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J Appl Physiol 93: 1660-1668, 2002. First published August 2, 2002; doi:10.1152/japplphysiol.00220.2002
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Vol. 93, Issue 5, 1660-1668, November 2002

Anion channels influence ECC by modulating L-type Ca2+ channel in ventricular myocytes

Shi-Sheng Zhou, Zhan Gao, Ling Dong, Yan-Feng Ding, Xiao-Dong Zhang, Yue-Min Wang, Jian-Ming Pei, Feng Gao, and Xin-Liang Ma

Department of Physiology, The Fourth Military Medical University, Xi'an 710032, China


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Anion channels are extensively expressed in the heart, but their roles in cardiac excitation-contraction coupling (ECC) are poorly understood. We, therefore, investigated the effects of anion channels on cardiac ventricular ECC. Edge detection, fura 2 fluorescence measurements, and whole cell patch-clamp techniques were used to measure cell shortening, the intracellular Ca2+ transient, and the L-type Ca2+ current (ICa,L) in single rat ventricular myocytes. The anion channel blockers 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and niflumic acid reversibly inhibited the Ca2+ transients and cell shortening in a dose-dependent manner. Comparable results were observed when the majority of the extracellular Cl- was replaced with the relatively impermeant anions glutamate (Glt-) and aspartate (Asp-). NPPB and niflumic acid or the Cl- substitutes did not affect the resting intracellular Ca2+ concentration but significantly inhibited ICa,L. In contrast, replacement of extracellular Cl- with the permeant anions NO<UP><SUB>3</SUB><SUP>−</SUP></UP>, SCN-, and Br- supported the ECC and ICa,L, which were still sensitive to blockade by NPPB. Exposure of cardiac ventricular myocytes to a hypotonic bath solution enhanced the amplitude of cell shortening and supported ICa,L, whereas hypertonic stress depressed the contraction and ICa,L. Moreover, cardiac contraction was completely abolished by NPPB (50 µM) under hypotonic conditions. It is concluded that a swelling-activated anion channel may be involved in the regulation of cardiac ECC through modulating L-type Ca2+ channel activity.

swelling-activated chloride channel; anion channel blocker; calcium transient; heart


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ANION CHANNELS, EXTENSIVELY expressed in the heart (17, 30), are mainly responsible for the transport of Cl-, the most abundant extracellular anion (14). The currents carried by Cl- are believed to play roles in the regulation of both the electrical activity of cardiac myocytes and cardiovascular functions (15, 17, 30). Previous studies have demonstrated that Cl- substitution has pronounced effects on electrical activities (12, 15, 29, 32) and contraction (13, 25) in cardiac myocytes. In addition, Cl- substitution or putative anion channel blockers (ACBs) have frequently been reported to significantly modify the activity of other cardiac ion channels, such as the hyperpolarization-activated current (1, 12), potassium channels (3, 39), and Na+ and Ca2+ channels (5, 24, 38). However, the mechanisms underlying the effects of ACBs and Cl- substitutes on other cardiac ion channels, which were hitherto attributed to nonspecific effects (5, 24, 38, 39) or unpredictable side effects (17), remain unclear.

In cardiac ventricular myocytes, the most important physiological event for cardiac function is excitation-contraction coupling (ECC). During membrane depolarization, extracellular Ca2+ enters the cell via L-type Ca2+ channels and triggers a Ca2+-induced Ca2+ release from sarcoplasmic reticulum (SR) (10, 40). The resultant cytosolic Ca2+ transient (CaT) activates myofilament proteins and initiates contraction. Although different anion channel currents have been characterized in the heart (17, 30), the effects of these channels on ECC in cardiac myocytes remain largely unknown. Therefore, the purpose of the present study was to investigate the roles of anion channels in the regulation of cardiac ECC.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell preparations. Ventricular myocytes were enzymatically isolated from the hearts of adult female Sprague-Dawley rats (200-250 g) by using essentially standard procedures, as reported previously (11). Briefly, the hearts were removed immediately after decapitation and retrogradely perfused with oxygenated Tyrode solution at 37°C for 5 min and then with Ca2+-free Tyrode solution for 5 min before the addition of 0.5 mg/ml collagenase (type I, Sigma Chemical, St. Louis, MO) and 1 mg/ml BSA to the same solution. After 35 min of digestion with the collagenase-containing solution, the hearts were perfused for 5 min with a Kraftbrühe (KB) (high K+) solution containing (in mM) 70 L-glutamic acid, 25 KCl, 20 taurine, 10 KH2PO4, 3 MgCl2, 0.5 EGTA, 10 glucose, and 10 HEPES-KOH (pH 7.35). Subsequently, the ventricular tissue was cut into small pieces in an oxygenated KB solution. After gentle stirring with a glass rod for 5 min, the myocyte-containing solution was filtered through a nylon mesh. The cells were maintained in KB solution at room temperature (23-25°C) for electrophysiological recordings. For measurements of cell shortening and CaT, after 30 min in KB solution, aliquots of cell suspension were sedimented by centrifugation at 100 g for 1 min and then resuspended in Ca2+-free Tyrode solution with 2% BSA. The Ca2+ concentration of the solution was gradually increased to normal (1.8 mM) within 30 min.

Whole cell patch-clamp experiments. Aliquots of cell suspension were transferred into a perfusion chamber on the stage of an inverted microscope (IMT-2, Olympus, Tokyo, Japan). Pipettes had tip resistances of 5-6 MOmega when filled with internal solution. Whole cell recordings were performed at room temperature by using a patch-clamp amplifier (Axopatch 200B, Axon Instruments, Foster, CA). Liquid junction potentials between external and pipette solutions were offset before the pipette touched the cell. In the anion substitution experiments, an agar-salt bridge was used as the reference electrode. To selectively activate the L-type Ca2+ current (ICa,L), the membrane potential was depolarized from -70 to -40 mV, where it was held for 100 ms to inactivate the Na+ and T-type Ca2+ channels. K+ currents were eliminated by internal tetraethylammonium chloride (20 mM) and by omission of K+ from pipette and bath solutions. To monitor the effects of drugs, ICa,L was elicited by a depolarization from -40 to 0 mV for 300 ms every 5 s. The current-voltage relationship was assessed by measuring currents at voltage pulses (300 ms) from -40 to +50 mV, applied in 10-mV increments. The current signals were low-pass filtered at 2 kHz and digitized with an analog-to-digital converter (Digidata 1322) and pCLAMP 8.1 software (Axon Instruments, Foster, CA) at a sampling rate of 10 kHz. ICa,L was calculated as the difference between the peak inward current and the holding current level.

Measurements of CaT and cell shortening. Cytosolic calcium was measured by the fluorescent calcium indicator fura 2 by using a dual-fluorescence, calcium ion-sensing system (IonOptix, Milton, MA). The myocytes, suspended in Tyrode solution, were incubated with 5 µM fura 2-acetoxymethyl ester (AM) (St. Louis, MO) for 30 min at room temperature and then washed three times with fura 2-AM-free Tyrode solution. The loaded cells were kept at room temperature (23-25°C) for 1 h before measurements of intracellular Ca2+ concentration ([Ca2+]i) to allow the fura 2-AM in the cytosol to deesterify. Loading with a low concentration of fura 2-AM at a relatively low temperature of 23-25°C was done to minimize the effects of the compartmentalization of fura 2 (28).

Fura 2-loaded myocytes were placed in a microperfusion chamber mounted on an inverted microscope (IX50, Olympus, Tokyo, Japan). A rod-shaped myocyte with clear striations and sharp edges was localized by microscopic observation, and contractions were elicited by field stimulation delivered at 0.25 Hz through two platinum electrodes mounted on either side of the chamber. Fura 2-loaded myocytes were alternately excited with a xenon lamp at wavelengths of 360 and 380 nm. The emission fluorescence was collected by the objective and reflected through a barrier filter to a photomultiplier tube, as previously described (26). Because each cell was used as its own control, [Ca2+]i was expressed as the 360-to-380 fura 2 ratio. Before myocyte [Ca2+]i was measured, the background fluorescence of the measuring area without a myocyte was set as zero. Cell length and contractile amplitude of myocytes were recorded with a video edge detector and a specialized data-acquisition software (SoftEdge Acquisition System and IonWizard, IonOptix, Milton, MA), as previously described (26).

All whole cell patch-clamp recordings and measurements of cell shortening and CaT were performed at room temperature (23-25°C).

Solutions. The normal Tyrode solution contained (in mM) 143 NaCl, 5.4 KCl, 0.5 MgCl2, 1.8 CaCl2, 0.3 NaH2PO4, 5 glucose, and 5 HEPES-NaOH (pH 7.4, 310 mosmol/kgH2O). The nominally Ca2+-free Tyrode solution was prepared by omitting CaCl2 from the normal Tyrode solution. To obtain the desired Cl- substitution solutions, the 148.4 mM Cl- in normal Tyrode solution was replaced by equimolar Glt-, Asp-, acetate (Ac-), NO<UP><SUB>3</SUB><SUP>−</SUP></UP>, SCN-, or Br-. In osmotic stress experiments, 90 mM mannitol was added to the normal Tyrode solution to obtain a hypertonic bath solution (400 mosmol/kgH2O). For the hypotonic experiments, a control isotonic solution (310 mosmol/kgH2O) was made by replacing the 35 mM NaCl in the normal Tyrode solution with 70 mM mannitol, and a hypotonic solution (240 mosmol/kgH2O) was obtained by omitting mannitol from the control isotonic solution.

In whole cell patch-clamp recordings, the standard bath solution contained (in mM) 140 NaCl, 1.8 CaCl2, 0.5 MgCl2, 10 glucose, and 5 HEPES-NaOH (pH 7.4; 300 mosmol/kgH2O). The control pipette solution contained (in mM) 110 aspartic acid, 10 NaCl, 5 Mg-ATP, 5 EGTA, 20 tetraethylammonium chloride, 5.5 glucose, and 10 HEPES-CsOH (pH 7.3). In some whole cell patch-clamp experiments, the 140 mM Cl- in the standard bath solution was replaced by equimolar Glt-, Asp-, Br-, or SCN-, and Asp- in the pipette solution was replaced by equimolar Cl-. In the osmotic stress experiments, 100 mM mannitol was added to the standard bath solution to make a hypertonic solution (400 mosmol/kgH2O). In hypotonic experiments, a control isotonic bath solution (300 mosmol/kgH2O) was prepared by replacing the 40 mM NaCl in the standard bath solution with 80 mM mannitol, and a hypotonic solution (220 mosmol/kgH2O) was made by omitting mannitol from the control isotonic solution.

The free Ca2+ concentration in bath solutions was calculated by using the CaBuf program (provided by Dr. G. Droogmans, Katholieke Universiteit, Leuven, Belgium) by taking the values of pH, temperature, ionic strength, association constants, and Mg2+ into consideration (36).

Chemicals. The following chemicals, purchased from Sigma Chemical, were added to the bath solutions: 0.1-100 µM 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and 0.1-500 µM niflumic acid (NFA). Stock solutions of fura 2-AM (0.5 mM), NPPB (100 mM), and NFA (500 mM) in DMSO were diluted to the desired final concentrations immediately before use. DMSO at a final concentration of <= 0.1% in the bath solution had no effect on contraction, CaT, or ICa,L (data not shown).

Statistical analysis. The data are presented as means ± SD. Statistical differences in the data were evaluated by Student's t-test and were considered significant at values of P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of ACBs on cardiac CaT and contraction. Simultaneous measurements of CaT and cell shortening showed that exposure of the myocytes to 50 µM NPPB decreased CaT and cell shortening by 92.9 ± 12.3 and 98.1 ± 4.6% (n = 13, P < 0.001, Fig. 1A), respectively. Concomitant with the reduction in CaT, the diastolic (resting) length increased by 2.5 ± 1.6 µm (n = 13, P < 0.05, Fig. 1A, bottom). The reduced CaT and cell shortening recovered on withdrawal of NPPB. Similar effects were observed with NFA. Bath application of NFA (100 µM) decreased the amplitude of CaT and cell shortening by 36.8 ± 12.8 and 83.6 ± 7.7%, respectively (n = 12, P < 0.001, Fig. 1B). Both compounds suppressed the contraction in a dose-dependent manner with EC50 values of 12.3 µM (NPPB) and 44 µM (NFA) (Fig. 1C). Neither NPPB (50 µM, n = 4) nor NFA (100 µM, n = 4) had detectable effects on resting [Ca2+]i (data not shown). These results indicate that the putative ACBs, NPPB and NFA, have negative inotropic effects on rat cardiac ventricular myocytes as a result of decreased Ca2+ release from the SR and/or decreased Ca2+ influx.


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  		Fig. 1.   Effects of 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and niflumic acid (NFA) on electrically induced Ca2+ transient (CaT) and contraction in fura 2-loaded cardiac myocytes. Changes in Ca2+ fluorescence ratio and cell shortening were recorded simultaneously. A: representative traces of CaT (top) and cell contraction (bottom) after electrical field stimulation at 0.25 Hz in the absence and presence of NPPB. B: representative traces of electrically induced CaT (top) and cell contraction (bottom) recorded before and after application of NFA. n = 13 (A) and 12 (B) experiments. C: concentration-response relations for the effects of NPPB () and NFA () on the electrically driven contraction of rat ventricular myocytes. Values are means ± SD; nos. in parentheses indicate the no. of cells observed.

Effects of relatively impermeant Cl- substitutes on cardiac CaT and contraction. To further explore the mechanism of the effects of NPPB and NFA on cardiac ECC, we investigated the effects of the relatively impermeant Cl- substitutes Glt- and Asp- on the CaT and contraction of rat cardiac ventricular myocytes. Replacement of 148.4 mM extracellular Cl- by equimolar Glt- reversibly reduced the amplitudes of the electrically induced CaT and cell shortening by 69.9 ± 17.9 and 94.7 ± 6.2%, respectively (n = 7, P < 0.001, Fig. 2A). Asp- substitution decreased the amplitude of CaT and cell shortening by 34.8 ± 16.4 and 64.3 ± 22.4%, respectively (n = 9, P < 0.001, Fig. 2B). These data showed that the inhibitory effects of Asp- substitution were weaker than those of Glt- substitution, although there was no significant alteration in the free [Ca2+]i after replacement of 148.4 mM extracellular Cl- with either equimolar Glt- (free Ca2+ = 1.77 mM) or Asp- (free Ca2+ = 1.75 mM). Neither Glt- (n = 3) nor Asp- (n = 3) induced noticeable changes in resting [Ca2+]i (data not shown). To further determine the role of extracellular anions on the ECC, we compared the effects of Glt- and Asp- with those of Ac-, a relatively small anion. In all 15 cells observed, Ac- substitution for Cl- provoked a biphasic response: an initially transient decrease in CaT and cell contraction followed by an increase (Fig. 2C). The second phase was also sensitive to NPPB (n = 3, Fig. 2C). The inhibitory effects of the putative ACBs and the relatively impermeant Cl- substitutes were comparable, suggesting that their actions may share a common pathway.


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  		Fig. 2.   Effects of glutamate, aspartate, and acetate substitution for Cl- on electrically induced CaT and contraction of cardiac myocytes. Representative recordings are shown of electrical field stimulation-induced CaT (top) and cell contraction (bottom) before and after replacement of 148.4 mM extracellular Cl- with equimolar Glt- (A), Asp- (B), and Ac- (C). The CaT traces were obtained at times corresponding to that indicated by a-c (B) or a-d (C) in bottom traces. Replacement of extracellular Cl- in the bath solution by Glt- (A), Asp- (B), and Ac- (C) is indicated by the horizontal bars.

Effects of ACBs and relatively impermeant Cl- substitutes on ICa,L. The sarcolemmal L-type Ca2+ channels are crucial in ECC, because Ca2+ influx through the L-type Ca2+ channels triggers Ca2+ release from the SR (40). Therefore, the effects of the ACBs, NPPB and NFA, and impermeant Cl- substitutes on ICa,L were investigated. Bath application of NPPB (50 µM) and NFA (100 µM) reversibly decreased the peak ICa,L by 60.1 ± 6.7% (n = 6, P < 0.01) and 45.4 ± 11.5% (n = 5, P < 0.01), respectively (Fig. 3, A and B). NPPB and NFA reduced the amplitude of ICa,L without altering the reversal potential for ICa,L (Fig. 3, C and D). Similarly, replacement of 140 mM extracellular Cl- by Glt- or Asp- suppressed the peak ICa,L by 78.2 ± 19.2% (n = 5, P < 0.01, Fig. 4A) and 36.5 ± 11% (n = 5, P < 0.01, Fig. 4B), respectively. The inhibitory effects of Glt- (n = 3) on ICa,L were not altered when the intrapipette Cl- was increased from the conventional level (30 mM) to 140 mM (data not shown). These data suggest that suppression of the L-type Ca2+ channel is responsible for the inhibition of cardiac ECC induced by the ACBs and the relatively impermeant Cl- substitutes.


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  		Fig. 3.   Effects of NPPB and NFA on L-type Ca2+ current (ICa,L) in rat ventricular myocytes. Changes in ICa,L were monitored by applying a depolarizing pulse (300 ms in duration) from -40 to 0 mV every 5 s. A and B: current traces in top panels were obtained at times indicated by a-c in bottom panels. To obtain current-voltage (I-V) curves of ICa,L, whole cell ICa,L was elicited by 300-ms step depolarizations between -40 and +50 mV from a holding potential of -40 mV in increments of 10 mV. C and D: I-V curves of ICa,L obtained in the absence and presence of NPPB (50 µM) and NFA (100 µM), respectively. Values are means ± SD (n = 5 cells).



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  		Fig. 4.   Effects of glutamate and aspartate substitution for Cl- on ICa,L in rat ventricular myocytes. ICa,L was elicited by applying a depolarizing pulse (300 ms in duration) from -40 to 0 mV every 5 s. Right: replacement of 148.4 mM extracellular Cl- with equimolar Glt- (A) and Asp- (B) is indicated by the horizontal bars. Left: current traces were obtained at times indicated by a-c in the right panels. A and B each represent 5 experiments.

Effects of substitution of permeant Cl- substitutes on cardiac CaT, contraction, and ICa,L. To understand the role of the anion channels in the modulation of ICa,L, we examined the effects of the permeant inorganic anions Br-, NO<UP><SUB>3</SUB><SUP>−</SUP></UP>, and SCN- on CaT and contraction in rat ventricular myocytes. In contrast to the effects of the relatively impermeant Cl- substitutes (Fig. 2), replacement of 148.4 mM extracellular Cl- with equimolar Br- increased cell shortening by 48.48 ± 17.5% (n = 7, P < 0.01, Fig. 5A, bottom). NO<UP><SUB>3</SUB><SUP>−</SUP></UP> and SCN- substitution for extracellular Cl- supported the cell contraction and CaT. NPPB (50 µM) abolished the electrically induced CaT and cell shortening after extracellular Cl- was substituted by Br-, NO<UP><SUB>3</SUB><SUP>−</SUP></UP>, or SCN- (Fig. 5). In whole cell patch-clamp experiments, replacement of 140 mM extracellular Cl- with equimolar Br- significantly increased the peak ICa,L in six of seven cells observed (Fig. 6A). SCN- substitution for extracellular Cl- supported the ICa,L (Fig. 6B). It seemed that the maintenance of L-type Ca2+ channel activity depends, to a large degree, on anion channels.


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  		Fig. 5.   Effects of replacement of extracellular Cl- by Br-, NO<UP><SUB>3</SUB><SUP>−</SUP></UP>, and SCN- on electrically induced CaT and contraction of cardiac myocytes. Changes in Ca2+ fluorescence ratio and cell shortening were recorded simultaneously. Top: representative traces of electrically induced CaT before and after replacement of 148.4 mM extracellular Cl- by equimolar Br- (A), NO<UP><SUB>3</SUB><SUP>−</SUP></UP> (B), and SCN- (C). Bottom: representative traces of the cell contraction. Application of NPPB (50 µM) is indicated by the horizontal bars. n = 7 (A), 5 (B), and 5 (C) experiments.



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  		Fig. 6.   Effects of replacement of extracellular Cl- by Br- (A) and SCN- (B) on cardiac ICa,L. Left: representative traces of ICa,L obtained at times indicated by a and b in the right panels. Right: time courses of changes in the amplitude of the ICa,L peak. Replacement of 148.4 mM extracellular Cl- with equimolar Br- (A) and SCN- (B) is indicated by the horizontal bars. n = 7 (A) and 6 (B) experiments.

Effects of osmotic stress on cardiac contraction and ICa,L. When the myocytes were perfused with hypotonic solution (240 mosmol/kgH2O), the amplitude of cell shortening increased by 28.4 ± 7.6% (n = 5, P < 0.01). The enhanced contraction in hypotonic conditions was also inhibited by NPPB (Fig. 7A). Hypotonic solution (220 mosmol/kgH2O) induced a significant increase in four of eight cells and counteracted the rundown of the ICa,L in the other four cells (Fig. 7B). On the contrary, exposure of the myocytes to hypertonic solutions (400 mosmol/kgH2O) decreased the cell shortening and ICa,L by 66.9 ± 17.6% (n = 4, P < 0.01, Fig. 7C) and 53.6 ± 7.2% (n = 6, P < 0.01, Fig. 7D), respectively. These results are in good agreement with the previous observation that hyperosmolality reduces the amplitude of ICa,L (27). Moreover, cell diastolic length in hypertonic solution decreased by 4.3 ± 2.2 µm due to cell shrinkage (n = 4, P < 0.05, Fig. 7C).


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  		Fig. 7.   Effects of osmotic stress on cardiac contraction and ICa,L. A: representative trace of cell shortening. Perfusion with hypotonic solution and bath application of NPPB (50 µM) are indicated by the horizontal bars. B: representative traces of ICa,L in left panel were obtained at times indicated by a-c in the right panel. C: representative recording of cell shortening in isotonic and hypertonic bath solutions. D: representative traces of ICa,L in the left panel were obtained at times indicated by a-c in the right panel. Application of hypertonic bath solution is indicated by the horizontal bar. ICa,L was elicited by applying a depolarizing pulse (300 ms in duration) from -40 to 0 mV every 5 s.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The major findings of the present study are that the putative ACBs, NPPB and NFA, and the relatively impermeant Cl- substitutes, Glt- and Asp-, suppress the CaT and contraction of isolated rat cardiac ventricular myocytes. The effects of NPPB and NFA on cardiac contraction were similar to those obtained with other ACBs, the disulfonic stilbene derivatives (25).

It is well known that the sarcolemmal L-type Ca2+ channel plays a crucial role in ECC, because Ca2+ influx through the L-type Ca2+ channels triggers Ca2+ release from the SR (40). Although little is known concerning the effects of NPPB and NFA on ECC of cardiac myocytes, a handful of studies have revealed that NPPB and NFA inhibit the contraction of vascular smooth muscles, probably mediated by Ca2+ channels (6-8). The present study suggests that NPPB- and NFA-induced depression of the cardiac ICa,L is mainly responsible for the decrease of the CaT and contraction.

ACBs are frequently reported to have profound effects on some other ion channels (1, 5, 24, 39), including the L-type Ca2+ channels, in both vascular smooth muscles (8) and cardiac ventricular myocytes (5, 38). These unpredictable effects of ACBs were attributed to nonspecific effects, unrelated to Cl- channel block (5, 8, 19, 24, 38, 39). However, these interpretations are challenged by the idea that the effect of NFA on vascular smooth muscles is related to a Cl- channel, which may lead to the opening of a calcium channel (6, 7, 22). In cardiac myocytes, Minocherhomjee et al. (25) have found that the disulfonic stilbene ACBs inhibit the uptake of both 36Cl and 45Ca. This evidence suggests that Cl- and Ca2+ movements may be interrelated in some way. In the present experiments, we found that Glt- mimicked the inhibitory effects of NPPB and NFA on cardiac ECC and that Asp- induced partial inhibition of the CaT and contraction, whereas Ac- substitution for Cl- produces a transient inhibition followed by a NPPB-sensitive rebound response. The effects of Glt-, Asp-, and Ac- could not be attributed to their chelation of Ca2+, because there was no significant alteration in the free Ca2+ after replacement of most of the extracellular Cl- by these substitutes. The parallelism between the effects of the ACBs and the relatively impermeant Cl- substitutes suggests that they may share a common mechanism. It is likely that the effects of the ACBs and the relatively impermeant Cl- substitutes on ICa,L may involve either Cl- per se or anion channels.

It is arguable that intracellular Cl- per se may play a role in cardiac ECC, because application of ACBs or relatively impermeant Cl- substitutes may result in intracellular Cl- depletion. However, the inhibitory effects of replacement of extracellular Cl- by Glt- remained unchanged while intracellular Cl- concentration was clamped at 140 mM. Moreover, permeant Cl- substitutes supported the contraction, CaT, and ICa,L of the cells (Figs. 5 and 6) but did not alter the inhibitory effects of NPPB (Fig. 5). These data suggest that the reduction in peak ICa,L is an intracellular Cl--independent effect. It appears that activation of the L-type Ca2+ channel needs the support of anion channel activity.

The driving force for Cl- movement through the sarcolemmal membrane via anion channels depends on the equilibrium potential of Cl-. In the present electrophysiological experiments with the gradient of 144.6 mM extracellular Cl- concentration/30 mM intracellular Cl- concentration, the equilibrium potential of Cl- estimated by the Nernst equation was approx -41 mV. If some Cl- moved across the membrane during the ICa,L measurements (from the holding potential of -40 to +50 mV), the current carried by Cl- would be expected to produce an outward-going current opposite in direction to the ICa,L due to Cl- influx. If so, inhibiting anion channels would be expected to increase the amplitude of the inward current. However, blockage of anion channels with ACBs inhibited ICa,L instead of enhancing it, an effect that was mimicked by the relatively impermeant Cl- substitutes, whereas permeant Cl- substitutes supported the L-type Ca2+ channels. These observations suggest that regulation of the L-type Ca2+ channel by an anion channel is not merely a question of an anion channel-mediated chloride conductance. The regulation may involve a channel-channel interaction mechanism, which has been well established among epithelial channels (4, 21, 33).

Three major types of Cl- channels have been detected in adult mammalian heart (30). Among these, the cystic fibrosis transmembrane regulator (CFTR), a Cl- channel activated by protein kinase A, is the least likely to be involved in modulating the L-type Ca2+ channel because CFTR is not detectable in adult rat ventricle (9). The Ca2+-activated Cl- channel (ICl,Ca) may play a role in the modulation of ICa,L; however, buffering by high intracellular EGTA prevents the activation of cardiac ICl,Ca (43). In the present patch-clamp experiments, ICl,Ca was expected to be eliminated by chelating internal free calcium with high EGTA (5 mM). Swelling-activated Cl- channel (ICl,swell), expressed ubiquitously in mammalian cells, is known to be blocked by NPPB, NFA, stilbene disulfonates, and diphenylamine-2-carboxylate (20, 31, 34, 35, 42). The present study found that the cardiac contraction and ICa,L were inhibited not only by NPPB and NFA but also by hypertonic stress, whereas hypotonic challenge increased the contraction and supported the ICa,L. These results suggest that the anion channel involved in the regulation of ICa,L is a ICl,swell. Because blockage of ICl,swell by NPPB and NFA under isotonic conditions produces pronounced effects on cardiac contraction and ICa,L, it seems that basal ICl,swell activity is important for maintaining the activation of the cardiac L-type Ca2+ channel.

Anion channels are ubiquitously present in cells. If there are channel-channel interactions between anion channels and other channels (such as cation channels), blocking anion channels with their inhibitors would be expected to alter the activities of other related channels. In epithelia, CFTR, an anion channel, is found to modulate other channels via channel-channel interaction (4, 21, 33). For this reason, inhibition of CFTR with ACBs must have a profound influence on such ionic channels as those regulated by CFTR. Therefore, this channel-channel interaction may be responsible, at least partially, for the "nonspecific effects" of ACBs observed in epithelial cells. In heart, it is frequently reported that the potency of inhibition of cardiac cation channels by ACBs that differ in structure seems to be similar to that of reduction of anion channels (1, 5, 24, 38). However, the possibility of direct interaction between cardiac anion channels and cation channels is often ignored. Although some studies mentioned the possible relation between the cation channels and some anion channel before they attributed the effects of ACBs on ICa,L to nonspecificity (24, 38), the limitation of those studies is that some other important anion channels in heart (such as ICl,swell) were not taken into consideration. Moreover, the nonspecific effects of ACBs observed in heterologous expression experiments should be carefully explained before excluding the possibility that the heterologously expressed ionic channels may be influenced by the intrinsic anion channels of host cells. The present study suggests that the mechanism of influence of ICa,L by ACBs and Cl- substitution may be mediated by direct channel-channel interaction between anion channels and L-type Ca2+ channels.

Although the present study found that either ACBs, or impermeant Cl- substitutes, or hyperosmolality significantly inhibit the ICa,L, which may be mainly responsible for the decrease in Ca2+-induced Ca2+ release, some other factors may also be involved in the changes of cardiac ECC, because ACBs and Cl- substitution also influence other cardiac ion channels (5, 39) apart from ICa,L. Our primary study found that cardiac Na+ current was also significantly inhibited by either NPPB or relatively impermeant Cl- substitutes, Glt- and Asp-, although the resting membrane potential was not changed significantly under those conditions (data not shown). The mechanism of the ACB- and Cl- substitution-induced changes in Na+ current and the role of these changes in cardiac ECC are under investigation.

Several studies reported that ACBs increased K+ currents in expressed cell systems or noncardiac cells. For example, NFA at low concentration (10 µM) increased a slow voltage-activated K+ current expressed in oocytes, but inhibited the slow voltage-activated K+ current at higher concentration (100 µM) (2). In smooth muscle cells from rabbit portal vein, NFA increased the noradrenaline-evoked Ca2+-activated K+ current but had no effect on spontaneous Ca2+-activated K+ current (16). In contrast, in rat heart, the transient outward K+ current (Ito), a major repolarizing ionic current in ventricular myocytes, was inhibited by either chloride channel blockers or relatively impermeant Cl- substitutes (23). In agreement with the previous observation, our experiments also verified that aspartate substitution for external Cl- strongly inhibited Ito (n = 3, data not shown). Moreover, Kv4.3, an important K+ channel responsible for Ito in rat cardiac cells, was significantly decreased by NFA, flufenamic acid, and disulfonic stilbenes (39). In addition, a volume-sensitive basolateral K+ current was blocked by NPPB in HT-29/B6 cells (18). It is well known that inhibition of Ito leads to a significant prolongation of the action potential duration (37). Accordingly, the cell contractility is expected to increase in this condition. Thus it seems unlikely that the strongly inhibitory effects of Cl- channel blockers and relatively impermeant Cl- substitutes on CaTs and cardiac contractility are due to increased K+ currents. The effects of ACBs and Cl- substitution on cardiac K+ currents should be subjected to further investigation.

Taken together, our data demonstrate that anion channels play an important role in cardiac ECC and suggest that the cardiac ICl,swell might be involved in the regulation of cardiac ECC mainly by modulating the L-type Ca2+ channel. The present study provides insight into the mechanisms of the control of both electrical and contractile activities in cardiac ventricular myocytes. The L-type Ca2+ channel is crucial to cardiac ECC under physiological conditions. Swelling-activated Cl- current may increase during myocardial ischemia-reperfusion due to cell swelling (41). Therefore, investigation of the interaction between the ICl,swell and the L-type Ca2+ channel may have profound physiological and clinical significance.


    ACKNOWLEDGEMENTS

The authors thank Dr. Iain C. Bruce for reviewing the manuscript, Dr. G. R. Li for helpful discussion, and Dr. G. Droogmans for the kind gift of the CaBuf program.


    FOOTNOTES

This research is funded by the National Natural Science Foundation of China (nos. 39870318 and 39970807) and the Foundation for University Key Teachers by the Ministry of Education of China.

Address for reprint requests and other correspondence: S. S. Zhou, Dept. of Physiology, The Fourth Military Medical Univ., No. 17, Chang-Le West Road, Xi'an, Shaanxi 710032, China (E-mail: sszhou{at}fmmu.edu.cn).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

August 2, 2002;10.1152/japplphysiol.00220.2002

Received 14 March 2002; accepted in final form 20 July 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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J APPL PHYSIOL 93(5):1660-1668
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