© 1997 American Heart Association, Inc.
Articles |
Phosphatase-Mediated Enhancement of Cardiac cAMP-Activated Cl- Conductance by a Cl- Channel Blocker, Anthracene-9-Carboxylate
From the Department of Cellular and Molecular Physiology (S.-S.Z., M.T., Y.O.), National Institute for Physiological Sciences, Okazaki, Japan, and the Department of Physiology (A.T.), School of Medicine, Nagoya (Japan) University.
Correspondence to Y. Okada, MD, PhD, Department of Cellular and Molecular Physiology, National Institute for Physiological Sciences, Okazaki 444, Japan. E-mail okada{at}nips.ac.jp
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Abstract |
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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.
Key Words: anion channel • cystic fibrosis transmembrane conductance regulator • Cl- channel blocker • dephosphorylation • cardiac myocyte
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A variety of Cl- currents have been identified in mammalian cardiac ventricular myocytes.1 2 3 Among these, stimulation of the ß-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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cardiac Preparations
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.
Electrophysiological Experiments
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 M
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 ß-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, 
400, of
p-nitrophenol at 400 nm and a given pH is well described by
the following equation:
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400max) 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.
Chemicals
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.
Statistical Analysis
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.
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Results |
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Prominent increases in Cl- conductance were induced by ß-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
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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.
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)
and inward (
) currents recorded at +100 and -100 mV,
respectively, after stimulation with 1 µmol/L
ISO. For enhancing and suppressing effects, the half-maximal
concentrations were 13.4 µmol/L and 0.942
mmol/L, and the Hill coefficients were 1.01 and 2.14,
respectively. B, Concentration-response relations for ISO effects on
outward currents recorded at +100 mV in the absence (
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
3.49x10-8 and 2.95x10-8 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 ß-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.
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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.
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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.
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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.
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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.
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, 
represent the control data, data with 10 mmol/L
tartrate, 1 mmol/L BrT, and 1 mmol/L
orthovanadate, respectively. Each point represents the average
of two to four values. B, Concentration-response relation for
suppressing effects of 9-AC in the presence of tartrate, BrT, and OA at
pH 7.4. The dotted curve represents the best fit to the Hill
equation with a Hill coefficient of 1.00. Each point represents
the average of 3 to 14 values. Vertical bars indicate SEM.
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).
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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).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 ß-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.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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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.
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Footnotes |
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Presented in part as preliminary data in abstract form (Jpn J Physiol. 1996;46[suppl]:S95).
Received November 12, 1996; accepted May 14, 1997.
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References |
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