1 Renal Section, In previous
studies, our laboratory has utilized a cell line derived from the rat
inner medullary collecting duct (IMCD) as a model system for mammalian
renal epithelial cell acid secretion. We have provided evidence, from a
physiological perspective, that acute cellular acidification stimulates
apical exocytosis and elicits a rapid increase in proton secretion that
is mediated by an H+-ATPase. The
purpose of these experiments was to examine the effect of acute
cellular acidification on the distribution of the vacuolar H+-ATPase in IMCD cells in vitro.
We utilized the 31-kDa subunit of the
H+-ATPase as a marker of the
complete enzyme. The distribution of this subunit of the
H+-ATPase was evaluated by
immunohistochemical techniques (confocal and electron microscopy), and
we found that there is a redistribution of these pumps from vesicles to
the apical membrane. Immunoblot evaluation of isolated apical membrane
revealed a 237 ± 34% (P < 0.05, n = 9) increase in the 31-kDa subunit
present in the membrane fraction 20 min after the induction of cellular
acidification. Thus our results demonstrate the presence of this pump
subunit in the IMCD cell line in vitro and that cell acidification
regulates the shuttling of cytosolic vesicles containing the 31-kDa
subunit into the apical membrane.
acid secretion; hydrogen-adenosinetriphosphatase; cell pH; exocytosis; protein trafficking
IN PREVIOUS STUDIES, our laboratory has utilized a cell
line derived from the rat inner medullary collecting duct (IMCD) as a
model system for mammalian renal epithelial cell acid secretion (17,
18). We have provided strong evidence, from a physiological perspective, that acute cellular acidification stimulates apical exocytosis and elicits a rapid increase in proton secretion that is
mediated by an H+-ATPase (17).
More recently, in these cells, we demonstrated the presence of proteins
known to be associated with vesicle docking and fusion, the SNARE
proteins. Cleavage of these SNARE proteins with clostridial toxin
inhibited proton secretion induced by an acute cellular acid load (1).
Taken together, these data strongly suggest that acid secretion by this
IMCD cell line is regulated by an exocytic process. It follows then
that with cellular acidification there is translocation of
H+-ATPase from vesicles to the
apical membrane.
The purpose of these experiments was to examine the effect of acute
acidification on the distribution of the vacuolar
H+-ATPase in IMCD cells in vitro.
In antibody studies, the polarized expression and redistribution of
H+-ATPase to the plasma membrane
has not been demonstrated in cultured renal epithelial cells (5). In
these studies, we utilized the 31-kDa subunit of the
H+-ATPase as a marker of the
complete enzyme. Previous studies demonstrated a similar staining
pattern with antibodies to the 70-, 56-, and 31-kDa subunits of the
kidney H+-ATPase (4). Our results
demonstrate the presence of this pump subunit in this acid-secreting
cell line. In addition, we found that acute cellular acidification
markedly increases the amount of this subunit in the apical membrane.
Thus cell acidification regulates the shuttling of cytosolic vesicles
containing the 31-kDa subunit into the apical membrane.
Solutions and reagents.
The following solutions were used: NaCl HEPES buffer (NHB; in mM: 110 NaCl, 50 HEPES acid, 5 KCl, 1 MgCl2, 1 CaCl2, and 5 glucose, pH 7.2) and
choline chloride HEPES buffer (CHB; identical to NHB except that 110 mM
choline chloride and 5 mM potassium acetate were substituted for
NaCl and KCl, pH 7.2). Buffers were titrated to the desired pH using
NaOH (for NHB), KOH (for CHB), or HCl.
Cell culture.
IMCD cells were obtained from rat papillae as described previously (17,
18). Aliquots of these isolations have been preserved at
Antibodies.
A synthetic polypeptide
(NH2-GANANRKFLD) corresponding to
the COOH-terminal 10 amino acid residues of the bovine 31-kDa sequence with the addition of an
NH2-terminal Cys was prepared. The
peptide was covalently coupled to maleimide-activated keyhole limpet
hemocyanin before immunization of New Zealand White rabbits. High-titer
sera were obtained after four boosts at 2-wk intervals (Charles River Biopharm, Westboro, MA). Sera were tested by peptide ELISA. The antibody against E-cadherin (uvomorulin), which was a rat monoclonal antibody (DECMA-1), and the antibody for the Preparation of tissue homogenate.
Confluent IMCD cells were washed three times in cold PBS and placed in
NHB or CHB for 20 min, scraped from the growth surface, and pelleted by
centrifugation at 1,000 g for 10 min.
The pellet was suspended in four volumes of ice-cold homogenizing
buffer containing 10 mM Tris · HCl, 150 mM NaCl, 5 mM
EDTA, and 1% Nonidet P-40, to which 4 mM phenylmethylsulfonyl
fluoride, 0.5 µg/µl aprotinin, 2 µg/µl
N-tosyl-L-phenylalanine
chloromethyl ketone, 5 µg/ml DNase, and 5 µg/ml RNase were added
just before use. The suspended pellet was homogenized by 10 1-s strokes
in a Teflon homogenizer. To remove intact cells and nuclei, this
homogenate was centrifuged for 10 min at 1,000 g at 4°C for 10 min.
Apical membrane isolation.
The apical membrane was selectively isolated by a modification of a
method that induces vesiculation of this membrane (19, 20). IMCD cells
grown to confluence in 150-mm culture dishes were washed three times
with PBS (pH 7.4) and then exposed to either NHB (control) or CHB
(acid) for 20 min. The monolayers were then incubated for 90 min at
37°C in vesiculation medium (in mM: 1 CaCl2, 1 MgCl2, 50 paraformaldehyde, and 2 dithiothreitol). At the end of the incubation period, vesiculation
medium was filtered through a 37-µm nylon mesh to remove whole cells,
and then the filtrate was centrifuged at 25,000 rpm at 4°C in an
RC5B Sorval centrifuge for 1 h to pellet the vesicles. The pellet was
dissolved in SDS sample buffer, and aliquots were saved for protein and immunoblot analysis. Nine separate experiments were performed and analyzed.
Immunoblot.
Whole cell homogenates and isolated plasma apical membrane samples
prepared as described above were heated at 100°C for 5 min before
loading on a 12-15% polyacrylamide-SDS gel and were run under
reducing conditions (9). Protein was electrophoretically transferred to
nitrocellulose filters that were washed in 150 mM NaCl, 100 mM
Tris · HCl (pH 7.5), and 0.05% Tween 20 (TBST) and
blocked for 1 h in TBST containing 5% wt/vol nonfat powdered milk
(TBSTM) before incubation with a primary antibody (1:1,000 in TBST-1%
BSA) at 4°C overnight. The filters were washed three times with
TBST and incubated in secondary antibody (horseradish peroxidase-goat
antibody, 1:2,000 in TBSTM) for 2 h at room temperature with agitation.
After three washes, bound antibody was detected using the enhanced
chemiluminescence system (ECL, Pierce, Rockville, IL).
Immunohistochemistry.
IMCD cells, grown on coverslips, were washed three times with PBS (pH
7.4) and then exposed to either NHB (control) or CHB (acid) for 20 min.
The bathing solution was removed, and the cells were fixed in
paraformaldehyde-lysine-periodate (PLP) solution for 20 min. They were
then washed three times for 5 min each time in PBS, exposed to Triton
X-100 for 4 min, and rinsed three times for 5 min each time in PBS. The
cells were then rinsed twice with 1% BSA for 5 min each time, drained,
and exposed for 1 h to the primary antibody at 20°C. Then, after
three 5-min rinses with PBS, they were exposed for 1 h to the secondary
antibody, a goat anti-rabbit antibody coupled to indocarbocyanine (CY3;
1:800), washed three times for 5 min each in PBS, and then mounted on a
glass slide with Vectorshield. Specimens were examined by confocal microscopy and photographed.
Immunogold electron microscopy.
Cells were incubated in NHB or CHB (pH 7.2) for 20 min, fixed in PLP
for 2 h, and washed in PBS. Cryosubstitution was in methanol, and
embedding was in Lowicryl HM20 (Polysciences, Warrington, PA) over a
3-day period using a Leica AFS freeze substitution machine. After
ultraviolet polymerization for 2 days at Preembedding immunoperoxidase staining.
Cells grown to confluence in 55-mm plastic petri dishes were incubated
in NHB or CHB (pH 7.2) for 20 min and then fixed for 1 h in 4%
paraformaldehyde plus 1% glutaraldehyde at room temperature. The
dishes were rinsed several times in PBS, and then the cells were
permeabilized for 1 h at room temperature on a shaker in 1% BSA, 0.2%
animal gelatin, and 0.05% saponin (this solution was also used in all
rinsing steps unless otherwise noted). The 31-kDa antibody described
above was applied to the dishes 1:100 in PBS-1% BSA in a humidified
chamber, overnight at 4°C on a shaker. One dish each of control and
acidotic cells had no primary antibody as a negative control. After
being rinsed, the cells were incubated in biotinylated goat-anti-rabbit
IgG (1:100 in PBS-BSA) for 2 h at room temperature. The cells were then
rinsed and incubated with Vector ABC reagent solution in PBS-BSA for 2 h at room temperature (Vector Laboratories, Burlingame, CA). After
several rinses in PBS, the cells were additionally fixed in 1%
glutaraldehyde in PBS plus 5% sucrose for 30 min at room temperature.
They were then rinsed in PBS plus 5% sucrose, followed by
0.05 M Tris (pH 7.6) plus 7.5% sucrose. The
3,3'-diaminobenzidine (DAB) reaction was carried out as follows.
The cells were incubated for 5 min at room temperature in a solution of
0.1% DAB (Electron Microscopy Sciences) in Tris-sucrose, and then
hydrogen peroxide was added to a final concentration of 0.01%, and the
incubation was continued for 10 min in the dark. The cells were then
rinsed in Tris-sucrose and then in Tris alone. The cells were then
rinsed in 0.1 M sodium cacodylate buffer (pH 7.4) and postfixed for 1 h
in 1% osmium tetroxide in 0.1 M sodium cacodylate buffer at room
temperature (EMS). After being rinsed in sodium cacodylate and
distilled water, the cells were stained en bloc for 1 h in 2% aqueous
uranyl acetate at room temperature. The cells were rinsed in distilled
water, dehydrated through a graded series of ethanol, and infiltrated in a 1:1 100% ethanol-EPON solution overnight on a shaker. The following day, after two changes of 100% EPON, a thin layer of fresh
EPON (Ted Pella) was added to the dishes and they were polymerized at
60°C overnight. Small pieces of the EPON, with cells embedded in
it, were cut out of the dish and reembedded in the tips of flat-embedded molds. Thin sections were collected onto Formvar-coated slot grids using a Rechert ultramicrotome, and the sections were examined on a Phillips CM 10 electron microscope and photographed.
To acidify cells, they were bathed in CHB for 20 min. As previously
demonstrated (21), this reduced intracellular pH
(pHi) from 7.35 ± 0.04 to
6.49 ± 0.05.
Immunostaining.
Confocal microscopy of the same control cells revealed
intracellular punctate labeling of the 31-kDa subunit of the
H+-ATPase that was most intense in
the perinuclear region, presumably the Golgi apparatus (Fig.
1A; cell
center), while there was minimal apical membrane staining (Fig.
1B; cell apex). After
acute cellular acidification, the Golgi continued to stain positively
(Fig. 1C), but, in addition,
immunostaining of the apical membrane was strikingly positive (Fig.
1D). Control immunohistochemical
studies with preimmune serum instead of the primary antibody did not
result in the staining of any structures. In addition, when
ABSTRACT
Top
Abstract
Introduction
METHODS
RESULTS
DISCUSSION
References
INTRODUCTION
Top
Abstract
Introduction
METHODS
RESULTS
DISCUSSION
References
METHODS
Top
Abstract
Introduction
METHODS
RESULTS
DISCUSSION
References
70°C and activated as needed. Cells from
passages
6-12
were grown to confluence in 75-cm2
plastic flasks or on 12 × 12-mm glass coverslips in
DMEM plus 10% FCS and penicillin and streptomycin in an atmosphere of
95% air-5% CO2.
-subunit of the coat
protein coatomer (
-COP), clone M3A5, were both obtained from Sigma Chemical (St. Louis, MO). The anti-GP-135 antibody, a mouse
monoclonal antibody, was a gift of Dr. G. K. Ojakian.
40°C and 1 day at
0°C, ultrathin sections were cut on a Reichert Ultracut E
ultramicrotome and were picked up on Formvar-coated nickel grids for
immunolabeling. Grids were floated, section side down, on drops of
distilled water for 10 min and then blocked for 1 h at 4°C on drops
of PBS containing 5% normal goat serum and 1% BSA (Sigma Chemical).
Grids were then incubated overnight at 4°C in a humidified chamber
on drops of a 1:100 dilution of a polyclonal antiserum raised against a
COOH-terminal peptide from the 31-kDa subunit of the
H+-ATPase. The diluent was PBS
containing 1% normal goat serum and 1% BSA, which was also used for
subsequent rinses. Preimmune serum (1:100) and diluent alone served as
negative controls. After rinsing, the grids were incubated for 1 h at
room temperature on drops of goat-anti-rabbit IgG coupled to 10-nm gold
particles (Ted Pella, Redding, CA; 1:25 in rinse buffer). Grids were
then rinsed in PBS rinse buffer, followed by rinses in PBS alone and
then in distilled water. The grids were then fixed for 10 min at room temperature on drops of 1% glutaraldehyde in distilled water (Electron Microscopy Sciences, Fort Washington, PA). Sections were rinsed in
distilled water and stained with 2% uranyl acetate for 7 min, followed
by 2 min in lead citrate. Sections were examined and photographed at 80 kV on a Philips CM10 electron microscope.
RESULTS
Top
Abstract
Introduction
METHODS
RESULTS
DISCUSSION
References
-COP, a
Golgi marker (8), was examined, only immunostaining of the Golgi
apparatus in control and acid cells was seen, without apical membrane
or vesicular staining. Double labeling with
-COP and 31-kDa
antibodies revealed partial overlap (not shown).
View larger version (76K):
[in a new window]
Fig. 1.
Immunolocalization of 31-kDa subunit of
H+-ATPase by confocal microscopy
in same cells: control at center of cell
(A) and at cell apex
(B) and after acute acidification at
center of cell (C) and at cell apex
(D). See text for descriptions.
Arrows identify the same cells. Scale bars, 1 µm.
Electron microscopic immunocytochemistry.
Further confirmation of the localization of the 31-kDa subunit is seen
in the representative electron micrographs in Fig. 2. In control cells, there is little 31-kDa
staining of the apical membrane (Fig.
2A), but there is staining of the
cytosolic structures and the Golgi (Fig. 2,
A and
B). After acute cellular
acidification, there is a marked increase in the
H+-ATPase staining of the apical
membrane and the microvilli (Fig. 2C). In addition, with immunogold
staining (Fig. 2D), the 31-kDa subunit was also demonstrated in the microvilli and apical membrane of
acid-loaded IMCD cells.
|
Apical membrane analysis.
To confirm and quantify these immunocytological observations, we
isolated apical membrane and analyzed it for
H+-ATPase subunits by immunoblot.
Apical membrane was isolated using an adaptation of a vesiculation
technique (19, 20). With this technique, we have been able to obtain a
preparation that is enriched in apical membrane and has no demonstrable
basolateral membrane contamination. The degree of purification was
demonstrated by Western blot analysis of apical vesicles and whole cell
homogenate (Fig. 3). The amount of GP-135,
an apical protein marker (14), was enriched 9.2 ± 0.4-fold
(P < 0.05, n = 9) in the vesicle fraction compared with the whole cell homogenate whereas, E-cadherin, a basolateral marker, was barely detectable in the vesicle preparation but was present in whole cell homogenate. With this method, we have
identified the 31-kDa subunit of the
H+-ATPase in the apical membrane.
In addition, after cells were acidified to stimulate proton secretion,
there was a striking increase in the amount of the 31-kDa subunit of
the H+-ATPase detected in the
apical membrane fraction in all nine experiments. Densitometric
evaluation of these data revealed an increase of 237 ± 34% in 31-kDa subunit (P < 0.05, n = 9), whereas there was no change in
GP-135 when control and acid incubated cells were compared.
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DISCUSSION |
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These experiments, utilizing immunohistochemical and immunoblot techniques, provide new information that IMCD cells, in culture, express the 31-kDa subunit of the proton pump and that an acute acid challenge to these cells initiates a redistribution of these pumps to the apical membrane. We have previously demonstrated that these cells exhibited pHi-mediated exocytosis that is enhanced by cell acidification (17) and that disruption of microtubules or microfilaments reduced this exocytic response (17). In addition, we have recently demonstrated that the exocytotic response to acid is probably mediated by the vesicle membrane SNARE (v-SNARE) and target membrane SNARE (t-SNARE) system (1), a mechanism for exocytosis best described in neural cells (2, 6). The present data provide additional support for the thesis that proton secretion by this IMCD cell line is mediated by an H+-ATPase and that the rate of proton transport is regulated by exocytic insertion and endocytic retrieval of this H+-ATPase from the apical membrane (4, 10, 16, 17).
In the control cells, examined by immunohistochemistry with confocal microscopy, the H+-ATPase was not detectable in the apical membrane, and positive staining was found only in the perinuclear region of the cells, including the Golgi apparatus. However, with the more sensitive techniques of electron microscopy and immunoblot of the isolated apical membrane, some H+-ATPase was detectable in the apical membrane even in control cells. This is consistent with the likelihood that proton pump-containing vesicles are continuously recycled from the cytosol to the apical membrane and participate in constitutive acid secretion (7).
The response to acute cellular acidification was striking. We have previously shown that exposure to CHB reduces pHi to 6.5 and that recovery occurs at 0.05-0.07 pH units/min (1, 17, 21). In the present studies under these same conditions, a markedly increased staining was noted in the apical membrane by immunohistological methods, and this increase was greater than 200% when quantified on Western blot of isolated apical membrane. Thus acute cell acidification appears to be a signal for shuttling of the 31-kDa subunit of the H+-ATPase from internal structures (cytosolic vesicles) to the apical membrane. Whether acidification also increased trafficking of proton pumps from the Golgi apparatus cannot be determined from our experiments.
These data are consistent with findings previously demonstrated in
other acid secretory cells such as the mitochondrion-rich cell of the
turtle bladder and the cortical collecting duct A or -intercalated
cell of the rat or rabbit. In these cell types, acidification leads to
an increase in the surface area of the apical membrane because of an
insertion of acidic vesicles containing proton pumps (12, 22). It is of
interest that we have previously demonstrated acidic vesicles in our
IMCD cell line also (17).
Studies examining the effect of acute and chronic acidosis on the distribution of H+-ATPase in cortical collecting duct tissue sections are also consistent with our findings (3, 15). Sabolic et al. (15) found that, after 6 h of metabolic acidosis in rats, there was a redistribution of H+-ATPase as demonstrated by an enhancement in apical membrane staining in type A intercalated cells (15). Bastani and colleagues (3) examined the effect of chronic acid feeding on the distribution of the 31-kDa subunit in tissue sections of rat IMCD. The shortest period they studied was 3-5 days of acid feeding. They found that acid loading produced a marked redistribution of the H+-ATPase staining. Under control conditions the proton pump was predominantly found in the cytosolic vesicles, whereas in acid-fed animals the apical membrane staining was much more prominent. These data are also consistent with the present findings. Although it is possible that in their chronic studies there was time for redistribution of new pumps to the apical locus, this issue remains highly controversial (11). In our studies, given the rapidity of response, only the redistribution of existing pumps could have occurred.
One alternative hypothesis that would also be consistent with the present data is the concept that some degree of regulation of the proton pump occurs through assembly of pump units upon cellular acidification. The 31-kDa subunit is thought to be a part of the stalk domain of the H+-ATPase that connects the catalytic unit (V1) to the transmembrane (Vo) domain of the protein. The appearance of the 31-kDa subunit in the apical membrane after acidification could represent, at least in part, pump assembly at this location.
These data demonstrate that the proton pump can be identified in cultured IMCD cells and that these cells respond to acidification in a manner that is consistent with that observed in intercalated cells of animals with acute or chronic acid feeding in vivo. The data provide further support for the proposal that changes in cell pH regulate acid secretion by insertion of proton pump units into the apical membrane of acid-secreting collecting duct cells. In addition, they provide further support for the use of this cell line as a model for renal cell acidification.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-28164 (to E. A. Alexander) and DK-42956 (to D. Brown).
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FOOTNOTES |
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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: E. A. Alexander, Evans 401, One Boston Medical Center Place, Boston, MA 02118-2908.
Received 1 October 1998; accepted in final form 1 December 1998.
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REFERENCES |
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---|
1.
Alexander, E. A.,
T. Shih,
and
J. H. Schwartz.
H+ secretion is inhibited by clostridial toxins in an inner medullary duct cell line.
Am. J. Physiol.
273 (Renal Fluid Electrolyte Physiol. 42):
F1054-F1057,
1997
2.
Augustine, G. J.,
M. E. Burns,
W. M. DeBello,
D. L. Pettit,
and
F. E. Schweizer.
Exocytosis: proteins and perturbations.
Annu. Rev. Pharmacol. Toxicol.
36:
659-701,
1996[Medline].
3.
Bastani, B.,
H. Purcell,
P. Hemken,
D. Trigg,
and
S. Gluck.
Expression and distribution of renal vacuolar proton-translocating adenosine triphosphatase in response to chronic acid and alkali loads in the rat.
J. Clin. Invest.
88:
126-136,
1991[Medline].
4.
Brown, D.,
S. Hirsch,
and
S. Gluck.
Localization of a proton-pumping ATPase in rat kidney.
J. Clin. Invest.
82:
2114-2126,
1988[Medline].
5.
Brown, D.,
and
J. L. Stow.
Protein trafficking and polarity in kidney epithelium: from cell biology to physiology.
Physiol. Rev.
76:
245-297,
1996
6.
Calakos, N.,
and
R. H. Scheller.
Synaptic vesicle biogenesis, docking, and fusion: a molecular description.
Physiol. Rev.
76:
1-29,
1996
7.
Dixon, T. E.,
C. Clausen,
D. Coachman,
and
B. Lane.
Constitutive and transport related endocytic pathways in the turtle bladder.
J. Membr. Biol.
102:
49-58,
1988[Medline].
8.
Duden, R.,
V. Allan,
and
T. Kreis.
Involvement of -COP in membrane traffic through the Golgi complex.
Trends Cell Biol.
1:
14-19,
1991.
9.
Emami, A.,
J. H. Schwartz,
and
S. C. Borkan.
Transient ischemia or heat stress induces a cytoprotectant protein in rat kidney.
Am. J. Physiol.
260 (Renal Fluid Electrolyte Physiol. 29):
F479-F485,
1991
10.
Gluck, S.,
C. Cannon,
and
Q. Al-Awquati.
Exocytosis regulates urinary acidification in turtle bladder by rapid insertion of H+ pumps into the luminal membrane.
Proc. Natl. Acad. Sci. USA
79:
4327-4331,
1982[Abstract].
11.
Gluck, S. L.,
D. M. Underhill,
M. Iyori,
L. S. Holliday,
T. Y. Kostrominova,
and
B. S. Lee.
Physiology and biochemistry of the kidney vacuolar H+-ATPase.
Annu. Rev. Physiol.
58:
427-445,
1996[Medline].
12.
Madsen, K. M.,
J. W. Verlander,
and
C. C. Tisher.
Relationship between structure and function in distal tubule and collecting duct.
J. Electron Microsc. Tech.
9:
187-208,
1988[Medline].
13.
Obrador, G.,
H. Yuan,
T. Shih,
W. H. Wang,
M. A. Shia,
E. A. Alexander,
and
J. H. Schwartz.
Characterization of anion exchangers in an inner medullary duct cell line.
J. Am. Soc. Nephrol.
9:
746-754,
1998[Abstract].
14.
Ojakian, G. K.,
R. Schwimmer,
and
R. E. Herz.
Polarized insertion of an intracellular glycoprotein pool into the apical membrane of MDCK cells.
Am. J. Physiol.
258 (Cell Physiol. 27):
C390-C398,
1990
15.
Sabolic, I.,
D. Brown,
S. L. Gluck,
and
S. L. Alper.
Regulation of AE1 anion exchanger and H+-ATPase in rat cortex by acute metabolic acidosis and alkalosis.
Kidney Int.
51:
125-137,
1997[Medline].
16.
Schwartz, G. J.,
and
Q. Al-Awqati.
Carbon dioxide causes exocytosis of vesicles containing H+ pumps in isolated perfused proximal and collecting tubules.
J. Clin. Invest.
75:
1638-1644,
1985[Medline].
17.
Schwartz, J. H.,
S. A. Masino,
R. D. Nichols,
and
E. A. Alexander.
Intracellular modulation of acid secretion in rat inner medullary collecting duct cells.
Am. J. Physiol.
266 (Renal Fluid Electrolyte Physiol. 35):
F94-F101,
1994
18.
Selvaggio, A. M.,
J. H. Schwartz,
H. H. Bengele,
F. D. Gordon,
and
E. A. Alexander.
Mechanisms of H+ secretion by inner medullary collecting duct cells.
Am. J. Physiol.
254 (Renal Fluid Electrolyte Physiol. 23):
F391-F400,
1988
19.
Scott, R. E.,
and
P. B. Maercklein.
Plasma membrane vesiculation in 3T3 and SV3T3 cells. II. Factors affecting the process of vesiculation.
J. Cell Sci.
35:
245-252,
1979[Abstract].
20.
Scott, R. E.,
R. G. Perkins,
M. A. Zschunke,
B. J. Hoerl,
and
P. B. Maercklein.
Plasma membrane vesiculation in 3T3 and SV3T3 cells. I. Morphological and biochemical characterization.
J. Cell Sci.
35:
229-243,
1979[Abstract].
21.
Slotki, I. N.,
J. H. Schwartz,
and
E. A. Alexander.
Na+-H+ exchange is stimulated by protein kinase C activation in inner medullary collecting duct cells.
Am. J. Physiol.
259 (Renal Fluid Electrolyte Physiol. 28):
F666-F671,
1990
22.
Stetson, D. L.,
and
P. R. Steinmetz.
Role of membrane fusion in CO2 stimulation of proton secretion by turtle bladder.
Am. J. Physiol.
245 (Cell Physiol. 14):
C113-C120,
1983