The Acid–Base Balance of the Outer Mantle Epithelium of Anodonta Cygnea

The outer mantle epithelium (OME) of Anodonta cygnea produces a spontaneous electrical potential in vitro (Istin and Kirschner, 1968). Under short-circuit conditions it generates a current (I_sc) which, when expressed as a molecular flux, has a magnitude the same as that of the simultaneously measured [¹⁴C]bicarbonate net flux from shell to haemolymph side (Coimbra et al. 1988). Both the net flux of bicarbonate and I_sc are abolished by 4-acetamido-4′ -isothiocyamatostilbene-2,2 ′ - disulphonic acid (SITS) and 4,4-diisothiocyanostilbene 2,2-disulphonic acid (DIDS). The current is blocked by iodoacetamide, partially blocked by diamox and is very sensitive to exogenous CO₂. To prevent the accumulation of protons (acid) within the cell (with potentially harmful effects), the net flux of bicarbonate must be accompanied by a net flux of protons to either the haemolymph or shell side. If protons are transported to the shell side, then the net flow of bicarbonate across the basolateral membrane, the net flow of protons across the apical membrane and I_sc should have the same magnitude, when expressed in the same units. The results presented here support this hypothesis. Within experimental error the three quantities are similar under a variety of conditions.

Specimens of Anodonta cygnea were collected from the Lagoon of Mira in northern Portugal and kept in the laboratory in aerated dechlorinated water for up to 2 weeks. The OME was dissected out, mounted in an Ussing-type chamber and continuously short-circuited as previously described (Coimbra et al. 1988).

The usual solution bathing both sides of the preparation (control solution) had the following composition (in mmol l⁻¹): Na⁺, 11; K⁺, 7; Cl⁻, 19; Mg²⁺, 0.5; Ca²⁺, 1; bicarbonate, 2. Both half-chambers were filled with 2 ml of this solution and gassed with a humidified mixture of CO₂ (5 % ) and oxygen (95 % ). In some experiments the haemolymph side of the preparation was bathed with control solution while ionic replacements were performed on the solution bathing the shell side (shell solution). When chloride was replaced by gluconate, isethionate, thiocyanate or sulphate, the osmolality of the solution was maintained by the addition of sucrose. When the effect of pH on the short-circuit current (I_sc) was studied, the shell solution contained 1 mmol 1⁻¹ NaH₂PO₄ instead of 2 mmol 1⁻¹ bicarbonate. Since most of the experiments were performed in the presence of exogenous CO₂ the pH-stat method was not used (Sanders et al. 1973). The amount of acid or base delivered into the baths over a period of 20–30 min was measured by titrating a known amount of fluid from each half-chamber, previously equilibrated with humidified nitrogen to remove all the dissolved CO₂. The titrant (HCl) was delivered from a precision syringe driven by a micrometer until all the bicarbonate had been removed from the solution. Each time a small amount of titrant was delivered there was an initial fall in pH followed by a slower rise (Fig. 1). This pattern results from the slowness of the hydration of CO₂ and of the dehydration of H₂CO₃. To avoid an overshoot, the amount of titrant delivered each time was progressively reduced. The titration was stopped when there was no rise in pH after the addition of acid (Fig. 1, second arrow). 50 samples of the bathing solution containing 2 mmol l⁻¹ bicarbonate yielded a value of 2.03±0.002mmol l⁻¹ (mean±s.e.). When inhibitors were used the standards included the same amount of inhibitor as the samples. The inhibitors used were applied at the following final concentrations: amiloride (3,5-diamino-6-chloropyrazinoylguanidine), 1 mmol 1⁻¹; diamox, 1 mmol 1⁻¹; vanadate 1 mmol l⁻¹; dinitrophenol (DNP), 1 mmol 1⁻¹; DIDS, 0.5 mmol 1⁻¹; oligomycin 200μg ml⁻¹; dicyclohexylcarbodiimide (DCCD), 50μg ml⁻¹.

To compare the rate of alkalinization of the haemolymph compartment, or rate of acidification of the shell compartment, with I_sc, the amount of base, or acid, delivered by the epithelium, expressed in μmolcm⁻² s⁻¹ was multiplied by the Faraday (96500 C equiv⁻¹).

The I_sc across the OME, which is known to be equal to the rate of bicarbonate transport to the haemolymph side when expressed in the same units (Coimbra et al. 1988), was also found to be equal to the rate of transport of acid to the shell side (Fig. 2). Similar observations have been reported for the amphibian gastric mucosa (Teorell, 1951) and for the turtle urinary bladder, in which the transepithelial sodium transport had been blocked (Husted et al. 1979).

I_sc in the OME is inhibited by diamox, DIDS, DNP, amiloride and iodoacetamide (Coimbra et al. 1988). Diamox, DIDS and DNP induced changes in the rates of secretion of base (haemolymph side) and acid (shell side) which were very similar to the concurrent changes in I_sc (Fig. 3). Amiloride induced a decrease in the amounts of acid and base secreted and had almost no effect on I_SC during the same period. This agrees with the observation (Coimbra et al. 1988) that the inhibition of I_sc by amiloride is very slow.

These results show that over a wide range of transport rates and within experimental error there is good agreement between and the net transport of acid and base, both under control conditions and when the preparation is treated with diamox, DIDS or DNP. Thus, provided the current is stable, it can be used to monitor the transport of acid (towards the extrapallial compartment) and base (towards the haemolymph compartment).

The dependence of I_sc on O₂ was assessed. Six preparations were gassed with O₂ and CO₂ until a steady state was attained. Three of the preparations were then gassed with a mixture of N₂ and CO₂; the current fell continuously (albeit slowly) reaching a relative value of 0.5 after 30 min (Fig. 4). The inset shows the results of similar experiments (N=6) in which I_sc and the rates of acidification and alkalinization of the shell and haemolymph compartments, respectively, were measured simultaneously. Both under control conditions (C, three 30-min periods for each preparation) and under anoxia (A, four consecutive 30-min periods) there was good agreement between the three quantities.

When CO₂ was removed from the mixture gassing the shell side of the preparation, the I_sc fell to 40% of the control value (Fig. 5). Under these conditions I_sc was almost insensitive to the concentration of bicarbonate on the same side in the range 10–1.25 mmol 1⁻¹. When the shell solution was nominally bicarbonate-free, I_sc fell to a relative value of less than 0.1. However, an effect of pH on I_sc cannot be ruled out in these experiments.

In another series of experiments the effect on I_sc of the pH of the solution on the shell side was studied (Fig. 6). When the shell solution was nominally bicarbonate-free, the preparation was gassed with pure oxygen. I_sc was measured with the shell solution pH adjusted (with HCl) to different values (9, 8, 7, 6 or 5). For comparison, a phosphate-free, nominally bicarbonate-free shell solution was also used. I_sc was pH-sensitive, in particular below pH7 (Fig. 6)..

I_sc was insensitive to vanadate (1 mmol l⁻¹) added to both sides (control 55.6±4.37μAcm⁻²;+vanadate 62.0±1.84μAcm⁻², N=6) and to oligomycin (200μg ml⁻¹) also added to both sides (control 42.0±12.4μAcm⁻²; + oligomycin 39.3±12.6μAcm^-2, N=6). DCCD (50μg ml⁻¹) caused a slow and moderate reduction of I_SC under anoxia when added to the shell side and was ineffective from the haemolymph side (Fig. 7). Tributyltin oxide (TBTO) was ineffective when added on the haemolymph side, but caused an almost complete inhibition of I_sc within less then 10 s when added to the shell solution to give final concentrations in the range 10–100 nmol 1⁻¹. Such an effect could be observed when the main anion in the solution was isethionate, gluconate, sulphate, thiocyanate or chloride (Fig. 8). TBTO reduces transepithelial conductance by 40% (Machado et al. 1989).

Our earlier work suggested that the short-circuit current of the OME under our experimental conditions cannot be due to a net flux of chloride, potassium or calcium and that the small net flux of sodium measured was in the opposite direction and corresponded, at most, to less then 15 % of I_sc (Coimbra et al. 1988). We have now shown that, under short-circuit conditions, the rates of accumulation of acid in the shell solution and of base in the haemolymph solution are numerically equal to l_sc, when expressed in the same units. These observations are also consistent with the observation that the transepithelial net flux of [¹⁴C]bicarbonate (towards the haemolymph compartment) is also equal to I_SC(Coimbra et al. 1988).

I_sc and the rates of delivery of acid and base by the epithelium have a similar oxygen dependence, and drugs such as iodoacetamide and DNP, which inhibit the rate of ATP production, are potent inhibitors of I_sc (Coimbra et al. 1988 and present work).

Transmembrane hydrogen ion movements are difficult to characterize, both because proton secretion is often indistinguishable from uptake of hydroxyl or bicarbonate ions and because the proton concentration can, by affecting the degree of ionization of proteins, modify the membrane proton permeability and some transport systems, in particular those responsible for the regulation upward of intracellular pH (Vaughan-Jones, 1988). A further complication is that proton movements may be modulated by intracellular pH (Boron, 1986), membrane potential, hormones, growth factors and osmotic challenges (Grinstein and Rothstein, 1986).

In the OME the tight links between I_sc and the acidification and alkalinization of the bathing fluids, together with the sensitivity of these processes to diamox, suggest the involvement of bicarbonate at some stage. Three alternative mechanisms may explain our observations: (1) a transepithelial transport of acid or simply of protons towards the shell side; (2) a transepithelial transport of base (perhaps bicarbonate) in the opposite direction; (3) or the simultaneous extrusion of base across the basolateral membrane and of protons across the apical membrane. Since the flux of [¹⁴C]bicarbonate, I_sc and the rates of alkalinization and acidification of the bathing fluids are sensitive to SITS and DIDS acting from the haemolymph side, an extrusion of bicarbonate towards the haemolymph in exchange for chloride (Boron, 1983) seems to be present. Such a process explains why intracellular chloride concentration rises when the basolateral side is bathed by a nominally bicarbonate-free solution (Coimbra et al. 1988) and entails a recirculation of chloride across the basolateral membranes. The low sensitivity of I_sc to the replacement of chloride on the haemolymph side may be due to a high affinity of the anion exchanger for chloride, as was observed in the turtle urinary bladder (Fischer et al. 1983).

The sensitivity of I_sc to bicarbonate and CO₂ on the shell side suggests that the absorption of bicarbonate across the apical membranes may be part of the process responsible for the I_sc. However, when the shell side is bathed by a solution nominally free of CO₂ and bicarbonate and adjusted to a pH of 7.2, currents of the order of 6 μ A cm^{− 2} can still be measured.

Alternatively, protons may be transported across the apical barrier towards the shell solution. Protons can be translocated across cell membranes by a variety of mechanisms, such as a Na⁺/H⁺ antiport, a K⁺/H⁺ pump or an electrogenic pump (Steinmetz and Andersen, 1982). The Na⁺/H⁺ antiport has been identified in a large number of cell systems from vertebrates and invertebrates and in particular in epithelia (Grinstein and Rothstein, 1986). The OME I_sc was sensitive to amiloride acting from the basolateral side, but, as the inhibition was very slow and as the replacement of sodium by choline in the bath of the same side has a small effect on I_sc (Coimbra et al. 1988), the contribution of this antiport (if it exists in this preparation) to the proton exchanges across the basolateral barrier is probably small. I_sc was insensitive to SITS, DIDS or amiloride added to the shell side or to the replacement of sodium or potassium on this side (Coimbra et al. 1988). It is thus unlikely that the acid secretion to the shell side was due to a Na⁺/H⁺ antiport, to a K⁺/H⁺ pump or to the uptake of bicarbonate across the apical membrane involving Na⁺/HCO₃⁻ cotransport (Boron, 1986) and a recirculation of bicarbonate across the same barrier. Furthermore, since the resistance of the apical barrier is 4 – 10 times that of the basolateral barrier and is insensitive to the replacement of sodium, potassium or chloride by impermeant ions on the shell side, it is unlikely that there is a direct link, or an electrical coupling, between the fluxes of protons and the fluxes of sodium, potassium or chloride ions across the apical barrier (Coimbra et al. 1988). In addition, I_sc is quickly and dramatically inhibited by DNP and by iodoacetamide, drugs that affect the rate of ATP production. We suggest that the acid secretion towards the extrapallial compartment is, at least in part, due to the operation of an electrogenic proton pump. Its location at the apical barrier is identical to that of turtle urinary bladder (Steinmetz and Andersen, 1982).

There is no specific inhibitor of the electrogenic proton pump found in epithelia. However, the inhibition of by I_sc DCCD (in anoxic conditions) and by TBTO from the shell side suggests the presence of a proton pump (Goffeau and Boutry, 1986) and the speed and nature of the TBTO effect indicate that it inhibits an electrogenic process by blocking a conductive channel rather than by creating an electrically silent anion exchange path (Linnet and Beechey, 1979). The observed sensitivity of I_sc to the pH of the shell solution is also compatible with the behaviour expected from an apical proton pump. Further characterization of this system requires measurements of intracellular pH under a variety of conditions.

We thank Mrs L. Santos and Mr U. Santos for invaluable technical collaboration. This work was supported by the Calouste Gulbenkian Foundation, Lisbon, and by the Junta Nacional de Investigação Cientifica e Tecnológica of Portugal.