ATP Free Energy Research Articles

Qdot Labeled Actin Super-Resolution Motility Assay Measures Step-Size for a Low Duty Cycle Muscle Myosin

Myosin powers contraction in heart and skeletal muscle and is a leading target for mutations implicated in inheritable muscle diseases. During contraction, myosin transduces ATP free energy into the work of muscle shortening against resisting force. Muscle shortening involves the relative sliding of myosin and actin filaments that is sometimes modeled in vitro with a motility assay quantitating actin filament translation over a myosin coated surface. Skeletal actin filaments were fluorescence labeled with a streptavidin conjugate quantum dot (Qdot) binding biotin-phalloidin on actin. Single Qdot's were imaged in time as they translated with actin over the skeletal heavy meromyosin (HMM) coated surface using total internal reflection fluorescence microscopy. Qdots were spatially localized to a few nanometers using a super-resolution algorithm and tracked over time. Average Qdot-actin velocity matched measurements with rhodamine-phalloidin labeled actin. The Qdot-actin velocity histogram contained low velocity events corresponding to actin translation in quantized steps of ∼5 nm. The quantization was independent of myosin surface concentration. Skeletal or cardiac myosin is a low duty cycle motor presenting challenges for a single molecule assay because actomyosin dissociates quickly and the freely moving element diffuses away. The in vitro motility assay enables modestly more actomyosin interactions to sustain the complex while preserving a subset of encounters that do not overlap in time on a single actin filament. A single myosin step is isolated in time and space then characterized using super-resolution. The approach opens the way for quick, quantitative, and inexpensive step-size measurement in low duty cycle muscle myosins.This research was supported by NIH Institutes: NIAMS R01 AR049277 and NHLBI HL095572 and by the Mayo Foundation.

Pyruvate-fortified resuscitation stabilizes cardiac electrical activity and energy metabolism during hypovolemia

To test the hypothesis that fluid resuscitation with Ringer's solution enriched with pyruvate (PR), a physiological antioxidant and energy substrate, affords protection of myocardial metabolism and electrophysiological performance superior to lactated Ringer's (LR) during hypovolemia and hindlimb ischemia-reperfusion. Male domestic goats (25-30 kg) were exsanguinated to a mean arterial pressure of 48 ± 1 mmHg. Right hindlimb ischemia was imposed for 90 min by applying a tourniquet and femoral crossclamp. LR or PR, infused iv, delivered 0.05 mmol/kg per minute L-lactate or pyruvate, respectively, from 30 min hindlimb ischemia until 30 min post-ischemia. Time controls (TC) underwent neither hemorrhage, hindlimb ischemia nor resuscitation. Goats were sacrificed and left ventricular myocardium biopsied at 90 min fluid resuscitation (n = 6 per group) or 3.5 h later (n = 9 LR, 10 PR, 8 TC). Myocardial 8-isoprostane content, phosphocreatine phosphorylation potential, creatine kinase activity, and heart rate-adjusted QT interval (QTc) variability were evaluated at 90 min resuscitation and 3.5 h post-resuscitation. PR sharply lowered pro-arrhythmic QTc variability vs LR (P < 0.05); this effect persisted 3.5 h post-resuscitation. PR lowered myocardial 8-isoprostane content, a product of oxidative stress, by 39 and 37% during and 3.5 h after resuscitation, respectively, vs LR. Creatine kinase activity fell 42% post-LR vs TC (P < 0.05), but was stable post-PR (P < 0.02 vs post-LR). PR doubled phosphocreatine phosphorylation potential, a measure of ATP free energy state, vs TC and LR (P < 0.05); this energetic enhancement persisted 3.5 h post-resuscitation. By augmenting myocardial energy state and protecting creatine kinase activity, pyruvate-enriched resuscitation stabilized cardiac electrical function during central hypovolemia and hindlimb ischemia-reperfusion.

3PT160 ABCトランスポーターのヌクレオチド結合ドメインに対するATP結合自由エネルギー計算(日本生物物理学会第50回年会(2012年度))

3PT160 ABCトランスポーターのヌクレオチド結合ドメインに対するATP結合自由エネルギー計算(日本生物物理学会第50回年会(2012年度))

Single Myosin Cross-Bridge Orientation in Cardiac Papillary Muscle Detects Lever-Arm Shear Strain in Transduction

Myosin motors transduce ATP free energy into mechanical work. Transduction models allocate specific functions to motor structural domains beginning with ATP hydrolysis in the active site and ending in a lever-arm rotating power-stroke. Myosin light chains, regulatory (RLC) and essential (ELC), bind IQ-domains on the lever-arm and track its movement. Strong evidence exists that light chains stabilize the lever-arm and that light chain mutation undermines stability. Human ventricular RLC tagged with photoactivatable GFP (HCRLC-PAGFP) replaces native RLC in porcine papillary muscle fibers, restores native contractility, and situates PAGFP for single molecule orientation tracking within the crowded fiber lattice. The spatial emission pattern from single photoactivated PAGFP tagged myosins was observed in z-stacks fitted simultaneously to maximize accuracy in estimated dipole orientation. Emitter dipole polar and azimuthal angle pair scatter plots identified an area where steric and molecular crowding constraints depopulated orientations unfavorable for actin interaction. Transitions between pre- and post-power-stroke states represent the lever-arm trajectory sampled by the data and quantify lever-arm shear strain in transduction at three tension levels. These data identify forces acting on myosin in the in situ fiber system due to crowding, steric hindrance, and actomyosin interaction. They induce lever-arm shear strain observed with single molecule orientation detection. A single myosin work histogram reveals discretized power-stroke substates reminiscent of the Huxley-Simmons model for myosin based contraction [Huxley and Simmons ( 1971 ) Nature 233 , 533]. RLC or ELC mutation, should it impact lever-arm shear strain, will be detected as changes in single myosin shear strain or power-stroke substate distribution.

Revisiting the Thermodynamic Theory of Optimal ATP Stoichiometries by Analysis of Various ATP-Producing Metabolic Pathways

The stoichiometry of ATP-producing metabolic pathways had been analysed theoretically by several authors by using evolutionary arguments and optimality principles. Waddell et al. (Biochem Educ 27:12-13, 1999) analysed (lactate-producing) glycolysis and used linear irreversible thermodynamics. The result was that half of the free-energy difference should be converted into free-energy of ATP and the remaining half should be used to drive the pathway. The calculated stoichiometry is in agreement with the observed yield of two moles of ATP per mole of glucose. Using the same approach, we here analyse eight other metabolic pathways. Although the deviation is not very large, the calculated values do not fit as nicely as for glycolysis as leading to lactate. For example, for O₂ respiration, the theoretical ATP yield equals 27.9. The real value varies among organisms between 26 and 38. For mixed-acid fermentation in Escherichia coli, the theoretical and experimental values are 2.24 and 2, respectively. For arginine degradation in M. pneumoniae, the calculated value is 2.43 mol of ATP, while in vivo only one mole is produced. During evolution, some pathways may not have reached their optimal ATP net production because energy yield is not their only function. Moreover, it should be acknowledged that the approach by linear irreversible thermodynamics is a rough approximation.

Increased Reactive Oxygen Species Production and Lower Abundance of Complex I Subunits and Carnitine Palmitoyltransferase 1B Protein Despite Normal Mitochondrial Respiration in Insulin-Resistant Human Skeletal Muscle

OBJECTIVEThe contribution of mitochondrial dysfunction to skeletal muscle insulin resistance remains elusive. Comparative proteomics are being applied to generate new hypotheses in human biology and were applied here to isolated mitochondria to identify novel changes in mitochondrial protein abundance present in insulin-resistant muscle.RESEARCH DESIGN AND METHODSMitochondria were isolated from vastus lateralis muscle from lean and insulin-sensitive individuals and from obese and insulin-resistant individuals who were otherwise healthy. Respiration and reactive oxygen species (ROS) production rates were measured in vitro. Relative abundances of proteins detected by mass spectrometry were determined using a normalized spectral abundance factor method.RESULTSNADH- and FADH2-linked maximal respiration rates were similar between lean and obese individuals. Rates of pyruvate and palmitoyl-dl-carnitine (both including malate) ROS production were significantly higher in obesity. Mitochondria from obese individuals maintained higher (more negative) extramitochondrial ATP free energy at low metabolic flux, suggesting that stronger mitochondrial thermodynamic driving forces may underlie the higher ROS production. Tandem mass spectrometry identified protein abundance differences per mitochondrial mass in insulin resistance, including lower abundance of complex I subunits and enzymes involved in the oxidation of branched-chain amino acids (BCAA) and fatty acids (e.g., carnitine palmitoyltransferase 1B).CONCLUSIONSWe provide data suggesting normal oxidative capacity of mitochondria in insulin-resistant skeletal muscle in parallel with high rates of ROS production. Furthermore, we show specific abundance differences in proteins involved in fat and BCAA oxidation that might contribute to the accumulation of lipid and BCAA frequently associated with the pathogenesis of insulin resistance.

Myosin individualized: single nucleotide polymorphisms in energy transduction.

BackgroundMyosin performs ATP free energy transduction into mechanical work in the motor domain of the myosin heavy chain (MHC). Energy transduction is the definitive systemic feature of the myosin motor performed by coordinating in a time ordered sequence: ATP hydrolysis at the active site, actin affinity modulation at the actin binding site, and the lever-arm rotation of the power stroke. These functions are carried out by several conserved sub-domains within the motor domain. Single nucleotide polymorphisms (SNPs) affect the MHC sequence of many isoforms expressed in striated muscle, smooth muscle, and non-muscle tissue. The purpose of this work is to provide a rationale for using SNPs as a functional genomics tool to investigate structurefunction relationships in myosin. In particular, to discover SNP distribution over the conserved sub-domains and surmise what it implies about sub-domain stability and criticality in the energy transduction mechanism.ResultsAn automated routine identifying human nonsynonymous SNP amino acid missense substitutions for any MHC gene mined the NCBI SNP data base. The routine tested 22 MHC genes coding muscle and non-muscle isoforms and identified 89 missense mutation positions in the motor domain with 10 already implicated in heart disease and another 8 lacking sequence homology with a skeletal MHC isoform for which a crystallographic model is available. The remaining 71 SNP substitutions were found to be distributed over MHC with 22 falling outside identified functional sub-domains and 49 in or very near to myosin sub-domains assigned specific crucial functions in energy transduction. The latter includes the active site, the actin binding site, the rigid lever-arm, and regions facilitating their communication. Most MHC isoforms contained SNPs somewhere in the motor domain.ConclusionsSeveral functional-crucial sub-domains are infiltrated by a large number of SNP substitution sites suggesting these domains are engineered by evolution to be too-robust to be disturbed by otherwise intrusive sequence changes. Two functional sub-domains are SNP-free or relatively SNP-deficient but contain many disease implicated mutants. These sub-domains are apparently highly sensitive to any missense substitution suggesting they have failed to evolve a robust sequence paradigm for performing their function.

Superoxide Production by Rat Skeletal Muscle Mitochondria: Influence of High Fat Diet

Chronic positive energy and fat balances increase the risk for the development of skeletal muscle insulin resistance. Reactive oxygen species may be a consequence and/or a contributing factor to this pathology. The purpose of this study was to evaluate superoxide (O2.) production by mitochondria (MITO) isolated from gastrocnemius muscle of rats fed standard chow (C) or a high fat (HF) diet. MITO were respired under either a static condition (oligomycin, OLIGO) or at an experimentally established ATP free energy corresponding to about 50% of maximal respiration (flow condition). Respiratory substrates were provided at saturating concentrations and O2. production was followed. Under the OLIGO condition MITO oxidizing pyruvate + malate (P+M) generated very low rates of O2. production irrespective of diet. In contrast, P+M supplemented with the cytosolic-mitochondrial electron shuttle substrates glycerol-3-phosphate and glutamate elicited O2. generation rates severalfold greater than P+M alone. Moreover, MITO from HF animals produced much higher O2. production than C rats when these products of the glycolytic pathway were available as respiratory fuels. For both C and HF MITO, the flow condition drastically reduced O2. production for all fuels and combinations. These findings suggest that excessive fuel and carbohydrate availability under sedentary conditions may promote the generation of reactive oxygen species. Supported by NSF IBN-0116997

Conformational Fluctuations of the Ca2+-ATPase in the Native Membrane Environment: EFFECTS OF pH, TEMPERATURE, CATALYTIC SUBSTRATES, AND THAPSIGARGIN

Digestion with proteinase K or trypsin yields complementary information on conformational transitions of the Ca(2+)-ATPase (SERCA) in the native membrane environment. Distinct digestion patterns are obtained with proteinase K, revealing interconversion of E1 and E2 or E1 approximately P and E2-P states. The pH dependence of digestion patterns shows that, in the presence of Mg(2+), conversion of E2 to E1 pattern occurs (even when Ca(2+) is absent) as H(+) dissociates from acidic residues. Mutational analysis demonstrates that the Glu(309) and Glu(771) acidic residues (empty Ca(2+)-binding sites I and II) are required for stabilization of E2. Glu(309) ionization is most important to yield E1. However, a further transition produced by Ca(2+) binding to E1 (i.e. E1.2Ca(2+)) is still needed for catalytic activation. Following ATP utilization, H(+)/Ca(2+) exchange is involved in the transition from the E1 approximately P.2Ca(2+) to the E2-P pattern, whereby alkaline pH will limit this conformational transition. Complementary experiments on digestion with trypsin exhibit high temperature dependence, indicating that, in the E1 and E2 ground states, the ATPase conformation undergoes strong fluctuations related to internal protein dynamics. The fluctuations are tightly constrained by ATP binding and phosphoenzyme formation, and this constraint must be overcome by thermal activation and substrate-free energy to allow enzyme turnover. In fact, a substantial portion of ATP free energy is utilized for conformational work related to the E1 approximately P.2Ca(2+) to E2-P transition, thereby disrupting high affinity binding and allowing luminal diffusion of Ca(2+). The E2 state and luminal path closure follow removal of conformational constraint by phosphate.

An Unusual Transduction Pathway in Human Tonic Smooth Muscle Myosin

The motor protein myosin binds actin and ATP, producing work by causing relative translation of the proteins while transducing ATP free energy. Smooth muscle myosin has one of four heavy chains encoded by the MYH11 gene that differ at the C-terminus and in the active site for ATPase due to alternate splicing. A seven-amino-acid active site insert in phasic muscle myosin is absent from the tonic isoform. Fluorescence increase in the nucleotide sensitive tryptophan (NST) accompanies nucleotide binding and hydrolysis in several myosin isoforms implying it results from a common origin within the motor. A wild-type tonic myosin (smA) construct of the enzymatic head domain (subfragment 1 or S1) has seven tryptophan residues and nucleotide-induced fluorescence enhancement like other myosins. Three smA mutants probe the molecular basis for the fluorescence enhancement. W506+ contains one tryptophan at position 506 homologous to the NST in other myosins. W506F has the native tryptophans except phenylalanine replaces W506, and W506+(Y499F) is W506+ with phenylalanine replacing Y499. W506+ lacks nucleotide-induced fluorescence enhancement probably eliminating W506 as the NST. W506F has impaired ATPase activity but retains nucleotide-induced fluorescence enhancement. Y499F replacement in W506+ partially rescues nucleotide sensitivity demonstrating the role of Y499 as an NST facilitator. The exceptional response of W506 to active site conformation opens the possibility that phasic and tonic isoforms differ in how influences from active site ATPase propagate through the protein network.

Identification of sources and functions of metabolic capacitance in the ATP metabolic network in muscle

Muscle contraction is activated by oscillatory calcium signals. Since calcium is known to boost oxidative ATP supply, mitochondrial ATP output could well become oscillatory during muscle activity. If so, this could interfere with calcium homeostasis given the high sensitivity to ATP free energy potential concomitant with full kinetic reversibility of calcium pumps. Here, we used computational modeling to identify and test sources of metabolic capacitance in the ATP metabolic network in muscle with respect to low-pass filtering of mitochondrial ATP output. We built a lumped, kinetic model of calcium-activated oxidative ATP metabolism in muscle with a calcium-sensing mitochondrion (Ngyen & Jafri 2005) at its core. Cytosolic components included basal- and calcium-activated ATPases, adenylate and creatine pools and kinases, and an oscillatory calcium input signal. Computations were performed in MATLAB and solved by imposing mass balance constraint. Physiological endpoints such as matrix- and cytosolic redox and ATP free energy potentials were computed for validation. We observed the classic creatine kinase capacitance function buffering amplitude and rate of ATP potential drop during activation of metabolism by the calcium input signal. This function was lost after in silico knock out. However, the network failed to filter the oscillation of the input signal, resulting in a 2 kJ/mole oscillation of cytosolic ATP free energy potential. These results suggest that metabolic capacitance is needed inside the mitochondrion to protect calcium homeostasis in muscle.

Computational model of excitable cell indicates ATP free energy dynamics in response to calcium oscillations are undampened by cytosolic ATP buffers

Mitochondria in excitable cells are recurrently exposed to pulsatile calcium gradients that activate cell function. Rapid calcium uptake by the mitochondria has previously been shown to cause uncoupling of oxidative phosphorylation. To test (i) if periodic nerve firing may cause oscillation of the cytosolic thermodynamic potential of ATP hydrolysis and (ii) if cytosolic adenylate (AK) and creatine kinase (CK) ATP buffering reactions dampen such oscillations, a lumped kinetic model of an excitable cell capturing major aspects of the physiology has been developed. Activation of ATP metabolism by low-frequency calcium pulses caused large oscillation of the cytosolic, but not mitochondrial ATP/ADP, ratio. This outcome was independent of net ATP synthesis or hydrolysis during mitochondrial calcium uptake. The AK/CK ATP buffering reactions dampened the amplitude and rate of cytosolic ATP/ADP changes on a timescale of seconds, but not milliseconds. These model predictions suggest that alternative sources of capacitance in neurons and striated muscles should be considered to protect ATP-free energy-driven cell functions.

Mitochondrial affinity for ADP is twofold lower in creatine kinase knock‐out muscles

Adaptations of the kinetic properties of mitochondria in striated muscle lacking cytosolic (M) and/or mitochondrial (Mi) creatine kinase (CK) isoforms in comparison to wild-type (WT) were investigated in vitro. Intact mitochondria were isolated from heart and gastrocnemius muscle of WT and single- and double CK-knock-out mice strains (cytosolic (M-CK-/-), mitochondrial (Mi-CK-/-) and double knock-out (MiM-CK-/-), respectively). Maximal ADP-stimulated oxygen consumption flux (State3 Vmax; nmol O2 x mg mitochondrial protein(-1) x min(-1)) and ADP affinity (K50ADP; microM) were determined by respirometry. State 3 Vmax and of M-CK-/- and MiM-CK-/- gastrocnemius mitochondria were twofold higher than those of WT, but were unchanged for Mi-CK-/-. For mutant cardiac mitochondria, only the of mitochondria isolated from the MiM-CK-/- phenotype was different (i.e. twofold higher) than that of WT. The implications of these adaptations for striated muscle function were explored by constructing force-flow relations of skeletal muscle respiration. It was found that the identified shift in affinity towards higher ADP concentrations in MiM-CK-/- muscle genotypes may contribute to linear mitochondrial control of the reduced cytosolic ATP free energy potentials in these phenotypes.

Pyruvate and citric acid cycle carbon requirements in isolated skeletal muscle mitochondria.

Carbohydrate depletion precipitates fatigue in skeletal muscle, but, because pyruvate provides both acetyl-CoA for mainline oxidation and anaplerotic carbon to the citric acid cycle (CAC), the mechanism remains obscure. Thus pyruvate and CAC kinetic parameters were independently quantified in mitochondria isolated from rat mixed skeletal muscle. Mitochondrial oxygen consumption rate (Jo) was measured polarographically while either pyruvate or malate was added stepwise in the presence of a saturating concentration of the other substrate. These substrate titrations were carried out across a physiological range of fixed extramitochondrial ATP free energy states (DeltaGP), established with a creatine kinase energy clamp, and also at saturating [ADP]. The apparent Km,malate for mitochondrial Jo ranged from 21 to 32 microM, and the apparent Km,pyruvate ranged from 12 to 26 microM, with both substrate Km values increasing as DeltaGP declined. Vmax for both substrates also increased as DeltaGP fell, reflecting thermodynamic control of Jo. Reported in vivo skeletal muscle [malate] are >10-fold greater than the Km,malate determined in this study. In marked contrast, the K(m,pyruvate) determined is near the [pyruvate] reported in muscle approaching exhaustion associated with glycogen depletion. When data were evaluated in the context of a linear thermodynamic force-flow (DeltaGP-Jo) relationship, the DeltaGP-Jo slope was essentially insensitive to changes in [malate] in the range observed in vivo but decreased markedly with declining [pyruvate] across the physiological range. Mitochondrial respiration is particularly sensitive to variations in [pyruvate] in the physiological range. In contrast, physiological [malate] exerts very little, if any, influence on mitochondrial pyruvate oxidation measured in vitro.

Regulation analysis of contractile ATPase flux in skeletal muscle.

J The [Ca2+] regulation of contractile ATPase flux, Jp, in skeletal muscle was analysed by computation of the Response R(Jp) Ca2+ for a 10 Hz range of electrical stimulation frequencies. Results of our analysis of the kinetic controls in ATP free energy metabolism in a network model of contracting muscle (J.A.L. Jeneson, H.V. Westerhoff and M.J. Kushmerick (2000) Am. J Physiol. 279, C813-C832) formed the basis for the computations of R(Jp) Ca2+. We found that neural regulation of sustained force generation via simple [Ca2+]cyto frequency encoding in the network was robust for frequencies up to 2 Hz. Above 2 Hz, however, this regulation design broke down because of a shift in contractile ATPase flux control from the Ca2+-sensitive contractile filaments to mitochondria with low Ca2+ sensitivity. The role of glyco(geno)lytic ATP production at high contraction workloads is discussed in the context of this result.

Congestive Heart Failure: Fifty Years of Progress

Congestive Heart Failure: Fifty Years of Progress

A metabolic control analysis of kinetic controls in ATP free energy metabolism in contracting skeletal muscle.

A system analysis of ATP free energy metabolism in skeletal muscle was made using the principles of metabolic control theory. We developed a network model of ATP free energy metabolism in muscle consisting of actomyosin ATPase, sarcoplasmic reticulum (SR) Ca(2+)-ATPase, and mitochondria. These components were sufficient to capture the major aspects of the regulation of the cytosolic ATP-to-ADP concentration ratio (ATP/ADP) in muscle contraction and had inherent homeostatic properties regulating this free energy potential. As input for the analysis, we used ATP metabolic flux and the cytosolic ATP/ADP at steady state at six contraction frequencies between 0 and 2 Hz measured in human forearm flexor muscle by (31)P-NMR spectroscopy. We used the mathematical formalism of metabolic control theory to analyze the distribution of fractional kinetic control of ATPase flux and the ATP/ADP in the network at steady state among the components over this experimental range and an extrapolated range of stimulation frequencies (up to 10 Hz). The control analysis showed that the contractile actomyosin ATPase has dominant kinetic control of ATP flux in forearm flexor muscle over the 0- to 1.6-Hz range of contraction frequencies that resulted in steady states, as determined by (31)P-NMR. However, flux control begins to shift toward mitochondria at >1 Hz. This inversion of flux control from ATP demand to ATP supply control hierarchy progressed as the contraction frequency increased past 2 Hz and was nearly complete at 10 Hz. The functional significance of this result is that, at steady state, ATP free energy consumption cannot outstrip the ATP free energy supply. Therefore, this reduced, three-component muscle ATPase system is inherently homeostatic.

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.

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.

Myocardial cell energetics.

Energy transformation at the main energy consuming processes of the myocardium takes place with high efficiency, i.e., with relatively small differences between the free energy level provided and the free energy level required for the two coupled processes. Thus, the free energy of ATP is only moderately higher than that of various ATP dependent processes. Under energy deficiency caused by hypoxia, free energy of ATP can drop to a level that critically affects subsequent steps. Detailed evaluation of cell energetics was carried out with the following approach: Cell shortening, oxygen consumption and intracellular calcium transients of isolated rat cardiomyocytes which were investigated under the influence of inotropic interventions. Increased extracellular Ca2+ and isoproterenol reduced the economy of contraction (contraction amplitude/VO2), whereas Ca-sensitizing agent EMD 57033 did not. This seems to be a consequence of the increased costs of ion cycling under the effect of Ca2+ and isoproterenol. Our current investigation suggests that alterations of ion transport processes and crossbridge kinetics have substantial impact on myocardial energetics.