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Pharmacological and Molecular Characterisation of P2y Receptors in Endothelial and Epithelial Cells

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Pharmacological and Molecular Characterisation of P2y Receptors in Endothelial and Epithelial Cells PHARMACOLOGICAL AND MOLECULAR CHARACTERISATION OF P2Y RECEPTORS IN ENDOTHELIAL AND EPITHELIAL CELLS VIJAY KENNETH D’SOUZA B.Pharm., MSc. A thesis submitted in partial fulfilment of the requirements of the University of Wolverhampton for the degree of Doctor of Philosophy 2008 This work or any part thereof has not previously been presented in any form to the University or to any other body whether for the purposes of assessment, publication or for any other purpose (unless otherwise indicated). Save for any express acknowledgments, references and/or bibliographies cited in the work, I confirm that the intellectual content of the work is the result of my own efforts and of no other person. The right of Vijay Kenneth D’Souza to be identified as author of this work is asserted in accordance with ss.77 and 78 of the Copyright, Designs and Patents Act 1988. At this date copyright is owned by the author. Signature: Vijay Kenneth D’Souza Date: 1st February 2008 ABSTRACT In light of the significant modulation of receptor activity previously shown by a peptide (designated L247), designed to mimic the third extracellular loop of the human P2Y2 receptor, the aim of this study was to use this peptide as an immunogen to generate and fully characterise polyclonal rabbit antibodies to the P2Y2 receptor. Other aims of this study were to characterise epithelial and endothelial cells for a thorough expression profile of P2Y receptor mRNA transcripts in order to provide a rapid screen for the molecular determinants of these receptors in these cells. These studies also aimed to confirm previously published pharmacology, thus, to set the basis for western blot studies using P2Y receptor antibodies. Bovine aortic endothelial cells that co-express P2Y1 and P2Y2 receptors; EAhy926, a human endothelial fusion cell line, that express P2Y2 receptors; and ECV304 human bladder cancer cell line, known to express P2Y2-like and P2Y11-like receptors were used in this study. The dose dependent accumulation of inositol phosphates and cAMP response to potent P2Y11 agonists and RT-PCR studies confirmed the functional expression of both P2Y2 and P2Y11 receptors in ECV304 cells. Likewise, the dose dependent accumulation of inositol phosphates in response to potent P2Y2 and P2Y6 agonists and the presence of mRNA transcripts confirmed the expression of functional P2Y2/4- like and P2Y6- like receptors in EAhy926 cells. Polyclonal antiserum raised against L247 peptide was affinity purified and the purified fractions showed strong immunoreactivity with immobilised immunogenic antigen in ELISA. In western blot analysis L247 rabbit polyclonal anti-P2Y2 antibody detected strong bands in ECV304 and EAhy926 cells. On pre-absorption with the immunogenic peptide these responses were abolished suggesting that this antibody is antigen specific. Agonist induced P2Y2 receptor desentisation studies in ECV304 cells showed that prolonged agonist incubation caused the receptor sequestration. The loss of bands caused by P2Y2 receptor desensitisation and sequestration in membrane enriched fractions of agonist incubated ECV304 cells confirmed the specificity of L247 antibody. This antibody also showed no immunoreactivity in 1321N1 human brain astrocytoma cells devoid of any P2Y receptor subtypes cells. Deglycosylation studies revealed that the P2Y2 receptors are glycosylated in ECV304 cells. i The polyclonal rabbit anti-P2Y2 receptor antibodies obtained from commercial sources produced completely different immunoreactive profiles with multiple bands even in 1321N1 cells. Furthermore, in comparison to L247 anti-P2Y2 antibody the commercial antibody showed no difference between normal and agonist incubated cells suggesting that this antibody may not be recognising the P2Y2 receptors in ECV304 cells. Likewise polyclonal rabbit antibodies to other P2Y receptors either showed no response or showed strong immunoreactive profile with multiple bands even in 1321N1 cells suggesting that these antibodies may not have been extensively characterized. Furthermore, immunofluorescence studies with commercial anti-P2Y2 antibodies showed that they may be only recognising non-denatured receptors. These studies suggest that the L247 anti-P2Y2 antibody raised against peptide designed to mimic specific region in the third extracellular loop of human P2Y2 receptor is highly specific and sensitive and provides an important tool to study endogenously expressed P2Y2 receptors in both non-denatured and denatured state. These studies indicate that this strategy of generating antibodies may be used to generate highly specific antibodies to other P2Y receptor subtypes. ii CONTENTS Page Abstract i Table of contents iii Acknowledgements xi Dedication xii Abbreviations xiii Amino acid codes xxi List of figures xxii List of Tables xxvii Chapter 1-General Introduction 1 1.1 Functional human P2Y receptor subtypes 2 1.2 Ligands for P2Y receptors 4 1.3 Molecular biology of P2Y receptors 7 1.4 P2Y receptors and binding domains 12 1.5 Antibodies to P2Y receptors 15 1.6 P2Y receptors and intracellular signaling transduction pathways 19 1.7 Tissue distribution of P2Y receptor subtypes 25 1.8 Diagnostic and therapeutic potential for P2Y receptors 27 1.8.1 Cystic fibrosis and chronic obstructive lung diseases 27 1.8.2 P2Y receptors and cancer 30 1.8.3 P2Y receptors and the vascular system 34 1.8.4 P2Y receptors and diabetes 38 1.8.5 P2Y receptors and hepatocytes 39 1.8.6 P2Y receptors and dry eye syndrome 39 iii 1.8.7 P2Y receptors and host defence 40 1.8.8 P2Y receptor and bone 43 1.8.9 P2Y receptors and neuroinflammation 44 1.9 Cell types used in this study 46 1.9.1 1321N1 46 1.9.2 Human bladder cancer cell line, ECV304 46 1.9.3 EAhy926 47 1.9.4 Bovine aortic endothelial cells (BAEC) 47 1.9.5 BRIN-BD11 48 1.10 The basis for this research project 50 1.11 Aims of this project 51 Section 2- Materials and Methods 52 2.1 Materials 53 2.2 Cell culture 62 2.2.1 Isolation of bovine aortic endothelial cells 62 2.2.2 Tissue culture of 1321N1, ECV304, EAhy926, bovine aortic endothelial (BAE) and BRIN-BD11 cells 64 2.3 Measurement of second messengers in cells 64 2.3.1 Measurement of total inositol (poly) phosphates 64 2.3.2 Measurement of cAMP production 65 2.4 RT-PCR studies 66 2.4.1 Primer design 66 2.4.2 RT-PCR studies for P2Y (P2Y1-14) receptor mRNA transcripts 66 2.4.3 PCR product purification 67 2.4.4 DNA sequencing 67 iv 2.5 Purification of antibody 68 2.5.1 L247 Polyclonal antibody production 68 2.5.2 Purification of L247 polyclonal antibodies 68 2.5.3 Dialysis 68 2.5.4 Protein quantification 68 2.5.5 Indirect ELISA 71 2.6 Western blot studies 71 2.6.1 Protein extraction 71 2.6.2 Protein quantification 72 2.6.3 SDS-PAGE and western blot analysis 72 2.6.4 Antibodies 73 2.6.5 Enzymatic N- deglycosylation 76 2.6.6 Protein sequencing 76 2.6.7 Receptor desensitisation 76 2.7 Immunofluorescence analysis 77 Chapter 3-Pharmacological characterisation of P2Y receptors in ECV304 and EAhy926 cells 78 3.1 Introduction 79 3.1.1 Human bladder cancer epithelial cell line ECV304 79 3.1.2 Endothelial Eahy926 cells 80 3.1.3 Bovine aortic endothelial (BAE) cells 80 3.2 Materials and Methods 82 3.2.1 Tissue culture 82 3.2.2 Measurement of total inositol (poly) phosphates 82 3.2.3 Measurement of cAMP production 82 v 3.2.4 Statistical analysis 82 3.3 Results 83 3.3.1 Measurement of inositol phosphate accumulation in ECV304 cells in response to BzATP and ATPγS 83 3.3.2 Measurement of cAMP production in ECV304 cells in response to ADPβS and ATPγS 84 3.3.3 Measurement of total inositol (poly) phosphates in ECV304 cells in response to UTP, ATP and ADP 85 3.3.4 Measurement of total inositol (poly) phosphates in EAhy926 cells in response to BzATP 86 3.3.5 Measurement of total inositol (poly) phosphates in EAhy926 cells in response to UTP and ATP 87 3.3.6 Measurement of total inositol (poly) phosphates in EAhy926 cells in response to 2MeSATP 88 3.3.7 Measurement of total inositol (poly) phosphates in EAhy926 cells in response to ADP 89 3.3.8 Measurement of total inositol (poly) phosphates in EAhy926 cells in response to UDP 90 3.3.9 Measurement of total inositol (poly) phosphates in BAE cells in response to UTP 91 3.3.10 Measurement of total inositol (poly) phosphates in BAE cells in response to 2MeSADP and 2MeSATP 92 3.4 Discussion 94 vi Chapter 4- Establishing the molecular identity of P2Y receptors in ECV304 and EAhy926 cells 98 4.1 Introduction 99 4.2 Materials and Methods 101 4.2.1 Tissue culture of 1321N1, ECV304, EAhy926, BAE and BRIN-BD11 cells 101 4.2.2 Primer design 101 4.2.3 RT-PCR 103 4.2.4 Agarose gel electrophoresis 103 4.2.5 PCR product purification 103 4.2.6 DNA sequencing 103 4.2.7 Database search 103 4.3 Results 104 4.3.1 RT-PCR studies on 1321N1, ECV304, EAhy926, BAEC and BRIN-BD11 cells 104 4.3.2 Determining the presence of P2Y1 transcripts 105 4.3.3 Determining the presence of P2Y2 transcripts 106 4.3.4 Determining the presence of P2Y4 transcripts 107 4.3.5 Determining the presence of P2Y6 transcripts 108 4.3.6 Determining the presence of P2Y11 transcripts 109 4.3.7 Determining the presence of P2Y12 transcripts 110 4.3.8 Determining the presence of P2Y13 transcripts 111 4.3.9 Determining the presence of P2Y14 transcripts 112 4.3.10 Database search 114 4.4 Discussion 116 vii Chapter 5- Purification of polyclonal antibodies 120 5.1 Introduction 121 5.2 Materials and Methods 124 5.2.1 Buffer preparation 124 5.2.2 Sample preparation 124 5.2.3 Affinity chromatography 124 5.2.4 Dialysis 124 5.2.5 Bicinchoninic acid protein assay 124 5.2.6
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