Distinct Ca2+- and
cAMP-dependent anion conductances in the apical membrane of
polarized T84 cells
Didier
Merlin1,4,
Lianwei
Jiang2,4,
Gregg R.
Strohmeier1,4,
Asma
Nusrat1,4,
Seth L.
Alper2,4,
Wayne I.
Lencer3,4, and
James L.
Madara1,4
1 Division of Gastrointestinal
Pathology, Department of Pathology, Brigham and Women's Hospital,
2 Molecular Medicine and Renal
Units, Beth Israel Deaconess Medical Center,
3 Combined Program in Pediatric
Gastroenterology and Nutrition, Department of Medicine, Children's
Hospital Medical Center, 4 Harvard Medical
School and Harvard Digestive Diseases Center, Boston, Massachusetts
02115
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ABSTRACT |
Monolayers of the human colonic epithelial cell line T84 exhibit
electrogenic Cl
secretion
in response to the Ca2+ agonist
thapsigargin and to the cAMP agonist forskolin. To evaluate directly
the regulation of apical Cl
conductance by these two agonists, we have utilized amphotericin B to
permeabilize selectively the basolateral membranes of T84 cell
monolayers. We find that apical anion conductance is stimulated by both
forskolin and thapsigargin but that these conductances are
differentially sensitive to the anion channel blocker DIDS. DIDS
inhibits thapsigargin-stimulated responses completely but forskolin
responses only partially. Furthermore, the apical membrane anion
conductances elicited by these two agonists differ in anion selectivity
(for thapsigargin, I
> Cl
; for forskolin,
Cl
> I
). However, the
DIDS-sensitive component of the forskolin-induced conductance response
exhibits anion selectivity similar to that induced by thapsigargin
(I
> Cl
). Thus
forskolin-induced apical anion conductance comprises at least two
components, one of which has features in common with that elicited by
thapsigargin.
intestinal epithelium; thapsigargin; forskolin; chloride channels; amphotericin B; human intestinal cell line T84
 |
INTRODUCTION |
CHLORIDE SECRETION in the intestine requires the
activation of specific ion transporters and channels located in apical
and basolateral membranes of polarized enterocytes. Basolateral
transporters, which include the
Na+-K+-ATPase,
the Na+-K+-2Cl
cotransporter,
and K+ channels, act coordinately
to elevate intracellular Cl
concentration to levels above its electrochemical equilibrium potential, so that enterocytes are primed to secrete
Cl
through apical anion
channels. Agonists that elicit elevations in intracellular
concentrations of Ca2+ and cAMP
regulate the activities of these transporters and channels and thus
regulate secretion (7, 17, 28, 38, 39).
Established human intestinal cell lines, such as T84, maintain a
secretory phenotype and have been used widely to examine the physiology
and regulation of intestinal
Cl
secretion (14).
Activation of apical Cl
conductances by cAMP agonists in intact T84 cell monolayers has been
well documented. The Cl
effluxes induced by cAMP are presumed to represent
Cl
transport through the
cystic fibrosis transmembrane conductance regulator (CFTR) (1). CFTR is
highly expressed in T84 cells (18). Activation of
Cl
secretion by
Ca2+-dependent agonists in T84
cells has also been well studied (3-5, 24), but the molecular
identity of the channel or channels mediating this response remains
undefined (1, 2, 9, 22).
Two biophysically distinct anion conductances have been described in
nonpolarized human T84 and parental HT-29 cell lines. Whole cell
voltage-clamp experiments have shown that isolated T84 cells grown on
glass coverslips contain
Ca2+-stimulated
Cl
currents that exhibit
outward rectification. cAMP-induced currents from the same cells
displayed linear current-voltage relationships (9, 32). Ion
selectivities of the
Ca2+-stimulated
Cl
currents in T84 cells
differed from the ion selectivities exhibited by channels activated by
cAMP (1). Experments using
125I
to trace Cl
secretion
similarly indicated that cAMP- and
Ca2+-induced
Cl
effluxes were mediated
by two separate pathways. Experimental measurements of
Cl
transport in
well-differentiated, polarized T84 and HT-29cl.19A cells grown on
permeable supports estimated by the
Cl
-sensitive fluorophore,
6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ) (22), microelectrodes (2), or
125I
efflux (16) also support the conclusion that T84 cells express Ca2+-regulated
Cl
channels that differ
from Cl
channels regulated
by cAMP.
Not all studies, however, have found evidence for the expression of
Ca2+-sensitive apical membrane
Cl
channels on
well-differentiated polarized HT-29cl.19A or T84 cells. For example,
when basolateral membranes of HT-29cl.19A or T84 cells were
permeabilized selectively by nystatin to examine ion conductances
across the apical membrane, no evidence for
Ca2+-stimulated
Cl
conductance was found
(1, 12, 34). These authors concluded that
Ca2+-stimulated
Cl
conductances, although
present on nonpolarized cells, were not expressed on apical membranes
of well-differentiated and polarized monolayers (1). However, another
study using this same approach found an increase of apical
Cl
conductance by
carbachol, suggesting that
Ca2+-sensitive
Cl
channels may be present
on apical membranes of T84 cells (22). Thus the presence of
Ca2+-sensitive
Cl
channels in polarized
colonic epithelial monolayer cultures has remained controversial.
Our aim in the present study was to reexamine the mechanisms of
conductive Cl
transport
across apical membranes of polarized T84 cells. For these studies, we
selectively permeabilized basolateral membranes of confluent monolayers
grown on permeable supports with the ionophore amphotericin B (25).
This allowed estimation of apical membrane conductances from
measurements of transepithelial conductance. Our data show that
well-differentiated human intestinal T84 cells express at least two
distinct apical membrane Cl
conductances. Although one can be activated only by the
Ca2+ agonist thapsigargin, both
appear to be activated by the cAMP agonist forskolin.
 |
METHODS |
Cell culture.
T84 cells (American Type Culture Collection, passages
45-98), a human colonic carcinoma cell line that
functionally and morphologically resembles crypt intestinal epithelia,
were grown as confluent monolayers in a 1:1 mixture of Dulbecco's Vogt
modified Eagle's medium and Ham's F-12 medium supplemented with 15 mM
HEPES buffer (pH 7.5), 14 mM
NaHCO3, 40 µg/ml penicillin, 90 µg/ml streptomycin, and 5% newborn calf serum. Monolayers were
subcultured every 7 days by trypsinization with 0.1% trypsin and 0.9 mM EDTA in Ca2+- and
Mg2+-free PBS and grown on
collagen-coated permeable supports (area 0.3 cm2, pore size 0.4 µm). All
experiments described in this study were performed on cells between
passages 65 and
92.
Electrophysiology.
Studies were carried out at either 37 or 4°C with confluent
monolayers plated on collagen-coated permeable supports (14) and
examined 7-16 days postplating as previously described (38). Before all studies, inserts were washed with
HCO
3-free medium (see Table
1) warmed to 37°C or cooled to 4°C
and transferred to new 24-well tissue culture plates containing the
experimental medium. To determine currents, transepithelial potentials,
and conductances, a commercial voltage clamp (Bioengineering Dept., University of Iowa, Iowa City, IA) was interfaced with equilibrated pairs of calomel electrodes submerged in saturated KCl and with paired
Ag-AgCl electrodes submerged in the experimental medium. Before each
experiment, a blank filter was used to compensate for both the fluid
resistance and resistance of the filter. In some experiments,
transepithelial voltage and short-circuit current (Isc) were
continuously recorded with the aid of an analog-to-digital converter
(MacLab, World Precision Instruments) and a microcomputer.
Measurement of Cl
conductance of
apical plasma membrane.
To evaluate the ion conductance of the isolated apical plasma membrane,
the polyene ionophore amphotericin B (25, 30, 31, 36) was added to the
basolateral solution at a concentration of 100 µM, the lowest
concentration that gave a maximal change in steady-state current as
determined in preliminary experiments. Only the plasma membrane facing
the amphotericin-containing solution (basolateral membrane in this
case) incorporates this ionophore, thus electrically isolating the
opposing (apical) plasma membrane. To study the
Cl
conductance of the
apical plasma membrane so isolated, the voltage across the monolayer
was then sequentially stepped from a holding voltage of 0 mV to values
between
80 and +80 mV over a period of ~20 s. The protocol was
performed before and 5 min after addition of forskolin and
thapsigargin. The compositions of all solutions used in these
experiments are shown in Table 1. Positive currents correspond to anion
secretion and/or cation absorption.
cAMP measurement.
Forskolin (100 µM) or thapsigargin (10 µM) was added to
basolaterally permeabilized monolayers at 4 or 37°C. Seven minutes after stimulation, monolayers (surface area 5 cm2) were removed from their
reservoirs and immersed in Hanks' balanced salt solution
(HBSS+) at 4°C to terminate
the reaction. The monolayers were rapidly cut from their plastic
supports and placed in Microfuge tubes containing extraction buffer
[66% ethanol, 33% HBSS+,
and 1 mM phosphodiesterase inhibitor IBMX (Sigma)] at 4°C.
Monolayers were compacted and extracts cleared of cellular debris by
centrifugation. An aliquot (100 µl) of cleared cell extract was
withdrawn for cAMP RIA (Du Pont-NEN) as previously described (41).
Cytosolic
Ca2+
measurement.
T84 cells cultured at subconfluent density on prepared collagen-coated
5-cm2 glass coverslips (200 µl
of collagen/glass coverslip) were loaded with the fluorescent
Ca2+ indicator dye fura 2 by
incubation with 5 µM of its AM derivative, fura 2-AM, in
HBSS+ at 37°C for 60 min.
Extracellular fura 2-AM was removed by washing twice with normal medium
(see Table 1). The monolayer was then mounted on a modified Leiden
chamber in which the coverslip constituted the bottom. Before the start
of the Ca2+ measurements, the
coverslip mounted in the Leiden chamber was maintained at either 37 or
0°C. To the chamber was added 1 ml of buffer solution at either 37 or 0°C. The unclamped temperature decreased ~4°C from
37°C and increased 4°C from 0°C during the course of each
experiment.
Intracellular free Ca2+
concentration
([Ca2+]i)
was measured by fluoresence ratio imaging with an Image-1 digital ratio
imaging system (Universal Imaging, West Chester, PA) equipped with an Olympus IMT-2 inverted microscope, a Dage-MTI CCD7 series videocamera, a Genisys image intensifier, a Pinnacle REO-650 optical disk drive, and
a color video monitor-printer, as described previously (23). Fura 2 fluorescence images were monitored at 510-nm emission with alternating
excitation at 340 and 380 nm. Fura 2-loaded T84 cells were selected by
individual cell fluorescence images and were well defined for a region
within each cell. Ca2+ response
was observed in cells near the center of confluent "islands." Images and 340-to-380-nm ratio values calculated on a pixel-by-pixel basis were collected for data processing. With the same experimental settings for the imaging system, fura 2 ratio values can be calibrated in vitro (19) to Ca2+
concentration. Fura 2 free acid (2 µM) was dissolved in
Ca2+-free HEPES buffer solution
[110 mM KCl, 10 mM NaCl, 1 mM
MgCl2, 25 mM HEPES, and 1.5 mM
EGTA (pH 7.0)], and variable total
Ca2+ was added in quantities
calculated to yield free
[Ca2+] in the range of
36 nM to 40 µM. The minimal fluorescence ratio (Rmin) was determined at zero
Ca2+ (free
Ca2+ < 10 nM), and the maximal
fluorescence ratio (Rmax) at 4 mM total Ca2+. The equilibrium
constant (Kb)
was determined by fitting experimental fluorescence ratio (R) values at
various free Ca2+ concentrations
by using the following equation (19)
where
the factor
Sf2/Sb2
corrects for fura 2 ion sensitivity at 380 nm.
Multipoint in situ calibration could not be completed because of loss
of tight junctional integrity and cell adhesion to substratum during
the period of cell equilibration in
ionophore-Ca2+-EGTA solutions of
low free [Ca2+].
Consequently, reported measurements of
[Ca2+]i
in T84 cells are based on in vitro calibration of the fura 2 fluorescence ratio. However, in situ fluorescence ratio calibration on
intact adherent cells was successfully performed at nominal [Ca]i values of 100 and 200 nM. The ratios measured in intact cells at these two
concentrations did not differ significantly from those determined by
the in vitro calibration procedure at the same values of free
[Ca2+]
(P > 0.2).
 |
RESULTS |
DIDS sensitivity of thapsigargin- and forskolin-stimulated
Isc.
In intact monolayers, 10 µM thapsigargin added to the basolateral
reservoir increases the transepithelial
Isc and the
conductance by 12.6 ± 1.3 µA/cm2 and 2.8 ± 0.6 mS/cm2
(n = 8), respectively (Fig.
1). As reported in previous studies, the
stimulation of adenylate cyclase by forskolin increases
Isc and
conductance to a greater degree 48.4 ± 5.7 µA/cm2 and 6.1 ± 0.35 mS/cm2
(n = 8), respectively (Fig. 1). These
data document stimulation of
Isc consistent
with electrogenic Cl
secretion but do not discriminate between the apical
Cl
efflux pathways
activated by thapsigargin on one hand and forskolin on the other.

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Fig. 1.
Transepithelial Cl
secretion was activated by addition of thapsigargin or forskolin. Cells
were grown to confluence on collagen-coated permeable supports, with
conductances of <1 mS/cm2.
Short-circuit current
(Isc) was
measured under voltage clamp at 0 mV (see
METHODS). Arrows indicate addition
of 10 µM thapsigargin ( ) or 100 µM forskolin ( ) to
basolateral compartment. Note that subsequent addition of 100 µM DIDS
to apical compartment reduced by ~40% forskolin-dependent
Isc ( ) and by
100% thapsigargin-dependent
Isc ( ). Data
are means of 8 experiments, each performed in triplicate.
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Our first approach to distinguish the possible presence of two
different channels was to use the
Cl
channel inhibitor DIDS.
This Cl
channel inhibitor
does not affect the function of the CFTR (27). As shown in Fig. 1, DIDS
(100 µM added to apical solution) inhibited by nearly 100% the
Cl
secretory response
induced by thapsigargin (Fig. 1, peak
Isc was 13.6 ± 0.8 and 0.4 ± 0.5 before and after addition of DIDS respectively, n = 8). In contrast,
DIDS at 100 µM and up to 1 mM (data not shown) reduced by only 40%
the Cl
secretion induced by
forskolin (Fig. 1, peak
Isc was 41.5 ± 6.5 and 25.4 ± 1.7 µA/cm2 before and after addition
of DIDS respectively, n = 8). These data indicate that Cl
secretions stimulated by thapsigargin or forskolin exhibit different sensitivities to DIDS.
Basolateral permeabilization does not affect integrity of apical
plasma membrane and leaves tight junctions intact.
To directly measure conductance changes that occur in the apical plasma
membrane, we used amphotericin B to permeabilize selectively basolateral membranes. Only monovalent ions traverse the amphotericin pore. Because of its requirement for cholesterol, amphotericin permeabilizes only the plasma membrane domain in direct contact with
the amphotericin B solution as previously demonstrated (25). Thus this
procedure leaves the apical membrane and tight junctions intact as
judged by measurement of transepithelial conductances (before
amphotericin B addition,
Gintact = 0.583 ± 0.032 vs. after amphotericin addition,
Gapical= 2.1 ± 0.6 mS/cm2,
n = 20; see Fig.
2). The observed increase in conductance
reflects removal of electrical resistance due to the basolateral
membrane and provides indirect evidence that apical membranes and
intercellular tight junctions remain intact. As previously reported
(30, 31, 36), changes in Cl
conductances of luminal plasma membranes under these conditions are
reflected as changes in
Isc when an
electrochemical gradient is established to drive
Cl
across the permeabilized
monolayer.

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Fig. 2.
Effect of amphotericin B treatment on intact T84 conductances and
effect of thapsigargin and forskolin (+, present; , absent) on
basolaterally permeabilized T84 conductances;
n = 20-30 monolayers for each
condition.
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Several tests were carried out to ensure that basolateral plasma
membranes were sufficiently permeabilized by amphotericin B. In these
studies, both intact and basolaterally permeabilized monolayers were
exposed to an apically directed
Cl
gradient
(buffer A in basolateral and
buffer B in apical reservoirs, Table
1) and voltage clamped at +10 mV. In a first approach, we utilized
bumetanide to inhibit basolateral membrane
Na+-K+-2Cl
transporters, which represent the rate-limiting step for
Cl
uptake in intact T84
cell monolayers. Bumetanide applied to intact monolayers (10 µM)
inhibited Cl
currents
(Isc) induced
by forskolin or thapsigargin by 55 and 65%, respectively. In contrast,
when applied to monolayers basolaterally permeabilized by amphotericin
B, bumetanide had no effect on
Isc (Table
2). These data show that
Cl
uptake via
Na+-K+-2Cl
transporters in permeabilized monolayers was no longer rate limiting. In a second approach, we utilized
Ba2+ (2 mM) or charybdotoxin to
inhibit basolateral K+ channels.
Basolateral K+ channels are
required to establish and maintain membrane potential in
Cl
secretory epithelia at
rest and during the secretory response (36). Although both reagents
inhibited cAMP- and Ca2+-induced
Isc in intact
monolayers (49% inhibition of forskolin-stimulated Isc by
Ba2+ and 46% inhibition of
thapsigargin-stimulated
Isc by
charybdotoxin), neither of these reagents inhibited
Isc in
basolaterally permeabilized monolayers (Table 2). These data show that
selective permeabilization of basolateral membranes with amphotericin B
effectively removed any rate-limiting contribution of basolateral
membrane K+ transport to the
secretory response. Finally, in separate studies, we have recently
shown that the K+ channel blocker
clotrimazole also had no effect on apical membrane Cl
conductances in
basolaterally permeabilized monolayers (36). In T84 cells, clotrimazole
blocks both K+ channels activated
by cAMP and those activated by
Ca2+ (13, 35, 36). Taken together,
these data provide direct evidence that basolateral membranes are
rendered freely permeant to monovalent ions by treatment with
amphotericin B. Thus currents recorded in monolayers treated with
basolaterally applied amphotericin B represent the sum of apical
membrane conductance and the unknown contribution of conductance
through intact tight junctions.
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Table 2.
Paired measurements in absence and presence of each inhibitor were
performed in intact basolaterally permeabilized monolayers
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Forskolin but not thapsigargin activates an apical conductance at
0-mV membrane potential.
We next examined the effects of forskolin and thapsigargin on apical
membrane Cl
currents in
permeabilized monolayers. After permeabilization of basolateral
membranes with amphotericin B, a
Cl
gradient was established
across the electrically isolated apical membrane by placing "normal
Cl
medium"
(buffer A) in the basolateral
reservoir and "low Cl
medium" (buffer B) in the apical
reservoir (Na+ present in
buffers A and
B at 137 mM). These conditions are
essentially identical to those established by Anderson and Welsh (1).
When the monolayer was voltage clamped at 0 mV, thapsigargin did not elicit a detectable increase in apical
Cl
conductance. In
contrast, forskolin stimulated a sizable
Cl
current under these same
conditions (Fig.
3A).
These data reproduce the results of previous studies and show
that, at 0-mV membrane potential, only forskolin stimulates a
Cl
conductance. However,
when the same experiment was performed at +15 mV potential (inside
negative), both thapsigargin and forskolin elicited a clear increase in
apical anion conductance (Fig. 3, B
and C). These data suggest that,
under appropriate conditions, apical membranes of T84 cells may exhibit
both cAMP- and
Ca2+-sensitive
Cl
conductances.

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Fig. 3.
A: at 0-mV transmembrane potential
(V = 0 mV), forskolin but not
thapsigargin stimulates
Isc across apical
plasma membrane. A defined basolateral-to-lumen
Cl gradient was established
across monolayer by substitution of Na-gluconate for NaCl in solution
bathing luminal side and addition of 100 µM amphotericin B to
basolateral solution (normal
Cl medium). First
thapsigargin (10 µM) and later forskolin (100 µM) were added to
basolateral solution. Under same conditions but at +15 mV transmembrane
potential (V = 15 mV), both
10 µM thapsigargin (B) and 100 µM forskolin (C) stimulate
Isc. Each tracing
is representative of results for 3 experiments with 4 different filters
in each condition.
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Thapsigargin and forskolin activate apical
Cl
conductances.
To test this hypothesis, the basolaterally permeabilized T84 monolayers
were studied in symmetrical buffers containing
Cl
as the major permeant
ion; monovalent cations were in the millimolar range
(buffer E, Table 1). Figure
4,
A and
B, shows that, under symmetrical
Cl
concentrations, the
relationships between Cl
current induced by either thapsigargin or forskolin were linear. Currents induced by forskolin were much larger than currents induced by
thapsigargin. Monolayer conductances induced by thapsigargin were 1 ± 0.2 mS/cm2 and by forskolin
3 ± 0.4 mS/cm2 at
+40 mV (mean of 20 independent experiments, see Fig. 2). Same results have been found in buffers containing
Cl
as the sole permeant ion
(choline chloride, 143 mM; CaSO4,
1.25 mM; thapsigargin and forskolin stimulated a
Cl
current under these
conditions, 30 and 100 µA/cm2 at
+40 mV, respectively). To verify that the thapsigargin- and forskolin-induced currents were specific to
Cl
transport,
Cl
was replaced by
gluconate in the continued presence of low
Na+ (buffer
F, Table 1). Under these conditions, the currents
normally stimulated by thapsigargin and forskolin were inhibited by
nearly 100% (Fig. 4, A and
B). These experiments suggest that
thapsigargin and forskolin stimulate a
Cl
current.

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Fig. 4.
Current-voltage relationships in basolaterally permeabilized monolayers
of T84 treated with thapsigargin (A)
or forskolin (B).
Basolateral-to-apical reservoir
Cl concentration ratios (in
mM) were 143.50/143.50 mM
Cl ,
Na+ present in both sides at 0.5 mM (thapsigargin, A, ; forskolin,
B, ); with presence of a
Cl gradient between
basolateral (143.50 mM
Cl ) and apical
compartment (6.61 mM Cl ),
Na+ present in both sides at 137 mM (thapsigargin, A, ; forskolin,
B, ); with a low
Cl concentration in both
apical and cytosol basolateral compartment (6.61 mM
Cl ),
Na+ present in both sides at 0.5 mM (thapsigargin, A, ×;
forskolin, B, ×). With presence
of a Cl gradient between
basolateral (143 mM Cl )
and apical compartment (43 mM), in absence of
Na+ in both sides (thapsigargin,
C, ; forskolin,
C, ).
Ordinates give difference current
between stimulated and unstimulated cells. Tracings are representative
of results from 10 different filters for each condition.
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When shifted from symmetrical to asymmetrical
Cl
solutions (see Table 1,
buffers A and
B;
Na+ present at 137 mM), apparent
reversal potentials
(Vrev) for
agonist-induced currents shifted negatively as predicted but by
different magnitudes [by 34 ± 5.8 mV
(n = 12) for forskolin-stimulated
currents (Fig. 4B, open circles), but
only by 1.2 ± 0.6 mV (n = 10) for
thapsigargin-stimulated currents (Fig.
4A, open squares)]. The observed
reversal potentials do not approximate theoretical values in this model
system, suggesting that a
Cl
conductance in the
apical plasma membrane is shunted by one or more pathways (tight
junction and/or apical plasma membrane with nonspecific
permeability). We examined whether the postulated anion channels had
different relative gluconate conductances. We then studied monolayers
exposed to symmetrical (apical and basolateral) buffers containing
gluconate as the major charge-carrying anion [143 mM
N-methyl-D-glucammonium
(NMDG) gluconate; 1.25 mM CaSO4
both sides]. We found that, under these conditions, thapsigargin and forskolin had no effect on gluconate currents (results not shown).
Thus neither the forskolin- nor thapsigargin-induced currents represent
gluconate transport. However, in symmetrical buffers containing
Na+ as the major permeant cation
(Na+ 143 mM; 1.25 mM
CaSO4 both sides), thapsigargin
and forskolin stimulated a sizable
Na+ current under these conditions
[8.2 and 14.2 µA/cm2
(n = 2) at +40 mV,
respectively]. Because we still do not know whether the
Na+ current is paracellular or
transcellular, it will be the focus of further study. We then studied
monolayers bathed by asymmetrical Cl
solutions in absence of
monvalent cations (apical: 43 mM choline chloride, 100 mM NMDG
gluconate, 1.25 mM CaCl2;
basolateral: 143 mM choline chloride, 1.25 mM
CaCl2);
Vrev for agonist-induced currents shifted
negatively as predicted but by different magnitudes [by 20 ± 5.2 mV (n = 5) for
forskolin-stimulated currents and by 10 ± 2 mV
(n = 5) for thapsigargin-stimulated
currents (Fig. 4C, open circles, for
forskolin; Fig. 4C, open squares, for
thapsigargin)]. The observed
Vrev levels
approximate theoretical values in this model system and demonstrate
that thapsigargin and forskolin stimulate a
Cl
conductance in the
apical plasma membrane.
Together, these results raise the possibility that these macroscopic
currents found under Cl
gradient conditions and in presence of monovalent cations (thapsigargin and forskolin dependent) could be the sum of both the cation and Cl
currents
(Na+ current < Cl
current), explaining our
observed Vrev
under these last conditions.
Thapsigargin- and forskolin-activated anion conductances display
different ion selectivities.
The anion selectivities of apical membrane conductances induced by
thapsigargin or forskolin were measured directly in basolaterally permeabilized monolayers. Current-voltage relationships were examined in asymmetrical buffers containing either
I
or
Cl
as the major permeant
anion (buffers A and
B or
C and
D,
Na+ present in
buffers A-D at 137 mM, Table 1).
Figure 5 shows that thapsigargin stimulated a greater increase in
Isc (anion
conductance) in buffers containing
I
[Gapical = 2.4 ± 0.3 mS/cm2
(n = 6); Fig.
5A]
than in those containing Cl
[Gapical = 0.8 ± 0.2 mS/cm2
(n = 6); Fig.
5A]. In contrast, forskolin
induced a greater increase in
Isc when buffers
contained Cl
rather than
I
as the major permeant ion
[Cl
:
Gapical = 6 ± 0.9 mS/cm2;
I
: 3.3 ± 1.0 mS/cm2
(n = 6)]. Both
Cl
and
I
currents, whether induced
by thapsigargin or forskolin, were linear (Fig. 5,
A and
B).

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Fig. 5.
Effects of thapsigargin (A) and
forskolin (B) on currents in
basolaterally permeabilized T84 monolayers in presence of iodide
solutions. Cytosol basolateral-to-apical reservoir
Cl concentration ratio was
143.50/6.6.0 mM
Cl ,
Na+ present in both sides at 137 mM (thapsigargin, ; forskolin, ). Cytosol basolateral-to-apical
reservoir I concentration
ratio for thapsigargin and forskolin was 136.89/6.60 mM
I ,
Na+ present in both sides at 137 mM (thapsigargin, ; forskolin, ).
Ordinates show difference current
between stimulated and unstimulated cells. Tracings are representative
of results from 6 different filters for each condition.
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From the directions of the shifts in apparent reversal potentials, it
can be deduced that thapsigargin stimulates an anion conductance which
displays a greater permeability for
I
than for
Cl
[I
:
Vrev =
6 ± 0.4 mV vs. Cl
:
electrode reversal potential
(Erev) =
2 ± 0.6 mV (n = 6)]
and that forskolin stimulates a conductance which displays greater permeability for Cl
than
for I
[Cl
:
Vrev =
32 ± 4.2 mV; I
:
Erev =
21 ± 3.6 mV (n = 6)]. These
results provide direct evidence that anion conductances induced by
thapsigargin and forskolin display different anion selectivities and
that T84 cell apical membranes exhibit at least two distinct anion
conductances.
DIDS affects Cl
conductances
stimulated by both thapsigargin and forskolin.
As shown in Fig. 1, the addition of DIDS to the apical compartment of
intact T84 monolayers inhibited differentially transepithelial Isc stimulated by
thapsigargin or forskolin (100 vs. 40%, respectively). We found nearly
the same differential inhibition of DIDS on thapsigargin- or
forskolin-induced
Isc in
basolaterally permeabilized monolayers studied in symmetrical buffers
containing Cl
as the major
permeant ion (buffer E). DIDS
inhibited completely Isc elicited by
thapsigargin
[
Gapical = 1.2 ± 0.2 mS/cm2 vs.
Gapical = 0.1 ± 0.1 mS/cm2 with DIDS
(n = 5); Fig.
6A].
In contrast, DIDS inhibited forskolin-induced Cl
currents by 57%
[
Gapical = 4.1 ± 0.7 mS/ cm2 vs.
Gapical = 1.6 ± 0.8 mS/cm2 with DIDS
(n = 5); Fig.
6B]. DIDS had a similar effect
on Isc induced by
1 mM 8-bromo-cAMP or 10 mM forskolin applied to permeabilized monolayers (data not shown). This last result verifies the initial analysis using 100 µM forskolin. Thus the anion conductances observed are not attributable to an artifact seen only at 100 µM forskolin concentration. In the presence of transepithelial
Cl
gradients in
basolaterally permeabilized monolayers, basal
Cl
currents were not
affected by apical DIDS treatment [
24 ± 4 vs.
20.3 ± 2.7 µA/cm2 at
+40 mV (n = 6)]; these results
are in accordance with previous studies (8, 40). Thus the effect of
thapsigargin on Cl
current
cannot easily be attributed to the swelling-activated Cl
channel (29). Taken
together, these data confirm results obtained on intact monolayers and
provide further evidence that T84 apical membranes exhibit distinct
Cl
conductances.

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Fig. 6.
Effect of apical addition of 100 µM DIDS on 10 µM thapsigargin
(A)- and 100 µM forskolin
(B)-induced conductances in
basolaterally permeabilized monolayers of T84
(ICl). In these
experiments, apical and cytosol basolateral buffers contained
Cl as major permeant ion,
Na+ present in both sides at 0.5 mM (see Table 1). Ordinates show
difference currents between stimulated and unstimulated cells in
absence (thapsigargin, ; forskolin, ) and presence of DIDS
(thapsigargin, ; forskolin, ). Tracings are representative of
results from 5 different filters for each condition.
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Because DIDS has no or little effect on
Cl
transport through CFTR
(15, 27), these data suggested that the DIDS-sensitive Isc induced by
forskolin may represent Cl
transport through another channel distinct from CFTR, possibly the
apical channel activated by thapsigargin. If so, the DIDS-sensitive component of the forskolin-induced current should display anion selectivities characteristic of the thapsigargin-induced channel. To
test this hypothesis, we studied the ion selectivities of
forskolin-induced currents in the presence and absence of DIDS by
applying apically directed
Cl
gradients to
permeabilized monolayers (buffer A
apical and buffer B basolateral).
Figure 7 shows the current-voltage
relationships for forskolin-induced
Cl
conductances in the
presence and absence of apical DIDS. In the presence of asymmetrical
buffers, a clear shift in
Vrev was induced by DIDS, indicating that the residual (DIDS-insensitive) apical membrane conductance displayed greater selectivity for
Cl
[Vrev in
absence of DIDS =
32 ± 2 mV; in presence of DIDS =
40 ± 5 mV (n = 10); Fig. 7].
These results can be interpreted to indicate that forskolin activates
at least two anion conductances. One conductance displays high
selectivity for Cl
and
insensitivity to DIDS and probably represents CFTR. The other conductance displays lower selectivity to
Cl
and complete sensitivity
to DIDS. In these properties, it resembles the thapsigargin-regulated
Cl
conductance. However, we
do know that forskolin is not likely to induce a purinergic
receptor-activated DIDS-sensitive current by activating the secretion
of ATP via CFTR (37), since the addition of hexokinase (5 U/ml) to
apical reservoirs had no effect on forskolin-induced
Isc
[Isc
without hexokinase 43.9 ± 2.6 vs. 47.4 ± 2.3 µA/cm2 with hexokinase
(n = 4)].

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Fig. 7.
Effect of apical addition of 100 µM DIDS on 100 µM
forskolin-induced conductances in basolaterally permeabilized T84
monolayers. Basolateral-to-apical reservoir
Cl concentration ratio (in
mM) was 136.89/6.60, Na+ present
in both sides at 0.5 mM. Ordinate
shows difference current between stimulated and unstimulated cells in
absence ( ) and presence of DIDS ( ). Tracings are representative
of results from 10 different filters for each condition.
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|
Low temperature affects Cl
efflux
stimulated by forskolin but not by thapsigargin.
Previous studies have shown that activation of cAMP-dependent apical
membrane Cl
channels in
permeabilized T84 monolayers was markedly attenuated by reducing
incubation temperatures to 5°C (11, 21), perhaps via prevention of
channel recruitment to the plasma membrane (11). The effects of
temperature on Ca2+-sensitive
channels have not been similarly studied. Thus, to further compare the
apical membrane Cl
conductances in T84 cell monolayers, we examined the effect of temperature on thapsigargin-induced and forskolin-induced
Isc in
permeabilized monolayers. Permeabilized monolayers were incubated in
symmetrical buffers containing
Cl
as the major permeant
ion; monovalent cations were in the millimolar range
(Cl
: 143.25;
K+: 0.43;
Na+: 0.34 mM; see
buffer E, Table 1) at 4 or 37°C
for 90 min before treatment with thapsigargin or forskolin. At 4°C,
T84 cells maintain intact tight junctions as determined by low
transepithelial conductances (0.8 ± 0.4 mS/cm2). The ultimate magnitude
of the thapsigargin-induced
Cl
secretory response was
not affected at 4°C (Fig.
8A). In
contrast, the forskolin-induced
Cl
secretory response was
diminished by incubation at 4°C as previously reported (Fig.
8B and Ref. 11). Thus the activation
of Cl
conductances by
thapsigargin and forskolin also differ in temperature sensitivity.
Interestingly, the magnitude of the residual forskolin-activated current at 4°C was similar to that of the temperature-insensitive thapsigargin-stimulated current, and both currents were inhibited by
DIDS to equivalent degree (Fig. 8, A
and B). Permeabilized monolayers
were incubated in symmetrical buffers containing
Cl
or
I
(see Table 1,
buffer A or
C); the ion selectivity of the
DIDS-sensitive component of forskolin-induced current at 4°C is
I
> Cl
(Fig.
9). These data demonstrate that the
fraction of the forskolin-activated conductance which is DIDS sensitive
and cold resistant may represent the same
Cl
conductance as that
stimulated by thapsigargin.

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Fig. 8.
Effect of temperature on thapsigargin
(A)- and forskolin
(B)-induced
Cl conductances in
basolaterally permeabilized T84 monolayers. In these experiments,
apical and cytosol basolateral buffers contained
Cl as major permeant anion,
Na+ present in both sides at 0.5 mM (see Table 1). Ordinates show
difference currents between stimulated and unstimulated cells treated
with agonist at 37°C (thapsigargin, ; forskolin, ) and
4°C (thapsigargin, ; forskolin, ) in absence and presence of
100 µM DIDS; addition inhibits completely thapsigargin-stimulated
current at 4°C (thapsigargin, A,
×; forskolin, B, ×).
Tracings are representative of results from 3 different filters for
each condition.
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Fig. 9.
Effects of 100 µM forskolin on currents in basolaterally
permeabilized T84 monolayers measured in iodide solutions at 4°C.
Basal Na+ present in both sides at
0.5 mM; basolateral-to-apical reservoir
Cl concentration ratio (in
mM) was 143.50/6.6.0, Na+ present
in both sides at 137 mM ( ). Cytosol basolateral-to-apical reservoir
I concentration ratio (in
mM) was 136.89/6.60, Na+ present
in both sides at 137 mM ( ).
Ordinate shows difference currents
between stimulated and unstimulated cells. Tracings are representative
of results from 3 different filters for each condition.
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|
Effects of thapsigargin and forskolin on intracellular cAMP and
Ca2+
concentrations.
In some epithelial cells, stimulation of cAMP also results in elevated
intracellular Ca2+ (10). To
investigate the possibility that forskolin may activate a
Ca2+-sensitive
Cl
conductance by raising
cytosolic Ca2+ (in addition to
cAMP), the effect of forskolin treatment on
[Ca2+]i
was examined. T84 cells grown on glass coverslips and loaded with the
fluorescent indicator fura 2 were used for these studies (35). Cells
incubated at 4 and 37°C were studied.
Ca2+ response was
observed in cells near the center of confluent islands. As shown in
Fig. 10, resting levels of
[Ca2+] were between
100 and 150 nM (133 ± 16 nM; n = 28 cells from 3 different coverslips). Application of forskolin at 37 or 4°C caused small but readily detected and consistently observed
intracellular Ca2+ elevations (33 ± 3 nM; n = 28 cells from 3 different coverslips). As expected, stimulation with thapsigargin (10 µM) raised
[Ca2+]i
approximately twofold above resting levels at both temperatures (at
37°C,
[Ca2+]i = 165 ± 12 nM vs.
[Ca2+]i = 150 ± 14 nM at 4°C; n = 28 cells from 3 different coverslips), although the time required to reach
maximal [Ca2+] levels
was greater at 4°C. Thapsigargin addition before or after forskolin
addition produced similar increases in
[Ca2+]i.
Thapsigargin had no effect on intracellular cAMP at either temperature
(Fig. 11), whereas forskolin increased
cAMP 30-fold above resting levels at 37°C and 3-fold above resting
levels at 4°C. These data provide direct evidence that forskolin
treatment can induce a small but detectable increase in intracellular
Ca2+ at both 4 and 37°C. These
data are consistent with previous studies in different cell types (10)
but differ from previous studies performed in isolated T84 cells (43).
The small Ca2+ response that we
have found after forskolin addition might be due to the polarized state
of T84. Thus the ability of forskolin to produce a small elevation in
[Ca2+]i
may contribute to its induction of DIDS-sensitive
temperature-insensitive anion conductance.

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Fig. 10.
Effects of sequential addition of forskolin (Fors) and thapsigargin
(Thap) on cytosolic
[Ca2+]
([Ca2+]i)
in T84.
[Ca2+]i
was measured at either 37 ( ) or 4°C ( ) in fura 2-loaded cells
on glass coverslips. Ca2+ response
was observed in cells near center of confluent "islands." A
single representative experiment for each temperature (37 and 4°C)
is given, and data are those of 14 cells from same coverslip. Tracings
are representative of results from 3 different coverslips.
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Fig. 11.
Effect of temperature on generation of cAMP by basolaterally
permeabilized T84 cells treated with forskolin (100 µM) and with
thapsigargin (10 µM). Apical and basolateral buffers contained
Cl as major permeant ion.
Experiments were carried out at 37 (A) or 4°C
(B). Ctrl, resting level of cAMP;
Thap and Fors, level of cAMP after addition of thapsigargin and
forskolin, respectively. Data are presented as picomoles of cAMP
generated per monolayer of T84 cells and represent 3 filters each
assayed in duplicate.
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|
 |
DISCUSSION |
The results of these studies show that selective permeabilization of
basolateral membranes of T84 cells permits effective electrical
isolation of apical membranes for analyses of anion conductances after
agonist stimulation, as previously described (30, 31, 36). Such
preparations avoid the complexities associated with regulation of
various basolateral transporters, pumps, and channels that contribute
to agonist-elicited
Isc and also
permit analyses of apical membrane conductance in the polarized state. We find that apical anion conductance is stimulated by both
thapsigargin and forskolin. However, the anion currents induced by
forskolin or thapsigargin exhibit different magnitudes and anion
selectivities. Furthermore, they are differentially sensitive to the
anion channel blocker DIDS and cold temperature, indicating that T84
cells exhibit two forms of apical membrane anion channels. Our results
also show that the ion selectivity
(Cl
< I
) and temperature
dependence of the DIDS-sensitive component of the forskolin-induced
Isc are similar
to the ion selectivity and temperature dependence of the
thapsigargin-induced
Isc. Thus the forskolin response appears to represent apical anion conductance of two
types, one of which has features common to the response elicited by
thapsigargin.
The T84 colon carcinoma cell has frequently been used as a model for
Cl
secretion, and
considerable efforts have been directed at defining Cl
channels by using
different methods such as patch-clamp, microelectrode, or
permeabilization techniques. Whole cell voltage-clamp studies with T84
cells reveal two different
Cl
conductances, one
Ca2+ dependent and the other one
cAMP dependent (1, 9). In addition, the anion selectivity of the
Ca2+-stimulated
Cl
conductance in T84 cells
differs from the that of cAMP-dependent Cl
conductance (1). The use
of the 125I efflux technique in
subconfluent cells and recently on T84 cell monolayers also suggests
that Ca2+ and cAMP induce two
different anion efflux pathways (5, 16). In contrast, results from
studies using a technique in which the basolateral membranes of
confluent T84 cells are permeabilized by nystatin are contradictory (1,
22). A speculative conclusion has been made that
Ca2+-stimulated
Cl
conductances observed in
cells grown on permeable supports are absent in cells grown on glass
coverslips (1). On the other hand, studies using intracellular
microelectrodes or using fluorescent dye (SPQ) have detected evidence
favoring the presence of both Ca2+- and cAMP-stimulated
Cl
conductances in T84
cells grown to confluence on permeable supports (2, 22).
The present study shows that intracellular elevation of
Ca2+ or cAMP activates distinct
Cl
conductances in
basolaterally permeabilized monolayers. In symmetrical buffers, the
conductances stimulated by forskolin or by
Ca2+ each have linear
relationships over the range ± 80-mV membrane potential. The
conductances, however, display different biophysical characteristics.
Experiments on the halide permselectivity between Cl
and
I
indicate that
Ganion(cAMP) has
the permeability sequence,
Cl
> I
. These results are in
accordance with previous studies (1, 6) and suggest that the observed
conductance probably represents transport through CFTR (42). In
contrast,
Ganion(Ca) has
the opposite permeability sequence,
I
> Cl
. We also show that
GCl(cAMP) is only
partially inhibited by DIDS in contrast to the
GCl(Ca), which is
completely inhibited by the addition of DIDS. Thus these data are
consistent with the presence of at least two distinct
Cl
conductances in T84
monolayers, as also suggested by previous patch-clamp and
microelectrode studies (2, 9).
In previous studies, apical
Cl
conductances in
confluent T84 monolayers grown on permeable supports were measured by
permeabilizing basolateral membranes with nystatin and then applying an
apically directed gradient of
Cl
across the monolayer
with the voltage clamped to 0 mV. Under these conditions in the
presence of amphotericin rather than nystatin, we observed similar
results to those reported, finding that
Cl
secretion was activated
by the addition of forskolin but not thapsigargin (1). When, however,
the transmonolayer potential was clamped at +15 mV, currents were
activated both by thapsigargin and forskolin. Our results suggest the
possibility that there is less discrimination between
Na+ and
Cl
currents at 0 mV than at
other membrane potentials. Thus, in our studies when higher driving
forces were applied, a thapsigargin-induced Cl
conductance was readily
apparent. The Cl
conductances induced by thapsigargin, however, were much smaller than
those observed for Cl
transport through forskolin-induced conductances. In other words, when
stronger driving forces were applied,
Cl
transport through the
small thapsigargin-induced conductance became detectable.
The finding that thapsigargin induced an elevation of the intracellular
Ca2+ without affecting cAMP levels
raises the possibility that elevations of intracellular
Ca2+ activate apical
Cl
conductances in T84
cells. Because low temperatures did not affect the
Ca2+-dependent
Cl
conductance or the
intracellular Ca2+ increase, it is
reasonable to suggest that Ca2+
could directly activate this
Cl
conductance. In
addition, our observation that the cAMP-stimulated Cl
conductance was
partially DIDS sensitive in basolaterally permeabilized as well as in
intact monolayers raises the possibility that cAMP may stimulate two
distinct Cl
conductances.
We show that the temperature-insensitive component of forskolin-induced
current, like the thapsigargin-induced current, was DIDS sensitive and
displayed a permselectivity of
I
Cl
. These results are
consistent with the possibility that a fraction of the
Cl
conductance induced by
forskolin represents transport through a
Ca2+-activated, DIDS-sensitive
Cl
channel. In T84 cells,
forskolin can thus activate, in addition to CFTR, the
Ca2+-sensitive conductance
activated by thapsigargin alone. These results are consistent with
previous studies showing that cAMP activates a
Cl
conductance which is
DIDS sensitive (20, 26) and shows preference for
I
over
Cl
. Our data, however, do
not allow us to distinguish the possibility that T84 cells may contain
two distinct DIDS-sensitive conductances, one activated by forskolin
and the other by thapsigargin.
Our data do not identify a mechanism by which forskolin might activate
the Ca2+-sensitive conductance.
Although forskolin induces a small increase in intracellular
Ca2+ at both 4 and 37°C, these
data do not exclude the possibility that cAMP may activate the
DIDS-sensitive conductance directly or through activation of protein
kinase(s).
From the results of these and previous studies (2, 5, 16, 22, 33), we
propose the following working model of
Cl
secretion in T84 cells
via two apical conductive pathways (Fig. 12). The first is activated by cAMP and
not sensitive to inhibition by DIDS. This conductance exhibits
temperature sensitivity (inhibition at 4°C), a large
Cl
conductance, ion
selectivity of Cl
> I
, and presumably
represents CFTR. The second is activated both by thapsigargin and by
forskolin. This conductance exhibits little or no temperature
sensitivity, a much smaller
Cl
conductance, and ion
selectivity of I
> Cl
. Forskolin may
activate the DIDS-sensitive conductance via cAMP or
Ca2+ second messengers. Further
studies, however, are needed to identify the mechanism by which
forskolin may activate this
Ca2+-sensitive conductance.

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Fig. 12.
Cellular model for regulation of
Cl secretion in T84.
Ca2+ activates a
Cl channel that is
completely inhibited by DIDS and insensitive to cold temperature.
Forskolin activates via protein kinase A (PKA), a
cold-temperature-sensitive
Cl channel only partially
inhibited by DIDS, as well as a second, temperature-insensitive anion
channel that is completely DIDS inhibited and of different anion
selectivity. Activation of this 2nd channel occurs by an unknown
pathway that may involve Ca2+
and/or cAMP and/or other second messengers.
PCl and
PI,
Cl and
I permeabilities; CFTR,
cystic fibrosis transmembrane conductance regulator.
|
|
 |
ACKNOWLEDGEMENTS |
This work was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grants DK-35932 and DK-47622 to J. L. Madara, DK-48106 to W. I. Lencer, DK-51059 to S. L. Alper, and
DK-02130 to A. Nusrat. W. I. Lencer is a recipient of the Samuel J. Meltzer Award from the American Digestive Health Foundation. S. L. Alper is an Established Investigator of the American Heart Association.
D. Merlin is a recipient of National Institute of Diabetes and
Digestive and Kidney Diseases National Research Service Award DK-09800.
We also acknowledge the support of the Harvard Digestive Diseases
Center (National Institute of Diabetes and Digestive and Kidney
Diseases Grant DK-34854).
 |
FOOTNOTES |
Present address and address for reprint requests: D. Merlin, Dept. of
Pathology and Laboratory Medicine WMRB, 1639, Emory University, 1639 Pierce Dr., Atlanta, GA 30322.
Received 9 July 1997; accepted in final form 16 April 1998.
 |
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