ABSTRACT
Sodium transport processes in the branchial epithelium of euryhaline crustaceans have been investigated using a perfused preparation of gills isolated from Chinese crabs Eriocheir sinensis acclimated to dilute (FW) and to concentrated (SW) media.
The results clearly establish the existence of a functional difference between the different pairs of branchiae with respect to their participation in the regulation of the blood Na+ content.
In FW-acclimated animals, the Na+ active uptake which counterbalances the salt loss along the concentration gradient is mostly achieved across the three posterior pairs of gills. Conversely, the Na+ fluxes measured in the three anterior pairs are essentially passive and carrier-mediated.
Further characterization of the Na+ uptake system present in the posterior gills by means of inhibitors like ouabain and ethacrynic acid indicates the existence of at least two spatially separated components of the Na+ carrying system.
It is shown that NH4+ may be used as co-ion for Na+ but that such a coupling can only account for a very small part of the Na+ actively transported inward. The existence of an electrogenic mechanism or of another coupled system has thus to be postulated but remains at present a matter of speculation.
To study FW-to-SW and SW-to-FW acclimation, Na+ fluxes were measured in isolated gills of SW-acclimated crabs and of FW crabs perfused and incubated in SW conditions.
During acclimation to SW the Na+ active uptake in the posterior gills is abolished primarily as a result of inhibition of the Na+carrier activity.
INTRODUCTION
It is well established that the salt loss by euryhaline crustaceans when in diluted media is counterbalanced by an active absorption of Na+ and Cl− occurring essentially through the gill tissue (for review, see for instance Krogh, 1939; Potts & Parry, 1964; Gilles, 1975, 1979; Kirschner, 1979; Evans, 1980). Nevertheless, the information about the Na+ movements effectively taking place at that level is very scanty, most experiments having indeed been carried out on whole animals.
In an attempt to provide more complete information about the part played by the gills in the ionic anisosmotic regulation of crustacean body fluids, we recently perfected a perfused preparation of isolated gills from the euryhaline Chinese crab Eriocheir sinensis. Preliminary results obtained with this preparation (Péqueux & Gilles, 1978 a, b) show important differences in extracellular space, tissue and intracellular ionic contents as well as in transepithelial potential of the so-called ‘anterior’ and ‘posterior’ gills. The data indicate that the ‘posterior’ gills may show physiological specialization in iono-regulation. The purpose of the present work has been to test this hypothesis and to characterize the Na+ movements in both types of gills.
MATERIAL AND METHODS
Animals
Experiments were performed on gills isolated from Chinese crabs Eriocheir sinensis acclimated in the laboratory either to fresh water or to sea water of the following Na+, K+and Cl− concentrations (m-equiv/1). FW: 0·63 Na+, 0·07 K+, 1·07 Cl−; SW: 474 Na+, 11 K+, 318 Cl−.
Gills were cut off at their base and prepared for perfusion according to a method already described (Péqueux & Gilles, 1978 a). The so-called ‘anterior’ and ‘posterior’ gills correspond respectively to the three most anterior and the three most posterior located pairs of large gills in the branchial chamber.
Salines
Gills isolated from FW-acclimated crabs were handled in and perfused by a ‘perfusion FW saline’ corresponding to the blood composition of FW-acclimated animals. This saline contained 240 mM NaCl, 5 mM KC1, 5 mM MgCl2 and 12·5 mM CaCl2. The pH was adjusted to 7·6, borate buffer (9 mm). Gills from SW crabs were perfused by a ‘perfusion SW saline’ which was twice as concentrated. The pH was kept at 7’6 with borate buffer (9 mM).
The perfused gills from FW crabs were incubated at 22 °C in an’ incubation saline’ obtained by diluting the perfusion FW saline 250 times while keeping constant its pH and buffer concentration. According to the experimental scheme, NaCl was added to this medium up to the required concentration. As far as SW crabs are concerned, incubations were run either in a saline identical to the perfusion SW saline, or in the ‘incubation saline’ described above.
Transepithelial potential
Potential differences across the gill epithelium were measured by means of a Keithley electrometer and two Calomel electrodes respectively connected to the incubation and to the perfusion salines with agar-KCl bridges.
Na+, Na+ flux measurements
Na+ inward and outward movements were estimated by adding 22Na+ (0·25 μ Ci/ml) to the incubation saline or perfusion saline and by measuring its appearance on the other side in 0·25 or 1 ml samples; samples were collected each 15 min for 60·90 min Sample radioactivity was measured with a γ scintillator. At the end of the incubation period, the gills were blotted on filter paper and weighed. Flux measurements were expressed as μequiv Na+/g wet wt.h.
Simultaneous influx-efflux experiments were performed according to the doubletracer method using 22Na+ and 24Na+ and following the above described scheme.
NH4+ measurements
NH4+ concentrations and effluxes were determined by colorimetry according to Nessler’s reaction, using the ready-to-use Sigma ammonia Color reagent (Sigma 14-2). Fluxes were expressed in μ equiv NH4+/g wet wt.h.
RESULTS
A. Na+fluxes in isolated gills from FW-acclimated crabs
Fig. 1 gives Na+ influx and efflux data obtained with anterior gills and measured as a function of the Na+ concentration in the incubation saline. The fluxes are of the same order of magnitude at each external Na+ level tested except for concentrations lower than 25 m-equiv/1. At these low concentrations the efflux becomes significantly larger than the influx.
The situation is quite different in the posterior gills. In this tissue, the Na+ efflux remains undetectable, whatever the Na+ concentration in the incubation saline (range from 0·96 to 250 m-equiv/1). In contrast, increasing external Na+ concentration leads to an increase in Na+ influx. As in the case of the anterior gills, this process shows saturation kinetics with a maximum influx value reached for an outside Na+ content of less than 50 m-equiv/1 (Fig. 2). The results allow calculation of an apparent Km for the Na+carrier of 13·7 mm, with a maximum influx fmax of 331 γequiv/g wet wt.h (fig. 2).
B. Nature of the Na+ fluxes
(1) Comparison of measured and calculated flux ratio
From the results described above, it is clear that the influx recorded in the posterior gills of FW-acclimated crabs must be of an active nature; no detectable efflux can be shown in these gills, whatever the amplitude of the Na+ gradient applied across the epithelium.
(2) Effect of inhibitors of the Na+ active transport
The effects of ouabain 10−3 M on the transepithelial influx of Na+ have been tested on both the anterior and posterior gills from FW-acclimated crabs. The cardiac glycoside remains without any significant effect on the anterior gills. Fig. 3 shows that ouabain severely lowers the Na+ influx of the posterior gills when added to the incubation saline only. Addition of the drug to the perfusion saline has no significant effect.
Ethacrynic acid 10−3 M has also been used in an attempt to identify other components of the active Na+ influx in the posterior gills which could be insensitive to ouabain. Fig. 4 shows that ethacrynate is only effective when added to the perfusion medium.
C. Further characterization of the Na+ influx in posterior gills
1. Effect of the ionic concentration of the perfusion saline
Changing the FW perfusion saline for an SW perfusion saline leads to an important decrease in the Na+influx recorded at the level of the posterior gills from FW-acclimated crabs. This is true whatever the Na+ concentration of the incubation saline (Table 2). In such conditions, both the apparent Km and particularly, fmax(the maximum influx) are considerably decreased (Km 5·6 mM, fmax 26·7 μequiv Na+/ g wet wt. h) when compared with the data obtained upon perfusion with an FW saline containing 240 m-equiv Na+/1 (refer to Fig. 2). It is worth noticing that, for an outside Na± level of 500 m-equiv/1, the influx becomes undetectable. In this situation, similar to that of the gills of animals living in sea water, we have also been unable to detect any significant efflux (see Table 3). There is thus no significant Na+ movement at all in these gills. This is very similar to the situation found in the posterior gills of SW-acclimated crabs, where no significant fluxes can be recorded (see part D below).
(2) Na+ influx and ammonia efflux
In other communications (Péqueux & Gilles, 1978,b; Gilles, 1978), we briefly reported on a dependency of the ammonia efflux in posterior gills on the Na+ level in the incubation saline, as well as on the fact that NH4+ can be a very good substitute for K+ in stimulating the activity of the Na+/K+ ATPase extracted from these fills. Such results are in agreement with the idea of a coupling between Na+ and HH4+ movements. This possibility is explored further in this section.
Fig. 5 confirms that the ammonia efflux in posterior gills depends on the Na+ level in the incubation saline. However, there is a considerable discrepancy between the magnitudes of Na+ influx and NH4+ efflux at any external Na+ level we have studied. Even at an external Na+ concentration giving maximum NH4+ efflux, the recorded Na+ influx remains some 10 times larger. On the other hand, addition of NH4C1 up to 10-3 M to the perfusion saline fails to induce any significant modification of the Na+ influx (Fig. 6). In the same way, addition of large amounts of proline, considered as a potent NH4+ donor in the gills after deamination, remains without significant effect on the Na+ influx (Fig. 6).
In another approach to a possible Na+/NH4+ coupling, the effects of ouabain and ethacrynic acid on the ammonia efflux have been studied. As shown in Fig. 7, ouabain, shown to lower the Na+ influx when added to the incubation medium (section B, 2), induces a decrease in the efflux of ammonia when added on that side of the epithelium. Conversely, ethacrynate when added to the perfusion saline fails to induce any significant effect on the NH4+ efflux while, as we have previously seen, it severely decreases the Na+ influx in the same conditions.
D. Na+ fluxes in isolated gills from SW-acclimated crabs
The Na+ fluxes in gills of SW crabs incubated and perfused with salines having Na+ levels similar to those found respectively in acclimation medium and blood are reported in Table 3. These results show that both influx and efflux are very large in the anterior gills. It is worth noticing that relatively high values can also be recorded in anterior gills from FW-acclimated crabs in similar conditions (Table 3).
In posterior gills, on the contrary, no significant Na+ efflux can be detected. As far as the Na+ influx is concerned, the limited specific activity of the incubation saline we could prepare and the limited volume of the sampling allowed by the perfusion technique result in a low accuracy of measurement and thus in large standard deviations. The obtained flux values can hardly be considered as statistically significant, however.
It is moreover interesting to notice here that the Na+ influx measured in posterior gills of SW-acclimated crabs is not significant-not only at the high external Na+ level corresponding to a normal SW medium (Table 3) but also at all the other much lower outside levels of Na+ we have tested (0·96, 25 and 100 m-equiv/1). Similarly, the Na+ influx in these gills remains not significant when the SW perfusion saline is replaced by an FW one (Na+: 480 m-equiv/1 to Na+: 240 m-equiv/1-Table 3).
DISCUSSION
Koch et al. (1954) suggested that the anterior and posterior gills of the euryhaline crab Eriocheir sinensis may differ in their ability to transport ions. Further arguments corroborating this view have been presented in only a few other studies (King & Schoffeniels, 1969; Schoffeniels & Gillies, 1970; Péqueux & Gilles, 1978,a). The results of this paper clearly establish the existence of a functional difference between these pairs of branchiae with respect to their participation in the blood osmoregulation that this species is able to achieve when in diluted media. It must be remembered that the Chinese crab Eriocheir sinensis is a typical hyperosmoregulator, which can maintain a pronounced blood hyperosmotic state when in diluted medium, and whose blood remains isosmotic to the external medium when the animal is acclimated to sea water (Berger, 1931; Conklin & Krogh, 1938; Krogh, 1939; Schoffeniels & Gilles, 1970). In the hyperosmoregulators, active Na+; uptake at the gill level appears to be an essential mechanism in counterbalancing the salt loss in diluted media. Our results show that in E. sinensis the active Na+ influx is mostly achieved by the three posterior pairs of gills. Considerations based on the comparison of the measured ratio of fluxes with those calculated according to the Ussing’s equation for passive ion movements (Ussing, 1949), lead indeed to the conclusion that the fluxes of Na+ in the anterior gills are essentially passive. The study of the Na+ fluxes as a function of the external Na+ level in these gills further reveals saturation kinetics indicating that the movements of Na+, though passive, are carrier-mediated and not the result of a simple diffusional process. It is also worth noticing that the Na+efflux decreases as the external Na+ concentration decreases, in spite of the fact that the Na+ gradient across the epithelium is increasing: this can be explained by considering that the permeability of the anterior gills decreases at low external Na+levels. Such a decrease in permeability has also been considered in fish gills (Maetz, 1971; Kirschner, 1979) and is of interest, since it should decrease the salt loss occurring in the animals acclimating to low salinities.
As far as the posterior gills are concerned we failed to demonstrate any significant Na+ efflux whatever the Na+ gradient applied across the epithelium. This shows that the Na+ permeability of these branchiae is extremely low. Consequently, the large Na+ influx which can be measured must be of an active nature. On the other hand, the magnitude of the Na+ influx is dependent on the external concentration of Na+, revealing saturation kinetics which allows calculation of an apparent for the transepithelial Na+ carrying system(s) of 13·7 m-equiv/1. This value is rather close to the one (20 mM) reported by Shaw (1961) when studying Na+ uptake in whole specimens of another marine euryhaline decapod Carcinus maenas, but is high when compared to those available for freshwater crustaceans (0·2 mMAstacus fluviatilis, Shaw, 1959a; 0·1 mM, Potamon niloticus, Shaw, 1959b; 0·15 mM, Gammarus pulex, Shaw & Sutcliffe, 1961). It must however be considered that the fmax which can be calculated for these marine euryhaline decapods is higher than the one which can be measured in freshwater species (331 μequiv/g wet wt.h E. sinensis, this study; 10mM/kg.h, C. maenas, Shaw, 1961; 0·15 mM/kg.h Astacus fluviatilis, Shaw, 1959a). It may thus be that the rather low affinity of the carrier for Na+ found in the marine euryhaline crustaceans is, at least partly, compensated by a high number of pumping sites.
The study of the effects of ouabain and ethacrynic acid, inhibitors of two different components of Na+ transport in cell membranes, allows some further characterization of the Na+ uptake system present in the posterior gills. It is interesting to consider that the effect of these agents is dependent on the side of the epithelium on which it is applied. Ouabain is effective only when added outside, in the incubation saline, while ethacrynate appears to be active only from the internal side of the epithelium. These results can be considered as indicating the existence of, at least, a two-component Na+ carrying system. One of these (the ouabain-sensitive one) would be restricted to the outside-facing membrane and would be linked to the activity of an ATPase sensitive to this cardiac glycoside. Inhibition by ouabain is classically considered to be indicative of an Na+/K+ coupled carrier system. Such a coupling is however unlikely in our preparation, for uptake of Na+ in such conditions would implicate loss of K+ to the environmental medium. Evidence has been provided in recent years showing that NH4+ is a very good substitute for K+ in stimulating the ouabain-sensitive ATPase in crustacean gill tissues (Towle, 1974; Mangum et al. 1976; Péqueux & Gilles, 1977; Gilles, 1978). The effect of ouabain on the Na+ influx in our preparation could thus be considered as an effect on an ATPase-mediated Na+/NH4+ coupled transport located on the outward-facing membranes of the epithelium. The fact that ouabain also inhibits the efflux of ammonia in the posterior gills is in agreement with this idea. On the other hand, the obtained results do not exclude the possibility of the existence of other Na+ transport mechanisms on the external membrane. This point will be considered later on in the discussion. It is interesting to consider here that ouabain does not induce any significant inhibition of the transepithelial Na+ uptake when added to the perfusion saline, while ethacrynic acid is only effective when added to that saline. This might indicate that there is no Na+/K+ transport system on the inside-facing membrane of the epithelium. In this view, the expulsion of Na+ from the cells would be achieved through another, ethacrynate-sensitive, Na+ transport system. This is however rather unlikely in the context of the regulation of the intracellular level of both K+ and Na+, which must involve control of the movements of these ions between blood and the intracellular fluid. Another possibility is that the transepithelial Na+ uptake is achieved through pathways different from those implicated in the expulsion of Na+ normally diffusing from blood to cells following its concentration gradient. In this view, a Na+/K+ coupled, ouabain-sensitive transport could be related to the expulsion from the cells of the Na+ diffusing from the blood, while another pumping mechanism, ethacrynate-sensitive, would be involved in driving the Na+ implicated in the transepithelial movement. In such a system, one could consider that the Na+ implicated in the transepithelial movement is largely moving through a tissue pool different from the cellular one controlled by the Na+/K+ coupled transport. It is more likely that the contribution of the Na+/K+ coupled, ouabain-sensitive transport to the total efflux from the cells is relatively small when compared to that of the transepithelial transport system. Both hypotheses may nevertheless account for the fact that ouabain has no significant effect on the Na+ influx when added to the inside medium. Experiments are in progress in this laboratory to bring more light on this problem.
Let us now come back to the uptake system located at the external side of the epithelium. As already discussed, the fact that the transepithelial flux of Na+ decreases when ouabain is added to the incubation medium led us to consider the possibility of an Na+/NH4+ coupled transport. The existence of such a process has already been postulated on the basis that it would account for the increase in ammonia output observed in euryhaline crustaceans upon acclimation to diluted media (Shaw, 1960; Gilles, 1975, 1978; Mangum et al. 1976). The fact that, in our preparation, the ammonia efflux depended on the external level of Na+ is in agreement with the idea of such a coupling. Various results, however, prompt us to minimize the importance of this mechanism. As a matter of fact, though both Na+ influx and NH4+ efflux depended on the external Na+ level, the nature of this dependency is quite different for each of the ionic species considered (see Fig. 5). Furthermore, the Na+ influx is much larger than the ammonia efflux. This might be explained on the basis of an Na+:NH4+ stoichiometry that is not 1:1. The important difference between the fluxes, however, makes this hypothesis unlikely, and it is more reasonable to assume that a large part of the active Na+ uptake is not coupled to NH4+ excretion. This conclusion is further substantiated by results showing that increasing the concentration of NH4C1 in the perfusion saline fails to induce any significant modification of the Na+ influx. Therefore, one has to consider that there is an Na+ pump on the external side of the epithelium, either working according to an electrogenic scheme or requiring a counter-ion different from NH4+. In this view, an Na+/H+ coupled system, as postulated by different authors (Ehrenfeld, 1974; Evans, 1980), would appear as a good candidate. Actually, both possibilities remain essentially a matter of speculation and need further investigation. The main conclusions up to now M summarized in Fig. 8.
Let us now consider the fluxes recorded in gills of SW-acclimated E. sinensis and of FW-acclimated ones perfused with salines, the Na+ level of which is similar to the one found in the blood of SW animals. These results may lead to a better understanding of the processes involved in the FW-to-SW or SW-to-FW acclimation.
As shown in Table 3, the fluxes of Na+ reach a very high level in the anterior gills of SW-acclimated animals while, as we have seen previously, they are much lower in the FW-acclimated crabs and dependent on the external concentration of Na+, a decrease in external Na+ resulting in a decrease in both influx and efflux. It thus appears that the passive, carrier-mediated fluxes of Na+ in the anterior gills result in high Na+ turnover when the animal is in SW and much lower exchange of Na+ when in FW.
On the other hand, increasing the Na+ concentration in the perfusion saline of posterior gills from FW animals to a level similar to that found in the blood of SW animals leads to an immediate decrease in the Na+ influx, whatever the concentration of Na+ in the incubation medium. As shown in Tables 2 and 3, the influx falls to undetectable values even when the concentration of Na+ in the incubation saline reaches a level similar to that of SW. In such conditions of high Na+ in both perfusion and incubation salines, the fluxes of Na+ in the posterior gills of SW-acclimated crabs are also barely significant. Acclimation to SW appears thus to result in an important decrease, even in the virtually complete abolition of the active Na+ uptake in the posterior gills; such an effect seems to be mainly related to the increase in Na+ blood level. From the important and instantaneous drop in fmax observed upon perfusion of posterior gills from FW crabs with an SW perfusion saline it can be reasonably concluded that the decrease in Na+ uptake activity might be mainly due to a rapid decrease in the number of active carrier sites. This decrease being extremely fast, it can be considered as resulting primarily from an inhibition of the Na+ carrier(s) activity rather than from a decrease in the synthesis of carrier(s) molecules. Whether or not such a repression of carrier(s) molecules synthesis may occur in long-term acclimation to sea water remains to be demonstrated. Experiments are in progress in this laboratory in order to bring more insight to these questions.
ACKNOWLEDGEMENTS
This work has been supported by grants ‘Crédit aux Chercheurs’ from the FNRS to the authors and by a grant no. 2.4511.76 from the FRFC to R. Gilles.