Proton-Equivalent Ion Transfer in Sipunculus Nudus as a Function of Ambient Oxygen Tension: Relationships With Energy Metabolism

The metabolic adaptation of facultative anaerobic marine invertebrates to environments of variable oxygen availability has been the subject of various studies resulting in a complex model of anaerobic energy metabolism (for a review see Grieshaber et al. 1988). Studies in the marine worm Sipunculus nudus L. have focused in particular on the effects of anaerobic energy production on the acid-base status. Theoretical considerations of acid-base relevant effects of (an)aerobic metabolic pathways (Pörtner et al. 1984a; Pörtner, 1987a, 1989) were closely resembled by the pattern of intra-and extracellular acid-base changes actually found during utilization of the respective metabolic pathways (Pörtner et al. 1984a,b, 1986a,b). This close agreement allowed a quantitative analysis based on the interrelationships between metabolic proton production and changes in acid-base variables to be conducted (Pörtner et al. 1986a,b; Pörtner 1987a,b).

The quantitative analysis of the acid-base changes in aerobic, anaerobic and post-anaerobic specimens of Sipunculus nudus suggested that the acid-base status may be regulated differently during aerobic and anaerobic energy production. It also became evident that the relative contributions of intra-and extracellular fluid compartments to the buffering of protons (and correlated intracellular/extracellular ion transfer processes) could vary greatly depending on the anaerobic metabolic rate (Pörtner et al. 1986b; Pörtner, 1986, 1987b).

Acid-base regulation in Sipunculus nudus, however, may not be confined to acid-base relevant transfer processes between body fluid compartments, but may also include net transfer of surplus protons between the animal and the environmental water, a mechanism well-known in acid-base regulation in fish under stress (e.g. Heisler, 1984, 1986b). This mechanism is also indispensable during steady-state acid-base regulation to counteract the continuous acid-base relevant load produced by aerobic substrate catabolism (Heisler, 1984, 1986b; Pörtner, 1989). The particular stress of long-term anaerobiosis, during which large amounts of acid-base relevant metabolic end products are produced, could be significantly ameliorated by such ionic transfer, although anaerobiosis in the natural environment of intertidal animals usually coincides with the restricted availability of water during low tide. Data obtained on Mytilus edulis during anaerobiosis suggested that transfer of protons to the water was unimportant for acid-base regulation in these mussels (Booth et al. 1984).

The present study in Sipunculus nudus was designed to investigate the contribution of acid-base relevant transepithelial ion transfer mechanisms during aerobic and anaerobic metabolic conditions associated with changing environmental oxygen partial pressures. The central aim was to correlate the effects of such conditions in terms of changes in metabolism with the effects on the acid-base

Ion transfer processes and metabolism in Sipunculus 23 status in intracellular and extracellular body fluid compartments and the environmental water.

Specimens of Sipunculus nudus L. were dug up close to the low water line of intertidal flats at Locquemeau, Brittany, France, in March and September (referred to as ‘spring’ or ‘autumn’ animals, respectively). Large animals (26–74g) were selected for experimentation and kept in tanks with a 10–20 cm deep layer of sand from the original habitat. The holding tank was supplied with filtered and recirculated artificial sea water at 10–15 °C. Net release of base equivalents by the spring animals during long-term holding caused an increase of water bicarbonate levels from 2.3 to 3 mmol ^l−1, whereas the [HCO₃⁻] for autumn animals was adjusted by titration with HC1 to a constant level of about 2.3 mmol l⁻¹.

The experiments were conducted in a closed seawater recirculation system (Fig. 1), with dimensions chosen according to the size of the animal to mimic a natural burrow (Pörtner, 1982). The system contained between 300 and 470 ml of artificial sea water and was thermostatted to 15±0.05 °C. The initial seawater bicarbonate level was adjusted to 2.3 mmol l⁻¹, but was allowed to rise up to 3 mmol l⁻¹ during the experiment as a result of the release of bicarbonate equivalent ions by the animal. Matching sizes of animals and chambers prevented the animals from turning around and allowed us to use a minimal water volume. The chamber was connected to a gas exchange column via a 2.5 mm mesh grid. To prevent evaporative loss of water the gases feeding the gas exchange column were thermostatted to 15 °C and humidified. Circulation of the water was provided at a rate of 100 ml min⁻¹ by a roller pump (type MP-GE, Ismatec, Zürich, Switzerland). The water flow was directed at the animal from the anterior, and flowed along the body surface (Pörtner, 1982). As well as mimicking the water flow in the natural habitat, this flow direction also kept the animals from exploring and clogging the lateral water inlet of the chamber. The animal tube and aeration column were darkened to minimize any stimulus for muscular activity.

Water from the aeration column was continuously sampled by means of a second roller pump at a rate of 6.6 ml min⁻¹ (Ismatec IP 4, Zürich), feeding a system for detecting changes in bicarbonate levels before being returned to the animal system (Heisler, 1978, 1984, 1989). During passage through this ‘A-HCO3-’ system the water was thermostatted to 30±0.05°C and equilibrated in a series of three glass columns with fritted bottoms with humidified gas at constant (1% CO2, delivered by a gas-mixing pump: M303/a-F, Wosthoff, Bochum, FRG). pH was measured by means of a glass electrode and a double electrolyte bridge Ag/AgCl reference with sleeve diaphragm connected to a high-impedance isolation amplifier (model 87, Knick, Berlin, FRG). The recording system was completely floating in electronic terms with the environmental water and was grounded via a platinum electrode close to the electrodes. After amplification and conditioning, the signal was recorded on a chart recorder and, after A/D conversion, fed into a microcomputer system for on-line analysis (PSI 98, Kontron).

This type of apparatus and water conditioning (30 °C) provided a sufficiently fast electrode response and a low drift of the electrode chain (<0.001 pH units per 24 h; see Heisler, 1989). The equilibration gas was chosen to give a pH close to 7, a range where non-bicarbonate buffering is negligible in sea water (Heisler, 1986a, 1989). At constant and temperature, changes in pH result exclusively from changes in water bicarbonate concentration (Heisler, 1986a, 1989). The accuracy of the respective calculations (Heisler, 1986a) was checked using a calibration procedure including the addition of known amounts of HC1 and NaHCO₃ to the system and by analysis of total CO₂ in water sampled from the A-HCO₃⁻system.

Prior to experimentation the animals were catheterized by introducing a 2–3 cm length of PE 60 or PE 90 tubing (75 cm) into the body cavity after puncturing the posterior end of the body. The tubing was secured with cyanoacrylate glue (type 7432, Bostik, Oberursel, FRG.). After placing the animal in the chamber as described above (Fig. 1), the cannula was fed out of the system through the grid and the aeration column. After 24h of acclimation at values between 13.3 and 16kPa (100–120mmHg) and between 0.053 and 0.093 kPa (0.4–0.7mmHg), the control rate of H⁺-equivalent ion release was determined for another 24 h. Hypoxic conditions (⁠<0.4kPa=<3mmHg) were introduced within 15-30 min by bubbling the water with normocapnie nitrogen during passage through the gas exchange column. 0.03% CO₂ in pure N₂ was provided by gas-mixing pumps. After 24 h of hypoxic incubation, normoxic conditions (see above) were restored by aerating the water. This led to an increase of water to above 12 kPa (90 mmHg) within 5 min. Recovery from anaerobiosis was followed for 48 h after the end of hypoxic incubation.

During the whole period of aerobiosis, anaerobiosis and subsequent recovery, coelomic fluid was sampled anaerobically via the indwelling catheter at the intervals indicated in the figures. System water was exchanged with fresh sea water at intervals of 24 h. At these times, 50–100 ml samples were taken for the analysis of volatile fatty acids until it became evident that they did not accumulate in the recirculated water because of the vigorous gas flow in the gas exchange and equilibration columns. Water samples taken at shorter intervals for measurement of water ammonium concentration were replaced with fresh sea water to maintain water levels in the aeration column. Thus, the release of protonated end products did not contribute to net H⁺-equivalent ion transfer to the water. The dilution of the environmental water with fresh sea water was taken into account during evaluation.

Coelomic fluid samples (about 700 μl from a total volume of 13.5–18 ml) were analyzed for extracellular pH, and using a thermostatted microelectrode assembly (15±0.1 °C, BMS3, 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. Total CO₂ in coelomic plasma was determined after centrifugation and analysis of supernatant plasma samples. The CO₂ contents of plasma and water samples were analyzed by means of a Capnicon III apparatus (Cameron Instruments, Port Aransas, TX, USA), calibrated with NaHCO₃ standards. The resulting apparent bicarbonate values were compared with those obtained by calculation based on the Henderson-Hasselbalch equation using values for CO₂ solubility and pKi‴ derived from the polynomials of Heisler (1984, 1986a). (Note: the last line term of the a-formula in Heisler, 1984, is misprinted and should read ‘+ ‘.) In order not to deplete the animals of coelomic fluid, the samples used for blood gas analyses (180–50 μl) were re-infused into the animal.

Water bicarbonate levels were monitored as described above. Ammonium concentrations in water and plasma samples were determined enzymatically using standard tests (Bergmeyer, 1974). Acetate and propionate levels in water samples were analyzed by high performance liquid chromatography, as previously reported (Pörtner et al. 1984c).

The first series of experiments with autumn animals (collected in September, see above), performed approximately 2 months after collection, was focused on steady-state acid-base parameters and the control rate of H⁺-equivalent ion exchange during long-term incubation. The acid-base status remained essentially unaffected during 1 week of incubation in aerated sea water. Coelomic pH (plasma pH, pH_pl) varied insignificantly between 7.87±0.10 after 24h and 7.92±0.07 after 162h; plasma bicarbonate (7.4±0.3 to 8.7±0.7mmoll⁻¹) and plasma (0.28±0.04 to 0.29±0.03 kPa, 2.1±0.3 to 2.2±0.2mmHg) did not change significantly (Fig. 2A). The apparent bicarbonate concentrations derived from measurements of total CO₂ and those calculated according to Heisler (1984, 1986a; see above) were essentially identical (Table 1), confirming the validity of the respective equations for the body fluids of this invertebrate. Average coelomic P_O2 varied insignificantly between 3.7 and 4.8kPa (28 and 36 mmHg) (Fig. 2B), and coelomic ammonium levels increased slightly, but also insignificantly (from 0.16 to 0.28 mmol l⁻¹; Fig. 2B), during these control experiments.

Bicarbonate and ammonium levels in the ambient water increased steadily in this series of experiments ( ΔNH₄⁺_w=0.041 mmol kg⁻¹ h⁻¹; ΔHCO₃⁻_W= 0.080 mmol h⁻¹ kg⁻¹ body mass). The resulting average net H⁺-equivalent ion transfer from animals to water was accordingly −0.039 mmol h⁻¹ kg⁻¹ (Fig. 3).

Experiments on proton movements during anaerobiosis were performed about 4 (autumn animals) or 5–6 months (spring animals) after animal collection. Since volatile fatty acids did not accumulate in the ambient water, determination of net proton transfer did not include the release of protonated metabolic end products. Upon exposure to anoxia, both autumn and spring animals exhibited an extracellular alkalosis, with pH rising 0.17 units above the control value of 7.81±0.01 in autumn animals and by 0.13 units above the control value of 7.99±0.07 in spring animals (Figs 4A, 6). No acidosis occurred until 12h after exposure of the animals to anoxia; after 24 h the depression of pH was much more pronounced in autumn animals (ApH=−0.21) than in spring animals (ΩpH=−0.10). These changes in pH were linked to a drop in plasma apparent bicarbonate levels (from 5.2±0.3 to 2.6±0.2mmoll⁻¹, autumn animals; 9.2±0.8 to 4.9±0.7 mmol l⁻¹, spring animals) and to a fall in coelomic ⁠, which reached its minimum after 8–16h of anaerobiosis [from 0.25±0.01 to 0.15±0.01kPa (1.9±0.1 to 1.1 ±0.1 mmHg) in autumn animals and from 0.30±0.03 to 0.2±0.03kPa (2.3±0.2 to 1.5±0.2mmHg) in spring animals after 8 h of anaerobiosis].

Upon return to normoxia (recovery), increased significantly above control levels. pH was further reduced by 0.23 units in spring animals and by 0.26 units in autumn animals. Minimal values were attained after recovery times of 3 h in autumn animals and 6h in spring animals. Peak values of were attained after 6–12 h, with rising by 0.25 kPa (1.9mmHg) in autumn and 0.17 kPa (1.3 mmHg) in spring animals. In autumn animals, remained above control values even after 72 h of recovery, so that plasma bicarbonate levels stayed slightly above control values even though pH was restored to its original level within 24 h. In spring animals, slowly started to decline after 12 h of recovery and and plasma bicarbonate concentration reattained control values after 48 h of recovery (Figs 4A, 6).

values in the coelomic fluid of autumn animals (Fig. 4B) followed the changes in water ⁠. Upon initiation of environmental anoxia, fell from 6.5±0.44kPa (48.7±3.3mmHg) to zero, but was elevated to only 2.9kPa (22 mmHg) upon return to normoxia. Plasma ammonium levels (0.23± 0.02 mmol l⁻¹) were significantly reduced during the initial 8h of anaerobiosis (to 0.13±0.01 mmol l⁻¹), but were later restored to control levels. Upon return to normoxia, the ammonium concentration was significantly reduced (to 0.18±0.02mmoll⁻¹; 12h) and then slightly elevated above control values (0.38±0.1mmoll⁻¹) after 48 h of recovery.

During the control period before anoxic exposure the ammonium and bicarbonate concentrations of the ambient water rose steadily (by 0.079 and 0.080 mmol kg⁻¹ h⁻¹, respectively), but the rate of H⁺-equivalent ion transfer was much lower (−0.001 mmol kg⁻¹ h⁻¹) than that measured during the control experiments (Fig. 3), possibly because of uptake of CaCO₃ from the sand (see also Discussion). During anaerobiosis, the rates of both bicarbonate and ammonium accumulation were reduced compared with the control period. These changes were significant and reflected an increased net base release during the first 16 h of anaerobiosis, before the process was reversed, indicating a net proton release during the last 8h of anaerobiosis in autumn animals (Fig. 5).

In spring animals (approximately 5–6 months after collection) the rate of net H⁺-equivalent ion transfer under control conditions was much higher (−0.032 mmol kg⁻¹ h⁻¹) than in autumn animals. In spring animals the rate of net bicarbonate release was also higher (0.105 mmol kg⁻¹ h⁻¹), whereas the rate of ammonium release was slightly lower (0.073 mmol kg⁻¹ h⁻¹). In spring animals the net base release reversed to a net release of protons immediately upon exposure to anoxia, with an additional rate increase during the last 8 h of anaerobiosis (Fig. 7).

In both spring and autumn animals, the rate of H⁺-equivalent ion transfer to the ambient water was greatly stimulated during the first hours of recovery (maximal rates: +0.320mmolkg⁻¹h⁻¹, autumn animals; +0.087 mmol kg⁻¹ h⁻¹, spring animals). This rise was primarily due to a considerable reduction in the rate of release of bicarbonate into the water, whereas the rate of ammonium release returned to control values in autumn animals (0.076 mmol kg⁻¹ h⁻¹) or slightly reduced values in spring animals (0.057 mmol kg⁻¹ h⁻¹). The control rate of bicarbonate release was reattained between 48 and 72 h of recovery in autumn animals (Fig. 5), whereas in spring animals this rate was still below the control rate at 72 h (Fig. 7). Accordingly, the net H⁺ transfer in autumn animals was close to the control value after 48 h of recovery, whereas this variable did not reattain control values in spring animals for the entire recovery period (Fig. 7).

Determination of transepithelial H⁺-equivalent ion transfer requires that the experimental animals are kept in a system with relatively high water flow and minimal volume. These somewhat unphysiological conditions applied during our experiments, however, did not significantly affect the status of the animals. Analysis of blood gases and acid-base parameters for 1 week under control conditions clearly demonstrated the ability of Sipunculus to maintain a steady state for prolonged experimental periods under these conditions. The rate of net H⁺ transfer between animals and ambient water was also rather constant and may be taken as an indication of steady-state conditions. Net transfer of protons I Sipunculus, however, is from the animal to the water and accordingly opposite in direction to that in fish. In fish, the net proton release is due mainly to aerobic degradation of sulphur-and phosphate-containing protein and nucleic acids (Heisler, 1986b). The reverse fluxes observed in Sipunculus may be linked to differences in energy substrates utilized. Metabolic processes leading to proton consumption at aerobic steady state include degradation of carboxylic acids (for a recent review of the relationships between metabolism and acid-base regulation see Pörtner, 1989). Organic acids originating from the diatom content of sand ingested by the animals may be of some importance in this respect. Protein or amino acid catabolism, however, is evidently also a major route of metabolism, as indicated by ammonium accumulation in the ambient water. Autumn animals kept for 2 months before experimentation exhibited lower rates of ammonium production than animals from the same pool pre-acclimated in the laboratory for 4 months. The oxygen consumption/nitrogen excretion (O/N) ratio changed considerably from 27.8 after 2 months to 14.4 after 4 months. A ratio of 15.6 was measured for spring animals after 5–6 months of acclimation (based on oxygen consumption measurements by Pörtner et al. 1985). In squid, which depend largely on protein catabolism for energy production, the O/N ratio was reported to be 14.9 (Hoeger et al. 1987). This suggests that after 4 months the (autumn) animals had switched to protein catabolism for a larger fraction of energy production because of depletion of potentially basic nutrients from the sand.

Another process possibly involved in promoting a net base release by the animals may be mobilization of carbonate deposits in the sand. A lowering of pH of the gut contents during passage through the digestive tract from the naturally rather alkaline sea water to values close to coelomic pH may well support resorption of basic equivalents originating from carbonate mobilization. These, in turn, have to be eliminated from the animal in soluble form. This source of basic equivalents will be depleted after the animals have used the same sand for two or more feeding cycles (especially in those tanks where water bicarbonate levels were kept low by the addition of acid), leading to an apparently more acidotic overall output of metabolism (as seen in autumn animals acclimated for 4 rather than for 2 months) (cf. Figs 3 and 5).

Exposure to an anoxic environment and accordingly anaerobiosis led to an initial alkalosis in both spring and autumn animals. This alkalization is mainly attributable to the reduced production of CO₂ during anaerobiosis and the resulting lower coelomic ⁠, whereas the non-respiratory component is likely to be attributable to hydrolysis of phospho-L-arginine (Pörtner et al. 1984b). The production of acidic anaerobic metabolic end products leads to normalization and finally to a considerable acidification of coelomic plasma pH after 16–24 h of anoxia. This acidosis is further aggravated upon return to normoxia and aerobiosis, partly as a result of the pronounced elevation of during the first 12 h of recovery indicative of the repayment of an oxygen debt (Pörtner et al. 1986b). This interpretation is supported by the observation that, owing to the elevated metabolic rate, coelomic values remain low for the entire recovery period (Fig. 4B). The non-respiratory component of the acidosis is related to the resynthesis of phospho-L-arginine (see below). Coelomic bicarbonate levels are restored and even elevated above normal in the course of the recovery period. The elevation during late recovery is related to the increased and serves to restore plasma pH.

The changes in plasma ammonium levels are probably governed to some extent by the changes in of the environmental water, which are closely correlated with the procedure of flushing the system with fresh sea water. Flushing was performed at the beginning of the anoxic and the recovery periods, and is clearly accompanied by reductions in plasma [NH₄⁺] levels (Fig. 4B). The plasma ammonium level returned to normal at the end of the anoxic period, but rose considerably above control values during the second half of the recovery period. This is probably due to the considerable elevation of metabolic rate associated with repayment of the oxygen debt. Two other factors should be mentioned which may also be involved in the initial reduction of plasma ammonium levels upon return to normoxia: a severe intracellular acidosis developing in parallel with the extracellular acidosis will cause considerable trapping of ammonium in the tissues, and amino acid stores depleted during anaerobiosis may be replenished. There is, however, little direct evidence for such speculation.

In autumn animals, the alkalosis during the first 16 h of anoxia resulted in a very slight enhancement of the normoxic rate of base release (negative net H⁺ flux). A reversal of the net H⁺ flux occurred only at the end of the anoxic period. The offset of the cumulative ammonium release curve during recovery from the extrapolated normoxic control curve (Fig. 5) -due to an almost complete halt in the elimination of ammonium -was essentially matched by a similar reduction in net bicarbonate release. Since plasma ammonium levels were not enhanced during anoxia and tissue storage of ammonium is naturally limited, the rate of elimination of ammonium clearly reflects a reduction in metabolic ammonium production. This reduction indicates a switch to glycogen as the main substrate for anaerobic metabolism (see Grieshaber et al. 1988, for the pathways involved). The observed transepithelial transfer of H⁺ equivalents during anaerobiosis is closely linked to changes in plasma pH, a base release during alkalosis being reversed to a net proton release during the period of progressive metabolic acidosis. This finding supports the conclusion that acid-base regulation is not completely terminated during anaerobiosis, but that ion exchange rates are probably reduced (see below).

The relationship between coelomic pH and net transepithelial ion transfer observed in autumn animals is not duplicated in spring animals. A net extrusion of protons takes place during the early stages of anoxia (Fig. 7). This phenomenon could be explained if the initial alkalosis were reduced owing to a decrease in the rate of phospho-L-arginine depletion compared to that in autumn animals. Another possible explanation depends on the anatomical arrangement of muscle tissues in 5. nudus. Intracellular muscle compartments represent a large fraction of the fluid space interfacing with the ambient water. Intracellular muscle pH, rather than coelomic fluid pH, may accordingly be the controlling factor for transepithelial ion transfer processes, especially if disequilibria between coelomic fluid and intracellular fluid pH occur. Further study is required to clarify whether the vertebrate model of bulk extracellular fluid as a ‘second defence’ line for intracellular compartments is applicable to the fluid compartment system in S. nudus.

Another explanation for the discrepancy between coelomic fluid pH (alkalotic) and the apparent net extrusion of protons from the body fluids may be sought in the difficulty of comparing extrapolated normoxic control fluxes with fluxes obtained using different substrates under completely different metabolic states. During anaerobiosis, metabolism is shifted towards the breakdown of carbohydrates, which during aerobiosis would produce no acid-base relevant effect. Thus, the normoxic control rate should not be taken into consideration during anaerobiosis, and the amount of protons released to the water would have to be determined as the difference between bicarbonate and ammonium release rates alone. On this basis, the average net acid-base relevant H⁺ release during anaerobiosis would be similar in spring (0.44mmolkg⁻¹24h⁻¹, Fig. 7) and in autumn (0.31 mmol kg⁻¹24 h⁻¹, Fig. 5) animals. These low rates would be independent of metabolic rate and in good accordance with the limited extent of acid-base disturbances during anoxia in both groups of animals.

Significant amounts of H⁺ were net transferred only during recovery, when more than 4 mmol kg⁻¹ body mass was eliminated from the autumn animals (Fig. 5, see below). This was based exclusively on a net reduction of the water bicarbonate level. Immediately upon return to normoxia, ammonium release was restored to control rates, indicating that ammonium metabolism was not involved in repayment of the oxygen debt. Rather, the enhanced metabolism was probably based on the breakdown of some of the accumulated intermediary products.

During recovery, the net release of protons to the water is accelerated, coinciding with the development of an acidosis in the extracellular space. The non-respiratory fraction of the additional extracellular acidosis is quantitatively explained by resynthesis of phospho-L-arginine (Meyerhof and Lohmann, 1928), depleted during early anaerobiosis. This resynthesis of phosphagen, restoring the high-energy stores, results in a delayed release of metabolic protons transiently associated with inorganic phosphate during anaerobiosis (Pörtner et al. 1986a,b). Previous experiments have indicated (Pörtner et al. 1986b) that intracellular pH remains constant during the initial phases of recovery, which may mean that the acid-base relevant transepithelial ion transfer is mainly governed by the extracellular acid-base status. At this stage, however, intracellular pH (pHi) is still well below control values (Pörtner et al. 1986b), and may contribute to eliciting high rates of net H⁺ release into the water. These observations clearly demonstrate that transmembrane transfer of protons is a valuable (though relatively inefficient) mechanism during anaerobiosis, but that transepithelial H⁺ translocation, in particular during early recovery, is the prime mechanism for acid-base regulation in S. nudus.

The large increase in the rate of transepithelial elimination of protons after the re-establishment of normoxia coincides with an increase in the coelomic acidosis, induced by increased metabolic CO₂ production and release of metabolic protons from resynthesis of phosphagen (Meyerhof and Lohmann, 1928). The rate increase, however, is larger than would be expected from the extent of the additional acidosis. The deflection of pH roughly doubles, whereas the H⁺ extrusion rate rises by a much larger factor to 320μmolkg⁻¹h⁻¹, a rate even higher than that observed during environmental hypercapnia with a larger pH deflection (110 μmolkg^_1h⁻¹; H. O. Pörtner and N. Heisler, unpublished results). This suggests that, under these conditions, either the intracellular muscle compartments experience a more severe acidosis than the extracellular fluid or other factors, such as the reavailability of aerobic energy, are involved in facilitating an extremely fast transfer.

Even after 72 h of normoxic recovery the curves of cumulative net H⁺ transfer (Figs 5 and 7) did not approach the extrapolation of the control curve, but were displaced in parallel by about 4 mmol kg⁻¹ H⁺. This pattern is different from that found in fishes after extensive activity associated with severe lactacidosis (Heisler, 1984, 1986b). Part of this offset may be attributable to the different metabolic substrates used under aerobic and anaerobic conditions (see above). The magnitude of the displacement, however, is too large to be explained on this basis alone. A more probable interpretation is that the anoxia-induced production of organic acids has not yet been reversed by aerobic metabolism. Previous experiments have indicated that anoxia-induced acetate accumulation is not totally reversed during 24 h of recovery (Pörtner et al. 1986a). Quantitatively more important is that strombine levels remain high more or less constant during 24 h of recovery. This is due to the characteristics of the respective opine dehydrogenase, which rapidly catalyzes the formation of this intermediate (Grieshaber and Kreutzer, 1986). Succinate and propionate were also found at relatively high concentrations, even after 24 h of recovery (Pörtner et al. 1986a). The amount of protons released from by these intermediates is actually very similar to the offset (4 mmol kg⁻¹) of the net H⁺ transfer curves (Figs 5 and 7) from the extrapolation of the control rate of H⁺ release (Table 2). The extent to which these intermediates remain at elevated levels in the body fluids or the environmental water, even after 48 h of recovery, is unknown and a more detailed evaluation is impossible until further data are available.

The pronounced differences found between the regulatory pattern in autumn and spring animals are probably attributable to differences in anaerobic and post-anaerobic metabolic rate (see also Pörtner, 1987b). The initial alkalosis is much greater in autumn animals, indicating a faster degradation of phospho-L-arginine than in spring animals. Differences in metabolic rates are also indicated by the rates of ammonium accumulation during recovery and from the observation that larger quantities of anaerobic end products are metabolized in autumn, as compared to spring, animals (Pörtner et al. 1986a). The higher metabolic rate of autumn animals is evidently linked to higher rates of ionic exchange, resulting in significantly earlier recovery.

Generally, re-establishment of aerobic (and overall much faster) metabolism after anaerobiosis is also correlated with a considerable elevation in the rate of H⁺ transfer. This high rate of H⁺ extrusion is required to facilitate repletion of phosphagen by establishing the appropriate homeostatic conditions. Resynthesis of phospho-L-arginine, however, results in release of large quantities of protons, which in turn have to be eliminated in order not to disturb the recovery process. The energy demand of the active translocation mechanism could thus explain part of the observed increase in the metabolic turnover.

This positive correlation between ionic transfer and metabolic rate will not provide any limitation for ionic transfer as long as sufficient energy can be provided without exceeding a certain ratio of gained energy/simultaneously produced protons. If the amount of protons produced by a metabolic process exceeds the amount that can be translocated, then the resulting acidosis cannot be corrected and further metabolism may even be inhibited. This may be the case during anaerobiosis, when the energy yield from substrates is small compared to that in aerobic conditions.

We conclude that, during long-term anaerobiosis, the rate and efficiency of ionic pH regulation in Sipunculus nudus are considerably diminished. pH regulation is primarily performed through a minimization of proton production from metabolism. The contribution of transmembrane acid-base relevant ion transfer is small and not sufficient to improve the homeostatic conditions of intracellular body compartments significantly. Transepithelial ion transfer processes are greatly reduced during anaerobiosis, and are closely correlated with the reduction of energy turnover reflected by a decrease in metabolic rate. The observed restriction to closed-system organismal pH regulation during anaerobiosis may accordingly be related to the reduction of energy consumption by epithelial net H⁺ transfer.

The tremendous increase in transepithelial ion transfer after return to normoxia facilitates the establishment of the homeostatic conditions required for the restoration of high-energy stores and sufficient energy production, coupled with a lower H⁺ yield than during anaerobiosis. The observed increase in metabolic rate forms the basis for the energy-consuming transfer mechanisms for acid-base relevant ions, which are capable of restoring the acid-base status long before the original stress factors, the protons dissociating from anaerobic intermediates, have been removed from the body fluids by further oxidation.

The authors gratefully acknowledge the generous support during animal collection by the members of the Station Biologique de Roscoff. Supported by Deutsche Forschungsgemeinschaft Gr456.