Calcitonin Gene-Related Peptide Characterization of Binding Sites and Structure-Activity Relationships in the Eye and Effects on Intraocular Pressure

…………………………………………………………………………………… 8 1 INTRODUCTION …………………………………………………………………………… 10 2 REVIEW OF THE LITERATURE ………………………………………………………… 12 2.1 The Eye ………………………………………………………………………………… 12 2.2 Blood Circulation in the Eye …………………………………………………………… 12 2.2.1 Regulation of Blood Circulation ………………………………………………… 13 2.3 Aqueous Humour Dynamics and Intraocular Pressure ………………………………… 14 2.4 Glaucoma ………………………………………………………………………………… 15 2.5 Calcitonin Peptide Family ……………………………………………………………… 16 2.5.1 CGRP ……………………………………………………………………………… 16 2.5.1.1 Localization and Function of CGRP ……………………………………… 17 2.5.1.2 CGRP Receptors ………………………………………………………… 18 2.5.1.3 Signalling Pathways of CGRP …………………………………………… 19 2.5.2. Amylin …………………………………………………………………………… 20 2.5.2.1 Amylin Receptors ………………………………………………………… 21 2.5.2.2 Signalling Pathways of Amylin …………………………………………… 22 2.6 Prostaglandins …………………………………………………………………………… 22 3 AIMS OF THE STUDY ……………………………………………………………………… 25 4 MATERIALS AND METHODS …………………………………………………………… 26 4.1 Materials ………………………………………………………………………………… 26 4.1.1 Reagents and Test Compounds …………………………………………………… 26 4.1.2 Anesthetics and Equipment ……………………………………………………… 26 4.1.3 Tissues and Experimental Animals ……………………………………………… 27 4.2 Methods ………………………………………………………………………………… 27 4.2.1 Autoradiography ………………………………………………………………… 27 4.2.2 Synthesis of Analogs ……………………………………………………………… 28 4.2.3 Binding Assay …………………………………………………………………… 28 4.2.4 Adenylate Cyclase Assay ………………………………………………………… 29 4.2.5 General Anesthesia and Intracameral Administration of Test Compounds ……… 29 4.2.6 Intravitreal Administration of Test Compound and Pneumatonometer IOP Measurement ……………………………………………………………………… 30 4.2.7 Measurement of IOP and Aqueous Humour Outflow after Intracameral and Intravitreal Administration of Test Compounds ………………………………… 30 4.2.8 Aqueous Humour Protein Concentration ………………………………………… 31 4.2.9 Statistical Analysis ……………………………………………………………… 31 5 RESULTS …………………………………………………………………………………… 32 5.1 CGRP Binding Sites in the Eye of Monkey, Pig, Cat and Guinea Pig (I) ……………… 32 5.1.1 Monkey …………………………………………………………………………… 32 5.1.2 Pig ………………………………………………………………………………… 32 5.1.3 Cat ………………………………………………………………………………… 32 5 5.1.4 Guinea pig ………………………………………………………………………… 32 5.2 Displacement of I-CGRP Binding by Cat, Human and Rat Amylins in the Eye of Monkey, Cat and Pig (II) …………………………………………………………… 33 5.3 Structure-Activity Relationship of CGRP Molecule (III) ……………………………… 33 5.3.1 Binding Affinity …………………………………………………………………… 34 5.3.2 Stimulation of Adenylate Cyclase ………………………………………………… 34 5.4 Intracameral and Intravitreal Injections of CGRP (IV) ………………………………… 34 5.4.1 IOP and Aqueous Humour Outflow after Intracameral Injection of CGRP ……… 34 5.4.2 IOP after Intravitreal Injection of CGRP ………………………………………… 35 5.4.3 IOP and Aqueous Humour Outflow after Intravitreal Injection of CGRP ………… 35 5.4.4 Protein Concentration in Aqueous Humour after Intravitreal Injection of CGRP … 35 5.5 Effects of Simultaneous Intracameral Injection of CGRP and PGF2α on IOP and Aqueous Humour Outflow in Rabbit (V) ………………………………………………………… 35 6 DISCUSSION ……………………………………………………………………………… 37 6.1 CGRP Binding Sites in the Eye ………………………………………………………… 37 6.1.1 Ciliary Muscle …………………………………………………………………… 37 6.1.2 Anterior Chamber Angle ………………………………………………………… 37 6.1.3 Ciliary Processes and Iris ………………………………………………………… 37 6.1.4 Limbal Conjunctiva ……………………………………………………………… 38 6.1.5 Choroid and Retina ……………………………………………………………… 38 6.1.6 Autoradiography as a Method …………………………………………………… 38 6.2 The Displacement of I-CGRP by Cat, Human and Rat Amylins …………………… 39 6.3 Structure-Activity Relationship of CGRP Molecule …………………………………… 39 6.3.1 Binding Affinity ………………………………………………………………… 39 6.3.2 Stimulation of Adenylate Cyclase ……………………………………………… 40 6.4 The Effect of CGRP on IOP and Outflow Facility ……………………………………… 41 6.5 The Effect of Simultaneous Injection of CGRP and PGF2α on Outflow Facility ……… 41 7 CONCLUSIONS …………………………………………………………………………… 43 8 ACKNOWLEDGEMENTS ………………………………………………………………… 44 9 REFERENCES ……………………………………………………………………………… 46