Departments of 1 Internal Medicine, 3 Surgery, and 2 Cellular and Molecular Physiology, Yale University, New Haven, Connecticut 06520
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ABSTRACT |
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Two distinct
colonic
H+-K+-adenosinetriphosphatase
(H+-K+-ATPase)
isoforms can be identified in part on the basis of their sensitivity to
ouabain. The colonic
H+-K+-ATPase
-subunit (HKc
) was recently cloned, and its message and protein
are present in surface (and the upper 20% of crypt) cells in the rat
distal colon. These studies were performed to establish the spatial
distribution of the ouabain-sensitive and ouabain-insensitive
components of both
H+-K+-ATPase
activity in apical membranes prepared from surface and crypt cells and
K+-dependent intracellular pH
(pHi) recovery from
an acid load both in isolated perfused colonic crypts and in surface
epithelial cells. Whereas
H+-K+-ATPase
activity in apical membranes from surface cells was 46% ouabain
sensitive, its activity in crypt apical membranes was 96% ouabain
sensitive. Similarly, K+-dependent
pHi recovery in isolated crypts
was completely ouabain sensitive, whereas in surface cells
K+-dependent
pHi recovery was insensitive to
ouabain. These studies provide compelling evidence that HKc
encodes the colonic ouabain-insensitive H+-K+-ATPase
and that a colonic ouabain-sensitive
H+-K+-ATPase
isoform is present in colonic crypts and remains to be cloned and
identified.
K+-dependent intracellular pH regulation; rat colon; H+-K+-adenosinetriphosphatase
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INTRODUCTION |
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ACTIVE ABSORPTION of
K+ is a novel function of the
mammalian distal colon and is likely energized by an apical membrane
H+-K+-adenosinetriphosphatase
(H+-K+-ATPase)
(1). The -subunit of the colonic
H+-K+-ATPase
(HKc
) has recently been cloned and sequenced (7, 11) and is a member
of a gene family of P-type ATPases that also includes Na+-K+-ATPase
and gastric
H+-K+-ATPase.
Given the 50-60% shared homology between these ATPases, it is not
surprising that the pharmacological properties of the colonic
H+-K+-ATPase
overlap those of the ouabain-sensitive,
omeprazole/SCH-28080-insensitive Na+-K+-ATPase
and the ouabain-insensitive, omeprazole/SCH-28080-sensitive gastric
H+-K+-ATPase.
Physiological studies of active K+
absorption in the rat distal colon and pharmacological characterization
of colonic
H+-K+-ATPase
provide compelling evidence for two different colonic H+-K+-ATPases
(9, 15), one that is insensitive to ouabain and stimulated by
aldosterone and a second that is sensitive to ouabain but nonresponsive
to aldosterone. Which of these two -subunits is encoded by HKc
cDNA is not known.
Expression studies of HKc have provided important information
regarding its segmental and spatial distribution but have yielded conflicting data regarding its sensitivity to ouabain (5, 6, 9, 13).
First, in situ hybridization studies localized HKc
message to the
surface (and the upper 20% of crypt) cells of the distal colon (11).
Localization of the HKc
message to the proximal colon of normal
animals has not been reported. Second, antibodies raised to a fusion
protein developed from a segment of HKc
cDNA identified a protein
only in the apical membrane of surface cells of the distal colon (13).
Third, infection of Sf9 cells with baculovirus transfected with HKc
cDNA resulted in expression of ouabain-insensitive
H+-K+-ATPase
activity (13). This activity in Sf9 cells was completely inhibited by
orthovanadate and was inhibited 18% by SCH-28080. H+-K+-ATPase
activity in apical membranes isolated from epithelial cells of rat
distal colon, which are predominantly surface cells, is 100% vanadate
sensitive, 55% ouabain insensitive, and 18% SCH-28080 sensitive (13);
it was therefore concluded that HKc
cDNA most likely encoded the
ouabain-insensitive isoform (13). Fourth, in contrast to these studies,
expression studies of HKc
cRNA in
Xenopus oocytes provide evidence of
both ouabain-sensitive and relatively ouabain-insensitive
86Rb uptake (5, 6).
We have recently developed methods to study specific functions of
surface and crypt cells independently (16). This present study was
therefore designed to determine the ouabain sensitivity of two
different colonocyte functions, apical membrane
H+-K+-ATPase
activity and K+-dependent recovery
of intracellular pH (pHi) from
an acid load both in surface and in crypt cells. These results
establish that these two functions in crypt cells are completely
ouabain sensitive. Because HKc message and protein are localized to
surface cells but not crypt cells, these studies provide compelling
evidence that HKc
message encodes the ouabain-insensitive
H+-K+-ATPase
-subunit isoform in surface cells of rat distal colon.
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METHODS |
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H+-K+-ATPase Determinations
Apical membrane preparation.
Apical membranes from surface cells and from crypt cells were prepared
from the distal colon of normal male Sprague-Dawley rats (200 g), as
described previously (17). In brief, surface cells and crypt glands
were isolated by divalent cation chelation technique using EDTA and
utilized for apical membrane preparation (16). Surface cells and crypt
glands were homogenized with Omni Mix (Waterbury, CT) at 2,500 and
4,000 rpm, respectively. Brush-border caps were isolated by Percoll
gradient (10%) centrifugation. Apical membranes were purified by
disrupting the brush-border caps by homogenization (Polytron, Westbury,
NY) and centrifugation (Sorvall RC5B, SS34 rotor, 10,999 rpm, 15 min).
Apical membranes used for enzyme assays were resuspended in 50 mM
tris(hydroxymethyl)aminomethane (Tris) · HCl buffer
(pH 7.4) containing 250 mM sucrose. Resuspended membranes were stored
at 70°C until further use. Purity of the surface cell apical
membrane was assessed by 10- to 12-fold enrichment of
H+-K+-ATPase
activity (9), whereas crypt cell apical membrane purity was assessed by
the presence of a
Cl
-dependent
Na+/H+
exchange activity (18).
Enzyme assay. H+-K+-ATPase activity was measured by the method of Forbush (10) as described previously (9). In brief, 500 µl reaction mixture (50 mM Tris · HCl, 5 mM MgCl2, 20 mM KCl, 5 mM ATP, and 10 µg protein) was incubated for 30 min at 37°C. The reaction was arrested by adding 1 ml of ice-cold stop solution (3% ascorbic acid, 1% sodium dodecyl sulfate, and 0.5% ammonium molybdate in 0.5 N HCl), and the color was developed by adding 1.5 ml of 1% sodium arsenate in 5% acetic acid. Mg2+-ATPase activity was measured in the absence of KCl. H+-K+-ATPase activity was measured in the presence of both Mg2+ and K+. H+-K+-ATPase activity was calculated by subtracting the Mg2+-ATPase activity from the activity obtained in the presence of both Mg2+ and K+. H+-K+-ATPase activity was also measured in the presence of 1 mM ouabain. H+-K+-ATPase activity measured in the presence of ouabain represents ouabain-insensitive H+-K+-ATPase activity. Ouabain-sensitive H+-K+-ATPase activity was calculated by subtracting H+-K+-ATPase activity in the presence of ouabain from H+-K+-ATPase activity in the absence of ouabain. ATPase activity is presented as micromoles Pi liberated per milligram protein per minute. Results are presented as means ± SE of three different membrane preparations; eight animals were used for each preparation. Protein was measured by the method of Lowry et al. (14).
K+-Dependent pHi Determinations
Measurement of pHi in isolated crypts.
pHi of individual colonic cells of
isolated perfused crypts was determined as previously described (16).
Briefly, individual crypts were isolated using a hand dissection
technique and perfused in vitro as previously described (19). After
isolation, the individual crypts were transferred to a thermostatically
controlled chamber mounted on the stage of an inverted microscope.
After transfer the crypts were double cannulated using a series of
concentric glass pipettes. The crypts were then perfused from both the
basolateral and apical membrane with a Ringer buffer, pH 7.0. The
luminal perfusate was then exchanged for a solution of 10 µM
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-acetoxymethyl ester (BCECF-AM) (Molecular Probes, Eugene, OR), the precursor of the
fluorescent isoform of the pH-sensitive dye BCECF. During perfusion
from the lumen there was preferential dye uptake in the
non-mucus-secreting cells along the length of the crypt. After a period
of 10 min of dye loading the luminal solution was exchanged for a
control -free Ringer solution that contained 5 mM K+, for a period of
5 min. The crypt cells were imaged with an intensified charge-coupled
device camera connected to an imaging system. Individual records of pHi vs. time within
single cells were obtained using a ratiometric analysis of the
individual fluorescent images in a manner similar to that previously
described for rabbit proximal colon cells (22).
Measurement of pHi in polarized surface
cells.
A preparation has also been developed that permits imaging studies of
surface cell pHi. These imaging
studies were performed in situ in full-thickness rat distal colon with
simultaneous independent superfusion of the serosal and mucosal aspects
of the tissue. After rats were killed, ~3 cm of full-thickness distal
colon was removed and opened, and fecal debris was removed with
-free Ringer. With a series of
O-rings, the full-thickness segment was mounted in a two-sided chamber
that was used for fluorescence imaging; the basic design of this
chamber was adapted from those described in recent studies of colonic
mucosa (4, 8, 12). This chamber is bound by two glass coverslips with a
volume of ~100 µM per side and was mounted on the stage of a Zeiss
IM-35 inverted microscope, equipped for epi-illumination (20). Luminal and basolateral (i.e., "bath") solutions were switched via
computer-controlled five-way valves with zero dead space. Chamber
temperature was monitored and maintained at 37°C by prewarming the
bath solution with a flow rate of ~3 ml/min.
Solutions. The HEPES-Ringer solution contained (in mM) 125 NaCl, 5 KCl, 1.0 CaCl2, 1.2 MgSO4, 2 NaH2PO4, 10.2 glucose, and 32 HEPES. KCl replaced NaCl (and vice versa) in selected experiments. The pH of all solutions was 7.40 at 37°C unless otherwise indicated. High-K+/nigericin calibration solution contained (in mM) 10 µM nigericin, 105 KCl, 1.0 CaCl2, 1.2 MgSO4, 2 H3PO4, 10.5 glucose, 32.2 HEPES, and 46.4 N-methyl-D-glucamine (NMDG); pH was adjusted with HCl or NMDG. All solutions were equilibrated with air and adjusted to an osmolality of 300-310 mosmol/kg with NaCl or mannitol.
Optical system. A solid-state intensified TV system, with digital image acquisition and analysis, was used in these studies, as described previously (20). Briefly, fluorescence emission or intensity of emitted light (I) was monitored at 530 nm while dye was alternately excited at 440 and 490 nm. Between excitation periods, the cells were in the dark, minimizing photobleaching of dye and photodynamic damage. Data pairs of 440/490 were acquired as often as one per 2.5 s, but the sampling rate (typically once per 5-30 s) was matched to the speed of pHi changes. A program was used to outline the boundaries of individual surface cells and to group pixels by cells. Typically, two to four dye-loaded surface cells were selected randomly for analysis from each preparation. For each cell, in each sample, I490 and I440 values were obtained, thus providing the I490/I440 ratio. Each experiment was concluded with a single-point calibration (3), using the nigericin/high-K+ technique to clamp pHi at pH 7.00 (21).
Statistics
One-tailed Student's t-test was used, with P < 0.05 considered statistically significant. ![]() |
RESULTS |
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Distribution of H+-K+-ATPase Isoform Activity in Surface and Crypt Cells
Surface cells.
Both ouabain-sensitive and ouabain-insensitive
H+-K+-ATPase
activities have previously been identified in apical membranes isolated predominantly from surface cells (9). HKc message and protein level
were expressed only in surface (and the upper 20% of crypt) cells (11,
13). It is not known whether both ouabain-sensitive and
ouabain-insensitive isoforms of
H+-K+-ATPase
are expressed solely in surface cells or in both surface and crypt
cells; therefore,
H+-K+-ATPase
activity was assayed in apical membranes isolated from surface and
crypt cells. As presented in Fig. 1,
H+-K+-ATPase
activity (94.0 ± 11.3 nmol Pi
liberated · mg
protein
1 · min
1)
in surface cell apical membrane was partially inhibited by 1 mM ouabain
(50.9 ± 7.0 nmol · mg
protein
1 · min
1).
The ouabain-insensitive fraction of
H+-K+-ATPase
activity was 54% of the total activity. These results are similar to
those previously presented (9).
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Crypt cells.
H+-K+-ATPase
activity was also measured in apical membranes isolated from crypt
cells and is presented in Fig. 1.
H+-K+-ATPase
activity of crypt cell apical membranes was substantially lower than
that of surface cell apical membranes (24.0 ± 4.2 vs. 94.0 ± 11.3 nmol · mg
protein1 · min
1).
In contrast to surface cell apical membranes,
H+-K+-ATPase
of crypt cell apical membranes was almost completely (96%) inhibited
by 1 mM ouabain. These results indicate that the ouabain-insensitive H+-K+-ATPase
isoform is expressed only in apical membranes of surface cells and that
the ouabain-sensitive isoform is the only
H+-K+-ATPase
isoform expressed in apical membranes from crypt cells. Because
ouabain-insensitive
H+-K+-ATPase
is present in surface but not in crypt cell apical membranes, a spatial
distribution identical to that of HKc
message and protein (11, 13),
it is likely that HKc
encodes the ouabain-insensitive H+-K+-ATPase
-subunit.
K+-Dependent Regulation of pHi
Crypt cells. To study K+-dependent regulation of pHi in crypt cells, a recently developed preparation of isolated colonic crypts (16, 19) was used that permits separate perfusion of apical (lumen) and basolateral (bath) membranes. An acid load was induced by removing Na+ from both lumen and bath solutions while maintaining lumen K+ concentration at 5 mM.1 Figure 2 demonstrates that the addition of 20 mM K+ to the lumen solution increased pHi to its baseline value. The presence of 0.5 mM ouabain before the readdition of 20 mM K+ completely prevented K+-dependent pHi recovery (Fig. 3A). These studies demonstrate that K+-dependent pHi recovery from an acid load in crypt cells is exclusively ouabain sensitive (Table 1) and is consistent with the presence of the ouabain-sensitive H+-K+-ATPase isoform in crypt cell apical membranes.
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Surface cells.
Apical K+-dependent acid extrusion
was also studied in polarized surface colonocytes, using a newly
developed preparation. After an
NH4Cl-induced acid
load1 (2),
pHi recovered rapidly to resting
pHi values in the presence of both
Na+ and
K+. In the absence of both
Na+ and
K+,
pHi did not alkalinize toward
resting pHi values until either K+ or
Na+ was returned to the mucosal
solution. pHi recovery was
initiated after the addition of 5 mM mucosal
K+, and further recovery was
observed on increasing mucosal K+
concentration to 20 mM (Fig. 3B).
Recovery in the presence of either 5 or 20 mM mucosal
K+ was not inhibited by 1.0 mM
mucosal ouabain (Fig. 3B and Table 1).
Because HKc message and protein are localized to surface cells but
not crypt cells, these studies of
K+-dependent regulation of
pHi provide further support for
the high probability that the functional activities encoded by HKc
isoform are not ouabain sensitive.
Role of voltage. To exclude the possibility that K+-dependent recovery of pHi in response to an acid load is a result of membrane depolarization and not apical membrane electroneutral H+/K+ exchange, experiments were performed with barium in both surface and crypt cells. Two separate sets of experiments were performed. In one series of experiments the effect of luminal 5 mM barium on basal pHi was determined. Barium will depolarize the apical membrane, so if K+-dependent recovery of pHi were a result of membrane depolarization, the addition of barium should have caused an intracellular alkalization. Table 2 demonstrates that the luminal addition of barium to either surface or crypt cells did not alter basal pHi.
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DISCUSSION |
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Physiological studies of active K+ absorption in the distal colon of the rat have revealed the presence of two distinct K+ transport processes (9, 15). In these studies of active K+ absorption, which were performed across isolated colonic mucosa under voltage-clamp conditions, K+ absorption consists of mucosal Na+-sensitive and mucosal Na+-insensitive components (15). Aldosterone stimulated the mucosal Na+-sensitive, but not the mucosal Na+-insensitive, component of active K+ absorption (15). The mucosal Na+-sensitive component was also ouabain insensitive, whereas the mucosal Na+-insensitive component was ouabain sensitive. In addition, two distinct H+-K+-ATPase fractions, ouabain sensitive and ouabain insensitive, were also identified in studies with apical membranes isolated from rat distal colon (9). Neither of these studies was designed, however, to identify the spatial localization of either the active K+-absorptive process or H+-K+-ATPase (9, 15).
In contrast, these present studies were planned to establish whether these different K+-dependent processes were exclusively present in surface and/or in crypt epithelial cells. The results of these studies provide compelling evidence that a single K+-dependent transport process is present in crypt epithelial cells. K+-dependent pHi recovery from an acid load in crypt cells was completely ouabain sensitive (Figs. 2 and 3A), whereas H+-K+-ATPase activity in apical membranes isolated solely from crypt cells was 96% inhibited by ouabain (Fig. 1). Thus it appears that there is but a single K+-dependent transport process in crypt epithelial cells, one that is ouabain sensitive. These studies also suggest that it is likely that only one K+-dependent pHi transport process is present in surface epithelial cells and that, in contrast to that in crypt cells, this K+-dependent pHi transport process is ouabain insensitive (Fig. 3B).
The results of these physiological studies must be interpreted in
relation to the recent cloning and localization of the colonic H+-K+-ATPase.
HKc cDNA was cloned from rat colon and is a member of a gene family
of related P-type ATPases (7, 11). Both HKc
message and protein have
been localized to surface (and to 20% of upper crypt) epithelial cells
of the rat distal colon, and Western blot analysis and
immunocytochemical studies have established that HKc
protein is
present in the apical membrane of surface cells (11, 13). Controversy
exists regarding the ouabain sensitivity of the
H+-K+-ATPase
that is encoded by this HKc
cDNA (5, 6, 13). The
H+-K+-ATPase
activity expressed in Sf9 cells by HKc
cDNA was ouabain insensitive
(13). In contrast, the ouabain sensitivity of
86Rb uptake after the injection of
HKc
cRNA into Xenopus oocytes depended on the specific
-subunit cRNA that was coinjected.
Coinjection of an amphibian bladder
-subunit resulted in 100%
ouabain-sensitive 86Rb uptake (6),
whereas the ouabain-sensitive fraction of
86Rb uptake by oocytes coinjected
with either gastric
H+-K+-ATPase
-subunit cRNA or
Na+-K+-ATPase
1-subunit cRNA was less than
50% of total 86Rb uptake (5).
Although the ouabain sensitivity of
H+-K+-ATPase
activity and that of K+-dependent
pHi recovery in crypt cells
manifest excellent concordance, there was modest dissociation of their
inhibition by ouabain in surface cells. Because
pHi recovery in surface cells was
measured by video fluorescent microscopy in individual cells, ouabain
insensitivity of pHi recovery
undoubtedly represents a surface cell function. In contrast, it is not
unlikely that the apical membranes isolated from surface cells are
partially contaminated with ouabain-sensitive H+-K+-ATPase
activity from crypt apical membranes. This suggestion is based on our
recent demonstration (16) that
Cl-dependent
Na+/H+
exchange is the sole
Na+/H+
exchange in crypt apical membrane vesicles, whereas in surface cell
apical membrane vesicles
Cl
-dependent
Na+/H+
exchange is 27% of total
Na+/H+
exchange.
In these studies an intracellular acid load was induced either by the removal of lumen and bath Na+ in crypt cells or by an NH3/NH4Cl prepulse in surface cells.1 We established in both surface and crypt cells that in the absence of lumen Na+, recovery from an acid load required lumen K+. Although this represents the first identification of a K+-dependent recovery from an acid load in small or large intestinal epithelia, we do not as yet know whether this represents an important control mechanism to regulate pHi in colonocytes or is merely an expression of H+/K+ exchange that mediates transepithelial K+ movement.
The results of these present studies, combined with the recent
localization of HKc message and protein to surface epithelial cells
(11, 13), provide compelling evidence that HKc
cDNA encodes a
protein that is ouabain insensitive and that the ouabain-sensitive H+-K+-ATPase
isoform is both exclusively localized in crypt cells and encoded by a
cDNA that has not as yet been cloned.
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ACKNOWLEDGEMENTS |
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This research was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-18777 and by Mentored Clinical Investigator Award DK-02410.
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FOOTNOTES |
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1 Use of an identical method to acid load both crypt and surface cells was not possible, as the luminal membrane of crypt cells is essentially impermeable to NH3/NH4Cl (20) and the chamber used to study surface cells does not permit ready access to the basolateral aspect. As a consequence, bilateral Na+ removal and an NH3/NH4Cl prepulse were used for acid loading in crypt and surface cells, respectively.
Address for reprint requests: H. J. Binder, Yale Univ. School of Medicine, Dept. of Internal Medicine, Section of Digestive Diseases, 333 Cedar St., 89 LMP, New Haven, CT 06520-8019.
Received 7 May 1997; accepted in final form 10 November 1997.
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REFERENCES |
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---|
1.
Binder, H. J., and G. I. Sandle.
Electrolyte transport in the mammalian colon. In:
Physiology of the Gastrointestinal Tract (3rd ed.),
edited by L. R. Johnson. New York: Raven, p. 2133-2172.
2.
Boron, W. F.,
and
P. DeWeer.
Intracellular pH transient in squid giant axons caused by CO2, NH3, and metabolic inhibitors.
J. Gen. Physiol.
67:
97-112,
1976.
3.
Boyarsky, G.,
M. D. Ganz,
R. B. Sterzel,
and
W. F. Boron.
pH regulation in single glomerular mesangial cells. I. Acid extrusion in absence and presence of .
Am. J. Physiol.
255 (Cell Physiol. 24):
C844-C856,
1988
4.
Chu, S.,
and
M. H. Montrose.
Extracellular pH regulation in microdomains of colonic crypts: effects of short chain fatty acids.
Proc. Natl. Acad. Sci. USA
92:
3303-3307,
1995[Abstract].
5.
Codina, J.,
B. C. Kone,
J. T. Delmas-Mata,
and
T. D. DuBose, Jr.
Functional expression of the H+,K+-ATPase subunit.
J. Biol. Chem.
271:
29759-29763,
1996
6.
Cougnon, M.,
G. Planelles,
M. S. Crowson,
G. E. Shull,
B. C. Rossier,
and
F. Jaisser.
The rat distal colon P-ATPase -subunit encodes a ouabain-sensitive H+,K+-ATPase.
J. Biol. Chem.
271:
7277-7280,
1996
7.
Crowson, M. S.,
and
G. E. Shull.
Isolation and characterization of a cDNA encoding the putative distal colon H+,K+-ATPase. Similarity of deduced amino acid sequence to gastric H+,K+-ATPase and mRNA expression in distal colon, kidney and uterus.
J. Biol. Chem.
267:
13740-13748,
1992
8.
Dagher, P. C.,
R. W. Egnor,
and
A. N. Charney.
Effect of intracellular acidification on colonic NaCl absorption.
Am. J. Physiol.
264 (Gastrointest. Liver Physiol. 27):
G569-G575,
1993
9.
Del Castillo, J. R.,
V. M. Rajendran,
and
H. J. Binder.
Apical membrane localization of ouabain-sensitive K+-activated ATPase activities in rat distal colon.
Am. J. Physiol.
261 (Gastrointest. Liver Physiol. 24):
G1005-G1011,
1991
10.
Forbush, B., III.
Assay of Na,K-ATPase in plasma membrane preparations: increasing the permeability of membrane vesicles using sodium dodecyl sulfate buffered with bovine serum albumin.
Anal. Biochem.
128:
159-163,
1983[Medline].
11.
Jaisser, F.,
N. Coutry,
N. Farman,
H. J. Binder,
and
B. C. Rossier.
A putative H,K-ATPase is selectively expressed in surface epithelial cells of rat distal colon.
Am. J. Physiol.
265 (Cell Physiol. 34):
C1080-C1089,
1993
12.
King, G. G.,
W. E. Lohrmann,
J. W. Ickes, Jr.,
and
G. M. Feldman.
Identification of Na+/H+ exchange on the apical side of surface colonocytes using BCECF.
Am. J. Physiol.
267 (Gastrointest. Liver Physiol. 30):
G119-G128,
1994
13.
Lee, J.,
V. M. Rajendran,
A. S. Mann,
M. Kashgarian,
and
H. J. Binder.
Functional expression and segmental localization of rat colonic K-ATPase.
J. Clin. Invest.
96:
2002-2008,
1995[Medline].
14.
Lowry, O. H.,
N. J. Rosebrough,
A. N. Farr,
and
R. J. Randall.
Protein measurement with the Folin phenol reagent.
J. Biol. Chem.
193:
265-275,
1951
15.
Pandiyan, V.,
V. M. Rajendran,
and
H. J. Binder.
Mucosal ouabain and Na+ inhibit active Rb+ (K+) absorption in normal and sodium-depleted rat colon.
Gastroenterology
102:
1846-1853,
1992[Medline].
16.
Rajendran, V. M.,
J. Geibel,
and
H. J. Binder.
Cl-dependent Na-H exchange: a novel mechanism of Na transport in colonic crypts.
J. Biol. Chem.
270:
11051-11054,
1995
17.
Rajendran, V. M.,
M. Kashgarian,
and
H. J. Binder.
Aldosterone induction of electrogenic sodium transport in the apical membrane vesicles of rat distal colon.
J. Biol. Chem.
264:
18638-18644,
1989
18.
Rink, T. J.,
R. Y. Tsien,
and
T. Pozzan.
Cytoplasmic pH, and free Mg2+ in lymphocytes.
J. Cell Biol.
95:
189-196,
1982[Abstract].
19.
Singh, S.,
H. J. Binder,
W. F. Boron,
and
J. P. Geibel.
Fluid absorption in isolated perfused colonic crypts.
J. Clin. Invest.
96:
2373-2379,
1995[Medline].
20.
Singh, S. K.,
H. J. Binder,
J. Geibel,
and
W. Boron.
An apical permeability barrier to NH3/NH+4 in isolated perfused colonic crypts.
Proc. Natl. Acad. Sci. USA
92:
11573-11577,
1995[Abstract].
21.
Thomas, J. A.,
R. N. Buchsbaum,
A. Zimniak,
and
E. Racker.
Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ.
Biochem. J.
18:
2210-2218,
1979.