Novel effect of CP55,940, a CB1/CB2 cannabinoid receptor agonist, on intracellular free Ca ²⁺ levels in bladder cancer cells

The study was undertaken to explore the effect of CP55,940 ((-)-cis-3-[2-Hydroxy-4-(1,1-dimethylheptyl) phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol), a drug commonly used as a CB1/CB2 cannabinoid receptor agonist, on intracellular free Ca ²⁺ levels ([Ca ²⁺ ] _i ) in several cell types. [Ca ²⁺ ] _i was measured in suspended cells by using the fluorescent dye fura-2 as an indicator. At concentrations between 1-50 μM, CP55,940 increased [Ca ²⁺ ] _i in a concentration-dependent manner with an EC ₅₀ of 8 μM. The [Ca ²⁺ ] _i signal comprised an initial rise, a slow decay, and a sustained phase. CP55940 (10 μM)-induced [Ca ²⁺ ] _i signal was not altered by 5 μM 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 Ca ²⁺ removal decreased the maximum value of the Ca ²⁺ signals by 50%. CP55,940 (10 μM)-induced [Ca ²⁺ ] _i increase in Ca ²⁺ -free medium was inhibited by 80% by pretreatment with 1 μM thapsigargin, an endoplasmic reticulum Ca ²⁺ pump inhibitor. Conversely, pre-treatment with 10 μM CP55,940 in Ca ²⁺ -free medium for 6 min abolished thapsigargin-induced [Ca ²⁺ ] _i increase. Nifedipine (10 μM) and verapamil (10 μM) did not alter CP55,940 (10 μM)-induced [Ca ²⁺ ] _i increase. CP55, 940 (10 μM)-induced Ca ²⁺ release was not affected when phospholipase C was inhibited by 2 μM U73122 thapsigargin-sensitive pools and by causing Ca ²⁺ entry. The CP55,940's action appears to be dissociated from stimulation of cannabinoid receptors.