Non-bicarbonate intracellular pH buffering of reptilian muscle

Summary Non-bicarbonate intracellular pH buffering values of skeletal and cardiac muscles were measured for 16 species of Australian reptiles from four orders (snakes, skelctal 19–36 slykes, cardiac 9–17 slykes; lizards, skeletal 25–54 slykes, cardiac 17–19 slykes; turtles, skeletal 25–43 slykes, cardiac 11–24 slykes; crocodile, skeletal 43 slykes). Although a positive correlation between pH buffering capacity and dependence on anaerobic muscle work was found, even the highest reptilian pH buffering values were low relative to equivalent white anaerobic muscles of fish, birds, and mammals. The low non-bicarbonate intracellular pH buffering capacity of reptilian muscle arises through lower contributions from proteins (10–14 slykes), non-protein histidine (7–18 slykes) and phosphate (5–15 slykes). It is concluded that while other vertebrates depend on these intracellular buffers for regulating muscle pH during anaerobic muscle work, reptiles rely less on buffering and instead may tolerate greater pH fluctuations.Abbreviations intracellular pH buffering capacity - EDTA ethylenediaminetetra-acetic acid - HPLC high performance liquid chromatography - I.D. internal diameter - LDH lactate dehydrogenase     

BUFFERING CAPACITY OF THE LOCOMOTOR MUSCLE IN CETACEANS: CORRELATES WITH POSTPARTUM DEVELOPMENT, DIVE DURATION, AND SWIM PERFORMANCE

Skeletal muscles of marine mammals must support the metabolic demands of exercise during periods of reduced blood flow associated with the dive response. Enhanced muscle buffering could support anaerobic metabolic processes during apnea, yet this has not been fully investigated in cetaceans. To assess the importance of this adaptation in the diving and swimming performance of cetaceans, muscle buffering capacity due to non-bicarbonate buffers was measured in the longissimus dorsi of ten species of odontocete and one mysticete. Immature specimens from a subset of these species were studied to assess developmental trends. Fetal and neonatal cetaceans have low buffering capacities (range: 34.8–53.9 slykes) that are within the range measured for terrestrial mammals. A lengthy developmental period, independent of muscle myoglobin postnatal development, is required before adult levels are attained. Adult cetacean buffering capacities (range: 63.7–94.5 slykes) are among the highest values recorded for mammals. Cetacean species that demonstrate extremely long dive durations or high burst speed swimming tend to have greater buffering capacities. However, the wide range of body size across cetaceans may complicate these trends. Enhanced muscle buffering capacity may enable small-bodied species to extend breath-hold beyond short aerobic dive limits for foraging or predator evasion when necessary.     

Buffering capacity of vertebrate muscle: Correlations with potentials for anaerobic function

Summary Buffering capacities (), measured in slykes (moles of base required to titrate the pH of one gram wet weight of muscle by one pH unit, over the pH range of pH 6 to pH 7) due to non-bicarbonate buffers were measured in locomotory muscles from a variety of terrestrial and marine mammals and teleost fishes (Tables 1 and 2). The highest buffering capacities were found in muscles capable of either intense, burst glycolytic function or prolonged, low-level anaerobic function. Marine mammals had higher muscle buffering capacity on the average than terrestrial mammals. Among the fishes studied, warm-bodied species had the greatest values of all animals examined (Table 2). Deep-sea fishes and shallow-living fishes with sluggish locomotory abilities had low values. Fish white muscle displayed higher buffering capacity than red muscle (Fig. 1; Table 2), in keeping with the more aerobic poise and higher capillary density of the latter type of muscle. Strong correlations were found between (1) and muscle myoglobin concentrations in the mammalian species (Table 1; Fig. 2), and (2) and muscle lactate dehydrogenase (LDH) activities in both the mammals and the fishes (Tables 1 and 2; Fig. 3). No correlation was found between and the activity of a citric acid cycle indicator enzyme, citrate synthase, in the mammalian species. While strongly correlated with buffering capacity, the amounts of myoglobin and LDH in a muscle are not the principal determinants of . The results indicate that muscle intracellular buffering capacity is especially critical in locomotory muscles which must function under conditions (burst locomotion and prolonged, low-level anaerobic function) where circulatory perfusion is inadequate to rapidly remove the acidic end-products such as lactic acid that are produced by anaerobic glycolysis. In this respect, the locomotory muscle of diving mammals and the white skeletal muscles of teleost fishes face a common acid-base regulatory problem and utilize a common biochemical strategy to resolve it.     

Neural Control of Myofibrillar ATPase Activity in Rat Skeletal Muscle

THE limb muscles of mammals such as the cat and rat can be divided into the fast-twitch muscles and the slow-twitch muscles. While the absolute contraction speeds vary from species to species the isometric twitch time (the time taken from the start of contraction until the instant of peak tension development) of a slow-twitch muscle is always about three times longer than the isometric twitch time of a fast-twitch muscle. Thus, at 37° C, the isometric twitch time of cat soleus muscle (a slow-twitch muscle) is approximately 70 ms while the isometric twitch time of the flexor hallucis longus muscle (a fast-twitch muscle) is approximately 20 ms. In the rat, the contraction times of the corresponding muscles would be of the order of 36 ms and 12 ms respectively.     

Cloning of a neonatal calcium atpase isoform (SERCA 1B) from extraocular muscle of adult blue marlin (Makaira nigricans)

Complete cDNAs for the fast-twitch Ca2+ -ATPase isoform (SERCA 1) were cloned and sequenced from blue marlin (Makaira nigricans) extraocular muscle (EOM). Complete cDNAs for SERCA 1 were also cloned from fast-twitch skeletal muscle of the same species. The two sequences are identical over the coding region except for the last five codons on the carboxyl end; EOM SERCA 1 cDNA codes for 996 amino acids and the fast-twitch cDNAs code for 991 aa. Phylogenetic analysis revealed that EOM SERCA 1 clusters with an isoform of Ca2+ -ATPase normally expressed in early development of mammals (SERCA 1B). This is the first report of SERCA 1B in an adult vertebrate. RNA hybridization assays indicate that 1B expression is limited to extraocular muscles. Because EOM gives rise to the thermogenic heater organ in marlin, we investigated whether SERCA 1B may play a role in heat generation, or if 1B expression is common in EOM among vertebrates. Chicken also expresses SERCA 1B in EOM, but rat expresses SERCA 1A; because SERCA 1B is not specific to heater tissue we conclude it is unlikely that it plays a specific role in intracellular heat production. Comparative sequence analysis does reveal, however, several sites that may be the source of functional differences between fish and mammalian SERCAs.     

Social buffering of the stress response: insights from fishes

Social buffering of stress refers to the effect of a social partner in reducing the cortisol or corticosterone response to a stressor. It has been well studied in mammals, particularly those that form pair bonds. Recent studies on fishes suggest that social buffering of stress also occurs in solitary species, gregarious species that form loose aggregations and species with well-defined social structures and bonds. The diversity of social contexts in which stress buffering has been observed in fishes holds promise to shed light on the evolution of this phenomenon among vertebrates. Equally, the relative simplicity of the fish brain is advantageous for identifying the neural mechanisms responsible for social buffering. In particular, fishes have a relatively small and simple forebrain but the brain regions that are key to social buffering, including the social behaviour network, the amygdala and the hypothalamic–pituitary–adrenal/interrenal axis, are functionally conserved across vertebrates. Thus, we suggest that insight into the mechanistic and evolutionary underpinnings of stress buffering in vertebrates can be gained from the study of social buffering of stress in fishes.     

Predicting the swimming and diving behaviour of penguins from muscle biochemistry

Energy metabolism in the pectoralis and supracoracoideus muscles of seven species of penguins was investigated by determining muscle fibre diameter, myoglobin content, pH buffering capacity and the distribution and properties of lactate dehydrogenase isoenzymes.The penguins can be arranged as follows in order of increasing anaerobic capabilities of the muscles: little < rockhopper and royal < gentoo < Adelie, emperor and king.As a good correlation exists between muscle biochemistry and known diving behaviour of emperor, king, gentoo and little penguins, predictions can be made about the behaviour of species for which only the biochemical data are available.     

Effect of beta-alanine supplementation on muscle carnosine concentrations and exercise performance

High-intensity exercise results in reduced substrate levels and accumulation of metabolites in the skeletal muscle. The accumulation of these metabolites (e.g. ADP, Pi and H⁺) can have deleterious effects on skeletal muscle function and force generation, thus contributing to fatigue. Clearly this is a challenge to sport and exercise performance and, as such, any intervention capable of reducing the negative impact of these metabolites would be of use. Carnosine (β-alanyl-l-histidine) is a cytoplasmic dipeptide found in high concentrations in the skeletal muscle of both vertebrates and non-vertebrates and is formed by bonding histidine and β-alanine in a reaction catalysed by carnosine synthase. Due to the pKa of its imidazole ring (6.83) and its location within skeletal muscle, carnosine has a key role to play in intracellular pH buffering over the physiological pH range, although other physiological roles for carnosine have also been suggested. The concentration of histidine in muscle and plasma is high relative to its K _m with muscle carnosine synthase, whereas β-alanine exists in low concentration in muscle and has a higher K _m with muscle carnosine synthase, which indicates that it is the availability of β-alanine that is limiting to the synthesis of carnosine in skeletal muscle. Thus, the elevation of muscle carnosine concentrations through the dietary intake of carnosine, or chemically related dipeptides that release β-alanine on absorption, or supplementation with β-alanine directly could provide a method of increasing intracellular buffering capacity during exercise, which could provide a means of increasing high-intensity exercise capacity and performance. This paper reviews the available evidence relating to the effects of β-alanine supplementation on muscle carnosine synthesis and the subsequent effects on exercise performance. In addition, the effects of training, with or without β-alanine supplementation, on muscle carnosine concentrations are also reviewed.     

Buffering capacity of deproteinized human vastus lateralis muscle

The in vitro deproteinized vastus lateralis muscle buffer capacity, carnosine, and histidine levels were examined in 20 men from 4 distinct populations (5 sprinters, 800-m runners; 5 rowers; 5 marathoners; 5 untrained). Needle biopsies were obtained at rest from the vastus lateralis muscle. The buffer capacity was determined in deproteinized homogenates by repeatedly titrating supernatant extracts over the pH range of 7.0-6.0 with 0.01 N HCl. Carnosine and histidine levels were determined on an amino acid AutoAnalyzer. Fast-twitch fiber percentage was determined by staining intensity of myosin adenosinetriphosphatase. High-intensity running performance was assessed on an inclined treadmill run to fatigue (20% incline; 3.5 m X s-1). Significantly (P less than 0.01) elevated buffer capacities, carnosine levels, and high-intensity running performances were demonstrated by the sprinters and rowers, but no significant differences existed between these variables for the marathoners vs. untrained subjects. Low but significant (P less than 0.05) interrelationships were demonstrated between buffer capacity, carnosine levels, and fast-twitch fiber composition. These findings indicate that the sprinters and rowers possess elevated buffering capabilities and carnosine levels compared with marathon runners and untrained subjects.     

Comparative analysis of fiber-type composition in the iliofibularis muscle of phrynosomatid lizards (Squamata).

The lizard family Phrynosomatidae comprises three subclades: the closely related sand and horned lizards, and their relatives the Sceloporus group. This family exhibits great variation in ecology, behavior, and general body plan. Previous studies also show that this family exhibits great diversity in locomotor performance abilities; as measured on a high-speed treadmill, sand lizards are exceptionally fast sprinters, members of the Sceloporus group are intermediate, and horned lizards are slowest. These differences are paralleled by differences in relative hindlimb span. To determine if muscle fiber-type composition also varies among the three subclades, we examined the iliofibularis (IF), a hindlimb muscle used in lizard locomotion, in 11 species of phrynosomatid lizards. Using histochemical assays for myosin ATPase, an indicator of fast-twitch capacity, and succinic dehydrogenase, denoting oxidative capacity, we classified fiber types into three categories based on existing nomenclature: fast-twitch glycolytic (FG), fast-twitch oxidative-glycolytic (FOG), and slow-twitch oxidative (SO). Sand lizards have a high proportion of FG fibers (64-70%) and a low proportion of FOG fibers (25-33%), horned lizards are the converse (FG fibers 25-31%, FOG fibers 56-66%), and members of the Sceloporus group are intermediate for both FG (41-48%) and FOG (42-45%) content. Hence, across all 11 species %FOG and %FG are strongly negatively correlated. Analysis with phylogenetically independent contrasts indicate that this negative relationship is entirely attributable to the divergence between sand and horned lizards. The %SO also varies among the three subclades. Results from conventional nested ANCOVA (with log body mass as a covariate) indicate that the log mean cross-sectional area of individual muscle fibers differs among species and is positively correlated with body mass across species, but does not differ significantly among subclades. The log cross-sectional area of the IF varies among species, but does not vary among subclades. Conversely, the total thigh muscle cross-sectional area does not vary among species, but does vary among subclades; horned lizards have slimmer thighs. Muscle fiber-type composition appears to form part of a coadapted suite of traits, along with relative limb and muscle sizes, that affect the locomotor abilities of phrynosomatid lizards.     

Biotope prioritisation in the Central Apennines (Italy): species rarity and cross-taxon congruence

The conservation status of invertebrates is usually lesser known than that of vertebrates, and strategies to identify biotopes to preserve invertebrate diversity are typically based on a single surrogate taxon, or even on the use of vertebrates as surrogates. Aim of this research is to illustrate a method for biotope prioritisation that can be easily adapted to different animal groups and geographical contexts. A two-step protocol for biotope prioritisation is proposed on the basis of a multidimensional characterisation of species vulnerability. Firstly, species vulnerability is estimated from rarity measures which include geographical range, abundance and biotope specialisation. Then, these values of vulnerability are used to rank biotopes. The method was applied here to the tenebrionid beetles, the butterflies, the birds and the mammals of the Central Apennines, a montane area of high conservation concern for South Europe. This study provides evidence for the importance of including insects in conservation decisions, because vertebrates are poor surrogates for insects. Conservation efforts in the reserves included in the study area are mostly focused on vertebrates, for which woodlands are considered particularly important. However high altitude open biotopes are crucial for both tenebrionids and butterflies, and preservation of such kind of biotopes would be beneficial also for vertebrates. The approach applied here demonstrates that (1) vertebrates are poor surrogates for insects, and (2) measures of species rarity, typically used in vertebrate conservation, can be obtained also for insects, for which a veritable amount of data are hidden in specialised literature and museum collections.     

Mechanics of biting in great white and sandtiger sharks

Although a strong correlation between jaw mechanics and prey selection has been demonstrated in bony fishes (Osteichthyes), how jaw mechanics influence feeding performance in cartilaginous fishes (Chondrichthyes) remains unknown. Hence, tooth shape has been regarded as a primary predictor of feeding behavior in sharks. Here we apply Finite Element Analysis (FEA) to examine form and function in the jaws of two threatened shark species, the great white (Carcharodon carcharias) and the sandtiger (Carcharias taurus). These species possess characteristic tooth shapes believed to reflect dietary preferences. We show that the jaws of sandtigers and great whites are adapted for rapid closure and generation of maximum bite force, respectively, and that these functional differences are consistent with diet and dentition. Our results suggest that in both taxa, insertion of jaw adductor muscles on a central tendon functions to straighten and sustain muscle fibers to nearly orthogonal insertion angles as the mouth opens. We argue that this jaw muscle arrangement allows high bite forces to be maintained across a wider range of gape angles than observed in mammalian models. Finally, our data suggest that the jaws of sub-adult great whites are mechanically vulnerable when handling large prey. In addition to ontogenetic changes in dentition, further mineralization of the jaws may be required to effectively feed on marine mammals. Our study is the first comparative FEA of the jaws for any fish species. Results highlight the potential of FEA for testing previously intractable questions regarding feeding mechanisms in sharks and other vertebrates.     

Cardiac metabolism in the Myxinidae: physiological and phylogenetic considerations

Cardiac muscle hearts of Atlantic hagfish continuously function under hypoxic conditions that would lead to cardiac failure in most other vertebrates. Contractile performance of hagfish systemic hearts is resistant to anoxia and respiratory poisons but shows a significant decrement when carbohydrate catabolism is blocked by 0.5 mM iodoacetic acid. Enzyme activity profiles of hagfish ventricle reveal a robust capacity for glycolysis of carbohydrate in comparison to that for general aerobic metabolism and catabolism of alternate metabolic fuels. Isolated working hagfish ventricles preferentially oxidize radiolabeled glucose even when fatty acid fuels are present in the incubation medium. Work output of the isolated ventricular preparation is maintained only in the presence of exogenous glucose. The results indicate that energy metabolism of the hagfish myocardium is predominantly carbohydrate-based and that energy demand of the tissue can be sustained by anaerobic glycolysis during extended periods of extreme hypoxia. Cardiac metabolism of this primitive species is compared with that of hearts from higher vertebrates and an evolutionary hypothesis relating cardiac workload to preferred metabolic fuel is discussed.     

Determination of buffering capacity of rat myocardium during ischemia

To determine the buffering capacity of ischemic rat myocardium, lactate production was altered by glycogen depletion prior to total global ischemia. Lactate production was monitored by 1H-NMR spectroscopy in perfused rat hearts and determined by enzymatic assay of freeze-clamped tissue extracts. Intracellular pH was measured by 31P-NMR spectroscopy. The relationship between total lactate produced and pH varied considerably, depending on the final pH reached. At pH greater than 6.4 this relationship is linear with a total buffering capacity (delta lactate/delta pH) of 25 mumol H+/g wet weight per pH unit. At lower pH values (pH less than 6.4), the total buffering capacity increases progressively. Since ischemia is invariably accompanied by ATP and phosphocreatine (PCr) hydrolysis, the proton production/consumption during high-energy phosphate hydrolysis must be considered when evaluating the intrinsic buffering capacity of the myocardium against proton loads produced by lactate production from glucose and glycogen. Schemes are presented which allow an estimation of the contribution of ATP and PCr hydrolysis and the buffering by the CO2/HCO3- system during ischemia. At pH greater than 6.4, the majority (about 60%) of buffering is due to hydrolysis of adenosine triphosphate, phosphocreatine in the heart, and neutralization of sodium bicarbonate in the perfusate. At pH less than 6.4 an increasing proportion of cardiac buffering is from intrinsic cardiac buffers, most likely from intracellular proteins. After correction for these contributions to the observed total cardiac buffering capacity, the intrinsic buffering capacity of the myocardium can be accounted for by a high capacity (170 mumol/g wet weight) but low pKa (5.2) buffering system.     

燕辽生物群脊椎动物的多样性及与其他生物群的对比分析*

燕辽生物群已发现脊椎动物54属58种, 包括鱼类、两栖类、爬行类、哺乳类等, 但其脊椎动物多样性及其成因机制还未有详细研究。本文对该生物群脊椎动物进行统计分析, 并与同时代的其他生物群脊椎动物类型进行对比, 这为认识燕辽生物群脊椎动物的多样性及其成因提供了重要的证据。早期代表道虎沟生物群与晚期代表玲珑塔生物群虽存在时代上的传承关系, 但生物组合特征明显不同。对比燕辽生物群与相近时代的新疆五彩湾动物群和四川大山铺恐龙动物群, 脊椎动物组合特征差异显著。燕辽生物群恐龙类群主要以小型兽脚类恐龙为主, 还包括一些小型鸟臀类恐龙。另外还具有非常丰富的翼龙和哺乳动物。脊椎动物生态多样性高, 适应飞行、树栖、水生、穴居等多种生活方式, 但是脊椎动物的类型与同时代的相近地区明显不同。翼龙、恐龙和哺乳动物等类群都展现出独特的生物组合特征。有证据表明该时期东亚地区与其他地区可能存在一定程度的地理隔离, 结合陆生脊椎动物组合特征推测燕辽生物群脊椎动物与外界可能存在一定的交流障碍。     

Temperature,metabolic power and the evolution of endothermy

Endothermy has evolved at least twice, in the precursors to modern mammals and birds. The most widely accepted explanation for the evolution of endothermy has been selection for enhanced aerobic capacity. We review this hypothesis in the light of advances in our understanding of ATP generation by mitochondria and muscle performance. Together with the development of isotope‐based techniques for the measurement of metabolic rate in free‐ranging vertebrates these have confirmed the importance of aerobic scope in the evolution of endothermy: absolute aerobic scope, ATP generation by mitochondria and muscle power output are all strongly temperature‐dependent, indicating that there would have been significant improvement in whole‐organism locomotor ability with a warmer body. New data on mitochondrial ATP generation and proton leak suggest that the thermal physiology of mitochondria may differ between organisms of contrasting ecology and thermal flexibility. Together with recent biophysical modelling, this strengthens the long‐held view that endothermy originated in smaller, active eurythermal ectotherms living in a cool but variable thermal environment. We propose that rather than being a secondary consequence of the evolution of an enhanced aerobic scope, a warmer body was the means by which that enhanced aerobic scope was achieved. This modified hypothesis requires that the rise in metabolic rate and the insulation necessary to retain metabolic heat arose early in the lineages leading to birds and mammals. Large dinosaurs were warm, but were not endotherms, and the metabolic status of pterosaurs remains unresolved.     

LIPID COMPOSITION OF THE CENTRAL NERVOUS SYSTEM OF MARINE VERTEBRATES

—The lipid composition of the central nervous system of some marine vertebrates and two mammalian species (rat and man) was analysed by one- and two-dimensional quantitative thin-layer chromatography, and the cerebroside fatty acids were analysed by gas chromatography. The concentrations of sphingomyelin and cerebrosides are higher in mammals than in fishes, while no significant differences are observed for other lipid classes. Furthermore, in mammals the ratio between hydroxy and normal fatty acids in the cerebrosides is much higher than in fishes. The cerebrosides of mammals contain more very long chain fatty acids than those of marine vertebrates.     

The ontogeny of contractile performance and metabolic capacity in a high-frequency muscle

High-performance muscles such as the shaker muscles in the tails of western diamond-backed rattlesnakes (Crotalus atrox) are excellent systems for studying the relationship between contractile performance and metabolic capacity. We observed that shaker muscle contraction frequency increases dramatically with growth in small individuals but then declines gradually in large individuals. We tested whether metabolic capacity changed with performance, using shaker muscle contraction frequency as an indicator of performance and maximal activities of citrate synthase and lactate dehydrogenase as indicators of aerobic and anaerobic capacities, respectively. Contraction frequency increased 20-fold in 20-100-g individuals but then declined by approximately 30% in individuals approaching 1,000 g. Mass-independent aerobic capacity was positively correlated with contractile performance, whereas mass-independent anaerobic capacity was slightly but negatively correlated with performance; body mass was not correlated with performance. Rattle mass increased faster than the ability to generate force. Early in ontogeny, shaker muscle performance appears to be limited by aerobic capacity, but later performance becomes limited equally by aerobic capacity and the mechanical constraint of moving a larger mass without proportionally thicker muscles. This high-performance muscle appears to shift during ontogeny from a metabolic constraint to combined metabolic and mechanical constraints.     

Key metabolic enzymes and muscle structure in triplefin fishes (Tripterygiidae): a phylogenetic comparison

Metabolic potential and muscle development were investigated relative to habitat and phylogeny in seven species of New Zealand triplefin fishes. Activity was measured in three principal glycolytic enzymes (lactate dehydrogenase, pyruvate kinase and phosphofructokinase) and two oxidative enzymes (citrate synthase and L3-hydroxyacyl CoA:NAD(+) oxidoreductase). The non-bicarbonate buffering capacity of caudal muscle was also estimated. Phylogenetic independent contrast analyses were used to reduce the effects of phylogenetic history in analyses. A positive relationship between metabolic potential and the effective water velocity at respective habitat depths was found only after the exclusion from analyses of the semi-pelagic species Obliquichthys maryannae. O. maryannae showed high glycolytic enzyme activities, and displayed double the activity of both oxidative enzymes relative to the six benthic species. Histochemically stained sections taken immediately posterior to the vent showed that adult O. maryannae and larval Forsterygion lapillum had significantly more red muscle, and smaller cross-sectional areas of white and red muscle fibres, than adults of benthic species. The distribution of red muscle in adult O. maryannae resembled that of larval F. lapillum, and differed from the typical teleost pattern seen in adults of the six benthic species. Both adult O. maryannae and larval F. lapillum have an expansive lateralis superficialis muscle, typical of larval fish, which encompasses much of the caudal trunk. Results suggest that anaerobic potential in New Zealand triplefins: (a) increases with the locomotory requirements of different habitats, and (b) displays a negative relationship with depth-dependent water velocities in benthic species. O. maryannae appears to have increased aerobic potential for sustained swimming by paedomorphic retention of larval muscle architecture.