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Interaction between NCX and SERPA in Ca²⁺ signaling in human endothelial cells Chan, Lally Lai Yee 2004

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Interaction between N C X a n d S E R C A in C a s i g n a l i n g in h u m a n e n d o t h e l i a l c e l l s by LALLY LAI Y E E CHAN B . S c , Simon Fraser University, 2001 A THESIS SUBMITTED IN PARITAL FULFILMENT O F THE REQUIREMENTS FOR THE D E G R E E O F MASTER O F SCIENCE in T H E FACULTY OF GRADUATE STUDIES Department of Pharmacology & Therapeutics We accept this thesis as conforming to the required standard T H E UNIVERSITY OF BRITISH COLUMBIA April 2004 © Lally Lai Yee Chan, 2004 Library Authorization In presenting this thesis in partial fulfillment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Title of Thesis: iMoiqiUoA {Of\vo<?M /QfTX auc\ A LAUK/ LAI yeB CHfthS Name of Author (please print) Date (dd/mm/yyyy) The University of British Columbia 7 ' Vancouver, BC Canada ABSTRACT The interaction between sodium-calcium-exchanger (NCX) and the endoplasmic reticulum (ER) with respect to C a 2 + signaling was studied using fura-2 fluorescence imaging microscopy in human endothelial cells. The inflammatory agonist histamine was used to increase the intracellular C a 2 + concentration. Under resting conditions, the endothelial NCX serves to unload some of the C a 2 + content accumulated in the ER. This unloading is important in order to maintain the buffer barrier function of the peripheral ER. Application of histamine (1 uM) in the presence of extracellular C a 2 + caused a long lasting C a 2 + response. This maintained response is dependent on the state of ER C a 2 + content, which is at least partly refilled by C a 2 + entry via NCX working in the reverse mode during the course of agonist stimulation. After cessation of agonist stimulation, a major part of the increased C a 2 + is cleared by sarcoplasmic/endoplasmic reticulum C a 2 + -ATPase (SERCA) and NCX working in a serial configuration. In summary, in human endothelial cells, NCX can unload or refill the ER and thereby modulate the C a 2 + level. ii T A B L E OF CONTENTS Abstract ii Table of contents iii List of Figures vi List of abbreviations vii Acknowledgements x C H A P T E R 1 The C a 2 + signaling in vascular endothelium 1.1. Introduction 1 1.2. Endothelial barrier function & permeability 2 1.3. Releasing of different factors for relaxation & contraction 3 1.3.1. Nitric oxide 3 1.3.2. Prostacyclin 5 1.3.3. Endothelium-derived hyperpolarizing factor.. 6 1.3.4. Endothelin 7 1.4. Ca 2* signaling 8 1.4.1. C a 2 + entry pathway 9 1.4.1.1. Receptor-operated channels 10 1.4.1.2. Store-operated channels or capacitative C a 2 + entry 11 1.4.1.3. Nonselective cation channels 14 1.4.1.4. Ca 2 +leak channels 14 1.4.1.5. Mechanosensitive Ca2+-permeable channels 15 1.4.1.6. Voltage-operated C a 2 + channels 15 1.4.1.7. Na + -Ca 2 + exchanger 16 1.4.1.8. K+channels 17 1.4.2. C a 2 + release from ER 18 1.4.2.1. IPs-mediated C a 2 + release 18 1.4.2.2. Ca2+-induced C a 2 + release 19 1.4.3. Ca 2 +release from mitochondria 19 1.4.4. C a 2 + extrusion pathway 21 1.4.4.1. Plasma membrane Ca 2 +-ATPase 21 1.4.4.2. Na + -Ca 2 + exchanger 22 1.4.5. Ca2+sequestration 24 1.4.5.1. Endoplasmic reticulum 24 iii 1.4.5.2. Mitochondria 25 1.4.6. The "Superficial Buffer Barrier" hypothesis 27 1.5. Cytoskeleton 29 CHAPTER 2 Ca 2 + signaling in human endothelial cells 2.1. Research focus 30 2.2. Hypothesis and specific aims 31 2.3. Methods and materials 32 2.3.1. Cell preparation 32 2.3.2. Fluorescent dye loading and experimental setup 32 2.3.3. Instrumentation for monitoring the fluorescence signal 33 2.3.4. [Ca2+]i measurement 34 2.3.5. Electronmicroscopy 35 2.3.6. Solutions & chemicals 36 2.3.7. Data analysis & statistics 36 2.4. Results 38 2.4.1. Interaction between NCX and SERCA during C a 2 + signaling 38 2.4.1.1. Under resting conditions 38 2.4.1.1.1. NCX unloads the ER C a 2 + store 38 2.4.1.1.2. Buffer barrier function 39 2.4.1.2. During histamine stimulation 42 2.4.1.2.1. Ca 2 +cycling 42 2.4.1.2.2. NCX refills ER C a 2 + store 44 2.4.1.3. Upon termination of agonist stimulation 44 2.4.1.3.1. Ca 2 +removal 44 2.4.2. Ultrastructure of PM and ER 52 2.5. Discussion 54 2.5.1. Depletion of ER C a 2 + by NCX before agonist stimulation 54 2.5.2. C a 2 + removal during histamine stimulation .....58 2.5.2.1. Cycling of C a 2 + by SERCA and ER C a 2 + release channels 58 2.5.2.2. The reverse mode of NCX refills the ER Ca2 +during stimulation...59 2.5.3 C a 2 + removal after stimulation—SERCA and NCX work in series 60 2.5.4. Limitations of cell preparation & methods 62 2.5.4.1. Cell type 62 iv 2.5.4.2. Fura-2 measurement 62 2.6. Summary 63 2.7. Future directions 63 2.8. References 65 v LIST O F FIGURES F i g u r e 1 Multiplicity of endothelium-derived relaxing and contracting factors 4 F i g u r e 2 Schematic representation of the ion transport pathways underlying calcium homeostasis in vascular endothelial cells 9 F i g u r e 3 Capacitative calcium entry 12 F i g u r e 4 Schematic representation of the Ga 2 + transport pathways of mitochondria 20 F i g u r e 5 Schematic diagram of the superficial buffer barrier 28 F i g u r e 6 Experimental setup for fluorescence measurements 34 F i g u r e 7 NCX contributes to ER calcium unloading under resting conditions 40 F i g u r e 8 NCX unloads ER C a 2 + and maintains buffer barrier function 41 F i g u r e 9 Histamine-induced C a 2 + signals in HUVEC 43 F i g u r e 10 "Calcium cycling" during histamine stimulation 45 F i g u r e 11 Effect of the inhibition of reverse mode NCX on histamine induced C a 2 + responses 46 F i g u r e 12 C a 2 + removal in HUVEC after histamine stimulation 47 F i g u r e 13 Rates of C a 2 + removal as a function of [Ca2+]i 49 F i g u r e 14 C a 2 + removal in HUVEC during histamine stimulation 50 F i g u r e 15 Effect of NCX in the presence of extracellular C a 2 + 51 F i g u r e 16 ER and PM junction in HUVEC visualized with EM 53 F i g u r e 17 Models for C a 2 + cycling and extrusion in HUVEC 57 vi ABBREVIATIONS AA: arachidonic acid Ach: acetylcholine AIF: apoptosis inducing factor AM: acetoxymethyl ester [Ca 2 +]j: free intracellular cytoplasmic C a 2 + concentration [Ca 2 +] e r: free C a 2 + concentration in the endoplasmic reticulum [Ca 2 + ] m : free C a 2 + concentration in the mitochondrial matrix C C E : capacitative C a 2 + entry CHO: Chinese hamster ovary CICR: Ca 2 +-induced C a 2 + release CIF: Ca 2 + influx factor CPA: cyclopiazonic acid DMEM: Dulbecco's modified eagle medium DMSO: dimethyl sulfoxide E m : membrane potential Ec a : reversal potential of C a 2 + E N a / c a : reversal potential of NCX E C : endothelial cells E-C coupling: excitation-contraction coupling EDCF: endothelium-derived constricting factor EDRF: endothelium-derived relaxing factor EDHF: endothelium-derived hyperpolarizing factor EDTA: ethylenediaminetetraacetic acid EGTA: ethylene glycol fc>/s-(p-aminoethylether)N,N,N',N'-tetraacetic acid EM: electron miscroscopy ER: endoplasmic reticulum ET: endothelin FBS: fetal bovine serum vii GFP: green-fluorescent protein HEPES: hydroxyethylpiperazine ethansulphonic acid HUVEC: human umbilical vein endothelial cells H 2 0 2 : hydrogen peroxide IP3: inositol-1,4,5-trisphosphate IP3R: inositol-1,4,5-trisphosphate receptor k: kinetic parameter K A: transient outward K + channel K A C h : muscarinic gated K + channel K A TP: ATP-sensitive K + channel Kc a : Ca 2 +-activated K + channel MLC: myosin light chain NCX: N a + - C a 2 + exchanger NMDG: N-Methyly D-Glucamine NO: nitric oxide NOS: nitric oxide synthase N S C C : nonselective cation channels 0 2 ~: superoxide nPSS: normal physiological saline soluntion P N a : sodium permeability Pc s : cesium permeability Pc a : calcium permeability PGI 2: prostacyclin PKC: protein kinase C PLC: phospholipase C PM: plasma membrane PMCA: plasma membrane C a 2 + ATPase PSS: physiological salt solution PTP: permeability transition pore RaM: rapid uptake mode ROC: receptor-operated channels viii ROS: reactive oxygen species RyR: ryanodine receptor SBB: superficial buffer barrier SEM: standard error of the mean SERCA: sarcoplasmic/endoplasmic reticulum Ca 2 + -ATPase SMC: smooth muscle cells S O C : store-operated channels SR: sarcoplasmic reticulum Trp: transient receptor potential channel VOC: voltage-operated C a 2 + channels VSM: vascular smooth muscle VSMC: vascular smooth muscle cells ix A C K N O W L E G E M E N T S There are many people that I would like to take a moment to give thanks during my study. I would like to express my gratitude to Dr. Casey van Breemen and Dr. Xiaodong Wang, my supervisors, for their support, generosity, and guidance in each step of my research endeavors. An appreciation goes to my research committee members, Dr. Ismail Laher and Dr. Chun Seow for their time and effort in providing advices and feedback during the course of study. A special thank goes to Dr. Roshanak Rahimian as my research mentor for her inspiring character and passion toward research. I am deeply grateful to all my friends for their spiritual and emotional support during all the good times and difficult times, particularly my best friend, David for all the encouragement he gives along the years. I want to express my deepest gratitude to my parents for their unwavering love and support. Without all these key people in my life that make this study impossible. x CHAPTER 1 THE VASCULAR ENDOTHELIUM 1.1. Introduction The vascular endothelium serves not merely as a passive monolayer (Augustin et al., 1994) barrier but uses its strategic location to participate in all aspects of vascular homeostasis. Being the interface between blood and tissue, it plays a pivotal role in modulating vascular tone, adjusting blood-tissue permeability, contributing to angiogenesis and vessel repair, inhibiting inflammation, blood coagulation and smooth muscle cells (SMC) proliferation, and regulating blood pressure. Disruption of the functional integrity of the vascular endothelium is a key event in the development of vascular disorders such as atherosclerosis, coronary vasoconstriction, and probably also myocardial ischemia. Endothelium lies between the lumen and the vascular smooth muscle. Despite its one-cell-layer thickness, it is able to sense changes in hemodynamic forces, or blood-borne signals, by membrane receptor mechanisms, and also responds to humoral, neural, and mechanical stimuli by synthesizing and releasing vasocative substances, thrombosis regulatory molecules, and growth factors. Among these substances, nitric oxide (NO) and prostacyclin have received considerable attention, since both have potent immediate actions on the underlying smooth muscle or myocardium and on platelets in the blood stream. In addition, they may play an important role in inflammation and vascular pathology. 1 1.2. Endothelial barrier function & permeability As an interracial layer, endothelium is the main physical barrier between blood and underlying tissue and actively plays an important role in maintaining homeostasis in circulation. As a barrier, there exists at least five different pathways for molecules to pass through the endothelium (van Nieuw Amerongen etal., 2003): 1) a limited number of molecules can diffuse across the membranes and the cytoplasm of endothelial cells (EC); 2) small lipophilic molecules diffuse along the endothelial membrane; 3) macromolecules, hormones, and solutes are transferred or transported across the E C by vesicular transporters, exchanging through endothelial fenestrations 4) and are transferred through endothelial clefts at cell junctions; 5) leukocytes can pass the endothelium via disruptions in endothelial cell-cell junctions. Under pathological conditions, vascular permeability of this monolayer barrier can increase by various inflammatory mediators (van Nieuw Amerongen al., 2003). Findings have demonstrated that a variety of inflammatory stimuli increase [Ca 2 +]j (Lum et al., 1994; Dudek et al., 2001). Since C a 2 + is critical for maintaining the integrity of EC , numerous studies have established a link between C a 2 + signaling and increased endothelial permeability (Sandoval era/., 2001a, 2001b; Nguyen et al., 1997; van Nieuw Amerongen et al., 1998). Recently, studies have also suggested that the store depletion activated C a 2 + entry through PM cation channels is the critical determinant of increased endothelial permeability. An increase in the intracellular C a 2 + concentration 2 ([Ca ]j) and protein kinase C (PKC) activation leads to the activation of Ca27calmodulin-dependent myosin light chain (MLC) kinase by which actin-myosin interaction is promoted (Garcia et al. 1995; Wysolmerski et al., 1900) and cytoskeleton reorganization is facilitated (Lum etal. 1994, Dudek et al., 2001) to induce E C shape change. The C a 2 + and PKC signaling can also mediate junctional disassembly by disrupting the VE-cadherin complex (Sandoval et al., 2001a, 2001b). Therefore, modulation of C a 2 + entry might be responsible for the regulation of permeability of E C (He etal., 1994). 1.3. Releases of different factors for relaxation & contraction The endothelium-dependent vasorelaxation has become one of the broadly applicable indicators of endothelial function (Ludmer et al., 1986). In 1980, Furchgott and Zawadzki discovered the essential role of endothelium in vasorelaxation, by stimulating the isolated aortic ring with acetylcholine (Ach) (Furchgott etal., 1980). Since then, many other stimuli were found to require the presence of the endothelium to produce partial or complete relaxation of arteries, vein and microvessels (Figure 1). 1.3.1. Nitric oxide The unstable relaxing substance released by endothelium as reported by Furchgott (endothelium-derived relaxing factor, EDRF) was identified to be nitric oxide (NO) in 1987 (Ignarro et al., 1987; Pamler et al., 1987). Hence, NO production plays a key role in determining the functional profile of EC. NO is also 3 Figure 1. Multiplicity of endothelium-derived relaxing and contracting factors. AA=arachidonic acid; ACh=acetylcholine; ATII=angiotensin II; BK=bradykinin; COX=cyclooxygenase; ECE=endothelin converting enzyme; EDHF=endothelium-derived hyperpolarizing factor; ET=endothelin-1; 0 2 " " =superoxide anions; P=purines; PGI2=prostacyclin; NO=nitric oxide; NOS=NO synthase; T=thrombin; TA2/Endo=TP-receptor; VP=vasopressin; TXA2=thromboxane A 2 ; 5-HT=5-hydroxytryptamine (serotonin); a =alpha-adrenergic. (Adapted from Vanhoutte, P. M., 2001) E N D O T H E L I U M - D E P E N D E N T R E S P O N S E S (not present in -II blood vessels) Short term regulation Long term regulation Blood Blood a potent inhibitor of platelet adhesion and aggregation (Radomski et al., 1987a, 1987b; Radomski et al., 1990). In addition to these effects on the vasculature, endothelial-derived NO inhibits leukocyte adhesion to the endothelium (Kubes, et al., 1991; De Caterina et al., 1995) and inhibits SMC migration (Marks et al., 1995) and proliferation (Garg etal., 1989). NO is derived from the amino acid L-arginine. The conversion of L-arginine to L-citrulline and NO is catalyzed by a family of enzymes termed NO 4 synthases (NOSs). This family of enzymes is comprised of three members: neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS). eNOS is constitutively expressed in endothelium and provides a constant, dynamic source of NO. A decrease in NO production and/or bioactivity contributes to the pathophysiology of several major diseases of the cardiovascular system (Arnal etal., 1999). 1.3.2. Prostacyclin Prostacyclin (PGI2) is another vasoactive substance which was first identified in endothelial-derived vascular SM relaxation (Campbell et al., 1996). PGI2 is a metabolite of arachidonic acid (AA). It is neither constitutively present in resting human E C nor stored within the cells. Its synthesis is induced by humoral and mechanical stimuli (Meade et al., 1996). PGI 2 can be rapidly released from E C (Weksler et al., 1977) and has a relatively short half-life (Campbell et al., 1996). Unlike NO, PGI2 appears at sites of vascular perturbation when E C are stimulated, rather than regulating basal systemic vascular tone (FitzGerald et al., 1983). As it is locally generated, it causes vasodilatation and altered regional blood flow. In addition to mediating and regulating vasodilatation, PGI 2 acts primarily to retard platelet aggregation and deposition. Due to its effect on blood flow and relevant cell-cell interactions, PGI2 may influence local inflammatory responses as well. The identification of an inducible form of a key enzyme in PGI 2 formation, prostaglandin H synthase-ll (PHS-II), provides a mechanism by 5 which the production of PGI 2 and other eicosanoids can be sustained in chronic states of inflammation and vascular injury (Cines etal., 1998). 1.3.3. Endothelium-derived hyperpolarizing factor In many blood vessels, it has become increasingly clear that the endothelial-dependent relaxation, in response to stimulation, is not completely lost in the result of the pharmacological inhibition, or genetic/knockout', of the synthesis of NO and PGI2. This endothelium-dependent, but NO- and PGI2-independent, vasodilation has also been reported in human vessels (Wallerstedt et al., 1997). The non-NO/PGI 2 relaxation is usually most notable in the resistance vasculature (Shimokawa et al., 1996; Quilley et al., 1997; Feletou et al., 1999) in which the action of this non-NO/PGl2 mediator is associated with the endothelium-dependent hyperpolarization of vascular smooth muscle cells (VSMC), hence the factor has been termed as the endothelium-derived hyperpolarizing factor, EDHF (Triggle etal., 1999; Ding etal., 2000). There are three explanations that are favored by current experimental evidence for the EDHF-mediated responses: 1) the increase in endothelial [Ca 2 + ] i triggers the synthesis of cytochrome P450 metabolites; 2) the increase in extracellular K + is released from the E C via K C a channels; 3) E C hyperpolarization is transmitted to the VSM via myoendothelial cell gap junctions. Although reactive oxygen species (ROS) is an indicator of cellular damage or a byproduct of metabolism, hydrogen peroxide (H2O2) is also suggested recently as a valid candidate of EDHF (Matoba et al., 2000, 2003). Despite the 6 considerable body of evidence suggesting cellular mechanisms of the EDHF-mediated response, in addition to the heterogeneity of EDHF between species and vascular beds, the chemical identity of EDHF remains controversial. 1.3.4. Endothelin E C not only release relaxing factors, but can also initiate endothelium dependent contractions of the underlying vascular smooth muscle cells. Among the endothelial-derived constricting factors (EDCF), endothelin (ET) is the most potent vasoconstrictor currently known (Agapitov et al., 2002). The peptide was identified by Masaki and colleagues in 1988 (Yanagisawa etal., 1988). Under normal conditions, ET produced in E C would be offset, as studies have suggested that NO may normally suppress the action/production of ET (Boulanger et al., 1990; Liischer et al., 1992). NO shortens the duration of the effect of ET by accelerating the restoration of intracellular C a 2 + to basal levels (Goligorsky et al., 1994). This feedback inhibition of the production and the action of endothelin is not limited to NO. The release of other EDRF such as prostacyclin and EDHF are also reported to contribute to this inhibition (Nakashima et al., 1993; Goodwin et al., 1998). The augmenting release of endothelin indicates the imbalance between the vasorelaxing and vasoconstricting factors. This imbalance between EDRF and endothelin contributes to the symptoms of aging and vascular disease. 7 1.4. C a 2 + signaling C a 2 + , the divalent cation, as a major signaling molecule that regulates energy output, cellular metabolism, and contraction, and secretion in cell. Most endothelial functions depend on changes in [Ca 2 +] to various extents. However, a prolonged high level of C a 2 + may lead to cell death. Therefore, the C a 2 + level in the cell has to be carefully maintained. In order to control cellular C a 2 + homeostasis and C a 2 + signaling processing, cells are equipped with sophisticated transport and sequestration mechanisms. These sophisticated mechanisms are involved in the control of C a 2 + entry into the interior of a cell across the plasma membrane and redistribution of C a 2 + from the cytosol into the intracellular organelles. The total [Ca 2 +] in extracellular biological fluids such as blood serum ranges from 1.6 - 2.0 mM (Kass et al., 1999). Within the cells, the cytosolic free [Ca 2 +] is around 0.1 uM and even the level of C a 2 + in the intracellular C a 2 + stores is in the range of the millimolar. To keep from being flooded by C a 2 + from the extracellular and intracellular stores, cells contain Ca2+-transporting systems in the plasma membrane (PM), in the endoplasmic reticulum (ER), and in the mitochondria. Typically, the PM in the E C contains three systems (Figure 2): the C a 2 + entry channels, specific Ca 2 + -ATPases, and sodium calcium exchanger (NCX). The ER contains a specific Ca 2 + -ATPase and different types of C a 2 + release channels whereas the mitochondria contain an electrophoretic uniporter that is exclusively designed for C a 2 + uptake and NCX for C a 2 + release from the mitochondrial matrix to the cytosol. These systems have different affinities of 8 Figure 2. Schematic representation of the ion transport pathways underlying calcium homeostasis in vascular endothelial cells. (Adapted from Adams, D. J . etal., 1989) Amilonde / J A Ha* 3Na* AW ( ) I ( Ma K-CI 3Na interaction with C a 2 + , and different total C a 2 + handling capacities. The C a 2 + -ATPase transports C a 2 + with high interaction affinity whereas the exchangers, channels, and uniporter are all low-Ca2+affinity systems. 1.4.1. Ca 2 + entry pathway The C a 2 + can gain access into cells through several mechanisms across the PM. One such mechanism involves the channels which are tightly controlled by receptors (receptor-operated channels, ROC) or dependent on the depletion of the intracellular store (store-operated C a 2 channels, SOC). Some even appear 9 to be nonselective cation channels, NSCC, while others are via passive permeability, via mechanosensing properties, or gated by the membrane potential (voltage-operated C a 2 + channels, VOC). Alternatively, hyperpolarization can also enhance C a 2 + entry as well as exchanging for N a + by the NCX in the NCX (Figure 2). 1.4.1.1. Receptor-operated channels One of the classes of C a 2 + permeable channels responsible for the C a 2 + influx during stimulation is ROC. The classical idea of ' R O C suggests that binding agonist to its transmembrane receptor directly gates the C a 2 + channel which mediates C a 2 + entry (Whorton et al., 1984; Johns, et al., 1987). The existence of R O C permeable to C a 2 + has been demonstrated in the E C of different types such as rabbit aortic EC , rabbit cardiac valves endothelium and cultured human umbilical vein E C (HUVEC) (Wang & van Breemen, 1997; Li & van Breemen, 1996; Jacob, 1990; Hallam & Rink, 1985). Most of these channels are activated via signaling cascades involving the activation of phospholipase C (PLC) (Wang & van Breemen, 1997; Li & van Breemen, 1996), but the second messenger which directly activates ROC remains unknown. 1.4.1.2. Store-operated channels or capacitative Ca 2 + entry S O C are present in the PM of almost all 'non-excitable' and some 'excitable' animal cells (Berridge, 1995; Putney & Bird, 1993; Birnbaumer et al., 1996; Friel, 1996; Putney, 1986). In general, S O C is much more selective for 10 Cad+ than R O C (Friel, 1996). This channel was first introduced in the late 1980's, showing that intercellular C a 2 + store depletion stimulated C a 2 + influx across the PM to aid the refilling of the stores (Putney, 1986). The exact mechanism of activation for S O C is still in debate. The observations of S O C C a 2 + inflow replenishing ER C a 2 + stores with little observable increase in [Ca2 +} (Mogami et al., 1997; Hofer et al., 1998) suggest that the physiological role of S O C may be to discretely supply C a 2 + to the ER in order to maintain an adequate free [Ca 2 +] in the ER ([Ca2 +]e r). Though it remains controversial whether ER depletion is the physiological link between receptor activation and C a 2 + entry in native endothelium, new evidence has shown that the inositol-1,4,5-trisphosphate receptor (IP3R) is a crucial component for mediating and maintaining coupling between store emptying and opening of S O C (Ma etal., 2000). The ER-PM interactions have been shown to be involved and required in S O C activation as well (Ma etal., 2000). The rise in [Ca 2 +]j during stimulation is usually biphasic which consists of an initial transient component followed by a sustained one. However, a small and transient rise in [Ca 2 +]j is observed under Ca 2 +-free condition. It has been hypothesized that PM cation channels (i.e. S O C or C a 2 + release activated channels) are activated by IP3-sensitive C a 2 + store depletion and can cause C a 2 + influx. This process was first proposed by Putney as capacitative C a 2 + entry (CCE) (Putney et al., 1986) (Figure 3A). C C E probably is the main mechanism of agonist-induced C a 2 + entry in E C (Dolor et al., 1992; Lambert et al., 1986; Schilling et al., 1992). Three main hypotheses have been proposed on the 11 F i g u r e 3. C a p a c i t a t i v e C a 2 + e n t r y . ( A ) PM cation channels (i.e. SOC or C a 2 + release activated channels) are activated by IP3-sensitive C a 2 + store depletion and can cause C a 2 + influx. ( B ) Diffusive messenger hypothesis. (C) Conformational coupling. (D) Vesicle secretion hypothesis. (Adapted from Dutta, D., 2 0 0 0 ) Ca* Store Operated Channel (SOC) PM P M Cytosol SOC X B . Diffusible messenger hypothesis Cytosol C . Conformational coupling hypothesis P M Cytosol Ca 2 * _ D . Vesicle secretion hypothesis 12 mechanism of coupling store depletion to the activation of Ca influx: 1) a diffusible messenger, 2) conformational coupling and 3) vesicle secretion. The diffusible messenger hypothesis proposes that the generation of a messenger molecule activates C a 2 + entry in response to store depletion ( F i g u r e 3B). Numerous candidate molecules such as small G proteins, ATP, GTP, product of cytochrome P450, AA, and C a 2 + influx factor (CIF) have been reported as diffusible messengers (Putney et al., 1999; Kwan et al., 2000). Among the candidates, CIF has been in favor recently due to a few evidence of its existence in yeasts cells and in mammalian smooth muscle cells (Petersen & Berridge, 1996; Trepakova etal., 2000). Conformational coupling hypothesis postulates that there is a physical coupling between the IP3R with the S O C ( F i g u r e 3C). Lievremont's group has provided evidence in favour of a close association of IP3R and S O C from cell fractionation studies showing that IP3R co-purifies with PM fractions (Lievremont etal., 1994). In addition to the speculation that S O C and IP3R interact physically, more studies have shown that the site of capacitative current entry is in close proximity to the site of inositol-1,4,5-trisphosphate (IP3) induced C a 2 + release (Petersen & Berridge, 1996; Jaconi etal., 1997). The third hypothesis of vesicle-mediated channel insertion explains that store depletion leads to the fusion of preformed S O C proteins with the PM (Patterson etal., 1999; Yao etal., 1999) ( F i g u r e 3D). 13 1.4.1.3. Nonselective cation channels N S C C are a diverse group of ion channels characterized by their low discrimination between cations. Their main biophysical properties include theirs voltage-independent activation and permeability sequencing PNa > PCs » Pea (Nilius et al., 1997). Although the gating mechanism of N S C C is still unclear, various gating signals for N S C C in the endothelium have been discussed, such as increase in [Ca2 +]i (Bregestovski etal., 1988; Baron etal., 1996; Nilius, 1990), mechanical forces (Lansman e r a / . , 1987; Popp & Gogelein, 1992), depletion of intracellular C a 2 + stores (Zhang etal., 1994; Pasyk etal., 1995; Sharma & Davis, 1995; Davis & Sharma, 1997), oxidant stress (Koliward e r a / . , 1996a, 1996b), and receptor activation (Nilius e r a / . , 1997; Kamouchi e r a / . , 1999a). These channels are believed to play a role in the C a 2 + influx pathway in endothelium (Baron etal., 1996; Kamouchi e r a / . , 1999b; Nilius, 1990; Nilius etal., 1993b; Nilius e r a / . , 1997) which is responsible for the maintained elevation of [Ca2 +]i during agonist stimulation. 1.4.1.4. Ca 2 + leak channels A component of C a 2 + entry into non-stimulated E C was observed under physiological conditions. This passive non-regulated C a 2 + leak, that crosses the plasmalemma and is driven by the electrochemical gradient for C a 2 + (Em-E C a, Em:membrane potential; E C a : the equilibrium potential for Ca 2 + ) , is present and increases [Ca2 +]i in E C (Johns e r a / . , 1987; Schilling, 1989; Demirel etal., 1993). Such a leak could be a candidate for providing an increased C a 2 + influx by cell 14 hyperpolarization (Nilius et al., 1993a). The nature of the leak pathway in E C is unknown, but it may play an important role in the basal and stimulated release of EDRF and other vasoactive mediators in physiological or pathological situations. 1.4.1.5. Mechanosensitive Ca2+-permeable channels Many E C responses are modulated by changes in blood flow and blood pressure (Lansman et al., 1987; Popp & Gogelein, 1992). Mechano-sensitive channels have been identified in vascular E C and presumably act as sensors for hemodynamic forces that induce C a 2 + influx or cell hyperpolarization (Davies, 1995; Lansman etal., 1987; Oleson etal., 1988). These types of channels could change E C E m , thus providing a sufficient driving force for passive C a 2 + entry (Nilius etal., 1994). Recent studies have indicated that the mechanically induced C a 2 + entry may be activated by the depletion of the IP3-sensitive stores (Nakao et al., 1999; Niggel etal., 2000; Oike etal., 1994). Since E C plays a crucial role in regulating smooth muscle tone, this may be the possible mechanism in response to haemodynamic stimuli by the synthesis of releasing of EDRF (Rubanyi et al., 1990). However, the mechanism of mechanotransduction whereby the E C induces vasoregulatory responses, is still poorly understood. 1.4.1.6. Voltage-operated Ca 2 + channels Although E C are non-excitable, several reports have shown the incidence of V O C in both cultured and freshly isolated E C (Guiet-Bara & Bara, 2002; Vinet & Vargas, 1999; Bkaily et al., 1993; Bossu et al., 1989). These V O C , which 15 share some similarities with classical L- and T-type Ca channels, have been described in freshly isolated capillary E C from bovine adrenal glands (Vinet & Vargas, 1999; Bossu etal., 1989; Bossu etal., 1992a, 1992b). However, with the slow and often small changes in E m in the cells, other studies have provided evidence against the presence of V O C in E C (Bregestovski etal., 1988; Li etal., 1999; Cannell & Sage, 1989; Jacob et al., 1988; Sturek et al., 1991). Their argument is that the role of E m in regulating C a 2 + entry is simply to determine the driving force for C a 2 + entry. Therefore, in most cases, depolarization decreases, while hyperpolarization increases [Ca 2 +] i in EC. It may be possible that putative V O C may be present in E C in situ are lost during the preparation of cells. However, the functional role of these channels remains hypothetical in EC. 1.4.1.7. Na + -Ca 2 + exchanger The NCX can function in two modes, such that C a 2 + can be transported in either direction (forward or reverse) across the PM in exchange for Na + . These operations depend on the electrochemical gradients of Na + and C a 2 + on both sides of the membrane (Blaustien, 1977). The net C a 2 + movement mediated by NCX is governed by the difference between E m , the reversal potential of the exchanger (E N a /ca) and the kinetic parameter (k) that controls the rate of exchange: Jca(Na/ca) = kNa/ca ( E m - ENa/ca)- If the ENa/c a is lower than the E m , the exchanger in vivo will operate in C a 2 + influx mode (reverse); if the E N a / c a is higher than the E m , the exchanger will operate in C a 2 + efflux mode (forward). 16 Modulation of C a signaling in E C via NCX is still an unresolved issue. Several groups have demonstrated that NCX-mediated C a 2 + entry, in which the Na + gradient is reduced, increases [Ca 2 +]i (Sage era/., 1991; Sedova & Blatter, 1999; Sturek et al., 1991; Teubl et al., 1999). Evidence suggests that the NCX may shape C a 2 + transients activated by vasoactive agonists in which [Ca 2 + ] i are modulated by changing Na + gradients. This could be achieved either by loading E C with N a + using the ionophore monensin, or applying NCX blockers (eg. 3', 4' dichlorobenzamil, La 3 +) (Sedova & Blatter, 1999). This C a 2 + influx via NCX was also suggested to have a role in cardiac excitation-contraction coupling (Hume et al., 1991; LeBlanc & Hume, 1990) and a major influence on sarcooplasmic reticulum (SR) C a 2 + release (Wasserstrom & Vites, 1996; Litwin etal., 1996), but the physiological significance still remains to be defined (Sipido, etal., 1997). 1.4.1.8. K + channe ls K + channels are recognized in many cell types to establish and regulate membrane potential (Adams et al., 1989). Membrane depolarization reduces agonist-stimulated C a 2 + influx in E C (Adams et al., 1989; Laskey et al., 1990; Luckhoff & Busse, 1990). At least four types of K + channels are present in EC: inwardly rectifying voltage-gated K + channel (K I R ), Ca 2 +-activated K + channel ( K C A ) , muscarinic gated K + channel ( K A c h ) , and transient outward K + channel ( K A ) . In addition, an ATP-sensitive channel (KATP) (Janigro et al., 1993) was also described in E C . All of these K + channels hyperpolarize the E C to control the resting E m . This hyperpolarization by K I R and K A c h has been demonstrated to 17 increase C a 2 + entry (Adams etal., 1989; Busse etal., 1988). Therefore, it is not mandatory for E C to activate C a 2 + entry channels upon various stimuli, but to provide a sufficiently large inwardly driving force for C a 2 + . K + channels would be one type of the channels that influence electrogenesis in E C that are important for regulating C a 2 + and Ca 2 +-dependent E C functions (Nilius & Droogmans, 2001). 1.4.2. Ca 2 + release from ER Upon agonist stimulation, a transient release of EDRF associated with a transient [Ca2+]\ increase was observed in the absence of extracellular C a 2 + (Freay et al., 1989). This agonist-induced C a 2 + release clearly indicates from intracellular Ca 2 + stores. Two types of C a 2 + release channels have been identified in the intracellular store, ER (Mignery et al., 1989). Both are referred to by their receptor properties: IP3R (Berridge, 1993, 1997) and the ryanodine receptor (RyR) (Wagenknecht et al., 1989). These channels play crucial roles in C a 2 + -mediated signaling. 1.4.2.1. IP3-mediated Ca 2 + release from ER Intracellular IP3 is generated by the phosphotidylinositol cascade that is released into the cytoplasm following the hydrolysis of phosphatidylinositol 4, 5-biphosphate (PIP2) by the membrane-associated enzyme, PLC. The PLC is activated by the G-protein in which PIP 2 is phosphorylated to IP3 and diacylglycerol. IP3 activates the IP3R to open Ca 2 +-permeable channels on the ER membrane, serving as an important second messenger in C a 2 + signaling. 18 1.4.2.2. Ca2+-induced Ca 2 + release C a 2 + itself is capable of triggering the release of C a 2 + by activating either the RyR or the IP3R to amplify the C a 2 + signal. This process is called C a 2 + -induced C a 2 + release (CICR). CICR can operate in two ways: 1) as a triggering role by linking either V O C or R O C to the release channels on the ER; 2) as a regenerative role by linking together the release channels on the ER to set up the intracellular waves that spread the signal throughout the cells (Berridge, 1997). The former function is particularly evident in excitable cells such as cardiac muscle and neurons (Verkhratsky, 2002; Cheng & Wang, 2002). CICR has also been observed in E C (Graier etal., 1998; Domenighetti etal., 1998). 1.4.3. Ca 2 + release from mitochondria C a 2 + efflux from mitochondria into the cytosol occurs via three different mechanisms (Figure 4) including Na+-dependent and Na+-independent transport processes that exchange C a 2 + for Na + or H + , respectively. C a 2 + efflux via the permeability transition pore (PTP) forms the third pathway. The Na+-dependent mechanism is electrogenic (Baysal etal., 1994) and is known as mitochondrial NCX. The less common C a 2 + efflux from mitochondria occurs via the Na + -independent pathway and is behaving as an active C a 2 + / 2 H + exchanger (Gunter et al., 1994). PTP is a regulated pore, which has two different open states and 19 Figure 4 . Schematic representation of the C a 2 + transport pathways of mitochondria. UP, C a 2 + uniporter; RaM, "rapid uptake mode", Ex, exchangers; ELTC, electron transport chain; PTP, permeability transition pore; VDAC, voltage dependent anion channel; IMM, inner mitochondrial membrane; OMM outer mitochondrial membrane. (Adapted from Vandecasteele, G. etal., 2001) 1 t fir i i ft i C a 2 + N a + I M M O M M can open transiently (Ichas etal., 1997). Under normal cellular conditions, the first two of the mitochondrial C a 2 + release pathways, as mentioned above, are involved to allow C a 2 + efflux in exchange for Na + or H + . The PTP is normally closed but can be opened under some pathophysiological conditions. It is widely believed that a high intramitochondrial [Ca 2 +] is one of the major factors that promotes pore opening (Bernardi etal., 1999). In its open configuration, the pore allows release of C a 2 + from the matrix. The permeation of the flux of ions and water dramatically changes the membrane's permeability, collapses mitochondrial membrane potential (A*Fm), and uncouples ATP synthesis, resulting in massive mitochondrial swelling and loss of transmembrane potential (Gunter & Pfeiffer, 1990; Zoratti & Szabo, 1995; Broekemeier etal., 1989; Van Der Heiden etal., 1997). 20 In addition to the release of Ca , the irreversible opening of PTP also leads to the release of mitochondrial matrix content such as different apoptosis mediators (cytochrome c, apoptosis inducing factor (AIF)-nuclear fragmentation protein, and procaspase) (Gunter etal., 1994; Lemasters etal., 1998; Shimizu et al., 1999; Susin etal., 1999). Though the mechanisms mediating PTP activation are yet to be clarified, there is reasonable agreement that the change of mitochondria permeability transition has been known to be a common and crucial mechanism in cell death (Lemasters etal., 1998). 1.4.4. C a 2 + e x t r u s i o n p a t h w a y 1 .4 .4 .1 . P l a s m a m e m b r a n e C a 2 + - A T P a s e Plasma membrane Ca 2 + -ATPase (PMCA), is the ATP-driven C a 2 + pump that transports C a 2 + against its electrochemical gradients across PM out of the cell. This C a 2 + pump has high C a 2 + binding affinity, but transports C a 2 + with a low total capacity compared to the NCX (Furukawa et al., 1988). It is generally agreed that the PMCA is predominantly responsible for maintaining resting [Ca2 +]i by restoring small and moderate deviations of [Ca2+]j from the control levels (Sedova & Blatter, 1999). PMCA is mostly responsible for extruding C a 2 + in many non-excitable cells (Monteith & Roufogalis, 1995). The C a 2 + efflux mediated by the PMCA occurs via the countertransport of C a 2 + and H + (Dixon & Haynes, 1989; Smallwood et al., 1983; Milanick, 1990). Hence, C a 2 + efflux is paralleled by the entry of H + into the cytoplasm. Although PMCA is essential for the maintenance 21 of the low resting-state [Ca ]i, it may also participate in dynamic events such as local C a 2 + signaling and regulation of C a 2 + spikes (Monteith & Roufogalis, 1995; Penniston & Enyedi, 1998; Garcia & Strehler, 1999). A model for C a 2 + oscillation by Berridge proposed that C a 2 + efflux is required for Ca 2 + sp ike recovery (Berridge, 1991). Others also demonstrated pulsating increases in C a 2 + extrusion that corresponded to [Ca 2 +]i spikes (Tepikin et al., 1992). Therefore, PMCA may be important in cytosolic acidification and regulation of [Ca 2 +]j oscillations, in addition to regulating the efflux of C a 2 + after agonist stimulation. Previous studies have demonstrated that the localization of PMCA in the caveolae (Fujimoto, 1993) may enable its above mentioned physiological significance. Since PMCA and other signal transduction regulators (Chang et al., 1994) are localized in the caveolae (Chang et al., 1994), it has given a new perspective into the possible new mechanisms for C a 2 + pump regulation. 1.4.4.2. Na + -Ca 2 + exchanger NCX is known as a high-velocity, low-affinity antiporter that has been shown to play a crucial role in regulating [Ca 2 +]i in several cell types including cardiac myocytes, SM and neurons (Chapman & Noble, 1989; Batlle etal., 1991; Reuter & Porzig, 1995). As it is named, NCX can mediate both C a 2 + entry and C a 2 + exit depending on the net electrochemical driving force on the exchanger. Despite its relatively low affinity for cytosolic C a 2 + , there is broad agreement that the exchanger plays an important role in the extrusion of C a 2 + in many cell types. 22 One feature of the exchanger that comes to light is its localization in particular domains of the PM. This may contribute to its role in C a 2 + extrusion. Early studies have shown that NCX was shown to be restricted to regions in close anatomical apposition with SR in gastric and vascular smooth muscle (Moore et al., 1993; Juhaszova et al., 1994). Its activity has been suggested to be functionally linked to SR to continuous unload the superficial SR C a 2 + (Nazer & van Breemen, 1997; Hisamitsu era/., 2001; Bradley era/., 2002) in different cell types such as rabbit inferior vena cava and guinea-pig colonic SM. There was also a suggestion that NCX and S E R C A of SMC function in series (van Breemen era/., 1997). However, there is little information available on the NCX modulating C a 2 + homeostasis in E C . Though the existence of the NCX in E C was demonstrated 10 years ago (Hansen et al., 1991; Sage et al., 1991; Li & van Breemen, 1995), the physiological role in NCX still remains to be explored in E C , particularly the contribution of NCX in the C a 2 + removal mechanism and its role with other transporters or C a 2 + pumps. The physiological role of NCX was investigated in human aortic E C by Goto in 1996. The results suggested that the existence of NCX has a physiological role in maintaining the resting level of [Ca 2 +] i (Goto etal., 1996). It was observed in bovine vascular E C that the removal of C a 2 + by NCX becomes more significant when [Ca 2 +] i exceeds levels higher than ~ 150 nM. Until recently, its mechanism in physiological condition is demonstrated by various groups. The C a 2 + removal by S E R C A was dependent on the forward mode of NCX in unloading the C a 2 + to the extracellular space by using different cell types: bovine 23 aortic E C (Paltauf-Doburzynska et al., 1999), cultured HUVEC (Paltauf-Doburzynska etal., 2000) and rabbit aortic E C (Wang etal., 2002). 1.4.5. Ca 2 + sequestration 1.4.5.1. Endoplasmic reticulum The intracellular store, ER, plays a curial role in C a 2 + removal pathway. ER is the most important organelle in the regulation of [Ca 2 +] i, and accounts for approximately 75 % of the total intracellular C a 2 + reserve in E C (Tran etal., 2000). Fusion-protein constructs of green-fluorescent protein (GFP) and blue-fluorescent protein, named 'cameleons', can be targeted to the lumen of the ER to measure the [Ca 2 + ] e r (Miyawaki et al., 1997). Using this approach, the free [Ca 2 + ] e r is shown to range from 60 uM to 400 uM when the C a 2 + stores are full. This intracellular C a 2 + sink is not only responsible for supplying C a 2 + for signal transduction, but also serves for C a 2 + sequestration. Sacroplasmic/endoplasmsic reticulum Ca 2 + -ATPase (SERCA), the major protein in ER membrane may represent up to 90 - 95 % of the total protein of the SR (Carafoli, 1987). These Ca 2 + -ATPases pump excess C a 2 + into the ER lumen, serving both to reduce the C a 2 + overload in cell and to refill the store for the next stimulation. Evidence suggests that the structure of the ER extends like a net throughout the entire cytoplasm (Subramanian & Meyer, 1997; Dayel etal., 1999) and that the peripheral and deep ER may have different functions. The peripheral ER may function as a buffer barrier for C a 2 + entry at the resting state, 24 limiting its access to the deeper part of the cytoplasm in VSM as first reported (van Breemen et al., 1995). In order to prevent SR from saturating and to maintain its capacity to buffer C a 2 + , C a 2 + is required to be released from the SR lumen and to be extruded to the extracellular space (van Breemen etal., 1986). 1.4.5.2. Mitochondria Mitochondria, where the cellular energy ATP is generated, were found in the 1960's to have a large capacity to take up C a 2 + . However theirs physiological significance was neglected since mitochondria only contained low affinity mitochondrial C a 2 + uptake mechanisms. Not until recently have novel approaches of directly measuring of the [Ca 2 +] in the mitochondrial matrix ([Ca 2 +]m) reversing the old belief of its marginal role in physiological intracellular C a 2 + homeostasis. One approach involved using Ca 2 +-sensitive photoproteins aequorin to target mitochondria. The other widely used approach involved loading fluorescent C a 2 + tracer into the organelle. Beside the conventional mitochondrial C a 2 + uptake via unporter, a rapid uptake mode (RaM) for C a 2 + entry has been described recently that seems to represent a second entry pathway for the C a 2 + (Figure 4). RaM is driven by the electrochemical C a 2 + gradient as is the uniporter, but it has a more efficient C a 2 + uptake. It is also observed that RaM occurs when mitochondria are exposed to trains of C a 2 + pulses of physiological concentration (-400 nM) (Sparagna et al. 1995) and is rapidly inhibited following its initiation. These mitochondrial C a 2 + uptakes are driven mainly by the negative potential which lies between -150 and 25 -180 mV, across the inner mitochondrial membrane. The negative membrane potential is created by the proton extrusion along the electron transport chain from the matrix into the inner mitochondrial membrane. Recent studies have shown that the [Ca 2 + ] m can reach up to 500 uJvl (Montero, et al., 2000). Due to the high Ca 2 +-binding ratio ([Ca]m, t otai/[Ca]m,free) within the matrix of > 2000 as compared to the typical values of 50 - 1000 for the cytosol (Babcock et al., 1997; Magnus & Keizer, 1997), mitochondria may accumulate extraordinary amount of C a 2 + . In addition, the phosphate in the matrix can reversibly bind to and precipitate C a 2 + (David, 1999) in which the mitochondria can maintain a low free [Ca 2 +] without triggering the PTP to open, which would lead to programmed cell death by releasing the mitochondrial matrix contents (Green & Reed, 1998; Kroemer etal., 1998). Given the biochemical properties of the mitochondrial C a 2 + transporters, mitochondria were believed to be relatively insensitive to the rise of global cytosolic [Ca 2 +] of 500 nM -1 uM (Hajnoczky et al., 2000). High C a 2 + microdomains have been found to be a prerequisite for mitochondrial C a 2 + uptake (Gallitelii etal., 1999; Park etal., 2002; Pacher etal., 2002). On the other hand, experiments have demonstrated that agonist-evoked C a 2 + release is associated with increases of [Ca 2 + ] m simultaneously (Rizzuto et al., 1992, 1993, 1994; Ramesh et al., 1998; Szalai et al., 2000). This evidence suggests that mitochondria sense local domains of high [Ca 2 +] and pointed to a close coupling of IP3R- and RyR- mediated C a 2 + release to mitochondrial C a 2 + uptake. This implies an existence of a close contact between mitochondria and ER. In fact, 26 cumulative evidence using different experimental approaches has supported this view. Electronmicroscopy (EM) (Satoh etal., 1990; Takei etal., 1992) and wide-field fluorescence microscopy combined with digital deconvolution (Rizzuto etal., 1998) have revealed a interconnected dynamic network of mitochondria, and by expressing variant G F P targeted to the organelles that has given an estimation of 5 - 2 0 % of the mitochondrial surfaces are in close apposition to the ER (Rizzuto et al., 1998). Since an increase in cytosolic [Ca 2 +] causes mitochondrial C a 2 + elevation, mitochondria may act as a local C a 2 + buffering system and play a role in shaping intracellular C a 2 + responses (Baumann & Walz, 2001). 1.4.6. The "Superficial Buffer Barrier" hypothesis Cells have to constantly regulate C a 2 + entry during basal or stimulated conditions. In preventing overload with C a 2 + and shaping the [Ca 2 +]i signal, cells possess a number of homeostasis mechanisms for maintaining a relatively low [Ca 2 +] and regulating the C a 2 + gradient. In 1977, the superficial buffer barrier (SBB) hypothesis was proposed by van Breemen regarding the regulation of C a 2 + entry (van Breemen, 1977). It was based on the observation in SM. The essence the "SBB hypothesis" states that (van Breemen etal., 1995) C a 2 + enters the cells across the plasmalemma and is, in part, pumped into the superficial ER before it exerts its biological function in the cytoplasm. C a 2 + accumulation by the S E R C A in superficial ER contributes to the C a 2 + extrusion from the cells. This unloading of C a 2 + , due to the vectorial release of ER C a 2 + 27 towards a restricted space near the inner surface of the plasmalemma allows it to be extruded by the NCX or PMCA on the PM. The peripheral C a 2 + gradients are generated beneath the plasmalemma, depending on the combined effects of the C a 2 + influx and the C a 2 + uptake by the superficial ER. Thus the peripheral cytoplasm has a higher [Ca 2 +]i than in the deeper part of the cell in the resting state ( F i g u r e 5). F i g u r e 5. S c h e m a t i c d i a g r a m o f t h e s u p e r f i c i a l b u f f e r b a r r i e r . Ca entry through the basal C a 2 + leak and ligand-, voltage- or stretch-gated channels is partially sequestered by the superficial endoplasmic reticulum (ER) from a restricted subplasmalemmal space via ER Ca 2 + -ATPase (SERCA). The superficial ER functions as a Ca buffer barrier. (Adapted from van Breemen C. etal., 1 9 9 5 ) 28 1.5. Cytoskeleton The cytoskeleton is a key regulator in maintaining endothelial integrity and cell signaling (Lee & Gotlieb, 2003). Studies have provided evidence that the cytoskeleton participates in C a 2 + signaling. In the signaling of C C E , communication between ER and PM is suggested to depend on a structural connection between these two cellular sites (Berridge, 1995; Rossier etal., 1991; Irvine, 1990). This structural connection is confirmed by morphological studies showing that the ER is closely juxtaposed to the PM (Berridge, 1995), including vascular E C (Lesh et al., 1993). In this case, the cytoskeleton is proposed to function as a three dimensional framework that holds the ER near the PM (Holda & Blatter, 1997). Because the cytosketetal network (e.g. microfilaments, microtubules, and intermediate filaments) can potentially form a physical link between the PM and organelle membranes, it is possible that this physical link is involved in transmitting important regulatory signals. Different studies have shown that by disrupting the cytoskeleton, IP3 sensitivity of IP3R may alter (Bourguignon era/. , 1993), agonist-induced C a 2 + release (Ribeiro et al., 1997; Bozem et al., 2000), agonist-induced C a 2 + entry (Sabala et al., 2002), and the C a 2 + removal rate may affect (Wang et al., 2002) as well. Therefore, the cytoskeleton plays an important role in regulating the C a 2 + signaling, in addition to maintain endothelial integrity by controlling the production of EDRF. The disruption and dysfunction of the cytoskeleton may result in the impairment of endothelial functions that may lead to different vascular diseases. 29 C H A P T E R 2 C A L C I U M S I G N A L I N G I N H U M A N E N D O T H E L I A L C E L L S 2.1. R e s e a r c h f o c u s Many studies have suggested that the endothelial dysfunction is marker of vascular disease and plays an important role in their initiation and progression. The maintenance of vascular homeostasis is dependent on the release of vasoactive substances from a healthy endothelium. As the release of vasoactive substances is disrupted, the vascular tone as well as the proliferation and the migration of different cell types present in the blood vessel wall could be influenced. It is commonly known that the vasoactive substances (i.e. NO, PGI2, EDHF) are dependent on changes of [Ca 2 +]i in the cell. Therefore, it is crucial to have mechanisms for Ca 2 + vascular homeostasis by vasoactive substances. Previous work performed by our laboratory has shown that a defect in the endothelial C a 2 + removal systems may play an extremely important role in the process of vascular disease. Using a rat heart transplant model, data suggested that the defect of the C a 2 + removal system precedes a full-blown endothelial dysfunction (Skarsgard etal., 2000). Though the C a 2 + removal mechanisms have been identified in vascular E C , the relative contribution of each of the components to the overall C a 2 + extrusion and sequestration remains to be elucidated. A recent study of the recombinant expression of NXC in Chinese hamster ovary (CHO) cells has shown that the NCX not only operates in the resting cells, but its activity also directly regulates the [Ca 2 +] in the intracellular stores (Brini et 30 al., 2002). In addition, a functional coupling between NCX and ER has also been identified in freshly isolated bovine (Paltauf-Doburzynska et al., 1999) and rabbit E C (Wang et al., 2002), which allows efficient C a 2 + unloading from the ER to the extracellular space. In the following sections of the dissertation, I will present my data which will address two issues: 1) The C a 2 + removal system is essential to protect against excess [Ca 2 +]j levels in human endothelial cells. 2) The spatial arrangement of the C a 2 + transporters is crucial for modulating C a 2 + signaling. 2.2. Hypothesis and specific aims Based on the above mentioned evidence, the central hypothesis of my thesis work is that there is a functional link between the NCX and S E R C A in modulating the subplasmlemmal and cyclic [Ca 2 +] in human endothelial cells. The following aims are addressed experimentally: 1) To determine the mechanism of C a 2 + clearance in HUVEC, with particular focus on the physiological functions of NCX and its role in modulating [Ca 2 + ] i together with S E R C A under three conditions: a) at rest, b) during the agonist stimulation, and c) at the termination of agonist stimulation. 2) To demonstrate the ultrastructure of PM and ER in HUVEC 31 2.3. Methods and materials 2.3.1. Cell Preparation The human umbilical vein endothelial cell line, EA.hy926 (Edgell et al., 1983) was a generous gift from Dr. Cora Edgell (University of North Carolina, Chapel Hill, NC, USA). Dulbecco's modified Eagle's medium (DMEM) containing, 10% fetal bovine serum (FBS), streptomycin (10,000 units/mL) and penicillin (10,000 ug/mL) was used for cell culture. The endothelial cells were maintained at 37 ° C in a humidified incubator with 95 % air and 5 % C 0 2 . Cells between passage 10 to 30 were used for the C a 2 + imaging experiment. After reaching 80 % confluence, HUVEC was trypsinized with 0.25 % trypsin-EDTA solution for 1 min at 37 ° C and resuspended with 10% FBS-DMEM. Then HUVEC were grown on glass coverslips for experimental use. 2.3.2. Fluorescent dye loading and experimental setup HUVEC were loaded with a fluorescent dye fura-2 fluorescence in a loading solution at room temperature in the dark for 30 min. The loading solution was made of normal physical saline solution (nPSS) containing 1 u.M of membrane-permeant fura-2 acetoxylmethyl ester (fura/AM) and pluronic acid (F-127, 1 |iM). The fura-2/AM stock solution (1 mM) was made up by dissolving 50ug fura-2/AM in 50 uL dimethyl sulfoxide (DMSO) so that the final concentration of DMSO in the loading solution would be 0.1 % by volume. At this concentration, DMSO neither influenced the fluorescence intensity of fura-2 nor 32 caused a shift of the excitation maxim. The fura-2/AM permeates the plasma membrane and is cleaved by intracellular esterase to the polarized free acid which is trapped inside the cells and available for C a 2 + binding (Grynkiewicz etal., 1985). Although cells would be loaded with fura-2 more rapidly at 37 ° C , fura-2 extrusion towards the extracellular space or sequestration into intracellular organelles is greatly reduced at room temperature. The coverslip was mounted by two plastic discs with a screw system to secure the position of the coverslip. The coverslip was mounted on a Nikon inverted microscope (Nikon, Diaphot). The experimental solution was infused through polyethylene tubing via gravity. The fluid level was maintained by a suction pump that continuously removed the perfusion solution. Three mL of solution was used for each solution exchange to assure complete wash out. The time of each exchange was less than 15 s. 2.3.3. Instrumentation for monitoring the fluorescence signal The cells were exposed to alternating 340- and 380- nm (bandwidth 10 nm) ultraviolet light (1/s) that was passed through a 510-nm (bandwidth 40 nm) cut-off filter before acquisition by an intensified charge-coupled device camera (model 4093G, 4810 series). Fluorescence signals were recorded as digital image data using the software program Northern Eclipse by Empix Imaging Systems housed in a Pentium processor personal computer. The composition of this imaging microscopy system is shown in Figure 6. The single line trace in each figure is 33 the average of the simultaneously measured fluorescence ratio (F340/F380) of individual or groups of cells in the chosen field. Figure 6. Experimental setup for fluorescence measurement solution vacuum xenon lamp 340 nm 380 nm 360 nm filters ICCD camera Recorder F340 F380 Ratio __[ (F340/F380) F360 510 nm filter computer. 2.3.4. [Ca2+]j measurement F340/F380 signals were collected every 7 s and plotted as background-subtracted ratio value versus time on line during the experimental procedure. The cells were then excited alternately at 340 and 380 nm and the fluorescence measured at 510 nm. The ratio of the two intensities after excitation at 340 and 380 nm (F340/F380) was taken as a relative measurement of [Ca 2 +]j. Calibration 34 was not attempted because the dissociation constant of fura-2 in the intracellular milieu was not known. 2.3.5. Electronmicroscopy HUVEC were resuspended with 0.25 % trypsin and 1 mM EGTA for 1 min at 37 ° C and transferred from the culture flask to a plastic centrifuge tube. After centrifugation at 1000 rpm for 5 min, the supernatant was quickly removed and replaced with the primary fixative solution, containing 1.5 % glutaraldehyde, 1.5 % paraformaldehyde, and 1 % tannic acid in 0.1 M sodium cacodylate buffer (37 °C) . The centrifuge tube was stored on ice (4 °C) for 2 h. After the primary fixation, the cells were washed with 0.1 M sodium cacodylate buffer and followed by a secondary fixation with 1 % osmium tetroxide (Os0 4) in 0.1 M sodium cacodylate buffer at room temperature for 1 h. After washing with distilled H 2 0 the cells were stained with 1 % uranyl acetate for 1 h, followed by three washes with distilled H 2 0 , and then dehydrated in an ethanol series (50, 70, 80, 90, 95 and 100 %) for 10 min each . The cells were then infiltrated in the resin (TAAB 812 mix, medium hardness) over night and embedded in molds. Sections (100 nm) were cut on an ultramicrotome, collected on 400 mesh copper grids, stained with 1 % uranyl acetate and Reynolds lead citrate for 4 and 3 min, respectively and examined with a Phillips 300 electron microscope. 35 2.3.6. Solutions & Chemicals The composition of different solutions used is shown in Table 1. Normal PSS (nPSS) contains (in mM): NaCI 140, KCI 5, CaCI2 1, MgCI2 1, Glucose 10, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) 5 (pH 7.4). nPSS was adjusted to a pH of 7.4 by using NaOH. 0Ca 2 + PSS was almost the same as nPSS except that CaCI2 was omitted from the solution. In 0Ca 2 + PSS, 0.2 mM ethylene glycol-bis(P-amino-ethyl ether) N,N,N',N'-tetraacetic acid (EGTA) replaced 1 mM CaCI2. External Na + substitution was achieved by equimolar substitution of external Na + with Li + (Li+-PSS) or N-methyl-D-glucamine (NMDG-PSS). NMDG-PSS was adjusted to a pH of 7.4 by using HCI. Analytical grade reagents for PSS, histamine, cyclopiazonic acid (CPA), NMDG, DMSO and, EGTA were obtained from Sigma-Aldrich Co. (Oakville, Ontario, Canada). Fura-2/AM was obtained from Molecular Probes (Eugene, OR, USA). All agents were dissolved in their respective stock solutions. The stock solution of CPA was made in DMSO; HIST was made in double distilled H 2 0. Serial dilutions of all chemicals were made in buffered PSS. The experimental agents used did not affect the fura-2 fluorescence under the experimental conditions. The maximal concentration of any vehicle to which preparations were exposed was 0.1 %, which had no effect on the fura-2 signal. 2.3.7. Data analysis & Statistics All cells with the same preparation responded similarly as the cell chosen. The single trace line shown in each figure represents simultaneously measured 36 F340/F380 of individual of endothelial cells (1 - 64 cells) of interest chosen in the field of the same preparation. Drugs were applied as indicated by the horizontal bars in each figure. All data has been normalized to the maximal response in [Ca 2 + ] i elevation elicited by histamine (1 u.M), which was observed to be consistent among all tested cells. Cells were allowed to recover for at least 15 min in nPSS solution in between each protocol and the order of application was randomly selected where possible to eliminate artifacts. The C a 2 + levels during the time course of each treatment are expressed as percentages of the histamine response in normal PSS. To determine the rates of C a 2 + removal, the difference between neighboring C a 2 + levels are divided by the time interval between the two points in the decline phase (A [Ca 2 + ]/Af ) . To be able to compare between different experimental protocols, all the data are binned at certain C a 2 + levels (5, 10, 20, 40, and 60 % of the maximal responses) and then averaged. Statistics were performed on C a 2 + removal rates expressed as percentages of control. Data from multiple experiments are given as mean ± S.E.M. f-Test and ANOVA are performed to determine the statistical significance. Comparisons of the results were made by using the t test. P < 0.05 is considered as significantly different. 37 2 . 4 R e s u l t s The inflammatory agonist histamine was used throughout the entire study to induce [Ca 2 + ] i increase. In this cell preparation, histamine (1 uJvl) consistently induced [Ca 2 + ] i elevation as detected by rapid increases of the fura-2 fluorescence ratio. 2 . 4 . 1 . I n t e r a c t i o n b e t w e e n N C X a n d S E R C A d u r i n g C a 2 + s i g n a l i n g 2 . 4 . 1 . 1 . U n d e r r e s t i n g c o n d i t i o n s 2 . 4 . 1 . 1 . 1 . N C X u n l o a d s t h e E R C a 2 + s t o r e The first set of experiments was designed to determine the functional role of NCX at the resting state. Human E C were incubated in the 0 C a 2 + PSS for 20, 100, 200, 400, or 600 s, before 1 u.M histamine was applied. The peak of histamine-induced C a 2 + release from the ER was detected to assess the content of the ER C a 2 + histamine releasable store at different times. The experiment showed that the ER C a 2 + content was reduced in a time-dependent manner ( F i g u r e 7 A , control) where the cells were incubated in 0 C a 2 + PSS. The contributions of ER and NCX were then eliminated, one at a time, by the S E R C A inhibitor C P A (Cyclopiazonic acid, 10 u,M) and the 0Na + PSS respectively. The ER C a 2 + store contents after different incubation times was assessed accordingly by the histamine-induced [Ca 2 + ] i in 0 C a 2 + PSS. 3 8 The inhibition of S E R C A dramatically reduced the ER C a 2 + content after 100 s of incubation with CPA. However, the C a 2 + content of the ER was initially higher and decayed more slowly after removing the extracellular Na + , which inhibited the NCX. All the peak levels of histamine response are normalized to the histamine response in nPSS, which represents 100 % of the histamine-sensitive ER C a 2 + store (Figure 7B). 2.4.1.1.2. Buffer barrier function After we established that NCX unloads ER C a 2 + content under resting conditions in 0 C a 2 + PSS as shown above, the next experiment was to show whether NCX functions when extracellular C a 2 + was present. As shown in Figure 8A, the intracellular C a 2 + level was stable only for a short period, then started to increase slowly when Na + was removed from the nPSS. Also shown in Figure 8A, the selective reverse mode NCX blocker, KB-R7943, had no effect on the slow increase, eliminating the possibility that 0Na + conditions could cause C a 2 + influx via NCX working in the reverse direction (see also Figure 11). Figure 8B shows another experiment where the ER C a 2 + content was tested in 0Na + conditions. The E C were first stimulated with 1 uM histamine in 0 C a 2 + PSS for a short time and the peak level was taken as a measurement of C a 2 + content in the ER. After the first stimulation, the cells were allowed to recover either in nPSS (solid trace) or 0Na + PSS (dashed trace) for 15 min and tested again with histamine. In nPSS, the second histamine response was slightly smaller than the first one, probably due to receptor desensitization. However, the second 39 Figure 7. NCX contributes to ER calcium unloading under resting conditions. (A) Histamine (1 |xM) was applied as indicated by the arrows after cells were incubated in 0 C a 2 + PSS for different time periods. The peak histamine response was taken as a measurement of ER C a 2 + content in HUVEC (n = 31). (B) Each data point represents the ER C a 2 + content as assessed by histamine induced C a 2 + peak level from panel A. All data are normalized to the histamine response in nPSS. The decay of ER C a 2 + content was plotted for: Control (o), with C P A (A ) or 0Na + PSS(«) respectively for comparing the ER C a 2 + store after different incubation times: 20, 100, 200, 400, and 600 s. Data are given as mean + S .E .M . Results were analyzed by t-test and ANOVA to determine the statistical significance. *P< 0.01 compared with control. A. 120 (0 O 100 80 | 60 o & 40 3 20 N CO E k. o z o -20 i i i J 100 200 300 400 500 Time (s) 600 700 800 B . 40 F i g u r e 8. N C X u n l o a d s E R C a 2 + a n d m a i n t a i n s b u f f e r b a r r i e r f u n c t i o n . ( A ) Extracellular Na + was removed from the bath as indicated by the solid line. The intracellular C a 2 + level remained stable for a short period and then started to increase slowly. The selective reverse-mode NCX blocker KB-R7943 had no effect on the C a 2 + increase (dashed line). The C a 2 + level returned to baseline upon adding back of extracellular Na + (n = 31). (B) Solid trace shows an representative experiment where HUVEC were stimulated with a short pulse of histamine in OCa PSS. After first stimulus, the cells were allowed to recover in nPSS (n = 21) for 15 min. Histamine was applied for a second time to assess the ER C a 2 + content. Dashed trace, same experiment was repeated except that extracellular Na + was removed during the 15 min recovery time in order to block NCX(n =49). *P<0.01. A . 0Na+ PSS KB-R7943 1.8 o 8 1.6 ] 1.2 100s B . ? , 2 0 1 2Q0JL Histamine 10CH -20 J OCa^  ,2+ OCa' i_ ,2+ 41 response after 0Na + PSS incubation was significantly higher than the first response, indicating that more C a 2 + was accumulated in the ER, when NCX was blocked. 2.4.1.2. During histamine stimulation 2.4.1.2.1. Ca2 +cycling The endothelial agonist histamine was applied in nPSS to evoke [Ca 2 + ] i increase in HUVEC. Histamine (1 uM) induced intracellular [Ca 2 + ] i elevation as detected by the rapid increase of the fluorescence ratio from 1.51 ± 0.02 to 2.25 ± 0.01 (Figure 9A, solid trace). The histamine induced response was maintained up to 20 min as long as the extracellular C a 2 + was present. In the absence of extracellular C a 2 + , the histamine-induced intracellular C a 2 + transient returned to baseline after 400 s (Figure 9A, dotted trace). To determine the role of ER in the histamine-induced C a 2 + response, we repeated the same experiment in the absence of extracellular C a 2 + and the continuous presence of CPA, a S E R C A blocker. As shown in Figure 9B, the application of C P A (10 u.M) caused a small increase in [Ca 2 + ] i by itself. Subsequent application of histamine and C P A together gave a rapid, but more transient response compared to the response induced by histamine alone. The slower decay phase in the control experiment (Figure 9B, dotted trace), then seen in the presence of CPA suggests that a large portion of the released C a 2 + is being taken back into the ER by S E R C A , and subsequently re-42 F i g u r e 9. H i s t a m i n e - i n d u c e d C a 2 + s i g n a l s i n H U V E C . ( A ) Histamine (1 uJvl) induced maintained [Ca 2 +] i response in nPSS (solid, n = 34), and transient response in 0 C a 2 + PSS (dotted, n = 34). ( B ) S E R C A blocker, C P A (10 jxM) (solid, n = 32) was applied in 0 C a 2 + PSS. Histamine was applied 200 s after CPA. The dotted trace represents the same control histamine response in 0 C a 2 + PSS from panel A . o 00 co 2.4 -, 2.2 2 % 1.8 1.6 1.4 -1.2 -200 Histamine B . released via ER C a release channels during agonist stimulation. This continuous cycling of C a 2 + would delay the decay phase of the response in the absence of extracellular C a 2 + . To determine the role of ER in continuous cycling 43 of intracellular C a in the presence of histamine, a sequence of histamine application/removal was performed. The time interval in between the application and removal was 100 s (Figure 10A). The trace shows that the re- application of agonist releases an amount of C a 2 + from the ER, which is largely restored to the ER by a Ca 2 +-clearing mechanism following agonist removal. As expected, C P A diminished the subsequent histamine response by abolishing the C a 2 + cycling mechanisms (Figure 10B). 2.4.1.2.2. NCX refills ER Ca 2 + store To investigate the contribution of NCX to the histamine-induced maintained C a 2 + response in nPSS, we conducted an experiment in the absence of extracellular Na + . As shown in Figure 11 A, the histamine-induced C a 2 + response declined to a new plateau below the level seen in nPSS (Figure 9A). We repeated the same experiment in the presence of KB-R7943, a selective agent that blocks the NCX in its reverse mode (Iwamoto etal. 1996; Ladilov etal., 1999). In Figure 11B, the plateau of the C a 2 + response declined to a lower level in the presence of KB-R7943 (1 uJvl) as was the case for 0Na + PSS shown in Figure 11 A. This data suggests that the NCX facilitates C a 2 + influx by working in the reverse mode during agonist stimulation. 2.4.1.3. Upon termination of agonist stimulation 2.4.1.3.1. Ca 2 + removal 44 Figure 10. "Calcium cycling" during histamine stimulation. (A) Histamine (1 ixM) was repeatedly applied and removed in intervals of 100 s (n - 64). (B) The same protocol as in panel A was applied after SERCA was blocked by CPA (n = 20). Experiments in panel A and B were both performed in 0Ca 2 + PSS. A-o n n Histamine 2.6! - u u In the next set of experiments, we determined how elevated Ca is cleared by the various C a 2 + transporters and pumps after cessation of histamine stimulation. Figure 12 illustrates the Ca2+extrusion experiment used in this study. Cells were stimulated with 1 uM histamine in nPSS. After the [Ca2+]i reached a 45 Figure 1 1 . Effect of the inhibition of reverse mode NCX on histamine induced Ca 2 + responses. Representative traces of histamine applied: (A) in the absence of the extracellular Na + (dotted, n = 134); (B) in the presence of KB-R7943 (1 u,M) (dotted, n = 37). All experiments were compared to the histamine-induced C a 2 + response in nPSS as control (solid trace). A. o co co 2.4 2.2 £ 1.8 1.6 1.4 200 Control 0Na + PSS Histamine B. 46 Figure 12. Ca 2 + removal in HUVEC after histamine stimulation. (A) Histamine (1 uM) was applied in nPSS to induce C a 2 + elevation. Histamine and extracellular C a 2 + were removed as the [Ca 2 +] i response reached a plateau in order to determine the speed of C a 2 + clearance. Cells were then allowed to recover in the nPSS for 10 min and the same protocol was repeated in the 0Na + PSS to inhibit the function of NCX (n = 150). (B) The same protocol was used in presence of C P A to inhibit the S E R C A (n = 80). (C) The same protocol was used in presence of 0Na + PSS with CPA to inhibit both NCX and S E R C A (n = 40). All dotted traces are represented the decline phase from nPSS. 47 peak, we removed both histamine and extracellular Ca to monitor the speed of the [Ca 2 + ] i decline. The same protocols were then repeated either in the presence of C P A (10 uM) in order to eliminate the contribution of S E R C A , or in the 0Na + PSS solution to stop the function of NCX. The contribution of each C a 2 + removal component to the removal process was then expressed as the rate of decline plotted against the [Ca 2 + ] i . As shown in Figure 13A, both 0Na + PSS and C P A significantly reduced the rate of C a 2 + removal. However, applying both 0Na + PSS and C P A together did not lower the rate of C a 2 + removal any further than the application of C P A alone. This agrees with previous finding obtained in fresh rabbit E C (Wang, et al., 2002), which suggests that NCX and S E R C A are functioning in series. Therefore, blocking S E R C A alone was sufficient to eliminate the contribution of NCX to the overall C a 2 + removal process. To exclude any possible side effect caused by 0Na + PSS such as pH change, we confirmed the role of NCX in C a 2 + removal by using a NCX blocker, bepridil (Watanabe & Kimura, 2001). As shown in Figure 13B, bepridil (10 uM) again significantly reduced the overall rate of C a 2 + removal comparable to that seen in Figure 13A. In contrast, we observed two different results when histamine was left in bath solution after removing extracellular C a 2 + in the C a 2 + extrusion protocol (Figure 14). First, the rate of C a 2 + removal was significantly slower than in experiments where histamine and extracellular C a 2 + were not present, shown in Figure 12 and 13; and second, in the presence of histamine the removal of extracellular N a + had no effect on the rate of C a 2 + clearance. This is consistent with the idea that when both histamine and C a 2 + are present, NCX works in the 48 Figure 13. Rates of Ca 2 + removal as a function of [Ca2+]j. (A) The C a 2 + removal rates were calculated from the data of Figure 12 by taking A[Ca 2 +]/At in the decay phase. All values of [Ca 2 +] i were normalized to the peak of histamine response in nPSS. The rates were binned at fixed level of [Ca 2 +] as shown. *P < 0.01 compared with control. (B) Bepridil (10 uM) was used to inhibit the NCX. The same protocol and calculations were used as described in Figure 11 and Figure 12 (A) respectively (n = 61). *P< 0.01 compared with control. A. —o— Control -* -0Na 0 10 20 30 40 50 Normalization of [Ca2+]j (%) B. 49 Figure 14. Ca 2 + removal in HUVEC during histamine stimulation. (A) Left panel, 1 uM histamine was applied in nPSS. Extracellular C a 2 + was removed at the time indicated. In contrast to the experiment in shown Figure 12, histamine was kept in the solution after extracellular C a 2 + removal. The dotted trace represents the trace from Figure 12, where both C a 2 + and histamine were removed. Right panel, same experiment was repeated in 0Na + PSS. When histamine was present the 0Na + protocol did not affect the rate of C a 2 + removal (n = 38). (B) Same protocol as described in (A), was repeated in the presence of S E R C A inhibitor, CPA (10 uM) (n = 30). The dotted trace was from Figure 12B, where C P A was present but histamine was removed together with C a 2 + removal. 50 reverse mode and does not contribute to the net Ca removal. The slowdown of the C a 2 + removal rate was probably caused by C a 2 + cycling as described in Figure 9 & 10. Indeed, in the presence of CPA (Figure 14B), the rate of C a 2 + removal was, upon switching to 0Ca 2 + PSS, identical with or without histamine. In the previous two sets of experiments (Figure 12-14), the extracellular C a 2 + was removed during the decline phase in the protocol, in order to eliminate any possible remaining C a 2 + entry and allow a more accurate estimation of the C a 2 + removal rate. The question remains whether NCX also effectively removes Ca 2 + when extracellular C a 2 + is present. In Figure 15, we therefore have Figure 15. Effect of NCX in the presence of extracellular Ca 2 + . Cells were stimulated with 1 u,M histamine in nPSS (solid trace, n = 17). Histamine was removed after stimulation as indicated. The C a 2 + level returned back to baseline. The removal of extracellular Na + slowed down the removal rate (dotted trace, n = 33). Histamine CC z -20 J 100 51 repeated the C a extrusion protocol in the presence of extracellular C a . Again the 0Na + protocol significantly slowed down the C a 2 + removal rate and stabilized at a new plateau. 2.4.2. Ultrastructure of PM and ER The existence of the peripheral ER was demonstrated in a morphological study. Examples of electron micrographs of cross sections of isolated E C from the same cell line show close appositions of the ER with the PM (Figure 16). The physical orientation of the ER and PM supports the concept of C a 2 + and N a + accumulation in this restricted space. The distance between the PM and the ER was estimated to be 20 - 40 nm. 52 F i g u r e 1 6 . E R a n d P M j u n c t i o n i n H U V E C v i s u a l i z e d w i t h E M . ( A ) and ( B ) the close apposition of PM (indicated by white arrow heads) and ER (indicated by black arrow heads) is shown in different sections of HUVEC. The junctional distance is about 20 - 40 nm. Scale bar: 200 nm. A . B . 53 2.5. Discussion This study presents a systematic investigation of the interactions between NCX and S E R C A before, during and after agonist stimulation in a human E C preparation. Two novel conclusions were drawn from the presented data. First, NCX unloads part of the ER C a 2 + , before and after agonist stimulation, thereby, facilitating C a 2 + removal from the cytoplasm and maintaining the superficial buffer barrier function. During agonist stimulation, however, NCX functions in the reverse mode refilling the ER and thereby slowing down the C a 2 + removal process. Second, S E R C A was shown to be able to exert two different effects on cytosolic [Ca 2 +]. Under resting conditions, peripheral ER serves as a buffer barrier to prevent C a 2 + from reaching the deep cytoplasm. During agonist stimulation, however, C a 2 + cycles through S E R C A and ER C a 2 + release channels to effectively maintain the elevation of [Ca2+]|. This cycling mechanism retains the free C a 2 + in the cytoplasm and competes with the process of C a 2 + extrusion by other C a 2 + pumps such as PMCA. 2.5.1. Depletion of ER Ca 2 + by NCX before agonist stimulation The contribution of NCX to the C a 2 + removal process under resting conditions is still somewhat controversial in the E C . The first study in bovine E C suggested that the NCX did not appear to be of primary importance in the maintenance of resting [Ca 2 +] i (Sage etal., 1991). However, more recent studies indicated that the NCX could serve as the major mechanism for extruding C a 2 + (Nakao ef al., 2000; Domotor et al., 1999). Previous data also implied that the 54 NCX might be relatively dominant under resting conditions in the intact endothelium of the rabbit cardiac valve (Li & van Breemen, 1995). Due to its low C a 2 + affinity binding, the NCX should not play any role in the C a 2 + removal process at resting [Ca 2 +] of the bulk cytoplasm, except in the so-called C a 2 + micro-domains, which have been proposed and observed in many cell types including vascular E C (Hansen etal., 1991). In this study, we used a depletion protocol to define the contribution of NCX to C a 2 + homeostasis. As shown in Figure 7, cells were placed in a 0 C a 2 + PSS and subsequently tested for the ER calcium store content by applying histamine at different time points. The data in the figure shows a loss of C a 2 + from the ER which is probably due to either a basal leak in the ER membrane (Hofer etal., 1996; Lomax etal., 2002; Mogami etal., 1998) or release from the ER toward the PM with subsequent extrusion across the PM. CPA, a S E R C A inhibitor, accelerated the process of depleting ER C a 2 + , revealing the dynamic state of the ER C a 2 + . Removal of extracellular Na + significantly slowed down the depletion process because C a 2 + released from the ER under these conditions fails to be extruded by the NCX and is re-sequestered by S E R C A (Nazer & van Breemen, 1998). It is therefore reasonable to conclude that the NCX functions to unload some of the ER C a 2 + content under resting conditions. This finding is in agreement with those experiments in Chinese hamster ovary cells expressed with cardiac NCX, which has shown that NCX decreased the ER [Ca 2 +] in resting cells (Brini etal., 2002). 55 Other studies also claimed that the NCX could be active under resting conditions (Fang et al., 1998, 1999). This process most likely takes place in the so-called restricted or junctional space, where the peripheral ER membrane is located in close proximity to the PM. The [Ca 2 +] i in the restricted space (Figure 16) could reach well into the micro-molar range due to its small volume and restriction to diffusion. In addition, the finding that the [Ca 2 +] of the subplasmalemmal region appears to be higher than the bulk cytosol in unstimulated cells measured by the targeted aequorin technique (Marsault et al., 1997) also supports such a hypothesis. Under resting conditions, this configuration could function as an effective barrier for preventing possible basal C a 2 + entry through S O C , N S C C or C a 2 + leak channels to reach the deep cytoplasm (Figure 17A). The unloading of the ER C a 2 + by NCX is needed to ensure that the superficial ER is not saturated with C a 2 + and can continuously buffer C a 2 + entry. Such phenomenon was indeed observed in this study. As shown in Figure 8, when NCX was blocked in 0Na + nPSS, the intracellular C a 2 + level remained stable only for a short period, and then started to increase slowly. Figure 17. Models for Ca 2 + cycling and extrusion in HUVEC (A) Under resting conditions, basal C a 2 + entry is mainly sequestered by the S E R C A of the peripheral ER. NCX unloads some of the ER C a 2 + to prevent saturation thereby maintaining the buffer barrier function. (B) Upon agonist stimulation, C a 2 + released from ER is actively taken up by S E R C A resulting in C a 2 + cycling in the deeper cytoplasm. The entry of Na + through the non specific cation channels in the PM drives the NCX into the reverse mode to refill the ER. (C) After stimulation, S E R C A actively reduces the [Ca 2 +] i in the deeper cytoplasm. NCX returns to its normal mode and extrudes C a 2 + which was vectorially released from the ER towards PM. 56 Extracellular j C a 2 + entry channel (SOC/NSCC) 57 This indicates that when NCX stopped working, the ER was only able to take up C a 2 + which entered from the outside for a limited time and started to lose its buffer barrier function when it became full. 2.5.2. Ca 2 + removal during histamine stimulation As discussed above, NCX and SERCA, under resting conditions, operate in series to extrude C a 2 + and to prevent C a 2 + from reaching the deep cytoplasm. However, when agonists are applied to the EC, the two C a 2 + transporters, which are supposed to remove cytosolic C a 2 + , have rather the opposite function in adding C a 2 + t o and slowing down C a 2 + clearance from the cytoplasm. 2.5.2.1. Cycling of Ca 2 + by SERCA and ER Ca 2 + release channels As shown in Figure 9A, when E C were stimulated with histamine (1 uM) in the presence of extracellular C a 2 + , [Ca 2 +] i was elevated and maintained at this level for a long time. Removal of the extracellular C a 2 + did not significantly affect the response for the first couple hundreds of seconds. The response in 0 C a 2 + PSS lasted about 400 s. However, if the same experiment was repeated while S E R C A was inhibited by C P A (10 uM), the histamine induced response returned back to baseline within about 100 s (Figure 9B). In this case, therefore, the action of S E R C A is to slow down the decline of the C a 2 + signal. This can be best explained by the idea that during agonist stimulation, a major portion of the C a 2 + taken up into the ER by S E R C A is re-released through ER C a 2 + release channels (Myers and Larkins, 1989; Loeb et al., 1988). Thus C a 2 + appears to be cycling 58 between SERCA and Ca release channels and retained in the cytosol, rather than extruded through the PM. To confirm this idea, we designed a different protocol as shown in Figure 10A. In this case, the cells were exposed to a short pulse of histamine stimulation (100 s) in 0Ca 2 + PSS. After 100 s of recovery in 0Ca 2 + PSS, the cells were stimulated again with histamine. We were able to repeat at least seven times the histamine induced C a 2 + transients in the same cells. However, when SERCA was blocked, the cells completely lost their response after only two stimulations. This confirms that during the recovery phase, a major portion of the cytosolic C a 2 + is recycled to the ER by SERCA. Only a small portion is lost as shown by the slow decrease in the histamine response, probably due to extrusion by PMCA. 2.5.2.2. The reverse mode of NCX refills the ER Ca 2 + during stimulation In contrast to the situation before stimulation, NCX changes its behavior during agonist stimulation. As described before, histamine stimulation in the presence of extracellular C a 2 + initiates a long lasting C a 2 + response. However, when we stimulated the cells with histamine after removing extracellular Na+, the response was not maintained at peak levels but instead declined to a new plateau. This suggests that the long-lasting response we observed in our control experiment is not only dependent on C a 2 + influx through membrane C a 2 + channels, but is also dependent on NCX activity. During the histamine response, NCX operates in the reverse mode, mediating C a 2 + entry. Agonist stimulation 59 opens S O C / R O C or N S C C permeable to Na + , which leads to an accumulation of N a + in the restricted space providing the driving force for C a 2 + entry via NCX. This confirms the finding by Graier (Paltauf-Doburzynska et al., 2000) in the same cell preparation and is in full agreement with the mechanism proposed in the arterial myocytes (Arnon et al., 2000) and VSM (Lee et al., 2001) which suggests that NCX serves to partly refill the Ca store by working in the reverse mode. Furthermore, we observed the same inhibitory effect of the histamine-induced C a 2 + response as seen in 0Na + PSS, after the application of KB-R7943, a selective reverse mode NCX inhibitor. We concluded that the long-lasting response is, in part, dependent on the ER C a 2 + content, and that the reversal mode of NCX during histamine stimulation serves to refill the ER C a 2 + stores (see Figure 17B). 2.5.3 Ca 2 + removal after stimulation—SERCA and NCX work in series Shortly after the histamine stimulation, NCX returned again to its normal mode as expected, as shown in Figure 12 and 13. To demonstrate this, we used the extrusion protocol in which we monitored the [Ca 2 + ] i decline after terminating histamine stimulation. The experiments were performed in 0 C a 2 + PSS to eliminate any remaining C a 2 + influx. The data showed that either the removal of extracellular N a + or the blockade of S E R C A with CPA significantly reduced the rate of C a 2 + extrusion. However, when the two conditions are applied together (e.g. C P A in 0Na + PSS), we did not observe any additive effect of the two transporters and pumps. This again confirms that NCX and S E R C A are 60 functioning in series to extrude a large part of the excess Ca (Figure 17C) This agrees with previous finding from freshly isolated rabbit aortic E C (Wang et al., 2002). If we only removed extracellular C a 2 + , but left histamine in solution (Figure 14), we observed that the rate of C a 2 + removal was significantly slower as compared to the control when both extracellular C a 2 + and histamine were removed in nPSS. This is due to the C a 2 + cycling mechanism described above, since now the IP3-sensitive C a 2 + release channels remain open. Repeating this protocol in 0Na + PSS showed no change in the removal rate. This is explained in Figure 17B, where in the presence of histamine, NCX functions in reverse mode and cannot contribute to the net C a 2 + removal process. In conclusion, this study shows that NCX and S E R C A can have different effects on [Ca 2 + ] i depending on their ultrastructural configuration which determines their relation to other C a 2 + regulatory pathways such as the ER C a 2 + release channels. In human EC, NCX in normal mode functions in tandem with S E R C A to maintain a low intracellular [Ca 2 +] in the absence of agonist stimulation. On the other hand, if cells are stimulated with the inflammatory agonist histamine, and presumably other agonists as well, NCX works in the reverse mode serving to refill the ER C a 2 + store. The C a 2 + thus taken up by S E R C A is again released through the open ER C a 2 + release channels. This special configuration has the net effect of maintaining C a 2 + at a high level. This schematic model implies that any alteration in the ultrastructural or molecular configuration of the various C a 2 + regulatory proteins would significantly modify fluctuations in [Ca 2 + ] i and subsequent physiological and patho-physiological functions. 61 2.5.4. Limitations of cell preparation & methods 2.5.4.1. Cell type It is always a concern to use cultured cells for experiments that the phenotype no longer reflects native EC. However, there are also problems using native cells usage problems, such as the presence of contaminating cells, the difficulty in obtaining a larger number of cells, and the progressive loss of cell viability and expression of endothelial markers. Thus, we chose to use the particular cultured cell line, EA.hy926, because it is the most frequently used and best characterized permanent human vascular E C line (Bou'i's et al., 2001). In addition to its extensive characterization and stability in its endothelial traits, EA.hy926 is a true immortalized cell, yielding more consistent results than primary cells. 2.5.4.2. Fura-2 measurement The use of the fluorescent dye, fura-2, allows us to measure the global level of C a 2 + within the E C in this study. However, this technique is limited and does not allow us to measure the changes in [Ca 2 +] that occurred in defined cell compartments (organelles, cytosolic subregions, the microenvironment of C a 2 + -sensitive proteins, etc.). However, the advantage of using ratiometric dye is quantitative irrespective of concentration of dye, but exact calibration is difficult. 62 2 . 6 . S u m m a r y Recent findings indicate that cellular C a 2 + overload or perturbation of intracellular C a 2 + compartmentalization can cause cytotoxicity and is associated with endothelial dysfunction. C a 2 + removal mechanisms in this case play an essential role to prevent the excess [Ca2 +} in the EC. Our study has indicated that there is a tight functional interaction between NCX and S E R C A in modulating [Ca 2 +]j in human endothelial cells. Under the basal conditions and after agonist stimulation, NCX unloads the ER C a 2 + content from C a 2 + saturation in order to maintain the buffer barrier function of the ER. Interestingly, the NCX works in the reverse mode to refill ER with C a 2 + during agonist stimulation. This contributes to the long sustained agonist-induced C a 2 + response and to maintain sufficient [Ca 2 +]i for different cellular processes. These pieces of new information bring us a step closer to understanding the regulation of C a 2 + removal mechanism in human EC. Hopefully we can apply this knowledge to prevent the cellular C a 2 + overload under pathophysiological conditions that may be associated with different vascular diseases and even cell death. 2 . 7 . F u t u r e d i r e c t i o n s To complete the determination of C a 2 + removal mechanisms in human E C , additional studies are needed to focus on investigating the rest of the components, such as PMCA and mitochondrial C a 2 + uniporter. PMCA was reported to be largely responsible for the extrusion of C a 2 + in many non-excitable cells (Monteith et al., 1995). We know that PMCA and NCX contribute to C a 2 + 63 extrusion in bovine vascular E C (Sedova & Blatter, 1999). However, most studies have examined its physiological role independently in the E C (Goto etal., 1996; Moccia et al., 2002; Wang et al., 2002), instead of reporting on their interplay with other C a 2 + removal components. In addition, mitochondria have become the focus of attention in the regulation of programmed cell death or apoptosis. The study of C a 2 + removal mechanisms is relevant because C a 2 + overload has been proposed to be a major apoptotic signal. After understanding the C a 2 + removal mechanisms in the physiological conditions, our ultimate goal is to understand how these mechanisms play a role under pathophysiological conditions. In animal models, endothelial dysfunction occurs in association with increased ROS in numerous disease conditions (Cai & Harrision, 2000), due to inactivation of NO by superoxide (02~). Though the actual molecular mechanisms associated with oxidant-induced E C damage remain unknown, substantial evidence suggests that oxidative stress alters cellular C a 2 + homeostasis mechanisms (Elliott et al., 1989; Franceschi et al.. 1990; Hirosumi et al., 1988; Orrenius et al., 1989) in the early stages of the dysfunction of E C . 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