Novel effects of a sleep-inducing lipid, oleamide, on Ca²⁺ signaling in renal tubular cells

The effect of oleamide, a sleep-inducing endogenous lipid in animal models, on intracellular free levels of Ca²⁺ ([Ca²⁺]i) in Madin-Darby renal tubular cells was examined using fura-2 as a fluorescent dye. Oleamide (5-50 μM) increased [Ca²⁺]i in a concentration-dependent fashion with an EC50 value of 20 μM. The [Ca²⁺]i signal comprised an initial rise and an elevated phase and was reduced by removing extracellular Ca²⁺ by 50%. After pretreatment with 5-50 μM oleamide in Ca²⁺-free medium, addition of 3 mM Ca²⁺ increased [Ca²⁺]i in a manner dependent on the concentration of oleamide. In Ca²⁺-free medium, pretreatment with thapsigargin (1 μM), an endoplasmic reticulum Ca²⁺ pump inhibitor, abolished [Ca²⁺]i increases induced by 20 μM oleamide; conversely, pretreatment with 20 μM oleamide reduced 1 μM thapsigargin-induced [Ca²⁺]i increases by 50%. Suppression of the activity of phospholipase C with 2 μM U73122 abolished 20 μM oleamide-induced Ca²⁺ release. Collectively, these data demonstrate that oleamide induced significant [Ca²⁺]i increases in renal tubular cells by a phospholipase C-dependent release of Ca²⁺ from thapsigargin-sensitive stores and by inducing Ca²⁺ entry via store-operated Ca²⁺ entry.