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Changes in the Activity and mRNA Levels of Phospholipase D During Ceramide‐Induced Apoptosis in Rat C6 Glial Cells
Author(s) -
Yoshimura Shinichi,
Sakai Hideki,
Ohguchi Kenji,
Nakashima Shigeru,
Banno Yoshiko,
Nishimura Yasuaki,
Sakai Noboru,
Nozawa Yoshinori
Publication year - 1997
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.1997.69020713.x
Subject(s) - ceramide , microbiology and biotechnology , apoptosis , lipid signaling , gtp' , biology , phospholipase d , guanosine , lysophosphatidylcholine , phospholipase c , messenger rna , biochemistry , enzyme , phospholipid , phosphatidylcholine , membrane , gene
N ‐Acetylsphingosine (C 2 ‐ceramide), a membrane‐permeable analogue, induced apoptosis in C6 glial cells. Phase‐contrast micrographs showed that the round cells appeared 3 h after exposure to 25 µ M C 2 ‐ceramide and the number of floating cells increased time‐dependently. Staining with Hoechst 33258 dye showed condensed or fragmented nuclei in round cells at 12 h. DNA fragmentation was also observed by agarose gel electrophoresis at 12 h. To understand the mechanism underlying glial cell death induced by C 2 ‐ceramide treatment, changes in phospholipase D (PLD) activity in response to guanosine 5′‐ O ‐(3‐thiotriphosphate) (GTPγS) and expression of mRNA levels of PLD isozymes were examined. In cell lysate, GTPγS‐dependent PLD activity was down‐regulated after ceramide treatment in a time‐dependent manner. In the in vitro PLD assay, membrane‐associated PLD activation in response to recombinant ADP‐ribosylation factor 1 was greatly suppressed. Furthermore, levels of rPLD1a and rPLD1b mRNAs were found to be down‐regulated, whereas the level of rPLD2 mRNA increased gradually, peaking at 3 h, followed by a slow decrease, as inferred by reverse transcription‐polymerase chain reaction. Decreases in GTPγS‐dependent PLD activity were well correlated with those in rPLD1a and rPLD1b mRNAs levels. Taken together, these data suggest that levels of PLD enzymes might be decreased by ceramide treatment.