Phenylarsine oxide induces mitochondrial permeability transition, hypercontracture, and cardiac cell death.

The mitochondrial permeability transition (MPT) is implicated in cardiac reperfusion/reoxygenation injury. In isolated ventricular myocytes, the sulfhydryl (SH) group modifier and MPT inducer phenylarsine oxide (PAO) caused MPT, severe hypercontracture, and irreversible membrane injury associated with increased cytoplasmic free [Ca(2+)]. Removal of extracellular Ca(2+) or depletion of nonmitochondrial Ca(2+) pools did not prevent these effects, whereas the MPT inhibitor cyclosporin A was partially protective and the SH-reducing agent dithiothreitol fully protective. In permeabilized myocytes, PAO caused hypercontracture at much lower free [Ca(2+)] than in its absence. Thus PAO induced hypercontracture by both increasing myofibrillar Ca(2+) sensitivity and promoting mitochondrial Ca(2+) efflux during MPT. Hypercontracture did not directly cause irreversible membrane injury because lactate dehydrogenase (LDH) release was not prevented by abolishing hypercontracture with 2,3-butanedione monoxime. However, loading myocytes with the membrane-permeable Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM) prevented PAO-induced LDH release, thus implicating the PAO-induced rise in cytoplasmic [Ca(2+)] as obligatory for irreversible membrane injury. In conclusion, PAO induces MPT and enhanced susceptibility to hypercontracture in isolated cardiac myocytes, both key features also implicated in cardiac reperfusion and reoxygenation injury.

Regulation of the mitochondrial permeability transition by matrix Ca(2+) and voltage during anoxia/reoxygenation.

We studied the interplay between matrix Ca(2+) concentration ([Ca(2+)]) and mitochondrial membrane potential (Deltapsi) in regulation of the mitochondrial permeability transition (MPT) during anoxia and reoxygenation. Without Ca(2+) loading, anoxia caused near-synchronous Deltapsi dissipation, mitochondrial Ca(2+) efflux, and matrix volume shrinkage when a critically low PO(2) was reached, which was rapidly reversible upon reoxygenation. These changes were related to electron transport inhibition, not MPT. Cyclosporin A-sensitive MPT did occur when extramitochondrial [Ca(2+)] was increased to promote significant Ca(2+) uptake during anoxia, depending on the Ca(2+) load size and ability to maintain Deltapsi. However, when [Ca(2+)] was increased after complete Deltapsi dissipation, MPT did not occur until reoxygenation, at which time reactivation of electron transport led to partial Deltapsi regeneration. In the setting of elevated extramitochondrial Ca(2+), this enhanced matrix Ca(2+) uptake while promoting MPT because of less than full recovery of Deltapsi. The interplay between Deltapsi and matrix [Ca(2+)] in accelerating or inhibiting MPT during anoxia/reoxygenation has implications for preventing reoxygenation injury associated with MPT.

Thapsigargin directly induces the mitochondrial permeability transition.

High concentrations of thapsigargin (TG) have been used to study the process of necrotic cell death, which involves mitochondria in the cell rapidly undergoing the mitochondrial permeability transition (MPT). We therefore investigated the effects of TG on MPT in isolated liver and heart mitochondria. Using a matrix swelling assay in combination with a novel enzymatic method based on inner membrane permeability to citrate synthase substrates, TG induced MPT in a concentration-dependent manner, independent of extramitochondrial [Ca2+] and inhibitable by cyclosporin A. Evidence from alamethicin-permeabilized mitochondria suggests that TG induces MPT by causing Ca2+ release from mitochondrial matrix Ca2+-binding sites. These findings suggest that the MPT-inducing effect of TG may contribute to its pro-necrotic and pro-apoptotic effects in various cell types.

Mitochondrial Ca2+ uptake, efflux, and sarcolemmal damage in Ca2+-overloaded cultured rat cardiomyocytes.

The purpose of this study was to determine mitochondrial Ca2+ accumulation and its possible role in initiation of mitochondrial permeability transition (MPT) and sarcolemmal damage in Ca2+-overloaded cardiomyocytes. Cellular Ca2+ overload, generated secondary to ouabain or p-chloromercuribenzoate-stimulated cell Na+ concentration increase, induced Ca2+ accumulation in mitochondria (approximately 3/4 of total net uptake) as identified by kinetic analysis and verified by use of mitochondrial inhibition. Mitochondrial Ca2+ uptake was followed by a rapid Ca2+ efflux ( approximately 1 mmol . kg dry wt-1 . min-1) that can be best explained by efflux via Ca2+-dependent nonspecific pores. Cell ATP concentration was stable during mitochondrial Ca2+ uptake and decreased in parallel with Ca2+ efflux. In addition, sarcolemmal damage was not related to the increase in mitochondrial Ca2+ concentration per se, but rather connected with the extent of Ca2+ efflux from the mitochondria. A decrease in the rate of this Ca2+ efflux, indicating also a decrease in a subpopulation of mitochondria with open pores, was followed by decreased sarcolemmal damage. Both dithiothreitol and cyclosporin A decreased rapid Ca2+ efflux and inhibited sarcolemmal damage, implicating MPT as an important component in the mechanism of sarcolemmal damage.

Effect of creatine phosphate on the contractile activity in acutely failing rat heart.

The hypothesis was tested that infusion of a solution containing creatine phosphate (CP) into rats with acutely failing hearts would enhance recovery of cardiac function. The acutely failing heart was produced by constricting the ascending aorta. This overload produced failure in approximately 25 min. At the point of failure the constriction was removed and solutions containing sterile physiological saline (PSS), PSS and CP, PSS and creatine, or PSS and creatine plus phosphate were infused. Cardiac function was assessed from systolic and diastolic blood pressure, +/- dp/dt, heart rate, and cardiac work. Ca2+ uptake by isolated sarcoplasmic reticulum and the concentrations of selected blood and tissue metabolites were measured. Normal cardiac function was restored in the PSS-CP infused rats whereas all other treatments did not restore cardiac function. Adenosine triphosphate and CP had declined in the myocardium of the failing hearts while lactate was elevated. The concentrations of these metabolites were normal in the PSS-CP infused animals. The glycogen concentration in the myocardium was reduced following the constriction. Ca2+ uptake by isolated sarcoplasmic reticulum was depressed in the failed hearts but normal in the hearts of CP-infused animals. These results demonstrate that the infusion of CP into animals with failing hearts can be effective in restoring cardiac function.

Factors limiting adenosine triphosphatase function during high intensity exercise. Thermodynamic and regulatory considerations.

It is widely accepted that a structural organisation favouring interaction between functionally-related enzymes is required for the economy and efficiency of metabolic reactions. Many functionally-related enzymes have been shown to be reversibly bound to cellular structures and to other enzymes at the sites where they are required. Resulting from this binding, close structural proximity and concentration of enzymes, a microenvironment is generated where the product of one enzyme is the substrate of the other. This reduces the diffusion distance for the substrate, saturates binding sites with maximal speed and, as a final outcome, increases the efficiency and economy of function behind these metabolic reactions. Available data indicate that the above-described association between adenosine triphosphatase (ATPase) and enzymes regenerating ATP has an important role in the regulation of ATPase function. A general consensus exists among published studies that the concentration of ATP ([ATP]) is not significantly decreased in fatigued muscle, even in those with severely diminished power output. However, in studies with isolated perfused hearts it has been possible to significantly reduce [ATP] in muscle cells without compromising mechanical activity. An explanation for this discrepancy is connected with local ATP regeneration in the vicinity of ATPase. Furthermore, when ATP regeneration is unable to balance ATP consumption a critical drop in the free energy of ATP hydrolysis is avoided by down-regulation of ATP consumption. The main function of local ATP regeneration is to maintain a low concentration of adenosine diphosphate ([ADP]), and the ADP/ATP ratio in the vicinity of the ATP-binding site of ATPase that is a prerequisite for high thermodynamic efficiency of ATP hydrolysis. Close proximity of creatine kinase and glycolytic enzymes to ATPase and high-affinity binding of substrates generate an ATPase microenvironment, where ADP and ATP are not in free equilibrium with those adenine nucleotides in the surrounding medium. In the physiological range of operation for important cellular ATPases (free energy change of 55 to 60 kJ/mol ATP) only a small fraction of energy, available in ATP, can be utilised, provided that no ATP regeneration takes place. However, ATP regeneration allows utilisation of most of the regenerating capacity, before ATP hydrolysis drops below the critical 55 kJ/mol. The importance of local ATP regeneration increases in parallel with an increase in the rate of ATPase turnover.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of calcium pump function in back inhibited vesicles by calcium-ATPase ligands.

OBJECTIVE: The aim was to evaluate the effect of changes in substrate-product concentrations of the overall sarcoplasmic reticular Ca(2+)-ATPase reactions on calcium pump function with an emphasis on those factors that modify back inhibition of the pump by intrareticular free calcium. METHODS: Sarcoplasmic reticular vesicles were isolated from the rabbit heart. Calcium uptake by the vesicles was measured with a calcium sensitive minielectrode and was related to ATPase activity, which was measured by the rate of inorganic phosphate (Pi) production or NADH oxidation. Back inhibition was varied by changing [oxalate]. RESULTS: At the high level of back inhibition and low calcium transport efficiency, calcium uptake by sarcoplasmic reticulum was stimulated by pH decrease, hydrophobic compounds, dimethyl sulphoxide, and ATP regeneration. These factors apparently modify either calcium binding to the low affinity binding site of Ca(2+)-ATPase or the effect derived from the binding of calcium to these sites. Under conditions where back inhibition was avoided and calcium transport efficiency was high, the same factors had a depressive effect on calcium uptake. Inorganic phosphate had a dual effect on the rate of calcium uptake supported by low [oxalate]: Pi < 2-3 mM strongly inhibited calcium uptake while further increase in [Pi] reversed this inhibition. At the lower level of back inhibition higher [Pi] was required to inhibit calcium transport. CONCLUSIONS: Changes in [H+], [ADP], and [Pi] can significantly affect calcium pump function, but the effect is dependent on the extent of back inhibition of calcium transport. Changes in the sarcoplasmic reticular Ca(2+)-ATPase environment which mimic those expected to take place at the start of reperfusion (pH increase to 7.0-7.1, high myoplasmic [Ca2+]) may have a depressive effect on the efficiency of calcium transport, provided that intrareticular free calcium is increased. Under those conditions factors able to decrease the inhibitory effect derived from the calcium binding to the low affinity binding sites are expected to improve the efficiency of calcium transport.

The importance of ATPase microenvironment in muscle fatigue: a hypothesis.

A broadly held opinion is that fatigue is not due to an insufficient supply of ATP to the energy consuming mechanisms because tissue [ATP] always remains at least one order of magnitude higher than Km for ATP of any ATPase. In general these findings also suggest that ATP consumption is well balanced with ATP regeneration even in the fatigued muscles. This balance is achieved by down-regulation of ATP consumption. Potentially this down-regulation could be accomplished by any product of the ATPase reaction and the role of Pi and H+ accumulation in this regulation has been discussed in the literature. The purpose of this paper is to describe known compartmentalization of ATP regeneration systems in muscle cell, their importance in the regulation of [adenine nucleotide] in the vicinity of ATPases and how such local ATP regeneration maybe important in the etiology of muscle fatigue. Available experimental evidence suggests that the binding of creatine kinase and glycolytic enzymes in the vicinity of sites where ATP is hydrolyzed and functional coupling between these ATP regenerating mechanisms and ATPase can generate ATPase microenvironments that have an important role in the regulation of ATPase function. Main function of this ATP regeneration is to keep the local ADP/ATP ratios favorable for ATPase function, which seems to be especially important when ATPase turnover is high. Unfortunately, the maximum rate of local ATP regeneration relative to that of ATP hydrolysis in vivo is not known, mainly because in vitro determinations underestimate this value due to a decrease in the fractional of loosely abound enzyme to the preparation during isolation procedure.(ABSTRACT TRUNCATED AT 250 WORDS)

Local ATP regeneration is important for sarcoplasmic reticulum Ca2+ pump function.

Ca2+ pump function of skeletal muscle sarcoplasmic reticulum (SR) vesicles was measured by monitoring Ca2+ uptake and efflux with a Ca(2+)-sensitive minielectrode and adenosinetriphosphatase (ATPase) activity of the same preparation under the same conditions. The efficiency of Ca2+ transport into SR vesicles, defined by the amount of Ca2+ transported per ATP hydrolyzed (coupling ratio), varied significantly depending on assay conditions. Coupling ratio increased in parallel with increase in precipitating anion concentration, which is supposed to decrease accumulation of free Ca2+ inside vesicles and its subsequent efflux. Membrane-bound creatine kinase-creatine phosphate (CK-CP) system, acting as a ADP sensor and local ATP regenerator, significantly improved Ca2+ pump function when the pump worked with low efficiency (coupling ratio < 1). The effect of CK-CP system on Ca2+ pump function was also dependent on extravesicular Ca2+ concentration ([Ca2+]o), the effect being most significant at high initial [Ca2+]o. Under conditions in which SR vesicles were allowed to decrease [Ca2+]o, as occurs also during muscle relaxation, plateau values of Ca(2+)-ATPase activity were reached at significantly higher [Ca2+]o (54 +/- 5.7, n = 6), compared with leaky vesicles or the condition in which [Ca2+]o was maintained. By preventing local accumulation of ADP, generated in ATPase reactions, CK-CP system also inhibited Ca2+ efflux under conditions in which this efflux was stimulated by the increase of free Ca2+ inside vesicles. This effect was at least partially responsible for the CK-CP-supported increase in Ca2+ uptake and coupling ratios that were more expressed at low precipitating anion concentration. We hypothesize that local ATP regeneration by CK-CP system is one mechanism the cell can use to improve Ca2+ uptake by SR in emergency conditions, where excessive increase in cytoplasmic [Ca2+] may have deleterious effects.

Iron effects on myocardial enzymes depend on redox state.

Rapid depression of Ca(2+)-uptake by sarcoplasmic reticulum (SR) vesicles and inhibition of the activity of creatine kinase (CK) and pyruvate kinase (PK) was observed during incubation of enzymes with micromolar concentrations of iron in the presence of adenine nucleotides. This effect of iron was dependent on the redox state of the iron as determined by the redox state of the environment. Redox conditions that generated an Fe2+/Fe3+ ratio close to 1 were most effective in depressing Ca(2+)-uptake by SR vesicles. Redox conditions that decreased the Fe2+/Fe3+ ratio by oxidizing iron were most effective in depressing CK activity while redox conditions that significantly increased the Fe2+/Fe3+ ratio by reducing iron were most effective in depressing PK activity. All iron sensitive enzymes possessed N-etylmaleimide (NEM) sensitive sulphydryl groups that are essential for their activity. The sensitivity to inhibition by NEM increased in the order: PK < Ca(2+)-uptake < CK. Iron initiated depression of CK and PK activities were reversible with dithiothreitol (DTT). This indicated that modification of SH groups was an important step in the mechanism by which iron depressed enzyme activity. Iron initiated depression of Ca(2+)-uptake and of the activity of CK and PK was prevented by not allowing the critical Fe2+/Fe3+ ratio to be reached and by binding of iron with desferroxamine and EDTA. These results, together with data from the literature, led us to suggest that changes in the redox state of cellular micro-environments, inevitably taking place during ischemia and reperfusion, may increase the availability of "low molecular weight iron" and, through changes in the redox state of this iron, selectively initiate reversible depression of several enzymes which contain SH groups essential for their activity.

The effect of changes in iron redox state on the activity of enzymes sensitive to modification of SH groups.

Iron ions in micromolar concentrations induced a rapid and selective inhibition of the activity of skeletal muscle creatine kinase (CK), sarcoplasmic reticulum (SR) Ca(2+)-ATPase, and pyruvate kinase (PK). This effect of iron was dependent on the presence of adenine nucleotides and on the redox state of iron. Changing the redox state of the media created different Fe2+/Fe3+ ratios which selectively depressed different enzymes: depression of PK activity occurred when iron was predominantly in its reduced form and, consequently, when there was a high Fe2+/Fe3+ ratio; depression of SR Ca2+ uptake and SR Ca(2+)-ATPase activity occurred when the Fe2+/Fe3+ ratio was close to 1; depression of CK activity occurred when iron was predominantly in its oxidized form and the Fe2+/Fe3+ ratio was low. All iron-sensitive enzymes possessed sulfhydryl groups, accessible to N-ethylmaleimide (NEM), which were essential for their activity. The rate of inhibition of enzyme activity with NEM increased in the order PK < Ca(2+)-ATPase < CK. Iron-induced depression of CK and PK activities was reversible by dithiotreithol. Results suggest that changes in the redox state of cellular microenvironments, which inevitably occur during reperfusion of ischemic tissue or rapid increase in tissue oxygen consumption, may selectively depress the activity of several enzymes bearing SH groups that are sensitive to modifications and that are essential for their activity. Iron-induced depression of enzyme activity depends on the availability of iron bound to adenine nucleotides and possibly to other low molecular weight chelators and on the Fe2+/Fe3+ ratio generated by the induced redox change.

Functional coupling between sarcoplasmic-reticulum-bound creatine kinase and Ca(2+)-ATPase.

We investigated the role of creatine kinase bound to sarcoplasmic reticulum membranes of fast skeletal muscle in the local regeneration of ATP and the possible physiological significance of this regeneration for calcium pump function. Our results indicate that ADP produced by sarcoplasmic reticulum Ca(2+)-ATPase is effectively phosphorylated by creatine kinase in the presence of creatine phosphate. This phosphorylation is an important function of the membrane-bound creatine kinase because accumulation of ADP has a depressive effect on Ca(2+)-uptake by sarcoplasmic reticulum vesicles. The concentration-dependent depression of Ca(2+)-uptake by ADP was especially pronounced when there was strong back inhibition by high intravesicular [Ca2+]. ATP regenerated by endogenous creatine kinase was not in free equilibrium with the ATP in the surrounding medium, but was used preferentially by Ca(2+)-ATPase for Ca(2+)-uptake. Efficient translocation of ATP from creatine kinase to Ca(2+)-ATPase, despite the presence of an ATP trap in the surrounding medium, can be explained by close localization of creatine kinase and Ca(2+)-ATPase on the sarcoplasmic reticulum membranes. These results suggest the existence of functional coupling between creatine kinase and Ca(2+)-ATPase on skeletal muscle sarcoplasmic reticulum membranes. Several factors (amount of membrane-bound creatine kinase, oxidation of SH groups of creatine kinase, decrease in [phosphocreatine]) can influence the ability of creatine kinase/phosphocreatine system to support a low ADP/ATP ratio and fuel the Ca(2+)-pump with ATP. These factors may become operative in the living cells, influencing functional coupling between creatine kinase and Ca(2+)-ATPase and may have an indirect effect on Ca(2+)-pump function before Ca(2+)-ATPase itself is affected.

The effect of physical exertions on heart sensitivity to ischemia and metabolic conditioning of these changes.

With increasing duration of swimming exercise the heart becomes less sensitive to ischemia, as evaluated by the rate of development of ischemic contracture immediately after the exertion. This delay in the development of ischemic contracture was apparently due to metabolic changes directed to decrease the heart energy consumption in conditions where the capacity for glycolytic ATP production was decreased. A decrease in: 1) the amount of rapidly exchangeable Ca2+, which is bound to anionic sites on the sarcolemmal membrane and 2) the myofibrillar Ca2+ sensitivity seems to play an important role. Regular swimming exercise, which is characterized by a significant cardiac hypertrophy and enhanced heart glycogen content, increased the sensitivity of energy mobilizing processes to catecholamine action. These changes accelerated ATP depletion and the development of an irreversible injury when the heart was made ischemic after catecholamine stimulation. Obtained results together with data from literature underline the importance of regular testing of cardiac function, including echocardiography, in young sportsmen undergoing high-intensity training.

The effect of regular physical exercise on sensitivity to ischaemia in the rat's heart.

The effect of different training regimes (three programmes of both swimming and running exercise) on the heart hypertrophy index and some biochemical indices was evaluated and compared individually with the sensitivity of the corresponding heart to ischaemia in order to elucidate the significance of training intensity and observed changes in the development of heart ischaemic injury. The sensitivity of the heart to ischaemia, evaluated by the rate of development of ischaemic contracture 48 h after completing the exercise programme, increased in parallel with an increase in the heart hypertrophy index. Experiments with different swimming programmes showed that the extent of cardiac hypertrophy increased together with an increase in the duration of everyday swimming bouts. Hypertrophied hearts from trained rats were characterized by greater mobilization of glycogen and increased incorporation of 32P into ATP when investigated 10 min after isoprenaline administration. During total ischaemia the development of ischaemic contracture was accelerated in catecholamine-stimulated trained hearts due to more rapid hydrolysis of ATP compared with that in the hearts from sedentary animals. It is suggested that the observed difference between hearts from sedentary and trained animals is, at least partially, connected with the higher sensitivity of myofibrils to Ca2+ in trained hearts.

The effect of isoprenaline and physical exertion to exhaustion on the mechanism of glucocorticoid action in the rat heart.

The effect of ATP and factors known to alter cellular ATP level on the properties of cytosolic glucocorticoid receptor of the heart were investigated. We measured the number of binding sites, binding affinity, receptor transformation and nuclear binding. Experiments performed on the cardiac cytosol of adrenalectomized rats labeled with hormone in vitro showed that both isoprenaline treatment and physical exertion to exhaustion decreased the number of triamcinolone acetonide binding sites, but had no significant effect on the binding affinity. Although ATP had an activating effect on the cytosolic steroid-receptor complexes, the results of phase partitioning and DEAE-cellulose chromatography indicated that neither isoprenaline nor exertion to exhaustion had any apparent effect on the equilibrium between the activated and unactivated forms of complexes. The amount of translocated complexes was dependent on the cytosolic receptor concentration. Experiments performed on the heart cytosol labeled with the hormone in vivo, showed that exertion to exhaustion changed the equilibrium between activated and unactivated complexes in favour of the latter form and at the same time decreased the amount of complexes bound to the nuclei and chromatin. It is not clear whether these changes reflect alterations in receptor activation and its accumulation in the nuclei, or whether they are connected with the release of the nuclear receptor due to the decrease in receptor affinity to chromatin or some other nuclear component. Speculatively, the data suggest that glucocorticoid action in heart cells may depend on the ability of cells to maintain a certain level of ATP.

ATP-dependent activation of glucocorticoid-receptor complexes from the rat's heart.

The effect of ATP on the cytosolic rat heart glucocorticoid receptor was studied by employing different methods for evaluation of the changes in molecular properties of the receptor, induced by activation. Incubation of triamcinolone acetonide labelled cytosol at 25 degrees C or with 10 mM ATP at 4 degrees C leads to the increase in the partition coefficient of the receptor in the aqueous dextran-polyethylene glycol two-phase system and also nuclear uptake of the complexes. The effect of ATP on the partition coefficient was more pronounced, compared with that of thermal treatment or dilution of the cytosol and totally inhibited by 10 mM sodium molybdate. The activating effect of ATP on the glucocorticoid-receptor complexes and sensitivity of this activation to sodium molybdate was also confirmed by DEAE-cellulose chromatography of cytosolic receptor preparations. The results suggest that ATP may be involved in the glucocorticoid receptor activation and through this regulates the translocation of complexes into the nucleus under in vivo conditions.

Binding constants and stability of dexamethasone-binding protein from rat heart.

A pronounced decrease in specific dexamethasone binding capacity of heart cytosol was observed. The inactivation of unbound glucocorticoid receptor was not due to the action of proteases since the rat of albumin digestion was very slow in the particulate preparations. The rate of inactivation of glucocorticoid binding capacity depends on cytosol protein concentration and temperature. Addition of hormone and glycerol to cytosol markedly slow binding capacity inactivation. The rapid loss of binding activity, especially in the cytosol with high protein concentration, makes difficult to perform equilibrium studies for simultaneous determination of binding affinity and a number of binding sites. Under various conditions the apparent dissociation constants were measured and compared with the dissociation constants, calculated from the rate of constants of association and dissociation, in order to find out optimal conditions for simultaneous determination of KD and [R0].