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GPCR Regulation of ATP Efflux from Astrocytes

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G PROTEIN-COUPLED RECEPTOR REGULATION OF ATP RELEASE FROM ASTROCYTES by ANDREW EDWARD BLUM Thesis advisor: Dr. George R. Dubyak Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Department of Physiology and Biophysics CASE WESTERN RESERVE UNIVERSITY May, 2010 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of _____________________________________________________ candidate for the ______________________degree *. (signed)_______________________________________________ (chair of the committee) ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ (date) _______________________ *We also certify that written approval has been obtained for any proprietary material contained therein. Dedication I am greatly indebted to my thesis advisor Dr. George Dubyak. Without his support, patience, and advice this work would not have been possible. I would also like to acknowledge Dr. Robert Schleimer and Dr. Walter Hubbard for their encouragement as I began my research career. My current and past thesis committee members Dr. Matthias Buck, Dr. Cathleen Carlin, Dr. Edward Greenfield, Dr. Ulrich Hopfer, Dr. Gary Landreth, Dr. Corey Smith, Dr. Jerry Silver have provided invaluable guidance and advice for which I am very grateful. A special thanks to all of the past and present members of the Dubyak lab who made the lab a home away from home. My gratitude extends to my friends and family members for their love and support. Finally, I would like to dedicate this thesis to my parents, David and Natalie Blum. 1 Table of Contents List of Tables 4 List of Figures 5 List of Abbreviations 8 Abstract 10 1. Introduction 1.1 ATP as an Extracellular signal 13 1.1.1 P2 Receptors and Extracellular ATP 13 1.1.2 Metabolism of Extracellular ATP 14 1.1.3 Compartmentalization of ATP release and 15 Issues of Experimental Measurement 1.2 Functions of ATP signaling in Astrocytes 16 1.2.1 Ca2+ wave Propagation 18 1.2.2 Response to Metabolic Changes and Ischemia 19 1.2.3 Cell Volume Homeostasis 20 1.3 G protein-Coupled Receptors and ATP Release 22 1.3.1 Protease-Activated Receptor (PAR) 23 1.3.2 Lysophosphatidic Acid Receptors (LPAR) 26 1.3.3 Muscarinic Receptors 28 1.3.4 Non G protein-Coupled Receptor Stimulated 28 ATP release 1.4 Pathways of ATP release 30 1.4.1 Conductive Pathways 31 1.4.1.1 Gap-junction Hemichannels 32 1.4.1.2 Maxi-Anion Channels 37 1.4.1.3 Volume-Sensitive Organic Anion Channels 40 1.4.2 Exocytosis 43 1.5 Aims of Study 45 2. Experimental Methods 59 3. Rho-Family GTPases Modulate Ca2+-Dependent ATP Release from Astrocytes ABSTRACT 75 INTRODUCTION 77 RESULTS 81 DISCUSSION 89 2 4. Extracellular Osmolarity Modulates G protein-Coupled Receptor Dependent ATP Release from 1321N1 Astrocytes ABSTRACT 116 INTRODUCTION 118 RESULTS 124 DISCUSSION 131 5. Multiple Pathways of ATP release from 1321N1 cells ABSTRACT 160 INTRODUCTION 161 RESULTS 163 DISCUSSION 165 6. Conclusions and Future Directions 184 References 200 3 Tables Table 1.1 Agonist Selectivity and Signaling Systems of the 47 P2 Nucleotide Receptors Table 1.2 Pharmacology of Candidate ATP release Channels 49 Table 4.1. Osmolarities and [NaCl] of basal salt solutions used in 141 ATP release experiments. 4 Figures Figure 1.1 Structure of adenine nucleotide. 51 Figure 1.2 Pharmacologic inhibitors of Connexin Hemichannels, 53 Pannexin Hemichannels, VSOAC, and maxi-anion channels Figure 1.3 Release of ATP to Extracellular Compartments. 55 Figure 1.4 Autocrine / Paracrine ATP mediated Ca2+ wave. 57 Figure 3.1 PAR1 mediated ATP release is sensitive to 100 BAPTA and ToxB. Figure 3.2 Rho-GTPase activity is correlated with thrombin 101 induced ATP release. Figure 3.3 Inhibition of ROCKI/II and MLCK does not affect 103 thrombin induced ATP release. Figure 3.4 Effects of ToxB and BAPTA-loading on ATP release 105 from 1321N1 astrocytes in response to LPA and Carbachol. Figure 3.5 Rho-GTPase activity is correlated with LPA- but not 107 carbachol- induced ATP release. Figure 3.6 Neither toxin treatment nor BAPTA affect 109 extracellular ATPase activity. Figure 3.7 ATP release is attenuated by brefeldin A and 111 carbenoxolone. Figure 3.8 CBX inhibition of thrombin-stimulated ATP release is 113 not correlated with changes in hemichannel activity or PAR1 signaling. Figure 4.1 Kinetics of basal and thrombin-stimulated ATP 143 release from 1321N1 astrocytes in isotonic or hypertonic media. Figure 4.2 Basal and thrombin-stimulated ATP release from 145 1321N1 astrocytes is inversely correlated with extracellular osmolarity. 5 Figure 4.3 Concentration-response relationships for 147 thrombin-stimulated ATP release and Ca2+ mobilization in isotonic, hypotonic, or hypertonic media. Figure 4.4 Concentration-response relationships for 149 thrombin-stimulated ATP release 1321N1 cells preincubated for 30 min in isotonic, hypotonic, or hypertonic media. Figure 4.5 Differential inhibitory effects of BAPTA and 151 Clostridial Toxin B on ATP release stimulated by thrombin versus strong hypotonic stress. Figure 4.6 Concentration-inhibition relationships for the effects of 153 dideoxyforskolin or carbenoxolone on ATP release by thrombin versus strong hypotonic stress. Figure 4.7 Concentration-inhibition relationships for the effects of 155 probenicid on ATP release by thrombin versus strong hypotonic stress. Figure 4.8 The maxi-anion channel inhibitor Gd3+ does not inhibit 157 thrombin-dependent or hypotonic stress induced ATP release from 1321N1 astrocytes. Figure 5.1 Transient ATP release induced by Thrombin and 170 Hypotonic Stress Contrasts with Sustained ATP release elicited by LDS. Figure 5.2 Reduced Temperature inhibits LDS, but not 172 thrombin-dependent or hypotonic stress induced ATP release from 1321N1 astrocytes. Figure 5.3 1321N1 astrocytes express pannexin 1 and 174 Connexin 43 mRNA. Figure 5.4 CBX blocks ATP release in response to thrombin, 176 hypotonic stress, or LDS. Figure 5.5 FFA blocks ATP release in response to thrombin, 178 hypotonic stress, or LDS. Figure 5.6 PB blocks ATP release in response to thrombin, 178 but not in response to hypotonic stress, or LDS. Figure 5.7 Gadolinium does not affect ATP release in 182 response to thrombin, hypotonic stress, or LDS. 6 Figure 6.1 Hypothetical scheme of the intracellular signaling 194 pathways contributing to GPCR-induced and osmotically-dependent activation of the putative volume-sensitive organic anion channel (VSOAC) pathway. Figure 6.2 Intracellular Ca2+ mobilization, but not PKC 196 activation elicits ATP release from 1321N1 astrocytes. Figure 6.3 Hypotonic stress, but not thrombin elicits 198 ATP release from HEK-293 cells. 7 ABBREVIATIONS ACh Acetylcholine ADA Adenosine deaminase ADP Adenosine-5’-diphosphate ATP Adenosine-5’-triphosphate AMP Adenosine-5’-monophosphate AMPK AMP-activated protein kinase AVD Apoptotic volume decrease BAPTA 1,2-bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid βγ-meATP Beta, gamma-methyleneATP BSS Basal saline solution cAMP Cyclic AMP or 3'-5'-cyclic adenosine monophosphate CBX Carbenoxolone CNS Central nervous system CSD Cortical spreading depression DAG Diacylglycerol DCPIB 4-(2-butyl-6,7-dichloro-2-cyclopentylindan-1-on-5-yl)oxybutyric acid ddF 1,9-dideoxyforskolin DIDS 4,4′-Diisothiocyanatostilbene-2,2′-disulfonic acid ecto-NDPK Ecto-nucleotide diphosphokinase EDG Epidermal differentiation gene FAK Focal adhesion kinase FFA Flufenamic acid GDP Guanosine diphosphate GEF Guanine nucleotide exchange factor GRK G-protein receptor kinase GPCR G protein-coupled receptor GPN Glycylphenylalanine 2-napthylamide GST Glutathione S-transferase GTP Guanosine triphosphate IP3 Inositol triphosphate LDS Low divalent solution LPA Lysophosphatidic acid LPAR Lysophosphatidic acid receptors MAPK Mitogen-activated protein kinase MMP Matrix metalloprotease NMDA N-methyl-D-aspartic acid NPP Ecto-nucleotide pyrophosphatase/phophodiesterase NPPB 5-Nitro-2-(3-phenylpropylamino)benzoic acid NTPD Ecto-nucleotide 5’-triphosphate diphosphohydrolase PAR Protease activated receptor Pi Inorganic phosphate PI3K Phosphoinositide 3-kinases PKC Protein kinase C PLA Phospholipase A 8 PLC Phospholipase C PMA Phorbol myristate acetate PPi Pyrophosphate PTX Pertussis toxin RLU Relative light unit RVD Regulatory volume decrease RVI Regulatory volume increase ROCK Rho-associated coiled-coil-forming protein kinase ROS Reactive oxygen specie RT Room temperature (20-22oC) RT-PCR Reverse transcriptase-polymerase chain reaction SITS 4-Acetamido-4′-isothiocyanato-2,2′-stilbenedisulfonic acid SNAP Soluble NSF attachment protein SNARE Soluble NSF attachment protein receptor TRAP Thrombin receptor activating peptide TRBD Rhotekin Rho-binding domain VDAC Voltage-dependant anion channel VRAC Volume-regulated anion channel VSOAC Volume-sensitive organic anion channel VSOR Volume-sensitive outwardly rectifying anion channels 9 G Protein-Coupled Receptor Regulation of ATP Release from Astrocytes Abstract By ANDREW EDWARD BLUM Extracellular nucleotides contribute to a complex autocrine / paracrine signaling network in most tissues by activating members of the P2 receptor family. While stimulated ATP release has been demonstrated in a variety of mammalian cells, how ATP is released remains poorly understood. This dissertation illustrates the ability of G protein-Coupled Receptor (GPCR) activation
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  • Activation of Pannexin 1 Channels by ATP Through P2Y Receptors and by Cytoplasmic Calcium

    Activation of Pannexin 1 Channels by ATP Through P2Y Receptors and by Cytoplasmic Calcium

    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector FEBS Letters 580 (2006) 239–244 Activation of pannexin 1 channels by ATP through P2Y receptors and by cytoplasmic calcium Silviu Locovei, Junjie Wang, Gerhard Dahl* Department of Physiology and Biophysics, University of Miami, School of Medicine, P.O. Box 016430, 1600 NW 10th Avenue, Miami, FL 33136, USA Received 28 September 2005; accepted 1 December 2005 Available online 12 December 2005 Edited by Maurice Montal in astrocytes of Cx43 null mice propagate at the same speed as Abstract The ability for long-range communication through intercellular calcium waves is inherent to cells of many tissues. in wild type cells, where Cx43 is the major gap junction protein A dual propagation mode for these waves includes passage of [15,16]. IP3 through gap junctions as well as an extracellular pathway A series of mysteries shroud the extracellular wave propaga- involving ATP. The wave can be regenerative and include tion scheme. What molecules sense a stimulus such as mechan- ATP-induced ATP release via an unknown mechanism. Here, ical stress? What links the stimulus to ATP release from the we show that pannexin 1 channels can be activated by extracel- cell? What is the ATP release mechanism in the stimulated cell? lular ATP acting through purinergic receptors of the P2Y group Does the same ATP release mechanism operate in the non- as well as by cytoplasmic calcium. Based on its properties, stimulated cells? If so, how is ATP release in the non-stimu- including ATP permeability, pannexin 1 may be involved in both lated cell activated? initiation and propagation of calcium waves.