Search

Teleost fishes are diverse and successful, comprising almost half of all extant vertebrate species. It has been suggested that their success as a group is related, in part, to their unique O 2 transport system, which includes pH-sensitive hemoglobin, a red blood cell β-adrenergic Na + /H + exchanger (RBC β-NHE) that protects red blood cell pH, and plasma accessible carbonic anhydrase which is absent at the gills but present in some tissues, that short-circuits the β-NHE to enhance O 2 unloading during periods of stress. However, direct support for this has only been examined in a few species of salmonids. Here, we expand the knowledge of this system to two warm-water, highly active marine percomorph fish, cobia (Rachycentron canadum) and mahi-mahi (Coryphaena hippurus). We show evidence for RBC β-NHE activity in both species, and characterize the Hb-O 2 transport system in one of those species, cobia. We found significant RBC swelling following β-adrenergic stimulation in both species, providing evidence for the presence of a rapid, active RBC β-NHE in both cobia and mahi-mahi, with a time-course similar to that of salmonids. We generated oxygen equilibrium curves (OECs) for cobia blood and determined the P 50 , Hill, and Bohr coefficients, and used these data to model the potential for enhanced O 2 unloading. We determined that there was potential for up to a 61% increase in O 2 unloading associated with RBC β-NHE short-circuiting, assuming a - 0.2 ∆pH a-v in the blood. Thus, despite phylogenetic and life history differences between cobia and the salmonids, we found few differences between their Hb-O 2 transport systems, suggesting conservation of this physiological trait across diverse teleost taxa., (Copyright © 2021 Elsevier Inc. All rights reserved.)

In the early 20th century, August and Marie Krogh settled one of the most controversial questions in physiology, showing through elegant experiments that oxygen (O 2 ) uptake at the lung is driven by passive diffusion alone. Krogh's later work, on the regulation of local blood flow and capillary recruitment at the tissues, was awarded with the Nobel Prize in 1920. A century later it is still undisputed that O 2 moves across tissues by diffusion, however, animals use active mechanisms to regulate and facilitate the passive process. Teleost fishes have evolved a mechanism by which adrenergic sodium-proton-exchangers (β-NHEs) on the red blood cell (RBC) membrane actively create H + gradients that are short-circuited in the presence of plasma-accessible carbonic anhydrase (CA) at the tissue capillaries. The rapid acidification of the RBC reduces the O 2 affinity of pH-sensitive haemoglobin, which increases the O 2 diffusion gradient to the tissues. When RBCs leave the site of plasma-accessible CA, β-NHE activity recovers RBC pH during venous transit, to promote renewed O 2 loading at the gills. This mechanism allows teleosts to unload more O 2 at their tissues without compromising O 2 diffusion gradients and therefore, to use the available O 2 carrying capacity of the blood to a greater degree. In Atlantic salmon, β-NHE short-circuiting reduces the requirements on the heart by up to 30% during moderate exercise and even at rest, with important ecological implications. Thus, in some teleosts, the RBCs participate in regulating the systemic O 2 flux by actively altering the passive diffusion of O 2 that Krogh discovered., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Blood-O2 affinities (P50 ) were measured over a physiologically relevant pH range at 31 (highest temperature average of Rio Negro over the last 8 years), 33 and 35° C for 10 species of the Rio Negro, aiming to test the acute effects of temperature foreseen by the IPCC (Intergovernmental Panel on Climate Change) for coming years. The animals were collected during an expedition to the Anavilhanas Islands of the Rio Negro, 110 km upstream from Manaus (2° 23' 41″ S; 60° 55' 14″ W). Hoplias malabaricus showed higher blood-O2 sensitivity to pH changes (Bohr effect, Φ = Δlog10  P50  ΔpH(-1) ) at both 31° C (Φ = -0·44) and 35° C (Φ = -0·26) compared to Osteoglossum bicirrhosum (Φ = -0·54 at 31° C and Φ = -0·58 at 35° C), but lower P50 under most conditions, and a greater sensitivity of P50 to temperature. Two out of the 10 analysed species had significant increases of P50 (lower blood-O2 affinity) at the highest temperature throughout the pH range tested. For all other species, a minor increase of P50 over the assay-tested temperatures was observed, although all presented a normal Bohr effect. Overall, a diversity of intensities of pH and temperature effects on blood-O2 affinities was observed, which seems to be connected to the biological characteristics of the analysed species. Thermal disturbances in their habitats, likely to occur due to the global warming, would impair blood-O2 binding and unloading in some of the analysed fish species. Copyright © 2016 John Wiley & Sons, Ltd., (© 2016 The Fisheries Society of the British Isles.)

The teleost fishes represent over half of all extant vertebrates; they occupy nearly every body of water and in doing so, occupy a diverse array of environmental conditions. We propose that their success is related to a unique oxygen (O2) transport system involving their extremely pH-sensitive haemoglobin (Hb). A reduction in pH reduces both Hb-O2 affinity (Bohr effect) and carrying capacity (Root effect). This, combined with a large arterial-venous pH change (ΔpHa-v) relative to other vertebrates, may greatly enhance tissue oxygen delivery in teleosts (e.g., rainbow trout) during stress, beyond that in mammals (e.g., human). We generated oxygen equilibrium curves (OECs) at five different CO2 tensions for rainbow trout and determined that, when Hb-O2 saturation is 50% or greater, the change in oxygen partial pressure (ΔPO2) associated with ΔpHa-v can exceed that of the mammalian Bohr effect by at least 3-fold, but as much as 21-fold. Using known ΔpHa-v and assuming a constant arterial-venous PO2 difference (Pa-vO2), Root effect Hbs can enhance O2 release to the tissues by 73.5% in trout; whereas, the Bohr effect alone is responsible for enhancing O2 release by only 1.3% in humans. Disequilibrium states are likely operational in teleosts in vivo, and therefore the ΔpHa-v, and thus enhancement of O2 delivery, could be even larger. Modeling with known Pa-vO2 in fish during exercise and hypoxia indicates that O2 release from the Hb and therefore potentially tissue O2 delivery may double during exercise and triple during some levels of hypoxia. These characteristics may be central to performance of athletic fish species such as salmonids, but may indicate that general tissue oxygen delivery may have been the incipient function of Root effect Hbs in fish, a trait strongly associated with the adaptive radiation of teleosts.

Aim: The ability of high carbon dioxide tensions or low pH to reduce blood oxygen binding even at high oxygen tensions, first observed by August Krogh and Isabella Leitch in 1919 and now known as the Root effect, was studied in red blood cells and haemoglobin solutions of several fish species., Methods: Red blood cells in physiological saline were acidified at atmospheric oxygen tension by increasing carbon dioxide tensions and the percentage decrease in oxygen content was used to quantify the Root effect. Haemoglobin was incubated in air-equilibrated citrate buffers between pH 5 and 7 and the percentage decrease in oxygen saturation relative to pH 8 determined by spectral deconvolution., Results: The maximal magnitude of the Root effect in citrate-buffered haemoglobin solutions closely matched the value in blood or red blood cells of 11 vertebrates over a Root effect range between 3 and 80%. Contrary to previous reports, there was no evidence for a significant Root effect in red blood cells or haemoglobin solutions of the wels catfish, but a significant Root effect under both conditions in the Siberian sturgeon., Conclusions: Under the conditions employed in this study, the maximal Root effect of citrate-buffered haemoglobin solutions closely resembles the maximal Root effect in red blood cells. This strengthens previous studies on the evolution of the Root effect and its role in oxygen concentration at the retina and swimbladder of a large number of fishes that were based on Root effect measurements in haemoglobin solutions., (© 2011 The Authors. Acta Physiologica © 2011 Scandinavian Physiological Society.)

Influence of temperature in the range of 1-15 degrees C on oxygen binding properties of blood of thermophilic--golden mullet (Liza aurata), anchovy (Engraulis encrasicolus), and cold-tolerant--sardelle (Clupeonella cultriventris) fishes has been investigated under experimental conditions. Heat dependence of oxygenation reaction in thermophilic fish blood at temperature below 10 degrees C considerably increases, which is evidenced by high deltaH values. That is accompanied by a substantial increase of blood oxygen affinity and complicates blood deoxygenation at the tissue level. This reaction is apparently determined by the change of hemoglobin interaction with intraerythrocyte medium. The concentration of NTP in erythrocytes increases, that partially compensates negative changes of blood oxygen affinity (parameter P50 is raised) under long-term maintenance of fishes at 5 degrees C. However this reaction is not observed at low temperatures (1-2 degrees C).

The Root effect, a reduction in blood oxygen (O(2)) carrying capacity at low pH, is used by many fish species to maximize O(2) delivery to the eye and swimbladder. It is believed to have evolved in the basal actinopterygian lineage of fishes, species that lack the intracellular pH (pH(i)) protection mechanism of more derived species' red blood cells (i.e., adrenergically activated Na(+)/H(+) exchangers; betaNHE). These basal actinopterygians may consequently experience a reduction in blood O(2) carrying capacity, and thus O(2) uptake at the gills, during hypoxia- and exercise-induced generalized blood acidoses. We analyzed the hemoglobins (Hbs) of seven species within this group [American paddlefish (Polyodon spathula), white sturgeon (Acipenser transmontanus), spotted gar (Lepisosteus oculatus), alligator gar (Atractosteus spatula), bowfin (Amia calva), mooneye (Hiodon tergisus), and pirarucu (Arapaima gigas)] for their Root effect characteristics so as to test the hypothesis of the Root effect onset pH value being lower than those pH values expected during a generalized acidosis in vivo. Analysis of the haemolysates revealed that, although each of the seven species displayed Root effects (ranging from 7.3 to 40.5% desaturation of Hb with O(2), i.e., Hb O(2) desaturation), the Root effect onset pH values of all species are considerably lower (ranging from pH 5.94 to 7.04) than the maximum blood acidoses that would be expected following hypoxia or exercise (pH(i) 7.15-7.3). Thus, although these primitive fishes possess Hbs with large Root effects and lack any significant red blood cell betaNHE activity, it is unlikely that the possession of a Root effect would impair O(2) uptake at the gills following a generalized acidosis of the blood. As well, it was shown that both maximal Root effect and Root effect onset pH values increased significantly in bowfin over those of the more basal species, toward values of similar magnitude to those of most of the more derived teleosts studied to date. This is paralleled by the initial appearance of the choroid rete in bowfin, as well as a significant decrease in Hb buffer value and an increase in Bohr/Haldane effects, together suggesting bowfin as the most basal species capable of utilizing its Root effect to maximize O(2) delivery to the eye.

We examined for the first time the hemoglobin components of the blood of the Australian lungfish, Neoceratodus forsteri and their functional responses to pH and the allosteric modulators adenosine triphosphate (ATP), guanosine triphosphate (GTP), 2,3-bisphosphoglyceric acid (BPG) and inositol hexaphosphate (IHP) at 25 degrees C. Lysates prepared from stripped, unfractionated hemolysate produced sigmoidal oxygen equilibrium curves with high oxygen affinity (oxygen partial pressure required for 50% hemoglobin saturation, p(50)=5.3 mmHg) and a Hill coefficient of 1.9 at pH 7.5. p(50) was 8.3 and 4.5 mmHg at pH 6 and 8, respectively, which corresponded to a modest Bohr coefficient (Delta log p(50)/Delta pH) of -0.13. GTP increased the pH sensitivity of oxygen binding more than ATP, such that the Bohr coefficient was -0.77 in the presence of 2 mmol L(-1) GTP. GTP was the most potent regulator of hemoglobin affinity, with concentrations of 5 mmol L(-1) causing an increase in p(50) from 5 to 19 mm Hg at pH 7.5, while the order of potency of the other phosphates was IHP>ATP>BPG. Three hemoglobin isoforms were present and each contained both alpha and beta chains with distinct molecular weights. Oxygen affinity and pH-dependence of isoforms I and II were essentially identical, while isoform III had a lower affinity and increased pH-dependence. The functional properties of the hemoglobin system of Neoceratodus appeared consistent with an active aquatic breather adapted for periodic hypoxic episodes.

The African (Protopterus sp.) and South American lungfish (Lepidosiren paradoxa) inhabit shallow waters, that seasonally dry out, which induces aestivation and cocoon formation in Protopterus. Differently, L. paradoxa has no cocoon, and it aestivates in a simple burrow. In water PaCO(2) is 21.8+/-0.4mmHg (mean values+/-S.E.M.; n=5), whereas aestivation for 20 days increased PaCO(2) to as much as 37.6+/-2.1mmHg, which remained the same after 40 days (35.8+/-3.3mmHg). Concomitantly, the plasma [HCO(3)(-)]-values for animals in water were 22.5+/-0.5mM, which after 20 days increased to 40.2+/-2.3mM and after 40 days to 35.8+/-3.3mM. Initially in water, PaO(2) was 87.7+/-2.0mmHg, but 20 days in aestivation reduced the value to 80.5+/-2.2 and later (40 days) to 77.1+/-3.0mmHg. Meanwhile, aestivation had no effect on pHa and hematocrit. The blood pressures were equal for animals in the water or in the burrow (P(mean) approximately 30mmHg), and cardiac frequency (f(H)) fell from 31beats min(-1) to 22beats min(-1) during 40 days of aestivation. The osmolality (mOsmkgH(2)O(-1)) was elevated after 20 and 40 days of aestivation but declined upon return to water. The transition from activity to aestivation involves new set-points for the variables that determine the acid-base status and PaO(2) of the animals, along with a reduction of cardiac frequency.

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.

The effects of experimental alterations of aerial O2 partial pressure (PO2,air) on bimodal gas exchange and air-breathing behaviour were investigated in the aquatic air-breathing fish Trichogaster leeri in normoxic water. Fish responded to increasing PO2,air by decreasing air-breathing frequency, increasing aerial O2 consumption rate (VO2), increasing mean O2 uptake per breath (VO2/breath) and decreasing aquatic VO2 to maintain a constant total VO2. The rate of oxygen uptake from the air-breathing organ (ABO) during apnoea (VO2,ap) was derived on a breath-by-breath basis from VO2/breath and apnoea duration. VO2,ap and estimates of ABO volume were used to calculate the PO2 in the ABO at the end of apnoea. This increased with increasing PO2,air, suggesting that ABO-PO2 is not regulated at a constant level by internal chemoreceptors. Furthermore, mean VO2,ap increased with increasing PO2,air, indicating that the observed increase in VO2/breath with increasing PO2,air was facilitated not only by an increase in apnoea duration but also by an increase in the air-blood PO2 gradient.

The relationship between whole blood-oxygen affinity (P(50)) and pH-dependent binding (i.e., cooperativity and the Bohr [ Phi ] and Root effects) was examined statistically under standardized conditions (10.0 degrees Celsius) in four unrelated cold-temperate marine fishes that differ widely in their swimming performance and their expected responses to hypoxia: cod (Gadus morhua), herring (Clupea harengus), mackerel (Scomber scombrus), and plaice (Pleuronectes platessa). An unexpected difference in blood-oxygen affinity was found (herring>plaice>mackerel>cod), and this was independent of both swimming performance and the predicted low O(2) response of each species. The ecotype of the four marine species was also unrelated to pH-dependent binding because no difference in the Bohr effect was apparent ( Phi varied insignificantly from -0.90 to -1.06), and differences in the magnitude of the cooperative binding reaction were associated only with the presence of the Root effect. Although several reviews propose a generalized link between blood-oxygen affinity and pH-dependent binding, our results advise against overestimating the adaptive functional properties of hemoglobin across unrelated species.

The blood oxygen affinity of vertebrates is regulated, in part, through changes in red cell phosphate levels and increased oxygen affinity during reductions in inspired oxygen and is a well-described and common feature. However, during anaemia, when oxygen delivery is compromised by a reduction in blood oxygen carrying capacity, a lowering of blood oxygen affinity will facilitate oxygen unloading in the tissues, while oxygen loading at the gas exchange organ is not impaired. The present study investigated the effects of artificially induced anaemia in vivo on the blood oxygen affinity and red cell nucleoside triphosphate (NTP) concentrations in the turtle, Chrysemys picta. Blood was obtained from conscious animals through an arterial catheter and oxygen equilibrium curves were determined using the Tucker method while NTP concentrations were analyzed spectrophotometrically. Before induction of anaemia haematocrit averaged 23% and P50 was 18.5 +/- 0.7 with a NTP/Hb of 0.20 +/- 0.01 (mmol/mmol). After the haematocrit had been reduced to approximately 10% by bleeding (48-96 h) (blood volume was maintained by re-infusion of plasma and Ringer) there were no effects on P50 or red cell NTP concentrations. Thus, in contrast to fish and mammals, turtles do not exhibit a change in blood oxygen affinity during anaemia.

Cooperative ligand binding by tetrameric vertebrate hemoglobins (Hbs) makes possible the delivery of oxygen at higher pressures than would otherwise occur. This cooperativity depends on changes in dimer-dimer interactions within the tetramer and is reflected in a 50 000-fold increase in the tetramer-dimer dissociation constant in human Hb upon oxygenation at pH 7.4, from approximately 2x10(-11)mol l-1 to approximately 10(-6)mol l-1. Hbs that undergo such ligand-dependent changes in association are widespread in non-vertebrates, where the mechanisms are very different from those in vertebrates. Oligomeric Hbs have been identified in organisms in five phyla (molluscs, echinoderms, annelids, phoronids and chordates) that dissociate to subunits upon oxidation of the heme iron and reassociate with the binding of ferric iron ligands such as CN-, N3- or NO2-. Thus, the valence and ligand state of the heme iron control the stability of a critical subunit interface. The broad distribution of this phenomenon suggests a common mechanism of communication between heme and interface that may be almost universal among non-vertebrate Hbs. This interaction may be similar to that known for the homodimeric Hb of the mollusc Scapharca inaequivalvis. Although muscle tissue Hbs or myoglobins (Mbs) are usually monomeric, with non-cooperative O2 binding, the radular muscles of gastropod molluscs and chitons have homodimeric Mbs that bind O2 cooperatively. Cooperative non-muscle tissue Hbs have also been identified. These include the neural Hb of the nemertean worm Cerebratulus lacteus and the Hb of the diving beetle Anisops assimilis, which exhibit deoxygenation-dependent self-association of monomers that is associated with high Hill coefficients. Calculations suggest that the 2-3 mmol l-1 concentration of Hb on a heme basis in the brain of Cerebratulus should substantially extend the time as an active predator in an anaerobic or hypoxic environment. Oxygen from the Hb of Anisops is delivered to a gas bubble and thereby controls the buoyant density. Many Hbs of amphibians, reptiles, birds and some embryonic mammals exhibit a further 'supercooperativity' of O2 binding which depends on reversible deoxygenation-dependent tetramer-tetramer association to form an assemblage with a very low affinity for O2. This phenomenon results in steeper O2-binding curves than exhibited by tetramers alone. The increased cooperativity should result in an increase in the amount of O2 delivered to the tissues and should be especially valuable for avian flight muscles.

Two major strategies are apparent for the regulation of gas transport by vertebrate blood except in the myxinoids, which seem to have little scope for such regulation. In lampreys and teleost fish, haemoglobins have low buffering capacities and large Bohr/Haldane effects. Na+/H+ exchange plays an important role in the control of haemoglobin oxygen-affinity in these vertebrate groups. The large Bohr/Haldane effect also facilitates carbon dioxide transport: the blood (or erythrocyte) pH increases upon deoxygenation, thus increasing the concentration of bicarbonate formed at a given carbon dioxide tension. In lampreys, the bicarbonate permeability of the erythrocyte membrane is low. As a consequence, extracellular acid loads cannot be buffered by haemoglobin. In contrast, teleost erythrocytes possess a functional anion exchange, allowing extracellular proton loads to be buffered by haemoglobin. However, because the buffering capacity of teleost haemoglobins is low, buffering of extracellular acid loads is less effective in teleost fish than in elasmobranch fish and in air-breathing vertebrates whose haemoglobins have a high buffering capacity. However, the high buffering capacity of the haemoglobins diminishes the possibility of regulating haemoglobin oxygen-affinity via secondarily active Na+/H+ exchange, because intracellular pH changes, caused by proton efflux, remain small.

The catecholamines, adrenaline and noradrenaline, are released into the circulation of fish during a variety of physical and environmental disturbances that share the common feature of a requirement for enhanced blood oxygen transport. Indeed, the dominant factor controlling the mobilization of catecholamines from chromaffin tissue is a depression of blood oxygen content usually coinciding with a reduction of hemoglobin-O2 (Hb-O2) binding to 50-60% saturation. The elevation of plasma catecholamine levels, under such conditions, activates a beta-adrenergic cyclic AMP-dependent Na+/H+ exchanger on the red blood cell (rbc) membrane. The adrenergic responsiveness AMP-dependent Na+/H+ exchanger on the red blood cell (rbc) membrane. The adrenergic responsiveness of the rbc Na+/H+ exchanger to catecholamines varies both within and between species. Such inter- and intra-specific differences may reflect, in part, the availability of cell surface beta-adrenoceptors that are functionally coupled to adenylate cyclase. The activation of rbc Na+/H+ exchange and the accompanying profound adjustments of intracellular and extracellular acid-base status, nucleoside triphosphate (NTP) levels, and cooperativity of Hb-O2 binding have important consequences on both O2 and CO2 transfer and transport in the blood that vary markedly at the sites of oxygenation (the gill) and deoxygenation (the tissues) thereby enabling simultaneous amelioration of O2 loading and unloading. At the gill, oxygen transfer is enhanced owing to increases in Hb-O2 affinity and capacity while at the tissues, oxygen delivery is facilitated by a reduction of Hb-O2 affinity. This reduction in affinity at the tissues is a consequence of the combined effects of increased cooperativity of Hb-O2 binding and a rise in venous PCO2 (PvCO2) caused by the titration of HCO3- by H+ extruded by the rbc Na+/H+ exchanger. This elevation of PvCO2 may contribute to the rise in arterial PCO2 (PaCO2) observed after adrenergic activation of rbc Na+/H+ exchange that is caused primarily by impairment of rbc CO2 excretion related to modification of the intracellular acid-base status.

Arterial and mixed venous blood were sampled through chronically implanted cannulae from rested and swimming hagfish. PaO2 remained high when hagfish were swum for 15 min at a velocity of 20 cm s-1. PvO2 fell from 17.2 mmHg at rest to 3.5 mmHg after swimming, and the arteriovenous pH difference increased from 0.15 to 0.25 pH units. Whole blood oxygen equilibrium curves were essentially hyperbolic (Hill's n value = 1.38) and gave a half-saturation PO2 (P50) value of 12.3 mmHg at pH 7.8 and 16 degrees C. A CO2-Bohr factor (phi = delta logP50/delta pH) of -0.43 and a limited buffering capacity of the blood, amounting to approx. 4 slykes, were observed. The role of the blood in transporting oxygen and carbon dioxide both at rest and after swimming is established by in vivo blood gas measurements and in vitro oxygen-binding data. The low internal PvO2 at rest is close to the P50 measured under similar conditions and the hyperbolic equilibrium curve permits further oxygen unloading when PvO2 falls during swimming.

Oxygen binding and buffer properties of the blood of the sheatfish, Silurus glanis, were investigated in vitro at 20 and 10 degrees C. The O2 binding curves were hyperbolic with P50 = 10.1 mm Hg (20 degrees C, pH = 7.5) and 4.6 (10 degrees C, pH = 7.5). There was a very large Bohr effect with an average delta log P 50/delta pH of - 1.14. At 20 degrees C this value tended to be higher than at 10 degrees C. As a consequence the apparent heat of oxygenation depended on pH. The mean value of delta H was -10.4 kcal/mol. The Haldane effect was pronounced too (delta pH/delta S = -0.14) as was the Root effect. Isoelectric focussing revealed 3 major hemoglobin fractions with isoionic points in a more alkaline region than in carp hemoglobin. The non-bicarbonate buffer value was -10 mmol . 1-1. pH -1. The intraerythrocytic pH depended on the extracellular pH and the O2 saturation: pH = (0.87 - 0.14 S) (pHe -6.68 + 6.48). Delta pH/delta t for a constant CO2 content was -0.0166.

Continuous recordings have been made of pH, PO2, and PCO2 of arterial blood (pHa, PaO2, PaCO2) in an extracorporeal circulation during periods of hypoxia (inspired PO2 45-10 Torr) in sea bass, Morone labrax. During moderate hypoxia hyperventilation was accompanied by an increase in pHa. Continuation of moderate hypoxia for periods up to 24 h produces an increase in lactate and a consequent decrease in pHa. During deep hypoxia there is a very brief alkalosis as ventilation increases but a marked decrease in pHa as lactate levels rise. Recovery from hypoxia is associated with an increase in lactate concentration reaching values of more than 6 meq X l-1 following deep hypoxia, and pHa falls to 7.73. PaO2 recovers rapidly, but recovery of PaCO2 is not so rapid and together with the residual hyperventilation indicates that the fish is paying off an O2 debt.

A mathematical model for gas exchange in fish gills is presented, which makes allowance for the non-linear nature of the oxygen and carbon dioxide equilibrium curves of blood, for the uneven distribution of diffusion conductance along the secondary lamellae, and for coupling of oxygen and carbon dioxide exchange through the Bohr and Haldane effects. The model demonstrates that for oxygen loading in the gill the sigmoid equilibrium curve is superior to a linear one, whereas the non-linearity of the carbon dioxide equilibrium curve does not significantly affect carbon dioxide exchange and that the Bohr and Haldane effects have importance only for carbon dioxide exchange. It is also shown that the arterial and expired gas tensions and concentrations are unaffected by whether the bulk of the diffusion conductance is at either the afferent or the efferent end of the individual lamellae, provided that the total conductance is unchanged.

The oxygen consumption of Sarotherodon niloticus L. was found to decline below a critical oxygen concentration of about 2 mg O2/l. An important influence of CO2 on the oxygen affinity of whole blood was observed at all temperatures between 20 and 35 degrees C for gas mixtures containing 5.6% CO2. Purified hemolysate showed extremely high oxygen affinities (p50 = 1.08 mmHg at pH 8.2 and 20 degrees C). Low cooperativity was observed at all temperatures from 20 to 35 degrees C, and pH values between 6.5 and 8.2. The Bohr effect proved to be important at pH values lower than pH 7.5 (phi = delta log P50/delta pH = -0.58 between pH 6.5 and 7.0 at 35 degrees C). The oxygen affinities show high thermal sensitivity without a marked pH influence (delta H value for overall oxygenation at pH was -71.7 kJ/mol). The obtained results are interpreted as adaptations to diurnal variations in ambient temperature and oxygen availability.

Teleosts living in some mid-water pelagic regions of the Pacific are hypoxic or anaerobic during most of the day and become aerobic only during their diurnal migrations to and from the sea surface. The blood oxygen capacities of these fishes are among the lowest ever reported, and the oxygen dissociation curves show a very low affinity for oxygen.

1. The kinetics of the Bohr effect and the role of carbonic anhydrase were studied in a series of representative vertebrates using a continuous flow rapid reaction apparatus. 2. The rates of the Bohr effect in vertebrates are very similar, and differences among classes are manifestations of the ambient temperature. 3. Complete carbonic anhydrase inhibition causes a fifteen to fortyfold reduction in the rate of the Bohr effect, sufficient to abolish its occurrence within capillary transit. 4. There is a twenty-three-fold (duck) to 360-fold (man) excess of carbonic anhydrase activity in vertebrate red cells for the normal generation of the Bohr effect. 5. When carbonic anhydrase is inhibited and CO2 hydration becomes rate limiting, the stoichiometry of the Bohr effect (delta log pO2/delta pH) is revealed in the ratio of the rates of proton formation in red cells to O2 release from haemoglobin.

The electrophoretic patterns of hemoglobin (Hb) and the functional properties of blood of seven migratory fish species, in two different kinds of habitat during both the rainy and dry seasons, were studied. All species showed multiple hemoglobins and very similar electrophoretic patterns except for Schizodon nasutus, which showed two cathodal components. The functional properties of blood reflect those electrophoretic patterns, since all species showed very little or no Bohr effect during each situation studied. Data on Leporinus obtusidens blood (during the rainy season), from both lotic and lentic environments, showed a normal Bohr effect (phi = -0.073). Similarly, Schizodon nasutus and Leporinus friderici from a lotic environment during the dry season were compared and showed a reverse Bohr effect (phi = 0.042). In the other 14 situations, only Leporinus elongatus from a lentic environment during the rainy season showed a statistically significant difference in log P50 values from the other fishes.

Blood respiratory properties have been studied in awake and estivating African lungfish, Protopterus amphibius. Fish had been estivating 28-30 months when blood was sampled. Hematocrit, O2 capacity and blood hemoglobin concentration increased by about 50% during estivation. Red cell Hb concentration (MCHC) was unaltered. Total blood concentration of nucleoside triphosphates (NTP) was reduced 50% in estivation. Thin-layer chromatography showed that the change in NTP resulted from a GTP/Hb (guanosine triphosphate) reduction to 20% of the value in awake fish. ATP (adenosine triphosphate) concentration remained largely unaltered. GTP/Hb changes were accompanied by marked changes in O2 affinity. The P50 value in blood from awake fish was 33 mm Hg at pH 7.5 compared to 9 mm Hg for the estivating fish. The n-value changed insignificantly. An increased Bohr effect was present in estivating fish, but its importance will depend on circulating pH in the two states. Standard bicarbonate ranged from a low of 8.6 mMol-L-1 (P plasma) at pH 7.5 in an awake fish to 49.6 mMol-L-1 (P) in an estivating fish. CO2 dissociation curves showed a variable Haldane effect having its highest value in the in vivo range of PCO2. The striking increase in O2-Hb affinity during estivation is regarded as an adaptation to a reduced alveolar O2 availability associated with estivation. Altered mechanics of pulmonary ventilation and reduced ambient O2 availability caused by the subterranean habitat may have been selection pressures for the development of the high O2 availability caused by the subterranean habitat may have been selection pressures for the development of the high O2 affinity. The mechanism behind it rests with the recorded change in erythrocytic concentration of GTP.

1. Acute air exposure of an air-breathing fish Channa argus immediately induced hypercapnic acidemia while total CO2 content of blood remained unchanged. Upon reimmersion, paco2 and pHa quickly restored to pre-exposure levels followed by gradual rise of [HCO-3]. 2. Artificial air ventilation of air-exposed fish restored acid-base status and greatly depressed voluntary air ventilatory movements. We conclude that the major cause of acid-base disturbances occurring during air exposure is the reduced air convection.

1. An electrolytic method was used to plot the oxygen dissociation curves of whole blood from both the larva and adult of the lamprey Lampetra fluviatilis at a temperature of 10 degrees C and over a pH range of 6-5-8-1. 2. Larval blood has a far higher affinity for oxygen than that of adults, the respective calculated P50's at a pH of 7-75 being 1-9 and 10-7 mmHg. 3. The high affinity of larval blood is of use to a relatively sedentary animal living in burrows, and the increased oxygen delivery pressure brought about by the shift of the curve to the right in the adult is of advantage to an animal exhibiting greater activity. 4. The n value obtained from the Hill plots increased with increasing saturation and were lower in larvae than adults at the same level of blood saturation. 5. The Bohr effect in larvae at 10 degrees C over the pH range 6-5-8-1 was --0-25, a value which did not differ significantly from the -0-22 found in adults.

Oxygen equilibrium curves of the purified hemoglobin component I from the Atlantic bluefin tuna (Thunnus thynnus) have been determined between pH 6.5 and 8.75 at 25 degrees C, and for five temperatures between 10 and 30 degrees C at pH 7.0 and 7.5. From the equilibrium data oxygen equilibrium constants for four oxygenation steps, Ki (i = 1 to 4) were estimated. The number of the Bohr protons released on the ith oxygenation (delta Hi+), and the enthalpy and entropy changes at each oxygenation step (delta Hi and delta Si) were calculated. The Hill plot for oxygenation below neutral pH is biphasic; the top asymptote lies to the right of the bottom one and the linking limb between them exhibits a slope less than unity, exhibiting apparent negative co-operativity. The values of K1 and K2 exhibit little pH dependence, while those of K3 and K4 increase by two orders of magnitudes as the pH is changed from 6.5 to 8.75. In consequence, oxygen equilibrium above neutral pH exhibits a normal positive co-operativity. The oxygen equilibrium at lower temperature is biphasic as is that below neutral pH. The shape of the Hill plot is temperature-dependent. The affinity at low saturation decreases, and that at high saturation increases upon raising the temperature from 10 to 30 degrees C, resulting in crossing of the middle portion of the equilibrium curves at different temperatures. The delta H1 and delta H2 values are negative as are those of most other hemoglobins, but the delta H3 and delta H4 values are positive. Consideration of these results in a framework of the allosteric model extended to take account of differences between subunits has indicated that the deoxy quaternary structure is stabilized at low pH or low temperature, and that subunit heterogeneity gives rise to the biphasic oxygen equilibrium curve. An analysis of delta Hi+ suggests that the large number of the Bohr groups is responsible for the biased allosteric equilibrium towards the deoxy quaternary structure. The positive delta H3 and delta H4 values are also considered to arise from the large endothermic contribution of the Bohr protons released at the third and fourth steps of oxygenation.

Oxygen transport characteristics and phosphate compounds were measured in the blood of reedfish, Erpetoichthys calabaricus, a bimodal breather. Blood from reedfish possessed the following values (mean +/- SD): hematocrit (21.7 +/- 0.4%), hemoglobin concentration (7.53 +/- 1.75 g%), red blood cell count (0.45 +/- 0.10 X 10(6)/mm3) and oxygen capacity (10.1 +/- 2.3 vol%). Although hematocrit, hemoglobin concentration, red blood cell count and oxygen capacity were all highly intercorrelated (P less than 0.01 in all cases), none of these parameters were significantly correlated with sex, weight or length in our sixteen fish sample. Erythrocyte volumes equalled 480 micrometers3, showed less variation (CV = 10.4%) and did not correlate with any other measured variable. Blood oxygen dissociation curves were sigmoidal and the P50's equalled 17.34 +/- 3.04 at 1% CO2 and 25 degrees C. Mean Bohr shift (delta log P50/delta pH) was -0.274 +/- 0.087. Temperature strongly influenced blood oxygen affinity. At 1% CO2, delta log P50/delta T equalled 0.026 +/- 0.006 (mean +/- SD). These hematological properties indicate that the blood of reedfish is similar to those of other tropical air-breathering species. Concentrations of total phosphate in the erythrocytes and percentage of total phosphate bound as nucleotide triphosphates were high. Surprisingly, 2,3diphosphoglycerate was found which has been reported in the erythrocytes of only two other fish species. Blood characteristics of reedfish exposed to air for 4 hr with one exception (Hill numbers) were not significantly different from water exposed controls. This suggests that the reedfish does not possess blood respiratory mechanisms to facilitate respiration solely by air-breathing.

Fundulus heteroclitus decreases erythrocyte adenosine triphosphate and increases blood hematocrit when acclimated to hypoxic conditions. A defined medium has been developed which allows isolated F. heteroclitus erythrocytes to be maintained for several hours without an appreciable loss of cellular ATP. The effect of oxygen tension, pH and metabolic inhibitors on the cellular concentration of ATP of fish red cells has been investigated as an in vitro model to explain in vivo responses to environmental changes. The isolated red cells significantly decrease their ATP/Hb molar ratio when exposed either to anaerobiosis or metabolic inhibitors. It is concluded that the in vivo response is mediated at the red cell level via decreased oxidative phosphorylation in the presence of low environmental oxygen. The length of time necessary to elicit the responce both in vivo and in vitro is also discussed.

Exposure of the elasmobranch Scyliorhinus stellaris to environmental hyperoxia (PO2 of 500 mm Hg) resulted in a considerable rise of arterial PCO2 from 1.9 mm Hg during normoxia to about 11 mm Hg after 6 days as an expression of the primarily oxygen-oriented regulation of gill ventilation. In contrast to the typical pattern during environmental hypercapnia, however, arterial plasma pH was hardly affected by the considerable hyperoxia-induced hypercapnia. At elevated arterial PO2 values (200-300 mm Hg) gill ventilation was apparently not adjusted exclusively for the oxygen demands of the organism, but was matched to the requirements of acid-base regulation such that the rise in PCO2 could be compensated for by a net gain of bicarbonate-equivalent ions from the environment. This fine adjustment of gill ventilation to the bicarbonate-equivalent uptake rate extended the process of adaptation to about 6 days and resulted in an almost complete pH compensation during the entire process of PCO2 increase. These data suggest that during conditions of reduced oxygen-related respiratory drive the regulation of gill ventilation is primarily dependent upon the acid-base parameters.

Oxygen equilibrium curves of blood from four antarctic fishes were obtained at subzero temperatures. Oxygen contents were measured directly and the results on two of these fishes agreed favorably with previous studies in which oxygen electrodes were used.

The oxygen-binding characteristics and the multiplicity of the stripped hemoglobiin from active lungfish Protopterus amphibius, are the same as in specimens that have been estivating for about 30 months, showing that alteration in the hemoglobin molecules is not involved in the earlier reported increase in oxygen affinity of whole blood during estivation (Johansen et al., '76). At pH 7.0 and 26 degrees C the hemolysates show a high oxygen affinity (P50 = 3.1 Torr), a Bohr factor (delta log P50/delta pH) of - 0.33, and a cooperativity coefficient (n) of 1.7. Between 15 and 26 degrees C, the apparent heat of oxygenation (delta H) is - 8.6 Kcal-mole-1 at pH 7.0, corresponding with data for other fish. A low sensitivity of oxygen affinity to urea appears to be adaptive to the high urea concentrations in estivating lungfish. The salt sensitivity is, however, similar to human hemoglobin. The hemoglobin consists of two major (electrophoretically anodal) components, which differ slightly in oxygen affinity but are both sensitive to pH and nucleoside triphosphates (NTP). Guanosine triphosphate (GTP), the major erythrocytic organic phosphate, however, depresses the oxygen affinity of the composite and separated hemoglobins more effectively than ATP suggesting that GTP is the primary modulator of oxygen affinity. Comparative measurements reveal only one major hemoglobin component in P. annectens which has a markedly lower oxygen affinity and phosphate sensitivity than P. amphibius hemoglobins and thus seems less pliable to phosphate-mediated variation in oxygen affinity. The data are discussed in relation to the hemoglobin systems of other fish.

1. A detailed account is given of an electrolytic method for determining the oxygen dissociation curve of fish blood using a single sample of 50-100 mul for the whole curve. The accuracy and some of the problems arising from its uses are discussed. 2. Oxygen dissociation curves have been determined for trout blood and human blood at temperatures of 15 and 37 degrees C. The relationship between P50 and temperature is similar to that obtained using other methods. Absolute values of P50 are generally lower than those obtained by other methods, especially in the case of fish blood. 3. The effect of PCO2 and pH on the oxygen dissociation curve of trout blood is tested and it is shown that PCO2 has a more marked effect than pH when the other factor is maintained at a constant level. The Bohr factor (delta log P50/delta pH) appears to be approximately the same and independent of the PCO2. 4. The P50 of ray blood determined from fish during and after an operation showed an increased Bohr factor.

For Labeo capensis (Smith) the increase in blood organic phosphates (ATP, GTP) in winter fish exceeds the increase in summer fish. Blood chlorides, blood osmotic pressure, hematocrit and hemoglobin concentration decrease significantly in winter fish when compared to summer fish. Four effectors, viz. temperature, organic phosphates, H+ and Cl-, could lower the oxygen affinity of whole blood, especially in summer fish.

Blood pH in the antarctic cod (Dissostichus mawsoni) and in two Trematomus species, occlrring at --1-9 degrees C, is extremely high (approximately 8-2 to 8-3). This supports and extends Rahn's (1966) model for the temperature-pH relationship in cold-blooded vertebrates. The blood of D. mawsoni shows a low oxygen affinity (P50 approximately equal to 14-5 mmHg at pH 8-16 and -1-9 degrees C). Despite normal in vitro temperature and pH sensitivities, blood P50 increases only slightly when live fish are temperature-stressed (+ 4-0 degrees C), or become acidotic as a result of agitational stress (blood pH 7-71), primarily as a result of compensatory decreases in blood ATP levels. Oxygen-binding properties of 'stripped' (cofactor-free) solutions of D. mawsoni haemoglobin were measured in attempts to elucidate the molecular mechanisms involved in the function of the pigment.

Previous studies have demonstrated that acclimation of mammals and fish to hypoxic conditions elicits opposite responses in red cell organic phosphate levels (an increase and a decrease, respectively). This investigation focuses on the "reverse" response - when the benthic fish Pleuronectes platessa, which occurs locally under hypoxic conditions, is subjected to higher oxygen tensions. Compared to the hypoxic (PO2 =30 mm Hg) situation the ATP:Hb ratio in increased (1.1 to 1.8) and oxygen affinity is increased (i.e. P50 changed from 18 to 30 mm, at pH 7.7 and 15 degrees C) by acclimation to Po2 of 150 mm Hg. Under hyperoxic conditions (Po2 =300 mm Hg) the ATP:hemoglobin ratio and blood P50 showed a surprising decrease and returned to values comparable to those in hypoxic fish (to 1.3 and 18 mm Hg, respectively). When "stripped" of organic and inorganic ions hemoglobin solutions prepared from fish acclimated at 30 and at 150 mm Hg have the same P50 and Bohr effect values, showing that the intra-erythrocytic factor rather than changes in the hemoglobin molecules, are involved. The pure hemoglobin shows high sensitivity to ATP at physiological concentrations. There is evidence that apart from the direct helcotropic effects, ATP decreases oxygen affinity in the red cells through modification of the Donnan distribution of protons across the red cell membrane. Fish acclimated to 30 and 150 mm Hg oxygen tensions showed the same oxygen consumption rates at the same oxygen tensions.