Sphingosylphosphorylcholine generates reactive oxygen species through calcium-, protein kinase Cdelta- and phospholipase D-dependent pathways.
Sphingosylphosphorylcholine (SPC) is a bioactive lipid molecule involved in numerous biological processes. Treatment of MS1 pancreatic islet endothelial cells with SPC increased phospholipase D (PLD) activity in a time- and dose-dependent manner. In addition, treatment of the MS1 cells with 10 microM SPC induced stimulation of phospholipase C (PLC) activity and transient elevation of intracellular Ca2+. The SPC-induced PLD activation was prevented by pretreatment of the MS1 cells with a PLC inhibitor, U73122, and an intracellular Ca2+-chelating agent, BAPTA-AM. This suggests that PLC-dependent elevation of intracellular Ca2+ is involved in the SPC-induced activation of PLD. The SPC-dependent PLD activity was also almost completely prevented by pretreatment with pan-specific PKC inhibitors, GF109203X and RO-31-8220, and with a PKCdelta-specific inhibitor, rottlerin, but not by pretreatment with GO6976, a conventional PKC isozymes-specific inhibitor. Adenoviral overexpression of a kinase-deficient mutant of PKCdelta attenuated the SPC-induced PLD activity. These results suggest that PKCdelta plays a crucial role for the SPC-induced PLD activation. The SPC-induced PLD activation was preferentially potentiated in COS-7 cells transfected with PLD2 but not with PLD1, suggesting a specific implication of PLD2 in the SPC-induced PLD activation. SPC treatment induced phosphorylation of PLD2 in COS-7 cells, and overexpression of the kinase-deficient mutant of PKCdelta prevented the SPC-induced phosphorylation of PLD2. Furthermore, SPC treatment generated reactive oxygen species (ROS) in MS1 cells and the SPC induced production of ROS was inhibited by pretreatment with U73122, BAPTA-AM, and rottlerin. In addition, pretreatment with a PLD inhibitor 1-butanol and overexpression of a lipase-inactive mutant of PLD2 but not PLD1 attenuated the SPC-induced generation of ROS. These results suggest that PLC-, Ca2+-, PKCdelta-, and PLD2-dependent pathways are essentially required for the SPC induced ROS generation.
Jeon
Eun Su
ES
Department of Physiology and the Medical Research Institute, College of Medicine, Pusan National University, 1-Ga, Ami-Dong, Suh-Gu, Busan 602-739, Republic of Korea.
Kang
Yong Jung
YJ
Song
Hae Young
HY
Im
Dong-Soon
DS
Kim
Hyeon Soo
HS
Ryu
Sung Ho
SH
Kim
Yong Keun
YK
Kim
Jae Ho
JH
D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000378:metabolism* / Q000502:physiology; D002460:Cell Line; D002522:Chlorocebus aethiops; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D010739:Phospholipase D / Q000378:metabolism*; D010766:Phosphorylation; D010767:Phosphorylcholine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D011493:Protein Kinase C / Q000378:metabolism* / Q000502:physiology; D051745:Protein Kinase C-delta; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015398:Signal Transduction; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D010738:Type C Phospholipases / Q000378:metabolism
D017382:Reactive Oxygen Species; C005356:sphingosine phosphorylcholine; D010767:Phosphorylcholine; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; D010738:Type C Phospholipases; C105606:phospholipase D2; D010739:Phospholipase D; D013110:Sphingosine; D002118:Calcium