Inhibitory Effects of Glibenclamide on Cystic Fibrosis Transmembrane Regulator, Swelling-Activated, and Ca²⁺-Activated Cl⁻ Channels in Mammalian Cardiac Myocytes

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 MΩ 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 GΩ) 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.

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)×Aⁿ]/(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.

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).

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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).

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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%.

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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%.

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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.

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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.

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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.