Am J Physiol Cell Physiol 306: C687–C696, 2014.
First published January 29, 2014; doi:10.1152/ajpcell.00174.2013.
Glucose stimulates calcium-activated chloride secretion in small
intestinal cells
Liangjie Yin,1 Pooja Vijaygopal,1 Gordon G. MacGregor,2 Rejeesh Menon,1 Perungavur Ranganathan,1
Sreekala Prabhakaran,3 Lurong Zhang,1 Mei Zhang,1 Henry J. Binder,4 Paul Okunieff,1
and Sadasivan Vidyasagar1
1
Department of Radiation Oncology, University of Florida Shands Cancer Center, Cancer and Genetics Research Complex,
Gainesville, Florida; 2Department of Biological Sciences, Shelby Center for Science and Technology, University of Alabama
in Huntsville, Huntsville, Alabama; 3Department of Pediatrics, Pediatric Pulmonary Division, University of Florida,
Gainesville, Florida; and 4Department of Internal Medicine, Section of Digestive Diseases, Yale School of Medicine, New
Haven, Connecticut
Submitted 12 June 2013; accepted in final form 23 January 2014
glucose-stimulated sodium absorption; glucose-stimulated chloride
secretion; SGLT1; calcium
SODIUM-COUPLED GLUCOSE TRANSPORTER-1 (SGLT1) is present on
the apical plasma membrane of small intestinal epithelial cells
and plays a critical role in sodium and glucose absorption (17,
51). SGLT1 has a stoichiometric ratio of 2:1, transporting 2
sodium ions for 1 glucose molecule, producing the driving
force for passive absorption of water from the lumen (8). The
Address for reprint requests and other correspondence: S. Vidyasagar, Dept.
of Radiation Oncology, Univ. of Florida Shands Cancer Center, Cancer and
Genomic Research Complex, 2033 Mowry Rd., Box 103633, Gainesville, FL
32610 (e-mail: [email protected]).
http://www.ajpcell.org
observation that cyclic adenosine monophosphate (cAMP)
does not inhibit glucose-stimulated sodium absorption provided the physiological basis for oral rehydration solution
(ORS) (1). The introduction of glucose-based ORS in the
1970s provided a significant breakthrough in the treatment of
cholera and other acute diarrheal conditions and saved the lives
of millions of children and adults.
Despite substantial enhancement of glucose-stimulated fluid
absorption and correction of dehydration and metabolic acidosis, ORS does not dramatically decrease stool output, which is
one of the reasons for the underutilization of ORS (14).
Although an adequate explanation for this dichotomy has not
been established, it could be explained if glucose also stimulated anion secretion. Efforts to modify and improve ORS to
decrease stool output have been only modestly successful (29,
31–33, 37). We initiated a series of experiments that systematically examined the effect of glucose on both sodium and
chloride movement across the mouse small intestine and in
human colorectal adenocarcinoma (Caco-2) cells. We found
that glucose not only enhanced sodium absorption via SGLT1,
a well-established phenomenon, but also stimulated active
chloride secretion via an increase in intracellular Ca2⫹. These
observations permit the speculation that in those clinical conditions in which there is villous atrophy and impaired glucosestimulated sodium absorption, glucose may have deleterious
effects on overall fluid and electrolyte movement by virtue of
its stimulation of active chloride secretion.
MATERIALS AND METHODS
Animal preparation. Eight-week-old, male, NIH Swiss mice were
fed a normal diet and housed at four mice per cage. Following CO2
inhalation, mice were humanely euthanized by cervical dislocation.
Following exsanguination, the ileal mucosa was obtained as previously described (54, 55). All experiments were approved by the
University of Florida Institutional Animal Care and Use Committee.
This study also received Insitutional Review Board approval
(IRB201200129 and IRB201200058).
Bioelectric measurements and flux studies. Stripped ileal sheets
were mounted between two halves of an Ussing chamber with 0.3 cm2
of exposed surface area (P2304; Physiologic Instruments, San Diego,
CA). The Ringer solution contained the following (mmol/l): 140 Na⫹,
⫺
2⫹
119.8 Cl⫺, 5.2 K⫹, 2.4 HPO⫺
, 1.2 Ca2⫹, and
4 , 0.4 H2PO4 , 1.2 Mg
25 HCO⫺
3 ; was bubbled with 95% O2 and 5% CO2 bilaterally; and
was maintained at 37°C. After the tissues were allowed to stabilize
for 45 min, the basal short-circuit current (Isc; expressed as
␮eq·h⫺1·cm⫺2) and conductance (G; expressed as mS/cm2), were
0363-6143/14 Copyright © 2014 the American Physiological Society
C687
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Yin L, Vijaygopal P, MacGregor GG, Menon R, Ranganathan P,
Prabhakaran S, Zhang L, Zhang M, Binder HJ, Okunieff P, Vidyasagar S. Glucose stimulates calcium-activated chloride secretion in
small intestinal cells. Am J Physiol Cell Physiol 306: C687–C696, 2014.
First published January 29, 2014; doi:10.1152/ajpcell.00174.2013.— The
sodium-coupled glucose transporter-1 (SGLT1)-based oral rehydration solution (ORS) used in the management of acute diarrhea does
not substantially reduce stool output, despite the fact that glucose
stimulates the absorption of sodium and water. To explain this
phenomenon, we investigated the possibility that glucose might also
stimulate anion secretion. Transepithelial electrical measurements and
isotope flux measurements in Ussing chambers were used to study the
effect of glucose on active chloride and fluid secretion in mouse
small intestinal cells and human Caco-2 cells. Confocal fluorescence laser microscopy and immunohistochemistry measured intracellular changes in calcium, sodium-glucose linked transporter,
and calcium-activated chloride channel (anoctamin 1) expression. In
addition to enhancing active sodium absorption, glucose increased
intracellular calcium and stimulated electrogenic chloride secretion.
Calcium imaging studies showed increased intracellular calcium when
intestinal cells were exposed to glucose. Niflumic acid, but not
glibenclamide, inhibited glucose-stimulated chloride secretion in
mouse small intestines and in Caco-2 cells. Glucose-stimulated chloride secretion was not seen in ileal tissues incubated with the intracellular calcium chelater BAPTA-AM and the sodium-potassium-2
chloride cotransporter 1 (NKCC1) blocker bumetanide. These observations establish that glucose not only stimulates active Na absorption, a well-established phenomenon, but also induces a Ca-activated
chloride secretion. This may explain the failure of glucose-based ORS
to markedly reduce stool output in acute diarrhea. These results have
immediate potential to improve the treatment outcomes for acute
and/or chronic diarrheal diseases by replacing glucose with compounds that do not stimulate chloride secretion.
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GLUCOSE TRANSPORT-MEDIATED ANION SECRETION
Ileal sacs for immunohistochemistry and brush border membrane
vesicle preparation. Ileal segments were tied at one end and filled with
200 ␮l of 1) Ringer or 2) glucose-containing Ringer solution. The sac
was then placed in a beaker containing O2-saturated Ringer solution at
37°C. The beaker containing the oxygenated Ringer solution with the
sac was slowly agitated in a water bath at 37oC and continuously
bubbled with 95% O2-5% CO2. After incubating for 60 min, the sacs
were removed from the beaker and the tissues either rapidly fixed for
immunohistochemistry or the mucosa was scraped for brush border
membrane vesicle (BBMV) preparation. BBMV were prepared using
a modified methods from Stieger et al. (44) and Binder et al. (3) and
stored in a buffer containing a protease inhibitor mixture (10 mM of
iodoacetamide, 1 mM of phenylmethylsulphonyl fluoride and 2 ␮g/ml
of leupeptin) with a pH of 7.4. Protein concentrations were determined in samples using the Bradford assay.
Caco-2 cell culture. To determine whether a glucose-stimulated
increase in the Isc was also present in other tissues, we performed
parallel studies in Caco-2 cells (American Type Culture Collection,
Manassas, VA) (4, 7, 15, 30). Caco-2 cells were cultured in Eagle’s
minimal essential medium supplemented with 10% fetal bovine serum
and 1% nonessential amino acids at 37°C and 5% CO2. Cells were
passaged 20 –25 times, seeded (2 ⫻ 105 cells/dish) in 5-cm Petri
dishes, and grown to 80% confluence; thereafter, the fetal bovine
serum concentration was changed to 5%. Cells were grown for
another 10 days before they were used for functional studies. Immunohistochemisty and Western blot analysis were performed to determine the presence of SGLT1 and a calcium-activated chloride channel. Inhibitors were added to the apical side of the permeable insert.
Colorimetric cAMP measurements. Freshly isolated ileal epithelial
cells obtained by mucosal scrapping and subsequently washed three
times in a Ringer solution containing 1.2 mM of Ca2⫹ at 37°C were
used to measure intracellular cAMP. Washed cells were then divided
into two groups and treated with either saline or 6 mM of glucose and
incubated for 45 min. The cell lysates were then used for a cAMP
direct immunoassay (Calbiochem, EMD Millipore, Billerica, MA).
cAMP levels were measured, as described in a previous publication
(55) (expressed in pmol/mg protein). Forskolin-treated cells were
used as a positive control. Glucose-treated and forskolin-treated
cells were incubated for 45 min. All assays were performed in
triplicate and repeated on ileal cells from four different mice to
obtain n ⫽ 4.
Confocal Ca2⫹ fluorescence microscopy. Caco-2 cells grown in a
25-mm, round coverslip were mounted on a RC-21BR bath chamber
attached to a Series 20 stage adapter (Warner Instruments, Hamden,
CT). A single-channel tabletop temperature controller (TC-324B;
Warner Instruments) maintained cells at 37°C. Cells were loaded with
0.5 ␮M of the fluorescent calcium indicator Fluo-8 AM dye (cat no.
0203; TEF Laboratory, Austin, TX) and incubated for 45 min at room
temperature. Confocal laser scanning microscopy was performed
using an inverted Fluoview 1000 IX81 microscope (Olympus, Tokyo,
Japan) and a U Plan S-Apo ⫻20 objective. Fluorescent images were
captured with a scanning confocal microscope fitted with argon lasers
with an excitation of 488 nm and emission of 515 nm wavelengths.
Solutions of Ringer, glucose-containing Ringer, or BAPTA-AMcontaining glucose-Ringer were added to the bath chamber using a
multivalve perfusion system (VC-8; Warner Instruments). Cells were
washed with Ringer solution, and the experiment was repeated with
3-OMG and carbachol.
Immunohistochemistry. Immunohistochemistry was performed using methods described previously (54, 55). Ileal tissues were collected
from ileal sacs incubated with Ringer or Ringer containing 8 mM of
glucose and were fixed in formalin (10%) for 20 h at room temperature and then embedded in paraffin. Tissue sections (4 ␮m) were
obtained using microtome (Leica RM2245; Leica Microsystems, Buffalo Grove, IL) and then fixed onto glass slides. Primary antibody
[SGLT-1 and anoctamin 1 (ANO1)] diluted in TBS (1:500) containing
1% BSA was incubated overnight at 4°C. Thereafter, the slides were
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recorded using a computer-controlled voltage/current clamp device
(VCC MC-8; Physiologic Instruments) as previously described (54,
55). To determine if an apical membrane sodium channel contributed
to the basal Isc, amiloride (10 ␮M) was added to the lumen side. The
addition of amiloride (10 ␮M) did not significantly decrease the Isc
(1.6 ⫾ 0.1 vs. 1.4 ⫾ 0.1 ␮eq·h⫺1·cm⫺2; n ⫽ 15), confirming the
absence of amiloride-sensitive electrogenic sodium absorption in the
ileum. The basal Isc was, therefore, primarily attributed to electrogenic
chloride and/or HCO⫺
3 secretion via a chloride channel. Epithelial
sodium channels were similarly excluded in tissues stimulated with
glucose. Amiloride did not inhibit the glucose-stimulated increase in
the Isc (8.0 ⫾ 0.5 vs. 7.8 ⫾ 0.6 ␮eq·h⫺1·cm⫺2; n ⫽ 15; P ⫽ NS),
ruling out the role for epithelial sodium channels. Anion channel
activity in the presence of glucose was studied by adding 100 ␮M of
5-nitro-2-(3-phenylpropylamino) benzoate (NPPB), a nonspecific anion channel blocker, to the lumen side. Glibenclamide was used as a
cystic fibrosis transmembrane conductance regulator (CFTR) channel
blocker, although we are aware that the inhibitor also blocks potassium channels and that its inhibitory effects are not specific to CFTR.
In these present experiments and in our previous publications (2, 3, 5),
we had results with glibenclamide that were comparable to those
achieved with CFTR(inh)-172. We also observed that the inhibition of
the Isc achieved with glibenclamide was relatively rapid compared
with that achieved with CFTR(inh)-172; however, the degree of
inhibition of these two inhibitors was identical. This rapidity of
inhibition was felt to be critical during 36Cl flux studies. In forskolinstimulated tissues, glibenclamide (7.5 ⫾ 0.5 vs. 0.6 ⫾ 0.2
␮eq·h⫺1·cm⫺2; P ⬍ 0.001) and CFTR(inh)-172 (7.3 ⫾ 0.7 vs. 1.8 ⫾
0.2 ␮eq·h⫺1·cm⫺2; P ⬍ 0.001) resulted in similar levels of inhibition.
For flux studies, radioisotopes of sodium (22Na) and chloride (36Cl)
were used to study sodium and chloride fluxes across the ileal mucosa
in the presence and absence of glucose as previously described (47,
55). 22Na activity was measured using a gamma counter (Wizard 2,
2480 Automatic Gamma Counter; Perkin Elmer), while 36Cl was
measured using a liquid scintillation counter (LS 6500 Multipurpose
Scintillation Counter; Beckman Coulter, Brea, CA). The presence of
basal electroneutral Na-H exchange activity was determined by using
10 ␮M of 5-(N,N-hexamethylene) amiloride (HMA), a selective
inhibitor of NHE3 (27). Basal Cl-HCO3 exchange activity was inhibited by 100 ␮M of 4=-diisothiocyano-2,2=-stilbenedisulfonic acid
(DIDS), an anion exchange inhibitor (9, 19, 20). Glibenclamide (100
␮M), a relatively specific CFTR channel blocker, was added to the
lumen side after glucose to study the presence of a CFTR channel
activity. In separate experiments, niflumic acid (10 ␮M), a calciumactivated chloride channel blocker, was added to the lumen side to
determine the presence of calcium-activated chloride channel activity
in the presence of glucose (23, 46, 48).
Glucose or 3-O-methylglucose-stimulated saturable kinetics. Saturation kinetics were studied after increasing concentrations of glucose
(up to 8 mM) were added to the lumen side in the presence of 140 mM
of sodium, and changes in the Isc were recorded. Tissues were allowed
to equilibrate for 10 min after each addition. The peak current for each
glucose concentration was used for fitting the data. The nonlinear
curve fit with the Michaelis-Menten model for enzyme kinetics was
used to calculate the Km and Vmax. Phlorizin (50 ␮M; Santa Cruz
Biotechnology, Santa Cruz, CA), a reversible competitive inhibitor of
SGLT1 (11, 25, 49), was used to determine whether glucose transport
via SGLT1 is essential for the glucose-stimulated increase in the Isc.
It was first added to the lumen side before increasing concentrations
of glucose were added.
To determine whether a metabolizable form of glucose is essential
for the glucose-stimulated increase in the Isc, saturation kinetic studies
were repeated in the presence of 3-O-methylglucose (3-OMG), a
nonmetabolizable form of glucose (10, 28). These studies were similar
to those for glucose.
GLUCOSE TRANSPORT-MEDIATED ANION SECRETION
RESULTS
Effects of glucose on the Isc and unidirectional and net fluxes
of Na. Ileal tissues mounted in the Ussing chamber exhibited a
basal Isc of 1.7 ⫾ 0.1 ␮eq·h⫺1·cm⫺2. Glucose (8 mM) added to
the lumen side of ileal tissues led to a significant increase in the
Isc (8.0 ⫾ 0.5 ␮eq·h⫺1·cm⫺2; Fig. 1A).
Basal sodium absorption (1.6 ⫾ 0.3 ␮eq·h⫺1·cm⫺2) was
abolished in the presence of HMA (27) (0.1 ⫾ 0.1
␮eq·h⫺1·cm⫺2; n ⫽ 15; P ⬍ 0.001), thereby confirming that
basal sodium absorption represented Na-H exchange. Glucose
(8 mM) added to the lumen side of the tissues resulted in a
significant increase in net sodium flux (JnetNa; 5.0 ⫾ 0.5
␮eq·h⫺1·cm⫺2; Fig. 1B) and was due to a significant increase
in mucosal-to-serosal flux (Jms; Table 1). HMA was added to
the lumen side and resulted in significant inhibition (3.2 ⫾ 0.3
␮eq·h⫺1·cm⫺2), indicating an NHE3-mediated net flux of 1.8
␮eq·h⫺1·cm⫺2 comparable to that identified in the absence of
glucose (1.5 ␮eq·h⫺1·cm⫺2). Thus the HMA-insensitive portion of the JnetNa represents glucose-stimulated sodium absorption.
The glucose-stimulated increase in JnetNa of 3.4
␮eq·h⫺1·cm⫺2 over basal JNetNa (5.0 ⫾ 0.5 - 1.6 ⫾ 0.3
␮eq·h⫺1·cm⫺2) was significantly less than the change in the
glucose-stimulated Isc [6.3 ␮eq·h⫺1·cm⫺2 (8.0 ⫾ 0.5 to 1.7 ⫾
0.1 ␮eq·h⫺1·cm⫺2); Fig. 1, A and B] and raised the possibility
that glucose also stimulated chloride secretion. In addition, the
change in JnetNa (3.2 ⫾ 0.3 ␮eq·h⫺1·cm⫺2) in the presence of
HMA in tissues exposed to glucose was considerably less than
the glucose-induced increase in the Isc (6.3 ␮eq·h⫺1·cm⫺2).
Effect of chloride channel blockers on the glucose-stimulated increase in the Isc. The addition of NPPB to tissues with
a glucose-stimulated increase in the Isc resulted in a significant
decrease in the Isc, suggesting anion channel activity (8.0 ⫾ 0.5
vs. 2.6 ⫾ 0.1 ␮eq·h⫺1·cm⫺2; n ⫽ 15; P ⬍ 0.001). Glibenclamide did not affect the glucose-stimulated Isc, but 10 ␮M of
niflumic acid added to the lumen side resulted in significant
inhibition of the glucose-stimulated increase in Isc (Fig. 1C).
The calcium-activated chloride channel blocker CaCC(inh)A01 was used in limited studies to inhibit the glucose-stimulated increase in the Isc (6.2 ⫾ 0.4 vs. 2.8 ⫾ 0.5 ␮eq·h⫺1·cm⫺2;
P ⬍ 0.001); the results were similar to those achieved with
niflumic acid (Fig. 1C). Since there was not a substantial
difference in the degree of inhibition of the glucose-stimulated
current in the presence of niflumic acid or CaCC(inh)-A01, we
Fig. 1. Dot diagram showing the effect of
mucosal addition of glucose. A: glucose (8
mM) added to the lumen side resulted in a
significant increase in the short-circuit current (Isc). B: 22Na flux studies showed net
sodium absorption in the basal state. Glucose
led to a significant increase in net sodium
flux. C: glibenclamide (100 ␮M) had no
effect on the glucose-stimulated Isc, whereas
niflumic acid (10 ␮M) led to a significant
decrease in Isc. D: 36Cl flux studies showed
net chloride absorption in the basal state.
Glucose resulted in significant chloride secretion. Niflumic acid (NFA) but not glibenclamide inhibited glucose-stimulated chloride secretion. The numbers shown in parenthesis represent n number of tissues, each dot
represents a data point from a tissue, and the
horizontal bars indicate the means ⫾ SE.
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incubated with fluorophore-conjugated secondary antibody (Alexa
Fluor 647 goat anti-rabbit IgG, A-21245; Life Technologies, Carlsbad, CA) at 1:1,000 dilution in TBS. The slides were then imaged
under the confocal laser scanning microscopy (Fluoview 1000 IX81
microscope; Olympus, Tokyo, Japan) using a 633-nm laser.
Western blot analysis. Western blot was performed using BBMV
prepared from intestinal sacs incubated with Ringer or with glucoseRinger solutions. Thirty micrograms of mouse ileal protein obtained
from tissues incubated with glucose or saline were resolved by
electrophoresis through SDS-7.5% polyacrylamide gels, as described
by Ranganathan et. al (36). Blots were subsequently reacted with
rabbit anti-ANO1 antibody (Aviva Systems Biology, San Diego, CA)
at a 1:500- to 1,000-fold dilution, followed by peroxidase-coupled
secondary antibody at a 1:3,000-fold dilution. Immunoreactive bands
were visualized by enhanced chemiluminescence (Pierce ECL Western blotting substrate; Thermo Scientific) and autoradiography. Blots
were stained with Ponceau S solution to confirm comparable sample
loading and transfer.
Statistics. Results are presented as means ⫾ SE. Statistical analyses were
performed with paired and unpaired t-tests and Bonferroni’s one-way analysis of variance post hoc test. P ⬍ 0.05 was considered significant.
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Table 1. Sodium and chloride flux, conductance, and short-circuit current
Sodium
Jms
Basal
Glucose*
Glucose ⫹
glibenclamide†
Glucose ⫹ NFA
†
Jsm
Chloride
JNet
Jms
Jsm
JNet
G
Isc
14.3 ⫾ 0.5 (15) 12.8 ⫾ 0.6 (15) 1.5 ⫾ 0.3 (15) 13.4 ⫾ 0.3 (19) 11.7 ⫾ 0.4 (19)
1.6 ⫾ 0.2 (19) 50.4 ⫾ 1.9 (68) 1.7 ⫾ 1 (68)
18.6 ⫾ 0.8 (15) 13.6 ⫾ 0.7 (15) 5.0 ⫾ 0.5 (15) 12.9 ⫾ 0.2 (19) 16.4 ⫾ 0.4 (19) ⫺3.5 ⫾ 0.4 (19) 53.3 ⫾ 2.1 (68) 7.7 ⫾ 0.3 (68)
P ⬍ 0.002
NS
P ⬍ 0.001
NS
P ⬍ 0.002
P ⬍ 0.001
NS
P ⬍ 0.001
20.1 ⫾ 0.7 (7)
NS
14.7 ⫾ 0.5 (8)
P ⬍ 0.002
15.5 ⫾ 0.5 (7)
NS
12.5 ⫾ 0.5 (8)
NS
4.5 ⫾ 0.9 (7)
NS
2.3 ⫾ 0.6 (8)
P ⬍ 0.002
12.1 ⫾ 0.3 (9) 15.3 ⫾ 0.6 (9) ⫺3.2 ⫾ 0.4 (9) 55.1 ⫾ 2.3 (32) 7.8 ⫾ 0.5 (32)
NS
NS
NS
NS
NS
13.2 ⫾ 0.7 (10) 12.4 ⫾ 0.5 (10)
0.7 ⫾ 0.4 (10) 55.6 ⫾ 1.9 (36) 2.5 ⫾ 0.2 (36)
NS
P ⬍ 0.002
P ⬍ 0.001
NS
P ⬍ 0.002
Values are means ⫾ SE. Numbers in parentheses represent number of tissues per group. Jms, mucosal to serosal flux; Jsm, serosal to mucosal flux; Jnet, net
flux of Na or Cl; G, conductance; Isc, short-circuit current; NFA, niflumic acid; NS, not specified. *Glucose-treated tissues were compared to basal tissues.
†Tissues treated with inhibitors were compared with tissues treated with glucose.
into the cell and is blocked by serosal addition of a potent
NKCC1 inhibitor, 100 ␮M of bumetanide. Bumetanide was
added both before and after stimulation with glucose. Tissues
preincubated with bumetanide showed a small but significant
increase in the Isc following the addition of glucose (Fig. 2B).
Subsequent addition of niflumic acid (10 ␮M) to the lumen
side did not result in a significant change in the Isc (Fig. 2B).
Bumetanide added after glucose stimulation showed a significant decrease in the Isc, which was not further altered by
niflumic acid (Fig. 2C).
Effects of bumetanide and BAPTA-AM on unidirectional and
net flux of chloride in the presence of glucose. To further
confirm whether bumetanide or BAPT-AM inhibited glucosestimulated, niflumic acid-sensitive chloride secretion, ileal tissues in the Ussing chamber were incubated with NKCC1
blocker or the intracellular Ca2⫹ chelater (50 ␮M BAPTAAM) for 45 min. Ileal tissues in the presence of bumetanide
showed a net absorption (1.4 ⫾ 0.3 ␮eq·h⫺1·cm⫺2). Addition
of glucose to the lumen side in the continued presence of
bumetanide on the serosal side did not result in a significant
change in JNetCl (1.9 ⫾ 0.2 ␮eq·h⫺1·cm⫺2). Subsequent addition of 10 ␮M of niflumic acid did not alter JNetCl (2.0 ⫾ 0.2
␮eq·h⫺1·cm⫺2; Fig. 2D). Similarly, the addition of glucose to
tissues incubated with BATA-AM did not result in a significant
change in JNetCl (1.3 ⫾ 0.4 vs. 1.8 ⫾ 0.3 ␮eq·h⫺1·cm⫺2).
Further addition of niflumic acid to the lumen side did not
result in a significant change in JNetCl (1.4 ⫾ 0.3
␮eq·h⫺1·cm⫺2; Fig. 2D and Table 2).
Effect of glucose on saturable kinetics of the Isc. The addition
of increasing concentrations of glucose (up to 8 mM) to the
lumen side revealed the saturation kinetics of the Isc (Fig. 3A). The
glucose studies demonstrated early signs of saturation at low
concentrations (0 – 0.6 mM); however, subsequent addition of
glucose resulted in a further increase in the Isc that saturated at
high concentrations of glucose (0.6 – 8 mM). Because the Isc
response suggested the possible presence of more than one
transporter with different Michaelis constant (Km) values at
low and high concentrations of glucose, the Km was calculated
for low- and high-glucose concentrations. The Km was 0.7 ⫾
0.2 mM at the low-glucose concentration and 1.9 ⫾ 0.1 mM at
the high concentration. The corresponding Vmax values were
2.5 ⫾ 0.5 and 5.0 ⫾ 0.7 ␮eq·h⫺1·cm⫺2, respectively. The
significant difference (n ⫽ 9; P ⬍ 0.001) between the Km
values suggests that more than one transporter may account for
the glucose-stimulated increase in the Isc.
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continued to use niflumic acid. These studies suggest the
possibility that a niflumic-sensitive chloride channel was
linked to an increase in the Isc (Fig. 1C).
Effects of glucose on unidirectional and net fluxes of Na and
Cl. JnetCl determined in the absence of glucose demonstrated
net absorption (1.6 ⫾ 0.2 ␮eq·h⫺1·cm⫺2) that was inhibited by
DIDS (9, 19, 20), (0.3 ⫾ 0.1 ␮eq·h⫺1·cm⫺2; P ⬍ 0.001) and
HMA (1.6 ⫾ 0.2 vs. 0.2 ⫾ 0.1 ␮eq·h⫺1·cm⫺2). These results
confirm that basal chloride absorption likely represents a
chloride-anion exchange. Since basal JnetNa (1.6 ⫾ 0.3
␮eq·h⫺1·cm⫺2) was equal to that of chloride absorption (1.6 ⫾
0.2 ␮eq·h⫺1·cm⫺2; P ⫽ NS) and inhibition of Na-H exchange
by HMA resulted in inhibition of Cl absorption, basal sodium
and chloride absorption likely represented parallel and coupled
Na-H and Cl-HCO3 exchanges. Glucose (8 mM) added to the
mucosal side stimulated net chloride secretion (⫺3.4 ⫾ 0.4
␮eq·h⫺1·cm⫺2) (Fig. 1D) and was a result of a significant
increase in serosal-to-mucosal flux of chloride (JsmCl) (11.7 ⫾
0.4 vs. 16.4 ⫾ 0.4 ␮eq·h⫺1·cm⫺2; Table 1). These studies
indicate that glucose stimulates a significant rate of chloride
secretion.
Mucosal addition of glibenclamide (100 ␮M) had no effect
on unidirectional or net fluxes of chloride in the presence of
glucose (Table 1 and Fig. 1D) (38, 42, 43). However, JsmCl
and JnetCl were significantly inhibited by niflumic acid (10
␮M) (Table 1). These studies indicate that glucose stimulates
anion secretion via a niflumic acid-sensitive apical membrane
chloride channel.
Effects of BAPTA-AM on glucose-stimulated increase in the
Isc. BAPTA-AM (50 ␮M) was added as a cell permeant Ca2⫹
chelater to the lumen side under basal conditions and then
exposed to 8 mM of glucose to determine if intracellular
calcium was essential for the glucose-stimulated increase in the
Isc. The addition of glucose resulted in a significant increase in
the Isc, which was significantly lower compared with glucose
stimulation in the absence of BAPTA-AM (Figs. 1A and 2A).
Further addition of 10 ␮M of niflumic acid did not have a
significant effect on the Isc. These studies suggest that glucose
activation of the niflumic acid-sensitive chloride channel is
associated with an increase in intracellular calcium.
Effects of bumetanide on glucose-stimulated increase in the
Isc. Movement of chloride across the apical membrane in the
presence of glucose requires chloride entry at the basolateral
side. Sodium-potassium-2 chloride cotransporter 1 (NKCC1) is
known to transport chloride across the basolateral membrane
GLUCOSE TRANSPORT-MEDIATED ANION SECRETION
C691
Effect of phlorizin on glucose-stimulated increase in Isc. The
Isc was completely inhibited by the mucosal addition of phlorizin (8.0 ⫾ 0.5 vs. 0.2 ⫾ 0.1 ␮eq·h⫺1·cm⫺2; n ⫽ 15; P ⬍
0.001). Saturation kinetics in the presence of phlorizin showed
that the increase in the Isc was abolished at all glucose concentrations, suggesting that a functional SGLT1 is essential
both for glucose-stimulated sodium absorption and for chloride
secretion (Fig. 3A).
Effect of 3-OMG on the Isc and 36Cl flux. Increasing concentrations of 3-OMG added to the lumen side revealed saturation kinetics with a Vmax of 2.3 ⫾ 0.1 ␮eq·h⫺1·cm⫺2 and a
Km of 0.2 ⫾ 0.1 mM (Fig. 3A). 36Cl flux studies in the presence
of 3-OMG did not show a significant difference from that of
the basal JnetCl (1.6 ⫾ 0.2 vs. 1.7 ⫾ 0.2 ␮eq·h⫺1·cm⫺2),
whereas 22Na flux studies showed a significant increase in
JNetNa (1.6 ⫾ 0.3 vs. 2.9 ⫾ 0.3 ␮eq·h⫺1·cm⫺2). These results
Table 2. Chloride flux
Chloride Flux
Bumetanide
Bumetanide ⫹
glucose
Bumetanide ⫹
NFA
BAPTA-AM
BAPTA-AM ⫹
glucose
BAPTA-AM ⫹
NFA
Jms
Jsm
JNet
12.6 ⫾ 0.7 (8)
11.2 ⫾ 0.6 (8)
1.4 ⫾ 0.3 (8)
12.5 ⫾ 0.9 (8)
10.6 ⫾ 0.9 (8)
1.9 ⫾ 0.2 (8)
13.6 ⫾ 1.0 (8)
11.4 ⫾ 0.6 (8)
11.6 ⫾ 1.0 (8)
10.1 ⫾ 0.6 (8)
2.0 ⫾ 0.2 (8)
1.3 ⫾ 0.4 (8)
12.1 ⫾ 0.7 (8)
10.3 ⫾ 0.5 (8)
1.8 ⫾ 0.3 (8)
13.6 ⫾ 0.7 (8)
12.2 ⫾ 0.6 (8)
1.4 ⫾ 0.3 (8)
Values are means ⫾ SE.
indicate that 3-OMG stimulates active Na absorption but not
active Cl secretion.
Effect of serosal addition of glucose on the Isc. To confirm
that the apical addition of glucose stimulated an increase in the
Isc, glucose was added to the bath (serosal) side. The bath
addition of 8 mM of glucose did not stimulate an increase in
the Isc.
Effect of low concentrations of glucose on unidirectional net
flux of sodium and chloride. 22Na and 36Cl flux studies performed at a low glucose concentration (0.6 mM) revealed a
small but significant increase in the JnetNa and a larger increase
in net chloride secretion (Fig. 3, B and C). Net chloride
secretion in the presence of glucose was due to an increase in
the unidirectional serosal to mucosal flux of chloride.
Effect of glucose on the Isc in Caco-2 cells. To determine if
a similar glucose-stimulated increase in the Isc was seen in
human epithelial cells with intestinal differentiation, studies
were performed on Caco-2 cells that were grown on permeable
inserts. Caco-2 cells exposed to glucose exhibited a significant
increase in the Isc (Fig. 3D). The addition of niflumic acid
resulted in a significant decrease in the glucose-stimulated
increase in the Isc (Fig. 3D), thereby suggesting the activation
of a calcium-activated anion channel. Glibenclamide had no
effect on the glucose-stimulated increase in the Isc.
Effect of glucose on intracellular cAMP. Cell lysates incubated with glucose (0.6 or 6 mM) or with 3-OMG (0.6 or 6
mM) did not induce a significant increase in intracellular
cAMP, compared with the cell fractions not treated with either
3-OMG or glucose (Fig. 4A). A positive control with forskolin
confirmed an increase in intracellular cAMP. Consequently,
the results do not support a role for intracellular cAMP con-
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Fig. 2. Effect of BAPTA-AM or bumetanide
on the Isc and net chloride flux (JNetCl).
A: incubation of tissues with BAPTA-AM
(50 ␮M) for 45 min had no effect on the
basal Isc. Glucose (8 mM) added to the
lumen side resulted in a significant increase
in the Isc. Niflumic acid (10 ␮M) added to
the lumen side did not change the glucosestimulated increase in the Isc. B: incubation
of tissues with bumetanide for 45 min had no
effect on the basal Isc. Glucose (8 mM)
added to the lumen side resulted in a significant increase in the Isc. Niflumic acid (10
␮M) added to the lumen side had no effect
on the glucose-stimulated increase in Isc (8
mM). C: bumetanide (100 ␮M) was added
after glucose. Bumetanide resulted in a significant decrease in the Isc that was not
further inhibited by niflumic acid. D: 36Cl
flux studies in tissues incubated with bumetanide or BAPTA-AM for 45 min in Ussing
chamber. The addition of glucose or subsequent additions of niflumic acid had no significant effect on JNetCl. The values are from
n ⫽ 8 pairs of tissues, each dot represents a
data point from a tissue, and the horizontal
bars indicate the means ⫾ SE.
C692
GLUCOSE TRANSPORT-MEDIATED ANION SECRETION
centration in glucose-mediated stimulation of net chloride
secretion.
Effect of glucose on intracellular Ca2⫹ in mouse small intestinal
cells and Caco-2 cell lines. Small intestinal cells incubated with
glucose (6 mM; see Supplemental Video S1; Supplemental Material
for this article is available at the Journal website) showed a significant
increase in Fluo-8 fluorescence (F/Fo; Fig. 4B), suggesting an
increase in intracellular Ca2⫹ concentration ([Ca2⫹]i). Postconfluent Caco-2 cells were used for subsequent [Ca2⫹]i measurements as the mouse ileal cells did not adhere to the coverslips
for extended periods, and stable recordings were not possible.
Glucose (0.6 mM) added to the bathing medium initiated
intracellular Ca2⫹ oscillations (Fig. 4D). The amplitude of each
oscillation was sustained for at least 200 s (Fig. 4B). The mean
peak amplitude of F/Fo was calculated as 1.32 ⫾ 0.1 (n ⫽ 10).
To determine if the increase in intracellular Ca2⫹ was due to
glucose metabolism, 3-OMG (10, 28) was also used (Fig. 4C).
3-OMG (0.6 mM) did not induce a similar increase in Ca2⫹
oscillations (1.0 ⫾ 0.1; n ⫽ 10) and suggested that the increase
in intracellular Ca2⫹ required a metabolizable form of glucose.
Preincubating the cells with 50 ␮M of BAPTA-AM, an intracellular calcium chelater, for 45 min abolished glucose-stimulated Ca2⫹ oscillations (1 .0 ⫾ 0.1; n ⫽ 10; Fig. 4C). These
results confirm that glucose is associated with an increase in
intracellular Ca2⫹. A higher glucose concentration (6 mM) also
increased the amplitude of Ca2⫹ oscillations (1.85 ⫾ 0.20 vs.
1.32 ⫾ 0.10) (Fig. 4D). 3-OMG (6 mM) added to the bathing
medium failed to elicit an increased [Ca2⫹]i response at concentrations of 0.6 mM or 6 mM (1.0 ⫾ 0.1 vs. 1.0 ⫾ 0.2; Fig. 4C).
Immunostaining for SGLT1 and ANO1 expression in mouse
small intestines. Immunostaining using a mouse-specific
SGLT1 antibody demonstrated maximal staining on the villi
tips, along the brush border membrane (Fig. 5, A and B) and
minimal expression on crypt cells in tissues from ileal sacs
incubated with 8 mM of glucose. Immunostaining using a
mouse-specific antibody for ANO1, a calcium-activated chloride channel expressed on the apical membrane of small
intestinal epithelial cells (6, 16, 53), revealed expression in the
upper one third of the villous and lower one third of the crypt
cell regions (Fig. 5, C and D). However, immunohistochemistry from tissues incubated with glucose (8 mM) demonstrated
increased ANO1 expression along the upper villous, midvillous, and low-villous regions and along the brush border
membrane (Fig. 5, E and F). Minimal expression was seen in
the crypt cell region (Fig. 5, E and F).
Western blot analysis for ANO1 in mouse ileum. Tissue
samples incubated with glucose showed increased ANO1 protein levels at 80 kDa, compared with Ringer controls (Fig. 5G).
These results are consistent with immunohistochemistry and
isotopic flux data that suggest glucose-activation of a calciumdependent chloride channel.
DISCUSSION
Stimulation of active electrogenic sodium absorption by
glucose via SGLT1 in the mammalian small intestine is a
well-established physiological process that has been extensively studied over the past 40 years (8, 17, 50 –52). Although
glucose enhances intestinal sodium and water absorption,
which is the physiological basis for the development of ORS in
the treatment of acute diarrhea, ORS is not associated with a
dramatic reduction in diarrhea (i.e., stool output) (14). Our
results demonstrated that the glucose-stimulated increase in the
Isc was considerably greater than the enhancement of active
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Fig. 3. Glucose and 3-O-methylglucose (3-OMG) saturation kinetics and 22Na and 36Cl fluxes on low concentration of glucose in mouse ileum and the effect of
glucose on the Isc response in Caco-2 cells. A: addition
of increasing concentrations of glucose or 3-OMG to
the lumen side showed a dose-dependent increase in the
Isc with saturable kinetics. At lower concentrations of
glucose, the saturation curve showed early signs of
saturation, but with increasing glucose concentration it
showed a further increase in Isc (). The addition of
increasing lumen 3-OMG resulted in a concentrationdependent increase in the Isc (). Tissues (n ⫽ 6)
pretreated with phlorizin and increasing concentrations
of glucose showed no response to glucose (Œ). B and
C: effect of a low concentration of glucose (0.6 mM) on
unidirectional and net flux of Na and Cl. In the absence
of glucose there was net sodium flux (JNetNa) and JNetCl
absorption. The addition of glucose to the lumen side
resulted in increased JNetNa absorption and a JNetCl
secretion. Values are from n ⫽ 8 tissues. D: addition of
glucose to Caco-2 cells grown on 0.4-mm permeable
inserts resulted in a significant increase in the Isc. The
addition of niflumic acid (10 mM) and not glibenclamide to the apical side resulted in significant inhibition
of the glucose-stimulated Isc. *P ⬍ 0.001, compared
with basal group after addition of glucose. Each dot
represents an insert, and the horizontal bars indicate the
means ⫾ SE. The values represented in parenthesis
represent n number of culture inserts.
GLUCOSE TRANSPORT-MEDIATED ANION SECRETION
C693
sodium absorption, as measured by 22Na fluxes. The JnetNa is
the sum of electrogenic (sodium-coupled glucose transport)
and electroneutral (Na-H exchange) sodium absorption. NHE3
exchange is the predominant Na-H exchange in the basal state
and in the presence of glucose (18, 24, 26, 56). Mucosal HMA
in the presence or absence of glucose resulted in comparable
inhibition of NHE3-dependent sodium absorption (5.0 ⫾ 0.5 3.2 ⫾ 0.3 ␮eq·h⫺1·cm⫺2 and 1.6 ⫾ 0.3 vs. 0.1 ⫾ 0.1
␮eq·h⫺1·cm⫺2), respectively. Thus HMA-insensitive sodium
flux (3.2 ⫾ 0.3 ␮eq·h⫺1·cm⫺2) reflects SGLT1-mediated electrogenic sodium absorption. In contrast, glucose also stimulates
active chloride secretion such that the increases in electrogenic
chloride secretion and electrogenic sodium absorption are
equivalent to its increase in the Isc. The JnetCl is the sum of
electrogenic chloride secretion and electroneutral the ClHCO⫺
3 exchange-mediated chloride absorption. Net chloride
secretion did not result in a significant change in JmsCl (Table
1), thereby suggesting that Cl- HCO⫺
3 exchange persists in the
presence of glucose. Thus the net change in chloride flux was
equal to basal chloride absorption (1.6 ⫾ 0.2 ␮eq·h⫺1·cm⫺2)
plus the net chloride secretion in the presence of glucose (⫺3.5 ⫾
0.4 ␮eq·h⫺1·cm⫺2 or 5.1 ␮eq·h⫺1·cm⫺2). These results established that the glucose-stimulated increase in the Isc (8.0 ⫾ 0.5
␮eq·h⫺1·cm⫺2) was equal to the sum of electrogenic chloride
secretion (5.1 ␮eq·h⫺1·cm⫺2) and electrogenic sodium absorption (3.2 ⫾ 0.3 ␮eq·h⫺1·cm⫺2).
The present studies provide compelling evidence that glucose stimulates active chloride secretion, which is mediated by
an increase in intracellular calcium. First, the glucose-stimulated Isc was inhibited by NPPB, a nonspecific anion channel
inhibitor. Second, niflumic acid, a calcium-activated chloride
channel blocker, but not glibenclamide, a CFTR channel inhibitor, prevented glucose-stimulated chloride secretion. Third,
glucose increased intracellular calcium but not intracellular
cAMP levels. Fourth, in tissues incubated with BAPTA-AM or
bumetanide, the glucose-stimulated increase in the Isc was
approximately equal to JNetNa. BAPTA-AM chelated the increase in intracellular calcium secondary to glucose stimulation, resulting in the subsequent failure to observe calciumactivated chloride secretion (Fig. 2A). Bumetanide inhibited
the NKCC1-mediated chloride entry into the cell at the basolateral side, which is essential for its apical exit and prevents a
chloride secretory response to luminal glucose (Fig. 2D). Thus,
by preventing the glucose-stimulated increase in intracellular
calcium or by preventing the chloride entry into the cell at the
basolateral side, we demonstrated that the observed glucoseinduced increase in the Isc is not completely accounted for by
JNetNa and represents chloride secretion, which requires an
increase in intracellular calcium for its activation. Finally,
glucose induced ANO1 protein expression on the brush border
membrane, a potential calcium channel protein. The molecular
identity of the calcium-activated chloride channels was largely
unknown until the recent identification of anoctamins 1
through 10 (6, 16, 53). Of these anoctamins, ANO1 is broadly
expressed in mammalian tissues, including the gastrointestinal
tract (35). Like ANO1, mCLCA6 is another calcium-activated
chloride channel that is expressed in mouse small intestines
(12). mCLCA6 is an integral apical membrane protein and
colocalizes with CFTR (5). However, further studies will be
essential to determine whether the chloride secretory response
occurs via ANO1 and /or mCLCA6.
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Fig. 4. Effect of glucose on intracellular
cAMP and Ca2⫹. A: cells incubated with
forskolin (FSK) showed increased intracellular cAMP levels. In contrast, neither glucose
nor 3-OMG increased cAMP levels. B: glucose resulted in a significant increase in fluorescence intensity in epithelial cells isolated
from the upper one-third of the villous. C: 0.6
mM and 6 mM of glucose led to a significant
increase in fluorescence, compared with the
control, and fluorescence at 6 mM was
greater than at 0.6 mM. Preincubating the
cells (45 min) with BAPTA prevented the
glucose-stimulated increase in intracellular
Ca2⫹. 3-OMG showed little increase in glucose-stimulated intracellular Ca2⫹. D: representative trace shows that 0.6 mM and 6.0
mM of glucose-stimulated increases in intracellular Ca2⫹. The solid arrows show the time
point when 0.6 and 6 mM glucose was added.
The doted arrow represents the time point
when Ringer solution was used to flush glucose off the cells. Columns represent the
mean values, and bars show the SE. The
values are from n ⫽ 6 different mice repeated
in triplicate. *P ⬍ 0.001, compared with
saline control (A and B) and glucose (C). NS,
not significant between the saline-treated and
glucose-treated villous cells.
C694
GLUCOSE TRANSPORT-MEDIATED ANION SECRETION
A metabolizable hexose that is transported across the apical
membrane is required for stimulation of active Cl secretion in
the mouse ileum as neither glucose in the presence of phlorizin,
an inhibitor of glucose uptake across the apical membrane;
3-OMG, a glucose metabolite that is transported via SGLT1
across the apical membrane but not metabolized; nor glucose
added to the serosal bathing solution stimulated active Cl
secretion (Fig. 3A). These present observations are not the
initial demonstration that glucose enhances Cl secretion in the
mammalian small intestine (2, 34, 41, 45). The initial detailed
studies that demonstrated glucose stimulation of Na absorption
were performed in the rabbit ileum (39 – 41). These studies by
Schultz and Zalusky (40) and Schultz et al. (41) did not provide
any suggestion that glucose modified Cl transport. Other studies that were performed in the 1970s in both hamster and rat
small intestines provided evidence that glucose might stimulate
active Cl secretion but did not reveal a consistent pattern (2, 34,
41, 45). For example, in the hamster ileum, active Na-Cl
secretion was demonstrated in the absence of glucose, and the
addition of glucose stimulated active Na-Cl secretion (2).
Similar to our present observations, neither 3-OMG nor serosal
glucose stimulated Cl secretion (2). Studies in rat small intestines also have indicated that glucose may stimulate Cl secretion, in addition to its well-established enhancement of Na
absorption (41, 45). None of these older studies examined a
link between glucose and Cl secretion (e.g., second messengers
or the mechanism by which glucose might stimulate active Cl
secretion). However, the well-established phenomenon in pancreatic islet cells that glucose increases insulin release via an
increase in intracellular calcium may be of relevance to our
present observations (13, 21).
While we found that active sodium absorption was higher at
8 mM of glucose compared with 0.6 mM (Figs. 1, A and B and
3B), active chloride secretion was similar in magnitude at these
two glucose concentrations (Fig. 1D and 3C). Since SGLT1
and ANO1 are predominantly located along the brush border
membrane of the villi (Fig. 5, A–F), we suspect that in clinical
or experimental conditions glucose-stimulated chloride secretion mostly occurs in the villous cell region. Further studies
are, however, essential to determine the role of the crypt in the
glucose-stimulated increase in chloride secretion. Indeed, studies show that rehydration with ORS is generally less successful
for rotavirus-induced diarrhea, which is associated with the
calcium-dependent enterotoxin NSP4 and modest changes in
villous morphology, than for cholera-induced diarrhea, which
is not associated with any histological abnormalities (22). We
speculate that simple dietary modifications and/or changes in
the ORS formulation, such that glucose is replaced with compounds that do not stimulate chloride secretion, could significantly alter the outcome of the disease process.
ACKNOWLEDGMENTS
We thank Dr. David Yule in the Department of Pharmacology and
Physiology at the University of Rochester Medical Center for help with
data analysis of intracellular calcium imaging studies. We also thank Kate
Casey-Sawicki at the University of Florida for preparing this manuscript
for publication.
GRANTS
The work was partially funded through a research agreement with the
University of Florida.
DISCLOSURES
Drs. S. Vidyasagar, P. Okunieff, and L. Zhang have shares and stocks in
Enterade USA.
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Fig. 5. Immunohistochemistry section showing sodium-coupled glucose transporter-1 (SGLT1) and anoctamin-1 (ANO1) expression in mouse small intestines.
A: SGLT1 expression occurs along the brush border membrane of villous epithelial cells (⫻20 objective). B: magnified view of villous region to show SGLT1
expression. C: immunohistochemistry shows minimal ANO1 expression in villous region of tissues isolated from ileal sacs incubated with Ringer solution.
D: magnified view of the villous region to show minimal expression of ANO1 protein along the brush border membrane (white arrows). E: strong immunostaining
of the villous and along the brush border membrane (white arrows) in sections taken from ileum incubated with glucose. F: magnified view of villous to show
ANO1 expression along the brush border membrane. G: Western blot showing increased ANO1 protein levels in tissue isolated from mice incubated with glucose
(80 kDa).
GLUCOSE TRANSPORT-MEDIATED ANION SECRETION
AUTHOR CONTRIBUTIONS
Author contributions: L.Y., P.V., R.M., P.R., S.P., and M.Z. performed
experiments; L.Y., P.V., G.G.M., R.M., P.R., S.P., L.Z., M.Z., P.O., and S.V.
analyzed data; L.Y., P.V., and S.V. drafted manuscript; L.Y., P.V., G.G.M.,
R.M., P.R., S.P., L.Z., M.Z., H.J.B., P.O., and S.V. approved final version of
manuscript; G.G.M. and S.V. conception and design of research; H.J.B. edited
and revised manuscript; S.V. interpreted results of experiments.
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