title: Multiple sphingolipid abnormalities following cerebral microendothelial hypoxia.
abstract: Hypoxia has been previously shown to inhibit the dihydroceramide (DHC) desaturase, leading to the accumulation of DHC. In this study, we used metabolic labeling with [3H]-palmitate, HPLC/MS/MS analysis, and specific inhibitors to show numerous sphingolipid changes after oxygen deprivation in cerebral microendothelial cells. The increased DHC, particularly long-chain forms, was observed in both whole cells and detergent-resistant membranes. This was reversed by reoxygenation and blocked by the de novo sphingolipid synthesis inhibitor myriocin, but not by the neutral sphingomyelinase inhibitor GW-4869. Furthermore, oxygen deprivation of microendothelial cells increased levels of dihydro-sphingosine (DH-Sph), DH-sphingosine1-phosphate (DH-S1P), DH-sphingomyelin (DH-SM), DH-glucosylceramide (DH-GlcCer), and S1P levels. In vitro assays revealed no changes in the activity of sphingomyelinases or sphingomyelin synthase, but resulted in reduced S1P lyase activity and 40% increase in glucosylceramide synthase (GCS) activity, which was reversed by reoxygenation. Inhibition of the de novo sphingolipid pathway (myriocin) or GCS (EtPoD4) induced endothelial barrier dysfunction and increased caspase 3-mediated cell death in response to hypoxia. Our findings suggest that hypoxia induces synthesis of S1P and multiple dihydro-sphingolipids, including DHC, DH-SM, DH-GlcCer, DH-Sph and DH-S1P, which may be involved in ameliorating the effects of stroke . Progressive hypoxia leads to the accumulation of several dihydrosphingolipids in cerebral microendothelial cells. Hypoxia also increases sphingosine-1-phosphate and the activity of glucosylceramide (Glc-Cer) synthase. These changes reverse by inhibiting the de novo sphingolipid synthesis, which worsens hypoxia-induced endothelial barrier dysfunction and apoptosis, suggesting that the identified sphingolipids may be vasculoprotective.
authors: Department of Neurology and Rehabilitation, University of Illinois at Chicago, Chicago, Illinois, USA.
authors: Fernando D
authors: FD
authors: Testai
authors: John P
authors: JP
authors: Kilkus
authors: Evgeny
authors: E
authors: Berdyshev
authors: Irina
authors: I
authors: Gorshkova
authors: Viswanathan
authors: V
authors: Natarajan
authors: Glyn
authors: G
authors: Dawson
mesh_terms: D016923:Cell Death / Q000187:drug effects; D015687:Cell Hypoxia / Q000187:drug effects / Q000502:physiology*; D002461:Cell Line, Transformed; D002518:Ceramides / Q000378:metabolism; D002855:Chromatography, Thin Layer; D017097:Electric Impedance; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D005963:Glucosylceramides / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry; D011412:Propanolamines / Q000494:pharmacology; D011759:Pyrrolidines / Q000494:pharmacology; D013107:Sphingolipids / Q000032:analysis / Q000378:metabolism*; D013109:Sphingomyelins / Q000378:metabolism
chemical_list: C404730:3',4'-ethylenedioxyphenyl-2-palmitoylamino-3-pyrrolidino-1-propanol; D002518:Ceramides; D004791:Enzyme Inhibitors; D005963:Glucosylceramides; D011412:Propanolamines; D011759:Pyrrolidines; D013107:Sphingolipids; D013109:Sphingomyelins
keywords: ceramide; cerebral endothelium; dihydroceramide; hypoxia; sphingolipids