Carbachol induces oscillations in membrane potential and
intracellular calcium in a colonic tumor cell line, HT-29
P.
Sand1,2,
T.
Svenberg2, and
B.
Rydqvist1
1 Department of Physiology and
Pharmacology, Karolinska Institutet, S-171 77 Stockholm; and
2 Department of Surgical Science,
Karolinska Sjukhuset S-171 76 Stockholm, Sweden
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ABSTRACT |
The patch-clamp technique was used to study the effects of
carbachol (CCh) on HT-29 cells. During CCh exposure, the cells (n = 23) depolarized close to the
equilibrium potential for
Cl
(
;
48 mV) and the membrane potential then started to oscillate
(16/23 cells). In voltage-clamp experiments, similar oscillations in
whole cell currents could be demonstrated. The whole cell conductance
increased from 225 ± 25 pS in control solution to 6,728 ± 1,165 pS (means ± SE, n = 17). In
substitution experiments (22 mM
Cl
in bath solution,
= 0 mV), the reversal potential changed from
41.6 ± 2.2 mV
(means ± SE, n = 9) to
3.2 ± 2.0 mV (means ± SE, n = 7).
When the cells were loaded with the calcium-sensitive fluorescent dye,
fluo 3, and simultaneously patch clamped, CCh caused a synchronous
oscillating pattern of fluorescence and membrane potential. In
cell-attached patches, the CCh-activated currents reversed at a
relative membrane potential of 1.9 ± 3.7 mV (means ± SE,
n = 11) with control solution in the
pipette and at 46.2 ± 5.3 mV (means ± SE,
n = 10) with a 15 mM
Cl
solution in the pipette.
High K+ (144 mM) did not change
the reversal potential significantly (P
0.05, n = 8). In inside-out patches,
calcium-dependent Cl
channels could be demonstrated with a conductance of 19 pS
(n = 7). It is concluded that CCh
causes oscillations in membrane potential that involve
calcium-dependent Cl
channels and a K+ permeability.
perforated whole cell technique; nystatin; chloride current; potassium current; fluo 3; chloride channels
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INTRODUCTION |
THE COLONIC MUCOSA exhibits the ability to
absorb but also to secrete electrolytes and water, the former mainly by
the surface cells, whereas the latter is thought to occur in the
colonic crypts (23), although there are reports stating that surface
cells also are capable of secretion (12). There is normally a net absorption of fluid from the intestinal contents, the result being formed fecal material in the rectum. In some pathological conditions, however, there is a net secretion of fluid resulting in more or less
severe diarrhea. The expulsion of chloride ions from the apical parts
of the crypt cells is thought to be the driving force for this
secretion. The movement of chloride ions cannot occur without a number
of different ion channels and transport systems. A previously suggested
(1) simplified secretory model consists of an apical
Cl
channel, a basolateral
2Cl
-K+-Na+
cotransport system, a basolateral
K+ channel, the
Na+-K+-ATPase,
and intercellular transport of sodium ions. The
Cl
secretion can be
activated through different intracellular pathways, e.g., via
activation of an adenylate cyclase, which increases the levels of
adenosine 3',5'-cyclic monophosphate (cAMP), and mobilization of intracellular calcium via the inositol
1,4,5-trisphosphate system. In this study we have used the colon
carcinoma cell line HT-29, which is a commonly used model for studies
of Cl
secretion, to
investigate the effects of carbachol (CCh), a parasympatheticomimetic agent known to increase intracellular calcium. With the perforated whole cell mode of the patch-clamp technique, we were able to show that
CCh caused a pattern of depolarizing oscillations of the membrane
potential, which was generated by
Cl
and
K+ permeabilities. With the
fluorescent dye fluo 3 we could further show that the intracellular
calcium concentration
([Ca2+]i)
also exhibited a pattern of oscillations, linking these events to the
changes in membrane potential. In inside-out patches, calcium-dependent Cl
channels could be
demonstrated.
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MATERIALS AND METHODS |
Cells.
The colon carcinoma cell line HT-29 was grown in Dulbecco's
modification of Eagle's medium supplemented with bovine calf serum (5% vol/vol), L-glutamine (2 mM), penicillin (105 U/l), and streptomycin (100 mg/l) and
was kept in an atmosphere of 5%
CO2 at 37°C. The cells were
seeded in petri dishes (35 mm, Corning Glass, Corning, NY). Only
isolated cells, grown for up to 36 h, were used for the patch-clamp
experiments (with or without simultaneous calcium measurements). For
calcium measurements on cell monolayers, experiments were performed up
to 7 days after subculture. The experiments were performed at room
temperature (20-22°C).
Solutions.
The normal extracellular bathing solution had the following composition
(in mM): 140 NaCl, 4 KCl, 1.5 CaCl2, 1 MgCl2, 5 glucose, and 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid (HEPES), adjusted to pH 7.4 with NaOH. In the substitution
experiments, Cl
was
replaced with glucuronate on a mole per mole basis. All other solutions
are described in RESULTS. Junction
potentials with different combinations of solutions were calculated
using the Axoscope program from Axon Instruments (Foster City, CA). The
membrane potentials were corrected for after the experiments were
performed. The nystatin solution was made as a stock solution with 5 mg
of nystatin dissolved in 100 µl of dimethyl sulfoxide (DMSO). From
this stock solution 10 µl were dissolved in 3 ml of pipette solution,
giving a final nystatin concentration of 180 µmol/l.
Whole cell measurements.
The pipettes were pulled from aluminosilicate glass (Hilgenberg,
Malsfeld, Germany; OD 1.6 mm, ID 1.28 mm) on a two-stage vertical
puller. The tips were covered with Sylgard (Dow Corning, Seneffe,
Belgium) and fire polished before use. The pipette resistance was
1.5-5 M
when filled with the different intracellular solutions. To obtain a stable whole cell recording and to minimize the disturbance of the intracellular solution, the perforated patch technique was used
with nystatin as the perforating agent (10). The tips were filled with
a nystatin-free solution, whereas the rest of the pipette was
backfilled with the nystatin solution described above. After formation
of a giga-seal (>2 G
), the capacitative current induced by a test
pulse slowly increased and became more rapid. From this current, the
series resistance could be calculated. The experiments started when the
series resistance was <30 M
, which was usually achieved within 10 min. The final series resistance was 7.5-30 M
, depending on the
size of the pipette. Before the experiments started, the series
resistance and whole cell capacitance were compensated for.
Conventional whole cell experiments, i.e., with light suction applied
to break the cell membrane, was not successful because of resealing of
the membrane with accompanying increase in access resistance to values
>50 M
. During the experiments, the chamber was continually
perfused hydrostatically with normal extracellular solution or one of
the test solutions. The perfusion system allowed an exchange of
solution within 5-15 s. Whole cell voltages and currents were
recorded using an Axopatch 200A patch-clamp amplifier together with the
pCLAMP (5.5 or 6.03) and Axotape software (Axon Instruments). The data
were also displayed on a pen recorder.
Cell-attached and inside-out experiments.
The experimental procedure was the same as described above concerning
pipettes, perfusion, data handling, and so forth. The pipettes were of
similar size as in the whole cell experiments.
Patch-clamp conventions.
In all experiments, a positive current is a membrane outward current,
i.e., positive ions moving from the inside of the membrane to the
outside or negative ions moving the opposite way.
Calcium measurements.
The
[Ca2+]i
was measured using the fluorescent calcium indicator fluo 3 (Molecular
Probes, Eugene, OR). The cells were incubated at room temperature in
8-10 µM fluo 3 containing 0.04% Pluronic and 0.4% DMSO for
30-60 min. The concentration and time used for incubation did not seem
to be critical for the loading of fluo 3. The fluorescence was
recorded using an inverted microscope (IM-35, Zeiss, Oberkochen,
Germany) equipped with an epifluorescent system. A 75-W halogen lamp
was used for fluorescence excitation, and a filter combination of 480, 40×/505/510-565 nm (bandwidth, dichroic, barrier; Chroma
Technology, Brattleboro, VT) was used for fluorescence excitation and
emission of fluo 3. The emitted fluorescent light was monitored using
a photomultiplier (R56000U, Hamamatsu, Shizuoka-ken, Japan) attached to
one of the microscope outlets. The signal from the photomultiplier was
fed into a current-voltage converter, and the potential was amplified
(10×) and low-pass filtered (3 Hz) using a direct current
amplifier (Princeton Applied Research, Princeton, NJ). The light
signal, together with the current or potential signal, was acquired and
stored in a computer using the pCLAMP acquisition system together with
the Axotape 2.0 software (Axon Instruments).
Chemicals.
All chemicals were purchased, unless otherwise indicated, from Sigma
Chemical (St. Louis, MO).
Data are presented as means ± SE unless otherwise stated.
Student's t-test was used for
statistical evaluation of mean values.
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RESULTS |
General properties.
The whole cell capacitance can be used as a measure of cell size. The
cells in these experiments had a capacitance of 11.5 to 23.8 pF,
corresponding to a cell diameter of 19.1-27.5 µm, assuming a
spherical cell and a specific membrane capacitance of 1 µF/cm2. The mean diameter as
measured from 23 cells was 22.6 ± 9.3 µm (means ± SD).
Membrane potential changes.
The resting membrane potential in 23 cells was
69.2 ± 12.4 mV (means ± SD). When the cells were exposed to 100 µM CCh, they depolarized to a mean value of
46.4 ± 8.6 mV (means ± SD, n = 23). This initial
depolarization was, in 16 of 23 cells, followed by a pattern of
membrane potential oscillations, i.e., the membrane potential
oscillated between the resting value and a depolarized value. Figure
1 shows six cells with different patterns
of oscillation. Some cells oscillated to the same depolarized potential
during the entire CCh exposure, whereas others showed oscillating peaks that subsided with time. The frequency of the oscillations varied between two and eight per minute. The depolarized value was close to
the equilibrium potential for
Cl
(
48 mV as
calculated with the Nernst equation, pipette solution in Fig. 1). To
further investigate if this depolarization was caused by a
Cl
permeability, the
following sets of experiments were performed.

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Fig. 1.
Oscillations of membrane potential
(Em) when cells
were exposed to 100 µM carbachol (CCh; as indicated by bars). Six
cells exhibited different patterns of oscillation (represented as
A-F). Cell D oscillates to same potential
(approximately 45 mV) during entire exposure to CCh, whereas
cell B shows only an initial
depolarizing peak to 41 mV and depolarizing peaks then decrease
in amplitude. Equilibrium potential for
Cl
( ) = 48 mV in these experiments. Note also that effect of CCh can
be reproduced in a single cell (B).
Pipette solution (in mM): 76 K2SO4,
10 KCl, 10 NaCl, 1 MgCl2, and 10 HEPES, adjusted to pH 7.25 with KOH. Junction potential correction
9 mV.
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Whole cell voltage-clamp experiments.
The same type of oscillations was also seen in 10 of 11 current
recordings. The frequency varied between one and six per minute. Figure
2 shows two cells voltage clamped froma
holding potential of
60 mV to the equilibrium potentials for
K+ and
Cl
(
90 and 0 mV,
respectively, see inset in Fig. 2)
(20). In this subset of experiments, the pipette solution contained (in mM) 145 KCl, 1.5 CaCl2, 1 MgCl2, and 10 HEPES to further
widen the gap between the equilibrium potentials for
K+ and
Cl
. Figure 2 shows that
there is both a Cl
and a
K+ current (at
90 and 0 mV,
respectively). Measured from 11 cells, the mean peak
Cl
current was 1,540 ± 174 pA, and the K+ current was 195 ± 32 pA. The onset of the two components differed with a mean time
to peak current of 5.7 ± 3.1 s
(Cl
current) and 18.5 ± 3.9 s (K+
current).

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Fig. 2.
Membrane current response of 2 cells exposed to CCh (100 µM). Cells
were voltage clamped between a holding potential ( 60 mV) and
equilibrium potentials for K+ and
Cl ( 90 mV and
0 mV, respectively). Pulse sequence
(inset) was delivered once every
second. There is both an inward
Cl current and an outward
K+ current, the former being
dominant, indicating a net outflow of
Cl .
Left cell shows an oscillating pattern
of holding ( 60 mV) current similar in frequency to potential
oscillations (see Fig. 1). Pipette solution (in mM): 144 KCl, 1.5 CaCl2, 1 MgCl2, and 10 HEPES adjusted to pH
7.25 with KOH. Junction potential correction was not made in these
experiments.
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Figure 3 shows the effect of 100 µM CCh
on the membrane currents when the cell was clamped using a 200-ms
voltage pulse protocol. The slope conductance between
80 and
40 mV increased from 200 pS in control solution to 10.4 nS in
CCh, and the reversal potential changed from
62 to
43 mV,
which is close to the equilibrium potential for
Cl
(
)
(Fig. 3B). The current shows outwardly rectifying properties in the negative voltage range. The
resting conductance of 17 cells in the voltage range
80 to
40 mV was 225 ± 25 pS, which increased to 6,728 ± 1,165 pS when the cell was exposed to 100 µM CCh. These findings (Figs. 2
and 3) suggest that a CCh-induced
Cl
permeability, together
with a K+ permeability, is
generating the oscillating response in HT-29 cells.

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Fig. 3.
Current-voltage relationship during CCh (100 µM) exposure. Cell was
clamped from a holding potential of 69 mV in 10-mV steps from
129 to 11 mV. Current was measured 40 ms from start of pulse. A: during CCh exposure, cells
exhibited a dramatic increase in current. A slow inactivation can be
seen during the first negative voltage steps.
B: current-voltage curves in control
solution ( ) and in CCh ( ). Slope conductance between 80
and 40 mV increased from 200 pS in control solution to 10.4 nS
in CCh solution.
Im, membrane
current. Pipette solution same as described in Fig. 1 legend. Junction
potential correction 9 mV.
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To further verify the existence of a CCh-induced
Cl
permeability, the
CCh-induced membrane currents at different potentials were measured in
normal and low Cl
. Figure
4A shows a
cell exposed for 10-20 s to 100 µM CCh at potentials from
109 to +37 mV. In control solution, the current changes
direction at approximately
44 mV, close to
(
48 mV). When the extracellular solution was changed with
respect to Cl
, from 149 to
22 mM (i.e., equal to the pipette solution), the reversal potential
changed to
5 mV, close to 0 mV, which could be expected with
equally high concentrations of
Cl
on both sides of the
membrane. The mean values for the reversal potentials were
41.6 ± 2.2 mV (control solution, n = 9)
and
3.2 ± 2.0 mV (low
Cl
solution,
n = 7). These values do not
differ statistically from the equilibrium potentials for
Cl
in the control solution
(
48 mV) and low-Cl
solution (0 mV; P < 0.02).

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Fig. 4.
Effect of low external Cl
on CCh-induced membrane currents. A:
cell was clamped to different potentials and exposed to 100 µM CCh
for 20 s in control solution (149 mM
Cl ) and low-
Cl solution (22 mM
Cl , equal to pipette
concentration; Cl replaced
with glucuronate), as indicated. B:
current-voltage relationships for evoked currents from 9 cells in
normal extracellular solution ( ) and 7 cells in
low-Cl solution ( ). In
normal extracellular solution, current reverses at 41.6 ± 2.2 mV, close to
.
In low-Cl solution,
reversal potential changed to 3.2 ± 2.0 mV.
Pipette solution same as described in Fig. 1 legend. Junction potential corrections were 9 mV (control solution) and 3 mV
(low-Cl solution).
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Calcium measurements.
Previous studies in HT-29 cell monolayers have shown that CCh causes an
increase in intracellular calcium. When calcium was omitted in the
extracellular solution during CCh exposure,
[Ca2+]i
decreased (5, 16). The same type of response in monolayers was seen in
the present study. Cells incubated with the fluorescent agent fluo 3
exhibited a dramatic increase in fluorescence at a wavelength of 525 nm, consistent with an increase in intracellular calcium when exposed
to 100 µM CCh (n > 50, not shown). In the majority of cells exposed to CCh investigated using
fluo 3, different types of oscillation in
[Ca2+]i
were observed. We were able to make simultaneous measurement of the
membrane potential and the intracellular calcium in six cells. There
was a close correlation between the changes in fluorescent-calcium signal and membrane potential (Fig.
5A).
Close scrutiny of the recordings showed that the increase in
fluorescence preceded the depolarization by a few seconds (Fig.
5B). These results suggest that the
underlying mechanism of the oscillating currents-potential changes
could be openings and closures of calcium-dependent
Cl
channels triggered by
CCh-induced calcium oscillations in the cytoplasm.

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Fig. 5.
Simultaneous measurements of
Em and
fluorescent signal from fluo 3
(F525; an indicator of
intracellular Ca2+) from an
isolated cell during exposure to CCh.
A: when exposed to 10 µM CCh
(indicated by bar),
fluorescent-Ca2+ signal and
Em oscillate with
same frequency. B: figure shows first
depolarizing peak (from A) on an
expanded time scale. It can be seen that the
Ca2+ signal precedes the potential
change by a few seconds. Pipette solution same as described in Fig. 1
legend. Junction potential correction 9 mV.
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Cell-attached experiments.
In this mode, and with standard extracellular solution in the pipette,
CCh induced membrane currents in 11 of 16 patches. To determine the
nature of this current, further experiments were performed with
different pipette solutions. Figure 6,
A and
B, shows two cells exhibiting
CCh-induced currents. In the first, the pipette contained normal
extracellular solution, and it can be seen that the current reverses at
~0 mV. With a low-Cl
solution in the pipette (15 mM), the reversal potential should, ideally, change 58 mV in the positive direction. However, in these experiments, the reversal potential changed to 46.2 ± 5.3 mV. The
use of 144 mM KCl in the pipette, on the other hand, did not change the
reversal potential. It can also be seen that the patch current
oscillates with approximately the same frequency as in the whole cell
experiments (cf. Figs. 1-2). Figure
6C summarizes the results using a
different pipette solution. The data is consistent with a
Cl
current being activated
by CCh in these patches.

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Fig. 6.
Cell-attached recordings during CCh (100 µM) exposure.
A: with standard extracellular
solution in pipette, current reversed at a pipette potential of about 0 mV. B: with a
low-Cl solution in pipette
(15 mM Cl , Cl replaced with
glucuronate), current reversed at a relative Em
(Vrel) of ~45 mV. Increasing
K+ (144 mM) in pipette solution
did not change reversal potential significantly
(P 0.05).
C: diagram summarizes findings with different pipette solutions.
Vrel = Vm Vrest, where
Vm is patch membrane
potential and Vrest is
resting membrane potential. Junction potential correction was 11 mV (15 mM Cl ) or 4 mV (144 mM
K+).
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Single-channel experiments.
To directly study the presence of calcium-activated
Cl
channels, we performed
experiments on inside-out patches. In excised patches, channel activity
could be seen in 19 of 20 patches. Figure 7
shows current from one of four patches exposed to solutions with
different calcium concentrations. The patch contained at least four
channels that opened and closed randomly when exposed to the normal
bathing solution. With the patch being clamped to 67 mV, the current
direction is positive, indicating a flow of chloride ions from the
pipette to the bath solution. When the calcium concentration was
changed to 50 nM [intracellular resting calcium level (5,
16)], the channels closed and stayed closed until the calcium was
again increased to 1,000 nM. Five hundred nanomolar calcium was not
sufficient to activate the channels. The current-voltage relationship
for the channels observed in the four patches exposed to low
calcium (as shown in Fig. 8), together with three patches not exposed to low calcium but examined at
several potentials, gave a channel conductance of 19 pS (regression analysis, r = 0.99). The current reversed at
2 mV, which is close to but statistically not equal
(P < .05) to the equilibrium potential for
Cl
(6 mV) using these
solutions. The equilibrium potential for
K+ was 90 mV.

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Fig. 7.
Continuous current trace of an inside-out patch with several
Cl channels. Membrane was
clamped to 67 mV. Lowering Ca2+
(to 50 nM) in normal extracellular solution facing inside of membrane
closed channels. Channels started to open again when Ca2+ was increased to 1,000 nM.
Inset: amplitude histogram from 1st part of 4th trace. Pipette solution (in mM): 110 KCl, 30 KOH, 1.5 CaCl2, 1 MgCl2, 11 EGTA, and 10 HEPES,
adjusted to pH 7.25 with KOH. Junction potential correction 7 mV.
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Fig. 8.
Current-voltage diagram of unitary currents from 7 patches with
Cl channels. Open symbols,
patches exposed to normal
Ca2+-containing extracellular
solution only. Filled symbols, 4 patches exposed to different
Ca2+ solutions (see Fig. 7).
Conductance 19 pS. Bath contained normal extracellular solution.
Pipette solution same as described in Fig. 7 legend. Junction potential
correction 7 mV.
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DISCUSSION |
Secretion in the large intestine is thought to be maintained by an exit
of chloride ions through Cl
channels in the apical membrane of the epithelial cells. The Cl
channels can be
stimulated by a variety of secretagouges, e.g., acetylcholine and
vasoactive intestinal peptide. These substances bind to receptors that
control intracellular second messengers such as calcium or cAMP through
G protein systems. The second messengers subsequently control the
Cl
channels.
HT-29 has been shown to contain several of the components necessary for
secretion, e.g., different types of
Cl
channels (6-9, 14,
15, 18, 19), basolateral
Na+-K+-Cl
exchange mechanisms and a
Na+-K+
pump (11). It has further been shown to contain muscarinic M3 receptors
(13) to respond with an increase in intracellular calcium and to
depolarize when exposed to the parasympatheticomimetic drug CCh (5, 16,
17).
When the cells in this study were exposed to CCh, the membrane
potential started to oscillate (in 16 of 23 cells) between the resting
value and a value close to the
(
48 mV). The type of oscillation varied between different cells (Fig. 1), but no systematic correlation between membrane potential, cell resistance, or cell capacitance and type of oscillation could be
revealed. One reason for the difference in behavior could be that cells
do not follow a uniform differentiation and that the cholinergic
receptor and/or ion channels have different properties. Intracellular stores of calcium might also differ between cells.
Oscillations of membrane potential have been reported in another
colonic tumor cell line, T84 (4). When exposed to CCh, T84 exhibited
oscillations, although in the hyperpolarizing direction, which was
caused by a calcium-induced K+
current.
The same authors could in a subsequent paper (3), using the perforated
patch-clamp technique, show that CCh actually activated three different
currents: an outward K+ current,
an outward nonselective cation current and, an inward Cl
current.
In this study, we have shown that the membrane potential oscillations
in the HT-29 cells were generated by a
Cl
conductance
(depolarizing phase) and a K+
conductance (repolarizing phase), as shown in Figs. 2 and 3. The
reversal potential for the CCh current (Figs. 3 and 4) was close to the
equilibrium potential for
Cl
(
48 mV). When the
extracellular Cl
was
lowered to 22 mM (equal to the pipette solution), the reversal potential changed to a value close to 0 mV (Fig. 4), in agreement with
the Nernst equation for a cell with a predominant
Cl
conductance. Using the
cell-attached mode with different pipette solutions, we could confirm
the macroscopic current in membrane patches. However, results from
cell-attached patches should be interpreted with caution, since the
membrane potential cannot be controlled and the intracellular ionic
composition is unknown. Bearing these cautions in mind, we conclude
that the currents seen in these patches are indeed carried by chloride
ions, since the reversal potential for the current changed ~45 mV
when the pipette concentration of
Cl
was lowered (from 149 to
15 mM) but did not change when the pipette concentration of
K+ was increased (from 4 to 144 mM). In most of the patches, it was difficult to see any distinct
conductance levels, either due to the existence of several different
channels being active at the same time, activation of low-conductance
channels, rapid transition between the open and closed state, or the
existence of multistate channels. Furthermore, as mentioned above, the
membrane potential cannot be controlled and any change in the membrane
potential will influence the current through the channels. At the
resting membrane potential, one would expect a larger current, since
the electrical driving force for chloride ions is greater than during the depolarized peaks when there is a very small net driving force.
In isolated patch experiments, with inside-out patches containing
multiple channels, the channels showed a strong dependence on the
calcium concentration with no activity at all in low calcium (50-500 nM) solutions. The conductance of the
Cl
channel was 19 pS (Fig.
8), and the reversal potential was
2 mV. The expected reversal
potential is 6 mV. The reason for this discrepancy is not clear. One
possible, but rather unlikely reason, is that the channel is not
completely selective for
Cl
, but, on the other hand,
there are no other anions present. Another possible explanation to this
deviation is that the junction potential correction used (based on the
Henderson equation) is not satisfactory, but at present we have no
alternative method. A third explanation is the presence of a
nonselective cation channel, but such a channel has, to our knowledge,
not been reported in HT-29.
It cannot be excluded that several calcium-dependent
Cl
channels are present in
this cell line. In earlier papers, a number of different
Cl
channels have been
reported. Hayslett et al. (9) found two types of
Cl
channels in
DBcAMP-stimulated cells. A small channel with a conductance of 15 pS
was found as well as a large rectifying channel with a conductance of
50 pS at positive clamp voltages and 32 pS at negative clamp voltages.
Kunzelmann et al. (15) reported the presence of a small (
4 pS)
Cl
channel, activated by
calcium, hypotonic cell swelling, and 8-BrcGMP. Morris and Frizzell
(18, 19) described a 15-pS
Cl
channel as the basis for
the whole cell-activated Cl
current. They also found the presence of a large multistate channel in
excised patches that was only poorly affected by calcium. The channel
presented in this study is probably the same as the one described by
Morris and Frizzell (18, 19).
In earlier work on HT-29 cells, it has been shown that CCh, at
concentrations similar to the ones used in this study, induces a
transient calcium peak followed by a plateau (16), but oscillations have, to our knowledge, not been reported before. It was concluded in
the earlier work that the transient peak was caused by release from
intracellular stores and the plateau level was dependent on an influx
from the extracellular solution (5, 16). However, in those experiments,
measurements were usually made on cell monolayers, and, in such a
situation, many cells contribute to the fluorescent signal, and
oscillations in an individual cell might be lost in the overall
fluorescence. Calcium oscillations have been demonstrated in T84 cells
by Devor et al. (2). They showed that oscillations were indeed seen in
isolated cells but not in cells in a subconfluent monolayer. They
suggested that this could be due to reorganization of cellular
organelles influencing the spatial relationship of the sinks and
sources within the cell or alterations in the availability of second
messengers during monolayer formation. Apart from HT-29 and T84 cells,
calcium oscillations are known to occur in other tissue types, e.g.,
mouse pancreatic acinar cells (22) and rat hepatocytes (24). In
experiments in which we simultaneously measured membrane potential and
intracellular calcium, it could be demonstrated that these two changed
concomitantly, the calcium signal preceding the potential signal by a
few seconds, linking the changes in intracellular calcium to membrane
potential.
The functional importance of the calcium oscillations in these cells
are not clear. In a study by Woods et al. (24), calcium oscillations
were observed in rat hepatocytes. They suggested that the frequency of
the calcium transients could determine the cellular response to the
hormone that initiated the calcium signal. From an energetic point of
view, calcium transients, compared with a long-lasting response,
decrease the intracellular calcium load and thus decrease the need for
ATP-consuming calcium pumps in the membrane of calcium-storing
organelles to be activated. It is also well known that sustained
increases of
[Ca2+]i
can be damaging to the cell (21).
In summary, we have found that CCh causes oscillations of the membrane
current in both the whole cell mode as well as in the cell-attached
mode. With a fluorescent technique we could also show that
intracellular calcium oscillated synchronously with the membrane
potential. The current had two components, an inward Cl
current and an outward
K+ current, the former being the
predominant one. Calcium-dependent Cl
channels (19 pS), the
ion channel probably responsible for the Cl
current, could be seen
in isolated patches.
 |
ACKNOWLEDGEMENTS |
The HT-29 cell line was kindly provided by Dr. J. Björk,
Dept. of Internal Medicine, Gastrointestinal Section, at the Karolinska Hospital.
 |
FOOTNOTES |
This work was supported by grants from Medical Research Foundation
Project 6838, Ruth och Richard Julins Fond, Glaxo, and grants from Karolinska Institutet.
Address for reprint requests: P. Sand, Dept. of Physiology and
Pharmacology, Karolinska Institutet, S-171 77 Stockholm, Sweden.
Received 21 October 1996; accepted in final form 12 June 1997.
 |
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