Biomedical subjects

E M Bastiaanse

Publications and source records attributed to E M Bastiaanse.

9 recordsLinked to original sources

PubMed1995

Metabolic inhibition of cardiomyocytes causes an increase in sarcolemmal fluidity which may be due to loss of cellular cholesterol.

We examined whether metabolic inhibition (5 mM NaCN + 10 mM 2-deoxyglucose) affects sarcolemmal fluidity in cultured neonatal cardiomyocytes. As a measure of sarcolemmal fluidity we determined the fluorescence steady-state anisotropy (rss, which is reciprocally related to membrane fluidity) of cardiomyocytes labeled with 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene, p-toluenesulfonate. During metabolic inhibition, membrane fluidity increased progressively: after 30 min rss had fallen by 6.7 +/- 1.2% (mean +/- SE; n = 9; P < 0.05) compared to baseline values, and after 90 min by 14.5 +/- 3.5% (P < 0.05; n = 5). Beyond 90 min rss did not decrease any further. During control incubations (without metabolic inhibition), no significant changes in rss were observed. During metabolic inhibition cellular free cholesterol content declined: after 30 min free cholesterol content had decreased by 12.2 +/- 3.1% (P < 0.02; n = 4), compared to baseline values, and after 90 min by 31.1 +/- 8.3% (P < 0.02; n = 4). We conclude that metabolic inhibition induces an increase in sarcolemmal fluidity, which may be caused by a decrease in sarcolemmal free cholesterol content.

Animals↗

PubMed1995

Glutathione disulfide as an index of oxidative stress during postischemic reperfusion in isolated rat hearts.

The objectives of this study were to determine 1) whether reactive oxygen species generated upon postischemic reperfusion lead to oxidative stress in rat hearts, and 2) whether an exogenous prooxidant present in the early phase of reperfusion causes additional injury. Isolated buffer-perfused rat hearts were subjected to 30 min of hypothermic no-flow ischemia followed by 30 min of reperfusion. Increased myocardial content of glutathione disulfide (GSSG) and increased active transport of GSSG were used as indices of oxidative stress. To impose a prooxidant load, cumene hydroperoxide (20 microM) was administered during the first 10 min of reperfusion to a separate group of postischemic hearts. Reperfusion after 30 min of hypothermic ischemia resulted in a recovery of myocardial ATP from 28% at end-ischemia to 50-60%, a release of 5% of total myocardial LDH, and an almost complete recovery of both coronary flow rate and left ventricular developed pressure. After 5 and 30 min of reperfusion, neither myocardial content of GSSG nor active transport of GSSG were increased. These indices were increased, however, if cumene hydroperoxide was administered during early reperfusion. After stopping the administration of cumene hydroperoxide, myocardial GSSG content returned to control values and GSH content increased, indicating an unimpaired glutathione reductase reaction. Despite the induction of oxidative stress, reperfusion with cumene hydroperoxide did not cause additional metabolic, structural, or functional injury when compared to reperfusion without cumene hydroperoxide. We conclude that reactive oxygen species generated upon postischemic reperfusion did not lead to oxidative stress in isolated rat hearts. Moreover, even a superimposed prooxidant load during early reperfusion did not cause additional injury.

Adenine Nucleotides↗

PubMed1995

Role of calcium-activated neutral protease (calpain) in cell death in cultured neonatal rat cardiomyocytes during metabolic inhibition.

Calcium-activated neutral protease (CANP), also known as calpain, has been implicated in the development of cell death in ischemic hearts. CANP is thought to be activated by the calcium overload that develops during ischemia. We studied the involvement of CANP in cell death in cultured neonatal rat cardiomyocytes during metabolic inhibition (5 mmol/L NaCN + 10 mmol/L 2-deoxyglucose). First, we isolated CANP using ion exchange and affinity chromatography. Then the efficacy of the CANP inhibitors calpain I inhibitor, leupeptin, and E64 to inhibit isolated CANP activity was tested with the use of fluorescently labeled beta-casein as a substrate. The IC50 for the inhibitors was between 2.1 and 56 mumol/L. Uptake of the inhibitors by intact cells was assessed with the use of 99mTc-radiolabeled inhibitors. The calculated intracellular inhibitor concentrations were sufficiently high to yield substantial inhibition of intracellular CANP activity. Intracellular CANP activity was measured directly with the use of the cell-permeant fluorogenic CANP-specific substrate N-succinyl-Leu-Leu-Val-Tyr-7-amido-4-methyl-coumarin. During metabolic inhibition, intracellular CANP activity was increased compared with control incubation. The time course of CANP activation was compatible with that of the rise in [Ca2+]i, as measured by fura 2 and digital imaging fluorescence microscopy. Calpain I inhibitor and leupeptin inhibited intracellular CANP activity both during metabolic inhibition and control incubation, whereas E64 did not. Despite their substantial inhibition of intracellular CANP activity, calpain I inhibitor and leupeptin did not attenuate cell death during metabolic inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Separation of VLDL subfractions by density gradient ultracentrifugation.

To assess the presence and composition of very-low-density lipoprotein (VLDL) in various types of hyperlipoproteinemia, a method of density gradient ultracentrifugation has been developed. After 2 hours of density gradient ultracentrifugation, human serum VLDL is separated into two distinct VLDL cholesterol peaks (VLDL1 and VLDL2). The two VLDL subfractions were detected in the serum samples from all subjects in the study, including subjects with normolipidemia (n = 10), familial dysbetalipoproteinemia (n = 12), and type IIa (n = 8), type IIb (n = 12), and type IV/V (n = 10) hyperlipoproteinemia. The cholesterol profiles obtained by the density gradient ultracentrifugation technique resembled the band patterns after electrophoresis of identical serum samples on 2% to 16% nondenaturing polyacrylamide gradient gel: VLDL1 represents relatively large VLDL particles (diameter of about 67 nm) and VLDL2 represents relatively small VLDL particles (diameter of about 38 nm). Recentrifugation of isolated VLDL1 and isolated VLDL2 did not result in any change in their density distribution. In all groups studied, the fluidity of VLDL1 was significantly higher than that of VLDL2, in accordance with the finding that VLDL1 particles were relatively rich in triglycerides and VLDL2 particles were relatively rich in cholesteryl esters. These results indicate that the two VLDL subfractions isolated represent distinct VLDL subclasses. The density gradient ultracentrifugation technique presented in this study allows the rapid isolation and characterization of VLDL subfractions from the serum samples of normolipidemic individuals and patients with hyperlipoproteinemia.

Adult↗

PubMed1994

The effect of sarcolemmal cholesterol content on intracellular calcium ion concentration in cultured cardiomyocytes.

In this study the relationship between sarcolemmal free cholesterol content and intracellular calcium ion concentration ([Ca2+]i) was explored. In cultured neonatal rat cardiomyocytes the cellular free cholesterol content was modulated by treatment with liposomes. Using cholesterol-rich or cholesterol-free liposomes, sarcolemmal free cholesterol content was raised or diminished, respectively. An increased sarcolemmal free cholesterol content resulted in a decreased sarcolemmal fluidity, whereas cholesterol depletion resulted in an increase in sarcolemmal fluidity. Cholesterol enrichment was associated with an increased [Ca2+]i, while cholesterol depletion resulted in a decreased [Ca2+]i. The membrane mobilizing agent 2-(2-methoxyethoxy)ethyl 8-(cis-2-c-octylcyclopropyl)-octanoate (A2C) caused an increase in sarcolemmal fluidity, and an increased [Ca2+]i. Thus, although sarcolemmal cholesterol depletion as well as A2C treatment increased sarcolemmal fluidity, their effects on [Ca2+]i are opposite. These results indicate that the effect of sarcolemmal free cholesterol content on [Ca2+]i is not mediated by sarcolemmal fluidity. The mechanisms responsible for the observed results are: (i) activated Ca2+ channels when the sarcolemma is enriched with cholesterol, (ii) most likely a stimulated Ca(2+)-ATPase activity when the sarcolemma is depleted of cholesterol, and (iii) inhibited Na+/Ca2+ exchanger activity when A2C is incorporated in the sarcolemma.

Animals↗

PubMed1994

Simvastatin-sodium delays cell death of anoxic cardiomyocytes by inhibition of the Na+/Ca2+ exchanger.

When incubated under anoxic conditions, cultured neonatal cardiomyocytes undergo cell necrosis. Simvastatin-sodium, the bioactive metabolite of simvastatin (a potent serum cholesterol-lowering drug), delayed the anoxia-induced myocyte necrosis in a dose-dependent manner. This beneficial effect of simvastatin-sodium could not be attributed to its cholesterol-lowering properties. We found that simvastatin-sodium, at concentrations of 20 and 50 microM, attenuated the rise in intracellular Ca2+ concentration ([Ca2+]i) measured with Fura-2 in anoxic cardiomyocytes. In a test of sarcolemmal Na+/Ca2+ exchange activity, simvastatin-sodium attenuated the rise of [Ca2+]i upon incubation in sodium-free buffer, which normally causes a reversal of Na+/Ca2+ exchange and cellular calcium overload. The inhibitory action of simvastatin-sodium on the sarcolemmal Na+/Ca2+ exchanger could well explain the cardioprotective effect of the drug on myocytes subjected to anoxia.

Animals↗

PubMed1994

The effect of sarcolemmal cholesterol content on the tolerance to anoxia in cardiomyocyte cultures.

To determine whether the sarcolemmal-free cholesterol content influences the tolerance to anoxia in cultured neonatal rat cardiomyocytes, we modulated the free cholesterol content of the cultures, and determined the time course of anoxia-induced cell death. Incubation for 5 h with liposomes having a free cholesterol to phospholipid molar ratio of 2, 0.5 and 0, resulted in a change of cellular free cholesterol content by +54.7 +/- 5.8% (P < 0.001), by -5.2 +/- 6.3% (n.s.), and by -22.1 +/- 4.9% (P < 0.01), respectively, when compared to cultures incubated without liposomes (control). In cells which were loaded with cholesterol, it was verified that the extra cholesterol was transferred predominantly to the sarcolemma, which was associated with a decrease in sarcolemmal fluidity. In cells which underwent cholesterol reduction it was verified that free cholesterol was retracted selectively from the sarcolemma, associated with an increase in sarcolemmal fluidity. Cellular enrichment with free cholesterol caused a delay of anoxia-induced release of lactate dehydrogenase (LDH): the time at which half-maximal release of LDH was reached (LDH50%) occurred later by 35.5 +/- 3.4 min (P < 0.001) compared to anoxic control cultures. Cholesterol reduction diminished the tolerance to anoxia: LDH50% occurred earlier by 36.4 +/- 7.8 min (P < 0.01), compared to anoxic control cultures. Cultures which were incubated with liposomes having a free cholesterol to phospholipid molar ratio of 0.5 had unchanged sarcolemmal free cholesterol content, unchanged sarcolemmal fluidity, and an unchanged LDH50% during anoxia in comparison to control cultures, excluding any effect of the incubation with liposomes per se. The present study indicates that the sarcolemmal free cholesterol content of cardiomyocytes is a determinant of their tolerance to anoxia.

Animals↗

PubMed1994

A novel two-compartment culture dish allows microscopic evaluation of two different treatments in one cell culture simultaneously. Influence of external pH on Na+/Ca2+ exchanger activity in cultured rat cardiomyocytes.

A new type of culture dish containing two separate compartments is described, that can be used in high-magnification microscopy. Using the dish, two halves of a single-cell culture, grown on a standard coverslip, can be exposed to different treatments simultaneously, allowing the effect of one treatment to be compared with that of the other treatment in the same culture. This way, the natural variability that might exist between different individual cultures is circumvented. In addition, by simultaneously conducting two experiments per dish, the number of experiments needed can be decreased. This both reduces the time to complete a series of experiments and allows the optimal use of specimens that are difficult to obtain, such as human material. We found there is an excellent barrier between the two compartments for lipophilic and hydrophilic compounds, and for low-molecular-mass cations. To illustrate the use of the dish we describe the influence of external pH on the activity of the Na+/Ca2+ exchanger in intact cultured neonatal rat ventricular cardiomyocytes. The intracellular free calcium concentration ([Ca2+]i) in the cardiomyocytes, measured using fura-2 and imaging fluorescence microscopy, was studied during sodium-free incubation. The resulting rise in [Ca2+]i at pH 7.4 in one compartment was compared with that in the other compartment in which the pH was either 6.0, 7.0, 7.4 or 8.0. It was found that below pH 7.4, Na+/Ca2+ exchanger activity was diminished, whereas at pH higher than 7.4 the Na+/Ca2+ exchanger activity was increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Heptanol-induced decrease in cardiac gap junctional conductance is mediated by a decrease in the fluidity of membranous cholesterol-rich domains.

To assess whether alterations in membrane fluidity of neonatal rat heart cells modulate gap junctional conductance (gj), we compared the effects of 2 mM 1-heptanol and 20 microM 2-(methoxy-ethoxy)ethyl 8-(cis-2-n-octylcyclopropyl)-octanoate (A2C) in a combined fluorescence anisotropy and electrophysiological study. Both substances decreased fluorescence steady-state anisotropy (rss), as assessed with the fluorescent probe 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) by 9.6 +/- 1.1% (mean +/- SEM, n = 5) and 9.8 +/- 0.6% (n = 5), respectively, i.e., both substances increased bulk membrane fluidity. Double whole-cell voltage-clamp experiments showed that 2 mM heptanol uncoupled cell pairs completely (n = 6), whereas 20 microM A2C, which increased bulk membrane fluidity to the same extent, did not affect coupling at all (n = 5). Since gap junction channels are embedded in relatively cholesterol-rich domains of the membrane, we specifically assessed the fluidity of the cholesterol-rich domains with dehydroergosterol (DHE). Using DHE, heptanol increased rss by 14.9 +/- 3.0% (n = 5), i.e., decreased cholesterol domain fluidity, whereas A2C had no effect on rss (-0.4 +/- 6.7%, n = 5). Following an increase of cellular "cholesterol" content (by loading the cells with DHE), 2 mM heptanol did not uncouple cell pairs completely: gj decreased by 80 +/- 20% (range 41-95%, n = 5). The decrease in gj was most probably due to a decrease in the open probability of the gap junction channels, because the unitary conductances of the channels were not changed nor was the number of channels comprising the gap junction. The sensitivity of nonjunctional membrane channels to heptanol was unaltered in cholesterol-enriched myocytes. These results indicate that the fluidity of cholesterol-rich domains is of importance to gap junctional coupling, and that heptanol decreases gj by decreasing the fluidity of cholesterol-rich domains, rather than by increasing the bulk membrane fluidity.

Alcohols↗