Department of Physiology, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, Tennessee 37614
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ABSTRACT |
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Wortmannin is a potent inhibitor of
phosphatidylinositol 3-kinase (PI3K) and membrane trafficking in many
cells. To test the hypothesis that cystic fibrosis transmembrane
conductance regulator (CFTR) traffics into and out of the plasma
membrane during cAMP-stimulated epithelial Cl
secretion, we have studied the effects of wortmannin on
forskolin-stimulated Cl
secretion by the human
colonic cell line T84. At the PI3K inhibitory concentration of 100 nM,
wortmannin did not affect significantly forskolin-stimulated
Cl
secretion measured as short-circuit current
(ISC). However, 500 nM wortmannin significantly
inhibited forskolin-stimulated ISC. cAMP activation
of apical membrane CFTR Cl
channels in
-toxin-permeabilized monolayers was not reduced by 500 nM
wortmannin, suggesting that inhibition of other transporters accounts
for the observed reduction in T84 Cl
secretion.
Forskolin inhibits apical endocytosis of horseradish peroxidase (HRP),
but wortmannin did not alter forskolin inhibition of apical HRP
endocytosis. In the absence of forskolin, wortmannin stimulated HRP
endocytosis significantly. We conclude that, in T84 cells, apical fluid
phase endocytosis is not dependent on PI3K activity and that CFTR does
not recycle through a PI3K-dependent and wortmannin-sensitive membrane compartment.
wortmannin; epithelia; short-circuit current; cystic fibrosis transmembrane conductance regulator; endocytosis
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INTRODUCTION |
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CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR
(CFTR) is a cAMP-activated Cl channel that mediates
the Cl
secretory activity of many different
epithelia (19, 25). The rate of epithelial Cl
secretion is determined in part by the gating of apical membrane resident CFTR Cl
channels and may be regulated
additionally by insertion and retrieval of CFTR or other
Cl
channels at the apical membrane. This type of
mechanism is analogous to regulation of gastric acid secretion and
renal water transport by membrane insertion and retrieval of proton
pumps and water channels, respectively (reviewed in Ref. 7). In many
epithelial tissues and cell lines, cAMP stimulation of
Cl
secretion is accompanied by changes in
endocytosis and exocytosis rates. A model of CFTR insertion and
retrieval at the plasma membrane has been proposed to explain the link
between cAMP-stimulated Cl
secretion and altered
plasma membrane turnover (7, 25). This model may not be universal and
its applicability may depend on the precise transport functions of
specific epithelia (20).
Evidence supporting CFTR insertion and retrieval at the apical membrane
comes from studies in human bronchial and shark rectal gland epithelia,
CFTR mRNA-injected Xenopus oocytes, and the cell lines T84,
Madin-Darby canine kidney (MDCK), and A6 (6). The human adenocarcinoma
cell line T84 is often used as a model for studies of colonic fluid
secretion and CFTR function. These cells maintain a high degree of
phenotypic differentiation characteristic of colonic crypt cells,
express high levels of CFTR, and have a large capacity for
cAMP-stimulated epithelial Cl secretion (2, 14). In
quiescent T84 cells, plasma membrane CFTR undergoes continuous
endocytosis and recycles back to the plasma membrane with rapid
kinetics (33). The presence of functional CFTR in clathrin-coated
vesicles and endosomal membranes isolated from T84 cells has been
demonstrated (5, 9). cAMP stimulation of CFTR-catalyzed
Cl
secretion causes a concomitant decrease in fluid
phase and CFTR endocytosis (11, 33) and a doubling of the CFTR content
of T84 apical membranes (39). How CFTR recycles to the plasma membrane following clathrin-mediated endocytosis and the effects of cAMP on CFTR
recycling rates have not been defined. Thus the relative contributions
of decreased endocytosis and potentially increased exocytosis to CFTR
abundance in the apical membrane following cAMP are unknown. However,
it has been shown in T84 cells that cAMP stimulates mucin secretion and
apical secretion of glucosaminoglycans (18, 22). The membrane vesicles
that carry these products may provide a vehicle for CFTR return to the
plasma membrane, or CFTR may recycle in other, yet to be defined,
transport vesicles.
Many membrane proteins cycle between the plasma membrane and
intracellular organelles. Examples include receptor proteins such as
the transferrin, low-density lipoprotein (LDL), and mannose 6-phosphate
(Man-6-P) receptors and transporting proteins such as the
insulin-stimulated glucose transporter GLUT-4, the
Na+-pumping Na+-K+-ATPase, the
Na+/H+ exchanger NHE3, and, in some cells,
CFTR. Extensive studies have shown that, for many of the recycling
membrane proteins, one or more steps in the recycling pathway is
dependent on phosphatidylinositol 3-kinase (PI3K) activity. Wortmannin,
a fungal metabolite, is a potent inhibitor of PI3K activity
[inhibitor constant (Ki) 5 nM], and it blocks the movement of many membrane proteins at some point in the endosome-to-plasma membrane recycling pathway (31,
36, 38). One effect of wortmannin is to reduce the plasma membrane
content of recycling proteins by trapping them in an endosomal
compartment. For instance, wortmannin blocks exocytosis of GLUT-4
without affecting its endocytosis (13). Similarly, wortmannin inhibits
exocytic recycling of transferrin receptors while at the same time
increasing the rate of endocytosis (37). The net effect is a reduction
in the steady-state level of plasma membrane transferrin receptors.
Additionally, wortmannin causes accumulation of NHE3 in an endosomal
compartment in NHE3-transfected Chinese hamster ovary (CHO) cells (27).
Reports from a number of cell systems suggest that wortmannin inhibits
trafficking within the endosomal system at multiple steps. In mouse L
cells, wortmannin has no effect on the internalization of the
Man-6-P receptor or its ligand,
-glucuronidase. However,
wortmannin inhibits recycling of the Man-6- P receptor back to
the trans-Golgi network as well as
-glucuronidase delivery
to lysosomes (25). Thus, while there may be multiple steps in vesicular
trafficking sensitive to wortmannin, a common observation is wortmannin
inhibition of trafficking out of the endosomal compartment (34).
Finally, it is important to keep in mind that wortmannin has additional
affects on vesicular trafficking beyond inhibition of postendosomal
sorting and recycling. Wortmannin is reported to inhibit the
endocytosis of plasma membrane proteins such as
Na+-K+-ATPase
-subunits in
dopamine-stimulated renal proximal tubules (12).
We have tested the hypothesis that CFTR cycles into and out of the
apical plasma membrane through a PI3K-dependent compartment by studying
the effects of wortmannin on cAMP-stimulated Cl
secretion in monolayers of T84 cells. We reasoned that wortmannin might
block plasma membrane insertion of CFTR, analogous to its effects on
insulin-stimulated GLUT-4 exocytosis in adipose and skeletal muscle, or
wortmannin might trap CFTR in endosomes, analogous to its effects on
NHE3 recycling in NHE3-transfected CHO cells. It has already been shown
that T84 cells express a wortmannin-inhibitable PI3K activity, which
can be stimulated by epidermal growth factor (EGF) (40). Wortmannin
inhibits EGF activation of PI3K and blocks EGF regulation of
Ca2+-activated Cl
secretion in T84 cells
(40). However, there have been no reports of wortmannin effects on
cAMP-stimulated Cl
secretion in these cells.
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MATERIALS AND METHODS |
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Materials. Cell culture medium, antibiotics, and newborn calf
serum (NBCS) were from Sigma Chemical (St. Louis, MO). The permeable supports used to culture T84 cells as monolayers for electrophysiology were Millicell-HA from Millipore (Bedford, MA). Nystatin, forskolin, cAMP, horseradish peroxidase (HRP; type II, 200 U/mg protein), and
3,3',5,5'-tetramethylbenzidine (TMB) were from Sigma (St. Louis, MO). Wortmannin, LY-294002, thapsigargin, and EGF were from
Calbiochem (La Jolla, CA). -Toxin was from GIBCO BRL (Grand Island,
NY). All other chemicals and reagents were from Sigma.
Cell culture. The nutrient medium used in this study was DMEM/F-12 supplemented with 15 mM HEPES, 2 mM glutamine, 5% NBCS, 100 U/ml penicillin, and 100 µg/ml streptomycin. For electrophysiology, 5 × 105 T84 cells were seeded into Millicell-HA culture plate inserts (12 mm diameter; 0.6 cm2 surface area). For HRP endocytosis studies, T84 cells were grown in 12-well plates at an initial plating density of 2.5 × 105 cells/well, and cultures were used at confluence. All cultures were fed fresh medium every other day and the day before use.
Monolayer development and integrity in Millicell-HA cultures were
determined by measuring transepithelial electrical resistance (Rte) with the epithelial volt-ohm meter (EVOM)
apparatus (World Precision Instruments, Sarasota, FL). To maintain
sterility before use, EVOM electrodes were soaked in 70% ethanol and
equilibrated in sterile PBS. Maximal Rte
(1,200-2,000 · cm2) developed
between days 7 and 12 of culture.
Transepithelial electrophysiology. In initial experiments, the EVOM apparatus was used to measure the effect of wortmannin on basal Rte. Measurement of short-circuit current (ISC) was conducted as previously described (16). Briefly, Millicells were mounted in water-jacketed Ussing chambers (Jim's Instrument Manufacturing, Iowa City, IA) and bathed in bicarbonate-buffered T84 Ringer solution (in mM: 115 NaCl, 5 KCl, 25 NaHCO3, 1.5 CaCl2, 1.5 MgCl2, and 5 glucose, as well as 0.005% phenol red) with continuous bubbling of 95% O2-5% CO2. The potential difference (PD) across the cell monolayer was measured with calomel electrodes in 3 M KCl and monitored with a Physiologic Instruments (San Diego, CA) current-voltage clamp. Monolayer PD was clamped to 0 mV with Ag-AgCl electrodes connected to the Ussing chamber by agar bridges. At intervals of 20-30 s, a biphasic pulse of 5 mV was applied, and the resulting current deflection was used to calculate monolayer conductance (G) by Ohm's law. All values for ISC, PD, and G were recorded on a Pentium-based microcomputer equipped with a DATAQ data acquisition board and running Acquire and Analyze software (Physiologic Instruments).
Apical plasma membrane Cl channel activity was
measured in
-toxin-permeabilized T84 monolayers. For this, the
basolateral side of Millicell cultures was treated with 500 U/ml of
-toxin for 30 min before being mounted in Ussing chambers. When
mounted in Ussing chambers, monolayers were bathed in Ringer solutions formulated to produce an apical-to-basolateral Cl
gradient [apical Ringer composition (in mM): 135 NaCl, 2.4 K2HPO4, 0.6 KH2PO4, 1.2 CaCl2, 1.2 MgCl2, 10 glucose, and 10 HEPES, as well as 0.005% phenol red; basolateral Ringer composition (in mM): 30 NaCl, 100 sodium gluconate, 2.4 K2HPO4, 0.6 KH2PO4, 0.1 CaCl2, 3 MgCl2, 10 glucose, and 10 HEPES, as well as 0.005% phenol red]. Ringer solution was bubbled continuously with 100%
O2. Preliminary experiments demonstrated that 40 µM cAMP
added to the basolateral hemichamber produced maximal
Cl
current (ICl), which was
insensitive to bumetanide but was blocked by apical diphenylamine
carboxylic acid (DPC).
Basolateral membrane Na+ pump activity was measured in T84
monolayers permeabilized with apical nystatin by methods similar to
those described previously (16). Briefly, monolayers were mounted in
Ussing chambers and bathed in symmetrical HEPES-buffered T84 Ringer (in
mM: 125 NaCl, 5 KCl, 2.4 K2HPO4, 0.6 KH2PO4, 1.5 CaCl2, 1.5 MgCl2, 5 glucose, and 10 HEPES, as well as 0.005% phenol red) and bubbled with 100% O2. Nystatin was diluted in
Ringer and added to the apical hemichamber by solution exchange at a final concentration of 350 µg/ml (0.7% DMSO). Gassing of the apical hemichamber was reduced to minimize frothing. Addition of nystatin produced a rapid increase in current (INa). This
current was completely ( 95%) inhibited by 10
4 M
ouabain added to the basolateral hemichamber, indicating that it
resulted from Na+-K+-ATPase activity.
HRP endocytosis. T84 cells were grown in 12-well plates and used on the day they reached full confluence. Cultures were washed twice with warm (37°C) DMEM/F-12 and cultured for an additional 2 h in DMEM/F-12 that contained inhibitors as indicated. Cells were then cultured with 0.5 mg/ml HRP in DMEM/F-12 + 1% BSA to minimize nonspecific HRP binding. Uptake was terminated by cooling to 4°C and washing three times for 5 min each with DMEM/F-12 + 1% BSA followed by three 5-min washes in DMEM/F-12. Cells were extracted in 1 ml PBS + 0.2% Triton X-100 for 30 min at 4°C with constant gentle agitation on an orbital shaker. HRP activity in 2-µl samples of cell lysate was determined in a microtiter plate assay (3-5 wells/lysate) using TMB as substrate. The reaction was terminated at 5 min by addition of 1 N H2SO4 and the absorbance determined at 450 nM using a Bio-Tek Instruments (Winooski, VT) 312e plate reader controlled by a microcomputer running KinetiCalc software. In preliminary experiments, HRP uptake was linear for at least 2 h, and nonspecific HRP binding (defined as cell-associated HRP activity in cultures maintained at 4°C) was <20% of the activity in vehicle control (0.1% DMSO) cultures maintained at 37°C. The protein concentration of cell lysates was determined by the bichinchoninic assay (Pierce, Rockford, IL) using BSA as standard.
Statistical analysis. Unless noted otherwise, all values are
reported as means ± SE. Statistical significance (P 0.05)
was determined using Student's t-test.
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RESULTS |
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Wortmannin blocks EGF inhibition of thapsigargin-stimulated
Cl secretion.
EGF inhibits thapsigargin-stimulated Cl
secretion by
T84 cells, an effect shown to be due to EGF activation of PI3K (40). Wortmannin (50 nM) blocked the effects of EGF on
thapsigargin-stimulated Cl
secretion (40). We have
replicated these experiments to verify that our T84 cells are sensitive
to wortmannin inhibition of PI3K activity and to verify the
concentration of our wortmannin stock solutions. Figure
1 illustrates our results.
Thapsigargin stimulated ISC (peak
ISC = 8.5 ± 1.4 µA/cm2, n = 6). Within 15 min of administration, 20 nM EGF inhibited the
thapsigargin-stimulated ISC (peak
ISC = 4.2 ± 0.8 µA/cm2, n = 11, P < 0.05). At 50 nM, wortmannin rapidly (within 30 min) and completely blocked the inhibitory effects of EGF on
thapsigargin-stimulated ISC (Fig. 1). In our
experiments, as in those reported by Uribe et al. (40), wortmannin
enhanced the ISC response to thapsigargin even in
the presence of EGF (Fig. 1).
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Low concentrations of wortmannin do not inhibit forskolin-stimulated
Cl secretion.
Addition of up to 500 nM wortmannin to forskolin-stimulated monolayers
did not reduce stimulated ISC (not shown). Acute
addition of up to 500 nM wortmannin to T84 monolayers mounted in Ussing chambers did not have an immediate effect on basal transepithelial resistance or basal ISC values (not shown).
However, beginning within a few minutes of addition, 100 and 500 nM
wortmannin caused a gradual, small, and sustained increase in basal
ISC (Fig. 2). After 30 min in the Ussing chambers, the basal ISC of 0.1%
DMSO control monolayers rose by 0.5 ± 0.1 µA/cm2
(n = 14), while at the same time the ISC in
100 nM wortmannin-treated monolayers rose by 2.6 ± 0.2 µA/cm2 (n = 9, P < 0.01). Currents in
the 500 nM wortmannin-treated monolayers rose to 1.5 ± 0.1 µA/cm2 (n = 10), which was significantly greater
than the DMSO controls (P < 0.01) but significantly less than
those with 100 nM wortmannin (P < 0.01).
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Wortmannin does not inhibit apical membrane cAMP-activated
Cl channels.
In a separate series of experiments we determined the time dependence
for inhibition of forskolin-stimulated ISC at
concentrations of wortmannin >100 nM. Using 200 nM wortmannin we
found that inhibition increased to ~50% after 2 h and remained at
this level for up to an additional 24 h (not shown). Also after 2 h,
the Rte values of 200 nM wortmannin-treated
monolayers were reduced by 30% compared with 0.1% DMSO-treated
monolayers (not shown). We chose to use a 2-h exposure to
wortmannin as an experimental system for investigating the mechanisms
of wortmannin inhibition at concentrations >100 nM.
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High wortmannin inhibits the basolateral membrane
Na+ pump.
We have used the monovalent ionophore antibiotic nystatin to
permeabilize the apical membranes of T84 monolayer cultures and measure
the resulting ouabain-inhibitable Na+ current
(INa). This was done to address whether the
basolateral membrane Na+-pumping
Na+-K+-ATPase is a target for wortmannin.
Permeabilization was carried out in symmetric Ringer solutions to
eliminate current flow through tight junctions or active ion channels
present in basolateral membranes. The magnitude of the
ouabain-inhibitable INa following nystatin
permeabilization was significantly lower in monolayers treated with 500 nM wortmannin (Fig. 4). This result
suggests that inhibition of forskolin-stimulated
ISC by 500 nM wortmannin results, at least in part,
from inhibition of basolateral membrane Na+ pump activity.
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DISCUSSION |
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The data presented in this paper demonstrate that cAMP-stimulated
Cl secretion by T84 cells is independent of
wortmannin- and LY-294002-inhibitable PI3K activity. We have confirmed
a previous report that 50 nM wortmannin completely blocks
EGF-stimulated and PI3K-dependent downregulation of
thapsigargin-stimulated Cl
secretion in T84 cells
(40). In addition, we have shown that a second PI3K inhibitor,
LY-294002, blocked EGF inhibition of thapsigargin-stimulated
Cl
secretion. However, under the same conditions
that these PI3K inhibitors blocked EGF effects, wortmannin and
LY-294002 produced only small and insignificant effects on
forskolin-stimulated Cl
secretion. In a recent
report, EGF was shown to inhibit carbachol-stimulated 86Rb+ efflux but not
125I
efflux from T84 cells (3).
Consistent with its effects on Cl
secretion,
wortmannin blocked the effect of EGF on 86Rb+
efflux without altering 125I
efflux.
These results are consistent with the results presented here, both of
which suggest that wortmannin has no effect on the activity of CFTR or
other anion channels in T84 cells. Using clones of the HT-29 human
colonic cell line, Merlin et al. (28) showed that 100 nM wortmannin had
no effect on forskolin-stimulated Cl
secretion,
although it did inhibit purinergic stimulation of mucin granule fusion
in these cells. These results further support our conclusion that PI3K
activity is not required for cAMP activation of CFTR-mediated
Cl
secretion.
The observation that wortmannin blocks EGF effects on
86Rb+ efflux (40) offers an explanation for
wortmannin and LY-294002 stimulation of basal ISC
and potentiation of thapsigargin-stimulated ISC in
T84 monolayers, as reported here and by Uribe et al. (40). We and
others routinely culture T84 cells in media that contain serum, which
in turn contains EGF and other growth factors that stimulate PI3K
activity. By inhibition of growth factor-stimulated PI3K, wortmannin
might remove a tonic inhibition of basolateral K+ channels,
which would hyperpolarize the cell and increase the driving force for
Cl exit through channels open in the basal state. In
addition, removal of this tonic inhibition would be expected to
increase the sensitivity of basolateral K+ channels to
elevated intracellular Ca2+ following thapsigargin
stimulation, thereby supporting an increased rate of
Cl
secretion.
At higher wortmannin concentrations (200-500 nM) and with
prolonged exposure (2 h), we observed significant inhibition of forskolin-stimulated Cl secretion in intact
monolayers. However, under these conditions there was no difference in
the cAMP-stimulated Cl
conductive properties of T84
monolayer apical membranes. This suggests that the inhibition observed
with high wortmannin concentrations is not due to inhibition of CFTR
function and that it must be due to inhibition of another transporter
contributing to Cl
secretion. Here and in a
preliminary report (15), we have shown that 500 nM wortmannin inhibits
Na+ pump activity in T84 monolayers. Inhibition of the
Na+ pump must account for some of the inhibition of
forskolin-stimulated ISC observed in 500 nM
wortmannin-treated monolayers. In addition, wortmannin above 100 nM is
known to cause inhibition of other enzymes including myosin light chain
kinase and mitogen-activated protein kinase (17, 30). It is likely that
these or other wortmannin-sensitive enzymes are responsible for
inhibition of Cl
secretion, alone or in combination
with PI3K inhibition.
Stimulation of T84 cell Cl secretion is accompanied
by an increase in the apical membrane content of CFTR (33, 39). This may result from increased rates of CFTR exocytosis, although the possibility that it reflects reduced CFTR endocytosis without a change
in CFTR exocytosis has not been fully addressed. However, cAMP does
stimulate the exocytosis of mucin-containing secretory granules,
glucosaminoglycan-containing constitutive secretory vesicles, and
vesicles involved in the recycling of lectin receptors in T84 cells
(10, 18, 22). One or more of these may mediate delivery of CFTR to the
apical membrane. It is unlikely that mucin granules or constitutive
secretory vesicles mediate the return of CFTR to the apical membrane
during its continuous recycling in unstimulated cells, but they may
deliver additional CFTR to the apical membrane during times of
stimulated Cl
secretion. Studies in mucin-secreting
clones of the HT-29 cell line suggest that mucin granules contain
Cl
channels that contribute to Cl
secretion under some conditions but that these are not CFTR
Cl
channels (28). If cAMP does stimulate exocytosis
of CFTR-containing vesicles, whatever their origin, our data suggest
that this process is independent of PI3K activity.
A detailed itinerary for endocytosed CFTR is lacking. We know that
internalization of CFTR in T84 cells occurs by clathrin-mediated endocytosis (8, 9). A putative tyrosine-based internalization sequence,
which targets other recycling proteins to coated pits, has been
identified in the carboxy-terminal tail of CFTR and has been shown to
be required for efficient internalization of CFTR/transferrin receptor
chimeras (32). Consistent with this is the observation that CFTR
expressed by cells of the human airway line Calu-3 is present in
clathrin-coated vesicles but excluded from caveolae (8). How CFTR gets
from clathrin-coated endocytic vesicles back to the plasma membrane is
unknown. It is unlikely that CFTR enters the sorting/recycling endosome
system in T84 cells for two reasons. First, CFTR is endocytosed and
recycled to the apical membrane with a half time of 1-2 min (33).
Although some cells exhibit similarly rapid rates of membrane protein
recycling (23), the recycling of proteins in most cell types is slower,
with reported half times of 10-30 min (29). Second, the amount of
CFTR in the apical membrane, as assayed by Cl
channel function, is unaltered by as much as 500 nM wortmannin for 2 h,
whereas the recycling of membrane proteins in other cells is
significantly reduced within minutes by 50-100 nM wortmannin (13,
26, 27, 34, 37). It may be that, in cell systems exhibiting rapid CFTR
recycling, CFTR endocytic vesicles return and fuse directly with the
plasma membrane and not with early endosomes.
In summary, we show that the functional expression of CFTR
Cl channels in the apical membrane of T84 cells is
insensitive to inhibition of PI3K activity by wortmannin and LY-294002.
The recycling of CFTR in the apical membrane of
Cl
-secreting epithelia is often compared with the
recycling of GLUT-4 and the transferrin receptor (6, 25). Membrane
recycling of these proteins is wortmannin sensitive (13, 37), and so our observations suggest that similarities between CFTR recycling and
GLUT-4 or transferrin receptor recycling are limited. A factor underlying the dissimilarities may be the recycling of CFTR
specifically to the apical membrane. Epithelial apical membranes are
compositionally distinct from basolateral membranes and from the plasma
membranes of nonpolarized cells (adipose, muscle, fibroblasts, etc.).
Delivery of newly synthesized proteins to the apical membrane involves sorting and vesicular trafficking mechanisms that are biochemically distinct from those that govern delivery to the basolateral membrane (24). Similarly, recycling of apical proteins may be governed by
sorting and trafficking mechanisms distinct from those operating at the
basolateral membrane or in nonpolarized cells. In this regard,
wortmannin inhibits transcytosis of the polymeric immunoglobulin receptor (pIgR) from basolateral to apical membrane in MDCK cells but
has no effect on apical recycling of pIgR (21). We suggest that a full
understanding of the molecular regulation and physiological significance of CFTR recycling necessitates development and pursuit of
models of protein recycling specifically at the apical membrane and
less reliance on recycling proteins expressed at the basolateral membrane or in nonpolarized cells.
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ACKNOWLEDGEMENTS |
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We are grateful to Thomas Wiegand for technical assistance and to Drs. R. Wondergem and B. Rowe for critical reading of the manuscript.
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FOOTNOTES |
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This work was supported by a grant from the American Heart Association (TN97N74) to T. W. Ecay.
A portion of this work has been published in abstract form (15).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: T. W. Ecay, Dept. of Physiology, East Tennessee State Univ., PO Box 70576, Johnson City, TN 37614 (E-mail: ecay{at}etsu.edu).
Received 30 August 1999; accepted in final form 15 November 1999.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Anderson, MP,
and
Welsh MJ.
Calcium and cAMP activate different chloride channels in the apical membrane of normal and cystic fibrosis epithelia.
Proc Natl Acad Sci USA
88:
6003-6007,
1991[Abstract].
2.
Barrett, KE.
Integrated regulation of intestinal epithelial transport: intercellular and intracellular pathways.
Am J Physiol Cell Physiol
272:
C1069-C1076,
1997
3.
Barrett, KE,
Smitham J,
Traynor-Kaplan A,
and
Uribe JM.
Inhibition of Ca2+-dependent Cl secretion in T84 cells: membrane target(s) of inhibition is agonist specific.
Am J Physiol Cell Physiol
274:
C958-C965,
1998
4.
Bell, CL,
and
Quinton PM.
Asymmetric permeabilization of an epithelium: a tool to study a single membrane in isolation.
J Tissue Cult Methods
14:
165-171,
1992.
5.
Biwersi, J,
and
Verkman AS.
Functional CFTR in the endosomal compartment of CFTR-expressing fibroblasts and T84 cells.
Am J Physiol Cell Physiol
266:
C149-C156,
1994
6.
Bradbury, NA.
Intracellular CFTR: localization and function.
Physiol Rev
79:
S175-S191,
1999[Medline].
7.
Bradbury, NA,
and
Bridges RJ.
Role of membrane trafficking in plasma membrane solute transport.
Am J Physiol Cell Physiol
267:
C1-C24,
1994
8.
Bradbury, NA,
Clark JA,
Watkins SC,
Widnell CC,
Smith HS, IV,
and
Bridges RJ.
Characterization of the internalization pathways for the cystic fibrosis transmembrane conductance regulator.
Am J Physiol Lung Cell Mol Physiol
276:
L659-L668,
1999
9.
Bradbury, NA,
Cohn JA,
Venglarik CJ,
and
Bridges RJ.
Biochemical and biophysical identification of cystic fibrosis transmembrane conductance regulator chloride channels as components of endocytic clathrin-coated vesicles.
J Biol Chem
269:
8296-8302,
1994
10.
Bradbury, NA,
Jilling T,
Berta G,
Sorcher EJ,
Bridges RJ,
and
Kirk KL.
Regulation of plasma membrane recycling by CFTR.
Science
256:
530-532,
1992[ISI][Medline].
11.
Bradbury, NA,
Jilling T,
Kirk KL,
and
Bridges RJ.
Regulated endocytosis in a chloride secretory epithelial cell line.
Am J Physiol Cell Physiol
262:
C752-C759,
1992
12.
Chibalin, AV,
Zierath JR,
Katz AI,
Berggren P-O,
and
Bertorello AM.
Phosphatidylinositol 3-kinase-mediated endocytosis of renal Na+,K+-ATPase subunit in response to dopamine.
Mol Biol Cell
9:
1209-1220,
1998
13.
Clarke, JF,
Young PW,
Yonezawa K,
Kasuga M,
and
Holman GD.
Inhibition of the translocation of GLUT1 and GLUT4 in 3T3-L1 cells by the phosphatidylinositol 3-kinase inhibitor, wortmannin.
Biochem J
300:
631-635,
1994[ISI][Medline].
14.
Dharmsathaphorn, K,
and
Madara JL.
Established intestinal cell lines as model systems for electrolyte transport studies.
Methods Enzymol
192:
354-389,
1990[Medline].
15.
Dickson, JL,
Conner TD,
and
Ecay TW.
Wortmannin inhibition of epithelial chloride secretion (Abstract).
Mol Biol Cell
9:
120,
1998.
16.
Ecay, TW,
and
Valentich JD.
Lovastatin inhibits cAMP- and calcium-stimulated chloride secretion by T84 cells.
Am J Physiol Cell Physiol
265:
C422-C433,
1993
17.
Ferby, IM,
Waga I,
Hoshino M,
Kume K,
and
Shimizu T.
Wortmannin inhibits mitogen-activated protein kinase activation by platelet-activation factor through a mechanism independent of p85/p110-type phosphatidylinositol 3 kinase.
J Biol Chem
271:
11684-11688,
1996
18.
Forstner, G,
Zhang Y,
McCool D,
and
Forstner J.
Regulation of mucin secretion in T84 adenocarcinoma cells by forskolin: relationship to Ca2+ and PKC.
Am J Physiol Gastrointest Liver Physiol
266:
G606-G612,
1994
19.
Grubb, BR,
and
Boucher RC.
Pathophysiology of gene-targeted mouse models for cystic fibrosis.
Physiol Rev
79:
S193-S214,
1999[Medline].
20.
Guggino, WB.
Focus on "Exocytosis is not involved in activation of Cl secretion via CFTR in Calu-3 airway epithelial cells."
Am J Physiol Cell Physiol
275:
C911-C912,
1998
21.
Hansen, SH,
Olsson A,
and
Casanova JE.
Wortmannin, an inhibitor of phosphoinositide 3-kinase, inhibits transcytosis in polarized epithelial cells.
J Biol Chem
270:
28425-28432,
1995
22.
Huflejt, ME,
Blum RA,
Miller SG,
Moore H-P H,
and
Machen TE.
Regulated Cl transport, K and Cl permeability, and exocytosis in T84 cells.
J Clin Invest
93:
1900-1910,
1994[ISI][Medline].
23.
Iacopetta, BJ,
and
Morgan EH.
The kinetics of transferrin endocytosis and iron uptake from transferrin in rabbit reticulocytes.
J Biol Chem
258:
9108-9115,
1983
24.
Ikonen, E,
and
Simons K.
Protein and lipid sorting from the trans-Golgi network to the plasma membrane in polarized cells.
Semin Cell Dev Biol
9:
503-509,
1998[ISI][Medline].
25.
Jilling, T,
and
Kirk KL.
The biogenesis, traffic, and function of the cystic fibrosis transmembrane conductance regulator.
Int Rev Cytol
172:
193-241,
1997[ISI][Medline].
26.
Kundra, R,
and
Kornfeld S.
Wortmannin retards the movement of the mannose 6-phosphate/insulin-like growth factor II receptor and its ligand out of endosomes.
J Biol Chem
273:
3848-3853,
1998
27.
Kurashima, K,
Szabo EZ,
Lukacs G,
Orlowski J,
and
Grinstein S.
Endosomal recycling of the Na+/H+ exchanger NHE3 isoform is regulated by the phosphatidylinositol 3-kinase pathway.
J Biol Chem
273:
20828-20836,
1998
28.
Merlin, D,
Guo X,
Martin K,
Laboisse C,
Landis D,
Dubyak G,
and
Hopfer U.
Recruitment of purinergically stimulated Cl channels from granule membrane to plasma membrane.
Am J Physiol Cell Physiol
271:
C612-C619,
1996
29.
Murkherjee, S,
Ghosh RN,
and
Maxfield FR.
Endocytosis.
Physiol Rev
77:
759-803,
1997
30.
Nakanishi, S,
Catt KJ,
and
Balla T.
Inhibition of agonist-stimulated inositol 1,4,5-trisphosphate production and calcium signaling by the myosin light chain kinase inhibitor, wortmannin.
J Biol Chem
269:
6528-6535,
1994
31.
Powis, G,
Bonjouklian R,
Berggren MM,
Gallegos A,
Abraham R,
Ashendel C,
Zalkow L,
Matter WF,
Dodge J,
Grindey G,
and
Vlahos CJ.
Wortmannin, a potent and selective inhibitor of phosphatidylinositol-3-kinase.
Cancer Res
54:
2419-2423,
1994[Abstract].
32.
Prince, LS,
Peter K,
Hatton SR,
Zaliauskiene L,
Cotlin LF,
Clancy JP,
Marchase RB,
and
Collawn JF.
Efficient endocytosis of the cystic fibrosis transmembrane conductance regulator requires a tyrosine-based signal.
J Biol Chem
274:
3602-3609,
1998
33.
Prince, LS,
Workman RB, Jr,
and
Marchase RB.
Rapid endocytosis of the cystic fibrosis transmembrane conductance regulator chloride channel.
Proc Natl Acad Sci USA
91:
5192-5196,
1994[Abstract].
34.
Reaves, BJ,
Bright NA,
Mullock BM,
and
Luzio JP.
The effect of wortmannin on the localization of lysosomal type I integral membrane glycoproteins suggests a role for phosphoinositide 3-kinase activity in regulating membrane traffic late in the endocytic pathway.
J Cell Sci
109:
749-762,
1996
35.
Santos, GF,
and
Reenstra WW.
Activation of the cystic fibrosis transmembrane regulator by cyclic AMP is not correlated with inhibition of endocytosis.
Biochim Biophys Acta
1195:
96-102,
1994[ISI][Medline].
36.
Shepherd, PR,
Reaves BJ,
and
Davidson HW.
Phosphoinositide 3-kinase and membrane traffic.
Trends Cell Biol
6:
92-97,
1996[ISI].
37.
Spiro, DJ,
Boll W,
Kirchhausen T,
and
Wessling-Resnick M.
Wortmannin alters the transferrin receptor endocytic pathway in vivo and in vitro.
Mol Biol Cell
7:
355-367,
1996[Abstract].
38.
Toker, A,
and
Cantley LC.
Signalling through the lipid products of phosphoinositide-3-OH kinase.
Nature
387:
673-676,
1997[ISI][Medline].
39.
Toussan, A,
Fuller CM,
and
Benos DJ.
Apical recruitment of CFTR in T84 cells is dependent on cAMP and microtubules but not Ca2+ or microfilaments.
J Cell Sci
109:
1325-1334,
1996
40.
Uribe, JM,
Keely SJ,
Traynor-Kaplan AE,
and
Barrett KE.
Phosphatidylinolitol-3-kinase mediates the inhibitory effect of epidermal growth factor on calcium-dependent chloride secretion.
J Biol Chem
271:
26588-26595,
1996
41.
Vlahos, CJ,
Matter WF,
Hui KY,
and
Brown RF.
A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002).
J Biol Chem
269:
5241-5248,
1994