The Effect of External Sodium and Calcium Concentrations on Sodium Fluxes by Salt-Depleted and Non-Depleted Minnows, Phoxinus Phoxinus (L.)

Most freshwater animals studied so far respond to sodium depletion by increasing K in equation 1, i.e. more carrier molecule is apparently synthesized, but its affinity for sodium (K_m) is not altered.

During investigation of the effects of some heavy metals on ionic regulation in the minnow Phoxinus phoxinus the characteristics of its sodium balance mechanisms were studied in relation to external sodium and calcium concentrations. The unusual characteristics of these mechanisms form the subject of this paper.

The terms influx and efflux refer in what follows to unidirectional movements of ions measured with radioactive tracers, and the terms uptake and loss refer to net movements of ions.

Minnows were collected by net from the River Wansbeck, near Scot’s Gap in Northumberland. The fish were kept in large (1·5 m×0·3 m×0·45 m) glass holding tanks at 5-20°C. These had gravel bottoms and contained rooted and floating aquatic plants; they were filled with Newcastle-upon-Tyne tap water and were aerated via air stones. The tanks were lit by ‘natural light’ strip lights using a 12h: 12h light:dark regime. The fish were fed daily on re-wetted freeze-dried tubifex and kept at low densities as disease spread rapidly in crowded conditions. Individual fish were kept for short periods and frequent collecting was necessary. Fish were kept in the aquarium for at least 1 week to adapt before being used in experiments.

The minnows were depleted of ions by treating them, in 2-1 polystyrene boxes, with three changes of distilled water. Each change lasted about 36h. During depletion the fish were not fed and were maintained at a density of about 1 g of fish to 75 ml of water. Non-depleted fish were fed and kept for about 5 days in a small aquarium filled with tap water.

After the period of adaptation the fish were netted, rinsed in distilled water and placed in a sodium chloride solution containing ²²Na, at a specific activity of about 30 × 10⁶ counts min⁻¹ mmol⁻¹ (approximately 100 μCi mmol 1⁻¹) for 10min. Experiments on sodium influx with time showed that adsorbtion of Na had no effect on the values for sodium influx over a 10-min period. These experiments also showed that disturbing the fish had no effect on sodium influx since there was no change in influx over a 30-min period, a period long enough for sodium regulation to return to normal after disturbance in Carassius auratus (Tarbit, 1967).

On removal from the radioactive solution the fish was rinsed quickly in three changes of distilled water, which took less than 30 s. The excess water was dried from the fish’s surface and opercular cavity with filter paper and the fish was killed by severing the spinal column just behind the head.

The fish were weighed (average mass approximately 0·2g), dried to constant weight at 60°C and then ashed at 450°C for 12 h in platinum or zirconium crucibles. The ash was dissolved in 0 5 ml of dilute Analar hydrochloric acid and transferred by teat pipette to a 1 ml steel planchette. The crucible and pipette were rinsed onto the planchette and the liquid was evaporated off. 100μl of 0·9 mol 1⁻¹ dextrose in 10% Teepol were used on the planchette as a spreader. The activity on the planchette was counted with a low-background end-window Geiger counter used in conjunction with a Betaplan 50 automatic sample changer, a Panax scaler and Kienzle printer.

The experiments were designed so that: (1) the ratio of external to internal sodium was large (between 5 and 100), (2) the animals were in the radioactive solution for only a short time (10min), (3) changes in internal and external sodium concentrations were small, less than 0·5 %.

These conditions ensured that (a) influx was not affected by changes in external sodium concentration and (b) back movement of tracer was negligible, so that the accumulation of radioactivity in the animal effectively measured influx only.

Larger fish (0·8g) were used in these experiments than in the influx experiments. However, no significant differences were observed between large and small fish. Rates of influx and efflux per gram were similar. The sodium concentrations established following transfer to distilled water were also similar.

Efflux measurements were originally performed by loading the fish for 48 h in tap water labelled with ²²Na (the specific activity of the whole fish increased very slowly after this time), rinsing them and then leaving them in 400 ml of unlabelled tap water for 8 h. However, this caused a large diuresis over the first 30 min. To avoid this, fish were loaded, depleted (for experiments on depleted fish only), then individual fish were placed in 400 ml of distilled water (depleted fish) or unlabelled tap water (nondepleted fish) for 2-3 h. The water was then siphoned off to a level just deep enough to allow the fish to swim (about 75 ml). The experimental solution was then slowly added to increase the volume to about 500ml. The process was repeated twice; the volume was finally adjusted to 400ml and the fish left for about 5h. Results are therefore from undisturbed fish and the large volume ensured minimum change in the external solution and little back flux, hence efflux only was measured. A sample of the external solution was then evaporated at 100°C and its radioactivity counted with a Beckman liquid scintillation counter.

Not all the internal sodium is readily exchangeable with external sodium. The specific activity of that fraction of internal sodium which is readily exchangeable was determined in the following way. Fish were loaded and then depleted (depleted fish only), then placed for 5 h in distilled water in polypropylene beakers. The sodium concentration and radioactivity of the solution were measured and the specific activity of the exchanging sodium was calculated. Specific activities found in this way were used to calculate sodium efflux from the fish. Influx and efflux were expressed as mmol kg⁻¹ wet mass h⁻¹.

Fig. 1 shows the effect of increasing external sodium concentration on sodium influx in non-depleted fish; the graph shows a saturable uptake resembling the Michaelis-Menten curve of enzyme kinetics. However, saturation was not reached until the external sodium concentration was above 10 mmol 1⁻¹ -at least 20 times higher than that of the fish’s natural environment. The value was about 3 mmol 1⁻¹, indicating a relatively poor affinity of the sodium uptake mechanism for sodium in comparison with other freshwater animals (Wright, 1975). Fig. 2 shows the results obtained at low external sodium concentrations. These results fit well with those in Fig. 1.

There was no significant difference in the maximum influx (K) in depleted and non-depleted fish with increasing external sodium concentrations, at 10, 20, 30 and 40 mmol 1⁻¹ (P>0·05, t-test) (Fig. 1). Linear regression analysis was carried out on data from depleted and non-depleted fish between 1·5 and 5 mmol 1⁻¹. This portion of the curve approximates to a straight line and shows that there is no significant difference (P>0·06) in the rise in influx between these concentrations in depleted and non-depleted fish, although the fluxes are at different levels.

Sodium influx in depleted fish at low external sodium concentrations was very different from that in non-depleted fish (Fig. 2). Linear regression analysis on data between 0·125 and 1·0 mmol 1⁻¹, where the curve approximates to a straight line, showed that while the data for non-depleted fish gave a significant slope above the horizontal (P< 0·001) the data for depleted fish did not (Fig. 2), with the regression line for these data sloping down slightly.

At lower sodium concentrations the levels of influx in depleted fish were much higher than in non-depleted fish. However, the maximum sodium influx was unaltered by salt depletion. This resulted in a very small change in the overall K_m of the two curves. However, the K_m for the curve for depleted fish below 1 mmol 1⁻¹ sodium is as low as 0·05 mmol 1⁻¹. The overall results for depleted fish, therefore, have a stepped appearance as shown in Fig. 3. Possible explanations for this are discussed later.

It is apparent from these results that sodium depletion causes a large increase in the affinity of the sodium uptake mechanism in the range of sodium concentrations found in the fish’s natural environment.

At low external sodium concentrations sodium efflux was unaltered by changes in external concentration (Fig. 4). At high external sodium concentrations efflux increased rapidly as the external sodium concentration increased (Fig. 4). This is probably not a direct effect of sodium on the carrier mechanism. In high external sodium concentrations (and indeed in low ones in the case of depleted fish) the fish will gain a considerable amount of sodium since influx levels will be high and the procedure takes several hours. For example, a 1-g fish in 10 mmol 1⁻¹ NaCl would have a net influx of over 3 mmol kg⁻¹ h⁻¹ resulting in an increase in internal sodium of 0·012mmol after 5h, a 27 % increase in the total sodium content of the fish. It seems likely that the observed increase in efflux is due to an increase in internal sodium levels, although a link between influx and efflux cannot be ruled out. This is supported by the fact that efflux from depleted fish, which will have lower internal sodium levels, into distilled water (0·45 ± 0·04 mmol kg⁻¹ h⁻¹, ±S.E.M., N= 18) was lower than that from non-depleted fish (0·70 ± 0·05 mmol kg⁻¹ h⁻¹, N = 60).

Sodium efflux could be reduced still further to 0·29 ± 0·04mmol kg⁻¹ h⁻¹, N – 12 by depleting the fish in 1 mmol 1⁻¹ calcium chloride. However calcium had no effect on sodium efflux when non-depleted or depleted (in distilled water) fish were placed in calcium chloride solutions of increasing concentration (Fig. 5).

Most freshwater animals respond to sodium depletion by increasing the maximum sodium influx (K), while the affinity of the uptake mechanism (K_m) for sodium remains unchanged. However, ammocoete Lampetra planeri show a change in K_m for sodium (Morris & Bull, 1970), and Carassius auratus show a change in K_mfor chloride (De Renzis & Maetz, 1973). In this respect these animals are similar to phoxinus, for which more extensive data (Figs 1, 2) are available. Since changes in are now known in a cyclostome and two species of teleost, it seems possible that such changes may be more widespread in fish than had been thought.

The relationship between sodium influx and external sodium concentration in Phoxinus is complex (Fig. 3). Several possible models for the observed pattern of sodium influx in depleted and non-depleted Phoxinus may be suggested. Any model must account for: (i) the high influx in depleted fish at low external sodium concentrations; (ii) the fact that fluxes in depleted and non-depleted fish are very similar above about 2 mmol 1⁻¹ ; (iii) the approximately Michaelis-Menten nature of the relationship between influx and external concentrations; (iv) the apparently prompt change in the K_m of the sodium uptake mechanism of depleted fish resulting from changes in external sodium concentration.

The possible models fall into two categories: (1) a single carrier molecule which is modified with respect to its affinity (K_m) for sodium, or is modified with respect to both K_m and the number of sodium attachment sites; (2) a second sodium carrier, with a much higher affinity for sodium than that of the normal carrier, which is activated in response to low internal sodium levels.

The second model seems more probable. It is difficult to imagine how the affinity of a single carrier could be instantaneously increased by changes in internal sodium level and instantaneously reduced by changes in external sodium level.

The assumption that there are two sodium carriers working in parallel across the gill of the depleted fish does not explain the stepped appearance of the curve of Fig. 3, since the result of adding two Michaelis-Menten curves is simply to produce another Michaelis-Menten-type curve.

To arrive at a stepped curve it is necessary to postulate a rapid inhibition of the second (high-affinity) carrier at external concentrations greater than 0·1mmol1⁻¹, the level portion of the curve (L in Fig. 3) resulting from a balance between the decreasing effect of the high-affinity carrier and the increasing effect of the low-affinity carrier as external sodium concentration increases.

The results from the experiments on sodium efflux show that external sodium concentration has no effect on sodium efflux. The results do, however, suggest that internal sodium concentration has a strong influence on sodium efflux.

The investigation of the effect of calcium on sodium efflux produced apparently contradictory results since depletion in 1 mmol 1⁻¹ calcium chloride reduced sodium efflux to a lower level than that in fish depleted in distilled water, while calcium added to the experimental solutions had no effect on sodium efflux from depleted and non-depleted fish. Similar results were obtained by Cuthbert & Maetz (1972) for Carassius auratus. Their explanation, which follows, seems also to be applicable to Phoxinus. Calcium in the gill epithelium reduces the permeability of the gill to ions such as sodium. When the calcium is removed, by depletion in distilled water, the permeability of the gill increases and more sodium is lost. When the depleted fish are placed in a solution containing calcium, the calcium is only slowly replaced in the gill epithelium, and so sodium efflux remains at its elevated level.

Since calcium has no effect on sodium influx it would appear that in Phoxinus changes of gill permeability do not involve the sodium influx mechanisms.

I would like to thank Dr R. H. Stobbart for his help and advice throughout all phases of this work. I am also grateful to Mrs D. Weightman of the Medical Statistics Department for her advice on the statistical analyses of the results. I am indebted to Professor J. Shaw, who made facilities available for me to work in the Zoology Department and to SERC for providing financial support.