Normal aging in rats and pathological aging in human Alzheimer's disease decrease FAAH activity: modulation by cannabinoid agonists.

Anandamide is an endocannabinoid involved in several physiological functions including neuroprotection. Anandamide is synthesized on demand and its endogenous level is regulated through its degradation, where fatty acid amide hydrolase plays a major role. The aim of this study was to characterize anandamide breakdown in physiological and pathological aging and its regulation by CB1 and CB2 receptor agonists. Fatty acid amide hydrolase activity was analyzed in an independent cohort of human cortical membrane samples from control and Alzheimer's disease patients, and in membrane and synaptosomes from adult and aged rat cerebral cortex. Our results demonstrate that fatty acid amide hydrolase activity decreases in the frontal cortex from human patients with Alzheimer's disease and this effect is mimicked by Aß(1-40) peptide. This activity increases and decreases in aged rat cerebrocortical membranes and synaptosomes, respectively. Also, while the presence of JWH-133, a CB2 selective agonist, slightly increases anandamide hydrolysis in human controls, it decreases this activity in adults and aged rat cerebrocortical membranes and synaptosomes. In the presence of WIN55,212-2, a mixed CB1/CB2 agonist, anandamide hydrolysis increases in Alzheimer's disease patients but decreases in human controls as well as in adult and aged rat cerebrocortical membranes and synaptosomes. Although a similar profile is observed in fatty acid amide hydrolase activity between aged rat synaptic endings and human Alzheimer's disease brains, it is differently modulated by CB1/CB2 agonists. This modulation leads to a reduced availability of anandamide in Alzheimer's disease and to an increased availability of this endocannabinoid in aging.

Age-related changes in the metabolization of phosphatidic acid in rat cerebral cortex synaptosomes.

In this study, phosphatidic acid (PA) metabolization is found to generate diacylglycerol (DAG), monoacylglycerol (MAG) and glycerol by the sequential action of lipid phosphate phosphatase (LPP), diacylglycerol lipase (DAGL), and monoacylglycerol lipase (MAGL) in cerebral cortex (CC) synaptosomes. It is also demonstrated that PA is metabolized by phospholipases A (PLA)/lysophosphatidic acid phosphohydrolase (LPAPase) in synaptic endings. Age-related changes in the metabolization of PA have been observed in rat cerebral cortex synaptosomes in the presence of the alternative substrates for LPP, namely LPA, sphingosine 1-phosphate (S1P) and ceramide 1-phosphate (C1P). In addition, LPA and C1P up to concentrations of about 50 microM favor the metabolism in the direction of MAG and glycerol in aged and adult synaptosomes, respectively. At equimolecular concentrations with PA, LPA decreases DAG formation in adult and aged synaptosomes, whereas S1P decreases it and C1P increases it only in aged synaptosomes. Sphingosine (50 microM) or ceramide (100 microM) increase PA metabolism by the pathway that involves LPP/DAGL/MAGL action in aged membranes. Using RHC-80267, a DAGL inhibitor, we could observe that 50% and 33% of MAG are produced as a result of DAGL action in adult and aged synaptosomes, respectively. Taken together, our findings indicate that the ageing modifies the different enzymatic pathways involved in PA metabolization.

Diacylglyceride lipase activity in rod outer segments depends on the illumination state of the retina.

We have demonstrated that the competition between phosphatidic acid (PA) and lysophosphatidic acid (LPA), sphingosine 1-phosphate (S1P) and ceramide 1-phosphate (C1P) for lipid phosphate phosphatases (LPP) generates different levels of diacylglycerol (DAG) depending on the illumination state of the retina. The aim of the present research was to determine the diacylglyceride lipase (DAGL) activity in purified rod outer segments (ROS) obtained from dark-adapted retinas (DROS) or light-adapted retinas (BLROS) as well as in ROS membrane preparations depleted of soluble and peripheral proteins. [2-(3)H]monoacylglycerol (MAG), the product of DAGL, was evaluated from [2-(3)H]DAG generated by LPP action on [2-(3)H]PA in the presence of either LPA, S1P or C1P. MAG production was inhibited by 55% in BLROS and by 25% when the enzymatic assay was carried out in ROS obtained from dark-adapted retinas and incubated under room light (LROS). The most important events occurred in DROS where co-incubation of [2-(3)H]PA with LPA, S1P or C1P diminished MAG production. A higher level of DAGL activity was observed in LROS than in BLROS, though this difference was not apparent in the presence of LPA, S1P or C1P. DAGL activity in depleted DROS was diminished with respect to that in entire DROS. LPA, S1P and C1P produced a similar decrease in MAG production in depleted DROS whereas only C1P significantly diminished MAG generation in depleted BLROS. Sphingosine and ceramide inhibited MAG production in entire DROS and stimulated its generation in BLROS. Sphingosine and ceramide stimulated MAG generation in both depleted DROS and BLROS. Under our experimental conditions the degree of MAG production depended on the illumination state of the retina. We therefore suggest that proteins related to phototransduction phenomena are involved in the effects observed in the presence of S1P/sphingosine or C1P/ceramide.

Age-associated changes of insulin action on the hydrolysis of diacylglycerol generated from phosphatidic acid.

Age-related changes in insulin action on diacylglycerol (DAG) degradation was studied in rat cerebral cortex synaptosomes. The generation of monoacylglycerol (MAG) and water soluble products (WSP, glycerol plus glycerol-3-phosphate) from DAG was studied in cerebral cortex (CC) synaptosomes from adult (4-month-old) and aged (28-month-old) rats. Additionally, the effect of porcine insulin and tyrosine phosphorylation was evaluated in the same group of animals. In this study we demonstrate that the age-related increase in WSP generation was accompanied by unmodified MAG levels. In the presence of diacylglycerol lipase (DAG lipase) inhibitor, RHC-80267, a lower inhibitory effect on MAG production was observed in CC synaptosomes from aged rats with respect to that in adult membranes. Under these experimental conditions, WSP formation was only diminished in aged membranes. Insulin stimulated MAG and WSP formation at long incubation times (30 min) in adult animals, while it had an inhibitory effect in aged animals. Insulin plus vanadate (as tyrosine-phosphatase inhibitor) inhibited MAG production at short incubation times whereas the same effect was observed in aged animals at long times of incubation. WSP formation was stimulated by insulin plus vanadate both in adult and aged animals at 30 min of incubation. Our results show that insulin differentially modulates MAG and WSP production from exogenous PA in CC synaptosomes from aged rats compared with adult rats.

Phospholipase D and phosphatidate phosphohydrolase activities in rat cerebellum during aging.

Aging is a process that affects different organs, of which the brain is particularly susceptible. PA and DAG are central intermediates in the phosphoglyceride as well as in the neutral lipid biosynthetic pathway, and they have also been implicated in signal transduction. Phospholipase D (PLD) and phosphatidate phosphohydrolase (PAP) are the enzymes that generate PA and DAG. The latter can be transformed into MAG by diacylglycerol lipase (DGL). In the present study, we examine how aging modulates the PLD, PAP, and DGL isoforms in cerebellar subcellular fractions from 4- (adult), 28-, and 33-mon-old (aged) rats. PI-4,5-bisphosphonate (PIP2)-dependent PLD, PAP1, and DGL1 were distributed in different percentages in all cerebellum subcellular fractions. On the other hand, PAP2 and DGL2 activities were observed in all subcellular fractions except in the cytosolic fraction. Aging modified the enzyme distribution pattern. In addition, aging decreased nuclear (45%), mitochondrial-synaptosomal (55%), and cytosolic (71%) PAP1 activity and increased (28%) microsomal PAP1 activity. DGL1 activity was decreased in nuclear (85%) and mitochondrial-synaptosomal (63%) fractions by aging. On the other hand, PIP2-dependent PLD activities were increased in the mitochondrial-synaptosomal fraction. PAP2 and DGL2 were increased in the microsomal fraction by 87 and 114%, respectively, and they were decreased in the nuclear fraction. The changes observed in cerebellum PAP1 and DGL1 activities from aged rats with respect to adult rats could be related to modifications in lipid metabolism. Differential PA metabolization during aging through PIP2-dependent PLD/PAP2/DGL2 activities could be related to alterations in the neural signal transduction mechanisms.

Synthesis of retinal ganglion cell phospholipids is under control of an endogenous circadian clock: daily variations in phospholipid-synthesizing enzyme activities.

Retinal ganglion cells (RGCs) are major components of the vertebrate circadian system. They send information to the brain, synchronizing the entire organism to the light-dark cycles. We recently reported that chicken RGCs display daily variations in the biosynthesis of glycerophospholipids in constant darkness (DD). It was unclear whether this rhythmicity was driven by this population itself or by other retinal cells. Here we show that RGCs present circadian oscillations in the labeling of [32P]phospholipids both in vivo in constant light (LL) and in cultures of immunopurified embryonic cells. In vivo, there was greater [32P]orthophosphate incorporation into total phospholipids during the subjective day. Phosphatidylinositol (PI) was the most 32P-labeled lipid at all times examined, displaying maximal levels during the subjective day and dusk. In addition, a significant daily variation was found in the activity of distinct enzymes of the pathway of phospholipid biosynthesis and degradation, such as lysophospholipid acyltransferases (AT II), phosphatidate phosphohydrolase (PAP), and diacylglycerol lipase (DGL) in cell preparations obtained in DD, exhibiting differential but coordinated temporal profiles. Furthermore, cultures of immunopurified RGCs synchronized by medium exchange displayed a circadian fluctuation in the phospholipid labeling. The results demonstrate that chicken RGCs contain circadian oscillators capable of generating metabolic oscillations in the biosynthesis of phospholipids autonomously.

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.

Effect of light on phosphatidate phosphohydrolase activity of retina rod outer segments: the role of transducin.

The aim of the present paper is to evaluate the modulation of phosphatidate phosphohydrolase (PAPase) and diacylglyceride lipase (DGL) activities in bovine rod outer segment (ROS) under dark and light conditions and to evaluate the role of transducin (T) in this phenomenon. In dark-adapted ROS membranes exposed to light, PAPase activity is inhibited by 20% with respect to the activity found under dark conditions. To determine whether the retinal G protein, T, participates in the regulation of PAPase activity in these membranes, the effects of GTPgammaS and GDPbetaS on enzyme activity were examined. Under dark conditions in the presence of GTPgammaS, which stabilizes T in its active form (Talpha + Tbetagamma), enzyme activity was inhibited and approached control values under light conditions. GDPbetaS, on the other hand, which stabilizes the inactive state of T (Talphabetagamma), stimulated PAPase activity by 36% with respect to control light conditions. ADP-ribosylation by cholera and pertussis toxin was also studied. In ADP-rybosilated ROS membranes with pertussis toxin under dark conditions, PAPase activity was 36% higher than the activity found under control light conditions. ADP-ribosylation by CTx, on the other hand, inhibited PAPase activity by 22%, with respect to dark control conditions, mimicking light effect. The effects of GTPgammaS and GDPbetaS and conditions of ADP-ribosylation by PTx and CTx on DGL activity were similar to those of PAPase activities. Based on NEM sensitivity we have also demonstrated that the PAPase present in ROS is the PAP 2 isoform. Our findings therefore suggest that light inhibition of PAP 2 in ROS is a transducin-mediated mechanism.

A simple method to obtain retinal cell preparations highly enriched in specific cell types. Suitability for lipid metabolism studies.

The neural retina is a highly complex tissue composed of excitatory and inhibitory neurons and of glial cells. The biosynthesis of lipids that occurs in the retina may be distinctly regulated in one neuronal type of cells with respect to another. To study the cell-type-specific aspects of lipid metabolism, a method for the separation of different retinal cell populations is needed. Herein, we describe a very simple procedure to isolate preparations highly enriched in specific retinal cell types that are suitable for in vitro biochemical assays. The method consists of selectively obtaining photoreceptors (PRC) and retina ganglion cells (RGC) from lyophilized chicken retinas using Scotch tape to assess, then, the in vitro incorporation of labeled precursors into phospholipid moieties. When their metabolic capability was assayed, it was found that these cell preparations maintain their enzyme activities intact to incorporate (32)P-phosphate into phospholipids in vitro at a similar rate as observed in fresh tissue after 1 h incubation. The highest proportion of labeling was observed in phosphatidylethanolamine (PE), followed by phosphoinositides (PIPs), phosphatidylcholine (PC) and phosphatidic acid (PA). Phosphatidate-phosphohydrolase (PAPase), a key enzyme of glycerolipid metabolism, exhibits similar levels of activity when assessed in fresh or frozen cell preparations, indicating that the lyophilization procedure does not significantly affect this activity. It is concluded that different cell populations obtained by the experimental procedure described herein, are useful to study the cellular metabolism and its regulation.

Light exposure activates retina ganglion cell lysophosphatidic acid acyl transferase and phosphatidic acid phosphatase by a c-fos-dependent mechanism.

We previously reported that the biosynthesis of phospholipids in the avian retina is altered by light stimulation, increasing significantly in ganglion cells in light and in photoreceptor cells in dark. In the present work, we have determined that light significantly increases the incorporation of [3H]glycerol into retina ganglion cell glycerophospholipids in vivo by a Fos-dependent mechanism because an oligonucleotide antisense to c-fos mRNA substantially blocked the light-dark differences. We also studied in vitro the enzyme activities of phosphatidate phosphohydrolase (PAPase), lysophosphatidate acyl transferase (AT II), and phosphatidylserine synthase from retinas of chickens exposed to light or dark. Higher PAPase I and AT II activities were found in incubations of retinal ganglion cells from animals exposed to light; no increase was observed in preparations obtained from light-exposed animals treated with the c-fos antisense oligonucleotide. No light-dark differences were found in phosphatidylserine synthase activity. These findings support the idea that a coordinated photic regulation of PAPase I and AT II is taking place in retina ganglion cells. This constitutes a reasonable mechanism to obtain an overall increased synthesis of glycerophospholipids in stimulated cells that is mediated by the expression of Fos-like proteins.

Phosphorylation of rod outer segment proteins modulates phosphatidylethanolamine N-methyltransferase and phospholipase A2 activities in photoreceptor membranes.

The activities of enzymes involved in lipid metabolism--phospholipase A2 (PLA2) and phosphatidylethanolamine N-methyltransferase (PE N-MTase)--were found to be differently affected by pre-incubation of rod outer segments (ROS) under protein phosphorylating or dephosphorylating conditions. Exposure to cAMP-dependent protein kinase (PKA), under dark or light conditions, produced a significant increase in PE N-MTase activity, whereas PLA2 activity decreased. Under standard protein kinase C (PKC) phosphorylating conditions in light, PE N-MTase activity was stimulated and PLA2 activity was not affected. When the assays were performed in the dark, both enzymatic activities were unaffected when compared to the corresponding controls. Incubation of ROS membranes in light in the presence of PKC activators phorbol 12,13-dibutyrate (PDBu) and dioctanoylglycerol (DOG) resulted in the same pattern of changes in enzyme activities as described for standard PKC phosphorylating condition. Pre-incubation of membranes with the PKC inhibitor H-7 reduced the stimulation of PDBu on PE N-MTase activity, and had no effect on PLA2 activity in ROS membranes incubated with the phorbol ester. Pre-treatment of isolated ROS with alkaline phosphatase resulted in decreased PE N-MTase activity and produced a significant stimulation of PLA2 activity under dark as well as under light conditions when compared to the corresponding controls. These findings suggest that ROS protein phosphorylation and dephosphorylation modulates PE N-MTase and PLA2 activities in isolated ROS, and that these activities are independently and specifically modulated by particular kinases. Furthermore, dephosphorylation of ROS proteins has the opposite effect to that produced by protein phosphorylation on the enzymes studied.

Can phosphorylation and dephosphorylation of rod outer segment membranes affect phosphatidate phosphohydrolase and diacylglycerol lipase activities?

Phosphatidate phosphohydrolase (PAPase) and diacylglycerol lipase (DGL) enzymatic activities were found to be differently affected by preincubation of rod outer segments (ROS) under protein phosphorylation or dephosphorylation conditions in darkness or in light. Under protein kinase C (PKC) phosphorylation conditions, PAPase and DGL were inhibited in darkness and in light. The inhibitory effect on PAPase and DGL activities by PKC phosphorylation in the presence of light was more pronounced when the activities were compared with the activities in control membranes determined in the presence of EGTA. The addition of PKC activators such as phorbol-12,13-dibutyrate and dioctanoylglycerol (DOG) instead of DG produced the same pattern of changes in enzymatic activities. Pretreatment of ROS membranes with cAMP-dependent protein kinase (PKA) produced a significant increase in both enzymatic activities in the presence of light. No changes were observed when ROS proteins were phosphorylated by PKA in the dark. Dephosphorylation of ROS membranes with alkaline phosphatase resulted in a decrease in PAPase activity that was more marked under light than under dark conditions. DGL activity was not modified under dephosphorylation conditions. These findings suggest that the metabolization of phosphatidic acid in isolated ROS is differently affected by protein phosphorylation and dephosphorylation reactions.

Lipid metabolism in photoreceptor membranes: regulation and mechanisms.

Lipid metabolism in photoreceptor rod outer segments has attracted considerable attention because of its importance in providing the appropriate environment for supporting an efficient phototransduction mechanism. Recent studies suggest that lipid metabolism in these membranes is involved in the generation of second messengers and in signal transduction mechanisms. Phospholipid turnover is tightly regulated by phosphorylation-dephosphorylation reactions and light, and provides, in turn, with molecules capable of activating protein kinases and cellular processes such as membrane fusion or light-adaptation. These findings suggest that photoreceptor membrane lipids are more than just important structural components of the visual cell rod outer segment.

Differential properties of phosphatidate phosphohydrolase and diacylglyceride lipase activities in retinal subcellular fractions and rod outer segments.

1. The effect of magnesium and dl-propranolol on phosphatidate phosphohydrolase (PAPase) and diacylglycerol lipase (DGL) activities in isolated rod outer segments (ROS) and of the former on subcellular fractions from bovine retina was investigated. 2. Mg(2+)-independent PAPase activity was found in ROS, whereas in the other subcellular fractions PAPase activities both dependent on and independent of Mg2+ were detected. 3. The membrane-bound PAPase activity was stimulated at low concentrations of Mg2+ and inhibited at higher concentrations. The soluble activity was always stimulated by the ion. 4. dl-Propranolol (1000 microM) exerted a slight stimulatory effect on PAPase in ROS whereas total PAPase activity of microsomal fraction was not affected. 5. Mg2+ (0.2 mM) stimulated DGL activity (30%) whereas it was inhibited at higher concentration. 6. DGL lipase activities, both dependent on and independent of Mg2+, were detected in subcellular fractions of bovine retina. 7. DGL properties in ROS are also described.