Glutamate stably enhances the activity of two cytosolic forms of phospholipase A2 in brain cortical cultures.
The mechanisms by which glutamatergic neurotransmitters modulate neuronal lipid metabolism are not well established. We have directly measured phospholipase A2 (PLA2) enzymic activity in cell-free extracts from cortical neuronal cultures from rat brain and have found that the PLA2 activity is up-regulated after cells are exposed to glutamate. Brief exposure to a calcium ionophore or phorbol 12-myristate 13-acetate (PMA) stably enhanced PLA2 activity. Down-regulation of protein kinase C activity partially blocked glutamate's effects. Two Ca(2+)-and pH-dependent forms of PLA2 were identified in cytosolic extracts. Activation of both forms of PLA2 was enhanced by prior exposure of the cultures to glutamate. One of the two forms had chromatographic characteristics on heparin-Sepharose, Mono Q and Superose 12 columns similar to the 100 kDa cytosolic PLA2 (cPLA2), and was recognized by an antibody raised to pig spleen cPLA2. The second form was similar in size to Group-I and -II PLA2s but differed in chromatographic characteristics. It was not inhibited by dithiothreitol, and did not react with antibodies to pancreatic Group-I PLA2, features that distinguish it from Group-I and -II PLA2. In extracts from cells pretreated with glutamate, the activity-Ca2+ concentration dose-response relationship of the 13.5 kDa form of PLA2 was shifted to the left with activation at lower Ca2+ concentration as the result of stable modification of the enzyme induced by glutamate. Thus glutamate-induced stable enhancement of PLA2 activity, by processes involving calcium and protein kinase C activation, is a potential molecular switch probably mediating changes in synaptic function and contributing to excitotoxicity.
Kim
D K
DK
Medical Service, Massachusetts General Hospital, Boston 02114, USA.
Rordorf
G
G
Nemenoff
R A
RA
Koroshetz
W J
WJ
Bonventre
J V
JV
D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology / Q000201:enzymology*; D002850:Chromatography, Gel; D003600:Cytosol / Q000201:enzymology*; D004789:Enzyme Activation; D018698:Glutamic Acid / Q000378:metabolism / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D009474:Neurons / Q000187:drug effects / Q000201:enzymology; D010741:Phospholipases A / Q000302:isolation & purification / Q000378:metabolism*; D054467:Phospholipases A2; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D007527:Isoenzymes; D000001:Calcimycin; D018698:Glutamic Acid; D010741:Phospholipases A; D054467:Phospholipases A2; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium