Leakage Of Cytosolic Lactate Dehydrogenase Research Articles

Eriodictyol protects H9c2 cardiomyocytes against the injury induced by hypoxia/reoxygenation by improving the dysfunction of mitochondria.

Myocardial infarction is a leading cause of mortality worldwide, while myocardial ischemia and timely reperfusion contribute to myocardial injury. The mitochondria are involved in the injury and mediate the apoptosis of cardiomyocytes. In order to develop novel therapeutic approaches for myocardial infarction, the present study evaluated the myocardial protective effects of eriodictyol and investigated relevant mechanisms in H9c2 cardiomyocytes. As a result, eriodictyol was observed to improve the H9c2 cardiomyocyte viability and block the leakage of cytosolic lactate dehydrogenase under hypoxia/reoxygenation. In addition, the dysfunction of mitochondria induced by hypoxia/reoxygenation was ameliorated by eriodictyol through suppressing the overload of intracellular Ca2+, preventing overproduction of reactive oxygen species, blocking mitochondrial permeability transition pore opening, increasing mitochondrial membrane potential level and decreasing ATP depletion. Finally, the apoptosis of H9c2 cardiomyocyte induced by hypoxia/reoxygenation was prevented by eriodictyol through upregulation of the expression of B-cell lymphoma-2 (Bcl-2) and downregulation of the expression levels of Bcl-2-associated X protein and caspase-3. These results provided evidence for further investigation on myocardial protection and the treatment of myocardial infarction using eriodictyol.

Auranofin, a thioredoxin reductase inhibitor, causes platelet death through calcium overload

Platelets are central to normal hemostasis and must be tightly controlled to prevent thrombosis. However, drug treatments that also affect platelets could lead to unwanted side effects on hemostasis or thrombosis. In this study, the effect of auranofin on platelets was tested. Auranofin, a gold-based thioredoxin reductase (TRXR) inhibitor, has been previously used in arthritis. Recently, auranofin and other inhibitors of the thioredoxin system have been proposed as novel anti-cancer therapies. TRXR is an important part of the antioxidant defenses in many cells that maintain intracellular proteins in their reduced state.TRXR activity in platelets could be completely inhibited by auranofin. Auranofin-treated platelets showed several features of cell death, including the inability to aggregate in response to thrombin, leakage of cytosolic lactate dehydrogenase, and surface exposure of procoagulant phosphatidylserine. Auranofin increased platelet reactive oxygen species production and intracellular calcium concentration. DTT, a sulfydyl reducing agent, and BAPTA-AM, which chelates intracellular calcium, prevented auranofin-induced phosphatidylserine exposure. These data suggest that TRXR is an important part of the platelet antioxidant defense. TRXR inhibition by auranofin triggers oxidative stress and disrupts intracellular calcium homeostasis, leading to platelet necrosis. The use of auranofin or other TRXR inhibitors could therefore lead to unwanted side effects.

Protective effects of carnosic acid against mitochondria-mediated injury in H9c2 cardiomyocytes induced by hypoxia/reoxygenation.

Myocardial ischemia and reperfusion occurs in myocardial infarction. Timely reperfusion will exacerbate the injury through the mitochondria-mediated apoptosis in cardiomyocytes due to the accumulation of excessive reactive oxygen species (ROS). In order to identify novel therapeutic approaches, the cardioprotective effects of carnosic acid and the underlying mechanisms were investigated in the present study in H9c2 cardiomyocytes injured by hypoxia/reoxygenation in vitro. The viability of H9c2 cardiomyocytes was detected by MTT assay and further confirmed by the detection of intracellular lactate dehydrogenase (LDH) release. The mitochondrial function in H9c2 cardiomyocytes was evaluated using fluorescence methods. The proteins related to apoptosis, including caspase-3, B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax) were analyzed by western blot analysis, and the activity of caspase-3 was determined using a colorimetric method. As a result, carnosic acid was demonstrated to improve the viability of H9c2 cardiomyocytes and suppress the leakage of cytosolic lactate dehydrogenase under hypoxia/reoxygenation. In addition, the overproduction of intracellular ROS and intracellular calcium overload were ameliorated in the presence of carnosic acid. The dysfunction of mitochondria in H9c2 cardiomyocytes was also attenuated by carnosic acid through blocking the collapse of mitochondrial membrane potential (MMP) and mitochondrial permeability transition pore (mPTP) opening. Furthermore, the apoptosis of H9c2 cardiomyocytes resulted from hypoxia/reoxygenation was inhibited by carnosic acid through the upregulation of Bcl-2 and the downregulation of Bax and caspase-3 levels. These results provided evidence for further investigation that would assist in the development of novel therapeutic approaches for myocardial infarction.

The Effect of Microarc Oxidation Ceramic Coating on Sensory Reconstruction around Implants Using Cultured Schwann Cells

Osseointegrated titanium implants have been widely used in clinics for replacement of missing teeth. However, sensory perception thresholds of implants were 10 to 100 times higher than those of natural teeth. The purpose of this study was to evaluate the effect of the ceramic coating generated by microarc oxidation (MAO) on sensory reconstruction around dental implants. The MAO coating was characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), and scanning probe microscope (SPM). Moreover, the chemical composition was analyzed with an energy dispersive spectroscope (EDS). Using smooth titanium discs as a control, Schwann cell responses to the coating were evaluated by SEM, MTT assay, total protein content and leakage of cytosolic lactate dehydrogenase (LDH) activity. In addition, the amount of brain-derived neurotrophic factor (BDNF) secreted by Schwann cells was measured by Enzyme-Linked Immunosorbent Assay (ELISA). It was observed that the porous coating had separated and homogeneous micropores in the diameter of 1-2 μm. The concentrations of Ca and P in the layer were 8.63% and 5.23% respectively. The cell morphology, proliferation and secretion of BDNF were unaffected. The results indicated that the MAO ceramic coating developed on the surface of pure titanium had good biocompatibility with Schwann cells for sensory reconstruction around dental implants.

Biocompatibility of Nano-TiO<sub>2</sub>/HA Bioceramic Coating for Nerve Regeneration around Dental Implants

Sensory perception thresholds of implants were much higher than those of natural teeth. The purpose of this study was to evaluate the biocompatibility of TiO2/HA-biocoated dental implants for nerve regeneration using cultured Schwann cells. The nano-TiO2/HA composite bioceramic coating was developed on the surfaces of commercially pure titanium discs by sol-gel route, and characterized by X-ray diffraction (XRD) and scanning electron microscopy (SEM). Using smooth titanium discs as a control, Schwann cell responses to the coating were evaluated by SEM, MTT assay, total protein content and leakage of cytosolic lactate dehydrogenase (LDH) activity. In addition, the amount of brain-derived neurotrophic factor (BDNF) secreted by Schwann cells was measured by Enzyme-Linked Immunosorbent Assay (ELISA). It was observed that the coating had fine crystallites and homogeneous micropores in the diameter of 5-10 um. The cell morphology, proliferation and function were unaffected. The results indicate that the TiO2/HA bioceramic coating developed on the surface of pure titanium by sol-gel route had good biocompatibility with Schwann cells for nerve regeneration around dental implants.

Biocompatibility of NGF-grafted GTG membranes for peripheral nerve repair using cultured Schwann cells

We previously developed a biodegradable composite with potentially good biocompatibility composed by tricalcium phosphate and gluataraldehyde cross-linking gelatin (GTG) with good mechanical property feasible for surgical manipulation. The purpose of this study was to evaluate the feasibility of immobilizing nerve growth factor (NGF) onto the composite (GTG) with carbodiimide (GEN composite). Cultured Schwann cells were seeded onto the GTG and GEN composites. For comparison, GTG membrane soaked in NGF solution without carbodiimide (GN composite) as cross-linking agent was also used to culture Schwann cells. Cell morphology was observed by a scanning electron microscope. Cell survival, cytotoxicity and cellular metabolism on the NGF-grafted GTG membrane were assessed quantitatively in terms of cell protein content, leakage of cytosolic lactate dehydrogenase (LDH) activity and by the well-established MTT assay, respectively. The result of LDH study did not show significant difference among GTG, NGF-modified GTG and control group. This indicated that GTG composite, whether cross-linking with NGF or not, has little cytotoxic effect. Comparing the protein content and MTT assay among GEN, GN composite and control group, the data confirmed more attachment of Schwann cells on GEN composite. Although GTG cross-linking with NGF did not promote Schwann cell proliferation, the techniques we used in this study provided a method to fabricate a novel biomaterial incorporation of Schwann cells and covalently immobilized NGF.

In vitro effects of lithium and nickel at different levels on Neuro-2a mouse Neuroblastoma cells

Lithium and nickel present low toxicity, but are able to cause alterations in different tissues. The toxic effects of lithium and nickel at different cellular levels were assessed using two inorganic chemical species: lithium chloride and nickel(II) chloride. Mouse neuroblastoma cell cultures (Neuro-2a) were exposed to both compounds for 24 h. The cytotoxic effects evaluated were cell proliferation by quantification of total protein content, cytoplasmic membrane integrity to cytosolic lactate dehydrogenase leakage, and lysosomal hexosaminidase release. Metabolic markers were lactate dehydrogenase activity and mitochondrial succinate dehydrogenase activity. Lysosomal markers were relative neutral red uptake by lysosomes, and lysosomal hexosaminidase sphingolipid degradation activity. Acetylcholinesterase activity on intact cells was also quantified. Nickel was found to be 36 times more toxic than lithium to neuroblastoma cell proliferation (EC 50= 0.29 and 10.5 m m, respectively), but the relative extent of other alterations differed. Lithium stimulated nearly all the indicators studied, particularly lactate dehydrogenase, mitochondrial succinate dehydrogenase and acetylcholinesterase activities, as well as hexosaminidase release. In contrast, nickel mainly stimulated hexosaminidase release and inhibited lactate dehydrogenase activity. The stabilization of the cytoplasmic membrane to lactate dehydrogenase leakage simultaneously with the secretion of lysosomal hexosaminidase for both compounds also shows that functional metabolic alterations produced by lithium and nickel are more important than cytoplasmic damage.

Cytotoxicity in pig hepatocytes induced by 8-quinolinol, chloramine-T and natamycin.

The potential cytotoxic effects of the compounds 8-quinolinol, chloramine-T and natamycin have been studied in isolated pig hepatocytes. The relative cytotoxicity of these compounds was evaluated on the basis of the leakage of cytosolic lactate dehydrogenase (LDH), 3-(4,5 dimethyl)thiazol-2-yl,-2,5-diphenyl tetrazolium bromide (MTT) reduction by mitochondrial dehydrogenases, uptake of neutral red (NR) by cytosolic lysosomes, glutathion (GSH) depletion and oxidized glutathion (GSSG) efflux after 24 h exposure. Evaluation of the 20%, 50% and 80% reduced absorbance data obtained from the parameters NR20, NR50, and NR80, and MTT20, MTT50 and MTT80 enabled us to rank these compounds in decreasing order of cytotoxicity: 8-quinolinol > natamycin > chloramine-T. Also for the parameters LDH and GSH, chloramine-T appears to be less cytotoxic than natamycin and 8-quinolinol. Our study demonstrated that pig hepatocytes may be a useful model for examining cytotoxic events of drugs to be used in pigs, therefore avoiding possible extrapolation problems due to species differences.

The effect of conjugated linoleic acid on the antioxidant enzyme defense system in rat hepatocytes.

Short-term effects of physiological concentrations of conjugated linoleic acid (CLA) on membrane integrity, metabolic function, cellular lipid composition, lipid peroxidation, and antioxidant enzymes were examined using rat hepatocyte suspension cultures. Incubation with CLA (5-20 ppm) for 3 h decreased the ability of hepatocyte plasma membranes to exclude trypan blue by approximately 25%, and caused leakage of cytosolic lactate dehydrogenase (LDH) into the medium. The significant decrease (P< 0.02) in hepatocyte viability as measured by LDH leakage during cell incubation with 10 and 20 ppm CLA was not associated with significant changes in cellular ATP content. Protein synthesis in hepatocytes was elevated (P < 0.05) in the presence of 5 and 10 ppm CLA, but at a higher concentration (20 ppm), protein synthesis was similar to that of control cells. Gluconeogenesis was maintained in cells incubated with lower concentrations of CLA (5 and 10 ppm) but was decreased (P < 0.02) at the higher concentration. Incubation with 20 ppm CLA for 3 h did not affect the specific activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-limiting enzyme of cholesterol synthesis. Both cis-9,trans-11/trans-9,cis-11, and cis-10,trans-12/trans-10,cis-12 isomers of CLA were incorporated to a similar level into hepatocytes. Levels ranged from 3.9 to 4.1%, respectively, of total fatty acids in neutral lipids, and from 0.7 to 0.8%, respectively, of total fatty acids in phospholipids. Cellular lipid peroxidation remained unchanged in the presence of CLA (5-20 ppm), despite significant inhibition (P < 0.05) of superoxide dismutase. Catalase activity was maintained near control levels in the presence of 5 and 10 ppm CLA but was significantly decreased in the presence of 20 ppm CLA. Glutathione peroxidase activity was significantly decreased in the presence of 10 ppm CLA. The apparent sensitivity of the antioxidant enzyme defense system of liver cells to CLA, coupled with the lack of effect of CLA on lipid peroxidation in cells, suggests that cytotoxic effects of CLA as described by LDH leakage and decreased gluconeogenesis were not mediated by a prooxidant action in hepatocytes.

Antidotal Efficacy of Antioxidants against Cyanide Poisoning in vitro.

Cyanide is a potent homicidal, genocidal and chemical warfare agent. Besides, its known inhibitory effects on various enzyme Systems, its other pronounced toxic effects include lipid peroxidation (LPx), particularly in the central nervous system or neuronal cells in vitro. The present study assessed the cytotoxicity of potassium cyanide (KCN) in two non-neuronal mammalian cell cultures, viz., human embryonic lung epithelium (L-132) and baby hamster kidney (BHK-21 ) cells. In addition, the cytoprotective potential of two antioxidant agents, namely, curcumin (CMN) and N-acetylcysteine (NAC) against KCN (2 and 4 mM) in vitro was evaluated. In both the cell lines, KCN reduced cell viability as indicated by trypan blue dye exclusion, leakage of cytosolic lactate dehydrogenase and neutral red uptake. Protein content was unaffected in L-132 cells while cellular respiration determined by MTT assay) was impaired in both the cells. A dose-dependent glutathione mediated LPx was observed in BHK-21 cells alone. The above cytotoxic changes produced by KCN were more effectively minimised by NAC as compared to CMN. Efficacy of CMN and NAC have therapeutic implications as adjuncts to existing cyanide antidotes.

Mutagenicity and dark toxicity of the second-generation photosensitizer bacteriochlorin a

Bacteriochlorin a (BCA) is an effective second-generation photosensitizer both in vitro and in vivo. BCA has a high molecular absorption coefficient (32 000 M −1 cm −1) at 760 nm. At this wavelength tissue penetration of light is almost optimal and melanin absorption is relatively low. BCA is preferentially retained in a number of tumour model systems and is rapidly cleared from non-cancerous tissues, thus inducing no or minor skin photosensitivity. Mutagenicity of BCA has been tested using the Salmonella typhimurium strains TA97, TA98, TA100, TA102 and TA104. In all tester strains used, BCA induces, in the dark, a minute increase in the number of revertants. No linear correlation between the number of revertants and the BCA dose is observed. Incubation of isolated rat hepatocytes with BCA, in the dark, does not result in increased cell death as measured by leakage of cytosolic lactate dehydrogenase. A convenient bioassay to test possible genotoxicity in vivo is the established Somatic Mutation and Recombination Test (SMART) on Drosophila melanogaster. Bacteriochlorin was tested for induction of loss of heterozygosity in the white/white + eye mosaic assay, which predominantly measures homologous mitotic recombination in somatic cells of Drosophila after treatment of larval stages. BCA did not induce loss of heterozygosity above the level of the incorporated controls, with or without illumination. Based on these results, obtained in prokaryotic and eukaryotic cells and in vivo, we are inclined to conclude that the dark toxicity and mutagenic properties of BCA, as measured by the applied bioassays, are negligible.

Effects of cobalt on mouse neuroblastoma cells cultured in vitro

To compare the effects of cobalt at different cellular levels, cultured mouse neuroblastoma cells (Neuro-2a) were exposed for 24 hr to cobalt(II) chloride. The following toxicity indicators were assessed: cell proliferation by quantification of total protein content; cytoplasmic membrane integrity to cytosolic lactate dehydrogenase leakage; lysosomal hexosaminidase release; lactate dehydrogenase activity (LDH); mitochondrial succinate dehydrogenase activity (SDH); relative neutral red uptake by lysosomes (RNRU); lysosomal hexosaminidase activity (HEX), and acetylcholinesterase activity (AChE). The effect of cobalt on the various indicators differed. Cobalt was not very toxic to neuroblastoma cell proliferation (EC 50 = 200 μ m). Cytoplasmic membrane permeability was not specifically increased, and LDH leakage occurred only at high concentrations, prior to the stimulation of HEX activity, an enzyme involved in sphingolipid degradation. In contrast, cobalt was lysosomotropic, with HEX release. The effects on lysosomal function were also studied with the RNRU, showing stimulation at low concentrations and inhibition at high concentrations. Neural AChE was decreased after an initial stimulation at low concentrations. LDH and SDH intracellular activities were both stimulated from low concentrations, mitochondrial SDH activity being the most sensitive marker studied. Metabolic stimulatory effects induced by cobalt were, therefore, more marked than changes in cytoplasmic and lysosomal membrane permeability.

Sodium lauryl sulphate increases tiludronate paracellular transport using human epithelial caco-2 monolayers

The potential effect of the common pharmaceutical wetting agent, sodium lauryl sulphate (SLS), on the transport of the hydrophilic bisphosphonate, tiludronate, was investigated both by performing physico-chemical determinations of the SLS-tiludronate interaction and by measuring the paracellular transport of tiludronate (Boulenc et al., Biochem. Pharmacol., 46 (1993) 1591–1600) across the in vitro human intestinal epithelium model, i.e., Caco-2. SLS did not affect the contact angles determined with different liquids (glycerin, dioxane, sulphuric acid, water, mercury, heptane and decane) on tiludronate tablets. SLS influenced neither the disintegration of tiludronate tablets nor tiludronate solubility. However, both the efficiency and effectiveness of SLS, in reducing surface tension, were affected by tiludronate. Hence, the presence of 0.48 g/l tiludronate in a water solution changed the efficiency of SLS in reducing the surface tension from 1 to 0.3 g/1. Before evaluating tiludronate transport across Caco-2 monolayers, the reversible (absorption enhancement of orally administered drugs) and irreversible (cell cytotoxicity) effects of SLS on the viability of Caco-2 monolayers were investigated. Following a 1 h exposure of well-differentiated Caco-2 cells to SLS concentrations above 100 mg/l, mitochondrial dehydrogenase activity decreased in a concentration-dependent manner, cytosolic lactate dehydrogenase leakage occurred, mannitol transport was irreversibly increased and a structural separation of the tight junctions was observed by electron microscopy. SLS concentrations up to 80 mg/l did not affect mitochondrial dehydrogenase and cytosolic lactate dehydrogenase activities, while both a reversible increase in mannitol paracellular transport and tight junction opening were observed. Under these incubation conditions, tiludronate paracellular transport was increased in a concentration-dependent manner. These studies demonstrate that SLS increased tiludronate paracellular transport through its specific and transient effect on the permeability of the intercellular space.

Comparative in vitro effects of sodium arsenite and sodium arsenate on neuroblastoma cells

The toxic effects of arsenic at different cellular levels were assessed using two inorganic chemical species: sodium arsenite and sodium arsenate, representing the trivalent and pentavalent states of arsenic, respectively. Mouse neuroblastoma cell cultures (Neuro-2a) were exposed for 24 h, and cytotoxic effects evaluated were: cell proliferation by quantification of total protein content; cytoplasmic membrane integrity to cytosolic lactate dehydrogenase leakage; lysosomal hexosaminidase release; lactate dehydrogenase activity; mitochondrial succinate dehydrogenase activity; relative neutral red uptake by lysosomes; lysosomal hexosaminidase sphingolipid degradation activity; and acetylcholinesterase activity. As(III) was found to be five times more toxic than As(V) to neuroblastoma cell proliferation, but the relative extent of other alterations differed. Special sensitivity was detected for lactate dehydrogenase inhibition. Hexosaminidase activity was also very susceptible, being inhibited at low concentrations and stimulated at high concentrations. Less sensitive were the inhibition of cell proliferation, relative neutral red uptake, and acetylcholinesterase activity. As(III) was lysosomotropic, with secretion of hexosaminidase, but the release was decreased by As(V). Mitochondrial succinate dehydrogenase was inhibited by As(III) and stimulated by As(V). Minor sensitivity to cytoplasmic lactate dehydrogenase leakage for both compounds also shows that functional metabolic alterations produced by arsenic are more important than structural damage.

In vitro effects of mercuric chloride and methylmercury chloride on neuroblastoma cells

An in vitro model system has been developed to establish dose-response relationships of mercuric chloride (HgCl 2) and methylmercuric chloride (HgCH 3Cl). Mouse neuroblastoma cell cultures (Neuro-2a) were exposed for 24 hr and cytotoxic effects evaluated with eight different endpoints. Toxic indicators assessed in the in vitro test system were as follows: cell proliferation by quantification of total protein content; cytoplasmic membrane integrity by cytosolic lactate dehydrogenase leakage; lysosomal membrane stability by hexosaminidase release; lactate dehydrogenase activity; mitochondrial succinate dehydrogenase activity; relative neutral red uptake by lysosomes; lysosomal hexosaminidase sphingolipid degradation activity; acetylcholinesterase activity. The toxicity of the two chemical species of mercury on neuroblastoma cells differed. HgCl 2 inhibited LDH activity specifically and very potently. Gross disruption of cytoplasmic membrane was accompanied by stimulation of hexosaminidase. HgCH 3Cl was 50 times more toxic than HgCl 2 to cell proliferation and also caused important alterations in both membrane stability and metabolic activities over a narrow range of doses. The data suggest that HgCl 2 acts mainly on cell membranes and LDH, whereas, although HgCH 3Cl is more cytotoxic, it does not affect any of the above-mentioned endpoints as specifically.

Toxicity of valproic acid in liver slices from sprague-dawley rats and domestic pigs

The reason for sensitivity to valproic acid (VPA) hepatotoxicity in humans is not known and requires further investigation. We investigated two in vitro animal models that might represent the unpredictably sensitive and the predictably non-sensitive populations of patients. VPA-induced hepatotoxicity was evaluated in vitro using precision-cut liver slices prepared from adult Sprague-Dawley rats and 4-wk-old domestic pigs. Protein synthesis, K + retention and cytosolic lactate dehydrogenase leakage in the slices were used as parameters of viability, with protein synthesis being the most sensitive indicator of viability. Exposure to 300 or 500 μg/ml produced damage in the rat liver slices after 24 hr. However, these VPA concentrations produced damage after 12 hr in slices from rats in which hepatic metabolism had been induced by administering phenobarbital. Damage to liver slices from domestic pigs was more severe than in those from rats. Slices from non-induced pigs showed damage 8 hr after culturing in the presence of 100, 300 or 500 μg VPA/ml. These data suggest that these two animal models may illustrate the different profiles of VPA-induced hepatotoxicity that are seen in the human population.

Isolated rat cardiac myocytes as an experimental tool in the study of anoxic cell injury. Effect of reoxygenation — A preliminary report

Rat cardiac myocytes were isolated by heart perfusion in the presence of collagenase and incubated in the absence or presence of oxygen. As a result of anoxia, there was a gradual increase in plasma membrane permeability, noted as a decrease in trypan blue exclusion frequency, leakage of cytosolic lactate dehydrogenase and intracellular accumulation of the isotope compound 99Tc m-gluconate. The changes in plasma membrane permeability properties were preceded by a marked decrease in cellular ATP level and an increased proportion of contracted myocytes. The ability of the myocytes to resynthesize ATP and to recover from the anoxic injury upon reoxygenation decreased gradually with the length of initial anaerobic incubation during the first 25 min and disappeared after 30 min of anoxia, indicating that the anoxic injury to the isolated rat cardiac myocytes becomes irreversible after 25 – 30 min of anoxia. It is suggested that a decreased energy level is of primary importance for the initiation of cell injury in anoxia and that it is followed by cell contracture and subsequently by a disturbed plasma membrane function, cell swelling and death. This experimental model system of isolated viable rat cardiac myocytes is suitable for problems dealing with reversibility of myocytic injury.

Interrelationships between ATP levels, enzyme leakage, gluconate uptake, trypan blue exclusion and length/width ratio of isolated rat cardiac myocytes subjected to anoxia and reoxygenation

Rat cardiac myocytes were isolated by perfusion of the heart with collagenase and subsequently incubated in the absence or presence of oxygen. As a result of anoxia, there was a gradual increase in plasma membrane permeability, as indicated by a decrease in trypan blue exclusion, leakage of cytosolic lactate dehydrogenase, and intracellular accumulation of the isotope [ 99Tc m]gluconate. These changes in plasma membrane permeability were preceded by a marked decrease in cellular ATP levels and an increased proportion of contracted myocytes. After an initial anaerobic incubation for 10 min, there was complete replenishment of ATP followed by signs of recovery from anoxic cell injury upon reoxygenation. Cell viability could also be followed on the basis of changes in the length/width ratio of myocytes upon anoxia and reoxygenation. The ability of myocytes to resynthesize ATP and to recover from anoxic injury upon reoxygenation decreased in proportion to the length of the initial anaerobiosis during the first 25 min and disappeared completely after 30 min of anoxia. The present model system is a convenient tool for evaluation of interrelationships between energy metabolism, plasma membrane integrity, and the morphology of myocytes subjected to anoxia and reoxygenation.