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LP can»; . 4.5.”. 7.. 57,.~...;..A..n Mai». r '0' I.( 2 'Zi’uww LIBRARY Michigan State University This is to certify that the dissertation entitled MECHANISM OF CANNABINOlD-MEDIATED ELEVATION OF INTRACELLULAR CALCIUM IN T CELLS presented by Gautham Karkala Rao has been accepted towards fulfillment of the requirements for the PhD. degree in Pharmacology and ToxicoLOL / 7 Major Professor’s Signature 10/, We 3" Date MSU is an Affinnative Action/Equal Opportunity Institution PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 2/05 p:/ClRCIDateDue.indd-p.1 MECHANISM OF CANNABINOID-MEDIATED ELEVATION OF INTRACELLULAR CALCIUM IN T CELLS By Gautham Karkala Rao A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Pharmacology and Toxicology 2005 ABSTRACT MECHANISM OF CANNABINOID-MEDIATED ELEVATION OF INTRACELLULAR CALCIUM IN T CELLS By Gautham Karkala Rao Cannabinoids are a class of over sixty structurally-related plant-derived compounds present in the marijuana plant, Cannabis sativa. Apart from their psychoactive and behavioral effects, cannabinoids are widely known to possess immunomodulatory properties. Generally, it is believed that cannabinoids exert their effects on physiology through the G protein-coupled cannabinoid receptors, CB] and CB2, expressed primarily in the brain and immune system, respectively. In T cells, cannabinoid treatment has been demonstrated extensively to modulate T cell activation- related events, especially the expression of interleukin (IL)-2. The regulation of IL-2 transcription is critically dependent on intracellular calcium ([Ca2+]r), and the Ca2+- dependent transcription factor, nuclear factor of activated T cells (N FAT). Prior results from this laboratory have found a strong correlation between the modulation of IL-2 expression by cannabinoids and the modulation of NFAT activation and nuclear translocation. However, little is known about the cellular mechanism by which cannabinoids exert their effects on T cells. The present dissertation project, therefore, first examined the hypothesis that the suppression of IL-2 expression in T cells by cannabinoids was dependent on the cannabinoid receptor-mediated elevation of [Ca2+]i. The first objective of the present studies was to examine the effect of cannabinoids, A9- THC and CP55,940, on IL-2 expression and [Ca2+]i elevation in the human T cell lines, HPB-ALL and Jurkat E6-1. It was found that while both A9-THC and CP inhibited the expression of IL-2, only A9-THC treatment resulted in the elevation of [Ca2+]i. Therefore, it was concluded that the modulation of [Ca2+]i and inhibition of IL-2 production by cannabinoids may be simultaneous, yet unrelated events. Regardless, the mechanism by which Ag-THC elevated [Ca2+]t was characterized as the second objective of the present studies in the HPB-ALL cells, as [Ca2+]i plays a critical role in many T cell processes including activation of enzymes, triggering of apoptotic pathways, and induction of T cell anergy. Results showed that A9-THC elevated [Ca2+]i in a cannabinoid receptor antagonist-sensitive and Ca2+ store-independent manner through the TRPCI class of receptor-operated cation channels (ROCCs). In addition, it was established that the robust induction of [Ca2+], influx in resting T cells was a characteristic unique to the tricyclic classical cannabinoid compounds, which was not observed with either bicyclic congeners or the eicosinoid-derived endocannabinoids. A final objective of the current dissertation project was to determine unequivocally whether the C31 and C82 receptors were involved in the mechanism by which tricyclic cannabinoid compounds elevated [Ca2+]3. Measurements of [Ca2+], performed in the wildtype and CBl"’/CBZ"' murine splenocytes demonstrated that the tricyclic cannabinoids induced a rise in [Ca2+]; independently of both CB] and CB2 receptors. Altogether, the studies from the present dissertation project suggest that tricyclic cannabinoid compounds elevate [Ca2+]i in a CB] and C32 receptor- and intracellular Ca2+ store-independent manner through ROCCs in T cells. The elevation of [Ca2+]t mediated by a novel cannabinoid receptor may partly explain the mechanism by which tricyclic cannabinoids modulate T cell function. .. AUM .. DEDICATION I dedicate this work to all my teachers, secular and spiritual, whose words of advice have been a constant source of inspiration throughout my dissertation process. In addition, I dedicate this dissertation to my parents and grandparents, who taught me the value of hard work, persistence and steadfastness. iv ACKNOWLEDGEMENTS First and foremost, I acknowledge my mentor, Dr. Norbert Kaminski, who has not only guided me along the arduous path toward the completion of my dissertation, but also provided me the freedom to carve my own niche within the framework of the project. His encouragement, positive reinforcement, and enthusiasm for science, in general, have been and will continue to be a constant source of inspiration for me. Second, I would like to thank the members of my dissertation committee, Dr. Peter Cobbett, Dr. Patty Ganey and Dr. John LaPres, for providing me with deep insights and expert advice from their respective fields towards the interpretation and development of my dissertation project. The recommendations and thought-provoking questions from my committee members have been invaluable to my understanding of the field, and progress as a scientist. Finally, I would like to thank the members of Dr. Kaminski’s laboratory, Katie Boland, John Buchweitz, Bob Crawford, Kim Hambleton, Dr. Barb Kaplan, Colin North, Dina Shnaider, and Dr. Alison Springs. It has been my privilege to work with these great individuals on a day-to-day basis. Each of them has contributed to my project either by teaching me the techniques or providing insightful advice. I would especially like to thank Dr. Tong-Rong Jan and Dr. Cheryl Rockwell, former members of the lab, who convinced me to pursue my dissertation project in the field of cannabinoids. TABLE OF CONTENTS LIST OF TABLES .................................................... LIST OF FIGURES ................................................... LIST OF ABBREVIATIONS ........................................... INTRODUCTION .................................................... I. II. Marijuana, cannabinoids and cannabinoid receptors .................. Marijuana .............................................. Physiological effects of cannabis ............................. Cannabinoids compounds ................................... Therapeutic potential of cannabinoids ......................... Cannabinoid receptors ..................................... Endogenous ligands ....................................... Cannabinoid receptor antagonists ............................ harmacological and biochemical effects of cannabinoids ............. Effects of cannabinoids on the central nervous system ............ 1. CNS neurons ........................................ 2. Astrocytes and microglial cells .......................... B. Effects of cannabinoids on the immune system .................. 1. Innate immunity ..................................... a. Macrophages ..................................... b. Neutrophils, NK cells and dendritic cells ............... 2. Adaptive immunity ................................... a. B cells and humoral immunity ....................... (i). Antibody production .......................... (ii). B cell proliferation . . . . . . L ..................... b. T cells and cell-mediated immunity ................... (i). T cell proliferation ............................. (ii). IL-2 suppression .............................. (iii). IL-2 enhancement ............................. (iv). In vivo cytokine regulation ...................... 3. Host resistance studies ................................ C. CB1 and CBZ receptor-independent effects .................... 1. Brain .............................................. 2. Peripheral vasculature ................................. 3. Immune cells ........................................ 4. Implications ......................................... D Effects of cannabinoids on intracellular calcium ................. 1. Inhibition of calcium elevation .......................... 2. Differential regulation calcium elevation .................. 3. Involvement of the C81 and CB2 receptors ................ QWWPOW? P'U vi X xi XV ©\)O\MWN—~—‘ 11 ll 11 12 14 15 15 l7 19 20 20 21 22 23 24 27 28 29 30 31 32 33 34 35 36 38 39 4. Non-CB1, non-CBZ-dependent calcium regulation .......... 39 III. Intracellular calcium and calcium channels ......................... 41 A. Calcium in T cells: CCE and CRAC channels ................... 4l 1. Calcium elevation in T cell activation .................... 4] 2. Properties of CRAC channels ........................... 42 a. Regulation ....................................... 42 b. Conduction ...................................... 43 c. Inactivation ...................................... 44 (1. Inhibition ........................................ 44 B. TRP channels: structure and function .......................... 45 1. Discovery and cloning of TRP channels ................... 46 2. Structure of TRP channels .............................. 46 3. Function of TRP channels .............................. 49 a. TRPC, TRPV and TRPM ........................... 50 b. TRPN, TRPML, TRPP and TRPA .................... 50 4. Candidates for CRAC channels .......................... 51 a. TRPCI and TRPC4 ................................ 52 b. TRPV6 .......................................... 53 C. TRPC subfamily: SOC and ROC channels ..................... 54 1. Group 1 — TRPC] .................................... 55 2. Group 3 — TRPC 3/6/7 ................................ 56 a. TRPC3 .......................................... 57 b. TRPC6 .......................................... 57 c. TRPC7 .......................................... 58 3. Group 4— TRPC4/5 ................................... 59 D. TRPC channels and lymphocytes ............................. 60 IV. Objective .................................................... 62 MATERIALS AND METHODS ......................................... 64 I . Cannabinoid compounds ........................................ 64 II. Reagents ..................................................... 64 III. Animals ..................................................... 65 IV. Preparation of splenocytes ....................................... 65 A. Whole splenocytes .......................................... 65 B. T cell isolations ............................................ 66 V. Cultured cell lines ............................................. 66 VI. IL-2 ELISAs and mRNA quantification ............................. 67 A. Cell treatments ........................................... 67 B. IL-2 protein quantification .................................. 68 C. IL-2 mRNA quantification .................................. 68 VII. Intracellular calcium determination ................................ 69 VIII. Kinase assays ................................................. 70 A. Sample preparation ........................................ 70 B. Substrate activity determination .............................. 71 IX. Western analysis .............................................. 72 A. Sample preparation ........................................ 72 vii B. Electrophoresis and blotting ................................. 72 X. RT-PCR .................................................... 73 A. CB1 and CB2 ............................................ 73 B. TRPC1-7 ............................................... 74 XI. DNA sequencing .............................................. 74 A. CB2 receptor sequence ..................................... 74 B. TRPC] alternative splice ................................... 76 XII. siRN A knockdown of TRPC] ................................... 77 A. Transfection of TRPC] siRNA .............................. 77 B. Detection of TRPC] knockdown ............................. 77 XIII. Radioligand binding analysis .................................... 78 XIV. Statistical analysis ............................................. 80 EXPERIMENTAL RESULTS ........................................... 81 I. Effects of Ag-THC and CP on IL-2 expression in HPB-ALL and Jurkat E6-1 cells .............................................. 81 A. Differential effects of A9-THC and CP on IL-2 secretion .......... 81 B. Characterization of the CB1 and CBZ receptors ................. 84 C. Cannabinoid 9receptor antagonists fail to antagonize the suppression of IL- 2 by A 9-THC and CP .................................. 89 D PTx treatment does not attenuate the A 9-THC-induced suppression of IL-2 secretion ............................................ 96 E. A9-THC suppresses IL- 2 mRNA production in HPB-ALL cells ...... 96 11. Effects 9of cannabinoids on 2[Ca2 ].1n T cells ......................... 99 A. A9-THC elevates +2[Ca ].1n T cells, but CP does not .............. 99 B. Removal of [Ca2 ]e and pretreatment with BAPTA- AM attenuate the rise in [Ca2+]| elicited by A9-THC ............................. 103 C. Cannabinoid receptor antagonists attenuate the A 9-THC-mediated rise in [Ca2 ]I ............................................ 107 D. PTx does not attenuate the A9 -THC-mediated rise in [Ca2 ]. ........ 112 E. HU-210 and CBN elevate [Ca2 ]I ............................. 114 F. Removal of [Ca2+]c attenuates the rise in [Ca2+]t elicited by HU-210 and CBN ................................................ 118 G. Cannabinoid receptor antagonists attenuate the HU-210 and CBN- mediated rise in [Ca2 ]l .................................... 118 H. CBD elevates [Ca2+]. ...................................... 122 I. Effect of other cannabinoids on [Ca 23'] ........................ 126 III. Mechanism of A9-THC induced [Ca2 ]. elevation .................... 126 A. Effect of Ca2+ channel inhibitors on the rise in [Ca2 ]. elicited by A9 -THC and TG .......................................... 126 B. A 9-THC-mediated elevation in [Ca2 ].' 18 not abolished upon TG or 8- Br-cADPR pretreatment .................................. 134 C. 09AG elevates [Ca2 ]I In HPB- ALL cells independently of PKC ..... 137 D. A9—-THC induced elevation of [Ca: :]. 15 independent of CaMKIl. . . 146 E A9-THC- induced elevation of [Ca2 ]l 13 independent of PLC, PI3K, viii IV. and soluble tyrosine kinases ................................. 149 F. HPB-ALL cells express transcripts for TRPCI .................. 153 G. Knockdown of TRPCl in HPB-ALL cells attenuates the A9-THC- mediated rise in [Ca2+], ..................................... 157 H. A9-THC elevates [Ca2+]. in the TRPC1”' SPLC .................. 157 Effect of tricyclic cannabinoids and cannabinoid antagonists in the CB 1"” /CB2'/‘ SPLC ................................................. 160 A. Ag-THC, HU-210 and CBN elevate [Ca2+]i in CBl"'/CB2"' SPLC . . 160 B. SR1 and SR2 antagonize the tricyclic cannabinoid-mediated elevation in 3[Ca2+]i in the CBl"'/C132"' SPLC .................. 167 C. Binding of[ H]-SR1 to CBl"‘/CB2"' SPLC .................... 167 DISCUSSION ....................................................... 181 I. Effect of Ag-THC and CP on IL-2 expression ........................ 181 II. Effect of A9-THC and CP on [Ca2+]i in T cells ....................... 183 III. Effect of [Ca2+]: removal and BAPTA-AM on the A9-THC-induced [Ca2+]; elevation .................................................... 185 IV. Effect of other cannabinoids on [Ca2+]i ............................. 190 V. Effect of Ca2+ channel inhibitors and store-depletion on the A9-THC- induced [Ca2+]i elevation ........................................ 193 VI. Effect of TRPC channel activators and inhibitors on the A9-THC-induced [Ca2+]; elevation ............................................... 195 VII. Involvement of TRPC] channels in the A9-THC-induced [Ca2+]t elevation . 197 VIII. Effect of tricyclic cannabinoids and the cannabinoids antagonists on [Ca2+], in the CBl'/'/CB2"' SPLC ................................. 199 IX. Significance and relevance ...................................... 202 LITERATURE CITED ................................................. 208 ix 2A. ZB. LIST OF TABLES Sequences of primers used for RT-PCR reactions ...................... 75 Real-time PCR primers sequences for TRPC] and B-actin ............... 79 Oligonucleotide sequences for TRPC] siRNA ........................ 79 Summary of the effects of cannabinoid agonists on IL-2 secretion and [Ca2+]i in various cell models used ....................................... 186 Summary of the effects of antagonists on cannabinoid-induced suppression of IL-2 secretion and [Ca2+], elevation in various cell models used ......... 187 10. 11. 12. l3. I4. 15. 16. 17. LIST OF FIGURES Structures of classical cannabinoid compounds ........................ 4 Structures of arachidonic acid and the endocannabinoids ................ 8 Structures of the cannabinoid receptor antagonists ..................... 10 Ca2+ signaling in T cell activation and concomitant IL-2 gene expression . . . 26 Proposed structure of TRPC channels ............................... 48 Ag-THC suppresses the secretion of IL-2 from PMA/Io-stimulated HPB-ALL cells .......................................................... 82 A9-THC does not suppress the secretion of IL-2 from PMA/Io—stimulated Jurkat E6-1 cells ................................................ 83 CP suppresses the secretion of IL-2 from PMA/Io-stimulated HPB-ALL cells . 85 CP suppresses the secretion of IL-2 from PMA/Io-stimulated Jurkat E6-1 cells 86 RT-PCR for CB1 and C82 in the HPB-ALL and Jurkat E6-l cells ........ 88 SR2 does not antagonize the A9-THC-induced suppression of IL-2 secretion in HPB-ALL cells ............................................... 90 SR2 does not antagonize the CP-induced suppression of IL-2 secretion in HPB-ALL cells ................................................. 91 SR2 does not antagonize the CP-induced suppression of IL-2 secretion in Jurkat E6-1 cells ................................................ 92 SR1 does not antagonize the A9-THC-induced suppression of IL-2 secretion in I-IPB-ALL cells ............................................... 93 SR1 does not antagonize the CP-induced suppression of IL-2 secretion in HPB-ALL cells ................................................. 94 SR1 does not antagonize the CP-induced suppression of IL-2 secretion in Jurkat E6-1 cells ................................................ 95 PTx does not reverse the A9-THC-induced suppression of IL-2 secretion in HPB-ALL cells ................................................. 97 xi 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. A9-THC suppresses IL-2 mRNA production in HPB-ALL cells ............ 98 A9-THC elevates [Ca2+]t in HPB-ALL cells, but CP does not ............. 100 A9-THC elevates [Ca2+], in murine splenic T cells, but CP does not ........ 101 Ag-THC modestly elevates [Ca2+]; in Jurkat E6-l cells, but CP does not . . . . 102 CP does not antagonize the elevation of [Ca2+]i elicited by A9-THC in HPB-ALL cells ................................................. 104 Removal of [Ca2+].: severely abrogates the elevation of [Ca2+]i elicited by A9-THC in murine splenic T cells .................................. 105 Removal of [Ca2+]e severely abrogates the elevation of [Ca2+], elicited by A9-THC in HPB-ALL cells ....................................... 106 Pretreatment with BAPTA-AM attenuates the elevation of [Ca2+]i elicited by A9-THC in HPB-ALL cells ..................................... 108 SR1 and SR2 antagonize the Ag-THC-mediated elevation in [Ca2+]t in murine splenic T cells ............................................ 109 SR1 and SR2 antagonize the A9-THC-mediated elevation in [Ca2+]i in HPB-ALL cells ................................................. 110 SR1 and SR2 antagonize the A9-THC-mediated elevation in [Ca2+], in Jurkat E6-1 cells ................................................ 111 SR2 antagonizes the modest elevation in [Ca2+]; elicited by A9-THC in the absence of [Ca2+]c in HPB-ALL cells ................................ 113 PTx does not attenuate the elevation in [Ca2+]i elicited by A9-THC in HPB-ALL cells ................................................. 115 HU-210 elevates [Ca2+]i in HPB-ALL cells ........................... 116 CBN elevates [Ca2+]i in HPB-ALL cells ............................. 117 Removal of [Ca2+]c severely abrogates the elevation of [Ca2+], elicited by HU-210 and CBN in HPB-ALL cells ............................... 119 SR1 and SR2 antagonize the HU-210- and CBN-mediated elevation'in [Ca2+]. in HPB-ALL cells ......................................... 121 xii 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. CBD elevates [Ca2+]t in HPB-ALL cells ............................. 123 Removal of [Ca2+]c abrogates the second phase of CBD-induced elevation of [Ca2+]i in HPB-ALL cells ....................................... 124 SR2 does not antagonize the CBD-induced elevation in [Ca2+]; in HPB-ALL cells .......................................................... 125 Effect of SKF on the elevation of [Ca2+], induced by A9-THC and TG ...... 128 Effect of 2-APB on the elevation of [Ca2+], induced by A9-THC and TG . . . . 131 Effect of LaCl3 on the elevation of [Ca2+], induced by A9-THC and TG ..... 133 Effect of SKF on the elevation of [Ca2+]; induced by HU-210 and CBN . . . . 136 Pretreatment with TG does not abrogate the A9-THC induced elevation in [Ca2+]t ........................................................ 138 Pretreatment with 8-Br-cADPR does not abrogate the A 9-THC induced elevation in [Ca2+]i .............................................. 139 Treatment of HPB-ALL cells with OAG induces an elevation in [Ca2+], . . . . 141 Pretreatment with OAG abolishes the A 9-THC-elicited rise in [Ca2+]i ...... 142 Downregulation of PKC does not affect the abrogation the A 9-THC-elicited rise in [Ca2+], by OAG ........................................... 144 Pretreatment with PMA modestly attenuates the A 9-THC induced elevation in [Ca2+]; ...................................................... 145 A 9-THC treatment does not result in the activation of PKC .............. 147 Pretreatment with KN-93 and KN-92 attenuates the A 9-THC-induced elevation in [Ca2+]i .............................................. 148 A 9-THC treatment does not result in the activation of CaMKII ............ 150 A9-THC treatment does not result in the autophosphorylation of CaMKII . . . 152 Pretreatment with Et-18-OCH3, LY294002 and PP2 does not attenuate the A9-THC-induced elevation in [Ca2+], ................................ 155 RT-PCR analysis of TRPCl-7 in HPB-ALL and Jurkat E6-I cells ......... 156 xiii 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. siRNA against TRPC] knocks down the mRNA expression of TRPCl and the [Ca2+]; elevation elicited by A9-THC ............................. 159 The elevation in [Ca2+]t by A9-THC is maintained in the TRPC1"' SPLC . . . . 161 RT-PCR analysis for TRPC1-7 in WT and TRPC1"’ SPLC .............. 162 The elevation in [Ca2+]i by A 9—THC is maintained in the CBl"’/CBZ"' SPLC 164 The elevation in [Ca2+]i by HU-210 is maintained in the CBl’/'/CBZ'/' SPLC 165 The elevation in [Ca2+]i by CBN is maintained in the CB1"'/CBZ"' SPLC . . . 166 The elevation in [Ca2+]i by A 9-THC in CB 1"'/CBZ"' and WT SPLC is antagonized by SR1 and SR2 ...................................... 169 The elevation in [Ca2+]. by HU-210 in CBl"'/CBZ”’ and WT SPLC is antagonized by SR1, but not SR2 ................................... 171 The elevation in [Ca2+]i by CBN is antagonized by SR1 and SR2 in the CB1"'/CB2"° SPLC, but not WT SPLC .............................. 173 Total and non-specific binding of [3H]-SR1 to WT and CBl'l'lCBZ'l' SPLC . 176 Specific binding of [3H]-SR1 to WT SPLC ........................... 178 Specific binding of [3H]-SR1 to CBI"'/CB2"' SPLC .................... 180 Putative model of cannabinoid-mediated [Ca2+]; regulation and suppression of IL-2 expression in the HPB-ALL cells ............................. 189 xiv AA Abn-CBD AEA 2-AG ANKTM] 2-APB AP-l APC BCR 8-Br-cADPR [Caylt [Ca2+]. cADPR CaM CaMKII cAMP CBl CBl"' CB1"'/CBZ"‘ CB2 LIST OF ABBREVIATIONS arachidonic acid abnormal cannabidiol; (-)-4-(3-3,4-trans-p- menthadien-[l ,8]-yl)-olivetol N-arachionoylethanolamide (anandamide) 2-arachidonoy1glycerol ankyrin-like with transmembrane domains 1 2-aminoethoxydiphenylborate activator protein-l antigen presenting cell B cell receptor 8-bromo-cyclic-adenosine diphosphate ribose intracellular calcium extracellular calcium cyclic adenoside diphosphaste ribose calmodulin calmodulin-dependent kinase II cyclic adenosine monophosphate cannabinoid receptor 1 cannabinoid receptor 1 null cannabinoid receptor 1 and 2 null cannabinoid receptor 2 XV C32" CBD CBN CCE CD CD4+ CD84r CGRP Con A CP CP56,667 CPC CRAC CREB DAG DC DNP—ficoll cannabinoid receptor 2 null cannabidiol; 2-[3-methyl-6-(1-methylethenyl)-2- cyclohexen— 1-yl]—5-pentyl-l ,3 ~benezenediol cannabinol; 6,6,9-trimethyl-3-pentyl-6H-dibenzo [b,d]pyran- 1 -ol capacitative calcium entry cluster of differentiation helper T cells cytotoxic T cells calcitonin-gene regulatory peptide concanavalin A CP55,940; (-)-cis-3-[2-hydroxy-[3,5-3H]-4-(1,l- dimethylheptyl)phenyl]-trans—4-(3-hydroxypropyl) cyclohexanol (+)-cis-3-[2-hydroxy-[3,5-3H]-4-(1,l-dimethyl heptyl)pheny1]-trans-4-(3-hydroxypropyl) cyclohexanol capsaicin calcium release-activated calcium cAMP response element-binding protein diacylglycerol dendritic cell dinitrophenyl-ficoll xvi DSI EBD ELISA ERK-MAP Et-18-OCH3 FKBP Fura-2/AM GABA GM-CSF HPB-ALL HU-210 IFN IL INAD iNOS Io 1P3 depolarization-induced suppression of inhibition ERM (ezrin-radixin-moesin) binding domain enzyme-linked immunosorbant assay extracellular signal-regulated mitogen-activated protein edelfosine; 1-O-octadecy1-2-O-methy1—rac-g1ycero- 3-phosphorylcholine FK506 binding protein 1 -[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5- oxy]-2-(2-amino-5-methylphenoxy)-ethane-N,N, N,N-tetraacetic acid pentaacetoxymethyl ester gamma-amino butyric acid granulocyte-macrophage cell stimulating factor human peripheral blood acute lymphoid leukemia (6aR,1 OaR)-3-( l ,1 -dimethylbutyl)-6a,7, 1 0,10a- tetrahydro-6,6-dimethyl—6H-dibenzo[b,d]pyran-9- methanol interferon interleukin inactivation-no-afierpotential D inducible nitric oxide synthase ionomycin inositoltrisphosphate xvii JWH-l33 KN92 KN93 LPS LY294002 Meth-AEA MHC MIP NFKB NFAT NHERF NK cell NOMPC 0-1918 OAG l l-OH-A9-THC PDZ (6aR, 1 OaR)-3-( l ,1-dimethylbutyl)-6a,7,10, 1 Oa- tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran 2-[N-(4'-methoxybenzenesulfony1)]amino-N-(4'- chlorophenyl)-2-propenyl-N-methylbenzylamine phosphate salt N-[2-[N-(4-chlorocinnamyl)-N-methylamino methyl]pheny1]-N-(2-hydroxyethyl)-4-methoxy benzenesulfonamide phosphate salt lipopolysaccharide 2-(4-morpholinyl)-8-phenyl- 1 (4H)-benzopyran- 4-one hydrochloride methanandamide major histocompatibility complex macrophage inflammatory protein nuclear factor KB nuclear factor of activated T cells Na+/H+ exchange regulatory factor natural killer cell no mechanoreceptor potential C (-)-4-(3-3 ,4-trans-p-menthadien-( 1 ,8)-y1)-orcinol 1 -oleoy1—2-acety1-sn-glycerol 1 1—hydroxy-A9-tetrahydrocannabinol PSD (postsynaptic density protein)-95, DLG xviii PHA PIP; PI3K PKA PKC PMA PMCA PP2 PTx RANTES ROCC RPMI RT-PCR RyR SCID SERCA siRN A SOC SOCC SOCE (discs large), zone occludens-l phytohemagglutinin phosphotidylinositol-bisphosphate phosphotidylinositol-3-kinase protein kinase A protein kinase C phorbol- l 2-myristate-1 3 -acetate plasma membrane calcium-ATPase AG 1879; 4-amino-5-(4-chlorophenyl)-7-(t—butyl) pyrazolo[3,4-d] pyrimidine pertussis toxin regulated upon activation, normally t-expressed, and presumably secreted receptor-operated cation channel Roswell Park Memorial Institute reverse transcriptase polymerase chain reaction ryanodine receptor severe combined immunodeficiency smooth endoplasmic reticulum calcium-ATPase small interference RNA store-operated calcium store-operated calcium channel store-operated calcium entry xix SPLC SKF SR1 SR2 sRBC TCR Th 1 Th2 TO TOP A9-THC TRP TRPA TRPC TRPCZ"’ TRPC4"’ splenocytes SK&F 96365; 1-[_-[3-(4-methoxyphenyl)propoxy]- 4-methoxyphenethyl]- 1 H-imidazole SR141 71 6A; N-(piperidin- 1 -yl)-5-(4-chlorophenyl) - 1 -(2,4-dichlorphenyl)-4-methyl-H-pyrazole-3 carboxyamidehydrochloride SR144528; N-[(1 S)-endo-l ,3,3,-trimethyl-bicyclo [2,2,1] heptan-2-yl]-5-(4-chloro-3-methylphenyl)- 1 - (4-methylbenzyl)-pyrazole-3-carboxamide sheep red blood cells T cell antigen receptor T helper cell subtype 1 T helper cell subtype 2 thapsigargin transforming growth factor (-)-A9-tetrahydrocannabinol; tetrahydro-6,6,9- trimethyl-3-penty1-6H-dibenzo[b,d]pyran-1 -01 tumor necrosis factor transient receptor potential transient receptor potential ANKTMl transient receptor potential canonical transient receptor potential canonical 2 null transient receptor potential canonical 4 null XX TRPC6”' TRPM TRPML TRPP TRPV vocc VR-l WIN-2 WIN55,212-3 WT transient receptor potential canonical 6 null transient receptor potential melastatin transient receptor potential mucolipin transient receptor potential polycystin transient receptor potential vanilloid voltage-operated calcium channel vanilloid receptor-1 WIN55,212-2; (R)-(+)-[2,3-dihydro-5-methyl-3[(4- morpholinyl)methyl]pyrrolo[1 ,2,3-de]-1 ,4- benzoxazinyl]-( 1-naphthalenyl) methanone mesylate salt. (S)-(-)-[2,3-dihydro-5-methyl-3-[(morpholinyl) methyl]pyrrolo[1 ,2,3-de]- 1 ,4-benzoxazin-6-yl]-(1 - naphthalenyl) methanone mesylate salt wildtype xxi INTRODUCTION 1. Marijuana, cannabinoids and cannabinoid receptors A. Marijuana Marijuana is the common name for Cannabis sativa, the hemp plant that is native to temperate and tropical climates. The recorded use of cannabis in Asia, both recreational and medicinal, spans several millennia. From Asia and the Middle East, marijuana use spread into North Africa, Europe, and the Americas (Nahas, 1972). Western medicine was first introduced to marijuana by the Irish physician William O’Shaughnessy in the 18405, as a treatment for rheumatism and appetite stimulation (Joy et al., 1999). Until the 19305, cannabis preparations were routinely included in British and American pharmacopoeias for treatment of convulsive disorders and as analgesics. However, with the development of more effective synthetic drugs, cannabis preparations lost their medical attention (Berdyshev, 2000). As the medicinal use of cannabis declined, its use as a recreational drug increased, leading to successive laws in most industrialized nations prohibiting the possession and supply of cannabis. Although still illegal, marijuana is currently the third most common drug of abuse in the United States and most European countries, following nicotine and alcohol (Baker et al., 2003). Nearly 50% of all l8-year-olds in the US and most European countries admit to having tried cannabis at least once in their lifetime (Iversen, 2003). Despite the endemic recreational use, a growing body of scientific literature and anecdotal evidence has shed new light on the potential therapeutic benefits of cannabis. B. Physiological effects of cannabis Smoked cannabis has been shown to have a wide array of effects on human physiology. Acute effects of smoked cannabis include mild euphoria, relaxing intoxication, slight changes in psychomotor activity, altered cognitive function, amotivational syndrome, tachycardia, peripheral vasodilation, appetite stimulation, and dizziness (Baker et al., 2003). Furthermore, marijuana smoke also produces inflammation, edema, and cellular injury in tracheobronchial mucosa of smokers (Sarafian et al., 1999). Long-term use of cannabis has been associated with bronchitis and emphysema, and may lead to changes in attention, memory, and ability to process complex information (Ashton, 2001). Some regular cannabis users also develop tolerance and dependence to the drug. Upon cessation of chronic intake, cannabis users who have become dependent may experience somatic withdrawal syndrome characterized by restlessness, insomnia, anxiety, increased aggression, anorexia, muscle tremor, and other autonomic effects (Ashton, 2001). As with other drugs of abuse such as cocaine, the development of tolerance, dependence, and withdrawal syndrome is attributed to the effects of cannabis on the nucleus accumbens, and reduction of neuronal activity in the mesolimbic dopaminergic pathway in the brain (Diana et al., 1998; Iversen, 2003). Apart from exerting psychoactive and behavioral effects, cannabis also exhibits immunomodulatory properties. The first immune cells that come into contact with cannabis smoke are the resident phagocytic cells in the lungs known as the alveolar macrophages. In alveolar macrophages, marijuana smoke has been shown to suppress antimicrobial activity, cytokine production and responsiveness to cytokines (Klein et al., 2003). Long-term use of cannabis may also be associated with an increased incidence of bacterial and viral infections due to suppression of alveolar macrophage and circulating lymphocyte function. Despite years of investigation into the physiological changes elicited by cannabis smoke, sufficient studies in chronic cannabis smokers have not yet been performed. In particular, a full assessment of the precise cellular mechanism by which cannabis may alter immune function in human subjects remains to be performed. C. Cannabinoid compounds The flowering tops and leaves of Cannabis sativa contain a family of structurally- related compounds known as cannabinoids. In the lay literature, cannabinoids are often described as the active constituents in cannabis smoke. The plant-derived cannabinoid family comprises over sixty dibenzopyran tricyclic ring structures consisting of a phenolic ring attached to a five carbon alkyl chain; a central pyran ring; and a monounsaturated cyclohexyl ring (Howlett et al., 2004). Although the presence and the biological effects of cannabinoids were long known, the structure of the primary psychoactive cannabinoid, A9-tetrahydrocannabinol (A9-THC), was not discovered until 1964 (Felder and Glass, 1998; Klein et al., 2000a). Together with A9-THC, cannabinol (CBN) and cannabidiol (CBD), which exhibit little and no psychoactivity respectively, form a triad of the most-often experimentally utilized plant-derived cannabinoids (figure 1). Following the discovery of the chemical structure of A9-THC, a variety of synthetic cannabimimetic compounds were developed as potential non-opioid analgesics (Howlett et al., 2004). The first generation of synthetic cannabinoids, such as CP55,940 (CP) and HU-210, were modeled after the chemical structure of A9-THC (figure 1), the prototypical cannabinoid, with small changes to phenolic, cyclohexyl and alkyl nonsense”. Eoosnéno owes; one @233 «-23sz one SEE .Euv ovofimmv $285988 wo>tovtfifiq 2m Qmu 98 750 .025? 55:52—53 Sofia—«:53 _3_mma_o no non—52.3w A 0.53% Q N..-N.mm7:>> cvafimA—U o E emu O :0 functional groups. However, later classes of cannabinoids such as the aminoalkylindoles, typified by WIN55,212-2 (WIN-2; figure 1), represent a continuing evolution of cannabimimetic compounds that came into being as advances in cannabinoid biology were made. D. Therapeutic potential of cannabinoids The initial observation that marijuana exerts effects on different branches of human physiology fueled a search for potential therapeutic benefits of cannabinoid compounds. Over the past few decades, plant-derived as well as synthetic cannabinoids have been shown to possess therapeutic potential as treatments in many human diseases and conditions. Cannabinoids have been proposed as treatments for nausea associated with cancer chemotherapy, neuropathic pain, spasticity in multiple sclerosis, migraine headaches, epilepsy, glaucoma, hypertension, AIDS-induced wasting syndrome, bronchioalveolar constriction associated with asthma, and allergic airway diseases (Baker et al., 2003; Felder and Glass, 1998; Jan et al., 2003; Jan et al., 2002). Currently, the FDA-approved cannabinoid-based drug, Marinol®, an oral preparation of A9-THC, is marketed in the United States for the treatment of cachexia observed in patients with AIDS or undergoing cancer chemotherapy (Felder and Glass, 1998). Additionally, Cesamet®, an oral preparation of a synthetic cannabinoid known as nabilone, is approved for treatment of cachexia in the United Kingdom (Pertwee, 2002). More recently, ®, an oral spray of cannabis extract containing low amounts of psychoactive Sativex cannabinoids, has been approved in Canada for the treatment of neuropathic pain associated with multiple sclerosis, and is currently awaiting approval in the United Kingdom. Moreover, since 1996 several states in the US including Alaska, Arizona, California, Colorado, Hawaii, Maine, Nevada, Maryland, Montana, Oregon and Washington have passed legislation that removes or softens state-level penalties for medicinal use of marijuana if prescribed by a physician. The legalization of medical marijuana in above-mentioned states has provided impetus for the fledgling medicinal marijuana movement in the US. The ever-growing body of scientific literature on the therapeutic potential of cannabinoids coupled with the medicinal marijuana movement among the lay public necessitates a comprehensive understanding of cannabinoid pharmacology and toxicology. E. Cannabinoid receptors The field of cannabinoid biology took a major leap forward upon the discovery of the cannabinoid receptors. To date, two cannabinoid receptors, CB] and CBZ, have been identified and cloned in mammalian tissues (Devane et al., 1988; Matsuda et al., 1990; Munro et al., 1993). Both CB] and CB2 belong to the G protein-coupled hexahelical transmembrane receptor superfamily, and are believed to mediate many of the pharmacological effects produced by A9-THC, as well as the synthetic cannabinoids. The CBI receptor is expressed primarily in neurons of the central nervous system, and has been shown to mediate most of the psychoactive and behavioral effects of cannabinoids (Zimmer et al., 1999). The highest expression of CB1 receptors was found in the GABAergic interneurons in the cerebral cortex, basal ganglia, amygdala, and hippocampus (Iversen, 2003). Apart from the central nervous system, the CBI receptor is also expressed in several peripheral tissues including the testes, uterus, vascular smooth muscle, and spleen (Pertwee, 1999b). The CBZ receptor, on the other hand, is absent from CNS, but expressed in cells of the immune system including B cells, natural killer (NK) cells, monocytes, neutrophils, CD8+ T cells and CD4+ T cells (Berdyshev, 2000). The CB2 receptor is the predominant cannabinoid receptor expressed in the immune system, although most immune cells also express CBl (Bouaboula et al., 1993; Munro et al., 1993; Schatz et al., 1997). Due to the higher levels of C32 receptor expression in the immune system, it is generally believed that the majority of immunological effects of cannabinoids are mediated by the CB2 receptor. The CB] and CB2 receptors bind to the plant-derived cannabinoids with moderately high affinity and pharmacological efficacy. However, the synthetic cannabinoid congeners have been subsequently shown to exhibit higher binding affinities and higher pharmacological efficacies than the plant-derived cannabinoids at both receptors (Klein etal., 2003; Pertwee, 1999b). F. Endogenous ligands Following the discovery of the cannabinoid receptors in mammalian cells, search for endogenous ligands for the cannabinoid receptors ensued. Soon after, the eicosinoid- derivatives, N-arachidonoylethanolamide (AEA or anandamide) (Devane et al., 1992), and 2-arachiodonoylglycerol (2-AG) (Lee et al., 1995; Mechoulam et al., 1995; Sugiura et al., 1995), were identified as endogenous ligands for the cannabinoid receptors, and labeled the endocannabinoids (figure 2). Both ABA and 2-AG exhibit moderately high affinities and pharmacological efficacies at the cannabinoid receptors, and are the most widely recognized endocannabinoids. In addition to these molecules, several other fatty acid and eicosinoid derivatives, including N-arachidonoyldopamine, 0- Arachidonic Acid (AA) O O OH OH 2-Arachidonoylglycerol (2-AG) Figure 2. Structures of arachidonic acid and the endocannabinoids. N- arachi donoylethanolamide (ABA) and 2-archidonoyl glycerol (2-AG) are endogenous cannabinoid compounds and arachi donic acid (AA) is the parent eicosinoid compound. arachidonoylethanolamine (virodhamine), docosatetraenylethanolamine, and 2- arachidonoylglycerol-ether (nolandin ether) have been found to exhibit binding affinity to the cannabinoid receptors (Baker et al., 2003). However, the role of the newer putative endocannabinoids remains obscure. The production of endocannabinoids, in particular ABA and 2-AG, has also been reported to occur in tissues expressing higher densities of cannabinoid receptors, such as the brain and the spleen (Klein et al., 2003). G. Cannabinoid receptor antagonists At approximately the same time as the discovery of the endocannabinoids, search for antagonists for the cannabinoid receptors was also initiated. High-throughput screening techniques by Sanofi Recherche led to the discovery of the biarylpyrazole compounds, SR141716A (SR1) and SR144528 (SR2), the CB1 and C82 receptor antagonists respectively (Rinaldi-Carmona et al., 1994; Rinaldi-Carmona et al., 1998). Both SR1 and SR2 (figure 3) are selective to their cognate receptors, but not specific; and exhibit binding affinities in the nanomolar concentration range. The discovery of the cannabinoid receptor antagonists revolutionized the field of cannabinoid biology, enabling the differentiation of cannabinoid receptor-dependent and ~independent modes of action. It was soon discovered, however, that both SR1 and SR2 elicit inverse agonistic properties at both CB] and CB2 (Bouaboula et al., 1999; Shire et al., 1999), confounding their use as exclusive neutral antagonists at either cannabinoid receptor. In addition, at higher concentrations, SR1 has been since shown to exhibit binding affinity at the CB2 receptor, and may exert antagonistic effects independent of both the C81 and CB2 receptors (Pertwee, 2005). SR2 also may bind and antagonize the CB] receptor at Cl SR141716A (SR1) O l | 1‘ SR144528 O ‘Sm’ Figure 3. Structures of the cannabinoid receptor antagonists. SR141716A (SR1) is a CB1 receptor-selective antagonist and SR144528 (SR2) is a CB2 receptor- selective antagonist. 10 higher concentrations (Pertwee, 1999b). Recently, novel classes of more selective cannabinoid receptor antagonists such as the CBZ-selective antagonist, AM630, have been developed, but extensive characterization of the newer classes of antagonists has yet to take place. 11. Pharmacological and biochemical effects of cannabinoids A. Effects of cannabinoids on the central nervous system 1. CNS neurons Since the discovery of the cannabinoid receptors, the endogenous ligands, and the cannabinoid receptor antagonists, a multitude of cannabinoid effects on various cell types have been reported. Perhaps the most extensive studies of cannabinoid effects have been performed in the brain and central nervous system (CNS). Cannabinoids of all classes have been shown to modulate the secretion of most major vertebrate neurotransmitters including glutamate, GABA, glycine, norepinephrine, serotonin, dopamine, acetylcholine and neuropeptides (Baker et al., 2003). A vast majority of the inhibitory effects of cannabinoids on CNS neuronal function are mediated by C81 receptors, which are expressed on axons and nerve terminals (Wilson and Nicoll, 2002). The GABAergic interneurons in the cerebral cortex, basal ganglia, amygdala and hippocampus express the highest density of CB1 receptors in the CNS (Iversen, 2003). The complex effects of cannabinoids on psychomotor behavior, learning, memory, and cognitive function are attributed to the inhibition of the GABAergic interneurons in the above-mentioned brain regions (Iversen, 2003). Under normal physiological conditions, it is hypothesized that upon depolarization of postsynaptic neurons rapid production of endocannabinoids 11 ensues. Endocannabinoids subsequently act as retrograde messengers to traverse the synaptic cleft and bind CBl receptors on presynaptic nerve terminals. This phenomenon known as depolarization-induced suppression of inhibition (DSI) results in the inhibition of presynaptic neurons, thereby attenuating neurotransmitter release (Howlett et al., 2004). The major mechanism by which endogenous and exogenous cannabinoids inhibit the secretion of neurotransmitters involves the modulation of ion channels. Under experimental conditions, various classes of cannabinoids have been shown to inhibit the function of voltage-operated calcium (Ca2+) channels (VOCCs), as well as to activate potassium (K+) channels. In particular, activation of neuronal CBl receptors leads to the inhibition of VOCCs type N, P and Q, as well as the activation of type A K+ channels (Glass and Northup, 1999; Twitchell et al., 1997). Together, the inhibition of VOCCs and activation K+ channels attenuate the presynaptic release of neurotransmitters. The changes in behavior and psychoactivity associated with cannabis use are physiological manifestations of the combined inhibitory effects of cannabinoids on the mammalian CNS. 2. Astrocytes and microglial cells Apart from neurons in the CNS, cannabinoids have also been demonstrated to affect the function of astrocytes and other glial cells. Glial cells play an important role in the mammalian CNS by controlling the microenvironment of nerve cells, and modulating many neuronal functions. Astrocytes are a unique class of glial cells, which provide the support structure for neurons, and control proliferation and repair following nerve injury. Another class of glial cells are the microglia, which are involved in removal and 12 phagocytosis of debris in the CNS. In general, cannabinoids have inhibitory effects on the function of glial cells. Specifically, cannabinoids inhibit the production of cytotoxins and cytokines by astrocytes and microglia. Using A9-THC as well as the synthetic cannabinoids, WIN-2 and CP, suppression of the production of several inflammatory cytokines, such as interleukin (IL)-lB, IL-la, IL-6, and tumor necrosis factor (TNF)—0t, has been shown in activated astrocytes (Berdyshev, 2000; Sheng et al., 2005). Furthermore, under conditions of neuroinflammation, endocannabinoid synthesis is induced in both microglia and astrocytes. The elevated levels of endocannabinoids are believed to act in an autocrine or paracrine manner to inhibit the activation of astrocytes and microglia, thus preventing the propagation of neuroinflammation and further cell damage due to the release of inflammatory mediators (Stella, 2004). The inhibition of astrocyte function has been demonstrated experimentally with methanandamide (meth- AEA), a non-hydrolysable analog of ABA, which inhibits astrocyte function by attenuating agonist-induced intracellular calcium ([Ca2+]i) rise (Venance et al., 1997). Interestingly, whereas most inhibitory effects of cannabinoids on CNS neurons can be attributed to CBl-dependent mechanisms, several studies have shown cannabinoid-mediated inhibition of glial cell function to be independent of the CBI receptor. For example, the inhibition of cyclic adenosine monophosphate (CAMP) formation in rat astrocytes by both WIN-2 and AEA was mediated by a G protein- coupled receptor distinct from €81 (Sagan et al., 1999). Similarly, the suppression of pro-inflammatory cytokine production by CP in rat microglia could be replicated by an enantiomer of CP, CP56,667, which does not bind either CBl or CB2, and was insensitive to either SR1 or SR2 (Berdyshev, 2000). Also, it is notable that microglial 13 cells, unlike CNS neurons, may express CB2 receptors. Immunofluorescent staining for C81 and CB2 in cultured rat microglia revealed that whereas the CB1 receptor was localized to intracellular compartments, the CBZ receptor was expressed throughout the cell surface (Felder and Glass, 1998; Stella, 2004; Walter et al., 2003). However, the expression of CBZ in the mammalian CNS remains highly controversial. B. Effect of cannabinoids on the immune system Aside from the CNS, cannabinoid effects have also been widely characterized in immune cells. In general, cannabinoids differentially modulate the immune system depending on cell type used, mode of activation, and functional response being measured. Although cannabinoids have significant immunomodulatory effects when immune cells are exposed directly, acute intake of cannabinoids by means of smoked cannabis has negligible effects on systemic immunity (Klein et al., 2003). However, cannabinoids have significant suppressive effects on immune cells when directly exposed to cannabis smoke. Previous in vivo immunological studies performed in rodents have revealed that exposure to cannabinoids results in altered innate, humoral and cell-mediated immune responses (Yahya and Watson, 1987). Preliminary studies have also revealed that long— term exposure to cannabinoids in humans may result in an increased incidence of viral and bacterial infections, and modulation of cannabinoid receptor expression in immune cells (Klein et al., 2003). A caveat to the aforementioned observation is that only a few controlled studies on the immune effects of cannabinoids in humans have been conducted. No comprehensive study has been performed to date investigating the immune effects of heavy and chronic use of marijuana in human subjects. 14 l. Innate immunity a. Macrophages The innate immune response is critical in the first phase of infections until the adaptive immune response is mounted after a delay of 4—7 days. A majority of the studies of cannabinoid effects on innate immune responses have been limited to macrophages. Macrophages are resident phagocytic cells in many tissues and are available to combat a wide array of pathogens. Isolated alveolar macrophages from marijuana smokers show a suppressed ability to secrete pro-inflammatory cytokines, and to generate nitric oxide (NO) (Roth et al., 2002). Similarly, the suppression of pro-inflammatory cytokines has also been demonstrated in non-alveolar macrophages treated with cannabinoids. In cultured macrophage cell lines activated with lipopolysaccharide (LPS), A9-THC and AEA inhibited the secretion of the pro-inflammatory cytokine, TNF-oc (Berdyshev, 2000; Cabral and F ischer-Stenger, 1994). The suppression of TNF-a secretion was not a result of attenuation in TNF-or transcription, but represented an suppression of TNF-(x maturation from its pre-secretory form (Cabral and F ischer-Stenger, 1994). Apart from acting as an inflammatory mediator, TNF-a has a secondary role in the tumoricidal activity of macrophages. Therefore, the suppression of TNF-a processing and secretion by cannabinoids may also result in the attenuation of the macrophage tumoricidal activity (McCoy et al., 1995). Cannabinoids also affect the bactericidal function of macrophages by inhibiting the production of NO. CP and A9-THC have both been demonstrated to inhibit the production of NO in macrophages stimulated with LPS (Jeon et al., 1996; Newton et al., 1998; Ponti et al., 2001). The inhibition of NO production by cannabinoids may involve 15 the cannabinoid receptors, as well as inducible nitric oxide synthase (iNOS). Macrophages activated with LPS upregulate the transcription of iNOS, which in turn is responsible for the generation of NO. Treatment of macrophages with A9-THC prior to LPS activation resulted in an inhibition of iNOS upregulation (Jeon et al., 1996). In another study the cannabinoid-mediated inhibition of LPS-activated NO production was reversed upon pretreatment with either SR1 or SR2 (Ponti et al., 2001). The observation that effects of cannabinoids on macrophages may be dependent on the cannabinoid receptors is not surprising given that macrophages express both CB1 and CB2, and are also known to produce endocannabinoids (Klein et al., 2000a). It is currently postulated that the macrophage-derived endocannabinoids may modulate macrophage function as well as the complex interactions between macrophages and other immune cells. Another important role fulfilled by macrophages is the activation of CD4+ T cells, leading to the onset of adaptive immunity. Upon trapping, engulfment and destruction of pathogens, macrophages process antigens to be presented to T cells in the context of major histocompatibility complex molecules (MHCs). Together with B7 co-stimulatory molecules, processed antigens in the context of MHC molecules provide a robust activation signal to CD4+ T cells. In this capacity, macrophages are known as antigen presenting cells (APCs). Numerous studies point to the macrophage-T cell interaction as a target for cannabinoid action. Early on, cannabinoids were determined to impair the function of macrophages to act as APCs. Studies by McCoy and coworkers demonstrated that the ability of a macrophage hybridoma to process antigens was attenuated in the presence of A9-THC (McCoy et al., 1995). The aforementioned study suggested that A9- THC mainly alters antigen processing, and not presentation of antigens to T cells. 16 Further studies demonstrated that the T cell response, as measured by IL-2 secretion, was diminished when macrophages were presented with unprocessed antigens, but not when presented with processed antigens in the presence of either A9-THC or CP (McCoy et al., 1999). Furthermore, SR2, but not SR1, attenuated the A9-THC-induced inhibition of antigen-dependent activation of CD4+ T cells by macrophages. More recently, the involvement of the CBZ receptor in the co-stimulation of CD4}( T cells was examined using macrophages from CB2 null (CBZ'I') mice. Peritoneal macrophages isolated from wildtype (WT) mice exhibited a diminished ability to co-stimulate a T cell hybridoma in the presence of A9-THC, whereas macrophages from CBZ"' mice did not (Buckley et al., 2000). Taken together, the above studies indicate that one manner in which cannabinoids disrupt innate immune responses is by interfering with macrophage function. h. Neutrophils, NK cells and dendritic cells Apart from macrophages, several other important cell types such as neutrophils, NK cells, and dendritic cells (DCs) also contribute to innate immunity. It has been established previously that all three cell types mentioned above express CB] and CBZ receptors (Klein et al., 2003; Matias et al., 2002). However, studies of cannabinoid effects on innate immune cells, other than the macrophage, have been scant. The available few such studies suggest that cannabinoids negatively affect the function of all innate immune cells. Neutrophils play an important role in primary defense against bacteria. In particular, the process known as respiratory burst, which generates reactive intermediates such as superoxide and NO, is critical for neutrophil bactericidal activity. In isolated 17 human neutrophils, CP inhibited the production of superoxide anions induced by the peptide flVILP (Krafi et al., 2004). Interestingly, in the same system, AEA did not inhibit superoxide production. Furthermore, the effect of CP on superoxide production could not be reversed by either SR1 or SR2 (Kraft et al., 2004). Cannabinoids have similarly been shown to have deleterious effects on NK cells. NK cells belong to the lymphoid family of immune cells, and are involved in the recognition and killing of abnormal cells, especially cells that are tumorogenic or virally- transformed. Critical to the function of NK cells is the secretion of small soluble molecules known as chemokines and cytokines. In a human NK cell line, Ag-THC was shown to inhibit the constitutive secretion of the chemokines, macrophage inflammatory protein (MIP)-10t, MIP-lB, IL-8 and RANTES, as well as the secretion of cytokines, TNF-a, GM-CSF, and IFN-y (Srivastava et al., 1998). Furthermore, in vivo administration of A9-THC significantly inhibited NK cell cytolytic activity, which was partially reversed by SR1 and SR2 (Massi et al., 2000). A full characterization of the cannabinoid modulation of NK cell activity and the involvement of the cannabinoid receptors therein remains to be investigated. Very little is known about the effects of cannabinoids on DCs. DCs are the most important type of APC, and have a central role in the initiation of adaptive immune responses by providing activation signals to T cells. Although the effect of cannabinoids on antigen presentation by DCs has not been accessed, in vivo treatment of mice with A9- THC has been shown to deplete DCs in the spleen (Do et al., 2004). The depletion of DCs in the spleen is believed to be a result of cannabinoid receptor-dependent activation of the apoptotic pathway. Both A9-THC and AEA were found to induce apoptosis in a 18 CB1 and CB2 receptor-dependent manner in murine bone marrow-derived DCs (Do et al., 2004). In addition, LPS activation of DCs was found to induce synthesis of endocannabinoids (Matias et al., 2002). However, the role of endocannabinoids in the complex DC dynamics is unknown. 2. Adaptive immunity Adaptive immunity is a delayed, yet specific, immune response that oftentimes confers lifelong protection against re-infection by the very same pathogen. Adaptive immunity is generally divided into two parts: humoral immunity and cell-mediated immunity, mediated by B and T cells respectively. Whereas the cell-mediated immune response is important for combating intracellular pathogens, resistance to extracellular pathogens is conferred by the humoral immune response. Cannabinoid effects on the adaptive immune response have been widely documented over the past few decades. Early immunological experiments revealed that administration of A9-THC in mice suppressed the adaptive immune response induced by pathogenic microorganisms or by the antigen, sheep red blood cells (sRBCs) (Morahan et al., 1979; Zimmerman et al., 1977). Although the mechanism of cannabinoid—mediated immune suppression was not yet known, it was soon discovered that A9-THC treatment did not alter the total number of circulating B or T cells (Silverman et al., 1982). As efforts toward developing a more in-depth understanding of cannabinoid-mediated immune modulation continued, it was found that cannabinoids inhibited the proliferation of both B and T cells activated by mitogens in culture (Klein et al., 1985; Pross et al., 1987). A large body of literature has 19 since emerged examining the effects of cannabinoids on various endpoints associated with B and T cell activation. a. B cells and humoral immunity The secretion of antigen specific immunoglobulins (antibodies) by activated B cells characterizes the humoral immune response. Antibodies secreted by B cells bind specifically to pathogens or their toxic products thereby neutralizing them and enhancing their phagocytosis. Moreover, antibodies also enable complement factor-dependent opsinization of microbes by phagocytic cells. Normally, the production of antibodies is initiated when B cells come into contact with small peptide antigens, which they process and present on the cell surface in the context of MHC class II molecules. Subsequently, antigen-specific CD4+ T cells recognize the MHC-antigen complex and interact with the B cells to deliver activation and co-stimulatory signals. In response to the T cell signals, B cells undergo clonal expansion and differentiate into antibody-forming cells (AF Cs). (i). Antibody production Extensive work by Kaminski and coworkers examined the effect of various cannabinoids on the generation of AF Cs in murine splenocytes (SPLC) stimulated by the T cell-dependent antigen, sRBCs. The mechanism by which sRBCs stimulate nai‘ve B cells to clonally expand and differentiate into AF Cs involves the accessory function of macrophages, as well as the helper function of CD4+ T cells. In assays where sRBCs were used as a stimulus, both in vivo administration of A9-THC in mice, as well as the in vitro treatment of murine SPLC with A9-THC, CP, HU-210 or CBN resulted in a 20 selective and dose-dependent inhibition of AFC production (Herring et al., 1998; Kaminski et al., 1992; Schatz et al., 1993). However, due the ambiguity of the mechanism by which cannabinoids inhibited AFC production, further AFC response studies were performed using antigens that stimulate B cells independently of T cells. In particular, B cells were stimulated with dinitrophenyl-ficoll (DNP-ficoll), a synthetic antigen, which requires accessory function of macrophages; or LPS, a polyclonal B cell activator. Interestingly, AFC production induced by either DNP-ficoll or LPS was not inhibited by A9-THC (Schatz et al., 1993). The observation that AFC production stimulated by T cell-independent antigens was not affected by A9-THC suggested that cannabinoid-mediated alteration of AFC responses was not a direct effect on B cells. Therefore, the AFC studies indicated that both B cell effector function and macrophage accessory function for humoral responses were insensitive to cannabinoid treatment. (ii). B cell proliferation A few studies have demonstrated direct effects of cannabinoids on B cells. Initially, Klein et a1. examined the effect of A9-THC and 11-OH-A9-THC, the primary metabolite of A9-THC, on the LPS-stimulated proliferation of human B cells (Klein et al., 1985). Under these conditions, B cell proliferation was strongly inhibited by micromolar concentrations of both A9-THC and 11-OH-A9-THC. Similarly, 2-AG, but not ABA, inhibited the LPS-induced proliferation of murine splenic B cells (Lee et al., 1995). By contrast, in human tonsillar B cells stimulated by cross-linking of the B cell receptor or by ligation of the CD40 antigen, nanomolar concentrations of CP, WIN-2 and A9-THC enhanced the proliferation of B cells (Derocq et al., 1995). Moreover, the cannabinoid- 21 mediated enhancement of B cell proliferation could be reversed by pertussis toxin (PTx), implicating the involvement of G protein-coupled receptors in the process. Subsequently, a second group confirmed the enhancement of proliferation induced by CD40 ligation in virgin tonsillar and germinal center B cells using CP (Carayon et al., 1998). The enhancement of B cell proliferation mediated by CP was antagonized by SR2. B cells are known to express one of the highest densities of CB2 and CB1 receptors among immune cells (Carayon et al., 1998; Klein et al., 2003). However, the significance of the high cannabinoid receptor density in B cell function remains largely unknown. Based on the above studies, Carayon et al. have hypothesized that CBZ receptors may act as co- receptors of CD40-induced proliferation in B cells (Carayon et al., 1998). b. T cells and cell-mediated immunity Cell-mediated immunity is the second branch of the adaptive immune system and involves responses mediated by T cells. Whereas the humoral immune response confers resistance against extracellular pathogens, the cell-mediated immune response is responsible for the elimination of intracellular pathogens, including viruses and some bacteria. The elimination of intracellular pathogens depends on a direct interaction between T cells and cells bearing the antigen that the T cell recognizes. Two major types of T cells participate in cell-mediated immune responses: CD4+ (helper) T cells and the CD8+ (cytotoxic) T cells. CD4+ T cells recognize specific antigens in the context of MHC class II molecules that are presented by the APCs, i.e. macrophages, B cells and DCs. Upon recognition of antigens, CD4+ T cells interact with the APCs and secrete cytokines. CD4+ T cells play an important role in delayed-type responses, which involve 22 the activation of macrophages, and also participate in the activation of B cells leading to the production of antibodies. CD8+ T cells, by contrast, recognize specific antigens in the context of MHC class I molecules, which can be presented by almost any cell in the body. Upon recognition of a specific antigen, CD8+ T cells interact with the cells presenting antigen and secrete cytokines and cytotoxic factors to induce cell death in the infected cell. CD8+ T cells are critical in the induction of cell death in tumorogenic and virus infected cells. (i). T cell proliferation A majority of cannabinoid studies in T cells have been focused on T cell activation-associated endpoints. Preliminary investigation by Klein and coworkers on cannabinoid-mediated modulation of T cell activation examined the proliferation of T cells upon stimulation by T cell-specific mitogens, phytohemagglutinin (PHA) and concanavalin A (Con A). In murine T cells activated with either PHA or Con A, both A9- THC and ll-OH-Ag-THC inhibited cell proliferation in a dose-dependent manner (Klein et al., 1985). Furthermore, the effect of cannabinoids on T cell proliferation was dependent on the age of mice, as well as the lymphoid organ used. Splenic T cells from younger mice and immature thymic T cells from older mice appeared to be more susceptible to the effects of A9-THC (Pross et al., 1987; Pross et al., 1990). Based on the aforementioned observations, it was proposed that cannabinoids suppress the normal development of mature effector T cells from less mature precursor cells. The inhibition of T cell function was also demonstrated with CD8+ T cells. Both A9-THC and ll-OH- A9-THC inhibited the proliferation and cytolytic activity of CD8+ T cells (Klein et al., 23 1991; Pross et al., 1992). To date, the precise mechanism of cannabinoid inhibition of proliferative and functional T cells responses remains unclear. However, the modulation of the mobilization of [Cay], by A9-THC may underlie many cannabinoid effects on T cell activation-associated endpoints (Yebra et al., 1992). (ii). IL-2 suppression Recent studies of cannabinoid effects on T cell activation have focused on cytokine production and secretion. Cannabinoids have been shown to modulate the expression of several cytokines including interferon (lFN)-'y, transforming growth factor (TGF)-[3, TNF-a, IL-2, IL-4, IL-6, IL-10, IL-12, IL-13 and IL-15 in human and murine cell models (Berdyshev et al., 1997). However, the most extensive investigation of cannabinoid modulation of cytokine production has been conducted with lL-2. IL-2 is a critical T cell cytokine that is de novo synthesized and secreted upon the full activation of T cells. IL-2 acts as an autocrine or paracrine T cell growth factor and is responsible for the induction of T cell clonal expansion. A majority of studies of IL-2 regulation by cannabinoids have utilized robust activation stimuli that either bypass the T cell receptor using phorbol ester and ionomycin (PMA/lo), or polyclonally activate T cells using anti- CD3 antibodies, PHA or Con A. Under conditions of robust T cell stimulation, suppression of the production and secretion of IL-2 has been demonstrated with a wide array of cannabinoid compounds including A9-THC, CP, WIN-2, CBN, CBD, ABA, and 2-AG in murine SPLC, thymocytes, and T cell lines (Condie etal., 1996; Faubert Kaplan et al., 2003; Herring and Kaminski, 1999; Klein et al., 2000b; Ouyang and Kaminski, 1999; Rockwell and Kaminski, 2004). The suppression of IL-2 has also been 24 demonstrated in Con A-stimulated SPLC cultures from mice which were acutely treated with A9-THC (Massi et al., 1993). More recent studies of IL-2 regulation have found that cannabinoids interfere with the expression of IL-2 at the level of gene transcription. The inhibition of the DNA- binding activity of several transcription factors regulating IL-2 by cannabinoids has been reported. Some of the transcription factors modulated by cannabinoids include activator protein-1 (AP-l), cAMP response element-binding protein (CREB), nuclear factor KB (N FKB), and nuclear factor of activated T cells (N FAT) (Condie et al., 1996; Faubert and Kaminski, 2001; Herring and Kaminski, 1999; Herring et al., 1998; Koh et al., 1997; Ouyang et al., 1998; Yea et al., 2000). The mechanism by which cannabinoids attenuate the binding of the above-mentioned transcription factors may involve an inhibition of upstream signaling mechanisms. For example, in murine SPLC and thymocytes, CBN, A9-THC and AEA inhibited the production of CAMP by adenylate cyclase, as well as the concomitant activation of protein kinase A (PKA) (Condie et al., 1996; Koh et al., 1997). The cAMP-PKA pathway regulates the activation of NFKB and CREB, and may explain the attenuation of NF KB and CREB DNA binding by cannabinoids. Similarly, CBN treatment of PMA/Io-activated murine SPLC resulted in the inhibition of p42 and p44 ERK-MAP kinase activation, as well as the expression of AP-l proteins, c-jun and c-fos (Faubert and Kaminski, 2001). The activation of ERK-MAP kinase pathway is necessary for the upregulation of the expression of AP-l proteins, which in turn are involved in the upregulation of IL-2 transcription. Finally, alterations in NFAT activation and DNA binding to the lL-2 promoter have also been demonstrated with CBN and AEA in murine SPLC (Ouyang et al., 1998; Yea et al., 2000). NFAT is a critical transcription factor for 25 Coca £3646 ”Bot Bantu onE $33935 one» Nu: 23:53:99 6:: E5959: :3 a. 3 mum—«5?. zany .v 953% moo ommfix 9:83 A man: n.-._. 95% pure T cells as established by flow cytometry. V. Cultured cell lines The HPB-ALL cell line was generously provided by Dr. J. A. Ledbetter (Pacific Northwest Research Institute). The human T cell leukemia line, Jurkat E6-l clone, was obtained from the American Type Culture Collection (ATCC TIB 152). Both cell lines were cultured in RPMI 1640 medium supplemented with 100 units of penicillin/ml, 100 66 units of streptomycin/ml, 10% BCS (Hyclone, Logan, UT), 100 mM non-essential amino acids (Gibco, Grand Island, NY) and 1 mM sodium pyruvate (Gibco, Grand Island, NY). VI. IL-2 ELISAs and mRNA quantification A. Cell treatments For IL-2 ELISA treatments, HPB-ALL or Jurkat E6-l (5x105 cells/ml) were cultured in triplicate in 48-well plates at 0.8 ml/well in a 2% BCS in RPMI medium (100 units of penicillin/ml, 100 units of streptomycin/ml, 2% BCS, 100 mM non-essential amino acids, 1 mM sodium pyruvate). Antagonists or vehicle (VH; 0.1% DMSO) were added 60 min prior to cell stimulation and cannabinoids or VH (0.1% EtOH) were added 30 min prior to cell stimulation. Cells were stimulated using phorbol ester and ionomycin (PMA/Io; 80 nM and 1 uM respectively). Following a 24 h cell incubation at 37°C, plates were centrifuged at 270 x g for 10 min. Supematants were collected, aliquoted and stored at -80°C until the day of the assay. For IL-2 mRNA treatments, HPB-ALL cells (5x105 cells/ml) were cultured in triplicate in 6-well plates at 5 ml/well in a 2% BCS in RPMI medium. A9-THC or VH (0.1% EtOH) were added 30 min prior to cell stimulation. Subsequently, cells were stimulated with PMA/Io (80 nM/l 1.1M) for 8 h. Following an 8 h incubation, total RNA was isolated from the cells using SV Total RNA Isolation System (Promega, Madison, WI), quantified and stored at -80°C until the day of the assay. 67 B. IL-2 protein quantification IL-2 was quantified using a sandwich ELISA method. Immulon IV strip plates (Dynatech Laboratories, Chantilly, VA) were coated with l ug/ml of purified mouse anti- human IL-2 antibody (BD Pharmingen, San Diego, CA) overnight at 4°C. Immulon strip plates were washed with PBST (1.9 mM NaHzPO4, 8.1 mM NazHPO4, 154 mM NaCl, 0.02% tween-20) and ddeO between each incubation step. Wells were blocked for 30 min at 37°C with 3% BSA in PBST. To generate a standard curve, a recombinant human IL-2 (0-8000 pg/ml) standard (BD Pharmingen, San Diego, CA) was included in each assay. Where needed, samples were diluted in 2% BCS in RPMI medium. Each sample and standard was placed into Immulon strip plates and incubated at 37°C for 2 h. Sample wells were incubated for 1 h at 37°C with 1 ug/ml of biotinylated anti-human IL-2 antibody (BD Pharmingen, San Diego, CA), followed by a 1 h incubation at 37°C with 1.5 ug/ml streptavidin peroxidase (Sigma Chemical Company, St. Louis, MO). IL-2 was detected colorometrically using tetramethylbenzidine to begin the reaction, and 6N H2804 to terminate the reaction. ELISA plates were read at 450 nm with a Bio-Tek Instruments EL-808 plate reader. C. IL-2 mRNA quantification Real time PCR was performed on a PE Applied Biosystems PRISM 7000 Sequence Detection System (Applied Biosystems, Foster City, CA). The PCR reaction was performed using 10 ng of reverse transcribed cDNA template and TaqMan predeveloped primers and probes (Applied Biosystems, Foster City, CA) for IL-2 and 68 188 ribosomal RNA, as a loading control, as recommended by the manufacturer. The amplication of each sample was plotted as the change in fluorescence dye (6-FAM) versus amplification cycle number. The cycle number at which the amplified product of each sample reaches a set threshold was termed the C. value. The C. value for the target gene (IL-2) was subtracted from the Ct value of the loading control gene (188 ribosomal RNA) to yield the AC: value. The relative mRNA levels for the gene of interest were determined through subtraction of the AC. value of the control sample (NA) form the AC, values of each treated sample. The resultant difference AACt was used to calculate the ZMC', and graphed as a fold increase over NA. relative mRN A levels using the formula: VII. Intracellular calcium determination Whole SPLC, isolated splenic T cells or human T cell lines were washed twice in Ca2+-KREB buffer (129 mM NaCl, 5 mM KCl, 1.2 mM KHzPO4, 1.2 mM MgSO4, 1 mM CaClz, 5 mM NaHCO;, 10 mM HEPES, 2.8 mM glucose, 0.2% BSA, pH 7.4). All experiments, except for those with LaCl3, were performed in the Ca2+-KREB buffer. The experiments with LaCl3 were performed in modified HPSS buffer (120 mM NaCl, 5.3 mM KCl, 0.8 mM MgSO4, 1.8 mM CaClz, 20 mM HEPES, 11.1 mM glucose, 0.2% BSA, pH 7.4), which contained a lower concentration of anions (Zhu et al., 1996). For studies with PTx, cells were pretreated with 100 ng/ml PTx for 18 h at 37°C and washed twice in Ca2+-KREB buffer. Cells were incubated with cell-permeant fura-2 AM dye (1 11M, Molecular Research Products, Eugene, OR) for 30 min at 37°C in the dark. Cells were harvested, washed three times with Ca2+-KREB buffer to remove extracellular fura- 2 dye, and readjusted to 5x106 cells/ml (for murine splenic cells) or 5x105 cells/ml (for 69 human T cell lines) in Ca2+-KREB buffer. Cells were placed in a 3 ml quartz cuvette with constant stirring. [Ca2+]: determinations were performed at room temperature with a Beckman Spex 1681 0.22m Spectrometer with dual excitation at 340 and 380 nm and emission at 510 nm (all slit widths were 1 mm). [Ca2+], calculations were based on maximum and minimum calcium values, as assessed with use of 0.1% Triton-X and 500 mM EGTA, respectively. The dissociation constant for the fura-2-calcium complex was 1.45 X 10'7 M. For studies conducted in the absence of [Ca2+]c, the Ca2+-KREB buffer was prepared as above without CaClz and supplemented with 1 mM MgC12 and 20 11M EGTA. All compounds used in [Calm determination were screened for autofluorescence using fura-2 sodium salt containing Ca2+-KREB buffer. None of the compounds, with the exception of WIN-2, exhibited autofluorescence, nor did they interfere with fura-2 fluorescence. VIII. Kinase assays A. Sample preparation HPB-ALL cells were cultured in 60 mm2 tissue culture plates at a density of 5x105 cells/ml (10 ml/plate) and incubated at 37°C for 2 h. Cells were left untreated (NA) or treated with VH (0.1% EtOH), various concentrations of A9-THC (1-20 1.1M), or PMA (for PKC assays) for varying periods of time (0.5-60 min) at 37°C. Cells were harvested and centrifuged at 300 x g for 5 min, then rinsed with 1x ice-cold PBS (1.9 mM NaHzPO4, 8.1 mM NazHPO4, 154 mM NaCl) and centrifuged again. For CaMKIl assays, cells were resuspended in assay dilution buffer (20 mM MOPS, 25 mM B-glycerol phosphate, 1 mM CaClz, pH 7.2; supplemented with 1 mM dithiothreitol, 1 mM NaVO3, 70 2 mM NaF and 1x EDTA-free solution of protease-inhibitor cocktail tablets). For PKC assays, cells were resuspended in triton-X lysis buffer (25 mM Tris-HCl, 20 mM MgC12, 140 mM NaCl, 1% triton-X 100, 1x EDTA-free solution of protease-inhibitor cocktail tablets, pH 7.2). The cells were lysed by briefly sonicating twice for 10 s on ice. For CaMKII assays, the cell lysate was further centrifuged at 6700 x g to remove membrane fractions, and the supematants were stored at —80°C. The samples were assayed for protein using a BCA protein assay kit (Sigma Chemical Company, St. Louis, MO) per manufacturer’s instructions. B. Substrate activity determination The phosphotransferase activity of CaMKII or PKC was determined in the cell lysates using a CaM Kinase II Assay Kit (Upstate Biotech, Lake Placid, NY) or a PKC Assay Kit (Upstate Biotech, Lake Placid, NY). The assay reactions were performed per manufacturer’s instructions with 10 uCi [y-3ZP] ATP (specific activity 3000 Ci/mmol; PerkinElmer, Boston, MA) using buffers, inhibitors and substrates provided by the manufacturer for 10 min at 30°C. Each assay reaction was then spotted onto individual P81 phosphocellulose paper filters (Whatman International, Maidstone, England), allowed to dry, and washed three times in 0.75% H3P04. The sample filters were assayed for 32P using a Beckman LS] 801 scintillation counter in polyethylene vials containing 10 ml scintillation cocktail. 71 IX. Western analysis A. Sample preparation HPB-ALL cells were cultured in 60 mm2 tissue culture plates at a density of 5x105 cells/m1 (10 ml/plate) and incubated at 37°C for 2 h. Cells were left untreated (NA) or treated with VH (0.1% EtOH), or various concentrations of A9-THC (1-20 11M) for varying periods of time (1-60 min) at 37°C. Cells were harvested and centrifuged at 300 x g for 5 min, then rinsed with 1x ice-cold PBS and centrifuged again. Cells were resuspended in HEDG buffer (20 mM HEPES, 1 mM EDTA, 0.5 mM dithiothreitol, 10% glycerol, pH 7.4; supplemented with a 1x solution of protease-inhibitor cocktail tablets). The cells were lysed by briefly sonicating twice for 10 s on ice. The cell lysate was centrifuged at 6700 x g, and the supematants were stored at —-80°C. The samples were assayed for protein using a BCA protein assay kit per manufacturer’s instructions. B. Electrophoresis and blotting SDS polyacrylamide gel electrophoresis was performed using a glass plate assembly with 1.5 mm spacers. A separating gel solution (375 mM Tris-HCl, 10% acrylamide, 0.1% SDS, 0.03% ammonium persulfate, 0.001% TEMED) was poured between the glass plate assembly and allowed to polymerize for 2 h at RT with a layer of butanol-saturated ddeO. The separating gel was stacked with a stacking gel solution (125 mM Tris-HCl, 4% acrylamide, 0.1% SDS, 0.03% ammonium persulfate, 0.001% TEMED), fitted with a 1.5 mm comb, and allowed to polymerize for at least 1 h at RT. Cell lysates were prepared in HEDG buffer to a protein concentration of 50 ug and incubated at 70°C for 10 min with loading buffer (62.5 mM tris-HCl, 2% SDS, 10% 72 glycerol, 0.01% bromophenol blue and 1% B-mercaptoethanol). Samples were loaded into the wells on the prepared gel assembly and electrophoresed at 100 V for 2 h at RT. Proteins were transferred onto nitrocellulose at 20 V overnight at 4°C. The nitrocellulose membranes were blocked with a 4% milk, 1% BSA in TBST solution (10 mM tris-HCl, 150 mM NaCl, 0.1% tween-20) for 2 h at RT with shaking. Membranes were then incubated with a 1:500 dilution of rabbit anti-active pT286 CaMKII polyclonal antibody (Promega, Madison, WI) in a 5% BSA in TBST solution for 2 h at RT with shaking. Next, blots were incubated for 30 min at RT with a donkey anti-rabbit antibody conjugated with horseradish peroxidase (Amersham, Arlington Heights, IL). Antibody binding was detected by exposing the blot to Supersignal West Femto Maximum Sensitivity Substrate (Pierce Scientific, Rockford, IL). X. RT-PCR A. CB] and CB2 Total RNA from HPB-ALL or Jurkat E6-1 cells was isolated using Tn' Reagent (Sigma Chemical Company, St. Louis, MO). Isolated RNA samples were confirmed to be free of DNA contamination by the absence of product after PCR amplification in the absence of reverse transcriptase. RT-PCR was performed using 100 ng of total RNA from HPB-ALL and Jurkat E6-1 cells. The PCR master mixture consisted of PCR buffer, 2.5 mM MgC12, 1.25 units of Taq DNA polymerase and 6 pmol of forward and reverse primers for either CBl or CB2. Samples were heated to 94°C for 4 min and cycled 40 times at 94°C for 30 s, 57°C for 30 s, and 72°C for l min, after which an additional extension step of 72°C for 5 min was included. The forward and reverse primer 73 sequences, amplicon sizes and accession numbers for C8] and C82 are given in table 1. PCR products were resolved in a 3% NuSieve 3:1 agarose gel (FMC Bioproducts, Rockland, ME) and visualized with ethidium bromide staining. B. TRPC1-7 Total RNA from HPB-ALL cells, Jurkat E6-1 cells, C57BL/6J WT and C57BL/6J TRPC1"' SPLC was isolated using Tri Reagent. Isolated RNA samples were confirmed to be free of DNA contamination by the absence of product after PCR amplification in the absence of reverse transcriptase. The PCR master mixture consisted of Mg2+-free PCR buffer, 2.5 mM MgC12, 1.25 units of Taq DNA polymerase, 6 pmol of forward and reverse primers, and 400 ng of cDNA reverse transcribed from isolated total RNA. Samples were heated to 94°C for 4 min and cycled 40 times at 94°C for 30 s, 56°C for 30 s, and 72°C for 1 min, after which an additional extension step of 72°C for 5 min was included. The forward and reverse primer sequences, amplicon sizes and accession numbers for human and murine TRPCl—7 are given in table 1. PCR products were resolved in a 2% NuSieve 3:1 agarose gel and visualized with ethidium bromide staining. XI. DNA Sequencing A. CBZ receptor sequencing Total RNA from HPB-ALL or Jurkat E6-l cells was isolated using Tri Reagent. CBZ cDNA was obtained by reverse transcription with 5 ug of isolated total RNA using PowerScriptTM reverse transcriptase (BD Biosciences, Palo Alto, CA) and amplified by PCR using Advantage-HF 2 PCR Kit (BD Biosciences, Palo Alto, CA) as per 74 Table 1. Sequences of primers used for RT-PCR reactions. Gene Accession Direction Primer Sequence Size Number (5’ to 3’) 11m) hCBl XM0043 50 Forward GGCTGGAACTGCGAGAAACT 301 Reverse TGATCAACACCACCAGGATCA hCB2 XM0863 56 Forward TCCCAGGCACCTAGACACG 203 Reverse TGGTCTCTGGAGGATGCAGG hTRPCl NM003 3 04 Forward GGAGGTGAAGGAGGAGAATACGCTG 686 Reverse ATAATCCCCGTTTGTCAAGAGGCTCG hTRPC3 NM003 305 Forward TTCTACGCTTACGACGAGGACG 786 Reverse GAACCTGTCTGAGGCATTGAACAC hTRPC4 NM016179 Forward AACAGATGTGGGATGGCGGAC 5 87 Reverse GTTGAGTAGAACAACCAGAGAGATGAC hTRPC5 NM012471 Forward TAGTTCAGAGGTAGACAGCCTGCG 1 195 Reverse GCCAAATACAGACCAGAAGAGTGAC hTRPC6 NM004621 Forward ATGGTAACATCCCAGTGGTGCG 925 Reverse CCAATGGCAACAGCAAGGAC hTRPC7 NM023089 Forward GCAAGGATTI‘TGTAGTGGGCG 1 025 Reverse TGGGTTGTATTTGGCACCTCG mTRPC l NM01 1643 Forward GCCTCAGACATTCCAGGTTTCG l 1 29 Reverse TCATTGCTTTGCTGTTCGCAG mTRPC2 NMO l 1 644 Forward ATCCCGAATCAACACCTACCG 60 1 Reverse AGACTCTCCCAGCAAGAAGATAAG mTRPC3 NM01 95 10 Forward GAGCAGACCATCGCTATCAAGTG 703 Reverse TGTCCTTCACAGTCCTTCCAAGAG mTRPC4 NM016984 Forward GAACTCAGCAAGGTGGAGAACG 1 1 08 Reverse CCCCAACAAACTCAGTGAACTCG mTRPC5 NM009428 Forward GGTTCAACAACACCTTCTGTCCC 83 7 Reverse CCTCTCCCCAAGTTTCAAATACG mTRPC6 NMOI 3 83 8 Forward GCTGAAGGCAAAAGGTTAGCG 799 Reverse AAATGGTGAAGGAGGCTGCGTG mTRPC7 NMO l 203 5 Forward CACCCTAACTGTCAGCAGCA 647 Reverse GAGATGATCTGGGGGTCTGA 75 manufacturer’s instructions. Briefly, samples were heated to 94°C for 5 min and cycled 40 times at 94°C for 30 s, 60°C for 30 s, and 72°C for 90 s, after which an additional extension step of 72°C for 5 min was included. The PCR primers contained cleavage sites for restriction endonucleases, BamHI and HindIII, within the forward and reverse primers, respectively. The sequences for PCR primers from 5’ to 3’ were: CTGAAGGATCCACCCCATGGAGGAATGCTGGGTGAC (forward) and CCTCTCAAGCTTCCAGGGAGTGAACTGATTTCTGACTTGAG (reverse). The PCR products were then cloned into pCMV-Tag-l (Stratagene, La Jolla, CA) and sequenced with T3 and T7 primers using an ABI PRISM® 3100 Genetic Analyzer at the Michigan State University Macromolecular Structure, Sequencing and Synthesis Facility (East Lansing, MI). The sequences for the T3 and T7 primers, respectively, from 5’ to 3’ were as follows: AATTAACCCTCACTAAAGGG and GTAATACGACTCACTATAGGGC. B. TRPC] alternative splice PCR products were resolved in a 1.2% NuSieve 3:1 agarose gel (FMC Bioproducts, Rockland, ME) and visualized with ethidium bromide staining. The bands for TRPCl were excised from the agarose gel and purified using Wizard® PCR preps DNA purification system (Promega, Madison, WI). Sequencing of TRPC] bands was performed with either TRPC] forward or reverse primers (see Table 1) using an ABI PRISM® 3100 Genetic Analyzer at the Michigan State University Macromolecular Structure, Sequencing and Synthesis Facility (East Lansing, MI). 76 XII. siRNA knockdown of TRPC] A. Transfection of TRPCl siRNA Chemically synthesized and pre-annealed siRNA for TRPCl and a non-silencing control sequence were custom synthesized from Ambion Inc. (Austin, TX). HPB-ALL cells (2.5x105 cells/ml) were transiently transfected with 20 nM siRNA specific for TRPCl or a non-silencing control sequence for 48 h. CodeBreakerTM siRNA Transfection Reagent (Promega, Madison, WI) was diluted in Opti-MEM® medium (Gibco, Grand Island, NY) according to manufacturer’s instructions. The mixture was incubated at RT for 20 min. siRNA specific for TRPCl or non-silencing control sequence was diluted in the prepared mixture and allowed to incubate at RT for a further 20 min. Transfection complexes were then dropped onto the cells, swirled to mix, and allowed to incubate at 37°C for 48 h. After 48 h, cells were harvested, washed and used for RNA isolation or [Ca2+]: determination. Sequences for siRNA oligonucleotides for TRPCl and non-silencing control are listed in table 2A. B. Detection of TRPC] knockdown Real time PCR was performed on a PE Applied Biosystems PRISM 7000 Sequence Detection System (Applied Biosystems, Foster City, CA). Briefly, total RNA was isolated from siRNA transfected HPB-ALL cells using SV Total RNA Isolation System (Promega, Madison, WI), and reverse transcribed. The PCR reaction contained 100 ng of cDNA template, 133 nM of forward and reverse primers, and the following components of the SYBR® Green PCR Core Reagents (Applied Biosystems, Foster City, CA): 0.025 units AmpliTaqTM Gold DNA polymerase, 1 mM dNTP mix, 3 mM Mng 77 and 1x SYBR Green PCR buffer. Each plate contained duplicate standards of purified PCR products of known template concentration that covered at least seven orders of magnitude to interpolate relative template concentrations of the experimental samples from the standard curves of log copy number versus threshold cycle (C.). In addition, no template controls were also included on each plate. The relative level of TRPCl mRNA was standardized to the housekeeping gene, B-actin, to control for differences in RNA loading, quality, and cDNA synthesis. Comparisons between vehicle (transfection reagent), non-silencing control and TRPC] siRNA treated groups were analyzed using a two-way analysis of variance followed by Dunnett’s post test. The forward and reverse primer sequences for TRPC] and B-actin are given in table 28. XIII. Radioligand-binding analysis Spleens from WT C57BL/6J or CBl'l'lCBZ'/' C57BL/6J mice were isolated, made into a single cell suspension and erythrocytes lysed. The cells were washed using Ca2+/Mg2+-free Hanks balanced salt solution (GIBCO, Grand Island, NY), and adjusted to a cell density of 1x108 cells/ml in binding buffer (38.45 mM Tris-HCl; 1 mM EDTA- Naz; 5 g/L BSA in Ca2+/Mg2+-free Hanks balanced salt solution; pH 7.4). The binding assay was performed in triplicate in 13 x 100 mm glass culture tubes pre-siliconized using a 1% Aquasil siliconizing solution (Pierce Scientific, Rockford, IL). Binding reactions were performed in a total of 1 ml volume of binding buffer, [3H]-SR1 (0.5 nM- 80 nM), 10 11M unlabeled SR1 (in half the samples for non-specific determination), and 100 ul of cells (1x107 cells). The reaction mixture was incubated at 37°C for 60 min. After the incubation, the reaction was stopped by adding 2 ml ice-cold washing buffer 78 Table 2A. Real-time PCR primer sequences for TRPCl and B—actin Gene Orientation Oligonucleotide Sequence Amplicon (5’ to 3’) Size (bp) TRPCI Forward GCCTTCCTCTCCATCCTCTT 83 Reverse TCAGCGTATTCTCCTCCTTCA B-actin Forward TCATGAAGTGTGACGTGGACATC 1 56 Reverse CAGGAGGAGCAATGATCTTGATCT Table 2B. Oligonucleotide sequences for siRNA Gene Orientation Oligonucleotide Sequence Corresponding (5’ to 3’) Nucleotides TRPC] Sense UGAACUUAGUGCUGAUUUA 612-630 Antisense AAAAUCAGCACUAAGUUCA Control Sense ACUUGUGACUGAAUAGUUU --- Antisense UAACUAUUCAGUCACAAGU 79 (38.45 mM Tris-HCl, 1 g/L BSA in ddHZO, pH 7.4). The reaction mixture was then passed through 2.4 cm GF/C glass microfiber filters (Whatman International, Maidstone, England), which were presoaked overnight in 0.1% polyethylenimine (Sigma Chemical Company, St. Louis, MO), and washed four times with 4 m1 washing buffer each time. The filters were transferred into 20 ml polyethylene scintillation vials containing 15 ml scintillation cocktail, and vortexed vigorously. The samples were assayed for 3 H using a Beckman LSlSOl scintillation counter. Specific binding of [3H]-SR1 was determined by subtracting non-specific binding from total binding. Saturation binding isotherms were plotted to demonstrate the relationship between total, non-specific and specific binding of [3H]-SR1 to WT C57BL/6J or CBl"‘/CB2'/' SPLC. Scatchard analysis was also performed to determine the K. (binding affinity) and Bmax (number of binding sites per cell). Scatchard analysis and binding isotherms were plotted using Graphpad Prism®. XIV. Statistical analysis The mean i standard error was determined for each treatment group performed in triplicate (for Ca2+ determinations) or individual (for all others) experiments by parametric analysis of variance. When significant differences were detected, treatment groups were compared with appropriate controls with the Dunnett’s two-tailed t test. 80 EXPERIMENTAL RESULTS 1. Effects of A9-THC and CP on IL-2 expression in HPB-ALL and Jurkat E6-l cells A. Differential effects of A9-THC and CP on IL-2 secretion Cannabinoids have been shown to modulate the expression of several T cell cytokines (Berdyshev, 2000; Klein et al., 2000a). IL-2 is of particular interest because it is a critical cytokine in the immune response and a hallmark of T cell activation. Previous research on IL-2 secretion has determined that synthetic, endogenous as well as plant-derived cannabinoids alter the secretion of IL-2 from activated murine T cells (Condie et al., 1996; Jan et al., 2002; Ouyang et al., 1998). Presently, the effect of A9- THC and CP on IL-2 secretion was investigated in the human T cell lines, HPB-ALL cells and Jurkat E6-1. The objective of the parallel studies in the HPB-ALL and Jurkat E6-1 cells was to elucidate the putative role of the C32 receptor in the modulation of IL- 2 by cannabinoids. The CB2 receptor expressed by Jurkat E6-l cells has previously been reported to be aberrant, and also to be dysfunctional (Schatz et al., 1997). Therefore, the present studies were performed with the hypothesis that cannabinoids would differentially modulate IL-2 secretion in the HPB-ALL vs. Jurkat E6-1 cells. HPB-ALL and Jurkat E6-1 cells were pretreated with various concentrations of A9-THC or CP (0.1- 20 uM) followed by cell stimulation with PMA/Io. After a 24 h incubation, IL-2 was assayed from each culture supernatant. The results showed that while A9-THC inhibited IL-2 secretion from HPB-ALL cells in a concentration-responsive manner (figure 6), A9- THC did not significantly inhibit the secretion of IL-2 in Jurkat E6-l cells (figure 7). In 81 10000 - I IL-2 T 7500— T T '72? e E0 55 50004 i N :4 4: :— * - a 2500— NA P/I VH 0.1 1.0 2.5 5.0 10.0 15.0 20.0 A9-THC (11M) PMA/IO Figure 6. A9-THC suppresses the secretion of IL-2 from PMA/Io-stimulated HPB- ALL cells. HPB-ALL cells were treated with various concentrations of Ag-THC or VH (0.1% EtOH) for 30 min followed by activation with PMA/Io (80 nM/l 11M) for 24 h. The supematants were harvested and assayed for IL-2 by ELISA. * p < 0.05 as compared to VH group. Results represent four separate experiments with three replicates per treatment group. ND indicates no IL-2 detected. 82 75000 1 I IL-2 60000— T T T g A T E 45000— T T T h, T 8 C‘.‘ a 30000— 15000- 0— ND NA P/Io VH 0.1 1.0 2.5 5.0 10.015.020.0 A9-THC (11M) PMA/Io Figure 7. Ag-THC does not suppress the secretion of IL-2 from PMA/Io-stimulated Jurkat E6-l cells. Jurkat E6-1 cells were treated with various concentrations of A9-THC or VH (0.1% EtOH) for 30 min followed by activation with PMA/Io (80 nM/l 11M) for 24 h. The supernatants were harvested and assayed for IL-2 by ELISA. Results represent four separate experiments with three replicates per treatment group. ND indicates no IL-2 detected. 83 contrast to the differential effects of A9-THC on IL-2 secretion, CP, the higher affinity CBl/CB2 non-selective ligand was equally efficacious at inhibiting the secretion of IL-2 in both cell lines (figure 8, 9). B. Characterization of the CB] and CBZ receptors Previous Northern analysis results in HPB-ALL and Jurkat E6-1 cells showed that while neither cell line expressed CBl transcripts, both expressed transcripts for CBZ (Schatz et al., 1997). Presently, the expression of CB] and CB2 was characterized by RT-PCR, a significantly more sensitive method. CBl mRNA expression was not detected by RT-PCR in either of the two cell lines (figure 10). Conversely, a single predicted 203 bp amplicon for CB2 was produced by RT—PCR from RNA derived from both cell lines (figure 10). Previous results have also suggested that the CB2 receptor in the Jurkat E6-1 cells was dysfunctional (Schatz et al., 1997). In order to elucidate whether the dysfunctionality of the CB2 receptor in the Jurkat E6-1 cells was due to putative mutations in the CBZ gene, CB2 cDNA was sequenced from both HPB-ALL and Jurkat E6-1 cells. Results showed that the CB2 coding sequence was 100% homologous between the two cells lines and 99.4% homologous as compared to the reported coding sequence for human CB2 (GenBank accession no. XM_086356; data not shown). In spite of the CBZ sequence homology between HPB-ALL cells and Jurkat E6-1 cells, CBZ does not negatively couple to adenylate cyclase in Jurkat E6-1 cells, which is in contrast to HPB-ALL cells and virtually all other cells known to express functional CB2 (Schatz etaL,1997) 84 5000 - l I IL-2 4000- A 31‘ E; 3000- 4 T T 8 ‘1‘ =3 2000- * 31‘. 1000- * 0 _ 14A PH \ni (101 (11 L0 25 51) 100 CP55,940 (uM) PMA/IO Figure 8. CP suppresses the secretion of IL-2 from PMA/Io-stimulated HPB-ALL cells. HPB-ALL cells were treated with various concentrations of CP or VH (0.1% EtOH) for 30 min followed by activation with PMA/Io (80 nM/l 11M) for 24 h. The supematants were harvested and assayed for IL-2 by ELISA. * p < 0.05 as compared to VH group. Results represent three separate experiments with three replicates per treatment group. ND indicates no IL-2 detected. 85 80000 « I IL-2 60000- E. 3, 40000- ‘7' :1 200004 0_ ND * NA P/I VH 0.01 0.1 1.0 2.5 5.0 10.0 CP55,940 (uM) PMA/Io Figure 9. CP suppresses the secretion of IL-2 from PMA/Io-stimulated Jurkat E6-l cells. Jurkat E6-1 cells were treated with various concentrations of CP or VH (0.1% EtOH) for 30 min followed by activation with PMA/Io (80 nM/l 11M) for 24 h. The supematants were harvested and assayed for IL-2 by ELISA. * p < 0.05 as compared to VH group. Results represent three separate experiments with three replicates per treatment group. ND indicates no IL-2 detected. 86 Figure 10. RT-PCR for CB1 and CB2 in the I-IPB-ALL and Jurkat E6-l cells. Total RNA was isolated from I-IPB-ALL and Jurkat E6-l cells. Three separate RNA isolates were assayed for expression of CB1 and CB2 mRNA transcripts by RT-PCR. The RNA samples were confirmed to be free of DNA contamination by the absence of product after PCR amplification in the absence of reverse transcriptase (RT). The results are representative of three independent experiments. 87 £8 Em 3.5—. .2: 4.51:: o... a 80 can So no. 5.3.: .2 3&3 “Ea:— Aéfimm ~23 15¢-mmm PM 3 PM 3 . .. . i . .. ... :45...» r. “GE: Alzfimm “Gag 412-9%.“ PM n+v PM 3 88 C. Cannabinoid receptor antagonists fail to antagonize the suppression of IL-2 by A9-THC and CP The above observation that both HPB-ALL and Jurkat E6-1 cells express only the CBZ receptor suggested that the differential modulation of IL-2 secretion by A9-THC and CP was putatively CB2-mediated. To test this hypothesis, lL-2 secretion studies were performed in the absence or presence of SR2 with A9-THC or CP. HPB-ALL or Jurkat E6-1 cells were treated with SR2 (1 11M) for 30 min, followed by varying concentrations of A9-THC and CP (0.1-20 11M) and then stimulated with PMA/Io. The resulting IL-2 measurements revealed that pretreatment of cells with SR2 failed to antagonize the suppression of IL-2 by A9-THC in the HPB-ALL cells (figure 11), and the suppression of IL-2 by CP in both cell lines (figure 12, 13). The lack of antagonism by SR2 of the A9- THC- and CP-mediated suppression of lL-2, however, was not surprising considering the prior observation that the suppression of IL-2 expression by cannabinoids in murine T cells was reported to be cannabinoid receptor-independent (Kaplan et al., 2003; Rockwell and Kaminski, 2004). In addition to SR2, the effect of SR1 on IL-2 suppression by A9- THC or CP was also examined. Although neither cell was found to express the CB1 receptor, SR1 has been demonstrated to cross react with a variety of other receptors and hence antagonize non-CB1 non-CB2 mediated cannabinoid actions (Pertwee, 2001, 2005). In the current experiments, SR1 (1 uM) pretreatment also failed to antagonize the suppression of IL-2 by A9-THC in the HPB-ALL cells (figure 14), and the suppression of IL-2 by CP in both HPB-ALL and Jurkat E6—1 cells (figure 15, 16). 89 25000 - T I Control I Vehicle 200004 T SR144528 A _ ’4 E 15000 ¢ 3 3° 2 / T 4': f 3‘ f 9 5’3 1" i- . 3 100004 Z p Z '55 :r / / / / a / / / / / 7 / / / / / / a a 6 6 a a 5000- g g g 2 g g / / / / / / / z a a z a 2 0_ \D /. A A /1 4 4 NA P/I VH 1.0 5.0 10.0 15.0 20.0 A9-THC (11M) PMA/Io Figure 11. SR2 does not antagonize the A9-THC-induced suppression of IL-2 secretion in HPB-ALL cells. HPB-ALL cells were pretreated with SR2 (1 11M) or VH (0.1% DMSO) for 30 min followed by various concentrations of A9-THC or VH (0.1% EtOH) for 30 min. The cells were then activated with PMA/Io (80 nM/l 11M) for 24 h. The supematants were harvested and assayed for IL-2 by ELISA. * p < 0.05 as compared to VH/V H group. Results represent three separate experiments with three replicates per treatment group. ND indicates no IL-2 detected. 90 15000- I Control I Vehicle T SR144528 10000~ - E a , 3 a a a a 5000- g é a Z / / / / / / / . / / / / . é % 3 ¢ 7 6 e a a a 0_ t. 4 /1 4 // é NA P/I VH 0.01 0.1 1.0 10.0 CP55,940(11.M) PMA/Io Figure 12. SR2 does not antagonize the CP-induced suppression of IL—2 secretion in HPB-ALL cells. HPB-ALL cells were pretreated with SR2 (1 11M) or VH (0.1% DMSO) for 30 min followed by various concentrations of CP or VB (0.1% EtOH) for 30 min. The cells were then activated with PMA/Io (80 nM/l 11M) for 24 h. The supematants were harvested and assayed for IL-2 by ELISA. * p < 0.05 as compared to VH/VH group. Results represent three separate experiments with three replicates per treatment group. ND indicates no IL-2 detected. 91 30001 I Control 2500- T T I Vehicle T 5’ SR141716A A2000- T g; g r E Z Z E" .. / / 45 5,1500 / g 7 6'] ¢ ¢ ¢ "' * :1- ‘=' - é % % T T. 1 1000 % ¢ ¢ :1 T / / / r’ '1' ¢ é % a 5' V 5004 g g g g g g / / ; é % / 0_ kl é g 4 Q g 2 NA PI VH 50 10.0 150 200 A9-THC(1~LM) PMA/Io Figure 13. SR2 does not antagonize the CP-induced suppression of IL-2 secretion in Jurkat E6-1 cells. Jurkat E6-1 cells were pretreated with VH (0.1% DMSO) or SR2 (1 uM) for 30 min followed by various concentrations of CP or VH (0.1% EtOH) for 30 min. The cells were then activated with PMA/Io (80 nM/l 11M) for 24 h. The supematants were harvested and assayed for IL-2 by ELISA. * p < 0.05 as compared to VH/VH group. Results represent three separate experiments with three replicates per treatment group. ND indicates no IL-2 detected. 92 10000 — I Control "' T I Vehicle 7500— l * SR141716A é * Te: 6 V 35000— g g I? g ‘i , / / / / :1 : ¢ ¢ ¢ / : / / / / * 2500— g g g g ./ é é a a a % a e a a 4 /, / é NA P/I VH 0.01 0.1 1.0 10.0 CP55,940 (pM) PMA/Io Figure 14. SR1 does not antagonize the A9-THC-induced suppression of IL-2 secretion in HPB-ALL cells. HPB-ALL cells were pretreated with SR1 (1 11M) or VH (0.1% DMSO) for 30 min followed by various concentrations of A9-THC or VH (0.1% EtOH) for 30 min. The cells were then activated with PMA/Io (80 nM/l uM) for 24 h. The supematants were harvested and assayed for lL-2 by ELISA. * p < 0.05 as compared to VH/V H group. Results represent three separate experiments with three replicates per treatment group. ND indicates no IL-2 detected. 93 80000 - I Control T T T ‘l’ I Vehicle 60000J 53 ¢ SR141716A A ’7’ 6 E % ¢ 31) ¢ / at S 40000 4 g g 3 4 I / / =‘ 4 ¢ / .4. 5 3 § '7 20000 4 g g Z Z / é / ¢ / / / / / / / / j 0 _ . g é g g // NA P/I VH 0.01 0.1 1.0 10.0 I CP55,94O (1.1M) PMA/Io Figure 15. SR1 does not antagonize the CP-induced suppression of IL-2 secretion in HPB-ALL cells. HPB-ALL cells were pretreated with SR2 (1 M) or VH (0.1% DMSO) for 30 min followed by various concentrations of CP or VH (0.1% EtOH) for 30 min. The cells were then activated with PMA/Io (80 nM/l uM) for 24 h. The supematants were harvested and assayed for IL-2 by ELISA. "‘ p < 0.05 as compared to VH/V H group. Results represent three separate experiments with three replicates per treatment group. ND indicates no lL-2 detected. 94 80000 - 'l' I Control I T 'l' : Vehicle 60000- >9 / 6 SR141716A I; / .. a a a a a be p / . 840000- ¢ g y E3 é a 6 i / .1: Z 4 9 '2 / / / / 20000- g g é % / / / / / é / a a a a . 0- k. 4 // A 4 71 NA P/I VH 0.01 0.1 1.0 10.0 | CP55,940(uM) PMA/Io Figure 16. SR1 does not antagonize the CP-induced suppression of IL-2 secretion in Jurkat E6-l cells. Jurkat E6-1 cells were pretreated with VH (0.1% DMSO) or SR1 (1 uM) for 30 min followed by various concentrations of CP or VH (0.1% EtOH) for 30 min. The cells were then activated with PMA/Io (80 nM/l 11M) for 24 h. The supematants were harvested and assayed for IL-2 by ELISA. * p < 0.05 as compared to VHNH group. Results represent three separate experiments with three replicates per treatment group. ND indicates no IL-2 detected. 95 D. PTx pretreatment does not attenuate the Ag-THC-induced suppression of IL-2 secretion Given the prior reports that both CB1 and CB2 are G-protein coupled receptors, the involvement of G proteins in the A9-THC-induced IL-2 suppression was examined. HPB-ALL cells were preincubated with PTx (100 ng/ml) or VH for 18 h for full ADP- ribosylation of Gal/G010, and then used for IL-2 studies. PTx-loaded cells were treated with varying concentrations of A9-THC (1-20 11M) followed by PMA/Io stimulation. As seen in figure 17, PTx treatment did not reverse the suppression of IL-2 secretion elicited by A9-THC, suggesting that A9-THC mediates the suppression of lL-2 secretion in a manner independent of Gal/G010. E. A9-THC suppresses IL-2 mRNA production in HPB-ALL cells In T cells, the production and secretion of IL-2 occurs de nova upon cellular stimulation. Previous investigations in murine T cells have revealed that the cannabinoid-mediated decrease of lL-2 expression occurred at the level of lL-2 gene transcription (Condie et al., 1996; Jan et al., 2002; Ouyang et al., 1998). To examine whether the suppression of IL-2 expression by cannabinoids was also due to the decrease of IL-2 transcription in human T cells, HPB-ALL cells were treated with A9-THC followed by PMA/Io stimulation for 8 b. Total mRNA from treated cells was isolated and assayed for lL-2 by real-time PCR. Treatment of the HPB-ALL cells with varying concentrations of A9-THC showed a concentration-dependent suppression of PMA/Io- stimulated IL-2 mRN A production (figure 18), consistent with prior observations made in murine T cells. 96 2000 - 15004 10004 IL-2 (pg/ml) 500- O _ . NA PI VH 1.0 5.0 10.0 15.0 20.0 | A9-THC (11M) PMA/IO Figure 17. PT}: does not reverse the Ag-THC-induced suppression of IL-2 secretion in HPB-ALL cells. HPB-ALL cells were preincubated with PTx (100 ng/ml) or VH (PBS) for 18 h. The cells were then pretreated with various concentrations of A9-THC or VH (0.1% EtOH) for 30 min, followed by activation with PMA/Io (80 nM/l 11M) for 24 h. The supematants were harvested and assayed for IL-2 by ELISA. * p < 0.05 as compared to VH/V H group. Results represent two separate experiments with three replicates per treatment group. ND indicates no IL-2 detected. 97 400 - A I IL-2mRNA <1: Z 5 g 300— 8 Cd 8 E. E 200- g 1 < * * a 100— T T f ‘7‘ d NA PI VH 1.0 5.0 10.0 15.0 20.0 A9-THC (11M) PMA/Io Figure 18. Ag-THC suppresses IL-2 mRNA production in HPB-ALL cells. HPB- ALL cells were pretreated with various concentrations of A9-THC or VH (0.1% EtOH) for 30 min, followed by activation with PMA/Io (80 nM/l 11M) for 8 h. The supematants were harvested and assayed for IL-2 by real time PCR for 40 cycles. Results are shown as percentage of mean IL-2 4: standard error of triplicate samples. * p < 0.05 as compared to VH group. Results are representative of three independent experiments. 98 11. Effects of cannabinoids on [Ca2+], in T cells A. A9-THC elevates [Ca2+]i in T cells, but CP does not Recent investigation of the modulation of IL-2 expression by cannabinoids has demonstrated a strong correlation between cannabinoid-mediated modulation of IL-2 gene expression and reciprocal changes in DNA binding and reporter gene activity of NFAT (Jan et al., 2002; Ouyang et al., 1998; Yea et al., 2000). Given that NFAT activation and nuclear translocation is tightly controlled by [Ca2+],, the effect of A9-THC and CP on [Ca2+], was examined in the HPB-ALL and Jurkat E6-1 cells. In addition, parallel studies were performed in primary murine splenic T cells as a comparative control. In both the HPB-ALL cells and murine splenic T cells, A9-THC induced a concentration-responsive elevation in [Ca2+], (figure 19, 20). Interestingly, the increase in [Ca2+], by A9-THC (10 uM) was robust in both HPB-ALL cells(883.0 a— 56.2 nM, n=3) and splenic T cells (1652.0 i 216.8 nM, n=3), but at concentrations of A9-THC concentrations below 10 1.1M, the elevation in [Ca2+], was small (HPB-ALL cells; figure 19) or negligible (splenic T cells; figure 20). Also in the splenic T cells the rise in [Ca2+], induced by A9—THC (10 11M) did not reach a plateau during the entire period of [Ca2+], measurement. By contrast, treatment of the Jurkat E6-1 cells with A9-THC (10 1.1M) led to a modest rise in [Ca2+], (101.7 i 12.1 nM, n=3; figure 21), which was small in comparison to the effect of A9-THC (10 1.1M) on [Ca2+],- in the murine splenic T cells and HPB-ALL cells. In the Jurkat E6-1 cells, the A9-THC-mediated rise in [Ca2+], was also comparatively slow and more concentration responsive. 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Interestingly, pretreatment of HPB-ALL cells with CP did not attenuate the increase in [Ca2+]i elicited by Ag-THC (figure 22). B. Removal of [Ca2+]. and pretreatment with BAPTA-AM attenuate the rise in [Ca2+]i elicited by Ag-THC In order to determine whether the Ag-THC-mediated rise in [Ca2+]i involved an influx of [Ca2+]e, [Ca2+]; measurements were performed either in the presence or absence of [Ca2+]e both in splenic T cells as well as in HPB-ALL cells. The results showed that absence of [Ca2+]e severely attenuated (>90%; n=3) the Ag-THC-mediated [Ca2+].- elevation, as compared to control conditions in the presence of [Ca2+]: in both splenic T cells (figure 23) and HPB-ALL cells (figure 24). In the absence of [Ca2+]e, A9-THC induced only a small and delayed rise in [Ca2+]i. To ascertain that stored Ca2+ from intracellular pools was not involved in the Ag-THC-mediated rise in [Ca2+];, HPB-ALL cells were preloaded with BAPTA-AM (10 uM) to chelate all [Ca2+]i for 30 min alongside fura-2. Pretreatment with BAPTA-AM failed to abrogate completely the rise in [Ca2+]i elicited by Ag-THC, and induced a significantly smaller elevation in [Ca2+]; 103 5 .0 - I Vehicle Z CP55,94O E" 5” P o o o l I l A Ratio (340 nm/3 80 nm)) y—A O l 0.0 F— ‘ VH A9-THC Figure 22. CP does not antagonize the elevation of [Ca2+]; elicited by A9-THC in HPB-ALL cells. A 3 ml aliquot of fura-2 loaded HPB-ALL cells was placed into a cuvette with constant stirring. CP (1 uM) or VH (0.1% EtOH) was added directly to the cuvette just prior to beginning [Ca2+]i measurements. At 300 s, Ag-THC (10 uM) or VH (0.1% EtOH) was injected into the cuvette and the increase in [Ca2+]i was measured for a total of 1600 s. [Ca2+]i changes are presented as the mean i SEM of the change in base to peak ratio of bound to free Ca2+ (340 nm/3 80 nm) of three independent experiments. 104 8.0- . + [C32+]e I a ' [ca2+]e A Ratio (340 nm/380 nm) 4‘ 9‘ <.= <3 i" o J 0.0 ......... VH A9-THC Figure 23. Removal of [Ca2+]. severely abrogates the elevation of [Ca2+]i elicited by Ag-THC in murine splenic T cells. A 3 ml aliquot of fura-2 loaded murine splenic T cells was resuspended in either Ca2+-KREB or Cay-free KREB buffer just prior to beginning [Ca2+]; measurements. At 300 s, Ag-THC (10 uM) or VH (0.1% EtOH) was injected into the cuvette and the increase in [Ca2+]iwas measured for a total of 1600 8. Results are presented as mean change :i: SEM in the 340 nm/3 80 nm fluorescence ratio of [Ca2+]i from base to peak of three independent experiments. * p < 0.05 as compared to A9-THC (+[Ca2+]¢) group. 105 .°‘ C I I +ICa2+]. A 5.0- a ’ [C32+]e g 4.0d E o 3 0- V G .O 5 2.0- <1 1.0-i a: 0.0 VH A9-THC Figure 24. Removal of [Ca2+]. severely abrogates the elevation of [Ca2+]. elicited by A9-THC in HPB-ALL cells. A 3 ml aliquot of fura-2 loaded HPB-ALL cells was resuspended in either Cay-KREB or Cay-free KREB buffer just prior to beginning [Ca2+]i measurements. At 300 s, A9-THC (10 0M) or VH (0.1% EtOH) was injected into the cuvette and the increase in [Ca2+]i was measured for a total of 1600 5. Results are presented as mean change :2 SEM in the 340 nm/380 nm fluorescence ratio of [Ca2+]i from base to peak of three independent experiments. * p < 0.05 as compared to A9-THC (+[Ca2+]e) grouv. 106 (figure 25). The significantly smaller elevation in [Ca2+], is attributed to the partial buffering of the influx of [Ca2+]e by BAPTA-AM present in the cytosol. C. Cannabinoid receptor antagonists attenuate the A9-THC-mediated rise in [Ca2+]: The role of the cannabinoid receptors in the Ag-THC-mediated elevation in [Ca2+]. was examined with the use of SR1 and SR2. In light of previous findings showing that both CB1 and CBZ receptor transcripts are expressed in murine splenocytes (Jan et al., 2002; Schatz et al., 1997), SR1 and SR2 were used either individually or in combination in studies with splenic T cells. The antagonists by themselves had no effect on [Ca2+]i. However, pretreatment of splenic T cells with SR1 and SR2 (1.0 or 5.0 uM of each), either individually or in combination, for 300 s, followed by Ag-THC (10 uM) treatment showed a marked attenuation of A9-THC-mediated rise in [Ca2+]; (figure 26). Interestingly, the magnitude of the attenuation in the Ag-THC-mediated [Ca2+], rise by SR1 and SR2, individually, was almost as great as when used in combination. Similarly, pretreatment of HPB-ALL cells with SR1 or SR2 (0.1-5.0 uM) also caused a marked inhibition of the rise in [Ca2+]i by Ag-THC (figure 27). The effect of both antagonists was concentration responsive, but SR2 was slightly more efficacious at inhibiting the A9- THC-mediated rise in [Ca2+], than was SR1. By contrast, the modest rise in [Ca2+], elicited by Ag-THC in the Jurkat E6-1 cells was not significantly inhibited by either SR1 or SR2 (1.0 — 5.0 uM; figure 28). To ensure that the cannabinoid receptor antagonists did not attenuate [Ca2+], 107 4.01 I Vehicle T BAPTA-AM ”€3.0- O 00 E i 2.0“ E / cu Dd <1 1.0- 00 ""1"!" VH A9-THC Figure 25. Pretreatment with BAPTA-AM attenuates the elevation of [Ca2+]i elicited by Ag-THC in HPB-ALL cells. HPB-ALL cells were coloaded with fura—2 only, or a combination of fura-2 and BAPTA-AM (10 uM). A 3 ml aliquot of fura-2 and/or BAPTA-AM loaded HPB-ALL cells was resuspended in Cay-KREB and treated with A9-THC (10 M) or VH (0.1% EtOH) at 300 s, and the increase in [Ca2+]; was measured for a total of 1600 5. Results are presented as mean change 3: SEM in the 340 nm/38O nrn fluorescence ratio of [Ca2+]i from base to peak of three independent experiments. * p < 0.05 as compared to VH/Ag-THC group. 108 8 '0 1 Vehicle SR144528 + SR141716A IE. SR144528 9‘ o 1 fl SR141716A *- -—l A Ratio (340 urn/380 nm) A 'o * a: \ \Il 2.0 4 .3; \I‘I :/:I * .3; at: 2:2; % * \ \ 0 0 _- I fi 33 , VH A9-THC 1.0 5.0 SR144528 :I: SR141716A (0M) Figure 26. SR1 and SR2 antagonize the A9-THC-mediated elevation in [Ca2+]iin murine splenic T cells. A 3 ml aliquot of fura-2 loaded murine splenic T cells was placed into a cuvette with constant stirring. SR2 (1-5 uM), SR1 (1-5 uM) and/or VH (0.1% DMSO) were added directly to the cuvette just prior to beginning [Ca2+]; measurements. At 300 s, A9—THC (10 0M) or VH (0.1% EtOH) was injected into the cuvette and the increase in [Ca2+], was measured for a total of 1600 s. [Ca2+]i changes are presented as the mean d: SEM of the change in base to peak ratio of bound to free Ca2+ (340 nm/380 nm) of three independent experiments. * p < 0.05 as compared to VII/A9- THC group. 109 3 '0 - I Vehicle B R442 254 8158 I I SR141716A l 3" o l q; l 2 A Ratio (340 nm/3 80 nm) 2; :2 .o u: l P o l l I VH A9-THC 0.1 SR144528 or SR141716A (uM) Figure 27. SR1 and SR2 antagonize the A9-THC-mediated elevation in [Ca2+]: in HPB-ALL cells. A 3 ml aliquot of fura-2 loaded HPB-ALL cells was placed into a cuvette with constant stirring. SR2 (0.1-5.0 uM), SR1 (0.1-5.0 uM) and/or VH (0.1% DMSO) was added directly to the cuvette just prior to beginning [Ca2+]imeasurements. At 300 s, A9-THC (10 0M) or VH (0.1% EtOH) was injected into the cuvette and the increase in [Ca2+]i was measured for a total of 1600 s. [Ca2+]i changes are presented as the mean :1: SEM of the change in base to peak ratio of bound to free Ca2+ (340 nm/380 nm) of three independent experiments. * p < 0.05 as compared to VII/A9-THC group. 110 0.8 - I Vehicle 2 SR144528 I 0-6‘ I SR141716A l .5 0.40 A Ratio (340 nm/380 nm) O i0 I I ‘ VH A9-THC 1.0 5.0 SR144528 or SR141716A (uM) Figure 28. SR1 and SR2 do not antagonize the Ag-THC-mediated elevation in [Ca2+], in Jurkat E6-1 cells. A 3 ml aliquot of fura-2 loaded Jurkat E6-1 cells was placed into a cuvette with constant stirring. SR2 (1.0-5.0 1.1M), SR1 (1.0-5.0 uM) and/or VH (0.1% DMSO) was added directly to the cuvette just prior to beginning [Ca2+]i measurements. At 300 s, Ag-THC (10 uM) or VH (0.1% EtOH) was injected into the cuvette and the increase in [Ca2+]i was measured for a total of 1600 s. [Cazm changes are presented as the mean :1: SEM of the change in base to peak ratio of bound to free Ca2+ (340 nm/38O nm) of three independent experiments. 11] responses by acting as Ca2+ channel blockers, [Ca2+], measurements were performed in HPB—ALL cells treated with SR1 or SR2 (1.0-5.0 uM) for 300 s followed by treatment with TG (1 11M). Under control conditions, TG induced a rapid elevation in [Ca2+], (348 i 53 nM, n=3), which was significantly reduced in magnitude in the absence of [Ca2+]e, demonstrating that the elevation of [Ca2+], by T6 was largely due to the influx of [Ca2+]e (data not shown). Pretreatment of cells with either SR1 or SR2 did not attenuate the TG- induced elevation of [Ca2+]; indicating that the cannabinoid receptor antagonists do not non-specifically block [Cay]e influx through Ca2+ channels (data not shown). Finally, [Ca2+]; measurements were also performed in HPB-ALL cells with SR2 in the absence of [Ca2+]e to investigate whether the small and modest rise in [Ca2+]; elicited by Ag-THC was attributable to a CB2 receptor-dependent mechanism. HPB-ALL cells were pretreated with SR2 (5 uM) for 300 s followed by Ag-THC (10 uM) in the absence of [Ca2+]... Interestingly, pretreatment with SR2 completely and significantly attenuated the small rise in [Ca2+], elicited by Ag-THC in the absence of [Ca2+]c (figure 29). D. PTx does not attenuate the A9-THC-mediated rise in [Ca2+]; Both C81 and CB2 receptors belong to the G protein-coupled receptor superfamily and have been demonstrated to couple to the PTx-sensitive G proteins, Geri/Get0 (Bouaboula et al., 1999; Glass and Northup, 1999). To investigate whether the Ag-THC-mediated increase in [Ca2+], occurred in a Gui/Gao-dependent manner, HPB- ALL cells were preincubated with PTx (100 ng/ml) or VH for 18 h to allow for a full ADP-ribosylation. Subsequently, cells were used for [Ca2+], measurements with Ag-THC 112 0.020.098 000002.000. 03. 000002.00 0000.0 +~00 2.... AS: owQE: 00m. $00 02.. 0. .0002. .0 0.000 0... 0. 00902.0 00 00.00002. 00 00wq0:0 £500. .0 com. .0 .08. 0 .8. 0200020 003 5.000. 0. 0000000. 0... .000 030.50 2.. 0.0. 00.00.... 003 9.0.“. $08 :> s 02: c: 0050 a. 8m 2. .0020 :08 :> no .21 .0 ~00 .200 5.3 0380 20 0582332: .000. 038.03 s coco 0:. 503 0000. 80-000 5 8050302 a; 0:8 0.2.0.5 8.02 we... .o 8:30 .8 m < 0:8 1.1.5.... 0. “.700“: .0 02.0000 2.. a. 03..-? .3 02.2.0 £300. 0. 00.3.5.0 .0000:— 0... 003003.00 $5 .3 0.5»... 0000300 .0. 0E_._. 010% e 80. 80. 80 8.. o r _ . h . F . < _ . a 0150 + 000 W. :> + N. 0 . o u..- .. ...;....:...._......W._:.1......uif .__._ ,..._,._...;_....1. .;. 4:01;...3 .2...‘53...._..u3.;jfimw.r: 143.30.... 03...}... 01.002.2i20 r O F) I> + :> 4 W. 0 . . _ U 015% + I> - W Go 8 0 U M I O. N 113 (10 uM). Preincubation with PTx, however, did not attenuate the Ag-THC-mediated increase in [Ca2+]. (figure 30). E. HU-210 and CBN elevate [Ca2+]i Given the observation that the tricyclic classical cannabinoid, Ag-THC, robustly elevated [Ca2+]; in T cells, but the bicyclic cannabinoid CP did not, the effect of classical tricyclic cannabinoids, HU-210 and CBN, were also investigated on [Ca2+], in the HPB- ALL cells. Both HU-210 and CBN elicited a robust and concentration-responsive elevation of [Ca2+]; in the HPB-ALL cells, with substantial elevation of [Ca2+]i at concentrations greater than 10 [1M (figure 31, 32). However, compared to Ag-THC, the magnitude of [Ca2+]i elevation by both HU-210 and CBN at all concentrations (1-20 11M) was modest (figure 31, 32). At a concentration of 20 1.1M, HU210 elicited a [Ca2+]i rise of 200.3 :t 13.9 nM (n=3), and CBN elicited a [Ca2+].- rise of 185.5 :1: 50.4 nM (n=4). Interestingly, the [Ca2+], rise elicited by all three classical tricyclic cannabinoid compounds followed a significant time delay after injection of the cannabinoid into the cuvette. The time delay to onset of [Ca2+], elevation varied depending on concentration and the cannabinoid employed. At a concentration of 20 “M, the time to onset of [Ca2+]i elevation by CBN was 195.2 i 8.0 5 (n=5), whereas the time to onset of [Ca2+], elevation by HU-210 was at 342.1 i 7.6 5 (n=5). By contrast, at a lower concentration, A9-THC (12.5 uM) time to onset of [Ca2+]i elevation was only 123.7 i 4.0 3 (n=8). It is intriguing to note that the time delay to onset of [Ca2+]i elevation by CBN (20 uM) and Ag-THC (12.5 11M) were similar, while HU-210 (20 HM) took significantly longer to induce an [Ca2+]i rise. 114 5.0- I Vehicle E“ 4.01 B PTx O 00 E 3.04 S 0 0 2.0- 35: <1 1.0- VH A9-THC Figure 30. PTx does not attenuate the elevation in [Ca2+]: elicited by A9-THC in HPB-ALL cells. HPB-ALL cells were preincubated with PTx (100 ng/ml) or VH (PBS) for 18 h, then washed and loaded with fura-2. A 3 ml aliquot of fura-2 loaded HPB-ALL cells was resuspended in Cay-KREB and treated with A9-THC (10 uM) or VH (0.1% EtOH) at 300 s, and the increase in [Ca2+], was measured for a total of 1600 3. Results are presented as mean change :1: SEM in the 340 nm/380 nm fluorescence ratio of [Ca2+]i from base to peak of two independent experiments. 115 3:08.098 .:00:0:00:. .50. 3:000:00: 00003 $00 0:... .AE: owQE: 3)... $00 00:. 3 0:03. .0 0.8: 05 :. 00w§0 mm 03:000.: 08 m0w§n0 Azao. .0 com. .0 .53 0 :0. 0050003 00>» A300. :. 00030:. 03 0:0 030.50 0... 3:. 00:00.3. 33 9.0... $0.6. ..> :0 A21 Wm: 0.....-2 5).... cm-.. o.N-D.. .0 0:0..0::00:00 30.?> .0 com .< .w:..:..m 3:30:00 5.3 030.50 0 3:. 0000.: 003 2.00 ...<-m.n... 00000. ~05. .0 .030...N .8 m < 0:00 4.37%.... :. 4:00. H“30.5.0 =.~-D= Am 0.5»... .0. 0E... 2%: com? come cow oov _ o _ _ _ _ . _ — _ 00 f -- r. 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Removal of [Ca2+]. attenuates the rise in [Ca2+]. elicited by HU-210 and CBN The effect of both HU-210 and CBN on [Ca2+]. elevation was further characterized by performing [Ca2+], measurements either in the presence or absence of [Ca2+]e. Analogous to the above observation for A9-THC, the absence of [Ca2+]e severely attenuated the elevation of [Ca2+]; by both CBN and HU-210 (figure 33). Compared to the control experiment in the presence of [Ca2+]e, removal of [Ca2+]e attenuated the magnitude of the rise in [Ca2+]. elicited by CBN and HU-210 by 85—88 % (n=3). Also in the absence of [Ca2+]e, both compounds elicited a very small and slow increase in [Ca2+]i. G. Cannabinoid receptor antagonists attenuate the HU-210- and CBN- mediated rise in [Ca2+]; To address further the mechanism by which HU-210 and CBN elicited the rise in [Ca2+]i, HPB-ALL cells were pretreated with the SR1 or SR2 (1-5 nM), for 300 seconds prior to addition of cannabinoids. Pretreatment with either SR1 or SR2 produced a concentration-responsive attenuation of both the HU-210- as well as the CBN-mediated rise in [Ca2+]; (figure 34). It was interesting to note that both antagonists were differentially sensitive at attenuating the elevation in [Ca2+]. elicited by HU-210 and CBN. Compared to VH control, SR2 (5 uM) attenuated the [Ca2+], rise elicited by CBN by 71.9 i 2.3% (n=3), whereas at the same concentration, SR2 attenuated the HU-ZlO- mediated [Ca2+]. rise by only 29.1 :1: 6.9% (n=4). Similarly, compared to VH control, SR1 (5 uM) attenuated the [Ca2+]. rise elicited by CBN by 70.9 :t 3.4 % (n=3), whereas at 118 2.00 I +[Ce.2+]e T A a _ 2+ E 15. [ca 1" o E o 1.0- v CL 3 0 94 . 4 0.5 a; r/ I VH HU-210 CBN Figure 33. Removal of [Ca2+]. severely abrogates the elevation of [Ca2+]; elicited by HU-210 and CBN in HPB-ALL cells. A 3 ml aliquot of fura-2 loaded HPB-ALL cells was resuspended in either Ca2+-KREB or Ca2+-free KREB buffer just prior to beginning [Ca2+]i measurements. At 300 s, HU-210 (20 11M), CBN (20 0M) or VB (0.1% EtOH) was injected into the cuvette and the increase in [Ca2+]; was measured for a total of 1600 3. Results are presented as mean change 3: SEM in the 340 nm/3 80 nm fluorescence ratio of [Ca2+]i from base to peak of three independent experiments. * p < 0.05 as compared to either HU-210 (+[Ca2+]¢) or CBN (+[Ca2*].) group. 119 Figure 34. SR1 and SR2 antagonize the HU-210- and CBN-mediated elevation in [Ca2+ ], in HPB-ALL cells. A 3 ml aliquot of fura-2 loaded HPB-ALL cells was placed into a cuvette with constant stirring. SR2 (1.0-5.0 uM), SR1 (1.0-5.0 1.1M) or VH (0.1% DMSO) was added directly to the cuvette just prior to beginning [Ca2+],measurements. At 300 s, HU-210 (20 0M; A), CBN (20 11M; B) or VH (0.1% EtOH) was injected into the cuvette and the increase in [Ca2+]i was measured for a total of 1600 s. [Ca2+]i changes are presented as the mean i SEM of the change in base to peak ratio of bound to free Ca2+ (340 nm/380 nm) of three (B) or four (A) independent experiments. * p < 0.05 as compared to either VH/HU-210 (A) or VH/CBN (B) group. 120 0:3 0.2-0:: .0 505. a. 505% 03033.20: 0.3 -200: 20 8.8035 $5 0.3 :5 .00 0.50.... 0.3 m .01 o..- m..L 9N1 9003:2200 8 03.300 ow o.. . . <2::% I 003420. N 0.0.:0> I Zm0 ..> .m. .23 0.223% 8 03.300 o6 o. o.~-Dw. ...> L «it-l \\\\\\\\\\\\V \\ \ 00.23% I ... 03.3% E 0.0.:0> I 9: J». O. v— C O (um 08 S/uru 017$) 01123 v I V? .— I Q N 121 the same concentration, SR1 potently attenuated the HU-ZlO-mediated [Ca2+], rise by 97.6 e 1.1 % (n=3). H. CBD elevates [of]i In addition to CBN and HU-210, the effect of the non-classical bicyclic cannabinoid, CBD, on [Ca2+]; was also assessed in the HPB-ALL cells. Unlike HU-ZlO, CBN and A9-THC, CBD is considered not to be an agonist at either the CB1 or CB2 receptors (Pertwee, 1999a). Nonetheless, treatment of HPB-ALL cells with CBD (1-20 uM) did lead to a small increase in [Ca2+], (figure 35). However, compared to the [Ca2+]. elevation profiles of HU-ZIO, CBN or A9-THC, the [Ca2+]; rise elicited by CBD was very modest, rapid, and not concentration-responsive. In addition, the CBD-induced [Ca2+]. rise occurred in two distinct phases — a rapid first phase and a slower second phase. Biphasic [Ca2+]; elevation has previously been established in lymphocytes to be a characteristic of SOCE (Parekh, 2003). To investigate the possibility that CBD was eliciting a release of stored Ca2+, cells were treated with CBD either in the presence or absence of [Cay]... The results showed that although the first phase of the CBD-induced [Ca2+]; rise was maintained in the absence of [Ca2+]e, the second phase was completely abolished (figure 36). The insensitivity of the first phase to lack of [Ca2+], indicated that CBD elicits a release of stored Ca2+, followed by [Ca2+]c influx. Finally, to ensure that CBD was not elevating [Ca2+]; by acting upon the CBZ receptor, cells were pretreated with SR2 (5 uM) for 300 5 prior to CBD addition. Pretreatment with SR2 did not attenuate the CBD-mediated elevation of [Ca2+]. (figure 37). 122 0303.098 30000003 000:... 300030: 0000.: +~00 0...... A3: 033.: 9%. $00 00:. 3 0:03. .0 0.30.: 0... :. 0300 00 0030003. 00 00:03.0 .7000. .m cow. .0 .033 0 :0. 0030003 003 _500. :. 00000:. 05 0:0 03030 03 3:. 00.00 .E. 00>» 500.”. $08 ..> :0 C21 om-.. Dm0 .0 300030300 0:0.0> .0. com .< .w:..:..m .:00:00 3.3 03030 0 3:. 0000.: 003 0:00 4.0.0... .0000. 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U +m 00 O m T l\ 124 0.5 - I Vehicle 44 E 0.4- E SR1 528 O 00 E 0.30 O 3 v 0.24 O E q 1- 0.1- 7' 0.. VH CBD Figure 37. SR2 does not antagonize the CBD-induced elevation in [Ca2+]; in HPB- ALL cells. A 3 ml aliquot of fura-2 loaded HPB-ALL cells was placed into a cuvette with constant stirring. SR2 (5 uM) or VH (0.1% DMSO) was added directly to the cuvette just prior to beginning [Ca2+]imeasurements. At 300 s, CBD (10 uM) or VH (O. 1% EtOH) was injected into the cuvette and the increase in [Ca2+]; was measured for a total of 1600 s. [Ca2+]i changes are presented as the mean i SEM of the change in base to peak ratio of bound to free Ca2+ (340 nm/3 80 nm) of three independent experiments. 125 I. Effect of other cannabinoids on [Ca2+]i Apart from CBN, HU-ZIO and CBD, WIN-2 and the high-affinity CBZ-selective agonist, JWH-133, were also tested for their ability to induce an influx in [Ca2+]i. The effect of WIN -2 on [Cay].- elevation could not be assessed since this compound interfered with fura-2 [Ca2+]; measurements. On the other hand, J WH-133 did not interfere with [Ca2+].- measurements, but interestingly did not induce an elevation of [Ca2+].- over a range of concentrations (0.1-20 uM) in the HPB-ALL cells (data not shown). In addition, ABA and 2-AG along with the precursor compound, arachidonic acid (AA), were also tested for their effect on [Ca2+]; elevation. Surprisingly, neither the endocannabinoids (1-20 uM) nor AA (1-50 uM) elevated [Ca2+]i in the HPB-ALL cells (data not shown). III. Mechanism of A9-THC-induced [Ca2+]; elevation A. Effect of Ca2+ channel inhibitors on the rise in [Ca2+]i elicited by A9- THC and TG In order to elucidate the identity of Ca2+ channels involved in [Ca2+]i elevation by Ag-THC, [Ca2+]i measurements were performed in HPB-ALL cells pretreated with Ca2+ channel inhibitors, SKF or 2-APB, followed by A9-THC treatment. In addition, [Ca2+]i experiments were also carried out to examine the effects of the Ca2+ channel inhibitors on the [Ca2+]; elevation elicited by TO (1 uM), a widely described activator of SOC current by way of Ca2+ store-depletion (Prakriya and Lewis, 2003; Putney, 2003). Pretreatment of HPB-ALL cells with SKF for 300 5 resulted in a concentration-dependent inhibition of the Ag-THC-mediated elevation in [Ca2+], with substantial inhibition at so uM SKF (figure 38A). On the other hand, SKF only modestly attenuated the TG-induced [Ca2+].- 126 Figure 38. Effect of SKF on the elevation of [Cay]. induced by A9-THC and TG. A 3 ml aliquot of fura-Z loaded HPB-ALL cells was placed into a cuvette with constant stirring. SKF (10-50 uM) or VH (dde) was added directly to the cuvette just prior to beginning [Ca2+],measurements. At 300 s, Ag-THC ( 12.5 M; A), TG (1 uM; B) or VH (0.1% EtOH) was injected into the cuvette and the increase in [Ca2+]iwas measured for a total of 1600 s. [Ca2+]i changes are presented as the mean :t SEM of the change in base to peak ratio of bound to free Ca2+ (340 nm/3 80 nm) of three independent experiments. * p < 0.05 as compared to VH/Ag-THC (A) or VHfI‘G (B) group. 127 ,5, 0m, 2; ' 5‘ M\\\\\\\\\\\\\‘§ . -E m if; ”—2 n E ' '0ng **\\\\\\\\\\\\\\\\\\\\\V 10 VH Al9-THC I o. v I I I O O O m’ 0i —-' (um oss/um ore) 01101 V q o 128 Figure 38. Effect of SKF on the elevation of [Ca2+]i induced by A9-THC and TG. rise at a concentration of 50 M (figure 38B). Conversely, 2-APB potently inhibited TG- induced [Ca2+]; elevation (figure 398) at a concentration of 25 11M, but was a weak inhibitor of the A9-THC-mediated elevation in [Ca2+]; even at a concentration of 100 uM (figure 39A). The contrasting effects of SKF and 2-APB on the Ag-THC and TG-induced rise in [Ca2+]i may be explained by the prior reports demonstrating that 2-APB is an SOCC inhibitor (Vazquez et al., 2003), while SKF is an ROCC inhibitor (Merritt et al., 1990). Additionally, 2-APB may also act non-specifically to inhibit Ca2+ channel signaling intermediates such as PLC in lymphocytic cells (Ma et al., 2003). To address the discrepancy of the contrasting effects of SKF and 2-APB on the A9-THC- and TG-induced elevation in [Ca2+]i, additional studies were conducted with LaCl3. La3+ cation is a potent inhibitor of SOCCs at submicromolar levels, and is effective at inhibiting a TG-induced [Ca2+]t rise (Aussel et al., 1996). In addition, lanthanide compounds have also been reported to inhibit TRPC channels, but at concentrations in the micromolar range (Beech et al., 2003; Trebak et al., 2003b). Presently, the effect of LaCl3 (20-250 uM) was examined on the Ag-THC- and TG- induced [Ca2+]i rise. Pretreatment of cells with LaCl3 markedly inhibited the Ag-THC- mediated elevation of [Ca2+]i, but only at concentrations greater than or equal to 100 11M (figure 40A). By contrast, LaCl3 was potent at attenuating the TG-induced rise in [Ca2+], at a lower concentration of 20 uM (figure 408). In light of the above observation that the ROCC inhibitor, SKF, but not the SOCC inhibitors, 2-APB or LaCl3, potently inhibited the [Cazm rise induced by A9-THC a further set of studies were performed to investigate the putative involvement of ROCCs in the [Ca2+]; rise induced by HU-ZIO and CBN. Cells were treated with 20-50 HM SKF 129 Figure 39. Effect of 2-APB on the elevation of [012*]I induced by A’-THC and TG. A 3 ml aliquot of fura-2 loaded HPB-ALL cells was placed into a cuvette with constant stirring. 2-APB (25-100 uM) or VB (0.1% EtOH) was added directly to the cuvette just prior to beginning [Ca2+]imeasurements. At 300 s, A9-THC (12.5 uM; A), TO (1 M; B) or VH (0.1% EtOH) was injected into the cuvette and the increase in [Caz*]i was measured for a total of 1600 s. [Ca2+]i changes are presented as the mean 1 SEM of the change in base to peak ratio of bound to free Ca2+ (340 urn/380 nm) of three independent experiments. * p < 0.05 as compared to VII/A9-THC (A) or VH/I‘G (B) group. 130 o 0 o G. <9. V M N '— O L L J OE WE: m I—— \ 3 * \ \tn “‘0': TG :St_ 75 2-APB (uM) RR\\\\\\\\\\\\\\\\\\\\\\_ 3 R\\\\\\\\\\\\\\\\\\\\\\\\\\\\\ (mu 08 S/wu 017$) 01193 V 131 Figure 39. Effect of 2-APB on the elevation of [Ca2+]i induced by A9-THC and TG. Figure 40. Effect of LaCl3 on the elevation of [Ca’*]I induced by A9-THC and TG. A 3 ml aliquot of fura-Z loaded HPB-ALL cells in HPSS buffer was placed into a cuvette with constant stirring. LaCl3 (20-250 uM) or VH (ddeO) was added directly to the cuvette just prior to beginning [Ca2+]imeasurements. At 300 s, A9-THC (12.5 “M; A), TO (1 uM; B) or VH (0.1% EtOH) was injected into the cuvette and the increase in [Ca2+], was measured for a total of 1600 s. [Ca2+], changes are presented as the mean :9: SEM of the change in base to peak ratio of bound to free Ca2+ (340 urn/3 80 nm) of three independent experiments. * p < 0.05 as compared to VH/Ag-THC (A) or VH/T G (B) group. 132 .00, 0.; 05-00 E 083:0 .55. .8 50.3% 20 0e «.63 .e .800 .3 950.: O... 9.3903 cm om o... I, _ m 0 o..- o.Nt 9m- 003 B 0.0.:0> I 0.0- ...> .m. 02$ 0.03 cmm _ co. m 0:50 m> — § * rem in W o m.03 E 0.0E0> I c'». <'= c‘z 3:. o. 0- m N .—t o (qu ass/um ove) 0m V l q In I O c. \o 133 for 300 s before treatment with cannabinoids. The results showed SKF did indeed inhibit the rise in [Ca2+]i elicited by both HU-210 and CBN (figure 41A, B). In accord with the antagonist studies with HU-210 and CBN, SKF too had differential effects on the HU- 210- and CBN-induced rise in [Car]. At a concentration of 50 nM, SKF potently inhibited the CBN-induced elevation in [Ca2+]; by 96.7 i 1.2 % (n=3). At the same concentration, SKF was not as efficacious and inhibited the HU-ZIO-mediated rise in [Ca2+]; by only 47.5 :1: 13.6 % (n=3). Another rather unusual effect of SKF pretreatment was that it reduced the time delay to onset of the HU-210-induced [Ca2+]i elevation. Compared to VH control, the time delay to onset of [Ca2+]i elevation for HU-21O (15 uM) in the presence of SKF (50 11M) was reduced by 165.3 0 1.2 5 (n=3; data not shown). B. Ag-THC-mediated elevation in [Ca2+]: is not abolished upon TG or 8- Br—cADPR pretreatment The experiments outlined above suggested that the A9-THC-mediated elevation in [Ca2+].- was dependent on SKF-sensitive ROCCs, but was independent of SOCCs. To eliminate the possibility that A9-THC was releasing stored Ca2+ pools and to examine the effect of Ca2+ store-depletion on the Ag-THC-mediated [Ca2+]i rise, [Ca2+]; measurements were performed with sequential addition of TG, followed by A9-THC. In these studies, cells were first treated with TG (1 uM) at 300 s, and followed by addition of either TG (1 nM), or A9-THC (12.5 11M) at 1200 s. As seen in figure 42, an initial addition of TO to the cells led to a rapid and robust increase in [Ca2+]g, indicating an influx of [Ca2+]e following depletion of stored intracellular Ca2+, and the subsequent addition of TG did 134 Figure 41. Effect of SKF on the elevation of [Ca2+]. induced by HU-210 and CBN. A 3 ml aliquot of fura-2 loaded HPB-ALL cells was placed into a cuvette with constant stirring. SKF (20-50 11M) or VH (dde) was added directly to the cuvette just prior to beginning [Ca2*],measurements. At 300 s, HU-210 (20 M; A), CBN (20 M; B) or VH (0.1% EtOH) was injected into the cuvette and the increase in [Ca2+],was measured for a total of 1600 s. [Ca2+]i changes are presented as the mean :1: SEM of the change in base to peak ratio of bound to free Ca2+ (340 urn/3 80 nm) of three independent experiments. * p < 0.05 as compared to VH/HU-ZIO (A) or VH/CBN (B) group. 135 od- QT WT 9N: .25 E: ESE E 332156.... 82.3% 2.. .3 55 S .35 .3. 9...»:— 225 $5 om om ZmU .NNw. _ * w H “Emmy o_oEo> I A2355 _ +\_ \\ . 3 m m s & m 2 * w -3 m .m 0.0M” .< rod (mu 08 e/uru 0179 011821 136 not lead to a further increase in [Ca2+],. In contrast, if cells were initially treated with TG to deplete stored Ca2+ and then treated with A9-THC, the addition of Ag-THC led to a further elevation of [Ca2+], over the TG-induced [Ca2+], rise (figure 42). It has previously been reported that there are two intracellular Ca2+ stores: the IP3- sensitive and -insensitive Ca2+ pools, which are depleted by TG; and a ”PG-insensitive RyR-gated Ca2+ pool depleted by cADPR (Guse et al., 1995). To ascertain that Ag-THC did not elicit the increase in [Ca2+].- following depletion of cADPR-sensitive Ca2+ pools, HPB-ALL cells were pretreated with 8-Br-cADPR (20-75 11M), a cell-permeant antagonist of the RyR, for 300 s followed by Ag-THC. The resulting Aq-THC-mediated [Ca2+], increase was not attenuated by 8-Br-cADPR treatment even at concentrations as high as 75 uM (figure 43). C. OAG elevates [Ca2+]i in HPB-ALL cells independently of PKC In light of the observation that A9-THC-mediated elevation in [Ca2+], was independent of store-depletion and was sensitive to high concentrations of LaCl3, the hypothesis that Ag-THC-induced elevation in [Ca2+], was mediated through ROCCs in the TRPC subfamily was tested. Recently, various groups have concluded that TRPC channels may operate either as ROCCs or SOCCs (Beech et al., 2003; Philipp et al., 2003; Singh et al., 2000; Trebak et al., 2003b; Vazquez et al., 2003). Moreover, analogs of DAG have been found to activate TRPCl, 3, 6 and 7 (Beech et al., 2003; Gamberucci et al., 2002; Hofmann et al., 1999; Lintschinger et al., 2000; Trebak et al., 2003b). In the present investigation, presence of DAG-gated channels in HPB-ALL cells was confirmed by treatment with OAG. Treatment of HPB-ALL cells with increasing concentrations of 137 .mEoEtogxo E02335 ooh: E8032 moon: +~mU 2E. .98 owQEc ova +~w0 out 8 953 we 2:2 05 E mowqfio mm Biomoa 2m mowSEu gang .m comm mo 38 a do.“ 3532: 33 5%2 a 3855 ofi .m 82 a :21 2: 05% 5 221 s E 5.3 8:8? 2.83. a 3 85:8 £23 2: 2.: 38.3 83 foam $2: m> 8 A21 3 OH .m com E .chE #5328 55 8325 a 9E 3039 we? £8 Aldermmm powwo— méhc .«o 8:33 is m < .__+~a0_ 5 5595—0 coo—6.: 2:94. of Snack? .2. 83. UP .53 «no—5.3.50.5 .9 9.59% OIPa®< E as: a 9 2 8mm 88. 82 L 88 com _ o _ _ r r _ _1 I> e e + I> P 7 L r» LLLLLLL a... -o.m cum .2... r L . - or 9 .. W. .‘ .... 3 4 _ 1 \.C,lv . _ . 1k I IV a... 1 0 015% + ..._ m . -o.© U 8 O .H- a + 0% u a - M -3: 138 4.01 I Vehicle T 1- ’ T E2» Z é Z a a a % / % I 8-Br-cADPR (uM) Figure 43. Pretreatment with 8-Br-cADPR does not abrogate the Ag-THC induced elevation in [Ca2+].. A 3 ml aliquot of fura-Z loaded HPB-ALL cells was placed into a cuvette with constant stirring. 8-Br-cADPR (20-75 uM) or VH (ddH20) was added directly to the cuvette just prior to beginning [Ca2+]i measurements. At 300 s, Ag-THC (12.5 uM) or VH (0.1% EtOH) was injected into the cuvette and the increase in [Ca2+], was measured for a total of 1600 s. [Ca2+], changes are presented as the mean i SEM of the change in base to peak ratio of bound to free Ca2+ (340 nm/380 nm) of two independent experiments. 139 OAG (50-300 uM) resulted in a rapid elevation of [Ca2+], (figure 44), which was, however, smaller in magnitude when compared to the Ag-THC-mediated rise of [Ca2+]i. Next, the hypothesis that Ag-THC and OAG both putatively induced [Ca2+], elevations through the same TRPC channel was examined in HPB-ALL cells. Cells were sequentially treated with OAG (300 11M) for 600 s, followed by either a second addition of OAG (300 nM) or Ag-THC (12.5 HM) at 1200 s. As expected, treatment of cells initially with OAG rapidly elevated [Ca2+],. However, as can be gleaned from figure 45, the subsequent addition of OAG did not further elevate [Ca2+]i. Interestingly, in cells initially treated with OAG, a subsequent addition of A9-THC also failed to elicit a further elevation in [Ca2+]i. Apart from gating several members of the TRPC channel subfamily, OAG is a well-known activator of PKC. To eliminate the possibility that OAG inhibits a subsequent A9-THC-mediated elevation in [Ca2+], by activating PKC, cells were preincubated with VH or PMA (500 nM) for 20 h. Treatment of cells with high concentrations of PMA for extended periods of time has been demonstrated to downregulate PKC (Bordin et al., 2003; Driessens et al., 1997). However, downregulation of PKC did not affect the ability of OAG pretreatment to block the subsequent elevation in [Ca2+]; elicited by Ag-THC (figure 46A, B), suggesting that OAG prevents the elevation of [Ca2+]i by Ag-THC independently of PKC. Nevertheless, PKC may still be involved in the negative regulation of the TRPC channels activated A9-THC. HPB-ALL cells treated with PMA (20-80 nM) for 300 s, followed by Ag-THC showed a modest, yet significant and concentration-responsive, attenuation of the resulting elevation in [Ca2+]i (figure 47). The observation that PKC may negatively regulate TRPC 140 .mEoEtomxo EoccoQuUE 025 :53th moon: 350 at. A8: owQE: ovO +Nm0 8cm 8 9509 mo 03% 2: E mowfiso mm voucomoa 3a muwfifio 5%“; .m coc— mo 38 a c8 3532: no? Hana E 0322: 05 98 Stan E cows—mu 52:3 8825 05 85 88...? 83 50am $md -_ .8 I> 8 A21 oomémv O 3 21 om N I 9 n7. 2: u -3 w W I. 1 com 8 . 4 r 0 fit I u 21 03...... (w\ l O. CO 141 .mucoEtoaxo 302533: 025 Homage mount +~mU BE. AS: owQE: owmv +~wo 02% 8 953 we can“ 05 E mama—Eu mm Bianca 0.8 mama—SB Hana .m 83 mo 38 a 8m @2388 83 5%8 E 09385 BC. .m 82 an 63.—Had 8.“ EOE £16 mO .8 Oz: m.~C 03.704 .321 80 O s 93 83 9.6 .m com a. has; 2588 as, 3.33 a as 88.3 as 2.8 fi< -mm: @350- Néua no “056:“ :b m < .__+~aU_ 5 9mm.— fioflfl-ounvflhnaaw 2: magma—can U+I> _. -21 i- : , - - - _. H oEdfog - m... ...: w -..+H.w....tr:...uss.fll. ....J..l.: \0) .. + 9.0 8 .7 9.0 + :> .3. M m 8 8 u 0 0:51. > u (w -3 I42 Figure 46. Downregulation of PKC does not affect the abrogation the A9-THC- elicited rise in [Ca2+]. by OAG. HPB-ALL cells were preincubated with VH (0.05% DMSO; A) or PMA (500 nM; B) for 20 h to downregulate PKC. The cells were then loaded with fura-2 and used for [Ca2”]i measurements. At 300 s, OAG (300 nM) or VH (0.3% EtOH) was injected into the cuvette without dilution, followed by a second addition of OAG (300 nM), A9-THC (12.5 M) or VH (0.3% EtOH for OAG; 0.1% EtOH for Ag-THC) at 1200 s. The increase in [Ca2+]iwas measured for a total of 1800 s. [Ca2+]i changes are presented as changes in the ratio of bound to free Ca“ (340 nm/380 nm). The Ca“ traces represent two independent experiments. 143 .35 3 f5. 5 3.: 33965-2 2.. nexus...“ 2.. .85 .2. 8% SE a. 5:232:35 .3 953... OIhum< 96 82 Amv 95... 82 8m 96 o P _ _ a _ o... e e z>+_._> V . o+I> _ , _:.,."...._V...,.§;_.,..,..__‘.: me I\ 29. 20 _ if. a if; _ I; \ . - < i . . E m 56 _ s 0920 #8 o <5.N 144 EM)- 7T I E - % 1"- Em / / / / PMA (nM) Figure 47. Pretreatment with PMA modestly attenuates the Ag-THC induced elevation in [Ca2+]i. A 3 ml aliquot of fura-2 loaded HPB-ALL cells was placed into a cuvette with constant stirring. PMA (20-80 nM) or VH (0.1% DMSO) was added directly to the cuvette just prior to beginning [Ca2+]i measurements. At 300 s, A9-THC (12.5 nM) or VH (0.1% EtOH) was injected into the cuvette and the increase in [Ca2+]i was measured for a total of 1600 s. [Ca2+]i changes are presented as the mean :t SEM of the change in base to peak ratio of bound to free Ca2+ (340 urn/380 nm) of three independent experiments. * p < 0.05 as compared to VH/Ag-THC group. 145 channels activated by Ag-THC is consistent with previous reports which have shown that PKC inhibits several TRPC channels (Trebak et al., 2003a; Venkatachalam et al., 2004). Finally, the effect of A9-THC on PKC activation was also assessed. It has been previously determined that several cannabinoids, including A9-THC, independently activate Ca2+-dependent isoforms of PKC in a murine brain preparation (Hillard and Auchampach, 1994). Here, the effect of Ag-THC on PKC activity was measured using a PKC substrate activity assay. HPB-ALL cells were treated with varying concentrations of Ag-THC (1-15 nM), VH, or PMA (80 nM) as a positive control, for 15 min. Cells were then harvested, lysed and assayed for PKC activity in the whole cell fraction. The results clearly showed that whereas the PMA treatment increased the activity of PKC, A9- THC treatment over a wide concentration range did not result in an increase in PKC activity over VH (figure 48). D. A9-THC-induced elevation of [Ca2+]. is independent of CaMKII To examine whether the elevation in [Ca2+], by Ag-THC was dependent on CaMKII, [Ca2+]; measurements were performed with the CaMKII inhibitor, KN-93, or the negative control analog, KN-92. Cells were pretreated with KN-93 or KN-92 (1-10 11M) for 300 s, followed by Ag-THC (figure 49). Interestingly, the elevation of [Ca2+]i by Ag-THC was inhibited by both compounds in a concentration-dependent manner. KN-93 was slightly more potent at inhibiting the Ag-THC-mediated rise of [Ca2+]; than was KN- 92, suggesting putatively that A9-THC treatment resulted in CaMKII activation. Therefore, the effect of A9-THC on CaMKII activity was measured using a CaMKII substrate activity assay. HPB-ALL cells were treated with Ag-THC (12.5 11M) or VH and 146 80- -125 I PKC Activity Control I E0 -100 E 60— e? E — 75 3‘ 40— .9 .3 - 50 O < U 20‘ if — 25 0- D - 0 NA VH 1.0 6.25 12.5 15.0 91’ 96° A9-THC (nM) $59 $0 ‘3‘” Figure 48. Ag-THC treatment does not result in the activation of PKC. HPB-ALL cells were left untreated (NA) or treated with VH (0.1% EtOH) or various concentrations of Ag-THC (1-15 11M), or PMA (80 nM) as a positive control for 15 min. Cells were then harvested, lysed and PKC phosphotransferase activity was determined in the whole cell fraction. Assay reactions were performed with 10 uCi [y—32P]-ATP for 10 min at 30°C. The assay included two negative controls performed in the absence of protein sample (no enz) or in the absence of substrate (no sub). Reactions were spotted on phosphocellulose filter papers and assayed for 32P using a scintillation counter. The results are presented as pmol of [y—32P]-ATP incorporated per min per mg of protein added. The results are representative of two independent experiments. ND indicates none detected. 147 5.0- . Vehicle A 4.0- E KN-92 E KN-93 o T E 3.0- * O 5’. :3; 2.0- Q <1 1.0- a: , * f 7 / / 00 . 4% d . , f l VH A9-THC 1.0 5.0 10.0 KN-92 or KN-93 (nM) Figure 49. Pretreatment with KN-93 and KN-92 attenuates the Ag-THC-induced elevation in [Ca2+]i. A 3 ml aliquot of fura-Z loaded HPB-ALL cells was placed into a cuvette with constant stirring. KN-93 (1-10 uM), KN-92 (1-10 nM) or VH (0.1% DMSO) was added directly to the cuvette just prior to beginning [Ca2+]imeasurements. At ‘300 s, A9-THC (12.5 nM) or VH (0.1% EtOH) was injected into the cuvette and the increase in [Ca2+]i was measured for a total of 1600 s. [Ca2+]ichanges are presented as the mean :1: SEM of the change in base to peak ratio of bound to free Ca2+ (340 nm/3 80 nm) of three independent experiments. * p < 0.05 as compared to VH/ Ag-THC group. 148 harvested at a variety of time points (0.5-30 min). The cells were lysed and assayed for CaMKII activity in the soluble fraction. The assay included a positive control, in which an aliquot of the naive protein sample was preincubated with Ca2+/CaM (1 mM/50 [lg/ml). The results clearly showed that whereas the Ca2+/CaM treatment strongly increased the activity of CaMKII, A9-THC treatment did not result in an increase in CaMKII activity over a period of 30 min (figure 50). The activation of CaMKII by A9-THC was also examined by Western analysis. The CaMKII family of enzymes is unique in that upon CaZ+/CaM binding, a high level of autonomous kinase activity is induced as a result of autophosphorylation on threonine 286 (Bui et al., 2000). Presently, HPB-ALL cells were treated with VH or A9-THC (10 nM) for varying periods of time (1-60 min). Cells were harvested, lysed, and CaMKII activation was determined in the soluble fraction using Western analysis. CaMKII activation was detected using an anti-CaMKIIme antibody. To account for loading discrepancies, the blot was further incubated with an anti-B-actin antibody. The results showed that treatment with A9-THC did not lead to an increase in the autophosphorylation of CaMKII, as compared to the corresponding VH control and as normalized with a B-actin loading control (figure 51). E. Ag-THC-induced elevation of [Ca2+]i is independent of PLC, PI3K and soluble tyrosine kinases The activation of TRPC channels has been reported previously to be dependent on the activation of several different enzymes including PLC, PI3K and src family tyrosine kinases (Trebak et al., 2003b). To examine whether the A9-THC-induced [Ca2+]i rise 149 200 _ I Vehicle ' 300 Ag-THC bl kl! O l ._. O O l CaMKH Activity (pmol/min/mg) NA 0.5 1.0 2.0 5.0 15.0 30.0 Time(min) 2911’5‘3300“ Figure 50. Ag-THC treatment does not result in the activation of CaMKII. HPB- ALL cells were left untreated (NA) or treated with VH (0.1% EtOH) or Ag-THC (12.5 nM) for various periods of time (0.5-30 min). Cells were then harvested, lysed and CaMKII phosphotransferase activity was determined in the soluble fraction. The assay reactions were performed with 10 uCi [y-3ZP]-ATP for 10 min at 30°C. The assay included two negative controls performed in the absence of protein sample (-enz) or in the absence of substrate (—sub); and a positive control in the presence of Ca2+/CaM (l mM/SO jig/ml). Reactions were spotted on phosphocellulose filter papers and assayed for 32P using a scintillation counter. The results are presented as pmol of [y-SZP]-ATP incorporated per min per mg of protein added. The results are representative of three independent experiments. ND indicates none detected. 150 Figure 51. A9-THC treatment does not result in the autophosphorylation of CaMKII. HPB-ALL cells were left untreated (NA) or treated with VH (0.1% EtOH) or Ag-THC (12.5 nM) for various periods of time (5-60 min). Cells were then harvested, lysed and CaMKII autophosphorylation was determined by Western analysis using an anti-CaMKIIpT286 antibody (A). In addition, blots were also probed with an anti-B-actin antibody as a loading control (B). The optical density of the resulting bands was quantified using a densitometer and graphed as the optical density of CaMKIIpT286 normalized by B-actin (C). The results are representative of three independent experiments. 151 5min I 10 min L 30 min L 60 min l l l NA v T v T v T v T Lag CaMKIIpT286 antibody ”:1. "'1' “h “'5 “" ‘“ “.m A. .- - B-actin antibody B. 2.0 - C. I Vehicle EH A9-THC 1.54 Optlcal DenSIty (pCaMKII/B-actin) § .9 u: 1 .0 o p NA 5 min 10 min 30 min 60 min Time (min) Figure 51. A9-THC treatment does not result in the autophosphorylation of CaMKII. 152 was dependent on the activation of PLC, PI3K or src family tyrosine kinases, inhibitors of each were used in [CaZ+]i measurements. HPB-ALL cells were pretreated with Et-18- OCH3, LY294002, PP2 (1-20 uM) and/or VH for 300 s, to inhibit PLC, PI3K or src family tyrosine kinases respectively, followed by A9-THC addition. It was found that the A9-THC-induced [Ca2+]i was not inhibited upon pretreatment with either Et-18-OCH3, LY294002 or PP2 suggesting that Ag-THC elevated [Ca2+]i independently of the aforementioned kinases (figure 52A, B). F. HPB-ALL cells express transcripts for TRPC] Results from studies of the effect of OAG on [Ca2+]i suggested that both OAG and Ag-THC functioned through a common mechanism putatively involving TRPC channels. In order to elucidate the putative channel involved in the Ag—THC-mediated [Ca2+]i elevation, RT-PCR was performed for TRPCl-7 in the HPB-ALL cells and the Jurkat E6- 1 cells, as a comparison control. RT-PCR results demonstrated that whereas Jurkat E6-l cells expressed transcripts for TRPCl, 3 and 6, the HPB-ALL cells expressed transcripts only for TRPCl (figure 53). Interestingly, in addition to the 686 bp predicted TRPCl amplicon, RT-PCR results demonstrated the presence of a second smaller amplicon in both the HPB-ALL and Jurkat E6-1 cells. The smaller TRPC] amplicon was sequenced from both HPB-ALL and Jurkat E6-1 cells using TRPC] forward and reverse primers. Sequencing confirmed that the smaller 532 bp product was an alternative splice variant of TRPCl, which is spliced between nucleotides 309-463 within the TRPC] coding sequence (data not shown). However, splicing of the 154 bp from within the TRPC] coding sequence shifts the open reading frame of TRPC] resulting in a premature stop 153 Figure 52. Pretreatment with Et-l8-OCH3, LY294002 and PP2 does not attenuate the A9-THC-induced elevation in [Ca2+].. A 3 ml aliquot of fura-2 loaded HPB-ALL cells was placed into a cuvette with constant stirring. (A) Et-l8-OCH3 (1-20 nM) or VB (0.1% EtOH); or (B) LY294002 (1-20 uM), PP2 (1-20 nM) or VB (0.1% DMSO) was added directly to the cuvette just prior to beginning [Ca2+]imeasurements. At 300 3, A9- THC (12.5 nM) or VB (0.1% EtOH) was injected into the cuvette and the increase in [Ca2+]i was measured for a total of 1600 s. 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(w. 10.0 165 000000 0000000 00000000000000 02.0 0000000000 00000 +000 00:. .9000 00.28: 009 +000 00000 00 000000 .00 0000 0.0 E 00w0000 00 0000000000 000 00w000~0 07.008 .0 002 .00 00000 0 000 00000008 003 07.008 5 0000005 05 000 0000300 05 005 00000.80 003 0005 :0 .8 => 8 02: as 200 .0 80 E 050000 0:588 as, 202,8 0 3.: 0820 as 808 0.030000 03 8 0.800100 520 0000 3008. 0.20 0o 080:“ 0s m < .0000 0.800.000 2: 0.. 0.23.02. 0 200 E .0000. :0 802% 2F .00 2:000 m 9:. com? 83 A V L. com zmo o F _ _ F _ O.—. 0 Eéz> . 1 iii: . .. ..¢ao¢lt!.t.31.,;.,: ». -0Nmo\.1mo I> Ea 200 (wu Des/um ova) ones 0.803.500 200 Io.m 166 cells and HPB-ALL cells, the bicyclic cannabinoid, CP, failed to elicit an elevation in [Ca2+]i in either WT or CBl"’/CBZ"‘ SPLC (data not shown). B. SR1 and SR2 antagonize the tricyclic cannabinoid-mediated elevation of [Ca2+]i in the CBl"'/CB2"‘ SPLC To pursue the hypothesis that SR1 and SR2 antagonize the tricyclic cannabinoid- induced [Ca2+]i elevation in non-CB1 non-CBZ mediated manner, further [Ca2+]i measurements were performed in WT and CB1"’/CB2"' SPLC. SPLC were treated with SR1, SR2 (1-5 nM) or VH for 300 s, followed by A9-THC (12.5 uM), HU-210 (20 nM), or CBN (20 uM). Similar to the prior observations in the HPB-ALL cells, the results in the WT and CBl'I'lCBZ'/' SPLC showed that SR1 and SR2 were differentially sensitive at antagonizing the [Ca2+], rise induced by different tricyclic cannabinoids. While the A9- THC-mediated [Ca2+], rise was sensitive to both SR1 and SR2 (figure 60), the HU-210- induced [Ca2+], rise could be antagonized only by SR1, but not SR2, in both WT and CBl"'/CBZ'/' SPLC (figure 61). Also, while SR1 was equally sensitive at antagonizing the HU-ZlO-mediated [Ca2+], elevation in both models (figure 61), the Ag-THC-induced [Ca2+], rise was slightly more sensitive to SR1 than SR2 in the CBl'/'/CB2'/' SPLC (figure 60). More interestingly, both SR1 and SR2 equally and significantly inhibited the CBN- induced [Ca2+]i elevation only in the CB1"'/CBZ"’ SPLC, but not the WT SPLC (figure 62). C. Binding of [3HI-SR1 to the CB1"'/CBZ"' SPLC 167 Figure 60. The elevation in [Ca2”]I by A’-THC in CB1"'/CB2"' and WT SPLC is antagonized by SR1 and SR2. A 3 ml aliquot of fura-Z loaded SPLC from WT (A) or CBl"'/CB2"' (B) C57BL/6J mice was placed into a cuvette with constant stirring. SR1 (1-5 uM), SR2 (1-5 nM) or VH (0.1% DMSO) was added directly to the cuvette just prior to beginning [Ca2+]imeasurements. At 300 s, Ag-THC (12.5 nM) or VB (0.1% EtOH) was injected into the cuvette and the increase in [Ca2+], was measured for a total of 1600 s. [Ca2+], changes are presented as the mean :9: SEM of the change in base to peak ratio of bound to free Ca2+ (340 urn/380 nm) of three independent experiments. * p < 0.05 as compared to the respective VH/A9-THC group. 168 o0: oNi ed. 904 023 0003.000 5 0.22300 023 0003.000 8 $02200 o6 Q~ .__ m: 0.0003000 .0 USP. 0 05-00 00> 0.0 0.0 — fl $022.00 N 0003000 I 000.0; 00 00> _ .000 0.3 000 S 808035 00 0000 0.800.100 0.8 0.3 :0 0000.-.? 0.0 0:000. :0 5026.0 20.0 .8 2:000 H34 I O- O. " to V c'>. N (um oat/um 017g) aim l ‘9 «a I ‘9 ‘- l '9 ‘1) M9 Figure 61. The elevation in [Cafi]. by HU-210 in CB1”’/CB2”’ and WT SPLC is antagonized by SR1, but not SR2. A 3 ml aliquot of fura-2 loaded SPLC from WT (A) or CB l"’/CB2"' (B) C57BL/6J mice was placed into a cuvette with constant stirring. SR1 (1'5 HM), SR2 (1-5 nM) or VH (0.1% DMSO) was added directly to the cuvette just Prior to beginning [Ca2+],measurements. At 300 s, HU-ZlO (20 nM) or VH (0.1% DMSO) was injected into the cuvette and the increase in [Ca2+],was measured for a total of 1600 s. [Ca2+], changes are presented as the mean 1 SEM of the change in base to peak ratio of bound to free Ca2+ (340 urn/380 nm) of three independent experiments. * p < 0.05 as compared to the respective VH/HU-ZIO group. 170 .000 .2. 0.5 .000 3 0.305055 0 0000 0.803.000 0:: 0.3 :0 000-000 E 0.300. 5 500.3% 2:. 00 2:000 (WI1 08 Wu 0175) 01101 v 023 0003000 023 0003000 .8 0.002200 00 $02300 0.0 0.0 000-000 m> 0.0 000.00 :> . 0 0 . 0.0 NH 1 md H.” -2 - 0.0 -00 0.~i E «.0530 .00 0.? N 0003000 i 0.0 .\.NmU\.\.— m0 .m I 0_0E0> E .< er im.m 171 Figure 62. The elevation in [Cali]I by CBN is antagonized by SR1 and SR2 in the CBl"’/CB2‘" SPLC, but not WT SPLC. A 3 ml aliquot of fura-2 loaded SPLC from WT (A) or CB1"‘/CB2”' (B) C57BL/6J mice was placed into a cuvette with constant stirring. SR1 (1-5 uM), SR2 (1-5 nM) or VH (0.1% DMSO) was added directly to the cuvette just prior to beginning [Ca2+],measurements. At 300 s, CBN (20 nM) or VH (0.1% EtOH) was injected into the cuvette and the increase in [Ca2+], was measured for a total of 1600 s. [Ca2+]i changes are presented as the mean :t SEM of the change in base to peak ratio of bound to free Ca2+ (340 nm/3 80 nm) of three independent experiments. * p < 0.05 as compared to the respective VH/CBN group. 172 .0000 0.3 .2. 0.5 .0000 000000.000 2.. :0 ~00 0.5 000 0.0 805055 00 200 E 0:000. :0 .3003... 2:. .00 2:000 0210 0003000 905 0003000 8 $5200 8.022.100 .0 3 200 00> . _ _ 0.0 O o.m A: 750 E L l C. v—t (11111 08 s/wu 017$) 01101 V *- §‘\\\ E 0.002200 ’— l O N E 0003.000 t\tNmU\t\t fl mo .m .r I 2038/ E .< 0.? to...” 173 The observation that both SR1 and SR2 attenuate the tricyclic cannabinoid-induced [Ca2+]; elevation, albeit with differential sensitivities, suggested that the tricyclic cannabinoids and the cannabinoid receptor antagonists may exert their effects on [Ca2+], by binding to a novel cannabinoid receptor. To test the above hypothesis, radioligand binding assays were performed using [3H]-SR1 in WT and CBl'/‘/CBZ'/' SPLC. SPLC isolated from WT and CBl'/'/CBZ'/' spleens were treated with various concentrations of [3H]-SR1 (0.5—8O nM) and allowed to bind for 60 min at 30°C either in the presence (non-specific binding) or absence (total binding) of unlabeled SR1 (10 nM) as a competitor. The resulting total and non-specific isotherms of WT and CBl'/'/CBZ'/' SPLC are shown in figure 63A and 63B. The amount of non-specific binding of [3H]- SR1 in WT and CB1'/'/CB2"' SPLC was high and closely overlapped total binding. Therefore, the difference between total and non-specific binding, i.e. specific binding, was negligible. The specific binding isotherms of [3H]-SR1 for WT and CBl"'/CBZ"' SPLC are shown in figure 64 and 65, respectively. The specific binding of [3H]-SR1 to WT and CBl'/'/CBZ'/' SPLC was very modest, and failed to reach saturability even at a concentration of 80 nM. 174 Figure 63. Total and non-specific binding of [3H]-SR1 to WT and CBl"'/CB2"' SPLC. SPLC isolated from WT (A) and CB1"‘/CB2"’ (B) C57BL/6J mice were treated with various concentrations of [3H]-SR1 (0.5-80 nM). The SPLC were allowed to bind [3H]-SR1 for 60 min at 30°C either in the presence (for non-specific binding) or absence (for total binding) of unlabeled SR1 (10 11M) as a competitor. Reactions were filtered through glass fiber filters and assayed for 3H using a scintillation counter. The resulting total and non-specific binding isotherms were fitted to a linear regression and graphed as bound [3H]-SR1 vs. free [3H]—SR1 (unbound - bound). 175 1:33: - Total binding 1200- A Non-Specific binding 1 100- :1 000‘ 900- 3; 800- 700% 600- 500- 400- 300- mo Bound A. WT O 10 20 30 40 50 60 70 80 Free [3H]-SR141716A (HM) 2250' I Total Binding 2000- A Non-Specific Binding 1750- ‘ 1 500- 1 250- 1 000- Bound(flnon I 3 " O 3 l J g 4 A B. CBN/032"- 10 20 30 40 50 60 70 Free [3H]-SR141716A (nM) O C Figure 63. Total and non-specific binding of PHI-SR1 to WT and CBl"' /CB2"‘ SPLC. 176 Figure 64. Specific binding of [3H]-SR1 to WT SPLC. The non-specific binding of [3H]-SR1 (in the presence of 10 uM unlabeled SR1) was subtracted from the total binding of [3H]-SR1 to yield the specific binding in WT SPLC. The resulting specific binding isotherm was fitted to a non-linear regression and graphed as specific binding of [3H]- SR1 vs. free [3H]-SR1 (unbound - bound). To estimate the Ema, (total number of binding sites) and Kd (binding affinity), Scatchard analysis was performed. The resulting Scatchard plot is also shown embedded within the specific binding isotherm. 177 .0000 03 8 000-000 0.. 0:0—Es 200300 .00 2:000 $5 502300-020 oeu. om on 00 on 00 om ON 0.. cost 05050 oEoo00 8 s o... a a e e ears/puma 0_0>.0c< 000:0000m T .80 8.00 u .20 .2: 00.00 H av. 178 Figure 65. Specific binding of [3H]-SR1 to CB1”'/CB2"' SPLC. The non-specific binding of [3H]-SR1 (in the presence of 10 uM unlabeled SR1) was subtracted from the total binding of [3H]-SR1 to yield the specific binding in CBl"‘/CB2”' SPLC. The resulting binding isotherm was fitted to a non-linear regression and graphed as specific binding of [3H]-SR1 vs. free [3H]-SR1 (unbound - bound). To estimate the BM, (total number of binding sites) and Kd (binding affinity), Scatchard analysis was performed. The resulting Scatchard plot is also shown embedded within the specific binding isotherm. 179 .0000 .+80\+80 3 80-000 0o 0:00.03 #00800 .00 2:000 $5 $5300.01; 8.0 am 0 e a... a... 0 e 0... cost 05050 oEoo00 00.0 03 000 2.00 2.: 0 l 8 .62. . m m 0.. m .00N .. on 0_0>_0:< 0005000..” I .000 u .000 .95 0.000 ".20 .2: 08.0 "ex teem (lowl) Bumwa ollloeds 180 DISCUSSION The immunomodulatory effects of cannabinoids have been widely established (Berdyshev, 2000; Condie et al., 1996; Klein et al., 1998; Newton et al., 1998; Schatz et al., 1993). Ag-THC, the most extensively characterized cannabinoid, exhibits a broad range of immunomodulatory activity including direct effects on T cell function as evidenced by altered mitogen-induced cell proliferation, suppressed accessory cell function in T cell-dependent antibody responses, and altered production of several T cell- derived cytokines (Condie et al., 1996; Klein et al., 2000a; Newton et al., 1998; Schatz et al., 1993). Although widely studied, the specific mechanism responsible for altered T cell function by cannabinoids remains poorly understood. Therefore, the overall objective of the present dissertation project was to examine the role of the cannabinoid receptors and [Ca2+], in the mechanism of altered T cell function by cannabinoid compounds. I. Effect of A9-THC and CP on IL-2 expression Work from several laboratories has demonstrated previously that treatment of activated T cells with cannabinoids results in the suppression of IL-2 production and secretion (Berdyshev, 2000; Condie et al., 1996; Klein et al., 2000a; Ouyang and Kaminski, 1999). Based on previous reports showing that HPB-ALL cells express normal sized CBZ transcripts, whereas the Jurkat E6-1 cells express aberrantly sized and putatively non-functional CBZ transcripts, the present set of studies aimed to elucidate the effect of cannabinoids on IL-2 secretion in both HPB-ALL and Jurkat E6-1 cells. In 181 particular, it was hypothesized that due to putative non-functionality of the CBZ receptor in the Jurkat E6-1 cells, cannabinoids would differentially modulate IL-2 expression in the two cell lines. In accord with the aforementioned hypothesis, A9-THC suppressed IL- 2 secretion only in the HPB-ALL cells and had no effect on IL-2 secretion in the Jurkat E6-1 cells. By contrast, the observation that CP, a high-affinity ligand at the C82 receptor, did not differentially modulate IL-2 secretion suggested that the mechanism of IL-2 suppression by A9-THC was likely independent of any differences in the CB2 receptor between the two cell lines. In fact, sequencing data confirmed that there was 100% homology in the CB2 coding sequence between the HPB-ALL and Jurkat E6-1 cells. Perfect homology of the CB2 coding region in the two cell lines essentially eliminated the possibility that the functional difference between HPB-ALL and Jurkat 136-] cells in their sensitivity to A9-THC could arise as a result of mutations or alternative splicing of CB2. Despite the early indications that the suppression of IL-2 by Ag-THC and CP was likely independent of the C82 receptor, IL-2 studies were performed to assess the effects of cannabinoid receptor antagonists. It was found that neither SR1 nor SR2 antagonized the decrease in IL-2 secretion by Ag-THC or CP. The lack of sensitivity of A9-THC- and CP-mediated suppression of IL-2 secretion to either cannabinoid receptor antagonist again indicated that both cannabinoids function via non-CB1 non-C82 mediated mechanisms. The failure of PTx to attenuate the suppression of IL-2 secretion by A9- THC further supported the non-involvement of the CB] and CBZ receptors in the suppression of IL-2 by cannabinoids. 182 Altogether several important insights can be gleaned from the studies of the effect of cannabinoids on IL-2 expression in HPB-ALL and Jurkat E6-1 cells. First of all, it is apparent that there is some functional difference between the HPB-ALL and Jurkat E6-1 cells in their sensitivity to A9-THC, but not CP. The present observation that the Jurkat E6-l cells are insensitive to A9-THC is not unique, and is consistent with a previous report showing that A9-THC failed to inhibit the forskolin-induced cAMP production (Schatz et al., 1997). More importantly, it is also clear that the differential effects of A9- THC on IL-2 secretion in HPB-ALL and Jurkat E6-1 cells cannot be attributed to variations in the CB2 receptor or the associated G proteins. Finally, the observation that Ag-THC, but not CP, had differential effects on IL-2 secretion in HPB-ALL and Jurkat E6-l cells indicates that the two cannabinoids exert their effect on T cells via divergent mechanisms of action, neither of which involve the C82 receptor. 11. Effect of A9-THC and CP on [Ca2+], in T cells Recent studies investigating the effects of cannabinoids on T cell activation and IL-2 gene expression have identified alterations in NF AT DNA binding and transcriptional activity (Jan et al., 2002; Ouyang et al., 1998; Yea et al., 2000). The regulation of NFAT in T cells is critically dependent on a sustained elevation in [Ca2+]i induced upon T cell activation (Rao et al., 1997). The critical role for [Ca2+], in T cell activation coupled with previous findings that cannabinoid treatment results in disrupted activation of NFAT prompted a comprehensive investigation of the effects of A9-THC and CP on [Ca2+], in resting T cells. An initial characterization showed that Ag-THC produced a robust and concentration-responsive rise in [Ca2+], in purified murine splenic 183 T cells and HPB-ALL cells, but only a modest rise in [Ca2+];in Jurkat E6-1 cells. Moreover, the high affinity cannabinoid agonist, CP, failed to elicit a rise in [Ca2+], in all three cell models utilized. It was interesting to note that CP also failed to act as a neutral antagonist on the [Ca2+], rise elicited by A9-THC. Together, the failure of CP both to induce an independent [Ca2+]i elevation and to act as a neutral antagonist on the A9-THC- induced [Ca2+]i elevation indicated that the mechanism by which Ag-THC elevates [Ca2+]i was likely independent of the CB2 receptor. Nevertheless, [Cay].- measurements were performed with A9-THC in the presence or absence of SR] and SR2 in the splenic T cells, HPB-ALL cells and Jurkat E6-1 cells. The ability of SR1 and SR2 to antagonize the elevation of [Ca2+], elicited by Ag-THC in the splenic T cells and the HPB-ALL cells was surprising given the high affinity cannabinoid ligand, CP, did not elicit a [Ca2+], rise in either model. Even more surprising was the finding that SR1 and SR2 did not significantly attenuate the small [Ca2+]; rise elicited by Ag-THC in the Jurkat E6-1 cells. At first glance the inability of SR1 and SR2 to antagonize the [Ca2+]i rise elicited by A9-THC in the Jurkat E6-1 cells appears to implicate the involvement of the cannabinoid receptors in the Ag-THC-mediated [Ca2+]i rise. However, it must also be noted that antagonism by SR1 and SR2 does not necessarily imply the involvement of CB] or CB2. Both SR1 and SR2 at concentrations greater than 1 uM may have effects on receptors other than C81 and CB2 (Pertwee, 1999b, 2005). Moreover, it was observed that both SR1 and SR2 inhibited the rise in [Ca2+].- by A9-THC in the HPB-ALL cells, despite the fact that the CB] receptor is not expressed by HPB-ALL cells. Lastly, the elevation of [Ca2+]: elicited by Ag-THC could not be attenuated upon the inactivation of G proteins known to couple the cannabinoid 184 receptors. Together, the aforementioned studies indicate that the effect of Ag-THC on [Ca2+], is very likely independent of both CB1 and CBZ receptors. A salient point that arises out of the studies of [Ca2+]; elevation by A9-THC and CP is that there may be no functional link between the rise in [Ca2+], by cannabinoids and the suppression of lL-2, as initially conjectured. Evidence for the lack of functional link between the two aforementioned effects comes from the observation that while only A9- THC elevated [Ca2+]i, both Ag-THC and CP suppressed IL-2 secretion in the HPB-ALL cells (table 3, 4). If [Ca2+], elevation was critical to the mechanism of IL-2 suppression, CP would not suppress IL-Z secretion. Moreover, the sensitivity of the Ag-THC-mediated [Ca2+]i rise to both SR1 and SR2 further severs any possible connection between the elevation of [Ca2+], and the suppression of IL-2, as the decrease in IL-2 by both A9-THC and CP was insensitive to either SR1 or SR2 (table 3, 4). The present data argue, therefore, that there are at least two modes of action for cannabinoids in T cells: one leading to the suppression of IL—2 and another leading to the elevation of [Ca2+], (figure 66). Additionally, it is apparent that only certain cannabinoids are able to function through the latter mode. All successive studies with 119-THC and other cannabinoids were performed in an attempt to characterize the second mode of action, i.e. the mechanism of [Ca2+]i elevation. 111. Effect of [Ca2+]. removal and BAPTA-AM on the A9-THC induced [Ca2+]i elevation In an effort to elucidate the manner in which Ag-THC robustly elevates [Ca2+], in the splenic T cells and HPB-ALL cells, [Ca2+], measurements were performed either in 185 Table 3. Summary of the effects of cannabinoid agonists on IL-2 secretion and [Ca2+], in various cell models used _A_gonist Response Measured Cell Model Effect PMA/Io-stimulated IL-2 secretion HPB-ALL cells Suppression Jurkat E6-l cells No effect Splenic T cells Elevation Ag-THC HPB-ALL cells Elevation [C32+]i Jurkat E6-l cells Elevation WT SPLC Elevation CBl"‘/CBZ"’ SPLC Elevation PMA/Io-stimulated IL-2 secretion HPB-ALL cells Suppression Jurkat E6-1 cells Suppression Splenic T cells No effect CP HPB-ALL cells No effect [C32+]i Jurkat E6-1cells No effect WT SPLC No effect CBl"'/CB2"’ SPLC No effect HPB-ALL cells Elevation CBN [Ca2+]. WT SPLC Elevation CBl"'/CB2'/' SPLC Elevation HPB-ALL cells Elevation HU-ZIO [Ca2+]. WT SPLC Elevation CBl"‘/CBZ"' SPLC Elevation AEA [Ca2+]; HPB-ALL cells No effect 2-AG [Ca2+]. HPB—ALL cells No effect JWH-l33 [CF]. HPB-ALL cells No effect CBD [Ca2+], HPB-ALL cells Small elevation 186 Table 4. Summary of the effects of antagonists on cannabinoid-induced suppression of IL-2 secretion and [Ca2+]i elevation in various cell models used Antagmist Response Measured Cell Model Effect E-THC-induced IL-2 suppression HPB-ALL cells No effect CP-induced IL-2 suppression HPB-ALL cells No effect Jurkat E6-1 cells No effect Splenic T cells Inhibition HPB-ALL cells Inhibition A9-THC-induced [Ca2+]i elevation Jurkat E6-1 cells No effect WT SPLC Inhibition SR144523 CEl"'/Cl32"' SPLC Inhibition HPB-ALL cells Inhibition HU-ZlO-induced [Ca2+]; elevation WT SPLC Inhibition CBl"'/CB2"' SPLC Inhibition HPB-ALL cells Inhibition CBN-induced [Ca2+]i elevation WT SPLC Inhibition CBl"'/CBZ"' SPLC Inhibition CBD-induced [Ca2+]t elevation HPB-ALL cells No effect A9-THC-induced IL-2 suppression HPB-ALL cells No effect CP-induced IL-2 suppression HPB-ALL cells No effect Jurkat E6-l cells No effect Splenic T cells Inhibition HPB-ALL cells Inhibition A9-THC-induced [Ca2+]; elevation Jurkat 136-] cells No effect SR141716A WT SPLC Inhibition CBl"'/CBZ"' SPLC Inhibition HPB-ALL cells Inhibition HU-210-induced [Ca2+], elevation WT SPLC Inhibition CBl"’/CBZ"' SPLC Inhibition HPB-ALL cells Inhibition CBN-induced [Ca2+]i elevation WT SPLC Inhibition CB1"'/CB2"' SPLC Inhibition 187 Figure 66. Putative model of cannabinoid-mediated [Caz“]I regulation and suppression of IL-2 expression. Based on the findings in the present dissertation, a model by which tricyclic cannabinoids regulate [Ca2+]i is proposed. CBx represents a novel cannabinoid receptor at which tricyclic cannabinoids are agonists and SR1 and SR2 are neutral antagonists. Prot X represents a putative protein that may form a functional link between CB, and TRPCl. Some of the possible effects that may result due to a premature elevation in [Ca2+], in T cells are also listed. In addition, the model also depicts the severing of any direct link between elevation of [Ca2+], and suppression of IL-2 expression. The current model does not illustrate a mechanism by which cannabinoids suppress IL-2 expression, as both the receptor and the precise downstream pathways involved remain elusive. 188 i=3 nus—«Tam: 2: E 003035.:— Nt‘: .00 0389.003 can 0060—03.. 4300— eoufluofitEofiaaanau .00 .2002 .3 ensure cozmzcfiod 07.902? 0239.98 NH: >993 0o :o_mmm._aq:m cor—grow =00 ._. :o muootm 0 g )\4 x095 /v> . 93 V00 my _ ‘ w \J _ A 96 IV / \, / Nam mEoEnmccmu Nam "020030203 Em __< Em 2.2.2: 189 the absence of [Ca2+].3 or in the presence of BAPTA-AM. In both splenic T cells and the HPB-ALL cells, the removal of [Ca2+]. potently abrogated the Aq-THC-mediated [Ca2+]; rise, leaving behind a small and delayed [Ca2+]i rise. Interestingly, SR2 completely attenuated the small [Cay]; rise elicited by A9-THC in the absence of [Ca2+]e, suggesting the rise in [Ca2+]t was an influx of residual [Ca2+]... Furthermore, the characteristics of the delayed [Ca2+]; rise elicited by Ag-THC in the absence of [Ca2+]. were incongruous with Ca2+-store release-mediated Ca2+ entry in lymphocytes (Lewis, 2001). In addition, the observation that A9-THC-induced a [Ca2+]t rise in BAPTA-AM loaded cells also suggested that the mechanism of Ag-THC-mediated [Ca2+]; elevation was independent of Ca2+ stores. The significantly smaller elevation in [Cay]; elicited by Ag-THC in BAPTA- AM loaded cells signifies Ca2+ entry from the extracellular space, which is buffered upon entry into the cytosol by free BAPTA-AM. Together, the studies of Ag-THC in the absence of [Ca2+]... and the presence of BAPTA-AM strongly allude to the understanding that the A9-THC-mediated elevation of [Ca2+]i occurs entirely through [Ca2+]. influx, and . . + 15 Independent of Ca2 -store release. IV. Effect of other cannabinoids on [Ca2+]t Given the above observation that the tricyclic cannabinoid, Ag-THC, but not the bicyclic cannabinoid, CP, elevated [Ca2+]t, additional studies were performed to test the hypothesis that only tricyclic cannabinoids elicit an [Ca2+]i elevation in T cells. It was found that treatment of HPB-ALL cells with HU-210 and CBN, classical tricyclic cannabinoids with structural similarity to A9-THC, resulted in a [Ca2+]; elevation. There were several similarities between the [Ca2+]; elevations elicited by A9-THC, HU-210 and 190 CBN. First, the elevation of [Ca2+]i by all three cannabinoids was concentration- responsive. Second, the removal of [Ca2+]c strongly abrogated the [Cap] rise elicited by HU-210 and CBN to a similar degree to that observed with Ag-THC. Next, the [Ca2+]i elevation elicited by Ag-THC, HU-ZlO and CBN occurred after a significant time delay to onset. Finally, the [Ca2+]i rise elicited by the three cannabinoids was sensitive to antagonism by SR1 and SR2. It is intriguing that Ag-THC, HU-210 and CBN all elevate [Ca2+]i in an SRl- and SR2-sensitive manner in the HPB-ALL cells, especially since the HPB-ALL cells do not express the CBI receptor. The ability of SR1 to attenuate [Ca2+]i elevation indicates that both SR1 and SR2 antagonize some yet unknown cannabinoid receptor in T cells. The inability of CP, AEA, 2-AG and the CB2-selective agonist, JWH-l33, to elevate [Ca2+]t in the HPB-ALL cells further implies that the rise in [Ca2+]t elicited by tricyclic cannabinoids is mediated by a novel cannabinoid receptor. Despite the apparent similarities between the profile of [Ca2+]; elevation of A9- THC, HU-ZlO and CBN, many differences also exist. First and foremost, the time delay to onset of [Ca2+]i elevation for HU-210 was significantly longer than for either CBN or A9-THC at all concentrations used. The significance of the varying time delays to onset of [Ca2+]i rise is presently unclear, but it is speculated that the time to onset may represent a signaling delay needed for gating a cell-surface Ca2+ channel. The significantly longer delay to onset for HU-210 in comparison with CBN or A9—THC may also suggest that HU-210 is less efficacious at coupling with an effector critical for the induction of a [Ca2+]; rise. It is interesting and important to note, however, that HU-ZIO has not previously been reported to be less efficacious at either CB1 or CB2 as compared with any other cannabinoid. Another factor differentiating the [Ca2+]g responses of HU-ZlO 191 and CBN from that elicited by Ag-THC was the differential sensitivity to SR1 and SR2. While the Ag-THC and CBN-induced [Ca2+]g elevation was attenuated by both antagonists with similar sensitivities, the HU-ZlO-mediated [Ca2+]i elevation was more sensitive to SR1 than to SR2. It is currently not known why the SR1 and SR2 are differentially sensitive in antagonizing the [Ca2+]i elevation elicited by the aforementioned cannabinoids. It is tempting to speculate, given the [Ca2+]t responses of A9-THC, CBN and HU- 210, that only cannabinoid compounds possessing tricyclic structures with a central pyran ring can elicit a rise in [Ca2+]i in T cells. The inability of CP, ABA and 2-AG to elevate [Ca2+]i in HPB-ALL cells support the aforementioned hypothesis. However, it must also be noted that existence of a tricyclic structure and a pyran ring are, by themselves, insufficient to induce a [Ca2+]i elevation, as established with the tricyclic cannabinoid, JWH-l33. Currently, it is postulated that only cannabinoids that possess A9-THC-like three ring structures are able to robustly increase [Ca2+]i. Clearly, the modifications to the aliphatic chain and the additional functional groups on the mono-unsaturated cyclohexyl ring present on HU-ZlO did not produce an increased efficacy to elevate [Ca2+]i, over A9-THC. Moreover, it is also apparent that there are other non-SRl/SRZ- sensitive mechanisms of [Ca2+]i elevation in T cells. CBD, which is neither an agonist for the cannabinoid receptors nor a tricyclic cannabinoid, modestly elevated [Ca2+]i, but through a mechanism distinct from the tricyclic cannabinoids. Compared to the tricyclic cannabinoids, the magnitude of the CBD-induced elevation of [Ca2+]i was very modest. In addition, the CBD-induced [Ca2+]i rise was not concentration-responsive, rapid and biphasic, involving both Ca2+ store-release and [Ca2+]e influx phases. Evidently, further 192 in-depth studies are necessary to understand the complex structure-activity of cannabinoid compounds on [Ca2+]; elevation in T cells. V. Effect of Ca2+ channel inhibitors and store-depletion on the A9-THC-induced [Ca2+]t elevation The primary mechanism of Ca2+ influx in T cells is by SOCE and is mediated by the CRAC channels (Lewis, 2001). However, preliminary studies with cannabinoids found that the mechanism of [Ca2+]; entry was independent of Ca2+ store-depletion. Therefore, additional studies were performed to investigate the mechanism of [Ca2+]; elevation by Ag-THC in the HPB-ALL cells. The first set of studies aimed at elucidating the class of Ca2+ channels involved in the elevation by A9-THC. To gain additional insight and as a comparison, studies utilizing Ca2+ channel inhibitors were performed side-by-side with TG and Ag-THC. Interestingly, while SKF proved to be a potent inhibitor of the A9-THC-mediated elevation in [Ca2+]i, SKF only modestly inhibited the TG-induced [Ca2+]i rise. Conversely, 2-APB potently inhibited the TG-induced [Ca2+]i elevation, but was rather weak at inhibiting the Ag-THC-mediated [Ca2+]ielevation. Together, the results from the Ca2+ channel inhibitor studies suggested that whereas TG elicits an [Ca2+]i rise through the SOCCs, A9-THC likely utilizes the ROCCs. To address further the contrasting effects of SKF and 2-APB on the Ag-THC- and TG-induced [Ca2+]; elevation, studies were performed with LaCl3. While generally, lanthanide cations are considered to be SOCC inhibitors, they are also known to inhibit TRPC channels at high concentrations (Aussel et al., 1996; Beech et al., 2003; Trebak et al., 2003b). Presently, the finding that LaCl3 markedly inhibited the TG-induced rise in 193 [Ca2+]i at low concentrations, and the Ag-THC-mediated elevation of [Ca2+]t at higher concentrations, confirmed that TG functions through SOCCs and that A9-THC utilizes the ROCCs, putatively from within the TRPC subfamily. Having observed that the A9-THC-mediated [Ca2+]i elevation was sensitive to SKF, the effect of SKF on HU-210- and CBN-induced [Ca2+]; elevation was also assessed. SKF significantly inhibited the HU-210— and CBN-mediated elevation in [Ca2+]i suggesting again that all three tricyclic cannabinoids activate ROCCs. Once again, the HU-ZlO-induced rise in [Ca2+]t was differentially sensitive to SKF than that induced by A9-THC or CBN. While SKF potently inhibited A9-THC- and CBN-mediated [Ca2+]i rise, SKF was only partially inhibitory on the HU-210-mediated rise in [Ca2+]i even at high concentrations. It is unclear why the HU-ZlO-induced [Ca2+]i rise was less sensitive to treatment with SKF, but the reason may be related to the previous observation that HU-210 is less efficacious at eliciting a [Ca2+]i rise then either A9-THC or CBN. Nevertheless, experiments with SKF illustrate that the tricyclic cannabinoids elicit a rise in [Ca2+]i, at least partially, through ROC channels in T cells. Apart from the effect of Ca2+ channel inhibitors, the effect Ca2+ store-depletion was also assessed on the Ag-THC-mediated [Ca2+]; rise. Store-depletion was induced with TG and followed by A9-THC treatment. The results showed that A9-THC elicited a [Ca2+]i rise that was additive with the store-depletion induced [Ca2+]e influx. The additive nature of the Ag-THC- and TG-induced [Ca2+]t rise strongly argues that the effects of Ag-THC and TG on [Ca2+]i were mutually exclusive and unrelated. A corollary of the aforementioned result is that A9-THC treatment does not lead to releases of TG- sensitive intracellular Ca2+ pools. Moreover, the lack of inhibition of the A9-THC- 194 induced increase in [Ca2+]; upon pretreatment with 8—Br-cADPR also argues against the release of any non-TG-sensitive intracellular Ca2+ pools by Ag-THC. Recent studies investigating the store-dependent and -independent mechanisms of Ca2+ entry in non-excitable cells have focused on the TRP channel superfamily, and specifically on the TRPC channel subfamily. Various groups have conjectured that TRPC channels may operate either as ROCCs or SOCCs (Beech et al., 2003; Mori et al., 2002; Philipp et al., 2003; Singh et al., 2000; Sydorenko et al., 2003; Trebak et al., 2003b; Vazquez et al., 2003). Consistent with the results from the present studies, several reports have demonstrated that TRPC channels are sensitive to high concentrations of both lanthanide compounds, as well as 2-APB (Beech et al., 2003; Trebak et al., 2002; Trebak et al., 2003b). Along with the aforementioned reports, the results from the current investigation demonstrating the partial sensitivity of A9-THC- and TG-induced [Ca2+]i elevation to SOC and ROC inhibitors, respectively, may suggest that the signaling mechanisms regulating SOC and ROC entry may indeed converge onto the same channel. Nevertheless, it remains to be fully resolved whether the SOCCs and ROCCs are distinct proteins, or the same channels regulated by alternate signaling mechanisms. VI. Effect of TRPC channel activators and inhibitors on the A9-THC-induced [Ca2+]; elevation In light of the finding that A9-THC-mediated elevation in [Ca2+]i was independent of store-depletion and sensitive to SKF and high concentrations of LaCl3, the involvement of TRPC channels therein was examined. Recently, analogs of DAG have 195 been demonstrated to activate four members of the TRPC subfamily, namely TRPCl, 3, 6 and 7 independently of PKC (Beech et al., 2003; Gamberucci et al., 2002; Hofmann et al., 1999; Lintschinger et al., 2000; Trebak et al., 2003b). Presently, the rapid and robust elevation in [Ca2+]i produced by OAG confirmed the presence of DAG-gated channels in HPB-ALL cells. In addition, the non-additivity of OAG and Ag-THC upon sequential addition implied that both compounds function through a common TRPC channel. Furthermore, the possibility that OAG inhibited a subsequent A9-THC-mediated [Ca2+]t rise by activating PKC was eliminated by the lack of abrogation of the non-additivity of OAG and A9-THC under conditions PKC downregulation. The non-involvement of PKC in the OAG-induced [Ca2+]i rise is consistent with a prior report demonstrating that the activation of TRPC3 by OAG was independent of PKC (Trebak et al., 2003a). Nonetheless, the TRPC channel activated by A9-THC is clearly sensitive to PKC activation, as short-term activation of PKC with PMA modestly attenuated the Ag-THC- induced [Ca2+]i elevation. In sum, it is evident that non-additivity of OAG and Ag-THC does not result due to the activation of PKC, but very likely implies that both compounds function through the same TRPC channel (figure 66). Utilizing enzyme inhibitors, the involvement of various enzymes in the elevation of [Ca2+]i by A9-THC was examined. [Ca2+]i measurements performed with Et-18-OCH3, PP2, LY294002, KN-93 and KN-92 showed that only the CaMKII inhibitors, KN-93 and KN-92, were effective at attenuating the A9-THC-induced [Ca2+]; increase. These results implied that while PLC, PIBK and src family tyrosine kinases were not involved in the regulation of the TRPC channels by A9-THC, CaMKII was putatively involved. Although intriguing, CaMKII has not previously been implicated in the activation or 196 inhibition of TRPC channels. Upon examination of CaMKII activity utilizing both an activation assay as well as Western analysis, it was found that Ag-THC treatment does not result in CaMKII autophosphorylation or activation. It is proposed that the sensitivity of the A9-THC-induced [Ca2+]; increase to both KN-93 and KN-92 implies that CaM, and not CaMKII, is involved in the regulation of TRPC channels activated by Ag-THC. The KN compounds were originally developed to inhibit CaMKII by way of competing for the CaM binding domain on the enzyme (Sumi et al., 1991). While CaMKII is not implicated in TRPC channel function, CaM has been reported to bind and negatively regulate all members of the TRPC subfamily (Vennekens et al., 2002). Therefore, it is proposed that KN-93 and KN-92 competitively associate with the CaM binding domains within the TRPC channels and thereby inhibit the A9-THC-induced [Ca2+]: increase. Collectively, studies of TRPC channel activators and inhibitors provide pharmacological evidence that the HPB-ALL cells express ROCCs in the TRPC subfamily. The TRPC channels activated by Ag-THC are gated by OAG in a manner independent of PKC. PLC, PI3K and src family tyrosine kinases, enzymes previously implicated in the regulation of TRPC channels, are clearly uninvolved in the mechanism of A9-THC-induced [Ca2+]i elevation. Nevertheless, the non-additivity of OAG- and A9- THC-induced [Ca2+]i elevations, coupled with the inhibitory effects upon PKC activation or treatment with KN -93 and KN-92 strongly implicate the involvement of ROC channels in the TRPC subfamily in the [Ca2+]; rise mediated by Ag-THC (figure 66). VII. Involvement of TRPC] channels in the Ag-THC-induced [Ca2+]. elevation 197 With strong pharmacological evidence that the Ag-THC-induced [Ca2+]; elevation was mediated by a member of the TRPC subfamily, RT-PCR was performed in the HPB- ALL and Jurkat E6-1 cells for TRPC1-7. RT-PCR analysis for TRPC subfamily genes showed that while Jurkat E6-1 cells express transcripts for TRPCl, 3 and 6; HPB-ALL cells express transcripts only for TRPCl. The endogenous expression of TRPCl in the absence of other TRPC subfamily members was, in itself, somewhat surprising because TRPC] is generally thought to function in tandem with other TRPC proteins (Beech et al., 2003). Further, siRNA-mediated knockdown of TRPC] demonstrated that both the expression of TRPCl mRNA and the A9-THC-mediated rise in [Ca2+]i were attenuated. These results established a clear role for TRPCl in the A9-THC-mediated rise in [Ca2+].- in the HPB-ALL cells. However, the above knockdown results do not eliminate the possibility that other proteins are involved in the elevation of [Ca2+]; by Ag-THC because transfection of siRNA against TRPCl did not result in attenuation of mRNA expression or [Ca2+]; rise by more than 50%. Regardless, the sole expression of TRPCl in HPB- ALL cells coupled with the TRPCl knockdown studies strongly indicate that the A9- THC-induced elevation of [Ca2+]; is mediated, at least in part, by TRPCl functioning as a ROC channel. Interestingly, whereas in the HPB-ALL cells A9-THC-induced elevation of [Ca2+],- was clearly dependent on the TRPCl channels, no difference was observed in the [Ca2+]; rise elicited by Ag-THC in the WT or TRPC1"' SPLC. A lack of difference in the [Ca2+],- elevation by Ag-THC in the WT and TRPC1"' SPLC suggested that TRPCl was critical for A9-THC-induced [Ca2+]; elevation only in the HPB-ALL cells, and that this finding did not hold true across different cell models. It is possible that in the absence of TRPC] , 198 Ag-THC—induces an elevation of [Ca2+]; through alternative TRPC proteins. In fact, the expression of several other TRPC subfamily proteins was documented in both the WT and TRPCl'/' SPLC. It is proposed that other TRPC proteins may compensate for TRPCl in the TRPCI"' SPLC. Alternatively, under conditions where several TRPC proteins are expressed, Ag-THC may normally activate one of the other TRPC proteins instead. Taken together, it is apparent that although TRPC] channels are involved in the Ag-THC-induced increase in [Ca2+]t, TRPCl does not fully account for the effects of A9- THC on [Ca2+]t. Several observations support the above conclusion. First, because the siRNA knockdown of TRPC] did not fully attenuate TRPCl mRNA expression or the [Ca2+]; rise by A9-THC, it cannot be unequivocally stated the TRPCl solely mediates the effects of Ag-THC on [Ca2+]i. Second, no difference was observed in the Ag-THC- mediated [Ca2+]; rise between the WT and TRPCl"' SPLC. Finally, in Jurkat E6-1 cells, which express TRPCl, 3 and 6, the magnitude of [Ca2+]; elevation by Ag-THC was modest in comparison with the HPB-ALL cells. Therefore, the overall conclusion from the studies of the mechanism of [Ca2+]; elevation by A9-THC is that OAG-sensitive TRPCl channels are at least partially responsible for the A9-THC-induced [Ca2+]; elevation in the HPB-ALL cells. In other cell models, however, other TRPC channels or alternative mechanisms of TRPC channel regulation may be involved. VIII. Effect of tricyclic cannabinoids and the cannabinoid antagonists on [Ca2+]i in the cart/car" SPLC 199 Preliminary experiments in the human and murine T cells with various cannabinoids and cannabinoid receptor antagonists provided circumstantial evidence to suggest that the effects of the tricyclic cannabinoids on [Ca2+]i, although sensitive to SR1 and SR2, may not be mediated by either CB1 or CB2. Therefore, the present set of studies using SPLC from WT or CBl'l'lCBZ'l' mice aimed to elucidate whether the [Ca2+]; elevation induced by Ag-THC, HU-210 and CBN occurred independently of CB1 and CB2. The finding that the Ag-THC-, HU-210— and CBN-induced [Ca2+]; elevation was not significantly different between the WT and CB1"'/CB2"' SPLC provided conclusive evidence that the classical tricyclic cannabinoids function independently of both cannabinoid receptors to elicit [Ca2+]i. Furthermore, studies of A9-THC-, HU-210- and CBN-induced [Ca2+]i elevation with either SR1 or SR2 in the WT and CB1"'/CB2'/' SPLC showed that the cannabinoid receptor antagonists can antagonize the [Ca2+]; response, even in the absence of CB1 and CB2. The results from the [Ca2+]; measurements with SR1 and SR2 provided a clear and unequivocal indication both SR1 and SR2 antagonize the cannabinoid-induced [Ca2+], elevation via action at sites distinct from CB1 and CB2. Several broad insights can be gained from the [Ca2+]; studies performed with the tricyclic cannabinoid compounds and the cannabinoid receptor antagonists in the WT and CEl"'/CE2"' SPLC. First and foremost, A9-THC, HU-210, CBN, SR1 and SR2 all have activity at sites distinct and unrelated to CB1 or CB2. Second, antagonism of cannabinoid responses with SR1 and SR2 does not necessarily imply dependence on CB1 or CB2 receptors. Although SR1 and SR2 have been illustrated previously to exert effects on non-cannabinoid receptors, the present dissertation work provides the first such 200 report in lymphocytes. Next, the parallel observations in the WT and CB1'/'/CB2"' SPLC as well as the HPB-ALL cells showing that the tricyclic cannabinoid-mediated [Ca2+]i elevation is differentially sensitive to SR1 and SR2 may imply that the different classical tricyclic cannabinoids have varying affinities, efficacies or mechanisms of action at the novel cannabinoid receptor. Another insight gained from the WT and CBl'l'lCBZ'l' SPLC, coupled with previous investigation in the HPB-ALL cells, is that CP, ABA and 2- AG, the non-tricyclic cannabinoid agonists, likely do not have activity at the novel cannabinoid binding site in T cells. The final extrapolation from the studies of [Ca2+], in the CB1'/'/CB2'/' SPLC is that any difference in the sensitivity to cannabinoids, or in the [Ca2+]; rise elicited by A9-THC in the HPB-ALL and Jurkat E6-l cells cannot be attributed to the CB2 receptor. In short, the present results strongly suggest that A9-THC and structurally-related tricyclic cannabinoids mediate their action on [Ca2+]i via non- CBl non-CB2 receptors in T cells, and that SR1 and SR2 can act as neutral antagonists at the novel cannabinoid receptors (figure 66). With the assumption that SR1 and SR2 can act as neutral antagonists at the novel cannabinoid receptor, radioligand binding analysis was performed with [3H]-SR1 in WT and CBl'/'/CB2"’ SPLC. Unfortunately, [3H]-SR1 failed to exhibit significant specific and saturable binding in either WT or CBl'/'/CB2'/' SPLC. The low amount of specific binding of [3H]-SR1 is attributed to the high degree of non-specific binding of [3H]-SR1 in both WT and CBl"'/CB2'/' SPLC. The failure of [3H]-SR1 to display the characteristics of a specific ligand for the novel cannabinoid receptor may also be attributed to the high lipophilicity of [3H]-SR1, high [3H]-SR1 concentrations required to elicit binding, and relatively low density of cannabinoid receptor expression in SPLC. 201 Collectively, the present dissertation work supports the conclusion that A9-THC and related tricyclic classical cannabinoids function through a novel cannabinoid binding site, distinct from CB1 and CB2, to elicit a robust influx of [Ca2+].3 in T cells. However, the identity of the new cannabinoid receptor in T cells remains to be resolved. IX. Significance and relevance The significance of the work presented in the current dissertation project to the broader field of cannabinoid biology is multifold. The present project demonstrates systematically that tricyclic cannabinoids, and A9-THC in particular, elicit an elevation in [Ca2+]; in T cells through [Ca2+]. influx in a manner independent of store-release, which is, at least partially, dependent on TRPCl. Several novel and unique findings are implicit in the above-mentioned demonstration. First, the finding that Ag-THC and other tricyclic cannabinoids can elevate [Ca2+]; independently of Ca2+ stores in T cells is surprising, where the primary mechanism of [Ca2+]i elevation involves SOCE (Lewis, 2001). Although non-SOCE-mediated [Ca2+]t elevation has been previously reported in lymphocytes (Gamberucci et al., 2002), the present studies are the first to demonstrate that cannabinoid compounds may elevate [Ca2+]; through a non-SOCE mechanism. In addition to providing evidence that tricyclic cannabinoids elevate [Ca2+].- independent of SOCE, the present dissertation also offers valuable insights into one putative mechanism of receptor-operated Ca2+ entry in T cells. A second significant finding in the present dissertation with regard to cannabinoid biology is the demonstration of the structure-activity relationship of cannabinoids and [Ca2+]i elevation in T cells. The current studies are the first to show that tricyclic 202 cannabinoids have effects on [Ca2+]; that cannot be reproduced with non-tricyclic cannabinoids. With consideration to cannabinoid biology, the differential effects of tricyclic and non-tricyclic cannabinoids on T cells imply that a uniform mechanism of action cannot be applied to all cannabinoid compounds. Work presented in the current project suggests that cannabinoid compounds of differing structures act via divergent mechanisms to elicit their effects on lymphocytes. The divergent mechanisms of action may converge to elicit the same end result, as seen with IL-2 suppression by A9-THC and CP in HPB-ALL cells, or alternatively may be completely independent of one another, as demonstrated by the effects of Ag-THC and CP on [Ca2+]i. The implications of the finding that different cannabinoids may act via divergent mechanisms are far-reaching particularly because many of the early studies with cannabinoid compounds presupposed that all cannabinoids act via the same mode of action. The present project shows indisputably that all cannabinoids do not act alike, and that the initial assumption of a uniform mechanism of action for structurally diverse cannabinoids may have to be reexamined. Another central finding presented in the current work is that the tricyclic cannabinoid-induced [Ca2+].- increase was independent of either CB1 or CB2, the two known and widely-studied cannabinoid receptors. The existence of a structure-activity relationship of cannabinoids on [Ca2+]; coupled with the ability of tricyclic cannabinoids to elicit a [Ca2+]; elevation in the CBl'/'/CB2’/' mouse SPLC strongly suggests that receptors other than CB1 and CB2 are present in lymphocytes which mediate the actions of cannabinoids. Studies in other organ systems have already suggested the existence of receptors other than CB1 and CB2 (Breivogel et al., 2001; Offertaler et al., 2003; Mo et 203 al., 2004; Wiley and Martin, 2002). Whereas the novel cannabinoid receptors described in the brain and peripheral vasculature were described to be PTx-sensitive and, therefore, G protein-coupled, the insensitivity of the A9-THC-induced [Ca2+], elevation to PTx in the HPB-ALL cells suggests that the novel cannabinoid receptor in T cells is perhaps a non-G protein-coupled receptor. Nevertheless, the current studies support the emerging view in the field of cannabinoid biology that additional unknown cannabinoid receptors mediate cannabinoid effects. The ability of SR1 and SR2 to antagonize the actions of the tricyclic cannabinoids on [Ca2+]; presents a fourth critical finding with implications to the field of cannabinoid biology. The demonstration that both SR1 and SR2 can antagonize the elevation on [Ca2+]; by Ag-THC, CBN and HU-21O at concentrations of 5 lLM or below reveals that the cannabinoid receptor antagonists are not as selective for CB1 or CB2 as originally reported. Moreover, the finding the SR1 and SR2 were able to antagonize the effects of tricyclic cannabinoids in the CB1'/'/CB2"' mouse SPLC further suggests that both antagonists have activity at receptors distinct from CB1 and CB2. Since their discovery SR1 and SR2 have been widely used to demonstrate that cannabinoid compounds elicit their actions through CB1 and/or CB2. Given the present findings, some previously documented cannabinoid effects, particularly in lymphocytes, attributed to CB1 and CB2, solely on the basis of antagonism by l ttM or higher concentrations of SR1 and SR2 respectively, may have to be reevaluated. A final point of significance of the present studies lies in the finding that the A9- THC-induced [Ca2+]; rise in the HPB-ALL cells was mediated by a ROCC in the TRPC subfamily. The regulation of channels in the TRPC subfamily by cannabinoid 204 compounds has not been previously reported. Therefore, the finding that TRPCl is involved in the elevation of [Ca2+].- by Ag-THC is novel. Apart from showing that TRPC] may be regulated by Ag-THC upon binding to an unknown cannabinoid receptor in T cells, the very finding that TRPCl channels may be gated upon ligand-receptor interaction in lymphocytes is also novel. Previous work in other cell systems has shown that TRPCl is a channel gated primarily by Ca” store-release, and that it is expressed in conjunction, and oligomerizes with other TRPC subfamily members, particularly TRPC4 and TRPC5 (Beech et al., 2003). The demonstration that the HPB-ALL cells express only TRPCl and that the Ag-THC-induced [Ca2+]; elevation is dependent on the TRPC] channel also suggests that TRPCl can putatively form a homotetramer, which has not been previously demonstrated in any cell type. Therefore, the work presented in the current dissertation has important implications not only to cannabinoid biology, but also to the field of TRPC channels. Overall, the data and interpretations in the present dissertation project contribute significantly to the current understanding of the fields of cannabinoids, T cell activation and TRPC channels. Therefore, the work presented in the current dissertation is deemed relevant to all three fields. The finding that cannabinoid treatment results in an [Ca2+]; rise independent of activation stimuli in T cells suggests that one mechanism by which cannabinoids interfere with T cell activation is by eliciting a premature [Ca2+]; elevation. Premature elevations in [Cay]; may result in apoptosis of immune cells (Howe et al., 2003). Interestingly, treatment of murine SPLC with cannabinoids, including A9-THC, in the absence of activation stimuli has been previously demonstrated to induce apoptosis (McKallip et al., 2002a; McKallip et al., 2002b). Alternatively, a premature elevation of 205 [Ca2+]; may also induce T cell anergy, a state in which T cells become unresponsive to cellular stimulation (F aubert Kaplan et al., 2003; Harding et al., 1992; Nakayama et al., 1992; Nghiem et al., 1994; Schwartz, 1992). Another point of relevance of the studies presented in the current dissertation is that the tricyclic cannabinoid-mediated [Ca2+]; elevation is a response likely mediated by a non-CB1 non-CBZ cannabinoid receptor in T cells. The above finding is relevant to the field of cannabinoids because although the existence of non-CB1 non-CB2 cannabinoid receptors has been conjectured in multiple tissues, little is known about the functional responses or endpoints that may be measured to assess the activity of cannabinoid compounds at the novel receptor. Therefore, it is suggested that one pharmacological method to assess the activity of the non-CB1 non-CB2 cannabinoid receptor in T cells is the measurement of [Ca2+]i. Moreover, the present studies indicate that initial characterization of the non-CB1 non-CB2 cannabinoid receptor in T cells may also be performed in the presence of SR1 and SR2, both of which putatively act as neutral antagonists at the novel receptor. Lastly, the present studies, particularly those performed in the HPB-ALL cells, are the first to demonstrate not only that Ag-THC treatment can activate TRPCl channels, but also that endogenously expressed TRPCl proteins putatively homomultimerize to form OAG-gated ROCCs. The aforementioned finding contributes significantly to the field of TRPC channels because most studies of TRPC channel structure and function have been performed in transfected cells which overexpress TRPC proteins, and in the presence of other TRPC channels. The HPB-ALL cells, therefore, provide a unique model for studying endogenously expressed TRPC] channels in the absence of other 206 TRPC subfamily members. Perhaps the most important and relevant finding from the present project that contributes to the field of TRPC channels is that A9-THC regulates TRPC] independently of both store-depletion and DAG. 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