© 1997 American Heart Association, Inc.
Articles |
Inhibitory Effects of Glibenclamide on Cystic Fibrosis Transmembrane Regulator, Swelling-Activated, and Ca2+-Activated Cl- Channels in Mammalian Cardiac Myocytes
From the Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno.
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Abstract Recent studies have provided evidence that sulfonylureas, in addition to blocking ATP-sensitive K+ (KATP) channels, also inhibit cystic fibrosis transmembrane regulator (CFTR) Cl- channels in epithelial and cardiac cells. The purpose of this study was to test whether the sulfonylurea glibenclamide might also inhibit other types of cardiac Cl- channels. Whole-cell patch-clamp techniques were used to compare the effects of glibenclamide on CFTR Cl- currents in guinea pig ventricular myocytes, swelling-activated Cl- currents in guinea pig atrial myocytes, and Ca2+-activated Cl- currents in canine ventricular myocytes. Glibenclamide markedly inhibited CFTR Cl- currents in a voltage-independent manner at 22°C, with estimated IC50 values of 12.5 and 11.0 µmol/L at +50 and -100 mV, respectively. The outwardly rectifying swelling-activated Cl- current in atrial cells was less sensitive to glibenclamide, and the block exhibited voltage dependence. At 22°C, the estimated IC50 values were 193 and 470 µmol/L at +50 and -100 mV, respectively, and block was enhanced at 35°C. Macroscopic Cl- currents activated by a rise in intracellular Ca2+, induced by either Ca2+-induced Ca2+ release or by external application of the Ca2+ ionophore A23187, were also markedly inhibited at 22°C by glibenclamide in a voltage-independent manner. The estimated IC50 values were 61.5 and 69.9 µmol/L at +50 and -100 mV, respectively. These results suggest that glibenclamide, an inhibitor of cardiac CFTR Cl- channels, also inhibits swelling-activated and Ca2+-activated Cl- channels at higher concentrations. The results also suggest that studies attributing the beneficial or deleterious effects of sulfonylurea compounds in the heart solely to blockade of KATP channels should use submicromolar concentrations of these agents to minimize possible secondary interactions with cardiac Cl- channels.
Key Words: Cl- channel • cystic fibrosis transmembrane regulator • swelling • Ca2+ • glibenclamide
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Introduction |
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The physiological significance of cardiac Cl- channels has attracted much attention, since cAMP-activated Cl- currents, which are now known to be due to the expression of an isoform of CFTR,1 2 3 were discovered in guinea pig ventricular myocytes.4 5 Other types of Cl- currents have been reported in the heart, including swelling-activated currents,6 7 8 Ca2+-activated current,9 10 protein kinase C–activated current,11 12 and ATP-activated current.13 14 15 The pharmacology of these channels is somewhat distinct. For instance, cAMP-activated Cl- currents are sensitive to anthracene-9-carboxylic acid and acrylaminobenzoates, such as diphenyl carboxylic acid and 5-nitro-2-(3-phenyl-propylamino)-benzoate, but not to stilbene derivatives, such as DIDS.16 17 18 19 On the other hand, swelling-activated Cl- currents and Ca2+-activated Cl- currents are inhibited by DIDS.18 20 The development of specific antagonists for the different types of cardiac Cl- channels is important, since such agents could prove to be instrumental in revealing their physiological role in the heart. In addition, the possibility has been raised that activation of Cl- current might be arrhythmogenic, since it can cause shortening of action potential duration and induction of repetitive activity.17 Therefore, specific blockers of Cl- channels may prove to be potentially useful as antiarrhythmic agents.
Previously, Sheppard and Welsh21 have shown that the sulfonylureas glibenclamide and tolbutamide are effective inhibitors of epithelial CFTR. Glibenclamide has also been shown to inhibit CFTR Cl- currents in guinea pig ventricular myocytes.22 Although these compounds are blockers of KATP channels,23 24 CFTR has nucleotide-binding domains and is included in the ATP-binding cassette superfamily.25 26 Recently, the sulfonylurea receptor, the target of glibenclamide and tolbutamide, was shown to be a member of the ATP-binding cassette superfamily.27 Therefore, in analogy to the KATP channel, it is presumed that sulfonylureas may modulate the ATP-binding site of the CFTR Cl- channel. However, the selectivity of such compounds for CFTR compared with other types of Cl- channels is uncertain.
The purpose of the present study was to compare the effectiveness of glibenclamide as an inhibitor of cAMP-activated CFTR Cl- currents and swelling-activated and Ca2+-activated Cl- currents in mammalian heart cells. Our data provide evidence showing that glibenclamide inhibits all three types of Cl- channels, although the potency and mechanism of block may be different. A preliminary report of these results has been published as an abstract.28
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Materials and Methods |
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Isolation of Guinea Pig and Canine Myocytes
Guinea pig ventricular and atrial myocytes were isolated using an enzyme dispersion technique. Hearts were quickly excised from adult male guinea pigs (350 to 450 g, 4 to 5 weeks old). The aorta was cannulated, and the heart was retrogradely perfused at a constant flow of 12 to 15 mL/min for >5 minutes with PSS-1 containing (mmol/L) NaCl 120, KCl 4.8, CaCl2 1.5, MgSO4 5.0, NaH2PO4 1.2, NaHCO3 25, taurine 13, and glucose 10, aerated with 95% O2/5% CO2 at 35°C. The solution was then switched to nominally Ca2+-free PSS-1 (5 minutes) and changed to the 50 µmol/L Ca2+-containing solution supplemented with collagenase (270 U/mL, class 2, Worthington) and protease (1 U/mL, type XXIV, Sigma Chemical Co), and the perfusate was recirculated for 15 to 17 minutes. Enzyme was removed by switching to 50 µmol/L Ca2+-containing PSS-1 without enzyme for 5 minutes. The heart was then cut down. The ventricular or atrial tissue was cut into small pieces and teased apart gently with forceps. Ca2+ was slowly reintroduced into the cell suspension to give a final concentration of 1.5 mmol/L Ca2+.
Adult mongrel dogs of either sex were anesthetized with pentobarbital sodium (45 mg/kg IV), and their hearts were quickly removed and placed in PSS-2 containing (mmol/L) NaCl 115, KCl 4.4, CaCl2 1.5, MgCl2 5, NaH2PO4 1, taurine 15, creatine 5, sodium pyruvate 5, HEPES 12, and glucose 15, pH 7.4 at 22°C. All cell-dispersion steps were carried out at room temperature. A section of the right ventricle was removed, and 1- to 2-mm-thick shavings of tissue were dissected from the epicardial region. Shavings of tissue were gently stirred in Ca2+-free PSS-2 for 20 minutes. Then tissue was minced and stirred in 100 µmol/L Ca2+-containing PSS-2 supplemented with collagenase (160 to 270 U/mL, class 2, Worthington) and protease (0.5 to 1 U/mL, type XXIV, Sigma) for 40 to 60 minutes. The pieces were then washed free of enzyme and resuspended in PSS-2 containing 100 µmol/L Ca2+, and single cells were obtained by gentle trituration. Ca2+ was slowly reintroduced into the cell suspension to give a final concentration of 1.5 mmol/L Ca2+.
Electrophysiological Techniques
Membrane currents were recorded using the whole-cell variant
of the patch-clamp technique.29 Patch pipettes were made
from borosilicate glass capillaries, pulled on a vertical micropipette
puller (model PP-83, Narishige), and fire-polished using a microforge
(model MF-83, Narishige). The pipettes had tip resistances of 2 to 4
M
when filled with internal solution.
Dissociated cells were placed in a chamber on the stage of an inverted
microscope and superfused with an external solution at 1.5 mL/min. The
Ag/AgCl wire and pellet were immersed in the pipette and the chamber,
respectively, connected to a patch-clamp amplifier (EPC-7, List
Electronic). A 3 mol/L KCl agar salt bridge between the bath and
Ag/AgCl reference electrode was used to minimize changes in liquid
junctional potential during the experiment to examine the
Cl- dependence. Junction potentials were zeroed before
formation of membrane-pipette seals. After a gigaohm seal (>10 G)
was obtained, the membrane was ruptured by application of negative
pressure. To avoid contamination by Na+ and
Ca2+ currents, the potential was usually held at 0 mV;
however, in experiments in which Ca2+-activated
Cl- currents were studied using double voltage-clamp
steps, the holding potential was -50 mV. In some experiments, to
facilitate the determination of concentration-response relations, a
rapid solution changer (RSC-100, Bio-Logic Co) was used. This device
allowed two or three cumulative doses of glibenclamide to be tested on
each cell, and steady state block was usually achieved within 2 to 3
minutes of drug exposure. To obtain the Cl- I-V
relation, whole-cell current was recorded during voltage pulses
(150 milliseconds) from the holding potential to potentials ranging
from -110 to +50 mV. In some cases, I-V relations were
measured using hyperpolarizing voltage ramps at -18.75 mV/s after a
1-second voltage step to +50 mV. Experiments were performed at room
temperature (
22°C) or 35°C. To keep the temperature at 35°C,
the external solution was heated in the bath chamber by a
servocontrolled heating device (Cell MicroControls). Data were filtered
at a frequency of 5 kHz and digitized on-line at 1 kHz using an
IBM-AT–compatible computer and pCLAMP 5.5 software (Axon
Instruments).
Solutions
The external and internal solutions used are listed in the
Table
. In most experiments, external and internal
solutions contained approximately equimolar Cl-. To
eliminate K+ currents, external K+ was replaced
with Cs+, and internal K+ was replaced with
Cs+ and tetraethylammonium
chloride. In order to elicit swelling-activated
Cl- currents, hyposmotic solutions (240 mOsm/kg
H2O) were made by removal of D-mannitol from
the normosmotic solution (300 mOsm/kg H2O). Osmolarity was
measured by using a freezing point depression osmometer (µOSMETTE,
Precision System Inc). In experiments measuring
Ca2+-activated Cl- currents, external
Ca2+ was raised to 2.5 or 5.0 mmol/L, and internal
EGTA was reduced to 0.4 or 0.8 mmol/L to increase the availability
of internal Ca2+. 4-Aminopyridine was added
externally to prevent 4-aminopyridine–sensitive
K+ current. When required, the anion substitute for
external Cl- was methane sulfonate.
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Glibenclamide, niflumic acid, and DIDS (all from Sigma) were prepared as 100 mmol/L stock solutions in DMSO. A23187 (Calbiochem) was prepared as a 10 mmol/L stock solution in ethanol. These stock solutions were diluted to the desired final concentration immediately before use. The final concentration of DMSO and ethanol was <0.1%, which, by itself, did not affect Cl- currents. All other compounds were purchased from Sigma.
Analysis of Data
The concentration-response curve for glibenclamide was
analyzed by fitting the following logistic equation:
R=[(Rmax-Rmin)xAn]/(IC50n+An)+Rmin,
where R is the degree of inhibition by glibenclamide, Rmax
is the maximal effect, Rmin is the minimal effect, A is the
concentration of glibenclamide, IC50 is the dose of
glibenclamide giving half-maximal inhibition, and n is the slope
factor.
Data are expressed as arithmetic mean±SEM. Statistical analysis was made by unpaired t test. A value of P<.05 was considered to be statistically significant.
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Results |
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Glibenclamide Inhibition of Cardiac CFTR Cl- Currents
Glibenclamide has been shown to block epithelial CFTR and cAMP-activated Cl- currents in cardiac cells. Using external and internal solutions with a symmetrical Cl- gradient, we first tested the effect of this compound in guinea pig ventricular myocytes at 22°C. Whole-cell current was recorded during the 150-millisecond step pulses applied from 0 mV to potentials ranging from -110 to +50 mV.
Isoproterenol (1 µmol/L) activated a time-independent
Cl- current (Fig 1A). The onset of the
effect of isoproterenol was within 2 minutes after switching the
perfusion solution. Within several minutes, the current reached a
plateau. The I-V relation obtained from five different cells
is shown in Fig 1B. The isoproterenol-induced current showed no
significant rectification, and the I-V reversed near 0 mV,
which is expected, since the Cl- gradient was symmetrical
(ECl, -0.1 mV). The maximal difference current was
1.91±0.34 and -3.33±0.55 pA/pF at positive (+50 mV) and negative
(-100 mV) potentials, respectively (n=5). Under these conditions, the
isoproterenol-induced current remained stable for at least 20 minutes
(103.7±3.5% at +50 mV and 96.2±10.6% at -100 mV [n=4] of
currents measured at 4 minutes).
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Glibenclamide (100 µmol/L) inhibited the isoproterenol-induced
Cl- current at both positive and negative membrane
potentials (Fig 1A
). The I-V curve from a total of five
cells is shown in Fig 1B. Glibenclamide (100 µmol/L) inhibited
outward and inward Cl- currents by an average of
84.9±3.1% and 85.1±4.8% at +50 and -100 mV, respectively. Fig 1C
shows the concentration-dependent block of isoproterenol-induced
Cl- current by glibenclamide with IC50 values
of 12.5 µmol/L at +50 mV and 11.0 µmol/L at -100 mV,
suggesting that block was voltage independent over this range of
potentials. These experiments confirm that glibenclamide is an
effective inhibitor of cardiac CFTR Cl-
channels.22
Glibenclamide Inhibition of Swelling-Activated
Cl- Currents
Experiments examining the effects of glibenclamide on
swelling-activated Cl- currents in guinea pig
atrial myocytes were performed using symmetrical Cl-
concentrations, comparable to the experimental conditions used to study
the isoproterenol-induced Cl- currents. Significant
background currents were not observed in normosmotic solutions (300
mOsm/kg H2O) in the atrial cells. Exposure to hyposmotic
solutions (240 mOsm/kg H2O) activated outwardly
rectifying currents (Fig 2). The time course of the
activation of the swelling-induced currents was variable from cell
to cell. In most cases, the time required for currents to
activate was 25 to 35 minutes at 22°C. Once the
swelling-activated current began to develop, currents continued
to increase until the solution was changed to normosmotic or
hyperosmotic solution (360 mOsm/kg H2O). After exposure to
hyperosmotic solution, the swelling-induced current was completely
diminished (data not shown). In four cells at 22°C, the mean reversal
potential of the current activated by hyposmotic solutions was
-5.4±2.3 mV, close to the predicted values of ECl under
these conditions (ECl, -2.5 mV).
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Addition of glibenclamide (100 µmol/L) caused a small inhibition
of the swelling-activated currents (Fig 2A and 2B). It
inhibited the outward current at +50 mV by 30.9±8.1% (n=4), whereas
it inhibited the inward current at -100 mV to a lesser degree
(12.1±4.4%, n=4). Thus, inhibition was significantly smaller than
that observed for isoproterenol-induced Cl- currents (Fig 1, P<.001). Fig 2C shows the concentration-response
relation for glibenclamide on swelling-activated
Cl- currents at positive and negative potentials. The
IC50 values were 193 µmol/L at +50 mV and 470
µmol/L at -100 mV. We also examined the effect of DIDS, which is
known to be a blocker of the swelling-activated
Cl- current. After washout of glibenclamide, 100
µmol/L DIDS caused marked inhibition of the
swelling-activated Cl- current. At positive
potentials, the blockade was more prominent than at negative potentials
(96.7% inhibition at +50 mV, 45.1% inhibition at -100 mV). Since the
swelling-induced current did not reach steady state completely during
these studies, the percent block by glibenclamide and DIDS may actually
be underestimated. Therefore, the values given represent a
lower limit for the true blocking effect of these compounds.
We also examined the effects of glibenclamide on the
swelling-activated Cl- current at 35°C. As shown
in Fig 3, glibenclamide (100 µmol/L) inhibited
the swelling-induced Cl- current more effectively at
35°C compared with 22°C, although inhibition of inward
Cl- currents at -100 mV was also smaller than that of
outward Cl- currents at +50 mV. Voltage-dependent block by
100 µmol/L DIDS was also observed, similar to that obtained at
22°C. The I-V relations for currents activated by
swelling before and after exposure to glibenclamide in three different
cells are also shown (Fig 3B). The swelling-induced Cl-
currents reversed near 0 mV and exhibited outward rectification in the
symmetrical Cl- solutions. Glibenclamide (100
µmol/L) inhibited the outward current at +50 mV by 81.6±4.1% and
inhibited the inward current at -100 mV by 54.5±5.9%.
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Glibenclamide Inhibition of Ca2+-Activated
Cl- Currents
Two approaches were used to study macroscopic
Ca2+-activated Cl- currents in canine
ventricular myocytes.9 10 The first approach
involved an examination of transient outward Cl- currents
activated by a rise in intracellular Ca2+ produced
by Ca2+-induced Ca2+ release triggered by
Ca2+ entry through voltage-dependent Ca2+
channels. In these experiments, 4-aminopyridine was
used to block transient outward K+ currents, and a double
voltage-clamp pulse was used to initially activate
Ca2+ currents (0 mV), followed by a step to +55 mV, near
ECa, to measure the outward Cl- tail current.
To facilitate activation of Cl- currents using this
protocol, low EGTA (0.4 to 0.8 mmol/L) was included in the
pipette, and two brief conditioning depolarizing pulses (10
milliseconds, +10 mV) were applied before each double-step test pulse
to ensure adequate filling of internal Ca2+ stores. The
second approach used was to directly elevate intracellular
Ca2+ by using the Ca2+ ionophore A23187 in
cells pretreated with BDM to prevent cell contraction. An advantage of
the latter technique was the ability to measure macroscopic
Ca2+-activated Cl- currents over a
wide range of potentials, allowing an examination of any potential
voltage dependence of block by glibenclamide.
Fig 4 shows the effects of glibenclamide on outward
Cl- currents activated by the double voltage-pulse
protocol in canine ventricular myocytes. Under control
conditions, outward Cl- currents were observed at +55 mV
after the activation of Ca2+ current at 0 mV. Glibenclamide
(100 µmol/L) caused partial inhibition of the outward
Cl- current and had little effect on Ca2+
currents activated at 0 mV. Subsequent addition of 100
µmol/L niflumic acid (n=4) or 100 µmol/L DIDS (n=1, not shown)
caused complete block of Ca2+-activated
Cl- currents. The glibenclamide-sensitive difference
current is shown in the right panel of Fig 4. In four cells using this
protocol, glibenclamide (100 µmol/L) inhibited the
Ca2+-activated Cl- current by an
average of 43.0±13.4%.
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We next examined the effects of glibenclamide on
Ca2+-activated Cl- currents directly
activated by external application of CaCl2 (5
mmol/L) and the Ca2+ ionophore A23187 (2 µmol/L) in
canine ventricular myocytes. In external solutions that
contained A23187 and were nominally Ca2+ free, the
application of voltage clamp steps ranging from -100 to +50 mV from a
holding potential of 0 mV activated only small leak currents
(Fig 5A). However, after exposure to 5 mmol/L
CaCl2, larger membrane currents were elicited by the same
voltage-clamp protocol, and outward currents were reduced and inward
currents were increased when the [Cl-]o was
reduced from 132 to 24 mmol/L. As shown in the I-V
plots in Fig 5B, the Ca2+-activated currents in
nearly symmetrical Cl- solutions were linear and reversed
(0.1±2.9 mV, n=3) near the estimated value of ECl (-1.5
mV). The linear I-V relation shown here resembles that
previously shown for small-conductance single
Ca2+-activated Cl- channels under
symmetrical Cl- conditions in the same
preparation.9 Reduction of external Cl- to
24 mmol/L caused the I-V to become inwardly rectifying,
and the reversal potential shifted to +47.0±10.9 mV (n=3), close to
the predicted value of ECl (44.8 mV). These data suggest
that the Ca2+-activated membrane currents observed
under these conditions exhibit properties consistent with a
significant Cl- permeability.
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Fig 6 shows that exposure of cells to glibenclamide
significantly reduced these Ca2+-activated
Cl- currents at 22°C under identical experimental
conditions using nearly symmetrical Cl- solutions. Fig 6A
shows the typical time courses of membrane currents at +50 and -100 mV
from two separate experiments activated by the addition of
5 mmol/L CaCl2 in the presence of A23187. Each panel
shows the result with or without subsequent exposure to glibenclamide
(100 µmol/L). Glibenclamide inhibited both outward and inward
Ca2+-activated Cl- currents. After
washout, the currents were recovered to some extent. The I-V
plots summarize the data obtained from six myocytes (Fig 6B). After
exposure to external CaCl2, the mean Cl-
current density was 3.46±0.48 pA/pF at +50 mV and
-6.99±0.48 pA/pF at -100 mV. In the presence of
glibenclamide (100 µmol/L), mean current density was
significantly (P<.05) reduced to 1.39±0.34 pA/pF at +50 mV
and -2.20±1.30 pA/pF at -100 mV. Fig 6C shows the
concentration-dependent block of Ca2+-activated
Cl- currents with IC50 values of 61.5
µmol/L at +50 mV and 69.9 µmol/L at -100 mV, suggesting no
apparent voltage dependence to the block.
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The main goal of the present study was to investigate whether sulfonylureas, in addition to their known ability to block cardiac KATP and CFTR Cl- channels, might also be effective inhibitors of other types of cardiac Cl- channels. To this end, we compared the effects of glibenclamide on cAMP-, swelling-, and Ca2+-activated Cl- currents in mammalian cardiac myocytes, using the conventional whole-cell patch-clamp technique. The results show that glibenclamide effectively inhibits cardiac CFTR Cl- channels and also inhibits swelling- and Ca2+-activated Cl- channels, albeit at higher concentrations. These results are consistent with an earlier report30 showing that glibenclamide, in addition to blocking CFTR Cl- channels, also inhibits outwardly rectifying intermediate-conductance Cl- channels in human intestinal carcinoma cells. This compound, therefore, over a narrow concentration range, may be a useful pharmacological probe for distinguishing CFTR from other types of cardiac Cl- channels.
Our results further suggest that some degree of caution should be exercised in some physiological and pathophysiological studies in the heart, which may attribute the beneficial or deleterious effects of sulfonylureas solely to their actions on KATP channels. Although the reported Kd for glibenclamide binding to KATP channels in heart ranges from 0.2 to 3.0 nmol/L,31 higher concentrations, well within the micromolar to 10-µmol/L range, have frequently been used in functional studies of ischemia,32 33 and the actual tissue concentrations achieved after in vivo intravenous administration of glibenclamide are uncertain.34 35 36 The relative potency of glibenclamide for inhibition of KATP channels compared with Cl- channels will be dependent on a number of factors, including temperature. At physiological temperatures, the potency of glibenclamide as a Cl- channel blocker will be expected to be even greater than the estimated IC50 values determined in the present study at 22°C. It is noteworthy that the functional consequences of blocking cardiac Cl- channels will be very similar to those produced by the blockade of KATP channels, specifically, a prolongation of action potential duration.22
Glibenclamide has previously been shown to block epithelial21 and cardiac22 CFTR channels. The IC50 values for the epithelial and cardiac channels were 22 and 25 to 38 µmol/L, respectively, which was close to the IC50 values estimated in the present experiment (11 to 12 µmol/L). The previous data are consistent with our own observations that 100 µmol/L glibenclamide causes nearly complete inhibition of cardiac CFTR Cl- currents at all membrane potentials examined. There is similarity between CFTR and KATP channels in that they are both regulated by intracellular ATP levels,23 37 38 and CFTR and the sulfonylurea receptor have been shown to possess an ATP-binding site.26 39 Therefore, the possibility exists that glibenclamide may bind to the ATP-binding domain of CFTR.21 Recently, blockade of single epithelial CFTR Cl- channels by the sulfonylurea compounds tolbutamide40 and glibenclamide41 has been attributed to their interactions with the nucleotide bound open state of the channel to cause a fast flickery type of channel block. Thus, the voltage-independent block of macroscopic CFTR Cl- currents by glibenclamide that we observed is consistent with the observation that unitary CFTR channels exhibit little, if any, voltage dependence of open probability.42
We also examined the effects of glibenclamide on swelling-activated Cl- currents in guinea pig atrial myocytes. At 22°C, glibenclamide caused a small inhibition of the current, which was potentiated at 35°C. The constructed concentration-response relation indicates that >100 µmol/L of glibenclamide is required to inhibit the swelling-activated Cl- currents at 22°C. In contrast to the block of cAMP-activated Cl- currents, glibenclamide blockade of the swelling-activated current was voltage dependent, with greater inhibition of current at positive compared with negative potentials at any concentration tested. Such voltage-dependent block resembles that produced by other inhibitors of swelling-activated Cl- currents like DIDS and dinitrostilbene disulfonic acid.20 43 The similarity among these blockers suggests that they may inhibit swelling-activated currents through a common mechanism. It is possible that the observed voltage dependence of glibenclamide block may be related to the outwardly rectifying properties of these channels. Although several volume-sensitive anion channels have been described in recent years, their electrophysiological and molecular characteristics are only beginning to be understood,44 and little is presently known about the mechanism responsible for rectification. Recent data from heart suggest that a 60-pS outwardly rectifying Cl- channel45 46 similar to that described in other tissues47 48 may be responsible for swelling-activated Cl- currents. Since these channels do not exhibit any noticeable voltage dependence of open probability,46 49 it seems unlikely that the voltage dependence of glibenclamide block observed can be attributed to an open channel–blocking mechanism. Alternatively, the voltage dependence of glibenclamide block might be related to the existence of a channel inactivation process that occurs at positive membrane potentials and is relieved at negative potentials.50 51 It is tempting to speculate that glibenclamide block of swelling-activated Cl- channels in heart might involve an intracellular nucleotide-binding site with homology to that found in CFTR and KATP channels. A role for intracellular ATP has yet to be demonstrated for swelling-activated Cl- currents in heart; however, such a site has been previously suggested for volume-sensitive Cl- channels in endothelial52 and rat glioma53 cells.
Two types of experimental protocols were used to examine the effect of glibenclamide on Ca2+-activated Cl- currents in canine ventricular cells, which are known to express in high-density Ca2+-activated Cl- channels9 10 but few, if any, CFTR Cl- channels.54 55 The first approach involved an examination of transient outward Cl- currents activated by a rise in intracellular Ca2+ produced by Ca2+-induced Ca2+ release triggered by Ca2+ entry through voltage-dependent Ca2+ channels. The cells were dialyzed with low Ca2+-buffering internal solution (low EGTA) and given a set of conditioning stimuli to refill Ca2+ stores. This procedure is important, since Ca2+ influx via voltage-dependent Ca2+ channels has been reported to be insufficient to cause activation of these channels.10 The outward Cl- currents activated using this procedure were significantly inhibited by 100 µmol/L glibenclamide. However, in these experiments, it is possible that the apparent block by glibenclamide observed may be due to a reduction in Ca2+ entry or Ca2+ release rather than a direct interaction with the channel. Therefore, we examined the effect of glibenclamide on Ca2+-activated Cl- currents directly activated by external application of CaCl2 and the Ca2+ ionophore A23187 in canine ventricular myocytes. The I-V relations and their sensitivity to changes in external Cl- verify that Cl- is the main charge carrier of these Ca2+-activated currents. Since glibenclamide also significantly inhibited Ca2+-activated Cl- currents under these conditions, we conclude that such an effect is likely due to a direct interaction of the compound with the channel and not due to an alteration in [Ca2+]i availability.
Similar to CFTR Cl- currents, with symmetrical [Cl-], the Ca2+-activated Cl- current I-V relations were linear over the voltage range examined (-100 to +50 mV), and the block by glibenclamide did not appear to exhibit any apparent voltage dependence at all concentrations tested. The apparent lack of voltage dependence of these macroscopic Ca2+-activated Cl- currents is consistent with the behavior of small-conductance (1.0- to 1.3-pS) Ca2+-activated Cl- channels recently identified in the same preparation.9 These channels resemble small-conductance Ca2+-activated Cl- channels, which have been identified in a number of smooth muscle and other types of cells (see Reference 5656 for review). It seems somewhat premature to speculate on whether a potential glibenclamide-binding site on Ca2+-activated Cl- channels may be analogous to the cytoplasmic nucleotide binding present in CFTR and KATP channels, since little is presently known about the molecular structure of these channels. However, the recent description of a cloned, 25- to 30-pS, niflumic acid–insensitive, Ca2+-activated Cl- channel from bovine tracheal epithelial cells57 failed to reveal any homologous nucleotide-binding site. Future studies should provide interesting new data on the nature of the glibenclamide-binding site of Ca2+-activated Cl- channels.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by National Institutes of Health grant HL-52803 (Dr Hume); canine ventricular myocardium was generously provided by National Institutes of Health Program Project DK-41315. We would like to thank Dr Lingyu Ye for expert assistance with the enzymatic dispersion of canine cardiac myocytes.
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Footnotes |
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Reprint requests to Joseph R. Hume, PhD, Department of Physiology & Cell Biology, University of Nevada School of Medicine, Manville Building/351, Reno, NV 89557-0046.
Previously published as preliminary results in abstract form (Circulation. 1996;94[suppl I]:I-641).
Received December 10, 1996; accepted May 1, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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