Acid-base effects on electrolyte transport in CA II-deficient mouse colon

David S. Goldfarb1, William S. Sly2, Abdul Waheed2, and Alan N. Charney1

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 [HCO-3] 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.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.

Unidirectional fluxes of Na+ and Cl- were measured by adding 2 µCi of 22Na+ and 1 µCi of 36Cl- (100 Ci/g specific activity; New England Nuclear, Boston, MA) to the mucosal side of one member of each tissue pair and the serosal side of the other. Mucosal-to-serosal (Jms) and serosal-to-mucosal (Jsm) fluxes expressed as microequivalents per square centimeter per hour were measured for 40 min after an initial 30-min equilibration. Twelve minutes were allowed for each new steady state. Net flux (Jnet) was calculated as Jms - Jsm.

Solutions and acid-base conditions. Reagent grade chemicals were obtained from Sigma Chemical unless otherwise indicated. All solutions were maintained at 37°C. The HCO-3 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.

The effects of CA inhibitors on ion flux were examined by the addition to both the mucosal and serosal reservoirs of 0.1 mM MTZ or 0.5 mM BNZ (kindly provided by Drs. W. Brechue and T. Maren). Preliminary experiments using 1 mM concentrations of these drugs resulted in similar findings.

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.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Table 1.   Carbonic anhydrase activities in ileum and colon

Membrane-associated isoenzyme CA IV was analyzed by SDS sensitivity, because unlike the soluble isoenzymes (CA I and II), CA IV is resistant to SDS. We found that ~60% of total CA activity in the control ileum is SDS resistant (i.e., CA IV), whereas in control colon only 3% of total CA activity is resistant to SDS. Because total CA activity in colon is >10 times that in ileum, the actual contribution of CA IV to total CA activity per milliliter of tissue extract was similar in these segments.

In Car-20 mice, total CA activity was reduced 30-40% (0.8 U/mg) in the ileum and 80% (3.1 U/mg) in the colon and was sensitive to ATZ. NaI inhibited total CA activity ~85% in ileum and colon, indicating that CA activity in Car-20 mice is mostly due to CA I. Colonic CA I activity appeared slightly reduced in Car-20 mice compared with controls. However, this was caused by 15-20% inhibition of CA II in addition to complete inhibition of CA I by NaI (and therefore overestimation of CA I) in control animals. SDS-resistant CA activity was approximately the same in the ileum and colon of Car-20 and control mice.

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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Fig. 1.   Western blot of carbonic anhydrase (CA) from ileum and colon of control mice (C57BL/6J and DBA/2J). Tissue homogenates equivalent to 100 µg of protein were subjected to SDS-PAGE. After electrophoretic transfer to polyvinylidene difluoride membranes, membranes were treated with rabbit anti-rat CA I antiserum (A), rabbit anti-rat CA II antiserum (B), rabbit anti-rat CA IV antiserum (C), or rabbit preimmune serum (D) followed with goat anti-rabbit IgG peroxidase conjugate. Arrows indicate major polypeptides cross-reacting with CA I, CA II, and CA IV antisera. Apparent molecular masses of standard proteins and polypeptides are indicated in kDa.

Ileum and colon from both control strains showed a polypeptide of 39 kDa as the predominant protein reacting with CA IV antiserum (Fig. 1C). The signal in the ileum of the C57BL/6J mouse was weaker. Several weak signals for lower-molecular-weight polypeptides were seen in C57BL/6J colon, which could be due to deglycosylated CA IV and proteolytic fragments of CA IV. The specificity of the reaction with the antiserum to CA IV was verified by using preimmune serum instead of antiserum (Fig. 1D). Preimmune serum did not react with any polypeptides when used under identical conditions.

Figure 2 shows that there was no reaction to CA II antiserum in ileum or colon from the Car-20 mouse. CA I antiserum reacted with both ileum and colon. Because identical enzyme units were loaded from the ileum and colon in this immunoblot, these bands are of similar intensity. The signal for CA IV was similar in both ileum and colon, although the enzyme in the ileum appears slightly larger. A cross-reaction of CA IV antisera with CA I and a nonspecific protein also were seen in the Car-20 mouse colon.


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Fig. 2.   Western blot of CA from ileum and colon of Car-20 mouse. Tissue homogenates equivalent to 0.1 U of NaI-inhibitable CA activity were subjected to SDS-PAGE followed by Western blotting. Immunoblots were developed using rabbit anti-rat CA I, CA II, or CA IV. Polypeptides for CA I and CA IV are marked. Arrowhead indicates a nonspecific protein.

To directly compare the expression of CA I in the ileum and colon of Car-20 and control mice, immunoblots were performed on identical quantities of protein. Figure 3 shows that CA I protein expression was very similar in ileum and colon of Car-20 and control mice.


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Fig. 3.   Western blot of CA I from ileum and colon of control and Car-20 mice. Tissue homogenates equivalent to 100 µg of protein were subject to SDS-PAGE followed by Western blotting. Immunoblots were developed using rabbit anti-rat CA I.

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- fluxes across distal colon. At PCO2 21 mmHg, net Na+ and Cl- absorption was observed and a serosa-positive transmural PD was present. When PCO2 was increased to 69 mmHg, JNanet and JClnet increased markedly primarily because of increases in Jms. Decreases in PD and Isc accompanied these changes. The effects of CO2 were stable for several hours at the altered PCO2 and were completely reversible (data not shown).

                              
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Table 2.   Effect of increasing PCO2 and methazolamide on colonic electrolyte transport in control mice

The dependence of the CO2-induced changes in Na+ and Cl- absorption on CA was tested by measuring the effects of 0.1 mM MTZ, a membrane-permeant CA inhibitor. As shown in Table 2, net Na+ and Cl- absorption rates were ~50% lower at PCO2 21 mmHg in the presence than in the absence of MTZ. MTZ also prevented the CO2-stimulated increments in JNams, JNanet, and JClms. An increase in JClnet of 1.8 ± 0.3 µeq · cm-2 · h-1 was observed because of a decrease in JClsm of 1.5 ± 0.5 µeq · cm-2 · h-1 when PCO2 was increased. This same finding was made in rat colon under similar experimental conditions (15).

By contrast, the membrane-impermeant CA inhibitor BNZ did not inhibit the CO2-stimulated increase in Na+ and Cl- absorptive fluxes [Delta JNams 1.9 ± 0.4, Delta JNanet 2.1 ± 0.5, Delta JClms 3.1 ± 0.6, and Delta JClnet 3.4 ± 0.3 µeq · cm-2 · h-1; n = 17, P = not significant (NS) compared with Delta  values for increased PCO2 in Table 2]. However, BNZ did decrease Na+ absorption at both low and high PCO2. At PCO2 69 mmHg, BNZ decreased JNams 2.7 ± 0.5 and JNanet 2.0 ± 0.6 µeq · cm-2 · h-1 (n = 10, P < 0.01). Chloride fluxes were not affected: Delta JClms -1.2 ± 0.7 and Delta JClnet = -1.0 ± 0.8 µeq · cm-2 · h-1 (n = 10, P = NS).

We then examined whether the effect of CO2 was specific for this acid-base variable. As shown in Table 3, decreasing pH by titration of a CO2/HCO-3-free HEPES-Ringer solution from 7.61 to 7.09 increased JNams and JNanet but had no effect on the absorptive flux of Cl-. Decreasing pH to a similar degree in HCO-3 Ringer solution by titrating the [HCO-3] from 39 to 11 mM (at constant PCO2 of 35 mmHg) stimulated both Na+ and Cl- absorption (Table 3).

                              
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Table 3.   Effect of decreasing pH on colonic electrolyte transport in control mice

To confirm that CO2 had no specific effect, we tested whether changing PCO2 in the absence of change in pH would affect Na+ and Cl- absorption. We found that Isc, PD, G, and the unidirectional and net fluxes of Na+ and Cl- were all not significantly different at PCO2 21 mmHg, [HCO-3] 11 mM, pH 7.34 and at PCO2 74 mmHg, [HCO-3] 39 mM, pH 7.35 (n = 6, P = NS by unpaired Student's t-test, data not shown). These findings suggest that Na+ absorption in normal mouse distal colon responds to bathing solution pH and Cl- transport responds to changes in pH but only in HCO-3 Ringer solution (see DISCUSSION).

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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Table 4.   Effect of increasing PCO2 and methazolamide on colonic electrolyte transport in Car-2° mice

Membrane-impermeant BNZ did not inhibit the pH-stimulated increase in Na+ and Cl- absorptive fluxes:Delta JNams 1.3 ± 0.5, Delta JNanet 1.7 ± 0.6, Delta JClms 2.1 ± 0.9, and Delta JClnet 3.6 ± 1.6 µeq · cm-2 · h-1 (n = 9, P = NS compared with Delta  values for increased PCO2 in Table 4). Nevertheless, BNZ decreased both Na+ and Cl- absorption at both low and high PCO2. At PCO2 69 mmHg, BNZ decreased JNams 3.5 ± 0.6, JNanet 1.8 ± 0.9, JClms 3.6 ± 1.3, and JClnet 3.8 ± 1.8 µeq · cm-2 · h-1 (n = 9, P < 0.05).


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 [HCO-3]. 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.


    ACKNOWLEDGEMENTS

The authors thank Richard W. Egnor for assistance with the performance of the experiments and preparation of the manuscript.


    FOOTNOTES

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.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Gastroint Liver Physiol 278(3):G409-G415