Novel effect of CP55,940, a CB1/CB2 cannabinoid receptor agonist, on intracellular free Ca2+ levels in bladder cancer cells.
The study was undertaken to explore the effect of CP55,940 ((-)-cis-3-[2-Hydroxy4-(1,1-dimethylheptyl) phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol), a drug commonly used as a CB1/CB2 cannabinoid receptor agonist, on intracellular free Ca2+ levels ([Ca2+]i) in several cell types [Ca2+]i was measured in suspended cells by using the fluorescent dye fura-2 as an indicator. At concentrations between 1-50 microM, CP55,940 increased [Ca2+]i in a concentration-dependent manner with an EC50 of 8 microM. The [Ca2+]i signal comprised an initial rise, a slow decay, and a sustained phase. CP55940 (10 microM)-induced (Ca2+]i signal was not altered by 5 microM of two cannabinoid receptor antagonists (AM-251, N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide; AM-281, 1-(2,4-Dichlorophenyl)-5-(4-iodophenyl)-4-m3thyl-N-4-morpholinyl-1H-pyrazole-3-carboxamide). Extracellular Ca2+ removal decreased the maximum value of the Ca2+ signals by 50%. CPS5,940 (10 microM)-induced [Ca2+]i increase in Ca2+-free medium was inhibited by 80% by pretreatment with 1 microM thapsigargin, an endoplasmic reticulum Ca2+ pump inhibitor. Conversely, pretreatment with 10 microM CP55,940 in Ca2+-free medium for 6 min abolished thapsigargin-induced [Ca2+]i increase. Nifedipine (10 microM) and verapamil (10 microM) did not alter CP55,940 (10 microM)-induced [Ca2+]i increase. CP55, 940 (10 microM)-induced Ca2+ release was not affected when phospholipase C was inhibited by 2 microM U73122 (1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione). CP55,940 (5 microM) also increased [Ca22+] in Madin-Darby canine kidney cells, MG63 human osteosarcoma cells, and IMR-32 neuroblastoma cells. Collectively, CP,55940 induced significant [Ca2+]i increases in several cell types by releasing store Ca2+ from thapsigargin-sensitive pools and by causing Ca2+ entry. The CP55,940's action appears to be dissociated from stimulation of cannabinoid receptors
Jan
Chung-Ren
CR
Department of Medical Education and Research Kaohsiung Veterans General Hospital, Taiwan. crjan@isca.vghks.gov.tw
Lu
Yih-Chau
YC
Jiann
Bang-Ping
BP
Chang
Hong-Tai
HT
Su
Warren
W
Chen
Wei-Chung
WC
Huang
Jong-Khing
JK
D002118:Calcium / Q000378:metabolism*; D003511:Cyclohexanols / Q000494:pharmacology*; D006801:Humans; D043882:Receptors, Cannabinoid; D011955:Receptors, Drug / Q000819:agonists*; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors; D001749:Urinary Bladder Neoplasms / Q000378:metabolism*
D003511:Cyclohexanols; D043882:Receptors, Cannabinoid; D011955:Receptors, Drug; C054649:3-(2-hydroxy-4-(1,1-dimethylheptyl)phenyl)-4-(3-hydroxypropyl)cyclohexanol; D010738:Type C Phospholipases; D002118:Calcium