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Originally published In Press as doi:10.1194/jlr.M200298-JLR200 on October 1, 2002

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Journal of Lipid Research, Vol. 44, 109-117, January 2003
Copyright © 2003 by Lipid Research, Inc.

Brain lipid metabolism in the cPLA2 knockout mouse

Thad A. Rosenberger1,*, Nelly E. Villacreses*, Miguel A. Contreras*, Joseph V. Bonventre{dagger} and Stanley I. Rapoport*

* Brain Physiology and Metabolism Section, National Institute on Aging, National Institutes of Health, Bethesda, MD 20892
{dagger} Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129

Published, JLR Papers in Press, October 1, 2002. DOI 10.1194/jlr.M200298-JLR200

1 To whom correspondence should be addressed. e-mail: plsetn{at}mail.nih.gov


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 Results
 Discussion
 REFERENCES
 
We examined brain phospholipid metabolism in mice in which the cytosolic phospholipase A2 (cPLA2, Type IV, 85 kDa) was knocked out (cPLA2-/- mice). Compared with controls, these mice demonstrated altered brain concentrations of several phospholipids, reduced esterified linoleate, arachidonate, and docosahexaenoate in choline glycerophospholipid, and reduced esterified arachidonate in phosphatidylinositol. Unanesthetized cPLA2-/- mice had reduced rates of incorporation of unlabeled arachidonate from plasma and from the brain arachidonoyl-CoA pool into ethanolamine glycerophospholipid and choline glycerophospholipid, but elevated rates into phosphatidylinositol. These differences corresponded to altered turnover and metabolic loss of esterified brain arachidonate. These results suggests that cPLA2 is necessary to maintain normal brain concentrations of phospholipids and of their esterified polyunsaturated fatty acids.

Reduced esterified arachidonate and docosahexaenoate may account for the resistance of the cPLA2-/- mouse to middle cerebral artery occlusion, and should influence membrane fluidity, neuroinflammation, signal transduction, and other brain processes.

Abbreviations: AA, arachidonic acid; CerP Cho, sphingomyelin; ChoGpl, choline glycerophospholipid; DHA, docosahexaenoic acid; DG, diacylglycerol; EtnGpl, ethanolamine glycerophospholipid; PAF, platelet activating factor; PLA2, phospholipases A2; cPLA2, cytosolic phospholipase A2; PlsCho, plasmenylcholine; PtdCho, phosphatidylcholine; PlsEtn, plasmenylethanolamine; PtdEtn, phosphatidylethanolamine; PtdIns, phosphatidylinositol; PtdSer, phosphatidylserine; PUFA, polyunsaturated fatty acid; sPLA2, secretory phospholipase A2; TG, triacylglycerol

Supplementary key words arachidonate • docosahexaenoate • phospholipase A2 • phospholipid • turnover • kinetics


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 Results
 Discussion
 REFERENCES
 
Phospholipases A2 (EC 3.1.1.4; PLA2) hydrolyze fatty acids from the sn-2 position of phospholipids to form non-esterified fatty acids and lysophospholipids. PLA2 are classified into eleven types, based on their catalytic activity, amino acid sequence, sequence homology, and mRNA splice variants (1, 2, 3). Types I, II, III, V, IX, X, and XI have low molecular weights, require Ca2+ for activity, and have a histidine residue at their catalytic site. Types IV, VI, VII, and VIII have higher molecular weights and are localized in the cell cytosol. These enzymes utilize a serine residue for catalytic activity and may or may not be Ca2+-dependent (4). In mammalian brain, mRNA levels indicate that PLA2 types IIA, IIC, IV, and VI PLA2 are widely expressed, whereas Type V PLA2 is expressed at low levels except in the hippocampus (5). The potential of the mammalian brain to modify the expression of the different PLA2 isoforms provides it with a certain redundancy in the regulation of fatty acid and phospholipid metabolism.

Activation of PLA2 can release arachidonic acid (AA; 20:4 n-6) or docosahexaenoic acid (DHA; 22:6 n-3) from the sn-2 position of membrane phospholipids in brain glia or neurons (4, 6–9). Activation can also produce lyso-platelet activating factor (lysoPAF), which when esterified at its sn-2 position becomes PAF, a potent neurotransmitter (10). PLA2 can be activated via membrane G proteins when certain neurotransmitters bind to specific neuroreceptors, or when Ca2+ enters cells following glutamate binding to NMDA receptors or acetylcholine binding to nicotinic receptors (11–15).

Because the different PLA2 isoforms in brain do not function interchangeably (4), knockout strategies might help to identify their separate roles (16). In this paper, we examine brain lipid metabolism in a mouse in which the 85 kDa type IVA cytostolic PLA2 (cPLA2) is absent (cPLA2-/- mouse) (16–18). cPLA2 is selective for AA over other fatty acids (1) and requires both Ca2+ and phosphorylation for full activation (3, 19). It is involved in neuroreceptor initiated signaling (7, 14), and its transcription is downregulated in rat brain by LiCl administration (7, 14, 20, 21).

The neurological development of the cPLA2-/- mouse is said to be normal (22). The female knockout mouse has a reduced reproductive ability, whereas adult males are more resistant to middle cerebral artery occlusion than are controls (17, 18, 22). The cPLA2-/- mouse is also more resistant to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)- induced dopamine depletion (23), and recovers more rapidly from allergen-induced bronchoconstriction. Peritoneal macrophages and mast cells in the cPLA2-/- mouse fail to produce eicosanoids in response to stimulation (22), suggesting a defective AA cascade (24, 25). Thus, by identifying alterations in brain lipid metabolism in the cPLA2-/- mouse, we may better understand the role of cPLA2 in brain structure and function.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
Results
 Discussion
 REFERENCES
 
Chemicals
[5,6,8,9,11,12,14,15-3H]Arachidonic acid ([3H]AA, 240 Ci· mmol-1, >=98% pure) was purchased from Moravek Biochemicals (Brea, CA). Scintillation counting and GC analysis confirmed tracer specific radioactivity. Phospholipid and neutral lipid standards were from Nu-Chek-Prep (Elysian, MN) and "fatty acid free" bovine serum albumin was from Sigma Chemicals (St. Louis, MO). HPLC grade n-hexane and 2-propanol were from EM Science (Gibbstown, NJ). Reagent grade chloroform, methanol, and other chemicals were from Mallinckrodt (Paris, KY) unless noted otherwise. A scintillation cocktail (Ready-Safe, Beckman, Fullerton, CA) containing 1.0% glacial acetic acid was used to determine radioactivity. Extracts were stored in n-hexane/2-propanol (3:2, v/v) + 5.5% H2O under N2 at -80°C.

Animals and surgery
Surgery was performed as previously described (26), following the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication no. 80–23), on 3-month-old control (C57Bl/6n) and cPLA2-/- mice (Charlestown Animal Unit, Massachusetts General Hospital, Boston, MA). Mice were anesthetized with 2–3% halothane (Halocarbon, River Edge, NJ). Polyethylene catheters (PE 10, Becton Dickinson, Sparks, MD) filled with saline containing sodium heparin (100 IU) were implanted into the right femoral artery and vein. The skin was closed and 1% lidocaine was applied as a local anesthetic. The animals were loosely wrapped in a fast-setting plaster body cast, taped to a wooden block, and allowed to recover from anesthesia for 3–4 h. Body temperature was maintained at 36.5°C using a feedback-heating device (Yellow Springs Laboratories, Yellow Springs, OH).

The mouse strains C57Bl/6n and 129/Sv have a naturally occurring defect in the gene encoding the group IIA secretory PLA2 (sPLA2), due to a frame shift mutation in exon 3 that results in a T-insertion at position 166 and terminates out of frame in exon 4 (27). The cPLA2-/- mouse was created using these strains and the 129/Sv cDNA library. Therefore, both the control and cPLA2-/- mice in this study were deficient in group IIA sPLA2 (22).

Infusion of [3H]AA
With an infusion pump (Harvard Apparatus, South Natick, MA), unanesthetized mice were infused intravenously for 5 min at a rate of 30 µl·min-1, with 150 µl isotonic saline containing 7.5 mCi·kg body wt-1 [3H]AA suspended in 0.06 mg bovine serum albumin. Arterial samples were collected at fixed times during infusion to determine the radioactivity and concentrations of non-esterified fatty acids and lipids in whole blood and plasma. Five min after starting infusion, mice were killed by an overdose of sodium pentobarbital (100 mg·kg body wt-1, iv) and immediately subjected to head-focused microwave irradiation to stop brain metabolism (5.5 kW, 1.2 s; Cober Electronics, Stamford, CT). The brain was excised, frozen on dry ice, and stored at -80°C.

Brain lipid extraction and chromatography
Total lipids from microwaved brains were extracted using n-hexane/2-propanol (3:2, by vol) in a glass Tenbroeck homogenizer (28). Standards and samples in chloroform were applied to Whatman silica gel 60A LK6 TLC plates and separated using chloroform-methanol-acetic acid-H2O (50:37.5:3:2, v/v/v/v) (29). Bands corresponding to ethanolamine glycerophospholipids (EtnGpl), phosphatidylinositol (PtdIns), phosphatidylserine (PtdSer), choline glycerophospholipids (ChoGpl), and sphingomyelin (CerP Cho) were scraped from the TLC plates. The EtnGpl and ChoGpl fractions were extracted from the silica gel using n-hexane/2-propanol (3:2, v/v) + 5.5% H2O, combined, and concentrated with N2 at 55°C. They then were subjected to mild acid hydrolysis over hydrochloric acid fumes to cleave the vinyl ether linkage of the plasmalogens. Phosphatidylethanolamine (PtdEtn), lysoplasmenylethanolamine representing plasmenylethanolamine (PlsEtn), phosphatidylcholine (PtdCho), and lysoplasmenylcholine representing plasmenylcholine (PlsCho) fractions were purified using HPLC (30). Samples were used to quantify fatty acids by GC, radioactivity by liquid scintillation counting, and phospholipid levels by measuring lipid phosphorus (31).

Quantification of labeled and unlabeled acyl-CoA
Acyl-CoA species were isolated from mouse brain extracts using oligonucleotide purification cartridges (Applied Biosystems, Foster City, CA) as previously described (32). Concentrations of acyl-CoA and associated radioactivity were measured using peak area analysis from HPLC chromatograms and liquid scintillation counting. These values were used to calculate the specific radioactivity of arachidonoyl-CoA.

Extraction and separation of plasma
Plasma lipids were extracted by the method of Folch (33). The neutral lipids were separated by TLC on silica gel 60 plates using the solvent system of heptane/diethyl ether/acetic acid (60:40:4, v/v/v) (34). Individual phospholipid classes were separated as previously described. Non-esterified fatty acids and esterified fatty acid in plasma were quantified using gas chromatography, and radioactivity was quantified using liquid scintillation counting.

Tracer identification
Tracer identification and separation was performed on phenacyl ester derivatives of plasma and brain extracted lipids as described previously (35, 36). Phenacyl esters were separated on a LunaTM C18 column (Phenomenex, Torrance, CA) using a linear gradient of acetonitrile and H2O (20% H2O at time zero to 8% H2O over 50 min) on a HPLC (Beckman, Fullerton, CA) equipped with an in-line UV/VIS detector ({lambda} = 242 nm, Gilson, Middleton, WI) and in-line scintillation counter (ß-RAM, IN/US System, Tampa, FL). The phenacyl ester of [3H]arachidonic acid was used to identify tracer radioactivity found in plasma and brain extracts.

Methylation of esterified and non-esterified acids
Esterified fatty acids in samples of the different phospholipid classes were methylated with 0.5 M methanolic potassium hydroxide at 37°C for 30 min. The reaction was stopped with methylformate, and the fatty acid methyl esters were extracted with n-hexane. The non-esterified fatty acids and the esterified fatty acids found in the CerP Cho fractions were methylated using 2% sulfuric acid in toluene-methanol (1:1, v/v) at 65°C for 2 h. The reaction was terminated with H2O and the fatty acid methyl esters were extracted with petroleum ether.

Gas chromatography of fatty acid methyl esters
Fatty acid methyl esters were quantified using a gas chromatograph (Trace 2000, ThermoFinnigan, Houston, TX) equipped with a capillary column (SP 2330; 30 m x 0.32 mm id, Supelco, Bellefonte, PA) and a flame ionization detector. Sample runs were initiated at 90°C with a temperature gradient to 230°C over 20 min. Fatty acid methyl ester standards were used to establish relative retention times and response factors. The internal standard, methyl heptadecanoate, and the individual fatty acids were quantified by peak area analysis. The detector response was linear with correlation coefficients of 0.998 or greater within the sample concentration range for fatty acid standards of differing chain length and degree of saturation.

Calculations
Radioactivity of a brain lipid of interest was calculated by correcting its net brain radioactivity for its intravascular radioactivity (the product of its whole blood radioactivity multiplied by brain blood volume, 2.0 x 10-2 ml·g-1) (37). Blood samples taken at the time of death (5 min after infusion began) were extracted and analyzed to make this correction.

The model for determining in vivo kinetics of brain fatty acids in rats has been described elsewhere (38, 39). Briefly, unidirectional incorporation coefficients, Ki*(ml·s-1·g-1) of [3H]AA from plasma into "stable" lipid compartments i were calculated as follows,

c*br,i(T ) (nCi·g-1) is brain radioactivity of lipid i at time T = 5 min (time of termination of experiment), t is time after beginning of infusion, and c*pl (nCi·ml-1) is the plasma concentration of labeled AA during infusion. Rates of incorporation of non-esterified AA from plasma into brain phospholipid i, Jin,i, and from the brain arachidonoyl-CoA into brain phospholipid i, JFA,i, were calculated as follows,


Cpl (nmol·ml-1) is the concentration of unlabeled non-esterified AA in plasma. The "dilution factor" {lambda} is defined as the steady-state ratio during [3H]AA infusion of the specific activity of the brain arachidonoyl-CoA pool to plasma specific activity,

where the numerator is the ratio of brain arachidonoyl-CoA radioactivity to the unlabeled brain arachidonoyl-CoA concentration. The fractional turnover rate of AA within phospholipid i, FFA,i (%·h-1), is defined as,

The half-life of the FA in i is defined as,

Data and statistics
Integrals of plasma radioactivity were determined by trapezoidal integration, and plasma half-lives were determined by fitting the following equation,

to plasma radioactivity, where c*pl(t=5) is steady-state plasma radioactivity and ß is a time constant (SigmaPlot, SPSS Science, Chicago, IL). Unpaired Student's t-tests (Instat® Ver. 3.05, GraphPad, San Diego, CA) were used to compare means between cPLA2-/- and control mice, where statistical significance was taken as P <= 0.05. Data are presented as mean ± SD.


   Results
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Results
Discussion
 REFERENCES
 
Plasma and brain non-esterified fatty acids, and brain acyl-CoAs
There was no statistically significant difference in the mean concentration of any plasma or brain non-esterified fatty acid between cPLA2-/- and control mice (Table 1). Additionally, there was no significant difference in the mean brain concentration of any long chain acyl-CoA between the two groups. The ratio of steady-state mean specific activity of arachidonoyl-CoA to plasma AA specific activity, represented by the term {lambda} (Eq. 4), also did not differ significantly between the groups (Table 1). A value for {lambda} equal to 0.04 indicates that about 4% of arachidonoyl-CoA is derived from plasma, with 96% derived by release from phospholipids (38, 39).


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  		TABLE 1. Plasma and brain non-esterified fatty acid, and brain acyl-CoA levels in control and cPLA-/- mice

 
Mean arterial plasma radioactivity profiles over time during intravenous [3H]AA infusion in cPLA2-/- and control mice are illustrated in Fig. 1 . Steady-state radioactivity, achieved after 180 and 300 s following the start of tracer infusion, equaled 453 ± 42 nCi·nmol-1 in cPLA2-/- mice and 640 ± 37 nCi·nmol-1 in controls (P < 0.001). As determined by HPLC analysis of phenacyl ester derivatives in plasma and brain fatty acids from the cPLA2-/- (Fig. 2A) and control (data not shown) mice, greater than 97% (n = 6) of the radioactivity in plasma and brain extracts was AA at 5 min (Fig. 2B, 2C, respectively). Integrated plasma radioactivity between 0 and 5 min was 27% less in the cPLA2-/- than in control mice (P < 0.01), consistent with a 35% shorter plasma half-life, 1.05 ± 0.16 min compared with 1.61 ± 0.46 min in the controls (P < 0.01).



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  		Fig. 1. Time course of plasma radioactivity (nCi·ml-1) in control and cPLA2-/- mice during intravenous infusion of 7.5 mCi·kg body wt-1 [3H]arachidonic acid over 5 min. Values are the mean ± SD (n = 6).

 


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  		Fig. 2. Representative chromatogram of phenacyl ester derivatives from brain extract (A) and the corresponding radioactivity profiles found in plasma fatty acids (B) and brain fatty acids (C) in cPLA2 knockout mice infused with 7.5 mCi·kg body wt-1 [3H]arachidonic acid. Abbreviations: 18:3n-3, alpha linolenic acid; 22:6n-3, docosahexaenoic acid; 20:4n-6, arachidonic acid; 16:1n-7, palmitoleic acid; 18:2n-6, linoleic acid; 16:0, palmitic acid; 18:1n-9, oleic acid; 18:0, stearic acid.

 
Brain phospholipids
Several statistically significant differences were found between cPLA2-/- and control mice in mean brain phospholipid concentrations, although the net brain phospholipid concentration did not differ between the two groups (Table 2). Mean concentrations of PtdIns, PtdSer, and CerP Cho were increased significantly by 12.5%, 12.8%, and 22.5%, respectively, in the cPLA2-/- mice, whereas mean concentrations of EtnGpl and ChoGpl did not differ from control values. Mean brain concentrations each of the PtdEtn, PlsEtn, PtdCho, and PlsCho fractions also did not differ between the groups (data not shown).


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  		TABLE 2. Brain phospholipid levels in control and cPLA2-/- mice

 
Esterified fatty acids
No significant concentration difference was found in any esterified fatty acid within EtnGpl (Table 3). In PtdIns esterified AA was reduced by about 368 nmol·g-1 (35%), whereas esterified stearate was increased by 459 nmol·g-1 and esterified palmitate by 70 nmol·g-1, apparently compensating for the AA decrease. In PtdSer, the only significant difference was a 30% increase in esterified stearate. In ChoGpl, differences were noted in all esterified fatty acids but oleate. Esterified linoleic acid (18:2 n-6) was reduced by about 99 nmol·g-1 (36%), AA by 671 nmol·g-1 (59%), DHA by 724 nmol·g-1 (66%), and stearate by 1,001 nmol·g-1. A compensatory increase of about 2,589 nmol·g-1 was evident in esterified palmitate. The esterified concentration differences in ChoGpl between the cPLA2-/- and control mouse could be ascribed to differences in PtdCho, as no significant concentration difference was noted in PlsCho (data not shown).


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  		TABLE 3. Esterified fatty acid concentrations in brain phospholipids of Control and cPLA2-/- mice

 
AA incorporation into and turnover within individual brain phospholipids
The significant reduction of esterified AA in brain PtdIns in the cPLA2-/- mouse was accompanied by an increased rate of incorporation Jin,i of AA from plasma (Table 4). This suggests that steady-state loss of AA from brain PtdIns by metabolism was accelerated. The increased Jin,i corresponded to increased AA incorporation from the arachidonoyl-CoA pool, JFA,I, and a 2-fold increase in AA turnover, FFA,i (Table 5).


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  		TABLE 4. Brain incorporation coefficients, net incorporation rates from plasma non-esterified arachidonic acid, and net incorporation rates from brain arachidonoyl-CoA in control and cPLA2-/- mice.

 

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  		TABLE 5. Turnover rates of arachidonic acid in brain phospholipids of control and cPLA2-/- mice

 
Although AA incorporation rates into ChoGpl from plasma and from the arachidonoyl-CoA pool were decreased in the cPLA2-/- mouse, AA turnover in ChoGpl was elevated, reflecting the disproportionate reduction of esterified AA (Table 5). The increases were found in the PtdCho but not the PlsCho fraction of ChoGpl, in which AA turnover actually was decreased (data not shown). AA incorporation rates into EtnGpl from both plasma and brain arachidonoyl-CoA were reduced about 3-fold in the cPLA2-/- mouse, consistent with a markedly reduced turnover. The reduction reflected reduced turnover in PtdEtn, as turnover in the PlsEtn fraction was increased (data not shown).


   Discussion
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Results
 Discussion
REFERENCES
 
Despite marked differences in brain phospholipid and fatty acid composition, and in the kinetics of AA within certain brain phospholipids, the cPLA2-/- mice demonstrated no significant difference from controls in plasma or brain concentrations of non-esterified fatty acids, brain levels of acyl-CoA, or the dilution factor {lambda} of brain arachidonoyl-CoA. Thus, measuring only the concentrations of plasma and brain non-esterified fatty acids or of brain acyl-CoA would have provided little evidence of the markedly altered brain lipid composition and AA kinetics in the knockout animal. The only evidence in plasma of altered AA metabolism was a more rapid rate of disappearance of injected [3H]AA than in controls, corresponding to a 35% shorter plasma half-life (Fig. 1).

Our kinetic fatty acid model showed increased rates of incorporation of non-esterified AA from plasma and from brain arachidonoyl-CoA into PtdIns in the cPLA2-/- mouse (Table 4), and increased turnover of AA within PtdIns and ChoGpl despite the lower concentrations of esterified AA. As Jin,i represents the rate of replacement by plasma AA of the brain esterified AA that is released and metabolized (40), esterified AA within PtdIns must be hydrolyzed and then metabolized more rapidly than controls. Our study also indicated that AA within ChoGpl turns over at a higher than normal rate in the cPLA2-/- mouse. Clearly, the absence of cPLA2 resulted in increased metabolic loss of brain AA, whether by conversion to eicosanoids or by other metabolic pathways (24).

These data suggest that cPLA2 is involved in the maintenance of esterified AA in PtdIns and ChoGpl. However, the increased incorporation and turnover rates of AA in PtdIns and turnover of ChoGpl in the cPLA2-/- mice, despite a reduced esterified AA concentration, suggest that other PLA2 isoforms may compensate for the absent cPLA2 activity in regulating metabolism of these phospholipid classes. Indeed, cPLA2 (41), as well as types IIA or V sPLA2, can augment immediate and delayed AA release in response to cytokines and fetal calf serum (42, 43). The different PLA2 isoforms can also hydrolyze AA from phospholipid with selectivity for different phospholipid classes (2, 4, 44). Furthermore, other lipolytic enzymes expressed in brain, specifically phosphatidylinositol-specific phospholipase C (45, 46) and phospholipase D (47), can selectively metabolize either PtdIns or PtdCho, respectively. Therefore, redundancy arising because of multiple isoforms of PLA2 and/or compensation by other lipolytic enzymes expressed in brain might contribute to the increased turnover of brain PtdIns and ChoGpl in cPLA2-/- mice.

Despite evidence that cPLA2 is more selective for AA than DHA (1), the absence of cPLA2 activity throughout life resulted in marked reductions in both esterified AA and DHA. This suggests that the metabolic pathways of each of these two polyunsaturated fatty acids (PUFAs) are interactive and in part regulated by cPLA2. For example, inhibition by DHA of the conversion of AA to eicosanoids by cyclooxygenase-2 or 5-lipoxygenase has been reported (48, 49). Competition between n-6 and n-3 PUFA acyl-CoA can also occur at the level of desaturation and elongation (50–52), in that during nutritional n-3 deprivation, brain AA will be converted to eicosapentaenoic acid (22:5 n-6) (53). However, the absence of esterified 22:5n-6 due to the lack of retrograde conversion or acyl chain elongation of [3H]AA in the brain of cPLA2-/- (Fig. 2) and control mice (data not shown) are consistent with normal concentrations of brain acyl-CoA and plasma non-esterified fatty acid in the cPLA2-/- mouse (Table 1).

Thus, the interaction between AA and DHA metabolism appears not to occur at the level of recycling within brain phospholipids. In this regard, rats subjected to nutritional n-3 PUFA deprivation for three generations have an reduced turnover of DHA but unaffected AA turnover within brain phospholipids (53), whereas chronic administration of LiCl to rats reduces AA turnover without affecting DHA turnover (54, 55). The independent recycling of AA and DHA within phospholipids is consistent with the presence of an AA-specific PLA2 (1, 56, 57) and an AA-specific arachidonoyl-CoA synthetase (58, 59). In the cPLA2-/- mouse, on the other hand, compensation from other phospholipid-metabolizing enzymes with differing acyl-chain specificity may account for the observed changes in both AA and DHA composition. Further studies are required to investigate the effects that the absence of cPLA2 has on the activity and expression of these enzymes, and on their ability to maintain plasma non-esterified fatty acid and lambda AA-CoA at control levels despite the apparent reduction in the plasma half-life of [3H]AA and the altered turnover rates of brain phospholipid. Additionally, we should determine incorporation and turnover rates of DHA in brain phospholipids of the cPLA2-/- mouse. The question remains as to what extent the alterations found in the knockout mouse are due to the lack of cPLA2, compared with compensatory effects by other enzymes.

The marked reductions in the cPLA2-/- mouse of esterified linoleic acid, AA, and DHA in brain ChoGpl and of AA in PtdIns, and abnormalities in brain phospholipid composition, would be expected to alter brain function and structure (60). Although neurological development is said to be normal in the cPLA2-/- mouse (22), sophisticated memory and behavioral tests have not been performed on these animals. In the absence of altered brain function, the involvement of cPLA2 and the relevance of the observed alterations in brain phospholipid metabolism to normal brain physiology are not clear. The apparent increase in the rate of incorporation and turnover of AA in PtdIns and increased turnover of ChoGpl, suggest that there are compensatory changes in other phospholipid-metabolizing enzymes. However, the reported resistance of the knockout mouse to middle cerebral artery occlusion (17, 18) may arise from reduced availability and release of esterified AA during insult as a result of the absence of cPLA2 activity (61–63). This interpretation is consistent with evidence that chronically administered LiCl in rats increases their resistance to cerebral ischemia (64), while decreasing brain cPLA2 expression and AA turnover (21, 54).

Differences in esterified brain PUFA concentrations in the cPLA2-/- mouse also would be expected to alter brain membrane fluidity, receptor function, membrane remodeling, neuroplasticity, and resistance to apoptosis, among other processes (10, 65, 66). The absence of cPLA2 can affect receptor-initiated signaling processes in which it participates to release AA and initiate the AA cascade (7, 14, 24, 25). The reported absence of eicosanoid formation in stimulated macrophages and mast cells from the cPLA2-/- (22) is consistent with this prediction. Alterations in the AA cascade may also account for the reduced female fertility in the cPLA2-/- mouse (18), as feto-placental development involves cPLA2 activation and prostaglandin formation (67). Therefore, the reported resistance of the cPLA2-/- mouse to middle cerebral artery occlusion, absence of eicosanoid formation, and reduced female fertility rates can be attributed to the loss of cPLA2 activity. However, whether the quantitative contribution of the observed changes in phospholipid metabolism found in these studies are due to the primary knockout or to secondary compensatory mechanisms is at this point unclear. Further studies measuring the changes in behavior, turnover of other brain fatty acids including DHA, and expression patterns of those enzymes associated with PtdIns and PtdCho metabolism will help us better understand the role that cPLA2 has in brain function.

This paper extends of our kinetic fatty acid method, developed in unanesthetized rats (38, 39) to unanesthetized mice. We found that the brain concentrations of phospholipids, esterified and non-esterified fatty acids, and acyl-CoA species in control mice were comparable to the respective concentrations reported in rats (53, 68). Derived kinetic parameters for AA were also comparable, with the exception of AA turnover within EtnGpl (53, 69). Control AA turnover within each of the phospholipid classes ranged from 4.8%·h-1 (EtnGpl) to 1.3%·h-1 (PtdIns) in the mouse brain (Table 4), equivalent to half-lives (Eq. 6) of 5.5 h (PtdIns) to 14 h (EtnGpl). In rat brain, turnover rates of 9.4·h-1, 6.0·h-1, 5.4·h-1, and 0.3%·h-1 have been reported for ChoGpl, PtdSer, PtdIns, and EtnGpl, respectively. In both species, calculated half-lives of hours are many fold shorter than half-lives days or weeks found when fatty acid recycling with brain phospholipids was ignored (38, 70, 71).

The 10-fold higher AA turnover in EtnGpl in control mice than in control rats may represent a species difference, as the distribution of radioactivity in brain EtnGpl and ChoGpl following the intracerebral injection of [1-14C]arachidonic acid (72) was similar to ours following intravenous injection. Alternatively, the difference may reflect that our control mice were deficient in group IIA sPLA2 (22, 27), since phosphatidylethanolamine may be the preferred phospholipid substrate for sPLA2 (73, 74). Another possibility is that the difference reflected a smaller fraction of white matter myelin in the mouse brain compared to the rat brain, and a more rapid turnover of EtnGpl in gray than white matter (68, 75).

In summary, a knockout animal may not provide a clear understanding of the normal role of a specific gene product, as its phenotype can result from primary gene loss as well as secondary changes during development and maturation (16). In this regard, a 50% reduction in brain cPLA2 transcription in the adult rat, caused by 6 weeks of LiCl administration, did not alter esterified brain concentrations of either AA or DHA, while reducing AA but not DHA turnover in brain phospholipids (20, 21, 54) and suggests that a conditional cPLA2 knockout, when available, might better elucidate the role of cPLA2 in brain than the lifetime knockout (16). Nevertheless, the absence of cPLA2 throughout development and maturation results in multiple changes involving brain membrane phospholipid composition, reductions in the levels of brain esterified AA and DHA, and alterations in the kinetics of brain AA metabolism. Finally, these results identify multiple ways in which the cPLA2-/- phenotype can be further examined so as to better understand how cPLA2 regulates brain structure, modulates signal transduction, as well as aid in future studies investigating its role in mechanisms associated with injury.

Manuscript received July 29, 2002 and in revised form September 3, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Results
 Discussion
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