Linoleamide, a brain lipid that induces sleep, increases cytosolic Ca²⁺ levels in MDCK renal tubular cells

Linoleamide is an endogenous lipid that has been shown to induce sleep in cats, rats and humans. However, its physiological function remains unclear. In this study the effect of linoleamide on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) in Madin Darby canine kidney (MDCK) tubular cells was examined, by using fura-2 as a Ca²⁺ probe. In a concentration-dependent manner, linoleamide induced increases in [Ca²⁺]_i between 10-500 μM with an EC₅₀ of 20 μM. The signal comprised a slow rise and a persistent phase, and was a result of internal Ca²⁺ release and external Ca²⁺ influx because it was partly inhibited by external Ca²⁺ removal. In Ca²⁺-free medium, depletion of the endoplasmic reticulum Ca²⁺ store with 1 μM thapsigargin abolished 100 μM linoleamide-induced internal Ca²⁺ release, and conversely, pretreatment with linoleamide prevented thapsigargin from releasing internal Ca²⁺. This demonstrates that the internal source of linoleamide-induced [Ca²⁺]_i increase is located in the endoplasmic reticulum. This discharge of internal Ca²⁺ caused capacitative Ca²⁺ entry because after incubation with 100 μM linoleamide in Ca²⁺-free medium for 8 min readmission of 3 mM CaCl₂ induced increases in [Ca²⁺]_i. After the formation of inositol-1,4,5-trisphosphate (IP₃) was blocked by the phospholipase C inhibitor U73122 (1 μM), linoleamide still induced an increase in [Ca²⁺]_i but the shape of the increase was altered. Similar results were found for another sleep-inducing lipid 9,10-octadecenoamide. Together, the present study shows that the endogenous sleep-inducing lipid linoleamide was able to cause significant increases in [Ca²⁺]_i in renal tubular cells, by releasing the endoplasmic reticulum Ca²⁺ store and triggering capacitative Ca²⁺ entry in a manner independent of IP₃.