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The function and the mechanism of agonist-induced asynchronous wave-like [Ca²⁺]i oscillations in the… Lee, Cheng-Han 2004

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The function and the mechanism of agonist-induced asynchronous wave- like [Ca2+]i oscillations in the in situ smooth muscle cells of the rabbit inferior vena cava by CHENG-HAN L E E B.Sc, The University of British Columbia, 1997 A THESIS SUBMITTED IN PARITAL FULFILMENT OF T H E REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in T H E F A C U L T Y OF GRADUATE STUDIES Department of Pharmacology & Therapeutics; Faculty of Medicine We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA Date: J"»"woy , ^QQ^ © Cheng-Han Lee, 2003 Abstract Background:' [Ca2+]j imaging studies of in situ vascular smooth muscle cells (VSMC) in agonist-stimulated arterial blood vessels have revealed asynchronous wave- like [Ca2 +]i oscillations as an important signaling mechanism. However, the functions and the mechanisms of these asynchronous wave-like [Ca ]i oscillations remain poorly defined. Given the importance of [Ca \ in determining vascular tone, understanding the functions and the mechanisms of this particular type of [Ca ]j oscillations is crucial to our understanding of excitation-contraction coupling in vascular smooth muscle (VSM). Methods: Confocal [Ca2+]j imaging of in situ VSMC from endothelium-denuded rabbit inferior vena cava (IVC) was employed to examine the subcellular C a 2 + responses to drugs. This was compared at times with whole-vessel spectrophotometric [Ca2 +]i measurement which assessed the averaged C a 2 + response from hundreds of VSMC. Isometric contraction studies were performed in parallel to examine the contractile effects of the same series of drugs. RT-PCR studies were utilized to verify the expression of implicated molecules. Furthermore, whenever applicable, parallel electron microscopy (EM) studies were also performed in parallel to correlate the disruption of V S M C ultrastructure to disruption in C a 2 + signaling. ii Results & Conclusions: The rabbit IVC is a large capacitance vessel that displays typical contractile dose-response curves for caffeine and PE. We observed that both caffeine and phenylephrine (PE) initially elicited C a 2 + waves in individual in situ V S M C of the IVC. The [Ca2+]i in cells challenged with caffeine subsequently returned to baseline while the [Ca ]i in cells challenged with PE exhibited repetitive asynchronous C a 2 + waves. The lack of synchronicity of the wave-like [Ca2+]i oscillations between V S M C can explain the observed tonic contraction at the whole-tissue level. The nature of these Ca waves was further characterized. For caffeine the amplitude was all-or-none in nature, with individual cells differing in sensitivity, leading to their recruitment at different concentrations of the agonist. This concentration-dependency of recruitment appears to form the basis for the concentration-dependency of caffeine-induced contraction. Furthermore, the speed of Ca waves correlated positively with the concentration of caffeine. In the case of PE, we observed the same characteristics with respect to wave speed, amplitude and recruitment. Increasing concentrations of PE enhance the frequency of the [Ca2+]j oscillations. We therefore concluded that PE stimulates whole-tissue contractility through differential recruitment of VSMCs and enhancement of the 9-t-frequency of asynchronous wave-like [Ca ]; oscillations once the cells are recruited. We then characterized the mechanisms of the asynchronous wave-like [Ca2+]j oscillations 9+ 9+ in response to PE. It was found that the Ca waves are initiated by Ca release from the ryanodine- and caffeine-sensitive sarcoplasmic reticulum (SR) Ca store via inositol-1,4,5-trisphosphate-sensitive SR C a 2 + release channels (IP3R) and that refilling of the SR iii Ca store through sarcoplasmic/endoplasmic reticulum Ca ATPase (SERCA) using C a 2 + derived from an extracellular source is required for maintained generation of the repetitive C a 2 + waves. Blockade of L-type voltage-gated C a 2 + channel (VGCC) with nifedipine reduced the frequency of PE-stimulated [Ca2+]j oscillations, while additional blockade of receptor-operated channel/store-operated channel (ROC/SOC) with SKF96365 abolished the remaining oscillations. Parallel force measurements showed that nifedipine inhibited PE-induced tonic contraction by 27% while SKF96365 abolished it. This indicates that stimulated C a 2 + entry refills the SR to support the recurrent waves of SR C a 2 + release and that both L-type V G C C and ROC/SOC contribute to this process. More interestingly, applications of Na + -Ca 2 + exchanger (NCX) inhibitors, 2',4'-dichlorobenzamil (forward- and reverse- mode inhibitor) and KB-R7943 (reverse-mode inhibitor) completely abolished the nifedipine-resistant component of [Ca2+]j oscillations and markedly reduced PE-induced tone. It thus appears that Na + entry through a putative non-selective cation channel (NSCC) result in elevated local [Na+] in a sub-plasmalemmal restricted space (PM-SR junctional space). The elevated junctional [Na+] subsequently facilitates Ca entry through the N C X operating in the reverse-mode which refills the SR and maintains PE-induced [Ca ]i oscillations. The nature of this putative NSCC remains unresolved as it could either be a ROC or a SOC. We performed additional studies using 2-APB to further characterize this putative NSCC. We showed that the application of 2-APB to antagonize IP3R-channels can prevent the initiation and abolish ongoing PE-mediated tonic constriction of the IVC by inhibiting the generation of these [Ca2+]i oscillations. The observed effects of 2-APB can iv only be attributed to its selective inhibition on the IPaR-channels, not to its slight inhibition on the L-type V G C C and the SERCA. Furthermore, 2-APB had no effect on the ryanodine-sensitive C a 2 + release channel (RyR) and SOC in the IVC. These results indicate that the putative NSCC involved in refilling the SR and maintaining the tonic contraction is most likely an SOC-type channel as it appears to be activated by IP3R-channel mediated SR C a 2 + release or store depletion. This is in accordance with its sensitivity to N i 2 + and L a 3 + (SOC blockers). More interestingly, RT-PCR analysis indicates that Trpl mRNA is strongly expressed in the rabbit IVC. The Trpl gene is known to encode a component of the store-operated NSCC. These data suggest that the activation of both the IP3R and the SOC (NSCC) are required for PE-mediated [Ca2+]i oscillations and constriction of the rabbit IVC. Furthermore, the activation of the SOC-type NSCC and the reversal of the N C X may depend on the presence of the PM-SR junctions. E M of V S M C from the rabbit IVC indicates about 14.2% of the plasma membrane (PM) is closely apposed by the prominent superficial SR network, forming a flattened PM-SR junctional space with a diameter reaching as long as 1 pm. Application of calyculin-A resulted in a concentration-dependent dissociation of the superficial SR and loss of PM-SR junctions. This progressive loss of the PM-SR junctions is correlated with progressive inhibition of the wave-like [Ca2+]i oscillations. These findings reveal the importance of the PM-SR junctions in the generation of wave-like [Ca ]\ oscillations, which underlie excitation-contraction coupling of the VSMC. In regard to the mechanism of PE-mediated wave-like [Ca ]; oscillations, we conclude OA- OA-that each Ca wave depends on initial SR Ca release via IP3R-channels followed by SR C a 2 + refilling through SERCA. A putative SOC-type NSCC is activated as a result of IP3R-mediated SR C a 2 + release or store depletion. Na + entry through a putative SOC-type NSCC then accumulates in a restricted sub-plasmalemmal space (PM-SR junctional space). The elevated junctional [Na+] facilitates C a 2 + entry through the N C X operating in the reverse-mode which refills the SR and maintains PE-induced [Ca2+]; oscillations. In addition some C a 2 + entry through L-type V G C C and NSCC serves to modulate the frequency of the oscillations and the magnitude of force development. vi TABLE OF CONTENTS ABSTRACT ii TABLE OF CONTENTS vii LIST OF TABLES x LIST OF FIGURES xi ABBREVIATIONS xiii PREFACE xiv ACKNOWLEDGEMENTS xviii CHAPTER I: INTRODUCTION 1 Role of VSM in the body physiology and pathophysiology 1 Role of Ca2+ in excitation-contraction coupling of VSM 2 Ca regulation in VSM 2 Biphasic model of agonist-mediated [Ca2+]i signaling and contraction in VSM 3 Pitfalls of single cell study and advantage of intact vessel study 4 Emergence of adrenoceptor-mediated asynchronous wave-like [Ca2*]t oscillations in intact arterial blood vessels 5 CHAPTER II: SPECIFIC AIMS 10 Specific aim #1: Examination of PE-mediated sub-cellular Ca2+ signal in in situ VSMC within the intact media of the rabbit IVC. 10 Specific aim #2: Correlation of PE-mediated asynchronous wave-like [Ca2+]i oscillations to contractile function in the rabbit IVC 10 Specific aim #3: Examination of the mechanism of PE-mediated asynchronous wave-like [Ca2+]i oscillations in the VSMC of the rabbit IVC 11 Specific aim #4: Examination the requirement for PM-SR junctions in the generation of wave-like [Ca2+]j oscillations in PE-stimulated rabbit IVC 11 CHAPTER III: MATERIALS AND METHODS. 12 Tissue isolation and preparation 12 Isometric contraction study 12 Spectrophotonmetric [Ca2+]j measurement 13 Laser scanning confocal [Ca2+]t imaging 13 Confocal image and Data analysis 14 RT-PCR study 15 Electron microscopy 17 Materials 17 CHPATERIV: EXAMINATION OF PE-MEDIATED VSMC C A 2 + SIGNAL AT WHOLE-TISSUE AND SUB-CELLULAR L E V E L OF THE RABBIT IVC 21 vii Removal of the endothelium 21 Examination of PE-induced and caffeine-induced Ca2* signal at whole-vessel Level 22 Examination of PE-mediated and Caffeine-mediated Ca2* signal at sub-cellular Level 23 CHAPTER V: CORRELATION OF PE-MEDIATED ASYNCHRONOUS WAVE-LIKE {CA2+]i OSCILLATIONS TO FORCE GENERATION 30 Concentration dependence of PE/caffeine-induced force generation 30 Concentration dependence of caffeine-induced Ccr + signal 31 Correlation of PE-induced asynchronous wave-like [Ca2*]! oscillations to force Generation 32 CHAPTER VI: ELUCIDATION OF THE MECHANISM OF PE-MEDIATED WAVE-LIKE [CA2 +]i OSCILLATIONS 43 Importance of the SR Ca2* store, SERCA, IP3R and Ca2* entry in PE-mediated wave-like [Ca2* ] i oscillations 43 Components of stimulated Ca2* entry 45 Characterization of the NSCC 48 CHAPTER VII: REQUIREMENT FOR SUPERFICIAL SR AND PM-SR JUNCTION IN PE-MEDIATED WAVE-LIKE [CA2 +]i OSCILLATIONS 77 CHAPTER VIII: DISCUSSION 86 Characteristics of PE-mediated [Ca2*]! signals 86 Correlation of [Ca2*], signals with the development of tension 87 Mechanism of PE-mediated wave-like [Ca2*]! oscillations 89 Requirement for superficial SR and PM-SR junctions in PE-mediated wave- like [Ca2*]j oscillations in the rabbit IVC 98 CHAPTER IX: OVERVIEW OF ASYNCHRONOUS WAVE-LIKE [CA2 +]i OSCILLATIONS AND THEIR FUNCTIONAL IMPLICATIONS IN VSMC 102 Ultra-structure of VSMC 104 Asynchronous Ca2* waves and vasoconstriction 106 Asynchronous Ca2* waves and vasodilatation 107 Mechanism of wave-like [Co2* ], oscillations 109 Possible advantage of wave-like [Ca2*], oscillations 112 Wave-like [Ca2*]t oscillations and mitochondrial Ca2* signaling 113 Wave-like [Ca2*], oscillations and nuclear Ca2* signaling 116 CHAPTER X: CONCLUSIONS AND FUTURE DIRECTIONS 121 BIBLIOGRAPHY 123 viii LIST O F T A B L E S Table 1: Oligonucleotide sequences of the primers used for RT-PCR 16 Table 2: Summary of reported asynchronous wave-like [Ca2+]j oscillations 103 in in situ VSMCs ix LIST O F FIGURES Figure 1: C a 2 + , calmodulin, M L C K and cross-bridge cycle in smooth 7 muscle Figure 2: C a 2 + translocating molecules in V S M C 8 Figure 3: Biphasic model of contractile activation 9 Figure 4: Schematic depiction of the experimental techniques 19 Figure 5: Derivation of Y-t plot, X-t plot and the 3 x 3 pixels region on the 20 confocal image Figure 6: Removal of endothelium confirmed with nuclei staining 25 Figure 7: Whole-vessel [Ca2+]i response to PE and caffeine 26 Figure 8: Fluo-3 loaded in situ VSMCs of the rabbit IVC visualized under 27 confocal microscopy. Figure 9: Sub-cellular [Ca2+]j signal elicited with PE and caffeine 28 visualized with confocal time-series images Figure 10: Contractile responses and concentration-dependence curves with 34 PE and caffeine Figure 11: Fixed-amplitude Ca waves and concentration-dependent 35 recruitment in response to caffeine Figure 12: Concentration-dependence of the apparent velocity of caffine- 37 induced C a 2 + wave. Figure 13: Fixed-amplitude Ca 2 +waves and concentration-dependent 38 recruitment in response to PE Figure 14: Concentration-dependent frequency of PE-induced wave-like 40 [Ca2+]i oscillations Figure 15: Concentration-dependence of the apparent velocity of PE- 42 induced recurring C a 2 + wave. Figure 16: Spatio-temporal pattern of C a 2 + signaling 55 Figure 17: Effects of SR depletion with caffeine or ryanodine on PE- 57 mediated [Ca2+]i oscillations Figure 18: Effects of CPA on PE-mediated [Ca2+]i oscillations 58 Figure 19: C a 2 + entry is required to sustain repetitive SR-mediated IP3R- 59 dependent C a 2 + waves. Figure 20: Effects of L-type V G C C blockade on 80mM [K +] e xt mediated 60 tonic contraction in the rabbit IVC. Figure 21: Effects of L-type V G C C blockade and ROC/SOC blockade on 61 PE-mediated [Ca2+]j oscillations. Figure 22: Effects of L-type V G C C blockade and ROC/SOC blockade on 63 PE-mediated tonic contraction. Figure 23: Effects of N C X inhibitor 2',4'-dichlorobenzamil on the 64 nifedipine-resistant component of PE-mediated [Ca2+]i oscillations and tonic contraction. Figure 24: Effects of selective reverse-mode N C X inhibitor KB-R7943 on 66 the nifedipine-resistant component of PE-mediated [Ca 2 +j\ oscillations and tonic contraction. x Figure 25. Effects of 75 pM 2-APB on the initiation and the maintenance of 68 PE-induced V S M C [Ca2+]j oscillations and venoconstriction in the rabbit JVC. Figure 26. Effects of 75 uM 2-APB on RyR, SERCA, L-type V G C C and 70 SOC in the rabbit rVC. Figure 27. Effects of N i 2 + and L a 3 + on the nifedipine-resistant component of 73 PE-mediated [Ca2+]j oscillations and tonic contraction of the rabbit IVC. Figure 28. Trpl and aic mRNA expression in the smooth muscle of the 75 rabbit IVC. Figure 29: Ultrastructure of V S M C of the rabbit IVC revealed with electron 82 microscopy. Figure 30. Correlation between the disruption of the PM-SR junctions and 84 the inhibition of PE-mediated wave-like [Ca2+]i oscillations in V S M C of the rabbit IVC. Figure 31. Hypothetical sequence of events during PE-mediated smooth 100 muscle [Ca2+]i oscillations in rabbit IVC. Figure 32: Myosin-poor space and myosin-rich myoplasm in V S M C of the 116 rabbit IVC. Figure 33: Model for C a 2 + movements during wave-like [Ca2+]i oscillations 118 and during resting state in V S M C . xi ABBREVIATIONS 2-APB: 2-aminoethoxydiphenyl borate [Ca2+]j: intracellular cytoplasmic C a 2 + concentration [Ca2+]jS: cytoplasmic C a 2 + concentration in the PM-SR junctional space 94- 94-[Ca ]n: Ca concentration in the nucleus cADPr: cyclic-ADP ribose CICR: Ca2+-induced C a 2 + release CPA: cyclopiazonic acid DMSO: dimethyl sulfoxide E-C coupling: excitation-contraction coupling E M : electron miscroscopy ER: endoplasmic reticulum IP3: inositol-1,4,5 -trisphosphate IP3R: inositol-1,4,5-trisphosphate-sensitive SR C a 2 + release channels IVC: inferior vena cava [K+]ext: extracellular K + concentration Mito: mitochondria M L C K : myosin light chain kinase [Na+]jS: cytoplasmic Na + concentration in the PM-SR junctional space NAADP: nicotinic acid-adenine dinucleotide phosphate NCX: Na + -Ca 2 + exchanger NSCC: non-selective cationic channel PE: phenylephrine PLC: phospholipase C PM: plasma membrane PMCA: plasma membrane C a 2 + ATPase PSS: physiological salt solution ROC: receptor-operated channel RT-PCR: reverse transcriptase polymerase chain reaction RyR: ryanodine-sensitive C a 2 + release channel SERCA: sarcoplasmic/endoplasmic reticulum C a 2 + ATPase SKF963 65: 1 - {0- [3-(4-methoxyphenyl) propoxy] -4-methoxy-phenethyl} -1H-imidazole hydrochloride SOC: store-operated channel SR: sarcoplasmic reticulum Tip: transient receptor potential channel VGCC: voltage-gated C a 2 + channel VSM: vascular smooth muscle VSMC: vascular smooth muscle cell xii P R E F A C E Material from this dissertation has been published or accepted in the following journals: • Lee CH, Poburko D, Kuo K H , Seow CY, van Breemen C. C a 2 + oscillations, gradients and homeostasis in vascular smooth muscle. American Journal of Physiology - Heart and Circulatory Physiology 2002;282:H1571-H1583. • Lee CH, Rahimian R, Szado T, Sandhu J, Poburko D, Behara T, Chan L, van Breemen C. Sequential opening of IP3-sensitive C a 2 + channels and SOC during ai-adrenergic activation of rabbit vena cava. American Journal of Physiology - Heart and Circulatory Physiology 2002;282: H1768-H1777. • Lee CH, Poburko D, Kuo K H , Seow CY, van Breemen C. Relationship between the sarcoplasmic reticulum and the plasma membrane. Novartis Foundation Symposium 2002;246:26-41 and discussion 41-7;48-51. • Lee CH, Poburko D, Sahota P, Sandhu J, Ruehlmann DO, van Breemen C. The mechanism of phenylephrine-mediated [Ca ]i oscillations underlying tonic contraction in the rabbit inferior vena cava. Journal of Physiology 2001;534:641 -650. • *Ruehlmann DO, *Lee CH, Poburko D, van Breemen C. Asynchronous Ca waves in intact venous smooth muscle. Circulation Research 2000;86(4):E72-9. (*: co-first-authors) Material from this dissertation has been submitted to the following journals: • Lee C H * , Kuo K H * , Seow CY, van Breemen C. The ultrastructural basis of phenylephrine-mediated [Ca2+]j oscillations in rabbit venous smooth muscle. Submitted to Circulation Research. (*: co-first-authors) Material from this dissertation has been presented in oral form at the following international and national meetings: • Lee CH, Crowley C M , van Breemen C. Calcium signaling in venous smooth muscle. 8th International Symposium on Mechanism of Vasodilation. May 31-June 3, 2001. Boston, Massachusetts. • van Breemen C, Lee C H * , Poburko D, Sandhu J, Sahota P. Mechanism and function of asynchronous C a 2 + waves in vascular smooth muscle. The 45 t h Biophysical xiv Society Annual Meeting. February 17-21, 2001. Boston. Massachusetts. (*: presenting author) • Lee CH, Sandhu J, Poburko D, Sahota P, van Breemen C. Asynchronous C a 2 + waves regulate vessel contractility. 29 th Western Student Medical Research Forum, Cardiovascular I. February 7-10, 2001. Carmel, California. • Lee CH, Poburko D, Ruehlmann DO, Sahota P, Sandhu J, van Breemen C. Asynchronous Ca waves regulate vascular contractility. Joint program for clinical scientist in training and MD-PhD students. Canadian Institutes of Health Research & Canadian Society for Clinical Investigation. September 21, 2000. Edmonton, Alberta. • Lee C H , Poburko D, Ruehlmann DO, van Bremen C. From Ca waves to tonic contraction in blood vessels. 28th Annual Western Student Medical Research Forum, Cardiovascular I. February 9-12, 2000. Carmel, California. Material from this dissertation has also been presented in poster form at the following international and national meetings: • Lee CH, Crowley C M , van Breemen C. Calcium signaling in venous smooth muscle. Joint program for clinical scientist in training and MD-PhD students. Canadian xv Institutes of Health Research & Canadian Society for Clinical Investigation. September 20, 2001. Ottawa, Ontario. Lee CH, Ruehlmann DO, Poburko P, van Breemen C. Asynchronous Ca waves in intact venous smooth muscle. First Conference on Arteriosclerosis, Thrombosis and Vascular Biology. American Heart Association & North American Vascular Biology Organization. May 20-22, 2000. Denver, Colorado. Lee CH, Nazer M A , van Breemen C. Temperature-sensitive patterns of sarcoplasmic reticulum calcium unloading in vascular smooth muscle. Joint program for clinical scientists in training and MD-PhD students. The Canadian Society for Clinical Investigations. September 23. 1999. Montreal, Quebec. xv i A C K N O W L E G E M E N T S I would first like to thank my supervisor Dr. Casey van Breemen for guiding me through this journey. The pursuit of a combined MD-PhD degree is typically a long and enduring journey, especially in the PhD portion. In my case, it has been fun. I owe this tremendously enriching experience to Casey, who always has been there for me, through the countless "brain gymnastics" sessions we had at his office in pharmacology, through the many delightful moments of scientific revelation and through the unwavering support that he has given me. To my supervisory committee, I am very grateful for the valuable advice and guidance that Dr. Edwin Moore, Dr. Xiaodong Wang and Dr.Chun Seow have provided. I would like to thank the individuals in the laboratory. I am especially grateful to Mark Nazer and Dr. Xiaodong Wang for helping me through my earlier days in the laboratory, Dr. Dietrich Ruehlmann for introducing me to the world of confocal imaging, Pauline Dan for her help with my immuno-histochemistry endeavor and her continual friendship, and Kuo-Hsin Kuo for his friendship and the work with electron microscopy. I thank the contributions by Damon Poburko, Paul Sahota, Jasmin Sandhu, Dr. Roshanak Rahimian , Tania Szado, Christine Crowley and Mike Keep with whom I had the opportunities to work with in research projects. Finally and perhaps more importantly, I would like to thank my parents, Lien-Hsin Lee and Chu-Miao Lee for providing me with a great environment to learn and pursue my dream, my sister Yow-Shan Lee and my brother Yow-Hann Lee for the continual quarrel xvii and support, and my fiancee Amy Chu for listening to me talking about Ca late at night on the phone. xvni C H A P T E R I: INTRODUCTION Role of VSM in the body physiology and pathophysiology Appropriate and coordinated vasoconstriction in response to endogenous agonists and stimuli is necessary for both the control of systemic and local hemodynamics. This allows the body to maintain proper organ perfusion and organ function. When the degree of vasoconstriction becomes excess and inappropriate, damage to the vasculature and the perfused organ can occur. Over time, this can result in serious morbidity as well as mortality for the affected individual. Vasoconstriction is a process mediated by the V S M C that reside in the media of a blood vessel and it can be induced by a-adrenergic neurotransmission, by vasoconstrictor hormone/chemicals released into the blood stream, by the overlying endothelium, and by an increase in luminal pressure inside the blood vessel. Depending on the types of blood vessels, the resulting vasoconstriction can either be sustained (tonic contraction) or phasic (vasomotion). For instance, in large vessels such as the IVC that is examined here, the contraction is typically of a tonic nature. By tonically constricting the IVC to different levels, the body can regulate the amount of venous return to the heart and thereby control the cardiac output of the heart as well as the arterial blood pressure. In order to provide effective relief to patients experiencing ailments caused or complicated by excessive vasoconstriction, it is imperative to study and to gain better understanding of the mechanism of contractile activation or excitation-contraction coupling in V S M at the cellular level. 1 Role of Ca + in excitation-contraction coupling of VSM VSMC, as mentioned, is the motor unit that drives the constriction of the blood vessel. It is capable of such mechanical output because it possesses contractile filaments that consist of myosin (thick) filaments and actin (thin) filaments along with a number of regulatory enzymes and proteins. Once activated, the thick and thin filaments slide pass each other in a process described as the cross-bridge cycle and force is generated due to the shortening effort by the V S M C (Karaki etal. 1997). Similar to its skeletal and cardiac counterparts, increase in [Ca ]j is the primary activating signal for the cross-bridge cycle in VSMC. The schematic diagram shown in Figure 1 illustrates how rise in [Ca2+]i can result in increased binding of C a 2 + to calmodulin and M L C K . Ca2+ regulation in VSM Given that the contractile process is sensitive to [Ca2+]i V S M C must be able to maintain its [Ca2+]j at a basal level to avoid unwanted contraction and to raise [Ca2+]j when contraction is desired. The resting [Ca2+]j in an un-stimulated VSMC is typically around 70nM. As depicted in Figure 2, V S M C possesses a number of C a 2 + translocating molecules. Elevation in [Ca2+]j can result when C a 2 + is introduced into the cytoplasm from the extracellular space where the free [Ca2+] is about 1.5mM, or from an intracellular source such as the SR which has a luminal free [Ca ] of around 200~400pM when replenished (Corbett & Michalak 2000). The different modes of regulated C a 2 + entry across the PM pertinent in V S M C include C a 2 + entry through the L-type V G C C , the ROC, the SOC and the N C X operating in the reverse mode (3 Na + out and 1 C a 2 + in). In addition, C a 2 + leak across the PM through a yet unidentified mechanism also appears to be an important mode of 2 Ca entry in VSMC. For Ca 2 release from the SR, the SR membrane of V S M C is known to contain functional RyR and IP3R (Karaki et al.1997). Both C a 2 + release channels can be activated by Ca 2 + , and their sensitivity for activation by C a 2 + can be enhanced by cADPr and IP3 04-respectively. There are also indications that other types of SR Ca release channels such as the 04- 04-NAADP-activated Ca release channels may exist in V S M C as well. Its role in Ca regulation or signaling in V S M C is however not understood. The removal of C a 2 + from the cytoplasm can occur in a number of different ways. C a 2 + can be extruded to the extracellular space via the PMCA and the N C X operating in the forward mode (3 Na + in and 1 C a 2 + out) (van Breemen et al. 1995, Karaki et al. 1997). In addition, cytoplasmic C a 2 + can also be uptaken by the SERCA on the SR membrane and C a 2 + - H + antiporter on the mitochondrial membrane. The PMCA and SERCA are high-affinity C a 2 + uptake mechanisms and consume ATP in the process. The N C X and the C a 2 + - H + antiporter are both low-affinity C a 2 + uptake mechanisms and require higher [Ca ] to be active. 04-In a V S M C at rest, there typically is basal Ca influx through the PM which is balanced by basal C a 2 + extrusion to the extracellular space. In this resting state, there are also constant C a 2 + 04- 04-discharges by the SR in the form of Ca sparks and even Ca waves (Jaggar & Nelson 2000, Lee et al. 2002). All these activities may be important in the maintenance of basal tone for the blood vessel. Thus, even though the resting [Ca2+] appears to be at an equilibrium state, there is active cycling of C a 2 + going in and out of the cell. 3 Biphasic model of agonist-mediated [Ca Ji signaling and contraction in VSM. Agonist (vasoconstrictor)-mediated [Ca2+]i signaling and its relation to force generation in V S M has been studied for several decades now. Based on earlier series of investigations, a classic model of agonist-mediated tonic contraction, which is widely prescribed in current medical texts and literature was proposed. This classical model for agonist-mediated tonic contraction of V S M involves the biphasic model of Ca2+-dependent activation (Karaki et al. 1997). In this model depicted in Figure 3, the initial force generation is mediated by a transient phase of Ca release (Phase 1) from the SR and the maintenance of the tonic contraction is the result of stimulated C a 2 + influx through the L-type V G C C , which directly activates the myofilaments (Phase 2). Supports for this model came largely from both whole-vessel [Ca2+]i measurements, which assess the averaged response of thousands of cells in a blood vessel, and single cell study involving cultured or freshly isolated preparations. However, as we will demonstrate later, the whole-vessel C a 2 + signal does not always reflect the C a 2 + signal at the individual cell level or the sub-cellular level and may therefore fail to reflect the true physiology of V S M C (Ruehlmann et al. 2000). Pitfalls of single cell study and advantage of intact vessel study Much of our understanding regarding [Ca2+]j signaling in V S M C has been based on studies using enzymatically isolated cells and cultured cells. Isolated and cultured VSMCs, when stimulated, can display a number of C a 2 + signaling patterns which range from the biphasic partem of [Ca2+]i elevation to cyclic elevations of [Ca2+]i that progress over the entire cell length, giving it the appearance of a wave (Berridge & Dupont 1994). Although these single-cell experiments have OA-greatly improved our knowledge of the mechanisms involved in Ca homeostasis, smooth muscle cells can present a very different phenotype once cultured (Thyberg 1996). For example, cultured 4 smooth muscle cells rapidly lose their L-type voltage operated channels (Gollasch et al. 1998) and undergo a reduction in the expression of a-actin (Campbell et al. 1989), hence changing from the contractile to the secretory or migrating phenotype (Owens 1995). In addition, ubiquitous chemicals leaching from cell culture plasticware can potently affect the C a 2 + homeostasis of vascular smooth muscle cells (Glossmann et al. 1993, Ruehlmann et al. 1998), and cell culture media with high glucose concentrations can reduce gap junction expression (Kurjiaka et al. 1998, Kuroki et al. 1998). Even when smooth muscle cells are freshly isolated, but not cultured, it is clear that proteolysis changes cellular characteristics, which are fundamental to physiologic function. More importantly, the presence of gap junctions (Little et al. 1995) between V S M C suggests that cells with intact intercellular communications may behave more like a syncytium and possibly very different from its behavior as isolated single cell. It therefore becomes necessary to verify mechanisms deduced from isolated or cultured cells by data obtained from smooth muscle cells in situ. Emergence of adrenoceptor-mediated asynchronous wave-like fCa2+h oscillations in intact arterial blood vessels 9+ Even though agonist-mediated [Ca ]j oscillations have been observed since the early 1990s in single VSMC preparations, the physiological relevance for the observed response is questionable because of the harsh isolation procedure and the phenotypic shifts that can occur during cell culturing discussed above. In 1994, with the application of confocal microscopy and fluo-3 (Ca 2 +-sensitive dye), lino et al observed that a-adrenergic stimulation of the intact rat tail artery generated recurring C a 2 + waves that resulted in cytoplasmic [Ca2+] oscillations in the in situ 9-t-V S M C (lino et al. 1994). Moreover, the generation of these Ca waves was not synchronized 5 between neighboring cells. Given that the most physiological intact vessel preparation was used here, this provides the first clear indication that elaborate C a 2 + signaling event that is more complex than that described by the biphasic model can occur in arterial VSMC. If this is true then there are serious concerns regarding our current state of understanding of how vascular tone is regulated at the level of VSMC. More importantly, this first report also prompted a series of questions crucial to our understanding of vascular physiology and contractile activation. For instance, do these asynchronous recurring C a 2 + waves observed in arterial SMC occur in venous SMC as well? What is the function of these recurring C a 2 + waves and how do these recurring C a 2 + waves modulate such function? What is the cellular mechanism for generating these recurring Ca waves? 6 Figure 1: Ca 2 + , calmodulin, M L C K and Cross-bridge cycle in smooth muscle A schematic diagram depicting the activation of the cross-bridge cycle in the smooth muscle cell is shown. 4Ca 2+ Ca2+-calmodulin-MLCK Actin Myosin Myosin-P Actin + activated myosin ATPase Smooth • • muscle contraction 7 Figure 2: Ca translocating molecules in V S M C Components involved in C a 2 + signaling in VSMC are schematically illustrated in this diagram and the movements of C a 2 + as well as Na+ are represented by arrows. NCX, sodium/calcium exchanger; SOC/ROC, store-operated channel/receptor-operated channel; SERCA, sarcoplasmic endoplasmic reticulum C a 2 + ATPase; L-type V G C C , L-type voltage-gated C a 2 + channel; PMCA, plasma membrane C a 2 + ATPase; IP3R, IP3-sensitive SR C a 2 + release channel; SR, sarcoplasmic reticulum. 8 Figure 3: Biphasic model of contractile activation The biphasic model for agonist-mediated tonic contraction of V S M is illustrated in this schematic diagram. The initial force generation is mediated by a transient phase of C a 2 + release (Phase 1) from the SR and the maintenance of the tonic contraction is the result of stimulated C a 2 + influx through the L-type V G C C , which directly activates the myofilaments (Phase 2). 9 C H A P T E R II: SPECIFIC AIMS The four primary aims are outlined below. Specific aim #1: Examination of PE-mediated Ca2+ signal in in situ VSMC within the intact media of the rabbit IVC. We will attempt to examine at high spatio-temporal resolution the C a 2 + signal elicited by PE in the in situ V S M C within the intact media of the rabbit IVC. For comparison, we will also examine the in situ V S M C C a 2 + signal elicited with caffeine (an activator of the RyR). We will also compare the C a 2 + signal observed at the level of individual in situ V S M C to the C a 2 + signal observed at the whole-vessel level (which reflects the signal averaged from hundreds of cells in the IVC segment). Specific aim #2: Correlation of PE-mediated asynchronous wave-like fCa2+li oscillations to contractile function in the rabbit IVC. Using various concentrations of agonists (PE or caffeine), we will attempt to correlate the concentration-dependence of various aspects of the agonist-induced C a 2 + signal to the concentration-dependence of force generation. In the case of PE which induces asynchronous recurrent C a 2 + oscillations in the in situ VSMC, the different aspects of the C a 2 + signal examined here include the recruitment of the in situ V S M C to initiate Ca signal, the amplitude of the recurrent C a 2 + waves (wave-like [Ca2+]j oscillations), the frequency of the recurrent C a 2 + waves (wave-like [Ca2+]j oscillations) and the velocity of the recurrent C a 2 + waves. 10 Specific aim #3: Examination of the mechanism of phenylephrine-mediated asynchronous wave-like fCa2+h oscillations in the VSMC of the rabbit IVC. The mechanism of PE-mediated wave-like [Ca2+]i oscillations and tonic contraction will be examined systematically with various pharmacological tools. RT-PCR studies will also be utilized to identify the expression of the molecules believed to be involved in the generation of the wave-like [Ca2+]i oscillations. Specific aim #4: Examination of the requirement for PM-SR junctions in the generation of wave-like fCa /,• oscillations in PE-stimulated rabbit IVC. Close apposition between the PM and the SR (PM-SR junctions) are required for proper E-C coupling in cardiac and skeletal muscle. With parallel E M and [Ca2+]i studies, we will examine the relationship between the disruption of the PM-SR junctions and the disruption of PE-mediated wave-like [Ca2+]i oscillations. 11 C H A P T E R III: M A T E R I A L S AND M E T H O D S Tissue isolation and preparation All the experiments and procedures were carried out in accordance with the guidelines of the University of British Columbia Animal Care Committee (protocol number: A990290). Male New Zealand White rabbits (1.5-2.5 kg, obtained from Animal Care, University of British Columbia) were sacrificed by CO2 asphyxiation and then exsanguinated. The IVC was removed, cleaned of surrounding connective tissue and then inverted. The endothelium was removed by gently swiping it with filter paper and the inverted vessel were then cut into multiple ring segments that were 4-5 mm in width. As depicted in Figure 4, the inverted rings were then ready for mounting onto the respective setups for contraction study, whole-vessel [Ca2+]j study and confocal subcellular [Ca2+]i imaging study. All experiments were performed at 37°C. Isometric contraction study Vessel segments attached to isometric force transducers were equilibrated with appropriate pretension at 37°C in PSS bubbled with 100% O2. Bath solution was exchanged with rapid draining and re-filling within 2-3s. Rings failing to contract after challenge with 80mM [K] e xt were excluded. Dose-response curves incremental, non-cumulative concentrations of agonists were performed. Data was acquired and analyzed using Chart v3.4.5 (ADIinstruments). All the contraction traces shown here represent findings from a minimum of five rings of IVC. One sample non-parametric tests (Wilcoxon Singed-Rank test) and two-sample non-parametric tests (Mann-Whitney U test) were used to assess statistical significance. Statistical significance is reached when p<0.05. 12 Spectrofluorimetric [Ca h measurement - whole-vessel/whole-tissue Ca signal The detailed methods to measure global [Ca2+]j in this preparation have been described elsewhere (Nazer & van Breemen 1998). Briefly, the tissue was mounted inside a 3 ml cuvette and placed in a spectrofluorimeter (Spex Fluorolog, Spex Industries, NJ). After subtraction of auto-fluorescence, the tissue was loaded with fura-2 A M (5 pM + 5 pM pluronic, 90 min at room temperature) and excited with alternating 340 and 380 nm. The ratio of these two wavelengths was taken as an indicator of [Ca2+]j. Fura-2 is a C a 2 + indicator with a high Ca2+-affinity that is typically used to monitor cytoplasmic [Ca2 +]. Such a measured [Ca2+]j represents the [Ca2+]j averaged among hundreds of the VSMCs in the intact media of the IVC. We refer to this averaged [Ca2+]j response as the whole-vessel/whole-tissue C a 2 + signal. Laser scanning confocal [Ca2+Ji imaging - Subcellular Ca2+ signal For confocal experiments, inverted rings were loaded with Fluo-3 A M (5 pM, with 5 pM Pluronic F-127, 90 minutes, 25°C) followed by a 30 minute equilibration period in normal PSS. Fluo-3 is a C a 2 + indicator with a high Ca2+-affinity and is typically used to monitor cytoplasmic [Ca2 +]. The vessel ring was isometrically mounted on a purpose-built microscope stage. Solution changes were achieved with rapid draining and refilling of the bath. Observation of [Ca2+]j changes was made using a Noran Oz laser scanning confocal microscope with a 100 pm slit through either an air 60x (numerical aperture : 0.7) or an air 20x (numerical aperture : 0.45) lens on an inverted Nikon microscope. The tissue was illuminated using the 488nm line of an Argon-Krypton laser and a high-gain photomultiplier tube collected the emission after it had passed through a 525/52 BP filter. Such a filter will only allow light from a narrow band of 525+25nm to pass through 13 (525nm is the emission wavelength of Fluo-3). All parameters (laser intensity, gain etc.) were left unchanged during the experiment. Generally, acquisition speed was set to 66 ms/frame with 2-frame integration resulting in an effective frame rate of 133ms/frame. Comparisons between recordings made at 66ms/frame and 133ms/frame were made when necessary to exclude sampling artifacts. The scanned region corresponds to a 232pmx217um area on the tissue and yields a 512x479 pixels image. All image analysis was performed in ImagePro Plus using customized routines as described below. Confocal image and Data analysis All data analysis was performed in ImageProPlus using customized routines written in Visual Basic. To obtain data on recruitment of cells during drug stimulation, a 3 pixel-wide scan line was drawn across multiple cells and propagated through the time stack. The resulting image (Y-t plot, Figure 5A) revealed the number of cells responding (expressed as percentage of the cells responding to the highest drug concentration) as well as the degree of heterogeneity between and oscillation frequency within cells. Further analysis of wave parameters was performed using a 3-pixel wide line along the longitudinal axis of a single cell. The resulting X-t plot (Figure 5B) revealed the point of origin as well as the progression speed of the apparent 'wave'. All experimental traces shown represent the averaged fluorescence signals from a 3x3 pixels region of 1.36pm in a single cell (Figure 5C). The representative experimental fluorescence traces shown reflect the averaged fluorescence signals from such a 3x3 pixels region in a single cell. Changes in this regional fluorescence level (F525) directly reflect changes in the C a 2 + concentration in this region of the cell. Prior to stimulation, the basal fluorescence allows for delineation of the outline of the ribbon-shaped vascular smooth muscle cell. The 3x3 pixels region was positioned towards 14 the midline of the ribbon-shaped smooth muscle cell (Figure 5C). Given that the focus of this study is on cytoplasmic C a 2 + signals, the region was positioned away from the highly fluorescent nuclear region of the cell, as Fluo-3 is known to accumulate in this region of the cell (Perez-Terzic et al. 1997). All the numerical data were analyzed in Excel and Sigma Plot. One sample non-parametric tests (Wilcoxon Singed-Rank test) and two-sample non-parametric tests (Mann-Whitney U test) were used to assess statistical significance. All the fluorescence traces shown represent findings from a minimum of 40 cells in four different tissues and all the contraction traces shown represent findings from a minimum of eight different tissues. RT-PCR study Total cellular RNA from endothelium-denuded rings of rabbit IVC was extracted using a RNeasy mini kit™ according to manufacturer's instructions. It is important to note that all the dissection equipment was pre-treated with RNAseZap before use. RNA was quantified by measuring absorbance spectrophotometrically at 260 nm and its integrity was assessed after electrophoresis in nondenaturing 1% agarose gels stained with ethidium bromide. Reverse transcription of 5 pg total RNA was performed in 60 pi reaction volumes containing 200 units of Superscript II™ reverse transcriptase, 60 units RNase inhibitor, 3 mM MgCb, 1 x Buffer II (Sigma) and 0.3 pg random primers and ImM dNTP for 50 min at 42°C. Contaminating genomic DNA present in the RNA preparations was removed by digesting the reaction with 5 units of DNase I for 45 minutes at 37°C prior to the addition of reverse transcriptase. 5 pi of the RT product was used in each 100 pi PCR reaction. The PCR mixture contained 250 pM dNTP, 2 mM MgCb, lx volume of Buffer and 2.5 unit Hotstar™ Taq polymerase, and 1 pi of forward and 1 pi 15 of reverse primers. The temperature program for the amplification was 40 cycles of 1 min at 94°C, 1 min at 55°C and 1 min at 72°C. The final extension was completed at 72°C for 7 min. 10 pi of 6x loading buffer (containing 0.25% bromothymolblue, 0.25% xylene cyanol FF, and 15% Ficoll type 400, Pharmacia, in DEPC-treated distilled water) was added to the PCR products. 20 pi of PCR products were then analyzed by electrophoresis on 2% agarose gels stained with ethidium bromide and gels were photographed under U V light. 18S ribosomal RNA expression was used as an internal control. The exemplary gels shown in this report represent findings from a minimum of 5 rabbits. Both rabbit and rat brains were used as a positive control for the expression of Trpl, 2, 3, 4, 5, 6, 7. Primers used for different amplifications were designed from published reports (McDaniel et al. 2001, Walker et al. 2001) or sequences available in Genbank (Table 1). Amplified PCR products from rabbit tissue were isolated from agarose gel, sequenced and found to be 100%o identical to the authentic sequences of rabbit Trpl~7 and aic cDNA. Table 1. Oligonucleotide sequences of the primers used for RT-PCR Channel GenBank Accession No. Predicted Size, bp Sense/ Antisense Location, nucleotides mTrpI U40980 372 b ' -CAAGAI I I I G G G A A A I I I C I G G - 3 ' 5 ' -TTTATCCTCATGATTTGCTAT -3' 1-22 352-372 rTrp2 AF136401 487 5 ' - C A G T T T C A C C C G A T T G G C G T A T - 3 ' 5 - C T T T G G G G A T G G C A G G A T G T T A -3' 1606-1627 2071-2092 hTrp3 U47050 331 5 ' - A T T A T G G T G T G G G T T C T T G G - 3 ' 5 ' - G A G A A G C T G A G C A C A A C A G C -3' 1483-1502 1795-1814 mTrp4 U50922 265 5 ' - C A A G G A C A A G A G A A A G A A T - 3 ' 5 ' - C C T G T T G A C G A G T A A T T T C T -3' 2535-2553 2781-2800 mTrp5 AF029983 419 5 ' - C C T C G C T C A T T G C C T T A T C A - 3 , 5 ' - T G G A C A G C A T A G G A A A C A G G -3' 675-694 1075-1094 mTrp6 U49069 410 5 ' -CTG C T A C T C A A G A A G G A A A A C - 3 ' 5 ' - T T G C A G A A G T A A T C A T G A G G C -3' 738-758 1128-1148 mTrp7 AF139923 260 5 ' - T G A C A G C C A A T A G C A C C T T C A - 3 ' 5 ' - G C A G G T G G T C I I I G T T C A G A T -3' 2397-2417 2637-2657 ralC M59786 371 5 ' - A T C C C C A A G A A C C A G C A C - 3 ' 5 - G G T G A T G G A G A T G C G G G A G T T -3' 3882-3900 4233-4253 Tip, transient rceptor potential; m, mouse; r, rat; h, human. 16 Electron microscopy The primary fixative solution contained 1.5% glutaraldehyde, 1.5% paraformaldehyde and 2% tannic acid in 0.1 M sodium cacodylate buffer that was pre-warmed to the same temperature as the experimental buffer solution (37 °C). The rings of rabbit IVC were fixed at 37 °C for 30 minutes, then cut into small blocks, approximately 1 x 0.5 x 0.2 mm in dimension and put in the same fixative for 2 hrs at 4 °C on a shaker. The blocks were then washed three times in 0.1 M sodium cacodylate (30 min). In the process of secondary fixation, the blocks were put in 1% OsG"4, 0.1 M sodium cacodylate buffer for 2 hours followed by three washes with distilled water (30 min). The blocks were then further treated with 1% uranyl acetate for 1 hour (en bloc staining) followed by three washes with distilled water. Increasing concentrations of ethanol (50%>, 70%, 80%>, 90% and 95%) were used (ten minutes each) in the process of dehydration. 100% ethanol and propylene oxide were used (three 10-min washes each) for the final process of dehydration. The blocks were left overnight in the resin (TAAB 812 mix, medium hardness) and then embedded in molds and place in an oven at 60 °C for 8 hrs. The embedded blocks were sectioned on a microtome using a diamond knife. The thickness of the sections was ~80 nm. The sections were then placed on 400-mesh cooper grids, stained with 1% uranyl acetate and Reynolds lead citrate for 4 and 3 min respectively. Images of the cross-sections of the muscle cells were obtained with a Phillips 300 electron microscope. Materials Normal physiological salt solution (PSS) containing (in mM) NaCl 140, KC1 5, CaCl 2 1.5, MgCl 2 1, glucose 10, HEPES 5, (pH 7.4 at 37°C) was used for all the studies. Zero C a 2 + PSS contains no 17 CaCl 2 but 1 mM EGTA. High K + (80rnM [K+]ext) PSS is identical in composition to normal PSS with the exception of (in mM) NaCl 65 and KC1 80. All the reagents were purchased from Sigma and were of the highest analytical grade. Fura-2 A M , Fluo-3 A M , pluronic F-127, Hoechst 33342 and 2',4'-dichlorobenzamil were purchased from Molecular Probes(Eugene, OR) and were dissolved in dimethyl sulfoxide (DMSO), so was ryanodine (Sigma). PE (Sigma), caffeine (Sigma), thapsigargin (Sigma), phentolamine (Sigma)), NiCl 2 (Sigma), LaCb (Calbiochem) and SKF96365 (Calbiochem) were prepared in normal PSS. Stocks of KB-R7943 (Tocris) and cyclopiazonic acid (CPA, Calbiochem) were prepared in DMSO while stocks of nifedipine (Sigma) and diphenylboric acid 2-aminoethyl ester (2-APB, Sigma) were prepared in ethanol and methanol respectively. For the RT-PCR study, Superscript.il™ reverse transcriptase, RNase inhibitor and random primers were obtained from Gibco/BRL Canada). Buffer II (lOx) was obtained from Sigma/Aldrich (Canada). MgCl 2 , dNTP, 10X volume PCR Buffer, Hotstar™ Taq polymerase and RNeasy mini kit™ were purchased from Qiagen (Canada). Ribosomal RNA (18S) and RNAseZap were purchased from Ambion Inc. (TX, USA). All live tissue experiments were performed at 37°C. 18 Figure 4: Schematic depiction of the experimental techniques. See text for details. Figure 5 : Derivation of Y-t plot, X-t plot and the 3 x 3 pixel region on the confocal image The Y-t plot, X-t plot and 3><3 pixel region are derived as shown below from time-series confocal images. A) Y-t plot B) X-t plot C) 3x3 pixel region 20 CHAPTER IV: EXAMINATION OF PE-MEDIATED VSMC C A ' + SIGNAL AT WHOLE-TISSUE AND SUB-CELLULAR L E V E L OF THE RABBIT IVC. Removal of the endothelium For the purpose of our current study, the endothelium was removed for three reasons. Firstly, the endothelium which is a monolayer of cells with tight intercellular junction was removed to improve the penetration of fluo-3 or fura-2 dyes into the media layer where the V S M C s reside. Secondly, in whole-vessel C a 2 + measurement studies with the spectrophotometer, removal of the endothelial cells ensures that the fluorescence signal can arise only from the V S M C s . Thirdly, many of the drugs used by us to target the V S M C can also result in the release of chemicals such as nitric oxide or endothelium-dependent hyperpolarising factor (EDHF) by the endothelial cells. The removal of the endothelium thus prevents these confounding effects. To confirm endothelial cell removal, denuded and control vessels were incubated with the selective D N A stain Hoechst 33342 (300 pg/mL). Nuclei were visualized using the 360nm laser illumination and in control vessels, a continuous layer of oblong nuclei, with their long axis oriented parallel to the direction of the flow, was observed indicating an undamaged endothelial cell layer (Figure 6). After denudation, the same staining protocol revealed only elongated nuclei oriented transversely and at right angles to the direction of the flow, consistent with vascular smooth muscle cell nuclei characterized by Daly et al (Daly et al. 1992). 21 Examination of PE-induced and caffeine-induced Ca signal at whole-vessel level. One of the methods commonly used to examine the changes in [Ca 2 +]j that occur in the V S M 9+ inside intact vessels during agonist stimulation is the whole-vessel spectrophotometer [Ca ]i measurement using Ca 2 +-sensitive dyes such as fura-2 (cytoplasmic C a 2 + indicator). However, as eluded to earlier with whole-vessel [Ca 2 +]j measurement, the C a 2 + signal measured reflects a whole-vessel response averaged from hundreds of V S M C inside the intact tissue. Interpretation of this type of measurement often involves the assumption that every V S M C responds identically in terms of its C a 2 + signal. The whole-vessel [Ca 2 + ] ; signal elicited by P E and caffeine were examined in this segment of the study. P E is an ct-adrenergic agonist and its application w i l l lead to tonic constriction of the I V C . Caffeine is an activator of R y R and its application w i l l constrict the I V C only transiently. Caffeine is used here for contrast with PE . As shown in Figure 7, P E (15uM) induced a rapid rise 9+ ' in whole-vessel [Ca ]\. The peak value, 145 ± 22% of basal F340/380 was reached 62 ± 9s after the onset of the response (n=4 rings from 4 animals). Whole-vessel [Ca 2 + ] i was maintained; 180s after the maximal level was reached as it remained at 142 ± 19% of the basal F340/380. In comparison, a similar fast initial rise was observed with caffeine (25mM). 136 ± 6 % of basal F340/F380 was reached after 53 ± 7s (n=4 rings from 4 animals) but in contrast to P E , the ratio returned to baseline after 180s (Figure 7). These findings indicate that P E stimulation resulted in maintained whole-vessel [Ca 2 + ] i elevation while caffeine stimulation resulted in transient whole-vessel [Ca 2 +]j elevation in the rabbit I V C . Thus, these patterns of the whole tissue [Ca 2 +]j signals appear to match the contractile profile of P E (sustained contraction) and caffeine (transient contraction) in the rabbit I V C . 22 Examination of PE-mediated and Caffeine-mediated Ca2+ sisnal at sub-cellular level A s mentioned, whole-vessel [Ca 2 + ] i signal is a population average among hundreds of in situ V S M C s in the intact vessel and may not necessarily reflect what occurs in individual in situ V S M C s . It thus is important to examine the sub-cellular [Ca 2 + ] ; signal of individual V S M C s in situ which is made possible with the advent of confocal [Ca 2 + ] i imaging. In this segment of the study, we w i l l examine the [Ca 2 + ] i signal elicited by P E and caffeine at sub-cellular levels of the in situ V S M C s . The pseudo-colored image displayed in Figure 8 shows the fluo-3 loaded in situ V S M C inside a ring of rabbit I V C mounted isometrically on the stage. The basal fluo-3 fluorescence reveals the morphology of these in situ V S M C s . These are ribbon-shaped cells that are approximately 3pm wide and 100pm long. With the fast laser scanning confocal [Ca 2 + ] ; imaging, a change in [Ca 2 +]j in any sub-cellular region of a ribbon-shaped V S M C w i l l be reflected by a change in fluorescence in that same region. Within the physiological range of cytoplasmic [Ca 2 + ] i fluctuations (50nM to 1 uM) , the change in fluorescence output of fluo-3 (cytoplasmic Ca indicator) w i l l be directly proportional in amplitude to the changes in [Ca 2 + ] ; in the region. Figure 9 shows representative time-series sequence of the changes in [Ca 2 + ] i visualized with fluo-3 fluorescence. Initially, both caffeine (25mM) and P E (1.5uM) elicited a rapid rise in [Ca 2 + ] i that appeared in the form of a calcium wave propagating along the longitudinal axis of the V S M C s (Figure 9 A and B , first frame). With time, [Ca 2 + ] i in the in situ V S M C s challenged with caffeine returned to baseline despite the continual presence of the drug (Figure 9B). The transient nature of 23 the caffeine-induced sub-cellular Ca signal (a single Ca wave) agrees with the transient whole-tissue [Ca 2 + ] i signal and contraction. In the same vena cava P E stimulated repetitive smooth muscle C a 2 + waves were not synchronized between adjacent cells. (Figure 9A, arrows to cell 1,2 and 3). This lack of synchronicity between neighboring V S M C s explains how summation of the individual cell [Ca 2 + ] i oscillations can lead to sustained macroscopic [Ca 2 + ] i elevation and tonic contraction of the whole-vessel. The above findings indicate the following. Firstly, sub-cellular [Ca ]i in in situ V S M C of intact I V C can be successfully measured using fluo-3 and confocal microscopy. Secondly, whole-vessel [Ca 2 + ] i signal (the averaged signal of hundreds of in situ V S M C ) does not always reflect the [Ca 2 + ] i signal that occurs at individual V S M C level or sub-cellular level. Thirdly, similar to what occurs in the in situ V S M C of the arterial vessel, a-adrenergic stimulation also induced asynchronous wave-like [Ca 2 +]j oscillations in the in situ V S M C of the venous vessel. 24 Figure 6: Removal of endothelium confirmed with nuclei staining In control sections (A) , Hoechst 33342 staining identifies both endothelial (oblong) and smooth muscle cell nuclei (elongated). Denudation (B) resulted in exclusive staining of smooth muscle cell nuclei confirming complete removal of endothelial cells. 25 Figure 7: Whole-vessel [Ca ]i response to PE and caffeine 2"r" • P E and caffeine evoke persistent and transient elevation in whole tissue [Ca ]j respectively in the intact rabbit I V C preparation. 26 Figure 8: Fluo-3 loaded in situ VSMCs of the rabbit IVC visualized under confocal microscopy. See text for descriptions. Figure 9: Sub-cellular [Ca ]j signal elicited with PE and caffeine visualized with confocal time-series images Intact I V C smooth muscle cells loaded with fluo-3 and challenged with either P E (A, 1.5uM) or caffeine (B, 25mM) showed an initial rapid rise in [Ca 2 +]j. In the continuous presence of the agonist, PE-exposed smooth muscle cells showed non-synchronous oscillations (arrows to cell 1,2 and 3 outlined), whereas the response to caffeine declined without any subsequent oscillations. 28 15 umol/L P E 25 mmol /L caffeine CHAPTER V: CORRELATION OF PE-MEDIATED ASYNCHRONOUS WAVE-LIKE [CA2+]i OSCILLATIONS TO FORCE GENERATION OF THE IVC Given that P E induces asynchronous recurring Ca waves and caffeine induces a single Ca wave in the in situ V S M C s of the rabbit I V C , how these C a 2 + signals at the sub-cellular level relate to constriction of the blood vessels remains undefined. More specifically, i f these agonists signal for contraction of the V S M by these C a 2 + waves, how is the graded contraction elicited by different concentrations of the agonists modulated. In order to provide insights into these questions, we compared the concentration dependency of PE/caffeine induced contraction with the concentration dependency of selected parameters of PE/caffeine induced C a 2 + signals. In order for the C a 2 + signal to be involved in modulating the degree of vasoconstriction, certain aspects of the C a 2 + signal must exhibit concentration dependence within the same range of agonist concentrations that elicit graded vasoconstriction. For the caffeine-induced single C a 2 + wave, the [caffeine]-dependence of the percentage recruitment of in situ V S M C s (to initiate Ca signal), wave amplitude and wave velocity were assessed. For PE-mediated asynchronous wave-like [Ca 2 + ]i oscillations, the [PE]-dependence of the percentage recruitment of in situ V S M C s , oscillation amplitude (recurring waves amplitude), oscillation frequency (recurring waves frequency) and wave velocity were assessed. Concentration dependence of PEIcaffeine-induced force generation To characterize the contractile responses of the I V C to caffeine and P E , the tissues were isometrically suspended and non-cumulatively exposed to increasing concentrations of either 30 caffeine or P E . Representative traces are shown in Figure 10 (A and B , inset). P E (15pM) induced a maintained increase in contractile force of 1.79 ± 0.42g (mean + S E M , n=8 rings from 4 animals). Such effect by P E (15pM) was, as expected mediated through activation of the cti-adrenergic receptor since it can be prevented by the application of the ai-adrenergic receptor antagonist, phentolamine (10 p M , data not shown). Caffeine (25mM) evoked a transient contraction; 5s after the onset of the response 0.42 ± 0.09g (n=l 1 rings from 4 animals) of tension was reached and subsequently relaxed completely to the basal level after 120s. Increasing concentrations of the agonists enhanced the contractile force until a plateau value was reached. These typical concentration-response curves are shown in Figure 10. The dose-response for P E fell within the concentration range of 0.0015 to 150pM, while caffeine increased force over a much narrower concentration range of 1 to l O m M . Concentration dependence of caffeine-induced Ca2+ signal To understand the cellular basis of the concentration-response curves illustrated in Figure 2, we analyzed the nature of the agonist-induced C a 2 + waves. A s shown by the representative traces in Figure 11C and the Y - t plot in Figure 1 I B , caffeine stimulates a single C a 2 + wave in the in situ V S M C s . Whenever a C a 2 + wave was initiated by caffeine, the amplitude of the [Ca 2 +]j signal was constant irrespective of concentrations applied (Figure 1 ID). This indicates an all-or-none phenomenon that excludes amplitude as a modulator of tissue contractility. However, we observed that the sensitivity of V S M C to caffeine varies between cells leading to an increased recruitment at higher caffeine concentrations (Figure 11C). This concentration dependence of recruitment resembles that of whole-tissue contractility (Figure 10B) and appears to form the basis for the concentration-dependence of caffeine-induced contraction 31 In addition, we also examined the concentration dependence of the velocity of caffeine-induced waves. The velocity of wave propagation illustrated in Figure 12 shows a strong dependence on the concentrations of caffeine. A t the highest concentrations wave propagation speeds reached 126.22 ± 7.31pm/s with caffeine (n=15 cells from 3 rings from 3 animals). In addition, as illustrated in Figure 12A, caffeine commonly elicited Ca waves at multiple foci within a given cell, which then eventually collided. Despite of the observed concentration-dependence, it is unclear as to how velocity of the C a 2 + waves may help to modulate force generation by the V S M C . Correlation of PE-induced asynchronous wave-like [Ca Ji oscillations to force generation In the case of P E , we observed similar characteristics with respect to amplitude and recruitment (Figure 13). A s shown in Figure 13 B (Y-t plot) and C (representative trace), P E induces [Ca 2 + ] i oscillations that are not synchronized between neighboring in situ V S M C s . The amplitude displayed an all-or-none character (Figure 13D) and the concentration-dependency of recruitment (Figure 13E) appeared to correlate with the concentration-dependency o f the PE-induced contraction (Figure 10A) at the lower concentration range of 0.015 to 1.5pM. However, the continued oscillations of [Ca 2 + ] i allow for more elaborate modulation of contractile force. Figure 14 shows that as [PE] was increased, the frequency increased. The frequency of [Ca 2 + ] i oscillations was determined with the X - t plot (Figure 14B). The X - t plot represents fluorescence recordings of a 3-pixels wide line drawn along the longitudinal axis of a single cell propagated through time. A n oscillatory C a 2 + wave traveling through the longitudinal axis of the cell can be readily identified on the X - t plot (arrows). Each arrow represents an oscillatory C a 2 + wave and the 32 frequency was derived by dividing the number of oscillatory Ca waves over a given time period. At the highest concentration used (150 p M PE), the frequency reached 0.511 ± 0.025Hz (n=15 cells from 4 animals). A s reflected in the representative traces from Figure 14A, the increased frequency of [Ca 2 +]j oscillations coincides with shortened inter-spike intervals. A t high [PE], the inter-spike [Ca 2 + ]i level was elevated above the basal level but [Ca 2 +]j peaks never fused. The concentration-dependence of the frequency of PE-induced [Ca 2 +]j oscillations appears to correlate with the concentration-response curve of PE-induced contraction at a higher concentration range of 0.15 to 150pM. Finally, we examined the concentration dependence of the apparent velocity of PE-induced Ca waves. The velocity of wave propagation illustrated in Figure 15 shows a strong dependence on the concentrations of P E . A t the highest concentrations wave propagation speeds reached 89.6 ± 7.4 pm/s (n=15 cells from 4 animals) with P E . In addition, as illustrated in Figure 15, P E commonly elicited C a 2 + waves at multiple foci within a given cell, which then eventually collided. Despite of the observed concentration-dependence, it is unclear as to how velocity of the C a 2 + waves may help to modulate force generation by the V S M C 33 Figure 10: Contractile responses and concentration-dependence curves with PE and caffeine P E and caffeine evoke persistent and transient contractions respectively in the intact rabbit I V C preparation (A and B insets show representative traces for P E and caffeine respectively). The concentration-response curves are shown for P E in A and caffeine in B . 34 Figure 11: Fixed-amplitude Ca 2 + waves and concentration-dependent recruitment in response to caffeine. In intact rabbit I V C preparation, caffeine (25mM) elicited transient [Ca 2 +]j signals in both cell 1 and 2 (A) as shown in the propagated Y - t line-scan (B) from which the respective traces in (C) were derived (images are noise-filtered and contrast-enhanced; the intensity level reflects [Ca The traces shown represent the averaged fluoresence signals from a 3x3 pixel region in a single cell. After the initial sharp rise, [Ca ]; returned to base level as reflected in (B) and (C). (D) The amplitude of the caffeine-induced C a 2 + transients were insensitive to agonist concentration (n=15 cells from 3 animals). N o [Ca ]i signals were observed at 0.25mM caffeine and thus no amplitude information was obtained. Amplitude was determined by subtracting pre-stimulation baseline from the peak value of the single Ca transient and the units are arbitrary on an 8-bit scale. (E) Caffeine recruited V S M C s in a concentration-dependent manner (n=3 preparations from 3 animals). Recruitment started at 1 m M and reached maximal level at l O m M caffeine. The number of cells firing is expressed as a percentile of cells responding to the maximal concentration (*) 35 B Sum C e l M Cel l 2 5 sec Ce"2 f^P50""^ 25 mmol /L caffeine 120 I " i o o c 3. 80 o> 1 60 1 40 4 1 20 0.1 1 10 [Caffeine] (mmol/L) co 100 E 4 = C 80 to ! 60 An o c 4 0 a E | 20 O £ oJ 0.1 1 10 [Caffeine] (mmol/L) 36 Figure 12: Concentration-dependence of the apparent velocity of caffine-induced Ca wave. Selected frames (A) show the propagation patterns of caffeine-induced Ca waves. Distinct wave origins were observed even at maximal agonist concentration (B). (C) The apparent propagation speed of these C a 2 + waves was correlated to the drug concentration (n=15 cells from 3 animals). 37 Figure 13: Fixed-amplitude Ca waves and concentration-dependent recruitment in response to PE In intact rabbit I V C preparation, P E (1.5uM) elicited [Ca ]i oscillations in both cell 1 and 2 as shown in the propagated Y - t line-scan (B) from which the respective traces in (C) were derived (images are noise-filtered and contrast-enhanced; the intensity level reflects [Ca 2 +]j). The traces shown represent the averaged fluoresence signals from a 3x3 pixel region in a single cell. (D) The amplitude of PE-induced [Ca 2 +]j oscillations did not change with increasing [PE] (n=15 cells from 4 animals). N o [Ca 2 + ] ; signals were observed at P E concentrations below 0.015pM. The amplitude was determined by subtracting the pre-stimulus baseline from the averaged peak values of all [Ca ]i oscillations observed and the units are arbitrary on an 8-bit scale. It is important to note that the averaged peak values were used because the amplitudes of [Ca 2 +]j oscillations from the 3x3 pixels region in a single cell do vary with time, which is due to the variability of stochastic noises recorded from the same 3x3 pixels region over time. Averaging of the peak values thus minimizes the effects of the noise. (E) A greater percentage of V S M C generated [Ca 2 + ]i signals as the P E concentration increased. This recruitment occurred between 0.015uM and 1.5pM P E with maximal recruitment achieved at 1.5uM P E in all the tissues examined (n=4 rings from 4 animals). The number of cells firing is expressed as a percentile of cells responding to the maximal concentration (*). 38 Amplitude (units) o -o us Figure 14: Concentration-dependent frequency of PE-induced wave-like [Ca ]i oscillations. The frequency of PE-induced [Ca 2 +]j oscillations increased with increasing [PE] as demonstrated in both the representative traces (A). The frequency of [Ca ]i oscillations was determined with the X - t plot (B). The X - t plot represents fluorescence recordings of a 3-pixels wide line drawn along the longitudinal axis of a single cell propagated through time. A n oscillatory C a 2 + wave traveling through the longitudinal axis of the cell can be readily identified on the X - t plot (arrows). Each arrow represents an oscillatory C a 2 + wave and the frequency was derived by dividing the number of oscillatory Ca waves over a given time period. C shows the concentration-frequency relationship of this response (n=15 cells from 4 animals). With increasing frequency, the inter-spike intervals were shortened and at high concentrations of PE , the inter-spike [Ca 2 + ] i level was elevated above pre-stimulation baseline. N o oscillations were observed between 0.0015 to 0.015 p M P E within the recording intervals (45-60s) and a frequency of zero is assigned to these concentrations. 40 Figure 15: Concentration-dependence of the apparent velocity of PE-induced recurring Ca 2 + wave. Selected frames (A) show the propagation patterns of P E -induced C a 2 + waves. Distinct wave origins were observed even at maximal agonist concentration (B). (C) The apparent propagation speed of these C a 2 + waves was correlated to the drug concentration (PE, n=15 cells from 4 animals). Os 0.01 0.1 1 10 100 [PE] fumol/L) 42 CHAPTER VI: ELUCIDATION OF THE MECHANISM OF PE-MEDIATED WAVE-LIKE [CA2+]i OSCILLATIONS With the understanding that PE-mediated asynchronous wave-like [Ca 2 +]j oscillations signal for and modulate the tonic constriction o f the rabbit I V C , its mechanism remains undefined. In this segment of the study, we examined the mechanism of the wave-like [Ca 2 +]j oscillations in relation to the force generation. In addition to pharmacological approaches, R T - P C R analysis of m R N A expression were also used to gain insights into the mechanism of the wave-like [Ca 2 +]j oscillations. Importance of the SR Ca2+ store. SERCA. IPiR and Ca2+ entry in PE-mediated wave- like fCa2+l oscillations We have described caffeine- and PE-stimulated C a 2 + waves in V S M C within the intact vessel wall of the rabbit I V C (Ruehlmann et al. 2000). Figure 16 shows a comparison of this wave-like pattern generated by caffeine and P E with the non-wave-like pattern of [Ca 2 +]j elevation initiated by high K depolarization. After addition of 2 5 m M caffeine to the bathing solution, Ca waves originate from distinct intracellular loci and travel along the longitudinal axis of the long ribbon-OA-shaped vascular smooth muscle cells, probably as the consequence of regenerative SR Ca release. This wave-like pattern is very different from the non-wave-like rise in [Ca 2 +]j along the length of the cells when the L-type V G C C are activated by elevating [K + ] e x t to 80mM. The OA-difference between these spatiotemporal patterns of the increments in [Ca ]; suggests that when the I V C is stimulated by P E , the initial event is intracellular C a 2 + release, rather than C a 2 + influx from the extracellular space. 43 Depletion of caffeine-sensitive and ryanodine-sensitive C a z + stores with either 2 5 m M caffeine (Figure 17) or lOOuM ryanodine (Figure 17) completely abolished PE-induced oscillations. These findings indicate that the SR is the immediate source of C a 2 + sustaining the observed PE-induced [Ca 2 +]j oscillations. Similarly, as shown in Figure 18, S E R C A blockade with l O p M C P A or 2 u M thapsigargin (data not shown) also abolished the waves while the [Ca 2 + ] i remains elevated just below wave peak value, confirming the critical role of the SR in generation of the repetitive C a 2 + waves. It thus appears that the wave-like [Ca ]j oscillations are the results o f repetitive cycles o f SR C a 2 + release followed by SR C a 2 + refill. During [Ca 2 +]j oscillations when [Ca 2 +]j is elevated above resting level, a significant amount of cytoplasmic Ca w i l l be extruded to the extracellular space via the P M C A or the plasma membrane N a + - C a 2 + exchanger ( N C X ) (Nazer & van Breemen 1998). In the case o f I V C , stimulated C a 2 + entry may be required to compensate for the loss of C a 2 + from the smooth muscle cells in order to adequately replenish the SR C a 2 + store and to sustain the [Ca 2 +]j oscillations. In 2+ 2+ order to determine whether Ca entry is necessary for maintaining these PE-induced [Ca ]j oscillations, extracellular C a 2 + was removed along with the addition of I m M E G T A . Under these conditions, the PE-induced [Ca ]j oscillations was transient and persisted for 28.25 ± 2.58s (n = 84 cells from 4 animals) before dissipating. We then pretreated the I V C with a combination of nifedipine (L-type V G C C blocker) and SKF96365 ( R O C / S O C blocker) to inhibit all known means of stimulated C a 2 + entry into the V S M C . Such pretreatment caused a delayed inhibition of the [Ca 2 + ]i oscillations and maintained the [Ca 2 +]j near the basal value (Figure 19). These results indicate that stimulated C a 2 + entry is required to refill the S R in order to maintain the periodic C a 2 + release, which is responsible for each up-stroke of the PE-induced [Ca 2 + ] i oscillations. Thus, 44 in the absence of stimulated C a 2 + entry, PE-induced [Ca 2 + ] i oscillations can only persist for a few cycles during which C a 2 + is lost from the SR to the extracellular space. The transient period of [Ca 2 + ] ; oscillations elicited by P E in the absence of stimulated C a 2 + entry (Figure 19) must be completely due to repetitive cycles of SR-mediated C a 2 + release. To test the hypothesis that the C a 2 + waves originate from the opening of IP3R channel, 2 - A P B (a putative IP3R channel inhibitor) (Ascher-Landsberg et al. 1999, M a et al. 2000) was added to the pre-incubation solution before stimulating with 5 p M PE . Figure 19 shows that blockade of the IP3R channel prevents all PE-induced C a 2 + waves. These results indicate that PE-induced [Ca 2 +]j oscillations are a direct consequence of C a 2 + release from the SR via I P 3 R channels and, in addition, rely on stimulated C a 2 + entry for refilling of the SR. Components of stimulated Ca2+ entry Given that stimulated Ca entry appears to be necessary for the maintenance of PE-mediated wave-like [Ca 2 +]j oscillations, we proceeded to define the modes of C a 2 + entry that are important here. Nifedipine (L-type V G C C blocker) and SKF96365 ( R O C / S O C and L-type V G C C blocker) 9+ were employed to investigate the routes of Ca entry. However, due to the ability of SKF96365 to block both R O C / S O C and L-type V G C C , its effects on PE-induced [Ca 2 + ] ; oscillations and tonic contraction were only assessed in tissues pretreated with l O p M nifedipine. A s shown in Figure 20, l O p M nifedipine abolished 80mM [K + ] e x t - induced tonic contraction (n= 8 rings) in rabbit I V C pretreated with l O p M phentolamine (an a-adrenergic receptor antagonist used to inhibit the actions of any norepinephrine released from the nerve endings). However, as shown in Figure 21, l O p M nifedipine pretreatment failed to abolish the asynchronous oscillatory C a 2 + 45 waves elicited by 5 u M P E . This is similar to what has been reported by M i r i e l et al in the rat mesenteric artery (Mir ie l et al. 1999). Furthermore, in a detailed analysis of the spatiotemporal characteristics of the oscillations before and after addition of nifedipine, we found that nifedipine pretreatment significantly reduced the frequency of the oscillations from 0.44 ± 0.02 H z to 0.28 ± 0.02 H z (mean ± S E M , n=86 cells). In contrast, nifedipine had no significant effect on the amplitude of the oscillations and the velocity of the Ca waves. These results suggest that there is a relatively large nifedipine-insensitive component of C a 2 + entry that is capable of refilling the SR and maintaining PE-induced [Ca 2 + ] i oscillations. It is clear from Figure 21 that blockade of R O C / S O C with SKF96365 (50pM) abolished the nifedipine-insensitive component of the P E -induced C a 2 + signal. In a parallel contraction study using identical protocols, it was found that nifedipine pretreatment significantly (p<0.05) reduced PE-induced tonic contraction by 27 ± 6 % (mean ± S E M , n=15 rings) (Figure 22) while addition of SKF96365 completely abolished the remaining contraction. This observation again supports the existence of a nifedipine-insensitive, SKF96365-sensitive component of C a 2 + entry which is responsible for 73 ± 6 % (mean ± S E M , n=15 rings) of tonic contraction. Arnon et al. (Arnon et al. 2000) have recently shown that in the mesenteric artery agonists stimulate C a 2 + entry via the reverse-mode N C X as a consequence of N a + entry through a non-selective cationic SOC (see discussion). We therefore tested the possibility that at least part of the SKF963 65-sensitive component of stimulated C a 2 + entry in the I V C is mediated by the N C X . Two distinct inhibitors of N C X , 2',4'-dichlorobenzamil (forward- and reverse-mode inhibitor) 46 (Blaustein & Lederer 1999) and KB-R7943 (selective reverse-mode inhibitor) (Ladilov et al. 1999) were employed in parallel [Ca ]j and contraction studies. For this series of studies all tissues were pretreated with l O u M nifedipine to eliminate Ca entry through L-type V G C C . In a series of contraction studies, it was found that 2',4'-dichlorobenzamil (2,4-DCB) at both l O u M (data not shown) and lOOpM (Figure 23A) significantly (p<0.05) reduced PE-induced tonic contraction in tissues pretreated with nifedipine by 81 ± 6 % (n=8 rings) and 80 ± 2 % (n=16 rings) respectively. The remaining 20% of contraction was completely inhibited with 5 0 p M SKF96365. In a parallel [Ca 2 + ] ; study, it was found that 2',4'-dichlorobenzamil (lOOpM) abolished PE-induced [Ca 2 +]j oscillations in tissues pretreated with nifedipine (Figure 23B), but did not reduce the steady-state [Ca 2 + ] i to the baseline level. However, subsequent addition o f 94-SKF96365 did reduce the level of non-oscillating [Ca ]; to the baseline level. These results indicate that the [Ca 2 + ] i oscillations are, for a large part, dependent on C a 2 + entry via the N C X . To test i f the N C X operating in the reverse-mode provides C a 2 + to sustain PE-mediated [Ca 2 +]j oscillations and tonic contraction in I V C pretreated with nifedipine, we used KB-R7943 which specifically blocks this mode of N a + / C a 2 + exchange. A s shown in Figure 24A, l O p M KB-R7943 also significantly reduced PE-induced tonic contraction in vessels pretreated with nifedipine by 75 + 5% (mean ± S E M , n=9 rings) while the remaining 25% of contraction was completely inhibited by 5 0 p M SKF96365. In a parallel [Ca 2 + ]i study (Figure 24B) the same concentration of KB-R7943 also abolished PE-induced [Ca 2 +]j oscillations. Thus, the fact that two structurally-unrelated inhibitors of N C X similarly abolished the PE-induced [Ca 2 +]j oscillations indicates that there is a large reverse-mode N C X component of C a 2 + entry which is required for refilling the SR 47 and allowing the [Ca 2 +]j oscillations to persist. Interestingly, there is a relatively smaller D C B -insensitive and KB-R7943-insensitive component within the SKF96365-sensitive component, which may reflect C a 2 + entry through the R O C / S O C . The findings that the N C X operating in the reverse mode may be coupled to a R O C / S O C indicate that such a putative R O C / S O C channel must be a non-selective cationic channel (NSCC) . Furthermore, stimulated C a 2 + entry through the L-type V G C C and the NSCC-coupled N C X operating in the reverse mode are important in maintaining PE-mediated wave-like [Ca 2 + ] i oscillations most probably by ensuring adequate refilling of the SR. Characterization of the NSCC The potential candidates for the putative non-selective cationic channels are the receptor-operated channel (ROC) and the store-operated channels (SOC). Given recent reports that certain transient receptor potential (Trp) molecules which have been shown to be the molecular substrates for the SOC are expressed in mammalian blood vessels (McDaniel et al. 2001, X u & Beetch 2001) and given that the [Ca 2 +]j oscillations involve repetitive cycles of SR store-emptying followed by store-refilling (Shmigol et al. 2001), it is highly likely that a non-selective cationic S O C may also be present in the rabbit I V C and may be involved in refilling the SR C a 2 + store to sustain the [Ca 2 + ]i oscillations and tonic contraction in the PE-stimulated rabbit I V C . A s mentioned, in order to maintain the asynchronous [Ca 2 +]j oscillations that signal for the tonic contraction, stimulated C a 2 + entry from the extracellular space is required for the repetitive refilling of the SR C a 2 + store. C a 2 + entry involved in the maintenance of [Ca 2 +]j oscillations and 48 venoconstriction is mediated by a putative non-selective cationic channel (NSCC) component coupled in series with the N C X , and a L-type voltage-gated C a 2 + channel ( V G C C ) component. In this portion of the study, we used the putative IP3R-channel blocker, 2 - A P B to characterize the putative N S C C that is crucial to the maintenance of PE-mediated [Ca 2 + ] i oscillations and constriction of the rabbit I V C . I f a store-operated N S C C is involved here, its activation w i l l be dependent either on IP3R-mediated C a 2 + release or IP3R-mediated SR store depletion. In this scenario, the addition of 2 - A P B should antagonize the activation of the store-operated N S C C . This should then result in the inhibition of PE-mediated [Ca 2 +]j oscillations and venoconstriction. However, the use of this compound has been recently criticized for its non-specific effects on other ion transport mechanisms in cultured cell preparations (Broad et al. 2001, Gregory et al. 2001, M a et al. 2001, Missiaen et al. 2001). For proper interpretation o f the results, we also examined the selectivity of 2 - A P B in V S M of the rabbit I V C . The concentration of 2 - A P B used in all the following experiments is 75 p M , which is above its IC50 for the inhibition of H^R-channels (Missiaen et al. 2001, W u et al. 2000). When the rings of rabbit I V C were pre-treated with 2 - A P B , P E (5 p M ) failed to induce any measurable C a 2 + signal (Figure 25A) as compared to the PE-induced [Ca 2 +]j oscillations observed prior to the pre-treatment. In terms o f force generation, as shown in Figure 25B, P E (5uM) typically elicits a significant sustained increase in tension with an average amplitude of 1.00 ± 0.01 g (mean ± standard error, n=16 rings from 4 rabbits) in the I V C . Such effect was, as expected, mediated through activation of the cti-adrenergic receptor since it can be prevented by the application of the ai-adrenergic receptor antagonist, phentolamine (10 p M , data not shown). Interestingly, P E failed to elicit any significant increase in tension (0.03 ± 0.02g, n=16 rings from 49 4 rabbits) following 2 - A P B pretreatment in the same vessels (solid line in Figure 25B). These results indicate that 2 - A P B prevents the development of PE-induced venoconstriction by blocking the initiation of PE-induced [Ca 2 + ] i oscillations. We then proceeded to examine whether 2 - A P B can disrupt ongoing PE-induced venoconstriction and [Ca 2 +]j oscillations as well . A s shown in Figure 25C and 25D, introduction of 2 - A P B immediately halted ongoing PE-mediated [Ca 2 +]j oscillations and fully relaxed the PE-mediated contraction (1.03 ± 0.05g) to baseline (0.03 ± 0.04g, n=16 rings from 4 rabbits). These findings clearly show that 2 - A P B at 75 p M can effectively prevent or abolish the tonic contraction induced by P E and such inhibition is mediated by preventing or abolishing PE-induced [Ca 2 +]j oscillations. It therefore appears that the opening of IP3R-channel is required for PE-mediated venoconstriction. However, before such a conclusion is reached, we had to examine the selectivity of 2 - A P B (75 p M ) in the rabbit I V C , especially with regards to important C a 2 + translocators such as RyR, S E R C A , S O C and the L-fype V G C C . A s shown in Figure 26A, pretreatment of I V C with 75 p M 2 - A P B did not significantly affect the peak amplitude of the second caffeine-induced C a 2 + transient (105.9 ± 13.1 % of the control, n=l 1 rings from 4 rabbits, p=0.66) and therefore appears to be inactive against RyR. Furthermore, the fact that 2 - A P B pretreatment did not affect the amplitude nor the profile of the caffeine-induced C a 2 + transient implies that it had no significant effect on the plasma membrane C a 2 + extrusion system responsible for removing the excess cytoplasmic C a 2 + released from the SR. The ability of S E R C A to replenish the SR C a 2 + store was assessed by examining the extent of refilling of the caffeine-sensitive store in the presence of 75 p M 2 - A P B in tissues that have been depleted of their SR C a 2 + stores (with 25 m M caffeine). Figure 26A shows that 2 - A P B only marginally affected the refilling of the caffeine-sensitive SR C a 2 + store as the presence of 2 - A P B reduced the peak 50 amplitude of the third caffeine-induced C a 2 + transient slightly but non-significantly by 14.3 ± 9.7g % (n=l 1 rings from 4 rabbits, p=0.12). Our finding indicates that 75 p M 2 - A P B may partially inhibit the S E R C A , as has been shown by Missiaen et al (Missiaen et al 2001). Complete inhibition of S E R C A by high concentration of C P A or thapsigargin w i l l , as we have reported earlier, abolish the [Ca ]; oscillations (Lee et al. 2001). However, it w i l l also lead to a sustained elevation in [Ca ]i presumably due to the opening of S O C as a result of store depletion. Such elevation in [Ca ]i was not observed following IP3R-channel blockade as the application of 75 p M 2 - A P B during P E stimulation promptly abolished ongoing [Ca 2 + ] i oscillations and returned the [Ca 2 + ] i to the baseline. Our result reveals that the SR was not depleted at this point as caffeine stimulated a large C a 2 + transient (Figure 26B), even though C a 2 + release by P E was completely blocked. Therefore, such disruption of [Ca 2 + ]i oscillations must be the consequence of the potent inhibition of IPaR-channel opening, rather than the weak inhibition of S E R C A . To test for direct effects on the Ca entry pathways, we examined the effects of 2 - A P B on the L -type V G C C and the S O C , two plasmalemmal channels important in PE-mediated [Ca 2 +]j oscillations. The L-type V G C C provides a portion of the C a 2 + used to refill the SR C a 2 + store and to sustain the tonic contraction. Given its voltage-dependence, we stimulated it with 80mM [K + ] PSS, which resulted in tonic contraction that can be completely abolished with l O p M nifedipine (Figure 20). Figure 26C shows that 75 u M 2 - A P B pretreatment inhibited high K-induced tonic contraction by 12.9 ± 5.0 % (n=16 rings from 4 rabbits, p=0.039). However, this slight inhibition of L-type V G C C cannot account for the complete inhibition of the force generation by 2 - A P B because we know that only 27% of the I V C constriction induced by P E is contributed by Ca influx through the L-type V G C C (Figure 22). Most notably, this result indicates that 7 5 p M 2-51 A P B exerts only marginal direct inhibition on the L-type V G C C . Therefore, during a i-adrenergic stimulation 2 - A P B must be inhibiting Ca influx through the L-type V G C C indirectly, by inhibiting the upstream activation mechanism(s). In addition to the L-type V G C C , a putative N S C C is important for sustaining the [Ca 2 + ] i oscillations and mediates nearly 73% of tonic contraction induced by P E . Its sensitivity to 2 - A P B observed here implies that it is most probably a SOC-type channel. To test for the presence of S O C in the rabbit I V C we used 5 p M P E to discharge C a 2 + from the S R and then applied l O p M C P A to inhibit S E R C A . A s shown by the representative trace (n = 30 cells from 3 rings of I V C ) in Figure 26D, this resulted in a maintained elevation o f [Ca 2 + ] i above baseline [Ca 2 + ]; . This time the elevated [Ca ]j could not be returned to baseline by 2 - A P B , because closing of the IP3R did not lead to refilling since S E R C A was blocked. However, the [Ca ]j returned to baseline upon subsequent addition of 5 0 p M of the R O C / S O C blocker SKF96365. This suggests that this maintained elevation o f [Ca 2 +]j is due to increased C a 2 + influx through the S O C , which is opened as a consequence of depletion of the SR C a 2 + store with P E and C P A . Theoretically, 2 - A P B can prevent activation of SOC either by preventing SR C a 2 + depletion, or by blocking the S O C channel directly. However, the representative trace depicted in Figure 26D shows that 2 - A P B did not affect the maintained elevation in [Ca 2 + ]; following SR store depletion with P E and C P A , even though SKF96365 completely abolished it. This finding indicates that in the V S M C of the rabbit I V C 2 - A P B does not inhibit the SOC directly. In light of these new findings, we can rule out direct inhibition on the RyR-channels, the S E R C A , the L-type V G C C and the S O C as the primary mechanism of inhibition by 2 - A P B of PE-mediated 52 [Ca 2 + ]i oscillations and tonic contraction. The prevention on the generation of any C a z + signal or force with 2 - A P B pre-treatment indicates that IPsR-channel-mediated SR C a 2 + release is crucial. In addition to inhibiting C a 2 + release, 2 - A P B also prevented stimulated C a 2 + entry through both the N S C C component and the L-type V G C C component because no force can be generated or maintained in the presence of 2 - A P B . This indicates that the putative N S C C is most probably a SOC-type channel, which as we demonstrated earlier, does appear to exist in the rabbit I V C . This speculated involvement of SOC in PE-mediated [Ca ]i oscillations and constriction of the rabbit I V C is also consistent with the findings that the nifedipine-resistant, SKF96365-sensitive component of [Ca 2 + ] i oscillations and tonic contraction mediated by the putative N S C C is also sensitive to N i 2 + and L a 3 + ions, agents commonly used to block the S O C (L iu et al. 2000, McDanie l et al. 2001, Shmigol et al. 2001). A s shown in Figure 27A, application o f 2 m M NiCl2 or 300 p M LaCl3 in I V C pretreated with nifedipine (10 pM) completely abolished the [Ca 2 +]j oscillations stimulated with 5 p M PE . In vessels pretreated with nifedipine, application of 2 m M NiCi2 reduced PE-mediated tonic contraction (0.71 ± 0.03 g) to a baseline level of 0.02 ± 0.0 l g (n=9 rings from 4 rabbits). Similarly, application of 300 p M L a C ^ decreased PE-mediated tonic contraction (0.70 ± 0.04 g) to 0.01 ± 0 .0lg (n=8 rings from 4 rabbits) (Figure 27B). It should be noted that even though L a 3 + and N i 2 + are commonly used to block the S O C , they are not selective for the SOC. In this context, these results do help to characterize this putative N S C C as L a 3 + - and Ni 2 +-sensitive. These characteristics, together with the sensitivity of the P E response to 2 - A P B , indicate that this putative N S C C is a SOC-type channel. Given that the functional evidence points to the non-selective cationic S O C as a crucial component for PE-mediated [Ca 2 +]j oscillations and tonic contraction in the rabbit I V C , we then proceeded to 53 determine whether S O C m R N A is expressed in the smooth muscle of the rabbit I V C by R T - P C R study. The most well characterized genes that encode the SOC belong to the family of Trp genes with a large subfamily of Trp 1-7 (Harteneck et al. 2000, McDanie l et al. 2001). We therefore examined the m R N A expression of Trp 1-7 genes in the smooth muscle of the rabbit I V C . It should be noted here that due to the fact that the rabbit equivalent o f Trp 1-7 have not been sequenced, we used primers designed based on mouse or rat sequences. However, since there is high inter-species sequence homology when comparing between mouse and rat sequences for the same type of Trp channels, it is highly plausible that the primers that we use can identify rabbit Trp channels as well . A s depicted in Figure 28A, only a single band of the predicted size (372 bp) of Trp 1 was detected in the smooth muscle of the rabbit I V C (n=5 I V C s from 5 rabbits). In parallel, both rabbit and rat brains were used as positive controls for Trp 1-7 expression. With the same primers, only T r p l , 3 and 4 m R N A were detected in the rabbit brain (n=3 rabbit brains) while all Trp 1-7 m R N A were detected in the rat brain (n=3 rat brains). Furthermore, because C a 2 + influx via the L-type V G C C has been shown to be important for PE-mediated [Ca 2 +]j oscillations in the rabbit I V C , we also tested the expression of ctic-subunit (the pore-forming unit) of the L -type V G C C in the rabbit I V C (Gustafasson et al. 2001). R T - P C R analysis of the I V C (n=5 IVCs from 5 rabbits) showed m R N A expression for the ctic subunit of the L-type V G C C (Figure 28A). Sequencing of the Trp 1-7 and a i c subunit amplification products revealed 100% homology with the respective sequences obtained from GenBank. 54 Figure 16: Spatio-temporal pattern of C a z + signaling Sets of time-series images (set 1,2 and 3) are displayed to identify the spatiotemporal patterns of 2+ [Ca ]j rise elicited by different stimuli. Still-images of a field of smooth muscle cells is shown to illustrate the rectangular regions (outlined in white in the leftmost images) from which each set of time-series images are derived. The rectangular regions contain a segment o f one representative ribbon-shaped smooth muscle cell and the rectangular regions are enlarged and contrast-enhanced to facilitate the visualization of the spatiotemporal patterns of [Ca 2 +]j rise in these time-series. Variations in fluorescence intensity (F525) directly reflects changes in [Ca 2 +]j . In addition, the changes in F525 over time from selected areas (area 1, 2 and 3 outlined in white) spaced out across the longitudinal axis of each depicted smooth muscle cell are illustrated on the right in the F525-time traces. SR C a 2 + release with 25mM caffeine caused an initial [Ca 2 + ] ; elevation that was initiated at a distinctive intracellular locus (time 1.07s) and subsequently propagated along the longitudinal axis o f the smooth muscle cell in a regenerative, wave-like fashion. The F525-time traces (Set 1) from the three intracellular areas indicate a sequential rise in [Ca 2 +]j in time as the C a 2 + signal was initiated at area 1 and subsequently propagated in a wave-like fashion through area 2 and finally to area 3. Stimulation by 5 p M P E also elicited a wave-like C a 2 + signal as the F525-time traces (Set 2) demonstrated a sequential rise of [Ca 2 +]j in time in the three intracellular areas. In contrast, C a 2 + influx across the plasma membrane (PM) stimulated with 8 0 m M extracellular K + PSS (80mM [ K + ] e x t ) , caused an initial C a 2 + elevation that appears to be non-wave-like in nature. Such a non-wave-like pattern is clearly demonstrated by the F525-time traces (Set 3) which indicate a synchronized rise in [Ca 2 + ] i from the three intracellular areas. Thus, the wave-like pattern of the [Ca 2 +]j rise within a series of PE-induced [Ca 2 + ] i oscillations resembles that seen 55 in response to caffeine. This suggests that individual Ca spikes elicited by P E are the result of regenerative SR C a 2 + release. The scale bars shown represent 5pm distance. 25mM caffeine - wave-like o c I co CN ' • 2 n i 1.86s 2). 5|JM phenylephrine - wave-like LU OL 3). 80mM [K +] e x t - non-wave-like 5 E o o o _ 0 3 02 1D Os _ 1.00s J ! 1.40s 9 1.80s 122 2.20s . t i 150 c 3 ^—'100 LO il CM W 50 li . ^ 1 5 0 "E 3 100 LO CM LO Li. 50 Time (s) 1 2 3 Time (s) 56 Figure 17: Effects of SR depletion with caffeine or ryanodine on PE-mediated [Ca2+] oscillations (A) I V C smooth muscle cells show distinct C a 2 + oscillations in the presence of P E (15pM) Pretreatment with caffeine (25mM, B) or ryanodine ( lOOpM, C) completely abolishes the oscillations (n=84 cells from 4 animals) suggesting that the oscillations are maintained by repetitive SR discharge. Control 10 units 10 s 15umol/LPE B 10 units 10 s Pre-treated with 25 mmol/L caffeine 15umol/L PE Pre-treated with 100 umol/L ryanodine -4 15 Mmol/L PE 10 units 10 s Figure 18: Effects of CPA on PE-mediated [Ca z]i oscillations Representative C a 2 + trace is shown that displays the temporal changes in [Ca 2 + ] i determined in a 1.36 p m 2 cytoplasmic region of a single smooth muscle cell from the rabbit I V C . S E R C A blockade with l O p M C P A resulted both in the abolishment of PE-induced [Ca ]; oscillations and a sustained elevation in [Ca 2 + ] i . 58 Figure 19: Ca entry is required to sustain repetitive SR-mediated IP3R-dependent Ca waves. PE-elicited transient [Ca 2 +]j oscillations in tissues pretreated with 10 p M nifedipine (L-type V G C C blocker) and 50 p M SKF96365 ( R O C / S O C blocker) and additional pretreatment with 75 p M 2 - A P B (IP3R channel blocker) abolished this transient Ca signal. Pretreated with 5LIM P E 10LIM nifedipine 5 0 L i M S K F 9 6 3 6 5 V * * L * * » * ^ ^ 7 5 M M 2 - A P B 5LJM P E 10(JM nifedipine 50LIM S K F 9 6 3 6 5 59 Figure 20: Effects of L-type VGCC blockade on 80mM [K+]ext mediated tonic contraction in the rabbit IVC. A representative tension trace showing that l O p M nifedipine completely abolished an 80mM [K+]ext-mediated tonic contraction in rings of rabbit I V C pretreated with l O u M phentolamine. 1.0 -I 0.8 • 0.6 • 0.4 • 0.2 • 0.0 • 10LIM Phentolamine 80mM [K+] ext 10|jM Nifedipine 0 5 10 Time (min) 15 60 Figure 21: Effects of L-type VGCC blockade and ROC/SOC blockade on PE-mediated [Ca2+]i oscillations. A s shown in the representative traces which indicate temporal changes in [Ca 2 +]j from a 1.36 p m 2 sub-cellular region, L-type V G C C blockade with l O u M nifedipine (Nit) did not abolish P E -induced [Ca 2 + ] i oscillations. It reduced the frequency of PE-induced [Ca 2 +]j oscillations, but did not affect the amplitude nor the apparent velocity of the oscillatory C a 2 + waves. R O C / S O C blockade with 5 0 p M SKF96365 (SKF) in addition to L-type V G C C blockade abolished P E -induced [Ca 2 + ]i oscillations completely. The frequency of the [Ca 2 +]j oscillations was derived by counting the number of C a 2 + waves generated in a single smooth muscle cell over a time period of 30~60s. A s for the estimation of the apparent velocity of the C a 2 + wave, two subcellular 1.36 p m 2 regions separated by distance x (Ax) were selected and the time lag (At) in the onset of Ca rise between the two regions was determined. The fraction of Ax over At yields the apparent velocity of the C a 2 + wave. (*: statistical significance with Mann-Whitney U test). 61 Subcellular [Ca2+], ( 5 M M ) + Nif ( 1 0 L I M ) + Nif ( 1 0 L J M ) + S K F ( 5 0 L J M ) 20 unitj 5s N 0 5 £ 0 . 4 >* £0 .3 SO.2 g> 0.1 ±S 50 c 340 0) •g 30 = 20 a E 10 < o -ST 35 "g 30 •.25 % 15 O 101 © 5 ^ 0 V*? PE +Nif +SKF&Nif T T . l l l l l lIBliii P i P i l l l t J PPf 111!!! PE +Nif +SKF&Nif ^ 3 3 PE +Nif +SKF&Nif 62 Figure 22: Effects of L-type VGCC blockade and ROC/SOC blockade on PE-mediated tonic contraction. L-type V G C C blockade with l O p M nifedipine partially inhibited a PE-mediated tonic contraction (P < 0.05, Wilcoxon Singed-Rank test), while additional R O C / S O C blockade with 5 0 p M SKF96365 completely abolished the remaining tonic contraction. — control • • • Preincubation -8 -4 0 4 8 12 16 Time (min) 63 Figure 23: Effects of NCX inhibitor 2',4'-dichlorobenzamil on the nifedipine-resistant component of PE-mediated [Ca2+]j oscillations and tonic contraction. Blockade of C a 2 + entry through N C X with lOOpM 2',4'-dichlorobenzamil (2,4-DCB) led to a large inhibition of PE-induced tonic contraction (A) and a complete inhibition of PE-induced [Ca 2 +]j oscillations (B) in rings pretreated with l O p M nifedipine. The remaining contraction was abolished by application of 5 0 p M SKF96365 (SKF) while the non-oscillating [Ca 2 + ] ; was correspondingly reduced . (*: control refers to the amplitude of PE-mediated tonic contraction in rings pretreated with l O p M nifedipine). 64 A Tissue Contraction 100 Nifedipine (10pM) P E (5pM) 2,4-DCB (100LJM) 20 0 mi SKF (50pM) x B Subcellular [Ca2+] i P E (5MM) Nifedipine (10pM) P E (5pM) Nifedipine (10pM) 2,4-DCB (100pM) 10 s 50pM SKF96365 65 Figure 24: Effects of selective reverse-mode NCX inhibitor KB-R7943 on the nifedipine-resistant component of PE-mediated [Ca2+]j oscillations and tonic contraction. Blockade of the N C X operating in the reverse with l O p M KB-R7943 led to a large inhibition of PE-induced tonic contraction (A) in rings pretreated with l O u M nifedipine and complete inhibition of PE-induced [Ca 2 +]j oscillations (B). The remaining contraction can be abolished with 5 0 p M SKF96365 (SKF) . (*: control refers to the amplitude of PE-mediated tonic contraction in rings pretreated with l O u M nifedipine). 66 T i s s u e contraction 0.2 g 500 s Nifedipine(IOpM) PE(5pM) KB-R7943(10pM) S K F ( 5 0 L I M ) 100I 1 80 S ~ 60 S 40 * 20 0 ISP ' m f fy X B P E (5pM) Nifedipine (10pM) P E (5pM) Nifedipine (10pM) KB-R7943 (10pM) Subcel lular [Ca2+]j 20 uni t [ 1 0 s 67 Figure 25. Effects of 75 uM 2-APB on the initiation and the maintenance of PE-induced VSMC [Ca2+]i oscillations and venoconstriction in the rabbit IVC. A ) . The still-frame image to the left shows the ribbon-shaped fluo-3 loaded V S M C that resides in the vessel wall of the rabbit I V C visualized with confocal microscopy. Two sub-cellular regions from two different V S M C in the field of view were chosen and the changes in fluorescence units over time in these regions are depicted by the representative traces to the right. Changes in fluorescence reflects changes in [Ca 2 +]j. Applications of P E initiated [Ca 2 + ] ; oscillations in the cells from the control group (dotted line) while it resulted in no measurable C a 2 + signal in the cells from the 2 - A P B pretreated group (solid line). The experimental control and 2 - A P B pre-treated traces are representative of the results in 70 cells from 4 different rings of rabbit I V C . B) . Application of P E stimulated tension development in the control I V C (dotted line) but elicited no response in the 2 - A P B pretreated I V C (solid line). C). Application of 2 - A P B immediately OA-abolished ongoing [Ca ]j oscillations mediated by P E . The experimental trace is representative of the results in 70 cells from 4 different rings of rabbit I V C . D). Application of 2 - A P B abolished ongoing tonic contraction mediated by P E . 68 75 uM 2-APB 75 U M 2-APB 69 Figure 26. Effects of 75 uM 2-APB on RyR, SERCA, L-type VGCC and SOC in the rabbit IVC. A). Three pulses of caffeine are applied with a 5-minute interval between each pulse. The maximum amplitude of the caffeine-induced C a 2 + transient from the first pulse reflects control SR C a 2 + level as C a 2 + from the SR is released through the opened RyrR-channel. The bar graph to the right compares the average maximum amplitude of the second and the third pulses to the first pulse (n=l 1 rings from 4 rabbits). The error bars represent standard error. Following the addition of 2 - A P B , the second pulse of caffeine resulted in a single C a 2 + transient whose maximum amplitude is similar to the first pulse, indicating that 2 - A P B did not interfere with the opening of RyrR-channels. The third pulse of caffeine resulted in C a 2 + transient whose maximum amplitude was slightly but not significantly diminished as compared to the first pulse, indicating that the 2-A P B may weakly inhibit the S E R C A which mediates refilling of the S R C a 2 + store. B). Following complete inhibition of PE-mediated [Ca 2 + ]j oscillations by 2 - A P B , application of caffeine resulted in a C a 2 + transient with a comparable maximum amplitude to that of the control condition prior to 7-4-P E stimulation (dotted line), indicating that the SR Ca store was replenished. The experimental traces shown are representative of the results obtained in 45 cells from 3 different rings of rabbit I V C . C). Pretreatment of rabbit I V C with 2 - A P B (solid line) significantly reduced high K + (80 m M extracellular K +)-mediated tonic contraction when compared to the control (dotted line). High K + contractions in both the control and the 2 - A P B treated vessels were performed in the presence of 10 p M phentolamine which is used to block of effects of neurotransmitters released by the nerve endings. The experimental trace shown is representative of findings in 16 rings from 4 rabbits. D). Application of 5 p M P E followed by l O u M C P A resulted in a maintained elevation in [Ca 2 + ]i o f V S M C (black solid line). Application of 7 5 p M 2 - A P B did not affect this plateau 70 response while the addition of 5 0 u M SKF96365 abolished the maintained [Ca ]; elevation and returned the [Ca ]j to pre-stimulation baseline level (indicated by gray solid line). The representative trace shown is typical of the responses obtained in 30 cells from 3 rings of I V C . 71 ( A ) , X ' 20 units 2 I . ios i ? 1 0 0 ^ S5 a. 2 0 75 MM 2 - A P B * I 0 25 mM caffeine uM 2 -  25 mM caffeine 25 mM caffeine 75 uM 2 - A P B 25 mM caffeine ( G ) 10 uM CPA & 5pM PE 75 uM 2-APB • • 50 MM SKF96365 V % % \ %• V % % % Y 029L_ 120 s 80 mM extracellular K* 72 Figure 27. Effects of Ni and La on the nifedipine-resistant component of PE-mediated [Ca2+]i oscillations and tonic contraction of the rabbit IVC. A ) . The representative trace to the left shows that applications of 2 m M N i C b to the bath abolished the nifedipine (Nif, 10 pM)-resistant component of the [Ca ]i oscillations stimulated with P E . Similarly, in a different cell from a different set of experiments, the representative trace to the right shows that application of 300 u M LaCL, abolished nifedipine-resistant component of PE-mediated [Ca 2 + ] i oscillations as well . These experimental traces are representative of the results in 60 cells from 4 different rings of rabbit I V C . B) . The representative contraction traces shows that the application of either 2 m M N i C L . (n=9 rings from 4 rabbits) or 300 p M LaCl3 (n=8 rings from 4 rabbits) to the bath completely inhibited the nifedipine-resistant component of the tonic contraction elicited by P E . 73 20 un i t s | 10 s IOUM /IJyDy 5uM P E 10uMNif 2mM NiCI2 • 10uM nifedipine • 5|JM P E 2mMNiCI2 20 un i t s | 10s 5uMPE 10uM Nif 5 u M P E 10uM Nif »'»»•'• >v«»W 300(JM LaCI3 • 10uM nifedipine • 5|JM P E • 300^ M LaCI3 Figure 28. Trpl and die mRNA expression in the smooth muscle of the rabbit IVC. Exemplary agarose gel electrophoresis from R T - P C R analysis of transient receptor potential (Trp) channel family members and a i c m R N A s in the rabbit I V C smooth muscle (A) , rabbit brain (B) and rat brain (C). P C R products were generated through the use of specific primers for Trpl~Trp7 and a i c subunit of the L-type V G C C . Only genes for T r p l (372 bp) and otic (371 bp) were found to be expressed in the smooth muscle of the rabbit I V C . In the rabbit brain positive control, only m R N A for T r p l (372bp), Trp3 (331bp) and Trp4 (265bp) were detected. In contrast, Trp l~7 and a i c m R N A expression were detected in the rat brain as positive controls. The expression of 18S ribosomal R N A was used as an internal control. R T - P C R reactions run in the absence of reverse transcriptase (-RT) or c D N A ( -cDNA) were used as negative controls. The gels shown are representative of findings in a minimum of 3 animals. 75 Rabbit IVC Trpl Trp2 Trp3 Trp4 Trp5 Trp6 Trp7 cti c 18S RT -cDNA Rabbit brain (positive control) Trpl Trp2 Trp3 Trp4 Trp5 Trp6 Trp7 a i c 18S Rat brain (positive control) Trpl Trp2 Trp3 Trp4 Trp5 Trp6 Trp7 <xic 18S CHAPTER VII: REQUIREMENT FOR SUPERFICIAL SR AND PM-SR JUNCTION IN PE-MEDIATED WAVE-LIKE [CA 2 +h OSCILLATIONS E - C coupling of muscle cells in the cardiovascular system typically involves Ca as the central signal for rapid contractile activation. In cardiac muscle cells, the generation of the C a 2 + signal is believed to occur through the mechanism of Ca 2 + -induced C a 2 + release whereby C a 2 + influx through the dihyodropiridine (DHP)-sensitive L-type V G C C activates the R y R (Bers 2000, Wier & Balke 1999). This results in massive Ca release by the SR into the adjacent myoplasm. Structurally, close apposition between the SR cistemae and the plasmalemmal T-tubule systems in the diads is critical for proper E - C coupling in the cardiac myocytes (Takeshima et al. 2000). Such specific ultrastructural arrangement between the P M and the S R allows the R y R to be positioned very close to the DHP-sensitive L-type V G C C in such a manner that the R y R can be exposed to and activated by the high [Ca 2 + ] microdomain near the mouth of the opened V G C C . Unlike its cardiac counterpart, V S M C do not possess the same elaborate ultrastructure. However, electron microscopic images of V S M C do reveal a similar type of arrangement between its P M and the superficial or peripheral SR (Somlyo 1985). What has been widely observed is that the superficial SR frequently comes into close contact (~20nm) with the intracellular surface of the P M . Even though not yet quantified, such close apposition can span hundreds of nanometers on the P M . This, in effect, creates a narrow space sandwiched between the closely apposing superficial SR and the P M and this space is generally referred to as the restricted space or P M - S R junctional space. Given that similar P M - S R junctions in the diads of cardiac myocytes function to 77 facilitate E - C coupling, there is increasing speculation that the P M - S R junction may serve important functions such as E - C coupling in the V S M C as well . A s demonstrated by us and others in various arterial and venous blood vessels, agonist-induced E -C coupling of V S M is mediated by wave-like [Ca 2 + ]i oscillations in individual in situ V S M C (Ruehlmann et al. 2000, Lee et al. 2001). In this case, the contractile myofilaments are activated by repetitive waves of SR C a 2 + release. Our findings have revealed that the opening of the I P 3 R is critical for the generation of these recurring C a 2 + waves as well as for the activation of various C a 2 + entry pathways for the refilling of the SR C a 2 + store (Lee et al. 2001). A critical component involved in mediating C a 2 + entry as we have demonstrated is a store-operated N S C C . It is generally believed that the activation of certain types of S O C requires a close association between the SR and the P M (Putney el al. 2001). In addition, reverse-mode N a -Ca exchange coupled in series with the N S C C was also identified as a main component mediating the refilling the SR C a 2 + store as shown earlier. It was found that N a + entering through the N S C C could accumulate in the P M - S R junctional space and drive the N C X into its reverse-mode of operation (Arnon et al. 2000, Lee el al. 2001). In this chapter, by correlating ultrastructure to function, we attempt to examine whether the presence of the superficial SR and P M - S R junctional space is required for the generation of the wave-like [Ca 2 + ]i oscillations which underlie E - C coupling in PE-stimulated rabbit I V C . Electron microscopy (EM) was used to examine the ultrastructure of the V S M C . A s shown in Figure 29A, the V S M C s of the rabbit I V C possess prominent superficial SR network and P M - S R junctions. The serial E M sections (Figure 29B) and the 3D schematic illustration (Figure 1 A ) depict the 78 geometry of these P M - S R junctions. The SR network in the I V C appears to be more sheath-like than tubular. A t the P M - S R junction, the superficial SR sheath becomes closely apposed to the intracellular surface of the P M . This forms a flattened narrow space that is referred to as the P M -SR junctional space. The P M - S R junction oftentimes extends to the neck of the caveolaes and occasionally to the apices of the caveolaes. Quantitatively, about 14.2±0.7% of the P M is closely associated (within 30nm) with the superficial SR in the V S M C of the rabbit I V C (n=22 cells). Even though the diameter of these P M - S R junctions can reach as long as 1pm (Figure 29A), the distance between the two apposing membranes is small, averaging 19±1 nm (n=22 cells). Thus, the flat P M - S R junctional space sandwiched by the P M and the superficial SR sheath occupies less than 0.5% of the total volume of the V S M C . Following the characterization of the P M - S R junction in the rabbit I V C , we employed cytoskeleton-disrupting agents (calyculin-A and nocodazole) in an attempt to disrupt the P M - S R junction in these V S M C s . Calyculin-A, a serine/threonine phosphatase inhibitor is known to result in condensation of existing F-actin filaments onto the P M (Patterson et al. 1999). Though not yet shown, this is believed to physically disrupt the association between the P M and the peripheral E R . Nocodazole is an activator for microtubule depolymerization (Platts et al. 1999). A s shown by the representative E M images in Figure 29C, 2-hour treatment with lOOnM calyculin-A resulted in the dissociation of the superficial SR away from the P M while caveolaes remained prominent. In this instance, the superficial SR appears to recede towards the middle of the V S M C and inspection of the periphery of 22 cells revealed little or no superficial SR and P M -SR junction present. Moreover, as depicted by the bar graph indicating the percentage of the P M closely (within 30nm) associated with the SR (Figure 30B), increasing concentrations of 79 calyculin-A caused increasing disappearance of P M - S R junctions. In contrast, cells that underwent 2-hour treatment with vehicle (0.1% D M S O ) control (n=22 cells) or l O u M nocodazole (n=12 cells) still displayed prominent superficial SR and P M - S R junctions similar to the vehicle control (Figure 30B). In parallel confocal [Ca 2 +]j imaging study, following a 2-hour treatment with vehicle control, 5 p M 94-P E stimulated wave-like [Ca ]j oscillations in in situ V S M C of rabbit I V C with a average frequency of 0.42±0.02Hz (n=l 13 cells). This is similar to what has been reported by us previously (Lee et al. 2001). A s shown in Figure 3OB, there were no significant changes in the frequency of PE-mediated [Ca ]i oscillations following a 2-hour treatment with l O p M nocodazole when compared to the vehicle control. In contrast, after a 2-hour treatment period, calyculin-A caused a concentration-dependent reduction in the frequency of PE-mediated [Ca 2 + ] j oscillations (Figure 30A and 30B). A t the highest concentration of calyculin-A used (lOOnM), 5 p M P E was only able to, in most instances stimulate a single C a 2 + wave within a 45 s recording period and the 94-actual frequency of [Ca ]; oscillations may thus be even lower than 0.03±0.02Hz (n=l 12cells) indicated. The fact that cells initially responded to P E with a C a 2 + wave indicates that the signal transduction pathway that leads to increased IP3 synthesis is fully functional. A s shown by the bar graph in Figure 30B, there is a positive correlation between the disappearance of the P M - S R junction and the reduction in the frequency of PE-mediated [Ca 2 +]j oscillations. Such positive correlation suggests that the dissociation of the superficial SR from the P M or the disappearance of the P M - S R junctions renders the cells incapable of generating the 94-wave-like [Ca ]j oscillations that underlie PE-induced E - C coupling in the rabbit I V C . This may 80 have occurred because a putative plasmalemmal SOC-type N S C C critical in mediating SR Ca refilling following each wave of C a 2 + release has been incapacitated by the disruption of the P M -SR junction. On this note, it is important to point out that the calyculin-A concentration-response curve for the inhibition of [Ca 2 + ] i oscillations in the I V C resembles that for the inhibition of the SOC in human platelets (Rosado & Sage 2000). The non-selective cationic S O C in human platelets has been identified as the T r p l channel. Coincidentally, earlier R T - P C R study also confirmed active m R N A expression of T r p l in the rabbit I V C as well . This would imply that the activation of this non-selective cationic SOC requires close spatial association between the superficial SR and the P M . This is also consistent with the conformation-coupling hypothesis and the exocytosis model for S O C activation (Barritt 1999, Putney et al. 2001). Alternatively, it is possible that IPaR-mediated C a 2 + release into the P M - S R junctional space may activate a C a 2 + release activated non-selective cationic channel on the P M and the dissociation of the superficial SR from the P M may disrupt the effectiveness of such functional coupling. Another consequence of dissociating the superficial SR from the P M may be that N a + entry through the S O C would be diluted into a space, which is now much larger than the original P M - S R junctional space and therefore the local [Na +] would be too low to reverse the N C X . These findings provide the first evidence for the importance of P M - S R junctions in the generation of wave-like [Ca ]j oscillations, which underlie E-C coupling in the V S M C . This has also raised the intriguing possibility that ultrastructural alterations of these P M - S R junctions in V S M C may occur and play a role in the pathogenesis of vascular disease. 81 Figure 29: Ultrastructure of VSMC of the rabbit IVC revealed with electron microscopy. A ) . These E M images illustrate various forms and sizes of the P M - S R junctions commonly seen in the V S M C of the rabbit I V C . These P M - S R junctions can reach a diameter as long as 1 pm (see arrows). The black scale bar indicated represents lOOnm of distance. The 3-D cartoon contains a stylized illustration of the close association between the superficial SR sheath, the perforating caveolae (C), the P M and a mitochondrion (M). B) . Serial cross-sections E M images of V S M C are shown in panel 1-6. Each section is ~80nm in thickness. These serial sections illustrates the close spatial apposition between the superficial SR sheath and the P M with the apices of the caveolaes perforating through the superficial SR sheaths to come into contact with the bulk cytoplasm. The close apposition between the superficial SR sheath, the P M and the caveolae creates a flattened narrow space that is referred to as the P M - S R junctional space. The black scale bar indicated represents lOOnm of distance. C). These E M images of two different cells shows lOOnmol/L calyculin-A mediated dissociation of the superficial SR sheaths from the P M (see arrows). The black scale bar indicated represents lOOnm of distance. 82 83 Figure 30. Correlation between the disruption of the PM-SR junctions and the inhibition of PE-mediated wave-like [Ca2 +]i oscillations in VSMC of the rabbit IVC. Tissues were pretreated for 2 hours with the vehicle (0.1% D M S O ) , lOpmol/L nocodazole (Noco) or various concentrations of calyculin-A (Caly-A; 3, 30, lOOnmol/L). A ) . A s shown by the representative fluorescence traces, pre-treatment with increasing concentrations of calyculin-A resulted in a concentration-dependent reduction in the frequency of PE-mediated [Ca 2 + ] ; oscillations. The experimental traces shown are representative of a minimum of 60 cells in each treatment group. B) . The bar graph shows a positive correlation between the loss of the P M - S R junctions (black bar) and the inhibition of pheylephrine (PE)-mediated [Ca ]j oscillations (grey bar) induced by various concentrations of calyculin-A. lOpmol/L nocodazole had no significant effect on either the frequency of PE-mediated [Ca 2 + ]j oscillations or the presence of the P M - S R junctions. The percentage of the P M associated with SR thus provides a quantitative assessment of the amount of P M - S R junctions present in the V S M C . The values indicated on the bar graph the averages derived from a minimum of 60 cells for the Ca study and a minimum of 12 cells for the E M study. The error bars indicate standard errors. 84 (A) Vehicle control 3 nmol/L Calyculin-A 30 nmol/L Calyculin-A 100 nmol/L Calyculin-A 5s 5 umol/L PE ^ o<^  N S; f / / / 85 CHAPTER VIII: DISCUSSION Characteristics of PE-mediated [Ca Ji signals The rabbit I V C is a large capacitance vein, whose tonic contractions determine venous return to the heart and therefore modulate the stroke volume and indirectly the blood pressure. Our observations on C a 2 + signaling in individual smooth muscle cells of this intact tissue shed new light on how wall tension is regulated in large veins. This response to agonists is typified by repetitive transient elevations in [Ca 2 + ]i which originate in certain locations and then spread as waves over the length of the cell (Figure 7 A - B ) . The cells respond independently of each other in that the [Ca 2 + ] i oscillations are not synchronized and that cells vary in their sensitivity to PE . Observing the summation of all smooth muscle cell activity in the tissue bath, reveals typical dose response curves for both force development and [Ca 2 +]; . Thus the asynchronous oscillations of tens of thousands of cells blend into tonic responses as proposed earlier by l ino for the behavior of the rat tail artery (lino et al. 1994). In the present study we have extended these observations to an actual comparison between the individual cellular responses and those of the whole vein. The repetitive [Ca ]j waves in the intact vena cava have the following characteristics: 1. the amplitude has a constant value regardless of the [PE] (Figure 13D), 2. the frequency increases with increasing [PE] (Figure 14C), 3. the velocity increases with increasing [PE] (Figure 15C) and 4. at high [PE] the inter-spike [Ca 2 + ]i becomes elevated but the peak values remain constant (Figure 13D and Figure 14A). In addition, the sensitivity of each cell to P E is variable, such that the recruitment of responding cells increases with increasing [PE] (Figure 13E). 86 Correlation of [Ca Ji signals with the development of tension Physiologically, a blood vessel controls blood flow through its lumen by producing varying degrees of vasoconstriction. Such regulation is reflected in the classical dose-response relationship whereby increasing concentration of endogenous vasoconstrictor agents elicit increasing levels of contractions at the whole-tissue level. A s demonstrated in Figure 10, rings of rabbit I V C display concentration-dependent contractions to P E . These graded isometric contractile responses must be mediated by certain concentration-dependent signaling mechanisms over the same range of agonist concentration. With regard to the Ca 2 +-dependent modulation of I V C contractility, we have identified distinctive, concentration-dependent signaling mechanisms both at the tissue level and at the individual cell level to account for the graded level of contraction produced. A t the tissue level, a concentration-dependent recruitment of V S M C initiating C a 2 + signals was observed in response to both P E and caffeine. The mechanism responsible for differential recruitment o f cells populating the same tissue is probably based on the intrinsic heterogeneity of V S M C (Frid et al. 1997). More specifically, our observations indicate that neighboring V S M C from I V C differ in their sensitivity to P E and caffeine. These differences may be due to variations in ultrastructure and/or basal enzyme activities (Frid et al. 1997). Regulation of contractile strength by caffeine appears to be mainly related to the recruitment process. Clearly, the "all-or-none" nature of the cellular [Ca 2 + ] i signal is incapable of yielding a graded dose response curve (Figure 1 I D and 13D). Thus, the magnitude of the [Ca 2 +]j signal at the cellular level does not encode information to modulate V S M C contractility. The velocity of the caffeine-induced C a 2 + wave, however, was found to be concentration dependent, especially at the higher concentration range between 2 .5mM caffeine to 87 25rnM caffeine (Figure 12). This observation is consistent with the concept that caffeine acts by increasing the sensitivity of R y R to C I C R (Rousseau & Meissner 1989, Sitsapesan & Willisams 04-1990, Rousseau et al. 1988). The Ca wave observed in response to caffeine reflects regenerative release of C a 2 + through R y R downstream from the origin of the C a 2 + wave. A s the concentration of caffeine applied increases, both C a 2 + sensitivity and open probability of R y R are further elevated, resulting in more rapid wave-like induction of C I C R (lino 1989). However, considering that the amplitude of the C a 2 + wave is constant, it seems unlikely that wave velocity modulates V S M C contractility. Therefore, given the close resemblance of the concentration-dependency relationships between tension generated (Figure 10) and the proportion of cells recruited (Figure 1 IE), the magnitude of caffeine-induced contraction is mainly determined by the level of V S M C recruitment. In contrast to caffeine, a comparison between the [PE]-response curves for contraction (Figure 10) and cell recruitment (Figure 13E) reveals a positive correlation between the degree of cell recruitment and developed force only at the lower concentrations ranging from 0.015pM to 1.5pM. This implies that additional mechanism(s) must be involved in regulating contractility, especially at the higher concentration range. We have shown that both frequency of oscillation 04-and the apparent velocity of the Ca waves are dependent on [PE] (Figures 14C and 15C), while the amplitude of oscillation is independent of [PE] (Figure 13D). Therefore, information governing the graded level of contraction at the higher [PE] range is encoded in the frequency and possibly the velocity domains. A s discussed above, it is unclear whether higher wave velocity can contribute to a greater degree of contraction. On the other hand, higher frequency of oscillations can enhance the contractile response by several possible mechanisms. Firstly, higher frequency of 88 fixed-amplitude C a oscillations due to shortening of the inter-wave periods results in higher average [Ca 2 + ]i over time and thus enhances myofilament activation. Secondly, increasing frequency of oscillation may lead to activation of certain frequency-sensitive enzymes that can potentially affect the level of contraction. A n example of this class of enzyme is Ca 2 + -calmodulin kinase II that was found to be sensitive to the frequency of Ca spikes in vitro (De Koninck & Schulman 1998). Thus, we speculate that oscillation frequency is an important regulator of contractility while the velocity of the C a 2 + waves remains of unknown physiological relevance. The observations presented and discussed herein emphasize that the classical concept of an SR C a 2 + release contribution to the initial phase of contraction followed by tonic force dependent on C a 2 + entry from the extracellular space to the myoplasm does not apply to a-adrenergic constriction of capacitance veins. Instead the entire tonic contraction is mediated by the SR beginning with its initial C a 2 + release followed by repeating cycles of C a 2 + uptake and release. Mechanism of PE-mediated wave-like [Ca2+Ji oscillations Functionally, tonic vasoconstriction is signaled by the asynchronous C a 2 + waves in individual V S M C s . But, how are they being generated and sustained? lino and collaborators (lino et al. 1994) proposed that agonist-induced C a 2 + waves are propagated by regenerative C a 2 + release from the SR network. This hypothesis is supported by our data on the inhibitory effect of 2 - A P B , 7-t-which shows that the generation of these Ca waves requires the opening of I P 3 R . However, this does not rule out that C a 2 + release via the RyR-channel may serve to amplify the C a 2 + signal (initiated by IP3R mediated C a 2 + release) through a C I C R mechanism. Typical for smooth 89 muscle is that the release of Ca from the SR is always accompanied by Ca extrusion from the cells and that in a C a 2 + free medium all C a 2 + released from the SR is irreversibly lost to the extracellular space (Leijten & van Breemen 1986). Thus, it follows that refilling of smooth muscle SR involves C a 2 + entry across the cell membrane. In this paper we present evidence to support the view that agonist-induced [Ca ]i oscillations are generated by cyclical movements of C a 2 + . This C a 2 + cycle starts with C a 2 + release from the SR through IP3R channels to the myoplasm. A large fraction of the elevated myoplasmic Ca is then extruded via the P M C A to the 94- 94-extracellular space, while the remaining fraction of the Ca is taken up by the S E R C A . Ca entry via the N C X operating in the reverse-mode and C a 2 + channels subsequently supplies C a 2 + to a sub-plasmalemmal, cytoplasmic microdomain from where it is pumped back into the SR via S E R C A . The mechanisms contributing to the individual components of this cyclical movement of C a 2 + are discussed below. Several investigators initially suggested that the SR could be directly filled either by R O C 94-(Fasolato et al. 1994), capacitative Ca entry (Casteels & Droogmans 1981, Putney 1986) or by V G C C (Gagov et al. 1994). However, the advent of the S E R C A blockers, like thapsigargin and C P A , provided evidence that refilling requires S E R C A activity (Deng & K w a n 1991). It was found that refilling of smooth muscle SR is not accompanied by the expected rise in global [Ca 2 + ]i and myofilament activation (Aaronson & van Breemen 1981, Casteels and Droogmans 1981). The results reported herein indicate that each upstroke of the [Ca 2 +]j oscillations is associated with release through IP3R channels rather than with the influx of C a 2 + , again suggesting that refilling does not proceed via the bulk of the cytoplasm. This and other functional evidence has lead to the hypothesis that refilling of smooth muscle SR takes place in part across a restricted 90 cytoplasmic space, or micro-domain, located between the cell membrane and the peripheral SR (for reviews see van Breemen et al. 1995 and Karaki et al. 1997). Although much of the evidence for cytoplasmic micro-domains is indirect and deduced from functional data, narrow spaces of approximately 15-20 nm in width between the plasma membrane (PM) and the SR have been visualized by electron microscopy (Figure 29) (Somlyo 1985). In addition, ion transport proteins residing in the P M have been co-localized with the superficial SR using deconvolution and confocal microscopy of fluorescent antibodies (Moore et al. 1993, Juhaszova et al. 1994, Juhaszova et al. 1997). A major finding in this report is that blocking the R O C / S O C with SKF96365 prevented the maintenance of repetitive C a 2 + waves. a-Adrenergic receptor activation has been reported to open R O C , which are cation-channels permeable to both C a 2 + and N a + (Fasolato et al. 1994). Since the a-adrenergic receptor is in itself not a channel, or channel activator, intermediate signalling possibly involving a second messenger ( G a , IP3, IP4 or C a 2 + ) is required (Fasolato et al. 1994; Barritt 1999). Another hypothetical mechanism, which links emptying of the SR store to opening of SOC, has been described for smooth muscle and non-excitable cells (Casteels & Droogmans 1981, Cauvin et al. 1984, Putney 1986). However the molecular mechanism, which may involve either a calcium-influx factor (Trepakova et al. 2000) or direct mechano-coupling with the IP3R (Putney 1999, M a et al. 2000), remains to be determined. It has also been reported that different modes of activation function in parallel to open a common channel identified as either R O C or SOC (Wang & van Breemen 1997). In any case the influx of N a + and C a 2 + through these N S C C s depolarizes the plasma membrane potential sufficiently to open L-type V G C C . This was confirmed by the inhibitory effect of nifedipine on the PE-induced contraction. 91 The effect of nifedipine was interesting in that it did not affect either the amplitude or velocity of the repetitive C a 2 + waves, but did reduce their frequency. There are two plausible mechanisms through which the frequency could be decreased. Firstly, the absence of stimulated Ca influx 94-through L-type V G C C may reduce the rate of refilling of the SR Ca store. Given that SR [Ca2 +]iuminai can regulate IP3R opening probability (Meldolesi & Pozzan 1998), a reduced rate of SR C a 2 + refilling can result in a decreased frequency of SR C a 2 + release at the wave initiation site. Secondly, C a 2 + influx through L-type C a 2 + channels can elevate [Ca 2 + ] i in regions adjacent to IP3R 9-4-which facilitates C I C R mediated by both types of SR Ca release channels. Thus, removal of C a 2 + entry through L-type V G C C can result in a delay of regenerative C I C R and thereby reduce the frequency of the [Ca 2 +]j oscillations. The nifedipine-insensitive component was not only blocked by SKF96365, but was also greatly inhibited by blockers of the N C X . This implicates the reverse-mode N C X as a physiological route of C a 2 + entry in the V S M C . Since the NCX-mediated C a 2 + entry is also sensitive to SKF96365, it follows that the reverse-mode N C X C a 2 + entry is dependent on the stimulation of R O C / S O C . While evidence indicates that at a resting [Na +]i o f ~ 1 0 m M the N C X typically operates in the forward C a 2 + extrusion mode, when [Na+]j is elevated above 20mM, the energetics favor the reverse-mode activity. In order to significantly raise [Na+]j, N a + must enter into the cell, putatively, via the N S C C (ROC/SOC) into a restricted space, which is considerably smaller than the bulk cytoplasm. In contrast to L-type V G C C (?Ca2+/?Na+ > 1000) (Bean 1989), R O C / S O C are believed to be much more permeable to N a + with a ?Ca2+/?Na+ < 10 (Philipp et al. 1996). With 140 m M of extracellular N a + as compared to 1.5 m M extracellular C a 2 + , opening of 92 R O C / S O C should result in a large N a + influx in addition to the C a 2 + influx. It has previously been proposed that N a + entering the cardiac myocyte accumulates in a restricted sub-plasmalemmal space (fuzzy space) formed by the close apposition between the superficial S R and the P M (Philipson & Nico l l 2000) similar to the one discussed above for smooth muscle. Thus, conditions may exist to temporarily reverse the balance between the N a + and C a 2 + gradients to promote C a 2 + entry via the N C X . In fact our findings support a mechanism, similar to that previously proposed by Blaustein and co-workers (Amon et al. 2000), whereby C a 2 + entry through the reverse-mode N C X is serially coupled to N a + entry through the activated R O C / S O C . Therefore, either blockade of the R O C / S O C or blockade of the N C X (reverse-mode) abolishes the [Ca ]j oscillations. This model is further supported by the structural findings that the plasmalemmal N C X and the high ouabain affinity isoform (a3) o f the N a + / K + ATPase appear to colocalize with the superficial S R and S E R C A in smooth muscles (Moore et al. 1993, Juhaszova et al. 1994; Juhaszova et al. 1997). 94-This close spatial arrangement would allow Ca entry through reverse-mode N C X to refill the superficial SR via its S E R C A pumps. Since the SR in S M C is believed to be an interconnecting tubular network, refilling o f the deeper SR would occur through S R C a 2 + redistribution from the superficial SR. We have previously shown that at rest the close association between the superficial SR and N C X functions in the forward-mode to unload the SR (Nazer & van Breemen 94-1998). These studies also provided evidence in support of Ca redistribution within the SR. The mechanisms described above may apply to other cell types, as Graier and coworkers recently reported that reverse-mode NCX-dependent endoplasmic reticulum C a 2 + refilling supports agonist-mediated [Ca 2 + ] ; oscillations in endothelial cell lines (Paltauf-Doburzynska et al. 2000). 93 C a 2 + entry through ROC/SOC-coupled reverse-mode N C X , L-type V G C C and R O C / S O C all 9-t-contribute to force generation. However, Ca entry through the R O C / S O C alone is incapable of supporting the recurrent C a 2 + waves. This may be due to the relative slow rate of C a 2 + entry through these N S C C , which is not able to refill the SR C a 2 + store to a sufficiently high level to maintain the repetitive firing of Ca waves. However, the slow Ca entry through the R O C / S O C does elevate steady state [Ca 2 +]j to support sub-maximal force development as shown in Figure 4. This first series of investigations showed that the tonic contraction of a large vein in response to a-adrenergic stimulation is maintained by repetitive, asynchronous Ca waves, which are initiated by opening of IP3-sensitive SR channels. This oscillating SR C a 2 + release is maintained by stimulated C a 2 + entry through NSCC-coupled reverse-mode N C X , L-type V G C C and receptor-operated/store-operated N S C C which in the I V C is responsible for 59%, 27% and 14% of the tonic contraction respectively. However, the nature of this putative nifedipine-insensitive and SKF-96365-sensitive N S C C remains to be better defined. The ensuing series of studies performed with 2 - A P B provided valuable insights into the nature of 94-this putative N S C C and the importance of IP3R opening in PE-mediated [Ca ]; oscillations that underlie the venoconstriction. Firstly, as suggested before (9), the generation of individual C a 2 + waves requires the opening o f IP3R. This is supported by the findings that the generation o f P E -mediated venoconstriction can be prevented with IP3R-channel blockade by 2 - A P B . These 94-observations indicate that during ai-adrenergic stimulation Ca is not delivered via plasmalemmal channels directly to activate the myofilaments in the rabbit I V C . Instead, the SR network in the aj-adrenergic stimulated I V C delivers C a 2 + directly to the myofilaments. One may 94 speculate that this may represent a more efficient and effective way of activating the myofilaments since the SR network penetrates deep into the myoplasm. The IP3R on the S R play a crucial role as Ca is released by the IP3R to activate the ca lmodul in -MLCK units tethered to the myofilaments (L in et al. 1997). The opening o f IP3R is thus required for PE-mediated [Ca 2 +]j oscillations and constriction of the rabbit I V C . Secondly, as discussed, stimulated Ca entry dependent on a putative nifedipine-resistant, SKF96365-sensitive, N S C C is crucial for sustaining PE-induced [Ca 2 + ] i oscillations and is responsible for nearly 73% of the force development. The complete inhibition of C a 2 + signal and force generation by 2 - A P B indicates that 2 - A P B , in addition to preventing C a 2 + release via the IP3R, also prevents the activation of this putative N S C C . However, when SR depletion is maintained by S E R C A blockade with C P A , 2 - A P B fails to block the N S C C , which subsequently could be blocked by either SKF96365, N i 2 + or L a 3 + . These findings strongly suggest that the putative N S C C is a SOC-type channel. Even though the activation o f this non-selective cationic SOC is dependent on IF^R-mediated SR C a 2 + release, its precise mechanism of activation remains undefined at this time. The observation made in Figure 26D, however, does exclude the conformational coupling model (Ma et al. 2000) as the mechanism of activation since the SOC in store-depleted V S M C s remains activated in the presence o f 2 - A P B . This channel thus may be similar to the calcium-influx factor activated SOC-l ike non-selective cationic channel that has been described in V S M C (Trepakova et al. 2000, Trepakova et al. 2001). Alternatively, it may be a Ca 2 +-release activated non-selective cationic channel which can be activated by C a 2 + release via the IP3R or by the built-up o f C a 2 + in the plasma membrane (PM)-SR junctional space following S E R C A blockade with C P A . Moreover, i f a SOC-type channel is involved here, the intermittent 95 C a 2 + release that produces the [Ca 2 +]j oscillations should activate this SOC-type N S C C intermittently. On this note, it is interesting to point out that oscillatory inward non-selective cationic current has been described in endothelin stimulated rat aorta, a large vessel that exhibits 2+ asynchronous wave-like [Ca ]j oscillations as well . In addition, our finding of Trp 1 m R N A expression in vascular smooth muscle of the rabbit I V C provides supporting evidence for the existence of a store-operated N S C C in this tissue (Harteneck et al. 2000). This observation of T r p l m R N A expression in rabbit V S M C from the I V C is consistent with a recent report by X u and Beech (Xu & Beech 2001) that Trp l protein is ubiquitously expressed in various human, rabbit and mouse vessels. T r p l is known to encode a component of the S O C (Zitt et al. 1996 Sinkins et al. 1998, L i u et al. 2000, Harteneck et al. 2000) and T r p l protein is the pore-forming component, which has been localized to the plasma membrane of rabbit V S M C ( X u & Beech 2001). Intriguingly, the S O C formed by the product of the T r p l gene has been shown to be a N S C C (Zitt et al. 1996, Sinkins et al. 1998). This is consistent with our earlier investigations, which suggested that N a + influx through this N S C C raises the local [Na +] in the restricted sub-plasmalemmal space which then drives the N a + - C a 2 + exchanger into its reverse mode of operation, bringing C a 2 + into the cell to refill the S R (Lee et al. 2001). It is important to note that even though only one particular T r p l m R N A was detected here, more Trp-type sub-units may also be expressed. Given that primers used to detect Trp 1-7 m R N A s were not of rabbit origin and that only T r p l , 3 and 4 m R N A s were positively identified in the rabbit brain, one can not dismiss the possibility that rabbit Trp2, 5-7 m R N A with distinct rabbit sequences were not detected in this study. Thirdly, our findings with 2 - A P B indicate that the activation of the nifedipine-sensitive, L-type V G C C component of C a 2 + entry which is responsible for 27% of PE-mediated tonic contraction is 96 dependent on IP 3R-channel opening as well . It is plausible that the L-type V G C C may be activated by the depolarizing inward cationic (Na + and C a 2 + ) current through the store-operated N S C C which is activated by IP3R-channel mediated S R C a release. In accordance with the functional data, the m R N A study showed that the a i c subunit of the L-type V G C C is expressed in the smooth muscle of rabbit I V C . Figure 31 depicts our current working model based on the evidence presented in this report and previous published results. It describes the sequence of events occurring during one cycle of SR store emptying and SR store refilling that when repeated gives rise to the observed [Ca 2 + ]i oscillations. Briefly, upon ai-adrenergic receptor stimulation, one of the earliest events is the opening of IP3R (Figure 31 A ) . The SR empties its C a 2 + through the IP3R and this gives rise to a C a 2 + wave (a single C a 2 + spike in a series o f [Ca 2 + ] i oscillations). The C a 2 + released by the IP3R not only elevates [Ca 2 + ] in the myoplasm to activate the myofilaments, it also rises [Ca 2 + ] near the IP3R to activate neighboring IP3R (Bezprozvanny et al. 1991, lino & Endo 1992). In the meantime, the release or emptying of the SR through IP3R-mediated Ca release leads to the opening the putative store-operated N S C C , which may contain the T r p l subunit (Figure 3IB) . Some C a 2 + and a large amount of N a + then enter the P M - S R junctional space. This inward cationic current causes depolarization of the membrane potential which then activates the L-type V G C C . Meanwhile, [Na +] in the restricted sub-plasmalemmal P M - S R junctional space elevates. This drives the N C X into its reverse-mode of operation, bringing C a 2 + into the cell. With the IP 3 R now inactivated by the greatly elevated [Ca 2 + ] at the pore's outer surface and C a 2 + being supplied to S E R C A by N C X , L-type V G C C and SOC, the SR starts to refill (Figure 31C). Once the IP 3 R are closed and the SR C a 2 + store is refilled, the signal to activate the S O C terminates and C a 2 + 97 exchange or Ca influx from the extracellular space ceases (Figure 3 ID) . A s the SR is filled further, both the elevated SR luminal C a 2 + level and C a 2 + leakage from the SR raise activation of IP3R to threshold for regenerative opening (Missiaen et al. 1992). Requirement for superficial SR and PM-SR junctions in PE-mediated wave-like [Ca2+Ji oscillations in the rabbit IVC A s shown by the bar graph in Figure 2B, there is a positive correlation between the disappearance of the P M - S R junction and the reduction in the frequency of PE-mediated [Ca 2 + ] i oscillations. Such positive correlation suggests that the dissociation of the superficial S R from the P M or the disappearance of the P M - S R junctions renders the cells incapable of generating the wave-like [Ca ]i oscillations that underlie PE-induced E - C coupling in the rabbit I V C . This may have occurred because a putative plasmalemmal SOC-type N S C C critical in mediating SR C a 2 + refilling following each wave of C a 2 + release has been incapacitated by the disruption of the P M - S R junction. On this note, it is important to point out that the calyculin-A concentration-response curve for the inhibition of [Ca ]i oscillations in the I V C resembles that for the inhibition of the SOC in human platelets (Rosado & Sage 2000). The non-selective cationic S O C in human platelets has been identified as the transient receptor potential channel type 1 (Trpl) . Coincidentally, unpublished work from our laboratory confirms active m R N A expression of Trp l as well . This would imply that the activation of this non-selective cationic S O C requires close spatial association between the superficial SR with the P M . This is also consistent with the conformation-coupling hypothesis and the exocytosis model for S O C activation (Barritt 1999, Putney et al. 2001). Alternatively, it is possible that IP3R-mediated C a 2 + release into the P M - S R junctional space may activate a C a 2 + release activated non-selective cationic channel on the P M 98 and the dissociation of the superficial SR from the P M may disrupt the effectiveness of such functional coupling. Another consequence of dissociating the superficial SR from the P M may be that N a + entry through the S O C would be diluted into a space, which is now much larger than the original P M - S R junctional space and therefore the local [Na +] would be too low to reverse the N C X . These findings provide the first evidence for the importance of P M - S R junctions in the generation of wave-like [Ca 2 + ] i oscillations, which underlie E - C coupling in the V S M C . It also 9-4-provides support for our model of PE-mediated wave-like [Ca ]i oscillations in the rabbit I V C (Figure 31), which requires the presence of the P M - S R junctions for proper activation and C a 2 + cycling. Furthermore, this has also raised the intriguing possibility that ultrastructural alterations of these P M - S R junctions in V S M C may occur and play a role in the pathogenesis of vascular disease. 99 Figure 31. Hypothetical sequence of events during PE-mediated smooth muscle [Ca ]j oscillations in rabbit IVC. The hypothetical sequence of events underlying smooth muscle [Ca ]i oscillations is shown here in the order from A to D as described in details in the Discussion. ( N C X : sodium/calcium exchanger; S O C : a non-selective cationic store-operated channel that contains Trpl-protein; 9+ 9+ S E R C A : sarcoplasmic endoplasmic reticulum Ca ATPase; L - V G C C : L-type voltage-gated Ca 9+ channel which contains the a i c sub-unit; IP3R: IP3-sensitive SR Ca release channel; E m : membrane potential.) 100 o NCX SOC L-VGCC (ct,c) .SERCA Ca 2* Ca2* Ca 2* Ca2* Ca 2* Ca 2* Ca2* Ca2* SERCA Ca 2 Ca 2* Ca 2* Ca 2* Ca 2 + Ca 2 IP3R ' (opened) 1 Ca 2 Ca 2* Myofilaments NCX (reverse-mode) Ca2* s o c (opened) Na* Na* (+) L-VGCC (o,c] • E,,, depolarization • M ( ° P e n e d ) Ca 2*Na* Na* N a + SERCA Ca2* • Na* Na* a + Ca2* Ca2* Ca 2 Ca2* Ca 2 Ca 2 V, I P 3 R (closed) Ca2* Ca2* Ca 2 Ca 2* Ca 2 Myofilaments Ca2* Ca2* B .SERCA SERCA Ca2* C a 2 + Myofilaments Ca 2* Ca 2* C a 2 + C a 2 * NCX 0= s o c (closed) 4 L-VGCC (one) I (closed) .SERCA Ca2* Ca 2* Ca 2* Ca 2 Ca2* Ca 2* Ca2* Ca2* 1 Ca 2 Ca2* Ca 2* Ca 2* Ca 2* Ca 2* I P 3 R (closed) Myofilaments CHAPTER IX: OVERVIEW OF ASYNCHRONOUS WAVE-LIKE [CA^h OSCILLATIONS AND THEIR FUNCTIONAL IMPLICATIONS IN VSMC. Since the first report by lino and co-workers in 1994 (27), asynchronous wave-like [Ca ]\ oscillations have emerged as a common mode of C a 2 + signaling in in situ V S M C s (Table 2). In these isolated blood vessel preparations confocal microscopy of intracellular Ca -sensitive dyes reveals recurrent intracellular C a 2 + waves traveling through the longitudinal axis of the ribbon-shaped V S M C s . These C a 2 + waves, which are usually but not always initiated by agonists, result from SR C a 2 + release and do not propagate between cells. Since the mechanism of the repetitive C a 2 + waves depends on interaction of the peripheral SR with the P M we w i l l first examine the ultra-structural evidence. 102 Table 2: Summary of reported asynchronous wave-like [Ca2+]i oscillations in in situ VSMCs Tissue/Reference Stimuli Ave. frequency Ave. wave velocity Inhibited by Rat tail artery (Iinoetal. 1994, Kasai et al. 1997, Asada et al. 1999) Noradrenaline 0.1 uM 0.3uM LOuM NA (increase with increasing [drug]) NA ~20um/s NA 30uM ryanodine, 50mM caffeine. Angiotensin Local release lOOnM ~0.13Hz ~0.19Hz 18.3+1.Oiim/s lOOnM U-73122, 5uM CPA. Rat mesenteric artery (Miriel et al. 1999, Mauban et al. 2001) Phenylephrine 300nM luM 5uM 0.051+0.002Hz 0.067~0.37Hz NA NA NA 32.4+1.7um/s 20uM CPA. Rat Aorta (Asada et al. 1999) Spontaneous (possibly local RAS) NA NA Rabbit inferior vena cava (Ruehlmann et al. 2000, Lee et al. 2001) Phenylephrine 0.15uM 1.5uM 15uM 150uM 0.044+0.01Hz 0.186+0.027Hz 0.442+0.044Hz 0.511+0.025Hz 16.8+1.3um/s 27.0+3.5um/s 70.9+5.5um/s 89.6+7.4um/s 100uM ryanodine, 25mM caffeine, 75uM 2-APB, lOuM CPA, 2uM thapsigargin. Rat mesenteric artery (Peng et al. 2001) Norepinephrine 0.1~0.5uM lOuM 0.05Hz NA ~36um/s NA lOmM caffeine, lOuM ryanodine, 1 uM thapsigargin. Rat cerebral artery (Jaggar & Nelson 2000, Jaggar2001) UTP lOuM 30uM NA (increase with increasing [drug]) NA 30+20Lim/s 1 OuM ryanodine. Luminal Pressure lOmmHg 60mmHg 0.15+0.03Hz 0.29+0.02Hz NA NA lOuM ryanodine, lOOnM thapsigargin, 25LIM diltiazem. High K + 6mM [K + ] e x t 30mM [ K + l e x t 0.14+0.02Hz 0.33+0.02Hz NA NA 25 uM diltiazem. NA: not available. RAS: renin-angiotensin system. 103 Ultra-structure of VSMC The SR is composed of an interconnected tubular and sheet like network the membranes of which bound the SR lumen. It extends throughout the spindle-shaped V S M C and is contiguous with the nuclear envelope (Somlyo 1985). It contributes to C a 2 + -signaling by virtue of active C a 2 + transport via the S E R C A from the cytoplasm to the SR lumen and C a 2 + release from the SR into the cytoplasm via IP3R and RyR. Its capacity for C a 2 + storage is greatly enhanced by the high capacity low affinity C a 2 + binding proteins, calsequestrin (Raeymaekers et al. 1993) and calreticulin (Milner et al. 1992). The SR has been classified according to its location as superficial or deep, with distinct functions being ascribed to the superficial SR. In domains where the SR apposes the P M it creates a narrow space, which extends on average in two dimensions for about 300-400 nm and has a depth of 15-20 nm. The structures responsible for this spacing have not yet been identified, although in some instances "feet" similar to those seen in triadic junctions in skeletal muscle have been reported (Somlyo 1985), and proteins called "junctophillins" have been isolated from the diads of cardiac muscle (Takeshima et al. 2000). The narrow cytoplasmic space between the junctional SR and P M is referred to as the P M - S R junctional space and is thought to present an imperfect barrier to diffusion of small molecules and ions, in particular C a 2 + and N a + . A s can be seen from the electron micrographs of serial sections of smooth muscle of the rabbit I V C and the schematic 3D interpretations in Figure 29A, the caveolaes are able to perforate the junctional SR sheet, such that their apices frequently remain in contact with the bulk cytoplasm. A s shown in Figure 29B (panel 1-6), the apices of the caveolaes oftentimes are close to the perpendicular or radial SR sheets that appear to arise from the superficial SR sheets. Because of these varying geometric arrangements it is plausible that different membrane domains perform specialized functions. For instance, the L-type V G C C s that supply the majority of the Ca 104 required for activation of actomyosin in small resistance arteries may be located at the apices of the caveolaes. This would allow entering Ca to bypass the P M - S R junction and to access the deeper cytoplasm where the myofilaments are located. In contrast, channels such as the store-operated channels (SOC), which function in refilling of the SR, are most probably located in the P M - S R junctional complex. C a 2 + influx mediated by the SOC could thus be selectively directed into the P M - S R junctional space from which S E R C A on the apposing SR membranes could efficiently take up C a 2 + into the SR lumen with minimal influence on the bulk [ C a 2 + ] i . Furthermore, it is important to note that the periphery of the V S M C is typically very low in density of myosin polymers as shown by the E M picture in Figure 32. This includes the P M - S R junctional space which is completely myosin-free and a peripheral zone of200~300nm in width here referred to as the myosin-poor space. If we assume that the V S M C is a cylindrical tube with a diameter of 3 pm and between 10 to 15% of its surface is closely apposed by the superficial SR, the P M - S R junctional space constitutes approximately 0.3% of the overall cellular volume of the V S M C . In comparison, the myosin-poor space would constitute between 25% and 36% of the total cellular volume. A s mentioned above, the junctional SR is connected to sheets of radial S R (Figure 29B), which extend from the P M through the myosin-poor space into the deeper myoplasm. In theory, these 9-1-perpendicular/radial SR sheets may provide an effective conduit for Ca to access the myosin-rich myosplasm for efficient activation of the myofilaments. In this context it is important to note that calmodulin is bound to the myosin light chain kinase, which is in rum tethered to the thin filaments and therefore in the appropriate place for direct activation by C a 2 + (Wilson et al. 2002). 105 In addition to forming close contacts with the P M , the SR network also comes into close contact with the mitochondria (Nixon et al. 1994, Rizzuto et al. 1998), forming yet another diffusionally restricted space, referred to as the Mi to -SR junctional space (Figure 29B). This less than 80nm wide space, sandwiched between the SR and mitochondrial membranes, also appears to be functionally important. A s the SR network penetrates deeper into the cell, it inserts into the nuclear membrane such that the lumen of the peri-nuclear SR network is continuous with the lumen of the nuclear envelope (Somlyo 1985). Asynchronous Ca2+ waves and vasoconstriction The main function of vascular smooth muscle is to distribute blood flow through selective vasoconstriction and vasomotion. The latter is clearly associated with oscillations in [Ca 2 + ] i , but it was long thought that tonic contraction was initiated by SR C a 2 + release and maintained by elevated C a 2 + influx. However confocal microscopy of intact blood vessels loaded with C a 2 + -sensitive dyes has shown that in many blood vessels agonist-induced contractions are maintained by asynchronous wave-like [Ca2 +]j oscillations in single smooth muscle cells, which summate to give a steady state elevation in [Ca2 +]j for the whole tissue (Ruehlmann et al. 2000). A s discussed, it appears that at the lower agonist concentrations force depends on the number of cells recruited to generate fixed amplitude [Ca ]; oscillations while at higher concentrations force is regulated by the frequency of the oscillations and perhaps also by the velocity of the recurring C a 2 + waves. In addition to causing tonic contraction, low frequency (< 0.05Hz) asynchronous wave-like [Ca2 +]j oscillations, which themselves are associated with only minimal development of tone, appear to be instrumental in the initiation of vasomotion in the rat mesenteric artery (Peng et al. 2001). In this proposed scenario asynchronous Ca waves can activate an unknown depolarizing current 106 through the plasma membrane of each V S M C . The activation of such depolarizing current can by chance occur synchronously in a sufficiently high fraction of V S M C s within the artery to entrain cycles of membrane depolarization and repolarization in electrically-coupled V S M C s . The resulting synchronized but intermittent activation of L-type V G C C in all V S M C s subsequently 94- • produces synchronized non-wave-like [Ca ]j oscillations which underlie vasomotion. Asynchronous Ca2+ waves and vasodilatation While C a 2 + waves can stimulate smooth muscle contraction in large vessels such as the rabbit I V C , similar C a 2 + waves have also been associated with the induction of dilatation of cerebral resistance arteries. In this case, the wave-like C a 2 + release is thought to stimulate C a 2 + activated K + channel (Kca) on the P M , such that the relaxing effect of the resulting hyperpolarization-induced closing of V G C C outweighs the local stimulation of contraction (Jaggar 2001). In accordance with this finding, activation of K c a has been reported following C a 2 + waves in smooth muscle cells (Young et al. 2001). Mechanistically, it is also important to point out that these recurrent C a 2 + waves are triggered by elevated [Ca 2 +]j that results from the maintained opening of the L-type V G C C . The higher surrounding [Ca 2 + ]i sensitizes the R y R to C I C R which initiates the C a 2 + waves repetitively (Mironneau et al. 2001). This is why diltiazem can completely abolish the occurrence of the recurring C a 2 + waves in the rat cerebral resistance artery (Table 1) even though the C a 2 + waves are initiated and produced by SR C a 2 + release. In rat cerebral resistance vessel the stimulus sensitizing the SR C a 2 + release channels to initiate C a 2 + waves is C a 2 + rather than IP3 as in the case of PE-stimulated rabbit I V C . 107 This dual function of Ca waves provides an intriguing example of vascular heterogeneity. How does apparently the same C a 2 + wave elicit contraction in one V S M C type and induce relaxation in another? The answer to this question may reside in the molecular makeup of the P M - S R junctional complex. We have shown previously (Nazer & van Breemen 1998) that blockade of Kca did not affect contractions of the rabbit inferior vena cava. This could be due to a lack of expression of these channels in this tissue; however, unpublished data from our laboratory indicate active large conductance K c a ( B K c a ) m R N A expression by the V S M C of the rabbit I V C . Alternatively, given that the activation of B K c a requires high [Ca 2 + ] i that can be achieved in the P M - S R junctional space, it is also possible that the B K c a in the rabbit I V C is not located in the P M - S R junctional complex and therefore not activated by each passing C a 2 + wave. In contrast, the cerebral resistance artery relies heavily on B K c a activation for the maintenance of membrane potential and regulation of pressure induced myogenic tone (Harder 1984, Nelson et al. 1990, Jaggar et al. 1998). Because of the low C a 2 + affinity of B K c a their open probability is regulated by the spontaneous opening of clusters of R y R releasing C a 2 + sparks near the P M (Bolton & Imaizumi 1996, Jaggar et al. 2000). Recent calculations place the B K c a within 20 nm of the R y R making it likely that in the resistance arteries both types of channels are localized within the P M -SR junctional complex (Perez et al. 2001). With this particular P M - S R junctional complex in the cerebral resistance artery containing B K c a , each Ca wave would induce membrane hyperpolarization, which in turn would inhibit the opening of the L-type V G C C and induce relaxation. We speculate that L-type V G C C must be located in the apices of the caveolae away from the P M - S R junction, such that the activating C a 2 + current would be delivered to the myoplasm. A s Ca originating from V G C C diffuses from the P M to the deeper myoplasm, the signal could be attenuated by S E R C A located in the peripheral SR, but outside the junctional 108 complex, or amplified by C I C R . Evidence suggests that in V S M the peripheral SR functions as a superficial buffer barrier (SBB) rather than amplifying through C I C R . Interestingly in the bladder myocytes where there is direct evidence for C I C R (Ganitkevich & Hirche 1996) the coupling between V G C C and R y R has been found to be of the "loose" type in support of the idea that the V G C C are not part of the P M - S R junctional complexes (Collier et al. 2000). This is further supported by the structural finding that the L-type V G C C is localized on caveolin-rich portion of the P M (Darby et al. 2000). The concept of two different P M - S R junctional complexes having opposite effects on smooth muscle contractility is illustrated in Figure 33. It is important to note that the interaction illustrated between the R y R and the B K c a represents only one aspect of the regulation of the membrane potential by the SR. Given the recent findings that revealed an inhibitory regulation by type 3 R y R on Ca release by other R y R isoforms (Lohn et al. 2001), it is likely that there are more complex interactions between different isoforms of R y R and B K c a in these junctional regions. Mechanism of wave-like [Ca /; oscillations Although rhythmic events have been observed in V S M for many decades their molecular mechanisms remain to be fully elucidated. One possible reason for our limited understanding is that blood vessels display different types of rhythmic activity, which impedes consensus between different laboratories. A t the present the molecular basis for agonist-induced [Ca 2 +]j oscillations has been resolved in some detail for smooth muscle of the inferior vena cava of the rabbit (Ruehlmarm et al. 2000, Lee et al. 2001), which may therefore serve as a basis for comparison with other types of blood vessels. In the case of the rabbit I V C , waves of SR-mediated C a 2 + release begin with the opening of IP3R, since they are prevented or instantly blocked by 2 - A P B , 109 which block IP3R (Figure 25). A s some of the released Ca is extruded to the extracellular space recurrence of the C a 2 + waves depends on replenishment of the SR by stimulated C a 2 + influx. This occurs through three different pathways: L-type V G C C , store-operated N S C C and the N C X operating in its reverse-mode. In this large vein the V G C C s play a relatively minor role since nifedipine w i l l only decrease the frequency of the oscillations and inhibit contraction by 27 %. Blockade of the N C X eliminates the oscillations, leaving a slightly elevated [ C a 2 + ] i , which can be reduced to baseline value by the receptor-operated channel/store-operated channel (ROC/SOC) blocker SKF96365 and also by 2 - A P B . These observations led to postulation of the following sequence of events, illustrated in Figure 33 A . 1) P E activates P L C which catalyzes the synthesis of 9-4-IP3, 2) activation of IP3R and Ca release from the SR near calmodulin tethered to the thin filaments, 3) opening of N S C C in the plasma membrane and influx of mainly N a + a n d some C a 2 + into the P M - S R junctional space, 4) depolarization, opening of the L-type V G C C and reversal of 9-4- 94-N C X resulting in Ca influx, 5) Ca uptake into the SR by S E R C A . The identity and the mode of activation of the N S C C are not yet resolved. The P M - S R junction complexes are likely the sites for interactions between N S C C , N C X and S E R C A during SR refilling and are thus crucial for the occurrence of the recurring C a 2 + waves. In addition the low Na +-affinity N a + / K + - A T P a s e isoforms ct2 and 013 have been localized to the junctional P M (Juhaszova & Blaustein 1997), which would promote elevated junctional [Na +] and reversal of the N C X (Arnon et al. 2000). A s mentioned, the importance of the P M - S R junctional complex in refilling of the SR was confirmed by the finding that dissociation of the superficial SR sheets from the P M by calyculin-A inhibits 9-4-maintenance of the agonist-induced wave-like [Ca ]j oscillations (Figure 29C and 30). In addition, as demonstrated, the smooth muscle of the rabbit I V C does express T r p l , which has been 94-shown to constitute a N S C C and to be activated by SR Ca release (Zitt et al. 1996, X u & Beech 110 2001), but direct evidence for its participation in the above events requires further experimentation with knock-out or anti-sense techniques. It is of interest to note that oscillatory inward non-selective cationic current has been described in endothelin-stimulated rat aorta, a large vessel that exhibits asynchronous wave-like [Ca 2 + ] i oscillations as well (Salter & Kozlowski 1998). The constant amplitude of the propagated C a 2 + wave indicates regenerative transient C a 2 + release 9+ from the SR. The regenerative nature depends on the positive feedback of increasing [Ca ]i on the IP3-sensitivity of IP3R and possibly recruitment of Ca -sensitive RyR. The propagation is due to elevation of [Ca 2 +]j to the threshold for activation of clusters of release channels in adjacent portions of the SR. In some smooth muscle the threshold value has been observed as the inflection point between a "foot" segment and the steep portion of the upstroke of [ C a 2 + ]j elevation. The relative involvement of IP3R and R y R appears to vary between different smooth muscle preparations. The delayed negative feedback on release, which is essential for oscillatory behaviour, has been ascribed to a number of mechanisms: 1) inhibition of IP3R type 1 isoform by high [Ca 2 + ]i (lino & Tsukioka 1994, Savineau & Marthan 2000) or inhibition of R y R by adaptation/inactivation mechanisms (Lamb et al. 2000, Sitsapesan & Will iams 2000), 2) inhibition 9+ of IP3R by low luminal SR Ca (Missiaen et al. 1992), and 3) time dependent inactivation of both IP3R (Hajnoczky & Thomas 1997). The latter mechanism would imply a maximal limit to the frequency of the wave-like [ C a 2 + ]; oscillations as it requires time for the channels in the inactivated state to return to the closed resting state. This is supported by the observation that [ C a 2 + ] i oscillations in the rabbit I V C appear to peak at a frequency of ~0.5Hz regardless of further increases in the agonist concentration (Ruehlmann et al. 2000). I l l A s the SR takes up Ca from the surrounding cytoplasm the store operated N S C C close and as a result of repolarization the V G C C also close. Stimulation of S E R C A , P M C A and possibly forward N C X by elevated [Ca ]; while release terminates, may be responsible for the down-stroke of the PE-induced [Ca 2 +]j oscillation (Nazer & van Breemen 1998). In this context it would be interesting to obtain evidence for N C X reversal during each cycle. The next Ca wave may start at the frequent discharge sites (Gordienko et al. 1998, Gordienko et al. 2001) when the SR luminal C a 2 + has been recharged and spillover from the SR raises the local [Ca 2 +]j to threshold once again. The observation, that waves tend to originate from the same area within the cell, has been explained by the observation that such sites posses a higher local density of SR near P M C a 2 + channels and are devoid of mitochondria (Gordienko et al. 2001). A conceptually simpler model of [Ca 2 +]j oscillations is one where [IP3]i oscillates (Hirose et al. OA-1999). The delayed negative feedback in this case is Ca activation of P K C , which then inhibits P L C . However, it is doubtful that the lower frequency [IP3]i oscillations can be responsible for the high frequency (~0.5Hz) [Ca 2 + ] j oscillations observed in these V S M C s . Possible advantage of wave-like [Ca2+]i oscillations It is clear that the additive effect of asynchronous C a 2 + waves is a steady state rise in the average tissue [ C a 2 + but is there a specific functional advantage to the oscillatory pattern of C a 2 + release? Several functions have been proposed: 1) several enzymes have been shown to respond to frequency of C a 2 + oscillations (see below), 2) maintenance of high [Ca 2 +]j may be damaging for the cell, 3) i f the C a 2 + sensor, in this case calmodulin, has a relatively fast C a 2 + on rate and a slow 112 off rate then the peak of the oscillation is the effective signal and signaling can be achieved at a lower average [Ca 2 + ] i and 4) SR C a 2 + release may be more effective in activating the contractile elements. The latter hypothesis was mentioned in the section on smooth muscle ultrastructure. It is clear that each wave depends on SR C a 2 + release into the myoplasm, while C a 2 + influx mainly serves to refill the SR. If the release occurs largely from the radial SR into the myosin rich space, where the calmodulin involved in the activation of M L C K is localized (Wilson et al. 2002), the flow of C a 2 + would then bypass the myosin poor or non-contractile space via the lumen of the radial SR and be more effective in local activation of the myofilaments. Consistent with this OA- OA-postulate is the observation that the entire E R is one continuous Ca pool and Ca can rapidly move and distribute inside the lumen of E R (Park et al. 2000). Blocking S E R C A would disrupt this process, allowing accumulation of C a 2 + in the myosin poor space and decrease the effectiveness of increase in average [Ca 2 +]j in stimulating contraction. This conclusion is supported by numerous reports that S E R C A blockade decreases the ratio of force/[Ca 2 +]j (Karaki etal . 1997,Tosunetal. 1998) Wave-like [Ca2+]i oscillations and mitochondrial Ca2+ signaling In addition to modulating cellular contractility, the recurring C a 2 + waves are likely to affect the regulation of numerous other cellular functions in the vascular smooth muscle. Mitochondrial metabolism is one such function that may be regulated by the recurring C a 2 + waves. The 0+ OA-mitochondria contain several Ca -sensitive dehydrogenases and a rise in mitochondrial [Ca ] can result in increased A T P synthesis (Hajnoczky et al. 1995, Robb-Gaspers et al. 1998). Mitochondria also possess low-affinity (Kd for C a 2 + is ~10-20pM) high-capacity C a 2 + uptake that OA-is minimally active in the sub-micromolar range of [Ca ] (Kroner 1986, Gunter & Pfeiffer 1990). 113 Given that physiological global [Ca ]i fluctuations are typically in the sub-micromolar range, it is not immediately clear how they could affect mitochondrial C a 2 + signaling of A T P synthesis. However, there is emerging evidence in both vascular smooth muscle as well as many other cell types to suggest that C a 2 + release from the E R / S R through either IP3R or R y R can raise mitochondrial [Ca 2 + ] to near lOOpM even though the corresponding elevation in global [Ca 2 + ]j is considerably lower (Robb-Gaspers et al. 1998, Csordas et al. 1999, Monteith & Blaustein 1999, Drummond & Tuft 1999, Gumey et al. 2000, Drummond et al. 2000, Nasser & Simpson 2000, Szalai et al. 2000). This apparent contradiction can be resolved i f one considers the possibility of a high [Ca 2 + ] microdomain generated by localized E R / S R C a 2 + release via the I P 3 R / R y R near the surface of the mitochondria. Such microdomains are the result of many areas of close contact between SR and mitochondria defining the Mi to -SR junctional space (Rizzuto et al. 1998). Although this space may be wider (<80nm) than the P M - S R junctional space, diffusion from it appears to be sufficiently restricted to support increases of local [Ca 2 + ] to 3 0 p M (Szalai et al. 2000). This would be sufficient to activate mitochondrial C a 2 + uptake and raise mitochondrial [Ca ]. Accordingly [Ca ]; oscillations in vascular smooth muscle cells have been found to cause 9+ oscillations in mitochondrial [Ca ] (Drummond & Tuft 1999). Furthermore, it has been shown in 94- 94-hepatocytes that ER-mediated oscillatory Ca signals can efficiently activate certain Ca -sensitive dehydrogenase in the mitochondria (Hajnoczky et al. 1995). Thus the evidence strongly 94-suggests that the SR-mediated asynchronous wave-like [Ca ]; oscillations seen in V S M can raise 94-mitochondrial [Ca ] and ultimately increase A T P generation to match the increased energy demand of contracting vascular smooth muscle. In addition, mitochondrial uptake of C a 2 + 94-released by the SR during wave-like [Ca ]i oscillations may also serve to ensure an adequate level of store-depletion for activation of the SOC. 114 Wave-like [Ca // oscillations and nuclear Ca signaling Since the nucleus is completely surrounded by the nuclear envelope its [Ca ] n has been found to be regulated differently than the [Ca 2 + ] j (Himpens et al. 1994). In particular [ C a 2 + ] n is lower than [ C a 2 + ]j during rest and higher during activation. Blockade of S E R C A also causes the [ C a 2 + ] n to rise in excess of [ C a 2 + ]i. Thus far no specific study has been made of [ C a 2 + ] n during [ C a 2 + ]j 94- 94-oscillations. It is possible that the recurring Ca waves or [Ca ]j oscillations may activate cellular functions other than contraction, such as smooth muscle hypertrophy, proliferation and synthesis of extracellular matrix indirectly by regulating at the level of gene expressions. Experiments in other cell types have revealed a number of Ca 2 +-sensitive cytosolic/nuclear enzymes whose activity is modulated by the frequency domain of fixed-amplitude [Ca 2 + ] i oscillations (De Koninck & Schulman 1998, Dolmetsch et al. 1998, Dupont & Goldbeter 1998, H u et al. 1999). This includes multi-purpose enzymes like Ca 2 + -calmodulin kinase II and transcription factors such as N F - K B , N F - A T and Oct /OAP. There likely exist more Ca 2 +-sensitive enzymes whose activity can 94-either be efficiently and/or selectively activated by [Ca ]j oscillations. Thus, even though not yet demonstrated, it is highly plausible that the wave-like [Ca 2 +]j oscillations observed in vascular smooth muscle can convey information in its frequency domain which can be "decoded" by these C a 2 + oscillation-sensitive enzymes to activate other cellular functions (Heist & Schulman 1998). Physiologically, this may represents an important adaptive mechanism for blood vessels. 115 Figure 32: Myosin-poor space and myosin-rich myoplasm in VSMC of the rabbit IVC. The electron micrograph image of the V S M C shows the position of myosin filaments (indicated by black arrows) in relation to the P M . It reveals a peripheral zone about 200-3OOnm in width that possess nearly no myosin filaments. This is referred to as the myosin-poor space which separates the P M and the myosin-rich myoplasm. The black scale bar indicated represents 200nm of distance. 116 Figure 33: Model for Ca movements during wave-like [Ca ]j oscillations and during resting state in VSMC. (A). The SR is composed of double membrane sheets separating its continuous lumen from the cytoplasm. The portion of the SR, which is closely apposed to the P M is called junctional SR (jSR). The j S R is separated from the P M by 15-20 nm, which creates an irregular narrow P M - S R -junctional space with a "diameter" of approximately 300 nm. Caveolae tend to perforate the jSR, such that the P M - S R junctional space includes the cytoplasm between the necks of the caveolae and the j S R and the tips of the caveolae face the peripheral cytoplasm. The peripheral cytoplasm has a low density of myosin filaments with average density reached approximately 200-300 nm from the P M . This myosin-poor space, which does not support contractile force development, comprises about 17% of the total cellular space or 25% of the myosin rich myoplasm, which does support force development. The P M - S R junctional space is much smaller comprising less than 0.4%o of the cell volume (see text for calculations). The data obtained in the inferior vena cava (IVC) of the rabbit support the model where during ct-adrenergic stimulation C a 2 + is transiently released from the radial S R through IP3R near the calmodulins tethered to the myofilaments. The subsequent depletion of the SR, which may be augmented by mitochondrial C a 2 + uptake across the Mi to -SR junctional space, opens S O C in the P M - S R junction allowing thousands of N a + and some C a 2 + to enter the P M - S R junctional space. N a + entry depolarizes the membrane to activate V G C C and drives the N C X backward to supply C a 2 + from the extracellular space to the junctional S E R C A for refilling of the SR. A s the SR is replenished the IP3-sensitized IP3R become once again activated by the locally raised [Ca 2 +]j to start the next wave of regenerative C a 2 + release through IP3R. The [Na +]j s is elevated at least transiently because diffusion from the P M - S R junctional space is restricted and the N a + / K + - A T P a s e isoform localized to the P M - S R junction is 118 of the low N a + affinity ct2 or 0.3 type. The junctional SR release channels would be inactivated by the high [Ca 2 + ] j s seen during activation. Such a junctional complex comprised of the SOC, the N C X and the S E R C A serves to facilitate smooth muscle contraction in the rabbit I V C . (B). In contrast, a different junctional complex comprised of the RyR, the S E R C A and the K c a functions to relax V S M C in the rat cerebral resistance artery. The recurring C a 2 + waves mediated by the R y R can elevate the [Ca 2 + ] in the P M - S R junctional space sufficiently high to activate K c a , leading to hyperpolarization of the membrane potential and inhibition of the L-type V G C C . (Ca, C a 2 + ; Na , N a + ; K , K + ; B K c a , Ca 2 +-activated K + channels; N C X , sodium/calcium exchanger; N C S S , non-selective cationic channel; S O C , store-operated channel; S E R C A , sarcoplasmic endoplasmic reticulum C a 2 + ATPase; V G C C , L-type voltage-gated C a 2 + channel; IP3R, IP3-04- 04-sensitive Ca release channel; RyR, ryanodine-sensitive Ca release channel; SR, sarcoplasmic reticulum; mito, mitochondria) 119 CHAPTER X: CONCLUSIONS AND FUTURE DIRECTIONS The focus of this thesis is to examine the function and the mechanism of asynchronous wave-like [Ca 2 + ]i oscillations observed in V S M C . The works presented here have provided significant insights into it. Functionally, the data indicates these asynchronous wave-like [Ca ]j oscillations underlie E - C coupling in PE-stimulated rabbit I V C . More specifically, the frequency of the wave-like [Ca 2 +]j oscillations determines the degree of tonic vasoconstriction. Mechanistically, the 9+ findings show that the generation of the wave-like [Ca ]j oscillations requires the presence of the P M - S R junctions, a concerted sequence of events of channels activation and repetitive cycling of C a 2 + through the IP 3 R, the store-operated N S C C , the L-type V G C C , the N C X operating in the reverse mode, the S E R C A and the P M C A (Figure 31). The works presented in this thesis has significantly improved our understanding of agonist-induced C a 2 + signaling in relation to E - C coupling in V S M . 9+ 9-f-Due to the versatility of Ca as a signaling molecule, the function of the wave- like [ G T ] i oscillations may not be restricted only to E - C coupling of the V S M . A s mentioned earlier, it is highly likely that these recurring Ca waves may also serve of an activating signal for increased gene expression, which may lead to hypertrophy and proliferation of V S M . The recurring waves of SR C a 2 + release may also be important in stimulating A T P generation in the mitochondria to match the energy demand of the contracting V S M . Because of these interesting potential functional implications, it is important to understand whether these asynchronous wave-like [Ca 2 + ]i oscillations serve any other special functions. Furthermore, the purpose for this particular type of C a 2 + signaling involving repetitive waves of SR C a 2 + release and oscillatory C a 2 + signal is 121 poorly understood as well . It is entirely possible that the SR-mediated oscillatory Ca signal protects the cell from non-selective over-stimulation by Ca , which is potentially cytotoxic. 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