cAMP-dependent exocytosis and vesicle traffic regulate

CFTR and fluid transport in rat jejunum in vivo

Nadia A. Ameen, Christopher Marino and Pedro J. I. Salas
Am J Physiol Cell Physiol 284:429-438, 2003. doi:10.1152/ajpcell.00261.2002

You might find this additional information useful...

This article cites 44 articles, 21 of which you can access free at:
http://ajpcell.physiology.org/cgi/content/full/284/2/C429#BIBL

This article has been cited by 7 other HighWire hosted articles, the first 5 are:
Rescue of Functional F508del Cystic Fibrosis Transmembrane Conductance Regulator by
Vasoactive Intestinal Peptide in the Human Nasal Epithelial Cell Line JME/CF15
S. Rafferty, N. Alcolado, C. Norez, F. Chappe, S. Pelzer, F. Becq and V. Chappe
J. Pharmacol. Exp. Ther., October 1, 2009; 331 (1): 2-13.
[Abstract] [Full Text] [PDF]

cAMP Stimulates Apical Exocytosis of the Renal Na+-K+-2Cl- Cotransporter NKCC2 in

the Thick Ascending Limb: ROLE OF PROTEIN KINASE A

Downloaded from ajpcell.physiology.org on November 24, 2010

P. S. Caceres, G. R. Ares and P. A. Ortiz
J. Biol. Chem., September 11, 2009; 284 (37): 24965-24971.
[Abstract] [Full Text] [PDF]

Rab11b Regulates the Apical Recycling of the Cystic Fibrosis Transmembrane

Conductance Regulator in Polarized Intestinal Epithelial Cells
M. R. Silvis, C. A. Bertrand, N. Ameen, F. Golin-Bisello, M. B. Butterworth, R. A. Frizzell and
N. A. Bradbury
Mol. Biol. Cell, April 15, 2009; 20 (8): 2337-2350.
[Abstract] [Full Text] [PDF]

Involvement of phosphatidylinositol 3-kinase in cAMP- and cGMP-induced duodenal

epithelial CFTR activation in mice
B. Tuo, G. Wen, Y. Zhang, X. Liu, X. Wang, X. Liu and H. Dong
Am J Physiol Cell Physiol, January 1, 2009; 297 (3): C503-C515.
[Abstract] [Full Text] [PDF]

Role of the scaffold protein RACK1 in apical expression of CFTR

M. Auerbach and C. M. Liedtke
Am J Physiol Cell Physiol, July 1, 2007; 293 (1): C294-C304.
[Abstract] [Full Text] [PDF]

Medline items on this article's topics can be found at http://highwire.stanford.edu/lists/artbytopic.dtl

on the following topics:
Biochemistry .. Membrane Conductance
Biochemistry .. Biotinylation
Physiology .. Exocytosis
Physiology .. Jejunum
Physiology .. Water Balance and Solute Transport
Physiology .. Rats
Updated information and services including high-resolution figures, can be found at:
http://ajpcell.physiology.org/cgi/content/full/284/2/C429

Additional material and information about AJP - Cell Physiology can be found at:
http://www.the-aps.org/publications/ajpcell

This information is current as of November 24, 2010 .

AJP - Cell Physiology is dedicated to innovative approaches to the study of cell and molecular physiology. It is published 12 times
a year (monthly) by the American Physiological Society, 9650 Rockville Pike, Bethesda MD 20814-3991. Copyright © 2003 by the
American Physiological Society. ISSN: 0363-6143, ESSN: 1522-1563. Visit our website at http://www.the-aps.org/.

cAMP-dependent exocytosis and vesicle traffic regulate
CFTR and fluid transport in rat jejunum in vivo
NADIA A. AMEEN,1 CHRISTOPHER MARINO,2 AND PEDRO J. I. SALAS3
Departments of 1Pediatrics and 3Cell Biology, University of Miami School of Medicine,
Miami, Florida 33101; and 2Department of Medicine (Gastroenterology),
University of Tennessee, Memphis, Tennessee 38104
Submitted 11 June 2002; accepted in final form 27 August 2002
Ameen, Nadia A., Christopher Marino, and Pedro
J. I. Salas. cAMP-dependent exocytosis and vesicle traffic
regulate CFTR and fluid transport in rat jejunum in vivo. Am
J Physiol Cell Physiol 284: C429–C438, 2003; 10.1152/ajp-
cell.00261.2002.—The cystic fibrosis transmembrane conduc-
tance regulator (CFTR) channel is regulated by cAMP-depen-
dent vesicle traffic and exocytosis to the apical membrane in
some cell types, but this has not been demonstrated in the
intestinal crypt. The distribution of CFTR, lactase (control), and
fluid secretion were determined in rat jejunum after cAMP
activation in the presence of nocodazole and primaquine to
disrupt vesicle traffic. CFTR and lactase were localized by
immunofluorescence, and surface proteins were detected by
biotinylation of enterocytes. Immunoprecipitates from biotinyl-
ated and nonbiotinylated cells were analyzed by streptavidin
detection and immunoblots. Immunolocalization confirmed a
cAMP-dependent shift of CFTR but not lactase from a subapical
compartment to the apical surface associated with fluid secre-
tion that was reduced in the presence of primaquine and no-
codazole. Analysis of immunoblots from immunoprecipitates
after biotinylation revealed a 3.8 ⫾ 1.7-fold (P ⬍ 0.005) increase
of surface-exposed CFTR after vasoactive intestinal peptide
(VIP). These measurements provide independent corroboration
supporting a role for vesicle traffic in regulating CFTR and
cAMP-induced fluid transport in the intestine.
cystic fibrosis transmembrane conductance regulator; intes-
tine
THE CYSTIC FIBROSIS transmembrane conductance regu-
lator (CFTR) is an apical membrane chloride channel
critical to the regulation of fluid, chloride, and bicar-
bonate transport in the intestine (15, 17, 32, 43). Mu-
tations in the gene encoding CFTR result in the disease
cystic fibrosis (CF) and are associated with the absence
or dysfunction of CFTR on the apical membrane of
epithelial cells, decreased cAMP-mediated fluid secre-
tion, increased mucus viscosity, and intestinal obstruc-
tion (15). On the other hand, overstimulation of the
secretory pathway and activation of luminal CFTR in
the small intestine are implicated in the pathogenesis
of toxigenic secretory diarrhea (11, 39).
Although cAMP- and protein kinase A-dependent
phosphorylation is recognized as a major signal trans-
Address for reprint requests and other correspondence: P. Salas,
Dept. of Cell Biology and Anatomy, R-124, Univ. of Miami School
of Medicine, PO Box 016960, Miami, FL 33101 (E-mail: psalas
@miami.edu).
http://www.ajpcell.org 0363-6143/03 $5.00 Copyright © 2003 the American Physiological Society
Am J Physiol Cell Physiol 284: C429–C438, 2003;
10.1152/ajpcell.00261.2002.
duction pathway for CFTR activation, other second
messenger pathways include PKC, Ca2⫹/calmodulin-
dependent kinase, and cGMP-dependent kinase signal
CFTR activation (12, 20). In some cell types, cAMP-
dependent vesicle traffic also regulates CFTR exocyto-
Downloaded from ajpcell.physiology.org on November 24, 2010
sis and insertion into the apical plasma membrane;
however, this remains a highly controversial issue (6,
18, 25, 37, 38, 40).
Although cultured cell models proved useful in iden-
tifying CFTR signal transduction pathways, the appli-
cability of these pathways to physiological events is not
assured because of inherent differences in the proper-
ties of transformed cells and those of tissues expressing
endogenous CFTR (8, 39). In the intestine, CFTR chan-
nel gating is regulated by cAMP- and cGMP-dependent
phosphorylation of protein kinases A and G (12).
Whether vesicle traffic regulates CFTR and fluid secre-
tion in the intestine is unclear. Published studies using
a variety of techniques to examine this question with
T84 cells (a widely used chloride-secreting colonic cell
line expressing CFTR) resulted in conflicting conclu-
sions (9, 28, 39), and there are no in vivo studies
addressing this issue except for our previous analysis
(4) of CFTR high expresser (CHE) cells in rat small
intestine.
In a recent study of isolated rat colonic crypts, mem-
brane capacitance was used to determine cAMP-stim-
ulated exocytosis of CFTR; however, the authors could
not demonstrate an increase in membrane surface area
on cAMP activation and therefore concluded that acti-
vation of CFTR by cAMP does not involve detectable
exocytosis (13). However, cAMP-dependent chloride se-
cretion in the rat colon has been shown to be dependent
on intact microtubules, the molecular motors of vesicle
transport (14). This latter evidence suggests a role for
cAMP-dependent vesicle traffic in regulating chloride
secretion in the rat intestine.
Although CHE cells are a predominantly villus cell
population with undefined ion transport properties,
the number of CFTR channels expressed on the surface
of these cells was demonstrated to be regulated by
cAMP-dependent exocytosis. The observation that
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
C429
C430 PHYSIOLOGICAL EXOCYTOSIS OF CFTR IN INTESTINE
CHE cells are also present in the human small intes-
tine suggests that cAMP-dependent exocytosis of
CFTR is also a potentially important regulatory mech-
anism in the human intestine (3, 34).
The extent to which vesicle traffic regulates fluid
secretion and the number of CFTR channels expressed
on the surface of the small intestinal lumen (the pre-
dominant site of CFTR-mediated fluid secretion) is
unknown. In previous studies (2), we used immuno-
electron microscopy to examine and quantify the sub-
cellular distribution of CFTR. These observations con-
firmed that CFTR was associated with subapical
vesicles and the plasma membrane of both crypt and
CHE cells. Furthermore, quantification of the subcel-
lular distribution in these cells supported a role for
CFTR regulation by vesicle traffic in the rat small
intestine (2). On the basis of these observations, we
used independent morphological and biochemical
methodologies in conjunction with fluid secretion mea-
surements in the current study to determine whether
cAMP and vesicle traffic regulate the exocytosis of
CFTR to the apical membrane of the crypts and the
whole mucosa of rat proximal small intestine.
MATERIALS AND METHODS
Animal preparation and fluid secretion measurements. The
study was approved by the Animal Research Committee of
the University of Miami School of Medicine. Male Sprague-
Dawley rats (Charles River Laboratories) weighing 250–300
g were fed standard chow. Rats were fasted overnight and
anesthetized with 45 mg/kg pentobarbital sodium adminis-
tered intraperitoneally. After anesthesia, a tracheotomy was
performed to maintain a patent airway. Rectal temperature
of 38.1°C was maintained with a thermostatically controlled
heating lamp. Rats were subjected to a laparotomy via a
midline incision, a 30-cm length of jejunum was identified
and cannulated proximally and distally, and the lumen was
washed with warm 0.9% NaCl. The saline was removed from
the loop by blowing through the proximal cannula. The distal
cannula was removed, and ligatures were placed to create
four intestinal loops each ⬃4 cm in length with a 2-cm length
of intestine separating each loop. Equal volumes (⬃0.5 ml) of
drug [0.1 mM primaquine ⫹ 1.0 mM 8-bromo-cAMP (8-
BrcAMP), 10 ␮g/ml nocodazole ⫹ 1.0 mM 8-BrcAMP, 1.0 mM
8-BrcAMP, or PBS] were delivered into each loop by a fluid-
filled syringe that was weighed before (A, g) and after (B, g)
delivery into each loop. The abdomen was closed, and the
animal was observed for 2 h. At the end of the study period,
the abdomen was opened and intestinal loops were excised,
blotted free of excess fluid, and weighed (C, g). The loops were
then cut open, drained, blotted, and reweighed (D, g). The
amount of fluid recovered was obtained by subtraction (C ⫺
D, g). The net movement of fluid into or from the loop was
calculated as [(C ⫺ D) ⫺ (A ⫺ B)] (g). Fluid absorption was
reflected by a net loss of fluid from the loop, (A ⫺ B) ⬎ (C ⫺
D), and secretion occurred if there was a net gain of fluid into
the loop, (C ⫺ D) ⬎ (A ⫺ B). Because the densities of the
solutions weighed are all approximately equal to that of
water, the fluid weights are assumed to be identical with
fluid volumes. Fluid movements in or out of the loops were
calculated as micrograms per centimeter of jejunal length per
minute. At the end of the study period, intestinal tissues
from each loop were embedded in OCT embedding medium,
AJP-Cell Physiol • VOL 284 • FEBRUARY 2003 •
frozen in liquid nitrogen-cooled isopentane, and prepared for
immunocytochemistry as described previously (3, 4).
Independent studies were performed to confirm our obser-
vations of CFTR distribution in the intestine after cAMP
stimulation with the receptor agonist vasoactive intestinal
peptide (VIP) as described previously (4). Thirty minutes
after VIP administration tissues from rat jejunum were re-
moved, embedded in OCT embedding medium, frozen, and
prepared for indirect immunofluorescence labeling, and en-
terocytes were isolated and used in immunoprecipitation and
surface biotinylation studies.
Reagents. All chemicals were obtained from Sigma (St.
Louis, MO) except where stated. R3194 and R3195 are affinity-
purified polyclonal antibodies raised against rodent CFTR
and were provided by C. R. Marino. The specificity of both
antibodies has been documented in rats (1, 2, 4, 44). The
previously characterized antibody to lactase, YBB 2/61, was a
gift from Dr. A. Quaroni (Cornell University, Ithaca, NY; Ref.
29). The antibody to AKAP149 was purchased from Alomone
Laboratories (Jerusalem, Israel).
Isolation of intestinal enterocytes. Segments of rat jejunum
Downloaded from ajpcell.physiology.org on November 24, 2010
were removed 30 min after VIP administration, and villus
and crypt enterocytes were isolated as described previously
(24). Suspensions of freshly isolated enterocytes were subject
to Trypan blue exclusion, immunofluorescence immunocyto-
chemistry, and surface biotinylation studies.
Surface biotinylation of isolated enterocytes. Suspensions
of freshly isolated cells from rat small intestine after VIP or
diluent infusion were biotinylated by rotating in the cold for
30 min in freshly prepared 1.0 mM sulpho-NHS biotin [bicin-
choninic acid (BCA) protein assay kit; Pierce Laboratories,
Rockford, IL] in PBS-CM (PBS supplemented with 1.3mM
Ca2Cl and 1.0 mM Mg2Cl). Control cells were incubated with
PBS-CM. After biotinylation, cells were washed in the cold
with 50 mM NH4Cl in PBS-CM to quench unreacted free
biotin and immunoprecipitation was performed. Surface bi-
otinylation of CFTR from the lumen of the intact intestine
was also attempted; however, complete diffusion into the
deep crypts (a major site of CFTR expression) could not be
ensured. Surface biotinylation was therefore performed in
isolated cells because the yield of isolated crypt and villus
enterocytes was high in our hands and polarity and mem-
brane preservation could be ensured in the majority of cells.
Indeed, it has been shown that isolated enterocytes remain
well polarized for hours (45), a fact that we tested further in
our experiments (see Fig. 3E).
Immunoprecipitation of CFTR. Enterocytes were lysed in
immunoprecipiation buffer (IP) containing 0.5% Triton
X-100, 0.1% SDS, and 0.5% sodium deoxycholate in PBS pH
7.4 supplemented with a protease inhibitor cocktail (Sigma).
Samples were homogenized and sonicated and then centri-
fuged for 15 min in the cold at 14,000 rpm. Protein content
was measured by UV absorption (Pierce Laboratories), and
immunoprecipitation was performed with a minimum of 1
mg/ml protein in supernatants. Supernatants were pre-
cleared with protein A-Sepharose beads (Amersham Phar-
macia, Piscataway, NJ) for 1 h in the cold. Immunoprecipi-
tations were performed with the anti-CFTR antibody R3194,
antibody to lactase YBB2/61, or nonspecific rabbit IgG. An-
tibody or serum was added to supernatants and incubated for
2 h at 4°C. Protein A agarose (5 mg/sample) was added to
samples and resuspended in IP buffer containing 1% (wt/vol)
globulin-free bovine serum albumin (BSA), and then samples
were rotated overnight in the cold. Samples were then cen-
trifuged (14,000 rpm), supernatants were discarded, and the
beads were washed in IP buffer supplemented with 0.5 M
KCl. The immunoprecipitates were eluted in 1% SDS, 4 M
www.ajpcell.org
 PHYSIOLOGICAL EXOCYTOSIS OF CFTR IN INTESTINE
urea, and 1 mM Tris 䡠 HCl, pH 6.8, precipitated on ice in
trichloroacetic acid, acetone extracted, and air dried. The
dried pellets were resuspended in sample buffer before anal-
ysis by Western blot.
Western blotting. Immunoprecipitates were analyzed by
SDS-PAGE using a 7.5% gel and proteins transferred to
polyvinylidene difluoride (PVDF) membranes by a semi-dry
transfer method. After transfer of proteins, membranes were
washed in deionized water, nonspecific proteins were blocked
in PBS-0.05% Tween 20 containing 5% nonfat dry milk for
2 h, and biotinylated proteins were detected by streptavidin
peroxidase binding (Sigma). Western blots of nonbiotinylated
immunoprecipitates were analyzed with antibodies to CFTR
(R3194) and lactase (YBB2/61). Western blots of CFTR or IgG
immunoprecipitates were also analyzed with a commercial
antibody to AKAP 149 (Alomone Labs). Detection of primary
antibodies was accomplished by using goat anti-rabbit (1:
10,000) or anti-mouse (1:8,000) peroxidase secondary anti-
bodies (Sigma). After immunodetection, membranes were
exposed to chemiluminescence (preflashed Hyperfilm ECL,
Amersham Pharmacia). Densitometric analysis of protein
bands was performed with a Kodak Image Station 440 CF
and IS440CF image analysis software. Bands were selected,
and signal was weighted as number of pixels ⫻ (average pixel
intensity in the band ⫺ average pixel intensity in the back-
ground).
Immunocytochemistry. Intestinal tissues from all experi-
ments were prepared for immunocytochemical localization
studies with indirect immunofluorescent immunolabeling of
cryostat sections from jejunum as described previously (1, 4),
and labeled sections were examined on a Leica epifluorescent
microscope. Confocal microscopy and image analysis of CFTR
fluorescence intensities were performed as described previ-
ously (4) with a Zeiss LSM 510 microscope equipped with
image analysis software. The apical domain of CFTR in
labeled sections was determined from images of perpendicu-
lar parallel sections labeled with fluorescent phalloidin and
measured ⬃1.5 ␮m in length from the luminal surface. The
CFTR signal below that depth was considered the subapical
compartment. Acquisition of parameters were adjusted with
the software so that the pixel intensity of the brightest
fluorescence was not saturated (⬎255 pixels). Data was col-
lected from an average of 30 cells in random sections (aver-
age 10 sections) from each tissue examined.
Indirect immunofluorescence labeling for apical mem-
brane markers (lactase and CFTR) was performed on freshly
isolated enterocytes from rat jejunum to confirm preserva-
tion of polarity. A drop of cell suspension was placed onto a
poly L-lysine-coated slide and allowed to air dry. Briefly, cells
were fixed in 2% paraformaldehyde for 10 min and washed in
50 mM ammonium chloride, and nonspecific proteins were
blocked in PBS-BSA 1% for 30 min. Cells were exposed to
primary antibody or PBS-BSA 1% for 1 h at room tempera-
ture in a moist chamber. Primary antibody was detected with
FITC-conjugated secondary antibody diluted in PBS-BSA
1%. After immunolabeling, slides were examined with a
Leica epifluorescent microscope.
RESULTS
Morphological distribution of CFTR in rat jejunum
after cAMP stimulation. Our previous light microscopic
localization of CFTR in rat proximal small intestinal
crypts revealed a subapical distribution for CFTR, sug-
gesting the presence of CFTR in a vesicular compart-
ment. Immunoelectron microscopic examination re-
vealed that although CFTR was detected on the apical
AJP-Cell Physiol • VOL
C431
membrane, the majority of CFTR was associated with
subapical vesicles, supporting a role for vesicle inser-
tion in regulating CFTR and anion secretion in the
crypt (2). VIP, a cAMP agonist, also induced a redistri-
bution of CFTR from the subapical compartment to the
apical domain in villus CHE cells as observed previ-
ously (4). On the basis of these observations, we exam-
ined the distribution of CFTR in the crypt after VIP
(Fig. 1E). We compared the distribution with that of
lactase, an integral apical membrane protein that is
not regulated by cAMP-dependent vesicle traffic (Fig.
1, A and B; Refs. 23, 26). Although lactase is absent in
proliferative undifferentiated crypt cells and the high-
est levels of CFTR are found in this compartment, both
lactase and CFTR are present on the apical pole of
newly differentiated crypt cells that are more superfi-
cially located (Fig. 1, A and D; Refs. 3, 29, 34). In fact,
lactase is mostly found in a subapical compartment in
the crypts (Fig. 1, A and B), whereas it is expressed in
Downloaded from ajpcell.physiology.org on November 24, 2010
the brush border in the villus (29). Examination of
crypt sections from rat jejunum revealed that lactase
did not redistribute to the cell surface after VIP admin-
istration (Fig. 1, B compared with A). CFTR, on the
other hand, was distributed in a broad subapical region
under control conditions (Fig. 1D) and redistributed to
the apical surface in a narrow band (correlating with
the region of phalloidin label) after VIP (Fig. 1E). To
further confirm that the effect of VIP was mediated by
cAMP, similar experiments were conducted after lumi-
nal 8-BrcAMP stimulation. Examination of labeled sec-
tions by confocal microscopy after administration of the
membrane-permeant agonist 8-BrcAMP similarly con-
firmed a redistribution of CFTR from a predominant
subapical compartment (Fig. 2A) to the region corre-
sponding to the apical microvilli of crypt epithelial cells
(Fig. 2B). The 8-BrcAMP-dependent shift of CFTR
from the subapical to apical domain corresponded with
an almost threefold increase in the ratio of apical to
subapical CFTR fluorescence (6.15 ⫾ 3.08) compared
with unstimulated PBS controls (2.14 ⫾ 1.03; P ⬍
0.001) and was associated with net fluid movement
into the lumen of the jejunum (Table 1).
To determine whether the cAMP-dependent shift of
CFTR from the subapical compartment to the apical
domain of crypt cells was dependent on vesicle traffic,
we examined whether the same shift of CFTR signal
occurred when vesicle traffic was interrupted. The an-
timalarial drug primaquine is a lysosomotropic amine
that inhibits vesicle trafficking and prevents the fusion
of vesicles with the plasma membrane (16). It was
recently shown to be a potent inhibitor of CFTR vesicle
traffic in oocytes (39). Examination of crypt sections
from jejunum labeled for CFTR after pretreatment
with 8-BrcAMP and primaquine (Fig. 2C) revealed a
prominent rim of CFTR fluorescence label (extending
⬃1.5 ␮m) beneath the apical microvilli, similar to the
subapical distribution of CFTR under control condi-
tions (Fig. 1A). The CFTR apical fluorescence was
reduced in the presence of primaquine both in terms of
pixel values and in the apical-to-subapical ratio (Table
1). Accordingly, the reduction in the shift of CFTR from
284 • FEBRUARY 2003 • www.ajpcell.org
C432 PHYSIOLOGICAL EXOCYTOSIS OF CFTR IN INTESTINE

Downloaded from ajpcell.physiology.org on November 24, 2010

Fig. 1. Vasoactive intestinal peptide (VIP) induces a redistribution of the cystic fibrosis transmembrane conduc-
tance regulator (CFTR), but not lactase, to the apical membrane of crypt cells. Lactase fluorescence labeling in
crypt cells under control conditions (A) reveals a distribution under the apical domain that does not change after
VIP stimulation (B). Control section labeled with nonimmune serum reveals lack of specific staining (C). Under
unstimulated conditions, CFTR fluorescence (D) is distributed in a broad band in the subapical region (arrows) and
redistributes to the apical surface after VIP stimulation (E). Control section labeled with CFTR antibody (R3195)
preincubated with peptide (F). Bars, 10 ␮m.

the subapical to the apical domain after pretreatment
with primaquine and 8-BrcAMP was associated with
an increase in CFTR signal detected in the subapical
compartment in the presence of primaquine. The shift
in the subcellular distribution of CFTR in the crypt
paralleled a functional effect of primaquine pretreat-
ment in blocking the fluid secretory response of
8-BrcAMP by 35% (Table 1). These observations are
consistent with an effect of primaquine in inhibiting
the 8-BrcAMP-dependent insertion of CFTR-contain-
ing vesicles from the subapical compartment into the
apical membrane and suggest that cAMP and vesicle
AJP-Cell Physiol • VOL
insertion of CFTR to the apical membrane regulates
fluid secretion in the jejunum.
Microtubules serve as molecular motors in the trans-
port of vesicles from the Golgi complex to the apical
domain of polarized cells and have been shown to play
a role in cAMP-dependent exocytosis of CFTR in T84
cells and in cAMP-dependent chloride secretion in rat
colon (14, 10, 21, 38). However, studies of polarized
Madin-Darby canine kidney (MDCK) cells and airway
epithelial cells could not confirm a role for microtu-
bules in regulating the exocytosis of CFTR (22, 25). We
examined the distribution of CFTR after 8-BrcAMP
284 • FEBRUARY 2003 • www.ajpcell.org
 PHYSIOLOGICAL EXOCYTOSIS OF CFTR IN INTESTINE
stimulation in the presence of nocodazole, an agent
that blocks microtubule polymerization and thereby
prevents vesicle transport in cells. Nocodazole also
blocked the subapical-to-apical shift in CFTR signal,
although to a lesser extent than primaquine, and re-
duced the 8-BrcAMP-induced fluid secretory response
by 33% (Table 1). Because the mechanisms of action of
primaquine and nocodazole are different and they have
in common their effect on exocytic membrane traffic,
these results strongly suggest a role of cAMP-induced
exocytosis of CFTR as a mechanism to regulate CFTR-
mediated anion transport and fluid secretion.
Detection of CFTR exocytosis by surface biotinylation
in vivo. Although immunofluorescence examination of
the distribution of CFTR in intestinal sections after
cAMP agonist treatment suggested a shift of CFTR
from a subapical compartment to the apical domain, we
wished to independently confirm that cAMP indeed
stimulated exocytosis of CFTR to the surface of intes-
tinal cells. Immunolocalization in toto could not con-
firm this because our antibodies were raised against
the cytoplasmic COOH terminus of CFTR. We there-
fore used the technique of surface biotinylation, a sen-
Table 1. CFTR fluorescence values
Apical Fl
PBS 190.19 ⫾ 59.41
8-BrcAMP 210.72 ⫾ 38.93
Nocodazole ⫹ 8-BrcAMP 166.05 ⫾ 70.03
8-BrcAMP ⫹ primaquine 125.58 ⫾ 89.42
Values for fluorescence (Fl) are expressed in pixels as means ⫾ SD determined from a minimum of 30 crypts and 10 random sections. n ⫽
4 animals per condition. A, apical; S, subapical; 8-BrcAMP, 8-bromo-CAMP; CFTR, cystic fibrosis transmembrane conductance regulator. P
values: * vs. PBS, † vs. 8-BrcAMP (t-test).
AJP-Cell Physiol • VOL
C433
Fig. 2. 8-Bromo-cAMP (8-BrcAMP)-de-
pendent redistribution of CFTR to the
apical surface of crypt cells is inhibited
by vesicle traffic interruption. Under
unstimulated conditions confocal im-
ages of labeled sections reveal that
CFTR is distributed in a wide band
extending beneath the apical surface
(A, arrows; as shown in Fig. 1D) and is
redistributed to the apical surface after
8-BrcAMP treatment (B, small arrows).
Treatment with 8-BrcAMP and prima-
quine results in an accumulation of
CFTR in the subapical compartment
(C, arrows). D: control section labeled
in the presence of preimmune serum
reveals no label in the crypts. L, lu-
men. Bar, 10 ␮m.
Downloaded from ajpcell.physiology.org on November 24, 2010
sitive method that is widely used to quantify surface
proteins in cells and to study membrane traffic of
proteins and has been used in studies examining CFTR
membrane traffic (28, 30).
Morphological examination of fixed intestinal seg-
ments after isolation confirmed that we could success-
fully retrieve most cells for biotinylation and immuno-
precipitation, including those from the crypts, within
30 min. Lack of damage to isolated enterocytes was
confirmed by Trypan blue exclusion in cell suspen-
sions. Furthermore, immunofluorescence labeling of
freshly isolated cells confirmed preservation of polarity
by the presence of apical markers (Fig. 3, E and F) in
isolated cells. Immunoprecipitations were performed
on freshly isolated cells with the CFTR antibody R3194
and nonspecific rabbit IgG as negative controls, and
immunoprecipitates were analyzed for CFTR by West-
ern blots using the same CFTR antibody. Western blot
analysis of immunoprecipitates from VIP-stimulated
or control cells with R3194 detected a broad band of
mature CFTR (band C) of molecular mass of 170–185
kDa (Fig. 3A, lane 2) and a smaller band of immature
CFTR of ⬃148 kDa in native rat tissues but not in IgG
Fluid,
Subapical Fl Ratio A/SA Fl ␮g 䡠 min⫺1 䡠 cm⫺1
98.93 ⫾ 36.56 2.14 ⫾ 1.03 ⫺0.29 ⫾ 0.04
43.33 ⫾ 23.33 6.15 ⫾ 3.08 ⫹0.105 ⫾ 0.03
P ⬍ 0.001* P ⬍ 0.005†
131.53 ⫾ 69.78 1.41 ⫾ 0.65 ⫹0.07 ⫾ 0.01
P ⬍ 0.001† P ⬍ 0.05†
159.54 ⫾ 71.95 0.97 ⫾ 0.92 ⫹0.04 ⫾ 0.01
P ⬍ 0.001† P ⬍ 0.005†
284 • FEBRUARY 2003 • www.ajpcell.org
C434 PHYSIOLOGICAL EXOCYTOSIS OF CFTR IN INTESTINE

Downloaded from ajpcell.physiology.org on November 24, 2010

Fig. 3. A: detection of CFTR in immunoprecipitates from isolated rat intestinal cells. Western blot analysis of
CFTR immunoprecipitates with the antibody R3194 (lane 2) reveals a broad band of 170–185 kDa consistent with
mature glycosylated CFTR (arrow) and a smaller band of ⬃148 kDa consistent with immature CFTR (band B),
neither of which are detected in IgG immunoprecipitates (lane 1). B: cAMP-dependent exocytosis of CFTR by
surface biotinylation. Rat jejunal enterocytes were isolated from animals infused with either vehicle or VIP,
biotinylated, and immunoprecipitated with anti-CFTR antibody R3194 or IgG. Streptavidin detection on blots
revealed a protein band of ⬃180 kDa (arrow) consistent with mature CFTR and 2 other bands (*) in cells
immunoprecipitated from VIP (lane 4) and in vehicle-treated animals with R3194 (lane 5) but not in nonimmune
IgG immunoprecipitates (lane 3). Although the top * band appears similar to band B of CFTR, it has a larger
molecular mass of ⬃162 kDa. Both antibody-specific bands appeared more prominent in VIP-treated immunopre-
cipitates (lane 4) than in controls (lane 5). Graph shows that densitometric analysis of biotinylated CFTR band
from VIP immunprecipitates (below lane 4) revealed an almost 4-fold increase over control (below lane 5). Data are
means ⫾ SD from 4 independent experiments; the difference between the bars was statistically significant (P ⬍
0.005). C: surface levels of biotinylated lactase do not change after cAMP stimulation. Streptavidin detection of
immunoprecipitates with an antibody to lactase (YBB2/61) after biotinylation reveals a single band of ⬃150 kDa
in cells from VIP (lane 8) and vehicle-treated (lane 9) rats consistent with lactase. No bands were identified in IgG
immunoprecipitates from either VIP (lane 6)- or vehicle (lane 7)-treated rats. D: normal rat jejunal enterocytes
were immunoprecipitated with R3194 (lane 11) or IgG (lane 10) and immunoblotted with an antibody to AKAP149.
A single band of ⬃149 kDa was detected in R3194 (lane 11) but not in IgG immunoprecipitates (lane 10).
Experiments shown in each panel were repeated at least 4 times. E: enterocytes from rat jejunum remain polarized
after isolation. Immunofluorescence label for lactase with the antibody YBB2/61 and detected with FITC goat
anti-mouse secondary antibody reveals apical labeling for lactase on the brush border of isolated enterocytes. F:
control enterocytes labeled in the absence of primary antibody. Bar, 30 ␮m.

controls (Fig. 3A, lane 1) as shown previously (1, 9).
These results confirmed the success of the immunopre-
cipitation.
Before surface biotinylation experiments, we con-
firmed that NHS-biotin was effective in surface bioti-
nylation of freshly isolated crypt and villus enterocytes
by immunofluorescence labeling with Texas red
streptavidin (not shown). Having confirmed that we
could detect CFTR in immunoprecipitates from iso-
lated enterocytes, we then proceeded with surface bi-
otinylation/immunoprecipitation studies on VIP-stim-
ulated and control cells. Streptavidin detection of
R3194 or IgG immunoprecipitates (Fig. 3B) after bioti-
nylation of VIP-stimulated (Fig. 3B, lane 4) and control
(Fig. 3B, lane 5) cells revealed a band consistent with
mature CFTR of ⬃175–183 kDa (Fig. 3B) that was
more intense in VIP-treated samples than control. The
total material from stimulated and control samples
was carefully normalized for total protein, and the
amounts of total immunoprecipitated CFTR were al-
most identical as determined in parallel immunoblots.
Densitometric analysis of the CFTR band in indepen-
dent experiments revealed a 3.8 ⫾ 1.7-fold (P ⬍ 0.005)
increase in surface biotinylated CFTR in immunopre-
cipitates after VIP treatment (Fig. 3B). In addition to
CFTR, we identified at least two other CFTR antibody-
specific biotinylated bands in both VIP and controls
(Fig. 3B), apparent molecular mass of ⬃162 and 110
kDa, that appeared more prominent in VIP-stimulated
immunoprecipitates. Although one of these bands ap-
pears at a level that may be confused with immature
CFTR, analysis of that band revealed it to be a polypep-
tide of ⬃162 kDa, ⬃14,000 kDa larger than the size of

AJP-Cell Physiol • VOL 284 • FEBRUARY 2003 • www.ajpcell.org

 PHYSIOLOGICAL EXOCYTOSIS OF CFTR IN INTESTINE
immature band B of CFTR in the same preparation
(Fig. 3A).
We explored the possibility that the results described
above are due to leaking of sulfo-NHS-biotin into the
cells and therefore labeling intracellular proteins in-
cluding CFTR. We tested this hypothesis by perform-
ing biotinylation/immunoprecipitation of CFTR in the
presence and absence of saponin. Streptavidin detec-
tion of R3194 immunoprecipitates after saponin treat-
ment and biotinylation revealed at least one additional
band that could not be identified in non-saponin-
treated immunoprecipiates. These experiments sug-
gest that in our system of isolated enterocytes, the
plasma membrane remains intact both in the presence
and absence of cAMP stimulation, preventing sulfo-
NHS-biotin from entering the cytoplasm unless the
cells are permeabilized with saponin.
We also entertained the possibility that cAMP may
be inducing a generalized exocytosis of membrane pro-
teins. To test the specificity of CFTR exocytosis, we
used the same biotinylation/immunoprecipitation pro-
cedure to analyze the apical membrane protein lactase
in VIP-stimulated and control immunoprecipitates
with the antibody YBB2/61 as shown in immunofluo-
rescence localization (Fig. 1, A and B). Streptavidin
detection of biotinylated immunoprecipitates of lactase
with the antibody YBB2/61 or IgG is shown in Fig. 3C.
Under the same conditions that we used to detect
CFTR, blot analysis of biotinylated lactase immuno-
precipitates revealed a single antibody-specific protein
band of ⬃150 kDa consistent with lactase in VIP and
unstimulated controls (Fig. 3, lanes 8 and 9) but not in
IgG immunoprecipitates of VIP-stimulated or control
(Fig. 3, lanes 6 and 7; Ref. 29). Densitometric analysis
revealed no difference in the intensities of the biotin-
ylated lactase band identified in VIP or control immu-
noprecipitates. These observations are consistent with
our immunofluorescence data indicating no change in
the distribution of lactase in intestinal cells on VIP
stimulation, and they suggest that cAMP stimulates
exocytosis of a specific population of apical membrane
proteins that comprises CFTR but not lactase. In ad-
dition, this result further supports the notion that the
two additional bands in CFTR immunoprecipitates
were true antibody-specific immunoprecipitating pep-
tides and not just contaminants.
In fact, we were puzzled by these two additional
biotinylated bands that coimmunoprecipitated with
CFTR and not with lactase under the same conditions.
One possible explanation for this observation is that
CFTR exists in a multiprotein complex that includes
other membrane proteins, as suggested by others (33,
35). CFTR was recently shown to be physically linked
to regulatory complexes containing PKA and PKA an-
choring proteins (AKAPs), sodium-hydrogen exchanger
regulatory factor (NHERF), and ezrin in a complex
insoluble scaffold (35). To test whether our immuno-
precipitation conditions preserve some of the protein-
protein interactions in that scaffold, we analyzed
whether AKAP coimmunoprecipitates with CFTR in
our system. Western blot analysis was performed using
AJP-Cell Physiol • VOL
C435
an antibody to AKAP 149, a PKA type II anchoring
protein that is highly expressed in the small intestine.
The antibody recognized a specific protein band of 149
kDa in CFTR immunoprecipitates (Fig. 3D, lane 11)
but not in IgG controls (Fig. 3D, lane 10), indicating
that we were indeed immunoprecipitating a multipro-
tein complex.
DISCUSSION
In the current study, two independent techniques
were used to confirm that physiological cAMP stimu-
lation and vesicle traffic regulate the number of CFTR
channels on the surface of the rat small intestinal
epithelium. This observation resolves the current con-
troversy regarding the role of membrane traffic in
regulating CFTR in the intestine. Although cAMP-
dependent exocytosis of CFTR to the apical membrane
has been demonstrated in villus CHE cells (4), the
Downloaded from ajpcell.physiology.org on November 24, 2010
physiological relevance of that observation remains
unknown. Our observation in this work that CFTR is
regulated in vivo by cAMP-dependent vesicle traffic
and channel insertion in both crypt and villus cells in
association with fluid secretion, however, provides
strong support for physiological membrane traffic reg-
ulation of CFTR and intestinal anion secretion.
Both receptor (VIP)- and non-receptor (8-BrcAMP)-
mediated cAMP agonists induced a redistribution of
CFTR from the subapical to the apical domain in the
jejunum. The lack of change in the distribution of
lactase in crypt cells after cAMP agonist stimulation
confirmed that the cAMP-dependent redistribution of
CFTR was specific, because lactase is not regulated by
cAMP-dependent vesicle insertion (23, 26). To deter-
mine whether the cAMP-induced redistribution of
CFTR from the subapical to the apical domain is reg-
ulated by vesicle traffic, we disrupted vesicle traffic in
vivo with primaquine and nocodazole. In rat jejunum,
primaquine (0.1 mM) was a potent inhibitor of the
cAMP-dependent shift of CFTR from the subapical to
the apical domain in the crypt and reduced the fluid
secretory response of 8-BrcAMP (Table 1). The accu-
mulation of CFTR in the subapical compartment in the
crypt in the presence of primaquine is consistent with
the observations by others of its effect in inhibiting
receptor recycling and in producing an intracellular
accumulation of endocytosed receptors, blocking the
exit of receptors from the early endosomes and recy-
cling to the plasma membrane (41). The reduction in
the fluid secretory response to 8-BrcAMP in vivo in the
presence of primaquine (⬃35%) supports the notion
that insertion of new CFTR channels into the mem-
brane contributes importantly to augmenting fluid se-
cretion.
These experiments, however, may allow an alterna-
tive albeit nonexclusive interpretation. If CFTR is con-
tinuously recycling between the apical domain and the
subapical compartment, primaquine may interrupt the
cycle and accumulate CFTR in the early endosomal
compartment. The fluorescence measurements in con-
focal images actually point to this scenario. In that
284 • FEBRUARY 2003 • www.ajpcell.org
C436 PHYSIOLOGICAL EXOCYTOSIS OF CFTR IN INTESTINE
case, we could conclude that the balance between exo-
cytosis and endocytosis of CFTR is almost as important
as channel gating as a regulatory factor. Our observa-
tions in the intestine support the results of recent
studies in oocytes demonstrating that primaquine
drastically reduced cAMP-dependent CFTR chloride
currents and effectively blocked vesicle and protein
traffic (40). Further studies will be necessary to assess
the relative contributions of exocytosis and endocytosis
to the steady-state levels of surface CFTR on cAMP
stimulation.
Although the effects of microtubule disruption on
CFTR distribution and fluid movement were less strik-
ing than those of primaquine, they also suggest a role
of membrane traffic in regulating the number of CFTR
channels on the apical surface and provide support for
the previous observation that cAMP-dependent chlo-
ride secretion in rat intestine is regulated by microtu-
bules (14).
Surface biotinylation, a well-established technique
used to assess the delivery of proteins to the plasma
membrane (30, 31), confirmed cAMP-stimulated CFTR
exocytosis. However, the finding that other unidenti-
fied polypeptides were coimmunoprecipitating with
CFTR and were also upregulated by cAMP, as shown
in Fig. 3, was unexpected. Our first interpretation was
that other proteins possessing at least one ectoplasmic
domain capable of biotinylation may be nonspecific
contaminants of the immunoprecipitation. This, how-
ever, was unlikely for the following reasons: 1) these
other biotinylated proteins were CFTR antibody spe-
cific in the immunoprecipitation and did not appear in
controls immunoprecipitated with nonimmune IgG
(Fig. 3B, lane 3); 2) the conditions for immunoprecipi-
tation were rather stringent, detergents were present
in all washes, and one of the washes was performed in
high salt (0.6 M KCl) conditions; and 3) the sucrase-
isomaltase immunoprecipitation experiments (Fig. 3C)
supported the notion that our immunoprecipitations
were “clean” (in those cases no additional bands were
observed).
Another potential artifact that could explain biotiny-
lation of multiple bands is damage to the plasma mem-
brane during the isolation of enterocytes before bioti-
nylation. This possibility was ruled out by verifying
Trypan blue exclusion in parallel cell suspensions and
by actually permeabilizing some cell suspensions with
saponin. The latter resulted in an increase in the
number of biotinylated bands, indicating that in the
absence of saponin the plasma membrane was tight.
The observation that AKAP coimmunoprecipitates
with CFTR (Fig. 3D) in the intestine supports the
notion that the physiological regulation of CFTR by
PKA involves a physical and functional association
with AKAP as demonstrated recently (19). The coim-
munoprecipitation of AKAP with CFTR indicated that
under the conditions of homogenization, detergent sol-
ubilization, and immunoprecipitation used here, the
NHERF-ezrin insoluble scaffold that normally holds
CFTR (35, 36) is at least partially preserved.
AJP-Cell Physiol • VOL 284 • FEBRUARY 2003 •
At least one other transmembrane protein, Na⫹/H⫹
exchanger (NHE), is known to be attached to this
scaffold in addition to CFTR (42). Although the appar-
ent molecular masses of the biotinylated bands that we
found do not correspond to that of NHE-3 (97 kDa; Ref.
5), it is conceivable that other membrane proteins are
also attached to the same scaffold. Furthermore, be-
cause some membrane proteins do not biotinylate and
because we cannot assert that the scaffold is totally
intact, the actual number of membrane proteins at-
tached to the same scaffold of CFTR may be actually
greater than three (CFTR and the 2 unknown proteins
found in this work). On the other hand, the data pre-
sented here do not rule out the possibility that the
additional unidentified proteins may be directly bound
to CFTR and not to a NHERF-type scaffold. Identifica-
tion of these proteins in future investigations will be
important before any mechanistic model can be postu-
lated.
Downloaded from ajpcell.physiology.org on November 24, 2010
In previous work from our laboratory (7) and others
(27), it was found that cAMP stimulates exocytosis of
apical membrane proteins at a post-Golgi step. The
lack of effect of cAMP stimulation on lactase would
suggest, however, that cAMP-dependent exocytosis is
restricted to a subpopulation of apical membrane pro-
teins. It has been generally accepted that cAMP oper-
ates by increasing membrane traffic (7, 27). If that is
the case, the results in this work would suggest that at
least two subpopulations of subapical vesicles must
exist, one that carries CFTR and other proteins regu-
lated by cAMP-dependent delivery and another cAMP-
independent pathway that facilitates the transport of
proteins such as lactase. Such a senario raises inter-
esting questions regarding potentially different path-
ways that may regulate the formation and sorting of
these two different subpopulations of vesicles.
Another alternative explanation that by no means
excludes differences in vesicle traffic pathways is that
cAMP may actually increase the number of binding
sites available in the scaffold itself. Because the scaf-
fold contains PKA and AKAP, it is conceivable that its
binding capacity may be modulated by cAMP. In that
scenario, cAMP stimulation would increase the num-
ber of surface molecules for all the membrane proteins
that bind to the same scaffold, disregarding the vesi-
cles that transport them to the cell surface. In other
words, retention in the apical domain would be respon-
sible for the increase of surface CFTR and some other
proteins. In both cases, the results of this study suggest
that the increase in the number of CFTR channels on
the surface of intestinal epithelial cells on cAMP stim-
ulation contributes substantially to regulating fluid
secretion and is regulated by vesicle traffic. The obser-
vations in this study should provide the basis for a
critical examination of membrane traffic in the patho-
genesis of CFTR-mediated diseases in the intestine.
We thank Dr. A. Quaroni for the generous gift of antibodies and
Dr. G. McLaughlin and M. Hernandez for technical assistance.
Present address of N. A. Ameen: Pediatric Gastroenterology and
Cell Biology, University of Pittsburgh School of Medicine, Pitts-
burgh, PA 15213.
www.ajpcell.org
 PHYSIOLOGICAL EXOCYTOSIS OF CFTR IN INTESTINE
REFERENCES
1. Ameen N, Alexis J, and Salas P. Cellular localization of the
cystic fibrosis transmembrane conductance regulator in mouse
intestinal tract. Histochem Cell Biol 114: 69–75, 2000.
2. Ameen N, vanDonselaar E, Posthuma G, deJonge H,
McLaughlin G, Geuze H, Marino C, and Peters P. Subcel-
lular distribution of CFTR in rat intestine supports a physiologic
role for regulation by vesicle traffic. Histochem Cell Biol 114:
219–228, 2000.
3. Ameen NA, Ardito T, Kashgarian M, and Marino CR. A
unique subset of rat and human intestinal villus cells express the
cystic fibrosis transmembrane conductance regulator. Gastroen-
terology 108: 1016–1023, 1995.
4. Ameen NA, Martensson B, Bourguinon L, Marino C, Isen-
berg J, and Mclaughlin EG. CFTR channel insertion to the
apical surface in rat duodenal villus epithelial cells is upregu-
lated by VIP in vivo. J Cell Sci 112: 887–894, 1999.
5. Bookstein C, DePaoil A, Xie Y, Niu P, Musch M, Rao M, and
Chang E. Na⫹/H⫹ exchangers, NHE-1 and NHE-3, of rat intes-
tine. Expression and localization. J Clin Invest 93: 106–113,
1994.
6. Bradbury N. Intracellular CFTR: localization and function.
Physiol Rev 79: S167–S191, 1999.
7. Brignoni M, Pignataro OP, Rodriguez ML, Alvarez A,
Vega-Salas D, Rodriguez-Boulan E, and Salas PJI. Cyclic
AMP modulates the rate of “constitutive” exocytosis of apical
membrane proteins in Madin-Darby canine kidney cells. J Cell
Sci 108: 1931–1943, 1995.
8. Chao AC, de Sauvage FJ, Dong YJ, Wagner JA, Goedde
DV, and Gardner P. Activation of intestinal CFTR chloride
channel by heat stable enterotoxin and guanylin via cAMP-
dependent protein kinase. EMBO J 13: 1065–1072, 1994.
9. Denning GM, Ostedgaard LS, Cheng SH, Smith AE, and
Welch MJ. Localization of the cystic fibrosis transmembrane
conductance regulator in chloride secretory epithelia. J Clin
Invest 89: 339–349, 1992.
10. Fuller CM, Bridges RJ, and Benos DJ. Forskolin- but not
ionomycin-evoked Cl⫺ secretion in colonic epithelia depends on
intact microtubules. Am J Physiol Cell Physiol 266: C661–C668,
1994.
11. Gabriel SE, Brigman KN, Koller BV, Boucher RC, and
Stutts MJ. Cystic fibrosis heterozygote resistance to cholera
toxin in the cystic fibrosis mouse model. Science 266: 107–109,
1994.
12. Gadsby D and Nairn A. Control of CFTR channel gating by
phosphorylation and nucleotide hydrolysis. Physiol Rev 79: S77–
S107, 1999.
13. Greger R. Role of CFTR in the colon. Annu Rev Physiol 62:
467–491, 2000.
14. Grotmol T and VanDyke RW. Prostaglandin- and theophyl-
line-induced Cl secretion in rat distal colon is inhibited by
microtubule inhibitors. Dig Dis Sci 37: 1709–1717, 1992.
15. Grubb B and Boucher R. Pathophysiology of gene-targeted
mouse models for cystic fibrosis. Physiol Rev 79: S193–S214,
1999.
16. Hiebsch RR, Raub TJ, and Wattenberg BW. Primaquine
blocks transport by inhibiting the formation of functional trans-
port vesicles. J Biol Chem 266: 20323–20328, 1991.
17. Hogan D, Crombie DL, Isenberg JI, Svendsen P, Schaf-
falitzky-de-Muckadell OB, and Ainsworth MA. Acid-stimu-
lated duodenal bicarbonate secretion involves a CFTR-mediated
transport pathway in mice. Gastroenterology 113: 533–541,
1997.
18. Howard M, Jiang X, Stolz D, Hill W, Johnson J, Watkins S,
Frizzell R, Bruton C, Robbins P, and Weisz O. Forskolin-
induced apical membrane insertion of virally expressed, epitope-
tagged CFTR in polarized MDCK cells. Am J Physiol Cell Physiol
279: C375–C382, 2000.
19. Huang P, Trotter K, Boucher R, Milgram S, and Stutts M.
PKA holozyme is functionally coupled to CFTR by AKAPs. Am J
Physiol Cell Physiol 278: C417–C422, 2000.
AJP-Cell Physiol • VOL 284 • FEBRUARY 2003 •
C437
20. Kunzelman K and Mall M. Electrolyte transport in the mam-
malian colon: mechanisms and implications for disease. Physiol
Rev 82: 245–289, 2001.
21. Lafont F and Simons K. The role of microtubule-based motors
in the exocytic transport of polarized cells. Semin Cell Dev Biol 7:
343–355, 1996.
22. Loffing J, Moyer B, McCoy D, and Stanton B. Exocytosis is
not involved in activation of Cl⫺ secretion via CFTR in calu-3
airway epithelial cells. Am J Physiol Cell Physiol 275: C913–
C920, 1998.
23. Mantei N, Villa M, Enzler T, Wacker H, Boll W, James P,
Hunziker W, and Semenza G. Complete primary structure of
human and rabbit lactase-phlorizin hydrolase: implications for
biosynthesis, membrane anchoring and evolution of the enzyme.
EMBO J 7: 2705–2713, 1988.
24. McNicholas CM, Brown CDA, and Turnberg LA. Na⫹-K⫹-
Cl⫺ cotransport in villus and crypt cells from rat duodenum.
Am J Physiol Gastrointest Liver Physiol 267: G1004–G1011,
1994.
25. Moyer BD, Loffing J, Schwiebert EM, Loffing-Cueni D,
Halpin PA, Karlson KH, Ismailov IL, Guggino WB, Lang-
ford GM, and Stanton BA. Membrane trafficking of the cystic
fibrosis gene product, cystic fibrosis transmembrane conduc-
Downloaded from ajpcell.physiology.org on November 24, 2010
tance regulator, tagged with green fluorescent protein in Madin-
Darby canine kidney cells. J Biol Chem 273: 21759–21768, 1998.
26. Naim HY and Naim H. Dimerization of lactase-phlorizin hy-
drolase occurs in the endoplasmic reticulum, involves the puta-
tive membrane spanning domain and is required for an efficient
transport of the enzyme to the cell surface. Eur J Cell Biol 70:
198–208, 1996.
27. Pimplikar SW and Simons K. Activators of protein kinase A
stimulate apical but not basolateral transport in epithelial Ma-
din-Darby kidney cells. J Biol Chem 269: 19054–19059, 1994.
28. Prince LS, Tousson A, and Marchese RB. Cell surface label-
ing of CFTR in T84 cells. Am J Physiol Cell Physiol 264: C491–
C498, 1993.
29. Quaroni A and Isselbacher K. Study of intestinal cell differ-
entiation with monoclonal antibodies to intestinal cell surface
components. Dev Biol 111: 267–279, 1985.
30. Rodriguez-Boulan E, Salas PJ, Sargiacomo M, Lisanti M,
Lebivic A, Sanbury Y, Vega-Salas D, and Graeve L. Meth-
ods to estimate the polarized distribution of surface antigens in
cultured epithelial cells. Methods Cell Biol 32: 37–56, 1989.
31. Sargiacomo M, Lisanti M, Graeve L, Bivic AL, and Rodri-
guez-Boulan E. Integral and peripheral protein composition of
the apical and basolateral membrane domains in MDCK cells. J
Membr Biol 107: 277–286, 1989.
32. Seidler U, Blumenstein I, Kretz A, Viellard-Baron D, Ross-
man H, Colledge WH, Evans M, Ratcliff R, and Gregor M.
A functional CFTR protein is required for mouse intestinal
cAMP, cGMP and Ca2⫹-dependent HCO3⫺ secretion. J Physiol
505: 411–423, 1997.
33. Short DB, Trotter KW, Reczek D, Kreda SM, Bretscher A,
Boucher R, Stutts JM, and Milgram SL. An apical PDZ
protein anchors the cystic fibrosis transmembrane conductance
regulator to the cytoskeleton. J Biol Chem 273: 19797–19801,
1998.
34. Strong TV, Boehm K, and Collins FS. Localization of the
cystic fibrosis conductance regulator mRNA in the human gas-
trointestinal tract by in situ hybridization. J Clin Invest 93:
347–354, 1994.
35. Sun F, Hug MJ, Bradbury N, and Frizzell R. Protein kinase
A associates with cystic fibrosis transmembrane conductance
regulator via an interaction with ezrin. J Biol Chem 275: 14360–
14366, 2000.
36. Sun F, Hug MJ, Lewarchik CM, Yun CC, Bradbury NA,
and AFR. E3KARP mediates the association of ezrin and pro-
tein kinase A with the cystic fibrosis transmembrane conduc-
tance regulator in airway cells. J Biol Chem 275: 29539–29546,
2000.
37. Takahashi A, Watkins SC, Howard M, Frizzell RA. CFTR-
dependent membrane insertion is linked to stimulation of the
www.ajpcell.org
C438 PHYSIOLOGICAL EXOCYTOSIS OF CFTR IN INTESTINE
CFTR chloride conductance. Am J Physiol Cell Physiol 271:
C1887–C1894, 1996.
38. Tousson A, Fuller CM, and Benos DJ. Apical recruitment of
CFTR in T84 cells is dependent on cAMP and microtubules but
not calcium or microfilaments. J Cell Sci 109: 1325–1334, 1996.
39. Vaandrager A, Bot AGM, and deJonge HR. Guanosine 3⬘,5⬘-
cyclic monophosphate-dependent protein kinase II mediates
heat-stable enterotoxin-provoked chloride secretion in rat intes-
tine. Gastroenterology 112: 437–443, 1997.
40. Weber WM, Segal A, Simaels J, Vankeerberghen A, Cassi-
man JJ, and Driessche WV. Functional integrity of the vesicle
transporting machinery is required for complete activation of
CFTR expressed in Xenopus laevis oocytes. Pflügers Arch 441:
850–859, 2001.
41. Weert AV, Geuze H, Groothuis B, and Stoorvogel W. Pri-
maquine interferes with membrane recyling from endosomes to
the plasma membrane through a direct interaction with endo-
somes which does not involve neutralisation of endosomal pH
AJP-Cell Physiol • VOL 284 • FEBRUARY 2003 •
nor osmotic swelling of endosomes. Eur J Cell Biol 79: 394–399,
2000.
42. Weinman E, Minkoff C, and Shenolikar S. Signal complex
regulation of renal transport proteins: NHERF and regulation of
NHE3 by PKA. Am J Physiol Renal Physiol 279: F393–F399,
2000.
43. Welch MJ, Tsui LC, Boat TF, and Beaudet AL. Cystic fibro-
sis. In: The Metabolic and Molecular Basis of Inherited Diseases.
New York: McGraw-Hill, 1995, p. 3799–3876.
44. Zeng W, Lee MG, Yan M, Diaz J, Benjamin I, Marino CR,
Kopito R, Freedman S, Cotton C, Muallem S, and Thomas
P. Immuno and functional characterization of CFTR in subman-
dibular and pancreatic acinar and duct cells. Am J Physiol Cell
Physiol 273: C442–C445, 1997.
45. Ziomek CA, Schulman S, and Edidin M. Redistribution of
membrane proteins in isolated mouse intestinal epithelial cells.
J Cell Biol 86: 849–857, 1980.
Downloaded from ajpcell.physiology.org on November 24, 2010
www.ajpcell.org