1. A technique is described for recording the electrical potential differences across the epithelium (epithelial potential) of isolated podobranch gills of Austropotamobius pallipes continuously perfused with Ringer solution in various external media.

  2. In a medium of 0·01 Ringer, in which the animals had previously been kept, the mean epithelial potential ± standard deviation was —60 ±12 mV. (Sign defines potential of body fluid with respect to external medium.) Chloride, sodium and potassium must be actively transported into the body fluid against an electrochemical gradient. Calcium and magnesium ions appear to be approximately in equilibrium.

  3. The steady-state membrane potentials were recorded in various external concentrations of Ringer solution. The potential is about zero with Ringer solution outside and rises to a maximum with 0·01 Ringer outside.

  4. Changes of the electrical potential were recorded when the concentration of a single electrogenic ion was changed in the external medium (0·01 Ringer), and were used to define an apparent transport number of the ion in the outer cell membrane.

  5. There was no correlation between the transport numbers and the epithelial potential.

  6. There was a continuous gradation of gill types from a predominantly cation-permeable type towards a more chloride permeable type. There is a correlation between the type of gill and the position in the gill series.

  7. The properties of the epithelial cells of Austropotamobius gill are significantly different from those of the epithelial cells of frog skin. It is suggested that in Austropotamobius a chloride pump is situated in the outer cell membrane.

There has been a considerable amount of work on the electrical potential differences and active transport processes of ions across various vertebrate epithelia (Ussing, 1960). Little work of this type has been attempted on invertebrate material. As the crustacean gill epithelium has only a single layer of cells separating the body fluid and the external medium, it should be particularly suitable for such studies. Crustacea living in hypo-osmotic media take up NaCl from the medium and it has been assumed that this uptake is across the gill epithelium. In the case of Eriocheir it has been shown that isolated gills can take up NaCl rapidly from a dilute NaCl solution (Koch, 1954). There have been measurements of the electrical potential difference between the haemolymph and dilute outer medium in whole Austropotamobius. The values range from —4·1 to — 6·6 mV. in tap water (Bryan, 1960) and — 5 to — 47 mV. in a medium of 0·3 mM/1. KC1 (Shaw, 1960). The sign of the potential is that of the haemolymph with respect to the medium. These results indicate that both chloride and sodium must be actively transported into the haemolymph against an electrochemical potential difference. In this paper measurements of the electrical potential difference across the epithelium of isolated perfused gills of Austropotamobius pallipes are reported.

The total electrical potential difference across the epithelium between the outer medium and the inner medium (body fluid) measured using concentrated KC1 solution bridges will be termed epithelial potential (EE). Using the sign convention adopted here it defines the electrical potential of the body fluid with respect to the outer medium. The epithelial potential is the sum of the membrane potentials of the outer and inner epithelial cell membranes.

If an ion j is in passive equilibrium between two phases (e.g. body fluid and outer medium), the electrical potential difference between the two phases (Ej) is given by the Nernst equation
where and are the concentrations (strictly activities) of ion j in the two phases. The extent to which the calculated Nernst potential for ion j differs from the actual epithelial potential indicates the extent to which ion j is out of equilibrium. A fuller discussion of this is given by Dainty (1962).
An actual membrane potential is usually regarded as a diffusion potential and can be related to ionic concentration differences and selective permeability properties of the membrane. A fuller discussion of this is also given by Dainty (1962). Information concerning these permeability properties can be obtained if the change of membrane potential following a change to a new medium on one side of the membrane is determined. One approach that has been used is to apply the Goldman constant field equation, but this equation cannot be applied in this study of this gill epithelium where there are two cell membranes in series as there is no information concerning the ionic concentrations in the epithelial cell fluid. A different approach is to consider the transport numbers of the ions in the cell membranes. The approach given below is an extension of that given by Hodgkin and Horowicz (1959). An expression for the membrane potential for an epithelium can be written
where Tj is the transport number of ion j in the outer (O) and inner (Z) cell membrane and Ej the Nernst potential of the ion across the outer (O) and inner (Z) cell membrane. If the concentration of a single permeable (electrogenic) ion i is varied in the outer medium and the ‘instantaneous’ potential change is determined (i.e. before changes in the ionic concentrations in the cell fluid have occurred), then
If the transport numbers are constants or if all the ions to which the membrane is significantly permeable are in equilibrium across the cell membrane , then the equation simplifies to
Further if finite changes are considered

Strictly this integration is only approximate as the transport number of an ion in a membrane will be a function of the concentration of the ion in the membrane. Also the ions are unlikely to be in equilibrium across the outer cell membrane of an epithelium across which ions are being transported. In view of this, the transport numbers defined by equation (5) should really be termed apparent transport numbers. They should give valuable information concerning the relative ease with which an ion can cross passively the outer cell membrane.

The permeability coefficient and transport number of an ion in a membrane are of course related. The relationship is considered by Hodgkin & Horowicz (1959).

Specimens of the British crayfish Austropotamobius pallipes were kept in a medium of crayfish Ringer solution diluted to 0·01 Ringer and to which was added a little solid CaCO3. The animals were kept in this medium in large aerated tanks at 9−10° C. and survived excellently. Intermoult animals only were used. In fact the relatively low temperature virtually suppressed moulting. Only the podobranch gills from the 2nd maxilliped to 4th pereiopod inclusive were used in these experiments. Whole gills were cut off between the basal plate and the insertion on the appendage and placed in crayfish Ringer solution until required.

In the experiments reported here the gill was continuously perfused with Ringer solution (van Harreveld, 1936) containing 1 mg. Evans Blue/100 ml. The apparatus is represented diagrammatically in Fig. 1. The Ringer solution was contained in an all-glass syringe driven by a Palmer slow injection apparatus. The cannulae were made by drawing out polythene cannula tubing. The afferent cannula with a tip diameter of ca. 0·2 mm. was inserted into the afferent vessel and the efferent cannula with a tip diameter of ca. 0·5 mm. was inserted into the large efferent vessel. The cannulae were held in place by a fine nylon ligature just below the basal plate. The gill was perfused at ca. 0·4 μl./sec. The dye was added to check that the gill filaments were properly perfused.

Fig. 1.

Diagram of apparatus for determining the epithelial potential of a perfused gill in various external media.

Fig. 1.

Diagram of apparatus for determining the epithelial potential of a perfused gill in various external media.

The medium was held in a small funnel (ca. 5·5 ml. capacity) connected by a multiway tap to 5 1. polythene reservoirs containing aerated solutions. The gill was lowered into the medium until the filaments were immersed. During the experiments the medium was flowing continuously at ca. 1 ml./sec. The multiway tap enabled the solution to be changed rapidly.

The electrodes were standard Pye Hg-calomel-sat. KC1 electrodes (type 11161).

One electrode was inserted in the afferent line. The reference electrode was attached by a rubber collar to a 2 mm. diameter glass tube drawn out to a fine rounded off tip containing sat. KC1 agar, and was placed in the medium. When not in use the electrode tips were kept immersed in sat. KC1 solution. The electrodes were connected to the input of a Pye Master pH meter-millivoltmeter and the output of this was connected via a 100 Ω potentiometer to a Kipp BD1 Micrograph pen recorder. Both sides of the input were isolated from earth. After each experiment the gill was removed and the zero was taken by immersing the afferent cannula and reference electrode in Ringer solution. With some reference electrode tips there was instability and shift of potential when the flow was turned on. This was particularly noticeable with the most dilute external solutions. Care was taken to select an electrode tip in which this effect was minimal. The experiments were carried out at room temperature (ca. 17° C.).

In all the experiments the electrical potential was measured with 0·01 Ringer, the medium in which the animals had previously been kept, flowing past the gill. The results are summarized in Fig. 2. The mean epithelial potential and standard deviation for all the gills in this medium is —60+12 mV. (40 gills from 13 animals).

Fig. 2.

Epithelial potential (mean ± s.D. ± s.E.) compared with position of gill in gill series. Outer medium 0·01 Ringer solution. Sign of potential is that of body fluid with respect to medium. Podobranch insertion : 2 M, 3 M, maxillipeds ; 1P (chela), 2 P, 3 P, 4 P, pereiopods.

Fig. 2.

Epithelial potential (mean ± s.D. ± s.E.) compared with position of gill in gill series. Outer medium 0·01 Ringer solution. Sign of potential is that of body fluid with respect to medium. Podobranch insertion : 2 M, 3 M, maxillipeds ; 1P (chela), 2 P, 3 P, 4 P, pereiopods.

In one group of experiments (13 gills from 4 animals) the solution flowing past the gill was changed as follows: Ringer (R)-o·1R-0·01R-0·001R-0·01R-0·1R-R. The potential was taken when it had reached a steady value (< 30 sec.) and the mean value was taken when there were two measurements. The results are summarized in Fig. 3.

Fig. 3.

Epithelial potential (mean ± s.D. ± s.E.) in various external concentrations of Ringer solution. Sign of potential is that of body fluid with respect to medium.

Fig. 3.

Epithelial potential (mean ± s.D. ± s.E.) in various external concentrations of Ringer solution. Sign of potential is that of body fluid with respect to medium.

In the other group of experiments (27 gills from 9 animals) the concentration of individual physiologically important ions were varied in turn using benzene-sulphonate or choline as assumed non-permeating and therefore non-electrogenic counter ions. The solutions used are given in Table 1. The concentrations of univalent ions were varied by × 5 and that of calcium by × 10. The potential was recorded with solution A (0·01 R) flowing until a steady value was obtained, and then the solution was changed to one of the experimental solutions. The solutions were changed as follows :
Table 1.

Composition of some experimental solutions

Composition of some experimental solutions
Composition of some experimental solutions

Some examples of records are given in Fig. 4.

Fig. 4.

Examples of recordings of epithelial potentials. Letters refer to the solutions flowing (see Table 1). Upper record is a 3rd maxilliped podobranch and lower record is a and pereiopod podobranch.

Fig. 4.

Examples of recordings of epithelial potentials. Letters refer to the solutions flowing (see Table 1). Upper record is a 3rd maxilliped podobranch and lower record is a and pereiopod podobranch.

The ‘instantaneous’ change of potential was taken as the change in 10 sec. after the recorder pen had begun to move. The results expressed as apparent transport numbers are summarized in Figs. 5, 6 and 7.

Fig. 5.

Apparent transport numbers of ions in relation to the epithelial potential immediately preceding the experimental determination.

Fig. 5.

Apparent transport numbers of ions in relation to the epithelial potential immediately preceding the experimental determination.

Fig. 6.

Relation between apparent transport numbers of the other ions and the apparent transport number of chloride.

Fig. 6.

Relation between apparent transport numbers of the other ions and the apparent transport number of chloride.

Fig. 7.

Relation between the apparent transport numbers (mean ± s.E.) of sodium and chloride and position of gill in gill series. Sodium, closed circles; chloride open circles. Podobranch insertion: 1M, 3M, maxillipeds; 1P, aP, 3P, 4P, pereiopods.

Fig. 7.

Relation between the apparent transport numbers (mean ± s.E.) of sodium and chloride and position of gill in gill series. Sodium, closed circles; chloride open circles. Podobranch insertion: 1M, 3M, maxillipeds; 1P, aP, 3P, 4P, pereiopods.

It is considered that the main advantage of using a preparation perfused with Ringer solution is that the ionic concentrations are known precisely and experimental variations due to individual variation in haemolymph composition are avoided. As the Ringer solution is very similar to the haemolymph composition (Lockwood, 1961) the conclusions drawn from these experiments are considered relevant for the normal gill.

The results summarized in Fig. 2 show that in all cases the body fluid is considerably negative with respect to the medium. The potential differences are considerably larger than those of Bryan (1960) for whole animals, suggesting possibly that short circuiting had occurred in his experiments. There appears to be no significant difference between the mean values of the epithelial potential of gills from different positions in the gill series. Although significant differences were frequently found between individual gills taken from a single animal, it should be remembered that in an intact animal the potential difference across all the gills will tend to be clamped at a similar value by the presence of common conducting media on each side of the epithelium.

The effect of the gill epithelium on the potential differences between Ringer solution and 0·01 R is even clearer when it is realized that the diffusion potential between these two solutions without any membrane is about + 26 mV.

It is simple to compare the Nernst potentials of the individual ions with the epithelial potential of Austropotamobius gills as the concentration of each ion differs by ·100 as between the two solutions. The results are :

Clearly, the electrochemical potential of chloride in the body fluid is very much greater than that in the external medium. The electrochemical potentials of sodium and potassium are also considerably above those in the medium. All these ions must therefore be actively transported from the medium into the body fluid across the gill epithelium. The divalent ions appear to be more or less in electrochemical equilibrium and thus there is no evidence to imply active transport. No conclusion will be drawn in the case of bicarbonate as there is no information concerning the bicarbonate concentration in the haemolymph which may be considerably different from that in the Ringer solution.

A similar approach to that described above has been applied by House (1963) in considering active transport across the gill epithelium of the brackish-water teleost Blennius pholis. But there are several papers purporting to decide the extent to which the ionic concentrations in the body fluid differ from equilibrium with the medium in which no attempt was made to measure the epithelial potential (Robertson, 1957, 1960; Webb, 1940). Conclusions arrived at in this way are fallacious.

The steady values of potential difference summarized in Fig. 3 show that the potential varies considerably in different external concentrations of Ringer solution. The potential difference was approximately zero with Ringer solution on both sides of the epithelium. This would be expected if the epithelium were a single membrane, and perhaps it suggests that under these conditions the composition of the cell fluid is similar to that of the Ringer solution. In more dilute external solutions the epithelial potential appears to rise to a maximum in 0·01 R. Although this maximum is not statistically significant when the mean data are considered, a maximum was in fact shown by a significant number of the individual gills (8 out of 13 gills) from which the data in Fig. 3 were derived. There was apparently no correlation between the type of response and the position of the gill in the gill series. This suggests that in external media more concentrated than 0·01 R, the epithelium (considered as a single membrane) is behaving as a selectively cation-permeable membrane, and that in media more dilute than about 0·01 R some change occurs in the permeability properties. The epithelial potential is maximal in the medium to which the animal had previously been adapted, and at this point the potential is relatively insensitive to changes in external concentration. The significance of this is uncertain.

The situation in the crayfish gill described above contrasts sharply with that found in amphibian skin. In frog skin the inside solution is normally positive with respect to the external solution (Steinbach, 1933; Ussing, 1960). Considered as a single membrane frog skin appears to behave as a selectively chloride-permeable membrane. Normally in frog skin the uptake of chloride appears to be a purely passive movement down an electrochemical potential gradient. However, in certain cases, particularly when the external solution is 3 mM./l. KC1 solution, there is also evidence of an active uptake of chloride across anuran skin (Jorgensen, Levi & Zerahn, 1954). Another case where there is an active transport of chloride across an external epithelium is that of the gill epithelium of the brackish-water teleost Blennius pholis (House, 1963).

The results of the experiments in which the outer concentration of an (assumed) single permeable ion was changed are difficult to interpret. Due to the complex filamentous and lamellar structure of the gills it is probably impossible to obtain a very rapid change of the medium at the outer surface of the epithelial cells even with the fast flow rates used. Furthermore, as the epithelial cells are thin, varying from ca. 1 μ up to as much as 30μ around nucleus (paraflin sections after fixation in 1 % OsO4 in Ringer solution), rapid changes in the cell fluid composition could occur The ‘instantaneous’ potential change was in fact taken as the change in 10 sec. after the pen had begun to move. In a number of cases the potential had reached, or nearly reached, a steady value in this time (an exception to this being solution E(Ca) where the potential usually changed more gradually). Thus even if the steady potential values in the various media had been used in place of the 10 sec. values the appearance of Fig. 5 would be little different.

In fact no clear picture emerges from Fig. 5. There is no correlation between the membrane potential with 0·01R outside and the transport number of any ion in the outer cell membrane. This is rather surprising. It must be remembered that our epithelial potential is the sum of the outer and inner cell membrane potentials, and the results may suggest that the outer cell membrane potential has a much lower value than the inner cell membrane potential, which would then act as the major determinant of the overall epithelial potential and effectively randomize the relation plotted in Fig. 5. Another point to be considered in the interpretation of Fig. 5 is that the gill filaments are divided into two populations with different histochemical properties (Curra & Croghan, in preparation) and the electrical measurements were made on these two populations. A point worth making is that the lower potentials are not associated with damaged leaking gills as the transport numbers would be much lower if short-circuiting had occurred.

The transport numbers indicate that the gills are mainly cation-permeable (especially to Na), which is in keeping with the results obtained with various dilutions of Ringer solution outside the gills. It should be stressed that these transport numbers apply to an outer medium of 0·01 R. As a transport number is a function of the concentration of the ion in the membrane, the values must be considered in relation to the absolute values of the concentration of the ion on either side of the membrane and may change considerably in different external media.

If we consider the data of Fig. 5 in terms of individual gills correlations do become apparent. Fig. 6 indicates a marked inverse correlation particularly of the transport numbers of sodium and calcium with the chloride transport number. An inverse correlation would be expected as it is a property of transport numbers that The mean value and standard deviation of the sum of the transport numbers of the ions considered in Fig. 6 is 0·97 ± 0·2. There appears to be a continuous gradation of gill properties from a mainly cation-permeable towards a more chloride-permeable gill type. Also Fig. 7 indicates that the transport numbers of the two major ions, sodium and chloride, are correlated with the position of the gill in the gill series. The anterior gills contrast sharply with the posterior gills. No regular correlation was found with calcium. The significance of the difference of properties between the anterior and posterior podobranchs is uncertain. It can be mentioned, however, that Koch, Evans & Schicks (1954) found that the three posterior gills of Eriocheir appear to take up sodium more rapidly than the anterior gills.

These results may be compared with those found in other material. Hodgkin & Horowicz (1959) were able to determine the transport numbers of potassium and chloride in the cell membrane of single muscle fibres. However, this study was simplified because these ions are in electrochemical equilibrium (at least approximately) in muscle cells (EK = Ec1) and these are the only ions to which the membrane is appreciably permeable. In these circumstances equation (4) holds exactly. Further, because of the simple geometry of their preparation, Hodgkin & Horowicz (1959, 1960) were able to get a virtually instantaneous change to a new medium. In experiments where the internal composition could vary there were drifts of membrane potential over long periods. Results of this precision, however, can scarcely be expected in such a complex organ as the crayfish gill. Experiments involving changes in the composition of the bathing solutions have also been carried out on frog skin (Koefoed-Johnsen & Ussing, 1958). Here, however, only the steady values of the potentials after equilibration in various media are given and the possible effects of variations of the concentration in the cell fluids are ignored, in spite of the fact that variations of the volume of the epithelial cells were observed in some cases. Furthermore these experiments were made under conditions where chloride conductance had been artificially reduced. Nevertheless, their conclusions concerning the relative permeability of the outer and inner membranes to sodium and potassium have been confirmed by MacRobbie & Ussing (1961) using more critical methods.

It is tempting to try to construct a model for the epithelial cell of the gills of Austro-potamobius similar to that developed in the above-mentioned papers for the epithelial cell of frog skin, and in particular to decide in which membranes the various pumps are situated. Such an attempt is really premature. But in general it can be considered logical that a specific ion pump will only be found in a membrane relatively impermeable to that ion. Thus considering sodium and chloride, the two major ions in the medium (0·01R), the transport number of chloride in the outer cell membrane is in the majority of gills (particularly the posterior gills) significantly less than the transport number of sodium (Figs. 6, 7). Thus tentatively the chloride pump could be placed in the outer cell membrane and, by analogy with frog skin, the sodium pump in the inner cell membrane.

However, these and related problems need to be investigated more critically using other techniques. Valuable information could be obtained by studying the effects of changes of the internal medium, which is technically possible with the perfused gill preparation. Even more valuable would be to obtain information on the chemical composition of the epithelial cell fluid and to split the overall epithelial potential into separate outer and inner membrane potentials by introducing a micro-electrode tip into the epithelial cell.

We wish to thank Prof. J. Dainty for many helpful discussions. We also wish to thank the Medical Research Council for a grant to one of us (P. C. C.) for equipment.

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A recent paper by Biehwski, J. (1964), ‘Chloride transport and water intake into isolated gills of crayfish’ (Comp. Biochem. Physiol. 13, 423), has demonstrated directly a net transport of chloride from dilute outer medium into the haemolymph in isolated crayfish gills.