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The role of the SR in Ca2+ extrusion from venous smooth muscle Nazer, Mark A. 2004

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T H E R O L E OF T H E SR IN C a  / +  EXTRUSION F R O M VENOUS SMOOTH M U S C L E by MARK A. NAZER  B.Sc, The University of British Columbia, 1994 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF T H E REQUIREMENTS FOR T H E D E G R E E OF DOCTOR OF PHILOSOPHY in T H E 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  T H E UNIVERSITY OF BRITISH C O L U M B I A September 2004 © Mark A . Nazer, 2004  ABSTRACT  Ca  z+  extrusion from rabbit inferior vena cava (IVC) smooth muscle (SM) was studied  using ratiometric fura-2 fluorimetry. Concomitant blockade ofthe plasma membrane (PM) Ca -adenosinetriphosphatase (PMCA), N a - C a 2+  +  2+  exchanger, and sarco-endo-  plasmic reticulum Ca -ATPase (SERCA) completely prevented the decline in the 2+  intracellular C a  ([Ca ]0 normally observed when C a 2+  concentration  2 +  the extracellular space (ECS) after stimulated C a  2 +  2+  is removed from  influx. Blockade of the N a - C a +  exchanger by removal of external N a reduced the rate of +  [Ca ]j 2+  2+  decline by 47%.  Blockade of SERCA with cyclopiazonic acid (CPA) reduced it by 23%, and this was not additive to the effects of Na removal. The loss of SR Ca  was determined by measuring  the decay of caffeine (CAF)-induced fura-2 fluorescence transients. Removal of C a from the ECS caused a rapid loss of SR C a  2 +  and a decline of  [Ca ]j 2+  2 +  preventing the  sarcoplasmic reticulum (SR) from reloading. Simultaneous removal of extracellular N a greatly inhibited the rate of this SR C a  2+  loss. A rapid loss of SR C a  2 +  +  was induced by 20  uM CPA, regardless ofthe presence or absence of extracellular N a or C a . These +  2+  effects were not influenced by alterations in membrane potential owing to activity of Ca -activated K channels (Kc channels) since 3mM or higher of T E A (or 70 nM 2+  +  a  Iberiotoxin) had no effect on the rate of C a when C a Na -Ca +  2+  2 +  2+  loss from the SR. These results indicate that  is removed from the ECS, it induces C a  2 +  release from the SR towards the P M  exchanger which subsequently translocates it from the junctional cytoplasmic  space to the ECS. When the N a - C a +  2+  exchanger is arrested by removal of extracellular ii  N a and C a , C a +  2+  2 +  released from the SR is re-sequestered by the SERCA. However,  when both the N a - C a +  2+  exchanger, and the SERCA are blocked, C a  2 +  released from the  SR is extruded from the cells by the PMCA. From these results and the additional information provided by electron micrographs of the adult IVC, we conclude that the SR not only contributes to C a  2 +  extrusion by way of coupling of the SERCA to the N a - C a +  2+  exchanger but also forms part of systematic hierarchy of interaction between the different Ca  2 +  transporters in the SR and cell membranes.  iii  T A B L E OF CONTENTS ABSTRACT T A B L E OF CONTENTS LIST OF T A B L E S LIST OF FIGURES .PREFACE Poster Presentation Venues Oral Presentations Publications ; ACKNOWLEDGEMENTS LIST OF ABBREVIATIONS Chapter I. B A C K G R O U N D A. Introduction V S M Cell Structure B. Ca Transport a) C a Influx-VOCCs, ROCs and SOCs b) C a Extrusion-PMCA and N a - C a exchange i) The P M C A ii) The Na -Ca Exchanger c) The SR in C a Transport i) SR C a Uptake-SERCA ii) SR C a Release-IP R and R Y R 1) IP Receptor 2) Ryanodine Receptor C. Ca Signaling a) Excitation-Contraction Coupling: Basic C a Signaling Function i) Contraction ii) Vasodilatation b) C a Signaling between the SR and PM i) The Superficial Buffer Barrier ii) Ca -Induced Relaxation C a Sparks and STOCs iii) The Nucleus and Mitochondria Chapter II. E X P E R I M E N T A L DESIGN, METHODS A N D RESERVATIONS A. Experimental Design a) Aims of This Thesis b) Specific Protocols B. Methods a) Animal Care b) Tissue Preparation c) Force/Tension Measurement d) Measuring the Loss of Endothelium from Intact Smooth Muscle 2+  2+  2 +  +  +  2+  2+  2 +  2 +  2 +  3  3  2+  2 +  2 +  2+  2 +  ii iv vi vii viii viii viii viii x xi 1 1 2 4 4 11 11 13 18 19 20 21 .23 24 24 24 27 28 28 29 31 33 36 36 36 38 43 43 43 44 44 iv  e) Tissue Immobilization 45 f) Spectrofluorimeter Set-Up 46 g) Autofluorescence Measurement 47 h) Fura-2 Loading 47 i) [Ca ]i Measurement 48 j) Fura-2 Calibration 48 k) Solutions and Chemicals 49 1) Statistics of Experiments 51 m) Curve-Fitting Analysis 51 C. Reservations 52 a) Fura-2 52 b) Temperature 54 c) Intracellular pH 55 d) In-Situ Preperations 56 Chapter III. RESULTS 58 A. Concomitant blockade of N a - C a exchange, SERCA, and the PMC A abolishes C a efflux 58 B. N a - C a exchanger and SERCA blockers attenuate the rate of [Ca ]i decline in C a loaded cells 61 a) Na -Ca exchanger 61 b) SERCA 63 c) SERCA plus N a - C a exchanger 66 C. Evaluating the decline of [Ca ]i under varied C a extrusion conditions 66 D. SR-mediated C a extrusion 70 a) Ca -freePSS 70 b) Na -Ca exchanger 72 c) SERCA 74 d) SERCA plus N a - C a exchanger 74 E. Comparing Caffeine-Induced [Ca ]i Peaks Following Blockade of the N a - C a exchanger and/or SERCA 77 F. Schematic Diagram #1-The Process of SR Buffering and Ca Extrusion 79 G. Ranking the order of access of the N a - C a exchanger, SERCA, and the P M C A to C a released from the SR under resting conditions 79 H. Schematic Diagram #2-Ca Cycling Dynamics 91 I. Effects of Ca -sensitive K currents on the C a signal 91 Chapter IV. DISCUSSION 97 A. Proof that the N a - C a exchanger is implicated in SR-mediated C a extrusion 97 B. Update and New Directions in V S M Research 106 REFERENCES 112 2+  +  +  2+  2 +  2+  +  2+  2+  2+  +  2+  2+  2 +  2+  2+  +  2+  +  2+  2+  +  +  2+  2 +  2+  2+  +  +  2+  2 +  2 +  2+  LIST OF T A B L E S  Table 1: Rate of [Ca ]j decline fitted to the equation, f=ae" +ce" (see methods) with blockade of the Na -Ca exchanger and/or SERCA 53 2+  +  bx  dx  2+  vi  LIST OF FIGURES Fig. 1. Electron micrograph ofthe adult IVC 3 Fig. 2. Voltage-operated C a channel topology 6 Fig. 3. Schematic diagram ofthe protein structure ofthe P M C A / S E R C A embedded within the plasmalemmal/SR membranes 12 Fig. 4. Structure ofthe N a - C a exchanger 15 Fig. 5. Excitation-contraction coupling vs. relaxation and agonist-induced C a sensitization 26 Fig. 6. Schematic diagram illustrating B K c channel topology 30 Fig. 7. Proposed model for SR-mediated C a extrusion 37 Fig. 8. Sample trace and corresponding protocol for C A F experiments 42 Fig. 9. Metabolic and transport blockade prevents C a extrusion 59 Fig. 10. Blockade of Na -Ca exchanger attenuates the rate of decline in [Ca ]i 62 Fig. 11. Blockade of the SERCA attenuates the rate of decline in [Ca ]i 65 Fig. 12. The addition of S E R C A blockade on top of Na -Ca exchanger blockade has no further affect on the rate of decline in [Ca ]j 67 Fig. 13. Comparison ofthe effects of CPA, 0Na t and their combination on the rate of 2 +  +  2+  2 +  a  2 +  2 +  +  2+  2+  2+  2+  +  ex  [Ca ]i decline  69  2+  Fig. 14. The SR refills only in the presence of extracellular C a (0Ca PSS treatment prevents refilling) 71 Fig. 15. Na -Ca exchanger blockade enhances SR refilling 73 Fig. 16. SERCA blockade depletes the SR 75 Fig. 17. N a - C a exchange + SERCA blockade prevents the SR from refilling 76 Fig. 18. Comparison ofthe refilling of the SR in 0 C a solution under conditions of SERCA and N a - C a exchange blockade 78 Fig. 19. The process of SR buffering and C a extrusion 80 Fig. 20. SR C a decay after abolishment of C a influx 82 Fig. 21. N a - C a exchange inhibition modulates the rate of decay in SR C a content. 83 Fig. 22. Exponential Ca decay in cytoplasmic Ca concentration under control and 0 Na conditions 85 Fig. 23. Comparison ofthe effects of C a removal, and Ca +Na removal on the SR C a content (CAF/80K amplitude, % ratio) 86 Fig. 24. SERCA blockade does not accelerate the decay of SR C a after C a removal. 88 Fig. 25. N a - C a exchanger + SERCA blockade does not change the decay of SR C a content from Fig. 24 89 Fig. 26. Comparison of the decay in SR C a content 90 Fig. 27. Diagram of C a cycling dynamics 92 Fig. 28. K channel blockade by T E A is insensitive to [Ca ]j 94 Fig. 29. Effects of T E A following treatment with CPA 95 2 +  +  2+  +  2+  2+  2+  +  2+  2+  2 +  +  2 +  2+  2+  +  2 +  2 +  2+  +  +  2 +  +  2+  2 +  2 +  2+  2 +  +  2+  Vll  PREFACE  Poster Presentation Venues Health Sciences Student Research Forum. The University of British Columbia, Oct. 16,1996. Inaugural Symposium, PEARL-Cardiovascular (a Hong Kong University collaboration) St. Paul's Hospital, Vancouver, B C , May 26, 1997 1997 Fred Fay Smooth Muscle Summer Research Conference (FASEB) Copper Mountain, Colorado, June 22-27, 1997. XIX Annual Meeting of the International Society for Heart Research Vancouver, B C , July 23-27, 1997. 9th International Symposium on Vascular Neuroeffector Mechanisms Porto, Portugal, Aug. 2-5, 1998.  Oral Presentations Department of Pharmacology and Therapeutics Graduate Student Seminar Series The University of British Columbia, 1996, 1997, 1998, 2003, and 2004. Regulation of C a entry and vascular smooth muscle tone. C. van Breemen, T. Szado, M . Nazer, C-H. Lee, G. Lagaud, E. Lam, I. Laher. Vascular Research Meeting. Honolulu, Hawaii (absent). 2 +  Publications Abstracts The Role of the Sarcoplasmic Reticulum in Regulating Calcium Extrusion in Smooth Muscle Cells of Rabbit Inferior Vena Cava. M . Nazer, and C. van Breemen. Health Sciences Student Research Forum, The University of British Columbia, October 16, 1996. The Role of the Sarcoplasmic Reticulum in C a Extrusion from Smooth Muscle Cells of Rabbit Inferior Vena Cava. M . Nazer, and C. van Breemen. Journal of Molecular and Cellular Cardiology, 29:A220, 1997. 2 +  Papers A Role for the Sarcoplasmic Reticulum in C a Extrusion from Rabbit Inferior Vena Cava Smooth Muscle, M . A . Nazer and C. van Breemen. American Journal of Physiology{Heart and Circulatory Physiology), 274:H123-H131, 1998. 2 +  A Role For the Sarcoplasmic Reticulum and N a - C a Exchange in Vascular Smooth Muscle C a Cycling. M . Nazer, and C. van Breemen. Pharmacology & Toxicology, 83(Suppl. l):54-56, 1998. +  2+  2 +  Functional Linkage of N a - C a Exchange and Sarcoplasmic Reticulum C a Release Mediates C a Cycling in Vascular Smooth Muscle, M . A . Nazer, and C. van Breemen. Cell Calcium, 24(4):275-283, 1998. +  2+  2 +  2 +  Paper Submitted For Publication Ontogeny of Plasma Membrane-Sarcoplasmic Reticulum Junctions in Smooth Muscle of the Rabbit Inferior Vena Cava, M . A . Nazer, K - H . Kuo, C-H. Lee, and C. van Breemen. 2004.  ix  ACKNOWLEDGEMENTS  I am deeply indebted to Dr. Casey van Breemen - supervisor, advocate, mentor, colleague and friend. His passionate drive to help me succeed and to continue on with his highly coveted research inspired me and permitted me to persevere even in the face of adversity. Constructive and healthy criticism was also part of the agenda and spawned many novel ideas to which I could apply to my experimentations. I am also very appreciative of Dr. Xiaodong Wang who guided me through my earlier days of research and was a sounding board of enlightenment for me. With ease, Dr. Wang could fluently provide insight into some of the more difficult concepts in such a way that I could understand. Furthermore, I am thankful for Dr. van Breemen and Dr. Wang for the impulsive brainstorming sessions and question periods. I am also particularly greatful of my supervisory committee whose members, Dr. van Breemen, Dr. Edwin Moore, Dr. Jim McClarnon, and Dr. Issy Laher listened to me and provided me with feedback. I thank my friend, Philipp Langer who helped me obtain data which was used toward a publication and as part of my thesis in fulfillment of the degree of Doctor of Philosophy. I willingly and openly exercised my knowledge of C a regulation on my colleague Dr. Cheng-Han Lee who has continued on with my research under the iCaptur e banner. 2 +  4  x  LIST O F ABBREVIATIONS  ACh 2-APB ATP BKca Ca [Ca ] cADPR CAF cAMP cGMP CICR CIF CPA DAG DCB DMSO ECS EGTA EC FCCP fura-2 A M HEPES IAA IICR 2 +  2+  ;  IP R IVC KB-R7943 3  Kca M L C 2 0  MLCK MLCP MT NA NCX nm NMDG NO NPSS PE pHi  Acetylcholine 2-aminoethyl diphenylborinate adenosine triphosphate large conductance Ca -sensitive K channel calcium ion intracellular calcium concentration cyclic ADP-ribose caffeine adenosine 3',5'-cyclic monophosphate guanosine 3',5'-cyclic monophosphate calcium-induced calcium release calcium influx factor cyclopiazonic acid diacylglycerol 3',4'-dichlorobenzamil dimethyl sulfoxide extracellular space 2+  +  ethylene glycol-bis-(P-aminoethyl ether) N , N , N', N'-tetraacetic acid endothelial cell carbonyl cyanide p-(tri-fluoromethoxy) phenyl-hydrazone hydrophobic form of fura-2 with an attached acetoxy methyl ester group N-[2-hydroxyethyl] piperazine-N'-[2-ethanesulfonic acid] iodoacetic acid IP3-induced calcium release IP3 receptor-operated C a channel Inferior vena cava 2-[2-[4-(4-nitrobenzyloxy) phenyl]ethyl]isothiourea methanesulfonate Ca -sensitive K channel myosin light chain myosin light chain kinase myosin light chain phosphatase mitochondria Noradrenaline N a - C a exchanger nanometres N-methyl-D-glucamine chloride nitric oxide normal physiological saline solution Phenylephrine intracellular pH 2 +  2+  +  +  2+  xi  PKA PKC PKG PLC PIP PM PMCA ROC RYR s SBB SERCA SM SOC SR STOCs TEA 2  voce VSM XIP  protein kinase A protein kinase C protein kinase G phospholipase C phosphoinositol bisphosphate plasma membrane plasma membrane C a ATPase receptor-operated channel ryanodine receptor-operated C a channel seconds superficial buffer barrier sarco-endo-plasmic reticulum C a ATPase smooth muscle store-operated channel sarcoplasmic reticulum spontaneous transient outward currents tetraethyl ammonium chloride voltage-operated C a channel vascular smooth muscle exchanger inhibitor peptide 2 +  2 +  2 +  2+  xii  Chapter I. BACKGROUND  A. Introduction Blood vessels supply all living cells in our body with the vital nutrients and hormones needed to survive. The blood vessel is comprised of a single layer of endothelium (EC), single or multiple layers of smooth muscle (SM) and an outer adventitia. The smooth muscle layer is responsible for generating vessel tone and vascular resistence required to regulate blood flow and distribution. When the degree of vasoconstriction remains above the normal tone as in the case of coronary vasospasm or-malignant hypertension, damage to vital organs can occur. In order to understand these diseases and devise effective means of reversing the excessive vasoconstriction, the mechanism of excitationcontraction coupling in vascular smooth muscle (VSM) must be carefully elucidated. Given the importance of C a  2+  as a signaling molecule for contractile activation in V S M ,  examination of the basic mechanisms for C a  2 +  homeostasis is fundamental to the  understanding of excitation-contraction coupling in V S M . In the last decade, more advanced technologies have provided us with extremely useful and highly accurate techniques to assess changes in [Ca  ]j  2+  in real time. In this study, we employed the  spectroflourimetric method to measure intracellular C a on the putative mechanisms that facilitate C a  2+  2+  concentrations. Our focus was  efflux mediated by interactions between  the SR and plasmalemmal membranes. This interaction has in recent years become 1  increasingly recognized as an important one in regulating [Ca ]i and vascular tone. In 2+  this chapter we will briefly visit the structure of V S M cells, the function of Ca and C a  2 +  regulation including influx, efflux, and C a  2 +  in V S M ,  cycling.  VSM Cell Structure The source of the SM for this study was the rabbit inferior vena cava (IVC) which is a vein with a thin media of three to four layers of circumferentially arranged vascular muscle cells. The IVC is considered a capacitance vessel characterized by a high capacity for pooling blood. In contrast to the uterine lining and intestinal SM where action potentials generated by the SM cell produce rhythmic or phasic contractions, IVC, like most other vasculature generates myogenic tone augmented by agonist-induced cell depolarization. Contractions are relatively slow and less intense than those generated by striated muscle. Individual SM cells are ribbon-shaped and measure about 50p.m in length by 4 urn in diameter (Orallo, F., 1996). The outer cellular membrane or plasma membrane (PM) is punctuated by small 'flask-shaped' invaginations or caveolae that increase the surface area of the cell by about 75 % (see Fig. 1 for IVC ultrastructure). Internal organelles like the nucleus, mitochondria and sarcoplasmic reticulum (SR) act to compartmentalize Ca  (see sections on "The SR in Ca  Transport" and "The Nucleus  and Mitochondria" for more details). Myofilaments comprised of actin and myosin are located throughout the S M cells and are the intrinsic contractile machinery for force development. The actimmyosin ratio in SM is 13:1 as opposed to 2:1 in striated muscle  Fig. 1. Electron micrograph of the adult IVC. P M - S R j u n c t i o n a l c o m p l e x e s a r e p r e s e n t t h r o u g h o u t the S M a n d a r e typically associated with the necks of the caveolae. S R = s a r c o p l a s m i c reticulum; C = c a v e o l a e ; ECS=extracellular s p a c e ; M T = m i t o c h o n d r i a . P h o t o g r a p h e d by Dr. K - H . K u o .  3  (Somlyo, A.P., and Somlyo, A . V . , 1990).  B. C a  z r  Transport  Significant ultrastructural features of S M include the appearance of docking ofthe 9-4-  plasmalemma to the SR with Ca transporting mechanisms playing an essential role in the cross-talk between these two barriers. Cross-talk exists because of a variety of C a signaling cascades in which the C a  2+  2 +  signal diverges at the receptor/channel level and  amplifies further downstream to affect several different internal targets. It is based on regulated C a of C a  2 +  2 +  flux across the plasmalemma and SR membranes via: 1) active transport  across the P M and the SR, 2) ion exchange (Na -Ca ) across the PM, 3) +  2+  transport into the nucleus and mitochondria, 4) excitable but energetically passive influx via channels such as voltage-operated C a  2 +  channels (VOCCs), receptor-operated  channels (ROCs) and store-operated channels (SOCs) and, 5) to a lesser extent, a passive Ca  2 +  leak (Orallo, F., 1996).  a) Ca Influx- VOCCs, ROCs and SOCs 2+  2+  Ca  is ubiquitous in excitable cells and most other cells and is the single most  important ion in cell signaling. The P M tightly regulates Ca -dependent cellular 2+  functions because it's permeability to Ca  is continuously being adjusted according to  membrane-bound ion channels that undergo rapid transistions from resting to open state. Membrane depolarization induced by a change in electrical potential due to activation of 4  non-selective cation channels as in the case of the inward flux of cations down their concentration gradient stimulates the activation of VOCCs which are highly specific for C a . There are several types of V O C C but the two most prevalent in V S M are the T2+  type (transient) and L-type (long-lasting) channels. The L-type VOCCs are by far the most important because they are responsible for the main C a  2 +  currents involved in S M  contraction (Ertel, E.A., et al., 2000). They require strong depolarization (i.e. high voltage) for activation and generate intermediate conductances of approximately 25pS (Receptor and ion channel nomenclature, 1998). These channels are comprised of several protein subunits designated cti, ct -8, (3, and y which in different combinations form an 2  oligomeric complex e.g. a , a -5, (3, y; a i , a -5, p, (-Hy?); a m , a -8, p, (-Hy?); or cti , + ] S  2  C  2  2  F  accessory subunits. The a - and 8-subunits are linked by a disulphide-bridge (Receptor 2  and ion channel nomenclature, 1998). The cti subunit surrounds the ion-selective pore and is the binding site for agonists and antagonists (Receptor and ion channel nomenclature, 1998). It is found largely in SM as a a i c subunit. The P subunit promotes cell surface expression but does not span the P M perhaps because it lacks hydrophobic a helices or glycosylation sites (Receptor and ion channel nomenclature, 1998). Each of the subunits is composed of four covalently-linked homologous repeats, I, II, III, and IV, each containing 6 a-helical transmembrane segments (Si-Se) as shown in Fig. 2. The S4 segment is a voltage sensor that responds to membrane depolarization by opening the ion channel, while the S5 and  segments and the peptide chain connecting the two surround  the pore (Katz, A . M . , 1997). Sustained channel openings occur only in a small window  5  Fig. 2. Voltage-operated C a channel topology. 2+  Representation covalently  of  the  linked  a subunit r  domains  each  transmembrane segments (S1-S6). ( a , P, y, 5) the 2  of  the  VOCC  consisting  containing  6  of  4  a-helical  A l o n g with o t h e r s u b u n i t t y p e s  f o r m s a n o l i g o m e r i c c o m p l e x ; a v o l t a g e s e n s o r is  c o n t a i n e d in the S 4 d o m a i n of the a s u b u n i t .  BTZ = benzodiazepine;  D H P = dihydropyridine; P A A = phenylalkylamines.  R e p r o d u c e d from  W i e l o w i e y s k i , P . A . et a l . , 2 0 0 1 .  6  current which is determined by the voltage activating and inactivating mechanisms (Cohen, N . M . et al., 1987). L-type channels are blocked by high [Ca ]i, as well as by the 2+  Ca  channel antagonists, nifedipine (a dihydropyridine class of blocker), verapamil (a  phenylalkylamine), and diltiazem (a benzothiazepine), or calciseptine all of which bind to the a, subunit (Katz, A . M . , 1997; Ertel, E.A. et al., 2000). There is a small number of Ttype Ca channels populating V S M but they tend to operate out of the range of membrane potential attained in the IVC and therefore would appear to remain inactivated (Nilsson, H . , 1998). Sustained C a  2+  influx can also be triggered by a number of  physiological stimuli (i.e. neurotransmitters) that bind to and activate receptor molecules (ROCs) on the cell surface which act to open non-selective cation channels and also have an excitatory effect on L-type channels by generating protein kinase C (PKC) (see section "SR C a  2+  Release-IP R and RYR" for more details). Such ROCs are variably activated 3  by: (1)  Noradrenaline (NA) i.e. oc-adrenergic transmission and ATP released from nerve endings located mostly in the adventitia;  (2)  Endothelial-derived agents such as the vasoconstrictors, endothelin and some arachidonic acid metabolites or vasodilators e.g. N O and prostacyclin;  (3)  Blood-borne agents which diffuse from the bloodstream into the endothelium and SM layers e.g. products secreted by endocrine, blood and neural cells some of which are vasoconstrictors like adrenaline, vasopressin, angiotensin II, serotonin, thromboxane A2 or vasodilators such as histamine, atrial natriuretic factor, and bradykinin; 7  (4)  Blood physico-chemical factors such as O2, C O 2 , pH and temperature;  (5)  Myogenic factors i.e. pressure and stretch (Orallo, F., 1996).  There are several different categories of C a  2+  permeable channel that are activated by  agonists. In some cases, the receptor molecule itself functions as a channel which opens as the agonist binds to it, activating N a and C a +  2 +  entry that depolarizes the cell and  therefore activates VOCCs. Or, in most cases receptor binding initiates a signaling cascade (i.e. the phospholipase C pathway) which, through diffusion of intracellular second-messengers, impinges on a separate channel, affecting its activity in various ways. These types of channels are activated by D A G , and IP4, a metabolite of IP3. There is yet another class of ROC exclusively activated by SR store depletion which is normally referred to as the store-operated channel (SOC) and was originally discovered in SM by Casteels and van Breemen and studied further in-depth by the groups of Putney, Casteels, and more recently by Bolotina. Much less is known about this C a  2 +  release-activated  type channel (CRAC) than of the ROC but it was found to share similar molecular makeup to a transient receptor gene (trp) channel identified in drosophila (note that many trp sub-families exist). The biggest difference is it's activation by C a not by C a  2 +  store depletion and  release from internal stores. The SOC is thought to mediate capacitative C a  entry via a process of permeabilization of the P M to C a N a to rapidly refill the SR and sustain +  2+  [Ca ]j. 2+  2 +  2 +  and monovalent cations like  SR store depletion by IP -induced IP R 3  3  activation generates a signal to open the SOC via a soluble messenger (Randriamampita, C , and Tsien, R.Y., 1993; Trepakova, E.S. et al., 2000) or an IP3R/SOC physical 8  interaction (Irvine, R.F., 1990). The capacitative Ca  entry model originally proposed  by Casteels and Droogmans (Casteels, R. and Droogmans, G., 1981) is based on a tightly regulated cross-talk between the SR and P M in which depletion of an intracellular C a pool can activate Ca  2+  influx via a plasmalemmal pathway. According to their  observations in the rabbit ear artery, it was found that refilling of an internal C a  2 +  previously depleted by agonists is dependent on the presence of extracellular C a  2 +  store of  which higher concentrations did not elicit a contraction, suggesting there is a direct pathway for C a  2+  to refill the SR from the ECS, thereby bypassing the bulk cytoplasm.  Further evidence to support the capacitative model was derived from work by Takemura and Putney (Takemura, H. and Putney, J.W., Jr., 1989) in non-excitable cells. Their "Ca  2+  to C a  overshoot experiments" revealed a marked elevation in the permeability of the P M 2 +  in response to agonist-induced depletion of the intracellular C a  agonist in the absence of extracellular C a increase in  [Ca ]j upon 2+  2 +  2 +  store by the  which then caused a rapid and significant  the re-introduction of external C a  2 +  i.e. the C a  ofthe first mechanisms demonstrating store depletion-induced C a  2 +  2 +  overshoot. One  influx was revealed  by Putney (Putney, J.W., Jr., 1986; Putney, J.W., Jr., 1990) and Putney and Bird (Putney, J.W., Jr. and Bird, G.S., 1993; Putney, J.W., Jr. and Bird, G.S., 1994) who proposed either a direct functional or physical interaction of nVsensitive C a  2 +  stores to molecules  embedded in the PM. A conformational change in the depleted C a  2+  store (or a change in  the cytoskeletal structure) which when linked to the P M would directly signal to the P M to open a specific C a  2 +  channel such as the SOC. Other types of membrane-membrane  interactions were further postulated and may include calcium influx factor (CIF), a 9  proposed messenger that is released following depletion of the intracellular C a and which diffuses toward the P M to stimulate Ca  stores  2 +  influx or a G-protein coupled  pathway. Trepakova et al. (Trepakova, E.S. et al., 2000; Trepakova, E.S. et al., 2001) recently examined SOC activation by CIF in V S M and found that it was a contributing factor in capacitative C a  2 +  influx. They postulated that pre-formed CIF is released  following depletion of the SR and binds to phospholipase A , thereby displacing calmodulin and disinhibiting the enzyme. This, in turn, catalyzes the production of lypophospholipid, activating the SOC which induces membrane depolarization and further C a Na -Ca +  2+  2+  influx by VOCCs. Furthermore, N a influx through the SOC could drive the +  exchanger in its reverse mode to refill the SR. De La Fuente et al. (De La  Fuente, P.G. et al., 1995) found that in a Ca -free bath when SR C a 2+  2 +  uptake was  inhibited by the SERCA blocker, CPA, subsequent addition of N A did not have any 9+  effect, however, after adding back Ca  to the medium there was a marked increase in  SM tone since the SR component of capacitative Ca  entry was effectively disabled.  Moreover, this response was not inhibited by verapamil, nifedipine, or very low external N a concentrations, leading us to believe that the influx was not due to VOCCs or +  reverse-mode N a - C a +  2+  exchange but by a non-selective cation channel that is C a  2 +  permeable which in good probability is SOC. SKF 96365 is the most selective SOC inhibitor currently available, however the SOC can also be non-specifically blocked by N i , M n , L a , and C o . Interestingly, partial depletion of IP3-sensitive C a 2 +  2 +  3+  2+  2+  stores by  9+  caffeine (CAF) or ryanodine does not trigger Ca  influx, but with complete depletion of  the IP3-sensitive stores by N A or acetylcholine (ACh) in 0 Ca -PSS there is C a 2+  2+  influx 10  upon addition of extracellular C a . 2+  b) Ca  2+  Extrusion-PMCA and Na -Ca +  2+  exchange  i) The P M C A The high-affinity, low-capacity plasma membrane Ca ATP to actively pump 1 C a  2+  ATPase (PMCA) utilizes 1  ion from the cytosol into the extracellular space (ECS) for  every 2 hydrogen ions pumped inward, it is therefore considered electroneutral. The P M C A ( K for C a D  2 +  = 0.91 u.M) consists of four or more gene products: P M C A 1-4 with  'a' and 'b' variants each composed of 10 membrane spanning helices. According to Fig. 3, the sequence homology of the P M C A highly resembles that of the sarco-endo-plasmic reticulum C a  2+  ATPase (SERCA), but with respect to levels of expression the P M C A  represents a higher percentage of membrane protein content. The P M C A lb isoform (M.W. « 140 kD) is the most common in SM and has 10 times the affinity for Ca /Calmodulin than P M C A la. Both 'a' and 'b' variants of P M C A 1 are expressed in 2+  rabbit V S M . M g  2 +  is required to catalyze the pump, while C a  2+  binds to several sites on  the transmembrane domains (Marin, J. et al., 1999). The C-terminal of the P M C A has calmodulin binding sites and substrates for cAMP- and cGMP-dependent protein kinases, Ca /calmodulin-dependent protein kinase, and PKC. Calmodulin-induced pump activation increases the affinity for binding C a  2+  as well as increasing pump velocity  while phosphorylation via various protein kinases increases the maximum C a  2+  uptake 11  Fig. 3. Schematic diagram of the protein structure of the PMCA/SERCA embedded within the plasmalemmal/SR membranes. T h e r e a r e 10 t r a n s m e m b r a n e h e l i c e s c o n n e c t e d by 5 s h o r t p e p t i d e c h a i n s o n t h e S R l u m e n / E C S s i d e (large s e g m e n t s a r e o n the c y t o s o l i c s i d e ) . N o t e the s i m i l a r A T P b i n d i n g site for both p u m p s p l u s t h e p h o s p h o l a m b a n b i n d i n g d o m a i n for S E R C A a n d the c a l m o d u l i n b i n d i n g d o m a i n for the P M C A . T h e longer C-terminus and Nt e r m i n u s of t h e P M C A attributes to the h i g h e r m o l e c u l a r w e i g h t of the P M C A ( M . W . a b o u t 1 3 0 - 1 4 0 k D a ) a s c o m p a r e d to the S E R C A ( M . W . about 110 kDa). C a b i n d s to a n u m b e r of s i t e s within t h e protein s t r u c t u r e a n d is t r a n s p o r t e d v i a the r e a c t i o n of a n a c y l p h o s p h a t e b o n d to a n a s p a r t y l r e s i d u e f o l l o w e d by h y d r o l y s i s . P h L = b i n d i n g site for p h o s p h o l i p i d s . R e p r o d u c e d f r o m M a r i n , J . et a l . , 1 9 9 9 . 2 +  12  rate (Marin, J. et al., 1999). P M C A activity also depends on the presence of acidic phospholipids which increase the affinity of the pump for C a  2+  thus, phospholipases  which cleave phospholipids will inhibit the P M C A (Marin, J. et al., 1999). Phospholamban is a regulatory protein that binds to and inhibits the SERCA; it is highly homologous to the C-terminal site of the P M C A , eventhough it does not bind there (Katz, A . M . , 1997). Carboxyeosin supposedly blocks the P M C A but it was not proven to be very specific. L a  3 +  and vanadate are non-selective blockers of the P M C A that bind to its  cytosolic face. Two potentially useful, novel P M C A blocking compounds, C10bisphenol and C12-bis resorcinol (care of Dr. Colin C. Duke of the The University of Sydney, Australia) were tested in our laboratory but were found to have chromatic properties which interfered with the fluorescent signal of fura-2. Even after further dilution and solubilization this characteristic did not change. One promising lead for P M C A inhibitors is the production of antibodies that are raised against external peptide sequences which can block the extracellular structure of the PMCA. Because the P M C A is dependent on ATP for its primary fuel source, one effective way to block it is to prevent synthesis of ATP by interrupting the PM-associated glycolysis cycle using iodoacetate (IAA) or other metabolic inhibitors simultaneously with mitochondrial uncouplers such as carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) which inhibits mitochondrial Ca  ii) The N a - C a +  The N a - C a +  2+  2+  uptake and, therefore ATP synthesis.  Exchanger exchanger is a high velocity P M C a  2+  anti-porter (its V  m a x  for C a  2+  13  pumping is 5-30 times greater than the PMCA) with a low-affinity for C a  2 +  exposure to [Ca ]; exceeding 1 uM to mediate C a  2+  2+  exchanges 3 N a for 1 C a +  2 +  2 +  that requires  extrusion (KD for Ca =5-12uM). It  and is effective in removing C a  2+  from the cell providing it is  localized to the restricted space between the SR and P M where high C a  2+  gradients  accumulate. This enables the exchanger to transport significant amounts of C a short time thereby greatly altering the SR C a  2 +  2 +  within a  content. Under resting conditions, N a is +  driven into the cell down its electrochemical gradient, releasing energy in the process that is used to transport C a  2+  up its concentration gradient and out of the cell. The N a - C a  exchanger can also move C a  +  2 +  2+  in the inward direction provided that there is an increase in  intracelluar N a or a decrease in extracelluar Na . There are three isoforms ofthe N a +  Ca  2 +  +  +  exchanger, N C X 1-3, each containing: two homologous regions (cci repeats-  extracellular and a.2 repeats-cytosolic) tucked within the nine transmembrane segments that may be involved in ion translocation, a large intracellular loop consisting of the exchanger inhibitor peptide (XIP) region that is involved in the inactivation process (and also where alternative splicing occurs), and a Ca -binding site involved in regulation of 2+  the exchanger (Philipson, K . D . et al., 2002) (Fig. 4). Protein folding transforms transmembrane segments 2, 3, 7, and 8 into the transport pore and the remaining segments form an outer ring (Schulze, D . H . et al., 2003). Bound N a may be transported +  or can cause the exchanger to inactivate by binding to intracellular transport sites. Conversely, non-transported C a , by binding to high-affinity sites on the large 2+  intracellular loop can cause conformational changes in protein folding that stimulate the Na -Ca +  2+  exchanger. The N a and C a +  2 +  binding sites are interactive and designated as two 14  Fig. 4. Structure of the Na -Ca exchanger. +  2+  It is c o m p o s e d of 9 t r a n s m e m b r a n e s e g m e n t s a n d a l a r g e intracellular loop. T h e X I P ( e x c h a n g e inhibitor p e p t i d e ) r e g i o n is i n v o l v e d in i n a c t i v a t i o n ; t h e C a b i n d i n g site is i n v o l v e d in r e g u l a t i o n b y i n t r a c e l l u l a r C a . R e p r o d u c e d f r o m P h i l i p s o n , K . D . et a l . , 2002. 2 +  2 +  15  types of regulation for N C X 1 (NCX 1 is the N a - C a +  2+  exchanger isoform expressed in  cardiac tissue) (Philipson, K.D. et al., 2002). Phosphorylation of the loop by cAMPdependent protein kinase and P K C activates the exchanger while the Na -K -ATPase +  +  provides regulation. PIP2 may putatively remove Na -dependent inactivation by binding +  to XIP peptide since the XIP region is implicated in the Na -dependent inactivation +  process (Philipson, K.D. et al., 2002). In order for the N a - C a +  any current at all, a low intracellular level of regulatory C a  2 +  2+  exchanger to produce  must be present (Nicoll,  +  2+  D.A. et al., 2002). In the past, the main function of the Na -Ca exchanger was thought to be in extruding excess C a  2 +  following contraction when intracellular C a  2 +  levels would  reach 1 uM which is dependent on the respective ionic gradients; the nernst equation shows that equilibrium can be attained when [Ca ] / [Ca ]i = ([Na ] / [Na ]i ) exp (2+  2+  +  e  EF/RT). However, more recently, the N a - C a +  2+  +  3  e  exchanger was found not only to be  activated by cytoplasmic Ca but also to play an important role in the modulation of SR Ca  2 +  content as will be shown in this thesis. Blocking the N a - C a +  achieved by removing external N a along with external C a +  reverse-mode N a - C a +  2+  2 +  2+  exchanger can be  to inhibit both forward- and  exchange or by using selective inhibitors such as amiloride and  its derivatives (e.g. 3',4'-Dichlorobenzamil [DCB]) including KB-R7943 (2-[2-[4-(4nitrobenzyloxy) phenyl]ethyl]isothiourea methanesulfonate) which blocks the reverse•+•  2+  mode Na -Ca exchange, is sensitive to washing, has high activity on the extracellular side of the PM, but it tends to inhibit Na , C a +  2+  and K channels. XIP is a synthetic  peptide which is derived from the cardiac N a - C a +  +  2+  exchanger amino acid sequence; it is  a potent and reversible exchange inhibitor (Li, Z. et a l , 1991) but its drawbacks include 16  interacting with calmodulin which severely affects the activity of the P M C A and being hydrophilic as well as binding to the cytoplasmic surface ofthe exchanger making it necessary to inject into the cell. More recent developments have yielded a potentially selective and potent inhibitor, SEA0400 which has no effect on Na , C a , or K channels +  2+  +  (Tanaka, H . et al., 2002). Another promising drug is anti-sense oligodeoxynucleotide, developed to knockdown the N a - C a +  exchanger in cultured arterial myocytes  2+  (Slodzinski, M . K . and Blaustein, M.P., 1998). Its mode of action is by targeting N C X mRNA, specifically and reversibly. But, overall, N a substitution still remains key to +  blockade of the N a - C a +  exchanger assuming we acknowledge its general blocking  2+  effect on the N a - K pump and N a - H exchange. Common N a substitutes are N M D G +  +  +  +  +  and LiCI. We used LiCI because it has been proven to be an effective replacement ion for N a (van Breemen, C. et al., 1979). In the past, sucrose, choline, and hydrazine were +  also used as N a substitutes but they were found to induce contractions and cause large +  increases in intracellular Ca  concentrations. LiCI, on the other hand, produces minimal  contractions and does not influence intracellular and extracellular C a inhibit Ca  2+  levels but does  efflux (van Breemen, C. et al., 1979). Ganitkevich and Isenberg  (Ganitkevich, V.Ya. and Isenberg, G., 1993) also found that N M G was no better than +  L i as a substitute for Na . +  +  With the advent of more specific exchange blockers there is therapeutic value in cardioprotection, and antiarrhythmic and antihypertensive effects.  17  c) The SR in Ca Transport The SR makes up approximately 1.5 to 7.5 % of a cell's volume and is an extensive membranous system that contains tubules and sheets (or cisternae) in a stacked configuration appearing mostly at the nuclear poles with a rough (ribosomal) or smooth membrane. The peripheral SR contains fenestrated cisternae where the SR and P M come to within 20 nm of each other and occasionally exhibit electron-dense bridging structures (Fig. 1). This portion of the SR, extending below the P M , is referred to as the superficial or peripheral SR while the deep SR extends to the central bulk of the cytoplasm. According to the most current studies, the SR has three basic functions: 1) as a C a 2) as a C a  2 +  source, 3) as a cyclical mechanism in which C a  2 +  2+  sink,  is sequestered, extruded  and re-sequestered. In order to buffer C a , the SR must maintain a static reserve or 2+  steady-state level of total C a  2 +  by balancing C a  2 +  fluxes between the cytoplasm, SR  lumen, and the ECS through the action of the SERCA, the ryanodine receptor (RYR) and IP receptor (IP3R), P M C A , and N a - C a +  2+  3  exchange (I excluded the mitochondria and  nucleus here because it is beyond the scope of my study, but my lab is presently examining their effects on intracellular C a  2 +  fluxes). In tonic V S M , like the aorta, there  is a large deep SR volume of 5-7.5% per cell but a more limited peripheral SR whereas, in phasic V S M such as the portal vein, vas deferens, and ureter there is a smaller SR volume per cell of 1.5-2% but a more abundant peripheral SR (Nixon, G. et al., 1994). At rest, SERCA with contribution from the P M C A and N a - C a +  100,000 fold C a  2 +  2+  exchanger, maintains a  gradient between the lumen and cytoplasm.  18  i) SR Ca  z+  Uptake-SERCA  SERCA ( K for C a D  2 +  = 0.58 uM) like the P M C A is an ATP-dependent pump of the P-  type cation transport ATPases with three genes encoding SERCA 1-3 (SERCA 2 isoforms possess "a" and "b" variants). SM expresses mostly SERCA 2b with negligible SERCA 2a expression, a cardiac-type SR C a  2+  pump (Wu, K-D. and Lytton, J., 1993), while  SERCA 3 is expressed primarily in stomach SM and has a much lower affinity for Ca . SERCA 1 is not expressed in SM. The molecular structure of SERCA (M.W. « 110 kDa) is highly homologous with the P M C A with ten transmembrane spanning helices connected on the luminal side by five short peptides (Fig. 3). There is a binding site for phospholamban and a site for phosphorylation by ATP. SERCA has a high affinity for Ca  2 +  and the most accepted stoichiometry for pumping is Vi mol ATP hydrolyzed per 1  Ca  2 +  pumped in for every K ion and H ion pumped out; it is considered 'electroneutral' +  +  because of a balanced transfer of charge across the SR membrane. The SERCA switches between a high- and low-affinity C a  2 +  binding conformation that is held in a state of  equilibrium until either ligand binding or ATP utilization shifts the balance (Marin, J. et al., 1999). Once within the SR lumen, C a are Ca  2+  binds to calreticulin and calsequestrin which  buffering proteins that are uniformly dispersed within the deep SR but conform  to a 1:5 ratio of distribution in the peripheral SR. SERCA is uniform within the SR membrane and may constitute up to 90 % of the protein content there (Orallo, F., 1996). It lacks a calmodulin-binding site, but contains a site for phospholamban. In its dephosphorylated state phospholamban physically blocks SERCA but when phosphorylated by protein kinases such as PKC, cAMP- and cGMP- dependent protein 19  kinases or Ca /calmodulin-dependent protein kinase (Marin, J. et al., 1999) it becomes z+  inert and can no longer bind to the SERCA. When agonists bind to the P-adrenergic receptor, for example, they utilize a complex protein kinase signaling pathway to inactivate phospholamban. There are numerous blockers for the SERCA, some poorly selective ones like the vanadates and lanthanides (SERCA is less sensitive than P M C A to these), and others, very highly selective such as cyclopiazonic acid (CPA) which was used in our study to selectively and reversibly inhibit the pump and, also thapsigargin which has a higher pump affinity but is irreversible and can inhbibit VOCCs (tBHQ is another alternative). Pump inhibitors do not interfer with phospholamban (Paul, R.J., 1998). The SERCA functions in conjunction with the P M C A and the N a - C a +  exchanger to maintain cytoplasmic C a  2+  2+  at specifically low levels as is the case when S M  relaxation or the modulation of steady-state [Ca ]i is required. 2+  ii) SR C a  2 +  Release-IP^ and R Y R  9+  SR Ca  store depletion can be achieved pharmacologically by inhibiting SERCA or  physiologically and pharmacologically by activating SR C a  2 +  release channels, namely  the IP3R and RYR. There is ample evidence to suggest that IP3RS and RYRs co-exist in all the regions of the SR albeit in different ratios or as two separate yet overlapping IPssensitive and ryanodine-sensitive stores (Missiaen, L . et al., 1992). Invariably, V S M exhibits an asymmetrical distribution in which IP3RS reside in the deep SR and RYRs are more superficial (Orallo, F., 1996). Other types of SR C a NAADP-activated Ca  2+  release channels such as the  release channels may also exist in V S M although their role in 20  Ca  regulation is not clear. There are several ways to stimulate SR Ca  release, but  physiological stimulation involves second messengers to convert agonist binding into an actual signal involving stimulation of a receptor molecule (ROC) that is coupled to PLC and subsequent activation of a G-protein that triggers further cascading causing PLC activation. Then, the P M phospholipid phosphatidyl inositol-4,5-bisphosphate (PIP2) is cleaved into IP3 and D A G . IP3 diffuses into the cytoplasm where it binds to the IP R and 3  induces C a  2 +  release (IICR). The rapid (on the order of seconds) increase in [Ca ] to 1-2 2+  uM near the SR membrane in turn may induce further C a R Y R (CICR). Thus, the SR C a activated by C a  2 +  2+  release via activation by the  release mechanism involves CICR and IICR which is  and IP3, respectively. The positive feedback by C a  2 +  cause a rapid release of C a  2 +  2 +  can potentially  from the SR, but, depending on SM type, C a  2 +  release can  also act as a negative feedback mechanism to inhibit CICR. D A G does not directly 2"f"  activate SR Ca  release but will activate PKC by binding to the regulatory domain of  PKC. Once activated, PKC may phosphorylate voltage-dependent C a P M leading to more channel openings and C a  2+  2 +  channels on the  influx. It will also phosphorylate the  myofilament regulatory proteins caldesmon and calponin to enhance myofilament C a  2+  sensitivity.  1) IP Receptor 3  Three genes encode IP R: IP R 1-3. The IP3 receptor channel is a pore-forming 3  3  homotetramer (i.e. 4 subunits [M.W. « 313 kDa for each subunit] each containing a  21  cytosolic domain and two a-helical transmembrane segments) with several C a binding 2 +  sites, an IP3 binding domain, and a central aqueous channel for the passage of C a  2 +  from  the SR lumen. The R Y R and IP3R are, by amino acid sequence, highly homologous and both form a C a  2 +  release channel from four identical or different subunits (Ogawa, Y . et  al., 2000). In order to release C a , IP3 must bind to the cytosolic surface of the IP3R. 2+  The release is considered biphasic because it is composed of an initial quick and large release of C a  2+  followed by a slow release component and then channel closure. The  shift from fast to slow release may arise from either spontaneous inactivation or conformational change of the receptor or regulation by a change in C a amounts of C a  2 +  2 +  store levels (low  can induce a partially open state) (Marin, J. et al., 1999). Cytosolic C a  2 +  can also regulate the IP3R in the following situations: increasing the [Ca ]i to as high as 2+  300 nM sensitizes IP3 to C a  2 +  release, whereas a [Ca ]; greater than 300nM has the 2+  opposite effect (lino, M . , 1990). Sensitization of the IP3R to C a  2+  can change the  threshold for channel activation and may cause the IP3R to inactivate while maintaining R Y R activation, however this depends on the spatial location of the two receptors on the SR membrane. In addition to activation of the IP3R by IP3, protein kinases such as PKC and cAMP-dependent protein kinase can activate the receptor through phosphorylation; ATP is a receptor modulator (Marin, J. et al., 1999). IP3R 1 and 2 have a single-channel Ca  2 +  conductance of about 70 pS and IP3R 3 has a C a  2 +  conductance of about 88 pS.  Despite there being few blockers of the IP R channel, there is an abundance of 3  antagonists of the IP3 receptor including: xestospongin C, caffeine, heparin (nonmembrane permeable), decavanadate, and PIP2 for IP3R 1; heparin and decavanadate for 22  IP3R 2; and IP3 and cytosolic C a  2 +  for IP3R 3 (Receptor and ion channel nomenclature,  1998). 2-aminoethyl diphenylborinate (2-APB) which is partially reversible has had useful application in the blockade of IP3R channels (especially type 1IP3R) without blocking IP3 binding, however it tends to block the activation of some P M channels such as the V O C C , ROC and SOC, the latter, at concentrations around 50 uM; at concentrations of 200 uM and above, 2-APB tends to block the SERCA 2b isoform (Bilmen, J.G., et al., 2002). In a B-cell line, low doses of 2-APB can have a stimulatory effect on the SOC but this requires that it be coupled in some way to the IP3R, since the effect is not observed if the coupling is disrupted (Ma, H-T. et al., 2002).  2) Ryanodine Receptor RYR receptor homology resembles the IP R and as such is composed of a 3  homotetramer (monomer M.W. is about 565 kDa) and expressed as three receptor 9+  subtypes: R Y R 1-3, each the product of a different gene (Ca  single channel  conductance is 90 pS, 90 pS and 140 pS, respectively) (Receptor and ion channel nomenclature, 1998). The R Y R forms a "four-leaf clover" or quatrefoil surrounding a central C a  2+  pore (Marin, J. et al., 1999). Compared to the IP3R, the R Y R has a greater  role in cardiac and skeletal muscle than it does in SM, but, typically the R Y R is responsible for a large release of activator C a  2 +  and different isoforms can co-express in  the same cells. Under physiological conditions, SR C a , a low cytosolic C a , and ATP 2+  2+  at intracellular concentrations of 3 to 9mM can activate channel openings (Ogawa, Y . et 23  al., 2000), but when the cytosolic [Ca ] rises above 100 u M (RYR1) or 1 mM (RYR2, z+  RYR3), channel openings are inhibited, this dual effect being attributed to the presence of high-affinity activating C a  2+  binding sites (A-sites) and low-affinity inactivating C a 2"t-  sites (I-sites) on the receptor molecule. Intracellular Mg  2+  2"r"  (0.5mM) is a Ca  concentration-dependent R Y R blocker by virtue of its competitive antagonism to C a  2 +  at  2+  the A-binding site and agonism with Ca  at the I-site. (Ogawa, Y . et al., 2000).  Pharmacologically, RYRs can be activated by the plant alkaloid, ryanodine, at low concentrations, which locks the channel into an open state or by C A F (RYR 1-3), neomycin (RYR2), or suramin (RYR1, RYR2). Calsequestrin is functionally and structurally associated to the R Y R and may facilitate channel openings (Marin, J. et al., 1999). There is even supporting evidence to suggest that in some cells, endogenous cyclic ADP ribose (cADPR) in connection with calmodulin can increase the open probability of the R Y R (Marin, J. et al., 1999). R Y R channel blockers include: ryanodine, at high doses (>100 p:M), ruthenium red (a polycationic compound), and procaine (RYR1, RYR2).  C . Ca Signaling 2+  a) Excitation-Contraction Coupling: Basic Ca  Signaling Function  i) Contraction V S M contraction is the result of several excitatory processes that together act to 24  enhance Ca release from internal Ca  stores and Ca  influx across the PM; the Ca  spreads in a wave-like fashion over the cytoplasm to rapidly increase [Ca ]i and evoke a 2+  contractile response. Calmodulin regulates contractility by complexing with 4 C a  2+  ions  to undergo a conformational change that causes it to bind to and activate myosin light chain kinase (MLCK), followed by M L C K phosphorylation of the regulatory myosin light chain (i.e. the 20 kD light chain) of myosin II, triggering cross-bridge cycling and force development, as shown in Fig. 5. The degree of myosin light chain  (MLC20)  phosphorylation determines the extent to which SM contracts but M L C 2 0 phosphorylation does not always parallel the [Ca ]i and can occur even in the absence of C a 2+  et al., 2001). For equivalent [Ca ]i, 2+  M L C 2 0  2+  (Fukata, Y .  phosphorylation or the development of force  is greater during agonist stimulation than in cell depolarization, a phenomenon commonly referred to as C a  2+  sensitization (Brozovich, F.V., 2003). Agonist-induced activation of  P K C by the binding of D A G to its regulatory domain can increase the sensitivity of myofilaments to C a  2 +  through inhibition of M L C phosphatase (Fukata, Y . et al., 2001).  2+  Ca  sensitization also occurs through direct activation of Rho kinase via arachidonic acid  release or by way of the agonist-induced Rho/Rho-kinase pathway. Rho is dependent on Ca  for its activation and is thought to be able to sustain force maintainance through a  signal cascade that originates in the P M and is coupled to G-proteins such as by agonistinduced stimulation (Brozovich, F.V., 2003). In greater detail, Rho is a GTP-binding GTPase protein that acts as a switch, cycling between an inactive GDP-bound and an active GTP-bound form. It is targeted to the cell membrane by Gan/n-associated RhoGEF (Rho-specific guanine nucleotide exchange factor) which catalyzes the exchange of 25  Ca'  Agonist  Fig. 5. Excitation-contraction coupling vs. relaxation agonist-induced Ca * sensitization.  and  2  In SM an increase in cytoplasmic C a enhances binding to calmodulin (CaM) which enables myosin light-chain (MLC o) to be phosphorylated by myosin light-chain kinase (MLCK); this promotes the interaction of actin and myosin and causes the muscle to contract. A fall in [ C a ] i relaxes the SM through myosin light-chain phosphatase (MLCP or MP)-induced MLC o dephosphorylation. Modulation of myosin phosphorylation can be achieved through the process of C a sensitization. In such a case, agonists e.g. PE and 5-HT increase intracellular C a leading to rho activation. In turn, rho inhibits MLCP, preventing myosin dephosphorylation and thus efficiently maintaining contractility. Reproduced from Fukata, Y. et al., 2001. 2+  2  2+  2  2+  2+  26  Rho-GDP for Rho-GTP (Wettschureck, N., and Offermanns, S., 2002). Activated Rho (or Rho-GTP) binds to Rho-kinase, activating it in the process and allowing it to bind to and phosphorylate the myosin-binding subunit of the M L C phosphatase enzyme which, in the phosphorylated state, is inhibited, and therefore cannot dephosphorylate  MLC20  (Fukata, Y . et al., 2001; Wettschureck, N . and Offermanns, S., 2002). It is also possible that changes in calmodulin concentrations affect the C a  2 +  sensitivity of M L C  phosphorylation (Karaki, H. et al., 1997) Actin-binding proteins such as caldesmon, calponin and M L C K may also be involved in the agonist-induced increase in C a  2+  sensitivity (Karaki, H . et al., 1997).  ii) Vasodilatation The calmodulin-MLCK complex dissociates when the [Ca ]i falls below 200 nM, 2+  generating the inactive form of M L C K and dephosphorylated myosin (via cleavage of phosphate by myosin light chain phosphatase (MLCP)) (Fig. 5). Muscle relaxation is caused by the simple removal of the stimulus or by the action of vasodilatory agents like nitric oxide (NO) or by direct interference with the mechanisms involved in V S M contraction which include: V O C C blockade with C a  2 +  antagonists (electromechanical  uncoupling), cell hyperpolarization by K channel agonists, and R O C blockade by +  antagonists (pharmaco- and electro-mechanical uncoupling). Vasodilators such as sodium nitroprusside, NO, atrial natriuretic factor, and prostacyclin as well as inhibitors of phosphodiesterase can individually elevate cAMP and cGMP to mediate smooth muscle relaxation through cAMP- and cGMP- dependent protein kinases, respectively 27  (Schulz, R. and Triggle, C.R., 1994; Sonnenburg, W.K. and Beavo, J.A., 1994). P K A and P K G also cause relaxation by 1) phosphorylating K c channels to induce membrane a  hyperpolarization (Gollasch, M . et al., 2000), 2) phosphorylating the SERCA inhibitor, phospholamban, to activate SR Ca  uptake (Raeymaekers, L . et al., 1988), 3) stimulating  the P M C A (Furukawa, K.I. et al., 1988) and N a - C a +  1991) to activate C a  2 +  2+  exchanger (Furukawa, K.I. et al.,  extrusion, 4) phosphorylating the IP3R to inhibit SR C a  2+  release  and IP3 formation (Komalavilas, P.K. and Lincoln, T . M . , 1994), and 5) phosphorylating VOCCs to inhibit I  Ca  (Lorenz, J.N. et al., 1994). cAMP and cGMP can also induce  relaxation by reducing the C a  2 +  sensitivity ofthe actin-myosin complex by  phosphorylating and inhibiting M L C K (cAMP) or phosphorylating and inhibiting Rho (cGMP). As well, calmodulin can accelerate relaxation by stimulating the P M C A and +  24~  Na -Ca exchanger (Orallo, F., 1996). The contraction-relaxation cycle is highly regulated and gives rise to a constantly changing basal blood vessel tone.  b) Ca Signaling between the SR and PM 2+  i) The Superficial Buffer Barrier The superficial buffer barrier (SBB) theory was first postulated by my supervisor, Dr. C. van Breemen in the late 1970's (van Breemen, C , 1977; van Breemen, C. et al., 1979). The SR was thought to retain C a intracellular C a  2 +  2+  but little was known of its role in regulating  gradients and in limiting diffusion of C a  2 +  into the bulk ofthe  myoplasm until our lab discovered that the superficial SR acts as a barrier to C a by taking up a fraction of this C a  2 +  2 +  entry  before it can reach the myofilaments in the deeper 28  myoplasm. In order to maintain this buffering function the superficial SR would require a pathway to the ECS to unload excess amounts of C a . Evidence based on morphology, 2+  has shown that parts of the P M are physically coupled to the superficial SR and that these junctions contain N a - C a +  2+  exchangers (Moore, E.D.W. et al., 1993). The SBB predicts a  system of sequential coupling of SR C a  2+  extruding mechanisms to transport Ca  release channels to plasmalemmal C a  2+  from the junctional space underneath the P M to  the extracellular space (for further reading, refer to: van Breemen, C. et al., 1995). The nomenclature given for such transfer from one carrier or channel to another is "linked Ca  2 +  transport" (Poburko, D. et al., 2004). The purpose of my thesis was to determine the  mechanical underpinnings of SR-mediated C a Na -Ca +  2+  exchange in unloading C a  determine whether N a - C a +  2+  2 +  2 +  extrusion which I propose hinges on the  from the SR. I have conducted several tests to  exchange is coupled in-series or is additive to the SR.  ii) Ca -Induced Relaxation A major K channel in SM is the high conductance (100-250 pS) Ca -sensitive +  2+  K channel or B K c channel. Less abundant are the IKc , an intermediate conductance +  a  a  channel (18-50 pS), and SKc , a small conductance channel (6-14 pS) (Receptor and ion a  channel nomenclature, 1998). B K c channels are constructed of four pore-forming aa  subunits (each subunit consisting often transmembrane segments) plus additional accessory P-subunits which can alter C a  2 +  sensitivity (Receptor and ion channel  nomenclature, 1998) (Fig. 6); for instance, p-subunit knock-out mice exhibit significantly increased blood pressure and cardiac hypertrophy (Brenner, R. et al., 2000). The B K  C a  29  BKg, p-subunit  BK  C a  a-subunit  rr"zr COO"  s S 7_  s 9  L o w  S 10 X  Fig. 6. Schematic diagram illustrating BK channel topology. Ca  The  BK  C a  c h a n n e l is c o n s t r u c t e d of a t e t r a m e r of p o r e - f o r m i n g ot-  s u b u n i t s with a n a d d i t i o n a l m e m b r a n e - s p a n n i n g p - s u b u n i t w h i c h m a y modify C a  2 +  sensitivity.  B e t w e e n s e g m e n t s 9 a n d 10 of the a - s u b u n i t  t h e r e is a s i t e i n v o l v e d in C a  2 +  regulation.  The B K  C a  c h a n n e l is  e n c o d e d b y a n hslo g e n e . R e p r o d u c e d f r o m V e r g a r a , C . et a l . , 1 9 9 8 .  30  channel is voltage-sensitive as well as being activated by Ca  at a minimum  concentration of 3-10 uM and by vasodilators, arachidonic acid epoxides, and carbon monoxide. cAMP- and cGMP-dependent protein kinases modulate the B K c channel a  while Iberiotoxin, Charybdotoxin, noxiustoxin, penitrem-A, or the tetraethylammonium ion (TEA) represent relatively selective B K c channel blockers except for T E A which is a  known to block a variety of K channels. Unlike the B K c channel, IKc channels may +  a  a  inactivate with depolarization, while, SKc channels, particularly SKc 3, an endothelial a  a  isoform, may not be expressed in SM (Burnham, M.P. et al., 2002). Voltage-sensitive K channels (inward and outward rectifiers) which are not dependent on C a are also present in V S M but are not part of my hypothesized C a  Ca  2 +  2+  2 +  +  for activation  extrusion model.  Sparks and STOCs  Spontaneous and highly localized release of C a  2 +  from the peripheral SR caused by the  random opening and closing events of clustered RYRs ("Ca sparks") or IP3RS ("Ca 2+  puffs") (Boittin, F.X. et al., 2000; Bootman, M . et al., 1997) can propagate into C a  2+  2 +  waves or stimulate B K c currents on the P M giving rise to spontaneous transient-outward a  currents (STOCS) first hypothesized by Benham and Bolton (Benham, C D . and Bolton, T.B., 1986) in visceral and vascular SM. In cerebral arteries and stomach SM, STOCs have a hyperpolarizing effect on the entire cell, closing voltage-gated C a thereby reducing Ca  2 +  channels,  influx and contraction and they are specifically blocked by  Iberiotoxin (Nelson, M.T. et al., 1995; ZhuGe, R. et al., 1999). Bursts or sparks of C a  2 +  31  derived from clusters of RYRs localized within the SR, as measured by ultra-fast digital imaging microscopy using fluo-3, provide the stimulus for B K c channels adjacent to the a  peripheral SR (ZhuGe, R. et al., 1999). If SR C a and C a  2 +  2+  release is inhibited, both the STOCs  sparks will diminish causing depolarization, C a  global [Ca ]i (ZhuGe, R. et al., 1999). C a 2+  2 +  2+  influx, and an increase in  sparks have been noted in the majority of  SM including arteries, the portal vein, airway, ileum and esophagus as well as in cardiac and skeletal muscle. A single C a Ca  2 +  2+  spark does not contribute significantly to cytosolic  and incidentally does not significantly activate the contractile apparatus but it does  contribute to local stimulation of B K c channels which in turn would lead to a global a  reduction in cytosolic C a  2 +  through inactivation of V O C C , however this depends on the  94-  location of Ca  release relative to K c channels. In cerebral arteries, Perez et al. (Perez, a  G.J. et al., 1999) found that approximately 96% of detectable C a with a STOC, strongly suggesting that C a  2 +  2+  sparks are associated  sparks cause STOCs and that they are 94-  functionally coupled (Nelson, M.T. et al., 1995). In SM, Ca spark amplitudes rise to 94-  average heights of between 30 and 300 nM as sensed by the Ca  indicator dye fluo-3 or 94-  10-30 p.M using the amplitude of B K c currents to sense a local rise in Ca and have a a  duration of about 100ms (Wellman, G.C. and Nelson, M.T., 2003). In the IVC, these Ca  2+  sparks can superimpose into propagating C a  Ca  2 +  oscillations for tightly regulated force development. This in contrast to cerebral  2+  waves which can produce efficient  94-  resistence arteries where Ca waves induce dilation rather than contraction due to activation of B K c currents. This is indicative of a symmetrical arrangement of RYRs to a  K channels within the PM-SR junction which is different from the vena cava. It is also32 +  important to note that during wave propogation in the cerebral arteries IP3R Ca  release  will secondarily activate RYRs rather than exclusively mediate repetitive SR refilling and emptying. In addition to K channels , Ca -sensitive Cl" channels are also present in SM and like +  2+  the B K c a channels are activated by intracellular C a , however in contrast to the B K c 2+  a  channels which when activated by the release of SR C a , hyperpolarize the cell 2+  membrane, they evoke cell depolarization when activated by C a . 2+  iii) The Nucleus and Mitochondria The nuclear envelope is composed of a double membrane containing a perinuclear luminal space that is continuous with the cisternae of the SR and of the golgi complex all forming part of a single membrane system. Within the nuclear envelope are numerous nuclear pores providing direct contact between the cytoplasm and nucleoplasm to enable free diffusion of C a  2+  and other ions. In ferret portal vein, significant amounts of C a  2+  have been noted in the nucleus of resting V S M (Papageorgiou, P. and Morgan, K . G . , 1990) and following C a  2 +  mobilization, nuclear C a  2 +  levels have been reported to fall.  However, Himpens et al. (Himpens, B . et al., 1992;) have reported just the opposite, showing that at rest, the nucleus contains less Ca than the cytoplasm. These results, 2+  though conflicting, indicate that C a  2+  is heterogeneously distributed between the nuclear  and cytosolic compartments possibly due to different rates of nuclear Ca ATPase2+  mediated C a  2 +  uptake (Nicotera, P. et al., 1989) and release of nuclear C a  2 +  by IP3RS  within the nuclear envelope. 33  The mitochondria (MT) are different from the nuclear envelope in that their lumen is not confluent with the SR. They are mainly synthesizers of ATP and will also store large amounts of C a . However, M T overloaded with C a 2+  2 +  may cause pathological conditions  such as overproduction of oxidative radicals which may precede programmed cell death or apoptosis. But recent examination of mitochondrial interactions has revealed a regulatory role on bi-directional C a  2+  transport between the SR and mitochondrial  membranes, utilizing cytoplasmic microdomains much like the PM-SR junction. This requires having the SR IP Rs (Rizzuto, R. et al., 1993), RYRs (Montero, M . et al., 2000), 3  M T ion channels and a low affinity M T C a  2+  uniporter ( K for C a D  juxtaposed. With the help of a mitochondrial N a - C a +  Ca  2 +  2+  uptake by the M T can be reversed until the SR C a  2+  = 10 uM)  exchanger and N a - H exchanger, +  2+  +  store is refilled (Arnaudeau, S.  et al., 2001). There is strong evidence in support of MT-SR couplings based on the fact that significant mitochondrial C a or enhance the rate of SR C a  2 +  2 +  uptake and subsequent C a  2 +  release can either retard  release (Poburko, D. et al., 2004), and moreover, C a  2+  hotspots have been observed between the SR and M T where the [Ca ] reaches levels 10 2+  times higher than the bulk cytoplasm (Pozzan, T. et al., 2000). The mitochondria therefore play an important role in shaping the C a phase through rapid C a  2 +  2 +  signal by modifying the recovery  uptake and delayed release toward the SR (Berridge, M.J.,  2002). Mitochondria situated more superficially may take up C a thereby competing with the SR for C a  2+  2+  directly from the P M  influx, providing evidence for a PM-SR-MT  junction (Poburko, D. et al., 2004). Functional coupling is corroborated by structural and morphological studies in which the SR, PM, and mitochondria, in certain regions ofthe 34  cell, are co-localized and thus form close contacts of lOOnm or less (Szado, T. et al., 2003). If mitochondria largely take-up Ca RYRs release C a  2 +  from the microdomain where IP3R.S and  then this can increase or decrease channel release activity because of  the regulation by C a  2 +  on the cytosolic face of the receptor resulting in altered C a  2 +  release kinetics, or in the event of P M - M T junctions, modulation in V O C C , SOC, and Na -Ca exchanger activity. Moreover, mitochondria can become clustered and act as an immobile buffer, attenuating or delaying the propogation of signals from one area of a cell to another (Pozzan, T. et al., 2000). Yet, an abnormal accumulation of C a  2+  in the  mitochondrial matrix may induce formation of a mitochondrial permeability transition pore and the destruction of the mitochondrial membrane potential along with a corresponding activation of a caspase cascade which would invariably lead to the onset of apoptosis (Berridge, M.J., 2002).  35  Chapter II. EXPERIMENTAL DESIGN, METHODS AND RESERVATIONS  A. Experimental Design  a) Aims of This Thesis The purpose of my thesis was to demonstrate the functional linkage between the +  2+  superficial SR and the Na -Ca exchanger in the PM. Here I document the supporting evidence for such a mechanism.  The schematic in Fig. 7 is intended to illustrate the logic of the experiments performed in this thesis. 1. The classical view of excitation-contraction coupling in S M involved two independent functions for P M and SR (A). 2. In 1977, it was postulated that the P M and superficial SR interacted directly without the involvement of the bulk cytoplasm. This superficial buffer barrier theory proposed that the superficial SR sequestered part of C a  2+  influx before it equilibriated with the bulk  cytoplasm and that this mechanism is important in regulating [Ca ]j (B). 2+  3. To maintain the SBB function Dr. van Breemen postulated that the SR can discharge its C a  2 +  to the ECS again without mixing with the bulk cytoplasm as illustrated in (C). In  this thesis I present [Ca ]j experiments which establish that this SR discharge takes place 2+  36  Fig. 7. Proposed model for SR-mediated C a  A  2 +  extrusion.  P M  Activation Model  Ca Efflux P a t h w a y y R O C / S Q C y V O C C | PMCA \ \ Ca * Ca",2* z+  2  \  activation=influx+release  B  PM  C l a s s i c a l V i e w for Relaxation  NCX  New Model for Ca * 2  Ca  Relaxation  2 +  relaxation=uptake+extrusion  37  via SR C a  2 +  release channels and the N a - C a +  2+  exchanger. I then went on to show that  this coupling between superficial SR and N a - C a +  SERCA to C a  2+  2+  exchange leads to a contribution by  extrusion.  From this model I have devised two specific aims:  Aim #1: To determine whether the SR actually mediates C a Ca  2 +  exchange mechanism or if there are other C a  2+  2 +  extrusion through a Na +  transporters such as the P M C A that  contribute to this. The approaches we used are: Using fura-2 A M to measure [Ca ]i in the IVC we observed the rate of [Ca ]i decline 2+  prior to and following N a - C a +  2+  2+  exchanger and/or SERCA and P M C A blockade in order  to determine the relative contribution of each to total C a  2 +  removal and if their function  overlaps or is complementary.  Aim #2: If the N a - C a +  2+  exchanger is in fact functionally coupled to the superficial SR  then do alterations in its C a  2 +  translocation function somehow modulate intra-luminal  Ca ? We examined exchanger-SR interactions according to these methods: 2+  Simultaneous measurements of [Ca ]i and SR C a 2+  2+  content were performed, again using  the fura-2 A M method, to determine any disparity in Ca Na -Ca +  2+  accumulation due to changes in  exchanger and/or SERCA function.  b) Specific Protocols 94-  There are several ways to observe Ca  extrusion from SM. Our lab was specially  equipped for the spectrofluorometrical method of monitoring changes in [Ca ]i using 38 2+  fura-2 A M as opposed to more complicated techniques such as radiotracing. Altered Ca  2 +  extrusion rates were estimated by comparing the rate of decay of [Ca ]i (using a bi2+  exponential fit) in cells containing maximal [Ca ]j after having blockaded the SERCA 2+  and the Na -Ca exchanger, individually or concomitantly, or including all three, the SERCA, N a - C a +  2+  exchange and the P M C A together (please refer to  RESULTS section).  I first recorded basal [Ca ]j and then when the baseline stabilized I switched to a solution 2+  of 80mM K physiological saline solution (PSS) containing 20 uM PE to induce both +  Ca  2 +  influx and release which caused an initial rapid rise in [Ca ]i followed immediately 2+  thereafter by a maintained plateau phase. At this point, the stimulus was removed and a solution containing Ca -free PSS and 10 u.M phentolamine was added to abolish C a 2+  influx and therefore repolarize the cell and as well, to antagonize PE-induced C a from the SR. When these conditions were attained the C a  2 +  2 +  2 +  release  decay curve was clearly  defined and thus I was able to properly and mathematically calculate the C a rate. High K and PE are not only needed to load the cell with C a +  2+  2 +  extrusion  but they also prime  the SR by activating its buffer barrier function and are good determinants of cell viability as well as serving as internal controls. Each time one or more of the C a to be blocked, the 0 C a  2+  +  2 +  transporters is  PSS+phentolamine washout solution is modified; we prepared  four different solutions in total: 0 N a 0 C a CPA+0 C a  2 +  2+  PSS to block the N a - C a +  PSS to block SERCA, CPA+0 N a 0 C a +  exchanger, or FCCP+IAA+CPA+0 N a 0 C a +  2+  2+  2+  exchanger,  PSS to block SERCA and the  PSS to block the P M C A , SERCA, and the  exchanger, for periods of up to 600 seconds to determine the rate of decline in [Ca ]i 2+  corresponding to changes in C a  2+  transport activity. Although FCCP was used to block  39  the M T C a  2 +  uniporter, the role ofthe mitochondria in C a  2 +  uptake from the cytoplasm  was omitted from this study simply because at the time little was known about the C a  2+  buffering action of the mitochondria (see Poburko, D. et al., 2004 for an update).  After having conducted experiments to test the various ways of blocking C a  2+  removal  from the cytoplasm I then proceeded to examine the effect of blockade on SR C a  2 +  content to better understand the interaction of the superficial SR to the P M in which extrusion by the N a - C a +  look at C a  2+  2+  exchanger is most likely thought to be involved. Therefore, to  accumulation by the SR, it is essential to measure its C a  content which is  2 +  accomplished by using caffeine (CAF) to induce a release response from the SR; I then took the amplitude of this [Ca ]; transient to reflect the SR C a 2+  amplitude is associated with a greater release of C a  2 +  2 +  content so that a higher  from the SR. C A F is a xanthine  compound that binds to the R Y R (i.e. activates CICR) and depletes the SM SR of all its Ca  2 +  if exposure is at least 200 seconds, thus producing large [Ca ]j transients in just one 2+  dose. In the first set of C A F experiments the goal was to determine if the SR C a content can actually be modulated by either the SERCA and/or the N a - C a +  2+  2 +  exchanger.  The following protocol was applied: 1) attainment of a stable baseline, 2) administration of a bolus of 25mM C A F in 0 C a adding C a  2+  2+  PSS, 3) re-adjustment ofthe baseline upward by  back to compensate for the under shoot effect, 4) stabilization of the baseline  before applying stimulation to the tissue to begin C a PSS and PE stimulus, 6) replacement with 0 C a or with 0 N a 0 C a +  2 +  PSS, CPA+0 C a  2+  2 +  2 +  loading, 5) application of high K  +  PSS+phentolamine to serve as a control  PSS or CPA+0 N a 0 C a +  2+  PSS for periods of up 40  to 600 seconds or until a new baseline was established, and finally, 7) administration of a second bolus of C A F in 0 C a  2 +  PSS. The peak amplitude ofthe 1 and 2 st  nd  CAF  responses were then ratioed for quantitative analysis.  In the second set of C A F experiments, my goal was to focus more on measuring the kinetics underlying the decay in SR C a  2+  content observed in the first set of C A F  experiments. This was done by blocking C a and applying the C a  2+  2 +  influx by exposure to Ca -free solutions 2+  decay model to cells in which the N a - C a +  2+  exchanger and/or  SERCA activity was blocked (see Fig. 8 for exact drug/solution combination). This involved stimulating the S M with 80 mM K P S S then washing it out with NPSS +  followed by C a  2 +  transport blockade for 0, 150, 300 or 600 seconds after which C A F in  0 C a T S S (25mM) was then administered. I took the peak amplitude of the CAF-induced z  response following blockade and divided into the height ofthe initial high K response. +  The C A F peaks were compared to one another by their relative C A F to High K ratios. +  As shown in Fig. 8 this C A F protocol is 2-dimensional in that there are at least two variables, one being the type of solution (of which there are four as mentioned above) and the other being the time variable (0-600 s). In keeping with the proper statistical procedure, one variable is kept constant while the other is changed in a way that the combination of events is randomized allowing us to make better use of the experiments while keeping costs down and eliminating possible interference due to the effect of decay in tissue responsiveness over time.  41  Os  Time(s) 70 Protocol O NPSS © • © I © •  160 80K • • • +  300 ' PSS NPSS • I . •  0-600  >300  0Ca 0Na ,0Ca CPA+0Ca CPA+0Na\0Ca 2 +  +  CAF+0Ca • +0Na ,0Ca I +CPA+0Ca • +CPA+0Na ,0Ca 2 +  2 +  +  2+  2 +  2+  2 +  Fig. 8. Sample trace and corresponding experiments.  +  :  protocol for CAF  E x p o s u r e to c a f f e i n e f o l l o w i n g 0, 1 5 0 , 3 0 0 , a n d 6 0 0 s in C a - f r e e s o l u t i o n with e a c h t i m e point b e i n g r e p e a t e d at l e a s t f o u r t i m e s , a n d e a c h t i m e c o u r s e g r o u p ( 0 - 6 0 0 s) s u b j e c t e d to t r e a t m e n t by e i t h e r 0 Ca P S S (control), 0 N a \ 0 C a PSS, C P A + 0 Ca P S S , or C P A + 0 Na\ 0 Ca P S S [ d e n o t e d by : P r o t o c o l © , © , © , © , r e s p e c t i v e l y ] , after initial s t i m u l a t i o n by 8 0 K P S S , a n d w a s h o u t with N P S S . A fictional s a m p l e t r a c e r e p r e s e n t i n g the e f f e c t s of p e r f u s i o n with t h e s e d r u g - s o l u t i o n c o m b i n a t i o n s is p i c t u r e d a b o v e the c o r r e s p o n d i n g protocol. 2 +  2 +  2 +  2 +  2 +  +  42  It is important to note that C A F has effects other than stimulating the opening of RYRs which includes inhibiting UVinduced C a  2+  release channels (Missiaen, L. et al.,  1994) and IP3 formation (Toescu, E.C. et al., 1992), inhibiting L-type Ca  channels, and  voltage-sensitive N a channels and voltage- and ATP-sensitive K channels plus +  +  inhibition of glucose uptake, and adenosine and G A B A receptor blockade (Shi, D. et al., 2003). Another known effect of C A F is that it can produce [Ca ]i undershoots following 2+  evoked [Ca ]; transients, particularly in Ca -free solutions, although this was reported to have no quenching effect on fura-2 (Rembold, C M . et al., 1995).  B. Methods  a) Animal Care New Zealand White rabbits were fed with Land O' Lakes chow and given water ad libitum; the temperature was kept at a constant, 20°C, with a 0700-1900 hr light schedule in effect. They were cared for in strict accordance with the guidelines published by the Canadian Council on Animal Care.  b) Tissue Preparation Female New Zealand White rabbits (1.5-2.5kg) were killed by a 95% C 0 / 5% 0 2  2  asphyxiation mixture then exsanguinated. The IVC was promptly excised and visible  43  connective and adipose tissues were removed in a petri dish containing-normal PSS (NPSS), pH 7.4, at room temperature (22-25°C). The tissue was opened longitudinally, cut into a 25 by 7mm rectangular sheet and the endothelium was removed using wetted filter paper. A rectangular plastic frame with a channel of silicon elastomer near the perimeter was used to pin the smooth muscle strip into place.  c) Force/Tension Measurement The procedure for tissue preparation is as stated in the section above except that the tissue was not cut into sheets but, rather, cut into rings approximately 6mm in length. The water jacket and internal bath temperatures were adjusted to 37°C and gassed with 95% O 2 / 5% C O 2 . The organ bath consists of four 25mL capacity chambers. A total of four rings, one in each chamber were suspended horizontally between two stainless steel hooks, the bottom one being attached to a stationary support and the top being sutured to a force transducer. Resting tension on the tissue was maintained at 0.5 grams. Equilibriation time was 45 minutes to allow development of a steady tone and reproducible responses. The signals were recorded on a Grass myograph.  d) Measuring the Loss of Endothelium from Intact Smooth Muscle It can be determined whether or not the mechanically denuded SM is completely devoid of endothelium. A n easy test for this is to prepare the tissue by cutting it into rings and suspending the rings with a force transducer to establish their contractility. N A ( l u M or 44  more) is then added to induce a sustained contraction which is followed by an effective dose o f A C h to induce relaxation in tissues having E C s present. E C s respond to A C h by releasing nitric oxide which has a relaxing effect on the V S M surrounding the endothelium and possibly reducing the sensitivity of C a  2 +  to the actin-myosin system in  the underlying S M . According to our results (data not shown), in the denuded tissue rings, there was little or no relaxation with A C h , thus confirming the absence o f endothelium or its lack o f response due to low numbers o f cells or the deleterious effect of cell lysis. Detergents can also be used to denude S M but they can cause damage to the S M wall.  e) Tissue Immobilization Isotonic contractions in the suspended I V C pose a problem because they can significantly affect [Ca ]i measurements. Thus, we tested the compound cytocholasin-D 2+  which is a tissue immobilizer that causes anchoring proteins within the cytoskeletal infrastructure to breakdown. Without proteins like ankyrin, microtubules, and microfilaments to stabilize intracellular structures and without the integrity o f actin and myosin, S M contraction is obliterated. I have shown that pre-treatment with cytocholasin-D decreases successive PE-induced contractile responses in a timedependent fashion compared to the control; however the disadvantages o f cytocholasin-D outweigh its benefits in that it is potentially toxic to S M , is irreversible and interacts with other compounds, and most importantly, does not leave C a  2 +  signaling intact. W i t h  45  improvements I made to the IVC support frame, lateral movement of the tissue was essentially eliminated and cytocholasin-D pre-treatment was therefore not used in the experiments reported on herein.  f) Spectrofluorimeter Set-Up A SPEX Fluorolog spectrofluorimeter (SPEX Industries, Inc., NJ) controlled by DM3000CM software was used to monitor and record the fluorescence from the intact tissue by a process known as "ratioing". A high-intensity xenon lamp (ozone-free) provides light to a beam splitter which directs the light through two monochromators that filter the incoming beam into wavelengths of 340 and 380nm (coinciding with the peak of fluorescence of calcium-bound and -unbound fura-2). A light chopper alternates the two sources via a circular mirror that is segmented. The alternating light is deflected by a series of concave mirrors onto the luminal side of the tissue and the emitted fluorescence calibrated to a wavelength of 505nm by another monochromator is detected by a photomultipliter tube which provides a count ofthe fluorescence in photons per second. For reference, a rhodamine-B light sensor located in the sample compartment monitors the excitation light intensity. To maintain proper illumination, the monochromator slits (both entrance and exit), and sensor supply voltages (for the rhodamine-B sensor and the photomultiplier tube) must be correctly adjusted; typical values are 1.5mm and 480 and 950 volts, respectively. The optimal emission signal level is up to 1 million counts. Light emission from the tissue is controlled by a deflector that can either be set to front46  face mode for whole specimens or right-angle for cell suspensions and cultured cells. Data collection, storage, and output is via an on-line PC computer and printer. The fluorescence intensities, F340 and F380, can be ratioed by mathematical options available on the SPEX software program. This is one virtue of fura-2 in that ratioing eliminates variables which are dependent on intensity.  g) Autofluorescence Measurement Background autofluorescence from naturally fluorescing proteins within the tissue was measured prior to incubation with fura-2. A n acrylic cuvette (4mL capacity) containing the suspended tissue was placed in the sample holder and excited with alternating wavelengths of 340nm and 380nm for at least 20 minutes depending on length of experiment.  h) Fura-2 Loading Immediately following measurement of tissue autofluorescence the preparation was placed in an oxygenated (100% 0 ), 18-22°C, NPSS solution containing 50ug fura-2 A M 2  and 625 u.g pluronic F-127 (to form more soluble micelles of fura-2) dissolved in dimethylsulfoxide (DMSO) to make up a 5\xM concentration of each in a final volume of lOmL. The incubation period was approximately 1.5 hours in the dark.  47  i) [Ca ]i Measurement z+  After loading, the tissue was washed in NPSS then transferred to a cuvette and placed in the sample chamber ofthe spectrofluorimeter. The bathing solutions (± drugs) were changed by a peristalstic pump maintaining a perfusion rate of 0.37mL/second and by vacuuming off the overflow. This would eliminate any artefacts in the fura-2 signal that often occur if the drugs solutions were delivered by quick injection. All experiments were carried out at room temperature (18-22°C).  j) Fura-2 Calibration Calibration tests should be conducted periodically to ensure precise operation ofthe spectrofluorimeter. The most important are the water rhamen and fura-2 free salt in increasing C a Ca  2 +  concentrations tests. The latter involves an excitation scan of graduated  2+  solutions in double distilled water, containing fura-2 pentapotassium salt (5uM).  The spectral range ofthe scan is normally 300-450nm. For in-situ calibration, Ionomycin 9+  (lOuM), a calcium ionophore, combined with a 20mM Ca determine  R  ; R m n i  max  was determined by switching to a Ca  solution was used to free (2mM ethyleneglycol-  bis(P-aminoethyl ether)-N,N,N',N'-tetraacetic acid [EGTA]), ionomycin-containing solution. The background autofluorescence at 340 and 380nm was substracted from experimental fluorescence intensities then the F340/F380 ratio (R) was converted to [Ca ]j using the equation of Grynkiewicz: 2+  [Ca ]i=KD(R-Rmin)/(Rmax-R)(So8o/Sb38o). 2 +  Fura-2 was assumed to have an apparent dissociation constant for C a  2+  (KD) of 200nM 48  (see McCarron, J.G. et al., 1994). R=F3407F380; R  m i n  , R  m a x  ,  and p=(S 8o/S 38o) were 0  b  0.95±0.04, 3.12±0.31, and 1.42±0.17 (n=4), respectively. A notable consideration is that fura-2's dissociation constant for Ca  ( K ) is variable. For example, the value published D  by Grynkiewicz et al. (Grynkiewicz, G. et al., 1985) (224 nM) refers to an artificial environment of fiira-2 in a high K solution, adjusted to 37°C, and a pH of 7.1 whereas, +  as mentioned above, our experiments were conducted in-situ, at room temperature. Published KD values vary widely over a range of 115-1300 nM depending on ionic strength, pH, protein binding (Henke, W. et al., 1996), and temperature, therefore calibration experiments can be performed to determine the K  in the particular tissue  D  chosen which involves plotting free [Ca ] versus (R-Rmin)/(Rmax-R)(Sf38o/Sb38o) to yield a 2+  slope equal to 1/K (Wan, B. et al., 1989). In our tissue the K value was extrapolated to D  D  the value obtained in toad gastric smooth muscle cells (200 nM at room temperature) in studies by Fred Fay and his co-workers (McCarron, J.G. et al., 1994; Williams, D.A. et al., 1985; Becker, P.L. et al., 1989).  k) Solutions and Chemicals The following table is a list of solutions we used to bath the tissue and to provide the vehicle for various pharmacological agents.  49  NPSS*  80mM  0Ca  2+  PSS*  K PSS| +  Compound KCI  0Na , 0 C a PSSf +  Conc.(mM) 5  80  5  5  1  1  1  1  NaCl  140  65  140  -  LiCI  -  -  -  140  HEPES  5  5  5  5  d-Glucose  10  10  10  10  CaCl  1.5  1.5  -  -  -  -  0.1  0.1  MgCl  2  2  EGTA  2+  pH 7.4 with *NaOH, or | K O H .  Cyclopiazonic acid (Research Biochemicals International), fura-2 and pluronic acid F127 (Molecular Probes), and FCCP and ionomycin (Sigma) were dissolved in DMSO. Caffeine and T E A (Sigma) were dissolved in PSS. PE, phentolamine, IAA, and iberiotoxin (Sigma) were dissolved in double distilled H 0 . Note: the concentrations of 2  C A F , PE, phentolamine, CPA, IAA, FCCP, and ionomycin used in this study were saturating.  50  I) Statistics of Experiments All experiments were reproducible, thus the term 'representative graph' was defined as a hand picked graph that best represented other graphs obtained by repeating the same experiment over at least three times. Where necessary, data were presented as means ± S.E. Differences of means between data points were compared by statistical analysis using the student t-test (for 2 samples) or one-way A N O V A (>2 samples). The nonparametric Newman-Keuls test was used to rank the differences in means from the A N O V A test.  m) Curve-Fitting Analysis Two curve-fitting equations (calculated by Jandel Scientific-SigmaPlot software) were used in the estimation of decline rates for [Ca ];. C a 2+  2 +  extrusion from the V S M cell was  modeled after a non-linear, bi-exponential curve fit of the following form: f=ae" +ce" bx  dx  where f=[Ca ]j (nM), a and c are compartment sizes, x^time (seconds), and b and d are the fast and slow rate constants (s" ), respectively (note:a single exponential did not 1  produce as a sufficient fit). The fast component of the curve corresponding to the rate constant, b, was used for comparison between the various experiments. The slow component, d, was consistently the same between control and experimental curves (see Table 1) and therefore was not considered in my calculation ofthe C a  2 +  extrusion rates.  Two components suggest that several different processes are involved in the extrusion of C a . Note that an alternative method of data collection was to calculate the linear slope 2+  51  of the C a  2 +  decline curve at a fixed [Ca ]i level rather than by exponential decay (data 2+  not shown), but the statistical results from these data were comparable to those found in Table 1. In order to determine the rate of decay in the basal cytoplasmic [Ca ] following the removal of extracellular Ca under 0Ca  2+  PSS, and 0Na , 0Ca +  we used the following single exponential decay curve 2+  PSS conditions: y=ae" , where, y=[Ca ] (nM), bx  2+  x=time (seconds), a=y at x (near 0), and, b=rate constant (s" ). The resulting rates of 1  decay ± S.E. were displayed in a bar graph as seen in Fig. 22.  C. Reservations  a) Fura-2 Fura-2 is a ratiometric fluorescent C a  2+  indicator that requires a complex  instrumentation set-up consisting of dual excitation monochromators and a single emission monochromator. The acetoxy methyl ester (AM) form of fura-2 crosses the P M and is enzymatically hydrolyzed by intracellular esterases. Some disadvantages of using fura-2 are that it can become compartmentalized in the nucleus, SR, and mitochondria; moreover, fura-2 can leak or be extruded from the cell, albeit lowering the temperature of the experiments can largely eliminate these problems. Other complications due to fiira-2 include photobleaching which can be avoided if the u.v. light source intensity is attenuated. Fura-2 can also become overloaded, particularly in thick tissues, but this is 52  Table 1. Rate of [ C a ] j decline fitted to the equation, f=ae" +ce (see Methods) with blockade of the Na -Ca exchanger and SERCA. bx  2+  +  Constants  Control (0Ca PSS)  0Na  +  2+  Control (0Ca PSS  2+  CPA  2+  Control (0Na PSS)  0Na +CPA  +  +  b(10" )s"'  7.64±1.24  4.09±0.72  14.23±1.94  10.85±1.07  5.46±1.07  4.76±0.91  dClO" )^  2.22±0.70*  1.78±0.72*  1.16±0.39*  0.62±0.02*  2.46±0.40*  2.53±0.47*  a(10 )nM 2  1.83±0.25  1.91±0.27  1.90±0.31  2.04±0.29  2.01±0.32  2.05±0.39  c(10 )nM  0.94±0.11  0.94±0.10  1.14±0.13  1.19=1=0.17  1.04±0.11  1.13±0.09  2  3  2  1  values expressed as means±S.E.M.; n>6; * A N O V A , PO.20.  53  minimized by limiting the fura-2 concentration to below lOOuM (Moore, E.D.W. et al., 1990). Incomplete de-esterification and photobleaching is a problem with fura-2 because the molecule converts to a highly fluorescent form that is insensitive to C a  2 +  and adds  significant background fluorescence to the spectral signal. Bathing solutions, naturally occuring fluorophores such as N A D H and riboflavin, and optical components within the spectrofluorimeter can contribute to noisy signals, but this can be eliminated by recording background fluorescence then subtracting it from the original signal.  Despite these problems, intact SM contains few exogenous contaminants as compared to singly-dispersed cells making it less likely for both real and artifactual signals to be superimposed upon each other. As with most other C a  2+  indicators, fura-2 naturally  binds to a variety of proteins such as histones, trypsin, and collagen (Bancel, F. et al., 1992) which along with cellular viscosity and lower temperatures can lead to a change in the "on" rate constant (KD) for fura-2/Ca complexation of up to several fold less in-situ 2+  due to slower kinetics (Kao, J.P.Y. and Tsien, R.Y., 1988). This conforms to our published K value of 200nM compared to the in solution value of 224nM published by D  Grynkiewicz et al. in 1985 (Grynkiewicz, G. et al., 1985).  b) Temperature Temperature dependency of biological processes is common and is implicated in S M function. Droogmans and Casteels (Droogmans, S. and Casteels, R., 1981) conducted 4 5  Ca  2 +  flux experiments in rabbit ear artery while lowering the bath temperature from  54  35°C to room temperature and they found that there was a decrease in resting tension and in the amplitude of the K -induced tonic contractions and decreased relaxation rates as +  well as a reduction in the rate of C a  2+  efflux and a decrease in the rate of SR C a  2+  release.  Furthermore, their report showed that a change in temperature did not have any appreciable effect on the N a - C a +  2+  exchanger, which differs from another report in  cardiac muscle where it was shown that SR-mediated C a  2 +  extrusion is clearly  temperature dependent and a rise in temperature (from 17°C to 37°C) was shown to increase SERCA and, thus N a - C a +  2+  exchanger activity (Marengo, F.D. et al., 1997).  Again, in contrast, in sheep hearts, cooling (from 23 °C to 5°C) was shown to cause an increase in SR C a  2 +  release channel currents (Sitsapesan, R. et al., 1991) which would  give rise to a reduced SR C a  2 +  content and may decrease overall total Ca i removal in 2+  part because of reduced activity of the N a - C a  2+  literature is that reduced temperature slows C a  2+  +  exchanger. The impression from the transport, but does not dramatically alter  basic Ca transport mechanisms.  c) Intracellular pH Intracellular pH (pHi) is Na -sensitive and will change according to N a - H antiporter +  +  +  activity (Weissberg, P.L. et al., 1987). A corresponding drop in pH; with removal of extracellular N a can produce a number of problems such as a reduction in C a +  2 +  influx  through reduced open probability and single channel conductance (lino, S. et al., 1994; Klochner, U . and Isenberg, G., 1994), reduced IP -induced C a 3  2 +  release (Tsukioka, M . et  55  al., 1994) and P M C A activity. In some SM there is a slow intracellular acidification of approximately 0.4 pH units after a five minute incubation in Na -free medium but with +  replacement of extracellular N a the acidosis is rapidly reversed (<12 seconds) +  (Weissberg, P.L. et al., 1987). pHi can potentially be affected by the N a - K ATPase and +  Na -Ca +  2+  +  exchanger, but the change appears nominal (Motley, E.D. et al., 1993).  Temperature is a compensatory mechanism which when increased induces a fall in pHj (Wray, S., 1988). Considering the lower temperature of our experiments a decrease in pHj may be attenuated.  d) In-Situ Preperations The complexity of multi-cellular preparations is obvious and adds to the difficulty in explaining the data but on the other hand there is considerable evidence to suggest that Ca  handling in intact S M is more representative of in vivo physiology. In fact, the  benefit of intact tissue preparations is that the [Ca ]i measurement reflects the [Ca ]i 2+  2+  from each cell with the value automatically averaged. In C a fluorimetry the beam of 2 +  light that illuminates the suspended tissue excites several hundred cells at one time thereby dramatically increasing the N value to provide a more accurate reading. In contrast, single cell preparations prepared by enzymatic digestion can affect the fura-2 signal, and moreover, these cells often remain contracted with repeated washing. Chemically isolated SM cells were also found to respond poorly to PE as evidenced by their inability to contract in a solution containing PE and Ca -free medium and their 2+  intracellular C a  2 +  stores were found to be partially depleted due to dysfunctional 56  sequestration (Kwan, C Y . et al., 1992). Cultured SM cells, on the other hand, can present with a very different phenotype, and, for example rapidly lose their L-type VOCCs and other channels or receptors.  Chapter III.  A . Concomitant blockade of N a - C a +  Ca  2 +  2 +  RESULTS  exchange, S E R C A , and the P M C A abolishes  efflux  M y first aim was to determine whether the SR actually mediates C a through a N a - C a +  2 +  exchange mechanism or if there are other C a  2 +  2 +  extrusion  transporters  such as the P M C A that contribute to this. The C a  2 +  transporters active in clearing C a +  2 +  from the cytoplasm as recognized in V S M  2-F  are the S E R C A , N a - C a Ca  2 +  exchanger, P M C A , and to a lesser extent, the mitochondrial  uniporter. In order to determine i f these mechanisms each play a role in C a  extrusion that when added together account for all the C a  2 +  2 +  removed from the I V C  cytoplasm, all three, including the mitochondrial uniporter, were blocked simultaneously to show cumulative effects (Fig. 9-representative trace) or independently (except for the P M C A for which no selective blockers were available at the time o f this study) to show their relative contributions (Fig. 10 to F i g . 12 - representative traces). In order to understand how the C a  2 +  transporters affect C a  kinetics. Firstly, intracellular-free C a uptake and C a  2 +  2 +  2 +  homeostasis we must focus on C a  levels depend on the sum o f C a  extrusion and this determines the direction o f net C a  2 +  2 +  2 +  influx, C a  2 +  flow providing 58  FCCP "I  r-  o  • • • />—*  i  i IAA 200 Sec.  500 _  400  ~ +  300  8  200 100  H  0  PE+80K  1  I  0Ca2+  CPA+IAA+FCCP  I  PE+80K 0Na ,0Ca +  200 Sec.  Fig. 9. Metabolic and transport blockade extrusion.  +  2+  prevents C a  [ C a ] i ( n M ) v s . t i m e ( s e c o n d s ) is r e c o r d e d d u r i n g s t i m u l a t i o n of C a 2 +  influx w i t h 8 0 m M K 0Ca  2 +  +  z+  2 +  P S S and 20^iM phenylephrine ( P E ) followed by  P S S c o n t a i n i n g 1 0 ( i M p h e n t o l a m i n e (left).  This  experiment  w a s r e p e a t e d in the p r e s e n c e of A T P inhibitors, 1 m M I A A , a n d 3 j i M F C C P , the S E R C A inhibitor, C P A ( 2 0 ^ M ) a n d r e m o v a l of e x t e r n a l Na  +  f r o m the C a - f r e e P S S (right).  elevated.  2 +  In this c a s e [ C a ] i 2 +  T h e s e t r a c e s a r e t y p i c a l of s i x e x p e r i m e n t s .  remains  In the i n s e t  a r e c o n t r o l s for the effect of I A A (solid line), a n d F C C P ( b r o k e n line). N P S S is the initial b a t h i n g s o l u t i o n for t h e s e c o n t r o l s (solid bar), later replaced by 0 C a  2 +  P S S ( b r o k e n bar).  D a t a f r o m the i n s e t a r e t y p i c a l  of t h r e e e x p e r i m e n t s . 59  there is an imbalance between influx and efflux (equation 1: net C a Ca  2 +  2 +  flux = Ca  z+  influx -  efflux). In order to determine one of the variables the other variable must be kept  constant; so, for example, with the C a  2 +  influx component totally removed as shown in  my experiments, there remains the mechanism of SR Ca The rate of C a  2 +  uptake, release and efflux.  removal from the cytoplasm is equivalent to the slope of the [Ca ]i 2+  decay when external C a  2 +  is removed from the tissue bath. This partially reflects C a  extrusion because total C a  2+  removed from the cytoplasm = C a  2 +  extrusion + C a  2 +  2+  sequestration (equation 2). I utilized standard procedures to perform all ofthe following experiments according to the design in the section on Specific Protocols in Chapter II.  As shown in the control (Fig. 9, left panel), the initial rise in [Ca ]j was due to the 2+  Ca -loading effect of high K and PE while the subsequent fall in [Ca ]i was assumed to 2+  +  be due to active C a  2+  2+  extrusion in the absence of a-adrenergic activation and of C a  2 +  influx. Pre-incubation ofthe IVC with CPA (20uM) to block SERCA, IAA (ImM), and FCCP (3uM) to block PMCA-associated glycolysis and the M T C a uniporter, 2 +  respectively, and subsequent replacement of the Ca -containing bath with N a and C a 2+  free PSS to block the N a - C a +  2+  +  exchanger produced a sustained C a  2+  2+  response that  reached a peak amplitude of 353±49.4nM, n=6 (compared to the peak amplitude of 202±32.2nM, n=6, in the control) and remained there until the end ofthe experiment even though Ca  influx was eliminated (Fig. 9, right panel). The prolonged plateau  phase and the absence of any C a  2 +  efflux indicates that all C a  2 +  uptake/extrusion  pathways were effectively blocked and there was negligible passive C a  2 +  leak outward. 60  To ensure that the blocking drug(s) had no deleterious side-effects on the fura-2 signal or on the tissue itself, or if they significantly affected the amplitude of the Ca response, I performed control experiments as demonstrated in the insets of Fig.9, 10, 11, and 14. IAA had no visible effect on the basal  [Ca ]j 2+  in NPSS (solid bar) or 0Ca PSS (broken 2+  bar). However, in the presence of FCCP there was a delayed upward deflection in [Ca  ]j  2+  94-  that was sustained even when external Ca was removed, possibly indicating inhibition of active C a  2+  removal from the cytoplasm via the mitochondrial C a  induced rise in  [Ca ]j 2+  2 +  uniporter. This  may be attributed to the higher experimental peak in the presence  of FCCP when compared to the control, although CPA and Na -free PSS also need to be +  tested for this (see inset of Fig. 10 and 11). The main point of this experiment was to show that total inhibition of C a Na -Ca +  2+  2 +  extrusion occurs when all active C a  2 +  transport and  exchange is inhibited.  B. Na -Ca +  2+  exchanger and SERCA blockers attenuate the rate of [Ca ]j decline in 2+  Ca -loaded cells 2+  a) Na -Ca +  2+  exchanger  I performed the next experiment in order to determine the individual contribution the I  94-  9_4-  Na -Ca exchanger made to Ca  9 j  extrusion by observing the change in the rate of [Ca ]j  decay following removal of high K and PE from the IVC and then replacing it by a N a +  +  and Ca -free solution to block the N a - C a 2+  +  2+  exchanger. Fig. 10, left panel, is the control  curve and Fig. 10, right panel, represents the experimental conditions. With  61  Fig. 10. Blockade of Na -Ca exchanger attenuates the rate of decline in [ C a ] j . +  2+  2+  T i m e c o u r s e ( s e c o n d s ) c h a n g e s in [ C a ] , ( n M ) w e r e m o n i t o r e d d u r i n g s t i m u l a t i o n with 8 0 m M K P S S a n d P E ( 2 0 > M ) t h e n by w a s h i n g out with 0 C a P S S a n d p h e n t o l a m i n e (lOjaM), a s s h o w n o n t h e left. O n t h e right, this e x p e r i m e n t is r e p e a t e d (for c o m p a r i s o n , the c o n t r o l d e c a y is s u p e r i m p o s e d a s s h o w n b y the b r o k e n line) with e x t e r n a l N a removed, which reveals Na -independent C a extrusion. T h e s e t r a c e s a r e t y p i c a l of e i g h t e x p e r i m e n t s . T h e effect of N a r e m o v a l in N P S S (solid bar) a n d in C a - f r e e P S S ( b r o k e n bar) a p p e a r s in the inset. 2+  +  2 +  +  +  2 +  +  2 +  62  Na -Ca +  2+  exchanger blockade, the fall in the rate of decline in [Ca ]j of 4.1±0.7 x 10" s" 2+  2  (n=8) was noticeably slower than the control value of 7.6± 1.2 x 10" s" (n=8) as represented by the broken line superimposed on the experimental curve, suggesting a significant role for the exchanger in C a notable aspect of N a - C a +  2+  2 +  extrusion from stimulated IVC. Another  exchange blockade is that the [Ca ]j was slower to return to 2+  the pre-blockade baseline in comparison to the control, yet another clue that the N a - C a +  2+  9+  exchanger regulates Ca \. A minor technicality is that both control and experimental curves have a distinct peak shape in comparison to those in Fig. 9 with no distinct plateau phase. This is indicative of C a  2 +  removal taking place during activation of SM.  To complete the blockade experiment it was necessary to test whether the procedure of removing external N a had any significant effect on basal [Ca ]j. As shown in the inset +  2+  graph of Fig. 10 (m=8), switching from NPSS to a Na -free solution (solid bar) caused a +  slow increase in  [Ca ]i 2+  of no more than 30 nM over a 200 second period after which  [Ca ]j remained steady; but when external C a 2+  2+  was also removed (broken bar) the  [Ca ]j slowly returned to baseline. This gradual increase in intracellular C a 2+  that the N a - C a +  2+  exchanger contributes to C a  2 +  2+  suggests  extrusion from the resting SM.  b) SERCA Next, I investigated the effect of SERCA inhibition. This experiment was similar to the one above except that C P A (20|aM) was pre-incubated with high K and PE. The +  63  addition of CPA resulted in a marked lag in the rate of decline of [Ca ]i (1.08+0.11 x 10" 1  s", n=7) compared to the decline in the control (1.42±0.19 x 10" s", n=7) shown in Fig. 1  1  1  11, left panel, and as a broken line superimposed on the experimental curve in Fig. 11, right panel. And, moreover, the peak amplitude (avg. peak value=291±38.0 nM, n=7) was approximately 50 nM greater in magnitude than that of the control (avg. peak value=244+29.4 nM, n=7) and the decay in [Ca ]j returned to baseline extremely slowly. 2+  The decrease in C a  2 +  removal by CPA suggests that SERCA does in fact remove C a  from the cytosol even though it may only contribute to uptake of the C a rather than being involved in the extrusion of C a  2 +  2 +  2+  by the SR  as will become clear from subsequent  experiments.  I ran a control experiment using only CPA in NPSS (solid bar) or in a Ca -free 2+  solution (broken bar) and recorded [Ca ]i, as shown in the inset of Fig. 11. I observed a 2+  delayed and gradual rise in [Ca ]j that gave rise to a plateau phase before falling back to 2+  baseline when external C a  2 +  was removed. The significant increase in [Ca ]j of about 2+  100 nM may be due to inhibition of C a  2+  extrusion, activation of store-operated channels  or both and may also explain the higher amplitude of the peak in the experimental curves. The delay in the initial increase of [Ca ]i might indicate that it takes time for CPA to 2+  exert its full effect, emphasizing the need to pre-treat the IVC with CPA.  64  I  I  200 Sec.  Fig. 11. [Ca ],.  Blockade of the SERCA attenuates the rate of decline in  24  CPA (20u.M) was pre-incubated for about 200 seconds to selectively block SERCA. The superimposed broken line on the right represents the control decay. These traces are typical of seven experiments. The effect of CPA on [Ca ]i in NPSS (solid bar) and after the removal of external C a (broken bar) appears in the inset. 2+  2+  65  c) SERCA plus Na -Ca +  2+  exchanger  In the next experiment, I tested for the possible additivity of effects of N a - C a +  exchange blockade and SERCA blockade on the rate of C a  2+  removal from the  2 +  cytoplasm. If the two are additive in nature with respect to their C a  2 +  extruding properties  then concomitant blockade should produce a greater attenuation in the decline of [Ca  ]j  2+  then either alone. Fig. 12 illustrates that the addition of CPA to a Na -free medium does +  not enhance the rate of decline over that seen in the presence of 0 N a alone. The rate of +  decline in the experimental curve (Fig. 12, right) was equal to 4.8±0.9 xlO" s" (n^) and 2  1  nearly identical to the control rate of decline of 5.4± 1.1 xlO" s" (n=6) (Fig. 12, left) 2  1  represented by the superimposed broken line. This result suggests that under these •f"  2+  conditions, the Na -Ca exchanger and SERCA do not function independently but appear to act in-series to extrude C a  2 +  from the cells. The larger peak amplitude of the  experimental curve (peak value=291±66.9 nM, n=6) when compared to the control (peak value=255±53.5 nM, n=6) is consistent with the effects seen of CPA in the previous figure.  C. Evaluating the decline of [Ca ]j under varied C a 2+  I compared the rates of cytoplasmic C a  2 +  2+  extrusion conditions  removal from the downward slope of the C a  response to determine if the SERCA and N a - C a +  2+  exchange co-ordinate C a  2 +  2 +  extrusion. 66  PE+80K 0Na ,0Ca2+ +  +  PE+80K ONa ,OCa +  +  CPA  200  Sec.  Fig. 12. The addition of SERCA blockade on top of Na -Ca exchanger blockade has no further affect on the rate of decline +  2+  in [Ca ]j. 2+  CPA and 0Na were added to the tissue simultaneously. The superimposed broken line represents the decay of the curve in the left panel. These traces are typical of six experiments. +  67  In order to calculate the Ca decay rates, I fitted each experimental curve using the following bi-exponential equation: f=ae" +ce~ bx  dx  where b and d are the fast and slow rate  time constants (s" ), respectively; b fits the initial C a 1  2 +  decay and d fits the tail ofthe C a  2+  decay curve. The b time constant contained the most relevant data for my study because it represented the initial slope, whereas, the d time constant was relatively constant under the various experimental conditions (refer to Table 1). As shown in Fig. 13, the rates of Ca  2 +  decline were obtained from three groups of data: CPA+0 C a  blockade; n=7), 0 N a , 0 C a +  0 Ca  2 +  2+  2 +  PSS (SERCA  PSS (Na -Ca exchanger blockade; n=8), or CPA+0 N a , +  2+  +  PSS (SERCA+Na -Ca exchanger blockade; n=6), expressed in terms of +  2+  percentages of control. SERCA blockade induced a 23% decrease in the rate of C a decline compared to the control. The remaining fraction, or 77% of C a presumed to be mediated by the P M C A and the N a - C a +  2+  2 +  2 +  extrusion was  exchanger which in this  treatment group were not blocked. This reduction in the rate of decline of [Ca ]i is 2+  indicative of an SR network that contributes to cytoplasmic C a of C a  2 +  uptake. With N a - C a +  2+  2 +  removal by the process  exchanger blockade, the rate of decline in [Ca ]j fell by 2+  47% which is double of that seen in SERCA blockade. The remaining fraction or 53% of extrusion is accounted for by the P M C A and possibly SERCA. The differences between these first two groups compared to the control group were significantly different (P<0.05). The rate of Ca  decline decreased by approximately the same amount (51%  compared to 47%) after the addition of CPA to 0 N a (group number three) and when +  these two groups were compared the differences were non-significant (P>0.05), suggesting that when the SERCA and N a - C a +  2+  exchanger are both active, the SERCA 68  100 c o o  80 60 -  ~o  0) "D + CNJ 05  o  o CD  40 20  H  03  0 0Na  CPA  0Na +CPA  +  +  Fig. 13. Comparison of the effects of CPA, 0Na combination on the rate of [Ca ]j decline.  + e x  t and their  2+  W i t h b l o c k a d e of S E R C A , the rate of [ C a ] i d e c l i n e w a s 7 8 ± 5 . 1 % of c o n t r o l , n=7. W i t h b l o c k a d e of Na -Ca e x c h a n g e the rate of d e c l i n e in [ C a ] j w a s further r e d u c e d to 5 3 ± 4 . 5 % of c o n t r o l , n=8, s i m i l a r to a v a l u e of 4 8 ± 3 . 5 % , n=6, w h i c h w a s o b t a i n e d by b l o c k i n g both of t h e s e C a extrusion m e c h a n i s m s . 8 P<0.0009 vs. control; * A N O V A , N e w m a n K e u l s test, P < 0 . 0 0 0 7 v s . C P A - v s . 0 N a - v s . 0 N a + C P A - t r e a t e d tissue. 2 +  +  2 +  2 +  2 +  +  +  69  functions in-series with the Na -Ca exchanger to extrude Ca ; however it appears that some of the plasmalemmal Na -Ca exchangers are not coupled to the superficial SR.  D. SR-mediated C a  2+  extrusion  M y second aim was to determine if the Na -Ca exchanger, coupled to the +  2+  superficial SR, somehow modulates the SR C a  2+  content.  a) Ca "-free PSS 2  The point ofthe experiment in Fig. 14 (representative trace) is to show that the downward slope of the C a  2+  response in 0 C a  2+  PSS reflects extrusion rather than SR C a  2 +  accumulation because the SR does not refill in the absence of external C a . With the use 2+  of C A F to induce C a  2 +  release from the SR by fully depleting it of C a  determine the relative amount of C a  2+  I was able to  stored in the SR by measuring the C a  ([Ca ]i versus time) after blocking the N a - C a 2+  2+  +  2+  2 +  transient  exchanger and/or SERCA.  In the following four experiments the IVC was exposed to a C A F (25mM)/0 C a  2 +  PSS  mixture (denoted by the downward arrows) prior to and following cell stimulation with high K and PE (see Specific Protocols in Chapter II). The amplitude of the 1 C A F +  induced C a Ca  2 +  st  2 +  response (the control) was determined to be proportional to the quantity of  within the SR under typical resting conditions; the 2  nd  C A F response represented the  70  Fig. 14. The SR refills only in the presence of extracellular Ca (0Ca PSS treatment prevents refilling). 2+  T h e first  [Ca  2 +  ]j  t r a n s i e n t w a s i n d u c e d by C A F ( 2 5 m M ) in 0 C a  2 +  PSS  ( d o w n w a r d arrow-peak-i). T h e u n d e r s h o o t w a s r e - a d j u s t e d b y a d d i n g Ca  b a c k to t h e b a t h i n g s o l u t i o n ( u p w a r d a r r o w ) , t h e n  2 +  with 8 0 m M K PSS  and  +  stimulation  P S S a n d P E ( 2 C V M ) f o l l o w e d b y w a s h o u t with 0 C a  phentolamine  (1CyM) was  performed  as  in  2 +  previous  e x p e r i m e n t s with a s u b s e q u e n t a d d i t i o n of C A F ( d o w n w a r d a r r r o w p e a k ) to d e t e r m i n e t h e d e p l e t o r y effect o n t h e S R . 2  T h i s figure  r e p r e s e n t s t h e c o n t r o l t r a c e a n d is t y p i c a l of f o u r e x p e r i m e n t s . In t h e i n s e t , a c o n t r o l for multiple e x p o s u r e s to c a f f e i n e w a s o b t a i n e d .  71  SR Ca  content during active extrusion in the absence of Ca  result in Fig. 14, following a short period in 0 C a  2 +  influx. According to the  PSS the amplitude of the 2  nd  CAF  response was drastically reduced compared to the 1 C A F response, indicating that the st  Ca  depletion caused by PE was not reversed upon removal of PE in the absence of  external C a . To test whether the presence of external C a 2+  2 +  is essential for re-loading the  SR, I performed a control experiment as illustrated in the inset of Fig. 14. After reapplying C a , as indicated by the upward arrow, I was able to induce a subsequent C A F 2+  response which was similar in magnitude to the first one.  One interesting feature of the C A F response is that it produces an undershoot in the C a  2 +  94-  signal if external Ca Ca  2 +  is removed from the C A F solution which may be due to overactive  extrusion by the N a - C a +  2+  exchanger and P M C A in the absence of C a  2 +  influx. The  undershoot can be overcome by re-adjusting the [Ca ]i baseline by adding back C a . 2+  b) Na -Ca +  2+  exchanger  2+  The purpose of this next experiment was to determine if the N a - C a +  modulates the SR C a  2 +  +  the N a - C a +  2+  2 +  exchanger  content. As shown by the representative trace in Fig. 15, the  removal of external N a from 0 C a SR to refill with C a  2+  2 +  PSS to block the N a - C a +  2+  exchanger allowed the  in spite of the absence of external C a . This indicates that when 2+  exchanger is blocked, the SR is able to refill with C a  2+  from the cytoplasm.  Note that in this representative trace, the second C A F peak compared to thefirstC A F peak was significantly larger which could be due to brief reversal of the N a - C a +  2+  72  300 —  250  >S 2 0 0 c f 150 CO  O  100 50 0  J  PE+80K+  J  200  0Na ,0Ca2+ +  Sec.  Fig. 15. Na -Ca exchanger blockade enhances SR refilling. +  2+  T h e e x p e r i m e n t in F i g . 1 4 w a s t h e n r e p e a t e d w i t h 0 N a \ 0 C a PSS to b l o c k t h e N a - C a exchanger. T h i s t r a c e is t y p i c a l of f o u r experiments. 2 +  +  2 +  73  exchanger before external Ca  is effectively buffered by E G T A or it could be the result  of L i substitution which is known to cause a delayed, reversible rise in baseline tension +  (and, thus would elevate [Ca ]j) in V S M . When the data were compiled from different trials ofthe same experiment and presented in a bar graph these effects were not as apparent (see Fig. 18).  c)  SERCA A third experiment was done to test if the SERCA blocker, CPA actually blocked the  SR from taking-up and releasing C a  2+  (Fig. 16-representative trace). Instead of removing  external Na , I added CPA (20 uM) to 0 C a +  2 +  PSS and observed the change in amplitude  ofthe CAF-induced Ca i transient with respect to the control. SERCA blockade caused 2+  nearly all ofthe SR to lose its store of C a ofthe 2  nd  2 +  in view of an approximately 100 % abolition  C A F response.  d) SERCA plus Na -Ca exchanger A fourth experiment showed that the preservation of SR C a was due to SERCA-mediated C a  2+  2 +  in 0 Na , 0 C a +  2 +  PSS  uptake. As can be seen from Fig. 17 (representative  trace), SERCA inhibition prevented refilling ofthe SR under these conditions.  74  Fig. 16. SERCA blockade depletes the SR. T h e e x p e r i m e n t in F i g . 14 w a s r e p e a t e d with C P A to b l o c k S E R C A . T h i s t r a c e is t y p i c a l of f o u r e x p e r i m e n t s .  the  75  Fig. 17. Na -Ca from refilling. +  2+  exchange + SERCA blockade prevents the SR  T h e e x p e r i m e n t in F i g . 14 w a s r e p e a t e d with C P A + 0 N a , 0 C a T h i s t r a c e is t y p i c a l of f o u r e x p e r i m e n t s . +  2 +  PSS.  76  E. Comparing Caffeine-Induced [Ca ]i Peaks Following Blockade ofthe Na -Ca +  2+  2+  exchanger and/or SERCA  For quantitative analysis, I compared each C A F experiment and presented the gathered data in a bar graph expressed, as peak2/peaki, % (mean ± S.E.) in Fig. 18. By ratioing the amplitudes of the two CAF-induced C a  2 +  transients I was able to eliminate  any variability between separate experiments, thereby reducing the standard error. Ratioing also normalized the data. There were four groups of C A F responses: 1) the control (0 C a PSS, 3) 0 N a , 0 C a +  2+  PSS, and 4) CPA + 0 Na , 0 C a +  correlated to the amplitude of the C a Ca  2 +  2 +  2+  2 +  PSS), 2) CPA in 0 C a  PSS. The SR C a  2+  2+  content was  transient induced by C A F . I found that when all  extruding pathways were intact (control), C a  2 +  was lost from the SR at a rate of 87  %/SR total within 300 seconds after removal of external C a . After SERCA blockade, 2+  the SR C a  2 +  content had decayed by about 98 % within that same time period. A similar  response was obtained after combined blockade of SERCA and N a - C a +  which SR Ca  2+  exchange in  had been depleted by approximately 95 %. These three groups are  statistically non-significantly different (P>0.05) and all involved uninhibited release of Ca  2 +  from the SR but unlike in the control, in the CPA-treated IVC, C a  abolished. After blocking the N a - C a +  2+  2 +  re-uptake was  exchanger an entirely different response was seen  wherein there were negligible differences in comparing peak2 to peaki, corresponding to  co  CD O. ~CNJ _^ (0  CD ^Q. CD CO CO Q) CD + CM CO  o"O  CD O TD _C CD C i  ig  CO  O <+— o  0Ca  CD  TD  2 +  PSS CPA 0Na  +  0Na +CPA +  "a. E <  Fig. 18. Comparison of the refilling of the SR in 0Ca solution under conditions of SERCA and Na -Ca exchange blockade. 2+  +  2+  T h e a m p l i t u d e ( b a s a l to p e a k l e v e l s ) of the s e c o n d C A F - i n d u c e d C a t r a n s i e n t ( p e a k ) w a s c o m p a r e d to t h e internal c o n t r o l ( p e a k ^ . T h e p o o l e d d a t a is e x p r e s s e d a s p e r c e n t ratio of the a m p l i t u d e of C A F induced C a r e l e a s e , p e a k / p e a k . In t h e a b s e n c e of e x t e r n a l C a ( c o n t r o l : 1 3 . 1 9 ± 6 . 7 5 % , n=4) N a - C a exchanger blockade induces refilling of the S R a s s h o w n by the C A F - i n d u c e d C a r e l e a s e ratio of 8 5 . 5 3 ± 1 3 . 4 6 % , n=4, w h e r e a s with the a d d i t i o n of C P A , the S R C a s t o r e is c o m p l e t e l y d e p l e t e d , c o r r e s p o n d i n g to a v a l u e of 5 . 0 8 ± 2 . 5 1 % , n=4. With N a - C a e x c h a n g e u n i n h i b i t e d , C P A still h a s a p p r o x i m a t e l y the s a m e effect (1.70 ± 1.70%, n=4). 8 P < 0 . 0 0 2 v s . 0Na -treated tissue; * A N O V A , P<0.3, 0 C a - P S S - vs. C P A - vs. 0 N a + C P A - treated tissue. 2 +  2  2 +  2 +  2  1  +  2 +  2 +  2 +  +  +  2 +  2 +  +  78  a loss of only 14 % of SR Ca  2  (P<0.05). These data lead to the hypothetical C a  2  extrusion model presented below.  F. Schematic Diagram #1-The Process of SR Buffering and C a  Fig. 19 illustrates a model for C a covers active C a and N a - C a +  2+  2 +  2+  Extrusion  2+  extrusion which is consistent with the results. It  buffering by the SERCA and extrusion toward the ECS via the P M C A  exchanger. As shown here, about half of all C a  2+  extruded from the IVC is  effected by the P M C A , whereas the other half is mediated by the N a - C a +  Upon further examination, it was found that of the C a Na -Ca +  2+  exchanger, half of it was due to C a  2+  2 +  2+  exchanger.  extruded from the cell by the  taken-up first by the SR that was  subsequently released into the space underneath the P M for removal by the N a - C a +  exchangers; whereas the other half was due to N a - C a +  2+  2+  exchangers unassociated with the  SR.  G. Ranking the order of access of the Na -Ca exchanger, SERCA, and the PMCA +  to C a  2+  2+  released from the SR under resting conditions  In the previous sections we discovered that SERCA and N a - C a +  2+  exchange function 79  E C S  Fig. 19. The process of SR buffering and Ca  2+  extrusion.  Illustration of the C a extrusion p r o c e s s a s mediated by the N a - C a e x c h a n g e r (filled o v a l s ) , the P M C A (filled c i r c l e ) , a n d the S E R C A (shaded circles). T h e P M C A e x t r u d e s 53% of t h e C a in t h e m y o p l a s m w h i l e the r e m a i n i n g C a is e x t r u d e d b y the N a - C a e x c h a n g e r s into t h e E C S ( e x t r a c e l l u l a r s p a c e ) , s o m e of w h i c h a r e a c t i v a t e d b y S R - r e l e a s e d C a . A b o u t half of t h e C a extruded by the N a - C a e x c h a n g e s y s t e m is in -series with C a uptake and r e l e a s e b y the S R , f o r m i n g a C a g r a d i e n t in the j u n c t i o n a l s p a c e , which permits C a j ( C a within the j u n c t i o n a l s p a c e ) to e x c e e d Ca ( m y o p l a s m i c C a ) . T h e i n c r e a s e in [ C a ] j a c t i v a t e s N a - C a e x c h a n g e , h o w e v e r , if the latter is b l o c k e d , C a is p u m p e d b a c k into the S R . 2 +  +  2 +  +  2 +  +  2 +  2 +  2 +  2 +  +  2 +  2 +  2 +  2 +  2 +  2 +  2 +  2 +  2+  m  2 +  80  in-series to extrude Ca  from the cytoplasm. The purpose of the next set of experiments  was to determine the time-course of decay in the SR C a  2 +  content following inhibition of  the N a - C a  2+  exchanger and/or SERCA in Ca -free PSS in order to consider the role of  the N a - C a  2+  exchanger in regulating the C a  +  +  2+  As before, CAF-induced C a Ca  2 +  2 +  2+  content of the SR.  release was assumed to reflect the SR C a  2 +  content. The  transient evoked by C A F was plotted as [Ca ]i (nM) versus time (seconds) of 2+  exposure to Ca -free solution (see Specific Protocols in Chapter II for more details). 2+  For analysis purposes I took this amplitude to be representative ofthe C a SR at the experimental time points. The cells were first loaded with C a  2 +  2 +  content of the  using high K  +  then washed in NPSS to obtain a control response (not shown), and then switched to 0 Ca  2 +  PSS for either 0, 150, 300, or 600 seconds before applying a bolus of C A F (refer to  Fig. 8). A control experiment was performed to determine the kinetics of decay of SR C a when C a  2 +  2 +  extrusion was uninhibited (see Fig. 20-representative trace). There was a  rapid decay in the C a  2+  response within the first 150 s, then the C a  2+  response flattened  out, closely resembling an exponential-like decay. The SR was fully depleted of C a  2 +  within 600 seconds. Next, I examined the effect of N a - C a +  by removing N a from the 0 C a +  induced C a  2 +  2 +  2+  exchanger blockade on the SR C a  2+  content  PSS. It was found that the amplitude of the C A F -  transient was sustained over the 600 second period, thus when the N a - C a  exchanger was blocked, the SR C a  +  2+  2+  content was maintained at high levels (see Fig. 2181  + CN  03  o  0) CO  c 03  + CN  03  o  "D 0 O  0  03  O  200  400  600  800  2+,  Time in OCa (sec.)  Fig. 20. SR Ca  decay after abolishment of Ca influx.  2+  2+  The S R C a c o n t e n t d e c a y s to v e r y l o w l e v e l s a s t h e t i s s u e is e x p o s e d to a C a - f r e e m e d i u m for 0, 1 5 0 , 3 0 0 , a n d 6 0 0 s e c o n d t i m e intervals. T i m e c o u r s e ( s e c o n d s ) c h a n g e s w e r e plotted a g a i n s t [ C a ] i ( n M ) . T h i s f i g u r e r e p r e s e n t s the c o n t r o l e x p e r i m e n t in w h i c h CAF-induced C a t r a n s i e n t s w e r e o b t a i n e d at the n o t e d t i m e i n t e r v a l s with C a r e m o v e d f r o m the e x t e r n a l m e d i u m . T h e s e t r a c e s a r e t y p i c a l of f o u r e x p e r i m e n t s . 2 +  2 +  2+  2 +  2 +  82  + CN  CO  O  c CD '(Jl  c  03  + CN  o 03  "O CD O Z3  TJ  o  200  0  03  400  600 800  Time in 0Na , 0Ca (sec.) +  2+  Fig. 21. Na -Ca exchange inhibition modulates the rate of decay in SR Ca content. +  2+  2+  T h e e x p e r i m e n t in F i g . 20 w a s r e p e a t e d e x c e p t that N a was r e m o v e d a l o n g with C a for the s a m e t i m e i n t e r v a l s . T h e S R C a c o n t e n t r e m a i n e d e l e v a t e d with b l o c k a d e of the e x c h a n g e r . T h e s e t r a c e s a r e t y p i c a l of f o u r e x p e r i m e n t s . +  2 +  83  representative trace) suggesting that the N a - C a +  2+  exchanger regulates SR C a  2+  and that  9+  the SR is capable of recycling its Ca without losing it to the P M C A .  It was possible to measure [Ca ]; at the same time as the CAF-induced C a 2+  by obtaining a measurement of the steady-state  [Ca ]i 2+  2+  transient  preceding the C A F spike. As  shown in Fig. 22, data from Fig. 20 and 21 were integrated and [Ca ]; (or cytoplasmic 2+  [Ca ]) in % nM was recorded against time at 0, 150, 300, and 600 s intervals (for 2+  normalization, the first value i.e. [Ca ]j at 0 s was assigned 100% and all subsequent data 2+  points by were divided by 100 and expressed in percentages of the control). Despite the SR being able to retain all its C a [Ca ]i 2+  after N a - C a  2+  +  was virtually identical to that in 0 C a  2 +  2+  exchanger blockade, the decay in  PSS. The inset of this figure shows the  marginal differences between the two groups of data when the rate of [Ca ]j decay in the 2+  two curves was calculated. In this situation the C a continuous SERCA-mediated Ca  To illustrate how the N a - C a +  9+  2+  2 +  may be confined to the SR by  re-uptake.  exchanger can modulate SR C a  was calculated from the CAF-induced C a  2+  2+  flux, SR C a  2 +  content  transient amplitude over the peak amplitude  of the high K response. The mean ± S.E. was taken from Fig. 20 and 21 and presented +  in Fig. 23. In 0 C a  2 +  PSS (control experiment, filled circles) SR C a  decayed, whereas N a - C a +  2+  2 +  exchange blockade preserved the SR C a  2 +  content quickly content (filled  triangles). Interestingly, re-activating the exchanger by adding back the N a at the 480 s +  84  140 ^ + CN  +  03  CM  o 03  oo  "E  120 100 80 -  Control  60 -  0Na  +  CO  03 7^  40 -  Q_ O >* 20 -  O  0  n  0  1  1  1  1  1  1—  100 200 300 400 500 600  Time in 0Ca (sec.) 2+  Fig. 22. Exponential Ca decay in cytoplasmic concentration under control and 0 Na* conditions. 2+  Ca2+  [ C a ] i w a s m e a s u r e d prior to e a c h of the f o u r a p p l i c a t i o n s of c a f f e i n e in F i g . 2 0 (n=4) a n d 21 (n=4). In th e inset, e x p o n e n t i a l r a t e s of d e c l i n e of c y t o p l a s m i c C a (s ) for c o n t r o l a n d 0 N a - t r e a t e d t i s s u e s are compared. 2+  2 +  +  85  o  '-4—<  03  Control 0Na +Na  o  © 120  +  1.E 100 03  +  80 -  + O oo  60 -  LL <  40  O c  CD  20  -t—>  C  o O +  0  CM CO  0  O  rr  100 200 300 400 500 600 9+  CO  T  i  m  e i  n OCa^  (  s  e  c  .  )  Fig. 23. Comparison of the effects of Ca removal, and Ca +Na removal on the SR Ca content (CAF/80K* amplitude, % ratio). 2+  2+  +  2+  T h e p r o t o c o l f r o m F i g . 2 0 a n d 21 w a s u s e d to d e t e r m i n e the m a g n i t u d e of the C a t r a n s i e n t s after 0, 1 5 0 , 3 0 0 , a n d 6 0 0 s e c o n d s in a C a - f r e e o r N a - , C a - f r e e b a t h i n g s o l u t i o n . In o n e s e t of e x p e r i m e n t s , after e x p o s u r e to N a - f r e e e x t e r n a l s o l u t i o n , at the 4 8 0 s e c o n d i n t e r v a l , N a w a s r e p l a c e d into the b a t h i n g s o l u t i o n . The s u b s e q u e n t rate of d e c l i n e in S R C a c o n t e n t is m u c h m o r e r a p i d t h a n in C a - f r e e s o l u t i o n a l o n e . 2 +  2 +  +  2 +  +  +  2 +  2 +  86  interval (filled squares) caused the S R C a previously obtained by 0 C a  store to rapidly deplete again to levels  P S S treatment at the 600 s interval, indicating the high  degree to which the S R and N a - C a +  2 +  exchanger are coupled.  I performed another two experiments to determine the precise direction C a  2 +  takes  after being released from the S R and then established a hierarchy by which the N a - C a  2 +  exchanger, S E R C A , and P M C A function and determined whether they compete for C a  2 +  +  within the junctional space. A s shown in Fig. 24 (representative trace), I examined the decay in S R C a  2 +  content as a function o f time (seconds) in C P A and 0 C a  2 +  P S S and  observed a rapid decay that depleted the S R within 300 seconds o f exposure, which is similar to that seen in the control experiment o f F i g . 20. Thus, provided that the N a +  Ca  2 +  exchanger remains activated, there is little i f any competition o f released C a  S E R C A . According to Fig. 25 (representative trace), blockade o f the N a - C a +  together with S E R C A blockade caused an rapid decay in the S R C a  2 +  2 +  2 +  by the  exchange  content which  depleted the S R within 600 seconds in Ca -free PSS. I compared the various decay 2+  curves by plotting the amplitude o f the CAF-induced C a  2 +  transient over the amplitude o f  the high K response (%) versus time (seconds), as shown in F i g . 26. A t time zero, the +  greatest apparent rate o f loss o f S R C a Ca  2 +  in terms o f S R C a  2 +  content versus time in 0  P S S occurs after blockade o f the S E R C A and N a - C a  2 +  exchanger (filled triangles).  2 +  +  The next highest initial C a  release occurred after S E R C A blockade (filled squares),  then lastly the control (filled circles). A t the 150, 300, and 600 second time points the magnitude o f C a  2 +  release was similar under all o f the respective conditions. 87  r^r  +  300  CN  03  O  250  0)  200  03  150  + CN  03  o TJ  0) O 13 "O  100 H  50 0 0  03  O  200  400  600  800  2+,  Time in CPA+OCa* (sec.)  Fig. 24. SERCA blockade does not accelerate the decay of SR Ca after Ca removal. 2+  2+  T i m e c o u r s e c h a n g e s (in s e c o n d s ) w e r e r e c o r d e d a g a i n s t [ C a ] i (nM). C A F - i n d u c e d C a t r a n s i e n t s w e r e o b t a i n e d at 0, 1 5 0 , 3 0 0 , a n d 6 0 0 s e c o n d s in t i s s u e i n c u b a t e d with C P A a n d 0 C a P S S . These t r a c e s a r e t y p i c a l of f o u r e x p e r i m e n t s . 2+  2 +  2 +  88  +  CN  CO  O  400 350 300 -  CD  "(/) C 03  250 -  +  150 -  CN  03  o  200 100 -  "O  50 -  "O  0 -  (D  O  0  03  O  200  400  600  800  Time in CPA+0Na , 0Ca (sec.) +  2+  Fig. 25. Na -Ca exchanger + SERCA blockade does not change the decay of SR Ca content from Fig. 24. +  2+  2+  T h e e x p e r i m e n t in F i g . 2 4 w a s r e p e a t e d e x c e p t that C P A a n d 0 N a \ 0Ca P S S w e r e c o - a d m i n i s t e r e d into t h e t i s s u e b a t h . T h e s e t r a c e s a r e t y p i c a l of f o u r e x p e r i m e n t s . 2 +  89  o CO CD TJ  13 -*—>  t-  160 140  CPA+0Na CPA Control  E cc 120  o  +  100  00  <  O -•—> c  CD  -4—*  c  o  O  +  OJ CO  O  80 60 40 20 0 0  100 200 300 400 500 600 T i m e  i n  0 C a  2  ( s e c . )  +  CD  Fig. 26. Comparison of the decay in SR Ca content. 2+  C o m p a r i s o n of the S R C a c o n t e n t to c o n t r o l (n=4) f o l l o w i n g S E R C A b l o c k a d e b y C P A (n=4), a n d further b l o c k a d e of t h e Na -Ca e x c h a n g e r (n=4) b y r e m o v i n g e x t e r n a l N a ( C A F / 8 0 K a m p l i t u d e , % ratio). 2 +  +  +  2 +  +  90  H. Schematic Diagram #2-Ca  Cycling Dynamics  I have demonstrated that the N a - C a +  exchangers, some of which are coupled to the  2+  SERCA, as well as the P M C A are involved in C a illustrate C a  2 +  2+  extrusion from the IVC. In Fig. 27,1  cycling which is achieved by the buffering of intracellular C a  When the superficial SR releases C a  2 +  2 +  by the SR.  into the junctional space it is either extruded into  the ECS or re-sequestered by the SERCA.  In panel A , I propose a superficial C a  2 +  cycle that is due to SR C a  subsequent extrusion via closely coupled N a - C a +  by SERCA-mediated C a  2 +  facilitated by the N a - C a +  Ca  2 +  exchange, then C a  uptake. In panel B, active removal of C a  2+  takes place when both C a  2+  2 +  2 +  2 +  release and 2 +  influx followed  from the SR is  exchanger. In panel C, SERCA-mediated SR C a influx and the N a - C a +  re-uptake. In panel D, blocking N a - C a +  2+  2+  2 +  uptake  exchanger are inhibited resulting in  exchange and SERCA favours C a  2 +  extrusion via the P M C A which is likely located outside the junctional space. Therefore, a hierarchy has been established in the IVC to explain how C a transporters. The sequential order of access to the SR C a Na -Ca +  2+  2+  2+  cycles between the various  release channels is first the  exchanger, then SERCA, and lastly the P M C A .  I. Effects of Ca -sensitive K currents on the C a 2+  +  2+  signal 91  Control Na+/Ga / Exchange*^  0 'Ca  B  w  J+  PM  junctional*  \  0 Na , 0 Ca +  PM  A PMCA  ¥  2  space - "  restricted space  CPA, 0 Na , 0 Ca +  4  PMCA  Ca *Channel  Junctional 1  2+  2+  4  Na vCa V Exchange iQr +  PM  Nat/Ca Exchange 2+  ! Ca^Channel  l+  PMCA  junctional space C*. restricted  Fig. 27. Diagram of Ca cycling dynamics. 2+  In t h e c o n t r o l ( A ) , C i s p r e d o m i n a n t , i n v o l v i n g preferentially, asymmetrical C a release from the S R C a channels, IP R, a n d , R Y R , toward the E C S through N a - C a exchangers closely apposed to t h e j u n c t i o n a l s p a c e . T h e S E R C A i s l e s s s t r o n g l y c o u p l e d to t h e S R r e l e a s e c h a n n e l s a n d i s , t h e r e f o r e , t h e s e c o n d a r y c o m p o n e n t in c y c l i n g , b e i n g i n v o l v e d in r e - u p t a k e o f C a derived from the E C S , a n d t h e restricted s u b p l a s m a l e m m a l s p a c e . T h e least favoured p a t h w a y for C a * removal is via the P M C A , which is localized outside the n a r r o w d o m a i n s o f the S R a n d P M . T h e t i m e c o u r s e o f S R [ C a ] is s h o w n b e l o w a n d c o r r e s p o n d s with t h e a c t i v e c y c l i n g o f C a o c c u r i n g u n d e r r e s t i n g c o n d i t i o n s . In 0 C a P S S (B), C a re-uptake is a b o l i s h e d (i.e. C is a b s e n t ) , e v i d e n t in t h e d e c a y in S R C a . In 0Na , 0 C a P S S (C), the N a - C a e x c h a n g e r is b l o c k e d , a n d , c o n s e q u e n t l y , the current cycling m e c h a n i s m is v i a S E R C A ( i . e . Q ) . W i t h t h e f u r t h e r a d d i t i o n o f C P A (D), h o w e v e r , c y c l i n g i s c o m p l e t e l y eliminated a n d C a j removal is b e a r e d by the P M C A . e  2 +  2 +  3  +  2 +  2 +  2  2 +  2 +  2 +  2 +  2 +  e  +  2 +  +  2 +  2 +  92  It has been reported that the effects of SERCA blockers in certain types of S M are partially due to blockage of sparks and STOCs which may lead to cell depolarization and opening of VOCCs.  In this section, I tested if such an effect may have complicated the interpretation of my experiments. B K  C a  channels which are found in SM are activated by C a  2 +  and inhibited  by Charybdotoxin, Iberiotoxin (IbTX), and TEA; IKc channels are inhibited by a  charybdotoxin and T E A , and SKc channels by apamin. I recorded changes in [Ca ]; 2+  a  (nM) versus time-course (seconds) following treatment with either T E A or IbTX.  As shown in Fig. 28, the IVC was first exposed to NPSS for approximately 175 seconds then switched to T E A (3mM) prior to adding CPA (20uM) to determine whether SERCA blockade had any effect on the C a  2 +  gradient between the SR and plasmalemma  which could significantly alter K c channel activity. With CPA there was a delayed a  increase in [Ca ]; followed by a sustained plateau phase (note that this response to CPA was similar to that in the inset of Fig. 11). T E A had neither an effect on the basal or the elevated [Ca ]j levels. Even at higher concentrations of T E A there was no apparent change in [Ca ]i (data not shown). When the sequence for adding T E A and CPA was 2+  reversed, there was a similar lack of response to T E A (Fig. 29). When IbTX (70nM) was substituted for T E A there was also no apparent change in [Ca ]j (data not shown). 2+  93  250  i  200 150 +  CM  03  O  100  50 0  3mM TEA  NPSS  20LIM C P A  0 100 200 300 400 500 600 700 800 T i m e ( s e c o n d s )  Fig. 28. K channel blockade by TEA is insensitive to +  [Ca ]j. 2+  T h e e f f e c t s of T E A w e r e d e t e r m i n e d b y a 3 m M a p p l i c a t i o n of T E A prior to S E R C A b l o c k a d e b y C P A ( 2 0 > M ) r e c o r d e d a s [ C a ] i ( n M ) 2+  versus  time  (seconds).  This  trace  is  representative  of  five  experiments.  94  250 200 150 +  co 100  CM  o  50  0  NPSS  20uM C P A 3mM T E A  i—  r  1  1  1  r  0 100 200 300 400 500 600 700 800  Time(seconds)  Fig. 29. Effects of TEA following treatment with CPA. T E A ( 3 m M ) w a s a d d e d following S E R C A b l o c k a d e . r e p r e s e n t a t i v e of five e x p e r i m e n t s .  This trace  In conclusion, T E A and I b T X did not significantly affect the C a  signal, indicating  that for the rabbit I V C preparation, under resting conditions, either K c channels were not a  activated or possible activation o f K c channels was not responsible for the nature ofthe a  data obtained.  96  Chapter IV. DISCUSSION  A. Proof that the Na -Ca exchanger is implicated in SR-mediated C a +  Ca  2+  extrusion  extrusion is a physiological process which is essential for keeping the resting  [Ca ]i low to balance C a 2+  2+  2 +  entry that is due to the activity of a variety of C a  the presence of a steep electrochemical gradient for C a  In my thesis I hypothesized that the N a - C a +  2+  2+  2+  channels in  across the PM.  exchanger serves to extrude C a  N  2 +  released by the superficial SR. The idea of such a novel mechanism was first considered in a study conducted by Dr. van Breemen in 1979 (van Breemen, C. et al., 1979). Then in 1981, Aaronson and van Breemen observed that upon the removal of extracellular N a there was a transfer of a large quantity of C a  2 +  +  from the ECS into guinea-pig taenia coli  S M cells without any effect on resting tone (Aaronson, P. and van Breemen, C , 1981). This, as well as other observations, led to my specific hypothesis. On the basis of Na  +  fluxes and force measurements also in the guinea-pig taeni coli, Brading et al. (Brading, A.F. et al., 1980) reached the conclusion that N a - C a +  2+  exchange between the SR and  ECS took place at the locations where the P M and SR membranes made contact. However, her theory held that the N a - C a +  2+  exchanger had direct access to SR C a . 2+  Junctional areas are most frequently observed between the caveolar P M and superficial SR in both visceral (Gabella, G., 1971), and vascular SM (Devine, C.E. et al., 1972). 97  Blaustein and co-workers (Ashida, T., and Blaustein, M.P., 1987; Blaustein, M.P. et al., 1992; Borin, M . L . et al., 1994) confirmed the role of the N a - C a +  regulating SR Ca intracellular C a  2 +  2+  exchanger in  content by showing that removal of extracellular Na loaded an pool which was releasable by C A F , thapsigargin, and the agonist, 5-  HT.  Finally, recent advances in imaging techniques have allowed for a direct visualization of co-localization of the N a - C a +  2+  exchanger and the superficial SR. Moore et al.  (Moore, E.D.W. et al., 1993), using digital imaging fluorescence microscropy and a constrained deconvolution algorithm for processing the data showed co-distribution and suggested functional coupling ofthe N a - C a +  2+  exchanger and the SR in SM. This was  more recently confirmed by Blaustein and co-workers (Juhaszova, M . et al., 1994; Juhaszova, M . et al., 1996).  In light of the SBB theory which holds that a portion of incoming C a  2 +  is sequestered  by the peripheral SR before it diffuses into the deeper myoplasm it has been postulated that in order to maintain steady-state conditions it is necessary that the SR continuously unloads C a  2 +  into the subplasmalemmal space from where it is subsequently extruded  toward the ECS (van Breemen, C. et al., 1986; Stehno-Bittel, L. and Sturek, M . , 1992). The N a - C a +  2+  exchanger likely plays a very significant role in this unloading process (van  Breemen, C. and Saida, K., 1989; Chen, Q. and van Breemen, C , 1992). Taggart and 98  Wray (Taggart, M.J. and Wray, S., 1997) confirmed this observation in pregnant rat uterine S M compared to non-pregnant rat uterus by showing that the amplitude of the carbachol-induced [Ca ]; response, which is largely due to SR C a 2+  average 160% higher in 0Na , 0 C a +  2+  2 +  release, was on  PSS, compared to that in 0Ca PSS. 2+  My aim in the first portion of this study was to investigate if the SERCA, N a - C a +  exchanger and P M C A contribute to total cytoplasmic C a relative contributions are. Total impairment of C a SERCA, and mitochondria) and N a - C a +  2+  2 +  2+  2+  removal and if so what their  pumping activity (i.e. P M C A ,  exchange completely abolished C a  2 +  extrusion  as seen by a plateau in the elevated steady state [Ca ]j following high K stimulation and 2+  +  the subsequent perfusion with a Ca -free bathing solution. P M C A blockade was 2+  achieved through the reduction of ATP synthesis (using FCCP and IAA) (Hardin, C D . et al., 1992) which may also inhibit SERCA (Twort, C H . and van Breemen, C , 1988) and is reported to markedly reduce the forward-mode operation of the N a - C a +  2+  exchanger in  the squid axon (DiPolo, R., 1978). In determining the relative contribution of each ofthe distinct C a  2 +  translocators we found that ofthe total C a  2 +  extrusion which could be  blocked by complete metabolic inhibition, 47% was extruded by the N a - C a +  2+  exchangers  and 53% via the P M C A . Fay and co-workers, using the stomach SM of Bufo marinus, observed a Na -dependent [Ca ]i decline accounting for 38-52% of the total C a +  2+  2 +  removal between a [Ca ]i of 300 and 500nM (McCarron, J.G. et al., 1994). 2+  Although there is excellent agreement between the results quoted above, it is 99  necessary to consider the experimental conditions before extrapolation to physiological conditions in vivo. One of the complexities includes the use of multi-cellular preparations which, unlike single cells, exhibit an averaged response, but on the other hand do not require isolation with digesting enzymes that may alter cell physiology. The multi-cellular preparation may have contributed to the requirement for two exponential components in fitting the rates of [Ca ]j decline. However, the fact that several 2+  mechanisms contribute to C a  2 +  extrusion and that cellular C a  2+  redistribution may take  place would also tend to complicate the kinetics. For this reason we adopted a second method by measuring the instantaneous rate of [Ca ]i decline at a fixed elevated [Ca ]j 2+  2+  value which led to conclusions identical to those derived from measuring changes in the fast rate constant (data not shown).  The presence of an extrusion pathway between the SR and P M was suggested by Chen and van Breemen (Chen, Q. and van Breemen, C , 1993) who found that inhibition of Ca  2 +  accumulation by the SR in the rabbit IVC increases [Ca ]j which was not 2+  accompanied by an increase in C a  influx. Suzuki et al. (Suzuki, M . et al., 1992) have  2+  observed reduced Ca -activated K channel activity (an indicator for localized C a 2+  +  increases derived from the SR) in ileum SM treated with CPA suggesting that C a uptake is immediately followed by extrusion of C a  2 +  2 +  2 +  toward the ECS. In another study  (Petkov, G.V. and Boev, K . K . , 1996) it was also concluded that this unique extrusion pathway may indeed exist. There is supporting evidence for the idea that C a  2+  taken up  into the SR is later extruded through an interaction between the SR and the P M and that 100  when SERCA is blocked, cytosolic Ca not involved in SR-mediated C a participation of the SR in C a  2 +  2 +  is removed by an alternate extrusion mechanism  extrusion (Low, A . M . et al., 1993). The proposed  extrusion from the V S M is consistent with the  observations made in this study where SERCA blockade reduced the rate of [Ca ]i 2+  decline by 23%. A n alternative interpretation of this result which does not involve a multi-step C a  transport between SERCA and N a - C a  2 +  +  is accumulating and retaining cytoplasmic C a  2+  2+  exchange would be that the SR  during exposure to zero external C a . In 2+  this case, SERCA would function additively to N a - C a +  2+  exchange and the P M C A .  However, our finding that the effects of CPA and zero external N a were not additive is +  not consistent with this interpretation. Other findings reported in the results section also diminish the likelihood of simple SR accumulation and retention as a possible explanation. We found that there was no significant difference in CAF-sensitive SR C a  2 +  content when comparing the [Ca ]i decline under control and C P A conditions. Removal 2+  of external N a greatly enhanced SR C a +  2 +  retention but its abolition by C P A did not  significantly slow the rate of [Ca ]i decline. In parallel experiments by Chen and van 2+  Breemen (Chen, Q. and van Breemen, C , 1993) (using thapsigargin as a SERCA inhibitor in place of CPA) similar data were obtained. One reason for the high SR C a content in 0Na media may be that C a +  2+  2 +  released from the SR into the junctional space is  unable to leave the cell because of the blocked N a - C a +  2+  exchanger and is quickly  pumped back into the SR without being detected by cytoplasmic fiira-2 as Wolska and Lewartowski (Wolska, B . M . , and Lewartowski, B., 1993) demonstrated in guinea-pig hearts. 101  The results presented here are best explained by the model presented in Fig. 19. This model assumes that a fraction (=50%) of the N a - C a +  2+  the peripheral SR and functions mainly to remove C a  2+  exchangers is positioned close to from the junctional space  between P M and SR membranes (denoted [Ca ]j). [Ca ]j is thought to be elevated 2+  2+  above the bulk cytoplasmic [Ca ] (see ref. Stehno-Bittel, L. and Sturek, M . , 1992) 2+  because of asymmetric release or impaired diffusion away from the release sites. The low affinity, high velocity N a - C a +  2+  exchanger transports C a  2 +  from the junctional space  to the extracellular space. This assumption is supported by our observation that inhibition of this process allows re-uptake of C a evidence in support of SR-mediated C a  2+  2+  into the SR via SERCA. Additional  extrusion is presented by Sturek and colleagues  (Stehno-Bittel, L . and Sturek, M . , 1992) who also demonstrated the presence of peripheral Ca  gradients by simultaneous measurement of outward currents mediated by  Ca -sensitive K channels and global [Ca ]i, using fura-2. A functional relationship 2+  +  2+  between the peripheral SR and the N a - C a +  2+  exchanger is based on ultrastructural data in  which calsequestrin, an indicator for superficial SR, is co-distributed with the P M N a +  Ca  2 +  exchanger. The pathway for SR unloading relies on an asymmetrical arrangement  of SERCA and SR C a  2 +  release sites such that the C a  2+  taken up from the myoplasm is  9+  preferentially released by Ca  channels facing the junctional space rather than by those  facing the myoplasm. Additional evidence suggests that the N a - C a +  have selective access to SR C a  2 +  2+  exchanger may  (Juhaszova, M . et al., 1996). It has also been suggested 9+  that local IP3 gradients may promote the asymmetrical Ca release by the peripheral SR. 102  In summary, I have shown that the Na -Ca exchanger contributes to Ca extrusion from the SR in the IVC after elevation of [Ca ]j by depolarization and receptor activation 2+  (Nazer, M . A . and van Breemen, C , 1998a). The mechanism envisioned for this process is sequential C a exchange of C a  2+  2 +  uptake by the SR and release into the junctional space followed by for extracellular N a within this specific region (van Breemen, C. et al., +  1979; van Breemen, C. and Saida, K., 1989; Moore, E.D.W. et al., 1991; van Breemen, C. et al., 1995). It is likely that cytoskeletal structures create a linkage between the SR Ca  2 +  release channel and the N a - C a +  2+  exchanger allowing it to function as a " C a  2+  release-exchange duo-porter".  In the second portion of my study I followed the decline of SR C a values after removal of extracellular C a mechanism(s) by which the N a - C a +  2+  2 +  2 +  from resting  in order to investigate the underlying  exchanger induces changes in SR C a  2+  accumulation and release. In keeping with the developments of previous studies our results provide evidence for C a  2+  cycling between the SR and ECS via the junctional and  restricted cytoplasmic micro-domains. The rapid loss of SR C a  2 +  into Ca -free medium 2+  observed under control conditions was dependent on extracellular Na . When the Na +  Ca  2 +  exchanger is blocked, C a  This implies that C a Ca  2 +  2 +  2 +  +  remains in the SR as long as SERCA is in operation.  release under resting conditions is preferentially toward the N a +  exchanger, but when the latter is blocked C a  2 +  spills over to the SERCA for re-  uptake into the SR. However, when both the N a - C a +  2+  exchanger and SERCA are 103  blocked, Ca released from the SR under resting conditions spills over past the Na -Ca 2+  +  2+  exchanger and the SERCA to be extruded by the PMCA. From this we can deduce that the order of proximity to the SR Ca release channels which are active at rest is as 2+  follows: Na -Ca exchanger>SERCA>PMCA. Previously, our laboratory proposed the +  2+  existance of three cytoplasmic domains: (1) the junctional space which is involved in unloading the SR; this narrow space would provide access to both SR Ca release 2+  channels and the Na -Ca exchanger; (2) the restricted subplasmalemmal space from +  2+  which incoming Ca is sequestered into the SR via the SERCA and, (3) the cytoplasmic 2+  space which may be in contact with the PMCA and which constitutes the bulk of the cytoplasm (van Breemen, C. et al., 1995). Thus, in resting SM bathed in NPSS, Ca  2+  cycles between the SR lumen and the ECS via the junctional space, PM, restricted space, and SERCA, respectively. As Fig. 27 illustrates, the process is hierarchical since rapid cycling between the SR and ECS (referred to by the symbol, C ) occurs more readily then 0  SERCA pumping the released Ca directly back into the SR (abbreviated, Cj) thereby, 2+  eliminating the extrusion component. At rest, the Na -Ca exchanger-driven cycle, C , +  2+  0  dominates over the 'deep' intracellular cycle, Q.  Miyashita et al. (Miyashita, Y. et al., 1997) recently proposed that p -adrenergic 2  stimulation with isoproterenol in rat tail artery decreases the CAF-releasable intracellular Ca pool by enhancing Ca extrusion via the Na -Ca exchanger. They based their 2+  2+  +  2+  hypothesis on the finding that removal of extracellular Na prevented the SR depleting +  effect of isoproterenol. Thus, it appears that p -adrenergic stimulation enhances SR2  104  mediated Ca^ removal from the cytoplasm by both cAMP-mediated stimulation of SERCA, and by the unloading of SR C a  2+  toward the ECS via activation of the N a - C a +  2+  exchanger by cAMP-mediated stimulation of the N a - K ATPase. On the other hand, it +  +  is well established that a-adrenergic stimulation results in IP3-mediated release of SR Ca  2 +  into the cytoplasm to induce contraction (Lepretre, N . et al., 1994).  The results presented and discussed, herein, thus raise the possibility that the direction of the slow, sustained release during rest (toward the junctional space) is different from the rapid SR C a  2 +  release during activation of phospholipase C which is toward the bulk  of the cytoplasm. It will be of great interest to investigate whether the asymmetry in release is based on specific distribution of the two types of C a  2 +  release channels (RYRs  and IP Rs). 3  My data strongly suggest differential localization ofthe two major C a mechanisms: P M C A and N a - C a +  2+  2+  extrusion  exchange. The preferential access of the N a - C a +  2+  exchanger to the superficial SR relegates it to the junctional areas. Since the P M C A only gained access to C a  2 +  released from the SR when both the N a - C a +  2+  exchanger, and the  SERCA were blocked it is probably located in areas of the P M which are not closely apposed to the SR. In this respect, it is of interest that in ECs the P M C A is reported to be absent from the narrow boundaries between the endoplasmic reticulum and the P M (Cabello, O.A. and Schilling, W.P., 1993); moreover in mesenteric arteries, the P M C A is not co-localized to DiOC staining which is an SR marker. The data presented in Fig. 23 105  suggest a process of redistribution of SR Ca . When removal of Ca junctional space by the N a - C a +  appeared to shift C a of N a - C a +  Ca  2 +  2+  2+  2+  exchanger was blocked, C a  2+  from the  cycling in the SR  from the deeper to the superficial SR since subsequent reactivation  exchange stimulated an accelerated loss of C a  2+  from the caffeine-releasable  pool.  In considering any specific mechanism investigated in a specific type of SM, it is best to remain cautious with respect to suggesting applicability to all types of SM. In the case of the rabbit IVC, the results could be interpreted in the manner described above, since we found little effect of blockade of Ca -sensitive K channels with either T E A or 2+  +  Iberiotoxin, suggesting an insignificant role for this channel in the junctional areas ofthe cell. Although experimental data support the interaction between the N a - C a +  and SR Ca  2+  2+  exchanger  transport, the detailed mechanism of this process is yet to be determined.  B. Update and New Directions in VSM Research My thesis research and publication of two articles was done during the period 1994 to 1998. At completion of research and articles my contribution to SM research consisted of identifying the N a - C a +  2+  exchanger as the P M C a  2+  transport protein that  communicated with the superficial SR and SERCA. In late October of 1998 I fell ill and was forced to take time off to recover from my symptoms brought on by mental illness. Approximately four years had passed before I was again able to complete the written 106  component of my thesis. Meanwhile, my research colleagues had made important steps toward better understanding the function of the SR in the IVC that includes a role for SOCs in inducing refilling of the SR with C a  2+  via direct activation of reverse N a - C a +  2+  exchange. I will review recent data from Dr. van Breemen's lab including confirmation of PM-SR junctional complexes through morphological data and resulting C a  2 +  oscillations.  Following my publications in 1998, there was re-newed interest in the V S M SR and with the lab's newly purchased confocal imaging microscope my colleagues were able to detect repetitive Ca release from the SR in the rabbit IVC. In so doing, Lee et al. (Lee, C-H. et al., 2002a) found that the inter-connected superficial SR and deep SR have distinct specialized functions and not only that, the former can shuttle C a  2+  to the latter  via the radial/perpendicular SR, thereby making the P M an ideal source of C a  2 +  for  uptake into the SR (via SERCA), making the myofilaments which are predominantly in the deep myoplasm, the receptor of C a and  IP3RS).  2 +  via C a  2 +  release from the deep SR (via RYRs  Agonist-induced stimulation (in which the SR was directly implicated) was  found to generate cyclic C a  2 +  waves that propogated throughout the whole length of a  V S M cell but were not synchronized with respect to neighboring cells (Ruehlmann, D.O. et al., 2000). Perturbation of the PM-SR junctional complexes with calyculin-A completely abolishes C a  2 +  waves, and thereby inhibits the  triggered by repetitive SR C a  2+  [Ca ]j 2+  oscillations which are  release and uptake (Lee, C-H. et al., 2002a). Cytoskeletal  scaffolding structures such as the putative "junctophilin" anchoring proteins found in 107  cardiac muscle (Takeshima, H. et al., 2000) (or "feet" injunctions of skeletal muscle) may be similar to proteins that stabilize the junctional complexes in V S M . In the rabbit IVC, the summation of asynchronous C a  2+  waves from underlying individual SM cells  evokes tonic contractions i.e. vasoconstriction, encompassing the entire blood vessel wall. In rat mesenteric artery with intact endothelium, these asynchronous waves can initiate rhythmic contractions or vasomotion by activating L-type VOCCs then repolarizing the P M simultaneously in a sufficient number of individual cells so as to become synchronized (Peng, H. et al., 2001). Agonist-induced [Ca ]i oscillations are 2+  primarily dependent on the SR for their maintainance since SR store depletion by SERCA blockade can abolish them. In order to initiate a C a  2+  wave, IP3RS on the SR  membrane must open-up, thereby releasing C a , some of which is extruded to the ECS. 2+  The partially depleted SR is replenished by several possible mechanisms, including the L-type V O C C , a non-selective cation channel we propose to be a SOC-type channel, and the Na -Ca exchanger operating in reverse-mode. In the adult IVC, the V O C C , a major source of C a  2 +  in resistence arteries, is not so relevant since nifedipine, an L-type V O C C  blocker, only reduced the frequency of [Ca ]i oscillations not the amplitude, whereas, the 2+  Na -Ca +  2+  exchanger and SOC play a much greater role in [Ca ]j oscillations since 2+  blockade of either one eliminates the oscillations (Lee, C-H. et al., 2002a). Lee et al. (Lee, C-H. et al., 2001; Lee, C-H. et al., 2002a; Lee, C-H. et al., 2002b) have postulated that [Ca ]i oscillations in the rabbit IVC are caused by the following order of events: 1) PE stimulation of IP3 release; 2) activation of the IP3R and release of C a  2 +  near the  myofilaments; 3) opening ofthe SOC to the inward diffusion of N a and C a +  2+  into the 108  junctional space in response to depletion of the SR; 4) cell membrane depolarization and opening of L-type VOCCs along with activation of reverse-mode N a - C a +  pump C a  2 +  from the ECS into the cell; and 6) SR uptake of junctional C a  SERCA. It is very likely that the SOC, N a - C a +  2+  2+  exchange to via the  2+  exchanger and SERCA are co-localized  in regions surrounding the PM-SR junction which would be crucial for the regenerative nature of C a  2 +  waves (i.e. refilling of the SR) (Lee, C-H. et al., 2002a). Notice that this  active cycle is essentially the reverse of the resting C a  2+  cycle described in my thesis  research. The downstroke of the [Ca ]; oscillation may be due to removal of C a 2+  the myoplasm via activation of the SERCA, forward-mode N a - C a +  2+  2+  from  exchange and the  P M C A as my experiments have shown (see Nazer, M . A . and van Breemen, 1998a; Nazer, M . A . and van Breemen, 1998b). At the end of the C a  2 +  oscillation cycle, the SR  is replenished and the SOC closes and repolarizes the P M causing the VOCCs to close. Spillover of C a  2 +  from the SR increases the [Ca ]j and once it has reached a certain 2+  threshold, IP3RS and possibly RYRs are activated, propogating another C a  2+  equal amplitude to the previous wave, indicating repetitive transient SR C a  2 +  wave of release  (Lee, C-H. et al., 2002a). [Ca ]j oscillations are beneficial for several reasons: 1) 2+  possibly because prolonged, high [Ca ]i damages cells, 2) SR C a 2+  Ca  2 +  influx is more efficient in providing C a  2+  2+  release, rather than  to the myofilaments which lie deeper  within the myoplasm taking advantage of the radial SR to transfer C a  2 +  from the  superficial SR to the deep SR, 3) many intracellular enzymes are responsive to the frequency of [Ca ]i oscillations, and, 4) lastly, C a 2+  2 +  sensors such as calmodulin are  dependent on the peak of the oscillation, providing signaling at relatively low C a  2 +  109  concentrations (Lee, C-H. et al., 2002a).  Dr. van Breemen's lab has, therefore shown that PE-induced contractions of the IVC are activated by recurrent C a influx through N a - C a +  2+  2 +  waves which involve C a  2 +  release from the IP3R and  exchange, SOC and V O C C .  Currently, I am investigating the nature of development of PM-SR junctions in the rabbit IVC beginning at birth and capturing the structural/functional organization that takes place when the IVC differentiates into a fully mature tissue. In experiments I conducted using the IVC of neonatal rabbits, I found that asynchronous C a  2+  waves were  not present. This is indicative of PM-SR junctional complexes that are either not yet fully formed or are altogether absent at this stage of development in the rabbit. This was supported by electron micrographs of IVCs of 10 day old rabbits taken by Dr. Kuo-Hsing Kuo (data not shown) which showed few extensions ofthe SR directly underneath the P M , although radial and deep SR was present; even caveolae which are thought to contain the N a - C a +  2+  exchanger and SOC were sparse especially on the non-luminal side  of the vessel. If the superficial SR is absent then C a  2 +  cannot be taken-up and released  toward the myofilaments, instead, it is believed the SR is bypassed and C a directly supplies the myofilaments with C a  2 +  2 +  entry  for contraction. I have shown that PE-  induced contractions of the adult IVC (60-180 days) are only partially sensitive to blockade of VOCCs by nifedipine, while this contraction in the neonate IVC (0-15 days) is completely blocked by this same agent (unpublished data). Thus, in the neonate IVC, 110  receptor activation is mostly dependent on the opening of L-type VOCCs. The difference between the neonate and adult IVCs becomes even more striking when comparing their responses to blockade of the IP3R by 2-APB. Blocking C a  2 +  release from the IP3R in the  adult (60 day) causes immediate relaxation while in the neonate (10 day), 2-APB can induce contraction (unpublished data). As reported before, blockade of the IP3R in the adult IVC prevents release of activating C a  2+  near the myofilaments and consequently,  opening of SOCs.  In conclusion, my findings support a role for the V S M SR in C a sequential coupling of SERCA, C a  2+  2 +  extrusion by a  release channels, and the N a - C a +  Furthermore, we found evidence in the rabbit IVC for rapid C a  2 +  2+  exchanger.  cycling, at rest, between  the ECS and SR lumen via vectorial release into the junctional space, extrusion to the ECS in exchange for N a influx, entry through the C a +  2 +  leak pathways, and finally, re-  uptake by SERCA. We have also proven, based on ultrastructure and functional data, that PM-SR junctional complexes are abundant in the adult IVC and are necessary for cross-talk between the N a - C a +  2+  exchanger and the SR C a  2 +  release channels in contrast  to the neonatal IVC which lacks developed junctions.  Ill  REFERENCES  1998 Receptor and ion channel nomenclature. 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