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¹ Institute of Basic Medical Sciences, Medical College, Dalian University, Dalian 116622, China ² Department of Physiology, Xinxiang Medical College, Xinxiang 453003, China

	Abstract

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The effects of monocarboxylic acid-derived Cl^– channel blockers on cardiac depolarization-activated K⁺ currents were investigated. Membrane currents in rat ventricular myocytes were recorded using the whole-cell configuration of the patch-clamp technique. 5-Nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) and niflumic acid (NFA) induced an outward current at 0 mV. Both NPPB and NFA failed to induce any current when used intracellularly or after K⁺ in the bath and pipette solutions was replaced by equimolar Cs⁺. Voltage pulse protocols revealed that NPPB and NFA enhanced the steady-state K⁺ current but inhibited the transient outward K⁺ current. Genistein, a tyrosine kinase (PTK) inhibitor, inhibited NPPB- and NFA-induced outward current. Another PTK inhibitor, lavendustin A, produced a comparable effect. In contrast, the inactive analogue of genistein, daidzein, was ineffective. Orthovanadate, a tyrosine phosphatase inhibitor, markedly slowed the deactivation of the outward current induced by NPPB and NFA. The protein kinase A (PKA) inhibitor H-89 inhibited NPPB-induced outward current at 0 mV. In contrast, the protein kinase C (PKC) inhibitor H-7 was without significant effect on the action of NPPB. Pretreatment of the myocytes with genistein or H-89 prevented the enhancing effect of NPPB. Increasing intracellular Cl^– from 22 to 132 mM slightly reduced NPPB-induced outward current at 0 mV. These results demonstrate that the monocarboxylic acid-derived Cl^– channel blockers NPPB and NFA enhance cardiac steady-state K⁺ current, and suggest that the enhancing effect of the Cl^– channel blockers is mediated by stimulation of PKA and PTK signalling pathways.

(Received 15 January 2007; accepted after revision 12 February 2007; first published online 15 February 2007)
Corresponding author S-S. Zhou: Institute of Basic Medical Sciences, Medical College, Dalian University, Dalian 116622, China. Email: zhouss{at}dlu.edu.cn

	Introduction

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Cardiac action potentials reflect changes in membrane conductance governed by numerous different ion channels and electrogenic exchangers. Among the cardiac ion channels, depolarization-activated K⁺ channels play important roles in the repolarization of the action potential of cardiac myocytes. Changes in the function of depolarization-activated K⁺ channels may affect cardiac action potential repolarization and lead to the generation of life-threatening arrhythmias (Nerbonne & Kass, 2005). Therefore, depolarization-activated K⁺ channels are potent targets for drugs.

Chloride channel blockers are notorious for their non-specific effect, i.e. they can profoundly affect cation channels (Hume et al. 2000). In the heart, Cl^– channel blockers are found to inhibit Ca²⁺-independent transient outward K⁺ current (I_to; Lefevre et al. 1996), L-type Ca²⁺ current (I_Ca,L; Conforti et al. 1994; Zhou et al. 2002) and Na⁺ current (Conforti et al. 1994; Zhou et al. 2005). However, how Cl^– channel blockers affect cation channels is still unclear. Monocarboxylic acid-derived Cl^– channel blockers, such as 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) and niflumic acid (NFA), are frequently used in the investigation of cardiac Cl^– channels. Both NPPB and NFA were found to inhibit cardiac I_Ca,L (Zhou et al. 2002) and Na⁺ channels (Zhou et al. 2005). Moreover, Cl^– substitution also has a profound influence on cardiac I_to (Lefevre et al. 1996; Lai et al. 2004) and I_Ca,L (Zhou et al. 2002). Based on these observations, a possible mechanism of involvement of Cl^– channel in the non-specific effects of Cl^– channel blockers and Cl^– substitution has been proposed (Zhou et al. 2002; Lai et al. 2004). Baker et al. (2004) recently found that a voltage-dependent anion-selective channel, located in the plasma membrane (Lawen et al. 2005), can function as an enzyme. This finding raises the possibility that the Cl^– channel per se may affect intracellular signalling pathways.

In the present study, we further investigated the effects of monocarboxylic acid-derived Cl^– channel blockers on the depolarization-activated outward K⁺ currents. We found that the monocarboxylic acid-derived Cl^– channel blockers NPPB and NFA enhanced the steady-state component (I_SS) of the delayed rectifier K⁺ current (I_K), but inhibited I_to in rat ventricular myocytes. The enhancing effect of NPPB and NFA was antagonized by the protein tyrosine kinase (PTK) inhibitors genistein and lavendustin A and by the protein kinase A (PKA) inhibitor H-89. Inhibition of protein tyrosine phosphatase (PTP) with sodium orthovanadate (VO₄) markedly slowed the deactivation of the enhanced outward K⁺ conductance after withdrawal of NPPB and NFA. Elevation of intracellular Cl^– concentration ([Cl^–]_i) slightly reduced NPPB-induced outward current at 0 mV. We conclude that NPPB and NFA activate cardiac I_SS, probably by stimulating PKA and PTK signalling pathways.

	Methods

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Cell preparations

All experiments were conducted in accordance with the guidelines of the local ethics committee. Adult Sprague–Dawley rats (200–250 g) were anaesthetized with urethane (1.5 g kg^–1 I.P.). The trachea was intubated to permit artificial ventilation with room air using a ventilator (model I, Jiangwan, Shanghai, China). The chest was opened under artificial ventilation, and the aorta was cannulated in situ. The hearts were excised rapidly and retrogradely perfused at 37°C, using methods similar to those previously described (Zhou et al. 2002), with the following solutions in turn: Tyrode solution (5 min); Ca²⁺-free Tyrode solution (5 min); Ca²⁺-free Tyrode solution with 0.5 mg ml^–1 collagenase Type II (Gibco, Grand Island, NY, USA) and 1 mg ml^–1 bovine serum albumin (BSA; 35 min); and Kraftbrühe (KB; high-K⁺) solution (5 min). After dissociation and collection, the cells were kept in KB solution at room temperature (23–25°C) for electrophysiological recordings.

Whole-cell patch-clamp experiments

Aliquots of cell suspension were transferred into a perfusion chamber on the stage of an inverted microscope. Pipettes had tip resistances of 2.0–2.5 M when filled with internal solution. Whole-cell recordings were performed at room temperature (23–25°C) using an EPC-10 patch-clamp amplifier and Pulse software (HEKA Elektronik, Lambrecht, Germany). The offset potentials between both electrodes were zeroed before the pipette touched the cell. The liquid junction potential between the pipette and the bath solutions was calculated by using the JPCalc program within Clampex 8.1 (Axon Instruments, Inc.) and corrected for each recording. The I_to was elicited by 300 ms pulses from a holding potential of –50 mV to test potentials ranging from –40 to +50 mV in 10 mV increments. Since currents activated from a holding potential of –20 mV reflect activation of I_SS in rat ventricular myocytes (Himmel et al. 1999; Komukai et al. 2002), in the present study, I_SS was activated from a holding potential of –20 mV to test potentials ranging from –70, –90 or –100 to +50 mV in 10 mV increments. In most experiments, single cardiac myocytes were voltage clamped at 0 mV to continuously monitor the effects of NPPB and NFA. The current signals were low-pass filtered at 5 kHz and stored in the hard disk of a compatible computer. Internal application of NPPB and NFA was performed by using the intrapipette perfusion technique as described in a previous study (Zhou et al. 1997).

Solutions

The Tyrode solution contained (mM): NaCl, 143; KCl, 5.4; MgCl₂, 0.5; CaCl₂, 1.8; NaH₂PO₄, 0.3; glucose, 5; and Hepes, 5 (pH was adjusted to 7.4 with NaOH). The nominally Ca²⁺-free Tyrode solution was made by omitting CaCl₂ from the standard Tyrode solution. The KB solution contained (mM): potassium glutamate, 70; KCl, 25; taurine, 20; KH₂PO₄, 10; MgCl₂, 3; EGTA, 0.5; glucose, 10; and Hepes, 10 (pH was adjusted to 7.35 with KOH). The standard pipette solution for recording cardiac K⁺ currents contained (mM) potassium aspartate, 110; KCl, 20; MgCl₂, 1; Na₂-phosphocreatine, 5; Mg-ATP, 5; EGTA, 5; and Hepes, 10 (pH was adjusted to 7.2 with KOH). The bath solution contained (mM): NaCl, 140; KCl, 5; MgCl₂, 1; KH₂PO₄, 0.4; CaCl₂, 1.8; CdCl₂, 0.5; glucose, 10; and Hepes, 5 (pH was adjusted to 7.4 with NaOH). Cadmium chloride was used to inhibit the I_Ca,L and the Ca²⁺-activated Cl^– current, and Ba²⁺ (1 mM) was added to the bath solution to block inward rectifier K⁺ current (I_K1; Zygmunt et al. 1997). In some experiments, K⁺ in the pipette and bath solutions was replaced by equimolar Cs⁺ to eliminate K⁺ currents, and aspartate in the standard pipette solution was replaced by equimolar Cl^–.

Chemicals

All chemicals were purchased from Sigma (St Louis, MO, USA) except for H-7 (RBI, Natick, MA, USA) and H-89 (Biomol Research Laboratory Inc., Plymouth Meeting, PA, USA). Stock solutions of NPPB (100 or 500 mM), NFA (100 mM), genistein (100 mM), daidzein (100 mM), lavendustin A (20 mM), H-89 (20 mM) and H-7 (40 mM) in DMSO were diluted to the desired final concentrations immediately before use. DMSO (= 0.2%) alone did not affect the cardiac membrane conductance.

Statistical analysis

The data are presented as means ± S.D. Statistical differences in the data were evaluated by Student's paired t test or ANOVA as appropriate, and were considered significant at values of P < 0.05.

	Results

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Effects of monocarboxylic acid-derived Cl^– channel blockers on the membrane conductance of rat ventricular myocytes

When the myocytes were held at 0 mV, bath application of monocarboxylic acid-derived Cl^– channel blocker NPPB (25 µM) significantly increased the outward conductance from a basal level of 1.1 ± 0.5 pA pF^–1 to a peak of 50.3 ± 26.4 pA pF^–1 (n = 6, P < 0.01, Fig. 1A). A similar result was obtained by using another monocarboxylic acid-derived Cl^– channel blocker, NFA (Fig. 1B). After exposure of the myocytes to 50 µM NFA, the outward conductance at 0 mV developed from a basal level of 2.1 ± 1.7 pA pF^–1 to a peak of 44.3 ± 22.8 pA pF^–1 (n = 7, P < 0.01). Moreover, internal application of a high concentration of NPPB (500 µM, n = 5, Fig. 1C) or NFA (100 µM, n = 4, Fig. 1D) did not induce any significant change in the membrane conductance. To identify the charge carrier of the current induced by NPPB and NFA, we replaced K⁺ in the internal and external solutions with equimolar Cs⁺. The present results showed that either NPPB (n = 4, Fig. 1E) or NFA (n = 4, Fig. 1F) did not induce any change in the membrane conductance at 0 mV after Cs⁺ substitution for K⁺. These data suggest that the outward current induced by NPPB and NFA is a K⁺ current.

View larger version (13K): [in this window] [in a new window]   Figure 1.  Effects of monocarboxylic acid-derived Cl– channel blockers NPPB and NFA on the membrane conductance of rat ventricular myocytes Whole-cell current was continuously monitored with the membrane potential held at 0 mV. A, representative current trace recorded in the absence and presence of 25 µM NPPB. B, representative current trace recorded before and after application of 50 µM NFA. C and D, representative current traces showing the effects of intracellular application (filled bar) and extracellular application (open bar) of NPPB and NFA, respectively. E and F, typical current traces showing that neither NPPB nor NFA elicited any outward current when K+ in the bath and pipette solutions was replaced by equimolar Cs+. The data in A, B, C, D, E and F represent 6, 7, 5, 4, 4 and 4 experiments, respectively.

We then observed the effect of NPPB and NFA on depolarization-activated outward currents by using a depolarizing step-pulse protocol. The data showed that NPPB (n = 6) and NFA (n = 4) abolished I_to but enhanced the sustained component of outward K⁺ current (Fig. 2). To further identify the current enhanced by NPPB and NFA, we observed the effect of the Cl^– channel blockers on the outward current after I_to was inactivated by using a holding potential of –20 mV (see references in Komukai et al. 2002). The data showed that NPPB increased the outward current from 7.1 ± 6.3 to 53.6 ± 19.4 pA pF^–1 at +50 mV (n = 6, P < 0.01, Fig. 3A), and that NFA enhanced the outward current from 2.2 ± 1.0 to 45.2 ± 25.2 pA pF^–1 at +50 mV (n = 6, P < 0.01, Fig. 3B). The enhanced current produced by NPPB and NFA exhibited an outward rectifying property (Fig. 3Ad and Bd). Substitution of K⁺ in the bath and pipette solutions with equimolar Cs⁺ abolished the enhancing effect of NPPB (n = 4, Fig. 3Cb) and NFA (n = 4, Fig. 3Cc) on the outward conductance. We further examined the shift of the reversal potential of NPPB- and NFA-enhanced current recorded in different intracellular K⁺ concentrations ([K⁺]_i). In control conditions ([K⁺]_i = 130 mM, [K⁺]_o = 5.4 mM), NPPB- and NFA-enhanced currents reversed at –76.2 ± 9.5 mV (n = 9) and –75.7 ± 3.6 mV (n = 6), respectively (Fig. 4E and F), which were near the K⁺ equilibrium potential given by the Nernst equation (–82.2 mV). Reduction of [K⁺]_i resulted in a reduction of NPPB- and NFA-induced current, with a rightward shift of the reversal potential. After intracellular K⁺ was decreased to 65 or 30 mM by partly replacing intracellular K⁺ with Cs⁺, NPPB-sensitive current reversed at –55.5 ± 3.5 (n = 12) and –33.6 ± 4.7 mV (n = 5), respectively (Fig. 4A, B and E), and NFA-sensitive current reversed at –54.2 ± 4.2 (n = 7) and –37.8 ± 2.8 mV (n = 5), respectively (Fig. 4C, D and F), which were close to the predicted K⁺ equilibrium potentials (–64.3 mV in 65 mM [K⁺]_i and –44.3 mV in 30 mM [K⁺]_i). These data indicate that the outward current sensitive to NPPB and NFA is I_SS.

View larger version (17K): [in this window] [in a new window]   Figure 2.  Effects of NPPB and NFA on depolarization-activated K+ currents Outward currents were elicited by 300 ms depolarizing pulses from a holding potential of –50 mV to test potentials ranging from –40 to +50 mV in 10 mV increments. IK1 was blocked by 1 mM Ba2+. A, representative current traces recorded before application of NPPB (a), in the presence of NPPB (b) and after 5 min washout of NPPB (c). B, a typical set of current traces showing the effect of NFA: control (a); after application of 50 µM NFA (b); and after 5 min washout of NFA (c). The dashed lines in A and B indicate the zero current level. The data in A and B represent 6 and 4 experiments, respectively.

View larger version (20K): [in this window] [in a new window]   Figure 3.  Effects of NPPB and NFA on IK IK was elicited by 300 (A) or 400 ms depolarizing pulses (B and C) from a holding potential of –20 mV to test potentials ranging from –90 (A) or –70 mV (B and C) to +50 mV in 10 mV (A and B) or 20 mV increments (C). IK1 was blocked by Ba2+ (1 mM). A, representative current traces recorded in the absence (a) and presence of NPPB (b) and after washout of NPPB (c); d, current–voltage relationship measured at the end of test pulses for recordings shown in Aa and b. B, representative current traces recorded before application of NFA (a), in the presence of NFA (b) and after removal of NFA (c); d, current–voltage relationship measured at the end of test pulses for recordings shown in Ba and b. The dashed lines in A and B indicate the zero current level. C, an example showing that NPPB (b) and NFA (c) were ineffective on the membrane conductance after K+ in the pipette and bath solutions was replaced by equimolar Cs+: a, control; b, after application of NPPB; and c, in the presence of NFA. The data in A, B and C represent 6, 6 and 4 experiments, respectively.

View larger version (28K): [in this window] [in a new window]   Figure 4.  Effects of NPPB and NFA on membrane conductance in different intracellular K+ concentrations Currents were elicited by 400 ms depolarizing pulses from a holding potential of –20 mV to test potentials ranging from –100 to +50 mV in 10 mV increments. IK1 was blocked by Ba2+ (1 mM). A and C, representative current traces recorded in the absence and presence of NPPB and NFA, respectively, in 65 mM [K+]i. Intracellular K+ was partly replaced by Cs+ ([K+]i:[Cs+]i = 65:65 mM). B and D, representative current traces recorded before and after application of NPPB and NFA, respectively, in 30 mM [K+]i ([K+]i:[Cs+]i = 30:100 mM). The dashed lines in A–D indicate the zero current level. The data in A, B, C and D represent 12, 5, 7 and 5 experiments, respectively. E and F, current–voltage relationships showing the shift of reversal potential of NPPB- and NFA-enhanced currents under different intracellular K+ concentrations.

To explore the mechanism of action of the Cl^– channel blockers, we observed whether protein tyrosine phosphorylation was involved in the process. The results showed that genestein, a PTK inhibitor, decreased NPPB- and NFA-induced outward current at 0 mV by 87.7 ± 2.4 (n = 5, P < 0.01, Fig. 5A) and 78.7 ± 3.2% (n = 5, P < 0.01, Fig. 5C), respectively. In contrast, the inactive analogue of genistein, daidzein, was without significant effect (n = 4, Fig. 5B). Pretreating the myocytes with the PTK inhibitor genistein (100 µM) prevented the enhancing effect of NPPB (Figs 5D, n = 3). To further examine the involvement of PTK, we tested the effect of another PTK inhibitor, lavendustin A. The results showed that lavendustin A (100 µM) reduced NPPB- and NFA-enhanced outward current by 83.8 ± 12.5 (n = 4, P < 0.01, Fig. 6A) and 71.7 ± 17.4% (n = 5, P < 0.01, Fig. 6B), respectively. It seems that the enhancing effect of NPPB and NFA on I_SS is mediated by PTK activation. If that is the case, inhibition of PTP may affect the deactivation of the enhanced K⁺ conductance after withdrawal of the compounds. To test this possibility, we observed the effect of PTP inhibitor orthovandate (VO₄) on the deactivation of NPPB- and NFA-induced outward current at 0 mV. In control conditions, the enhanced outward K⁺ conductance returned to the basal level within 3 min after washout of NPPB or NFA (Fig. 1A and B). In contrast, in the presence of extracellular and intracellular VO₄ (1 mM), the deactivation of the K⁺ current became incomplete after washout of NPPB and NFA (Fig. 7). These data suggest that a PTK signalling pathway may be involved in the enhancement of the outward current induced by the Cl^– channel blockers.

View larger version (12K): [in this window] [in a new window]   Figure 5.  Effect of genistein and daidzein on NPPB- and NFA-elicited outward current Whole-cell current at 0 mV was continuously monitored. A and C, representative current traces showing the inhibitory effect of genistein on the outward current induced by NPPB (A) and NFA (C). B, representative current trace showing that daidzein is without significant effect on NPPB-induced current. D, an example showing the effect of pre-application of genistein on the enhancing action of NPPB. The data in A, B, C and D represent 5, 4, 5 and 3 experiments, respectively.

View larger version (8K): [in this window] [in a new window]   Figure 6.  Effect of tyrosine kinase inhibitor lavendustin A on NPPB- and NFA-induced current Whole-cell current at 0 mV was continuously monitored. A and B, representative current traces showing the inhibiting effect of lavendustin A on the outward current induced by NPPB (A) and NFA (B). The data in A and B represent 4 and 5 experiments, respectively.

View larger version (7K): [in this window] [in a new window]   Figure 7.  Effect of tyrosine phosphatase inhibitor orthovanadate on the deactivation of NPPB- and NFA-induced outward current Whole-cell current at 0 mV was continuously monitored. A and B, representative current traces showing that extracellular and intracellular application of sodium orthovanadate inhibited deactivation of the outward current induced by either NPPB (A) or NFA (B). The dashed lines in A and B indicate the zero current level. The data in A and B are representative of 6 and 4 experiments, respectively.

To determine whether other protein kinases play a role in the action of NPPB, we examined the effect of H-89, a potent PKA inhibitor, on the action of NPPB and NFA. The present results showed that H-89 significantly antagonized the enhancing effect of NPPB (Fig. 8A) and NFA (Fig. 8C). The H-89 (20 µM) reduced the amplitude of NPPB- and NFA-induced outward current at 0 mV by 87.2 ± 9.9 (n = 4, P < 0.01) and 95.2 ± 1.2% (n = 4, P < 0.01), respectively. In contrast, bath application of H-7, a PKC inhibitor, neither significantly inhibited NPPB-induced outward current at 0 mV (n = 4, Fig. 8D), nor prevented the enhancing effect of NPPB (n = 3, Fig. 8E). Moreover, NPPB failed to induce any significant effect on the membrane conductance at 0 mV after pretreatment of the myocytes with H-89. In contrast, withdrawal of H-89 unmasked the enhancing effect of NPPB (n = 4, Fig. 8B).

View larger version (14K): [in this window] [in a new window]   Figure 8.  Effect of PKA and PKC inhibitors on NPPB action A, an example of an original current trace showing the inhibitory effect of H-89 on NPPB-induced outward current at 0 mV. B, an example showing that NPPB produced no significant effect on the membrane conductance monitored at 0 mV after the myocyte was pretreated with 20 µM H-89; in contrast, removal of H-89 unmasked the enhancing effect of NPPB. C, an example of a typical current trace showing the inhibitory effect of H-89 on NFA-induced outward current at 0 mV. D and E, representative current traces showing that H-7 neither significantly inhibited the NPPB-induced outward current at 0 mV, nor prevented the simulating action of NPPB. The data in A, B, C, D and E represent 4, 4, 4, 4 and 3 experiments, respectively.

Accumulating evidence indicates that Cl^– may modify ion channels (Pusch et al. 1999; Yuan et al. 2000; Bekar et al. 2005). Since blockade of the Cl^– channel may alter [Cl^–]_i, we tested whether [Cl^–]_i may play a role in the effect of NPPB. The present results showed that NPPB-induced outward current at 0 mV in high-Cl^– (132 mM)-dialysed myocytes (39.8 ± 15.8 pA pF^–1, n = 34) was slightly reduced compared with those in normal-Cl^– (22 mM)-dialysed myocytes (55.7 ± 23.8 pA pF^–1, n = 34; P < 0.05, Fig. 9).

View larger version (9K): [in this window] [in a new window]   Figure 9.  Effect of [Cl–]i on the action of NPPB Histogram showing the mean density of NPPB-induced outward current at 0 mV with standard Cl– (22 mM, open bar, n = 34) or high Cl– (132 mM, hatched bar, n = 34) in the pipette solution. *P < 0.05 versus standard [Cl–]i. The data were from 6 hearts. Equal numbers of normal [Cl–]i- and high [Cl–]i-dialysed myocytes were observed in each heart.

	Discussion

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Effects of monocarboxylic acid-derived Cl^– channel blockers on depolarization-activated K⁺ currents

Several lines of evidence indicate that Cl^– channel blockers have profound influences on a variety of K⁺ currents. For example, NFA is found to enhance the presynaptic voltage-activated K⁺ current (Miralles et al. 1996), Ca²⁺-activated K⁺ current (Miralles et al. 1996), human ERG (ether-a-go-go related gene) current (Malykhina et al. 2002) and lipid-sensitive mechano-gated 2P domain K⁺ current (Takahira et al. 2005). In the present study, we found that the monocarboxylic acid-derived Cl^– channel blockers NPPB and NFA activated a macroscopic outward current at 0 mV in rat ventricular myocytes. The NPPB- and NFA-sensitive current reversed near the predicted K⁺ equilibrium potential, suggesting that the current is a K⁺ current.

The I_K is composed of multiple components, including I_Kr (I_K,rapid), I_Ks (I_K,slow), I_Kur (I_K,ultrarapid) and I_SS. Evidence has revealed that the Kv1.5 probably encodes I_Kur and I_K,slow1, and that the ERG encodes I_Kr (Nerbonne & Kass, 2005). Studies indicate that Kv1.5 and human ERG channels show significant Cs⁺ permeability (Fedida et al. 1999; Zhang et al. 2003). If the channel activated by NPPB and NFA is I_Kur or I_Kr, there should be a Cs⁺ current after replacement of intracellular and extracellular K⁺ with Cs⁺. However, the present study found that Cs⁺ substitution for K⁺ completely abolished the enhancing effect of NPPB and NFA. Therefore, it seems unlikely that NPPB and NFA target these two channels.

In rat ventricular myocytes, currents activated from a holding potential of –20 mV reflect activation of I_SS, the non-inactivating component of I_K that is sensitive to blockade by Cs⁺ (Himmel et al. 1999; Komukai et al. 2002). The present results indicated that, after replacement of intracellular and extracellular K⁺ with Cs⁺, NPPB and NFA neither induced any significant effect on the membrane conductance at 0 mV, nor enhanced the outward current activated from a holding potential of –20 mV (see Figs 1D and 3C). These results suggest that NPPB- and NFA-induced enhancement of I_K is a result of activation of the I_SS component. To our knowledge, this study provides the first evidence that Cl^– channel blockers NPPB and NFA can simulate cardiac I_SS.

Involvement of protein kinases in the effects of NPPB and NFA on cardiac K⁺ currents

Protein phosphorylation is a common mechanism for regulating K⁺ channel activity (Levitan, 1994; Davis et al. 2001). It is becoming apparent that tyrosine phosphorylation may modulate the activity of a variety of K⁺ channels (Davis et al. 2001), including I_K channels in cardiac myocytes (Gao et al. 2004; Missan et al. 2006). The present study found that the stimulating effect of NPPB and NFA on the outward current was markedly inhibited by the PTK inhibitors genistein and lavendustin A, but not by the inactive analogue of genistein, daidzein. Moreover, inhibition of PTP with VO₄ significantly slowed the deactivation of NPPB- and NFA-induced outward current at 0 mV (Fig. 6). So it seems that the effect of NPPB and NFA on I_SS involves the PTK signalling pathway.

It is well known that PKA-dependent phosphorylation plays an important role in regulation of a variety of cardiac K⁺ channels. This PKA-dependent phosphorylation can upregulate I_K (Walsh & Kass, 1988; Huang et al. 1994). In the present study, we found that the enhancing effect of NPPB and NFA on the outward current was also inhibited by the PKA inhibitor H-89, but not by the PKC inhibitor H-7. These results suggest that, besides PTK, PKA may also be involved in the action of NPPB and NFA on I_SS. The effect of NPPB and NFA on I_SS is most likely to result from enhancement of PTK and PKA activity. Moreover, the present results demonstrate that either presuppression of PTK with genistein (Fig. 4D) or pre-inhibition of PKA with H-89 (Fig. 7B) prevented the enhancing effect of NPPB on the outward current. Therefore, it seems that a cross-talk between PKA and PTK pathways is required for the enhancing effect of NPPB. Although there are clues to suggest that PKA may regulate the activity of PTKs and PTPs (Wilson & Kaczmarek, 1993; Park et al. 2000), the relationship between these two signalling pathways in the regulation of cardiac I_SS is not known from the present study.

It is known that both PKA and PTK are involved in modulation of cardiac I_Ca,L. Activation of PKA can upregulate I_Ca,L (van der Heyden et al. 2005). If NPPB and NFA can stimulate the PKA signal pathway, they may also enhance I_Ca,L. Thus, it seems that our previous study indicating that NPPB and NFA inhibit I_Ca,L (Zhou et al. 2002) is inconsistent with the present finding of activation of the PKA signalling pathway by NPPB and NFA. However, it should be noted from the present study that NPPB and NFA seem also to stimulate the PTK signalling pathway, which is known to downregulate I_Ca,L (Boixel et al. 2000; Schroder et al. 2004; van der Heyden et al. 2005). In this case, the response of I_Ca,L to NPPB and NFA may depend on which kinase plays a dominant role in modulating the I_Ca,L channel. Therefore, it is not strange that NPPB and NFA may induce I_Ca,L inhibition.

It has long been known that ₁-adrenoceptor stimulation can inhibit I_to (Apkon & Nerbonne, 1988; Ravens et al. 1989). A recent study reveals that ₁-adrenoceptor stimulation inhibits I_to via a cAMP/PKA-mediated pathway (Gallego et al. 2005). It is generally accepted that the voltage-gated K⁺ channels Kv4.2 and Kv4.3 are the most likely molecular correlates for I_to (Dixon et al. 1996; Nerbonne & Kass, 2005). Recent evidence demonstrates that -adrenoceptor stimulation-induced inhibition of I_to has been correlated with phosphorylation of the Kv4.2 and Kv4.3 (van der Heyden et al. 2006). It seems obvious that the cAMP/PKA-dependent pathway may play an important role in downregulating the I_to channel. In contrast, whether PTK plays a role in the regulation of cardiac I_to is still uncertain. Gao et al. (2004) recently reported that genistein-sensitive PTK may positively modulate I_to of rat ventricular myocytes. Although the present study implies that NPPB and NFA may enhance the activity of both PKA and PTK, the effect of NPPB and NFA on I_to is inhibition. Thus, it seems that even if PTK is involved in the regulation of I_to, PKA may play a dominant role in the regulation of the I_to channel.

Does the action of NPPB and NFA on cardiac K⁺ channels involve Cl^– or Cl^– channel?

Chloride has been found to play roles in cell function. It affects the gating of some Cl^– channels (Pusch et al. 1999; Pusch, 2004), interferes with non-chloride channels (Yuan et al. 2000; Bekar et al. 2005), and even influences protein phosphorylation (Yang et al. 2000). Thus, there is a possibility that the effect of the Cl^– channel blockers NPPB and NFA on cardiac K⁺ channels may be a Cl^–-mediated reaction, since blockade of the Cl^– channel may alter [Cl^–]_i. The present results indicated that increasing [Cl^–]_i from 22 to 132 mM slightly attenuated the action of NPPB on the outward current. It seems that the change in [Cl^–]_i is not the main reason for the effect of NPPB.

It is well established that the action potential of cardiac myocytes is the result of harmonious activity of dozens of channels (Nerbonne & Kass, 2005). However, the relationship among the ion channels is still unclear. Lefevre et al. (1996) compared the effect of Cl^– substitution with that of Cl^– channel blockers, and found that the effect of the Cl^– channel blocker SITS (4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid) on I_to was similar to that substitution of the less permeant anion aspartate for Cl^–. A comparable effect of Cl^– channel blockers and less permeant anions on cardiac I_Ca,L has also been observed in rat ventricular myocytes (Zhou et al. 2002). Based on these observations, we therefore speculate that the Cl^– channel may be involved in the non-specific effect of less permeable anions and Cl^– channel blockers on cardiac cation channels (Zhou et al. 2002; Lai et al. 2004). Indeed, Baker et al. (2004) recently found that voltage-dependent anion-selective channel 1, located in the mitochondrial outer membrane and the plasma membrane, can function as an enzyme (Lawen et al. 2005). This finding raises the possibility that the Cl^– channel per se may play a direct role in modulating intracellular signalling events. Thus, it seems possible that any factors (including Cl^– channel blockers) that affect the Cl^– channel may alter the channel-mediated responses, such as intracellular signalling. The present data showed that intracellular application of NPPB and NFA did not have any effect on the outward current. These results demonstrate that NPPB and NFA influence the outward current from the outside of the cell membrane, and suggest that it is unlikely that these Cl^– channel blockers stimulate I_SS through a direct action on PKA or cytoplasmic PTKs. Thus, there is a possibility that NPPB and NFA may exert a direct action on cardiac I_SS channels. However, the present study found that the enhancing effect of the Cl^– channel blockers on I_SS is antagonized by the inhibitors of either PKA or PTK. Therefore, it is also unlikely that NPPB and NFA act directly on I_SS channels. Chloride may affect the gating of Cl^– channels (Pusch et al. 1999; Pusch, 2004), and Cl^– channel blockers may alter Cl^– flux through Cl^– channels. Thus, these factors may change the Cl^– channel activity and consequently Cl^– channel-mediated intracellular signalling. Therefore, the possibility of involvement of the Cl^– channel in the effect of the Cl^– channel blockers on I_SS cannot be ruled out.

In conclusion, the present study demonstrates that the monocarboxylic acid-derived Cl^– channel blockers NPPB and NFA can stimulate cardiac steady-state K⁺ current, which is probably mediated by stimulation of the PKA and PTK signalling pathways.

	Footnotes

 
S-S. Zhou and L-B. Zhang contributed equally to this work.

	References

Top Abstract Introduction Methods Results Discussion References

 
Apkon M & Nerbonne JM (1988). ₁-Adrenergic agonists selectively suppress voltage-dependent K⁺ current in rat ventricular myocytes. Proc Natl Acad Sci U S A 85, 8756–8760.[Abstract/Free Full Text]

Baker MA, Lane DJR, Ly JD, De Pinto V & Lawen A (2004). VDAC1 is an NADH-ferricyanide reductase. J Biol Chem 279, 4811–4819.[Abstract/Free Full Text]

Bekar LK, Loewen ME, Forsyth GW & Walz W (2005). Chloride concentration affects Kv channel voltage-gating kinetics: importance of experimental anion concentrations. Brain Res Bull 67, 142–146.[CrossRef][Medline]

Boixel C, Tessier S, Pansard Y, Lang-Lazdunski L, Mercadier JJ & Hatem SN (2000). Tyrosine kinase and protein kinase C regulate L-type Ca²⁺ current cooperatively in human atrial myocytes. Am J Physiol Heart Circ Physiol 278, H670–H676.[Abstract/Free Full Text]

Conforti L, Sumii K & Sperelakis N (1994). Diphenylamine-2-carboxylate blocks voltage-dependent Na⁺ and Ca²⁺ channels in rat ventricular cardiomyocytes. Eur J Pharmacol 259, 215–218.[CrossRef][Medline]

Davis MJ, Wu X, Nurkiewicz TR, Kawasaki J, Gui P, Hill MA & Wilson E (2001). Regulation of ion channels by protein tyrosine phosphorylation. Am J Physiol Heart Circ Physiol 281, H1835–H1862.[Abstract/Free Full Text]

Dixon JE, Shi W, Wang HS, McDonald C, Yu H, Wymore RS, Cohen IS & McKinnon D (1996). Role of the Kv4.3 K⁺ channel in ventricular muscle. A molecular correlate for the transient outward current. Circ Res 79, 659–668.[Abstract/Free Full Text]

Fedida D, Maruoka ND & Lin S (1999). Modulation of slow inactivation in human cardiac Kv1.5 channels by extra- and intracellular permeant cations. J Physiol 515, 315–329.[Abstract/Free Full Text]

Gallego M, Setién R, Puebla L, Boyano-Adánez MC, Arilla E & Casis O (2005). ₁-Adrenoceptors stimulate a G_s protein and reduce the transient outward K⁺ current via a cAMP/PKA-mediated pathway in the rat heart. Am J Physiol Cell Physiol 288, C577–C585.[Abstract/Free Full Text]

Gao Z, Lau CP, Wong TM & Li GR (2004). Protein tyrosine kinase-dependent modulation of voltage-dependent potassium channels by genistein in rat cardiac ventricular myocytes. Cell Signal 16, 333–341.[CrossRef][Medline]

Himmel HM, Wettwer E, Li Q & Ravens U (1999). Four different components contribute to outward current in rat ventricular myocytes. Am J Physiol Heart Circ Physiol 277, H107–H118.[Abstract/Free Full Text]

Huang XY, Morielli AD & Peralta EG (1994). Molecular basis of cardiac potassium channel stimulation by protein kinase A. Proc Natl Acad Sci U S A 91, 624–628.[Abstract/Free Full Text]

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

Komukai K, Brette F, Pascarel C & Orchard CH (2002). Electrophysiological response of rat ventricular myocytes to acidosis. Am J Physiol Heart Circ Physiol 283, H412–H422.[Abstract/Free Full Text]

Lai XG, Yang J, Zhou SS, Zhu J, Li GR & Wong TM (2004). Involvement of anion channel(s) in the modulation of the transient outward K⁺ channel in rat ventricular myocytes. Am J Physiol Cell Physiol 287, C163–C170.[Abstract/Free Full Text]

Lawen A, Ly JD, Lane DJ, Zarschler K, Messina A & De Pinto V (2005). Voltage-dependent anion-selective channel 1 (VDAC1) – a mitochondrial protein, rediscovered as a novel enzyme in the plasma membrane. Int J Biochem Cell Biol 37, 277–282.[CrossRef][Medline]

Lefevre T, Lefevre IA, Coulombe A & Coraboeuf E (1996). Effects of chloride ion substitutes and chloride channel blockers on the transient outward current in rat ventricular myocytes. Biochim Biophys Acta 1273, 31–43.[Medline]

Levitan IB (1994). Modulation of ion channels by protein phosphorylation and dephosphorylation. Annu Rev Physiol 56, 193–212.[CrossRef][Medline]

Malykhina AP, Shoeb F & Akbarali HI (2002). Fenamate-induced enhancement of heterologously expressed HERG currents in Xenopus oocytes. Eur J Pharmacol 452, 269–277.[CrossRef][Medline]

Miralles F, Marsal J, Peres J & Solsona C (1996). Niflumic acid-induced increase in potassium currents in frog motor nerve terminals: effects on transmitter release. Brain Res 714, 192–200.[CrossRef][Medline]

Missan S, Linsdell P & McDonald TF (2006). Tyrosine kinase and phosphatase regulation of slow delayed-rectifier K⁺ current in guinea-pig ventricular myocytes. J Physiol 573, 469–482.[Abstract/Free Full Text]

Nerbonne JM & Kass RS (2005). Molecular physiology of cardiac repolarization. Physiol Rev 85, 1205–1253.[Abstract/Free Full Text]

Park JH, Park JK, Bae KW & Park HT (2000). Protein kinase A activity is required for depolarization-induced proline-rich tyrosine kinase 2 and mitogen-activated protein kinase activation in PC12 cells. Neurosci Lett 290, 25–28.[CrossRef][Medline]

Pusch M (2004). Structural insights into chloride and proton-mediated gating of CLC chloride channels. Biochemistry 43, 1135–1144.[CrossRef][Medline]

Pusch M, Jordt SE, Stein V & Jentsch TJ (1999). Chloride dependence of hyperpolarization-activated chloride channel gates. J Physiol 515, 341–353.[Abstract/Free Full Text]

Ravens U, Wang XL & Wettwer E (1989). -Adrenoceptor stimulation reduces outward currents in rat ventricular myocytes. J Pharmacol Exp Ther 250, 364–370.[Abstract/Free Full Text]

Schroder F, Klein G, Frank T, Bastein M, Indris S, Karck M, Drexler H & Wollert KC (2004). Src family tyrosine kinases inhibit single L-type: Ca²⁺ channel activity in human atrial myocytes. J Mol Cell Cardiol 37, 735–745.[CrossRef][Medline]

Takahira M, Sakurai M, Sakurada N & Sugiyama K (2005). Fenamates and diltiazem modulate lipid-sensitive mechano-gated 2P domain K⁺ channels. Pflugers Arch 451, 474–478.[CrossRef][Medline]

van der Heyden MA, Wijnhoven TJ & Opthof T (2005). Molecular aspects of adrenergic modulation of cardiac L-type Ca²⁺ channels. Cardiovasc Res 65, 28–39.[Abstract/Free Full Text]

van der Heyden MA, Wijnhoven TJ & Opthof T (2006). Molecular aspects of adrenergic modulation of the transient outward current. Cardiovasc Res 71, 430–442.[Abstract/Free Full Text]

Walsh KB & Kass RS (1988). Regulation of a heart potassium channel by protein kinase A and C. Science 242, 67–69.[Abstract/Free Full Text]

Wilson GF & Kaczmarek LK (1993). Mode-switching of a voltage-gated cation channel is mediated by a protein kinase A-regulated tyrosine phosphatase. Nature 366, 433–438.[CrossRef][Medline]

Yang T, Park JM, Arend L, Huang Y, Topaloglu R, Pasumarthy A, Praetorius H, Spring K, Briggs JP & Schnermann J (2000). Low chloride stimulation of prostaglandin E₂ release and cyclooxygenase-2 expression in a mouse macula densa cell line. J Biol Chem 275, 37922–37929.[Abstract/Free Full Text]

Yuan A, Dourado M, Butler A, Walton N, Wei A & Salkoff L (2000). SLO-2, a K⁺ channel with an unusual Cl^– dependence. Nat Neurosci 3, 771–779.[CrossRef][Medline]

Zhang S, Kehl SJ & Fedida D (2003). Modulation of human ether-a-go-go-related K⁺ (HERG) channel inactivation by Cs⁺ and K⁺. J Physiol 548, 691–702.[Abstract/Free Full Text]

Zhou SS, Gao Z, Dong L, Ding YF, Zhang XD, Wang YM, Pei JM, Gao F & Ma XL (2002). Anion channels influence ECC by modulating L-type Ca²⁺ channel in ventricular myocytes. J Appl Physiol 93, 1660–1668.[Abstract/Free Full Text]

Zhou SS, Takai A, Tominaga M & Okada Y (1997). Phosphatase-mediated enhancement of cardiac cAMP-activated Cl^– conductance by a Cl^– channel blocker, anthracene-9-carboxylate. Circ Res 81, 219–228.[Abstract/Free Full Text]

Zhou SS, Yang J, Li YQ, Zhao LY, Xu M & Ding YF (2005). Effect of Cl^– channel blockers on aconitine-induced arrhythmias in rat heart. Exp Physiol 90, 865–872.[Abstract/Free Full Text]

Zygmunt AC, Goodrow RJ & Antzelevitch C (1997). Sodium effects on 4-aminopyridine-sensitive transient outward current in canine ventricular cells. Am J Physiol Heart Circ Physiol 272, H1–H11.[Abstract/Free Full Text]

	Acknowledgements

 
This research was supported by the National Natural Science Foundation of China (no. 30570665). The authors thank Qing Chi for technical assistance and Ms Li-Xia Wang for secretarial assistance.

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