Respiratory Properties of Blood From Voluntarily and Forcibly Submerged Xenopus Laevis

Xenopus laevis is an aquatic anuran which relies on periodic episodes of air breathing for the major portion of its oxygen uptake. This behaviour causes substantial oscillations in respiratory gases in lungs, blood and tissues (Shelton & Boutilier, 1982). It has been argued that oscillations of this type are an inevitable consequence of the evolution of air-breathing in fishes and early tetrapods (Shelton, Jones & Milsom, 1985). It also seems likely that the early evolution of a partially divided, double circulation is linked to the use of the blood as a store for respiratory gases (Shelton, 1985). If these views are to be sustained, it is important to understand the effects of intermittent ventilation on all parts of the gas exchange and storage systems.

InXenopus, short (10 min) voluntary dives cause blood O₂ partial pressures to fall from approximately 80 to 40 Torr (Emilio & Shelton, 1974). At times, however, the animal will remain voluntarily submerged for periods of up to 1 h (Boutilier, 1984), though the corresponding details of blood gases are not known. Ina recent study in which Xenopus was allowed to dive voluntarily but then restricted from reaching the surface, Emilio & Shelton (1980) found that a mild respiratory acidosis developed over the first 10 min. Longer dives of this sort, up to 30 min duration, caused a combined respiratory and metabolic acidosis, the latter persisting for some hours after the animal surfaced and breathed. Associated with this acidosis was a considerable haemoconcentration resulting from a loss of plasma water to some extravascular compartment. Similar observations have been made on other forcibly submerged amphibians (Jones, 1972; Jones & Mustafa, 1973; Emilio, 1974; Lillo, 1978). These and other studies have led to a view that anaerobic pathways are an integral part of these animals’ normal diving metabolism. However, there is an increasing amount of evidence to show that periods of voluntary submergence end before O₂ stores are fully depleted (Lenfant & Johansen, 1967; Toews, 1971; Toews, Shelton & Randall, 1971; Emilio & Shelton, 1974, 1980). In the present study, we examine the effects of forced and voluntary dives on the oxygen-carrying and acidbase properties of Xenopus blood in order to understand the changes that can be produced in this important gas transport and storage medium as the animal breathes intermittently.

Male and female specimens of the South African clawed toad, Xenopus laevis, were either obtained commercially or selected from a laboratory reared stock. All animals were kept in large communal tanks (20–25 °C) and fed on chopped liver twice weekly. At least 1 week prior to experimental procedures, the animals were transferred to laboratory tanks (25 ± 2°C) where feeding was suspended.

After anaesthesia, induced by immersion in a 0·06% solution of tricaine methanesulphonate (MS-222, Sandoz; titrated to pH7·0 with NaOH), a catheter was chronically implanted in the femoral artery of each animal (Boutilier, 1984). The toads were then artificially ventilated until they had recovered and they were given at least 24h in which to acclimate to the experimental chambers (25 ± 0·5°C). The of the water was >140 Torr at all stages of the experiments, <0·5 Torr (Radiometer electrodes and meters).

Acid-base and oxygenation characteristics of whole blood and plasma were studied by in vitro tonometry at 25°C. Individual blood samples were drawn from the femoral cannulae of several animals, pooled to the required volumes, and stored at 4°C for less than 1 h before use. Aliquots having the same haematocrit as the parent blood pool were transferred to tonometers (50 ml round bottom glass flasks) and equilibrated with a wide range of gas mixtures produced by Wösthoff pumps (Bochum, FRG). Blood samples for analyses were drawn after a predetermined 30-min equilibration time, into positive displacement gas-tight Hamilton syringes.

Whole blood oxygen equilibrium curves were constructed at several (and thus pH) levels using the mixing method (Haab, Piiper & Rahn, 1960) as detailed further by Scheid & Meyer (1978). Blood samples were equilibrated against gas mixtures containing various concentrations of CO₂ in air and CO₂ in nitrogen. Samples of known O₂ saturation were analysed for pH and haematocrit, with replicate samples from the 2% CO₂/air-equilibrated tonometer being used for measures of total blood O₂-carrying capacity (i.e. at approximately 14 ·5 Torr and 155 Torr ⁠). The oxygen content of separated plasma was determined also on samples equilibrated with gas mixtures containing 2% CO₂-balance air. The relationship between blood O₂-carrying capacity and haematocrit was assessed over a wide range on blood whose haematocrit was experimentally altered to high and low levels by centrifugation and resuspension at various red blood cell to plasma volume ratios.

In vitro acid—base relationships were determined using the same apparatus as above. Measurements of pH, ⁠, lactate concentrations, total CO₂ concentrations and haematocrit were made on blood equilibrated against humidified gas mixtures containing various proportions of CO₂ in either air or nitrogen. The true plasma of each blood sample, obtained by centrifugation of blood in a sealed capillary tube, was analysed for total CO₂ concentration. To allow comparison, the above procedures were carried out on blood obtained from unrestrained, freely-diving animals (67–101 g, N=31) and from animals at the 30min mark of an enforced dive (62–93g, N= 19).

Twelve toads (80–120 g) were prepared with arterial catheters and allowed to recover as before. Blood samples were drawn initially from each animal as it was freely diving and surfacing in air-saturated water. By lowering a Perspex lid over the water surface, the animal was forced to remain submerged for 30 min at which time a second blood sample was taken. The lid was then removed from the surface and additional blood samples (400 μl) were drawn at +1, +2 and +4h following the ‘dive’. The determinations were carried out on groups of three animals; the blood samples (400 μl animai⁻¹ stage⁻¹) were pooled, stored on ice, and within lh the blood pool was divided equally between an oxygenated and deoxygenated tonometer. Following equilibration with 2% CO₂/air or 2% CO₂/N₂ gas mixtures, known blood O₂ saturations were prepared by the mixing method (approximately 10, 50 and 90% saturations). Measurements made on each of these prepared samples included ⁠, pH, haematocrit and true plasma concentrations of both total CO₂ and lactate.

Methodologies for the measurement of haematocrit, pH, total CO₂, PO₂ and lactate have been detailed previously (Boutilier, Randall, Shelton & Toews, 1977; Boutilier, McDonald & Toews, 1980; Boutilier & Toews, 1981; Boutilier, 1984). Oxygen contents were determined with a Lex-O₂-Con analyser (Lexington Instruments, MA) using, for whole blood, a sample volume of 20 μ1. For low O₂ contents (i.e. plasma), a calibration curve between 0 and 2 vols% was constructed by injecting known volumes of O2/N2 equilibrated distilled water.

Bicarbonate concentrations ([HCO3⁻]) in true plasma were estimated from our measured values of total CO₂ concentration ([CO₂]) with the equation ⁠, where αCO₂ is the plasma carbon dioxide solubility (in mmol l⁻¹ Torr⁻¹) at 25°C (Reeves, 1976). The proportion of the total blood oxygencarrying capacity which could be attributed to oxyhaemoglobin binding was obtained by subtracting the dissolved fraction of O₂ in plasma from the blood O₂ capacity (note that all O₂ capacity measurements were made at approximately 14·5 Torr and 155 Torr ⁠).

The effective increase in haemoglobin concentration of blood from forcibly submerged Xenopus laevis was reflected in vitro by large differences in oxygen and carbon dioxide combining properties when compared with blood samples from unrestrained animals (Table 1).

Oxygen equilibrium curves for whole blood samples drawn from animals engaged in voluntary diving-emergence behaviour and for those following a 30-min enforced dive are illustrated in Fig. 1. Individual curves contributing to each of the averaged curves shown were determined at six O₂ saturation levels (approximately 10, 20, 50, 60, 70 and 90%). For any given level of ⁠, the P₅₀ value of blood from forcibly submergedXenopus was greater than that determined for the freely diving animal, as were the concentrations of lactate and free hydrogen ions (Table 1; Fig. 1). The large haematocrit increase following a forced dive and the corresponding increase in blood O₂-carrying capacity, meant that this blood in vitro carried greater amounts of chemically bound oxygen for any given level of ⁠.

The CO₂ Bohr effect (Fig. 2) was determined by linear regression analysis of all data points obtained at 50% saturation from the oxygen equilibrium curves of both voluntarily and forcibly submerged animals (Fig. 1). Though the slope of the ΔlogP₅₀/ΔpH relationship was evidently reduced in blood taken from involuntarily diving toads (Fig. 2), statistical comparisons of the slopes are not offered as they represent data collected over two different pH ranges.

The relationship between whole blood O₂-carrying capacity (C_bO₂) and haematocrit (Fig. 3) was described by the equation; C_bO₂ = 0·339 (haematocrit)+0·61 (correlation coefficient, r = 0·985, N=45). As expected, the y-intercept of this equation is virtually identical to the measurement of the O₂-carrying capacity of plasma (Table 1; Fig. 3). Data in Fig. 3 are values for freely diving and forcibly submerged animals with additional points at high and low haematocrit values being obtained by experimentally preparing various red blood cell to plasma volume ratios. Note that the points for each group of animals are clustered on discrete portions of the line, the higher values being attributable to the haemoconcentration which occurred during a 30-min enforced dive (Table 1).

An increase in causes the red blood cells to swell in Xenopus as in mammals. This swelling is reflected as an increased haematocrit as Fig. 4 shows. Determinations of oxyhaemoglobin-carrying capacity made at the same time as the haematocrit showed that it did not change with equilibration gas ⁠, confirming that the haematocrit variations were due to cell swelling. Plasma trapping during centrifugation can amount to 2–3% in packed red cells of Xenopus (Emilio & Shelton, 1980) and, though this may change as a result of increased red cell size, the effects will be much smaller than those measured here.

Xenopus whole blood exhibited a Haldane effect: at any given level, the whole blood total CO₂ content and pH were significantly lower in oxygenated than deoxygenated blood (Fig. 5). The mean difference in total CO₂ content between oxygenated and deoxygenated blood was found to be 1·4 mmol l ⁻¹ (0·045 –0·070 pH units difference). Accordingly, the Haldane effect is expressed as the ratio of the change in blood total CO₂ content between oxygenated and deoxygenated blood over the corresponding change in blood O₂ content. In Fig. 5, the change in blood O₂ content is equal to the O₂-carrying capacity of the oxygenated blood (8·5 vols% or 3·8mmol O₂l⁻¹ of blood). The ratio (i.e. Haldane effect) amounts, therefore, to 0·37 mol CO₂mol⁻¹ O₂.

Compared with voluntarily diving animals, the non-bicarbonate buffering capacity of true plasma from forcibly submerged animals increased, due to the haemoconcentration effect (Table 1). Analyses of the individual contributing slopes showed that the true plasma buffering capacity (Δ[HCO₃⁻]/ΔpH) is linearly related to the blood O₂-carrying capacity (C_bO₂) by the equation Δ[HCO₃⁻]/ΔpH = l·46C_bO₂+l·02 (r=0·90).

The data in Fig. 6 summarize the time course and nature of the changes in blood characteristics following an enforced 30-min dive. Hydrogen ions from metabolic sources were clearly present after a dive because pH fell at a constant of 14·5 Torr. True plasma bicarbonate concentrations of blood tonometered at 14·5 Torr also declined during the enforced dive (Fig. 6). Using a HCO₃-pH diagram and the relationship between haematocrit and plasma buffering capacity, each of these post-dive bicarbonate concentrations was backtitrated along its respective buffer slope to the level which would be present at the pre-dive control pH (cf. Wood, McMahon & McDonald, 1977; McDonald, Boutilier & Toews, 1980).

The difference in bicarbonate concentration between the control pH and that after backtitration is a measure of the concentration change in metabolically originating hydrogen ions (Woodbury, 1974). Fig. 7 is a plot of the changes in true plasma concentrations of metabolic acid (in mmol l⁻¹) and lactate following enforced dives of the animals in Fig. 6. These measurements were made on blood samples prepared at 10% O₂ saturation (for time zero) and 90% O₂ saturation (for times B, +1, +2 and + 4h) which are realistic estimates of the state of oxygenation of the blood at these times in the experimental cycle. The Haldane effect of blood O₂ saturation on CO₂ carriage (Fig. 5) is thus taken into account in the plot of metabolic acid equivalents in Fig. 7. Changes in true plasma concentrations of metabolic acid and lactate were similar in direction but quantitatively quite different over the first 2 h following an enforced dive (Fig. 7). During that time, the lactate concentration was approximately 50% that of the metabolic acid, both subsequently declining to pre-dive levels by the fourth hour of recovery.

The blood O₂-carrying capacity of voluntarily diving Xenopus is similar to that observed in other amphibians (Lenfant & Johansen, 1967; Wood, Weber, Maloiy & Johansen, 1975). The comparatively higher O₂-carrying capacity forXenopus blood, as reported by Jokumsen & Weber (1980), is probably explicable in terms of acute blood sampling methods since both the blood pH and haematocrit were evidently affected in the same fashion as the forcibly submerged animals of the present study. Oxyhaemoglobin affinities (at 25°C) of the semi-terrestrial anurans Rana catesbeiana (P₅₀ = 42Torr at pH7·79), Rana brevipoda (P₅₀ = 52Torr at pH7·72; Tazawa, Mochizuki & Piiper, 1979) and Bufo marinus (P₅₀ = 53Torr at pH 7·75; Boutilier & Toews, 1981) are very much lower than those found at comparable temperatures and pH levels in blood from voluntarily diving Xenopus (Table 1; Fig. 1). The P₅₀ value of 29·6Torr at pH7·732 (Fig. 1) is similar, however, to that found in the aquatic urodele, Amphiuma tridactylum (P₅₀ = 27Torr at 22°C; Lenfant & Johansen, 1967), an animal which, likeXenopus, punctuates long periods of diving with comparatively short outbursts of ventilation (Toews et al. 1971). Furthermore, the Bohr coefficient of blood fromXenopus (−0·37; Table 1; Fig. 2) is much higher than those seen in the ranids and Bufo (−0·18 to −0·27; Tazawa et al. 1979; Boutilier & Toews, 1981). The Haldane coefficient in Xenopus blood (0·37) is identical to that of the Bohr effect, this being the prediction of the linkage equation (Wyman, 1964; Heck, 1970). Though comparable data on other anurans are not available, the linkage between coefficients suggests that a higher Bohr effect in Xenopus, relative to other more terrestrial anurans, will mean that the Haldane effect in Xenopus is also higher than in the other species. We could find no evidence for a Root effect in Xenopus whole blood, despite its apparent presence in haemoglobin solutions (Perutz & Brunori, 1982). All other interrelationships between haematocrit and O₂ and CO₂ carriage resemble conventional mammalian patterns.

The in vitro O₂ and CO₂ combining properties of blood samples taken during periods of lung ventilation were identical with those taken at various stages of voluntary dives lasting 10–30 min. In contrast, enforced 30-min dives led to a large haemoconcentration and blood acidosis (Table 1; Fig. 6). The rise in blood lactate concentrations in the forcibly submerged animal (Fig. 7) indicated that the diving O₂ store was not adequate to supply the full energetic demands of the dive and that a switch to anaerobiosis had occurred.

Significant differences between the true plasma concentration changes in metabolic acid and lactate following an enforced dive (Fig. 7) are reminiscent of the postactivity changes seen in the blood of other species of amphibians (McDonald et al. 1980; Boutilier et al. 1980) and some fishes (Wood et al. 1977; Turner, Wood & Hobe, 1983). In all instances, the concentration of metabolic acid equivalents was significantly higher than lactate for some hours following the enforced behaviour. The most obvious resolution of this discrepancy would be if hydrogen ion production occurred via metabolic pathways whose end-products were other than lactate, though evidence for this is scarce (cf. Bennett, 1978). The generation of protons by metabolic processes and their degree of stoichiometry with fermentable substrates is complex and controversial (Gevers, 1977; Hochachka & Mommsen, 1983; Portner, Heisler & Grieshaber, 1984). The most favoured possibility for the discrepancies observed in this and other studies is that protons and lactate anions are released to and/or removed from the blood at different rates.

Experiments on working gastrocnemius muscles of Xenopus have shown in vivo and in vitro that the haemoconcentration of an enforced dive may be explained in part by plasma water uptake into the hypoxic cells of lactate-enriched muscle tissue (Boutilier, Emilio & Shelton, 1986). Presumably, a small component of the raised haematocrit in vivo results from CO₂-induced red cell swelling (Fig. 4), since arterial levels are elevated by approximately 10 Torr during a 30-min enforced dive (Emilio & Shelton, 1980). There is much recent evidence that red blood cell swelling also occurs as a result of hormone-sensitive ion transport systems in erythrocytic membranes and that the associated changes in pHi, Donnan ratio, and dilution of organic polyanions can influence the respiratory properties of the blood (Palfrey & Greengard, 1981; Weber, 1982; Nikinmaa, 1983; Baroin, Garcia-Romeu, Lamare & Motais, 1984). Certainly, red cell swelling cannot account for the changes in haematocrit which lead to an elevated O₂-carrying capacity of the blood and an increased non-bicarbonate buffering ability. Thus, the rise in haematocrit observed here must be largely due to haemoconcentration, increasing the buffer slope during an enforced dive (Table 1; Fig. 6) and reducing the change in blood pH that would otherwise occur. In addition, the delivery of oxygen to hypoxic tissues will be facilitated by the decreased oxyhaemoglobin affinity (Fig. 1).

Amphibians are noted for their ability to tolerate wide internal fluctuations in body oxygenation, acid-base balance and anaerobic end-product concentrations. In diving forms, these capabilities have often been viewed as metabolic adaptations that allow diving times to be greatly extended as a part of the animals’ natural behaviour. Though a shift to anaerobic pathways may at times become necessary as a short-term solution to increased energy demands, this is probably avoided during most voluntary diving conditions (Shelton & Boutilier, 1982; Boutilier, 1984).

The respiratory properties of blood taken from freely moving animals seem well matched to their predominantly aquatic existence and periodic nature of air breathing. For example, the high oxyhaemoglobin affinity and Bohr and Haldane effects (Figs 1, 2, 5), relative to more terrestrial anurans (cf. Wood et al. 1975; Boutilier & Toews, 1981), can be viewed as adaptations aimed at reducing the amount of time required for effective blood gas turnover whilst ventilating at the surface. In Xenopus, these biochemical adaptations appear in unison with a cardiovascular arrangement which permits high blood flow to the lung during breathing (Shelton, 1970, 1976; Emilio & Shelton, 1972; Shelton & Boutilier, 1982). This is consistent with the observation that all movements of the buccal pump in Xenopus are concerned with lung filling and emptying whereas much of the ventilatory pattern of ranid and bufonid species involves non-pulmonary buccal oscillations (Dejongh & Gans, 1969; Macintyre & Toews, 1976; West & Jones, 1975); Xenopus precedes each inspiration phase with a single expiration, thereby ensuring a more complete renewal of the lung gas store (Brett & Shelton, 1979). Taken together, these factors seem most appropriate to an exclusively aquatic animal whose acquisition of fresh air may become limited to rather brief episodes at the water surface (Boutilier, 1984).

One obvious outcome of intermittent ventilation in Xenopus is that it leads to considerable oscillations in the respiratory gas tensions in blood (Emilio & Shelton,1974; Shelton & Boutilier, 1982). During a dive, however, the changes in are far less than those of ⁠, owing to the differences in the capacitances of the respiratory gases in water surrounding the permeable skin. As a result, the animal is able to operate over a wide range of blood ⁠, and thus blood O₂ contents, without becoming severely acidotic (Emilio & Shelton, 1974; Shelton & Boutilier, 1982; Boutilier, 1984). From the oxygen equilibrium curves in Fig. 1, it is clear that the blood O₂ store will change in a complex way throughout the period of diving, as it depends not only on the oxygen utilization by the animal but also on the magnitude of the changes in and thus pH (Fig. 2). In addition, the lungs of Xenopus are used as an additional storage site for oxygen during a dive and may be selectively perfused so as to supplement an ever decreasing store of blood oxygen (Shelton & Boutilier, 1982). Just as the deoxygenated blood of Xenopus may be used as an effective storage site for bulk delivery of carbon dioxide to the lungs during breathing, the comparatively high Bohr effect (Table 1) represents an adaptation which will facilitate the delivery of the blood O₂ store to the tissues during a dive. It seems likely that these biochemical adaptations are evolutionarily linked to the changes in respiratory blood flow during intermittent breathing which are made possible by the undivided ventricle. Thus, the Haldane effect occurs in unison with high blood flow to the lungs (Shelton, 1970), whereas the Bohr effect appears to be linked to an increasing movement of blood into the systemic circulation as apnoea progresses (Shelton & Boutilier, 1982).

Financial support from the Commonwealth Scholarship Commission is gratefully acknowledged.