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COMPARATIVE AND EVOLUTIONARY PHYSIOLOGY

Ouabain-sensitive bicarbonate secretion and acid absorption by the marine teleost fish intestine play a role in osmoregulation

Rosenstiel School of Marine and Atmospheric Sciences, University of Miami, Miami, Florida

Submitted 22 November 2005 ; accepted in final form 16 May 2006

	ABSTRACT

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The gulf toadfish (Opsanus beta) intestine secretes base mainly in the form of HCO₃^– via apical anion exchange to serve Cl^– and water absorption for osmoregulatory purposes. Luminal HCO₃^– secretion rates measured by pH-stat techniques in Ussing chambers rely on oxidative energy metabolism and are highly temperature sensitive. At 25°C under in vivo-like conditions, secretion rates averaged 0.45 µmol·cm^–2·h^–1, of which 0.25 µmol·cm^–2·h^–1 can be accounted for by hydration of endogenous CO₂ partly catalyzed by carbonic anhydrase. Complete polarity of secretion of HCO₃^– and H⁺ arising from the CO₂ hydration reaction is evident from equal rates of luminal HCO₃^– secretion via anion exchange and basolateral H⁺ extrusion. When basolateral H⁺ extrusion is partly inhibited by reduction of serosal pH, luminal HCO₃^– secretion is reduced. Basolateral H⁺ secretion occurs in exchange for Na⁺ via an ethylisopropylamiloride-insensitive mechanism and is ultimately fueled by the activity of the basolateral Na⁺-K⁺-ATPase. Fluid absorption by the toadfish intestine to oppose diffusive water loss to the concentrated marine environment is accompanied by a substantial basolateral H⁺ extrusion, intimately linking osmoregulation and acid-base balance.

bicarbonate transport; chloride absorption; epithelial water transport; seawater ingestion; pH-stat titrations

However, in addition to these cotransport systems, apical anion exchangers have recently been associated with Cl^– and water absorption across the marine teleost intestine (15) and appear to contribute significantly to overall Cl^– absorption. Evidence for intestinal anion-exchange activity, indicated by highly alkaline intestinal fluids with high concentrations of total CO₂ or large cation-anion gaps when total CO₂ was not measured, is abundant in the literature. The earliest report of high total CO₂ in intestinal fluids dates back three-quarters of a century (33), and observations now include a large number of species, including elasmobranchs and sturgeon (14, 31, 39, 43, 46), suggesting that alkaline luminal fluids are common to fish inhabiting marine environments. Experiments on isolated intestinal preparations demonstrate that the source of luminal total CO₂ is HCO₃^– secretion, which may occur along the entire length of the intestine (13–15, 46). A recent study revealed that the intestinal epithelium performs secondary active secretion of HCO₃^–, which accounts for the high luminal total CO₂ concentrations while providing for the active absorption of Cl^– via apical anion exchange in seawater-acclimated European flounder (15). A prediction of strong temperature dependence of this secondary active transport system in the gulf toadfish (Opsanus beta) was tested in the present study by direct measurement of HCO₃^– secretion using a pH-stat approach at 15, 25, and 35°C. Furthermore, whether aerobic energy metabolism and, thus, cellular ATP are required for the putative active HCO₃^– transport was examined.

Studies on the source of HCO₃^– secreted by the intestinal epithelium range from demonstrating that extracellular HCO₃^– transported across the intestinal epithelium provides the majority of HCO₃^– secreted into the intestinal lumen (1) to showing that endogenous epithelial CO₂ constitutes the main source of luminal HCO₃^– (15, 45). Therefore, one goal of the present study was to determine the contribution of endogenous metabolic CO₂ and extracellular HCO₃^– to the overall apical HCO₃^– secretion by the intestine of a marine teleost, the gulf toadfish. To the extent that endogenous CO₂ provides for apical HCO₃^– secretion, cytosolic CO₂ hydration must occur. This prompted examination of the potential involvement of carbonic anhydrase (CA) in intestinal HCO₃^– secretion. Our findings demonstrate that hydration of endogenous CO₂ provides for apical secretion of HCO₃^–, which led to investigations of the fate of H⁺ from CO₂ hydration. The prediction was that H⁺ from CO₂ hydration would be eliminated from the epithelial cells to maintain cellular pH and to prevent reversal of the hydration reaction. Furthermore, because the epithelium exhibits net base secretion, it was predicted that this H⁺ extrusion would occur across the basolateral membrane. The possibility that basolateral H⁺ extrusion by the intestinal epithelium of the gulf toadfish is mediated by Na⁺/H⁺ exchange (NHE), as in the rat pancreatic ducts (38), was examined by characterization of the dependence of apical HCO₃^– secretion on serosal Na⁺ and by pharmacological manipulations of Na⁺ gradients. Finally, the high apical HCO₃^– secretion rates and the resulting need to eliminate H⁺ produced from the CO₂ hydration reaction prompted direct measurements of basolateral H⁺ extrusion. The osmoregulatory role of the intestinal epithelium of marine teleost fish is salt and water absorption to maintain water balance, whereas the gill extrudes salt from the intestine (25). Thus the water absorption across the intestinal epithelium is obligately associated with the need for branchial salt secretion. Our observations of high basolateral H⁺ secretion rates in the water-absorbing intestinal epithelium prompted measurement of net water transport by isolated intestinal epithelia, which ultimately allowed for calculation of the pH in the absorbed fluids. These measurements revealed that intestinal fluid absorption in marine teleosts is associated with a significant acid load in addition to the unavoidable salt gain.

	MATERIALS AND METHODS

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Experimental animals. Gulf toadfish (Opsanus beta) from Biscayne Bay, FL, were obtained from shrimp fishermen during spring and early summer 2005 and transferred to holding facilities at the University of Miami Rosenstiel School of Marine and Atmospheric Sciences. Procedures were approved by our Institutional Animal Care and Use Committee. On arrival, the fish received a prophylactic treatment for ectoparasites, as previously described (27). The fish were provided short lengths of polyvinylchloride tubing as shelters in 80-liter glass aquaria that received a continuous flow of filtered seawater (salinity 27–30 ppt, 22–26°C, from Bear Cut, FL). Fish were fed frozen squid twice weekly, but food was withheld 48 h before experimentation.

pH-stat experiments: general experimental protocol. Segments of the anterior intestine of toadfish (25–40 g body wt) were obtained by dissection, cut open, and placed in tissue holders exposing 0.7-cm² surface area with the serosal muscle layer intact. The true exposed surface area of the intestinal epithelium clearly exceed 0.7 cm² because of the presence of villi and microvilli, but all transport rates are reported as a function of this known gross surface area. The intact intestinal epithelium was subsequently mounted in Ussing chambers (model P2300; Physiological Instruments) containing 1.6 ml of appropriate, pregassed saline in each half-chamber (Table 1). Mixing was achieved by gassing with O₂ through airlifts unless otherwise stated. Pregassing of the luminal saline and continued gassing with CO₂-free gas in the luminal half-chamber ensured stable titration curves throughout the experiments. The Ussing chambers were mounted in chamber holders connected via a pump to a thermostatic water bath maintained at 25°C unless otherwise stated. Current and voltage electrodes connected to amplifiers (model VCC600; Physiological Instruments) recorded the transepithelial potential (TEP) differences under current-clamp conditions at 0 µA. At 60-s intervals, 3-s 50-µA pulses were passed from the mucosal to the serosal side. The TEP measurements were logged on a personal computer using BIOPAC systems interface hardware and Acqknowledge software (version 3.8.1). TEP values are reported with a luminal reference of 0 mV. The luminal half-chamber (unless otherwise stated) was fitted with a combination pH electrode (model PHC4000.8; Radiometer) and a microburette tip, both of which were connected to a pH-stat titration system (model TIM 854 or 856; Radiometer). The pH-stat titration system was grounded to the amplifier to allow for pH readings during current pulsing. The pH-stat titrations were performed on luminal saline solutions at pH 7.800 throughout all experiments, with pH values and rate of acid addition logged on personal computers using Titramaster software (versions 1.3 and 2.1). Luminal pH was generally maintained within ±0.003 pH unit around the set point throughout the experiments. Base secretion rates were calculated from the rate of addition and concentration of titrant (0.0005 N HCl). Common to all experiments was an initial control period of 60 min with stable TEP and base secretion rates before the manipulations outlined below.

Longevity of toadfish intestinal epithelium. Two series of control experiments were performed to assess the temporal performance of the preparation, with gut saline on the luminal side and HCO₃^–/CO₂ saline on the serosal side of the epithelium. In the first series of control experiments, three subsequent 60-min flux periods were separated by a change of the serosal saline. In the second series of control experiments, an initial 60-min control period was followed by two 120-min flux periods. The initial 60-min flux period and the two 120-min flux periods were again separated by serosal saline changes. The serosal saline in the first of the two 120-min flux periods contained 0.1% DMSO, which served as a vehicle control for some of the pharmacology experiments (see below).

Is aerobic energy metabolism required for HCO₃^– secretion? The intestinal HCO₃^– secretion in marine teleosts appears to be of a secondary active nature (15) and can, therefore, be expected to rely on aerobic energy metabolism and, thus, cellular ATP. Cellular ATP levels were suppressed by N₂ gassing of mucosal and serosal saline solutions. Also, for these experiments, the serosal HCO₃^–-free saline was replaced after 60 and 120 min with saline preequilibrated for >90 min with N₂ or O₂, respectively. From 60 to 120 min, gassing of mucosal saline solutions was changed from O₂ to N₂.

Temperature dependence of HCO₃^– secretion. A strong temperature dependence of luminal HCO₃^– secretion was hypothesized to be due to the secondary active transport characteristics of luminal alkalinization. To test this hypothesis, measurements were performed after an initial 60-min control period at 25°C and for 120 min at 15 or 35°C, then the temperature was returned to 25°C for an additional 120-min recovery period. Exposure temperatures were verified by measurements of saline temperature in the Ussing chambers, which revealed that up to 30 min were required for a complete change in temperature from 25°C to 15 or 35°C.

Determining the source of luminal HCO₃^– secretion. To assess whether secreted HCO₃^– is derived from endogenous epithelial CO₂ or is dependent on serosal CO₂ and/or HCO₃^–, experiments were performed to compare secretion rates obtained with serosal HEPES-saline gassed with O₂ with rates obtained with serosal saline containing 5 mM HCO₃^– and gassed with 0.3% CO₂ in O₂. In these experiments, an initial 60-min control period with a HEPES-O₂ serosal saline solution was followed by a 60-min period with the above-mentioned serosal HCO₃^–/CO₂-containing saline solution before a final 60-min control period with HEPES-O₂ serosal saline solution. The HCO₃^– concentrations and CO₂ levels resulted in pH and CO₂ partial pressure typical of the extracellular fluids of toadfish.

Does CA mediate CO₂ hydration? Having established that hydration of endogenous CO₂ is the main source of luminal HCO₃^– secretion, the potential involvement of CA was examined by application of the lipophilic CA inhibitor etoxzolamide (10^–3 M in 0.1% DMSO) to the luminal saline. The CA inhibitor was added after a 60-min control period, and measurements were continued for 120 min thereafter. These experiments were performed with HEPES-O₂-containing serosal saline.

Involvement of serosal H⁺ secretion. To test the hypothesis that serosal H⁺ export is important for luminal HCO₃^– secretion, luminal HCO₃^– secretion was measured during manipulation of serosal pH and, thereby, H⁺ gradients across the basolateral membrane, with the prediction that reduced serosal pH would inhibit luminal HCO₃^– secretion. In these experiments, which were performed with HEPES-O₂-containing serosal saline, an initial 60-min control period at serosal pH 7.800 was followed by a 60-min period with reduced serosal pH and, finally, a 60-min recovery period at serosal pH 7.800. A total of three series were performed on three sets of preparations with serosal pH reduced to 7.4, 7.0, and 6.6.

Rate of basolateral H⁺ extrusion. Considering that CO₂ hydration within the intestinal epithelium provides the main source for luminal HCO₃^– secretion and that basolateral proton extrusion is critical for normal rates of apical HCO₃^– secretion, we tested the prediction that basolateral H⁺ secretion rates across the basolateral membrane would be high. In these experiments, pH-stat titrations were performed with the electrode and burette tip in the serosal, rather than the mucosal, half-chamber, and 0.0005 N NaOH, rather than HCl, was employed as the titrant. The serosal saline for these experiments contained neither HEPES nor HCO₃^– (but was osmotically compensated with mannitol; Table 1) and was gassed with O₂. These titrations were also, in this case, performed with a titration set point of pH 7.800 (normal teleost fish blood plasma pH). Experiments were continued until stable titration and electrophysiological values were obtained for 30 min.

Na⁺ dependence of serosal H⁺ secretion: involvement of NHE. The possibility that secretion of H⁺ across the basolateral membrane occurred via an Na⁺-H⁺ antiport was examined by measuring luminal HCO₃^– secretion under conditions with no serosal HCO₃^– and reduced serosal Na⁺. In these experiments, an initial 60-min control period was followed by a 60-min period in which the serosal saline Na⁺ concentration was reduced to 11 mM and a final 60-min recovery period under control conditions. In the low-Na⁺ saline, the majority of NaCl was replaced with choline chloride. The pH and osmolality were adjusted to match those of the serosal control saline.

Subsequently, experiments with the Na⁺-H⁺ antiport inhibitor ethylisopropylamiloride (EIPA) were performed. In these experiments, the initial 60-min control period was followed 120 min during which serosal saline contained 10^–3 M EIPA in a final concentration of 0.1% DMSO.

Na⁺-K⁺-ATPase inhibitor studies. To further examine whether serosal H⁺ extrusion and, thereby, luminal HCO₃^– secretion rely on Na⁺ gradients, experiments with the Na⁺-K⁺-ATPase inhibitor ouabain were performed. A 60-min control period was followed by a 120-min experimental period in which the serosal saline contained 10^–3 M ouabain. Ouabain was dissolved by sonication in a low volume of serosal saline.

Rates of fluid absorption. For measurement of rates of fluid absorption in the anterior intestine, the fish were killed by an overdose of tricaine methanesulfonate (MS-222; 0.25 g/l), and the anterior segment of the intestine was obtained by dissection. A short length of heat-flared polyethylene tubing was tied in the anterior end of the intestinal segment with double silk ligatures, and the segment was flushed with 20 ml of mucosal saline (Table 1). The proximal end of the 30-mm-long segments was subsequently closed with double silk ligatures. These intestinal sacs were then filled with gut saline containing ¹⁴C-labeled polyethylene glycol (PEG; 0.01 µCi/ml), and a sample of the mucosal saline was obtained as follows: the sac preparations were moderately overfilled, and the saline from the preparations was drawn from the sacs through the polyethylene catheter into a syringe and flushed back and forth three times before a sample was obtained. This procedure ensured that the sample was completely mixed and that this initial sample of gut saline was truly representative of the content of the sac preparation. The catheter was sealed, and the preparations were rinsed in serosal saline before they were submerged individually in 20 ml of serosal saline in glass vials. The serosal saline for these studies contained HCO₃^– and was gassed with the 0.3% CO₂-in-O₂ gas mixture for 90 min before and during experimentation. At the beginning and end of a 3-h flux period, samples were taken from the serosal saline. Finally, at the end of the flux, a sample of the [¹⁴C]PEG-labeled mucosal saline was obtained, the intestinal sac was cut open, and the gross surface area of the exposed epithelial surface was determined from a trace of its outline on graph paper. This type of preparation has been employed in the past and exhibits stable transport characteristics for 6 h (15). Water absorption was determined from the increase in the ¹⁴C radioactivity of the luminal saline during the 3 h of experimentation. Measurements of ¹⁴C radioactivity in the serosal saline solutions confirmed that all [¹⁴C]PEG was retained in the mucosal saline, despite considerable rates of fluid absorption.

Data presentation and statistical analysis. Values are means ± SE of five to eight observations. Statistical evaluation revealed that the data were normally distributed and was performed by paired t-tests with Bonferroni's multisample comparison correction to evaluate the difference between individual time points after treatment and an average control value (34). This average control value was based on the last 30 min of the corresponding initial 60-min control flux. Samples were considered statistically significantly different at P < 0.05.

	RESULTS

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On the basis of relatively stable HCO₃^– secretion rates, TEP, and conductance, the anterior toadfish intestine appears viable and stable for >5 h under the conditions employed in the present study and is not influenced by 0.1% DMSO (Fig. 1). Control experiments of 3-h duration with saline changes every 60 min also revealed stable transport rates and electrophysiological parameters (data not shown). In these initial experiments in the presence of serosal HCO₃^–, HCO₃^– secretion rates were 0.3–0.5 µmol·cm^–2·h^–1 (Fig. 1), whereas TEP and conductance consistently were approximately –20 mV and 10 mS/cm² for all control experiments. Compared with an overall mean control base secretion rate of 0.25 µmol·cm^–2·h^–1 (n = 42) in the absence of serosal HCO₃^–, double end-point titration determination of HCO₃^– equivalents building up in the mucosal saline in the absence of titration revealed an HCO₃^– secretion rate of 0.15 µmol·cm^–2·h^–1 (Table 2), accounting for 60% of overall base secretion. For these experiments, perfusion of the luminal chamber, rather than gas-lift mixing, did not influence TEP or conductance (Table 2, Fig. 1).

View larger version (24K): [in this window] [in a new window]   Fig. 1. Bicarbonate (HCO3–) secretion, transepithelial potential (TEP), and conductance (G) of isolated anterior toadfish intestine under asymmetric conditions mimicking in vivo intestinal fluid chemistry. Serosal saline solutions were replaced after 60 and 180 min and contained 0.1% DMSO at 60–180 min. On the basis of secretion rates and electrophysiological parameters, preparation appears stable for >5 h. Average values for the last 30 min of the initial control period for HCO3– secretion, TEP, and conductance were 0.37 ± 0.01 µmol·cm–2·h–1, –18.1 ± 0.1 mV, and 11.6 ± 0.1 mS/cm2. Values are means ± SE (n = 6).

View larger version (21K): [in this window] [in a new window]   Fig. 2. HCO3– secretion, TEP, and conductance of isolated anterior toadfish intestine under normoxic and anoxic conditions. Serosal and mucosal saline solutions were gassed with O2 at 0–60 min and 120–180 min and with nitrogen at 60–120 min. Average values for the last 30 min of the initial control period for HCO3– secretion, TEP, and conductance were 0.35 ± 0.01 µmol·cm–2·h–1, –19.1 ± 0.1 mV, and 10.0 ± 0.2 mS/cm2. Values are means ± SE (n = 5). *Significantly different from control values, i.e., average of the last 30 min of the initial control period (by paired Student's t-test).

View larger version (34K): [in this window] [in a new window]   Fig. 3. HCO3– secretion, TEP, and conductance of isolated anterior toadfish intestine at 15 and 35°C. Left: initial 60-min period at 25°C followed by 120 min of gradual temperature decrease to 15°C and a final 120 min of return to 25°C (n = 7). Right: initial 60-min period at 25°C followed by 120 min of gradual temperature increase to 35°C and final 120 min of return to 25°C (n = 6). Average values for the last 30 min of the initial control period for HCO3– secretion, TEP, and conductance were 0.31 ± 0.01 µmol·cm–2·h–1, –23.0 ± 0.1 mV, and 11.6 ± 0.1 mS/cm2 for the 15°C series and 0.31 ± 0.01 µmol·cm–2·h–1, –19.5 ± 0.3, and 11.1 ± 0.1 mS/cm2 for the 35°C series. Values are means ± SE (n = 6). *Significantly different from control values (see Fig. 2 legend).

View larger version (21K): [in this window] [in a new window]   Fig. 4. HCO3– secretion, TEP, and conductance of isolated anterior toadfish intestine in the presence and absence of serosal HCO3– and CO2. During the initial 60 min and the last 60 min at 120–180 min, serosal saline solutions contained no HCO3– and were gassed with pure O2. At 60–120 min, serosal saline solutions contained 5 mM HCO3– and was gassed with 0.3% CO2 in O2. Both serosal saline solution contained HEPES and were adjusted to pH 7.800. Average values for the last 30 min of the initial control period for HCO3– secretion, TEP, and conductance were 0.18 ± 0.01 µmol·cm–2·h–1, –17.3 ± 0.1 mV, and 6.3 ± 0.1 mS/cm2. Values are means ± SE (n = 7). *Significantly different from control values (see Fig. 2 legend).

View larger version (22K): [in this window] [in a new window]   Fig. 5. HCO3– secretion, TEP, and conductance of isolated anterior toadfish intestine under control conditions and in the presence of the carbonic anhydrase inhibitor etoxzolamide in luminal saline. Because etoxzolamide slightly reduced luminal pH (0.2 pH unit), HCO3– secretion could not be measured during the first 10–15 min until luminal pH recovered to 7.8. Thus no HCO3– secretion data are reported for the first 2 time intervals. Values are means ± SE (n = 7). Average values for the last 30 min of the initial control period for HCO3– secretion, TEP, and conductance were 0.21 ± 0.01 µmol·cm–2·h–1, –17.3 ± 0.1 mV, and 6.3 ± 0.1 mS/cm2. *Significantly different from control values (see Fig. 2 legend).

View larger version (32K): [in this window] [in a new window]   Fig. 6. HCO3– secretion, TEP, and conductance of isolated anterior toadfish intestine under control conditions (serosal pH 7.800) and serosal pH 7.400, 7.000, and 6.600. Bottom right: fractional maximal inhibition of mucosal HCO3– secretion as a function of serosal pH. Average values for the last 30 min of the initial control period for HCO3– secretion, TEP, and conductance were 0.23 ± 0.01 µmol·cm–2·h–1, –19.0 ± 0.2 mV, and 10.7 ± 0.1 mS/cm2 for pH 7.4, 0 0.22 ± 0.01 µmol·cm–2·h–1, –20.9 ± 0.2 mV, and 10.0 ± 0.1 mS/cm2 for pH 7.0, and 0.24 ± 0.01 µmol·cm–2·h–1, –19.4 ± 0.2 mV, and 10.9 ± 0.3 mS/cm2 for pH 6.6. Values are means ± SE (n = 5–8). *Significantly different from control values (see Fig. 2 legend).

View larger version (14K): [in this window] [in a new window]   Fig. 7. Mucosal HCO3– secretion (n = 42) and serosal H+ extrusion (n = 6) of isolated anterior toadfish intestine at luminal and serosal pH 7.800. Values are means ± SE.

View larger version (20K): [in this window] [in a new window]   Fig. 8. HCO3– secretion, TEP, and conductance of isolated anterior toadfish intestine under control (162 mM serosal Na+) and low-Na+ (11 mM) conditions. Average values for the last 30 min of the initial control period for HCO3– secretion, TEP, and conductance were 0.16 ± 0.01 µmol·cm–2·h–1, –21.2 ± 0.2 mV, and 8.9 ± 0.1 mS/cm2. Values are means ± SE (n = 7). *Significantly different from control values (see Fig. 2 legend).

View larger version (23K): [in this window] [in a new window]   Fig. 9. HCO3– secretion, TEP, and conductance of isolated anterior toadfish intestine under control conditions and in the presence of the Na+/H+ exchange inhibitor ethylisopropylamiloride (EIPA) in serosal saline. Average values for the last 30 min of the initial control period for HCO3– secretion, TEP, and conductance were 0.35 ± 0.01 µmol·cm–2·h–1, –22.8 ± 0.2 mV, and 10.3 ± 0.2 mS/cm2. Values are means ± SE (n = 6). *Significantly different from control values (see Fig. 2 legend).

View larger version (22K): [in this window] [in a new window]   Fig. 10. HCO3– secretion, TEP, and conductance of isolated anterior toadfish intestine under control conditions and in the presence of the Na+-K+-ATPase inhibitor ouabain in the serosal saline. Average values for the last 30 min of the initial control period for HCO3– secretion, TEP, and conductance were 0.35 ± 0.01 µmol·cm–2·h–1, –20.7 ± 0.2 mV, and 9.3 ± 0.3 mS/cm2, respectively. Values are means ± SE (n = 6). *Significantly different from control values (see Fig. 2 legend).

	DISCUSSION

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The isolated toadfish intestine displays stable HCO₃^– transport and electrophysiological characteristics over 5 h, demonstrating that this preparation, in agreement with previous work on European flounder (15), is suitable for the experimental protocols employed in the present study. Comparisons of base secretion rates obtained using pH-stat techniques with rates obtained by direct double-end-point titration measurements of HCO₃^– and CO equivalents revealed that the majority of intestinal base secretion occurs in the form of HCO₃^–. These measurements permit use of the term "HCO₃^– secretion," rather than "base secretion."

As predicted, luminal HCO₃^– secretion appears to rely on aerobic energy metabolism and, thus, ATP. This observation is in agreement with previous observations of reduced H⁺ secretion, which fuels Cl^– uptake (via anion exchange) across anuran skin under anaerobic conditions (21–23), and with findings of HCO₃^– transport in cortical collecting ducts relying on oxidative metabolism (17). Strong temperature dependence is expected for biological reactions mediated by enzymatic reactions and transporters. Typically, increases in reaction velocity of >1.5-fold per 10°C increase are ascribed to biological, rather than physiochemical, processes (19). A 10°C reduction in temperature resulted in a 1.8- to 3.0-fold reduction in luminal HCO₃^– secretion, clearly indicating that intestinal HCO₃^– secretion in the gulf toadfish is biologically mediated. Experiments with increased temperature revealed a lower HCO₃^– secretion response and, perhaps, a general deterioration, as indicated by substantial nonrecoverable reduction in TEP. This latter response may reflect that 35°C is approaching the thermal maximal tolerance for the gulf toadfish, which experience temperatures ranging from 13 to 35°C in its natural environment (3). In any case, the apparent dependence of HCO₃^– secretion on aerobic energy metabolism and, thus, ATP and the strong reduction in secretion rates observed with reduced temperature support previous conclusions that intestinal HCO₃^– secretion in marine teleosts is of a secondary active nature (15).

The continued, although reduced, HCO₃^– secretion in the absence of serosal CO₂ and HCO₃^– demonstrates that endogenous CO₂ provides a significant (50%) source for luminal HCO₃^– secretion under resting conditions, with transepithelial HCO₃^– transport or serosal CO₂ accounting for the remaining secretion. These observations are in agreement with reports of 30–60% of the luminal HCO₃^– secretion being sustained by endogenous epithelial CO₂ in the European flounder and the goby (8, 15, 45) but are in contrast to findings from the Japanese eel, where serosal HCO₃^– seems to fully account for luminal HCO₃^– secretion (1). However, the studies on the Japanese eel employed hyperphysiological serosal HCO₃^– levels [25 mM vs. 5–8 mM normally present in teleost fish extracellular fluids (25)]. Therefore, it seems that endogenous epithelial CO₂ generally contributes significantly to intestinal HCO₃^– secretion in marine teleosts, at least under resting, nonstimulated conditions. The remainder of the studies presented here were designed to investigate the transport processes associated with HCO₃^– secretion from hydration of endogenous CO₂.

The HCO₃^– secretion rates measured in the present study agree well with previous results from marine teleost fish (1, 8, 15, 45, 46), which seem to be on the order of 0.5 µmol·cm^–2·h^–1 under resting conditions in the presence of serosal HCO₃^–. These intestinal HCO₃^– secretion rates from marine teleost fish are 10-fold higher than the only other resting HCO₃^– secretion rate reported from an exothermic vertebrate, the bullfrog (11), and are comparable to the range (0.5–1 µmol·cm^–2·h^–1) of resting duodenal HCO₃^– secretion rates reported from mammals at 37°C (6, 20, 40, 41). The high intestinal HCO₃^– secretion rates in the marine teleost, often at temperatures much below 37°C, and the significant contribution of endogenous CO₂ to luminal alkalinization led to the hypothesis that CA might be involved in accelerating CO₂ hydration to fuel the apical anion exchange. The lipophilic CA inhibitor etoxzolamide clearly inhibited luminal HCO₃^– secretion, although a gradual recovery controlled secretion rates, despite the continued presence of the inhibitor. We interpret this as an immediate depletion of cytosolic HCO₃^– caused by CA inhibition followed by a gradual recovery mediated by noncatalyzed cellular CO₂ hydration, perhaps fueled by an increased cellular partial pressure of CO₂. An increase in partial pressure of CO₂ might be expected in a situation with sustained metabolic CO₂ production and without CA activity. What appears to be a complete inhibition of HCO₃^– secretion in the first 10 min after etoxzolamide addition is caused in part by a slight acidification of the buffer-free mucosal saline in response to drug addition. However, luminal pH of 7.80 was fully recovered in all preparations after 10–15 min of drug addition, when a 50% reduction in luminal HCO₃^– secretion rates persisted. This observation is in agreement with previous reports of reduced, but not fully inhibited, intestinal HCO₃^– secretion by marine teleosts in the presence of CA inhibitors (8, 44) and with the strong physical and functional association between CA and anion exchangers in mammals (35–37).

The H⁺ arising from hydration of CO₂ must be extruded from the epithelial cells to maintain intracellular pH, and furthermore, whereas HCO₃^– is excreted across the apical membrane, the H⁺ must be extruded preferentially across the basolateral membrane, because the intestinal epithelium exhibits strong net base secretion. This polarization of HCO₃^– and H⁺ secretion is demonstrated for the toadfish intestinal epithelium by the reduced HCO₃^– secretion when serosal H⁺ concentration is increased, presumably leading to reduced basolateral H⁺ extrusion, and by direct measurements of basolateral H⁺ secretion. A similar polarization of HCO₃^– and H⁺ secretion is characteristic of the pancreatic duct, which secretes HCO₃^– to reach concentrations of 70–140 mM (38), values similar to HCO₃^– concentrations in marine teleost intestinal fluids (14, 25, 26, 46).

Manipulation of the H⁺ gradient across the basolateral membrane by reduction of serosal pH revealed a concentration-dependent inhibition of luminal HCO₃^– secretion that appeared to be at least partly reversible within 60 min after return to control conditions. Serosal pH as low as 6.6 did not influence epithelial integrity, as indicated by unaltered TEP and conductance. A likely explanation for these observations is that basolateral H⁺ extrusion is reduced by the increased serosal H⁺ concentrations and that this in turn slows the cellular CO₂ hydration and, thus, depletes the cytosolic HCO₃^– available for apical anion exchange. Direct evidence for a full polarization of HCO₃^– and H⁺ secretion come from measurements of H⁺ secretion to serosal fluids, which match those of the luminal HCO₃^– secretion. The basolateral H⁺ secretion is important for the secondary active apical HCO₃^– secretion and must be carrier mediated, because it occurs against an electrochemical gradient. Under serosal HCO₃^–-free conditions, the involvement of a basolateral Na⁺-dependent Cl^–/HCO₃^– exchanger (5) in H⁺ extrusion can be excluded, leaving NHE and H⁺-ATPase as two possible mechanisms of basolateral H⁺ excretion. The marked reduction in apical HCO₃^– secretion in response to lowered serosal Na⁺ concentrations clearly indicates the involvement of an Na⁺-dependent H⁺ extrusion mechanism in HCO₃^– secretion by the marine teleost intestine and is consistent with transport mechanisms involved in rat pancreatic duct HCO₃^– secretion (29, 38). However, although the dependence of luminal HCO₃^– secretion on serosal Na⁺ strongly suggests the involvement of a basolateral NHE-like protein, this could not be confirmed pharmacologically, because neither serosal amiloride nor EIPA at 10^–3 M influenced luminal HCO₃^– secretion. The inhibitor concentrations should be sufficient to inhibit H⁺ extrusion via EIPA-sensitive NHE isoforms (4), but the lack of amiloride and EIPA effects does not necessarily exclude the involvement of NHE, inasmuch as certain NHE isoforms or spliced variants of NHE isoforms are EIPA insensitive (2, 47).

Serosal addition of the Na⁺-K⁺-ATPase inhibitor ouabain (10^–3 M) results in an 70% inhibition of luminal HCO₃^– secretion after 120 min. The reduced HCO₃^– secretion is likely related to reduced basolateral H⁺ extrusion via NHE resulting from the ouabain-induced reduction in basolateral electrochemical Na⁺ gradient.

The basolateral H⁺ extrusion is necessary for apical HCO₃^– secretion, as discussed above, and this basolateral H⁺ extrusion seems to occur via an EIPA-insensitive NHE mechanism. The basolateral NHE is driven by the electrochemical Na⁺ gradient established by the basolateral Na⁺-K⁺-ATPase, which thereby fuels the apical, secondary active HCO₃^– secretion (Fig. 11).

View larger version (16K): [in this window] [in a new window]   Fig. 11. Schematic transport model of HCO3– secretion by the gulf toadfish intestinal epithelium. Model includes polarized apical Cl–/HCO3– exchange and basolateral EIPA-insensitive Na+/H+ exchange, which rely on the Na+ gradient established by Na+-K+-ATPase. Hydration of endogenous CO2, partly catalyzed by carbonic anhydrase (CA), supplies the majority of the cellular substrate for apical anion exchange. Basolateral HCO3– uptake, possibly via Na+-HCO3– cotransport, may be involved in apical HCO3– secretion.

The substrate for luminal HCO₃^– secretion by the gulf toadfish intestine is concluded to be a combination of endogenous epithelial CO₂ hydration and serosal CO₂ and/or HCO₃^–, with endogenous CO₂ hydration accounting for 50% of the total HCO₃^– secretion rates under resting, in vivo-like conditions. The present study demonstrates that the polarized extrusion of H⁺ and HCO₃^– (arising from cellular CO₂ hydration) accounts for 50% of the net epithelial secretion of HCO₃^–. The nature of the remaining 50% of the overall HCO₃^– secretion, which relies on serosal HCO₃^– and, possibly, CO₂, offers an interesting area for further studies. An Na⁺-HCO₃^– cotransporter is a likely candidate for basolateral HCO₃^– uptake by intestinal epithelial cells (Fig. 11) and could provide for apical anion exchange, as in the case of the rat, pig, and guinea pig pancreatic ducts (38), but this possibility remains to be investigated.

High rates of intestinal anion exchange represent a significant contribution to marine teleost osmoregulation and, thus, can be expected to be regulated, depending on salt and water balance. In addition, intestinal HCO₃^– secretion in fish likely serves the same functions as in mammals, where duodenal HCO₃^– secretion protects against acidic gastric effluent (6) and can be potently stimulated by multiple factors. The characteristics of the gulf toadfish intestinal HCO₃^– secretion described in the present study apply to unstimulated, resting epithelia, and HCO₃^– secretion by stimulated epithelia may display different characteristics. The involvement of an H⁺-ATPase in H⁺ extrusion and a more significant contribution of transepithelial HCO₃^– transport to overall luminal HCO₃^– secretion may apply to stimulated intestinal epithelia.

In a recent study, it was suggested that basolateral H⁺ extrusion from marine teleost intestine may account for a substantial fraction of intestinal H⁺ absorption and that the H⁺ secretion from the epithelium across the basolateral membrane could be as high as 0.3 µmol·cm^–2·h^–1 (15). In the present study, this suggestion was verified by direct measurement of basolateral H⁺ extrusion at 0.3 µmol·cm^–2·h^–1, which, combined with direct measurements of water absorption, yields a theoretical H⁺ concentration of 76.5 mM in the absorbed fluid. From this calculation and previous reports (15), it is clear that intestinal fluid absorption by marine teleosts is accompanied by acid absorption (equivalent to a theoretical pH of 1.1 in the absorbed fluid with the assumption of no buffer capacity) and that maintenance of systemic acid-base balance requires compensation for the acid load associated with vital intestinal water absorption.

Interestingly, elevated extraintestinal excretion of acidic equivalents when intestinal base secretion is stimulated has been reported recently (45), illustrating this intimate link between osmoregulation and acid-base balance.

Perspectives

The marine teleost intestine, which is capable of secondary active HCO₃^– secretion, resulting in luminal concentrations >100 mM in some cases, displays polarized apical HCO₃^– and basolateral H⁺ secretion arising from endogenous CO₂ hydration. CA-catalyzed CO₂ hydration provides significantly for the apical anion exchange, whereas the electrochemical Na⁺ gradient fuels basolateral H⁺ extrusion via an EIPA-insensitive NHE mechanism. Basolateral H⁺ extrusion is required for continued secondary active HCO₃^– secretion, and the energy for this transport process is ultimately supplied from the basolateral Na⁺-K⁺-ATPase, as illustrated by ouabain-sensitive HCO₃^– secretion. Under nonstimulated conditions, transepithelial HCO₃^– transport, possibly via Na⁺-HCO₃^– cotransporter-like proteins, appears to contribute 50% to overall secretion rates. The intestinal apical anion exchange contributes significantly to Cl^– and, thereby, water absorption across marine teleost intestine, and a consequence of the CO₂ hydration required for this transport pathway is a highly acidic absorbate. If we consider that the immense diversity among marine teleosts and possible diversity in epithelial transport mechanisms, it is likely that the macroscopic intestinal epithelium may provide useful models for mammalian HCO₃^– secreting and/or fluid-absorbing epithelia.

	GRANTS

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This work was supported by National Science Foundation Grant 0416440 to M. Grosell.

	FOOTNOTES

 

Address for reprint requests and other correspondence: M. Grosell, Rosenstiel School of Marine and Atmospheric Sciences, Univ. of Miami, 4600 Rickenbacker Causeway, Miami, FL 33149-1098 (e-mail: mgrosell{at}rsmas.miami.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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