Potential impact of metabolic acidosis on the fixed-acid Bohr effect in snapper (Pagrus auratus) following angling stress.

Oxygen equilibrium data were obtained for intact erythrocytes from Pagrus auratus and the pH-dependence of the fixed-acid Bohr and Root effects described. Indicators of aerobic-anaerobic poise were then measured following a period of strenuous exercise induced by angling capture. Haematological indices and plasma metabolites were evaluated with respect to their potential impact on the blood oxygen transport mechanism. An increase in post-capture haemoglobin content, blood lactate, and glucose were observed. The fall in caudal venous pH from 7.53 to 7.41, reflecting the acid-base status of blood draining the working tail musculature, remained within the operational zone of the maximal fixed-acid Bohr effect (Phi(7.4-7.8)=-0.95), but above the critical pH at which the Root effect suppresses oxygen transport capacity. These stress-induced changes reflect a strategy of optimal O(2) unloading to tissues with the Root effect unlikely to be expressed in the swimming vasculature.

Thermal effects on the blood respiratory properties of southern bluefin tuna, Thunnus maccoyii.

Thermal effects on the blood respiratory properties of southern bluefin tuna (Thunnus maccoyii) at 10, 23 and 36 degrees C, and at 0.5 and 1.5% CO(2) were investigated. A reversed temperature effect occurred as the oxygen partial pressure required for 50% haemoglobin saturation (P(50)) at 0.5% CO(2) decreased from 2.9 kPa at 10 degrees C to 1.7 kPa at 23 degrees C (apparent heat of oxygenation, DeltaH degrees , =+27 kJ mol(-1)). However, oxygen binding was essentially independent of temperature at warmer temperatures (P(50)=1.7-2.0 kPa from 23-36 degrees C at 0.5% CO(2); DeltaH degrees =-6.5 kJ mol(-1)). Hill's coefficient (n(H)) ranged from 1.3 to 1.6, and there was a large effect of temperature on the Bohr factor (DeltalogP(50)/DeltapH=-1.6 at 10 degrees C and -0.9 at 36 degrees C). This is the first study of whole blood to demonstrate the thermal dependence of DeltaH degrees itself, whereby the oxygen equilibrium curve is more sensitive to temperature in the lowest thermal range examined. We suggest that the functional basis for these observations lies in the necessity to ensure a sufficient oxygen supply to all tissues, including the heart and liver, without suffering from premature or excessive oxygen unloading around the heat exchanger prior to delivery of oxygen to organs and tissues that lie efferent to the exchanger.

A new method for the detection of hydroxyl radical production by phagocytic cells.

Benzoic acid, a specific scavenger of hydroxyl radical (OH.) is known to be oxidized as the result of a reaction with OH.. We have determined that the decarboxylation of benzoic acid can be used to detect OH. generated in cell-free systems and human granulocytes. Benzoic acid is oxidized by the xanthine-xanthine oxidase enzyme system. This system is known to generate O2-, H2O2 and OH.. This oxidation is inhibited by superoxide dismutase, catalase and mannitol. Therefore, the oxidation of benzoic acid occurs by a mechanism similar to that reported for the oxidation of methional to ethylene and involves OH.. Resting granulocytes do not oxidize benzoic acid. However, marked oxidation of this substrate occurs during the phagocytosis of opsonized zymosan particles, indicating the production of OH. by these cells. The reaction can be inhibited by superoxide dismutase, catalase, azide and mannitol. Therefore, the production of OH. in the cell may be similar to that observed in the cell-free system. The granulocytes of a patient with known chronic granulomatous disease did not oxidase benzoic acid, indicating a defect in the generation of OH. by these cells.

On-line detection of reversible myocardial ischemic injury by measurement of myocardial electrical impedance.

The metabolic and physiological alterations associated with changes in myocardial tissue electrical resistivity during ischemia were characterized to assess the feasibility of using such resistivity as an on-line indicator of the onset of ischemic injury. Twelve anesthetized dogs underwent rapid cardiac extirpation; 5 served as untreated controls, and 7 were pretreated with metoprolol tartrate. Beta blockade was used to alter the time course of ischemic injury as demonstrated previously in studies using this experimental model. In vitro measurement of myocardial resistivity, the detection of ischemic contracture, and serial measurements of tissue adenosine triphosphate (ATP) and lactate were obtained from totally ischemic left ventricles at 37 degrees C. Myocardial resistivity began to increase significantly before onset of ischemic contracture in the untreated control group (resistivity at 42.3 +/- 3.1 minutes, contracture at 53.8 +/- 3.7 minutes; p less than 0.025) as well as the metoprolol group (resistivity at 50.7 +/- 1.5 minutes, contracture at 70.0 +/- 3.5 minutes; p less than 0.005). As expected, ischemic contracture was delayed in the beta-blocked group compared with controls (p less than 0.01). Similarly, the onset of myocardial resistivity increase was delayed in the beta-blocked group (p less than 0.025). ATP and lactate levels at the onset of myocardial resistivity increase were consistent with severe but reversible injury. Resistivity changes during ischemia correlated linearly with simultaneous ATP depletion and lactate accumulation (r = 0.88 to 0.98; p less than 0.05). Furthermore, during global ischemia studied in 3 anesthetized dogs in vivo, the onset of myocardial resistivity increase occurred after 20 minutes. Finally, 6 anesthetized dogs underwent 60 minutes of in vivo regional ischemia by coronary artery occlusion, followed by 60 minutes of reperfusion. Myocardial resistivity in the ischemic region increased immediately and steadily after coronary occlusion, followed by a rapid decrease during subsequent reperfusion. These data show that myocardial resistivity may be useful for identifying severe but still reversible ischemic injury in on-line fashion during regional and global myocardial ischemia.

Antagonistic effect of urea on oxygenation-linked binding of ATP in an elasmobranch hemoglobin.

The O2 affinity of "stripped" (cofactor-free) hemoglobin (Hb) of the elasmobranch, Squalus acanthias is decreased by ATP, the main erythrocytic phosphate cofactor but increased by urea at physiological concentration. When both compounds are present, as in life, urea decreases the ATP sensitivity, indicating that previous Hb oxygenation studies in the absence of urea overestimate the modulator role of phosphate cofactors in sharks. Whereas ATP decreases the O2 association equilibrium constant of the deoxygenated pigment, urea raises those of both the deoxy and the oxygenated states. Possible mechanisms for the urea-protein interactions i.e. binding at carboxy-termini or carbamylation of amino-termini of the protein chains, are discussed.

Evidence that OH. production by human PMNs is related to prostaglandin metabolism.

Recent studies indicate that human granulocytes generate OH. during the phagocytosis of zymosan particles. Several theoretical considerations suggested to us that this OH. production might be related to prostaglandin metabolism, particularly the observation that OH. is generated by the reducation of hydroperoxides in microsomal systems. In our studies, we tested the importance of prostaglandin metabolism in the production of OH. by human granulocytes (PMNs). Indomethacin and aspirin at concentrations known to impair cyclooxygenase activity decreased OH. production by PMNs during the phagocytosis of zymosan particles. Phenol, which is known to alter prostaglandin metabolism, ablated OH. completely. None of these drugs at the concentrations used impaired the generation of O-2 or H2O2 by PMNs, as indicated by their failure to diminish significantly the generation of chemiluminescence. Thus, the decrement in OH. production by these drugs could not be attributed to a nonspecific effect on the production of O-2 or H2O2. These experiments therefore, indicate that the model for OH. production observed during prostaglandin synthesis with microsomal systems applies to human granulocytes.

Metabolic characteristics of muscles in the spiny lobster, Jasus edwardsii, and responses to emersion during simulated live transport.

The metabolic characteristics of five muscle groups in the spiny lobster Jasus edwardsii were examined in order to compare their anaerobic and oxidative capacities. Enzyme activities of phosphorylase, phosphofructokinase, pyruvate kinase, and lactate dehydrogenase were highest in abdominal muscles supporting anaerobic burst activity. Hexokinase, citrate synthase, and HOAD activities in the leg and antennal muscles indicated higher aerobic potential. Arginine kinase activities were high in all muscle groups indicating that muscle phosphagens are an important energy reserve. Arginine phosphate concentrations in 4th periopod and abdominal flexor muscle from lobsters sampled in the field were higher than any values from captive animals, and approximately five times those for ATP. Muscle lactates were high in captive animals. Responses to emersion during simulated live transport appear to exploit the capacity for functional anaerobiosis and further differentiated the muscle groups. Abdominal muscles were especially sensitive and after 24 h showed significant increases in lactate, glucose, ADP, and AMP. ATP levels appeared to be maintained by muscle phosphagens and raised doubts about the efficacy of the adenylate energy charge in evaluating the emersion response. Haemolymph glucose, lactic acid, and ammonia peaked after 24 h emersion and were largely restored following re-immersion. We propose that arginine phosphate concentrations in the 4th periopod are an appropriate index of metabolic stress, and could lead to improved commercial handling protocols.

Ontogenetic changes in enzyme activities associated with energy production in the spiny lobster, Jasus edwardsii.

The larval life of the spiny lobster Jasus edwardsii is one of the longest and most complex of any marine organism and is poorly understood due to the difficulty of studying cryptic, pelagic organisms. Hence, the capacity for active swimming in the phyllosoma, puerulus and juvenile stages and the use of possible metabolic fuel reserves was inferred from a number of enzyme activities, including citrate synthase, lactate dehydrogenase, and HOAD. High activities of CS and LDH in abdominal tissues of Stage 11 phyllosoma and pueruli are consistent with a capacity to commence active on-shore movement. The activities of LDH and HOAD showed positive allometry while CS was independent of body mass. The body mass dependence of LDH activity may reflect the developing ability of the lobster to initiate brief escape manoeuvres, and the scaling of HOAD reflects an increased use of lipid fuel reserves. Aerobic enzyme activities were higher in abdominal tissues than in cephalic tissues of pelagic pueruli, but high activities appear in the cephalic tissues of juveniles. These changes mirror a developmental shift in activity from pelagic oceanic swimming to a benthic existence on the seabed of the near shore. The low LDH activity in pueruli confirmed previous findings that they have limited feeding capacity, with carbohydrate contributing little towards the major energy reserves. The highest LDH activities occur in the abdominal muscles of juveniles and correlate with rapid tail-flicking escape behaviour. The activities of HOAD increased throughout development, and in the abdominal tissues of juveniles, may reflect lipid transformation and accumulation as an energy reserve. Enzyme activities, therefore, provide useful information concerning migratory behaviour that is presently unavailable from ecological studies.

Partitioning of respiration between the gills and air-breathing organ in response to aquatic hypoxia and exercise in the pacific tarpon, Megalops cyprinoides.

The evolution of air-breathing organs (ABOs) is associated not only with hypoxic environments but also with activity. This investigation examines the effects of hypoxia and exercise on the partitioning of aquatic and aerial oxygen uptake in the Pacific tarpon. The two-species cosmopolitan genus Megalops is unique among teleosts in using swim bladder ABOs in the pelagic marine environment. Small fish (58-620 g) were swum at two sustainable speeds in a circulating flume respirometer in which dissolved oxygen was controlled. For fish swimming at 0.11 m s(-1) in normoxia (Po2 = 21 kPa), there was practically no air breathing, and gill oxygen uptake was 1.53 mL kg(-0.67) min(-1). Air breathing occurred at 0.5 breaths min(-1) in hypoxia (8 kPa) at this speed, when the gills and ABOs accounted for 0.71 and 0.57 mL kg(-0.67) min(-1), respectively. At 0.22 m s(-1) in normoxia, breathing occurred at 0.1 breaths min(-1), and gill and ABO oxygen uptake were 2.08 and 0.08 mL kg(-0.67) min(-1), respectively. In hypoxia and 0.22 m s(-1), breathing increased to 0.6 breaths min(-1), and gill and ABO oxygen uptake were 1.39 and 1.28 mL kg(-0.67) min(-1), respectively. Aquatic hypoxia was therefore the primary stimulus for air breathing under the limited conditions of this study, but exercise augmented oxygen uptake by the ABOs, particularly in hypoxic water.

Polycythaemia associated with a new haemoglobin variant: haemoglobin Palmerston North beta 23 (B5) val----phe.

A new haemoglobin variant, with increased oxygen affinity, has been identified in a patient with a long history of polycythaemia. This new haemoglobin, Hb Palmerston North, has an amino acid substitution of valine to phenylalanine at position beta 23 (B5). The increased oxygen affinity is accompanied by a decrease in globin stability which was responsible for the laboratory detection of the haemoglobinopathy.

Syntheses and in-vitro evaluation of novel adamantane based γ-secretase inhibitors.

Abnormal processing of amyloid precursor protein (APP) by ß - and γ -secretases to produce excess amyloid-ß-peptide is believed to contribute to the pathophysiological cascade that results in Alzheimer's disease. γ -Secretase inhibition or modulation therefore represents a rational approach to the prevention and/or management of AD. Here, we present the discovery and SAR of a class of novel adamantanyl sulfonamide based γ -secretase inhibitors. Activity evaluation was conducted on cell lines overexpressing APP (wild type and Swedish mutation). Our results suggest size threshold and hydrogen bond formation are necessary for inhibitory activity. There was no correlation between compound activity, Log P, and the electronic effect of substituents on the aromatic ring. These compounds possess desirable drug like properties and results of the study can guide a pharmacophore based design of γ -secretase inhibitors.

Changes in cardiac output during swimming and aquatic hypoxia in the air-breathing Pacific tarpon.

Pacific tarpon (Megalops cyprinoides) use a modified gas bladder as an air-breathing organ (ABO). We examined changes in cardiac output (V(b)) associated with increases in air-breathing that accompany exercise and aquatic hypoxia. Juvenile (0.49 kg) and adult (1.21 kg) tarpon were allowed to recover in a swim flume at 27 degrees C after being instrumented with a Doppler flow probe around the ventral aorta to monitor V(b) and with a fibre-optic oxygen sensor in the ABO to monitor air-breathing frequency. Under normoxic conditions and in both juveniles and adults, routine air-breathing frequency was 0.03 breaths min(-1) and V(b) was about 15 mL min(-1) kg(-1). Normoxic exercise (swimming at about 1.1 body lengths s(-1)) increased air-breathing frequency by 8-fold in both groups (reaching 0.23 breaths min(-1)) and increased V(b) by 3-fold for juveniles and 2-fold for adults. Hypoxic exposure (2 kPa O2) at rest increased air-breathing frequency 19-fold (to around 0.53 breaths min(-1)) in both groups, and while V(b) again increased 3-fold in resting juvenile fish, V(b) was unchanged in resting adult fish. Exercise in hypoxia increased air-breathing frequency 35-fold (to 0.95 breaths min(-1)) in comparison with resting normoxic fish. While juvenile fish increased V(b) nearly 2-fold with exercise in hypoxia, adult fish maintained the same V(b) irrespective of exercise state and became agitated in comparison. These results imply that air-breathing during exercise and hypoxia can benefit oxygen delivery, but to differing degrees in juvenile and adult tarpon. We discuss this difference in the context of myocardial oxygen supply.

Plasma lactate and glucose flushes following burst swimming in silver trevally (Pseudocaranx dentex: Carangidae) support the "releaser" hypothesis.

Silver trevally (Pseudocaranx dentex) are highly athletic marine teleosts inhabiting the tropical waters of the Great Barrier Reef, Australia. Burst swimming increased plasma lactate from 1.6 +/- 0.4 S.D. to 21.6 +/- 3.3 mM (N = 6), among the highest values reported for functional hypoxia in fish. These data support the hypothesis that elite swimmers release lactate produced in the myotome into the circulation following anaerobic burst activity. The fish further developed a hyperglycaemic response to burst exercise with plasma glucose increasing from 6.6 +/- 2.0 to 13.2 +/- 2.3 mM (N = 6). Post-exercise erythrocyte swelling also occurred, but nucleoside triphosphate levels remained unaltered and do not provide a mechanism to modulate haemoglobin function during exercise. Metabolism of the blood cells appeared to be fuelled by both lactate and glucose.

Red blood cell function and haematology in two tropical freshwater fishes from Australia.

Salmon catfish and tarpon occur in habitats that periodically become deficient in oxygen resulting in high mortalities of other fish species. The water-breathing catfish, Arius leptaspis, and the facultative air-breathing tarpon, Megalops cyprinoides, both have high haemoglobin and haematocrit, and the oxygen carrying capacity in the air-breather is exceptionally high (15.6+/-1.2 vol%). Iso-pH oxygen equilibria of the red blood cells at 25 degrees C revealed high affinity (P(50)=9 mmHg, pH 7.4) and co-operativity (n(50)>2.2, pH 7.4) in the catfish, and contrasted with low affinity (P(50)=32 mmHg, pH 7.4) and co-operativity (n(50) approximately 1) in the air-breathing tarpon. Oxygen binding was further distinguished by relative pH insensitivity (Bohr factor, Ø=Deltalog P(50)/Deltalog pH=-0.22) in the catfish, compared with a significant Bohr effect in the tarpon (Ø=-0.96). The potential for modulation of haemoglobin-oxygen affinity was indicated by a high ratio of GTP to ATP in the erythrocytes of the catfish, whereas regulation in the tarpon appeared due to ATP alone. Differences in blood respiratory functions between the two species are likely to reflect reduced opportunity for activity under extreme hypoxia in the catfish.

The oxygen affinity of chicken haemoglobin in whole blood and erythrocyte suspensions.

The oxygen equilibrium of whole chicken blood has a P50 of 52.3 mm Hg and a Hill coefficient of 2.6 at pH 7.4 and 37 degrees C when determined with new microtechniques which are not vitiated by cellular respiration. The apparent failure of the haemoglobin to reach full saturation at arterial PO2 IS DISCUSSED IN RELATION TO THE HAEMoglobin-oxygen equilibrium concept. The low affinity observed is due to intraerythrocytic inositol pentaphosphate (IPP), and the affinity of a haemolysate "stripped" of IPP is greatly increased (deltalog P50 =1.25). Unlike the mamalian analogue 2, 3-diphosphoglycerate, IPP concentration of whole blood does not decrease after incubation for 10 hr at 37 degreesC.

Respiration of antarctic fish from McMurdo Sound.

1. Resting rates of oxygen uptake were measured for nine species of unstressed fish living at -1.8 degree C in McMurdo Sound, Antarctica (77-78 degrees S). Interspecific differences in VO2 were correlated with the habits and activity of the fish. 2. The cryopelagic Pagothenia borchgrevinki regulated oxygen uptake down to a critical PO2 of approximately 60 mmHg. The inactive benthic species Trematomus centronotus extracted oxygen to lower PO2 and appeared to have a lesser degree of oxyregulation when the data were analysed using a quadratic model. 3. Cutaneous oxygen uptake in the nototheniids T. bernacchii and P. borchgrevinki amounted to 9 and 17% of total VO2 under normoxic conditions which is less than that reported for scaleless Antarctic fish. 4. The contentious concept of metabolic cold adaptation in polar fish has been reviewed, and the opinion expressed that the phenomenon cannot be dismissed on the grounds of technically incompetent measurement, or through inappropriate extrapolation of data from fish at lower latitudes.

Evolution of haemoglobin function: molecular adaptations to environment.

1. Nearly 1000 mutations have been described for human haemoglobin (Hb), many of which result in subtle changes to the oxygen transport system. Similar changes have occurred over the course of animal evolution resulting in a diversity of functional attributes which appear to correlate the availability of oxygen in the environment with metabolic demand. 2. At an early stage in evolution, worm-like animals had large, polymeric aggregations of Hb subunits circulating through primitive circulatory systems and some possessed monomeric Hb in blood cells functioning as an oxygen store. 3. The circulating vertebrate red blood cell provides an environment allowing haem units to interact among themselves and with various organic phosphates to allow a responsive and highly regulated system of gas transport. During metazoan evolution the burden of physiological regulation has shifted from the cells to organ systems, as endothermy and aerial breathing permit a relatively constant environment. 4. An understanding of the adaptive possibilities of Hb has helped us to understand the ontogeny of oxygen transport and to interpret recently described functional properties of human embryonic haemoglobins.

A simple and inexpensive tonometry system for use with microlitre blood samples.

The construction of a blood tonometering system based on the vibrating action of a firebell is described. Its performance has been assessed with human blood at 37 degrees C and in use with Antarctic fish blood (Pagothenia borchgrevinki) at -1.5 degrees C.