1 Nephrology Section, Veterans Affairs Medical Center and New York University School of Medicine, New York, New York 10010; and 2 Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri 63104
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ABSTRACT |
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To
determine the role of carbonic anhydrase (CA) in colonic electrolyte
transport, we studied Car-20 mice, mutants
deficient in cytosolic CA II. Ion fluxes were measured under
short-circuit conditions in an Ussing chamber. CA was analyzed by assay
and Western blots. In Car-20 mouse colonic mucosa,
total CA activity was reduced 80% and cytosolic CA I and
membrane-bound CA IV activities were not increased. Western blots
confirmed the absence of CA II in Car-20 mice.
Normal mouse distal colon exhibited net Na+ and
Cl absorption, a serosa-positive PD, and was
specifically sensitive to pH. Decrease in pH stimulated active
Na+ and Cl
absorption whether it was
caused by increasing solution PCO2, reducing HCO
3 concentration, or
reducing pH in
CO2/HCO
3-free HEPES-Ringer
solution. Membrane-permeant methazolamide, but not impermeant
benzolamide, at 0.1 mM prevented the effects of pH.
Car-20 mice exhibited similar basal transport rates
and responses to pH and CA inhibitors. We conclude that basal and
pH-stimulated colonic electrolyte absorption in mice requires CA I. CA
II and IV may have accessory roles.
pH; carbon dioxide tension; carbonic anhydrase isoenzymes; methazolamide; benzolamide
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INTRODUCTION |
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AS DEMONSTRATED in a number of species, absorption of
Na+ and Cl in ileum and colon is
modulated by acid-base variables (3, 5, 12). Among the most thoroughly
studied is the rat colon, in which the responses to extracellular and
intracellular pH (pHi), PCO2, and
HCO
3 concentration
([HCO
3]) have been
characterized in vivo and in vitro (2, 4, 15). Increases in
PCO2 cause reversible increases in
Na+ and Cl
absorption mediated through
effects on apical membrane and electroneutral Na+/H+ and
Cl
/HCO
3 exchangers.
The effect on Na+ absorption is not mediated by cytoplasmic
pHi as expected but by the pH of a poorly characterized
subapical domain or process sensitive to CO2 and perhaps
short-chain fatty acids (2, 8, 9). CO2 effects on
Cl absorption are mediated through changes in
intracellular [HCO
3] ([HCO
3]i) (7,
10, 24). Thus all means of changing pHi, extracellular pH
(pHe), and extracellular
HCO
3 concentration
[HCO
3]e, to
the extent that they change
[HCO
3]i, will
affect Cl
absorption.
These transport effects are also dependent on carbonic anhydrase (CA), an enzyme with relatively high activity in the colon and isoenzymes distributed in the membrane (CA IV) and cytosol (CA I and II) (14, 17). Inhibition of CA with acetazolamide (ATZ) or methazolamide (MTZ) largely abolishes the effects of CO2 on Na+ absorption (6, 15). Of note, ATZ and MTZ are membrane-permeant sulfonamides and inhibit all CA isoenzymes. We were therefore interested to find that inhibition of membrane-bound CA IV by relatively impermeant benzolamide (BNZ) had no effect on colonic ion fluxes (8, 19a). Another way to examine the role of CA, described here, is to study the relative importance of the CA isoenzymes in the Car-20 mouse, a mutant lacking CA II activity.
The Car-20 mouse was developed by breeding animals exposed to the mutagen N-ethyl-N-nitrosourea (16). Homozygous progeny exhibit renal tubular acidosis, growth retardation, and vascular calcifications (1, 16, 21). These mice and nonmutated controls offered us the opportunity to examine 1) the response of the normal mouse colon to acid-base variables, 2) the importance of CA II in mediating this response, and 3) the degree to which other CA isoenzymes may function as substitutes for CA II.
We addressed these issues by measuring the activity of the CA
isoenzymes in the colon (and ileum for comparison) and the relative importance of ambient pH, PCO2, and
[HCO3] in mediating
colonic electrolyte transport in vitro. The effects of permeant and
relatively impermeant CA inhibitors on ion fluxes were studied as well.
We found that although colonic CA activity in normal mouse was similar
to the rat, the electrolyte transport response to acid-base variables
differed. Furthermore, Car-20 exhibited electrolyte
transport characteristics identical to those of control mice despite
the demonstrable absence of colonic CA II activity.
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METHODS |
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Approval of the Department of Veterans Affairs Subcommittee for Animal Studies was obtained for study of male and female control (C57BL/6J and DBA/2J) and Car-20 mice. Two controls were used because Car-20 mice were derived from mating C57BL/6J mice to DBA/2J mice exposed to a mutagen and then backcrossing (16). All animals were 3-5 mo old, were maintained on a standard diet with free access to water, and weighed 25-35 g at the time of study. Under pentobarbital sodium anesthesia (5 mg/100 g body wt), the distal ileum and entire colon were removed and rinsed with 0.9% saline.
Preparation of cell homogenate.
Cell pellets from ileum or whole colon were suspended in 200-400
ml of 10 mM Tris · SO4, pH 7, containing
1 mM benzamidine and homogenized by ultrasonication with two 10-s
bursts on ice. The cell homogenates were stored at 70°C. The
protein concentration was determined by the micro-Lowry procedure using
bovine serum albumin as a standard (19).
CA assay. CA activity was measured according to Maren (18), as described previously (22). NaI-sensitive CA was determined by incubating 5 mM NaI in the reaction tube during CA assay. Membrane-associated CA IV was determined by incubating the enzyme sample with 0.2% SDS at room temperature for 30 min before CA assay. The average of four measurements in each of two experiments was used for the calculation of enzyme activity [enzyme units (U) per mg protein]. The maximum deviation from lowest to highest value in any measurement set was <5%.
Electrophoresis and immunoblotting. Antibodies against rat CA I, II, and IV were raised in rabbits as described previously (26). Goat anti-rabbit IgG-peroxidase was purchased from Sigma Chemical. SDS-PAGE was carried out in 12% acrylamide. After electrophoretic transfer of the polypeptides from the gel to Immobilon-P membranes, the membranes were treated first with antibodies against rat CA I, II, or IV and then with goat anti-rabbit IgG peroxidase conjugate. (For details, see Refs. 14 and 20.)
Ion flux measurements.
Details of the method (as performed in rat colon) were described
previously (9, 15). Pairs of resected distal colonic segments were
mounted in modified Ussing half-chambers exposing 0.38 cm2
of surface area. Tissues were studied under short-circuit conditions. Periodic bipolar pulses of 0.5 mV yielded electrical current values that were used to calculate tissue conductance (G). Tissues
were paired for ion flux studies only when differences in G
were 25%. The short-circuit current
(Isc) divided by G yielded the active
transport potential difference (PD), which was referenced to the
mucosal side.
Solutions and acid-base conditions.
Reagent grade chemicals were obtained from Sigma Chemical unless
otherwise indicated. All solutions were maintained at 37°C. The
HCO3 Ringer solution contained (in mM)
10 glucose, 96 NaCl, 4 KCl, 2.4 Na2HPO4, 0.4 NaH2PO4, 1 CaSO4, 1.2 MgSO4, 21 NaHCO3, and 18 Na+
gluconate. [HCO
3] was
adjusted to 11, 21, and 39 mM by reciprocal alterations in
Na+ gluconate to keep the osmolality constant. These
solutions were gassed with 3% CO2-97% O2, 5%
CO2-95% O2, or 11% CO2-89%
O2 to obtain various pH and
PCO2 values. In several experiments a
nominally CO2/HCO
3-free
Ringer solution was used in which 21 mM NaHCO3 was replaced
with 21 mM HEPES Na+ salt. HEPES-Ringer solution was gassed
with 100% O2, and pH was titrated using 2 M
H2SO4 or 1 M NaOH. Bathing solution pH and PCO2 were measured with a Radiometer
BMS 3 Mk 2 system with a PHM 73 acid-base analyzer (The London Company,
Cleveland, OH).
[HCO
3]e was
computed using the Henderson-Hasselbalch equation. The
pK' and CO2 solubility were 6.115 and 0.0306, respectively.
Statistics. Calculated values for the flux measurements are expressed as means ± SE. Statistical analyses consisted of paired and unpaired two-tailed Student t-tests. A P value of <0.05 was considered significant.
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RESULTS |
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CA activity.
As shown in Table 1, total CA activity in
the ileum of both control strains was ~1 U/mg protein. In control
colon, CA activity was near 14.5 U/mg. CA activity in both ileum and
colon was sensitive to 1 mM ATZ, although residual CA activity was
present in control colon. In the presence of the CA I inhibitor NaI,
46-63% of CA activity in the ileum and 38-44% of CA
activity in the colon were inactivated. Thus about one-half of total CA
activity in control ileum and colon is due to the CA II and/or CA IV
isoenzymes.
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Immunoblots of CA isoenzymes.
The various isoenzymes of CA were characterized by immunoblotting using
specific antibodies against rat CA I, II, and IV. Preimmune serum was
used to assess the specificity of the cross-reacting polypeptides. As
shown in Fig. 1, A and B,
there was a signal for the soluble isoenzymes CA I and II in the ileum
of both control strains, but it was very weak. Signals for CA I and II
in the colon of both strains were very strong, appearing as a doublet with apparent molecular masses of ~30 kDa. The
faster-migrating species of the doublet appears to be CA I.
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Colonic electrolyte transport in control mice. Transport measurements for all control mice were pooled. This was based on similar flux rates and, as described above, similar CA isoenzymes and activities in male and female mice of both control strains.
Table 2 shows the effect of increasing PCO2 on Na+ and Cl
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Colonic electrolyte transport in Car-20 mice.
Table 4 shows the effects of pH and MTZ on
distal colonic transport in Car-20 mice. At
PCO2 21 mmHg, net Na+ and
Cl absorption and a serosa-positive luminal PD were
observed. Comparison with Table 2 indicates that the transport rates
were similar to those in controls. We also found that reducing pH by
increasing PCO2 stimulated
JNams,
JNanet, JClms, and
JClnet and decreased
Isc and PD. The changes in
JNanet and
JClnet appeared smaller
than in controls but were not statistically different. At
PCO2 21 mmHg, MTZ reduced
JNanet to near zero by inhibiting JNams and caused net
Cl
secretion by decreasing
JClms and increasing JClsm. Notably,
JNams and
JClms were 3 µeq · cm
2 · h
1
lower than in control mice. MTZ also prevented the proabsorptive effects of decreasing pH, and as in control mice, increases in PCO2 in the presence of MTZ decreased
JClsm and thereby increased
JClnet.
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DISCUSSION |
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The development of tools to examine the tissue and cellular location
and function of the CA isoenzymes has enabled investigators to examine
their specific physiological roles. In the rat colon, the effects of
CO2 on colonic Na+ and Cl
absorption depend on CA activity. In previous studies we found that
both basal and CO2-stimulated Na+ absorption
are reduced in the presence of the permeant CA inhibitors ATZ and MTZ
but not in the presence of relatively impermeant BNZ (6, 8, 9, 15).
This suggests that cytoplasmic CA I and/or CA II rather than
membrane-bound CA IV mediate the effects of CO2 on colonic
Na+ absorption.
CA II-deficient mice offer another means of examining this issue. Such mice exhibit total absence of CA II in all tissues that have been examined including the colon, brain, stomach, and kidney (1, 16). The fact that nonmutated controls for Car-20 mice were available permitted us to study two groups of animals, with the single difference being the presence or absence of CA II. In addition, we could determine whether in the absence of CA II, the colonic isoenzymes I and IV increase their activity or possibly change their functional roles.
One potentially important systemic effect of CA II deficiency that
could have affected our results is the presence of chronic metabolic
acidosis (i.e., renal tubular acidosis type 1) (1, 16). This
abnormality may be responsible for the slower growth of
Car-20 mice. In intact animals, on the basis of our
data (Table 3), we would expect metabolic acidosis to stimulate colonic
Na+ and Cl absorption. This contrasts
with the effects of a model of chronic metabolic acidosis in rat colon
that mimics renal tubular acidosis (13). In this model, in which the
lumen of anesthetized animals was perfused, colonic Na+
absorption was unaffected and Cl
absorption and net
HCO
3 secretion were reduced. In
resected colon studied in the Ussing chamber, however, the bathing
solutions are determined by the experimental protocol and have effects
on the cellular environment and transporters that may supercede in situ
acid-base conditions. Indeed, in our study, colonic Na+ and
Cl
fluxes were similar in Car-20
and control mice (compare Tables 2 and 4) despite far-different in vivo
metabolic states (1).
The CA assays and immunoblots confirmed the absence of CA II in
Car-20 mouse intestine and showed no compensatory
increase in CA I or CA IV activities. That is, total CA activity
was reduced by 30-40% in the ileum and 80% in the colon in
these mice, consistent with the results of the NaI and SDS assays.
Although colonic CA I activity appeared somewhat decreased in
Car-20 mice, this was because NaI-sensitive
CA activity overestimated CA I activity by 15-20% in control
mice. The normal levels of colonic Na+ and
Cl absorption observed in Car-20
mice therefore suggest that CA II activity is not involved in the
absorptive process(es) or that other CA isoenzymes also participate in
this process.
We examined whether CA I or IV was important in colonic absorption by
studying the effects of permeant (MTZ) and relatively impermeant (BNZ)
CA inhibitors (19a). We found that in both control and
Car-20 mice MTZ reduced absorption at high pH (low
PCO2) and prevented the pH-induced
increase in Na+ and Cl absorption. By
comparison, BNZ reduced absorption at baseline but did not affect the
pH-stimulated increase in absorption. This suggests that CA I mediates
the colonic transport changes due to pH. In addition, depending on the
degree to which BNZ is truly membrane impermeant, CA IV may be
important in maintaining normal basal levels of colonic absorption.
We cannot conclude that CA II has no role in electrolyte transport.
Possibly, both CA I and CA II mediate the effects of pH and the absence
of one isoenzyme is not sufficient to decrease basal or stimulated
absorption. Evidence that CA II plays some role was provided by the
greater Na+ and Cl absorptive fluxes in
control than in Car-20 mice in the presence of MTZ.
This MTZ-insensitive flux was likely due to CA II, as suggested by the
residual CA activity in control but not in Car-20
mice in the presence of ATZ (Table 1). Although unusual and of
interest, the partial inhibition of CA II (in vivo and in vitro) was
not studied further.
A role for any of the CA isoenzymes is remarkable considering that
distal colonic absorption in the mouse is specifically sensitive to
bathing solution pH rather than PCO2
or [HCO3]. As described
above, colonic Na+ and Cl
absorption
increased equivalently when pH was reduced by increasing PCO2, reducing
[HCO
3], or reducing pH in
the nominal absence of CO2 and
HCO
3 (in HEPES-Ringer solution).
Presumably pH-sensitive transport processes, which proceed without the
requirement for catalyzed CO2 hydration and indeed without
medium CO2, should not require CA activity. In the rat
colon, which is uniquely CO2 sensitive, ATZ and MTZ reduce
Na+ and Cl
absorption at low
PCO2 and decrease the
CO2-induced increment in absorption (6, 9, 15). By
contrast, ATZ does not affect the pH-stimulated increment in
Na+ and Cl
absorption in rat ileum,
which, like mouse colon, is specifically responsive to pH (23, 25).
The mechanism of pH action to stimulate colonic Na+
absorption is uncertain. In the rat ileum, there is no correlation
between net Na+ absorption measured in vivo or in vitro and
pHi (23, 25). However, Cl absorption and
net HCO
3 secretion vary with [HCO
3]i and a
similar relation appears to be present in mouse colon. That is,
Cl
absorption increased when
PCO2 was increased or when [HCO
3] was reduced but not
when pH was decreased in HCO
3-free
HEPES-Ringer. These changes in PCO2
and [HCO
3] increase the
driving force for apical membrane
Cl
/HCO
3 exchange by
increasing the
[HCO
3]i/[HCO
3]e gradient, as described previously (9). The pH change in HEPES buffer
does not affect this gradient.
We conclude from our data that the normal mouse distal colon is
pH sensitive, has relatively high CA activity, and requires CA activity
to absorb Na+ and Cl optimally. CA I is
the isoenzyme primarily responsible for this effect, but CA II and IV
may have accessory roles. CA II may function like CA I, but its
presence does not enhance electrolyte transport. That is, CA
II-deficient and normal mice have equivalent basal and stimulated rates
of Na+ and Cl
absorption. Both CA II and
membrane-bound CA IV may support basal Na+ absorption, but
this conclusion rests on partial MTZ and ATZ insensitivity of CA II and
the complete membrane impermeability of BNZ in mouse colon, neither of
which were tested here. The mechanism by which CA supports colonic
transport in the mouse is uncertain. On the basis of the dramatic
effects of CA inhibition in the non-CO2-responsive mouse
colon observed here, we must consider the possibility that CA has a
noncatalytic role in ion transport.
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ACKNOWLEDGEMENTS |
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The authors thank Richard W. Egnor for assistance with the performance of the experiments and preparation of the manuscript.
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FOOTNOTES |
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This work was supported by the Office of Research and Development, Medical Research Service, Department of Veterans Affairs, and by National Institutes of Health Grants GM-34182 and DK-40163.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: A. N. Charney, Nephrology Section/111, VA Med. Ctr., 423 E. 23rd St., New York, NY 10010.
Received 30 July 1999; accepted in final form 9 November 1999.
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