Effect of D609 on phosphatidylcholine metabolism in the nuclei of LA-N-1 neuroblastoma cells: a key role for diacylglycerol.

In our previous studies, TPA treatment of LA-N-1 cells stimulated the production of diacylglycerol in nuclei, probably through the activation of a phospholipase C. Stimulation of the synthesis of nuclear phosphatidylcholine by the activation of CTP:phosphocholine cytidylyltransferase was also observed. The present data show that both effects were inhibited by the pretreatment of the cells with D609, a selective phosphatidylcholine-phospholipase C inhibitor, indicating that the diacylglycerol produced through the hydrolysis of phosphatidylcholine in the nuclei is reutilized for the synthesis of nuclear phosphatidylcholine and is required for the activation of CTP:phosphocholine cytidylyltransferase.

Neurochemical consequences of kainate-induced toxicity in brain: involvement of arachidonic acid release and prevention of toxicity by phospholipase A(2) inhibitors.

In kainate-induced neurotoxicity, the stimulation of kainate receptors results in the activation of phospholipase A(2) and a rapid release of arachidonic acid from neural membrane glycerophospholipids. This process raises arachidonic acid levels and produces alterations in membrane fluidity and permeability. These result in calcium influx and stimulation of lipolysis and proteolysis, production of lipid peroxides, depletion of ATP, and loss of reduced glutathione. As well as the above neurochemical changes, stimulation of ornithine decarboxylase, altered activities of protein kinase C isozymes, and expression of immediate early genes, cytokines, growth factors, and heat shock proteins have also been reported. Kainate-induced stimulation of arachidonic acid release, calcium influx, accumulation of lipid peroxides and products of their decomposition, especially 4-hydroxynonenal (4-HNE), along with alterations in cellular redox state and ATP depletion may play important roles in kainate-induced cell death. Thus the consequences of altered glycerophospholipid metabolism in kainate-induced neurotoxicity can lead to cell death. Kainate-induced neurotoxicity initiates apoptotic as well as necrotic cell death depending upon the intensity of oxidative stress and abnormality in mitochondrial function. Other neurochemical changes may be related to synaptic reorganization following kainate-induced seizures and may be involved in recapitulation of hippocampal development and synaptogenesis.

Plasmalogens: workhorse lipids of membranes in normal and injured neurons and glia.

Plasmalogens are unique glycerophospholipids because they have an enol ether double bond at the sn-1 position of the glycerol backbone. They are found in all mammalian tissues, with ethanolamine plasmalogens 10-fold higher than choline plasmalogens except in muscles. The enol ether double bond at the sn-1 position makes plasmalogens more susceptible to oxidative stress than the corresponding ester-bonded glycerophospholipids. Plasmalogens are not only structural membrane components and a reservoir for second messengers but may also be involved in membrane fusion, ion transport, and cholesterol efflux. Plasmalogens may also act as antioxidants, thus protecting cells from oxidative stress. Receptor-mediated degradation of plasmalogens by plasmalogen-selective phospholipase A2 results in the generation of arachidonic acid, eicosanoids, and platelet activating factor. Low levels of these metabolites have trophic effects, but at high concentration they are cytotoxic and may be involved in allergic response, inflammation, and trauma. Levels of plasmalogens are decreased in several neurological disorders including Alzheimer's disease, ischemia, and spinal cord trauma. This may be due to the stimulation of plasmalogen-selective phospholipase A2. A deficiency of plasmalogens in peroxisomal disorders and Niemann-Pick type C disease indicates that this deficiency may be due to the decreased activity of plasmalogen synthesizing enzymes that occur in peroxisomes.

Plasmalogens, phospholipase A2, and docosahexaenoic acid turnover in brain tissue.

Plasmalogens are glycerophospholipids of neural membranes containing vinyl ether bonds. Their synthetic pathway is located in peroxisomes and endoplasmic reticulum. The rate-limiting enzymes are in the peroxisomes and are induced by docosahexaenoic acid (DHA). Plasmalogens often contain arachidonic acid (AA) or DHA at the sn-2 position of the glycerol moiety. The receptor-mediated hydrolysis of plasmalogens by cytosolic plasmalogen-selective phospholipase A2 generates AA or DHA and lysoplasmalogens. AA is metabolized to eicosanoids. The mechanism of signaling with DHA is not known. The plasmalogen-selective phospholipase A2 differs from other intracellular phospholipases A2 in molecular mass, kinetic properties, substrate specificity, and response to glycosaminoglycans, gangliosides, and sialoglycoproteins. A major portion of [3H]DHA incorporated into neural membranes is found at the sn-2 position of ethanolamine glycerophospholipids. Studies with a mutant cell line defective in plasmalogen biosynthesis indicate that the incorporation of DHA is reduced in this RAW 264.7 cell line by 50%. In contrast, the incorporation of AA remains unaffected. This is reversed completely when the growth medium is supplemented with sn-1-hexadecylglycerol, suggesting that DHA can be selectively targeted for incorporation into plasmalogens. We suggest that deficiencies of DHA and plasmalogens in peroxisomal disorders, Alzheimer's disease (AD), depression, and attention deficit hyperactivity disorders (ADHD) may be responsible for abnormal signal transduction associated with learning disability, cognitive deficit, and visual dysfunction. These abnormalities in the signal-transduction process can be partially corrected by supplementation with a diet enriched with DHA.

Effect of retinoic acid on the Ca2+-independent phospholipase A2 in nuclei of LA-N-1 neuroblastoma cells.

LA-N-1 neuroblastoma cell cultures contain Ca2+-independent phospholipases A2 hydrolyzing phosphatidylethanolamine and ethanolamine plasmalogens. These enzymes differ from each other in their molecular mass, substrate specificity, and kinetic properties. Subcellular distribution studies have indicated that the activity of these phospholipases is not only localized in the cytosol but also in non-nuclear membranes and in nuclei. The treatment of LA-N-1 neuroblastoma cell cultures with retinoic acid results in a marked stimulation of Ca2+-independent phospholipases A2 hydrolyzing phosphatidylethanolamine and plasmenylethanolamine. The increase of the activities of both enzymes was first observed in nuclei followed by those present in the cytosol. No effect of retinoic acid on either phospholipase activity could be observed in non-nuclear membranes. The stimulation of these enzymes may be involved in the generation and regulation of arachidonic acid and its metabolites during differentiation.

Differential effects of calcium-dependent and calcium-independent phospholipase A(2) inhibitors on kainate-induced neuronal injury in rat hippocampal slices.

Brain tissue contains multiple forms of intracellular phospholipase A(2) (PLA(2)) activity that differ from each other in many ways including their response to specific inhibitors. The systemic administration of kainic acid to rats produces a marked increase in cPLA(2) activity in neurons and astrocytes. This is associated with increased lipid peroxidation as evidenced by accumulation of 4-hydroxynonenal (4-HNE) modified proteins. The present study describes the effect of specific inhibitors of Ca(2+)-dependent or Ca(2+)-independent PLA(2) on kainite-induced excitotoxic injury in rat hippocampal slices. Specific inhibitors of Ca(2+)-dependent PLA(2) prevented the decrease of a neuronal marker, GluR1, and increase in cPLA(2) and 4-HNE immunoreactivities in slices treated with kainate. This shows that cPLA(2) plays an important role in kainite-induced neurotoxicity and that cPLA(2) inhibitors can be used to protect hippocampal slices from damage induced by kainate.

Deacylation and reacylation of neural membrane glycerophospholipids.

The deacylation-reacylation cycle is an important mechanism responsible for the introduction of polyunsaturated fatty acids into neural membrane glycerophospholipids. It involves four enzymes, namely acyl-CoA synthetase, acyl-CoA hydrolase, acyl-CoA: lysophospholipid acyltransferase, and phospholipase A2. All of these enzymes have been purified and characterized from brain tissue. Under normal conditions, the stimulation of neural membrane receptors by neurotransmitters and growth factors results in the release of arachidonic acid from neural membrane glycerophospholipids. The released arachidonic acid acts as a second messenger itself. It can be further metabolized to eicosanoids, a group of second messengers involved in a variety of neurochemical functions. A lysophospholipid, the second product of reactions catalyzed by phospholipase A2, is rapidly acylated with acyl-CoA, resulting in the maintenance of the normal and essential neural membrane glycerophospholipid composition. However, under pathological situations (ischemia), the overstimulation of phospholipase A2 results in a rapid generation and accumulation of free fatty acids including arachidonic acid, eicosanoids, and lipid peroxides. This results in neural inflammation, oxidative stress, and neurodegeneration. In neural membranes, the deacylation-reacylation cycle maintains a balance between free and esterified fatty acids, resulting in low levels of arachidonic acid and lysophospholipids. This is necessary for not only normal membrane integrity and function, but also for the optimal activity of the membrane-bound enzymes, receptors, and ion channels involved in normal signal-transduction processes.

Glycerophospholipids in brain: their metabolism, incorporation into membranes, functions, and involvement in neurological disorders.

Neural membranes contain several classes of glycerophospholipids which turnover at different rates with respect to their structure and localization in different cells and membranes. The glycerophospholipid composition of neural membranes greatly alters their functional efficacy. The length of glycerophospholipid acyl chain and the degree of saturation are important determinants of many membrane characteristics including the formation of lateral domains that are rich in polyunsaturated fatty acids. Receptor-mediated degradation of glycerophospholipids by phospholipases A(l), A(2), C, and D results in generation of second messengers such as arachidonic acid, eicosanoids, platelet activating factor and diacylglycerol. Thus, neural membrane phospholipids are a reservoir for second messengers. They are also involved in apoptosis, modulation of activities of transporters, and membrane-bound enzymes. Marked alterations in neural membrane glycerophospholipid composition have been reported to occur in neurological disorders. These alterations result in changes in membrane fluidity and permeability. These processes along with the accumulation of lipid peroxides and compromised energy metabolism may be responsible for the neurodegeneration observed in neurological disorders.

Immunocytochemical localization of cPLA2 in rat and monkey spinal cord.

Rat spinal cord contains a high level of calcium-dependent cytosolic phospholipase A2 (PLA2) activity. A dense immunoreactivity is present in motor neurons from cervical, thoracic, lumbar, and sacral regions of rat spinal cord. Under normal conditions, this enzyme liberates arachidonic acid, a polyunsaturated fatty acid that is a second messenger itself, and a precursor for eicosanoids. However, under pathological conditions during spinal cord injury, intracellular calcium increases so the cytosolic PLA2 may also be involved in the release and accumulation of arachidonic acid, eicosanoids, and lipid peroxides.

Distribution of cytoplasmic phospholipase A2 in the normal rat brain.

The calcium-dependent cytoplasmic PLA2 (cPLA2) is an 85-kDa cytosolic enzyme that has been detected in cytosolic fractions from rat brain. With immunocytochemical methods, this cPLA2 is distributed throughout rat brain. Very dense immunostaining is observed in the superior olivary nucleus, periolivary nucleus, facial motor nucleus and dorsal cochlear nucleus in hindbrain whereas light immunostaining is seen in forebrain and midbrain areas. Assays of cPLA2 activity in forebrain, midbrain and hindbrain show the highest specific activity in the hindbrain. The distribution of cPLA2 coincides with that of protein kinase C activity in rat brain. The presence of cPLA2 and PKC in hindbrain suggests that these enzymes play a central role in neurotransmitter release, long-term potentiation and neuritogenesis in this area under normal conditions.

Health benefits of docosahexaenoic acid (DHA)

Docosahexaenoic acid (DHA) is essential for the growth and functional development of the brain in infants. DHA is also required for maintenance of normal brain function in adults. The inclusion of plentiful DHA in the diet improves learning ability, whereas deficiencies of DHA are associated with deficits in learning. DHA is taken up by the brain in preference to other fatty acids. The turnover of DHA in the brain is very fast, more so than is generally realized. The visual acuity of healthy, full-term, formula-fed infants is increased when their formula includes DHA. During the last 50 years, many infants have been fed formula diets lacking DHA and other omega-3 fatty acids. DHA deficiencies are associated with foetal alcohol syndrome, attention deficit hyperactivity disorder, cystic fibrosis, phenylketonuria, unipolar depression, aggressive hostility, and adrenoleukodystrophy. Decreases in DHA in the brain are associated with cognitive decline during aging and with onset of sporadic Alzheimer disease. The leading cause of death in western nations is cardiovascular disease. Epidemiological studies have shown a strong correlation between fish consumption and reduction in sudden death from myocardial infarction. The reduction is approximately 50% with 200 mg day(-1)of DHA from fish. DHA is the active component in fish. Not only does fish oil reduce triglycerides in the blood and decrease thrombosis, but it also prevents cardiac arrhythmias. The association of DHA deficiency with depression is the reason for the robust positive correlation between depression and myocardial infarction. Patients with cardiovascular disease or Type II diabetes are often advised to adopt a low-fat diet with a high proportion of carbohydrate. A study with women shows that this type of diet increases plasma triglycerides and the severity of Type II diabetes and coronary heart disease. DHA is present in fatty fish (salmon, tuna, mackerel) and mother's milk. DHA is present at low levels in meat and eggs, but is not usually present in infant formulas. EPA, another long-chain n-3 fatty acid, is also present in fatty fish. The shorter chain n-3 fatty acid, alpha-linolenic acid, is not converted very well to DHA in man. These longchain n-3 fatty acids (also known as omega-3 fatty acids) are now becoming available in some foods, especially infant formula and eggs in Europe and Japan. Fish oil decreases the proliferation of tumour cells, whereas arachidonic acid, a longchain n-6 fatty acid, increases their proliferation. These opposite effects are also seen with inflammation, particularly with rheumatoid arthritis, and with asthma. DHA has a positive effect on diseases such as hypertension, arthritis, atherosclerosis, depression, adult-onset diabetes mellitus, myocardial infarction, thrombosis, and some cancers.

Inhibitors of intracellular phospholipase A2 activity: their neurochemical effects and therapeutical importance for neurological disorders.

Intracellular phospholipases A2 (PLA2) are a diverse group of enzymes with a growing number of members. These enzymes hydrolyze membrane phospholipids into fatty acid and lysophospholipids. These lipid products may serve as intracellular second messengers or can be further metabolized to potent inflammatory mediators, such as eicosanoids and platelet-activating factors. Several inhibitors of nonneural intracellular PLA2 have been recently discovered. However, nothing is known about their neurochemical effects, mechanism of action or toxicity in human or animal models of neurological disorders. Elevated intracellular PLA2 activities, found in neurological disorders strongly associated with inflammation and oxidative stress (ischemia, spinal cord injury, and Alzheimer's disease), can be treated with specific, potent and nontoxic inhibitors of PLA2 that can cross blood-brain barrier without harm. Currently, potent intracellular PLA2 inhibitors are not available for clinical use in human or animal models of neurological disorders, but studies on this interesting topic are beginning to emerge. The use of nonspecific intracellular PLA2 inhibitors (quinacrine, heparin, gangliosides, vitamin E) in animal model studies of neurological disorders in vivo has provided some useful information on tolerance, toxicity, and effectiveness of these compounds.

Identification and characterization of alkenyl hydrolase (lysoplasmalogenase) in microsomes and identification of a plasmalogen-active phospholipase A2 in cytosol of small intestinal epithelium.

A lysoplasmalogenase (EC 3.3.2.2; EC 3.3.2.5) that liberates free aldehyde from 1-alk-1'-enyl-sn-glycero-3-phospho-ethanolamine or -choline (lysoplasmalogen) was identified and characterized in rat gastrointestinal tract epithelial cells. Glycerophosphoethanolamine was produced in the reaction in equimolar amounts with the free aldehyde. The microsomal membrane associated enzyme was present throughout the length of the small intestines, with the highest activity in the jejunum and proximal ileum. The rate of alkenyl ether bond hydrolysis was dependent on the concentrations of microsomal protein and substrate, and was linear with respect to time. The enzyme hydrolyzed both ethanolamine- and choline-lysoplasmalogens with similar affinities; the Km values were 40 and 66 microM, respectively. The enzyme had no activity with 1-alk-1'-enyl-2-acyl-sn-glycero-3-phospho-ethanolamine or -choline (intact plasmalogen), thus indicating enzyme specificity for a free hydroxyl group at the sn-2 position. The specific activities were 70 nmol/min/mg protein and 57 nmol/min/mg protein, respectively, for ethanolamine- and choline-lysoplasmalogen. The pH optimum was between 6.8 and 7.4. The enzyme required no known cofactors and was not affected by low mM levels of Ca2+, Mg2+, EDTA, or EGTA. The detergents, Triton X-100, deoxycholate, and octyl glucoside inhibited the enzyme. The chemical and physical properties of the lysoplasmalogenase were very similar to those of the enzyme in liver and brain microsomes. In developmental studies the specific activities of the small intestinal and liver enzymes increased markedly, 11.1- and 3.4-fold, respectively, in the first approximately 40 days of postnatal life. A plasmalogen-active phospholipase A2 activity was identified in the cytosol of the small intestines (3.3 nmol/min/mg protein) and liver (0.3 nmol/min/mg protein) using a novel coupled enzyme assay with microsomal lysoplasmalogenase as the coupling enzyme.

A light and electron microscopic study of cytoplasmic phospholipase A2 and cyclooxygenase-2 in the hippocampus after kainate lesions.

The systemic administration of kainate (10 mg/ml) into adult Wistar rats produces seizures and neurodegeneration. We have studied the effect of kainate administration on cPLA2 and COX-2 immunoreactivities after 3 days and 1, 2, 4 and 11 weeks. The cPLA2 immunoreactivity was increased in hippocampal neurons at 1 and 3 days after kainate injection suggesting that PLA2 may be involved in neurodegeneration. Increased cPLA2 and COX-2 immunoreactivities in astrocytes at 1, 2, 4 and 11 weeks after kainate injection indicate an adaptive astrocytic response that may be associated with gliosis.

Lipid peroxides in the free radical pathophysiology of brain diseases.

1. Polyunsaturated fatty acids are essential for normal cell membrane functioning because many membrane properties, such as fluidity and permeability, are closely related to the presence of unsaturated and polyunsaturated side chains. Lipid peroxidation results in loss of membrane polyunsaturated fatty acids and oxidized phospholipids as polar species contributing to increased membrane rigidity. 2. Polyunsaturated fatty acids are released from membrane phospholipids by a number of enzymic mechanisms involving the receptor-mediated stimulation of phospholipase A2 and phospholipase C/diacylglycerol lipase pathways. 3. The overstimulation of excitatory amino acid (EAA) receptors stimulates the activities of lipases and phospholipases, and this stimulation produces changes in membrane phospholipid composition, permeability, and fluidity, thus decreasing the integrity of plasma membranes. 4. Alterations in properties of plasma membranes may be responsible for the degeneration of neurons seen in neurodegenerative diseases. Two major processes may be involved in neuronal injury caused by the overstimulation of EAA receptors. One is a large Ca2+ influx and the other is an accumulation of free radicals and lipid peroxides as a result of neural membrane phospholipid degradation. It is suggested that calcium and free radicals act in concert to induce neuronal injury in acute trauma (ischemia and spinal cord injury) and in neurodegenerative diseases.

Effects of maturation on the phospholipid and phospholipid fatty acid compositions in primary rat cortical astrocyte cell cultures.

Phospholipid and phospholipid fatty acid compositional changes were studied in rat cortical astrocytes during dibutyryl cyclic adenosine monophosphate (dBcAMP, 0.25 mM) treatment starting after 14 days in culture (DIC). After 15 DIC, ethanolamine- and choline glycerophospholipid levels were increased 1.2- and 1.3-fold, respectively in treated compared to control cells. However, after 21 and 28 DIC, these levels were not significantly different between groups. Both groups had an increase in phosphatidylserine levels with increasing time in culture. Similarly, ethanolamine plasmalogen levels were transiently elevated after 21 DIC, but returned to previous levels after 28 DIC. The phospholipid fatty acid compositions for the acid stable and labile ethanolamine- and choline glycerophospholipids indicated that in dBcAMP treated cells, 20:4 n-6 and 22:6 n-3 proportions were elevated with increasing time in culture relative to control cells. As 20:4 n-6 proportions increased, there was a concomitant decrease in 20:3 n-9 proportions, suggesting an up regulation of n-6 series elongation and desaturation. In contrast, in control cells, the 20:4 n-6 proportions decreased with a corresponding increase in the 20:3 n-9 proportions. Thus, in treated cells, the cellular phospholipid fatty acid composition was dramatically different than control cells, suggesting that dBcAMP treatment may act to increase fatty acid elongation and desaturation.