Development of cell-based assays for adenine receptors and selected purine receptor subtypes [Elektronische Ressource] : receptor characterization and search for novel ligands / vorgelegt von Aliaa Mahmoud Mohamed Eltayb Abdelrahman

Aliaa Mahmoud Mohamed Eltayb Abdelrahman

Sujets

• Naturwissenschaften
• Gesundheit

Informations

• Publié par: rheinische_friedrich-wilhelms-universitat_bonn
• Publié le: 01 janvier 2010
• Nombre de lectures: 39
• Langue: Deutsch
• Poids de l'ouvrage: 3 Mo

Extrait

Development of Cell-Based Assays for
Adenine Receptors and Selected
Purine Receptor Subtypes:
Receptor Characterization and Search
for Novel Ligands







Dissertation
zur
Erlangung des Doktorgrades (Dr. rer. nat.)
der
Mathematisch-Naturwissenschaftlichen Fakultät
der
Rheinischen Friedrich-Wilhelms-Universität Bonn




vorgelegt von
Aliaa Mahmoud Mohamed Eltayb Abdelrahman

aus
Ägypten




Bonn 2010
































Angefertigt mit Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät
der Rheinischen Friedrich-Wilhelms-Universität Bonn
























1. Referent: Prof. Dr. Christa E. Müller
2. Referent: Prof. Dr. Michael Wiese

Tag der Promotion: 20.07.2010

Erscheinungsjahr: 2010































Die vorliegende Arbeit wurde in der Zeit von April 2006 bis April 2010 am
Pharmazeutischen Institut, Pharmazeutische Chemie I, Bonn unter der Leitung von
Frau Prof. Dr. Christa E. Müller durchgeführt.






















Mein besonderer Dank gilt Frau Prof. Dr. Christa E. Müller für Ihre fortwährende
Unterstützung, Ihre stetige Diskussionsbereitschaft und Ihre zahlreichen Anregungen,
die maßgeblich zum Gelingen dieser Arbeit beigetragen haben.

Ebenso bedanke ich mich an dieser Stelle bei Prof. Dr. Michael Wiese für die
freundliche Übernahme des Koreferates.






























To My Parents,
To Ali, Aia and Khaled





























Table of contents I
Table of contents

1. Introduction………………………………………………………………….. 1
1.1. Purinergic receptor and its subtypes………………………………………….. 1
1.1.1. P2 receptors…………………………………………………………………… 1
1.1.1.1. P2X receptors…………………………………………………………………. 1
1.1.1.1.1. Homomeric P2X receptor……………………………………………………. 6 2
1.1.1.1.2. Homomeric P2X and heteromeric P2X receptors ……………………........ 7 3 2/3
1.1.1.1.3. Homomeric P2X receptor ………………………………………………........ 9 4
1.1.1.2. P2Y receptors…………………………………………………………………. 10
1.1.1.2.1. P2Y receptors ………………………………………….... …………………. 14 1
1.1.1.2.2. P2Y receptors ……………………………………………................. 15 11
1.1.1.2.3. P2Y receptors …………………………………………………..................... 15 12
1.1.1.2.4. P2Y receptors ………………………………………………………………. 17 13
1.1.2. P1 receptor……………………………………………………………………. 20
1.1.3. P0 receptor ……………………………………………………….................... 22
1.2. Radioligand binding studies……………………………………………........... 24
1.2.1. Basic concepts in radioligand binding studies………………………………... 24
1.2.2. Basic types of receptor binding experiments…………………………………. 25
1.2.2.1. Saturation experiments……………………………………………………….. 25
1.2.2.2. Competition experiments……………………………………………………... 27
1.2.2.3. Kinetic experiments…………………………………………………………... 28
1.3. G protein-coupled receptors…………………………………………………... 30
1.4. Functional studies……………………………………………………….......... 33
1.4.1. Regulation of intracellular 3´,5´-cyclic adenosine monophosphate levels........ 33
1.4.1.1. Quantitative analysis of 3´,5´-cyclic adenosine monophosphate levels……… 34
1.4.1.1.1. Accumulation assays………………………………………………………….. 34
1.4.1.1.2. Reporter-gene assays for 3´,5´-cyclic adenosine monophosphate levels
detection………………………………………………………………………. 35
1.4.2. Measurement of intracellular calcium or inositol 1,4,5-trisphosphte levels
(IP )………………………………………………………………………........ 39 3
1.4.3. Measurent of extracellular signal-regulated kinase 1/2 levels (ERK )…........ 41 1/2
1.4.4. β-Arrestin assays ……………………………………………………………. 45
1.5. Retroviral gene transfer technology principle……………………………........ 46
2. Scope of investigation……………………………………………….............. 49
2.1. Adenine receptors (P0 receptors)……………………………………............... 49
2.1.1. Human adenine receptors…………………………………………................... 49
2.1.2. Mouse adenines…………………………………………................... 50
2.1.3. Hamster (CHO cells) and rat (PC12 cells) adenine receptors……………........ 51
3. Results and discussion…………………………………………..................... 52
3.1. Human adenine receptors …………………………………………….............. 52
3.1.1. Pharmacology and test system at the human adenine receptors ……………... 52
3.1.1.1. Radioligand binding studies………………………………………………….. 52
Table of contents II
33.1.1.1.1. [ H]Adenine saturation experiments (saturation experiments for whole 52
cells)………………………………………………………………...................
3.1.1.1.2. Kinetic studies for whole cells………………………………………………... 53
33.1.1.1.3. [ H]Adenine competition assays ……………………………………………... 53
3.1.1.2. Functional studies…………………………………………………………….. 55
3.1.1.2.1. cAMP accumulation assay induced by human adenine receptors in HEK293
cells ……………………………………………………................................... 55
3.1.1.2.2. Investigation of extracellular signal-regulated kinase (ERK) phosphorylation
for human adenine receptors.............................................................................. 56
3.1.2. Structure-activity relationships of adenine derivative at human and rat
adenine receptors............................................................................................... 58
3.1.2.1. Investigation of agonistic properties of adenine and adenine derivatives at the
rat adenine receptor ........................................................................................... 74
3.1.2.1.1. cAMP accumulation assays............................................................................... 74
3.1.2.1.2. cAMP-dependent luciferase assay.................... 76
3.1.2.2. Extracellular signal-regulated kinase (ERK) phosphorylation assay................. 78
3.1.3. Investigation of the affinity of selected adenine derivatives for rat adenosine
A and A receptors.......................................................................................... 81 1 2A
3.1.4. Characterisation of the mouse adenine receptor (mAde2R).............................. 84
3.1.4.1. cAMP accumulation assay at the mAde2R........................................................ 84
3.1.4.2. Radioligand binding assays…………………………....................................... 85
3.1.5. Investigation of adenine receptors natively expressed in Chinese hamster
ovary cells (CHO K1) …………....................................................................... 90
3.1.5.1. Accumulation cAMP assay for CHO K1 or CHO flp-in…………................... 90
33.1.5.2. [ H]Adenine saturation experiments………………………………………….. 91
3.1.6. Homologous competition assays....................................................................... 92
3.1.6.2. Homs competition binding curves for characterization of endogenous
rat adenine receptors in pheochromocytoma cells and human melanoma 1539
cells.................................................................................................................... 93
3.1.6.2.1. Functional studies for endogenous rat adenine receptors in PC12 cells............ 94
3.1.6.2.2. mRNA localization studies for endogenous rat adenine receptors in PC12
cells……………………………………………………………….................... 96
3.2. Adenosine A receptors…………………………………………………........ 97 2B
3.2.1. Evaluation of adenosine A receptors expressed in CHO cells........................ 97 2B
3.2.1.1. Adenosine A receptors……………………………………............................ 97 2B
3.2.1.2. cAMP accumulation assay for CHO cells stably expressing the human
adenosine A receptor………………………………….................................. 98 2B
3.2.2. Modification of a protein-binding method for rapid quantification of cAMP
in cell-culture supernatants ……………………………................................... 99
3.2.3. Adenosine A receptor agonists……………………………………………... 101 2A
3.2.3.1. cAMP accumulation assay for CHO cells stably expressing the human
adenosine A receptor……………………………………………………….. 101 2A
3.2.4. Adenosine A receptor antagonist………………………………………........ 103 2A