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J A Post

Publications and source records attributed to J A Post.

At least 19 recordsLinked to original sources
PubMed1999

Validation of the peroxidative indicators, cis-parinaric acid and parinaroyl-phospholipids, in a model system and cultured cardiac myocytes.

cis-Parinaric acid is increasingly being used in eukaryotic cells as a very sensitive marker for the initial stages of lipid peroxidation. Despite the increased application of this probe, no extensive validation, especially in cellular systems, has been performed. cis-Parinaric acid can either be inserted freely into biomembranes or incorporated (bio)synthetically into lipids (parinaroyl-lipid). Therefore, a direct comparison was made between the peroxidative behaviour of the two parinaroyl probes and the endogenous polyunsaturated fatty acids arachidonic and linoleic acid, in both an artificial lipidic system and in cultured neonatal rat heart cells. Three different radical generating systems were used, i.e., hydrogen peroxide, cumene hydroperoxide and the thermo-labile 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH). The data demonstrate that the peroxidation rate of cis-parinaric acid is higher than that of the parinaroyl, arachidonoyl and linoleoyl lipids. The latter three displayed comparable peroxidation rates, showing that the peroxidative decay of parinaroyl-lipid is a good marker for the degradation of endogenous polyunsaturated fatty acids. Experimental results using the freely inserted cis-parinaric acid could potentially lead to an overestimation of the inflicted damage and should be interpreted with care. In addition, a comparison was made with the measurement of conjugated dienes and malon dialdehyde as thiobarbituric acid reactive substances. The results demonstrate that measurement of conjugated dienes and malon dialdehyde only provide information on peroxidative processes in vitro, but are not suitable for in-depth studies in cultured cells. In contrast, the use of the parinaroyl probes is a suitable, straightforward, sensitive and reproducible method for detecting the initial stages of lipid peroxidation in living cells.

Animals↗
PubMed1998

pHe, [Ca2+]e, and cell death during metabolic inhibition: role of phospholipase A2 and sarcolemmal phospholipids.

This study measures cellular lactate dehydrogenase (LDH) release during metabolic inhibition as a monitor of sarcolemmal integrity as affected by variation of external pH (pHe) and Ca2+ concentration ([Ca2+]e). The sigmoidal relationship between pHe and LDH release and pHe and net Ca2+ uptake was essentially identical with the 50% maximal value occurring at pH 7.0 for both. This suggests that a process(es) sensitive to both pHe and [Ca2+]e plays a role in cell lysis during the course of metabolic inhibition. Variation of pHe during metabolic inhibition did not alter the decline in cellular ATP, nor did it affect changes in sarcolemmal phospholipid topology. Intracellular pH followed changes of pHe with a few minutes lag. Cell lysis increased in a graded manner as pHe and [Ca2+]e were increased, but pHe was the sole determinant of lysis, i.e., [Ca2+]e level had no effect, at the lowest (6.2) and the highest (8.0) pHe levels. pHe variation did not affect the release of radiolabeled arachidonic acid, nor did inhibitors of phospholipase A2 (PLA2) affect cell lysis at varying pHe. Therefore, cellular PLA2 activation could not be implicated for a role in cell lysis in the present model of metabolic inhibition. Alternatively, we propose that Ca2+ binding to the cytoplasmic leaflet, in combination with membrane alterations secondary to the metabolic insult, combine to destabilize the sarcolemma (20). This Ca2+ binding to the negatively charged phosphatidylserine results in the expression of the bilayer destabilizing effect of phosphatidylethanolamine. This Ca2+ binding is greatly diminished by lowered pH, resulting in an attenuation of cell lysis.

Adenine Nucleotides↗
PubMed1997

The use of cis-parinaric acid to measure lipid peroxidation in cardiomyocytes during ischemia and reperfusion.

cis-Parinaric acid (PnAc), a fluorescent, polyunsaturated fatty acid, was used to measure lipid peroxidation during simulated ischemia and reperfusion in cultured neonatal rat cardiomyocytes. PnAc was used both as free fatty acid, inserted in the membranes following cultivation of the cells, as well as constituent of the cellular complex lipids by metabolically integrating the fatty acid during growth. In the insertion experiments a pre-incubation with DL-aminocarnitine, an inhibitor of beta-oxidation, was necessary to prevent loss of fluorescent signal. Such a pre-incubation resulted in an enrichment of PnAc in the sarcolemma: In pre-treated cells 57 +/- 1.3% of total inserted PnAc is present in the sarcolemma compared to 27 +/- 5.7% in cells containing the integrated probe. Both methods to introduce PnAc into the cells were compared with respect to their sensitivity for an externally applied oxidative stress and thereafter lipid peroxidation during simulated ischemia and reperfusion was assayed. Going from normoxic to ischemic conditions lipid peroxidation did not increase and remained at a low level. When the ischemic cells were subsequently subjected to reperfusion (reintroduction of both oxygen and glucose), large scale lipid peroxidation was obvious. When, on the other hand, oxygen alone was reintroduced (reoxygenation) no increased lipid peroxidation was observed. These observations led to the conclusion that ischemia does not lead to an enhanced lipid peroxidation and that resumption of metabolic activity during reperfusion is necessary to induce lipid peroxidation.

Animals↗
PubMed1996

Release of proteins from isolated neonatal rat cardiomyocytes subjected to simulated ischemia or metabolic inhibition is independent of molecular mass.

This study addressed the question whether the molecular mass of proteins influences their release from isolated rat neonatal cardiomyocytes subjected to simulated ischemia (SI) or metabolic inhibition (MI). During these interventions cellular ATP content and the relative releases of several proteins, ranging in molecular mass from 15 to 140 kDa, were determined. After 180 min of normoxia, cellular ATP content was about 90% of the initial value, and cellular protein loss was about 1%. During either SI (180 min) or MI (120 min) the cellular ATP content decreased to less than 5% of the initial value. After 180 min of SI the release of soluble cytoplasmic proteins from the cells had increased to about 35%, and after 120 min of MI to about 90%. There were no major differences in the release pattern of four cytoplasmic proteins, during both SI and MI. A soluble mitochondrial and a partly mitochondrial protein, however, showed delayed release patterns. These data indicate that the release of proteins from damaged isolated neonatal rat cardiomyocytes is not related to the molecular mass of the proteins. It is concluded that protein release from damaged cardiomyocytes is not a sieving process in which small proteins are preferentially lost. In contrast, our data suggest that sarcolemmal disruption is a relatively fast process resulting in the simultaneous release of all soluble cytoplasmic proteins, irrespective of their molecular mass.

Adenosine Triphosphate↗
PubMed1996

Cultured neonatal rat heart cells can be preconditioned by ischemia, but not by heat shock. The role of stress proteins.

In this study we attempted to induce tolerance to simulated ischemia in beating cultured neonatal rat cardiomyocytes by subjecting them either to elevated temperatures or to a short period of simulated ischemia. This was done to investigate whether development of tolerance can be observed at the isolated, contracting myocyte level, as has been already described for intact organs: and whether the effect of preconditioning already become apparent during ischemia alone, as opposed to ischemia-reperfusion protocols. We find that no preconditioning can be achieved by a preceeding heat treatment. On the other hand, subjecting the cells to a non-lethal period of ischemia significantly reduces myocyte death during a second more severe ischemic insult. Both pretreatments induce elevated levels of the major fully inducible species of the hsp70 family, hsp68, making it unlikely that the presence of this protein is the sole determinant during preconditioning. The mRNA levels of several heat shock proteins (hsps) are increased by both treatments. However, these mRNAs are induced with different patterns, the most notable difference being the induction of hsp60 mRNA by ischemia, and the absence of this induction by heat shock. We further find that, contrary to some earlier reports, simulated ischemia activates the heat shock transcription factor (HSF) rapidly, as is the case for heat shock.

Animals↗
PubMed1995

Organization and function of sarcolemmal phospholipids in control and ischemic/reperfused cardiomyocytes.

The topic of this review is the lipidic part of the sarcolemma, the plasma membrane of the myocardial cell, and its role in (dis)function of the cardiomyocyte. First the isolation of the sarcolemma and its lipid composition are discussed. These phospholipids are not randomly distributed over the two monolayers of the lipid bilayer and negatively charged phospholipids are exclusively present in the cytoplasmic leaflet of the sarcolemma, which also contains the majority of phosphatidylethanolamine. This distribution is most likely caused by an active transport of these lipids and by an interaction of the headgroup of these lipids with the cytoskeleton. Subsequently the physicochemical properties of sarcolemmal phospholipids are discussed, where it is shown that certain phospholipids prefer non-bilayer phases, and the effects of sarcolemmal phospholipids on trans-sarcolemmal ion fluxes and calcium compartmentation are discussed. In the second part the effect of ischemia on sarcolemmal phospholipids is discussed with regard to: transbilayer distribution, hydrolysis, lateral distribution and sarcolemmal bilayer stability. In our view, onset of ischemia initiates a sequence of events leading to a loss of normal sarcolemmal phospholipid distribution with an outward migration of phosphatidylethanolamine. There follows, as ischemia progresses, loss of sarcolemmal bilayer stability due to the expression of the non-bilayer behavior of phosphatidylethanolamine, leading to irreversible disruption of the sarcolemma and cell death.

Animals↗
PubMed1995

Aggregation of myocardial sarcolemmal transmembrane proteins is not hindered by an interaction with the cytoskeleton. Possible implications for ischemia and reperfusion.

Heart myocytes subjected to ischemia show a clustering of the sarcolemmal proteins. In the erythrocyte membrane, a system in which intramembranous particle (IMP) aggregation is extensively studied, it is found that an IMP aggregation can in principle only occur upon removal of the membrane skeleton of spectrin and actin by rather drastic experimental conditions. With regard to phospholipid composition and topology the sarcolemma and the erythrocyte membrane show large similarities and therefore it was proposed that a loss of the interaction of the IMPs and the cytoskeleton is also a prerequisite for the sarcolemmal IMP aggregation (Verkleij et al., 1990). Freezing myocardial tissue, both from adult and neonatal rat, from temperatures lower than 37 degrees C resulted in an aggregation of the sarcolemmal IMPs. The aggregation is proportional to the degree of lowering of the temperature at which the tissue is cryofixed. This in contrast to the erythrocyte membrane, where lowering the temperature only induces moderate IMP aggregation. The IMP aggregation in the sarcolemma is reversible upon a subsequent increase in incubation temperature. The results clearly demonstrate that the interaction between the sarcolemmal proteins does not hinder aggregation of the IMPs, as proposed previously, and suggest that loosening of this complex does not have to proceed the aggregation of the sarcolemmal intramembranous particles during ischemia.

Age Factors↗
PubMed1995

Phosphatidylethanolamine and sarcolemmal damage during ischemia or metabolic inhibition of heart myocytes.

Phosphatidylethanolamine (PE) is a nonbilayer-preferring and fusogenic phospholipid. It is kept in the bilayer configuration by interaction with other phospholipids in biologic membranes. However, reorganization of the membrane phospholipids could lead to expression of the nonbilayer nature of PE and induce bilayer instability. During ischemia a transbilayer reorganization of sarcolemmal PE is observed, and results have been published that suggest a lateral phase separation in the inner sarcolemmal leaflet phospholipids. These reorganizations and the subsequent expression of the nonbilayer behavior of PE are proposed to form the basis for sarcolemma destabilization and destruction. Lowering the PE content of myocytes, especially of the sarcolemma, is then expected to attenuate myocyte damage after simulated ischemia or metabolic inhibition. Culturing neonatal rat heart myocytes in the presence of N,N-dimethylethanolamine resulted in the synthesis of the bilayer-preferring N,N-dimethyl-PE and a lowering of the ratio between nonbilayer- and bilayer-preferring phospholipids from 0.58 to 0.30. This change in phospholipid composition did not impair cell functioning but did result in a strong attenuation of cell damage on ischemia or metabolic inhibition. A good correlation between the nonbilayer-preferring phospholipid content and the degree of cell damage was obtained (r = 0.98). These results provide further evidence that physicochemical properties of the sarcolemmal phospholipids play a crucial role in the sarcolemmal disruption during prolonged ischemia and/or reperfusion.

Adenosine Triphosphate↗
PubMed1993

A discrete Na+/Ca2+ exchange dependent, Ca2+ compartment in cultured neonatal rat heart cells. Characteristics, localization and possible physiological function.

A pool of calcium has been identified in cultured neonatal rat heart cells whose exchange is dependent on extracellular sodium and calcium. In the absence of extracellular sodium and calcium this pool retains at least 760 mumol calcium/kg dry weight, which is released upon the introduction of sodium and calcium, with a t1/2 of 4.4 s. Video microscopy experiments of non-stimulated cells showed that the cells, despite the retention of 760 mumoles calcium/kg dry weight, do not develop contracture, indicating that the majority of the pool is not retained in the general cytoplasm. Neither is the pool displaced by lanthanum. Several probes for the sarcoplasmic reticulum were used. Ryanodine (1 microM) had no significant effect on the size of the pool. Caffeine reduced the size of the pool by 60% and thapsigargin, an inhibitor of the sarcoplasmic reticulum Ca-ATPase reduced the content of the pool by 70%. High concentrations of ryanodine (1 mM) reduced the pool even further. The experimental data, in association with recently developed concepts of the diadic region [1], indicate that the pool may reside in the sub-sarcolemmal space between the sarcoplasmic reticulum and the sub-sarcolemma. The physiological significance of this pool is discussed.

Animals↗
PubMed1993

Sarcolemmal phospholipid asymmetry and Ca fluxes on metabolic inhibition of neonatal rat heart cells.

The present study examines the hypothesis that during depletion of high-energy phosphates a change will occur in the phospholipid topology and in Ca fluxes in cultured neonatal cells and that these two events may be causally related. A combination of 2-deoxyglucose and iodoacetic acid was used to produce graded changes in the adenine nucleotides in the cells. An on-line technique for 45Ca measurement was used to follow Ca uptake and compartmentation by the cells, and chemical and enzymatic probes were used to study sarcolemmal phospholipid topology. After 15 min of metabolic inhibition (ATP = 10% of control) an increase in cellular Ca occurs, which progresses with time. Over 70% of this Ca accumulates in the mitochondria. After 30 min of metabolic inhibition (ATP < 10% of control) a change in the phospholipid topology is observed, and an increased amount (two times control) of sarcolemmal phosphatidylethanolamine is present in the outer monolayer of the sarcolemma. This change in phospholipid topology was independent of the extracellular Ca concentration. The sequence of altered Ca fluxes and distribution followed by the altered phospholipid topology is discussed in terms of its possible role in the pathogenesis of sarcolemmal disruption.

Animals↗
PubMed1993

Cationic amphiphiles prevent calcium leak induced by ATP depletion in myocardial cells.

Excessive calcium influx is important in the irreversible injury of cardiac myocytes and other cell types. The mechanism is unknown, but possibilities include L-type channels, Na(+)-Ca2+ exchange, sarcolemmal (SL) defects, and calcium leak channels. In this study, metabolic inhibition was used to induce ATP depletion and augmented calcium influx in cultured cardiac myocytes. Inhibition of the L-type calcium channel and Na(+)-Ca2+ exchanger had no significant effect on the calcium leak. There was no significant lactate dehydrogenase release, indicating that the leak did not occur through major SL defects. No alterations in the asymmetric distribution of SL phospholipids were demonstrated. Phospholipid rearrangements were therefore not responsible. The leak was unaffected by 0.5 mM cadmium and 1 microM nifedipine but was augmented by 50 microM nifedipine, characteristics in common with calcium leak channels. Insertion of the cationic amphiphiles dodecyltrimethylammonium bromide or polymyxin B sulfate into the SL had a profound inhibitory effect on the calcium leak. The anionic amphiphile sodium dodecyl sulfate had the opposite effect, and the neutral amphiphile lauryl acetate had no effect. These results suggest that an alteration in the SL surface charge affects calcium leak. It is proposed that the augmented calcium influx occurs via calcium leak channels and that these can be modulated by charged amphiphiles.

Adenosine Triphosphate↗