Outwardly rectifying
Cl
channel in guinea pig
small intestinal villus enterocytes: effect of
inhibitors
Alan S.
Monaghan1,
Gerard M.
Mintenig2, and
Francisco V.
Sepúlveda3,4
1 Department of Child Health,
Ninewells Hospital and Medical School, University of Dundee, Dundee DD1
9SY, United Kingdom; 2 Departament
de Ciències Mèdiques Bàsiques, Facultat de Medicina,
Universitat de Lleida, 25198 Lleida, Spain;
3 Departamento de Medicina
Experimental, Facultad de Medicina, Universidad de Chile, Casilla
70058, Santiago-7; and 4 Centro de
Estudios Científicos de Santiago, Casilla 16443, Santiago-9,
Chile
 |
ABSTRACT |
Previous studies in enterocytes isolated from
the villus region of small intestinal epithelium have identified a
macroscopic current carried by
Cl
. In this work a
single-channel patch-clamp study was carried out in the same cells, and
a spontaneously active, outwardly rectifying Cl
channel was identified
and proposed to underlie the whole cell current. The channel had
conductances of 62 and 19 pS at 80 and
80 mV, respectively, in
symmetrical Cl
solutions in
excised patches. Similar activity was seen in cell-attached patches,
but only outward currents could be discerned in this configuration. The
activity of the channel, measured as open probability, was independent
of intracellular calcium levels and voltage. The selectivity sequence
for different anions was
SCN
> I
> Br
> Cl
> F
> (gluconate,
glutamate,
). The channel was
inhibited by 5-nitro-2-(3-phenylpropylamino)benzoic acid
(NPPB), verapamil, and 4-hydroxytamoxifen (but not by
tamoxifen), with potencies similar to those observed for
Cl
channels previously
described in other cells. Inhibition by trinitrophenyladenosine 5'-triphosphate was also observed but only at
depolarized potentials. At 50 mV the half-maximal inhibitory
concentration was 18 nM. It is proposed that this channel plays a role
in transepithelial Cl
transport and certain regulatory
Cl
fluxes.
epithelium; chloride transport; trinitrophenyladenosine
5'-triphosphate
 |
INTRODUCTION |
OUTWARDLY RECTIFYING
Cl
channels (ORCCs) were
first identified in respiratory epithelial cells (16, 56) and later
found in many cell types, including renal and small intestinal
epithelium, pancreas, colon, human skin fibroblasts, and lymphocytes
(2, 5, 8, 10, 17, 20). Also, they have been described in several cell
lines of diverse origin (29). ORCCs have been proposed to have
important roles in cell homeostasis, volume regulation, and secretory
and absorptive processes in epithelial cells (15, 38).
Most studies of Cl
channels
in epithelia have been done using secretory epithelial cells (15); in
contrast, very little is known about
Cl
channels in absorptive
epithelial cells. A 30-pS inwardly rectifying anion channel of
basolateral membranes of mammalian urinary bladder cells with a high
open probability
(Po) in the
physiological voltage range has been described (23). A linear 40-pS
adenosine 3',5'-cyclic monophosphate (cAMP)-activated
Cl
channel in the
basolateral membrane of the thick ascending limb of the mouse kidney is
proposed to be involved in the basolateral Cl
exit step of NaCl
absorption (37). A similar role has been proposed for an ORCC (13/96
pS) present in the basolateral membrane of rabbit renal cortical
collecting duct cells (11).
Little is known about how
Cl
that has been absorbed
across the apical membrane of absorptive enterocytes exits across the basolateral membrane. Whole cell patch-clamp experiments, using small
intestinal villus enterocytes isolated from guinea pig, have shown the
presence of an outwardly rectifying
Cl
current (43). This
spontaneously active current was proposed to be mediated by basolateral
membrane Cl
channels, whose
function would be to allow the basolateral exit of
Cl
during NaCl absorption.
The single-channel configurations of the patch-clamp technique have
been used here to find and characterize basolaterally located
Cl
channels in small
intestinal villus enterocytes isolated from guinea pigs. An outwardly
rectifying channel was observed that appeared to be spontaneously
active in cell-attached patches. Its selectivity and sensitivity to
pharmacological agents have been explored. On this basis it is proposed
to underlie the macroscopic currents previously described in the same
cells. We hypothesize that this channel mediates
Cl
, and perhaps nutrient,
exit across the enterocyte basolateral membrane.
 |
METHODS |
Cell isolation.
Guinea pig villus enterocytes were isolated by methods developed
previously (9), with only minor modifications. Adult male guinea pigs
(weighing 250-400 g) were starved for 24 h and killed by cervical
dislocation. The first 30-40 cm of the small intestine (after the
duodenum) were immediately excised and placed into ice-cold saline. The
intestinal lumen was rinsed through with three 30-ml volumes of the
cold saline solution and then with an intracellular-like solution
[solution I, composition (in
mM): 7 K2SO4,
44 K2HPO4,
9 NaHCO3, 15 Na3 citrate, 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, and 180 glucose, pH 7.4, with
tris(hydroxymethyl)aminomethane]. The lumen was filled with
solution I, clamped at both ends, and incubated at 37°C for 8 min. After this time, the intestinal lumen was emptied and refilled with a similar solution containing 1 mM
dithiothreitol and 0.2 mM EDTA. The intestine was again incubated at
37°C for 3 min and then gently palpated for 3 min, and its contents
were emptied into 20-30 ml of ice-cold Dulbecco's modified Eagle's medium (DMEM; Sigma, Poole, UK). The process was repeated and
the contents were pooled with those from the first palpation. The
collected contents were centrifuged at 50 g for 4 min, and the resulting cell
pellet was resuspended in 10 ml of DMEM containing ~1,000 U of
collagenase type 1A. The cells were incubated, with gentle agitation,
at 37°C for 15 min and then diluted with an equal volume of
ice-cold DMEM and filtered through nylon mesh (60- and then 30-µm
pore size) to remove clumps of cells and aggregated debris. After
centrifugation, cells were resuspended in 20 ml of ice-cold DMEM,
plated out into 35-mm plastic Petri dishes, and left on ice for 1 h to
settle. Cells isolated by the above procedure have been shown
previously to be mainly of villus origin (9, 49). Villus cells were
15-20 µm in diameter and could be identified easily by their
clear brush border and basolateral domains located at opposite poles,
as shown in the micrograph (Fig. 1). Cell
viability assessed by trypan blue exclusion was >95%. Viable cells
viewed with phase-contrast optics were birefringent and hence easily
identified even without the use of trypan blue.

View larger version (168K):
[in this window]
[in a new window]
|
Fig. 1.
Phase-contrast micrograph of isolated villus enterocytes used in this
study, illustrating the apparent basolateral approach employed to
obtain all membrane patches used in the present work. Calibration bar,
15 µm.
|
|
Patch-clamp recordings.
Single-channel currents were recorded at room temperature using the
patch-clamp technique (22). Micropipettes were fabricated from
thin-walled borosilicate (hard) glass tubing of external diameter 1.5 mm and internal diameter 1.17 mm (Clark Electromedical, Pangbourne,
UK). Pipettes were pulled using a two-stage vertical pipette puller
(PP-83, Narishige, Japan) and fire-polished on a microforge. When
filled with the appropriate intracellular or extracellular solution
they had resistances of 5-10 M
(2-3 M
in whole cell
recording experiments). The petri dish containing the cells was
continuously superfused with normal saline (see Table
1), and excess solution was
removed using a peristaltic pump. Nonstandard solutions were applied
using a local microperfusion device similar to one described previously
(50). This device allowed the directing of a small jet of the desired
solution at the cell or membrane patch without greatly altering the
bulk solution of the chamber. Current measurements were carried out
using an EPC 7 (List Electronic, Darmstadt, Germany) amplifier. Whole
cell recordings were performed as described previously (44).
Enterocytes were viewed on an inverted microscope (Nikon Diaphot),
equipped with phase-contrast optics, at a total magnification of
×400. Seals of 10-100 G
were routinely obtained. Excised
inside-out patches were obtained from cell-attached patches either by
simply pulling the patch pipette away from the cell or (for cells not attached to the bottom of the petri dish) by using the microperfusion to blow the cell away from the pipette tip. For recording in the outside-out or whole cell configuration, a short burst of strong suction was applied before excision to break the patch membrane. The
reference (ground) electrode consisted of a silver-silver chloride
pellet connected to the bath solution by an agar bridge filled with
saline solution. Liquid junction potentials, which occurred as a result
of bath solution changes during an experiment, were calculated (4), and
current-voltage relations were corrected accordingly. Voltage-pulse
protocols were generated using an IBM-AT microcomputer connected to the
patch-clamp amplifier through a Cambridge Electronic Design 1401 laboratory interface (Cambridge, UK). Current and voltage signals from
the patch-clamp amplifier were filtered at 10 kHz and stored on digital
audiotapes using a modified digital audiotape recorder (Sony).
Acquisition of signals into the computer was done off-line at a rate of
1-5 kHz after the signal was filtered at 0.5-1 kHz with an
eight-pole Bessel filter. Acquisition and analysis of single-channel
data were carried out using a patch-clamp analysis (PAT) or
voltage-clamp analysis (VCAN) program. The software was kindly provided
by Dr. John Dempster (University of Strathclyde, Glasgow, UK).
Unless otherwise indicated, all chemicals were from Sigma (UK).
5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) was a kind gift from
Prof. R. Greger (Freiburg, Germany).
Errors, where given, are ±SE.
 |
RESULTS |
Membrane patches were obtained from what, under phase-contrast optics,
looked like a smooth (basolateral) surface as opposed to a fuzzy,
presumably brush border, membrane (see Fig. 1). The general morphology
of the cells was reminiscent of what has been termed bilobulated or
figure-eight cells for epithelial cells isolated from kidney proximal
tubule and gallbladder, respectively (42, 52). These cells have been
demonstrated to maintain in suspension both structural and functional
polarity. Structural polarity at the level of the electron microscope
has been demonstrated in the enterocytes used here (9), but no further
functional tests for polarity have been performed.
An ORCC is active in cell-attached patches. Figure
2A shows
traces recorded from a cell-attached patch on a guinea pig enterocyte with a pipette containing an NaCl-rich solution and normal saline in
the bath (see Table 1 for composition of solutions). A single channel
was seen when the patch membrane was depolarized, but no inward
transitions were observed on hyperpolarization down to
120 mV.
Average slope conductances were 96 ± 6 pS
(n = 24) at 110 mV and 45 ± 4 pS
(n = 17) at 50 mV (command
potentials). Excision of patches into an NaCl-rich (see Table 1) bath
solution gave the activity illustrated in Fig.
2B. The outward currents seen at
depolarized potentials were very similar to those in cell-attached patches. However, in excised patches small inward currents were seen at
hyperpolarizing voltages. Figure 2C
shows that the current-voltage relation for this and other similar
channels in both the cell-attached and inside-out configurations was
strongly outwardly rectifying. The mean chord conductance in 16 patches
was 62 ± 3 pS at 80 mV and 19 ± 1 pS at
80 mV. Excision
of cell-attached patches containing outward currents, such
as those in Fig. 2A,
always gave rise to both outward and inward currents. The
Cl
channel was seen in 97 of 162 patches successfully recorded. Twelve of these appeared to
correspond to single channels.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 2.
Outwardly rectifying Cl
currents in cell-attached and excised inside-out patches.
A: current traces at indicated command
voltages in cell-attached patch. Arrows indicate closed-current level.
Patch pipette contained NaCl-rich solution, and bath contained normal
saline. B: currents of the same
channel type after excision (inside-out) into NaCl-rich solution.
C: current-voltage
(I-Vc)
relation for Cl currents
recorded from cell-attached ( ) or excised inside-out ( ) patches
as described in A and
B, respectively.
|
|
Comparison of current-voltage relations for the cell-attached and
excised inside-out configurations (Fig.
2C) revealed that the cell-attached
curve was shifted by ~15 mV to the right of the excised-patch
relation. Thus, if Cl
is
assumed to be the charge carrier, the intracellular anion should be 15 mV above equilibrium. If a membrane potential of
40 to
50
mV is assumed [hence a reversal potential
(Erev) of
25 to
35 mV], an intracellular
Cl
concentration
([Cl
]i)
value of 37-55 mM can be calculated.
[Cl
]i
measured in Necturus enterocytes with
ion-selective electrodes was ~30 mM and was above equilibrium by
~10 mV (18).
Experiments to determine the kinetics of the ORCC were very difficult
to carry out because patches from the isolated villus enterocytes often
did not stand up to the lengthy time periods at different voltages
required for such analysis. Also, most patches contained multiple
channels. Po was
~0.8 at positive potentials. A greater variation was observed at
negative potentials, but on average
Po remained
potential independent (in 4 experiments
Po values were
0.86 ± 0.09 and 0.76 ± 0.10 at 60 and
60 mV,
respectively). Po
did not depend on intracellular
Ca2+, which varied between 1.3 mM
and no added Ca2+ plus 1 mM
ethylene glycol-bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic acid (not shown).
Selectivity of guinea pig enterocyte
Cl
channel.
Replacing Na+ in the pipette by
the impermeant cation
N-methyl-D-glucamine
(NMG+) had no effect on the
current-voltage relation. The slope conductance at 110 mV was 102 ± 12 pS (41 ± 5 pS at 50 mV) in five separate experiments; these
values do not differ from those obtained with Na+-rich pipette solutions. When
cell-attached patches were excised into either NMG-Cl- or NaCl-rich
bath solutions, there was no difference in the current-voltage
relations obtained, and in both cases the current reversed at 0 mV
(data not shown).
Figure 3 illustrates an
experiment in an excised inside-out patch in which the pipette solution
was NaCl rich (see Table 1) and the bath solutions were
Na+ rich but contained different
anions. Traces of currents measured while the potential was held at 40 or 0 mV are illustrated in Fig. 3. When
Cl
-rich solutions bathed
both sides of the membrane, outward currents could be seen at 40 mV and
no currents could be detected at 0 mV. Outward currents were seen at
both voltages when
or gluconate
was the main anion in the intracellular medium. When SCN
was used as the main
intracellular anion, inward currents were seen at 0 mV and outward
currents (reduced in size compared with the equivalent situation in
symmetrical Cl
) were seen
at 40 mV. Only outward currents could be measured accurately at a range
of voltages when either
or
gluconate was used as the main intracellular anion in inside-out patches (not shown).

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 3.
Anion selectivity of the outwardly rectifying
Cl channel (ORCC) of guinea
pig enterocytes. Current traces were recorded from an excised
inside-out patch with an NaCl-rich pipette solution at indicated
voltages and bathing solutions containing indicated main anion. Arrows
indicate zero-current level.
|
|
The permeability of the channel for different anions can be expressed
as a ratio of the Cl
permeability, i.e.,
Panion/PCl,
and was calculated using the Goldman-Hodgkin-Katz (GHK) equation.
Concentration values were used instead of activities because, for
monovalent salts, activity coefficients are all very similar. Table
2 shows the
Erev
values obtained for the different anions relative to the
Erev obtained with a Cl
-rich bath
solution. Also shown in Table 2 are the
Panion/PCl values calculated for the different anions.
Glutamic acid can permeate the enterocyte ORCC.
Figure 4C
illustrates current-voltage relations obtained from an excised
inside-out patch in which the pipette solution was NMG-Cl rich and the
bath solutions were as indicated. When the main intracellular anion was
switched from Cl
to
glutamate, the inward currents were much smaller and the
Erev shifted in
the negative direction. Thus, although glutamate permeated the channel
much less easily than Cl
,
it appears that the channel does nevertheless allow glutamate to
traverse it.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 4.
Activity of ORCCs in outside-out patch configuration: effect of
replacement with glutamic acid. Traces are from an outside-out patch
with a pipette containing corresponding NaCl-rich solution (see Table
1). A: traces at 80 and 0 mV where
bath solution was NaCl rich. B: traces
at 80 and 0 mV where bath solution was 500 mM
N-methyl-D-glucamine
(NMG) glutamate. C: current-voltage
relations for the outside-out patch in
A ( ) and
B ( ). Data for NMGCl-rich ( ) and
500 mM NMG glutamate ( ) solution replacements in an inside-out patch
are also shown.
|
|
To get a more accurate measure of the channel permeability to
glutamate, we decided it was more practical to look at the effect of
substituting glutamate for
Cl
at the extracellular
side of the patch membrane. Thus the effects were on the larger,
clearer outward currents. The
Cl
channel was therefore
recorded in outside-out patches. The mean chord conductance in four
separate experiments was 70 ± 3 pS at +80 mV and 19 ± 1 pS at
60 mV. These values are similar to those derived from
experiments on inside-out patches. Figure
4A shows traces recorded from an
excised outside-out patch in which the pipette (intracellular) solution
was NaCl rich and the bath (extracellular) solutions had a similar
composition (see Table 1). Traces are shown at 80 and 0 mV,
respectively. At 80 mV, outward currents were seen; the unitary current
was 5.3 pA. At 0 mV, no currents were seen. In Fig.
4B the bath solution had been changed
to 500 mM NMG glutamate. Again outward currents were seen at 80 mV, but this time the unitary current was only 2 pA (note change in scale from
Fig. 4A). At 0 mV, small inward
currents were seen.
Current-voltage relations obtained from the experiment illustrated are
shown in Fig. 4C. When
Cl
was the main bath anion,
Erev was ~0 mV.
When glutamate was the main bath anion,
Erev shifted to
28 mV. Application of the GHK equation with the
Erev obtained for
glutamate gave a
PGlu/PCl of 0.09; similar results were obtained in three separate experiments. The corresponding permeability ratio found by Banderali and Roy (3) in
MDCK cells was 0.18.
Effects of pharmacological agents on the enterocyte ORCC.
Many Cl
channel blockers
act primarily on the extracellular surface of the membrane. Because of
this, the effects of blockers at the single-channel level were studied
on excised outside-out patches.
Whole cell recordings from isolated guinea pig villus enterocytes have
shown that the blocker NPPB at a concentration of 10 µM strongly
inhibits the outwardly rectifying
Cl
conductance. Figure
5A shows
traces recorded from an outside-out patch held at 50 mV. In the top
trace of Fig. 5A, current fluctuations in a patch containing two ORCCs are shown. The bottom trace of Fig.
5A was recorded after the addition of
50 µM NPPB to the bath solution. Under control conditions the two
channels were seen to be open with high
Po. The addition
of NPPB resulted in a flickery-type blockade that reduced the current
going through the patch by 82%; similar results were obtained in two
separate experiments. These results are consistent with those
previously obtained in the whole cell configuration (43).

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 5.
Effects of inhibitors on guinea pig enterocyte ORCCs.
A: traces recorded from an excised
outside-out patch held at 50 mV. Pipette solution was NaCl rich. Top
trace: bath solution was normal saline; bottom trace: 50 µM
5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) had been added to
the bath solution. Arrows indicate closed level.
B and
C: traces recorded as in
A but from different patches and with
blockers indicated.
|
|
It has been shown that the Ca2+
channel blocker verapamil inhibits an outwardly rectifying
Cl
conductance modulated by
changes in cellular volume (12, 53). Verapamil also inhibits similar
Cl
channels in colonic
carcinoma cells (6). The effect of this compound on the guinea pig
enterocyte Cl
channel was
explored. Figure 5B shows traces
recorded from an outside-out patch before and after verapamil addition.
The compound caused a strong flickery-type blockade. The inhibition of
the Cl
channel was
reversible (results not shown). 1,9-Dideoxyforskolin (DDFSK), like
verapamil, has recently been shown to inhibit volume-activated outwardly rectifying Cl
conductances (12, 53). Figure 5C shows
traces recorded from an excised outside-out patch with pipette and bath
solutions rich in NaCl. As with verapamil, addition of DDFSK to the
bath solution caused a flickery-type blockade. The channel inhibition
by DDFSK was reversible (results not shown).
Anti-estrogen interaction with guinea pig enterocyte ORCCs.
The anti-estrogen tamoxifen was used to explore further the
relationship between the ORCCs in guinea pig villus enterocytes and
volume-activated ORCCs. Tamoxifen has been shown to block completely
the Cl
conductance
modulated by cell volume increase at concentrations
10 µM (54, 60).
When 10 µM tamoxifen was added at the extracellular surface of
excised outside-out patches from isolated guinea pig villus
enterocytes, the patches rapidly deteriorated and broke. Thus it was
not possible to look at the effect of tamoxifen on the
Cl
channel at this
concentration. Figure
6A shows
traces recorded from an excised outside-out patch in which the pipette
solution was NaCl rich and the bath solution was normal saline. In Fig. 6A, the top trace shows channel
activity under control conditions, whereas the bottom trace shows
channel activity after the addition of 5 µM tamoxifen to the bath
solution. There was no effect on either the
Po or current
amplitude of the channel. In two separate experiments with 5 µM
tamoxifen, three experiments with 2 µM tamoxifen, and three
experiments with 1 µM tamoxifen, no effect was seen on the
Cl
channel. Although
tamoxifen failed to block the guinea pig enterocyte Cl
channel, the derivative
4-hydroxytamoxifen (4-OH-tamoxifen) was tested to see if it would have
any effect. This compound has been shown to inhibit volume-activated
outwardly rectifying Cl
conductance and, like tamoxifen, does so at concentrations in the low
micromolar range. As shown in Fig. 6B
the same is true for the Cl
channel from guinea pig enterocytes. The top trace in Fig.
6B shows the control condition, in
which no 4-OH-tamoxifen had been added to the bath solution. There were
two active channels in the patch, both of which had very high
Po values. The
middle and bottom traces of Fig. 6B
show the effect of adding 2 and 10 µM 4-OH-tamoxifen to the bath
solution. It can be seen that this resulted in an increasingly marked
flickery-type blockade of the Cl
channel. There was also
a slight decrease in the current amplitude in the presence of
4-OH-tamoxifen. The current amplitude under control conditions was 3.0 pA, and it decreased to 2.4 pA at 10 µM tamoxifen. A dose-response
curve for the inhibition of the Cl
channel by
4-OH-tamoxifen is shown in Fig. 7. Even at
10 µM 4-OH-tamoxifen the channel was only inhibited by ~38%. Using
higher concentrations of 4-OH-tamoxifen caused damage to excised
outside-out patches. The line is a fit of a rectangular hyperbola to
the data. From this fit the maximal inhibition was 50%; for
4-OH-tamoxifen, the concentration required for half-maximal inhibition
of the channel (IC50) was 3.2 µM.

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 6.
Effects of inhibitors on guinea pig enterocyte ORCCs.
A: traces recorded from an excised
outside-out patch held at 50 mV. Pipette solution was NaCl rich. Top
trace: bath solution was normal saline; bottom trace: 5 µM tamoxifen
had been added to bath solution. Arrows indicate closed level.
B: traces recorded as in
A, but from a different patch and with
4-hydroxytamoxifen (4-OH-tamoxifen) at indicated concentrations.
|
|

View larger version (11K):
[in this window]
[in a new window]
|
Fig. 7.
Dose-response curves for guinea pig enterocyte ORCC inhibition. Percent
inhibition of the Cl
current at indicated concentrations of 4-OH-tamoxifen ( ),
1,9-dideoxyforskolin (DDFSK; ), and verapamil ( ). Effect of
NPPB ( ) at a single concentration is also shown. Values are means ± SE.
|
|
Figure 7 also shows the concentration dependence for the effects of
verapamil and DDFSK. The continuous lines are fits of rectangular
hyperbolas to the data. For verapamil inhibition, maximal blockade of
the channel deduced from the fit was 78%. The
IC50 for
Cl
channel inhibition by
verapamil was 12 µM. The dose-response curve for the inhibition of
the Cl
channel by DDFSK
gave an IC50 of 52 µM.
Blockade of guinea pig enterocyte ORCCs by trinitrophenyl-ATP.
ATP and some derivatives can block ORCCs.
Trinitrophenyladenosine 5'-triphosphate (TNP-ATP), reported to be
a powerful blocker (55), has been used here. Figure
8A shows
traces recorded from an outside-out patch maintained at 50 mV
throughout the experiment. The patch, seen in the control condition in
the top trace of Fig. 8A, contained
three active ORCCs. The middle and bottom traces of Fig.
8A were recorded after addition of 10 and then 50 nM TNP-ATP to the bath solution, respectively. The addition
of TNP-ATP resulted in a strong inhibition, which greatly reduced
Po without any
effect on the single-channel current. In Fig.
8B a rectangular hyperbola has been
fitted to the concentration dependence of TNP-ATP inhibition, yielding
an IC50 value of 18 nM.

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 8.
Effects of trinitrophenyladenosine 5'-triphosphate (TNP-ATP) on
guinea pig enterocyte ORCCs. A: traces
recorded from an excised outside-out patch held at 50 mV. Pipette
solution was NaCl rich. Top trace: bath solution was normal saline;
bottom traces: recordings were from the same patch after perfusion with
TNP-ATP. Arrows indicate closed level.
B: dose-response curve for TNP-ATP
effect. Results are means ± SE from 4 experiments.
|
|
The high potency of blockade of TNP-ATP would make it an excellent tool
to investigate the role of the ORCC in enterocyte physiology. As a way
to link the whole cell currents previously described in villus
enterocytes (43) and the single-channel activity studied here, the
effect TNP-ATP was studied. Figure 9A shows
whole cell recordings measured with voltage protocols clamping the cell
between
120 and 120 mV in 40-mV steps from a holding potential
of 0 mV, with Cl
-rich
intra- and extracellular solutions (see Table 1 for composition). As
described previously, an outwardly rectifying current was obtained with
little evidence for voltage dependence except for the most hyperpolarizing pulse. Surprisingly, addition of 100 nM ATP to the
medium did not affect the currents markedly. This is seen in Fig.
9B, in which the corresponding
current-voltage relations are shown. An experiment, also shown in Fig.
9B, in low
Cl
concentration (gluconate
replacement, see Table 1) confirmed the anionic nature of the charge
carrier in these experiments, as there was a marked decrease in outward
current and a displacement of the
Erev to the
right. If the channels described above were nevertheless responsible
for the macroscopic current, a possible explanation of the lack of
effect could be a marked voltage dependence of TNP-ATP inhibition
coupled to a slow on-rate. This was tested by increasing the length of
depolarizing pulses. Figure 9C shows that by the end of an 80-mV pulse a marked inhibition by 100 nM TNP-ATP
could be observed. With 1 µM TNP-ATP complete inhibition could be
observed after 2 s. When the same current-voltage protocols as in Fig.
9A were applied from a holding
potential of 80 mV, inhibition was observed at all potentials. This is
shown in Fig. 9D, in which it can be
seen that inhibition by 100 nM TNP-ATP was voltage dependent (i.e., it
was more marked as the cell was more depolarized). The
voltage dependency of TNP-ATP blockade was also evident in experiments
(not shown) in which a conditioning pulse was given before voltage was
returned to 80 mV and the tail currents were measured. Little
inhibition was seen with a
120-mV prepulse, but blockade became
progressively more marked as the conditioning prepulses became more
positive. The voltage dependence of blockade by 100 nM TNP-ATP could be
described reasonably well by a Boltzman distribution with effective
valence of
0.8 and 50% inhibitory voltage of 14 mV.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 9.
Effect of TNP-ATP on whole cell
Cl currents.
A: whole cell
Cl currents obtained under
the indicated conditions. Cell was held at 0 mV and pulsed to
potentials from 120 to 120 mV in 40-mV steps. Bath and pipette
solutions were NMGCl rich (see Table 1).
B: current-voltage relations, where
result of partial replacement of extracellular
Cl by gluconate is also
shown. C: effect of long pulses to 80 mV under control conditions or in the presence of 2 concentrations of
TNP-ATP. D: current-voltage relations
for whole cell currents measured with same protocol and solutions as in
A, but at a holding potential of
80 mV.
|
|
That similar voltage dependence was present at the single-channel level
could be observed in outside-out patches as shown in Fig.
10. ORCC activity was studied by pulsing
for 5 s to
80 mV and then to 80 mV for 20 s under control
conditions (Fig. 10A) or after
perfusion with 100 nM (Fig. 10B) and
then 1 µM TNP-ATP (Fig. 10C). A
similar degree of activity was observed at
80 mV regardless of
the presence of the nucleotide. On application of the depolarizing
step, progressive blockade was apparent, which resulted in complete
inhibition by the end of the pulse. In Fig. 10D averaged traces are shown, which
show no differences between control and in the presence of the
inhibitor at
80 mV. On switching to 80 mV in the presence of
TNP-ATP, rapid blockade occurred that was virtually complete after 5 s
at 80 mV. Similar results were obtained in five separate patches. These
observations link the single ORCC activity and the macroscopic
Cl
current in enterocytes,
although they might correspond to different channels coincidentally
affected by TNP-ATP in a similar fashion.

View larger version (11K):
[in this window]
[in a new window]
|
Fig. 10.
Time course of the effect of TNP-ATP on guinea pig enterocyte ORCCs.
A-C: traces were recorded from an
excised outside-out patch held at 0 mV and pulsed to 80 mV for 5 s and then switched to 80 mV for a further 20 s. Pipette and bath
solutions were NaCl rich. Control traces and traces in the presence of
TNP-ATP are from same patch. D:
averaged traces obtained from 22 sweeps (control) and 15 sweeps each in
the presence of 100 nM or 1 µM TNP-ATP.
|
|
 |
DISCUSSION |
Cell-attached patches on villus enterocytes isolated from guinea pig
small intestine contain a spontaneously active
Cl
channel. Outward
currents through this channel were seen by depolarizing the membrane
patch. However, in the cell-attached configuration, inward currents
could not be resolved. A spontaneously active Cl
channel of intermediate
conductance (35-40 pS) that did not rectify has been observed in
cell-attached patches from isolated rat colonic enterocytes (10). ORCC
activity in cell-attached patches has been reported, but only after
some form of stimulation. ORCC activity has been seen in cell-attached
patches of HT-29 cells after strong depolarizing voltage pulses (14)
were applied, in MDCK cells after exposure to hypotonicity (2), and in
colonic T84 cells under the action of cAMP-mediated secretagogues (21).
Most reports describe activation of ORCCs after excision of previously
silent cell-attached patches, often requiring strong depolarization
(20, 21, 33, 57). Kunzelmann et al. (31) found that the ORCC activated
instantaneously on excision of cell-attached patches at 37°C,
irrespective of the clamp potential. It was later shown that the
cytosol of some cells (including HT-29 cells, T84 cells, and CFPAC-1
cells) can inhibit the ORCC, and this might explain "excision
activation" of ORCCs (28, 32). Although it appears that the channel
studied here does not require any form of activation to be recorded,
some unintentional stimulus might have arisen during the isolation
procedure. If the enterocyte ORCC were sensitive to cell swelling or
direct or indirect mechanical stimulation, it is conceivable that this
type of activation might have taken place during the isolation
procedure. It is not possible to say whether the volume of the cells is
at a normal level after isolation. Similarly isolated cells, however,
can respond to anisotonicity or changes in volume consequent to active
transport with regulatory volume adjustments (34, 35) and have
near-physiological ion gradients (9).
Thus the spontaneously active
Cl
channel reported here in
cell-attached patches is unusual in two ways: it required no apparent stimulation in order to activate, and only outward currents could be
seen for this channel in the cell-attached mode. It is conceivable that
inward currents (Cl
leaving
the cell) are not detectable, as Goldman-Hodgkin-Katz rectification
would further decrease the already small inward currents at negative
potentials. Another explanation would be that some intracellular
component blocks Cl
exit
(inward current). This may be related to the proposed cytoplasmic inhibitor of this type of channel (28, 32). A possible physiological role for this channel might be to allow the efflux of
Cl
from the cell during the
Na+-coupled absorption of
nutrients (34, 35).
When cell-attached patches containing outward
Cl
currents were excised,
both outward and inward currents were always seen. There were no
exceptions to this observation, suggesting that the outward and inward
currents were through the same channel and not through two different
channel types. In symmetrical 145 mM
Cl
solutions the chord
conductance for outward currents (at 80 mV) was ~62 pS and for inward
currents (at
80 mV) was ~19 pS. These values are typical for
intermediate-conductance ORCCs (51). Interestingly, a recent report
(26) has demonstrated that ORCC activity probably accounts for
volume-regulated anion currents as previously proposed (46, 58).
The anion permeability sequence for the
Cl
channel, derived from
shifts in the
Erev in response
to bathing excised inside-out patches in different anion-containing
solutions, was very similar to selectivity sequences reported for other
epithelial Cl
channels (15,
17, 19). Earlier whole cell experiments in guinea pig villus
enterocytes had shown that the outwardly rectifying Cl
conductance had
Panion/PCl
values of 2.57, 0.75, and 0.27 for SCN
,
F
, and gluconate,
respectively (43). The permeability sequence obtained was
SCN
> I
> Br
> Cl
> F
> (gluconate,
glutamate,
). This
sequence corresponds to Eisenman's sequence I (59), suggesting that
the channel pore contains weak binding sites.
The strong outwardly rectifying nature of the
Cl
channel described here,
together with its high selectivity for anions over cations and its
anionic permeability sequence, suggests that this channel may be the
channel responsible for the whole cell
Cl
currents reported
previously in isolated guinea pig small intestinal villus cells (43).
Roy and Sauvé (40) have reported that after exposure to hypotonic
medium, MDCK cells undergo a regulatory volume decrease (RVD) that
involves the loss of K+,
Cl
, and amino acids from
the cells. It was later found that the pathway for amino acid loss
during RVD was selective for neutral and anionic amino acids over
cationic amino acids (39). In MDCK cells (2) the loss of
K+ during RVD occurs by activation
of a highly selective K+ channel;
loss of Cl
was found to be
through the activation of an ORCC that was not highly selective among
anions. Single-channel patch-clamp recordings from MDCK cells have now
shown that the ORCC will allow the permeation of the amino acids
glutamate, taurine, and aspartic acid (3), and it is proposed that
neutral and anionic amino acid (and some sugar) losses observed during
RVD occur via the ORCC (see Ref. 48 for a recent comprehensive review
on the subject).
It has been demonstrated in this report that the ORCC in guinea pig
villus enterocytes will allow the large anion gluconate and the anionic
amino acid glutamate to permeate. The permeability of glutamate
relative to that of Cl
was
0.09 and that of gluconate relative to
Cl
was 0.23. These values
suggest that the permeation of organic molecules through the ORCC could
be of physiological importance.
Although it is well known that the role of villus enterocytes is to
absorb nutrients from the gut lumen, relatively little has been
reported on how these nutrients leave the cell at the basolateral
membrane. Sugars and amino acids exit across the basolateral membrane
of absorptive cells by what appears to be either simple diffusion or
facilitated diffusion (7, 47). As these molecules do not readily cross
the lipid bilayer, one could speculate that the enterocyte ORCC would
be a possible route for the exit of nutrients (both amino acids and
sugars) after Na+-dependent uptake
at the brush border membrane. Banderali and Roy (3) postulate that for
MDCK cells a sudden increase in amino acid uptake at the apical
membrane would lead to an important rise in cell amino acid content and
cell volume increase that would trigger the opening of the
basolaterally located ORCC, thus permitting a rapid exodus of amino
acids (45). Thus it may be that cells that have a physiological
capability to absorb nutrient across the apical membrane require the
ORCC in the basolateral membrane as an exit route for the absorbed
nutrients.
In conclusion, cell-attached and excised patches from guinea pig villus
enterocytes have been used to demonstrate the presence of a
Cl
channel, whose
characteristics suggest that it is responsible for the whole cell
Cl
currents reported
previously (43). This channel has many of the characteristics of ORCCs
present in other epithelial cells. The guinea pig villus enterocyte
ORCC is novel in that it is spontaneously active in cell-attached
patches.
Blockade by NPPB of the ORCC in HT-29 colonic cells has been studied
extensively (6, 13, 14, 24, 51). NPPB is a potent blocker of the HT-29
ORCC, and it acts by binding to the extracellular aspect of the
channel. Swelling-activated outwardly rectifying
Cl
currents and ORCC
single-channel activity are also blocked by NPPB, with
IC50 values of 2-25 µM (11,
12, 17, 30). Whole cell recordings from guinea pig small intestinal
villus enterocytes have previously shown that the outwardly rectifying
Cl
currents present were
strongly and reversibly inhibited by 10 µM NPPB (43). When applied to
the extracellular surface of an enterocyte excised outside-out patch,
50 µM NPPB caused a >80% blockade of the ORCC, suggesting that
IC50 lies in the low micromolar range.
The Ca2+ channel blocker verapamil
caused a potent, dose-dependent, fully reversible inhibition of the
ORCC of guinea pig small intestinal villus enterocytes. DDFSK, an
analog of forskolin that has no effect on cAMP levels, fully blocks the
enterocyte ORCC with an IC50 of 50 µM. The ORCC of HT-29 (D4) cells was inhibited with an
IC50 of 100 µM by verapamil
added to the extracellular surface of excised outside-out patches (6).
Verapamil and DDFSK also inhibit the volume-activated outwardly
rectifying Cl
currents (12,
41, 53) with IC50 values <100
µM. Thus the spontaneously active ORCC of guinea pig small intestinal
villus enterocytes is blocked to a similar extent by verapamil and
DDFSK as volume-activated outwardly rectifying
Cl
currents are blocked.
The anti-estrogen tamoxifen is a potent blocker of volume-activated
outwardly rectifying Cl
currents, which are inhibited with an
IC50 of 0.3 µM when tamoxifen is
added extracellularly (60). Tamoxifen has no effect on
either the cAMP-activated or
Ca2+-activated
Cl
currents of T84 cells
(54). The fact that both the guinea pig small intestinal villus
enterocyte ORCC and volume-activated
Cl
currents are blocked by
verapamil and DDFSK suggested that these currents might be related. One
might expect that tamoxifen would be a potent blocker of the guinea pig
small intestinal villus enterocyte ORCC, but it was without effect.
4-OH-tamoxifen, which also blocks volume-activated
Cl
currents (60), caused a
dose-dependent blockade of the guinea pig small intestinal villus ORCC
with IC50 of 3.2 µM.
4-OH-tamoxifen caused a flickering blockade and also slightly decreased
the current amplitude. It is surprising that 4-OH-tamoxifen can block
the ORCC, whereas tamoxifen, of similar chemical structure, does not. It has been shown, however, that 4-OH-tamoxifen has a 100-fold greater
affinity for the estrogen receptor than tamoxifen (27), showing that a
small change in the chemical structure can lead to dramatic changes in
properties. The finding that 4-OH-tamoxifen but not tamoxifen blocks
the guinea pig villus enterocyte ORCC suggests that this family of
potential blockers may also be used to distinguish between different
types of Cl
channel.
Voltage-dependent blockade of various
Cl
channels by ATP has been
reported (1, 25, 36), and an ORCC from rat colonic crypts was blocked
with high affinity by TNP-ATP (55). An even more potent blockade than
previously reported was observed for the small intestinal enterocyte
ORCC by TNP-ATP in the present work, with an
IC50 of 18 nM
compared with 270 nM in the rat colonic channel. Inhibition was voltage
dependent, making TNP-ATP a poor inhibitor at physiological voltages
and therefore decreasing the value of its use in functional intact cell
work. The compound probably exerts its effect crossing part of the
membrane field and should be valuable as a probe for the channel
structure, although it is not known whether it inhibits other channel
types. The similarity in TNP-ATP effects on whole cell current and
ORCC, coupled with the similarity in permeability sequence, outward
rectification, and sensitivity to NPPB, make the ORCC a
good candidate to mediate the macroscopic currents described before.
In summary, an ORCC of spontaneous activity has been described in
enterocytes from small intestinal villus of the guinea pig. It is
proposed that it underlies the
Cl
currents previously
described in these cells and could serve to provide an exit pathway for
Cl
taken up across the
apical membrane (43). In addition, it is speculated that it might be
the pathway responsible for
Cl
, and perhaps nutrient,
efflux from enterocytes swollen osmotically or by nutrient uptake.
 |
ACKNOWLEDGEMENTS |
This work was supported by Fondecyt (Chile) Grant 1961208 and by a
grant from the Volkswagen Stiftung (Germany). Institutional support to
Centro de Estudios Científicos de Santiago from a group of Chilean private companies (Compañía
Manufacturera de Papeles y Cartones S.A., Compañía
General de Electricidad Industrial, Corporación Nacional del
Cobre de Chile, Compañía de Petróleos de Chile
S.A., Minera Escondida Limitada, Nova Gas International, Business
Design Associates, and Xerox de Chile S.A.) is also
acknowledged. A. S. Monaghan was supported by a Research
Studentship from the Agricultural and Food Research Council of the UK
(AFRC), and G. M. Mintenig was supported by AFRC Grant
LRG-111. F. V. Sepúlveda is in receipt of an
International Fellowship of the Howard Hughes Medical Institute and a
Cátedra Presidencial en Ciencias.
 |
FOOTNOTES |
Address for reprint requests: F. V. Sepúlveda, Departamento de
Medicina Experimental, Facultad de Medicina, Universidad de Chile,
Casilla 70058, Santiago-7, Chile.
Received 13 January 1997; accepted in final form 8 August 1997.
 |
REFERENCES |
1.
Ackerman, M. J.,
J. H. Widdicombe,
and
D. E. Clapham.
Hypotonicity activates a native chloride current in Xenopus oocytes.
J. Gen. Physiol.
103:
153-179,
1994[Abstract].
2.
Banderali, U.,
and
G. Roy.
Activation of K+ and Cl
channels in MDCK cells during volume regulation in hypotonic media.
J. Membr. Biol.
126:
219-234,
1992[Medline].
3.
Banderali, U.,
and
G. Roy.
Anion channels for amino acids in MDCK cells.
Am. J. Physiol.
263 (Cell Physiol. 32):
C1200-C1207,
1992[Abstract/Free Full Text].
4.
Barry, P. H.
JPCalc, a software package for calculating liquid junction potential corrections in patch-clamp, intracellular, epithelial, and bilayer measurements and for correcting junction potential measurements.
J. Neurosci. Methods
51:
107-116,
1994[Medline].
5.
Bear, C. E.
Phosphorylation-activated chloride channels in human skin fibroblasts.
FEBS Lett.
237:
145-149,
1988[Medline].
6.
Champigny, G.,
B. Verrier,
and
M. Lazdunski.
Ca2+ channel blockers inhibit secretory Cl
channels in intestinal epithelial cells.
Biochem. Biophys. Res. Commun.
171:
1022-1028,
1990[Medline].
7.
Cheeseman, C.
Role of intestinal basolateral membrane in absorption of nutrients.
Am. J. Physiol.
263 (Regulatory Integrative Comp. Physiol. 32):
R482-R488,
1992[Abstract/Free Full Text].
8.
Chen, J. H.,
H. Schulman,
and
P. Gardner.
A cAMP-regulated chloride channel in lymphocytes that is affected in cystic fibrosis.
Science
243:
657-660,
1989[Medline].
9.
Del Castillo, J. R.
The use of hyperosmolar intracellular-like solutions for the isolation of epithelial cells from guinea-pig small intestine.
Biochim. Biophys. Acta
901:
201-208,
1987[Medline].
10.
Diener, M.,
W. Rummel,
P. Mestres,
and
B. Lindemann.
Single chloride channels in colon mucosa and isolated colonic enterocytes of the rat.
J. Membr. Biol.
108:
21-30,
1989[Medline].
11.
Dietl, P.,
and
B. A. Stanton.
Chloride channels in apical and basolateral membranes of CCD cells (RCCT-28A) in culture.
Am. J. Physiol.
263 (Renal Fluid Electrolyte Physiol. 32):
F243-F250,
1992[Abstract/Free Full Text].
12.
Díaz, M.,
M. A. Valverde,
C. F. Higgins,
C. Rucareanu,
and
F. V. Sepúlveda.
Volume-activated chloride channels in HeLa cells are blocked by verapamil and dideoxyforskolin.
Pflügers Arch.
422:
347-353,
1993[Medline].
13.
Dreinhofer, J.,
H. Gögelein,
and
R. Greger.
Blocking kinetics of Cl
channels in colonic carcinoma cells (HT-29) as revealed by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB).
Biochim. Biophys. Acta
260:
C51-C63,
1991.
14.
Fischer, H.,
K.-M. Kreusel,
B. Illek,
T. E. Machen,
U. Hegel,
and
W. Clauss.
The outwardly rectifying Cl
channel is not involved in cAMP-mediated Cl
secretion: evidence for a very low conductance Cl
channel.
Pflügers Arch.
422:
159-167,
1992[Medline].
15.
Frizzell, R. A.,
and
D. R. Halm.
Chloride channels in epithelial cells.
In: Current Topics in Membranes and Transport, edited by S. I. Helman,
and W. Van Driessche. New York: Academic, 1990, p. 247-282.
16.
Frizzell, R. A.,
G. Rechkemmer,
and
R. L. Shoemaker.
Altered regulation of airway epithelial cell chloride channels in cystic fibrosis.
Science
233:
558-560,
1986[Medline].
17.
Giraldez, F.,
K. J. Murray,
F. V. Sepúlveda,
and
D. N. Sheppard.
Characterization of a phosphorylation-activated Cl
selective channel in isolated Necturus enterocytes.
J. Physiol. (Lond.)
416:
517-537,
1989[Abstract].
18.
Giraldez, F.,
F. V. Sepúlveda,
and
D. N. Sheppard.
A chloride conductance activated by adenosine 3',5'-cyclic monophosphate in the apical membrane of Necturus enterocytes.
J. Physiol. (Lond.)
395:
597-623,
1988[Abstract].
19.
Gögelein, H.
Chloride channels in epithelia.
Biochim. Biophys. Acta
947:
521-547,
1988[Medline].
20.
Gray, M. A.,
A. Harris,
L. Coleman,
J. R. Greenwell,
and
B. E. Argent.
Two types of chloride channel on duct cells cultured from human fetal pancreas.
Am. J. Physiol.
257 (Cell Physiol. 26):
C240-C251,
1989[Abstract/Free Full Text].
21.
Halm, D. R.,
G. R. Rechkemmer,
R. A. Schoumacher,
and
R. A. Frizzell.
Apical membrane chloride channels in a colonic cell line activated by secretory agonists.
Am. J. Physiol.
254 (Cell Physiol. 23):
C505-C511,
1988[Abstract/Free Full Text].
22.
Hamill, O. P.,
A. Marty,
E. Neher,
B. Sakmann,
and
F. J. Sigworth.
Improved patch-clamp techniques for high-resolution current recording from cell and cell-free membrane patches.
Pflügers Arch.
391:
85-100,
1981[Medline].
23.
Hanrahan, J. W.,
W. P. Alles,
and
S. A. Lewis.
Single anion-selective channels in basolateral membrane of a mammalian tight epithelium.
Proc. Natl. Acad. Sci. USA
82:
7791-7795,
1985[Abstract].
24.
Hayslett, J. P.,
H. Gögelein,
K. Kunzelmann,
and
R. Greger.
Characteristics of apical chloride channels in human colon cells (HT29).
Pflügers Arch.
410:
487-494,
1987[Medline].
25.
Jackson, P. S.,
and
K. Strange.
Characterization of the voltage-dependent properties of a volume-sensitive anion conductance.
J. Gen. Physiol.
105:
661-676,
1995[Abstract].
26.
Jackson, P. S.,
and
K. Strange.
Single-channel properties of a volume-sensitive anion conductance. Current activation occurs by abrupt switching of closed channels to an open state.
J. Gen. Physiol.
105:
643-660,
1995[Abstract].
27.
Jordan, V. C.
Biochemical pharmacology of antiestrogen action.
Pharmacol. Rev.
36:
245-276,
1984[Medline].
28.
Krick, W.,
J. Disser,
A. Hazama,
G. Burckhardt,
and
E. Fromter.
Evidence for a cytosolic inhibitor of epithelial chloride channels.
Pflügers Arch.
418:
491-499,
1991[Medline].
29.
Krouse, M. E.,
C. Haws,
Y. Xia,
R. H. Fang,
and
J. J. Wine.
Dissociation of depolarization-activated and swelling-activated Cl
channels.
Am. J. Physiol.
267 (Cell Physiol. 36):
C642-C649,
1994[Abstract/Free Full Text].
30.
Kubo, M.,
and
Y. Okada.
Volume-regulatory Cl
channel currents in cultured human epithelial cells.
J. Physiol. (Lond.)
456:
351-371,
1992[Abstract].
31.
Kunzelmann, K.,
H. Pavenstadt,
and
R. Greger.
Properties and regulation of chloride channels in cystic fibrosis and normal airway cells.
Pflügers Arch.
415:
172-182,
1989[Medline].
32.
Kunzelmann, K.,
M. Tilmann,
C. P. Hansen,
and
R. Greger.
Inhibition of epithelial chloride channels by cytosol.
Pflügers Arch.
418:
479-490,
1991[Medline].
33.
Li, M.,
J. D. McCann,
C. M. Liedtke,
A. C. Nairn,
P. Greengard,
and
M. J. Welsh.
Cyclic AMP-dependent protein kinase opens chloride channels in normal but not cystic fibrosis airway epithelium.
Nature
331:
358-360,
1988[Medline].
34.
MacLeod, R. J.,
and
J. R. Hamilton.
Separate K+ and Cl
transport pathways are activated for regulatory volume decrease in jejunal villus cells.
Am. J. Physiol.
260 (Gastrointest. Liver Physiol. 23):
G405-G415,
1991[Abstract/Free Full Text].
35.
MacLeod, R. J.,
and
J. R. Hamilton.
Volume regulation initiated by Na+-nutrient cotransport in isolated mammalian villus enterocytes.
Am. J. Physiol.
260 (Gastrointest. Liver Physiol. 23):
G26-G33,
1991[Abstract/Free Full Text].
36.
Manning, S. D.,
and
A. J. Williams.
Conduction and blocking properties of a predominantly anion-selective channel from human platelet surface membrane reconstituted into planar phospholipid bilayers.
J. Membr. Biol.
109:
113-122,
1989[Medline].
37.
Paulais, M.,
and
J. Teulon.
cAMP-activated chloride channel in the basolateral membrane of the thick ascending limb of the mouse kidney.
J. Membr. Biol.
113:
253-260,
1990[Medline].
38.
Paulmichl, M.,
M. Gschwentner,
E. Woll,
A. Schmarda,
M. Ritter,
M. Kanin,
H. Ellemunter,
W. Waitz,
and
P. Deetjen.
Insight into the structure-function relation of chloride channels.
Cell. Physiol. Biochem.
3:
374-387,
1993.
39.
Roy, G.,
and
C. Malo.
Activation of amino acid diffusion by a volume increase in cultured kidney (MDCK) cells.
J. Membr. Biol.
130:
83-90,
1992[Medline].
40.
Roy, G.,
and
R. Sauvé.
Effect of anisotonic media on volume, ion and amino-acid content and membrane potential of kidney cells (MDCK) in culture.
J. Membr. Biol.
100:
83-96,
1987[Medline].
41.
Rugolo, M.,
T. Mastrocola,
M. De Luca,
G. Romeo,
and
L. J. Galietta.
A volume-sensitive chloride conductance revealed in cultured human keratinocytes by 36Cl
efflux and whole-cell patch clamp recording.
Biochim. Biophys. Acta
1112:
39-44,
1992[Medline].
42.
Segal, A. S.,
E. L. Boulpaep,
and
A. B. Maunsbach.
A novel preparation of dissociated renal proximal tubule cells that maintain epithelial polarity in suspension.
Am. J. Physiol.
270 (Cell Physiol. 39):
C1843-C1863,
1996[Abstract/Free Full Text].
43.
Sepúlveda, F. V.,
F. Fargon,
and
P. A. McNaughton.
K+ and Cl
currents in enterocytes isolated from guinea-pig small intestinal villi.
J. Physiol. (Lond.)
434:
351-367,
1991[Abstract].
44.
Sheppard, D. N.,
M. A. Valverde,
F. Giraldez,
and
F. V. Sepúlveda.
Potassium currents of isolated Necturus enterocytes: a whole-cell patch-clamp study.
J. Physiol. (Lond.)
433:
663-676,
1991[Abstract].
45.
Simmons, N. L.
The effect of hypo-osmolarity upon transepithelial ion transport in cultured renal epithelial layers (MDCK).
Pflügers Arch.
419:
572-578,
1991[Medline].
46.
Solc, C. K.,
and
J. J. Wine.
Swelling-induced and depolarization-induced Cl
channels in normal and cystic fibrosis epithelial cells.
Am. J. Physiol.
261 (Cell Physiol. 30):
C658-C674,
1991[Abstract/Free Full Text].
47.
Stevens, B. R.,
J. D. Kaunitz,
and
E. M. Wright.
Intestinal transport of amino acids and sugars: advances using membrane vesicles.
Annu. Rev. Physiol.
46:
417-433,
1984[Medline].
48.
Strange, K.,
F. Emma,
and
P. S. Jackson.
Cellular and molecular physiology of volume-sensitive anion channels.
Am. J. Physiol.
270 (Cell Physiol. 39):
C711-C730,
1996[Abstract/Free Full Text].
49.
Sundaram, U.,
R. G. Knickelbein,
and
J. W. Dobbins.
pH regulation in ileum: Na+-H+ and Cl
-
exchange in isolated crypt and villus cells.
Am. J. Physiol.
260 (Gastrointest. Liver Physiol. 23):
G440-G449,
1991[Abstract/Free Full Text].
50.
Suzuki, S.,
M. Tachibana,
and
A. Kaneko.
Effects of glycine and GABA on isolated bipolar cells of the mouse retina.
J. Physiol. (Lond.)
421:
645-662,
1990[Abstract].
51.
Tilmann, M.,
K. Kunzelmann,
U. Frobe,
I. Cabantchik,
H. J. Lang,
H. C. Englert,
and
R. Greger.
Different types of blockers of the intermediate-conductance outwardly rectifying chloride channel in epithelia.
Pflügers Arch.
418:
556-563,
1991[Medline].
52.
Torres, R.,
G. A. Altenberg,
J. A. Copello,
G. Zampighi,
and
L. Reuss.
Preservation of structural and functional polarity in isolated epithelial cells.
Am. J. Physiol.
270 (Cell Physiol. 39):
C1864-C1874,
1996[Abstract/Free Full Text].
53.
Valverde, M. A.,
M. Díaz,
F. V. Sepúlveda,
D. H. Gill,
S. C. Hyde,
and
C. F. Higgins.
Volume-regulated chloride channels associated with the multidrug resistance P-glycoprotein.
Nature
355:
830-833,
1992[Medline].
54.
Valverde, M. A.,
G. M. Mintenig,
and
F. V. Sepúlveda.
Differential effects of tamoxifen and I
on three distinguishable chloride currents in T84 intestinal cells.
Pflügers Arch.
425:
552-554,
1993[Medline].
55.
Venglarik, C. J.,
A. K. Singh,
R. Wang,
and
R. J. Bridges.
Trinitrophenyl-ATP blocks Cl
channels in planar phospholipids bilayers. Evidence for two nucleotide binding sites.
J. Gen. Physiol.
101:
545-569,
1993[Abstract].
56.
Welsh, M. J.
An apical-membrane chloride channel in human tracheal epithelium.
Science
232:
1648-1650,
1986[Medline].
57.
Welsh, M. J.,
and
C. M. Liedtke.
Chloride and potassium channels in airway epithelia.
Nature
322:
467-470,
1986[Medline].
58.
Worrell, R. T.,
A. G. Butt,
W. H. Cliff,
and
R. A. Frizzell.
A volume-sensitive chloride conductance in human colonic cell line T84.
Am. J. Physiol.
256 (Cell Physiol. 25):
C1111-C1119,
1989[Abstract/Free Full Text].
59.
Wright, E. M.,
and
J. M. Diamond.
Anion selectivity in biological systems.
Physiol. Rev.
57:
109-156,
1977[Abstract/Free Full Text].
60.
Zhang, J. J.,
T. J. Jacob,
M. A. Valverde,
S. P. Hardy,
G. M. Mintenig,
and
F. V. Sepúlveda.
Tamoxifen blocks chloride channels. A possible mechanism for cataract formation.
J. Clin. Invest.
94:
1690-1697,
1994[Medline].
AJP Gastroint Liver Physiol 273(5):G1141-G1152
0193-1857/97 $5.00
Copyright © 1997 the American Physiological Society