The Absorption of Sodium Ions by the Crayfish Astacus Pallipes Lereboullet: III. The Effect of other Cations in the External Solution

It is now well established that many aquatic animals can selectively absorb sodium ions from their environment. In sea water sodium is quantitatively the most important cation, but in natural fresh waters sodium ions may form only a small part of the total cation content. Calcium and magnesium ions, especially, may be present in concentrations considerably higher than that of sodium. It is important, therefore, to see what effect, if any, these other cations have on the rate of absorption of sodium by freshwater animals. In laboratory experiments on sodium uptake ammonium ions may also be present in relatively high concentrations since these are often excreted by aquatic animals and tend to accumulate in the limited volume of external solution used for the experiments. The effects of most of these cations on sodium absorption is not known. It is firmly established that sodium can be selectively absorbed from mixtures of sodium and potassium salts in the frog and goldfish (Krogh, 1939) and in cases where sodium influx has been measured by the tracer technique it has been found that the influx is not affected by the presence of potassium in the external solution (Koch & Evans, 1956, for Eriocheir; Ussing, 1954, for the frog skin; and Treherne, 1954, for Aedes)

The object of the work reported in this paper was to see to what extent sodium influx in the crayfish was influenced by the cation composition of the external solution.

These were generally the same as described in previous papers (Shaw, 1959, 1960a). All the animals were previously salt-depleted by treating them with de-ionized water. To determine the effect of a given cation, the sodium influx from a sodium chloride solution of known concentration was first measured in the manner described previously (Shaw, 1960a). After a sufficient time had elapsed the cation in the form of solution of its chloride or sulphate was added to bring its concentration to the desired value. The experiment was then continued and the influx was measured again. The new influx (i.e. after the addition of the cation) was expressed as a percentage of the initial influx.

The effects of the addition of potassium salts on the sodium influx from 0·05 mM./l. NaCl solutions are shown in Fig. 1. 1 mM./l. concentrations of potasium chloride or sulphate had no effect in any of the experiments despite the fact that the concentration of potassium was twenty times that of sodium. In the higher potassium concentrations the results were less consistent but there was no general tendency for potassium to increase or decrease the influx. This behaviour is in accord with the effects of potassium on sodium influx found in other animals, and once again illustrates the selectivity of the sodium-transporting system.

The effect of calcium on sodium influx is shown in Fig. 2. At calcium concentrations lower than 1 mM./l. no effects were observed. At higher concentrations the influx was reduced to as low as 60% of the initial rate in certain cases, but this effect was not consistently observed. Over the range studied there were, in fact, more cases in which the influx was completely unaffected than in which it was reduced. There is no evidence that the reduction in influx is greater as the concentration increases and it is unlikely, therefore, that the occasional effects of calcium are due to the ions competing with sodium ions for transport. It may be that in high calcium concentrations the adsorption of calcium ions at the surface is sufficient in some cases to block some of the transporting sites. At the concentrations of calcium normally found in fresh water this ion is unlikely to affect sodium influx.

The effect of magnesium ions on sodium influx is shown in Fig. 3. Again the results were rather inconsistent. In many cases magnesium was without effect but generally the sodium influx was definitely increased. In the most pronounced cases the new influx was practically three times the original rate, but, on the average, the increase was much smaller—amounting to about 30% of the initial rate. The inconsistency in the response often applied even to individual animals. Thus the influx in one animal might sometimes be affected by magnesium and yet at other times not affected at all. It was generally noted that an increase in the influx occurred if the initial influx was rather below normal and it is possible that the magnesium ions exert their effect by stimulating the activity of the transporting cells. The presence of magnesium in the external solution may, therefore, be important in maintaining the normal level of activity of the sodium-transporting system, although at the concentrations of magnesium normally found in fresh water this effect is not likely to be a very important one.

The effect of free ammonium ions in the external solution on the sodium influx from 0·05 mM./l. sodium chloride is shown in Fig. 4. Compared with the effects of the other cations studied, the effect of ammonium ions was most marked. Ammonium ions produced a marked inhibition of sodium influx which at ammonium concentrations of 1 mM./l. amounted to a reduction of the influx to about 20 % of its normal value. The effect of ammonium ions on the influx is clearly related to the external ammonium concentration, the influx decreasing with increasing concentration. It is interesting to note that ammonium ions were more effective than lithium ions at the same concentration in reducing the sodium influx. The effect of a 1 mM./l. lithium chloride solution is also shown in Fig. 4. At this concentration the sodium influx was reduced to between 50 and 70% of the initial value compared with a reduction to between 5 and 30 % produced by ammonium ions. Lithium ions have been shown to reduce sodium influx in Eriocheir (Koch & Evans, 1956) and partially to replace sodium ions for transport in the frog skin (Zerahn, 1955) and there is little doubt that lithium can compete with sodium for transport. It may be argued that the same is true for ammonium ions in the crayfish, but before this idea can be considered it is necessary to rule out the possibility that the ammonia is simply exerting a toxic effect on the transporting cells. This seems unlikely for the following reasons:

(a) the inhibition is produced by very low concentrations of ammonia—lower in fact than that recorded for free ammonia in crayfish blood (Florkin & Frappez, 1940), (b) the same concentration of ammonium ions has no effect on the active transport of chloride ions (Shaw, 1960b), and (c) the inhibition can be abolished by simply increasing the external sodium concentration. This latter effect is illustrated in two animals in fig. 5. In these animals, with an external sodium concentration of 0·05 mM./l., 0·8 mM./l. ammonium chloride reduced the sodium influx to 20 % and 40 % of the normal respectively. As the external sodium concentration was increased the degree of inhibition became smaller until at a sodium concentration of 1 mM./l. the effect was almost negligible. It would thus appear that the ammonium ion must exceed the sodium ion concentration in the external solution by a ratio of 10:1 or more for the inhibition to be effective. This fact, together with the fact that ammonium ions exert their effect specifically on sodium influx (i.e. they are ineffective in the case of chloride influx) gives weight to the suggestion that ammonium ions are competing with sodium ions for available transport sites. If this is so then the transporting system can be characterized by its affinity for cations in the following order : sodium > ammonium > lithium.

Whether ammonium ions are actually transported inwards or not cannot be decided from these experiments—it is possible that they simply interfere with the normal attachment of sodium ions to the transporting system.

Ammonium ions only affect the sodium influx. They have no effect on the rate of sodium loss even when the influx is reduced to a low level. This is illustrated in Table 1 for two animals. The loss rate was calculated either by equating it with the influx at the external equilibrium concentration or by the difference between the influx and the rate of net uptake of sodium when this took place. Although the presence of ammonium ions had reduced the influx to 20–30 % of its normal value, there was no significant effect on the rate of sodium loss.

Sodium and ammonium ions resemble each other quite closely in electronic structure but the ammonium ion is complicated by the presence of the three protons from the hydrogen atoms. It is of interest to see the effect of compounds closely related to ammonia and differing only in the fact that one or more of the protons are replaced by larger nuclei. The compounds used ranged from the simplest substituted ammonia, methyl ammonium chloride, to the completely substituted compounds, tetra-ethyl ammonium chloride and choline chloride. The results of these experiments are shown in Table 2 where the sodium influx from a 0·05 mM./l. sodium chloride solution in the presence of 1 mM of the substituted ammonium compound is shown as a percentage of the normal influx. No reduction in influx similar to that produced by ammonium ions was observed with any of the substituted compounds. In two out of five of the experiments with methyl ammonium chloride a slight reduction of the influx was found, although in the case where this inhibition was greatest (specimen no. 37) it was not found again when the experiment was repeated. No inhibition occurred with any of the other substituted compounds. With the fully substituted compounds the influx was generally slightly increased— an effect rather reminiscent of that produced by magnesium ions.

The action of hydrogen ions on a specific sodium-transporting system constitutes rather a special case since they may be expected to affect the system in more than one way. If they are present in high concentration they may combine with the system and displace sodium ions ; on the other hand, they may affect the configuration of the system in such a way that it no longer retains its specific property of combining with sodium ions. In the latter case the effect would be analogous to that of pH on the combining properties of an enzyme.

The effects of the pH of the external solution on the sodium influx are shown in Fig. 6. The pH has no effect in the range of pH 6–10 but below 6 the influx falls off sharply and at a pH 4 it is reduced to 20–30 % of the normal value. pH 4 corresponds to a concentration of free hydrogen ions of o-i mg. ions/1. and may be compared with the concentration of 1·0 mg. ions/1. required for ammonium ions to produce a similar degree of inhibition.

The effect of pH on the sodium influx is similar to that found for the isolated frog skin (Ussing, 1949; Schoffeniels, 1955). Ussing interpreted the effect of pH on the outside of the frog skin as being due to alterations in the permeability of the outside surface layer and suggested that this was a pace-setting factor for sodium influx. However, measurements of sodium outflux at low pH in the outside solution by Schoffeniels (1955) show that at pH 4 and above the outflux is little changed and at a lower pH it is greatly increased. Measurements of the loss rate in the crayfish, shown in Table 3, pointed to the fact that at pH 4 the loss was not decreased and, hence, presumably the surface permeability was also not decreased. These results are consistent with the view that the external pH directly affects the sodium-transporting system, but it is not possible in this way to distinguish between the effects of pH in interfering with sodium uptake directly, in changing the combining properties of the transporting system, or in depressing the general metabolism of the transporting cells.

It has been shown that sodium uptake in the crayfish is basically a process of ion exchange (Shaw, 1960a), but the effects of cations on sodium influx show that the process is quite dissimilar to that, for example, found for a typical ion-exchange resin. In this case cations are generally taken up according to the lyotropic series ; the sodium-transporting system has little affinity for calcium and magnesium, and. further, readily distinguishes between sodium and potassium. The specific nature of the sodium-transporting system is clearly demonstrated. The effects of hydrogen ions on the sodium influx are not unexpected, but the marked inhibition produced by ammonium ions is surprising. Evidence has been presented that the ammonium ions compete with sodium ions and it is curious that the obviously highly selective sodium transport system should be inhibited by the one common cation which is excreted in quantity by the animal itself. It is possible, of course, that this is purely a coincidence, but it is strange that the evolution of the highly selective system should stop short at extending selectivity to the ammonium ion. Since it has been shown that during sodium uptake (in the absence of a penetrating anion) the sodium ions are exchanged for ammonium or hydrogen ions which are normally excreted, it may be thought more probable that the effect of ammonium ions on the sodium influx is associated with this basic exchange mechanism. It would not be unexpected, that if sodium ions were exchanging for ammonium ions, high concentrations of the exchanging ion in the outside solution would inhibit the exchange. Thus, for example, if sodium transport is mediated by a carrier, as is popularly supposed, then a net transfer of sodium would take place if the affinity of the carrier for sodium and ammonium ions was different at the outside and inside surfaces of the transporting membrane. At the inside the carrier affinity would be high for ammonium and low for sodium so that sodium was released and exchanged for ammonium. At the outside the reverse situation would be found ; the affinity for sodium would be high and this ion would be taken up and displace ammonium. The presence of a high concentration of ammonium on the outside would prevent the ammonium ions on the carrier being displaced by sodium ions in the outside solution.

This work was assisted by a grant from the Royal Society.