Differential effect on inositol-phospholipid hydrolysis, cytosolic-free Ca2+ concentration, protein kinase C activity and protein phosphorylation of 1-oleoyl-2-acetyl-sn-glycerol growth-stimulated ascites tumor cells

This study shows that the membrane-permeable stereospecific 1-oleoyl-2-acetyl-sn-glycerol (OAG), which is the analog of the natural 1,2-diacylglycerol (DAG), can stimulate the growth of ascites tumor cells. OAG can fully replace high serum concentrations in the culture medium and stimulates DNA synthesis in a dose-dependent manner. Investigation of the protein kinase C (PKC) isolated from a Triton extract of a 100,000g membrane pellet revealed that OAG can directly activate this enzyme. Concomitantly the phosphorylation of several cytosolic proteins with the molecular weights of 26, 33, 49, 55, 64, and 90 kDa is observed which is also found in serum-stimulated cells. Since DAG as a second messenger molecule originates from the hydrolysis of phosphoinositides we have investigated the metabolism of these lipids after labeling the cells with [3H]inositol. In detail, we have measured the amount of radioactive inositol trisphosphate (IP3) and the phosphodiesterase hydrolyzing phosphatidylinositol-4,5-bisphosphate (PIP2). The decreased radioactivity level of IP3 in OAG-stimulated cells as compared to non-growing cells (1-2% serum) indicates a feedback regulation of PIP2 hydrolysis which is substantiated by a profound reduction of PIP2-specific phospholipase C activity. The reduced IP3 formation has apparently no inhibitory effect on the cytoplasmic free Ca2+ concentration of OAG-stimulated cells, suggesting that the Ca2+ release is not directly correlated to the amount of IP3, which is also demonstrated for the non-growing cells. These data indicate that OAG apparently has a duel effect on the inositol phospholipid-mediated signal transfer system.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serum Albumin Washing Specifically Enhances Arachidonate Incorporation into Synaptosomal Phosphatidylinositols

Arachidonate incorporation into synaptosomal phospholipids was shown to be affected by factors including the procedure for preparation of the membrane fractions and preincubation of synaptosomes prior to assay of incorporation of arachidonate into both phosphatidylcholine (PC) and phosphatidylinositol (PI). However, the inhibition toward incorporation into PIs, but not PCs, was fully reversed when the membranes were washed with bovine serum albumin. A twofold increase in arachidonate incorporation into PIs was also observed when freshly prepared synaptosomes were washed with serum albumin immediately before assay of incorporation activity. The inhibitory action is thought to be due to an increase in polyunsaturated fatty acids and/or their oxidation products which may then elicit a special effect on the acyltransferase responsible for transferring arachidonate into phosphatidylinositols. The differences in fatty acid uptake and response to serum albumin also suggest the presence of different acyltransferase for acyl transfer to PIs and PCs.     

Effects of acute hypoxia on incorporation of [1-14C]arachidonic acid into glycerolipids of rat brain

The effects of 1 min of acute hypoxic treatment (1% O₂ in N₂) on incorporation of [1-¹⁴C]arachidonic acid into brain lipids of 16-day-old rats were investigated at 3, 6, and 12 min after intracerebral injection of the labeled fatty acid. The hypoxic-hypoxia condition associated with convulsive seizures caused a decrease in the conversion of labeled arachidonate to its acyl-CoA as well as incorporation of the label into the brain phospholipids. Among the phospholipids, there was a specific decrease in the labeling of diacylglycerophosphoinositol (GPI), and this change was accompanied by an increase in labeling of the diacylglycerols. These results indicate that metabolism of the long-chain fatty acids and some glycero-lipids in brain are vulnerable to acute hypoxic treatment.     

Metabolism of oleoyl-CoA in rat brain synaptosomes

The hydrolysis of acyl-CoA by acyl-CoA hydrolase (EC 3.1.2.2.) in brain synaptosomes was inhibited by calcium. This inhibition was partly due to interaction of Ca²⁺ with the acyl-CoA, which was present in the soluble form, and partly due to complex formation among acyl-CoA, Ca²⁺ and membrane phospholipids. The inhibition of acyl-CoA hydrolase activity, as well as the complex formation. could be reversed if incubation was carried out in the presence of Ca²⁺ chelating agents. Synaptosomes isolated from brain samples after 1 min of postdecapitative treatment showed a decrease in oleoyl-CoA hydrolase activity. The physiological implication of acyl-CoA metabolism in relation to synaptic function is discussed.Abbreviations FFA Free fatty acids - GPC glycerophosphocholines - GPE glycerophosphoethanolamines - GPI glycerophosphoinositols - GPS glycerophosphoserines     

Ca2+-Independent, Ca2+-Dependent, and Carbachol- Mediated Arachidonic Acid Release from Rat Brain Cortex Membrane

Synaptoneurosomes obtained from the cortex of rat brain prelabeled with [14C]arachidonic acid [( 14C]AA) were used as a source of substrate and enzyme in studies on the regulation of AA release. A significant amount of AA is liberated in the presence of 2 mM EGTA, independently of Ca2+, primarily from phosphatidic acid and polyphosphoinositides (poly-PI). Quinacrine, an inhibitor of phospholipase A2 (PLA2), suppressed AA release by about 60% and neomycin, a putative inhibitor of phospholipase C (PLC), reduced AA release by about 30%. An additive effect was exhibited when both inhibitors were given together. Ca2+ activated AA release. The level of Ca2+ present in the synaptoneurosomal preparation (endogenous level) and 5 microM CaCl2 enhance AA liberation by approximately 25%, whereas 2 mM CaCl2 resulted in a 50% increase in AA release relative to EGTA. The source for Ca(2+)-dependent AA release is predominantly phosphatidylinositol (PI); however, a small pool may also be liberated from neutral lipids. Carbachol, an agonist of the cholinergic receptor, stimulated Ca(2+)-dependent AA release by about 17%. Bradykinin enhanced the effect of carbachol by about 10-15%. This agonist-mediated AA release occurs specifically from phosphoinositides (PI + poly-PI). Quinacrine almost completely suppresses calcium-and carbachol-mediated AA release. Neomycin inhibits this process by about 30% and totally suppresses the effect of bradykinin. Our results indicate that both phospholipases PLA2 and PLC with subsequent action of DAG lipase are responsible for Ca(2+)-independent AA release. Ca(2+)-dependent and carbachol-mediated AA liberation occurs mainly as the result of PLA2 action. A small pool of AA is probably also released by PLC, which seems to be exclusively responsible for the effect of bradykinin.     

Metabolism of [14C]arachidonic acid-labeled lipids in quiescent and OAG-stimulated ascites tumor cells.

1. A rapid uptake and esterification of [14C]arachidonic acid during the first 4 hr of cultivation of ascites cells in serum-deprived medium was observed followed by a fast turnover of the fatty acid. 2. Labeling and turnover of esterified arachidonate in individual phospholipid classes was in the order: phosphatidylcholine (PC) greater than phosphatidylinositol (PI) much greater than phosphatidylinositol-4-phosphate (PIP) and -4,5-bisphosphate (PIP2) greater than phosphatidylethanolamine (PE) greater than PE-plasmalogens. 3. In cells stimulated with 1-oleoyl-2-acetyl-sn-glycerol a transient course of arachidonic acid incorporation into PC, PI, PIP and PIP2 was determined peaking 30 min after stimulation, indicating both esterification and release under these conditions. 4. The release of arachidonate was blocked by quinacrine which is a specific inhibitor of phospholipase A2.     

Docosahexaenoic acid and tetracyclines as promising neuroprotective compounds with poly(ADP-ribose) polymerase inhibitory activities for oxidative/genotoxic stress treatment

The human genome is exposed to oxidative/genotoxic stress by several endogenous and exogenous compounds. These events evoke DNA damage and activate poly(ADP-ribose) polymerase-1 (PARP-1), the key enzyme involved in DNA repair. The massive stress and over-activation of this DNA-bound enzyme can be responsible for an energy crisis and neuronal death. The last data indicated that product of PARP-1, i.e. poly(ADP-ribose) (PAR), acts as a signalling molecule and plays a significant role in nucleus-mitochondria cross-talk. PAR translocated to the mitochondria can be involved in mitochondrial permeability, the release of an apoptosis-inducing factor (AIF). Its translocation into the nucleus leads to chromatin condensation, fragmentation and cell death. The exact mechanism of this novel death pathway has not yet fully been understood.     

Alteration of Phosphoinositide Degradation by Cytosolic and Membrane-Bound Phospholipases after Forebrain Ischemia - Reperfussion in Gerbil: Effects of Amyloid Beta Peptide

The reperfusion of previously ischemic brain is associated with exacerbation of cellular injury. Reperfusion occasionally potentates release of intracellular enzymes, influx of Ca²⁺, breakdown of membrane phospholipids, accumulation of amyloid precursor protein or amyloid -(like) proteins, and apolipoprotein E. In this study, the effect of reperfusion injury on the activity of cerebral cortex enzymes acting on phosphatidyl [³H] inositol (PI) and [^l4C-arachidonoyl] PI was investigated. Moreover the effect of amyloid 25–35 on PI degradation by phospholipase(s) of normoxic brain and subjected to ichemia-reperfussion injury was determined. Brain ischemia in gerbils (Meriones unguiculatus) was induced by ligation of both common carotid arteries for 5 min and then brains were perfused for 15 min, 2 h and 7 days. Statistically significant activation of enzyme(s) involved in phosphatidylinositol degradation in gerbils subjected to ischemia-reperfusion injury was observed. Nearly all gerbils showed a higher activity of cytosolic PI phos-pholipase C (PLC) at 15 min after ischemia. Concomitantly, the significant enhancement of the level of DAG and AA radioactivity at this short reperfusion time confirmed the active PI degradation by phospholipase(s) in cerebral cortex and hippocampus. After a prolonged reperfusion time of 7 days after ischemia, both cytosolic and membrane-bound forms of PI-PLC were activated. The question arises if alteration of membranes by the degradation of phospholipids occurring after an ischemic episode potentates the effect of A on membrane-bound enzymes. A neuro-toxic fragment of amyloid, A 25–35, incubated in the presence of endogenous Ca²⁺, increased significantly the PI-PLC activity of normoxic brain. In its non-aggregated form, A 25–35 activates PI-PLC but in the aggregated form the enzymatic activity decreased. Thus, A 25–35 exerts a similar effect on the membrane-bound PI-PLC from normoxic brain or subjected to ischemia reperfussion injury. We conclude that the degradation of phosphatidylinositol by cytosolic phosphoinositide-phospholipase C may contribute to the pathophysiology of delayed neuronal death following cerebral ischemia. Thus, a specific inhibitor of this enzyme(s) may offer therapeutic strategies to protect the brain from damage triggered by ischemia. Ischemia-reperfusion injury had no effect on A-evoked alterations of synaptic plasma membrane-bound PI-PLC.     

Effect of amyloid beta peptide on poly(ADP-ribose) polymerase activity in adult and aged rat hippocampus

It is suggested that the fibrillar amyloid beta peptide (A beta) in brain plays a direct role in neurodegeneration in Alzheimer's disease, probably through activation of reactive oxygen species formation. Free radicals and numerous neurotoxins elicit DNA damage that subsequently activates poly(ADP-ribose) polymerase (PARP, EC 2.4.2.30). In this study the effect of neurotoxic fragment (25-35) of full length A beta peptide on PARP activity in adult and aged rat hippocampus was investigated. In adult (4 month old) rat hippocampus the A beta 25-35 peptide significantly enhanced PARP activity by about 80% but had no effect on PARP activity in cerebral cortex and in hippocampus from aged (24-27 month old) rats. The effect of A beta peptide was reduced by half by the nitric oxide synthase inhibitor N-nitro-L-arginine. Stimulation of glutamate receptor(s) itself enhanced PARP activity by about 80% in adult hippocampus. However, A beta 25-35 did not exert any additional stimulatory effect. These results indicate that A beta, through NO and probably other free radicals, induces activation of DNA bound PARP activity exclusively in adult but not in aged hippocampus.     

The Molecular Mechanism of Amyloid β42 Peptide Toxicity: The Role of Sphingosine Kinase-1 and Mitochondrial Sirtuins

Our study focused on the relationship between amyloid β 1–42 (Aβ), sphingosine kinases (SphKs) and mitochondrial sirtuins in regulating cell fate. SphK1 is a key enzyme involved in maintaining sphingolipid rheostat in the brain. Deregulation of the sphingolipid metabolism may play a crucial role in the pathogenesis of Alzheimer’s disease (AD). Mitochondrial function and mitochondrial deacetylases, i.e. sirtuins (Sirt3,-4,-5), are also important for cell viability. In this study, we evaluated the interaction between Aβ_1–42, SphKs and Sirts in cell survival/death, and we examined several compounds to indicate possible target(s) for a strategy protecting against cytotoxicity of Aβ_1–42. PC12 cells were subjected to Aβ_1–42 oligomers and SphK inhibitor SKI II for 24–96 h. Our data indicated that Aβ_1–42 enhanced SphK1 expression and activity after 24 h, but down-regulated them after 96 h and had no effect on Sphk2. Aβ_1–42 and SKI II induced free radical formation, disturbed the balance between pro- and anti-apoptotic proteins and evoked cell death. Simultaneously, up-regulation of anti-oxidative enzymes catalase and superoxide dismutase 2 was observed. Moreover, the total protein level of glycogen synthase kinase-3β was decreased. Aβ_1–42 significantly increased the level of mitochondrial proteins: apoptosis-inducing factor AIF and Sirt3, -4, -5. By using several pharmacologically active compounds we showed that p53 protein plays a significant role at very early stages of Aβ_1–42 toxicity. However, during prolonged exposure to Aβ_1–42, the activation of caspases, MEK/ERK, and alterations in mitochondrial permeability transition pores were additional factors leading to cell death. Moreover, SphK product, sphingosine-1-phosphate (S1P), and Sirt activators and antioxidants, resveratrol and quercetin, significantly enhanced viability of cells subjected to Aβ_1–42. Our data indicated that p53 protein and inhibition of SphKs may be early key events responsible for cell death evoked by Aβ_1–42. We suggest that activation of S1P-dependent signalling and Sirts may offer a promising cytoprotective strategy.