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Role of sarcoplasmic reticulum and mitochondria in Ca2+ signaling in vascular smooth muscle Szado, Tania 2002

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ROLE OF SARCOPLASMIC RETICULUM AND MITOCHONDRIA IN Ca 2 + SIGNALING IN VASCULAR SMOOTH MUSCLE. by TANIA SZADO B.Sc, The University of British Columbia, 1998 A THESIS SUBMITTED LN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE F A C U L T Y OF G R A D U A T E STUDIES Department of Pharmacology & Therapeutics; Faculty of Medicine We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September 2002 © Tania Szado, 2002 UBC Rare Books and Special Collections - Thesis Authorisation Form Seite 1 von 1 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an advanced degree a t t h e U n i v e r s i t y o f B r i t i s h C o lumbia, I agree t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u rposes may be g r a n t e d by t h e head o f my department o r by h i s or her r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f The U n i v e r s i t y o f B r i t i s h Columbia Vancouver, Canada Date Oct. I* o 3 -Abstract Superficial sarcoplasmic reticulum (SR) regulates smooth muscle force development directly and indirectly. In the rabbit basilar artery (BA), relative contributions of direct effects and those mediated through activation of K c a were evaluated by measuring force and intracellular C a 2 + concentration ([Ca2+]j) in response to the SR-depleting agents thapsigargin (Tg) and ryanodine and the large conductance Kc a (BKca) blockers iberiotoxin (IbTx) and tetraethylammonium ion (TEA). It appears that a significant fraction of K c a remains activated in the absence of SR function and that SR contributes to relaxation after blockade of Kc a . We found that depletion of SR before stimulating C a 2 + influx through voltage-gated C a 2 + channels markedly reduced force development rate and that thapsigargin abolished this effect. We conclude that the SR of rabbit cerebral arteries modulates constriction by direct and indirect mechanisms. Next, we investigated the role of mitochondria (MT) in calcium signaling in a primary culture of rat aortic smooth muscle cells. We have used the calcium photoprotein, aequorin, selectively targeted to the mitochondrial matrix to measure [Ca2 +] in this organelle. Our results reveal that smooth muscle cell stimulation with 1 mM ATP or 1 uM vasopressin (AVP) causes a large, transient increase in mitochondrial [Ca ] ([Ca 2 + ]m). This large transient can be'blocked with 100 uM cyclopiazonic acid (CPA) or 1 uM Tg, suggesting a close relationship between the SR and MT. Thus, in addition to SR, MT are also important in C a 2 + homeostasis of smooth muscle. Finally, gene expression studies using RT-PCR were performed in 3 types of smooth muscle; rabbit BA , inferior vena cava (IVC), and a rat aortic smooth muscle cell (RASMC) line. Expression of B K c a channels, and V G C C differed between rabbit B A ii and IVC, and was compared to functional data using various inhibitors. Taken together, this data suggests an association of RyR to BKc a in BA, and store-operated C a 2 + channels and IP3 in IVC. We hypothesize that SR and MT interactions with channels and pumps on the P M , and with each other, are critical in the formation of cytoplasmic C a 2 + microdomains, contributing to the diversity of Ca signaling in different smooth muscles. iii T A B L E O F C O N T E N T S A B S T R A C T II T A B L E OF C O N T E N T S IV LIST OF T A B L E S VII LIST OF F IGURES VIII A B B R E V I A T I O N S X LIST OF PUBL ICAT IONS AND A B S T R A C T S XIII A C K N O W L E D G E M E N T S X V C H A P T E R I: INTRODUCTION 1 1.1. SMOOTH MUSCLE 1 7.7.7. The structure of smooth muscle 2 1.1.2. Role of the endothelium 3 1.1.3. Excitation-contraction coupling in vascular smooth muscle 4 1.2. C A L C I U M SIGNALING IN V A S C U L A R SMOOTH MUSCLE 7 7.2.7. Plasma membrane receptors 7 7.2.2. TheSR 16 1.2.3. The mitochondrion 24 1.2.4. Model for Ca2+ signaling in smooth muscle 30 1.2.3. Targeted aequorin technology 32 1.3. RATIONALE, PURPOSE AND SPECIFIC AIMS 3 9 Specific aim #1: Role of the SR in Ca2+ signaling in cerebral arteries 39 Specific aim #2: Role of the mitochondria in Ca2+ homestasis in vascular smooth muscle 39 Specific aim #3: Smooth muscle heterogeneity 39 C H A P T E R II: M A T E R I A L S AND M E T H O D S 41 2.1 . ISOLATION OF RABBIT CEREBRAL ARTERIES AND MYOGRAPHY 41 2.2. FURA-2 IMAGING OF INTACT RABBIT BASILAR ARTERIES 42 2.3. SMOOTH MUSCLE CELL CULTURE 43 iv 2.4. AEQUORIN TECHNOLOGY 43 2.4.1. Expression vector and its amplification 43 2.4.2. Smooth muscle cell line transiently expressing mt-Aeq 47 2.4.3. Mitochondrial Ca2* measurement 47 2.4.4. Cytosolic Ca2+ measurement 48 2.5. RT-PCR 49 92.5.1. RNA extraction 49 2.5.2. Semi-Quantitative RT-PCR 49 2.6. CONFOCAL MICROSCOPY 52 2.7. ELECTRON MICROSCOPY 54 2.8. SOLUTIONS AND PHARMACOLOGICAL AGENTS 55 2.9. DATA ANALYSIS 56 2.9.1. Statistical analysis 56 2.9.2. Curve analysis 57 C H A P T E R III: SR B U F F E R I N G OF CA 2 + IN RABBIT B A S I L A R A R T E R Y 60 3.1. INTRODUCTION 60 3.2. RESULTS 62 3.2.1. Thapsigargin dose-response curve 62 3.2.2. Sequential blockade of BKa, and SERCA 65 3.2.3. Simultaneous force/Ca2+ recording 71 3.2.4. Contribution of all Kca channels 72 3.2.5. Mn2+quenching 75 3.2.6. SR buffering 77 Model 80 3.3. DISCUSSION 79 C H A P T E R IV: AGONIST INDUCED M I T O C H O N D R I A L C A 2 + TRANSIENTS IN S M O O T H M U S C L E 87 4.1. INTRODUCTION 87 4.2. RESULTS 89 4.2.1. Agonist-induced mitochondrial Ca2+ transients 89 4.2.2. Role ofL-type VGCC and FCCP uncoupling 91 v -Confocal images of mito-GFP transfected smooth muscle cell 93 4.2.3. Effect of SERCA blockade on mitochondrial transients 94 4.2.4. Role of IP 3R 94 4.2.5. Involvement of the Na+/Ca2+ exchanger 99 4.2.6. Electron Microscopy 102 Model 104 4.3. DISCUSSION 106 C H A P T E R V : S M O O T H M U S C L E H E T E R O G E N E I T Y 115 5.1. INTRODUCTION 115 5.2. RESULTS 117 5.2.1. ET contractions in rabbit BA and IVC 117 5.2.2. Confocal imaging of intact arteries 122 5.2.3. Effect of KCa channel blockade in rabbit IVC and BA 123 5.2.4. KCa expression in rabbit IVC and BA 125 5.2.5. Expression of L- and T- type VGCC in rabbit IVC and BA 125 5.2.6. Rat aortic smooth muscle cells (RASMC) 126 5.2.7. Trp channel expression 129 Model 138 5.3. DISCUSSION 134 C H A P T E R VI : G E N E R A L CONCLUSIONS AND F U T U R E DIRECTIONS 143 A P P E N D I X I. L U M I N O M E T R Y SETUP 148 B I B L I O G R A P H Y 149 vi List of Tables TABLE 1. TARGETED AEQUORINS/GFP 36 TABLE 2. OLIGONUCLEOTIDE SEQUENCES OF THE PRIMERS USED FOR RT-PCR 52 vii List of Figures FIGURE 1.1. MORPHOLOGY OF SMOOTH M U S C L E 2 FIGURE 1.2. ENDOTHELIAL VASOACTIVE FACTORS 3 FIGURE 1.3. T H E M E C H A N I S M OF SMOOTH M U S C L E CONTRACTION 6 FIGURE 1.4. TOPOLOGICAL STRUCTURE OF THE A-1 SUBUNIT OF T H E V G C C OR C A V 1.2 8 FIGURE 1.5. TOPOLOGICAL STRUCTURE OF THE CA 2 + -SENSITIVE K + CHANNELS 12 FIGURE 1.6. C A 2 + EXTRUSION MECHANISMS IN SMOOTH M U S C L E 15 FIGURE 1.7. SCHEMATIC CARTOON REPRESENTING THE MAJOR IMPORTANT PATHWAYS IN MITOCHONDRIAL FUNCTION 2 7 FIGURE 1.8. C A 2 + H A N D L I N G IN SMOOTH M U S C L E 31 FIGURE 1.9. RECOMBINATION OF AEQUORIN 33 FIGURE 1.10. SCHEMATIVE REPRESENTATION OF THE TARGETED AEQUORINS (AEQ) C U R R E N T L Y A V A I L A B L E 37 FIGURE 2.1 . PLASMID M A P OF PCDNAI-BASED AEQUORIN EXPRESSION VECTOR 44 FIGURE 2.2. DIGESTION OF MITOAEQ/PCDNAI PLASMID WITH ECORI. 45 FIGURE 2.3. STEPS INVOLVED IN PREPARING SMOOTH M U S C L E CELLS FOR LUMINOMETRY 47 FIGURE 3.1. THAPSIGARGIN CONCENTRATION RESPONSE C U R V E 64 FIGURE 3.2. SEQUENTIAL B L O C K A D E OF B K C A FOLLOWED B Y SERCA INHIBITION 67 FIGURE 3.3. EFFECT OF B K C A C H A N N E L B L O C K A D E IN RABBIT B A 68 FIGURE 3.4. SEQUENTIAL B L O C K A D E OF SERCA FOLLOWED B Y B K C A INHIBITION 70 FIGURE 3.5. SIMULTANEOUS F O R C E / C A 2 + RECORDING IN INTACT RABBIT BASILAR ARTERY 71 FIGURE 3.6. APPLICATION OF R Y FOLLOWED B Y TEA 73 FIGURE 3.7. CONTRIBUTIONS OF S K C A , I K C A , A N D B K C A B L O C K A D E S TO ADDITIONAL CONTRACTIONS AFTER SERCA B L O C K A D E 74 FIGURE 3.8. M N 2 + QUENCHING IN A N ENDOTHELIUM-DENUDED RABBIT BASILAR A R T E R Y 76 FIGURE 3.9. SR BUFFERING IN RABBIT BASILAR ARTERIES 78 FIGURE 3.10. SCHEMATIC REPRESENTATION OF THE VARIOUS SR R E L A T E D MECHANISMS INVOLVED IN REGULATION OF M E M B R A N E POTENTIAL A N D GENERATION OF FORCE IN V A S C U L A R SMOOTH M U S C L E 80 FIGURE 4.1. AGONIST-INDUCED RISE IN MITOCHONDRIAL [CA ] ([CA ]M) IN SMOOTH MUSCLE CELLS 90 FIGURE 4.2. FCCP INHIBITS ATP-1NDUCED TRANSIENT BUT BLOCKADE OF L-TYPE VGCC HAS NO EFFECT 92 FIGURE 4.3. CONFOCAL IMAGES OF SMOOTH MUSCLE CELLS TRANSFECTED WITH MT-GFP 93 FIGURE 4.4. SERCA BLOCKADE INHIBITS TRANSIENT [CA 2 + ] M RISE INDUCED BY ATP 95 FIGURE 4.5. SUMMARY OF SERCA BLOCKADE ON PEAK [CA 2 + ] M . . . 96 FIGURE 4.6. PLC AND IP3 ANTAGONIST PARTIALLY INHIBIT TRANSIENT ATP-INDUCED INCREASE IN [CA 2 + ] M 97 FIGURE 4.7. SUMMARY DATA FOR EFFECTS OF PLC AND IP3 ANTAGONISTS ON ATP-INDUCED MITOCHONDRIAL TRANSIENT : 98 FIGURE 4.8. NA + REMOVAL RE-ESTABLISHES ATP TRANSIENT IN 0 C A 2 + SOLUTIONS 100 FIGURE 4.9. SUMMARY DATA FOR THE EFFECT OF 0 C A 2 + AND 0 NA + /0 C A 2 + SOLUTION ON ATP INDUCED MITOCHONDRIAL C A 2 + TRANSIENTS 101 FIGURE 4.10 ELECTRON MICROSCOPY OF SMOOTH MUSCLE CELLS 103 FIGURE 4.11 MODEL FOR C A 2 + MOVEMENTS IN VASCULAR SMOOTH MUSCLE CELLS 104 FIGURE 5.1. ET REPONSES IN RABBIT BA 119 FIGURE 5.2. ET RESPONSES IN RABBIT IVC 120 FIGURE 5.3. BIPHASIC AGONIST-INDUCED RESPONSE IN FLUO-4 LOADED RABBIT BASILAR ARTERY 122 FIGURE 5.4. SPECTROPHOTOMETRIC MEASUREMENT OF C A 2 + IN RABBIT INFERIOR VENA CAVA 124 FIGURE 5.5. EXPRESSION OF THE L-TYPE VGCC, B K C A AND T-TYPE CALCIUM CHANNELS IN DIFFERENT ARTERIES 127 FIGURE 5.6. EXPRESSION OF VOLTAGE-GATED CALCIUM CHANNELS IN SEMI- AND FULLY-CONFLUENT CELLS 128 FIGURE 5.7. TRPCl AND CtlC MRNA EXPRESSION IN THE SMOOTH MUSCLE OF THE RABBIT i v c 131 FIGURE 5.8. TRP CHANNEL EXPRESSION IN RABBIT BASILAR ARTERY 132 FIGURE 5.9. TRP CHANNEL EXPRESSION IN SMOOTH MUSCLE CELLS 133 FIGURE 5.10. MODEL FOR C A 2 + SIGNALING PATTERNS IN RABBIT IVC (A) AND BA (B) 138 i x Abbreviations ACh acetylcholine A C R acceptor control ratio A E activity extent Aeq aequorin APO apoaequorin ATP adenosine triphosphate A V P [ A r g" 8Vasopressin A U C Area under the curve 2-APB 2- aminoethoxydiphenyl borate BSA bovine serum albumin BQ610 (homopiperidenyl-CO-Leu-D-Trp (CHO)-D-Trp-OH) BQ788 N-cis-2,6-dimethylpiperidinocarbonyl L-[gamma]-MeLeu-D-Trp (COOCH 3)-Nle 5-HT 5 -hy droxytryptamine [Ca 2 +]i intracellular (cytosolic) free C a 2 + concentration [Ca 2 + ] m mitochondrial calcium concentration Caf caffeine [Ca 2 +] 0 extracellular free C a 2 + concentration A N O V A analysis of variance ADP adenosine diphosphate ATP adenosine trisphosphate C a 2 + calcium ion cGMP guanosine 3',5'-cyclic monophosphate CGP 37157 7-chloro-5-(2-chlorophenyl)-l,5-dihydro-4,l-benzothiazepin-2(3H) CICR Ca2+-induced Ca2+-release CPA cyclopiazonic acid CTx charybdotoxin CRAC C a 2 + release-activated C a 2 + channel D A G diacylglycerol DCB dichlorobenzamil DMSO dimethylsulfoxide A ¥ m mitochondrial membrane potential E-C coupling excitation-Contraction coupling EDHF endothelium-derived hyperpolarising factor EDRF endothelium-derived relaxing factor EGTA ethyleneglycol-bis(P-aminoethyl ether)-N,N,N',N'-tetraacetic acid eNOS endothelium-derived nitric oxide synthase ER endoplasmic reticulum ET endothelin FCS fetal calf serum FCCP carbonyl cyanide jt?-trifluoromethoxyphenylhydrazone GFP green fluorescent protein X GTP guanosine triphosphate HA1 hemagglutinin HEPES 4-(2-hydroxyethyl)piperazine-1 -ethanesulfonic acid High K + high K + depolarization solution IbTx iberiotoxin IICR HVinduced C a 2 + release IP3 inositol 1,4,5-trisphosphate K + potassium ion KB-R7493 2-[2-[4-(4-Nitrobenzyloxy)phenyl] ethyl] isothiourea L -NAME N^-nitro-L-arginine methyl ester M A P K mitogen-activated protein kinase mPTP mitochondrial permeability transition pore mtAeq aequorin targeted to mitochondria M L C K myosin light chain kinase M L C P myosin light chain phosphatase Mn manganese MT mitochondria N A noradrenalin Na + sodium ion nAChR nicotinic acetylcholine receptor N C X Na + / C a 2 + exchanger NSCC non-selective cation channel Ni f nifedipine NO nitric oxide PE phenylephrine PIP 2 phosphoinositol bisphosphate PKC protein kinase C PLC phospholipase C P M plasma membrane PSS physiological salt solution ROC/ROCC receptor-operated cation channel ROS reactive oxygen species RyR ryanodine receptor TEA tetraethylammonium ion Tg thapsigargin SEA0400 2-[4-[(2,5-difluorophenyl) methoxy]phenoxy] -5 -ethoxyaniline SEM standard error of the mean SERCA sarcoplasmic-endoplasmic reticulum Ca 2 +-ATPase SK&F-96365 1 - {a-[3-(4-methoxyphenyl) propoxy]-4-methoxy-phenethyl} -1 H-imidazole hydrochloride SNAP-25 synaptosome-associated protein of 25 kDa SNARE soluble N-ethylmaleramide-sensitive factor (NSF) attachment protein receptor SNP sodium nitroprusside xi SOC/SOCC store-operated cation receptor SR sarcoplasmic reticulum TrpC transient receptor potential channel V G C C voltage-gated C a 2 + channel 20-HETE 20-hydroxyeicosatetraenoic acid List of Publications and Abstracts Material from this dissertation has been accepted for publication: . T. Szado, M. McLarnon, X.Wang, and C. van Breemen. Role of the Sarcoplasmic Reticulum in Tonic Contraction of Rabbit Basilar Artery. American Journal of Physiology: Heart and Circulatory Division; AJP - Heart 2001 281: H1481-89 C.Lee, R. Rahimian, T. Szado, J. Sandhu, D. Poburko, T.Behra, L. Chan, and C. van Breemen. Requirement for the opening of fiVsensitive C a 2 + channels and SOC in ai-adrenoceptor mediated venoconstriction. AJP - Heart 282: pg. H1768-77 T. Szado, Kuo-Hsing Kuo, K. Bernard-Helary, Poburko D, Cheng-Han Lee, Chun Seow, U.T. Ruegg, and C. van Breemen. Agonist-induced Mitochondrial C a 2 + Transients in Smooth Muscle. FASEB J. In Press. Material from this dissertation has been submitted for publication: . D. Poburko, T. Szado, R. Rahimian, T. Behra, P. Lhote, C. van Breemen, and U.T. Ruegg. C a 2 + Leak Channels in Smooth Muscle. Cell Calcium. Submitted October 2002. Material from this dissertation has been presented in oral form at the following meeting: . C. van Breemen , T. Szado, M. Nazer, C. Lee, G. Lagaud, E. Lam, I. Laher. SR Regulation of C a 2 + entry and vascular smooth muscle tone. Vascular research meeting. Invited speaker. Honolulu. Hawaii. Material from this dissertation has also been presented in abstract form at the following international and local meetings: . T. Szado, C. van Breemen. Sarcoplasmic Reticulum Regulation of Calcium-Activated Potassium Channels in Rabbit Basilar Arteries. FASEB, Experimental Biology 2000 meeting. Poster presentation. April 2000. San Diego, California. . T. Szado, X . Wang, C. van Breemen. Superficial SR Regulation of K C a in Rabbit Basilar Arteries. FASEB smooth muscle summer conference. Poster presentation. July 2000. Snowmass, Colorado. xm T. Szado, M. McLarnon, X . Wang, C. van Breemen. K c a activation by the SR in rabbit cerebral arteries. FASEB Experimental Biology 2001 and Pharmacology Vancouver 2001. Poster presentation. March 25-29, 2001 and March 31-April 4. Orlando, Florida and Vancouver, British Columbia. T. Szado, K . Bernard-Helary, U.T. Ruegg, and C. van Breemen. Agonist-induced Mitochondrial C a 2 + Transients in Smooth Muscle. FASEB Experimental Biology 2002. Material from this dissertation has also been presented orally for the Graduate Student Seminars Series in the Department of Pharmacology and Therapeutics at UBC, and in research meetings for the Vancouver Vascular Biology Research Centre. xiv Acknowledgements My time as a graduate student has only been a positive one. My greatest thanks goes to the most wonderful supervisor that a graduate student could have, Dr. Casey van Breemen. Not only has he shared his immense knowledge with me, but he has allowed me to explore many aspects of science and to apply all these techniques to a common goal. He presented me with the incredible opportunity to travel to Switzerland for six months to learn a novel method and to experience a new culture, a trip that I will never forget. He is also a great friend and someone whose opinion I will always trust. Thank you Casey. Also, to all my colleagues in Lausanne: Olivier, Valerie, Katy, Phillipe, Timo and Alexandra, thank you for your endless help, and continuing friendship. To Dr. Urs Ruegg, I thank you for giving me the opportunity to work in you laboratory. It was a truly fantastic time and I thank you for your support. I was also lucky enough to have a number of students work with me, particularly Megan McLarnon, thank you for your hard work and for filling the lab with laughter, and Tasniem Behra for teaching me some important molecular biology methods and working so hard on this project. In addition, all of my labmates, from UBC, St. Paul's and Children's, I thank you for your friendship, valuable discussions and various collaborations. Thanks to Vicki Noble, for always being there, and Cor de Wit for proofreading this thesis. Thanks to the Heart and Stroke Foundation of Canada for funding my thesis work over the past years. Finally, to my ever-supportive family who continually encouraged me, I thank you for teaching me patience, hard-work and, always to be humble and contrite of heart. xv CHAPTER 1 • Introduction 1.1. SMOOTH MUSCLE 1.1.1. The structure of smooth muscle There are 3 types of muscle: cardiac, skeletal and smooth muscle. Although these muscles all differ in their organization and function, they share some properties, namely the basic mechanism of contraction. Initiation of contraction of all 3 types of muscle occurs via a Ca2+-dependent mechanism. Smooth muscle is different from the other two in the most visible way due to a lack of striations and hence, is called smooth. Smooth muscle comprises the medial layers in the walls of most hollow organs, including the gastrointestinal tract, urogenital tract, and vasculature, and are a subject of study due to their ability to regulate lumen diameter (Halayko et al, 1997). These spindle shaped single cells are generally 50-100 pm long and 2-5 pm thick (See Figure 1.1.). Smooth muscle cells are connected via gap junctions (channels which span two closely apposed membranes and are composed of connexin proteins), specifically connexins 43 and 40 have been identified in vascular smooth muscle (Christ et al, 1996), and have been shown to be important in receptor mediated contraction. In smooth muscle, T-tubules are absent, there are no elaborate neuromuscular junctions, the sarcoplasmic reticulum is poorly developed and the calcium pump is present but it is slower acting as compared to skeletal muscle. These differences allow smooth muscle to contract using less energy than skeletal muscle, however, it takes about 30 times as long to contract and relax. 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). 1 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). The contractile state of vascular smooth muscle determines peripheral vascular resistance. Moreover, proliferation of smooth muscle cell's is a critical determinant of the resulting arterial remodelling. This thesis will concentrate on the regulation of contraction and relaxation of arterial smooth muscle via Figure 1.1. Morphology of Smooth Muscle A. Diagrammatic representation of smooth mucle cells connected via gap junctions. Activation occurs via binding of, for example, neurotransmitters to their receptors. B. Microscopic image of smooth muscle cells in the small intestine. (B taken from http://www.uoguelph.ca/zoology/devobio/210labs/muscle1.html.) 2 1.1.2. Role of the endothelium Although the role of the endothelium is not the focus of this thesis, since smooth muscle and endothelial cells (EC) are intrinsically connected, possibly via gap ACh F i g u r e 1.2. E n d o t h e l i a l v a s o a c t i v e agen ts . Shown here are the most important mechan isms of endothel ium-dependent relaxation of vascular smooth muscle. Nitric oxide (NO) is produced from the binding of an agonist, i.e. acetylchol ine (ACh) to its receptor on the E C membrane. This activates nitric oxide synthase ( N O S ) which converts L-Arg to N O and subsequent ly increases c G M P in the s m c caus ing relaxation. Prostacycl in (PGI 2 ) is produced from arachadonic acid (AA) via cyclooxygenase-1 ( C O X - 1 ) , diffuses to the smc , increases c A M P and causes relaxation. Finally, the unknown endothel ium-derived hyperpolarizing factor ( E D H F ) is most likely a cytochrome p450 metabolite and a lso diffuses to the s m c where it activates potassium (K + ) channels , caus ing hyperpolarization and relaxation. Adapted from Farac i 1998. 3 junctions (Yashiro and Duling, 2000), one must consider the vasoactive agents released from the EC when studying whole arterial preparations. The main mediators of smooth muscle relaxation derived from the endothelium are shown in Figure 1.2. 1.1.3. The mechanism of smooth muscle contraction Contraction of smooth muscle cells reduces the lumen of arteries and veins and can therefore, regulate blood flow. 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 near the plasma membrane via intermediate filaments and do not depend on motor neurons to be stimulated. Smooth muscle contraction is regulated by Ca2+-dependent myosin light chain kinase (MLCK) activation 9+ and myosin light chain phosphatase (MLCP) (Kamm and Stull, 1985). Once Ca enters the smooth muscle cell and its concentration in the cytoplasm reaches approximately 10"5 M, it binds to calmodulin, a calcium binding protein. This Ca2 +-calmodulin complex then activates myosin light chain kinase (MLCK), which in turn, leads to phosphorylation of the myosin light chain. This phosphorylation allows myosin to bind to actin which leads to muscle contraction (Allen and Walsh, 1994) (Figure 1.3.). Recently, a lot of evidence has been brought to light about regulation of the force/Ca 2 + ratio (Ca 2 + sensitivity) of the myofilaments. The C a 2 + sensitivity of the contractile apparatus varies and the force/Ca 2 + ratio is generally higher during receptor activation (i.e. lower C a 2 + levels produce higher force) than by a depolarization-induced increase in C a 2 + (Somlyo and Somlyo, 1994). Various increases and decreases in force without accompanying changes in C a 2 + may be due to phosphorylation or dephosphorylation processes. Multiple enzymes, like a 4 heterotrimeric GTP-binding protein (G-protein), cyclic nucleotide dependent kinases (PKA and PKG), protein kinase C (PKC), arachidonic acid, rho kinase and tyrosine kinases seem to be involved in this regulation (Nishimura and van Breemen, 1989; Nishimura et al, 1990; Somlyo and Somlyo, 1994; Amano et al, 1996; Hollenberg, 1994; Jinsi etal, 1996). 5 C a V C a M MLCK A M oooperatr/e aiscacfifimeril: AIM •Gai*\-CalMl' MLCK — — — — — > M LCP ATP ATP A D P ' - P , A C ' P ' P , Ca J ' 'CaM-MILCK ; pr. Mp M L C P Figure 1.3. The mechanism of smooth muscle contration. Once C a 2 + enters the cell it can bind to calmodulin (CaM). This Ca -CaM complex can then bind to myosin light chain kinase (MLCK). The combination of these three elements triggers contraction via phosphorylation of the myosin head, thereby allowing myosin to bind to actin and slide past each other in the typical 'sliding filament' fashion. The end of contraction, or relaxation, of smooth muscle is achieved by dephosphorylation of myosin where it can no longer slide past actin filaments. Myosin light chain phosphatase (MLCP) is responsible for this relaxation step. 1.2 CALCIUM SIGNALING IN VASCULAR SMOOTH MUSCLE 1.2.1. Plasma membrane receptors Modulation of the concentration of free cytoplasmic calcium, [Ca2+]j is a ubiquitous signalling system used by neurotransmitters, hormones, and other external signals in a wide variety of cell types to regulate such diverse cellular activities as exocytosis, ion transport, contraction, and motility. There are numerous channels and exchangers in smooth muscle which are critical for C a 2 + homeostasis. These will be described individually below. Voltage-gated Ca2+ channels (VGCC) The most important C a 2 + channel is the voltage-operated channel on the P M in arterial smooth muscle, also known as the dihydropyridine receptor. Of the six subtypes of voltage-gated C a 2 + channels, only the L-type C a 2 + channel (referred to as VGCC) is considered to be a major C a 2 + influx pathway (Kuriyama et al, 1995) in smooth muscle. This channel is composed of a-subunits (See Figure 1.4.) with several different accessory subunits that may be important in pore formation, gating or kinetics (Sanders, 2001). This 6 membrane spanning channel consists of several subunits designated as a i , 012, (3, y and 5, forming an oligomeric complex of 430 kDa (Campbell et al, 1988). The voltage sensor is thought to be contained in the S4 domain of the a-subunit and is activated by depolarization, similar to other voltage gated ion channels (Catterall, 1995). Due to both voltage activating and inactivating mechanisms a window current is defined by the voltages at which V G C C are capable of sustained openings and inward current (Cohen and Lederer, 1987). Although this sustained C a 2 + current is smaller than the initial 7 transient currents, it still generates a large C a 2 + influx relative to the cell volume (Sanders, 2001) and may be a component of basal C a 2 + entry described below. Further V G C C are modulated by several signaling systems, particularly activated by vasoconstrictors that activate the PKC pathway (Hughes and Bolton, 1995). Additionally, vasodilators that stimulate the production of cAMP and activate PKA have been reported to both activate and inhibit these channels (Jackson, 2000). These channels are inhibited by increases in [Ca ];, dihydropyridines such as nifedipine and other antagonists including diltiazem. In addition, T-type V G C C have been identified in some smooth muscles and are characterized by a low voltage activation and small unitary conductance (Tsien, 1999; Nargeot, 2000). These channels can be blocked non-specifically by mibefradil (Hermsmeyer et al., 1997). Figure 1.4. Topological structure of the a1 -subunit of the VGCC or Cav1.2. The topology of the full-length cardiac C a v 1.2 channel. Reproduced from Wielowieyski P.A., era/. 2001. Abbreviations: BTZ, benzothiazepines; DHP, dihydropyridines; PAA, phenylalkylamines. 8 Ca leak A C a 2 + leak, or non-specific influx of C a 2 + , is also a possible C a 2 + influx pathway (van Breemen et al, 1972) although its precise mechanism is still elusive. This Ca leak has been demonstrated in a number of cell types, including smooth muscle cells where it has been shown to substantially contribute to basal influx (Mayer et al, 1972; Casteels and van Breemen, 1975), and therefore, may play a role in the regulation of contractile activity. Receptor (ROCC) Operated and Store Operated Cation Channels (SOCC) The concept of ROCCs in smooth muscle was introduced over 20 years ago (Somlyo and Somlyo, 1968; Bolton, 1979; van Breemen et al, 1978; Bolton and Large, 1986). Receptor-activation results in the opening of intracellular-messenger activated non-selective cation channels (NSCCs). Agonists open VGCCs by depolarising the cell membrane through activation of ROCCs, inhibition of K + channel and/or activation of a Cl" channel (Karaki et al, 1997; Pacaud and Bolton, 1991). ROCCs are defined as C a 2 + channels located in the plasma membrane other than VGCCs that are opened as a result of the binding of an agonist to its receptor and in which channel opening does not involve depolarisation of the P M (Barritt, 1999). For example, ATP, a purinergic agonist is thought to bind to P2X receptors which are ROCCs expressed in smooth muscle (Brake et al, 1994; Nori et al, 1998; Valera et al, 1994). Additionally, SOCCs are a major subfamily in this class of receptors. First, it is important to distinguish between ROCCs and SOCCs. The two main characteristics of SOCCs are: 1) they are not activated by C a 2 + release from stores, but 2) store depletion, regardless of how, activates the store-operated current (McFadzean and 9 Gibson, 2002). The relative importance of SOCC or Ca release activated channels (CRAC) in smooth muscle cells is still controversial. However, there is ample evidence to suggest the existence of some kind of SOCC activated upon depletion of intracellular stores which has also been called capacitative C a 2 + entry (Putney and Ribeiro, 2000). Many groups believe that release of C a 2 + from the SR stimulates activation of these channels and further influx of C a 2 + in order to sustain [Ca 2 +]j (Clapham, 1995). Although there is yet no clear evidence to explain the mechanism of activation of these channels, 2 main hypotheses are proposed. Firstly, a diffusible messenger could be produced upon store depletion and then subsequently activate SOCC (Putney, 1990). Secondly, depletion of the SR stores may confer a conformational change in the SR membrane proteins placing the IP3R in close contact with SOCC (Berridge, 1990; Irvine, 1990; Rosado and Sage, 2000). These hypotheses are difficult to prove as no selective SOCC blockers exist. Nonetheless SKF-96365, an inhibitor of the putative SOCC, is used extensively. Recently, a lot of attention has been placed on the molecular identification of these S O C C s . In fact, a transient receptor gene (trp) identified in Drosphila shows several SOCC-like characteristics and encodes a non-voltage sensitive Ca channel. The products of these genes in C. elegans have been divided into 3 TRP channel subfamilies: short, long, and Osm (Harteneck et al, 2000). The mammalian equivalents have been called C, V, and M respectively (Clapham et al, 2001). TrpC has gained the most attention as putative SOCC channel proteins although a member of the TRPV subfamily, CaT l , may also be involved (Yue et al, 2001). Seven TrpCs, TrpC 1-7, have been identified thus far (Philipp et al, 2000) and TrpCl has been recently shown to 10 encode a component of the SOCC (Sinkins et al, 1998; Zitt et al, 1996). In addition, TrpCl is the pore-forming component which has been localized to the plasma membrane of rabbit V S M C (Xu and Beech, 2001; Inoue et al, 2001; Welsh et al, 2002). TRP channels are tetramers assembled from subunits with six membrane-spanning domains (Clapham et al, 2001). Some are linked to the PIP2 and are permeable to C a 2 + and monovalent cations but, their function remains largely unknown. Further, stretch activated non-selective cation channels, activated by mechanical stretch are believed to open a C a 2 + permeable channel in some smooth muscles (Setoguchi et al, 1997; van Breemen and Saida, 1989). Gadolinium is often used as a non-selective inhibitor of these channels. Potassium channels Potassium channels are critical in maintaining the membrane potential of the cell. Opening of potassium channels leads to an efflux of potassium, membrane hyperpolarization, and a reduced C a 2 + entry through V G C C , thereby causing vasodilation (Clapp and Tinker, 1998). There are several classes of K + channels and the most important ones in smooth muscles are the delayed rectifier K v , the inward-rectifier K i r , the ATP-sensitive K A T P , the Ca -sensitive Kc a . For a detailed review of the role of K channels in smooth muscle see (Nelson et al, 1995; Brayden, 1996; Beech, 1997; Clapp and Tinker, 1998). Briefly, the delayed rectifier or voltage gated K + channels, are important in establishing resting membrane potential. They are activated by depolarization and blocked by 4-aminopyridine (4-AP), intracellular calcium rises and vasoconstrictors. The channel has been identified at the molecular level as K v1.5, 1.1. 11 BKCa fj-subunit B K C a a-subunit s S S S 7_ J3 9 1, —. «i 10 to C O Q -Figure 1.5. Topological structure of Ca2+-sensitive K+ channels. The highly conserved region betweeen S9-S10 is a potential site involved in C a 2 + regulation. The p-subunit has also been shown to confer C a 2 + sensitivity (see text). Reproduced from Vergara C. etal. 1998. and/or 1.2. The Kj r identified as Kjr2.0. and/or 1.0. is activated by membrane hyperpolarization and found in endothelium in addition to the smooth muscle of resistance vessels. It is blocked by barium and high doses of 4-AP and functions in potassium-induced dilation and establishing resting membrane potential. K A T P channels are a complex of sulphonylurea receptor (SUB) subunits and are voltage-independent, nonactivating channels blocked by high intracellular ATP concentration. They can also be blocked specifically with glibenclamide and function in maintaining local blood flow. Finally, and most importantly in regards to this thesis, the Kc a channels consist of large-(BK) intermediate (IK), and small (SK) conductance subtypes. The B K c a channels (200-250 pS) are encoded by a hslo gene and can be modulated by P K A and PKG. These are 12 the most abundant potassium channels in vascular smooth muscle and high levels of Ca , from 3-10 uM, are required for channel activity in the physiological range of membrane potentials (-60 to -30 mV) in relaxed cells (Jackson, 1998). The channel complex is made up of a tetramer of pore-forming a subunits and an additional (31 subunit (see Figure 1.5.). The P subunit is thought to be important in regulation of the C a 2 + sensitivity of the channel. This p i subunit has been recently knocked out in mice by a targeted deletion of 2_|_ the gene, with the physiological consequences being a decrease in Ca sensitivity of the BKc a channels, a reduction in functional coupling of C a 2 + sparks (see section on C a 2 + sparks below) to B K c a channel activation, and increases in arterial tone and blood pressure (Brenner et al, 2000). This channel can be blocked specifically by iberiotoxin and functions as a negative regulator of myogenic tone (Nelson et al, 1995) and vasoconstriction in hypertension (Rusch and Liu, 1997). Additionally, it can be activated by vasodilators acting through cGMP and cAMP (Nelson et al, 1995; Paterno et al, 1996) , epoxides of arachidonic acid (Campbell et. al, 1996) and CO (Wang and Wu, 1997) . 20-OH arachidonic acid produced by cytochrome P4504a, may close these channels (Lange et al, 1997). IKc a channels (20-50 pS) are blocked by 2 mM TEA and charybdotoxin at high concentrations. Unlike B K c a channels, IKc a tend to inactivate with depolarization. Also, in contrast to B K c a channels which require uM [Ca2 +]i to 1 activated, IKc a channels have been proposed to possibly activate by modest increases in [Ca2 +]i and channel opening may persist longer (Vogalis et al, 1998). The SKcachannels (10 pS) have been cloned from brain (Kohler et al, 1996), are activated by cGMP and blocked by apamin. They function in responding to dilation possibly as a result of the recently emerging evidence that they reside on endothelial cells rather than smooth 13 muscle cells and therefore, seem to be involved in the release of EDHF rather than being effectory of EDHF action on smooth muscle cells (Burnham et al, 2002). Chloride channels Two types of Cl" channels have been identified in smooth muscle: Ca2+-activated Cl" channels (Clca) (Large and Wang, 1996) and volume regulated Cl" channels (CIVR) (Nelson et al, 1997; Yamazaki et al, 1998). Some reports show that Clc a are activated by agonist (Large and Wang, 1996) while others disregard these observations due to the hypothesis that Clc a could have little effect on membrane potential due to high densities of B K c a (Nelson, 1998). However, a role for CIVR has been shown to potentially regulate resting membrane potential and myogenic tone (Zimmermann et al, 1997). Therefore to establish a role for the involvement of these channels in regulating vascular tone requires further investigation. Extrusion mechanisms Due to the large chemical gradient of extracellular [Ca2 +] (2 mM) to intracellular [Ca2 +]i (0.1 pM) in smooth muscle, it is critical for the cell to remove C a 2 + and prevent an accumulation of C a 2 + that may lead to cellular dysfunction. 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 an electrical driving force for C a 2 + into the cell and thus further emphasizes the need for C a 2 + extrusion mechanisms. 14 1- Ca 2 +-ATPase 2- Na + /Ca 2 + exchanger 3- Na +/K +-ATPase pump Figure 1.6. C a 2 + Extrusion Mechanisms in Smooth Muscle. Adapted from Klabunde 2002. There are only two major pathways to extrude C a 2 + from smooth muscle cells, namely the P M Ca 2 +-ATPase (PMCa) and the Na + /Ca 2 + exchanger (NCX), the latter of which is also regulated by the Na + /K +-ATPase (Figure 1.6.). The Na+/Ca2 +-exchanger may mediate Ca influx in some smooth muscle preparations under physiological conditions (Karaki et al, 1997; Laporte and Laher, 1997) and can be blocked by DCB in the forward mode and KB-R7493 in the reverse mode. Recently, a more specific N C X inhibitor has been reported, named SEA0400, which is said to be more specific and potent than previous inhibitors (Tanaka et al, 2002). The N C X exchanges 1 C a 2 + out of the cell for 3 Na + ions in. Additionally, Na + influx through ROCCs may stimulate the influx of C a 2 + via the reverse mode of the Na + /Ca 2 + exchanger as demonstrated in the rabbit inferior vena cava (Lee et al, 2001) and in conditions when the activity of the Na + /K +-ATPase is decreased. The role and importance of N C X in smooth muscle appears to vary between smooth muscle preparations. 15 The P M C A is activated by binding of calmodulin to the COOH-terminal end; this increases transport rate and its affinity for C a 2 + (Marin and Rodriguez-Martinez, 1999). Once C a 2 + is pumped out via hydrolysis of ATP, 2 H + are pumped in, which are in turn regulated via a N a + - H + exchanger. The P M C A has no specific blockers to date, but L a 3 + and vanadate block it non-specifically (Carafoli, 1991). It should be noted here that extrusion is one of the main mechanisms for long term maintenance of the gradient of C a 2 + inside and outside of the cell. In addition to extrusion, C a 2 + binding sites in the cytoplasm and uptake of C a 2 + into the SR via SERCA transiently contribute to [Ca2 +]i homeostasis and are discussed below. 1.2.2. TheSR In terms of C a 2 + regulation, the SR is the most important organelle in smooth muscle. It acts as a 'superficial buffer barrier' to C a 2 + entry and in so doing, regulates contraction and relaxation of arteries (van Breemen et al, 1995). C a 2 + initially enters the cell via V G C C or ROCC channels. This influx of C a 2 + is sensed by the SERCA which then pumps C a 2 + into the SR. The buffer barrier model further suggests the existence of three important restricted spaces; superficial SR, myoplasm, and the junctional space (van Breemen et al, 1986 and Figure 1.8, page 31), (for review see van Breemen et al, 1995). C a 2 + which accumulates in the superficial space can be buffered by the SR whereas C a 2 + in the myoplasm will bind to calmodulin and start the cycle for smooth muscle contraction (See Figure 1.3.). C a 2 + accumulation in the junctional space may activate a number of Ca2+-sensitive channels on the P M and SR. The SR has been shown to be a vast intracellular network with a volume estimated from 1.5-7.5% of the cell 16 depending on the smooth muscle cell type (Sanders, 2001) and is continuous with the nuclear envelope (Somlyo, 1985). Most notably, in the cerebral vasculature, a clear role for the involvement of SR 9+ 9+ release channels and K c a channels has been demonstrated. 'Ca sparks' (Ca release from the SR, see C a 2 + sparks section below) activate the B K c a channels on the P M (Nelson et al, 1995) producing a spontaneous transient outward current or STOC (Benham and Bolton, 1986). In addition, stimulation of Ca2+-sensitive Cl" channels has also been reported to be activated by C a 2 + sparks (Zhuge, 1998). Activation of these channels by C a 2 + sparks causes a depolarization of the membrane and subsequent opening of the V G C C . In addition, other junctional and restricted spaces seem to exist, specifically a restricted space between the SR and mitochondrial membranes. This will be discussed more thoroughly in Chapter 4. Ca2+ sparks The existence of a junctional restricted space between the P M and SR has important implications for the regulation of Ca -sensitive pumps and global Ca regulation. Deconvolution microscopy has revealed co-localization of the N C X and SR release channels (Moore et al., 1993) further adding to the wealth of pharmacological evidence for this restricted space. A breakthrough for the importance of this microdomain was demonstrated by Nelson et al. (Nelson et al, 1995) in rat resistance cerebral arteries. Line scanning confocal microscopy and parallel membrane potential recordings revealed that release of C a 2 + from the SR stimulated a spontaneous transient outward current or STOC, which was identified some time earlier (Benham and Bolton, 1986). More specifically, C a 2 + release from RyR released a C a 2 + spark towards the P M , 17 activating BKc a , as demonstrated by specific blockade with iberiotoxin. This activation caused membrane hyperpolarization due to the K+-efflux out of the cell, which inhibited VGCC. In this way, the authors concluded that cerebral arteries possess a negative feedback mechanism essential in regulating constriction and relaxation through modulation of the amplitude and frequency of Ca sparks (Knot and Nelson, 1995). Since this initial discovery, many reports have confirmed this finding and discovered further regulators of C a 2 + sparks including many second messengers (for a review of C a 2 + sparks see Jaggar 2000). Additionally, a recent report shows similar C a 2 + sparks acting as a negative feedback mechanism in human vascular smooth muscle (Wellman et al, 2002). Some unique properties of a C a 2 + spark include a required opening of some 10-20 RyRs acting in a concerted way. Intriguingly, while the [Ca2 +] in the restricted space reaches 10-100 uM in only approximately 1% of the total cell volume (Jaggar et al, 1998), global [Ca 2 +]j rarely increases to over 200 nM. Thus, due to this large, local release of C a 2 + from the SR, B K c a channels are activated (by uM Ca 2 + ) even under physiological conditions (Perez et al, 1999), and can induce a hyperpolarization of -20 mV in single cells (Ganitkevich and Isenberg, 1990). The distance between release sites responsible for C a 2 + sparks and BKc a has been estimated to be 20 nm by mathematical modelling (Neher, 1998; Perez et al, 1999). Recently, proteins termed junctophilins have been isolated from cardiac muscle diads. They belong to a novel family of junctional membrane complex proteins, and may be involved in linking SR and P M elements in such a close spatial apposition (Takeshima et al, 2000). Similar proteins may exist in smooth muscle, but as yet have not been identified. 18 Ca puffs are defined as Ca transients released through IP3R.S and have also been shown to affect BKc a and SKc a channels in colonic myocytes (Sanders, 2001). SERCA The SR is the main C a 2 + store within the cell and is surrounded by a membrane not permeable to C a 2 + at rest. A Ca 2 +-ATPase, i.e. the sarco-endoplasmic reticulum C a 2 + -ATPase (SERCA) is located on this SR membrane and is essential for pumping Ca from the cytoplasm into the SR. There is a 10,000-fold gradient between the SR lumen and the cytosol emphasizing the importance of these SERCAs in pumping C a 2 + from the bulk cytoplasm into the SR. There are 3 genes encoding for SERCA, of which SERCA2b and SERCA3 have been identified in smooth muscle (Wu et al, 1995). A l l SERCAs encode a cytoplasmic region that contains the catalytic site and a transmembrane domain that forms a channel-like structure allowing C a 2 + translocation across the membrane (Engelender and De Meis, 1996; Zhang et al, 1998). The catalytic and transport cycle of SERCAs begins with high-affinity binding of two C a 2 + per ATPase molecule, whereby the enzyme is shifted from an inactive to an activated state (El). The active enzyme forms a phosphorylated intermediate with the terminal phosphate of ATP, corresponding to a second activated state (E2). The two bound C a 2 + ions are thus displaced, followed by hydrolytic cleavage of Pi (Zhang et al, 2000). Phospholamban is a small protein that negatively regulates SERCA; upon phosphorylation via PKC or P K G (Raeymaekers et al, 1990), this 2_|_ inhibition is relieved and SERCA is activated thereby pumping Ca into the SR. 19 Calreticulin and calsequestrin, proteins located within the SR, can bind large amounts of C a 2 + at low affinity and in this way C a 2 + is stored in the SR. The total concentration of C a 2 + 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, CPA and thapsigargin (Tg) respectively, have been instrumental in understanding the importance of SERCA and the SR in regulating contraction and relaxation of smooth muscle. Ryanodine and IP3 receptors In addition to being an intracellular C a 2 + storage site, the SR also releases C a 2 + upon activation (Loutzenhiser and Epstein, 1985) via two different mechanisms: C a 2 + -induced C a 2 + release (CICR) (Bolton et al, 1999; Gordienko et al, 1998; Putney, Jr., 1993) and IP3-induced C a 2 + release (IICR) (Putney, Jr., 1993; Berridge, 1993). CICR is activated by an increase in [Ca2 +]i, which regulates the opening of the RyR, and IICR is induced by IP3 and Ca 2 +(Karaki et al, 1997). The IP3 receptor is mainly found in smooth muscle and nonmuscle cells, while the RyR is most important in striated muscle, but also plays a role in smooth muscle, and neurons. RyR's Named due to their binding to the plant alkaloid, ryanodine (from the American plant Ryania speciosa), the RyRs are endogenously regulated by [Ca \x in the sub-micromolar range (Endo, 1977; lino, 1989) which opens RyRs while mM [Ca2 +]i inhibits opening of these channels (Hayek et al, 2000). This regulation is due to the presence of high- and low- affinity binding sites on the RyR for C a 2 + (Marx et al, 2000). In addition, the Ca2 +-binding protein, calsequestrin located in the SR possibly positioned close to 20 RyRs (Berchtold et al., 2000;Parys et ai, 2000) via junction and triadin proteins (Guo and Campbell, 1995; Zhang et al., 1997), may enhance opening of the RyRs (Szegedi et al., 1999) when phosphorylated. Three isoforms of these channels exist, although only RyR2 and RyR3 have been identified in smooth muscle. Xanthines, such as caffeine are a tool often used to empty the SR, as it directly activates the RyR. Additionally, ryanodine at high concentrations, locks the RyR into a subconductance state thus eventually emptying the SR store (Hymel et al., 1988). Ryanodine at low concentrations activates the channel and ruthenium red, a polycationic agent, blocks RyRs. The physiological importance of RyRs, and specifically CICR seems to be limited to specific smooth muscles that have high current densities through VGCCs and the necessary spatial associations with the RyR (Sanders, 2001). This introduces the concept of smooth muscle heterogeneity that will be investigated in this thesis. Many studies have noted that the same stimulus applied to two different smooth muscles elicits different Ca2+-mediated responses. For example, hypoxia causes resistance pulamonary arteries to constrict while conduit arteries dilate (Michelakis et al., 1997). Evidence suggests that morphology of the smooth muscles, in particular the arrangement and interactions of P M transport proteins with SR, MT and nuclear membranes are crucial in determining the mechanism of C a 2 + regulation in varying smooth muscles. In particular the formation of diffusional barriers resulting from the close proximity of organellar and P M membranes defines different types of junctional spaces within the cytoplasm (Lee C H et al., 2002a). This will be addressed more directly in Chapter 5. 21 IPsR's Since the demonstration of receptor-mediated phospholipase C activation, Ca release in response to IP3, and the purification and cloning of JiVsensitive Ca release channels, inositol phosphate signalling has been rapidly accepted as an important cellular second messenger system (for a review, see Berridge, 1993). G protein-linked receptors activate G proteins in the plasma membrane which stimulate phospholipase C-fl (PLCfl) to split phosphatidyl 4,5-bisphosphate (PIP2) into diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). D A G activates protein kinase C (PKC), and IP3 binds to and gates a distinct class of intracellular, endoplasmic reticulum-bound IP3 receptor (IP3R) channels. Once the IP3R channel is open, C a 2 + flows down the large gradient from the SR into the cytoplasm. The C a 2 + concentration in the cytoplasm rises rapidly, within seconds, to 1-2 u.M levels. C a 2 + diffuses to adjacent I P 3 R S (and ryanodine receptors in many cells) channels, initiating the release of more C a 2 + . The exact details of the propagative release of C a 2 + depend on the cell type. There is a biphasic relationship between the open probability of IP 3R and Ca release (Bezprozvanny et al, 1991; lino, 1990; Mak et al, 1998). High concentrations of intracellular C a 2 + actually decrease the activity of the IP 3R although a rise of C a 2 + to ~300 nM increases the potency of IP3 in activating channel openings (Sanders, 2001). However, due to the C a 2 + sensitivity of the IP 3R, 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 (lino, 1999). Heparin is a specific antagonist of IP3R, but is nonpermeable to the cell membrane. Recently, the permeable inhibitors Xestospongin C (Gafni et al, 22 1997), and 2-aminophenylborate (2-APB) (Maruyama et al, 1997) have been identified and these are important pharmacological tools in determining the role of IP3R in C a 2 + signaling. However, caution must be used when interpreting results using both Xestospongin C and 2-APB, as their selectivity has been questioned in recent studies (Ma et al, 2002; Bilman and Michelnageli, 2002) Myogenic Tone Arteries and arterioles exist in a state of partial contraction, the myogenic tone, which is dependent on the level of intravascular pressure. This mechanism participates in local blood flow regulation, setting of peripheral vascular resistance and regulation of capillary hydrostatic pressure. Importantly, the level of constriction provided by myogenic tone enables the vessel to respond to vasodilatory stimuli. The exact signal transduction pathways involved are still uncertain. However, it is clear that this phenomenon resides in vascular smooth muscle and involves, as other mechanisms of contraction, Ca2+/calmodulin-dependent phosphorylation of the myosin light chain and subsequent interaction of myosin and actin (for a review see Hi l l et al, 2001). A key player in myogenic tone is extracellular C a 2 + , its influx resulting in the elevation of [Ca2+]j. Although VGCCs are a major player in C a 2 + influx, other mechanisms may contribute, e.g. stretch activated cation channels, and they may be critically involved in initiating the response. The role of C a 2 + release is relatively poorly defined, but some evidence indicates a role for C a 2 + release via second messengers like IP3. Additional important regulatory mechanisms contribute to myogenic tone and these can be summarized as C a 2 + sensitization or desensitization pathways. One example is the hypothesis that enhanced phosphorylation of the myosin light chains by activation of 23 PKCct and/or RhoA may be key mechanisms for the Ca sensitization (Yeon et al, 2002). Further, inhibitors of the formation of 20-HETE block the myogenic response of renal, cerebral, and skeletal muscle arterioles in vitro and autoregulation of renal and cerebral blood flow in vivo (Roman, 2002). 1.2.3. The mitochondrion Mitochondria have long been known as the energy producers in the cell via oxidative phosphorylation. Pyruvate, NAD+-isocitrate and 2-oxoglutarate dehydrogenase are specific mitochondrial enzymes that regulate the production of ATP via activation of the tricarboxylic acid cycle. These enzymes are directly regulated by an increase in mitochondrial C a 2 + (McCormack and McCormack, 1994; De Giorgi et al, 1999; McCormack and Denton, 1999). Therefore, it is intrinsically important for the mitochondrion to regulate C a 2 + in order to match the energy demand of the cell. Additionally, recently emerging evidence from various cell lines shows the importance of mitochondria in global C a 2 + homeostasis. It has now become clear that in many cell lines, delivery of nyRyR-mediated [Ca2 +]i spikes to the mitochondria is due to the strategic localization of the mitochondria at C a 2 + release sites (Rizzuto et al, 1993a; Rizzuto et al, 1994;Rizzuto et al, 1998; Hajnoczky et al, 1995; Brini et al, 1997; Simpson et al, 1997; Csordas et al, 1999; Sharma et al, 2000). It was well known that the mitochondria possess a C a 2 + uniporter with a fCj for C a 2 + of approximately 10-20 pM. Cytoplasmic C a 2 + rises to about 1 pM in response to agonist stimulation. The low agonist induced global [Ca2 +]i increases paired with the large fCj for C a 2 + required by the mitochondrial uniporter was initially believed to prevent mitochondria from playing a key role in regulating C a 2 + at basal levels. However, emerging evidence using specifically targeted 24 molecular probes and Ca -sensitive dyes, both of which are localized to the mitochondria, have revealed that the mitochondria are capable of sensing C a 2 + signals initiated by the SR. Close apposition of SR and MT membranes has been reported in HeLa cells (Rizzuto et al, 1998) and thus, agonist stimulation can generate a large C a 2 + burst from the SR, supplying a restricted microdomain in which C a 2 + increases to levels required for activation of the mitochondrial uniporter. Therefore, a role for mitochondria in regulating C a 2 + release in smooth muscle also seems plausible and is confirmed with aequorin targeting experiments in Chapter 4 of this thesis. Some of the other important roles of mitochondria in the cell include production of reactive oxygen species, and apoptosis. C a 2 + may be an important trigger in cell apoptosis as calcineurin and calpains, two Ca2+-sensitive proteins are thought to be involved in cell death (Klee et al., 1998; Carafoli and Molinari, 1998; Nicotera and Orrenius, 1998). To fulfill all these functions mitochondria contain many pumps and channels located in the inner mitochondrial membrane while the outer membrane is freely permeable to small molecules. However, the genes of the various C a 2 + transporters have yet to be cloned. Nonetheless, the 3 most important C a 2 + regulating channels in the mitochondria are the C a 2 + uniporter, PTP and N C X (See Figure 1.7.). Ca2+ uniporter The driving force for this gated channel is due to the negative mitochondrial membrane potential generated by the respiratory chain (Litsky and Pfeiffer, 1997). It has a low affinity for C a 2 + with a ICj of greater than 10 uM (Pozzan et al, 2000) and therefore has been previously ignored as an important intracellular organelle in the 25 regulation of Ca . Recently however, using mitochondrial GFP in both hepatocytes and HeLa cells, it has been shown that the SR/ER membranes and the mitochondrial membranes are in close spatial arrangements (Hajnoczky et al, 1995; Rizzuto et al, 1998) thereby suggesting the existence of a Ca microdomain which sees sufficiently high [Ca 2 +]j to activate the uniporter. This uniporter can be blocked exogenously by ruthenium red and its derivatives, Ru360 (Matlib et al, 1998) and other external nucleotides (Litsky and Pfeiffer, 1997). In addition, it is regulated endogenously by spermine and M g 2 + (Gunter and Gunter, 1994). The second C a 2 + entry pathway in addition to the C a 2 + uniporter is the rapid uptake mode (RaM) (Gunter et al, 1998) which is transiently activated by high [Ca ]j but its exact mechanisms and relative function in smooth muscle is as yet, unknown. 26 Figure 1.7. Schematic cartoon representing the major important pathways in mitochondrial function. The most important role of the mitochondrion is to produce A T P via oxidative phosphorylation. This occurs via electron transport through the electron transport chain (ETC) which consists of complexes I, an NADH: ubiquinone oxidoreductase, II, a succinate: ubiquinone oxidoreductase, III, an ubiquinol: cytochrome c oxidoreductase, and IV, a cytochrome c oxidase and ultimately these electrons are transferred to oxygen, and produce H 2 0 . Production of A T P results from the re-pumping of protons through complex V, a F ^ o - A T P synthase (the subunit is the A T P synthase while the F 0 subunit is the proton channel) and is thereafter transported out of the cell via the adenine nucleotide translocator (ANT). In addition, mitochondria are capable of taking up C a 2 + via the electrogenically driven mitochondrial C a 2 + uniporter, (U), which can be inhibited by ruthenium red. C a 2 + can be extruded from the mitochondrial matrix by a mitochondrial N a + - C a exchanger, (mNCX) or a Na +-independent pathway not illustrated here. Increases in [Ca 2 + ] m or substrates such as acetyl C o A may activate enzymes involved in the tricarboxylic acid cycle, (TCA) and subsequent production of NADH. The permeability transition pore, (PTP), located at both the inner and outer mitochondrial membrane, may play a role in C a 2 + regulation within the mitochondria, but this is still uncertain. Note that the ANT forms part of the m P T P in addition to cyclophilin D (cyp D). Opening of the PTP releases cytochrome c (cyt c) and apoptosis-inducing factor (AIF), both of which cause cell death. The anti-apoptotic factor Bcl2 and the pro-apoptotic factor Bax regulate this process. Finally, the mitochondria are also responsible for the generation of toxic oxygen radicals. When complex I is inhibited, electrons accumulate and are able to bind with oxygen in the mitochondrial matrix producing superoxide (0 2 *") and later hydrogen peroxide 27 ( H 2 0 2 ) with the help of superox ide d i smu tase ( S O D ) . Howeve r , H 2 0 2 c a n be detoxi f ied by glutathione pe rox idase . If iron is present , the highly react ive hydroxyl rad ica l (OH*) c a n a l so be fo rmed. C o Q , c o e n z y m e Q10 or ub iqu inone; C s A , cyc lospor ine A ; F C C P , ca rbony l cyan ide p-t r i f luoromethoxypheny lhydrazone (abo l ishes the proton gradient) ; AY™ mi tochondr ia l m e m b r a n e potent ial ; C N - , cyan ide ; C O , cobalt . Mitochondrial NCX Inasmuch as the mitochondria requires C a 2 + , too much C a 2 + has been shown to cause apoptosis via activation of the mPTP (Hunter et ah, 1976) and subsequent release of Bax, an apoptotic factor. The association of the mPTP, Bax and its pore forming activity are prevented by the anti-apoptotic factor Bcl-2 (Kluck et ah, 1997; Yang and Yang, 1997; Somlyo et ah, 2000). Accumulation of C a 2 + is balanced by C a 2 + extrusion from the mitochondria by two mechanisms, N C X and a Na+-independent C a 2 + efflux. It is as yet unknown which one is predominant in smooth muscle although there are reports in other cells showing that the mNCX is dominant (Crompton and Roos, 1985; Sorimachi et ah, 1999; Gunter and Gunter, 1994). Normally, the N C X works as a passive electroneutral exchanger however, under certain conditions it can be electrogenic (3Na + / lCa 2 +) or an active electroneutral (2Na + / lCa 2 +) exchanger (McCormack and Denton, 1999). It can be specifically blocked with CGP 37157, a benzothiazepine derivative of clonazepam (Cox et ah, 1993). 28 The Mitochondrial Permeability Transition Pore (mPTP) For a review of the mPTP refer to Bernardi 1999 (Bernardi, 1999). The mPTP is a high conductance, non-selective channel which is dependent on matrix C a 2 + . A typical characteristic of mPTP is a sudden increase in the permeability of the mitochondrial inner membrane to small ions and molecules, leading to a complete collapse of the membrane potential and to swelling of the mitochondrial matrix. The mPTP has a voltage dependency similar to voltage-dependent anion channels and has a very large conductance (Litsky and Pfeiffer, 1997; Gunter and Gunter, 1994). Its opening is stimulated by [Ca 2 + ] m in excess of 20pM according to (McCormack and McCormack, 1994) and >100pM according to (Gunter and Gunter, 1994), oxidative stress or markedly reduced energy capacity. The roles of the mPTP are still controversial, but a role in apoptosis (via activation of Bax and Bcl2 as shown in Figure 1.7.) is gaining much attention and has been recently reviewed (Marzo et al, 1998; Bernardi et al, 1999; Kroemer and Reed, 2000). Cyclosporin A (CsA) is an inhibitor of the mPTP (Duchen, 1999). A role for the transient opening of the mPTP in the regulation of basal Ca levels has also been proposed (Ichas et al, 1997) and thus it may not only be activated under conditions of stress, but also under physiological conditions. It has also been hypothesized to function as a physiological mitochondrial C a 2 + release channel in vivo (Bernardis et al, 1996), however, whether the mPTP is important in smooth muscle cell C a 2 + regulation is yet to be determined. 29 1.2.4. A model for Ca signalling in vascular smooth muscle Figure 1.8. summarizes the main C a 2 + pathways, channels, and intracellular organelles involved in smooth muscle Ca regulation. 30 cytosol acting myoplasm myosin 31 Figure 1.8. C a 2 + handling in smooth muscle . The main Ca 2 + pathways present in smooth muscle are shown. Arrows represent normal movements of Ca 2 + . Ca 2 + enters the cell via DHPR and is sequestered via SERCA into the SR where it is tightly bound. A restricted subplasmalemmal space is crucial for this buffering to occur. In addition, a junctional space between the SR and PM exists where Ca 2 + release via RyR (Ca2 + sparks), or release via IP3R activates Ca2+-sensitive K+ and Cl" channels (BKC a/SKC a and CIC a respectively) in addition to NCX. Another restricted diffusional space exists between the SR and MT and release of Ca 2 + through SR release channels activates the mitochondrial uniporter. Ca 2 + is extruded from the MT via a mNCX or a Na+-independent efflux and possibly the mPTP, although the role for the latter channel is still unknown. Ca 2 + can also diffuse to the deeper cytosol where it may bind to calmodulin and initiate contraction via activation of myosin light chain kinase. Other Ca 2 + entry mechanisms include agonist-activated NSCC, activated by NE stimulation in this figure and CCE channels. The amount of Ca 2 + entry through NSCC is controversial, but these channels yield depolarization that activates DHPRs. ATP binds to receptors coupled to G proteins (Gq/G11) and activates PLC which produces IP3 via hydrolysis of PIP2. IP3 binds to receptors in the SR membrane and causes Ca 2 + release. This can sum with Ca 2 + entry mechanisms and contribute to global Ca 2 + transients. Finally, in addition to NCX, the PMCA is important in removal of Ca 2 + from the cell. Abbreviations: BKC a, large conductance Ca2+-activated K+ channel; CCE, capacitative Ca 2 + entry channel; CIC a, Ca-sensitive chloride channel; DHPR, dihydropyridine receptor; IP3R, IP3 receptor; mNCX, mitochondrial NCX; mPTP, mitochondrial permeability transition pore; MT, mitochondrion; NCX, Na7Ca 2 + exchanger; NE, norepinephrine; PIP2, phosphoinositol bisphosphate; PLC, phospholipase C; P2 YR, purinergic receptor; PM, plasma membrane; PMCA, plasmalemmal Ca2+-ATPase; RyR, ryanodine receptor; SERCA, sarcoplasmic Ca 2 + -ATPase; SKC a , small conductance Ca2+-activated K+ channel; SR, sarcoplasmic reticulum; Aym, mitochondrial membrane potential; ?, uncertain physiological role of the mPTP. Adapted from Challet, thesis 2001; Sanders 2001; Lee etal. 2002. 1.3. RECOMBINANT AEQUORIN AS A TOOL FOR MEASURING CALCIUM Aequorin This 22-kDa protein, derived from the Pacific jellyfish, Aequorea victoria, is a photoprotein dependent on the presence of C a 2 + to emit light or photons based on the reaction shown in Figure 1.9. (Prendergast, 2000). 32 Coelenterazine Aequorin Coelenteramide Figure 1.9. Recombination of Aequorin. Upon addition of the prosthetic group, CoE (coelenterazine), the apoaequorin (APO) will form the fuctional 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. Aeq is composed of 189 amino acids and has 3 E-F hand structures which are thought to bind C a 2 + although only two occupied sites seem to be required to initiate activation (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 blue light (466 nm) in the presence of small amounts of C a 2 + and therefore can be used as a C a 2 + indicator. A functional aequorin molecule is only obtained after the apoaequorin has been incubated with its prosthetic group, coelenterazine and oxygen which forms a stable intermediate (see Figure 1.9.). When C a 2 + binds to the complex a fast, irreversible photochemical reaction occurs, converting coelenterazine (CoE) to coelenteramide which ultimately emits light at 466 nm which can be detected and quantified. Thus, once C a 2 + binds to the functional aequorin, it can only emit one photon. Reconstitution with additional CoE and removal of C a 2 + will regenerate 33 the functional photoprotein. In addition since aequorin (Aeq) has been cloned (Inouye et al, 1985; Prasher et al, 1985), it is now possible to genetically engineer the apoaequorin in Escherichia coli. This recombinant protein can be transfected into cells and reconstitution of the functional Aeq is possible by simply incubating cells with the hydrophobic prosthetic group, CoE which is membrane permeable (Rizzuto et al, 1994). Thus, it is now possible to more easily introduce this photoprotein into mammalian cells 2+ where previously microinjection was necessary due to its large size. However, other Ca 2+ indicator dyes such as fura 2 or fluo 4, are better suited for measurements of global Ca . In the last 10 years, Aeq has been used to specifically measure C a 2 + concentrations in various cellular compartments, such as. mitochondria and SR, where previously only slightly selective membrane-permeable C a 2 + indicators were useful. This is due to Aeq fusing with molecular targeting sequences. Targeted aequorins have been constructed for mitochondria, ER, SR, P M , cytosol and the nucleus (See Table 1 and Figure 1.9., and refer to (Chiesa et al, 2001) for details of recombinant AEQs). This targeting is achieved by an N-terminal fusion 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 aeq cDNA allows immunolocalization. In addition, a GFP targeted to the same mitochondrial sequence as used in the mtAeq plasmid, has been made to allow for simpler identification of correct localization by 2_|_ imaging with confocal microscopy. Moreover, photoproteins with differing Ca 34 sensitivities have been created by mutation of specific amino acids. For example, changing low affinity Aeq by one amino acid (Asp 119 to Ala) modifies the low affinity Aeq to a high affinity one which when incubated with n-CoE, showed rapid millimolar C a 2 + transients in a new subpopulation of mitochondria (Montero et al, 2000). Thus, not only can the Aeq be mutated, but CoE analogs regulate the Ca sensitivity of the functional photoprotein additionally regulating membrane permeability and regeneration rate (Shimomura et al, 1989; Shimomura et al, 1990; Shimomura et al, 1993). This is 2_|_ especially important in organelles such as the SR or ER where Ca content is high and a low affinity Aeq is absolutely required as it is immediately consumed with such high [Ca2+]i. 35 Target Targeting Sequence Reference Cytosol, cytAEQ None (Brini et al., 1995) ER, erAEQ CH1 domain of lgG2 which binds to BiP (an ER protein) (Montero etal., 1995b) Mitochondria, mtAEQ Subunit VIII of human cytochrome c oxidase (Rizzuto etal., 1992) Mitochondrial intermembrane space, mimsAEQ Glycerol phosphate dehydrogenase (Brini etal., 1994) Nucleus, nuAEQ Glucocorticoid receptor nuclear localization signal (Brini etal., 1994) Plasma membrane, pmAEQ SNAP-25 (Marsault etal., 1997) SR, srAEQ Calsequestrin (Robert etal., 1998) SNAP-25, S N A P -G F P SNAP-25 (Marsault etal., 1997) G F P , MT-GFP Subunit VIII of human cytochrome c oxidase (Pozzan et al., 2000) Golgi, goAEQ Siayltransferase (Pinton etal., 1998) Gap Junctions Connexins (Martin etal., 1998) Secretory Vesicles 2-synaptobrevin (Mitchell et al., 2001) Outer surface of secretory vesicles Phogrin (granule protein) (Pouli et al., 1998) Table 1. Targeted Aequorins/GFP Note: T h e n u / c y t A E Q is loca l i zed in the cytosol until it is t rans loca ted to the nuc leus by the addit ion of g lucocor t ico ids (Brini etal. 1994). Tab le adap ted from (Chal le t , 2001). Abbrev ia t ions : B ip , heavy cha in binding protein; S N A P , synap togen a s s o c i a t e d protein. 36 HP\ Aequorin H . J H h c y t - A E Q 100 bp i—i COX 3 m t - A E Q m i m s - A E Q e r - A E Q s r - A E Q g o - A E Q p m - A E Q c y t / n u - A E Q — n u - A E Q F i g u r e 1.10. S c h e m a t i c r e p r e s e n t a t i o n o f the t a r g e t e d a e q u o r i n s ( A E Q ) c u r r e n t l y a v a i l a b l e . The blue region is the apoaequorin while the green region has the appropriate targeting sequence or none at all as is the case for cytoplasmic (cytAEQ). The hemagglutinin (HAI) tag is represented as a small black box. In the case of nuclear vs. cytoplasmic targeting, nu-AEQ refers to the nucleus only whereas cyt/nu-AEQ can be directed to the nucleus or cytoplasm depending on the presence or absence of glucocorticoids. Abbreviations: cyt, cytoplasm; er, endoplasmic reticulum; go, golgi apparatus; mims, mitochondrial intermembrane space; mt, mitochondrial; nu, nucleus; pm, plasma membrane. From Chiesa etal., 2001. 37 In summary, there are 3 main ways in which AEQ can be modified to change its 2"T" affinity. Firstly, one can mutate the Ca binding site, i.e., as for the ER-probe, done by mutating Asp-119 to Ala which decreases the C a 2 + affinity 20-fold (Chiesa et al, 2001). Secondly, one can use a modified prosthetic group, such as coelenterazine n which further decreases the affinity of the mutated photoprotein (Barrero et al, 1997). Finally, the substitution of Sr 2 + for C a 2 + will drastically decrease aeq light emission (Montero et al, 1995a). Therefore a number of different constructs are currently available for specific detection of C a 2 + in various organelles and restricted spaces. However, there are several limitations of aequorin that limit its use. Firstly, because Aeq is rapidly and irreversibly consumed by high C a 2 + , experimental duration and design are limited. Further, detection of Aeq in single cells is extremely difficult as the low light output is difficult to detect (Rutter et al, 1996; Hofer et al, 2000). Lastly, as cells must be lysed with digitonin permeabilization at the end of each experiment for accurate calibration, no further experiments can be performed. Nevertheless, Aeq offers many advantages over other standard C a 2 + imaging dyes. The most important being the specificity of the probe and direct C a 2 + measurement in the area of interest. Additionally, Aeq measurements are virtually free of any background, are extremely sensitive and are not weakened by autofluorescence (Haugland, 1995). Furthermore, since the concentration of the functional Aeq is around 100 nM, the buffering capacity of aequorin is negligible (Brini et al, 1995). Because aequorin is targeted, any leakage problems are circumvented and there are no side products of Aeq. 38 AIMS OF THE PRESENT WORK To better understand Ca homeostasis in vascular smooth muscle, I utilized various systems to study C a 2 + movements throughout the cell. I had 3 specific aims: > To characterize the importance of the SR in buffering Ca in the basilar artery and to demonstrate its relationship to Ca2+-sensitive channels in the plasma membrane. The approaches used are: . in vitro myography using intact basilar artery segments in order to measure contractile force in these vessels upon application of various pharmacological agents. • fura 2 imaging done in parallel with contraction studies to directly measure [Ca ]; levels • manganese quenching experiments to test for the existence of a SOCC. > To evaluate the role of mitochondria in cellular C a 2 + homeostasis I utilized a rat aortic smooth muscle cell line. I: transfected targeted mitochondrial aequorin into the smooth muscle cell line and 2_|_ were thus able to directly measure mitochondrial Ca levels upon application of different stimuli. • performed parallel fura 2 experiments to measure [CaZT]i in the same smooth muscle cell line. . carried out E M studies of single smooth muscle cells in order to gain insight into the specific morphology of intracellular organelles in this cell line model. > To examine smooth muscle heterogeneity between three types of smooth muscle: rabbit basilar artery, rabbit inferior vena cava, and rat aortic smooth muscle cells. I achieved this by: 39 measurement of contractile force via a myograph in conjunction with measurement of [Ca 2 +]i via fura 2, and confocal microscopy upon various stimuli in both rabbit B A and IVC RT-PCR studies of channel expression 40 CHAPTER 2 • MATERIALS AND METHODS 2.1. Tissue preparation and myography. Adult New Zealand White rabbits weighing 2.0-2.5 kg were killed by C O 2 asphyxiation followed by exsanguination from the carotid arteries. The brain was removed, immediately placed in cold physiological saline solution (PSS), and placed in the refrigerator. The basilar artery was dissected from the brain in cold PSS continually bubbled with 95% 02-5% C O 2 , cut into approximately four 2-rnm segments, and mounted on a wire myograph (Multi Myograph 610M, Danish Myo Technology) with two 30-pm stainless steel wires, 2.5 cm in length. The tissues were constantly kept at 37°C and continually bubbled with 95% 0 2 -5% C 0 2 in 5 ml of PSS in their respective chambers. The wire myograph was connected to a personal computer and data were recorded and analyzed via myodaq and myodata (LabView, National Instruments) software. To account for differences in length and contractility, values were normalized to a maximal contraction obtained in the presence of high potassium (80 mM) and graphs were produced by Graph Pad Prism. Although many of the responses had an initial transient component, all increases in force were recorded at plateau levels and these values were used in calculation of the mean data, represented by bar graphs in Figs. 3.2.-3.4. Error bars represent means ± SEM. Results shown are representative of at least three experiments. Note: in Figure 3.2., experimental conditions varied; some tissues were pre-treated with L -NAME and Indo prior to pre-tension and others were adjusted to their determined optimal pre-tension and then L -NAME and Indo was added. Therefore, the level of force acting on the tissues was different depending on which protocol was employed. However, in both cases the trends observed were the same and therefore, the 41 data was pooled. Data were analyzed as unpaired data using a minimum of 5 tissues from 5 rabbits. 2.2. [Ca 2 +]i measurement in rabbit basilar arteries. The rabbit basilar artery was inverted with forceps to expose the endothelial cell side, and the endothelium was removed by gently rubbing the inverted vessel on filter paper. The tissue was placed in cold HEPES in a specially designed chamber mounted on two stainless steel rods and stretched slightly. Loading solution was added to the chamber with the following composition: HEPES with 5 pM membrane-permeant fura 2-acetoxymethyl ester (fura 2-AM; 1 mM stock in DMSO) and 5 p M pluronic acid to facilitate loading for 2 h in the dark at room temperature. After incubation with fura 2, the tissue was washed two to three times with HEPES and left for approximately 20 min to remove excessive external fura 2-AM. The chamber, maintained at 37°C, was placed on the stage of an inverted microscope (Nikon Diaphot). The bottom of the basilar artery was exposed to alternating 340- and 380-nm (bandwidth 10 nm) ultraviolet light (1/s) that was passed through a 510-nm (bandwidth 40 nm) cut-off filter before acquisition by an intensified charge-coupled device camera (model 4093G, 4810 series). Fluorescence signals were recorded as digital image data using the software program Northern Eclipse by Empix Imaging Systems housed in a Pentium processor personal computer. The single line trace in each figure is the average of the simultaneously measured fluorescence ratio (F340 /F380) of individual or groups of smooth muscle cells in the chosen field of the basilar artery preparation. Traces shown in Figs. 3.1.-3.9. are each representative of similar responses obtained from at least three experiments. Each trace was obtained from 42 the same basilar artery preparation where noted. Drugs were applied as indicated by the horizontal bars or arrows in each figure. Data were analyzed as paired observations using tissues from 3 rabbits. Simultaneous force/Ca 2 + measurements were done similarly except the tissues were mounted in a specially designed myograph (Danish Myotechnologies) and force was measured by a separate PC. 2.3. Smooth Muscle Cell Culture A primary culture of smooth muscle cells was previously isolated from fetal rat aortae (Lo et al, 1996), grown up as a feeder culture, and stored in aliquots in liquid nitrogen, in 90% D M E M , 10% DMSO. Cells were used between passages 5-11, and were grown in D M E M supplemented with 10% fetal calf serum, 100 units/ml penicillin G, 100 u.g/ml streptomycin, 0.25u.g/ml amphotericin B, M E M vitamin solution, and M E M amino acid solution (Life Technologies). Cells were incubated at 37°C in a humidified atmosphere of 5% CO2. 2.4. Aequorin Technology 2.4.1. mtAeq/pcDNAI expression vector audits amplification The expression vector for mitochondrial-targeting aequorin, mtAeq/pcDNAI, has been previously described (Rizzuto et al, 1992) and was provided by Dr. Tullio Pozzan (University of Padua, Italy). 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 (Fig. 2.1.). 43 Figure 2.1. Plasmid map of pcDNAI-based aequorin expression vector. m t A e q / p c D N A I s h o w s the ~770bp D N A insert ( E C O R I f ragment) with mi tochondr ia l target ing p r e s e q u e n c e ( M P S ) of human cy tochrome c o x i d a s e subuni t V l l l , HA1 ep i tope and apoaequor in cod ing s e g m e n t s (Haug land , 1999). F r o m Cha l le t thes is , 2 0 0 1 . 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 amber-mutant 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 (Haugland, 1999). The amplification protocol was, in brief, the following. Competent bacteria were heat shocked in the presence of lOOng mtAeq/pcDNAI. They were grown in LB medium for 30min at 37°C and were seeded on LB-Agar plate containing 50pg/ml ampicillin. 44 After an overnight incubation at 37°C, colonies of bacteria were isolated and were further grown overnight in 200ml LB medium with 50pg/ml ampicillin. After bacterial suspension was centrifuged, plasmid was purified using the kit Nucleobond® AX500 (Machery-Nagel, Oensingen, CH). The identification of the purified plasmid was checked by enzyme digestion. ECORI restriction enzyme could be used alone (Fig. 2.2.) or in combination with Hindll l (Pharmacia Biotech, Dubendorf, CH). F i g u r e 2.2. D i g e s t i o n o f m t A e q / p c D N A I p l a s m i d w i t h ECORI . T h e plasid m tAeq /pcDANI was ampl i f ied v ia a max ip rep . App rox ima te l y 1 u.g of D N A w a s a d d e d to e a c h lane from 4 different s a m p l e s . A l l s a m p l e s were d igested with E C O R I . T h e lowest band in all s a m p l e s ind icates the D N A insert of m tAeq marked with an arrow. A 100-bp ladder is s h o w n on both s i d e s a s a re ference. 45 2.4.2. Smooth Muscle Cell Line Transiently Expressing Aequorin Targeted to Mitochondria Smooth muscle cells were transiently transfected with a pcDNAI expression vector containing a cDNA encoding aequorin targeted to the mitochondria (Rizzuto et al, 1992). In brief, smooth muscle cells were seeded on Matrigel-coated Thermanox™ coverslips of 13mm diameter in 4-well plates (Nunc, Life Technologies). After one day in culture, cells in 4-well plates were transfected with the following solution: 4pg mtAeq/pcDNAI ( lpg per well) in 12.5 pL of effectene reagent in serum-containing medium (see Materials). Cells were used after 3 days in culture (See Figure 2.3.) Photon quantification after digitonin (100 pM) permeabilization showed that cells exhibited sufficient mtAeq expression for quantitative [Ca ]m analysis and was performed after each experiment. 46 + hv (466nm) Coelenterazine Aequorin Coelenteramide I 4) Luminometry (for setup of luminometer see Appendix 1) Figure 2.3. Steps involved in Preparing Smooth Muscle Cells for Luminometry. 1) The specific plasmid must be amplified by maxiprep and cut with restriction enzymes to ensure correct plasmid has been obtained. Next, 2) smc are transfected with effectene for 20-30 hours. On the day of experimentation, 3) CoE is added 2 hours prior to manipulation and is active for a maximum of 4 hours. Finally, 4) cells are placed in luminometer (see appendix I) and photons of emitted light are obtained as digital data. 2.4.3. Mitochondrial [Ca J Measurement Smooth muscle cells were seeded on Matrigel-coated Thermanox coverslips of 13-mm diameter (Nunc, Life Technologies, Inc.). After 3 days in culture, [Ca ]m was measured in a population of smooth muscle cells (about 60,000 cells/coverslip) as 47 follows. The mtAeq was reconstituted with coelenterazine (5 uM) in D M E M for 2-4 h before the experiment. 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 a rate of 1 ml/min with salt solution (PSS, in mM: NaCl 145, KC1 5, M g C l 2 1, Hepes 5, glucose 10, and CaCb 1.2, pH 7.4). Stimuli were usually applied for 5 min (unless otherwise stated) in PSS. Emitted luminescence was detected by a photomultiplier apparatus (EMI 9789, Thorn-EMI, UK) and recorded every second using a computer photon-counting board (EMI C660) as described previously (Kennedy et al, 1996). As published (Allen and Blinks, 1978; Cobbold et al, 1983; Cobbold and Rink, 1987; Brini et al, 1995), the relationship between recorded counts and [Ca ] is shown in Equation 1. [Ca 2 +]m(M)=VL/Lm a x.10- y (Eq. 1) where L are the recorded photons/s and Z m a x the remaining photons which correspond to the total light output during the whole experiment minus the photons emitted up to the measured point. Total light output was obtained by exposing cells to 10 mM CaCb, after permeabilization with 100 uM digitonin, to consume all aequorin. 2.4.4. Cytosolic [Ca ] Measurement Smooth muscle cells were seeded on glass coverslips of 22 mm diameter. After 40 min loading in 5 uM Fura-2/AM in the dark at room temperature, cells were washed twice with PSS. The coverslip was placed in a thermostated chamber at 37 °C on the stage of a fluorescence microscope (Nikon Diaphot, Kiisnacht, Switzerland). After 3 min of stabilization in PSS, smooth muscle cells were excited at alternative wavelengths of 48 340 and 380 nm, and emission was recorded at 510 nm. The PhoCal software (Life Science Resources Ltd., Cambridge, UK) was used to analyze the collected data. The ratio R between the emitted light at 340 and 380 nm permitted calculation of [Ca 2 +] c according to the equation formulated by Grynkiewicz et al. {Grynkiewicz 1985} shown in Eq. 2. [ C a 2 + ] c = : f f - K d - [ ^ " R ^ ] (Eq.2) L K m a x _ K J where Kd is the apparent dissociation constant for C a 2 + (224nM at 37°C), R is the ratio between the emitted fluorescence at 340nm (F34o) and the emitted fluorescence at 380nm (F380) at the measured point, R m i n is the ratio at zero free C a 2 + (obtained after EGTA (50mM) addition), R m a x is the ratio at saturating free C a 2 + (obtained after ionomycin (lOpM) addition) and p is a correction factor which is equivalent to the ratio F 3 8 0 m i t / F 3 8 0 m a x - The sample autofluorescence determined with 25mM MnCl2 was subtracted from the fluorescence measurements. Calibration was done at the end of each experiment. As no perfusion was available, stimuli were directly added in the PSS bathing cells. [Ca 2 + ] i measurements were done independently of the [Ca2+],„ measurements on separate coverslips. 2.5. RT-PCR 2.5.1. RNA extraction Total cellular R N A from endothelium-removed rings of rabbit IVC, BA or smooth muscle cells were extracted using a RNeasy mini kit™ according to manufacturer's instructions. It is important to note that all the dissection equipment was 49 pre-treated with RNAseZap before use. RNA was quantified by measuring absorbance spectrophotometrically at 260 nm and its integrity was assessed after electrophoresis in nondenaturing 1% agarose gels stained with ethidium bromide. 2.5.2. Semi-quantitative RT-PCR Reverse transcription of 5 u.g total RNA was performed in 60 ul reaction volumes containing 200 units of Superscript II™ reverse transcriptase, 60 units RNase inhibitor, 3 mM MgCb, lx Buffer II (Sigma) and 0.3 u.g random primers and ImM dNTP for 50 min at 42°C. Contaminating genomic DNA present in the RNA preparations was removed by digesting the reaction with 5 units of DNase I for 45 minutes at 37°C prior to the addition of reverse transcriptase. 5 pi of the RT product was used in each 100 ul PCR reaction. The PCR mixture contained 250 uM dNTP, 2 mM MgCl 2 , lx volume of buffer and 2.5 unit Hotstar™ Taq polymerase, and 1 pi of forward and 1 pi of reverse primers. The temperature program for the amplification was 40 cycles of 1 min at 94°C, 1 min at 55°C and 1 min at 72°C. The final extension was completed at 72°C for 7 min. 10 ul of 6x loading buffer (containing 0.25% bromothymolblue, 0.25% xylene cyanol FF, and 15% Ficoll type 400, Pharmacia, in DEPC-treated distilled water) was added to the PCR products. 20 ul of PCR products were then analyzed by electrophoresis on 2% agarose gels stained with ethidium bromide and gels were photographed under U V light. 18S ribosomal R N A expression was used as an internal control. The exemplary gels shown in this report represent findings from a minimum of 5 rabbits. Pieces of rat or rabbit brain were homogenized and used as a positive control for the expression of TrpCl , 2, 3, 4, 5, 6, 7. Primers used for different amplifications were designed from published reports 50 (McDaniel et al, 2001; Walker et al, 2001) or sequences available in Genbank (Table 1). Amplified PCR products from rabbit tissue were isolated from agarose gel, sequenced and found to be 100% identical to the authentic sequences of rabbit trpl~7, otic , ctiD, a n d hslo cDNA. The comparison of expression levels of target genes were performed only after initial normalization to the amount of 18S ribosomal R N A found with PCR. The 18S ribosomal RNA was chosen as a standard for normalization of used R N A and PCR cycling efficacy. Moreover, amplification cycle optimization was carried out to ensure that the results were taken in the log phase of amplification and not in the saturated phase. To verify endothelium denudation of the tissues used for RT-PCR, rabbit B A and IVC were stained with Hoechst 33342, a dye commonly used to stain the nuclei of living cells. Endothelial cell nuclei were oblong shaped and smooth muscle cell nuclei appeared elongated (data not shown). After endothelium-denudation, only smooth muscle nuclei were present thus confirming complete endothelium removal. However, the presence of other contaminating cells, such as fibroblasts was not excluded. 51 Channel GenBank Accession No. Predicted Size, bp Sense/ Antisense Location, nucleotides mTrpI U40980 372 5'-CAAGATTTTGGGAAATTTCTGG-3' 5'-TTTATCCTCATGATTTGCTAT -3' 1-22 352-372 rTrp2 AF136401 487 5'-CAGTTTCACCCGATTGGCGTAT-3' 5'-CTTTGGGGATGGCAGGATGTTA -3' 1606-1627 2071-2092 hTrp3 U47050 331 5'-ATTATGGTGTGGGTTCTTGG-3' 5'-GAGAAGCTGAGCACAACAGC -3' 1483-1502 1795-1814 mTrp4 U50922 265 5'-CAAGGACAAGAGAAAGAAT-3' 5'-CCTGTTGACGAGTAA I I I CT -3' 2535-2553 2781-2800 mTrp5 AF029983 419 5'-CCTCGCTCATTGCCTTATCA-3' 5'-TGGACAGCATAGGAAACAGG -3' 675-694 1075-1094 mTrp6 U49069 410 5'-CTGCTACTCAAGAAGGAAAAC-3' 5'-TTGCAGAAGTAATCATGAGGC -3' 738-758 1128-1148 mTrp7 AF139923 260 5'-TGACAGCCAATAGCACCTTCA-3' 5'-GCAGGTGGTCTTTGTTCAGAT -3' 2397-2417 2637-2657 hsio - 277 5'-GGACTTAGGGGATGGTGGTT-3' 5'-GGGATGGAGTGAACAGAGGA-3' 3237-3256 3533-3514 raig AF027984 221 5'-GAACGTGAGGCCAAGAGT-3' 5'-GCTTGTATGCGTTCCCCT-3' 3910-3918 4113-4130 r a i c M59786 371 5'-ATCCCCAAGAACCAGCAC-3' 5'-GGTGATGGAGATGCGGGAGTT -3' 3882-3900 4233-4253 Table 2. Oligonucleotide sequences of the primers used for RT-PCR Trp, transient receptor potential; m, mouse; r, rat; h, human. 2.6. Confocal Microscopy Confocal microscopy of the smooth muscle cells in Chapter 4 was achieved with an Olympus BX50WI microscope using a 60x water dipping lens (numerical aperture of 0.90) and an Ultraview Nipkow confocal disk. Data were acquired and analyzed with Ultraview 4.0 software. Confocal microscopy experiment in Chapter 5 was performed in collaboration with Cheng-Han Lee, a MD-PhD student in Dr. Casey van Breemen's laboratory; details are given below. 52 Data acquisition Detailed methods have been previously described regarding confocal [Ca2 +]i imaging of in situ vascular smooth muscle cells within intact blood vessels (Ruehlmann 2_|_ et al, 2000). Briefly, inverted rings of rabbit B A were loaded with the Ca -binding dye fluo-4 A M (10 uM, with 10 uM Pluronic F-l27, dissolved in PSS for 90 min at 25°C), followed by a 30 minute equilibration period in normal PSS. The rings were 2_j_ , , isometrically mounted on a custom-made microscope stage. [Ca ]; imaging was accomplished with the use of a Noran Oz™ laser scanning confocal microscope (Noran Instruments, Middleton, WI) with a 100 urn slit through an air 20X lens (numerical aperture 0.45) on an inverted Nikon microscope. The 488 nm line of an argon-krypton laser illuminated the lumen side of the vessel, while a high-gain photo-multiplier tube collected the emission after it had passed through a 525/25nm bandpass filter. A l l parameters (eg, laser intensity, gain) were left unchanged during the experiment. The scanned region corresponds to a 232 pm x 217 pm area on the tissue and yields an image 512 pixels x 479 pixels in size. Image acquisition was set at a rate of 1.07 frames/s. A higher image acquisition rate was unnecessary, as it did not yield any additional information. The experimental trace shown represents the averaged fluorescence signals from a 3x3 pixel region (1.36 pm2) in a single cell. Changes in fluorescence intensity 2_|_ directly reflect changes in the [Ca ]j. Data analysis Data analysis was performed in Image-Pro Plus© (Media Cybernetics; Silver Spring, MD) using customised macros. The representative experimental fluorescence traces reflected the average fluorescence signal from a region of 3 x3 pixels (or 1.36um ) 53 in size within a single cell. The changes in fluorescence (F525) in this region directly reflected changes in [Ca 2 +]j. The 1.36pm2 region was positioned towards the midline of the spindle-shaped smooth muscle cell that was delineated by the basal fluorescence level prior to stimulation. 2.7. Electron Microscopy The primary fixative solution contained 1.5% glutaraldehyde, 1.5% paraformaldehyde and 2% tannic acid in 0.1 M sodium cacodylate buffer that was pre-warmed to the same temperature as the experimental buffer solution (37 °C). Cultured aortic smooth muscle cells were re-suspended (with 0.25% trypsin and ImM EGTA for lmin at 34'C) and transferred from the culture flask to a glass centrifuge tube. After centrifugation at 1000 rpm for 5 min, the supernatant was quickly removed and replaced with the primary fixative solution. The fixed supernatant was then cut into small blocks, approximately 1 x 0.5 x 0.2 mm in dimension and put in the same fixative for 2 hrs at 4 °C on a shaker. The blocks were then washed three times in 0.1 M sodium cacodylate (30 min). In the process of secondary fixation, the blocks were put in 1% OsO^, 0.1 M sodium cacodylate buffer for 2 hours followed by three washes with distilled water (30 min). The blocks were then further treated with 1% uranyl acetate for 1 hour (en bloc staining) followed by three washes with distilled water. Increasing concentrations of ethanol (50%, 70%, 80%, 90% and 95%) were used (ten minutes each) in the process of dehydration. 100% ethanol and propylene oxide were used (three 10-min washes each) for the final process of dehydration. The blocks were left overnight in the resin (TAAB 812 mix, medium hardness) and then embedded in molds and place in an oven at 60 °C for 8 hrs. The embedded blocks were sectioned on a microtome using a diamond knife. 54 The thickness of the sections were ~80 nm. The sections were then placed on 400-mesh copper grids, stained with 1% uranyl acetate and Reynolds lead citrate for 4 and 3 min respectively. Images of the cross-sections of the muscle cells were obtained with a Phillips 300 electron microscope. 2.8. Solutions and Pharmacological Agents For myograph experiments and experiments using whole tissue the following solutions and drugs were used: PSS contained (in mM) 119 NaCl, 4.7 KC1, 2.5 CaCl 2 , 1.17 MgS0 4 , 1.18 K H 2 P 0 4 , 24.9 NaHC0 3 , and 11.1 glucose (bubbled continuously with 95% 0 2 -5% C 0 2 , pH 7.4 at 37°C). For high extracellular K + solution (80 K + PSS) is identical in composition to normal PSS with the exception of (in mM) 43.7 NaCl and 80KC1. HEPES-PSS contained (in mM) 140 NaCl, 5 KC1, 1.5 CaCl 2 , 1 MgCl 2 , 10 glucose, and 5 HEPES (pH 7.4 with 1 M NaOH at 37°C). For high extracellular K + solution (HEPES-80 K), 75 mM NaCl was replaced with equimolar KC1 to make the final concentration equal to 80 mM. Experimental solutions used for the experiments illustrated in Fig. 3.9. consisted of Krebs solution with only 0.5 mM CaC^ and, in cases where 0 C a 2 + was used, no C a 2 + was added and 0.1 mM EGTA was included in the solution. Analytical grade reagents for PSS, HEPES-PSS, N-nitro-L-arginine methyl ester (L-NAME), histamine, bradykinin, acetylcholine (ACh), indomethacin (Indo), thapsigargin, diltiazem, nifedipine, iberiotoxin (IbTx), apamin, charybdotoxin (CTx), M n 2 + , tetraethylammonium ion (TEA), ryanodine, and DMSO were obtained from Sigma (St. Louis, MO). Fura 2-AM was obtained from Molecular Probes (Eugene, OR). The stock solutions of Indo, nifedipine, ryanodine, and thapsigargin were dissolved in DMSO. TEA was dissolved in PSS or HEPES-PSS; all others were dissolved in double-distilled 55 water. Serial dilutions of all chemicals were made in buffered PSS. The maximal concentration of any vehicle to which preparations were exposed was 0.1%, which had no effect on contraction or fura 2 signals. For mitochondrial experiments and experiments using smooth muscle cell culture, the following solutions and drugs were used: Buffers: PSS contained in mM: HEPES, 5; KC1 5; MgCl 2 , 1; NaCl 145; CaCl 2 , 1.2; Glucose, 10, pH 7.4 at 37°C. 0 C a 2 + solution was the same as PSS without CaCl 2 and included 0.1 mM EGTA. 0 N a + solution was the same as 0 C a 2 + solution except N a + was replaced with an equimolar concentration of N-methyl-D-glucamine (NMDG). Osmolarity was measured for the latter solution with a vapor pressure osmometer (Wescor 5500, Logan, USA) and was approximately 300mOsm. Chemicals: Coelenterazine, Fura-2/AM, and Mitotracker were from Molecular Probes (Eugene, OR). Carbonyl cyanide /?-trifluoromethoxyphenylhydrazone (FCCP) was purchased from Fluka (Buchs, Switzerland). Thapsigargin, N-methyl-D-glucamine (NMDG), adenosine 1,4,5 trisphosphate (ATP), vasopressin (AVP), nifedipine, 2-aminophenylborate (2-APB), cyclopiazonic acid (CPA), and U73122 were from Sigma. Digitonin was from Calbiochem. Effectene was from Qiagen. Matrigel was from Fischer Scientific (Wohlen, Switzerland). 2.9. Data Analysis 2.9.1. Statistical analysis Results are shown as means ± SEM and are analyzed for significance by paired or unpaired t-tests where noted for Results in Chapters 3 and 5. For Chapter 4, where applicable, values are expressed as means ± S.E.M., and significance of difference was 56 calculated by one-way analysis of variance and two-tailed Student's t test for unpaired data in GraphPad Prism. Traces are representative of at least 3 experiments. The off-line [Ca 2 +] c and [Ca 2 + ] m data analysis was performed with the Matlab environment version 5.3 (MathWorks, Gumlingen, Switzerland) to determine the area under the curve (AUC). 2.9.2. [Ca2+]m curve analysis The Matlab® environment version 5.3 (MathWorks, Gumligen, CH) was used to perform off-line data analysis and the Matlab scripting and visualization capabilities were applied. Several parameters, i.e. synthetic values and synthetic views, were determined but only A U C values were utilized in this study. The input and acquisition protocols are defined below. Input protocol Smooth muscle cells were supervised with PSS for 100-150s. They were then stimulated with ATP or A VP for 5 min and after were washed for at least lmin. Acquisition protocol The acquisition protocol was as follows: Photon counting Filtering y n = Zn-l + Zn + Z„+1 where x n, y n and z n are discrete data at time n and Q j ^ is the curve corresponding to type of experiment (KC1, ATP, ...), group j (group of type i experiments done on the same day) and experiment k (coverslip #1 in the group j of type i experiment, ...). The sampling frequency (fe) was 1Hz. Frequencies < 0.5Hz were assumed to be negligible 57 according to the sampling theorem ( f m a x < f e / 2 , where f e is the sampling frequency and fmax the maximal frequency allowed in the observations producer). Synthetic values The following synthetic value (by group j) was determined: A U C Mean of area under the curve ( A U C ) [uM-s] n=1 n=N ^.i.k(n) This mean value is related to the following parameters: AUCIJJC = ){ciJtk{t)-B)lt = fXCuM-Bij*)-A"}[^M-s] o « = | where N = T/AT + 1 with AT = period = l/fe= Is, and n runs from onsetjj;k (n=l) to recoveryij;k (n=N). The approximation used is the discrete rectangular approximation. Onsetijtk = max {n = 1 ...peak time | C^n) = std(Bij,k)} [s] Recoveryij^k = min {n = peak time...dij?k I Cy,*(w) = std(B* y-*)} [s] where B* is the basal after cell activity, std is the standard deviation and d; jsk the dimension of the experiment^. Activity Extent = AE/j^ - Recoveryijk - Onsetijik+ 1 [s] 58 Median values of this curve of a type i experiment were calculated after aligning all curves with respect to their peak. For each sampling time, all the activity values of any curve were used to process the median value. This approach was used to avoid modifying the crude data collected and erasing useful information. The closest curve to the median was determined in the mean-square sense. Microsoft Excel was used for generating figures. 59 CHAPTER 3 • SR BUFFERING OF Ca 2 + IN RABBIT BASILAR ARTERY 3.1. INTRODUCTION Since Devine et al.'s first description (Devine et al, 1972) of sarcoplasmic reticulum morphology in vascular smooth muscle, many functions have been attributed to this organelle. The sarcoplasmic reticulum is specialized in C a 2 + storage, functioning both to relax smooth muscle by removing C a 2 + via sarco(endo)plasmic reticulum Ca 2 +-ATPase (SERCA) from the cytoplasm and activation by release through D-myo-inositol 1,4,5-trisphosphate and ryanodine receptors. Subsequently, more refined aspects of these general functions were discovered. lino et al. (lino et al, 1988) and Saida and van Breemen (Saida and van Breemen, 1983) showed there was calcium-induced calcium release where the C a 2 + entry signal is amplified by opening of the Ca2+-sensitive ryanodine channels in the superficial sarcoplasmic reticulum. This was later confirmed by Ganitkevich and Isenberg (Ganitkevich and Isenberg, 1992) by showing that sarcoplasmic reticulum depletion decreases C a 2 + signals accompanying opening of voltage-gated C a 2 + channels (VGCC). Another indirect mechanism potentially regulating vascular constriction is activation of C a 2 + entry through sarcoplasmic reticulum depletion (Casteels and Droogmans, 1981; Cauvin et al, 1984; Putney, Jr., 1986; Trepakova et al, 2000). On the other hand, van Breemen (van Breemen, 1977) showed that the superficial sarcoplasmic reticulum could also attenuate the VGCC-mediated C a 2 + signal by sequestering C a 2 + from a restricted subsarcolemmal space. This was made all the more plausible because of Somlyo and Franzini-Armstrong's (Somlyo and Franzini-Armstrong, 60 1985) distinction between the superficial and deep sarcoplasmic reticulum. In fact, Somlyo and colleagues (Devine et al, 1972; Somlyo and Somlyo, 1971; Somlyo et al, 1971) showed specialized junctional areas where the two membranes are separated by only 10-15 nm. In the 1980s, Bulbring and Tomita (Bulbring and Tomita, 1987) suggested that localized release of C a 2 + could have a relaxing action on intestinal smooth muscle as seen during a :adrenergic stimulation. This mechanism was established on a firm basis by Benham and Bolton (Benham and Bolton, 1986), who showed localized activation of clusters of K + channels by spontaneous C a 2 + release from the sarcoplasmic reticulum, also termed spontaneous transient outward currents (STOCs). More recently, Nelson et al. (Nelson et al, 1995) provided direct evidence for such local C a 2 + release by imaging C a 2 + sparks. It is of particular interest that sparks more frequently occur peripherally and that there seem to be specific sites that function as hot spots (Imaizumi et al, 1998) or frequent discharge sites (Benham and Bolton, 1986; Perez et al, 1999). This work led to the suggestion that, at least in small cerebral arteries, the main function of the sarcoplasmic reticulum is to relax the blood vessels as a consequence of sparks and STOCs (Nelson, 1995). This hypothesis fits well with the earlier evidence that resistance arteries are mainly activated by C a 2 + influx rather than by intracellular C a 2 + release (Cauvin et al, 1988; Nelson et al, 1995). In addition, this concept may have clinical relevance in that the activity and expression of Ca2+-activated K + channels (Kca) are enhanced in genetically hypertensive rats. The enhanced Ca -sensitive K current may function as a compensatory mechanism to balance the enhanced activity of V G C C 61 observed in vascular smooth muscle cells of another hypertensive rat model (Liu et al., 1995). Considering all of these possible mechanisms of sarcoplasmic reticulum-mediated regulation, I was interested in determining the relative contributions of each of these in a conduit, cerebral blood vessel, the basilar artery. I approached this problem by pharmacologically manipulating C a 2 + pumps and channels in the sarcoplasmic reticulum and plasmalemma and by measuring force and intracellular C a 2 + concentration ([Ca2+]j) in rabbit basilar arteries. I found that both activation of K+ channels by sarcoplasmic reticulum C a 2 + release and buffering of C a 2 + influx are important mechanisms in maintaining the basilar artery in a relaxed state. I found no evidence for store-operated cation channels (SOCCs). 3.2. RESULTS 3.2.1. Thapsigargin Dose Response Curve To examine the contribution of the sarcoplasmic reticulum to C a 2 + activation of Kca, it was important to determine the maximal concentrations of thapsigargin required for depletion of the store. Therefore, a concentration-response curve for thapsigargin was determined by measuring depletion of the sarcoplasmic reticulum Ca content as reflected by inhibition of a caffeine response (Fig. 3.1.). In Fig. 3.1.^ 4, I show reproducible contractile responses to l O m M caffeine that are initiated by C a 2 + release from the sarcoplasmic reticulum and are used in subsequent experiments as a control measure of sarcoplasmic reticulum C a 2 + content. The extent of sarcoplasmic reticulum 62 depletion is measured by the ratio of the caffeine response after a 10-min exposure to thapsigargin (Fig. 3.1.B) divided by the initial control response (Fig. 3.1.^ 4). By varying the concentrations of thapsigargin, a range of values for the percentage of emptying of the sarcoplasmic reticulum could be determined, and a concentration-response curve was constructed (Fig. 3.1.C). From this curve, it was concluded that a 10-min exposure to 1 uM thapsigargin completely depleted the caffeine-sensitive store, and, therefore, this was the minimum concentration used in subsequent experiments. 63 E S 4.5 4 3.5 3 g, 2.5 ] o O 21 1.5 1 Q S 0 0.0 1 1 0 7 13 2.0 0.2 0 9 16 2 2 Time (minutes) - 120 < O a i co O c a 60 S3 40 2 £ 20 0 B 4.5 2 3 5 I 3 ' 25 j y 2 5 L U- 1.5 f 1 0.5 • -e.5 -7.3 -6.3 -60 5 3 Log [TG concentration] (M) A. • 0.0 0 7 13 2 0 2 7 0.3 1.0 Time (minutes) Figure 3.1. T h a p s i g a r g i n Concen t ra t i on -Response C u r v e . A: two consecutive applications of 0 Ca 2 + / 0 .1 mM E G T A / 1 0 mM caffeine (arrows). The second concentration of caffeine was applied after removing it from the solution by a 10-min washout in physiological saline solution (PSS) indicated by the slashed bars in the time scale. 8: same as A but 1 uM T G was applied to all solutions. C: T G concentration-response curve constructed from the ratio of second caffeine responses in 6 divided by control caffeine responses over time in A. A/-nitro-L-arginine methyl ester ( L -NAME, 200 uM) and indomethacin (Indo, 10 uM) were added 30 min before the experiment. Note that 1-2 pM T G was found to completely deplete sarcoplasmic reticulum (SR) stores. S E R C A , sarco(endo)plasmic reticulum C a 2 + A T P a s e . n=9 different t issues from 3 different rabbits from each dose. To eliminate the endothelial contributions to the data obtained in subsequent experiments, tissues were exposed to 200 uM L -NAME [inhibits synthesis of nitric oxide (NO)] and 10 uM Indo (inhibits synthesis of prostacylin) for either 30 min before experimentation or incorporated into the initial bathing solution during the experimental time period. These concentrations were used because they eliminated relaxing responses 64 of the basilar artery to 10 pM ACh and 10 uM bradykinin (data not shown). Other studies have shown that all endothelium-dependent relaxations in rabbit basilar artery can be blocked with NO synthase and prostacyclin inhibitors and that there is no indication for the action of an endothelium-derived hyperpolarizing factor other than NO or a prostanoid in this artery (Mackert et al, 1997). Mechanical removal of the endothelium also inhibited vasodilatory responses to ACh and bradykinin, but these tissues were not used in these experiments because of compromised smooth muscle function. 3.2.2. Sequential blockade of BKca and SERCA After establishing a procedure for the complete depletion of the sarcoplasmic reticulum, I compared the effects of this procedure with direct blockade of large B K c a with IbTx. The rationale for this experiment is that if the only functional consequence of abolishing sarcoplasmic reticulum function is inactivation of B K c a , then the two procedures should be indistinguishable and nonadditive (Brenner et al, 2000). Blockade of the BKca channel with 100 nM IbTx added to the bath solution produced a sustained contraction presumably due to depolarization (Fig. 3.2.A, left). Subsequent additive application of 2 pM thapsigargin tended to further increase contraction, although not significantly due to large variability in individual tissue responses (Fig. 3.3.). Those tissues which contracted close to maximal (as compared to 80 mM K + application) to initial IbTx application often showed no additional contraction (4 out of 10 tissues). However, in other vessels, exhibiting less contraction after initial IbTx (6 out of 10 tissues), additional application of Tg induced a further, significant increase in force, indicating an action at sites other than B K c a (Fig. 3.2.^, right). Figure 3.2.5 examines the C a 2 + signals measured in parallel with the above contraction experiments. IbTx alone 65 caused an increase in [Ca ]j (Fig. 3.2.5, left) and additional subsequent blockade of SERCA with thapsigargin increased [Ca2 +]i further (Fig. 3.2.5, right). In contrast to force, this increase in C a 2 + was significant. In a time control series, the level of C a 2 + fluorescence did not rise over a similar time course (Fig. 3.2Q. Note that IbTx response started to rise upon application but took approximately 10 minutes to reach maximal increase in [Ca2 +]i in Fig. 3.25. This may be due to the fact that drugs were added to the bathing solution (there was no perfusion) and therefore rate of response was propotional to rate of diffusion of the drug. 66 A l O O n M l b T * o IH I 16 IS 14 13 1? B 1 M T G 2 .2 E o 3 0 o 2 oo •£ 20 vP w 10 t0 20 Time (minutes) 100 nM IbTx 2 uM Tg 0 075 a ooo 23.3 35.0 46.7 Time (min) 58.3 70.0 100 nM i&Tx 2 | iM TG 0.7 oo 0.6 co U . o co 0.5 0.0 6.7 13.3 20.0 26.7 33.3 40.0 46.7 53.3 T ime (min) Figure 3.2. Sequential blockade of B K C a followed by SERCA inhibition. Isometric tension traces in response to Ca 2 + -act ivated K + channel (K C a ) antagonist, iberiotoxin (IbTX), followed by application of T G in rabbit basilar arteries. L - N A M E (200 |JM) and Indo (10 uM) were included in the bathing solution. A, left: representative trace of at least 10 tissues from at least 3 rabbits. Right, summary of data compared 80 mM K + contractions with control for tissue variability. No significant additional increases in force after KCa blockade were observed, although the trend was a slight increase in force. S: fura 2 spectrofluorometry of inside-out rabbit basilar arteries denuded of endothelium. Left, IbTX is added first, followed by blockade with T G resulting in an increase in [Ca 2 + ] , which is inhibited by further addition of 10 uM diltiazem. Right, summary of data compared with increases from the initial baseline. *P < 0.05 resulting from a paired f-test. Results represent at least 5 different tissues from at least 3 rabbits. F340/F380, f luorescence ratio. C) Representative trace showing baseline levels, without any stimulation, over 54 minutes in rabbit basilar artery. 6 7 A Figure 3.3. Effect of B K C a channel blockade in Rabbit BA. A ,B . Representat ive traces showing increase in force to application of 100 n M iberiotoxin in rabbit B A under basal condit ions. Result is representative of a minimum of 10 different t issues. Refer to Materials and Methods for myograph experimental conditions. In the reverse situation where SERCA was blocked initially followed by blockade of BKca, I again would expect that the two procedures would be nonadditive if the 2_|_ sarcoplasmic reticulum is solely responsible for releasing Ca toward the B K c a . Abolishing the sarcoplasmic reticulum function first with 2 pM thapsigargin resulted in 68 an increase in tension (Fig. 3.4.^ 4) paralleled by an increase in [Ca ]j (Fig. 3.4.5). When BKca was then blocked in the absence of sarcoplasmic reticulum function, there were 2_j_ additional large increases in tension (Fig. 3.4.^ 4) and [Ca ]j (Fig. 3.4.5). These additional increases induced by IbTx application reached values of significance in the case of force development and [Ca 2 +]i (Figs. 3.4., A, right, and B, right). The additional contraction of IbTx was not only seen in these vessels exhibiting high levels of tone (Fig. 3.4.), but also in tissues in which pre-tension was adjusted after treatment with L - N A M E and indomethacin resulting in lower amounts of force (Fig. 3.7). The level of tone depended on the experimental conditions used as described in Materials & Methods. These results suggest that even though the sarcoplasmic reticulum is empty and SERCA is blocked, BKc a remain active. Therefore, BKc a must also be activated by C a 2 + from sources other than the sarcoplasmic reticulum. 69 2 u M T G | u E o LL B o CO rt 20 18 lh M 13 in 0 B 0.56 0.52 0.48 0 44 0,4 100 nM 8>r« 100 10 15 Time (minutes) 2 M M T G tOOriMfcTx <rt . _ eg 41 0 2 , . I : • I f 11 21 T i m e (m inu tes ) o n 9 n si <u n 0)0. E E £ o g u 0.0 2..V 'G 2 I I M T G IDOnM IbTx +2 MM T G 100 nM IbTx +2 uM T G F igure 3.4. Sequent ia l b l ockade of S E R C A fo l lowed by B K C a inh ib i t ion. A: isometric tension traces in response to the S E R C A antagonist T G followed by application of the large conductance K C a ( B K C a ) blocker IbTX in rabbit basilar arteries. L - N A M E (200 pM) and Indo (10 uM) were included in the bathing solution. Left, representative trace of at least 19 t issues from at least 7 different rabbits. Note that even after the S R has been emptied with T G , IbTX additionally increases force. Right, summary of data compared 80 mM K + contractions with control for tissue variability. **P < 0.005 resulting from an unpaired f-test. 6: fura 2 spectrofluorometry of inside-out rabbit basilar arteries denuded of endothelium. Similar trends are observed compared with force measurements. Left, T G is added first, followed by blockade with IbTX resulting in an increase in [ C a 2 +]|. Right, summary of data compared with increases from initial baseline. *P < 0.05 resulting from a paired f-test. Results represent at least 5 different tissues from at least 5 different rabbits. 70 3.2.3. Simultaneousforce/Ca2+ recording 9+ Additionally, simultaneous force/Ca measurements were performed. These experiments confirmed the data shown in Figure 3.4. As seen previously, 2 | iM Tg increased C a 2 + , and therefore force (Figure 3.4.). Subsequent application of TEA led to a further increase in Ca and force. This could be completely abolished by 10 uM diltiazem. 14 12 10 SOmMK* 80mMK+ 2uM Thapsigargin 3mMTEA I •ml 10uM Diltiazerh • Force (mN) Fluorescence (units) 1.200 1.000 0.800 0.600 9. 0.400 0.200 0.000 -0.200 14200 Figure 3.5. Simultaneous Force/Ca 2* recording in intact rabbit basilar artery. Intact arteries were mounted in a specially designed chamber as described in Material & Methods. Denudation of endothelium was achieved by inverting the vessel and gently rubbing on filter paper. Two applications of 80 mM K + are shown as control responses. 2 uM Tg raised both force and [Ca2*], levels. Subsequent application of 3 mM TEA further increased both parameters. Finally, both responses could be abolished completely with 10 uM diltiazem. These results are representative of 2 tissues. 71 When the previous experiments were repeated with 10 uM ryanodine in place of thapsigargin, the same trends were observed. Ryanodine alone increased force and subsequent application of 3 mM TEA additionally increased tension (Fig. 3.6.^ 4). [Ca2+]j measurements confirmed that the increases in tension were due to increases in [Ca2+]j. Subsequent addition of 3 mM TEA, after the sarcoplasmic reticulum was no longer functional, further increased [Ca2+]j (Fig. 3.6.5). These results were repeated with 100 nM IbTx (n=3), and the same trends were observed (data not shown). Therefore, regardless of the mode of inactivation of the sarcoplasmic reticulum, i.e., either with thapsigargin (via SERCA blockade) or ryanodine (via depletion due to opening of ryanodine receptors), there still seemed to be activation of B K c a independent of the sarcoplasmic reticulum. Contribution of all Kca channels Because at 3 mM, TEA is a non-specific blocker of all Kc a , it was important to determine the relative contribution of each Kc a channel when blocked with this agent. Therefore, the contribution of small conductance K c a (SKc a) was tested with 100 nM apamin, a selective inhibitor of SKc a (Hinrichsen, 1993). When added subsequently to treatment with ryanodine or thapsigargin and IbTx (blocks B K c a specifically), apamin had no additional effect (Fig. 3.7.), suggesting that in the absence of a functional sarcoplasmic reticulum, SKc a do not play a significant role. Intermediate conductance K c a (IKca) were also examined by the addition of 100 nM CTx after IbTx and apamin were present in the bathing solution. 72 10 uM Ryanodine 6 5 2 4 QJ 3 I 2 ] 1 0 B 3 mM T E A 11 M Time (minutes) 21 10 uM Ryanodine 3 mM T E A 100-75H 50 25H 0.2 0.1 12 18 24 30 36 42 Time (minutes) 0.0 10 uM Ry 1 0 u M R y 3 m M T E A 3 mM T E A Figure 3.6. Application of Ry followed by TEA. Ryanodine (Ry, 10 uM) produces trends similar to those observed with 2 uM TG. A, left: isometric tension measurements in rabbit basilar arteries show an increase in force with 10 uM Ry and an additional increase when 3 mM tetraethylammonium ion (TEA) was subsequently added. Results represent at least 5 tissues from at least 5 different rabbits. L-NAME (200 uM) and Indo (10 uM) were included in the bathing solution. Right, summary of results compared with 80 mM K* contractions. *P < 0.05 resulting from an unpaired r-test. 6: fura 2 spectrofluorometry in inside-out rabbit basilar arteries denuded of endothelium. Left, 10 uM Ry increases [Ca2*], and additional blockade of K C a further increases [Ca2*];. 10 uM diltiazem brings [Ca2+], back to baseline. Right, summary of [Ca2+], data comparing increases from baseline and analyzed with a paired Mest. The more-specific blocker of BK C a , IbTX, has the same effect as TEA, indicating that BK C a is active without a functional SR. 2 pM Tg 100 nM IbTx 100 nM CTx 10 20 30 40 50 60 70 80 90 100 Time (min) F igu re 3.7. C o n t r i b u t i o n s of S K C a , I K C a , a n d B K C a b l o c k a d e s to add i t i ona l con t rac t i on after S E R C A b l o c k a d e . Blockade with TG increased force and additional blockade with IbTX additionally increased force. Apamin had no additional effects, whereas charybdotoxin (CTx) slightly increased force. Rabbit basilar arteries were pretreated with 200 uM L-NAME and 10 (JM Indo. Results represent 8 tissues from 3 different rabbits. Although CTx blocks both IKc a and BKc a , its addition after blockade of SKc a with apamin and B K c a with IbTx would test specifically for the involvement of IKc a. Addition of CTx did appear to have a small effect (Fig. 3.7.), and, therefore, IKc a may also contribute to the regulation of membrane potential (Em) under these conditions. In addition, experiments performed with 3 mM TEA tended to show slightly larger effects than in the presence of 100 nM IbTx, which may be explained by a non-specific action on IKc a (data not shown). Thus, although TEA is not entirely specific (and may additionally block KATP or K v channels), I was confident that the major portions of the responses observed after depletion of the sarcoplasmic reticulum using ryanodine were due to 74 activation of B K c a . This idea was supported by use of the specific B K c a blocker, IbTx, in experiments using thapsigargin. The increases in [Ca2+]j and force observed with sarcoplasmic reticulum-depleting agents or B K c a blockers were completely abolished by application of 1 uM nifedipine or 10 uM diltiazem (see figures 3.2B, 3.3 and 3.5 for representative figures, n=>10 tissues from 10 rabbits; note, in all experiments performed with rabbit basilar artery, nifedipine or diltiazem always completely inhibited responses when applied after initial stimulus or stimuli). The effects of all sequences of SERCA and K c a blockers were completely reversed with V G C C blockers (data not shown). In addition, preincubation with 1 p M nifedipine for 20 min almost completely prevented a high K + response (data not shown). These data support the notion that cerebral vessels rely solely on the V G C C as a source of C a 2 + as has been shown in smaller, resistance-sized cerebral vessels (Gollasch et al, 2000; Cauvine'a/., 1988). 3.2.5. Mn2+quenching To test for a possible contribution by SOCC, 5 uM M n 2 + was added to a nominally Ca2+-free PSS and fura 2 fluorescence recorded at the isobestic wavelength of 360 nm. There was no change in slope when either 10 uM ryanodine and/or 2-5 uM thapsigargin were added to the superfusate, even though subsequent high K + depolarization caused a fourfold increase in the rate of M n 2 + entry (Fig. 3.8.). These data are consistent with the presence of V G C C and absence of functional SOCC in the basilar artery of the rabbit. 75 o to C3 1.8 1.6 1.4 1.2 • 1 0.8 0.6 • 0.4 0.2 -0 -r 5 u.M M n 2 + 2JIM TG and 10 Ryanodine 8 0 m M K e V B OOO-i -025 c ° c C c *S »- p o > o O l O ^ -0.50 o a o c/) CO -0.75 7 10 13 Time (minutes) 17 20 5pM Mn 2p.M TG B0 mM K+ Figure 3.8. Mn 2 + quenching in an endothelium-denuded rabbit basilar artery. The tissue was kept in a 0 C a 2 + H E P E S solution before the addition of M n 2 + (see MATERIALS AND METHODS) . Fluorescence is measured at a wavelength of 360 nM (F360), the isobestic point of fura 2. A: representative trace of F360 in an endothelium-denuded rabbit basilar artery. Note that the addition of T G and/or Ry had no effect on the slope indicating no additional entry of C a 2 + . B: summary of F360 data constructed from the tangent of the slope during application of various agents. K + (80 mM) increased the steepness of the slope and is included as a positive control. The tangent slope was measured within the first 2 min of high K + application as it quenches fura 2 completely. Each bar represents at least 4 tissues from 3 different rabbits. *P < 0.05 resulting from an unpaired f-test. SR buffering Finally, it was demonstrated in these cerebral arteries that the sarcoplasmic reticulum is capable of actively buffering C a 2 + influx. A l l solutions for this experiment contained low CaCl 2 (refer to MATERIALS AND METHODS) to slow down the rate of force 2_j_ development. Figure 3.9. shows rate of force development when Ca is restored and the K + concentration is elevated in the superfusate of basilar arteries with a depleted sarcoplasmic reticulum. Before the increase in extracellular [K+] the sarcoplasmic reticulum was depleted by caffeine in 0 C a 2 + PSS and the caffeine then washed out for 10 min. Under these conditions, addition of thapsigargin to the high-K + solution markedly enhances the rate of force development (Fig. 3.9.B), presumably because it prevents C a 2 + uptake into the sarcoplasmic reticulum as it enters the smooth muscle cells through VGCC. 77 80 m M K f B o 14 •, 12 10 E s a a 6 o u. 4 2 0 80 mM K* 12 uM TG -r-10 1 Time (minutes) 11 1 2 io • 8 6 4 2 0 -2 uM TG control 0.0 0.2 0.3 0.5 0.7 Time (minutes) 08 1 0 Figure 3.9. SR buffering in rabbit basilar arteries. First, 0 C a 2 + / 0 . 1 m M EGTA/caf fe ine was applied to deplete the Ry- sensi t ive store followed by washout of caffeine with 0 C a 2 + / 0 . 1 mM E G T A solution for at least 10 min. A: subsequent application of 80 m M K + produced results as shown by the first trace. The second trace was treated similarly, except 2 p M T G was added to all solutions. 6 : 80 m M K + responses of arteries after depletion with caffeine and with or without T G are super imposed on an expanded time sca le . In the absence of T G (control), the increased tension due to depolarization is markedly s lower compared with arteries in the presence of T G (+2 pM TG) . L - N A M E (200 pM) and Indo (10 uM) were also included in all solut ions. Refer to M A T E R I A L S A N D M E T H O D S for composi t ion and concentrations of solutions. Note that in the presence of T G , tension increases more quickly than under control condit ions. Resul ts represent at least 5 t issues from 3 rabbits. S lashed l ines indicate a break in time for recovery of the t issue in P S S and subsequent S R depletion. 7 8 3.3. DISCUSSION The major findings in this chapter are 1) the effects of thapsigargin or ryanodine and blockade of K c a on force development and [Ca 2 +]i are additive; 2) depletion of sarcoplasmic reticulum does not enhance M n 2 + influx; and 3) inhibition of SERCA enhances force developed during high K+-induced depolarization. After SERCA is blocked with thapsigargin, K c a blockers additionally increased [Ca 2 +]i and force, suggesting that K c a is at least partially activated in the absence of a functional sarcoplasmic reticulum. In addition, when Kc a is blocked first, subsequent SERCA blockade still increases [Ca 2 +]i and force. These data indicate that in the rabbit basilar artery 1) B K c a activity is not totally dependent on functional sarcoplasmic reticulum; and 2) SERCA lowers [Ca 2 +]j at least in part by mechanisms other than regulation of E m , most likely by buffering C a 2 + entry. It has been reported that the C a 2 + sensitivity of K c a is too low to respond to global [Ca 2 +]j (Ganitkevich and Isenberg, 1996; Tsukamoto et al, 1995) and, therefore, requires local [Ca 2 +]j elevation by virtue of release of quanta of C a 2 + from the sarcoplasmic reticulum in the form of sparks (Bolton and Imaizumi, 1996; Nelson et al, 1995). Yet, after complete blockade of SERCA, which has been shown to eliminate sparks and STOCS (Perez et al, 1999; Yoshikawa et al, 1996), K C a of the basilar artery is still activated. We postulate that, under control conditions, SERCA removes C a 2 + from a restricted subplasmalemmal space and that its blockade leads to accumulation of C a 2 + in this space (see Fig. 3.10.). The portion of K c a that faces these restricted cytoplasmic regions would then be activated upon SERCA blockade. However, it is also feasible that B K c a are simply active because of a prolonged elevation of Ca in the sub-sarcolemmal space in the absence of SERCA. 79 F i g u r e 3.10. S c h e m a t i c r ep resen ta t i on of the v a r i o u s S R re la ted m e c h a n i s m s i n v o l v e d in g e n e r a t i o n o f f o r c e in v a s c u l a r s m o o t h m u s c l e . The arrows indicate C a 2 + movements . The dashed arrows fol low a path that C a 2 + ions may take through a basi lar artery smooth musc le cel l . Note that C a 2 + may accumula te in the junctional space between the S R and p lasma membrane where interaction with other C a 2 + -sensi t ive channe ls and pumps may occur. V G C C , vol tage-gated C a 2 + channe ls ; N C X , N a + / C a 2 + exchanger ; R y R , Ry receptor. These observations were made in the rabbit basilar artery. Although this artery is not strictly speaking a resistance artery, it is an important conduit cerebral artery, which has many characteristics in common with cerebral resistance arteries. For example, vasoconstriction is completely dependent on Ca influx through V G C C , the smooth muscle displays spontaneous activity, and both are myogenically active vessels. Smooth muscle cells isolated from the basilar artery of the rat have been used in the study of sarcoplasmic reticulum function in small, cerebral arteries (Perez et al, 1999). In 80 addition, Omoteand Mizusawa (Omote and Mizusawa, 1996) have studied fluctuations in basilar artery myogenic responses generated by variable activity of V G C C . They proposed that this C a 2 + entry activated B K c a , leading to hyperpolarization of the membrane and, thus, providing negative feedback on the opening of C a 2 + channels. Similar mechanisms have been suggested in cerebral arteries from spontaneously hypertensive rats (Osol and Halpern, 1988) and in rabbit mesenteric arteries (Omote and Mizusawa, 1994). In addition, this latter study suggested that the endothelium does not contribute to myogenic activity because its removal did not affect the oscillations. This may further implicate a role for B K c a because there is some recent evidence suggesting that B K c a is not active in the endothelium (Kohler et al, 2000). I chose the rabbit basilar artery as an appropriate model for the investigation of the roles the sarcoplasmic reticulum plays in a cerebral artery. The main question to be resolved is: What is the function of the sarcoplasmic reticulum in a blood vessel in which force development is totally dependent on C a 2 + entry through VGCC? It appears unlikely that even agonist-induced contractions have a significant component of sarcoplasmic reticulum C a 2 + release because histamine contractions are almost completely blocked by nifedipine. In peripheral resistance arteries, noradrenalin-induced contractions are readily abolished by removal of extracellular C a 2 + (Cauvin et al, 1984). C a 2 + sequestration by the sarcoplasmic reticulum without interaction between the plasma membrane and sarcoplasmic reticulum would also not be able to explain the results obtained in this study because such a putative function would cease on saturation of the sarcoplasmic reticulum and, therefore, be transient. In 1995, Nelson et al (Nelson et al, 1995) proposed that in cerebral resistance arteries, the sole function of the sarcoplasmic reticulum is to 81 hyperpolarize the plasma membrane by releasing Ca in the vicinity of K c a . The authors based their conclusion on the observation that the effects of sarcoplasmic reticulum depletion and K c a blockade on diameter, [Ca2+]j, and E m were identical and nonadditive. Because they subsequently reported on spark activation of K c a in cerebral resistance vessels of other species, including humans, their hypothesis appeared to have universal validity. There is, indeed, an abundance of recent evidence suggesting the importance of C a 2 + sparks in regulating E m and vascular tone specifically in resistance-sized vessels (Jaggar et al, 1998; Miriel et al, 1999; Mironneau et al, 1996; Nelson et al, 1995) (for a review see (Jaggar et al, 2000). However, in the rabbit basilar artery, it appears that activation through C a 2 + sparks may not be the only way the E m is regulated in these larger cerebral vessels. The idea that localized C a 2 + release could cause relaxation though activation of K c a was first proposed by Bulbring and Tomita (Bulbring and Tomita, 1987) to explain a-adrenergic relaxation of intestinal smooth muscle (Liu et al, 2001). The existence of this mechanism was established by the studies of Benham and Bolton (Benham and Bolton, 1986) showing that STOCs arose from transient C a 2 + releases by the sarcoplasmic reticulum. Nelson et al.'s elegant and extensive studies (Nelson et al, 1995) of sparks and related events in cerebral resistance arteries have established their contribution as a feedback regulatory mechanism. The B K c a contributes to resting memebrane potential in arteries with myogenic tone as inhibition with IbTx or T E A causes membrane depolarization and vasoconstriction (Nelson et al, 1995, Nelson and Quayle, 1995). In contrast, the basal activitiy of B K c a is silent in the microcirculation as blockade with IbTx or TEA has no effect on diameter of these vessels in vivo (Jackson 2000). Some 82 general characteristics of the B K c a channels include activation via increases in intracellular C a 2 + and membrane depolarization. The voltage dependence of the channel is e-fold per 12-14mV depolarization and the K D for C a 2 + is 1.8uM at -11 mV and lOuM at -83 mV (Jaggar, 2000). Due to the voltage dependence of this channel, future experiments including membrane potantial measurements may more directly show that a change in E m is not responsible for the results reported in this chapter. Evidence also exists for other sarcoplasmic reticulum-mediated mechanisms for vascular smooth muscle, namely buffering of C a 2 + influx by the superficial sarcoplasmic reticulum and the activation of SOCC by sarcoplasmic reticulum depletion. Clearly, the significant thapsigargin-induced increase in [Ca2 +]i by blockage of B K C a by IbTx or TEA supports the involvement of either of these two latter mechanisms. In this instance, C a 2 + entry through V G C C would be enhanced by the depolarization caused by blockade of Kc a . To test for the activity of SOCC in the rabbit basilar artery, I measured the effect of thapsigargin on M n 2 + quenching of intracellular fura 2. The negative outcome of this experiment makes the SOCC possibility unlikely, although it cannot be completely ruled 9+ • out at this point due to the fact the Mn permeability is variable among different SOCCs, and M n 2 + can enter through channels other than those that C a 2 + would enter through. Therefore, further experiments are required to determine whether an SOCC exists and is 2_j_ active in rabbit basilar artery. In some cells other than smooth muscle, Ca release-activated channels have been described, which were highly selective for Ca (Hoth and Penner, 1992; Parekh and Penner, 1997). However, in our experiments, the effects of thapsigargin were completely blocked by nifedipine (at 1 or 10 uM), which is not known 83 to inhibit SOCC. In contrast, Mn entry through V G C C was clearly observed. Additionally, it has been shown previously that either cadmium or diltiazem (both C a 2 + channel blockers) inhibited M n 2 + influx in aortic smooth muscle cells isolated from rats (Liu et al, 1995) and in venous smooth muscle of the rabbit (Chen and van Breemen, 1993) respectively. Having thus exhausted all the other possible mechanisms, I conclude that the most likely candidate for explaining the actions of thapsigargin and ryanodine after blockade of Kca is inhibition of the Ca2+-buffering action of the superficial sarcoplasmic reticulum. This is supported by our observation that thapsigargin enhanced the rate of force development in response to C a 2 + entry through V G C C . In the experiment reported herein, an effect on E m was ruled out by the large depolarization induced by the 80 mM K + solution. This result would also not be explained by inhibition of calcium-induced calcium release, because this would have the opposite effect. Nevertheless, perhaps these differences reflect the role of the sarcoplasmic reticulum in buffering non-physiological levels of C a 2 + influx as Tg has no effect on pressure-induced contractions in rat cerebral arteries (Oyabe 2000) and therefore this requires further investigation. In addition, our results support the idea of Guia et al. (Guia et al, 1999) that in rabbit coronary artery smooth muscle cells, there may be coupling of V G C C and B K c a . C a 2 + influx through V G C C may directly cause accumulation of C a 2 + in the junctional space and directly activate B K c a as demonstrated by whole cell patch-clamp and simultaneous [Ca 2 +]; measurements in vascular smooth muscle cells (Guia et al, 1999). Guia etal. (Guia et al, 1999) showed that abolishing the sarcoplasmic reticulum function with CPA or ryanodine had no effect on the transient opening of B K c a . In fact, the 84 transient stimulation of K c a they observed appeared to be independent of sarcoplasmic 2_|_ reticulum function. Our data are consistent with the notion that local Ca entry may directly activate at least a fraction of B K c a . This leads to the concept of the existence of cytoplasmic microdomains within the smooth muscle cell. In some areas of vascular smooth muscle, it has been observed that the relative distance of the sarcoplasmic reticulum from the plasma membrane is on the order of 20 nm (Jaggar et al, 2000). Therefore, colocalization and functional interactions of various channels and ion pumps within this small, restricted space seems probable. Although our data are consistent with those of Guia et al. (Guia et al, 1999), suggesting a direct functional interaction between V G C C and Kc a > I cannot rule out the possibility that blockade of C a 2 + removal from a restricted space may lead to accumulation of C a 2 + and activation of K c a in the absence of C a 2 + sparks. Thus, i f SERCA (located in the superficial sarcoplasmic reticulum) removes C a 2 + from the restricted cytoplasmic space (Fig. 3.10.), it would not only decrease the flow of C a 2 + into the deeper cytoplasmic space, but it would also lower [Ca 2 +]i in the vicinity of Kc a . Blocking SERCA would then not only abolish sparks and their activation of K c a but, in addition, raise [Ca 2 +] near the plasma membrane, which would partially activate Kc a . The model presented in Fig. 3.10., which proposes a closer interaction between sarcoplasmic reticulum C a 2 + release channels and K C a than between V G C C and K p a and, in addition, C a 2 + removal from a subplasmalemmal space, would explain all the data reported herein. In conclusion, our results demonstrate for the first time that in an intact cerebral artery, B K c a remains active in the absence of C a 2 + release from the sarcoplasmic reticulum and that the superficial sarcoplasmic reticulum buffers C a 2 + entry through 8 5 V G C C . Additionally, our data suggest that SOCC do not significantly contribute to elevation of [Ca 2 +]i in the basilar artery. 86 CHAPTER 4 • AGONIST-INDUCED MITOCHONDRIAL Ca TRANSIENTS IN SMOOTH MUSCLE CELLS 4.1. INTRODUCTION In smooth muscle cells, the SR is the most important organelle in the regulation of [Ca2+]j; it releases C a 2 + during receptor activation, buffers or releases C a 2 + during influx, and removes C a 2 + from the cytosol during relaxation. However, it appears that this organelle may not be solely involved in accumulating and releasing Ca 2 + . A role for the mitochondria in C a 2 + homeostasis has been shown in a number of cell types (for a review see (Duchen, 1999). Although mitochondrial C a 2 + transport has been studied for many years, its role in the shaping of spatio-temporal Ca signaling patterns has not been thoroughly examined in vascular smooth muscle. The low-affinity high-capacity C a 2 + uniporter, which is driven by the large electrical potential across the inner membrane (due to proton extrusion via the respiratory chain (Gunter and Pfeiffer, 1990) and located in the inner mitochondrial membrane, has a Kd for C a 2 + of 10-20 u M and is minimally active in the sub-uM range (Gunter and Pfeiffer, 1990; Kroner, 1986). Therefore, it was previously thought that the mitochondria would not sense [Ca2+]j under physiological conditions. However, with the discovery of various SR related cytoplasmic microdomains such as in the superficial buffer barrier (SBB) (van Breemen et al, 1995) and transient localized C a 2 + sparks activating Ca2+-activated potassium channels (Nelson et al, 1995) it also seemed likely that such microdomains for C a 2 + might be related to other organelles. This idea was in fact proven by the elegant experiments done by 87 Pozzan and co-workers (for a review see (Pozzan et al, 2000) using targeted aequorin technology to specifically localize a C a 2 + sensing probe to the mitochondrial matrix of HeLa cells (Rizzuto et al, 1993b; Rizzuto et al, 1996). Aequorin is exquisitely suited for measuring calcium concentrations in the range where mitochondria are active as it is sensitive between 0.1 and 10 p M (Blinks et al, 1978) and can be accurately calibrated. Mitochondria contain many Ca2+-sensitive enzymes, mainly dehydrogenases, such that uptake of C a 2 + by mitochondria (after agonist stimulation) increases cellular ATP production in anticipation of cellular needs (McCormack et al, 1990; Hajnoczky et al, 1995). In addition, it has been shown that these organelles are capable of taking up C a 2 + under physiological conditions if high concentrations of local C a 2 + are sensed by the uniporter during stimulation by a variety of IP3 generating agonists (Miyata et al, 1991). These results have been validated by subsequent electron microscopy and GFP studies (Rizzuto et al, 1998), which have revealed close appositions of the ER and MT membranes in HeLa cells. In an earlier study Challet et al. have examined the role of the mitochondria in regulating C a 2 + in a skeletal muscle cell line (Challet et al, 2001); however, few have looked specifically in vascular smooth muscle (VSM) as it is difficult to transfect large plasmids, such as aequorin in this tissue. However, with new methods for transfection, this technique can now be extended to cultured smooth muscle cells in order to directly examine the contribution that mitochondria may make in regulating [Ca ]\. Using the above approach I examined mitochondrial C a 2 + signals in response to ATP stimulation of vascular smooth muscle cells in culture transiently transfected with a mitochondrially targeted aequorin. The data generated indicates that mitochondria share 88 cytoplasmic microdomains with the SR, and during SR Caz+ release have local [Caz+] ten fold greater than the average [Ca2+];. The Na+/Ca2+-exchanger (NCX) indirectly modulates the C a 2 + signals of half of the mitochondria. 4.2. R E S U L T S 4.2.1. Agonist-induced mitochondrial Ca2* transients G-protein coupled receptor agonists, ATP or A V P both induced [Ca ] m transients (Figure 4.1A,C), although ATP stimulation produced a much larger response in these smooth muscle cells. ATP may bind to one or both of the 2 purinergic receptor isoforms identified in smooth muscle, namely P 2x and P 2 Y , . However, the majority of the response is thought to be mediated via P 2 y receptor activation and subsequent production of IP3. 1 mM ATP was used to obtain highly reproducible results. Signals were also obtained at lower concentrations but with less consistency thus, ATP potency seems to be diminished during culture. ATP increased [Ca 2 + ] m to 4-5 uM, whereas A V P only produced increases of [Ca 2 + ] m up to 1 uM (For statistics, see Figure 4.5). The [Ca 2 + ] m responses were always transient. In contrast, ATP and A V P induced the 'typical' biphasic agonist induced increase in [Ca 2 +]i as measured by fura 2 fluorescence in parallel experiments in the same cell type (Figure 4.1B,D). In contrast to the M T responses the cytoplasmic [Ca2 +]j responses were similar for ATP and A V P . Repeated ATP stimulation produced very small and often negligible second responses (Figure 4. IE). 89 B 5000 "to 4000 O !ii 3000 TD O 2000 0 2 4 6 8 10 12 14 16 Time [min] Figure 4.1. Agonist-induced rise in mitochondrial [Ca2+] ([Ca2+]m) in smooth muscle cells. A. 1 mM ATP was applied for 5 minutes followed by a washout periods in PSS for 10 minutes. All calculations and solutions are provided in Material & Methods. B. Fura 2 loaded smooth muscle cells were stimulated with topical application'of 1 mM ATP for approximately 1 minute and washed out with PSS as indicated. C. 1 uM vasopressin (AVP) was applied for 5 minutes. D. Fura 2 loaded smooth muscle cells were stimulated with 1 pM vasopressin. E: A second ATP stimulation after a 10 minute washout in PSS often produced much smaller and often negligible [Ca 2 + ] m transients. Results are representative of at least 3 independent experiments. Note: For calibration of both fura-2 and aequorin signals please refer to Methods section. Briefly, for calibration of the mtAeq signals, a computer algorithm developed by Rizzuto et al. to convert aequorin luminescence to [Ca 2 + ] m , has been used which is based on the calculated fractional rate of consumption of aequorin and the C a 2 + response curve at physiological conditions of pH, temperature, ionic strength and [Mg ] (refer to Brini et al, 1995 for specific details). Additionally, since the recombinant aequorin is expressed at a concentration in the range of 0.1-1 p M (2-3 fold less than other dyes), it has a very low buffering capacity and thus, will affect intracelluar C a 2 + homeostasis less than other fluorescent indicators. Further, Aeq can accurately measure [Ca2 +]i in the 0.5-10 p M range (Brini et al, 1995 and 1999). Therefore, mtAeq can be precisely calibrated. 4.2.2. Role of L-type VGCC and FCCP uncoupling 1 p M nifedipine had no effect on the ATP-induced [Ca 2 + ] m transient. (Figure 4.2.B,C). This suggests that the C a 2 + supplied to the mitochondria comes from source other than L-type L-Type V G C C . Pre-incubation with 2 p M FCCP in PSS 10 minutes prior to application of ATP (Figure 4.2.A,C) or A V P (data not shown) completely inhibited the responses. This was not due to altered timing as a time control revealed a robust ATP-induced [Ca 2 + ] m transient after a 10 minute pre-incubation in PSS (data not shown). It may therefore be concluded that the [Ca 2 + ] m transient is dependent on the intact function of the mitochondrial C a 2 + uniporter. This also confirms specific localization of the mitochondrial-aequorin plasmid which is more directly confirmed using a targeted MT-GFP. Confocal images in Figure 4.3. clearly exhibit a vast mitochondrial network similar to data published in HeLa cells and convincingly show correct localization of the mitochondrial targeted probe. 91 A ^ 5 o XI c o o o 6000 5000 4000 3000 2000 1000 0 Digitonin 2 uM F C C P 1 mM ATP B ^_ 6000 r%3 o TO T3 C o o o 5 c ro O 5000 4000 3000 2000 1000 0 6000 5000 4000 2 4 6 8 10 12 14 Time [min] 1 >M nifedipine 1 mM ATP 2 4 6 8 10 12 Time [min] 14 ie ro 3000 "D O 2000 o o |j 1000 ATP 1 MM nif+ ATP 2 uM FCCP+ ATP Bgd Figure 4.2. FCCP inhibits ATP-induced transient but blockade of VGCC has no effect. A. 2 uM F C C P was added for 10 minutes prior to stimulation with 1 mM A T P . There was no change in initial baseline with F C C P alone but response to A T P was almost completely abolished. Final application of digitonin and C a C I 2 as described in Material & Methods shows that there was sufficient aequorin. B. 1 uM nifedipine (nif) was added for 10 minutes prior to stimulation with 1 mM A T P . Analysis showed no difference in peak or area under the curve (AUC) of the A T P response as compared to control. Results are representative of a minimum of 5 independent experiments. C. Summary data for peak responses from 5 different populations of cells on 5 different days. Data were analysed by A N O V A followed by Bonferroni's post-hoc test. There was no significant difference between A T P and nif+ATP or FCCP+nif and Bgd. 92 Figure 4.3. Confocal images of smooth muscle cells transfected with MT-GFP. Smooth muscle cells were transfected with the same plasmid as the MT-AEq, except the apoaequorin was exchanged for GFP. Mitochondria exist as a tubular network in smooth muscle cells. Panels A& B are two different images from different plates of cells. Panel C shows a deconvolved image and Panel D is a 3D reconstruction. All images are taken at 60x magnification. n=10 different cells from 5 different populations. 93 4.2.3. Effect of SERCA blockade on mitochondrial transients The effects of SERCA inhibitors, CPA and Tg, were tested on agonist-induced [Ca 2 + ] m transients. Either 100 uM CPA or 1 p M Tg completely inhibited the ATP response. (Figure 4.4.A,B, page 95). This implicates the SR of being the source for the [Ca 2 + ] m transient. In addition, Tg blockade with little or no store depletion slightly enhanced the [Ca 2 + ] m transient possibly due to inhibition of C a 2 + reuptake by SERCA (Figure 4.4.C), but [Ca2+]i measurements are required in order to verify this preliminary hypothesis. Tg or CPA alone caused a small and slow [Ca ] m transient. (Figure 4.4.B,C). A summary of these results is shown in Figure 4.5. on page 96. 4.2.4. Role of IP3R ATP binds to a P2Y G-protein coupled receptor which, through the hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2), generates IP3. Inhibition of phospholipase C (PLC), the enzyme which catalyses the hydrolysis of PIP2 to IP3 and diacylglycerol, with 1 ixM U73122 partially inhibited the ATP-induced mitochondria transient, suggesting pathways other than PLC are involved or that ATP is also acting on P2X receptors (Figures 4.6. and 4.7. and Discussion). 94 2 6000 5000 O 4000 rial 3000 •o o 2000 o o 1000 i 100uMCPA 1 mM ATP B 6 8 10 Time [mini „ 6000 £L 5000 4000 o "g 2000 o O 1000 o 1 uMTg 1 mM ATP Time [min] o "ro <= o O O 1 \sM Tg 1 mM ATP J 8000 7000 6000 5000 4000 3000 2000 ^ 1000 0 0 2 4 6 8 Time [min] Figure 4.4. SERCA blockade inhibits transient [Ca 2 +]m rise induced by ATP. A, B. 100 uM CPA or 1 uM Tg were added 10 minutes prior to stimulation with 1 mM ATP. CPA and TG produced similar results in all experiments and only slightly and transiently increased baseline. There was never an additional response to ATP after SERCA blockade. Results are representative of a minimum of 3 independent experiments. C. 1 uM Tg was added for 1 minute prior to stimulation with 1 mM ATP. The peak amplitude was slightly, but not significantly higher as compared to ATP control. Result is representative of two independent experiments. 95 6000 Figure 4.5. Summary of S E R C A blockade on peak [Ca 2 + ] m . Basal levels represent initial calibrated values upon PSS perfusion before the beginning of each experiment. Note: Values here were calculated from peak [Ca 2 + ] m responses. Data were analysed by ANOVA followed by Bonferroni's post-hoc test. Significant decreases as compared to the ATP control were achieved in the presence of CPA or Tg. Results are representative of a minimum of three independent experiments. C P A and TG were added 10 minutes prior to application of ATP or AVP. *P<0.001 as compared to peak ATP control responses. §P<0.05 compared to AVP. 96 6000 O "ro •c C o .n o o o • " r o C o o o ,-fci 5000 4000 3000 2000 1000 1 u.M U73122 1 mM ATP 8 10 Time [min] 12 14 16 6000 -, 5000 H 4000 J 3000 2000 1000 75 U M A P B 1 mM ATP 8 10 Time [min] 12 14 16 Figure 4.6. PLC and IP3 antagonist partially inhibit transient ATP-induced increase in [Ca 2 +]m . A: 1 pM U73122 was added 10 minutes prior to stimulation with 1 mM ATP. All subsequent doses were also applied for a minimum of 10 minutes prior. B. 75 pM A P B was added 10 minutes prior to application of 1 mM ATP. Results are representative of a minimum of 3 independent experiments. This concentration is effective since the IC5o of U73122 for inhibition of PLC has been reported to be 0.12pM (Cho et al, 2001). The IP 3R inhibitor, 2-aminophenylborate (2-APB) inhibited the [Ca 2 + ] m response to the same extent as seen with maximal PLC inhibition with U73122. Data are presented as area under the curve measurements 97 (AUC) analysed as described in Materials & Methods. A summary graph for dose-dependent 2-APB and U73122 inhibition of the [Ca 2 + ] m transients induced by ATP is shown in Figure 4.7. Figure 4.7. Summary Data for effects of PLC and IP 3 Antagonists on ATP-induced mitochondrial transient. Maximal inhibition by A P B appears to be reached at 50 pM. At this concentration the A T P -induced C a 2 + transient is significantly reduced as compared to control by an unpaired t-test. 1 pM U73122 also significantly decreases ATP-induced transient to levels similar to A P B . Results are representative of at least 3 independent experiments for each column of values. Results were analyzed by one-way A N O V A and further with the Dunnett's Multiple Comparison post-hoc test. * P < 0.001 # P < 0.05 as compared to A T P control response. 98 4.2.5. Role of the Na+-Ca2+ exchanger The above data show that mitochondrial C a 2 + uptake is initiated by C a 2 + release from the SR. When the cells are exposed to C a 2 + free medium the [Ca 2 + ] m response to ATP decreases presumably because C a 2 + is lost from the SR (Figure 4.8.A). However, this effect is not seen when, in addition to Ca 2 + , N a + is removed from the bathing solution as well (Figure 4.8.B). The results are summarized in Figure 4.9., which shows that the M T C a 2 + response declines by approximately 50% in the absence of Ca 2 + . However, the response is not significantly attenuated when the N C X is blocked by Na + removal. 99 0 C a 2 + / 0 . 1 mM E G T A Dig o • " r o X3 c o sz o o o " r o " O o . c o o 6000 5000 4000 3000 -j 2000 1000 0 1 mM A T P 6000 5000 4000 ^ 3000 2000 -1000 -0 2 4 6 8 10 Time [min] 0NaV0Ca 2 70 .1 mM E G T A 1 mM A T P 12 14 8 10 12 Time [min] Figure 4.8. Na + Removal re-establishes ATP transient in 0 C a 2 + solutions. Solid line trace is a control ATP response. The dotted trace was obtained in cell incubated for 200 sec in 0 C a 2 + solutions (0Ca 270.1 mM EGTA). A. In N a + containing 0 C a 2 + solution subsequent stimulation with 1 mM A T P for 5 minutes induced only a small transient. Washout was performed with P S S containing 1.2 mM C a 2 + and the observed small C a 2 + transient was always evident. B. N a + was substituted with NMDG to produce a 0 Na + /0 Ca 2 + /0 .1 mM E G T A solution. The A T P -induced transient observed was similar to those seen in normal P S S . Digitonin response is shown which was performed at the end of all experiments and used for calibration of the aequorin signal (See Material & Methods). Results are representative of at least 3 independent experiments. 100 140000 120000 20000 A T P O s 200s 600s 1000s OCa 2 * * A T P O s 200s 600s 1000s ONa+OCa 3* • Figure 4.9. Effect of 0 C a 2 + and 0 Na+/0 Ca 2 * solution on ATP induced mitochondrial C a 2 * transients. Data are given as area under the curve. Control responses upon A T P obtained in normal P S S is shown for comparison (1st column). The subsequent 4 columns show the A U C for the A T P response generated after the time shown in 0 C a 2 + solution prior to agonist application. The next block of columns shows the response to A T P after incubation for the indicated time points in 0 Na+/0 C a 2 + solution. Both 0 NaVO C a 2 + and 0 C a 2 + solutions contained 0.1 mM E G T A . There was no significant difference in any of the A U C measurements where N a + was removed as compared to control. However, there was a significant difference with all 0 C a 2 + as compared to control A T P but no significant differences along the varying time points. Each column is representative of a minimum of 3 independent experiments. Results were analyzed by one-way A N O V A and further with the Dunnett's Multiple Comparison post-hoc test. * P < 0.001 as compared to A T P control response. 101 4.2.6 Electron Microscopy Electron micrograph images from fixed single smooth muscles cells from the same cell line show close appositions of the SR with the plasma membrane and also with mitochondria (Figure 4.1 OB). The membranous structures in close proximity to mitochondria shown in this electron micrograph are smooth, do not contain vesicles and are not associated with stacks of rough endoplasmic reticulum. This indicates that they are SR rather than part of the Golgi apparatus. Mitochondrial-SR junctional spaces are clearly visible (Figure 4.10B,C) supporting the notion of C a 2 + accumulation in this restricted space which appears to be on the order of 20 nm in width. In addition, it is important to notice SR elements in contact with both the plasma membrane (PM) and mitochondria and separate regions of only MT-SR interactions in the deeper cytoplasm. Thus, two populations of mitochondria can be distinguished: those associated with SR-P M elements and those located deeper in the cytoplasm interacting only with SR elements. The frequency of these MT-SR junctional spaces was calculated to be 73 ± 2% (standard error, S.E) from 15 different cells and the average distance between the M T and SR was 18.8 ± 0.8 nm (S.E.) calculated from an average of 20 cells. Based on the experimental evidence presented in this communication, a model for the major C a 2 + signaling events in vascular smooth muscle is depicted in Figure 4.11. 102 Figure 4.10. Two populat ions of mitochondria in cultured rat aortic smooth musc le cel ls visual ized with electron microscopy. Panel A electron micrograph shows the whole cell. Panel B shows an enlargement of the box indicated in A and indicates the presence of numerous mitochondria (MT) and the nucleus (Nuc) in the cytoplasm of a smooth muscle cell. Dashed arrows represent plasma membrane (PM)-SR-MT associations whereas full arrows show SR-MT junctions. In both cases, the S R membrane becomes closely apposed (within 20nm) to the outer mitochondrial membrane. Panel C is an additional enlargement of an area showing the very close apposition of the MT and SR membranes. 103 104 Figure 4.11. Model for Ca movements in vascular smooth muscle cells. ATP activates IP3 release which binds to its receptor on the SR membrane. Opening of the I P 3 R releases C a 2 + towards the C a 2 + uniporter, U , in a restricted space allowing C a 2 + to reach p M levels and sufficient for activation of U thereby allowing the M T to accumulate Ca 2 + . N C X , located on the plasma membrane (PM), normally functions to remove C a 2 + from the restricted space between the SR-PM. When N a + is removed, the N C X is inactive and C a 2 + accumulates in the SR-PM junctional space. This C a 2 + is sensed by SERCA, also located in this region, and is pumped back into the SR. Thus, upon stimulation with an IP3-generating agonist in a 0 Na + environment, the M T receive the same amount of C a 2 + as under normal conditions. However, only M T associated closely with both SR and P M elements are thought to exhibit this response. A second population of mitochondria create close MT-SR junctions but appear to not be influenced by P M pumps or channels. (Ca, Ca 2 + ; Na, Na + ; N C X , sodium/calcium exchanger; SERCA, sarcoplasmic endoplasmic reticulum Ca 2 +-ATPase; V G C C , L-type voltage-gated C a 2 + channel; IP3R, IP3-sensitive C a 2 + release channel; P2Y, purinergic G-protein coupled receptor; P 2 X , purinergic ligand-gated ion channel; RyR, ryanodine-sensitive C a 2 + release channel; SR, sarcoplasmic reticulum; MT, mito, mitochondria; U , mitochondrial Ca2+-uniporter) (adapted from Lee et al, 2002). 105 4.3. DISCUSSION This is the first report on the use of targeted aequorin to record M T C a 2 + signals in a primary culture of smooth muscle cells. I used ATP and A V P to stimulate these cells and demonstrate C a 2 + transients in mitochondria. As expected, the transients required a functional mitochondrial uniporter since they were prevented by FCCP. The fact that the signal was blocked by Tg and CPA, but not by nifedipine, demonstrated that C a 2 + was supplied to the uniporter by SR C a 2 + release channels. However, the magnitude of the global increase in [Ca 2 +] i during agonist activation was more than an order of magnitude 2"f" 2"1" below the K D for mitochondrial Ca uptake. Therefore the M T Ca signals could only be explained by the presence of a cytoplasmic microdomain within the 80 nm wide gap between the SR and the MT, where the local [Ca 2 +]MT-SR would increase by about 10 fiM during opening of SR release channels. The role of mitochondria in regulating [Ca2+]j has been studied intensively in a number of cells over the past decade (for a review see Rizzuto, 2001). It has been shown that C a 2 + uptake by neighbouring M T controls the kinetics of the P M C a 2 + channels and modulates the excitotoxic effect of glutamate in neurons (Stout et al, 1998; Friel, 2000). Additionally, release of C a 2 + from intracellular pools plays an important role in controlling process such as neutrite growth, synaptic plasticity, secretion and neurodegeneration (Rizzuto 2001). Furthermore, in neurons and chromaffin cells, M T act as rapid and reversible C a 2 + buffers during cell stimulation (White et al., 1997; Park et al., 1996) and in the clearance of large C a 2 + loads (Xu et al., 1997). Finally, it was shown that [Ca 2 + ] m can reach the mM range during stimulation of chromaffin cells and this large M T C a 2 + uptake regulates the availability of C a 2 + for the secretory machinery (Montero et 106 a l , 2000). These authors also show a tight functional coupling of V G C C , RyR and MT. Thus, an important role for M T in regulating C a 2 + signals is well established in some cell types. However, the current evidence for a role of mitochondria in C a 2 + regulation in smooth muscle is still controversial. There are many studies using a variety of techniques that, for the most part, show convincing evidence for an important role of mitochondria in sequestering Ca 2 + . For example, in rabbit aortic smooth muscle, 4 5 C a 2 + fluxes have shown that the mitochondria take up C a 2 + when the extracellular K + is increased (Karaki and Weiss, 1981). Also, the fluorescent indicator, rhod-2 (a lipophilic cationic dye), has been used to measure mitochondrial [Ca 2 +]; in pulmonary artery myocytes simultaneously with fura 2 measurements of [Ca2 +]j. Results from this study show that C a 2 + release from the SR increases [Ca 2 + ] m via RyR or IP 3R (Drummond and Tuft, 1999). Another low affinity Ca2+-sensitive fluorescent indicator, Mag-Fura, which was thought to mainly localize in the SR, has been used in rabbit aortic myocytes and revealed compartmentalization of the dye in the mitochondria in addition to SR elements. The authors report that mitochondrial inhibitors profoundly effected C a 2 + released from the SR and thereby suggest a functional integration between SR and mitochondria in aortic smooth muscle (Gurney et al, 2000). In addition, they show that mitochondrial C a 2 + remained elevated for minutes after stimulation as opposed to [Ca 2 +]; which showed a rapid decline upon removal of the agonist. A l l of the agonist-mediated mitochondrial responses were transient. However, this discrepancy may be due to the different modes of [Ca 2 + ] m measurement, rhod-2 versus targeted aequorin, although experiments done with rhod-2 by Hajnoczky et al. agree with targeted aequorin measurements (Hajnoczky 107 et al, 1995). Another explanation for these varied results and maintenance of the M T response may reflect the lack of a Na+-dependent C a 2 + efflux pathway in the mitochondria of some smooth muscles (Crompton et al, 1978). There is also indirect evidence using classical pharmacological approaches that suggest a role for mitochondria in regulating C a 2 + in smooth muscle cells (Drummond and Fay, 1996; Greenwood et al, 1997), and in guinea-pig colonic smooth muscle (McCarron and Muir, 1999). Finally, studies in A10 smooth muscle cells using the same targeted mitochondrial aequorin but, in cells permeabilized with digitonin prior to experimentation, find that Ca2+-induced C a 2 + release. (CICR) at the RyR generates microdomains of elevated C a 2 + that are sensed by adjacent mitochondria (Nassar and Simpson, 2000). These authors suggest that CICR is required for generating sufficient elevation of mitochondrial Ca 2 + . However, this study also confirms data from many other cell types supporting the notion that mitochondria can sense C a 2 + released directly from the SR via IP3 channels. Therefore release from both IP3R and RyR may be important in producing the large, local increases in [Ca2+JMT-SR. Additionally, a recent article suggests that in rat tail artery, mitochondrial inhibition 2_j_ reduces cti-adrenoceptor stimulated force by 50-80% but no reduction in global [Ca ]i is observed. Also, confocal imaging showed that mitochondrial inhibitors increased the frequency but reduced the amplitude of asynchronous cellular C a 2 + waves induced by cirazoline. Thus, in a more physiological setting, it has been shown that mitochondrial inhibition influences C a 2 + wave activity most probably due to the close spatial arrangement of SR and M T membranes (Sward et al, 2002). I present evidence for a role of IP3R in directly supplying C a 2 + sensed by the M T uniporter. The PLC inhibitor, U73122 and the IP 3R inhibitor, 2-APB, both caused a 50% 108 decrease in the ATP-induced [Ca ] m transient (Figure 4.6.). However, recent articles suggest a non-specific action of 2-APB and therefore these results should be interpreted with caution (Bootman et al, 2002). Additionally, ATP may also activate P2X receptors, which are ligand gated ion channels, in addition to P2Y receptors thereby activating a non-selective cation current which may also explain the partial inhibition (Burnstock, 2002). Nevertheless, it is possible and likely that half of the C a 2 + supplied to the M T is released from the SR through IP 3R. The remaining C a 2 + is also released from the SR, since 2_|_ SERCA blockade abolishes the entire [Ca ] m transient. Presumably ryanodine receptors (RyR) are involved in this process although the mechanism is not clear at this time. Coupling between [Ca 2 + ] m transients to RyR-mediated [Ca 2 +]i signals in smooth muscle cells has been reported by Drummond and co-workers (Drummond and Tuft, 1999). In addition a study in which caged C a 2 + was released in smooth muscle cells from portal vein supports the concept of cooperativity between IP 3R and RyR (Boittin et al, 1998). Recent reports have shown that the second messengers, cyclic-ADP ribose and nicotinic acid-adenine dinucleotide phosphate, activate RyR (Li et al, 2001; Yusufi et al, 2001). It is therefore possible that the mitochondria may receive signals from release through RyR and I P 3 R , both of which may be in close spatial apposition to the M T uniporter. This study shows that M T can sense activity of N C X by virtue of close apposition to the SR. Our results reveal that at 0 Ca 2 + , low EGTA conditions, stimulation with 1 mM ATP generates a smaller transient response as compared to control ATP applications. Interestingly, these responses do not diminish completely even after 1000 sec. in a 0 C a 2 + environment, but decline only to approximately half of a control ATP transient. On the contrary, substitution of Na + by N M D G prevented any short term 109 decline in the ATP-induced [Ca 2 + ] m transient (Figure 4.8.). However, removal of Na + may also cause intracellular acidification due to inhibiton of the N a + / H + exchanger and therefore, further studies are needed to investigate this possible effect. In addition, removal of C a 2 + may alter cell function, specifically by changing membrane potential. Therefore, future experiments should include measurements of membrane potential in these smooth muscle cells in 0Ca 2 + and 0Ca 2 + / 0Na + solutions. Nevertheless, Lee et al. have previously shown that in vascular smooth muscle, the N C X located at PM-SR junctions mediates transfer of C a 2 + between the SR lumen and the extracellular space (Lee C H et al, 2002a). If mitochondria are located close to that portion of the SR which is depleted via the N C X , their responses would show a parallel decline. Thus our results could be explained by assuming the existence of two distinct populations of mitochondria: those associated with peripheral SR elements that form junctional complexes with the P M and other mitochondria situated deeper in the cytoplasm, which are associated with SR elements that do not form junctions with the P M . This hypothesis seems all the more plausible by examination of electron micrographs of our smooth muscle cells. In a single cell, SR-MT junctions are obvious, and the organellar membranes are only separated by 20 nm in many instances. Moreover, peripheral SR elements are observed which are also in close contact with MT. In addition, deeper in the cell exist SR-MT junctions without any close association with the P M . Thus, two populations of M T are visible with E M and possibly explain our functional findings. Interestingly after a prolonged period of exposure to Ca free conditions, replenishment of C a 2 + was always accompanied by a smaller [Ca 2 + ] m transient. This may be related to enhanced Ca2+-permeability of the P M due to activation of store-110 operated cation channels (SOCC) and non-specific membrane destabilization (Cauvin and van Breemen, 1985). It is not likely that this [Ca 2 + ] m transient was due exclusively to SOCC supplying MT, because SERCA blockade only transiently increased [Ca 2 + ] m , while the SOCC would remain activated. Whether the mitochondrial C a 2 + signal following C a 2 + replenishment is also mediated by SR C a 2 + release, remains to be investigated. Finally, digitonin permeabilization at the end of all experiments often exhibited a biphasic response. Although many other processes may be involved, it is possible that this biphasic response is due to the existence of two different populations of mitochondria. Although ATP and A V P produce almost equal increases in [Ca2+]j as measured by fura-2, they show marked differences in C a 2 + accumulation by the mitochondria. ATP shows a greater than 10-fold increase in [Ca 2 + ] m as compared to [Ca 2 +]i. On the other hand, A V P only shows a doubling of [Ca 2 + ] m as compared to [Ca2+]j. Although the cause for this remains unclear it could be related to the fact that different agonists rely to different extents on C a 2 + influx and release in order to elevate [Ca2 +]j. In this study I have shown that [Ca 2 + ] m is much more sensitive to release than influx. Thus i f the influx/release ratio were higher in A V P than in ATP the mitochondrial C a 2 + signal would be proportionally smaller. Furthermore, the ATP-induced biphasic response obtained with fura-2 may also suggest that some C a 2 + influx is mediated via the P2X receptor and requires further investigation. Although it is clear the mitochondria are accumulating Ca upon agonist stimulation, it is as yet, unclear as to why this may be beneficial for the cell. Firstly, 111 mitochondria contain many Ca -dependent enzymes (Hansford, 1985; Rutter and Denton, 1988), therefore much of this C a 2 + may be used to signal production of ATP via oxidative phosphorylation. SR-mediated C a 2 + release can raise the mitochondrial [Ca2 +] which in turn, increases ATP generation to match the increased energy demands required for smooth muscle cell contraction. In addition, the mitochondria may act as a secondary buffer upon receiving a signal from the SR. This additional buffering capacity of the cell may act to delay or spread the C a 2 + signal to the rest of the cell as has been shown in pancreatic acinar cells (Tinel et al, 1999). Furthermore, this close apposition between SR and M T membranes suggests that there may be SR-MT cross-talk in this restricted space. For example, in hepatocytes, mitochondrial C a 2 + uptake suppressed the positive effects of C a 2 + on the IP3R, therefore reducing C a 2 + release at submaximal doses of agonist (Csordas et al, 1999). The C a 2 + sensitivity of the IP3R is complicated, as a small increase in [Ca2+]j opens type I receptors but micromolar increases in [Ca2+]j closes type IIP3R (Ehrlich, 1995) and opens type II and III receptors (Mignery et al, 1992). In this way, buffering of the C a 2 + signal by mitochondrial uptake may act to enhance or restrict the evolution of [Ca2+]j signals (Duchen, 2000). Mitochondria also contain extrusion mechanisms which release the C a 2 + (albeit more slowly than the uptake of C a 2 + via the uniporter) back into the cytosol or SR, namely a mitochondrial N C X (mNCX) and a Na+-independent pathway via H + / C a 2 + exchange (Montero et al, 2001). However, recently emerging evidence using CPG-37157, a specific blocker of mNCX, in primary cultures of rat brain capillary endothelial cells suggests that the mNCX is the dominant extrusion mechanism. Blockade of the mNCX greatly enhanced the ATP-induced transient and the authors conclude that accumulation of C a 2 + by the mitochondria is 112 limited by the N C X thereby allowing Ca1+ cycling to occur during [Ca / + ] m transients (Gerencser A A and Adam-Vizi, 2001). Once the mitochondria have removed C a 2 + from the cytoplasm via the uniporter, they can return it more slowly to the cytoplasm to prolong the activity of high affinity Ca2+-dependent process and/or refill the SR by a secondary active transport (Babcock et al, 1997). Additionally, the mitochondrial permeability transition pore (PTP) may be critical in maintaining C a 2 + in the mitochondria at reasonable levels. The PTP opens upon high [Ca 2 + ] m which triggers a fast release of C a 2 + and has been implicated in the release of apoptotic factors. The PTP has also been suggested to behave as a [Ca2+]i-activated C a 2 + release channel under certain conditions (Ichas et al, 1997) and requires further investigation. Therefore, multiple mechanisms exist in which the mitochondria play a role in C a 2 + homeostasis both within the mitochondria itself and also in shaping the spatio-temporal global C a 2 + signal via rapid uptake and slow release of Ca 2 + . The existence of a MT-SR restricted space in vascular smooth muscle cells allows accumulation of uM C a 2 + which is sensed by the mitochondrial C a 2 + uniporter. Only release of C a 2 + from SR channels and not influx contribute to this large, local increase in 9+ • Ca . In addition, half of the mitochondria are also associated with the P M , and can be indirectly affected by the activity of the N C X . Therefore, mitochondrial C a 2 + regulation is dependent on the filling state of the SR and as a result, the possible functions of mitochondria in smooth muscle are 1) dampening and prolongation of the cytoplasmic C a 2 + response, 2) refilling of the SR, and 3) regulation of oxidative phosphorylation. Future experiments with other specifically targeted aequorins will provide further insight 9+ into the complex Ca signals regulating vasoconstriction and relaxation in vascular 113 smooth muscle. Finally, studies in single cells and intact tissues using specifically targeted molecular probes may lead to a better understanding of the physiological role of mitochondria in C a 2 + signaling, and the development of new drug targets in conditions where mitochondrial C a 2 + regulation is disrupted. 114 CHAPTER 5 • SMOOTH MUSCLE HETEROGENEITY 5.1. Introduction Although all smooth muscles require C a 2 + to contract, the way that C a 2 + is regulated differs in smooth muscle from different vascular beds, from arteries to veins, and in different animals. This heterogeneity may be due to differences in ion channels, calcium pump distribution, second messenger systems, and/or the spatial organization of the SR, mitochondria, and contractile filaments. A l l of these aformentioned factors are thought to play an important role in determining the local and global transmission of the C a 2 + signal as well as the frequency of the signals within the cell (Pabelick et al, 2001). In addition, the plasticity of the smooth muscle cell phenotype in combination with the temporal and spatially diverse development and differentiation, appears to produce divergent smooth muscle cell populations between and within tissues (Halayko et al, 1997). For example, small cerebral arteries require fine control to maintain a constant blood flow to and within the brain. This is achieved via a negative feedback mechanism; upon activation and influx of Ca 2 + , the SR subsequently releases C a 2 + via RyR or IP3R. Release of C a 2 + via RyR, a ' C a 2 + spark', will activate a K c a channel on the P M (Jaggar et al, 2000). Activation of this K c a channel induces a spontaneous transient outward current (STOC) (Benham and Bolton, 1986; Hisada et al, 1990; Hume and Leblanc, 1989; Nelson et al, 1995; Ohya et al, 1987), hyperpolarizes the P M (Ganitkevich and Isenberg, 1992), and ultimately closes L-type V G C C . On the contrary, larger conduit vessels, such as the rabbit IVC, may not require this fine level of control and thus K Q , does not appear to be an important channel regulator of C a 2 + in these vessels. In addition, under resting conditions, tonically active arterial smooth muscle is more dependent on 115 Ca 2 influx via L-type V G C C rather than the SR for global [Ca^ ]i (Knot et al, 1998; Szado et ai, 2001). However, in urinary and venous smooth muscle, the SR C a 2 + release is more significant (Ganitkevich and Isenberg, 1992; Lee et al., 2001). The interaction 2_|_ between L-type V G C C and SERCA and between N C X and SR Ca release channels across a restricted sub-plasmalemmal space have been recently reviewed (Lee C H et al, 2002a). However, we now know that such local interactions also occur between RyR and Kca, Na +/K +ATPase and N C X , SR C a 2 + release channels and Ca2+-sensitive Cl" channels and L-type V G C C and Kc a . These localized ionic transport interactions may be one mechanism contributing to smooth muscle heterogeneity as activation of one type of channel versus another may completely change the final physiological function. For example, coupling RyR to K c a will cause SR-dependent hyperpolarization, while coupling RyR to Clc a would cause SR-dependent depolarization. Therefore, the local microstructure of plasmalemmal and organellar membranes may and most likely will 9+ determine the specific characteristics of the Ca signaling systems in vascular smooth 2+ muscle of various sub-types (Lee C H et al, 2002a). The regulation of this Ca in different types of smooth muscle is still elusive since each type seems to be regulated by the location, and association of various channels and transporters within defined restricted spaces. Therefore, smooth muscle heterogeneity still remains a somewhat unknown, yet real phenomenon. I have studied smooth muscle heterogeneity in three different types of smooth muscle: rabbit inferior vena cava, rabbit basilar artery, and rat aortic smooth muscle cells. We have compared the first 2 tissues pharmacologically measuring changes in force via myography and in C a 2 + with the use of a confocal microscope upon histamine 116 stimulation. Further, I have measured RNA expression in all three types of smooth muscle by testing with specific primers for various channels using RT-PCR. 5.2. Results 5.2.1. ET responses in Rabbit BA and IVC Endothelin (ET) responses were tested by measuring force using either myography (for basilar arteries) or conventional organ bath procedures (for the rabbit inferior vena cava). ET elicited a large contraction in both tissues (Figures 5.1. and 5.2.). However, blockade of L-type V G C C with 10 p M nifedipine only partially blocked this agonist-induced response in the IVC while completely abolishing it in the B A . The remainder of the response in rabbit IVC could be abolished with addition of the ROCC/SOCC antagonist, SKF-96365. A summary of these results is presented in Figure 5.IB. and 5.2B. for rabbit B A and IVC respectively. Therefore, in the IVC it appears that activation of ROCCs/SOCCs are extremely important in maintaining agonist-induced contractions in addition to C a 2 + influx through L-type V G C C . In contrast, the B A appears to depend solely on L-type V G C C as observed in previous studies (Szado et al, 2001) and is required for maintenance of the ET response. In addition, because ET is known to activate receptors on both endothelial and smooth muscle cells, B A studies were performed in the presence of L - N A M E and indomethacin, whereas IVC was denuded of endothelium mechanically using filter paper. Furthermore, since E T B receptors are thought to be present mainly on endothelial cells and E T A receptors on smooth muscle cells, I blocked both using the selective inhibitors BQ788 and BQ610 respectively. Figures 5.1C. and 5.2C. show that BQ788 had no effect 117 on ET-induced contraction suggesting that our preparations were indeed devoid of endothelium. In contrast, the ETA-receptor antagonist, BQ610, reduced ET-induced contractions to baseline levels in both B A and IVC thereby supporting the notion of ETA-receptor expression specifically on smooth muscle cells. A summary of these results is shown in Figure 5.ID and 5.2D. 118 10 nM ET B 16 10 uM Nifedipine 12 z 8 E LL 4 10 nM ET Nif S K F 10 nMET 10 \M BQ788 10 uM BQ610 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 Time (min) 23 z 13 E I 10 n M E T 1 0 p M B Q 7 8 8 10 u M B Q 6 1 0 Figure 5.1. Endothelin (ET) Responses in Rabbit BA. A) Representative trace of force generation by 10 nM ETwh i ch produces large, sustained contraction. This contraction was completely abolished with 10 \M nifedipine (Nif). Summary of results from 5 different tissues and 5 rabbits shown in B. C) ET B-antagonist, BQ788, had no effect on ET-induced contraction whereas specific E T A inhibtior, BQ610, returned force to baseline levels. Summary of results from a minimum of 5 t issues from 3 rabbits is shown in D. 119 B 10 nM ET 10 u.M Nifedipine 50 uM S K F 2 0 Time (min) i o 1 4 1 2 1 0 8 6 4 2 0 10 nM ET S K F 10 nM ET 10 uM BQ788 10 pM BQ610 20 30 Time (min) 10 nM ET 10pM BQ788 10pM BQ610 Figure 5.2. Endothelin (ET) Responses in Rabbit IVC. A) Representative trace of force generation by 10 nM ET. This contraction was decreased by approximately 25% with 10 [M nifedipine (Nif) and the remainder of the response could be blocked with subsequent addition of the S O C C inhibitor, SKF-96365 (SKF). Summary of results from 5 different tissues and 5 rabbits shown in B. C) ET B-antagonist, BQ788, had no effect on ET-induced contraction whereas specific E T A inhibitor, BQ610, returned force to baseline levels. Summary of results from a minimum of 7 tissues from 7rabbits is shown in D. 120 5.2.2. Confocal microscopy of intact arteries [Ca 2 + ]i imaging in the rabbit basilar artery was accomplished after loading with fluo-4 and subsequent confocal microscopy in an intact arterial segment (with focus on individual smooth muscle cells within the intact tissue) denuded of endothelium by inverting the vessel and rubbing with filter paper (see Methods). Histamine, a G-protein coupled agonist, induced an increase in [Ca 2 + ] i and tonic contraction in the rabbit basilar artery (Figure 5.3.). The [Ca 2 + ]i increases observed in the rabbit basilar artery had a very slow time course (Figure 5.3.) and were not wave-like (similar to the C a 2 + flashes described by lino et al, 1994). In contrast, Lee et al. recently published results showing that phenylephrine, an a-adrenergic G-protein coupled agonist, induced faster, wave-like [Ca 2 + ] i oscillations in the rabbit IVC (refer to Lee et al, 2001 for details). Additionally, preliminary experiments suggest that the [Ca 2 +]j increases can be almost completely abolished by application of the L-type V G C C inhibitor, nifedipine in rabbit B A (data not shown). However, in rabbit IVC at the whole tissue and single smooth muscle level, nifedipine only decreases the agonist-induced responses by 25%, whereas the remainder of the response is abolished by SKF-96365 or alternatively an IP3 antagonist, 2-APB (refer to Lee C H et al, 2002b). Accordingly, these results would corroborate our findings with tissue segments using myography which show that agonist-mediated increases in force can be abolished with nifedipine in rabbit B A (Figure 5.1.) while SKF-96365 is required, in addition to nifedipine, to abolish response in rabbit IVC (Figure 5.2). This suggests a major function of L-type VGCCs and a very minor or no role for ROCCs/SOCCs on agonist-induced responses in rabbit B A . The opposite is true for the 121 rabbit IVC where evidence suggests a minor role of L-type V G C C upon agonist stimulation, and a large role for ROCCs/SOCCs in this venous tissue. lOpM histamine I O L I M histamine Figure 5 . 3 . Bi phasic agonist-induced response in fluo-4 loaded rabbit basilar artery. Tissue was stimulated with 10uM histamine. Analysis at the single cell le\el revealed no oscillations, but rather a typical biphasic response. From the average whole-cell view, 3 cells were selected and are shown here as Cells 1,2,3. Traces from single cells are similar to the whole-field signal and also to other individual cells suggesting that in this tissue, the cells are responding in a simultaneous fashion. 122 5.2.3. Effect of Kca channel blockade in rabbit IVC and BA Nazer and van Breemen (Nazer and van Breemen, 1998b) previously showed that blockade of K c a channels with 3 mM TEA had no effect on [Ca 2 +]; in IVC (n=3 tissues) (Figure 5.4A.). This is in striking contrast to rabbit B A in which blockade of K c a with 3 mM T E A largely increased [Ca ]; over the same time period (n= 5 tissues) (Figure 5.4B.). Experiments using the specific B K c a antagonist, iberiotoxin also produced no increase in [Ca 2 +]i in rabbit IVC over a 10 minute time period (unpublished observation), whereas a large increae in force and [Ca 2 +]i was observed in rabbit B A (see Chapter 3, Figures 3.2 & 3.3). In other experiments, both tissues were treated under the same experimental conditions exhibiting similar levels of tension using the more specific B K c a antagonist, iberiotoxin instead of TEA, and force was measured. These results are shown in Figure 5.4.C&D. It is apparent that IVC does not contract to IbTx, under basal conditions, whereas the B A shows a large, sustained contraction. Furthermore, as indicated in the previous section, nifedipine completely abolished the response in B A whereas in IVC, SKF-96365 in addition to nifedipine was necessary to reduce force back to baseline levels. This nonresponsiveness may be due to non-active Krj a channels or due to the absence of these channels in IVC. This led us to investigate K c a channel expression in both rabbit IVC and B A using semi-quantitative RT-PCR technology. 123 A C Figure 5.4. Effect of B K C a channel blockade in Rabbit IVC. A. 3 m M T E A was applied for 3 minutes without response in rabbit IVC (adapted from Nazer 1998 with permission). Subsequent addition of 20 uM C P A shows a large increase in [Ca 2 +]j. Results are representative of three experiments. Data provided by M. Nazer with permission. For detailed Material and Methods, refer to Nazer et. al., 1998. B. 3 mM T E A increases [Ca 2*], within minutes of application in rabbit BA loaded with fura 2. Refor to Methods for details. Result is representative of 5 tissues from 5 rabbits. C Representative trace showing that 100 nM iberotoxin has no effect on contraction in rabbit IVC at basal levels. Result is representative of 4 t issues. D. Representative trace showing increase in force to application of 100 nM iberiotoxin in rabbit BA under basal conditions. Result is representative of a minimum of 10 different t issues. Refer to Materials and Methods for myograph experimental conditions. Note: C. Is the same trace as shown in Chapter 3, Figure 3.3. 124 5.2.4. Kca expression in rabbit IVC and BA Whole tissues were isolated from rabbits, de-endothelialized, and R N A was extracted as described in Materials and Methods (Greenwood et al, 1997; McCarron and Muir, 1999). I examined B K c a channel expression as this K c a channel is reportedly the most dominant in smooth muscle and is functionally the most important in rabbit B A as shown in Chapter 3. It is encoded specifically by the hslo gene (Hinrichsen, 1993). Figure 5.5. shows that the non-responsiveness of rabbit IVC to T E A was not due to the absence of B K c a as this channel is expressed in both IVC and B A . 5.2.5. Expression of L-and T- type VGCCs in rabbit IVC and BA Since C a 2 + influx via the L-type V G C C has been shown to be important for PE-mediated [Ca2+]j oscillations in the rabbit IVC (Lee et al, 2001) and also because the rabbit B A seems to be driven solely through activation via L-type V G C C (Szado et al, 2001), I tested the expression of both L- and T- type V G C C . Voltage-sensitive N a + and C a 2 + channels have a single cti subunit composed of four repeats of six transmembrane segments (Brereton et al, 2000), which has a high sequence homology to many other transmembrane channels. Therefore, I used primers for the otic subunit to specifically identify the L-type C a 2 + channel (Ohya et al, 2001; Gustafsson et al, 2001). In addition, I used the otig subunit which is specific for the T-type calcium channel (Mitterdorfer et al, 1998). Figure 5.5. shows that an RNA transcript for the alpha subunit of the L-type V G C C is present in both rabbit B A and IVC, but a transcript for the alpha subunit of the T- type channel is only present in rabbit B A (Ohya et al, 2001; Gustafsson et al, 2001). 125 A l l RT-PCR results are representative traces of the same results from 5 tissues of 5 rabbits. 5.2.6. Rat aortic smooth muscle cells (RASMC) The difference in channel expression found between rabbit IVC and B A may be due to the local environment of the respective tissue, i.e., stretch due to different pressure levels in different vascular beds. Therefore, I examined in addition to intact tissues, channel expression in RASMCs. This is highlighted by the fact that loss of expression of L-type calcium channels during the culturing process has been a common problem. However, Ruegg et al. have provided functional evidence for the existence of L-type channels in the smooth muscle cells used herein (Ruegg et al, 1985). I have further confirmed this finding specifically using RT-PCR. These smooth muscle cells are those used in Chapter 4 of this thesis, therefore it was important to determine that L - and T-type C a 2 + channels were present in this cell line as evidence suggests that cells may lose some channels at different levels of competence. 126 Rabbit basilar artery 600 bp 500 bp 400 bp a 1 c a 1 G hslo 18S Fig. 5.5. Expression of VGCC, BKC a and T-type calcium channels in rabbit BA and IVC. Exemplary agarose gel electrophoresis from R T - P C R analysis of a 1 c and a 1 G , and hslo mRNAs in A) rabbit basilar artery and B) rabbit inferior vena cava. P C R products were generated through the use of specific primers for a 1 c , a 1 G , and hslo. A 100-bp DNA ladder was used to estimate the size of the amplified products. Genes for a 1 c (371 bp), and hslo (277 bp) were found to be expressed in both BA and IVC whereas a 1 G (221 bp) was only detected in BA. The expression of 18S ribosomal R N A was used as a loading control. n= 5 tissues from 5 different rabbits which all showed the same results. (A) Low confluency S M C s F i g . 5.6. E x p r e s s i o n o f vo l t age -ga ted c a l c i u m c h a n n e l s in s e m i - a n d f u l l y - con f l uen t c e l l s . Exemplary agarose gel electrophoresis from R T - P C R analys is of a 1 c and a 1 G m R N A s in the R A S M C s in A) low and B) high confluent states. P C R products were generated through the use of speci f ic primers for a 1 c and a 1 G . A 100-bp D N A ladder was used to est imate the s ize of the amplif ied products. G e n e s for a 1 c (371 bp) and a 1 G (221 bp) were found to be expressed in the both confluency states of the cultured R A S M C . The express ion of 18S r ibosomal R N A was used as a loading control. n= 5 independent exper iments. 128 5.2.7. Trp channel expression As a result of these findings of differential channel expression in two different rabbit tissues, I moved on to examine other important SOCC channel components. It is now coming to light that the transient receptor potential, or Trp channels may be components of SOCC associated channels. Seven mammalian genes have been identified and cloned, namely TrpCl-7 (Harteneck et al, 2000; Walker et al, 2001; Clapham, 1995). Because I had pharmacological data to show that the ROCC/SOCC blocker SKF-95365 was important in the rabbit IVC, but not in the rabbit B A , I extended the expression studies to include these important proteins. Since, also other functional evidence from our laboratory points to SOCC as a crucial component for PE-mediated [Ca2+]j oscillations and tonic contraction in the rabbit IVC (Lee et al, 2001) I proceeded to determine which TrpC channels are expressed in different smooth muscles. The results were different for all three smooth muscle types and therefore will be reported separately below. It should be noted here that due to the fact that the rabbit equivalent of Trp 1-7 have not been sequenced, we used primers based on mouse or rat sequences. However, because there is high interspecies sequence homology when comparing mouse and rat sequences for the same type of Trp channels, it is highly plausible that the primers that we use can also identify rabbit Trp channels. It should also be noted that rat and rabbit brain were used as a positive controls for trpl-7 expression in all smooth muscles done as experiments in parallel. Sequencing of the trpl-7 amplification products revealed 100% homology with the respective sequences obtained from GenBank. 129 Rabbit IVC Interestingly, trpl was the only TrpC expressed in rabbit IVC (Figure 5.7.)- The band of the predicted size (372 bp) for trpl was detected in 5 rabbits. Thus, both pharmacological and molecular evidence suggests the involvement of a SOCC in rabbit IVC. With the same primers, only trp 1, 3, and 4 mRNA were detected in the rabbit brain (« = 3 rabbits), whereas all trp 1-7 mRNA were detected in the rat brain (n = 3 rats). Rabbit Basilar Artery trp 1 and 4 were expressed in rabbit B A as shown by a single band of the predicted sizes for trp 1 (372 bp) and trp 4 (265 bp) in Figure 5.8 (n=5 rabbits). However, I have yet to show a functional role of these channels to support their involvement in regulating C a 2 + levels in rabbit B A . Rat aortic smooth muscle cells A single band of the predicted sizes of trpl (372 bp), trp 4 (265 bp), and trp 6 (410 bp) was detected in the smooth muscle cells from both low- and high- confluency cultures (n = 6 and 4 lysates, respectively). In addition, a low level of trp 2 (487 bp) gene was detected in low confluency cells (Figure 5.9.) (n=5 popoulations of cells). Thus, cultured smooth muscles cells are also suitable for studies on SOCC/ROCC, although the TrpC channels components expressed differ to either tissue preparation observed herein. Additionally, confluency does appear to have an effect on channel expression in these smooth muscle cells. Preliminary evidence from our lab suggests a role for SOCC in these cells (Poburko, unpublished). 130 B Rabbit IVC 600 bp 500 bp 400 bp 300 bp 200 bp Trpl Trp2 Trp3 Trp4 Trp5 Trp6 Trp7 1BS - R T - c D N A Rabbit brain (positive control) 506 bp 396 bp 344 b p 298 b p 220 bp 201 bp 600 bp 500 bp 400 bp 300 b p 200 bp Trpl Trp2 Trp3 Trp4 Trp5 Trp6 Trp7 uu 18S Rat brain (positive control) Trpl Trp2 Trp3 Trp4 Trp5 Trp6 Trp7 a 1 c 18S Figure 5.7. TrpC1 expressed in the smooth muscle of the rabbit IVC. Exemplary agarose gel electrophoresis from RT-PCR analysis of Trp channel family members and a1C-mRNAs in the rabbit IVC smooth muscle {A), rabbit brain (B), and rat brain (C). PCR products were generated through the use of specific primers for Trp1-Trp7. Only trp 1 (372 bp) was found to be expressed in the smooth muscle of the rabbit IVC. In the rabbit brain-positive control, only mRNA for trpl (372 bp), trp 3 (331 bp), and trp 4 (265 bp) were detected. In contrast, trp1~7 was detected in the rat brain as a positive control. Expression of 18S ribosomal RNA was used as an internal control. RT-PCR reactions run in the absence of reverse transcriptase (-RT) or cDNA (-cDNA) were used as negative controls. Gels shown are representative of findings in a minimum of 3 animals. 131 B Rabbit basilar artery 600 bp 500 bp 400 bp 300 bp 200 bp -> Trpl Trp2 Trp3 Trp4 Trp5 Trp6 Trp7 18 S Rat brain (positive control) 600 bp S00 bp 400 bp 300 bp 200 bp Trp1 Trp2 Trp3 Trp4 Trp5 Trp6 Trp7 F i g . 5.8. T rpCI e x p r e s s i o n in rabbi t bas i la r ar tery. Exemplary agarose gel electrophoresis from R T - P C R analys is of transient receptor potential (TrpC) channel m R N A s in (A) rabbit basi lar artery and (B) rat brain. P C R products were generated through the use of specif ic primers for trp1~trp7. G e n e s for trp 1 (372 bp) and trp 4 (265 bp) were expressed in B A . A 100-bp D N A ladder was used to est imate the s ize of the amplif ied products. The express ion of 18S r ibosomal R N A was used as a loading control. n=5 t issues from 5 different rabbits. 132 B 600 bp 500 bp 400 bp 300 bp 200 bp 600 bp 500 bp 400 bp 300 bp 200 bp Low confluency smooth muscle cells Trp1 Trp2 Trp3 Trp4 Trp5 Trp6 Trp7 18S High confluency smooth muscle cells 600 bp 500 bp 400 bp 300 bp 200 bp Trp1 Trp2 Trp3 Trp4 Trp5 Trp6 Trp7 18S Rat brain (positive control) Trp1 Trp2 Trp3 Trp4 Trp5 Trp6 Trp7 Figure 5.9. TrpC channel e x p r e s s i o n in RASMC. Exemplary gel electrophoresis from R T - P C R analysis of transient receptor potential (TrpC) channel mRNAs in R A S M C s both in A) low, B) high confluency states and C) rat brain. P C R products were generated through the use of specific primers for trp1-7. A 100-bp DNA ladder was used to estimate the size of the amplified products. Genes for trp1 (372 bp), trp 4 (265 bp) and trp 6 (410 bp) were found to be expressed in both confluency states of R A S M C . In addition, low levels of trp 2 (287 bp) were detected in low confluency cells. In contrast, trp1-7 were detected in the rat brain as positive controls. The expression of 18S ribosomal RNA was used as an internal control. n= 5 independent experiments. DISCUSSION Smooth muscle heterogeneity in the rabbit IVC and B A was observed. The latter functions to supply blood to all cerebral arteries in the brain, and is subject to clinical conditions of subarachnoid haemorrhage and subsequent development of delayed cerebral vasospasm. On the other hand, the vena cava is a capacity, conduit vessel physiologically important in regulating venous return and ultimately cardiac output through venoconstriction and venodilation. Force measurements of intact arteries reveal that the B A accumulates Ca mainly or only via L-type V G C C whereas this channel only accounts for approximately 25% of an ET-induced response in IVC. The remainder of the response appears to be via ROCC/SOCC activation as SKF-96365 returns the force values to baseline. Additionally, the K c a channel is not active or important in the IVC whereas its function in the B A is critical. Further molecular examination shows that although BKca and L-type V G C C are expressed in both tissues, but quantitative Western blot analysis would be necessary to validate whether the respective protein is also expressed equally in both tissues. In addition, molecular identification of the putative ROCC/SOCC channel components, the short TrpC family (TrpC 1-7) show TrpCl and 4 expression in B A , but only TrpCl expression in IVC. This latter observation fits with a model proposed by Lee et al. (Lee et al, 2001) showing the functional importance of the SOCC in refilling the SR and subsequent maintenance of oscillations in IVC. However, no functional evidence has yet been shown to prove a role of these channels in the rabbit B A . 134 Finally, the relevance of smooth muscle cell lines as a model for the study of Ca 9+ signaling was also examined and RT-PCR revealed the presence of all major Ca regulating channels although differences exist in TrpC channel subunits as compared to both B A and IVC. However, cultured cells did loose TrpC2 at higher levels of confluency. Therefore, this smooth muscle line is a good choice for the study of basic C a 2 + signaling mechanisms, since they express TrpCl , 4 and 6. It must be stressed, however, that the expression pattern in tissues may be different to the cultured cell system. Taken together, the data propose that smooth muscle heterogeneity exists regarding the expression of TrpC channels in rabbit IVC, B A and a smooth muscle cell line. Somlyo et al. have characterized smooth muscle into 3 groups due to differences in innervation, depolarization and responses to excitatory stimuli (Somlyo and Somlyo, 1994b); phasic (i.e. gastrointestinal smooth muscle), tonic (i.e. arterial smooth muscle), and intermediate (i.e. airway smooth muscle). In addition to these differences, molecular and biochemical evidence is also known to underlie smooth muscle heterogeneity within these groups. Differential expression of proteins which make up receptors, ion channels, or the contractile apparatus and those involved in signal transduction have been reported (Somlyo and Somlyo, 1994; Daemen and De Mey, 1995; Halayko et al, 1996; Archer, 1996; Walker et al, 2001). For instance, the mesenteric arterial bed constricts whereas the skeletal muscle arterial bed dilates upon adrenergic stimulation. This is partially explained by differential expression of and a- and (3-receptors. Further, regional differences within arterial beds to contraction via adrenergic agonists has also been shown, which is due to differential expression of a l - and al- receptor subtypes 135 (Vanhoutte, 1978; Kolbeck and Speir, Jr., 1987). Finally, a great diversity in the electrophysiological properties among various smooth muscles has also been documented, for example, K + channel diversity among cells taken from the same segment of pulmonary artery (Archer, 1996). A l l of these differences are necessary for specialized smooth muscle cells to fulfill their functional role in different environments. In this communication, I show that pharmacological data points to the importance of BKca in rabbit B A while rabbit IVC shows no response to blockade of these receptors. However, RT-PCR shows that the hslo gene, which encodes the B K c a channel, is expressed in both tissues. It has to be kept in mind that RT-PCR is only semi-quantitative and requires verification, e.g. by quantitative Western blotting and/or real-time RT-PCR. BKca requires high levels of C a 2 + in order to be activated in a restricted space between the P M and SR membranes. Therefore, it is possible that the B K c a is not located in this restricted space in the rabbit IVC or C a 2 + levels under resting conditions are not high enough to activate this channel (Lee C H et al, 2002a). In contrast, in rabbit B A , I propose that a SR-PM junctional space exists because the B K c a is active in the unstimulated tissue. In order to accumulate the large amounts of Ca required for B K c a activation probably via C a 2 + sparks, this restricted junctional space is necessary. As a result, the membrane would hyperpolarize, thereby inhibiting the L-type V G C C , and inducing relaxation or attenuating constriction. However, the data presented cannot rule out the possibility that B K c a is activated also in IVC upon agonist stimulation. Figure 5.1 OA shows a predicted model for C a 2 + signaling in the rabbit IVC with a large role for the SOCC located in the SR-PM junctional space. In contrast, Figure 5.10B represents a predicted model for C a 2 + signaling patterns in rabbit B A . In this case, the B K c a channel 136 is present in the SR-PM restricted space and thus smooth muscle heterogeneity links structure to function in these two vasculatures. Elevated [Ca ]j in the SR-PM space would activate a SOCC in rabbit IVC causing contraction whereas in the rabbit B A , this increase in C a 2 + would activate the B K c a leading to an efficient servo-brake mechanism opposing contraction. The L-type C a 2 + channel is, as expected, expressed in both tissues. Normalized to the internal standard, it seems that rabbit B A expressed higher levels of this channel. Moreover, a T-type channel transcript was found only in rabbit B A , but not in IVC. T-type C a 2 + channels are low-voltage activated channels that have been reported to be expressed in smooth muscle and may contribute to Ca entry (Sanders 2001). Further pharmacological experiments are required in order to determine the importance of T-channels in rabbit B A . If further experiments confirm that higher expression of L-type C a 2 + channels is indeed the case in rabbit B A , this would not be completely unexpected. 2"T" As previously shown, the rabbit B A is solely dependent on the L-type V G C C for Ca influx with little or no role yet shown for an SOCC as M n 2 + quenching experiments showed no increased permeability to C a 2 + in the presence of SERCA inactivation (Chapter 3 and (Szado et al, 2001), althought this experiment can not exclude their possible activity as discussed in Chapter 3. This is further confirmed by confocal imaging of the intact B A which reveals a typical biphasic response upon stimulation with histamine and small or no subsequent oscillations all of which can be completely abolished with the L-type V G C C inhibitor, nifedipine, whereas oscillations observed likewise in intact rabbit IVC are not blocked by nifedipine, but instead require SKF-137 96365 for complete inhibition (for further details of oscillations in rabbit IVC see (Lee et al, 2001). A 1 OOnm Figure 5.10. Model for C a 2 + signaling patterns in rabbit IVC (A) and rabbit BA (B). A) C a 2 + is released from IP 3 R and subsequent depletion of the SR opens S O C located on the P M in the SR-PM junctional space, allowing N a + to depolarize the membrane, activate V G C C and drive N C X in the reverse mode to supply C a 2 + to the junctional S E R C A for refilling of the SR. B) Contrastingly, the arrangment of the channels in the rabbit BA suggests that the RyR can release C a 2 + towards B K C a loacted on the PM, in the junctional space, thereby hyperpolarizing the membrane, inhibiting V G C C , and causing relaxtion. Abbreviations: B K C a , large conductance Ca 2 +-activated K + channel; NCX, sodium/calcium exchanger; N C S S , non-selective cation channel; S O C , store-operated channel. Adapted with permission from Lee et al. 2002. In addition to the L-type V G C C , 3 putative ROCC/SOCC channel proteins are expressed in rabbit B A . TrpCl appears to encode a NSCC channel whereas TrpC4 has been shown as store- and C a 2 + sensitive. TrpCl, 2, 4 and 5 appear to be sensitive to Ca2+-store depletion (Golovina et al, 2001; Philipp et al, 1998; Philipp et al, 2000; Zitt et al, 1996; Philipp et al, 2000) whereas TrpC3, 6, and 7 appear to be activated by receptor 138 stimulation (Hofmann et al, 1999; Hashimoto et al, 1999). There is also evidence showing TrpCl and 6 as NSCCs (Inoue et al, 2001). However, there is much evidence to suggest that TrpC proteins can form both ROCC and SOCC channels, yet is it still unclear whether these channels underlie the native currents in smooth muscle (McFadzean and Gibson, 2002). Additionally, it is likely that the functional channels are formed by a tetrameric assembly of TrpC proteins which includes the likelihood of hetero-tetrameric assembly. In fact, co-expression of TrpC5 with either TrpCl (Clapham et al, 2001) or TrpC4 (Schaefer et al, 2000) forms NSCC activated by G-protein coupled receptors, but not store depletion in HEK-293 cells. TrpC6 acts much the same (Boulay et al, 1997; Inoue et al, 2001) and appears to be a component of native current as demonstrated by phenylephrine-induced current in rabbit portal vein which act similarly to H E K cells expressing TrpC6 (Inoue et al, 2001). Thus, in the case of rabbit B A , TrpCl and 4 expression may not be related to a SOCC function but rather a NSCC or ROCC function due to combination of TrpC proteins forming a functional channel. However, it can not be ruled out that the TrpC channels expressed in rabbit B A do combine to form active TrpC channels and thus further investigation of the role of TrpC channels in smooth muscle is required. Furthermore, more TrpC subunits may also be expressed in both rabbit B A and IVC. Given that primers used to detect trpl~7 mRNAs were not of rabbit origin and that only TrpCl , 3, and 4 mRNAs were positively identified in the rabbit brain, one cannot dismiss the possibility that rabbit TrpC2, and 5-7 mRNA with distinct rabbit sequences are present but are not detected in this study. The identification of the expression of TrpCl in the vascular smooth muscle of the rabbit IVC suggest the possible existence of a store-operated non-selective cation 139 channel in this tissue (Harteneck et al, 2000). A recent report shows that TrpCl is ubiquitously expressed in various human, rabbit and mouse vessels (Xu and Beech, 2001). TrpCl is known to encode a component of the SOCC (Sinkins et al, 1998; Zitt et al, 1996) and TrpCl protein is the pore-forming component which has been localized to the plasma membrane of rabbit V S M C (Xu and Beech, 2001). In addition, the SOCC formed by the product of the TrpCl gene has been shown to be a non-selective cationic channel (Sinkins et al, 1998; Zitt et al, 1996). These results correlate with other investigations which suggested that Na + influx through this non-selective cationic channel can raise the local [Na+] in the restricted sub-plasmalemmal space. The elevated local [Na+] then drives the Na + -Ca 2 + exchanger into its reverse mode of operation, thereby bringing C a 2 + into the cell to refill the SR (Lee et al, 2001). This data suggests that because the SR and P M are in close association (van Breemen et al, 1995), and often also associated with caveolae, a signal may be passed from the depleted store (via IP 3R activation) to the TrpC channel protein (see review by (Putney, Jr. et al, 2001) thereby activating a NSCC (Figure 5.10A.). I have also reported here that R A S M C express both the L-type and T-type calcium channels at both confluencies equally. This is important to verify as it has been reported that smooth muscle cells may lose L-type V G C C during cell culture. The molecular presence confirms earlier functional data suggesting a role for the L-type V G C C in this smooth muscle cell preparation (Doyle and Ruegg, 1985). Despite the comparable expression level of the L-type C a 2 + channel at different confluency states, there are indications that C a 2 + influx is different in high vs. low confluency states (unpublished observations). However, it has to be kept in mind that the actual regulation 140 of VGCCs may vary depending on the growing state of the cells and requires further investigation. In addition, we have yet to test whether transfection of cells alters the expression of L-type VGCCs. Poburko et al. have shown clearly that the L-type calcium channel is involved in the basal calcium influx (Poburko et al, 2002). I show that these smooth muscle cells express the L-type specific a ic transcript of the voltage gated cation channel (Figure 5.6.). However, our unpublished results indicate that less than half of basal calcium influx is carried by the L-type channel. Thus, the TrpC proteins, as shown here to be expressed additionally may account for the Ca 2 +-influx normally referred to as ROCC/SOCC-mediated. Taken together, these smooth muscle cells seem to be a good system to study C a 2 + pathways and SOCC/ROCC-related phenomenon although the expression pattern of TrpC channels differs compared to B A and IVC smooth muscles. Thus, cell systems should be used only as a model and related back to the in vitro and in vivo conditions, if possible. It has previously been stated that a complete understanding of inter-smooth muscle heterogeneity and the factors controlling its manifestation are critical for the development of interventions and therapies aimed to act at exact locations in specific smooth muscle cells (Halayko et al, 1997). Herein, I report one more factor that may help to further our understanding of the differences in contractility between smooth muscles; namely, differential TrpC channel expression. However, there is much work to be done in order to specifically determine the complex ionic interactions governing Ca homeostasis in different smooth muscles. With the development of new molecular biological tools and imaging techniques, a clearer picture may arise in the future 141 identifying some of the key components governing vascular smooth muscle heterogeneity. 142 CHAPTER 6 • General conclusions and future outlook This thesis emphasizes the importance of intracellular organelles in the regulation of Ca signaling in vascular smooth muscle; in particular, the spatial arrangement of both the mitochondria and SR in relation to each other, the plasma membrane and possibly the nucleus. The expression of channel receptors on the plasma membrane may contribute to the production of specific microdomains within different vascular tissues and therefore, may contribute to smooth muscle heterogeneity. These restricted diffusional spaces, particularly the SR-PM and SR-MT junctional complexes, are critical under physiological conditions in buffering C a 2 + from the bulk cytosol, shifting C a 2 + from one compartment to the other, and in the regulation of membrane potential. C a 2 + uptake into the M T is vital for activation of different enzymes therein essential in the production of energy, and thus crucial for the smooth muscle cell to match energy demands to its needs under constantly changing conditions. Due to the large extracellular C a 2 + concentration and thus large gradient for C a 2 + to flow into cells, powerful C a 2 + extrusion mechanisms are critical for maintaining [Ca 2 +]i at reasonable levels. This is accomplished through extrusion mechanisms located on the P M , namely N C X and P M C A , the former of which is shown to be important in regulation of C a 2 + in rat aortic cell cultures in Chapter 4 of this thesis. Although the role of N C X in smooth muscle has been controversial, data from our laboratory in intact tissues demonstrate its importance in Ca homeostasis (Nazer and van Breemen, 1998b; Nazer and van Breemen, 1998a; Lee et al, 2001; Lee C H et al, 2002b). However, in addition to extrusion, other intracellular organelles, specifically the SR which is critical in regulating [Ca 2 +]j as shown in Chapter 3, and the MT, which also appears to play a role in 143 sequestering and releasing C a 2 + as shown in Chapter 4, are important. The SR is important in buffering of Ca 2 + , and in releasing Ca 2 + , via IP 3Rs or RyRs, upon agonist stimulation attenuating or initiating contractile responses. In addition, the SR releases C a 2 + sparks which activates B K c a and induces hyperpolarization of the cell membrane. However, in Chapter 3 I have shown here that the B K c a can be activated by C a 2 + from other sources, presumably the L-type V G C C , and initiate relaxation. I now also could show that the M T is capable of accumulating C a 2 + in the uM range, upon agonist stimulation as demonstrated in Chapter 4. This C a 2 + may be important in the activation of key mitochondrial enzymes to produce ATP, but M T accumulation of C a 2 + may also play a role in modulating agonist-induced responses. Additionally, our data suggests the existence of a restricted space between the SR and MT. Nonetheless, under basal conditions the SR is the most important organelle for buffering of Ca 2 + . However, as the SR spills C a 2 + towards the M T uniporter, under conditions of stress or receptor activation, it allows more C a 2 + to be accumulated via SERCA. Thus, the M T may act as a secondary storage site for C a 2 + thereby prolonging the activation phase of agonists and perhaps maintenance of C a 2 + oscillations due to slow release back into the cytosol via the mNCX. As yet this is only a hypothesis, but offers interesting possibilities. These possibilities need to be further verified in cells and proven to be active in tissues and intact vessels. Therefore, there are still uncertainties surrounding M T Ca uptake and its involvement in shaping the spatiotemporal C a 2 + signal. Thus, future directions should include further analysis for the role of M T in C a 2 + signalling in smooth muscle. Specifically, the extrusion mechanism in the M T is not well defined, but with the specific mNCX inhibitor, CPG 37157 coupled with mitochondrial-targeted aequorin, the role for 144 mNCX in smooth muscle could be determined. In addition, further application of the targeted aequorin technology could be applied, specifically with a variety of targeted intracellular probes, i.e. the nucleus and plasma membrane, which are readily available. Therefore, transfection of these targeted plasmids into various diseased, knock-out cells and/or normal vascular smooth muscle cells would give us valuable insight into possible C a 2 + deregulation in disease with comparison to normal C a 2 + homeostasis mechanisms. In addition, possible incorporation of this technique in a smooth muscle organ culture would provide a more physiologically relevant model for the study of C a 2 + homeostasis if transfection protocols could be perfected. Furthermore, with the explosion of molecular biology and gene sequencing, various targeted fluorophores are now available. Some examples include camelons, camgaroos and most recently, pericams. The latter is a C a 2 + sensitive probe that can be specifically targeted and is fused with GFP. Several mutations have been made and the only report published yet utilizing these pericams shows encouraging results (Robert et al, 2001). With the production of such molecules it may now be possible to measure C a 2 + in these aforementioned restricted spaces thereby enhancing our knowledge of C a 2 + movements within smooth muscle and the relationship of the organelles to each other and the P M . C a 2 + levels within the SR-PM microdomain, critical in the production of C a 2 + sparks and subsequent activation of K + channels, may be measured with a targeted aequorin which is fused to SNAP-25, a plasma membrane associated receptor. With all this novel technology and with the advances in imaging systems, deconvolution technology in combination with electron microscopy a clear image for C a 2 + movements in and out of organelles and within restricted spaces is now feasible. 145 Finally, the topic of smooth muscle heterogeneity has been studied with regards to expression of various P M channels in addition to functional contraction data and C a 2 + imaging. It is clear that differential channel expression is certainly one of many components responsible for coupling heterogeneity to function. However, there are many unanswered questions. In the case of rabbit IVC and B A it is intriguing that the B K c a appears to be non-active in IVC under resting conditions. However, it is expressed within this tissue suggesting that the functional difference may lie in the association of the various channels within these microdomains and/or caveolae. However, it may also be that due to the low transmural pressure in this vessel as opposed to B A , the IVC is more hyperpolarized and therefore the K c a is silent. Membrane potential measurements in both intact tissues would help to answer this question. Although we often think of these restricted spaces as ubiquitous within smooth muscle, we often fail to realize that these restricted spaces may be very different in a cerebral versus mesenteric artery. Therefore, future studies in a variety of vascular tissues from different regions of the body, both functionally and at the molecular level will give further insights into the most important factors regulating this diversity. Furthermore, many structural elements are involved in placing organelles in their spatial arrangement within the cell, namely the recently identified junctophilins, integrins, formation of caveolae, and the actin cytoskeleton which all require further investigation. In addition, electron micrographs and confocal imaging of these microdomains will be central in determining the various important channels and pumps which regulate ionic concentration within these restricted spaces, where these microdomains are, i.e. within caveolae or between organelles or both, and which PM-SR-MT-Nuclear interactions may or may not be occurring in a given tissue. 146 The ultimate goal of all these studies is to provide information on how our circulation is regulated. Therefore, the more we understand about C a 2 + signaling within the cells that make up all of our arteries and veins, the more possible therapeutic targets are available in the treatment of diseases such as hypertension and stroke. In addition, by studying the mechanisms of smooth muscle heterogeneity, we may be able to specifically design therapeutic agents acting exclusively within a certain vascular bed thereby increasing effectiveness, and hopefully, decreasing side-effects. 147 APPENDIX I p o w e r s u p p l y p e r f u s i o n Luminometry setup. The cell chamber is constantly thermostated at 37°C 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 specially designed chamber. Photons emitted by the cells are deteced with a low noise photomultiplier tube (pmt, Thorn-EMI 9789A) connected to a high voltage power supply (Thorn-EMI, PM28B) . 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