Elena B. Rodriguez de Turco

Coupling Among Energy Failure, Loss of Ion Homeostasis, and Phospholipase A2 and C Activation During Ischemia

Abstract— The objective of the present experiments was to correlate changes in cellular energy metabolism, dissipative ion fluxes, and lipolysis during the first 90 s of ischemia and, hence, to establish whether phospholipase A2or phospholipase C is responsible for the early accumulation of phospholipid hydrolysis products. Ischemia was induced for 15–90 s in rats, extracellular K+ (K+e) was recorded, and neocortex was frozen in situ for measurements of labile tissue metabolites, free fatty acids, and diacylglycerides. Ischemia of 15‐and 30‐s duration gave rise to a decrease in phosphocreatine concentration and a decline in the ATP/free ADP ratio. Although these changes were accompanied by an activation of K+ conductances, there were no changes in free fatty acids until after 60s, when free arachidonic acid accumulated. An increase in other free fatty acids and in total diacylglyceride content did not occur until after anoxic depolarization. The results demonstrate that the early functional changes, such as activation of K+ conductances, are unrelated to changes in lipids or lipid mediators. They furthermore suggest that the initial lipolysis occurs via both phospholipase A2 and phospholipase C, which are activated when membrane depolarization leads to influx of calcium into cells.

Synergy by Secretory Phospholipase A2 and Glutamate on Inducing Cell Death and Sustained Arachidonic Acid Metabolic Changes in Primary Cortical Neuronal Cultures

Secretory and cytosolic phospholipases A2 (sPLA2 and cPLA2) may contribute to the release of arachidonic acid and other bioactive lipids, which are modulators of synaptic function. In primary cortical neuron cultures, neurotoxic cell death and [3H]arachidonate metabolism was studied after adding glutamate and sPLA2 from bee venom. sPLA2, at concentrations eliciting low neurotoxicity (≤100 ng/ml), induced a decrease of [3H]arachidonate-phospholipids and preferential reesterification of the fatty acid into triacylglycerols. Free [3H]arachidonic acid accumulated at higher enzyme concentrations, below those exerting highest toxicity. Synergy in neurotoxicity and [3H]arachidonate release was observed when low, nontoxic (10 ng/ml, 0.71 nM), or mildly toxic (25 ng/ml, 1.78 nM) concentrations of sPLA2 were added together with glutamate (80 μM). A similar synergy was observed with the sPLA2 OS2, from Taipan snake venom. The NMDA receptor antagonist MK-801 blocked glutamate effects and partially inhibited sPLA2 OS2 but not sPLA2 from bee venom-induced arachidonic acid release. Thus, the synergy with glutamate and very low concentrations of exogenously added sPLA2 suggests a potential role for this enzyme in the modulation of glutamatergic synaptic function and of excitotoxicity.

Docosahexaenoic Acid Uptake and Metabolism in Photoreceptors: Retinal Conservation By an Efficient Retinal Pigment Epithelial Cell-Mediated Recycling Process

After 18:3 omega 3 is obtained from the diet, it is accumulated by the liver, where it is esterified and temporarily stored as triacylglycerols. As it is required, 18:3 omega 3 is elongated and desaturated to 22:6 omega 3, then released into the circulation with lipoprotein carriers. RPE cells remove the 22:6 omega 3 from the choriocapillaris and subsequently release it to the retina proper. In the frog, all 22:6 omega 3 input to the photoreceptors occurs by way of the RPE cells. After passing through the interphotoreceptor matrix, it is selectively taken into the myoid region of photoreceptor cells where it is immediately activated and esterified onto position 2 (and sometimes also position 1) of a glycerol molecule. Some phospholipids are passed through the endoplasmic reticulum and Golgi apparatus, while others are not. Generally, transport to the outer segments seems to be independent of the Golgi apparatus. Addition to rod outer segments occurs in two ways: i) a general diffuse pathway, probably common to all fatty acids, which rapidly labels the entire outer segment; and ii) a specific dense pathway, utilized only by 22:6 omega 3-containing phospholipids, which become locked into the matrix of disc membranes along with opsin. There appears to be no exchange between these two forms of label. Accumulation of newly synthesized basal discs pushes older, 22:6 omega 3-laden discs apically until the outer segment tips, high in 22:6 omega 3-phospholipids (the dense form of outer segment label), are shed into the RPE cytoplasm. There, as the 22:6 omega 3 fatty acids are released from the disc membranes during degradation, a recycling mechanism immediately directs these essential fatty acids back into the interphotoreceptor matrix, thus conserving this molecule in the retina, and permitting it to be again selectively taken up by the photoreceptors for photomembrane synthesis. The process of 22:6 omega 3 handling and trafficking by the retina is specifically orchestrated around a conservation mechanism that is regulated by the RPE cells and that ensures, through a short feedback loop from the phagosomes to the interphotoreceptor matrix, adequate levels of 22:6 omega 3 for photoreceptors at all times.

Systemic fatty acid responses to transient focal cerebral ischemia: influence of neuroprotectant therapy with human albumin

Human albumin therapy is highly neuroprotective in focal cerebral ischemia. Because albumin is the main carrier of free fatty acids (FFA) in plasma, we investigated the content and composition of plasma FFA in jugular vein (JV), femoral artery (FA) and femoral vein (FV) of rats given intravenous human albumin (1.25 g/kg) or saline vehicle (5 mL/kg) 1 h after a 2 h middle cerebral artery occlusion (MCAo) or sham surgery. Arachidonic acid was the only FFA significantly increased by MCAo in all plasma samples prior to albumin administration, remaining at the same level regardless of subsequent treatments. Albumin treatment induced in both MCAo‐ and sham‐groups a 1.7‐fold increase in total plasma FFA (mainly 16:0, 18:1, 18:2n‐6) during 90‐min reperfusion. MCAo selectively stimulated the albumin‐mediated mobilization of n‐3 polyunsaturated fatty acids (PUFA), with an early increase in 22:5n‐3 and 22:6n‐3 in the FA prior to detectable changes in the JV. In the MCAo‐albumin group, the lower level of FFA in JV as compared with FA and FV suggests an albumin‐mediated systemic mobilization and supply of FFA to the brain, which may favor the replenishment of PUFA lost from cellular membranes during ischemia and/or to serve as an alternative source of energy, thus contributing to albumin neuroprotection.

Retinal pigment epithelial cells play a central role in the conservation of docosahexaenoic acid by photoreceptor cells after shedding and phagocytosis.

The involvement of retinal pigment epithelial (RPE) cells in the recycling of docosahexaenoic acid (DHA), from phagocytized disc membranes back to the retina, was studied in frogs subsequent to injection of [3H]DHA via the dorsal lymph sac. Rod outer segments (ROS) gradually accumulated [3H]DHA as a dense, heavily labeled region that arrived at the distal tips by 28 days post-injection. Autoradiographic analysis at the time of maximal shedding and phagocytosis (1-2 hr after the onset of light) showed diffusely (before 28 days) and heavily (after 28 days) labeled phagosomes in RPE cells. Biochemical analysis of the [3H]DHA-containing lipids of discs that contribute to the labeling of RPE cells after phagocytosis was also performed. Between 27 and 34 days, when 12% of retinal [3H]DHA-lipids present in disc membranes are phagocytized by RPE cells, total retinal labeling remained unchanged. Taken together, these data suggest that the [3H]DHA of the densely labeled region of the ROS was recycled back to the photoreceptor cells only after it had reached the RPE cells following 28 days post-injection. We conclude that, following daily phagocytosis of ROS tips, RPE cells play a central role in the conservation and redelivery of ROS-derived DHA back to photoreceptor cells through the interphotoreceptor matrix.

Chapter 17 Role of phospholipase A2 and membrane-derived lipid second messengers in membrane function and transcriptional activation of genes: implications in cerebral ischemia and neuronal excitability

Publisher Summary The membrane phospholipids of brain and retina are highly enriched in polyunsaturated fatty acids (PUFA), especially arachidonate and docosahexaenoate. Release of free arachidonic acid is the rate-limiting step in the cascade that results in the formation of the biologically active eicosanoids. Phospholipase A 2 (PLA 2 ) also leads to the synthesis of platelet-activating factor (PAF), an alkyl–ether phospholipid that is a potent mediator of inflammatory and immune processes. The chapter discusses the nature of the changes in membrane phospholipid-derived second messenger generation elicited by ischemia and convulsions; describes synaptosome-specific activation of phospholipases A 2 , A 1 and C and the arachidonic acid cascade; and examines a hypothesis on how PAF might, in addition to its short-term effects, be involved in long-term changes in neuronal phenotype via activation of nuclear proto-oncogenes. These latter effects may link selective membrane phospholipid hydrolysis with synaptic remodeling and plasticity changes through the modulation of gene expression.

Neuronal damage by secretory phospholipase A2: modulation by cytosolic phospholipase A2, platelet-activating factor, and cyclooxygenase-2 in neuronal cells in culture.

Activation of cytosolic phospholipase A(2) (cPLA(2)) is an early event in brain injury, which leads to the formation and accumulation of bioactive lipids: platelet-activating factor (PAF), free arachidonic acid, and eicosanoids. A cross-talk between secretory PLA(2) (sPLA(2)) and cPLA(2) in neural signal transduction has previously been suggested (J Biol Chem 271:32722; 1996). Here we show, using neuronal cell cultures, an up-regulation of cPLA(2) expression and an inhibition by the selective cPLA(2) inhibitor AACOCF3 after exposure to neurotoxic concentrations of sPLA(2)-OS2. Pretreatment of neuronal cultures with recombinant PAF acetylhydrolase (rPAF-AH) or the presynaptic PAF receptor antagonist, BN52021, partially blocked neuronal cell death induced by sPLA(2)-OS2. Furthermore, selective COX-2 inhibitors ameliorated sPLA(2)-OS2-induced neurotoxicity. We conclude that sPLA(2)-OS2 activates a neuronal signaling cascade that includes activation of cPLA(2), arachidonic acid release, PAF production, and induction of COX-2.

Decreased Electroconvulsive Shock‐Induced Diacylglycerols and Free Fatty Acid Accumulation in the Rat Brain by Ginkgo biloba Extract (EGb 761): Selective Effect in Hippocampus as Compared with Cerebral Cortex

Abstract: The effect of Ginkgo biloba extract (EGb 761) treatment (100 mg/kg/day, per os, for 14 days) on electroconvulsive shock (ECS)‐induced accumulation of free fatty acids (FFA) and diacylglycerols (DAG) was analyzed in rat cerebral cortex and hippocampus. EGb 761 reduced the FFA pool size by 33% and increased the DAG pool by 36% in the hippocampus. These endogenous lipids were unaffected in cerebral cortex. During the tonic seizure (10 s after ECS) the fast accumulation of FFA, mainly 20:4, was similar in sham‐ and EGb 761 ‐treated rats, in both the cerebral cortex and hippocampus. However, further accumulation of free 18:0 and 20:4, observed in the hippocampus of sham‐treated rats during clonic seizures (30 s to 2 min after ECS), did not occur in EGb 761‐treated animals. The rise in DAG content triggered in the cortex and hippocampus by ECS was delayed by EGb 761 treatment from 10 s to 1 min, when values similar to those in sham animals were attained. Moreover, in the hippocampus the size of the total DAG pool was decreased by 19% during the tonic seizure. At later times, DAG content showed a faster decrease in EGb 761‐treated rats. By 2 min levels of all DAG acyl groups decreased to values significantly lower than in sham animals in both cortex and hippocampus. This study shows that EGb 761 treatment affects, with high selectivity, lipid metabolism and lipid‐derived second messenger release and removal in the hippocampus, while affecting to a lesser extent the cerebral cortex.

Light stimulates in vivo inositol lipid turnover in frog retinal pigment epithelial cells at the onset of shedding and phagocytosis of photoreceptor membranes

We have developed an experimental model to study in vivo inositol lipid metabolism in frog retinal pigment epithelial (RPE) cells, including the effect of light on phospholipase C-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate. RPE cells were rapidly isolated after either brief light or dark periods. Light and electron microscopy showed complete detachment of the retina from the RPE cells, and that the RPE cell suspensions were devoid of photoreceptor cell outer segments. Frog tissues were labeled in vivo for 20 hr by intravitreal injection of [3H]inositol (4 microCi, 4 microliters per eye) within a 24-hr constant illumination period. Following 1 hr of darkness (priming period), frogs were intravitreally injected with LiCl (0.5 M, 4 microliters per eye) 15 min before the onset of either 30-min light stimulation or an additional 30 min of darkness (controls). In order to preserve endogenous inositol phosphate pools present after dark and light exposure, the RPE cells were harvested in the shortest time possible, at low temperatures (18-20 degrees C), and in the presence of 10 mM LiCl. Total [3H]inositol-labeled water-soluble products (inositol plus inositol phosphates) were increased by 86% after 30 min of light. Inositol trisphosphate (IP3) showed the highest accumulation (a 5.5-fold increase), followed by inositol bisphosphate (1.9-fold increase) and inositol monophosphate (1.4-fold increase). Free [3H]inositol also accumulated (2.8-fold increase), reflecting only a partial inhibition of phosphomonoesterase by LiCl. These changes were paralleled by a 12% decrease in 3H-labeled phosphatidylinositol with no significant difference in the labeling of polyphosphoinositides.(ABSTRACT TRUNCATED AT 250 WORDS)

EGb 761 inhibits stress-induced polydipsia in rats

The effect of daily treatments with Ginkgo biloba extract (EGb 761, IPSEN, France) on body weight and water intake of rats was followed for 15 days. During this period, two groups of rats, under slight ether anesthesia, were intubated and fed either EGb 761 (100 mg/kg b.wt. in 5% ethanol) or, for sham controls, 5% ethanol alone (6.6 ml/kg b.wt.). The increase in body weight was similar for the control and experimental groups. However, during the same period of time, the water intake, ml water/g b.wt./24 h, increased 37% in the controls. In EGb 761-treated rats, water intake remained unchanged. This suggests that EGb 761 treatment inhibits the development of polydipsia due to the stress of daily handling and intubation.