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 microM with an EC50 of 20 microM. 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 microM thapsigargin abolished 100 microM 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 microM 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 microM), 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.
Huang
J K
JK
Department of Surgery, Kaohsiung Veterans General Hospital, School of Medicine, National Yang Ming University, Taipei, Taiwan. crjan@isca.vghks.gov.tw
Jan
C R
CR
D000818:Animals; D002118:Calcium / Q000378:metabolism* / Q000493:pharmacokinetics / Q000494:pharmacology; D002460:Cell Line; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004285:Dogs; D004553:Electric Conductivity; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005456:Fluorescent Dyes; D016257:Fura-2; D007684:Kidney Tubules / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D008041:Linoleic Acids / Q000494:pharmacology*; D009829:Oleic Acids / Q000494:pharmacology
D005456:Fluorescent Dyes; D008041:Linoleic Acids; D009829:Oleic Acids; C420162:linoleamide; C029407:oleylamide; D002118:Calcium; D016257:Fura-2