1. Department of Neurology and Rehabilitation, University of Illinois at Chicago, , Chicago, Illinois, USA;2. Department of Pediatrics, University of Chicago, , Chicago, Illinois, USA;3. Department of Medicine, University of Illinois at Chicago, , Chicago, Illinois, USA;4. Department of Pharmacology, University of Illinois at Chicago, , Chicago, Illinois, USA
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 .
