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Alternative pathways for phospholipid synthesis in different brain areas during aging
M.G. Ilincheta de Boschero et al. / Experimental Gerontology 35 (2000) 653–668
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Experimental Gerontology 35 (2000) 653–668 www.elsevier.nl/locate/expgero
Alternative pathways for phospholipid synthesis in different brain areas during aging M.G. Ilincheta de Boschero, M.E. Roque, G.A. Salvador, N.M. Giusto* Instituto de Investigaciones Bioquı´micas de Bahı´a Blanca (INIBIBB), Universidad Nacional del Sur, CONICET, Camino La Carrindanga Km 7 B8000FWB, Bahı´a Blanca, Buenos Aires, Argentina Received 7 December 1999; received in revised form 16 February 2000; accepted 4 April 2000
Abstract Morphological and biochemical changes take place in the membrane of aged brain. In particular, studies on aged rats report alterations in brain phospholipid synthesis and in phospholipid-specific fatty acid composition. However, no significant changes in main phospholipid class content have been reported in aged brain, possibly owing to alterations in the alternative pathways for phospholipid synthesis during aging. Therefore, the present study was designed to determine the effect of aging on the enzyme activities responsible for phospholipid synthesis by alternative pathways. Indifferent brain areas of adult (3.5-month-old) and aged (28.5-month-old) rats we examined: 1) the activity of base exchange enzymes, which is a calcium-dependent, energy-independent and calcium stimulated enzymatic pathway; 2) phosphatidylethanolamine (PE) synthesis by phosphatidylserine decarboxylase activity (PSD); 3) phosphatidylcholine (PC) synthesis by transfer of methyl groups to endogenous PE by phosphatidylethanolamine N-methyltransferase activity (PEMT); 4) the synthesis of phosphatidylglycerol (PG) through phospholipase D (PLD) activity. Because the dependence on and the stimulation by calcium of base-exchange reactions is a well known mechanism and alterations in calcium levels in rat brain have been reported, we decided to investigate PS synthesis in the presence of endogenous and exogenous calcium (2.5 mM). PS synthesis increased in cerebral cortex (CC) and cerebellum (CRBL) of aged rats with respect to adult rats in basal conditions (without the addition of exogenous calcium), but more significant changes were observed in serine base exchange activity during aging when exogenous calcium was added. PEMT activity in aged CC increased by 100%, the principal modification being observed in the first methylated product of the sequential reaction. Futhermore, the transphosphatidyl reaction was higher in aged brain as indicated by the increased PG synthesis. Our findings allow us to conclude that age affects some alternative pathways for phospholipid synthesis in the central nervous system, and indicate the presence of a * Corresponding author. Tel.: 154-291-4861201; fax: 1 54-291-4861200. E-mail address: [email protected] (N.M. Giusto). 0531-5565/00/$ - see front matter q 2000 Elsevier Science Inc. All rights reserved. PII: S0531-556 5(00)00104-2
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compensatory mechanism to provide a pool of phospholipid classes for the maintenance of cellular membrane lipid composition during aging. q 2000 Elsevier Science Inc. All rights reserved. Keywords: Aging; Brain; Lipid metabolism; Phospholipids; Phospholipid metabolism
1. Introduction Age inevitably affects all organs, but the brain seems particularly susceptible and is of course central to the problem. The vulnerability of the brain increases rapidly with age, and its deterioration is made manifest in many ways. One intriguing feature of the aging process is the modification of cellular membrane properties. It has been observed that brain membrane lipid fatty acid composition and consequently membrane fluidity change with increasing age (Kumar et al., 1999) and that alterations in brain specific fatty acid binding protein levels in synaptosomal plasma membranes and synaptosomal cytosol may be important factors modulating neuronal differentiation and function (Pu et al., 1999). Several enzyme activities are modified during the aging process. The plasma membrane Ca 21-ATPase plays a critical role in Ca 21 homeostasis, and its kinetic properties change in aged rat brain, affecting the regulation of free intracellular calcium (Zaidi and Michaelis, 1999). Delta-9 desaturase activity is reduced up to 50% with age, such decrease is known to cause alterations in membrane fluidity and to affect cellular signaling pathway (Kumar et al., 1999). Age-related alterations in antioxidant enzyme has also been reported recently (Yargicoglu et al., 1999). All these age-related changes seem to correlate with the aging of the nervous system. Studies carried out on rats up to 18 months old, indicate that the synthesis of phospholipids such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in the brain is decreased during aging (Gaiti et al., 1981, 1982a). Further evidence using labeled CDPcholine as PC precursor in brain microsomes suggests that synthesis is impaired mainly by the modified diacylglycerol (DG) composition rather than by CDP-base availability (Brunetti et al., 1979). Furthermore, PC synthesis from [ 3H]choline in a minced tissue suspension of cerebral cortex (CC) from 21.5-month-old rats is lower than in 3.5-monthold rats (unpublished results). PC content in CC from 21.5- and 28.5-month-old rats was found to be similar to that in adult CC (Lo´pez et al., 1995), possibly owing to the presence of PC synthesis pathways other than the Kennedy pathway. This may mean that a phospholipid rather than DG is partially a PC precursor. PC synthesis through phosphatidylethanolamine N-methyltransferase (PEMT) activity in rat brain synaptosomes has been studied (Crews et al., 1980). PC synthesis via PE methylation involves a multistep reaction with the intermediate formation of phosphatidyl-N-monomethylethanolamine (PMME) and phosphatidyl-N,N-dimethyl-ethanolamine (PDME). In the brain, this alternative pathway for PC synthesis could provide a source of choline for acetylcholine synthesis in a controlled manner, suggesting a role in the functions of synapses (Crews et al., 1980). In the present study, the level of PEMT activity measured in post-mitochondrial fraction from brain homogenates of aged (28.5-monthold) and adult (3.5-month-old) rats was analyzed. PE can be synthesized from free ethanolamine through the CDP-ethanolamine pathway or by base exchange with the base moiety of preexisting phospholipids. A third pathway
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Fig. 1. Alternative pathways for phospholipid synthesis in the brain.(a) The calcium-dependent, energy-independent phospholipid synthesis through the incorporation of serine or ethanolamine (PSS1 and PSS2 respectively). (b) phosphatidylserine decarboxylase activity (PSD) was also measured through serine incorporation into phosphatidylserine (PS) and its transformations in phosphatidylethanolamine (PE); (c) the synthesis of phosphatidylcholine (PC) by successive transfer of methyl groups from SAM to endogenous PE, with the intermediate formation of phosphatidyl-N-monomethylethanolamine (PMME) and phosphatidyl-N,N-dimethylethanolamine (PDME); and (d) PC breakdown by phospholipase D (PLD) yielding PA or phosphatidylglycerol in the presence of glycerol.
involves the formation of PE from free serine by base exchange, followed by removal of the C1 carboxyl group by decarboxylation (Fig. 1). This activity is mediated by PS decarboxylase (PSD) and is mainly localized in the mitochondrial fraction of rat brain where it seems to be responsible for the presence of mitochondrial ethanolamine phospholipids (Butler and Morell, 1983; Voelker, 1997). Phosphatidylserine biosynthesis in mammals takes place by an energy-independent, calcium-dependent base exchange reaction between l-serine and preexisting endogenous phospholipids (Kanfer, 1972; Porcellati et al., 1971). This reaction is localized in the microsomal fraction, with PE, PC, or PS serving as the phospholipid substrate. Different enzymes with specificity toward choline, ethanolamine and l-serine were isolated from rat brain (Vance, 1998). In this base exchange reaction, l-serine replaces choline and ethanolamine moieties in PC and in PE respectively, to produce PS and free choline or ethanolamine by PSS1 and PSS2 enzyme activities, respectively (Vance, 1998). We examined PS synthesis through [ 3H]l-serine (PSS1 activity) and [1,2- 14C]ethanolamine incorporation (measured as PSS2 activity to take into account the reversibility of this reaction) in homogenates of different brain areas of adult and aged rats. We considered it of interest to explore the effect of aging on PLD activity. It is known that PLD possesses both hydrolytic and transphosphatidylation activities (Chalifour et al., 1980). Furthermore, recent evidence has implied that PLD activation is generated by a wide variety of stimuli. Several PLD isozymes differing in their pH optima and responses
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to calcium, phosphatidylinositolphosphates, oleate, detergents, and substrate specificity were purified from rat brain (Exton, 1997, 1999; Frohman, 1999]. In the present paper, transphosphatidylation activity of PLD was studied in CC homogenates from adult and aged rats. On the basis of the changes reported above, we have carried out a series of enzyme assays to examine the activity of alternative pathways for the synthesis of phospholipids in different brain areas during aging. Serine and ethanolamine base exchange, and PEMT, PSD, and PLD activities were studied in the brain of adults and aged rats (Fig. 1). 2. Materials and methods Wistar-strain male rats were kept under constant environmental conditions and were fed on a standard pellet diet. [ 3H]l-Serine (specific activity 21,7 Ci/mmol); [1,2- 14C]ethanolamine (specific activity 4 mCi/mmol); S-adenosyl-l-[methyl- 3H] methionine (73.8 Ci/mmol); [2- 3H]glycerol (specific activity 5–10 Ci/mmol) and Omnifluor were purchased from New England Nuclear-Dupont (Boston, MA, USA). All other reagents were of analytical grade. 2.1. Homogenates Homogenates were prepared from whole brain, CC, SWM, and CRBL of 3.5-(adult) and 28.5-month-old (aged) rats. Rats were killed by decapitation and all brain areas were immediately dissected (2–4 min after decapitation). Brain homogenates were prepared in the following way: 1) 20% (w/v) in 0.32 M sucrose, 1 mM EDTA, 5 mM buffer HEPES (pH 7.4) (homogenates for serine baseexchange activity); 2) 10% (w/v) in 100 mM Tris-glycylglycine buffer (pH 8.5), 10 mM MgCl2, 5 mM DTT, 0.1 mM PMSF (homogenates for PEMT assay); and 3) 20% (w/v) in a medium containing 0.32 M sucrose, 1 mM EDTA, 50 mM Tris-HCl (pH 7.4) (homogenates for PLD assay). Because PLD and base exchange enzymes are located in different subcellular fractions, total homogenates are the most adequate medium for assaying both enzyme activities. 2.2. Post-mitochondrial supernatants Post-mitochondrial supernatants were prepared from CC total homogenates. Homogenates were centrifuged at 1000 × g for 10 min. The pellets were discarded and the supernatants (S1) were centrifuged at 17 000 × g for 15 min. The resulting supernatant was used as post-mitochondrial supernatant (enriched with microsomal and cytosolic fraction). PEMT, a predominantly microsomal enzyme, was assayed in this subcellular fraction. 2.3. Assay for base-exchange activity Serine base exchange activity was assayed by measuring the incorporation of [ 3H]lserine into phospholipids from brain homogenates in the presence of calcium chloride (Buchanan and Kanfer, 1980). We employed brain homogenate, as this allowed us to measure base exchange enzyme and PSD activity, present mainly in mitochondrial
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fraction. We showed the effect of aging on PSD activity through [ 3H]l-serine incorporation into PS and its transformation in PE. The assay medium contained 50 mM HEPES pH 8, 2.5 calcium chloride or 5 mM EGTA, 2 mg protein in a final volume of 500 mL. The assay was started by adding [ 3H]l-serine (specific activity 21.7 Ci/mmol) and carried out in a thermostated shaking bath for 60 min. Comparative studies with [ 3H]l-serine and [1,2- 14C]ethanolamine were carried out at 2.5 mM of serine and ethanolamine under the above-described conditions. The calcium-independent incorporation into lipids was measured in the presence of 5 mM EGTA. Blanks were prepared in the same way as the corresponding samples except that membrane suspensions were boiled for 5 min before use. In all cases incubations were stopped by adding chloroform/methanol (2:1: v/v). Lipids were extracted according to the method of Folch et al. (1957). The chloroform phase was extensively washed with theoretical upper phase containing 0.05% calcium chloride to eliminate water-soluble radioactive products. The chloroform phase was then evaporated to dryness under N2 and the residue was dissolved in chloroform/methanol (2:1, v/v). Individual phospholipids were isolated by monodimensional pre-coated TLC (Holub and Skeaff, 1987). Lipids were visualized by exposure of the chromatograms to iodine vapors, and scraped off to a vial for counting by liquid scintillation after the addition of 0.4 ml water and 10 ml 5% omnifluor in toluene/triton X-100 (4:1, v/v). 2.4. Assay for phosphatidylethanolamine N-methyltransferase PEMT activity was assayed by measuring the incorporation of [ 3H]methyl groups into endogenous PE from post-mitochondrial supernatant in the presence of S-adenosyl-l[methyl- 3H]methionine ([ 3H]SAM). The buffer assay contained 100 mM Tris-glycylglycine buffer (pH 8.5), 10 mM MgCl2, 5 mM DTT, 0.1 mM PMSF, and 300 mg protein of post-mitochondrial supernatant, at a final volume of 250 mL (Roque and Giusto, 1995). The assay was started by adding 200 mm (10 mCi) [ 3H]SAM (specific activity 1 mCi/ mmol) and carried out in a thermostated shaking bath for 60 min. Incubation was stopped by adding chloroform/methanol/HCl (2:1:0.02%, v/v). The sample was applied on a silicagel G plate and developed by two-dimensional TLC using chloroform/methanol/water (65:25:4, v/v) in the first dimension, and n-propanol/propionic acid/acetic acid/water (2:2:1:1, v/v), in the second. PMME and PDME were added as internal standards and simultaneously chromatographed. Blanks were prepared in the same way as the corresponding samples except that the membrane suspensions were boiled for 5 min before use. Lipids were visualized by exposure of the chromatograms to iodine vapors, and scraped off for counting by liquid scintillation after the addition of 0.4 ml water and 10 ml 5% omnifluor in toluene/triton X-100 (4:1, v/v). 2.5. Assay for phospholipase D PLD activity was assayed by the incorporation of [2- 3H]glycerol (0.15 mCi/mol) into endogenous phospholipids. The buffer assay contained 40 mM HEPES, pH 6.8, 25 mM potassium fluoride; 1 mM DTT, 10 mCi [2- 3H]glycerol (0,15 mCi/mol) and 350 mg of CC homogenates in a total volume of 250 mL. Incubations were carried out for 90 min at 378C. Reactions were stopped by adding 5 ml of chloroform/methanol (2:1,v/v) and lipids were extracted according to the procedure of Folch et al. (1957), using first the upper phase
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containing 0.1 M sulfuric acid and then the upper phase containing water (Casola and Possmayer, 1981). [ 3H]PG was separated by thin layer chromatography in precoated plates using chloroform/acetone/methanol/acetic acid/water (50:20:10:10:5, v/v) as phase. Lipids were visualized by exposure of chromatograms to iodine vapors, and scraped off the plate for counting by liquid scintillation. 2.6. Other methods Protein and lipid phosphorus were determined according to Lowry et al. (1951) and Rouser et al. (1970), respectively. 2.7. Statistical analysis Statistical analysis was performed using Student’s t-test, with the values representing the mean ^ S.D. of the total number of samples indicated in each legend (Johnson and Kotz). 3. Results 3.1. Incorporation of [ 3H]l-serine into phospholipids of different areas of CNS from adult and aged rats The incorporation of [ 3H]serine into phospholipids from homogenates of CC, CRBL and SWM was measured without the addition of exogenous calcium (basal condition) and in a medium supplemented with 2.5 mM calcium. EGTA 5 mM was used to measure calcium-independent synthesis. Lipid synthesis from [ 3H]serine was found to be absent in the presence of EGTA in all brain areas (data not shown). When calcium was present, PS and PE were found to be labeled by the precursor. The effect of aging and calcium ions on PS synthesis from different brain areas is shown in Fig. 2. Under basal conditions, [ 3H]serine incorporation into PS from CRBL was higher than in CC and SWM. In adult rats, 2.5 mM concentration of calcium ions increased PS synthesis one- or 2-fold (P , 0.001) with respect to that found under basal conditions in all brain areas. Under basal conditions PS labeling in CC and CRBL of aged rats was slightly higher than that in adult rats, whereas inhibited PS synthesis was observed in aged rat SWM. It is interesting to note that a stimulatory effect on PS synthesis was observed in CRBL and CC from aged rats at 2.5 mM calcium (P , 0.001). Experiments under base-exchange assay conditions at high base concentration (2.5 mM) were also carried out in CC and CRBL homogenates from adult and aged rats with [1,2- 14C]ethanolamine and [ 3H]serine (Fig. 3). Previously reported data indicate that Km values rise to 2 mM in adult brain (Porcellati et al., 1971). In both tissues, [1,2 14C]ethanolamine and [ 3H]serine incorporation into PE and PS, respectively, was negligible in the presence of EGTA (Fig. 3). In the presence of 2.5 mM calcium, labeled PE in CC and CRBL was one- and 2-fold higher (P , 0.001) than labeled PS, respectively. Data shown in Fig. 3 also show that the incorporation of [1,2 14C]ethanolamine into PE from aged rat CC was similar to that in adult rats, whereas a slight inhibition (19%) was observed in
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Fig. 2. Effect of aging on serine base exchange activity in cerebral cortex (CC), cerebellum (CRBL) and subcortical white matter (SWM) homogenates from adult A and aged rats o . Brain homogenates were prepared in the following way: a) 20% (w/v) in 0.32 M sucrose, 1 mM EDTA, 5 mM buffer HEPES pH 7.4. The assay medium contained 50 mM HEPES pH 8, 2.5 calcium chloride, or 5 mM EGTA, 2 mg protein (100 mL homogenate) in a final volume of 500 mL. The assay was started by adding [ 3H]l-serine (specific activity 21.7 Ci/ mmol) to 12 mm l-serine concentration and carried out in a thermostated shaking bath for 60 min. Incubation was stopped by adding chloroform/methanol (1:1: v/v). Lipids were isolated as described in Section 2. Each experiment was performed twice, using on each occasion three control rats and three senile rats. Data are the mean ^ SD of the six samples from each experiment. **P , 0.025, ***P , 0.001 (*adults versus aged); 111 P , 0.001 (1 basal versus calcium conditions).
CRBL. As shown in Fig. 2, at a lower serine concentration (12 mmm), PS synthesis increased with aging in both brain areas (12 and 24%, respectively). As previously indicated, PE was also synthesized from radioactive serine by PSD activity in CC, CRBL, and SWM homogenates with and without calcium addition (basal condition) (Table 1). PE synthesis in all brain areas was absent when calcium was chelated with EGTA (data not shown).The level of PSD activity exhibited the following order CRBL . CC . SWM and represented 17, 16, and 10% of that of PS, respectively (Table 1). Addition of 2.5 mM calcium increased PE synthesis from serine by PSD about 2-fold (P , 0.001) with respect to the values observed under basal conditions. This stimulatory effect of calcium was enhanced in SWM (approximately a 4-fold increase with respect to basal conditions). The changes on PSD activity seems to be dependent with increased PS availability,
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Fig. 3. Effect of aging on serine and ethanolamine base exchange activities in cerebral cortex (CC) and cerebellum (CRBL) homogenates from adult A and aged rats o . Brain homogenates were prepared at 20% (w/v) in 0.32 M sucrose, 1 mM EDTA, 5 mM buffer HEPES pH 7.4. The assay medium contained 50 mM HEPES pH 8, 2.5 calcium chloride or 5 mM EGTA, 2 mg protein (100 mL homogenate) in a final volume of 500 mL. The assay was started by adding [ 3H]l-serine (specific activity 21.7 Ci/mmol) or [1,2- 14C]ethanolamine (specific activity 4 mCi/ mmol) at 2.5 mM base concentration and was carried out in a thermostated shaking bath for 60 min at 378C. Incubation was stopped by adding chloroform/methanol (1:1: v/v). Lipids were isolated as described in Section 2. Each experiment was performed twice, using on each occasion three control rats and three senile rats. Data are the mean ^ SD of the six samples from each experiment. *P , 0.05; **P , 0.025; ***P , 0.001 (*adults versus aged).
resulting from the calcium stimulatory action in base exchange activity. This was observed when synthesized PE was expressed as a percentage of PS labeling (25.2% and 16.9% in the presence of exogenous calcium and under basal conditions, respectively). PE syntheTable 1 Phosphatidylethanolamine synthesis through phosphatidylserine decarboxylase in cerebral cortex, cerebellum and subcortical white matter from adult and aged rats (Homogenates at 20% (w/v) in 0.32 M sucrose, 1 mM EGTA, 5 mM buffer HEPES pH 7.4, were prepared from cerebral cortex (CC), cerebellum (CRBL), and subcortical white matter (SWM) of 3.5- and 28.5-month-old rats (adult and aged, respectively). Assays for serine base exchange with or without calcium were made in a medium containing 50 mM HEPES pH 8 without or with 2.5 mM calcium chloride, 2 mg protein (100 mL homogenate) in a final volume of 500 mL. The assay was started by adding [ 3H]l-serine (specific activity 21.7 Ci/mmol) and carried out in a thermostated shaking bath for 60 min. Lipid extracts and PE isolation were made as described in Section 2. Synthesized PE is expressed as pmol/mol of total lipid P. Data in parentheses correspond to synthesized PE relative to PS synthesized in ratiox 100. Each experiment was performed twice, using on each occasion three control rats and three senile rats. Data are the mean ^ SD of the six samples from each experiment) CC
CRBL
Adult Basal condition Ca 21 2.5 mM
3.6 ^ (16.9 ^ 13.0 ^ (25.2 ^
Aged 0.9 2) 1.2 1.6)
4.5 ^ (17.3 ^ 15.1 ^ (24.6 ^
SWM
Adult 1.1 4.7) 2.6 2.4)
5.7 ^ (16.6 ^ 14.5 ^ (22.8 ^
Aged 1.8 4.6) 1.9 2.4)
5.8 ^ (14.3 ^ 17.2 ^ (23.6 ^
Adult 1.9 3.7) 0.7 1.9)
2.4 ^ (9.7 ^ 10.3 ^ (19.1 ^
Aged 0.1 0.3) 0.3 0.5)
1.8 ^ (10.4 ^ 10.7 ^ (18.5 ^
0.3 1.3) 1.0 2.0)
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Fig. 4. Phosphatidylethanolamine N-methyltransferase activity in post-mitochondrial fraction of cerebral cortex (CC) homogenates from adult and aged rats. Homogenates of CC were prepared in 10% (w/v) in 100 mM Trisglycylglycine buffer (pH 8.5), 10 mM MgCl2 5 mM DTT, 0.1 mM PMSF. Post-mitochondrial supernatant was obtained after 17 000 × g centrifugation during 15 min. The suspension was incubated with 10 mM [ 3H]SAM (2.5 mCi), in 100 mM Tris-glycylglycine buffer (pH 8.5), 10 mM MgCl2 5 mM DTT, at a final volume of 250 mL. Incubations were stopped and lipids were extracted by addition of 2 ml chloroform/methanol/HCl (2:1:0.2, v/v). Lipids were isolated as described in Section 2. Each experiment was performed twice, using on each occasion three control rats and three senile rats. Data are the mean ^ SD of the six samples from each experiment. *P , 0.05; **P , 0.025; ***P , 0.001. Total methylated lipids (TML PMME 1 PDME 1 PC), phosphatidyl monomethylethanolamine (PMME); phosphatidyl dimethylethanolimine (PDME) and phosphatidylcholine (PC). **P , 0.025; ***P , 0.001).
sized by this pathway in CRBL and SWM were similarly affected by calcium ions. Data on CC, CRBL, and SWM from aged rats reveal that PE synthesized from serine by PSD maintained adult rat values. (Table 1, parentheses). 3.2. Phosphatidylethanolamine N-methyltransferase activity in CC of aged and adult rats The post-mitochondrial fraction of CC from aged and adult rats was incubated with 10 mm [ 3H]SAM at pH 8.5 in the absence of exogenously added phospholipid acceptor for 60 min. Under these conditions, the major methylated product formed in adult rats was PMME, followed by PC and PDME (Fig. 4). An increased incorporation of methyl groups into endogenous PE was observed in the post-mitochondrial fraction of CC from 28.5 month old rats with respect to adult rats, and total methylated products were 52% higher in aged rats (P , 0.001) with respect to adult rats. PMME increased the most in aged with respect to adult rats (94%) (P , 0.025). 3.3. Phospholipase D activity in CC of aged and adult rats Phospholipase D activity was measured by transphosphatidylation reactions on endogenous phospholipids. Homogenates from CC of adult and aged rats were used as enzymatic source. At all incubation times (30, 60, and 90 min) radioactive PG was measured only as a lipid reaction product. PG labeling in CC homogenates from adult rats is shown in the insert of Fig. 5. The time course of the incorporation of [ 3H]glycerol into PG was seen to increase up to 90 min incubation. [ 3H]glycerol incorporation into PG at 30 and
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Fig. 5. Phospholipase D activity in homogenates from cerebral cortex (CC) from A adult and aged rats o . Homogenates were prepared from CC from adults (3.5 months of age) and aged (28.5 months of age) rats in 20% in a medium containing 0.32 M sucrose, 1 mM EDTA, 50 mM Tris-HCl (pH 7.4). The assay was started by addition of CC homogenates (350 mg protein) in a medium containing 5 mCi of [2- 3H]glycerol (0.15 mCi/mol), 40 mM HEPES, pH 6.8, 25 mM potassium fluoride, 1 mM DTT in a total volume of 250 mL. Incubations were carried out for 90 min at 378C. Reactions were stopped by addition of 5 ml of chloroform/methanol (2:1), extracts were purified and lipids were isolated as described in Section 2. Each experiment was performed twice, using on each occasion three control rats and three senile rats. Data are the mean ^ SD of the six samples from each experiment. **P , 0.025.
90 min incubation in adult and aged rats shows that aging increases PLD activity by 60% (P , 0.025).
4. Discussion Phospholipids are mainly synthesized by the following pathways in mammalian tissues: 1) the well-known Kennedy pathway, which are compartamentalized and independently metabolized, lead to PE and PC synthesis; 2) a remodeling pathway that represents a calcium-dependent and -stimulated incorporation of serine, ethanolamine, choline, and other amino alcohols into pre-existing endogenous phospholipids; 3) a pathway by which PC is formed by stepwise PE methylation. An additional mechanism is PLD enzyme activity. PLD, and base exchange in particular, remove the amino alcohol substituent yielding PA (PLD activity) or substitute the amino alcohol on preexisting phospholipids with another amino alcohol (base exchange). A common feature of these enzymes is their ability to modify the polar portion of phospholipids. Although a lower age-related de novo choline or ethanolamine phospholipid synthesis measured by CDP-base incorporation in vivo or in vitro was previously reported, the level of phospholipid content was never-
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theless normal (Brunetti et al., 1979; Gaiti et al., 1981, 1982a; Lopez et al., 1995). This could be due to the increased activity of an enzymatic pathway other than the Kennedy pathway for PC and PE provision. We thus focused on a putative enzymatic mechanism that could become modified in normal aging. Previous work from our laboratory has shown significant changes in acyl group composition in aging rat brain, and it was suggested that such changes are related to modifications in the properties of neural membranes (Lopez et al., 1995; Crews et al., 1980). It has also been suggested that the degree of phospholipid polyunsaturation plays a role in the modulation of membrane protein function (as a signaling system) (Litman and Mitchell, 1996). It is interesting to note that all these enzymes, base exchange (PSS1 and PSS2), PEMT, and PLD, seem to be firmly embedded in the matrix of membranes and show a marked preference for endogenous substrates that are components of their environment. In this context, modifications in these enzymatic activities are likely to be related to changes in the membrane microenvironment. However, at this stage we cannot discard the possibility that serine base exchange could be activated by age-induced alteration in calcium availability. Calcium dependence and calcium stimulation of base-exchange reactions are well known (Vance, 1998). Although alteration of calcium homeostasis levels in senescence neurons has not been fully confirmed (Verhrastky and Toescu, 1998), age-related inhibition of microsomal Ca 21ATPase activity and neuronal calcium levels in rat brain has been recently reported (Hanahisa and Yamaguchi, 1997). Furthermore, an age-related decrease in the expression of a neuronal calcium binding protein, calbindin D28k, which could act as an intraneuronal calcium buffering protein, has also been reported. As previously suggested, the loss of calcium binding proteins may therefore result in the disturbance of intracellular Ca 21 concentration (Kishimoto et al., 1998). It is interesting to note that CRBL, in which serine base exchange was found to mainly increase with aging, is the neural tissue most affected by a decrease in the content of calbindin D28k (50% to 68% of adult mRNA expression) (Baimbridge et al., 1992). In agreement with a previous report, we have also found that ethanolamine-exchange (PSS2) is one- or 2-fold higher than serine-exchange in rat brain (Holbrook and Wurtman, 1988). Furthermore, ethanolamine incorporation into PE was not modified in CC or CRBL from aged rats with respect to the values observed in adult rats. However, serine base exchange activity was selectively increased by aging, CRBL being the most affected. In this context, it is interesting to note the heterogeneity of the exchange system as evidenced by kinetic responses of the enzymes to membrane fluidity and polyvalent cations (Holbrook and Wurtman, 1988). It is known that base exchange reaction primarily forms hexaenoic and tetraenoic phosphatidylserine species in rat liver (Bjerve, 1984). It has also been suggested that this could reflect either the availability of the corresponding phospholipid substrates or a specificity of the serine base exchange enzyme toward the fatty acid composition of the phospholipid substrate. It seems that considerably more hexaenoic species are formed than those accounted for by the composition of the available phospholipid substrate (Bjerve, 1982). In addition, a purified serine base exchange enzyme from rat brain showed some specificity toward the fatty acid composition of the lipid substrate (Taki and Kanfer, 1978). Present results on the increased selective serine-base
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exchange activity in normal aging suggest that aged brain is able to increase phospholipid synthesis, thus contributing to polyunsaturated fatty acid supply to membranes. Several reports show that age has differential effects on neuronal populations as exemplified by: the age-dependent loss of dopaminergic neurons in the nigrostriatal system (Cardozo-Pelaez et al., 1999); changes in mitochondrial respiratory activities in several brain areas during aging (Ojaimi et al., 1999); the chronological changes in the gene expression for cytosolic fatty acid-binding proteins in rat brain (Owada et al., 1996). These findings could imply a similar behavior for base-exchange enzymes in different brain areas during aging. Neurological alterations in aging brain have partially attributed to declining cellular energy. The decreased intracellular ATP levels reported in rat CC may be associated with pathological changes of overall mechanisms involved in the senescent brain (Joo et al., 1999). The increased serine base exchange activities in CC shown in our results may constitute an alternative source for energy independent lipid biosynthesis. It was later reported that base exchange activities and also PLD activity of rat brain are modified during brain development (Kobayashi et al., 1988). During this period, i.e. between embryonic day 17 and postpartum days 14 and 30, drastic modifications in brain lipid fatty acid composition take place (Cunnane and Chen, 1992). This phenomenon could be related to the modification of enzyme activities intrinsic to membranes such as PLD and base exchange enzymes. Although previous results show a decrease in in vivo serine incorporation into PS of CC and hypocampus of aged rats, such decrease seems not to be related to base exchange activity. It has been suggested that an age-related decrease in serine uptake (as with other low-molecular-weight molecules) by neural tissue could be responsible (Gatti et al., 1989). It has been previously reported that PSD significantly contributes to PE synthesis in adult brain, this activity being mainly located in the mitochondrial fraction (Voelker, 1997; Percy et al., 1983). The formation of PE by PSD has also been observed in cultures of cerebral hemispheres and in myelinating cultures of neonatal rat cerebellum (Yavin and Zeigler, 1983; Bradbury, 1984). It is known that base exchange activities are mainly located in the microsomal fraction; because in our experiments homogenates were employed as enzymatic source, simultaneous PSD could be measured through labeled PS as precursor. PE synthesis through PSD using [ 3H]serine as precursor under base exchange assay conditions allowed us to determine whether any changes take place in PSD activity during brain aging. As shown in Table 1, PE synthesis through PSD activity in the CC of adult and aged rats was found to be similar. In addition, calcium produced an increase in PE synthesis by PSD activity in adult and aged rats. Whereas PE labeling represented about 17% of labeled PS under basal conditions, it increased to 25% at a 2.5 mM calcium concentration in adult and aged rats. In preliminary experiments using minced tissue suspension from cerebral hemispheres, increased PE synthesis by decarboxylation was observed in aged brain with respect to adults (unpublished result). This phenomenon could be explained by the different availability of free calcium in the tissue. PC synthesis from S-adenosyl-l-[methyl- 3H] methionine by successive transfer of methyl groups from endogenous PE was higher in the brain of aged rats than in that of
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adult rats. In agreement with our results, Crews et al. (1981) have reported a significant aged-related increase in rat brain PEMT I activity. Although a net increase in the sum of methyl [ 3H] products of PEMT could be seen, the stimulatory effect, in homogenates of the neural tissue was mainly observed in the first compound, PMME. Previous studies on the effect of aging on the acyl composition of brain and liver phospholipid revealed a decrease in polyunsaturated acyl chains sterified to phospholipids. However, this effect is mainly observed in brain lipids rather than in liver lipids. In addition, it has also been reported that PE, rather than PC, is modified by aging (Lo´pez et al., 1995). It has been demonstrated that N-methylation produces PC enriched in arachidonic acid in tissues other than brain, confirming that their precursors (PMME and PDME) also contain high amounts of polyunsaturated fatty acids in rat brain (Tacconi and Wurtman, 1985). One could expect PC synthesized from PE to incorporate polyunsaturated species to the preexisting pool. It is possible that in the brain, the PC formed by N-methylation is deacylated and then reacylated with very long-chain polyunsaturated fatty acids, or that PEMT preferentially uses PE molecular species enriched in long- and very long-polyunsaturated fatty acids. This alternative PC source could also be present in aged brain but seems not to be sufficient to mask the partial polyunsaturated PC depletion previously observed (Lo´pez et al., 1995). Given that these enzyme activities are firmly embedded in the membrane, the significant changes in the acyl group composition of phospholipids in aging brain may be responsible for the modification of such activities through changes in the physico-chemical properties of membrane domains. On the other hand, previous functional studies suggest that important biological properties of membranes are dependent on PC/PE ratios (Cerbon and Calderon, 1995). Furthermore, the increase in the PEMT activity shown in aged brain may play an important role in biological signal transduction. It has been reported that myocardium membranes from aged rats show a significantly lower phospholipid methylation than adult rats. Furthermore, it was suggested that this decrease is connected with the age-related diminution in Ca 21 pump activity as a consequence of decreased membrane fluidity (Heyliger et al., 1988). In the diaphragm, there is a good correlation between the decrease in phospholipid methylation and the decline in its contractile performance as age progresses (Sastry et al., 1982). It is likely that different tissue-specific isoforms could be differently affected by aging. In our experiments, the fatty acid composition of myocardium phospholipids from 28-month-old rats shows no changes with respect to adult rats (unpublished data). It is believed that brain tissue lacks the enzymatic capability to synthesize acetylcholine during aging and that choline present in phosphatidylcholine may represent the reservoir of choline for ACh synthesis. The present findings showing increased PEMT and PLD activities with aging could be related to the supply of choline for ACh synthesis. It has been demonstrated that ACh synthesis in rat brain synaptosomes is stimulated by PC breakdown catalyzed by PLD (Hattori and Kanfer, 1985). In addition, it was observed that methylated intermediates, which have been demonstrated to be good substrates for PLD activity (Jacobs et al., 1988), increase during aging. The findings of the present study reveal the ability of aged brain to increase the activity of enzymatic mechanisms involved in the modification of a portion of phospholipid molecules. Although some authors have reported that de novo phospholipid biosynthesis seems to
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decrease with age (Brunetti et al., 1979; Gaiti et al., 1981, 1982a; Yargicoglu et al., 1999), energy-independent alternative pathways are stimulated. These pathways, made evident through the activation of serine base-exchange (PSS1 and PSS2), PSD, PE N-Mtase, and PLD activities, modify the phospholipid hydrophilic head group and may serve to provide a good availability of unsaturated phospholipid species. This phenomenon, which was observed in in vitro assays with neural membranes from aged rats, could be interpreted as a sort of “recycling” of different phospholipid pools under conditions where precursors and activators (e.g. calcium) are appropriately supplied. An interesting point worthy of further exploration is whether or not this apparent compensatory mechanism is beneficial for aged neural membranes.
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Tacconi, M., Wurtman, R., 1985. Phosphatidylcholine produced in rat synaptosomes by N-methylation is enriched in polyunsaturated fatty acids. Proc. Natl. Acad. Sci. 82, 4828–4831. Taki, T., Kanfer, J., 1978. A phospholipid serine base exchange enzyme. Biochem Biophys Acta 528, 309–317. Vance, J.E., 1998. Eukaryotic lipid biosynthetic enzymes: the same but not the same. Trends Biochem. Sci. 23, 423–428. Verhrastky, A., Toescu, E.C., 1998. Calcium and neuronal ageing. Trends Neurosci. 21, 2–7. Voelker, D.R., 1997. Phosphatidylserine decarboxylase. Biochim. Biophys. Acta. 1348, 236–244. Yargicoglu, P., Agar, A., Gumuslu, S., Bilmen, S., Oguz, Y., 1999. Age-related alterations in antioxidant enzymes, lipid peroxide levels, and somatosensory-evoked potentials: effect of sulfur dioxide. Arch. Environ. Contam. Toxicol. 37, 554–560. Yavin, E., Zeigler, B., 1983. Regulation of phosphatidylserine metabolism in differentiating cells from rat brain cerebral hemispheres in culture. J. Biol. Chem. 252, 260–267. Zaidi, A., Michaelis, M.L., 1999. Effects of reactive oxygen species on brain synaptic plasma membrane Ca 21ATPase. Free Radic. Biol. Med. 27, 810–821.
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Alternative pathways for phospholipid synthesis in different brain areas during aging M.G. Ilincheta de Boschero, M.E. Roque, G.A. Salvador, N.M. Giusto* Instituto de Investigaciones Bioquı´micas de Bahı´a Blanca (INIBIBB), Universidad Nacional del Sur, CONICET, Camino La Carrindanga Km 7 B8000FWB, Bahı´a Blanca, Buenos Aires, Argentina Received 7 December 1999; received in revised form 16 February 2000; accepted 4 April 2000
Abstract Morphological and biochemical changes take place in the membrane of aged brain. In particular, studies on aged rats report alterations in brain phospholipid synthesis and in phospholipid-specific fatty acid composition. However, no significant changes in main phospholipid class content have been reported in aged brain, possibly owing to alterations in the alternative pathways for phospholipid synthesis during aging. Therefore, the present study was designed to determine the effect of aging on the enzyme activities responsible for phospholipid synthesis by alternative pathways. Indifferent brain areas of adult (3.5-month-old) and aged (28.5-month-old) rats we examined: 1) the activity of base exchange enzymes, which is a calcium-dependent, energy-independent and calcium stimulated enzymatic pathway; 2) phosphatidylethanolamine (PE) synthesis by phosphatidylserine decarboxylase activity (PSD); 3) phosphatidylcholine (PC) synthesis by transfer of methyl groups to endogenous PE by phosphatidylethanolamine N-methyltransferase activity (PEMT); 4) the synthesis of phosphatidylglycerol (PG) through phospholipase D (PLD) activity. Because the dependence on and the stimulation by calcium of base-exchange reactions is a well known mechanism and alterations in calcium levels in rat brain have been reported, we decided to investigate PS synthesis in the presence of endogenous and exogenous calcium (2.5 mM). PS synthesis increased in cerebral cortex (CC) and cerebellum (CRBL) of aged rats with respect to adult rats in basal conditions (without the addition of exogenous calcium), but more significant changes were observed in serine base exchange activity during aging when exogenous calcium was added. PEMT activity in aged CC increased by 100%, the principal modification being observed in the first methylated product of the sequential reaction. Futhermore, the transphosphatidyl reaction was higher in aged brain as indicated by the increased PG synthesis. Our findings allow us to conclude that age affects some alternative pathways for phospholipid synthesis in the central nervous system, and indicate the presence of a * Corresponding author. Tel.: 154-291-4861201; fax: 1 54-291-4861200. E-mail address: [email protected] (N.M. Giusto). 0531-5565/00/$ - see front matter q 2000 Elsevier Science Inc. All rights reserved. PII: S0531-556 5(00)00104-2
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compensatory mechanism to provide a pool of phospholipid classes for the maintenance of cellular membrane lipid composition during aging. q 2000 Elsevier Science Inc. All rights reserved. Keywords: Aging; Brain; Lipid metabolism; Phospholipids; Phospholipid metabolism
1. Introduction Age inevitably affects all organs, but the brain seems particularly susceptible and is of course central to the problem. The vulnerability of the brain increases rapidly with age, and its deterioration is made manifest in many ways. One intriguing feature of the aging process is the modification of cellular membrane properties. It has been observed that brain membrane lipid fatty acid composition and consequently membrane fluidity change with increasing age (Kumar et al., 1999) and that alterations in brain specific fatty acid binding protein levels in synaptosomal plasma membranes and synaptosomal cytosol may be important factors modulating neuronal differentiation and function (Pu et al., 1999). Several enzyme activities are modified during the aging process. The plasma membrane Ca 21-ATPase plays a critical role in Ca 21 homeostasis, and its kinetic properties change in aged rat brain, affecting the regulation of free intracellular calcium (Zaidi and Michaelis, 1999). Delta-9 desaturase activity is reduced up to 50% with age, such decrease is known to cause alterations in membrane fluidity and to affect cellular signaling pathway (Kumar et al., 1999). Age-related alterations in antioxidant enzyme has also been reported recently (Yargicoglu et al., 1999). All these age-related changes seem to correlate with the aging of the nervous system. Studies carried out on rats up to 18 months old, indicate that the synthesis of phospholipids such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in the brain is decreased during aging (Gaiti et al., 1981, 1982a). Further evidence using labeled CDPcholine as PC precursor in brain microsomes suggests that synthesis is impaired mainly by the modified diacylglycerol (DG) composition rather than by CDP-base availability (Brunetti et al., 1979). Furthermore, PC synthesis from [ 3H]choline in a minced tissue suspension of cerebral cortex (CC) from 21.5-month-old rats is lower than in 3.5-monthold rats (unpublished results). PC content in CC from 21.5- and 28.5-month-old rats was found to be similar to that in adult CC (Lo´pez et al., 1995), possibly owing to the presence of PC synthesis pathways other than the Kennedy pathway. This may mean that a phospholipid rather than DG is partially a PC precursor. PC synthesis through phosphatidylethanolamine N-methyltransferase (PEMT) activity in rat brain synaptosomes has been studied (Crews et al., 1980). PC synthesis via PE methylation involves a multistep reaction with the intermediate formation of phosphatidyl-N-monomethylethanolamine (PMME) and phosphatidyl-N,N-dimethyl-ethanolamine (PDME). In the brain, this alternative pathway for PC synthesis could provide a source of choline for acetylcholine synthesis in a controlled manner, suggesting a role in the functions of synapses (Crews et al., 1980). In the present study, the level of PEMT activity measured in post-mitochondrial fraction from brain homogenates of aged (28.5-monthold) and adult (3.5-month-old) rats was analyzed. PE can be synthesized from free ethanolamine through the CDP-ethanolamine pathway or by base exchange with the base moiety of preexisting phospholipids. A third pathway
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Fig. 1. Alternative pathways for phospholipid synthesis in the brain.(a) The calcium-dependent, energy-independent phospholipid synthesis through the incorporation of serine or ethanolamine (PSS1 and PSS2 respectively). (b) phosphatidylserine decarboxylase activity (PSD) was also measured through serine incorporation into phosphatidylserine (PS) and its transformations in phosphatidylethanolamine (PE); (c) the synthesis of phosphatidylcholine (PC) by successive transfer of methyl groups from SAM to endogenous PE, with the intermediate formation of phosphatidyl-N-monomethylethanolamine (PMME) and phosphatidyl-N,N-dimethylethanolamine (PDME); and (d) PC breakdown by phospholipase D (PLD) yielding PA or phosphatidylglycerol in the presence of glycerol.
involves the formation of PE from free serine by base exchange, followed by removal of the C1 carboxyl group by decarboxylation (Fig. 1). This activity is mediated by PS decarboxylase (PSD) and is mainly localized in the mitochondrial fraction of rat brain where it seems to be responsible for the presence of mitochondrial ethanolamine phospholipids (Butler and Morell, 1983; Voelker, 1997). Phosphatidylserine biosynthesis in mammals takes place by an energy-independent, calcium-dependent base exchange reaction between l-serine and preexisting endogenous phospholipids (Kanfer, 1972; Porcellati et al., 1971). This reaction is localized in the microsomal fraction, with PE, PC, or PS serving as the phospholipid substrate. Different enzymes with specificity toward choline, ethanolamine and l-serine were isolated from rat brain (Vance, 1998). In this base exchange reaction, l-serine replaces choline and ethanolamine moieties in PC and in PE respectively, to produce PS and free choline or ethanolamine by PSS1 and PSS2 enzyme activities, respectively (Vance, 1998). We examined PS synthesis through [ 3H]l-serine (PSS1 activity) and [1,2- 14C]ethanolamine incorporation (measured as PSS2 activity to take into account the reversibility of this reaction) in homogenates of different brain areas of adult and aged rats. We considered it of interest to explore the effect of aging on PLD activity. It is known that PLD possesses both hydrolytic and transphosphatidylation activities (Chalifour et al., 1980). Furthermore, recent evidence has implied that PLD activation is generated by a wide variety of stimuli. Several PLD isozymes differing in their pH optima and responses
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to calcium, phosphatidylinositolphosphates, oleate, detergents, and substrate specificity were purified from rat brain (Exton, 1997, 1999; Frohman, 1999]. In the present paper, transphosphatidylation activity of PLD was studied in CC homogenates from adult and aged rats. On the basis of the changes reported above, we have carried out a series of enzyme assays to examine the activity of alternative pathways for the synthesis of phospholipids in different brain areas during aging. Serine and ethanolamine base exchange, and PEMT, PSD, and PLD activities were studied in the brain of adults and aged rats (Fig. 1). 2. Materials and methods Wistar-strain male rats were kept under constant environmental conditions and were fed on a standard pellet diet. [ 3H]l-Serine (specific activity 21,7 Ci/mmol); [1,2- 14C]ethanolamine (specific activity 4 mCi/mmol); S-adenosyl-l-[methyl- 3H] methionine (73.8 Ci/mmol); [2- 3H]glycerol (specific activity 5–10 Ci/mmol) and Omnifluor were purchased from New England Nuclear-Dupont (Boston, MA, USA). All other reagents were of analytical grade. 2.1. Homogenates Homogenates were prepared from whole brain, CC, SWM, and CRBL of 3.5-(adult) and 28.5-month-old (aged) rats. Rats were killed by decapitation and all brain areas were immediately dissected (2–4 min after decapitation). Brain homogenates were prepared in the following way: 1) 20% (w/v) in 0.32 M sucrose, 1 mM EDTA, 5 mM buffer HEPES (pH 7.4) (homogenates for serine baseexchange activity); 2) 10% (w/v) in 100 mM Tris-glycylglycine buffer (pH 8.5), 10 mM MgCl2, 5 mM DTT, 0.1 mM PMSF (homogenates for PEMT assay); and 3) 20% (w/v) in a medium containing 0.32 M sucrose, 1 mM EDTA, 50 mM Tris-HCl (pH 7.4) (homogenates for PLD assay). Because PLD and base exchange enzymes are located in different subcellular fractions, total homogenates are the most adequate medium for assaying both enzyme activities. 2.2. Post-mitochondrial supernatants Post-mitochondrial supernatants were prepared from CC total homogenates. Homogenates were centrifuged at 1000 × g for 10 min. The pellets were discarded and the supernatants (S1) were centrifuged at 17 000 × g for 15 min. The resulting supernatant was used as post-mitochondrial supernatant (enriched with microsomal and cytosolic fraction). PEMT, a predominantly microsomal enzyme, was assayed in this subcellular fraction. 2.3. Assay for base-exchange activity Serine base exchange activity was assayed by measuring the incorporation of [ 3H]lserine into phospholipids from brain homogenates in the presence of calcium chloride (Buchanan and Kanfer, 1980). We employed brain homogenate, as this allowed us to measure base exchange enzyme and PSD activity, present mainly in mitochondrial
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fraction. We showed the effect of aging on PSD activity through [ 3H]l-serine incorporation into PS and its transformation in PE. The assay medium contained 50 mM HEPES pH 8, 2.5 calcium chloride or 5 mM EGTA, 2 mg protein in a final volume of 500 mL. The assay was started by adding [ 3H]l-serine (specific activity 21.7 Ci/mmol) and carried out in a thermostated shaking bath for 60 min. Comparative studies with [ 3H]l-serine and [1,2- 14C]ethanolamine were carried out at 2.5 mM of serine and ethanolamine under the above-described conditions. The calcium-independent incorporation into lipids was measured in the presence of 5 mM EGTA. Blanks were prepared in the same way as the corresponding samples except that membrane suspensions were boiled for 5 min before use. In all cases incubations were stopped by adding chloroform/methanol (2:1: v/v). Lipids were extracted according to the method of Folch et al. (1957). The chloroform phase was extensively washed with theoretical upper phase containing 0.05% calcium chloride to eliminate water-soluble radioactive products. The chloroform phase was then evaporated to dryness under N2 and the residue was dissolved in chloroform/methanol (2:1, v/v). Individual phospholipids were isolated by monodimensional pre-coated TLC (Holub and Skeaff, 1987). Lipids were visualized by exposure of the chromatograms to iodine vapors, and scraped off to a vial for counting by liquid scintillation after the addition of 0.4 ml water and 10 ml 5% omnifluor in toluene/triton X-100 (4:1, v/v). 2.4. Assay for phosphatidylethanolamine N-methyltransferase PEMT activity was assayed by measuring the incorporation of [ 3H]methyl groups into endogenous PE from post-mitochondrial supernatant in the presence of S-adenosyl-l[methyl- 3H]methionine ([ 3H]SAM). The buffer assay contained 100 mM Tris-glycylglycine buffer (pH 8.5), 10 mM MgCl2, 5 mM DTT, 0.1 mM PMSF, and 300 mg protein of post-mitochondrial supernatant, at a final volume of 250 mL (Roque and Giusto, 1995). The assay was started by adding 200 mm (10 mCi) [ 3H]SAM (specific activity 1 mCi/ mmol) and carried out in a thermostated shaking bath for 60 min. Incubation was stopped by adding chloroform/methanol/HCl (2:1:0.02%, v/v). The sample was applied on a silicagel G plate and developed by two-dimensional TLC using chloroform/methanol/water (65:25:4, v/v) in the first dimension, and n-propanol/propionic acid/acetic acid/water (2:2:1:1, v/v), in the second. PMME and PDME were added as internal standards and simultaneously chromatographed. Blanks were prepared in the same way as the corresponding samples except that the membrane suspensions were boiled for 5 min before use. Lipids were visualized by exposure of the chromatograms to iodine vapors, and scraped off for counting by liquid scintillation after the addition of 0.4 ml water and 10 ml 5% omnifluor in toluene/triton X-100 (4:1, v/v). 2.5. Assay for phospholipase D PLD activity was assayed by the incorporation of [2- 3H]glycerol (0.15 mCi/mol) into endogenous phospholipids. The buffer assay contained 40 mM HEPES, pH 6.8, 25 mM potassium fluoride; 1 mM DTT, 10 mCi [2- 3H]glycerol (0,15 mCi/mol) and 350 mg of CC homogenates in a total volume of 250 mL. Incubations were carried out for 90 min at 378C. Reactions were stopped by adding 5 ml of chloroform/methanol (2:1,v/v) and lipids were extracted according to the procedure of Folch et al. (1957), using first the upper phase
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containing 0.1 M sulfuric acid and then the upper phase containing water (Casola and Possmayer, 1981). [ 3H]PG was separated by thin layer chromatography in precoated plates using chloroform/acetone/methanol/acetic acid/water (50:20:10:10:5, v/v) as phase. Lipids were visualized by exposure of chromatograms to iodine vapors, and scraped off the plate for counting by liquid scintillation. 2.6. Other methods Protein and lipid phosphorus were determined according to Lowry et al. (1951) and Rouser et al. (1970), respectively. 2.7. Statistical analysis Statistical analysis was performed using Student’s t-test, with the values representing the mean ^ S.D. of the total number of samples indicated in each legend (Johnson and Kotz). 3. Results 3.1. Incorporation of [ 3H]l-serine into phospholipids of different areas of CNS from adult and aged rats The incorporation of [ 3H]serine into phospholipids from homogenates of CC, CRBL and SWM was measured without the addition of exogenous calcium (basal condition) and in a medium supplemented with 2.5 mM calcium. EGTA 5 mM was used to measure calcium-independent synthesis. Lipid synthesis from [ 3H]serine was found to be absent in the presence of EGTA in all brain areas (data not shown). When calcium was present, PS and PE were found to be labeled by the precursor. The effect of aging and calcium ions on PS synthesis from different brain areas is shown in Fig. 2. Under basal conditions, [ 3H]serine incorporation into PS from CRBL was higher than in CC and SWM. In adult rats, 2.5 mM concentration of calcium ions increased PS synthesis one- or 2-fold (P , 0.001) with respect to that found under basal conditions in all brain areas. Under basal conditions PS labeling in CC and CRBL of aged rats was slightly higher than that in adult rats, whereas inhibited PS synthesis was observed in aged rat SWM. It is interesting to note that a stimulatory effect on PS synthesis was observed in CRBL and CC from aged rats at 2.5 mM calcium (P , 0.001). Experiments under base-exchange assay conditions at high base concentration (2.5 mM) were also carried out in CC and CRBL homogenates from adult and aged rats with [1,2- 14C]ethanolamine and [ 3H]serine (Fig. 3). Previously reported data indicate that Km values rise to 2 mM in adult brain (Porcellati et al., 1971). In both tissues, [1,2 14C]ethanolamine and [ 3H]serine incorporation into PE and PS, respectively, was negligible in the presence of EGTA (Fig. 3). In the presence of 2.5 mM calcium, labeled PE in CC and CRBL was one- and 2-fold higher (P , 0.001) than labeled PS, respectively. Data shown in Fig. 3 also show that the incorporation of [1,2 14C]ethanolamine into PE from aged rat CC was similar to that in adult rats, whereas a slight inhibition (19%) was observed in
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Fig. 2. Effect of aging on serine base exchange activity in cerebral cortex (CC), cerebellum (CRBL) and subcortical white matter (SWM) homogenates from adult A and aged rats o . Brain homogenates were prepared in the following way: a) 20% (w/v) in 0.32 M sucrose, 1 mM EDTA, 5 mM buffer HEPES pH 7.4. The assay medium contained 50 mM HEPES pH 8, 2.5 calcium chloride, or 5 mM EGTA, 2 mg protein (100 mL homogenate) in a final volume of 500 mL. The assay was started by adding [ 3H]l-serine (specific activity 21.7 Ci/ mmol) to 12 mm l-serine concentration and carried out in a thermostated shaking bath for 60 min. Incubation was stopped by adding chloroform/methanol (1:1: v/v). Lipids were isolated as described in Section 2. Each experiment was performed twice, using on each occasion three control rats and three senile rats. Data are the mean ^ SD of the six samples from each experiment. **P , 0.025, ***P , 0.001 (*adults versus aged); 111 P , 0.001 (1 basal versus calcium conditions).
CRBL. As shown in Fig. 2, at a lower serine concentration (12 mmm), PS synthesis increased with aging in both brain areas (12 and 24%, respectively). As previously indicated, PE was also synthesized from radioactive serine by PSD activity in CC, CRBL, and SWM homogenates with and without calcium addition (basal condition) (Table 1). PE synthesis in all brain areas was absent when calcium was chelated with EGTA (data not shown).The level of PSD activity exhibited the following order CRBL . CC . SWM and represented 17, 16, and 10% of that of PS, respectively (Table 1). Addition of 2.5 mM calcium increased PE synthesis from serine by PSD about 2-fold (P , 0.001) with respect to the values observed under basal conditions. This stimulatory effect of calcium was enhanced in SWM (approximately a 4-fold increase with respect to basal conditions). The changes on PSD activity seems to be dependent with increased PS availability,
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Fig. 3. Effect of aging on serine and ethanolamine base exchange activities in cerebral cortex (CC) and cerebellum (CRBL) homogenates from adult A and aged rats o . Brain homogenates were prepared at 20% (w/v) in 0.32 M sucrose, 1 mM EDTA, 5 mM buffer HEPES pH 7.4. The assay medium contained 50 mM HEPES pH 8, 2.5 calcium chloride or 5 mM EGTA, 2 mg protein (100 mL homogenate) in a final volume of 500 mL. The assay was started by adding [ 3H]l-serine (specific activity 21.7 Ci/mmol) or [1,2- 14C]ethanolamine (specific activity 4 mCi/ mmol) at 2.5 mM base concentration and was carried out in a thermostated shaking bath for 60 min at 378C. Incubation was stopped by adding chloroform/methanol (1:1: v/v). Lipids were isolated as described in Section 2. Each experiment was performed twice, using on each occasion three control rats and three senile rats. Data are the mean ^ SD of the six samples from each experiment. *P , 0.05; **P , 0.025; ***P , 0.001 (*adults versus aged).
resulting from the calcium stimulatory action in base exchange activity. This was observed when synthesized PE was expressed as a percentage of PS labeling (25.2% and 16.9% in the presence of exogenous calcium and under basal conditions, respectively). PE syntheTable 1 Phosphatidylethanolamine synthesis through phosphatidylserine decarboxylase in cerebral cortex, cerebellum and subcortical white matter from adult and aged rats (Homogenates at 20% (w/v) in 0.32 M sucrose, 1 mM EGTA, 5 mM buffer HEPES pH 7.4, were prepared from cerebral cortex (CC), cerebellum (CRBL), and subcortical white matter (SWM) of 3.5- and 28.5-month-old rats (adult and aged, respectively). Assays for serine base exchange with or without calcium were made in a medium containing 50 mM HEPES pH 8 without or with 2.5 mM calcium chloride, 2 mg protein (100 mL homogenate) in a final volume of 500 mL. The assay was started by adding [ 3H]l-serine (specific activity 21.7 Ci/mmol) and carried out in a thermostated shaking bath for 60 min. Lipid extracts and PE isolation were made as described in Section 2. Synthesized PE is expressed as pmol/mol of total lipid P. Data in parentheses correspond to synthesized PE relative to PS synthesized in ratiox 100. Each experiment was performed twice, using on each occasion three control rats and three senile rats. Data are the mean ^ SD of the six samples from each experiment) CC
CRBL
Adult Basal condition Ca 21 2.5 mM
3.6 ^ (16.9 ^ 13.0 ^ (25.2 ^
Aged 0.9 2) 1.2 1.6)
4.5 ^ (17.3 ^ 15.1 ^ (24.6 ^
SWM
Adult 1.1 4.7) 2.6 2.4)
5.7 ^ (16.6 ^ 14.5 ^ (22.8 ^
Aged 1.8 4.6) 1.9 2.4)
5.8 ^ (14.3 ^ 17.2 ^ (23.6 ^
Adult 1.9 3.7) 0.7 1.9)
2.4 ^ (9.7 ^ 10.3 ^ (19.1 ^
Aged 0.1 0.3) 0.3 0.5)
1.8 ^ (10.4 ^ 10.7 ^ (18.5 ^
0.3 1.3) 1.0 2.0)
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Fig. 4. Phosphatidylethanolamine N-methyltransferase activity in post-mitochondrial fraction of cerebral cortex (CC) homogenates from adult and aged rats. Homogenates of CC were prepared in 10% (w/v) in 100 mM Trisglycylglycine buffer (pH 8.5), 10 mM MgCl2 5 mM DTT, 0.1 mM PMSF. Post-mitochondrial supernatant was obtained after 17 000 × g centrifugation during 15 min. The suspension was incubated with 10 mM [ 3H]SAM (2.5 mCi), in 100 mM Tris-glycylglycine buffer (pH 8.5), 10 mM MgCl2 5 mM DTT, at a final volume of 250 mL. Incubations were stopped and lipids were extracted by addition of 2 ml chloroform/methanol/HCl (2:1:0.2, v/v). Lipids were isolated as described in Section 2. Each experiment was performed twice, using on each occasion three control rats and three senile rats. Data are the mean ^ SD of the six samples from each experiment. *P , 0.05; **P , 0.025; ***P , 0.001. Total methylated lipids (TML PMME 1 PDME 1 PC), phosphatidyl monomethylethanolamine (PMME); phosphatidyl dimethylethanolimine (PDME) and phosphatidylcholine (PC). **P , 0.025; ***P , 0.001).
sized by this pathway in CRBL and SWM were similarly affected by calcium ions. Data on CC, CRBL, and SWM from aged rats reveal that PE synthesized from serine by PSD maintained adult rat values. (Table 1, parentheses). 3.2. Phosphatidylethanolamine N-methyltransferase activity in CC of aged and adult rats The post-mitochondrial fraction of CC from aged and adult rats was incubated with 10 mm [ 3H]SAM at pH 8.5 in the absence of exogenously added phospholipid acceptor for 60 min. Under these conditions, the major methylated product formed in adult rats was PMME, followed by PC and PDME (Fig. 4). An increased incorporation of methyl groups into endogenous PE was observed in the post-mitochondrial fraction of CC from 28.5 month old rats with respect to adult rats, and total methylated products were 52% higher in aged rats (P , 0.001) with respect to adult rats. PMME increased the most in aged with respect to adult rats (94%) (P , 0.025). 3.3. Phospholipase D activity in CC of aged and adult rats Phospholipase D activity was measured by transphosphatidylation reactions on endogenous phospholipids. Homogenates from CC of adult and aged rats were used as enzymatic source. At all incubation times (30, 60, and 90 min) radioactive PG was measured only as a lipid reaction product. PG labeling in CC homogenates from adult rats is shown in the insert of Fig. 5. The time course of the incorporation of [ 3H]glycerol into PG was seen to increase up to 90 min incubation. [ 3H]glycerol incorporation into PG at 30 and
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Fig. 5. Phospholipase D activity in homogenates from cerebral cortex (CC) from A adult and aged rats o . Homogenates were prepared from CC from adults (3.5 months of age) and aged (28.5 months of age) rats in 20% in a medium containing 0.32 M sucrose, 1 mM EDTA, 50 mM Tris-HCl (pH 7.4). The assay was started by addition of CC homogenates (350 mg protein) in a medium containing 5 mCi of [2- 3H]glycerol (0.15 mCi/mol), 40 mM HEPES, pH 6.8, 25 mM potassium fluoride, 1 mM DTT in a total volume of 250 mL. Incubations were carried out for 90 min at 378C. Reactions were stopped by addition of 5 ml of chloroform/methanol (2:1), extracts were purified and lipids were isolated as described in Section 2. Each experiment was performed twice, using on each occasion three control rats and three senile rats. Data are the mean ^ SD of the six samples from each experiment. **P , 0.025.
90 min incubation in adult and aged rats shows that aging increases PLD activity by 60% (P , 0.025).
4. Discussion Phospholipids are mainly synthesized by the following pathways in mammalian tissues: 1) the well-known Kennedy pathway, which are compartamentalized and independently metabolized, lead to PE and PC synthesis; 2) a remodeling pathway that represents a calcium-dependent and -stimulated incorporation of serine, ethanolamine, choline, and other amino alcohols into pre-existing endogenous phospholipids; 3) a pathway by which PC is formed by stepwise PE methylation. An additional mechanism is PLD enzyme activity. PLD, and base exchange in particular, remove the amino alcohol substituent yielding PA (PLD activity) or substitute the amino alcohol on preexisting phospholipids with another amino alcohol (base exchange). A common feature of these enzymes is their ability to modify the polar portion of phospholipids. Although a lower age-related de novo choline or ethanolamine phospholipid synthesis measured by CDP-base incorporation in vivo or in vitro was previously reported, the level of phospholipid content was never-
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theless normal (Brunetti et al., 1979; Gaiti et al., 1981, 1982a; Lopez et al., 1995). This could be due to the increased activity of an enzymatic pathway other than the Kennedy pathway for PC and PE provision. We thus focused on a putative enzymatic mechanism that could become modified in normal aging. Previous work from our laboratory has shown significant changes in acyl group composition in aging rat brain, and it was suggested that such changes are related to modifications in the properties of neural membranes (Lopez et al., 1995; Crews et al., 1980). It has also been suggested that the degree of phospholipid polyunsaturation plays a role in the modulation of membrane protein function (as a signaling system) (Litman and Mitchell, 1996). It is interesting to note that all these enzymes, base exchange (PSS1 and PSS2), PEMT, and PLD, seem to be firmly embedded in the matrix of membranes and show a marked preference for endogenous substrates that are components of their environment. In this context, modifications in these enzymatic activities are likely to be related to changes in the membrane microenvironment. However, at this stage we cannot discard the possibility that serine base exchange could be activated by age-induced alteration in calcium availability. Calcium dependence and calcium stimulation of base-exchange reactions are well known (Vance, 1998). Although alteration of calcium homeostasis levels in senescence neurons has not been fully confirmed (Verhrastky and Toescu, 1998), age-related inhibition of microsomal Ca 21ATPase activity and neuronal calcium levels in rat brain has been recently reported (Hanahisa and Yamaguchi, 1997). Furthermore, an age-related decrease in the expression of a neuronal calcium binding protein, calbindin D28k, which could act as an intraneuronal calcium buffering protein, has also been reported. As previously suggested, the loss of calcium binding proteins may therefore result in the disturbance of intracellular Ca 21 concentration (Kishimoto et al., 1998). It is interesting to note that CRBL, in which serine base exchange was found to mainly increase with aging, is the neural tissue most affected by a decrease in the content of calbindin D28k (50% to 68% of adult mRNA expression) (Baimbridge et al., 1992). In agreement with a previous report, we have also found that ethanolamine-exchange (PSS2) is one- or 2-fold higher than serine-exchange in rat brain (Holbrook and Wurtman, 1988). Furthermore, ethanolamine incorporation into PE was not modified in CC or CRBL from aged rats with respect to the values observed in adult rats. However, serine base exchange activity was selectively increased by aging, CRBL being the most affected. In this context, it is interesting to note the heterogeneity of the exchange system as evidenced by kinetic responses of the enzymes to membrane fluidity and polyvalent cations (Holbrook and Wurtman, 1988). It is known that base exchange reaction primarily forms hexaenoic and tetraenoic phosphatidylserine species in rat liver (Bjerve, 1984). It has also been suggested that this could reflect either the availability of the corresponding phospholipid substrates or a specificity of the serine base exchange enzyme toward the fatty acid composition of the phospholipid substrate. It seems that considerably more hexaenoic species are formed than those accounted for by the composition of the available phospholipid substrate (Bjerve, 1982). In addition, a purified serine base exchange enzyme from rat brain showed some specificity toward the fatty acid composition of the lipid substrate (Taki and Kanfer, 1978). Present results on the increased selective serine-base
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exchange activity in normal aging suggest that aged brain is able to increase phospholipid synthesis, thus contributing to polyunsaturated fatty acid supply to membranes. Several reports show that age has differential effects on neuronal populations as exemplified by: the age-dependent loss of dopaminergic neurons in the nigrostriatal system (Cardozo-Pelaez et al., 1999); changes in mitochondrial respiratory activities in several brain areas during aging (Ojaimi et al., 1999); the chronological changes in the gene expression for cytosolic fatty acid-binding proteins in rat brain (Owada et al., 1996). These findings could imply a similar behavior for base-exchange enzymes in different brain areas during aging. Neurological alterations in aging brain have partially attributed to declining cellular energy. The decreased intracellular ATP levels reported in rat CC may be associated with pathological changes of overall mechanisms involved in the senescent brain (Joo et al., 1999). The increased serine base exchange activities in CC shown in our results may constitute an alternative source for energy independent lipid biosynthesis. It was later reported that base exchange activities and also PLD activity of rat brain are modified during brain development (Kobayashi et al., 1988). During this period, i.e. between embryonic day 17 and postpartum days 14 and 30, drastic modifications in brain lipid fatty acid composition take place (Cunnane and Chen, 1992). This phenomenon could be related to the modification of enzyme activities intrinsic to membranes such as PLD and base exchange enzymes. Although previous results show a decrease in in vivo serine incorporation into PS of CC and hypocampus of aged rats, such decrease seems not to be related to base exchange activity. It has been suggested that an age-related decrease in serine uptake (as with other low-molecular-weight molecules) by neural tissue could be responsible (Gatti et al., 1989). It has been previously reported that PSD significantly contributes to PE synthesis in adult brain, this activity being mainly located in the mitochondrial fraction (Voelker, 1997; Percy et al., 1983). The formation of PE by PSD has also been observed in cultures of cerebral hemispheres and in myelinating cultures of neonatal rat cerebellum (Yavin and Zeigler, 1983; Bradbury, 1984). It is known that base exchange activities are mainly located in the microsomal fraction; because in our experiments homogenates were employed as enzymatic source, simultaneous PSD could be measured through labeled PS as precursor. PE synthesis through PSD using [ 3H]serine as precursor under base exchange assay conditions allowed us to determine whether any changes take place in PSD activity during brain aging. As shown in Table 1, PE synthesis through PSD activity in the CC of adult and aged rats was found to be similar. In addition, calcium produced an increase in PE synthesis by PSD activity in adult and aged rats. Whereas PE labeling represented about 17% of labeled PS under basal conditions, it increased to 25% at a 2.5 mM calcium concentration in adult and aged rats. In preliminary experiments using minced tissue suspension from cerebral hemispheres, increased PE synthesis by decarboxylation was observed in aged brain with respect to adults (unpublished result). This phenomenon could be explained by the different availability of free calcium in the tissue. PC synthesis from S-adenosyl-l-[methyl- 3H] methionine by successive transfer of methyl groups from endogenous PE was higher in the brain of aged rats than in that of
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adult rats. In agreement with our results, Crews et al. (1981) have reported a significant aged-related increase in rat brain PEMT I activity. Although a net increase in the sum of methyl [ 3H] products of PEMT could be seen, the stimulatory effect, in homogenates of the neural tissue was mainly observed in the first compound, PMME. Previous studies on the effect of aging on the acyl composition of brain and liver phospholipid revealed a decrease in polyunsaturated acyl chains sterified to phospholipids. However, this effect is mainly observed in brain lipids rather than in liver lipids. In addition, it has also been reported that PE, rather than PC, is modified by aging (Lo´pez et al., 1995). It has been demonstrated that N-methylation produces PC enriched in arachidonic acid in tissues other than brain, confirming that their precursors (PMME and PDME) also contain high amounts of polyunsaturated fatty acids in rat brain (Tacconi and Wurtman, 1985). One could expect PC synthesized from PE to incorporate polyunsaturated species to the preexisting pool. It is possible that in the brain, the PC formed by N-methylation is deacylated and then reacylated with very long-chain polyunsaturated fatty acids, or that PEMT preferentially uses PE molecular species enriched in long- and very long-polyunsaturated fatty acids. This alternative PC source could also be present in aged brain but seems not to be sufficient to mask the partial polyunsaturated PC depletion previously observed (Lo´pez et al., 1995). Given that these enzyme activities are firmly embedded in the membrane, the significant changes in the acyl group composition of phospholipids in aging brain may be responsible for the modification of such activities through changes in the physico-chemical properties of membrane domains. On the other hand, previous functional studies suggest that important biological properties of membranes are dependent on PC/PE ratios (Cerbon and Calderon, 1995). Furthermore, the increase in the PEMT activity shown in aged brain may play an important role in biological signal transduction. It has been reported that myocardium membranes from aged rats show a significantly lower phospholipid methylation than adult rats. Furthermore, it was suggested that this decrease is connected with the age-related diminution in Ca 21 pump activity as a consequence of decreased membrane fluidity (Heyliger et al., 1988). In the diaphragm, there is a good correlation between the decrease in phospholipid methylation and the decline in its contractile performance as age progresses (Sastry et al., 1982). It is likely that different tissue-specific isoforms could be differently affected by aging. In our experiments, the fatty acid composition of myocardium phospholipids from 28-month-old rats shows no changes with respect to adult rats (unpublished data). It is believed that brain tissue lacks the enzymatic capability to synthesize acetylcholine during aging and that choline present in phosphatidylcholine may represent the reservoir of choline for ACh synthesis. The present findings showing increased PEMT and PLD activities with aging could be related to the supply of choline for ACh synthesis. It has been demonstrated that ACh synthesis in rat brain synaptosomes is stimulated by PC breakdown catalyzed by PLD (Hattori and Kanfer, 1985). In addition, it was observed that methylated intermediates, which have been demonstrated to be good substrates for PLD activity (Jacobs et al., 1988), increase during aging. The findings of the present study reveal the ability of aged brain to increase the activity of enzymatic mechanisms involved in the modification of a portion of phospholipid molecules. Although some authors have reported that de novo phospholipid biosynthesis seems to
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decrease with age (Brunetti et al., 1979; Gaiti et al., 1981, 1982a; Yargicoglu et al., 1999), energy-independent alternative pathways are stimulated. These pathways, made evident through the activation of serine base-exchange (PSS1 and PSS2), PSD, PE N-Mtase, and PLD activities, modify the phospholipid hydrophilic head group and may serve to provide a good availability of unsaturated phospholipid species. This phenomenon, which was observed in in vitro assays with neural membranes from aged rats, could be interpreted as a sort of “recycling” of different phospholipid pools under conditions where precursors and activators (e.g. calcium) are appropriately supplied. An interesting point worthy of further exploration is whether or not this apparent compensatory mechanism is beneficial for aged neural membranes.
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