The impact of endurance training on arterial plasma K+ levels and swimming performance of rainbow trout

Arterial plasma K+ and lactate concentrations ([K+]a and [lactate]a), as well as blood oxygenation status, were measured in relation to increasing swimming speeds in rainbow trout Oncorhynchus mykiss. Neither [K+]a nor [lactate]a changed at swimming speeds below 1.5 BL s-1, where BL is total body length. Between 1.5 and 2.0 BL s-1, [K+]a started to increase, and above 2.0 BL s-1 both [K+]a and [lactate]a increased with swimming speed. Training shifted the onset of these increases to higher swimming speeds and increased the critical swimming speed (Ucrit) from 2.4 to 3.0 BL s-1. Blood oxygen content showed no changes in control fish, whereas in trained fish it increased by 22 % at the final swimming speed. From the [K+]a data, we suggest that no loss of K+ occurred from the working muscle at low swimming speeds, allowing an unlimited endurance, whereas moderate and higher speeds were probably associated with a loss of K+ from the working muscles, indicating a limited endurance.

Catecholamine-induced changes in oxygen affinity of carp and trout blood.

Carp and trout blood maintained at low constant oxygen and carbon dioxide tensions was beta-stimulated. This activated the Na+/H(+)-exchanger of the red cell membrane, leading to increases in red cell pH (pHi) and cell water content, the latter resulting in dilution of hemoglobin and organic phosphates. The increase in pHi was rapid and maintained throughout the experimental period, the trout red cells showing the largest increase. Likewise swelling of the red cells was larger in trout than in carp blood. As a consequence of beta-stimulation the oxygen affinity of the blood increased. In trout the intracellular Bohr factor of unstimulated blood combined with the pHi increase upon stimulation could account for 85% of the increase in oxygen affinity, whereas it only covered 65% of the increase in carp blood. We therefore conclude that blood oxygen affinity is dependent on the red cell hemoglobin concentration in both species, the effect being more marked in carp.

Role of protein phosphorylation in control of K flux pathways of trout red blood cells.

The role of protein phosphatases in the regulation of K flux pathways of the trout red blood cell has been investigated using the phosphatase inhibitors calyculin A and okadaic acid. Both inhibitors completely blocked an oxygenation-activated Cl-dependent K flux with a 50% inhibitory concentration of 17 and 675 nmol/l, respectively, but not the hypotonically activated Cl-independent K uptake. N-ethylmaleimide (NEM) and staurosporine caused an increase in the Cl-dependent flux. In both cases preincubation with calyculin A blocked activation but, when added during activation, it prevented any further increase with NEM but abolished the staurosporine-induced uptake. K uptake that was activated by NEM and "clamped" by calyculin A was volume sensitive, indicating a dual influence on this pathway. Chelerythrine, a protein kinase inhibitor, activated a Cl-independent K uptake that was unaffected by calyculin A. It is concluded that activation and deactivation of both Cl-dependent and Cl-independent pathways require changes in the phosphorylation of an as yet unidentified target protein(s), although with different sets of protein kinase and/or phosphatases. These observations also suggest a complex model of kinase-phosphatase regulation and provide drugs for the pharmacological definition and manipulation of Cl-dependent and Cl-independent K flux pathways.

THE RELATIVE CONTRIBUTIONS OF RED AND WHITE BLOOD CELLS TO WHOLE-BLOOD ENERGY TURNOVER IN TROUT

Previous studies addressing energy turnover in fish blood have ignored the possible influence of white cells. The present investigation quantified the contribution of white and red cells to whole-blood energy turnover in trout (Oncorhynchus mykiss) before and after adrenergic stimulation. All experiments were carried out on cells kept in their native plasma. White cells were found to have an almost twenty times higher rate of oxygen consumption than red cells. Furthermore, white cells were responsible for essentially all whole-blood lactate production. Our data therefore show that white cells account for almost half of the energy turnover in trout blood. Adrenergic stimulation elicited a significant increase in total as well as ouabain-sensitive (a Na+/K+-ATPase inhibitor) red cell oxygen consumption. However, the ouabain-sensitive red cell oxygen consumption amounted to approximately 23 % of the total red cell oxygen consumption, regardless of adrenergic stimulation. Therefore, energy-consuming processes other than Na+/K+-ATPase activity are probably involved in the increased red cell oxygen uptake after adrenergic stimulation.

Oxygenation-activated K fluxes in trout red blood cells.

The effect of oxygenation on the dissipative fluxes of K in trout red blood cells has been determined. Unidirectional influx under low oxygen tension (PO2 = 1 kPa) was 0.56 +/- 0.07 mmol.l-1 packed cells.h-1. Within a few minutes of equilibration with high oxygen tension (PO2 = 120 kPa), influx was increased 14-fold, and this was associated with a progressive loss of KCl and a cell shrinkage. K influx progressively declined over the following 3 h to levels close to those characteristic of cells at low oxygen tension. Replacement of medium Cl by NO3- or methane sulfonate inhibited the stimulation due to high oxygen as did furosemide and low extracellular pH. The oxygenation-stimulated influx was highly volume sensitive, being increased by up to 100% by osmotic swelling and decreased by osmotic shrinkage. By contrast, the small influx under low oxygen tension was unaffected by either Cl replacement or by shrinkage and increased only with extreme swelling. Thus high oxygen tension activated a Cl-dependent and furosemide-sensitive K flux. Once activated, the mechanism was rapidly deactivated on transfer back to low oxygen tension but slowly deactivated when maintained at high PO2. The oxygenation-stimulated flux mechanism promotes a rapid and more complete volume regulatory decrease than in cells at low oxygen tension.

Changes in plasma and erythrocyte K+ during hypercapnia and different grades of exercise in trout.

Trout were exposed to hypercapnia, two levels of aerobic exercise, or three successive periods of supramaximal exercise to evaluate the effects on erythrocyte and plasma K+. During aerobic exercise, plasma K+ increased slightly with the intensity of work, while no change was found in the erythrocyte K+ content. In contrast, both hypercapnia and supramaximal exercise induced a net erythrocyte K+ uptake. This uptake changed to a net loss of K+ as arterial pH and hemoglobin-bound oxygen saturation returned to control values during recovery. The maximal rates of net K+ uptake found during hypercapnia and supramaximal exercise corresponded to 195 and 350 mumol.kg fish-1.h-1, respectively, and the maximal rates of net K+ loss found during recovery corresponded in both cases to approximately 130 mumol.kg fish-1.h-1. Hypercapnia had only a minor effect on plasma K+, but return to normocapnic conditions induced a 0.8 mM rise in plasma K+. Of this increase, approximately 70% could be accounted for by the simultaneous net release of erythrocyte K+. Each period of supramaximal exercise induced an elevated plasma K+ level, resulting in accumulation of plasma K+ despite slight decreases in plasma K+ in between the exercise periods. At the same time the net erythrocyte K+ uptake caused an estimated reduction in plasma K+ of 1.5 mM. It is concluded that both hypercapnia and supramaximal exercise cause profound net changes in the erythrocyte K+ content with significant effects on plasma K+.

In vitro effects of pH and hemoglobin-oxygen saturation on plasma and erythrocyte K+ levels in blood from trout.

Plasma and erythrocyte K+ were monitored during storage and tonometry of blood samples taken from resting rainbow trout, Oncorhynchus mykiss. During storage of arterial blood samples, plasma K+ concentration increased by 38% in 12 min. During extended tonometry of blood with a pH near 7.9 and full hemoglobin-bound oxygen (HbO2) saturation the erythrocytes showed a net loss of K+. Plasma K+ concentration increased from 2.9 mM to a near steady-state value of 5.6 mM. When tonometered at a pH near 7.2 and a HbO2 saturation at approximately 4% the erythrocytes took up K+, leading to a dramatic reduction in plasma K+ concentration to 0.2 mM. This net uptake was stimulated by isoprenaline and was inhibited by ouabain. It is concluded that net erythrocyte K+ uptake and loss can be induced in trout by changes in blood pH or HbO2 saturation in vitro.

Potency of adrenaline and noradrenaline for beta-adrenergic proton extrusion from red cells of rainbow trout, Salmo gairdneri.

The red cell adrenoceptor affinity for the unspecific agonists adrenaline and noradrenaline and the specific beta-agonist isoprenaline was studied in vitro on whole blood of rainbow trout, Salmo gairdneri at 15 degrees C. The erythrocytic adrenoceptors could be pharmacologically characterized as beta-receptors of the 'noradrenaline'-type (beta 1-type), with an order of potency of isoprenaline greater than noradrenaline much greater than adrenaline. The adrenoceptor affinities, expressed as agonist concentrations for 50% response (EC50), were 1.3 X 10(-8) and 7.6 X 10(-7) mol l-1 for noradrenaline and adrenaline, respectively. Winter fish showed a red cell adrenergic response identical to that of summer-acclimated fish. It is concluded that most red cell beta-adrenergic responses in vivo are exclusively elicited by noradrenaline.

Effects of graded exercise on blood gas tensions and acid-base characteristics of rainbow trout.

A swim tunnel respirometer and an extracorporeal blood circulation technique allowed continuous collection of data from exercising fish below and near their critical speed. Swimming at speeds below maximum showed no changes in plasma Na+, Cl- and lactate concentrations but increased levels of blood hemoglobin and plasma K+. PaO2 and PaCO2 showed an exponential decrease and increase respectively and related to swimming speed. Increased swimming speed changed the acid-base status toward a mixed respiratory and metabolic acidosis. Upon reaching maximum speed sudden and large increases in plasma Na+, K+ and lactate concentrations occurred associated with a large metabolic acidosis.

Acute exposure of rainbow trout to mild and deep hypoxia: O2 affinity and O2 capacitance of arterial blood.

Respiratory properties and pH of blood were followed during acute exposure of rainbow trout to three levels of environmental hypoxia at 15 degrees C. In a first stage, the blood oxygen affinity was preserved (mild hypoxia, PwO2 = 60 mm Hg) or slightly increased (deep hypoxia, PwO2 = 35 mm Hg), despite a simultaneous drop in arterial pH within the first 5-10 min. This is possibly caused by a catecholamine induced increase in red cell pH. The second stage showed for the mild hypoxia group a temporary increase in affinity followed by a recovery within 60 min, correlating with the changes in arterial pH. The deep hypoxia group, however, further increased the blood oxygen affinity, due to a rapid decrease in the ATP:Hb4 and GTP:Hb4 molar ratios within the following 1-2 h. This was associated with a complete pH recovery. Very deep hypoxia (PwO2 = 30 mm Hg) furthermore elicited a 20% increase in blood hemoglobin concentration within 20 min. This group showed a more pronounced drop in blood pH, without a complete recovery. Calculated values of the arterial blood oxygen capacitance, beta bO2, are discussed in the context of the very different responses of trouts acutely subjected to mild and deep hypoxia, respectively.

Temperature acclimation and oxygen binding properties of blood of the European eel, Anguilla anguilla.

Temperature acclimation of the European eel, Anguilla anguilla, resulted in red cell GTP/Hb molar ratios of 1.20, 1.77 and 0.80 at 2, 17 and 29 degrees C, respectively. A small increase in blood oxygen capacity was present in 29 degrees C acclimated eels. The CO2 Bohr effect and the shape of the oxygen binding curve (n-Hill) were invariant with both temperature and GTP/Hb. The significant differences in the GTP/Hb ratio corresponded with a strong enhancement of the temperature effect on blood oxygen affinity between 2 and 17 degrees C and a similarly strong compensation between 17 and 29 degrees C. Predicted in vivo P50 values were 3.0, 13.8 and 17.6 mmHg at 2 degrees C, 17 and 29 degrees C, respectively. The adaptational value of these findings are discussed in relation to standard metabolic rates at the various temperatures. A tentative hypothesis is proposed that the present study confirms and expands earlier work and supports the contention that adjustments in blood oxygen affinity of thermally acclimated teleosts serve to provide them with an unloading O2 tension for diffusion closely matching the standard oxygen requirements at the various temperatures.

Seasonal variations in hematocrit, red cell hemoglobin and nucleoside triphosphate concentrations, in the European eel Anguilla anguilla.

Eels acclimatized in nature show a significant annual variation in erythrocytic guanosine triphosphate (GTP) concentration, temperature range 0.5-17 degrees C. A similar but smaller annual variation is also present in eels acclimated in the laboratory at constant temperature, 17 degrees C. Hematocrit and blood oxygen capacity showed no seasonal variation. Natural minimal and maximal red cell GTP concentrations were found at the end of the dormancy period (March) and in the late summer, respectively. Furthermore, a chronological connection of the erythrocytic GTP values versus ambient temperature, in the natural environment, demonstrates a hysteresis. This allows for a prediction of a slowly progressing enhancement of the temperature effect on Hb-O2 binding throughout autumn, whereas a relatively fast and pronounced enhancement predictably takes place in spring (April-May) coincident with the "awakening" of the eels.

The significance of the linkage between the Bohr and Haldane effects in cephalopod bloods.

This study concerns the physiological implications of the theoretical identity between the Bohr and Haldane effects in the hemocyanin containing blood from the cephalopods Loligo forbesii and Sepia officinalis. The Bohr factor is for both species lower than -1.0. The functional Haldane coefficient (delta ctCO2/delta cHcO2)pH ranges from -2.0 to -0.9 mMCO2/mMO2 in S. officinalis and from -1.2 to -0.7 mMCO2/mMO2 in L. forbesii. This implies for S. officinalis that there may not be enough protons produced from aerobic metabolism to facilitate the release of O2 from HcO2 via the pH sensitivity of this binding. The amount of anaerobically produced protons are predictably of little consequence for release of Hc bound O2 in cephalopods since octopine as the primary product of anaerobic metabolism is a much weaker acid than lactic acid at physiological pH. It is proposed that during burst swimming powered by the ventilatory current, such as practiced by many squids, the overall requirement for ventilation may increase causing a reduced pCO2 and bring the functional Haldane effect below unity. This implies that more H+ are produced by aerobic metabolism than can be found to hemocyanin, hence a shift of the O2 equilibrium curve to the right and improved O2 unloading potential. The advantage of the very large (pH sensitivity) Bohr shift in benthic forms like S. officinalis, both tolerant of and frequently experiencing hypoxic water, may rest with a marked increase in O2 affinity and improved O2 loading in response to respiratory alkalosis produced by hyperventilation.

An O2-Hb "paradox' in frog blood? (n-values exceeding 4.0).

O2-Hb dissociation curves have been determined for the anurans, Rana temporaria and Rana catesbeiana, and compared with human blood for the specific purpose of defining the n-value (hill's cooperativity coefficient) at high O2 saturations. The P50 values at normal conditions (see fig. 1) for each species were 24.6 mm Hg for human blood, 37.0 mm Hg for Rana temporaria blood and 53.5 mm Hg for Rana catesbeiana. For human blood the n-value was 2.7 at saturations from 36% to 98% levelling off at the highest saturations. For Rana temporaria Hill plots between saturations from 20% to 98% showed 3 segments. The average n-value increased from 1.6 to 2.4 at about 50% saturation, increasing again at about 80% saturation to 7.3. The pattern in Rana catesbeiana blood was similar with the n-value changing from 1.6 to 3.1 at about 50% saturation, averaging 3.5 between 53% and 89% saturation. Additionally the Bohr effect in Rana temporaria blood more than doubled when compared at 50% and 90% saturation. The data show that for R. temporaria blood the free energy of interaction associated with the binding of 02 to Hb is displaced to the upper segment of the dissociation curve.

Training and exercise change respiratory properties of blood in race horses.

Effects of training and exercise on blood respiratory properties were investigated in standard-bred race horses. Training caused an increase in the circulating O2 capacity at rest from 18.4 to 21.0 vol%, and in the O2 capacity during exercise from 24.9 to 30.3 vol%. An increase in the in vitro oxygen affinity [P50(PH 7.4, 37.9 degrees C)] of about 2 mm Hg correlated with a decrease in the red cell concentration of 2,3-diphosphoglycerate (DPG) from 6.35 mM-1-1(E), erythrocytes. Trained horses also showed an acute lowering of the red cell DPG concentration after maximal exercise. The physiological significance of the increases in O2 capacity and O2 affinity are discussed and a hypothesis presented to explain the possible relationship between changes in spleenic size, total red cell volume and red cell DPG concentration.

Oxygen-binding properties of hemoglobins from estivating and active African lungfish.

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.

Blood respiratory properties in the naked mole rat Heterocephalus glaber, a mammal of low body temperature.

Respiratory properties of whole blood and Hb solutions have been studied in Heterocephalus glaber, a fossorial rodent, having a low body temperature (30.0-32.0 degrees C) and poor thermoregulatory ability. For comparison similar, measurements were made on laboratory mice, Mus musculus. Whole blood showed a distinctly higher O2 affinity for Heterocephalus at both 30 and 37 degrees C.P50 values were 23.3 mm Hg and 33.0 mm Hg at 37 degrees C for Heterocephalus and Mus, respectively, while at 30 degrees CP50's were 18.8 mm Hg and 24.9 mm Hg, all values at pH (b) 7.4. deltaH values (expressive of the effect of temperature on P50) were -5.8 kcal-mol-1 for Heterocephalus and -7.5 kcal-mol-1 for Mus. The CO2 Bohr effects (omega) were -0.43 and -0.50 for Heterocephalus at 37 and 30 degrees C. Corresponding values for Mus were -0.65 and -0.56. Both species had a Hill's n-value of 2.6. Red cell concentrations of 2,3-DGP were closely similar in the species being 7.3 mmol-L-1 rbc for Heterocephalus and 7.4 mmol-L-1 rbc for Mus. Stripped Heterocephalus Hb had a very high O2 affinity, at pH 7.25, 37 degrees C,P50 was 8.0 mm Hg whereas the corresponding value for Mus was 11.3 mm Hg. Addition of DPG to stripped Hb from the two species decreased O2 affinity to the same degree. The high O2 affinity of Heterocephalus blood is viewed as a possible adaptation to its burrowing habits. Its basis is inherent to the hemoglobin molecule itself and not dependent upon cofactor influence or the temperature sensitivity of the O2-Hb binding.

Respiratory properties of blood in awake and estivating lungfish, Protopterus amphibius.

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.

Physiological properties of eel haemoglobin: hypoxic acclimation, phosphate effects and multiplicity.

Unlike the whole body oxygen affinity, which adapts readily to environmental oxygen tensions, haemoglobins, prepared from normoxic- and hypoxic-accimated eels (Anguilla anguilla) show no adaptive changes in oxygenation properties or in multiplicity. Hypoxic acclimation is, howeveer, accompanied by a strong decrease in red cell nucleoside triphosphates, particularly guanosine triphospphate (GTP), which depresses oxygen affinity of the composite and component haemoglobins more strongly than does the concurring ATP. The effects of pH, temperature and salts on the oxygenation properties of the (isolated) haemoglobins are reported, discussed in relation to the varying environmetal conditions encountered by eels, and compared with data on American and Japanese eels (A. rostrata and A. juponica, respectively.