Modulation of the activity of cytosolic phospholipase A2alpha (cPLA2alpha) by cellular sphingolipids and inhibition of cPLA2alpha by sphingomyelin.
We examined the effect of the cellular sphingolipid level on the release of arachidonic acid (AA) and activity of cytosolic phospholipase A2alpha (cPLA2alpha) using two Chinese hamster ovary (CHO)-K1-derived mutants deficient in sphingolipid synthesis: LY-B cells defective in the LCB1 subunit of serine palmitoyltransferase for de novo synthesis of sphingolipid species, and LY-A cells defective in the ceramide transfer protein CERT for SM synthesis. When LY-B and LY-A cells were cultured in Nutridoma medium and the sphingolipid level was reduced, the release of AA stimulated by the Ca(2+) ionophore A23187 increased 2-fold and 1.7-fold, respectively, compared with that from control cells. The enhancement in LY-B cells was decreased by adding sphingosine and treatment with the cPLA2alpha inhibitor. When CHO cells were treated with an acid sphingomyelinase inhibitor to increase the cellular SM level, the release of AA induced by A23187 or PAF was decreased. In vitro studies were then conducted to test whether SM interacts directly with cPLA2alpha. Phosphatidylcholine vesicles containing SM reduced cPLA2alpha activity. Furthermore, SM disturbed the binding of cPLA2alpha to glycerophospholipids. These results suggest that SM at the biomembrane plays important roles in regulating the cPLA2alpha-dependent release of AA by inhibiting the binding of cPLA2alpha to glycerophospholipids.
Nakamura
Hiroyuki
H
Laboratory of Chemical Pharmacology, Graduate School of Medicine, Chiba University, Chuo-ku, Chiba, Japan. ropi@p.chiba-u.ac.jp
Wakita
Shigeo
S
Suganami
Akiko
A
Tamura
Yutaka
Y
Hanada
Kentaro
K
Murayama
Toshihiko
T
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism*; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004791:Enzyme Inhibitors / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D020404:Glycerophospholipids / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D054513:Group IV Phospholipases A2 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008081:Liposomes / Q000378:metabolism; D009154:Mutation; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding; D021381:Protein Transport / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D013107:Sphingolipids / Q000378:metabolism*; D013109:Sphingomyelins / Q000378:metabolism*; D013110:Sphingosine / Q000031:analogs & derivatives / Q000494:pharmacology
D004791:Enzyme Inhibitors; D020404:Glycerophospholipids; D008081:Liposomes; D011993:Recombinant Fusion Proteins; D013107:Sphingolipids; D013109:Sphingomyelins; C481931:erythro-(2R,3S)-sphingosine; D049452:Green Fluorescent Proteins; D016718:Arachidonic Acid; D048049:Extracellular Signal-Regulated MAP Kinases; D054513:Group IV Phospholipases A2; D013110:Sphingosine