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M R Prasad

Publications and source records attributed to M R Prasad.

At least 55 records · Page 3Linked to original sources
PubMed1991

Role of phospholipases A2 and C in myocardial ischemic reperfusion injury.

We investigated the role of phospholipase A2 (PLA2) and phospholipase C (PLC) in myocardial phosholipid degradation and cellular injury during reperfusion of ischemic myocardium. For this purpose, isolated rat hearts were perfused with isotopic arachidonic acid to label its membrane phospholipids. Hearts preperfused with antiphospholipase A2 (anti-PLA2) retained a significantly higher amount of radiolabel in phosphatidylcholine and phosphatidylinositol and a corresponding lower amount of radiolabel in lysophosphatidylcholine and nonesterified fatty acids (P less than 0.05) after 30 min of reperfusion following 30 min of normothermic global ischemia compared with hearts preperfused with nonimmune immunoglobulin G. In similar experiments, antiphospholipase C (anti-PLC)-treated hearts were associated with significantly (P less than 0.05) higher radiolabel in all phospholipids and lower radiolabel in diacyglycerol compared with nonimmune immunoglobulin G-treated hearts. Measurement of phospholipase activity in subcellular organelles of these hearts showed decreased PLA2 activity in cytosol, mitochondria, and microsomes of anti-PLA2-treated hearts and decreased PLC activity of microsomes in anti-PLC-treated hearts. Furthermore, both the antiphospholipases attenuated the release of creatine kinase and lactate dehydrogenase into perfusate and increased contractility as well as coronary flow in the reperfused hearts. Results of this study suggest that both PLA2 and PLC are involved in the degradation of phospholipids and cellular injury that occur during reperfusion of ischemic myocardium.

Animals↗
PubMed1990

Polymorphonuclear leucocytes as potential source of free radicals in the ischaemic-reperfused myocardium.

The feasibility of polymorphonuclear leucocytes as a potential source of free radicals during reperfusion of ischaemic myocardium was evaluated. Isolated rat heart was perfused in the presence of f-Met-Leu-Phe-activated and normal polymorphonuclear leucocytes for 30 min. To judge the degree of cellular injury which might result from activated polymorphonuclear leucocytes during perfusion, isolated hearts were also perfused with superoxide anions, hydroxyl radicals, and hypochlorous acid-generating systems in the absence or presence of their corresponding scavengers, superoxide dismutase plus catalase, dimethylthiourea, and allopurinol, respectively. Activated polymorphonuclear leucocytes stimulated the release of lactate dehydrogenase, a biological marker of cellular injury, and malondialdehyde, a presumptive marker for lipid peroxidation; increased tissue injury, as evidenced by morphologic examinations using light and electron microscopy; decreased dry/wet ratios of heart, signifying oedema formation; and reduced myocardial adenosine triphosphate and creatine phosphate content as well as coronary flow, indicating decreased myocardial performance. These biological, physiological, and morphologic parameters were reversed significantly, but not completely, by treating the heart with scavengers, superoxide dismutase plus catalase or allopurinol, but were reversed completely by simultaneous treatment with superoxide dismutase, catalase, and allopurinol. Comparable results were obtained when the hearts were treated with each of these free radical-generating systems and their corresponding scavengers. Generation of free radicals was confirmed either by cytochrome c reduction or by examining the chemiluminescence response using a luminometer. These results indicate that activated polymorphonuclear leucocytes can cause myocardial cellular injury equivalent to the damage caused by free radicals and oxidants which are present in an ischaemic-reperfused heart, suggesting that polymorphonuclear leucocytes may be a potential source of free radicals in the reperfused heart.

Animals↗
PubMed1990

Role of iron on membrane phospholipid breakdown in ischemic-reperfused rat heart.

Oxygen-derived free radicals have been implicated in causing degradation of myocardial membrane phospholipids associated with ischemia and reperfusion. Since iron is known to catalyze the hydroxyl radical formation responsible for cellular injury, this study was designed to relate the role of iron with phospholipid breakdown in ischemic-reperfused heart. Isolated rat heart perfused by the Langendorff technique was subjected to 30 min of normothermic ischemia followed by 30 min of reperfusion. The experimental group received 0.6 mM deferoxamine, an iron chelator, before reperfusion of ischemic myocardium. Deacylation and reacylation of membrane phospholipids were monitored by using [14C]arachidonic acid (AA), whereas the de novo phospholipid synthesis was evaluated by using [3H]glycerol in the perfusate. In the deferoxamine group, the loss of [14C]phosphatidylcholine (PC) and the corresponding accumulation of isotopic lysophosphoglycerides as well as AA was significantly lower compared with the control. The incorporation of radioactivity for [14C]AA and [3H]glycerol into phospholipids was significantly increased in the treated group compared with the untreated group. In addition, decreased malonaldehyde formation and lactate dehydrogenase release, a higher recovery of high-energy phosphate compounds, and myocardial contractility were noticed in the deferoxamine-treated hearts. These results indicated that postischemic administration of an iron chelator such as deferoxamine can preserve membrane phospholipids and reduce myocardial dysfunction associated with reperfusion of ischemic heart.

Acylation↗
PubMed1989

Effects of oxyradicals on oxymyoglobin. Deoxygenation, haem removal and iron release.

We have examined the effects of O2-derived free radicals on oxymyoglobin, the myocardial intracellular protein involved in the storage and transport of O2. The oxyradicals generated by the xanthine/xanthine oxidase system decreased the concentration of oxymyoglobin. Based on the decreases in absorbance peaks at 581 nm and 415 nm it is estimated that out of a 10 nmol decrease in oxymyoglobin, 5 nmol appears to be oxidized to ferrimyoglobin (deoxygenation), while haem was removed from the other 5 nmol of haem protein. These processes were inhibited by both catalase alone and superoxide dismutase in combination with catalase, but not by either superoxide dismutase alone or deferoxamine. These results suggest that among H2O2, OH. and O2.-, only H2O2 causes the removal of haem and the oxidation of oxymyoglobin. Furthermore, the oxyradicals also released 3 microM free iron from oxymyoglobin, which is at least 5-fold less than the 15 nmol loss of oxymyoglobin. The loss of oxymyoglobin also preceded the release of free iron. These results indicate that oxymyoglobin oxidation and haem removal occur before the removal of free iron. Thus myoglobin appears to be highly susceptible to free radical attack, and this may represent yet another mechanism of free radical-mediated cellular injury.

Animals↗
PubMed1989

Topography of rat hepatic microsomal enzymatic components of the fatty acid chain elongation system.

The orientation of the condensing enzyme, the beta-hydroxyacyl-CoA dehydrase, and the trans-2-enoyl CoA reductase within the rat liver microsomal membrane was investigated by the use of impermeant inhibitors of enzyme activity: trypsin, chymotrypsin, subtilisin, mercury-dextran, and anti-beta-hydroxyacyl-CoA dehydrase IgG. The activity of the condensing enzyme was inhibited more than 70% by various proteases and was completely inhibited by 80 microM mercury-dextran. Similar results were obtained for the trans-2-enoyl-CoA reductase activity. On the other hand, in the absence of detergent, proteases inhibited beta-hydroxyacyl-CoA dehydrase activity by 25-40%, while in the presence of detergent the inhibition increased to 65-90%. Furthermore, anti-beta-hydroxyacyl-CoA dehydrase IgG, which in the absence of detergent produced no inhibition, in the presence of detergent inhibited beta-hydroxyacyl-CoA dehydrase activity by more than 80%; under identical conditions, preimmune IgG caused a 13% inhibition. Microsomes used throughout this study displayed greater than 90% latency with respect to mannose-6-phosphatase activity, indicating that the microsomes were intact. Latency was not affected by the proteases, by mercury-dextran, or by the presence of the enzyme assay components. These results suggest that both the condensing enzyme and the reductase are present on the cytoplasmic surface of the membrane, whereas the beta-hydroxyacyl-CoA dehydrase is embedded in the microsomal membrane.

Acyl-CoA Dehydrogenases↗
PubMed1989

Improvement of ischemia-reperfusion-induced myocardial dysfunction by modulating calcium-overload using a novel, specific calmodulin antagonist, CGS 9343B.

The present paper explores the mechanism of calcium-overloaded cardiac cell exocytosis during reperfusion of ischemic myocardium. A novel specific inhibitor of calmodulin, CGS 9343B, was used to pretreat an ischemic heart in an effort to enhance myocardial preservation. The experimental model employed an isolated in situ pig heart subjected to 120 min of ischemic insult by reversibly occluding the left anterior descending coronary artery, the last 60 min being superimposed with global hypothermic cardioplegic arrest. This ischemic episode was followed by 60 min of revascularization. CGS 9343B enhanced post-ischemic myocardial recovery, as judged by improved regional as well as global myocardial functions, better preservation of high-energy phosphate compounds, and reduced release of creatine kinase. Since this compound blocks calmodulin without inhibiting protein kinase C, the results of this study suggest that calmodulin-dependent kinase, rather than protein kinase C, is primarily involved in expressing calcium-overloaded cell exocytosis, and a specific calmodulin antagonist such as CGS 9343B can be used to salvage an ischemic heart from reperfusion injury.

Animals↗
PubMed1989

Mechanism of membrane phospholipid degradation in ischemic-reperfused rat hearts.

We investigated the mechanism of membrane phospholipid degradation during reperfusion of ischemic myocardium using isolated and perfused rat hearts. Thirty min of myocardial reperfusion after 30 min of normothermic global ischemia resulted in a significant decrease of phosphatidylcholine (PC) content associated with a small but significant increase in lysophosphatidylcholine (LPC) content. Myocardial ischemia for up to 60 min caused no significant loss of any of the major phospholipids. Isotopic incorporation of [14C]arachidonic acid (AA) as well as [3H]-glycerol into PC was significantly attenuated in the ischemic-reperfused heart compared with the normally perfused heart, suggesting that both reacylation and de novo pathways for PC synthesis were inactivated during reperfusion. In the heart prelabeled with [14C]AA, the radiolabeled PC was decreased significantly during reperfusion, associated with a small but significant increase in [14C]AA accumulation. The decreases of PC content and incorporation of [14C]AA into PC, as well as the increases of LPC content and the [14C]AA during reperfusion, were prevented by reperfusion with low Ca2+ (50 microM) buffer or by pretreatment with trifluoperazine (10 microM) or mepacrine (50 microM), but not with verapamil (1 microM). The inhibition of loss of PC was associated with significant diminution of creatine kinase release from the reperfused hearts. The present study indicates that the net loss of membrane phospholipids, especially with respect to PC during reperfusion, may result from 1) inhibition of reacylation of AA, 2) inhibition of de novo synthesis, and 3) stimulation of phospholipase activity. These results are consistent with an influx of Ca2+, although other interpretations are also possible.

Animals↗
PubMed1989

Free radical scavenging by myocardial fatty acid binding protein.

Recent investigations have indicated the presence of a fatty acid binding protein (FABP) in mammalian heart. This protein binds free fatty acids and their esters with high affinity, however, its physiological role remains unknown. Since FABP constitutes a significant amount of cystolic protein, it is likely that it would be a target for free radical attack. To test this hypothesis, FABP was examined for scavenging against free radicals such as the superoxide anion (O2-), hydroxyl radical (OH.) and hypochlorite radical (OCl.) which may be present in an ischemic reperfused heart. Our results suggest that FABP scavenges O2-, OH. and OCl. as indicated by the FABP inhibition of O2- -dependent reduction of cytochrome c, OH.-dependent hydroxybenzoic acid formation and OCl.-mediated chemiluminescence response. FABP was found to be a more potent scavenger of these free radicals compared to bovine serum albumin. Furthermore, FABP was more effective in scavenging OH. than O2-, and inhibited OH. mediated lipid peroxidation process. These results indicate that FABP can scavenge free radicals which may be present in an ischemic/reperfused heart and, thus, may play a significant physiological role in the heart during ischemia and reperfusion.

Albumins↗
PubMed1989

Effects of free radicals on the fluidity of myocardial membranes.

Free radicals, including superoxide anions (O2.-), hydroxyl radical (HO.), and hypohalite radical (OCl.), as well as oxidants such as hydrogen peroxide (H2O2) and hypochlorous acid (HOCl), have been indicated in the pathogenesis of myocardial ischemic and reperfusion injury. In this report, we compared the integrity of the myocardial membrane when exposed to these free radicals/oxidants. Isolated rat heart membrane preparations were exposed to chemically generated free radicals with or without their respective scavengers. Membrane fluidity was monitored by fluorescence polarization using the diphenylhexatriene probe, as well as by electron spin resonance (ESR) spectroscopy using 2,2,6,6-tetramethyl piperidine-n-oxyl as the spin labeling agent. HO., H2O2, and OCl. + HOCl increased the fluorescence polarization (FP) and microviscosity significantly by 1.7-fold, 1.8-fold, and 1.7-fold, respectively, as compared to an only 1.2-fold increase in FP by O2.-. O2.- did not alter the fatty acid profiles of the membrane phospholipids. However, HO. and H2O2 reduced the arachidonic acid contents in phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylinositol (PI). These radicals also stimulated the lipid peroxidation by several-fold, while that by O2.- was only insignificant. These results suggest that HO. and H2O2 decreased the membrane fluidity and induced lipid peroxidation by releasing the arachidonic acid from PC, PE, and PI.

Animals↗
PubMed1989

Effect of oxygen-derived free radicals and oxidants on the degradation in vitro of membrane phospholipids.

The abilities of chemically generated hydroxyl radical (OH.), superoxide anion (O.-) and hydrogen peroxide (H2O2) to degrade rat myocardial membrane phospholipids previously labeled with [1-14C]arachidonic acid were studied. HO. and H2O2, but not O2.-, caused the degradation of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylinositol (PI). With OH. and H2O2, the loss of radiolabel in PC was accompanied by an increase in the radiolabel of lysophosphatidylcholine (LPC), but not in that of free fatty acid (FFA). These results suggest the hydrolysis of 1-oxygen ester bond of PC by HO. and that H2O2 and that HO. and H2O2, but not O.-, are detrimental to the structure and function of membrane phospholipids. However, since microM amounts of HO. and mM amounts of H2O2 were necessary to affect the membrane phospholipids, it is likely that in the reperfused myocardium only HO., but not H2O2, may directly cause the breakdown of membrane phospholipids.

Animals↗
PubMed1989

Steroid-induced myocardial preservation is associated with decreased cell membrane microviscosity.

Reperfusion of ischemic myocardium is associated with phospholipid degradation and corresponding changes in membrane fluidity. Dexamethasone (1.25 mg/kg i.v.) was evaluated in the pig, with pretreatment of the animal 1 1/2 hours before ischemic insult. The isolated perfused in vivo pig heart model was subjected to 60 minutes of regional ischemia of the left anterior descending coronary artery. The ischemic heart was then subjected to 60 minutes of global hypothermic cardioplegic arrest followed by 60 minutes of reperfusion, including reperfusion of the ischemic left anterior descending coronary artery region. Phospholipase A2, arachidonic acid, total free fatty acids, myocardial microviscosity, coronary blood flow, myocardial oxygen consumption, creatine kinase release, and regional and global myocardial function were measured. Dexamethasone pretreatment resulted in dramatic inhibition of phospholipase A2 activity accompanied by a reduction in arachidonic acid and total free fatty acid levels. Myocardial microviscosity (the inverse of membrane fluidity) was significantly increased only in untreated animals. Coronary blood flow and myocardial oxygen consumption were maintained at preischemic levels only in the dexamethasone-treated animals and were significantly reduced in the control group. Creatine kinase release increased nearly six times in control animals only while remaining stable in the dexamethasone-treated group, and regional and global myocardial contractility and compliance were improved dramatically in the dexamethasone-treated animals. These results indicate that dexamethasone enhances myocardial function by preserving membrane structure through inhibition of phospholipase activation, thereby preventing phospholipid degradation and maintaining membrane integrity and fluidity.

Animals↗
PubMed1988

Separation of fatty acid binding protein by high-performance mixed-mode chromatography.

Fatty acid binding protein (FABP) (14 kDa), can regulate the levels of tissue free fatty acids by binding them with high affinity. Since free fatty acids are known to accumulate in the ischemic myocardium, it is likely that FABP has a significant role in regulating their concentration in ischemic heart. FABP has recently been purified from other proteins, but the method requires several hours and special techniques. In this report, we describe a rapid high-performance liquid chromatographic method for separating and isolating the FABP from myocardial tissue biopsies. About 25-50 micrograms of rat heart cytosol was incubated with 2 nmol of the potassium salt of [9,10-3H]oleate (25,000 cpm) for 10 min at 37 degrees C. This was then injected onto a Bio-Rad (Richmond, CA, U.S.A.) TSK-125 column. The sample was run using a low-salt isocratic mobile phase containing 10 mM potassium phosphate buffer (pH 7) and 1 mM dithiothreitol, and at a flow-rate of 0.8 ml/min. The heart cytosol, when incubated with isotopic oleate, was resolved into two radioactive peaks, one eluting in the area of serum albumin (retention time 9.6 min) and the other corresponding to a retention time of 12.9 min. The sodium dodecyl sulfate polyacrylamide gel electrophoretic profile of the later peak revealed a major protein band of ca. 14 kDa. Rat heart FABP purified by gel filtration and ion-exchange chromatography coeluted with the second radioactive peak.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Analysis of tissue free fatty acids isolated by aminopropyl bonded-phase columns.

Gas chromatographic analysis revealed that polyunsaturated fatty acids such as arachidonic acid and total tissue free fatty acids isolated from an aminopropyl bonded-phase column yield a two- to three-fold higher recovery of arachidonic acid as compared to those isolated from thin-layer chromatographic plates. This method was further improved by packing the aminopropyl bonded phase in glass columns, since the glass column significantly eliminated the other contaminants (from polypropylene columns) coeluting with fatty acids in both a neutral lipid thin-layer chromatographic system and on a 5% DEGS-PS column of gas chromatographic analysis. In aminopropyl bonded-phase columns, the standard triglycerides and phospholipids were completely separated from free fatty acids as judged by gas chromatographic analysis. These results warrant the use of an aminopropyl bonded-phase column for the isolation of free fatty acids to obtain better recovery of polyunsaturated fatty acids.

Animals↗
PubMed1988

Improved myocardial performance induced by clofibrate during reperfusion after acute myocardial infarction.

The increase of cellular fatty acids appears to be one of the causes of the myocardial injury during ischemia and reperfusion. This study was designed to examine whether a hypolipidemic drug such as clofibrate can reduce the myocardial injury during ischemia and reperfusion. Clofibrate was fed to experimental pigs for 9 days. Isolated in situ hearts from both experimental and control pigs were subjected to 60 min of regional ischemia induced by occluding the left anterior descending coronary artery, followed by 60 min of global ischemia by hypothermic cardioplegic arrest and 60 min of reperfusion. The clofibrate feeding resulted in the better cardiac performance as judged by increased coronary blood flow, improved left ventricular function, and reduced myocardial injury as judged by creatine kinase release. Although the clofibrate-fed animals contained higher levels of thiobarbituric reactive materials, the free fatty acid levels of plasma and myocardium were much lower compared with control animals. The clofibrate feeding was also associated with increased peroxisomal catalase and beta-oxidation of fatty acids. These results suggest that decreased levels of free fatty acids in the plasma and the myocardium and increased catalase activity induced by antilipolytic therapy appear to provide beneficial effects to the myocardium during ischemia and reperfusion.

Animals↗
PubMed1988

Enhanced phosphodiesteratic breakdown and turnover of phosphoinositides during reperfusion of ischemic rat heart.

In this study, we examined phosphoinositide metabolism during ischemia and reperfusion using an isolated and perfused rat heart. When myocardial phosphoinositides were prelabeled with [3H]inositol, reperfusion after 30 minutes of normothermic global ischemia resulted in significant accumulations of radiolabeled inositol phosphate, inositol bisphosphate, and inositol trisphosphate. Isotopic incorporation of [3H]inositol into phosphatidylinositol, phosphatidylinositol-4-phosphate, and phosphatidylinositol-4,5-bisphosphate was increased significantly in the heart reperfused with [3H]inositol after 30 minutes of ischemia compared with that perfused with [3H]inositol after 30 minutes of nonischemic perfusion. However, isotopic incorporation of [3H]glycerol into diacylglycerol, phosphatidic acid, and all of the three phosphoinositides was diminished in the reperfused hearts. Reperfusion of the ischemic heart prelabeled with [14C]arachidonic acid resulted in significant increases in [14C]diacylglycerol and [14C]phosphatidic acid. The enhanced accumulations of [3H]inositol phosphates during reperfusion were not affected by treatment with prazosin plus atropine or indomethacin, but were inhibited by hypoxic reperfusion, reperfusion with Ca2+-free buffer, or by mepacrine. These results suggest that myocardial reperfusion stimulates phosphodiesteratic breakdown and turnover of phosphoinositides, and increased Ca2+ influx caused by reperfusion may be involved in the mechanism of stimulation of phosphatidylinositol-specific phospholipase C activity in the rat heart.

Animals↗
PubMed1988

Effect of ischemia and reperfusion of the myocardium on in vitro beta-oxidation of fatty acids.

The in vivo oxidation of perfused [14C]-labeled fatty acids has been shown to decrease dramatically in hypoxic hearts. This study addresses the influence of ischemia and reperfusion on the enzymic activities of beta-oxidation of fatty acids in mitochondria and of peroxisomal origin. The rate of beta-oxidation of fatty acids in the isolated mitochondria from myocardium of swine fed control diet declined about 20% by the ischemic insult induced by hypothermic cardioplegic arrest. Upon reperfusion, the rate of mitochondrial beta-oxidation returned to a normal level. In clofibrate-fed animals, the rate of mitochondrial beta-oxidation did not vary significantly between control, ischemic, and perfused tissues. Furthermore, neither in control nor in clofibrate-fed animals did the rates of peroxisomal beta-oxidation of fatty acids vary significantly in the ischemic or reperfused tissues as compared to that of preischemic controls. These results suggest that ischemia does not contribute to any loss of enzymic activity in beta-oxidation of fatty acid cycles either in mitochondria or peroxisomes. Furthermore, the feeding of 0.5% (w/w) clofibrate to pigs increased the rate of mitochondrial beta-oxidation of fatty acids only by 50% while that of peroxisomes increased threefold. A similar threefold increase in catalase activity was also produced by clofibrate feeding. These results suggest that the heart plays a role in the hypolipidemic action of clofibrate.

Animals↗
PubMed1987

Role of xanthine oxidase inhibitor as free radical scavenger: a novel mechanism of action of allopurinol and oxypurinol in myocardial salvage.

Xanthine oxidase (XO) has been hypothesized to be a potential source of oxygen-derived free radicals during reperfusion of ischemic myocardium based on the fact that allopurinol, a XO-inhibitor, can reduce reperfusion injury. In this communication we report that both allopurinol and oxypurinol, the principle metabolite of allopurinol, prevent the reperfusion injury in isolated pig heart. However, we found that neither pig heart nor pig blood contain any XO activity. Our study showed a direct free radical scavenging action of these XO-inhibitors during ischemia and reperfusion, as judged by the reduction of free radical signals when compared using an Electron Paramagnetic Resonance Spectrometer. Using a Luminometer, we also confirmed that both allopurinol and oxypurinol can scavenge ClO2, HOCl, and significantly inhibit free radical signals generated by activated neutrophils. These XO-inhibitors, however, failed to scavenge O2. and OH. radicals. Our results suggest that these XO-inhibitors salvaged the ischemic-reperfused myocardium by scavenging free radicals, and not by inhibiting XO in the pig heart.

Adenosine Triphosphate↗