Effect of chronic ethanol exposure on mouse brain arachidonic acid specific phospholipase A2.
The enzyme phospholipase A2 (PLA2), which catalyzes the hydrolysis of an ester bond at the sn-2 position of 1,2-sn-diacylglycerols, has been suggested to play an important role in regulating cellular functions. Although ethanol (EtOH)-induced activation of PLA2 activity was reported previously by us in mouse brain (Hungund et al., Neurochem Int 25: 321-325, 1994), its subcellular localization and biochemical properties have not been investigated. Therefore, in the present study, we examined the subcellular localization and characterization of EtOH-activated PLA2 activity in mouse brain. The results indicated that EtOH treatment decreased the specific activity of PLA2 for the first 48 hr, and then the activity increased and reached a peak level in both cytosol (1.6-fold) and membrane (1.7-fold) fractions at 96 hr of exposure. Specific activity was found to be higher in the membrane fraction than in the cytosol. Using differential density gradient centrifugation, subcellular localization of the membrane-associated PLA2 revealed that most of the EtOH-activated PLA2 specific activity was associated with the synaptic membrane (44%) followed by the nuclear membrane (13%). No significant increase in the PLA2 specific activity of mitochondrial and microsomal membranes was observed. No activity was detected in the myelin membrane. PLA2 specific activity of membranes from control and EtOH-exposed mouse brain exhibited preference for arachidonic acid over linoleic acid at the sn-2 position of glycero-3-phosphocholine (PC). No detectable PLA2 specific activity was found when PC containing oleic acid at the sn-2 position was used as a substrate. The present results also indicated that the PLA2 specific activity of membrane from control and EtOH-exposed mouse brain was insensitive to dithiothreitol, strongly stimulated by Ca2+, enhanced by glycerol, and inhibited by the cytosolic PLA2 (cPLA2) inhibitor methyl arachidonyl fluorophosphonate with an IC50 value of 3.33 microM. In summary, results suggest that the properties of EtOH-activated PLA2 activity found in mouse brain membrane fraction are similar to those of cPLA2 found in variety of cells, including mammalian brain.
Basavarajappa
B S
BS
Division of Analytical Psychopharmacology, New York State Psychiatric Institute, Orangeburg, NY 10962, USA.
Cooper
T B
TB
Hungund
B L
BL
D000437:Alcoholism / Q000201:enzymology*; D000818:Animals; D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000494:pharmacology; D001921:Brain / Q000187:drug effects* / Q000201:enzymology*; D002118:Calcium / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D000431:Ethanol / Q000633:toxicity*; D005990:Glycerol / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D051379:Mice; D063065:Organophosphonates / Q000494:pharmacology; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000378:metabolism*; D054467:Phospholipases A2; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013570:Synaptic Membranes / Q000201:enzymology
D001095:Arachidonic Acids; D004791:Enzyme Inhibitors; D063065:Organophosphonates; D013439:Sulfhydryl Reagents; C104810:methyl arachidonylfluorophosphonate; D016718:Arachidonic Acid; D000431:Ethanol; D010741:Phospholipases A; D054467:Phospholipases A2; D005990:Glycerol; D002118:Calcium; D004229:Dithiothreitol