title: Sphingomyelinase and cell-permeable ceramide analogs increase the release of plasminogen activator inhibitor-1 from cultured endothelial cells.
abstract: We investigated the effect of exogenous staphylococcal sphingomyelinase (SMase) on the release of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) from cultured human umbilical vein endothelial cells (HUVEC). Addition of SMase (2 units/ml) to the culture medium induced an approx. 15-fold increase in the extracellular level of PAI-1 antigen at 3 h. No significant increase in the level of t-PA antigen was detected. Treatment of HUVEC with SMASE (2 units/ml) for 3 h resulted in a significant decrease in the cellular sphingomyelin (SM) level, accompanied by a corresponding increase in the ceramide level. Cell-permeable ceramide analogs also enhanced the release of PAI-1 from cultured HUVEC in concentration- and time-dependent manners. A 6-fold increase in PAI-1 antigen level was observed after incubation for 3 h with 10 microM N-acetylsphingosine. Similar effect was noted as early as 2 h with 10 microM N-hexyanoylsphingosine. Addition of sphingosine failed to affect the release of PAI-1 from cultured HUVEC, indicating that the effects of ceramide analogs were independent of sphingosine generation. Pretreatment with cycloheximide or actinomycin D abated the response of HUVEC to N-acetylsphingosine in the increased levels of both extracellular and intracellular PAI-1. These results suggest that ceramide, generated via "SM cycle", acts as a lipid mediator of PAI-1 release from vascular endothelial cells, and may contribute to a better understanding of the pathogenesis of the PAI-1-associated thrombotic disorders.
authors.affiliation: Department of Biochemistry, Faculty of Pharmaceutical Sciences, Fukuoka University, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Soeda
authors.forename: O
authors.initials: O
authors.lastname: Honda
authors.forename: H
authors.initials: H
authors.lastname: Shimeno
authors.forename: A
authors.initials: A
authors.lastname: Nagamatsu
mesh_terms: D002463:Cell Membrane Permeability / Q000187:drug effects*; D002518:Ceramides / Q000494:pharmacology*; D003513:Cycloheximide / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D013107:Sphingolipids / Q000378:metabolism; D013108:Sphingomyelin Phosphodiesterase / Q000494:pharmacology*; D014471:Umbilical Veins / Q000166:cytology
chemical_list: D002518:Ceramides; D017395:Plasminogen Activator Inhibitor 1; D011500:Protein Synthesis Inhibitors; D013107:Sphingolipids; D003609:Dactinomycin; D003513:Cycloheximide; D013108:Sphingomyelin Phosphodiesterase