QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.104.074369v1 312/1/61    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Lee, S.-Y. Articles by Lee, C. O. Search for Related Content PubMed PubMed Citation Articles by Lee, S.-Y. Articles by Lee, C. O.

CARDIOVASCULAR

Inhibition of Na⁺-K⁺ Pump and L-Type Ca²⁺ Channel by Glibenclamide in Guinea Pig Ventricular Myocytes

Department of Life Science, Pohang University of Science and Technology, Pohang, Kyung-buk, Republic of Korea

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
Glibenclamide, a potent cystic fibrosis transmembrane conductance regulator (CFTR) Cl^– channel blocker, is frequently used to study function and regulation of CFTR Cl^– channels. In this study, the effects of glibenclamide on intracellular Na⁺ concentration ([Na⁺]_i), contraction, Ca²⁺ transient, and membrane potential were investigated in isolated guinea pig ventricular myocytes. Glibenclamide increased [Na⁺]_i and decreased contraction and Ca²⁺ transient. However, glibenclamide did not change membrane potential. To determine whether inhibition of Na⁺-K⁺ pumps and L-type Ca²⁺ channels is responsible for the increase of [Na⁺]_i and the decrease of contraction, we tested the effects of glibenclamide on Na⁺-K⁺ pump current and L-type Ca²⁺ current (I_Ca,L). Glibenclamide decreased Na⁺-K⁺ pump current and I_Ca,L in a concentration-dependent manner. In the presence of Cl^– channel inhibitors, glibenclamide depolarized diastolic membrane potential and reduced action potential duration. This result suggests that the reason for lack of effect of glibenclamide on membrane potential might be due to its combined inhibitory effects on the Na⁺-K⁺ pump, the L-type Ca²⁺ channel, and Cl^– channels, which may have opposing effects on membrane potential. These results indicate that glibenclamide increases [Na⁺_i] by inhibiting the Na⁺-K⁺ pump and decreases contraction and Ca²⁺ transient, in addition, by blocking the L-type Ca²⁺ channel.

Glibenclamide (Glib), a potent cystic fibrosis transmembrane conductance regulator (CFTR) Cl^– channel blocker, is an anion with large hydrophobic components (Sheppard and Robinson, 1997; Hume et al., 2000; Gupta and Linsdell, 2002). It is frequently used to study regulation of the CFTR Cl^– channel (Yamamoto-Mizuma et al., 2004) and CFTR Cl^– channel pore (Gupta and Linsdell, 2002; Zhou et al., 2002). However, several studies have revealed that glibenclamide, in addition to blocking the CFTR Cl^– channels, also inhibits ATP-sensitive K⁺ channels (Ashcroft and Gribble, 2000), human ether-a-go-go-related gene (HERG) channels (Rosati et al., 1998), transient outward K⁺ currents (Hernandez-Benito et al., 2001), swelling-activated Cl^– channels (Hume et al., 2000), and Ca²⁺-activated Cl^– channels (Hume et al., 2000) in cardiac myocytes.

Despite a large number of studies examining the effect of glibenclamide on cardiac channels, there have been very few studies that looked at the effect of the drug on [Na⁺]_i, contraction, Ca²⁺ transient, and membrane potential in cardiac muscle. Thus, the objective of the present study was to determine the effect of glibenclamide on [Na⁺]_i, contraction, Ca²⁺ transient, and membrane potential in guinea pig ventricular myocytes. Our study indicates that glibenclamide increases [Na⁺]_i by inhibiting the Na⁺-K⁺ pump and decreases contraction and Ca²⁺ transient by blocking the L-type Ca²⁺ channel. However, glibenclamide does not change diastolic membrane potential and action potential duration.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Cell Isolation
To get single ventricular myocytes, guinea pigs (300–500 g) were anesthetized with pentobarbital (50 mg/kg i.p.), and then their hearts were removed. The heart was enzymatically dissociated into single myocytes by using a method previously described (Lee and Levi, 1991). The isolated myocytes were kept at room temperature (20–23°C) until they were used (within 8–10 h). Experiments were performed in accordance with the national ethical guidelines as described previously (Gadsby and Nakao, 1989).

Electrical Recording
Myocytes in the experimental chamber were continuously superfused at 37°C with Tyrode's solution containing 140 mM NaCl, 4.4 mM KCl, 1.8 mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES, and 10 mM glucose (pH 7.4 with NaOH).

Measurement of Membrane Potential. To measure membrane potential, we used conventional microelectrodes pulled from filamented thin-wall glass (TW150F-6, 1.5-mm o.d.; World Precision Instruments, Inc., Sarasota, FL). They were filled with 300 mM KCl and had a resistance between 25 and 40 M. The electrode resistance and capacitance were compensated to about to 90% of their initial value. Microelectrode potential was measured with an Axoclamp 2A amplifier (0.1-gain head stage; Axon Instruments Inc., Union City, CA). Action potentials were elicited at 0.5 Hz by 2-ms depolarizing current pulse passed down the microelectrode.

Voltage-Clamp Procedure with Conventional Microelectrodes. To record the L-type Ca²⁺ current (I_Ca,L), the voltage-clamp procedure was performed using the amplifier's discontinuous switch-clamp mode with conventional microelectrodes filled with 300 mM KCl and 20 mM tetraethylammonium chloride (TEA-Cl) and having about 20 M tip resistance. The electrode resistance and capacitance were compensated to about to 90% of their initial value. The I-V relationship of peak I_Ca,L was constructed using 500-ms voltage pulses to the potentials of 10-mV increment between –40 mV and +40 mV from the holding potential of –40 mV (to inactivate the Na⁺ current). TEA-Cl (5.4 mM) was added to the Tyrode's solution to block K⁺ currents. Voltage-clamp protocol and data acquisition were performed with an Axoclamp 2A amplifier, an analog-to-digital converter (CED 1401; Cambridge Electronic Design, Cambridge, UK), and the software WCP (written by John Dempster of Strathclyde University, Glasgow, Scotland, UK).

The biophysical properties of I_Ca,L were then analyzed in N-methyl-D-glucamine-containing solutions to efficiently isolate I_Ca,L from the contamination of other currents (Na⁺, K⁺, Na⁺-Ca²⁺ exchanger). After obtaining the whole-cell configuration, myocytes were perfused with an extracellular Na⁺- and K⁺-free I_Ca,L solution: 144 mM N-methyl-D-glucamine-Cl, 5.4 mM CsCl, 1 mM MgSO₄, 1.8 mM CaCl₂, and 10 mM HEPES (pH 7.4). Patch pipettes (1–2 M) contained 120 mM CsCl, 20 mM TEA-Cl, 1 mM MgSO₄, 5 mM EGTA, 5 mM Mg-ATP, 0.2 mM GTP, and 10 mM HEPES (pH 7.3 with CsOH). The use of these solutions allowed recording of stable I_Ca,L currents with minimal rundown (Bett et al., 2002). Myocytes were voltage clamped to a holding potential of –70 mV. The steady-state inactivation and activation relationships were determined using a gapped double-pulse protocol once every 2 s; a 500-ms prepulse to potentials between –60 and +70 mV was followed by a 10-ms return to the holding potential and then a fixed 500-ms test pulse to +10 mV.

Whole-Cell Recording. To measure the Na⁺-K⁺ pump current, whole-cell currents were recorded via wide-tipped pipettes with resistance of about 1 M as described (Gadsby and Nakao, 1989). The pipette-to-bath liquid junction potential was small (–3.5 mV) and was uncorrected. Membrane capacitance (the time integral of the capacitive response to a 10-mV hyperpolarizing pulse from a holding potential of 0 mV, divided by the voltage drop) averaged 131.5 ± 24.5 pF (n = 10). The holding potential was set to 0 mV to inactivate Na⁺ and Ca²⁺ channels. Whole-cell currents were recorded in response to 50-ms voltage pulses to potentials from –100 to +40 mV in 20-mV steps. The steady-state current was plotted against the test potential. All patch-clamp protocol and data acquisition were performed with an Axopatch-200A amplifier, an analog-to-digital converter (Digidata1200; Axon Instruments Inc.), and the software pClamp 5 (Axon Instruments Inc.). The modified Ca²⁺-free Tyrode's solution used in the whole-cell current-recording experiments contained: 145 mM NaCl, 5.4 mM KCl, 1 mM MgCl₂, 2 mM BaCl₂, 1 CdCl₂, 5.5 mM glucose, and 5 mM HEPES (pH 7.4 with NaOH). The pipette solution contained: 50 mM NaOH, 76 mM CsOH, 90 mM aspartic acid, 20 mM TEA-Cl, 3 mM MgCl₂, 5.5 mM glucose, 10 mM EGTA, 10 mM Mg-ATP, 5 mM Tris-creatine phosphate, 5 mM pyruvic acid, 10 mM HEPES, and 4 mM CaCl₂ (for free Ca²⁺ 50 nM) (pH 7.3 with CsOH).

Simultaneous Measurement of Intracellular Na⁺ or Ca²⁺ and Contraction
To measure [Na⁺]_i or [Ca²⁺]_i and contraction simultaneously, myocytes were loaded with the fluorescent Na⁺-sensitive indicator SBFI-AM or fluorescent Ca²⁺-sensitive indicator Fura-2-AM as described previously (Lee and Levi, 1991; Woo and Lee, 1999). Myocytes loaded with the indicator were then moved to the experimental chamber and illuminated with ultraviolet light (75-W xenon lamp) applied via an epifluorescence microscope. A filter wheel in front of the UV light was rotated continuously at 20 Hz (for SBFI) or 75 Hz (for Fura-2), and excitation filters of 340 and 380 nm were selected alternatively (Cairn Research, Kent, UK). The ratio of the light emitted at 340-nm excitation to that emitted at 380-nm excitation (the 340:380 ratio) is a direct index of the level of intracellular sodium or calcium. After an experiment, in situ calibration of the [Na⁺]_i was performed as described by Lee and Levi (1991). At the same time, contraction was measured optically with a video edge detector system (Crescent Electronics, Windsor, ON, Canada) that has been previously described (Steadman et al., 1988).

Chemicals and Statistics
SBFI-AM and Fura-2-AM were obtained from Molecular Probes (Eugene, OR). All other chemicals including glibenclamide were purchased from Sigma-Aldrich (St. Louis, MO). Glibenclamide was prepared as 0.1 or 0.2 M stock solution in dimethyl sulfoxide (DMSO). The maximum concentration of glibenclamide applied was 500 µM, which was the maximum concentration (Tominaga et al., 1995). Anthracene-9-carboxylic acid (A9C), niflumic acid (NFA), and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) were prepared in DMSO as a stock solution so that the desired final concentration was achieved by 1:1000 dilution with the external control solution. The final concentration of DMSO did not exceed 0.3%, which had no effect under the conditions of our experiments.

All data are presented as means ± S.E.M., and statistical differences were determined by paired Student's t tests. Numerical data were analyzed with Sigmaplot 2001 for Windows (SPSS Inc., Chicago, IL) and Origin 6.1 (OriginLab Corp., Northampton, MA). Differences were considered statistically significant at P < 0.05.

	Results

Top Abstract Materials and Methods Results Discussion References

 
Effects of Glibenclamide on [Na⁺]_i, Contraction, Ca²⁺ Transient, and Membrane Potential. Figure 1A shows the effects of glibenclamide on [Na⁺]_i and membrane potential (V_m) in a single guinea pig ventricular myocyte that was driven at a rate of 0.5 Hz. Application of 200 µM glibenclamide increased [Na⁺]_i by 3.9 ± 0.4 mM (n = 14, P < 0.01) and did not change diastolic membrane potential. After washing off glibenclamide, the [Na⁺]_i was recovered to the control level. Interruption of the stimulation slightly decreased [Na⁺]_i. Figure 1B illustrates the concentration dependence of glibenclamide's effect on [Na⁺]_i. The recordings of [Na⁺]_i were obtained from myocytes treated with 1,10, 100, and 200 µM glibenclamide. The increase in [Na⁺]_i by glibenclamide was fitted by the Hill function (1+(IC₅₀/[Glib])^p)^–1, and Hill number p was 0.9 ± 1.3. Glibenclamide increased [Na⁺]_i at concentrations >1 µM. Figure 1C summarized the effect of glibenclamide on membrane potential. Application of 200 µM glibenclamide produced no change in diastolic membrane potential and action potential amplitude (top, n = 15). Glibenclamide at concentrations of 1, 10, 100, and 200 µM also did not alter action potential duration (APD) (bottom, n = 6, 10, 6, 15, respectively).

View larger version (33K): [in this window] [in a new window]   Fig. 1. Effects of Glib on [Na+]i and membrane potential in an isolated guinea pig ventricular myocyte stimulated at 0.5 Hz. A, continuous recordings of [Na+]i (top) and Vm (bottom). After washing off Glib, the myocyte was calibrated for [Na+]i using an in situ calibration method in which intracellular Na+ was equilibrated with extracellular Na+ using a calibration solution containing 2 and 10 mM. Inset, superimposed action potentials were recorded at the times indicated by 1 (control) and 2 (Glib). B, superimposed recordings show changes in [Na+]i caused by Glib at concentrations of 1, 10, 100, and 200 µM (top). Concentration-response curve for the increases in [Na+]i caused by Glib is shown in bottom of B. Data were fitted by the Hill function (see text for parameters). Each data point represents 3 to 14 cells. C, summary data showing: the effect of Glib on diastolic membrane potential (top, n = 15), action potential amplitude (top, n = 15), and normalized APD (bottom, n = 6–15). Data are shown as mean value ± S.E.M.

Figure 2, A and B, show effects of glibenclamide on Ca²⁺ transient (Fura-2 ratio) and contraction ( cell length) in a single myocyte that was driven at a rate of 0.5 Hz. The application of 200 µM glibenclamide decreased Ca²⁺ transient by 22.0 ± 0.8% (n = 9, P < 0.05) and contraction by 44.8 ± 2.4% (n = 21, P < 0.01), respectively. After washing off glibenclamide, both Ca²⁺ transient and contraction were recovered to the control level. Figure 2C shows the concentration-response curves for the decrease in contraction and Ca²⁺ transient caused by glibenclamide. The decreases in contraction and Ca²⁺ transient by glibenclamide were fitted by the Hill function (1+(IC₅₀/[Glib])^p)^–1, and the Hill numbers were 0.6 ± 0.3 and 0.4 ± 0.3 for contraction and Ca²⁺ transient, respectively. Glibenclamide decreased contraction and Ca²⁺ transient at concentrations > 1 µM.

View larger version (45K): [in this window] [in a new window]   Fig. 2. Effects of Glib on Ca2+ transient and contraction in an isolated guinea pig ventricular myocyte stimulated at 0.5 Hz. A, continuous recordings of Ca2+ transient (Fura-2 ratio) and contraction ( cell length). B, recordings of averaged Ca2+ transients of three consecutive beats (top) and contraction (bottom) on an expanded time scale from the same cell (in A). C, concentration-response curves for the decreases in contraction and Ca2+ transient caused by Glib. Data were fitted by the Hill function (see text for parameters). Data are shown as mean value ± S.E.M. decreases in contraction or Ca2+ transient (percentage of control). Each data point represents 4 to 21 cells.

To examine the role of sarcoplasmic reticulum (SR) Ca²⁺ load in the glibenclamide-induced reduction in magnitude of Ca²⁺ transient and glibenclamide-induced increase in diastolic Ca²⁺ levels, SR Ca²⁺ content was assessed by caffeine-induced Ca²⁺ transient. After steady-state SR Ca²⁺ load was achieved, application of 20 mM caffeine at the end diastole resulted in a large [Ca²⁺]_i increase due to SR Ca²⁺ release. Figure 3 shows no significant differences in caffeine-sensitive SR Ca²⁺ stores before and during the exposure to glibenclamide. Results also show that glibenclamide did not significantly increase diastolic Ca²⁺ levels (from 0.44 ± 0.02 to 0.46 ± 0.02, n = 11; Fig. 3B). These results indicate that decrease in contraction by glibenclamide is due to reduction of the magnitude of Ca²⁺ transient without change in SR Ca²⁺ content.

View larger version (27K): [in this window] [in a new window]   Fig. 3. Effects of glibenclamide on SR Ca2+ content in an isolated guinea pig ventricular myocyte stimulated at 0.5 Hz. A, SR Ca2+ content monitored by applying 20 mM caffeine. B, summarized data of basal Ca2+ levels (n = 11) and SR Ca2+ content (n = 10).

Effects of Glibenclamide on Na⁺-K⁺ Pumps. To determine whether the inhibition of Na⁺-K⁺ pumps was responsible for the increase in [Na⁺]_i, we tested the effects of glibenclamide on Na⁺-K⁺ pump current. Figure 4A illustrates the effect of glibenclamide on the Na⁺-K⁺ pump current in a ventricular myocyte. Na⁺-K⁺ pump current was estimated as cardiac steroid-sensitive current (Gadsby and Nakao, 1989). We used 0.5 mM strophanthidin (stro), a cardiac steroid, as an inhibitor of Na⁺-K⁺ pump. Strophanthidin-sensitive currents represent the difference between the membrane currents in the presence and absence of strophanthidin. Na⁺-K⁺ pump current was decreased 43.8 ± 3.0% (n = 8, P < 0.05) by 200 µM glibenclamide at 0 mV membrane potential. After washing off glibenclamide and strophanthidin, the Na⁺-K⁺ pump current was almost recovered to the control level. The membrane currents (Fig. 4A, bottom) recorded at the times indicated by corresponding numbers show steady state. Figure 4B shows normalized plots of the steady-state membrane current (I) against membrane voltage (V). The steady-state levels of whole-cell current determined before (Fig. 4A, 1 and 3) and during (Fig. 4A, 2 and 4) the applications of strophanthidin. Figure 4C shows the average strophanthidin-sensitive I-V relationships obtained by subtracting steady-state current levels in strophanthidin from those determined just before exposure to strophanthidin. Glibenclamide reduced strophanthidin-sensitive current at all voltages (n = 5).

View larger version (27K): [in this window] [in a new window]   Fig. 4. Effects of Glib on Na+-K+ pump current. A, change in membrane current at 0-mV holding potential. The vertical lines indicated by one, two, three, four, five, and six mark applications of 50-ms voltage step pulses from the 0-mV holding potential between –100 and +40 mV. Current recordings labeled 1 (control), 2 (stro), 3 (Glib), and 4 (Glib + stro) were taken at the times indicated by the corresponding numbers in the upper panel of A. Applications of stro or Glib were indicated above the current recording. B, average relationships between steady-state membrane current and voltage. The membrane currents indicated by open symbols ( and ) were determined before application of stro. The membrane currents indicated by filled symbols ( and ) were determined during application of 0.5 mM stro. C, average relationships between stro-sensitive current and voltage from five cells.

Figure 5 shows the relation between the decrease of Na⁺-K⁺ pump current and the concentration of glibenclamide. As concentration of glibenclamide increased, the Na⁺-K⁺ pump current decreased. The Na⁺-K⁺ pump current began to decrease at 1 to 10 µM of glibenclamide. The decrease in Na⁺-K⁺ pump current by glibenclamide was fitted by the Hill function (1+(IC₅₀/[Glib])^p)^–1, and Hill number p was 1.0 ± 0.5.

View larger version (14K): [in this window] [in a new window]   Fig. 5. Concentration-response curve for decrease in Na+-K+ pump current by Glib at 0-mV membrane potential. Data were fitted by the Hill function (see text for parameters). Data are shown as mean value ± S.E.M. decreases in Na+-K+ pump current (percentage of the control). Each data point represents three to eight cells.

Effects of Glibenclamide on L-Type Ca²⁺ Channels. To determine whether inhibition of L-type Ca²⁺ channels was responsible for the decrease of contraction, we tested effects of glibenclamide on I_Ca,L. Figure 6A shows the time course of change in I_Ca,L when glibenclamide was applied. Application of 200 µM glibenclamide decreased I_Ca,L and the decreased I_Ca,L recovered to the control level after washing off glibenclamide. In six myocytes tested, 200 µM glibenclamide decreased peak I_Ca,L by 33.1 ± 4.0% (P < 0.01) at 0-mV membrane potential. Figure 6B represents the average I_Ca,L-V relationship in the absence and presence of 200 µM glibenclamide. The effects of glibenclamide on kinetics and steady-state activation and inactivation of I_Ca,L were then examined. Glibenclamide did not show significant effects on the macroscopic biexponential inactivation kinetics of I_Ca,L recorded at 0 mV (Fig. 6C). Mean values (n = 6 myocytes) were as follows: ₁, control 8.9 ± 1.0 ms, glibenclamide 10.5 ± 1.5 ms; and ₂, control 88.9 ± 14.3 ms, glibenclamide 95.8 ± 13.9 ms. To determine whether change in voltage dependence of steady-state activation and inactivation of I_Ca,L was involved in the decrease of I_Ca,L, effect of glibenclamide on the steady-state activation (d) and inactivation (f) relationship was determined using a gapped double-pulse protocol in Na⁺- and K⁺-free solutions. Figure 6D shows that glibenclamide caused a 12-mV hyperpolarizing shift in holding potential (V_h) of f (from –22.9 ± 4.6 mV, n = 5, in control, to –34.7 ± 1.3 mV, n = 4, P < 0.01) without a significant change in the slope factor (k; control, 7.6 ± 3.9 mV; glibenclamide, 5.0 ± 1.1 mV). Results also show that glibenclamide did not cause a significant change in V_h of d (from –16.5 ± 1.8 mV, n = 6, in control, to –20.4 ± 1.5 mV, n = 6) and in k values (control, 7.6 ± 3.9 mV; glibenclamide, 5.0 ± 1.1 mV). The glibenclamide-induced hyperpolarizing shift in f could account for the reduced I_Ca,L.

View larger version (24K): [in this window] [in a new window]   Fig. 6. Effects of Glib on L-type Ca2+ current (ICa,L). A, normalized peak ICa,L measured at every 30 s are plotted as a function of time (in min). B, average ICa,L-V relationships in the absence and presence of Glib. Test potentials ranged from –40 to 40 mV in 10-mV steps. C, current recordings (left) at 0-mV test potential from a holding potential of –40 mV labeled 1 (control), 2 (Glib), and 3 (washout). Macroscopic inactivation kinetics of ICa,L recorded at 0 mV (right). D, steady-state inactivation and activation of ICa,L. Steady-state inactivation (f) and activation (d) of ICa,L were determined using gapped double-pulse protocol (see Materials and Methods) and curve fit by Boltzmann equations as follows: I/Imax = 1/[1 + exp(V – Vh)/k], where Vh is the half-maximum inactivation potential and k is the slope factor for f, and G/Gmax = 1/{1 + exp[– (V – Vh)/k]}, where Vh is the half-maximum activation potential for d, G is conductance, and Gmax is maximal conductance. Data represent mean value ± S.E.M. from four to six experiments.

Figure 7 shows the concentration dependence of glibenclamide's effect on I_Ca,L. In the concentration range tested, inhibition of I_Ca,L by glibenclamide was dependent on the concentration of glibenclamide. Decrease in I_Ca,L was observed concentration greater than 1 µM glibenclamide. The maximum concentration of glibenclamide applied was 500 µM, which was the maximum concentration used in our study. The decrease in I_Ca,L by glibenclamide was fitted by the Hill function (1+(IC₅₀/[Glib])^p)^–1, and Hill number p was 0.7 ± 0.3.

View larger version (14K): [in this window] [in a new window]   Fig. 7. Concentration-response curve for decrease in ICa,L current by Glib at 0-mV test potential. Data were fitted by the Hill function (see text for parameters). Data are shown as mean value ± S.E.M. decreases in ICa,L (percentage of the control). Each data point represents three to five cells.

Effects of Glibenclamide on Membrane Potential in the Presence of Cl^– Channel Inhibitors. Our results revealed that glibenclamide had lack of effect on the membrane potential (Fig. 1C). However, it is well recognized that inhibition of the Na⁺-K⁺ pump and the L-type Ca²⁺ channel reduces APD (Lee and Levi, 1991; Qu et al., 1993). glibenclamide also blocks several kinds of Cl^– channels (Yamazaki and Hume, 1997). Activation of CFTR Cl^– channels and swelling-activated Cl^– channels has a significant effect on membrane potential (Hume et al., 2000). Inhibition of the CFTR Cl^– channels and swelling-activated Cl^– channels could lengthen the APD and hyperpolarize the diastolic membrane potential. Therefore, it is possible that glibenclamide might have lengthened the APD by inhibition of the Cl^– channels and shortened it by inhibitions of the Na⁺-K⁺ pumps and the L-type Ca²⁺ channels, which together might result in no change in diastolic membrane potentials and APD.

To determine whether the inhibition of the Cl^– channels was responsible for the lack of effect on APD, we tested the effect of glibenclamide on APD in the presence of Cl^– channel inhibitors, NFA, NPPB, and A9C. All of these compounds, except glibenclamide, result in partial inhibition of CFTR Cl^– channels (Sorota, 1999). Figure 8A shows the effect of 200 µM glibenclamide on membrane potential in the presence of 50 µM NFA. The application of 50 µM NFA hyperpolarized diastolic membrane potential and then addition of 200 µM glibenclamide slightly depolarized diastolic membrane potential by 2.0 ± 1.6 mV (n = 6). Addition of glibenclamide to the superfusion solution containing NFA decreased APD₂₀ from 104.5 ± 18.3 to 67.7 ± 9.8 ms by 27.7 ± 7.6% (n = 6, P < 0.05) and APD₉₀ from 253.1 ± 32.6 to 192.2 ± 15.9 ms by 21.3 ± 4.9% (n = 6, P < 0.05), respectively. Figure 8B shows the effect of 200 µM glibenclamide on membrane potential in the presence of 10 µM NPPB. In the presence of NPPB, glibenclamide depolarized diastolic membrane potential by 0.8 ± 0.4 mV (n = 5, P < 0.05) and reduced APD₂₀ from 79.9 ± 4.8 to 53.8 ± 5. 5 ms by 32.9 ± 4.7% (n = 5, P < 0.01) and APD₉₀ from 170.3 ± 7.0 to 141.8 ± 12.0 ms by 17.2 ± 4.7% (n = 5, P < 0.05), respectively. In the presence of A9C, glibenclamide also depolarized diastolic membrane potential by 1.0 ± 0.5 mV (n = 4) and reduced APD₂₀ by 22.5 ± 4.8% (n = 4, P < 0.05) and APD₉₀ by 9.1 ± 1.7% (n = 4, P < 0.05). We tested effects of 0.3% DMSO on diastolic membrane potential and APD as a control. The contribution of the Na⁺-K⁺ pumps to resting membrane potential is expected to be <1 mV (Levi et al., 1997). This suggests that depolarization by glibenclamide in the presence of Cl^– channel inhibitor is small. The effects of glibenclamide on resting membrane potential and APD in the presence of NFA, NPPB, A9C, and DMSO were summarized in Fig. 8, C and D. Therefore, the results suggest that glibenclamide lengthened the APD by inhibition of Cl^– channels and shortened it by inhibition of Na⁺-K⁺ pumps and L-type Ca²⁺ channels, which together might result in no change in diastolic membrane potentials and APD.

View larger version (58K): [in this window] [in a new window]   Fig. 8. Effects of Glib on membrane potential in the presence of Cl– channel inhibitors. A and B, continuous recordings of Vm in the presence of 50 µM NFA (A) and 10 µM NPPB (B). The magnified diastolic membrane potentials are shown in bottom. Inset, superimposed action potentials were recorded at the times indicated by 1 (NFA), 2 (NFA + Glib), 3 (NPPB), and 4 (NPPB + Glib). C, summarized effects of Glib on resting membrane potential in the presence of NFA (n = 6), NPPB (n = 5), A9C (n = 4), and DMSO (n = 3). D, summarized effects of Glib on APD in the presence of NFA (n = 6), NPPB (n = 5), A9C (n = 4), and DMSO (n = 3). Data are shown as mean value ± S.E.M. *, P < 0.05; **, P < 0.01.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
In the present study, we report that glibenclamide increases [Na⁺]_i, decreases contraction and Ca²⁺ transient but does not alter diastolic membrane potential and APD in isolated guinea pig ventricular myocytes. Furthermore, it was revealed that the increase of [Na⁺]_i was caused by inhibition of the Na⁺-K⁺ pumps and that the decrease of contraction and Ca²⁺ transient were due to blockage of the L-type Ca²⁺ channels. The reason for the lack of effect on membrane potential by glibenclamide might be due to its combined inhibitory effects on the Na⁺-K⁺ pump, the L-type Ca²⁺ channel, and Cl^– channels, which may have opposing effects on membrane potential.

Effects of Glibenclamide on Na⁺-K⁺ Pumps. Maintenance of a low [Na⁺]_i is important for electrophysiological functions of cardiac muscle. Changes in [Na⁺]_i can affect the [Ca²⁺]_i, the contractile force, action potential, and intracellular pH of heart cells (Lee and Levi, 1991; Levi et al., 1997). The increase of [Na⁺]_i induced by glibenclamide is considered to be an indirect evidence of inhibition of the Na⁺-K⁺ pump. We tested the effect of glibenclamide on the Na⁺-K⁺ pump current that provides direct evidence. Glibenclamide decreased the Na⁺-K⁺ pump current in a concentration-dependent manner (Figs. 3 and 4), which strongly supports that glibenclamide increased [Na⁺]_i via inhibition of the Na⁺-K⁺ pump.

The rise of [Na⁺]_i leads to increases in [Ca²⁺]_i via Na⁺-Ca²⁺ exchange (Lee and Levi, 1991; Levi et al., 1997). In the present study, however, glibenclamide increased [Na⁺]_i, but it decreased Ca²⁺ transient and contraction of ventricular myocytes (Figs. 1 and 2). The decrease of contraction could be due to the decrease of L-type Ca²⁺ current by glibenclamide, which reduced [Ca²⁺]_i. We propose that the reduction in Ca²⁺ transient, despite the increase in Na⁺-Ca²⁺ exchange activity, is partially explained by that the reduction of [Ca²⁺]_i exceeds the increase of [Ca²⁺]_i via the Na⁺-Ca²⁺ exchange.

Inhibition of the Na⁺-K⁺ pump depolarizes diastolic membrane potential and shortens APD of the myocytes because the Na⁺-K⁺ pump is electrogenic (Lee and Levi, 1991; Glitsch, 2001), and increased [Na⁺]_i produces outward current via the Na⁺-Ca²⁺ exchange (Levi et al., 1997). However, it was reported that 100 µM glibenclamide had no effect on APD in anesthetized guinea pig (Chen et al., 2000), and 1 to 10 µM glibenclamide did not alter APD significantly in rat and rabbit heart (Smallwood et al., 1990; Doggrell and Bishop, 1996; Light et al., 1999). Similarly, our study shows that glibenclamide produced no change in APD of the myocytes (Fig. 1).

The well known effect of glibenclamide is inhibition of ATP-sensitive K⁺ channels in various types of cell and organs (Ashcroft and Gribble, 2000). However, the ATP-sensitive K⁺ channel is closed by ATP with half-maximal closure at 20 to 100 µM of ATP in heart. Normal physiological intracellular ATP concentration is in the range of 5 to 10 mM (Nichols and Lederer, 1991; Grover and Garlid, 2000). Actually, in our experiments, there was no significant difference in glibenclamide's effect on membrane potential between no ATP and 20 mM ATP in the pipette solution of the microelectrode having 12 to 15 M tip resistance (data not shown). Therefore, the contribution of ATP-sensitive K⁺ channels to glibenclamide's effect on membrane potential could be ruled out. Glibenclamide is also known to block several kinds of Cl^– channels (Yamazaki and Hume, 1997). Inhibition of Cl^– channels only, especially CFTR Cl^– channels and swelling-activated Cl^– channels, could lead to an increase in APD (Hume et al., 2000). Actually, application of A9C increased the APD (data not shown). When Cl^– channels were inhibited by several kinds of Cl^– channel inhibitors, glibenclamide reduced the APD (Fig. 7), suggesting that inhibition of the Na⁺-K⁺ pump and the L-type Ca²⁺ channel by glibenclamide decreased the APD. Thus, our findings suggest that the reason for the lack of effect of glibenclamide on APD might be due to its dual inhibitory effects in which APD prolongation by inhibition of Cl^– channels offsets APD shortening by inhibition of Na⁺-K⁺ pump and L-type Ca²⁺ channel.

Rosati et al. (1998) reported that 100 µM glibenclamide prolonged APD by about 9.4%. They suggested that the increase of APD resulted from the inhibition of the HERG channels. They applied glibenclamide for 30 s, which was too short to induce any changes in [Na⁺], contraction, Ca²⁺_i transient, and membrane potential in our experiment. We applied glibenclamide at least for 2 min to wait for the stabilization of that effect. The lack of effect on APD by glibenclamide was might be interpreted by its opposing inhibitory effects on the Na⁺-K⁺ pump, the L-type Ca²⁺ channel, and Cl^– channels. Nevertheless, it is possible that inhibition of the HERG channels caused by glibenclamide could partially contribute to the no change in APD by cooperating with the Cl^– channels against the Na⁺-K⁺ pump and the L-type Ca²⁺ channel.

Effects of Glibenclamide on L-Type Ca²⁺ Channels. L-type Ca²⁺ channels play an essential role in cardiac excitability and in E-C coupling. Depolarizing current through L-type Ca²⁺ channels contributes to the plateau phase of the cardiac action potential, and it is well known that I_Ca,L triggers calcium release from SR, which induces twitch force (Ferrier and Howlett, 2001).

The strength of contraction can be changed by altering the amplitude or duration of the Ca²⁺ transient and by altering the sensitivity of myofilaments to Ca²⁺ (Bers, 2002). glibenclamide decreased the magnitude of Ca²⁺ transient, which reduced contraction (Fig. 2). Sulfonylureas including glibenclamide have not been reported to change the sensitivity of myofilaments to Ca²⁺. Also, our study shows no evidence that glibenclamide might change the sensitivity of the contractile proteins.

Glibenclamide's effect on the L-type Ca²⁺ channels is tissue-specific (Bian and Hermsmeyer, 1994; Kim et al., 1997; Ashcroft and Gribble, 2000). In pancreatic cells, 0.2 to 1 µM glibenclamide inhibits ATP-sensitive K⁺ channels, which in turn produces depolarization of the membrane and opening of the voltage-gated Ca²⁺ channel. The resulting Ca²⁺ influx leads to insulin secretion (Luzi and Pozza, 1997; Ashcroft and Gribble, 1999, 2000). Similarly, it was reported that 10 to 100 µM glibenclamide activated the voltage-dependent Ca²⁺ channel by depolarizing membrane potential and regulated secretion of mechanically activated atrial natriuretic peptide in atrial cells (Kim et al., 1997). In the present study with ventricular myocytes, 10 to 200 µM glibenclamide inhibited the I_Ca,L and reduced the contraction. However, glibenclamide did not cause change in transmembrane potential of the myocytes (Fig. 1). Similar inhibitory effect of 10 to 100 µM glibenclamide on the I_Ca,L was also observed in rat aortic smooth muscle (Yoshitake et al., 1991; Bian and Hermsmeyer, 1994).

Some investigators did not observe a negative inotropic effect of glibenclamide in the heart (Sykes et al., 1977; Del Valle et al., 2001). They applied glibenclamide via oral administration or intravenous injection, and the concentration of glibenclamide in plasma might be around 0.5 to 2.5 µM (Del Valle et al., 2001). Our results also revealed that glibenclamide had no significant effect on contraction at concentrations < 1 µM, and the threshold concentration for the effect was between 1 and 10 µM (Fig. 2).

Glibenclamide and CFTR Cl^– Channel. Cardiac CFTR Cl^– channels have been demonstrated to play a role in action potential shortening during hypoxia and ischemia, thereby limiting intracellular Ca²⁺ overload and cell damage (Hume et al., 2000). Recent evidence suggests a potential role of the CFTR Cl^– channel in ischemic preconditioning (Chen et al., 2004). Therefore, it is important to characterize CFTR Cl^– channel inhibitors as probes of CFTR Cl^– function. glibenclamide is widely used to study the CFTR Cl^– channel because it is relatively potent among CFTR Cl^– channel inhibitors (Sorota, 1999). However, several studies reported that glibenclamide, in addition to blocking the CFTR Cl^– channels, also inhibits ATP-sensitive K⁺ channels (Ashcroft and Gribble, 2000), HERG channels (Rosati et al., 1998), transient outward K⁺ currents (Hernandez-Benito et al., 2001), swelling-activated Cl^– channels (Hume et al., 2000), and Ca²⁺-activated Cl^– channels (Hume et al., 2000) in cardiac myocytes. In our study, glibenclamide also inhibited the Na⁺-K⁺ pump and the L-type Ca²⁺ channel. Recently, Muanprasat et al. (2004) reported a new compound, glycine hydrazide. The IC₅₀ values of glibenclamide and glycine hydrazide for CFTR Cl^– channel are 11 to 12.5 µM (Yamazaki and Hume, 1997) and 1.4 to 5.6 µM (Muanprasat et al., 2004), respectively. The glycine hydrazide also has high water solubility and rapidity of action (Muanprasat et al., 2004). This compound may be useful to examine the function and regulation of the CFTR Cl^– channel.

	Acknowledgements

 
We thank Eun-Mi Hur for reading and editing this manuscript.

	Footnotes

 
This work was supported by the Korea Research Foundation (Grant KRF 2002-015-DS0035) and the Korea Science and Engineering Foundation (KOSEF 98-0401-02).

Published abstract on parts of the work is as follows: Lee SY and Lee CO (2003) Effects of glybenclamide on Na⁺-K⁺ pump and L-type Ca²⁺ channel in guinea pig ventricular myocytes. Biophys J 84:409a.

doi:10.1124/jpet.104.074369.

ABBREVIATIONS: [Na⁺]_i, intracellular Na⁺ concentration; Glib, glibenclamide; CFTR, cystic fibrosis transmembrane conductance regulator; HERG, human ether-a-go-go-related gene; I_Ca,L, L-type Ca²⁺ current; TEA-Cl, tetraethylammonium chloride; SBFI, sodium-binding benzofuran isophthalate; AM, acetoxymethyl ester; DMSO, dimethyl sulfoxide; A9C, anthracene-9-carboxylic acid; NFA, niflumic acid; NPPB, 5-nitro-2-(3-phenylpropylamino)-benzoic acid; V_m, membrane potential; APD, action potential duration; SR, sarcoplasmic reticulum; [Ca²⁺]_i, intracellular Ca²⁺ concentration; V_h, holding potential; APD₉₀, APD at 90% repolarization; stro, strophanthidin.

Address correspondence to: Chin O. Lee, Department of Life Science, Pohang University of Science and Technology, Pohang, Kyung-buk, 790-784, Republic of Korea. E-mail: colee{at}postech.ac.kr

	References

Top Abstract Materials and Methods Results Discussion References

 

Ashcroft FM and Gribble FM (1999) ATP-sensitive K⁺ channels and insulin secretion: their role in health and disease. Diabetologia 42: 903–919.[CrossRef][Medline]

Ashcroft FM and Gribble FM (2000) Tissue-specific effects of sulfonylureas: lessons from studies of cloned K_ATP channels. J Diabetes Complications 14: 192–196.[CrossRef][Medline]

Bers DM (2002) Cardiac excitation-contraction coupling. Nature (Lond) 415: 198–205.[CrossRef][Medline]

Bett GC, Dai S, and Campbell DL (2002) Cholinergic modulation of the basal L-type calcium current in ferret right ventricular myocytes. J Physiol 542: 107–117.[Abstract/Free Full Text]

Bian K and Hermsmeyer K (1994) Glibenclamide actions on the dihydropyridine-sensitive Ca²⁺ channel in rat vascular muscle. J Vasc Res 31: 25–64.[Medline]

Chen CC, Lin YC, Chen SA, Luk HN, Ding PY, Chang MS, and Chiang CE (2000) Shortening of cardiac action potentials in endotoxic shock in guinea pigs is caused by an increase in nitric oxide activity and activation of the adenosine triphosphate-sensitive potassium channel. Crit Care Med 28: 1713–1720.[CrossRef][Medline]

Chen H, Liu LL, Ye LL, McGuckin C, Tamowski S, Scowen P, Tian H, Murray K, Hatton WJ, and Duan D (2004) Targeted inactivation of cystic fibrosis transmembrane conductance regulator chloride channel gene prevents ischemic preconditioning in isolated mouse heart. Circulation 110: 700–704.[Abstract/Free Full Text]

Del Valle HF, Lascano EC, Negroni JA, and Crottogini AJ (2001) Glibenclamide effects on reperfusion-induced malignant arrhythmias and left ventricular mechanical recovery from stunning in conscious sheep. Cardiovasc Res 50: 474–485.[Abstract/Free Full Text]

Doggrell SA and Bishop BE (1996) Effects of potassium channel blockers on the action potentials and contractility of the rat right ventricle. Gen Pharmacol 27: 379–385.[Medline]

Ferrier GR and Howlett SE (2001) Cardiac excitation-contraction coupling: role of membrane potential in regulation of contraction. Am J Physiol Heart Circ Physiol 280: H1928–H1944.[Abstract/Free Full Text]

Gadsby DC and Nakao M (1989) Steady state current-voltage relationship of the Na⁺-K⁺ pump in guinea-pig ventricular myocytes. J Gen Physiol 94: 511–537.[Abstract/Free Full Text]

Glitsch HG (2001) Electrophysiology of the sodium-potassium-ATPase in cardiac cells. Physiol Rev 81: 1791–1826.[Abstract/Free Full Text]

Grover GJ and Garlid KD (2000) ATP-Sensitive potassium channels: a review of their cardioprotective pharmacology. J Mol Cell Cardiol 32: 677–695.[CrossRef][Medline]

Gupta J and Linsdell P (2002) Point mutations in the pore region directly or indirectly affect glibenclamide block of the CFTR chloride channel. Pflügers Arch 443: 739–747.[CrossRef][Medline]

Hernandez-Benito MJ, Macianskiene R, Sipido KR, Flameng W, and Mubagwa K (2001) Suppression of transient outward potassium currents in mouse ventricular myocytes by imidazole antimycotics and by glybenclamide. J Pharmacol Exp Ther 298: 598–606.[Abstract/Free Full Text]

Hume JR, Duan D, Collier ML, Yamazaki J, and Horowitz B (2000) Anion transport in heart. Physiol Rev 80: 31–81.[Abstract/Free Full Text]

Kim SH, Cho KW, Chang SH, Kim SZ, and Chae SW (1997) Glibenclamide suppresses stretch-activated ANP secretion: involvements of K⁺_ATP channels and L-type Ca²⁺ channel modulation. Pflügers Arch 434: 362–372.[CrossRef][Medline]

Lee CO (1981) Ionic activities in cardiac muscle cells and application of ion-selective microelectrodes. Am J Physiol 241: H459–H478.

Lee CO and Levi AJ (1991) The role of intracellular sodium in the control of cardiac contraction. Ann NY Acad Sci 639: 408–427.[Abstract]

Levi AJ, Dalton GR, Hancox JC, Mitcheson JS, Issberner J, Bates JA, Evans SJ, Howarth FC, Hobai IA, and Jones JV (1997) Role of intracellular sodium overload in the genesis of cardiac arrhythmias. J Cardiovasc Electrophysiol 8: 700–721.[Medline]

Light PE, Cordeiro JM, and French RJ (1999) Identification and properties of ATP-sensitive potassium channels in myocytes from rabbit Purkinje fibres. Cardiovasc Res 44: 356–369.[Abstract/Free Full Text]

Luzi L and Pozza G (1997) Glibenclamide: an old drug with a novel mechanism of action? Acta Diabetol 34: 239–244.[CrossRef][Medline]

Muanprasat C, Sonawane ND, Salinas D, Taddei A, Galietta LJ, and Verkman AS (2004) Discovery of glycine hydrazide pore-occluding CFTR inhibitors: mechanism, structure-activity analysis and in vivo efficacy. J Gen Physiol 124: 125–137.[Abstract/Free Full Text]

Nichols CG and Lederer WJ (1991) Adenosine triphosphate-sensitive potassium channels in the cardiovascular system. Am J Physiol 261: H1675–H1686.

Qu Y, Campbell DL, Whorton AR, and Strauss HC (1993) Modulation of basal L-type Ca²⁺ current by adenosine in ferret isolated right ventricular myocytes. J Physiol 471: 269–293.[Abstract/Free Full Text]

Rosati B, Rocchetti M, Zaza A, and Wanke E (1998) Sulfonylureas blockade of neural and cardiac HERG channels. FEBS Lett 440: 125–130.[CrossRef][Medline]

Sheppard DN and Robinson KA (1997) Mechanism of glibenclamide inhibition of cystic fibrosis transmembrane conductance regulator Cl^– channels expressed in a murine cell line. J Physiol 503: 333–346.[CrossRef][Medline]

Smallwood JK, Ertel PJ, and Steinberg MI (1990) Modification by glibenclamide of the electrophysiological consequences of myocardial ischaemia in dogs and rabbits. Arch Pharmacol 342: 214–220.

Sorota S (1999) Insights into the structure, distribution and function of the cardiac chloride channels. Cardiovasc Res 42: 361–376.[Abstract/Free Full Text]

Steadman BW, Moore KB, Spitzer KW, and Bridge JH (1988) A video system for measuring motion in contracting heart cells. IEEE Trans Biomed Eng 35: 264–272.[CrossRef][Medline]

Sykes CA, Wright AD, Malins JM, and Pentecost BL (1977) Changes in systolic time intervals during treatment of diabetes mellitus. Br Heart J 39: 255–259.[Abstract/Free Full Text]

Tominaga M, Horie M, Sasayama S, and Okada Y (1995) Glibenclamide, an ATP-sensitive K⁺ channel blocker, inhibits cardiac cAMP-activated Cl^– conductance. Circ Res 77: 417–423.[Abstract/Free Full Text]

Woo SH and Lee CO (1999) Role of PKC in the effects of alpha1-adrenergic stimulation on Ca²⁺ transients, contraction and Ca²⁺ current in guinea-pig ventricular myocytes. Pflügers Arch 437: 335–344.[CrossRef][Medline]

Yamamoto-Mizuma S, Wang GX, and Hume JR (2004) P2Y purinergic receptor regulation of CFTR chloride channels in mouse cardiac myocytes. J Physiol 556: 727–737.[Abstract/Free Full Text]

Yamazaki J and Hume JR (1997) Inhibitory effects of glibenclamide on cystic fibrosis transmembrane regulator, swelling-activated and Ca²⁺-activated Cl^– channels in mammalian cardiac myocytes. Cir Res 81: 101–109.[Abstract/Free Full Text]

Yoshitake K, Hirano K, and Kanaide H (1991) Effects of glibenclamide on cytosolic calcium concentrations and on contraction of the rabbit aorta. Br J Pharmacol 102: 113–118.[Medline]

Zhou Z, Hu S, and Hwang TC (2002) Probing an open CFTR pore with organic anion blockers. J Gen Physiol 120: 647–662.[Abstract/Free Full Text]

This article has been cited by other articles:

				K. Fukuzaki, T. Sato, T. Miki, S. Seino, and H. Nakaya Role of sarcolemmal ATP-sensitive K+ channels in the regulation of sinoatrial node automaticity: an evaluation using Kir6.2-deficient mice J. Physiol., June 1, 2008; 586(11): 2767 - 2778. [Abstract] [Full Text] [PDF]

				S.-Y. Lee, B.-H. Choi, E.-M. Hur, J.-H. Lee, S.-J. Lee, C. O. Lee, and K.-T. Kim Norepinephrine activates store-operated Ca2+ entry coupled to large-conductance Ca2+-activated K+ channels in rat pinealocytes Am J Physiol Cell Physiol, April 1, 2006; 290(4): C1060 - C1066. [Abstract] [Full Text] [PDF]

				J. M. Evans, A. K. Allan, S. A. Davies, and J. A. T. Dow Sulphonylurea sensitivity and enriched expression implicate inward rectifier K+ channels in Drosophila melanogaster renal function J. Exp. Biol., October 1, 2005; 208(19): 3771 - 3783. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.104.074369v1 312/1/61    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Lee, S.-Y. Articles by Lee, C. O. Search for Related Content PubMed PubMed Citation Articles by Lee, S.-Y. Articles by Lee, C. O.