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(Circulation Research. 1997;81:101-109.)
© 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

Jun Yamazaki, , Joseph R. Hume

From the Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno.

	Abstract

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Abstract Recent studies have provided evidence that sulfonylureas, in addition to blocking ATP-sensitive K⁺ (K_ATP) 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 K_ATP 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

	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 K_ATP 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 K_ATP 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

	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.

View this table: [in this window] [in a new window]   Table 1. External and Internal Solutions

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=[(R_max-R_min)xAⁿ]/(IC₅₀ⁿ+Aⁿ)+R_min, where R is the degree of inhibition by glibenclamide, R_max is the maximal effect, R_min 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.

	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 (E_Cl, -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).

View larger version (19K): [in this window] [in a new window]   Figure 1. Glibenclamide (glib) inhibition of CFTR Cl- currents activated by isoproterenol (iso) in guinea pig ventricular myocytes. A, Raw membrane currents activated during 150-millisecond voltage steps from 0 mV to potentials ranging from -100 to 50 mV (10-mV increments) before (a) and after exposure to 1 µmol/L iso (b) and after subsequent exposure to 1 µmol/L iso and 100 µmol/L glib (c). Vertical and horizontal calibration is 400 pA and 20 milliseconds, respectively. B, I-V relations before and after exposure to glib (100 µmol/L). Difference currents were obtained by subtracting current before exposure to iso (1 µmol/L) from current after each treatment. Each data point represents mean±SEM (n=5). Mean cell capacitance was 139.3±22.1 pF. C, Concentration-response relations for the inhibition of CFTR Cl- currents by glib at +50 mV and -100 mV. Each data point represents mean±SEM. Numbers in parentheses indicate the number of cells tested at each concentration.

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 E_Cl under these conditions (E_Cl, -2.5 mV).

View larger version (20K): [in this window] [in a new window]   Figure 2. Effect of glibenclamide (glib) on swelling-activated Cl- currents in guinea pig atrial myocytes at 22°C. Cell swelling was induced by a hyposmotic solution (240 mOsm/kg H2O). A, Time course of the effect of 100 µmol/L glib and 100 µmol/L DIDS on Cl- currents at potentials of +50 and -100 mV. B, I-V relations in hyposmotic solution (240 mOsm/kg H2O) before and after exposure to glib (100 µmol/L). Membrane currents were activated by voltage ramps applied from 50 to -100 mV at the times indicated in panel A. C, Concentration-response relations for the inhibition of swelling-activated Cl- currents by glib at +50 and -100 mV. Each data point represents mean±SEM. Numbers in parentheses indicate the number of cells tested at each concentration.

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%.

View larger version (23K): [in this window] [in a new window]   Figure 3. Effect of glibenclamide (glib) on swelling-activated Cl- currents in guinea pig atrial myocytes at 35°C. Cell swelling was induced by a hyposmotic solution (240 mOsm/kg H2O). A, Time course of the effect of 100 µmol/L glib and 100 µmol/L DIDS on Cl- currents at potentials of +50 and -100 mV. B, I-V relations in the hyposmotic solution (240 mOsm/kg H2O) before and after exposure to glib. The currents were measured during 150-millisecond voltage steps from 0 mV to potentials ranging from -90 to 70 mV (10-mV increments). Difference currents were obtained by subtracting current before exposure to hyposmotic solution from current after each treatment. Each data point represents mean±SEM (n=3). Mean cell capacitance was 36.1±6.9 pF.

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 E_Ca, 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%.

View larger version (13K): [in this window] [in a new window]   Figure 4. Inhibition of Ca2+-activated Cl- current by glibenclamide (glib) in canine ventricular myocytes. In this experiment, outward Cl- currents were elicited by Ca2+-induced Ca2+ release triggered by activation of Ca2+ current. Left, Ca2+ current was elicited by applying 10-millisecond voltage steps to 0 mV from a holding potential of -50 mV. Subsequent application of a voltage step to +55 mV elicited an outward Cl- current. Superimposed traces were recorded from the same cell before and after 100 µmol/L glib and after 100 µmol/L niflumic acid. Right, Glib-sensitive difference current is shown. 4-Aminopyridine (2 mmol/L) was present throughout.

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 E_Cl (-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 E_Cl (44.8 mV). These data suggest that the Ca²⁺-activated membrane currents observed under these conditions exhibit properties consistent with a significant Cl^- permeability.

View larger version (23K): [in this window] [in a new window]   Figure 5. Cl- dependence of the Ca2+-induced currents in canine ventricular myocytes exposed to the Ca2+ ionophore A23187 and Ca2+. The cells were exposed to A23187 (2 µmol/L) for >10 minutes in the nominally Ca2+-free BDM-containing solution. Thereafter, CaCl2 (5 mmol/L) was added to the bath solution. A, Raw membrane currents measured during 150-millisecond voltage steps from 0 mV to potentials ranging from -100 to 50 mV (10-mV increments) with [Ca2+]o=0 mmol/L and [Cl-]o=132 mmol/L (left), [Ca2+]o=5 mmol/L and [Cl-]o=132 mmol/L (middle), and [Ca2+]o=5 mmol/L and [Cl-]o=24 mmol/L (right). Vertical and horizontal calibrations are 400 pA and 20 milliseconds, respectively. B, I-V relations for Ca2+-activated currents with [Cl-]o=132 mmol/L and [Cl-]o=24 mmol/L. The currents were measured during 150-millisecond voltage steps from 0 mV to potentials ranging from -100 to 50 mV (10-mV increments) in the 132 mmol/L Cl- solution, followed by the 24 mmol/L Cl- solution. Difference currents were obtained by subtracting the current in the absence of Ca2+ from the current in the presence of Ca2+. Each data point represents mean±SEM (n=3). Mean cell capacitance was 166.4±19.7 pF.

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.

View larger version (34K): [in this window] [in a new window]   Figure 6. Effect of glibenclamide (glib) on Ca2+-activated Cl- currents in canine ventricular myocytes exposed to the Ca2+ ionophore A23187 at 22°C. The cells were exposed to A23187 (2 µmol/L) for >10 minutes in the nominally Ca2+-free BDM-containing solution. Thereafter, CaCl2 (5 mmol/L) was added to the bath solution. A, Time courses of membrane currents at +50 and -100 mV, activated by 5 mmol/L Ca2+ with or without 100 µmol/L glib. [Cl-]o was 132 mmol/L throughout. B, Mean I-V relations of the Ca2+-induced Cl- currents in the absence and presence of 100 µmol/L glib (n=6). These currents were measured during 150-millisecond voltage steps from 0 mV to potentials ranging from -100 to 50 mV (10-mV increments). Difference currents were obtained by subtracting the current in the absence of Ca2+ from the current in the presence of Ca2+. Each data point represents mean±SEM. Mean cell capacitance was 131.0±8.1 pF. C, Concentration-response relations for the inhibition of Ca2+-activated Cl- currents by glib at +50 and -100 mV. Each data point represents mean±SEM. Numbers in parentheses indicate the number of cells tested at each concentration.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The main goal of the present study was to investigate whether sulfonylureas, in addition to their known ability to block cardiac K_ATP 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 K_ATP channels. Although the reported K_d for glibenclamide binding to K_ATP 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 K_ATP 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 K_ATP 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 K_ATP 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^- channel^{45 46} similar to that described in other tissues^{47 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 K_ATP 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^- channels^{9 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 K_ATP 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.

	Selected Abbreviations and Acronyms

 

BDM = 2,3-butanedione monoxime CFTR = cystic fibrosis transmembrane regulator DMSO = dimethyl sulfoxide ECl, ECa = Cl- and Ca2+ equilibrium potential I-V = current-voltage KATP channel = ATP-sensitive K+ channel

	Acknowledgments

 
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.

	Footnotes

 
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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