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Squid (Illex illecebrosus, Loligo pealei) were cannulated in the vena cava and swum in a Beamish-type respirometer. Gas tensions and acid-base variables as well as octopine levels were estimated in samples of the mantle and of venous blood collected from quiescent, exercised, and recovered animals. When exhausted, both species exhibited a decrease in vena cava oxygen tensions and a slight alkalosis. With high swimming speeds prior to exhaustion in Illex a slight acidosis developed in the blood, which was linked to a severe intracellular acidosis. Generally, the drop in intracellular pH was linearly correlated with octopine accumulation in this species. Metabolic proton (and end-product) release from the mantle, however, was minimal, thus protecting arterial oxygen binding. High PCO2 values in the mantle of both species lead to the conclusion that the vena cava values analyzed in this and all literature studies on unrestrained cephalopods may not reflect the scope of respiratory acid-base changes in venous blood. Although metabolic changes in blood acid-base status are negligible, the respiratory acidification of venous mantle blood may allow for a classical function of Bohr and Haldane effects in these animals.

Mean intracellular pH (pHi) and PCO2 (PiCO2) have been analysed based on pH and total CO2 measurements in tissue homogenates. Tissues were sampled from undisturbed worms (Sipunculus nudus), squid (Illex illecebrosus), trout (Salmo gairdneri), toads (Bufo marinus), and rats. Homogenate metabolism was inhibited by the addition of potassium fluoride and nitrilotriacetic acid (NTA). Model calculations revealed that the influence of dilution, medium buffers, and contamination by extracellular fluids was negligible. In white muscle tissue the resulting pHi values were virtually the same as found in studies using DMO (dimethyloxazolidinedione). If large fractions of mitochondria were present (e.g. in heart muscle), DMO derived pHi values were considerably higher, probably representing overestimates. Homogenate derived pHi values are concluded to represent the effective mean pHi by taking into account pH gradients, and the volumes and buffering of cellular compartments. High time resolution and small variability make this method especially useful to assess rapid changes in pHi, e.g. in exercising animals.

Non-bicarbonate buffer values have been analysed in homogenates of squid mantle muscle and toad gastrocnemius muscle using the PCO2 equilibration technique. Homogenate metabolism was inhibited by the addition of potassium fluoride and nitrilotriacetic acid. After an initial change in the concentration of organic phosphates a new metabolic equilibrium was achieved which was insensitive to pH changes during PCO2 variations. Inaccurate measurements of homogenate buffer values could, thereby, be prevented. The accumulation of inorganic phosphate during the initial metabolic shift caused an increase in homogenate buffer values. Levels of unbound inorganic phosphate, which are effective in buffering, were estimated by enzymatic analysis in homogenate ultrafiltrates. Based on the evaluation of apparent dissociation constants pK', and buffer values for fluoride and inorganic phosphate, the non-bicarbonate, non-phosphatei (i: inorganic) buffer value of the tissue was calculated. The level of free inorganic phosphate in the resting tissue was considered for an estimate of the intracellular non-bicarbonate buffer value under control conditions.

Proton balance is analysed in relation to the anaerobic and aerobic metabolism of carbohydrates, carbonic acids, amino acids and fat by considering oxidation, carboxylation, decarboxylation and phosphorylation reactions, as well as the influence of ammonium, on the acid-base status of animal tissues. The functional role of the adenylates, phosphagens and inorganic phosphate in acid-base balance is investigated with respect to differences in the physicochemical properties of organic and inorganic phosphates. General principles are established for different anaerobic metabolic pathways in species from several phyla. It is concluded that proton release from the substrate, which is always involved in substrate-level phosphorylations, is essential for the mechanism of ATP formation. Anaerobic metabolism, which is characterized by incomplete oxidation of carbon chains and an accumulation of acidic groups, supports pH regulation in facultative anaerobes by minimizing the amount of accumulated protons. High levels of phosphagens mean high proton absorption during hydrolysis and an increase in the intracellular buffer value. Decarboxylation reactions in catabolic pathways are equivalent to proton consumption. The degradation of carbonic acids during anaerobiosis, therefore, contributes to pH regulation. Release of ammonia or ammonium ions in catabolism is also linked to the buffering of protons originating from the formation of carboxyl groups and net cleavage of ATP. Net disposal of amino groups or ammonium ions by transamination, reductive amination or ion exchange does not change this general picture. The proton, bicarbonate and CO2 turnover in metabolic pathways is discussed with respect to the interrelationships between pH and metabolic regulation.

Oxygen consumption, anaerobic metabolism, and oxygen supply of inner tissues were analysed in Sipunculus nudus at different oxygen tensions. Oxygen consumption, energy expenditure, and the PO2 in the coelomic fluid decreased linearly with declining ambient PO2. Below a certain range of PO2, which was a function of the size of the animals, the rate of oxygen consumption deviated progressively from the linear PO2/MO2 function. In the same range of ambient PO2 the coelomic PO2 levelled off. Anaerobic glycolysis, phosphagen degradation, and the succinate-propionate pathway became apparent with concentration changes of anaerobic metabolites first occurring in inner tissues. In extension of the conventional definition (Prosser, 1973; Dejours, 1981) the term critical PO2 (Pc) is applied to the oxyconforming Sipunculus nudus. The Pc is redefined as the steady-state PO2 below which environmental oxygen availability becomes insufficient for complete aerobic metabolism (as indicated by the onset of anaerobic energy production). It is discussed to be closely linked to the oxygen supply of inner tissues. This redefined critical PO2 is shifted to higher partial pressures with increasing size of the animals because of the diffusion distance related decrease in coelomic PO2. Accordingly, with decline of ambient PO2, oxygen starts to be released from haemerythrin at higher ambient PO2 values in larger animals. The pigment, which is likely to function as an oxygen store, defers anaerobiosis and, thereby, supports compensation of a higher Pc in large individuals by means of an increased haematocrit. The Pc is discussed as crucial factor for survival of individual animals in intertidal oxygen-depleted environments.