Biochemical, histological, and inhibitor studies of membrane
carbonic anhydrase in frog gastric acid secretion
Erik R.
Swenson1,
Timothy W.
Tewson2,
Per J.
Wistrand3,
Yvonne
Ridderstrale4, and
Chingkuang
Tu5
1 Department of Medicine, Department of Veterans Affairs
Medical Center, and 2 Department of Radiology, University of
Washington, Seattle, Washington 98108; 3 Department of
Neuroscience, Uppsala University, SE-751 24 Uppsala;
4 Department of Animal Physiology, Swedish University of
Agricultural Sciences, SE-750 07 Uppsala, Sweden; and
5 Department of Pharmacology, College of Medicine, University of
Florida, Gainesville, Florida 32610
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ABSTRACT |
Gastric acid
secretion is dependent on carbonic anhydrase (CA). To define the role
of membrane-bound CA, we used biochemical, histochemical, and
pharmacological approaches in the frog (Rana pipiens). CA activity and inhibition by membrane-permeant
and -impermeant agents were studied in stomach homogenates and
microsomal fractions. H+ secretion in the
histamine-stimulated isolated mucosa was measured before and after
mucosal addition of a permeant CA inhibitor (methazolamide) and before
and after mucosal or serosal addition of two impermeant CA inhibitors
of differing molecular mass: a 3,500-kDa polymer linked to
aminobenzolamide and p-fluorobenzyl-aminobenzolamide (molecular mass, 454 kDa). Total CA activity of frog gastric mucosa is
2,280 U/g, of which 10% is due to membrane-bound CA. Membrane-bound CA
retains detectable activity below pH 4. Histochemically, there is
membrane-associated CA in surface epithelial, oxynticopeptic, and
capillary endothelial cells. Methazolamide reduced H+
secretion by 100%, whereas the two impermeant inhibitors equally blocked secretion by 40% when applied to the mucosal side and by 55%
when applied to the serosal side. The presence of membrane-bound CA in
frog oxynticopeptic cells and its relative resistance to acid
inactivation and inhibition by impermeant inhibitors demonstrate that
it subserves acid secretion at both the apical and basolateral sides.
stomach; methazolamide
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INTRODUCTION |
CARBONIC
ANHYDRASE IV (CA IV) is present in the plasma membranes of
endothelial cells and numerous secretory cells (3, 25,
39). Unlike cytosolic CA II, which catalyzes the interconversion of CO2 and HCO
within the cell, CA IV is
anchored externally to the cell membrane by a
glycosylphosphatidylinositol tail (7). Positioned in this
fashion, the catalytic activity of CA IV is available to the adjacent
extracellular space. Histochemical and cell fractionation studies
(7, 13, 15, 16, 20, 21, 32, 39) have revealed considerable
membrane-bound CA activity in both surface epithelial and parietal
cells of mammalian stomach, constituting 5-35% of total CA
activity. Studies (3, 7) using isozyme-specific CA IV
antibodies have revealed that CA IV represents part of this activity.
Inhibition of gastric CA with diffusible permeant sulfonamide
inhibitors markedly suppresses acid secretion (see Ref. 32 for review). However, the role of CA IV or membrane-bound CA has not
been established. Its function can be investigated using impermeant sulfonamides that remain extracellular by either marked hydrophilicity imparted by an ionized state at physiological pH [benzolamide (37) and quaternary ammonium sulfonamide
(9)] or high molecular mass. However, benzolamide is not
absolutely impermeant, and the toxicity and weak inhibiting activity of
quaternary ammonium sulfonamide limits its use to isolated cell and
organ studies. Earlier large molecular mass inhibitors such as the
dextran-linked sulfonamides of Tinker et al. (36) and
Karlmark et al. (11) had certain problems, including
dissociation of the drug from the polymer (8) and
anaphylactic reactions to dextrans.
The problems of earlier sulfonamide-linked polymers stimulated the
synthesis of a 3,500-kDa polymer linked to aminobenzolamide (F3500);
aminobenzolamide irreversibly bound to a nontoxic polymer of the
dicarboxy derivative of polyoxyethylene (6). In vivo studies (17) using this polymer have demonstrated
successful tight sulfonamide binding and lack of toxicity. F3500 has
revealed the significant contribution of membrane-bound CA in renal
bicarbonate reabsorption. Recently, we (34) synthesized an
analog of benzolamide, p-fluorobenzyl-aminobenzolamide (pFBAB), which is very
hydrophilic and only slightly larger than its parent compound. It
retains high affinity for membrane-bound CA (33) and is
considerably easier to synthesize than F3500.
In the present study, we examined the role of membrane-bound CA and
cytoplasmic CA in H+ secretion in the frog stomach using
the impermeant inhibitors F3500 and pFBAB and methazolamide, a highly
diffusible and permeant inhibitor. To better understand and correlate
the function of membrane-bound CA in frog gastric acid secretion, we
performed histochemical studies in the frog stomach. In addition, given the highly acidic milieu in which an apical membrane-bound CA operates,
we tested the pH resistance of CA IV in vitro.
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MATERIALS AND METHODS |
CA isozymes and inhibitors.
Purified CA II was obtained from human blood by an affinity
chromatography technique (12). Membrane-bound CA IV was
prepared as a microsomal suspension from bovine kidneys
(39).
Methazolamide was obtained from Lederle Laboratories (Pearl River, NY).
F3500 was synthesized according to a previously published method
(6). The synthesis of pFBAB was achieved by reductive alkylation of aminobenzolamide with p-fluorobenzaldehyde
(34).
Enzyme activity and sensitivity to pH and inhibitors.
The inhibitory potency of the CA inhibitors toward cytosolic and
membrane-bound CA fractions of the frog gastric mucosa (prepared as
described below) was determined at 0°C by monitoring the catalysis of
CO2 hydration by canine red cell CA (5).
Briefly, the method involves the incubation of two enzyme units (EU) of
canine red cell CA with known amounts of inhibitor in distilled water
containing 25 mg/l bromthymol blue indicator at 0°C for a minimum of
2 min. The solution is then saturated with 100% CO2, and
the hydration reaction is initiated by the addition of 2 ml of 50 mM
barbital buffer at pH 7.9. The catalyzed time
(tc) is recorded to obtain a color change of the
indicator, approximately pH 6. The reaction is also run in the
absence of inhibitor to obtain the uncatalyzed time
(tuc). The number of enzyme units in the system
is given by EU = (tuc
tc)/tc. The
IC50 for a drug is the molar amount that reduces the number
of enzyme units by 50%.
Gastric mucosa and red cells from Rana pipiens were obtained
from anesthetized frogs after a 5-min partial immersion in a 1.5%
solution of the methanesulfonate salt of 3-aminobenzoic acid ethyl
ester. The abdomen was opened, and the stomach was perfused free of
blood with an infusion of isotonic saline via the aorta. When the
mesenteric effluent was clear, the stomach was excised and opened
laterally, and the mucosa was separated from muscle with a no. 15 surgical blade. The mucosa was weighed, cut into small pieces, and
homogenized with 2 vol of a buffer containing 25 mM triethanolamine
sulfate, 50 mM sodium sulfate, and 1 mM benzamidine. This mixture was
filtered through cheesecloth and spun at 20,000 g for 30 min
at 4°C. An aliquot of the supernatant was assayed for total CA
activity and residual hemoglobin (Sigma, St. Louis, MO). The clear
supernatant was spun at 240,000 g for 1 h at 4°C to
obtain the microsomal pellet, which was washed with 10 vol of the above
solution. The pellet was suspended in an equal volume of solution and
used for activity and inhibition studies as described above.
The special situation of determining the pH sensitivity of CA IV
activity required a method capable of measuring enzymic activity outside the practical pH limits of the above assay system. For this
purpose, we used the 18O exchange method, which measures
the ratio of the catalytic rate (kcat) to the
Michaelis constant (Km) of CA at chemical
equilibrium. In this method, the exchange of 18O between
CO2 and water and the exchange of 18O between
12C- and 13C-labeled species of CO2
are continuously measured by membrane inlet mass spectrometry
(29). This method can be used to determine the rate of
interconversion of CO2 and bicarbonate at chemical equilibrium, a rate that is described by the equation
k
= [CO2][EO]/(Keff + [CO2]) where k
is the
maximal rate constant for interconversion of CO2 and
HCO
, [CO2] is the concentration of
CO2, [EO] is the total enzyme concentration, and Keff is the effective binding constant of
substrate to enzyme. At values of [CO2]
Keff, the rate of the interconversion of CO2 and HCO
is determined by
k
/Keff. This
ratio is equivalent in theory and practice to
kcat/Km determined at
steady state.
The rate of exchange of 18O between CO2 and
HCO
was measured using a total concentration of 10 mM for all species of CO2 at 10°C. Purified murine CA IV
prepared as described by Hurt et al. (10) was added at a
concentration of 80-120 nM. No buffers were added. Under these
conditions and for a pH of 6.7, Keff of the
equation is too large to measure but is estimated at
Keff > 100 mM based on the substrate
dependence of the interconversion rate (10). The lower
temperature was used to slow the uncatalyzed 18O exchange
relative to the catalyzed exchange and allow more precise measurements
of rates at the lower values of pH at which the uncatalyzed exchange is
very rapid (17). Testing below pH 3.5 was not possible because of the very fast uncatalyzed time, even at 10°C, which depletes the isotope before measurements can be taken.
Gastric CA histochemistry.
The gastric mucosa from Rana pipiens was obtained from
anesthetized frogs as described above except that they were not
perfused free of blood. The isolated mucosa was then placed in a
buffered physiological solution (2) at 25°C and
continuously gassed with 95% O2-5% CO2. After
a 30-min incubation period, the mucosa was then rinsed three times with
the physiological solution. Fixation was accomplished by transferring
the mucosa to a 2.5% gluteraldehyde solution at pH 7.2, containing 19 mM NaCl and 4 mM Na2HPO4, and incubating with
frequent agitation for 6 h at 3-4°C. Afterward, this tissue
was rinsed with cold 6 mM phosphate buffer at pH 7.2 to remove the gluteraldehyde.
The histochemical technique used in this study was the resin version of
the cobalt-precipitation technique described previously by Ridderstrale
(23, 24). The samples were dehydrated by increasing concentrations of ethanol and embedded in a water-soluble glycol methylcrylate (Historesin, Kulzer, Heidelberg, Germany). The tissue samples were infiltrated with a mixture of resin monomer and ethanol for 3 h, followed by resin monomer for 3 h, before embedding
and polymerization at room temperature. Sections (2-µm thick) were cut with a microtome (RM 2165, Leica Instruments) using glass knives.
Sections were incubated, floating in the incubation medium, for 3 and 6 min. The incubation medium contained (in mM) 3.5 CoSO4, 53 H2SO4, 11.7 KH2PO4, and
157 NaHCO3. After incubation, sections were rinsed in 0.67 mM phosphate buffer (pH = 5.9), transferred to 0.5%
(NH4)2S, and finally rinsed twice with
distilled water. Before mounting, some sections were counterstained
with azure blue. Reaction specificity was checked by adding
10
5 M acetazolamide to the incubation medium and
incubating sections as described above. The procedure results in a
black precipitate at sites of CA activity. Sections incubated with
acetazolamide showed no significant staining, except for weak shading
of the nuclei.
Gastric acid secretion measurements.
Isolated frog gastric mucosa was obtained as described above and then
mounted in an Ussing-type chamber. The details of the basic chamber
construction and the solution compositions used on the apical and
basolateral sides were as given previously by Rehm et al.
(22). The solution composition used on the basolateral (nutrient) side was (in mM) 102 Na+, 4 K+, 0.8 Mg2+, 81 Cl
, 0.8 SO
, 25 HCO
, 1 phosphate, and 10 glucose. The apical
solution was made NaCl free by replacement of choline for sodium and
sulfate for chloride with sucrose added to make up any osmotic
difference (2). In all studies, apical and basolateral
solutions (temperature, 25°C) were gassed continuously with 95%
O2-5% CO2. Histamine (0.1 mM) was added to the
basolateral solution to stimulate secretion. Apical H+
secretory rates were measured over times ranging up to 10 min. The
apical (secretory) solution was titrated with 2.5 mM NaOH to maintain a
pH of 5, utilizing a voltage of 100 mV with the basolateral side
positive. Three to four measurements were made. A plot of NaOH (in
µeq) added vs. time yielded a straight line, the slope of which was
the secretory rate. Figure 1 shows a
representative example of a control histamine-stimulated stomach. For
inhibition with methazolamide, appropriate aliquots of a 5 mM
stock solution were added to both apical and basolateral sides. For
inhibition with F3500 and pFBAB, a weighed quantity of either drug was
dissolved in the apical or basolateral solution before addition to the
chamber, and this was allowed to equilibrate with the tissue for 5 min before initiation of stimulated secretion.

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Fig. 1.
Representative example of cumulative luminal NaOH addition to
maintain a luminal pH of 5 after stimulation of a control stomach by
histamine (see MATERIALS AND METHODS for details).
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RESULTS |
Inhibitor and enzyme activity measurements.
The structures, molecular masses, and inhibitory activities of the
three compounds against purified mammalian CA II and IV isoenzymes are
given in Fig. 2. The inhibition against
both isozymes in vitro roughly follows a molecular size relationship
with methazolamide showing the greatest potency, followed by pFBAB, and
then F3500. All drugs show a 20- to 25-fold greater activity against CA
II than CA IV. When the data in Fig. 2 are compared with the inhibition constants for the frog tissues in Table
1, they are roughly four- to fivefold
higher. This largely reflects the temperature differences at
which the assays were run (25°C vs. 0°C) and the expected
temperature-related decrease in inhibitor affinity.

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Fig. 2.
Structures, molecular mass (in kDa), and inhibitory constants
[Ki; against purified mammalian carbonic
anhydrase (CA) II and CA IV] of the permeant and impermeant
sulfonamide CA inhibitors used to suppress frog gastric acid
secretion. F3500, 3,500-kDa polymer linked to aminobenzolamide;
pFBAB, p-fluorobenzyl-aminobenzolamide.
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Table 1.
CA activity and sensitivity to CA inhibitors of frog gastric mucosal
homogenate and microsomes and red cells
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To demonstrate the effective exclusion of pFBAB from intracellular
(cytosolic) CA, we tested pFBAB penetration into human red
cells. Erythrocytes are a simple cell system that permits quantitative determination of intracellular drug accumulation without
the complication of membrane binding because red cells have no
membrane-bound CA. In five experiments at 37°C, with blood of 50%
hematocrit and 500 µM external concentration, red cell pFBAB
concentration was only 5 ± 2 µM (SD) at 1 h. This compares with 35 ± 18 µM for benzolamide (a relatively impermeant
inhibitor), 225 ± 31 µM for methazolamide (a permeant
inhibitor), and a value below the detection limit (<2 µM) for F3500,
the 10-fold larger impermeant polymer.
Table 1 shows the CA activity in frog red cells and mucosal homogenate
and in a preparation of mucosal microsomes along with the inhibitory
potencies of methazolamide, F3500, and pFBAB. Analysis of residual
hemoglobin as a marker of contaminating red cells showed that only 30 U
of activity could be attributed to red cell CA. The CA activity in
mucosal microsomes (180 EU/g) was tested for residual cytosolic enzyme
by treatment with 1% SDS, a concentration known to inactivate CA II
but not CA IV (26). There was no change in the CA activity
(data not shown). Thus ~10% of the overall CA activity in frog
gastric mucosa resides in the membrane fraction with a resistance to
SDS denaturation similar to mammalian CA IV.
The pH dependence of log
(kcat/Km) under acid
conditions is shown in Fig. 3. The solid
line shows that the data are consistent with a
pKa near 7 for the zinc-bound water at the
active site of the enzyme. The slope of unity at pH <6 remains
consistent with the single ionization of the zinc-bound water to a pH
as low as 3.5.

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Fig. 3.
pH sensitivity of purified CA IV in the 18O
exchange assay. kcat, catalytic rate;
Km, Michaelis constant.
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Gastric CA histochemistry.
Figure 4 shows views of the frog gastric
mucosa comprising the outer surface epithelium and the underlying
glandular zone, consisting of oxynticopeptic cells. Staining for CA
activity is evident in nuclei and cytoplasm of some surface epithelial
cells. All capillaries are heavily stained, as are occasional red cells trapped within. Staining in the oxynticopeptic cells is seen along cell
borders, with obvious staining apparent along the basolateral membranes. The small surface area of the apical membrane in these narrow unstimulated gland lumens is unstained. Weak cytoplasmic staining is present together with heavily stained nuclei.

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Fig. 4.
CA
histochemistry of the gastric mucosa of the frog (Rana
pipiens). A: survey picture with surface epithelium at
top and glandular zone with its combined oxynticopeptic
cells below. Strong cytoplasmic and nuclear CA activity and occasional
basolateral and apical (arrowhead) cell membrane staining in the
surface epithelium. The oxynticopeptic cells have weak cytoplasmic and
strong nuclear CA activity and stained cell membranes. Strongly stained
capillaries are identified (arrow). B: higher magnification
of transversely cut glands with oxynticopeptic cells. The apical cell
membranes facing the narrow lumen (*) are unstained, and the
basolateral membranes are stained for CA activity (arrowheads).
Capillaries (arrow) and nuclei are strongly stained.
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Acid secretion measurements.
Table 2 shows the H+
secretory rates for the histamine-stimulated frog gastric mucosa along
with dose-response data for the permeant inhibitor methazolamide, as
well as the secretory rates after inhibition by the two
membrane-impermeant inhibitors when applied to either the apical or
basolateral side. Both impermeant inhibitors are capable of decreasing
acid secretion, but compared with the control secretory rate of
0.051 µeq · min
1 · cm
2,
F3500 and pFBAB gave only ~40% reductions in H+
secretion when applied to the apical side and a slightly greater reduction (55%) when applied to the basolateral side. These results should be contrasted with the total abolition of H+
secretion that occurred when intracellular and membrane-bound CA was
inhibited by 100 µM methazolamide, in agreement with Carrasquer and
Schwartz (2) who used acetazolamide, another permeant
inhibitor.
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Table 2.
Inhibition of gastric acid secretion in isolated frog stomach by CA
inhibitors of differing membrane permeability
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 |
DISCUSSION |
This is the first study to demonstrate a membrane-bound CA
function in gastric acid secretion. Our main findings are that the frog
gastric mucosa contains a cell membrane-associated mammalian-like CA IV
activity, as shown by histochemical and biochemical techniques. This
membrane-bound CA represents almost 10% of total mucosal CA activity,
and it retains significant activity in an acid milieu in contrast to
other CA isozymes. Using two different size membrane-impermeant drugs
with high activity against CA IV applied to the apical or basolateral
surface, we find that this CA activity subserves almost 40% and 55%,
respectively, of stimulated H+ secretion.
Presence and characteristics of membrane-bound CA.
Membrane-bound CA activity in the gastric mucosa of many vertebrate
species has been amply documented by histochemical and biochemical
techniques in the surface epithelial cells and gastric glands
(13, 15, 16, 20, 21, 32, 38). In mammalian parietal cells,
apical and basolateral membranes stain positively with the
histochemical technique (7, 13, 20, 30). The staining in
the frog (Fig. 4) is more prominently found at the basolateral
(antiluminal) border of the oxynticopeptic cells. The lack of evident
staining in the apical membranes is not entirely explicable because the
nondiffusible inhibitors were active when applied to the luminal side.
Because these histochemical results were obtained in unstimulated
stomachs, it may be possible that CA is moved into the apical membrane
with the onset of stimulated acid secretion. In mammals, this activity
has been ascribed to CA IV (3, 7) using isozyme-specific
antibodies. However, a recent report (19) of a second
membrane-bound CA isoenzyme in renal proximal tubules, designated CA
XIV, suggests that not all membrane-bound CA activity is necessarily CA
IV. This may explain the curious finding (1) that in pig
parietal cell tubulovesical membranes, which are heavily enriched in
H+-K+-ATPase and have high CA activity, the
~30-kDa protein suspected to be a CA did not stain with an anti-CA IV antibody.
Our histochemical findings confirm CA activity in cell membranes of
oxynticopeptic cells of the gastric mucosa of Rana pipiens, similar to staining observed in other frog species (21,
31). In contrast to histochemical results in mammals, in which
cytoplasmic staining is heavy (21, 31), cytoplasmic
staining is weaker in amphibian stomach, and membrane staining appears
accentuated against this weaker background. The isozymes present in
nonmammalian species have not been rigorously delineated by gene or
protein analysis, but it appears that in frog stomach, membrane-bound CA activity has similarities to CA IV. The data (Fig. 2 and Table 1)
show that membrane-bound activity is 20 times less sensitive to CA
inhibitors than the cytosolic enzyme and is not denatured by SDS,
characteristics in mammals that distinguish cytosolic CA II from CA IV
(5). In distinct contrast to other CA isozymes, which lose
all activity by denaturation pH <5, CA IV retains significant activity
in the acid pH range (Fig. 2) over which it must function at the
extracellular apical surface (see below).
The results of CA inhibition in the isolated frog gastric mucosa
provide further evidence of membrane-bound and extracellularly oriented
CA. Two different, but otherwise very impermeant, potent CA-inhibiting
sulfonamides gave nearly similar maximal levels of inhibition when
applied luminally or antiluminallly. Our data establish that apical and
basolateral membrane-bound CA are responsible for almost 40% and 55%
of gastric acid secretion, respectively, with the remainder dependent
on intracellular CA II, which could only be accessed by methazolamide.
Functions of gastric CA in acid secretion.
Figure 5 provides an overview of cell and
membrane events relevant to the acid secretion and possible roles of CA
in parietal or oxynticopeptic cells of the stomach. How apical
membrane-bound CA in the stomach subserves H+ secretion is
not totally answered by our studies. In the presence of
HCO
(swallowed in saliva, contained in food, and
secreted by surface epithelial cells), membrane-bound CA permits rapid
dehydration of H2CO3 to CO2 as
luminal HCO
is titrated by H+ secretion.
The newly generated CO2 then readily diffuses across the
apical membrane (see Fig. 5, reaction 1). This would be
operative at the start of secretion until the pH fell below 6.

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Fig. 5.
Schematic representation of cell and membrane events in gastric
acid secretion by parietal or oxynticopeptic cells of the stomach.
B , buffering groups on mucus. Circled numbers denote
possible reactions subserved by membrane-bound and cytosolic CA in
support of acid secretion (see DISCUSSION for details).
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We have shown that CA IV is functional in an acidic milieu in which
bicarbonate is no longer quantitatively present (pH < 6).
Although we could not extend our measurements below a pH of 3.5 for
technical reasons, this may be a moot point. Studies utilizing confocal
microscopy and pH-sensitive fluorescent dyes (4) or small
pH microelectrodes (28) capable of resolving pH down to the epithelial cell layer and gastric crypts have shown pHs no lower
than 3.5 despite bulk luminal values <1 under stimulated conditions.
However, it must be realized that it is not yet established how low the
extracellular pH is in the immediate vicinity of apical membrane-bound
CA. Schreiber and Scheid (28) have advanced the novel but
controversial concept that special buffering and H+ release
properties of gastric mucus form a vehicle for proton transport toward
the gastric lumen. In this model (28), newly secreted
protons are highly buffered by cosecreted mucus. As the mucus migrates
outward, protons are released into the bulk luminal fluid as pepsin
acts on and reduces the buffering capacity of mucus. Mucus production
and its H+ buffering capacity may not accommodate all
stimulated gastric acid secretion, but to the extent that it can
facilitate acid secretion, perhaps apical membrane-bound CA also
permits rapid buffering of secreted H+ by gastric mucus
(Fig. 5, reaction 2).
We also studied the function of basolateral membrane-bound CA,
which we and others (21, 31) observed
histochemically, by application of membrane-impermeant inhibitors
to the serosal side. Our data showing a 55% reduction of
gastric acid secretion is in accord with Loveridge et al.
(14) who used an antibody with CA-inhibiting properties
from the serum of patients with pernicious anemia. When the antibodies
were applied to the basolateral side they suppressed acid secretion by
nearly 80% in the stimulated frog stomach. Basolateral membrane-bound
CA may subserve acid secretion in two ways. The extrusion of
H+ across the apical membrane by
H+-K+-ATPase generates OH
, which
in the presence of CO2 and cytosolic CA II rapidly reacts to form bicarbonate. The HCO
is then extruded across
the basolateral membrane by either a
Cl
/HCO
anion exchanger
(35) or an electrogenic
Na+-HCO
cotransporter (27).
In the extracellular space (Fig. 5, reaction 3), the
extruded HCO
and available H+ are
catalytically reacted to regenerate CO2. Thus a potential rate-limiting buildup of extracellular HCO
in the
vicinity of the anion transporters is minimized by the activity of a
basolateral membrane CA.
The function of cytosolic CA is crucial to H+ secretion.
This is evident in our data with methazolamide in the frog stomach and
many other studies using permeant inhibitors (see Ref. 32 for review). Cytosolic CA supports the high turnover of
H+-K+-ATPase by permitting rapid conversion to
HCO
of OH
produced in the hydrolysis
of ATP and translocation of H+ across the apical membrane
(Fig. 5, reaction 4). The equimolar intracellular generation
of the hydroxyl ion as a H+ is translocated to the lumen
must be dissipated quickly to forestall a rate-limiting alkalinity on
the ATPase reaction.
In conclusion, high rates of gastric H+ secretion are
dependent on both membrane-bound and cytosolic CA. The relatively
acid-resistant apical membrane CA operates in the acidic environment of
the gastric gland lumen in HCO
reabsorption at the
onset of stimulated secretion and then possibly to facilitate rapid
H+ binding to gastric mucus. Cytosolic CA subserves high
H+-K+-ATPase turnover by catalyzing
OH
conversion to HCO
. Basolateral
membrane and capillary endothelial CA subserve rapid transfer of
cytosolic HCO
to the blood by membrane anion exchangers and Na+-coupled extrusion.
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ACKNOWLEDGEMENTS |
We thank Dr. Curtis Conroy for careful measurements of gastric
H+ secretion and Dr. David Silverman for helpful advice in
the CA IV measurements and careful review of the manuscript.
 |
FOOTNOTES |
This work was supported by National Heart, Lung, and Blood Institute
Grant HL-45571.
Address for reprint requests and other correspondence: E. R. Swenson, Pulmonary Section, S-111-Pulm, Dept. of Veterans Affairs Medical Center, 1660 South Columbian Way, Seattle, WA 98108 (E-mail: eswenson{at}u.washington.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 30 June 2000; accepted in final form 12 February 2001.
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