Effect of oleamide on Ca²⁺ signaling in human bladder cancer cells

The effect of oleamide, a sleep-inducing endogenous lipid in animal models, on intracellular free levels of Ca²⁺ ([Ca²⁺]_i) in non-excitable and excitable cells was examined by using fura-2 as a fluorescent dye. [Ca²⁺]_i in pheochromocytoma cells, renal tubular cells, osteoblast-like cells, and bladder cancer cells were increased on stimulation of 50 μM oleamide. The response in human bladder cancer cells (T24) was the greatest and was further explored. Oleamide (10-100 μM) increased [Ca²⁺]_i in a concentration-dependent fashion with an EC₅₀ of 50 μM. The [Ca²⁺]_i signal comprised an initial rise and a sustained plateau and was reduced by removing extracellular Ca²⁺ by 85 ± 5%. After pre-treatment with 10-100 μM oleamide in Ca²⁺-free medium, addition of 3 mM Ca²⁺ increased [Ca²⁺]_i in a manner dependent on the concentration of oleamide. The [Ca²⁺]_i increase induced by 50 μM oleamide was reduced by 100 μM La³⁺ by 40%, but was not altered by 10 μM nifedipine, 10 μM verapamil, and 50 μM Ni²⁺. In Ca²⁺-free medium, pre-treatment with thapsigargin (1 μM), an endoplasmic reticulum Ca²⁺ pump inhibitor, abolished 50 μM oleamide-induced [Ca²⁺]_i increases; conversely, pretreatment with 50 μM oleamide reduced 1 μM thapsigargin-induced [Ca²⁺]_i increases by 50 ± 3%. Suppression of the activity of phospholipase C with 2 μM U73122 failed to alter 50 μM oleamide-induced Ca²⁺ release. Linoleamide (10-100 μM), another sleep-inducing lipid with a structure similar to that of oleamide, also induced an increase in [Ca²⁺]_i. Together, it was shown that oleamide induced significant [Ca²⁺]_i increases in cells by a phospholipase C-independent release of Ca²⁺ from thapsigargin-sensitive stores and by inducing Ca²⁺ entry.