Hyperglycemic Damage to Mitochondrial Membranes During Cerebral Ischemia: Amelioration by Platelet-Activating Factor Antagonist BN 50739

Abstract: The Pulsinelli-Brierley four-vessel occlusion model was used to study the consequences of hyperglycemic ischemia and reperfusion. Rats were subjected to either 30 min of normo- or hyperglycemic ischemia or 30 min of normo- or hyperglycemic ischemia followed by 60 min of reperfusion. In some animals, 2 mg/kg BN 50739, a platelet-activating factor receptor antagonist, was administered intraarterially either before or after the ischemic insult. The changes in mitochondrial membrane free fatty acid levels, phosphatidylcholine fatty acyl composition, and thiobarbituric acid-reactive material (TBAR) content plus the mitochondrial respiratory control ratio (RCR) were monitored. When the platelet-activating factor antagonist was present during normoglycemia, (a) the mitochondrial free fatty acid release both during and after ischemia was slowed, (b) reacylation of phosphatidylcholine following ischemia was promoted, and (c) TBAR accumulation during and following ischemia was decreased. The detrimental effects of hyperglycemia were muted when BN 50739 was present during ischemia. The RCR was preserved and phosphatidylcholine hydrolysis during ischemia was decreased. TBAR levels were consistently higher in hyperglycemic brain mitochondria both during and after ischemia. The RCR correlated directly with mitochondrial phosphatidylcholine polyunsaturated fatty acid content during ischemia and reperfusion. BN 50739 protection of mitochondrial membranes in brain may be influenced by tissue pH.     

The effect of a free radical scavenger and platelet-activating factor antagonist on FFA accumulation in post-ischemic canine brain

The effects of the platelet-activating factor antagonist BN 50739 and a free radical scavenger dimethyl sulfoxide on the accumulation of free fatty acids in post-ischemic canine brain are reported. Following 14 min of complete normothermic ischemia and 60 min of reperfusion, the total brain FFAs were approximately 150% higher than in the control group (p<0.05). Perfusion with the platelet-activating factor antagonist BN50739 in its diluent dimethyl sulfoxide during 60 min of post-ischemic reoxygenation resulted in a 61.8% (p<0.01) reduction in the total brain free fatty acid accumulation. Palmitic, stearic, oleic, linoleic, and arachidonic acids decreased by 53.8%, 63.5%, 69.0%, 47.4%, and 57.2%, respectively. Although dimethyl sulfoxide alone caused stearic and arachidonic acids to return to the normal concentration range, BN 50739 had a significant influence on recovery of palmitic, oleic, and linoleic acids and was previously shown to provide significant therapeutic protection against damage to brain mitochondria following an ischemic episode. Because free fatty acid accumulation is one of the early phenomena in cerebral ischemia, this study provides evidence to support the hypothesis that both platelet-activating factor and free radicals are involved in initiating cerebral ischemic injury.     

Ischemia-Induced Changes in Cerebral Mitochondrial Free Fatty Acids, Phospholipids, and Respiration in the Rat

Abstract: Changes in the free fatty acid pool size and fatty acyl chain composition of mitochondrial membrane phospholipids and their relation to disruption of mitochondrial function were examined in rat brains after 30 min of cerebral ischemia (Pulsinelli-Brierley model) and 60 min of normoxic reoxygenation. During ischemia, significant hydrolysis of polyunsaturated molecular species from diacyl phosphatidylcholine, particularly fatty acyl 20:4 (arachidonic acid; 20% decrease) and 22:6 (docosahexaenoic acid; 15% decrease), was observed. Thirty minutes of ischemia caused a 16% loss of 18:2 (linoleic acid) from phosphatidylethanolamine. Recirculation for 60 min did not return the polyunsaturated fatty acid content of phospholipids to normal. Total content of free fatty acids increased during ischemia, particularly 18:2 and 22:6, which exhibited the most dramatic rise. The free fatty acid pool size continued to increase during 60 min of recirculation. The respiratory control ratio decreased significantly during 30 min of ischemia with no apparent recovery following 60 min of reoxygenation. The degree of free radical-mediated lipid peroxidation in mitochondria was significantly increased during ischemia and reperfusion. It was concluded that (a) 30 min of cerebral ischemia caused differential degradation in each of the phospholipid classes and preferential hydrolysis of the polyunsaturated molecular species and (b) 60 min of normoxic reperfusion failed to promote reacylation of the mitochondrial phospholipids and restoration of normal respiration.     

Recovery of Postischemic Brain Metabolism and Function Following Treatment with a Free Radical Scavenger and Platelet-Activating Factor Antagonists

We have studied the metabolic and functional effects of two new platelet-activating factor (PAF) antagonists (BN 50726 and BN 50739) and their diluent (dimethyl sulfoxide; DMSO) during reoxygenation of the 14-min ischemic isolated brain. Blood gases, EEG, auditory evoked potentials, cerebral metabolic rate for glucose (CMRglc), and cerebral metabolic rate for oxygen (CMRO2) were monitored throughout the study. Frozen brain samples were taken for measurement of brain tissue high-energy phosphates, carbohydrate content, and thiobarbituric acid-reactive material (TBAR, an indicator of lipid peroxidation) at the end of the study. Following 60 min of reoxygenation in the nontreated 14-min ischemic brains, lactate, AMP, creatine (Cr), intracellular hydrogen ion concentration [H+]i), and TBAR values were significantly higher and ATP, creatine phosphate (PCr), CMRglc, CMRO2, and energy charge (EC) values were significantly lower than the corresponding normoxic control values. PCr and CMRO2 values were significantly higher, and glycogen, AMP, and [H+]i values were significantly lower in the BN 50726-treated ischemic brains than in DMSO-treated ischemic brains. In brains treated with BN 50739, ATP, ADP, PCr, CMRO2, and EC values were significantly higher, and lactate, AMP, Cr, and [H+]i values were significantly lower than corresponding values in the DMSO-treated ischemic brains. TBAR values were near control levels in all brains exposed to DMSO. There was also marked recovery of EEG and auditory evoked potentials in brains treated with DMSO. Treatment with BN 50726 or BN 50739 in DMSO appeared to improve brain mitochondrial function and energy metabolism partly as the result of DMSO action as a free radical scavenger.(ABSTRACT TRUNCATED AT 250 WORDS)     

Involvement of platelet-activating factor (PAF) in cerebral post-ischemic phase in Mongolian gerbils

Platelet-activating factor (PAF) is a potent mediator of anaphylaxis and shock. In addition, evidence for PAF participation in gastric, intestinal and heart post-ischemic phase has been recently demonstrated. Ginkgo biloba extracts improve cerebral metabolism and protect brain against hypoxic damage in various models of cerebral ischemia. Potent and specific antagonists of PAF have been found in Ginkgo biloba and termed Ginkgolides: BN 52020, BN 52021, BN 52022, BN 52024. We therefore undertook the investigation of the role of Ginkgolides in cerebral ischemia obtained by bilateral ligature of the common carotid for 10 min and 6 h of recirculation in male Mongolian adult gerbils. Given preventively (one week treatment 10 mg/kg/day orally) or at the time of clamping, BN 52021 and related Ginkgolides dose-dependently antagonize morbidity assessed by the stroke-index. Similarly the mitochondrial respiration evaluated by the respiratory control ratio is significantly improved. In both determinations, the range of activity: BN 52021 greater than, BN 52020 greater than BN 52022 greater than BN 52024 shows that the effect of Ginkgolides in cerebral ischemia are correlated with their PAF antagonistic properties. Given curatively, 1 h after declamping, BN 52021 is able to reverse the cerebral impairment trend. Kadsurenone and brotizolam, two other chemically unrelated PAF antagonists led to similar recovery. Therefore PAF appears to play an important role in the post-ischemic phase after bilateral carotid ligation in Mongolian gerbils.     

Brain free fatty acids, edema, and mortality in gerbils subjected to transient, bilateral ischemia, and effect of barbiturate anesthesia

Brain free fatty acids (FFAs) and brain water content were measured in gerbils subjected to transient, bilateral cerebral ischemia under brief halothane anesthesia (nontreated group) and pentobarbital anesthesia (treated group). Mortality in the two groups was also evaluated. In nontreated animals, both saturated and mono- and polyunsaturated FFAs increased approximately 12-fold in total at the end of a 30-min period of ischemia; during recirculation, the level of free arachidonic acid dropped rapidly, while other FFAs gradually decreased to their preischemic levels in 90 min. In treated animals, the levels of total FFAs were lower than the nontreated group during ischemia, but higher at 90 min of reflow, and the decrease in the rate of free arachidonic acid was slower in the early period of reflow. Water content increased progressively during ischemia and recirculation with no extravasation of serum protein, but the values were consistently lower in the treated group. None of the nontreated animals survived for 2 weeks; in contrast, survival was 37.5% in the treated group. It is suggested that barbiturate protection from transient cerebral ischemia may be mediated by the attenuation of both membrane phospholipid hydrolysis during ischemia and postischemic peroxidation of accumulated free arachidonic acid.     

Platelet-Activating Factor Antagonist BN52021 Decreases Accumulation of Free Polyunsaturated Fatty Acid in Mouse Brain During Ischemia and Electroconvulsive Shock

We have investigated the effects of the specific platelet-activating factor (PAF; 1-alkyl-2-acetyl-glycerophosphocholine) antagonist BN52021 on free fatty acid (FFA) and diacylglycerol (DG) accumulation and on the loss of fatty acids from phosphatidylinositol-4,5-bisphosphate (PIP2) in mouse brain. Mice were pretreated with BN52021 (10 mg/kg, i.p.) 30 min before electroconvulsive shock (ECS) or postdecapitation ischemia. These procedures cause rapid breakdown of PIP2 and accumulation of FFA and DG. Lipid extracts were prepared from microwave-fixed cerebrum and fractionated by TLC, and the fatty acid methyl esters were prepared by methanolysis and quantified by capillary GLC. In saline or vehicle (dimethyl sulfoxide)-treated mice, ECS caused marked accumulation of FFA and DG and loss of mainly stearic (18:0) and arachidonic (20:4) acids from PIP2. BN52021 pretreatment of ECS-treated mice decreased the accumulation of free palmitic (16:0), 18:0, 20:4, and docosahexaenoic (22:6) acids with no effect on the fatty acids in DG or the loss of PIP2. BN52021 had no effect on basal levels of FFA, DG, or PIP2. One minute of postdecapitation ischemia induced PIP2 loss and accumulation of FFA and DG. BN52021 attenuated the accumulation of free 20:4 and 22:6 acids, decreased the content of oleic (18:1), 20:4, and 22:6 acids in DG, but had no effect on PIP2 loss. These data indicate that BN52021 reduces the injury-induced activation of phospholipase A2 and lysophospholipase, which mediate the accumulation of FFA in brain, while having a negligible effect on phospholipase C-mediated degradation of PIP2.     

Cyclic AMP Concentrations in Rat Neocortex and Hippocampus During and Following Incomplete Ischemia: Effects of Central Noradrenergic Neurons, Prostaglandins, and Adenosine

The concentrations of cyclic AMP, noradrenaline, glycogen, glucose, lactate, pyruvate, labile phosphate compounds, and free fatty acids were investigated in the rat neocortex and hippocampus during and following cerebral ischemia. An incomplete ischemia of 5 and 15 min duration was induced by bilateral carotid clamping combined with hypotension. The postischemic events were studied after 5, 15, and 60 min of recirculation. Five minutes of ischemia did not significantly alter the neocortical or hippocampal concentrations of cyclic AMP. After 15 min of ischemia the neocortical levels decreased significantly below control values. In the recirculation period following ischemia a significant elevation of the cyclic AMP concentrations was observed. Following 5 min of recirculation after 5 min of ischemia the levels increased from 2.53 +/- 0.21 nmol X g-1 to 5.18 +/- 0.09 nmol X g-1 in the neocortex and from 2.14 +/- 0.16 nmol X g-1 to 3.52 +/- 0.35 nmol X g-1 in the hippocampus. Five minutes of recirculation following 15 min of ischemia led to a significant increase in the levels of cyclic AMP, to 12.86 +/- 1.43 nmol X g-1 in the neocortex to 5.58 +/- 0.57 nmol X g-1 in the hippocampus. With longer recirculation periods the cyclic AMP levels progressively decreased and were similar to control values after 60 min. Depletion of cortical noradrenaline by at least 95% was performed by injections of 6-hydroxydopamine into the ascending axon bundles from the locus ceruleus. The lesion did not significantly change the ischemic or post-ischemic neocortical and hippocampal levels of cyclic AMP, glycogen, or free fatty acids including arachidonic acid.(ABSTRACT TRUNCATED AT 250 WORDS)     

Involvement of Platelet-Activating Factor in Cell Death Induced Under Ischemia/Postischemia-Like Conditions in an Immortalized Hippocampal Cell Line

Abstract: The involvement of platelet-activating factor (PAF) in cell damage induced by ischemia/postischemia-like conditions was studied in a hippocampus-derived cell line, HN33.11. Cells exposed to N₂-saturated glucose-free HEPES-buffered saline (ischemia) for 5 h followed by 18 h of incubation in serum-free control medium (postischemia reincubation) remained 67.4 ± 2.4% viable in comparison with sham-treated cells. Analysis of DNA fragmentation in combination with Hoechst 33258 staining indicates that apoptosis is the dominant mode of cell death in the present model. PAF level during 10 h of ischemia was unchanged. However, an increase in PAF accumulation was found early during the reincubation period that followed 5 h of ischemia. Peak PAF concentrations were noted at 2 h after initiation of reincubation and rapidly declined to control level after 7 h of reincubation. Consistent with a role of PAF in mediating cell death under ischemia/postischemia reincubation in this model, the PAF antagonist BN 50739 exerted a dose-dependent protective effect. Maximal protection (85.7 ± 5.4%) of the cells from ischemia/reincubation-induced cell damage was achieved at 0.1 µM BN 50739. The PAF antagonist lacked any protective effect against ischemia-induced cell death. On the other hand, the addition of the stable PAF analogue 1-O-hexadecyl-2-N-methylcarbamyl-sn-glycero-3-phosphocholine (MC-PAF) at the onset of ischemia potentiated ischemia/reincubation-induced apoptosis—an effect that was blocked by BN 50739. Pretreatment of HN33.11 cells with the Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid acetoxymethyl ester (BAPTA-AM) also provided a protective effect against ischemia/reincubation-induced cell damage. BAPTA-AM increased cell viability by 50%. Pretreatment with BAPTA-AM also decreased ischemia/reincubation-induced PAF accumulation in HN33.11 cells. The results suggest that PAF, acting via a PAF receptor, is at least in part mediating apoptosis under ischemia/postischemia-like conditions in HN33.11 cells.     

Inhibition of very long chain acyl-CoA dehydrogenase during cardiac ischemia

The heart utilizes primarily fatty acids for energy production. During ischemia, however, diminished oxygen supply necessitates a switch from beta-oxidation of fatty acids to glucose utilization and glycolysis. Molecular mechanisms responsible for these alterations in metabolism are not fully understood. Mitochondrial acyl-CoA dehydrogenase catalyzes the first committed step in the beta-oxidation of fatty acids. In the current study, an in vivo rat model of myocardial ischemia was utilized to determine whether specific acyl-CoA dehydrogenases exhibit ischemia-induced alterations in activity, identify mechanisms responsible for changes in enzyme function, and assess the effects on mitochondrial respiration. Very long chain acyl-CoA dehydrogenase (VLCAD) activity declined 34% during 30 min of ischemia. Loss in activity appeared specific to VLCAD as medium chain acyl-CoA dehydrogenase activity remained constant. Loss in VLCAD activity during ischemia was not due to loss in protein content. In addition, activity was restored in the presence of the detergent Triton X-100, suggesting that changes in the interaction between the protein and inner mitochondrial membrane are responsible for ischemia-induced loss in activity. Palmitoyl-carnitine supported ADP-dependent state 3 respiration declined as a result of ischemia. When octanoyl-carnitine was utilized state 3 respiration remained unchanged. State 4 respiration increased during ischemia, an increase that appears specific to fatty acid utilization. Thus, VLCAD represents a likely site for the modulation of substrate utilization during myocardial ischemia. However, the dramatic increase in mitochondrial state 4 respiration would be predicted to accentuate the imbalance between energy production and utilization.     

Dissociation of cytochrome c from the inner mitochondrial membrane during cardiac ischemia

Mitochondria isolated from ischemic cardiac tissue exhibit diminished rates of respiration and ATP synthesis. The present study was undertaken to determine whether cytochrome c release was responsible for ischemia-induced loss in mitochondrial function. Rat hearts were perfused in Langendorff fashion for 60 min (control) or for 30 min followed by 30 min of no flow ischemia. Mitochondria isolated from ischemic hearts in a buffer containing KCl exhibited depressed rates of maximum respiration and a lower cytochrome c content relative to control mitochondria. The addition of cytochrome c restored maximum rates of respiration, indicating that the release of cytochrome c is responsible for observed declines in function. However, mitochondria isolated in a mannitol/sucrose buffer exhibited no ischemia-induced loss in cytochrome c content, indicating that ischemia does not on its own cause the release of cytochrome c. Nevertheless, state 3 respiratory rates remained depressed, and cytochrome c release was enhanced when mitochondria from ischemic relative to perfused tissue were subsequently placed in a high ionic strength buffer, hypotonic solution, or detergent. Thus, events that occur during ischemia favor detachment of cytochrome c from the inner membrane increasing the pool of cytochrome c available for release. These results provide insight into the sequence of events that leads to release of cytochrome c and loss of mitochondrial respiratory activity during cardiac ischemia/reperfusion.     

Ischemia-Induced Alterations in Lipid Metabolism of the Gerbil Cerebral Cortex: I. Changes in Free Fatty Acid Liberation

Abstract: Does the impaired lipid metabolism during nonlethal transient ischemia truly recover within a few hours after recirculation? In an attempt to answer this question, we first investigated the time course of the changes in the amount and composition of free fatty acids (FFAs) accumulated during 5-min ischemia and after various postischemic recirculation durations (3 min, 1 h, 24 h, 3 days, and 6 days) in the gerbil cerebral cortex. Then those of FFAs liberated in response to the second 5-min ischemia at various recirculation intervals (3 min, 1 h, 3 days, and 6 days) following the initial one were also measured to evaluate the changes in the cellular response. The former study disclosed that the FFA levels transiently returned to the control levels at 1-h recirculation, increased again a few days after the onset of recirculation, followed by the final return to the control levels after 6-day recirculation. The latter study disclosed that the cellular response to the second ischemia was quite different from that to the initial one even after 6-day recirculation, suggesting that membrane lipid metabolism had not yet been recovered even at such a late period. We discuss the significance of the alterations in lipid metabolism.     

Temperature response of oxygen uptake in brain mitochondria from normal and ischemic rats

Mitochondria isolated from rat brains following 30 min of complete (decapitation) ischemia showed a 3-fold increase in free fatty acid content, but no significant decreases in the total fatty acid or phospholipid content.This free fatty acid increase was associated with an altered mitochondrial function: a 50% inhibition of state 3 (+ ADP) respiration and a decrease in the respiratory control ratio from 5.5 to 3 to 24°C. The P:O ratio remained unchanged at 3, and there was no increase in stage 4 respiration. When glutamate and malate supported respiration was determined as a function of temperature in control mitochondria, the resulting Arrhenius plot of the state 3 respiration was biphasic with a transition temperature around 30°C, while ischemic mitochondria exhibited a linear Arrhenius plot with energy of activation (approximately 10 kcal/mol) similar to that of control mitochondria below the transition temperature.The difference in temperature response between control and ischemic mitochondria reflects a change in mitochondrial lipid composition, and is therefore a functional manifestation of the altered cerebral lipid composition commonly observed during ischemia.     

Brain Cortical Fatty Acids and Phospholipids During and Following Complete and Severe Incomplete Ischemia

Abstract: To explore the possibility that peroxtdative degradation of brain tissue lipid constituents is an important mechanism of irreversible ischemic damage, we measured cortical fatty acids and phospholipids during reversible brain ischemia in the rat. Neither complete nor severe incomplete ischemia (5 and 30 min) caused any measurable breakdown of total or individual fatty acids or phospholipids. Except for a small (and reversible) decrease of inositol plus serine phosphoglycerides in the early postischemic period following 30 min of incomplete ischemia, there were no significant losses of fatty acids or phospholipids during recirculation. Since peroxidation, induced in brain cortical tissue in vitro , characteristically involves degradation of polyenoic fatty acids (arachidonic and docosahexaenoic acids) and of ethanolamine phosphoglycerides, the present in vivo results fail to support the hypothesis that peroxidation of membrane lipids is of primary importance for ischemic brain cell damage. Both complete and severe incomplete ischemia caused a similar increase in the tissue content of free fatty acids (FFA). Thus the FFA pool increased by about 10 times during a 30-min ischemic period, to constitute 1 - 2% of the total fatty acid pool. Since there was a relatively larger increase in polyenoic FFA (especially in arachidonic acid) than in saturated FFA, the release of FFA may be the result of activation of a phospholipase A₂ unbalanced by reesterification. Increased levels of FFA persisted during the initial recirculation period, but a gradual normalization occurred and the ischemic changes were essentially reversed at 30 min after restoration of circulation. The pathophysiological implications of the changes in FFA are discussed with respect to mitochondrial dysfunction, formation of cellular edema and prostaglandin-mediated deterioration of postischemic circulation.     

Free Fatty Acids and Energy Metabolites in Ischemic Cerebral Cortex with Noradrenaline Depletion

Abstract: We tested whether cerebral noradrenaline (NA) may play a central role in mediating the increased production of free fatty acids (FFAs) during cerebral ischemia. Levels of FFAs, cyclic AMP, and NA, as well as ATP, ADP, and AMP, were measured in cerebral cortex during decapitation ischemia in rats 2 weeks after unilateral locus ceruleus lesion. Comparisons were made between the results obtained from the contralateral cortex with normal NA content and the NA-depleted ipsilateral cortex. Although NA depletion was associated with a diminished transient rise of cyclic AMP in response to ischemia, it failed to influence the magnitude of FFA increase or the decline of energy state within the 15-min period of ischemia. A more than twofold increase of total FFAs (sum of palmitic, stearic, oleic, arachidonic, and docosahexaenoic acids) was observed in both hemispheres at 1 min after decapitation, when energy failure became manifest. The increased production of FFAs continued throughout the 15 min of ischemia, with a preferential rise in the levels of stearic and arachidonic acids. There was an inverse correlation between FFA levels and total adenylate pool. The results do not support a major role for NA and cyclic AMP in increasing cortical FFAs during complete ischemia. Instead, they are consistent with the view that impaired oxidative phosphorylation activates deacylating enzymes. Disturbance of reacylation due to energy depletion is probably another factor contributing to the continuous increase of FFAs during prolonged ischemia.     

Effect of the first window of ischemic preconditioning on mitochondrial dysfunction following global cerebral ischemia

Rats may develop sustained tolerance against lethal cerebral ischemia after exposure to a sublethal ischemic insult (ischemic preconditioning (IPC)). Two windows for the induction of tolerance by IPC have been proposed, one that occurs within 1h following IPC, and the other one that occurs 1-3 days after IPC. An important difference between these two windows is that in contrast to the second window, neuroprotection against lethal ischemia is transient in the first window. We tested the hypothesis that rapid IPC would reduce or prevent ischemia-induced changes in mitochondrial function. IPC and ischemia were produced by bilateral carotid occlusions and systemic hypotension (50 mmHg) for 2 and 10 min, respectively. The non-synaptosomal mitochondria were harvested 30 min following the 10 min 'test' ischemia. Mitochondrial rate of respiration decreased by 10% when the substrates were pyruvate and malate, and 29% when the substrates were ascorbic acid and N,N,N',N'-tetramethyl-p-phenylenediamine ( P< 0.01). The activities of complex I-III decreased in ischemic group by 16, 23 (P < 0.05) and 24%, respectively. IPC was unable to prevent decreases in the rate of respiration and activities of different complexes. These data suggest that rapidly induced IPC is unable to protect the integrity of mitochondrial oxidative phosphorylation following cerebral ischemia, perhaps explaining why IPC only provides transitory protection in the 'first window'.     

Mechanism of Arachidonic Acid Liberation During Ischemia in Gerbil Cerebral Cortex

Once brain ischemia was induced in the gerbil cerebral fronto-parietal cortex, serial changes occurred in energy metabolites and various lipids. The amounts of inositol-containing phospholipids began to decrease immediately after energy failure, followed by an increase in the amount of 1,2-diacylglycerol with a subsequent liberation of arachidonic acid and other free fatty acids. The fatty acid compositions of inositol-containing phospholipids, of 1,2-diacylglycerols produced by ischemia, and of free fatty acids liberated during ischemia were quite similar. The amount of stearic acid liberated was much larger than that of arachidonic acid between 30 s and 1 min of ischemia. On the other hand, there was no significant decrease in the amount of the other phospholipids except for phosphatidic acid. Furthermore, there was also no change in the fatty acid composition of phosphatidylcholine or phosphatidylethanolamine throughout 15 min of ischemia. The amount of cytidine-monophosphate reached a peak (36.7 nmol/g wet wt) at 2 min of ischemia. These results indicated that arachidonic acid was predominantly liberated from inositol-containing phospholipids by phospholipase C, and by the diglyceride lipase and monoglyceride lipase system rather than from phosphatidylcholine or phosphatidylethanolamine by phospholipase A2 or plasmalogenase or choline phosphotransferase during the early period of ischemia.     

Cerebral Phosphoinositide, Triacylglycerol, and Energy Metabolism in Reversible Ischemia: Origin and Fate of Free Fatty Acids

Levels of phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol (PI), phosphatidic acid, diacylglycerol (DAG), triacylglycerol (TAG), and free fatty acids (FFAs), as well as their fatty acid composition, were determined in rat forebrain during ischemia and postischemic recirculation. Cerebral energy state and electroencephalograms (EEGs) were also studied. Fifteen minutes of ischemia resulted in a decrease in PIP2 and PIP contents but not in PI content, concurrent with an enlargement of the FFA and DAG pools. The latter were enriched in stearate and arachidonate. Prolongation of ischemia did not produce further changes in content of any of the inositol phospholipids, but the increase in levels of FFAs and DAG continued. At the end of 45 min of ischemia, levels of both PIP2 and PIP decreased by 45-50%, and the total phosphoinositide content (PIP2 + PIP + PI) decreased by 21%, whereas levels of FFAs and DAG increased to 14- and 3.6-fold of control levels, respectively. During ischemia, the TAG-palmitate level decreased, but the TAG-arachidonate level increased; the tissue energy state deteriorated severely; and the EEG was suppressed. A 30-min recirculation period after 15 or 45 min of ischemia led to increases in PIP2, PIP, and total phosphoinositide contents, whereas levels of FFAs and DAG promptly decreased toward control values. The TAG-arachidonate level peaked and the TAG-palmitate level returned to a low control value during early recirculation. The ischemic changes in tissue lipids were completely reversed within 3 h of recirculation after both periods of ischemia. Adenylates were fully phosphorylated with as little as 30 min of reflow. The EEG activity partially recovered during reflow after 15 min of ischemia, whereas it remained depressed after prolonged ischemia. Thus, phosphodiesteric cleavage of PIP2 and PIP followed by deacylation of DAG is likely to contribute to the production of FFAs in early ischemia. Deacylation of undetermined lipids plays a role for the increment in levels of FFAs in the later period of ischemia. The rapid postischemic increase in levels of PIP2 and PIP indicates active synthesis not only from existing PI, but probably also by means of accumulated FFAs and DAG. These results indicate that the impaired resynthesis of inositol phospholipids cannot be a cause of the poor EEG activity after prolonged ischemia. Degradation and resynthesis of polyphosphoinositides and formation of TAG-arachidonate may be important for modulation of free arachidonic acid levels in the brain during temporary ischemia.     

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.     

Involvement of acyl exchange between acyl-CoA and phosphatidylcholine in the remodelling of phosphatidylcholine in microsomal preparations of rat lung

Microsomal membrane preparations from rat lung catalyse the incorporation of radioactive linolenic acid from [14C]linolenoyl-CoA into position 2 of sn-phosphatidylcholine. The incorporation was stimulated by bovine serum albumin and free CoA. Free fatty acids in the incubation mixtures were not utilised in the incorporation into complex lipids. Fatty acids were transferred to the acyl-CoA pool during the incorporation of linolenic acid into phosphatidylcholine. An increase in lysophosphatidylcholine occurred in incubations containing both bovine serum albumin and free CoA and in the absence of acyl-CoA. The results were consistent with an acyl-CoA: lysophosphatidylcholine acyltransferase operating in both a forwards and backwards direction and thus catalysing the acyl exchange between acyl-CoA and position 2 of sn-phosphatidylcholine. In incubations with mixed species of acyl-CoAs, palmitic acid was the major fatty acid substrate transferred to phosphatidylcholine in acyl exchange, whereas this acid was completely selected against in the acylation of added lysophosphatidylcholine. The selectivity for palmitoyl-CoA was particularly enhanced when the mixed acyl-CoA substrate was presented to the microsomes in molar concentrations equivalent to the molar ratios of the fatty acids in position 2 of sn-phosphatidylcholine. During acyl exchange, the predominant fatty acid transferred to phosphatidylcholine from acyl-CoA was palmitic acid, whereas arachidonic acid was particularly selected for in the reverse reaction from phosphatidylcholine to acyl-CoA. A hypothesis is presented to explain the differential selectivity for acyl species between the forward and backward reactions of the acyltransferase that is based upon different affinities of the enzyme for substrates at high and low concentrations of acyl donor. Acyl exchange between acyl-CoA and phosphatidylcholine offers, therefore, a possible mechanism for the acyl-remodelling of phosphatidylcholine for the production of lung surfactant.