Purinergic receptor expression in neuronal, bladder smooth muscle and urothelial cells: characterization and inhibition by low molecular weight antagonists

.....................................................................................................3 Acknowledgements ...................................................................................4 Publications arising from this thesis .......................................................5 Prologue .....................................................................................................9 Chapter 1: Introduction.........................................................................13 Abstract .............................................................................................................14 Acknowledgement ............................................................................................14 Introduction ......................................................................................................15 Homomeric P2X1 channels .........................................................................18 Key Messages.........................................................................................18 Localization and Function of P2X1 Channels ........................................18 Activation of P2X1 Channels .................................................................20 Homomeric P2X2 channels .........................................................................23 Key Messages.........................................................................................23 Localization and Function of P2X2 Channels ........................................23 Activation of P2X2 Channels .................................................................25 Inhibition of P2X2 Channels ..................................................................27 Homomeric P2X3 and heteromeric P2X2/3 channels...................................28 Key Messages.........................................................................................28 Localization and Function of P2X3 and P2X2/3 Channels ......................28 Activation of P2X3 and P2X2/3 Channels ...............................................30 Inhibition of P2X3 and P2X2/3 Channels ................................................33 Homomeric P2X4 channels .........................................................................34 Key Messages.........................................................................................34 Localization and Function of P2X4 Channels ........................................35 Activation of P2X4 Channels .................................................................36 Inhibition of P2X4 Channels ..................................................................37 Homomeric P2X5 and heteromeric P2X1/5 channels...................................38 Key Messages.........................................................................................38 Localization and Function of P2X5 and P2X1/5 Channels ......................38 Activation of P2X5 and P2X1/5 Channels ...............................................39 Inhibition of P2X5 and P2X1/5 Channels ................................................42 Homomeric P2X6 and heteromeric P2X2/6 and P2X4/6 channels ................43 Key Messages.........................................................................................43 Localization and Function of P2X6, P2X2/6 and P2X4/6 Channels .........43 Activation of P2X6, P2X2/6 and P2X4/6 Channels...................................44 Inhibition of P2X6, P2X2/6 and P2X4/6 Channels....................................45 Homomeric P2X7 channels .........................................................................46 Key Messages.........................................................................................46 7 Localization and Function of P2X7 Channels ........................................46 Activation of P2X7 Channels .................................................................48 Inhibition of P2X7 Channels ..................................................................50 Recent Advances.........................................................................................54 Chapter 2: Methods................................................................................60 Drug substances and Materials ...................................................................61 Cell Culture .................................................................................................62 Cloning and transfection .............................................................................64 Cytosolic Calcium Measurements ..............................................................65 Fura-2 .....................................................................................................66 Pharmacological Selectivity........................................................................67 Radioligand Binding ...................................................................................67 Whole Cell Voltage Clamp Electrophysiology...........................................69 Tissue Bath Studies .....................................................................................70 RNA extraction and quantitative real-time PCR.........................................71 Western blotting..........................................................................................72 Immunocytochemistry.................................................................................72 Measurement of ATP release ......................................................................74 Spinal Electrical Stimulation-Evoked Intravesical Pressure Change in Pithed Rats ..................................................................................................75 Pharmacokinetics ........................................................................................75 Animals ..................................................................................................75 Blood and Urine Collection....................................................................75 Plasma Protein Binding..........................................................................75 Determination of Brain to Plasma Ratio ................................................76 Pharmacokinetic Analysis ......................................................................76 Data analysis ...............................................................................................76 Chapter 3: RO-4, A Potent Orally Bioavailable P2X3/P2X2/3 Antagonist............................................................................................78 Abstract .............................................................................................................79 Acknowledgements...........................................................................................79 Introduction ......................................................................................................80 Results ...............................................................................................................82 Discussion..........................................................................................................90 Chapter 4: Pharmacological Characterization of RO-1, A Selective P2X1 Antagonist................................................................................102 Abstract ...........................................................................................................103 Acknowledgements.........................................................................................103 Introduction ....................................................................................................104 Results .............................................................................................................106 Discussion........................................................................................................109 Chapter 5: Expression And Function Of Rat Urothelial P2Y Receptors ...........................................................................................118