Phospholipase Cβ (PLCβ) cleaves phosphatidylinositol-4,5-bisphosphate (PIP₂) into the second messengers inositol-1,4,5-triphosphate (IP₃) and diacylglycerol (DAG). IP₃ increases intracellular Ca²⁺, while DAG remains in the membrane, and together with increased Ca²⁺, activates protein kinase C (PKC). PLCβ has low basal activity but is activated following stimulation of G_i- and G_q-coupled receptors through direct interactions with Gα_q and Gβγ. PLCβ is essential for normal cardiomyocyte and vascular smooth muscle function and regulates cell proliferation, survival, migration, and differentiation. However, increased PLCβ activity and expression results in arrhythmias, hypertrophy, and heart failure. PLCβ must interact with the cell membrane for its activity. While heterotrimeric G proteins stimulate PLCβ, they are insufficient for full activation, suggesting the membrane itself contributes to increased lipid hydrolysis, potentially via interfacial activation. However, how the composition of the membrane and its resulting properties, such as surface charge, contribute to adsorption and interfacial activation is not well-established. Furthermore, whether or how interfacial activation also impacts other regulatory elements in PLCβ and Gα_q-dependent activation is unknown. Using an innovative combination of atomic force microscopy on compressed lipid monolayers and biochemical assays, we are beginning to understand how the membrane itself, PLCβ autoinhibitory elements and Gα_q regulate PLCβ activation. These studies provide the first structure-based approach to understanding how the cell membrane regulates the activity of this essential effector enzyme.

Gliotransmitters dérivés de l'astrocyte glutamate et l'ATP modulent l'activité neuronale. Cependant, il reste à savoir comment les astrocytes contrôlent la libération et coordonnent les actions de ces gliotransmetteurs. Dans la première partie de ma thèse, en utilisant l'expression transgénique de la canalrhodopsine 2 (ChR2) sensible à la lumière dans les astrocytes, nous avons observé que la photostimulation augmentait de manière fiable le potentiel d'action des neurones pyramidaux de l'hippocampe. Cette excitation repose principalement sur une libération de glutamate dépendant du Ca2+ par les astrocytes qui active les NMDR neuronaux extrasynaptiques. Remarquablement, nos résultats montrent que l'augmentation de Ca2+ induite par ChR2 et la libération ultérieure de glutamate sont amplifiées par l'activation autocrine induite par l'ATP/ADP des récepteurs P2Y1 sur les astrocytes. Ainsi, l'excitation neuronale est favorisée par une action synergique de la signalisation glutamatergique et purinergique autocrine dans les astrocytes. Ce nouveau mécanisme peut être particulièrement pertinent pour les conditions pathologiques dans lesquelles la concentration extracellulaire d'ATP est augmentée et agit comme un signal de danger majeur. Dans la seconde partie de ma thèse, nous rapportons que la photostimulation sélective des astrocytes ChR2 dans le gyrus denté facilite la transmission synaptique excitatrice sur les cellules granulaires via l'activation des NMDR pré-synaptiques contenant GluN2B. De plus, nous avons découvert que l'élévation intracellulaire du Ca2+ induite par l'ATP et dérivée de l'ATP contrôlait étroitement la libération du glutamate par les astrocytes au cours de la photostimulation des astrocytes. Nos résultats fournissent des preuves d'une relation étroite entre l'ATP dérivé d'astrocyte et le glutamate. / Astrocyte-derived gliotransmitters glutamate and ATP modulate neuronal activity. It remains unclear, however, how astrocytes control the release and coordinate the actions of these gliotransmitters. In the first part of my thesis, using transgenic expression of the light-sensitive channelrhodopsin 2 (ChR2) in astrocytes, we observed that photostimulation reliably increases action potential firing of hippocampal pyramidal neurons. This excitation relies primarily on a Ca2+-dependent glutamate release by astrocytes that activates neuronal extra-synaptic NMDRs. Remarkably, our results show that ChR2-induced Ca2+ increase and subsequent glutamate release are amplified by ATP/ADP-mediated autocrine activation of P2Y1 receptors on astrocytes. Thus, neuronal excitation is promoted by a synergistic action of glutamatergic and autocrine purinergic signaling in astrocytes. This new mechanism may be particularly relevant for pathological conditions in which ATP extracellular concentration is increased and acts as a major danger signal. In the second part of my thesis, we report that selective photostimulation ChR2 positive astrocytes in dentate gyrus facilitates excitatory synaptic transmission onto granule cells via the activation of pre-synaptic GluN2B-containing NMDRs. Moreover, we discovered that astrocyte-derived ATP-mediated intracellular Ca2+ elevation tightly controls glutamate release from astrocytes during astrocyte photostimulation. Our results provide evidence for a close relationship between astrocytic-derived ATP and glutamate.

Yau Ho Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 131-144). / Abstracts in English and Chinese. / DECLARATION --- p.I / ACKNOWLEDGEMENTS --- p.II / ENGLISH ABSTRACT --- p.III / CHINESE ABSTRACT --- p.VI / Chapter Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- Oxidative Stress --- p.1 / Chapter 1.1.1 --- Historical Background of reactive oxygen/nitrogen species --- p.1 / Chapter 1.1.2 --- What is Oxidative Stress? --- p.3 / Chapter 1.1.3 --- Reactive Oxygen Species (ROS) --- p.4 / Chapter 1.1.3.1 --- Superoxide anion (02-) --- p.4 / Chapter 1.1.3.2 --- Hydrogen peroxide (H202) --- p.5 / Chapter 1.1.3.3 --- Hydroxyl radical --- p.6 / Chapter 1.1.3.4 --- Nitric oxide (NO) --- p.7 / Chapter 1.2 --- Cardiovascular System --- p.8 / Chapter 1.2.1 --- Enzymatic and Non-enzymatic Sources of ROS in Cardiovascular System --- p.8 / Chapter 1.2.1.1 --- NADPH oxidase --- p.8 / Chapter 1.2.1.2 --- Hypoxanthine-Xanthine oxidase (HX-XO) --- p.9 / Chapter 1.2.1.3 --- Nitric oxide synthase (NOS) --- p.10 / Chapter 1.2.1.4 --- Mitochondrial electron transport chain (ETC) --- p.11 / Chapter 1.2.1.5 --- Cyclooxygenase --- p.11 / Chapter 1.2.1.6 --- Lipoxygenae --- p.12 / Chapter 1.2.1.7 --- Endoplasmic reticulum --- p.12 / Chapter 1.2.2 --- ROS/RNS Scavenging Systems --- p.13 / Chapter 1.2.2.1 --- Superoxide dismutase (SOD) --- p.13 / Chapter 1.2.2.2 --- Catalase --- p.14 / Chapter 1.2.2.3 --- Glutathione peroxidase --- p.15 / Chapter 1.2.2.4 --- Non-enzymatic antioxidants --- p.15 / Chapter 1.2.3 --- Factors that stimulate ROS production in cardiovascular system --- p.18 / Chapter 1.2.3.1 --- Oxygen tension --- p.18 / Chapter 1.2.3.2 --- "Flow, Shear, and Stretch as an initial stimulus for endothelial oxidant signalling" --- p.18 / Chapter 1.2.3.3 --- Activation of rennin-angiotensin system promote oxidative stress in cardiovascular system --- p.19 / Chapter 1.2.3.4 --- Regulation of vascular ROS production by vasoactive substances --- p.19 / Chapter 1.2.4 --- Regulation of vascular tone in Cardiovascular System by ROS/RNS --- p.20 / Chapter 1.2.4.1 --- Regulation of vascular tone --- p.20 / Chapter 1.2.5 --- Pathophysiological Effects of ROS --- p.23 / Chapter 1.2.5.1 --- Cellular injury by lipid peroxidation --- p.23 / Chapter 1.2.5.2 --- Role of ROS in immune defence --- p.23 / Chapter 1.2.5.3 --- Redox regulation of cell adhesion --- p.24 / Chapter 1.2.6 --- Evidences from Clinical Studies of Oxidative Stress-Related Vascular Diseases --- p.25 / Chapter 1.2.6.1 --- Hyperlipidaemia --- p.25 / Chapter 1.2.6.2 --- Hypertension --- p.25 / Chapter 1.2.6.3 --- Chronic heart failure (CHF) --- p.26 / Chapter 1.2.6.4 --- Chronic renal failure (CRF) --- p.26 / Chapter 1.2.6.5 --- Atherosclerosis --- p.27 / Chapter 1.2.6.6 --- Ischemia/reperfusion (I/R) injury --- p.27 / Chapter 1.2.7 --- Role of Vascular Endothelium in Oxidative Stress --- p.29 / Chapter 1.2.8 --- Role of Ca in oxidative stress in cardiovascular system --- p.29 / Chapter 1.2.8.1 --- Calcium Signaling in Vascular Endothelial Cells --- p.30 / Chapter 1.2.9 --- ROS effect on endothelial Ca2+ --- p.31 / Chapter 1.2.9.1 --- Multiple targets of ROS on intracellular Ca2+ mobilization --- p.32 / Chapter 1.2.9.2 --- Reports of H202-induced Ca2+ release in various cell types --- p.33 / Chapter 1.2.9.3 --- Reported effects of H202 on agonist-induced Ca2+ signal --- p.34 / Chapter 1.2.9.4 --- Differences between macrovessels and microvessels --- p.34 / Chapter 1.3 --- TRP Channel --- p.41 / Chapter 1.3.1 --- Discovery of Drosophila TRP --- p.41 / Chapter 1.3.2 --- Mammalian TRP subfamily --- p.41 / Chapter 1.3.3 --- General topology of TRP channel --- p.42 / Chapter 1.3.4 --- Interactions of oxidative stress with TRP channels --- p.44 / Chapter 1.3.5 --- The role of TRPC3 and TRPC4 in oxidative stress --- p.44 / Chapter 1.3.6 --- TRPM subfamily --- p.44 / Chapter 1.3.6.1 --- Expression of TRPM2 --- p.45 / Chapter 1.3.6.2 --- Dual Role of TRPM´2ؤChannel and Enzyme --- p.45 / Chapter 1.3.6.3 --- Regulatory mechanisms of TRPM2 --- p.46 / Chapter 1.3.6.3.1 --- ADP-ribose (ADPR) directly regulating --- p.46 / Chapter 1.3.6.3.2 --- NAD regulating --- p.46 / Chapter 1.3.6.3.3 --- Oxidative stress regulating independent of ADPR or NAD --- p.47 / Chapter 1.4 --- Cell Death Induced by Oxidative Stress --- p.48 / Chapter 1.4.1 --- Redox status as a factor to determine cell death --- p.48 / Chapter 1.4.2 --- Role of TRPM2 in oxidative stress-induced cell death --- p.48 / Chapter 1.5 --- Aims of the Study --- p.49 / Chapter Chapter 2: --- Materials and Methods --- p.50 / Chapter 2.1 --- Functional Characterization of TRPM2 by Antisense Technique --- p.50 / Chapter 2.1.1 --- Restriction Enzyme Digestion --- p.50 / Chapter 2.1.2 --- Purification of Released Inserts and Cut pcDNA3 Vectors --- p.51 / Chapter 2.1.3 --- "Ligation of TRPM2 Genes into Mammalian Vector, pcDNA3" --- p.52 / Chapter 2.1.4 --- Transformation for the Desired Clones --- p.52 / Chapter 2.1.5 --- Plasmid DNA Preparation for Transfection --- p.53 / Chapter 2.1.6 --- Confirmation of the Clones --- p.53 / Chapter 2.1.6.1 --- Restriction Enzymes Strategy --- p.53 / Chapter 2.1.6.2 --- Polymerase Chain Reaction (PCR) Check --- p.54 / Chapter 2.1.6.3 --- Automated Sequencing --- p.55 / Chapter 2.2 --- Establishing Stable Cell Lines --- p.56 / Chapter 2.2.1 --- Cell Culture --- p.56 / Chapter 2.2.2 --- Geneticin Selection --- p.57 / Chapter 2.3 --- Expression of TRPM2 in Transfected and non-Transfected H5V Cells --- p.57 / Chapter 2.3.1 --- Protein Sample Preparation --- p.57 / Chapter 2.3.2 --- Western Blot Analysis --- p.58 / Chapter 2.3.3 --- Protein Expression Analysis --- p.59 / Chapter 2.4 --- "Immunolocalization of TRPM2 in Human Heart, Cerebral Artery, Renal, Hippocampus and Liver" --- p.59 / Chapter 2.4.1 --- Paraffin Section Preparation --- p.59 / Chapter 2.4.2 --- Immunohistochemistry --- p.60 / Chapter 2.5 --- [Ca2+ ］i Measurement in Confocal Microscopy --- p.62 / Chapter 2.5.1 --- Cytosolic Ca2+ measurement --- p.62 / Chapter 2.5.2 --- Measuring the Ca2+ in the Internal Calcium Stores --- p.63 / Chapter 2.5.3 --- Data Analysis --- p.64 / Chapter 2.6 --- Examining Cell Death Induced by H2O2 by DAPI Staining --- p.65 / Chapter 2.6.1 --- DAPI Staining --- p.65 / Chapter Chapter 3: --- Results --- p.66 / Chapter 3.1 --- Superoxide Anion-Induced [Ca 2+]i rise in H5V Mouse Heart Microvessel Endothelial Cells --- p.66 / Chapter 3.1.1 --- Superoxide Anion-induced [Ca2+ ]i Rise --- p.66 / Chapter 3.1.2 --- Effect of Catalase on the Superoxide Anion-induced [Ca2+]i］］ Rise --- p.66 / Chapter 3.1.3 --- IP3R inhibitor Inhibits Superoxide anion-induced [Ca 2+]i Rise --- p.67 / Chapter 3.1.4 --- Effect of Phospholipase A2 Inhibitor on Superoxide anion- induced [Ca2+]i Rise --- p.67 / Chapter 3.1.5 --- Effect of Hydroxyl Radical Scavenger on Superoxide Anion- induced [Ca2+]i Rise --- p.68 / Chapter 3.2 --- Hydrogen Peroxide-induced Ca2+ Entry in Mouse Heart Microvessel Endothelial Cells --- p.74 / Chapter 3.2.1 --- Hydrogen Peroxide Induces [Ca2 +]i rise in H5V Mouse Heart Microvessel Endothelial Cells --- p.74 / Chapter 3.2.2 --- Hydrogen Peroxide Induces [Ca 2+]i rise in two phases (Rapid and Slow response) --- p.74 / Chapter 3.2.3 --- Hydrogen Peroxide Induces [Ca 2+]i rise in a Extracellular Ca + Concentration Dependent Manner --- p.77 / Chapter 3.3 --- Hydrogen Peroxide Reduces Agonist-induced [Ca2+]i rise --- p.79 / Chapter 3.3.1 --- Hydrogen Peroxide Reduces ATP-induced [Ca2+ ]i rise in a H2O2 Concentration Dependent Manner --- p.79 / Chapter 3.3.2 --- Hydrogen Peroxide Reduces ATP-induced [Ca 2+]i rise in a H2O2 Incubation Time Dependent Manner --- p.79 / Chapter 3.3.3 --- Hydrogen Peroxide Reduces the ATP-induced Intracellular Ca2+ Release --- p.80 / Chapter 3.3.4 --- XeC Inhibited H202-induced [Ca2+]i rise --- p.80 / Chapter 3.3.5 --- Hydrogen Peroxide Partially Depletes Internal Ca2+ Stores --- p.81 / Chapter 3.4 --- Dissecting Signal Transduction Pathways in H202-induced [Ca2+]i rise --- p.82 / Chapter 3.4.1 --- Effect of Phospholipase C Inhibitor on H202-induced [Ca2 +]i rise --- p.82 / Chapter 3.4.2 --- Effect of Phospholipase A2 Inhibitor on H202-induced [Ca 2+]i rise --- p.83 / Chapter 3.4.3 --- Effect of hydroxyl radical scavenger on H2O2-induced [Ca 2+]i rise --- p.83 / Chapter 3.5 --- Functional Role of TRPM2 Channel in H202-induced [Ca2+]i Rise in H5V Cells --- p.92 / Chapter 3.5.1 --- Expression of TRPM2 and the Effect of TRPM2 Antisense Construct on TRPM2 Protein Expression --- p.92 / Chapter 3.5.2 --- Effect of Antisense TRPM2 on H202-induced Ca2+ Entry --- p.94 / Chapter 3.6 --- H202-induced Cell Death --- p.101 / Chapter 3.7 --- Expression Pattern of TRPM2 Channel in Vascular System --- p.104 / Chapter 3.7.1 --- Immunolocalization of TRPM2 in Human Cerebral Arteries --- p.104 / Chapter 3.7.2 --- Immunolocalization of TRPM2 in Human Cardiac Muscles --- p.105 / Chapter 3.7.3 --- Immunolocalization of TRPM2 in Human Kidney --- p.105 / Chapter Chapter 4: --- Discussion --- p.113 / Chapter 4.1 --- Oxidative modification of Ca2+ homeostasis --- p.113 / Chapter 4.2 --- Pathophysiological effects of ROS on endothelium --- p.113 / Chapter 4.3 --- Effects of ROS on microvascular endothelial Ca2+ reported by other investigators --- p.115 / Chapter 4.4 --- Studies of the effect of HX-XO on cytosolic [Ca2+]i --- p.116 / Chapter 4.4.1 --- Role of 0´2Ø- and H202 in HX-XO-induced [Ca2+]i elevation --- p.116 / Chapter 4.4.2 --- IP3R involvement in HX-XO-evoked Ca + movements in H5V cells --- p.118 / Chapter 4.4.3 --- PLA2 involvement in HX-XO experiment --- p.119 / Chapter 4.5 --- Studies of the effect of direct H202 application on cytosolic [Ca2+]i --- p.120 / Chapter 4.5.1 --- Hydrogen Peroxide Induced [Ca2 +]i rise in a Extracellular Ca2 + Concentration Dependent Manner --- p.120 / Chapter 4.5.2 --- Hydrogen Peroxide Induced [Ca 2+]i rise in two phases (Rapid and Slow response) --- p.121 / Chapter 4.6 --- Effect of H202 on ATP-induced Ca2+ response --- p.121 / Chapter 4.6.1 --- H202 inhibited ATP-induced Ca2+ release in a concentration and time dependent manner --- p.121 / Chapter 4.6.2 --- IP3R involvement and store depletion in H202 experiment --- p.123 / Chapter 4.7 --- Dissecting Signal Transduction Pathways in H202-induced [Ca2+]i rise --- p.124 / Chapter 4.7.1 --- PLC involvement in H2O2 experiment --- p.124 / Chapter 4.7.2 --- PLA2 involvement in H2O2 experiment --- p.125 / Chapter 4.7.3 --- Hydroxyl radical did not involve in H2O2 experiment --- p.125 / Chapter 4.8 --- Functional Studies of TRPM2 --- p.127 / Chapter 4.8.1 --- Expression of TRPM2 in H5V on protein level --- p.127 / Chapter 4.8.2 --- TRPM2 involvement in the Ca2+ signalling in response to H2O2 in H5V cells --- p.127 / Chapter 4.9 --- H202 concentration in my projec´tؤphysiological or pathological? --- p.128 / Chapter 4.10. --- H20´2ؤTRPM´2ؤCell death --- p.129 / Chapter 4.11 --- Expression of TRPM2 in human blood vessels and other tissues --- p.130 / References --- p.131

Optogenetics has emerged as a branch of biotechnology that combines genetic engineering with optics to observe intracellular changes as well as control cellular function. Despite recent progress, there still remains the need for an optogenetic tool that can specifically control Ca2+. Such a tool would greatly facilitate the study of highly Ca2+-dependent cellular processes that are regulated both spatially and temporally. Ca2+ signaling regulates many cellular processes in both healthy and diseased cells. The ability to modulate the shape, duration, and amplitude of Ca2+ signaling is important for elucidating mechanisms by which endogenous Ca2+ concentrations are maintained. In this thesis, we used optogenetic approaches to explore a number of strategies to control Ca2+ influx through store-operated Ca2+ entry (SOCE) mediated by Stim1 and Orai1. To better study Ca2+ signaling in live cells, protein-based biosensors can be developed to monitor intracellular Ca2+ changes. To aid in this, we developed a computational modeling tool called FPMOD to improve both new and existing biosensor designs. Although FPMOD was initially intended for evaluating biosensor designs, other research groups have since used it to construct models of other proteins to answer questions related to protein conformation. We next studied the modulation of SOCE by using drug-inducible fusion proteins to study the regulation of Stim1 puncta formation. Interestingly, recruiting a Ca2+-buffering protein to Stim1 led to puncta formation, a previously unknown means of inducing puncta. These results suggest Stim1 may additionally be regulated by cytoplasmic Ca2+ levels. Finally, we developed LOVS1K, an optogenetic tool to directly activate Orai1 channels and specifically control Ca2+ influx. Photo-sensitive LOVS1K was used to generate both local Ca2+ influx at the membrane and global cytoplasmic Ca2+ signals. As proof of concept, LOVS1K was further used to modulate engineered Ca2+-dependent proteins. Ca2+ is a remarkably versatile intracellular messenger. The combination of high spatiotemporal control of irradiation and the ability of LOVS1K to generate both local and global Ca2+ changes provides a promising platform to study cellular processes that are highly dependent on different Ca2+ signals. Together, biosensors and engineered Ca2+-modulating tools can be used to study the many different aspects of Ca2+ signaling and controllably manipulate endogenous Ca2+ signaling pathways.

Optogenetics has emerged as a branch of biotechnology that combines genetic engineering with optics to observe intracellular changes as well as control cellular function. Despite recent progress, there still remains the need for an optogenetic tool that can specifically control Ca2+. Such a tool would greatly facilitate the study of highly Ca2+-dependent cellular processes that are regulated both spatially and temporally. Ca2+ signaling regulates many cellular processes in both healthy and diseased cells. The ability to modulate the shape, duration, and amplitude of Ca2+ signaling is important for elucidating mechanisms by which endogenous Ca2+ concentrations are maintained. In this thesis, we used optogenetic approaches to explore a number of strategies to control Ca2+ influx through store-operated Ca2+ entry (SOCE) mediated by Stim1 and Orai1. To better study Ca2+ signaling in live cells, protein-based biosensors can be developed to monitor intracellular Ca2+ changes. To aid in this, we developed a computational modeling tool called FPMOD to improve both new and existing biosensor designs. Although FPMOD was initially intended for evaluating biosensor designs, other research groups have since used it to construct models of other proteins to answer questions related to protein conformation. We next studied the modulation of SOCE by using drug-inducible fusion proteins to study the regulation of Stim1 puncta formation. Interestingly, recruiting a Ca2+-buffering protein to Stim1 led to puncta formation, a previously unknown means of inducing puncta. These results suggest Stim1 may additionally be regulated by cytoplasmic Ca2+ levels. Finally, we developed LOVS1K, an optogenetic tool to directly activate Orai1 channels and specifically control Ca2+ influx. Photo-sensitive LOVS1K was used to generate both local Ca2+ influx at the membrane and global cytoplasmic Ca2+ signals. As proof of concept, LOVS1K was further used to modulate engineered Ca2+-dependent proteins. Ca2+ is a remarkably versatile intracellular messenger. The combination of high spatiotemporal control of irradiation and the ability of LOVS1K to generate both local and global Ca2+ changes provides a promising platform to study cellular processes that are highly dependent on different Ca2+ signals. Together, biosensors and engineered Ca2+-modulating tools can be used to study the many different aspects of Ca2+ signaling and controllably manipulate endogenous Ca2+ signaling pathways.

Cardiac myofibroblasts participate in post-myocardial infarct (MI) wound healing, infarct scar formation, and remodeling of the ventricle remote to the site of infarction. The role of intracellular calcium handling in cardiac myofibroblasts as a modulator of cellular motility, contractile responses, and proliferation is largely unexplored. We have investigated the role of sodium calcium exchange (Na Ca exchange or NCX1.1) and non-selective cation channels (NSCCs) in regulation of myofibroblast function using a pharmacological inhibitor approach in vitro. Primary myofibroblasts were stimulated with PDGF-BB and cellular chemotaxis, contraction and proliferative responses were characterized using standard bioassays (Costar Transwell apparatuses, pre-formed collagen type I gel deformation assays, and 3H-thymidine incorporation). Stimulated cellular responses were compared to those in the presence of AG1296 (PDGFβR inhibitor), KB-R7943 (NCX inhibitor), gadolinium, nifedipine or ML-7. Immunofluorescence was used to determine localized expression of αSMA, SMemb, NCX1.1, and Cav1.2a in cultured myofibroblasts. Motility of myofibroblasts in the presence of PDGF-BB was blocked with AG1296 treatment. Immunoblotting and immunocytochemical studies revealed expression of NCX1.1 in fibroblasts and myofibroblasts. Motility (in the presence of either PDGF-BB or CT-1), contraction (in the presence of either PDGF-BB or TGFβ1), and proliferation (in the presence of PDGF-BB) were sensitive to KB-R7943 treatment of cells (7.5 and 10 μM for motility, 5 and 10 μM for contractility, and 10 μM for proliferation). Proliferation (in the presence of PDGF-BB), and contractility (in the presence of either PDGF-BB or TGFβ1) but not motility (in the presence of PDGF-BB) are sensitive to nifedipine treatment, while gadolinium treatment was associated only with decreased motility of cells (in the presence of either PDGF-BB, CT-1, or LoFGF-2). We found that ML-7 treatment inhibited cellular chemotaxis, and contraction. Thus cellular chemotaxis, contractile, and proliferation responses were sensitive to different pharmacologic treatment. Regulation of transplasmalemmal calcium movements may be important in cytokine and growth factor receptor-mediated cardiac myofibroblast motility, contractility, and proliferation. Furthermore, our results support the hypothesis that activation of specific calcium transport proteins is an important determinant of physiologic responses. / May 2006

Cardiac myofibroblasts participate in post-myocardial infarct (MI) wound healing, infarct scar formation, and remodeling of the ventricle remote to the site of infarction. The role of intracellular calcium handling in cardiac myofibroblasts as a modulator of cellular motility, contractile responses, and proliferation is largely unexplored. We have investigated the role of sodium calcium exchange (Na Ca exchange or NCX1.1) and non-selective cation channels (NSCCs) in regulation of myofibroblast function using a pharmacological inhibitor approach in vitro. Primary myofibroblasts were stimulated with PDGF-BB and cellular chemotaxis, contraction and proliferative responses were characterized using standard bioassays (Costar Transwell apparatuses, pre-formed collagen type I gel deformation assays, and 3H-thymidine incorporation). Stimulated cellular responses were compared to those in the presence of AG1296 (PDGFβR inhibitor), KB-R7943 (NCX inhibitor), gadolinium, nifedipine or ML-7. Immunofluorescence was used to determine localized expression of αSMA, SMemb, NCX1.1, and Cav1.2a in cultured myofibroblasts. Motility of myofibroblasts in the presence of PDGF-BB was blocked with AG1296 treatment. Immunoblotting and immunocytochemical studies revealed expression of NCX1.1 in fibroblasts and myofibroblasts. Motility (in the presence of either PDGF-BB or CT-1), contraction (in the presence of either PDGF-BB or TGFβ1), and proliferation (in the presence of PDGF-BB) were sensitive to KB-R7943 treatment of cells (7.5 and 10 μM for motility, 5 and 10 μM for contractility, and 10 μM for proliferation). Proliferation (in the presence of PDGF-BB), and contractility (in the presence of either PDGF-BB or TGFβ1) but not motility (in the presence of PDGF-BB) are sensitive to nifedipine treatment, while gadolinium treatment was associated only with decreased motility of cells (in the presence of either PDGF-BB, CT-1, or LoFGF-2). We found that ML-7 treatment inhibited cellular chemotaxis, and contraction. Thus cellular chemotaxis, contractile, and proliferation responses were sensitive to different pharmacologic treatment. Regulation of transplasmalemmal calcium movements may be important in cytokine and growth factor receptor-mediated cardiac myofibroblast motility, contractility, and proliferation. Furthermore, our results support the hypothesis that activation of specific calcium transport proteins is an important determinant of physiologic responses.

Cardiac myofibroblasts participate in post-myocardial infarct (MI) wound healing, infarct scar formation, and remodeling of the ventricle remote to the site of infarction. The role of intracellular calcium handling in cardiac myofibroblasts as a modulator of cellular motility, contractile responses, and proliferation is largely unexplored. We have investigated the role of sodium calcium exchange (Na Ca exchange or NCX1.1) and non-selective cation channels (NSCCs) in regulation of myofibroblast function using a pharmacological inhibitor approach in vitro. Primary myofibroblasts were stimulated with PDGF-BB and cellular chemotaxis, contraction and proliferative responses were characterized using standard bioassays (Costar Transwell apparatuses, pre-formed collagen type I gel deformation assays, and 3H-thymidine incorporation). Stimulated cellular responses were compared to those in the presence of AG1296 (PDGFβR inhibitor), KB-R7943 (NCX inhibitor), gadolinium, nifedipine or ML-7. Immunofluorescence was used to determine localized expression of αSMA, SMemb, NCX1.1, and Cav1.2a in cultured myofibroblasts. Motility of myofibroblasts in the presence of PDGF-BB was blocked with AG1296 treatment. Immunoblotting and immunocytochemical studies revealed expression of NCX1.1 in fibroblasts and myofibroblasts. Motility (in the presence of either PDGF-BB or CT-1), contraction (in the presence of either PDGF-BB or TGFβ1), and proliferation (in the presence of PDGF-BB) were sensitive to KB-R7943 treatment of cells (7.5 and 10 μM for motility, 5 and 10 μM for contractility, and 10 μM for proliferation). Proliferation (in the presence of PDGF-BB), and contractility (in the presence of either PDGF-BB or TGFβ1) but not motility (in the presence of PDGF-BB) are sensitive to nifedipine treatment, while gadolinium treatment was associated only with decreased motility of cells (in the presence of either PDGF-BB, CT-1, or LoFGF-2). We found that ML-7 treatment inhibited cellular chemotaxis, and contraction. Thus cellular chemotaxis, contractile, and proliferation responses were sensitive to different pharmacologic treatment. Regulation of transplasmalemmal calcium movements may be important in cytokine and growth factor receptor-mediated cardiac myofibroblast motility, contractility, and proliferation. Furthermore, our results support the hypothesis that activation of specific calcium transport proteins is an important determinant of physiologic responses.

Ionized calcium (Ca2+), acting as an intracellular messenger, controls numerous biological processes that are essential for life. However, it is also able to convey signals that result in cell death. The fidelity of Ca2+ as a universal second messenger therefore depends on mechanisms that specifically and dynamically regulate its levels within a cell, as well as maintain resting intracellular Ca2+ concentration ([Ca2+]i) very low. One such mechanism for Ca2+ signaling and homeostasis is the plasma membrane Ca2+-ATPase (PMCA), which is a primary active Ca2+ transporter that translocates Ca2+ from a low intracellular Ca2+ environment to a high extracellular environment. There are four mammalian PMCA isoforms (PMCA1−4), which are differentially expressed depending on tissue or cell type. PMCA isoforms possess different sensitivities to biochemical regulation of Ca2+ efflux activity and are also able to subtly alter the dynamics of Ca2+ signals. These properties suggest that the PMCA is not merely a trivial mechanism for Ca2+ extrusion but is influential in contributing to the Ca2+ signaling requirements and unique physiology of different cells. The indispensable nature of Ca2+ signaling in organs such as the brain, heart and skeletal muscle has been the studied extensively but little is known about the roles and regulation of Ca2+ in the mammary gland. This is despite the fact that the mammary gland is a site of extensive Ca2+ flux during lactation. However, cumulating evidence indicates that upregulation of PMCA2 expression in the mammary gland is a major mechanism for milk Ca2+ enrichment. Therefore, the PMCA is likely to be an important mediator of bulk Ca2+ homeostasis in the mammary gland. Studies in other model systems also suggest that PMCAs may regulate other cellular processes such as cell proliferation, differentiation and apoptosis that are required for normal mammary gland physiology. These basic cellular processes are also disturbed in breast cancer and hence deregulation of PMCA expression in the mammary gland may have pathophysiological consequences. Previous studies show that PMCA1 expression is greater in tumorigenic MCF-7 and MDA-MB-231 human breast cancer cells compared to non-tumorigenic MCF-10A human breast epithelial cells. Furthermore, the expression of PMCA1b and PMCA4b is lower in human skin and lung fibroblasts neoplastically transformed by simian virus 40, compared to non-transformed counterparts. It is therefore hypothesized that regulation of PMCA isoform expression is disrupted in breast cancer and that inhibition of PMCA expression in an in vitro model of breast cancer has important effects in modulating intracellular Ca2+ homeostasis, cell proliferation, differentiation and apoptosis. This thesis describes the use of real time RT-PCR to compare PMCA isoform mRNA expression in tumorigenic and non-tumorigenic mammary gland epithelial cells. It demonstrates that particular breast cancer cell lines overexpress PMCA2, an isoform with restricted tissue distribution and which is present in abundant amounts in the lactating rat mammary gland. Thus, some breast cancers may be characterized by the overexpression of Ca2+ transporters that are normally upregulated during the physiological course of lactation. The pathophysiological significance of PMCA2 overexpression in breast cancer is uncertain and future investigations should look at whether levels of PMCA isoform expression correlate with malignancy, prognosis or survival. To address the second hypothesis of this thesis, a stable MCF-7 Tet-off human breast cancer cell line able to conditionally express PMCA antisense was generated. This strategy was necessary due to the current lack of specific pharmacological inhibitors of the PMCA. This thesis shows that PMCA antisense expression significantly inhibits PMCA protein expression, while subtly affecting PMCA-mediated Ca2+ efflux without causing cell death. However, it also reveals that inhibition of PMCA expression has major effects in mediating cell proliferation and cell cycle progression. Moderate changes in PMCA expression and PMCA-mediated Ca2+ transport result in dramatic consequences in MCF-7 cell proliferation. These studies not only support the supposition that modulation of Ca2+ signaling is a viable therapeutic approach for breast cancer but also suggest that PMCAs are possible drug targets. Alternatively, inhibitors of the PMCA may act as adjuvants to augment the efficacy of other anti-neoplastic agents like tamoxifen that have been shown to modulate Ca2+ signaling. Since the discovery of a new family of primary active Ca2+ transporters, which are related to PMCAs, the opportunities in this field of research are very promising.

Ionized calcium (Ca2+), acting as an intracellular messenger, controls numerous biological processes that are essential for life. However, it is also able to convey signals that result in cell death. The fidelity of Ca2+ as a universal second messenger therefore depends on mechanisms that specifically and dynamically regulate its levels within a cell, as well as maintain resting intracellular Ca2+ concentration ([Ca2+]i) very low. One such mechanism for Ca2+ signaling and homeostasis is the plasma membrane Ca2+-ATPase (PMCA), which is a primary active Ca2+ transporter that translocates Ca2+ from a low intracellular Ca2+ environment to a high extracellular environment. There are four mammalian PMCA isoforms (PMCA1−4), which are differentially expressed depending on tissue or cell type. PMCA isoforms possess different sensitivities to biochemical regulation of Ca2+ efflux activity and are also able to subtly alter the dynamics of Ca2+ signals. These properties suggest that the PMCA is not merely a trivial mechanism for Ca2+ extrusion but is influential in contributing to the Ca2+ signaling requirements and unique physiology of different cells. The indispensable nature of Ca2+ signaling in organs such as the brain, heart and skeletal muscle has been the studied extensively but little is known about the roles and regulation of Ca2+ in the mammary gland. This is despite the fact that the mammary gland is a site of extensive Ca2+ flux during lactation. However, cumulating evidence indicates that upregulation of PMCA2 expression in the mammary gland is a major mechanism for milk Ca2+ enrichment. Therefore, the PMCA is likely to be an important mediator of bulk Ca2+ homeostasis in the mammary gland. Studies in other model systems also suggest that PMCAs may regulate other cellular processes such as cell proliferation, differentiation and apoptosis that are required for normal mammary gland physiology. These basic cellular processes are also disturbed in breast cancer and hence deregulation of PMCA expression in the mammary gland may have pathophysiological consequences. Previous studies show that PMCA1 expression is greater in tumorigenic MCF-7 and MDA-MB-231 human breast cancer cells compared to non-tumorigenic MCF-10A human breast epithelial cells. Furthermore, the expression of PMCA1b and PMCA4b is lower in human skin and lung fibroblasts neoplastically transformed by simian virus 40, compared to non-transformed counterparts. It is therefore hypothesized that regulation of PMCA isoform expression is disrupted in breast cancer and that inhibition of PMCA expression in an in vitro model of breast cancer has important effects in modulating intracellular Ca2+ homeostasis, cell proliferation, differentiation and apoptosis. This thesis describes the use of real time RT-PCR to compare PMCA isoform mRNA expression in tumorigenic and non-tumorigenic mammary gland epithelial cells. It demonstrates that particular breast cancer cell lines overexpress PMCA2, an isoform with restricted tissue distribution and which is present in abundant amounts in the lactating rat mammary gland. Thus, some breast cancers may be characterized by the overexpression of Ca2+ transporters that are normally upregulated during the physiological course of lactation. The pathophysiological significance of PMCA2 overexpression in breast cancer is uncertain and future investigations should look at whether levels of PMCA isoform expression correlate with malignancy, prognosis or survival. To address the second hypothesis of this thesis, a stable MCF-7 Tet-off human breast cancer cell line able to conditionally express PMCA antisense was generated. This strategy was necessary due to the current lack of specific pharmacological inhibitors of the PMCA. This thesis shows that PMCA antisense expression significantly inhibits PMCA protein expression, while subtly affecting PMCA-mediated Ca2+ efflux without causing cell death. However, it also reveals that inhibition of PMCA expression has major effects in mediating cell proliferation and cell cycle progression. Moderate changes in PMCA expression and PMCA-mediated Ca2+ transport result in dramatic consequences in MCF-7 cell proliferation. These studies not only support the supposition that modulation of Ca2+ signaling is a viable therapeutic approach for breast cancer but also suggest that PMCAs are possible drug targets. Alternatively, inhibitors of the PMCA may act as adjuvants to augment the efficacy of other anti-neoplastic agents like tamoxifen that have been shown to modulate Ca2+ signaling. Since the discovery of a new family of primary active Ca2+ transporters, which are related to PMCAs, the opportunities in this field of research are very promising.