O2 transport and its interaction with metabolism; a systems view of aerobic capacity.

This commentary demonstrates that VO2max depends, in part, on diffusive O2 transport; exercise hyperemia is necessary but not sufficient. Experiments and new mathematical models place the principal site of resistance to O2 diffusion between the surface of a red cell and the sarcolemma. The large drop in PO2 over this short distance is caused by high flux density and absence of heme protein O2 carrier in this region. PO2 gradients within red myocytes are shallow at high VO2 because myoglobin acts as O2 carrier and PO2 buffer. At high VO2 cell PO2 is less than 5 torr, the myoglobin P50. Low cell PO2 relative to blood PO2 is essential to a) maintain the driving force on diffusion as capillary PO2 falls, and b) to increase myoglobin-facilitated diffusion and the overall O2 conductance. O2 per se does not limit mitochondrial ATP production under normal circumstances because the low O2 drive on electron transport is compensated by greater phosphorylation and redox drives. These metabolic adaptations support transcapillary diffusion by defending VO2 at the low cell PO2 required to extract O2 from blood. Thus aerobic capacity is a distributed property, dependent on the interaction of transport and metabolism as a system.

Interaction of blood flow, diffusive transport and cell metabolism in isovolemic anemia.

1) High blood flow can compensate for half-normal hematocrit, leaving the rate at which O2 is offered to the capillaries unchanged. Nevertheless, intracellular PO2 is lower in anemia, indicating impaired diffusive transport. 2) Anemia increases O2 flux per red cell and decreases functional capillary surface area. These changes increase flux density and the extracellular component of resistance to diffusive O2 transport, in accord with current theory (Federspiel and Popel, 1986; Groebe, 1990; Hellums, 1977). 3) Maintenance of diffusive flux in presence of anemia required a larger delta PO2 between Hb and Mb, and higher intracellular O2 conductance brought about by greater Mb-facilitated diffusion. Both compensations depend on lower PmbO2. 4) PmbO2 and creatine charge fall with increasing VO2 and ATP demand. These responses, as well as adaptive changes in redox help maintain VO2 in the presence of a lower O2 drive on electron transport. 5) Greater engagement of reserves of both transport and metabolism limits the range of aerobic performance in anemia. 6) The match between the transcapillary and mitochondrial O2 fluxes depends on interaction of transport and metabolism as a system.

Regulation of muscle carbohydrate metabolism during exercise.

This review examines the mechanisms that regulate muscle carbohydrate metabolism during exercise. Muscle carbohydrate utilization is regulated primarily by two factors, namely, delivery of substrate to the glycolytic pathway either from glycogenolysis or from transport of extracellular glucose into the fibers, and formation of triosephosphate by phosphofructokinase. The regulation involves the integration of the glycolytic controls with other metabolic controls and the needs of the whole muscle in meeting the physiological demand. The controls operating in the glycolytic sequence in vivo appear to couple glycolytic recruitment to signals from the rate of energy demand, the TCA cycle state, and the mitochondrial redox state so as to satisfy the major regulatory goal of maintaining the supply of ATP for tension development.

A simple model of aerobic metabolism: applications to work transitions in muscle.

Adding kinetics to the model of the phosphate energy system [Connett. Am. J. Physiol. 254 (Regulatory Integrative Comp. Physiol. 23): R949-R959, 1988], we provide a framework for analyzing metabolic transients in muscle tissue. We modify the formalism of the earlier model and introduce a buffering factor, which measures buffering of adenine nucleotides by phosphocreatine. The time course of the phosphate energy state can be calculated given the following: 1) adenosinetriphosphatase (ATPase) rate, 2) pH, and 3) a mitochondrial driving function, i.e., ATP production in terms of the phosphate energy state. We use mitochondrial driving functions derived from steady-state measurements to predict the time courses for rest-work transitions. Predictions for transitions in the rat gastrocnemius muscle agree with published values. The model is used to test different existing hypotheses of oxygen consumption (VO2) regulation. Each hypothesis generates a specific mitochondrial driving function, which in turn generates a specific time course of phosphate energy state during transitions. A mitochondrial driving function based on enzyme kinetics with ADP as a substrate leads to time courses not matching the data. Mitochondrial driving functions that are linear with phosphocreatine, Pi, phosphorylation potential, or the pool of high-energy phosphate bonds (phosphate potential energy) gave good agreement with the data.

Defining hypoxia: a systems view of VO2, glycolysis, energetics, and intracellular PO2.

The necessity for defining hypoxia as O2-limited energy flux rather than low partial pressure is explored from a systems perspective. Oxidative phosphorylation, the Krebs cycle, glycolysis, substrate supply, and cell energetics interact as subsystems; the set point is a match between ATP demand and aerobic ATP production. To this end the transport subsystem must match the transcapillary and mitochondrial O2 fluxes. High transcapillary O2 flux requires intracellular PO2 in the range 1-10 Torr. In this range the O2 drive on electron transport must be compensated by adaptive changes in the phosphorylation and redox drives. Thus the metabolic subsystem supports diffusive O2 transport by maintaining O2 flux at intracellular partial pressures required for O2 release from blood. Since responses to stress are distributed according to the state of the entire system, several simultaneous metabolic measurements, including intracellular PO2 (or a known direction of change in intracellular PO2) and the O2 dependence of a measurable function are required to judge the adequacy of O2 supply. ATP demand and aerobic capacity must also be evaluated, because the hypoxic threshold depends on the ratio of ATP demand to aerobic capacity. The application and limitation of commonly used criteria of hypoxia are discussed, and a more precise terminology is proposed.

In vivo control of phosphofructokinase: system models suggest new experimental protocols.

There is still uncertainty as to how much control of in vivo rates of glycolysis by phosphofructokinase (PFK) depends on cytosolic phosphate energy state. Three models of PFK kinetics incorporating sensitivity to pH, adenine nucleotides, and inorganic phosphate (Pi) were embedded in the physiological "phosphate energy system" of creatine-containing tissues [Connett, R.J. Am. J. Physiol. 254 (Regulatory Integrative Comp. Physiol. 23): R949-R959, 1988]. Effects of changes in phosphate energy state and total adenine nucleotide and phosphate pools on steady-state kinetics were examined. Analyses mimicking in vitro experiments indicated no activity at the pH and [ATP] of working muscles. When tested using the coordinated changes in Pi and adenine nucleotides expected in vivo, all models showed reasonable activity. Control was dominated by [Pi] in the normal physiological range of energy states. The almost linear response to phosphate energy state, measured by creatine charge (phosphocreatine/total creatine), is insensitive to the absolute size of the adenine nucleotide pool. A step to almost full activation occurred when phosphocreatine buffering of [ATP] was exceeded. Several experimental studies are suggested.

Regulation of VO2 in red muscle: do current biochemical hypotheses fit in vivo data?

Observations used to test biochemical models of the regulation of O2 consumption (VO2) by cytosolic phosphate energy state must include a change in intracellular pH and/or a change in the adenine nucleotide or phosphate pools [Connett, R. J. Analysis of metabolic control: new insights using a scaled creatine kinase model. Am. J. Physiol. 254 (Regulatory Integrative Comp. Physiol. 23): R949-R959, 1988]. Data were collected over a wide range of energy turnover from canine muscles in situ. Intracellular PO2, glycolytic intermediates, adenine nucleotides, creatine, phosphocreatine (PCr), phosphate, and intracellular pH were determined for each muscle. PO2 was used to eliminate muscles in which VO2 could have been O2 limited (PO2 less than 0.5 Torr). This removed an important source of heterogeneity. Because adenine nucleotide and phosphate pools were constant relative to the creatine pool, discrimination among models depended solely on pH. The observed pH range from 7.2 to 5.9 did not permit separation of [PCr] from log[( ATP4-]/[ADP3-][H2PO4-]) (phosphorylation potential) as a regulatory parameter for VO2. However, [ADP] could be eliminated as an independent regulator. Because 90% of variability in VO2 was accounted for by phosphate energetics, an independent redox component must be small when intracellular PO2 greater than 0.5 Torr.

How phosphocreatine buffers cyclic changes in ATP demand in working muscle.

1. Spatial buffering is hardly necessary for maintaining ATP concentration. 2. Temporal buffering may be essential to defend concentration of products of ATP hydrolysis. ADP would vary 4-fold and AMP would vary 16-fold during a single twitch without buffering. 3. The principal function of the transphosphorylating reaction may be to buffer temporally the concentrations of controlling reactants.

Analysis of metabolic control: new insights using scaled creatine kinase model.

The creatine kinase-adenylate kinase equilibria equations are given a dimensionless form by normalizing to total creatine concentration. Analysis with appropriate equilibrium and cation-binding constants identified two sharply separated phases of energy depletion. In the "buffering" phase, energy is derived from phosphocreatine. In the "depleting" phase, adenine nucleotides are the source of energy. Defining the state of the adenine nucleotide pool requires only pH, phosphocreatine, and creatine concentrations. Analysis of data from skeletal muscle, heart, brain, and smooth muscle demonstrated that the [free adenine nucleotide]/[total creatine] and [total phosphate]/[total creatine] are essentially constant over the greater than 20-fold concentration range among tissues and species. This result permits quantitative evaluation of cell energetics with data scaled to the total phosphate, as obtained with nuclear magnetic resonance studies, or to total creatine, as obtained in chemical analysis of freeze-trapped tissue. By applying the stability of the tissue parameters to the equations, it is demonstrated that unique identification of a hypothesis describing the recruitment of O2 uptake requires testing at several pH values.

Glycolytic regulation during an aerobic rest-to-work transition in dog gracilis muscle.

Glycogen phosphorylase activity and several glycolytic intermediates were measured at rest and after 5, 10, 15, 30, 60, and 180 s of twitch stimulation at 4 Hz in fast-frozen samples of gracilis muscle. During an initial burst of glycolysis (0-5 s) only 3-phosphoglycerate and lactate accumulate. These changes are reversed during the period of low glycolytic flux (5-30 s). During a second burst of glycolysis (30-60 s) most glycolytic intermediates increase. The levels of glycogen phosphorylase a changes in parallel with the initial burst of glycolysis but remain at resting levels throughout the second burst. The phosphoglycerate mutase-enolase steps deviate from equilibrium during the initial burst of glycolysis, suggesting a transiently rate-limiting role. Analysis using a model of phosphofructokinase kinetics indicates that combined changes in cytosolic pH (R. J. Connett, J. Appl. Physiol. 63: 2360-2365, 1987) and free [ADP] and [AMP] can account for the initial burst of glycolysis. The second burst of glycolysis requires other regulatory factors. It is concluded that an initial alkalization is a major regulatory factor in the early burst of glycolysis during a rest-to-work transition in red muscle.

Cytosolic pH during a rest-to-work transition in red muscle: application of enzyme equilibria.

Muscles sampled from a vascularly isolated autoperfused dog gracilis by fast freezing techniques at 5, 10, 15, 30, 60, and 180 s after the initiation of twitch contractions at 4 Hz were analyzed for phosphocreatine, creatine, ATP, lactate, pyruvate, 3-phosphoglycerate, and dihydroxyacetonephosphate contents. Metabolite concentrations were used with equilibrium constants of triosephosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, lactate dehydrogenase, and creatine kinase to estimate cytosolic pH changes during the rest-to-work transition. Magnesium and hydrogen binding were taken into account. Limits to this approach include errors in the intermediate measurements and uncertainties in values of the equilibrium constants. The former leads to maximum errors of +/- 0.15 pH units, whereas the latter affects the absolute pH value but not estimates of the changes in pH. The estimated pH increases from a resting value of 7.05 to approximately 7.8 by 5 s of stimulation and then falls to a pH value of approximately 6.5 after 3 min of stimulation. The results are consistent with previous studies but permit identification of a larger early alkaline shift. Potential causes for the pH changes are discussed.

How large is the drop in PO2 between cytosol and mitochondrion?

The subject of this brief review is the size of the local drops in PO2 around consuming mitochondria. We show that large drops (several Torr or more) are in conflict with the predictions of basic diffusion theory, when one uses accepted values for relevant parameters. In particular, oxygen diffusion coefficients must be reduced by at least a factor of 10 below measured values to reconcile Fick's law with large PO2 drops. Experimental evidence offered for large drops is often ambiguous because of system heterogeneities. In those cases where tractable models of heterogeneous systems can be developed, the experimental data are consistent with drops in PO2 on the order of a few hundredths of a Torr between cytosol and mitochondrion.

Minimum intracellular PO2 for maximum cytochrome turnover in red muscle in situ.

Probability distributions of myoglobin (Mb) saturation and intracellular PO2 were determined with subcellular spatial resolution in dog gracilis muscles during steady-state twitch contraction at 5-100% of maximal rate of O2 consumption (VO2). Calculations (Clark, A., and P. A.A. Clark. Biophys. J. 48: 931-938, 1985) and measurements (Gayeski, T. E. J., and C. R. Honig. Adv. Exp. Med. Biol. 200: 487-494, 1986) indicate that the PO2 in equilibrium with Mb is virtually identical to the PO2 at cytochrome aa3. Median intracellular PO2 and PO2 in the lower tails of probability distributions were poorly correlated with VO2. The variability of cell PO2 was greatly diminished when median PO2 was less than the PO2 for half saturation of MB, since Mb acts as a PO2 buffer. The lower tails of PO2 distributions contained almost no anoxic loci even when median PO2 was less than 1 Torr. VO2 was well correlated with the concentration ratio of phosphocreatine to free creatine (PCr/Crf) over a wide range of PO2. PO2 greater than or equal to 0.5 Torr supported maximal VO2 and energy demand. We conclude that 1) the mechanism of action of cytochrome aa3 is the same in red muscle in vivo as in mitochondria in vitro, and 2) an upper bound on the apparent Michaelis constant for maximal VO2 of red muscle is approximately 0.06 Torr.

Changes in the pattern of phospholipid synthesis during the induction by cytokinin of cell division in soybean suspension cultures.

A method is described for preparing fully viable, cytokinin-starved soybean (Glycine max (L.) Merr. cv. Acme) cells from a suspension-culture of callus tissue. The cells respond to kinetin treatment by re-initiating cell division. We present evidence, from the pattern of incorporation of (32)P-labelled inorganic phosphate into individual phospholipids during the first hour of this response, that the synthesis of phosphatidylinositol (PI) and of phosphatidic-acid head-groups is affected within 15 min. The polyphosphoinositide phosphatidylinositol 4-phosphate, but not phosphatidylinositol 4,5-bisphosphate, was detected in the tissue. The characteristics of cytokinin-induced PI synthesis in cytokinin-starved soybean cells appear to resemble the 'PI response' of animal cells.