The Absorption of Sodium Ions by the Crayfish, Astacus Pallipes Lereboullet: I. The Effect of External and Internal Sodium Concentrations

The fact that many freshwater animals can absorb salts from the very low concentrations found in their environment was first demonstrated conclusively by Krogh (1937, 1938). He showed this by first reducing the salt content of the animal by washing, often for long periods, in continuously running distilled water. He was then able to demonstrate the uptake of sodium and chloride from dilute solutions and, in some cases, the concentration of these solutions was reduced below the limit of detection by his techniques. It was not possible to decide from his experiments whether ion absorption was a mechanism which came into action under conditions of salt depletion or whether it was a process which normally operated to maintain the salt balance of the animal.

The advent of the use of radioactive tracers provided another method for the measurement of ion uptake. The absorption of sodium ions has been demonstrated by this technique in the axolotl (Jorgensen, Levi & Ussing, 1946) and in the frog (Jorgensen, 1950; Jorgensen, Levi & Zetahn, 1954). More recently the long-lived sodium isotope, ²²Na has been used to describe sodium uptake in the goldfish (Meyer, 1951; Sexton & Meyer, 1955) and in the crab, Eriocheir sinensis (Koch & Evans, 1956). In all these animals sodium ions are continually absorbed under normal conditions and the absorption is balanced by an equal loss of sodium from the excretory organs and through the integument.

Before completely accepting the results on tracer studies it is necessary to establish that the ion influx measured by the tracer technique is a true measure of the ion uptake rate. In the case of the isolated frog skin, which is the only material which has been thoroughly investigated from this point of view, the evidence strongly favours the identity of tracer influx and true uptake rate (see, for example, Ussing, 1954). Although this is encouraging, it cannot be argued, without further evidence, that it is necessarily true of the intact frog or of other animals. However, if tracer measurements are always accompanied by chemical analyses of the ion in question it is generally possible to assess the validity of the tracer measurements.

Although the uptake of ions has now been established in many aquatic animals, little is known of the factors which influence the rate of uptake. The extent to which this is modified by environmental factors may be of great importance in the survival of the animal in any particular environment. On the other hand, there is evidence that the uptake rate may be regulated by the animal itself. Thus Jorgensen (1950) found that for both frogs and toads washing in distilled water for prolonged periods increased the sodium influx from 3 mM./1. NaCl solutions by a factor of 2 or more. Furthermore, Jorgensen, Levi & Zerahn (1954) found in the frog that sodium and chloride uptake mechanisms could be activated independently by sodium and chloride depletion, respectively.

The work described in this paper was designed to measure the normal uptake of sodium in the freshwater crayfish, Astacus pallipes, and to study the influence of one environmental factor—the external sodium concentration—on this. The quantitative effects of changes in the internal sodium content of the animal on the rate of uptake has also been studied in relation to the regulation of the uptake of sodium for the maintenance of sodium balance.

Some of these results have previously been briefly reported elsewhere (Shaw, 1958).

Previous knowledge of the absorption of ions by the crayfish is very limited. Krogh (1939, p. 91), quoting otherwise unpublished experiments, first showed the uptake of chloride by a single salt-depleted crayfish. He found that this animal absorbed chloride at a rate of 2·3μM./40 g. body weight/hr. and at a maximum rate of 6 μM./hr. Wikgren (1953) also reports the uptake of chloride in the crayfish, Potamobius fluviatilis, to be generally below 10μM./100 g./hr. although higher values were found. His results are based mainly on measurements of conductivity of the external solution, and the interpretation of these changes in terms of chloride movement requires great caution. The uptake of cations by the crayfish has not been previously reported.

The crayfishes were obtained from the Freshwater Biological Association, Ambleside, and were kept in a running tap-water aquarium until used. For each experiment a single crayfish, weighing about 10 g., was transferred to a 1 1. Pyrex beaker containing the experimental solution, maintained at a constant temperature (12-13⁰ C.). In all cases the experimental solutions consisted of de-ionized water or water containing pure sodium chloride. The effect of other ions on sodium uptake is reserved for consideration in another paper of this series. The volume of the experimental solution into which each crayfish was placed had to be carefully chosen. For animals weighing about 10 g. volumes of solution of the order of 50 ml. invariably resulted in low values for the uptake rate and this is probably due to the effects of accumulation of excretory products and the removal of oxygen from the water. On the other hand, if very large volumes were used, the accuracy of the sodium uptake measurements was considerably reduced. A compromise was adopted with a standard volume of 250 ml. for a crayfish of about 10 g.

Measurements of the sodium concentration of the external solution and of the blood were made by means of an EEL Flame Photometer.

Sodium influx was measured by using ²⁴Na as a tracer. A crayfish was placed in 250 ml. of de-ionized water and ²⁴NaCl was added to bring the total radioactivity of the solution to about 3μ.c. Then sufficient non-radioactive NaCl was added to raise the total external sodium concentration to the desired level. A 1 ml. sample of the experimental solution was removed to a planchet, together with a drop of strong dextrose solution, and dried in an oven. The radioactivity of the sample was measured by means of an end-window Geiger counter in the usual manner. At approximately half-hourly periods during the experiment further samples were removed and measured in the same way. Generally, each experiment was continued for a total period of 4−6 hr. The radioactivity counting errors were less than ± 2 % and the counting rate was corrected for the decay of the ²⁴Na during the experiment. Because large differences in the concentration of sodium between the blood and the solution were always maintained, the build-up of radioactivity in the blood during the experimental period was never great enough for back-diffusion of radioactive ions to have a significant effect on the calculation of the sodium influx.

In every experiment measurements of the total sodium concentration of the external solution were made at the beginning and at the end, and, in certain cases, during the course of the experiment. Generally, the conditions of the experiment were arranged so that only a small change in the external sodium concentration occurred.

To calculate the sodium influx, the logarithm of the measured radioactivities was plotted against the time at which the sample was taken. Under these experimental conditions this relationship was always satisfactorily linear as is shown in a typical example in Fig. 1. The sodium influx can be calculated from the slope of this line in the manner described by Jørgensen, Levi & Ussing (1946) in their studies on the uptake of sodium by the axolotl.

In presenting the results of these experiments the following terminology will be used. Sodium influx (M) refers to the influx of sodium ions as measured by the radioactive tracer technique. Uptake rate(U) is the true influx of sodium ions. This cannot be measured directly and it may or may not be equal to M. Net uptake is the difference between the uptake rate and the loss rate and this can be measured by chemical analysis. Loss rate (L) refers to the total loss of sodium ions, by outward diffusion, through the excretory organs and by any other means. The equilibrium concentration is the external sodium concentration at which a steady state is reached between the solution and the animal. The animal is in sodium balance and there is no net loss or gain of sodium.

In regard to the relation between the sodium influx and the uptake rate it is worth while making two observations at this point. First, the loss of radioactivity from the external solution can be accounted for approximately in terms of transfer of sodium ions across the integument to the blood: its disappearance cannot be ascribed to any adsorption phenomena. Measurements of the radioactivity of blood samples removed at the end of an experiment gave values consistent with the view that those radioactive ions which were lost from the external solution had been accumulated in the blood. These results are discussed in detail below. Secondly, although the very low external sodium concentrations used ensure that inward diffusion can play only a small part in the measured sodium influx, the possibility that exchange diffusion (Ussing, 1947) may account for a variable proportion of the sodium influx has always to be considered. However, the simultaneous measurement of sodium net uptake together with a knowledge of the loss rate make it possible to assess the importance of this type of interchange. This is also discussed below in the appropriate section.

The aquarium water contained 0·3 mM./l. Na. To measure the normal sodium influx, animals were transferred from the aquarium to experimental solutions containing this concentration of sodium chloride. The results of these measurements are shown in Table 1. The mean value is 1·5μM./10g./hr.

If the sodium influx approximates to the uptake rate and if the animal is in sodium balance, then the sodium influx should be matched by an equal rate of sodium loss. The loss rate was estimated in the following way. When a crayfish was placed in 250 ml. of de-ionized water with no added sodium chloride, sodium was initially lost to the water. The concentration of sodium in the water gradually increased until it reached a level at which it remained more or less constant for several days. The results of several experiments of this kind are shown in Fig. 2. The increase in sodium concentration of the water over the first few hours was used to calculate the rate of loss of sodium from the animal. Table 2 shows loss rates calculated in this way for a number of different individuals. The mean value for the loss rate (1·5 μM./10 g./hr.) is the same as that of the influx and, where the same individuals have been used for both measurements, there is generally fairly good agreement for the two values. This estimate of the loss rate should not be accepted without qualification. It is only strictly valid for loss into de-ionized water—it does not necessarily follow that the loss rate will be the same at all external concentrations or in all physiological states of the animal. This question is discussed again in more detail below.

It is interesting to see where the main loss of sodium occurs. Krogh (1939) calculated that 0·17μM./10 g./hr. of chloride was lost through the excretory organ. If the sodium loss is of the same order, then it is clear that only about one-tenth of the sodium could come out by this means. The remaining 90% presumably diffuses out through the integument, probably largely through the gills. It is not certain if the same is true for the behaviour of the chloride ion, since the situation for this ion is far from clear. In a single experiment Krogh found the rate of loss of chloride to distilled water was not much greater than the expected loss through the excretory organ. On the other hand, Wikgren (1953) states that the ratio of the loss of chloride through the body surface to that through the urine is 10-20:1, but the data from which he calculated this ratio is by no means clearly presented.

Returning now to Fig. 2, it is clear that the equilibrium concentration for those animals transferred from the aquarium to de-ionized water is not necessarily the same as the sodium concentration of the aquarium water. Indeed, it is almost always rather lower. Fig. 2 shows a number of individuals which have come into sodium balance with an external sodium concentration of between 0·1 and 0·2 mM./l. Furthermore, if animals which have already come into balance after placing in deionized water are transferred to a new volume of de-ionized water, then the new final equilibrium concentration will be lower than before. Progressive loss of sodium gradually reduces the equilibrium concentration further until, finally, a minimum equilibrium concentration, or threshold, is reached. Further removal of sodium cannot reduce the equilibrium concentration below this minimum value. In Table 2 the minimum equilibrium concentrations for a number of individuals are listed. The variability is fairly large, but the values generally lie below 0·1 mM./l. and the mean value is 0·04 mM./l.

It is noteworthy that the minimum equilibrium concentration on no occasion approached zero, as recorded by Krogh after the absorption of chloride from a dilute Ringer solution by a salt-depleted crayfish. This reason for this difference is not clear. Although it is possible that lower minimum sodium equilibrium concentrations could result if larger volumes of de-ionized water were used, this is not the explanation of the divergence from Krogh’s result. It can be calculated from his figures that he must have used only 70 ml. of the Ringer solution for a 40 g. crayfish. It may well be that there is a real difference in the behaviour of the mechanisms controlling the uptake of sodium and chloride respectively, but other possibilities, such as species differences, cannot be ruled out.

The attainment of sodium balance at low external concentrations after salt depletion is probably a feature common to many freshwater animals, although the value of the minimum concentration is variable. Thus in the freshwater crab, Eriocheir sinensis, Koch & Evans (1956) found that the minimum equilibrium concentration could not be reduced below between 0·2·0·5 mM./l. On the other hand, the East African freshwater crab, Potamon niloticus (Shaw, 1959), closely resembles the crayfish in this respect, attaining balance at a minimum concentration of about 0·05 mM./l.

The facts that the crayfish comes into balance with a certain external sodium concentration, and that this concentration varies according to the sodium content of the animal, make it clear that both the external sodium concentration and the internal sodium content must exert some influence over either the rate of sodium uptake or the rate of loss, or over both. The effect of these two factors on the sodium uptake rate will now be considered.

The sodium influx at different external sodium concentrations was measured by the method described above, although, since it was necessary to make several measurements on a single individual, a number of precautions had to be taken. In the first place, to avoid back-diffusion of radioactive ions, a period of time (generally i day) was allowed to elapse between each influx measurement. During this time the radioactivity of the blood had decayed to a suitable level. This meant that only a few influx measurements on each individual could be made in 1 week and the complete experiment had to be extended over several weeks. In the second place, during an influx measurement at a high external concentration there was a considerable net uptake of sodium : this increased the internal sodium content and this, in its turn, reduced the influx (see below). To get over this difficulty the following experimental procedure was adopted. The animal was first subjected to sodium loss by treatment in de-ionized water until the animal was in balance with its minimum equilibrium concentration. Further sodium was then removed and in this situation it was found that the influx was not affected by small changes in the internal sodium content. As an additional precaution, at the end of each influx measurement at a high external concentration the animal was placed in de-ionized water to remove the sodium which had been absorbed. Care was also taken that the influx measurements were not made with progressively increasing or decreasing external concentrations; the external concentrations were selected at random.

The adoption of the above procedure made it possible to obtain consistent and reproducible measurements of sodium influx over a wide range of external concentrations. The results obtained for a number of individual animals are shown in Figs. 3 and 4. It might be supposed that as the external concentration is increased, the number of sodium ions making contact with the sodium-transporting system will also be increased and the sodium influx will increase in proportion. If such were the case, there would be a linear relation between the external concentration and the sodium influx. Such a relation clearly does not exist except perhaps at very low external concentrations : in all cases the influx levels off to approach a maximum rate at an external concentration of about 1 mM./l.

This phenomenon has not been previously observed in any intact animal, but it does have a parallel in the transport of sodium by the isolated frog skin. Ussing (1949) first observed that the relation between the sodium influx and the external sodium concentration was non-linear, although he found no maximum even with external concentrations of the order of 100 mM./l. The question was later reinvestigated by Kirschner (1955) who, calculating the influx by the difference between the outflux and the short-circuit current, found a definite maximum at an external concentration of about 35 mM./l. At a higher external concentration the influx appeared to decline. Kirschner sought to explain the non-linear relation in terms of the mediation of a sodium carrier which formed an unionized complex with sodium ions and which became saturated at high external sodium concentrations. On this assumption he devised an equation relating the influx to the external concentration, but this was not completely adequate to account for his experimental results. Both Ussing and Kirschner used external concentrations which were much in excess of the normal physiological range.

In the crayfish the non-linear relation between influx and concentration becomes apparent at concentrations well below that found for the frog skin. The maximum influx is approached at a concentration within the normal physiological range. Without making any assumptions of the presence of sodium carrier complexes, it seems probable that in the crayfish sodium ions are transported inwards by a mechanism which is limited in its rate of operation and therefore becomes saturated at higher external concentrations. This type of relation recalls the relationship between the rate at which an enzyme breaks down its substrate and the concentration of the substrate—a situation which has been successfully described by the well-known Michaelis equation. It is interesting to see if this equation is applicable in the case of the sodium influx. Fig. 4 shows the relation between the sodium influx and the external concentration in one animal on which a large number of influx measurements had been made. On the same figure the dotted line represents the expression, M=K_c/(K_m + c), where Mis the influx, c the external sodium concentration, and K and K_m are constants with arbitrarily chosen values of 7 and 0·25, respectively. This expression clearly provides a reasonable approximation of the relation between M and c over the range which has been studied. However, there may well be other expressions which would fit equally well, or even better, and no particular significance is attached to this one. It does seem likely, though, that the true relation would (like the Michaelis equation) contain a factor determining the maximum influx and another, the rate at which the maximum was reached. On these grounds it may be suggested that sodium ions are transported inwardly by a saturable and rate-limited system which is characterized by a very high affinity for sodium ions.

Before proceeding with the next stage of the analysis, we must now examine the question of the equality between the sodium influx, as measured by the tracer technique, and the true uptake of sodium against the concentration gradient. This has been investigated by comparing the measured influx with the net uptake (or loss) which occurred during the experiment. If the influx correctly measures the total sodium uptake, then the difference between the influx and the net uptake must equal the rate of loss. The results of these experiments on one animal are shown in Fig. 5 and further examples are shown in Table 3 (specimens 10, 11 and 1).

We can first consider in detail the results which were obtained on crayfish no. 14 (body weight 7·2 g.), which are those shown in Fig. 5. The conclusions drawn concerning the behaviour of this animal are valid also for those animals listed in Table 3. Over the lower part of external concentration range the value of the influx minus the net uptake is about 0·8μM./hr. (this corresponds to a value of 1·1μM./hr./10 g. body weight), and this agrees well with the rate of loss as measured by the loss of sodium into de-ionized water (see Table 2, specimen no. 14). However, as the external concentration increases, the value of the influx minus the net uptake also increases to a value of about 1·8μM./hr. Now these results could obviously be explained on the grounds that the loss rate was not constant but increased at the higher external concentrations. Fortunately, this hypothesis can be tested in the following way : if the influx at the high external concentrations is reduced to a value below about 1·8μM./hr. then a net loss of sodium should occur. A reduction in the influx was brought about by two different methods—(1) by the increase in the internal sodium content of the animal and the consequent adaptation to a higher equilibrium concentration (the effect of the internal sodium content in reducing the influx is discussed in detail in the following section; here it is used simply to lower the influx at the high concentrations), (2) by the use of an influx inhibitor in the external solution (in fact, a mixture of 5 % CO₂ in air was bubbled through the solution). The application of these two methods to crayfish no. 14 are shown in the lower half of Table 3 (Exps. 14b and d−g). At the higher external concentrations (for example, 0·31 mM./l. in Exp. 14d and 0·445 mM./l. in Exp. 14g), despite the lowered influx (compared with that shown in Fig. 5 for these concentrations), a net uptake of sodium still occurred. The value of the influx minus the net uptake (0·53 and 1·0μM./hr. for 14d and 14g, respectively) had fallen from the previous values (shown in Fig. 5) and now approximated to the normal loss rate (0·8μM./hr., Fig. 5).

Now it might be argued that the reduction in the influx, although effected by two different methods (i.e. by the use of CO₂ as in 14d, or by the increase in internal sodium content as in 14g), was always accompanied by a decrease in the true loss rate as well. If this was so, then one would expect that at the lower external concentrations (for example, Exps. 14b and 14f in Table 3) the value of the influx minus the net uptake (or loss) would be less than the normal loss rate. In fact these values are not significantly different from the normal loss rate (146b, 0·7 and 1·0 μ M./hr. respectively, compared with the normal loss rate of 0·8 μ M./hr.). On these grounds it seems reasonable to suppose that the true rate of loss is practically constant over the range of external concentrations studied.

It must therefore follow that whereas at the lower range of external concentrations the influx is a reasonable measure of the true uptake rate, at the higher concentrations the influx is distinctly greater. In the case of crayfish no. 14 (Fig. 5) at an external sodium concentration of 0·77 mM./l. the sodium influx is 4·5μM./hr., whereas the uptake rate is only 3·5μM./hr. (i.e. the net uptake, 2·7μM./hr. + loss rate, 0·8μM./hr.)—some 22% lower. This difference can probably be explained in terms of the type of interchange between radioactive and non-radioactive ions which Ussing (1947) has called ‘exchange diffusion’. It is significant that the divergence between the influx and the uptake rate becomes apparent only when the transporting system approaches saturation. It is probably analogous to a leaky pump which works efficiently at low rates of delivery but develops a considerable leak-back when overworked.

An increase in the sodium outflux (measured by a tracer technique) with increasing external sodium concentration has been observed in the isolated frog skin by Ussing (1949) and Kirschner (1955). Kirschner attempted to explain this phenomenon on the grounds that the outwardly diffusing sodium ions interacted with a hypothetical sodium carrier system and that some of them were carried back again. If the rate of backward movement is proportional to the concentration of free carrier present, then it will be smaller at the higher external concentrations, and hence the outflux will be greater. This hypothesis is not applicable to the loss of sodium by the crayfish. If it was, then a reduction in the influx from low external concentrations should result in a considerable increase in the rate of loss of sodium and this is not found (see Table 3, 14b, f).

When sodium is lost from an animal by treatment in de-ionized water the consequent decrease in the internal sodium content has a pronounced effect on the sodium influx. Table 4 shows the influxes from 0·3 mM./l. NaCl measured in animals taken from the aquarium and also after they have lost sufficient internal sodium to reduce their external equilibrium concentrations to the minimum level. The influx was increased from three to five times.

These observations reveal little about the quantitative aspect of the effect of the internal sodium content. This was analysed by measuring the influx when known amounts of sodium had been introduced or removed from the animal. Artificial methods of changing the sodium content, such as by the injection of sodium chloride solutions, were avoided and changes were induced by physiological means. Thus, to increase the sodium content, the animal was allowed to absorb a measured amount of sodium from an external solution more concentrated than the equilibrium concentration to which the animal was adapted; and conversely, to decrease the sodium content, the animal was placed in de-ionized water until the required amount of sodium had leaked out. The method may be illustrated by reference to a typical experiment. An animal was first adapted to an external equilibrium concentration of, say, 0·3 mM./l. NaCl. The influx was then measured at a known external sodium concentration. The animal was then placed in a known volume of de-ionized water and allowed to lose a measured amount of sodium (calculated from the increase in sodium concentration of the external solution). The influx was now measured again at the same external sodium concentration as before. The animal was again placed in de-ionized water and a further amount of sodium removed. The influx was measured once more. The removal of sodium and the measurement of the new influx was repeated several more times. Between each new influx measurement 24 hr. was allowed to elapse to ensure that the new influx rate was fully established.

In an alternative form of the experiment the animal was first adapted to a very low equilibrium concentration. The influx was measured as before and then the animal was allowed actively to absorb a measured amount of sodium. The new influx was again recorded and then the procedure repeated several times more.

The results of these experiments are shown in Fig. 6. It can be seen that a small decrease in the internal sodium content causes a steep rise in the influx until a maximum is reached : after that further sodium loss has no effect on the influx. A loss of only 10μM. Na often affected the influx, whereas the loss of 100μM. or less was generally sufficient to change the influx from the normal to the maximum level.

We must now establish what these amounts represent in terms of the total sodium content of the animal, or, rather, the total sodium which is free to exchange rapidly with the external sodium. This can be calculated from the volume of fluid in the animal which contains this sodium (the ‘sodium space ‘) and its sodium concentration. The ‘sodium space ‘consists, of course, largely of blood, but the two volumes may not necessarily be identical, since some tissues may also contain freely exchangeable sodium. The ‘sodium space’ was calculated from tracer experiments. An animal was placed in a known volume of solution containing some ²⁴Na and some of this was absorbed into the blood. The radioactivity which was lost from the outer solution was measured, after correction for decay, and compared with the specific activity of a blood sample. From this the volume of fluid into which the radioactive ions must have been transferred and dispersed was calculated. The blood sodium concentration was also measured and it was assumed that the sodium concentration was uniform throughout the whole of the ‘sodium space’. From these two measurements the total freely exchangeable sodium content of the animal was calculated.

The results of measurements of this kind on several individuals are given in Table 5. The measurements of blood sodium concentration agree with those reported in the literature previously (see, for example, Bogucki, 1934). The sodium space (mean value = 37·2% of body weight) is probably not much larger than the true blood volume. No figures for this appear to be available for Astacus, but Prosser & Weinstein (1950) recorded the blood volume of the crayfish, Cambarus virilis as 25·1 % of the body weight. However, the body weights range from 10 to 48 g. and if only the smaller specimens are considered (16 g. or less) a much higher value of 34·5% is obtained. A value similar to this has also been found for the blood volume of Carcinus maenas (Webb, 1940) and for Eriocheir sinensis (Krogh,1939).

With a knowledge of the total internal sodium content, we may now refer back to Fig. 6, where this is also recorded. It is now possible to see that changes in the influx can be induced by sodium losses which represent only 1 % of the total sodium content. Further, the influx may be changed from the normal to the maximum value by the removal of only 5·10% of the internal sodium.

The next question that arises is whether the changes in internal sodium content correspond to changes in the internal sodium concentration (i.e. whether the ‘sodium space’ remains constant). This was tested by first measuring the blood sodium concentration of an animal with a normal influx and in balance with an external concentration of 0·3 mM./l. NaCl, and then measuring the concentration again after sodium had been removed from the animal and the maximum influx attained. These results are shown in Table 6. In all cases a measurement of the blood concentration at maximum influx was obtained before and after the measurement at normal influx. The blood concentration at normal influx is always higher than at maximum influx. Furthermore, the difference between the concentrations is of the same order (i.e. about 10%) as the percentage change in sodium content required to alter the influx from one extreme to the other. It is, therefore, almost certain that it is the internal sodium concentration which is the factor responsible for determining the influx.

The effect of the external sodium concentration on the influx is characterized by its immediate and reproducible action, but the responses to changes in the internal sodium concentration appear to be of a somewhat different nature.

In the first place, the removal of sodium from, or its addition to, the animal does not have a completely reversible and reproducible effect on the influx. Fig. 7 shows, in several individuals, the internal sodium changes inducing successive increases and decreases in the influx. It will be seen that there is not complete coincidence of the influxes at any given sodium level and that hysteresis is often in evidence. It is possible that it is not the absolute internal sodium concentration which determines the influx but the change from one concentration to another. The rate at which this occurs may be of some importance.

In the second place, the change from one level of influx to another, induced by a change in the internal sodium content, is rather slow. An estimate of the time for the adaptation to a new rate was made in the following way: an animal with the influx at the maximum level was placed in a solution of higher external concentration (about 0-6 mM./l. NaCl) containing some ²⁴Na. A continuous record of the fall of activity and the change in sodium concentration of the external solution was made. Net uptake of sodium occurred and this increased the internal sodium content. The experiment was continued over a period of 26 hr. and the time taken for the increase in the sodium content to affect the influx was noted. The results obtained for two individuals are shown in Fig. 8. Here the actual records are shown and the influx is indicated by the slope of the upper curves representing the decrease in radioactivity of the external solution. After the first 6 hr. little change in the influx had occurred, although a considerable amount of sodium had been absorbed. In crayfish no. 10, from 6 to 14 hr. a gradual change in the influx took place and at the end of this time the final new rate was established and the external concentration had fallen to the new equilibrium level. In crayfish no. 11, the influx remained unaltered for a longer period and then changed over to the new rate between 9 and 15 hr. after the start.

Since both the internal and external sodium concentrations affect the sodium influx, sodium balance must be achieved at an external concentration, where the combined effect of these two factors produces sodium influx which just equals the loss rate. Changes in the internal sodium concentration must, therefore, affect the external equilibrium concentration. Table 7 illustrates, quantitatively, the effects of the removal or addition of sodium on the external equilibrium concentration. As would now be expected, the losses or gains of sodium required to change the equilibrium concentration from a low level to a normal level (and, in the case of specimen no. 1, back again) are of the same order as those which produce a change in the influx from the normal to the maximum level.

In the analysis described above the behaviour of the crayfish in pure sodium chloride solutions only has been considered—the effects of other ions will be analysed later. With this limitation, it is possible to build up a picture of sodium balance in the crayfish, within its normal physiological range of sodium concentrations. In the system under consideration there are only three important variables ; (a) the sodium uptake rate, (b) the external sodium concentration, and (c) the internal sodium concentration. The rate of loss, for reasons given above, can probably be considered as a constant. The three variables may be related to each other in the form of a family of characteristic curves, such as is shown diagrammatically in Fig. 9. This, of course, is an oversimplification, since the precise nature of the curves has not yet been fully determined, but they serve for a qualitative demonstration of the balance conditions. The effect of a change in one of the variables can be followed. Let A represent the uptake rate (U) at a certain external sodium concentration (A) and let the animal be in sodium balance (i.e. A’ the loss rate, L). If the external concentration should now fall to B, then the uptake rate falls along the curve to B’. Now U is less than L, sodium is lost from the animal and the internal sodium concentration is decreased. The uptake rate is now increased to B’ on the curve which corresponds to the new internal sodium concentration and balance is re-established at the new external sodium concentration. The system is clearly a self-balancing one. Because a small change in the internal sodium concentration may bring about a large change in the uptake rate, a tenfold fall in the external sodium concentration may be compensated by a decrease in the internal sodium of less than 10%

There seems little reason to doubt that the effect of the external sodium concentration on the sodium transporting system is a direct one, exerting its action by increasing or decreasing the number of sodium ions which come in contact with the transport sites. The effect of the internal sodium concentration is more difficult to explain. The fact that its action may be delayed and is also somewhat unpredictable suggests that it may not affect the transporting system directly. It is possible that changes in the blood sodium concentration may produce an indirect effect. It might, for example, stimulate the liberation of a hormone, rather in the same way as, in the mammal, changes in the blood osmotic pressure provoke the liberation of the antidiuretic hormone which, in its turn, regulates the reabsorption of water by the kidney tubules (Vemey, 1947).

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