A distinct carbonic anhydrase in the mucus of the colon of humans and other mammals
1 Zentrum Physiologie, Medizinische Hochschule Hannover, D-30625 Hannover,
Germany
2 Department of Gastroenterology, Hepatology and Endocrinology, Medizinische
Hochschule Hannover, D-30625 Hannover, Germany
3 IPF PharmaCeuticals GmbH, Feodor-Lynen-Strasse 31, D-30 625 Hannover,
Germany
4 Human Genetics Center, University of Texas School of Public Health at
Houston, Texas, USA
5 Institute of Medical Technology, University of Tampere and Tampere
University Hospital, FIN-33014 Tampere, Finland
* Author for correspondence (e-mail: gros.gerolf{at}mh-hannover.de)
Accepted 15 November 2004
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Summary |
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Key words: human, guinea pig, rat, mouse, gastrointestinal mucus, carbonic anhydrase, colon, stomach
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Introduction |
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Materials and methods |
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Animals
Guinea pigs
Male guinea pigs (Cavia aperea f. porcellus Bohlken 1961, body
weight 550700 g) from Pietsch (Hohlenberg, Germany) were used.
Maintenance and treatment of the animals was as described previously
(Engelhardt et al., 1994).
After decapitation, between 8.009.00 a.m., the abdomen was opened and
the large intestine removed, cut into the different segments (caecum, proximal
colon and distal colon) and rinsed with 25 mmol l-1
Tris-SO4 pH 8.7 to remove luminal contents. Mucus samples were
obtained from the second rinse of the intestinal segments with the same buffer
solution. This washing containing diluted mucus was microscopically free of
epithelial cells. After the second washing, the pieces of intestine were
rinsed several more times before they were used to prepare tissue homogenates.
For the latter purpose, to 1 g of tissue we added 3 ml buffer (20 mmol
l-1 Tris-HCl, 250 mmol l-1 sucrose, 2 mmol
l-1 EDTA, 5 mmol l-1 DTT, 0.2 mmol l-1 PMSF,
pH 7.4) and then homogenized the suspension with an Ultra Turrax 3x30 s
on ice at maximal speed. Subsequently, the homogenate was centrifuged at
4°C for 1 h at 100,000 g to obtain the supernatant.
Mice
Normal and CAII-deficient mice (Mus domesticus L.) were bred in
cooperation with the central animal facility of the Medical School. Breeding
was started with two pairs of mice (C57 BL/6J Car 2°) from the
Jackson Laboratory (Bar Harbor, USA) as first described elsewhere
(Lewis et al., 1988).
Characterization of the genotype of the animals was achieved by measuring
carbonic anhydrase (CA) activity in red cell lysate. Normal animals have an
activity of about 60,00080,000 units (in undiluted red cells), whereas
the homozygous CAII-deficient animals have an activity of only 5001000
units. Heterozygous animals have activities of 30,00050,000 units. This
allows one to distinguish clearly between the three groups on the basis of CA
activity measurements.
Mice were anesthetized with ether and killed by neck dissection. Mucus samples were obtained as described for guinea pigs, with buffer solution containing 5 mmol l-1 imidazole pH 7.4, from the second rinsing of the intestinal segments. After repeated rinsing of these segments, the tissues were minced, diluted 1:4 with ice-cold buffer solution, and homogenized three times for 30 s with an Ultra Turrax. After centrifugation at 4°C and 100,000 g for 1 h we obtained the cytosolic supernatant of the pieces of large intestine. Samples of saliva of mice were obtained by rinsing the mouth of the animals with buffer solution.
Rats
We used female Wistar rats (Rattus norvegicus albinos Berkhaut
1769) purchased from Charles River (Sulzfeld, Germany). Germ-free rats were
bred in the central animal facility of the Medical School. Animals were
anesthetized and killed as described above and mucus samples and saliva were
also obtained as described for mice
Rabbits
Chinchilla bastards (Chinchilla lanigera Molina 1782) from Charles
River (Sulzfeld, Germany) were used for immunization to raise antibodies
against CAI and CAII from guinea pigs.
Human material
Mucus samples of human gastric and colonic mucosa were taken during
endoscopies performed for diagnostic purposes by the Dept of Hepatology and
Gastroenterology, Medical School in Hannover. Colonic mucus was obtained from
the solution used to rinse the large intestine prior to endoscopy. It was not
possible, therefore, to distinguish between mucus from the proximal and the
distal colon. The large intestine was rinsed with Oralav (Braun, Melsungen
Germany) until no stool could be seen macroscopically. The solution from the
rinse following thereafter was collected. The samples were concentrated by
ultrafiltration using a membrane type PM10. After concentrating the samples
about 10-fold, they were dialyzed against 5 mmol l-1 imidazole, pH
7.4, using dialysis tubing (Sigma Lot 26H1022) before carrying out
measurements. The temperature during concentration and dialysis was
4°C.
Methods
Purification of cytosolic carbonic anhydrases
Purification of CA from lysed red cells and colonic mucus of guinea pigs
was carried out by affinity chromatography. For the purification of CAI and
CAII, first a chloroformethanol-extraction of the lysed red cells was
performed as described by Bernstein and Schraer
(1972). The fraction containing
CA was applied to an affinity column and washing as well as elution steps were
carried out as described by Whitney
(1974
). To purify the CAI
fraction, we performed a re-chromatography step as described by Whitney
because this fraction was contaminated with CAII, whereas the CAII fraction
was highly pure after the first affinity chromatography step. The purified
fractions of CAI and CAII were concentrated about 10-fold by ultrafiltration
using a membrane type PM10. After concentrating, the samples were dialyzed
against 25 mmol l-1 Tris-SO4 pH 7.4.
The diluted mucus from guinea pigs was purified and concentrated in two
filtration steps. Initially, a membrane with a pore diameter of 0.45 µm was
used to remove particles from the mucus samples. The filtrate was concentrated
about 50-fold using a membrane type PM10. Finally, the sample was dialysed
against 25 mmol l-1 Tris-SO4 pH 8.7 before it was
applied to the affinity column. Washing steps were carried out as described by
Whitney (1974). The mucus CA
was eluted with 7.5 mmol l-1 sodium azide pH 5.7. Concentration and
dialysis were performed as described for CAI and CAII.
Production of polyclonal antibodies
To obtain polyclonal antibodies against CAI and CAII of guinea pig, rabbits
were immunized with the purified enzyme fractions. For the immunization about
50 µg protein in complete Freund's adjuvant were injected. The booster
injection followed 4 weeks later in incomplete Freund's adjuvant. The antisera
were obtained from a blood sample 2 weeks after the booster injection. Before
immunization, 5 ml blood were taken to obtain pre-immune serum.
Protein determination
Determination of protein concentration was carried out by the method of
Lowry et al. (1951) modified
by Peterson (1977
) using the
protein assay kit from Sigma (Procedure No. P 5656). The samples were
solubilized with sodium deoxycholate, and protein was precipitated with
trichloroacetic acid before the assay.
Measurement of carbonic anhydrase activity
CA activity was determined according to the micromethod of Maren
(1960) as modified by Bruns
and Gros (1991
). The principle
of this method is to determine the (acid) change of pH caused by
CO2 hydration in the reaction vessel following the addition of
alkaline barbital buffer to the CO2-saturated assay volume. The pH
change is visualized by the pH indicator phenol red. The time needed after
addition of barbital buffer to reach the pH at which the indicator turns from
red to yellow is measured at 0°C. In the presence of CA this time is
reduced. One enzyme unit is defined as the final enzyme concentration in the
assay volume that halves the uncatalyzed reaction time. Specific CA activity
is obtained by dividing the CA units of a sample by its protein
concentration.
Measurements with the CA inhibitors acetazolamide and KI were carried out by adding the inhibitor into the reaction vessel and allowing 2 min incubation of sample and inhibitor at 0°C before the barbital was added. To determine the inhibitory effect of SDS, samples were preincubated for 30 min at room temperature with a final concentration of 0.2% (w/v) SDS before being added into the reaction vessel where an identical final SDS concentration was established.
Triton X-114 phase separation
The phase separation with Triton X-114 was carried out by a method similar
to that of Bordier (1981):
equal volumes of ice-cold Triton X-114 solution (2.2% (w/v) Triton X-114, 300
mmol l-1 NaCl, 20 mmol l-1 Tris-HCl, pH 7.4) and sample
were mixed in an Eppendorf test tube. The mixture was shaken, incubated for 4
min at 31°C and centrifuged for 3 min at 2000 g at room
temperature in an Eppendorf centrifuge (type 5414, Eppendorf, Germany). After
centrifugation the supernatant (aqueous phase No. 1) was removed and mixed
again with Triton X-114 solution (4.5% Triton X-114, 150 mmol l-1
NaCl, 10 mmol l-1 Tris-HCl pH 7.4) to a final Triton concentration
of 0.85% (w/v) before the procedure was repeated. After removing the new
supernatant (aqueous phase No. 2), the two sediments (Triton phases) were
combined. Determination of total volumes and CA activities was carried out for
each phase and used to estimate the distribution of the total amount of CA
activity between the two phases.
Immunoblotting
After SDS polyacrylamide gel electrophoresis (separating gel: 15%, stacking
gel: 5%), proteins were electrophoretically transferred to nitrocellulose
using a semi-dry blotting system at 0.8 mA cm-2 for 1.5 h. The
blocking of non-specific binding was carried out with BSA (15 g 500
ml-1 in PBS/Tween). For the immunostaining, polyclonal antibodies
against CAI, CAII and CAVI were used at a dilution of 1:500 and those against
CAIV at 1:1000. Secondary antibodies were labeled with peroxidase and used in
same dilution as the primary antibody. Detection was carried out using DAB as
substrate for peroxidase (CAI, CAII, CAVI) or the ECL system according to the
manufacturer's recommendations (CAIV).
Sequence analysis
Isolated human mucus CA was sequenced as follows. After treatment with
trypsin (0.036 g l-1) the cleavage products were separated by
µHPLC, fractions were sequenced by Edman degradation and mass spectra were
obtained by MALDI-TOFMS analysis.
µHPLC separation of tryptic cleavage products
Prior to the chromatographic separation of 20 µl of sample solution, 125
µl of the incubation mixture were lyophilized and dissolved in 25 µl
0.06% (v/v) aqueous TFA. Chromatography was carried out at 30°C (215 nm)
using a Reprosil-Pur C18-AQ column (250 mmx1 mm i.d.; 3 µm; A.
Maisch, Ammerbuch, Germany) and a rising acetonitrile gradient from 10 to 60%
(v/v) eluent B within 50 min (20 µl min-1) collecting fractions
of 1 min each. The mobile phase comprised eluent A (H2O, 0.06% v/v
TFA) and eluent B (ACN, 0.05% v/v TFA). The HPLC system consisted of a solvent
delivery system 140B, a programmable absorbance detector 785A and an
oven-injector 112A from Applied Biosystems (Weiterstadt, Germany). UV
absorbance was monitored by a Chromjet Integrator (Spectra-Pysics, Fremont,
CA, USA).
MALDI-TOFMS analysis
MALDI mass spectra were obtained on a Voyager DE Pro mass spectrometer
(Applied Biosystems, Weiterstadt, Germany) in the positive linear operation
mode. The matrix comprised -cyano-4-hydroxycinnamic acid mixed with
L-fucose (2.5 mg ml-1 each, both Sigma-Aldrich,
Steinheim, Germany) dissolved in a 50% (v/v) mixture of ACN/0.1% (v/v) aqueous
TFA. Equal volumes of 1 µl of the sample solution and of the matrix were
mixed on a stainless steel multiple sample tray according to the dried droplet
technique. Data acquisition and analysis were performed using the Voyager
control software and Data Explorer version 4.0 software supplied by the
manufacturer.
Amino acid sequence analysis
N-terminal sequencing was performed on a Procise 494 sequencer (Applied
Biosystems, Weiterstadt, Germany) by Edman degradation with on-line detection
of phenylthiohydantoin-amino acid using the standard protocol recommended by
the manufacturer. Sequence search and identification was performed using the
MS-Edman 2.2.1. software from ProteinProspector 3.2.1.
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Results |
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To investigate whether the mucus CA in the large intestine is a soluble or
a membrane-bound isoenzyme, phase separation experiments with Triton X-114
were carried out (Fig. 1).
Soluble proteins like CAI and CAII from red cell lysate partition into the
water phase in this experiment (left-hand column), whereas membrane-bound CA
from heart sarcolemmal vesicles partitions into the Triton phase (right-hand
column in Fig. 1; data from
Bruns and Gros, 1992).
Figure 1 shows the results for
mucus samples from caecum and colon of guinea-pigs. As in red cell lysate,
<10% of CA activity is found in the Triton >80% is found in the water
phase. These results show that mucus CA of the large intestine is a soluble
isoenzyme, indicating that it cannot be identical with membrane-bound
CAIV.
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Table 2 shows the sensitivity of mucus CA towards SDS. The membrane-bound CA IV is the only CA isoform that is known to resist SDS as is exemplified for sarcoplasmic reticulum (SR) vesicles from rat skeletal muscles in the last line of Table 2. Mucus CA of gastric mucosa and of colon and caecum as well as CA of red cell lysate are fully inhibited after incubation with 0.2% SDS. Like the phase separation experiments, these results argue against the existence of a membrane-bound CAIV in the mucus of the gastrointestinal tract.
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To investigate whether the mucus CA in the large intestine is identical to
one of the known cytosolic isoenzymes, CAI or CAII, we carried out experiments
with CAII-deficient mice. Results are shown in
Table 3: CAII-deficient mice
show a large reduction of CA activity in red cell lysate from about 71,000
units for wild-type mice to about 900 units. This can be explained by the lack
of the high-activity isoform CAII. The remaining CA activity is likely to be
mostly due to CAI; the latter conclusion derives from: (1) the expected very
low contribution of CA III to intra-erythrocytic red cell CA activity
(Carter et al., 1984); and (2)
the (line 2 of Table 3)
complete inhibition of the CA activity of red cells from CAII deficient mice
by 7 mmol l-1 iodide, when in contrast iodide at the same
concentration inhibits CA activity in wild-type mouse red cells by about 44%
only. This finding tallies with the known difference in anion sensitivity of
CAI and CAII (Maren et al.,
1976
).
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In the cytosolic supernatant of the intestinal homogenates, we obtain
results similar to those in red cell lysate. Compared with the wild-type mice,
the cytosolic supernatant of intestinal segments of CAII-deficient mice shows
a considerable reduction of specific CA activity. As in red cell lysate, 7
mmol l-1 iodide caused a complete inhibition of CA activity in the
cytosolic supernatant of CAII-deficient mice, whereas inhibition in wild-type
mice was only about 60%. By analogy to the red cell lysates, these data
suggest that the intestinal epithelium of normal mice contains cytosolic CAII
as well as CAI, while only CAI is expressed in the CAII-deficient mice. This
agrees with early studies of Carter and Parsons
(1970), which showed the
existence of both CAI and CAII in the epithelial cells of the large
intestine.
Conversely, Table 3 shows unaltered or even increased CA levels in the mucus of the CAII-deficient mice. This clearly indicates that the CA activity in the mucus of mice is not due to CAII. In addition, 7 mmol l-1 iodide could not completely inhibit the CA activity in the mucus of either CAII-deficient or normal mice. Iodide caused a similar fractional inhibition in the mucus of both genotypes by about 50%. Similarly, 7 mmol l-1 iodide inhibited the CA in colonic mucus samples from humans and guinea pig by about 40% only. The mucus isozyme therefore is only moderately anion-sensitive, which shows that mucus CA activity is not due to CAI.
Western blots with available anti-human CA isoform antibodies were carried out to characterize further the CA in the mucus of the gastrointestinal tract. Figure 2 shows the results for human samples. Identical amounts of CA activity were applied onto the lanes with colonic mucus and the corresponding control lanes with positive control samples, respectively. Antibodies against human CAI, CAII, CAIV and CAVI show no reaction with human colonic mucus whereas the same amount of CA activity in the corresponding positive control lanes produces reactions at the expected molecular weights. The western blots confirm for human mucus the above conclusions, i.e. that mucus CA is not identical to either CAI, CAII nor CAIV. In addition, Fig. 2 provides evidence that it is not CAVI that is responsible for the CA activity in the human colonic mucus.
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It may be added that we have also studied human gastric mucus by western
blotting with anti-human CAVI antibody (results not shown). In agreement with
the results of Parkkila et al.
(1997), we find CAVI in some
samples of gastric mucus, but not in others. In each case, the intensity of
the immunostaining signal of the bands of gastric mucus was less than that of
control samples of human saliva containing identical amounts of CA activity.
This suggests that the gastric mucus contains not only (small variable amounts
of) CAVI but also some other CA isozyme, which may possibly be the same
isozyme that we describe here in the colonic mucus.
To study whether guinea pig mucus CA like human and mouse mucus CA has properties distinct from the intraepithelial CAI and CAII, we isolated CA from guinea pig colonic mucus for further characterization of the enzyme. The molecular weight was determined to be 30 kDa by SDS polyacrylamide electrophoresis. Results of inhibition studies with the isolated mucus enzyme from guinea pigs agree well with the results obtained from inhibition experiments with mucus samples. The isolated CA could be fully inhibited by SDS and the inhibition with 7 mmol l-1 KI is about 36%, i.e. guinea pig mucus CA is moderately anion-sensitive as is mouse mucus CA (Table 3). Western blot analyses with anti-guinea pig CAI and CAII are shown in Figs 3 and 4. It is apparent that there is some reaction of mucus samples with both antibodies. However, it is clear that at identical amounts of CA activity applied to the gel, the bands seen for CA in whole mucus are with anti-CAI as well as with anti-CAII markedly weaker than those seen with isolated CAI or CAII, respectively. It can be hypothesized that mucus samples are either contaminated to a minor extent with CAI and CAII, or that both antibodies cross react with mucus CA. In view of the other evidence presented below, the latter possibility appears more likely. That isolated mucus CA as well as the corresponding positive controls do not react with the two antibodies is due to the very the low amounts of enzyme applied to the gel in these cases (see legend to Figs 3 and 4); this could not be improved upon because of lack of sufficient amounts of purified mucus CA.
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To decide whether it may be cytosolic CAIII that occurs in mucus, we
determined the sensitivity of mucus CA for sulfonamides. CAIII is known to be
present in type I skeletal muscle fibers and exhibits a uniquely high
resistance towards sulfonamides. We find that mucus CA from guinea pig
possesses a high sensitivity towards these CA inhibitors: Ki-values
of guinea pig colonic and caecal mucus for acetazolamide are about
6x10-9 mol l-1, whereas Ki of CAIII for
acetazolamide is between 10-4 and 10-3 mol
l-1 (Gros and Dodgson,
1988). We conclude that the CA activity in the mucus of the
gastrointestinal tract is not due to CAIII. The partial sequences of human
mucus CA (see below) are also not compatible with CAIII.
To investigate whether the mucus CA is synthesized by the tissues of the
gastrointestinal tract or by gastrointestinal bacteria, we compared the CA
activities in the mucus of normal and germ-free rats. As seen in
Fig. 5, we find no significant
decrease in the mucus CA activity of germ-free compared to normal rats in
caecum and colon. We conclude that the CA observed in the mucus of the
gastrointestinal tract is not due to bacteria. It may be noted that studies of
Lönnerholm et al. (1988)
have shown that there is no difference in intracellular CA activity in the
epithelial cells of colon and caecum between normal and germ-free rats.
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To characterize further the human mucus CA, we obtained partial sequences of the isolated enzyme. A comparison of these sequence fragments with human CAI, CAII, CAXIII and CAIII is shown in Fig. 6. It is apparent that there are remarkable differences from CAII, excluding this enzyme as a candidate, and also from cytosolic CAXIII and CAIII. The differences from CAI, on the other hand, are much smaller.
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Discussion |
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Identity of mucus carbonic anhydrase
The results of the described experiments seem to exclude the presence of
five CA isoenzymes in the mucus of the gastrointestinal tract of some of the
investigated species. (1) CAI is excluded for human and for guinea pig colonic
mucus by the absence of reactivity with anti-CAI antibodies (except for some
possible cross-reactivity in the case of guinea pig mucus), and by the lack of
complete inhibition by KI in the cases of the mucus of normal and
CAII-deficient mice and of human and guinea pig colonic mucus. Conversely, the
partial sequences of the human mucus CA might be taken to suggest that mucus
CA has some similarity although not identity with the CAI isoform. (2) CAII as
a mucus isozyme is ruled out by the absence of reactivity with anti-CAII
antibody (in the case of human colonic mucus and except for a possible
minor cross-reactivity also guinea pig colonic mucus), by the partial
sequences of human mucus CA, and by the finding of an unaltered CA activity in
the gastrointestinal mucus of CAII-deficient mice. (3) CAIII cannot account
for the CA activity in mucus because it possesses a low sensitivity towards
sulfonamides while mucus CA exhibits high sensitivities for these CA
inhibitors (guinea pig mucus). Also, the sequence of
Fig. 6 is not compatible with
that of human CAIII. (4) CAIV is a known apical membrane-bound CA isoenzyme.
Its presence in mucus would be conceivable if it was cleaved from its
GPI-anchor at the apical epithelial membrane and retained in the mucus layer.
However, CAIV is resistant against 0.2% SDS while mucus CA is fully inhibited
by this concentration of SDS. This is shown here for gastrointestinal mucus CA
of guinea pigs, mice and humans. In addition, intestinal mucus CA from guinea
pigs is found in the water phase during phase separation experiments with
Triton X-114, while CAIV is normally found in the Triton phase. Lastly, in the
case of human colonic mucus, there is no immunoreactivity with anti-CAIV and
the partial sequences of Fig. 6
are not compatible with human CA IV. (5) CAVI is known to be secreted into
saliva and might pass intact through the gastrointestinal tract into caecum
and colon. We report here lack of immunoreactivity of human colonic mucus in
western blots with anti-human CAVI, eliminating CAVI as a possible major
colonic mucus CA. In addition, the partial sequences of the human colonic
mucus CA are not compatible with the sequence of human CA VI.
We conclude therefore that colonic mucus CA in humans and likely in several other species as well does not appear to be identical with any of the tested CA isoforms. The interpretation of the partial sequences obtained for human mucus CA is not entirely clear. They may suggest an isoform similar to CAI; the lack of CAI antibody to react with human mucus CA, however, argues against this view. In the case of mouse, human and guinea pig mucus the moderate anion sensitivities in addition are clearly not compatible with the high anion sensitivity known for CAI. It should be mentioned that the mucus CA sequences shown in Table 6 are also incompatible with the isoenzymes CAVA, CAVB, CAVII, CAIX, CAXII, CAXIII, CAXIV and CAXV as well as the `acatalytic' CA-related protein isoforms CA-RP VIII, CA-RP X and CA-RP XI. It appears likely therefore that mucus CA represents a hitherto unknown CA isoenzyme.
A search in the human genome databases for the mucus CA sequences of Table
6 was not successful. It may be noted that Chegwidden et al.
(1995) and Chegwidden et al.
(2001
) reported a variant
CAI-like DNA sequence encoding an isozyme which they named CAIB. Their
inferred CAIB protein sequence is quite distinct from the mucus-CA peptide
sequences shown in Fig. 6. Like
our sequence, however, it cannot presently be found in the human genome or
other DNA sequence databases.
We have constructed an evolutionary tree
(Fig. 7) using just the part of
the alignment covering the three peptides (68 amino acids) that have been
characterized in the human mucus CA (Fig.
6). We included CAI from the Old World monkey Macaca
nemestrina (Hopkins et al.,
1995), and from mouse, sheep and turtle, plus human and mouse
CAXIII, CAII, CAIII, CAVA, CAVB, CAVII and (as root) CAIV. This tree indicates
that the mucus CA I-like gene most likely resulted from a gene duplication
between 30 and 75 million years ago, i.e. it is probably restricted to
primates although it might be absent from the earliest diverging primate
lineages (lemurs and lorisoids). A less straightforward possibility, however,
is that the gene duplication predated the mammalian radiation generating a
pair of CAI genes, one of which became expressed in all mammalian intestinal
mucosae. The close similarity of the gene pair in primates could have arisen
by intergenic sequence exchange, i.e. gene conversion of the mucus CAI gene by
the well characterized CAI gene, giving a more recent apparent date for the
gene duplication based on the limited peptide sequence data presently
available.
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Possible physiological significance of mucus carbonic anhydrase
What might be the physiological function of CA in gastrointestinal mucus?
The apical membrane of the colon of the guinea pig, as well as other species,
is a membrane through which several acid-base-relevant transport processes
occur: Na+-H+ exchange (proximal colon),
K+-H+ pump (distal), HCO3-
secretion by a short-chain-fatty-acid/HCO3- exchanger
and possibly by a Cl-HCO3- exchanger, proton
consumption on the apical surface by the uptake of undissociated short chain
fatty acids through the apical membrane (for overview see
von Engelhardt et al., 1994).
These transport processes can pose a challenge to the apical surface pH; but
the latter can in addition be challenged by high
PCO2 values in the colonic lumen or by high
loads of fixed acid associated with low pH values in the luminal contents. It
has been shown that under a large variety of luminal conditions the pH in the
mucus layer, the so-called pH microclimate that exists in a layer
0.5 mm
thick on the apical epithelial surface, is held rather constant, indicating
that there is a regulatory system for this surface pH
(Rechkemmer 1981
;
McNeil et al., 1987
;
Rechkemmer et al., 1986
;
Said et al., 1986
). Most,
although not all, of the molecules involved in challenging as well as
regulating the apical surface pH are the molecules and ions that participate
in the CO2 hydration reaction: H+,
HCO3-, and CO2. In view of rapidly changing
luminal conditions and in view of high transcellular fluxes of acidbase
relevant ions, it would appear very beneficial for maintaining a constant pH
on the apical surface that these three species can rapidly achieve chemical
equilibrium. When it takes about 1 min for the uncatalysed
hydrationdehydration reaction to reach chemical equilibrium, as would
be the case in the absence of carbonic anhydrase, pH regulatory transport
processes cannot be expected to re-establish the pH microclimate after a
challenge faster than this. It is likely, therefore, that an extracellular CA
in the intestinal mucus contributes to maintaining the pH microclimate.
Experimental proof demonstrating this has yet to be provided.
This hypothesis does not answer the question whether such an extracellular
CA would have necessarily to be located in the mucus layer rather than
directly on the membrane surface as a membrane-bound CA on the apical
membrane. In fact, it would appear logical to have a CA right on the external
surface of the membrane across which acidbase transports occur, because
this would ensure that the reaction partners establish chemical equilibrium
immediately after they have crossed the membrane. Conversely, we have recently
shown (Endeward et al., 2003)
that a CA distributed throughout the entire mucus layer may be useful for
another possible function of the epithelial barrier. We have demonstrated
theoretically that due to the continuous mucus production (and mucus flow from
the apical membrane across the mucus layer towards the lumen) the
CO2 partial pressure at the apical membrane may be considerably
lower than that which prevails in the lumen. This is accomplished by the flow
of mucus in conjunction with the very high buffer power of the mucus and the
presence of a substantial CA activity all across the entire mucus layer. Mucus
CA may, therefore, have an important role in protecting the epithelium against
the very high CO2 partial pressures that can occur in the lumina of
various sections of the gastrointestinal tract.
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Acknowledgments |
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References |
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