Clinical concentrations of doxorubicin inhibit activity of myocardial membrane-associated, calcium-independent phospholipase A(2).

Use of the anticancer antibiotic doxorubicin continues to be limited by its cumulative dose-related cardiotoxicity. Our study reports inhibition of myocardial intracellular calcium-independent phospholipase A(2) (iPLA(2)) activity by clinically relevant concentrations of the drug. The effect was first shown in vitro using suspensions of freshly isolated rat and rabbit cardiomyocytes. Addition of 0.1-10 microM doxorubicin to these cells led to a concentration- and time-dependent inhibition of total iPLA(2), as measured using (16:0, [(3)H]18:1) plasmenylcholine and phosphatidylcholine substrates in the presence or absence of calcium. Subcellular fractionation into cytosolic and membrane fraction revealed that the drug selectively inhibits membrane-associated iPLA(2) activity, without altering activity of the cytosolic enzyme. Doxorubicin treatment of cells prelabeled with [H(3)]arachidonic acid led to a depression of baseline arachidonic acid release levels, corroborating iPLA(2) inhibition. Reducing agents blocked PLA(2) inhibition in cardiomyocyte suspensions, suggesting that the doxorubicin effect is mediated by oxidation of susceptible cysteines. In vivo experiments, in which adults rats were i.v. injected with a bolus dose of 4 mg/kg doxorubicin, confirmed in vitro findings, revealing a 2-fold decrease in membrane-associated Ca(2+)-independent iPLA(2) activity in the heart tissue of treated animals. The observed phenomenon has important implications for myocyte signaling cascades and membrane remodeling.

Localized injury in cardiomyocyte network: a new experimental model of ischemia-reperfusion arrhythmias.

We developed a new experimental approach to study the effects of local injury in a multicellular preparation and tested the ability of the method to induce reperfusion arrhythmias in cardiomyocyte monolayers. A small region of injury was created using geometrically defined flows of control and ischemia-like solutions. Calcium transients were acquired simultaneously from injured, control, and border zone cells using fluo 4. Superfusion with the injury solution rapidly diminished the amplitude of calcium transients within the injury zone, followed by cessation of cell beating. Reperfusion caused an immediate tachyarrhythmic response in approximately 17% of experiments, with a wave front propagating from a single cell or small cell cluster within the former injury zone. Inclusion of a gap junction uncoupler (1 mM heptanol) in the injury solution narrowed the functional border and sharply increased the number of ectopic foci and the incidence of reperfusion arrhythmias. The model holds a potential to reveal both micro- and macroscopic features of propagation, conduction, and cell coupling in the normal and diseased myocardium and to serve as a new tool to test antiarrhythmic protocols in vitro.

Oxidant-induced inhibition of myocardial calcium-independent phospholipase A2.

We discovered the acute inhibition of myocardial phospholipase A2 activity by micromolar concentrations of tert-butyl hydroperoxide and hydrogen peroxide. Specifically, freshly isolated adult rat cardiomyocytes were treated with the oxidants for 30 min, and phospholipase A2 activity was assessed in cell subcellular fractions using (16:0, [3H]18:1) plasmenylcholine and phosphatidylcholine substrates in the absence or presence of calcium. Calcium-independent phospholipase A2 activity was inhibited by approx 50% in both the cytosolic and membrane fractions by the oxidants. The inhibition of the phospholipase A2 activity was concentration dependent and preceded detectable changes in cell viability as assessed by lactate dehydrogenase release and rod-shaped morphology. Taking into account that oxidized sn-2 fatty acyl groups must first be hydrolyzed by phospholipase A2 to be repaired by glutathione peroxidase, we suggest that the observed inhibition of phospholipase A2 activity by oxidants compromises the myocyte's ability to deal with lipid peroxidation. This conclusion was further validated by the experiments in which pretreatment with the calcium-independent phospholipase A2 inhibitor bromoenol lactone exacerbated cardiotoxicity of tert-butyl hydroperoxide in myocyte cultures.

Localization of dichlorofluorescin in cardiac myocytes: implications for assessment of oxidative stress.

Localization and staining features of the oxidant-sensitive fluorescent probe 2'7'-dichlorofluorescin (DCFH) were evaluated in isolated cardiac muscle cells. Cardiomyocytes rapidly accumulated the probe and retained steady levels of DCFH and its highly fluorescent oxidized product dichlorofluorescein (DCF) in probe-free medium for 1.5 h. DCF was associated with mitochondria and was released by the proton ionophore carbonyl cyanide m-chlorophenylhydrazone but not by saponin, which permeabilizes the plasma membrane. A mitochondrial distribution of DCF was also suggested by experiments with the mitochondrial marker MitoTracker Red, in which quenching was observed between DCF and MitoTracker Red in live cells. Isolated cardiac mitochondria rapidly accumulated DCF, and high micromolar concentrations of the probe inhibited ADP-stimulated respiration rate. The study provides an information base essential for the interpretation and design of experiments with DCF as a marker of oxidative stress in cardiac muscle and reveals preferential localization of the probe in mitochondria.

Interactions between benzylamiloride and fura-2: studies in vitro and in cardiac myocytes.

Amiloride derivatives are commonly used inhibitors of Na+/H+- and Na+/Ca2+-exchange. Because they are fluorescent molecules the use of benzylamiloride (BZA), an inhibitor of Na+/Ca2+ exchange, in conjunction with Fura-2, a commonly used fluorescent Ca2+ indicator, might complicate interpretation of fluorescence data obtained. In vitro data show that BZA decreases the Fura-2 fluorescence at all useful wavelengths in a concentration-dependent manner. The Fura-2 ratio 340/380 (used to estimate intracellular Ca2+ ([Ca2+]in)) also decreased with increasing BZA concentrations. The Stern-Volmer relation suggests that this phenomenon is due to either static or dynamic quenching. Varying temperatures from 4 to 37 degreesC did not alter Stern-Volmer constants, consistent instead with fluorescence resonance energy transfer (FRET). The in situ relevance of these interactions was evaluated in adult rat cardiac myocytes which exhibit Na+/Ca2+ exchange reflected by rapid [Ca2+]in increase following Na+ removal. Pretreatment with BZA >/= 25 microM decreased the magnitude of Fura-2 changes induced by Na+ removal. Analysis of the individual Fura-2 useful wavelengths indicated that >/= 25 microM BZA altered the Fura-2 signal in a manner consistent with the quenching effects noted in vitro. Together, these data show that BZA interacts with Fura-2 in vitro and in situ and suggest caution when interpreting Fura-2 fluorescence data derived in conjunction with BZA.

A new approach to assess viability of adult cardiomyocytes: computer-assisted image analysis.

OBJECTIVE: to develop a computerized procedure to obtain the percentage of rod-shaped cells in preparations of isolated adult cardiomyocytes. METHODS: (1) isolated adult rat myocytes were either pretreated with a fixative (0.2% glutaraldehyde, 0.25% Triton X-100 and 0.1% trypan blue) or monitored via phase-contrast optics without fixation; (2) an image field with several hundred cells was captured by a microscope-mounted video camera, which was connected to a frame grabber, and saved as a TIFF file; (3) the image was processed using Mocha software. Analysis consisted of setting the image threshold, an object count, automatic measurements of objects and their classification. RESULTS: though the software could measure many geometrical characteristics, a combination of two parameters, object size and shape factor, was found to be the most efficient. With these parameters, four classes of sample objects were constructed including rod-shaped myocytes, round damaged cells, overlapping cell clusters, and small debris. Test objects were classified automatically based on which sample object their parameters matched the most. Excellent correlation was obtained between manual counts and computer analysis of cell viability (r=0.98). CONCLUSION: a more efficient method, based on computer-assisted classification of objects, has been developed to assess the viability of isolated adult cardiomyocytes.

A point mutation in Galphao and Galphai1 blocks interaction with regulator of G protein signaling proteins.

Regulator of G protein-signaling (RGS) proteins accelerate GTP hydrolysis by Galpha subunits and are thought to be responsible for rapid deactivation of enzymes and ion channels controlled by G proteins. We wanted to identify and characterize Gi-family alpha subunits that were insensitive to RGS action. Based on a glycine to serine mutation in the yeast Galpha subunit Gpa1(sst) that prevents deactivation by Sst2 (DiBello, P. R., Garrison, T. R., Apanovitch, D. M., Hoffman, G., Shuey, D. J., Mason, K., Cockett, M. I., and Dohlman, H. G. (1998) J. Biol. Chem. 273, 5780-5784), site-directed mutagenesis of alphao and alphai1 was done. G184S alphao and G183S alphai1 show kinetics of GDP release and GTP hydrolysis similar to wild type. In contrast, GTP hydrolysis by the G --> S mutant proteins is not stimulated by RGS4 or by a truncated RGS7. Quantitative flow cytometry binding studies show IC50 values of 30 and 96 nM, respectively, for aluminum fluoride-activated wild type alphao and alphai1 to compete with fluorescein isothiocyanate-alphao binding to glutathione S-transferase-RGS4. The G --> S mutant proteins showed a greater than 30-100-fold lower affinity for RGS4. Thus, we have defined the mechanism of a point mutation in alphao and alphai1 that prevents RGS binding and GTPase activating activity. These mutant subunits should be useful in biochemical or expression studies to evaluate the role of endogenous RGS proteins in Gi function.

Determinants of gi1alpha and beta gamma binding. Measuring high affinity interactions in a lipid environment using flow cytometry.

G protein heterocomplex undergoes dissociation and association during its functional cycle. Quantitative measurements of alpha and betagamma subunit binding have been difficult due to a very high affinity. We used fluorescence flow cytometry to quantitate binding of fluorescein-labeled Gi1alpha (F-alpha) to picomolar concentrations of biotinylated G beta gamma. Association in Lubrol solution was rapid (kon = 0.7 x 10(6) M-1 s-1), and equilibrium binding revealed a Kd of 2.9 +/- 0.8 nM. The binding showed a complex dependence on magnesium concentration, but activation of F-alpha with either GDP/aluminum fluoride or guanosine 5'-O-(3-thiotriphosphate) completely prevented formation of the heterocomplex (Kd > 100 nM). The binding was also influenced by the detergent or lipid environment. Unlabeled betagamma reconstituted in biotinylated phospholipid vesicles (pure phosphatidylcholine or mixed brain lipids) bound F-alpha approximately 2-3-fold less tightly (Kd = 6-9 nM) than in Lubrol. In contrast, beta gamma in ionic detergents such as cholate and 3-[(cholamidopropyl)diethylammonio]-1-propanesulfonate exhibited substantially lower affinities for F-alpha. Dissociation of F-alpha from beta gamma reconstituted in lipid vesicles was observed upon addition of aluminum fluoride or excess unlabeled alpha subunit, indicating that myristoylated alpha subunit has only a weak interaction with lipids without the beta gamma subunit. The kinetics of aluminum fluoride-stimulated dissociation were slower than those of the alpha subunit conformational change detected by intrinsic fluorescence. These results quantitatively demonstrate G protein subunit dissociation upon activation and provide a simple but powerful new approach for studying high affinity protein/protein interactions in solution or in a lipid environment.

Effects of oxidants on properties of fluorescent calcium indicators.

An increasing number of studies use calcium-sensitive fluorescent dyes to address the relationship between elevated levels of intracellular calcium and free-radical-mediated damage in a variety of pathophysiological phenomena. The present study evaluates the effects of reactive oxygen species on the spectral properties of widely used calcium probes such as Fura-2 and Fluo-3. We found that both Fura-2 and Fluo-3 are rapidly inactivated by hydroxyl radicals and enzymatically inactivated by peroxidase/H2O2. This results in a decrease in the dynamic range of sensitivity of both dyes to Ca2+, as well as in a decrease in the affinity of Fluo-3 for Ca2+. The data suggest that oxidation of the calcium probes affects the measurement of calcium in vitro and may alter the interpretation of in vivo data since the absence of or small changes in the calcium fluorescence signal can be the result of probe deactivation by free oxygen radicals rather than the lack of actual Ca2+ changes.

Ligand-sensitive interactions among the transmembrane helices of Na+/K+-ATPase.

An extensively trypsin-digested Na+/K+-ATPase, which retains the ability to bind Na+, K+, and ouabain, consists of four fragments of the alpha-subunit that contain all 10 transmembrane alpha domains, and the beta-subunit, a fraction of which is cleaved at Arg142-Gly143. In previous studies, we solubilized this preparation with a detergent and mapped the relative positions of several transmembrane helices of the subunits by chemical cross-linking. To determine if these detected helix-helix proximities were representative of those existing in the bilayer prior to solubilization, we have now done similar studies on the membrane-bound preparation of the same digested enzyme. After oxidative sulfhydryl cross-linking catalyzed by Cu2+-phenanthroline, two prominent products were identified by their mobilities and the analyses of their N termini. One was a dimer of a 11-kDa alpha-fragment containing the H1-H2 helices and a 22-kDa alpha-fragment containing the H7-H10 helices. This dimer seemed to be the same as that obtained in the solubilized preparation. The other product was a trimer of the above two alpha-fragments and that fraction of beta whose extracellular domain was cleaved at Arg142-Gly143. This product was different from a similar one of the solubilized preparation in that the latter contained the predominant fraction of beta without the extracellular cleavage. The cross-linking reactions of the membrane preparation, but not those of the solubilized one, were hindered specifically by Na+, K+, and ouabain. These findings indicate that (a) the H1-H2 transmembrane helices of alpha are adjacent to some of its H7-H10 helices both in solubilized and membrane-bound states, (b) the alignment of the residues of the single transmembrane helix of beta with the interacting H1-H2 and H7-H10 helices of alpha is altered by detergent solubilization and by structural changes in the extracellular domain of beta, and (c) the three-dimensional packing of the interacting transmembrane helices of alpha and beta are regulated by the specific ligands of the enzyme.

Visualization of doxorubicin-induced oxidative stress in isolated cardiac myocytes.

The paper presents high-resolution fluorescence images obtained using laser-scanning confocal microscopy. Isolated cells from adult rat hearts were preloaded with 2',7'-dichlorofluorescin (an oxidant-sensitive fluorescent probe) and exposed to doxo- rubicin, an important anticancer drug with prominent cardioxicity. Fluorescence images were collected from live cells simultaneously on two channels: 1) 515-530 nm emission range was used to monitor an increase in dichlorofluorescein, the oxidized product of dichlorofluorescein, and 2) emission > 610 nm was used to visualize the intracellular distribution of doxorubicin. The images reveal intracellular oxidation close to the mitochondria after only 20 min of exposure of isolated cardiomyocytes to 40-160 microM doxorubicin. The data confirm an oxidative mechanism of doxorubicin cardiotoxicity and demonstrate the capability of a new technique to monitor intracellular oxidation in living cardiomyocytes.

Intersubunit and intrasubunit contact regions of Na+/K(+)-ATPase revealed by controlled proteolysis and chemical cross-linking.

To identify interfaces of alpha- and beta-subunits of Na+/K(+)-ATPase, and contact points between different regions of the same alpha-subunit, purified kidney enzyme preparations whose alpha-subunits were subjected to controlled proteolysis in different ways were solubilized with digitonin to disrupt intersubunit alpha,alpha-interactions, and oxidatively cross-linked. The following disulfide cross-linked products were identified by gel electrophoresis, staining with specific antibodies, and N-terminal analysis. 1) In the enzyme that was partially cleaved at Arg438-Ala439, the cross-linked products were an alpha,beta-dimer, a dimer of N-terminal and C-terminal alpha fragments, and a trimer of beta and the two alpha fragments. 2) From an extensively digested enzyme that contained the 22-kDa C-terminal and several smaller fragments of alpha, two cross-linked products were obtained. One was a dimer of the 22-kDa C-terminal peptide and an 11-kDa N-terminal peptide containing the first two intramembrane helices of alpha (H1-H2). The other was a trimer of beta, the 11-kDa, and the 22-kDa peptides. 3) The cross-linked products of a preparation partially cleaved at Leu266-Ala267 were an alpha,beta-dimer and a dimer of beta and the 83-kDa C-terminal fragment. Assuming the most likely 10-span model of alpha, these findings indicate that (a) the single intramembrane helix of beta is in contact with portions of H8-H10 intramembrane helices of alpha; and (b) there is close contact between N-terminal H1-H2 and C-terminal H8-H10 segments of alpha; with the most probable interacting helices being the H1,H10-pair and the H2,H8-pair.

A comparative study of Na+/K(+)-ATPases of duck salt gland and canine kidney: implications for the enzyme's reaction mechanism.

Highly purified preparations of duck salt gland and canine kidney Na+/K(+)-ATPases with comparable specific activities were used to clarify the causes of previously reported differences between the substrate-velocity curves of these enzymes. When assays were done under identical conditions (pH 7.4; 37 degrees C), and a wide range of closely spaced ATP concentrations were used, the curves of both enzymes exhibited intermediary plateaus, as noted before for the salt gland enzyme. The two enzymes also had the same numbers of phosphorylation and ouabain binding sites, and their catalytic subunits were of the alpha 1 isoform type as revealed by immunostaining with specific antibodies. The findings suggest that the substrate-velocity curves of all widely used Na+/K(+)-ATPases may contain an intermediary plateau which is diagnostic of reaction mechanisms that generate rate equations containing powers of substrate concentration greater than two, e.g., a mechanism involving an oligomer with more than two protomers.

Role of intracellular SOD in oxidant-induced injury to normal and copper-deficient cardiac myocytes.

Previous studies have shown that susceptibilities of hepatocytes and endothelial cells to H2O(2)-induced injury are altered by changes in the intracellular activity of Cu,Zn-containing superoxide dismutase (CuZn-SOD). To evaluate the role of intracellular CuZn-SOD in oxidant-induced injury to rat cardiac myocytes, cells with reduced CuZn-SOD activity but normal ATP content were either isolated from the hearts of adult copper-deficient rats or obtained by treatment of normal isolated adult myocytes with diethyldithiocarbamate. These myocytes and controls with normal CuZn-SOD activity were exposed to either reagent H2O2 or oxidants generated by extracellular glucose oxidase plus glucose or xanthine oxidase plus xanthine. It was shown that myocytes with CuZn-SOD activities reduced by 70-90% were equally susceptible to H2O2 and the two oxidant-generating systems as the control myocytes. The findings suggest that in adult cardiac myocytes, in contrast to the situation in some other cells, intracellular CuZn-SOD may not have a significant defensive role against acute H2O(2)-induced injury. The possibility remains, however, that changes in the activity of this enzyme, e.g., in copper deficiency, may be relevant to the ability of myocytes to cope with chronic oxidative stress resulting from imbalance between intracellular oxygen radical-generating and -scavenging systems.

Effects of doxorubicin on cardiomyocytes with reduced level of superoxide dismutase.

Our previous studies have shown that isolated adult rat cardiomyocytes with normal and reduced Cu/Zn SOD activities are equally susceptible to extracellularly generated oxidants (hydrogen peroxide, glucose oxidase/glucose and xanthine oxidase/xanthine systems). In the present study we exposed myocytes with reduced SOD activity to doxorubicin (adriamycin). Cardiotoxicity of doxorubicin has been attributed to the production of superoxide anion inside the cell. Cardiomyocytes with reduced SOD activity, but normal ATP content and viability, were obtained by the treatment of isolated cells with diethyldithiocarbamate (DDC). DDC-treated myocytes were significantly less resistant to doxorubicin than controls. Doxorubicin-stimulated superoxide anion formation, measured by the rate of SOD-inhibitable acetylated cytochrome C reduction, was significantly higher in the cytosolic fraction of DDC-treated cells compared to controls. These results indicate that for isolated cardiac myocytes an essential part of cytotoxicity of doxorubicin can be explained by the formation of superoxide anion and that the level of intracellular SOD activity should be considered as a significant factor for cell protection.

Differential regulation of superoxide dismutase in copper-deficient rat organs.

The effects of dietary copper deprivation on the activities, immunoreactive protein concentrations, and mRNA abundance of copper/zinc- and manganese-superoxide dismutase (Cu/Zn- and Mn-SOD) were examined in liver, heart, and brain of weanling rats fed a Cu-deficient diet for 4 weeks. Hepatic Cu/Zn-SOD activity, enzyme content, and mRNA abundance were significantly reduced, and, conversely, the activity, protein, and mRNA levels of Mn-SOD were significantly elevated in Cu-deficient rats. In Cu-deficient heart, the activity and protein content for Cu/Zn-SOD were reduced, whereas those for Mn-SOD were increased; the levels of mRNAs for these two enzymes was unaffected. Dietary Cu deficiency was without effect on the activities, enzyme contents, and mRNA abundance of brain Cu/Zn- and Mn-SOD. These results indicate that SODs from liver, heart, and brain exhibit differential sensitivities to dietary Cu deprivation, and that different mechanisms (transcriptional, posttranscriptional, or posttranslational) may be involved in their regulation.