Temperature-Dependence of Permeability to Water and To Sodium of the Gill Epithelium of the Eel Anguilla Anguilla

Teleosteans maintain a constant internal osmotic pressure of between 250 and 300 m-osmole. l⁻¹ whether the external medium is hypo-or hypertonic to the internal medium. As a result of the osmotic gradients, positive or negative, net water movements occur across the outer epithelia which are compensated by specialized osmoregulatory organs such as the kidney and the digestive tract.

Experiments on the eel have shown that it is the gill rather than the skin which is the principal water-exchange surface (Motais et al. 1969). Thus, by canalizing the afferent and efferent branchial vessels the clearance of tritiated water can be studied and it can be seen that virtually all of the diffusion flux passes through the gills.

Recent work has been concerned with the analysis of the water-permeability characteristics of the branchial epithelium. The osmotic permeability has been shown to be a function of the salinity of the adaptation medium, being lowest when the transepithelial osmotic gradient is highest (Motais et al. 1969). This adaptive mechanism is of obvious physiological significance. On the other hand, the diffusion permeability, measured with tritiated water, does not seem to be systematically modified by the intensity of the osmotic gradient (Potts & Evans, 1967; Evans, 1967–9; Motais et al. 1969).

A comparison of the coefficients of osmotic permeability (P_os) and of diffusion (P_dif) under various experimental conditions is of interest in throwing light on the mechanism of water transport across the gill. Results obtained by Motais et al. (1969) showed that in freshwater fish P_os is always higher than P_dif, the ratio varying from 2·5 to 6, while in marine fish it is around 1. From thermodynamic considerations these results may indicate that water crosses the gill of marine fish by simple diffusion and not by bulk flow. The very low osmotic permeability of the branchial epithelium of these fish would thus seem to be related to the absence of ‘water-filled’ pores. Similarly, when a euryhaline fish is transferred from fresh water to sea water the lowering of osmotic permeability observed during adaptation may be interpreted as the result of a structural modification of the epithelial membrane, the ‘water-filled channels’ disappearing and being replaced by molecular pores.

It should be pointed out, however, that the comparison of P_os and P_dif on which these hypotheses are based involves the determination of the degree of interaction between solute (salt) and solvent. In calculating the coefficients we had assumed that this interaction was negligible. In view of the intensity of the branchial ionic exchanges in marine fish (Mullins, 1950; Motais, 1961 – 7; Potts, 1968) this assumption may not be justified.

The aim of the present work is to evaluate the importance of a possible solutesolvent interaction in the branchial epithelium of the seawater-adapted eel. Sodium and water permeabilities were studied at different temperatures, since a comparison of the temperature coefficients yields information concerning the independence of salt and water movements. The water permeability of the gill of the freshwater-adapted eel was also studied.

The eels (Anguilla anguilla) used averaged 60 g in weight and came from the estuary of the Rhône. They were kept together at a constant temperature (± 0·5 °C) in aquaria with closed water circulations. They were considered adapted after a month at a given temperature and salinity (sea water or fresh water).

The experimental procedure and calculations have been described by Motáis et al. (1969).

To study the effects of rapid temperature changes on the sodium and water outfluxes the following procedure was adopted: 1 h after intraperitoneal injection of isotope (tritium or ²⁴Na) the appearance rate of the tracer was recorded during three successive short periods (15 min), the first and third of which were control measurements at the temperature of acclimation, and the second the test period at a higher or lower temperature. The temperature change was generally 10 °C. The volume of external medium was identical in the three periods. The method of outflux calculation has already been described (Motais, 1967).

It should be pointed out, however, that E can only be determined if one assumes that the surface, A, and Δx do not change when the temperature varies.

Fig. 1 illustrates results obtained with three seawater-adapted eels adapted to 5, 15 and 25 °C respectively. The left side of the figure gives the curves of the appearance of HTO in the external medium as a function of time, and the right side gives the mathematical analysis permitting the determination of K, which is proportional to the turnover rate, λ, of the body water. It can clearly be seen that the rate of water renewal is greater the higher the temperature. Similar results were obtained with freshwater-adapted eels. The unidirectional fluxes can be calculated from K, and also the waterdistribution space. From Table 1 it can be seen that the water distribution is on the average 70 ml (100 g)⁻¹ and does not vary with either temperature or salinity. This value, which is similar to that obtained by Thorson (1961) in various species of fish and by Lahlou & Sawyer (1969) in Carassius, was used in calculating the diffusion fluxes.

Table 1 summarizes the results obtained from seawater-adapted and freshwater-adapted eels acclimated at the three temperatures. At each temperature there was no significant difference between the renewal rates in fresh water and sea water, although differences had previously been recorded at 19 °C (Motais et al. 1969).

The Q₁₀ between 25 and 15 °C was 1·6 in sea water and 1·5 in fresh water : between 15 and 5 °C it was 2·5 and 2·1, respectively.

Table 2 summarizes the values net flux of water obtained indirectly from the differences between drinking rates and urinary flows, which are also given. The net flux was positive in the freshwater-adapted eel and negative in the seawater-adapted eel.

It is possible that the values of drinking rate are slightly too high since it is assumed that all the water entering the digestive tract is in fact absorbed. For comparison, the net-flux values which should theoretically be found if the water movements are purely diffusional (see Materials and Methods) are also given in Table 2. Calculation of these net fluxes takes into account the difference in water concentration between the internal and external media.

In the seawater-adapted eel the drinking rate falls rapidly with declining temperatures to become practically zero at 5 °C. Thus, at this temperature, in spite of an osmotic gradient of 850 m-osmoles. l⁻¹, it can be shown experimentally that the fish loses practically no water through the gills. Furthermore, the theoretical diffusion net fluxes at 15 and 5 °C are much higher than the measured osmotic net fluxes. This result is paradoxical in the sense that at any given osmotic gradient, the diffusion net flux is theoretically the minimum net flux (see Discussion).

The drinking rates of the freshwater-adapted eels recorded here are lower than those obtained by Maetz & Skadhauge (1968) using a technique with phenol red marked with ¹³¹I, while the urinary flows are similar to those published by Motais et al. (1969) if allowance is made for the temperature difference of the experiments. It should be noted that in fresh water the diffusion net flux is always lower than the osmotic net flux.

Table 3 gives the permeability coefficient values assuming a reflexion coefficient equal to 1. First, in the freshwater-adapted eel the ratio of the permeability coefficients is always considerably greater than 1. This ratio, P_os/P_dif, is 3·9 at 2·5 °C, becoming about 2 at a lower temperature. This agrees with the 3·16 at 20 °C found by Motais et al. (1969). Secondly, in the seawater-adapted eel the ratio P_os/P_dif is about 1 at 25 °C (Motais et al. 1969 had found 1·05 at 20 °C) but considerably lower than 1 at the lower temperatures.

Fig. 2 illustrates these effects. The experiment was carried out on seawater-adapted (2a) and freshwater-adapted (2b) eels acclimated at 5, 15 or 25 °C. It can be seen that when a fish is exposed to a temperature T₁ lower than its adaptation temperature the immediate water flux is lower than that of an animal adapted to this temperature T₁. Conversely, when it is exposed to a higher temperature, T₂, the immediate water flux is higher than that of an acclimated fish at T₂.

In seawater-adapted eels the ratio of the water flux at a given temperature to that measured immediately after transfer to a temperature 10 °C lower, which we have defined as the ‘temperature coefficient’, varies from 2 to 3·2 (Table 4) and the activation energies vary from 12 to 20 (Table 5).

Similar results were obtained from freshwater-adapted eels (Table 4), although at 5 °C the water fluxes were much more affected by temperature, the temperature coefficient being 5.

The distribution space and the rate of renewal of internal sodium were measured in seawater-adapted eels at 5, 15 and 25 °C. From these data the sodium fluxes (in m-equiv. h⁻¹(100 g)⁻¹) and the sodium permeability coefficient of the gill (in cm. sec⁻¹) were calculated (Table 6).

It can be seen that the fraction of internal sodium renewed per unit time, A, decreases with decreasing temperature in a similar way to the rate of internal water renewal (see Table 1). However, although the water distribution space was not changed at different acclimation temperatures, the sodium distribution space increased as the adaptation temperature diminished, probably as a result of inactivation of active transport in the tissues. The internal sodium concentration also increased. The resulting increase of the internal sodium pool partly compensates for the λ decrease, so that the sodium fluxes appear relatively little influenced by the temperature changes. Indeed, reported Q₁₀ values range from 1·2 to 1·6, as would be expected in the case of a diffusion process.

These are given in Table 4. The temperature coefficients are lower than those obtained for water, values ranging from 1·5 to 2, with activation energies of 6·5 to 11·8 kcal.M⁻¹ (Table 5).

Diffusional processes are not very temperature-sensitive. The tritiated water fluxes measured at 5, 15 and 25 °C would not be so very different were it not for modification of the permeability characteristics of the gill by acclimation. It has been pointed out that in both freshwater and seawater eels the water fluxes increase with the acclimation temperatures (Table 1, Fig. 1), these increases being much greater than would be expected for a simple diffusional process: thus, between 5 and 15 °C the Q₁₀ of acclimation are above 2 (2·1 and 2·5). Similarly, when the temperature is changed rapidly, there is an immediate variation in the water diffusional fluxes : a rise in temperature resulting in an increased flux and vice versa (Fig. 2). These variations are greater than would be expected for a diffusional process : the temperature coefficients are even higher than the Q₁₀ of acclimation, ranging from 2 to 5 (Table 4). For example, when a seawater-adapted eel is changed abruptly from 15 to 25 °C (Fig. 2) an initial overshoot is found (pattern 1); then partial acclimation occurs (pattern 2). When the eel, acclimated to 25 °C is transferred from 25 to 15 °C, an undershoot is observed (pattern 3), followed by an acclimation reaction (pattern 4).

One cannot conclude, however from these data that temperature modifies the effective permeability of the branchial epithelium. Thus, referring to the mathematical formula describing the unidirectional water flux,f_out = P_dif. A. (H₂O)_in, one can see that an increased flux may be caused not only by an increase of the effective permeability of the branchial barrier (P_dif), but also by an increase of the surface accessible to diffusion (A), or by a simultaneous variation of these two parameters. The plasma water concentration, (H₂O)_in, may be considered as constant. A change in the surfacearea of the gill, resulting probably from circulation shifts towards preferential circuits, must be considered a possibility. Thus Steen & Kruysse (1964) have presented evidence that the branchial microcirculation comprises two parallel pathways: one traverses the lamellae while the other provides a shunt through the filament core. The latter allows blood to go directly from afferent to efferent arterioles, by-passing the respiratory surface. At a high temperature, when the oxygen demand is high, a large volume of blood must pass through the lamellae and consequently diffusive transfer of water occurs. At a low-temperature oxygen consumption would be markedly lower, and the blood shunt through the by-pass in the filament core would minimize the diffusive transfer of water. Thus the flux variations recorded in steady-state eels may be the result of modifications of the exchange surface in relation to the adaptation temperature. According to Kirschner (1969) such modifications explain the fact that when he perfused eel gills at a constant perfusion rate the perfusion pressure was highest at low temperatures. As the lamellar vessels are opened by adrenaline, and the shunt by-pass increased by acetylcholine (Maetz & Rankin, 1968), it is possible that the rapid variations in water flux caused by sudden temperature changes may also be explained in terms of this dual-pathway model of branchial circulation.

It is thus impossible at present to affirm that the variations of the diffusional water fluxes brought about by temperature changes result from true permeability modifications or simply from variations of the exchange surface.

Assuming that no instantaneous variation of exchange surface occurs when measurements of temperature coefficients are being made during rapid temperature changes, the activation energies necessary for diffusion of water and sodium can be calculated.

The values obtained for water (Table 5) vary from 11·8 to 19·9 kcal.M⁻¹. The activation energy for the diffusion of tritiated water in water is 4·6 kcal.M⁻¹ (Wang, Robinson & Edelman, 1953). The resistance to water diffusion across the branchial epithelium is thus high.

The values for sodium (Table 5) are considerably lower, varying from 6’5 to 11·8 kcal.M^-1. The activation energy for the free diffusion of the sodium ion in water is known to be identical to that required for the diffusion of water in water (Longs-worth, 1955). If sodium crosses the branchial membrane as an aqueous solution, the activation energy for the solute would be at least equal to that measured for the solvent, and probably even higher, in view of the possible restriction incurred by the charge and dimensions of the ion (Samoilov, 1951). In point of fact, it has been found to be lower; in other words, sodium crosses the branchial epithelium more easily than water. This result furnishes evidence in favour of a certain degree of dissociation between the movements of water and sodium. This does not, however, imply that sodium does not cross the membrane as an aqueous solution, but simply that part of the water moving does so without solute and encounters a high-energy barrier.

A comparison of the rates of exchange of water and sodium in the gill of the seawater-adapted eel is of interest for assessing any possible solute-solvent interaction.

Unidirectional ionic fluxes across the gills of teleosteans have frequently been recorded as spectacularly high (Mullins, 1950; Motais, 1961, 1967; Maetz, 1971; Potts & Evans, 1967; Evans, 1967). Indeed, at 25 °C 52% of the exchangeable sodium mass of the eel is renewed per hour, and at 15 °C 32% (Table 6). Table 1 shows that body water is also renewed very rapidly (56% and 35% respectively), and as the water pool is much larger than the sodium pool, the water flux is three orders of magnitude faster than the sodium flux. For example, at 25 °C the sodium outflux is only 2 m-equiv. h⁻¹ while the water outflux is 2200 mm. h⁻¹.

At the gill surface, therefore, water transfers are much greater than sodium transfers, although this does not necessarily imply that the branchial epithelium is more permeable to water than to sodium. These rates of exchange are simply a result of the concentrations of water and sodium and of the surface for exchange accessible to the different types of molecule.

The seawater-adapted eel adapted to 25°C has a plasma concentration of 138m-equiv.l⁻¹ sodium and 55200mm.l⁻¹ water while the outfluxes are 2 m-equiv.h⁻¹ and 22000mm. h⁻¹ respectively. The concentration ratio of 400 cannot thus entirely account for the flux ratio of 1100. Considering first the hypothesis that the same surface is accessible to sodium and to water, the permeability to water would thus appear to be three times greater than to sodium. This ratio of the permeability coefficients of sodium and water is given in Tables 3 and 6,⁠.When the adaptation temperature diminishes the ratio falls, to become about 1 at 5 °C. This value indicates that at 5 °C the branchial epithelium becomes equally permeable to sodium and to water. The change of the ratio is brought about by a slight diminution of the permeability to sodium, such as would be expected with a diffusional process, and a much more important diminution of the permeability to water as indicated by the Q₁₀ and the temperature coefficients (Tables 1, 4). The occurrence of a high temperature coefficient indicates that the conductance of the membrane is low and that the molecules must possess a high activation energy to cross it. A comparative analysis of the Q₁₀ and temperature coefficients for sodium and water thus shows that the conductance of the epithelial barrier is higher for sodium than for water, which is not in accordance with the preceding conclusion that at 25 °C the branchial permeability to water is three times that to sodium. One is forced to conclude that not all the epithelial surface accessible to water is also accessible to sodium ; a hypothesis which has already been advanced above in paragraph 2.

One may consider, therefore, that certain exchange surfaces are not accessible to sodium. As the chloride cells are generally considered to be the sites of electrolyte exchange (Copeland, 1948 ; Mizuhira et al. 1969 ; Philpott, 1965 ; Shirai & Utida, 1970) they may well be responsible for most of the diffusive movements of sodium, while the diffusive movements of water would occur not only in the chloride cells but also in the respiratory epithelium which, being permeable to gases, is also permeable to water.

The components of the water balance of the eel have been defined by Smith (1932). As a result of the osmotic gradient between the internal and external media, water enters through the gills of freshwater-adapted fish (and is compensated for by the kidney), whereas it is lost to the external medium through the gill of the seawater-adapted fish (and compensation is effected by the intestinal absorption of sea water). Direct measurement of these branchial fluxes can be made in vivo, but it is also possible to calculate them indirectly by measuring the compensatory fluxes, i.e. renal excretion or intestinal absorption.

The importance of these osmotic net fluxes through the gill depends not only on the osmotic gradient but also on the structure of the membrane. If a lipoid membrane possesses ‘pores’ whose diameters are that of a water molecule, water can only transverse these pores by diffusion. In this case the osmotic net flux, a function of the difference of water concentrations on the two sides of the membrane, may be called the ‘diffusion net flux’. If, on the other hand, a lipoid membrane possesses ‘water-filled channels’, the osmotic pressure difference across the membrane will cause a bulk flow of water. As the resistance to the flow of water molecules is less in these conditions, given the same exchange surface and osmotic gradient, the net flux of water will be greater through the latter membrane than through the former.

Thus for a given membrane, the theoretical diffusion net flux, which can be calculated from the diffusion outflux, represents the minimum net flux, which is directly proportional to P_dif. The net flux actually measured across biological membranes (and which is directly proportional to P_os) is often greater than the theoretical diffusion net flux. In other words, the ratio P_os/P_dif is generally high and only exceptionally around 1 (Gutknecht, 1968).

In the present work the theoretical diffusion net flux was calculated under each experimental condition, and these values are given in Table 2 beside the measured net fluxes. In the freshwater-adapted eel it can be seen that at all temperatures the diffusion net flux is much lower than the measured osmotic net flux. The ratio P_os/P_dif varies from 2 to 4 (Table 3). This would suggest that the branchial membrane of the freshwater-adapted eel contains water-filled pores, a conclusion already reached by Motais et al. (1969).

The results obtained with seawater-adapted eels, however, indicate a curious phenomenon. Surprisingly, the diffusion net flux was found at 25 °C to be slightly higher than the measured net flux. It is even probable that this difference should really be greater, because all the water drunk by the fish is not necessarily absorbed (Oide & Utida, 1968; Hickman, 1968). Nevertheless, bearing in mind that the flux measurements are indirect and not very precise and that it is impossible to eliminate the possibility of interaction between water flow and ion flow, this difference can scarcely be considered significant. One should then assume that the osmotic net flux and the diffusion net flux are equal, i.e. that P_os/P_dif= 1·This would signify that water crosses the gill of the seawater-adapted eel solely by diffusion. Working at 19 °C, and from similar results, Motais et al. (1969) had already proposed that the low water permeability of the branchial epithelium of marine fish was related to the absence of water-filled pores.

The results obtained from experiments at lower temperatures, especially at 5 °C, however, do not support this hypothesis. At 5 °C the fish should theoretically lose 151 μl.h⁻¹ (100 g)⁻¹ of water, but experimentally they were found to lose virtually none, the compensation flux being extremely low (24 µl) and the P_os/P_dif ratio 0·17 (Table 3). Recently, Kirsch (1971) has also shown, with a completely different technique, that the drinking rate of a seawater-adapted eel at 15 °C is practically zero when the fish is kept completely undisturbed for a long period (several months).

These results show that the unstressed seawater-adapted eel, or one rendered unstressable by low temperature, is to all intents purposes in osmotic equilibrium with the external medium. The observed water loss is considerably lower than expected from the permeability characteristics of the gill to tritiated water and the osmotic pressure difference between sea water and blood. This can only be explained by inferring either that the osmotic pressure difference does not represent the true osmotic gradient across the membrane, or that a particular region of the gill exists for water reabsorption against the activity gradient, a reabsorption linked with solute movements.

Motais & Garcia-Romeu (1972) have recently suggested that the osmotic water losses at the respiratory epithelium are in part compensated by an absorption of water by the chloride cells. The functional model of the chloride cells proposed by these authors is based on the particular structure of these cells, the localization of ATPase and the observed rate of ionic exchanges in sea water. It illustrates the hypothesis that water movements across the chloride cell are driven by a system of recycling ions within the cell, and resembles the model proposed for the rectal pad of insects (Phillips, 1970). The model assumes a partial dissociation of salt and water movements across the respiratory epithelium-a hypothesis which is also supported by the results discussed above.