Cannabinoids and endocannabinoids: two sides of one Title 6-10 September coin 2010 Supramolecular Systems in Chemistry and Biology Lviv, Ukraine Piotr BREGESTOVSKI Mediterranean Institute of Neurobiology Marseille, France Euphoria Cannabis sativa Short history of cannabis Marijuana (Cannabis sativa) has been used for thousands of years. It is one of the oldest multi-purpose commercial plants grown for its fibre, edible seeds and psychotropic substances. China, India, Greece, Roman, Arabic countries > 5 000 years • Pain • whooping cough • asthma • as sebative/soporific • Euphoria (Shen-Nung, China’s Emperor) Avicenna - arabian physician described the medical uses of cannabis Cannabis sativa Bad and Good cannabis Marijuana (Cannabis sativa) has on the brain and human body many powerful effects, including: M Bad cannabis Good cannabis ? • pain sensitivity decrease • lowering blood pressure • asthma • sebative effects • decreased intraocular pressure • Impared motor coordination • Tremor • Decreased body temperature • Altered perception, memory • Hallucinations Euphoria We will discuss today: Evidences on existance endocannabinoid (EC) system Types of cannabinoid receptors (CBRs) Distribution of cannabinoid receptors in CNS Main classes of cannabinoids Synthesis and degradation of endocannabinoids Evidences on retrograde action of ECs Contemporary view on mechanisms of endocannabinoid signaling in CNS 1964 - discovery of D9-Tetrahydrocannabinol (D9-THC) the major psychoactive ingredient of Cannabis sativa Gaoni Y. & Mechoulam Cannabis sativa THC Discovery of D9-Tetrahydrocannabinol (D9-THC) OH O D9-Tetrahydrocannabinol Raphael Mechoulam Numerous related compounds have been synthesized or isolated, and together they form a class of drugs called the CANNABINOIDS. Endocannabinoid system in biological organisms: Main Evidences • Specific receptors for cannabinoids (CBRs) • Endogenous cannabinoids (ECs) • Specific distribution of CBRs in the brain • Formation and degradation of ECs in neurons 20 years later after discovery of D9-Tetrahydrocannabinol Short history of cannabinoid system 1964 - discovery of D9-Tetrahydrocannabinol (D9-THC) the major psychoactive ingredient of Cannabis sativa Gaoni Y. & Mechoulam 1988 & 90 - identification & cloning of the first (CB1) receptor (rat) Devane, Howlett et al., 1988; Matsuda et al., 1990 1993- cloning of the second cannabinoid receptor (CB2) Munro et al. CB1 & CB2 cannabinoid receptors • Preservation in evolution: human, rat and mouse CB1 receptors have 97–99% amino acid sequence identity • CB1 & CB2: a low (45%) overall homology • CB1: 472 aminoacids, highly expressed in the CNS • CB2: 360 aminoacids, mainly expressed in cells of the immune system. • CB1: - adenylite cyclase inhibition; - N/Q- Ca2+ channels inhibition; - K+ conductance stimulation Endocannabinoid system in biological organisms: Main Evidences • Specific receptors for cannabinoids • Endogenous cannabinoids • Specific distribution of receptors in the brain Major Endocannabinoids First endocannabinoid, 1992 Anandamide - “internal bliss” (Sansrit) Lumir O. Hanus R. Mechoulam Anandamide THC & 2-AG Main brain endocannabinoid, 1995 Plant cannabinoid Major Endocannabinoids Anandamide Arachidonic acid Lumir O. Hanus R. Mechoulam Anandamide THC & 2-AG Plant cannabinoid Other possible endocannabinoids KANO ET AL., 2009 Endocannabinoids regulate multiple physiological and pathological conditions • Suppress nociceptive processing through activation of CB1 • • • • • and CB2 receptors Regulates food intake Immunomodulation Inflammation Addictive behavior Epilepsy Short history of cannabinoid system 1964 - discovery of D9-Tetrahydrocannabinol (D9-THC) the major psychoactive ingredient of Cannabis sativa Gaoni Y. & Mechoulam 1988 & 90 - identification & cloning of first cannabinoid (CB1) receptor Devane et al., 1988; Matsuda et al., 1990 1993- cloning of second cannabinoid receptor (CB2) Munro et al. 1992 - discovery of the first endocannabinoid (anantamide) Devane, Hanus & co-authors 1995 - identification of 2-arachidonoyl-glycerol (2-AG) main endocannabinoid in CNS Mechoulam et al.; Sugiura et al. Endocannabinoid system in biological organisms: Main Evidences • Specific receptors for cannabinoids • Endogenous cannabinoids • Specific distribution of receptors in the brain How to see disribution of receptors in the brain? • Radioactively labeled ligands • Specific antibodies Distribution of cannabinoid CB1 receptors in rat brain Revealed by an autoradiograph of thebinding of radioactively labeled CP-55940 (a high af®nity CBR ligand) Iversen, 2003 Antibodies for CB1 receptor Ken Mackie, Univ.Washington, Seattle Distribution of CB1 receptors in the brain 1mm Kano et al.2009 Distribution of CB1 receptors in the brain CB1 receptors are located presynaptically on axon terminals CB1 receptors are localized presynaptically From Katona et al (1999) J Neurosci 19:4544-58 CB1 Endocannabinoid system in biological organisms. Main evidences • Specific receptors for cannabinoids (CBRs) • Endogenous cannabinoids (ECs) • Specific distribution of CBRs in the brain • Formation and degradation of ECs in neurons Formation of 2-AG Phosphatydilinositol (PIP2) Phospholipase C (PLC) Diacylglycerol (DAG) lipase Diacylglycerol lipaseDG (DAG) 2-arachidonoilglycerol (2-AG) Formation of anantamide in neurons + Arachidonic acid Endocannabinoid system in biological organisms: Main Evidences • Specific receptors for cannabinoids (CBRs) • Endogenous cannabinoids (ECs) • Specific distribution of CBRs in the brain • Formation and degradation of ECs in neurons 1964-2009: 45 years Main classes of cannabinoids -Herbal cannabitoins (phytocannabinoids) -Synthetic cannabinoids -Endocannabinoids Phytocannabinoids Cannabis sativa Cannabis sativa: - contains 70 phytocannabinoids - 11 classes Cannabidiol THC Agonist Antagonist Endocannabinoids For CB1 EC50 = 240 nM For CB2 EC50 = 440 nM For CB1 EC50 = 3425 nM For CB2 EC50 = 1200 nM Amount of 2-AG in the brain is >100-fold higher then anandamide Synthetic cannabinoids Natural agonist Agonist Synthetic agonists and antagonists CB1 EC50 = 40 nM CB2 EC50 = 30 nM Agonist CB1 EC50 = 1.3 nM CB2 EC50 = 1 nM Antagonist IC50 = 7.5 nM Effects of cannabinoids (human): Psychological effects (2-10 mg): 1. Effective: - euphoria and elation; - cheerfulness (feeling of happiness); - sedation - transient hallucinations 2. Sensoric: - increased perception of external stimuli 3. Somatic: - feeling of the body floating or falling 4. Cognitive: - disturbed time perception (one min like several); - memory failure; - troubles with concentration Effects of cannabinoids (human): Psychological effects (>10 mg): - anxiety and panic; - impaired attention; - disturbed coordination - increased risk of traffic accidents Good cannabis Long- lasting use: Bad cannabis - increased myocardial infraction - chronic bronchitis - disturbance in the ability to organize complex information - “amotivational syndrome” - smaller whole brain volume (hippocampus, amigdala) - worsen symptoms of schizophrenia Therapeutic effects of cannabinoids: Positive effects of cannabinoids: - side effects of anti-tumor therapy (nausea and vomiting) -increase appetite (against anorexia) - agonists -decrease appetite (against obesity) - antagonists -glaucoma - antiallergic - antiinflammation - sleep disorders Bad cannabis -anxiety disorders Positive effects of cannabinoids: Good cannabis - protective role in atherosclerosis progression - decreased intraocular pressure - decreasing the ischemic-reperfusion injury Medical applications - 1 Cesamet Lilly Indianapolis, Indiana, USA Treatment of cancer patients after chemotherapy: nausea and vomiting Marinol or Dronabinol Solvay, Marietta, USA - Cancer patients after chemotherapy: nausea and vomiting. - Appetite stimulant for treatment of cachexia (weight loss and wasting) in AIDS patients Medical applications-2 Agonist Sativex GW pharmaceuticals, Salisbury, Wiltshire, UK + Antagonist Relief of neuropathic pain Cannabidiol Mechanism of cannabinoids action in the brain Synapses in mammalian brain Excitatory Glutamate AMPA/Kainate Inhibitory GABA or/and Glycine GABAR GABAAR GlyR NMDA r-rs Endocannabinoid-mediated control of synaptic transmission Modulation of Ca2+ influx: - Inactivation of Ca2+ channels - Depletion of extracellular Ca2+ - Action potential failure CB Pre CB R-R Ca2+ Post Presynaptic receptors: - Metabotropic - Adeosine - Noradrenaline - mAChR - Cannabinoid receptors Modulation of postsynaptic receptors: - Sensitivity - Desensitization Discovery of Depolarization-induced Suppression of Inhibition: DSI Stim Whole-cell Pre Ca2+ Post Discovery of Depolarization-induced Suppression of Inhibition: DSI I. Llano & A. Marty, 1991 cerebelum Example of hippocampal DSI Depo Depo Depo Depo Depo Depo Depo Depo Depo Whole-cell Pre Ca2+ Post Pitler and Alger, 1992, hippocampus Depolarization-induced suppression of inhibition (DSI) DSI Features DSI in cerebellum & hippoampus Llano et al, 1991 cerebelum Stim Pitler and Alger, 1992, hippocampus Whole-cell Pre Ca2+ Post - results from decrease of neurotransmitter release -needs elevation of Ca in postsynaptic cell - involves retrograde signaling Depo Ca2+ Retrograde signals for DSI: endocannabinoids Wilson & Nicoll, 2001 Stim Whole-cell Pre Ca2+ Post Kreitzer & Regehr, 2001 Ohno-Shosaku et al., 2001 Diana et al., 2002 Ca2+-induced retrograde signaling by endocannabinoids AM281 - Antagonist B Depolarization Wilson & Nicoll, 2002 Short-term synaptic plasticity Depo Chevaleyre et al, 2006 Depo Hippocampal DSI is absent in CB1knock-out mice CB+/+ Depo Depo Depo Depo Depo 1mm Depo DSI and DES - general phenomenon CB1 CB1 CB1 Inhibitory synapses: GABAA- and glycinergic GlyR GlyR channels Ionic channels are key proteins of electrochemical transformation Short-term plasticity in glycinergic synapses DSI in glycinergic synapses Marat Mukhtarov Mukhtarov, Ragozzino, Bregestovski, J. Physiol., 2005 Davide Ragozzino Action of 2-AG in excitatory synapses Presynaptic cell AMPAR NMDAR mGluR Postsynaptic cell Synapses on dendrite & spine organization Sheng and Hoogenraad, 2007 Distribution of AMPA & NMDA receptors in synapses Somogyi et al., 1998 Distribution of mGlu receptors in synapses AMPAR NMDAR mGluR5 Area of perisynaptic signaling machinery Synapses on dendrite & spine organization mGluR5 DG lipase Formation of 2-AG Phosphatydilinositol (PIP2) Phospholipase C (PLC) Diacylglycerol (DAG) lipase Diacylglycerol lipaseDG (DAG) 2-arachidonoilglycerol (2-AG) Low activity Postsynapse Presynapse Activation of perisynapric signaling machinery Katona & Freund, 2008 High activity Suggested mechanism of 2-AG action in excitatory synapses QuickTime™ and a Photo - JPEG decompressor are needed to see this picture. Remember Bad cannabis Good cannabis Endocannabinoid system is an ubiquitous lipid signalling system Endocannabinoids are small molecules derived from arachidonic acid. They bind to G-protein-coupled cannabinoid receptors Two cannabinoid receptors have been cloned, CB1R and CB2R. Both are receptors coupled to the pertussis toxin–sensitive G protein, Gi/o. The CB1R are among the most highly expressed G proteincoupled receptors in the CNS, and they are responsible for most of the psychoactive effects of cannabinoids. Endocannabinoids regulate multiple physiological and pathological conditions: food intake, immunomodulation, inflammation, analgesia, cancer, addictive behavior, epilepsy and others In the brain, most CB1Rs are found on axon terminals. Its activation leads to presynaptic inhibition of transmitter release. Suggested reading Alger BE. Retrograde signa ling in the regu la tion of syn aptic trans mis sion: focus on endoc annab inoids. Prog Neurobiol. 2002 Nov;68(4):247 -86. Chevaleyr e V, Takaha shi KA, Castill o PE. Endo cannab inoid-me diated synap tic plastic ity in the CNS. Annu Rev Neurosci. 2006;29:37-76 Diana MA, Breges tovski P. Calcium and endocannab inoids in the modulation o f inhibit ory synap tic transmi ssion. Cell Calcium. 2005 May;37(5):497-505. Howlett AC, Breivoge l CS, Chil ders SR, Deadwyler SA, Hampson RE, Porrino LJ. Cannab inoid phys iology and pha rmacology: 30 yea rs of progre ss. Neuropha rmacology. 2004;47 Suppl 1:345 -58. Kano M, Ohno -Shosaku T, Hashimotodani Y, Uchigash ima M, Watanabe M. Endocannab inoid-mediated con trol of synap tic transmi ssion . Physiol Rev. 2009 Jan;89(1):309-80. Onaivi ES. Cann abinoid receptors in b rain: pha rmacogenetics, neu ropha rmacology, neu rotoxicology, and po tential t herapeutic applications . Int Rev Neurobiol. 2009;88:335-69.