Energy Change Of ATP Hydrolysis Research Articles

Structure of Dimeric F1F0-ATP Synthase

The structure of the dimeric ATP synthase from yeast mitochondria was analyzed by transmission electron microscopy and single particle image analysis. In addition to the previously reported side views of the dimer, top view and intermediate projections served to resolve the arrangement of the rotary c(10) ring and the other stator subunits at the F(0)-F(0) dimeric interface. A three-dimensional reconstruction of the complex was calculated from a data set of 9960 molecular images at a resolution of 27 Å. The structural model of the dimeric ATP synthase shows the two monomers arranged at an angle of ∼45°, consistent with our earlier analysis of the ATP synthase from bovine heart mitochondria (Minauro-Sanmiguel, F., Wilkens, S., and Garcia, J. J. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 12356-12358). In the ATP synthase dimer, the two peripheral stalks are located near the F(1)-F(1) interface but are turned away from each other so that they are not in contact. Based on the three-dimensional reconstruction, a model of how dimeric ATP synthase assembles to form the higher order oligomeric structures that are required for mitochondrial cristae biogenesis is discussed.

Inferring crossbridge properties from skeletal muscle energetics

Work is generated in muscle by myosin crossbridges during their interaction with the actin filament. The energy from which the work is produced is the free energy change of ATP hydrolysis and efficiency quantifies the fraction of the energy supplied that is converted into work. The purpose of this review is to compare the efficiency of frog skeletal muscle determined from measurements of work output and either heat production or chemical breakdown with the work produced per crossbridge cycle predicted on the basis of the mechanical responses of contracting muscle to rapid length perturbations. We review the literature to establish the likely maximum crossbridge efficiency for frog skeletal muscle (0.4) and, using this value, calculate the maximum work a crossbridge can perform in a single attachment to actin (33 x 10(-21) J). To see whether this amount of work is consistent with our understanding of crossbridge mechanics, we examine measurements of the force responses of frog muscle to fast length perturbations and, taking account of filament compliance, determine the crossbridge force-extension relationship and the velocity dependences of the fraction of crossbridges attached and average crossbridge strain. These data are used in combination with a Huxley-Simmons-type model of the thermodynamics of the attached crossbridge to determine whether this type of model can adequately account for the observed muscle efficiency. Although it is apparent that there are still deficiencies in our understanding of how to accurately model some aspects of ensemble crossbridge behaviour, this comparison shows that crossbridge energetics are consistent with known crossbridge properties.

An examination of the metabolic processes underpinning critical swimming in Atlantic cod (Gadus morhua L.) using in vivo31P-NMR spectroscopy

Traditionally, critical swimming speed has been defined as the speed when a fish can no longer propel itself forward, and is exhausted. To gain a better understanding of the metabolic processes at work during a U(crit) swim test, and that lead to fatigue, we developed a method using in vivo (31)P-NMR spectroscopy in combination with a Brett-type swim tunnel. Our data showed that a metabolic transition point is reached when the fish change from using steady state aerobic metabolism to non-steady state anaerobic metabolism, as indicated by a significant increase in inorganic phosphate levels from 0.3+/-0.3 to 9.5+/-3.4 mol g(-1), and a drop in intracellular pH from 7.48+/-0.03 to 6.81+/-0.05 in muscle. This coincides with the point when the fish change gait from subcarangiform swimming to kick-and-glide bursts. As the number of kicks increased, so too did the Pi concentration, and the pH(i) dropped. Both changes were maximal at U(crit). A significant drop in Gibbs free energy change of ATP hydrolysis from -55.6+/-1.4 to -49.8+/-0.7 kJ mol(-1) is argued to have been involved in fatigue. This confirms earlier findings that the traditional definition of U(crit), unlike other critical points that are typically marked by a transition from aerobic to anaerobic metabolism, is the point of complete exhaustion of both aerobic and anaerobic resources.

Temperature-dependent pH regulation in eurythermal and stenothermal marine fish: an interspecies comparison using 31P-NMR

Temperature-induced pH changes in white muscle tissue of three eelpout populations with different levels of eurythermy (the cold stenothermal Antarctic species Pachycara brachycephalum and the temperate eelpout Zoarces viviparus from the North Sea and the Baltic Sea) were monitored online by use of in vivo 31P-NMR in unrestrained, unanaesthetized fish. An intracellular pH (pH i) change of around −0.015 pH units/°C was observed in all eelpout populations in accordance with the α-stat hypothesis. The pH change was completed earlier (within 4 h) in the stenothermal Antarctic eelpout than in the Baltic population (within 8 h) and latest (not within 12 h) in the eurythermal North Sea population. These findings confirm the hypothesis that the kinetics of temperature-dependent pH i regulation is reflected by the relative contribution of active and passive processes to a temperature-induced pH change. The extent of passively induced pH i changes is in line with the general hypothesis that the temperature-dependent adjustment of pH i occurs mostly by active mechanisms in eurythermal animals, whereas in stenothermal animals pH changes are largely elicited by passive processes. Temperature changes had no influence on high-energy phosphates like phosphocreatine and ATP or on the Gibbs free energy change of ATP hydrolysis (Δ G/Δ ξ).

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.

Role of endogenous opioids in ischemic preconditioning but not in short-term hibernation in pigs.

Endogenous opioids are involved in ischemic preconditioning (IP) in several species. Whether or not opioids are important for IP and short-term myocardial hibernation (STMH) in pigs is currently unknown. In 34 enflurane-anesthetized pigs, the left anterior descending coronary artery was flow constantly perfused. Subendocardial blood flow (Endo), infarct size (IS; percent area at risk), and the free energy change of ATP hydrolysis (DeltaG) were determined. After 90-min severe ischemia and 120-min reperfusion, IS averaged 28.3 +/- 5.4% (means +/- SE) (n = 8; Endo: 0.047 +/- 0.009 ml. min(-1) x g(-1)). IP by 10-min ischemia and 15-min reperfusion reduced IS to 9.9 +/- 3.8% (P < 0.05, n = 8; Endo: 0.044 +/- 0.009 ml. min(-1) x g(-1)). After naloxone (1 mg/kg iv followed by 2 microg x kg(-1) x min(-1)), IS averaged 25.8 +/- 7.0% (n = 6; Endo: 0.039 +/- 0.008 ml x min(-1) x g(-1)) without and 24.7 +/- 4.7% (n = 6; Endo: 0.044 +/- 0.006 ml x min(-1) x g(-1)) with IP. At 5-min moderate ischemia in the presence of naloxone, Endo decreased from 0.90 +/- 0.07 to 0.28 +/- 0.03 ml x min(-1) x g(-1)and DeltaG decreased from -58.6 +/- 1.0 to -52.6 +/- 0.4 kJ/mol. Prolongation of ischemia to 90 min did not alter Endo, but DeltaG recovered toward control values (57.7 +/- 1.1 kJ/mol), and the myocardium remained viable. These responses are identical to those of nonnaloxone-treated pigs. Endogenous opioids are involved in IP but not in STMH in pigs.

Metabolic performance of the squid Lolliguncula brevis (Cephalopoda) during hypoxia: an analysis of the critical PO2

Brief squid Lolliguncula brevis are regularly exposed to fluctuating oxygen levels in their shallow coastal environment. To assess hypoxia resistance, animals were exposed for two hours to ambient oxygen partial pressures ( P O 2 ) between 19.3 kPa (normoxia) and 6.6 kPa (34.2% air saturation) at 20±1°C. In a second set of experiments, the animals were subjected to a low P O 2 of 2.8±0.3 kPa (14.5±1.6% air saturation) for 15 to 60 min. Subsequently, metabolic, energy and acid–base status were analysed in the mantle tissue. Onset of anaerobic metabolism was observed between 9.4 and 7.9 kPa (48.7 and 40.9% air saturation), reflecting the critical oxygen tension for this species. The formation of octopine and acetate indicates a simultaneous onset of anaerobic metabolism in both the cytosol and the mitochondria during progressive hypoxia. Concomitantly, an intracellular acidosis developed. During exposure to oxygen partial pressures between 19.3 and 6.6 kPa, aerobic and anaerobic processes were sufficient to maintain energy status in the mantle musculature. No significant changes in ATP and phospho- l -arginine (PLA) concentrations were observed. In contrast, both ATP and PLA levels declined significantly after 15 min at an ambient P O 2 of 2.8±0.3 kPa. Concomitantly, the Gibb’s free energy change of ATP hydrolysis fell to a minimum value of about −44 kJ·mol −1 , a level suggested to reflect limiting energy availability for cellular ATPases. These results indicate that hypoxia at 2.8 kPa (14.5% air saturation) rapidly takes Lolliguncula brevis to the limits of performance. However, it is probably capable of withstanding longer periods of moderate hypoxia close to 50% air saturation (9.7 kPa), enabling the squids to cope with oxygen fluctuations in their shallow estuarine environment or to dive into hypoxic waters by use of their economic jetting strategy. Nonetheless, the critical P O 2 is considered to be high compared to other hypoxia tolerant animals, an observation likely related to the high metabolic rate of these squids.

Intracellular pH and energy metabolism in the highly stenothermal Antarctic bivalve Limopsis marionensis as a function of ambient temperature

Changes in oxygen consumption, ammonia excretion and in the acid-base and energy status of various tissues were investigated in the cold stenothermal Antarctic bivalve, Limopsis marionensis, and compared to similar data in the limpet, Nacella concinna, for an assessment of thermal sensitivity. Oxygen consumption of L. marionensis varied between −1.5 and 2°C with a Q 10 of 2.2. Ammonia excretion could only be detected in animals exposed to elevated temperature for periods in excess of 45 days and close to death and it is interpreted as the onset of protein and amino acid catabolism with starvation under temperature stress. In L. marionensis any change in temperature as well as starvation stress at constant temperature induced a decrease in phospho-l-arginine and ATP levels. However, only temperature stress resulted in a drop in the Gibb's free energy change of ATP hydrolysis. Intracellular pH rose in all tissues during upward or downward temperature changes of only 1.5 or 2°C for 24 h with a concomitant trend to accumulate succinate and acetate in the tissues. These changes are seen to reflect disturbances of the tissue acid-base and energy status with any under- or overshoot in aerobic metabolic rate during a temperature decrease or increase. Elevated temperature at 2°C during 2 weeks of incubation resulted in continued net ATP depletion, at low levels of ATP free energy. This indicates long-term stress, which was also mirrored in the inability to establish a new steady-state mean rate of oxygen consumption. Incubation at even higher temperatures of 4 and 7°C led to an aggravation of energetic stress and transition to an intracellular acidosis, as well as a fall in oxygen consumption. In N. concinna a drop in energy levels was also visible at 2°C but was compensated for during long-term incubation. In conclusion, L. marionensis will be able to compensate for a temperature change only in a very narrow range whereas the thermal tolerance window is much wider in N. concinna. The inability of the metabolic rate to rise continually and the concomitant transition to anaerobic metabolism and long-term energetic stress characterize the upper critical temperature. Stenothermality is discussed, not only as reflecting the permanent and very stable low temperature in the natural environment, but also regarding dif- ferences in the level of activity and aerobic scope.

Habitat as a factor involved in the physiological response to environmental anaerobiosis of White Sea Mytilus edulis

The ability of blue mussels Mytilus edulis L. to withstand severe environmental hypoxia was studied in mussels from an intertidal population and from a suspended cultured settlement. Specimens were exposed to air for 60 h at + 10°C. Tissues were analysed for the amount of anaerobic metabolic end products, adenylates, phosphagen, and inorganic phosphate, and for changes in intracellular pH (pH i ). Proton balance and Gibb's free energy of ATP hydrolysis were calculated. Under the experimental conditions applied succinate appeared to be the only anaerobic end product. Under control conditions pH i measured using the homogenate technique ranged between 6.78 and 6.85 in both groups. Very small decreases in pH i were observed after air exposure. Rapid breakdown of ATP and phosphoL-arginine (PLA) was accompanied by the accumulation of inorganic phosphate, free AMP and ADP. The Gibb's free energy change of ATP hydrolysis decreased from about -57 to -50 kJ mol 1 , showing the depletion of energy reserves in the tissues. The calculated ATP turnover rate was higher in intertidal mussels. It is concluded that mussels from the intertidal zone demonstrate lower abilities for metabolic depression under the conditions of air exposure than cultured (sublittoral) ones. The latter are characterised by higher initial PLA content. These differences may lie related to a difference in mitochondrial density depending on the habitat.

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.

Inorganic phosphate content and free energy change of ATP hydrolysis in regional short-term hibernating myocardium.

Short-term myocardial hibernation is characterized by an adaptation of contractile function to the reduced blood flow, the recovery of creatine phosphate content and lactate balance back towards normal, whereas ATP content remains reduced at a constant level. We examined the hypothesis that, despite the absence of ATP recovery, the short-term hibernating myocardium regains an energetic balance. An enzymatic method was modified for the measurement of inorganic phosphate (Pi) in transmural myocardial drill biopsies (about 5 mg). In 12 anaesthetized swine, moderate ischemia was induced by reduction of coronary inflow into the cannulated left anterior descending coronary artery to decrease regional myocardial function (sonomicrometry) by 50%. The development of short-term hibernation was verified by the recovery of creatine phosphate content, the persistence of inotropic reserve in response to dobutamine and the absence of necrosis (triphenyl tetrazolium chloride). At 5-min ischemia, Pi was increased from 3.6 +/- 0.3 (SD) to 8.1 +/- 1.1 mumol/gwet wt (p < 0.05). The free energy of ATP hydrolysis (delta GATP) was decreased from -57.8 +/- 0.8 to -52.2 +/- 1.4 kJ/mol (p < 0.05). The relationships between function and Pi (r = -0.81) and delta GATP (r = -0.83), respectively, during control and at 5-min ischemia became invalid at 90-min ischemia, as myocardial blood flow and function remained reduced at a constant level, but Pi decreased back to 4.9 +/- 0.9 mumol/g (p < 0.05 vs. control and 5-min ischemia), and delta GATP fully recovered back to -57.2 +/- 1.3 kJ/mol (p < 0.05 vs. 5-min ischemia). In short-term hibernating myocardium, myocardial inorganic phosphate content recovers partially and the free energy change of ATP hydrolysis returns to control values. Contractile function remains reduced by mechanisms other than an energetic deficit.

High-energy turnover at low temperatures: recovery from exhaustive exercise in Antarctic and temperate eelpouts.

Earlier work on Notothenioids led to the hypothesis that a reduced glycolytic capacity is a general adaptation to low temperatures in Antarctic fish. In our study this hypothesis was reinvestigated by comparing changes in the metabolic status of the white musculature in two related zoarcid species, the stenothermal Antarctic eelpout Pachycara brachycephalum and the eurythermal Zoarces viviparus during exercise and subsequent recovery at 0 degreesC. In both species, strenuous exercise caused a similar increase in white muscle lactate, a drop in intracellular pH (pHi) by about 0.5 pH units, and a 90% depletion of phosphocreatine. This is the first study on Antarctic fish that shows an increase in white muscle lactate concentrations. Thus the hypothesis that a reduced importance of the glycolytic pathway is characteristic for cold-adapted polar fish cannot hold. The recovery process, especially the clearance of white muscle lactate, is significantly faster in the Antarctic than in temperate eelpout. Based on metabolite data, we calculated that during the first hour of recovery aerobic metabolism is increased 6.6-fold compared with resting rates in P. brachycephalum vs. an only 2.9-fold increase in Z. viviparus. This strong stimulation of aerobic metabolism despite low temperatures may be caused by a pronounced increase of free ADP levels, in the context of higher levels of pHi and ATP, which is observed in the Antarctic species. Although basal metabolic rates are identical in both species, the comparison of metabolic rates during situations of high-energy turnover reveals that the stenothermal P. brachycephalum shows a higher degree of metabolic cold compensation than the eurythermal Z. viviparus. Muscular fatigue after escape swimming may be caused by a drop of the free energy change of ATP hydrolysis, which is shown to fall below critical levels for cellular ATPases in exhausted animals of both species.

Acid-base regulation, metabolism and energetics in sipunculus nudus as a function of ambient carbon dioxide level

Changes in the rates of oxygen consumption and ammonium excretion, in intra- and extracellular acid-base status and in the rate of H+-equivalent ion transfer between animals and ambient water were measured during environmental hypercapnia in the peanut worm Sipunculus nudus. During exposure to 1 % CO2 in air, intracellular and coelomic plasma PCO2 values rose to levels above those expected from the increase in ambient CO2 tension. Simultaneously, coelomic plasma PO2 was reduced below control values. The rise in PCO2 also induced a fall in intra- and extracellular pH, but intracellular pH was rapidly and completely restored. This was achieved during the early period of hypercapnia at the expense of a non-respiratory increase in the extracellular acidosis. The pH of the extracellular space was only partially compensated (by 37 %) during long-term hypercapnia. The net release of basic equivalents under control conditions turned to a net release of protons to the ambient water before a net, albeit reduced, rate of base release was re-established after a new steady state had been achieved with respect to acid-base parameters. Hypercapnia also affected the mode and rate of metabolism. It caused the rate of oxygen consumption to fall, whereas the rate of ammonium excretion remained constant or even increased, reflecting a reduction of the O/N ratio in both cases. The transient intracellular acidosis preceded a depletion of the phosphagen phospho-l-arginine, an accumulation of free ADP and a decrease in the level of Gibbs free energy change of ATP hydrolysis, before replenishment of phosphagen and restoration of pHi and energy status occurred in parallel. In conclusion, long-term hypercapnia in vivo causes metabolic depression, a parallel shift in acid-base status and increased gas partial pressure gradients, which are related to a reduction in ventilatory activity. The steady-state rise in H+-equivalent ion transfer to the environment reflects an increased rate of production of protons by metabolism. This observation and the reduction of the O/N ratio suggest that a shift to protein/amino acid catabolism has taken place. Metabolic depression prevails, with completely compensated intracellular acidosis during long-term hypercapnia eliminating intracellular pH as a significant factor in the regulation of metabolic rate in vivo. Fluctuating levels of the phosphagen, of free ADP and in the ATP free energy change values independent of pH are interpreted as being correlated with oscillating ATP turnover rates during early hypercapnia and as reflecting a tight coupling of ATP turnover and energy status via the level of free ADP.

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.

Metabolic depression during environmental stress: the role of extracellular versus intracellular pH in Sipunculus nudus

Environmental stresses such as hypoxia or hypercapnia are known to cause acid-base disturbances and in several organisms they lead to metabolic depression. The present study was undertaken to quantify the influence of these changes in acid&shyp;base parameters on metabolic rate. We determined the rate of oxygen consumption in a non-perfused preparation of the body wall musculature of the marine worm Sipunculus nudus at various levels of extra- and intracellular pH (pHe and pHi, respectively), PCO2 and [HCO3-]. The acid&shyp;base status of the tissue was modified and clamped by long-term exposure to media set to specific values of extracellular pH, PCO2 and [HCO3-]. At a pHe of 7.90, which is equivalent to the normoxic normocapnic in vivo extracellular pH, and an ambient PCO2 of 0.03 kPa (control conditions), pHi was 7.26&plusmn;0.02 (mean &plusmn; s.d., N=5). A reduction of extracellular pH from 7.90 to 7.20 resulted in a significant decrease of pHi to 7.17&plusmn;0.05 at 0.03 kPa PCO2 (normocapnia) and to 7.20&plusmn;0.02 at 1.01 kPa PCO2 (hypercapnia). At the same time, the rate of oxygen consumption of the tissue was significantly depressed by 18.7&plusmn;4.7 % and 17.7&plusmn;3.0 %, respectively. A significant depression of oxygen consumption by 13.7&plusmn;4.7 % also occurred under hypercapnia at pHe 7.55 when pHi was elevated above control values (7.32&plusmn;0.01). No significant changes in oxygen consumption were observed when pHe was either drastically elevated to 8.70 under normocapnia (pHi 7.36&plusmn;0.05) or maintained at 7.90 during hypercapnia (pHi 7.37&plusmn;0.03). ATP and phospho-l-arginine concentrations, as well as the Gibbs free energy change of ATP hydrolysis (dG/dATP), were maintained at high levels during all treatments, indicating an equilibrium between energy supply and demand. We conclude that the depression of aerobic energy turnover in isolated body wall musculature of S. nudus is induced by low extracellular pH. A model is proposed which could explain a reduced ATP cost of pHi regulation during extracellular acidosis, thus contributing to metabolic depression.

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)

Protection of energy status of hypoxic cardiomyocytes by mild acidosis.

The question was investigated whether mild acidosis can provide energetic protection on the hypoxic myocardial cell by mechanisms other than negative inotropy. The experimental model used was quiescent ventricular cardiomyocytes isolated from the adult rat. The extracellular pH (pHe) in modified hypoxic Tyrode's solution was varied by changing the concentration of bicarbonate at constant PCO2. The intracellular pH (pHi) was determined with 2',7'-bis-(carboxyethyl)5-(6')-carboxyfluorescein. When the pHe was varied between 6.6 and 7.4, the pHi varied only between 6.91 and 7.14. At pH 7.0, pHi and pHe were identical. Around pHe 7.0, the rate of hypoxic energy loss was lowest. Energetic differences were most prominent after 90 min hypoxia. At this time loss of ATP was 57.1 +/- 2.9% at pHe 6.95 vs. 83.5 +/- 4.1% at pHe 7.4 and 73.9 +/- 2.8% at pHe 6.6; the free energy change of ATP hydrolysis was reduced from 58.8 +/- 0.4 kJ/mol under control conditions to 52.0 +/- 1.1 kJ/mol at pHe 6.95 vs. 45.8 +/- 1.2 and 47.3 +/- 1.4 kJ/mol at pHe 7.4 and 6.6, respectively (means +/- S.D.). Anaerobic lactate production was not significantly altered in the investigated pHe range, indicating that glycolytic energy production remained constant. The results of the present study demonstrate that mild acidosis (pHe around 7.0) has a prominent energy-saving effect on the hypoxic cardiomyocyte due to a reduction in the demand of energy.

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.

31P NMR spectroscopy of hypertrophied rat heart: Effect of graded global ischemia

To investigate the cause for the greater susceptibility of hypertrophied hearts to ischemic injury, we determined the interrelations of total work output, contractile function and energy metabolism in isolated, perfused normal and hypertrophied rat hearts subjected to graded global ischemia. Cardiac hypertrophy was induced by giving rats seven daily injections of either triiodothyronine (0.2 mg/kg) or isoproterenol (5 mg/kg). All hearts were perfused at an aortic pressure of 100 mmHg in the isovolumic mode in an NMR spectrometer (7.05 Tesla). Heart rate, developed pressure, and coronary flow were monitored simultaneously with changes in pH, creatine phosphate, ATP and inorganic phosphate. During pre-ischemic perfusion, the total work output (rate-pressure product) of hyperthyroid hearts was 28% higher than that of control hearts, whereas hearts from isoproterenol-treated animals showed no difference. However, when related to unit muscle mass, work was normal in hyperthyroid hearts and 26% lower after isoproterenol. Contractile function per unit myocardium (developed pressure/g wet weight) was lower in the hypertrophied hearts. ATP content was the same in all groups. Creatine phosphate decreased 41% after triiodothyronine and 25% after isoproterenol. Inorganic phosphate levels and intracellular pH were similar in control and isoproterenol-treated rat hearts, but were higher in the hyperthyroid rat hearts. The phosphorylation potential and the free energy change of ATP hydrolysis were lowered by hypertrophy, the levels correlating with the depressed contractile function. At each ischemic flow rate, both work and contractile function per unit myocardium were the same for all hearts, but the relations between flow and phosphorylation potential were different for each type of heart. Thus, at low flow rates, hypertrophied hearts perform the same amount of work and have the same contractile function as control hearts, but with abnormal changes in energy metabolism, indicating that the relations of energy status to coronary flow, total work output and contractile function are altered during the process of hypertrophy.