Regulation of FFA by the acyltransferase pathway in focal cerebral ischemia-reperfusion

Cerebral insult is associated with a rapid increase in free fatty acids (FFA) and arachidonic acid release has been linked to the increase in eicosanoid biosynthesis. In transient focal cerebral ischemia induced by middle cerebral artery (MCA) occlusion, there is an inverse relationship between the increase in FFA and the decrease in ATP, both during the ischemia period and at later time periods after reperfusion. In this study, the focal cerebral ischemia model was used to examine incorporation of [¹⁴C]arachidonic acid into the glycerolipids in rat MCA cortex at different reperfusion times after a 60 min ischemia. The label was injected intracerebrally into left and right MCA cortex 1 hr prior to decapitation. Labeled arachidonic acid was incorporated into phosphatidylcholine, phosphatidylethanolamine and neutral glycerides. With increasing time (4–16 hr) after a 60 min ischemia, an inhibition of labeled arachidonate uptake could be found in the right ischemic MCA cortex, whereas the distribution of radioactivity among the major phospholipids was not altered. When compared to labeled PC, there was a 3–4 fold increase in incorporation of label into phosphatidic acid and triacylglycerols (TG) in the right MCA cortex, suggesting of an increase in de novo biosynthesis of TG. In an in vitro assay system, synaptosomal membranes isolated from MCA cortex 8 and 16 hr after a 60 min ischemia showed a significant decrease in arachidonoyl transfer to lysophospholipids, due mainly to a decrease in lysophospholipid:acylCoA acyltransferase activity. Assay of phospholipase A₂ activity with both syaptosomes and cytosol, however, did not show differences between left and right MCA cortex or with time after reperfusion. These results suggest that besides ATP availability, the decrease in acyltransferase activity may also contribute to the increase in FFA in cerebral ischemia-reperfusion.Abbreviations PC phosphatidylcholine - PE phosphatidylethanolamine - PEpl ethanolamine plasmalogen - PI phosphatidylinositol - PS phosphatidylserine - poly-PI polyphosphoinsoitide - DG diacylglycerol - TG triacylglycerol - FFA free fatty acids - PUFA polyunsaturated fatty acids - MCA middle cerebral artery - CCAs common carotid arteries - HPTLC high performance thin layer chromatography - GLC gas-liquid chromatography - PLA₂ phospholipase A₂ Special issue dedicated to Dr. Leon S. Wolfe.     

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.     

Amino acid and purine release in rat brain following temporary middle cerebral artery occlusion

Excitatory amino acid release and neurotoxicity in the ischemic brain may be reduced by endogenously released adenosine which can modulate both glutamate or aspartate release and depress neuronal excitability. The present study reports on the patterns of release of glutamate and aspartate; the inhibitory amino acids GABA and glycine; and of the purine catabolites adenosine and inosine from the rat parietal cerebral cortex during 20 and 60 min periods of middle cerebral artery (MCA) occlusion followed by reperfusion. Aspartate and glutamate efflux into cortical superfusates rose steadily during the period of ischemia and tended to increase even further during the subsequent 40 min of reperfusion. GABA release rose during ischemia and declined during reperfusion, whereas glycine efflux was relatively unchanged during both ischemia and reperfusion. Adenosine levels in cortical superfusates rose rapidly at the onset of ischemia and then declined even though MCA occlusion was continued. Recovery to pre-occulusion levels was rapid following reperfusion. Inosine efflux also increased rapidly, but its decline during reperfusion was slower than that of adenosine.     

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.     

Effects of an inhibitor of adenosine deaminase,deoxycoformycin, and of nucleoside transport,propentofylline, on post-ischemic recovery of adenine nucleotides in rat brain

The effects of an adenosine deaminase inhibitor (deoxycoformycin, 500 μg/kg) and of an inhibitor of nucleoside transport (propentofylline, 10 mg/kg) on adenosine and adenine nucleotide levels in the ischemic rat brain were investigated. The brains of the rats were microwaved before, at the end of a 20 min period of cerebral ischemia (4 vessel occlusion+hypotension), or after 5, 10, 45, and 90 min of reperfusion. Deoxycoformycin increased brain adenosine levels during both ischemia and the initial phases of reperfusion. AMP levels were elevated during ischemia and after 5 min of reperfusion. ATP levels were elevated above those in the non-treated animals after 10 and 45 min of reperfusion. ADP levels were elevated above the non-drug controls at 90 min. These increases in ATP, ADP and AMP resulted in significant increases in total adenylates during ischemia, and after 10 min and 90 min of reperfusion. Propentofylline administration resulted in enhanced AMP levels during ischemia but did not alter adenosine or adenine nucleotide levels during reperfusion in comparison with non-treated controls.     

Reassessment of Brain Free Fatty Acid Liberation During Global Ischemia and Its Attenuation by Barbiturate Anesthesia

Abstract: We previously reported that whole-brain free fatty acids (FFA) rose almost linearly for up to 1 h after decapitation of unanesthetized rats and was significantly attenuated by pentobarbital anesthesia. However, our values for total FFA and arachidonic, stearic, oleic, and palmitic acids were severalfold higher than those obtained by previous investigators. Based upon the suggestion that this may be due to FFAs released from di- and triglycerides in the quantitation of FFAs, we have now analyzed and improved our procedures for TLC separation of FFA and reassessed the accumulation of FFA in whole brain during decapitation ischemia in unanesthetized and pentobarbital-anesthetized rats. FFA levels in whole brain after 0.5 min of ischemia were one-half to one- fourth the levels previously reported after 1 min of ischemia. The rise in FFA between 0.5 and 60 min of ischemia was 9-fold for total FFA, and between 7 and 12-fold for each of the FFAs quantitated. Pentobarbital significantly attenuated the rise of all FFAs with, however, greater effects on oleic and palmitic acids than previously reported.     

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.     

Asymmetry of Diacylglycerol Metabolism in Rat Cerebral Hemispheres

Diacylglycerols (DGs) were found to be asymmetrically distributed between the two cerebral hemispheres of rat brain. The left cerebral hemisphere (LCH) contained 100% more DG than the right cerebral hemisphere (RCH). The lateralization was enhanced in animals subjected to depolarization induced by a single electroconvulsive shock (ECS). During the acute phase of the convulsion, the DG pool increased in both hemispheres, with the LCH attaining a concentration 180% higher than the RCH. Stearate and arachidonate were the principal DG-acyl groups accumulated in the RCH, whereas in the LCH stearate and palmitate were mainly involved. After the last of a series of five shocks (one per day) the lateralization of the "DG response" was less accentuated during the acute phase of the ECS. Whereas DG release was drastically reduced in the LCH, in the RCH it was minimally affected. The DG sidedness after five shocks was nevertheless maintained at the level of arachidonate-containing DGs, which showed a higher accumulation in the LCH than in the RCH. The kinetics of DG removal showed a rapid phase during the first minute following a single or five ECSs. Total DG levels returned to basal values in the RCH, whereas in the LCH they remained slightly increased with respect to the initial levels 1 min after the convulsive episode. Minimal changes occurred in the subsequent 4 min. Chronic ECS altered the endogenous DG content and composition. Thus, 24 h after the last of four ECSs, total levels of DGs diminished by 40% in the RCH, whereas they remained unchanged in the LCH.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Activation of Calpain,Cathepsin-b and Caspase-3 during Transient Focal Cerebral Ischemia in Rat Model

Calpains, cathepsins and caspases play crucial role in mediating cell death. In the present study we observed a cascade of events involving the three proteases during middle cerebral artery occlusion (MCAo) in Wistar rats. The rats were MCA occluded and reperfused at various time points. We observed a maximal increase in the levels of calpains during 1h and 12 h after reperfusion than permanently occluded rats. Further, these levels were reduced by 1st and 3rd day of reperfusion. Similarly the cathepsin-b levels were significantly increased during 1h and 12 h, of reperfusion, followed by activation of caspase-3 which reached maximal levels by 1st and 3rd day of reperfusion. The sequential activation of calpains, cathepsin-b and cleaved caspase-3 is evident by the Western blot analysis which was further confirmed by the cleavage of substrates like PSD-95 and spectrin. The differences in the regional distribution and elevation of these proteases at different reperfusion time periods indicates that differential mode of cell death occur in the brain during cerebral ischemia in rat model.     

Regional Studies of Changes in Brain Fatty Acids Following Experimental Ischaemia and Reperfusion in the Gerbil

Regional studies of brain phospholipid metabolism were carried out during a period of ischaemia induced in the gerbil by bilateral carotid occlusion for 60 min. The associated changes in free fatty acids (FFAs) during this period and following recirculation for up to 180 min were noted. Following ischaemia there was a generalised rise in the levels of all FFAs with no selective release of either the unsaturated (arachidonic and docosahexaenoic) or saturated (palmitic and stearic) fatty acids. There were no observed differences between the brain regions studied, which is in contrast to previously reported observations for prostaglandins. There was also no indication of any specific phospholipid fraction being involved in FFA release. This would indicate that the release of FFAs from phospholipids is a nonspecific event, probably due to the action of hydrolytic lipases. Restoration of the circulation resulted in a short, sharp increase (within 5 min) in all FFAs, but in contrast to the observations during ischaemia alone there was a relatively larger rise in the unsaturated FFAs as compared to the saturated FFAs. Following this increase there was a gradual general decline in all FFA levels until 180 min of reperfusion. Since there was no preferential depletion of unsaturated FFAs during reperfusion, when free radical attack is considered to be at its maximum, it is our opinion that free radical peroxidation is unlikely to explain the pathology described in our model.     

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.     

Stroke-induced progenitor cell proliferation in adult spontaneously hypertensive rat brain: effect of exogenous IGF-1 and GDNF

Progenitor cells in the dentate gyrus of hippocampus (DG) and the subventricular zone of lateral ventricles (SVZ) generate new neurons throughout the life of mammals. Cerebral ischemia increases this basal progenitor cell proliferation. The present study evaluated the time frame of proliferation, length of survival and the phenotypes of the new cells formed after transient middle cerebral artery occlusion (MCAO) in adult spontaneously hypertensive rats. Compared to sham controls, ischemic rats showed a significantly higher number of newly proliferated cells (as defined by BrdU immunostaining) in both the DG (by fourfold, p < 0.05) and the SVZ (by twofold, p < 0.05). DG showed increased proliferation only in the first week of reperfusion and 49% of the cells formed in this period survived to the end of third week. Whereas, SVZ showed a continuous proliferation up to 3 weeks after MCAO, but the cells formed survived for less than a week. In both DG and SVZ, at the end of the first week of reperfusion, majority of the BrdU-positive (BrdU+) cells were immature neurons (DCX positive). In the DG, 28% of the cells formed in the first week after MCAO mature into neurons (NeuN positive). The ischemic cortex and striatum showed several BrdU+ cells which were ED-1 positive microglia/macrophages. At 1 week of reperfusion, MCAO-induced progenitor cell proliferation in the ipsilateral DG was significantly increased by i.c.v. infusion of IGF-1 (by 127 +/- 14%, p < 0.05) and GDNF (by 91 +/- 5%, p < 0.05), compared to vehicle. In the growth factor treated rats subjected to transient MCAO, several BrdU+ cells formed in the first week survived up to the third week.     

Hydroxyl Radical Formation in Hyperglycemic Rats During Middle Cerebral Artery Occlusion/Reperfusion

Preexisting hyperglycemia is associated with enhanced reperfusion injury in the postischemic rat brain. The goal of this study was to evaluate whether the hyperglycemic exacerbation of brain injury is associated with enhanced generation of hydroxyl radicals in rats subjected to middle cerebral artery occlusion (2 h), followed by reperfusion (2 h). Magnetic resonance images revealed the exacerbation of focal brain injury in hyperglycemic rats. The salicylate trapping method was used in conjunction with microdialysis to continuously estimate hydroxyl radical production by measurement of the stable adducts 2,3- and 2,5-dihydroxybenzoic acid (DHBA) during ischemia/reperfusion. In normoglycemic rats, from a mean baseline level of 130 nmol/l, 2,3-DHBA levels surged to peak levels of 194 nmol/l 45 min into ischemia and to 197 nmol/l 15–30 min into the reperfusion period, returning to baseline by 2 h into reperfusion. A similar temporal profile was observed in hyperglycemic rats, except that absolute 2,3-DHBA levels were higher (165 nmol/l at baseline, 317 nmol/l peak during ischemia, 333 nmol/l peak during reperfusion), and levels remained significantly high (p < .05) throughout the reperfusion period. These results suggest that hydroxyl radical is an important contributor to the exacerbation of neuronal and cerebrovascular injury after focal ischemia/reperfusion in hyperglycemic rats.     

Recovery from Edema and of Protein Synthesis Differs Between the Cortex and Caudate Following Transient Focal Cerebral Ischemia in Rats

Postischemic recovery from brain edema and of protein synthesis was examined following 1 h of middle cerebral artery (MCA) occlusion in rats. Recovery from brain edema and of protein synthesis showed a good correlation until 7 days after reperfusion in each area (cerebral cortex or lateral caudate) in the occluded MCA side. However, regional differences in the above types of recovery in the cortex and in the lateral caudate were found for the first time in this experiment. A profound inhibition of protein synthesis and formation of brain edema began sooner in the lateral caudate than in the cortex and continued long after reperfusion. Grades of cerebral blood flow during ischemia and the early period of reperfusion were almost the same in the two regions. Therefore, the regional differences in the above recoveries may not be due to the difference in the blood flow during ischemia and reperfusion, but may be partly attributable to the imbalance of excitatory and inhibitory innervation in the above two areas of the brain, may be due to a distinctive response to ischemic stress, and may be caused also by the potentiative effect of free arachidonate on the excitotoxic mechanism.     

Polyphosphoinositides as a Probable Source of Brain Free Fatty Acids Accumulated at the Onset of Ischemia

The quantitative relationship between phosphoinositides and free fatty acids (FFAs) in brain ischemia was studied by measuring contents of individual fatty acids in phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol (PI), phosphatidic acid (PA), diacylglycerol (DAG), and the FFA pool. Various periods of complete ischemia (1, 3, 10, and 30 min) were produced by decapitation. Ischemia of 1-3 min caused rapid decreases in PIP2 and PIP content together with preferential production of stearic and arachidonic acids in the DAG and FFA pools. The decrement in levels of these fatty acid residues in polyphosphoinositides was sufficient to account for their increment in levels in the enlarged DAG and FFA pools. After 10 min of ischemia, levels of PIP2, PIP, and DAG approached plateau values, but levels of all FFAs continued to increase. The increases in content of DAG and FFAs at later ischemic periods could not be accounted for by the decreases in content of PIP2 and PIP, PI and PA levels showed only transient and subtle changes. These results indicate that, at the onset of ischemia, phosphodiesteric cleavage of PIP2 and PIP and subsequent deacylation by lipases are primarily responsible for the preferential increase in levels of free stearic and arachidonic acids and that, later, hydrolysis of other phospholipids plays a major role in the continuous accumulation of FFAs.     

Phospholipase D signaling in ischemic heart.

Phospholipase D (PLD) activity was found to be present in the membrane fraction of rat myocardial cells by in vitro assays (36.7 +/- 4.1 nmol/mg protein per h against 1-palmitoyl-2-arachidonoyl- phosphatidylcholine) and demonstrated in intact cells by the specific transphosphatidylation reaction (in the presence of 0.02% ethanol) quantitated using n-[1-14C]butanol (201.16 +/- 7.1 pmol/min per g dry weight in the whole heart). Both methods showed a significant increase in PLD activity (by 62 and 44%, respectively) in hearts subjected to reversible (30 min) global normothermic ischemia followed by reperfusion (30 min). In hearts prelabeled with [1-14C]arachidonic acid, ischemia/reperfusion induced a significant increase in the amount of radiolabel incorporated into phosphatidic acid (PtdOH) (by 49.6%) and diacylglycerol (DG) (by 259%). DG kinase inhibition by 100 microM dioctanoylethylene glycol did not affect the ischemia/reperfusion DG and PtdOH levels while PtdOH phosphohydrolase inhibition with 40 microM propranolol produced a further increase in PtdOH (to 2.36-fold the baseline level) and a reduction in DG (to only 145% over the baseline levels). Put together, all these results suggest an activation of PLD during myocardial ischemia/reperfusion generating intracellular PtdOH, part of which is converted by PtdOH phosphohydrolase to DG. We further investigated the possible pathophysiological significance of the observed PLD activation. Stimulation of PLD with sodium oleate (20 microM) induced a significant improvement of functional recovery of ischemic hearts during reperfusion (as monitored by coronary flow and left intraventricular pressure measurements) and an attenuation of cellular injury as expressed by lactate dehydrogenase and creatine kinase release in the coronary effluent during reperfusion. These results suggest a PLD-mediated signaling in the ischemic heart which may benefit functional recovery during reperfusion.     

Metabolic Turnover of Fatty Acids and Acylglycerols in Rat Sciatic Nerve

To explain the discrepancy between the low level and high metabolic activity of endoneurial free fatty acids (FFAs) and triacylglycerol (TG), levels of de novo synthesized FFA and acylglycerols were measured in rat sciatic endoneurium at various intervals after endoneurial microinjection of [14C]acetate. Soon after injection (less than 10 min), the [14C]acetate was metabolized to FFA and incorporated into diacylglycerol (DG), TG, sterols, ceramides, and various phospholipids. The proportions of 14C-labeled FFA, DG, TG, and ceramides to total 14C-labeled lipids decreased, whereas those of phospholipids and cerebrosides increased with time after injection. These findings suggest that rapid turnover of FFA and TG may contribute to their low level in sciatic endoneurium. The de novo synthesized fatty acids were largely incorporated into phosphatidylcholine (approximately 50% of total 14C-labeled phospholipids), probably via the cytidine nucleotide pathway using 1,2-DG as a metabolic intermediate. Hydrolysis of [14C]phosphatidylcholine revealed that fatty acids were labeled at both the C-1 (approximately 43%) and C-2 (approximately 57%) positions. On the other hand, a temporal association between decreased amounts of 14C-label in ceramides and increased amounts of 14C-label in sphingomyelin and galactocerebrosides supports the hypothesis that peripheral nerve galactocerebroside is derived, in vivo, from ceramide via acylation of sphingosine. This exclusive labeling of endoneurial lipids by endoneurial microinjection of labeled precursor provides a unique model for studying synthesis and metabolic turnover of membrane lipids in experimental neuropathies.     

Effects of focal cerebral ischemia on inositol 1,4,5-trisphosphate 3-kinase and 5-phosphatase activities in rat cortex.

Ins(1,4,5)P3 3-kinase and 5-phosphatase are important enzymes responsible for the metabolism of Ins(1,4,5)P3, a second messenger for mobilization of intracellular Ca2+ stores. Focal cerebral ischemia induced in Long Evans rats through occlusion of the right middle cerebral artery (MCA) and both common carotid arteries resulted in a time-dependent decrease in the 3-kinase activity but not the 5-phosphatase activity. Approximately 50% of the 3-kinase activity in the cerebral cortex of the right MCA territory disappeared after 60 min of ischemia, and the enzyme activity was not restored during reperfusion. Reperfusion for 24 hr after a 60 min ischemic insult almost abolished the 3-kinase activity but the 5-phosphatase activity remained unaltered. These results suggest that the Ins(1,4,5)P3 3-kinase is one of the target enzymes of cerebral ischemia. The changes in Ins(1,4,5)P3 metabolism may be associated with the changes in intracellular Ca2+ homeostasis that underlies the pathophysiology of neuronal cell death.     

Butylated hydroxyanisole inhibits the growth of HeLa cervical cancer cells via caspase-dependent apoptosis and GSH depletion

To evaluate the effects of in vitro ischemia/reperfusion on contractile response to field stimulation (FS), free fatty acid (FFA) content, phospholipid (PL) content, and malondialdehyde (MDA) levels of the rabbit urinary bladder. There is significant evidence that ischemia/reperfusion injury is linked to obstructive bladder dysfunction secondary to men with benign prostatic hyperplasia (BPH). Twelve New Zealand White male rabbits were separated into two groups of six rabbits each. Each rabbit was euthanized, and the bladder was surgically removed intact for whole bladder incubation. The bladders in Group 1 received a 3-h incubation under normal oxygenated physiological conditions. These bladders received electrical field stimulation (32?Hz) after 1 and 3?h. The bladders associated with Group 2 received a 1-h incubation under normal oxygenated physiological conditions. At the end of this 1-h period, the bladders were subjected to FS. After a maximal pressure response was recorded, the stimulation was turned off and the bath medium was changed to one equilibrated with 95% nitrogen, 5% oxygen without glucose (ischemic medium) and incubated for 1?h with field stimulations (32?Hz) occurring at 5-min intervals to represent overactive bladder dysfunction. At the end of this hour of ischemia with repetitive stimulation, the bath was changed to an oxygenated medium with glucose for a 1-h period after which the stimulation was repeated. At the end of the experimental period, each bladder was opened longitudinally and the muscle and mucosa separated by blunt dissection, frozen under liquid nitrogen, and stored at -80°C for biochemical analyses. Each tissue was fractionated by differential centrifugation into nuclear, mitochondrial, synaptosomal, and supernatant (cytosol) components. PL, FFA, and MDA content were analyzed for each fraction using standard biochemical techniques. The bladder contractile responses decreased during the period of in vitro ischemia and returned to only 30% of control after reperfusion. In vitro ischemia/reperfusion showed the following: (1) There was a modest but significant decrease in the FFA content of the microsomes of the muscle and significant increases in the FFA content of the nuclei and mitochondria of the mucosa. (2) There were decreases in the PL content of the homogenate and microsomes of the muscle and decreases in the PL content of the homogenate, microsomes, and supernatant of the mucosa. (3) Significant increases were observed in the MDA levels of the homogenate, mitochondria, and microsomes of both the muscle and mucosa. The significant increases in the lipid peroxidation of the bladder smooth muscle are consistent with the marked decrease in the contractile ability of the bladder following ischemia/reperfusion. The specific increased lipid peroxidation of the mitochondrial and microsomal components is consistent with the specific dysfunctions of the mitochondria and innervations observed following I/R in earlier published studies. The marked increases in lipid peroxidation in the mucosa associated with the loss of PL and FFA from this component are consistent with the significant dysfunction in both the antiadherence and antipermeability properties of the mucosa and may play a major role in the symptomatic nature of I/R-linked diseases of the bladder.