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

Linolcamide is an endogenous lipid that has been shown to induce sleep in c ats, rats and humans. However, its physiological function remains unclear. In this study the effect of linoleamide on cytosolic free Ca2+ concentratio ns ([Ca2+](i)) in Madin Darby canine kidney (MDCK) tubular cells was examin ed, by using fura-2 as a Ca2+ probe. In a concentration-dependent manner, l inoleamide induced increases in [Ca2+](i) between 10-500 muM with an EC50 o f 20 muM. 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 p artly inhibited by external Ca2+ removal. In Ca2+-free medium, depletion of the endoplasmic reticulum Ca2+ store with 1 muM thapsigargin abolished 100 muM linoleamide-induced internal Ca2+ release, and conversely, pretreatmen t with linoleamide prevented thapsigargin from releasing internal Ca2+ This demonstrates that the internal source of linoleamide-induced [Ca2+](i) inc rease is located in the endoplasmic reticulum, This discharge of internal C a2+ caused capacitative Ca2+ entry because after incubation with 100 muM li noleamide in Ca2+-free medium for 8 min readmission of 3 mM CaCl2 induced i ncreases in [Ca2+](i). After the formation of inositol-1,4,5-trisphosphate (IP3) was blocked by the phospholipase C inhibitor U73122 (1 muM), linoleam ide still induced an increase in [Ca2+](i) but the shape of the increase wa s altered. Similar results were found for another sleep-inducing lipid 9,10 -octadecenoamide. Together, the present study shows that the endogenous sle ep-inducing lipid linoleamide was able to cause significant increases in [C a2+](i) in renal tubular cells, by releasing the endoplasmic reticulum Ca2 store and triggering capacitative Ca2+ entry in a manner independent of IP 3. (C) 2001 Elsevier Science Inc. All rights reserved.