Tribute to R. G. Boutilier: the role for skeletal muscle in the hypoxia-induced hypometabolic responses of submerged frogs.

Much of Bob Boutilier's research characterised the subcellular, organ-level and in vivo behavioural responses of frogs to environmental hypoxia. His entirely integrative approach helped to reveal the diversity of tissue-level responses to O(2) lack and to advance our understanding of the ecological relevance of hypoxia tolerance in frogs. Work from Bob's lab mainly focused on the role for skeletal muscle in the hypoxic energetics of overwintering frogs. Muscle energy demand affects whole-body metabolism, not only because of its capacity for rapid increases in ATP usage, but also because hypometabolism of the large skeletal muscle mass in inactive animals impacts so greatly on in vivo energetics. The oxyconformance and typical hypoxia-tolerance characteristics (e.g. suppressed heat flux and preserved membrane ion gradients during O(2) lack) of skeletal muscle in vitro suggest that muscle hypoperfusion in vivo is possibly a key mechanism for (i) downregulating muscle and whole-body metabolic rates and (ii) redistributing O(2) supply to hypoxia-sensitive tissues. The gradual onset of a low-level aerobic metabolic state in the muscle of hypoxic, cold-submerged frogs is indeed important for slowing depletion of on-board fuels and extending overwintering survival time. However, it has long been known that overwintering frogs cannot survive anoxia or even severe hypoxia. Recent work shows that they remain sensitive to ambient O(2) and that they emerge rapidly from quiescence in order to actively avoid environmental hypoxia. Hence, overwintering frogs experience periods of hypometabolic quiescence interspersed with episodes of costly hypoxia avoidance behaviour and exercise recovery. In keeping with this flexible physiology and behaviour, muscle mechanical properties in frogs do not deteriorate during periods of overwintering quiescence. On-going studies inspired by Bob Boutilier's integrative mindset continue to illuminate the cost-benefit(s) of intermittent locomotion in overwintering frogs, the constraints on muscle function during hypoxia, the mechanisms of tissue-level hypometabolism, and the details of possible muscle atrophy resistance in quiescent frogs.

Effects of ambient PO2 and temperature on oxygen uptake in Nautilus pompilius.

This study employs closed-circuit respirometry to evaluate the effect of declining ambient oxygen partial pressure (PO2) and temperature on mass specific rates of oxygen uptake (VO2) in Nautilus pompilius. At all temperatures investigated (11, 16, and 21 degrees C), VO2 is relatively constant at high PO2 (oxyregulation) but declines sharply at low PO2 (oxyconformation). The critical PO2 below which oxyconformation begins (Pc) is temperature dependent, higher at 21 degrees C (49 mmHg) than at 11 degrees C or 16 degrees C (21.7 mmHg and 30.8 mmHg respectively). In resting, post-absorptive animals, steady-state resting VO2 increases significantly with temperature resulting in a Q10 value of approximately 2.5. The metabolic strategy of N. pompilius appears well suited to its lifestyle, providing sufficient metabolic scope for its extensive daily vertical migrations, but allowing for metabolic suppression when PO2 falls too low. The combination of low temperatures and low PO2 may suppress metabolic rate 16-fold (assuming negligible contributions from anaerobic metabolism and internal O2 stores), enhancing hypoxia tolerance.

Mitochondrial function in flying honeybees (Apis mellifera): respiratory chain enzymes and electron flow from complex III to oxygen.

The biochemical bases for the high mass-specific metabolic rates of flying insects remain poorly understood. To gain insights into mitochondrial function during flight, metabolic rates of individual flying honeybees were measured using respirometry, and their thoracic muscles were fixed for electron microscopy. Mitochondrial volume densities and cristae surface densities, combined with biochemical data concerning cytochrome content per unit mass, were used to estimate respiratory chain enzyme densities per unit cristae surface area. Despite the high content of respiratory enzymes per unit muscle mass, these are accommodated by abundant mitochondria and high cristae surface densities such that enzyme densities per unit cristae surface area are similar to those found in mammalian muscle and liver. These results support the idea that a unit area of mitochondrial inner membrane constitutes an invariant structural unit. Rates of O(2) consumption per unit cristae surface area are much higher than those estimated in mammals as a consequence of higher enzyme turnover rates (electron transfer rates per enzyme molecule) during flight. Cytochrome c oxidase, in particular, operates close to its maximum catalytic capacity (k(cat)). Thus, high flux rates are achieved via (i) high respiratory enzyme content per unit muscle mass and (ii) the operation of these enzymes at high fractional velocities.

Relationships between enzymatic flux capacities and metabolic flux rates: nonequilibrium reactions in muscle glycolysis.

The rules that govern the relationships between enzymatic flux capacities (Vmax) and maximum physiological flux rates (v) at enzyme-catalyzed steps in pathways are poorly understood. We relate in vitro Vmax values with in vivo flux rates for glycogen phosphorylase, hexokinase, and phosphofructokinase, enzymes catalyzing nonequilibrium reactions, from a variety of muscle types in fishes, insects, birds, and mammals. Flux capacities are in large excess over physiological flux rates in low-flux muscles, resulting in low fractional velocities (%Vmax = v/Vmax x 100) in vivo. In high-flux muscles, close matches between flux capacities and flux rates (resulting in fractional velocities approaching 100% in vivo) are observed. These empirical observations are reconciled with current concepts concerning enzyme function and regulation. We suggest that in high-flux muscles, close matches between enzymatic flux capacities and metabolic flux rates (i.e., the lack of excess capacities) may result from space constraints in the sarcoplasm.

Nitric oxide responses of air-breathing and water-breathing fish.

Nitric oxide (NO), exogenously administered or endogenously produced by NO synthase (NOS), is an important regulator of lung ventilation and perfusion in mammals. This study attempts to investigate the evolutionary history of this system in fish and its possible relationship to air breathing. The gas bladder of Hoplerythrinus unitaeniatus (air-breathing teleost) and Oncorhynchus mykiss (non-air-breathing teleost) and the lung of Lepidosiren paradoxa (air-breathing dipnoan) all exhibited elevated guanosine 3',5'-cyclic monophosphate (cGMP) levels in response to 1 microM sodium nitroprusside. Only the H. unitaeniatus gas bladder responded to 10 microM acetylcholine chloride (ACh) with increased cGMP levels. The ACh response was inhibited by N omega-nitro-L-arginine methyl ester, which inhibits NOS. These data suggest that although tissues from each species may respond to exogenous NO, only the gas bladder of H. unitaeniatus appears to synthesize NO through NOS. This is the first report of constitutive NOS outside of the central nervous system in a teleost. These results also imply that NOS did not necessarily coevolve with air breathing in fish.