for your kindness and intellect. You are the best mentor for me and I have learned a lot from you not only conducting research projects but also aspiration of my future scientific · career. I would like to give thanks to the entire Webb's lab members, including old and new members and Dr. Testes. Thank you for your assistance and motivation throughout my graduate school training. I have enjoyed a lot with you in our lab and I am really happy to know many people like you. I wish you all the best. I would like to thank the member of my advisory committee; Drs. Wendy Bollag, David Fulton, Tsugio Seki, and Mong-Heng Wang, for guiding me through the course of this project. And I would also like to thank the readers of my dissertation; Drs. Rita Testes and Richard White. Thank you for sparing your time for me and my dissertation. A special thanks to my family who always help and pray for me. I would not have gotten this far in my academic endeavors without your continued support and encouragement. Finally, God, thank you so much. You are the best supporter and everything in my life.

v TABLE OF CONTENTS

ACKNOWLEDGEMENTS ...... v LIST OF FIGURES ...... vii LIST OF TABLES ...... viii

CHAPTER 1. INTRODUCTION ...... 1 A. Statement of the problem and specific aims of the overall project ...... 1 B. Brief literature review and discussion of the rationale of the project ...... 4

CHAPTER2 ...... 13 S-NITROSYLATION INHIBITS PROTEIN KINASE C-MEDIATED CONTRACTION IN MOUSE AORTA ...... 13

CHAPTER 3 ...... 34 THIOREDOXIN REDUCTASE INHIBITION REDUCES RELAXATION BY INCREASING OXIDATIVE STRESS AND S-NITROSYLATION IN MOUSE AORTA ...... ~ ...... 34

CHAPTER 4 ...... 53 AUGMENTED S-NITROSYLATION CONTRIBUTES TO IMPAIRED RELAXATION IN ANGIOTENSIN II HYPERTENSIVE MOUSE AORTA: ROLE OF THIOREDOXIN REDUCTASE ...... 63

CHAPTER 5. DISCUSSION ...... 78

CHAPTER 6. SUMMARY ...... 90

REFERENCES ...... 91

vi LIST OF FIGURES

CHAPTER 1. Figure 1.1. Schematic of protein S-nitrosylation ...... 12 CHAPTER 2. Figure 2. 1. S-Nitrosylated proteins (SNO) are increased in the presence of DNCB, CysNO, or DNCB plus CysNO in mouse aorta ...... 21 2.2. Vascular contraction to PDBu is attenuated by S-nitrosylation ...... 22 2.3. PDBu-induced contraction in mouse aorta is abolished by increased S- nitrosylation ...... 24 2.4. S-Nitrosylation inhibits PKCa activity ...... 26 2.5. Phosphorylation of CPI-17 was decreased by S-nitrosylation ...... 28 2.6. Phosphorylation of caldesmon was reduced by S-nitrosylation ...... 29 CHAPTER 3. Figure 3.1. Vascular relaxation to ACh is attenuated by DNCB and auranofin ...... 44 3.2. DNCB increased H202 levels in mouse aorta ...... 45 3.3. Antioxidants normalized relaxation impaired by DNCB ...... 46 3.4. DNCB shifted vascular relaxation to SNP to the right ...... 48 3.5. sGC S-nitrosylation was increased and its activity was decreased by DNCB ...... 49 CHAPTER 4. Figure 4.1. Concentration-response relaxation to ACh was attenuated by DNCB only in sham aortic rings not in Angll ...... 64 4.2. Vascular relaxation to SNP was shifted to the right by DNCB in sham and Angll ...... 65 4.3. DNCB attenuated cGMP concentration in SNP-treated condition ...... 66 4.4. S-Nitrosylated proteins (SNO) were increased in Angll aorta compared to sham ...... 68 4.5. TrxR protein expression was not different between sham and Angll, but TrxR activity was diminished in Angll aorta compared to sham ...... 69 4.6. Increased TrxR normalized relaxation to ACh and SNP in Angll aortic rings ...... ······ ...... 71 4.7. CysNO decreased relaxation in Angll aortic rings ...... 73 CHAPTER 5. Figure 5.1. DNCB does not change PE-dependent contraction, but DNCB attenuates PDBu-induced contraction ...... 80 5.2. DNCB increases H202 levels in normotensive mouse (Sham) aorta ...... 85 5.3. Schematic of our conclusion under normal conditions ...... 88 5.4. Schematic of our conclusion under hypertensive conditions ...... 89

vii LIST OF TABLES

CHAPTER 3. Table 3.1. Emax and pD2 values for ACh and SNP-induced responses in aortic rings ... 43

CHAPTER 4. Table 4.1. Mean arterial pressure and body and heart weight in normotensive (sham) and Angll-infused mice ...... 62

viii 1

CHAPTER 1. INTRODUCTION

A. Statement of the problem and specific aims of the overall project

High blood pressure is a major risk factor for cardiovascular disease and is a main contributor to widespread morbidity and mortality [1 ]. In 2000, more than one quarter of the world's adult population, nearly one billion people, were afflicted with hypertension. It is estimated that this population will increase 29% to 1.56 billion people by 2025 [2]. Prevention of hypertension is one of the important therapeutic aims in the treatment of cardiovascular disease. Although current drugs are effective in many patients, a number of patients are unresponsive even to combination therapy. This lack of treatment efficacy in some patients clearly indicates that further advances in the field are required.

Vascular tone is determined by the balance between constrictor and dilator stimuli and it is essential to the regulation of blood pressure. Abnormal vascular reactivity, including enhanced constriction and reduced dilation, is a main characteristic of hypertension. However, the cellular and molecular mechanisms associated with vascular dysfunction require further study.

S-Nitrosylation is the post-translational modification linking nitric oxide (NO) to cysteine residues of proteins. These modified proteins include channels, transporters, kinases, signaling proteins, and transcription factors. In the cardiovascular system, NO is a principal mediator of vascular tone, and NO bioavailability can be regulated by S-nitrosylation. Studies on the effects of S­ nitrosylation in the vasculature, under both normal and pathological conditions, 2 are essential to understand the role of this post-translational modification in the regulation of vascular signaling. Dysregulation of S-nitrosylation may represent a common mechanism for the effects of hypertension, thereby contributing to increased peripheral vascular resistance. In our study, we evaluated whether augmented S-nitrosylation contributes to abnormal reactivity in mouse aorta with the following specific aims:

Specific aim 1: To test the hypothesis that S-nitrosylation of PKC in vascular smooth muscle inhibits contractile activity.

The approach utilized phorbol ester (PKC activator)-induced contraction in mouse aorta. PKC S-nitrosylation and function were measured in the presence or absence of modulators of S-nitrosylation. Specific downstream signaling of

PKC was also monitored to determine the function .of PKC S-nitrosylation.

Specific aim 2: To test the hypothesis that thioredoxin reductase inhibition decreases vascular relaxation via increased oxidative stress and S-nitrosylation.

The effects of a selective inhibitor of thioredoxin reductase (DNCB) on vascular relaxation were determined. The basic action of thioredoxin reductase as an antioxidant was confirmed in mouse aorta. The specific mechanism and signaling pathways by which S-nitrosylation modulates vascular relaxation were determined. 3

Specific aim 3: To test the hypothesis that increased S-nitrosylation is associated with reduced vasodilation in angiotensin II (Angll)-induced hypertensive mice.

Using aortas from Angll-induced hypertensive mice, experiments were performed to characterize changes in the protein levels of S-nitrosylation and to determine relaxation associated with increased S-nitrosylation in this condition.

Experiments were performed to determine whether Angll decreases the activity of a denitrosylation enzyme (thioredoxin reductase) and thereby increases S­ nitrosylation. 4

B. Brief literature review and discussion of the rationale of the project

Vascular dysfuncti~n and hypertension · Hypertension· results from complex interactions of genetic and environmental factors. These factors include neural, humoral, and cellular mechanisms that re~ulate blood pressure [3]. In hypertension, arteries undergo structural and functional changes, leading to reduced lumen size and increased peripheral resistanc~. Vascular changes are associated with abnormal function and growth of ce11J1ar components of the vessel wall. Functional alterations j

I include enhanced reactivity to vasoconstrictors and/or impaired vasodilation [4, 5].

Humoral, neural, and mechanical factors participate in signal transduction, resulting in vascular changes in hypertension. Humoral factors that regulate I i blood vessels in h~pertension are vasoconstrictors, such as angiotensin II and

i endothelin-1; vasodilators, such as NO and endothelium-derived hyperpolarizing factor; growth factoJs, such as insulin-like growth factor-1; and cytokines, such as transforming growth factor-beta [6].

Vascular dysfunction, including enhanced vasoconstriction and reduced endothelium-depen~ent dilation, is a main feature of hypertension. This has i been evidenced in ~everal hypertensive animal models. Vasoconstricting agents ! ' i and calcium responsiveness are enhanced in arteries and smooth muscle cells

i from hypertensive tats [4, 7]. Compromised endothelium-dependent vascular relaxation, referred ~o as endothelial dysfunction, has been demonstrated in both

I conduit arteries and resistance arteries. Endothelium-dependent vasodilation is I reduced in isolated arteries from spontaneously hypertensive rats [8, 9],

I I I 5 deoxycorticosterone acetate salt-treated animals [1 0, 11 ], Dahl salt-sensitive rats 1

[12], and renovasculkr hypertensive animals [13, 14].

In vascular smooth muscle cells, the rise and fall of intracellular free

I calcium are the principal mechanisms initiating contraction and relaxation, respectively. However, vascular smooth muscle contraction is mediated by both calcium-dependent and -independent mechanisms. Increases in . intracellular calcium from receptor or channel-activated pathways lead to activation of myosin light chain (MLC) kinase through thin-filament-associated proteins, such as calmodulin and calponin [15]. MLC kinase phosphorylates MLC, activating myosin ATPase andi increasing vascular smooth muscle contraction and vascular tone [15]. The regulator)! mechanisms of calcium-independent contraction include protein kinase C (PKC), mitogen-activated protein (MAP) kinase, and

RheA/Rho-kinase. , i

I NO is an endogenous vasodilator and a key regulator of vascular tone.

Due to the action: of endothelial NO synthase (eNOS) to produce NO in endothelial cells, eNOS is responsible for most of the NO production in the vasculature [16]. R~leased NO from endothelial cells activates soluble guanylyl cyclase (sGC) to generate cyclic 3',5'-guanosine monophosphate (cGMP).

1

! Increased cGMP l~ads to decreased intracellular calcium levels and activated

I potassium channelis, which induces membrane hyperpolarization [17]. In ~ I contrast to contra~tion, vascular smooth muscle relaxation is mediated by activation of MLC p~osphatase, which dephosphorylates MLC. Thus, increased

I I 6 cGMP, decreased cytosolic calcium levels, protein phosphorylation· (e.g. eNOS), and myosin light chain dephosphorylation result in relaxation.

Reactive oxygen species in the vasculature

Reactive oxygen species (ROS) are reactive derivatives of 02 metabolism found ubiquitously in all biological systems. Major ROS are superoxide anion

(02·), hydrogen peroxide (H202), and hydroxyl radical (·OH), and also contribute to an oxidative environment, the reactive nitrogen species, such as peroxynitrite

(ONoo-). ROS are generated when oxygen gains an electron, forming 02·- by various enzymatic reactions. These enzyme sources include NADPH oxidase,

NO synthase, xanthine oxidase, the respiratory chain of mitochondria, the arachidonic acid pathway enzymes lipoxygenase and cyclooxygenase, and cytochrome p450s. 0 2·- has an unpaired electron, which results in high reactivity, instability, and a short half-life. 02·- reacts with itself, and superoxide dismutase

(SOD) catalyzes the reaction to remove 02·- and form H202 [18, 19]. Unlike 02·-,

H202 is a more stable molecule, has the ability to cross the cell membrane and has a longer half-life. H202 is scavenged by catalase and glutathione peroxidase, and also, it can be reduced to produce the highly reactive ·OH in the presence of 2 metal-containing molecules, such as Fe + [18].

ROS from mitochondria and other cellular sources have been considered to be cellular by-products with the potential to damage DNA, proteins, and lipids

[20]. In addition, ROS participate in many intracellular signaling pathways leading to changes in gene transcription and protein synthesis, and consequently 7 in cell function [21 ]. In the vasculature, ROS are produced in a·ll vascular cell types, including endothelial cells, smooth muscle cells, and cells in the adventitia

[22, 23]. In the vasculature, 02·-, H202, NO, ONoo-, and ·OH are produced to various degrees under tight regulation by antioxidants [24]. These antioxidant systems include SOD, catalase, thioredoxin, glutathione, vitamins, and other small molecules. Under physiological conditions, ROS are maintained at low concentrations and function as signaling molecules to .regulate vascular contraction-relaxation and vascular smooth muscle cell growth [25-27]. However, under pathological conditions, ROS production is increased, leading to endothelial dysfunction, increased contractility, excessive vascular smooth muscle cell growth, monocyte migration, lipid peroxidation, inflammation, and other processes contributing to vascular damage [26, 28, 29].

Oxidative stress occurs when there is an increase in the generation of

ROS compared to the normal antioxidant defense systems and is defined as a disruption of redox signaling and control [30]. Increased oxidative stress is associated with reduced endothelium-dependent relaxation, and antioxidants have been shown to acutely improve such responses in animals [31, 32]. SOD plays a role in prevention of the reaction between 0 2·- and NO. In vascular tissues, SOD has been demonstrated to release NO from endothelium and protect NO during its diffusion from endothelium to the vascular smooth muscle

[33, 34]. In cardiovascular disease, impaired endothelium-dependent relaxation has been linked to decreased NO bioavailability. NO is degraded by its interaction with 02·- to form ONoo-, which is a weak vasodilator compared with 8

NO and has pro-inflammatory properties [35]. Increased ROS lead to augmented deposition of .extracellular matrix proteins, such as collagen and fibronectin. Thus, increased vascular ROS contribute to vascular dysfunction and structural remodeling, establishing a role for these molecules in increasing blood pressure [36-38].

Nitrosative stress and S-nitrosylation

NO is an important mediator in the cardiovascular system and is generated by NOS via the oxidation of L-arginine. NO has a beneficial function in maintaining proper physiological homeostasis. For example, NO regulates vascular tone and cellular adhesion and also has protective roles, such as an antioxidant and in inhibiting leukocyte adhesion. However, NO has been found to have cytotoxic properties in pathophysiological conditions, such as arthritis, atherosclerosis and stroke [39]. NO can interact directly with biological molecules and also can react indirectly via reaction with other radicals or oxygen, which generates reactive nitrogen species. Nitrosative stress is caused by increased reactive nitrogen species and can impair NO signaling. Nitrosative stress can result in disrupted cellular signaling and injury. Dysregulated reactive nitrogen species may be associated with a disturbance in the redox state [40].

Indirect effects of NO can be further subdivided into nitrosation, oxidation, and nitration chemistry [39]. Nitration adds a nitro triatomic group (-N02), generally on tyrosine residues to form nitrotyrosine. This modification is related to increasing ONoo- and is called tyrosine nitration [41]. Oxidation removes one 9 or two electrons from the substrate, and this chemical production includes DNA strand breaks, lipid peroxidation, and hydroxylation [39]. Lastly, nitrosation occurs when an equivaiJnt NO is added to a thiol, metal, amine, or hydroxyl aromatic group. The te+s nitrosation and nitrosylation is distinguished by the addition of the NO diatom1c group and nitrosyl group, NO, respectively. However, in the NO species, both Jroups are the same and inclusion of the '-yl-' in terms describing the post-tranflational modification is widespread. Accordingly, nitrosylation is used to describe both cases [42, 43]. Clearly, the prefix 'S-' refers to the incorporation of thi NO moiety to a sulfur atom to form S-NO bond: "S­ nitrosation" or "S-nitrosylafion" [42].

S-Nitrosylation is a redox-based modification of a cysteine residue on a I target protein by NO [43]. To trigger S-nitrosylation, NO is converted to dinitrogen trioxide (N203) with an electron acceptor such as oxygen, followed by partial dissociation into [ ON .. N02l As this reaction occurs, the nitrosonium

(NO+) moiety can interac with the nucleophile sulfur atom to form S-nitrosothiol

(SNO) to the protein's th~ol group [42, 44]. Additionally, as a transnitrosylation reaction occurs, an NO jquivalent (between a thiol and a SNO) is transferred from one molecule to another [44]. These reactions are achieved through the non-catalyzed chemical Jodification of a protein residue. Thus, in S-nitrosylation, the target specificity is determined by the chemical reactivity between the nitrosylating agent and thj target. Especially, NO-targeting depends on the 30 cysteine environment [45]. This effect is proved the studies in which a protein

. may have several cyst ines with a free-thiol, but the protein becomes S- 10 nitrosylated only on some cysteine residues [46, 47]. There are consensus motifs for SNO synthesis, but the most important component of the sequence is cysteine-(aspartate,glutamate); an acidic amino acid following cysteine [44, 48].

In addition, there are several factors involved in target specificity, such as concentration of nitrosylating agent and the protein and low pKa [42, 45].

The levels of S-nitrosylation are regulated indirectly or directly by several enzymatic systems. As an indirect enzymatic regulation, NOS produces NO, which can be converted to the NO+ which attacks the sulfur, forming SNO.

Increased production of SNO by NOS i's associated with the activation of all NOS isoforms [49, 50]. Direct enzymatic systems to regulate S-nitrosylation include denitrosylation enzymes which remove SNO. Denitrosylation is regulated by two different enzyme systems, thioredoxin/thioredoxin reductase [51, 52] and S­ nitrosoglutathione reductase (GSNOR) [53, 54]. Thioredoxin reductase is a redox-based enzyme that catalyzes the nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reduction of the oxidized redox protein, thioredoxin. The reduced form of thioredoxin denitrosylates S-nitrosylated proteins [52]. Stamler and colleagues [53, 55, 56] have investigated the effects of GSNOR, which is also known as glutathione (GSH)-dependent formaldehyde dehydrogenase or class Ill alcohol dehydrogenase 5 (ADH5). The GSNOR system comprises GSH and GSNOR, which degrades S-nitrosoglutathione

(GSNO) to oxidized GSH and ammonia and thus, lowers the levels of S­ nitrosylation. 11

Over 100 S-nitrosylated proteins, including channels, transporters, kinases, signaling proteins, and transcription factors, have been identified [57]. These proteins can be activated or inactivated by S-nitrosylation. For instance, the activity of p21 ras is increased by S-nitrosylation [58], but endothelial NOS (eNOS) activity is inhibited [59]. Proteins with an important role in vascular function are also targets for S-nitrosylation. For instance, protein kinase C (PKC) is a well characterized enzyme important to vascular function, and it phosphorylates many proteins involved in vascular contraction. PKC is a target of NO leading to functional inactivation [60, 61] and is also modified by S-nitrosylation [62]. As mentioned previously, eNOS is inactivated by S-nitrosylation and is an important enzyme producing NO in the vasculature. Additionally, soluble guanylyl cyclase

(sGC) is a critical enzyme mediating relaxation of vascular smooth muscle and is desensitized and inhibited by S-nitrosylation [63]. Vascular structural proteins, such as actin and myosin are also modified by S-nitrosylation [49, 64]. Although many proteins important in vascular function have been identified as targets for

NO and S-nitrosylation, there is a paucity of information on the effects of S­ nitrosylation in vascular signaling. Therefore, our study presents an innovative research approach to better understand S-nitrosylation-associated mechanisms that control vascular (dys)function and may also provide insight into novel therapeutic approaches. 12

Auranofin, DNCB j_

SH SNO

NOX (PANOate, CysNO)

Figure 1.1. Schematic of protein S-nitrosylation. NO sources induce S­ nitrosylation of a target protein and the thioredoxin/thioredoxin reductase system decreases the level of S-nitrosylation. Auranofin and DNCB (1-Chloro-2,4- dinitrobenzene) are thioredoxin reductase inhibitors. PANOate and CysNO are NO sources. PANOate; Propylamine propylamine NONOate, CysNO; S­ nitrosocysteine. 13

CHAPTER 2.

I i S-NITROSYLATION INHIBITS PROTEIN KINASE C-MEDIATED ! CONTRACTION IN MOUSE AORTA 14

ABSTRACT

S-Nitrosylation is a ubiquitous protein modification in redox-based signaling and uses nitric oxide (NO) tl form S-nitrosothiol (SNO) on cysteine residues. Dysregulation of (S)NO ~ignaling (nitrosative stress) leads to impairment of cellular function. Protein ~inase C (PKC) is an important signaling protein that plays a role in the regulati+ of vascular function. It is not known whether (S)NO affects PKC's role in vascqlar reactivity. We hypothesized that S-nitrosylation of

PKC in vascular smooth mluscle would inhibit its contractile activity. Aortic rings from male C57BL/6 mi~e were treated with auranofin or 1-chloro-2,4-

1 dinitrobenzene (DNCB) as pharmacological tools, which stabilize S-nitrosylation by inhibiting the removal of S-nitrosothiol, and propylamine propylamine

NONOate (PANOate) or sfnitrosocysteine (CysNO) as NO donors. Contractile responses of aorta to phorbol-12, 13-dibutyrate (PDBu), a PKC activator, were I I attenuated by auranofin, DNCB, PANOate, and CysNO. S-Nitrosylation of PKCa

I was increased by auranofin or DNCB and CysNO as compared to control. I . Augmented S-nitrosylation inhibited PKCa activity and subsequently downstream signal transduction. ThJse data suggest that PKC is inactivated by S-

[ nitrosylation and this modification inhibits PKC-dependent contractile responses. I Since S-nitrosylation of P~C inhibits phosphorylation and activation of target I proteins related to contraction, this post-translational modification may be a key player in conditions of decrlased vascular reactivity.

KEY WORDS: S-nitrosylatian, protein kinase C, vascular contraction 15

INTRODUCTION

S-Nitrosylation is tHe post-translational modification of a cysteine residue I on target proteins by nitric pxide (NO) [43]. In the S-nitrosylation reaction, NO is

i converted to dinitrogen trioXide (N203) with an electron acceptor such as oxygen, followed by formation of S-~itrosothiol (SNO) in the protein's cysteine thiol group. Additionally, as a transnitr~sylation reaction, a NO equivalent is transferred from

I one molecule to another (~rom a thiol and a SNO) [44]. There are over 100 S-

1

I nitrosylated proteins, includ,ing channels, transporters, kinases, signaling proteins, I and transcription factors [57]. These proteins are activated or inactivated by I SNO. For example, the activity of p21 ras is increased by S-nitrosylation [65], but I . endothelial NO synthase (eNOS) is inhibited [59].

I

I The levels of S-n~trosylation are regulated indirectly and directly by several enzymatic systems. NOS, for example, functions as an indirect enzymatic regulator via NO production, which is added to" the thiol group, leading to the formation of SNO. Direct enzymatic systems to regulate S-nitrosylation include enzymes for denitTsylation to remove SNO. Two enzymatic systems, thioredoxin/thioredoxin reductase [51, 52] and S-nitrosoglutathione reductase

(GSNOR) [53, 54], are infolved in the denitrosylation process. Thioredoxin reductase is a redox-based enzyme and catalyzes the nicotinamide adenine dinucleotide phosphate (NlDPH)-dependent reduction of the oxidized redox protein thioredoxin. Reduld thioredoxin denitrosylates S-nitrosylated proteins [52]. Stamler and collea~ues [53, 55, 56] have investigated the effects of 16

GSNOR, which degrades S-nitrosoglutathione (GSNO) to oxidized glutathione and ammonia, and lowers 'he levels of S-nitrosylation.

I Vascular smooth ~uscle contraction is associated with the regulation of

I vascular resistance and blood pressure, and its dysregulation can lead to hypertension. Protein kina~e C (PKC) is a well characterized enzyme in agonist­ induced vascular smooth muscle contraction [66-69]. In vascular smooth muscle, I several signal cascades ~ctivated by physiological agonists are regulated by

I PKC. Activated PKC ph:osphorylates CPI-17 (PKC-potentiated phosphatase inhibitor protein-17 kDa)! [70], which inhibits myosin light chain (MLC)

I

I phosphatase resulting in v~scular contraction. In addition, PKC phosphorylates other proteins such as calponin, caldesmon, or mitogen-activated protein kinase

I (MAPK) to cause vascula~ contraction [71, 72]. PKC also affects membrane­ ~ bound regulatory proteins: such as MARCKS (myristoylated, alanine-rich C- 1 kinase substrate) and in~ibitory GTP-binding protein Gi in vascular smooth ! l muscle [73, 74]. I

PKC is a target of NO, which leads to PKC's functional inactivation [60, 61]. It is possible that in t~is regulation, PKC is being modified to S-nitrosylated I I PKC. In the vasculature, the action of S-nitrosylation is unknown. We

I hypothesized that the PKCimediated vascular contractile response is inhibited by

I

I S-nitrosylation. To test lthis hypothesis, we determined whether vascular reactivity and PKC signal transduction is modified by increased levels of S- nitrosylation. 17

MATERIALS AND METHODS

I Drugs and Reagents

I Phorbol-12, 13-dibutyrate (PDBu) was purchased from Calbiochem (San Diego,

CA). Propylamine propylamine NONOate (PANOate) was obtained from

Cayman Chemical (Ann Arbor, Ml). S-nitrosocysteine (CysNO) was synthesized from L-cysteine using acidified nitrite. Auranofin was purchased from BioMol

I I (Plymouth Meeting, PA).: 1-Chloro-2,4-dinitrobenzene (DNCB) and other chemicals were obtained frbm Sigma-Aldrich (St. Louis, MO). ! Animals

i Male, 12-14 weeks-old, C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME) were used in this study. All procedures were performed in accordance with the

I Guiding Principles in the Care and Use of Animals, approved by the Medical

I I College of Georgia Committee on the Use of Animals in Research and Education.

! The animals were housed on a 12-hour lighUdark cycle and fed a standard chow

diet with water ad libitum. i

Isolation of aortic rings a~d functional studies i After mice were euthanize~, thoracic aorta were excised, cleaned from fat tissue

i and cut into 2 mm-length ri~gs in an ice-cold physiological salt solution consisting of the following: 130 mM: NaCI, 4.7 mM KCI, 1.18 mM KH2P04, 1.18 mM

MgS04·?H20, 1.56 mM CaCI2·2H20, 14.9 mM NaHC03, 5.6 mM glucose, and

I 0.03 mM EDTA. Aortic irings were mounted in a myograph (Danish Myo

Technology AIS, Aarhus, Dbnmark) containing warmed (37°C), oxygenated (95% I 02/5% C02) physiological·salt solution. The preparations were equilibrated for at I

I I 18 least 1 hour under a passiie force of 5 mN. After the equilibration period, arterial integrity was assessed b~ stimulation of vessels with 120 mM KCI and, after contraction reached a plat~au, the rings were washed. Subsequently, the rings were stimulated with phenylephrine (0.1 1-JM) followed by relaxation with acetylcholine (1 1-JM), whi9h was used as evidence of an intact endothelium.

I Cumulative concentration-response curves to PDBu using 1o- 9 to 1o- 6 M were ! performed. !

Detection of S-nitrosylaticl>n i The biotin-switch method to detect S-nitrosylation was performed with the S­ nitrosylated protein detection assay kit (Cayman Chemical, Ann Arbor, Ml) i I modified from the method 9f Jaffrey et al. [75] Biotin-labeled proteins were used for western blot analysis or pulled down by streptavidin agarose beads (Thermo

i Scientific, Rockford, IL) to specifically detect PKCa S-nitrosylation and then f

i western blot analysis was performed. Ten percent of biotin-labeled proteins were used to measure total PKCdx protein expression. ! Western blot analysis I I Aortas were treated with aqranofin (1 lJM) or DNCB (4 1-JM) for 1 h, and CysNO ! .

I (1.5 mM) for 30 min in Dulbecco's modified Eagle's medium (DMEM). Proteins I I (40 lJg) extracted from aorta in lysis buffer (20 mM Tris-HCI, pH 7 .4, 5 mM

Na2P207, 100 mM NaF, 2 ;mM Na3V04, 1o/o NP-40, protease inhibitor cocktail,

I and 1 mM PMSF) werej separated by electrophoresis on a 12% SDS-

1 polyacrylamide gel and transferred to a nitrocellulose membrane. The membranes were blocked lith 5% skim milk in Tris-buffered saline solution with I

I

I I I 19 I . I 0.1% Tween-20 for :1 h ayoom-temperature. Membranes were then incubated with primary antibodies ov~rnight at 4 °C. Antibodies were as follows: phospho­

CPI-17, CPI-17 (Millipore, Temecula, CA), phospho-caldesmon, caldesmon

(Santa Cruz Biotechnology, Santa Cruz, CA), PKCa (BD Biosciences, San Jose,

CA), and J3-actin (Sigma-Aldrich). After incubation with secondary antibodies, signals were visualized w.ith chemiluminescence followed by autoradiography and quantified densitometrically. I PKC activity assay

To obtain specific PKCa isoform activity, immunoprecipitation (IP) was performed before the activity assay [76]. Protein extracts (500 IJg) were incubated with 2 IJg of anti-PKCa antibody (BD: Biosciences) and 40 IJI of protein A/G plus-agarose

(Santa Cruz Biotechnology) at 4 oc, overnight. The beads were collected and extensively washed six times with phosphate buffered saline (PBS). A PKC activity assay was perforr:ned with the IP samples using a PKC assay kit

(Calbiochem).

Statistical analysis I Values are mean ± stand~rd error of the mean (SEM), and n represents the number of animals used i~ the experiments. Contractions were recorded as changes in the displacement (mN) from baseline and were expressed as percentage change normalized to 120 mM KCI-contraction values.

' i Concentration-respOr'1Se curves were fitted using a nonlinear interactive fitting program (Graph Pad Prism 4.0; GraphPad Software, San Diego, CA), and 2 I • i pharmacological parameters were obtained: the maximal effect generated by the

I I 20 i I agonist (or Emax) and. EC5o (molar concentration of agonist producing 50o/o of the I maximum response) .. Stati$tical differences were calculated by Student's t-test and a P value less than 0.05 was considered to be statistically significant.

RESULTS

Role of S-nitrosylation in PKC-induced contraction

We first determined the content of total S-nitrosylation proteins in mouse aorta. The vascular content of total S-nitrosylation proteins was increased after incubation with CysNO, an exogenous SNO source, or DNCB as a pharmacological tool leading to the stabilization of S-nitrosylation, as well as the combination of the two drugs (Figure 2.1 ).

To test the effect of S-nitrosylation generated by NO on vascular contraction, two exogenous SNO sources were used to induce S-nitrosylation.

PANOate is a NO donor and CysNO mediates transnitrosylation (the switch between SNO and a thiol) [77]. To measure PKC-dependent contraction, PDBu, a PKC activator, was used: in endothelium-intact aortic rings. Incubation with i CysNO significantly attenua~ed the PDBu-induced contractile responses in a concentration-dependent ma1nner, compared with vehicle control (Figure 2.2A).

In addition, contraction to iPDBu in PANOate-treated rings was decreased compared with vehicle ·(Figur~ 2.28). 21

B

MW(kDa)

c: 0 ~ >oco- "'ZeV') 37 .~­z I V)

25 20

P-actin

*·. ' •.. .. ':S· .. ·. : . . (,) ·, . . . c:c.'•1 '(5 z tn ca Q . 0 .· .I-

.Control · DNCB CysNO Control DNCB+CysNO

Figure 2.1. S-Nitrosylated proteins (SNO) are increased in the presence of DNCB, I CysNO, or DNCB plus CysNO in mouse aorta. A and 8: Representative western blot images of S-nitrosylated proteins (top) and corresponding bar graphs showing the relative content: of S-nitrosylated proteins after normalization to ~­ actin expression (bottom). R~sults are presented as mean ± SEM for n=3 in each experimental group.*, p<0.05 compared with control. I I

I I

I 22

A B

D Vehicle . Cl Vehicle 400 A CysNO (1mM): 400 ·A PANOate (0.2mM) . - V CysNb (1.5mf0) · -· • PANOate (2mM) . ~ .. 4t ·cysNO (2m~f 300 '* . .~·- 300 c . · ... c 0 ;; CJ ... . e· 2oo . *. t·~oo .· ..... :· c .: ... ·o: .. :::;.,:'.i .·· '* 0 .· . ~: ioo 0 100 . ·1:1' '#.

0 0

-9 . ~a -7 : -9 -8 -7 -6 log[PDBu] (M) ·log[PDBu] (M)

Figure 2.2. Vascular contraction to PDBu is attenuated by S-nitrosylation. Concentration-dependent contractile responses to PDBu (1 o-9 to 1o- 6 M) were measured after incubation with PANOate (A, n=6 to 8) or CysNO (B, n=6 to 8) at the indicated concentnations !for 15 minutes in mouse aorta. Experimental values I of contraction were calculated relative to the contractile response produced by 120 mM KCI, which w~s taken as 1 00°/o. Results are presented as mean ± SEM in each experimental group. ':*, p<0.05 compared with vehicle. I 23

To increase. S-nitr\sylation, we used two experimental approaches: increased exposure !Ito SNO sources and inhibition of denitrosylation enzymes. Thioredoxin reducta~e is o~e of the denitrosylation enzymes and two inhibitors of this enzyme, auranofin and !oNCB [51, 52], were used to demonstrate whether S- nitrosylation changes PK<;-mediated contractile responses. Auranofin and

DNCB significantly reduce'p PDBu-induced contractions in endothelium-intact

I aortic rings. To further incr~ase S-nitrosylation, aortic rings were incubated with i each inhibitor plus either PANOate or CysNO and the simultaneous addition of

! these compounds al$o dim,nished PDBu-induced contractions compared to the control group (Figure :2.3). 24

-·A -c Vehicl~' - ,- i -B IJ Vehicl_e • _Auranofin i . · 400 ·A Auranofin ff:- -v -cysNO . v PANQate -~-- __ - . . ~: AuraJ1dfin+Cy$N(),. -C3 - 300 ~ 300 c c 0 ;:: 0· (,)- -ns:e 200. E - ...... c . _- c o. ---0 0- 0 100 * * ~ ''#. 0 * 0 * #

. - .g -8 ·7 . ·.g- ·8 -7 -6

c IJ Vehicle D \ - 400 T DNCB+CysNO (SOJ.tM) ff • DNCB+CysNO (100J.tM) -~ _ • DNCB+PANOate. ~ 300 300 i: c 0 .2 * :eE -2oo_ - ---g.200 .- - ... - c 1:- -­o· 0 0 100 0 100 ?fl.. ?fl.

. .g -·.-6. .g. -. ·8 -7- -6 . ·log[PDBul(M) E 400 -C3 ~ 300 i: * 0 ..(,) ...ns 200 * c -o· 0 100 ?fl. -o

.g - -8 -.7 -6 .Jog[PDBu] (M) \- _

Figure 2.3. PDBu-induced contraction in mouse aorta is abolished by increased S-nitrosylation. Conc

I 25

Effect of S-nitrosylation on PKC activity

• 'I I Considering : that PKC-induced vascular contraction is modified with manipulations that increasl S-nitrosylation, we determined protein expression of I . total and S-nitrosylated PKC. The levels of S-nitrosylation were determined by I I I the biotin-switch method ahd western blot analysis as described in the Methods section. To confirm whether PKCa S-nitrosylation is associated with its activity, 1 I

PKCa activity was deter~ined with immunoprecipitated PKCa. CysNO plus i auranofin and CysNO plus pNCB increased PKCa S-nitrosylation and attenuated I PKCa activity (Figure 2.4). i 26

A

SNO-PKCa

PKCa

B c

*

.. ·Control . ·. DN~B-i-CysNO

Figure 2.4. S-Nitrosylation inhibits PKCa activity. Isolated mouse aortas were treated either with or without\auranofin (Aura; 1 fJM, 1 h) or DNCB (4 fJM, 1 h) plus CysNO (1.5 mM, 30 miri). A: PKCa S-nitrosylation (SNO-PKCa) was . I detected using the biotin-switch method, streptavidin-precipitation, and western blot analysis with anti-PKCa ~ntibody. Total PKCa expression (PKCa) was detected with biotin-labeled ~roteins using western blot analysis. B and C: PKCa activity assay. Treated aorta\ tissues were immunoprecipitated with anti-PKCa I antibody (2 f.,Jg) and then subjected to the protocol for activity assay. The vehicle- treated group, control, was s~t to one hundred and the bars represent means± SEM in each experimental grbup. *, p<0.05 compared with control (B, n=5 and C, n=4). 27

I Effect of PKC S-nitrosylatiT in vascular signaling proteins

PKC phosphorylates p1any downstream signaling proteins, such as CPI-

1 17 and caldesmon in vascul~r smooth muscle. Phosphorylation of CPI-17 was

I attenuated by treatment withl auranofin plus CysNO and DNCB plus CysNO in I endothelium-intact aortas (Fi~ure 2.5). Additionally, another regulatory protein, I smooth muscle-specific h-cal~esmon, was investigated, and phosphorylation of caldesmon was also reduced jby incubation with DNCB plus CysNO (Figure 2.6). 28

A

p-CPI-17

CPI-17

B-actin

.c 1;0·.

Controi .Control Df:JCB+CysNO

Coritrol· · Aurariofin:+CysNO Control DNCB+CysNO

D

*

p-CPI-17

CPI-17

B-actin

Figure 2.5. Phosphorylation o~ CPI-17 was decreased by S-nitrosylation. Isolated mouse aortas were treated with auranofin (Aura; 1 IJM, 1 h) or DNCB (4 IJM, 1 h) plus CysNO (A: 1.5 mM, D: 100I IJM, 30 mm)• and then analyzed by western blot 29 with p-CPI-17, CPI-17, and ~iactin antibodies. A and D: Western blot analysis. B and C: Bar graphs for A, sHowing the relative expression of phospho-CPI-17 and total proteins after norm~lization to ~-actin expression. E: Bar graphs for D. Results are presented as me~n ± SEM in each experimental group. *, p<0.05 vs. control (n=4). 1

i

! I I

! A I Control DNCB+CysNO

p-caldesmon

caldesmon

~ .. actin B c . ·~·- cp cc. ·oc ., ·. E *:·' tn .I. C1) ·-a;"C CJ I Q. - Control DNCB+CysNO i

I

I ! Figure 2.6. Phosphorylation of caldesmon was reduced by S-nitrosylation. Isolated mouse aortas were trbated with DNCB (4 ~M, 1 h) plus CysNO (1.5 mM, 30 min) and then subjected to\ immunoblot with p-caldesmon, caldesmon, and ~­ actin antibodies. A: Western t!>lot analysis. B: Bar graphs showing the relative expression of phosphorylation\ and total proteins after normalization to ~-actin expression. Results ar~ presented as means± SEM in each experimental group. *, p<0.05 vs. control (n=.4). 30

DISCUSSION

In this study, we investigated whether S-nitrosylation interferes with PKC-

\ mediated vascular contractiT. We used endothelium-intact aorta to define this cellular response under nqrmal physiological condition. NO bioactivity is I - mediated by soluble guanyl~l cyclase (sGC), with the subsequent activation of cyclic 3',5'-guanosine mon~phosphate (cGMP)-dependent signaling [78] or I I cGMP-independent actions. The cGMP-independent actions of NO include S- nitrosylation of biomoleculej to form SNO on target proteins [79]. Although cGMP-dependent NO bioactifity has been well studied in vascular function, the cGMP-independent mecha~isms involving S-nitrosylation have not been I demonstrated. Thus, as a inew approach, we investigated S-nitrosylation in terms of cGMP-independent ~0 action in PKC-dependent vasoconstriction.

Our results showed that increased S-nitrosylation abolished PDBu- induced contraction in mousy ·aorta (Figure 2.2 and 2.3). One of the phorbol

I esters, PDBu was used to m~asure PKC-mediated vascular contraction. Phorbol I esters are potent PKC activators and increase calcium-sensitivity, allowing inhibition of MLC phosphatajl in smooth muscle, to cause contraction [80, 81].

We used several types of drugs to increase S-nitrosylation in proteins (Figure 2.1) and to evaluate the effect Jf increased S-nitrosylation on vascular reactivity.

I Specifically, PANOate and C~sNO were used as exogenous SNO sources, but · I CysNO increases S-nittosylation in a more specific manner via transnitrosylation than PANOate, a NO c;tonor. Furthermore, CysNO is transported into the cells 31 with higher efficiency, suggesting that the source of SNO may not be just NO [77, I 82]. '

PKC is activated the second messengers or effectors bind to its wh~nI

i regulatory domain, such as ~he conserved C1 and C2 domains, typically at the ! plasma membrane [83, 84]. : Phorbol esters bind to the C1 domain, which has a

I cysteine-rich sequence, and :this sequence is essential for PKC to bind phorbol

I esters [85-87]. Since S-nitrosylation modifies cysteine residues of target proteins, I phorbol esters may not bind! to or affect the C 1 domain of S-nitrosylated PKC,

I resulting in less PKC activatic~n. I I PKC is known as a target of NO, and NO decreases PKC activity [60], I

I suggesting the possibility th,at PKC may be modified by S-nitrosylation. As

I I shown in Figure 2.4, we d~tected PKC S-nitrosylation and decreased PKC

I

I activity in mouse aortas incubated with auranofin or DNCB plus CysNO, ·

I indicating that S-nitrosylationl is associated with PKC inactivation. In mammals, I the PKC family has 12 diff~rent isoforms [88] and 6 isoforms are present in I vascuiSr smooth muscle cell~ [89]. Previous studies reported that different PKC

I isoforms have specific substtates and functions in different blood vessels and ! species [74, 90, 91]. The pr~sent study specifically determined S-nitrosylation of

PKCa, because PKCa belonJs to the conventional PKC subfamily. This enzyme ! i has calcium and diacylgly~erol binding regions, both of which signaling molecules represent key reJulators of vascular signal transduction. Several I studies have shown that PKCa enhances vascular contraction via calcium 32 sensitization mechanisms ~nd is translocated in response to elevated calcium i from the cytosol to the mem~rane [90, 92, 93].

To determine the re~ulation of target proteins by functional inactivation of

PKC, the expression and phosphorylation of CPI-17 and caldesmon were assessed. One of the major target proteins for PKC that is important in vascular contraction is CPI-17 [70]. PKC-mediated phosphorylation of CPI-17 inhibits

MLC phosphatase, which induces dephosphorylation of MLC, and results in vascular contraction. Caldesmon is another downstream signaling protein of

PKC and is an actin-binding protein. Two isoforms of caldesmon exist: a high molecular weight, smooth muscle-specific h-caldesmon, and a low molecular weight, non-muscle 1-caldesmon. Phosphorylation of caldesmon induces its detachment from actin, leading to the binding of actin and myosin, and results in vascular contraction. We found that these two proteins, CPI-17 and h-caldesmon, exhibited less phosphorylation when S-nitrosylation increased, further supporting the likely inactivation of PKC signaling pathway by S-nitrosylation. Noteworthy, incubation with a low concentration of CysNO (1 00 !JM) and DNCB reduced phosphorylation of CPI-17 only (Figure 2.50 and 2.5E), not caldesmon (data was not shown), suggesting that CPI-17 is an important downstream signaling protein i for PKC function related to vascular contraction and further that this protein is extremely sensitive to the redox state of the cell.

I In summary, in the present study we have confirmed that S-nitrosylation leads to decreased PKC function and signaling and impairs vascular contraction.

! This post-translational modification, and specifically S-nitrosylation of PKC, may 33 represent a key mechanism in conditions associated with abnormal vascular I i reactivity. 34

CHAPTER 3.

THIOREDOXIN REDUCTASE INHIBITION REDUCES RELAXATION BY

INCREASING OXIDATIVE STRESS AND S-NITROSYLATION IN MOUSE

AORTA 35

ABSTRACT i I I Oxidative stress is well khown to lead to vascular dysfunction. Thioredoxin reductase (TrxR) catalyzes the reduction of oxidized thioredoxin (Trx). Reduced

Trx plays a role in cellular antioxidative defense as well as in decreasing S- nitrosylation. It is not known whether TrxR affects vascular reactivity. We hypothesized that TrxR inhibition decreases vascular relaxation via increased oxidative stress and S-nitrosylation. Aortic rings from C57BL/6 mice were treated with the TrxR inhibitor, 1-chloro-2,4-dinitrobenzene (DNCB) or auranofin for 30 min. Vascular relaxation to acetylcholine (ACh) was measured in the rings contracted with phenylep~rine. DNCB and auranofin reduced relaxation compared to vehicle (vehicl.e Emax =71 ± 3 °/o, DNCB Emax = 53 ± 3 °/o; p<0.05).

The antioxidants, apocynin (NADPH oxidase inhibitor) and tempol (superoxide dismutase mimetic) normal.ized impaired relaxation by DNCB in aorta (DNCB

Emax = 53 ± 3 %, DNCB+tempol Emax = 66 ± 3 °/o; p<0.05). In addition, DNCB reduced sodium nitroprusside (SNP)-induced relaxation. DNCB increased soluble guanylyl cyclase (sGC) S-nitrosylation and decreased sGC activity.· i These data suggest that TrxR regulates vascular relaxation via antioxidant defense and sGC S-nitrosylation. TrxR may be a target for the development of I therapies,to treat vascular dysfunction and arterial hypertension.

KEYWORDS

I i vascular relaxation, oxidative stress, S-nitrosylation I I 36

INTRODUCTION I I I Reactive oxygen ~pecies (ROS) are small molecules derived from

molecular oxygen and are .known to play an important role in vascular function. CG

ROS are produced in all vascular cell types, including endothelial cells, smooth

muscle cells, and adventitial fibroblasts [22, 23]. Under physiological conditions, the generation of ROS in vascular cells and redox-dependent signaling are tightly

regulated; however, in pathological conditions, such as cardiovascular diseases,

ROS production is increased. Numerous studies indicate that ROS are

associated with various mechanisms related to impaired vascular function, such

as endothelial dysfunction, vascular inflammation, vascular smooth muscle cell

growth, and apoptosis [94, 95].

An imbalance betWeen ROS generation and antioxidant defense

mechanisms results in oxidative stress. Several enzymes and enzyme systems,

such as NADPH oxidase; xanthine oxidase, mitochondria, and uncoupled endothelial nitric oxide synt~ase (eNOS), produce ROS in the vessels. ROS are i scavenged and inactivate~ by several antioxidant mechanisms, such as I I superoxide dismutase, catal~se, glutathione peroxidase, and the thioredoxin (Trx) i system. The Trx system plays a role in the degradation of hydrogen peroxide I (H202) and has other cellular functions [96]. However, the role of the Trx divers~I ! system in the vasculature is unclear and is the focus of this study. i The Trx system con~ists of Trx and Trx reductase (TrxR). TrxR is a I . I redox-based enzyme and patalyzes the NADPH-dependent reduction of the

I oxidized redox protein Trx.\ Mammalian TrxR can reduce several substrates, i I 37 I I i such as glutaredoxin an, glutathione [97, 98], but reduces mainly Trx. In addition to an antioxidant\ mechanism, reduced Trx by TrxR functions in cell ' i growth by inhibiting apopto;sis and gene transcription [96]. Additionally, a newly defined function of TrxR is denitrosylation, which is removal of S-nitrosylation from target proteins [52]. S-Nitrosylation is a redox-based and post-translational modification of a cysteine residue of a target protein by nitric oxide (NO) to form

S-nitrosothiol (SNO) [43]. :

NO is a key regulator of vascular tone, which is determined by the balance of vasodilator and vasoconstrictor stimuli. In the vasculature during relaxation,

NO is released from endothelial cells and stimulates endothelium-dependent vasodilation via diffusion into the smooth muscle. NO activates soluble guanylyl cyclase (sGC) to form cyclic 3',5'-guanosine monophosphate (cGMP) from guanosine triphosphate (GTP), and cGMP results. in vascular relaxation via decreased cytosolic calciu~ levels, increased protein phosphorylation, and myosin light chain dephosph:orylation [17]. I In this study, the hyppthesis was that TrxR inhibition attenuates vascular I relaxation via increased oxid.ative stress and S-nitrosylation. In order to test this I I hypothesis, we examined w!hether TrxR plays a role in vascular reactivity and I i signaling by investigating vascular relaxation in mouse aorta in the presence or I absence of a TrxR inhibitor. , I I 38 I I I MATERIALS AND METHdDs I Drugs and reagents ! I 1-Chloro-2,4-dinitro8enzene (DNCB) and other chemicals were purchased

1 I from Sigma-Aldrich (St. Louis, MO). Auranofin was obtained from BioMol

(Plymouth Meeting, PA).

Animals

Male, 12-14 weeks-old, C57BL/6 mice (Jackson Laboratory, Bar Harbor,

ME) were used in this study. All procedures were performed in accordance with the Guiding. Principles in ·the Care and Use of Animals, approved by the

Institutional Animal Care and Use Committee (IACUC) and Georgia Health

Sciences University Committee on the Use of Animals in Research and

Education. The animals were housed on a 12-hour light/dark cycle and fed a standard chow diet with water ad libitum.

Isolation of aortic rings and functional studies

After mice were euthanized using carbon dioxide (C02), thoracic aortas I were excised, cleaned froml fat tissue and cut into 2 mm-length rings in an ice-

cold physiological salt soluti~n consisting of the following: 130 mmoi/L NaCI, 4.7 i mmoi/L KCI, 1.18 mtnoi/L !KH2P04, 1.18 mmoi/L MgS04·?H20, 1.56 mmoi/L I I CaCI2·2H20, 14.9 mmoi/L NaHC03, 5.6 mmoi/L glucose, and 0.03 mmoi/L EDTA. I

. I Aortic rings were mounted in a myograph (Danish Myo Technology A/S, Aarhus, I Denmark) containing wa~med (37°C), oxygenated (95% 0 2/5% C02) I physiological salt solution. T~e preparations were equilibrated for at least 1 hour I under a passive force of 5 mN. After the equilibration period, arterial integrity was I I 39

I assessed by stiniul~tion o~ vessels with 120 mmoi/L KCI and, after contraction ! reached a plateau, the hngs were washed. Subsequently, the rings were I I I stimulated with phenylephrine (PE, 0.1 IJmoi/L) followed by relaxation with acetylcholine (ACh, 1 1Jmoi/L), which was used as an evidence of an intact endothelium. Some aortic rings were incubated with DNCB (4 IJmoi/L) or auranofin (1 1Jmoi/L) for 30 min, apocynin (1 00 IJmoi/L) for 40 min, or tempol (1 mmoi/L) for 20 min. Endothelium-dependent relaxation was performed on PE­ contracted rings followed by cumulative concentration-response curves to ACh

(1 o-9 to 3x1 o-5 moi/L), and endothelium-independent relaxation was tested with sodium nitroprusside (SNP;. 1o- 11 to 1o- 6 moi/L).

Extracellular H202 assay

H202 production was measured using an Amplex red H202 assay kit

(Invitrogen, Carlsbad, CA) according to the manufacturer's protocol.[99] Briefly, cleaned aorta was equilibrated for 1 h at 37 DC in a modified Krebs-HEPES buffer

(containing 20 mmoi/L HEPES, 119 mmoi/L NaCI, 4.6 mmoi/L KCI, 1.0 mmoi/L

MgS04·?H20, 0.15 mmoi!L\Na2HP04, 0.4 mmoi/L KH2P04, 5 mmoi/L NaHC03, I 1.2 mmoi/L CaCb, and 5.5\ mmoi/L dextrose, pH 7.4) and then incubated with

I Amp lex red working solution (1 00 1Jmoi/L) at 37°C for 1 h. The supernatant was I then transferred to a 96-well\ plate, and absorbance was measured (561 nm) on a I . I IJQuant plate reader (Bio-Te1,k Instruments, Inc., Winooski, VT). A H202 standard i I curve constructed on the s~me 96-well plate was incubated with Amplex red

I I working solution (1 00 .IJmol/~) at 37°C at the same time as the tissues and was I I - used to determine H202 concentrations from samples. After each experiment, I I 40

I tissue total protein was determined with the BCA protein assay and used for I normalization.

Detection of S-nitrosylation

Aortas were treated with vehicle (ethanol) or DNCB (4 !Jmoi/L) for 1 h at

37 oc in Dulbecco's modified Eagle's medium (DMEM). The biotin-switch method to detect S-nitrosylation was performed with a S-nitrosylated protein detection assay kit (Cayman Chemical, Ann Arbor, Ml) modified from the method of Jaffrey et al [75]. Biotin;..labeled proteins were used for western blot analysis or pulled down by streptavldin-agarose beads (Thermo Scientific, Rockford, IL), to specifically detect sGC $-nitrosylation, followed by western blot analysis. Ten

percent of biotin-labeled proteins were used to measure total sGC protein expression.

Western blot analysis

After precipitation with streptavidin, proteins were eiuted by boiling in

Laemmli sample buffer (Bio-Rad, Hercules, CA). The eluted samples or biotin-

labeled proteins were s~parated by electrophoresis on a 10% SDS- 1 i polyacrylamide gel and t:ransferred to a nitrocellulose membrane. The I

! membranes were blocked with 5% skim milk in Tris-buffered saline solution with

! I 0.1% Tween-20 for 1 h at room-temperature. Membranes were then incubated with anti-sGCJ31 primary ant,ibody (Cayman Chemical), anti-phospho-eNOS (Ser

1177), or anti-eNOS antibody (Cell Signaling Technology, Inc., Danvers, MA) I I overnight at 4 °C. After ihcubation with secondary antibody, signals were 41

I developed with chemilu~minescence, visualized by autoradiography, and i quantified densitometricall~. cGMP assay

Cleaned aortas were incubated with vehicle (ethanol) or DNCB (4 J,.~moi/L) for 1 h at 37 oc in DMEM and then treated with SNP (3x1o-a moi/L) for 20 min. cGMP was measured using a commercially available cGMP enzyme-linked immunoassay (cGMP EIA kit) according to the manufacturer's instructions

(Cayman Chemical, Ann arbor, Ml). The measured cGMP level was normalized by protein concentration using BCA protein assay.

Statistical analysis

Values are means ± standard error of the mean (SEM}, and n represents the number of animals used in the experiments. Experimental values were calculated relative to the maximal changes from the contraction produced by PE in each segment, which was taken as Oo/o. The baseline tension before addition of PE was considered as 1OOo/o. Concentration-response curves were fitted using a nonlinear interactiv~ fitting program (Graph Pad Prism 4.0; GraphPad

Software, San Diego, CA), and two pharmacological parameters were obtained: the maximal effect gener~ted by the agonist (or Emax) and EC50 (molar concentration of agonist prqducing 50% of the maximum response) or pD2 (-log

EC50). Statistical differences were calculated by Student's t-test or one-way

AN OVA. A P value less than 0.05 was considered to be statistically significant. ! 42

RESULTS I i Effect of TrxR inhibition on endothelium-dependent relaxation I To test the hypothesis that inhibition of TrxR decreases vascular relaxation in mouse aorta, we first determined ACh-induced relaxation in the presence of the TrxR inhibitors, DNCB or auranofin [51, 52]. Aortic rings incubated with a TrxR inhibitor exhibited significantly attenuated relaxation to

ACh compared to the vehicle control group (Figure 3.1; Table 3.1 ).

TrxR inhibition and ROS

One of the functions of Trx and TrxR is to reduce ROS; therefore, we measured whether TrxR inhibition increases ROS in mouse aorta. Figure 3.2 shows that DNCB increased H202 production from aorta. To test whether increased ROS by TrxR in~ibition contributes to impaired relaxation to ACh, we used two antioxidants, a NADPH oxidase inhibitor (apocynin) and· a superoxide dismutase mimetic (tempo!), to attenuate ROS concentration. These antioxidants normalized ACh-induced relaxation decreased by DNCB (Figure 3.3;

Table 3.1). 44

A B

. ~· 20 -c. . ;;0 40 ca .. >< ca * ~ 60 * 80 o Vehicle 0 Vehicle 100 • DNCB • Auranofin

~9. -7. ·•. ~6· · i ~s -4 -9 -8 . -7 . -6 -5 l.og[AChf(M) ·. · log[Ach] (M)

Figure 3.1. Vascular relaxation to ACh is attenuated by DNCB and auranofin . . Concentration-dependent responses to ACh were measured after incubation with DNCB (4 J.Jmoi/L; A, n=S to 6) or auranofin (1 J.Jmoi/L; 8, n=S to 6) for 30 min in mouse aorta. Relaxation responses were calculated relative to the maximal PE contraction, which was taker as 100°/o. Results are presented as means± SEM in each experimental group.· *p<0.001 compared with vehicle. 43

I Table 3.1. Emax and pD2 ~alues for ACh and SNP-induced responses in aortic

rings

Vehicle DNCB Agonist Em ax pD2 Em ax pD2 ACh 71±3 7.3±0.2 53±3* 6.8±0.2 ACh + Apocynin 71±2 6.7±0.1 71±4t 6.7±0.1 ACh + Tempol 67±3 7.3±0.1 66±3t 7.4±0.1 SNP 98±2 9.0±0.1 95±2 8.7±0.1*

Values are mean ± SEM for n=5 to 7 in each group. Relaxation induced by ACh and SNP was calculated relative to the maximal changes from the contraction produced by PE and is represented as percentage of relaxation.

*p<0.05 vs vehicle in same agonist; tp<0.05 vs DNCB with ACh. 45

*

Control·

Figure 3.2. DNCB increased H202 levels in mouse aorta. H202 concentration -was measured in the presence or absence of DNCB (4 IJmoi/L, 1 h) in mouse aorta using Amplex red as described in the Methods. Results are presented as means ± SEM for n=3 in control and n=4 in DNCB group. *p<0.05 compared with control. 46

A B

w.c... 20 ·c- c 0 0 .. 40 cu -~.cu >

·9 -8 -7 . ·6 ·5 -4 ·9 -8 -7 ·6 ·5 log[ACh] (M) log[ACh] (M)

Figure 3.3. Antioxidants normalized relaxation impaired by DNCB. Concentration-dependent relaxation to ACh was measured after incubation of aortic rings with A: vehicle, DNCB (4 !Jmoi/L, 30 min), apocynin (1 00 !Jmoi/L, 40 min), and DNCB plus apocynin. B: vehicle, DNCB (4 !Jmoi/L, 30 min), tempo! (1 mmoi/L, 20 min), and DNCB plus tempo!. Relaxation responses were calculated relative to the maximal: PE contraction, which was taken as 100°/o. Results are presented as means ± ·SEM in each experimental group (n=6). *p<0.05 DNCB vs. DNCB plus apocynin (A) or DNCB plus tempo! (B). 47

Effect of TrxR inhibitio'n on endothelium-independent relaxation

Considering that TrxR inhibition contributes to relaxation via an endothelium-independent mechanism, we measured relaxation to SNP diffused directly into smooth muscle. DNCB shifted the concentration-response relaxation to SNP to the right in mouse aorta (Figure 3.4; Table 3.1 ).

TrxR and S-nitrosylation

In addition to its antioxidant effect, a denitrosylation function of TrxR was confirmed via measurement of S-nitrosylation levels in the previous study [62].

Using a TrxR inhibitor DNCB, protein S-nitrosylation is stabilized, and we measured S-nitrosylation levels of sGC as a relaxation signaling protein in mouse aorta. One of the sGC subunits, J31 showed increased S-nitrosylation modification in the presence of DNCB compared to control without DNCB (Figure

3.5A). cGMP, which is downstream of sGC, was measured in mouse aorta, and

SNP-stimulated cGMP levels were reduced by DNCB, not basal levels (Figure

3.58). 48

-w 20 Q. .. -c 0 40 ...cu )(cu ·"Qi 60 0:::. "#. 80

CJ . V~hiFie. 100 .· •: DNCB' ...

· · . ~9 ·• · .:a :;_log[SNPJ (M)

Figure 3.4. DNCB shifted vascular relaxation to SNP to the right. Concentration­ dependent relaxation to SNP was measured after incubation with DNCB (4 1Jmoi/L, 30 min) in aortic rings. Relaxation responses were calculated relative to the maximal PE contraction, which was taken as 100°/o. Results are presented as mean ± SEM in each experimental group. *p<0.05 compared with vehicle (n=6 to 7). 49

A 8

DNCB +

SNO-sGC~l

sGC~l

SNP

Control DNCB

Figure 3.5. sGC S-nitrosylation was increased and its activity was decreased by DNCB. A: sGC~t S-nitrosylation (SNO-sGCJ31) was detected using the biotin­ switch method, streptavidin precipitation, and western blot analysis with anti­ sGC~1 antibody. Total sGC~1 expression (sGC~1) was detected using 1:10 biotin-labeled proteins using western blot analysis. Representative western blot images (Top). Bar graphs showing the relative expression of SNO-sGC~1 after normalization to total sGCJ31 (Bottom). *p<0.05 compared to control (n=5). 8: cGMP levels were measured in mouse aorta to determine sGC activity. Aorta was incubated with DNCB (4 !Jmoi/L, 1 h) in the presence or absence of SNP (3x1 o-a moi/L, 20: min). Results are presented as means ± SEM in each experimental group. ~p<0.05 compared to control with SNP (n=4 to 6). 50

DISCUSSION

The main finding~ of the present study is that the inhibition of TrxR leads to

impairment in aortic relaxation via increased H202 and sGC S-nitrosylation .. TrxR

is an important a.ntioxidant enzyme and also has other cellular functions;

. however, the relationship between TrxR and vascular reactivity has not been

elucidated. In the· present study, we investigated whether TrxR plays a role in

vascular relaxation.

Several studies show that oxidative stress in cardiovascular disease

contributes to endothelial dysfunction [1 00-1 04], which refers to impaired

endothelium-dependent vascular relaxation. Increased ROS production

scavenges NO and generates oxidants, resulting in loss of NO bioactivity. TrxR

is an antioxidant enzyme that reduces ROS, and this function was confirmed

(Figure 3.2). ROS concentration is increased by TrxR inhibition; and thus, NO

may be scavenged rapidly by ROS. Additionally, since increased ROS impairs

vascular reactivity~ reducing ROS levels by treatment with apocynin and tempol

normalized the decreased endothelium-dependent relaxation observed with

inhibition of TrxR. These data indicate that TrxR regulates vascular relaxation

via an antioxidant function.

ACh acts 1as a vasodilator via NO production, which is induced by

activation of the ACh receptor and subsequently eNOS in endothelial cells. NO

activates sGC in :va~9ular smooth muscle to produce cGMP, and then, cGMP

plays a critical role to: lead to smooth muscle relaxation [1 05]. In Figure 3.1A and

3.4, a TrxR inhi~itor (DNCB) impaired vascular relaxation to ACh and SNP. 51

These data suggest th~t some factor(s) in vascular smooth muscle has/have important functions •related to TrxR. We propose that one of these factors may be sGC. Accordingly, TrxR is associated with sGC signaling in the relaxation of mouse aorta. In addition to its antioxidant effect, TrxR is an enzyme that decreases protein S-nitrosylation [52]. Studies show that sGC is desensitized and inhibited by increasing S-nitrosylation [63]. We confirmed this effect using the biotin-switch method and a cGMP assay. As shown in Figure 3.5, sGCJ31 S- nitrosylation ·was increased and sGC activity (cGMP levels) was reduced in the presence of DNCB, suggesting that vascular relaxation was attenuated by TrxR inhibition through sGC S-nitrosylation. This finding provides a new concept that, in addition to ROS function in vasculature, S-nitrosylation can affect cellular signaling in aorta.

NO bioactivity is mediated by sGC, with the subsequent activation of cGMP-dependent signaling [78], as well as by cGMP-independent mechanisms.

The cGMP-independent actions of NO include protein S-nitrosylation to form

SNO [79]. Although cGMP-dependent NO bioactivity has been well studied in vascular reactivity, the cGMP-independent mechanisms involving S-nitrosylation remain to be further investigated. Since NO and cGMP play an important role in transduction of signaling, the present study suggests that it is possible to manage vascular signaling through sGC via both cGMP-dependent and -independent mechanisms.

It is known that! S-nitrosylation modifies several target proteins including channels, transpo~ers,. kinases, signaling proteins, and transcription factors [57].

I 52

One of the important proteins in vascular signaling, eNOS is modified by S- nitrosylation and :its ,activity is inhibited [59]. In our results, eNOS phosphorylation at serine 1177, which represents eNOS activation, and S­ nitrosylation was not different between control and DNCB treatment groups (data not shown). These data suggest that while TrxR inhibition using DNCB stabilizes

S-nitrosylation, this is not sufficient to increase eNOS S-nitrosylation and inactivate eNOS.

Furthermore, we tested concentration-dependent contractile responses to

PE in the presence or absence of DNCB, a TrxR inhibitor, but DNCB did not change contraction to PE in mouse aorta (data not shown). Thus, we investigated relaxation using aortic rings contracted by PE in this study. In a previous study, we observed that protein kinase C-mediated contraction was slightly attenuated by DNCB [62]. Therefore, we speculate that TrxR effects on

S-nitrosylation are associated with several vascular signaling processes, and further study will be necessary to fully understand the mechanism of TrxR and vascular reactivity.

In summary, the present study shows that a TrxR inhibitor impaired both endothelium-dependent and -independent vascular relax~tion. This impaired relaxation by the TrxR inhibitor was due to increased oxidative stress and sGC S­ nitrosylation and infictivation. Accordingly, TrxR modulates vascular relaxation in

mouse aorta via j functions including an antioxidant and a denitrosylation mechanism. 53

CHAPTER4.

AUGMENTED S-NITROSYLATION CONTRIBUTES TO IMPAIRED

RELAXATION IN ANGIOTENSIN II HYPERTENSIVE MOUSE AORTA: ROLE

OF THIOREDOXIN REDUCTASE 54

ABSTRACT

Vascular dysfunction, including reduced endothelium-dependent dilation, is a main characteristic of hypertension. Nitric oxide (NO) is a critical factor in the cardiovascular system and modifies target proteins' cysteine residues via S­ nitrosylation. Thioredoxin (Trx) and Trx reductase (TrxR) play roles in limiting S­ nitrosylation. S-Nitrosylation and TrxR fuction are unstudied in hypertension, and we hypothesized that S-nitrosylation is associated with reduced vasodilation in hypertensive mice. Aortic rings from normotensive (sham) and angiotensin II

(Angll)-induced hypertensive C57BL/6 mice were treated with a TrxR inhibitor, 1- chloro-2,4-dinitrobenzene (DNCB) for 30 min, and relaxation to acetylcholine

(ACh) was measured in the rings following contraction with phenylephrine.

DNCB reduced relaxation to ACh compared with vehicle in sham aorta but not in

Angll (sham-vehicle Emax=77±2, sham-DNCB Emax=59±4, *p<0.05; Angll-vehicle

Emax=73±3, Angii-DNCB Emax=66±3). DNCB shifted the concentration-response curve for relaxation to sodium nitroprusside (SNP) to the right in both sham and

Angll aortic rings (sham-vehicle pD2=8.8±0.1, sham-DNCB pD2=8.4±0.1, *p<0.05;

Angll-vehicle pD2=8.S±0.1, Angii-DNCB pD2=8.3±0.1, *p<0.05). Soluble guanylyl cyclase (sGC) acti~ity. (cyclic GMP assay) was reduced by DNCB during activation with SNP. The effect of DNCB to increase S-nitrosylation was confirmed by the biotin-switch method and western blot analysis, and total protein S-nitrosylation was increased in Angll aorta (1.5-fold) compared to sham.

TrxR activity was in~ibit~d in Angll aorta compared to sham. These data suggest that increased S-nitrosylation is related to impaired vasodilation in Angll aorta. 55

Angll treatment resulted in inactivation of TrxR and increased S-nitrosylation, i indicating that TrxR could provide a link between increased oxidative stress and augmented S-nitrosylation in Angll-induced hypertension.

KEY WORDS: S-nitrosylation, thioredoxin reductase, vascular relaxation, hypertension 56

INTRODUCTION

I ! Vascular dysfunction, including reduced endothelium-dependent dilation and enhanced vasoconstriction, is a main feature of hypertension. This has been observed in hypertensive patients where forearm blood flow responses to vasodilators are impaired [1 06-1 08]. Compromised endothelium-dependent vascular relaxation, referred to as endothelial dysfunction, has been demonstrated in both conduit arteries and· resistance arteries in several hypertensive animal models. Endothelium-dependent vasodilation is reduced in isolated arteries from spontaneously hypertensive rats [8, 9], deoxycorticosterone acetate salt-treated animals [1 0, 11 ], Dahl salt-sensitive rats [12], and in animal models of renovascular hypertension [13, 14].

Nitric oxide (NO) is an important signaling molecule in the cardiovascular system and a regulator of vascular tone. NO bioactivity is mediated by soluble guanylyl cyclase (sGC), with the subsequent activation of cyclic 3',5'-guanosine monophosphate (cGMP)-dependent signaling [78] or by cGMP-independent signaling [79]. The cGMP-independent mechanisms include protein S- nitrosylation, which is a post-translational modification forming S-nitrosothiol

(SNO) on the cysteine residue of target proteins [79]. Although cGMP- dependent NO bioactivity is well understood in vascular function, the cGMP- independent signaling involving S-nitrosylation is not. S-Nitrosylation is regulated by denitrosylation to' decrease S-nitrosylation, and one of the enzymatic systems for denitrosylation iJ the thioredoxin (Trx) system. The Trx system consists of

Trx and Trx reductase (TrxR). TrxR is an enzyme that catalyzes the 57 nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reduction of the oxidized redox pJotein Trx. Reduction of Trx by TrxR removes S-nitrosylation

I from proteins [52]. ·Mammalian TrxR can reduce several substrates, such as glutaredoxin and glutathione [97, 98], but its main target is Trx. In addition to denitrosylation, the · reduced Trx serves in many cell functions, including antioxidant formation, cell growth, inhibition of apoptosis, and gene transcription

[96].

Abnormal S-nitrosylation is related to various human diseases, such as diabetes, heart failure, asthma and pulmonary hypertension [54, 109-111].

However, an association between hypertension and S-nitrosylation has not been demonstrated. Therefore, the present study aims to investigate a novel regulatory mechanism, S-nitrosylation, modulating vascular function during hypertension. In this study, we hypothesized that increased S-nitrosylation is associated with impaired vasodilation in hypertension. To investigate this hypothesis, we tested vascular relaxation in aorta from normotensive and angiotensin II (Angll)-induced hypertensive mice. Specifically, we focused on

TrxR function to demonstrate S-nitrosylation effects on vascular reactivity, using a TrxR inhibitor to stabilize the level of S-nitrosylation [51, 52].

METHODS

I Drugs and reagentS

Angll was obtained from Phoenix Pharmaceuticals (Burlingame, CA). 1-

Chloro-2,4-dinitrobenzene (DNCB) and other chemicals were purchased from 58

Sigma-Aldrich (St. Lquis, 'MO). S-Nitrosocysteine (CysNO) was synthesized from

L-cysteine using abdified nitrite. Auranofin was purchased from BioMol

(Plymouth Meeting, PA).

Animals

Male C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME), 12-14 weeks of age, were used in this study. To induce Angll hypertension, osmotic minipumps (Durect Corporation, Cupertino, CA) with Angll (3.6 !Jg/kg/min) were implanted subcutaneously into the dorsum for 14 days. Mean arterial pressure

(MAP) was measured in non-anaesthetized mice by the tail cuff method using a

CODA6 blood pressure system (Kent Scientific Corporation, CT). All procedures were performed in accordance with the Guiding Principles in the Care and Use of

Animals, approved by the Georgia Health Sciences University Committee on the

Use of Animals in Research and Education. The animals were housed on a 12- hour light/dark cycle and fed a standard chow diet with water or saline ad libitum.

Isolation of aortic ri.ngs and functional studies

After mice were euthanized, thoracic aorta were excised, cleaned from fat tissue and cut into 2 mm length-rings in an ice-cold physiological salt solution consisting of the following: 130 mmoi/L NaCI, 4. 7 mmoi/L KCI, 1.18 mmoi/L

NaHC03, 5.6 mmoi/L glucose, and 0.03 mmoi/L EDTA. Aortic rings were mounted in a myograph (Danish Myo Technology AIS, Aarhus, Denmark) containing warmed !(37°C), oxygenated (95% 02/6°/o C02) physiological salt solution. The preparations were equilibrated for at least 1 hour under a passive 59 force of 5 mN. After the equilibration period, arterial integrity was assessed by stimulation of vessdls with 120 mmoi/L KCI and, after contraction reached a plateau, the rings were washed. Subsequently, the rings were stimulated with phenylephrine (PE, 0.1 1Jmoi/L) followed by relaxation with acetylcholine (ACh, 1

1Jmoi/L), which was used as evidence of an intact endothelium. Some aortic rings were incubated with DNCB (4 IJmoi/L) for 30 min or CysNO (1 0 IJmoi/L) for 15 min. Endothelium-dependent relaxation was performed on PE-contracted rings followed by cumulative concentration-response curves to ACh (10-9 to 3x1o-5 moi/L) and endothelium-independent relaxation was tested with sodium nitroprusside (SNP, 10-11 to 10-6 moi/L). cGMP assay

Cleaned aortas were incubated with vehicle (ethanol) or DNCB (4 1Jmoi/L) for 1 h at 37 oc in Dulbecco's modified Eagle's medium (DMEM) and then treated with SNP (3x1 o-8 moi/L) for 30 min. cGMP was measured using a commercially available cGMP enz;yme-linked immunoassay (cGMP EIA kit) according to the

! manufacturer's instructions (Cayman Chemical, Ann Arbor, Ml).

Detection of S-nitrosylation

Aortas were treated with vehicle (ethanol) or DNCB (4 1Jmoi/L) for 1 h at

37 oc in DMEM. The biotin-switch method to detect S-nitrosylation was performed with a S:nitrosylated protein detection assay kit (Cayman Chemical,

Ann Arbor, Ml), modified from the method of Jaffrey et al [75]. Biotin-labeled proteins were used fbr western blot analysis. 60

I Western blot analysis

Proteins (40 [IJg), extracted from aorta or biotin-labeled proteins were eluted by boiling in Laemmli sample buffer (Bio-Rad, Hercules, CA) with or without mercaptoethanol, respectively. These proteins were separated by electrophoresis on a 1Oo/o SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. The membranes were blocked with 5°/o skim milk in

Tris-buffered saline solution with Tween-20 for 1 h at room-temperature.

Membranes for biotin-labeled proteins were then incubated with anti-biotin antibody conjugated with horseradish peroxidase (HRP) (Cayman Chemical) overnight at 4 °C. Other membranes were incubated with primary antibodies, such as TrxR 1 and TrxR2 (Abeam, Cambridge, MA) overnight at 4 °C. After incubation with secondary antibodies and washing of the membranes, signals were developed with chemiluminescence, visualized by autoradiography, and quantified densitometrically.

TrxR activity

Aortas were treated with vehicle (ethanol) or DNCB (4 !Jmoi/L) for 1 h at

37 oc in DMEM. TrxR activity was measured using a commercially available

TrxR assay kit (Sigma-Aldrich, St. Louis, MO) according to the manufacturer's instructions.

Recombinant protein delivery

I To deliver redombinant protein, TrxR (American Diagnostic Inc., Stamford,

I CT), into the aortic rings, Chariot protein delivery reagent (Active Motif, Carlsbad,

CA) was used according to the manufacturer's instruction as described 61

previously [112, 113]. This transfection reagent is able to deliver protein and

antibody into cells ahd h~s been used successfully to deliver proteins to lung and

vascular tissue [114, 115]. In brief, 6 J.JI of Chariot reagent in 100 J.JI of 40%,

dimethyl sulfoxide (DMSO) were mixed with 6 lJg of protein in 100 lJI of

phosphate-buffered .saline (PBS) and incubated at room temperature for 30

minutes. The aortas were transferred to 400 lJI of DMEM in a 24-well cell culture

plate, and 200 lJI of the protein/Chariot complexes were added. Aortas were

incubated for 1 hour at 37 oc in a C02 incubator, followed by addition of DMEM

(750 lJI) and further incubated for 3 hours at 37 oc (C02 incubator).

Subsequently, the aortic rings were mounted in the myograph,. and functional

studies were performed.

Statistical analysis

Values are means ± standard error of the mean (SEM), and n represents the number of animals used in the experiments. Experimental values were

calculated relative to the maximal changes from the contraction produced by PE

in each segment, which was taken as 0%. The baseline tension before addition

of PE was considered as 100°/o. Concentration-response curves were fitted

using a nonlinear interactive fitting program (Graph Pad Prism 4.0; GraphPad

Software, San Diego, CA), and two pharmacological parameters were obtained: the maximal effect generated by the agonist (or Emax) and ECso (molar concentration of agonist producing 50% of the maximum response) or pD2 (-log

ECso). Statistical di,fferencesI were calculated by Student's t-test or one-way

AN OVA. A P value 'ess than 0.05 was considered to be statistically significant.

I 62

RESULTS

Table 4.1. Mean arterial pressure and body and heart weight in normotensive

(sham) and Angll-infused mice.

Parameter Sham Angll MAP (mm Hg) 114.4 ± 3.8 149.3 ± 3.8* Body weight (g) 27.9 ± 0.6 24.5 ± 0.4* Heart weight (mg) 130.1 ±3.1 143.4 ± 3.8*

MAP; Mean arterial pressure.

Values are means ± SEM for n=7 in each group. *p<0.05, sham vs. Angll 63

Effects of TrxR inhibition on vascular relaxation

i ' Considering trat endothelium-dependent relaxation is impaired in mouse models of Angll-induced hypertension, we first measured relaxation to ACh, which was attenuated in the Angll compared to sham normotensive mouse aorta

(Figure 4.1A and 4.1 B). To test the hypothesis that TrxR inhibition contributes to vascular relaxation, ~e used a TrxR inhibitor, DNCB [51, 52]. In endothelium- intact aortic rings incubated with DNCB, relaxation to ACh was significantly reduced in sham, but not in Angll aortic rings (Figure 4.1 C and 4.1 D). In addition,

DNCB shifted the concentration-response relaxation to SNP to the right in both sham and Angll aortic rings (Figure 4.2).

Effect of TrxR on cGMP concentration

Figure 4.3 shows that basal cGMP levels without SNP were not different between control and DNCB-treated aorta. However, in the SNP-stimulated condition, DNCB decreased cGMP levels in aorta from both sham and Angll.

Also, SNP-treated aorta from Angll-induced hypertensive mice exhibited lower cGMP levels than sham aorta. 64

A B

0 we:. 20 c 0 40 ~ & 60 ?fl. 80 * 6 :•sham-Vehicle· · Vehicle . DNCB Vehicle .DNCB 100 o An~II:-V13hiclef: •.sham· . Angtl ·~~ .. -ii· . :.~~',.: ~~•. •. . •.. IO{J[ACh]

0 ...... w 20 w 20' a. !:.· -·c: c 0 40 0 40 ..~ >< ~ ~ ~ Q) 60' - 60 ·~ ~

~0 80 tJ 80 c Sham-Vehicle' o Angli-Vehicle 100 • Sham-DNCB : 100 • AngU:DNCB

~ ·~ ' ql ~· ~ -9 ·8 -7 . -6. -5 log[AChl(M) Jog[ACh] (M)- . i . .

Figure 4.1. Concentration-response relaxation to ACh was attenuated by DNCB only in sham aortic rings but not in Angll. A: Relaxation to ACh was measured in vehicle-treated ring~ from sham (n=6) and Angll (n=9). 8: Representative pD2 values from graphs! A, C, and D. C and D: Relaxation to ACh was measured after incubation with or without DNCB (4 tJmoi/L) for 30 min in sham (C; n=6) or

1 Angll (D; n=9) aortic rings. Relaxation responses were calculated relative to the maximal PE contraction, which was taken as 100%. Results are presented as means ± SEM in each experimental group. *p<0.05 compared with sham-vehicle. 65

A B

0

w 20 -a.·

...... ' . . · · ,.CI. ·sh~rt,.:vehid{ 100 · · o. Angll .. Vehicle Sham Angll

-9 -8 -7 log[SNP] {M) .

C D

0

c 0 40 . ..(U• '>( (U :60 :,;~· · .. ?fl. 80 . . . . . t( $h~m~vehicl~ o. An~u .. \iehicle · +· 100 100 IIi . Sham.. DNCj3 i .• Angir-DNCB

-10 ·9 ' ~8 ·1 -10 -9 -8 ·1 log[SNP] {M) log[SNP] {M)

Figure 4.2. Vascular relaxation to SNP was shifted to the right by DNCB in sham and Angll. A: Relaxation to SNP was measured in vehicle-treated rings from sham (n=5) and Angll (n=8). 8: Representative pD2 values from graphs A, C, and D. C and D: :Concentration-dependent relaxation to SNP was measured after incubation with1 vehicle or DNCB (4 IJmoi/L, 30 min) in sham (C; n=5 to 6) or Angll (D; n=8) aortiQ rings. Relaxation responses were calculated relative to the maximal PE contrattion, which was taken as 100%. Results are presented as means± SEM in each experimental group. *p<0.05 compared with sham-vehicle. tp<0.05 compared ~etWeen vehicle and DNCB in the Angll group. 66

· C::tCohtr~l . : .• :oNes·

t * *'

·sham Angll Sham Angll SNP

Figure 4.3. DNCB. attenuated cGMP concentration in SNP-treated condition. cGMP levels were measured in mouse aorta to determine sGC activity. Aortas were incubated with or without DNCB (4 lJmoi/L, 1 h) in the presence or absence of SNP (3x1o-s moi/L, 30 min). Results are presented as means± SEM in each experimental group. 1 *p<0.05 compared to control with SNP in each sham or Angll group. tp<0.05 compared to control without SNP in each sham or Angll group (n=3 to 6). 67

Contribution of Trx'R and S-nitrosylation in An gil

Total S-nitro~ylated proteins were increased in Angll aorta compared to sham (Figure 4.4 ). Considering that the level of protein S-nitrosylation is associated with TrxR, which decreases S-nitrosylation, TrxR protein expression and activity were measured. Protein expression of the cytosolic form TrxR1 and mitochondrial TrxR2 was not different between sham and Angll aorta (Figure

4.5A). However, TrxR activity was significantly less in Angll aorta when compared to sham (Figure 4.58). 68

A B

250 c * 150 :;:; CJ ca.. 100 ·c:c;. 0 .· 75 ·-z U) ··;s. 0 50 1-

SNO Sham An gil 37

25

20

Figure 4.4. S-Nitrosylated proteins (SNO) were increased in Angll aorta compared to sham) A: Representative western blot images of S-nitrosylated proteins. 8: Bar g~aphs showing the relative content of S-nitrosylated proteins after normalization ito [3-actin expression. Results are presented as means ± SEM in each experimental group. *p<0.05 compared with sham (n=4 to 5). 69

A B· 1:::1 Control - DNCB TrxR1

TrxR2'

13-actin

· ·Sham Angll

Sham Angll·

Figure 4.5. TrxR protein expression was not different between sham and Angll, but TrxR activity was diminished in Angll aorta compared to sham. A: Representative western blot images of TrxR1 and TrxR2 proteins in aorta (Top). Bar graphs. show the relative content of TrxR 1 or TrxR2 proteins after normalization to ~-actin expression (Bottom; n=4). B: TrxR activity was measured in mouse aorta as described in the Methods section. Results are presented as means ± SEM in each experimental group. *p<0.05 compared to control in sham (n=$ to 8). I 70

As an alternative approach to evaluate the role of TrxR in vascular relaxation, TrxR ~rotein delivery using the Chariot system resulted in I normalization of vascular relaxation to ACh and SNP in Angll aortic rings (Figure

4.6).

Effects of S-nitrosylation on vascular relaxation

CysNO is used to increase directly the S-nitrosylation of proteins [77]. We observed that concentration-dependent responses to ACh and SNP were attenuated in the presence of CysNO in Angll aortic rings (Figure 4.7). 71

A B

0 t

a.w 20 -c ~ 40 ~ . )( . ~ . & 60 '

· · · .so· · · · · ...... t{ Sham.;control· Control TrxR · Control . TrxR ·:1oo.· . ·a· :~nb.il.:c6ntr,61. Sham Angll ~1 ,. · ::·1 ·:: ,.·.,:.f~.. . I . ·.1. 9 -8;. . .. ~!· . :, ;.6 . ·5 · log[J.\Ch] (M)

c D

0 0

w 20 w. 20 ·e:. a. ·C -c .2. 0 ..., 40 :.;::; . 40 ~ ~ >< >< ~ ~ ·(i; 60 a; .60 ·~ ·0:: '';!. ....1, ?fe: 8() :>.·: .•.... :."; ·:· ;~::-!;;!.:: 80 . .. . .· :·o' ,Sham-coiitrol. ·•· · ~~~ .o Angii.:.Controf. ·. 100 · ill Sham-TrxR · · · ·100 · e AngH-TrXR · ··:

1- . ~. . . .r I .. . I " · -- :~I .. ., : : · I I I · ~9 . -8 ~t · · -6· . : ~s ·~·· ' ~~ ··~~ . : . ~~:' . ~~ .·5 -4· IQg[ACh] (M,:·· log[ACil] (M)

Figure 4.6. lncreas~d TrxR normalized relaxation to ACh and SNP in Angll aortic rings. Concentration-dependent relaxations to ACh (A-D) or SNP (E-H) were measured after inqubation of the aortic rings with empty chariot (control) or Chariot-TrxR complex (TrxR). B and F: Bar graphs are representative pD2 values from the .ACh or SNP-mediated relaxation curves, respectively. Relaxation responses were calculated relative to the maximal PE contraction, 1 which was taken as 1 00°/o. Results are presented as means ± SEM in each experimental group~ *p

! 72

E F

.0 * 20 t .a.w .··.~ c 0 -~ ca ·a::.Qi·· rft

Sham-Control Control · TrxR · Control TrxR 100 Angii-Control Sham Angll

~10 -9 I -8 -7 log[SNP] (M)

G H

.• Sham-Contr~l · .. ·~. f\hgii~Controt . t. • Sham-TrxR i · · • · AngJf.,TrxR:

,; ~10 .•g: -8 -7 ..g.. -8 ·log[SNPf (rv'!) log[SNP] (M)

Figure 4.6 (continued). 73

A . ·f B

0

20 . . 0,;.w . c c - o· :8 40 ca .·ca+i.. .. ~ . ··~~··· ·. ~ 60. ·:~: ·•?ft. * ... . ?f. . 80 1:1 Stiam-Vehide D • Shain.Vehidle · . , · · t ..:. . I v: Angii-Vehicl;e V · Angii~Vehicle 100 Y Angli-CysNO . T Angii-CysNO . . : .... ;

-9 -8 . ~7 -6· .. -5 -4 •10 •9 . . -8 . -7 lo~[ACh](M) lo.g[SNP](M)

Figure 4.7. CysNO decreased relaxation in Angll aortic rings. Concentration­ dependent relaxation to (A) ACh or (B) SNP was measured after incubation of the aortic rings with vehicle or CysNO (1 0 !Jmoi/L, 15 min). Relaxation responses were c~lculated relative to the maximal PE contraction, which was taken as 100°/o. R~sults are presented as means ± SEM in each experimental group. *p<0.05 compared with sham-vehicle. tp<0.05 compared with Angll­ vehicle (n=6). 74

DISCUSSION

The major finding of this study is that TrxR, through S-nitrosylation, plays

! a role in vascular relaxation during hypertension. Our results demonstrate that augmented S-nitrosylation is associated with impaired vasodilation in hypertensive mice. ~ TrxR has various cellular functions including S-nitrosylation, which is a redox-based post-translational modification, leading to the modification of several proteins and alteration in their activity. However, the functional importance of TrxR and S-nitrosylation has not been elucidated in hypertension.

Therefore, these findings provide a new concept, linking TrxR-mediated S- nitrosylation and impaired vascular reactivity in hypertension.

ACh stimulates relaxation of vascular smooth muscle by binding to the

I endothelial muscarinic receptor, which stimulates increased intracellular calcium to activate endothelial NO synthase (eNOS), ultimately leading to production of

NO in endothelial cells. NO then diffuses into the vascular smooth muscle and stimulates sGC to convert guanosine triphosphate (GTP) to cGMP, which acts as a second messenger, affecting ion channels and proteins, resulting in vascular relaxation [1 05]. Our data show that TrxR inhibition impaired endothelium- dependent relaxation in sham, but not in Angll aortic rings, suggesting that in

Angll, TrxR function is more important in vascular smooth muscle. Due to the hypertensive conditi.on, where endothelial dysfunction already exists [8-14], TrxR might already be inactivated in endothelial cells, and thus, DNCB did not change

ACh-induced relax~tion in hypertensive mice. In endothelium-independent relaxation, DNCB attenuated the concentration-response relaxation to SNP in 75 both sham and Anbll, suggesting that relaxation related to TrxR inhibition is associated with im~aired signaling of vascular smooth muscle proteins. As shown in Figure 4.3;·cGMP levels were reduced by DNCB in SNP-treated aortas, indicating that sGC activity was desensitized following inhibition of TrxR.

Additionally, we determined that the cGMP concentration was decreased in Angll aorta ·compared to sham, and these results demonstrate that endothelium- independent relaxation is impaired in Angll.

There are a large number of Trx and TrxR functions related to human disease, such as cancer, inflammation, viral infection, and cardiovascular diseases [116]. Further, the expression of the Trx system proteins has been found to be changed in many disease conditions [117]. In Angll-induced hypertension, the protein expression of TrxR in the aorta was not changed compared to normotensive aorta; however TrxR activity was less in Angll than sham. These data were consistent with · our protein S-nitrosylation results showing that in Angll, augmented S-nitrosylation is associated with inactivation of

TrxR.

In addition to' its denitrosylation function, TrxR protects against oxidative stress [96]. We investigated previously whether TrxR inhibition impairs vascular relaxation by increasing ROS. In normotensive mouse aorta, we determined that

TrxR contributes to antioxidant status and relaxation (unpublished data). Angll is known to produce; superoxide, and Angll-induced hypertension leads to increased ROS generation via activation of NADPH oxidase and/or degradation of endothelium-deri+ed NO [118-120]. Since in Angll-induced hypertension, 76

I TrxR function was 1 decreased and S-nitrosylation was increased, our results suggest a link betw~en increased oxidative stress and S-nitrosylation in Angll­ ! induced hypertensioh through TrxR.

Deficiencies and/or an excess of NO can lead to S-nitrosylation dysregulation of specific cellular targets and signaling pathways. In vascular reactivity, we show that increased S-nitrosylation impairs relaxation signaling in the vasculature in both sham and Angll, and further increasing S-nitrosylation in aorta from Angll mice could contribute to impaired relaxation. In addition to relaxation, augmented S-nitrosylation inactivates PKC and inhibits PKC-mediated vasoconstriction [62]. Accordingly, S-nitrosylation within the vasculature may be

I a critical regulatory step to investigate as well as a potential pharmacological target in disease conditions related to vascular dysfunction. The possibility for therapeutic alterations of S-nitrosylation levels is receiving increasing attention, and potential agents that affect S-nitrosylation are being explored [121]. Thus, research on the molecular mechanisms of dysregulated S-nitrosylation may be important for determ~ining potential therapeutic targets.

Research into S-nitrosylation and diseases has shown that the level of S- 1 nitrosylation is increased or decreased in various disease states. Also similarly,

S-nitrosylation of proteins is increased or decreased in various disease conditions. For example, in diabetes, the levels of S-nitrosylation are elevated and evidence has linked insulin resistance in type 2 diabetes with protein S- nitrosylation [11 0, 122]. Inducible NOS (iNOS)-mediated S-nitrosylation inactivates insulin SJgnaling proteins, such as protein kinase B (PKB, Akt) and I I 77

I I insulin receptor sub~trate 1 (IRS-1). On the other hand, protein S-nitrosylation is ,' ~,

I decreased in pulmdnary hypertension and heart failure [54, 109]. In the heart,

dysregulated S-nitrdsylation may explain some of the characteristic dysfunction

of the failing heart; because the cardiac ryanodine receptor/calcium channel

(l~yR2) and L-type calcium channel are known to be S-nitrosylated [123, 124].

Furthermore, as a .reservoir of NO, SNO promotes circulating low-molecular­

weight S-nitrosylatiqn mediated by serum albumin and lowers vasodilatory NO

levels in the vasculature [125]. Increased albumin S-nitrosylation can raise blood

pressure in preeclamptic patients via deficiency of ascorbate, releasing NO,

levels in plasma [126]. Additionally, increased SNO concentrations in plasma

predict cardiovascular risk in patients with end-stage renal disease [127].

In conclusion, to date the level of protein S-nitrosylation has been

incompletely examined in hypertension; however, the present study is the first to

demonstrate that protein S-nitrosylation is increased and related to impaired

relaxation in Angll-induced hypertensive mouse aorta. Specifically·, these effects

are accompanied by TrxR inactivation, which is important in mediating redox-

. based protein signaling. Thus, TrxR and S-nitrosylation may represent a key

mechanism and a new therapeutic approach in hypertension associated with

abnormal vascular reactivity. 78

CHAPTER 5. DISC:USSION !

I I In the preserh study, the main finding is that S-nitrosylation is associated i with abnormal vascular reactivity. Additionally, the specific findings are as follows: 1) S-nitrosylation inhibits PKC-mediated vascular contraction; 2) TrxR

inhibition attenuates vascular relaxation via increased ROS and sGC S-

nitrosylation; 3) augmented S-nitrosylation contributes to impaired vasodilation in aorta from Angll-induced hypertensive mice.

As demonstrated in Chapter 2, to consider whether S-nitrosylation contributes to the mechanisms of vascular contraction, we investigated the role of S-nitrosylation of :PKCa in mouse aorta [62]. PKC is inactivated by increased

S-nitrosylation. AlsO:, augmented S-nitrosylation reduces phosphorylation of CPI-

17 and caldesmon, ~which are PKC downstream signaling proteins, These data indicate that S-nitrosylation plays a critical role in regulating PKC activation, resulting in decreased PKC-induced contractile responses.

The PKC family is composed of structurally and functionally related serine/threonine kinases represented by at least 11 different isoforms. The PKC isoforms are classified as classic, novel, and atypical, depending upon the configuration of conserved and variable regions and the second messengers activating them. ·These enzymes regulate various intracellular signaling pathways including ;those for growth factors, hormones, neurotransmitters, and cytokines.

PKC is an illiportant enzyme in the vasculature. Activated PKC plays a I role in vascular s~ooth muscle cell growth [128], and several studies have I I I 79

I shown that PKC contributes to vascular smooth muscle contraction [129-132].

Both calcium-deperldent and -independent PKC isoforms induce vasoconstriction. I

Phorbol ester is a pbtent PKC activator and increases calcium-sensitivity, leading to the inhibition of MLC phosphatase in smooth muscle, causing contraction [80,

81]. Also, PKC inhibitors result in a significant decrease in agonist-induced vasoconstriction [132]. Additionally, in the neonatal pulmonary vasculature, PKC

inhibits SNP-stimulated cGMP production [133], suggesting that PKC could

contribute to reduced vasodilation.

Our data show that in contrast to concentration-dependent responses to

PE, those to PDBu were altered in the presence or absence of DNCB (Figure

5.1 ). Although inhibition of TrxR using DNCB leads to stabilization of S-

nitrosylation and inhibits PKC activity, DNCB did not change PE-induced

contractile responses, whereas this agent did inhibit phorbol ester-stimulated

contraction. These data suggest that PKC is sensitive to increased S-

nitrosylation, but since there are complicated mechanisms for PE-mediated

contraction, other signaling proteins can also apparently regulate

vasoconstriction. Thus, TrxR may not be related to PE-dependent contraction.

Further studies wm be necessary to clarify this mechanism, which signaling

proteins are modified by S-nitrosylation to affect vascular contraction. 80

A B

· tJ Sham-Vehicle · D Sham-Vehicle ·

1 • . Shanr:DNCB. . . ! · • Sham-DNCB

*

-9 ..a· -7 . •6 -5 -9 -~ -7 -6 'og[PE] (M) log[PDB.u] (M)

Figure 5.1. DNCB does not change PE-dependent contraction, but DNCB attenuates PDBu-in:duced contraction. Concentration-dependent contraction to I PE (A) or PDBu (B) :was measured after incubation of the aortic rings with vehicle or DNCB (4 l-JM, 40 min). Experimental values of contraction were calculated relative to the contractile response produced by 120 mM KCI, which was taken as 100%. Results are presented as means ± SEM in each experimental group. *, p<0.05 vs. vehicle. ,A; n=5, B; n=9 to 10. 81

Various PKC family members participate in diverse signal transduction

I

I processes, such as cellular proliferation, differentiation, and survival. Since PKC

1 isoforms have broad specificity and function, activation or disruption of different

PKC isoforms can differentially affect cellular systems. Abnormal PKC signaling has been implicated in the development of multiple human diseases, including metabolic and cardiovascular disorders, central nervous system dysfunction, neuronal degeneration, and cancer [134]. We investigated the function of a classic PKC member, specifically PKCa, and its relationship to S-nitrosylation, but our techniques can be applied to determine whether other PKC members are activated or inhibited by S-nitrosylation. Also, in disease condition, such as vascular disease, the contribution of PKC and S-nitrosylation could also be investigated.

To further demonstrate whether S-nitrosylation is related to abnormal vascular reactivity, we investigated vascular relaxation as shown in Chapters 3 and 4. One denitrosylation enzyme, TrxR, was investigated using specific inhibitors in mouse aorta. We observed that TrxR regulates vascular relaxation 1 via oxidative stress and S-nitrosylation. The Trx system consists of Trx, TrxR, and NADPH. TrxR is named for its ability to reduce oxidized Trx and is an enzyme belonging to the selenium-containing flavoprotein family. This enzyme family includes an FAD prosthetic group, a NADPH binding site, and redox-active sites, containing a conserved catalytic site and a C-terminal selenocysteine [96].

Mammalian TrxR is dependent upon a selenocysteine residue for reduction of 82 the active site disulfide in Trx. Beyond Trx reduction, TrxR also· has multiple

I properties and functions.

In the study Iof Chapter 3, we focused on two different functions of TrxR, which are antioxidant defense and dehitrosylation. However, there are more functions of TrxR, which could be studied further in the vasculature. First, the Trx system contributes. to DNA synthesis by reducing mammalian ribonucleotide reductase, which is: essential for DNA replication and repair [135-137].

Also, protein tyrosine phosphatase (PTP) is sensitive to inhibition by oxidation and this effect is related to H202 generation in response to growth factor signaling [138]. Thus, TrxR ac~ivity can reverse the inhibition of PTP activity via enzyme reduction. TrxR has been shown to support PTP .activity, thereby inhibiting signaling of src tyrosine kinase [139].

TrxR is also associated with transcription factor activities. This has been shown for several transcription factors, which are sensitive to redox disturbances.

These transcription, factors include NF-KB, AP-1 [140], Sp1 [141], and p53 [142].

However, it is unclear what events are physiologically required, or when the Trx system is necessarily involved in transcription factor regulation [137].

In addition to causing denitrosylation, interestingly, human Trx can itself be modified with S-hitrosylation. It contains two Cys residues (Cys32 and Cys35) as the active site and three structural Cys residues in positions 62, 69, and 73

[143]. Hashemy et al.. [143] showed that both Cys69 and Cys73 of reduced human Trx are nitrosylated and that the nitrosylated Trx is not a substrate for

TrxR. 83

Furthermore~ the main components of the eukaryotic cytoskeleton are well

I known to be redox: regulated. Oxidation of both non-muscle and muscle beta- and gamma-actin ihhibits actin polymerization, and the Trx system counteracts this inhibition [144].

In a different study, treatment of thrombocytes with DNCB, a TrxR inhibitor, induces a pronounced tendency for platelet aggregation [145], suggesting that

TrxR may be related to platelet coagulation. In addition, vascular endothelial growth factor (VEGF) is an important regulator of angiogenesis. Using inhibitors and selenium titratlons, it was shown that inhibition of TrxR activity increases

VEGF expression and angiogenesis in endothelial cells [146]. Taken together, examining a variety of TrxR functions in vascular function could be a suitable project for future studies.

As mentione'd above, the mammalian Trx system has a large number of functions, and thus, the components of this system could be related to disease.

These human diseases include cardiovascular and neurodegenerative diseases, diabetes, rheumatoid arthritis, cancer, and virus infection [116, 117]. The expression of Trx system proteins is changed under many disease conditions I and in aging [117]. Therefore, future studies could further elucidate the roles and

I mechanisms of the Trx system in cardiovascular diseases. Indeed, the Trx system may play a pivotal role in antioxidant protection in diseases.

Nitric oxide (NO) and superoxide are tightly regulated under physiological conditions. NO ~ignaling influences organ function via post-translational modifications of effJctor molecules, often through S-nitrosylation. S-Nitrosylation

I

I 84

I is a highly versat(le signaling mechanism that affects a number of biological events in a tighily regulated and reversible manner [57]. However, in pathophysiological\ conditions, oxidative stress disrupts NO signaling, and it is therefore appropriate to think of cellular redox balance in terms of the nitroso-

redox balance [14 7:]. In Chapters 3 and 4, we investigated an important enzyme for redox balance,i TrxR, as well as S-nitrosylation in a hypertensive animal model. Our data showed that the vascular content of S-nitrosylation is increased

in aorta from Angll-induced hypertensive mice as compared to normotensive

mice, suggesting that impaired relaxation is associated with increased S-

1 nitrosylation in Angll mouse aorta. Also, these data are speculated by

inactivation of TrxR in Angll. Thus, attenuated TrxR activity results in increased oxidative stress in the vasculature of Angll aorta (Figure 5.2). This result further demonstrates that TrxR plays an important role in increased ROS and S-

nitrosylation by Angll. However, further study is necessary to identify how Angll

affects TrxR activity. 85

1. * ·c::- 2 e 1. NO. 0 0') N E :I: ...... Q) . 0 0 . . E .._c::

Sham Angll

Figure 5.2. DNCB increases H202 levels in normotensive mouse (Sham) aorta. H202 levels are also increased in Angll control groups compared to sham control. H202 concentration was measured in the presence or absence of DNCB (4 1Jmoi/L, 1 h) in mouse aorta using Amplex red. Results are presented as mean± SEM for n=3 in control (Sham) and n=4 in other groups. *p<0.05 compared with control (Sham). 86

I In the cardioyascular system, S-nitrosylation regulates key processes in

I both the heart and ~he vasculature. In the heart, S-nitrosylation of ion channels maintains the calcium cycle within cardiac cells, which is responsible for normal systolic and diastoHc function [148]. Dysregulated S-nitrosylation may explain some of the characteristic dysfunction of the failing heart, because the cardiac ryanodine receptor/calcium channel (RyR2) and L-type calcium channel are known to be S-nitrosylated [123, 124]. A disruption in nitroso-redox signaling has the potential to contribute to congestive heart failure [14 7].

In some circumstances, such as acute ischemia, sepsis, or heart failure, the expression of NO synthase (NOS) is abnormally elevated. These situations result from the induction of inducible NOS (iNOS), leading to the adverse effect of nitrosative stress [148]. Santhanam et al. [149] showed that iNOS-dependent

S-nitrosylation causes endothelial dysfunction. Additionally, arginase 1 S- nitrosylation increases its activity and leads to endothelial NOS (eNOS) uncoupling, contributing to the nitroso-redox imbalance, endothelial dysfunction, and vascular stiffness observed in aging [149, 150]. Accordingly, a nitroso-redox imbalance can affect both cardiac performance and vascular tone.

In the blood, S-nitrosoalbumin (SNO-albumin) and S-nitrosohemoglobin

(SNO-Hb) constitute the major conduits for circulating NO bioactivity. As a reservoir of NO, S-r'!itrosothiol (SNO) promotes circulating low-molecular-weight

S-nitrosylation medi~ted by serum albumin and lowers vasodilatory NO levels in the vasculature, COJjltrolling vascular tone [125]. Increased SNO-albumin can raise blood pressure in preeclamptic patients via deficiency of ascorbate, which 87 can induce to rele~se NO, in plasma [126]. Additionally, an increased SNO concentration in piJsma predicts cardiovascular risk in patients with end-stage renal disease [127].

In diabetes, the levels of S-nitrosylation are elevated and evidence has linked insulin resistance in type 2 diabetes [11 0, 122]. iNOS-mediated S- nitrosylation inactivates insulin signaling proteins, such as protein kinase B (PKB,

Akt) and insulin receptor substrate 1 (IRS-1). On the other hand, protein S- nitrosylation is reduced in pulmonary hypertension [54, 109]. Several researchers have investigated abnormal S-nitrosylation levels in numerous pathophysiological states, suggesting that dysregulation of S-nitrosylation homeostasis may contribute to disease pathogenesis [121].

Ascorbate is a compound known to promote SNO degradation. In the review of May [151 ], ascorbate itself improves defective endothelial function in atherosclerosis, hypercholesterolemia, and preeclampsia. Thus, ascorbate might be a therapeutic supplement to combat abnormal SNO and disease processes

[152]. Further investigation is required to address the mechanistic basis and relevance of alterations in protein S-nitrosylation levels in disease and to identify therapeutic leads. 88

t NOx I ~ TrxR

t S-Nitrosylation / 'f' PKC-SNO 'f' sGC-SNO +PKC activity +sGC activity

Figure 5.3. Schematic of our conclusion under normal conditions. Augmented S-nitrosylation contributes to abnormal vascular reactivity. NO; nitric oxide, TrxR; thioredoxin reductas'e, PKC; protein kinase C, SNO; S-nitrosothiol, sGC; soluble guanylyl cyclase. 89

Angiotensin II

\ \ t 'TrxR I t S-Nitrosylation

! Hypertension

Figure 5.4. Schematic of our conclusion under hypertensive conditions. Angiotensin II and :. elevated arterial pressure increase protein S-nitrosylation, thereby contributing to impaired vascular relaxation. 90

i CHAPTERS. SU~MARY

Abnormal vascular reactivity, including augmented vasoconstriction and impaired vasodilation, is a main characteristic in hypertensive disease. However, the cellular and molecular mechanisms leading to vascular dysfunction is poorly understood. In this thesis study, the role of S-nitrosylation in the vasculature, under both normal and pathological conditions, is investigated to understand the regulation of vascular signaling. PKC is an important enzyme in vascular signaling and activated PKC causes vasoconstriction.· PKC S-nitrosylation leads to its inactivation and thus, PKC-mediated contractile responses are attenuated.

ROS play a major role in the development of vascular dysfunction, and we also showed that TrxR regulates vascular relaxation and signaling. Specifically, among TrxR functions, antioxidant defense and denitrosylation are important to mediate signaling in the vasculature. To date, the level of protein S-nitrosylation has not been determined in hypertension; however, the present study demonstrates that protein S-nitrosylation is increased and is related to impaired relaxation in Angll-induced hypertensive mouse aorta. Therefore, these research approaches to understand S-nitrosylation-associated mechanisms that control vascular dysfunction may provide novel insight for the development of therapeutics. 91

REFERENCES

1. Kannel WB: !Elevated systolic blood pressure as a cardiovascular risk factor. Am J Cardiel 2000;85(2):251-255.

2. Kearney PM, Whelton M, Reynolds K, Muntner P, Whelton PK, He J: Global burden of hypertension: analysis of worldwide data. Lancet 2005;365(9455):217 -223.

3. Carretero OA, Oparil S: Essential hypertension. Part 1: definition and etiology. Circulation 2000; 101 (3):329-335.

4. Touyz RM, Tolloczko 8, Schiffrin EL: Mesenteric vascular smooth muscle cells from spontaneously hypertensive rats display increased calcium responses to angiotensin II but not to endothelin-1. J Hypertens 1994; 12(6):663-673.

5. Feldman RD, Gras R: Impaired vasodilator function in hypertension: the role of alterations in receptor-G protein coupling. Trends Cardiovasc Med 1998;8(7):297-305.

6. Touyz RM: Molecular and cellular mechanisms regulating vascular function and structure--implications in the pathogenesis of hypertension. Can J Cardiol2000;16(9):1137-1146.

7. Satoh S, Kreutz R, Wilm C, Ganten D, Pfitzer G: Augmented agonist­ induced Ca(2+)-sensitization of coronary artery contraction in genetically hypertensive rats. Evidence for altered signal transduction in the coronary smooth muscle cells. J Clin Invest 1994;94(4):1397-1403.

8. Yang D, Gluais P, Zhang JN, Vanhoutte PM, Feletou M: Endothelium­ dependent contractions to acetylcholine, ATP and the calcium ionophore A 23187 in aortas from spontaneously hypertensive and normotensive rats. Fundam Clin Pharmacol2004;18(3):321-326.

9. Luscher TF, Aarhus LL, Vanhoutte PM: Indomethacin improves the impaired endothelium-dependent relaxations in small mesenteric arteries of the sponta~eously hypertensive rat. Am J Hypertens 1990;3(1 ):55-58.

10. Cordellini S: Endothelial dysfunction in DOCA-salt hypertension: possible involvement of prostaglandin endoperoxides. Gen Pharmacal 1999;32(3):315-320.

11. Jimenez R, Lopez-Sepulveda R, Kadmiri M, Romero M, Vera R, Sanchez M, Vargas F, O'Valle F, Zarzuelo A, Duenas M, Santos-Buelga C, Duarte J: Polyphenols restore endothelial function in DOCA-salt hypertension: role of endothelin-1 and NADPH oxidase. Free Radic Bioi Med I 2007;43(3):462-4 73. I 92

12. Zhou MS, Kosaka H, Tian RX, Abe Y, Chen QH, Yoneyama H, Yamamoto A, Zhang L: ,L-Arginine improves endothelial function in renal artery of hypertensive Dahl rats. J Hypertens 2001 ;19(3):421-429.

13. Heitzer T, Wenzel U, Hink U, Krollner D, Skatchkov M, Stahl RA, MacHarzina R, Brasen JH, Meinertz T, Munzel T: Increased NAD(P)H oxidase-mediated superoxide production in renovascular hypertension: evidence for an involvement of protein kinase C. Kidney lnt 1999;55(1 ):252-260.

14. Quaschning T, Hocher B, Ruhl S, Kraemer-Guth A, Tilgner J, Wanner C, Galle J: Vasopeptidase inhibition normalizes blood pressure a·nd restores endothelial function in renovascular hypertension. Kidney Blood Press Res 2006;29(6):351-359.

15. Somlyo AP, Somlyo AV: Signal transduction and regulation in smooth muscle. Nature 1994;372(6503):231-236.

16. Forstermann U, Closs El, Pollock JS, Nakane M, Schwarz P, Gath I, Kleinert H: Nitric oxide synthase isozymes. Characterization, purification, molecular cloning, and functions. Hypertension 1994;23(6 Pt 2): 1121-1131.

17. Chitaley K, Weber D, Webb RC: RheA/Rho-kinase, vascular changes, and hypertension. Cur~ Hypertens Rep 2001 ;3(2):139-144.

18. Fridovich 1: Superoxide anion radical (02-.), superoxide dismutases, and related matters. J Bioi Chern 1997;272(30):18515-18517.

19. Wolin MS: Interactions of oxidants with vascular signaling systems. Arterioscler Thromb Vase Biol2000;20(6):1430-1442.

20. Freeman BA, Crapo JD: Biology of disease: free radicals and tissue injury. Lab Invest 1982;47(5):412-426.

21. Griendling KK, Sorescu D, Lassegue B, Ushio-Fukai M: Modulation of protein kinase activity and gene expression by reactive oxygen species and their role in vascular physiology and pathophysiology. Arterioscler Thromb Vase Biol2000;20(10):2175-2183.

22. Grech ED, Dodd NJ, Jackson MJ, Morrison WL, Faragher EB, Ramsdale DR: Evidence for free radical generation after primary percutaneous transluminal coronary angioplasty recanalization in acute myocardial infarction. Am J Cardiol1996;77(2):122-127.

I I 23. White CR, Brock TA, Chang LY, Crapo J, Briscoe P, Ku D, Bradley WA, 1 Gianturco SH, Gore J, Freeman BA, .: Superoxide and peroxynitrite in atherosclero~is. Proc Natl Acad Sci U SA 1994;91 (3): 1044-1048. I ' 93

24. Touyz RMI Schiffrin EL: Reactive oxygen species in vascular biology: implications in hypertension. Histochem Cell Biol2004;122(4):339-352.

25. Cosentino F I Sill JCI Katusic ZS: Role of superoxide anions in the mediation of endothelium-dependent contractions. Hypertension 1994;23(2):229-235.

26. Rao GN I Berk BC: Active oxygen species stimulate vascular smooth muscle cell growth and proto-oncogene expression. Circ Res 1992;70(3):593-599.

27. Touyz RMI Schiffrin EL: Ang 11-stimulated superoxide production is mediated via phospholipase D iri human vascular smooth muscle cells. Hypertension 1999;34(4 Pt 2):976-982.

28. Harrison DG.: Cellular and molecular mechanisms of endothelial cell dysfunction. J Clin Invest 1997;100(9):2153-2157.

29. Berk BC: Redox signals that regulate the vascular response to injury. Thromb Haemost 1999;82(2):810-817.

30. Jones DP: Redefining oxidative stress. Antioxid Redox Signal 2006;8(9- 10): 1865-1879.

31. Aubin MCI Carrier M1 Shi YF 1 Tardif JCI Perrault LP: Role of probucol on endothelial dysfunction of epicardial coronary arteries associated with left ventricular hypertrophy. J Cardiovasc Pharmacol 2006;47(5):702-71 0.

32. Liu YH 1 You Y 1 Song Tl Wu SJI Liu LY: Impairment of endothelium­ dependent relaxation of rat aortas by homocysteine thiolactone and attenuation by captopril. J Cardiovasc Pharmacol 2007;50(2):155-161.

33. Abrahamsson Tl Brandt Ul Marklund SLI Sjoqvist PO: Vascular bound recombinant extracellular superoxide dismutase type C protects against the detrimental effects of superoxide radicals on endothelium-dependent arterial relaxation. Circ Res 1992;70(2):264-271.

34. Omar HAl Cherry POl Mortelliti MP I Burke-Wolin Tl Wolin MS: Inhibition of coronary artery superoxide dismutase attenuates endothelium-dependent and -independent nitrovasodilator relaxation. Circ Res 1991 ;69(3):601-608.

35. Szabo C: Mujltiple pathways of peroxynitrite cytotoxicity. Toxicol Lett 2003;140-14r :105-112.

36. Rodriguez-M~rtinez MAl Garcia-Cohen EC 1 Baena AB 1 Gonzalez R1

Salaices M 1 Marin J: Contractile responses elicited by hydrogen peroxide in aorta from !normotensive and hypertensive rats. Endothelial modulation and mechani~m involved. Br J Pharmacol1998;125(6):1329-1335.

I 94

37. Yang ZW, Zheng T, Wang J, Zhang A, Altura BT, Altura BM: Hydrogen peroxide induces contraction and raises [Ca2+ ]i in canine cerebral arterial smooth mus'cle: participation of cellular signaling pathways. Naunyn Schmiedebergs Arch Pharmacal 1999;360(6):646-653.

38. uch-Schwelk W, Katusic ZS, Vanhoutte PM: Contractions to oxygen­ derived free radicals are augmented in aorta of the spontaneously hypertensive rat. Hypertension 1989; 13(6 Pt 2):859-864.

39. Wink DA, Mitchell JB: Chemical biology of nitric oxide: Insights into regulatory, cytotoxic, and cytoprotective mechanisms of nitric oxide. Free Radic Bioi Med 1998;25(4-5):434-456.

40. Benhar M, Forrester MT, Stamler JS: Protein denitrosylation: enzymatic mechanisms and cellular functions. Nat Rev Mol Cell Bioi 2009; 10(1 0):721-732.

41. lschiropoulos H: Biological selectivity and functional aspects of protein tyrosine nitration. Biochem Biophys Res Commun 2003;305(3):776-783.

42. Martinez-Ruiz A, Lamas S: S-nitrosylation: a potential new paradigm in signal transduction. Cardiovasc Res 2004;62(1 ):43-52.

43. Stamler JS, Simon Dl, Osborne JA, Mullins ME, Jaraki 0, Michel T, Singel DJ, Loscalzo J: S-nitrosylation of proteins with nitric oxide: synthesis and characterization of biologically active compounds. Proc Natl Acad Sci U S A 1992;89(1 ):444-448.

44. Gaston BM, Carver J, Doctor A, Palmer LA: S-nitrosylation signaling in cell biology. Mol lnterv 2003;3(5):253-263.

45. Hao G, Derakhshan B, Shi L, Campagne F, Gross SS: SNOSID, a proteomic method for identification of cysteine S-nitrosylation sites in complex protein mixtures. Proc Natl Acad Sci US A 2006;103(4):1012- 1017.

46. Perez-Mate I, Castro C, Ruiz FA, Corrales FJ, Mato JM: Methionine adenosyltransferase S-nitrosylation is regulated by the basic and acidic amino acids surrounding the target thiol. J Bioi Chern 1999;274(24):17075-17079.

47. Sun J, Xin C,; Eu JP, Stamler JS, Meissner G: Cysteine-3635 is responsible for skeletal muscle ryanodine receptor modulation by NO. Proc Natl Ac~d Sci US A 2001 ;98(20):11158-11162.

48. Stamler JS, Tjoone EJ, Lipton SA, Sucher NJ: (S)NO signals: translocation, regulation, arnd a consensus motif. Neuron 1997;18(5):691-696. 95

49. Jaffrey SR, Erdjument-Bromage H, Ferris CD, Tempst P, Snyder SH: Protein S-nitrosylation: a physiological signal for neuronal nitric oxide. Nat Cell Bioi 200~ ;3(2):193-197.

50. Gow AJ, Chen Q, Hess DT, Day BJ, lschiropoulos H, Stamler JS: Basal and stimulated protein S-nitrosylation in multiple cell types and tissues. J Bioi Chern 2002;277(12):9637 -9640.

51. Holmgren A: Biochemistry. SNO removal. Science 2008;320(5879):1 019- 1020.

52. Benhar M, Forrester MT, Hess DT, Stamler JS: Regulated protein denitrosylation by cytosolic and mitochondrial thioredoxins. Science 2008;320(5879): 1050-1054.

53. Liu L, Van Y, Zeng M, Zhang J, Hanes MA, Ahearn G, McMahon TJ, Dickfeld T, Marshall HE, Que LG, Stamler JS: Essential roles of S­ nitrosothiols in vascular homeostasis and endotoxic shock. Cell 2004;116(4):617-628.

54. Whalen EJ, Foster MW, Matsumoto A, Ozawa K, Violin JD, Que LG, Nelson CD, Benhar M, Keys JR, Rockman HA, Koch WJ, Daaka Y, Lefkowitz RJ, Stamler JS: Regulation of beta-adrenergic receptor signaling by S-nitrosylation of G-protein-coupled receptor kinase 2. Cell 2007; 129(3):511-522.

55. Lima B, Lam GK, Xie L, Diesen DL, Villamizar N, Nienaber J, Messina E, Bowles D, Kontos CD, Hare JM, Stamler JS, Rockman HA: Endogenous . S-nitrosothiols protect against myocardial injury. Proc Natl Acad Sci U S A 2009; 106(15),:6297 -6302.

56. Liu L, Hausladen A, Zeng M, Que L, Heitman J, Stamler JS, Steverding D: Nitrosative stress: protection by glutathione-dependent formaldehyde dehydrogenase. Redox Rep 2001 ;6(4):209-210.

57. Stamler JS, Lamas S, Fang FC: Nitrosylation. the prototypic redox-based signaling mechanism. Cell 2001; 106(6):675-683.

58. Lander HM, Hajjar DP, Hempstead BL, Mirza UA, Chait BT, Campbell S, Quilliam LA: A molecular redox switch on p21 (ras). Structural basis for the nitric oxide-p21 (ras) interaction. J Bioi Chern 1997;272(7):4323-4326.

59. Erwin PA, Lin AJ, Golan DE, Michel T: Receptor-regulated dynamicS­ 1 nitrosylation of endothelial nitric-oxide synthase in vascular endothelial cells. J Bioi Chern, 2005;280(20): 19888-19894. 96 I

I 60. Kahlos K, Zhang J, Block ER, Patel JM: Thioredoxin restores nitric oxide­ induced inhi~ition of protein kinase C activity in lung endothelial cells. Mol Cell Biochem 2003;254(1-2):47-54.

61. Gopalakrishna R, Chen ZH, Gundimeda U: Nitric oxide and nitric oxide­ generating agents induce a reversible inactivation of protein kinase C activity and phorbol ester binding. J Bioi Chern 1993;268(36):27180-27185.

62. Choi H, Tastes RC, Webb RC: S-nitrosylation Inhibits Protein Kinase C­ mediated Contraction in Mouse Aorta. J Cardiovasc Pharmacol 2011 ;57(1 ):65-71.

63. Sayed N, Baskaran P, MaX, van den AF, Beuve A: Desensitization of soluble guanylyl cyclase, the NO receptor, by S-nitrosylation. Proc Natl Acad Sci US A 2007;104(30):12312-12317.

·64. Nogueira L, Figueiredo-Freitas C, Casimiro-Lopes G, Magdesian MH, Assreuy J, Sorenson MM: Myosin is reversibly inhibited by S-nitrosylation. Biochem J 2009;424(2):221-231.

65. Lander HM, Hajjar DP, Hempstead BL, Mirza UA, Chait BT, Campbell S, Quilliam LA: A molecular redox switch on p21 (ras). Structural basis for the nitric oxide-p21 (ras) interaction. J Bioi Chern 1997;272(7):4323-4326.

66. Nishimura J, Khalil RA, Drenth JP, van BC: Evidence for increas~d myofilament Ca2+ sensitivity in norepinephrine-activated vascular smooth muscle. Am J Physiol 1990;259(1 Pt 2):H2-H8.

67. Martinez MC, Randriamboavonjy V, Ohlmann P, Kamas N, Duarte J, Schneider F, Stoclet JC, Andriantsitohaina R: Involvement of protein kinase C, tyrosine kin«;ises, and Rho kinase in Ca(2+) handling of human small arteries .. Am J Physiol Heart Circ Physiol 2000;279(3):H1228-H1238.

68. Kitazawa T, Eto M, Woodsome TP, Brautigan DL: Agonists trigger G protein-mediated activation of the CPI-17 inhibitor phosphoprotein of myosin light chain phosphatase to enhance vascular smooth muscle contractility. J Bioi Chern 2000;275(14):9897-9900.

69. Sato K, Dohi Y, Suzuki S, Miyagawa K, Takase H, Kojima M, van BC: Role of Ca2+-.sensitive protein kinase C in phenylephrine enhancement of Ca2+ sensitivity in rat tail artery. J Cardiovasc Pharmacol2001 ;38(3):347- 355.

70. Wood some TP, Eto M, Everett A, Brautigan DL, Kitazawa T: Expression of CPI-17 and myosin phosphatase correlates with Ca(2+) sensitivity of protein kinas~ C-induced contraction in rabbit smooth muscle. J Physiol 2001 ;535(Pt ~):553-564. 97

71. Andrea JE, Walsh MP: Protein kinase C of smooth muscle. Hypertension 1992;20(5):q85-595.

72. Winder SJ, Walsh MP: Smooth muscle calponin. Inhibition of actomyosin MgATPase and regulation by phosphorylation. J Bioi Chern 1990;265(17):1 0148-10155.

73. Hartwig JH, Thelen M, Rosen A, Janmey PA, Nairn AC, Aderem A: MARCKS is an actin filament crosslinking protein regulated by protein kinase C and calcium-calmodulin. Nature 1992;356(6370):618-622.

7 4. Kanashiro CA, Khalil RA: Signal transduction by protein kinase C in mammalian cells. Clin Exp Pharmacal Physiol1998;25(12):974-985.

75. Jaffrey SR, Snyder SH: The biotin switch method for the detection of S~ nitrosylated proteins. Sci STKE 2001 ;2001 (86):11.

76. Lin D, Zhou J, Zelenka PS, Takemoto OJ: Protein kinase Cgamma regulation of gap junction activity through caveolin-1-containing lipid rafts. Invest Ophthalmol Vis Sci 2003;44(12):5259-5268.

77. Greco TM, Hodara R, Parastatidis I, Heijnen HF, Dennehy MK, Liebler DC, lschiropoulos H: Identification of S-nitrosylation motifs by site-specific mapping of the S-nitrosocysteine proteome in human vascular smooth muscle cells. Proc Natl Acad Sci US A 2006;103(19):7420-7425.

78. Denninger JW, Marietta MA: Guanylate cyclase and the .NO/cGMP signaling pathway. Biochim Biophys Acta 1999; 1411 (2-3):334-350.

79. Gow AJ, Farkouh CR, Munson DA, Posencheg MA, lschiropoulos H: Biological significance of nitric oxide-mediated protein modifications. Am J Physiol Lung Cell Mol Physioi2004;287(2):L262-L268.

80. Laher I, Vorkapic P, Dowd AL, Bevan JA: Protein kinase C potentiates stretch-induced cerebral artery tone by increasing intracellular sensitivity to Ca2+. Biochem Biophys Res Commun 1989;165(1):312-318.

81. Masuo M, Reardon S, lkebe M, Kitazawa T: A novel mechanism for the Ca(2+ )-sensit'izing effect of protein kinase C on vascular smooth muscle: inhibition of myosin light chain phosphatase. J Gen Physiol 1994; 104(2):~65-286.

82. Zhang Y, Hogg N: The mechanism of transmembrane S-nitrosothiol transport. Prof Natl Acad Sci US A 2004;101(21):7891-7896.

83. Nalefski EA, Newton AC: Membrane binding kinetics of protein kinase C betall mediated by the C2 domain. Biochemistry 2001 ;40(44):13216-

13229. ! 98

84. Oancea E, Meyer T: Protein kinase C as a molecular machine for decoding ca!ciu·m and diacylglycerol signals. Cell1998;95(3):307-318.

85. Hommel U, Zurini M, Luyten M: Solution structure of a cysteine rich domain of rat protein kinase C. Nat Struct Bioi 1994; 1(6):383-387.

86. Kazanietz MG, Wang S, Milne GW, Lewin NE, Liu HL, Blumberg PM: Residues in the second cysteine-rich region of protein kinase C delta relevant to phorbol ester binding as revealed by site-directed mutagenesis. J Bioi Chern 1995;270(37):21852-21859.

87. Ono Y, Fujii T, Igarashi K, Kuno T, Tanaka C, Kikkawa U, Nishizuka Y: Phorbol ester binding to protein kinase C requires a cysteine-rich zinc­ finger-like sequence. Proc Natl Acad Sci U SA 1989;86(13):4868-4871.

88. Mellor H, Parker PJ: The extended protein kinase C superfamily. Biochem J 1998;332 ( Pt 2):281-292.

89. Salamanca DA, Khalil RA: Protein kinase C isoforms as specific targets for modulation of vascular smooth muscle function in hypertension. Biochem Pharmacol2005;70(11):1537-1547.

90. Lieu YM, Morgan KG: Redistribution of protein kinase C isoforms in .association with vascular hypertrophy of rat aorta. Am J Physiol 1994;267(4 Pt 1):C980-C989.

91. Nishizuka Y: Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. Science 1992;258(5082):607-614.

92. Dessy C, Matsuda N, Hulvershorn J, Sougnez CL, Sellke FW, Morgan KG: Evidence for involvement of the PKC-alpha isoform in myogenic contractions of the coronary microcirculation. Am J Physiol Heart Circ Physiol 2000;279(3):H916-H923.

93. Khalil RA, Lajoie C, Morgan KG: In situ determination of [Ca2+]i threshold for translocation of the alpha-protein kinase C isoform. Am J Physiol 1994;266(6 Pt 1):C1544-C1551.

94. Wilcox CS: Oxidative stress and nitric oxide deficiency in the kidney: a critical link to hypertension? Am J Physiol Regul lntegr Camp Physiol 2005;289(4):R913-R935.

95. Zhang DX, Gwtterman DD: Mitochondrial reactive oxygen species­ mediated sigrlaling in endothelial cells. Am J Physiol Heart Circ Physiol 2007;292(5):~2023-H2031.

I 96. Mustacich D, Powis G: Thioredoxin reductase. Biochem J 2000;346 Pt 1:1-8. 99

97. Johansson¢, Lillig CH, Holmgren A: Human mitochondrial glutaredoxin reduces S-g!utathionylated proteins with high affinity accepting electrons from either glutathione or thioredoxin reductase. J Bioi Chern 2004;279(9)~7537 -7543.

I 98. Lillig CH, Lonn ME, Enoksson M, Fernandes AP, Holmgren A: Short interfering RNA-mediated silencing of glutaredoxin 2 increases the sensitivity of Hela cells toward doxorubicin and phenylarsine oxide. Proc Natl Acad Sci US A 2004;101(36):13227-13232.

99. Szasz T, Thompson JM, Watts SW: A comparison of reactive oxygen species metabolism in the rat aorta and vena cava: focus on xanthine oxidase. Am.J Physiol Heart Circ Physioi2008;295(3):H1341-H1350.

100. Watt PA, Thurston H: Endothelium-dependent relaxation in resistance vessels from the spontaneously hypertensive rats. J Hypertens 1989;7(8):661-666.

101. Williams SP, Shackelford DP, lams SG, Mustafa SJ: Endothelium­ dependent relaxation in estrogen-treated spontaneously hypertensive rats. Eur J Pharmacal 1988; 145(2):205-207.

102. Oyama Y, Kawasaki H, Hattori Y, Kanno M: Attenuation of endothelium­ dependent relaxation in aorta from diabetic rats. Eur J Pharmacal 1986; 132(1 ):75-78.

103. Winquist RJ, Bunting PB, Baskin EP, Wallace AA: Decreased endothelium-dependent relaxation in New Zealand genetic hypertensive rats. J Hypertens 1984;2(5):541-545.

104. Cai H, Harrison DG: Endothelial dysfunction in cardiovascular diseases: the role of oxidant stress. Circ Res 2000;87(1 0):840-844.

105. Carvajal JA, Germain AM, Huidobro-Toro JP, Weiner CP: Molecular mechanism of cGMP-mediated smooth muscle relaxation. J Cell Physiol 2000; 184(3):409-420.

106. Benndorf RA, Appel D, Maas R, Schwedhelm E, Wenzel UO, Boger RH: Telmisartan improves endothelial function in patients with essential hypertension. J Cardiovasc Pharmacol2007;50(4):367-371. ! 107. Panza JA, Quyyumi AA, Brush JE, Jr., Epstein SE: Abnormal endothelium-aependent vascular relaxation in patients with essential hypertension.' N Eng I J Med 1990;323(1 ):22-27.

108. Wid Iansky ME, Price DT, Gokce N, Eberhardt RT, Duffy SJ, Holbrook M, Maxwell C, Palmisano J, Keaney JF, Jr., Morrow JD, Vita JA: Short- and I 100

long-term COX-2 inhibition reverses endothelial dysfunction in patients with hypertension. Hypertension 2003;42(3):31 0-315.

109. McMahon TJ, Ahearn GS, Moya MP, Gow AJ, Huang YC, Luchsinger BP, Nudelman R, Van Y, Krichman AD, Bashore TM, Califf RM, Singel DJ, Piantadosi CA, Tapson VF, Stamler JS: A nitric oxide processing defect of red blood cells created by hypoxia: deficiency of S-nitrosohemoglobin in pulmonary hypertension. Proc Natl Acad Sci US A 2005;102(41):14801- 14806.

110. Carvalho-Filho MA, Ueno M, Hirabara SM, Seabra AB, Carvalheira JB, de Oliveira MG, Velloso LA, Curi R, Saad MJ: S-nitrosation of the insulin receptor, insulin receptor substrate 1, and protein kinase B/Akt: a novel mechanism of insulin resistance. Diabetes 2005;54(4):959-967.

111. Nozik-Grayck E, McMahon T J, Huang YC, Dieterle CS, Stamler JS, Piantadosi CA: Pulmonary vasoconstriction by serotonin is inhibited by S­ nitrosoglutathione. Am J Physiol Lung Cell Mol Physiol 2002;282(5):L 1057-L 1065.

112. Giachini FR, Chiao CW, Carneiro FS, Lima W, Carneiro ZN, Dorrance AM, Testes RC, Webb RC: Increased activation of stromal interaction molecule-1/0rai-1 in aorta from hypertensive rats: a novel insight into vascular dysfunction. Hypertension 2009;53(2):409-416.

113. Clarke CJ, Forman S, Pritchett J, Ohanian V, Ohanian J: Phospholipase C-delta1 modulates sustained contraction of rat mesenteric small arteries in response to noradrenaline, but not endothelin-1. Am J Physiol Heart Circ Physioi2008;295(2):H826-H834.

114. Aoshiba K, Yokohori N, Nagai A: Alveolar wall apoptosis causes lung destruction and emphysematous changes. Am J Respir Cell Mol Bioi 2003;28(5):555-562.

115. Keller M, Lidington D, Vogel L, Peter BF, Sohn HY, Pagano PJ, Pitson S, Spiegel S, Pohl U, Bolz SS: Sphingosine kinase functionally links elevated transmural pressure and increased reactive oxygen species formation in resistance arteries. FASEB J 2006;20(6):702-704.

116. Holmgren A, Lu J: Thioredoxin and thioredoxin reductase: current research with special reference to human disease. Biochem Biophys Res Commun 201 0;396(1 ): 120-124.

117. Lillig CH, Holrf1gren A: Thioredoxin and related molecules--from biology to health and disease. Antioxid Redox Signal2007;9(1):25-47. 101

118. Laursen JB,: Rajagopalan S, Galis Z, Tarpey M, Freeman BA, Harrison DG: Role of superoxide in angiotensin 11-induced but not catecholamine­ induced hypertension. Circulation 1997;95(3):588-593.

119. Landmesser U, Cai H, Dikalov S, McCann L, Hwang J, Jo H, Holland SM, Harrison DG: Role of p47(phox) in vascular oxidative stress and hypertension caused by angiotensin II. Hypertension 2002;40(4):511-515.

120. Wang HD, !xu S, Johns DG, Du Y, Quinn MT, Cayatte AJ, Cohen RA: Role of NADPH oxidase in the vascular hypertrophic and oxidative stress response to angiotensin II in mice. Circ Res 2001 ;88(9):947-953.

121. Foster MW, Hess DT, Stamler JS: Protein S-nitrosylation in health and disease: a current perspective. Trends Mol Med 2009; 15(9):391-404.

122. Kaneki M, Shimizu N, Yamada D, Chang K: Nitrosative stress and pathogenesis of insulin resistance. Antioxid Redox Signal 2007;9(3):319- 329.

123. Xu L, Eu JP, Meissner G, Stamler JS: Activation of the cardiac calcium release channel (ryanodine receptor) by poly-S-nitrosylation. Science 1998;279(5348):234-237.

124. Sun J, Picht E, Ginsburg KS, Bers DM, Steenbergen C, Murphy E: Hypercontractile female hearts exhibit increased S-nitrosylation of the L­ type Ca2+ channel alpha1 subunit and reduced ischemia/reperfusion injury. Circ Res 2006;98(3):403-411.

125. Rafikova 0, Rafikov R, Nudler E: Catalysis of S-nitrosothiols formation by serum albumin: the mechanism and implication in vascular control. Proc Natl Acad Sci U SA 2002;99(9):5913-5918.

126. Gandley RE, Tyurin VA, Huang W, Arroyo A, Daftary A, Harger G, Jiang J, Pitt B, Taylor RN, Hubel CA, Kagan VE: S-nitrosoalbumin-mediated relaxation is enhanced by ascorbate and copper: effects in pregnancy and preeclampsia plasma. Hypertension 2005;45(1 ):21-27.

127. Massy ZA, Fumeron C, Borderie D, Tuppin P, Nguyen-Khoa T, Benoit MO, Jacquot C, Buisson C, Drueke TB, Ekindjian OG, Lacour B, lliou MC: lncreasec;J pasma S-nitrosothiol concentrations predict cardiovascular outcomes among patients with end-stage renal disease: a prospective study. J 1;\m Soc Nephrol 2004; 15(2):470-476.

128. Assender JW, lrenius E, Fredholm BB: Endothelin-1 causes a prolonged protein kinase C activation and acts as a co-mitogen in vascular smooth muscle cells. Acta Physiol Scand 1996;157(4):451-460. 102

129. Horowitz A, Men ice CB, Laporte R, Morgan KG: Mechanisms of smooth muscle contraction. Physiol Rev 1996;76(4):967-1 003.

130. Nishizuka Y: Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. Science 1992;258(5082):607-614.

131. Kanashiro CA, Khalil RA: Signal transduction by protein kinase C in mammalian cells. Clin Exp Pharmacal Physic! 1998;25(12):974-985.

132. Dallas A, Khalil RA: Ca2+ antagonist-insensitive coronary smooth muscle contraction involves activation of epsilon-protein kinase C-dependent pathway. Am J Physiol Cell Physioi2003;285(6):C1454-C1463.

133. Johnson JA, Barman SA: Protein kinase C modulation of cyclic GMP in rat neonatal pulmonary vascular smooth muscle. Lung 2004; 182(2):79-89.

134. Fields AP, Gustafson WC: Protein kinase C in disease: cancer. Methods Mol Biol2003;233:519-537.

135. Holmgren A: The function of thioredoxin and glutathione in deoxyribonucleic acid synthesis. Biochem Soc Trans 1977;5(3):611-612.

136. Moore EC: Components and control of ribonucleotide reductase system of "­ the rat. Adv Enzyme_Regul1976;15:101-114.

137. Arner ES: Focus on mammalian thioredoxin reductases--important selenoproteins with versatile functions. Biochim Biophys Acta 2009; 1790(6):495-526. .· .·J

138. Finkel T: Redox-dependent signal transduction. FEBS Lett 2000;476(1- 2):52-54.

139. Shinozaki Y, Koizumi S, Ohno Y, Nagao T, Inoue K: Extracellular ATP counteracts the ERK.1/2-mediatE?d de~th-promoting signaling cascades in astrocytes. Glia 2006;54(6);606~618.

. . 140. Schenk H, Klein M, Erdbrugg~iW, Droge W, Schulze-Osthoff K: Distinct effects of thioredox'il1 and antioX:idants ori the activation of transcription factors NF-kappa B aqd AP-1 •. Proc Natl Acad Sci US A 1994;91(5):1672- 1676. t ; ' . . . . . I' . . 141. Bloomfield KL, Osborn? SA, l

p53 and for apoptosis induced by end genous electrophiles. Carcinogenesis 2006;27(12):2538-25 9.

143. Hashemy Sl, Holmgren A: Regulation of the catalytic activity and structure of human thioredoxin 1 via oxidation and S-nitrosylation of cysteine residues. J Bioi Chern 2008;283(32):21890-21898.

144. Lassing I, Schmitzberger F, Bjornstedt M, Holmgren A, Nordlund P, Schutt CE, Lindberg U: Molecular and structur~l basis for re9ox regulation of beta-actin. J Mol Bioi 2007;370(2):331-348.

145. Hill TO, White JG, Rao GH: Platelet hypersensitivity induced by 1-chloro-2, 4-dinitrobenzene, hydroperoxides and inhibition of lipoxygenase. Thromb Res 1989;53(5):447-455.

146. Streicher KL, Sylte MJ, Johnson SE, Sordillo LM: Thioredoxin reductase regulates angiogenesis by increasing endothelial cell-derived vascular endothelial growth factor. Nutr Cancer 2004;50(2):221-231.

147. Hare JM: Nitroso-redox balance in the cardiovascular system. N Engl J

., Med 2004;351 (20):2112-2114. ;/' ' 148. Hare JM: Nitric oxide and excitation-contraction coupling. J Mol Cell Cardiel 2003;35(7):719-729.

149. Santhanam L, Lim HK, Lim HK, Miriel V, Brown T, Patel M, Balanson S, Ryoo S, Anderson M, Irani K, Khanday F, Di CL, Nyhan 0, Hare JM, _./ / Christianson OW, Rivers R, Shoukas A, Berkowitz DE: Inducible NO synthase dependent S-nitrosylation and activation of arginase1 contribute to age-related endothelial dysfunction. Circ Res 2007;101(7):692-702.

150. Kim JH, Bugaj LJ, Oh YJ, Bivalacqua TJ, Ryoo S, Soucy KG, Santhanam L, VJebb A, Camara A, Sikka G, Nyhan D, Shoukas AA, !lies M, Christianson OW, Champion HC, Berkowitz DE: Arginase inhibition restores NOS coupling and reverses endothelial dysfunction and vascular .stiffness in old rats. J Appl Physiol2009;107(4):1249-1257. ·

151. May JM: How does ascorbic acid prevent endothelial dysfunction? Free Radic Bioi Med 2000;28(9):1421-1429.

152. Foster MW, Pawloski JR, Si~igel OJ, Stamler JS: Role of circulating S­ nitrosothiols in control9f bl.ooq pressure. Hypertension 2005;45(1):15-17 .

./

-·-::-