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1 July 2000 Sodium-Sulfate Symport by Aplysia californica Gut
George A. Gerencser, Randy Levin
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Sulfate transport across plasma membranes has been described in a wide variety of organisms and cell types including gastrointestinal epithelia. Sulfate transport can be coupled to proton, sodium symport or antiport processes involving chloride or bicarbonate. It had previously been observed in Aplysia gut that sulfate was actively absorbed. To understand the mechanism for this transport, short-circuited Aplysia californica gut was used. Bidirectional transepithelial fluxes of both sodium and sulfate were measured to see whether there was interaction between the fluxes. The net mucosal-to-serosal flux of Na was enhanced by the presence of sulfate and it was abolished by the presence of serosal ouabain. Similarly, the net mucosal-to-serosal flux of sulfate was dependent upon the presence of Na and was abolished by the presence of serosal ouabain. Theophylline, DIDS and bumetanide, added to either side, had no effect on transepithelial potential difference or short-circuit current in the Aplysia gut bathed in a Na2SO4 seawater medium. However, mucosal thiosulfate inhibited the net mucosal-to-serosal fluxes of both sulfate and Na and the thiosulfate-sensitive Na flux to that of sulfate was 2:1. These results suggest the presence of a Na-SO4 symporter in the mucosal membrane of the Aplysia californica foregut absorptive cell.


Gastrointestinal and renal transport of the divalent anion sulfate across epithelial apical membranes has been investigated in various vertebrate groups including mammals (Ahearn and Murer, 1984; Pritchard, 1987), teleost fish (Renfro and Pritchard, 1982, 1983) and the domestic chicken (Renfro et al., 1987). A number of mechanisms for brush-border carrier mediated sulfate transport across epithelial membranes have been proposed and include sodium-sulfate cotransport (Ahearn and Murer, 1984; Lucke et al., 1979), anion exchange (Renfro and Pritchard, 1982; Taylor et al., 1987) and pH gradient-dependent transfer (Schron et al., 1985). These processes contribute to transepithelial regulation of sulfate levels, and may affect acid-base balance and plasma osmolarity.

However, there are very few studies of sulfate transport across epithelia of invertebrates. A proton-stimulated sulfate/chloride exchanger has recently been described in apical membranes of lobster (Homarus americanus) hepatopancreatic epithelial cells (Cattey et al, 1992), while an oxalate/sulfate antiporter has also been described in the basolateral membranes of the same cells of lobster hepatopancreas (Gerencser et al, 1995). Many years ago, it was shown that Aplysia foregut could actively absorb sulfate (Gerencser, 1979), however the mechanism for transporting sulfate was not defined. In view of this observation, the present study was undertaken to determine the nature of the sulfate transporter in Aplysia gut. The present study uses isolated foregut from Aplysia californica to characterize a sodium/sulfate symporter that is located in the mucosal membrane of the gut cells and is inhibited by the thiosulfate and ouabain. This transport mechanism may contribute, in part, in maintaining sulfate homeostasis by Aplysia.



Aplysia californica were obtained from Marinus (Westchester, CA) and were maintained at 25°C in circulating filtered seawater. Adult Aplysia (600–1000 g) were used in these experiments and in most cases only animals that had been kept in the laboratory under the above conditions for ≤1 wk were used.

Incubation media for gut tissue

The formula for the standard seawater (Ringer's) solution used was: Na2SO4, 231 mM; MgSO4 · H20, 12.3 mM; K2SO4, 12.1 mM; NaHCO3, 2.4 mM; Ca (Gluconate)2, 11.4 mM; mannitol, 0.237 mM. A Na+-free medium was prepared by totally replacing Na+ with trishydroxyaminomethane+ using sulfate and bicarbonate salts. A sulfate-free medium was prepared by totally replacing sulfate and mannitol with gluconate. The total osmolality of the bathing media was 1010 mOsm/Kg and their pH was 7.8 at 25°C.

Experimental Procedures

The preparation and mounting of gut sheets between the two halves of a Lucite Ussing chamber that allowed measurement of transepithelial potential difference (ΨMS) and short-circuit current (SCC) across the gut have been described previously (Gerencser, 1978) (Fig. 1). Both the mucosal and serosal media were gassed with 100% O2, and both aspects of the gut were independently and continuously perfused by gravity with seawater medium at room temperature (25±1°C).

Fig. 1


The methods used to measure ΨMS and SCC were essentially similar to those employed for rabbit ileum by Schultz and Zalusky (1964), except that agar bridges from calomel half-cells, instead of Ag-AgCl electrodes, were used to apply external current to the system. The electrolyte content of these bridges was identical to that of the bathing solution in each experiment to minimize diffusion currents. The agar bridges from the potential-sensing electrodes contained saturated KCl because K+ and Cl have approximately equal mobility constants (Schultz and Curran, 1970). To minimize potential offset between these electrodes, the ends of these bridges were preequilibrated with the bathing medium for several hours before the experiment. Offset between the potential-sensing electrodes was measured at the beginning of the experiment and again at the end of the run following removal of the tissue and replacement of the bathing fluid. The potential drop between the potential-sensing electrodes due to the resistance of the bathing solution was compensated automatically by the voltage-clamp device as described by Rothe et al. (1969).

By use of 22Na and 35SO4 (New England Nuclear), unidirectional mucosal-to-serosal (JMS) and serosal-to-mucosal fluxes (JSM) of Na+ or SO4 were determined on paired pieces of tissue from the same animal when their respective SCC's were comparable in magnitude. In these radioisotopic experiments the tissue was allowed to equilibrate for 30–90 min in nonradioactive seawater solution. At this electrical steady-state time, a trace amount of isotope was directly added to the chamber. Thereafter, at timed intervals of approximately 20 min, 0.1 ml samples of solution were removed from the initially unlabeled half-chamber for counting. Fluxes observed during the early sampling stages, i.e., before specific activity equilibrium between tissue and bathing solution was achieved, were small. They increased to constant values by the end of the first hour following introduction of tracer. Therefore, only samples obtained following the first hr were used to estimate steady-state fluxes. Experiments were usually terminated 4–5 hr after addition of isotope. From the results obtained JMS and JSM of 22Na and 35SO4 were computed as described by Quay and Armstrong (1969). All data are reported as means±SEM. Differences between means were analyzed statistically using a Student's paired t-test.


The first group of experiments was designed to examine whether sulfate and/or ouabain had any effect on Na+ fluxes. As can be seen in Table 1, the mean net JMS of Na+ (JMSNET) is approximately equal to the average SCC with gluconate being the major anion in the bathing medium. However, upon replacing both the mucosal and serosal bathing media with a media containing sulfate as its major anion, there is a significant increase (P<0.05) in the JMSNET of Na+. This change in Na+ absorption is due to an increase in unidirectional JMS of Na+. The unidirectional JSM of Na+ did not significantly change in the sulfate-based medium. Also, the mean JMSNET of Na+, in the presence of sulfate, is significantly greater (P<0.05) than the corresponding average SCC. Serosal ouabain (10−4M) abolished both the basal and sulfate-dependent JMSNET of Na+ by inhibiting solely the unidirectional JMS of Na+. Ouabain also abolished the SCC.

Table 1

Na+ fluxes in various seawater media


The next group of experiments was designed to examine if Na+ and/or ouabain had any effect on sulfate fluxes. As can be seen in Table 2, the average net JMSNET of sulfate is almost absent when the gut was bathed in Na+-free bathing media. The corresponding average SCC is also close to zero. However, when the Na+-free sulfate bathing medium was replaced with a Na+-containing sulfate medium, the average JMSNET of sulfate increased significantly (P<0.05). This increase in the JMSNET of sulfate was entirely attributable to the increase in the unidirectional JMS of sulfate because there was no significant change in the undirectional JSM of sulfate in the presence of Na+. The average SCC, in the presence of Na+, was significantly greater than zero (P<0.05) and it was also greater than the JMSNET of sulfate. Serosal ouabain (10–4M) inhibited both the JMSNET of sul fate and the SCC. The unidirectional JMS of sulfate was the only flux of sulfate that was affected by serosal ouabain.

Table 2

Sulfate fluxes in various seawater media.


The next series of experiments were designed to exam ine the effects of thiosulfate on Na+ and sulfate fluxes in Aplysia gut. The addition of thiosulfate (10–2M) to the mucosal com partment of a Na2SO4 bathing medium inhibited the unidirectional JMS of sulfate, but not the JSM of sulfate, resulting in the complete depression of JMSNET of sulfate (Table 3). In contrast, the serosal addition of 10-2M thiosulfate to the serosal bathing solution had no effect on either the unidirectional JMS or JSM of sulfate [data not shown (n=3)]. The addition of 10–2M thiosulfate to the mucosal bathing solution also inhibited the unidirectional JMS of Na+ without affecting the unidirectional JSM of Na+. The ratio of the thiosulfate-sensitive Na+ and sulfate fluxes was 2:1 in both JMSNET and JMS. On the other hand, thiosulfate had no significant effect on SCC across the Aplysia gut.

Table 3

Effect of thiosulfate on Na+ and sulfate fluxes.


Theophylline (10–6M), bumetanide (10–5M) nor 10–5M 4,4′- diisothiocyano-2,2′-disulfonic stilbene (DIDS) added to either the mucosal or serosal bathing medium had no effect on JMS or SCC in the Aplysia gut preparation. Each of these chemical agents were used in three experiments.


In the current investigation we presented suggestive evidence for the existence of a carrier-mediated Na+-sulfate symport located in the apical membrane of Aplysia californica foregut epithelium. Sulfate carriers have been described in the apical membranes of several vertebrate epithelial tissues (Cattey et al., 1994). Both sodium-sulfate cotransport and sulfate-hydroxyl exchange mechanisms have been demonstrated in rabbit ileal brush border (Schron et al., 1985; Schneider et al., 1984). In avian renal apical membranes multiple pathways were shown to transport sulfate; sodiumsulfate cotransport, sulfate-bicarbonate exchange and protondependent sulfate transport (Renfro et al., 1987). Marine teleost renal tubule apical membranes have been shown to contain a sulfate-anion exchange mechanism which is most effective with bicarbonate (Renfro and Pritchard, 1983). In lobster hepatopancreatic apical membranes sulfate uptake was not stimulated by inwardly directed cation gradients of either Na+ or K+ (Cattey et al., 1992). However, intravesicular Cl stimulated the influx of radiolabeled sulfate which was interpreted as there being a SO4/Cl antiporter in the apical membrane.

When the Aplysia foregut was bathed in a sulfate-free (Table 1) or chloride-free (Gerencser, 1981; Gerencser, 1985) Na+-containing seawater media, the net active absorptive flux of Na+ was equivalent to the SCC. This observation is interpreted as Na+ being the only ion actively translocated, in a net sense, across the gut tissue. However, when sulfate replaced gluconate [a non-transportable anion (Cattey et al., 1992)] in the bathing media, the net active absorptive flux of Na+ increased solely due to the increase in the unidirectional JMS of Na+. This suggests that sulfate stimulates the absorptive flux of Na+. However, the JMSNET of Na+ is significantly greater than the corresponding SCC (Table 1). This disparity in JMSNET of Na+ and SCC could be accounted for by a net active absorptive flux of an anion such as sulfate. Serosally-applied ouabain inhibited both JMSNET of Na+ and the SCC, accompanied an inhibition of the unidirectional JMS of Na+ (Table 1). These observations suggest that Na+ transport and SCC are dependent on the activity of the Na+/K+-ATPase (Gerencser and Lee, 1985; Skou, 1965).

In a Na+-free seawater bathing medium there is no net transport of sulfate nor a SCC across the Aplysia gut (Table 2). However, upon replacing the Na+-free seawater medium with a medium containing Na+, there is a finite JMSNET of sulfate under short-circuited conditions. These observations suggest that active sulfate absorption is dependent upon the presence of Na+ and that there is coupling between these two ions in their transit from the mucosal to the serosal bathing solutions. This is because, in the presence of Na+, there is a finite SCC, part of which can be accounted for by the JMSNET of sulfate while the remainder of the SCC can be accounted for by a net mucosal-to-serosal movement of Na+ (Tables 1,2,3). The substantiation of Na+ as the co-transported ion species with that of sulfate is shown with the inhibition of both the unidirectional JMS of sulfate and the SCC by serosally-applied ouabain (Table 2). As previously stated ouabain specifically inhibits active Na+ transport (Skou, 1965; Schultz and Zalusky, 1964). Therefore, its inhibition of active sulfate absorption implies a degree of coupling between the two undirectional fluxes (JMS's) of both Na+ and sulfate.

Thiosulfate is a known inhibitor of sulfate transport (Schneider et al., 1984; Turner, 1984). In the present study, mucosally-applied thiosulfate inhibited the JMS of sulfate such that the active component of sulfate absorption was abolished (Table 3). In addition mucosally-applied thiosulfate also inhibited the unidirectional JMS of Na+ (Table 3). Together, these results strongly suggest a coupling between Na+ and sulfate transport, in their co-movement from mucosa to sersosa. The result that serosally-applied thiosulfate had no effect on either Na+ or sulfate transport suggests that the transporter for both ions resides in the apical membrane of the Aplysia foregut absorptive cell and not in the basolateral membrane. Since thiosulfate significantly inhibited both unidirectional JMS's of Na+ and sulfate, but did not significantly inhibit the corresponding SCC (Table 3), the decrease in coupled Na+-sulfate flux, from mucosa-to-serosa, must be electrically silent. In addition, as seen in Table 1, sulfate stimulated the JMS of Na+ without an increase in SCC. The SCC's under these different experimental conditions did not change. This suggested that the coupled Na+/sulfate cotransport, from mucosa-to-serosa was electrically neutral. Since Na+ is a univalent cation and sulfate is a divalent anion, the stoichiometry of coupled Na+/sulfate transport in the Aplysia gut could be two Na+ per one sulfate per cycle of transport, or some mathematical equivalent of 2 Na+ per 1 sulfate in order for electroneutrality to be maintained. In fact, the ratio of the thiosulfate-sensitive Na+ to sulfate fluxes was 2:1.

In summary, we have presented suggestive evidence for the existence of a Na-SO4 symporter located in the apical membrane of the Aplysia californica foregut absorptive cell that could be responsible for the net absorption of sulfate by this animal. This event could be beneficial for cellular viability of cellular metabolic reactions such as: 1) sulfur conjugation (Gerencser, 1996; Turner, 1984; and/or Pritchard, 1987) complexing with heavy metals such as what happens in lobster hepatopancreas (Gerencser et al., 1995). Sulfate homeostasis in the Aplysia is, at least, partly maintained by this luminal Na/SO4 symport transport mechanism.


This investigation was supported by grants from the Whitehall Foundation and the Eppley Foundation. We acknowledge the excellent technical assistance of F. Robbins.



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George A. Gerencser and Randy Levin "Sodium-Sulfate Symport by Aplysia californica Gut," Zoological Science 17(5), 579-583, (1 July 2000).
Received: 19 January 1999; Accepted: 1 December 1999; Published: 1 July 2000
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