On the mechanism of sodium-proton exchange in crayfish

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Abstract

In salt depleted crayfish net sodium and proton fluxes were coupled 1:1 as required by the frog skin–turtle bladder model. In addition, three proton pump inhibitors produced equal reductions of both fluxes. It is concluded that the model operates in these animals. Net Na+ and H+ fluxes were very small in tap water adapted animals, but regression analysis clearly showed that they were coupled, though perhaps not 1:1. Proton pump inhibitors, at concentrations that suppressed ++++fluxes in salt-depleted crayfish, had no measureable effect on proton movement in tap water-adapted animals. Two of them (dicyclocarbodiimide and N-ethyl maleimide), caused a small reduction in Na+ influx without affecting proton efflux. These experiments provide no support for operation of the frog–turtle system in adult crayfish adapted to tap water. A 2Na+H+ exchanger is considered from an energetic point of view. Such a system might be able to couple Na+ and H+ fluxes in dilute, near neutral solutions ([Na+]∼1–2 mM; pH 7).

Introduction

Due largely to the work of two laboratories, we have a compelling model for sodium uptake and proton extrusion in a pair of epithelial organs — the freshwater turtle urinary bladder and the frog skin. The model involves a diffusive sodium channel and an electrogenic proton pump in the apical membranes of specialized cells in the epithelia, as well as the Na+K+ pump in the basal membrane. The ion flows across the apical membrane, both electrogenic, are coupled by the apical membrane potential producing an obligatory 1:1 exchange (Ehrenfeld et al., 1985, Steinmetz, 1986, Harvey, 1992).

Although the frog and the turtle are freshwater animals both are semi-terrestrial, a lifestyle that might have consequences for the operation of an apical ion exchange. Recently, attempts have been made to find evidence of this system in fully aquatic animals (fish), but the results have been ambiguous. A proton-ATPase was shown to be present in homogenates of rainbow trout gills (Lin and Randall, 1993). Antibody staining showed that the proton pump was located on the apical membrane (Lin et al., 1994). Probably the most convincing data have been obtained in very young fish. Bafilomycin, a specific inhibitor of the proton pump, inhibited sodium uptake in tilapia and carp (Fenwick et al., 1999) and also silver influx in trout (Bury and Wood, 1999). In the latter it was presumed that silver enters the cells through the sodium channel and that its entry is coupled to proton extrusion.

On the other hand, amiloride (0.1 mM), had little or no effect on proton efflux in rainbow trout (Lin and Randall, 1991); sodium uptake is completely abolished at this concentration. In addition, it has been shown that sodium–proton coupling was 1:1 in salt-depleted, intermolt crayfish and that amiloride abolished both fluxes as required by the current model (Kirschner et al., 1973, Ehrenfeld, 1974). However, such coupling could not be observed in tap water-adapted animals. There was net uptake of sodium but also net uptake of protons (or excretion of base), in untreated animals. Amiloride abolished the sodium flux, but it had a much smaller effect on proton movement (Ehrenfeld, 1974).

In this paper we pursue the question of transport mechanisms in crayfish. Much of the early work on Na+H+ exchange in freshwater animals was done on salt-depleted animals. Since such animals must have an input of metabolic energy above that provided by the Na+K+ pump on the basal membrane (discussed later), it is likely that they employ the system found in frog skin and turtle bladder. Ion fluxes in salt-depleted crayfish should, therefore, provide a useful basis for comparison with those in animals adapted to normal tap water.

Section snippets

Animals

Early experiments, including a few that employed dicycohexylcarbodiimide (DCCD) and N-ethylmaleimide (NEM), were carried out on intermolt Orconectes spp. obtained from a lake in Montana. All other work used intermolt Procambarus clarkii obtained from a commercial source (ATCHAFALAYA, Raceland, LA, USA). Control Na+ fluxes were larger in P. clarkii, but there were no differences between net Na+ and proton fluxes. O. spp. weighed between 20 and 30 g, P. clarkii between 30 and 50 g. The animals

Results

Net fluxes for Na+, TA, NH4+ and ΣH+ were measured in 58 TW-adapted crayfish; Jin(Na) in 55 of them. Net Cl movement was measured in 28 of these animals. The data in Table 1 show that there was a significant Na+ influx. Net efflux of Na+ was significant (P<0.05), but very small, while Jnet (ΣH) was not significantly different from zero. The animals were obviously in, or very near, sodium and acid–base balance under these experimental conditions. However, there was a substantial Cl efflux as

Discussion

Although it does not bear on the mechanism(s) of Na+ and proton movements, the imbalance among fluxes of Na+, H+ and Cl in tap water-adapted animals requires explanation. The apparent inward movement of positive charge, shown in Table 1, is approximately equal to the efflux of Cl and indicates that the latter was accompanied by the efflux of an unmeasured cation — a reasonable candidate is Ca2+. Two studies have shown that there is a net efflux of Ca2+ in TW-adapted crayfish amounting to as

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