Actions of benzophenanthridine alkaloids and various synthetic compounds on the cannabinoid-1 (CB1) receptor pathway of mouse brain with particular reference to the

effects on [3H]CP55940 and [3H]SR141716A binding, interference with basal and

CP55940-stimulated [35S]GTPγS binding, and modification of WIN55212-2-dependent

inhibition of L-glutamate release from synaptosomes

by Amey S. Dhopeshwarkar

MSc., University of Abertay Dundee, 2007 B.Pharm., University of Pune, 2004

THESIS SUBMITTED IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

in the

Department of Biological Sciences

Faculty of Science

Amey S. Dhopeshwarkar 2012

SIMON FRASER UNIVERSITY Summer 2012

All rights reserved. However, in accordance with the Copyright Act of Canada, this work may

be reproduced, without authorization, under the conditions for “Fair Dealing.” Therefore, limited reproduction of this work for the

purposes of private study, research, criticism, review and news reporting is likely to be in accordance with the law, particularly if cited appropriately.

ii

Approval

Name: Amey S. Dhopeshwarkar Degree: Doctor of Philosophy (Biological Sciences) Title of Thesis: Actions of benzophenanthridine alkaloids and various

synthetic compounds on the cannabinoid-1 (CB1) receptor pathway of mouse brain with particular reference to the effects on [3H]CP55940 and [3H]SR141716A binding, interference with basal and CP55940-stimulated [35S]GTPγS binding, and modification of WIN55212-2-dependent inhibition of L-glutamate release from synaptosomes.

Examining Committee: Chair: Dr Julian Christians, Associate Professor

Dr Russell A. Nicholson Senior Supervisor Associate Professor

Dr Christopher Kennedy Supervisor Professor

Dr Francis C.P. Law Supervisor Professor

Dr Gordon Rintoul Internal Examiner Associate Professor Department of Biological Sciences, SFU

Dr Andrew Gifford External Examiner Scientist, Medical Department Brookhaven National Laboratory

Date Defended/Approved: August 15, 2012

iii

Partial Copyright Licence

Ethics Statement

The author, whose name appears on the title page of this work, has obtained, for the research described in this work, either:

a. human research ethics approval from the Simon Fraser University Office of Research Ethics,

or

b. advance approval of the animal care protocol from the University Animal Care Committee of Simon Fraser University;

or has conducted the research

c. as a co-investigator, collaborator or research assistant in a research project approved in advance,

or

d. as a member of a course approved in advance for minimal risk human research, by the Office of Research Ethics.

A copy of the approval letter has been filed at the Theses Office of the University Library at the time of submission of this thesis or project.

The original application for approval and letter of approval are filed with the relevant offices. Inquiries may be directed to those authorities.

Simon Fraser University Library Burnaby, British Columbia, Canada

update Spring 2010

iii

Abstract

Benzophenanthridine alkaloids (chelerythrine and sanguinarine) inhibited the binding of

[3H]CP55940 and [3H]SR141716A to mouse brain membranes (IC50s approx. 1-2 µM).

Piperonyl butoxide and (S)-methoprene were more potent inhibitors of [3H]CP55940

binding (IC50s: 8.2 µM and 16.4 µM respectively) than of [3H]SR141716A binding (IC50s:

21 µM and 63 µM respectively). Binding experiments demonstrated selectivity towards

the brain CB1 versus spleen CB2 receptor.

Benzophenanthridines reduced the Kd of [3H]CP55940 binding to brain membranes

whereas (S)-methoprene and piperonyl butoxide lowered Bmax. These study

compounds reduced the association of [3H]CP55940 and [3H]SR141716A, however

benzophenanthridines were consistently more effective.

In the presence of a saturating concentration of SR141716A, (S)-methoprene and

piperonyl butoxide increased dissociation of [3H]SR141716A above that observed with

SR141716A alone. All compounds activated [3H]SR141716A dissociation when assayed

alone, but (S)-methoprene was the least effective. In separate studies, phthalate

diesters reduced the Bmax of [3H]SR141716A without affecting Kd, and increased

[3H]SR141716A dissociation above a saturating concentration of AM251.

Benzophenanthridines antagonized CP55940-stimulated and basal binding of

[35S]GTPγS to the G-protein of mouse brain, whereas piperonyl butoxide and (S)-

methoprene inhibited CP55940-stimulated [35S]GTPγS binding only. Inhibition of

CP55940-stimulated binding of [35S]GTPγS was also demonstrated with phthalates.

4-Aminopyridine- (4-AP-) induced release of L-glutamate from mouse brain

synaptosomes was partially inhibited by WIN55212-2. The inhibitory effect of

WIN55212-2 was completely neutralized by AM251, (S)-methoprene, piperonyl butoxide

and phthalate diesters, whereas in the presence of WIN55212-2, the

benzophenanthridines enhanced 4-AP-induced L-glutamate release above that caused

by 4-AP alone.

iv

The [3H]CP55940 and [3H]SR141716A binding data suggest that the study compounds

modify radioligand binding allosterically. The [35S]GTPγS binding results suggest that

chelerythrine and sanguinarine are inverse agonists of G-protein-coupled CB1 receptors,

while piperonyl butoxide, (S)-methoprene and phthalate diesters are neutral lower

potency antagonists. Modulation 4-AP-evoked L-glutamate release from synaptosomes

by the study compounds with WIN-55212-2 present strongly supports this latter profiling.

Although these compounds exhibit lower potencies versus many conventional CB1

receptor inhibitors, further studies are warranted, given their potential to 1) modify CB1

receptor-dependent behavioral/physiological outcomes in the whole animal, and 2) serve

as starting structures for synthesis of novel/more potent G-protein-coupled CB1 receptor

blocking drugs.

Keywords: Benzophenanthridines; (S)-methoprene; piperonyl butoxide; [3H]CP55940; [35S]GTPγS; L-glutamate; synaptosomes;cannabinoid-1 (CB1) receptor

v

Dedication

To My Beloved Mom and Dad

vi

Acknowledgements

I wish to express my deepest gratitude and appreciation to my senior supervisor, Dr

Russell A. Nichoson for his guidance, patience and indefatigable support throughout my

graduate research career. I remember the days when Dr Nicholson spared his time

even on weekends and holidays to discuss my research and his invaluable suggestions

and encouragements have always made me feel confident about my research work.

Thorough discussion sessions with him about project and related scientific issues and

perspectives have enriched my knowledge in this field. Without Dr Nicholson’s support

and effort, I would not have completed my PhD research in time. I believe that I was

lucky to have such a knowledgeable senior supervisor and I am fortunate to be his last

graduate student.

I am very much thankful to Dr Chris Kennedy and Dr Francis C.P. Law for serving as my

committe members and their valuable time and inputs during my PhD. They have

always been supportive during my studies at SFU.

I am also thankful to Mr Saurabh Jain and Ms Kathleen M. Bisset for their help and

advice during my research.

Finally, I would like to thank my family for their love, support encouragement and always

believing in me.

vii

Table of Contents

Approval .......................................................................................................................... ii Abstract .......................................................................................................................... iii Dedication ....................................................................................................................... v Acknowledgements ........................................................................................................ vi Table of Contents .......................................................................................................... vii List of Tables ................................................................................................................. xii List of Figures................................................................................................................xiv Glossary ........................................................................................................................xxi

1. Introduction .......................................................................................................... 1 1.1. Historical significance of cannabis use and cannabinoids ....................................... 1

1.1.1. The early Chinese/Indian era ...................................................................... 1 1.1.2. The period encompassing the early Christian era through to the 18th

century ........................................................................................................ 2 1.1.3. The Western medicine era of the 19th and 20th centuries ............................. 2

1.2. Cannabinoids ......................................................................................................... 5 1.2.1. G protein-coupled receptors (GPCRs) and their activation cycle ................. 7 1.2.2. The [35S]GTPγS binding assay .................................................................... 8

1.3. Other cannabinoid receptors .................................................................................. 8 1.4. Cannabinoid-1 Receptors (CB1-Rs) ........................................................................ 9

1.4.1. The structure and activation of CB1-Rs ....................................................... 9 1.4.2. The distribution of CB1-Rs in mammalian brain ......................................... 17

1.5. CB1-R-mediated intracellular signaling pathways .................................................. 18 1.5.1. Inhibition of cyclic AMP (cAMP) ................................................................ 18 1.5.2. Stimulation of cAMP production ................................................................ 20 1.5.3. CB1-Rs and the modulation of Ca2+ fluxes and phospholipases C

and A ........................................................................................................ 21 1.5.4. CB1-R-dependent regulation of ion channels ............................................. 21 1.5.5. Involvement of CB1-Rs in the suppression of neurotransmitter

release ...................................................................................................... 22 1.6. Homodimerization and heterodimerization of CB1-Rs ........................................... 24 1.7. Constitutive activity of CB1-Rs .............................................................................. 25 1.8. The biochemistry of endocannabinoids ................................................................. 25

1.8.1. Anandamide biosynthesis ......................................................................... 28 1.8.2. 2-Arachidonoyl glycerol (2-AG) biosynthesis ............................................. 30

1.9. Degradation pathways for endocannabinoids ....................................................... 32 1.10. Transport of endocannabinoids ............................................................................ 33 1.11. Endocannabinoid-mediated short term depression (DSI and DSE) ....................... 35 1.12. Endocannabinoids as synaptic circuit breakers and retrograde messengers ........ 35 1.13. Mechanisms of endocannabinoid mediated short term depression (eCB-

STD) ..................................................................................................................... 38 1.13.1. CaER ........................................................................................................ 38 1.13.2. Basal RER ................................................................................................ 38 1.13.3. Ca2+-assisted RER .................................................................................... 39

1.14. Termination of eCB-STD ...................................................................................... 39 1.15. Endocannabinoid-mediated long term depression (eCB-LTD) .............................. 41

viii

1.16. Other important aspects of endocannabinoid signaling ......................................... 41 1.16.1. Regulation of excitability ........................................................................... 41 1.16.2. Basal activity of endocannabinoid signaling .............................................. 42 1.16.3. Plasticity of endocannabinoid signaling ..................................................... 42

1.17. Subcellular distribution of various signaling molecules involved in regulation of the endocannabinoid system ............................................................................ 42 1.17.1. Gq Protein α subunit .................................................................................. 42 1.17.2. Phospholipase Cβ (PLCβ) ......................................................................... 43 1.17.3. Diacylglycerol lipase (DAGL) ..................................................................... 43 1.17.4. N-acyl-phosphatidylethanolamine-hydrolyzing phospholipase D

(NAPE-PLD) ............................................................................................. 43 1.17.5. Monoacylglycerol lipase (MAGL) ............................................................... 44 1.17.6. Fatty acid amide hydrolase (FAAH) ........................................................... 44

1.18. Physiological roles of the endocannabinoid system .............................................. 44 1.18.1. Learning and Memory ............................................................................... 44 1.18.2. Anxiety ...................................................................................................... 45 1.18.3. Depression ................................................................................................ 45 1.18.4. Addiction ................................................................................................... 46 1.18.5. Appetite ..................................................................................................... 46 1.18.6. Pain .......................................................................................................... 46

1.19. Classification of ligands that bind to cannabinoid receptors .................................. 47 1.19.1. Cannabinoid receptor agonists .................................................................. 47

1.19.1.1. Classical cannabinoids ............................................................... 47 1.19.1.2. Non-classical cannabinoids ........................................................ 47 1.19.1.3. Aminoalkylindoles ....................................................................... 47 1.19.1.4. Eicosanoids/Endocannabinoids .................................................. 48

1.19.2. Cannabinoid receptor antagonists/ Inverse agonists ................................. 48 1.19.2.1. Diarylpyrazoles ........................................................................... 48 1.19.2.2. Other inverse agonists primarily active at CB1-Rs ....................... 48

1.20. Cannabinoid receptor 2 (CB2-R) ........................................................................... 53 1.20.1. CB2-R receptor signaling ........................................................................... 53

1.20.1.1. Adenylyl cyclase regulation......................................................... 53 1.20.1.2. Mitogen-activated protein kinase regulation ................................ 53

1.20.2. Therapeutic aspects of CB2-R modulators ................................................. 54 1.21. Brief overview of the test chemicals used in my research ..................................... 55

1.21.1. Benzophenanthridine alkaloids ................................................................. 55 1.21.2. Piperonyl butoxide (PBO) .......................................................................... 56 1.21.3. Methoprene ............................................................................................... 57 1.21.4. Phthalate esters ........................................................................................ 58 1.21.5. Tributyl tin (TBT) compounds .................................................................... 59

1.22. Rationale behind my research and the general approach ..................................... 62 1.22.1. Summary of objectives .............................................................................. 62

2. The actions of benzophenanthridine alkaloids, piperonyl butoxide and (S)-methoprene at the G-protein coupled cannabinoid CB1 receptor in vitro. .................................................................................................................... 64

2.1. Abstract ................................................................................................................ 64 2.2. Introduction .......................................................................................................... 65 2.3. Materials and Methods ......................................................................................... 67

ix

2.3.1. Radioligands, drugs and study compounds ............................................... 67 2.3.2. Animals ..................................................................................................... 67 2.3.3. Determination of the effects of study compounds on the binding of

[3H]CP55940 to CB1 receptors in mouse brain membranes....................... 67 2.3.4. Determination of the effects of study compounds on basal and

CP55940-stimulated [35S]GTPγS binding to mouse brain membranes ............................................................................................... 69

2.3.5. Data analysis ............................................................................................ 70 2.4. Results ................................................................................................................. 70 2.5. Discussion ............................................................................................................ 71 2.6. Figures and Tables ............................................................................................... 75

3. The G protein-coupled cannabinoid-1 (CB1) receptor of mammalian brain: Inhibition by phthalate esters in vitro. ................................................... 88

3.1. Abstract ................................................................................................................ 88 3.2. Introduction .......................................................................................................... 89 3.3. Materials and methods ......................................................................................... 91

3.3.1. Animals ..................................................................................................... 91 3.3.2. Investigation of the effects of phthalate esters on the binding of

[3H]CP55940 and [3H]SR141716A to CB1 receptors of mouse brain. ......... 92 3.3.3. Investigation of phthalate interference with CB1 receptor agonist-

stimulated [35S]GTPγS binding to the Gα-protein. ...................................... 93 3.3.4. Data analysis ............................................................................................ 95

3.4. Results ................................................................................................................. 95 3.4.1. Effects of phthalate esters on binding of [3H]CP55940 to CB1

receptors. .................................................................................................. 95 3.4.2. Effects of selected phthalate esters on binding of [3H]SR141716A to

CB1 receptors. ........................................................................................... 95 3.4.3. Influence of selected phthalates on the saturation binding of

[3H]SR141716A to CB1 receptors .............................................................. 96 3.4.4. Effects of selected phthalates on [3H]SR141716A kinetics ........................ 96 3.4.5. Effects of phthalates on CB1 receptor agonist-stimulated [35S]GTPγS

binding to the Gα-protein .......................................................................... 96 3.5. Discussion ............................................................................................................ 97 3.6. Note in added proof ............................................................................................ 100

3.6.1. Background ............................................................................................. 100 3.6.2. Experimental approach ........................................................................... 101 3.6.3. Results .................................................................................................... 101 3.6.4. Conclusion .............................................................................................. 101

3.7. Figures and Tables ............................................................................................. 102

4. Benzophenanthridine alkaloid, piperonyl butoxide and (S)-methoprene action at the cannabinoid-1 receptor (CB1-R) pathway of mouse brain: interference with [3H]CP55940 and [3H]SR141716A binding and modification of WIN55212-2-dependent inhibition of synaptosomal L-glutamate release. ............................................................................................ 115

4.1. Abstract .............................................................................................................. 115 4.2. Introduction ........................................................................................................ 116

x

4.3. Materials and Methods ....................................................................................... 118 4.3.1. Chemicals and supplies .......................................................................... 118 4.3.2. Animals ................................................................................................... 119 4.3.3. Isolation of membranes from mouse brain for binding studies ................. 119 4.3.4. Effects of benzophenanthridines, (S)-methoprene and piperonyl

butoxide on equilibrium binding of [3H]CP55940 and [3H]SR141716 to brain CB1 receptors ............................................................................. 120

4.3.5. Effect of benzophenanthridines, (S)-methoprene and piperonyl butoxide on the association and dissociation kinetics of [3H]CP55940 and [3H]SR141716A .......................................................... 121

4.3.6. Interaction of benzophenanthridines, methoprene and piperonyl butoxide with CB2 receptors in mouse spleen ......................................... 121

4.3.7. Preparation of synaptosomes from mouse whole brain ........................... 122 4.3.8. Release of L-Glutamate from synaptosomes........................................... 123 4.3.9. Analysis of radioligand binding data and glutamate release data ............ 124

4.4. Results ............................................................................................................... 124 4.4.1. Effects of benzophenanthridines, piperonyl butoxide and (S)-

methoprene on binding of [3H]SR141716A to CB1 receptors ................... 124 4.4.2. Influence of study compounds on the saturation binding of

[3H]SR141716A to CB1 receptors of mouse brain .................................... 125 4.4.3. Effects of sanguinarine, chelerythrine, piperonyl butoxide, and (S)-

methoprene on the kinetics of CB1 receptor-selective radioligand binding .................................................................................................... 125

4.4.4. Effects of study compounds on mouse spleen CB2 receptors as assessed by inhibition of [3H]CP55940 binding ....................................... 126

4.4.5. Effects of study compounds on WIN55212-2-dependent inhibition of 4-aminopyridine- (4-AP-) evoked release of L-glutamate from mouse brain synaptosomes ................................................................................ 126

4.5. Discussion .......................................................................................................... 127 4.6. Figures and Table .............................................................................................. 132

5. Effects of organotins on the CB1 receptor pathway of mouse brain in vitro. .................................................................................................................. 150

5.1. Introduction ........................................................................................................ 150 5.2. Materials and methods ....................................................................................... 151 5.3. Results ............................................................................................................... 152

5.3.1. Displacement of [3H]CP55940 binding to mammalian CB1 receptors by organotin compounds ......................................................................... 152

5.3.2. Basal and CP55940-stimulated [35S]GTPγS binding to the Gα subunit as influenced by tributyltin compounds ....................................... 152

5.3.3. Modulation by tributyltin acetate and phenylethynyl tributyltin of WIN55212-2-dependent inhibition of 4-aminopyridine-evoked release of L-glutamate from mouse brain synaptosomes ........................ 153

5.4. Discussion .......................................................................................................... 153 5.5. Figures and Table .............................................................................................. 156

xi

6. Conclusion and future prospects .................................................................... 162

References ................................................................................................................. 164

xii

List of Tables

Table 2.1 Inhibition of specific [3H]CP55940 binding to mouse brain membranes by isoquinoline type compounds and PMSF. Isoquinolines were present in the assay at 30 µM and PMSF was present at 0.5 mM. Data represent mean ± S.E.M. of 3 independent experiments. ...................................................................... 84

Table 2.2 Inhibition of 100 nM CP55940-stimulated and basal [35S]GTPγS binding to mouse brain membranes by AM251. Data represent mean ± S.E.M. of 3 independent experiments. ND = not determined. Results provided by Mr Saurabh Jain. ................................ 85

Table 2.3 Lack of effect of isoquinoline type compounds on CP55940-stimulated and basal [35S]GTPγS binding to mouse brain membranes. Study compounds were present in the assay at 40 µM. Data represent mean ± S.E.M. of 3 independent experiments. ........ 86

Table 2.4 Lack of effect of piperonyl butoxide and (S)-methoprene on the basal binding of [35S]GTPγS to mouse brain membranes. Values represent mean ± S.E.M. of 3 independent experiments. ....................... 87

Table 3.1 Inability of PMSF to influence the inhibitory effects of n-butylbenzylphthalate (nBBP) and di-n-butylphthalate (DnBP) on [3H]CP55940 binding to mouse brain membranes. Phthalate esters were present in the assay at 20 µM and PMSF was used at 50 µM. Each value represents the mean ± S.E.M. of 3-6 independent experiments. .................................................................... 113

Table 3.2 Inhibitory effects of n-butylbenzylphthalate (nBBP), di-n-butylphthalate (DnBP), diethylhexylphthalate (DEHP), mono-isohexylphthalate (MiHP) and mono-n-butyl phthalate (MnBP) on the specific binding of [3H]SR141716A to mouse brain membranes. Diesters were present at concentrations producing 50% inhibition of [3H]CP55940 binding. Each value represents the mean ± S.E.M. of 3 independent experiments. ..................................... 114

xiii

Table 4.1 Inhibitory effects of chelerythrine, sanguinarine, piperonyl butoxide and (S)-methoprene on spleen CB2 receptors as determined with [3H]CP55940. Each study compound was added at a concentration that achieved an IC50 for [3H]CP55940 binding to brain CB1 receptors (Dhopeshwarkar et al. 2011). All values represent mean percentage inhibition ± S.E.M. of at least 3 independent experiments. Parallel experiments with [3H]CP55940 corroborated our previously published IC50s at brain CB1 receptors (2.2 µM chelerythrine gave 49.03 ± 0.94 % inhibition, 1.2 µM sanguinarine gave 51.33 ± 0.49 % inhibition, 8.2 µM piperonyl butoxide gave 47.50 ± 1.17 % inhibition and 16.4 µM methoprene gave 50.22 ± 1.10 % inhibition). ...................................... 149

Table 5.1 Inhibitory effects of tributyl and triphenyltins on the binding of [3H]CP55940 to CB1 receptors in mouse brain. All values are as IC50s (with 95% confidence intervals in brackets) calculated from curves based on at least 3 independent experiments except for triphenyltin chloride where the IC50 was estimated from 2 independent experiments). ................................................................... 161

xiv

List of Figures

Figure 1.1 The spread of the use of cannabis across the globe (Adapted from Zuardi, 2006). ........................................................................................... 3

Figure 1-2 Structure of two important phytocannabinoids. Structures redrawn using ChemDraw Ultra 11.0 from structures reported in Pertwee et al. (2010). ................................................................................................. 6

Figure 1.3 Two dimensional representation of the CB1-R (Adapted from Shim et al., 2011). ........................................................................................... 12

Figure 1.4 Diagramatic representation of the C terminal domain of the CB1-R (Adapted from Stadel et al., 2011) .......................................................... 13

Figure 1.5 Structures of prominent endocannbinoids (All structures redrawn using ChemDraw Ultra 11.0 from Kano et al., 2009). ............................. 27

Figure 1.6 Transacylation-phosphodiesterase pathway for biosynthesis of anandamide (Adapted from Cadas et al., 1997). .................................... 29

Figure 1.7 Metabolic pathways for biosynthesis of 2-AG (Adapted from Kano et al., 2009). ........................................................................................... 31

Figure 1.8 Blockade of DSI by CB1-R antagonists. .................................................. 37

Figure 1.9 The pathway involved in the termination of endocannabinoid-mediated short term depression (eCB-STD) (Adapted from Kano et al., 2009). ........................................................................................... 40

Figure 1.10 Structures of ∆9-THC, ∆8-THC, HU210, DALN, CP47497, CP55244, CP55940, WIN55212-2, JWH015 and L-768242. All structures redrawn using ChemDraw 11.0 ultra from Howlett et al. (2002). ................................................................................................... 49

Figure 1.11 Structures of anandamide, 2-AG ether and 2-AG. All structures redrawn using ChemDraw Ultra 11.0 from Howlett et al. (2002). ............ 50

Figure 1.12 Structures of SR141716A, AM251, AM281, LY320135 and AM630. All structures redrawn using ChemDraw Ultra 11.0 from Howlett et al. (2002). .............................................................................. 51

Figure 1.13 Structures of (S)-methoprene, piperonyl butoxide, sanguinarine, chelerythrine, nBBP and DnBP. Structures redrawn using ChemDraw 11.0 from Dhopeshwarkar et al. (2011) and Bisset et al., (2011). .............................................................................................. 52

Figure 1.14 Structures of selected phthalate esters and tributyl tin compounds. All structures redrawn using ChemDraw Ultra 11.0. ............................... 61

xv

Figure 2.1 The structures of sanguinarine, berberine, papavarine and possible comparison of conformations of piperonyl butoxide and (S)-methoprene with anandamide and 2-arachidonoyl glycrol. Also possible comparison of sanguinarine and (S)-methoprene with ∆9-tetrahydrocannabinol and ∆9-tetrahydrocannabivarin. ............................ 76

Figure 2.2 Concentration-dependent inhibition of [3H]CP55940 binding to mouse brain CB1 receptors by sanguinarine and chelerythrine. Values represent mean ± S.E.M. of at least 3 independent experiments each performed in duplicate. Ki values were 0.38 µM (sanguinarine) and 0.57 µM (chelerythrine). ........................................... 77

Figure 2.3a Inhibition of A) CP55940-stimulated and B) basal binding of [35S]GTPγS to mouse brain membranes by chelerythrine. Values represent mean ± S.E.M. of 3 independent experiments each performed in triplicate. ............................................................................ 78

Figure 2.3b Inhibition of A) CP55940-stimulated and B) basal binding of [35S]GTPγS to mouse brain membranes by chelerythrine. Values represent mean ± S.E.M. of 3 independent experiments each performed in triplicate.Basal binding data provided by Mr Saurabh Jain. ....................................................................................................... 79

Figure 2.4a Inhibition of A) CP55940-stimulated and B) basal binding of [35S]GTPγS to mouse brain membranes by sanguinarine. Values represent mean ± S.E.M. of 3 independent experiments each performed in triplicate. ............................................................................ 80

Figure 2.4b Inhibition of A) CP55940-stimulated and B) basal binding of [35S]GTPγS to mouse brain membranes by sanguinarine. Values represent mean ± S.E.M. of 3 independent experiments each performed in triplicate. Basal binding data provided by Mr Saurabh Jain. ......................................................................................... 81

Figure 2.5a A) Concentration-dependent inhibition of [3H]CP55940 binding to mouse brain CB1 receptors by (S)-methoprene and piperonyl butoxide. Ki values were 2.13 µM (methoprene) and 4.25 µM (piperonyl butoxide). B) Inhibition of CP55940-stimulated binding of [35S]GTPγS to mouse brain membranes by (S)-methoprene and piperonyl butoxide. Values represent mean ± S.E.M. of 3 independent experiments each performed in triplicate. .......................... 82

Figure 2.5b A) Concentration-dependent inhibition of [3H]CP55940 binding to mouse brain CB1 receptors by (S)-methoprene and piperonyl butoxide. Ki values were 2.13 µM (methoprene) and 4.25 µM (piperonyl butoxide). B) Inhibition of CP55940-stimulated binding of [35S]GTPγS to mouse brain membranes by (S)-methoprene and piperonyl butoxide. Values represent mean ± S.E.M. of 3 independent experiments each performed in triplicate. .......................... 83

xvi

Figure 3.1 (a-f) The structures of phthalate diesters: n-butylbenzylphthalate (nBBP); di-n-hexylphthalate (DnHP); di-n-butylphthalate (DnBP); di-ethylhexylphthalate (DEHP); di-isooctylphthalate (DiOP) and di-n-octylphthalate (DnOP).(g-i) The structures of phthalate monoesters: mono-2-ethylhexyl-phthalate (M2EHP), mono-isohexyl-phthalate (MiHP) and mono-n-butyl-phthalate (MnBP). All structures have been redrawn from Bissett et al. (2011) using IsisDraw. .............................................................................................. 102

Figure 3.2 Inhibitory effects of phthalate esters (DnBP, nBBP, DnOP, MiHP and MnBP) on the binding of [3H]CP55940 to mouse brain CB1 receptors in vitro. Each point represents the mean ± SEM of 3 independent experiments. Results provided by Ms Kathleen M. Bisset. .................................................................................................. 103

Figure 3.3 Inhibitory effects of phthalate esters (DEHP, DnHP, DiOP and M2EHP) on the binding of [3H]CP55940 to mouse brain CB1 receptors in vitro. Each point represents the mean ± SEM of 3 independent experiments. Results provided by Ms Kathleen M. Bisset. .................................................................................................. 104

Figure 3.4 The effect of nBBP and DnBP (both at 35 µM) on the equilibrium binding of of [3H]SR141716A to CB1 receptors of mouse whole brain. Kd and Bmax values are displayed for each treatment and 95% confidence intervals were as follows: control (Kd 0.628 to 0.859. Bmax 0.303 to 0.343), nBBP (Kd 0.761 to 1.333. Bmax 0.176 to 0.229) and DnBP (Kd 0.624 to 0.846. Bmax 0.120 to 0.136). R2 values were 0.9877 (control), 0.9756 (nBBP) and 0.9887 (DnBP). Data points represent the means ± SEMs of 3 independent experiments (most SEM bars are obscured by data symbols). ............. 105

Figure 3.5a Influence of nBBP (35 µM) and DnBP (50 µM) on the time course of association of [3H]SR141716A with CB1 receptors of mouse brain. In a) membranes received the standard 15 min preincubation with phthalate esters prior to [3H]SR141716A addition. In b) the phthalate ester and [3H]SR141716A were applied simultaneously. Data points represent the means ± SEMs of 3 independent experiments (most SEM bars are obscured by data symbols). ...................................................................................... 106

Figure 3.5b Influence of nBBP (35 µM) and DnBP (50 µM) on the time course of association of [3H]SR141716A with CB1 receptors of mouse brain. In a) membranes received the standard 15 min preincubation with phthalate esters prior to [3H]SR141716A addition. In b) the phthalate ester and [3H]SR141716A were applied simultaneously. Data points represent the means ± SEMs of 3 independent experiments (most SEM bars are obscured by data symbols). ...................................................................................... 107

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Figure 3.6 Dissociation of the [3H]SR141716A:CB1 receptor complex (initiated by challenge with 5 µM AM251) in the absence (control) or in the presence of 35 µM nBBP or 50 µM DnBP. Data represent mean ± SEM of at least 3 independent experiments, each performed in triplicate. .......................................................................... 108

Figure 3.7 Inhibition of CP55940-stimulated binding of [35S]GTPγS to mouse whole brain membranes by phthalate esters. Phthalate esters were assayed at 75 µM throughout. Each column represents the mean, and error bar the ± SEM of 7 independent experiments. ............ 109

Figure 3.8 Relationship between the ability of study compounds to inhibit the binding of [3H]CP55940 and CP55940-stimulated binding of [35S]GTPγS in mouse whole brain membrane fractions. All assays were performed 75 µM; r2 = 0.7844. ..................................................... 110

Figure 3.9 With WIN55212-2 present, BBP (at 30 µM but not 5 µM) enhances 4-AP-evoked L-glutamate release above the level produced by 4-AP alone. ............................................................................................. 111

Figure 3.10 With WIN55212-2 present, MnBP (both at 30 µM and 5 µM) does not enhance 4-AP-evoked L-glutamate release above the level produced by 4-AP alone. ...................................................................... 112

Figure 4.1 Concentration dependency of inhibition by chelerythrine (open circles), sanguinarine (solid circles), piperonyl butoxide (solid triangles) and (S)-methoprene (squares) on [3H]SR141716A binding to mouse brain CB1 receptors. IC50 and 95% confidence interval values are provided in Section 4.4.1. ....................................... 132

Figure 4.2 Effect of chelerythrine (1 µM; open circles), sanguinarine (1 µM; solid circles), piperonyl butoxide (30 µM; solid triangles) and (S)-methoprene (60 µM; squares) on equilibrium binding of [3H]SR141716A to mouse brain CB1 receptors. Control data points are identified by the diamond symbols. Kd values (as nM): control 0.51 ± 0.04; chelerythrine 0.47 ± 0.08; sanguinarine 0.46 ± 0.04; (S)-methoprene 1.5 ± 0.6 and piperonyl butoxide 2.5 ± 1.1. Bmax values (as pmol [3H]SR141716A/mg protein): control 0.79 ± 0.02; chelerythrine 0.32 ± 0.02; sanguinarine 0.50 ± 0.01; (S)-methoprene 0.44 ± 0.08 and piperonyl butoxide 0.56 ± 0.13. ............... 133

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Figure 4.3 Effect of chelerythrine (2.5 µM; open circles), sanguinarine (1.5 µM; solid circles), piperonyl butoxide (10 µM; solid triangles) and (S)-methoprene (20 µM; squares) on equilibrium binding of [3H]CP55940 to mouse brain CB1 receptors. Control data points are identified by the diamond symbols. Kd values (as nM): control 0.36 ± 0.07; chelerythrine 2.32 ± 0.43; sanguinarine 2.28 ± 0.77; (S)-methoprene 1.37 ± 0.25 and piperonyl butoxide 0.34 ± 0.19. Bmax values (as pmol [3H]SR141716A/mg protein): control 0.6 ± 0.03; chelerythrine 0.65 ± 0.06; sanguinarine 0.63 ± 0.11; (S)-methoprene 0.25 ± 0.02 and piperonyl butoxide 0.35 ± 0.05. ............... 134

Figure 4.4a Influence of study compounds on the time course of association of [3H]SR141716A and [3H]CP55940 with CB1 receptors of mouse brain. In a) membranes received a standard 15 min preincubation with sanguinarine (2.5 µM), chelerythrine (2.5 µM), piperonyl butoxide (30 µM) and (S)-methoprene (30 µM) prior to [3H]SR141716A addition. .................................................................... 135

Figure 4.4b Influence of study compounds on the time course of association of [3H]SR141716A and [3H]CP55940 with CB1 receptors of mouse brain.The same study compound concentrations were applied simultaneously with [3H]SR141716A.. .................................................. 136

Figure 4.4c The effects of benzophenanthridines (5 µM), piperonyl butoxide (20 µM) and (S)-methoprene (20 µM) on the association of [3H]CP55940 under preincubation conditions are shown Symbols: diamonds = control; solid circles = sanguinarine; open circles = chelerythrine; triangles = piperonyl butoxide and squares = (S)-methoprene.Data points represent the means ± SEMs of 3 independent experiments (a number of SEM bars are obscured by data symbols) ....................................................................................... 137

Figure 4.5a The influence of study compounds on the dissociation of CB1 receptor-selective radioligands. Figure 4.5a shows the effects of piperonyl butoxide (30 µM) and (S)-methoprene (60 µM) on the dissociation of [3H]SR141716A when initiated by challenge with a saturating concentration (5 µM) of SR141716A. ................................... 138

Figure 4.5b The influence of study compounds on the dissociation of CB1 receptor-selective radioligands. Figure 4 5b, defines the effects of sanguinarine (5 µM), chelerythrine (5 µM), piperonyl butoxide (30 µM) and (S)-methoprene (60 µM) when added alone on the dissociation of [3H]SR141716A from the [3H]SR141716A:CB1 receptor complex .................................................................................. 139

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Figure 4.5c The influence of study compounds on the dissociation of CB1 receptor-selective radioligands. In Figure 4 5c, the effects of sanguinarine (5 µM), chelerythrine (5 µM), piperonyl butoxide (20 µM) and (S)-methoprene (60 µM) on the dissociation of [3H]CP55940 when initiated by application of a saturating concentration (5 µM) of CP55940 are given. Symbols: diamonds = control; solid circles = sanguinarine; open circles = chelerythrine; triangles = piperonyl butoxide and squares = (S)-methoprene. Data represent mean ± SEM of at least 3 independent experiments, each performed in triplicate......................... 140

Figure 4.6 Relationship between concentration of (S)-methoprene and inhibition at CB2 receptors of mouse spleen based on interference with [3H]CP55940 binding. .................................................................... 141

Figure 4.7a Inhibition of 50 µM veratridine-evoked release of L-glutamate from mouse brain synaptosomes by 5 µM tetrodotoxin (TTX) ...................... 142

Figure 4.7b Failure of 5 µM TTX to modify 3 mM 4-AP-evoked release of L-glutamate from synaptosomes. ............................................................ 143

Figure 4.8 Partial inhibition of 4-AP-evoked release of L-glutamate from synaptosomes by the CB1-R agonist WIN55212-2, and full relief of WIN55212-2-dependent inhibition by the CB1-R antagonist AM251. ................................................................................................ 144

Figure 4.9 With WIN55212-2 present, sanguinarine (at 2 µM but not 0.25 µM) enhances 4-AP-evoked L-glutamate release above the level produced by 4-AP alone. ...................................................................... 145

Figure 4.10 With WIN55212-2 present, chelerythrine (at 2 µM but not 0.25 µM) enhances 4-AP-evoked L-glutamate release above the level produced by 4-AP alone. ...................................................................... 146

Figure 4.11 With WIN55212-2 present, (S)-methoprene (at 25 µM but not 5 µM) enhances 4-AP-evoked L-glutamate release above the level produced by 4-AP alone. ...................................................................... 147

Figure 4.12 With WIN55212-2 present, piperonyl butoxide (at 25 µM but not 5 µM) enhances 4-AP-evoked L-glutamate release above the level produced by 4-AP alone. ...................................................................... 148

Figure 5.1 Structures of tributyl and triphenyltin compounds examined in the present investigation. Structures were constructed using Isis Draw. ................................................................................................... 156

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Figure 5.2 Concentration-dependent inhibition of specific [3H]CP55940 binding to mouse brain CB1 receptors by tributyltin benzoate, tributyltin acetate and phenylethynyl tributyltin. Each data point represents the mean ± S.E.M. inhibition of specific [3H]CP55940 binding for at least three independent assays, each performed in triplicate. Experiments conducted by Mr. Saurabh Jain. This figure was originally published in the M.Sc. thesis of Mr. Saurabh Jain (Simon Fraser University, 2011). .......................................................... 157

Figure 5.3 Concentration-dependent inhibition of CP55940 (100 nM)-stimulated [35S]GTPγS binding by tributyltin benzoate and phenylethynyl tributyltin. Each data point represents the mean ± S.E.M. percentage inhibition of CP55940 stimulated [35S]GTPγS binding determined by three independent assays each performed in triplicate. These experiments were conducted by Mr Saurabh Jain and this figure was originally published in the M.Sc. thesis of Mr. Saurabh Jain (Simon Fraser University, 2011). .............................. 158

Figure 5.4 Modulation of WIN55212-2-dependent inhibition of 4-aminopyridine (4-AP-)-evoked release of L-glutamate from mouse brain synaptosomes by tributyltin acetate (TBT acetate). Typical release profiles are displayed with mean % changes (± SEM) to 4-AP-evoked and control release in the adjacent table. ........................... 159

Figure 5.5 Modulation of WIN55212-2-dependent inhibition of 4-aminopyridine (4-AP-)-evoked release of L-glutamate from mouse brain synaptosomes by phenylethynyl tributyltin (TBPE tin). Typical release profiles are displayed with mean % changes (± SEM) to 4-AP-evoked and control release in the adjacent table. .......... 160

xxi

Glossary

2-AG 2-Arachidonyl glycerol

2-AGE 2-Arachidonyl glycerol ether

4-AP 4-Aminopyridine

AEA Anandamide

Bmax Maximum concentration of binding sites

BSA Bovine serum albumin

CBD Cannabidiol

CB1-R Cannabinoid receptor-1

CB2-R Cannabinoid receptor-2

CHEL Chelerythrine

DAGL Diacylglycerol lipase

DSE Depolarization-induced suppression of excitation

DSI Depolarization-induced suppression of inhibition

DMSO Dimethylsulfoxide

EDTA Ethylenediamine tetraacetic acid

EGTA Ethylene glycol-bis(2-aminoethyl)-N,N,N’,N’-tetraacetic acid

EPSCs Excitatory post synaptic currents

eCB-STD Endocannabinoid mediated short term depression

eCB-LTD Endocannabinoid mediated long term depression

FAAH Fatty acid amide hydrolase

GPCR G-protein coupled receptor

GTP Guanosine-5’-triphosphate

GDP Guanosine-5’-diphosphate

GABA γ-Aminobutyric acid

IC50 Concentration effective in producing 50% inhibition

IPSCs Inhibitory post synaptic currents

KCl Potassium chloride

Kd Dissociation constant

Lys Lysine

L-GLU L-glutamic acid

MAGL Monoacylglycerol lipase

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MnBP Mono-n-butyl phthalate

METHO Methoprene

NADA N-Arachidonyl dopamine

NAPE N-Arachidonyl-phosphatidylethanolamine

nBBP n-Butylbenzylphthalate

PLD Phospholipase D

PI Phosphatidyl inositol

PLA1 Phospholipase A1

PMSF Phenylmethane sulfonyl fluoride

PBO Piperonyl butoxide

SANG Sanguinarine

TTX Tetrodotoxin

TBT Tributyltin

∆9 -THC ∆9- Tetrahydrocannabinol

∆8-THC ∆8- Tetrahydrocannabinol

VGSCs Voltage-gated sodium channels

VTD Veratridine

1

1. Introduction

1.1. Historical significance of cannabis use and cannabinoids

Cannabis sativa and its preparations have been used throughout the millennia for

recreational and various therapeutic purposes (Hollister, 2001). Cannabis sativa is one of the oldest cultivated plants in the history of humankind dating back at least 10,000

years (Jiang, 2006).

The history of cannabis use can be broadly classified into three eras, the early

Chinese/Indian era, the early Christian era through to the 18th century, and the era of

Western medicine of the 19th and 20th centuries (Zuardi, 2006).

1.1.1. The early Chinese/Indian era

The earliest references to the use of different parts of the cannabis plant were

documented in the Han dynasty in China (Zuardi, 2006). Fibers obtained from the stem

were used for preparing ropes, strings and paper, while fruits were used as food by the

ancient Chinese (Li, 1973).

The world’s oldest pharmacopoeia, Pen-ts’ao ching documented the use of

cannabis as a medicine for the treatment of rheumatic pain, constipation and disorders

of the female reproductive system (Zuardi, 2006). Other evidence for the use of

cannabis in ancient China was reported by Jiang et al. (2006), where a clay bowl

containing cannabis was discovered in the 2500 old Yanghai tombs of Northwestern

China. It is believed that stores such as this were probably used for medicinal purposes

and psychomanupulation (Russo et al., 2008).

The ancient Indian culture (around 1000 years B.C.) regularly employed

cannabis for medicinal and religious reasons (Zuardi, 2006). In ancient Indian medicine,

2

the plant was used for various purposes including induction of analgesia and hypnotic

states and reducing the occurence of epileptic seizures. It was also used as an

antiparasitic, an antispasmodic, an antibiotic, an expectorant and an aphrodisiac (Zuardi,

2006).

1.1.2. The period encompassing the early Christian era through to the 18th century

During this period, the use of cannabis gained increasing acceptance throughout

the Middle East and Africa. Around 1000 A.D., Arabic medical compendiums described

the use of cannabis as a plant beneficial in the treatment of diuretic disorders and

gastrointestinal problems including flatulence (Zuardi, 2006). In the 16th century,

cannabis was introduced to South America through the arrival of African slaves, while

Arab traders introduced cannabis to the European sub-continent firstly in Spain and then

to various Mediterranean countries including Italy (Zuardi, 2006).

1.1.3. The Western medicine era of the 19th and 20th centuries

Pioneering scientific studies and several books published by the Irish physicist

William B. O’Shaughnessy and the French psychiatrist Jacques-Joseph Moreau

facilitated the rapid introduction of cannabis to Western medicine. In their books, they

documented a range of therapeutic uses as well as psychomimetic and experimental

manipulations based on the use of cannabis and hashish (cannabis resin) (Di Marzo,

2006).

Western medicine readily accepted many of their proposed uses of cannabis

since during this period there were very few realistic therapeutic options available for the

treatment of disorders such as rheumatism, muscular spasms, pain and convulsive

states (Zuardi, 2006). Figure 1.1 summarizes the global spread of cannabis use from

South East Asia through Africa and South America to Europe and the USA (as published

by Zuardi (2006)), while Table 1.1 details the main landmarks in cannabinoid research

up until the late 1990s (Novarro and Fonseca, 1998).

3

Figure 1.1 The spread of the use of cannabis across the globe (Adapted from Zuardi, 2006).

4

Table 1.1 Major advances in the use of cannabis and research on the cannabinoid system of mammalian brain (Adapted from Navarro and Fonseca, 1998).

Event Date

Medical, ceremonial and recreational uses of Cannabis 3000 B.C. onwards

Isolation of psychoactive cannabinoids 1964

Discovery of synthetic cannabinoids 1980 onwards

Discovery of the cannabinoid-1 receptor ( CB1-R) in mammalian brain

1988

Mapping of the CB1-R in mammalian brain 1990

Cloning of the CB1-R 1990

Neuropharmacology of the CB1-R 1988-1995

Discovery and isolation of a natural cannabinoid anandamide in brain

1992-1995

Synthesis of diarylpyrazole CB1 receptor antagonists, (e.g. SR141716A and AM251)

1994

Isolation and identification of 2-arachidonyl glycerol (2-AG) as another important endocannabinoid

1995-1997

Functional neuroanatomy of CB1 receptors 1996-1997

Delineation of anandamide biosynthesis and its mechanism of uptake

1997

5

1.2. Cannabinoids

Gaoni and Mechoulam (1964) identified ∆9-tetrahydrocannabinol (∆9-THC)

(Figure 1.2) as the main psychoactive component of Cannabis sativa, a discovery that

eventually led to the synthesis of various analogs of ∆9-THC (Howlett et al., 2002).

Compounds that mimic the actions of the cannabis derivative ∆9-THC are defined

as cannabinoids (Howlett et al., 2002). A critical advance in cannabinoid research

occured with the discovery of specific membrane receptors to which ∆9-THC actively

binds in brain tissue (Devane et al., 1988). Matsuda et al. (1990) cloned and

characterized the first cannabinoid-1 receptor (CB1-R) while a cannabinoid-2 receptor

(CB2-R) was identified by Munro et al. in 1993.

Before the discovery of these receptors, the psychoactive actions of ∆9-THC and

related cannabinoids were assumed to arise from their ability to 1) dissolve in lipids

(Seeman et al., 1972), 2) modify the fluidity of synaptic plasma membranes (Hillard et

al., 1985) and 3) intercalate with lipids and other components of neuronal plasma

membranes (Pertwee, 1988).

Both CB1-Rs and CB2-Rs belong to the rhodopsin-like subfamily of receptors

which are G protein-coupled receptors (GPCRs) with seven transmembrane spanning

domains (TMH1-7). CB1-Rs and CB2-Rs were found to be sensitive to inhibition by

Pertussis toxin treatment, indicating that the response to cannabinoid drugs was

mediated through the Gi/o family of G proteins (Howlett et al., 1986).

Moreover, both CB1-Rs and CB2-Rs are found to have varied tissue distributions

in vertebrates. CB1-Rs are densely located in many regions of the central nervous

system with much lower levels in kidney, testis, uterus, heart and vascular tissue. On

the other hand, CB2-Rs are abundantly expressed in tissues of the immune system,

including spleen, tonsils and haematopoietic cells, but are found at much lower levels in

central nervous system (CNS) (Kano et al., 2009; Brown, 2007).

6

∆9-Tetrahydrocannabinol (∆9-THC)

∆8-Tetrahydrocannabinol (∆8-THC)

Figure 1-2 Structure of two important phytocannabinoids. Structures redrawn using ChemDraw Ultra 11.0 from structures reported in Pertwee et al. (2010).

7

G protein-coupled CB1-Rs and G protein-coupled CB2-Rs are differentiated on

the basis of predicted amino acid sequence, signaling mechanisms, affinity towards

specific agonists and antagonists and tissue distribution. They each share 48% amino

acid sequence homology and both have their G proteins coupled to adenylyl cyclase and

mitogen-activated protein kinase (MAPK) (Howlett et al., 2002). The CB1-R is larger than

the CB2-R with 13 more amino acid residues on the C terminal, an extra 72 amino acid

residues on the N terminal and 15 additional residues on the third extracellular loop

(Childers, 2006).

These G protein-coupled cannabinoid receptors are activated by certain

cannabis-derived compounds as well as endogenous lipid molecules termed

endocannabinoids. The endocannabinoids, their receptors and associated biochemical

machinery (including precursors, critical biosynthetic enzymes, degradative enzymes,

mediators and transporters) collectively constitute the endocannabinoid system (ECS).

The ECS represents a highly conserved system within all vertebrate phyla as well as

some invertebrates, with subtle structural differences in the structure of receptors,

enzymes and other components, thus underscoring the importance of the ECS for

survival of many life forms (De Petrocellis et al., 2004).

1.2.1. G protein-coupled receptors (GPCRs) and their activation cycle

GPCRs are seven transmembrane spanning receptors and are coupled to

specific heterotrimeric guanine nucleotide-binding proteins (G proteins) (Drake et al.,

2006). G proteins transduce an extracellular signal to an intracellular effector (Drake et

al., 2006). These receptors represent an attractive target for drug discovery and it has

been estimated that nearly half of the drugs marketed today target GPCRs (Kroeze et

al., 2003).

G proteins are made up of a monomer (Gα subunit) and dimer (Gβ and Gγ

subunit). In their inactive state, the Gα subunit is bound to guanosine diphosphate (GDP)

and exists as Gα(GDP)βγ (Harrison and Traynor, 2003). When activated, the Gα subunit

exchanges GDP for guanosine-5’-triphosphate (GTP) and this binary complex (Gα-GTP)

then detaches from the Gβγ subunit to act on different effectors (Griffin et al., 1998;

8

Harrison and Traynor, 2003). Inactivation of GPCRs occurs by the intrinsic GTPase

activity of the Gγ subunit which hydrolyses GTP to GDP. Finally, Gα-GDP and Gβγ

subunits recombine to form the inactive Gα(GDP)βγ.

1.2.2. The [35S]GTPγS binding assay

The [35S]GTPγS binding assay is a functional assay that can be employed to

measure the extent of G protein activation following the binding of ligand(s) to the GPCR

(Breivogel and Childers, 2000; Breivogel et al., 2001; Harrison and Traynor, 2003). It is

an excellent assay to measure the primary functional event that immediately follows the

activation of the GPCR by its ligand (Harrison and Traynor, 2003).

This assay is characterized by the replacement of endogenous GTP by

[35S]GTPγS which binds to Gα subunit to form the Gα[35S]GTPγS complex. The γ-

thiophosphate bond on [35S]GTPγS is highly resistant to GTPase-mediated hydrolysis

and therefore inactivation of the GPCR cycle is blocked and the extent of activation can

be conveniently quantified by measuring the [35S]-label bound (Griffin et al., 1998;

Harrison and Traynor, 2003).

1.3. Other cannabinoid receptors

Besides CB1-Rs and CB2-Rs, other two GPCRs, GPR55 and GPR119 (Lambert

and Muccioli, 2007) have been proposed as novel cannabinoid receptors based on their

affinity towards endocannabinoids. There is much ongoing debate in the scientific

community regarding classification of these receptors as genuinely cannabinoid

selective (Okunu and Yokomizo, 2011). Oka et al., (2007) reported the activation of

GPR55 by lysophosphatidylinositol derivatives but not cannabinoids. In marked

contrast, Lauckner et al., (2008) found that AEA and ∆9-THC increased intracellular

calcium in a cell line expressing GPR55. Nevertheless, this review will be focused

mainly on CB1-Rs and CB2-Rs. Phylogenetic analysis by Brown (2007) revealed that

CB1-Rs and CB2-Rs belong to family of lipid receptors (formerly endothelial

differentiation gene receptors (EDG)) which are activated by acylethanolamide

9

analogues typified by 2-arachidonylglycerol (2-AG) and anandamide

(arachidonylethanolamide, AEA).

Sharir et al., (2010) described the work of researchers at AstraZeneca who found

that nanomolar concentrations of cannabinoid agonists stimulate [35S]GTPγS binding

and this response was antagonized by cannabidiol, a natural product cannabinoid

receptor antagonist. The ion channel, transient receptor potential vanilloid 1 (TRPV1)

share several similarities with cannabinoid receptors in terms of intracellular signaling,

shared ligand and tissue distribution, their role in pathophysiological conditions and

binding of the endocannabinoid/endovanilloid anandamide (AEA) (Starowicz et al.,

2007).

1.4. Cannabinoid-1 Receptors (CB1-Rs)

1.4.1. The structure and activation of CB1-Rs

The first cloning and expression of a 473 amino acid CB1-R from rat brain was

achieved by Matsuda et al. (1990) (Figure 1.3). The human homolog of 472 amino acids

was reported by Gerard et al. (1990) and a 473 amino acid sequence from mouse brain

was identified by Chakrabarti et al. (1995). Significantly these three CB1-Rs exhibit 97-

99% amino acid sequence homology (Kano et al., 2009). Like any GPCR, the CB1-R is

an integral membrane protein consisting of seven hydrophobic transmembrane helices

(7TMH) linked by three extracellular (E1-3) and three intracellular loops (I1-3) which are

flanked by an N-terminal on the periplasmic domain and a C terminal on the cytoplasmic

domain (Montero et al., 2005). Between the cytoplasmic extension of TMH7 and the proximal C terminus lies another helix designated helix 8, which runs parallel to the

cytosolic membrane surface (Patny et al., 2006). The cytoplasmic regions are

responsible for G protein binding, desensitization and cellular signal trafficking. Binding

of an agonist triggers activation of the heterotrimeric G proteins by exchanging GTP for

GDP on the α subunit. This leads to the dissociation of G proteins from receptors and

cleavage of α and β/γ subunits which in turn modulate downstream effectors (Stadel et

al., 2011). According to the two state model proposed for GPCRs, CB1-Rs exist in the

active (R) state and the inactive (R*) state (Samama et al., 1993; Gullapalli, 2010).

10

These states are in equilibrium in the absence of ligand and, following ligand binding, the

equilibrium can shift to either state. Thus, an agonist will actively bind to the active state

of this receptor while an inverse agonist will bind to the inactive state. A classical

antagonist will be overall neutral having an equal affinity towards both R and R* the state

of the receptors. These properties can be conveniently studied in CB1-Rs due to the

availability of selectively acting agonists (e.g. ∆9-THC, CP55940 and WIN55212-2),

antagonists (e.g. cannabidiol and AM251) and the inverse agonist (SR141716A)

(Gullapalli, 2010; Gatley et al., 1997; Herkenham et al., 1990; Pertwee, 2006; Rinaldi-

Carmona et al., 1994). Studies by various research groups have greatly improved our

understanding the role of CB1 transmembrane helices (TMH), extracellular loops (ECs)

in particular the E2 loop and the carboxyl terminus in cannabinoid binding and receptor

activation (Shim et al., 2011a; Shim et al., 2011b, McAllister, 2003; Ahn et al., 2009;

Stadel et al., 2011). However, despite much effort, little information is available on the

way in which ligands orient and dock at their respective active sites within the CB1-R

binding pocket. Chin et al. (1998) and Song and Bonner (1996) showed that the

hydrogen bonding interaction between residue K192 on the TMH and the phenolic

hydroxyl group of CP55940 and the carboxamide oxygen of the inverse agonist

SR141716A (Hurst et al., 2002, 2006) were critical for ligand binding. Moreover, residue

S383, which has been proposed to induce a bend in the TMH7, again appears essential

for agonist (CP55940) binding (Kapur et al., 2007). In addition, C386 (on TMH7) has

also been implicated as a critical amino acid for SR141716A binding (Fay et al., 2005).

McAllister et al., (2003) demonstrated that aromaticity at TMH5 (imparted by

residues such as F201, W280 and W357) was important for accomodating the agonist

CP55940 and inverse agonist SR141716A within the binding pocket of the CB1-R. By

employing molecular modeling, McAllister and associates also proposed that the binding

pocket for various ligands was primarily located in the hydrophobic transmembrane helix

bundle of the receptor. In related studies, Shi and Javitch (2004) found that the

extracellular loops also play a vital role regards ligand recognition, ligand sensitivity and

access of the ligand to the binding pocket of dopamine receptors (which are also a

GPCRs). This observation also proved relevant for CB1-Rs, since Ahn and coworkers

(2009) showed that the second extracellular loop (E2) was important for ligand binding

and receptor localization. They were also able to demonstrate that alanine (Ala)

11

mutations in the C terminal residues on E2 led to reduced agonist binding but had no

effect on the binding of inverse agonists. In addition, Bertalovitz et al. (2010) reported

that point mutations on the C terminal region of E2 can lead to loss of agonist and

antagonist binding capacity of CB1-Rs but have no effect on inverse agonist binding.

Shim et al. (2011) further elucidated the structure of the E2 loop by using a

combination of simulated annealing and molecular dynamics simulations. They studied

the molecular structure of E2 in two forms, disulphide (E2disulphide) and dithiol (E2dithiol). It

was found that that E2disulphide helical segment has a amphipathic alignment (at the

membrane:water interface) which stabilizes the receptor and imparts greater flexibility

compared to E2dithio. This further led to E2/TMH coupling and rearrangement of the TMH.

These coupling/interactions and TMH rearrangement are important for receptor

activation. However, the extent of this coupling was distinct in both the forms of E2.

Since in CB1 E2disulphide E2 offers more flexibility, the C terminal region of E2 inserts into

the extracellular H3/H5 region causing H5 to move away from H3 and H6 to move into

H3 at the extracellular region, leading to efficient coupling of E2 to TMH. However, in

CB1 E2dithiol, E2 has reduced flexibility leading to less insertion of E2 C terminal residues

and thus weak coupling of E2/TMH. This work confirmed the importance of the E2 C

terminal for receptor activation and also served as supporting evidence of the original

findings by Ahn et al. (2009).

12

Figure 1.3 Two dimensional representation of the CB1-R (Adapted from Shim et al., 2011).

13

Figure 1.4 Diagramatic representation of the C terminal domain of the CB1-R (Adapted from Stadel et al., 2011)

14

The C terminal is responsible for interaction with G proteins, CRIP 1a, protein

kinases, arrestins and with itself or other receptors to form a dimer (Ahn et al., 2009)

(Figure 1.4). Several characteristic features of the C terminal sequence such as

transmembrane interaction sites, palmitoylation sites, phosphorylation sites and the PDZ

binding domain make it a potential candidate for roles in biogenesis, receptor localization

and activity (Ahn et al., 2009).

The CB1-R carboxy terminus has 73 amino acid residues (R400-L472) and differs

from CB2-R by being 14 residues longer. Despite rough similiarities in length, the sharing

of a few ligands and participation in similar signal pathways, there is no significant

homology between the C termini of these receptors (Bramblett et al., 1995; Xie and

Chen, 2005; Choi et al., 2005). Hydropathy plot analyses conducted by Kyte and Dolittel

(1982) predicted the C terminus of the CB1-R to be less hydrophobic than the equivalent

region of CB2-R, but there remained difficulties in purifying the full length CB1 terminus

because of its high flexibility and relatively unstructured nature. Not until 2009 was this

problem overcome when successful purification of a peptide corresponding to the full

length CB1-R carboxy terminus was achieved. NMR analysis using doubly tagged (15N

and 13C) full length C terminus in dodecylphosphocholine revealed the presence of two

amphipathic α helical domains (Ahn et al., 2009). Importantly, Ahn and associates were

also successful in identifying the specific amino acids of these helical domains (S401-

F412 and A440-M461, respectively) thus suggesting an amphipathic role for each. They

also reported that the hydrophobic face of each helix was intimately associated with the

membrane surface, thus stabilizing the helical domains for binding with other proteins

involved in receptor function, while the polar face of the helices was able to project into

the cytosol (Ahn et al., 2009). Besides its role in receptor function, the C terminus has

been found to be a requirement for receptor exit from endoplasmic reticulum (Tai et al.,

1999; Bermak et al., 2001; Duvernay et al., 2004, Robert et al., 2005).

The cytoplasmic extension of TMH7 is characterized by the presence of a highly

conserved motif within the rhodopsin class A GPCRs, NPXXY and is termed Helix 8

(Patny et al., 2006; Tiburu et al., 20011). This amphipathic helical domain is an integral

part of the intracellular GPCR binding connection to G proteins (Tiburu et al., 2011;

Rosenbaum et al., 2009, Fritze et al., 2003) and is reported to play a crucial role both in

15

ligand recognition and signal transduction (Tiburu et al., 2011). The distal region on the

C-terminus carries another helical domain, Helix 9. However, Helix 9 has only recently

been identified within the CB1-R and very little work has so far been done in defining the

precise structure and role of this helix (Ahn et al., 2010).

TMH7/H8 region has always been an area of interest in probing various structural

determinants involved in activation of the GPCR cycle. Tiburu et al. (2011), using solid

state nuclear magnetic resonance (NMR) and site directed spin labeling-electron

paramagnetic resonance (SDSL/EPR) demonstrated short range electrostatic

interactions between TMH/H8 (with its conserved motif and proline kink) and the

phospolipid bilayer/membrane microenvironment. By employing local helix distortion

studies, they postulated that the conserved but flexible NPXXY motif likely plays a vital

role in ligand binding and signaling events involving TMH7/H8 (Tiburu et al., 2011;

Tiburu et al., 2009; Tyukhtenko, 2009; Hall et al., 2009). Furthermore, experiments

conducted by Tiburu et al. (2011) demonstrated dynamic functional interactions between

TMH7/H8 and the membrane phospholipid environment which modifies the membrane

bilayer structure at discrete loci. These findings hinted at a potential mechanochemical

role of the phospholipid bilayer in mediating CB1/GPCR signal transmission and hence

signal transduction.

Helix 8 is also reported to have a contributory role towards receptor signaling by

interaction with intracellular loops (ICs). This helix interacts with distal part of the C

terminus, offering it rigidity and potential to interact with the third intracellular loop (IC3).

This interaction is essential for proper receptor signaling (Ahn et al., 2010). Moreover,

Swift et al., (2006) identified various noncovalent interactions between helix 8, TMH 7

and IC1, which again were important for receptor signaling. Several studies in various

laboratories indicated a possible role of Helix 8 in receptor biosynthesis, folding and

trafficking (Oksche et al., 1998; Duvernay et al., 2004; Thielen et al., 2005).

Studies by Ahn et al., (2010) helped to understand the importance of this helical

domain (H8) for ligand binding and activation. As mentioned earlier, this helix is

amphipathic with leucines and/or phenylalanines and basic residues common to both

hydrophobic and hydrophilic faces. Ahn and coworkers employed point mutations in

several key residues on both faces. The first mutant involved substitution of the

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hydrophobic groups, Leu404, Phe408 and Phe412 with alanine to reduce hydrophobicity

while the second involved replacement of the basic residues, Lys402, Arg405 and

Arg409 with glutamine to remove positive charge. The first mutant yielded low Bmax

values (based on saturation binding isotherms), minimal Emax (from [35S]GTPγS binding

studies) and defective localization when compared to the wild type CB1-R. Intriguingly,

the second mutant was virtually identical when compared with the wild type with respect

to the same parameters as the first mutant. Circular dichroism spectroscopy further

revealed that intact hydrophobic residues were indeed vital for maintenance of the helix

while positively charged residues could be easily replaced by less polar or neutral

residues without affecting the structure/function of the helix. These data supported the

importance of the formation of this helical domain for receptor localization and hence

ligand binding and activation. Moreover, various other groups have independently

reported that a defective H8 leads to impaired receptor localization and β arrestin

translocation to the plasma membrane (Suvorova et al., 2009; Yasuda et al., 2009; Ahn

et al., 2010).

Apart from this, the L404F point mutation on H8 domain displayed faster agonist

induced internalization when compared to the wild type, thus underlying its importance in

CB1-R trafficking (Anavi-Goffer et al., 2007).

Helix 9 (H9) consists of charged residues which have been implicated in the

formation of contacts with the cytoplasmic helical extension of TMH5, TMH6 and IC3.

Schertler (2008) suggested the probable role of hydrophobic residues on H9 as a point

of contact with the G protein (Gq).

Besides, extracellular loops, TMH/H8 and H9, distal C terminus ((∆418-472) of

the CB1-R have also been implicated in receptor localization, receptor stability, G protein

binding, desensitization, intracellular sorting during internalization and cellular trafficking

of the receptor (Stadel et al., 2011; Ahn et al., 2009).

Truncation of the CB1 distal C terminal domain led to changes in the magnitude

and kinetics of Ca2+ current inhibition via G protein coupling, suggesting the role of this

domain in cellular signal regulation (Nia and Lewis, 2001). Chillakuri et al. (2007)

demonstrated that after deletion of this domain of the CB1-R expressed in insect cells,

17

Sf9 resulted in a two-fold increase in receptor production and increased basal activity as

compared to wild type. Moreover, residues 418-439 on this domain were found to be

important for receptor desensitization but not internalization, while internalization was

affected when residues within the 460-473 were phosphorylated (Hsieh et al., 1999; Jin

et al., 1999).

Despite the accumulation of much data concerning the role of various domains of

theCB1GPCR, much more research is needed to completely understand the subtle

complexities of this fundamental signaling unit.

1.4.2. The distribution of CB1-Rs in mammalian brain

Understanding the distribution pattern of cannabinoid receptors in brain was

made possible by employing ligand binding studies with the highly specific synthetic

agonist [3H]CP55940 (Herkenham et al., 1991; Herkenham et al., 1990; Mailleux and

Vanderhaeghen, 1992).

The binding of [3H]CP55940 was found to be widely distributed with intensities

dependent on the area of brain concerned, with the general pattern of binding conserved

across mammalian species. The inner most layers of the olfactory bulb, the

hippocampus (in particular the dentate molecular layer and the CA3 region), the lateral

striatum, globus pallidus, entopenduncular nucleus, substantia nigra, pars reticularis and

the cerebellar molecular layer displayed the highest levels of binding of [3H]CP55940,

while moderate levels were found in the cerebral cortex, septum amygdala,

hypothalamus, lateral subnucleus of interpeduncular nucleus, parabrachial nucleus,

nucleus of the solitary tract and the spinal dorsal horn. Low ligand binding was noted

particularly in the thalamus, and various other nuclei in the brain stem and the spinal

ventral horn (Kano et al., 2009; Herkenham, 1990).

The telencephalic and cerebellar regions, where high intensity binding of

[3H]CP55940 occurs, control motor and cognitive function which can explain the

profound effect of cannabinoids on motor and cognitive responses, while lower brain

stem which controls cardiovascular and respiratory functions exhibited low density

binding. This latter distribution is compatible with the fact that high doses of

cannabinoids are not fatal (Herkenham et al., 1991; Mailleux and Vanderhaeghen,

18

1992). Similarly, moderate binding in spinal dorsal horn is consistent with the analgesic

action of intrathecally administered cannabinoid. Moreover, the anti-anorexic and anti-

emetic actions of cannabinoid agonists are likely achieved by moderate binding in the

ventromedial hypothalamic nucleus (located in the hypothalamus and amygdala) which

forms a major part of the satiety center (Kano et al., 2009).

The CB1-R expression pattern has been further investigated by histochemical

analysis. Depending on the region of brain, two distinct forms or labelings of CB1-R

mRNA expression have been reported, specifically, uniform labeling and non-uniform

labeling (Mailleux and Vanderhaeghen, 1992; Matsuda et al., 1993). Uniform labeling

reflecting a large number of cells expressing high level of CB1-R mRNA was detected in

the striatum, thalamus, hypothalamus, cerebellum and lower brain stem. In contrast,

non-uniform labeling resulting from a low number of cells expressing high levels of CB1-

R mRNA was found in the cerebral cortex, amygdala and hippocampus (Kano et al.,

2009). Furthermore, CB1-R expression is invariably greater at inhibitory synapses than

excitatory synapses; however the density of receptors at inhibitory synapse varies

considerably according to brain region. CB1-R density as assessed by immunogold

labeling was 30 times higher on inhibitory synapses than excitatory ones for

hippocampal CA1 pyramidal cells, 6 times for Purkinje cells and nearly 4 times higher for

striatal medium spiny neurons (Kawamura et al., 2006; Uchigashima et al., 2007).

1.5. CB1-R-mediated intracellular signaling pathways

1.5.1. Inhibition of cyclic AMP (cAMP) Activation of the CB1-R following agonist binding triggers a cascade of multiple

signal transduction pathways, a finding supported by [35S]GTPγS assays and studies

examining Pertussis toxin sensitivity of cannabinoid-dependent effects (Pertwee, 1997).

The first characterized pathway for agonist-stimulated CB1-R activation was the

inhibition of adenylyl cyclase and this effect was completely blocked by Pertussis toxin

indicating that it was mediated through Gi/o proteins (Howlett, 1985; Howlett and Fleming,

1984; Howlett et al., 1986). Further evidence towards the role of CB1-R and Gi/o proteins

was supported by work of Derkinderen and co-workers (1996 and 2001) who reported

19

that cannabinoid receptor stimulation led to Tyr-phosphorylation of the focal adhesion

kinase in hippocampal slices and this effect was readily blocked by both SR141716A

and Pertussis toxin.

In their activated states, Gi/o proteins were found to regulate adenylyl cyclase

isoforms 1,3,5,6 or 8 since co-expression of these isoforms resulted in CB1-R-mediated

inhibition of cAMP accumulation (Rhee et al., 1998).

Depending upon the precise signal transduction pathway, different subtypes of

Gi/o were found to be activated by the same ligand, agonist, antagonist or inverse agonist

indicating a CB1-R-biased signaling mechanism (Kenakin, 2007; Houstan and Howlett,

1998; Glass and Northup, 1999; Mukhopadhyay et al., 2002).

In N18TG2 cells, WIN55212-2 displayed agonist behavior for Gi1, Gi2, and Gi3 while desacetyllevonantradol (a CB1-R agonist) was an agonist at Gi1, Gi2 and an inverse

agonist at Gi3. Methanandamide (an agonist and stable analog of anandamide) behaved

as an inverse agonist at Gi1 and Gi2, but it displayed full agonist activity at Gi3.

Interestingly, SR141716A acted as an inverse agonist at all three G protein subtypes

(Mukhopadhyay and Howlett, 2005; Turu and Hunyady, 2010). Furthermore, Houston

and Howlett (1998) suggested the existence of different affinity states for the CB1-R

representing different conformations. This proposal was supported by Georgieva and co-

workers (2008) where, using plasmon-waveguide resonance spectroscopy they found

that the CB1-R assumed distinctly different conformations when bound by CP55940 or

WIN55212-2.

Deadwyler et al. (1995) and Hampson et al. (1995) reported that in hippocampal

cells, agonist-mediated stimulation of the CB1-R decreased intracellular cAMP, lowered

net dephosphorylation of ion channels, activated A-type potassium currents and in turn

led to hyperpolarization of the membrane. The cyclic AMP-dependent protein kinase

(PKA) pathway as regulated by CB1-Rs is intimately involved in synaptic plasticity and

neuronal remodeling (Howlett, 2005).

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1.5.2. Stimulation of cAMP production

Intriguingly and in contrast to the above studies, CB1-R-mediated increases in

cAMP concentrations have also been observed in response to treatment with

cannabinoids (Howlett, 2005). Maneuf and Brotchie (1997) found an increase in basal

cAMP production in globus pallidus slice preparations treated with cannabinoid agonists

such as CP55940 and WIN55212-2.

Surprisingly, the order of potency of these agonists was similar in mediating this

effect through the CB1-R, and SR141716A was a competitive inhibitor for both inhibitory

and stimulatory components of this mechanism (Bonhaus et al., 1998).

Three mechanisms have been put forward by different research groups to

explain this observation. The first mechanism suggested the possibility of endogenous

synthesis of an adenylyl cyclase activator possibly CB-R-mediated synthesis of

prostaglandins (Burstein et al., 1986, 1994). Here prostaglandins might operate as

cellular stimulators for cannabinoid-mediated cAMP production (Hillard and Bloom,

1983).

The second possibility relates to potential differences in the type of adenylyl

cyclase isoforms expressed by the target cells and the capacity of expressed isoform to

respond to Gi/o-mediated regulation (Howlett, 2005). Agonist stimulation of recombinant

cannabinoid receptors co-expressing adenylyl cyclase isoforms of the 5/6 family or the

1/3/8 family displayed inhibition of adenylyl cyclase as a result of inhibition by Gi (α-

subunit), while those expressing isoforms of the 2/4/7 family led to stimulation of

adenylyl cyclase as a result of the increased Gs response by the Gi (βγ subunit) released

following cannabinoid receptor stimulation (Rhee et al., 1998).

The third mechanism could involve direct interaction between CB1-Rs and Gs. It

was reported that neurons and CHO cells expressing recombinant CB1-Rs pre-treated

with Pertussis toxin, supported cannabinoid agonist-mediated stimulation of cAMP

(Glass and Felder, 1997; Felder et al., 1998; Bonhaus et al., 1998)

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1.5.3. CB1-Rs and the modulation of Ca2+ fluxes and phospholipases C and A

Sugiura and co-workers (1996, 1997, 1999) using a fura-2 fluorescence assay,

reported cannabinoid and endocannabinoid-mediated increases in intracellular free

calcium in N18TG2 neuroblastoma and NG108-15 neuroblastoma-glioma hybrid cells.

This response was due to a CB1-R and Gi/o interaction since it was blocked by both

SR141716A and Pertussis toxin. Other data also support the ability of cannabinoid

receptors to signal through an inositol 1,4,5-triphosphate (IP3)-Ca2+ mobilization pathway

(Howlett,