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 exci table cells was examined by using fura-2 as a fluorescent dye. [Ca2+](i) in pheochromocytoma cells, renal tubular cells, osteoblast-like cells, and bl adder cancer cells were increased on stimulation of 50 muM oleamide. The re sponse in human bladder cancer cells (T24) was the greatest and was further explored. Oleamide (10-100 muM) increased [Ca2+](i) in a concentration-dep endent fashion with an EC50 of 50 muM. The [Ca2+](i) signal comprised an in itial rise and a sustained plateau and was reduced by removing extracellula r Ca2+ by 85 +/- 5%. After pre-treatment with 10-100 muM oleamide in Ca2+-f ree medium, addition of 3 nM Ca2+ increased [Ca2+](i) in a manner dependent on the concentration of oleamide. The [Ca2+](i) increase induced by 50 muM oleamide was reduced by 100 muM La3+ by 40%, but was not altered by 10 muM nifedipine, 10 muM verapamil, and 50 muM Ni2+. In Ca2+-free medium, pre-tr eatment with thapsigargin (1 muM), an endoplasmic reticulum Ca2+ pump inhib itor, abolished 50 muM oleamide-induced [Ca2+](i) increases; conversely, pr etreatment with 50 muM oleamide reduced 1 muM thapsigargin-induced [Ca2+](i ) increases by 50 +/- 3%. Suppression of the activity of phospholipase C wi th 2 muM U73122 failed to alter 50 muM oleamide-induced Ca2+ release. Linol eamide (10-100 muM), another sleep-inducing lipid with a structure similar to that of oleamide, also induced an increase in [Ca2+](i). Together, it wa s shown that oleamide induced significant [Ca2+](i) increases in cells by a phospholipase C-independent release of Ca2+ from thapsigargin-sensitive st ores and by inducing Ca2+ entry. (C) 2001 Elsevier Science Inc. All rights reserved.