The relationship between transepithelial sodium movement and potential difference in the larva of Camptochironomus tentans (Fabr.) and some observations on the accumulation of other ions

There is an increasing body of information about potential differences across epithelia that are concerned with ion transport. The work has largely concerned freshwater vertebrates, and has yielded valuable information regarding the nature of the ionic pumps, notably the sodium pump.

On one hand, considerable use has been made of isolated epithelial preparations, such as the amphibian bladder (Brodsky & Schilb, 1966; Herrera, 1968; Solinger et al. 1968) and skin (Linderholm, 1951; Koefoed-Johnsen & Ussing, 1957; Aceves, Erlig & Edwards, 1968). On the other hand, the potential difference across the body wall of whole animals has been the subject of much recent interest (Brown, 1962; Maetz & Campanini, 1966; Dietz, Kirschner & Porter, 1967; Alvarado & Stiffler, 1970; Kirschner, 1970).

Investigations of transepithelial potentials (hereafter referred to as TEPs) in invertebrates can also be divided into studies of intact animals (Smith, 1969a, b; Bryan, 1960; kStobbart, 1974) and isolated epithelial preparations (Croghan, Curra & Lockwood, 1965). A comparison of Bryan ‘s (1960) data on Astacus, with that of Croghan et al (1965) shows that there may be considerable differences between TEP measurements in whole animals and isolated epithelial preparations.

The large haemolymph compartment in chironomid larvae and their vermiform shape afforded a very good opportunity to study TEPs in intact animals, especially in view of the localized and extreme posterior position of the ion transporting membrane. Investigations were confined to the large fourth instar larvae of Camptochironomus tentons and were prompted by various points emerging in a previous paper (Wright, 1975b)

Data concerning the movement of Na⁺, K⁺ and Cl⁻ during net ionic uptake into the haemolymph and the body as a whole extended existing information on body compartmentation of these ions (Wright, 1975 a), and attention was paid to this.

Collection and maintenance of experimental animals was as described earlier (Wright, 1975 a), as was the method of salt depletion. Study was confined to fairly large specimens of fourth instar C. tentans, generally 20–30 mg in weight.

Animals were mildly anaesthetized with ether vapour, and laid ventral side uppermost on a bed of beeswax resin (Krogh & Weis-Fogh, 1951). A little of the surrounding resin was melted with a warm needle to hold the larva. This quickly cooled and hardened without damage to the animal. Control tests showed that salt-depleted animals treated in this way were able to take up sodium normally, if the posterior end was covered by a drop of 2 mm-NaCl solution. A high resistance electrode was inserted into the thorax of the larva. The weakest point of the cuticle was at the prothoracic legs, and it was found that, if the electrode was applied at this point, the larva could be pulled onto it like a glove, using fine forceps. The low resistance electrode was held in contact with a small pool (0–3 ml) of external medium which bathed the posterior end of the animal, including the anal papillae. The size of this pool did not appear to affect the TEP over the period measured. The experimental set-up used, is represented semi-diagramatically in Fig. 1. The electrodes were of the mercury-calomel-saturated KCl agar type, having a tip potential of less the 5 mV. This was checked regularly. Leakage of KCl from the electrodes was small (< 1 mμ-mole/min), and the addition of unnaturally high amounts of KCl to a 2 mm-NaCl external medium did not appreciably alter the TEP. Measurements were made with a Vibron Electrometer model 33B-2, with a high input impedance.

Once the thorax of the animal was pierced, drying and shrinkage occurred after a few minutes. This limited time available for TEP measurement, and it was decided to record the potential difference after a period of 2 min. In some cases a degree of drift in potential was noted, which varied in magnitude and sign in different specimens. In measuring the TEP in such cases, it was decided that recording the potential after a set time was preferable to integration of the curve produced on the trace, particularly as the large individual variation necessitated the use of large numbers of animals. The sign convention adopted in describing TEPs is that the potential of the inner solution (haemolymph) is measured with respect to the outer solution (medium).

These determinations were made as described earlier (Wright, 1975 a).

Sulphate penetration was measured in animals with and without their mouths blocked by beeswax resin (Krogh & Weis-Fogh, 1951). A 1 mm-Na₂SO₄ solution labelled with ³⁵S was used. The ³⁵S was supplied as a H₂³⁵SO₄ solution by the Radiochemical Centre, Amersham. At the specific activity used, the excess H⁺ ions in the experimental solution were negligible. Groups of eight animals were labelled for 2 h. For counting of radioactivity, animals were macerated with 0·2 ml of distilled water, using fine grinding sand and a glass rod. This continued until, when viewed under a microscope, no recognizably intact tissue was seen. A further 0-3 ml of distilled water was added, before spreading, drying, and counting as described earlier (Wright, 1975a)

As a control 0·2 ml of a labelled Na₂³⁵SO₄ solution of known specific activity was used during maceration In this way, it was established that a self-shielding factor reduced the count by 15·8 ± 2·5% (N = 4). A suitable correction was applied.

Table 1 gives values for the TEP in steady-state and depleted C. tentans larvae, and illustrates the variability encountered throughout this investigation. Despite considerable individual variation, a highly significant (P < 0·001) increase in TEP was found on depletion, the haemolymph becoming increasingly electronegative with respect to the external medium. Table 2 compares these TEPs with those measured in other freshwater animals. In this comparison the external sodium concentration was standardized as far as possible. It may be seen that C. tentans is similar to Aëdes, Astacus and the teleosts, in having the body fluid electronegative to the medium at low external salt concentrations.

The time course for net sodium and chloride uptake from 2 mm-NaCl is followed in Fig. 2. The concomitant change in TEP is shown in Fig. 3. This experiment was repeated using 1 mm-Na₂SO₄ (Figs. 4 and 5) and 2 mm-KCl (Figs. 6 and 7). In all cases, animals used for measurement of TEP were the same as those used for measurement of body ions. A smaller number of animals were set aside from each experimental batch for measurement of haemolymph Na⁺ during net uptake from 2 mm-NaCl haemolymph K⁺ during net uptake from 2 mm-KCl, and haemolymph Cl⁻ during net uptake from 2 mm-NaCl and 2 mm-KCl (Fig. 8). Net NaCl uptake from 2 mm-NaCl was accompanied by a drift in TEP from approximately –36 mV to about 0 mV. This change in TEP coincided reasonably well with the time course of net sodium uptake. By contrast, during net sodium uptake from 1 mm-Na₂SO₄ no clearly defined drift in TEP could be seen, and despite some scatter of the data, the TEP after 60 h (– 23·45 mV) Bad altered little from the value of –29·77 mV at time 0 h. This stability of TEP was associated with a very much reduced net sodium uptake. Similarly, no significant alteration in TEP was seen during net KCl uptake from a 2 mm-KCl solution. TEP values at o h and 49 h were –34·74 mV and –29·13 mV respectively.

The inhibition of net sodium uptake from 1 mm-Na₂SO₄ (Fig. 4) emphasizes the strong inhibitory influence of SO₄²⁻ on sodium movement noted earlier (Wright, 1975b) and reveals a further similarity, in this respect, with E. sinensis (Koch, 1965; Wright, 1975 b). Linear regression analysis of this data does indicate a significant uptake (P < 0·05) but this is obviously very slight. Penetration of SO₄²⁻ into the larvae from a 1 mm-Na₂³⁵SO₄ solution was followed (Table 3). There is some ingestion of SO₄²⁻ into the gut. However, if gut-blocked animals are considered, the influx of SO₄²⁻ is clearly very small.

The effect of external sodium concentration on TEP was studied in depleted animals placed in NaCl and Na₂SO₄, and in steady-state animals placed in NaCl (Fig. 9). The results obtained from depleted larvae are very similar to those obtained from salt-depleted specimens of the amphibians Ambystoma tigrinum (Dietz et al. 1967) and Ranapipiens (Kirschner, 1970). In both steady-state and depleted C. tentans the TEP remains stable at external NaCl concentrations below 1 mm, although that of depleted animals is displaced 30–40 mV negative in comparison with steady-state larvae. From 1 to 5 mm-NaCl in the external medium, there is a significant correlation (P < 0·01) between TEP and external NaCl concentration in steady-state animals. In depleted animals, the tendency towards a less negative TEP above an external sodium concentration of 1 mm is curtailed at higher NaCl concentrations. However, when SO₄²⁻ replaces Cl⁻, the TEP continues the positive trend, reaching about + 40 mV at higher external Na₂SO₄ concentrations.

Smith (1969b) shows how an asymptotic ionic flux-concentration curve may be obtained without necessarily postulating the influence of a carrier (Wright, 1975b). This situation results from passive movement of an ion, caused by TEP changes following alteration in concentration of the external medium, and therefore applies to an ion distributed, more or less, according to its electrochemical equilibrium. He) concedes, however, that where large concentration differences occur between haemolymph and external medium, such a curve would arise from a carrier mediated relationship, and so could be described by the Michaelis-Menten equation. Although, regarding sodium, the latter condition clearly exists in C. tentans, it was considered of interest to compare the actual flux-concentration curve observed in this species (taken from Wright, 1975 b) with that expected to result from passive movement caused by TEP changes resulting from alterations in concentration of the external medium. The object of this was to provide a quantitative estimate of the degree of disparity between the two curves.

An experiment was designed to investigate further the inhibitory role of lithium on the sodium pump, as indicated in an earlier paper (Wright, 1975b). Net sodium uptake from a solution containing 2 mm-NaCl +10 mm-LiCl was followed, along with concomitant TEP changes (Figs. 11 and 12). A Li⁺: Na⁺ ratio of 5 is known to be sufficient to inhibit sodium uptake almost completely. Control animals were placed in 2 mm-NaCl only. As mentioned earlier, the same animals were used for both total body sodium and TEP measurement. Control animals gave similar results to those described in Fig. 3. It is interesting to note, however, that while no net sodium uptake was possible from the NaCl/LiCl mixture, the TEP changes in these animals were similar to the controls (Fig. 12). A third batch of animals placed in 2 mm-LiCl displayed similar TEP changes (Fig. 12), although whole body lithium content was not followed.

Work on isolated epithelia and whole body preparations has led to the suggestion of a number of possible systems for sodium transport in freshwater animals. A consideration of certain features is useful in placing the data presented here within the framework of existing models.

Models for isolated epithelia are, essentially, modifications of Koefoed-Johnsen & Ussing ‘s (1957) frog skin model, and involve at least one cationic pump on the inner and outer borders of the epithelium. A saturable electrogenic sodium pump has been postulated at the outer, apical surface of such complex vertebrate epithelia (Biber & Curran, 1970), for which lithium may also compete (Frazier, 1964; Biber & Curran, 1970) and cause a TEP to be developed (Herrera, Egea & Herrera, 1971). Brodsky & Schilb (1966), working on turtle bladder membrane, note that in sodium-free mucosal solutions the serosal (blood) side becomes electronegative to the mucosa. When a sodium-rich mucosal solution is used, the serosal side becomes less electropositive to the mucosal side. Increasing serosal electropositivity with increase in external sodium is also noted in the frog skin (Brown, 1962). Net sodium transport across the turtle bladder membrane into the blood is associated with increased electropositivity of the serosal side followed by a period when this side becomes less electropositive. This may be considered in the light of work by Solinger et al. (1968), who showed that inhibition of the sodium pump in the turtle bladder by ouabain was accompanied by a reversal of TEP. Both effects may be explained in terms of electrogenic cation pump superimposed upon an electrogenic anion pump. The two pumps are apparently independent.

There are, then, two features widely found in freshwater vertebrate epithelia which could contribute to a model for C. tentans: an electrogenic sodium pump secreting sodium into the blood, and a high degree of independence of anion and cation transport. It seems that an electrogenic sodium pump causing transepithelial separation of charge in C. tentans is the best explanation of data from Figs. 2 – 7. When net sodium movement is restricted, so too is a change in TEP (Figs. 4 and 5). TEP also remains stable in the absence of sodium in the external medium. Data from Figs. 2 – 7 compare fairly closely with similar data for Aëdes aegypti (Stobbart, 1974). Probably the most significant difference concerns TEP changes in 1 mm-Na₂SO₄, where a significant shift in TEP in Aëdes was associated with net sodium uptake from this solution. The stability of TEP in depleted C. tentans placed in 1 mm-Na₂SO₄ (Fig. 5) probably reflects the greater inhibition of sodium movement by SO₄^{2 −} in this species, and is consistent with the conclusions drawn here.

Despite some possible mutual enhancement (Wright, 1975 b) it seems that the sodium and chloride pumps are capable of working quite independently of each other. Probably the best evidence of this comes from a consideration of net chloride uptake from NaCl and KCl (Figs. 2 and 6). Unlike the turtle bladder, carriage of charge by chloride does not appear to be implicated in C. tentans at these external chloride concentrations. It is possible that stabilization of TEP during net uptake from KCl could be due to a ‘balance’ of charges carried out by K⁺ and Cl ⁻. However, such a model would presuppose a separate electrogenic potassium pump, electrically equal to the chloride pump, and electroneutral chloride movement seems a more likely explanation. Dietz et al. (1967) found that a wide variation in external chloride concentration, in the absence of sodium, made no difference to the TEP in Ambystoma tigrinum. They concluded that chloride is transported in a non-electrogenic manner over a wide range of external concentrations. In both C. tentans and A. tigrinum it seems likely that the mechanism concerned is a closely coupled anion/anion exchange.

It seems, then, for depleted C. tentans placed is solutions containing the major ions (Na⁺, Cl⁻ and K⁺) at concentrations of 2 mm the TEP data can be explained in terms of a sodium pump woiking electrogenically, with chloride (and possibly potassium) being transported by an electroneutral pathway. The term ‘electrogenic’ is used here to describe a mechanism causing a very localized separation of charge, and is not intended to imply a strict quantitative link between TEP and ionic movement. The possibilities of electrogenic Cl⁻ movement, or indeed electroneutral Na⁺ movement at external concentrations other than 2 mm, have not been eliminated and are discussed later.

Data from Figs. 2, 6 and 8, although limited, suggest that the total amount of chloride taken up from NaCl was greater than that taken up from KCl, despite similar initial net uptake rates for chloride. It is interesting to note that such a phenomenon has been described in Rana esculenta (Krogh, 1937), Aëdes aegypti (Stobbart, 1967) and Ambystoma gracile (Alvarado & Dietz, 1970). The latter authors suggest that the Na : Cl ratio may be of some importance in ionic control, as suggested by Shaw (1960) for Astacus. Although it is possible that TEP changes during net NaCl uptake could create a more favourable gradient for chloride accumulation than in KCl, it is unlikely that this effect would be very great, due to the largely electroneutral and independent nature of the chloride pump. No significant overshoot in TEP during net NaCl uptake is seen in C. tentans, as was noted in the turtle bladder membrane (Brodsky & Schilb, 1966).

Stobbart (1967) noted that, in A. aegypti, increased chloride uptake was more effective in enhancing sodium uptake than vice versa. The effect was manifest as an increase in sodium influx, and a decrease in outflux. It has already been noted that the chloride uptake rate is similar in both KCl and NaCl. In an earlier paper (Wright, 1975b) it was implied that chloride does stimulate sodium influx to some extent. As net sodium uptake from 1 mm-Na₂SO₄ is very small (Fig. 4), the sodium efflux at this concentration may be taken as more or less equal to influx (Wright, 19756, Fig. 3). Comparing these values with similar data for sodium fluxes and net uptake in NaCl (Wright, 1975b), it appears that compared with sulphate, chloride promotes a very much increased sodium influx, although, unlike the situation in A. aegypti, efflux is also slightly increased. As was noted earlier (Wright, 1975b), it is always difficult to decide on a baseline for assessing ionic interactions. For example, it is impossible to say where inhibition of sodium movement by sulphate ‘ends’, and enhancement by chloride ‘begins’. Certainly the lack of sulphate penetration would create an unfavourable electrical gradient for inward sodium movement. It must be remenbered, however, that this may have little effect on active sodium movement and, bearing this in mind, it is interesting to consider the data relating TEP to the external sodium concentration.

In depleted animals there is a non-linear change in TEP with increasing external sodium, the haemolymph becoming less electronegative (Fig. 9). The stability of the TEP in C. tentans below 1 mm-NaCl may be due to a low sodium influx, perhaps coupled with a non-electrogenic mode of operation of the pump, such as a closely linked cation/cation exchange. In this respect C. tentans is similar to Austropotamobius pallipes, where stability of TEP was noted until the sodium concentration of the external medium rose above 2 mm (Croghan et al. 1965). In C. tentans above an external sodium concentration of 1 mm the TEP becomes concentration dependent. The most likely explanation for this appears to be a change in mode of operation of the pump involving the introduction of the electrogenic component, which is apparent in the net uptake experiments (Figs. 2 – 7). It is possible that an increase in sodium permeability may also be a contributory factor. At higher external NaCl concentrations, the rate of change of TEP in C. tentans falls off and diverges from the line followed in Na₂SO₄. In the amphibians Rana pipiens (Kirschner, 1970) and Ambystoma tigrinum (Dietz et al. 1967) there is a similar effect, and in fact the blood becomes less electropositive at higher NaCl concentrations. This effect has also been noted in isolated frog skin (Linderholm, 1951). Kirschner (1970) attributes this effect to an increase in permeability to chloride at high NaCl concentrations. As in C. tentans there is a continued tendency towards a more positive TEP at higher external NaCl concentrations (Fig. 9), it appears that despite a possible chloride influence at higher NaCl concentrations, transport of charge by sodium still remains ‘ahead’ of chloride. It may be noted that the levelling off of the TEP at higher NaCl concentrations starts at concentrations where the electrochemical gradient is still opposed to chloride accumulation. This could be explained by a chloride pump operating in an electro-genic mode at such concentrations. However, further work would be needed to elucidate this.

Table 3 indicates that C. tentans is almost completely impermeable to sulphate. This is similar to the situation in A. tigrinum (Dietz et al. 1967). As TEPs at very low concentrations of both NaCl and Na₂SO₄ are similar, it would appear that the effective chloride permeability at these low concentrations is also very low. It seems reasonable to assume, for depleted animals, that the logarithmic part of the graph relating external sodium (as Na₂SO₄) to TEP is due to electrogenic sodium movement. Although separation of charge appears to be unimpeded, the low influx and net sodium uptake rates from Na₂SO₄ suggest that SO₄^{2 −} strongly inhibits the release of Na⁺ from the inner side of the membrane.

Although in both steady-state and depleted animals the TEP below 1 mm-NaCl remains stable, in depleted larvae the TEP is displaced by about – 35 mV. This displacement of TEP is opposite in sign to that obtained by Dietz et al. (1967) for A. tigrinum. In C. tentans this probably represents a sodium diffusion potential, the mobility of chloride being reduced more than that of sodium during the depletion process. Between 1 mm and 5 mm-NaCl there is a significant correlation between TEP and external sodium (P < 0 · 01), which may represent electrogenic sodium movement at these concentrations. Above 5 mm-NaCl, the TEP again goes negative, and, at very high external NaCl concentrations coincides with the TEP for depleted animals. This, again, may be due to an alteration in the relative permeabilities of sodium and chloride.

As C. tentans has been reported from salinities of these high values (Palmen & Aho, 1966), clearly more work on sodium and chloride movements at such external concentrations would be valuable. Consideration of flux-concentration data in the light of observed TEPs (Fig. 10) indicates that the mechanism for sodium accumulation in C. tentans is clearly very different from Artemia (Smith, 1969a, b), although again a study of this relationship in C. tentans exposed to dilutions of seawater would provide a more valid comparison, and would seem to be a useful area of expansion of this investigation.

It appears from Figs. 11 and 12 that lithium competes with sodium for the same electrogenic mechanism. Once again, this compares with larval A. tigrinum (Dietz et al. 1967) where ‘… of the alkali and alkaline earth metals tested, only sodium and lithium generated a concentration dependent TEP (transepithelial potential)’.

Overall, then, the TEP data presented here, for C. tentans is broadly similar to that collected for whole animal preparations of freshwater amphibians (Dietz et al. 1967; Alvarado & Dietz, 1970; Alvarado & Stiffler, 1970), the larva of A. aegypti (Stobbart, 1974) and the isolated crayfish gill (Croghan et al. 1965).

A more detailed model of TEP components in C. tentans is difficult without access to the inner side of the membrane. However, if the transporting membrane proves to be a syncytium as in Aëdes larvae (Sohal & Copeland, 1964), the mechanism may be rather more simple than in complex vertebrate epithelia.

Reference to whole body and haemolymph sodium, potassium and chloride levels shown in Figs. 2, 6 and 8 indicates that these ions are considerably reduced in depleted animals. Sodium, potassium and chloride loss represent approximately 40, 32 and 60% of the body totals respectively. In both steady-state and depleted larvae it appears that approximately 60 % of the body chloride is contained in the haemolymph. As haemolymph and whole body chloride are restored proportionally, there is obviously free exchange of haemolymph and tissue chloride. Haemolymph potassium in depleted animals was not significantly different from steady-state animals (Wright, 1975 a). This is probably indicative of the small haemolymph potassium compartment (apparently only 10% of total body potassium) in these animals. During net uptake from 2 mm-KCl, total body potassium was slowly restored over a period of 50 h or more. However, during net uptake, a rise in haemolymph potassium was noted within a period of 5 h and this was apparently sustained even after restoration of total body potassium. It is possible that this replaced some of the haemolymph sodium.