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: Fernando D Testai, John P Kilkus, Evgeny Berdyshev, Irina Gorshkova, Viswanathan Natarajan, Glyn Dawson
• Journal: Journal of neurochemistry (J Neurochem) Vol. 131 Issue 4 Pg. 530-40 (Nov 2014) ISSN: 1471-4159 [Electronic] England
• PMID: 25060904 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)
• Copyright: © 2014 International Society for Neurochemistry.

Chemical References

• 3',4'-ethylenedioxyphenyl-2-palmitoylamino-3-pyrrolidino-1-propanol
• Ceramides
• Enzyme Inhibitors
• Glucosylceramides
• Propanolamines
• Pyrrolidines
• Sphingolipids
• Sphingomyelins

Topics

• Cell Death (drug effects)
• Cell Hypoxia (drug effects, physiology)
• Cell Line, Transformed
• Ceramides (metabolism)
• Chromatography, Thin Layer
• Electric Impedance
• Enzyme Inhibitors (pharmacology)
• Epithelial Cells (drug effects, metabolism)
• Glucosylceramides (metabolism)
• Humans
• Mass Spectrometry
• Propanolamines (pharmacology)
• Pyrrolidines (pharmacology)
• Sphingolipids (analysis, metabolism)
• Sphingomyelins (metabolism)