Expression of Opioid and Cannabinoid Receptors as a Basis for Drug Effects

1. Expression of Opioid and Cannabinoid Receptors as a Basis for Drug Effects Jürgen Kraus Summer School Bologna 27.6.2012

2. 1. Intro: Opioids and Cannabinoids 2 Constitutive Expression of Opioid and Cannabinoid Receptors 3. Inducible Expression of Opioid and Cannabinoid Receptors 3.1 Cytokines Physiological Consequences Pharmacological Consequences 3.1 Epigenetic Factors Physiological Consequences Pharmacological Consequences

3. Papaver somniferum "KING HYPNOS WAS FATHER OF A THOUSAND SONS - INDEED A TRIBE - AND OF THEM ALL, THE ONE HE CHOSE WAS MORPHEUS, WHO HAD SUCH SKILL IN MIMING ANY HUMAN FORM AT WILL. NO OTHER DREAM CAN MATCH HIS ARTISTRY IN COUNTERFEITING MEN: THEIR VOICE, THEIR GAIT, THEIR FACE - THEIR MOODS; AND, TOO, HE IMITATES THEIR DRESS PRECISELY AND THE WORDS THEY USE MOST FREQUENTLY...“ OVID, METAMORPHOSES Friedrich Wilhelm Sertürner, „Über das Morphium“ (1817)

10. Analgesia Euphoria Antitussivum Sedation Opioids

13. Analgesia Euphoria Antitussivum Sedation Opioids

14. Opioids Analgesia Euphoria Antitussivum Sedation

15. ! Heroin taken orally with a clear indication will not make you high

16. Analgesia Euphoria Antitussivum Sedation Opioids

17. Opioid Receptors m Inhibition of Adenylyl Cyclase Inhibition of Ca-Channels Activation of K-Channels d Opioids k

18. Commonly Used Opioids: Agonists Morphine, Methadon, Pethidin, Tilidin, Oxycodone classical analgetics Fentanyl, Sufentanil Short acting drugs > anaesthesiology Buprenorphin, Fentanyl good analgetics as plaster, little tolerance Tramadol, Tapentadol MOR agonist plus noradrenaline reuptake inhibitor > analgetic plus antidepressant > elderly

19. Commonly Used Opioids: Antagonists Naloxone, Naltrexon classical highly potent antagonists used in morphine/heroin intoxications

20. Opioid Peptide Precursors Höllt, 1983

21. Met-Enkephalin and Morphine

22. Mood Hallucinations Emotion Food Intake Effects of Cannabinoids

23. Mood Hallucinations Anandamide Emotion Neuronal Homeostasis Food Intake 2-Arachidonylglycerol Effects of Cannabinoids

24. Exogenous Cannabinoids 9-Tetrahydrocannabinol (THC) Cannabis sativa

25. Cannabinoid Receptors Inhibition of Adenylyl Cyclase Inhibition of Ca-Channels Activation of K-Channels CB1 CB2 Cannabinoids others

26. 1. Intro: Opioids and Cannabinoids 2 Constitutive Expression of Opioid and Cannabinoid Receptors 3. Inducible Expression of Opioid and Cannabinoid Receptors 3.1 Cytokines Physiological Consequences Pharmacological Consequences 3.1 Epigenetic Factors Physiological Consequences Pharmacological Consequences

27. For physiological and pharmacological effects - and unwanted side effects - the expression of drug receptors at a certain place and a certain time is important Opioid Receptors: Mu, Delta and Kappa OR throughout the central and peripheral nervous system in distinct cells Cannabinoid Receptors: CB1: highly abundant in nervous system CB2: „peripheral CB“. Immune effector cells

28. Mood Hallucinations Anandamide Emotion Neuronal Homeostasis Food Intake 2-Arachidonylglycerol Effects of Cannabinoids

29. Opioids and Cannabinoids: Activation of the Reward System

30. Cannabinoid Antagonists: Drugs against Obesity Cannabinoid Antagonist Rimonabant Cannabinoid Receptors Food-Reward

31. ! this could be expected - and indeed, it was argued by many pharmacologists

32. 1. Intro: Opioids and Cannabinoids 2 Constitutive Expression of Opioid and Cannabinoid Receptors 3. Inducible Expression of Opioid and Cannabinoid Receptors 3.1 Cytokines Physiological Consequences Pharmacological Consequences 3.1 Epigenetic Factors Physiological Consequences Pharmacological Consequences

33. 1. Intro: Opioids and Cannabinoids 2 Constitutive Expression of Opioid and Cannabinoid Receptors 3. Inducible Expression of Opioid and Cannabinoid Receptors 3.1 Cytokines Physiological Consequences Pharmacological Consequences 3.1 Epigenetic Factors Physiological Consequences Pharmacological Consequences

34. Effects of Opioids on Immune Cell Functions  Opioid addicts are more susceptible to infections compared to non-opioid users  Chronic morphine treatment of mice leads to lymphoid organ atrophy, and reduced natural killer cell activity  Morphine decreases antigen-induced production of IL-2, IL-6 and TNF-a, and the activity of the transcription factor NFkB  In thymocytes, selective µ-opioid receptor agonists induce chemotactic responsiveness  Morphine and ß-endorphin direct the balance between type 1 and type 2 T helper cells towards the Th2 direction with increased production of IL-4 and decreased production of IFN-

35. µ opioid receptors in immune cells as a structural basis for opioid-mediated immunomodulation Induction of µ-Opioid Receptor Transcripts in Immune cells in Response to IL-4

36. IL-4 STAT6

37. STATs - STAT is a family of transcription factors - (signal transducer and activator of transcription) - STATs mediate transcriptional regulation evoked by various cytokines and growth factors - the STAT transcription factor family comprises seven members: STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B and STAT6 - distinctive DNA sequences for the different STATs are largely unknown, all members bind to the core sequence 5‘-TTC(N)2-4GAA-3‘

38. ? How to localize transcription factor binding sites on a gene‘s promoter

39. Defining Regulatory Promoter Elements by Reporter Gene Analysis Stimulus Reporter Gene Promoter

40. Reporter Gene Reporter Gene Reporter Gene Reporter Gene Promoter Promoter Promoter Promoter X Defining Regulatory Promoter Elements by Reporter Gene Analysis Stimulus

41. Interleukin-4 response elements H MOR PCE1 tagTTCatgGAAgaa H MOR PCE2 cttTTCctaGAAttt H MOR PCE3 ttCTTCTcAGAAGca R MOR rMG2 tttTTCcaGAAtga R MOR rMG6 ctgTTCccaGAAgct R MOR rMG5 cCTTTCcagaGAAAGa

42. Localization of IL-4 Responsive Elements on µ-Opioid Receptor Genes

43. Localization of IL-4 Responsive Elements on µ-Opioid Receptor Genes

44. Interleukin-4 response elements H MOR PCE1 tagTTCatgGAAgaa H MOR PCE2 cttTTCctaGAAttt H MOR PCE3 ttCTTCTcAGAAGca R MOR rMG2 tttTTCcaGAAtga R MOR rMG6 ctgTTCccaGAAgct R MOR rMG5 cCTTTCcagaGAAAGa

45. ? How to further characterize transcription factor sites (to be sufficient for publication)

46. Gel Reaction Tube Electrophoretic Mobility Shift Assay (EMSA) Radioactive Probe

47. Gel Reaction Tube Electrophoretic Mobility Shift Assay (EMSA) Radioactive Probe Transcription Factor

48. Gel Electrophoretic Mobility Shift Assay (EMSA) Unlabeled Competitor Radioactive Probe Reaction Tube Transcription Factor

49. - - + + EMSA is not a suitable method to study STAT6/DNA interactions EMSA with Putative STAT6 Elements

50. STATs - STAT is a family of transcription factors - (signal transducer and activator of transcription) - STATs mediate transcriptional regulation evoked by various cytokines and growth factors - the STAT transcription factor family comprises seven members: STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B and STAT6 - distinctive DNA sequences for the different STATs are largely unknown, all members bind to the core sequence 5‘-TTC(N)2-4GAA-3‘