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Mechanisms of asynchronous Ca²⁺ oscillations and their role in (mal)function of vascular smooth muscle Syyong, Harley Thomas Tan 2010

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MECHANISMS OF ASYNCHRONOUS Ca2+ OSCILLATIONS AND THEIR ROLE IN (MAL)FUNCTION OF VASCULAR SMOOTH MUSCLE by HARLEY THOMAS TAN SYYONG B.Sc., University of British Columbia, 2005  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  in  THE FACULTY OF GRADUATE STUDIES (Pharmacology and Therapeutics)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  April 2010  © Harley Thomas Tan Syyong, 2010  ABSTRACT Contraction of vascular smooth muscle is regulated by fluctuations in the cytosolic concentration of Ca2+. The spatio-temporal regulation of Ca2+ relies on the subcellular architecture of the smooth muscle cell and the juxtaposition of the opposing plasmalemma, sarcoplasmic reticulum, and mitochondria. This thesis addresses two related aspects of Ca2+ signaling in vascular smooth muscle: 1) Reversal of the plasma membrane Na+/Ca2+ exchanger (NCX) during agonist-mediated stimulation in cultured rat aorta smooth muscle cells, and 2) the primary function of agonist-stimulated asynchronous Ca2+ waves and the signaling pathway(s) underlying them in the intact tissue. Evidence for functional coupling of reverse-mode NCX with canonical transient receptor potential channels (TRPC), specifically TRPC6, was provided in rat aortic smooth muscle cells by demonstrating that NCX reversal was increased following stimulation with ATP and 1Oleoyl-2-acetyl-sn-glycerol, a diacylglycerol analog. However, this was attenuated by blockade of non-selective cation channels with SKF-96365 and by activation of protein kinase C. These data are consistent with the known properties of TRPC6 and further support that functional coupling of TRPC6 and NCX occurs via a receptor-operated cascade. A combination of wire myography and confocal microscopy determined that uridine 5’triphosphate (UTP)-induced tonic contractions in rat basilar artery were associated with sustained repetitive oscillations in cytosolic Ca2+ which propagated along the length of the smooth muscle cells as Ca2+ waves. Pharmacological characterization of the mechanism of Ca2+ waves revealed that they are a result of repetitive cycles of sarcoplasmic reticulum (SR) Ca2+ release via inositol 1,4,5-trisphosphate-sensitive channels followed by the refilling of the SR.  ii  Plasmalemmal Ca2+ entry via the reverse-mode NCX coupled with the receptor-operated and Ltype Ca2+ channels is involved in replenishing the SR and supporting the ongoing Ca2+ waves. Finally, phenylephrine-stimulated vascular smooth muscle contraction in mesenteric arteries of a mouse model of Marfan syndrome was significantly inhibited and associated with reduced frequency of Ca2+ waves. In addition, endothelium-dependent and endothelium-independent vasodilation was impaired, and vessel stiffness was increased. Together, these vasomotor abnormalities in the resistance vessel may have a negative and detrimental impact on the overall cardiovascular function in Marfan syndrome.  iii  TABLE OF CONTENTS ABSTRACT................................................................................................................................... ii TABLE OF CONTENTS ............................................................................................................ iv LIST OF TABLES ...................................................................................................................... vii LIST OF FIGURES ................................................................................................................... viii LIST OF ABBREVIATIONS ..................................................................................................... xi ACKNOWLEDGEMENTS ...................................................................................................... xiii CO-AUTHORSHIP STATEMENT ......................................................................................... xiv CHAPTER 1: INTRODUCTION.................................................................................................1 1.1 Organization and function of the vasculature ......................................................................1 1.1.1 Endothelium................................................................................................................2 1.1.2 Overview of smooth muscle .......................................................................................3 1.1.3 Mechanism of smooth muscle contraction .................................................................4 1.2 Mechanisms of Ca2+ homeostasis and signaling in vascular smooth muscle ......................5 1.2.1 Voltage-gated Ca2+ channels.......................................................................................6 1.2.2 Receptor-operated and store-operated cation channels...............................................7 1.2.3 Ryanodine and IP3 receptors.....................................................................................11 1.2.3.1 Ryanodine receptors ........................................................................................12 1.2.3.2 IP3 receptors .....................................................................................................13 1.2.4 Ca2+ uptake and extrusion mechanisms........................................................................14 1.2.5 Recombinant aequorin as a method of measuring Ca2+ ............................................17 1.3 Nanodomains and Ca2+ signaling in vascular smooth muscle ...........................................20 1.3.1 Structural and functional considerations of the PM-SR junctional nanodomain......22 1.3.2 Ca2+ oscillations in smooth muscle...........................................................................25 1.3.3 Mechanism underlying Ca2+ oscillations ..................................................................26 1.3.4 Vasomotion ...............................................................................................................28 1.4 Marfan syndrome ...............................................................................................................30 1.4.1 Clinical manifestations and diagnostic criteria .........................................................31 1.4.2 Fibrillin, microfibrils and elastic fibers ....................................................................33 1.4.3 Molecular genetics and pathophysiology..................................................................35 1.4.4 Marfan syndrome and vasomotor function ...............................................................36 1.4.5 Mouse models of marfan syndrome..........................................................................38 1.5 Summary of proposed research objectives ........................................................................39 1.6 References..........................................................................................................................41  iv  CHAPTER 2: ATP PROMOTES NCX-REVERSAL IN AORTIC SMOOTH MUSCLE CELLS BY DAG-ACTIVATED NA+ ENTRY .........................................................................62 2.1 Introduction........................................................................................................................62 2.2 Methods..............................................................................................................................64 2.2.1 Smooth muscle cell culture .....................................................................................64 2.2.2 Expression of aequorin ...........................................................................................64 2.2.3 Measurement of mitochondrial [Ca2+] ....................................................................64 2.2.4 Experimental protocol.............................................................................................65 2.2.5 Chemicals................................................................................................................66 2.2.6 Statistical analysis...................................................................................................66 2.3 Results................................................................................................................................66 2.3.1 Mitochondrial Ca2+ uptake following 0Na+–PSS stimulation is due to NCX reversal ..........................................................................................................................................66 2.3.2 NCX reversal is increased upon purinergic receptor stimulation but inhibited by antagonists of NSCCs ............................................................................................68 2.3.3 Protein kinase C has an inhibitory effect on NCX reversal ....................................69 2.4 Discussion ..........................................................................................................................70 2.5 References..........................................................................................................................73 CHAPTER 3: MECHANISM OF ASYNCHRONOUS CA2+ WAVES UNDERLYING AGONIST-INDUCED CONTRACTION IN THE RAT BASILAR ARTERY.....................76 3.1 Introduction........................................................................................................................76 3.2 Methods..............................................................................................................................78 3.2.1 Tissue preparation.....................................................................................................78 3.2.2 Measurement of intracellular Ca2+ ............................................................................78 3.2.3 Measurement of isometric force ...............................................................................79 3.2.4 Statistics ....................................................................................................................80 3.2.5 Drugs, solutions, and chemicals................................................................................80 3.3 Results................................................................................................................................81 3.3.1 Relation between UTP-induced tonic contraction and UTP-induced Ca2+-waves ...81 3.3.2 Dependence of UTP-induced Ca2+-waves on extracellular Ca2+ influx ...................84 3.3.3 Dependence of UTP-induced Ca2+-waves on SR Ca2+ release .................................89 3.4 Discussion ..........................................................................................................................96 3.5 References........................................................................................................................106  CHAPTER 4: DYSFUNCTION OF ENDOTHELIUM AND SMOOTH MUSCLE CELLS IN SMALL ARTERIES OF A MOUSE MODEL OF MARFAN SYNDROME.................113 4.1 Introduction......................................................................................................................113 4.2 Methods............................................................................................................................114 4.2.1 Experimental animals and tissue preparation .........................................................114 4.2.2 Mechanical properties.............................................................................................115  v  4.2.3 Measurement of isometric force .............................................................................116 4.2.4 Measurement of intracellular Ca2+ ..........................................................................117 4.2.5 Detection of H2O2 production from endothelial cells .............................................118 4.2.6 Statistics ..................................................................................................................119 4.2.7 Drugs, solutions, and chemicals..............................................................................119 4.3 Results..............................................................................................................................119 4.3.1 Vessel stiffening and weakness in marfan mesenteric artery .................................119 4.3.2 Reduced contractile function of smooth muscle cells in marfan mesenteric artery ..... ..........................................................................................................................................123 4.3.3 Frequency of PE-induced Ca2+ waves is reduced in marfan syndrome..................126 4.3.4 Reduced endothelium-dependent and independent relaxation in marfan mesenteric artery ......................................................................................................................127 4.3.5 Nature of EDHF......................................................................................................130 4.3.6 Role of superoxide ..................................................................................................132 4.3.7 H2O2 production of endothelial cells ......................................................................133 4.4 Discussion ........................................................................................................................134 4.5 References........................................................................................................................144  CHAPTER 5: CONCLUSION AND FUTURE DIRECTIONS ............................................151 5.1 Overall summary and conclusions ...................................................................................151 5.1.1 Overview of NCX reversal in smooth muscle cells................................................152 5.1.2 Overview of UTP-induced Ca2+ oscillations in rat basilar artery ...........................152 5.1.3 Overview of dysfunction of endothelium and smooth muscle cells in small arteries of a mouse model of marfan syndrome .................................................................155 5.2 Significance of work and future directions......................................................................156 5.3 Concluding remarks .........................................................................................................161 5.4 References........................................................................................................................163 APPENDICES ............................................................................................................................166 Appendix A............................................................................................................................166 Appendix B ............................................................................................................................167 Appendix C ............................................................................................................................168 Appendix D............................................................................................................................169 Appendix E ............................................................................................................................170 Appendix F.............................................................................................................................172  vi  LIST OF TABLES Table 1 Effects of different treatments on potency and maximal acetylcholine-induced relaxation in control and marfan mice ............................................................................................132  vii  LIST OF FIGURES Figure 1.1  Ca2+, calmodulin, MLCK, and cross-bridge cycle in smooth muscle .....................5  Figure 1.2  Ion channels and receptors involved in regulating intracellular Ca2+ concentration in smooth muscle cells .............................................................................................6  Figure 1.3  Recombination of aequorin....................................................................................19  Figure 1.4  Electron microscope cross-sections of smooth muscle cells from rabbit inferior vena cava................................................................................................................24  Figure 1.5  Structure of fibrillin-1 protein................................................................................34  Figure 1.6  Mouse model of marfan syndrome ........................................................................39  Figure 2.1  Recovery kinetics of 0Na+-PSS stimulation in mitochondria-targeted aequorin...67  Figure 2.2  SKF-96365 (SKF) attenuates ATP- and 1-Oleoyl-2-acetyl-sn-glycerol (OAG)induced increase in NCX reversal..........................................................................69  Figure 2.3  Role of protein kinase C (PKC) in ATP-stimulated NCX reversal .......................70  Figure 3.1  Properties of uridine 5’-triphosphate (UTP)-induced Ca2+ waves underlying tonic contraction in rat basilar artery ..............................................................................82  Figure 3.2  Uridine 5’-triphosphate (UTP, 100 μM)-induced Ca2+ waves in rat basilar artery ... ................................................................................................................................83  Figure 3.3  Extracellular Ca2+ influx is required for maintenance of uridine 5’-triphosphate (UTP, 100 μM)-induced Ca2+ waves .....................................................................84  Figure 3.4  Effect of the L-type Ca2+ channel antagonist nifedipine and the receptor/storeoperated channel antagonist SKF-96365 on uridine 5’-triphosphate (UTP, 100 μM)-induced Ca2+ waves and tonic contraction ....................................................86  viii  Figure 3.5  Effect of the reverse-mode Na+/Ca2+ exchanger inhibitor KB-R7943 on uridine 5’-triphosphate (UTP, 100 μM)-induced Ca2+ waves and tonic contraction .........88  Figure 3.6  Effects of KB-R7943 on L-type Ca2+ channels and store/receptor-operated channels in rat basilar artery ..................................................................................89  Figure 3.7  Effect of blockade of the sarco(endo)plasmic reticulum Ca2+ ATPase by cyclopiazonic acid (CPA) on uridine 5’-triphosphate (UTP, 100 μM)-induced Ca2+ waves .............................................................................................................90  Figure 3.8  Effect of 2-aminoethoxydiphenylborate (2-APB) on uridine 5’-triphosphate (UTP, 100 μM)-induced Ca2+ waves and tonic contraction .............................................91  Figure 3.9  Effects of 2-aminoethoxydiphenylborate (2-APB) on the ryanodine-sensitive SR Ca2+ release channels, sarco(endo)plasmic reticulum Ca2+ ATPase, L-type Ca2+ channels, and store-operated channels in rat basilar artery....................................92  Figure 3.10  Effect of ryanodine, tetracaine, and caffeine-induced depletion of SR Ca2+ stores on uridine 5’-triphosphate (UTP, 100 μM)-induced Ca2+ waves and tonic contraction in rat basilar artery ..............................................................................94  Figure 4.1  Vessel elasticity during aging in mesenteric arteries...........................................121  Figure 4.2  Reversibility of mesenteric artery elasticity ........................................................122  Figure 4.3  Reversibility of contractile function of mesenteric arteries from control and marfan mice .........................................................................................................123  Figure 4.4  Isometric force measurement in response to KCl and PE in control and marfan mice......................................................................................................................125  Figure 4.5  Active force in control and marfan mice .............................................................125  ix  Figure 4.6  Endothelium-dependent relaxation in mesenteric arteries from control and marfan mice......................................................................................................................128  Figure 4.7  Endothelium-independent relaxation in mesenteric arteries from control and marfan mice .........................................................................................................129  Figure 4.8  Effects of Nω-Nitro-L-arginine methyl ester, indomethacin, and catalase on acetylcholine-induced relaxation in control and marfan mice.............................131  Figure 4.9  Effect of superoxide dismutase on phenylephrine (3 μM)-stimulated contraction and acetylcholine-mediated relaxation in mesenteric arteries from control and marfan mice .........................................................................................................133  Figure 4.10  Production of endothelial hydrogen peroxide......................................................134  Figure 5.1  Model for UTP-induced Ca2+ waves in smooth muscle cells of rat basilar artery ..............................................................................................................................154  Figure 5.2  Model of Ca2+ wave propagation from xenopus oocytes.....................................155  Figure 5.3  Ca2+ waves provide a more efficient stimulus for tonic contraction compared to steady elevation in average Ca2+ ..........................................................................158  x  LIST OF ABBREVIATIONS 2-APB [Ca2+]i [Ca2+]MT [Ca2+]o [Ca2+]subPM [Na+]o [Na+]subPM ACh ATP BIM BKCa cADPR cGMP cbEGF COX CPA DCF DMSO Em Emax ENCX EC50 EDHF EDTA EGTA ER FBN GFP HA1 HEPES H2O2 ICRAC INDO IP3 IP3R KB-R7943 KCl LAP L-NAME LTBP MLCK MLCP Na+/K+ ATPase NCX  2-aminoethoxydiphenylborate intracellular [Ca2+] mitochondrial matrix [Ca2+] extracellular [Ca2+] subplasmalemmal [Ca2+] extracellular [Na+] subplasmalemmal [Na+] acetylcholine adenosine triphosphate bisindolylmaleimide Ca2+-sensitive K+ channels cyclic ADP ribose cyclic guanosine monophosphate calcium-binding epidermal growth factor-like cyclooxygenase cyclopiazonic acid dichlorodihydrofluorescein diacetate dimethyl sulfoxide membrane potential maximal response NCX reversal potential half-maximum response endothelium-derived hyperpolarizing factor ethylenediaminetetraacetic acid ethylene glycol tetraacetic acid endoplasmic reticulum fibrillin green fluorescent protein hemagglutinin 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid hydrogen peroxide Ca2+-release activated Ca2+ current indomethacin inositol 1,4,5-trisphosphate inositol-1,4,5-triphosphate receptor 2-(2-(4-(4-Nitrobenzyloxy)phenyl)ethyl)isothiourea potassium chloride latency-associated protein Nω-Nitro-L-arginine methyl ester latent TGF-β binding protein myosin light chain kinase myosin light chain phosphatase Na+/K+ pump Na+/Ca2+ exchanger  xi  NMDA NO NSCC OAG pD2 PE PGI2 PI PIP2 PKC PKG PLC PM PMA PMCA PSS RyR ROC SEM SERCA SOC SOCE SOD SKF-96365 SNP SR STIM1 TGFβ TRP TRPC TRPM TRPV TRPP TRPML TRPN UTP VSMC  N-methyl-D-aspartic acid nitric oxide non-selective cation channel 1-Oleoyl-2-acetyl-sn-glycerol negative logarithm phenylephrine prostacyclin phosphoinositol phosphatidyl 4,5-bisphosphate protein kinase C cGMP-dependent protein kinase phospholipase C plasma membrane phorbol ester 12-tetradecanoylphorbol-13 acetate plasma membrane Ca2+ ATPase physiological saline solution ryanodine receptor receptor operated channel mean ± standard error sarco/endoplasmic reticulum Ca2+ ATPase store-operated channel store-operated Ca2+ entry superoxide dismutase 1-[β-(3-(4-Methoxyphenyl)propoxy)-4-methoxyphenethyl]-1H-imidazole hydrochloride, 1-[2-(4-Methoxyphenyl)-2-[3-(4methoxyphenyl)propoxy]ethyl]imidazole sodium nitroprusside sarcoplasmic reticulum stromal-interacting molecule 1 transforming growth factor beta transient receptor potential transient receptor potential canonical transient receptor potential melastatin transient receptor potential vanilloid transient receptor potential polycystic transient receptor potential mucolipin no mechanoreceptor potential C uridine 5’-triphosphate vascular smooth muscle cell  xii  ACKNOWLEDGEMENTS I would like to express my sincere gratitude to my co-supervisors Dr. Cornelis van Breemen and Dr. Kuo-Hsing Kuo, for their excellent scientific guidance, never-ending support, and personal help during my PhD studies. Their scientific knowledge and belief in my abilities have made this thesis possible. I am grateful to my supervisory committee members, Dr. David Fedida and Dr. Ed Moore, for all their insights and suggestions on my projects. I also owe a special thanks to Dr. Ada Chung for sharing her knowledge and experience with me, as well as providing technical and moral support. To all of my labmates at the Child and Family Research Institute, I thank you for all your support and for making my time in the laboratory an enjoyable one. Finally, I would also thank Dr. Chun Seow for giving me with an opportunity work in his laboratory for my directed studies project. I would like to recognize and thank my parents and my sister for all their love, encouragement, and continual support throughout the years. And to my fiancé Christine Cheung, I thank you for always being so patient and supportive of my work. Finally, I would like to acknowledge the Natural Sciences and Engineering Research Council of Canada and the Michael Smith Foundation for Health Research for their financial support during my training.  xiii  CO-AUTHORSHIP STATEMENT  CHAPTER 2: ATP PROMOTES NCX-REVERSAL IN AORTIC SMOOTH MUSCLE CELLS BY DAG-ACTIVATED NA+ ENTRY Damon Poburko, Harley Syyong, and Cornelis van Breemen developed the experimental design and protocols. Harley Syyong and Nicola Fameli performed all the experiments, and Harley Syyong analyzed the data. Harley Syyong and Damon Poburko contributed to the writing of the manuscript, which was revised by Cornelis van Breemen.  CHAPTER 3: MECHANISM OF ASYNCHRONOUS CA2+ WAVES UNDERLYING AGONIST-INDUCED CONTRACTION IN THE RAT BASILAR ARTERY Harley Syyong and Cornelis van Breemen developed the experimental design and protocols. Harley Syyong, Huei-Hsin Clarice Yang, Gina Trinh, and Christine Cheung performed all the animal experiments, and data was analyzed by Harley Syyong. KuoHsing Kuo provided an electron micrograph of the basilar artery smooth muscle cell. Harley Syyong wrote the manuscript which was revised by Cornelis van Breemen.  CHAPTER 4: DYSFUNCTION OF ENDOTHELIUM AND SMOOTH MUSCLE CELLS IN SMALL ARTERIES OF A MOUSE MODEL OF MARFAN SYNDROME Harley Syyong, Ada Chung, and Cornelis van Breemen developed the experimental design and protocols. Harley Syyong and Huei-Hsin Clarice Yang performed all the animal experiments. The data was analyzed by Harley Syyong, Ada Chung, and Cornelis van Breemen. Harley Syyong wrote the manuscript which was revised by Ada Chung and Cornelis van Breemen.  xiv  CHAPTER 1 – INTRODUCTION 1.1 Organization and function of the vasculature The innermost layer (tunica intima) of the vasculature consists of endothelial cells that provide a continuous lining against the blood. The endothelial cells control the leakage of fluid and proteins from the blood, prevent coagulation, and regulate smooth muscle contraction and differentiation through paracrine signaling (Ross, 1993; Toborek and Kaiser, 1999). The endothelium connects to a thin basal membrane, and in larger arteries there is also a subendothelial layer containing an extracellular mesh and some smooth muscle cells. The next layer is the tunica media, which contains smooth muscle cells enclosed in a basement membrane and suspended in a fibrous extracellular matrix. Elastic laminae are also located between smooth muscle cell layers in larger arteries. The matrix and laminae serve to withstand transmural pressure and to transmit tension to and from the smooth muscle cells. The matrix binds several enzymes and hormones and also influences smooth muscle cell properties by allowing adhesion (Carey, 1991; Ross, 1993). The thickness of the medial layer, the occurrence of elastic laminae, and the orientation of smooth muscle cells all vary between segments of the vasculature. Generally, smooth muscle cells have circular or helical orientation around the vessel lumen, while in some vessels a layer of smooth muscle is also oriented along the longitudinal axis of the cell. The outermost layer of the vascular wall (tunica adventitia) consist of loose connective tissue, longitudinal bundles of smooth muscle cells (primarily in large veins), minute blood vessels (vaso vasorum, in large arteries), and a network of autonomous nerve fibers (Wagenseil and Mecham, 2009). The main function of vascular smooth muscle is to control the blood flow to tissues and organs, providing the primary route of transportation for nutrients, immune cells, and signaling  1  molecules in the body. This is achieved by regulating the resistance to flow, which in turn is the basis for a maintained blood pressure. Interactions with other cells (nerve endings, endothelia, and smooth muscle cells), with humoral (circulating hormones and nutrients) and physical factors (blood pressure, blood flow) determine if the vascular smooth muscle will contract, relax, or maintain pressure. In their contractile phenotype, the basic physiological role of vascular smooth muscle is to regulate the diameter of the vessel lumen in order to control perfusion pressure and direct regional blood flow. The control of vascular tone is modulated by numerous factors, such as perfusion pressure, autonomic stimuli, paracrine and autocrine receptor ligands, and oxygen tension (Mchedlishvili, 1980; Zang et al., 2006). However, in damaged vessels, smooth muscle cells can differentiate into a secretory/proliferative phenotype to assist with blood vessel repair (Schwartz et al., 1986). In both phenotypes, changes in the free ionic concentration of Ca2+ in the cytosol play a central role in the regulation of multiple functions of smooth muscle. However, with increasing age, poor diet, and lack of exercise, regulation of vascular smooth muscle becomes increasingly prone to failure which can lead to hypertension, vasospasm, and other malfunctions (Ferrari et al., 2003, Proctor and Parker, 2006).  1.1.1 Endothelium The vascular endothelium consists of a continuous monolayer of cells, lining the luminal surface of the entire vascular system, which provides a structural and metabolic barrier between the blood and the underlying tissues. Endothelial cells are induced to migrate during the process of new capillary blood formation and during repair of the endothelial lining which result from injury of large vessels. The endothelium plays a central role in the regulation of the vascular tone (Furchgott and Zawadzki, 1980), releasing a variety of vasoactive mediators, including  2  prostaglandins, nitric oxide (NO), and endothelium-derived hyperpolarizing factor (EDHF) to regulate smooth muscle contractility and thus vascular smooth muscle tone (Ramsey et al., 1995; Boutouyrie et al., 1997; Wilkinson et al., 2002; Vanhoutte, 2004). Within vessels, the endothelial and the smooth muscle cells are separated by connective tissue and the internal elastic membrane. However, these two cell types also establish close contacts with each other, via myo-endothelial bridges that cross fenestration of the internal elastic lamina (Emerson and Segal, 2000; Sandow and Hill, 2000).  1.1.2  Overview of smooth muscle  Smooth muscle comprises the medial layers in the walls of most hollow organs, including the gastrointestinal tract, urogenital tract, and the vasculature, and is a subject of study due to its ability to regulate lumen diameter (Halayko et al., 1997). The term “smooth” refers to the histological appearance of the muscle, which in contrast to skeletal and cardiac muscle does not exhibit striations. The spindle shaped single cells are generally 50-100 μm long and 2-5 μm thick (Bolton et al., 1999). While, t-tubules and elaborate neuromuscular junctions are absent, the sarcoplasmic reticulum (SR) is still well-developed, occupying 1.5–7.5% of the total cell volume (Devine et al., 1972). Neurotransmitters such as acetylcholine or norepinephrine may activate or inhibit smooth muscle cells by diffusing and interacting with specific receptors on the plasma membrane (PM). Smooth muscle is important clinically as it plays a primary role in the pathogenesis of fibroproliferative disorders of the vascular wall associated with diseases such as atherosclerosis (Halayko et al., 1997).  3  1.1.3 Mechanism of smooth muscle contraction Each smooth muscle cell contains thick (myosin) and thin (actin) filaments that slide against each other to produce contraction of the cell. The thick and thin filaments are anchored to dense bodies throughout the cytosol and dense bands (or plaques) at the PM (Somlyo, 1985). Smooth muscle contraction is regulated by Ca2+-dependent myosin light chain kinase (MLCK) activation and myosin light chain phosphatase (MLCP) (Kamm and Stull, 1985). Cytosolic free ionic Ca2+ is the primary second messenger linking membrane excitation or stimulation to the contraction of smooth muscle cells. During smooth muscle activation, Ca2+ levels may increase rapidly to above 10-6 M (Sato et al., 1988; Karaki, 1997). This is due to the opening of Ca2+ channels in the PM and in intracellular Ca2+ stores, where Ca2+ is also present at high concentrations. Elevation of Ca2+ promotes the binding of 4 Ca2+ ions to calmodulin, forming a Ca2+-calmodulin complex which then binds to and activates MLCK. Activated MLCK then phosphorylates the myosin light chain and stimulate the acto-myosin cross-bridge cycling (Rembold and Murphy, 1990; Allen and Walsh, 1994). The activity of MLCK is opposed by MLCP, and the opposing actions of MLCK and MLCP on myosin phosphorylation can also be modulated by Ca2+-independent phosphorylation events, causing an apparent shift in the Ca2+ sensitivity of the myofilaments. For example, Rho-kinase can phosphorylate MLCP and impair its dephosphorylation of myosin, thereby favouring contraction (Sward et al., 2003). Phosphorylation of the actin-associated proteins, caldesmon and calponin, also regulate their inhibitory effect on cross-bridge cycling (Allen and Walsh, 1994; El-Mezgueldi, 1996). Relative to cardiac and skeletal muscles, smooth muscle can maintain tension with much less energy expenditure by virtue of a prolonged association of actin and myosin upon myosin dephosphorylation, known as “latch-state” (Hai and Murphy, 1988; Paul, 1990). This model has been expanded into an 8-state model, which  4  further accounts for the phosphorylation of the thin actin filament (Hai and Kim, 2005), and future iterations may include regulatory influences of proteins such as Rho-kinase.  Actin 4Ca2+·CaM  Ca2+-calmodulin-MLCK  ADP Myosin  Myosin - P  ATP Myosin phosphatase Actin + Activated myosin ATPase  Smooth muscle contraction  Figure 1.1: Ca2+, calmodulin, MLCK (myosin light-chain kinase), and cross-bridge cycle in smooth muscle. A schematic diagram depicting the activation of the crossbridge cycle in the smooth muscle cell is shown. 1.2 Mechanisms of Ca2+ homeostasis and signaling in vascular smooth muscle In order for cytosolic Ca2+ to increase and stimulate the contractile apparatus or other Ca2+ dependent processes, Ca2+ must enter the cytosol from the extracellular space or be released into the cytosol from the SR. This section describes the routes by which Ca2+ can enter the cytosol.  5  Ca2+/ Na+ Ca2+ ROC/SOC  3Na+ NCX  Ca2+  VGCC Ca2+  Ca2+  PMCA  RyR IP3R SR Ca2+  SERCA Ca2+  PM  Figure 1.2 Ion channels and receptors involved in regulating intracellular Ca2+ concentration in smooth muscle cells. (PM, plasma membrane; SR, sarcoplasmic reticulum; VGCC, voltage-gated Ca2+ channel; NCX, Na+/Ca2+ exchanger; ROC, receptor-operated channel; SOC, store-operated channel; PMCA, plasma membrane Ca2+ ATPase; IP3R, IP3 receptor; RyR, ryanodine receptor; SERCA, sarco-endoplasmic reticulum Ca2+-ATPase). 1.2.1 Voltage-gated Ca2+ channels The most important Ca2+ channels in vascular smooth muscle are the voltage-operated channels on the PM, of which there are six subtypes. However, only the L-type voltage-gated Ca2+ channel is considered to be a major Ca2+ influx pathway in smooth muscle (Kuriyama et al., 1995). Due to both voltage-activating and inactivating mechanisms, a window current is defined by the voltages at which L-type Ca2+ channels are capable of sustained openings and inward current (Cohen and Lederer, 1987). Although this sustained Ca2+ current is smaller than the initial transient currents, it still generates a large Ca2+ influx relative to the cell volume (Sanders, 2001). Furthermore, L-type Ca2+ channels are modulated by several signaling systems, 6  particularly activated by vasoconstrictors that activate the protein kinase C (PKC) pathway (Hughes and Bolton, 1995). Additionally, vasodilators that stimulate the production of cyclicAMP and activate protein kinase A have been reported to both activate and inhibit these channels (Jackson, 2000). These channels are inhibited by increases in [Ca2+]i, dihydropyridines such as nifedipine and other antagonists such as diltiazem (Triggle, 1999; Catterall, 2000).  1.2.2 Receptor-operated and store-operated cation channels The concept of receptor- and store-operated channels in smooth muscle was introduced over 30 years ago (van Breemen et al., 1978; Bolton, 1979; Casteels and Droogmans, 1981; Bolton and Large, 1986), and it has been known since 1981 that depletion of SR Ca2+ stores and activation of G-protein coupled receptors can activate Ca2+ influx by mechanisms independent of membrane depolarization through what have been termed store-operated channels (SOC) and receptor operated channels (ROC). ROCs are activated by agonists activating on a range of Gprotein coupled receptors, while SOCs are activated following depletion of the Ca2+ stores within the SR. It is important to note that receptor-operated currents have shown varying degrees of selectivity for Ca2+, so these channels are sometimes termed non-selective cation channels (NSCCs). Store-operated Ca2+ entry (SOCE) was proposed as a mechanism for receptor-regulated Ca2+ influx that allows refilling of the intracellular Ca2+ pool once agonist stimulation has finished (Rosado, 2006; Putney, 2007). SOCE involves a number of non-selective Ca2+ permeable channels with different biophysical properties. The first identified store-operated channel current, ICRAC (Ca2+-release activated Ca2+ current), is mediated through a non-voltage activated, inwardly rectifying channel that is highly selective for Ca2+ (Hoth and Penner, 1992; Zweifach and Lewis,  7  1993; Parekh and Putney, 2005). The nature of the channels that conduct ICRAC has been a matter of investigation and debate. Recently, the protein Orai1 has been proposed to form the pore of the channel mediating ICRAC (Feske et al., 2006; Peinelt et al., 2006; Prakriya et al., 2006; Vig et al., 2006; Zhang et al., 2006). Along with two other subtypes (Orai2 and Orai3), heteromultimetric complexes can be formed that share a high selectivity for Ca2+ (Lis et al., 2007). The channel formed by Orai1 has been reported to be regulated by Ca2+ store depletion with the participation of an intraluminal Ca2+ sensor, stromal-interacting molecule 1 (STIM1), a transmembrane protein located in the SR, which has been identified as the intraluminal Ca2+ sensor that communicates the amount of stored Ca2+ to the PM SOC channels (Liou et al., 2005; Roos et al., 2005). STIM1 physically moves from locations throughout the membrane of the Ca2+ stores to accumulate in regions close to the PM (Wu et al., 2006). The aggregation of STIM1 underneath the PM induces Orai1 clustering at discrete sites in the PM directly opposite to STIM1 clusters, resulting in the activation of SOCE (Xu et al., 2006; Barr et al., 2008). The transient receptor potential (TRP) proteins have also been suggested as components of SOCs. A number of mammalian homologues of TRP have been found and are classified into three major subfamilies closely related to TRP (transient receptor potential canonical, TRPC; transient receptor potential vanilloid, TRPV; transient receptor potential melastatin, TRPM), two subfamilies that are more distantly related to TRP (transient receptor potential polycystic, TRPP; transient receptor potential mucolipin, TRPML), and a less related no mechanoreceptor potential C (TRPN) group that is expressed in flies and worms (Montell et al., 2002). TRP channels are mostly nonselective for monovalent and divalent cations (PCa/PNa ≤ 10), with exceptions including TRPM4 and TRPM5, which shows a great selectivity for monovalent cations, and the  8  Ca2+-selective TRPV5 and TRPV6. As with Orai proteins, TRP channels lack voltage sensitivity (Venkatachalam and Montell, 2007). Particular attention has been paid to members of the TRPC subfamily, which has seven related members designated TRPC1-7. These can be divided subgroups: (i) TRPC3, TRPC6 and TRPC7 channels; and (ii) TRPC1, TRPC4 and TRPC5 channels, based on biochemical and functional similarities (Parekh and Putney, 2005), although TRPC2 is a pseudogene in humans and is not functional (Wes et al., 1995). TRPC1 is suggested to be involved in SOCE in both vascular smooth muscle and endothelial cells (Liu et al., 2000; Brough et al., 2001; Xu and Beech, 2001; Rosado et al., 2002). A role for TRPC3 was demonstrated when overexpression enhances SOCE (Zhu et al., 1996; Zhu et al., 1998), while suppression leads to the disappearance of SOCs (Kaznacheyeva et al., 2007). TRPC4 is suggested to be an important component of the channel supporting ICRAC-like currents, which are small currents activated by extracellular Ca2+ (Fatherazi et al., 2007). A role for TRPC6 in SOCE has also been proposed in human platelets (Hassock et al., 2002). Thus far, a role for TRPC7 has not been elucidated. The involvement of TRP proteins as components of SOC has received support from studies reporting that STIM1 directly or indirectly regulates all TRPC proteins, with the exception of TRPC7, in cells with depleted Ca2+ stores. For example, STIM1 and TRPC1 have been shown to interact upon Ca2+ store depletion (Huang et al., 2006; Lopez et al., 2006). STIM1 has also been reported to interact directly with TRPC2, TRPC4 and TRPC5. Furthermore, STIM1 mediates the interaction of TRPC1 with TRPC3 and of TRPC4 with TRPC6. TRPC hetermultimerization is enhanced by Ca2+-mobilizing agonists, suggesting that store depletion-induced clustering of STIM1 is required for the formation of heteromeric SOCs (Yuan et al., 2007). The number of putative SOCs formed by heteromultimerization of channel subunits has been considerably  9  enhanced by the demonstration that Orai proteins interact with TRPCs. The interaction of Orai proteins with TRPCs has been reported to confer STIM1-mediated store depletion sensitivity to SOCs (Liao et al., 2007). Additionally, TRPCs have also been suggested to play a role in receptor-mediated Ca2+ entry (Venkatachalam et al., 2002; Clapham, 2003; Freichel et al., 2004; Dietrich et al., 2005a; Dietrich et al., 2005b; Montell, 2005; Parekh and Putney Jr, 2005). Receptor-induced Ca2+ signals are crucial to the function of all cells (Berridge et al., 2003) and involve both the release of Ca2+ from stores and the entry of Ca2+ through channels in the PM (Venkatachalam et al., 2002; Berridge et al., 2003; Parekh and Putney Jr, 2005). ROCs are thought to be activated by diacylglycerol (DAG) that is generated following hydrolysis of phosphatdyl inositol by phospholipase C (Thebault et al., 2005). The first ROC to be described in vascular smooth muscle was a noradrenaline-evoked cation conductance in rabbit portal vein myocytes (Byrne and Large, 1988). The activation of these channels involves the classical G-protein-coupled phosphoinositol (PI) system, involving stimulation of PI-phospholipase C (PLC) and the production of DAG (Helliwell and Large, 1997). A surprising and novel result from the latter study was that the generation of endogenous DAG, as well as DAG analogs, activated ROCs via a PKC-independent mechanism. Gating by DAG of several NSCCs has been subsequently described, including TRPC3/6/7 channel proteins expressed in cell lines (Hofmann et al., 1999; Inoue et al., 2001; Estacion et al., 2004; Shi et al., 2004) and it has been shown that TRPC6 and TRPC3 proteins are components of native ROCs in portal vein and cerebral artery myocytes (Inoue et al., 2001; Reading et al., 2005). One functional characteristic distinguishing the two TRPC subgroups is the ability of DAG to activate TRPC3/6/7 channels, but not the TRPC1/4/5 channels (Hoffman et al., 1999;  10  Venkatachalam et al., 2003; Freichel et al., 2004; Dietrich et al., 2005a; Dietrich et al., 2005b; Parekh and Putney Jr., 2005). DAG appears to have an important dual role in TRPC channels; in addition to rapidly activating TRPC3 channel directly, DAG also mediates a slower PKCmediated deactivation of the TRPC3 channel (Venkatachalam et al., 2003; Estacion et al., 2006). This bimodal regulation may form the basis of a spectrum of regulatory phenotypes of expressed TRPC channels. Finally, although the opening of ROCs mediates Ca2+ influx, it should be noted that the extracellular concentration of Na+ is ~100 fold higher than that of Ca2+. Therefore, the opening of these channels also mediates substantial Na+ influx, changing the concentration gradient of Na+ to favour reversal of the Na+/Ca2+ exchanger and bring Ca2+ into the cell (Lee et al., 2001).  1.2.3 Ryanodine and IP3 receptors In addition to being an intracellular Ca2+ storage site, the SR also releases Ca2+ upon activation. Release of Ca2+ from the SR is mediated by two types of Ca2+ channels: ryanodinesensitive receptors (RyR) and inositol 1,4,5-trisphosphate (IP3)-sensitive receptors (IP3R), each of which is expressed in three isoforms. Both channels are essentially gated by local [Ca2+], such that cyclic ADP-ribose (cADPR) and IP3 alter the Ca2+ affinity of the RyR and IP3R, respectively. Release of SR Ca2+ induced by PLC-linked receptors, such as α-adrenergic receptors, is often associated with the production of IP3 and subsequent activation of IP3R (Karaki et al., 1997). In contrast, relatively little is known about linkage between ryanodine receptor activation and the production of cADPR by ADP ribose cyclase (Bai et al., 2005). Rather, the activation of RyR is most often attributed to Ca2+-induced Ca2+ release in response to local elevation of Ca2+ (Bolton et al., 1999; Gordienko et al., 1998; Putney, Jr., 1993). However,  11  despite the fact that both receptors release Ca2+ into the cytosol, evidence suggests that they may be localized to separate elements of the SR (Golovina and Blaustein, 1997; McGeown, 2004), which may allow each receptor to regulate specific processes in the cell. However, others have suggested that the IP3R and RyR both access a common SR Ca2+ store (Saida and van Breemen, 1984; Lepretre and Mironneau, 1994; McCarron and Olson, 2008).  1.2.3.1 Ryanodine receptors Named due to their binding to the plant alkaloid ryanodine, the RyRs are endogenously regulated by [Ca2+]i. Three isoforms of RyRs exist, although only RyR2 and RyR3 have been identified in smooth muscle. At low concentrations (<100 μM), ryanodine causes a persistent subconductance state of the channels which may lead to store depletion (Endo, 1977; Rousseau et al., 1987; Hymel et al., 1988; Kanmura et al., 1988; Iino, 1989; Xu et al., 1994), while higher concentrations lock RyRs in a closed state to inhibit Ca2+ release (Hayek et al., 2000; Fill and Copello, 2002). This regulation is due to the presence of high- and low- affinity binding sites on the RyR for Ca2+ (Marx et al., 2000). In addition, the Ca2+-binding protein calsequestrin located in the SR may be positioned close to and perhaps co-localized together with RyRs (Berchtold et al., 2000; Moore et al., 2004) via junction and triadin proteins (Guo and Campbell, 1995; Zhang et al., 1997) and may enhance opening of the RyRs (Szegedi et al., 1999) when phosphorylated. Xanthines such as caffeine are often used as a tool to empty the SR, as they increase the Ca2+ sensitivity of the RyR (Smith et al., 1988; Sitsapesan and Williams, 1990). The opening of RyRs in smooth muscle are also responsible for the production of Ca2+ sparks, localized Ca2+ transients which can reach concentrations of 1-10μM, while increasing global intracellular [Ca2+] by <2nM (Jaggar et al., 1998; Nelson et al., 1995; Jaggar et al., 2000).  12  Ca2+ sparks appear to represent the simultaneous activation of a cluster of RyRs, and were proposed to be responsible for the activation of a number of nearby Ca2+-sensitive K+ channels (BKCa) on the PM to cause a macroscopic current that was previously described as a spontaneous transient outward current (Benhan and Bolton, 1986; Nelson et al., 1995). BKCa are uniquely suited to respond to very high local [Ca2+] because they require micromolar [Ca2+] for their activity under physiological conditions (Perez et al., 1999). The opening of BKCa can hyperpolarize the cell membrane by up to 20mV, providing an important contribution to regulation of vascular tone (Ganitkevich and Isenberg, 1990). Ca2+ sparks can also activate spontenaous transient inward currents, which are caused by the opening of Ca2+-sensitive Clchannels, resulting in cell membrane depolarization (ZhuGe et al., 1998).  1.2.3.2 IP3 receptors Since the demonstration of receptor-mediated PLC activation, Ca2+ release in response to IP3 and the purification and cloning of IP3Rs, inositol phosphate signaling has been rapidly accepted as an important cellular second messenger system (Berridge, 1993). G protein-linked receptors activate G proteins in the PM which stimulate PLC to split phosphatidyl 4,5-bisphosphate (PIP2) into DAG and IP3. DAG activates protein kinase C (PKC), and IP3 binds to and gates a distinct class of intracellular, endoplasmic reticulum-bound IP3 receptor channels. The resulting Ca2+ release increases [Ca2+]i within seconds to micromolar concentrations. The Ca2+ diffuses to adjacent IP3Rs (and also RyRs in many cells) channels, initiating the release of more Ca2+. A biphasic relationship exists between the open probability of IP3R and Ca2+ release (Iino, 1990; Bezprozvanny et al., 1991; Mak et al., 1998), as [Ca2+]i of ~300 nM provides optimal activation of IP3R by IP3, while higher concentrations of [Ca2+]i decrease the activity of the IP3R  13  (Sanders, 2001). However, due to the Ca2+ sensitivity of the IP3R, the RyR may be activated when the IP3R is no longer active. This has important implications for the participation of the RyR and IP3R, one being able to activate the other depending on their spatial location on the SR membrane (Iino, 1999). Heparin is a specific antagonist of IP3R, but is impermeable to the cell membrane (Ghosh et al., 1988; Kobayashi et al., 1988; Supattapone et al., 1988). Permeable inhibitors such as Xestospongin C (Gafni et al., 1997), and 2-aminophenylborate (2-APB) (Maruyama et al., 1997) are important also pharmacological tools in determining the role of IP3R in Ca2+ signaling, although their selectivity has been questioned (Bilmen and Michelnageli, 2002; Ma et al., 2002).  1.2.4 Ca2+ uptake and extrusion mechanisms Due to the large chemical gradient of extracellular [Ca2+] (2 mM) to cytosolic [Ca2+] (0.1 μM) in smooth muscle, it is critical for the cell to remove Ca2+ and prevent accumulation of Ca2+, which may be cytotoxic. Smooth muscle cells have a resting potential between -40 and -60 mV when subjected to normal levels of intravascular pressure (Nelson and Quayle, 1995), meaning that there is also a large inwardly directed electrochemical gradient for Ca2+ into the cell. Ca2+ also tends to move into resting cells (Poburko et al., 2004c) and must be extruded by energy-dependent mechanisms. Calcium transporters such as the PM Ca2+-ATPase (PMCA) and the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) play a crucial role in maintaining Ca2+ homeostasis by maintaining low resting Ca2+ and restoring Ca2+ for relaxation of smooth muscle (Floyd and Wray, 2007). Storage of Ca2+ in cellular organelles provides important physiological regulation and the potential for release of Ca2+ during physiological signaling. The main Ca2+ storage organelle is  14  the SR, and has a major role in maintaining low [Ca2+]i. The SR is surrounded by a membrane which is not freely permeable to Ca2+, and on which SERCA pumps sit. SERCA actively sequesters cytosolic Ca2+ into the SR (Sanders, 2001) and maintains a 10,000-fold concentration gradient between the SR lumen and the cytosol. There are 3 genes encoding for SERCA, and in smooth muscle, the SERCA2b isoform is mainly expressed, with the SERCA2a and SERCA3 isoforms forming the remainder of the SERCA population (Lytton et al., 1989; Wuytack et al., 1989; Eggermont et al., 1990; Amrani et al., 1995; Trepakova et al., 2000; Wu et al., 2001). All SERCAs encode a cytoplasmic region that contains the catalytic site and a transmembrane domain that forms a channel-like structure allowing Ca2+ translocation across the membrane (Engelender and De Meis, 1996; Zhang et al., 1998). Phospholamban is a small protein that negatively regulates SERCA; upon phosphorylation via PKC or cGMP-dependent protein kinase (PKG) (Raeymaekers et al., 1990), this inhibition is relieved and SERCA is activated, thereby pumping Ca2+ into the SR. After Ca2+ is pumped into the SR, it is buffered and sequestered by proteins such as calreticulin and calsequestrin, which bind large amounts of Ca2+ (Milner et al., 1992; Raeymaekers et al., 1993). The total concentration of Ca2+ in the SR has been estimated to be as high as 10-15 mM (Nishimura et al., 1989). The specific reversible or irreversible inhibitors of SERCA, cyclopiazonic acid (CPA) and thapsigargin respectively, have been instrumental in understanding the importance of SERCA and the SR in regulating contraction and relaxation of smooth muscle. Mitochondria also play an important role in Ca2+ homeostasis, as they regulate Ca2+ release events from both IP3R and RyR which are important to the propagation of Ca2+ waves (Lee et al., 2002; Wellman and Nelson, 2003). Mitochondria can also modulate the activity of PM ion  15  channels such as SOCs, L-type Ca2+ channels, Ca2+-activated K+ channels, and Ca2+-activated Clchannels by buffering local Ca2+ gradients (Hoth et al., 1997; Montero et al., 2000; Thyagarajan et al., 2001; Malli et al., 2003a; Malli et al., 2003b). This type of regulation can indirectly affect SR refilling and consequently SR release. Two other pathways to extrude Ca2+ from smooth muscle cells are the PMCA and the Na+/Ca2+ exchanger (NCX), the latter of which is also regulated by the Na+/K+ pump (Na+/K+ATPase) (Blaustein and Lederer, 1999). The PMCA uses energy from ATP to pump Ca2+ up the electrochemical gradient from the cytosol to the extracellular space. This pump is electron neutral, as the Ca2+ pumped to the extracellular space is exchanged for two protons. Therefore, Ca2+ extrusion results in the uptake of H+ and must be compensated by other means, such as Na+/H+ exchange (Lucchesi and Berk, 1995). The NCX can operate in both the forward (Ca2+-efflux) mode and the reverse (Ca2+-influx) mode (Karaki et al., 1997; Laporte and Laher, 1997). Regardless of the mode of operation, the coupling ratio for the NCX is 3 Na+:1 Ca2+ (that is, 1 Ca2+ ion is exchanged for every 3 Na+ ions) (Bolton et al., 1999). Under normal resting conditions, the NCX almost always operates in the forward mode, although depending on the net electrochemical driving force, the NCX can also operate in reverse mode physiologically (Blaustein and Lederer, 1999; Lee et al., 2001). This may happen at restricted subplasmalemmal areas in the cell, such as the close apposition between the PM and superficial SR, where the concentrations of Na+, Ca2+, and other ions may be different from the cytosol. NCX activity is a function of membrane potential and the local [Ca2+] and [Na+] on each side of the PM. The reversal potential of the NCX is related by the following equation: ENCX = 3ENa – 2ECa  (1)  16  In general, EXz+ = RT/(zF) ln([Xz+]out/[Xz+]in), where R is the universal gas constant (8.3 J/mol•K), T the absolute temperature in Kelvin, Xz+ is a z-valent cation, and F is Faraday’s constant (9.65×104 J/V•mol). Under resting conditions, using Vm = −60 mV (Haddock and Hill, 2005), [Na+]o = 145 mM, [Ca2+]i = 10−4 mM, [Ca2+]o = 1.5 mM (Blaustein and Lederer, 1999; Alberts et al., 2002), ENa = +71.3mV and ECa = +128mV, which yields ENCX = -42 mV. For NCX reversal to occur, ENCX must be more negative than Vm. Therefore, because resting membrane potential is under the stated conditions is −60 mV, the NCX will generally operate in the forward (Ca2+-extrusion) mode. The NCX can be blocked by 2’,4’-dichlorobenzamil (2,4-DCB) in both the forward mode (Blaustein and Lederer, 1999), while KB-R7493 at concentrations of 10 μM or less selectively block the NCX in the reverse (Ca2+-influx) mode (Ladilov et al., 1999). Recently, a more specific and potent NCX inhibitor, SEA0400, has been developed (Tanaka et al., 2002).  1.2.5 Recombinant aequorin as a method of measuring Ca2+ Aequorin is derived from the Pacific jellyfish, Aequorea victoria, and is a photoprotein dependent on the presence of Ca2+ to emit light based on the reaction shown in Fig. 1.3 (Prendergast, 2000). Aequorin is composed of 189 amino acids and has 3 EF-hand structures which bind Ca2+ (Head et al., 2000; Hofer et al., 2000). In addition, a hydrophobic region exists where the protein may interact with its functional chromophore, coelenterazine (Inouye et al., 1985). This protein emits light (466 nm) in the presence of small amounts of Ca2+. However, a functional aequorin molecule is only obtained after the apoaequorin has been incubated with its prosthetic group, coelenterazine and oxygen which form a stable intermediate. When Ca2+ binds to the complex, coelenterazine is converted to coelenteramide in an irreversible photochemical  17  reaction and emits light at 466 nm, which then can be detected and quantified. Aequorin offers many advantages over other standard Ca2+ imaging dyes, the most important being the specificity of the probe and direct Ca2+ measurement in the area of interest. Additionally, aequorin measurements are virtually free of any background, are extremely sensitive and are not weakened by auto fluorescence (Rizzuto et al., 1994). Some limitations to the use of aequorin include its rapid and irreversible consumption by high Ca2+, which limits experimental duration and design, and relatively low light output which makes it difficult to detect in single cells (Rutter et al., 1996; Hofer et al., 2000). To transform luminescence values into [Ca2+] values, calibration requires knowledge of the total luminescence contained in the preparation. This relies on the relationship between Ca2+ and the ratio (L/Lmax) between light intensity recorded in physiological conditions, which represents aequorin consumption (L), and that which would have been recorded if all of the aequorin in the cell had been suddenly exposed to a saturating Ca2+ (Lmax). A good estimate of Lmax can be obtained from the total aequorin light output recorded from the cells after discharging of all the aequorin through the lysing of the cells. As aequorin is being consumed continuously, the value of Lmax is not constant and decreases steadily during the experiment. The value of Lmax to be used for Ca2+ calculations at every point along the experiment is then calculated as the total light output of the whole experiment minus the light output recorded before that point. Generally, as Ca2+ is raised from low levels to high levels, a calibration curve which plots L/Lmax vs. pCa generally yields a sigmoidal curve with a slope ranging from zero to a maximum of 3 before falling again to zero at saturating [Ca2+] levels. The steep cube-law relationship between aequorin consumption and [Ca2+] is due to the 3 Ca2+ binding sites on the aequorin molecule, and  18  it is between this range that aequorin can give the most accurate measurements (Allen et al., 1977; Cobbold and Rink; 1987; Brini et al., 1995).  Figure 1.3: Recombination of aequorin. Upon addition of the prosthetic group CoE (coelenterazine), the apoaequorin (APO) will form the function aequorin molecule upon exposure to oxygen. Calcium molecules bind to one of three EF-hand motifs on the aequorin molecule and upon binding produces coelenteramide, the apoaequorin, carbon dioxide, and light at a wavelength of 466 nm (Prendergast, 2000; Chiesa et al., 2001). Aequorin has been used to specifically measure Ca2+ concentrations in various cellular compartments, such as mitochondria and SR, where previously only slightly selective membrane-permeable Ca2+ indicators were useful. This is due to aequorin fusing with molecular targeting sequences. Targeted aequorins have been constructed for mitochondria, ER, SR, PM, cytosol and the nucleus (Chiesa et al., 2001). This targeting is achieved by an N-terminal fusion  19  of the photoprotein with a minimal targeting sequence as C-terminal fusions have been proven unsuccessful. These plasmids are cloned in vectors suitable for transfection, such as pcDNAI or pMT2. Charged liposomes have been used as the method of transfection into smooth muscle cells in this thesis. To ensure correct localization of the plasmids, a hemagglutinin epitope tag (HA1) between the targeting sequence and the aequorin cDNA allows immunolocalization. In addition, a green fluorescent protein (GFP) targeted to the same mitochondrial sequence as used in the aequorin plasmid targeted to the mitochondria has been made to allow for simpler identification of correct localization by imaging with confocal microscopy. Moreover, photoproteins with differing Ca2+ sensitivities have been created by mutation of specific amino acids. For example, changing low affinity aequorin by one amino acid (Asp 119 to Ala) modifies it to a high affinity aequorin, which when incubated with a coelenterazine analog, showed rapid millimolar Ca2+ transients in a new subpopulation of mitochondria (Montero et al., 2000). Thus, not only can the aequorin be mutated, but coelenterazine analogs can also regulate the Ca2+ sensitivity of the functional photoprotein by regulating membrane permeability and regeneration rate (Shimomura et al., 1989; Shimomura et al., 1990; Shimomura et al., 1993). This is especially important in organelles such as the SR or ER where Ca2+ content is high and a low affinity aequorin is required.  1.3 Nanodomains and Ca2+ signaling in vascular smooth muscle Ca2+ regulates nearly all fast processes in the body, including contraction, chemotaxis, secretion, synaptic transmission, and several slower processes, including fertilization, proliferation, learning, memory, and apoptosis. However, it is unclear how the fluctuations in Ca2+ can regulate a multitude of cellular processes. Although cells have developed a variety of  20  Ca2+ sensitive, signal-transducing proteins to tailor their cell-specific regulation of many diverse processes by Ca2+ (Berridge et al., 2003), this cannot entirely explain how multiple processes, such as cross-bridge cycling and myosin filamentogenesis in smooth muscle or endothelial nitric oxide and epoxyeicosatrienoic acid production, can be regulated simultaneously. One solution may rely on the physical and temporal separation of numerous targets for Ca2+, combined with the generation of localized cytoplasmic Ca2+ gradients (Poburko et al., 2004). For example, mitochondrial dehydrogenases, voltage-gated Ca2+ channels, IP3Rs, and DNAses are located in different subcellular regions and could be selectively activated by focal Ca2+ signals. Moreover, activation of certain target proteins by the Ca2+-calmodulin complex could be site-specific, despite the widespread intracellular distribution of calmodulin, since calmodulin can be tethered to effector complexes such as smooth muscle myofilaments (Wilson et al., 2002). In order to create Ca2+ signals with specific temporal and spatial characteristics, it is not sufficient to only have a large variety of ion transport molecules, but also to have these components be strategically clustered around cytoplasmic nanodomains. Analysis of the interaction of Ca2+ transport molecules in the PM, the SR, nuclear envelope, and mitochondria suggest that these interactions provide structural basis for the spatially and temporally encoded fluctuations in [Ca2+]i that are thought to mediate site-specific Ca2+ signaling. This interaction takes place in two fundamentally different ways: 1) directed Ca2+ supply to or removal from the Ca2+-sensing domains of signal transducing molecules and Ca2+ translocators, and 2) Ca2+ delivery from a transport site located in one membrane to a second Ca2+ transport site located in an apposing membrane. An example of the first type of interaction is the delay of Ca2+-mediated inhibition of L-type Ca2+ channels due to nearby mitochondria sequestering Ca2+. The second type of interaction is exemplified by coupling of Ca2+ entry through the NCX to SERCA during  21  store refilling (Lee et al., 2002). The latter process circumvents free diffusion throughout the cytoplasm and typically takes place at organellar junctions where physically restricted diffusion of Ca2+ within the narrow cytoplasmic domain is further slowed down by a high density of fixed, negatively charged Ca2+ binding sites (Bers, 2001). This second type of interaction occurs in nanodomains, which are formed at the sites where Ca2+ enters the cytoplasm at either the cell surface or at the internal stores, and are defined by the ultrastructural architecture of the cell, such as the close (nanometre-ranged) spatial association of the PM, SR and mitochondria (Poburko et al., 2008; Fameli et al., 2009). The resulting microstructural arrangements of the apposing membranes create diffusional barriers defining different types of junctional spaces within the cytoplasm. The diffusional limitations of these junctional spaces allow for accumulation of ions such as Na+ and Ca2+ in concentrations greatly exceeding that in the bulk cytoplasm (Rizzuto and Pozzan, 2006; van Breemen et al., 2006). These cytoplasmic nanodomains have important functional implications. For example, Ca2+sensitive ion channels selective for K+, Cl-, and Ca2+ and Ca2+-sensitive enzymes, such as PKC and PLC, which are located in membranes bordering the restricted space between the PM and the superficial SR, can be regulated separately from the myofilaments occupying the bulk of the cytoplasm (Berridge, 2006; Edwards and Pallone, 2007).  1.3.1 Structural and functional considerations of the PM-SR junctional nanodomain An important physiological nanodomain is the PM-SR junction, formed by the close apposition between the superficial SR and the PM. Much of the SR is associated with the cell membrane (Devine et al., 1972). The SR is composed of an interconnected tubular and sheet-like network with a volume estimated from 1.5-7.5% of the cell depending on the smooth muscle cell  22  type (Sanders, 2001) and is contiguous with the nuclear envelope (Somlyo, 1985; Nixon et al., 1994). The SR can be classified according to its location as superficial or deep, and electron microscopy shows that the superficial SR forms a flattened pedestal as it approaches the PM, at which point it creates a narrow space which extends on average in two dimensions for about 300400 nm and has a depth of 15-20 nm (Lee et al., 2002). In vascular smooth muscle cells of the rabbit inferior vena cava, 14.2 ± 0.7% of all the PM (including the necks of caveolae) is closely associated (within 30 nm) with the superficial SR, the junctional width averages 19 ± 1 nm (Lee et al., 2002), and the caveolae protrude through this thin fenestrated junctional SR (Fig 1.4) (Devine et al., 1972; Darby et al., 2000). The narrow cytoplasmic space between the junctional SR and PM is referred to as the PM-SR junctional space and is thought to present an imperfect barrier to diffusion of small molecules and ions, in particular Ca2+ and Na+. This is an important physiological mechanism as it allows Na+ influx through SOCs/ROCs to build up to a concentration high enough to allow NCX reversal (Lee et al., 2001; Poburko et al., 2006). The resulting Ca2+ influx following NCX reversal is essential for refilling of the SR and maintaining the Ca2+ waves (Lee et al., 2002). The structures responsible for the remarkably consistent spacing between the PM-SR junction have not yet been identified, although in some instances “feet” similar to those seen in triadic junctions in skeletal muscle have been reported (Somlyo, 1985), and proteins called junctophilins have been isolated from the diads of cardiac muscle (Takeshima et al., 2000). It is unknown what causes a particular portion of the PM to form a junction, although such junctions are common in caveolae-rich regions of the PM. The junctional PM might have specific chemical characteristics, such as those of cholesterol and sphingolipid-rich lipid rafts, which form platforms for signaling and transport molecules, or plasmalemmal receptors may be  23  physically linked to SR proteins such as the linkage of metabotropic glutamate receptors to SR release channels via Homer proteins (Feng et al., 2002). Interestingly, the α-1d adrenoceptor contains a consensus sequence for Homer protein binding (Zhong and Minneman, 1999).  Figure 1.4: Electron microscope cross-sections of smooth muscle cells from rabbit inferior vena cava. The superficial sarcoplasmic reticulum SR (arrows) comes into close contract (~20nm) with the plasma membrane (PM), forming a flattened narrow space termed the PM-SR junction. The PM-SR junctions typically extend to the necks of the caveolae (*) on either side. The black scale bar on the lower right indicates a distance of 200 nm. Image courtesy of Dr. KuoHsing Kuo.  24  1.3.2 Ca2+ oscillations in smooth muscle It was originally thought that the Ca2+ profile following agonist stimulation was a biphasic model, where agonist-mediated receptor activation initially released Ca2+ from the SR to initiate contraction and subsequently stimulated Ca2+ influx to maintain vascular tone (van Breemen et al., 1978; Bolton, 1979; Streb et al., 1983; Putney, 1986). However, Iino and colleagues (Iino et al., 1994) provided the first description of asynchronous Ca2+ oscillations in rat tail artery smooth muscle cells stimulated with norepinephrine, which occurred in the form of repetitive intracellular Ca2+ waves that propagated along the longitudinal axis of each smooth muscle cell. The described waves did not spread intercellularly and were not synchronized between neighbouring smooth muscle cells. Since this initial report, it has become apparent that fluctuations in the average arterial wall [Ca2+] observed previously (Jiang and Morgan, 1989; Meininger et al., 1991) are not representative of Ca2+ events within individual smooth muscle cells. Ca2+ oscillations have also been described in a variety of other smooth muscle preparations (Boittin et al., 1999; Miriel et al., 1999; Ruehlmann et al., 2000; Jaggar and Nelson, 2000; Bergner and Sanderson, 2002; Perez and Sanderson, 2005; Dai et al., 2007). In large veins and arteries agonist-mediated force development is regulated by recruitment of cells and the frequency of asynchronous, agonistinduced Ca2+ oscillations that are primarily generated by the release of Ca2+ from the SR (Evans and Sanderson, 1999; Ruehlmann et al., 2000). Agonist-induced contractions are maintained by asynchronous wave-like Ca2+ oscillations in single smooth muscle cells, which summate to give a steady-state elevation in Ca2+ for the whole tissue (Ruehlmann et al., 2000). The fact that asynchronous Ca2+ oscillations are observed in various smooth muscle cell types across different species has led to speculation of underlying physiological reasons for this  25  form of signaling. One physiological advantage may be related to the efficacy of Ca2+ oscillations in activating contraction. Contractile force is significantly decreased when Ca2+ oscillations are abolished by inhibition of SR Ca2+ re-uptake, although the average [Ca2+]i remains elevated (Lee et al., 2001; Sward et al., 2003). This observation indicates a higher forceto-Ca2+ ratio when smooth muscle cells are activated with asynchronous Ca2+ waves as compared to sustained Ca2+ elevation. It has also been proposed that wave-like Ca2+ oscillations may be more efficient in activating myofilaments because they utilize the SR network, which penetrates deep into the myoplasm, to deliver Ca2+ directly to the myofilaments. This is supported by the fact that myofilaments are not typically observed in the periphery of smooth muscle cells (Lee et al., 2002). Furthermore, mitochondria contain several Ca2+-sensitive dehydrogenases, and oscillatory Ca2+ signaling could serve to modulate frequency-encoded Ca2+ sensitive mitochondrial dehydrogenases and gene expression (Poburko et al., 2004a,b). Indeed, it has also been shown in hepatocytes that ER-mediated Ca2+ oscillations can efficiently activate certain Ca2+-sensitive dehydrogenases in the mitochondria (Hajnóczky et al., 1995). Finally, since an excessive amount of Ca2+ may produce deleterious effects in the cell, an oscillatory rise in Ca2+ with efficient delivery of Ca2+ to the target myofilaments may minimize the activation of unintended processes such as apoptosis, by avoiding overloading of the cell with an excessive amount of Ca2+ over a prolonged period of time.  1.3.3 Mechanism underlying Ca2+ oscillations Although Ca2+ oscillations have been observed in vascular smooth muscle for years, their molecular mechanism remains to be fully elucidated. This may be due to the fact that different  26  blood vessels display different types of rhythmic activity, which impedes consensus between different laboratories. So far, the most detailed description of the ionic mechanism of smooth muscle Ca2+ oscillations has been provided for adrenergically stimulated rabbit inferior vena cava (Lee et al., 2002). In this scenario, the initial α1-receptor stimulation activates PLC which catalyzes the synthesis of IP3. This leads to SR Ca2+ release from IP3R, which spreads across the cell from Ca2+ wave initiation sites. Stimulation of the α1-receptor or depletion of the SR also leads to opening of receptor-linked NSCCs permeable to Na+ and Ca2+ (Arnon et al., 2000a). The resultant inward current depolarizes the PM, activating L-type Ca2+ channels. At the same time, Na+ is postulated to enter the PM-SR junctional space through the NSCCs to increase the local [Na+] sufficiently to promote NCX reversal (Lee et al., 2002). Finally, the Ca2+ is taken back up into the SR by SERCA. The PM-SR junction complexes are postulated to be the sites for interactions among the NSCCs, NCX, and SERCA during SR refilling, and are thought to be crucial for the occurrence of the recurring Ca2+ waves. This theory is supported by the observation that the low-affinity NaK-ATPase isoforms α2 and α3 have been localized to the junctional PM (Juhaszova and Blaustein, 1997), which would promote elevated junctional [Na+] and NCX reversal (Arnon et al., 2000b). Finally, separation of the superficial SR from the PM with calyculin-A results in the abolishment of Ca2+ waves (Lee et al., 2005). This may disrupt SR refilling from Ca2+ coming from the PM, because as the junction separates the SERCA molecules are not able to capture as many Ca2+ molecules (Fameli et al., 2007). While there is general agreement that the initiation of oscillations and waves is a response to agonists acting on sarcolemmal receptors which releases Ca2+ from the SR via IP3Rs, controversy remains on whether or not Ca2+ release from the IP3Rs then activates RyRs to  27  generate further release by Ca2+-induced Ca2+-release and to propagate waves, or whether the entire release process arises from IP3Rs without significant RyR involvement (Mccarron et al., 2003). The former proposal is supported by studies which showed that drugs which block RyRs often abolish Ca2+ oscillations initiated by IP3-generating agonists (Hyvelin et al., 1998; Boittin et al., 1999; Jaggar and Nelson, 2000). This is possibly due to co-localization of RyRs and IP3Rs, which allows Ca2+ released locally by IP3R to activate adjacent clusters of RyR by Ca2+-induced Ca2+ release (Gordienko and Bolton, 2002). Interestingly, in some small vessels, asynchronous Ca2+ waves appear to cause vasodilation. For example, Jaggar (2001) showed in rat cerebral vessels that abolishment of these waves causes vasoconstriction. A proposed mechanism is that the asynchronous Ca2+ waves in these vessels may stimulate Ca2+-activated K+ channels, causing hyperpolarization of the membrane potential, inhibition of L-type Ca2+ channels, and muscle relaxation without significant contractile activation (Jaggar, 2001). It is plausible that these Ca2+ waves occur around the periphery or the subplasmalemmal region of the smooth muscle cells rather than in the deeper myoplasm, where the myofilaments are located.  1.3.4 Vasomotion Under certain conditions, such as during application of high concentrations of agonists, Ca2+ waves in vascular smooth muscle cells may become synchronized to initiate vasomotion, or spontaneous rhythmical contractions. Although its underlying mechanism and physiological importance is still unclear, these synchronized contractions are spread out over considerable distances and may assist in tissue perfusion, especially during periods of altered metabolism or perfusion pressure (Funk et al., 1983, Meyer et al., 2002). Vasomotion may also provide  28  oscillation of oxygen tension, which provides better tissue oxygenation (Misrahy et al., 1962). The observation that it is altered under pathophysiological conditions such as hypertension provides evidence of a protective effect (Stansberry et al., 1996). Vasomotion is associated with oscillations of membrane potential and cytosolic Ca2+ concentration (Gustafsson et al., 1993; Peng et al., 1998). The Ca2+ oscillations begin as asynchronous oscillations without the generation of tension before being entrained during agonist stimulation into synchronized oscillations which underlie the rhythmical contractions (Miriel et al., 1999; Peng et al., 2001). These oscillations result in the release of Ca2+ from IP3 stores in all forms of rhythmicity studied to date (Mauban et al., 2001; Peng et al., 2001; Haddock and Hill, 2002; Mauban and Wier, 2004). A model has been proposed by Aalkjaer and colleagues where in the presence of the endothelium, which produces a background level of NO to raise the level of cyclic guanosine monophosphate (cGMP), the periodic rise in [Ca2+]i activates cGMP-dependent, Ca2+-sensitive Cl- channels. These Cl- currents cause periodic depolarizations, which rapidly spread between smooth muscle cells (connected through gap junctions) to activate L-type Ca2+ channels that play a much more prominent role in small rather than large vessels (Rahman et al., 2005). The synchronization of Ca2+ oscillations is made possible as a result of cell-to-cell coupling via gap junctions (Christ et al., 1996; Koenigsberger et al., 2004), which in smooth muscle cells, the predominant connexin subtypes within gap junctions are Cx40 and Cx43 (Li and Simard, 1999). The spreading of electrical ions synchronizes oscillations in membrane potential, thereby oscillating Ca2+ influx through L-type Ca2+ channels, leading to the synchronized contractions associated with vasomotion (Peng et al., 2001; Aalkjaer and Nilsson, 2005). The resultant periodic Ca2+ influx facilitates Ca2+-induced Ca2+ release to initiate the next Ca2+ wave, which will then occur simultaneously in all the nearby  29  smooth muscle cells and generate oscillatory vasoconstriction (Peng et al., 2001, Aalkjaer and Nilsson, 2005; Rahman et al., 2005). The endothelium may also play an important regulatory role in vasomotion, particularly though the myoendothelial gap junctions which connect the endothelial cells to smooth muscle cells (Griffith et al., 2004; Rummery and Hill, 2004). These gap junctions consist of connexin proteins, enabling electrical coupling between the endothelium and smooth muscle cell layer, facilitating the synchronization of vasomotor activity (Haefliger et al., 2004; Haddock et al., 2006). The loss of connexin Cx40 has been associated with irregular vasomotion (de Wit et al., 2003), and myoepithelial gap junction inhibitors can prevent vasomotion in a number of vascular beds (Chaytor et al., 1997; Hill et al., 1999; Bonnet et al., 2001). Finally, myoendothelial gap junctions may also provide the pathway for release factors such as endothelial-dependent hyperpolarizing factor to modulate the contractile activity of the smooth muscle cells (Dora et al., 2003).  1.4 Marfan syndrome Marfan syndrome is an autosomal dominant disorder caused by mutations in the gene encoding for fibrillin-1, affecting multiple organ systems including cardiovascular, skeletal, ocular, and pulmonary, with a prevalence of around 2-3 in 10,000 individuals (Dietz et al., 1991; Pyeritz, 2000; Judge and Dietz, 2005). The clinical signs of Marfan syndrome were first described in 1875, although the disease was named for pediatrician Antoine-Bernard Marfan, who evaluated a 5-year-old patient in 1896 and described disproportionately long, thin limbs, narrow skull, tall stature, and long, slender digits. Upon reviewing more cases, Marfan also recognized Mendelian inheritance with co-segregating malfunction of mitral valve, congenital  30  displacement of the lens and excessively long limbs (Pyeritz, 2000; Judge and Dietz, 2005; Chaffins, 2007; Judge and Dietz, 2008). The involvement of the aorta was first described in 1943, and in 1955, the extent of cardiovascular abnormality in aortic dilatation and dissection, as well as aortic valve regurgitation, was documented (Judge and Dietz, 2008).  1.4.1 Clinical manifestations and diagnostic criteria Marfan syndrome is a pleiotropic disorder, with diverse manifestations in different organ systems resulting from one single mutation. The clinical presentations and severity of the disorder are different depending on the location of the mutation and expressivity in each individual. Due to the lack of genetic heterogeneity, diagnosis of Marfan syndrome is based on clinical features rather than molecular testing (Pyeritz, 2000; Judge and Dietz, 2005; Judge and Dietz, 2008). The first standard (Berlin nosology) for the diagnosis of Marfan syndrome was proposed in 1986, and the criteria focused on the three most prominent organ systems: the skeleton, eyes, and heart and aorta. In 1995, a revision (Ghent nosology) was proposed which recognized family history, included other organ systems, and placed greater emphasis on the skeletal findings. Moreover, the Ghent nosology contained classifications with more stringent and explicit criteria, helping to solve the problem of overdiagnosis or misdiagnosis with the Berlin nosology (Judge and Dietz, 2005; Chaffins, 2007). The skeletal features, mainly caused by the disproportionate overgrowth of the long bones, are the most striking and immediately evident manifestations of Marfan syndrome. The overgrowth gives rise to abnormally tall stature, arachnodactyly, dolichocephaly, and elongation of limbs which leads to an arm span greater than 1.05 times the height. Another prominent feature is scoliosis or kyphoscoliosis caused by vertebral deformities. Anterior chest deformity  31  can also result from overgrowth of the ribs (Gray and Davies, 1996; Judge and Dietz, 2005). The main manifestations in the ocular system include severe myopia and lens dislocation, while in the pulmonary system, the most frequently occurring presentation is spontaneous pneumothorax caused by widening of the distal air spaces. Restrictive lung disease is exacerbated in patients with severe chest deformity (Pyeritz, 2000). The skin can also be affected, with the presentation of “stretch marks.” Finally, another common manifestation is dural ectasia, an abnormal protrusion of dural membranes (Gray and Davies, 1996; Judge and Dietz, 2005). Cardiovascular complications are the major cause of morbidity and mortality in patients with Marfan syndrome (Ammash et al., 2008). In the heart, the mitral valves are often affected, which may progress to mitral valve prolapse and in some cases leads to mitral valve regurgitation (Gray and Davies, 1996). However, the most life-threatening manifestations are aortic dilatation and aneurysm, which could result in aortic rupture (Pyeritz, 2000; Judge and Dietz, 2005). Due to their proximity to the heart, aortic root and ascending aorta withstand the highest hemodynamic stress and are the most susceptible to dissection and dilatation (Gray and Davies, 1996). The changes in the walls of the elastic arteries (e.g. fragmentation and disarray of elastic fibers, a paucity of smooth muscle cells, separation of muscle fibers by collagen and mucopolysaccaride) are primarily in the media layer and result in the observed decrease in distensibility and increase in stiffness in the aorta (Pyeritz, 2000). In the clinical setting, pulse wave velocity, which is increased in patients with Marfan syndrome, is a well-established parameter to measure aortic wall stiffness; furthermore, an increase in stiffness is a marker for aortic dilatation and susceptibility to aortic rupture (Hirata et al., 1991; Marque et al., 2001; Vitarelli et al., 2006).  32  1.4.2 Fibrillin, microfibrils and elastic fibers Fibrillin assemblies (microfibrils) serve two key physiological functions: the function of a structural support that imparts tissue integrity and the function of a regulator of signaling events that instruct cellular performance (Ramirez et al., 2004; Hubmacher et al., 2006). Microfibrils are the product of the head-to-tail polymerization of fibrillin molecules with the addition of other proteins (Arteaga-Solis et al., 2000; Ramirez and Dietz, 2007). Microfibrils, without association with elastin, form fibrous aggregates to link different constituents of the extracellular matrix and hold tissue components in place (Ramirez et al., 1999). The head-to-tail polymerization gives rise to the bead-to-bead structure with extendibility and flexibility. Fibrillin, a 350kD connective tissue glycoprotein, is widely distributed in connective tissue matrices of skin, lung, kidney, vasculature, cartilage, tendon, muscle, cornea, and ciliary zonule (Sakai et al., 1986), most of which are later found to be affected in patients with Marfan syndrome. A mutation in the FBN1 gene, which contains 110kb with 56 exons and 10kb of coding sequence (Gray and Davies, 1996) and encodes for the fibrillin-1 protein, was later discovered to be responsible for the classic Marfan syndrome by Dietz and colleagues (Dietz et al., 1991; Pyeritz, 2000; Chaffins, 2007) Fibrillin proteins are mainly composed of Ca2+-binding epidermal growth factor-like (cbEGF) domains interspersed with domains with homology to transforming growth factor-β (TGF-β) binding proteins or unique cysteine-rich EGF-TGF hybrid domains (Fig 1.5) (Kielty and Shuttleworth, 1995; Arteaga-Solis et al., 2000; Kielty et al., 2002). The cbEGF repeats have six crucial cysteine residues that are vital for disulphide bonding to form stable β-sheets (Gray and Davies, 1996). Ca2+ binding sites within the cbEGF domains are also important for stabilizing cbEGFs into a linearly rigid structure, mediating fibrillin monomer interactions and lateral  33  packing of microfibrils, and organizing the macroaggregates and protecting them against proteolysis (Kielty and Shuttleworth, 1995; Ramirez et al., 1999; Arteaga-Solis et al., 2000). In the most common form (Type I or “Classic”) of Marfan syndrome, the mutation occurs in the cbEGF domain and thus reduces the Ca2+-binding affinity of fibrillin-1. The deficiency of Ca2+binding results in microfibril instability and increased susceptibility to proteolytic degradation by proteases such as matrix metalloproteinases, elastase, and thrombin (Kielty and Shuttleworth, 1995; Ramirez et al., 1999; Williams et al., 2008). In addition to microfibrillar aggregates, the fibrillin-rich microfibrils also participate in the elastic fibrillogenesis by acting as a template upon which tropoelastin (precursor of mature elastin) is deposited (Kielty et al., 2002). With the elastic and stretchable outer microfibrillar mantle and inner cross-linked elastin core, mature elastic fibers are organized into tissue-specific structures that reflect the mechanical demands of each system (Kielty et al., 2002; Ramirez and Dietz, 2007). For example, the loosely organized network of microfibrils and elastic fibers confers pliability in the skin (Ramirez and Dietz, 2007).  Figure 1.5: Structure of fibrillin-1 protein (Boileau et al., 2005).  34  1.4.3 Molecular genetics and pathophysiology Marfan syndrome is an autosomal dominant disorder with high penetrance but variable expressivity (Dietz et al., 1991). For classic Marfan syndrome, linkage analyses have mapped the locus to chromosome 15q21.1, where the gene encoding fibrillin-1 is located (Dietz et al., 1991; Pyeritz, 2000; Boileau et al., 2005; Judge and Dietz, 2005). Approximately 66 to 75% of people with Marfan syndrome inherited the disorder from their parents; however, 25% of the patients have de novo mutations (Chaffins, 2007). Currently, over 600 genetic mutations have been identified (Williams et al., 2008), which can be divided into two classes: nonsense and missense. Nonsense mutations account for 38.6% of the mutations and result in premature termination codons and shortened fibrillin-1 molecule; The severity of the disorder is determined by the quantity of mutant mRNA transcripts and the percentage of truncated proteins incorporated into microfibrils. Missense mutations are more common and account for 60% of the mutations. Moreover, 78% of the point mutations locate in the cbEGF modules and affects mostly the cysteine residues or amino acids involved in Ca2+ binding. Of the point mutations, 12% are recurrent and affect a region of the DNA where a cytosine nucleotide occurs next to a guanine nucleotide in the linear sequence of bases along its length (CpG) (Gray and Davies, 1996; Boileau et al., 2005). The CpG is a noted mutational hotspot, constituting the single most frequent mutational effect, and may suggest that mutations that cause Marfan syndrome could be truly recurrent (Ollila et al., 1996). Both haploinsufficiency and dominant negative mechanism, in which abnormal protein interacts and interferes with normal proteins, have been shown to be responsible for disease pathogenesis (Judge et al., 2004; Judge and Dietz, 2008). Haploinsufficiency for the wild-type protein could bring the amount of fibrillin-1 down to the threshold, resulting in phenotypic  35  consequences (Ramirez and Dietz, 2007). Furthermore, the mutant fibrillin-1 with dominantnegative potential or the recruitment of inflammatory cells by fibrillin-1 degradation products can promote progressive loss of fibrillin-1 with increased proteolytic clearance (Judge et al., 2004; Ramirez and Dietz, 2007). All the above-mentioned processes could lead to the loss of fibrillin-1 in the extracellular matrix and thus a breakdown of tissue integrity. Recently, an upregulation of TGF-β signaling has also been shown to be closely associated with disease progression and responsible for the pathogenesis of Marfan syndrome (Judge and Dietz, 2005; Habashi et al., 2006). Microfibrils containing fibrillin-1 have been shown to not only have structural function but also regulate the signaling pathway of TGF-β, a cytokine that regulates cell proliferation and migration, as well as tissue development (Judge and Dietz, 2005; Lacro et al., 2007). TGFβ is synthesized and secreted as a large latent complex which is composed of three proteins: the latent TGF-β binding protein (LTBP), the active form of TGF-β and the latency-associated protein (LAP) (Matt et al., 2008). The latter two are associated and sequestered by LTBP which provides safe harbour for TGF-β before its release (Byers, 2004). Fibrillin-1 shares a high degree of homology with the LTBP, and indeed, the LTBP of the large latent complex of TGF-β localizes to the microfibrils and interacts directly with fibrillin-1 (Judge and Dietz, 2005; Lacro et al., 2007). Therefore, it has been suggested that some clinical manifestations of Marfan syndrome may result from the failure of latent complex sequestration and subsequent excessive TGF-β release and downstream signaling.  1.4.4 Marfan syndrome and vasomotor function Marfan syndrome is associated not only with extensive degeneration of elastic fibers, but also with endothelial dysfunction and reduction of smooth muscle contractility in the vasculature  36  (Chung et al., 2007a,b). The alteration of the structural integrity of elastic fibers leads to reduced distensibility and elasticity (Bunton et al., 2001). Furthermore, alteration of fibrillin-1 may also disrupt the attachment of elastic fibers to the cells in the endothelial layer and impair endothelial permeability (Davis, 1994; Sheremet’eva et al., 2004). Although elastic fiber composition is gradually reduced along the arterial tree, elastin remains an important determinant of passive mechanical properties in mesenteric arteries (Dobrin, 1978; Milnor, 1989; Mulvany and Aalkjaer, 1990; Briones et al., 2003; Gonzalez et al., 2005). However, little is known about how Marfan syndrome affects vessel elasticity and vasomotor function in the resistance vasculature, although dysfunction of these vessels may have important clinical consequences. For example, aneurysms in peripheral and resistance vessels have been reported in patients with Marfan syndrome (Savolainen et al., 1993; Hatrick et al., 1998; Goffi et al., 2000; Lay et al., 2006), though no clear link has been established between resistance artery dysfunction and aortic dilatation and rupture (Jondeau et al., 1999). Furthermore, maximum forearm blood flow in response to acetylcholine is reduced in patients with Marfan syndrome (Nakamura et al., 2000), and impairment in flow-mediated vasodilation is also observed (Wilson et al., 1999). Marfan syndrome has been associated with decreased smooth muscle contractility in the aorta (Chung et al., 2007b). No mechanisms have been proposed, although reduced active force may be due to low intrinsic force generation of the contractile filaments or modifications in the coupling between the contractile elements and the cytoskeleton in smooth muscle cells (Rembold and Murphy, 1990). Additionally, decreased association between smooth muscle cells and elastic fibers would reduce the strain on the smooth muscle cells and blunt their response to agonist stimulation (Bunton et al., 2001). Furthermore, upregulation of matrixmetalloproteinase-2 and  37  matrixmetalloproteinase-9 in Marfan syndrome may inhibit Ca2+ entry from the extracellular space and reduce vessel contraction (Chew et al., 2004; Chung et al., 2007b; Chung et al., 2008), although further investigation is required to elucidate possible involvement of Ca2+ signaling and myofilament contractile mechanisms.  1.4.5 Mouse models of marfan syndrome Several mouse models with different mutations in the Fbn1 gene have been developed, and many of these mouse lines display typical manifestations of Marfan syndrome, including aortic root aneurysm, mitral valve thickening, lung emphysema, and long-bone overgrowth (Dietz and Mecham, 2000; Ramirez et al., 2004). A common mouse model of Marfan syndrome used experimentally is heterozygous for a cysteine substitution (C1039G) in the cbEGF-like domain in Fbn1 (Fbn1C1039G/+), the most common class of mutation observed in classic Marfan syndrome (Habashi et al., 2006; Judge et al., 2004; Ng et al., 2004). This mutant transgene harbors a naturally occurring human mutation (C1663R). In a patient with the missense mutation, normal synthesis of fibrillin-1 was observed; however, fibrillin-1 deposition was impaired (Judge et al., 2004). Similarly, in murine cells heterozygous for the mutation (C1039G), histological examination consistently demonstrated a reduction in the deposition of microfibrils (Judge et al., 2004). In addition to histological similarities, the Fbn1C1039G/+ mouse model also demonstrates similar clinical manifestations which are common to Marfan patients. For example, there is progressive deterioration of the aortic wall with elastic fiber fragmentation and disarray of vascular smooth muscle cells which eventually led to aortic dilatation (Judge et al., 2004). Furthermore, skeletal deformities common in Marfan syndrome, including kyphosis and overgrowth of the ribs, are also observed in this model.  38  A Control  Marfan  2 cm  B  Control  Marfan 0.25 cm  Figure 1.6: Mouse model of Marfan syndrome. A. The heterozygous (Fbn1C1039G/+) mouse, the mouse model used in this study, has phenotypes (e.g. kyphoscoliosis) similar to those of the patients with Marfan syndrome. B. The mouse carrying the Fbn1C1039G/+ mutation (Marfan mouse) developed an aneurysm in the aortic arch which was absent in the control. 1.5 Summary of proposed research objectives My projects have sought to better understand Ca2+ homeostasis and signaling using both cultured vascular smooth muscle cells and intact tissue. I had 3 specific aims:  ¾ To provide evidence for functional coupling of receptor-operated non-selective cation channels and the Na+/Ca2+ exchanger (NCX) in rat aortic smooth muscle cells, I: •  transfected cells with aequorin targeted to mitochondrial matrix to directly measure mitochondrial Ca2+ levels upon NCX reversal, with our lab having previously demonstrated that mitochondria buffer NCX-mediated Ca2+ entry.  39  •  determined whether Ca2+ entry through NCX reversal, stimulated by removal of extracellular Na+, was increased following stimulation with ATP and the diacylglycerol analog 1-Oleoyl-2-acetyl-sn-glycerol, both activators of receptor-operated channels.  ¾ To investigate the mechanism of agonist-stimulated Ca2+ waves and determine their relationship with tonic contraction in rat basilar artery smooth muscle, I: •  measured intracellular Ca2+ levels loaded smooth muscle cells with the Ca2+-sensitive dye Fluo-4AM and measured tonic contraction using a small vessel myograph.  •  characterized the properties of uridine-5’-triphosphate (UTP)-stimulated Ca2+ waves and contraction and measured the effect of various pharmacological inhibitors (nifedipine, SKF-96365, KBR-7943, cyclopiazonic acid, 2-APB, ryanodine, tetracaine).  ¾ To determine whether stiffness, vasomotor function, and Ca2+ signaling were affected in mesenteric resistance vessels in a mouse model of Marfan syndrome, I: •  used an accepted mouse model (Fbn1C1039G/+) of Marfan syndrome, with their littermates as controls, at 3, 6, and 10 months of age.  •  characterized vessel elasticity, smooth muscle cell contraction, calcium signaling, and endothelium-dependent and endothelium-independent relaxation using various pharmacological agents.  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Proc Natl Acad Sci U S A 90: 6295-9.  61  CHAPTER 2 - ATP PROMOTES NCX-REVERSAL IN AORTIC SMOOTH MUSCLE CELLS BY DAG-ACTIVATED NA+ ENTRY1 2.1 Introduction The Na+/Ca2+ exchanger (NCX) is important in maintaining Ca2+ homeostasis in vascular smooth muscle, operating both in the forward (Ca2+-efflux) and reverse (Ca2+-influx) modes (Blaustein and Lederer, 1999; Brini et al., 2002). NCX reversal has been shown by our laboratory and others to be responsible for Ca2+ entry following agonist stimulation in different cell types (Lee et al., 2001; Takai et al., 2004; Zhang et al., 2005; Poburko et al., 2006). Furthermore, reverse-mode NCX contributes to increased vascular tone and may be important in salt-sensitive hypertension (Iwamoto et al., 2005). Smooth muscle cell contraction depends on the regulation of cytosolic Ca2+. Following stimulation of phospholipase C (PLC)-linked receptors, the Ca2+ profile in the cytosol is characterized by a transient increase due to release of sarcoplasmic reticulum (SR) Ca2+ stores, followed by a sustained plateau due to Ca2+ influx across the plasma membrane (PM). The transient phase is due to the production of inositol-1,4,5-triphosphate (IP3) and the resulting opening of IP3-sensitive channels (IP3R) on the SR, while the sustained phase is due to the opening of receptor-operated channels (ROCs) and L-type voltage-gated Ca2+ channels. While the identity and mechanism of activation of ROCs remains to be fully elucidated, members of the transient receptor potential channel (TRP) family, especially from the ‘‘canonical’’ subfamily (TRPC), form non-selective cation channels (NSCCs) with many of the same properties as ROCs (Gudermann et al., 2004).  1  A version of this chapter has been published. Syyong HT, Poburko D, Fameli N, van Breemen C (2007). ATP promotes NCX-reversal in aortic smooth muscle cells by DAG-mediated Na+ entry. Biochem Biophys Res Commun. 357: 1177-1182.  62  Agonist-induced NCX reversal has been suggested to be due in part to localized elevation of Na+ at the subplasmalemmal junctions where both the NSCC and NCX are thought to co-localize (Arnon et al., 2000). Previous studies support both physical and functional coupling of TRPC3 with NCX in HEK-293 cells (Rosker et al., 2004). TRPC6 is closely related to TRPC3, and in rat aortic smooth muscle cells TRPC6 is expressed to greater levels than TRPC3 (Soboloff et al., 2005; Maruyama et al., 2006). TRPC6 is activated by diacylglycerol (DAG) and forms a NSCC with a Na+:Ca2+ permeability ratio of ~1:5 (Inoue et al., 2001; Estacion et al., 2006). Our laboratory has recently provided the first evidence of functional coupling between TRPC6 and reverse-mode NCX in rat aortic smooth muscle cells (Lemos et al., 2007). Following up on these findings, we now provide further evidence for functional coupling of TRPC6 and reverse-mode NCX by demonstrating that NCX reversal is potentiated following stimulation of rat aortic smooth muscle cells with ATP, which we know to elevate intracellular [Na+] (Poburko et al., 2006). We used mitochondria-targeted aequorin to monitor mitochondrial Ca2+ as an indirect, but localized, measure of NCX reversal in cultured rat aortic smooth muscle cells. Aequorin is a powerful tool used to measure rapid changes in cellular [Ca2+] and can be targeted to organelles such as the mitochondria to measure [Ca2+] (Rizzuto et al., 1992). Due to the close spatial association with the PM and superficial SR, a sub-population of mitochondria play an important role in Ca2+ homeostasis and can be stimulated to take up Ca2+ entry mediated by reverse-mode NCX following purinergic receptor stimulation; thereby buffering Ca2+ influx and preventing rapid diffusion throughout the cytosol (Szado et al., 2003; Dai et al., 2005; Poburko et al., 2006).  63  2.2 Methods 2.2.1 Smooth muscle cell culture Rat aortic smooth muscle cells were cultured as described previously (Szado et al., 2003; Poburko et al., 2006). Briefly, cells were stored in 90% DMEM / 10% DMSO in liquid nitrogen and used between passages 8 and 12. Cells were incubated at 37°C in a humidified atmosphere of 5% CO2. For experiments, cells were plated on 13 mm Thermanox coverslips (Nunc, Life Technologies).  2.2.2 Expression of aequorin Rat aortic smooth muscle cells were transiently transfected with a pcDNAI expression vector encoding apo-aequorin containing the amino terminal targeting sequence for human cytochrome oxidase VIII (mitoaequorin). Cells were allowed to grow on cover slips for 1 day before being washed with Ca2+/Mg2+ free phosphate-buffered saline (PBS) that was replaced with 500 μL of DMEM (Dulbecco’s modified Eagle’s media (Sigma, D1152), 10% FCS, 100 U/mL penicillin G, 100 μg/mL streptomycin, MEM vitamin solution, and MEM essential and non-essential amino acid solution) before transfection. Cells were transfected using TransFectin (Bio-Rad) as per manufacturer’s instructions (1 μg DNA per coverslip and a lipid:DNA ratio of 1:1) and were used for experiments the next day.  2.2.3 Measurement of mitochondrial [Ca2+] Mito-aequorin was reconstituted in coelenterazine (5 μM) in serum-free DMEM for 2–4 h before experiments. The coverslip was held in a 0.5-ml chamber heated constantly at 37 °C and was placed 5 mm from the photon detector. Cells were superfused at 1 mL/min with physiological  64  salt solution (PSS, in mM: NaCl 145, KCl 5, MgCl2 1, 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES) 5, glucose 10, and CaCl2 1.2, pH 7.6). For 0Na+–PSS, Na+ was replaced with 145 mM N-methyl-D-glucamine. Luminescence was detected by photomultiplier tubes (EMI 9789 and P25232, Electron Tubes Inc., USA) and photon emission was recorded at 1 Hz with EM6 photon-counting software (Electron Tubes Inc., USA). The EMI 9789 was coupled to an AD6 analog-digital converter and a CT-2 counting module (Electron Tubes Inc., USA), while the P25232 is a self-contained photon counting system. The offlinecalibration of the photon emission to [Ca2+] was performed as previously described using the method of Allen and Blinks (Allen et al., 1977; Allen and Blinks, 1978; Brini et al., 1995; Szado et al., 2003).  2.2.4 Experimental protocol Rat aortic smooth muscle cells were initially superfused with PSS for 2 min, followed by 0Na+–PSS for 3 min, 9 min with PSS (recovery period), 0Na+–PSS for 3 min, and finally, PSS for 3 min. At the end of the protocol, cells were permeabilized with digitonin (100 μM) and then exposed to 10 mM CaCl2 to determine total aequorin expression. The degree of recovery of NCX reversal was measured by taking the ratio of the two 0Na+–PSS mediated [Ca2+]MT peaks (0Na2/0Na1 ratio). To determine the effects of ATP (1 mM) or the diacylglycerol analog 1Oleoyl-2-acetyl-sn-glycerol (OAG, 100 μM) on the degree of recovery of NCX reversal, agonists were superfused (for 3 min) 3 min after the first 0Na+–PSS stimulation. To examine the effects of SKF-96365 (50 μM), a receptor-operated channel (ROC) antagonist, phorbol ester 12tetradecanoylphorbol- 13 acetate (PMA, 1 μM), a direct activator of protein kinase C (PKC), or bisindolylmaleimide I (BIM, 500 nM), an inhibitor of PKC, these drugs were added for 2 min (1  65  min after the first 0Na+–PSS stimulation) prior to the application of ATP or OAG and continuation of the regular protocol.  2.2.5 Chemicals All drugs were dissolved in DMSO to make stock solutions, except for ATP (distilled water). Drugs were then diluted to the appropriate concentration in PSS. 1-Oleoyl-2-acetyl-snglycerol (OAG) and bisindolylmaleimide I (BIM) were obtained from Calbiochem (San Diego, CA, USA). ATP, SKF-96365, and PMA were obtained from Sigma (St. Louis, MO, USA).  2.2.6 Statistical analysis Values are expressed as means ± standard error (SEM). Means were compared using the most robust test appropriate to each experimental design. Groups of three or more means were compared by ANOVA with pair-wise comparisons made by Bonferroni post hoc tests. Traces are representative of at least six independent experiments performed in duplicate. Data were compiled and analyzed using GraphPad Prism 4.0, in coordination with Microsoft Excel. NCSS was used to perform statistical tests.  2.3 Results 2.3.1 Mitochondrial Ca2+ uptake following 0Na+–PSS stimulation is due to NCX reversal NCX reversal induced by 0Na+–PSS results in an initial rapid increase in mitochondrial Ca2+ ([Ca2+]MT), which diminishes and is followed by a plateau phase (Fig. 2.1A). The transient increase is due to Ca2+-influx following reversal of the NCX, while the plateau is due to  66  inhibition of Ca2+ extrusion by blockade of forward-mode NCX (Poburko et al., 2006). The 0Na2/0Na1 ratio is dependent on both the recovery period (time between 0Na+ and PSS stimulations) as well as the duration of the 0Na+–PSS stimulation. During 3 or 5 min 0Na+–PSS stimulation, the 0Na2/0Na1 ratio is significantly smaller after 1 min of recovery compared to the 0Na2/0Na1 ratios after 3 or 9 min recovery, which does not significantly differ from each other (Fig. 2.2B). However, if 0Na+–PSS stimulation is limited to 30 s, which is still sufficient to reach peak [Ca2+]MT, then the 0Na2/0Na1 ratio is equivalently recovered after 1 or 3 min between stimulations (Fig. 2.1B).  30s stimulation 3 min stimulation 5 min stimulation  Figure 2.1: Recovery kinetics of 0Na+-PSS stimulation in mitochondria-targeted aequorin. A. The amplitude of the second 0Na+-mediated [Ca2+]MT peak (0Na2) was typically smaller than the first [Ca2+]MT peak (0Na1). B. The 0Na2/0Na1 ratio, indicating the degree of NCX reversal recovery, is dependent on the time of stimulation with 0Na+PSS. The 0Na2/0Na1 ratio is not significantly different after 1 or 3 min of recovery (time between consecutive 30 s 0Na+ stimulations). However, the 0Na2/0Na1 ratio is significantly lower after 1 min recovery (time between consecutive 3 or 5 min 0Na+ stimulations) compared to 3 and 9 min recovery time. Data were compared by one-way ANOVA, Bonferroni pairwise post-test. * P < 0.05, *** P < 0.001. 67  2.3.2 NCX reversal is increased upon purinergic receptor stimulation but inhibited by antagonists of NSCCs Stimulation of rat aortic smooth muscle cells with ATP (1 mM) between consecutive 0Na+– PSS stimulations always increased the second [Ca2+]MT peak compared to the first [Ca2+]MT peak (Fig. 2.2A). This is reflected in the increased 0Na2/0Na1 ratio compared to control (Fig. 2.2C). Furthermore, the DAG analog, 1-Oleoyl-2-acetyl-sn-glycerol (100 μM, OAG), similarly increased the 0Na2/0Na1 ratio (Fig. 2.2C). Based on reports that DAG can activate members of the TRPC family (Venkatachalam et al., 2003), we hypothesized that PLC-mediated DAG generation increases Na+ entry into the cells by opening NSCCs and thereby enhances NCX reversal upon removal of extracellular Na+. To test this hypothesis by inhibiting NSCCs, we applied SKF-96365 (50 μM) to the rat aortic smooth muscle cells prior to stimulation with ATP or OAG. SKF-96365 inhibited the ATP and OAGmediated increases in NCX reversal, but did not directly affect the 0Na2/0Na1 ratio (Fig. 2.2B,D).  68  Figure 2.2 SKF-96365 (SKF) attenuates ATP- and 1-Oleoyl-2-acetyl-sn-glycerol (OAG)-induced increase in NCX reversal. A. Representative trace when cells are stimulated with ATP (1 mM) between consecutive 0Na+ stimulations, showing a greater amplitude of the second 0Na+–PSS mediated [Ca2+]MT peak compared to the first. B. Representative trace when cells are incubated with SKF (50 μM) for 2 min prior to stimulation with ATP between consecutive 0Na+ stimulations, showing a reduced amplitude of the second 0Na+–PSS mediated [Ca2+]MT peak compared to A. C. Stimulation with ATP or OAG (100 μM) between consecutive 0Na+ stimulations significantly increases the 0Na2/0Na1 ratio. D. The increase in the 0Na2/0Na1 ratio due to ATP and OAG is attenuated in the presence of SKF (50 μM). Data were analyzed using paired t-tests. P-values are shown for specific comparisons. * P < 0.05. 2.3.3 Protein kinase C has an inhibitory effect on NCX reversal To determine the possible involvement of PKC in the purinergically activated cascade, rat aortic smooth muscle cells were pre-treated with the PKC inhibitor bisindolylmaleimide I (BIM, 500 nM, Fig. 2.3A,C). Pre-treatment of the rat aortic smooth muscle cells with BIM prior to ATP stimulation had no significant effect on the 0Na2/0Na1 ratio. When rat aortic smooth muscle cells 69  were pre-treated with PMA (1 μM) to activate PKC prior to ATP stimulation, the 0Na2/0Na1 ratio was significantly decreased (Fig. 2.3B,D).  Figure 2.3 Role of protein kinase C (PKC) in ATP-stimulated NCX reversal. A. Representative trace when cells are pre-incubated with bisindolylmaleimide I (BIM, 500 nM) for 2 min prior to stimulation with ATP (1 mM) between consecutive 0Na+ stimulations. B. Representative trace when cells preincubated with 12tetradecanoylphorbol-13 acetate (PMA, 1 μM) for 2 min prior to stimulation with ATP (1 mM) between consecutive 0Na+ and PSS stimulations. C. Inhibition of PKC with BIM (500 nM) alone or during stimulation with ATP (1 mM) does not change the 0Na2/0Na1 ratio. D. Direct activation of PKC by PMA (1 μM) alone does not change the 0Na2/0Na1 ratio, although PMA application prior to ATP stimulation (1 mM) significantly decreases the 0Na2/0Na1 ratio. Data were analyzed using paired t-tests. P-values are shown for specific comparisons. *** P < 0.001. 2.4 Discussion Acute removal of extracellular Na+ causes transient reversal of the NCX, resulting in Ca2+ entry, by reversing the plasmalemmal Na+-gradient (Poburko et al., 2006). The Ca2+ entry by  70  reverse-mode NCX eventually declines as the intracellular [Na+] decreases, explaining the transient nature of the mitochondrial Ca2+ response to 0Na+–PSS. Based on the observation that recovery of the 0Na+–PSS response is stabilized after 1 min following a 30 s stimulation, while requiring 3 min following 3 or 5 min stimulation, we concluded that the reduced response to a second 0Na+ stimulation was due to depletion of intracellular Na+, rather than the NCX being directly inhibited by Ca2+, as has been suggested (Opuni and Reeves, 2000). Diacylglycerol (DAG) has been shown to activate TRPC6 channels following agonist stimulation of G-protein coupled receptors (Estacion et al., 2006). In these smooth muscle cells, ATP activates metabotropic P2Y, G-protein coupled receptors resulting in the production of IP3 and DAG (Szado et al., 2003). Furthermore, ATP stimulation has also been demonstrated to increase cytosolic [Na+], which promotes NCX reversal (Poburko et al., 2006). Having previously shown that TRPC6 is expressed in these cells (Poburko et al., 2004) and that its activation is essential to agonist-induced Ca2+ entry via reverse-mode NCX (Lemos et al., 2007), our current findings suggest that DAG provides the stimulatory link between purinergic receptor activation and the opening of TRPC6-containing ROCs. Although DAG is an activator of TRPC6, DAG and its analogs are also well-known activators of protein kinase C (PKC) (Go et al., 1987, Lee and Severson, 1994; Albert and Large, 2004). PKC in turn has been reported to inhibit TRPC6 (Venkatachalam et al., 2003; Estacion et al., 2006). The IC50 of BIM for PKC subtypes a, b, and c (the most common Ca2+-dependent PKC subtypes) is reported to range from 16 to 20 nM, so 500 nM should completely inhibit PKC activity (Toullec et al., 1991; Albert et al., 2003). This suggests that PKC does not directly regulate the activation of NSCCs by ATP in these cells and is consistent with previous findings showing that PKC does not activate these channels (Venkatachalam et al., 2003). While PKC has  71  been reported to directly activate Ca2+ influx via reverse-mode NCX (Aiello et al., 2005), such an effect is unlikely under these conditions given the PMA-mediated decrease in the 0Na2/0Na1 ratio. Rather, our present results are consistent with recent observations demonstrating that OAG-induced cation entry mediated by TRPC6 channels is not activated by PKC, but rather can be inhibited by PKC if it is activated prior to stimulation with OAG or ATP (Venkatachalam et al., 2003). Our current results raise the question of why ATP stimulation results in a net increase in NCX reversal in response to 0Na+–PSS if DAG activates both TRPC6 and PKC. One simple explanation is that PKC activation during ATP stimulation is not sufficient to inhibit TRPC6, which is consistent with the lack of observable effects of BIM. On the other hand, two different PLC pathways might be activated upon ATP stimulation: one of which directly activates TRPC6, while the other activates PKC, as has been suggested in previous studies of TRPC6-like channels (Toullec et al., 1991; Lee and Severson, 1994; Albert et al., 2003; Albert and Large, 2004). In our system, ATP stimulation may not recruit the appropriate PKC isoforms to the cell membrane, while the application of the high concentration of PMA likely activates all PKC isoforms. However, further experiments are required to clarify whether and/or where PKC is activated during ATP stimulation in these cells. In conclusion, this study adds to our recently published work by providing further mechanistic insight into the functional linkage between reverse-mode NCX and TRPC6. We show that agonist-stimulated production of DAG is important to the increased Na+ entry that facilitates the reversal of NCX, which is an essential component of the agonist-mediated Ca2+ entry in rat aortic smooth muscle cells. We also show that Na+ entry can be inhibited by PKC activation, but that this effect is not sufficiently prominent during purinergic stimulation to abolish the stimulatory effect of DAG.  72  2.5 References Aiello EA, Villa-Abrille MC, Dulce RA, Cingolani HE, Pérez NG (2005). Endothelin-1 stimulates the Na+/Ca2+ exchanger reverse mode through intracellular Na+ (Na+i)-dependent and Na+i-independent pathways. Hypertension 45: 288-93. Albert AP, Piper AS, Large WA (2003). Properties of a constitutively active Ca2+-permeable non-selective cation channel in rabbit ear artery myocytes. J Physiol 549: 143-56. Albert AP, Large WA (2004). Inhibitory regulation of constitutive transient receptor potentiallike cation channels in rabbit ear artery myocytes. 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Feedback inhibition of sodium/calcium exchange by mitochondrial calcium accumulation. J Biol Chem 275: 21549-54. Poburko D, Lhote P, Szado T, Behra T, Rahimian R, McManus B, et al. (2004). Basal calcium entry in vascular smooth muscle. Eur J Pharmacol 505: 19-29. Poburko D, Potter K, van Breemen E, Fameli N, Liao CH, Basset O, et al. (2006). Mitochondria buffer NCX-mediated Ca2+-entry and limit its diffusion into vascular smooth muscle cells. Cell Calcium 40: 359-71. Rizzuto R, Simpson AW, Brini M, Pozzan T (1992). Rapid changes of mitochondrial Ca2+ revealed by specifically targeted recombinant aequorin. Nature 358: 325–327. Rosker C, Graziani A, Lukas M, Eder P, Zhu MX, Romanin C, et al. (2004). Ca(2+) signaling by TRPC3 involves Na(+) entry and local coupling to the Na(+)/Ca(2+) exchanger. J Biol Chem 279: 13696-704. Soboloff J, Spassova M, Xu W, He LP, Cuesta N, Gill DL (2005). Role of endogenous TRPC6 channels in Ca2+ signal generation in A7r5 smooth muscle cells. J Biol Chem 280: 39786-94. Szado T, Kuo KH, Bernard-Helary K, Poburko D, Lee CH, Seow C, et al. (2003). Agonistinduced mitochondrial Ca2+ transients in smooth muscle. FASEB J 17: 28-37.  74  Takai N, Yamada A, Muraki K, Watanabe M, Imaizumi Y (2004). KB-R7943 reveals possible involvement of Na(+)-Ca2+ exchanger in elevation of intracellular Ca2+ in rat carotid arterial myocytes. J Smooth Muscle Res 40: 35-42. Toullec D, Pianetti P, Coste H, Bellevergue P, Grand-Perret T, Ajakane M, et al. (1991). The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C. J Biol Chem 266: 15771-81. Venkatachalam K, Zheng F, Gill DL (2003). Regulation of canonical transient receptor potential (TRPC) channel function by diacylglycerol and protein kinase C. J Biol Chem 278: 29031-40. Zhang S, Yuan JX, Barrett KE, Dong H (2005). Role of Na+/Ca2+ exchange in regulating cytosolic Ca2+ in cultured human pulmonary artery smooth muscle cells. Am J Physiol Cell Physiol 288: C245-52.  75  CHAPTER 3 - MECHANISM OF ASYNCHRONOUS CA2+ WAVES UNDERLYING AGONIST-INDUCED CONTRACTION IN THE RAT BASILAR ARTERY2 3.1 Introduction Uridine 5’-triphosphate (UTP) is a potent constrictor of cerebral arteries which exerts its effects through purinergic P2Y receptors and the phospholipase C pathway (Urquilla, 1978; Strobaek et al., 1996; Horiuchi et al., 2001). Brain tissue is especially rich in UTP and cerebral vessels have greater reactivity to UTP compared with other vessels (Shirasawa et al., 1983; Hardebo et al., 1987). UTP may be involved in the regulation of cerebrovascular tone under both physiological conditions and pathophysiological reactions in disease states such as subarachnoid haemorrhage or migraine (Debdi et al., 1992; Boarder and Hourani, 1998; Burnstock, 1998). Smooth muscle contraction is initiated by an increase of intracellular Ca2+ ([Ca2+]i) from resting levels of ~100 nM to values up to 1 mM. In general, the [Ca2+]i profile following stimulation is biphasic, consisting of a rapid transient rise in [Ca2+]i from sarcoplasmic reticulum (SR) Ca2+ release followed by a plateau phase, which is mediated by Ca2+ entry from voltagegated Ca2+ channels and store/receptor-operated channels (van Breemen et al., 1978; Bolton, 1979; Streb et al., 1983; Putney, 1986). The advent of confocal microscopy has allowed the employment of physiological preparations to examine the Ca2+ signals in individual in situ vascular smooth muscle cells (VSMCs) of intact blood vessels. It has since become apparent that the average arterial wall [Ca2+]i observed previously is not representative of the Ca2+ signaling events within individual VSMCs, which are capable of generating Ca2+ signals with varying spatial and temporal patterns (Lee et al., 2002). Among these signals are Ca2+ waves, which are 2  A version of this chapter has been published. Syyong HT, Yang HH, Trinh G, Cheung C, Kuo KH, van Breemen C (2009). Mechanism of asynchronous Ca(2+) waves underlying agonist-induced contraction in the rat basilar artery. Br J Pharmacol 156: 587-600.  76  manifested as changes in [Ca2+]i which travel the length of VSMCs, and constitute a specialized form of agonist-induced Ca2+ signalling which appears to be involved in contractile regulation. Since 1994 when they were first described, Ca2+ waves have been observed in the smooth muscle fibres of a variety of intact blood vessels, including cerebral vessels (Iino et al., 1994; Asada et al., 1999; Miriel et al., 1999; Jaggar, 2001; Lee et al., 2001; Peng et al., 2001). Although there are likely to be underlying physiological reasons for signalling with Ca2+ waves (as opposed to steady state [Ca2+]i elevations), the mechanisms behind how Ca2+ waves within individual VSMCs signal for contraction remain poorly understood. Ca2+ waves in cerebral arteries can be induced by a variety of stimuli, including vasoconstrictor agonists such as UTP, pressure and alkaline pH (Jaggar and Nelson, 2000; Jaggar, 2001; Heppner et al., 2002). In cerebral arteries, UTP stimulation shifts Ca2+ sparks to Ca2+ waves through the differential regulation of inositol-1,4,5-triphosphate receptors (IP3Rs) and ryanodine receptors (RyRs) (Jaggar and Nelson, 2000). From studies in cultured basilar artery smooth muscle cells, it is generally accepted that UTP induces vasoconstriction by a combination of stimulated plasma membrane Ca2+ entry and SR Ca2+ release (Sima et al., 1997). However, little is known about the mechanism underlying between agonist-induced Ca2+ waves and their relationship to vasoconstriction in the cerebral vasculature. In our present study, we investigated the mechanism of UTP-induced Ca2+ waves in the rat basilar artery, focusing on the mode(s) of Ca2+ entry involved in sustaining the UTP-induced cyclical release of Ca2+ by identifying the Ca2+ transport molecules involved in the generation and maintenance of UTP-induced Ca2+ waves.  77  3.2 Methods 3.2.1 Tissue preparation Male Sprague-Dawley rats (250-350g) were obtained from Charles River and housed in the institutional animal facility (University of British Columbia, Child and Family Research Institute) under standard animal room conditions (12h light-12h dark, at 25ºC, 2 animals in a cage). All the experiments and procedures were carried out in accordance with the guidelines of the University of British Columbia. Rats were anesthetized with a mixture of ketamine hydrochloride (70 mg•kg-1) and xylazine hydrochloride (5 mg•kg-1) given intraperitoneally. The brain was quickly removed and placed in ice-cold, oxygenated (95% O2-5% CO2) Krebs solution. The basilar artery (180-280 μm in diameter) was removed, carefully cleaned, and cut into 2 mm segments. Endothelial denudation was achieved by gently rubbing the inside of the vessel with a 40μm tungsten wire.  3.2.2 Measurement of intracellular Ca2+ The arterial rings were loaded with Fluo-4AM (5 μM with 5 μM Pluronic F-127, 1 hr at 37ºC) and isometrically mounted, followed by a 30 min washout time in 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES)-buffered physiological saline solution (PSS). Sustained Ca2+ waves were induced by 100 μM UTP, and all mechanistic studies were done at this concentration. Images were acquired on an upright Olympus BX50WI microscope with a 60x water-dipping objective (NA 0.9) and equipped with an Ultraview confocal imaging system (Perkin-Elmer). The rate of image acquisition was 3 frames/s. The tissue was illuminated using the 488nm line of an Argon-Krypton laser and a high-gain photomultiplier tube collected the emission at wavelengths between 505 and 550 nm. The scanned regions correspond to a 91.685 x  78  66.68 μm area (or 248 x 328 pixels). The representative fluorescence traces shown reflect the averaged fluorescence signals from a region of 3 x 3 pixels (1.69 μm2) of the smooth muscle cell. The frequency of Ca2+ waves was determined by counting the number of waves occurring within a period of 50s. The measured changes in Fluo-4 fluorescence level are proportional to the relative changes in [Ca2+]i. All parameters (laser intensity, gain, etc) were maintained constant during the experiment. The confocal images were analyzed off-line with the Ultraview 4.0 Software (Perkin-Elmer). Fluorescence traces were extracted from the movies to exclude nuclear regions and traces were normalized to initial fluorescence values.  3.2.3 Measurement of isometric force Basilar artery segments were mounted isometrically in a small vessel wire myograph (A/S Danish Myotechnology, Aarhus N, Denmark), using two 40 μm tungsten wires, for measuring generated force. The chambers were kept at 37ºC and bubbled continuously with 95% O2-5% CO2 in Krebs solution. Optimal tension was determined in preliminary experiments by subjecting arterial segments to different resting tensions and stimulating with 60 mM KCl. The vessels were stretched to the optimal tension (obtained from preliminary experiments, the maximal force generation given in response to 60 mM KCl; 3mN) for 60 min. The vessels were challenged twice with 60 mM KCl before experiments were continued. The percent of contraction compared to the second 60 mM KCl-induced contraction was recorded at different concentrations of UTP and concentration-response curves were constructed. Tonic contraction was induced by 100 μM UTP and all mechanistic studies done at this concentration. The negative logarithm (pD2) of the concentration of UTP giving half-maximum response (EC50) was  79  assessed by linear interpolation on the semilogarithm concentration-response curve [pD2 = log(EC50)].  3.2.4 Statistics Values are expressed as mean ± standard error (SEM) from at least six independent experiments. Statistical analysis and construction of concentration-response curves were performed using GraphPad Prism 4.0 software (San Diego, CA, USA). Differences between groups were analyzed by Student’s two-tailed t-test. Statistical significance was defined as Pvalues <0.05.  3.2.5 Drugs, solutions, and chemicals HEPES-PSS containing (in mM) NaCl 140, glucose 10, KCl 5, HEPES 5, CaCl2 1.5, and MgCl2 1 (pH 7.4) was used for all confocal studies. Hi-K+ (60 mM extracellular K+) PSS was identical in composition to normal PSS with the exception of (in mM) NaCl 85 and KCl 60. Zero-Ca2+ PSS was prepared in the same way as normal PSS, but CaCl2 was replaced with 1 mM ethylene glycol tetraacetic acid (EGTA). Krebs solution containing (in mM) NaCl 119, glucose 11.1, KCl 4.7, CaCl2 1.6, KH2PO4 1.18, MgSO4 1.17, and ethylenediaminetetraacetic acid (EDTA) 0.023 (pH 7.4) were used for all isometric contraction studies. UTP, CPA, 2-APB, nifedipine, SKF-96365, tetracaine, and pluronic F-127 were obtained from Sigma-Aldrich (Oakville, Ontario, Canada). Ryanodine and KB-R7943 were obtained from Calbiochem (Gibbstown, NJ, USA). Fluo-4AM was purchased from Molecular Probes (Eugene, OR).  80  3.3 Results 3.3.1 Relation between UTP-induced tonic contraction and UTP-induced Ca2+-waves UTP produced tonic contraction in a concentration-dependent manner, with a pEC50 of 4.34 ± 0.13 and maximal response (Emax) of 105.5 ± 7.3% (normalized to contraction at 60 mM KCl, n = 9 animals, Fig. 3.1A,B). At 100 μM UTP, the average contraction was 70.3 ± 4.5% (n = 12 animals, normalized to contraction at 60 mM KCl). In parallel experiments, confocal microscopy was used to observe changes in [Ca2+]i within the smooth muscle cells following UTP stimulation. In the absence of UTP, asynchronous Ca2+ waves of low amplitude were observed in a small percentage (< 10%) of the cells, similar to the “Ca2+ ripples” described previously in rat tail artery (Asada et al., 1999). Application of UTP induced a large transient Ca2+ response which was followed by sustained repetitive oscillations in intracellular [Ca2+]i which propagated along the length of the smooth muscle cell as Ca2+ waves (Fig. 3.1C and 3.2). The frequency of Ca2+ waves increased in a concentration-dependent manner, closely paralleling the development of force (Fig. 3.1D, pEC50 = 4.74 ± 0.14, maximum frequency = 0.089 ± 0.007Hz, 109 cells from 12 animals). At 100 μM UTP, the average frequency of the Ca2+ waves was 0.082 ± 0.005 Hz (n = 48 cells from 8 animals). The number of cells displaying Ca2+ waves were also concentrationdependent; at 100 μM UTP, 91.34 ± 2.45 % of cells displayed at least one Ca2+ wave (n = 48 cells from 8 animals, Fig. 3.1E). The velocity of wave propagation, illustrated in Fig. 3.1F, also shows a strong correlation with UTP concentration; At the highest concentrations, wave propagation speeds reached 67.51 ± 3.62 μm/s (n = 10 cells from 4 animals). The Ca2+ waves originated from distinct intracellular foci and propagated down the longitudinal axis of the individual smooth muscle cells (Fig. 3.2). They did not appear to propagate intercellularly, and were sustained during the entire experimental period.  81  A  B 100μM UTP  2 mN 100s  % contraction (of 60mM Hi-K +)  125 100 75 50 25 0 -8  C  -7  -6 -5 log[UTP]  -4  -3  -4  -3  D  100μM UTP  Frequency (Hz)  0.10  0.5 F/Fo 50s  0.08 0.06 0.04 0.02 0.00 -8  E  -7  -6 -5 log[UTP]  F 80  Wave propagation (μm s-1)  Recruitment of cells (% max)  125 100 75 50 25  70 60 50 40 30 20 10 0  0 -8  -7  -6  -5 -4 log[UTP]  -3  -2  -8  -7  -6  -5 -4 log[UTP]  -3  -2  Figure 3.1: Properties of uridine 5’-triphosphate (UTP, 100 μM)-induced Ca2+ waves underlying tonic contraction in rat basilar artery. A. UTP-induced tonic contraction. Traces are representative of results from 6 animals. B. Concentrationresponse curve for UTP-induced tonic contraction. pEC50 = 4.24 ± 0.13 (n = 9 animals) C. In parallel experiments, application of UTP produced sustained Ca2+ oscillations which propagated along the cell as waves. Experimental Ca2+ traces are representative of results from 58 cells from 6 animals. D. Concentration-response curve for frequency of UTP-induced Ca2+ waves pEC50 = 4.74 ± 0.14 (n = 109 cells from 12 animals). E. A greater percentage of smooth muscle cells generated Ca2+ signals as UTP concentration increased. This recruitment occurred between 3 and 1000 μM, with maximal recruitment  82  achieved at 300 μM UTP (n = 90 cells from 10 animals). The number of cells firing is expressed as a percentage of cells responding to maximal concentration. F. The apparent propagation speed of the Ca2+ waves was correlated to increasing UTP concentration (n = 87 cells from 11 animals).  A  50s  AOI 1 0.5 F/Fo  AOI 2  100μM UTP  B  0s  2s  4s  6s  100μM UTP Figure 3.2: Uridine 5’-triphosphate (UTP, 100 μM)-induced Ca2+ waves in rat basilar artery. A. [Ca2+]i changes in 2 intracellular regions from 2 different smooth muscle cells upon UTP stimulation are depicted in the Ca2+ traces taken from the steady state of UTP-induced Ca2+ waves. It should be noted that the Ca2+ waves occurred at different frequencies. Experimental Ca2+ traces are representative of results from 58 cells in 6 animals. B. Intact rat basilar artery smooth muscle cells challenged with UTP displayed Ca2+ waves which originated from distinct intracellular foci and propagated along the longitudinal axis of the smooth muscle cells (indicated by AOI1 and AOI2). The area of AOI is 3x3 pixels (1.69 μm2). Scale bar = 10 μm.  83  3.3.2 Dependence of UTP-induced Ca2+-waves on extracellular Ca2+ influx There are two potential sources of Ca2+ that can contribute to the generation of UTP-induced Ca2+ waves: Ca2+ release from the intracellular stores and Ca2+ influx from the extracellular space. To determine the contribution of extracellular Ca2+ to the initiation and maintenance of UTP-induced Ca2+ waves, extracellular Ca2+ was removed prior to and during UTP stimulation, respectively. Removal of extracellular Ca2+ immediately prior to UTP stimulation reduced the Ca2+ signal to only a few transient Ca2+ waves (n = 39 cells from 6 animals, Fig. 3.3A), while UTP-induced Ca2+ waves were completely abolished in the absence of extracellular Ca2+ within 1 minute of treatment (n = 34 cells from 5 animals) (Fig. 3.3B), showing that extracellular Ca2+ influx was necessary for maintenance of Ca2+ waves.  A  B 0.5 F/Fo  0.5 F/Fo 50s  50s  100μM UTP  0Ca2+  0Ca2+  100μM UTP  Figure 3.3: Extracellular Ca2+ influx is required for maintenance of uridine 5’triphosphate (UTP, 100 μM)-induced Ca2+ waves. A. Removal of extracellular Ca2+ during ongoing UTP-induced Ca2+ waves results in their abolishment within 1 minute. Traces shown are representative of 39 cells from 6 animals. B. Removal of extracellular Ca2+ immediately prior to UTP stimulation limits Ca2+ signaling to transient Ca2+ waves. Traces shown are representative of 34 cells from 5 animals.  84  To further define the Ca2+ entry pathways involved in maintaining UTP-induced Ca2+ waves, nifedipine, a selective inhibitor of L-type Ca2+ channels, and SKF-96365, an inhibitor of receptor-operated and store-operated channels, were used. Nifedipine (10 μM) reduced the frequency of 100 μM UTP-induced Ca2+ waves to 59.25 ± 3.86 % of control, while the combined application of nifedipine and SKF-96365 (50 μM) completely abolished the Ca2+ waves (P < 0.001, n = 42 cells from 8 animals). In parallel, application of nifedipine (10 μM) significantly reduced tonic contraction to 52.14 ± 3.46 % of control (P < 0.001, n = 6 animals), while the combined application of nifedipine and SKF-96365 (50 μM) decreased tonic contraction to 2.17 ± 1.10 % of control (P < 0.001, n = 5 animals) (Fig. 3.4A,B). It should also be noted that nifedipine (10 μM) completely abolished the contraction induced by 60 mM KCl (data not shown).  85  A  *** *** 0.5 F/Fo  0.12  Frequency (Hz)  50s  100μM UTP  0.10 0.08 0.06 0.04 0.02 0.00  10μM Nifedipine  Nifedipine  50μM SKF-96365  SKF-96365  -  B  +  +  -  +  *** 2mN  ***  250s  100μM UTP 10μM Nifedipine 50μM SKF-96365  Contraction (of 100μM UTP)  125 100 75 50 25 0  Nifedipine SKF-96365  -  +  +  -  +  Figure 3.4: Effect of nifedipine and SKF-96365 on uridine 5’-triphosphate (UTP, 100 μM)-induced Ca2+ waves and tonic contraction. A. The frequency of Ca2+ waves is significantly reduced (59.25 ± 3.86% of control) following nifedipine (10 μM) application, but not abolished. The nifedipine-insensitive component is completely abolished following addition of SKF-96365 (50 μM). B. UTP-induced tonic contraction is significantly reduced to 52.14 ± 3.46 % of control by nifedipine (10 μM) and almost completely abolished (2.17 ± 1.10% of control) after SKF-96365 (50 μM). *** - P < 0.001. Dashed lines indicate a 2 minute interval. In addition to the conventional plasmalemmal Ca2+ permeable channels, the Na+/Ca2+ exchanger operating in the reverse-mode is also an important pathway for Ca2+ entry in smooth  86  muscle cells (Lee et al., 2002; Poburko et al., 2006; Fameli et al., 2007). KB-R7943, an inhibitor of reverse-mode NCX at low (≤ 10 μM) concentrations, was used to examine whether reversemode Na+/Ca2+ exchange is involved in supporting nifedipine-insensitive Ca2+ waves (Iwamoto et al., 1996; Ladilov et al., 1999). The application of KB-R7943 (10 μM) abolished nifedipineinsensitive Ca2+ waves (P < 0.001, n = 34 cells from 6 animals) and inhibited tonic contraction to 3.56 ± 1.00 % of control (P < 0.001, n = 7 animals) (Fig. 3.5A,B). Application of KB-R7943 (10 μM) by itself also abolished UTP-induced Ca2+ waves (P < 0.001, n = 29 cells from 4 animals) and tonic contraction (P < 0.001, n = 4 animals), suggesting that Ca2+ entry through reversemode Na+/Ca2+ exchange plays an important role in maintenance of Ca2+ waves even when Ltype Ca2+ channels are operative (Fig. 3.5C,D). Although KB-R7943 (10 μM) reduced 60 mM KCl induced tonic contraction by 10.6% ± 3.0%, this effect was not significant (P = 0.08, n = 5 animals) (Fig. 3.6A). KB-R7943 may also inhibit store-operated channels (Arakawa et al., 2000). To test for possible effects on store-operated channels, we used UTP (100 μM) to stimulate sustained Ca2+ waves and then applied cyclopiazonic acid (10 μM, CPA), an inhibitor of the sarco(endo)plasmic reticulum Ca2+-ATPase, to inhibit SR Ca2+ reuptake. This resulted in an elevation of cytosolic Ca2+ levels, on which KB-R7943 had no effect, but the addition of SKF96365 brought Ca2+ levels to baseline (Fig. 3.6B). This suggests that KB-R7943 did not abolish the Ca2+ waves through blockade of store/receptor-operated channels. It is also important to note that extracellular Na+-depletion with the use of zero-Na+ PSS also abolished the Ca2+ waves, which further supports the role of reverse-mode Na+/Ca2+ exchange (data not shown).  87  A  B 2mN  0.5 F/Fo  250s  50s  100μ M UTP  100μ M UTP 10μ M Nifedipine  10μ M Nifedipine 10μ M KB-R7943  C  10μ M KB-R7943  D 0.5 F/F o 50s  2mN 250s  100μ M UTP  100μ M UTP  10μ M KB-R7943  10μ M KB-R7943  Figure 3.5: Effect of the reverse-mode Na+/Ca2+ exchanger inhibitor KB-R7943 on uridine 5’-triphosphate (UTP, 100 μM)-induced Ca2+ waves and tonic contraction. A. Blockade of reverse (Ca2+-entry) mode Na+/Ca2+ exchange using KB-R7943 (10 μM) abolished the nifedipine-resistant UTP-induced Ca2+ waves (P < 0.001, n = 34 cells from 6 animals) B. Similarly, KB-R7943 (10 μM) also inhibited the nifedipine-insensitive tonic contraction to 3.56 ± 1.00 % of control (P < 0.001 n = 6 animals) C. Application of KB-R7943 (10 μM) alone reduced the frequency of UTP-induced Ca2+ waves, followed by complete abolishment (P < 0.001, n = 29 cells from 4 animals) D. KB-R7943 (10 μM) alone also abolished UTP-induced tonic contraction (P < 0.001, n = 5 animals).  88  A  B 0.5 F/Fo 2 mN 100s 60mM KCl 10μM KB-R7943  50s  UTP + CPA (+)  UTP + CPA (-)  10μM KB-R7943 50μM SKF-96365  Figure 3.6: Effects of KB-R7943 on L-type Ca2+ channels and store/receptoroperated channels in rat basilar artery. A. Application of KB-R7943 reduced tonic contraction induced by 60 mM KCl by 10.6% ± 3.0% (P = 0.08, n = 5 animals). B. Application of UTP (100 μM) followed by CPA (10 μM) resulted in a maintained elevation in Ca2+ (solid black line). Application of KB-R7943 (10 μM) did not affect this plateau response, whereas the addition of SKF-96365 (50 μM) abolished the maintained Ca2+ elevation and returned to pre-stimulation baseline level (solid gray line). Representative trace shown is typical of the responses obtained in 36 cells from 4 rats.  3.3.3 Dependence of UTP-induced Ca2+-waves on SR Ca2+ release The all-or-none wave-like nature of Ca2+ signal in the rat basilar artery suggests regenerative Ca2+ release from the SR. If this is the case, blockade of SR Ca2+ uptake should completely inhibit the Ca2+ waves. The application of CPA (10 μM) to ongoing UTP-induced Ca2+ waves resulted in a broadening of the Ca2+ waves followed by their complete abolishment, leaving a significant elevation in baseline [Ca2+]i corresponding to 35 ± 4 % of the peak [Ca2+] of the Ca2+ waves (P < 0.001, n = 38 cells from 6 animals) (Fig. 3.7A). In parallel, CPA (10 μM) also produced a 79.3 ± 1.7 % inhibition of the UTP-induced tonic contraction (Fig. 3.7B, P < 0.001, n = 5 animals).  89  A  B 0.5 F/Fo 50s  2mN 250s  100μM UTP  100μM UTP  10μM CPA  10μM CPA  Figure 3.7: Effect of blockade of the sarco(endo)plasmic reticulum Ca2+ ATPase by cyclopiazonic acid (CPA) on uridine 5’-triphosphate (UTP, 100 μM)-induced Ca2+ waves. A. Addition of CPA (10 μM) to ongoing UTP-induced Ca2+ waves completely abolished the oscillations, leaving a small but significant elevation in baseline Ca2+ which corresponds to 35 ± 4 % of the peak [Ca2+] of the asynchronous Ca2+ oscillations (P < 0.001, n = 28 cells from 5 animals) B. Application of CPA (10 μM) produced a 79.3 ± 1.7 % inhibition of the UTP-induced tonic contraction (P < 0.001, n = 5 animals). Ca2+ release from the SR can be mediated through either the inositol-1,4,5-triphosphate receptor (IP3R) and/or the ryanodine receptor (RyR). 2-aminoethoxydiphenylborate (2-APB, 100 μM), an inhibitor of IP3Rs in smooth muscle cells (Missiaen et al., 2001), was used to examine the role of IP3Rs in UTP-induced Ca2+ waves. Addition of 2-APB (100 μM) to ongoing Ca2+ waves immediately abolished them (n = 31 cells from 5 animals), and inhibited tonic contraction to 3.4 ± 0.7% of the control level (P < 0.001, n = 6 animals) (Fig. 3.8A,B). Furthermore, UTP (100 μM) failed to elicit a Ca2+ transient or contraction in basilar arteries pre-incubated for 30 minutes with 2-APB (Fig. 3.8C,D).  90  A  B 2 mN 0.5 F/Fo  100s  50s  100μM UTP  100μM UTP  100μM 2-APB  100μM 2-APB  C  D  100μM UTP  0.5 F/Fo  100μM UTP 2-APB (-)  50s  2 mN 2-APB (-)  100s 2-APB (+)  2-APB (+)  Figure 3.8: Effect of 2-aminoethoxydiphenylborate (2-APB) on uridine 5’triphosphate (UTP, 100 μM)-induced Ca2+ waves and tonic contraction. A. The application of 2-APB (100 μM) immediately abolished UTP-induced Ca2+ waves (n = 31 cells from 5 animals) B. 2-APB (100 μM) decreased UTP-induced tonic contraction to 3.4 ± 0.7% of the control level (P < 0.001, n = 6 animals). C. UTP stimulation after pretreatment of vessels with 2-APB (100 μM) for 30 minutes failed to elicit a Ca2+ response (solid gray line, P < 0.0001, n= 24 cells from 3 animals). In contrast, control vessels without 2-APB (100 μM) preincubation (solid black line) displayed Ca2+ waves after UTP stimulation. D. UTP stimulation after pretreatment of vessels with 2-APB (100 μM) for 30 minutes failed to induce contraction (solid gray line, P < 0.0001, n = 5 animals) compared to control vessels without 2-APB preincubation (solid black line).  91  A 0.5 F/Fo  100 μM 2-APB 25 mM caffeine  25 mM caffeine  % of the maximum amplitude of control  50s  Control  25 mM caffeine  B  120 110 100 90 80 70 60 50 40 30 20 10 0 2nd caffeine 3rd caffeine  C 0.5 F/Fo 2 mN 100s  UTP + CPA (+)  50s  UTP + CPA (-)  60 mM KCl  100 μM 2-APB  100 μM 2-APB  50 μM SKF-96365  Figure 3.9: Effects of 2-aminoethoxydiphenylborate (2-APB) on the ryanodinesensitive SR Ca2+ release channels, sarco(endo)plasmic reticulum Ca2+ ATPase, Ltype Ca2+ channels, and store-operated channels in rat basilar artery. A. Left: Three pulses of caffeine (25 mM) were applied with a 5-min interval between each pulse (dashed lines). Maximum amplitude of the caffeine-induced Ca2+ transient from the first pulse reflects control SR Ca2+ level as Ca2+ from the SR is released through the opened RyR channels. After the addition of 2-APB (100 μM), the second pulse of caffeine resulted in a single Ca2+ transient whose maximum amplitude is similar to the first pulse (103.3 ± 7.6% of control, P = 0.66, n = 6 animals). The third pulse of caffeine resulted in a Ca2+ transient whose maximum amplitude was slightly, but not significantly, diminished by 13.4 ± 4.1% compared with the first pulse (P = 0.11, n = 6 animals). Right: Bar graph comparing the average maximum amplitude of the second and third caffeine pulses to the third pulse (n = 6 animals). B. Application of 2-APB (100 μM) reduced tonic contraction induced by 60 mM KCl by 14.8 ± 4.3% (P = 0.041, n = 6 animals). (c) Application of UTP (100 μM) followed by CPA (10 μM) resulted in a maintained elevation in Ca2+ (black solid line). Application of 2-APB (100 μM) did not affect this plateau response, whereas the addition of SKF-96365 (50 μM) abolished the maintained Ca2+ elevation and returned to pre-stimulation baseline level (gray solid line). Representative trace shown is typical of the responses obtained in 30 cells from 4 rats.  92  It therefore appears that the opening of the IP3Rs is required for UTP-mediated vasoconstriction and Ca2+ waves. However, as the specificity of 2-APB has been questioned, it was important to examine the selectivity of 2-APB in our preparation, especially with regard to Ca2+ translocators such as RyRs, sarco(endo)plasmic reticulum Ca2+ ATPase, the store-operated channels, and the L-type Ca2+ channels. As shown in Fig. 3.9A, pre-treatment with 2-APB (100 μM) did not significantly affect the peak amplitude of caffeine (25 mM)-induced Ca2+ release (103.3 ± 7.6% of the control, P = 0.66, n = 5 animals), and therefore appears to be inactive against RyRs. Furthermore, 2-APB marginally affected SR refilling, as the peak amplitude of the third caffeine-induced Ca2+ transient was decreased slightly, but not significantly by 13.4 ± 4.1% (P = 0.11, n = 6 animals). To test for direct effects on Ca2+ entry pathways, the effects of 2-APB on L-type Ca2+ channels and store-operated channels, two plasmalemmal channels important to UTP-mediated Ca2+ waves, were examined. 2-APB (100 μM) reduced 60 mM KCl-induced tonic contraction by 14.8 ± 4.3% (Fig. 3.9B, P = 0.041, n = 6 animals). However, this slight inhibition of L-type Ca2+ channels cannot account for the complete inhibition of UTP-induced tonic contraction by 2-APB, as blockade of L-type Ca2+ channels with nifedipine only reduced force by 52%. In addition to L-type Ca2+ channels, store-operated channels are also important for maintaining the Ca2+ waves. 2-APB has been reported to have non-selective effects on storeoperated channels (Bootman et al., 2002). We stimulated the vessel with UTP (100 μM) to generate sustained Ca2+ waves and then applied CPA (10 μM) to inhibit SR Ca2+ reuptake, resulting in a maintained elevation of [Ca2+] and depletion of the SR (Fig. 3.9C). The application of 2-APB (100 μM) did not affect the [Ca2+]i plateau, although Ca2+ returned to baseline upon  93  the subsequent addition of the store-operated channel blocker SKF-96365 (50 μM), indicating that in this preparation 2-APB does not inhibit the store-operated channels directly.  A 0.5 F/Fo  B  50s  2 mN 100s  100μM UTP  100μM UTP  200μM ryanodine  200μM ryanodine  C  D 0.5 F/Fo 2 mN  50s 100s 100μM UTP  100μM UTP  100μM tetracaine  100μM tetracaine  E 0.5 F/Fo 50s  50μM ryanodine 25mM caffeine  25mM caffeine  25mM caffeine  100μM UTP  94  Figure 3.10: Effect of ryanodine, tetracaine, and caffeine-induced depletion of SR Ca2+ stores on uridine 5’-triphosphate (UTP, 100 μM)-induced Ca2+ waves and tonic contraction in rat basilar artery. A. Application of a high concentration (200 μM) of ryanodine did not affect ongoing UTP-induced Ca2+ waves (P = 0.67, n = 33 cells from 5 animals) B. Ryanodine (200 μM) also did not affect UTP-induced tonic contraction (P = 0.71, n = 5 animals). C. High-concentration (100 μM) tetracaine also did not affect ongoing UTP-induced Ca2+ waves (P = 0.71, n = 29 cells from 4 animals) D. Tetracaine (100 μM) had no significant effect on UTP-induced tonic contraction (P = 0.64, n = 5 animals). E. To determine effects of depletion of RyR-sensitive SR Ca2+ stores, the artery was exposed to three 1-minute treatments of caffeine (25 mM) in the continuous presence of ryanodine (50 μM) resulted in depletion of SR Ca2+ stores. The second stimulation of caffeine produced a much reduced Ca2+ transient, while the third stimulation produced no Ca2+ response. After depletion of SR Ca2+ stores, stimulation with UTP (100 μM) in the presence of ryanodine failed to elicit a Ca2+ response. Dashed lines indicate a 5 minute interval. Although the opening of IP3Rs are required for UTP-mediated Ca2+ waves and contraction, it does not rule out Ca2+ release through the RyR, another type of SR Ca2+ release channel which is functionally important in smooth muscle cells. To assess the involvement of RyRs, highconcentrations of ryanodine (200 μM) and tetracaine (100 μM) were used to lock RyRs in their closed configuration. Neither ryanodine nor tetracaine had any effect on the frequency of the ongoing UTP-induced Ca2+ waves (P = 0.67, n = 33 cells from 5 animals; P = 0.71, n = 29 cells from 4 animals, respectively) or tonic contraction (P = 0.64, n = 5 animals; P = 0.64, n = 6 animals) (Fig. 3.10A,B,C,D). This supported the notion that Ca2+ release from the RyRdependent SR store is not responsible for the generation of Ca2+ waves. To explore this issue further, RyRs were locked in the subconductance state by preincubation with ryanodine (50 μM) followed by a brief (1 min) exposure to caffeine (25 mM). The first caffeine exposure caused a transient Ca2+ response, whereas a second exposure elicited a much-reduced Ca2+ transient, and the third failed to elicit any Ca2+ transient at all (Fig. 3.10E). We interpreted the final lack of Ca2+ transient in response to caffeine to indicate that release of Ca2+ through RyRs on the SR was no longer possible due to depletion of SR Ca2+ content and/or the locking of RyRs in an open 95  state. UTP (100 μM) stimulation immediately after depletion of the RyR-sensitive store no longer elicited a Ca2+ response. These results suggest that IP3Rs and RyRs have access to a common SR Ca2+ store, but that opening of RyRs do not appear to be critical for the maintenance of UTP-induced Ca2+ waves.  3.4 Discussion The presence of agonist-induced Ca2+ waves in cerebral arteries has been documented by various groups (Jaggar and Nelson 2000; Jaggar, 2001; Heppner et al., 2002), but a detailed investigation of their underlying mechanisms has not yet been conducted. We have investigated the link between agonist-induced Ca2+ waves and tonic contraction using an in situ preparation of the rat basilar artery and have systematically studied the ionic mechanisms underlying these Ca2+ waves, which appear to be similar to those described in vascular smooth muscle from larger conduit blood vessels (Lee et al., 2002). Our studies of UTP-induced Ca2+ waves were performed in isometrically stretched arteries, which are similar to the conditions in which UTPinduced Ca2+ waves were first described (Jaggar and Nelson, 2000), and may shed new light on how wall tension may be regulated in the basilar artery. The response to UTP is typified by repetitive transient elevations in Ca2+ which originate in distinct intracellular foci and then spread out as waves over the length of the smooth muscle cell. The cells respond independently of each other in that the Ca2+ waves are asynchronous and that the cells vary in their sensitivity to UTP, such that recruitment of responding cells increases with increasing UTP concentration. Furthermore, the propagation velocity and frequency also increases with increasing UTP concentration and have similar dose-response relationships (Fig. 3.1C,D). Functionally, it appears that Ca2+ waves underlie tonic contraction, as their inhibition  96  with nifedipine, SKF-96365, KB-R7943, or 2-APB is association with complete inhibition of force (Fig. 3.4, 3.5, 3.8). Finally, the lack of synchronicity between neighbouring smooth muscle cells explains how summation of individual-cell Ca2+ waves can lead to tonic contraction, as the summation of Ca2+ signals in all the cells averages out to be a steady state Ca2+ increase in whole vessels (Ruehlmann et al., 2000; Mauban et al., 2001). The apparent importance of Ca2+ waves for tonic contraction is further demonstrated when their abolishment by CPA markedly reduces force by 80%, although the average [Ca2+]i remains significantly elevated above baseline (Fig. 3.7). This indicates a higher force-to-[Ca2+]i ratio when smooth muscle cells are activated with Ca2+ waves as compared with sustained [Ca2+]i, suggesting that Ca2+ waves represent a more efficient method to deliver Ca2+ to activate myosin light-chain kinase, which is tethered to the contractile filaments (Lee et al., 2001; Wilson et al., 2002). However, contraction is ultimately determined by the level of phosphorylation of myosin light chain, which is both Ca2+ dependent and Ca2+-independent (Weber et al., 1999). Currently, the way in which Ca2+ waves might be related to this level of phosphorylation is unknown. The UTP-induced Ca2+ waves appear to be propagated by regenerative Ca2+ release from the SR network, as depletion of SR Ca2+ stores with CPA abolishes the oscillations (Fig. 3.7). Extracellular Ca2+ influx appears to be critical for the maintenance of Ca2+ waves, although the ability of Ca2+ waves to persist for a time in the absence of Ca2+ is likely due to several Ca2+ transport mechanisms. In smooth muscle, a proportion of the Ca2+ released by the SR is inevitably extruded to the extracellular space by the actions of the plasma membrane Ca2+ATPase (PMCA), and in a Ca2+-free medium all Ca2+ release from the SR is irreversibly lost to the extracellular space (Leijten and van Breemen, 1986). However, removal of Ca2+ towards the extracellular space is in competition with SR Ca2+ reuptake through the sarco(endo)plasmic  97  reticulum Ca2+ ATPase, which allows the SR to continue releasing decreasing amounts of Ca2+ to sustain the Ca2+ waves. Finally, a third mechanism of Ca2+ unloading of the SR during Ca2+-free conditions is the transfer of Ca2+ release by the peripheral ryanodine receptors (RyRs) towards the forward-mode (Ca2+-extrusion) Na+/Ca2+ exchange, a mechanism which as been described in both smooth muscle cells and endothelial cells (Nazer and van Breemen, 1998; Liang et al., 2004). Therefore, without refilling of the SR, all of the Ca2+ is eventually extruded resulting in the disappearance of the Ca2+ waves. Influx of extracellular Ca2+ through L-type Ca2+ channels is central in the control of cerebrovascular arterial diameter (Nelson et al., 1990). However, it is interesting that the UTPinduced Ca2+ waves were not abolished by nifedipine, but only reduced in frequency. One mechanism through which frequency could be decreased is that the absence of stimulated Ca2+ influx through L-type Ca2+ channels may reduce the rate of refilling of the SR Ca2+ store. As SR luminal Ca2+ can regulate IP3R channel opening probability, a reduced rate of SR Ca2+ refilling can result in a decreased frequency of SR Ca2+ release at the wave initiation site (Meldolesi and Pozzan, 1998). Similarly, blockade of L-type Ca2+ channels in pressurized mouse mesenteric arteries, which abolished myogenic tone, also reduced the frequency of phenylephrine-induced Ca2+ oscillations (Zacharia et al., 2007). However, pressure-induced Ca2+ waves in small rat cerebral arteries were completely abolished by diltiazem (Jaggar, 2001). Although the larger cerebral vessels, such as the basilar artery, share some properties of resistance vessels (Toyoda et al., 1996), these differing observations may be the result of tissue differences, with respect to relative involvement of the various Ca2+ entry mechanisms. Furthermore, these apparent mechanistic differences may also be attributed to different physiological conditions, for example pressurization versus tension. For example, the development of myogenic tone may influence the  98  Ca2+ signal elicited by agonists (Zacharia et al., 2007). Consequently, comparisons between the mechanisms of Ca2+ waves must take differences in vascular beds and experimental preparations into consideration. Another major finding in this study is that UTP-induced Ca2+ waves are abolished by KBR7943, an inhibitor of reverse-mode Na+/Ca2+ exchanger-mediated Ca2+ entry across the plasma membrane (Iwamoto et al., 1996; Ladilov et al., 1999). The plasma membrane Na+/Ca2+ exchanger is a transmembrane protein that normally couples the influx of Na+ ions to the efflux of Ca2+ ions in a 3:1 ratio (Philipson and Nicoll, 2000). However, Na+ entry through receptorand store-operated channels which are functionally coupled to the Na+/Ca2+ exchanger may influence the dynamics Na+/Ca2+ exchange, as Na+ accumulates regionally in a restricted subplasmalemmal space between the superficial SR and the plasma membrane (Arnon et al., 2000; Poburko et al., 2004; Lemos et al., 2007). This build-up in subcellular Na+ was hypothesized to change the electrochemical gradient to favour Ca2+ influx through NCX reversal, which in turn refills the SR Ca2+ stores (Lee et al., 2001). This would explain our findings that the nifedipine-resistant Ca2+ waves and tonic contraction are similarly sensitive to both SKF96365, an inhibitor of store- and receptor operated channels, and KB-R7943. However, to achieve NCX reversal, the subplasmalemmal Na+ ([Na+]subPM) must reach at least the level of Km. Although the concentration of [Na+]subPM has not been measured in this preparation, we have predicted from our studies with rat aortic smooth muscle cells that reversemode Na+/Ca2+ exchange should occur when [Na+]subPM exceeds 23-25mM, assuming Em = 60mV, ENCX = 3ENa – 2ECa, [Ca2+]o = 1.2mM, [Ca2+]subPM = 500nM, and [Na+]o = 145mM (where [Ca2+]o = extracellular [Ca2+], [Ca2+]subPM = subplasmalemmal [Ca2+], and [Na+]o = extracellular [Na+]) (Poburko et al., 2006). Recently, Poburko and colleagues provided the first direct  99  demonstration of localized subcellular increases in Na+ through receptor-operated/store-operated channels to ≥ 30 mM (Poburko et al., 2007), which is consistent with estimates of Na+ ranging from 24 to 40mM in ventricular myocytes (Wendt-Gallitelli et al., 1993; Isenberg et al., 2003). In addition, the space constant for the subplasmalemmal Na+ gradient in ventricular myocytes is 28nm, which is highly consistent with the intermembrane separation (~20nm) in PM-SR junctions in the rat basilar artery preparation (unpublished observations). Furthermore, given that the resting membrane potential in rat basilar artery is approximately -43mV (Haddock and Hill, 2002), and that a more depolarized membrane decreases [Na+]subPM required for NCX reversal, it seems plausible that reverse-mode Na+/Ca2+ exchange is a physiological route of Ca2+ entry in cerebral arteries. This is especially relevant as our finding that KB-R7943 abolishes Ca2+ waves suggests that SR refilling is critically dependent on reverse-mode Na+/Ca2+ exchange during UTP stimulation (Fig. 3.5C). Although it remains to be investigated, this may serve as an example of privileged delivery of Ca2+ from a transport site located in one membrane to a second Ca2+ transport site in an apposing membrane, a process which serves to circumvent free diffusion throughout the cytoplasm (Poburko et al., 2004; Fameli et al., 2007). In addition to its inhibition of reverse-mode Na+/Ca2+ exchange, KB-R7943 has been also been reported to have effects on L-type Ca2+ and store-operated channels, neuronal nicotinic acetylcholine receptors, the Nmethyl-D-aspartic acid (NMDA) receptor, and norepinephrine transporter (Watano et al., 1996; Sobolevsky and Khodorov, 1999; Arakawa et al., 2000; Iwamoto, 2004). However, in our preparation, KB-R7943 does not significantly inhibit L-type Ca2+ channels or store-operated channels, which supported the notion that it is the reverse-mode Na+/Ca2+ exchange, which is important refilling the SR to maintain Ca2+ waves.  100  UTP exerts its effects on metabotropic purinergic P2Y receptors, and has been shown to augment Ca2+ release via an increase in cytoplasmic IP3 (Strobaek, 1996; Sima et al., 1997). To investigate the role of IP3Rs, we used 2-APB, a small molecular weight membrane permeable modulator of the IP3R (Missiaen et al., 2001). However, its use to block IP3Rs has been criticized for its nonspecific effects on other ion transport mechanisms, notably its inhibition of storeoperated channels (Broad et al., 2001; Ma et al., 2001; Ratz and Berg, 2006). Importantly in our preparation, 2-APB immediately abolished ongoing Ca2+ waves and tonic contraction, and did not affect caffeine-releasable Ca2+ stores, which is consistent with an action of 2-APB to block IP3Rs (Fig. 3.9A). Furthermore, preincubation with 2-APB did not elicit a Ca2+ response or contraction. It also had only a minor insignificant effect on SR Ca2+ reuptake. 2-APB had no significant inhibition on store-operated channels, and although it does affect L-type Ca2+ channels, the slight inhibition observed could not have accounted for the abolishment of Ca2+ waves (Fig. 3.9B,C). Therefore, our findings support the notion that opening of IP3R channels is not only responsible for the initial Ca2+ release, but is also required for subsequent regenerative release of Ca2+ underlying the propagation of the Ca2+ waves. Acetylcholine-induced Ca2+ waves in rat portal vein myocytes are also dependent on activation of IP3Rs, although interestingly the IP3R2 subtype, which is most sensitive to Ca2+, appears to be most important for the propagation of Ca2+ waves (Morel et al., 2003; Fritz et al., 2008). It should also be noted that the Ca2+ waves are maintained by the intrinsic sensitivity of the IP3R2 subtype to cytosolic [Ca2+]i, and not due to oscillation of IP3 levels (Fritz et al., 2008). A possible scenario in the rat basilar artery is that UTP-induced Ca2+ wave begins with elevation of IP3. The IP3 sensitizes the IP3R to Ca2+, and when Ca2+ reaches a threshold concentration the release channels open (Streb et al., 1983; Ferris et al., 1992). As the concentration of UTP is raised, the concentrations of IP3 and basal [Ca2+]i  101  are also raised, which shortens the time required for Ca2+ to reach threshold value for the initiation of the next wave. The regenerative nature depends on the positive feedback of increasing Ca2+ on the IP3 sensitivity of IP3R. This mechanism, combined with the fact that IP3 sensitizes the IP3R to Ca2+, ensures that both the frequency and velocity increase with increasing UTP concentration. However, knowledge of the IP3 dynamics in our preparation is required before this conclusion can be established. The observed effect of 2-APB indicates an essential role of IP3Rs in the initiation and maintenance of UTP-induced Ca2+ waves, but does not exclude involvement of RyRs. Although there is general agreement that the initiation of oscillations and waves is a response to agonists acting on sarcolemmal receptors which releases Ca2+ from the SR via IP3Rs, controversy remains whether or not Ca2+ release from the IP3Rs then activates RyRs to generate further release by Ca2+-induced Ca2+-release and to propagate waves, or whether the entire release process arises from IP3Rs without significant RyR involvement (Mccarron et al., 2003). The former proposal is supported by studies which showed that drugs which block RyRs often abolish Ca2+ oscillations initiated by IP3-generating agonists (Hyvelin et al., 1998; Boittin et al., 1999; Jaggar and Nelson, 2000). This is possibly due to co-localization of RyRs and IP3Rs, which allows Ca2+ released locally by IP3R activates adjacent clusters of RyR by Ca2+-induced Ca2+ release (Gordienko and Bolton, 2002). On the other hand, some preparations which lack a Ca2+-induced Ca2+ release mechanism still exhibit Ca2+ waves (DeLisle and Welsh, 1992; Lechleiter and Clapham, 1992). Furthermore, in pressurized rat mesenteric artery, RyRs do not appear to play a role in agoniststimulated Ca2+ waves (Lamont and Wier, 2004). It is important to note here that many studies which utilize pharmacological tools to inhibit RyRs, such as the plant alkaloid ryanodine, are complicated by the concentration-dependent effects in different tissues. For example, low  102  concentrations (< 100 μM) of ryanodine cause persistent opening of the channels which may lead to store depletion (Rousseau et al., 1987, Kanmura et al., 1988, Xu et al., 1994), while higher concentrations are reported to lock RyRs in a closed state to inhibit Ca2+ release (Fill and Copello, 2002). Furthermore, the drugs may also block IP3-mediated Ca2+ signals themselves (either directly or indirectly) without RyR involvement in Ca2+ increase. In our preparation, depletion of the RyR-sensitive Ca2+ stores using a combination of caffeine and low concentration of ryanodine to lock the RyRs in a subconductance state eliminated the ability of UTP to induce Ca2+ oscillations (Fig. 3.10). The concentration of ryanodine (50 μM) we used which is greater than the concentration which is known to lock RyRs in an open state in smooth muscle (Iino et al., 1988; Kanmura et al., 1988). This suggests that the IP3R and RyR both access a common SR Ca2+ store such that the depletion of RyR stores prevents Ca2+ oscillations, as has been demonstrated (Lepretre and Mironneau, 1994; McCarron and Olson, 2008), but does not prove that RyRs participate in the propagation of Ca2+ waves. Therefore, we used a high concentration of ryanodine (200 μM) to lock the RyRs in the closed-configuration and found that UTP-induced Ca2+ waves were not affected (Fig. 3.10). Additionally, we used tetracaine (100 μM), which is not dependent on the opening of RyRs to exert their effects, and also found that the Ca2+ waves were not affected (Györke et al., 1997). This is in contrast to the rat cerebral arteries, where ryanodine (10 μM) inhibited UTP-induced Ca2+ waves (Jaggar and Nelson, 2000). However, it should be noted that in the same preparation, ryanodine also inhibited Ca2+ sparks, likely as a result of SR Ca2+ store depletion. Additionally, another possibility is that in our preparation and in others, the RyRs do not play a role because they are not localized near the IP3Rs. However, well-controlled double-labeling of the IP3Rs and RyRs at electron microscopic resolutions is required before such a conclusion can be made.  103  It is interesting to note that the mechanism of UTP-induced asynchronous Ca2+ waves elicited in this study shares some similarities to the mechanism of Ca2+ oscillations underlying spontaneous vasomotion, as they were not abolished by nifedipine, dependent on a functional SR, and were abolished by antagonists of IP3 (Haddock and Hill, 2002; 2005). This is more significant in light of the fact that asynchronous Ca2+ waves often precede the rhythmic contraction of blood vessels, or vasomotion, which has been observed to occur spontaneously or in response to high concentrations of agonist stimulation, and may have physiological and pathophysiological importance (Gratton et al., 1998; Hudetz et al., 1998, Shimamura et al., 1999; Rücker et al., 2000). In agonist-stimulated vasomotion, asynchronous Ca2+ waves are first initiated without the generation of tension. In the presence of the endothelium, the periodic increases in [Ca2+]i activate cGMP-dependent, Ca2+-sensitive Cl- channels, which cause Clcurrents which depolarize the membrane periodically. The depolarization spreads rapidly through neighbouring cells and activates L-type Ca2+ channels, facilitating Ca2+ influx which facilitates Ca2+-induced Ca2+-release to initiate the next Ca2+ wave, which will then occur simultaneously in all the nearby smooth muscle cells and generate oscillatory vasomotion (Peng et al., 2001; Rahman et al., 2005). Similarly, with spontaneous vasomotion, the trigger for synchronicity of Ca2+ waves is thought to be due to the activation of a chloride-dependent Ca2+ channel (Haddock and Hill, 2002). The resulting depolarization then spreads quickly to neighbouring cells, such that L-type Ca2+ channels are simultaneously activated. The resulting Ca2+ influx then facilitates Ca2+induced Ca2+ release to initiate a synchronous Ca2+ release and contraction. In the study by Haddock and Hill (2002), synchronized Ca2+ waves were abolished upon blockade of L-type Ca2+ channels with nifedipine, but asynchronous Ca2+ oscillations persisted in individual  104  cells, which supports the hypothesis that the entrainment of L-type Ca2+ channels are important in synchronized Ca2+ oscillations. Synchronization of Ca2+ oscillations between VSMCs underlying vasomotion is critically dependent on the coordination of Ca2+ signals within individual VSMCs leading to synchronized Ca2+ responses and the development of simultaneous contractions along the vessel length (Christ et al., 1996). In small vessels, this coordination may be dependent on an intact endothelium, which may be one reason why we did not observe synchronized Ca2+ waves upon UTP stimulation in our preparation (Haddock and Hill, 2005). In summary, the data presented in this article show that multiple Ca2+ translocating proteins are involved in the generation of Ca2+ waves in UTP-stimulated smooth muscle cells of the rat basilar artery. These Ca2+ waves appear to be produced by repetitive cycles of SR Ca2+ release which are mediated by IP3Rs, followed by SERCA-mediated SR Ca2+ re-uptake of Ca2+ entry involving L-type Ca2+ channels, ROCs/SOCs, and reverse-mode NCX. In general, the mechanisms of the Ca2+ waves in the basilar artery are similar to those in the large conduit vessels, which may indicate a common Ca2+ signaling mechanism which initiates and sustains Ca2+ waves in the vasculature.  105  3.5 References Arakawa N, Sakaue M, Yokoyama I, Hashimoto H, Koyama Y, Baba A, et al. (2000). KBR7943 inhibits store-operated Ca(2+) entry in cultured neurons and astrocytes. Biochem Biophys Res Commun 279: 354-7. Arnon A, Hamlyn JM, Blaustein MP (2000). 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Am J Physiol Heart Circ Physiol 292: H152332.  112  CHAPTER 4 – DYSFUNCTION OF ENDOTHELIUM AND SMOOTH MUSCLE CELLS IN SMALL ARTERIES OF A MOUSE MODEL OF MARFAN SYNDROME3 4.1 Introduction Marfan syndrome is an autosomal dominant disorder caused by mutations in the gene encoding for fibrillin-1 and affects many tissues, including those of the cardiovascular, skeletal, ocular and pulmonary systems (Dietz et al., 1991; Pyeritz, 2000; Judge and Dietz, 2005). Fibrillin-1 is the structural glycoprotein for microfibrils, which act as scaffolding proteins for elastin deposition and formation of elastic fibres (Reinhardt et al., 1995). Abnormalities in the formation and integrity of elastic fibres in Marfan syndrome cause weakening of the blood vessel walls and are especially pronounced in the aorta due to its high (~50%) elastin content, which normally allows it to buffer pressure variations during the cardiac cycle and permit constant blood flow and organ perfusion (Rosenbloom, 1993; Safar and London, 1994). Weakening of the aortic wall leads to root dilatation, dissection and eventual rupture, the major cause of death in patients with Marfan syndrome (Murdoch et al., 1972). Marfan syndrome is associated not only with extensive degeneration of elastic fibres, but also with endothelial dysfunction and reduction of smooth muscle contractility in the vasculature (Chung et al., 2007a,b). The alteration of the structural integrity of elastic fibres leads to reduced distensibility and elasticity (Bunton et al., 2001). Furthermore, alteration of fibrillin-1 may also disrupt the attachment of elastic fibres to the cells in the endothelial layer and impair endothelial permeability (Davis, 1994; Sheremet’eva et al., 2004). Although elastic fibre composition is gradually reduced along the arterial tree, elastin remains an important determinant of passive mechanical properties in mesenteric arteries (Dobrin, 1978; Milnor, 1989; Mulvany and 3  A version of this chapter has been published. Syyong HT, Chung AW, Yang HH, van Breemen C (2009). Dysfunction of endothelial and smooth muscle cells in small arteries of a mouse mdoel of Marfan syndrome. Br. J Pharmacol. 158: 1597-1608.  113  Aalkjaer, 1990; Briones et al., 2003; González et al., 2005). However, little is known about how Marfan syndrome affects vessel elasticity and vasomotor function in the resistance vasculature, although dysfunction of these vessels may have important clinical consequences. For example, aneurysms in peripheral and resistance vessels have been reported in patients with Marfan syndrome (Savolainen et al., 1993; Hatrick et al., 1998; Goffi et al., 2000; Lay et al., 2006), although no clear link has been established between resistance artery dysfunction and aortic dilatation and rupture (Jondeau et al., 1999). Furthermore, maximum forearm blood flow in response to acetylcholine (ACh) is reduced in patients with Marfan syndrome (Nakamura et al., 2000), and impairment in flow-mediated vasodilation is also observed (Wilson et al., 1999). In the present study, we compared the stiffness and vascular function of resistance-sized mesenteric arteries from a mouse model of Marfan syndrome with those from their wild-type littermates. We conclude that during the progression of Marfan syndrome, mesenteric arteries show signs of increased stiffness. Furthermore, the contractile function of smooth muscle cells and endothelium-dependent and endothelium-independent vasorelaxation are all markedly impaired. Therefore, we suggest that Marfan syndrome should be considered as a disorder not only in the aorta, but also in the peripheral resistance vasculature.  4.2 METHODS 4.2.1 Experimental animals and tissue preparation Heterozygous (Fbn1C1039G/+) mice were mated to C57BL/6 mice to produce equal numbers of Fbn1C1039G/+ Marfan subjects and wild-type controls as described previously (Judge et al., 2004; Ng et al., 2004; Habashi et al., 2006; Chung et al., 2007a,b). Both strains were housed in the institutional animal facility (Child and Family Research Institute, University of British  114  Columbia) under standard animal room conditions (12h light-12h dark, at 25ºC, 2-5 animals in a cage), and all animal procedures were approved by the institutional Animal Ethics Board. Mice at ages 3 (n = 30), 6 (n = 30), and 10 (n = 30) months were anesthetized with a mixture of ketamine hydrochloride (80 mg·kg-1) and xylazine hydrochloride (12 mg·kg-1) intraperitoneally for experimentation. The mesenteric arcade was excised and placed in ice-cold oxygenated (95% O2-5% CO2) 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-buffered physiological saline salt (HEPES-PSS) solution. Second-order branches of the mesenteric artery with diameters of 130-150 μm were dissected and cut into 2 mm segments.  4.2.2 Mechanical properties "Vessel elasticity" was deduced from the stress–strain curves. In a small vessel myograph (A/S Danish Myotechnology, Aarhus, Denmark), a 2 mm mesenteric artery segment was stretched by increasing the distance between the 2 stainless wires (= increase in length of vascular smooth muscle cell) and held at each length for 1 min. The chambers were kept at 37ºC and bubbled continuously with 95% O2-5% CO2 in HEPES-PSS solution. Initially, 2 wires were adjusted to Lo, at which the vessel was not stretched. The inside circumference of the mesenteric segment was measured as twice the distance between 2 wires, plus the wire circumference, plus 2 wire radii (2x40 µm). The distance between the 2 wires was then increased by 25 µm, and the new length was denoted as "L." The developed force (mN) was divided by the surface area ( = inside circumference of the segment x length of the segment) of the blood vessel segment (mm2) to calculate the wall stress (mN/mm2). The procedure was repeated until the vessel was unable to maintain its tone. The L/Lo and the wall stress were fitted on an exponential curve. "Passive force" was measured by repeating the above procedures in a Ca2+-free HEPES-PSS solution  115  prepared by replacing CaCl2 with 320 μM ethylene glycol tetraacetic acid (EGTA) to eliminate smooth muscle cell contractility. "Total force" was determined by assessing the active contractility at each level of stretch in response to depolarization (60 mM KCl). To study the "reversibility of vessel contractility" after stretching, the mesenteric segment was stimulated with 60 mM KCl at the optimal tension ( L/L0 is approximately equal to 2.0, a value that gives the maximal force generation in response to KCl), then stretched to either L/L0 = 2.5, 3.0, 3.5 or 4.0 for 3 min, and restored to optimal tension. Contraction was induced after 3 min, and the percentage of developed force change compared to the optimal tension was calculated.  4.2.3 Measurement of isometric force Mesenteric artery segments were isometrically mounted in a small vessel wire myograph (A/S Danish Myotechnology, Aarhus N, Denmark) using two 40 μm tungsten wires for measuring generated force. The chambers were kept at 37ºC and bubbled continuously with 95% O2-5% CO2 in HEPES-PSS. Optimal tension (4 mN) was determined in preliminary experiments by subjecting arterial segments to different resting tensions and stimulating with 60 mM KCl. The vessels were stretched to the optimal tension (the maximal force generation given in response to 60 mM KCl, which were the same for control and Marfan mouse mesenteric arteries; 4 mN) for 60 min. The vessels were challenged twice with 60 mM KCl before experiments were continued. Tonic contraction was induced by 3 μM phenylephrine (PE) and all mechanistic studies were performed at this concentration. Concentration-response curves of PE-induced contraction were constructed, and the negative logarithm (pD2) of the concentration of PE giving  116  half-maximum response (EC50) was assessed by linear interpolation on the semilogarithm concentration-response curve [pD2 = -log(EC50)]. To determine endothelium-dependent and endothelium-independent relaxations, vessels were pre-contracted with 3 μM PE before making cumulative applications of acetylcholine (ACh) or sodium nitroprusside (SNP) (1 nM – 10 μM), respectively. Control concentration response curves to ACh were produced and compared to those in vessels pre-treated with Nω-Nitro-Larginine methyl ester (L-NAME, 200 μM), indomethacin (10 μM), or catalase (1000 U·mL-1) for 30 min. Other vessels were pre-treated with L-NAME (200 μM) and indomethacin (10 μM) for 30 min and concentration-response curves to ACh were produced. These responses were then repeated in the presence of catalase (1000 U·mL-1) or carbenoxolone (100 μM). Percent relaxation was calculated as the percent decrease in force with respect to the initial PE (3 μM)induced precontraction, and the percent relaxation was used to construct the concentrationresponse curves of ACh-induced relaxation.  4.2.4 Measurement of intracellular Ca2+ The arterial rings were loaded with Fluo-4AM (5 μM with 5 μM Pluronic F-127, 1 hr at 37ºC) and isometrically mounted, followed by a 30 min washout time in HEPES-PSS. Sustained Ca2+ waves were induced by 3 μM PE, and all mechanistic studies were done at this concentration. Images were acquired on an upright Olympus BX50WI microscope with a 40x water-dipping objective (NA 0.9) and equipped with an Ultraview confocal imaging system (Perkin-Elmer). The rate of image acquisition was 3 frames/s. The tissue was illuminated using the 488nm line of an Argon-Krypton laser and a high-gain photomultiplier tube collected the emission at wavelengths between 505 and 550 nm. The scanned regions correspond to a 91.69 x  117  66.68 μm area (or 248 x 328 pixels). The representative fluorescence traces shown reflect the averaged fluorescence signals from a region of 3 x 3 pixels (1.69 μm2) of the smooth muscle cell. The frequency of Ca2+ waves was determined by counting the number of waves occurring within 50s. The measured changes in Fluo-4 fluorescence level are proportional to the relative changes in [Ca2+]i. All parameters (laser intensity, gain, etc) were maintained constant during the experiment. The confocal images were analyzed off-line with the Ultraview 5.5 Software (Perkin-Elmer). Fluorescence traces were extracted from the movies to exclude nuclear regions and traces were normalized to initial fluorescence values.  4.2.5 Detection of H2O2 production from endothelial cells Mesenteric artery segments of control and Marfan mice were cut into rings and then opened longitudinally. The vascular strip was loaded with dichlorodihydrofluorescein diacetate (DCF, 5 μM), a peroxide-sensitive fluorescence dye (Ohba et al., 1994), for 10 min at 25°C. Images were acquired on an upright Olympus BX50WI microscope with a 60x water-dipping objective (NA 0.9) equipped with an Ultraview confocal imaging system (Perkin-Elmer, USA). The tissue was illuminated using the 488nm line of an argon-krypton laser and a high-gain photomultiplier tube collected the emission at wavelengths between 505 and 550 nm. The tissue was preincubated with indomethacin (10 μM) and L-NAME (200 μM) for 30 min, and then stimulated with ACh (10 μM). Additionally, the effect of catalase (1000 U·mL-1) on the ACh-induced increase in fluorescence intensities was also determined.  118  4.2.6 Statistics Values are expressed as mean ± standard error (SEM) from at least six independent experiments. Statistical analysis and construction of concentration-response curves were performed using GraphPad Prism 4.0 software (San Diego, CA, USA). Differences between control and Marfan groups were analyzed by Student’s two-tailed t-test. Statistical significance was defined as P-values <0.05.  4.2.7 Drugs, solutions, and chemicals HEPES-PSS containing (in mM) NaCl 130, HEPES 10, glucose 6, KCl 4, NaHCO3 4, CaCl2 1.8, MgSO4 1.2, KH2PO4 1.18, and ethylenediaminetetraacetic acid (EDTA) 0.03 (pH 7.4) was used for all studies. Hi-K+ (60 mM extracellular K+) HEPES-PSS was identical in composition to normal HEPES-PSS with the exception of (in mM) NaCl 74 and KCl 60. PE, SNP, L-NAME, catalase, carbenoxolone, indomethacin, and pluronic F-127 were obtained from Sigma-Aldrich (Oakville, Ontario, Canada). Fluo-4AM and dichlorodihydrofluorescein diacetate (DCF) were purchased from Molecular Probes (Eugene, OR). Superoxide dismutase was obtained from Calbiochem (San Diego, CA, USA)  4.3 RESULTS 4.3.1 Vessel stiffening and weakness in marfan mesenteric artery In the measurement of stiffness, stress increases exponentially as a function of vessel diameter. At 3 months of age, the fitted curves for the stress-strain relationship from control and Marfan vessels were not significantly different (Fig. 4.1A). However, at 6 and 10 months of age,  119  the slope of the stress-strain curves from the Marfan vessels was increased compared with that of the control vessels, which indicates increased stiffness (Fig. 4.1B,C). However, true elasticity also implies the capability to return to the original conformation or length, a situation which is analogous to an elastic band. To test this elasticity, we measured the “reversibility of mesenteric artery elasticity” by comparing 2 stress-strain curves from control and Marfan vessels at 6 and 10 months of age. Since we did not observe any increase in stiffness in the Marfan vessels at 3 months of age, these measurements were not done in this group. We found that the apparent vessel elasticity in the second measurement remained for the most part unchanged in the control vessels, but was highly increased in the Marfan vessels (Fig. 4.2). This may suggest a weakening of the vessel wall at 10 months of age, and a similar observation was also found at 6 months of age (data not shown). We also measured the effects of stretch on contractility in 6 and 10 month old mice. After distending to L/Lo = 2.5, the 60mM KClinduced contraction in the Marfan vessels at both 6 and 10 months of age (normalized to the optimal contraction recorded at L/Lo = 2.0) was not significantly decreased. However, distending to L/Lo = 3.0 reduced the Marfan vessel contraction to 74.0 ± 5.9% and 42.0 ± 15.8%, while contraction in the control vessels were reduced to 93.2 ± 1.5% and 69.1 ± 3.0% at 6 and 10 months of age, respectively (Fig. 4.3). Further distension to L/Lo = 3.5 reduced contractility in Marfan vessels to 43.5 ± 8.7% and 24.6 ± 9.6%, while in control contraction was reduced to 76.8 ± 0.3% and 51.7 ± 3.1%. Distention to L/Lo = 4.0 reduced contractility in Marfan vessels to 22.2 ± 5.0% and 5.3 ± 3.2%, while in controls it was 58.4 ± 3.1% and 29.0 ± 3.3%. Thus impairment due to stretch was significantly greater in the Marfan than the control mice.  120  A  B 75  Stress (mN/mm 2)  Stress (mN/mm 2)  75  50  25  0 0.0  0.5  1.0  1.5  2.0  2.5  3.0  ΔL/Lo  *  50  * 25  0 0.0  * 0.5  1.0  1.5  2.0  2.5  3.0  ΔL/Lo  C  *  75  Control  *  2  Stress (mN/mm )  *  *  50  Marfan  25  0 0.0  0.5  1.0  1.5  2.0  2.5  3.0  3.5  ΔL/Lo  Figure 4.1 Vessel elasticity during aging in mesenteric arteries. Elasticity was tested in Marfan (triangle) and control (filled square) mice from (A) 3, (B) 6, and (C) 10 months of age (* P < 0.05 vs. control, n = 8-12).  121  A  B 100  Stress (mN/mm 2)  Stress (mN/mm 2)  70 60 50 40 30 20 10 0 0  1  2  3  4  ΔL/Lo  First measurement  75 50 25 0 0  1  2  3  4  ΔL/L o  Second measurement  Figure 4.2 Reversibility of mesenteric artery elasticity. In (A) control and (B) Marfan mice at 10 months of age, reversibility of elasticity was tested by performing two consecutive stress-strain measurements. Vessel elasticity from the first (square) and second (triangle) measurement was compared in each group. Representative results are shown from 3 independent experiments.  122  % force generation normalized to optimal  A 110 100 90 80 70 60 50 40 30 20 10 0  * * *  2.5  3.0  3.5  4.0  ΔL/Lo  B % force generation normalized to optimal  100  *  75 50  * *  25 0 2.5  3.0  3.5  4.0  ΔL/Lo Control  Marfan  Figure 4.3 Reversibility of contractile function of mesenteric arteries from control and marfan mice. Contractile function was determined from both control and Marfan mice (A) 6 and (B) 10 months of age after stretching at L/Lo = 2.5, 3.0, 3.5, and 4.0. After being stretched for 3 minutes and restored to optimal tension, vessels were stimulated with 60 mM KCl (* - P < 0.05 vs. control, n = 6-8). Values (%) are changes of force generation normalized to that at the optimal tension.  4.3.2 Reduced contractile function of smooth muscle cells in marfan mesenteric artery To determine if smooth muscle contractile function is affected in Marfan mesenteric arteries, we stimulated the vessels with both KCl (60mM) depolarization and PE. From 3 months of age 123  onward, vasoconstriction in response to KCl-induced depolarization in Marfan vessels was significantly less (2.41 ± 0.21 mN) compared to that in age-matched controls (3.31 ± 0.23 mN), a 28% reduction (Fig. 4.4A). At 6 and 10 months of age, contractility of the Marfan vessels was reduced to only 3.15 ± 0.39 mN (30% reduction) and 2.51 ± 0.32 mN (52% reduction), respectively, compared to 4.43 ± 0.38 mN and 5.15 ± 1.06 mN in the age-matched control vessels. Application of PE produced tonic contraction in a concentration-dependent manner at all ages in both control and Marfan mice (Fig. 4.4B). Although there was no significant difference at 3 months of age, maximal contraction was significantly reduced at 6 and 10 months of age in the Marfan vessels, 3.29 ± 0.60 mN and 2.60 ± 3.1 mN, respectively, compared to 5.49 ± 0.62 mN and 4.20 ± 0.70 mN in age-matched controls (Fig. 4.4B). However, there were no significant differences in pEC50 values at all ages (3 months: Control 6.37 ± 0.17, Marfan 6.02 ± 0.25; 6 months Control 5.83 ± 0.21, Marfan 5.72 ± 0.21; 10 months Control 5.95 ± 0.23, Marfan 5.66 ± 0.42). To further examine smooth muscle contractile function, we measured the active force in response to KCl-depolarization. Control arteries at 3 months of age generated active force over a range of strain L/Lo, 0.2 to 1.7, while control arteries at 6 and 10 months of age generated active force over a range of strain L/Lo, 0.2 to 2.0 (Fig. 4.5). However, this range was markedly reduced in the Marfan vessels at all age groups. Furthermore, the maximum active force generated in the Marfan mesenteric vessels was also markedly reduced at all age groups by 52% (3 months), 56% (6 months), and 66% (10 months) compared with the age-matched controls, respectively.  124  A  *  *  B  *  6  7  Contraction (mN)  Contraction (mN)  7  5 4 3 2 1  *  *  6  10  6 5 4 3 2 1 0  0 3  6  3  10  Age (Months)  Age (Months)  Control  Marfan  Figure 4.4: Isometric force measurement in response to KCl and PE in control and marfan mice. Maximal force generated in response to (A) 60 mM KCl and (B) PE (3 μM) was compared between control and Marfan vessels (* - P < 0.05 vs. control, n = 812).  A  B 5  5  Active Force (mN)  Active Force (mN)  6  4 3 2 1 0 -1  0.25 0.50 0.75 1.00 1.25 1.50 1.75  ΔL/Lo  4 3  * *  2  *  1  *  0 0.5 -1  1.0  1.5  2.0  2.5  ΔL/Lo  C Active Force (mN)  7 6  Control  5 4 3  *  2  * *  Marfan  * *  1 0 -1  0.5  1.0  1.5  2.0  2.5  ΔL/Lo  125  Figure 4.5: Active force in control and marfan mice. Active force, the difference between total and passive force, was compared between control (filled square) and Marfan (triangle) vessels from (A) 3, (B) 6, and (C) 10 months of age (* - P < 0.05 vs. control, n = 8-12). 4.3.3 Frequency of PE-induced Ca2+ waves is reduced in marfan syndrome In both control and Marfan vessels, stimulation with PE (3 μM) induced a large transient Ca2+ response in both control and Marfan vessels at all ages, which was followed by repetitive transient elevations in Ca2+ which originate in distinct intracellular foci and then spread out as waves over the length of the smooth muscle cell (Appendix A). The Ca2+ waves were asynchronous and did not propagate intercellularly. The frequency of PE-stimulated Ca2+ waves increased in a concentration-dependent manner at all ages and closely paralleled the development of force in both control and Marfan mice (pEC50; 3 months: Control 6.49 ± 0.26, Marfan 6.28 ± 0.30 ; 6 months Control 6.01 ± 0.19, Marfan 5.79 ± 0.21; 10 months Control 6.03 ± 0.12, Marfan 6.01 ± 0.21). However, the average frequency of the PE-stimulated Ca2+ waves was significantly reduced in Marfan vessels compared to control vessels at 6 (Control 0.075 ± 0.005 Hz, Marfan 0.033 ± 0.004 Hz; P < 0.05) and 10 months (Control 0.052 ± 0.007 Hz, Marfan 0.031 ± 0.003 Hz; P < 0.05), but not at 3 months (Control: 0.068 ± 0.006 Hz, Marfan: 0.056 ± 0.005 Hz) of age (Appendix B). The average frequency of PE-stimulated Ca2+ waves was also reduced in control mice at 10 months compared to 6 months of age (0.052 ± 0.006 Hz vs. 0.075 ± 0.005 Hz; P < 0.05) (Appendix B). The number of cells displaying at least one Ca2+ wave was also significantly reduced in Marfan mice at 6 (Control: 74.51 ± 5.45%, Marfan: 49.71 ± 6.72%; P < 0.05) and 10 months (Control: 52.67 ± 5.93%, Marfan: 37.84 ± 5.52%; P < 0.05) of age, but not at 3 months (Control: 82.70 ± 3.68 %, Marfan 71.97 ± 5.42%) of age. At 6 months of age, more cells display  126  Ca2+ waves in response to PE-stimulation compared to at 10 months of age (71.68 ± 5.20% vs. 48.38 ± 6.65%) (Appendix B).  4.3.4 Reduced endothelium-dependent and independent relaxation in marfan mesenteric artery In PE (3 μM)-precontracted vessels from control and Marfan mice, addition of ACh resulted in a concentration-dependent relaxation at all age groups (Fig. 4.6). There was no difference in the maximal response (Emax) to ACh (10 μM)-induced relaxation between Marfan and control vessels in mice at 3 months of age, although Emax in Marfan vessels at 6 and 10 months of age were 69.7% and 44.9% of the controls, respectively. It should be noted that the maximal relaxation values did not tend to change with increasing age in the control animals. Values for pEC50 for ACh indicated that at 6 months of age, the Marfan vessels were less sensitive to ACh than the controls (6.49 ± 0.21 vs. 7.17 ± 0.30, respectively). This difference was not seen at 3 (7.17 ± 0.12 vs. 7.23 ± 0.11 in control and Marfan, respectively) and 10 months of age (6.03 ± 0.30 vs. 6.23 ± 0.16, Marfan and control, respectively).  127  A  B 100  % relaxation  % relaxation  100 75 50 25 0 -10  -9  -8  -7  -6  -5  -4  75  25 0 -10  -3  log[ACh]  *  50  -9  -8  -7  -6  -5  -4  -3  log[ACh]  C  % relaxation  100 75  *  Control  50  Marfan 25 0 -10  -9  -8  -7  -6  -5  -4  -3  log[ACh]  Figure 4.6 Endothelium-dependent relaxation in mesenteric arteries from control and marfan mice. Concentration-response curve of acetylcholine (ACh)-induced relaxation in phenylephrine (3 μM)-precontracted mesenteric arteries from control and Marfan mice at (A) 3, (B) 6, and (C) 10 months of age (* - P < 0.05 vs. control, n = 812). Endothelium-independent vasodilatation was studied by the addition of sodium nitroprusside (SNP), a nitric oxide (NO) donor which bypasses endogenous NO production by endothelial cells, and resulted in complete dilatation in PE-precontracted control and Marfan mesenteric vessels at 6 and 10 months of age (Fig. 4.7). Although there was no difference in Emax of SNPrelaxation between control and Marfan vessels, there was a significant increase in the pEC50 in Marfan vessels at 6 months (pEC50 = 5.64 ± 0.11, control pEC50 = 7.34 ± 0.04) and 10 months (pEC50 = 5.99 ± 0.07, control pEC50 = 6.99 ± 0.14), indicating that the Marfan vessels are less sensitive to NO at these ages. However there was no significant difference in pEC50 at 3 months of age (data not shown). 128  B 125  125  100  100  % relaxation  % relaxation  A  75 50 25 0  50 25 0  -9 -25  75  -8  -7  -6  -5  -4  -3  -9  -8  -25  log[SNP]  Control  -7  -6  -5  -4  -3  log[SNP]  Marfan  Figure 4.7 Endothelium-independent relaxation in mesenteric arteries from control and marfan mice. Concentration-response curve of sodium nitroprusside (SNP)-induced relaxation in phenylephrine (3 μM)-precontracted mesenteric arteries from control and marfan mice at (A) 6 and (B) 10 months of age (* - P < 0.05 vs. control, n = 8-12). We then assessed the ACh response in the presence of L-NAME (200 μM), an inhibitor of nitric oxide synthase. Because impairment of ACh-induced vasorelaxation was most evident in the Marfan mice at 10 months of age, we focused our experiments on this age group. L-NAME preincubation inhibited maximal relaxation in the control vessels to 57.1 ± 4.6% of control values and but did not change potency (pEC50: 5.88 ± 0.16). However, L-NAME neither significantly affect maximal relaxation (Emax: 38.7 ± 3.0%) to ACh nor potency (pEC50: 5.95 ± 0.20) in Marfan vessels. To determine the contribution of the cyclooxygenase (COX) pathway, vessels were preincubated with indomethacin (10 μM), a non-specific COX inhibitor. In control vessels, indomethacin significantly increased maximal relaxation (Emax: 99.4 ± 2.2%) and potency (pEC50: 6.58 ± 0.16), while in vessels from Marfan mice, maximal relaxation (Emax: 36.0 ± 3.3%) and potency (pEC50: 5.56 ± 0.17) was decreased (Fig. 4.8A,B).  129  Neither maximal contraction (Emax; absence of indomethacin: 4.20 ± 0.70 mN; presence of indomethacin: 3.92 ± 0.41 mN) nor potency to PE (pEC50; absence of indomethacin: 5.95 ± 0.23; presence of indomethacin: 6.12 ± 0.20) was significantly different in the presence of indomethacin in control mice. However, maximal contraction was significantly increased in the Marfan mice (Emax; absence of indomethacin: 2.60 ± 0.31 mN; presence of indomethacin: 4.62 ± 0.35 mN) but did not significantly change potency (pEC50; absence of indomethacin: 5.66 ± 0.42; presence of indomethacin: 5.78 ± 0.31) (data not shown).  4.3.5 Nature of EDHF The contribution of H2O2 in the EDHF-mediated relaxation was examined by the inhibitory effect of catalase (1000 U·mL-1), an enzyme that dismutates H2O2 to form water and oxygen. In the presence of indomethacin (10 μM) and L-NAME (200 μM), the addition of catalase to control vessels markedly reduced maximal relaxation (Emax) to 40.3 ± 7.8% of control, but did not significantly affect potency (pEC50: 6.13 ± 0.32). Similarly, the addition of catalase to Marfan vessels markedly reduced Emax to 14.3% ± 2.3% of control values and decreased the potency (pEC50: 5.27 ± 0.31) (Fig. 4.8C,D). To determine the role of gap junctions in the EDHFmediated relaxation response, we used carbenoxolone (100 μM), a derivative of glycyrrhetinic acid and uncoupler of gap junctions (Tare et al., 2002). In both control and Marfan mice, carbenoxolone had no inhibitory effect on the EDHF-mediated relaxation (Fig. 4.8C,D). In the presence of L-NAME alone, catalase reduced Emax to 42.3 ± 3.2% in control vessels but did not change potency (pEC50: 5.82 ± 0.23). In Marfan vessels, catalase and L-NAME reduced Emax to 18.6 ± 1.7% but did not affect potency (pEC50: 6.38 ± 0.24). Catalase alone did not change potency (pEC50: 5.99 ± 0.20) or maximal relaxation (Emax: 81.5 ± 5.6%) in control  130  mice, though in Marfan mice, catalase decreased potency to ACh (pEC50: 5.80 ± 0.14), and decreased maximal relaxation (Emax: 30.2 ± 3.3%) (Table 1).  Control  Marfan  A  B Control L-NAME Indo  75  * *  50 25 0 -10  -9  -8  -7  100  % relaxation  % relaxation  100  -6  -5  -4  50  * 25 0 -10  -3  Control L-NAME Indo  75  -9  -8  log[ACh]  -7  -6  -5  -4  -3  log[ACh]  C  D  75  100  L-NAME + Indo L-NAME + Indo + catalase L-NAME + Indo + carbenoxolone  50  *  25 0 -10  -9  -8  -7  -6  log[ACh]  -5  -4  -3  % relaxation  % relaxation  100  75  L-NAME + Indo L-NAME + Indo + catalase L-NAME + Indo + carbenoxolone  50 25 0 -10  * -9  -8  -7  -6  -5  -4  -3  log[ACh]  Figure 4.8 Effects of Nω-Nitro-L-arginine methyl ester (L-NAME), indomethacin (Indo), and catalase on acetylcholine (ACh)-induced relaxation in control and marfan mice. The concentration-response curves of ACh are shown. (A) Control and (B) Marfan vessels were preincubated with L-NAME (200 μM) or indomethacin (10 μM) for 30 min and then contracted with phenylephrine (3 μM). (C) Control and (D) Marfan vessels were preincubated with catalase (1000 U·mL-1) or carbenoxolone (100 μM) in the presence of L-NAME (200 μM) and indomethacin (10 μM) for 30 min and then contracted with phenylephrine (3 μM). (* - P < 0.05, n = 8-12).  131  Table 1 Effects of different treatments on potency (pEC50) and maximal (% of maximal response, Emax) acetylcholine (Ach)-induced relaxation in control and Marfan mice at 10 months of age  Strain  Control  Marfan  Emax No treatment L-NAME L-NAME + INDO L-NAME + INDO + catalase L-NAME + INDO + carbenoxolone L-NAME + catalase Catalase INDO  84.4 ± 4.8 57.1 ± 4.6 * 57.8 ± 4.3 40.3 ± 7.8 # 55.1 ± 5.2 42.3 ± 3.2 * 81.5 ± 5.6 * 99.4 ± 2.2 *  44.9 ± 3.2 38.7 ± 3.0 25.3 ± 4.1 * 14.3 ± 2.3 # 24.3 ± 2.8 18.6 ± 1.7 30.2 ± 2.4 * 36.0 ± 3.3 *  pEC50 No treatment L-NAME L-NAME + INDO L-NAME + INDO + catalase L-NAME + INDO + carbenoxolone L-NAME + catalase Catalase INDO  6.03 ± 0.30 5.88 ± 0.16 5.73 ± 0.18 6.13 ± 0.32 6.23 ± 0.24 5.82 ± 0.23 5.99 ± 0.20 6.58 ± 0.16*  6.23 ± 0.16 5.95 ± 0.20 5.50 ± 0.12 * 5.27 ± 0.31 # 5.58 ± 0.17 6.38 ± 0.24 5.80 ± 0.14 * 5.56 ± 0.17 *  Abbreviations: L-NAME, Nω-Nitro-L-arginine methyl ester, INDO, indomethacin * - P < 0.05 without inhibitors in respective groups, # - P < 0.05 with L-NAME + INDO in respective groups. 4.3.6 Role of superoxide To determine the role of reactive oxygen species, vessels were preincubated with superoxide dismutase (150 U·mL-1), an enzyme that converts superoxide to H2O2. In the control mice, superoxide dismutase had no significant effect on either the maximal contraction or potency in PE (3 μM)-induced contractions. Moreover, ACh-induced relaxation was also not affected. In contrast, superoxide dismutase potentiated the PE (3 μM)-induced contraction in the Marfan vessels and increased maximal contraction to control levels (Emax: 4.71 ± 0.61 mN).  132  Maximal relaxation to ACh was also improved (Emax: 74.5 ± 3.8%) and potency was also increased (pEC50; 6.71 ± 0.13) (Fig. 4.9).  A  *  6  B  8  4  pEC50  Emax (mN)  5  3 2  *  7  6  1 0  5  Marfan  D  100  *  75  pEC50  Emax (% relaxation)  C  Control  50  Control  Marfan  8  *  7  6  25 0  5 Control  Marfan  No Treatment  Control  Marfan  SOD (150 UymL-1)  Figure 4.9 Effect of superoxide dismutase on phenylephrine (3 μM)-stimulated contraction and acetylcholine-mediated relaxation in mesenteric arteries from control and marfan mice. Bar graphs show (A) Emax and (B) pEC50 in response to phenylephrine (PE, 3 μM) in the presence and absence of superoxide dismutase (SOD, 150 U·ml-1) at 10 months of age, while (C) Emax and (D) pEC50 show responses to acetylcholine (ACh, 10 μM-mediated relaxation. (* - P < 0.05 vs. control, n = 5-8, SOD, superoxide dismutase). 4.3.7 H2O2 production of endothelial cells H2O2 production by endothelial cells was detected in the experiments using a laser confocal microscope with DCF, a peroxide-sensitive fluorescence dye. ACh (10 μM) application caused a significant increase in the DCF fluorescence intensity in endothelial cells, which was  133  unaffected by pretreatment with indomethacin (10 μM) and L-NAME (200 μM). When the vessel was pre-treated with catalase (1000 U·mL-1) in the presence of both indomethacin and LNAME, the ACh-induced increase in the fluorescence intensity was abolished (Fig. 4.10).  10 μM ACh  0.5 F/Fo 50s (-) catalase  (+) catalase  Figure 4.10 Production of endothelial hydrogen peroxide. Acetylcholine (ACh, 10 μM)-induced production of hydrogen peroxide (H2O2) by the endothelium detected as an increase in fluorescence intensity in dichlorodihydrofluorescein diacetate (DCF)-loaded endothelial cells in small mesenteric arteries of mice. The ACh-induced increase in fluorescence intensity was abolished when the artery was preincubated with catalase (1000 U·mL-1). All experiments were performed in the presence of Nω-Nitro-L-arginine methyl ester (L-NAME, 200 μM) and indomethacin (10 μM). Representative traces shown are typical of the responses obtained in 23 cells from 4 mice. 4.4 Discussion Using a genetically defined and validated mouse model of Marfan syndrome, we demonstrated increased vessel stiffness, reduced smooth muscle contractile function associated with decreased frequency of Ca2+ waves, decreased resistance to mechanical stress, and impaired endothelium-dependent and endothelium-independent relaxation in the small mesenteric arteries. We used appropriate control littermates to distinguish between observations owing to the  134  pathogenesis of Marfan syndrome from those owing to the physiological process of aging. We concluded that Marfan syndrome is a genetic disorder which affects not only the aorta and other large blood vessels, but also the resistance vasculature. Degeneration of elastic fibers during the progression of Marfan syndrome is expected to decrease blood vessel elasticity. Indeed, vessel elasticity was decreased in Marfan vessels compared to the age-matched controls, although this was not apparent until 6 months of age (Fig. 4.2). This delayed effect may be due to the relative paucity of elastic fibers in the resistance vessels, as decreased vessel elasticity can always be seen in aorta from the same mouse model of Marfan syndrome starting at 3 months of age (Chung et al., 2007a). Secondly, vessel wall weakening was also indicated by irreversible changes in vessel wall elasticity (Fig. 4.3) and reduced contractility after exposure to stretching (Fig. 4.4). Reduced contraction and irreversible changes in elasticity after stretching may be indicative of the ‘breakage’ of the physical linkage between smooth muscle cells and elastic fibers, which results in the changing of the phenotype in smooth muscle cells (Bunton et al., 2001). It should be noted that aging is also associated with decreased vessel elasticity (Laurant et al., 2004). Indeed, the elasticity of control vessels progressively decreased during aging, although the differences in elasticity between Marfan and control vessels persisted. This suggested that the increased vessel wall stiffness was due to the progression of Marfan syndrome. The present study is the first to report aberrant contraction of smooth muscle cells in resistance vessels in Marfan syndrome. We showed that contraction in response to membrane depolarization and agonist-stimulation is suppressed. The decrease in the L/Lo range in which active force is generated suggests that at high distention, the association between smooth muscle cells and extracellular matrix might be disrupted (Fig. 4.4, 4.5), while reduced active force may  135  be due to low intrinsic force generation of the contractile filaments or modifications in the coupling between the contractile elements and the cytoskeleton in smooth muscle cells (Rembold and Murphy, 1990). Additionally, decreased association between smooth muscle cells and elastic fibers would reduce the strain on the smooth muscle cells and blunt their response to agonist stimulation (Bunton et al., 2001). Furthermore, upregulation of matrixmetalloproteinase-2 and matrixmetalloproteinase-9 in Marfan syndrome may inhibit Ca2+ entry from the extracellular space and reduce vessel contraction (Chew et al., 2004; Chung et al., 2007a; Chung et al., 2008), although further investigation is required to elucidate possible involvement of calcium signaling and myofilament contractile mechanisms. Finally, the rate of vascular smooth muscle cell apoptosis is increased in Marfan syndrome, which may lead to vessel wall weakness and decreased contractile force (Nataatmadja et al., 2003). We also show in Appendix B that PE-induced Ca2+ waves are associated with tonic contraction in both control and Marfan mice. This is similar to that reported in other vessel preparations, where the asynchronous nature of the Ca2+ waves explains how summation of individual-cell Ca2+ waves can lead to tonic contraction, as the summation of Ca2+ signals in all the cells averages out to be a steady state Ca2+ increase in whole vessels (Ruehlmann et al., 2000; Mauban et al., 2001). A strong relationship between frequency of agonist-induced Ca2+ waves and tonic contraction, as well as the relationship between the number of cells displaying Ca2+ waves and generation of force, has been documented in other vascular preparations (Lee et al., 2001; Dai et al., 2007). A higher frequency of Ca2+ waves can enhance the myofilament activation by increasing average [Ca2+]i over time. Furthermore, the activation of certain frequency-sensitive enzymes can potentially affect the level of contraction, such as Ca2+calmodulin kinase II, which has been found to be sensitive to the frequency of Ca2+ spikes in  136  vitro (De Koninck and Schulman, 1998). In mice at 6 and 10 months of age, the significant reduction in tonic contraction in the Marfan group is correlated with significantly decreased Ca2+ wave frequency and number of cells displaying Ca2+ waves. This implies that the decreased occurrence and/or frequency of Ca2+ waves are associated with decreased tonic contraction. The reduction in frequency of PE-stimulated Ca2+ waves in Marfan mesenteric vessels may suggest inhibition of SR refilling. This is because a mechanism by which Ca2+ wave frequency could be decreased is by a reduced rate of refilling of the SR Ca2+ store. Since SR luminal Ca2+ can regulate inositol-1,4,5-triphosphate receptor channel opening probability, a reduced rate of SR Ca2+ refilling can lead to decreased frequency of SR Ca2+ release at the wave initiation site (Meldolesi and Pozzan, 1998). In resistance vessels, extracellular Ca2+ influx through L-type Ca2+ channels is central in the control of vascular tone and plays a significant role in maintaining Ca2+ homeostasis and contraction (Mulvany and Aalkjaer, 1990; Nelson et al., 1990; Hughes, 1995). The upregulation of matrixmetalloproteinase-2 and matrixmetalloproteinase-9 enzymes in the same mouse model of Marfan syndrome used in these studies (Chung et al., 2007a; Chung et al., 2008), and the suggestion that both enzymes may inhibit Ca2+ influx through L-type Ca2+ channels (Chew et al., 2004). This inhibition may occur through their interactions with specific cell proteins such as intercellular adhesion molecule-1 or stimulate proteinase-activated receptors and activate signaling pathways, all of which could lead to blockade of Ca2+ channels (Macfarlane et al., 2001; Fiore et al., 2002; Marutsuka et al., 2002). It should also be noted that there is a significant reduction in PE-induced Ca2+ wave frequency and cell recruitment in control mice from 6 to 9 months of age, although maximal tonic contraction is not similarly affected. Since advancing age has generally not been associated with decreased contraction in response to PE stimulation in resistance arteries (Hüsken et al.,  137  1994; Moreau et al., 1998; Gros et al., 2002), and in mice at 30 months of age, neither maximal peak height of Ca2+ release in response to PE nor L-type Ca2+ current are affected (del Corsso et al., 2006), this may suggest that although Ca2+ wave signaling is affected during aging, tonic contraction is maintained by way of a compensatory mechanism. For example, no oscillatory Ca2+ signaling has been observed in humans, although PE-induced tonic contraction is supported by Rho-kinase (Crowley et al., 2002). How then, can the reduced frequency of Ca2+ waves during in the control animals be explained? It has been suggested that the close association between the superficial SR and the plasma membrane is essential for SR refilling (Lee et al., 2002; Fameli et al., 2007), and separation of this association has been shown to decrease frequency of Ca2+ waves presumably through reduced Ca2+ refilling (Lee et al., 2005). However, this hypothesis cannot be further investigated in this model without the use of electron microscopy. Nonetheless, the frequency of PE-induced Ca2+ waves is significantly reduced in the Marfan animals compared to their aged-matched controls at both 6 and 10 months of age, suggesting that the effects of Marfan syndrome play a greater role than the process of aging in this study. The endothelium releases a variety of vasoactive mediators, including prostaglandins, NO, and EDHF to regulate smooth muscle contractility and thus vascular smooth muscle tone (Ramsey et al., 1995; Boutouyrie et al., 1997; Wilkinson et al., 2002; Vanhoutte, 2004). The present study demonstrated that in Marfan mesenteric vessels, endothelium-dependent relaxation stimulated by ACh was significantly impaired at 6 and 10 months (Fig. 4.6), suggesting an impairment of NO release. In Marfan syndrome, the endothelium is a likely target as Fbn-1 rich microfibrils are present in the connective tissue immediately subjacent to arterial endothelial cells (Davis, 1994; Kielty et al., 1996). Marfan syndrome is also associated with elevated plasma  138  levels of homocysteine, which attenuate endothelial function and limits NO bioavailability (Giusti et al., 2003; Jiang et al., 2005). Endothelial Akt/eNOS phosphorylation and mechanosignaling can be compromised through increased vessel wall stiffness, further reducing endothelium-dependent vasorelaxation (Peng et al., 2003). However, there is conflicting data with regard to agonist-induced vasodilator responses in Marfan syndrome; Nakamura and colleagues (2000) showed inhibition of ACh-induced relaxation in the brachial artery of Marfan syndrome patients, while Wilson and colleagues (1999) showed that vasodilator responses to ACh and bradykinin were unaffected in the same artery. In addition, both groups found no impairment of the response to exogenous nitrovasodilators, while our studies demonstrated an approximately 100-fold reduction of sensitivity of smooth muscle cells of Marfan mice to NO at 6 months of age and a 10-fold reduction at 10 months of age (Fig. 4.7). This reduced sensitivity may be attributed to a number of factors. First of all, bioavailability of NO is decreased due to excessive amounts of reactive oxygen species in the pathogenesis of cardiovascular diseases (Cai and Harrison, 2000; Faraci and Didion, 2004). NO bioavailability is also decreased during ageing, which may explain the difference in SNP sensitivity between control and Marfan vessels at 6 and 10 months of age (Newaz et al., 2006; Donato et al., 2007). Finally, the predisposition of the medial layer to degeneration and fibrosis in Marfan syndrome may also physically inhibit the ability of the vessel to dilate (Dietz et al., 1991; Pyeritz, 2000). The findings from this study may have potentially important clinical implications, but should be viewed in the context of the existing in vivo data from human subjects. The apparent discrepancies highlighted above between human subjects and our studies may be explained by the following reasons: 1) relatively small groups of Marfan syndrome and non-Marfan syndrome patients were recruited for these studies (usually 20 or less), 2) a wide range of ages of Marfan  139  patients were recruited (from 11 to 61 years old), and 3) the vessels studied were not true resistance (<500 μm) vessels. As indicated by the data from the present study and others, pathogenesis of Marfan syndrome in the vasculature with respect to the functional properties varied during aging (Chung et al., 2007a). Therefore, combining both pediatric and adult patients in the same study may perturb the results and data interpretation. Furthermore, the conflicting results should also be viewed in the context of our mouse model. There are over 600 genetic mutations that have been identified to cause Marfan syndrome (Williams et al., 2008). While missense mutations account for slightly over 60% of the mutations, 78% of the point mutations locate in the cbEGF modules and affect Ca2+ binding. A further 12% of these mutations are recurrent and affect a mutation hotspot, CpG, for a cysteine residue, representing the most common mutation in classic Marfan syndrome and providing the basis of the mouse model used in our studies (Boileau et al., 2005; Gray and Davies, 1996). Therefore, although our model is useful to investigate the general pathogenesis of the most common type of Marfan syndrome, it may not be representative of all cases. Although advancing age is associated with derangement of endothelial cells leading to a decrease in NO production (Gerhard et al., 1996), the differences in ACh-induced relaxation between control and Marfan vessels persisted despite increasing age. It is unlikely that the effect of aging played a significant role in this study, as changes in ACh-mediated relaxation in control mice are not apparent until around 15 months of age (Bulckaen et al., 2008). The same AChrelaxation response in the control arteries between 3 and 10 month age-groups could be related to upregulation of alternative mechanisms to compensate for the decreasing NO bioavailability. EDHF has been shown to play a greater role in the face of reduced NO bioavailability during aging (McCullough et al., 1997; Nishikawa et al., 2000; Gaubert et al., 2007).  140  ACh-mediated vasorelaxation is primarily dependent on NO in the secondary branch of mouse mesenteric artery (McGuire et al., 2002; Ceroni et al., 2007). Preincubation with the nitric oxide synthase inhibitor L-NAME significantly reduced ACh-mediated vasorelaxation in control vessels, but interestingly did not have any effect on ACh-mediated vasorelaxation in the Marfan vessels, suggesting that the endothelial NO pathway is significantly compromised in Marfan syndrome. COX-derived prostanoids are also involved in the regulation of vasomotor function and prostacyclin (PGI2), produced from arachidonic acid through the COX-2 enzyme, is the most important of these (Smith et al., 2000). Upregulation of COX-2 expression may also be induced through the loss of vessel elasticity (Vitarelli et al., 2006), which leads to increased production of PGI2 (Chung et al., 2007c). In our study, blockade of the COX pathway with indomethacin inhibited ACh-mediated vasorelaxation in Marfan vessels. The upregulation of prostanoidmediated relaxation may thus represent a compensatory mechanism of the Marfan endothelial cells in the apparent absence of NO-mediated vasorelaxation. In addition to agonist-induced endothelium-dependent vasorelaxation, EDHF plays an important role in modulating vasomotor tone in the resistance vasculature through its hyperpolarization of smooth muscle cells (Shimokawa, 1999). However, its nature is still controversial; K+, gap junctions, epoxyeicosatrienoic acids, and H2O2 are all thought to be potential candidates and the contribution of each appears to vary depending on the species tested and vessels used (Vanhoutte, 2004). In mouse mesenteric arteries, there are conflicting reports about the role of H2O2 as an EDHF (Matoba et al., 2000; Ellis et al., 2003). However, in our preparation the addition of catalase, an enzyme which dismutates H2O2 to form water and oxygen and thus lowers H2O2 concentration, in the presence of L-NAME and indomethacin reduced ACh-mediated vasorelaxation in the Marfan vessels to a greater degree than in control  141  vessels. This greater inhibition may be due to the upregulation of EDHF as a compensatory or back-up mechanism, which occurs when endothelial production of NO is impaired (Kilpatrick and Cocks, 1994; Corriu et al., 1998). Although myoepithelial gap junctions have been suggested to provide the pathway for EDHF in mouse mesenteric artery (Dora et al., 2003), we did not observe significant inhibition of ACh-mediated relaxation with carbenoxolone, an uncoupler of gap junctions, in either control or Marfan vessels. Therefore, it is unlikely that gap junctions mediate the EDHF response in our preparation. It is well established that oxidative stress has a profound influence on vascular function, though the effect of oxidative stress on vasomotor response in the progression of Marfan syndrome has never been investigated. Oxidative stress has been reported to be involved in the pathogenesis of various cardiovascular diseases (Cai and Harrison, 2000; Faraci and Didion, 2004). Superoxide dismutase treatment was shown to reverse the hypersensitivity of the arteries in diabetic and hypertensive animal models and normalize the agonist-induced contraction to that of the control animals (Kanie and Kamata, 2000; Alvarez et al., 2008). Although the mechanism of action of reactive oxygen species on smooth muscle cell contractility is still unclear (Lyle and Griendling, 2006), reactive oxygen species have been proposed to have multiple effects on calcium signaling in both vascular endothelial and smooth muscle cells (Elmoselhi et al., 1996; Lounsbury et al., 2000; Walia et al., 2000). The impairment of the Ca2+ signaling pathway caused by oxidative stress may consequently lead to the alteration of vascular reactivity (Sener et al., 2004). Thus, the removal of superoxide with superoxide dismutase may restore calcium signaling and thereby the contractile responses. Furthermore, oxidative stress may cause endothelial dysfunction through several direct and indirect pathways, the most well-known of which is the scavenging of NO by superoxide. 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Circulation 99: 909–915.  150  CHAPTER 5: CONCLUSIONS AND RECOMMENDATIONS FOR FUTURE WORK 5.1 Overall summary and conclusions Vascular smooth muscle is essential to the regulation of blood pressure and dynamic regional blood flow, responding rapidly to the moment-to-moment metabolic demands of specific tissues and maintaining global vascular tone. This requires constant and minute control of cytosolic [Ca2+], which is achieved through spatial and temporal partitioning of Ca2+ signals such that Ca2+ can simultaneously modulate contraction and processes such as gene transcription and oxidative metabolism. The subcellular architecture and arrangement of the SR, mitochondria, and PM in the form of Nanodomains all contribute to this partitioning of cytosolic Ca2+ gradients and signals, which may take the form of waves, sparks, puffs, and others (Macrez and Mironneau, 2004). The deterioration of this intricate ultrastructure may have pathological consequences (Lee et al., 2005; Petersen et al., 2006). The studies presented in this thesis first examined the nature of agonist-induced mitochondrial Ca2+ elevations as indicators of localized cytosolic Ca2+ elevations due to reversemode NCX, providing important insights into the functional linkage between reverse-mode NCX and ROCs in vascular smooth muscle. This laid the foundation for examination of asynchronous wave-like Ca2+ oscillations in the basilar artery, in which reverse-mode NCX was found to be a crucial mediator of SR Ca2+ refilling to maintain ongoing Ca2+ oscillations. Finally, having established the importance of Ca2+ oscillations in maintaining tonic contraction in resistancesized vessels, we extended this knowledge to a mouse model of vascular disease in Marfan syndrome, where it was discovered that aberrant Ca2+ wave signaling is associated with reduced smooth muscle contractile function, thus giving clues to the importance of Ca2+ wave signaling in vascular health.  151  5.1.1 Overview of NCX reversal in smooth muscle cells It has become clear that reverse (Ca2+-influx)-mode NCX plays an important role in maintaining Ca2+ homeostasis in vascular smooth muscle cells (Lee et al., 2001; Fameli et al., 2007). This is made possible through functional and physical coupling of NCX with ROCs, particularly at the nanodomain where the SR comes into close association with the PM (Lee et al., 2002; Rosker et al., 2004). In vascular smooth muscle, TRPC6 proteins form an important component of these channels (Inoue et al., 2001; Lemos et al., 2007). I investigated the process of TRPC and NCX coupling in vascular smooth muscle cells, using aequorin targeted to the mitochondria as a way to indirectly measure agonist-induced NCX reversal. I show that agoniststimulated production of DAG is important to the increased Na+ entry through the TRPC6 channels, which facilitates the reversal of NCX, and also show that Na+ entry can be inhibited by PKC activation. However, this latter effect is not sufficiently prominent during purinergic stimulation to abolish the stimulatory effect of DAG. These observations are consistent with known properties of TRPC6 channels. Overall, we add to the growing body of work to show that ROCs are functionally coupled to reverse-mode NCX.  5.1.2 Overview of UTP-induced Ca2+ oscillations in rat basilar artery Although Ca2+ waves have been observed to underlie tonic contraction in vascular smooth muscle preparations, little is known about this mechanism in the cerebral vasculature. A detailed mechanistic study of agonist-induced Ca2+ waves has never been done in the cerebral arteries. Using fluorescent Ca2+ indicators in conjunction with confocal microscopy, I showed that the underlying mechanism of UTP-induced Ca2+ waves in basilar artery is similar to what has been described in the large conductance vessels such as the IVC (Lee et al., 2002). Importantly, I also  152  demonstrated a role for reverse-mode NCX. Multiple Ca2+ translocating proteins are involved as the Ca2+ waves appear to be produced by repetitive cycles of SR Ca2+ release which are mediated by IP3Rs, followed by SERCA-mediated SR Ca2+ re-uptake of Ca2+ entry involving L-type voltage-gated Ca2+ channels, ROCs, and reverse-mode NCX. As the inhibition of IP3Rs completely abolishes the Ca2+ waves while inhibition of RyRs has no effect, this suggests that the Ca2+ waves are induced from IP3Rs and propagate by sequential release from one cluster of IP3Rs to the next throughout the SR store. From studies conducted in Xenopus oocytes and from modeling simulations, IP3Rs are thought to be arranged in clusters of around 25-35 receptors, which are 300-800nm in diameter and are several micrometers apart from each other (Swillens et al., 1999; Shuai et al., 2006), although these measurements need to be verified in the intact smooth muscle. The rising phase of the Ca2+ wave is thought to consist of a localized “initiation” component derived from the release of Ca2+ from one IP3R in the cluster (a so-called Ca2+ puff), which is followed by an amplification component during which this release is augmented by Ca2+-induced Ca2+ release by positive feedback at other IP3Rs within the cluster. The Ca2+ which is released from the cluster then diffuses to the next cluster and initiates Ca2+ release, thus propagating Ca2+ release as a wave (McCarron et al., 2004). The wave declines as IP3Rs become inhibited due to the high localized Ca2+ concentration, as IP3Rs are activated at a narrow range of Ca2+ concentration (~300nM), while higher concentrations inhibit the receptor (McCarron et al., 2007; Fig 5.2). Alternatively, IP3Rs may also become desensitized due to depletion of local Ca2+ stores. Reverse (Ca2+ influx)-mode NCX is activated by the production of DAG, which then activates TRPC6 channels, initiating Na+ influx, which ultimately serves to refill the SR store via reverse-mode NCX. Functionally, the UTP-induced asynchronous Ca2+ waves appear to underlie  153  EC coupling, as their abolishment through inhibition of SR Ca2+ re-uptake dramatically decreased tonic contraction, although average Ca2+ remained unchanged. We also show that the frequency of the Ca2+ waves is an important regulator of force generated. In general, the mechanisms of the Ca2+ waves in the basilar artery are similar to those in the large conduit vessels, indicating a common Ca2+ signaling mechanism in the vasculature.  Extracellular space  Na+  Ca2+  Ca2+  Ca2+ NCX PM-SR junction  3Na+  (+)  TRPC6  PMCA L-VGCC  SR Ca2+  SERCA  IP3R Ca2+  Myofilaments  Figure 5.1: Model for UTP-induced Ca2+ waves in smooth muscle cells of rat basilar artery. (SR, sarcoplasmic reticulum; L-VGCC, L-type voltage-gated Ca2+ channel; NCX, Na+/Ca2+ exchanger; TRPC, canonical transient receptor potential; IP3R, IP3 receptor; RyR, ryanodine receptor; SERCA, sarco-endoplasmic reticulum Ca2+-ATPase; PMCA, plasma membrane Ca2+ ATPase).  154  Cytosol  Ca  Ca2 Ca2 Ca2 2  Ca2 Ca2  IP3R  IP3R  300 -800 nm  ~3 μm  300 -800 nm SR lumen  Figure 5.2: Model of Ca2+ wave propagation from Xenopus oocytes. Inositol 1,4,5trisphosphate-medated Ca2+ signals propagate by sequential release from one cluster of inositol 1,4,5-trisphosphate receptors (IP3Rs) to the next throughout the SR store. [Ca2+] release from one IP3R induces positive feedback Ca2+-induced Ca2+ release from within the same cluster. The increasing [Ca2+] then diffuses to an adjacent cluster (~3μm apart) and stimulates Ca2+-induced Ca2+ release, resulting in the propagation of the wave (SR, sarcoplasmic reticulum). Figure adapted from McCarron et al., 2007. 5.1.3 Overview of dysfunction of endothelium and smooth muscle cells in small arteries of a mouse model of marfan syndrome Marfan syndrome is associated with endothelial dysfunction and reduction of smooth muscle contractility in the vasculature, particularly in the aorta (Chung et al., 2007a,b), but little is known about how this disease affects the peripheral resistance vessels. Using a mouse model of Marfan syndrome, I determined that mesenteric artery smooth muscle cell contraction in response to phenylephrine and high potassium stimulation was reduced in Marfan mice compared to control, and more importantly, that reduced smooth muscle contractility in response to phenylephrine stimulation was associated with reduced recruitment of cells expressing  155  decreased frequency of Ca2+ waves. This observation is an important first step in drawing a possible link between vascular health and Ca2+ wave signaling. Although the specific mechanism explaining why there is reduced Ca2+ wave signaling needs to be examined, possible options include inhibition of Ca2+ entry through L-type Ca2+ channels from the extracellular space, which may be attributed to upregulation of matrixmetalloproteases (Chew et al., 2004; Chung et al., 2007a), as well as the disappearance of PM-SR junctions. Furthermore, I also showed that elasticity in mesenteric resistance vessels is decreased and endothelium-dependent and endothelium-independent relaxation is impaired.  5.2 Significance of work and future directions The importance of Ca2+ as a signaling ion in vascular smooth muscle is apparent from how it regulates a plethora of different processes, from contraction to apoptosis. The ultrastructure of the vascular smooth muscle cell, particularly at the nanodomains formed by the close spatial association of the PM, SR and mitochondria allow diffusional barriers to Ca2+ and to other ions, allowing them to interact with a variety of transporters and exchangers. Such nanodomains were responsible for the indirect measurement of NCX reversal through mitochondria-mediated Ca2+ uptake and also for UTP-induced Ca2+ wave signaling, described Chapter II and III, respectively. Furthermore, the breakdown of these nanodomains may be responsible for decreased frequency of Ca2+ waves in vascular smooth muscle of mice with Marfan syndrome, as found in Chapter IV. My work from this thesis suggests there are many future experiments to be attempted and several relevant future studies are outlined below. First of all, the study of NCX reversal mediated by Na+ influx from ROCs, presumably those composed of TRPC6, adds further evidence to the growing body of work supporting the  156  importance of the NCX as a Ca2+ entry mechanism. Indeed, Na+ influx following agonist stimulation has been directly visualized in cultured rat aortic smooth muscle cells (Poburko et al., 2007). However, cultured cells have many disadvantages over intact tissue, including the alteration of expression of ion channels, transporters, receptors, and contractile proteins (BerraRomani et al., 2008). Therefore, a logical next step would be attempting to directly image Na+ influx following agonist stimulation in the intact tissue, and to determine if these Na+ transients are inhibited after inhibition of TRPC channels expression. Electrophysiological experiments in enzymatically dissociated cells using patch-clamp techniques to determine NCX currents at rest and following agonist stimulation would also provide direct and more accurate measurement of NCX reversal. Secondly, the finding that Ca2+ waves underlie tonic contraction in the rat basilar artery, in addition to what is already known about the mechanism of Ca2+ waves in conduit vessels, may indicate that Ca2+ waves represent a common signaling mechanism in the vasculature. However, the question of why smooth muscle cells appear to have adopted Ca2+ waves as a way to activate contractile filaments is unknown, although there are a number of important considerations. First, Ca2+ as an intracellular signaling molecule is capable of activating a large number of effector molecules and processes. Ca2+ waves may be a mechanism to achieve high localized Ca2+ to allow effective activation of specific effector functions without a generalized activation of other Ca2+ sensitive pathways which may arise from a sustained local increase in Ca2+. Second, the peak Ca2+ level of the Ca2+ wave may be sufficient to signal for contractile activation because the rate of Ca2+ dissociation from the calmodulin is much slower than the the rate of Ca2+ binding (Johnson et al., 1996; Wilson et al., 2002). In this case, the average Ca2+ achieved during agonist stimulation is lower than when a sustained rise in Ca2+ is used to stimulate contration; this has  157  been noted in the studies from the rat basilar artery presented in this thesis (Fig 5.3). Finally, certain frequency-dependent enzymes may be activated to a greater extent by Ca2+ waves, such as calmodulin kinase II and the transcription factor NF-AT (de Koninck and Schulman, 1998; Dupont and Goldbeter, 1998; Hu et al., 1999). As Ca2+ waves represent a signaling mechanism which is widely utilized by a variety of smooth muscle cells to stimulate tonic contraction (Ruehlmann et al., 2000; Lee et al., 2002; Perez and Sanderson, 2005), it is unlikely that nature would adopt and preserve such a complex form of Ca2+ signaling without an underlying advantage. The role of Ca2+ waves in activating contraction may only be conclusively determined with direct observation of myosin light-chain kinase, as has been done with  125  0.125  100  0.100 ***  75  0.075 ###  50  0.050 ***  25  0.025  0  0.000 UTP  Force  Frequency of Ca2+ waves (Hz)  % Maximum  carbachol-induced elevation of Ca2+ in mouse bladder smooth muscle cells (Isotani et al., 2004).  UTP + Nifedipine  Peak [Ca2+]  UTP + CPA  Avg [Ca2+]  Frequency  Figure 5.3: Ca2+ waves provide a more efficient stimulus for tonic contraction compared to steady elevation in average Ca2+. The stimulation of rat basilar artery with UTP (100 μM) generates tonic contraction. The addition of nifedipine to UTP-induced Ca2+ waves significantly slows frequency and tonic contraction, but neither changes the Ca2+ peak of the oscillations nor average Ca2+. However, the application of CPA to ongoing UTP-induced Ca2+ waves results in a significantly decreased tonic contraction, which is associated with significantly decreased peak Ca2+, but elevated average Ca2+. *** - P < 0.001 vs. UTP-induced tonic contraction, ### - P < 0.001 vs. UTP-induced peak [Ca2+]. 158  Additionally, it was revealed that there is an important role for reverse (Ca2+-entry)-mode NCX in the maintenance of Ca2+ waves, presumably through refilling of the SR at the PM-SR junctions. The driving force (the difference between membrane potential, Vm, and NCX reversal potential, ENCX) determines the direction of net Ca2+ movement, and varies during agonist stimulation. NCX reversal (Ca2+ entry) occurs when ENCX < Vm. In the arterial smooth muscle, agonist stimulation depolarizes the myocytes through the opening of ROCs, which results in Na+ entry that builds up in the PM-SR junction. As [Na+] increases and Vm is depolarized, the thermodynamic driving force on the NCX begins to favour Ca2+ entry mode. Assuming for an activated cell, where Vm = -20mV, [Na+]o = 140 mM, [Ca2+]i = 10 μm, [Ca2+]o = 2 mM, around 30mM Na+ in the subplasmalemmal space is required before NCX reversal occurs, which is feasible given that such contentrations have been observed and mathematically predicted (Poburko et al., 2006; Fameli et al., 2007, Fameli et al., 2009). Then, as the Ca2+ is taken up by the SR and the leading edge of the wave begins to decline, the membrane begins to repolarize and the driving force Vm-ENCX again becomes negative and favours Ca2+ extrusion. However, information on the time-dependent changes of Vm during Ca2+ oscillations has never been obtained. The use of Vm-sensitive fluorescent dye such as DiBAC4(3) would allow visualization of the changes in Vm during Ca2+ oscillations, as the dye fluctuates in intensity in response to changes in Vm (Rottenberg 1979; Freedman and Novak, 1989; Rottenberg 1989; Sguilla et al., 2003). Another approach would require the enzymatic isolation of cells from the basilar artery and recording of Em with the patch clamp technique before and during application of UTP. This would assume that the enzymatically dissociated cells also display the same Ca2+ oscillations as in the intact tissue. In these same cells, Em could also be recorded by measuring  159  emitted fluorescence from DiBAC4(3), such that there will be two independent measurements and the means for calibration of the fluorescent measurements. Thirdly, the finding that decreased smooth muscle contraction in Marfan syndrome was associated with decreased Ca2+ wave frequency and cell displaying Ca2+ waves is one of the first to reveal how Ca2+ wave signaling can be affected by vascular disease. Although not examined directly in this thesis, these Ca2+ waves (as in other vascular smooth muscles) are likely supported by the underlying PM-SR junctional cellular ultrastructure, which are important for SR Ca2+ refilling (Lee et al., 2005; Fameli et al., 2007). More evidence to support this theory comes from the observation that phenylephrine elicited tonic contractions in both mouse and human mesenteric arteries, but asynchronous Ca2+ waves were only seen in the mouse mesenteric artery smooth muscle cells (which had abundant superficial SR and many PM-SR junctions), while the human mesenteric artery smooth muscle cells (which had far less peripheral SR and was almost devoid of PM-SR junctions) displayed only single transient Ca2+ signal (Dai et al., 2010). Finally, diabetes is also associated with separation of the superficial SR from the PM in the coronary artery (Witczak and Sturek, 2004). Therefore, the deterioration of PM-SR junctional complexes (and subsequent disappearance of oscillatory Ca2+ signaling) may be an important contributing factor in the etiology of vascular disease. Future studies which could directly test this hypothesis would involve a comparative study of the extent of PM-SR junctions between age-matched control and Marfan mice. Because the frequency of Ca2+ waves decreases as Marfan syndrome progresses, we speculate that there would be fewer PM-SR junctions in the diseased vessels compared to control. If, however, the deterioration of PM-SR junctions causes the disappearance of Ca2+ waves, it should logically follow that their restoration would enable Ca2+ wave signaling to return. This  160  raises the question whether it is possible to restore Ca2+ wave signaling and/or PM-SR junctions following treatment, pharmacological or otherwise, of vascular diseases. Interestingly, exercise restores the diabetes-induced impaired structure between the superficial SR and the PM in the coronary artery (Witczak and Sturek, 2004). Additionally, the treatment of Marfan syndrome using doxycyline was found to normalize vasomotor function (Chung et al., 2008). Does the restoration of PM-SR junctions following exercise induce Ca2+ waves, in the case of coronary artery? And does it follow that normalization of vasomotor function through doxycycline treatment is secondary to the restoration of PM-SR junctions? An interesting follow-up study to answer the latter question would be to compare control, doxycycline-treated, and untreated Marfan mice to determine if Ca2+ wave signaling is also normalized following treatment with doxycycline, with parallel electron microscopy studies to examine the cellular ultrastructure. A final unresolved mystery is the identity of the structural protein that is responsible for the regular spacing of the smooth muscle PM-SR junctions? Although the presence of connecting structures between the PM and SR have been known for many years (Somlyo, 1985), their composition remains unknown. The identification of these structures would be an important step in the possible development of therapeutic agents which would preserve their structure, Ca2+ wave signaling, and presumably vascular health.  5.3 Concluding remarks The goal of the studies contained in this thesis was to gain a more comprehensive understanding of Ca2+ signaling in smooth muscle of blood vessels. The contributions made in this thesis enhance our understanding of Ca2+ signaling in healthy and diseased animal cells and the intact blood vessels. In particular, this work provides in some detail the mechanism and  161  function of agonist-induced Ca2+ waves and their relation to tonic contraction, as well as their underlying mechanisms. Ca2+ waves are clearly an important part of the regulation of vascular smooth muscle contraction and their disappearance may be linked to declining vascular health. 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It should be noted that the Ca2+ waves occurred at different frequencies. Experimental Ca2+ traces are representative of results from 58 cells in 6 animals. (B) Intact vascular smooth muscle cells challenged with PE (3 μM) displayed Ca2+ waves which originated from distinct intracellular foci and propagated along the longitudinal axis of the smooth muscle cells, as indicated by area of interest (AOI) AOI1 and AOI2. The AOI is 3x3 pixels (1.69 μm2). Scale bar = 10 μm.  166  APPENDIX B  B 9 8 7 6 5 4 3 2 1 0 -10  Control Marfan  -9  -8  Frequency (Hz)  Force (mN)  A  -7  -6  -5  -4  0.10 0.09 0.08 0.07 0.06 0.05 0.04 0.03 0.02 0.01 0.00 -10  Control Marfan  -9  log[PE]  -8  -7  -6  -5  -4  log[PE]  C  D #  *  0.10 0.09 0.08 0.07 0.06 0.05 0.04 0.03 0.02 0.01 0.00  *  100  Recruitment of cells (% max)  Frequency (Hz)  #  3  6  *  *  6  10  75 50 25 0  10  3  Age (Months)  Age (Months)  Control  Marfan  Properties of phenylephrine (PE, 3 μM)-induced Ca2+ waves underlying tonic contraction in mouse mesenteric artery. Concentration-response curves for PE-induced (A) tonic contraction and (B) Ca2+ wave frequency in control and Marfan mice at 6 months of age. (C) Reduced frequency of PE (3 μM)-stimulated Ca2+ waves in Marfan vessels compared to control vessels. (D) A decreased percentage of vascular smooth muscle cells in Marfan vessels displayed at least one Ca2+ wave compared to control vessels when stimulated with PE (3 μM) (n = 90 cells from 10 animals). The number of cells firing is expressed as a percentage of cells responding to maximal concentration. *, # - P<0.05  167  APPENDIX C  Plasmid map of pcDNA1-based aequorin expression vector. mtAeq/pcDNA1 shows the ~770bp DNA insert (ECOR1 fragment) with mitochondrial targeting presequence (MPS) of human cytochrome c oxidase subunit VIII, HA1 epitope and apoaequorin coding segments (From Challet thesis, 2001). The mtAeq/pcDNAI contains sequences encoding the mitochondrial targeting peptide from subunit VIII of human cytochrome c oxidase fused to an HA1 antigen and to apoaequorin. To amplify this pcDNAI-based plasmid, which carries the supF suppressor tRNA gene, a special host bacterium, MC1061/P3 E. Coli (Invitrogen, Groningen, NL), had to be used. The P3 plasmid in this host carries a wild-type kanamycin resistance gene, plus ambermutant versions of both ampicillin and tetracyclin-resistance genes. Transformation of MC1061/P3 by a plasmid bearing a supf confers ampicillin and tetracyclin resistance by translational suppression of the defective genes. The amplification protocol was, in brief, the following. Competent bacteria were heat shocked in the presence of l00ng mtAeq/pcDNAI. They were grown in LB medium for 30min at 37°C and were seeded on LB-Agar plate containing 50ug/ml ampicillin. After an overnight incubation at 37°C, colonies of bacteria were isolated and were further grown overnight in 200ml LB medium with 50u.g/ml ampicillin. After bacterial suspension was centrifuged, plasmid was purified using the kit Nucleobond" AX500 (MachcryNagcl, Oensingen, CH). The identification of the purified plasmid was checked by enzyme digestion. ECORI restriction enzyme could be used alone or in combination with Hindlll (Pharmacia Biotech, Dubcndorf, CH).  168  APPENDIX D  Luminometry setup. The cell chamber is constantly thermostated at 37 degrees with a water bath and perfused via a peristaltic pump at a rate of 1 mL/min. The smooth muscle cells, on Thermanox coverslips, are placed into the specifically designed chamber. Photons emitted by the cells are detected with a low noise photomultiplier tube (pmt, Thorn-EMI 9789A) connected to a high voltage power supply (Thorn-EMI, PM28B). Acquired data are recorded every second using an amplifier discriminator (amp/disc, Thorn-EMI, C640A) and a computer photon counting board (Thorn-EMI C660). Reproduced from Chiesa et al., 2001.  169  APPENDIX E List of Publications and Abstracts The chapters in this thesis contain work that has been previously published in peer-reviewed journals. Material from this dissertation has been published in:  •  Syyong HT, Chung AWY, Yang HHC, van Breemen C (2009). Dysfunction of endothelial and smooth muscle cells in small arteries of a mouse model of Marfan syndrome. Br J Pharmacol 158: 1597-1608.  •  Syyong HT, Yang HH, Trinh G, Cheung C, Kuo KH, van Breemen C (2009). Mechanism of asynchronous Ca(2+) waves underlying agonist-induced contraction in the rat basilar artery. Br J Pharmacol 156: 587-600.  •  Syyong HT, Poburko D, Fameli N, van Breemen C (2007). ATP promotes NCX-reversal in aortic smooth muscle cells by DAG-activated Na+ entry. Biochem Biophys Res Commun 357: 1177-82.  Material from this dissertation has been presented in poster format at the following international meetings:  •  Syyong HT, Lemos VS, Poburko DT, Liao CH, Pillai R, Fameli N, Kuo KH, van Breemen C. ATP-induced reversal of sodium-calcium exchanger and the role of the canonical transient receptor potential (TRPC) subfamily. Canadian Cardiovascular Congress - Vancouver, BC October 21-25, 2006. Canadian Journal of Cardiology, 22(Suppl D):211D  170  •  Syyong, HT, Kuo, KH, and van Breemen, C. Mechanism of uridine 5'-triphosphate (UTP)induced calcium waves in rat basilar artery. Experimental Biology, Washington, DC, USA, April 28-May 2, 2007. FASEB J. 2007 21:873.7  •  Syyong, HT, Yang, C., Kuo, K.H., and van Breemen, C. Uridine 5'-triphosphate (UTP)induced Ca2+ oscillations underlie tonic contraction in rat basilar artery. Canadian Cardiovascular Congress - Quebec City, QC Oct 20-24, 2007. Canadian Journal of Cardiology, 23(Suppl C):217C.  •  Syyong, H, Yang, C, Trinh, G, Kuo, K.H., and van Breemen, C. Ultrastructural basis of asynchronous uridine 5'-triphosphate (UTP)-induced Ca(2+) waves in rat basilar artery. Experimental Biology - San Diego, CA, USA Apr 5-9, 2008. FASEB J. 2008 22:913.1.  Material from this dissertation has also been presented orally for the Graduate Student Seminar Series in the Department of Anesthesiology, Pharmacology and Therapeutics at UBC  171  APPENDIX F  Copies of the UBC animal care committee approval letters are included.  172  https://rise.ubc.ca/rise/Doc/0/1Q188B1DCF1KJ6QLOETKV09V1E/fromS...  THE UNIVERSITY OF BRITISH COLUMBIA  Application Number: A07-0255 Investigator or Course Director: Cornelis Van Breemen Department: Medicine, Faculty of Animals:  Rats Sprague-Dawley 220  Start Date:  July 1, 2007  Approval Date:  July 11, 2007  Funding Sources: Funding Agency: Funding Title:  Unfunded title:  Canadian Institutes of Health Research (CIHR) Calcium oscillations in vascular smooth muscle  N/A  The Animal Care Committee has examined and approved the use of animals for the above experimental project. This certificate is valid for one year from the above start or approval date (whichever is later) provided there is no change in the experimental procedures. Annual review is required by the CCAC and some granting agencies.  A copy of this certificate must be displayed in your animal facility.  Office of Research Services and Administration 102, 6190 Agronomy Road, Vancouver, BC V6T 1Z3 Phone: 604-827-5111 Fax: 604-822-5093 1 of 1  11/26/2009 8:10 PM  https://rise.ubc.ca/rise/Doc/0/BAALGC7BEB5KREP3QOS61GM306/fr...  THE UNIVERSITY OF BRITISH COLUMBIA  Application Number: A06-1459 Investigator or Course Director: Cornelis Van Breemen Department: Medicine, Faculty of Animals: Mice fibrilin 1 cys +/- mice, C1039G mutation; Fbn1 is on the Bl6 180  Start Date:  November 1, 2006  Approval Date:  November 10, 2006  Funding Sources: Funding Agency: Funding Title:  Unfunded title:  Heart and Stroke Foundation of B.C. & Yukon Cellular mechanisms of aortic pathogenesis in Marfan syndrome  N/A  The Animal Care Committee has examined and approved the use of animals for the above experimental project. This certificate is valid for one year from the above start or approval date (whichever is later) provided there is no change in the experimental procedures. Annual review is required by the CCAC and some granting agencies.  A copy of this certificate must be displayed in your animal facility.  Office of Research Services and Administration 102, 6190 Agronomy Road, Vancouver, BC V6T 1Z3 Phone: 604-827-5111 Fax: 604-822-5093  1 of 1  11/26/2009 8:12 PM  https://rise.ubc.ca/rise/Doc/0/MA0SQ8DP0M8K14VOK23FJA05F3/from...  The University of British Columbia  ANIMAL CARE CERTIFICATE BREEDING PROGRAMS  Application Number: A05-1480 Investigator or Course Director: Casey Van Breemen Department: Pharmacology & Therapeutics Animals:  fibrilin 1 cys +/- mice, C1039G mutation; Fbn1 is on the Bl6 180  Approval Date: December 8, 2005 Funding Sources: Funding Agency:  Canadian Marfan Association  Funding Title:  In-vitro assessment of endothelial and arterial smooth muscle function in a Marfan mouse model  Unfunded title:  N/A  The Animal Care Committee has examined and approved the use of animals for the above breeding program. This certificate is valid for one year from the above approval date provided there is no change in the experimental procedures. Annual review is required by the CCAC and some granting agencies.  A copy of this certificate must be displayed in your animal facility.  Office of Research Services and Administration 102, 6190 Agronomy Road, Vancouver, V6T 1Z3 Phone: 604-827-5111 Fax: 604-822-5093  1 of 1  11/26/2009 8:12 PM  

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