CDPcholine and CDpethanolamine prevent the release of free fatty acids during brain ischemia

CDPCHOLINE AND CDPETHANOLAMINE PREVENT THE RELEASE OF FREE FATTY ACIDS DURING BRAIN ISCHEMIA LLOYD A. HORROCKS, ROBERT V. nORMAN, ZBIGNIEW DABROWIECKI, GIANFRANCESCO GORACCI a n d GIUSEPPE PORCELLATI Department of Physiological Chemistry, The Ohio State University, Columbus, Ohio 43210 and Institute of Biochemistry, Faculty of Medicine, University of Perugia, Italy

Ischemia markedly increases the concentration of free fatty acids in rat brain. 2"4 After bilateral ligation of the carotoid arteries of gerbils, the content of free arachidonate increases linearly for about 5 rain to a 3-fold higher level) 1 The content of esterified arachidonate includes 1.8/zmoles in phosphatidylcholines, 1.8/~moles in ethanolamine plasmalogens, 0.17 pmoles in phosphatidylinositols, and 0.16/~moles in diacylglycerols. During the first 30 sec of ischemia, 0.10/~moles of arachidonate are lost from phosphatidylinositols. Ethanolamine plasmalogens are the source of 0.34/~moles between 30 and 60 sec followed by 0.40/anoles of arachidonate from phosphatidylcholines between 60 and 180 sec. The content of arachidonate in diacylglycerols does not change during this period. 11 Some of the released arachidonate is converted to oxygenated metabolites by cyclooxygenese and lipoxygenase pathways. 6 Some of these compounds may be associated with the pathogenesis of the inflammation, edema, and demyelination that result from ischemia. A new pathway 7'9 for release of free fatty acids, particularly from phosphatidylcholines, may be activated during ischemia. Choline phosphotransferase (Fig. 1) produces phosphatidylcholine. The equilibrium constant for this reversible reaction is: [phosphatidylcholine] [CMP] Keq = [CDPcholine] [diacylglycerols]" Under normal conditions, CMP is phosphorylated and thus, not available for the reverse reaction. However, during ischemia, the diacylycerols may be removed by diacylglycerol lipase a and the CMP may not be removed due to a decreased concentration of ATP.13 Additional CMP may arise from hydrolysis of CDPcholine. 11 Ethanolamine plasmalogens are hydrolyzed by plasmalogenase s and the activity of this enzyme increases markedly after ligation of the carotid arteries of the gerbil.I° The activity of phospholipase A2 with phosphatidylethanolamine increases during the first min of ischemia 5 but the activity is much lower than that of plasmalogenase and diacylglycerol lipase. If a major portion of the release of free fatty acids is due to reversal of phosphotransferases and the action of diacylglycerol lipase, the intracerebral injection of CDPcholine and CDPethanolamine should decrease this release. The fast turnover pools of Phosphocholinetronsferose,

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532

L.A. Horrocks et al.

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brain lipids were prelabeled by intracerebral injection of 37 nmol [3H]acetate (5.01 #Ci) into 21-23 day old male rats. Both nucleotides decreased the amount of radioactivity in the free fatty acid fraction at 1, 3 and 5 min after injection of 0.25 or 1.0/zmoles (Fig. 2). A mixture of both nucleotides was tested with a decapitation ischemia model. Compared with controls, ischemia decreased the labeling of choline glycerophospholipids by 22% and ethanolamine glycerophospholipids by 31%. The nucleotides stimulated labeling of these glycerophospholipids by 43% and 51%, respectively. In ischemic brains, the nucleotides prevented the loss of radioactivity from choline and ethanolamine glycerophospholipids (Fig. 3). Following complete ischemia, the radioactivity in free fatty acids 60 I

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FIG. 3. The effects of ischemia and of a mixture of CDPethanolamine and CDPcholine on the labeling of rat brain phospholipids. Intracerebral injections of PHJacetate were given 2 hr before decapitation. Five min before decapitation, intracerebral injections of saline or 0.6 gmoles each of CDPethanolamine and CDPcholine were given. The rat heads were either placed immediately into liquid nitrogen or were incubated for 5 rain at 37°C before freezing.

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was increased by 123% as expected from previous results (Fig. 4). The mixture of CDPcholine and CDPethanolamine reduced the labeling of flee fatty acids in iscbemic brains to 41% of the saline control value and to 18% of the ischemia control value. The nucleotides increased the labeling of monoglyceride and diglyceride fraction in both ischemic and control rat brains. The prevention of lipid alterations by the CDP-bases suggests that reversal of the choline and ethanolamine phosphotransferases is involved in ischemia (Fig. 5). Diglycerides may be liberatedand then react with diacylglycerollipase to releasefatty acids.The increased labeling of choline and ethanolamine glycerophospholipids after injection of CDP-bases also supports phosphotransferase involvement. Ansell and Chojnacki ~ have reported incorporation into these phospholipids after intracerebralinjection of [sZP]labeled CDPcholine and CDPethanolamine. Increased acylation may also take place after injectionof CDP-bases. The incorporation of glycero-3-phosphate into lipidis stimulated

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L.A. Horrocks et al.

by C D P c h o l i n c in t ' i t r o . ' 2 F u r t h c r e x p e r i m e n t s o n the elTects o f C D P - b a s e s o n p l a s m a l o 8¢n m c t a b o l i s m and o n the g e r b i l m o d e l o f ischemia are in prosress.

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