NO₂⁻ uptake and HCO₃₋ excretion in the intestine of the European flounder (Platichthys flesus)

Nitrite (NO₂⁻) is a potential contaminant in aquatic environments that receive nitrogenous waste and in recirculated aquaculture systems. Several studies have examined the toxicity and physiological effects of NO₂⁻ in fish (for reviews, see Lewis and Morris, 1986; Eddy and Williams, 1987; Jensen, 1995, 1996).

Most investigations have concerned freshwater teleosts, because NO₂⁻ is generally more toxic to freshwater than to marine species. However, we have shown that a marine teleost, the European flounder Platichthys flesus, accumulates NO₂⁻ in the plasma during exposure to 1 mmol l⁻¹ ambient NO₂⁻ and that this accumulation is associated with an increased blood methaemoglobin content (haemoglobin not available for oxygen transport) (M. Grosell and F. B. Jensen, in preparation). Whereas freshwater fish take up NO₂⁻ primarily across the gills, marine fish may be expected to have significant intestinal uptake as a result of the high rate of drinking required for osmoregulation (Jensen, 1995). Indeed, in vivo studies have demonstrated that the European flounder has high NO₂⁻ concentrations in the intestinal fluids during exposure to elevated ambient NO₂⁻ concentrations (M. Grosell and F. B. Jensen, in preparation). Furthermore, by using in situ perfusion of the intestine (M. Grosell, G. Deboeck, O. Johannsson and C. M. Wood, in preparation), we have demonstrated that intestinal NO₂⁻ uptake accounts for approximately two-thirds of the total whole-body uptake (M. Grosell and F. B. Jensen, in preparation).

Having established that the intestine is the primary site of NO₂⁻ uptake in the European flounder, we set out to investigate the mechanism of NO₂⁻ transport across the intestinal epithelium. In freshwater fish, it has been suggested that the branchial Cl⁻/HCO₃⁻ exchanger is the carrier mediating the apparent active uptake of NO₂⁻ (Bath and Eddy, 1980), but no attempts have been made to document this.

In the present study, insight into transport mechanisms was obtained by using preparations of freshly isolated intestinal segments, a modification of the technique described by M. Grosell, G. Deboeck, O. Johannsson and C. M. Wood (in preparation), which allowed us to manipulate the composition of both the apical (mucosal) and basolateral (serosal) media.

To evaluate the possible uptake of NO₂⁻via an intestinal Cl⁻/HCO₃⁻ exchanger, we applied 4,4′-diisothiocyanostilbene-2,2′-disulphonic acid (DIDS), a well-known blocker of Cl⁻/HCO₃⁻ exchange in erythrocytes and other cells (Nikinmaa, 1990). In a recent review of intestinal ion transport in marine teleost fish, Loretz (1995) proposed a model involving a Cl⁻/HCO₃⁻ exchanger in the basolateral membrane. However, only two studies (Dixon and Loretz, 1986; Ando, 1990) provide evidence for a basolateral Cl⁻/HCO₃⁻ exchanger, and Ando (1990) also reports evidence for apical Cl⁻/HCO₃⁻ exchange. Consequently, we applied DIDS both in the mucosal solution and in the mucosal and serosal solutions in combination to assess the potential importance of apical and basolateral Cl⁻/HCO₃⁻ exchangers for intestinal NO₂⁻ uptake.

A further possible route of uptake for NO₂⁻ would be the Na⁺/K⁺/2Cl⁻ cotransporter in the apical membrane of the intestine (Loretz, 1995). Accordingly, we have also used bumetanide in the mucosal solution to target this transporter and its possible role in NO₂⁻ uptake.

During the studies, double radiolabelling of Na⁺ and Cl⁻ allowed us to measure not only NO₂⁻ uptake but also unidirectional Na⁺ and Cl⁻ fluxes as indicators of the viability of the freshly isolated intestinal segments and of the action of pharmacological agents. These isotopic techniques, in combination with recordings of net transport of Na⁺, Cl⁻, HCO₃⁻ and water, gave insight into the general ion-transport properties of the intestinal epithelium of the European flounder.

European flounder (Platichthys flesus) were obtained from a local fisherman and brought to the Aquatic Research Center of Odense University in Kerteminde, Funen, Denmark. The fish were held in a 400 l polyvinylchloride tank with a flow of aerated Kerteminde sea water for at least 6 days prior to experiments. The fish were not fed during holding. Experimental fish were transferred to glass aquaria with a bottom surface area of 0.25 m² containing 25 l of Kerteminde sea water (static, 24 h renewal) adjusted to 20 ‰ salinity with sea salt (three fish per tank) at 6 °C and allowed to acclimate to this salinity and temperature for a minimum of 3 days prior to experiments.

Preparations were made from a total of 36 fish, ranging in body mass from 105 to 283 g. Fish were killed with an overdose of neutralized MS-222 (200 mg l⁻¹). One side of the body cavity was removed, exposing the gastrointestinal tract. The intestine was obtained by dissection, placed on a glass plate, and put on ice. An inflow catheter (a 10 cm length of PE90 tubing flared at the tip) was inserted at the anterior end of the intestine and tied in place using two silk ligatures. This catheter was used to flush the intestine gently with 20 ml of the gut saline to be used in the following experiments (see below) to displace any intestinal fluid and solids. The anterior, mid and posterior parts of the intestine were then separated. The mid and posterior parts were fitted with a filling catheter (PE90) at the anterior end as described above, and all three ‘gut bags’ were then closed at the posterior end using two silk ligatures. Subsequently, these ‘gut bags’ were filled with gut saline containing ²²Na and ³⁶Cl as well as NO₂⁻ (see below for composition) and transferred to glass vials containing 18 ml of aerated L-15 medium (Leibovitz’s L-15 medium, containing L-glutamine; Gibco). The L-15 medium was supplemented with 10 mmol l⁻¹ NaHCO₃, giving 10 mmol l⁻¹ total CO₂ and a pH of approximately 8.00 to mimic the in vivo situation for these fish at a similar temperature (M. Grosell and F. B. Jensen, in preparation). The vials were placed in a water bath maintained at a constant temperature of 6 °C. All solutions used in the experiments were at 6 °C. Immediately after filling the gut bag with the appropriate gut saline, a sample of 300–400 μl of gut saline was obtained via the catheter, which was subsequently plugged. The gut bags were carefully lifted out of the surrounding L-15 medium, blotted dry, weighed and again placed in the vial (time zero). Samples (0.5 ml) of the external L-15 medium were taken (in triplicate) at 0, 2 and 4 h and transferred to scintillation vials for analysis of ³⁶Cl and ²²Na. A fourth sample of L-15 medium was taken at the same times in the NO₂⁻-treated preparations and used for analysis of [NO₂⁻]. The L-15 medium removed by sampling was replaced with fresh medium. After 4 h, the gut bags were blotted dry and reweighed. Finally, a 300–400 μl sample of the internal gut saline was collected from the catheter. The gut saline samples taken at the start and at the end of the experiment were analysed for pH, total CO₂ content, [Na⁺], [Cl⁻], [NO₂⁻] and ³⁶Cl and ²²Na radioactivity. Each gut bag was opened using a longitudinal incision, the remaining gut saline was removed, and the intestinal tissue was blotted dry and reweighed. The exposed net gross surface area of each gut bag was measured using graph paper.

The internal volume of gut saline in each individual gut bag was determined by subtracting the mass of the empty gut bag from the corresponding initial mass. Net water transport was calculated from the difference in total mass over the experimental period, with the surface area of each gut bag and the time elapsed taken into account. Net fluxes of Na⁺, Cl⁻ and HCO₃⁻ were calculated by dividing the difference in the total number of moles of the given ion in the gut saline between the beginning and end of the experiment by the surface area and elapsed time.

Unidirectional mucosal-to-serosal Na⁺ and Cl⁻ fluxes were calculated from the increase in ²²Na and ³⁶Cl radioactivity in the surrounding L-15 medium during two successive 2 h periods using the means of corresponding specific activities at the start and at the end of the flux period. Unidirectional NO₂⁻ flux was calculated from the appearance of NO₂⁻ in the surrounding L-15 medium during the two successive 2 h periods. Unidirectional Na⁺ and Cl⁻ fluxes and net NO₂⁻ fluxes were related to corresponding gut bag surface area and elapsed time.

The gut saline used for flushing the intestine prior to experiments consisted of the following (in mmol l⁻¹); 24 NaCl, 15 MgCl₂, 2 CaCl₂, 1 KCl and 1 NaHCO₃, pH 7.4. The composition of the gut saline was based on actual measurements of gut fluid composition (for details, see M. Grosell and F. B. Jensen, in preparation). During experiments, 11.1 kBq of ³⁶Cl (Amersham) and 7.4 kBq of ²²Na (Amersham) were added for unidirectional measurements of Cl⁻ and Na⁺ fluxes, respectively. For the studies of NO₂⁻ transport, 1 mmol l⁻¹ NO₂⁻ (as NaNO₂) was added. Experimental treatments consisted of the following four series with control L-15 medium on the serosal side of the gut bags: control gut saline with 0.1 % dimethylsulphoxide (DMSO) (vehicle control), (2) gut saline with 10⁻⁴ mol l⁻¹ bumetanide, an inhibitor of the Na⁺/K⁺/2Cl⁻ cotransporter, dissolved in a final concentration of 0.1 % DMSO; (3) control gut saline with 1 % DMSO (vehicle control) and (4) 10⁻³ mol l⁻¹ DIDS, a blocker of Cl⁻/HCO₃⁻ exchange, dissolved in 1 % DMSO. Two additional experimental series were as follows: (5) 1 % DMSO in both the L-15 medium and the gut saline (vehicle control); and (6) 10⁻³ mol l⁻¹ DIDS dissolved in a final concentration of 1 % DMSO in both the L-15 medium (serosal) and the gut saline (mucosal).

Appropriate gut bag saline was made up freshly just prior to experiments from a stock solution of gut saline by adding ³⁶Cl, ²²Na, NaNO₂ (where appropriate) and bumetanide or DIDS with corresponding DMSO vehicle controls. L-15 medium containing DIDS and the corresponding DMSO control (experimental series 5 and 6) were also made up freshly prior to experiments.

γ-Radioactivity from ²²Na was measured in samples of the external L-15 medium and in samples of gut bag saline from the start and from the end of the flux period using a gamma-counter (1480 Wizard 3 Automatic, Wallac, Turku, Finland). Total β-radioactivity from ³⁶Cl and ²²Na was also measured in the same external L-15 medium samples and in the initial and terminal internal gut bag saline samples using a scintillation counter (Packard 2200 CA Tri-Carb, Packard, Groningen, Netherlands). The two counters were cross-calibrated for ²²Na radioactivity, and the contribution of ²²Na to the total β-radioactivity was subtracted from the total β-radioactivity to determine the ³⁶Cl β-radioactivity. NO₂⁻ concentrations in gut saline samples and in samples of L-15 medium were determined spectrophotometrically following the method of Shechter et al. (1972). For NO₂⁻ measurements in L-15 medium, the corresponding standard curve was obtained from known concentrations of NO₂⁻ in L-15 medium, since the red colour of the medium interfered with the absorbance of the NO₂⁻ assay. Similarly, at the relatively high concentrations used in the present investigation, DIDS was found to interfere with the NO₂⁻ assay. Consequently, the standard curves for NO₂⁻ measurements in experimental series 5 and 6 were derived from known NO₂⁻ concentrations in L-15 medium containing the appropriate DIDS concentration. Gut bag saline and L-15 medium pH was measured using Radiometer (Copenhagen, Denmark) BMS3 electrodes (BMS3 system) with the signal displayed on a PHM 73 blood gas monitor and REC 80 recorders. The total CO₂ concentrations of gut bag saline and L-15 medium were measured using the method of Cameron (1971). The Cl⁻ concentrations of gut bag saline samples were measured using a Radiometer CMT 10 chloride titrator, and [Na⁺] was measured by flame photometry (Instrumentation Laboratory 243).

All values are expressed as absolute values, means ± S.E.M. (N). Significant differences between values obtained from preparations with either bumetanide or DIDS and their corresponding vehicle controls were evaluated using unpaired Student’s t-test (two-tailed). Because of day-to-day variations in absolute flux values, experiments involving preparations from two control fish (vehicle control) and two fish from one of the above treatments were conducted simultaneously. Statistical comparisons with the corresponding control values were conducted on relative flux rates (percentage of the corresponding control value for the same day) using an ASIN conversion to ensure normal distribution of the data. Vehicle controls (0.1 % and 1 % DMSO) were compared with control (no DMSO) values by two-tailed t-test with Bonferroni multi-sample comparison correction. One-way analysis of variance (ANOVA) was used to compare both unidirectional and net flux rates between different segments of the intestine. In all cases, groups were considered significantly different at P<0.05.

The presence of NO₂⁻ in the gut lumen led to a significant NO₂⁻ uptake across the gut epithelium. The rate of NO₂⁻ influx correlated well with the rate of Cl⁻ influx (y=0.0035+0.0071x; P<0.05, r²=0.566) (Fig. 1). The ratio between unidirectional NO₂⁻ and Cl⁻ flux rates was approximately 1:100 at mean flux rates. Considering the change in concentration of these two anions in the gut saline over time, this ratio reflects the different luminal concentrations of NO₂⁻ and Cl⁻ ([NO₂⁻]:[Cl⁻] being 1:59 initially and 0.7:116 finally).

The rate of NO₂⁻ influx did not differ among the different segments of the intestine, but tended to be lower during the second 2 h flux period than during the first 2 h flux period (Fig. 2). Overall, the unidirectional rate of influx of NO₂⁻ was 8.9 nmol cm⁻² h⁻¹ in the control series. NO₂⁻ uptake was significantly inhibited by 10⁻⁴ mol l⁻¹ bumetanide added to the gut saline and thus contacting the mucosal side of the intestine (Fig. 2). Bumetanide (10⁻⁴ mol l⁻¹) caused an overall 47 % inhibition of NO₂⁻ uptake in all segments of the intestine compared with corresponding control values. DIDS (10⁻³ mol l⁻¹) applied to the mucosal side of the intestine did not influence NO₂⁻ influx (Fig. 2). When applied simultaneously to both the gut saline and the L-15 medium and thus acting on both the mucosal and serosal sides of the intestine, DIDS (10⁻³ mol l⁻¹) caused a slight increase in the initial rate of NO₂⁻ influx. This initial increase was only statistically significant in the mid part of the intestine (Fig. 2).

Vehicle control (saline + DMSO) unidirectional Cl⁻ (Fig. 3) and Na⁺ (Fig. 4) flux rates and net flux rates of HCO₃⁻ (Fig. 5) did not differ from corresponding control flux rates in preparations exposed to gut saline without DMSO and NO₂⁻(data not shown).

Overall, the unidirectional rate of Cl⁻ influx was 0.72 μmol cm⁻² h⁻¹. This rate tended to be slightly higher in the anterior part than in the mid and posterior parts of the intestine, but did not differ significantly between the first and the second 2 h flux periods (Fig. 3). Neither bumetanide (10⁻⁴ mol l⁻¹) nor DIDS (10⁻³ mol l⁻¹) applied to the mucosal side of the intestine caused significant changes in unidirectional Cl⁻ fluxes. Rates of Cl⁻ influx in the bumetanide-treated preparations, however, showed a general trend to be lower than their corresponding controls, but these differences were not significant (Fig. 3A). DIDS (10⁻³ mol l⁻¹) applied to both the mucosal and serosal sides of the preparation caused a significant 22 % inhibition of Cl⁻ influx in the anterior and posterior parts of the intestine in the first 2 h flux period (Fig. 3C).

The mean overall Na⁺ influx in control preparations was 0.66 μmol cm⁻² h⁻¹. As for Cl⁻, the rate of Na⁺ influx tended to be slightly higher in the anterior than in the mid and posterior parts of the intestine, but did not differ significantly between the first and the second 2 h flux period (Fig. 4). Bumetanide (10⁻⁴ mol l⁻¹) caused an overall 40 % inhibition of unidirectional Na⁺ influx. DIDS (10⁻³ mol l⁻¹) applied to the mucosal side of the intestine did not influence Na⁺ influx rates. When DIDS was applied to both the mucosal and the serosal sides, however, Na⁺ influx was significantly increased by 38 % compared with control values (Fig. 4C).

The net HCO₃⁻ flux averaged for all control flux periods was −0.28 μmol cm⁻² h⁻¹ (i.e an efflux) (Fig. 5). The rate of HCO₃⁻ efflux to the gut lumen, however, tended to be highest in the anterior part of the intestine (Fig. 5). All the blockers applied caused inhibition of HCO₃⁻ efflux (Fig. 5). Mucosal bumetanide (10⁻⁴ mol l⁻¹) resulted in an overall inhibition of 24 %, mucosal DIDS (10⁻³ mol l⁻¹) caused a 39 % inhibition, while DIDS applied to both the serosal and the mucosal sides resulted in 28 % inhibition. For both DIDS treatments, the inhibition was only significant in the anterior and posterior parts of the intestine (Fig. 5).

Net flux rates of Na⁺, Cl⁻ and water in control, vehicle control, bumetanide- and DIDS-treated preparations are given in Table 1. With the exception of water flux in the anterior segment of series 1, none of the net flux rates differed significantly between the control and the vehicle control (two different concentrations of DMSO). Overall, net flux rates of both Na⁺ and Cl⁻ were slightly negative (i.e. a net efflux of both these ions). For both Na⁺ and Cl⁻ fluxes, there were no apparent differences in net flux rates among the different intestinal segments. For water flux, however, the anterior part of the intestine exhibited the highest flux rate, and there was a progressive decrease in the rate of water uptake in the mid and posterior parts of the gut. DIDS applied to both the mucosal and serosal sides of the intestine caused an increased net flux of Na⁺ in the mid section (less negative). This is consistent with the effect of DIDS on unidirectional Na⁺ flux (Fig. 4) and was apparently associated with increased rates of water transport (Table 1).

Bumetanide appeared to have a significant effect on the rate of water transport in the anterior part of the intestine. It should be noted, however, that the control value for this treatment is unusually high (compared with the other control values) (Table 1).

The relatively high concentrations of DMSO needed to dissolve the high concentrations of both bumetanide and DIDS used in the present study did not influence net flux rates of Na⁺, Cl⁻ or water, indicating that there was no apparent effect of DMSO on membrane permeability at these concentrations. The intestinal epithelium is highly convoluted and rich in microvilli, and this probably explains the relatively high concentration of blockers needed to provide a local concentration sufficient to inhibit carrier-mediated ion fluxes. Typical concentrations of DIDS needed to inhibit Cl⁻/HCO₃⁻ exchange by 50 % (I₅₀) in erythrocytes are between 10⁻⁷ and 0.5×10⁻⁶ mol l⁻¹ (Nikinmaa, 1990). In comparison, Wilson et al. (1996) reported no effect of mucosal DIDS at 10⁻⁵ mol l⁻¹ on HCO₃⁻ excretion in seawater-adapted rainbow trout during in situ perfusion of the intestine, and Dixon and Loretz (1986) observed no effect of mucosal DIDS at 10⁻⁴ mol l⁻¹ on HCO₃⁻ transport in the goby (Gillichthys mirabilis). In the present study, 10⁻³ mol l⁻¹ DIDS applied to the mucosal medium caused a significant inhibition of the rate of HCO₃⁻ transport. The same concentration of mucosal DIDS was effective in reducing transepithelial potential and water flux in seawater-adapted Japanese eel (Anguilla japonica) (Ando, 1990). The value of I₅₀ for bumetanide is typically betweeen 10⁻⁷ and 0.5×10⁻⁶ mol l⁻¹ (Nikinmaa, 1990). Various isoforms of the Na⁺/K⁺/2Cl⁻ cotransporter, however, have different sensitivities to bumetanide; in the basolateral cotransporter of the operculum epithelium, for instance, the sensitivity can be low (Marshall and Bryson, 1998). We chose a concentration of 10⁻⁴ mol l⁻¹ in the present study. Similar mucosal concentrations of bumetanide have previously been used to study the apical Na⁺/K⁺/2Cl⁻ cotransporter in marine teleost intestines (Charney et al., 1988; Musch et al., 1982) and to study branchial Na⁺/K⁺/2Cl⁻ cotransport (Flik et al., 1997).

The present data suggest that NO₂⁻ is taken up across the intestine in the European flounder via the Na⁺/K⁺/2Cl⁻ cotransporter rather than via the Cl⁻/HCO₃⁻ exchanger. The mucosal Na⁺/K⁺/2Cl⁻ cotransporter was targeted with bumetanide, which caused a similar inhibition of NO₂⁻ and Na⁺ influx, and a tendency to reduce Cl⁻ influx. In contrast, DIDS applied to the mucosal medium did not inhibit NO₂⁻ uptake rates, although the rate of HCO₃⁻ excretion was significantly reduced. When DIDS was applied to both the mucosal and serosal sides of the epithelium, NO₂⁻ uptake was slightly elevated compared with controls. A similar effect was observed on unidirectional Na⁺ influx and net Na⁺ flux rates. The effects of DIDS when applied to both solutions differ from the situation where DIDS was applied to the mucosal solution only and can therefore be regarded as an effect of DIDS in the serosal solution. The mechanism(s) involved in the stimulation of unidirectional Na⁺ influx and net Na⁺ flux by serosal DIDS is unknown.

NO₂⁻ did not influence unidirectional Cl⁻ influx or net Cl⁻ flux. This contrasts with the situation in freshwater teleost fish and crustaceans, in which NO₂⁻ uptake across the gills induces net Cl⁻ loss with a consequent reduction in extracellular Cl⁻concentration (Jensen et al., 1987; Jensen, 1990; Harris and Coley, 1991). In freshwater fish, increased external Cl⁻ concentrations reduce toxicity by reducing the accumulation of NO₂⁻ in the plasma (Eddy et al., 1983; Bath and Eddy, 1980). Williams and Eddy (1986) demonstrated that NO₂⁻ acts as a competitive inhibitor of Cl⁻ uptake and vice versa, which explains the protective effect of ambient Cl⁻ against NO₂⁻ uptake and toxicity. The transport affinities (K_m) for NO₂⁻ and Cl⁻ were comparable. Taken together, these observations indicate that Cl⁻ and NO₂⁻ share the same uptake mechanism across the gills of freshwater fish and that the carrier involved has a similar affinity for both anions (i.e. does not distinguish between them). This means that the ratio of NO₂⁻ to Cl⁻ uptake across gills in freshwater fish will reflect the ratio of the ambient concentrations of the two anions. In the present study, the Cl⁻ concentration in the gut saline applied on the mucosal side was 59 mmol l⁻¹, and this increased during the 4 h of experimentation to approximately 116 mmol l⁻¹ (negative net Cl⁻ flux), whereas the corresponding initial NO₂⁻ concentration was 1 mmol l⁻¹, decreasing to approximately 0.7 mmol l⁻¹ during the experimental period (uptake from the lumen). The slope of the regression line in Fig. 1 (approximately 0.01) therefore suggests that Cl⁻ and NO₂⁻ transport rates across the intestine reflect the available concentrations of the two anions, as is the case for branchial transport in freshwater fish. However, experiments involving different NO₂⁻ and Cl⁻ concentrations are needed to determine conclusively whether the ratio of the concentrations of the two ions is determining the rate of uptake of NO₂⁻ in the intestine of marine fish. Considering the high [Cl⁻]:[NO₂⁻] ratio in the mucosal solution, a similar affinity of the Na⁺/K⁺/2Cl⁻ cotransporter for NO₂⁻ and Cl⁻ would imply that Cl⁻ influx should be inhibited by approximately by 1 % in the presence NO₂⁻. Such a low level of inhibition would not be detectable. The Na⁺/K⁺/2Cl⁻ cotransporter is also present in the gills of freshwater teleosts (Cutler et al., 1998; Flik et al., 1997) and, in the light of the present study, it seems relevant to investigate the relative roles of this transporter and of the Cl⁻/HCO₃⁻ exchanger (which was implicated by the study of Bath and Eddy, 1980) in NO₂⁻ uptake across the gills of freshwater fish.

Na⁺ and Cl⁻ influx in the presence of bumetanide was not completely inhibited. This could be due to an insufficient bumetanide concentration in the microenvironment of the transporter, but it could also be due to transport across the apical membrane by the bumetanide-insensitive Na⁺/Cl⁻ cotransporter, which is present at the apical membrane of the intestine of marine teleosts (Loretz, 1995). Whether NO₂⁻ is also transported across the apical membrane via the Na⁺/Cl⁻ cotransporter must be determined in further studies. However, assuming NO₂⁻ concentrations of 1 mmol l⁻¹ in the mucosal solution and 0.01 mmol l⁻¹ (typical NO₂⁻ concentration in the L-15 medium at the end of an experiment) in the serosal solution, the equilibrium potential of NO₂⁻ would be approximately −120 mV. This is much lower than the transepithelial potential recorded in asymmetrical in vivo conditions (−20 mV; Loretz, 1995), indicating that part of the NO₂⁻ uptake could be by simple diffusion.

The intestine of seawater-adapted rainbow trout has recently been identified as an important site for base excretion (Wilson et al., 1996). Most of the base equivalents transported across the intestinal epithelium are in the form of HCO₃⁻ (Dixon and Loretz, 1986; Wilson et al., 1996). Even though a significant part of the excreted HCO₃⁻ forms carbonate deposits within the intestinal lumen of various marine teleost fish (Walsh et al., 1991; Wilson et al., 1996; Wilson, 1997), high concentrations of HCO₃⁻ in the intestinal fluids have frequently been reported from a wide range of marine teleosts (Walsh, 1991; Wilson et al., 1996; Wilson, 1997; M. Grosell, G. Deboeck, O. Johannsson and C. M. Wood, in preparation; M. Grosell and F. B. Jensen, in preparation). In the European flounder, we have recently shown that HCO₃⁻ is the major anion in the fluids of at least the mid and posterior parts of the intestine (M. Grosell and F. B. Jensen, in preparation). The lumen-to-blood gradient of [HCO₃⁻] in the European flounder (M. Grosell and F. B. Jensen, in preparation) would require an equilibrium potential of −47 mV (lumen negative); in seawater-adapted rainbow trout the corresponding equilibrium potential is −63 mV (Wilson, 1997). Considering that the transepithelial potential is only a few millivolts serosa-negative even in asymmetrical conditions (Loretz, 1995), this means that HCO₃⁻ is not passively distributed and that HCO₃⁻ excretion must be active. An active Cl⁻/HCO₃⁻-ATPase was suggested by Wilson (1997). However, considering transmembrane potentials and electrochemical Cl⁻ gradients, HCO₃⁻ excretion can be accounted for by Cl⁻/HCO₃⁻ exchange. A strong electrochemical gradient for Cl⁻ favours Cl⁻ efflux across the basolateral membrane, and part of this efflux has been attributed to a basolateral non-conductive exchange with HCO₃⁻ (Dixon and Loretz, 1986). This exchange, driven by the electrochemical gradient for Cl⁻, could allow HCO₃⁻ to be taken up across the basolateral membrane against its electrochemical gradient. Subsequent HCO₃⁻ efflux across the apical membrane would be favoured by the membrane potential (−100 mV, cytosol negative; Loretz, 1995), which can potentially support a [HCO₃⁻] gradient of approximately 1:60 (cytosol:lumen). The apical HCO₃⁻ efflux could be via a conductive pathway, as suggested by Dixon and Loretz (1986). This suggestion, as well as the proposed involvement of a Cl⁻/HCO₃⁻-ATPase (Wilson, 1997), is, however, based on the absence of an effect of mucosal DIDS at concentrations of 10⁻⁴ and 10⁻⁵ mol l⁻¹, respectively. In the present study, and in the study of Ando (1990), mucosal DIDS at a concentration of 10⁻³ mol l⁻¹ (10–100 times more concentrated than in the previous studies) did reduce the rate of HCO₃⁻ transport, suggesting the presence of an apical Cl⁻/HCO₃⁻ exchanger.

Transepithelial HCO₃⁻ transport could therefore include both a basolateral Cl⁻/HCO₃⁻ exchanger, favoured by the electrochemical gradient for Cl⁻ (as reported by Dixon and Loretz, 1986), and an apical DIDS-sensitive carrier-mediated HCO₃⁻ transport, favoured by the electrochemical gradient for HCO₃⁻ (as suggested by the results of the present study). This model is supported by Ando (1990), who reported additional effects of mucosal and serosal DIDS. In contrast to Ando (1990), we found no effect of serosal DIDS in the present study. This discrepancy could be due to different experimental approaches. Unlike Ando (1990), we worked on intact intestinal segments, in which the serosal muscle layer could have reduced the access of DIDS to the serosal membrane and thus have caused the apparent lack of effect of serosal DIDS. Finally, species-specific differences in the mechanisms of HCO₃⁻ excretion cannot be excluded. Clearly, more studies of transepithelial HCO₃⁻ transport in either isolated epithelia or double-perfused (luminal and vascular) whole-intestine preparations are required to determine the mechanism of base excretion by marine teleost fish.

We thank Annie Bach for excellent technical assistance during the analytical work of the present study.