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Biomedical subjects

W B Weglicki

Publications and source records attributed to W B Weglicki.

At least 73 records · Page 4Linked to original sources
PubMed1990

Endothelial cells as a source of oxygen-free radicals. An ESR study.

Endothelial cells were subjected to anoxia/reoxygenation in order to simulate some of the free radical mechanisms occurring in ischaemia/reperfusion. With ESR and spin trapping using the spin traps 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) and 3,3,5,5-dimethyl-1-pyrroline-1-oxide (M4PO), the results show that upon reoxygenation of endothelial cells, following a period of anoxia, these cells generate superoxide (O2-.). Cytotoxicity of the spin traps was measured by standard trypan blue exclusion methods. Cell injury or death was measured at various times during reoxygenation by lactate dehydrogenase (LDH) release. Experiments using oxyupurinol, SOD, CAT and a combination of SOD and CAT show that while oxypurinol partially prevents spin adduct formation, the combination of SOD and CAT is more effective in doing so. These results suggest that the majority of the oxygen radicals produced by endothelial cells are done so exogenously. The results also suggest that endothelial cells are not only a source of oxygen radicals but also a target.

Animals↗
PubMed1989

Is the ATP-dependent protection of lysosomes against osmotic lysis a function of the lysosomal proton pump.

Rat liver lysosomes have been used to characterize further the effects of ATP on lysosomal stability during incubation at 37 degrees C at hypo-osmolarity. As previously reported, when the osmotically-supporting solute is the salt of a strong base (K+), ATP protects against lysis during incubation. However, if the osmotically-supporting solute is the salt of a weak base, e.g. Tris HCl or NH4Cl, ATP actually promotes lysis during incubation. Thus, ATP can exert destabilizing as well as protective effects on lysosomes. The destabilizing effect is eliminated by protonophores. The protective effect in the presence of potassium salts is not eliminated by protonophores. Moreover, when incubation is in the presence of a salt of a weak base, protonophores actually cause an ATP-dependent protective effect to be established. The destabilizing effect occurs at 37 degrees C, but not at 0 degrees C. The Mg++-dependence of the destabilizing effect was found to be similar to that found earlier for the ATP-dependent protective effect, insofar as only 1 mM MgCl2 in the presence of 1mM EDTA is sufficient for nearly maximal stimulation of both effects. The destabilizing effect may result from a H+ ion gradient across the lysosomal membrane which is maintained by the lysosomal ATP-dependent proton pump. The protective effect, on the other hand, does not depend on such a gradient being maintained; on the contrary, protonophores appear to act as enablers of the protective effect. The question that remains to be answered is: does the protective effect derive in some way from the same ATP-driven mechanism which constitutes the proton pump? Some possible answers to this question are considered.

Adenosine Triphosphate↗
PubMed1989

Inhibition of sarcolemmal carbon-centered free radical formation by propranolol.

The mechanism of propranolol-inhibited sarcolemmal membrane lipid peroxidation was investigated by electron spin resonance spin-trapping technique using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and 2-methyl-2-nitrosopropane (MNP). Highly purified canine myocytic sarcolemma were peroxidized by a superoxide-driven (from dihydroxyfumarate) and Fe3+-catalyzed free radical-generating system. Hydroxyl radicals (.OH), identified by electron spin resonance signals as DMPO-OH adducts, were generated in the aqueous phase. Propranolol up to 500 microM did not effectively reduce the intensity of the DMPO-OH adducts. When the sarcolemma were incubated with MNP before the addition of free radicals, MNP adducts characteristic of carbon-centered radicals were produced. Pretreatment of the membranes with propranolol (3-100 microM) decreased the intensity of the MNP adducts in a log concentration-dependent manner; the EC50 is about 7 microM. D- and L-propranolol were found equally effective. When protein-depleted sarcolemmal lipids were similarly incubated with MNP and the free radical system, identical MNP adducts were observed; this finding suggests that the adducts were lipid-derived products arising from lipid peroxidation. Furthermore, their formation was also inhibited by propranolol pretreatment. Since propranolol is not an effective scavenger of oxygen radicals in the aqueous phase, the data suggest that the antiperoxidative effect of propranolol is due to its lipophilic interaction with the membrane and thus subsequent interruption of the free radical chain reactions.

Animals↗
PubMed1989

Spin trapping of free radicals formed during peroxidation of sarcolemmal membranes (SLM).

The generation of free radicals in a superoxide (O2-.) driven Fe+3 catalysed reactions with isolated myocytic sarcolemma using electron spin resonance was investigated. Incubation of highly purified canine myocytic sarcolemma in the presence of the spin trap, 2-methyl-2-nitrosopropane (MNP), followed by the addition of dihydroxyfurmarate (DHF) and Fe+3-ADP resulted in the generation and detection of radical adducts of this spin trap. Spin trapping of the alkyl radicals with 2-methyl-2-nitrosopropane led to the identification of methyl radical adduct following exposure to DHF/Fe+3-ADP. With sarcolemma and the alkyl nitroso compound, the only radical product trapped was the methyl radical formed by beta-scission of alkoxyl radical. The participation of hydroperoxide-derived radicals in this system verified that the decomposition of unsaturated hydroperoxy fatty acid does proceed via a free radical mechanism.

Animals↗
PubMed1988

The effect of erythrocyte associated light scattering on membrane fluorescence polarization.

The apparent membrane fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene has been reported to be lower in intact erythrocytes than in isolated erythrocyte membranes. Although this difference was once suggested to be caused by the fluidizing effect associated with the loss of erythrocyte proteins during membrane isolation, it is currently thought to be an artifact resulting from intense light scattering properties of intact erythrocytes which overwhelm extrapolation methods of correcting for light scattering. This study confirmed that, at erythrocyte concentrations greater than 10(7) cells/ml, this difference was caused by intense light scattering; however, at erythrocyte concentrations less than 4.0 X 10(6) cells/ml, the anisotropy values for erythrocytes and isolated membranes are identical, demonstrating that intense light scattering can be overcome with dilute suspensions of cells.

Erythrocyte Membrane↗
PubMed1988

Abnormal electrical activity induced by free radical generating systems in isolated cardiocytes.

Oxygen free radicals may participate in a variety of pathological cardiac conditions which are associated with an increased incidence of arrhythmias. However, evidence that free radicals per se can alter the electrical function of the myocardium is not convincing. Physiological solutions containing 3 mM dihydroxyfumaric acid (DHF), a compound known to generate free radicals, were superfused over calcium-tolerant cells isolated from the adult canine ventricle. The time course for changes in transmembrane action potentials was monitored using conventional microelectrode techniques. Changes were observed which could be conveniently segregated into three stages. Initially during superfusion with DHF, the voltage of the action potential plateau became more positive and the action potential duration increased (stage 1). Continued superfusion was associated with the development of both early and delayed afterdepolarizations (stage 2), which occasionally produced triggered beats. Subsequently, some cells failed to repolarize beyond -40 mV following an action potential upstroke. In cells which maintained normal levels of resting membrane potential, early and delayed afterdepolarizations ceased concomitant with the development of an increasingly more negative plateau voltage. Action potential duration decreased and plateau potential "collapsed", eventually merging with the resting level of the membrane potential. Resting membrane potential then gradually depolarized to less than -40 mV and all cells became inexcitable within 6 to 20 min (stages 3). Exposure of cells to xanthine (2 mM): xanthine oxidase (0.01 U/ml), another system known to generate free radicals, produced similar results. Superfusion with DHF solutions containing either superoxide dismutase or catalase delayed the appearance and attenuated the development of the changes in the cardiocyte action potential. The results demonstrate that isolated cardiocytes exposed to free radical generating solutions can undergo changes in their electrophysiological activity that resemble those said to underlie disturbances of cardiac rate and rhythm in the clinical setting.

Action Potentials↗
PubMed1988

Protection by beta-blocking agents against free radical-mediated sarcolemmal lipid peroxidation.

The effects of beta-blocking and class I antiarrhythmic agents on free radical-mediated sarcolemmal lipid peroxidation were examined. Highly purified canine myocytic sarcolemmal membranes were pretreated with 10-800 microM of selected beta-blocking (propranolol, pindolol, metoprolol, atenolol, or sotalol) and class I (quinidine, lidocaine, procainamide, or diphenylhydantoin) antiarrhythmic agents at 37 degrees C for 10 minutes. Subsequently, a superoxide radical (derived from dihydroxyfumarate) driven, Fe3+-ADP catalyzed free radical generating system was added and incubated for up to 45 minutes. Lipid peroxidation of sarcolemma was determined by malondialdehyde formation. Pretreatment of the membranes with the five beta-blockers resulted in various degrees (20-95%) of inhibition of sarcolemmal peroxidation in a concentration- and time-dependent manner. All the class I agents were less effective (less than 20% inhibition). The order of potency of the beta-blockers was propranolol greater than pindolol greater than metoprolol greater than atenolol greater than sotalol and appeared to relate to their degree of lipophilicity. Propranolol, the most potent agent, achieved half-maximal inhibition of peroxidation at about 100 microM and achieved significance (p less than 0.01) at 20 microM. At pH 6.0, the efficacy of pindolol, metoprolol, atenolol, and sotalol diminished by 30-50% compared to pH 7.2, but the potency of propranolol remained unchanged. Since increased free radical production may occur during myocardial ischemia/reperfusion injury, the above findings suggest that the lipophilic beta-blockers may provide additional antiperoxidative protection of ischemic tissue.

Adrenergic beta-Antagonists↗
PubMed1987

Identification of free radicals in myocardial ischemia/reperfusion by spin trapping with nitrone DMPO.

The spin trapping ESR technique was applied to investigate oxygen-derived radicals in ischemic and post-ischemic rat hearts. Using 5,5'-dimethyl-l-pyrroline-N-oxide, carbon-centered radicals were identified during ischemia and oxy-radical adducts (superoxide anion radical, O.-2 and hydroxyl radicals, .OH) in post-ischemic rat heart. The formation of these spin adducts was inhibited by superoxide dismutase, suggesting that superoxide plays a role in the adducts' formation. The results demonstrate that oxygen derived free radicals are important byproducts of abnormal oxidative metabolism during myocardial ischemic and reperfusion injuries.

Animals↗
PubMed1987

Phospholipases of the myocardium.

The myocardium contains diverse cellular components and heterogeneous phospholipid-containing membranes. The major phospholipids are phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositnol, sphingomyelin and cardiolipin. The phospholipases capable of hydrolyzing these membrane lipids include phospholipase A, lysophospholipase, and phosphatidylnositol-specific phospholipase C. Early studies revealed that myocardial phospholipase A with an acid pH is localized to lysosomes; those with more alkaline and neutral activities are present in cytosol, microsomes, mitochondria and sarcolemma. Recently, we have identified phosphatidylinositol-specific phospholipase C activity in bovine myocardium with molecular weights ranging from 40,000 to 271,000. Interestingly, forms I, II and III, had pH optima ranging from 4.5 to 5.5; form III also had significant activity at pH 7.0. All activities were stimulated by calcium, suggesting that they are different from calcium-independent phospholipases C found in liver and brain. The pathophysiological significance of these four cytosolic forms of phospholipase C remains to be determined. Thus, under injury-promoting conditions, phospholipase C appears capable of hydrolyzing membrane-associated phosphatidylinositol and the polyphosphoinositides, whereas phospholipases A and lysophospholiphases appear to prefer non-inositol containing phospholipids. Finally, very recent studies suggest "free radical-triggered lipolysis" by phospholipases as a possible mechanism for production of lysophospholipids in myocardial membranes.

Animals↗
PubMed1987

Spin-trapping evidence that graded myocardial ischemia alters post-ischemic superoxide production.

The spin trapping agent 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was used to investigate oxy-radical production in post-ischemic rat hearts previously exposed to 20, 30, or 40 minutes of global ischemia. A hydroxyl spin adduct (DMPO-OH) was identified in coronary effluent during the initial seconds of reperfusion by Electron Spin Resonance (ESR) Spectroscopy. The intensity of the ESR signal in post-ischemic effluent increased as ischemic duration was prolonged; however, regardless of the duration of ischemia, maximal spin adduct detection occurred 3 minutes after initiation of reperfusion. Superoxide dismutase inhibited the formation of DMPO-OH, suggesting that superoxide anion was initially generated and is the principle source for the production on the hydroxyl adduct. Our investigations indicate that superoxide anion is produced during the early moments of reperfusion and that its production in the post-ischemic heart is related to the severity of ischemia.

Animals↗
PubMed1987

Spin trapping of oxygen and carbon-centered free radicals in ischemic canine myocardium.

Oxygen free radical injury has been postulated to occur during myocardial ischemia. We have used Electron Spin Resonance and Spin Trapping techniques to directly demonstrate the production of carbon-centered (R.) and oxygen-centered lipid radical (RO.) in ischemic canine heart. In addition, venous effluent from the ischemic region showed that conjugated dienes (lipid peroxidation products) increased with ischemic duration. Our results suggest that the formation of the oxygen-centered and carbon-centered lipid radical species during ischemia are a consequence of oxy-radical peroxidation of myocardial membrane lipids.

Animals↗
PubMed1986

Inhibition of cyclic AMP-dependent protein kinase activity by the cardiotonic drugs amrinone and milrinone.

Amrinone and milrinone are new cardiotonic drugs that have potent inotropic and vasodilatory properties. The mechanism of action of these agents is controversial, but the positive inotropic component is thought to be due to the inhibition of phosphodiesterase. Because amrinone and milrinone have been shown to be involved primarily in cyclic AMP-mediated processes, we examined the effect of these agents on cyclic AMP-dependent protein kinase. The results indicate that amrinone and milrinone inhibit cyclic AMP-dependent protein kinase activity by competing with ATP but not cyclic AMP binding sites. Dissociation constants (Ki) of amrinone and milrinone for ATP binding sites on protein kinase were calculated to be 100-300 microM and 842 microM, respectively. The phosphodiesterase inhibitor isobutylmethylxanthine (1 mM) had no effect on protein kinase activity. Amrinone and milrinone inhibited the catalytic subunit of protein kinase to the same degree as the holo-enzyme by competitively inhibiting the binding of ATP. Amrinone and milrinone had no effect on phospholipid-sensitive, calcium-dependent protein kinase indicating that there may be differences in the ATP binding sites on these two protein kinases. Inhibition of cyclic AMP-dependent protein kinase by amrinone and milrinone occurs at concentrations higher than those used clinically. However, because amrinone and milrinone are lipophilic drugs, they may be useful tools for the investigation of protein kinase mediated reactions.

1-Methyl-3-isobutylxanthine↗
PubMed1986

Potentiation of free radical-induced lipid peroxidative injury to sarcolemmal membranes by lipid amphiphiles.

The effects of naturally occurring lipid amphiphiles on free radical-mediated peroxidative injury in isolated canine sarcolemma were studied. Highly enriched canine myocytic sarcolemmal membranes were preincubated for 10 min at 37 degrees C with or without different amphiphilic lipids before the addition of a free radical-generating system consisting of dihydroxyfumarate and Fe3+-ADP. Lipid peroxidation, assayed as malondialdehyde formation, was catalyzed linearly up to 40 min in the control samples. Pretreatment of the sarcolemma with palmitoyl-CoA, palmitoylcarnitine, or lysophosphatidylcholine accelerated the initial rates (20 min) of peroxidation in a concentration-dependent manner (10-100 microM) and achieved maximal stimulation (240%, 160%, and 210%, respectively, of controls) at 50 microM concentrations of each of these amphiphiles. However, free fatty acids, CoA, and carnitine were without effect. These promoting effects of the amphiphiles persisted over a wide pH range (pH 6.0-7.8) and exhibited additive effects when lower levels of different amphiphiles were combined together. Associated with the accelerated rates of peroxidation produced by palmitoyl-CoA and palmitoylcarnitine were greater losses in the activity of sarcolemmal (Na,K)-ATPase. Since all three kinds of amphiphilic lipids accumulate during ischemia, this study suggests a novel mechanism of potentiation of sacolemmal membrane injury when free radicals are present.

Animals↗
PubMed1985

Superoxide dismutase: tissue, cellular, and subcellular distribution in adult canine heart.

Cell-free extracts of canine myocardial tissue were found to contain two biochemically and electrophoretically distinct superoxide dismutases (SOD), an enzyme that provides defense against the deleterious effect of superoxide radicals (O2.-). Polyacrylamide gel (7.5%) electrophoresis revealed two distinct bands of SOD activity: a slower moving band [retardation factor (Rf) = 0.4] resembling the manganese SOD found in bacteria and mitochondria (which is not inhibited by 2.5 mM cyanide) and a faster moving band (Rf = 0.75) that is sensitive to cyanide. In contrast, extracts from isolated adult canine cardiac myocytes were found to contain only the cyanide-insensitive SOD. Extracts of whole myocardium and isolated cardiac myocytes contain 22.3 +/- 1.2 and 27.0 +/- 1.5 U cyanide-insensitive SOD/mg protein, respectively. However, the activity of cyanide-sensitive SOD in these fractions is 7.9 +/- 2.0 (tissue) and 1.5 +/- 1.4 (cells) U/mg protein. Cardiac myocyte SOD activity was particulate in nature, and the major part of the SOD activity was associated with heavy mitochondrial fractions. The biologic significance of this higher activity of SOD in the heavier mitochondrial fraction remains to be elucidated.

Animals↗
PubMed1985

Inhibition of sarcolemmal Na+-K+-ATPase by palmitoyl carnitine: potentiation by propranolol.

Native sarcolemma (SL) from adult canine cardiac myocytes (Na+-K+-ATPase activity 74.2 +/- 3.0 mumol X mg-1 X h-1) was preincubated (10 min, 37 degrees C, pH 7.2) with 1) 20-600 microM palmitoyl carnitine, 2) 250 nM-2.5 mM propranolol, or 3) 20-600 microM palmitoyl carnitine plus propranolol at various concentrations (0.0, 0.025, 0.25, 0.5, 1.0, and 2.5 mM); after preincubation, Na+-K+-ATPase activity was assayed. Palmitoyl carnitine alone (series 1) had no effect on ATPase activity over the range of 20-400 microM but was inhibitory (30%) at 600 microM. Propranolol alone (series 2) did not alter ATPase activity at any concentration. When SL membranes were exposed to both palmitoyl carnitine and propranolol (series 3), a dose-dependent inhibition of ATPase activity was observed. The inhibitory effect was not reversed by 3.0% bovine serum albumin. Propranolol concentrations greater than 0.025 mM significantly inhibited the activity of SL exposed to palmitoyl carnitine (above 150 microM). Palmitoyl carnitine and propranolol do not have to be added simultaneously to produce combined inhibition. Activity was inhibited 50% when SL were pretreated with 100 microM palmitoyl carnitine followed by addition of 2.5 mM propranolol no inhibition occurred if preincubation conditions were reversed. Thus exposure of SL to propranolol and reported physiological levels of palmitoyl carnitine leads to irreversible inhibition of the Na+-K+-ATPase, which may be due to the combined membrane-perturbant actions of these amphipathic agents.

Alamethicin↗
PubMed1985

Response of canine cardiocyte lysosomes to ATP.

The present study was designed to evaluate the relationship of adenosine triphosphate (ATP) to maintenance of cardiac lysosome latency. Highly latent lysosomes were isolated from adult canine ventricular myocytes or cardiocytes and exhibited latency values (based on % free N-acetyl-beta-glucosaminidase, NAGA) of 29.3 +/- 4.7% (SE), minimal cross contamination by mitochondria (less than 2% cardiolipin by weight) and only 1.68% of the total cytochrome c oxidase activity; enzymatic enrichments ranged from 10-fold for NAGA to almost 50-fold for cathepsin B. Incubations of cardiocyte lysosomes at pH 7.0 and 25 degrees C for up to 1 h resulted in changes in the rate of loss in lysosomal latency when ATP levels were adjusted from 0.0 to 5.0 mM; under these conditions as ED50 of 0.62 mM ATP was determined. The protection afforded by ATP was reversed by addition of the H+ ionophore m-chlorocarbonylcyanide phenylhydrazone (CCCP) at 10 microM to the lysosomal suspensions. A significant increase in lysosomal membrane fluidity, measured by fluorescent polarization of diphenylhexatriene, was also seen in the absence of ATP. Adenosine diphosphate (ADP) afforded significant protection only at the very highest concentration (5.0 mM); inorganic pyrophosphate (PPi) did not protect against loss of latency at any concentration. Thus ATP exerts a significant stabilizing effect on cardio(myo)cyte lysosomes in vitro and may be one of the metabolites regulating lysosomal integrity in vivo.

Acetylglucosaminidase↗