Mitochondrial ATPase Inhibitor Research Articles

Isolation of protein subpopulations undergoing protein-protein interactions.

A new method is described for isolating and identifying proteins participating in protein-protein interactions in a complex mixture. The method uses a cyanogen bromide-activated Sepharose matrix to isolate proteins that are non-covalently bound to other proteins. Because the proteins are accessible to chemical manipulation, mass spectrometric identification of the proteins can yield information on specific classes of interacting proteins, such as calcium-dependent or substrate-dependent protein interactions. This permits selection of a subpopulation of proteins from a complex mixture on the basis of specified interaction criteria. The new method has the advantage of screening the entire proteome simultaneously, unlike the two-hybrid system or phage display, which can only detect proteins binding to a single bait protein at a time. The method was tested by selecting rat brain extract for proteins exhibiting calcium-dependent protein interactions. Of 12 proteins identified by mass spectrometry, eight were either known calcium-binding proteins or proteins with known calcium-dependent protein interactions, indicating that the method is capable of enriching a subpopulation of proteins from a complex mixture on the basis of a specific class of protein interactions. Because only naturally occurring interactions of proteins in their native state are observed, this method will have wide applicability to studies of protein interactions in tissue samples and autopsy specimens, for screening for perturbations of protein-protein interactions by signaling molecules, pharmacological agents or toxins, and screening for differences between cancerous and untransformed cells.

Deletion of mitochondrial ATPase inhibitor in the yeast Saccharomyces cerevisiae decreased cellular and mitochondrial ATP levels under non-nutritional conditions and induced a respiration-deficient cell-type.

T(1), a mutant yeast lacking three regulatory proteins of F(1)F(o)ATPase, namely ATPase inhibitor, 9K protein and 15K protein, grew on non-fermentable carbon source at the same rate as normal cells but was less viable when incubated in water. During the incubation, the cellular ATP content decreased rapidly in the T(1) cells but not in normal cells, and respiration-deficient cells appeared among the T(1) cells. The same mutation was also induced in D26 cells lacking only the ATPase inhibitor. Overexpression of the ATPase inhibitor in YC63 cells, which were derived from the D26 strain harboring an expression vector containing the gene of the ATPase inhibitor, prevented the decrease of cellular ATP level and the mutation. Isolated T(1) mitochondria exhibited ATP hydrolysis for maintenance of membrane potential when antimycin A was added to the mitochondrial suspension, while normal and YC63 mitochondria continued to show low hydrolytic activity and low membrane potential. Thus, it is likely that deletion of the ATPase inhibitor induces ATPase activity of F(1)F(o)ATPase to create a dispensable membrane potential under the non-nutritional conditions and that this depletes mitochondrial and cellular ATP. The depletion of mitochondrial ATP in turn leads to occurrence of aberrant DNA in mitochondria.

The region from phenylalanine-17 to phenylalanine-28 of a yeast mitochondrial ATPase inhibitor is essential for its ATPase inhibitory activity.

Mitochondrial ATP synthase (F(1)F(o)-ATPase) is regulated by an intrinsic ATPase inhibitor protein. In the present study, we investigated the structure-function relationship of the yeast ATPase inhibitor by amino acid replacement. A total of 22 mutants were isolated and characterized. Five mutants (F17S, R20G, R22G, E25A, and F28S) were entirely inactive, indicating that the residues, Phe17, Arg20, Arg22, Glu25, and Phe28, are essential for the ATPase inhibitory activity of the protein. The activity of 7 mutants (A23G, R30G, R32G, Q36G, L37G, L40S, and L44G) decreased, indicating that the residues, Ala23, Arg30, Arg32, Gln36, Leu37, Leu40, and Leu44, are also involved in the activity. Three mutants, V29G, K34Q, and K41Q, retained normal activity at pH 6.5, but were less active at pH 7.2, indicating that the residues, Val29, Lys34, and Lys41, are required for the protein's action at higher pH. The effects of 6 mutants (D26A, E35V, H39N, H39R, K46Q, and K49Q) were slight or undetectable, and the residues Asp26, Glu35, His39, Lys46, and Lys49 thus appear to be dispensable. The mutant E21A retained normal ATPase inhibitory activity but lacked pH-sensitivity. Competition experiments suggested that the 5 inactivated mutants (F17S, R20G, R22G, E25A, and F28S) could still bind to the inhibitory site on F(1)F(o)-ATPase. These results show that the region from the position 17 to 28 of the yeast inhibitor is the most important for its activity and is required for the inhibition of F(1), rather than binding to the enzyme.

The role of the mitochondrial ATPase inhibitor, IF1, in cardiac ATP conservation under hypoxic/ischemic conditions

The role of the mitochondrial ATPase inhibitor, IF1, in cardiac ATP conservation under hypoxic/ischemic conditions

Requirement for lysine-19 of the yeast mitochondrial ATPase inhibitor for the stability of the inactivated inhibitor-F1Fo complex at higher pH.

The ATPase inhibitor is a regulatory subunit of mitochondrial ATP synthase. In this study, the role of Lys19 of the yeast ATPase inhibitor was examined by site-directed mutagenesis. Two amino acids (Gln and Glu) were substituted for the Lys19. The purified mutant inhibitor (Lys19-->Gln) had similar ATPase inhibitory activity to that of the wild-type inhibitor at pH 6.5, but was less active at pH 7.4. ATP synthesis in mutant mitochondria was normally activated by the addition of ADP and succinate, but the inactivated ATPase complex in the mutant mitochondria was activated more readily than that in control cells by raising pH. These results show that Lys19 of the yeast ATPase inhibitor is not essential for ATPase inhibitory activity, but increases the stability of the inhibitor-F1Fo complex at higher pH.

Identification of genes regulated by UV/salicylic acid

Purpose : Previous work from the authors' group and others has demonstrated that some of the effects of UV irradiation on gene expression are modulated in response to the addition of salicylic acid to irradiated cells. The presumed effector molecule responsible for this modulation is NF-kappaB. In the experiments described here, differential-display RT-PCR was used to identify those cDNAs that are differentially modulated by UV radiation with and without the addition of salicylic acid. Materials and methods : Differential-display RT-PCR was used to identify differentially expressed genes. Results : Eight such cDNAs are presented: lactate dehydrogenase (LDH-beta), nuclear encoded mitochondrial NADH ubiquinone reductase 24kDa (NDUFV2), elongation initiation factor 4B (eIF4B), nuclear dots protein SP100, nuclear encoded mitochondrial ATPase inhibitor (IF1), a cDNA similar to a subunit of yeast CCAAT transcription factor HAP5, and two expressed sequence tags (AA187906 and AA513156). Conclusions : Sequences of four of these genes contained NF-kappaB DNA binding sites of the type that may attract transrepressor p55/p55 NF-kappaB homodimers. Down-regulation of these genes upon UV irradiation may contribute to increased cell survival via suppression of p53 independent apoptosis.

The Mitochondrial Permeability Transition Mediates Both Necrotic and Apoptotic Death of Hepatocytes Exposed to Br-A23187

A23187 and related Ca2+ionophores are widely used to study Ca2+-dependent cell injury. Here, using laser scanning confocal microscopy and parameter-indicating fluorophores, we investigated the role of the mitochondrial permeability transition (MPT) in Br-A23187 toxicity to cultured rat hepatocytes. After 10 μM Br-A23187, over 60% of hepatocytes lost viability within 1 h. This necrotic cell killing was preceded by increased mitochondrial free Ca2+, mitochondrial depolarization, and onset of the MPT. Cyclosporin A (CsA), a blocker of the permeability transition pore, prevented the MPT and cell killing but had no effect on increased mitochondrial free Ca2+and depolarization after Br-A23187. To determine whether Br-A23187-induced cell killing was linked to loss of cellular ATP supply, hepatocytes were incubated with fructose and oligomycin, a source of glycolytic ATP and an inhibitor of the uncoupler-stimulated mitochondrial ATPase, respectively. Fructose plus oligomycin prevented cell killing after Br-A23187 but not the MPT. When fructose plus oligomycin prevented necrotic cell killing, apoptosis developed after 10 h. When cells were treated additionally with CsA, these apoptotic changes were prevented. In conclusion, the MPT mediates Br-A23187 cytotoxicity. Acutely, the MPT causes mitochondrial uncoupling and profound ATP depletion, which leads to necrotic cell death. However, when glycolytic ATP generation is available, the MPT induces apoptosis. CsA blocks the MPT and prevents both necrotic and apoptotic cell killing after Br-A23187.

The carboxyl-terminal region of the yeast ATPase inhibitor is indispensable for the stability of the protein in mitochondria.

The role of the carboxyl-terminal region of the yeast mitochondrial ATPase inhibitor was investigated. Three progressive C-terminal deletion mutants of the inhibitor were constructed: (i) Ile58-->end; (ii) Ile51-->end; and (iii) Gln43-->end. The truncated inhibitor was detected in extracts of Ile58-->end mutant yeast cells. For the Ile51-->end mutant, the truncated inhibitor was only detected when the cells were grown on medium containing the membrane-permeable metal chelator, o-phenanthroline, which inhibits mitochondrial proteases. The most greatly truncated inhibitor protein, Gln43-->end, was never detected even in the cells grown in the presence of the metal chelator. The rates of ATP synthesis and hydrolysis in the mutant mitochondria containing the Ile51-->end inhibitor were similar to those in wild type control cells, while the Ile51-->end inhibitor protein was degraded in the cells unless they were incubated in the presence of the chelator. These results indicate that the carboxyl-terminal region of the ATPase inhibitor is not involved in the its inhibitory action on the F1Fo-ATPase, but is required for the stable conformation of the protein which is protected against degradation by proteases.

Induction of an ATPase inhibitor protein by propylthiouracil and protection against paracetamol (acetaminophen) hepatotoxicity in the rat.

1. The purpose of the present study was to test the following hypothesis: propylthiouracil (PTU) treatments of rats induces an increase in the concentration and activity of the mitochondrial ATPase (m-ATPase) inhibitor protein (IF1). The PTU-induced elevated baseline levels of this inhibitor protein inactivated m-ATPase, and prevented hepatotoxicity by a toxic dose of acetaminophen (AAP) (paracetamol), by maintaining hepatic adenosine 5'-triphosphate (ATP) levels. 2. Male Wistar rats were either gavaged with a toxic dose of AAP alone, or after pretreatment with PTU for periods of 3 and 12 days. 3. Twenty four hours after acetaminophen treatment alone, toxicity was manifested by: an approximately 10 fold increase in serum transaminase levels (serum glutamic oxaloacetic transaminase and serum glutamic pyruvic transaminase); depletion of hepatic reduced glutathione (GSH) and ATP levels; loss of inhibitor protein activity, and extensive pericentral necrosis of the hepatocytes. Propylthiouracil pretreatment for 12 days enhanced the concentration of the following metabolites in the liver: ATP (1.5 fold), ATPase inhibitor protein (IF1) (4.5 fold), and reduced glutathione (1.3 fold), while the activity of the inhibitor protein increased 2 fold. When the PTU treated rats were challenged with AAP, transaminases were not elevated, and only sporadic areas of necrosis were detected by histological examination of the liver tissue. In contrast to the 12 day treatment with PTU the 3 day treatment had no protection against AAP. No histological evidence of protection was manifested and the transaminases were not different from AAP treated controls. Most of the protective metabolites were depleted. 4. Our findings suggest that PTU-induced increased concentration of inhibitor protein and GSH, are contributing factors in the prevention of hepatotoxicity by maintaining hepatic m-ATP levels and reducing the harmful effect of the toxic metabolite of AAP.

Preconditioning in rat hearts is independent of mitochondrial F1F0 ATPase inhibition.

Mitochondrial F1F0 adenosinetriphosphatase (ATPase) is responsible for the majority of ATP synthesis during normoxic conditions, but under ischemic conditions it accounts for significant ATP hydrolysis. A previous study showed that preconditioning in isolated rat hearts is mediated by inhibition of this ATPase during ischemia. We tested this hypothesis in our isolated rat heart model of preconditioning. Preconditioning was accomplished by three 5-min periods of global ischemia separated by 5 min of reperfusion. This was followed by 20 min of global ischemia and 30 min of reperfusion. Preconditioning significantly enhanced reperfusion contractile function and reduced lactate dehydrogenase release but paradoxically reduced the time to onset of contracture during global ischemia. Myocardial ATP was depleted at a faster rate during the prolonged ischemia in preconditioned than in sham-treated hearts, which is consistent with the reduced time to contracture. ATP during reperfusion was repleted more rapidly in preconditioned hearts, which is consistent with their enhanced contractile function. Preconditioning significantly reduced lactate accumulation during the prolonged ischemia. We were not able to demonstrate that mitochondrial F1F0 ATPase (measured in submitochondrial particles) was inhibited by preconditioning before or during the prolonged ischemia. The mitochondrial ATPase inhibitor oligomycin significantly conserved ATP during ischemia and increased the time to the onset of contracture, which is consistent with inhibition of the mitochondrial ATPase. Our results show that preconditioning in rat hearts can be independent of mitochondrial ATPase inhibition as well as ATP conservation.

Increased Class Ib Antigen Display onTAP-2Mutant Cells by a Mitochondrial Function Inhibitor

Class I histocompatibility antigen display is defective in the RMA-S mutant cell line due to a mutation in theTap-2gene, which encodes a peptide transporter. Incubation of RMA-S cells with oligomycin, an inhibitor of mitochondrial ATPase, strongly increased lysis by cytotoxic T lymphocytes (CTL) specific for the class Ib antigen H2-M3, and lysis by Qa-1b-specific CTL was restored. Oligomycin did not affect normal class I display on RMA cells. Treatment of RMA-S cells with other inhibitors of mitochondrial function failed to increase lysis by anti-H2-M3 or Qa-1bCTL. Lysis by allogenic CTL specific for H-2bantigens was either not enhanced or only weakly increased, depending upon theH-2haplotype of the alloreactive effector cells used.

IF1 Function in Situ in Uncoupler-challenged Ischemic Rabbit, Rat, and Pigeon Hearts

Rabbit, rat, and pigeon are species representative of three cardiac muscle mitochondrial ATPase regulatory classes, a, b and c, respectively. Class a species contain a full complement of higher affinity ATPase inhibitor subunit, IF1, in their cardiac muscle mitochondria and show marked IF1-mediated mitochondrial ATPase inhibition during myocardial ischemia. Class b species contain low levels of higher affinity IF1 and show very little IF1-mediated ATPase inhibition during ischemia. Class c species contain a full complement of a lower affinity form of IF1 and show a low-to-moderate level of IF1- mediated ATPase inhibition during ischemia. In the present study we perfused hearts of a member of each regulatory class through the coronary arteries with the uncoupler, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), before making them ischemic. We then compared net rates of cell ATP depletion during ischemia in the FCCP-treated hearts to identically treated FCCP-free hearts. Thus, we tested the relative capacities of cardiac muscle mitochondria of the three species to avert a potentially greatly increased net rate of cell ATP depletion due to ATP hydrolysis by the fully uncoupled mitochondrial ATPase. We found that FCCP-uncoupling in situ had a relatively small effect on ATP depletion during ischemia in rabbit hearts, that it dramatically accelerated ATP depletion in ischemic rat hearts, and that it had an intermediate effect on ATP depletion in ischemic pigeon hearts. These results demonstrate for the first time the relative extents to which IF1-mediated mitochondrial ATPase inhibition can slow cell ATP depletion due to the fully uncoupled mitochondrial ATPase in these three classes of hearts. They show that, in contrast to the situation in rabbit hearts, the low level of higher affinity IF1 present in the cardiac muscle mitochondria of the rat is, under these conditions, essentially nonfunctional, while the full complement of the lower affinity form of IF1 present in the cardiac muscle mitochondria of the pigeon is partially functional in that it appeared to provide an intermediate level of protection against rapid cell ATP depletion.

Isoform-independent heart rate-related variation in cardiac myofibrillar Ca(2+)-activated Mg(2+)-ATPase activity.

In the present study, we compared the activities of the cardiac myofibrillar Ca(2+)-activated Mg(2+)-ATPase and the content of cardiac muscle mitochondrial ATPase inhibitor protein (IF1) of several mammalian species covering broad ranges of body mass and heart rate, i.e., from beef cattle to mouse. The cardiac myofibrillar ATPase from each species was assayed over a range of pCa values at pH 7.4. While the cardiac myofibrillar ATPase from all species examined showed essentially identical Ca2+ concentration dependencies with the ATPase in each species activating steeply between pCa 6.5 and 5.5, the maximal ATPase specific activity reached varied considerably from species to species, and this variation was largely independent of the predominant cardiac myosin ATPase isoform present. Thus, while adult beef cattle, pig, dog, and rabbit all contain predominantly the slow cardiac myosin ATPase isoform the cardiac myofibrillar ATPase specific activities of these four species varied over approximately a fourfold range. Moreover, there was a fairly smooth curvilinear relationship between maximum Ca(2+)-activated myofibrillar ATPase activity and median conscious heart rate for the slow cardiac myosin ATPase-possessing species examined. This smooth continuum also extended to include two species possessing the fast cardiac myosin ATPase isoform, rat and mouse. This relationship between myofibrillar ATPase activity and heart rate that appears to be applicable to a broad range of species suggests that the myofibrillar ATPase is specifically modeled or fine-tuned to the kinetic (heart rate) demand of each species and, within slow and fast heart rate ranges, is essentially independent of myosin ATPase isoform per se. Only hearts containing predominantly the slow myosin ATPase isoform contained functional levels of IF1. Finally, while it has been reported that the ratio of myosin Ca(2+)-ATPase to actomyosin Mg(2+)-ATPase activity is a good index of the percent of the fast myosin ATPase in rabbit myofibrillar preparations, we found that this relationship may be applicable to only some species.

Comparative Study of the Cytological Aspects of the Mode of Action of Destruxins and Other Peptidic Fungal Metabolites on Target Epithelial Cells

The cytopathological effects of cyclic fungal peptides, destruxins (dtxs) A and E, Cyclosporin A, and linear peptaı̈bols efrapeptins (=tolypin), on epithelial cells were studied byin vitroexperiments with preparations of insect organs. The treatment of Malpighian tubules and midgut revealed the induction by dtx E of the formation of vesicles on microvilli of Malpighian cells and on the apical surface of midgut cells. This morphological alteration of the brush border was a Ca2+-dependent process, very strongly inhibited in Ca2+-free medium or when a combined treatment with cadmium chloride + dtx E was applied. However, cadmium chloride did not inhibit all the cytotoxic effects of dtx E. The ability to trigger the formation of vesicular structures is a property of biologically active dtxs, the inducing capacity showing, however, a decreasing intensity from dtx E to dtx A and dtx B. On the contrary, the other peptides, Cyclosporin A and efrapeptins, did not induce the formation of vesicles. The most obvious intracellular alterations consist of a pycnosis of the nucleus, and in changes of mitochondria, the contents of which show a decrease in density and are comparable to those observedin vivo. If mitochondria are target organelles for the three different types of toxins tested, the structural changes induced at this level were specific for dtxs, Cyclosporin A, and efrapeptins, respectively. The cytological data obtained strongly suggested that dtxs have a unique mode of action among fungal peptides and do not act mainly as ionophores, inducing the formation of pores in cellular membranes, or as mitochondrial ATPase inhibitors.

Acidocalcisomes in Toxoplasma gondii tachyzoites.

Toxoplasma gondii tachyzoites were loaded with the fluorescent indicator fura 2 to investigate the transport mechanisms involved in maintaining their intracellular Ca2+ homoeostasis. The mitochondrial ATPase inhibitor oligomycin and the endoplasmic-reticulum Ca(2+)-ATPase inhibitor thapsigargin increased the intracellular Ca2+ concentration ([Ca2+]i), thus indicating the requirement for ATP and the involvement of the endoplasmic reticulum in maintaining intracellular Ca2+ homoeostasis. The effect of thapsigargin was more accentuated in the presence of extracellular Ca2+, clearly showing that, as occurs with other eukaryotic cells, depletion of intracellular Ca2+ pools led to an increase in the uptake of Ca2+ from the extracellular medium. In addition to these results, we found evidence that, in contrast with what occurs in mammalian cells, T. gondii tachyzoites possess a significant amount of Ca2+ stored in an acidic compartment, termed the acidocalcisome, as indicated by: (1) the increase in [Ca2+]i induced by bafilomycin A1 (a specific inhibitor of H(+)-ATPases), nigericin (a K+/H+ exchanger) or the weak base NH4Cl, in the nominal absence of extracellular Ca2+ to preclude Ca2+ entry; and (2) the effect of ionomycin, a Ca(2+)-releasing ionophore that cannot take Ca2+ out of acidic organelles and that was more effective after alkalinization of these compartments by addition of bafilomycin A1, nigericin or NH4Cl. Considering the relative importance of the ionomycin-releasable and the ionomycin + NH4Cl-releasable Ca2+ pools, it is apparent that T. gondii tachyzoites contain a significant amount of Ca2+ stored in acidocalcisomes.

The ATPase inhibitor protein from bovine heart mitochondria: the minimal inhibitory sequence.

The mitochondrial ATPase inhibitor subunit is a basic protein of 84 amino acids that helps to regulate the activity of F1F0-ATPase. In order to obtain structural information on the mechanism of inhibition, the bovine inhibitor subunit has been expressed in Escherichia coli and purified in high yield. The recombinant protein has a similar inhibitory activity to the inhibitor subunit isolated from bovine mitochondria. Progressive N-terminal and C-terminal deletion mutants of the inhibitor subunit have been produced either by overexpression and purification, or by chemical synthesis. By assaying the truncated proteins for inhibitory activity, the minimal inhibitory sequence of the inhibitor subunit has been defined as consisting of residues 14-47. The immediately adjacent sequences 10-13 and 48-56 help to stabilize the complex between F1F0-ATPase and the inhibitor protein, and residues 1-9 and 57-84 appear to be dispensable. At physiological pH values, the inhibitor subunit is mainly alpha-helical and forms monodisperse aggregates in solution. Smaller inhibitory fragments of the inhibitor protein, such as residues 10-50, seem to have a mainly random coil structure in solution, but they can adopt the correct inhibitory conformation when they from a complex with the ATPase. However, these latter fragments are mainly monomeric in solution, suggesting that the aggregation of the inhibitor subunit in solution may be due to intermolecular alpha-helical coiled-coil formation via the C-terminal region. The noninhibitory peptides consisting of residues 10-40 and 23-84 of the inhibitor protein can bind to F1F0-ATPase, and interfere with inhibition by the intact inhibitor subunit. The noninhibitory fragments of the inhibitor protein consisting of residues 22-46 and 44-84 do not compete with the inhibitor subunit for its binding site on F1F0-ATPase.

Mitochondrial membrane potential in single living adult rat cardiac myocytes exposed to anoxia or metabolic inhibition.

1. The relation between mitochondrial membrane potential (delta psi m) and cell function was investigated in single adult rat cardiac myocytes during anoxia and reoxygenation. delta psi m was studied by loading myocytes with JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'- tetra-ethylbenzimidazolylcarbocyanine iodide), a fluorescent probe characterized by two emission peaks (539 and 597 nm with excitation at 490 nm) corresponding to monomer and aggregate forms of the dye. 2. De-energizing conditions applied to mitochondria, cell suspensions or single cells decreased the aggregate emission and increased the monomer emission. This latter result cannot be explained by changes of JC-1 concentration in the aqueous mitochondrial matrix phase indicating that hydrophobic interaction of the probe with membranes has to be taken into account to explain JC-1 fluorescence properties in isolated mitochondria or intact cells. 3. A different sensitivity of the two JC-1 forms to delta psi m changes was shown in isolated mitochondria by the effects of ADP and FCCP and the calibration with K+ diffusion potentials. The monomer emission was responsive to values of delta psi m below 140 mV, which hardly modified the aggregate emission. Thus JC-1 represents a unique double sensor which can provide semi-quantitative information in both low and high potential ranges. 4. At the onset of glucose-free anoxia the epifluorescence of individual myocytes studied in the single excitation (490 nm)-double emission (530 and 590 nm) mode showed a gradual decline of the aggregate emission, which reached a plateau while electrically stimulated (0.2 Hz) contraction was still retained. The subsequent failure of contraction was followed by the rise of the emission at 530 nm, corresponding to the monomer form of the dye, concomitantly with the development of rigor contracture. 5. The onset of the rigor was preceded by the increase in intracellular Mg2+ concentration ([Mg2+]i) monitored by mag-indo-1 epifluorescence. Since under these experimental conditions intracellular [Ca2+] and pH are fairly stable, the increase in [Mg2+]i was likely to be produced by a decrease in ATP content. 6. The inhibition of mitochondrial ATPase induced by oligomycin during anoxia was associated with a rapid and simultaneous change of both the components of JC-1 fluorescence, suggesting that delta psi m, instead of producing ATP, is generated by glycolytic ATP during anoxia. 7. The readmission of oxygen induced a rapid decrease of the monomer emission and a slower increase of the aggregate emission. These fluorescence changes were not necessarily associated with the recovery of mechanical function.(ABSTRACT TRUNCATED AT 400 WORDS)

Mechanisms of Ischemic Preconditioning in Rat Myocardium

Adenosine has been proposed as one mediator for the preconditioning effect in the myocardium of some animals, but recent investigations have shown that this may not be the mechanism in the rat heart, although the effect itself is clearly demonstrable. The cellular energy state has been shown to be better in preconditioned hearts, and the role of ATP consumption has been discussed. The role of inhibition of mitochondrial F1F0-ATPase as a mechanism for the preservation of ATP in preconditioned hearts remains controversial. Three-minute global ischemia followed by 9 minutes of reperfusion was used to precondition Langendorff-perfused rat hearts, and control hearts were perfused under normoxic conditions for the same time. The duration of sustained ischemia in both groups of hearts was 21 minutes, after which the hearts were reperfused for 15 minutes to evaluate their mechanical and metabolic recovery. Separate experiments were performed for tissue metabolite determinations, mitochondrial ATPase activity measurements, and 31P nuclear magnetic resonance studies. The recovery of the rate-pressure product was better in the preconditioned group. Three-minute preconditioning ischemia caused inhibition of the mitochondrial ATPase that persisted throughout the 9-minute intervening reperfusion so that at the early stages of sustained ischemia the enzyme activity was still more inhibited in preconditioned hearts. ATP was better preserved in preconditioned hearts than in control hearts during sustained ischemia. The accumulation of adenosine and its degradation products during sustained ischemia was greater in the control group. More lactate and H+ ions accumulated in this group, indicating higher anaerobic glycolysis. Also, inhibition of mitochondrial ATPase by oligomycin slowed ATP depletion during ischemia. The results indicate that preconditioning causes inhibition of rat heart mitochondrial ATPase that persists during reperfusion so that the enzyme is inhibited from the very beginning of the sustained ischemia. This inhibition leads to sparing of high-energy phosphates and improves the time-averaged energy state during ischemia. Although a causal relationship is difficult to prove, this reversible inhibition may contribute to postischemic recovery of the heart.

Adaptation of the heart to ischemia by preconditioning: effects on energy equilibrium, properties of sarcolemmal ATPases and release of cardioprotective proteins.

Ischemic preconditioning of the heart is referred as a manifest increase in tolerance of the myocardium to otherwise damaging ischemic insult, achieved by one or few consequent initial short exposures to ischemia, each followed by reperfusion of the ischemic area. Several mechanisms such as opening of collateral vessels, the action of catecholamines, inositol phosphates, G-proteins and/or adenosine; inhibition of mitochondrial ATPase, the effects of different endogenous protective substances like heat stress or shock proteins, etc., are believed to cooperate in the mechanism of induction of preconditioning or in maintaining its effect. The present study is an attempt to extend the present knowledge about preconditioning from two aspects: i.) the peculiarities of energy equilibrium in preconditioned myocardium including adaptation of cardiac sarcolemmal ATPases to ischemia and/or hypoxia, and ii) participation of a new endogenous cardioprotective substance in the mechanism of preconditioning. The energy equilibrium in preconditioning is characterized by adaptation of cardiac energy demands to the capacity of energy production and delivery decreased by anaerobiosis and is manifested by constant ratios between ATP, ADP, AMP and the sum of ADN. Principles are proposed that may enable a prediction and mathematical modelling of the balanced energetic state in the preconditioned myocardium. These principles are based on thermodynamics and involve besides others a more economic handling of ATP by sarcolemmal ATPases. The latter enzymes adapt themselves to lowered availability of ATP by decreasing besides their Vmax also their values of Km (increase in the affinity) for ATP and some of them even adjust their activation energy (the anaerobiosis-induced elevation of Ea.t. is missing).(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation of the rat F 1-ATPase inhibitor gene and and its pseudogenes

Multiple mitochondrial ATPase inhibitor genes have been identified in the rat genome. The sequences of two genomic clones indicate that one encodes the functional gene, and the other is a processed pseudogene. The ATPase inhibitor gene isolated is about 1.5 kb long and the coding region contains three exons and two introns. The presence of multiple pseudogenes in the rat is suggested by this study and this is unique since in the bovine genome only a single gene has been found, which is also confirmed here. The presence of multiple inhibitor transcripts in the rat suggests that the functional gene might have multiple transcriptional start sites.