Division of Biomedical Sciences, University of California, Riverside, California 92521
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ABSTRACT |
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Na-K-2Cl cotransport and
Cl/HCO3 exchange are prominent mechanisms for
Cl uptake in
Cl
-secreting epithelial
cells. We used immunofluorescence microscopy to delineate the
distributions of Na-K-2Cl cotransporter-1 (NKCC1) and anion exchanger-2
(AE2) proteins in rat gastric mucosa (zymogenic zone). Parietal cells
(PCs) above the neck of the gastric gland contained abundant AE2 but
little or no NKCC1, whereas those in the neck and base contained high
NKCC1 but diminished AE2. Lower levels of NKCC1 were detected in
surface mucous cells and in cells comprising the blind ends of all
glands. Pulse labeling of proliferating cells with bromodeoxyuridine
indicated that new PCs originate in the isthmus with scant NKCC1; the
subset of PCs that migrate downward expresses NKCC1 abruptly on
entering the neck, within 7 days of cell division. Our results suggest
that downwardly migrating PCs replace one mechanism for
Cl
entry
(Cl/HCO3 exchange) with another (Na-K-2Cl cotransport).
gastric mucosa; hydrochloric acid secretion; anion exchanger-2; chloride transport
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INTRODUCTION |
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THE Na-K-2Cl cotransporters are membrane glycoproteins
that electroneutrally move salt and osmotically obliged water into cells. The Na-K-2Cl cotransporter-1 (NKCC1) isoform functions in most
animal cells to maintain cellular
Cl concentration and volume
near physiological set points. The same NKCC1 isoform is present at 10- to 30-fold higher levels in the basolateral membrane of various
Cl
-secreting epithelial
cells (19), where it maintains cytosolic Cl
concentration above
electrochemical equilibrium in opposition to rapid apical
Cl
efflux (23).
Although NKCC1 is prominently expressed in gastric mucosa (5, 19, 26, 29), its cellular distribution and physiological function in the stomach remain undefined. It has been proposed that NKCC1 participates in HCl secretion (1, 29) on the basis of studies of intact mammalian (1, 11) and amphibian (29) gastric mucosa showing that HCl secretion is partly dependent on serosal Na+ and blocked by Na-K-2Cl cotransport inhibitors such as furosemide and bumetanide.
The source of gastric acid is the parietal cell (10). The active
extrusion of HCl across the canalicular membrane of this cell requires
a concomitant and balanced uptake of
H+ equivalents and
Cl across the basolateral
membrane. HCl uptake is believed to be accomplished, largely if not
wholly, by a Cl/HCO3 exchange process (22, 24)
that displays a high transport capacity (31) and strong allosteric
activation by intracellular alkalinity (27), which has been attributed
to anion exchanger-2 (AE2), the main anion exchanger isoform in
parietal cells (13, 30).
Of interest is whether NKCC1 is abundant in parietal cells. To evaluate this possibility, we used double-label immunofluorescence microscopy to localize NKCC1, AE2, and H-K-ATPase (a marker for parietal cells) in rat gastric mucosa. NKCC1 labeling was exceptionally intense along the basolateral margin of parietal cells inhabiting the neck and base of the gastric gland. Parietal cells in and above the proliferative zone (isthmus) of the gland, in sharp contrast, were virtually devoid of NKCC1 yet rich in AE2. The abrupt acquisition of NKCC1 by downwardly migrating parietal cells was correlated with transit from isthmus to neck. A preliminary report of this work has appeared in abstract form (21).
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MATERIALS AND METHODS |
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Antibodies. The distribution of NKCC1
in gastric mucosa was examined by immunocytochemistry using antibodies
T4 and N1c. T4 is a mouse monoclonal antibody that was generated
against a fusion protein encompassing the carboxy terminus
(S760-S1212) of human NKCC1, as described previously (19). N1c is
a novel polyclonal rabbit antibody directed against the same fusion
protein. Both T4 and N1c prefer the SDS-denatured form of NKCC1 and
yield similar labeling patterns on aldehyde-fixed, SDS-treated sections
of various tissues. Both antibodies selectively recognize the same
broad band of NKCC1 protein centered at ~160 kDa on immunoblots of
rat gastric mucosal membrane protein (19) and, in some preparations of
gastric membranes, a diffuse smear running at lower molecular weight
that presumably represents proteolytic fragments. Parietal cells were
identified with a mouse monoclonal antibody (HK12.18) directed against
the -subunit of hog gastric H-K-ATPase (28), kindly provided by Drs.
David Scott and George Sachs (VA WLA Medical Center/UCLA). An
affinity-purified rabbit polyclonal antibody (
-SA6) raised against a
carboxy-terminal peptide (residues 1224-1237) of murine AE2 was
supplied by Drs. A. Stuart-Tilley and S. Alper (Beth Israel-Deaconess
Medical Center) and has been characterized previously (30).
Reagents. We obtained reagent-grade salts, adjuvant, protease inhibitors, 5-bromo-2-deoxyuridine (BrdU), and anti-BrdU antibody from Sigma; Hemo-De and Paraplast Plus from Fisher Scientific; and secondary antibodies from Vector Laboratories.
Tissue preparation. Adult (3-4 mo
old) male Sprague-Dawley rats given free access to food and water were
anesthetized with pentobarbital sodium. The stomach was removed
and rinsed with PBS containing a battery of protease inhibitors
[18 µg/ml
N-tosyl-L-phenylalanine chloromethyl ketone, 1 µg/ml pepstatin A, 1 µg/ml leupeptin,
2 µg/ml chymostatin, 0.004 units aprotinin, 50 µg/ml
4-(2-aminoethyl)benzenesulfonyl fluoride]. The stomach wall was
cut into 5-mm strips and placed in ice-cold PLP fixative (2%
paraformaldehyde, 75 mM lysine, 10 mM sodium periodate, 45 mM sodium
phosphate, pH 7.4) (20). One hour later, the slices were transferred to
fresh fixative and allowed to incubate for an additional 2 h on ice.
The tissue was rinsed with PBS and stored for 2-30 days in
cryoprotectant (2.3 M sucrose). For sectioning, the tissue was embedded
in tissue-freezing medium (Triangle Biomedical Sciences) and frozen at
35°C. Frozen sections of 5 µm thickness were cut on a
cryostat microtome (HM 500 OM, Microm Lamborgeraete), mounted on
Superfrost Plus slides (Fisher), and stored at
20°C in the
presence of desiccant. Every tenth slide was stained with hemotoxylin
and eosin for histological assessment.
Labeling of S phase cells. Adult male Sprague-Dawley rats fed ad libitum were killed 3 h or 7 days after receiving 100 mg BrdU by intraperitoneal injection. These times were chosen because precursory time course studies had established that new cells are adequately labeled within 3 h and that parietal cells express Na-K-2Cl cotransporter before they reach the age of 7 days. The stomach was excised, cut into 5-mm squares, and fixed by immersion in 4% paraformaldehyde for 30 min. The tissue was dehydrated in a series of graded alcohols (10 min each in 50, 75, 85, 95, and 100% ethanol), then rinsed three times in clearing agent (Hemo-De). For embedding, the tissue was incubated at 60°C in 50% paraffin and then in 100% paraffin. Sections 6-µm thick were cut and mounted on Fisher Superfrost Plus slides and baked at 60°C overnight. Every tenth slide was stained with hemotoxylin and eosin for histological assessment. Before use, slides were cleared in Hemo-De, rehydrated in a series of graded alcohols (twice each in 95% then 70% ethanol), and rinsed with K+-free PBS.
Single- and double-label
immunocytochemistry. All steps were carried out in a
humidified chamber at room temperature. Mounted frozen sections were
thawed and rehydrated in K+-free
PBS. To reduce autofluorescence, sections were treated with 1% sodium
borohydride for 10 min, then washed twice in
K+-free PBS. To improve antibody
labeling, sections were exposed to 1% SDS in
K+-free PBS for 10 min, then
washed three times with K+-free
PBS. Denatured sections were incubated for 1 h in a blocking solution
(BS+) consisting of PBS and 20%
goat serum, 0.2% BSA, 25 mM
NH4Cl, 25 mM glycine, and 25 mM
lysine (pH 7.4). Subsequent incubations were carried out in
BS (20% goat serum, 0.2%
BSA, in PBS). After one wash in
BS
, sections were incubated
with the primary antibody (diluted 1:1,000 or 1:2,000) for 2 h, except
for antibody T4, which gave optimal specific binding after only 2 min.
Sections were washed three times in
BS
and incubated for 1 h
with secondary antibody (biotinylated goat anti-rabbit IgG or
rat-adsorbed biotinylated horse anti-mouse IgG) diluted to 5 µg/ml in
BS
. Sections were washed
twice with PBS, then incubated for 1 h with fluorochrome-conjugated
avidin diluted to 5 µg/ml in PBS. For dual-label experiments,
incubations with different antibodies were carried out consecutively.
Slides were mounted in mounting medium (Vectashield) and viewed with a
Nikon UFX-DX epifluorescence microscope. For BrdU immunocytochemistry,
sections were treated with 1 N HCl for 1 h at 40°C (to denature
double-stranded DNA) before incubation with primary antibody (mouse
monoclonal anti-BrdU). Each immunolocalization result is representative
of at least three independent experiments conducted on different rats.
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RESULTS |
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The glandular mucosa of the rodent stomach is histologically divisible
into three distinct regions (16): a distal pure mucous zone, a middle
mucoparietal zone, and a proximal zymogenic zone (corpus). In the pure
mucous zone, the branched glands comprise mainly mucous cells and,
unlike other glands of the stomach, lack parietal cells. In this type
of gland, NKCC1 was detected at modest levels along the basolateral
margins of surface mucous cells and of cells comprising the blind end
of each gland (Fig.
1D). In the mucoparietal zone, NKCC1 was likewise detected in surface mucous
cells and in cells inhabiting the terminus of each gland. Glands in
this region contained a few scattered cells that stained with eosin and
antibody HK12.18, identifying them as parietal cells, but not antibody
T4 or N1c (data not shown); hence, parietal cells in the mucoparietal
zone lack detectable NKCC1.
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Glands in the zymogenic zone were longer and straight and characteristically populated by numerous parietal cells (Fig. 1A). On examination of fixed eosin-stained sections, parietal cells in the superficial one-third of the gland appeared larger than those at deeper levels of the mucosa, as described earlier for this species (9) and for rabbit (15). Single-label immunocytochemistry using antibody HK12.18 revealed intense labeling of parietal cells, in accord with the unique abundance of H-K-ATPase in these cells (not shown). Differences in H-K-ATPase labeling intensity between parietal cells at different positions along the gastric gland were not apparent, even when the HK12.18 antibody was titrated to concentrations giving submaximal staining. Double-label immunocytochemistry using antibodies T4 for NKCC1 and HK12.18 for H-K-ATPase indicated that the larger parietal cells in the superficial one-third of the gland expressed little or no NKCC1 protein (Fig. 1B). In striking contrast, parietal cells in the neck and base of the gland showed intense basolateral labeling with monoclonal antibody T4 (Fig. 1B). A similar labeling pattern was obtained with another antibody against NKCC1, polyclonal N1c (data not shown). Examination of NKCC1 immunostaining at higher magnification revealed a very sharp transition between the two populations of parietal cells over a small proportion of the gland length (Fig. 1E). Transitional cells with intermediate NKCC1 immunoreactivity were not apparent. The juncture marking the abrupt acquisition of NKCC1 was situated roughly one-third the distance down the gland. These results indicate that parietal cells express high levels of NKCC1 as they migrate into the basilar segment of the gastric gland.
NKCC1 was also detected, although at lower levels, in cells comprising the blind end of the zymogenic glands (Fig. 1F). These cells appear analogous to NKCC1-positive cells situated in the extreme terminus of pure mucous and mucoparietal glands (e.g., Fig. 1D).
To estimate the age at which parietal cells express NKCC1, rats were injected 3 h or 7 days before death with BrdU to pulse-label S phase cells (32); because unincorporated BrdU is cleared within hours by excretion and metabolism, only cells undergoing DNA synthesis shortly after injection become labeled. BrdU-positive cells were initially concentrated within the proliferative zone (isthmus) of each gastric gland (Fig. 1, BrdU, 3 h), confirming previous reports (32). None of the cells within the isthmus labeled strongly with antibodies against NKCC1. Within 1 wk, 7-day-old parietal cells, identified by colabeling for BrdU (nucleus stained green) and H-K-ATPase (red), had migrated into the lower pit and upper neck of each gland (Fig. 1, BrdU, 7 days). Those in the neck exhibited intense basolateral NKCC1 labeling; one such parietal cell, with a BrdU-labeled (green) nucleus, is designated by left arrow in Fig. 1 (in panel labeled BrdU, 7 days). Thus the abrupt acquisition of NKCC1 by downwardly migrating parietal cells was topographically correlated with transit from isthmus to neck, and this event occurred within 7 days of cell division. Upwardly migrating parietal cells, in contrast, did not express high levels of NKCC1.
In parietal cells, a major mechanism for basolateral HCl uptake is
Cl/HCO3 exchange (25), and analysis of
anion exchanger isoforms in parietal cells has implicated AE2 (13, 30).
To assess whether AE2 coexists with NKCC1 in basilar parietal cells, we
mapped its distribution in rat gastric mucosa using the AE2-selective antibody -SA6 (30). Labeling was most prominent along the
basolateral margin of parietal cells (Fig.
1C), particularly those situated in
the superficial one-third of the gland, confirming Stuart-Tilley et al.
(30). AE2 immunolabeling was progressively weaker in parietal cells
inhabiting deeper levels of the gland. The gradation of AE2 loss was
most conspicuous near the stratum, where parietal cells abruptly gain
NKCC1 (compare Fig. 1B and Fig.
1C). Thus, as parietal cells migrate
downward from the isthmus, they appear to replace one mechanism for
chloride entry (Cl/HCO3 exchange) with
another (Na-K-2Cl cotransport).
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DISCUSSION |
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The detection of abundant NKCC1 mRNA (5, 26, 29) and NKCC1 protein (19) in gastric mucosa has prompted speculation that Na-K-2Cl cotransport might play important roles in gastric secretion. Our results indicate that the NKCC1 signal in rat gastric mucosa is derived mainly from parietal cells, specifically those situated in the neck and base of the gastric gland.
The epithelium that lines the stomach is perpetually renewed through proliferation, migration-associated differentiation, and exfoliation/phagocytosis of its three principal cell lineages (parietal, pit, and zymogenic) (7, 14). New parietal cells arise from progenitors in the isthmus of the gastric gland, then embark on a bipolar migration toward the pit or base regions (14). This orderly cell migration creates a stratified system in which the successive stages of the parietal cell's life cycle can be correlated anatomically with the position along the gastric gland. Our results with the rat experimental model suggest that downward migration of parietal cells is associated with an abrupt and prodigious expression of NKCC1 protein along with a diminution of AE2 protein. As a result, parietal cells in the superficial one-third of the gland are rich in AE2 but not NKCC1, whereas those deeper in the gland have abundant NKCC1 but diminished AE2. The juncture marking the abrupt acquisition of NKCC1, like that marking the progressive loss of AE2, is near the border between the proliferate compartment (isthmus) and the neck of the gland.
The observed transformation could be programmed to occur in relation to parietal cell age or spatial position within the gland. It is interesting that glands in the mucoparietal region of the rat stomach contain only a few scattered parietal cells, and all of them, even those situated deep in the gland, lack detectable NKCC1. In this respect they resemble parietal cells inhabiting the isthmus of glands in the zymogenic region. This implies that the expression of NKCC1 by parietal cells is not dependent on time or spatial position per se but rather on signals or cell contacts present in zymogenic glands but absent in mucoparietal glands. In this regard, it is possibly important that the parietal cells that express NKCC1 (those below the isthmus) are almost always peripheral to zymogenic cells, as identified by cresyl fast violet staining (Fig. 1A) and pepsinogen immunocytochemistry (unpublished observations), whereas the parietal cells that do not express NKCC1 (those in mucoparietal glands and those above the neck of zymogenic glands) reside adjacent to mucous cells. This interrelationship suggests that the zymogenic cell lineage might influence the migration-associated differentiation program of parietal cells, just as parietal cells appear to modulate differentiation of zymogenic cells (17). NKCC1 expression might serve as an important positional marker for a key step in parietal cell differentiation.
What function might NKCC1 serve in basilar parietal cells? Few animal
cells contain comparably high levels of this transporter, and those
that do are uniquely specialized for
Cl secretion; examples
include epithelial cells of the salivary acinus, colonic crypt, sweat
gland secretory coil, trachea, shark rectal gland, and avian salt gland
(19). In these cells, basolateral NKCC1 units function to maintain
cytosolic Cl
concentration
above electrochemical equilibrium (18, 23). Stimulation of the cell
brings about rapid transepithelial fluxes of
Cl
and water that are
sustained as a result of Cl
entry via NKCC1 and Cl
exit
via apical channels. By analogy, NKCC1 in basilar parietal cells most
likely drives rapid transepithelial
Cl
flux. What remains
unclear is whether the cotransported
Cl
are used for the purpose
of acid secretion (HCl) or fluid secretion (NaCl or KCl).
Regional variations in parietal cells. Our findings add to a growing body of evidence that parietal cells above and below the neck of the gastric gland are not functionally equal. Changes in acid secretory activity in response to a meal are intimately associated with striking changes in parietal cell morphology (8). The transformation to the secreting configuration includes a massive redistribution of intracellular tubulovesicles to the canalicular membrane, resulting in the translocation of proton pumps to a greatly expanded luminal surface (6). Early ultrastructural studies of rodent stomach showed that parietal cells in the base, compared with those in the neck, have a less elaborate secretory surfaces and less rough endoplasmic reticulum (9, 12) and fail to assume the morphological features associated with acid secretion in response to feeding (12), prompting speculation that parietal cells in the gland base are less active in HCl secretion. Compelling support for this concept was provided by histochemical analyses of H-K-ATPase activity in mouse gastric mucosa, which indicated that the proton pumps in base parietal cells, although immunologically detectable, are nevertheless catalytically dormant (3) and entirely refractory to physiological stimulation by feeding (4). Weaker H-K-ATPase activity in base parietal cells is correlated with lower levels of mRNA encoding both H-K-ATPase subunits (2, 15). Unified evidence for parietal cell heterogeneity was obtained in recent studies of isolated rabbit gastric glands (15). Graded stimulation of parietal cells provoked parallel changes in acid production, tubulovesicular-to-canalicular H-K-ATPase translocation, and cellular morphology. Given the close association in parietal cells between membrane rearrangements and acid secretory state, morphometric analysis of parietal cells at different levels in gastric glands provided a visual index of their acid secretory state. This ultrastructural analysis revealed that base cells, in sharp contrast to more superficial cells, are morphologically unresponsive to acid secretagogues and, by inference, far less active in acid secretion.
These findings, together with our evidence that rat parietal cells
abruptly gain NKCC1 and begin to lose AE2 on migrating into the gland
neck, are consistent with earlier speculation that basilar parietal
cells have become reconfigured for some function other than acid
secretion (4). We hypothesize that parietal cells might undergo a
programmed conversion of their principal function from acidic chloride
(HCl) secretion to nonacidic chloride (NaCl or KCl) secretion on
migrating into the neck. The elaboration of a saline fluid volume by
base parietal cells might help flush the inactive form of pepsinogen
from where basilar zymogenic cells secrete it (in the blind end of the
gland lumen) toward the gastric pit, where it is rendered functional by
acid. However, because our immunocytochemical results demonstrate the
antigenic presence rather than ion-transporting activity of NKCC1, the
true function of this transporter in parietal cells remains
speculative. In which parietal cells, and under what physiological
circumstances, is NKCC1 active? Definitive answers should come from
measurements of vectorial
Cl transport and
intracellular Cl
homeostasis in single parietal cells inhabiting different levels of the
gastric gland.
In conclusion, immunolocalization of NKCC1 and AE2 in rat gastric mucosa distinguishes two types of parietal cells; those inhabiting the basilar two-thirds of the gastric gland possess abundant NKCC1, whereas those in the superficial one-third do not. The migration-associated expression of NKCC1 appears to be topographically correlated with a diminution of AE2 and with previously described ultrastructural (9, 12, 15) and cytochemical alterations (3, 4).
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ACKNOWLEDGEMENTS |
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We are indebted to Yeon Kim for expert technical assistance, Drs.
George Sachs and David Scott (Center for Ulcer Research and Education & UCLA) for HK12.18 antibody and substantive suggestions, Drs. A. Stuart-Tilley and S. Alper (Harvard Medical School) for providing
-SA6 antibody, and Dr. Mark Haas (University of Chicago) for
thoughtful discussions and critical review of the manuscript.
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FOOTNOTES |
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This work was supported by American Heart Association Grant 94015270.
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: C. Lytle, Div. of Biomedical Sciences, Univ. of California, Riverside, CA 92521 (E-mail: christian.lytle{at}ucr.edu).
Received 31 July 1998; accepted in final form 1 February 1999.
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