Actions of benzophenanthridine alkaloids and various synthetic compounds on the cannabinoid-1 (CB 1 ) receptor pathway of mouse brain with particular reference to the effects on [ 3 H]CP55940 and [ 3 H]SR141716A binding, interference with basal and CP55940-stimulated [ 35 S]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.
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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.
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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
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.
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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
1.4.1. 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
1.9. 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-
1.14. Termination of eCB-STD ...................................................................................... 39 1.15. Endocannabinoid-mediated long term depression (eCB-LTD) .............................. 41
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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
1.20.2. Therapeutic aspects of CB2-R modulators ................................................. 54 1.21. Brief overview of the test chemicals used in my research ..................................... 55
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
2.3.1. 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
3.3.1. 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
3.7. 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
4.3. 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
5.3.1. 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
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6. Conclusion and future prospects .................................................................... 162
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
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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
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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
xix
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
xx
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
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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
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
xxii
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
Unlike anandamide, 2-AG displays full agonism at the CB1-R. Electrical
stimulation (Stella et al., 1997) in hippocampus as well as ionomycin treatment in
N18TG2 neuroblastoma cells (Bisogno et al., 1997) leads to increased levels of 2-AG.
Several biochemical pathways have been suggested for biosynthesis of 2-AG
(Figure 1-7), however the more important pathway involves phospholipase C (PLC) and
diacylglycerol lipase (DAGL) (Kano et al., 2009). The first step involves formation of
diacylglycerol (DAG) (which has an arachidonic acid moiety) by enzymatic hydrolysis of
arachidonic acid containing membrane phospholipids (like phosphatidylinositol) by PLC.
In the second step, DAGL acts on DAG to generate 2-AG (Kano et al., 2009). Other
biosynthetic pathways of possible relevance include those reactions leading to the
production of lysophospholipid from membrane phospholipid initially mediated by
phospholipase A1 (PLA1) and final formation of 2-AG by the action of Lyso-PLC on PLA1
(Sugiura et al., 1995; Tsutsumi et al., 1994; Ueda et al., 1993). Nakane et al. (2002)
suggested that 2-AG could also be formed by the action of a phosphatase on 2-
arachidonoyl lysophosphatidic acid (2-arachidonoyl LPA), while the generation of 2-
arachidonoyl phosphatidic acid from 1-acyl-2-arachidonoylglycerol has been suggested
by Bisogno et al. (1999) and Carrier et al. (2004) (Figure 1.7).
31
Figure 1.7 Metabolic pathways for biosynthesis of 2-AG (Adapted from Kano et al., 2009).
32
1.9. Degradation pathways for endocannabinoids
After endocannbinoids are released they can be degraded by two main
mechanisms i.e. hydrolysis and oxidation (Vandevoorde and Lambert, 2007). The
hydrolytic pathway involves the breakdown of anandamide by the action of fatty acid
amide hydrolase (FAAH) and 2-AG by monoacylglycerol lipase (MAGL) while the
oxidative pathway includes oxidation of the arachidonic acid moiety of endocannabinoids
by cyclooxygenase (COX) and lipoxygenase (LOX).
FAAH was first identified, purified and cloned from rat liver by Cravatt et al.
(1996). Formerly named as ‘anandamide amidohydrolase’, FAAH was identified in the
brain and many other organs. FAAH was reported to sensitive to the serine protease
inhibitor phenylmethylsulfonyl fluoride (PMSF), and was also found to act on other fatty
acid amides but with anandamide as the preferred substrate (Cravatt et al., 1996;
McFarland and Baker, 2004). In vitro studies have revealed that FAAH has the ability to
hydrolyse the ester linkage of 2-AG; however, this activity was minimal in vivo (Cravatt et
al, 1996). However, the importance of FAAH in anandamide breakdown was underlined
further when FAAH knockout mice were reported to be more responsive towards
exogenously administered anandamide (Cravett et al, 2001).
Tornqvist and Belfrage (1976) were the first to describe MAGL but this enzyme
was not cloned until 1997 by Karlsson and co-workers (1997) who cloned it from a
mouse adipocyte cDNA library. In vivo, MAGL is found to be the main enzyme that
catalyzes the hydrolysis of 2-AG (Dinh et al., 2002; Dinh et al., 2004; Vandevoorde and
Lambert, 2007). MAGL has 303 amino acids and is present in various organs including
brain. This enzyme accounts for about 85% of 2-AG degradation, while the remainder is
attributed to two less characterized enzymes namely, ABHD6 and ABHD12 (Blankman
et al., 2007).
Among the three known forms of COX enzymes in mammalian tissues, COX-2
accepts anandamide as a substrate generating prostaglandin-ethanolamides
(Vandevoorde and Lambert, 2007). Anandamide and 2-AG also apparently serve as a
33
substrates for LOX, however much less work has been done in this area (Chen et al.,
1994).
1.10. Transport of endocannabinoids
A two step process mediates the extracellular removal of endocannabinoids, the
first being transport into the cells followed by enzymatic hydrolysis (McFarland and
Baker, 2004; Kano et al., 2009).
Anandamide (AEA) transport has been widely studied and compared to 2 AG.
AEA uptake has been reported in cortical neurons (Fegley et al., 2004), striatal neurons,
astrocytes (Di Marzo et al., 1994; Beltramo et al., 1997) and cerebellar granule cells
(Hillard et al., 1997). Various aspects of AEA transport have been noted by research
groups. It has been reported that AEA transport is (i) temperature sensitive (ii) inhibited
by certain fatty acid amide derivatives (iii) relatively fast (t1/2 approx. 2.5 minutes) (iv)
controlled by other signal transduction pathways and (v) saturable at 37oC (Di Marzo et
al., 1994; Beltramo et al., 1997; Hillard et al., 1997; Maccarrone et al., 1998; Maccarrone
et al., 2000; Rakshan et al., 2000).
Several mechanisms have been advanced to explain the cellular uptake of AEA
including a protein carrier-mediated process, facilitated diffusion regulated by FAAH,
AEA sequestration by cellular machinery and endocytotic uptake of AEA (McFarland and
Baker, 2004).
Accumulation of a substrate on the cis side of the membrane leads to carrier
protein accumulation on the trans side of the membrane thus causing movement of an
extracellular substrate into intracellular compartment against a concentration gradient, a
phenomenon termed as ‘trans flux coupling’ (McFarland and Baker, 2004). ‘Trans flux
coupling for AEA was observed in cerebellar granule neurons (Hillard and Jarrahian,
2000). The intracellular presence (or expression) of FAAH positively modulated the
cellular uptake of AEA (McFarland and Baker, 2004). Neuroblastoma or glioma cells
expressing FAAH when treated with the FAAH inhibitor,
methylarachidonylflurophosphonate (MAFP) display close to a 50% reduction in AEA
accumulation (Deutsch et al., 2001). Furthermore, Day et al., (2001) showed a 2-fold
34
increase in the uptake of AEA following FAAH transfection of HeLa cells (which are
otherwise devoid of FAAH activity). Glaser et al. (2003) suggested FAAH-dependent
facilitated diffusion as a mechanism for cellular uptake of AEA. Kinetic analysis by
Glaser et al. (2003) demonstrated that AM404 (an AEA transport inhibitor and FAAH
inhibitor, Hillard and Jarrahian, 2000), when added at the 5 min time point, significantly
decreased AEA cellular accumulation, while no such effect was reported at earlier time
points (25 sec and 45 sec). This observation was consistent in both neuroblatoma cells
(with FAAH activity) and astrocytoma cells (without FAAH activity) (Glaser et al. 2003).
Hence, these researchers concluded that AEA accumulation was saturable at 5 min time
and this coincided with the inhibition of downstream components of AEA uptake
especially FAAH (Glaser et al., 2003).
Besides the above mentioned mechanisms, Hillard and Jarrahian (2003)
hypothesized that AEA could be taken up through sequestration by cellular components.
They found that radiolabeled AEA reached intracellular concentrations that were higher
than those in the extracellular media (Hillard and Jarrahian, 2003). They suggested the
existence of two distinct intracellular pools of AEA, one was free AEA and the other was
AEA sequestered by a cellular component. This sequestration of AEA is saturable and
may involve certain membranous compartments that serve as reservoir for this lipophilic
molecule (Hillard and Jarrahian, 2003). Since the AEA that is sequestered or bound is
not available for free diffusion across plasma membrane, it generates a positive inward
concentration gradient of AEA that is maintained by FAAH (Hillard and Jarrahian, 2003;
McFarland and Lambert, 2004).
The final model or mechanism proposed for AEA degradation is through the
caveolae-related endocytotic process (McFarland et al., 2003; McFarland et al., 2004).
McFarland (2004) showed that by inhibiting the caveolae-related endocytosis process
(by treating cells with N-ethylmaleimide and tyrosine kinase inhibitor, genistein), there
was marked reduction of cellular accumulation of AEA (McFarland et al., 2004)
As mentioned earlier, few studies have been directed towards understanding the
cellular uptake and degradation of 2-AG (Kano et al., 2009). However, some reviews
suggest the existence of a very similar mechanism for 2-AG transport as for AEA
(Beltramo and Piomelli, 2000; Bisogno et al., 2001; Piomelli et al., 1999).
35
1.11. Endocannabinoid-mediated short term depression (DSI and DSE)
In 2001, four research groups demonstrated independently that
endocannabinoids mediate synaptic plasticity through retrograde signaling in the CNS
and play a vital role towards short term and long term synaptic tonicity (Wilson et al.,
2001; Kreitzer et al., 2001; Ohno-Shosaku et al., 2001; Maejima et al., 2001).
Endocannabinoids are biosynthesized de novo and then released into the synaptic cleft
either tonically under basal conditions or in an activity-dependent manner (Kano et al.,
2009, Katona and Freund, 2008). The endocannbinoids that are released then travel
retrogradely (unlike usual anterograde transmission) and activate presynaptically-located
CB1-Rs (Katona and Freund, 2008), thus causing suppression of transmitter release
either transiently (endocannbinoid mediated short term depression, eCB-STD) or over
extended duration (eCB-long term depression, eCB-LTD) (Kano et al., 2009).
1.12. Endocannabinoids as synaptic circuit breakers and retrograde messengers
Pitler and Alger (1994) reported that following a train of action potentials from the
post synaptic cell, the spontaneous inhibitory postsynaptic potentials (IPSPs) from CA1
pyramidal cells of hippocampal slices were suppressed, thus indicating a reduction of
GABA release from presynaptic nerve endings: a novel phenomenon termed
‘depolarization-induced suppression of inhibition’ (DSI).
It was found that depolarization of the post synaptic neuron triggers Ca2+ entry
through voltage-dependent Ca2+ channels which then elevates Ca2+ levels in post
synaptic neurons. Support for this mechanism was clear since cerebellar DSI is
inhibited by chelation of extracellular Ca2+ or by adding Cd2+ to the bath solution (Llano
et al., 1991). Moreover, DSI was enhanced by the L-type Ca2+ channel activator, BAY K
8644 while blocked by calcium chelators like BAPTA and EGTA (Pitler and Alger, 1992;
Vincent and Marty, 1993). An ultimate presynaptic locus for DSI expression was
36
confirmed by Llano et al. (1991) when they found that DSI correlated directly with the
decrease in frequency of IPSCs (IPSCs).
Almost the same time, Ohno-Shosaku et al. (2001) using rat hippocampal
cultures and Wilson and Nicoll (2001) using rat hippocampal slices, reported that DSI
was completely abolished by the CB1 receptor antagonists SR141716A, AM251 and
AM281, while a metabotropic glutamate receptor antagonist failed to do this, indicating
that the retrograde signaling mechanism was mediated through CB1-Rs.
Meanwhile, in cerebellar Purkinje cells, Kreitzer and Regehr (2001) reported a
phenomenon similar to DSI where they observed that following postsynaptic
depolarization, the excitatory transmission was transiently suppressed, an effect termed
as ‘depolarization-induced suppression of excitation’ or DSE. The presynaptic Ca2+
currents generated in response to stimulation of the excitatory climbing fibers were found
to be inhibited or suppressed during DSE, thus providing good evidence for a
presynaptic locus of this effect (Kreitzer and Regehr, 2001). Moreover, DSE was
abolished by treatment with the calcium ion chelator BAPTA and occluded by the CB1-R
antagonist, AM251. This effect was not blocked by mGlu, adenosine and GABA
receptor antagonists suggesting that like DSI, DSE is also mediated through the
endocannabinoid system (Kreitzer and Regehr, 2001).
37
Figure 1.8 Blockade of depolarization-induced suppression of inhibition (DSI) CB1-R antagonists by AM251 and SR141716A in rat hippocampal neurons (Kano et al., 2009).
A: Examples of inhibitory postsynaptic currents (IPSCs) (right panel) and
control results showing that DSI can be evoked repetitively without change in its
magnitude (left panel)
B: Example of IPSCs, control and after treatment with AM281 (left panel).
Average time courses for DSI before and after treatment with AM281 (right panel)
C: Example of IPSCs, control and after treatment with SR141716a (left
panel). Average time courses for DSI before and after treatment with SR141716A (right
panel)
38
1.13. Mechanisms of endocannabinoid mediated short term depression (eCB-STD)
Unlike classical neurotransmitters, endocannabinoids are de novo synthesized
on demand and released and not stored in vesicles. Precisely what initiates the
endocannabinoid production and hence eCB-STD in neurons has been the subject of
much scientific debate (Kano et al., 2009). Two different pathways have been put forth
i.e. a PLCβ-independent pathway triggered by large rise in intracellular Ca2+
concentration alone (CaER), while the other is a PLCβ-dependent pathway that is
activated by stimulation of basal (receptor-driven) endocannabinoid release, (basal
RER) or elevated calcium-driven endocannabinoid release (Ca2+-assisted RER).
1.13.1. CaER
In the hippocampus, influx of Ca2+ ions into the postsynaptic cell triggers DSI
thus indicating vital role of Ca2+ elevation in initiating DSI and hence eCB-STD (Wilson
and Nicoll, 2001). The main sources for postsynaptic Ca2+ elevation are through voltage
gated Ca2+ channels (Ohno-shosaku et al., 2007; Pitler and Alger, 1992) or through
release from an intracellular Ca2+ reservoir (Isokawa and Alger, 2006).
According to this model, micromolar concentrations of Ca2+ trigger the activation
of voltage-dependent calcium channels which then produce 2-AG, likely through a
DAGL-mediated pathway and a PLCβ-independent pathway (Kano et al., 2009). 2-AG
released then initiates DSI/DSE by activating CB1-Rs.
1.13.2. Basal RER
The G-protein coupled receptors (Gq/11) mGlu1/5, M1/M3 muscarinic receptors,
glucocorticoid receptors, oxytocin receptors and orexin receptors were found to induce
eCB-STD when strongly activated (Hashimotodani et al., 2007; Hashimotodani et al.,
2007; Maejima et al., 2005). The most likely pathway for induction of eCB-STD by this
pathway is thought to involve stimulation of postsynaptic G-protein-coupled receptors
39
and DAG generation (through a PLCβ-dependent pathway), which then generates 2-AG
by the action of DAGL. The 2-AG then mediates induction of DSI or DSE (Kano et al.,
2009).
1.13.3. Ca2+-assisted RER
This model proposed to understand eCB-STD is a combination of the above two
models except here a weak stimulation of postsynaptic Gq/11 receptors cause a small rise
in Ca2+ to submicromolar concentrations which in turn activates PLCβ and thus 2-AG
and DSI/DSE (Kano et al., 2009; Hashimotodani et al., 2007).
1.14. Termination of eCB-STD
The 2-AG biosynthesized by the above mechanisms may then be partially
degraded by the COX-2 enzyme which is located in the postsynaptic cell and the
remaining 2-AG then diffuses rapidly into extracellular space by lateral diffusion
ultimately binding and activating presynaptic CB1-Rs. Presynaptically-located MAGL can
then degrade the remaining 2-AG thus terminating eCB-STD signalling (Kano et al.,
2009).
40
Figure 1.9 The pathway involved in the termination of endocannabinoid-mediated short term depression (eCB-STD) (Adapted from Kano et al., 2009).
41
1.15. Endocannabinoid-mediated long term depression (eCB-LTD)
As the name indicates, eCB-LTD induces a prolonged suppression of
neurotransmitter release in the brain and this effect is mediated through presynaptically
located CB1-Rs. However, the precise location of the initiation of eCB-LTD in specific
regions of brain also plays an important role as a deciding factor for involvement of CB1-
Rs and/or presynaptic components (Kano et al., 2009). It was found that application of
the CB1-R agonist (CP55940) at both excitatory synapses in the nucleus accumbens
(Robbe et al., 2002) and inhibitory synapses in the hippocampus (Chevaleyre et al.,
2007) induces LTD, indicating that CB1-Rs alone are sufficient to initiate the LTD
response in these regions of brain. However, at excitatory synapses in the dorsal
striatum, LTD cannot be evoked by CB1-R activation alone (Ronesi et al., 2004) since it
requires simultaneous activation of CB1-Rs and low frequency presynaptic activity
(Singla et al., 2007).
Studies have also been directed towards seeking an understanding of the way in
which a relatively short activation of CB1-Rs triggers LTD. It was reported that
presynaptic inhibition of the cAMP/PKA cascade and P/Q type voltage-gated calcium
channels was required for induction of LTD in the nucleus accumbens (Mato et al.,
2008), while presynaptic cAMP/PKA signalling and RIM1α were needed for expression
of LTD in the hippocampus and amygdala (Chavaleyre et al., 2007).
1.16. Other important aspects of endocannabinoid signaling
1.16.1. Regulation of excitability
Endocannabinoids have been found to control neuronal firing in several brain
regions (Kano et al., 2009). Kreitzer et al. (2002) showed in that in rat cellebellar cells,
interneuronal excitability was reduced in a CB1-R-dependent manner when Purkinje cells
were depolarized. They suggested that depolarization triggers generation and release of
endocannabinoids that then bind to CB1-Rs causing opening of K+ channels and hence
hyperpolarization (Kreitzer et al., 2002; Kano et al., 2009).
42
1.16.2. Basal activity of endocannabinoid signaling
CB1-Rs expressed exogenously using recombinant expression systems have
been found to support constitutive activity, but the case for such activity in native
membranes is weak (Howlett, 2004). SR141716A has shown to behave as an inverse
agonist in many native brain membrane preparations by inhibiting basal G protein
binding. This effect of SR141716A however, was at micromolar concentrations, in
contrast to the competitive antagonist behaviour in electrophysiological systems which
can occur at nanomolar concentrations (Sim-Selley et al., 2001). This again opens the
door to the debate as to whether CB1-Rs are constitutively active or this inverse agonist
activity of SR141716A (in the micromolar range) can be interpreted as its action on the
constitutively active adenosine receptor (Savinainen et al., 2003).
Moreover, CB1-Rs have been found to display basal activity and this basal effect
can be attributed to the tonically-released endocannabinoids (Kano et al., 2009;
Hoffman, 2003).
1.16.3. Plasticity of endocannabinoid signaling
Endocannabinoid system is tonically regulated and can be up or down regulated
in different situations (Kano et al., 2009). Chen et al., (2007) showed that DSI can be
potentiated by tetanic stimulation in hippocampal slice preparations and this was
mediated through CB1-Rs. Similarly, chronic exposure of nucleus accumbens to ∆9-THC
or WIN55212-2 reduced the sensitivity of CB1-R and abolished eCB-LTD (Hoffman et al.,
2003).
1.17. Subcellular distribution of various signaling molecules involved in regulation of the endocannabinoid system
1.17.1. Gq Protein α subunit
So far four isoforms of Gq protein α-subunits have been identified by
immunochemical studies. These include Gαq, Gα11, Gα14 and Gα15/16. Among these,
Gαq and Gα11 are the main isoforms found mostly in brain (Tanaka et al., 2000) and they
43
also represnt the major ones that are attached to the extrasynaptic membrane
containing mGluR1 in Purkinje cells and mGluR5 in hippocampal pyramidal cells
(Tanaka et al., 2000).
1.17.2. Phospholipase Cβ (PLCβ)
All four isoforms (1-4) of PLCβ are abundantly expressed in the brain largely in a
nonoverlapping expression manner (Kano et al., 2009). PLCβ1 is mainly expressed in
the telencephalon, PLCβ2 in white matter, PLCβ3 in the caudal cerebellum, PLCβ4 in
the raustral cerebellum, thalamus and brain stem (Ross et al., 1989; Roustan, 1995;
Tanaka and Kondo, 1994; Watanabe et al., 1998).
1.17.3. Diacylglycerol lipase (DAGL)
DAGLα is widely distributed in various regions of brain with its highest density
found in cerebellar Purkinje cells, pyramidal cells in hippocampus and medium spiny
neurons in the striatum (Kano et al., 2009). However the distribution of DAGLα varies
from region to region. For example, DAGLα is at the highest amount in the spine neck
(as compared to spine head) in Purkinje cells (Yoshida et al., 2006), while in
somatodendritic membranes, DAGLα levels were highest in spine followed by the
dendritic shaft with lower amounts in the soma (Katona et al., 2006; Yoshida et al.,
2006).
1.17.4. N-acyl-phosphatidylethanolamine-hydrolyzing phospholipase D (NAPE-PLD)
NAPE-PLD is reported to be widely expressed in presynatic regions with the
highest levels in granule cells of the dentate gyrus and medium to low levels in CA3
pyramidal cells of the hippocampus, olfactory bulb, piriform cortex and thalamic nuclei
(Kano et al., 2009). Interestingly, the presence of NAPE-PLD in the axonal terminal may
indicate a presynaptic locus for anandamide synthesis which in turn may be functioning
as an anterograde messenger (Kano et al., 2009).
44
1.17.5. Monoacylglycerol lipase (MAGL)
Dinh et al. (2002) described the MAGL mRNA expression patterns in various
brain regions. They found the presence of this enzyme in the synapse-rich neuropil
region of the hippocampus, the amygdala and the cerebral cortex, where this enzymes is
mostly distributed as a seat for axon terminals (Dinh et al., 2002). Interestingly, MAGL
activity in brain was also found to be responsible for determining basal endocannabinoid
tonicity and retrograde signaling since presynaptically-located MAGL was reported to
breakdown the 2-AG released from the postsynaptic cell, thus restricting the
accumulation of 2-AG in and around the synaptic cleft (Hashimotodani et al., 2007).
1.17.6. Fatty acid amide hydrolase (FAAH)
FAAH presence is complementary to the expression of MAGL and CB1-Rs, for
example, FAAH is absent in the globus pallidus where CB1-Rs are abundantly expressed
while it is present at high density in somatodendritic elements of principal neurons but
mostly absent in interneurons (where CB1 and MAGL are widely expressed) (Kano et al.,
2009).
1.18. Physiological roles of the endocannabinoid system
Behavioural studies have helped enormously in exploring the physiological role
of the endocannabinoid system in a variety of brain functions like learning and memory,
depression, addiction, appetite and feeding behaviour, pain, as well as neuroprotection.
1.18.1. Learning and Memory
It has been now well established that phytocannabinoids and synthetic
cannabinoids cause memory and learning impairment in humans and laboratory animals
(Davies et al., 2002; Kano et al., 2002). In laboratory animals, Morris water maze tests
have been employed to investigate the effects of cannabinoid agonists. Mice (and rats)
failed to perform well in this test following systemic administration of CB1-R agonists
(Verval et al., 2001). Moreover, CB1 knockout mice and wild type mice treated with
SR141716A (CB1-R antagonist) exhibited similar performance in the fixed hidden
45
platform water maze task. Interestingly, when the same task was repeated with change
in location of the hidden platform there was a marked difference in the behaviour of both
animals with wild-type returning to the new location and CB1 knock out returning to the
old location of the hidden platform, thus indicating impairment of the extinction process
(Varvel and Lichtman, 2002).
1.18.2. Anxiety
There is increasing evidence suggesting a relationship between anxiety and
endocannabinoid tone. The effects of cannabinoid agonists on anxiety in laboratory
animals can be studied by employing different tests like elevated plus-maze, the light-
dark crossing test, the vocalization test and the social interaction test. The results are
quite complex to interpret although generally, cannabinoid agonists at low doses were
anxiolytic but at high doses were anxiogenic (Viveros et al., 2005). A cross talk
mechanism was suggested by Berrendero and Maldonaldo (2002) between the
cannabinoid and opioid systems since in the light-dark crossing test, the anxiolytic
effects of ∆9-THC were completely blocked by the opoid antagonist, naltrindole.
Moreover, in the plus-maze test, the anxiogenic effect of CP55940 was almost
completely blocked by an opioid antagonist, nor-binaltorphimine (Marin et al., 2003).
1.18.3. Depression
The endocannabinoid system has also been implicated in depressive episodes
(Kano et al., 2009). Hill and Gorzalka (2005) reported an antidepressant effect in
rodents following activation of the CB1-R. They found that in the rat forced swim test
(FST), HU210 (a CB1-R agonist) (5-25µg/kg i.p.) and AM404 (an anandamide transport
inhibitor) (5 mg/kg, i.p.) had antidepressant effects similar to the well-know
antidepressant drug, desipramine and this antidepressant effect was reversed by AM251
(a CB1-R antagonist) (Hill and Gorzalka, 2005).
Moreover recently, the CB1-R antagonist SR141716A (Rimonabant) was
withdrawn from European markets after reports of suicidal tendencies in some patients
employing this drug to treat obesity (Christensen et al., 2007).
46
1.18.4. Addiction
The endocannabinoid system has been known for its drug seeking and drug
reward effects and its likely involvement in addiction (De Vries and Schoffelmeer, 2005;
Fattore et al, 2007; Maldonado et al, 2006). Castane et al. (2002) showed that there
was a significant rewarding effect in wild type mice administered with nicotine (0.5 mg/kg
sc) but not in CB1 knockout mice. Furthermore, this effect was reduced by
administration of SR141716A (Le Foll and Goldberg, 2004).
However, the exact mechanism by which the endocannabinoid system
contributes to drug seeking and addiction behaviour is still rather obscure (Kano et al.,
2009).
1.18.5. Appetite
Food intake, appetite and feeding behaviour are precisely regulated through
involvement of the endocannabinoid system, and the mechanisms involved are subject
to ongoing investigation (Kano et al., 2009). Pagotto et al. (2006) showed that CB1-R
agonists increase food intake in a dose- dependent manner in laboratory animals while
CB1-R antagonists lead to decreased food intake in wild type mice but not in CB1
knockout mice (Di Marzo et al., 2001).
Rodent models and clinical studies strongly support the notion that CB1-Rs can
be targeted for the treatment of appetite disorders and obesity (Kano et al., 2009). ∆9-
THC has been employed effectively as an appetite stimulant in patients with HIV-
induced wasting syndrome and cancer while, as mentioned earlier, rimonabant
(SR141716A) was briefly used in European markets for the treatment of obesity
(Christensen et al., 2007; Kano et al., 2009).
1.18.6. Pain
The endocannabinoid system is intimately involved in pain modulation, and the
antinociceptive effects of cannbinoids have been reported to be at par with the opiates
(Hohmann and Suplita, 2006). Evidence for this came from the studies by Calignano et
al. (1998) who showed that SR141716A induced hyperalgesia in the Formalin test and
47
the hot plate test (Richardson et al., 1998). Also, analgesia induced by electrical
stimulation of periaqueductal gray matter was blocked by SR141716A (Walker et al.,
1999), thus indicating the role of the endocannabinoid system in pain modulation.
1.19. Classification of ligands that bind to cannabinoid receptors
1.19.1. Cannabinoid receptor agonists
1.19.1.1. Classical cannabinoids
This group encompasses derivatives of ABC-tricyclic benzopyran compounds
obtained from the Cannabis plant or synthetic analogs. The prototypical example of this
class is ∆9-THC (Figure 1.10). Others includes ∆8-THC, (6aR,10aR)- 9-(hydroxymethyl)-
The first cannabinoid receptor antagonist namely, SR141716A (CB1-R-selective)
and SR144528 (CB2-R-selective) were synthesized at Sanofi (Rinaldi-Carmona et al.,
1994 and 1998). Initially identified as cannabinoid antagonists, they were later found in
some preparations to exert effects opposite to cannabinoid agonists and hence were
classified as inverse agonists (Pertwee, 1999). Two other analogs in this diarylpyrazole
series include AM251 and AM281 (Figure 1.12).
1.19.2.2. Other inverse agonists primarily active at CB1-Rs
The Eli Lilly compound, LY320135 (a substituted benzofuran) has a higher
affinity for CB1-Rs then CB2-Rs and displays an inverse agonist profile similar to
SR141716A (Howlett et al., 2002). Similarly, another aminoalkylindole, 6-
iodopravadoline (AM630) was reported to be an inverse agonist at CB2-Rs (Howlett et
al., 2002) (Figure 1-12).
Dhopeshwarkar et al. (2011) found that (S)-methoprene and piperonyl butoxide
were antagonistic at CB1-R while sanguinarine and chelerythrine displayed inverse
agonist-like profiles. Bisset et al. (2011) reported that certain phthalate diesters (nBBP,
DnBP) were antagonist at CB1-Rs. However, all these compounds require low to
moderate micromolar concentrations for activity at CB1-Rs (for structures of these
compounds see Figure 1.13).
49
∆9-THC ∆8-THC HU210
DALN CP47497 CP55244
CP55940 WIN55212-2
JWH015 L-768242
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).
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).
51
SR141716A AM251 AM281
LY320135 AM630
Figure 1.12 Structures of SR141716A, AM251, AM281, LY320135 and AM630. All structures redrawn using ChemDraw Ultra 11.0 from Howlett et al. (2002).
52
Methoprene Piperonyl butoxide
Sanguinarine Chelerythrine
nBBP DnBP
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).
53
1.20. Cannabinoid receptor 2 (CB2-R)
1.20.1. CB2-R receptor signaling
Like CB1-Rs, CB2-Rs couple to Gi/o proteins and modulate adenylyl cyclase and
MAPK activity. However, unlike CB1-Rs, CB2-Rs do not couple to the Gs subunit of G
protein, and hence cannot modulate ion channel activity (Demuth and Molleman, 2006;
Felder et al., 1995; Kobayashi et al., 2001).
1.20.1.1. Adenylyl cyclase regulation
CB2-Rs are negatively coupled to adenylate cyclase thus they decrease cAMP
production. Slipetz et al. (1995) reported that in cell lines transfected with CB2-Rs,
forskolin-stimulated cAMP production was inhibited by CB2-R agonists and this effect
was concentration-dependent. Similar results were obtained by Felder et al. (1995) with
∆9THC and anandamide. Another line of evidence for coupling of CB2-R to Gi/o proteins
came from experiments where following pretreatment of CB2 transfected CHO cells with
Pertussis toxin was found to attenuate the inhibition of cAMP production (Pertwee,
1997).
1.20.1.2. Mitogen-activated protein kinase regulation
In common with CB1-Rs, CB2-Rs have been implicated in the regulation of MAP
kinase (Pertwee, 1997). CP55940 and WIN55212-2 were found to activate MAP kinase
in CHO cells transfected with CB2-Rs in a concentration-dependent manner (Bouaboula
et al., 1996). This effect of CP55940 was blocked by pretreatment of cells with Pertussis
toxin, indicating the involvement of Gi/o G proteins (Bouaboula et al., 1996). Bouaboula
and co-workers (1996) also found that MAP kinase regulation was mediated through
protein kinase C, since inhibitors of this enzyme decreased CB2-R-mediated activation of
MAP kinase. Interestingly, CHO cells stably transfected with the CB1-R showed
activation of MAP kinase but this activation of unaffected by treatment with a protein
kinase C inhibitor, indicating a distinct difference in the signal transduction mechanism of
each receptor (Bouaboula et al., 1996).
54
1.20.2. Therapeutic aspects of CB2-R modulators
Studies have indicated a potential role of CB2-R agonists in the treatment of
pH 7.4 with HCl). Membranes (154.3 + 3.5 µg protein) were then added to each tube
and the mixture vortexed and incubated for 15 minutes at room temperature. Following
addition of [3H]CP-55940 (added in 10 µl DMSO; final radioligand concentration 1.0 nM),
the tube contents were thoroughly mixed and incubations run for 90 min at 30 oC with
gentle shaking. Binding reactions were stopped by adding ice-cold wash buffer (0.9%
NaCl containing 2 mg/ml BSA; 1 ml) and membranes were collected by rapid vacuum
filtration on pre-soaked Whatman GF/C filters. Membranes trapped on the filter were
immediately washed (3 x 4 ml) with ice-cold wash buffer. Filters were thoroughly air
dried before adding scintillant (4 ml; BCS, Amersham Bioscience UK) and radioactivity
was quantitated using liquid scintillation counting. Non-specific binding, measured in the
presence of unlabeled CP55,940 or WIN55,212-2 (both at 10 µM), was subtracted from
total binding to yield the specific binding signal which averaged 80.9 + 4.7 % and 80.7 =
3.1% respectively. In each experiment, binding in the absence and presence of
unlabeled CP55,940 or WIN55212-2 was performed in triplicate and test compounds
were assayed in duplicate. A minimum of three experiments were conducted for every
treatment. All protein measurements were carried out as described by Peterson (1977).
69
2.3.4. Determination of the effects of study compounds on basal and CP55940-stimulated [35S]GTPγS binding to mouse brain membranes
The procedure for isolating brain membranes and measuring the effects of study
compounds on basal and agonist-stimulated [35S]GTPγS binding was adapted from that
of Breivogel et al., (2000). The isolation of brain membranes was carried out at 0-4 oC.
Immediately following the cervical dislocation procedure, whole brains were removed
from two mice and homogenized (Polytron Kinematica GmBH; speed setting 6 for 15
seconds) in isolation buffer (Trisma base (50 mM), MgCl2.6H20 (3 mM), EGTA (0.2 mM),
NaCl (100 mM) with pH adjusted to 7.4 with HCl). The homogenate was centrifuged in a
Beckman J2HS centrifuge (JA20 rotor) at 24,000 x g for 25 min, and the resulting pellet
was then resuspended in isolation buffer and re-centrifuged The final membrane pellet
was thoroughly homogenized in isolation buffer, the protein concentration adjusted to 7
mg/ml and aliquots transfered to the -80 oC freezer. After removal from storage at -80 oC, brain membranes were thawed on ice and thoroughly dispersed as described in
Section 2.3. [35S]GTPγS binding experiments were performed as follows. The test
compound (in DMSO; 5 µl) or DMSO control, as appropriate, was placed in the tube first
followed by assay buffer (500 µl; isolation buffer (pH 7.4) containing, bovine serum
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 (continued on page 76).
Figure 2.1 (continued) 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.
(a) - (e): The structures of sanguinarine, chelerythrine, berberine, papaverine and (-) (1R,9S)-β-hydrastine. (f) - (i): Comparisons of possible conformations of piperonyl butoxide and (S)-methoprene with anandamide and 2-arachidonoyl glycerol respectively. (j - m): Comparison of the pseudobase form of sanguinarine and a possible conformation of (S)-methoprene with ∆9-tetrahydrocannabinol and ∆9-tetrahydrocannabivarin.
All structures redrawn using IsisDraw from Dhopeshwarkar et al. (2011).
77
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).
78
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.
79
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.
80
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.
81
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.
82
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.
83
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.
84
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.
Compound Inhibition (%)
Berberine 12.05 + 2.2
1R, 9S-(-)-β-Hydrastine 4.09 + 1.64
Papaverine 17.56 + 0.5
PMSF 1.27 + 3.12
85
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.
AM251
(µM)
Inhibition of CP55940-stimulated
[35S]GTPγS binding (%)
Inhibition of
basal [35S]GTPγS
binding (%)
0.010 59.59 + 2.66 ND
1.0 98.93 + 1.62 ND
10.0 100 7.61 + 6.32
20.0 ND 10.01 + 1.37
9.21 + 0.76% encroachment of AM251 on the basal component of [35S]GTPγS
binding was observed in experiments involving 10 µM CP55940 agonist, as observed by
others with the closely related analog SR141716A (Selley et al., 1996; Petitet et al.,
1997).
86
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.
Compound
Inhibition of CP55940-
stimulated [35S]GTPγS
binding (%)
Effect on basal
[35S]GTPγS binding
(+ = % increase; - = % decrease)
Berberine 2.82 + 0.89 a 1.98 + 0.35
1R, 9S-(-)β-
Hydrastine
2.23 + 2.23 - 0.50 + 5.77
Papaverine 5.79 + 1.77 3.90 + 2.32
a represents a % increase in CP-55940-induced stimulation
87
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.
Compound
Concentration
(% increase)
Piperonyl butoxide 20 µM 8.93 + 6.20
30 µM 7.21 + 1.61
40 µM 1.47 + 0.47
(S)-Methoprene 25 µM 4.33 + 6.32
40 µM 2.80 + 0.80
50 µM 0.03 + 5.59
88
3. The G protein-coupled cannabinoid-1 (CB1) receptor of mammalian brain: Inhibition by phthalate esters in vitro.
3.1. Abstract
This research examines the in vitro interaction of phthalate diesters and
monoesters with the G protein-coupled cannabinoid 1 (CB1) receptor, a presynaptic
complex involved in the regulation of synaptic activity in mammalian brain. The diesters,
free; 3 mg/ml) adjusted to pH 7.4 with HCl). Brain membranes (170.67 + 0.84) µg
protein) were then placed in each tube and the suspension was then thoroughly
vortexed and pre-incubated at room temperature for 15 minutes. Additions of [3H]CP-
55940 (side chain-2,3,4-[3H]; sp. act. 174.6 Ci/ mmol; Perkin Elmer Life and Analytical
Sciences, Canada) to each tube were made in 10 µl DMSO (final radioligand
concentration 1.0 nM; final DMSO concentration 2.8%), and after careful mixing,
incubations were run for 1.5 h at 30 oC with gentle shaking. Incubations were stopped by
the addition of 1 ml of ice-cold wash buffer (0.9% NaCl containing 2 mg/ml BSA) and
membranes were quickly harvested by vacuum filtration on pre-soaked Whatman GF/C
93
filters. Membranes trapped on filters were immediately washed (3 x 4 ml) with ice-cold
wash buffer. Filters were completely dried (in a fume hood). Scintillation cocktail (BCS,
Amersham Bioscience UK) was then added and radioactivity was measured using liquid
scintillation counting. Non-specific binding, measured in the presence of unlabeled
WIN55,212-2 (10 µM), was subtracted from total binding to calculate the specific binding
signal (76.8 + 1.1% of total binding). This is very similar to the 80% obtained by Quistad
et al. (2002) using 1 µM WIN55212-2. Under the present assay conditions the IC50 for
WIN55212-2 was 6 nM and maximum displacement of [3H]CP55940 by WIN55212-2
was achieved at both 1 and 10 µM.
Selected phthalate esters were also evaluated in binding assays using the CB1
receptor antagonist [3H]SR141716A (sp. act. 56 Ci/ mmol; Perkin Elmer Life and
Analytical Sciences, Canada). For competitive displacement assays an identical
experimental procedure to that described above was used. [3H]SR141716A was
present at 1.2 nM and AM251 (at 2 µM) was introduced to estimate the specific binding
signal, which averaged (71.0 + 0.7%). For association experiments, membranes were
either preincubated with the phthalate ester for 15 min. before [3H]SR141716A addition
or received simultaneous application of phthalate and radioligand. Dissociations were
initiated on CB1 receptors equilibrated with [3H]SR141716A using either a saturating
concentration of AM251 or this concentration of AM251 plus the phthalate ester.
In each experiment with [3H]CP55940 or [3H]SR141716A, binding in the
absence and presence of unlabeled WIN55212-2 or AM251 was performed in triplicate
and test compounds were assayed in duplicate. A minimum of three independent
experiments were performed for every treatment. Protein measurements were
conducted according to Peterson (1977).
3.3.3. Investigation of phthalate interference with CB1 receptor agonist-stimulated [35S]GTPγS binding to the Gα-protein.
The method we used to isolate the mouse whole brain membrane fraction and
determine the effects of phthalates on agonist-stimulated [35S]GTPγS binding generally
followed the procedure published by Breivogel et al. (2000). Whole brains were quickly
removed from two mice and homogenized for 15 sec in 10 ml of ice-cold isolation buffer
94
(Trisma base (50 mM), MgCl2.6H20 (3 mM), EGTA (0.2 mM), NaCl (100 mM) with pH
adjusted to 7.4) using a tissue fragmenter (Polytron Kinematica GmBH; setting 6). The
suspension was centrifuged in a Beckman J2HS centrifuge (24,000 x g for 25 min at 2 oC) and the pellet was then resuspended in fresh ice-cold isolation buffer and re-
centrifuged. The washed membrane pellet was thoroughly dispersed in isolation buffer,
then protein concentration was adjusted to approx. 7 mg/ml before aliquots were
transferred to a -80 oC freezer. Prior to experimentation, the membrane fractions were
thawed on ice and completely dispersed as described in the previous section. This
procedure helped improve the reproducibility between replicates without obvious loss in
agonist-stimulated [35S]radioligand binding. Binding experiments were performed using
guanosine 5'-O-(g-γ35S]thio)-triphosphate ([35S]GTPγS) of sp. act. 1250 Ci/mmol
purchased from Perkin Elmer Life and Analytical Sciences, Canada.
The phthalate esters (dissolved in DMSO; 5 µl) or DMSO control (as required)
were placed in borosilicate glass tubes (13 x 100 mm; siliconized 24 h prior to assay with
Sigmacote [Sigma-Aldrich Canada]) and then 500 µl of isolation buffer (pH 7.4) was
added which contained fatty-acid free bovine serum albumin (1 mg/ml), guanosine
55.4 and 75.2) and efficacies were also comparatively low (55-65%). The critical nature
of the diester configuration for inhibition of [3H]CP55940 binding is emphasized by
comparison of nBBP and DnBP (which are amongst the most effective compounds
studied) with MnBP (a phthalate devoid of activity).
For the experiments with PMSF, we reasoned that using phthalates of
intermediate (DnBP) and higher (nBBP) potency at < IC50 would offer a sensitive basis
for assessment. Moreover, DnBP and nBBP (study compounds with alkyl and aryl
substituents respectively) might be expected to show different susceptibilities to
breakdown by serine hydrolases. Nonetheless, based on our experiments, there was no
evidence that serine hydrolases limit the inhibitory effect of either of these analogs in the
[3H]CP-55940 binding assay.
The equilibrium binding and dissociation data using [3H]SR141716A provide a
useful insight into the mechanism by which nBBP and DnBP inhibit radioligand binding.
The saturation isotherms demonstrate that these phthalates act by eliminating binding
sites for radioligand (i.e. Bmax is reduced), without affecting the affinity of radioligand for
the remaining sites (i.e. Kd is unchanged). Moreover, the dissociation experiments
strongly suggest that these compounds act allosterically with respect to the
[3H]SR141716A binding site, since under our assay conditions any access by phthalate
99
diesters to the radioligand binding site is completely prevented by the saturating levels of
AM251. The dissociation data also argue against an irreversible or tight binding of
phthalate esters to the [3H]SR141716A recognition site, another potential explanation of
the reduced Bmax and unchanged Kd. The time courses for dissociation of
[3H]SR141716A in the presence of nBBP and DnBP indicate that the binding of
phthalates to this allosteric binding site and subsequent negative modulation of
radioligand binding occurs very rapidly. Rapid engagement of phthalates with a site
coupled allosterically to the [3H]SR141716A binding site is also consistent with the
reduced levels of nBBP and DnBP binding in the association experiments. However, the
association profiles in the presence of nBBP and DnBP are likely markedly influenced by
the effect of these compounds on availability of receptors (Bmax) that can bind
[3H]SR141716A. Overall, our results indicate that a critical mechanism underlying
inhibition of [3H]SR141716A to CB1 receptors involves phthalates engaging with a site
that is distinct from but negatively coupled to the radioligand recognition site. The
proposed binding region for phthalate esters on the CB1 receptor may represent a novel
target that could be exploited therapeutically by phthalate ester analogs or other drugs to
produce downregulation of endocannabinoid action in the brain.
Unlike phthalate diesters, MEHP and other monoesters inhibit the binding of
follicle stimulating hormone (FSH) to G-protein coupled FSH receptors, an action that
may involve direct engagement of MEHP with the G-protein (Grasso et al., 1993). The
allosteric inhibition of [3H]SR141716A binding to the CB1 receptor by phthalate diesters
could also arise from a direct interaction with its G-protein as we have postulated for
chelerythrine and sanguinarine (Dhopeshwarkar et al. 2011). However, in contrast to the
findings of Grasso et al., (1993), we found that monoesters are, at best, exceptionally
weak inhibitors of CB1 receptor radioligand binding. Therefore, negative allosteric
coupling between a phthalate diester recognition site on the CB1 receptor and the
radioligand binding site is likely a more productive area for future exploration.
Phthalate diesters have potential to access the brain, since a number of these
compounds interfere with barbiturate-induced sleep duration following systemic
administration (Calley et al., 1966) and phthalate ester exposure in school children has
been associated with a behavioral (attention-deficit/hyperactivity) disorder (Kim et al.
2009). The presynaptic CB1 receptor plays a fundamental role at many synapses in
100
mammalian brain and activation of this complex by endocannabinoids promotes a
variety of physiological and behavioral responses. Moreover, downregulation of CB1
receptors and other components of the endocannabinoid system in human epilepsy is
associated with increased excitability in neuronal networks and has been linked to
reduced seizure thresholds (Ludanyi et al., 2008). A critical question is whether brain
CB1 receptors are exposed to phthalate diesters in vivo at concentrations that are
sufficient to interfere with the activation of this signaling pathway by endocannabinoids.
Phthalate esters undergo extensive ester cleavage in the gastrointestinal tract and
hydrolysis would be expected to limit the ability of diesters to reach the brain particularly
after acute oral exposure. However, individuals receiving higher exposure to phthalate
esters on a continuous basis (perhaps as a result of occupational exposure) or hospital
patients exposed to phthalates released from medical devices may be more likely to
accumulate these chemicals in the brain. In the present investigation, threshold inhibitory
effects of DEHP, DnOP, DiOP and nBBP on [3H]CP55940 binding are evident between 1
and 10 µM. Even concentrations within this range in brain may be sufficient to
antagonize endocannabinoid-mediated signaling at CB1 receptors to an extent that
causes low level synaptic perturbations and subtle pathophysiological and affective
responses.
Finally, it must be stressed that further studies aimed at determining 1) phthalate
ester levels in brain following short term systemic and chronic exposures and 2) the
ability of these compounds to modify critical effects of cannabinoid agonists in intact
animals are essential to improve our understanding of the potential phthalate diesters
might have in modulating the endocannabinoid system in vivo.
3.6. Note in added proof
3.6.1. Background
Subsequent to our Neurochemistry International publication appearing, I
established the L-glutamate release assay originally described by Nicholls et al. (1987)
in our laboratory. This assay uses purified synaptosomes and has been utilized by
Wang (2003) to study inhibition of 4-aminopyridine- (4-AP-) evoked release of L-
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glutamate by the CB1-R agonist WIN55212-2. Since inhibition by WIN55212-2 is
blocked by a diarylpyrazole CB1-R antagonist (Wang, 2003), this system provides a
rigorous functional test for compounds that might antagonize CB1-Rs in the brain.
3.6.2. Experimental approach
We selected two phthalates for investigation, BBP and MnBP. The former was
one of the more potent diesters, both for inhibition of [3H]CP55940 binding and inhibition
of CP55940-stimulated [35S]GTPγS binding. The latter, a monoester, gave no inhibition
of [3H]CP55940 binding and at best produced marginal (<20%) inhibition of [35S]GTPγS
binding. The methods for the isolation of synaptosomes and the fluorimetric
measurement of L-glutamate release are described in detail in Chapter 4.
3.6.3. Results
Consistent with the findings of Wang (2003), WIN55212-2 inhibited of 4-AP-
evoked release of L-glutamate and this effect was fully blocked by AM251. In agreement
with our [3H]CP55940 binding results and as predicted by the [35S]GTPγS binding data,
BBP at 30 µM (but not 5 µM) fully reversed the inhibition by WIN55212-2 (Figure 3.9).
Again in agreement with the [3H]CP55940 binding experiments and the [35S]GTPγS
assays, MnBP failed to modify the inhibitory effect of WIN55212-2 (Figure 3.10).
3.6.4. Conclusion
The L-glutamate assay results strongly supports the idea that 1) phthalate
diesters (as exemplified by BBP) exert antagonist actions at presynaptic CB1-Rs at low
to moderate micromolar concentrations in vitro, and 2) monoesters (as exemplified by
MnBP) are inactive.
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3.7. Figures and Tables
O
O
O
O
O
O
O
O
O
O
O
O
(a) nBBP (b) DnHP (c) DnBP
O
O
O
O
O
O
O
O
O
O
O
O
(d) DEHP (e) DiOP (f) DnOP
O
O
O
O
H
O
O
O
O
H
O
O
O
O
H
(g) M2EHP (h) MiHP (i) MnBP
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.
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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.
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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.
105
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).
106
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. (a) membranes received the standard 15 min preincubation with phthalate esters prior to [3H]SR141716A addition.
107
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. (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).
108
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.
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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.
110
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.
111
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.
112
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.
113
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.
Treatment Inhibition (%)
PMSF -2.82 + 3.18
nBBP 28.25 + 2.11
nBBP + PMSF 27.01 + 4.55
DnBP 20.50 + 1.40
DnBP + PMSF 22.61 + 3.31
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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.
Treatment Inhibition of specific binding (%)
nBBP (27 µM) 67.82 + 1.71
DnBP (46 µM) 72.30 + 3.23
DEHP (47 µM) 37.42 + 3.48
MiHP (100 µM) 33.23 + 4.15
MnBP (100 µM) 0
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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.
4.1. Abstract
Benzophenanthridine alkaloids (chelerythrine and sanguinarine) inhibited binding
of [3H]SR141716A to mouse brain membranes (IC50s: <1 µM). Piperonyl butoxide and
(S)-methoprene were less potent (IC50s: 21 and 63 µM respectively).
Benzophenanthridines and piperonyl butoxide were more selective towards brain CB1-
Rs versus spleen CB2-Rs.
All compounds reduced Bmax of [3H]SR141716A binding to CB1-Rs, but only
methoprene and piperonyl butoxide increased Kd (3-5-fold). Benzophenanthridines
increased the Kd of [3H]CP55940 binding (6-fold), but did not alter Bmax. (S)-methoprene
increased the Kd of [3H]CP55940 binding (by almost 4-fold) and reduced Bmax by 60%.
Piperonyl butoxide lowered the Bmax of [3H]CP55940 binding by 50%, but did not
influence Kd.
All compounds reduced [3H]SR141716A and [3H]CP55940 association with CB1-
Rs. Combined with a saturating concentration of SR141716A, only piperonyl butoxide
and (S)-methoprene increased dissociation of [3H]SR141716A above that of SR141716A
alone. Only piperonyl butoxide increased dissociation of [3H]CP55940 to a level greater
than CP55940 alone. Binding results indicate predominantly allosteric components to
the study compounds action.
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4-Aminopyridine- (4-AP-) evoked release of L-glutamate from synaptosomes was
partially inhibited by WIN55212-2, an effect completely neutralized by AM251, (S)-
methoprene and piperonyl butoxide. With WIN55212-2 present, benzophenanthridines
enhanced 4-AP-evoked L-glutamate release above 4-AP alone. Modulatory patterns of
L-glutamate release (with WIN-55212-2 present) align with previous antagonist/inverse
agonist profiling based on [35S]GTPγS binding. Although these compounds exhibit lower
potencies compared to many classical CB1 receptor inhibitors, they may modify CB1-R-
dependent behavioral/physiological outcomes in the whole animal and could offer
templates for synthesis of novel and more potent CB1-R blocking drugs.
Note: The research described in this chapter will be submitted shortly for
publication in an appropriate neurochemical/neuropharmacological journal. The
submission will adhere closely to the format laid out here.
4.2. Introduction
Cannabinoid-1 receptors (CB1-Rs) are present in numerous regions of
mammalian brain and are particularly abundant within the cerebral cortex, hippocampus,
cerebellum and basal ganglia (Herkenham et al., 1991; Tsou et al., 1998). CB1-Rs
couple to G-proteins in the plasma membrane of nerve terminals and together they
constitute the primary presynaptic element of the endocannabinoid signaling pathway
that regulates transmitter release through negative feedback (Howlett et al., 1986;
Katona et al., 1999; Kawamura et al., 2006). Endocannabinoids, generated in
postsynaptic neuronal cell bodies when synaptic activity intensifies, migrate retrogradely
and bind to presynaptic CB1-Rs. G-protein activation leads to inhibition of voltage-
sensitive Ca++ channels (Mackie and Hille, 1992; Twichell et al., 1997, Kushmerick et al.,
2004; Guo and Ikeda, 2004), negative modulation of adenylate cyclase (Howlett and
Fleming, 1984; Howlett, 1985) and activation of K+ currents (Deadwyler et al., 1993;
Mackie et al., 1995, Childers and Deadwyler, 1996; Guo and Ikeda, 2004). Since these
various signaling mechanisms reduce the ability of action potentials impinging on the
nerve ending to depolarize and activate calcium entry, transmitter release is adjusted
downwards, thus completing the negative feedback loop (Chevaleyre et al., 2006;
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Kreitzer and Regehr, 2001; Wilson and Nicoll, 2001; Howlett, et al., 2002; Freund et al.,
2003).
Certain plant natural products and synthetic drugs mimic endocannabinoid
activation of this signaling pathway by exerting potent (nanomolar) agonist actions at
CB1-Rs. Prominent xenocannabinoid agonists include ∆9-tetrahydrocannabinol, the main
psychoactive principle of Cannabis sativa (Razdan, 1986), CP55940 (Johnson and
Melvin, 1986) and the aminoalkylindole WIN55212-2 (Compton et al., 1992). Selective
high potency CB1-R antagonists, notably the phytocannabinoid ∆9-
tetrahydrocannabivarin and the diarylpyrazole antagonist/inverse agonists AM251 and
SR141716A, have also been reported (Rinaldi-Carmona et al., 1994; Lan et al., 1999;
Thomas et al., 2005).
There is considerable interest in possible therapeutic applications of CB1-R
modulators. Agonists and allosteric activators of agonist action have been considered in
the relief of pain, muscle spasms, anxiety states and depressive illness, and they can
also block emesis, improve sleep and stimulate appetite (Van Sickle et al., 2001;
Iversen, 2003, Ligresti et al., 2009; Bradshaw and Walker, 2005; Di Marzo, 2009). On
the other hand, CB1-R antagonists/inverse agonists such as the diarylpyrazole
rimonabant (SR141716A) have shown effectiveness in reducing body weight through
suppression of appetite (Colombo et al., 1998), but rimonabant use in human medicine
was curtailed due to adverse psychiatric side effects. Nevertheless, discovery of a CB1-
R inhibitor divorced of such unfavourable symptoms clearly remains of considerable
interest (Szabo et al., 2009; Wu et al., 2009; Riedel et al., 2009).
Research in our laboratory has focused on other natural products and synthetic
environmental chemicals capable of interacting with the endocannabinoid system.
Specifically we have demonstrated that at very low to moderate micromolar
concentrations, the benzophenanthridine alkaloids (sanguinarine and chelerythrine), the
pesticides (piperonyl butoxide and (S)-methoprene), certain phthalate dialkyl ester
plasticizers, as well as the more acutely toxic tributyltin derivatives (tributyltin acetate
and tributylethynyl tin) inhibit both the binding of [3H]CP55940 to CB1-Rs, as well as CB1-
R agonist-dependent activation of the G-protein (Dhopeshwarkar et al., 2011; Bisset et
[Trisma base (100 mM), EDTA (1 mM), adjusted to pH 9 with HCl; 1 brain per 10 ml
buffer]. The homogenate was centrifuged for 10 min at 900 x g in a JA20 rotor of a
Beckman J2HS centrifuge. Centrifugation of the supernatant at 11,500 x g for 25 min
produced a membrane pellet which was resuspended in ice-cold buffer [Trisma base (50
mM), EDTA (1 mM) and MgCl2.6H2O (3 mM); adjusted to pH 7.4 with HCl] at a protein
concentration of approx. 6.5 mg/ml. The membrane preparation was then frozen in
aliquots at -80 oC. Just prior to assay, the preparation was thawed on ice and carefully
120
dispersed by slowly moving the membrane suspension in and out of a syringe fitted with
a 18 gauge needle (6 times) followed by vortexing.
4.3.4. Effects of benzophenanthridines, (S)-methoprene and piperonyl butoxide on equilibrium binding of [3H]CP55940 and [3H]SR141716 to brain CB1 receptors
The saturation binding method of Steffens et al. (2004) was adopted (with
modifications as detailed below), and effects on radioligand saturation binding
characteristics were investigated by assaying the study compound (at ≥ IC50) with
different concentrations of [3H]CP55940 or [3H]SR141716A (0.032-3.5 nM). To achieve
these concentrations of [3H]CP55940 and [3H]SR141716A, radioligand specific activity
was reduced by addition of the required quantity of unlabelled CP55940 and
SR141716A. For assay, compounds formulated in DMSO (5 µl) or DMSO control (5 µl),
as appropriate, were rapidly injected into borosilicate glass tubes (13 x 100 mm; Kimble-
MgCl2.6H2O (3 mM), BSA (fatty acid free; 3 mg/ml) adjusted to pH 7.4 with HCl]. After
addition of brain membranes (227.09 + 0.66 µg protein), the mixture was gently vortexed
and the incubation continued for 15 minutes at room temperature. [3H]CP55940 or
[3H]SR141716A was then added at the required concentrations and incubations were
run for 90 min to ensure equilibration of radioligand. Incubations were concluded by
addition of 4 ml ice-cold wash buffer (0.9% NaCl containing 2 mg/ml BSA (fatty acid
free)) and membranes were harvested on Whatman GF/C filters (presoaked with wash
buffer) using a (Hoefer FH 225V) vacuum filtration system attached to a vacuum pump
(Hoefer FH 225V). Membranes collected on filters were washed (3 x 4 ml) with ice-cold
wash buffer. Each filter was removed from its filtration well and placed in a scintillation
vial and allowed to dry completely. After drying, 4 ml of scintillation cocktail (BCS,
Amersham Biosciences, UK) was added and radioactivity measured using liquid
scintillation counting. Specific binding was calculated by subtracting non-specific binding
(binding in presence of 10 µM CP55940 or 10 µM SR141716A) from total binding and
this was determined for each concentration of [3H]CP55940/CP55940 or
[3H]SR141716A/SR141716A in the absence and presence of study compound. Values
were used to construct equilibrium binding isotherms which allowed calculation of the Kd
(equilibrium dissociation constant) and Bmax (number of receptors available for
121
radioligand binding). At least three experiments were conducted for each treatment and
protein measurement was done as described by Peterson (1977).
4.3.5. Effect of benzophenanthridines, (S)-methoprene and piperonyl butoxide on the association and dissociation kinetics of [3H]CP55940 and [3H]SR141716A
Association studies were initiated by addition of study compound in DMSO (5 µl)
or DMSO control (5 µl), as appropriate, to 500 µl assay buffer in borosilicate glass tubes.
Membranes (approx. 230 µg protein) were then added and the system allowed to
incubate for 15 min at room temperature. Meanwhile, the Hoefer FH 225V filtration
apparatus was prepared by inserting pre-soaked Whatman GF/C filters and allowing the
vacuum to draw down a few minutes before the end of the preincubation. [3H]CP55940
or [3H]SR141716A (1 nM final concentration) was added at t = 15 min, and the brain
membrane suspensions filtered at various time points between 0 and 180 secs. After
three 4 ml washes, filters were dried and radioactivity associated with the membranes
quantified. The time course of radioligand association was also tracked after each study
compound (or DMSO control) was added a few seconds before the radioligand (defined
as co-treatment situation).
Dissociation studies were conducted by equilibrating brain membranes (approx.
230 µg protein) with [3H]CP55940 or [3H]SR141716A (1 nM final concentration) for 90
minutes at 37 oC with gentle shaking. At equilibrium, either a saturating (5 µM)
concentration of (CP55940) or AM251 (added in 10 µl DMSO), or study compound at ≥
IC50 (added in 5 µl DMSO) plus 5 µM (CP55940) or AM251 (added in 5 µl DMSO) was
added, Dissociation of radioligand was monitored over 300 seconds.
4.3.6. Interaction of benzophenanthridines, methoprene and piperonyl butoxide with CB2 receptors in mouse spleen
After evaluation of several methods, the method of Hillard et al. (1999) was
adopted for this investigation. Two mice were euthanized by rapid cervical dislocation
and the spleens were rapidly removed and homogenized in 10 ml TME buffer (Tris-HCl
(50 mM), EDTA (1 mM) and MgCl2.6H2O (3 mM), titrated to pH 7.4 with HCl) using a
pre-chilled motor driven homogenizer (10 strokes up and down, pestle rotation 1500
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rpm). The homogenate was then centrifuged at 500 x g for 10 minutes in a Beckman
J2HS centrifuge using JA-20 rotor. The pellet was discarded and the supernatant
centrifuged at 17,500 x g for 30 minutes. The fresh spleen membranes were
immediately used for experimentation. For assay, 500 µl of binding buffer (Tris-HCl (50
mM), EDTA (1 mM) and MgCl2. 6H2O (3 mM) and BSA (fatty acid free, 3 mg/ml), pH 7.4
with HCl) was transferred to borosilicate glass tubes (13 x 100 mm; Kimble-Chase).
Study compounds (in 5 µl DMSO) or DMSO controls, as appropriate, were then
introduced followed by spleen membranes (200.20 ± 2.07 µg of protein per tube). The
mixture was gently vortexed and incubated for 15 min at room temperature, whereupon
[3H]CP55940 (1 nM final concentration; added in 10 µl in DMSO) was injected, the tube
contents thoroughly mixed, and a 90 minute incubation at 30 oC with gentle shaking
carried out. The binding reactions were terminated by addition of ice-cold wash buffer
(0.9% NaCl containing 2 mg/ml BSA; 1 ml) and membranes were collected on pre-
adjusted to 7.4 with NaOH]. This dilution was performed over a 30 minute period
whereupon the suspension was centrifuged at 20,000 x g (at 4 0C) for 30 minutes. The
pellets containing purified synaptosomes were gently resuspended in 1 ml assay buffer
and 100 µl aliquots of pure synaptosomes [0.767 ± 0.11 µg of protein per aliquot as
determined by Peterson (1977)] were apportioned to 9 snap top vials which were held on
ice in the cold room until required.
4.3.8. Release of L-Glutamate from synaptosomes
The enzyme-linked fluorescence technique originally described by Nicholls et al.
(1987) was adopted with minor modifications to measure the release of endogenous
glutamate from mouse brain synaptosomes. Briefly, 100 µl synaptosomes were added
to 2 ml of ice-cold assay buffer and the suspension incubated in shaking water bath at
37 0C for 15 mins. The suspension was then transferred to stirred quartz cuvette
thermostated at 37 0C in a Perkin Elmer LS 50 spectrophotometer. NADP+ (1 mM),
glutamic dehydrogenase (100 units) and CaCl2 (as appropriate; 1.3 mM) were added
followed by the cannabinoid agonist (WIN55212-2) and/or antagonist (AM251) along
with study compounds, inhibitors, EGTA or solvent controls, as appropriate, and the
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system allowed to incubate for another 5 minutes. Fluorescence recording was started
and 4-AP (3 mM) was added at 100 secs to activate glutamate release from
synaptosomes and the experiment was terminated at 500 secs. The excitation
wavelength was set at 360 nm (slit width 5) and the emission was sampled at 460 nm
(slit width 5). Fluorescence output was measured at 1 sec intervals. The amount of
glutamate released was monitored as an increase in fluorescence due to NADPH
forming from NADP+ as a result of the oxidative deamination of released glutamate by
glutamate dehydrogenase. Standard glutamate was added to allow quantitation of the
released glutamate as nmol glutamate/mg synaptosomal protein.
All compound additions were done with microsyringes (Hamilton, USA).
4.3.9. Analysis of radioligand binding data and glutamate release data
Curve fitting and calculation of binding parameters was performed using Prism,
GraphPad Software Inc., San Diego, CA, USA). In the glutamate release assays,
fluorescence changes were measured with the vertical analysis tool of the Perkin Elmer
LS-50 software and the percentage change to the 4-AP-induced fluorescence signal
above the control (assay buffer added at 100 secs instead of 4-AP) was calculated.
Time resolved fluorescence data were also transferred to GraphPad Prism (5.0) and full
fluorescence traces constructed. Percentage changes induced by standard
pharmacological agents and study compounds were calculated as mean ± S.E.M. based
on 3-4 independent experiments.
4.4. Results
4.4.1. Effects of benzophenanthridines, piperonyl butoxide and (S)-methoprene on binding of [3H]SR141716A to CB1 receptors
Figure 4.1 shows the inhibitory effects of benzophenanthridines, piperonyl
butoxide and (S)-methoprene on specific binding of [3H]SR141716A to CB1 receptors in
the mouse brain membrane preparation. The benzophenanthridines, sanguinarine and
chelerythrine were the most potent inhibitors based on IC50 values of 732 nM (95%
confidence interval (CI) = 364-1470 nM) and 911 nM (95% CI = 713-1164 nM)
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respectively. Piperonyl butoxide and methoprene had weaker effects on [3H]SR141716A
binding, achieving IC50s of 21.1 µM (95% CI 17.4-25.7) and 62.8 µM (95% CI 48.4-81.5)
respectively. At maximum effect concentrations, the benzophenanthridines produced
>90% inhibition, in contrast to piperonyl butoxide (70-80%) and methoprene (approx.
50%).
4.4.2. Influence of study compounds on the saturation binding of [3H]SR141716A to CB1 receptors of mouse brain
The control saturation binding curve was constructed by measuring the specific
binding of [3H]SR141716A to CB1 receptors at equilibrium over a range of radioligand
concentrations (0.032 to 2.8 nM). Control experiments were always performed
concurrently with benzophenanthridines, piperonyl butoxide or (S)-methoprene assayed
at ≥ IC50 (Figure 4.2). All study compounds reduced the apparent Bmax, but only
methoprene and piperonyl butoxide increased the Kd of [3H]SR141716A binding (by
approx. 3- and 5-fold respectively). Analogous equilibrium binding experiments were
conducted with [3H]CP55940 (Figure 4.3) and differences between benzopenanthridines
and synthetic compounds were again indicated. The benzophenanthridines increased
the Kd of [3H]CP55940 binding by approximately 6-fold, but did not alter Bmax. In
contrast, methoprene increased the Kd of [3H]CP55940 binding by almost 4-fold and
reduced Bmax by approx. 60%. Piperonyl butoxide reduced the Bmax of [3H]CP55940
binding by close to 50%, but had no influence on the Kd.
4.4.3. Effects of sanguinarine, chelerythrine, piperonyl butoxide, and (S)-methoprene on the kinetics of CB1 receptor-selective radioligand binding
The effects of sanguinarine, chelerythrine, piperonyl butoxide, and (S)-
methoprene on the association of [3H]SR141716A with CB1 receptors over the initial
phase of the binding reaction are displayed in Figure 4.4. All compounds reduced the
ability of [3H]SR141716A to progressively bind to CB1 receptors, when applied both
before radioligand (Figure 4.4a) and together with radioligand (Figure 4.4b). In similar
experiments, all study compounds reduced the association of [3H]CP55940 (Figure
4.4c). When added together with a saturating concentration of SR141716A, piperonyl
butoxide (30 µM) and (S)-methoprene (60 µM) increased the dissociation of the
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[3H]SR141716A:CB1 receptor complex to a rate greater that induced by the saturating (5
µM) concentration of SR141716A alone (Figure 4.5a). Sanguinarine and chelerythrine
(at 5 µM) failed to increase SR141716A-induced dissociation under these conditions
(data not shown). However, when applied alone, all study compounds initiated
dissociation of [3H]SR141716A, (S)-methoprene being the least effective (Figure 4.5b).
In the presence of a saturating (5 µM) concentration of CP55940, only piperonyl
butoxide accelerated the dissociation of [3H]CP55940 to a level greater than that induced
by CP-55940 alone (Figure 4.5c).
4.4.4. Effects of study compounds on mouse spleen CB2 receptors as assessed by inhibition of [3H]CP55940 binding
After verifying the IC50s of sanguinarine, chelerythrine, piperonyl butoxide and
(S)-methoprene that we reported previously for [3H]CP55940 binding to mouse brain CB1
receptors (Dhopeshwarkar et al. 2011), we employed identical concentrations to
investigate the inhibitory actions of study compounds on [3H]CP55940 binding to CB2
receptors of mouse spleen. In contrast to (S)-methoprene, which showed 51% inhibition
of [3H]CP55940 binding to CB2 receptors, the three other compounds were very much
weaker, achieving 4% inhibition (chelerythrine), 14% inhibition (sanguinarine) and 21%
inhibition (piperonyl butoxide), (Table 4.1). Since (S)-methoprene was of very similar
inhibitory potency at brain CB1 as spleen CB2 receptors, the relationship between
concentration and inhibition of [3H]CP55940 binding to spleen CB2 receptors was
explored in more detail. The results demonstrated (S)-methoprene to be a partial
inhibitor, unable to produce greater than 50% inhibition of [3H]CP55940 binding (Figure
4.6).
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
In marked contrast to 50 µM veratridine-evoked release of L-glutamate from
synaptosomes, the release of L-glutamate induced by 3 mM 4-AP was completely
insensitive to inhibition by 5 µM tetrodotoxin (TTX; Figure 4.7). In these preliminary
experiments, we also verified that 4-AP-evoked release of L-glutamate is partially (28.4 +
1.59 %) inhibited by the CB1-R agonist WIN55212-2 (8 µM), that this inhibition by
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WIN55212-2 is completely relieved by the CB1-R antagonist AM251 (8 µM), and that
preincubation with AM251 alone has no influence on the amount of neurotransmitter
released by 4-AP (Figure 4.8). All study compounds were then tested at a low and high
concentration for their ability to modify WIN55212-2-dependent inhibition of L-glutamate
release from mouse brain synaptosomes. Sanguinarine and chelerythrine (both at 0.25
µM) and (S)-methoprene at 5 µM failed to affect inhibition by WIN55212-2 of 4-AP-
evoked release, however a weak (approx. 5%) reduction in inhibition by WIN55212-2
was observed with piperonyl butoxide at 5 µM (Figures 4.9-4.12). In the presence of
WIN55212-2, sanguinarine and chelerythrine (at 2 µM) enhanced the release of L-
glutamate over and above that of 4-AP alone (Figures 4.9 and 4.10), whereas (S)-
dependent inhibition of L-glutamate release without any tendency for a
benzophenanthridine-like overshoot (Figures 4.11 and 4.12). Like AM251, chelerythrine
(2 µM), sanguinarine (2 µM), (S)-methoprene (25 µM) and piperonyl butoxide (2 µM), did
not enhance the baseline release of L-glutamate from mouse brain synaptosomes
(Figures 4.9-4.12).
4.5. Discussion
In an earlier investigation the benzophenanthridine alkaloids (chelerythrine and
sanguinarine) and the pesticide formulation components ((S)-methoprene and piperonyl
butoxide) were found to inhibit the binding of [3H]CP55940 to CB1-Rs of mouse brain and
inhibit CB1-R agonist-dependent activation of [35S]GTPγS binding to the G protein Gα
subunit (Dhopeshwarkar et al., 2011). The present research provides insight into how
these compounds inhibit the binding of [3H]CP55940 and [3H]SR141716A to CB1-Rs of
mouse brain and explores putative antagonist-like actions further by investigating the
ability of study compounds to modify evoked (CB1-R-sensitive) release of L-glutamate
from mouse brain synaptosomes.
Present evidence indicates that [3H]CP55940 and [3H]SR141716A bind to
specific regions within the CB1 receptor binding pocket (Shim, 2010). Our initial
experiments of this investigation established IC50s for study compounds in the
[3H]SR141716A binding assay. When compared to [3H]CP55940 values
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(Dhopeshwarkar et al., 2011), the inhibitory potencies for [3H]SR141716A binding were 1
to 2-fold higher for benzophenanthridines and 2.5-fold and 3.8-fold lower for piperonyl
butoxide and (S)-methoprene respectively, indicating different capacities to impact on
radioligand-specific binding loci within the CB1-R binding pocket. Our saturation binding
and kinetic data for these radioligands support this and other mechanistic differences
between study compounds in their actions.
The saturation binding constants obtained for [3H]SR141716A agree closely with
those published by Rinaldi-Carmona et al. (1996) using a fraction from rat brain. We
found that chelerythrine and sanguinarine decrease the total number of binding sites
(Bmax) available to [3H]SR141716A without affecting the affinity (Kd). This may be
explained by the benzophenanthridines binding to an allosteric site and triggering a
profound conformational modification to the [3H]SR141716A recognition site such that
radioligand cannot bind. However our results do not exclude benzophenanthridines
binding irreversibly (or in a very slowly reversible manner) to the orthosteric site. At
similar concentrations to those used for the benzophenanthridines in this study,
Beausoleil et al., (2009) found that sanguinarine and chelerythrine competitively inhibit
the binding of a GTP fluoroprobe to Rac1b (a GTP binding protein), and this led us to
propose that benzophenanthridines may inhibit [3H]CP55940 binding to the CB1-R by
targeting the guanine nucleotide recognition site on its associated G-protein
(Dhopeshwarkar et al., 2011). However, while it is well recognized that GTP and its
GTP analogs allosterically dissociate [3H]CP55940 from the CB1 receptor (Devane et al.,
1988; Houston and Howlett, 1993), the binding of [3H]SR141716A is known to be
unaffected by 300 µM GTPγS (Rinaldi-Carmona et al., 1996). Nevertheless, our
saturation binding assays with [3H]CP55940 indicated that the benzophenanthridines
reduce radioligand binding affinity without changing Bmax, lending support to an apparent
competitive mechanism for inhibition of [3H]CP55940 binding by benzophenanthridine
alkaloids, with potential for allosteric involvement. The kinetic results support the idea
that benzophenanthridine-dependent changes to the equilibrium binding constants of
[3H]CP55940 and [3H]SR141716A can arise both through a slowing of the rate of
association and by increasing the rate of dissociation of these radioligands.
It is improbable that (S)-methoprene and piperonyl butoxide engage with the
[3H]SR141716A recognition site itself because the pattern of inhibition by these
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compounds is mixed, with both compounds affecting Bmax and Kd of [3H]SR141716A
binding. The possibility of an allosteric mechanism with respect to [3H]SR141716A
binding is supported because (S)-methoprene and piperonyl butoxide increase the
dissociation of equilibrated radioligand from its recognition site, irrespective of whether a
saturating concentration of unlabeled SR1417126A is present or not.
Our findings regarding the mechanism of interference with the binding of
[3H]CP55940 by (S)-methoprene are not so clear. Whereas a reduction in the initial rate
of formation of the radioligand:recognition site complex by (S)-methoprene, combined
with its failure to influence the dissociation of [3H]CP55940 lends strong support to a
simple competition, this compound clearly increases Kd and reduces Bmax in saturation
binding assays, an outcome in apparent conflict with such a mechanism. We have
hypothesized that (S)-methoprene may represent a flexible analog of ∆9-
tetrahydrocannabinol (Dhopeshwarkar et al., 2011), a phytocannabinoid that binds to the
same site region of the CB1-R binding pocket as CP55940 (Gatley et al., 1997; Thomas
et al., 2005). A model that involves binding of (S)-methoprene to the [3H]CP55940
binding region in two conformations, one that allows radioligand to bind but with reduced
affinity, and one that blocks access to [3H]CP55940, could reconcile these observations.
The manner in which (S)-methoprene and piperonyl butoxide interfere with
[3H]CP55940 binding are evidently different since the ability of piperonyl butoxide when
combined with a maximum effect concentration of unlabeled CP55940 to accelerate
dissociation of radioligand over and above the rate observed with a maximum effect
concentration of CP55940 alone suggests a prominent allosteric component. We
conclude that the reduction in the Bmax of [3H]CP55940 binding observed with piperonyl
butoxide in saturation binding experiments is most likely allosterically-mediated and
therefore not inconsistent with our premise (Dhopeshwarkar et al., 2011) that piperonyl
butoxide could bind to the endocannabinoid receptor within the CB1-R binding pocket.
The L-glutamate release experiments reported in this investigation provide
strong support to the pharmacological profiling we originally proposed for
benzophenanthridines, (S)-methoprene and piperonyl butoxide that was based on
inhibition of CB1-R agonist-activated [35S]GTPγS binding (Dhopeshwarkar et al., 2011).
In preliminary experiments, we confirmed, as found originally by Wang (2003), that 4-
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AP-dependent release of L-glutamate is inhibited by WIN55212-2, and that the inhibition
is relieved by a diarylpyrazole (AM251 in present studies). The lower level of inhibition
(approx. 28%) by WIN55212-2 observed in the present investigation aligns more closely
with the level of inhibition by WIN55212-2 of KCl-evoked L-glutamate release from
synaptosomes (Godino et al., 2007). We also found that this WIN55212-2-sensitive
component of release required extrasynaptosomal Ca++ (data not shown) and that 3 mM
4-AP-induced release of L-glutamate from synaptosomes was unaffected by
tetrodotoxin. This latter result confirmed a lack of participation of voltage-sensitive
sodium channels in the 4-AP response, a critical prerequisite because CB1-R drugs,
including WIN55212-2 and diarylpyrazoles inhibit voltage-sensitive sodium channels at
low micromolar concentrations (Nicholson et al. 2003; Kim et al., 2005; Liao et al., 2004),
and very similar concentrations of these drugs are necessary to reveal CB1-R-dependent
effects on L-glutamate release from synaptosomes.
A salient finding of the present study is that concentrations of sanguinarine,
chelerythrine, (S)-methoprene and piperonyl butoxide that have no effect on basal
release of L-glutamate are able to neutralize (reverse) the inhibitory effect of WIN55212-
2 on 4-AP-evoked release of this neurotransmitter. In parallel experiments, the classical
CB1-R antagonist AM251 displayed an identical profile in agreement with other studies
using the diarylpyrazole AM281 (Wang 2003; Godino et al., 2007). The effects of the
study compounds on WIN55212-2-dependent inhibition of 4-AP-evoked release are
concentration-dependent. Moreover, the concentrations of sanguinarine, chelerythrine,
(S)-methoprene and piperonyl butoxide that we show are capable of neutralizing the
inhibitory effects of WIN55212-2 on L-glutamate release are very similar to those needed
to produce significant inhibition of radioligand ([3H]CP55940 and [3H]SR141716A)
binding and inhibition of CB1-R agonist-stimulated [35S]GTPγS binding to the G protein.
In marked contrast to the neutral antagonist actions of (S)-methoprene and
piperonyl butoxide in the L-glutamate release assay, an inverse agonist-like action was
indicated for sanguinarine and chelerythrine since at 2 µM (and in the presence of 4-AP),
these alkaloids promote release of neurotransmitter that is greater than that achieved by
4-AP alone. Our results show that this latter phenomenon is exclusively dependent on
WIN 55212-2 being present, so for benzophenanthridines to act in this way in vivo, a
significant level of endocannabinoid tone would be necessary.
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In conclusion, we demonstrate that binding sites for [3H]CP55940 and
[3H]SR141716A within the CB1-R binding pocket are differentially influenced in the very
low micromolar range by benzophenanthridine alkaloids (chelerythrine and
sanguinarine), and in the low to moderate micromolar range by piperonyl butoxide and
(S)-methoprene. Certain structural features of these study compounds or their binding
sites may be useful points of consideration for discovery of more potent G-protein-
coupled CB1 receptor blocking drugs that might be capable of downregulating the central
effects of endocannabinoid agonists.
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4.6. Figures and Table
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.
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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.
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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.
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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. 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).
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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. b) The same study compound concentrations were applied simultaneously with [3H]SR141716A. 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 4.4c Influence of study compounds on the time course of association of [3H]SR141716A and [3H]CP55940 with CB1 receptors of mouse brain. c) 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).
138
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. Symbols: diamonds = control; triangles = piperonyl butoxide and squares = (S)-methoprene. Data represent mean + SEM of at least 3 independent experiments, each performed in triplicate.
139
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. 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 4.5c The influence of study compounds on the dissociation of CB1 receptor-selective radioligands. 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.
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Figure 4.6 Relationship between concentration of (S)-methoprene and inhibition at CB2 receptors of mouse spleen based on interference with [3H]CP55940 binding.
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Figure 4.7a Inhibition of 50 µM veratridine-evoked release of L-glutamate from mouse brain synaptosomes by 5 µM tetrodotoxin (TTX)
143
Figure 4.7b Failure of 5 µM TTX to modify 3 mM 4-AP-evoked release of L-glutamate from synaptosomes.
144
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.
145
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.
146
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.
147
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.
148
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.
149
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).
Compound Inhibition of [3H]CP55940 binding to CB2 receptors of mouse spleen
Chelerythrine (2.2 µM) 4.14 + 0.14
Sanguinarine (1.2 µM 14.34 + 0.23
Piperonyl butoxide (8.2 µM) 20.86 + 0.23
(S)-Methoprene (16.4 µM) 50.98 + 0.21
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5. Effects of organotins on the CB1 receptor pathway of mouse brain in vitro.
Note: The research described in this chapter will be submitted shortly for
publication in an appropriate neurochemical/neuropharmacological journal. The
submission will adhere closely to the format laid out here.
5.1. Introduction
This Chapter constitutes a report on the modulatory effects of eight tributyltins
and two triphenyltin compounds at G protein-coupled cannabinoid-1 receptors (CB1-Rs)
of mouse brain in vitro. Organotin compounds have been exploited extensively for their
ability to preserve lumber and prevent biofouling on marine structures. Concerns over
toxicity to many marine and terrestrial species has led to severe restrictions on organotin
use, however they continue to be used on large ocean-going cargo ships because of the
huge reductions in fuel costs (and CO2 output) that can be achieved by minimizing
biofouling and the associated frictional drag on hulls. Tributyltin derivatives have also
seen significant uses as chemical intermediates and also as catalysts for various
chemical reactions.
The nervous system is known to be a highly sensitive target of tributyltins and a
number of mechanisms appear to be involved in their toxic actions. For example, in vivo
administration of tributyltin chloride to pregnant mice elevates dopamine concentrations
in the striatum and 5-hydroxytryptamine (5-HT) concentrations in the medulla oblongata
of F1 offspring, while dams exhibit widespread decreases in brain 5-HT levels (Tsunoda
et al., 2006). Tributyltins have been reported to lower the binding of [3H]MK801 to NMDA
receptors in the cerebral cortex of mice both in vitro and in vivo (Konno et al., 2001).
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Tributlytins modulate glutathione levels in the striatum, hippocampus and cortex (Fortier
et al., 2010). Measurements of extracellular glutamate concentrations indicate that
tributyltins cause significant release of L-glutamate from neurons, leading to
excitotoxicity (Nakatsu et al., 2006).
The potential of tributyltins to interfere with the binding of [3H]CP55940 was
demonstrated originally as a result of experiments conducted by Dr. Chengyong Liao in
our laboratory. Mr. Sudip Ghose and Mr. Saurabh Jain of our laboratory went on to test
other organotin compounds in the [3H]CP55940 binding assay. Selected tributyltin
compounds were subsequently assayed for their capacity to modulate basal and
CP55940-stimulated binding of [35S]GTPγS to the G protein by Mr. Saurabh Jain (M.Sc.
Thesis Simon Fraser University, 2011) These assays suggested that some tributyltins
could possibly act as inverse agonists of CB1-Rs at low micromolar concentrations. My
task was to examine selected tributyltins for their ability to modify WIN55212-2-
dependent inhibition of evoked transmitter (L-glutamate) release from synaptosomes,
since we hypothesized that the inhibitory effect of WIN55212-2 should be neutralized
and transmitter release may possibly undergo further enhancement.
5.2. Materials and methods
All organotin compounds were purchased from Sigma-Aldrich. Methodological
details of the binding assays for [3H]CP55940 and [35S]GTPγS have been adequately
described in Chapter 2, Section 2.3.3 and 2.3.4 and Chapter 3, Section 3.3.2 and 3.3.3
of this thesis. The isolation of synaptosomes from mouse brain and the assay of L-
glutamate release was described in sufficient detail in the previous Chapter 4 (Section
4.3.7 and 4.3.8).
152
5.3. Results
5.3.1. Displacement of [3H]CP55940 binding to mammalian CB1 receptors by organotin compounds
Eight tributyltins and 2 triphenyltins were tested for their ability to interfere with
[3H]CP55940 binding to mammalian CB1 receptors. The two triphenyltins and all
tributyltins except two (tributyltin hydride and tributylphenyltin; both inactive) achieved
IC50s at low micromolar concentrations (Table 5.1). All except the two inactive
compounds gave well-defined sigmoidal inhibition curves. Apart from phenylethynyl
tributyltin (which had the highest IC50 value of the group of active compounds and
produced about 80% inhibition at its maximum effect concentration), all other active
compounds achieved full (93-100%) inhibition. Concentration:inhibition curves are
displayed for tributyltin benzoate, tributyltin acetate and phenylethynyl tributyltin (Figure
5.1).
5.3.2. Basal and CP55940-stimulated [35S]GTPγS binding to the Gα subunit as influenced by tributyltin compounds
The inhibitory effects of tributyltin benzoate and phenylethynyl tributyltin on
[35S]GTPγS binding to the Ga subunit are shown in Figure 5.3. Tributyltin benzoate
reached IC50 at 1.43 µM (95% CI = 1.35 - 1.53 µM) and produced complete inhibition of
agonist-stimulated [35S]GTPγS binding around 2.5 µM. In addition tributyltin benzoate
encroached 51.03 ± 3.24% into basal binding at 5 µM with agonist present.
Phenylethynyl tributyltin achieved IC50 at 1.87 µM (95% CI = 1.71 - 2.02 µM) and
reached complete inhibition of agonist-stimulated [35S]GTPγS binding around 4 µM with
48.98 ± 14.64% and 68.53 ± 8.81% encroachment into the basal binding signal at 10 µM
and 20 µM respectively. Tributyltin benzoate (on its own) reduced basal [35S]GTPγS
binding by 22.12 ± 3.60% (at 2.5 µM) and 50.87 ± 1.87% (at 5 µM). Phenylethynyl
tributyltin (on its own) also reduced basal binding signal by 63.06 ± 3.62% at 20 µM.
153
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
The capacity of tributyltin acetate and phenylethynyl tributyltin to modify CB1-R
agonist- (WIN55212-2-) dependent inhibition of 4-AP-evoked release of L-glutamate
from synaptosomes was explored to test the hypothesis that tributyltins negatively
modulate CB1-Rs by acting as inverse agonists. The fluorescence traces of L-glutamate
release from synaptosomes under these circumstances are displayed in Figures 5.4 and
5.5. Tributyltin acetate and phenylethynyl tributyltin (at 3 µM) fully relieved the inhibition
of 4-AP-evoked release caused by 8 µM WIN55212-2. This effect was identical to that of
the classical diarylpyrazole inverse agonist/antagonist AM251 at 8 µM. No relief of
WIN55212-2-dependent inhibition was observed at 0.5 µM with either compound. In
contrast to phenylethynyl tributyltin, tributyltin acetate (at 3 µM) actually enhanced the
release of L-glutamate over and above that observed with 4-AP alone. Little effect (2-8%
change) of the organotin compounds alone on background release of L-glutamate from
mouse brain synaptosomes was detected (Figures 5.4 and 5.5).
5.4. Discussion
This investigation demonstrates that a number of tin-containing compounds have
the capacity to inhibit the binding of [3H]CP55940 to CB1-Rs in mouse brain at very low
micromolar concentrations. However, the relationships between structural features and
inhibitory potencies are complex. Compounds containing a wide variety of substituents
attached to the central tin atom of the tributyltin system (including benzoate, acetate,
methoxide, hydroxide, bromide and trifluoromethane sulfonate), as well as the hydroxide
derivative of triphenyltin, all achieved IC50 between 2.0 and 3.3 µM. The chloride
derivative of triphenyltin and the phenylethynyl derivative of tributyltin were less potent
(IC50s approx. 5 and 15 µM respectively). Intriguingly, the highly bulky three phenyl
substituent system (in triphenyltin hydroxide and triphenyltin chloride) fails to reduce
inhibitory potency greatly. Furthermore, in the tributyltin series, if a single phenyl ring is
attached directly to the central tin atom (e.g. tributylphenyl tin), inhibitory activity is
eliminated. However, if an ethynyl or carboxylate spacer is inserted between the tin atom
154
and the phenyl ring (as in phenylethynyl tributyltin or tributyltin benzoate), the ability to
inhibit the binding of [3H]CP55940 to CB1-Rs dramatically recovers. In addition, within
the subtituted tributyltin series, the hydride is inactive; nevertheless, replacement of the
hydrogen with bromine produces the most potent organotin inhibitor we have found to
date.
The results of the [35S]GTPγS binding experiments (conducted by Mr. Saurabh
Jain) provided the first indication that tributyltins are able to interfere functionally with
CB1-R agonist-dependent activation of the CB1-R:G protein complex. For these
experiments, we focused specifically on tributyltin benzoate and phenylethynyl tributyltin.
Since the results demonstrated that both compounds inhibit basal binding of [35S]GTPγS,
CP55940-stimulated binding of [35S]GTPγS to the Gα subunit and they also inhibit the
basal [35S]GTPγS signal with CP55940 present, we infer that they likely act as inverse
agonists of CB1-Rs.
The possibility that tributyltins negatively modulate CB1-Rs by acting as inverse
agonists, was pursued further by investigating the ability of two analogs (tributyltin
acetate and phenylethynyl tributyltin) to modulate CB1-R agonist- (WIN55212-2-)
dependent inhibition of the release L-glutamate from synaptosomes following challenge
with 4-aminopyridine. This assay is well suited for such studies because it has been
shown by Wang (2003) and Godino et al. (2007) that inhibition of evoked L-glutamate
release from synaptosomes by WIN55212-2 is blocked by AM281, a classical
diarylpyrazole inverse agonist. Tributyltin acetate and phenylethynyl tributyltin fully
suppressed the inhibition of 4-AP-evoked release caused by WIN55212-2. However, in
contrast to phenylethynyl tributyltin, tributyltin acetate actually enhanced release over
and above that observed with 4-AP alone. Tributyltin acetate and phenylethynyl
tributyltin are certainly functional inhibitors of CB1-Rs as assessed by their modulatory
effects on presynaptic release of L-glutamate. Moreover, they closely mimic the standard
antagonist/inverse agonist AM281 in this assay. The profile of tributyltin acetate is
particularly consistent with an inverse agonist action at brain CB1-Rs. Given the findings
of this study, the ability of tributyltins to cause significant release of L-glutamate from
neurons and excitotoxicity (Nakatsu et al., 2006) may be caused in part by blockade of
endocannabinoid-dependent inhibition of L-glutamate release.
155
Although organotin compounds can be quite toxic to mammals, there may be
potential to develop from these structures a new class of drug that acts at CB1 receptors.
A practical approach may be to develop hybrid molecules that link the tin-containing
center with various pharmacophoric moieties present in the many classical CB1-R active
drugs. These hybrids may or may not be problematic from the toxicological standpoint.
Additionally, replacement of the central tetravalent tin atom with silicon or carbon could
generate useful structure activity data that could assist in drug design especially if tin-
TBT bromide Tributylphenyl tin Tributyl(phenyl ethynyl)tin
Sn
O
O
Sn
O
H
Sn
Cl
TBT benzoate Triphenyl tin hydroxide Triphenyl tin chloride
Sn
H
TBT hydride
Figure 5.1 Structures of tributyl and triphenyltin compounds examined in the present investigation. Structures were constructed using Isis Draw.
157
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).
158
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).
159
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.
160
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.
161
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).
Organotin IC50 with 95% confidence interval (µM)
TBT benzoate 2.6 (1.7 - 3.9)
TBT acetate 2.7 (2.3 - 3.3)
TBT methoxide 3.3 (2.7-3.8)
TBT bromide 2.0 (1.6 - 2.5)
Tributylstannyl-TMS 2.9(2.3- 3.6)
Triphenyltin hydroxide 2.6 (1.5-4.5)
Triphenyltin chloride 5.1
Tributylphenylethynyl tin 14.8 (9.8 - 22.2)
TBT hydride >100
Tributylphenyltin >100
The above IC50 values were calculated from concentration:inhibition experiments carried out by Dr. Chengyong Liao, Mr. Sudip Ghose and Mr. Saurabh Jain. TBT = tributyltin, TMS = trifluoromethane sulfonate
162
6. Conclusion and future prospects
The conclusions of my Ph.D. research have already been discussed in detail
In summary, I conclude that sanguinarine, chelerythrine, (S)-methoprene and
piperonyl butoxide exert inhibitory actions at CB1-Rs in mouse brain in vitro. These
compounds act at very low to moderate micromolar concentrations with an inhibitory
potency ranking (estimated from [3H]CP55940, [3H]SR141716A and [35S]GTPγS binding
data) of sanguinarine ~ chelerythrine > piperonyl butoxide > methoprene.
Based on my saturation binding and kinetic experiments I infer that these
compounds inhibit via predominently allosteric mechanisms with respect to [3H]CP55940
and [3H]SR141716A binding.
My experiments with mouse brain synaptosomes demonstrate that WIN-55212-2-
dependent inhibition of 4-AP-evoked L-glutamate release is blocked by sanguinarine,
chelerythrine, (S)-methoprene and piperonyl butoxide. The actions of (S)-methoprene
and piperonyl butoxide are indistinguishable from AM251 (a classical diarylpyrazole CB1-
R antagonist), demonstrating an antagonist action at CB1-Rs. In addition to blocking the
inhibitory effect of WIN55212-2 on evoked release of L-glutamate, sanguinarine and
chelerythrine (with WIN55212-2 present) increase the release of neurotransmitter to a
level greater than that caused by WIN55212-2 alone. This suggests an inverse agonist-
like action of the benzophenanthridines. The L-glutamate release results therefore align
with our previous profiling of these compounds in the [35S]GTPγS binding assay.
Building on the findings of Dr. Chengyong Liao, Ms. Kathleen M. Bisset and Mr.
Saurabh Jain in my laboratory, I further explored the pharmacological actions of
phthalate esters and tributyltins at brain CB1-Rs. Based on [35S]GTPγS binding and L-
163
glutamate release results I conclude that these common environmental pollutants are
antagonists of CB1-R function.
The environmental chemicals highlighted in this thesis represent a broad range
of structural classes. It is interesting that when their potential to modify functional
outcomes of the CB1-R signaling pathway was investigated (i.e. using [35S]GTPγS
binding and L-glutamate release) only inhibitory (antagonist or inverse agonist-like)
actions were revealed. From these observations I infer that environmental chemicals
possessing the structural features of a CB1-R agonist might be more rarely encountered.
It would be logically predicted that if the study compounds were able to enter the
brain and engage with CB1-Rs, they should reduce the effectiveness of
endocannabinoids (e.g. anandamide and 2-AG) in activating CB1-Rs. I recommend that
future studies examine the ability of these compounds to inhibit both [3H]anandamide
binding to CB1-Rs and anandamide-induced suppression of L-glutamate release from
synaptosomes. Another very important line of future research would be to examine how
in-vivo administration of study compounds might modify the classical behavioral
manifestations of CB1-R agonists. Should any compound show CB1-R antagonist or
inverse agonist-like effects in vivo, careful consideration should be given to its potential
as a prototype for rational design of more effective analogs. As mentioned in the
introduction, CB1-R antagonists are effective in body weight reduction. Despite of the
issue with Rimonabant (SR141716A) and the USFDA's recent approval of Lorcaserin (a
weight reducing 5-HT2C agonist), there is a huge demand for drugs with this property.
Certain study compounds (e.g. (S)-methoprene and piperonyl butoxide) would represent
very low acute toxicity starting points and I would also suggest that improvements in
potency to the level of SR141716A may not be needed as some CNS drugs are effective
and well tolerated at low micromolar concentrations.
164
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