Mechanisms of Acid-Base Adjustment in Dogfish (Scyliorhinus Stell Aris) Subjected to Long-Term Temperature Acclimation

The acid-base regulation observed in the Larger Spotted Dogfish with change of acclimation temperature is peculiar in comparison with that observed in air-breathing ectothermal vertebrates (Heisler et al. 1976). Most of the studied air-breathing ectotherms maintain a constant relative alkalinity (i.e. a constant [OH^-]/[H+] ratio) and thus a constant dissociation of imidazole in the extracellular compartment, by adjustment of while the bicarbonate concentration remains constant (Baumgardner & Rahn, 1967; Howell et al. 1970; Reeves, 1972; Crawford & Gatz, 1974; Jackson & Kagen, 1976). Also, intracellular pH was found in two air-breathing species to be maintained more or less at a constant relative alkalinity (Reeves & Wilson, 1970; Malan & Reeves, 1973 ; Malan, Wilson & Reeves, 1976). In dogfish, acclimation to a new temperature for 24 h has been shown to result in a change of extracellular pH that was produced by changes of both and extracellular bicarbonate concentration (Heisler et al. 1976), and was smaller than that to be expected on the basis of the rule of constant relative alkalinity (Rahn, 1966, 1967). Also, in two of three studied dogfish cell types pH changed with temperature much more (red muscle) or much less (heart muscle) than expected according to the rule of constant relative alkalinity.

There are, however, some doubts whether the observations of Heisler et al. were made in equilibrium conditions or whether the differences observed in comparison with other species can be attributed to a non-steady-state due to the relatively short acclimation period of only 24 h. To resolve this problem, we here report the effect upon acid-base parameters of acclimation for the relatively long time period, of more than 3 weeks, to three different temperatures.

In this paper we also investigated the relative importance of changes in and bicarbonate concentration in pH regulation. For this we raised plasma ⁠, by elevation of inspired ⁠, in dogfish acclimated to low temperature.

Specimens of Larger Spotted Dogfish (Scyliorhinus stellaris) were caught in the Bay of Naples and kept at temperatures of 14–17 °C in large, well aerated seawater tanks for two weeks to several months prior to the experiments. Then groups of 4–6 were acclimated to the desired temperatures for 22–28 days in thermostatted 400 1 PVC tanks. The tanks were flushed with fresh sea water at a rate of 40–80 1/h and the water was bubbled with air or air/CO₂ mixture at a rate of 10–15 l/min. Circulation of the water in the tanks was provided by mixing pumps immersed into the water of the tanks (20–30 1/min). The animals were fed daily during the adaptation time. Two experimental series were conducted:

Groups of four juvenile (0·25–1 kg body weight) and five adults (1·8–3·2 kg) were acclimated to 10, 15 or 20 °C. The tanks were aerated with room air. (Series performed during April and May 1976.)

Six adults (1·7–3·5 kg) were acclimated to 20 °C, in a tank aerated with room air. Five others (1·9–3·4 kg) were acclimated to 10 °C, in a tank aerated with a mixture of air and Co₂. The Co₂ concentration of the mixture was adjusted by an electronic regulation circuit, consisting of a -electrode in the tank water, a high impedance amplifier, a comparator and a solenoid valve, to produce a constant in the tank of about 1·9 mmHg. The regulation circuit was recalibrated twice a day and test measurements in the water showed that varied less than ± 0·2 mmHg. (Series performed during April and May 1978.)

Two days prior to the measurements indwelling catheters were placed in the dorsal aorta (see Heisler et al. 1976) and [¹⁴C]DM0 (5,5-dimethyl-2,4-oxazolidinedione) and [¹⁴H] inulin were injected for the determination of intracellular pH (Waddell & Butler, 1959) in white muscle (from fillets of the medium third of the body length).red muscle (from the side line red muscle layer, hind two thirds of body length) and heart muscle. Measurements and calculations of plasma pH, ⁠, bicarbonate concentration, and intracellular pH were carried out as described earlier (Heisler et al. 1976).

The mean extracellular pH values determined in 12 juvenile and 15 adult specimens acclimated to 10, 15 and 20 °C are presented in Fig. 1 (a). The values for change of pH with acclimation temperature (ΔpH/Δt, Fig. 1) are not significantly different from those determined earlier in 24 h temperature-acclimated specimens (Heisler et al. 1976). However, they were significantly different from those expected on the basis of a constant relative alkalinity (ΔpH/Δt, = –0.0184, Handbook of Chemistry and Physics, 1975).

The slight increase of ⁠, with temperature (Fig. 1,b) in juvenile fish was the same as in 24 h acclimated fish, but the increase in adult fish was slightly smaller than with short-term acclimation (Heisler et al. 1976). The decrease of calculated bicarbonate in juvenile fish and the increase in adults (Fig. 1 c) was again very similar to the pattern observed in 24 h acclimated fish (Heisler et al. 1976).

The mean intracellular pH values measured in white muscle, red muscle and heart muscle in the 27 specimens of this series are presented in Fig. 2, together with the determined temperature coefficients. None of the coefficients is significantly different from the corresponding value in 24 h temperature-acclimated fish (Heisler et al. 1976). The values for red muscle and heart muscle, however, are significantly different from that anticipated on the basis of constant relative alkalinity (ΔN/Δt = 0.0184, Handbook of Chemistry and Physics, 1975).

The fractional water contents and the fractional extracellular volumes (Q₆) of the three muscle types, determined by drying tissue samples and by application of the inulin distribution method respectively, are summarized in Table 1. None of the values is significantly different from the corresponding value measured in 24 h temperature-acclimated fish.

The results obtained in the 11 adult specimens of this series are presented in Figs. 3 and 4. Plasma (Fig. 3b) in specimens acclimated to 10 °C at elevated inspired is, at about 2.7 mmHg, markedly above the normal value of about 1 mmHg (series 1). This, however, does not influence the extracellular pH (Fig. 3 a) as the bicarbonate concentration (Fig. 3 c) is also markedly increased (from about 4 mm under normal conditions to about 10mm).

The intracellular pH of white muscle and heart muscle (Fig. 4) at 10 and 20 °C was slightly lower than in series 1, but the temperature coefficients (ΔpH₁ /Δt) of all three muscle types was not significantly affected by the manipulation.

The acid-base equilibrium observed in 22–28 days acclimated dogfish was almost the same as in 24 h acclimated specimens (Heisler et al. 1976). Apparently the temperature adaptation of the acid-base parameters is largely complete after 24 h. Consequently the peculiar pH/temperature regulation reported earlier (Heisler et al. 1976) is not the expression of non-steady-state conditions.

Relatively little is known about the regulatory mechanisms responsible for the temperature-dependent extracellular and intracellular acid-base regulation in poïkilothermie animals. The following mechanisms could be involved :

1. Changes of ⁠, due to changes in ventilation, changes of metabolic rate and/or limitations of the CO₂ elimination.

2. Changes in pK values of buffer substances (substances of special interest: imidazole-like and phosphate-like compounds, which are, according to their pK values, known to be almost exclusively responsible for non-bicarbonate buffering in the physiological pH range).

3. Changes in the concentration of substrates or metabolites according to changes of temperature-dependent reaction constants associated with release of H+ or OH^-.

4. Changes of the steady-state output of H+ or OH^- in the metabolism according to temperature-dependent changes of metabolic rate and pathways.

5. Exchange of either HCO₃^-, H+ or OH^- between body compartments and/or with the environment

   • passively due to changes in membrane permeabilities, chemical or electrical gradients,

   • actively by pump mechanisms for the adjustment of pH or of other parameters.

The imidazole alphastat hypothesis (Reeves, 1972), a model for acid-base regulation with temperature changes, seems to explain completely the observed extracellular and intracellular regulation in frog and turtle (Malan et al. 1976) based only on mechanisms (1) and (2) (Reeves & Malan, 1976). Also the majority of the other studied air breathing ectotherms fits reasonably well the proposed imidazole alphastat regulation (see Introduction). The essence of this model is that is regulated in such manner that the dissociation (alpha) of imidazole is kept constant. As imidazolehistidine is the predominant nonbicarbonate buffer in the extracellular compartment and the concentration of imidazole is considered to be fairly large also in the intracellular compartments (Reeves & Malan, 1976), the maintenance of a constant imidazole dissociation results in more or less constant bicarbonate concentrations with no additional mechanisms required.

This model, however, does not fit the obtained data in dogfish. Extracellular pH (ΔpH_e/Δt = –0·012) and intracellular pH in heart muscle (ΔpH_i/Δt = –0·007) change with temperature much less, and in red muscle (ΔpH_i/Δt = –0·03) much more, than required for a constant dissociation of imidazole (ΔpK_lm/Δt = – 0·018 to – 0·024, dependent on the type of imidazole compound; Edsall & Wyman, 1958). This complex pattern of pH regulation with different relationships of pH to acclimation temperature in all four studied compartments cannot be achieved by changes of only buffer pK values and ⁠. Actually, the bicarbonate concentrations in the extracellular space of both adult and juvenile specimens (Fig. 1 c) change considerably when temperature is changed. As recently shown, these changes in bicarbonate concentration have to be attributed to exchange processes between extracellular space, intracellular space and environmental sea water (Heisler, 1978). Thus at least mechanisms (1), (2) and (5) are involved in the acid-base regulation of dogfish.

It is quite evident from the results of the second series of experiments in the present study that changes of ⁠, artificially produced by increasing the inspired ⁠, have little influence on pH in extracellular and intracellular compartments. These data suggest that is not of major importance for the pH regulation in dogfish. This is also supported by the observation that even larger increases of in dogfish result only in very transient increases of gill ventilation (Randall, Heisler & Drees, 1976). In this context it must be realized that ventilation and thus can be varied in water breathing animals only within very narrow limits, because of the low oxygen content of water in comparison with air, while the bicarbonate concentration in the extracellular space has been shown to be increased by a factor of 4; from 5 to more than 20 mm (Heisler et al. 1976).

It has to be concluded that the acid-base equilibrium in dogfish is not characterized by imidazole alphastat regulation. Of the three mechanisms proposed to be involved, (1), (2) and (5), the third will be the most significant. The changes of buffer pK values are inevitably constant for a given change in temperature. The changes of are more or less predetermined according to metabolic rate and the gas exchange conditions and can only be modulated in a small range. Thus only the third mechanism, the bicarbonate transfer between body compartments and environment, is variable within sufficiently wide limits to account for the ultimate acid-base regulation in dogfish.

We gratefully acknowledge the support by the Stazione Zoológica at Naples and the skilful technical assistance of Mr G. Forcht and Miss S. Braun.