UNIVERSITÀ DEGLI STUDI DI PAVIA Department of Internal Medicine and Therapeutics Unit of Cellular and Molecular Pharmacology and Toxicology MPEP, a Metabotropic Glutamate Receptor 5 (mGluR5) negative allosteric modulator, protects from hepatic ischemic injury both in vitro and ex vivo Tutors: Prof. Plinio Richelmi Dott. Andrea Ferrigno PhD candidate: Clarissa Berardo Doctorate in Biomedical Sciences Curriculum Pharmacology XXX Cycle - A.A. 2014-2017 UNIVERSITÀ DEGLI STUDI DI PAVIA Department of Internal Medicine and Therapeutics Unit of Cellular and Molecular Pharmacology and Toxicology MPEP, a Metabotropic Glutamate Receptor 5 (mGluR5) negative allosteric modulator, protects from hepatic ischemic injury both in vitro and ex vivo Tutors: Prof. Plinio Richelmi Dott. Andrea Ferrigno Referees: Prof. Ferdinando Nicoletti Prof. Luca Fabris PhD candidate: Clarissa Berardo Doctorate in Biomedical Sciences Curriculum Pharmacology XXX Cycle - A.A. 2014-2017 Table of Contents List of Abbreviations ................................................................................................................ 9 AMINO ACID RESIDUES ................................................................................................... 11 GLOSSARY ............................................................................................................................ 12 ABSTRACT ............................................................................................................................ 13 INTRODUCTION .................................................................................................................. 20 1.1 Genetics.......................................................................................................................... 23 1.2 Distribution ................................................................................................................... 24 1.2.1 Distribution in the central nervous system ............................................................... 24 1.2.2 Distribution in peripheral tissues ............................................................................. 25 1.2.3 mGluR5 in the liver ................................................................................................. 28 1.2.4 Intracellular distribution ........................................................................................... 28 1.3 Structure ........................................................................................................................ 30 1.3.1 The Venus Flytrap Domain ...................................................................................... 30 1.3.2 The Cysteine-Rich Domain ...................................................................................... 32 1.3.3 The Transmembrane Domain .................................................................................. 32 1.3.4 The C-terminal Domain ........................................................................................... 35 1.4 Signaling ........................................................................................................................ 38 1.5 Desensitization, Endocytosis & Trafficking ............................................................... 40 1.6 Ligands .......................................................................................................................... 42 1.6.1 Orthosteric binding pocket ....................................................................................... 43 1.6.1.1 Orthosteric agonists ........................................................................................... 44 1.6.1.2 Orthosteric antagonists ...................................................................................... 46 1.6.2 Allosteric binding pocket ......................................................................................... 48 1.6.2.1 Positive allosteric modulators ........................................................................... 52 1.6.2.2 Negative allosteric modulators .......................................................................... 54 1.6.3. Molecular switches ................................................................................................. 57 1.7 Role ................................................................................................................................ 59 1.7.1 Physiological roles ................................................................................................... 59 1.7.2 Pathological roles ..................................................................................................... 59 1.7.2.1 Schizophrenia ........................................................................................................ 60 1.7.2.2 Fragile X syndrome ............................................................................................... 61 5 1.7.2.3 Anxiety disorders .................................................................................................. 63 1.7.2.4 Chronic pain .......................................................................................................... 64 1.7.2.5 Drug addiction ...................................................................................................... 66 1.7.2.6 Parkinson’s disease ............................................................................................... 67 1.7.2.7 Huntington’s disease ............................................................................................. 68 1.7.2.8 Pathological role in peripheral tissues .................................................................. 70 1.8 Glutamate in ischemia .................................................................................................. 71 1.9 Hepatic ischemia/reperfusion injury .......................................................................... 73 1.9.1 ATP depletion .......................................................................................................... 74 1.9.2 Intracellular acidosis ................................................................................................ 76 1.9.3 Calcium overload ..................................................................................................... 76 1.9.4 Kupffer cells activation ............................................................................................ 77 1.9.5 ROS .......................................................................................................................... 78 1.9.6 Nitric oxide .............................................................................................................. 78 1.9.7 Cell death ................................................................................................................. 79 AIM of THE STUDY ............................................................................................................. 82 Materials & Methods ............................................................................................................. 85 3.1 Materials ........................................................................................................................ 86 3.1.1 Animals .................................................................................................................... 86 3.1.2 Antibodies ................................................................................................................ 86 3.1.3 Drugs ........................................................................................................................ 87 3.2 In vitro experiments ...................................................................................................... 87 3.2.1 Hepatocyte isolation ................................................................................................. 87 3.2.1.1 Apparatus .......................................................................................................... 87 3.2.1.2 Solutions ............................................................................................................ 88 3.2.1.3 Isolation procedure ............................................................................................ 89 3.2.1.4 Pharmacological treatment ................................................................................ 90 3.2.1.5 Warm ischemia experiments on rat hepatocytes ............................................... 91 3.2.1.6 In vitro assays on hepatocytes ........................................................................... 92 3.2.1.6.1 Measurement of Cell Viability: trypan blue exclusion and LDH release ... 92 3.2.1.6.2 Measurement of ATP .................................................................................. 93 3.2.2 Mitochondrial isolation ............................................................................................ 94 3.2.2.1 Solutions ............................................................................................................ 94 6 3.2.2.2 Procedure ........................................................................................................... 95 3.2.2.3 Pharmacological treatment ................................................................................ 95 3.2.2.4 In vitro assays on mitochondria ........................................................................ 95 3.2.2.4.1 Mitochondrial membrane potential (ΔΨ) ................................................... 95 3.2.2.4.2 Respiratory Control