Changes In ATP Hydrolysis Research Articles

The AT-hook is an evolutionarily conserved auto-regulatory domain of SWI/SNF required for cell lineage priming

The SWI/SNF ATP-dependent chromatin remodeler is a master regulator of the epigenome, controlling pluripotency and differentiation. Towards the C-terminus of the catalytic subunit of SWI/SNF is a motif called the AT-hook that is evolutionary conserved. The AT-hook is present in many chromatin modifiers and generally thought to help anchor them to DNA. We observe however that the AT-hook regulates the intrinsic DNA-stimulated ATPase activity aside from promoting SWI/SNF recruitment to DNA or nucleosomes by increasing the reaction velocity a factor of 13 with no accompanying change in substrate affinity (KM). The changes in ATP hydrolysis causes an equivalent change in nucleosome movement, confirming they are tightly coupled. The catalytic subunit’s AT-hook is required in vivo for SWI/SNF remodeling activity in yeast and mouse embryonic stem cells. The AT-hook in SWI/SNF is required for transcription regulation and activation of stage-specific enhancers critical in cell lineage priming. Similarly, growth assays suggest the AT-hook is required in yeast SWI/SNF for activation of genes involved in amino acid biosynthesis and metabolizing ethanol. Our findings highlight the importance of studying SWI/SNF attenuation versus eliminating the catalytic subunit or completely shutting down its enzymatic activity.

Effect of gallic acid on purinergic signaling in lymphocytes, platelets, and serum of diabetic rats

Diabetes Mellitus (DM) is associated with an increased susceptibility to various infections, which might be attributed to changes in immune response owing to chronic hyperglycemia. Nucleoside triphosphate diphosphohydrolase (NTPDase), 5′-nucleotidase, and adenosine deaminase (ADA) are important enzymes involved in the generation of anti-aggregant and anti-inflammatory microenvironments. The aim of this study was to evaluate the effect of gallic acid (GA) on the hematological parameters and ectonucleotidase activities in platelets, lymphocytes, and serum of diabetic rats. Experimental rats were categorized into 4 groups: (i) control -saline, (ii) control - GA, (iii) diabetic -saline, and (iv) diabetic - GA. One week after induction of DM using streptozotocin (65 mg/kg), GA (30 mg/kg) or saline was orally administered to the rats for 21 days. Our results demonstrated that the concentration of mean corpuscular hemoglobin was decreased, whereas that of red cell distribution was increased in the diabetic group, however, GA could revert these alterations. Moreover, in diabetic rats, GA reverted the increase in ATP and ADP hydrolysis and ADA activity in lymphocytes, and it prevented the increase in NTPDase and ADA activities in platelets. A decrease in ATP hydrolysis and an increase in ADP and AMP hydrolysis were observed in the serum of diabetic rats; however, GA treatment could solely revert changes in ATP hydrolysis. Our study suggests that GA exhibits beneficial effects on immuno- and thrombo-regulatory responses in DM and that these effects may be related to the modulation of purinergic signaling.

Allosteric L5-Directed Inhibitors of Kinesin-5 Can Control Different Biochemical Intermediates

Kinesin-5 (Eg5) is an essential mitotic motor that couples ATP hydrolysis to different protein-protein association states with microtubules. Human Eg5 is a cancer chemotherapeutic target, having a unique allosteric site covered by loop-5 (L5). This site is capable of binding small-molecule inhibitors with > 100 different chemotypes. The prevailing biochemical model is that all L5-directed drugs inhibit ADP release, but it does not address the 107-fold difference in potency or the lack of chemical homology between inhibitor families. An alternative hypothesis is that the inhibitors act on different catalytic intermediates, which gives rise to their disparate potencies. Here we present our linear free energy relationship (LFER) study of Eg5, a method to determine whether an inhibitor can block the catalytic transition-state. Steady-state kinetic parameters for wildtype Eg5 and eight different L5 mutants were determined in the background of three different L5-directed inhibitors and a mock control. The predominant effect of the L5 residue substitution is alteration of substrate binding (Km), whereas principal outcome of allosteric drug is change in ATP hydrolysis (kcat). Second, our data showed that despite their use of the same binding pocket, one compound can inhibit the transition state and the other two do not. We conclude that the drugs are not synonymous: it is possible for one allosteric modulator to regulate ADP release and another to control transition-state formation. The significance is that this is the first demonstration of allosteric control of more than one catalytic intermediate for any drug target. Furthermore, these results may give insight into the disparity between increased inhibitor potency and success in clinical trials.This work is funded by the support of the National Institutes of Health (R01 GM097350; S.K.) and the LSU School of Graduate Studies (M.L.).

Cardiomyopathy Causing Mutations Stabilize an Intermediate State of Thin Filaments

Congenital cardiomyopathies are initiated by changes in ATP hydrolysis and result in hypertrophy, fibrosis, and myofibrillar disarray. We studied the mechanism by which mutations in troponin and tropomyosin change ATPase rates and have linked several mutants to inappropriate switching between the inactive and active states of the actin thin filament. We have shown that troponin I mutants mimicking protein kinase C phosphorylation stabilize the inactive state of actin filaments whereas the Δ14 TnT mutant stabilizes the active state. We have now shown that two mutations on troponin I, R146G and R146W, which cause cardiomyopathy produce complex effects on ATPase activity. These TnI mutations produced increased ATPase rates in the absence of calcium and decreased rates in the presence of calcium compared to wild type. These differences were maintained at high actin concentrations. Saturating concentrations of the activator NEM-S1 equalized the rates of both the mutants and wild type. The NEM-S1 data rule out alterations in rate constants of transitions (i.e. product release) along the active pathway. The results from the R146G and R146W mutants have implications for the function of the 3 structural states of regulated actin that have been observed. That is, the results can be explained most readily if the mutants stabilize an intermediate state in both calcium and EGTA with an activity between that of the inactive and active states. In the past we have assumed that the intermediate state had properties identical to the inactive state. Our current data show that while the intermediate more closely resembles the inactive state it has unique properties. Our present results, as well as previous results, indicate that inappropriate stabilization of any state of regulated actin can result in cardiac dysfunction.

Multiple Roles of Charged Amino Acids in Cytoplasmic Loop 7 for Expression and Function of the Multidrug and Organic Anion Transporter MRP1 (ABCC1)

Multidrug resistance protein MRP1 mediates the ATP-dependent efflux of many chemotherapeutic agents and organic anions. MRP1 has two nucleotide binding sites (NBSs) and three membrane spanning domains (MSDs) containing 17 transmembrane helices linked by extracellular and cytoplasmic loops (CL). Homology models suggest that CL7 (amino acids 1141-1195) is in a position where it could participate in signaling between the MSDs and NBSs during the transport process. We have individually replaced eight charged residues in CL7 with Ala, and in some cases, an amino acid with the same charge, and then investigated the effects on MRP1 expression, transport activity, and nucleotide and substrate interactions. A triple mutant in which Glu(1169), Glu(1170), and Glu(1172) were all replaced with Ala was also examined. The properties of R1173A and E1184A were comparable with those of wild-type MRP1, whereas the remaining mutants were either poorly expressed (R1166A, D1183A) or exhibited reduced transport of one or more organic anions (E1144A, D1179A, K1181A, (1169)AAQA). Same charge mutant D1183E was also not expressed, whereas expression and activity of R1166K were similar to wild-type MRP1. The moderate substrate-selective changes in transport activity displayed by mutants E1144A, D1179A, K1181A, and (1169)AAQA were accompanied by changes in orthovanadate-induced trapping of [alpha-(32)P]azidoADP by NBS2 indicating changes in ATP hydrolysis or release of ADP. In the case of E1144A, estradiol glucuronide no longer inhibited trapping of azidoADP. Together, our results demonstrate the extreme sensitivity of CL7 to mutation, consistent with its critical and complex dual role in both the proper folding and transport activity of MRP1.

Nitroxyl effects on myocardium provide new insights into the significance of altered myofilament response to calcium in the regulation of contractility

The idea that cardiac sarcomeres are not passive slaves to membrane related mechanisms modifying Ca2+ fluxes is generally accepted. There is substantial evidence that fundamental regulatory mechanisms including the Frank–Starling mechanism and neuro-humoral control of cardiac dynamics involve alterations in Ca2+ binding to troponin C (TnC) or transduction of the Ca–TnC signal to the promotion of ATP hydrolysis, force and shortening (Solaro, 2001; Kobayashi & Solaro 2005). In testimony to the recognition of the relative significance of these potential mechanisms in control of cardiac contractility, there has been a long-standing effort to develop inotropic agents acting directly on the myofilaments to modify their response to Ca2+ (Kass & Solaro, 2006). A paper by Dai et al. (2007) in this issue of The Journal of Physiology marks a new milestone in this search for ways to modify cardiac myofilament response to Ca2+. They demonstrated that a donor of nitroxyl (HNO) increases maximum tension and Ca2+ sensitivity of trabeculae sarcomeres functioning in situ. Although HNO also increased the peak amplitude of the Ca2+ transient, when compared to controls, the increase in tension was greater than expected from the rise in Ca2+. Except for the implication of thiols for functionally significant Cys residues in the sarcomeric proteins, the molecular mechanism of the effects of HNO remains obscure. An appealing property of HNO is that the enhancement of myofilament Ca2+ response occurs with a concomitant increase in Ca2+ flux into and out of the sarcoplasmic reticulum by a mechanism independent of β-adrenergic receptors, cAMP and cGMP. Other agents that increase myofilament response to Ca2+ have an associated inhibition of cAMP phospho-diesterases. An increase in cAMP increases the threat of arrythmias. The effects of HNO indicate a novel mechanism of enhancement of contractility without an effect on diastolic function and with a relative increase in the efficiency transduction of the Ca2+ binding signal. One aspect of this increase in efficiency is that the increase in myofilament Ca2+ response permits an increase in force generation while sparing the energy required in transporting Ca2+ to storage depots. There is also the possibility of increased economy in the unit change in ATP hydrolysis required for a unit change in tension (tension cost). Dai et al. (2007) reported no effect of HNO on ATPase rate of isolated myofibrils, but these preparations do not generate force. More rigorous tests are required in preparations bearing a load. The identification of the mechanism of the effect of HNO is necessarily couched in our understanding of the molecular mechanisms of the transitions of sarcomeres from diastolic to systolic states and how this transition is geared into the overt function of the heart to develop pressure, eject against the arterial impedance so as to satisfy metabolic needs of the body, and relax with a time course so as to not compromise filling for the next cycle. The critical parameter with regard to the relative role of the sarcomeres is not the kinetics of free Ca2+, detected by cytoplasmic probes, but the kinetics of Ca bound to TnC during the heart beat. Computations based on best estimates of the kinetics of Ca binding to the myofilaments during a beat of the ejecting ventricle indicate that Ca bound to TnC falls faster than systolic pressure and is near zero during isovolumic relaxation (Peterson et al. 1991; Hinken & Solaro, 2007). The mechanism that keeps the myofilaments activated with relatively little Ca bound resides in the ability of strongly bound crossbridges to promote Ca-binding affinity of TnC within a regulatory unit (actin: Tn complex: tropomyosin: myosin in a 7: 1: 1: 1 ratio) and to spread activation longitudinally along the thin filament by near neighbour cooperative mechanisms (Landesberg, 1996; Moss et al. 2004). Future investigation of HNO should include a detailed dissection of potential effects on thin and thick filament proteins as well as determination of effects on TnC–Ca and tension cost. Tropomyosin seems a likely target in view of its functionally significant and reactive Cys as well its key role as a determinant of Ca sensitivity of the number of cross-bridges reacting with the thin filament. It will also be of interest to know the effects of HNO in fast and slow skeletal muscle.

In vitro exposure of heavy metals on nucleotidase and cholinesterase activities from the digestive gland of Helix aspersa

Zinc, copper and cadmium are important environmental contaminants and differences in purinergic and cholinergic systems of invertebrates have been described when compared to characteristics of these signaling systems in vertebrates. Here we evaluate the effect in vitro of these metals on the ATPase, 5′-nucleotidase and cholinesterase (ChE) activities in the digestive gland of Helix aspersa. Zinc (500 and 1000 μM) promoted a significant decrease in 5′-nucleotidase activity. However, it did not induce changes in ATP hydrolysis. Copper (25 and 50 μM), inhibited significantly ATPase activity, but did not alter 5′-nucleotidase when compared to control (no metal added). In relation to effects of cadmium, an inhibitory effect on ATP hydrolysis has been observed at concentrations of 100, 500 and 1000 μM and a similar decrease of AMP hydrolysis was observed at 500 and 1000 μM. However, there were no significant changes in ChE activity from homogenates of the digestive gland of H. aspersa for all metals tested. This study demonstrated that zinc, cadmium and copper affect ATPase and 5′-nucleotidase in digestive gland, but not ChE, suggesting that the purinergic system may be a target related to toxicity induced by these metals and a possible indicator of biological impact of exposure to these contaminants.

Cardiac magnetic resonance spectroscopy.

This review describes recent advances in cardiac magnetic resonance spectroscopy (MRS). MRS allows noninvasive characterization of the metabolic state of cardiac muscle, in both animal and human models. Recent experimental MRS studies have allowed new insights into the essential role of energetics in heart failure. Various new studies suggest a rapidly growing role of MRS for phenotyping new genetically modified mouse models, and recent methodologic advances include development of absolute quantification of high-energy phosphates, measurement of ATP turnover rates and thermodynamic parameters (such as free ADP and free energy change of ATP hydrolysis), and improved acquisition sequences. New patient studies demonstrate the potential value of MRS as a clinical diagnostic tool in patients with ischemic heart disease, heart failure, cardiac transplantation, valve disease, and genetic cardiomyopathy.

High-Energy Phosphate Metabolism in Normal, Hypertrophied and Failing Human Myocardium

This chapter examines the role of cardiac high-energy phosphate metabolism in normal, hypertrophied and failing human myocardium. Myocardial biopsies allow analysis of ATP, total adenine nucleotides, creatine kinase activity and total creatine content, while non-invasive 31P-magnetic resonance spectroscopy can be used to determine phosphocreatine/ATP ratios and, most recently, absolute levels of ATP and phosphocreatine. The failing human myocardium is characterized by reduced phosphocreatine and total creatine levels, normal or slightly reduced ATP levels and reduced creatine kinase activity. These changes are consistent with, but do not prove, a role of high-energy phosphate metabolism as a contributing factor in heart failure. An answer to the precise functional role of high-energy phosphate metabolism necessitates analysis of free ADP levels, free energy change of ATP hydrolysis and creatine kinase reaction velocity; these measurements may become feasible in coming years. However, analysis of energy metabolism in a compartmentalized manner, i.e., in the compartments relevant for contractile function such as the perimyofibrillar space, will remain elusive for the foreseeable future.

Intracellular sodium homeostasis in relation to the effective free-energy change of ATP hydrolysis: studies of crayfish muscle fibers

We investigated the impact of reductions in the effective free-energy change of ATP hydrolysis (dG/dATP) on intracellular sodium homeostasis in bundles of fibers from the abdominal extensor muscle of the crayfish Procambarus clarkii. 31P nuclear magnetic resonance (NMR) spectroscopy was used to monitor high-energy phosphate levels and intracellular pH while interleaved 23Na-NMR spectra were acquired to monitor changes in sodium. Previous work has shown that the bulk of intracellular Na+ is NMR visible (see Ivanics et al. 1994). The 23Na-NMR spectra were diffusion-weighted which effectively filtered out signal contributions from extracellular sodium. The efficacy of this procedure was validated using a relatively non-toxic chemical shift reagent which allowed resolution of extracellular and intracellular Na+ signals. Metabolic inhibition (cyanide/iodoacetate) produced pronounced reductions in dG/dATP coincident with dramatic increases in intracellular Na+ levels ([Na+]i). However, the increases in [Na+]i occurred at dG/dATP values well above the threshold value of −46 kJ · mol−1 required by the existing Na+ gradient and the membrane potential. These results suggest that the global dG/dATP value may not reflect the dG/dATP value in the vicinity of the pump. Alternatively, other factors, including low molecular modulators of Na+, K+-ATPase activity, may be important in this context.

Metabolic and energy correlates of intracellular pH in progressive fatigue of squid (L. brevis) mantle muscle.

Squid (Lolliguncula brevis) were exercised at increasing swimming speeds to allow us to analyze the correlated changes in intracellular metabolic, acid-base, and energy status of the mantle musculature. Beyond a critical swimming velocity of 1.5 mantle lengths/s, an intracellular acidosis developed that was caused by an initial base loss from the cells, the onset of respiratory acidification, and, predominantly, octopine formation. The acidosis was correlated with decreasing levels of phospho-L-arginine and, thus, supported ATP buffering at the expense of the phosphagen. Monohydrogenphosphate, the actual substrate of glycogen phosphorylase accumulated, enabling glycogen degradation, despite progressive acidosis. In addition to octopine, succinate, and glycerophosphate accumulation, the onset of acidosis characterizes the critical velocity and indicates the transition to a non-steady-state time-limited situation. Accordingly, swimming above the critical velocity caused cellular energy levels (in vivo Gibbs free energy change of ATP hydrolysis) to fall. A minimal value was reached at about -45 kJ/mol. Model calculations demonstrate that changes in free Mg2+ levels only minimally affect ATP free energy, but minimum levels are relevant in maintaining functional concentrations of Mg(2+)-complexed adenylates. Model calculations also reveal that phosphagen breakdown enabled L. brevis to reach swimming speeds about three times higher than the critical velocity. Comparison of two offshore squid species (Loligo pealei and Illex illecebrosus) with the estuarine squid L.brevis indicates that the latter uses a strategy to delay the exploitation of high-energy phosphates and protect energy levels at higher than the minimum levels (-42 kJ/mol) characterizing fatigue in the other species. A more economical use of anaerobic resources and an early reduction in performance may enable L. brevis to tolerate more extreme environmental conditions in shallow estuarine waters and even hypoxic environments and to prevent a fatal depletion of energy stores.

The effect of osmolarity on human placental mitochondria function

Human placental explants survive large changes in osmolarity, but the mechanism for this property is unknown. The goal of this work was to examine the effect of osmolarity on human placental mitochondria. Mitochondria from human term placenta were isolated by differential centrifugation, and incubated in the presence of different concentrations of sucrose or KCl, to modify the osmolarity of the media. Rat liver mitochondria were used as control. The parameters studied were: respiration rate, adenine nucleotide hydrolysis, calcium transport, membrane potential, and mitochondrial morphology. Stimulation of the mitochondrial respiration rate and an increase in Ca 2+ transport was observed in the presence of K +. With sucrose, Ca 2+ transport showed a complex kinetic behavior, whereas the respiratory control was slightly diminished. Although the ATPase activity was enhanced in the absence of a respiratory substrate, no change in ATP hydrolysis due to osmolarity was observed. ADP hydrolysis was inhibited by a high K + concentration, but not by sucrose. The membrane potential was not modified by osmolarity, even in the absence of sucrose or K + in the medium. Mitochondria isolated with KCl showed aggregation, whereas dispersed mitochondria were observed with sucrose. This study showed that sucrose-induced changes in osmolarity, does not modify metabolic and transport properties of human placental mitochondria, whereas KCl-induced osmolarity changes does affect these functions.

Graded intracellular acidosis produces extensive and reversible reductions in the effective free energy change of ATP hydrolysis in a molluscan muscle.

Phosphorus nuclear magnetic resonance spectroscopy was used to evaluate the impact of experimental reductions of intracellular pH on in vitro preparations of the radula protractor muscle of the marine gastropod, Busycon canaliculatum. The intracellular pH of radula refractor muscle bundles superfused with buffered artificial sea water (pH = 7.8) was 7.29. It was possible to clamp muscle intracellular pH at various acidotic states by changing the superfusate to 5, 10, and 15 mmol.l-1 5,5-dimethyl-oxazolidine-2,4-dione in buffered artificial sea water (pH = 6.5). Consistent and temporally stable reductions of intracellular pH were achieved (intracellular pH = 6.98, 6.79, and 6.62, respectively). During the acidotic transitions, arginine phosphate concentrations decreased and inorganic phosphate concentrations increased in a reciprocal manner and remained essentially constant after the intracellular pH stabilized. The extent of changes in arginine phosphate and inorganic phosphate was directly proportional to the magnitude of the imposed acidosis. Total adenosine triphosphate concentrations remained unchanged in all treatments. However, the magnesium adenosine triphosphate to total adenosine triphosphate ratio declined in direct relation to the extent of the acidosis. Intracellular free Mg2+ fell incrementally with reduced intracellular pH. All of the above effects were rapidly reversed when the 5,5-dimethyl-oxazolidine-2,4-dione was washed out by changing the superfusate to buffered artificial sea water (pH = 7.8). Mg-adenosine diphosphate concentrations were calculated in all treatments using equilibrium constants for the arginine kinase reaction corrected for pH and intracellular free [Mg2+]. The metabolite, intracellular pH, and [Mg2+] data were used to estimate the effective free energy of hydrolysis of adenosine triphosphate (dG/d xi ATP) under most experimental conditions. Experimental acidosis resulted in dramatic reductions in dG/d xi ATP which were fully reversible upon wash-out of 5,5-dimethyl-dioxazolidine-2,4-dione and recovery to normal intracellular pH conditions. Acidosis resulted in net hydrolysis of arginine phosphate, likely via a complex mechanism involving enhancement of rate of adenosine triphosphate hydrolysis and/or inhibition of adenosine triphosphate synthesis.

Hypoxia-induced activation of KATP channels limits energy depletion in the guinea pig heart.

The functional role of ATP-dependent potassium (KATP) in hypoxic cardiac failure was investigated in isolated guinea pig hearts with glibenclamide and rimalkalim as inhibitor and activator, respectively. Monophasic action potential duration at 90% of repolarization (MAP50), left ventricular function, and cardiac energy status (31P nuclear magnetic resonance spectroscopy) were measured during normotoxic (95% O2) and hypoxic (20% O2) perfusion. In normoxic hearts, 1 microM glibenclamide did not affect MAP50, left ventricular function, and coronary flow (n = 4). In contrast, rimalkalim rapidly shortened MAP50 and left ventricular pressure (LVP) in a dose-dependent fashion (e.g., by 60.2 +/- 3.5 and 80.8 +/- 8.2%, respectively, with 0.6 microM rimalkalim). This latter effect was reversed by 1 microM (glibenclamide (n = 4). With hypoxic perfusion, a reduction in LVP was observed, along with a shortening of the action potential (MAP90; 202 +/- 13 vs. 164 +/- 9 ms) and an increase in coronary flow. Glibenclamide (1 microM) reversed the MAP90 shortening and the increase in coronary flow. In addition, glibenclamide increased LVP transiently (n = 4). When coronary flow of hypoxic hearts was kept constant, however, glibenclamide elicited a sustained positive inotropic effect (n = 7). After glibenclamide, an increase in LVP from 54 +/- 4 to 64 +/- 3 mmHg was observed, along with a reduction in the free energy change of ATP hydrolysis from -54.5 +/- 1.9 to -52.9 +/- 0.2 nJ/mol and a further increase in the coronary venous adenosine from 269 +/- 48 to 1,680 +/- 670 nmol/l.(ABSTRACT TRUNCATED AT 250 WORDS)

Adaptation speed of cardiac mitochondrial oxygen consumption decreases with higher heart rate

The purpose of the present study was to determine whether the mean response time of cardiac mitochondrial oxygen consumption after a step in metabolic demand is constant in heart muscle, as has already been found for skeletal muscle. The mean response time reflects the average delay between the change in ATP hydrolysis due to a heart rate step and mitochondrial ATP production. Isolated rabbit hearts with a water-filled balloon in the left ventricle were perfused according to Langendorff with a constant flow of Tyrode solution at 28 degrees C. The mean response time increased significantly from 7.6 s for a step in heart rate from 60 to 70 min-1 to 12.1 s for a step from 60 to 120 min-1. The mean response times for heart rate steps downward from 120 min-1 were all approximately 12 s, but for the step from 120 to 140 min-1 the response time was 16.8 s. These results demonstrate that the mean response time of cardiac mitochondrial oxygen consumption in most cases increases with heart rate. These findings are in contrast to those obtained in skeletal muscle, where the response time at which ATP synthesis adapts to a change in work load is constant.

Metabolic and functional responses of immature and mature rabbit hearts to hypoperfusion, ischemia, and reperfusion

Relationships between contractile function and cytosolic metabolism were examined in mature and immature rabbit hearts during graded hypoperfusion, global ischemia, and reperfusion. Differences in basal contractile function and metabolic demand in immature and mature hearts were paralleled by differences in energy substrate indexed by the phosphorylation potential and phosphocreatine (PCr) levels but not by differences in ATP, pH, or inorganic phosphate (Pi). During graded hypoperfusion, contractility declined comparably in both age groups. Relative changes in [ATP], [PCr], and [Pi] were similar in both groups, whereas pH declined to a greater extent in mature hearts, and [Mg2+] declined to a greater extent in immature hearts. Contractile function correlated most consistently with [Pi] and supports the notion that Pi (and H+) are primary determinants of function in underperfused immature and mature hearts. Aerobic efficiency declined in immature but not mature hearts during hypoperfusion, reflecting an improved ratio of ATP formation relative to ATP hydrolysis in the hypoperfused immature myocardium. Finally, the enhanced contractile recovery from global ischemia in immature compared with mature hearts (to 93 +/- 4 vs. 79 +/- 2% of basal function) was unrelated to recovery of ATP, Pi, pH, Mg2+, [ATP]/[ADP].[Pi], or free-energy change of ATP hydrolysis. Age-related differences in maintenance of ATP may be related to enhanced metabolic activation of glycolysis coupled with better buffering of pH and an improved match between ATP hydrolysis and ATP formation in immature heart.

Mitochondrial dehydrogenase activity affects adaptation of cardiac oxygen consumption to demand.

The effect of regulation of mitochondrial dehydrogenase activities on the mean response time of mitochondrial oxygen consumption, which characterizes the delay between changes in ATP hydrolysis and changes in oxygen consumption, was investigated in isolated rabbit hearts and perfused with Tyrode solution at 28 degrees C. Perfusion with ruthenium red (RR) blocks mitochondrial calcium uptake and thus decreases mitochondrial dehydrogenase activities. Perfusion with pyruvate increases pyruvate dehydrogenase activity. The mean response time was 11.8 +/- 0.7 s (means +/- SE) during control, 12.2 +/- 1.2 s during perfusion with 0.9 microgram/ml RR, and 20.7 +/- 3.4 s during perfusion with 2.1 micrograms/ml RR. Blockade with 0.9 microgram/ml RR, which is presumably partial, did not slow the response, suggesting that mitochondrial calcium uptake may not be rate limiting. Strong blockade of mitochondrial calcium uptake increases the mean response time, presumably due to decreased calcium activation of the mitochondrial dehydrogenases. Perfusion with pyruvate significantly decreased the mean response time to 10.0 +/- 1.4 s compared with 11.9 +/- 0.7 s during perfusion with glucose. This decrease with pyruvate is not compatible with a shift to regulation by high-energy phosphates but may reflect increased mitochondrial oxidative capacity caused by increased NADH levels.

Temporary contractile blockade prevents hypercontracture in anoxic-reoxygenated cardiomyocytes

Reoxygenation after 120-min substrate-free anoxia causes sudden hypercontracture in isolated rat cardiomyocytes. Reoxygenated-hypercontracted cardiomyocytes maintain their sarcolemmal integrity as indicated by the absence of enzyme release and reestablish a nearly normal free energy change of ATP hydrolysis within 15 min [Siegmund, B., A. Koop, T. Klietz, P. Schwartz, and H. M. Piper.Am J. Physiol. 258 (Heart Circ. Physiol. 27): H285-H291, 1990]. In the same model, it was now investigated whether a temporary contractile blockade by 20 mM 2,3-butanedione monoxime (BDM) can prevent reoxygenation-induced hypercontracture. When BDM was present during 120-min anoxia and the subsequent 15-min reoxygenation, hypercontracture could be prevented. The anoxic changes of high-energy phosphate contents, the free energy change of ATP hydrolysis, and the ultrastructure of the cells remained unaffected by the presence of BDM. When BDM was applied anoxically immediately before reoxygenation, it also prevented hypercontracture. Contracture still remained absent when BDM was washed out after the first 15 min of reoxygenation. These results demonstrate that a temporary contractile blockade (15 min) at the onset of reoxygenation prevents hypercontracture in anoxic-reoxygenated cardiomyocytes. This result, the energetic recovery, and the sarcolemmal integrity of cardiomyocytes in anoxia-reoxygenation demonstrate that reoxygenation-induced hypercontracture is not based on an already irreversible cell damage.

Na+-K+-ATPase and Changes in ATP Hydrolysis, Monovalent Cation Affinity, and K+ Occlusion in Diabetic and Galactosemic Rats

This study showed that steady-state kinetics of ATP hydrolysis by Na(+)-K(+)-ATPase are altered in the BB Wistar diabetic rat and experimental galactosemia. Four days after onset, this change was not evident if NaCNBH3 was omitted during enzyme preparations (indicating reversibility). Ninety days after onset, NaCNBH3 reduction was not necessary to see the change in ATP hydrolysis kinetics (indicating nonreversibility). The change in steady-state ATP hydrolysis was similar to that reported earlier for Na(+)-K(+)-ATPase of the lens epithelium and kidney medulla of diabetic individuals and for two in vitro glycosylation models. Our study also showed that the affinities of Na(+)-K(+)-ATPase for K+ are altered, and Na(+)-K(+)-ATPase-dependent K+ occlusion is inhibited in diabetic and galactosemic animals. Because K+ occlusion is required for efficient K+ transport, this finding supports previous in vitro studies that indicated that glycosylation inhibits pump-dependent K+ transport. Furthermore, our study suggested an irreversible impairment of Na(+)-K(+)-ATPase function in the diabetic BB Wistar rat as early as 15 days after onset, even when blood glucose was maintained at 6.7 mM by daily insulin injection.

Na(+)-K(+)-ATPase and changes in ATP hydrolysis, monovalent cation affinity, and K+ occlusion in diabetic and galactosemic rats

Na(+)-K(+)-ATPase and changes in ATP hydrolysis, monovalent cation affinity, and K+ occlusion in diabetic and galactosemic rats