Phosphatase-Mediated Enhancement of Cardiac cAMP-Activated Cl− Conductance by a Cl− Channel Blocker, Anthracene-9-Carboxylate
Abstract An aromatic carboxylate, anthracene-9-carboxylic acid (9-AC), is known as a Cl− channel blocker. However, variable 9-AC effects have hitherto been reported on the cardiac cAMP-activated Cl− conductance, when applied extracellularly. We have reexamined the 9-AC effect on the Cl− conductance activated by isoproterenol or forskolin in guinea pig ventricular myocytes under whole-cell patch-clamp conditions. The inward current was blocked by 9-AC at ≥0.5 mmol/L, but in contrast, the outward current was enhanced at much lower concentrations (ED50, ≈13 μmol/L). 9-AC applied by the intracellular perfusion technique increased both the inward and outward currents. In the presence of intracellular 9-AC, deactivation of the conductance after washout of isoproterenol or forskolin was largely prevented. 9-AC produced an enhancing effect, even after inhibiting the deactivation process by okadaic acid (OA), whereas it failed to produce additional effects in the presence of orthovanadate. Intracellular application of 9-AC together with OA virtually abolished the current deactivation. The 9-AC effects on the Cl− conductance were not dependent on intracellular Ca2+ or pH. Putative inhibitors of alkaline (bromotetramisole) and acid phosphatases (tartrate) were without effect. 9-AC failed to inhibit the activities of purified protein phosphatase (PP)-1, -2A, and -2C. In the extract of guinea pig ventricle, 9-AC (≥10 μmol/L for full action) significantly inhibited a fraction of endogenous phosphatase activity that was sensitive to orthovanadate but not to OA, bromotetramisole, and tartrate. It is concluded that 9-AC blocks cardiac cAMP-activated (cystic fibrosis transmembrane conductance regulator) Cl− conductance from the extracellular side but enhances the conductance from the intracellular side by inhibiting an orthovanadate-sensitive phosphatase distinct from PP-1, -2A, -2B, or -2C and alkaline or acid phosphatase.
- anion channel
- cystic fibrosis transmembrane conductance regulator
- Cl− channel blocker
- cardiac myocyte
A variety of Cl− currents have been identified in mammalian cardiac ventricular myocytes.1 2 3 Among these, stimulation of the β-adrenergic receptor leads to activation of a cAMP-activated Cl− channel.4 5 6 7 The structure and functional properties were found to be similar to the epithelial CFTR Cl− channel.8 9 10 11 12 Cardiac cAMP-activated CFTR, like epithelial CFTR, Cl− channels are known to be activated via phosphorylation mediated by protein kinase A and deactivated via dephosphorylation mediated by some phosphatases.10 13 In addition, our recent study14 has shown that regional differences in CFTR mRNA expression in the guinea pig heart are closely correlated with the regional differences in cAMP-activated Cl− current density. Moreover, a full-length cloning of a cDNA from rabbit ventricles has recently shown that an exon-5 splice variant of epithelial CFTR is actually responsible for the cardiac cAMP-activated Cl− channel.15
An aromatic carboxylate, 9-AC is a useful inhibitor of the voltage-gated Cl− channel of skeletal muscle.16 9-AC is known to reduce the epithelial Cl− conductance in the apical membranes of canine tracheal cells17 and rabbit colon18 and the cAMP-activated Cl− efflux pathway in a human colonic epithelial cell line.19 However, the reported effects of 9-AC applied to the extracellular solution on the cardiac cAMP-activated Cl− conductance are contradictory. In guinea pig ventricular myocytes, Cl− currents activated by ISO were found to be inhibited by 9-AC in a voltage-independent manner,20 21 whereas their voltage-dependent inhibition was observed only in the inward direction by Ehara and Matsuura.22 In contrast, ISO-induced Cl− currents were reported to be insensitive to 9-AC in guinea pig ventricular myocytes by Vandenberg et al.23
The initial purpose of the present study was to reexamine the effects of a Cl− channel blocker, 9-AC, on the cardiac cAMP-activated Cl− currents by application not only from the extracellular but also from the intracellular side. The data showed that intracellular 9-AC enhances cAMP-activated Cl− conductance by inhibiting a fraction of the endogenous activity of vanadate-sensitive and okadaic acid-, bromotetramisole-, tartrate-, and Ca2+-insensitive cardiac phosphatase, which, together with okadaic acid–sensitive phosphatases, is involved in deactivation of the CFTR Cl− channel in guinea pig ventricular myocytes.
Materials and Methods
Isolated ventricular myocytes were prepared from the heart, which had been dissected from adult female guinea pigs under pentobarbital anesthesia (50 mg/kg IP), by perfusing with Ca2+-free Tyrode’s solution containing 0.1 mg/mL collagenase, as previously described.11 The myocyte suspension was stored at 4°C in KB solution until use in electrophysiological experiments. The composition of Ca2+-free Tyrode’s solution was (mmol/L) NaCl 143, NaH2PO4 0.3, KCl 5.4, MgCl2 0.5, glucose 5.5, and HEPES 5 (pH 7.4 adjusted with NaOH). KB solution contained (mmol/L) l-glutamic acid 70, KCl 25, taurine 20, KH2PO4 10, MgCl2 3, EGTA (Nacalai Tesque) 0.5, glucose 10, and HEPES-KOH 10 (pH 7.35).
For the assay of endogenous phosphatase activities, the ventricle was dissected, blotted, weighed (wet weight), and minced after the heart was perfused with Ca2+-free Tyrode’s solution without collagenase. The tissue was homogenized with 3 vol (vol/wt) of an ice-cooled solution containing (mmol/L) KCl 60, MgCl2 20, DTT 1, and HEPES 50 (pH 7.0 adjusted with KOH). The homogenate was centrifuged at 15 000g for 10 minutes, and the resultant supernatant was used for assay.
Aliquots of myocyte suspension were added to a perfusion chamber on the stage of an understage microscope (TMD, Nikon). Borosilicate glass pipettes (Hilgenberg) were pulled using a puller (P-97, Sutter Instruments) and had a tip resistance of 1 to 1.5 MΩ when filled with the pipette solution. Whole-cell membrane currents were recorded using a patch-clamp amplifier (Axopatch 200A, Axon Instruments), as previously described.11 Data were acquired on-line by computer (PC9801DX, NEC) through a Bessel-type filter at 2 kHz and recorded on videotape by means of an AD convertor (PCM-501ES, Sony) for backup. Ramp voltage pulses (0 to ±100 mV, every 8 or 13 seconds) were generated using a signal generator (type 1915, NF Electronic Instruments), and square-shaped voltage pulses (0 to ±80 mV in 20-mV increments, 200-millisecond duration) were generated by an electric stimulator (SEN-3301, Nihon Kohden). To record selectively Cl− currents under whole-cell conditions, K+ currents were eliminated by internal TEA (20 mmol/L) and by omission of K+ from both pipette and bath solutions; Na+ and Ca2+ currents, by inactivating at a holding potential of 0 mV; any residual Ca2+ currents, by extracellular Cd2+ (1 mmol/L); Na+-K+ pump currents, by removal of external K+; and Na+-Ca2+ exchange currents, by the nominal absence of internal and external Ca2+. In some experiments, the intracellular solution was changed by the intrapipette perfusion technique reported by Soejima and Noma24 with slight modification. All the electrophysiological studies were performed at room temperature (23°C to 25°C).
The control bath solution contained (mmol/L) NaCl 150, MgCl2 0.5, CdCl2 1, glucose 5.5, and HEPES-NaOH 5 (pH 7.4). The control pipette solution contained (mmol/L) aspartic acid 85, EGTA 10, TEA-Cl 20, Na2-creatine phosphate 5, Mg-ATP 10, MgCl2 0.5, glucose 5.5, and HEPES-CsOH 10 (pH 7.35). During β-adrenoceptor stimulation, Na2-GTP (200 μmol/L) was added to the pipette solution to minimize fade of the Cl− conductance. In some experiments, the pH of the pipette solution was increased to 9 by adding CsOH or decreased to 5.85 or 6 by adding HEPES. For experiments under symmetrical Cl− conditions (106 mmol/L), 47 mmol/L NaCl was replaced with equimolar sodium gluconate in the bath solution and 85 mmol/L cesium aspartate with equimolar CsCl in the pipette solution.
Endogenous Phosphatase Activity Measurements
The endogenous phosphatase activities in the ventricular muscle extract were determined by measuring the rate of phosphate liberation from pNPP at 25°C using the buffer that contained (mmol/L) MES 40, Tris-HCl 40, glycine 40, DTT 1, MgCl2 1, and ZnCl2 0.1. In some experiments, 2 mmol/L EDTA was added after MgCl2 was removed. The pH was adjusted by titration with KOH at 6 to 11. The pH of reaction mixtures was little affected by adding the stock solution of 9-AC (0.1 mol/L in 0.1N KOH) when the final concentration of 9-AC was 0.1 to 2 mmol/L. Reactions were started by injecting the extract (10 μL) into the buffer (990 μL) containing 2 mmol/L pNPP as the substrate, and the initial steady state velocity of hydrolysis was measured by continuously tracing the increase in absorbance at 400 nm due to liberation of the reaction product (p-nitrophenol) with the use of a spectrophotometer (type 330, Hitachi Co). Preliminary experiments showed that the specific absorbance, ε400, of p-nitrophenol at 400 nm and a given pH is well described by the following equation: where the maximal absorbance at 400 nm (ε400max) is 16.7±0.1 (mmol/L)−1 · cm−1, and the acid dissociation constant (Ka) is 89.3±0.4 nmol/L (degrees of freedom=61).
The endogenous activity of PP-2C in the muscle extract was measured by essentially the same isotopic method as described below for the assay of purified PP-2C, except that the buffer containing 10 or 30 mmol/L magnesium acetate was supplemented with 10 μmol/L OA in order to minimize interference by activities of PP-1 and PP-2A (see Takai et al25 and Cohen et al26 ).
Purification and Assay of PPs
The catalytic subunits of PP-1 and PP-2A were purified from rabbit skeletal muscle by the method of Tung et al.27 PP-2C isolated from rat liver was kindly provided by Dr G. Mieskes (Göttingen, FRG). MLC was isolated from chicken gizzard by essentially the same method as described for isolation of cardiac MLC by Cummins and Lambert,28 and they were 32P-phosphorylated using chicken gizzard MLC kinase isolated by the method of Ngai et al.29 The MLC phosphatase activities of the purified phosphatases were measured at 25°C by the procedure described previously.30 Briefly, reactions were started by injection of 32P-phosphorylated MLC into the buffer containing one of the purified enzymes with or without the addition of a substance to be tested (eg, 9-AC), and the initial rate of 32P liberation was measured. Buffers used contained (mmol/L) Tris (base) 40, KCl 100, and DTT 1 (pH 7.4 at 25°C, adjusted with HCl). For the assays of PP-1 and PP-2C, 0.1 mmol/L MnCl2 and 30 mmol/L magnesium acetate were supplemented, respectively.
The following agents were added to the bath solutions: 0.1 nmol/L to 10 μmol/L ISO, 1 to 10 μmol/L FSK, 1 mmol/L IBMX, 3 mmol/L db-cAMP, 100 μmol/L H-89, or 100 μmol/L DIDS. In some experiments, 4 mmol/L cAMP, 1.5 mmol/L sodium orthovanadate, 10 μmol/L OA, 1 mmol/L BrT, or 4 mmol/L l(+)-tartaric acid was added to the pipette solution by means of intracellular perfusion system. 9-AC was added to bath or pipette solution at 0.1 to 2000 μmol/L. All the agents except for H-89 (Seikagaku Corp) and DIDS (Dojinkagaku) were purchased from Sigma Chemical Co. Stock solutions of ISO (10 mmol/L in distilled water), FSK (10 mmol/L in ethanol), IBMX (0.5 mol/L in DMSO), H-89 (0.1 mol/L in DMSO), OA (10 mmol/L in DMSO), and 9-AC (1 mol/L in DMSO for electrophysiology or 0.1 mol/L in 0.1N KOH for phosphatase activity assay) were diluted to the desired final concentrations immediately before use. Neither DMSO (≤0.2%) nor ethanol (≤0.1%) alone affected the cardiac cAMP-activated Cl− conductance. cAMP, db-cAMP, orthovanadate, DIDS, theophylline, BrT, and tartrate were added directly to the solutions.
Electrophysiological data were given as mean±SD, and biochemical data were given as mean±SEM in the text and tables. Statistical differences of the data were evaluated by Student’s t test and considered significant at a value of P<.05.
Prominent increases in Cl− conductance were induced by β-adrenoceptor stimulation with ISO (1 μmol/L) or direct stimulation of adenylate cyclase by FSK (1 μmol/L) under symmetrical (106 mmol/L, Fig 1A⇓) or asymmetrical (intracellular, 21 mmol/L; extracellular, 153 mmol/L; Fig 2A⇓) Cl− conditions. The I-V relation of the ISO- or FSK-induced component was linear under symmetric Cl− conditions (Fig 1B⇓) but exhibited outward rectification under the transmembrane Cl− gradient (Fig 2B⇓). The reversal potential was shifted from 1.2±1.8 mV (n=8) under the symmetrical Cl− conditions to −35.6±4.5 mV (n=8) under the asymmetrical Cl− conditions, indicating preferential activation of Cl−-selective conductance. The FSK-activated Cl− current exhibited time-independent kinetics in response to step voltage pulses (Fig 2C⇓) and was insensitive to 100 μmol/L DIDS (Fig 1C⇓). Pretreatment with 10 μmol/L H-89, which is a potent and selective inhibitor of protein kinase A,31 largely impaired ISO-induced augmentation of the current (n=3, not shown). Thus, it appears that ISO- or FSK-induced currents represent cAMP-activated (CFTR) Cl− conductance, as originally reported by Bahinsky et al4 and Harvey and Hume.5 6
Dual Effects of Extracellular Application of 9-AC on Cardiac cAMP-Activated Cl− Conductance
As shown in Figs 1A⇑ and 2A⇑, in a reversible manner, bath application of 0.5 or 1 mmol/L 9-AC suppressed the inward component but increased the outward component of cAMP-activated Cl− current, without discernible alteration in cell volume or morphology under the light microscope. The ISO- and FSK-induced current densities recorded at −100 mV were decreased from −8.26±4.01 to −3.83±1.96 nA/nF (n=13, P<.001) and from −9.53±4.37 to −4.65±2.60 nA/nF (n=8, P<.001), respectively, by 1 mmol/L 9-AC, whereas those at +100 mV were increased from 8.96±4.15 to 11.96±6.15 nA/nF (n=13, P<.001) and from 10.56±4.58 to 13.89±6.92 nA/nF (n=8, P<.001), respectively. The I-V relation became more outwardly rectified after attaining the steady state effect of 9-AC (Figs 1B⇑ and 2B⇑). The suppressing effect on the inward current became more apparent at larger negative potentials. The Cl− current in the presence of 9-AC was also time independent (Fig 2C⇑) and DIDS insensitive (Fig 1D⇑).
In contrast, bath application of 9-AC never affected the basal currents recorded at −100 to +100 mV under symmetrical (1 mmol/L, n=5, not shown) or asymmetrical Cl− (Fig 2D⇑) conditions.
Both the enhancing and suppressing effects of 9-AC were concentration dependent, as shown in Fig 3A⇓. The enhancing effect on outward currents was observed at low concentrations with half-maximum concentrations (ED50) of ≈13 μmol/L. The suppressing effect, overwhelming the enhancing effect, on inward currents was observed at higher concentrations (with an apparent ED50 of ≈0.94 mmol/L) even after saturation of the enhancing effect. Therefore, it appears that there is a distinct difference in the concentration-response relations for the enhancing and suppressing effects. This fact suggests that the two effects are based on different mechanisms.
9-AC Action Site Distal to cAMP-Induced Activation of the Cl− Conductance
Fig 3B⇑ shows the concentration dependence of ISO-induced currents before and after extracellular application of 0.5 mmol/L 9-AC. The apparent affinity for ISO action was not substantially altered by 9-AC. The ED50 values were 3.49×10−8 and 2.95×10−8 mol/L in the absence and presence of 9-AC, respectively. These results rule out the possibility that 9-AC acts through altering the apparent affinity with which ISO activates the β-adrenoceptor.
9-AC–induced enhancing effects were observed under stimulation with a maximal concentration of ISO or FSK (Fig 4A⇓). 9-AC brought about the enhancing effect even under stimulation with 10 μmol/L FSK, 3 mmol/L db-cAMP, and 1 mmol/L IBMX (Fig 4B⇓). 9-AC was still effective during intracellular perfusion with a high concentration (4 mmol/L) of cAMP (Fig 4C⇓). Thus, it appears that the site of 9-AC action is distal to cAMP-induced activation of the Cl− channel.
Enhancing Effects of Intracellular Application of 9-AC on Cardiac cAMP-Activated Cl− Conductance
As shown in Fig 5A⇓, intracellular perfusion with 1 mmol/L 9-AC consistently increased not only the outward components (from 7.89±2.90 to 9.71±2.86 nA/nF at +100 mV, n=10, P<.001) but also the inward components (from −6.82±2.93 to −7.84±2.80 nA/nF at −100 mV, n=10, P<.001) of ISO-induced Cl− currents. Essentially similar effects were observed with 0.3 mmol/L 9-AC (n=3, not shown). The I-V relation indicates that the intracellular 9-AC effect shows little dependence on voltages (Fig 5B⇓). Extracellular application of 1 mmol/L 9-AC during intracellular perfusion with 1 mmol/L 9-AC markedly suppressed the inward currents (from −8.41±3.26 to −3.77±0.89 nA/nF at −100 mV, n=6, P<.001) but did not produce a large effect on the outward currents (Fig 5C⇓ and 5D⇓). These results clearly indicate that 9-AC blocks cAMP-activated Cl− currents only from the extracellular side but enhances the currents from the intracellular side.
Inhibiting Effects of 9-AC on Phosphatase-Mediated Deactivation of the Cl− Channel
In the continued presence of 1 mmol/L 9-AC in the cytosol, the deactivation process of ISO-induced Cl− conductance, which was observed after washout of ISO, became prominently slowed (Fig 5E⇑). Similar incomplete deactivation after washout was also observed for FSK-induced Cl− conductance during the presence of intracellular 9-AC (Fig 5F⇑). However, deactivation was attained after withdrawal of intracellular 9-AC by intracellular perfusion with a 9-AC–free pipette solution (Fig 5F⇑). An inhibitor of protein kinase A (H-89) failed to affect the sustained activation of Cl− conductance in the presence of intracellular 9-AC (Fig 5F⇑). Since deactivation of cardiac cAMP-activated Cl− conductance is known to be caused by dephosphorylation of the CFTR Cl− channel,10 these results strongly suggest that a phosphatase involved in the dephosphorylation process of Cl− channel is inhibited by intracellular 9-AC.
OA (10 μmol/L), an inhibitor selective for PP-1 and PP-2A,30 added to the intracellular solution also enhanced FSK-induced Cl− conductance, as shown in Fig 6A⇓ (from 7.19±1.97 and −6.46±2.25 to 8.80±2.61 and −7.77±2.65 nA/nF at +100 and −100 mV, respectively; n=7, P<.001). OA made the deactivation process incomplete (Fig 6B⇓), as reported previously.32 Even in the continued presence of intracellular OA, however, bath application of 9-AC further induced sizable increases in the outward Cl− currents (to 9.28±2.59 nA/nF, n=7, P<.05), as shown in Fig 6B⇓. Intracellular application of 1 mmol/L 9-AC together with 10 μmol/L OA virtually abolished deactivation of the Cl− current after washout of FSK (Fig 6C⇓). These results suggest that deactivation of cAMP-activated Cl− conductance can be attained by both OA-sensitive phosphatases (PP-1 and PP-2A) and OA-insensitive phosphatases and that 9-AC enhances the cardiac CFTR Cl− conductance by inhibiting some OA-insensitive phosphatase.
Intracellular application of 1.5 mmol/L orthovanadate further increased the ISO-activated Cl− currents in both the outward direction (from 7.07±2.15 to 11.05±2.45 nA/nF at +100 mV, n=7, P<.0005) and inward direction (from −6.25±2.33 to −9.18±2.31 nA/nF at −100 mV, n=7, P<.001) and markedly slowed the deactivation process of the Cl− conductance after washout of ISO, as reported previously.33 In the presence of intracellular orthovanadate, 9-AC added to the extracellular solution failed to further increase the ISO-induced outward Cl− current (to 11.21±2.46 nA/nF at +100 mV, n=7, P>.05) but was still effective in blocking the inward current (to −4.29±1.49 nA/nF at −100 mV, n=7, P<.001).
Since alkaline phosphatase has been reported to be implicated in the dephosphorylation process of epithelial CFTR Cl− channels,34 there is a possibility that 9-AC inhibits alkaline phosphatase involved in regulation of cardiac cAMP-activated Cl− conductance. However, as shown in Fig 7A⇓, intracellular perfusion with BrT, which is a potent inhibitor for alkaline phosphatase,35 failed to affect the 9-AC effects on the ISO-induced Cl− conductance and deactivation process after washout of ISO. In the presence of intracellular BrT, the ISO-induced outward Cl− current was increased from 5.49±0.74 to 8.52±1.35 nA/nF (n=6, P<.001) at +100 mV, and the inward current decreased from −5.34±0.61 to −4.31±1.16 nA/nF (n=6, P<.05) at −100 mV by extracellular application of 9-AC (1 mmol/L). Similarly, no effects were observed with 10 mmol/L theophylline, which is known to inhibit some type of alkaline phosphatases36 (n=3, not shown). Intracellular application of tartaric acid (4 mmol/L), which is a potent inhibitor of acid phosphatase,37 did not affect the 9-AC effects on ISO-induced Cl− conductance (Fig 7B⇓). In the presence of tartrate, 9-AC (1 mmol/L) increased the ISO-induced outward current from 5.48±1.04 to 8.24±1.94 nA/nF (n=5, P<.001) at +100 mV and decreased the inward current from −5.07±1.25 to −3.76±1.02 nA/nF (n=5, P<.05). Also, the ISO-induced and 9-AC–induced responses were affected neither by increasing the pH in the intracellular (pipette) solution to 9 (Fig 7C⇓) nor by decreasing the pH to 5.85 (Fig 7D⇓). When the pH was either increased to 9 or decreased to 5.85 from 7.35, ISO-induced Cl− currents changed little (n=4 or 5). 9-AC (1 mmol/L) enhanced the ISO-induced outward current at +100 mV from 5.36±1.49 to 8.10±2.57 nA/nF (n=4, P<.001) at pH 9 or from 5.18±1.49 to 7.94±1.96 nA/nF (n=5, P<.001) at pH 5.85, whereas 9-AC suppressed the inward current at −100 mV from −4.61±1.63 to −2.81±0.90 nA/nF (n=4, P<.05) at pH 9 or from −4.58±1.43 to −3.13±1.34 nA/nF (n=5, P<.05) at pH 5.85.
Inhibiting Effects of 9-AC on Cardiac Phosphatase Activity
As shown in Fig 8A⇓ (open circles), the extract of guinea pig ventricular muscle exhibited a phosphatase activity toward pNPP (2 mmol/L) as the substrate in the presence of 1 mmol/L Mg2+ in the pH range between 6 and 11. Since it is known that PP-2A has a very high activity against pNPP,38 the experiments were carried out in the presence of OA (10 μmol/L) in order to minimize the interference by endogenous PP-2A. Of the three phosphatase inhibitors, tartrate, BrT, and orthovanadate, it was orthovanadate that exhibited the strongest inhibitory action. The pNPP phosphatase activity was completely abolished in the presence of 1 mmol/L orthovanadate over the pH range examined (Fig 8A⇓, open triangles). At pH lower than 7, the phosphatase activity was partially inhibited by 10 mmol/L tartrate (Fig 8A⇓, solid circles), which produced no inhibitory effect at pH higher than 8 (n=2, not shown). BrT (1 mmol/L) exerted a potent inhibitory action at pH higher than 9 (Fig 8A⇓, solid squares), whereas it had little or no effect at pH lower than 8 (n=2, not shown). These data indicate that the guinea pig ventricular muscle exhibits the endogenous activity of orthovanadate-sensitive phosphatases, which are distinct from PP-2A, alkaline phosphatase, and acid phosphatase, at pH 7 to 8.
The effect of 9-AC was examined on the fraction of the pNPP phosphatase activity in the presence of BrT (1 mmol/L), tartrate (10 mmol/L), and OA (10 μmol/L) at pH 7.4. The endogenous phosphatase activity was found to be inhibited by ≈27% by 0.1 mmol/L 9-AC (93.9±2.3 [n=14] to 68.5±4.9 [n=5] μmol · min−1 · kg wet wt−1, P<.005). Fig 8B⇑ shows the concentration-inhibition curves for the effects of 9-AC on the pNPP phosphatase activity in the presence of BrT, tartrate, and OA at pH 7.4. The concentration for full inhibition was ≥10 μmol/L, and the half-maximum concentration (ID50) was 0.225 μmol/L.
Table 1⇓ gives the activities of the purified PPs against 32P-phosphorylated MLC. The specific activities in the absence of 9-AC were within the same range as reported for these enzymes previously.30 9-AC exhibited no inhibitory effects on the activities of the catalytic subunits of PP-1 and PP-2A or on the activity of PP-2C measured in the presence of 30 mmol/L Mg2+ (Table 1⇓).
We also examined the effect of 9-AC on the endogenous activity of Mg2+-dependent PP toward MLC, ie, PP-2C, in the ventricular extract (Table 2⇓). The assay was carried out in the presence of 10 μmol/L OA, since it has been shown that this concentration of OA completely inhibits PP-1 and PP-2A but does not affect PP-2C.30 38 The ventricular extract exhibited the PP-2C activity that was almost completely inhibited by removing Mg2+. 9-AC (1 mmol/L) exhibited little inhibitory action on the activity in the presence or absence of 10 or 30 mmol/L Mg2+ (Table 2⇓).
The present patch-clamp study demonstrated that extracellular application of an aromatic carboxylic acid, 9-AC, suppresses cardiac cAMP-activated Cl− conductance in a voltage-dependent manner. Only the inward currents were blocked by extracellular 9-AC. This is in good agreement with a previous observation by Ehara and Matsuura.22 The 9-AC–induced suppression was more marked at larger negative potentials. In addition, the present study showed that the outward currents are, in contrast, enhanced by the bath application of 9-AC. The mechanisms for the enhancing and blocking effects of 9-AC should be different from each other because of a distinct difference in their concentration-response relations.
Intracellular application of 9-AC gave rise to augmentation of not only outward but also inward currents. Extracellular application of 9-AC on the top of intracellular 9-AC of a high concentration (1 mmol/L) failed to produce an additional enhancing effect on outward currents. Thus, it is likely that 9-AC applied to the bath solution suppresses the inward current only from the extracellular side but augments the outward current from the intracellular side after permeating the plasma membrane. It is possible that 9-AC applied to the bath solution may have rapidly leaked in, thereby inducing an early enhancing effect, which can be produced at lower intracellular concentrations, before inducing the suppressing effect at higher concentrations at the extracellular binding site. Actually, 9-AC–induced suppression of the inward current was, in some cases, preceded by transient enhancement. 9-AC applied to the intracellular solution may have leaked out and suppressed the inward current, although those must have been largely diluted by the bulk bathing solution. In fact, at larger potentials the enhancing effect of internally applied 9-AC on inward currents (by 15% at −100 mV) was slightly less prominent than that on outward currents (by 23% at +100 mV).
Since many types of Cl− channels exist in cardiac myocytes,1 2 3 there is a possibility that the enhancing effect of 9-AC could be due to activation of some Cl− channel other than the cAMP-activated one. However, activation of Cl− conductance dependent on Ca2+ or protein kinase C is unlikely, because intracellular Ca2+ was strongly chelated with 10 mmol/L EGTA. No visible cell volume change during 9-AC application could exclude activation of volume-sensitive Cl− conductance. 9-AC enhanced the Cl− conductance only after maneuvers that increase the intracellular cAMP level. Furthermore, 9-AC–sensitive Cl− currents exhibited functional properties characteristic of the CFTR Cl− channel, such as time-independent kinetics, linear I-V relation under symmetrical Cl− conditions, and DIDS insensitivity. Taken together, it is clear that the 9-AC–enhanced component of the Cl− current is cAMP-activated CFTR Cl− conductance.
9-AC did not affect the apparent affinity of the β-adrenoceptor for ISO. 9-AC could stimulate the Cl− conductance even under stimulation by cAMP. Sustained activity of FSK- or ISO-induced Cl− currents in the presence of intracellular 9-AC was totally insensitive to an inhibitor of protein kinase A (H-89). Therefore, it appears that 9-AC acts downstream from the protein kinase A–mediated phosphorylation step.
Both OA-sensitive PPs and OA-insensitive unidentified phosphatases have been reported to be involved in dephosphorylation of cAMP-activated Cl− channels in guinea pig ventricular myocytes.32 Even in the presence of OA, 9-AC exhibited additional effects on the cAMP-activated Cl− conductance. When given in combination with 9-AC and OA, deactivation of the Cl− current after washout of FSK was virtually abolished. In addition, 9-AC failed to significantly inhibit the purified preparation of OA-sensitive PP-1 and PP-2A in vitro. These results indicate that intracellular 9-AC enhances cardiac cAMP-activated Cl− currents by specifically inhibiting the activity of OA-insensitive cardiac phosphatase (such as PP-2B, PP-2C, alkaline phosphatase, and acid phosphatase), which, together with OA-sensitive phosphatases, is involved in dephosphorylation of the Cl− channel protein phosphorylated by cAMP-dependent protein kinase.
An involvement of PP-2B, which is dependent on Ca2+/calmodulin and insensitive to orthovanadate,39 is, however, unlikely, because intracellular Ca2+ was strongly chelated by 10 mmol/L EGTA under the whole-cell patch-clamp conditions and because the 9-AC effects were not observed in the presence of orthovanadate. Contribution of alkaline or acid phosphatase is also unlikely on the basis of observations of insensitivity of the 9-AC effect to BrT, theophylline, and tartaric acid as well as to pHi changes. The role of Mg2+-dependent PP-2C was difficult to assess by electrophysiological approaches, because there is no specific inhibitor for PP-2C and because the CFTR Cl− channel itself is Mg2+ dependent. However, the contribution of PP-2C is unlikely, because not only purified but also endogenous PP-2C activity in the ventricular extract was insensitive to 9-AC.
The present biochemical study showed that even in the presence of OA, BrT, and tartrate, the extract of guinea pig ventricle exhibits pNPP phosphatase activities totally sensitive to orthovanadate, which is known to inhibit enzymes that catalyze phosphotransfer reactions.40 Also, a fraction of the endogenous phosphatase in the ventricular extract was shown to be suppressed by 9-AC at micromolar concentrations. It would be feasible that the effective concentrations for enhancement by extracellular 9-AC application on the outward current recorded in vivo (ED50, ≈13 μmol/L) are higher than those for suppression by direct application on the phosphatase activity measured in vitro (ID50, ≈0.23 μmol/L). Thus, this endogenous cardiac phosphatase, which is orthovanadate sensitive and OA insensitive, is likely to be involved in the 9-AC effect, although this inference might be at variance with a recent report that orthovanadate may lock the channel open as a phosphate analogue.33
Cardiac cAMP-activated Cl− channels have recently be shown to be encoded by CFTR gene, the sequence of which is >90% identical to human epithelial CFTR cDNA.15 Regulation of the epithelial CFTR Cl− channel has been reported to involve PP-2A41 and alkaline phosphatase.34 The present study provided evidence that a phosphatase, which is sensitive to 9-AC or orthovanadate and distinct from PP-1, -2A, -2B, or -2C and alkaline or acid phosphatase, is implicated in dephosphorylation of the cardiac CFTR Cl− channel. To specify the 9-AC–sensitive cardiac phosphatase, however, further investigation is definitely required. Under autonomic nervous system control, activation of cAMP-activated Cl− channels is known to modulate cardiac action potentials and is thought to exhibit an arrhythmogenic action.20 42 Therefore, the cardiac 9-AC–sensitive phosphatase involved in deactivation of the CFTR Cl− channel may be a potential therapeutic target. There is a possibility that other functionally important cardiac proteins are the target of the 9-AC–sensitive phosphatase. In fact, our recent preliminary data (S.-S. Zhou and Y. Okada, unpublished data, 1996) showed that the 9-AC–sensitive phosphatase is also involved in the dephosphorylation of L-type Ca2+ channels in guinea pig ventricular myocytes. Thus, identification of the ventricular 9-AC–sensitive phosphatase is a next subject of physiological importance.
Selected Abbreviations and Acronyms
|CFTR||=||cystic fibrosis transmembrane conductance regulator|
|I-V||=||current (I)-voltage (V)|
|MLC||=||myosin light chain|
This study was supported by a grant-in-aid for scientific research (06404017), by a grant on the priority areas of “Channel-Transporter Correlation” (07276104) from the Ministry of Education, Science, and Culture of Japan, and by the Daiko Foundation Research Fellow Program. We thank Dr Andrew F. James for reading the earlier draft of the manuscript.
Presented in part as preliminary data in abstract form (Jpn J Physiol. 1996;46[suppl]:S95).
- Received November 12, 1996.
- Accepted May 14, 1997.
- © 1997 American Heart Association, Inc.
Hume JR, Harvey RD. Chloride conductance pathways in heart. Am J Physiol. 1991;261:C399-C412.
Hwang T-C, Gadsby DC. Chloride ion channels in mammalian heart cells. Curr Top Membr. 1994;42:317-346.
Harvey RD. Cardiac chloride currents. News Physiol Sci. 1996;11:175-181.
Harvey RD, Hume JR. Autonomic regulation of a chloride current in heart. Science. 1989;244:983-985.
Harvey RD, Hume JR. Isoproterenol activates a chloride current, not the transient outward current, in rabbit ventricular myocytes. Am J Physiol. 1989;257:C1177-C1181.
Levesque PC, Hart PJ, Hume JR, Kenyon JL, Horowitz B. Expression of cystic fibrosis transmembrane regulator Cl− channels in heart. Circ Res. 1992;71:1002-1007.
Horowitz B, Tsung SS, Hart P, Levesque PC, Hume JR. Alternative splicing of CFTR Cl− channels in heart. Am J Physiol. 1993;264:H2214-H2220.
Tominaga M, Horie M, Sasayama S, Okada Y. Glibenclamide, an ATP-sensitive K+ channel blocker, inhibits cardiac cAMP-activated Cl− conductance. Circ Res. 1995;77:417-423.
James AF, Tominaga T, Okada Y, Tominaga M. Distribution of cAMP-activated chloride current and CFTR mRNA in the guinea pig heart. Circ Res. 1996;79:201-207.
Hart P, Warth JD, Levesque PC, Collier ML, Geary Y, Horowitz B, Hume JR. Cystic fibrosis gene encodes a cAMP-dependent chloride channel in heart. Proc Natl Acad Sci U S A. 1996;93:6343-6348.
Palade PT, Barchi RL. On the inhibition of muscle membrane chloride conductance by aromatic carboxylic acids. J Gen Physiol. 1977;69:879-896.
Horvath PJ, Ferriola PC, Weiser MM, Duffey ME. Localization of chloride secretion in rabbit colon: inhibition by anthracene-9-carboxylic acid. Am J Physiol. 1986;250:G185-G190.
Mandel KG, Dharmsathaphorn K, McRoberts A. Characterization of a cyclic AMP-activated Cl− transport pathway in the apical membrane of a human colonic epithelial cell line. J Biol Chem. 1986;261:704-712.
Harvey RD, Clark CD, Hume JR. Chloride current in mammalian cardiac myocytes: novel mechanism for autonomic regulation of action potential duration and resting membrane potential. J Gen Physiol. 1990;95:1077-1102.
Vandenberg JI, Yoshida A, Kirk K, Powell T. Swelling-activated and isoprenaline-activated chloride currents in guinea pig cardiac myocytes have distinct electrophysiology and pharmacology. J Gen Physiol. 1994;104:997-1017.
Takai A, Troschka M, Mieskes G, Somlyo AV. Protein phosphatase composition in the smooth muscle of guinea-pig ileum studied with okadaic acid and inhibitor 2. Biochem J. 1989;262:617-623.
Cummins P, Lambert SJ. Myosin transitions in the bovine and human heart: a developmental and anatomical study of heavy and light chain subunits in the atrium and ventricle. Circ Res. 1986;58:846-858.
Ngai PK, Carruthers CA, Walsh MP. Isolation of the native form of chicken gizzard myosin light-chain kinase. Biochem J. 1984;218:863-870.
Bialojan B, Takai A. Inhibitory effect of a marine-sponge toxin, okadaic acid, on protein phosphatases: specificity and kinetics. Biochem J. 1988;256:283-290.
Chijiwa T, Mishima A, Hagiwara M, Sano M, Hayashi K, Inoue T, Naito K, Toshioka T, Hidaka H. Inhibition of forskolin-induced neurite outgrowth and protein phosphorylation by a newly synthesized selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide (H-89), of pC12D pheochromocytoma cells. J Biol Chem. 1990;265:5267-5272.
Hwang T-C, Horie M, Gadsby DC. Functionally distinct phospho-forms underlie incremental activation of protein kinase-regulated Cl− conductance in mammalian heart. J Gen Physiol. 1993;101:629-650.
Becq F, Jensen TJ, Chang X-B, Savoia A, Rommens JM, Tsui L-C, Buchwald M, Riordan JR, Hanrahan JW. Phosphatase inhibitors activate normal and defective CFTR chloride channels. Proc Natl Acad Sci U S A. 1994;91:9160-9164.
Farley JR, Ivey JL, Baylink DJ. Human skeletal alkaline phosphatase: kinetic studies including pH dependence and inhibition by theophylline. J Biol Chem. 1980;255:4680-4686.
Hollander VP. Acid phosphatases. In: Boyer PD, ed. The Enzymes. New York, NY: Academic Press; 1971:449-498.
Takai A, Mieskes G. Inhibitory effect of okadaic acid on the p-nitrophenyl phosphate phosphatase activity of protein phosphatases. Biochem J. 1991;275:233-239.
Berger HA, Travis SM, Welsh MJ. Regulation of the cystic fibrosis transmembrane conductance regulator Cl− channel by specific protein kinases and protein phosphatases. J Biol Chem. 1993;268:2037-2047.