Induction of group VIA phospholipase A2 activity during in vitro ischemia in C2C12 myotubes is associated with changes in the level of its splice variants.
The involvement of group VI Ca(2+)-independent PLA(2)s (iPLA(2)-VI) in in vitro ischemia [oxygen and glucose deprivation (OGD)] in mouse C2C12 myotubes was investigated. OGD induced a time-dependent (0-6 h) increase in bromoenol lactone (BEL)-sensitive iPLA(2) activity, which was suppressed by specific short interfering (si)RNA knockdown of iPLA(2)-VIA. OGD was associated with an increase in iPLA(2)-VIA protein levels, whereas mRNA levels were unchanged. The levels of iPLA(2)-VIB mRNA and protein were not increased by OGD. RT-PCR and Western blot analysis identified a mouse iPLA(2)-VIA homolog to catalytically inactive 50-kDa iPLA(2)-VIA-ankyrin variants previously identified in humans. Both the mRNA and protein levels of this approximately 50-kDa variant were reduced significantly within 1 h following OGD. In C2C12 myoblasts, iPLA(2)-VIA seemed to predominantly reside at the endoplasmatic reticulum, where it accumulated further during OGD. A time-dependent reduction in cell viability during the early OGD period (3 h) was partially prevented by iPLA(2)-VIA knockdown or pharmacological inhibition (10 microM BEL), whereas iPLA(2)-VIA overexpression had no effect on cell viability. Taken together, these data demonstrate that OGD in C2C12 myotubes is associated with an increase in iPLA(2)-VIA activity that decreases cell viability. iPLA(2)-VIA activation may be modulated by changes in the levels of active and inactive iPLA(2)-VIA isoforms.
Poulsen
K A
KA
Dept. of Molecular Biology, Univ. of Copenhagen, Universitetsparken 13, Copenhagen Ø DK-2100, Denmark. kapoulsen@aki.ku.dk
Pedersen
S F
SF
Kolko
M
M
Lambert
I H
IH
D000818:Animals; D001095:Arachidonic Acids / Q000378:metabolism; D015687:Cell Hypoxia; D002460:Cell Line; D002470:Cell Survival; D004721:Endoplasmic Reticulum / Q000201:enzymology; D004790:Enzyme Induction; D005947:Glucose / Q000172:deficiency / Q000378:metabolism; D054513:Group IV Phospholipases A2 / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D007511:Ischemia / Q000201:enzymology* / Q000235:genetics; D051379:Mice; D008970:Molecular Weight; D018485:Muscle Fibers, Skeletal / Q000187:drug effects / Q000201:enzymology*; D018482:Muscle, Skeletal / Q000098:blood supply*; D009281:Naphthalenes / Q000494:pharmacology; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D020033:Protein Isoforms; D011753:Pyrones / Q000494:pharmacology; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D013997:Time Factors