Differential modulation of phospholipase D and phosphatidate phosphohydrolase during aging in rat cerebral cortex synaptosomes.

Phosphatidylcholine (PC) hydrolysis generates two important second messengers: phosphatidic acid (PA) and diacylglycerol (DAG). Phospholipase D (PLD) and phosphatidate phosphohydrolase (PAPase) are involved in their generation and therefore are key enzymes in signal transduction. Specific isoforms of these enzymes are activated by receptor occupancy in brain. Phosphatidylinositol 4,5-bisphosphate-dependent PLD (PIP2-PLD) and N-ethylmaleimide-insensitive PAPase (PAP2) have been suggested to act in series to generate the biologically active lipids PA and DAG. In the present study we examine age-induced changes mainly in PIP2-PLD and PAP2 activities in cerebrocortical synaptosomes from adult (4 months) and aged (28 months) Wistar rats. Aging increases the activity of both enzymes. Guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) and cytosol (from cerebral cortex) stimulate PLD activity in adult and senescent synaptosomal membranes, the effect being greater in the latter. Under the same experimental conditions PAP2 activity was stimulated in aged membranes whereas in adult membranes GTPgammaS had no effect and cytosol showed a slight inhibitory effect. Diacylglycerol lipase (DGL) activity differs from that of PAP2 in aged rats and it was 21% inhibited with respect to synaptosomal membranes from adult rats. Increased sinaptosomal PLD activity in aged membranes appears to be independent of G protein regulation, whereas PAP2 activity is differentially regulated by GTPgammaS in aged membranes with respect to adult membranes. Our results suggest that under G-protein activation conditions, DAG production by the serial activation of PLD and PAP2 activities is increased in synaptosomal membranes from aged brain. The present paper demonstrates that PA generation (PLD activity) and degradation (PAPase activity) are differentially modulated during the aging process.

Age-associated changes in central nervous system glycerolipid composition and metabolism.

In this review, changes in brain lipid composition and metabolism due to aging are outlined. The most striking changes in cerebral cortex and cerebellum lipid composition involve an increase in acidic phospholipid synthesis. The most important changes with respect to fatty acyl composition involve a decreased content in polyunsaturated fatty acids (20:4n-6, 22:4n-6, 22:6n-3) and an increased content in monounsaturated fatty acids (18:1n-9 and 20:1n-9), mainly in ethanolamine and serineglycerophospholipids. Changes in the activity of the enzymes modifying the phospholipid headgroup occur during aging. Serine incorporation into phosphatidylserine through base-exchange reactions and phosphatidylcholine synthesis through phosphatidylethanolamine methylation increases in the aged brain. Phosphatidate phosphohydrolase and phospholipase D activities are also altered in the aged brain thus producing changes in the lipid second messengers diacylglycerol and phosphatidic acid.

Aging promotes a different phosphatidic acid utilization in cytosolic and microsomal fractions from brain and liver.

Among the morphological and biochemical changes taking place in the membranes of aged tissues, we reported in previous studies on alterations in phospholipid synthesis and phospholipid-specific fatty acid composition. Phosphatidic acid (PA) and diacylglycerol (DAG) are central intermediates in phosphoglyceride and neutral lipid biosynthetic pathways and have also recently been implicated in signal transduction. The present paper shows the effect of aging on phosphatidate phosphohydrolase (PAPase) activiy, which operates on phosphatidic acid to synthesize diacylglycerol. Two forms of mammalian PAPase can be indentified on the basis of subcellular localization and enzyme properties, one involved predominantly in lipid synthesis (PAP 1) and the other in signal transduction (PAP 2). Microsomal and cytosolic fractions of brain and liver from 3.5-month-old (adult) and 28.5-month-old (aged) rats were used. PAPase isoform activities were differentiated on the basis of N-ethylmaleimide (NEM) sensitivity and Mg(2+)-dependency. Our results demonstrate that aging caused PAP 2 to increase in brain microsomal fractions but did not affect PAP 1, whereas in brain cytosolic fractions, it caused a strong decrease in PAP 1 (57%). The distribution of enzymes between microsomes and cytosol changed in aged rats with respect to adult rats, showing a translocation of PAP 1 from cytosol to microsomes. In addition, an increase in the production of monoacylglycerol (MAG) was observed in microsomes from aged brain. PAP 2 activity in liver microsomal fractions from aged rats showed no changes with respect to adult rats whereas PAP 1 activity increased 228% in microsomal fractions and 76% in cytosolic fractions in this tissue. The distribution of PAP 1 activity between microsomal and cytosolic fractions in liver tissue was also affected in aged rats, indicating a translocation of this form of the enzyme from cytosolic to microsomal fractions. The production of monoacylglycerol in liver microsomes also increased, whereas there was a decrease in MAG formation from cytosolic fraction. The changes observed in the two PAPase forms in brain and liver of aged rats with respect to adult rats suggest that PA is differently utilized by the PAPase isoforms, probably generating aging-related DAGs different to those present in adults and required for specific cellular functions. The changes observed in liver PAP 1 from aged with respect to adult rats suggest that such changes could be related with modifications in lipid homeostasis induced by age-altered hormonal balance. However, PA-modified utilization during aging through PAP 2 activity could be related to alterations in neural signal transduction mechanisms.

Alternative pathways for phospholipid synthesis in different brain areas during aging.

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 (2.5mM). 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. Furthermore, 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 compensatory mechanism to provide a pool of phospholipid classes for the maintenance of cellular membrane lipid composition during aging.

Differential incorporation of precursor moieties into cerebral cortex and cerebellum glycerophospholipids during aging.

The incorporation of polar and non-polar moieties into cerebral cortex (CC) and cerebellum (CRBL) phospholipids of adult (3.5-month-old) and aged (21.5-month-old) rats was studied in a minced tissue suspension. The biosynthesis of acidic phospholipids through [3H]glycerol appears to be slightly increased with respect to that of zwitterionic or neutral lipids in CC of aged rats with respect to adult rats. On the contrary, the synthesis of phosphatidylcholine (PC) from [3H]choline was inhibited. However, the incorporation of [14C]serine into phosphatidylserine (PS) was higher in CC and CRBL in aged rats with respect to adult rats. The synthesis of phosphatidylethanolamine (PE) from PS was not modified during aging. Saturated ([3H]palmitic) and polyunsaturated ([3H]arachidonic) acids were incorporated successfully by adult and aged brain lipids. In addition [3H]palmitic, [3H]oleic and [3H]arachidonic acid were employed as glycerolipid precursors in brain homogenate from aged (28.5 month old) and adult (3.5 month old) rats. [3H]oleic acid incorporation into neutral lipids (NL) and [3H]arachidonic acid incorporation into PC, PE and phosphatidylinositol (PI) were increased in aged rats with respect to adult rats. Present results show the ability and avidity of aged brain tissue in vitro to incorporate unsaturated fatty acids when they are supplied exogenously. They also suggest a different handling of choline and serine by base exchange enzyme activities to synthesize PC and PS during aging.

Age-associated changes in the content and fatty acid composition of brain glycerophospholipids.

The fatty acid composition of total lipids and individual phospholipids and the ratio of cholesterol-to-phospholipid content were studied in cerebral cortex, subcortical white matter, cerebellum and medulla oblongata/pons in 4-, 21.5- and 28-month-old rats. The cholesterol-to-phospholipid molar ratio in subcortical white matter, medulla oblongata/pons and cerebellum in 28-month-old rats was found to be 17, 17 and 16% higher, respectively, than in adult rats. These alterations in the molar ratio were produced as a result of a net increase in cholesterol content rather than by changes in the total phospholipid content. The content of alkenylacylglycerophosphoethanolamine (alkenylacyl GPE) increased in 28-month-old rats with respect to 4-month-old rats, following the order cerebral cortex > cerebellum > medulla oblongata/pons > subcortical white matter. The fatty acid composition of total lipids showed an increase in monounsaturated fatty acids (MUFA) and a decrease in polyunsaturated fatty acid (PUFA) content with increasing age in all regions studied, such changes being more marked in cerebellum, medulla oblongata/pons and subcortical white matter than in cerebral cortex. The proportion of 22:6(n-3) in cholineglycerophospholipids in the different brain regions of 28-month-old rats showed a slight decrease with respect to that in adult rats following the order cerebellum > medulla oblongata/pons > subcortical white matter, whereas that of 20:4(n-6) decreased only in cerebellum. Ethanolamineglycerophospholipid fatty acid composition was modified in 28-month-old rats through a marked increase in monounsaturated fatty acids (18:1(n-9) and 20:1(n-9) specifically) in all brain areas. The MUFA content of alkenylacyl GPE increased with increasing age in all the regions studied, in the order cerebral cortex > medulla oblongata/pons > subcortical white matter > cerebellum. An increase in the MUFA content of diacylglycerophosphoethanolamine was observed in medulla oblongata/pons and cerebellum of aged rats with respect to control rats. PUFA content of alkenylacyl GPE decreased mainly in medulla oblongata/pons, cerebral cortex and subcortical white matter of aged rats. The PUFA content of serineglycerophospholipids was the most affected by aging. Changes occurred mainly in the content of 22:6(n-3), 22:4(n-6) and 20:4(n-6) in cerebellum and of 22:6(n-3) and 22:4(n-6) in subcortical white matter. Changes in the inositolglycerophospholipid fatty acid content of 28-month-old rats were observed mainly in medulla oblongata/pons, which showed a decrease in PUFA (22:4(n-6) and 22:6(n-3)) content and an increase in MUFA (18:1 and 20:1).

Effects of aging on the content, composition and synthesis of sphingomyelin in the central nervous system.

Sphingomyelin (SPH) content and composition in different regions of the brain were analyzed in 2.5, 21.5 and 26.5-month-old rats. SPH content increased in the cerebral hemispheres, cerebellum and medulla oblongata plus pons as age increased. The highest SPH content was observed in 26.5-month-old rats, with values increasing by 1.74, 2.75 and 0.88-fold, respectively, over 2.5-month-old rats. The SPH fatty acid composition of brains from aged rats was markedly different from that of adult rats. Between 2.5 and 26.5 months of age the monoenoic/saturated fatty acid ratio increased from 0.22, 0.30 and 0.54 to 0.54, 0.68 and 1.03 in cerebral hemispheres, cerebellum and medulla oblongata plus pons, respectively. The percentage and content of fatty acids longer than 22 carbon atoms esterified to SPH increased with age from 18, 26 and 44 to 48, 52 and 62 mole % in cerebral hemispheres, cerebellum and medulla oblongata plus pons in 26.5-month-old rats. In subcortical white matter from aged rats, monoenoic 22-26 carbon atom fatty acids increased more than the saturated ones in 21.5-month-old rats relative to 2.5-month-old rats. In vitro synthesis of SPH from [3H]choline and [3H]palmitic acid in cerebral cortex and cerebellum showed no significant differences between adult rats and those 21.5 months of age. In cerebellum and in cerebral cortex, [14C]serine incorporation increased in aged rats. The results suggest that aging induces increases in both SPH content and in the monoenoic/saturated fatty acid ratio. These increases are quantitatively different in all brain regions analyzed.

Phosphatidic acid and polyphosphoinositide metabolism in rod outer segments. Differential role of soluble and peripheral proteins.

The phosphorylation of endogenous diacylglycerol (DAG) and phosphoinositides by [tau-32P]ATP was studied in bovine rod outer segments (ROS) selectively depleted of soluble or peripheral and soluble proteins by treatment with moderate (100 mM) or low (5 mM) ionic strength medium, respectively. DAG kinase activity was similar in bleached and non-bleached ROS extracted with 100 mM medium, and amounted to 70% of that observed in the corresponding non-extracted ROS. Phosphatidic acid (PtdH) labelling in ROS extracted in the dark with low ionic strength medium was markedly lower than in those extracted in light. Thus, even when a major proportion of DAG kinase was associated to the membrane, a soluble form also occurred. Most of the membrane-bound fraction behaved as a peripherally associated protein, its binding to the membrane being modified by light. Ir ROS extracted at moderate ionic strength the labelling of inositides was similar to that in non-extracted ROS. A marked enhancement in polyphosphoinositide labelling was observed in ROS extracted in the dark with low ionic strength. Alkaline treatment of ROS also produced inhibition of polyphosphoinositide phosphorylation. A peripheral form of a type C phospholipase, or a peripheral protein-mediated activation of a particulate form thereof, is suggested. Labelled polyphosphoinositides were more actively hydrolyzed in the light and in the dark plus GTP tau S than in the dark-incubated membranes. The results of phosphorylation experiments in membranes where differential extraction of the alpha subunit of transducin was carried out suggest that alpha and beta tau subunits may play opposite modulating roles in PtdH and polyphosphoinositide metabolism.

Effects of aging on the composition and metabolism of docosahexaenoate-containing lipids of retina.

The amount of docosahexaenoate (22:6n-3)-containing phospholipid species decreases with aging in the rat retina. Most lipids, but especially choline and serine glycerophospholipids, show a significant fall in 22:6n-3, which is not compensated by increases in other polyenoic fatty acids. The decrease not only affects 22:6 but also various very long chain n-3 hexaenoic fatty acids which, in phosphatidylcholine, have up to 36 carbon atoms, and which are probably synthesized by successive elongations of 22:6n-3. The in vitro incorporation of [2-3H]glycerol into retinal lipids indicates that the de novo biosynthetic pathways are not impaired by aging. The incorporation of [1-14C]docosahexaenoate is significantly stimulated into all lipids of aged retinas, but to the largest extent in those showing the largest decreases in 22:6, especially in choline glycerophospholipids. The results indicate that the decreased levels of 22:6 with aging are due not to an impaired activity of the enzymes involved in the synthesis and turnover of phospholipids but to a decreased availability of this polyene in the retina. It is suggested that this may stem from a defect in some of the enzymatic steps that lead to the synthesis of 22:6n-3, probably that catalyzed by delta 4 desaturase, the effect on longer hexaenes being secondary to the decreased synthesis of 22:6.

Synthesis of polyphosphoinositides in vertebrate photoreceptor membranes.

Rod outer segments isolated from bovine retinas incorporated 32P into phospholipids after incubation with [gamma-32P]ATP in a Mg2+-containing medium. Only phosphatidylinositol 4-phosphate, phosphatidylinositol 4,5-bisphosphate, and phosphatidate were labelled. The incorporation of label into lipids was detected as early as 20 s after the start of incubation and the products were stable for at least 10 min. The reactions were time, protein and ATP-concentration dependent. Entire rod outer segments showed higher diacylglycerol kinase and lower phosphatidylinositol and phosphatidylinositol 4-phosphate kinase activities than the disc membranes obtained from them. Exogenously added phosphatidylinositol (up to 1 mM) in the presence of Triton X-100 increased phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate labelling in rod outer segments (8- and 6-fold, respectively). Triton X-100 at a concentration of 0.4% stimulated phosphorylation of endogenous phosphoinositides. Diacylglycerol kinase activity was largely suppressed by the detergent, but this effect was partially reversed by addition of phosphatidylinositol. It is suggested that the rod outer segments contain phosphatidylinositol kinase and phosphatidylinositol 4-phosphate kinase bound to disc membranes, as well as an active diacylglycerol kinase occurring either as a soluble or a peripherally bound protein in disc membranes.

Accumulation of phosphatidic acid in microsomes from propranolol-treated retinas during short-term incubations.

The pool size and synthesis of phosphatidic acid derived from [2-3H]glycerol were studied in bovine whole retinas and subcellular fractions. Microsomal preparations from retinas incubated with [2-3H]glycerol displayed the highest percentage labeling of phosphatidic acid at 5 min of incubation: labeling decreased rapidly thereafter. In drug-treated retinas, 0.5 mM propranolol increased the endogenous content of phosphatidic acid and stimulated [2-3H]glycerol labeling in whole retina and microsomal and postmicrosomal supernatant fractions. This effect was observed during short-term incubations and was reversible. In pulse-chase experiments, 60 min of reincubation greatly reduced the labeling effect, although propranolol still enhanced phosphatidic acid labeling. At the same time, endogenous phosphatidic acid accumulated, and reincubation without propranolol reversed the effect. During accumulation, the amount of palmitate increased and that of oleate decreased, whereas the relatively high level of docosahexaenoate in phosphatidic acid remained unchanged. It was concluded that this propranolol-induced effect is due to cationic amphiphilic drug activity in the endoplasmic reticulum that results in a partial inhibition of phosphatidic acid degradation and a stimulation of its de novo synthesis. Hence, net synthesis of phosphatidic acid can be assessed in the retina during short-term incubation with propranolol.

Reversibility of propranolol-induced changes in the biosynthesis of monoacylglycerol, diacylglycerol, triacylglycerol, and phospholipids in the retina.

The biosynthesis and metabolism of phospholipids and neutral glycerides were studied in the bovine retina. Radioactive glycerol was used as a precursor. Phentolamine and d- and dl-propranolol were found to produce similar effects on lipid metabolism in the retina. Marked stimulation of phosphatidylinositol (PhI) synthesis and maximal inhibition of phosphatidylcholine (PhC), diacylglycerol (DG), and triacylglycerol (TG) formation were observed within 5 min after exposure to 0.5 mM dl-propranolol. Pulse-chase experiments showed a high turnover rate in DG and a reversibility of the propranolol-induced changes produced during the synthesis of PhC, TG, DG, monoacylglycerol (MG), and phosphatidylserine. All reversals of the drug-induced biosynthetic profiles approached control values 60 min after incubation in drug-free medium. However, complete reversal was not achieved in any of the cases under these conditions. Propranolol appeared to inhibit both the formation of DG from phosphatidic acid and the further metabolism of DG, probably to MG. Phosphatidylethanolamine biosynthesis showed some recovery from this inhibition. Synthesis of PhI was greatly stimulated by preincubation with propranolol and was further enhanced by reincubation in the presence of propranolol. However, this effect was not reversed by reincubation without the drug. The active de novo biosynthesis of retinal phospholipids and glycerides is a very dynamic pathway that may be redirected by amphiphilic drugs. In addition, the partial reversal of modifications induced in the flux of [2-3H]glycerol through the lipids can occur during short-term reincubations of retinas in drug-free medium.

Biosynthesis of membrane lipids in the retina: Subcellular distribution and propranolol action on phosphatidic acid, phosphatidylserine and phosphatidylethanolamine.

The de novo biosynthesis of phospholipids and glycerides was followed in subcellular fractions from bovine retinas incubated during short periods with radioactive glycerol. The labeling of lipid classes was determined in the following fractions: rod outer segments, P(1), P(2), microsomes and soluble. A highly labeled phosphatidic acid was found in microsomal fractions with a maximum at 5 min relative to other lipids labeling. The labeling of phospholipids and glycerides from microsomes as well as from other fractions was rapid. The soluble contained minor quantities of phospholipids with high radioactivity. Using radioactive glycerol and amphiphilic drugs the rates of phosphatidic acid synthesis in microsomal membranes were assessed. Moreover, in agreement with previous studies from this laboratory showing that phosphatidic acid contains a high proportion of docosahexaenoate, it is demonstrated that this fatty acid is acylated in the biosynthetic route leading to phosphatidic acid formation. Phosphatidylserine is synthesized from radioactive serine and also through a Ca(2+) independent pathway not involving phosphatidylethanolamine. Phosphatidylethanolamine is also formed by serine decarboxylation. Amphiphilic drugs greatly stimulate phosphatidylserine synthesis. Extracellular Ca(2+) stimulates the synthesis of phosphatidylserine and its decarboxylation. Besides, Ca(2+) potentiates the phospholipid effect exerted by propranolol. The synthesis of phosphatidylinositol as well as that of other minor acidic phospholipids seems to operate in a coordinated manner under different experimental conditions. The microsomal system of the retina generates a docosahexaenoyl enriched phosphatidic acid at high rates. In addition to the turnover of different phospholipid moieties there are active pathways for the de novo biosynthesis of membrane lipids in the retina.