Recovery From Anaerobiosis in the Intertidal Worm Sipunculus Nudus II. Gas Exchange And Changes in the Intra- And Extracellular Acid-Base Status

Restoration of aerobic homeostasis after exposure to hypoxia in marine invertebrates not only includes the disposal of accumulated end-products and the replenishment of high energy phosphates (Ellington, 1983; Pörtner, Vogeler & Grieshaber, 1986), but also requires the readjustment of physiological parameters like and pH in body fluids and tissues. Anaerobic degradation of glycogen, for instance, always leads to an accumulation of protons, the amount of which may exceed the amount of protons absorbed by phosphagen hydrolysis (Pörtner, Heisler & Grieshaber, 1984b). In Sipunculus nudus, long-term experimental hypoxia was found to lead to a decrease of intra- and extracellular pH in vivo (Pörtner, Grieshaber & Heisler, 1984a), which must be reversed during postanaerobic recovery.

In this paper, gas exchange and changes in the intra- and extracellular acid-base status during recovery in Sipunculus nudus are described. Since the restoration of aerobic homeostasis in the energy metabolism has been investigated in the same animals (Pörtner et al, 1986), we can discover whether or not a correlation exists between recovery processes in energy metabolism and acid-base events. From theoretical considerations, oxidation of accumulated end-products, for instance, would lead to proton consumption, but resynthesis of the phosphagen should lead to an acidification of the cell (cf. Pörtner et al. 1984b). The latter might be important, especially in Sipunculus nudus, since cleavage of a high amount of phosphagen during anaerobiosis has been reported for the body wall musculature of this species (Pörtner et al. 1984c).

Specimens of Sipunculus nudus were collected in the intertidal zone near Morgat (small animals of 7–12 g) and Locquemeau (large animals 25–35 g), Brittany, France, and kept in Düsseldorf for several weeks before the experiments were started. Cannulation of the animals, acclimation to the experimental conditions, and exposure to hypoxia and postanaerobic normoxia are described in detail in the preceding paper (Pörtner et al. 1986).

Coelomic fluid was withdrawn anaerobically via the indwelling catheter. Samples were analysed for pH_e, and using a thermostatted (15 ± 0·1 °C) microelectrode assembly (BMS 3, Radiometer, Copenhagen). The electrodes were calibrated with precision phosphate buffers (Radiometer, Copenhagen) or humidified gas mixtures of N₂, CO₂ and O₂ provided by gas-mixing pumps (Type M 303/a-F, Wôsthoff, Bochum, FRG).

At the end of each experiment, the (March) animals were dissected quickly in the dorso-ventral plane. The left or right half of the musculature of each animal was utilized for pH, analysis in the body wall (cut into four pieces, introvert excluded) and in the introvert retractors (two retractors in one sample). The remaining half was utilized for the analysis of metabolites (Pörtner et al. 1986). Intracellular pH was determined by application of the DMO-distribution method (Waddell & Butler, 1959). Details of the procedure have been described previously (Heisler, Weitz & Weitz, 1976; Pörtner et al. 1984a).

In March animals, 24 h of anaerobiosis led to a non-significant decrease of extracellular but a significant drop of intracellular pH by 0·07 and 0·18 pH units, respectively, starting from pH_e = 8·21 ± 0·05 and pH_i = 7·37 ± 0·02 under normoxia. These pH reductions were accompanied by a decrease of intra- and extracellular bicarbonate concentrations and of coelomic (Fig.1). Furthermore, the oxygen stores of the animals were depleted during anaerobiosis, the being zero after 24 h (Fig. 2).

During postanaerobic recovery, more prominent pH changes occurred in the coelomic fluid (Fig. 1). Instead of returning to control values, pH_e continued to fall and reached a value of 7·85 ± 0·30 after 3 h of recovery. Such a decrease of pH was not observed in the intracellular compartment. pH, remained constant during the first 3 h of recovery, but then started to rise. Both pH_e and pH₁ approached control values within 24 h of recovery.

During initial recovery, the coelomic rose above control values, reaching a maximum after 3 h. After 1 h, the had increased to above 40 Torr (Figs 1,2). The bicarbonate content remained low in the coelomic fluid during the first hours of recovery, whereas it was elevated in the tissue. After 24 h of recovery, intra- and extracellular bicarbonate contents and had more or less reached control levels (Fig. 1).

Extracellular pH values and bicarbonate concentrations during long-term recovery were highly variable in small animals, which had to be killed for the determination of intracellular pH and tissue metabolite concentrations. Observed differences were sometimes not significant (cf. Fig. 1). Therefore, in a more sensitive design, the influence of postanaerobic recovery on the extracellular acid-base status was monitored individually in three large specimens of Sipunculus nudus by repeated sampling of coelomic fluid during the normoxic, hypoxic and posthypoxic incubation periods. This procedure is impossible in small specimens because of the limited volume of coelomic fluid. In every animal the pattern of results was essentially the same as described for small animals (Fig. 3). The mean pH_e decreased significantly by 0T4pH units during anaerobiosis, starting from 7·97 ± 0·04, and fell significantly by an additional 0·29 pH units during the first 3 h of recovery. After 6 h, pH_e began to rise and reached normoxic control values after 24 h of recovery. Extracellular bicarbonate concentration and dropped during anaerobiosis. rose during the first 3 h of recovery, whereas the bicarbonate content decreased slightly. From 6 to 24 h of recovery, and bicarbonate concentrations approached control levels.

Presentation of pH, and bicarbonate concentration in a pH-bicarbonate diagram allows evaluation as to whether the observed changes in the acid-base status are caused by respiratory or non-respiratory processes (Figs 4, 5). Buffer values of coelomic fluid and muscle tissue were adopted from Pörtner et al. (1984a). During 24 h of anaerobiosis, a non-respiratory acidosis developed in both intra- and extracellular compartments. In the intracellular space, pH started to increase after 3 h of recovery, when the had reached a maximum. The acidosis was compensated progressively, mainly by base equivalents of non-respiratory origin during 24 h (Fig. 4). In the coelomic fluid, the non-respiratory acidosis observed during 24 h of anaerobiosis was aggravated by protons of respiratory and mainly non-respiratory origins during 3–6 h of recovery (Fig. 5). Subsequently, non-respiratory processes led to an increase of pH as already pointed out for the intracellular space.

The changes in the intra- and extracellular acid-base status were analysed quantitatively (Fig. 6). Respiratory processes could be demonstrated to be of minor importance for the observed changes, exhibiting a slightly positive base excess during anaerobiosis and a negative base excess during initial recovery which turned into a positive base excess during long-term recovery. Plotting of the non-respiratory changes demonstrated the severe proton load in the extracellular compartment, especially during initial recovery, which was progressively reduced after 6h. The amount of protons expected from organic acids present in the coelomic plasma (measured in plasma samples of the same animals) changed independently of the amount of non-respiratory H⁺ ions (Fig. 6, dissociation of 1 mol H⁺ mol⁻¹ of acetate and propionate and 2mol H⁺mol⁻¹ of succinate is assumed). The concentration of organic acid anions had already decreased when the amount of non-respiratory protons rose during initial recovery.

The amounts of non-respiratory protons in the intra- and extracellular compartments were calculated from the mean values of pH and bicarbonate content in small March animals (Fig. 7). During anaerobiosis most of the protons were trapped intracellularly. During recovery, an increase of the amount of non-respiratory protons was observed only in the extracellular space.

In animals collected during October, metabolite concentrations (see Pörtner et al. 1986) and extracellular acid-base status were investigated (Table 1). pH_e decreased by 0·45 pH units during anaerobiosis, starting from 7·97 under normoxia, and fell by an additional 0·39 pH units to 7·03 during the first 3 h of recovery. The bicarbonate concentration fell from 4·4 to 0·7 mmol l⁻¹ and did not start to rise until after 3 h of recovery. Pco₂ reached a maximum of 1·7 Torr after 6h. After 24 h of recovery, pH, and bicarbonate concentration had approached normoxic values.

Comparison of March and October animals not only reveals differences in concentrations of anaerobic metabolites (Pörtner et al. 1986) but also differences in changes in the acid–base status during 24 h of anaerobiosis. In October animals, the higher levels of anaerobic end-products, predominantly of octopine and strombine, represent a higher amount of protons generated by anaerobic metabolism (Pörtner, 1982; Pörtner et al. 1984b). This obviously led to a drastic fall of extracellular pH during anaerobiosis. Since changes in extra- and intracellular pH correlate during experimental hypoxic exposure (cf. Pörtner et al. 1984a), a severe intracellular acidosis may be assumed to occur as well. These conclusions clearly demonstrate that the extent of the acidosis observed during environmental hypoxia depends on the amount of accumulated metabolites and, therefore, is correlated to the rate of anaerobic metabolism. Comparison of March and October data shows that an increase of the metabolic rate mainly means an increase of cytosolic glycolysis. This implies that the ratio of proton generation to ATP formation in metabolism is changed from a lower value towards unity. Apart from high expenditure of stored substrates, the ratio of 1 H⁺ per ATP formed is another disadvantageous feature of the Embden-Meyerhof pathway as compared to the succinate-propionate pathway (acetate and propionate formation from aspartate or glycogen, succinate and alanine formation from aspartate; cf. Pörtner et al. 1984b).

As already pointed out by Pörtner et al. (1984a), respiratory processes only negligibly changed the picture of variations in the acid–base status which were caused by the metabolic impact during experimental hypoxia, the more so since normocapnie conditions were maintained during the present study. This is also true for the recovery period. Respiratory protons transiently accumulated during the first 6h of recovery and slightly aggravated the extracellular non-respiratory acidosis (Fig. 6). The increase of above control values is very likely to be caused by an increase of oxygen consumption, indicating the repayment of an oxygen debt during initial recovery. Inhibition of gas exchange can be excluded as an improbable reason for this finding, because the animals were probably ventilating and the concentration of CO₂ was kept constant in the ambient water throughout the experiments. Possibly the increase in is also linked to an augmented ventilatory activity. Probably due to ventilation, the in the coelomic fluid quickly rose during initial recovery. Haemerythrin, which was found to be saturated with oxygen at 20 Torr (Pörtner, Heisler & Grieshaber, 1985), can be expected to have been completely loaded after 1 h of recovery in March and after 3 h in October animals. The increase of was higher after 6 h of recovery in October than in March animals, an observation that corresponds to the higher metabolic rate in October animals as derived from the concentrations of anaerobic metabolites (Pörtner et al. 1986).

The correlation between changes in the intra- and extracellular acid-base status during hypoxic exposure was not as clear as in the study of Pörtner et al. (1984a), who reported pH_i and pH_e to be similarly affected by hypoxia. In the present study, the mean intracellular pH dropped by 0·18 pH units, whereas extracellular pH fell insignificantly by only 0·07 pH units in the same animals. A closer examination reveals that, in the study of Pörtner et al. (1984a), pH_i and pH_e had both fallen during 24 h of anaerobiosis by 0·3–0·4 pH units as compared to control values, i.e. pH reduction was higher than in the March animals of the present study, presumably because of different metabolic rates. The discrepancy between pH_c and pH_i reductions in small March animals may be attributed to the kinetics of proton distribution. Non-respiratory protons are consumed or generated in the intracellular compartment, and there may be a delay in proton exchange with the extracellular fluid, which is more clearly seen in animals with low metabolic rates and minor pH changes.

During recovery, in contrast to the situation in anaerobiosis, there was no correlation between changes of pH_e and pH_i. During anaerobiosis most of the protons had been trapped in the intracellular space. With the onset of recovery under normoxia, however, additional non-respiratory protons appeared in the extracellular space. Since proton absorption or release by metabolism occurs in the intracellular compartment, the protons, which during 3–6 h of recovery were found in the coelomic plasma, have probably been released from the musculature into the extracellular space. Metabolic changes in the coelomic cells are minor compared with the amounts of protons observed (Pörtner, 1982). Consequently, the extracellular pH dropped by more than it had fallen during anaerobiosis. Proton release, however, was not accompanied by an accumulation of organic acid anions in the coelomic plasma. On the contrary, anions were removed from the coelomic plasma right from the beginning of recovery (Fig. 6). In accordance with the observations of Pörtner et al. (1984a), this finding leads to the conclusion that there is a discrepancy between the kinetics of proton and anion distribution. In addition, it becomes clear from the present study that intracellular pH is regulated at the expense of the extracellular acid-base status when oxygen is sufficiently available to the tissues.

In order to evaluate whether there was a quantitative correlation between proton release by phosphagen resynthesis and proton accumulation in the plasma, phospho-L-arginine, L-arginine and values are given in Table 2. Since the sum of the concentrations of all L-arginine derivatives was rather variable (cf. Table 1, Pörtner et al. 1986), but is assumed to be kept constant during anaerobiosis (cf. Pörtner et al. 1984c), the concentrations have been recalculated from the mean value of this sum for all animals investigated. The phosphagen resynthesized during 3h of recovery amounts to 7·4 μmolg⁻¹ wet weight, which is equivalent to a proton yield of 2·1 μmolg⁻¹ wet weight. The musculature, which may be assumed to be the only tissue exhibiting a high turnover of phosphagen, constitutes about 80% of the tissues, whereas 52% of the animal is coelomic fluid (Pörtner, 1982). The total extracellular fluid represents 62 % of the animal (calculated from fractional values of water content and extracellular space, Table 2), and of this portion approximately 15% is coelomic cells (H. O. Pörtner, unpublished data). In the coelomic cells, 1·34 ± 0·28 μmol phospho-L-arginine g⁻¹ fresh weight has been found during aerobiosis, which is totally depleted during 24 h of anaerobiosis (Pörtner, 1982) and which during resynthesis will only slightly influence the proton load of the plasma (0·06 mmol H⁺ l⁻¹, assuming pH_i = 7·2). An amount of l-5mmol H⁺ l⁻¹ plasma, however, may result from the resynthesis of phospho-L-arginine in the musculature of March animals, which is close to the amount of H⁺ evaluated from the observed changes in the acid-base status (1·6mmol H⁺l⁻¹ plasma). This balance is feasible only for a period of 3 h recovery, since afterwards ionic exchange with the ambient sea water significantly contributes to changing the amount of non-respiratory protons present in the extracellular compartment (H. O. Pörtner & N. Heisler, unpublished observations). In addition, during the first hours of recovery, concentration changes of other metabolites had only a minor influence on the acid-base status in March animals. A decrease of succinate and propionate contents, for instance, was accompanied by an accumulation of malate and aspartate and, therefore, did not lead to a marked reduction of the amount of carboxyl groups present (cf. Pörtner et al. 1984b).

In October animals, strombine appeared to increase by 5 μmol g⁻¹ wet weight, which during the first hour of recovery may have aggravated the acidosis provoked by replenishment of the phosphagen. Therefore, pH_c dropped further than in March animals and a transient decrease of pH_i cannot be excluded because of the higher metabolic rate observed.

A period of 24 h of recovery led to the restoration of values of pH₁, pH_e, and bicarbonate content typical for normoxia. The analysis of metabolism demonstrated, however, that strombine, alanine and, to a minor extent, acetate concentrations remained elevated in March animals. Formation of these metabolites from glycogen yields a stoichiometric amount of protons (Pörtner, 1982; Pörtner et al. 1984b: for the net proton balance of alanine formation, it is irrelevant whether the ammonium group originates from aspartate or from free ammonia, since the pattern of dissociation is the same in both cases). Therefore the organism obviously disposes of surplus non-respiratory protons which are not reabsorbed by gluconeogenesis or by oxidation to CO₂. Correspondingly, pH regulation during recovery could be demonstrated to occur also via ionic exchange with the ambient water (H. O. Pörtner & N. Heisler, unpublished observations).

In summary, restoration of aerobic homeostasis after a period of anaerobiosis in Sipunculus nudus proceeds with a rapid replenishment of the phosphagen and, thereby, restoration of the energy status. The priority of the resynthesis of arginine phosphate is such that even an additional acid-base disturbance is accepted for the extracellular fluid. Intracellular pH is regulated efficiently as soon as oxygen is available to the tissues. The plasma is the medium utilized as a sink for protons or base equivalents during ionic regulation of intracellular pH (cf. Pörtner et al. 1984a). During periods of high metabolic rate or energy consumption, which are likely to depend on the season (Pörtner et al. 1986), anaerobic glycolysis becomes involved during recovery and even aggravates the acidosis. Aerobic metabolism is then not sufficient to restore the energy status rapidly enough, although repayment of an oxygen debt is observed during the same period. During long-term recovery, restoration of the aerobic acid-base status is accomplished even if some anaerobic metabolites remain: a high concentration of these obviously has little effect on homeostasis in the sense that their disposal is of lower priority in the preparations for the next period of anaerobiosis.

The authors wish to thank C. Loh for technical assistance and S. Morris for critically reading the manuscript. Supported by Deutsche Forschungsgemeinschaft Gr 456/9.