Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada 89557
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ABSTRACT |
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Swelling-activated or volume-sensitive
Cl currents are found in
numerous cell types and play a variety of roles in their function; however, molecular characterization of the channels is generally lacking. Recently, the molecular entity responsible for
swelling-activated Cl
current in cardiac myocytes has been identified as ClC-3. The goal of
our study was to determine whether such a channel exists in smooth
muscle cells of the canine colon using both molecular biological and
electrophysiological techniques and, if present, to characterize its
functional and molecular properties. We hypothesized that ClC-3 is
present in colonic smooth muscle and is regulated in a manner similar
to the molecular entity cloned from heart. Indeed, the ClC-3 gene was
expressed in colonic myocytes, as demonstrated by reverse transcriptase
polymerase chain reaction performed on isolated cells. The current
activated by decreasing extracellular osmolarity from 300 to 250 mosM
was outwardly rectifying and dependent on the
Cl
gradient. Current
magnitude increased and reversed at more negative potentials when
Cl
was replaced by
I
or
Br
. Tamoxifen
([Z]-1-[p-dimethylaminoethoxy-phenyl]-1,2-diphenyl-1-butene; 10 µM) and DIDS (100 µM) inhibited the current, whereas 25 µM niflumic acid, 10 µM nicardipine, and
Ca2+ removal had no effect.
Current was inhibited by 1 mM extracellular ATP in a voltage-dependent
manner. Cl
current was also
regulated by protein kinase C, as phorbol 12,13-dibutyrate (300 nM)
decreased Cl
current
magnitude, while chelerythrine chloride (30 µM) activated it under
isotonic conditions. Our findings indicate that a current activated by
hypotonic solution is present in colonic myocytes and is likely
mediated by ClC-3. Furthermore, we suggest that the ClC-3 may be an
important mechanism controlling depolarization and contraction of
colonic smooth muscle under conditions that impose physical stress on
the cells.
chloride channels; adenosine 5'-triphosphate; protein kinase C; myogenic response; gastrointestinal motility
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INTRODUCTION |
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THE ABILITY OF MECHANICAL stimuli to elicit smooth muscle contraction has been realized for almost 100 years (2), and the first simultaneous recordings of smooth muscle tension and membrane potential were provided almost one-half century ago (3). During the past decade, the cellular mechanisms underlying depolarization and contraction in response to mechanical distension (i.e., the myogenic response) have begun to be elucidated (5, 6, 11). Of particular importance, mechanosensitive ion channels are thought to transduce mechanical forces into cellular signals such as Ca2+ influx (5, 6). Mechanosensitive ion channels are present in visceral smooth muscle and may regulate their electrical activity (18, 34, 36).
Mechanical stimulation, applied in the form of hypotonic cell swelling,
potentiates the nonselective cation current activated by muscarinic
stimulation in smooth muscle cells isolated from the guinea pig ileum
without effect on other voltage-dependent channels such as outwardly
rectifying K+ channels or L-type
Ca2+ channels (34). Activation of
these nonselective cation channels would be expected to cause membrane
depolarization and contraction of smooth muscle. Indeed, such a
mechanism has been shown to mediate a portion of the myogenic response
of small arteries (23). The nonselective cation channels of arterial
myocytes can be activated directly by stretching isolated cells with
glass microelectrodes attached at each end (5, 6). Along with
nonselective cation currents,
Cl currents may also
participate in the depolarization response of smooth muscle to
mechanical stimuli, as the Nernst equilibrium potential for
Cl
(ECl) is
positive to the resting membrane potential in visceral smooth muscles
(1).
Mammalian cardiac myocytes possess a swelling-activated
Cl conductance (29), which
has been shown to affect electrical activity (31). A molecular entity
responsible for a Cl
current activated by cell swelling has recently been cloned from guinea
pig heart and expressed in 3T3 fibroblasts (8). The ClC-3 gene encodes
a swelling-activated Cl
channel that is a member of the gene superfamily that includes ClC-2
(17), another ion channel proposed to play a part in cellular volume
regulation (10). Neither ClC-2 nor other previous candidates for the
swelling-activated Cl
channel such as P-glycoprotein (32) or pICln (26) possess properties
consistent with a role for these channels as the current carrier in
native cells. Evidence supporting ClC-3 as the cardiac swelling-activated Cl
channel includes (8) 1) the
magnitude of current through cloned ClC-3 increases and decreases as
cells are exposed to hypotonic and hypertonic solutions, respectively;
2) the reversal potential of this
conductance shifts with
ECl;
3) ClC-3 channels are outwardly rectifying and more permeable to
I
than
Cl
;
4) DIDS, tamoxifen, extracellular
ATP, and phorbol 12,13-dibutyrate (PDBu) inhibit ClC-3 current; and
5) single-channel properties of
ClC-3 are similar to those in native cardiac myocytes. These properties
are similar to the native cardiac swelling-activated Cl
current. In addition, a
single amino acid mutation in cloned ClC-3 changed the anion
selectivity and rectification properties, suggesting that ClC-3 is
responsible for swelling-activated
Cl
current in mammalian
cardiac myocytes (8).
Swelling-activated Cl
channels are thought to be distributed ubiquitously in mammalian cells,
but only recently has functional expression of swelling-activated
Cl
currents been
demonstrated in smooth muscle cells (36, 37). However, ClC-3 current
and gene expression per se have not been demonstrated in visceral
smooth muscle. Cl
current
activated by hypotonic solution was recently described in guinea pig
antral smooth muscle, and this conductance was suggested as a mediator
of volume regulation (36).
Cl
currents may also be
involved in the response to mechanical stimulation of canine pulmonary
and renal artery smooth muscle cells, which express the ClC-3 gene
(37). The present study tests the responses of smooth muscle cells of
the gastrointestinal (GI) tract to hypotonic solutions to determine
whether a Cl
conductance is
activated. We have also investigated whether the properties of the
current activated by hypotonic solution are consistent with ClC-3, the
swelling-activated Cl
channel in cardiac myocytes (8). Molecular studies were also performed
on isolated smooth muscle cells to determine whether the ClC-3 gene is
expressed in visceral myocytes.
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MATERIALS AND METHODS |
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Preparation of smooth muscle cells. Mongrel dogs of either sex were killed with an overdose of pentobarbital sodium (100 mg/kg), and the abdomen was opened along a midline incision. The stomach and proximal colon were removed and placed in Krebs solution (see Solutions and reagents). Sheets of the tunica muscularis were dissected from the overlying mucosal elements, and strips of muscle were cut with a double-bladed scalpel and pinned out in a dissection dish filled with Ca2+-free Hanks' solution (see Solutions and reagents). Smooth muscle cells from the circular muscle layer of the fundus and colon were isolated as described previously (19).
Isolated smooth muscle cells from mouse colon were also prepared. Adult BALB/c mice of either gender were anesthetized with chloroform and killed by cervical dislocation. A segment of proximal colon was removed, placed in Ca2+-free Hanks' solution, opened along the mesenteric border, and washed free of fecal contents. Mucosa and submucosa were removed by blunt dissection, and the remaining tunica muscularis was covered with an enzyme solution containing 230 U/ml collagenase (Worthington Biochemical, Freehold, NJ) and 100 U/ml elastase (Sigma Chemical, St. Louis, MO) in Ca2+-free Hanks' buffer. Tissues were incubated 20-30 min at 37°C, washed free of enzymes with Ca2+-free Hanks' solution, and cut into six to eight pieces that were ~10-15 mm2. Tissue pieces were then triturated to create a cell suspension. The cells were stored at 4°C and used within 8 h.Current measurements.
Drops of cell suspensions were placed on a glass coverslip forming the
bottom of a 300-µl chamber mounted on an inverted microscope. Cells
were allowed to adhere to the coverslip and then solutions were
suffused by gravity. The bath solution flowed at a rate of ~3 ml/min,
and therefore, the solution in the chamber was exchanged ~10 times
per minute. "Giga seals" were made with fire-polished glass
pipettes having tip resistances of 3-4 M. The whole cell, dialyzed configuration of the patch-clamp technique was used to record
ionic currents. The headstage ground wire was connected to the bath via
an agar bridge containing 3 M KCl to prevent junction potential errors
when the Cl
concentration
was altered. An Axopatch 1-D amplifier and CV-4 headstage (Axon
Instruments, Foster City, CA) were used to measure ionic currents. The
currents were sampled at 4 kHz and filtered at 1 kHz. A personal
computer running pCLAMP software (version 5.5.1, Axon Instruments) was
used to collect data. All recordings were performed at room temperature
(22-25°C).
Solutions and reagents.
Nicardipine, PDBu, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine
(H-7), and chelerythrine chloride were purchased from Research Biochemicals (Natick, MA). CaCl2,
KCl,
KH2PO4,
NaCl, NaHCO3,
Na2HPO4, sucrose, and glucose were from Fisher Scientific (Fair Lawn, NJ), and
all other chemicals were from Sigma. Krebs solution contained (in mM)
125 NaCl, 5.9 KCl, 2.5 CaCl2, 1.2 MgCl2, 15.5 NaHCO3, 1.2 Na2HPO4,
and 11.5 glucose, with pH adjusted to 7.3-7.4 by bubbling with
95% O2-5%
CO2.
Ca2+-free Hanks' solution
contained (in mM) 125 NaCl, 5.36 KCl, 15.5 NaHCO3, 0.336 Na2HPO4,
0.44 KH2PO4,
10 glucose, 2.9 sucrose, and 11 HEPES, pH adjusted to 7.4 with NaOH.
Isotonic bath solution contained (in mM) 125 NaCl, 5 CsCl, 1.2 MgCl2, 2 CaCl2, 10 HEPES, 5 Tris, and 10 glucose, with pH adjusted to 7.4 with NaOH. Osmolarity of
all solutions was checked with a freezing point depression osmometer
(model 5004, Precision Systems). Isotonic bath osmolarity was adjusted
to 300 mosM with mannitol. Hypotonic bath solution had the same
composition as isotonic bath solution except that the NaCl
concentration was reduced to 100 mM and adjusted to 250 mosM with
mannitol. Hypertonic bath solution was prepared by adding 50 mosM
mannitol to the normal isotonic bath solution, thus giving a final
strength of 350 mosM. Extracellular
Cl was removed by equimolar
replacement with aspartate, isethionate, Br
, or
I
as indicated in Figs.
1-10 or text. The pipette solution contained (in mM) 100 tetraethylammonium (TEA) chloride, 10 HEPES, 2 EGTA, 5 Mg-ATP, 1 Na2GTP, and 2.5 phosphocreatine
and was brought to pH to 7.1 with TEA hydroxide. The osmolarity of the
pipette solution was adjusted to 300 mosM by adding mannitol.
Molecular biology techniques.
Single smooth muscle cells isolated from the various species and
tissues were differentiated from other cell types by their characteristic morphology; that is, smooth muscle cells were taken to
be those that were spindle-shaped with a length of 50-100 µm and
a width of 5-10 µm. Smooth muscle cells were collected by aspirating them into a wide-bore patch-clamp pipette. Sixty cells from
each source were collected and frozen in liquid nitrogen for molecular
biological work. Total RNA was prepared from colonic and fundus muscle
cells and tissue isolated from dogs and mice by use of the SNAP Total
RNA Isolation kit (Invitrogen, Carlsbad, CA) as per the manufacturer's
instructions. First-strand RNA (1 µg) was reverse transcribed by use
of an oligo(dT) 12-18 primer (500 µg/µl). Specific ClC-3
primers were used that anneal to the sequence near the carboxy terminus
of the amino acid sequence forward, nucleotides 1891-1911, and
reverse, 2130-2150 (gene accession no. U83464). The housekeeping
gene control sequence -actin was amplified from the same cDNA by
using primers that anneal at nucleotides 2382-2400 or
3071-3090 (gene accession no. V01217). Sense and antisense primers
(20 µM) were combined with cDNA and 1 mM dNTP, 40 mM
Tris · HCl (pH 8.3), 100 mM KCl, 3 units
Taq (Promega, Madison, WI), 1 Ampliwax Gem 100 (Perkin
Elmer, Foster City, CA), and RNase-free water to a final volume of 50 µl. PCR was performed in a COY II Thermal Cycler under the following
conditions: 32 cycles at 94°C, 1 min; 57°C, 30 s; 72°C, 1 min; and then, incubated at 72°C, 10 min. PCR products were
separated by electrophoresis on a 2% agarose gel. Quantitative PCR
(Q-PCR) was performed by use of the PCR MIMIC Construction Kit
(Clontech, Palo Alto, CA), which is based on a competitive PCR
approach, where nonhomologous engineered DNA standards (referred to as
PCR MIMIC) compete with target DNA for the same gene-specific primers.
PCR MIMICs were constructed for ClC-3 and the control standard
(
-actin), and competitive PCR was carried out by titration of sample
cDNA with known amounts of the desired nonhomologous PCR MIMIC
constructs; serial dilutions of these constructs were added to PCR
amplification reactions. After PCR, products were separated by
electrophoresis on a 2% agarose gel and quantified by use of Molecular
Analyst (Bio-Rad, Hercules, CA). Experiments utilizing PCR to determine ClC-3 expression in visceral smooth muscle cells were performed on
cells isolated from at least three different animals. For Q-PCR, the
concentration of the target DNA was normalized to
-actin expression.
Statistical analyses.
Data are expressed as means ± SE from
n number of cells (or
n number of animals for Q-PCR).
Current-voltage
(I-V)
relationships were constructed from peak currents. Currents in isotonic
solutions were subtracted from currents in hypotonic solutions to
calculate difference currents. The time at which the difference current reached maximum at the most positive test potential was determined, and
all current amplitudes were measured at this time point. All statistical analyses were performed using SigmaStat 1.0 software (Jandel, San Rafael, CA).
I-V
relationships were compared by two-way repeated-measures ANOVA. In all
statistical analyses, values of P < 0.05 were considered statistically significant. When differences between
I-V
curves were found by ANOVA, Student-Newman-Keuls post hoc test was used
to identify voltages at which the
I-V
curves might differ. One-way ANOVA was used to compare reversal
potentials and maximum currents in
Cl replacement experiments,
and Bonferroni post hoc test was used to discern differences from
control. For Q-PCR experiments, one-way ANOVA was used to compare gene
expression among the groups, and Student-Newman-Keuls post hoc test was
then used to identify differences between the groups.
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RESULTS |
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Whole cell current activated by hypotonic solution.
When myocytes isolated from the circular layer of the canine colon were
exposed to hypotonic solution (osmolarity decreased from 300 to 250 mosM; pipette osmolarity 300 mosM), outwardly rectifying currents
developed. Experiments were performed to determine the nature of this
current. Cells were dialyzed with 100 mM TEA chloride to block
K+ currents and the outward flow
of monovalent cations through the nonselective cation channels that are
known to be present in colonic myocytes (21). Cells were held at
40 mV and stepped from
100 to +120 mV in 20-mV steps
before and after exposure to hypotonic solution. Subtraction of
currents in isotonic and hypotonic solution yielded difference currents
that reversed at an average of
9.8 ± 0.9 mV (Fig.
1B,
inset), close to the
ECl (calculated
to be
8 mV). A current with the same characteristics and
reversal potential was observed in identical experiments conducted on
isolated muscle cells from the circular layer of the canine fundus and
mouse proximal colon (n = 3 each, data
not shown). Hypotonic solution activated an outwardly rectifying
current that reversed at
10.2 ± 0.8 mV (n = 3) in the presence of a
symmetrical 100 mM TEA
(ECl =
8 mV), ruling out the possibility that nonselective cation channels carry
the current or that extracellular
Na+ influences the current. Also,
correcting solution osmolarity with sucrose produces the same effects
as mannitol; that is, hypotonic and hypertonic solutions made with
sucrose increase and decrease current amplitude, respectively
(n = 2, data not shown).
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Time and concentration dependence of the
Cl current.
Upon exposure to hypotonic solution,
Cl
current increased as a
function of osmolarity and time (Fig. 4).
Three cells were exposed to solutions ranging from 250 to 300 mosM.
During exposure to each solution, the cells were stepped from
40
mV to potentials ranging from
100 to +120 mV in 20-mV steps.
There was an inverse relationship between the osmolarity of the
solution and current magnitude, because lowering the osmolarity
increased Cl
current in a
concentration-dependent manner (Fig.
4B). After exposure to hypotonic
solution, Cl
current
developed with a half time of 2.1 ± 0.2 min and reached an apparent
steady-state level within 5-10 min. In four additional experiments, we studied the reversal of the activated
Cl
current when cells were
returned to isotonic solution. After full activation of the
Cl
current, the time for
recovery in isotonic solution was 10.3 ± 1.7 min. Experiments
showing the time course of the development of the
Cl
current are summarized
in Fig. 4, C and
D.
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Pharmacology of the Cl current
activated by hypotonic solution.
We tested the effects of known
Cl
channel inhibitors at
potentials from
100 to +120 mV on the current activated by
hypotonic solution. After induction of the
Cl
current, cells were
treated with tamoxifen (10 µM; n = 3), DIDS (100 µM; n = 3), or
niflumic acid (25 µM; n = 3). DIDS
and tamoxifen reduced the
Cl
current (Fig.
5). Niflumic acid, a cyclooxygenase
inhibitor and potent inhibitor of
Ca2+-activated
Cl
channels, did not
attenuate Cl
current (data
not shown). Tamoxifen, an antiestrogen compound, inhibits
swelling-activated Cl
channels. Tamoxifen decreased significantly the magnitude of the
Cl
current activated by
hypotonic solution at both positive and negative test potentials, as
has been shown in vascular smooth muscle (37). The tamoxifen-sensitive
Cl
current reversed at
8.8 ± 2.7 mV (Fig. 5B).
In contrast, DIDS, a stilbene derivative that antagonizes various anion
channels and transporters, produced a striking effect on the outward
Cl
current but much less
inhibitory effect on the inward current, as is shown in the example in
Fig. 5C and has been demonstrated previously (8, 36, 37). In the three experiments with DIDS, the
DIDS-sensitive Cl
current
reversed at
12.3 ± 1.5 mV. The pharmacology of
Cl
current induced by
hypotonic solution was also examined within the physiological range of
potentials by stepping cells in 6-mV increments from
80 to +10
mV (Fig. 5D). The tamoxifen- and
DIDS-sensitive Cl
currents
reversed at
10.8 ± 0.6 and
8.5 ± 2.7 mV,
respectively.
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Ca2+
independence of the Cl current
activated by hypotonic solution.
Ca2+-activated
Cl
channels are present in
numerous smooth muscle cell types (20), so we performed experiments to
test whether the Cl
current
activated in hypotonic solution was
Ca2+ dependent. Resistance of the
current activated by hypotonic solution to 25 µM niflumic acid
suggests that it is not mediated by
Ca2+-activated
Cl
channels; however, this
does not rule out some other dependence on
Ca2+. Replacement of extracellular
Ca2+ with
Mn2+ or addition of nicardipine
has been shown in previous studies to reduce currents through
Ca2+-dependent conductances in
canine colonic muscle cells (4). Replacement of extracellular
Ca2+ with equimolar
Mn2+ had no effect on the
Cl
current activated by
hypotonic solution (Fig. 6,
A and
B). The lack of dependence on
Ca2+ entry was confirmed in the
physiological range of membrane potentials, and data from these
experiments are displayed in Fig. 6B,
inset. Cl
current in the presence
of Ca2+ or
Mn2+ reversed at
11.7 ± 0.9 and
11.7 ± 1.9 mV, respectively. There was no
significant difference in the magnitude or the reversal potential of
the Cl
current in the
presence and absence of Ca2+.
Addition of nicardipine (10 µM) to inhibit
Ca2+ entry through L-type
Ca2+ channels in these cells (35)
did not affect the ability of hypotonic solution to activate the
Cl
current (Fig. 6,
C and
D).
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Inhibition of Cl current by
extracellular ATP.
ClC-3 was recently suggested as a candidate for volume-activated
Cl
current in mammalian
cells (8). Extracellular ATP has been shown to attenuate current
carried by ClC-3 (8, 37) and other volume-sensitive
Cl
channels (14-16).
We tested the effects of ATP on the
Cl
current activated by
hypotonic solution in canine colonic myocytes. ATP (1 mM) reduced the
amplitude of the Cl
current
activated by hypotonic solution (Fig.
7A). In
another volume-sensitive Cl
channel, pICln, this effect has been proposed to be due to ATP binding
to a nucleotide binding site in the area of the predicted pore region
(26). Jackson and Strange (15, 16) have suggested that ATP access to
such a nucleotide binding site would function as an open channel
blocker.
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Regulation of the Cl current by
protein kinase C.
ClC-3 has also been shown to be regulated by protein kinase C (PKC;
Ref. 8). We tested the effect of PKC activation on the current induced
by hypotonic solution in canine colonic myocytes (Fig.
8). PDBu (300 nM) reduced the amplitude of
the Cl
current recorded in
hypotonic solution (Fig. 8, A and
B). PDBu, a diacylglycerol mimetic,
activates PKC. Difference currents revealed that the current inhibited
by PDBu was an outwardly rectifying, inactivating current that reversed
at
10.3 ± 1.2 mV. Under isotonic conditions (bath and
pipette both 300 mosM), chelerythrine chloride (30 µM), a specific
inhibitor of PKC, activated a current similar to the
Cl
current induced by
hypotonic solution (Fig. 8, C and
D). The reversal potential of the
chelerythrine-activated Cl
current was
7.5 ± 2.6 mV. H-7 (100 µM), a broad-spectrum
kinase inhibitor, also activated a
Cl
current similar to the
current elicited by hypotonic solution (n = 2, data not shown).
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Evidence for basal activation of the
Cl current.
Under the control conditions of these studies (bath and pipette
solutions were both 300 mosM and of the same ionic composition as in
Fig. 1), we observed a current with the same properties as the
Cl
current activated by
hypotonic solution (250 mosM). Experiments were performed to determine
whether the basal current was due to the same
Cl
conductance activated
when cells were exposed to hypotonic solution. The current observed
under basal conditions was outwardly rectifying and reversed near
ECl (
8.8 ± 1.9 mV, n = 5). The basal
current was also dependent on the
Cl
gradient, as replacing
extracellular Cl
with
aspartate reduced current magnitude and shifted the reversal potential
to more positive values (15.0 ± 4.6 mV,
n = 5). Basal current was inhibited by
10 µM tamoxifen, and only the outward portion of the current was
blocked by DIDS (100 µM). The basal current, similar to the current
activated by hypotonic solution, was insensitive to niflumic acid (25 µM). ATP blocked the outward portion of the basal current in a
voltage-dependent manner. The magnitude of the basal
Cl
current was also reduced
when cells were exposed to hypertonic solution (increased to 350 mosM
with mannitol). Taken together, these data suggest that the same
population of channels responsible for
Cl
current elicited by
hypotonic solution were active under isotonic conditions. Results of
these experiments are shown in Fig. 9.
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Molecular studies indicating ClC-3 gene expression.
The electrophysiological studies suggest that phasic and tonic regions
of the GI tracts of two species possess a current that is activated by
exposure to hypotonic solution. This current has properties similar to
ClC-3 (8, 37). Molecular studies were therefore performed to determine
whether ClC-3 is expressed in colonic and fundus myocytes. RT-PCR was
performed on isolated smooth muscle cells. Qualitative RT-PCR indicated
that ClC-3 mRNA was present in colonic and fundus smooth muscle cells.
Quantitative RT-PCR was performed to determine the relative amount of
ClC-3 transcripts in mRNA isolated from murine and canine smooth
muscle; a representative gel used for digital analysis is shown in Fig. 10A.
These RT-PCR experiments were quantified by comparing ClC-3 gene
expression to the amount of -actin gene expression (37). These
quantitative RT-PCR experiments revealed significantly greater amounts
of ClC-3 transcripts in colonic smooth muscle than the fundus
(n = 3). No difference in ClC-3 gene
expression was observed between murine and canine colon. The normalized
values for ClC-3 expression are smaller (approximately one-tenth
magnitude) compared with those in canine renal and pulmonary arteries
(37); however, current density in canine colon is virtually identical
to that in canine vascular tissues. For example, current density for
the six cells shown in Fig. 1B was
5.0 ± 1.0,
2.1 ± 0.4, and 5.8 ± 0.9 pA/pF at
100,
40, and +40 mV, respectively. This apparent difference in ClC-3 gene expression between this and our previous study
is most likely due to the fact that more specific
-actin primers
were used here. However, at this time, differences in
-actin
isoforms between visceral and vascular smooth muscle cannot be
excluded, nor can an actual difference in gene expression.
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DISCUSSION |
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Volume-regulated or swelling-activated
Cl currents are present in
numerous cell types and play important roles in the control of cell
volume, pH, and membrane potential (9, 12). The molecular identity of
these anion channels has been a matter of debate (25). Recently, ClC-3
has been identified as the molecular entity that underlies the
swelling-activated Cl
current in guinea pig cardiac myocytes (8) and in canine vascular smooth muscle cells (37). A recent report has demonstrated a volume-sensitive Cl
current
in smooth muscle cells of the guinea pig gastric antrum (36), but there
have been no studies attempting to identify the molecular entity that
underlies swelling-activated
Cl
currents in visceral
smooth muscles. In this study, we demonstrated the presence, function,
and some aspects of the regulation of a
Cl
current activated by
hypotonic solution in GI smooth muscles. Tonic (canine fundus) and
phasic (murine and canine colon) muscles express ClC-3 and manifest a
Cl
current that was
1) activated by hypotonic solution,
2) outwardly rectifying and
demonstrated voltage-dependent inactivation,
3) selectively permeable such that
I
> Br
> Cl
,
4) blocked by DIDS and tamoxifen but
not affected by niflumic acid, 5)
inhibited by extracellular ATP, and
6) regulated by PKC. These are all
properties of ClC-3, suggesting that this molecular entity is
responsible for at least a portion of the response to hypotonic
solution and stretch in visceral myocytes.
Hypotonic solutions have been used to activate stretch-sensitive
currents in a number of studies. Osmotic perturbations may not
completely mimic the physical stresses imposed on smooth muscle cells
in the GI tract during distension; however, both cell swelling and
radial stretch could have similar effects on the surface membrane and
elements of the cytoskeleton. At present, little is known about how
cells sense and transduce changes in volume and respond by ionic
mechanisms (28). Davis and co-workers (5, 6) have mechanically
lengthened single smooth muscle cells and shown activation of
stretch-sensitive channels, but systematic studies comparing responses
of stretch-sensitive ionic channels to stimulation from osmotic
perturbation and radial stretch have not been performed in smooth
muscles. It is likely that the volume-sensitive
Cl current would be
activated by changes in cell shape, because this type of current may be
controlled by mechanisms involving deformation of cytoskeletal elements
(38).
We isolated the Cl current
activated by hypotonic solution under conditions that minimized
contributions from other ionic currents present in GI muscle cells:
K+ currents (removal of
intracellular and extracellular K+
by replacement with TEA), Ca2+
currents (replacement of extracellular
Ca2+ with
Mn2+ and addition of nicardipine),
and nonselective cation currents (replacement of
Na+ and
K+ with symmetrical TEA). Under
these conditions, the reversal potential of the current activated by
hypotonic solution shifted with the ECl,
demonstrating that the current is a
Cl
current. The current was
outwardly rectifying and possessed properties similar to the
swelling-activated Cl
current in cardiac myocytes and in fibroblasts transfected with ClC-3
(7, 8). A channel of ~40 pS was responsible for the current in those
studies. The single-channel conductance that mediates the
Cl
current activated by
hypotonic solution in smooth muscle cells has not yet been determined.
However, on the basis of the presence of ClC-3 gene transcripts in
vascular (37) and visceral (this study) smooth muscles and high degree
of similarity in characteristics to ClC-3 (7, 8), it is likely that
ClC-3 channels mediate a significant portion of the swelling-activated
current in smooth muscles.
The current attributed to ClC-3 was sensitive to known
Cl channel antagonists such
as tamoxifen and DIDS. DIDS was more effective at blocking outward
current than inward current as demonstrated in other studies (8, 36,
37). Tamoxifen, on the other hand, was an inhibitor of
Cl
current at all membrane
potentials as has been shown previously (37), suggesting that it may
provide a pharmacological tool to investigate the role of this
conductance in tissue experiments. Ca2+-activated
Cl
channels are present in
many smooth muscle cells (20); however, niflumic acid, nicardipine, and
Ca2+ replacement with
Mn2+ had no affect on the
Cl
current activated by
hypotonic solution, suggesting this conductance was not a
Ca2+-activated
Cl
current. In fact, there
is no evidence for a
Ca2+-activated
Cl
current in canine
colonic cells. Niflumic acid has been shown to inhibit volume-sensitive
Cl
channels in some other
cell types (33, 36); however, this difference might be explained by
cell and species differences.
Purinergic agonists are known to regulate the activity of numerous ion
channels. In some cases, extracellular ATP directly activates
conductances, such as P2x
receptors (30). In other cases, ATP regulates ion channels via changes
in intracellular Ca2+
concentration or through other second messenger systems. ATP inhibits
swelling-activated Cl
currents (8, 26, 37). At the present time, we do not know whether the
inhibition of ClC-3 by ATP is receptor mediated or due to direct access
of ATP to an intrinsic nucleotide binding site, as has been reported
for pICln and the anion channels of rat C6 glioma cells and skate
hepatocytes (14-16, 26). The physiological significance of the
effects of ATP on the swelling-activated
Cl
conductance is not
understood at present. ATP is proposed as inhibitory neurotransmitter
in many GI muscles (13), and it is possible that this agent could
participate in the regulation of myogenic activation in response to
distension.
PKC was shown to regulate cloned ClC-3 channels and the native
conductance found in cardiac muscle cells (8). PKC suppressed the
Cl current-activated
hypotonic solution in GI muscles in the present study, and inhibitors
of PKC (e.g., chelerythrine chloride and H-7) activated the current.
These results are consistent with the recently proposed role of
endogenous PKC and phosphatases in the regulation of ClC-3 channels by
cell volume (D. Duan, S. Cowley, B. Horowitz, and J.R. Hume,
unpublished observations). Thus agonists coupled to activation or
inactivation of PKC may have important effects on regulating responses
to stretch in the GI tract. For example, ClC-3 may provide negative
feedback to excitatory agonists coupled through
Gq and phospholipase C, such as
the neurotransmitters, acetylcholine, and tachykinins, since the net
inward current activated by stretch would be lessened by PKC inhibition
of the Cl
conductance.
Swelling-activated Cl
current in cardiac muscle cells may additionally be regulated by
tyrosine kinase (27). The role of tyrosine kinases in regulating ClC-3
in smooth muscle or in cells transfected with cloned ClC-3 has not been
evaluated.
It should be noted that myogenic tone and membrane potential of rat
cerebral arteries are sensitive to the same pharmacological agents that
inhibit ClC-3 current (24). DIDS and indanyloxyacetic acid 94 hyperpolarized and dilated pressurized arteries, whereas niflumic acid
was without effect. A similar effect may occur in GI smooth muscles;
that is, antagonists of ClC-3 may hyperpolarize and relax visceral
smooth muscles. This hypothesis remains to be tested and may require
ClC-3 to be activated by an agonist or stretch (analogous to conditions
in pressurized cerebral arteries) before an inhibitory effect of
Cl channel antagonists is
evident.
In summary, we have found that the ClC-3 gene is expressed in tonic and
phasic smooth muscles of the dog and mouse GI tracts. An outwardly
rectifying Cl current is
induced in visceral myocytes on exposure to hypotonic solution. This
current is strikingly similar to the swelling-activated Cl
conductance in mammalian
cardiac myocytes (7, 29) and the current resulting from the expression
of ClC-3 in fibroblasts (8). Our data suggest that ClC-3 channels
convey at least a portion of the response to changes in extracellular
osmolarity and possibly radial stretch in GI smooth muscle cells. In
the physiological range of membrane potentials, currents resulting from
ClC-3 activation are significant and as great in magnitude as currents
carried by L-type Ca2+ channels in
this tissue (35). Therefore, depending on the state of distension,
ClC-3 could provide a significant source of inward current in GI smooth
muscle cells and a depolarizing influence in intact muscles. This
conductance may have an important effect on the electrical activity of
GI muscles, as has been demonstrated in guinea pig cardiac myocytes and
rat cerebral arterioles (24, 31).
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ACKNOWLEDGEMENTS |
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We thank Drs. Dayue Duan, Jun Yamazaki, and Jim Kenyon for helpful advice during the completion of this study. The assistance of Nancy Horowitz in the collection of tissue and preparation of smooth muscle cells is appreciated.
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FOOTNOTES |
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This work was supported by National Institutes of Health Grants DK-41315 (to K. Sanders and G. M. Dick) and HL-49254 (to J. R. Hume and B. Horowitz). K. K. Bradley is a postdoctoral fellow of the American Heart Association, Nevada Affiliate.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address reprint requests to K. M. Sanders.
Received 16 April 1998; accepted in final form 1 July 1998.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Aickin, C. C.,
and
A. F. Brading.
Measurement of intracellular chloride in guinea-pig vas deferens by ion analysis, 36chloride efflux and micro-electrodes.
J. Physiol. (Lond.)
326:
139-154,
1982[Medline].
2.
Bayliss, W. M.
On the local reaction of the arterial wall to changes of internal pressure.
J. Physiol. (Lond.)
28:
220-231,
1902.
3.
Bülbring, E.
Correlation between membrane potential, spike discharge and tension in smooth muscle.
J. Physiol. (Lond.)
128:
200-221,
1955.
4.
Carl, A.,
H. K. Lee,
and
K. M. Sanders.
Regulation of ion channels in smooth muscles by calcium.
Am. J. Physiol.
271 (Cell Physiol. 40):
C9-C34,
1996
5.
Davis, M. J.,
J. A. Donovitz,
and
J. D. Hood.
Stretch-activated single-channel and whole cell currents in vascular smooth muscle cells.
Am. J. Physiol.
262 (Cell Physiol. 31):
C1083-C1088,
1992
6.
Davis, M. J.,
G. A. Meininger,
and
D. C. Zawieja.
Stretch-induced increases in intracellular calcium of isolated vascular smooth muscle cells.
Am. J. Physiol.
263 (Heart Circ. Physiol. 32):
H1292-H1299,
1992
7.
Duan, D.,
J. R. Hume,
and
S. Nattel.
Evidence that outwardly rectifying Cl channels underlie volume-regulated Cl
currents in heart.
Circ. Res.
80:
103-113,
1997
8.
Duan, D.,
C. Winter,
S. Cowley,
J. R. Hume,
and
B. Horowitz.
Molecular identification of a volume-regulated chloride channel.
Nature
390:
417-421,
1997[Medline].
9.
Franciolini, F.,
and
A. Petris.
Chloride channels of biological membranes.
Biochim. Biophys. Acta
1031:
247-259,
1990[Medline].
10.
Gründer, S.,
A. Thiemann,
M. Pusch,
and
T. J. Jentsch.
Regions involved in the opening of ClC-2 chloride channel by voltage and cell volume.
Nature
360:
759-762,
1992[Medline].
11.
Harder, D. R.
Pressure-dependent membrane depolarization in cat middle cerebral artery.
Circ. Res.
55:
197-202,
1984[Abstract].
12.
Hoffmann, E. K.,
and
L. O. Simonsen.
Membrane mechanisms in volume and pH regulation in vertebrate cells.
Physiol. Rev.
69:
315-382,
1989
13.
Hoyle, C. H. V.,
and
G. Burnstock.
Neuromuscular transmission in the gastrointestinal tract.
In: Handbook of Physiology. The Gastrointestinal System. Motility and Circulation. Bethesda, MD: Am. Physiol. Soc., 1989, sect. 6, vol. I, pt. 1, chapt. 13, p. 435-464.
14.
Jackson, P. S.,
K. Churchwell,
N. Ballatori,
J. L. Boyer,
and
K. Strange.
Swelling-activated anion conductance in skate hepatocytes: regulation by cell Cl and ATP.
Am. J. Physiol.
270 (Cell Physiol. 39):
C57-C66,
1996
15.
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].
16.
Jackson, P. S.,
and
K. Strange.
Characterization of the voltage-dependent properties of a volume-sensitive anion conductance.
J. Gen. Physiol.
105:
661-677,
1995[Abstract].
17.
Jentsch, T. J.
Chloride channels: a molecular perspective.
Curr. Opin. Neurobiol.
6:
303-310,
1996[Medline].
18.
Kirber, M. T.,
J. V. Walsh, Jr.,
and
J. J. Singer.
Stretch-activated ion channels in smooth muscle: a mechanism for the initiation of stretch-induced contraction.
Pflügers Arch.
412:
339-345,
1988[Medline].
19.
Langton, P. D.,
E. P. Burke,
and
K. M. Sanders.
Participation of Ca currents in colonic electrical activity.
Am. J. Physiol.
257 (Cell Physiol. 26):
C451-C460,
1989
20.
Large, W. A.,
and
Q. Wang.
Characteristics and physiological role of the Ca2+-activated Cl conductance in smooth muscle.
Am. J. Physiol.
271 (Cell Physiol. 40):
C435-C454,
1996
21.
Lee, H. K.,
C. W. Shuttleworth,
and
K. M. Sanders.
Tachykinins activate nonselective cation currents in canine colonic myocytes.
Am. J. Physiol.
269 (Cell Physiol. 38):
C1394-C1401,
1995
22.
Levitan, I.,
and
S. S. Garber.
Voltage-dependent inactivation of volume-regulated Cl current in human T84 colonic and B-cell myeloma cell lines.
Pflügers Arch.
431:
297-299,
1995[Medline].
23.
Meininger, G. A.,
and
M. J. Davis.
Cellular mechanisms involved in the vascular myogenic response.
Am. J. Physiol.
263 (Heart Circ. Physiol. 32):
H647-H659,
1992
24.
Nelson, M. T.,
M. A. Conway,
H. J. Knot,
and
J. E. Brayden.
Chloride channel blockers inhibit myogenic tone in rat cerebral arteries.
J. Physiol. (Lond.)
502:
259-264,
1997[Abstract].
25.
Okada, Y.
Volume expansion-sensing outward-rectifier Cl channel: fresh start to the molecular identity and volume sensor.
Am. J. Physiol.
273 (Cell Physiol. 42):
C755-C789,
1997
26.
Paulmichl, M.,
Y. Li,
K. Wickman,
M. Ackerman,
M. Peralta,
and
D. Clapham.
New mammalian chloride channel identified by expression cloning.
Nature
356:
238-241,
1992[Medline].
27.
Sorota, S.
Tyrosine protein kinase inhibitors prevent activation of cardiac swelling-induced chloride current.
Pflügers Arch.
431:
178-185,
1995[Medline].
28.
Strange, K.
Are all cell volume changes the same?
News Physiol. Sci.
9:
223-228,
1994.
29.
Tseng, G.-N.
Cell swelling increases membrane conductance of canine cardiac cells: evidence for a volume-sensitive Cl channel.
Am. J. Physiol.
262 (Cell Physiol. 31):
C1056-C1068,
1992
30.
Valera, S.,
N. Hussy,
R. J. Evans,
N. Adami,
R. A. North,
A. Surprenant,
and
G. Buell.
A new class of ligand-gated ion channel defined by P2X receptor for extracellular ATP.
Nature
371:
516-519,
1994[Medline].
31.
Vandenberg, J. I.,
G. C. Bett,
and
T. Powell.
Contribution of a swelling-activated chloride current to changes in the cardiac action potential.
Am. J. Physiol.
273 (Cell Physiol. 42):
C541-C547,
1997
32.
Vanoye, C. G.,
G. A. Altenberg,
and
L. Reuss.
P-glycoprotein is not a swelling-activated Cl channel: possible role as a Cl
channel regulator.
J. Physiol. (Lond.)
502:
249-258,
1997[Abstract].
33.
Voets, T.,
G. Droogmans,
and
B. Nilius.
Modulation of voltage-dependent properties of a swelling-activated Cl current.
J. Gen. Physiol.
110:
313-325,
1997
34.
Waniishi, Y.,
R. Inoue,
and
Y. Ito.
Preferential potentiation by hypotonic cell swelling of muscarinic cation current in guinea pig ileum.
Am. J. Physiol.
272 (Cell Physiol. 41):
C240-C253,
1997
35.
Ward, S. M.,
and
K. M. Sanders.
Upstroke component of electrical slow waves in canine colonic smooth muscle is due to nifedipine-resistant calcium current.
J. Physiol. (Lond.)
455:
321-337,
1992[Abstract].
36.
Xu, W. X.,
S. J. Kim,
I. So,
T. M. Kang,
J. C. Rhee,
and
K. W. Kim.
Volume-sensitive chloride current activated by hyposmotic swelling in antral gastric myocytes of the guinea-pig.
Pflügers Arch.
435:
9-19,
1997[Medline].
37.
Yamazaki, J.,
D. Duan,
R. Janiak,
K. Kuenzli,
B. Horowitz,
and
J. R. Hume.
Functional and molecular expression of volume-regulated chloride channels in canine vascular smooth muscle cells.
J. Physiol. (Lond.)
507:
729-736,
1998
38.
Zhang, J.,
T. H. Larsen,
and
M. Lieberman.
F-actin modulates swelling-activated chloride current in cultured chick cardiac myocytes.
Am. J. Physiol.
273 (Cell Physiol. 42):
C1215-C1224,
1997