Am J Physiol Regul Integr Comp Physiol 299: R62–R71, 2010.
First published April 21, 2010; doi:10.1152/ajpregu.00761.2009.
Modulation of NaCl absorption by [HCO⫺
3 ] in the marine teleost intestine is
mediated by soluble adenylyl cyclase
Martin Tresguerres,1 Lonny R. Levin,1 Jochen Buck,1 and Martin Grosell2
1
Department of Pharmacology, Weill Cornell Medical College, New York, New York; and 2Rosenstiel School of Marine
Atmospheric Sciences, University of Miami, Miami, Florida
Submitted 16 November 2009; accepted in final form 21 April 2010
Tresguerres M, Levin LR, Buck J, Grosell M. Modulation of
NaCl absorption by [HCO⫺
3 ] in the marine teleost intestine is mediated by soluble adenylyl cyclase. Am J Physiol Regul Integr Comp
Physiol 299: R62–R71, 2010. First published April 21, 2010;
secretion and
doi:10.1152/ajpregu.00761.2009.—Intestinal HCO⫺
3
NaCl absorption are essential for counteracting dehydration in marine
teleost fish. We investigated how these two processes are coordinated
in toadfish. HCO⫺
3 stimulated a luminal positive short-circuit current
(Isc) in intestine mounted in Ussing chamber, bathed with the same
saline solution on the external and internal sides of the epithelium.
The Isc increased proportionally to the [HCO⫺
3 ] in the bath up to 80
mM NaHCO3, and it did not occur when NaHCO3 was replaced with
Na⫹-gluconate or with NaHCO3 in Cl⫺-free saline. HCO⫺
3 (20 mM)
induced a ⬃2.5-fold stimulation of Isc, and this [HCO⫺
3 ] was used in
all subsequent experiments. The HCO⫺
3 -stimulated Isc was prevented
or abolished by apical application of 10 ␮M bumetanide (a specific
inhibitor of NKCC) and by 30 ␮M 4-catechol estrogen [CE; an
inhibitor of soluble adenylyl cyclase (sAC)]. The inhibitory effects of
bumetanide and CE were not additive. The HCO⫺
3 -stimulated Isc was
prevented by apical bafilomycin (1 ␮M) and etoxolamide (1 mM),
indicating involvement of V-H⫹-ATPase and carbonic anhydrases,
respectively. Immunohistochemistry and Western blot analysis confirmed the presence of an NKCC2-like protein in the apical membrane
and subapical area of epithelial intestinal cells, of Na⫹/K⫹-ATPase in
basolateral membranes, and of an sAC-like protein in the cytoplasm.
We propose that sAC regulates NKCC activity in response to luminal
⫹
HCO⫺
3 , and that V-H -ATPase and intracellular carbonic anhydrase
are essential for transducing luminal HCO⫺
3 into the cell by CO2/
HCO⫺
3 hydration/dehydration. This mechanism putatively coordinates
HCO⫺
3 secretion with NaCl and water absorption in toadfish intestine.
NKCC; cAMP; carbonic anhydrase; bumetanide; proton pump
THE INTESTINE OF MARINE FISH absorbs NaCl and water to compensate for dehydration in a hyperosmotic environment. This
hypoosmoregulatory mechanism relies in part on HCO⫺
3 secretion, in exchange for Cl⫺ uptake, resulting in intestinal luminal
⫺
concentration of HCO⫺
3 {[HCO3 ]} of ⬎ 100 mM (20, 65). The
resulting alkaline conditions lead to the formation of Ca2⫹ and
Mg2⫹ carbonate precipitates and a reduction of osmotic pressure (20, 65). Water is absorbed following transepithelial NaCl
absorption, and the carbonate precipitates are expelled with the
feces. Intestinal carbonate precipitation takes place in all marine bony fish species investigated so far, and it has been
argued that the combined piscine carbonates output contributes
up to 15% of total oceanic carbonate production (64).
In the intestine of marine fish, the major pathway for NaCl
absorption from the lumen and into the intestinal cells is
Address for reprint requests and other correspondence: M. Tresguerres,
Dept. of Pharmacology, Weill Cornell Medical College, 1300 York Ave., New
York, NY 10065 (e-mail: [email protected]).
R62
through apical Na⫹-K⫹-2Cl⫺ cotransporters (NKCC), also
known as bumetanide-sensitive cotransporter. In mammalian
epithelia, the apical NKCC isoform is predominantly present in
the thick ascending loop of Henle, and it is termed NKCC2 (10,
19, 28, 32, 39, 48, 49, 66). Several studies have confirmed the
involvement of an NKCC2-like protein in fish intestine. The
evidence includes a mucosal-positive transepithelial potential
difference under in vivo-like conditions, related to Cl⫺- dependent short-circuit current (Isc) (1), codependence of Na⫹
and Cl⫺ transport (12, 15, 38), dependence on an apical K⫹
conductance (14, 38), and, importantly, sensitivity to the loop
diuretic bumetanide (15, 38, 42, 43). The presence of intestinal
NKCC2 was confirmed by RT-PCR in seawater eels (7) and by
immunohistochemistry in mudskipper from brackish water
(65), killifish (34), and sea bass (31). Like many epithelia, the
driving force for NaCl absorption is provided by basolateral
Na⫹/K⫹-ATPases.
An additional pathway for apical Cl⫺ entry is apical anion
exchange, which can, in some cases, account for as much as
70% of the overall Cl⫺ absorption by the intestinal epithelium
(20). Interestingly, elevation of luminal NaCl results in a
reduction of HCO⫺
3 secretion, despite the increased concentration of luminal substrate (Cl⫺) for the process (23). This
response (reduction in anion exchange) to elevated luminal
NaCl, which would favor salt absorption by cotransport pathways, suggests that coordination of the multiple Cl⫺ uptake
pathways takes place in the marine teleost intestine.
Intestinal bicarbonate secretion and NaCl and water absorption are regulated by several factors. In the winter flounder,
atrial natriuretic factor and its downstream intracellular second
messenger cGMP inhibit the ion-absorbing Isc (41, 42), and
they also reduce the net inward flux of Na⫹ and Cl⫺ (41).
These effects may be related to the inhibition of NaCl and
water absorption when euryhaline fish enter freshwater (15,
37), but they also could be involved in acclimation to seawater
in eel (58). At least part of the cGMP effect is attributable to
inhibition of apical NKCC and K⫹ channels activity (51), a
regulation that also takes place in the mammalian thick ascending loop of Henle (2).
Addition of 8-Br-cAMP (an exogenous, phosphodiesteraseresistant cAMP analog) also affects Isc, but the inhibition is
incomplete and the underlying cellular mechanisms are poorly
understood. Some studies have applied 8-Br-cAMP in combination with theophylline, and they have found robust inhibition
of Isc and Na⫹ and Cl⫺ absorption (13, 15). However, theophylline is a nonspecific phosphodiesterase inhibitor that prevents
both cAMP and cGMP degradation. When applied alone,
8Br-cAMP increased both Cl⫺ absorption and secretion in a
manner that resulted in a reduction (although not statistically
significant) of net Cl⫺ absorption, together with inhibitions of
0363-6119/10 Copyright © 2010 the American Physiological Society
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sAC REGULATES NaCl ABSORPTION IN FISH INTESTINE
the outside-positive Isc and net K⫹ secretion and an increase in
transepithelial conductance (Gte) (51). Thus, it appears that
regulation of ion transport across fish intestine by cAMP
involves multiple, possibly opposing, pathways.
The anion bicarbonate is an overlooked factor that regulates
ion absorption in fish intestine. Reducing the saline’s [HCO⫺
3]
from 20 mM to 5 mM induces an ⬃60% reduction of Cl⫺
absorption and Isc, a 30% reduction of Na⫹ absorption, and a
significant increase in Gte (12, 13). HCO⫺
3 stimulation of ion
absorption has been attributed to increases in pH (12), which
stimulates NaCl and water absorption on its own (44). However, it is possible that both pH and HCO⫺
3 have synergistic
stimulatory effects via independent mechanisms not yet identified.
One of such potential mechanisms is the newly identified
soluble adenylyl cylase (sAC). sAC is an evolutionary conserved signaling enzyme whose activity is directly modulated
by HCO⫺
3 to produce cAMP (3, 6, 63). Unlike the classic,
hormonal- and G protein-regulated transmembrane ACs
(tmACs), sAC is present in the cell cytoplasm and in organelles
(67, 68). sAC has been proposed to be a universal sensor of
HCO⫺
3 , and indirectly, of pH and PCO2 (6). Furthermore, sAC
has recently been shown to be present in an elasmobranch fish,
the dogfish Squalus acanthias, where it is essential for activating branchial H⫹ absorption and HCO⫺
3 secretion to counteract
blood alkalosis (63). Like mammalian and cyanobacterial
sACs (6, 25, 55), dogfish sAC (dfsAC) is bicarbonate responsive and inhibited by two unrelated small molecules, 4-catechol estrogen (CE) and KH7 (63).
In the present paper, we have investigated whether sAC is
involved in regulating NaCl absorption across the intestinal
epithelium of a marine fish in response to elevated HCO⫺
3, a
feature that might explain previous results and may also apply
to other NaCl-absorbing epithelia.
METHODS
Animals. Gulf toadfish (Opsanus beta) 20 –30 g in size, from
Biscayne Bay, FL, were obtained from shrimp fishermen during the
winter and spring of 2008 and 2009 and transferred to holding
facilities at the University of Miami Rosenstiel School of Marine and
Atmospheric Sciences. Procedures were approved by the University
of Miami Animal Care and Use Committee. On arrival, the fish
received a prophylactic treatment for ectoparasites, as previously
described (35). 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, 22–25°C, from Bear Cut,
Florida). Fish were fed frozen squid twice weekly, but food was
withheld 48 h before experimentation. Only tissues from males and
nonovigerous females were used for the procedures outlined below.
Isc and Gte measurements. Toadfish were anaesthetized with MS222 (0.5 g/l; Argent Laboratories, Redmond, WA), and the spinal
chord was severed. After opening the abdominal cavity, the anterior
intestine (from the pyloric sphincter to ⬃1.5 cm distally) was dissected, cut open, and mounted in a tissue holder that exposed 0.7
cm⫺2 epithelial surface area (not considering villi and microvilli). The
tissue-containing holder was inserted in an Ussing chamber (model
P2300; Physiological Instruments, San Diego, CA) containing 2 ml of
pregassed saline. During the experiments, gas mixes were delivered to
each hemichamber through airlifts to maintain O2, CO2 and HCO⫺
3
levels constant and promote mixing in the bathing solutions. The
Ussing chambers were placed in chamber holders maintained at 25 ⫾
1°C throughout experimentation. Current and voltage electrodes
(model P2020-S; Physiological Instruments) connected to amplifiers
R63
(model VCC600; Physiological Instruments) recorded the Isc under
voltage-clamp conditions (0 mV). At 60-s intervals, the Gte was
calculated from an imposed 1-mV voltage pulse from the mucosal
to the serosal side and the resulting current deflection. Isc measurements were logged on a personal computer using a data acquisition
system (BIOPAC systems interface with AcqKnowledge software,
version 3.8.1).
The bathing saline was applied symmetrically into both hemichambers, and its composition was (in mM) 151 NaCl, 3 KCl, 0.88 MgSO4,
4.6 Na2HPO4, 0.48 K2HPO4, 1 CaCl2, 11 HEPES-free acid, 11
HEPES-free salt and 4.5 urea. All saline solutions were bubbled with
100% O2 or 0.3% CO2-99.7 O2 before adjusting osmolarity to 330
mOsm and pH to 7.8. Serosal saline also contained 3 mM glucose.
Mounted epithelia were initially bathed in this saline (notice the
absence of HCO⫺
3 ) gassed with 100% O2 until a stable Isc was
reached. To elevate [HCO⫺
3 ], different volumes of a 1 M NaHCO3
stock solution were pipetted into both hemichambers to avoid diffusive fluxes, and the gas was changed to 0.3% CO2-99.7 O2. Pharmacological inhibitors were injected into the luminal bath from concentrated stocks in DMSO, and the added volume of drug was 2 ␮l in all
cases. DMSO alone had no effect on Isc or Gte. After each change of
saline or addition of drug, it typically took 15–20 min for Isc and Gte
to reach new stable values. The Isc and Gte after at least additional 20
min of stable readings were used for statistical analyses.
Western blot analysis and immunofluorescence. The following
antibodies were used: mouse monoclonal ␣5 (against ␣1- and ␤1subunits of chicken Na⫹/K⫹-ATPase; 0.01 ␮g/ml in Western blot
analysis, 0.05 ␮g/ml in immunofluorescence), mouse monoclonal T4
(against the carboxy-term of human NKCC Met-902 to Ser-1212;
0.5 ␮g/ml in Western blot analysis, 0.2 ␮g/ml in immunofluorescence), rabbit polyclonal dfsAC antibodies [against an epitope in
dfsAC: INNEFRNYQGRINKC (63); 0.15 ␮g/m1 in Western blot
analysis, 70 ␮g/ml in immunofluorescence], and mouse monoclonal R7 [against an epitope in mammalian sAC: EIERIPDQRAVKV
(27); 0.1 ␮g/ml in Western blot analysis, 20 ␮g/ml in immunofluorescence]. The ␣5 and T4 antibodies developed by Douglas
Fambourgh (John Hopkins University, Baltimore, MD) and Christian Lytle (University of California Riverside, Riverside, CA),
respectively, were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child
Health and Human Development and maintained by Department of
Biological Sciences, The University of Iowa, Iowa City, IA.
For Western blot analysis, the anterior intestine was dissected, flash
frozen in liquid N2 and stored at ⫺80°C. Samples were homogenized
in lysis buffer (in mM: 150 NaCl, 5 EDTA, 50 Tris, 1 PMSF, 1 DTT,
and 10 ␮g/ml aprotinin, 10 ␮g/ml leupeptin, pH 7.5) by using a glass
Dounce homogenizer and were spun down (10 min, 16,000 g, 4°C).
Approximately 10 ␮l of the supernatant (15 ␮g total protein) was
combined with 2⫻ Laemmli buffer (30), heated (70°C, 10 min), and
used in PAGE-Western blot analysis. Purified recombinant rat sAC
protein, produced as previously described (6) was used as control for
the R7 antibody.
For immunofluorescence, the anterior intestine was dissected, cut
into ⬃2 mm sections, and immersed in fixative (4% paraformaldehyde
in 0.1 M PBS) overnight at 4°C. The fixative was replaced by ice-cold
15% (wt/vol) sucrose, incubated for 4 h at 4°C, then replaced by 30%
(wt/vol) sucrose, and stored at 4°C. Fixed tissues were sectioned at
4 – 8 ␮m using a cryostat (⫺15°C) and immunostained. Sections were
treated with 1% SDS in PBS, followed by 2⫻ 5 min washes in PBS
and blocking in 2% normal goat serum in PBS for 1 h at room
temperature. Incubation with the antibodies (in 2% normal goat serum
in PBS) was performed overnight at 4°C. After 3⫻ 5 min washes in
PBS, the secondary antibodies Alexa Fluor 488-conjugated antimouse IgG or Alexa Fluor 568-conjugated anti-rabbit IgG were
applied for 1 h at room temperature and subsequently washed 3⫻ 5
min in PBS. For double labeling, sections were first labeled with
dfsAC (overnight) and fluorescent anti-rabbit IgG (1 h), followed by
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sAC REGULATES NaCl ABSORPTION IN FISH INTESTINE
experiments, 20 mM NaHCO3 was used because it induced
significant changes on Isc and Gte and it was within or near
physiological values for both the luminal and the serosal sides.
Addition of 20 mM NaHCO3 raised the saline’s pH from 7.80
to 8.15. Since a pH of 8.15 did not change Isc significantly and
it only produced a small increase in Gte (Fig. 1C), the HCO⫺
3
stimulation of Isc by 20 mM HCO⫺
3 is independent of pH.
sAC in toadfish intestine. We hypothesized that sAC, a
recently discovered HCO⫺
3 -responsive signaling enzyme, mediates the HCO⫺
3 -stimulation of Isc in toadfish intestine. Polyclonal antibodies against dfsAC recognized a single specific
band of ⬃100 kDa in anterior intestine samples from toadfish
(Fig. 2A), which was absent in the peptide preabsorption and
preimmune serum controls (not shown). A mouse monoclonal
antibody (R7), raised against a different epitope in mammalian
sAC, also recognized a specific ⬃100 kDa band (Fig. 2B). sAC
in the toadfish intestine was detected by immunofluorescence
using dfsAC (Fig. 3, A, C, and E) and R7 (Fig. 3, D and E)
antibodies. sAC immunoreactivity was present throughout the
epithelial intestinal cells but not directly on the apical membrane. Incubation with preimmune serum (Fig. 3B), mouse
IgG, or dfsAC antibodies preabsorbed with specific peptide
(not shown) resulted in no signal.
Involvement of sAC in the stimulation of Isc by HCO⫺
3.
Micromolar concentrations of CE specifically inhibits all
known sAC and sAC-related enzymes, including CyaC from
cyanobacteria (55), dfsAC (63), and mammalian sAC (47, 55).
CE (30 ␮M) significantly reduced the Isc in the absence of
HCO⫺
3 in the bathing saline and slightly increased Gte (Fig.
4A). Subsequent addition of 20 mM HCO⫺
3 in the presence of
CE failed to stimulate Isc, but Gte increased similar to controls;
the CE inhibition of Isc was only partially reversible (Fig. 4A).
Application of CE after Isc had been stimulated by 20 mM
⫺
HCO⫺
3 completely abolished the HCO3 stimulation (Fig. 4B).
17␤-estradiol (30 ␮M), a compound related to CE that is inert
toward sAC-like cyclases (47, 55) did not significantly affect
Fig. 1. HCO⫺
3 stimulation of short-circuit current (Isc) and transepithelial
conductance (Gte) across toadfish anterior intestine: dose-response to NaHCO3
(A), dose-response to Na⫹-gluconate (B), and effect of 20 mM NaHCO3 and
the equivalent change in pH (C) . Bic, bicarbonate. Letters indicate different
levels of statistical significance (repeated-measures ANOVA, Tukey’s multiple-comparisons test, P ⬍ 0.05, n ⫽ 4 –5).
blocking and 1-h incubation at room temperature (T4) or overnight
incubation at 4°C (R7) and anti-mouse fluorescent IgG (1-h room
temperature) with 3⫻ 5 min washes in PBS in between. All sections
were mounted using 4=,6-diamidino-2-phenylindole dihydrochloride
(DAPI)-containing medium (Vector Labs).
Statistical analyses. All data sets were analyzed using one-way
repeated-measures ANOVA, followed by Tukey’s multiple-comparisons test using GraphPad Prism version 4.0a for Macintosh (GraphPad
Software, San Diego, CA). Differences between means were considered statistically significant when P ⬍ 0.05.
RESULTS
Bicarbonate stimulates Isc and Gte. Increasing the
[NaHCO3] in the bathing solutions resulted in dose-dependent
increases in the outside-positive Isc and in Gte (Fig. 1A). These
effects were not due to the extra Na⫹ or to increases in osmotic
pressure, since Na⫹-gluconate has no effect on Isc or Gte (Fig.
1B). Therefore, the observed stimulations were due to HCO⫺
3
itself and/or to the associated increase in pH. For subsequent
Fig. 2. Soluble adenylyl cyclase (sAC) in toadfish (TF) intestine. Western blot
analysis using antibodies (Ab) against dogfish sAC (dfsAC; A) and mammalian
sAC mouse monoclonal antibody R7 (B). A purified, 50-kDa isoform of rat
sAC (sACt) was used as control for R7. The second lane in each panel shows
preabsorption with peptides (pept) corresponding with the respective epitopes.
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Fig. 3. sAC in toadfish intestine. Immunofluorescence detection using antibodies against dfsAC (A
and C; red) and mammalian sAC (D; R7, green).
B: incubation with dfsAC preimmune serum.
Brightfield and fluorescent pictures (A and B) are
overlaid. E: double staining using dfsAC and R7
antibodies. Yellow indicates colocalization of signals. Nuclei are stained with 4=,6-diamidino-2phenylindole dihydrochloride (DAPI; blue) (A, B,
and E). Scale bars: 10 ␮M. F: sequences and
positions of antigen peptides in rat and dfsAC.
Conserved amino acid residues are shaded in
black, similar residues are in grey.
the HCO⫺
3 -stimulated Isc (Fig. 4C). Although similar concentrations of KH7, the most specific sAC inhibitor available (25),
failed to affect Isc, most KH7 precipitated immediately after
entering the bathing saline. Precipitation was exacerbated by
the continuous gas bubbling necessary during these experiments. To increase the KH7 concentration ([KH7]) in solution,
the nominal [KH7] was raised to 200 ␮M, which resulted in
significant inhibition of the HCO⫺
3 -stimulated Isc (Fig. 4D).
The real [KH7] in solution during these experiments is unknown, but based on the visible precipitate it certainly was
much lower than 200 ␮M.
Although the specificity of CE for sAC is much higher
than for tmACs, high concentrations of CE can also inhibit
tmACs (55). To test the potential involvement of tmACs, we
applied 2=5=-dideoxyadenosine (50 ␮M), which inhibits
tmAC-dependent cellular processes without affecting sAC
(50). Unlike CE, 2=5=-dideoxyadenosine increased the outside-positive HCO⫺
3 -stimulated I sc (Fig. 4E). These results
suggest that the CE effect is not due to cross-reaction with
tmACs.
NKCC and Na⫹-K⫹-ATPase in toadfish intestine. Two major players in fish intestinal NaCl absorption are apical
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Fig. 4. Involvement of sAC in the HCO⫺
3 stimulation (20 mM) of Isc and Gte across toadfish anterior
intestine. A: sAC inhibitor 4-catechol estrogen (CE;
30 ␮M) prevents the HCO⫺
3 stimulation of Isc.
B: CE inhibits the Isc previously stimulated by
HCO⫺
3 . C: 17␤-estradiol (Est) does not affect the
stimulated Isc. D: KH7 (200 ␮M), another sAC
inhibitor, also inhibits the Isc previously stimulated
by HCO⫺
3 . The real KH7 concentration is not known
because KH7 was only partially soluble in the bathing saline. E: 2=5=-dideoxyadenosine (Dideox, 50
␮M), a specific inhibitor of transmembrane adenylyl
cyclases, has the opposite effect on Isc compared
with CE and KH7. Letters indicate different levels
of statistical significance (repeated-measures
ANOVA, Tukey’s multiple-comparisons test, P ⬍
0.05; n ⫽ 5– 6).
NKCC and basolateral Na⫹/K⫹-ATPase. However, these
transporters have never been shown to be present in toadfish
intestine. T4 (against human NKCC) and ␣5 (against avian
Na⫹/K⫹-ATPase) monoclonal antibodies recognized single bands
of ⬃180 and ⬃100 kDa, respectively (Fig. 5, A and D), which
match previously published results from fish (9, 26, 60, 61). In
immunostained sections, both transporters were found in almost all epithelial cells, with the exception of larger, round
cells that are presumably goblet (mucus secreting) cells.
NKCC was predominantly present in the apical membrane and
subapical area (Fig. 5E), indicative of an NKCC2 isoform.
Weaker NKCC immunostaining was also found throughout
the cell (presumably an NKCC1 isoform), but the signal was
only observed after increasing the sensitivity of the detector
significantly. Na⫹/K⫹-ATPase was present throughout the
cells, presumably in basolateral infoldings typical of marine
fish intestine (12, 40, 53); Na⫹/K⫹-ATPase immunoreactivity was absent from the apical membrane (Fig. 5, B and C).
Because NKCC and sAC immunoreactivities overlapped in
the subapical region (Fig. 5F), we next tested whether the
HCO⫺
3 and sAC stimulation of I sc was due to activation of
NKCC.
Involvement of NKCC in the stimulation of Isc by HCO⫺
3.
Sensitivity to bumetanide is a hallmark of NKCC (66). Apical
application of bumetanide (10 ␮M) completely prevented (Fig. 6A)
or abolished (Fig. 6B) the HCO⫺
3 -stimulated Isc. The inhibitory
effects of bumetanide and of the sAC inhibitor CE were not
additive, regardless of the order of application (Fig. 6, A–C),
suggesting that sAC and NKCC belong in the same pathway. To
confirm that the Isc was indeed carried by Cl⫺ ions, we
conducted experiments in chloride-free bathing saline (chlorides substituted by gluconates). In this saline, 20 mM
HCO⫺
3 did not stimulate I sc or G te (Fig. 6D). Although 40
⫺
mM HCO⫺
3 did stimulate I sc in Cl -free conditions, the
stimulation was minor, and it was not inhibited by bumetanide (Fig. 6D), indicating the effect is different from that
studied in normal, Cl⫺-containing saline.
Involvement of V-H⫹-ATPase and carbonic anhydrase in the
⫹
stimulation of Isc by HCO⫺
3 . Apical addition of the V-H ⫺
ATPase inhibitor bafilomycin (1 ␮M) prevented the HCO3 stimulated Isc (Fig. 7A). Etoxolamide (1 mM), a permeable
inhibitor of carbonic anhydrase, also prevented the HCO⫺
3stimulated Isc, and it additionally reduced the basal Isc under
HCO⫺
3 -free conditions (Fig. 7B).
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Fig. 5. Na⫹/K⫹-ATPase (NKA), Na⫹-K⫹-2Cl⫺ cotransporter (NKCC) and sAC immunoreactivity in
toadfish intestine. NKA: Western blot analysis (n ⫽
3) (A); immunofluorescence (B) and brightfield
overlay (C). Nuclei have been stained with DAPI
(blue). Notice that NKA is present in basolateral infoldings and absent in the apical membrane. NKCC:
Western blot analysis (n ⫽ 3) (D); immunofluorescence (E). Notice that NKCC signal is strongly apical
and subapical. F: overlay of NKCC signal (green) and
sAC signal (red). Scale bars: 10 ␮M.
DISCUSSION
The intestine of marine fish is an excellent model to study
epithelial NaCl absorption because of the homogeneity of cell
types and its well-characterized electrophysiology. In fact, the
luminal-positive Isc across fish intestine has been established to
be equivalent to the asymmetrical net Na⫹ and Cl⫺ absorption
(reviewed in Ref. 37).
Our results demonstrate that the anion HCO⫺
3 stimulates
NaCl absorption across the anterior intestine of toadfish, an
effect that is independent of alkaline pH. Regulation of NaCl
Fig. 6. Involvement of NKCC in the HCO⫺
3 -stimulation (20 mM) of Isc and Gte across toadfish anterior
intestine. A: NKCC inhibitor bumetanide (Bum; 10
␮M) prevents the HCO⫺
3 stimulation of Isc, the sAC
inhibitor CE (30 ␮M) does not induce any further
inhibition. B: bumetanide inhibits the Isc previously
stimulated by HCO⫺
3 ; CE does not have any further
effect. C: same as in B, but the order of application
of CE and bumetanide are reversed. D: in the absence of chloride ions in the bathing saline (replaced
by gluconate), 20 mM NaHCO3 does not stimulate
Isc or Gte. NaHCO3 (40 mM) induced a small,
significant stimulation but it was not dependent on
NKCC. Letters indicate different levels of statistical
significance (repeated-measures ANOVA, Tukey’s
multiple-comparisons test, P ⬍ 0.05; n ⫽ 5– 6).
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Fig. 7. Involvement of V-H⫹-ATPase (VHA) and carbonic anhydrase (CA) in
the HCO⫺
3 stimulation (20 mM) of Isc and Gte across toadfish anterior intestine.
A: VHA inhibitor bafilomycin (Baf; 1 ␮M) prevents the HCO⫺
3 stimulation of
Isc. VHA inhibition was not reversible. B: permeable CA inhibitor etoxolamide
(Et; 1 mM) inhibits the basal Isc and prevents the stimulation by HCO⫺
3.
Bumetanide does not have any further effect. Letters indicate different levels
of statistical significance (repeated-measures ANOVA, Tukey’s multiplecomparisons test, P ⬍ 0.05; n ⫽ 5).
depend on cAMP. The driving force for NaCl absorption is
provided by basolateral Na⫹/K⫹-ATPases, which we have confirmed to be at the toadfish intestinal epithelium by Western blot
analysis and immunohistochemistry. The Na⫹/K⫹-ATPase is another potential target of sAC activity.
sAC in toadfish intestine. Based on immunodetection with
two different antibodies, an sAC-like protein is present in the
toadfish intestine. Unfortunately, we were unable to clone
toadfish sAC by a variety of approaches, including bioinformatic methods and homology cloning using degenerate primers. Based on our experience working with mammalian (3, 11)
and dfsAC (63), cloning the toadfish sAC ortholog is challenging because of the relatively low conservation of the sAC genes
between species and the typically low sAC mRNA abundance.
Nonetheless, immunodetection of a protein band of identical
molecular size and colocalization in microscopy sections using
two different antibodies, although heterologous, is good evidence for the presence of sAC in toadfish. The alternatives, that
a protein unrelated to sAC contains the two different epitopes
or that the antibodies recognize different proteins of identical
molecular size, are highly unlikely.
To our knowledge, this is the first study to attempt selective
inhibition of sAC and tmACs in an ion-transporting epithelium. Inhibition of sAC (with CE and KH7) had the opposite
effect of inhibition of tmACs (with 2=5=-dideoxyadenosine).
This finding has two profound implications. First, it suggests
that cAMP signaling in toadfish intestinal cells occurs in
defined intracellular microdomains (i.e., basolateral and apical
membranes, cytoplasm, etc.) and that each of them may exert
different, even opposing physiology. Second, it shows that
studies using permeable cAMP analogs should be interpreted
with caution, as the cAMP analogs are likely to have multiple
effects on cellular physiology through at least two different
cAMP signaling pathways. This situation is further complicated by the common use of nonspecific inhibitors of phos-
absorption by HCO⫺
3 /pH in the marine fish intestine is not
surprising considering the processes of carbonate precipitation
and NaCl-driven water absorption. While a portion of the
HCO⫺
3 secreted into the intestinal lumen by apical anion
exchangers of the Slc26a6 family (29) precipitates as CaCaO3
and MgCaO3, there is still significant (up to 100 mM) HCO⫺
3
dissolved in the luminal fluids (65). Our proposed model for
HCO⫺
3 -stimulated NaCl absorption is depicted in Fig. 8. Part of
⫹
the dissolved HCO⫺
3 combines with H pumped into the lumen
by V⫹-H⫹-ATPases, which is known to be present in the apical
membrane of toadfish intestinal cells (22). This reaction yields
CO2 and H2O, and, while it might be catalyzed by extracellular
carbonic anhydrase, the complete inhibition by apical bafilomycin suggests enzymatic catalysis is not necessary for the
stimulation of Isc. According to our proposed model, luminal
CO2 then diffuses inside the intestinal cells where it is hydrated
back into H⫹ and HCO⫺
3 by cytoplasmic carbonic anhydrase.
Interestingly, in intestinal cells of seawater-acclimated rainbow
trout, cytoplasmic carbonic anhydrase is located in the subapical region (21), which is also where sAC is most abundantly
present in toadfish intestinal cells. Therefore, it is likely that
intracellular HCO⫺
3 generated by carbonic anhydrase activates
sAC, which, in turn, activates apical NaCl absorption across
NKCCs in a yet undetermined manner, but which should
Fig. 8. Model for the activation of Isc by HCO⫺
3 in toadfish intestinal cells. The
anion exchanger secrets intracellular HCO⫺
3 [produced by cellular metabolism
and imported by NBC from the blood (59)] into the intestinal lumen. Luminal
2⫹
HCO⫺
and Mg2⫹ precipitation (not shown), a fraction of the
3 results in Ca
⫹
excess HCO⫺
pumped by the VHA to form CO2, which
3 combines with H
⫹
diffuses into the intestinal cells. CAcyt hydrates CO2 back into HCO⫺
3 and H
⫺
(not shown). HCO3 activates sAC, which stimulates (⫹) the activity of apical
NKCC to absorb NaCl via a cAMP-dependent mechanism yet to be determined. Putative apical K⫹ and basolateral Cl⫺ channels are not shown. CAcyt,
cytoplasmic carbonic anhydrase; NBC, Na⫹/HCO⫺
3 cotransporter.
AJP-Regul Integr Comp Physiol • VOL
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sAC REGULATES NaCl ABSORPTION IN FISH INTESTINE
phodiesterases, which could not only prevent the degradation
of cAMP from multiple sources, but also of cGMP.
Targets of the presumptive HCO⫺
3 /sAC pathway. The two
most likely mechanisms of NKCC activation are PKA-mediated insertion of NKCC from subapical vesicles into the apical
membrane and PKA-phosphorylation of NKCC already
present at the apical membrane. There is abundant evidence
supporting these two mechanisms, which are not mutually
exclusive, in other systems. cAMP has been shown to promote
the insertion of mammalian NKCC2 into the cell membrane
both in heterologous expression experiments in Xenopus oocytes (36) and in the native thick ascending loop of Henle (4,
46). The cAMP effect has been traditionally linked to hormones, such as vasopressin (reviewed in Ref. 16), but the
potential involvement of sAC has not yet been considered,
likely due to this signaling enzyme being discovered recently.
Notably, sAC mRNA is present in several NaCl and water
absorbing mammalian epithelia, including small intestine (17),
and sAC has been immunolocalized in the thick ascending loop
of Henle in the kidney (47).
Vasopressin induces NKCC2 activation in at least two ways:
increased translocation into the membrane and direct NKCC2
phosphorylation (18). However, a direct link between vasopressin, cAMP, PKA, and NKCC2 has not yet been established. Our immunohistochemical pictures from toadfish intestinal fragments (which were immediately fixed after dissection)
clearly show that NKCC is present both in the apical membrane and in the subapical region of intestinal epithelial cells,
leaving open both trafficking and direct phosporylation as
possible NKCC activating mechanisms in response to HCO⫺
3
via sAC.
Alternatively or additionally, the pathway might involve
activation of Na⫹/K⫹-ATPase. Because this enzyme is the
main driving force behind NaCl absorption via NKCC (20, 23),
a putative modulation of Na⫹/K⫹-ATPase by sAC would explain
why sAC and NKCC inhibitors did not show additive effects.
Interestingly, sAC has been shown to regulate Na⫹/K⫹-ATPase
catalytic activity in kidney collecting duct cells (24).
Some of our experimental conditions, inhibitors, and results
require a few clarifications. First, to avoid diffusive ion movement across intestinal segments mounted in Ussing chamber, it
is essential to apply symmetrical conditions in the saline at the
serosal and mucosal sides. We mimicked the composition of
the serosal side (blood) found in live fish, reasoning that it is
best for cell function. A [HCO⫺
3 ] of 80 mM, although normal
for the intestinal lumen, is much higher than the normal
[HCO⫺
3 ], pH, and osmolarity in the serosal side. Therefore, we
used 20 mM HCO⫺
3 in all subsequent experiments, which also
stimulated Isc significantly. We predict the greater Isc stimulated by higher [HCO⫺
3 ] to be at least partially dependent on
sAC. Second, HCO⫺
3 not only stimulated the absorptive Isc, but
it also increased Gte in a dose-dependent manner. None of the
inhibitors affected the stimulated Gte, although 20 mM HCO⫺
3
failed to stimulate Gte in Cl⫺-free conditions. It is difficult to
speculate about these results, but the most likely candidates are
transporters in the apical membrane (which typically dominates Gte) such as secretory K⫹ channels and electrogenic
Cl⫺/HCO⫺
3 exchangers (SLC26a6 and possibly SLC26a3),
which might be directly or indirectly modulated by HCO⫺
3
and/or pH . Third, inhibition of sAC or carbonic anhydrase
inhibited the basal Isc in HCO⫺
3 -free saline. This points to a role
R69
of endogenous metabolic CO2 as an additional modulator of
NaCl transport in toadfish intestine via carbonic anhydrase and
sAC.
Interaction between intestinal NaCl absorption and anion
exchange. Earlier observations demonstrate that conditions of
low luminal NaCl, which are unfavorable for salt absorption
via NKCC, results in elevated Cl⫺ absorption by anion exchange as evident from elevated HCO⫺
3 secretion (23). This
enhanced anion exchange appears to be, at least in part, via
SLC26a6 proteins (22, 29). The present study, on the other
hand, demonstrates that conditions more adverse to Cl⫺ absorption via anion exchange (high-luminal HCO⫺
3 ) stimulate
NaCl absorption via NKCC. It thus appears that the pathways
for Cl⫺ absorption by the intestinal epithelium in marine fish
are coordinated, depending on luminal conditions to allow for
Cl⫺ and thus water absorption. While sAC appears to be the
link between luminal HCO⫺
3 and NKCC activation, it remains
to be established how low NaCl stimulates anion exchange (or
how high NaCl inhibits anion exchange).
HCO⫺
3 as a modulator of epithelial ion transport. From an
ion-transporting perspective, HCO⫺
3 is typically only considered as a substrate for anion exchangers and Na⫹/HCO⫺
3
cotransporters (57), and any additional effects are usually
attributed to indirect changes in pH. However, HCO⫺
3 can also
act as a signaling molecule, which can gain specificity and be
amplified through HCO⫺
3 -responsive soluble adenylyl cyclase
(3, 6, 45, 56, 63).
Several studies have reported modulation of transepithelial
ion transport by HCO⫺
3 and alkaline pH in a variety of
epithelia, including rat small intestine (52), rat colon (8),
human jejunum (54), crab gills (62), fish intestine (5, 12, 13),
and frog stomach (33). These unexplained effects of HCO⫺
3
could depend on sAC, similar to the toadfish intestine, or to
⫺
other yet unidentified HCO3 modulation.
Perspectives and Significance
We have identified a novel regulatory pathway for epithelial
NaCl absorption, which depends on HCO⫺
3 , and it may involve
sAC and NKCC. Our results also suggest the existence of
cAMP signaling microdomains in ion-transporting epithelia.
Activation of NaCl absorption by HCO⫺
3 /sAC/NKCC may be
essential for intestinal carbonate precipitation and water absorption, a process that is essential for marine fish osmoregulation and which has implications for the ocean carbon cycle.
Moreover, the literature suggests this novel regulation may
exist in other epithelia.
ACKNOWLEDGMENTS
We thank the members of Martin Grosell’s, Lonny R. Levin’s, and Jochen
Buck’s labs for their assistance, in particular Dr. Josi Taylor for sample
fixation, Janet Genz, and Ed Mager. We are grateful to Dr. Larry Palmer for
critically reading the manuscript. Dr. Danielle McDonald kindly provided us
with a number of experimental fish.
GRANTS
This work was supported by the Journal of Experimental Biology Traveling
Fellowship to M. Tresguerres and National Science Foundation Grant
IOS0743903 (to M. Grosell) and National Institutes of Health Grants GM62328 and NS-55255 to L. R. Levin and J. Buck.
DISCLOSURES
AJP-Regul Integr Comp Physiol • VOL
No conflicts of interest, financial or otherwise, are declared by the author(s).
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sAC REGULATES NaCl ABSORPTION IN FISH INTESTINE
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