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INTRODUCTION |
Protein phosphorylation is a putative effector mechanism in the
infarct size-limiting effect of ischemic preconditioning (1), a
phenomenon whereby a brief period of ischemia and reperfusion can
protect the heart against subsequent prolonged ischemia and reperfusion
injury (2). Indeed, it was shown that phosphorylation levels decrease
during ischemia in nonpreconditioned hearts, whereas they increase
during ischemia in preconditioned hearts (3). A number of cellular
components, including cytoskeletal and stress proteins, have been
proposed as potential phosphorylation targets in the ischemic
preconditioned heart (4-6).
ATP-sensitive K+ channels (KATP
channels)1 in sarcolemma and
mitochondria are also modulated by phosphorylation (7, 8). The majority
of the studies on the contribution of phosphorylation to
KATP channel activity to the cardioprotective effects of
ischemic preconditioning have centered on the role of protein kinase C. Protein kinase C may act as a link between one or more
receptor-mediated pathways and increased KATP channel
activity and thus lead to ischemic preconditioning (9).
The release of endogenous substances, such as adenosine, bradykinin,
nitric oxide (NO), and prostacyclin (10-12), has been proposed as a
potential mechanism of ischemic preconditioning. These substances
increase cGMP via direct stimulation of myocardial cells or via the
endothelium. It was reported that the cGMP levels in preconditioned
hearts are higher than in nonpreconditioned hearts (13-15). Since cGMP
can induce protein phosphorylation via protein kinase G (PKG)
activation, the involvement of PKG-dependent phosphorylation in ischemic preconditioning is expected. To date, however, the role of PKG in ischemic preconditioning and the possible PKG target proteins in this process are not well understood.
There is evidence that KATP channels have potential
phosphorylation sites, including serine/threonine residues (16-19),
and that the channels are activated by phosphorylation of these
residues (20). Since PKG is a serine/threonine protein kinase, it is likely that PKG leads to phosphorylation of KATP channels
and ischemic preconditioning. In fact, the regulation of
KATP channels by cGMP was recently shown in
follicle-enclosed oocytes (21, 22), pancreatic
-cells (23, 24),
vascular smooth muscle cells (25, 26), and cardiac myocytes (24, 27),
and it was suggested that this effect was mediated by PKG activation. However, there is no direct evidence that PKG activates
KATP channels.
In this study, we investigated the role of PKG-dependent
phosphorylation in modulating KATP channels using isolated
rabbit ventricular myocytes. The experiments examined the signal
transduction pathways involved in PKG-dependent
phosphorylation. Our findings demonstrate that the
NO/cGMP/PKG-signaling pathway facilitates the activity of
KATP channels via phosphorylation. Our findings may be
important for understanding the mechanism by which PKG acts as a link
in one or more known receptor-mediated pathways to increase
KATP channel activity during ischemic preconditioning.
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EXPERIMENTAL PROCEDURES |
Materials--
ATP and glibenclamide were added to either the
extracellular or intracellular solution, following the experimental
protocols described below. Pinacidil (RBI, Natick, MA) was freshly
prepared before the experiments and diluted in the test solution to
obtain the indicated final concentrations.
S-Nitroso-N-acetylpenicillamine (SNAP) was
purchased from Calbiochem. Okadaic acid (OA) was purchased from RBI,
stored at a stock concentration of 100 µM in ethanol at
4 °C, and used at a final concentration of 5 nM. PP2A
was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). PKG
was obtained from Promega (Madison, WI); protein kinase A (PKA) from
BIOMOL (Plymouth Meeting, PA); and
(Sp)-8-Br-PET-cGMPS,
(Rp)-8-Br-PET-cGMPS, 8-pCPT-cGMP,
(Sp)-5,6-DCl-cBIMPS, and
(Rp)-8-pCPT-cAMPS from Biolog Life Science
Institute (Bremen, Germany). All other chemicals used in this study,
unless specified otherwise, were from Sigma. After the addition of
drugs to the test solution, the pH was readjusted to 7.4 with KOH.
Cell Isolation--
Single ventricular myocytes were isolated
from rabbit hearts by an enzymatic dissociation procedure, as discussed
previously (28). Briefly, in accordance with national animal care
guidelines, rabbits weighing 150-280 g were anesthetized with sodium
pentobarbital (50 mg/ml, 1 ml/kg of body weight) and heparin (300 IU/ml). After adequate anesthesia was achieved, sternostomy was
performed, and the heart was exposed. Artificial perfusion of the heart
was established by cannulation of the aorta. The heart was then removed
and placed in a Langendorff perfusion apparatus, and an enzymatic
method was used to isolate single ventricular cells for
electrophysiological experiments.
Electrophysiological Recording and Data Analysis--
Single
channel currents were measured in the cell-attached and inside-out
patch configurations of the patch clamp technique (29). Channel
activity was measured using a patch clamp amplifier (EPC-7, LIST,
Darmstadt, Germany; Axopatch-1D, Axon Instruments, Foster City, CA).
The DAD-12 superfusion system (Adams & List Associates, New York) was
used to rapidly exchange (within 100 ms) the bath solution and drugs in
most experiments. Experiments were done at a room temperature of
25 ± 2 °C. Membrane currents were filtered at 0.1-10 kHz and
digitized at 0.4-20 kHz. All current recordings were stored in
digitized format on digital audiotapes using a DTR-1200 recorder
(Biologic, Grenoble, France). To analyze single channel activity, the
data were transferred to a computer (IBM-PC, Pentium-III 450, Pusan,
Korea) with pCLAMP software (version 6.3; Axon Instruments Inc.,
Burlingame, CA) through an analog-to-digital converter interface
(Digidata-1200, Axon Instruments Inc., Burlingame, CA). The threshold
for judging the open state was set at half of the single channel
amplitude (30). The open time histogram was formed from continuous
recordings of more than 60 s. The open probability
(Po) was calculated using the following formula
in Equation 1,
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(Eq. 1)
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where tj is the time spent at current levels
corresponding to j = 0, 1, 2, ...N
channels in the open state; Td is the duration of
the recording; and N is the number of channels active in the
patch. The number of channels in a patch was estimated by dividing the
maximum current observed, during an extended period at zero ATP, by the
mean unitary current amplitude. Po was
calculated over 30-s records.
Solutions--
The normal Tyrode solution contained 143 mM NaCl, 5.4 mM KCl, 1.8 mM
CaCl2, 0.5 mM MgCl2, 5.5 mM glucose, and 5 mM HEPES, adjusted to pH 7.4 with NaOH. The solutions facing the outside of the cell membrane in the
excised patch recordings contained 140 mM KCl, 2 mM CaCl2, 1 mM MgCl2,
10 mM glucose, and 10 mM HEPES, adjusted to pH
7.4 with KOH. The solutions facing the inside of the cell membrane in
the excised patch recordings contained 127 mM KCl, 13 mM KOH, 1 mM MgCl2, 5 mM EGTA, 10 mM glucose, and 10 mM
HEPES, adjusted to pH 7.4 with KOH. The modified Kraft's Brühe solution had the following composition: 70 mM KOH, 50 mM L-glutamic acid, 40 mM KCl, 20 mM KH2PO4, 20 mM
taurine, 3 mM MgCl2, 10 mM HEPES,
0.5 mM EGTA, and 10 mM glucose, adjusted to pH
7.4 with KOH.
In order to exclude cross-activation between PKA and PKG during
measurements of the effects of PKG activation on KATP
channel activity, we performed the experiments under conditions in
which PKA was inhibited by the potent and selective PKA inhibitor
(Rp)-8-pCPT-cAMPS, unless otherwise stated.
(Rp)-8-pCPT-cAMPS (10 µM) has no
effect on KATP channels.
Statistical Analysis--
The data were statistically analyzed
using either Student's unpaired t test when two treatment
groups were compared or one-way analysis of variance followed by a
post hoc Student-Newman-Keuls test when all pairwise
comparisons among the different treatment groups were made. Tests were
considered significant when p < 0.05. All data are
presented as means ± S.E.
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RESULTS |
Effect of NO Donors on the KATP Channel Activity of
Rabbit Ventricular Myocytes--
KATP channels were
characterized by their conductance over a voltage range of
80 to
20
mV (77.8 ± 3.5 pS), over a unitary current range (2.8 ± 0.2 pA;
40 mV), and through their responses to potassium channel openers,
ATP, and glibenclamide.
In order to test the role of the PKG signaling pathway in the
regulation of KATP channels in ventricular myocytes, we
first investigated the effects of NO on KATP channels in
cell-attached patches. NO stimulates soluble guanylate cyclase and
produces its effects by increasing intracellular cGMP concentration,
possibly leading to the activation of PKG (31). We used SNP and SNAP, potent stimulators of cGMP formation, which are known NO donors (32,
33). After gigaseal formation, the pipette potential was set to
40
mV, and the bath solution was switched from normal Tyrode solution to
high K+ solution. Under these conditions, little single
channel activity was recorded. Subsequent application to the bath of
pinacidil (50 µM) opened KATP channels with a
single channel current amplitude of 2.9 ± 0.4 pA
(n = 7 patches). As the concentration of SNAP was
increased, KATP channel activity increased in a
concentration-dependent fashion (Fig.
1A). The addition of
glibenclamide (30 µM) immediately suppressed this channel
activity, confirming that the observed openings were due to
KATP channels. In Fig. 1B, the channel activity for each NO donor concentration was normalized using the equation y = (Po - Po,min)/(Po,max
Po,min), where y is the
relative open probability (Po),
Po,max is Po at a
given concentration of NO donor (1 mM), and Po,min is the Po
without an NO donor. The continuous line in the
graph is the curve fitted to the Hill equation using the least-square
method: Po = (Po,max
Po,min)[NO donor]n/(Kdn + [NO
donor]n) + Po,min, where [NO
donor] is the concentration of the NO donor, Kd is
the concentration of the NO donor at the half-maximal activation of the
channel, and n is the Hill coefficient. NO donors were found
to activate KATP channels with the following
Kd values: 7.9 ± 1.2 µM for SNAP
(n = 8 patches) and 66.9 ± 1.3 µM
for SNP (n = 11 patches). The preceding equations were
used throughout the remainder of the experiments to determine for each
compound used the concentration-response relationship for channel
activation.

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Fig. 1.
Effects of NO donors on the KATP
channel activity in rabbit ventricular myocytes. A,
continuous current traces from representative experiments showing the
effect of the sequential addition of SNAP to the bath solution in the
presence of 50 µM pinacidil and 10 µM
(Rp)-8-pCPT-cAMPS. The pipette potential was
held at 40 mV in cell-attached patches. B, the
relationships between the concentrations of the NO donors and relative
channel activity from a series of experiments similar to that shown in
A. The Po for each NO donor concentration was
normalized using the equation under "Results." The
solid lines in B were drawn from
calculations that are described under "Results." Data were
sampled at 20 kHz and filtered at 1 kHz. The dashed
line indicates the zero current level.
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The effects of NO donors on KATP channel activity were
examined using inside-out and outside-out patch configurations to
exclude the possibility that NO donors act on KATP channels
directly. Fig. 2A shows a
representative result obtained for an inside-out patch. The application
of NO donors to the bath failed to enhance the channel activity at
40
mV. The average Po values before and during the
addition of NO donors were as follows: 0.16 ± 0.07 and 0.17 ± 0.09 for 1 mM SNP (p > 0.05, n = 5 patches) and 0.18 ± 0.06 for 300 µM SNAP (p > 0.05, n = 5 patches). The application of NO donors to the extracellular surface of
the outside-out patch also failed to enhance the channel activity (Fig.
2B). The average Po values before and
during the addition of NO donors were as follows: 0.11 ± 0.07 and
0.13 ± 0.06 (p > 0.05, n = 3 patches) for 1 mM SNP (p > 0.05, n = 3 patches) and 0.12 ± 0.05 for 300 µM SNAP (p > 0.05, n = 3 patches).

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Fig. 2.
Effects of NO donors on the KATP
channel activity in inside-out (A) and outside-out
(B) patches. 1 mM SNP or 300 µM SNAP added to the perfusion medium did not activate
KATP channels. The membrane potential was held at 40 mV.
Data were sampled at 20 kHz and filtered at 1 kHz. The
dashed line indicates the zero current
level.
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In the experiments described in Fig. 3,
increasing the concentration of
(Rp)-8-Br-PET-cGMPS, the most potent known
inhibitor of PKG (34), inhibited the SNAP-induced KATP
channel activity (Fig. 3, A and C). In Fig.
3D, the channel activity for the
(Rp)-8-Br-PET-cGMPS concentration used was
normalized using the equation: y = (Po
Po,min)/(Po,max
Po,min), where y is the
relative open probability (Po),
Po,max is the Po
without (Rp)-8-Br-PET-cGMPS, and
Po,min is the Po at a
given concentration of 10 µM
(Rp)-8-Br-PET-cGMPS. The continuous
line in the graph is the curve fitted to the Hill equation
using the least-square method: Po = (Po,max
Po,min)
Kdn/(Kdn + [(Rp)-8-Br-PET-cGMPS]n) + Po,min, where
[(Rp)-8-Br-PET-cGMPS] is each concentration of
(Rp)-8-Br-PET-cGMPS, Kd is
the concentration of (Rp)-8-Br-PET-cGMPS at the
half-maximal inhibition of the channel, and n is the Hill coefficient. The Kd value for this inhibitory effect was 0.12 ± 0.01 µM (n = 5 patches).
The preceding equations were used throughout the remainder of the
experiments to determine the concentration-response relationship for
channel inhibition by the compounds used.
(Rp)-8-pCPT-cAMPS had no effect on the channel
(Fig. 3, B and C). In Fig.
4, we tested the effect of (Rp)-pCPT-cGMP, another potent inhibitor of PKG,
using cell-attached patches. The potentiating effect of SNP was
suppressed by (Rp)-pCPT-cGMP (100 µM) in a reversible manner in all of the cells tested
(n = 5 patches); the average Po
before and during the addition of (Rp)-pCPT-cGMP
was 0.24 ± 0.05 and 0.09 ± 0.05 (p < 0.05, n = 5 patches). These results suggest that NO donors
facilitate the pinacidil-induced KATP channel activity via
a cGMP/PKG-dependent mechanism.

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Fig. 3.
Effects of potent and selective inhibitors of
PKG and PKA on the NO donor-induced KATP channel
activity. Continuous traces showing single KATP
channel currents in cell-attached patches perfused, in the presence of
300 µM SNAP, with various concentrations of
(Rp)-8-Br-PET-cGMPS (A) and
(Rp)-8-pCPT-cAMPS (B) applied in the
sequence shown. The pipette potential was held at 40 mV in
cell-attached patches. C, the concentration-response
relationships for the effects of
(Rp)-8-Br-PET-cGMPS ( ) and
(Rp)-8-pCPT-cAMPS ( ) on KATP
channel activity from a series of experiments similar to that shown in
A and B. The Po for each
(Rp)-8-Br-PET-cGMPS and
(Rp)-8-pCPT-cAMPS concentration was normalized
by referring to the value in the absence of
(Rp)-8-Br-PET-cGMPS and
(Rp)-8-pCPT-cAMPS. D, the channel
activity for (Rp)-8-Br-PET-cGMPS was normalized
using the equation under "Results." The solid
line was drawn from calculations that are described under
"Results."
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Fig. 4.
The effect of
(Rp)-8-pCPT-cGMP on the SNP-induced
KATP channel activity. There are two types of channel,
one with a large conductance and fast open-close kinetics
(KATP channel) and the other with a smaller conductance and
a long opening time (the inward rectifier K+ channel).
Expansions of the records at the times marked by bars are
shown in the lower panels. a, channel
activity in the presence of 50 µM pinacidil (Po = 0.058). b, with 1 mM SNP in the bath solution,
Po was increased to 0.189. c, adding 100 µM (Rp)-pCPT-cGMP suppressed the
channel activity (Po = 0.063). d, wash-out of
(Rp)-pCPT-cGMP restored the channel activity
(Po = 0.192). Data were sampled at 20 kHz and filtered at 1 kHz. The dashed line indicates the zero current
level.
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Effects of PKG Activators on KATP Channels in Rabbit
Ventricular Myocytes--
The involvement of PKG in the regulation of
KATP channels was further confirmed in an experiment using
a potent activator of PKG, (Sp)-8-Br-PET-cGMPS
(Fig. 5, A and C).
It has been reported that (Sp)-8-Br-PET-cGMPS is
the only compound that displays significant selectivity for PKG over
PKA (34). In seven patches, (Sp)-8-Br-PET-cGMPS stimulated half-maximal KATP channel activity at a
concentration of 4.12 ± 0.96 µM (Fig.
5D). In five separate patches, the specific activator for
PKA, (Sp)-5,6-DCl-cBIMPS (35), had no effect on the channel (Fig. 5, B and C). In Fig.
5E, the pinacidil-induced KATP channel activity
was reversibly facilitated by another PKG activator, 8-pCPT-cGMP (36).
The average Po increased 1.78 ± 0.12 times following the
addition of 100 µM 8-pCPT-cGMP (Po = 0.17 ± 0.09) compared with the Po (0.09 ± 0.04) recorded before
the addition of 8-pCPT-cGMP (p < 0.05, n = 6 patches). The pinacidil-induced single channel
activity was inhibited by the subsequent application of 30 µM glibenclamide (Po = 0.002 ± 0.001).
Summarized data are shown in Fig. 3F.

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Fig. 5.
Effects of potent and selective
activators of PKG and PKA on pinacidil-induced single channel
activity. Continuous traces showing single
KATP channel currents in cell-attached patches perfused, in
the presence of 50 µM pinacidil and 10 µM
(Rp)-8-pCPT-cAMPS, with various concentrations
of (Sp)-8-Br-PET-cGMPS (A) and
(Sp)-5,6-DCl-cBIMPS (B) applied in
the sequence shown. The pipette potential was held at 40 mV in
cell-attached patches. C, the concentration-response
relationships for the effects of
(Sp)-8-Br-PET-cGMPS ( ) and
(Sp)-5,6-DCl-cBIMPS ( ) on KATP
channel activity from a series of experiments similar to that shown in
A and B. The Po for each
(Sp)-8-Br-PET-cGMPS and
(Sp)-5,6-DCl-cBIMPS concentration was normalized
by referring to its value in the presence of 100 µM
(Rp)-8-Br-PET-cGMPS or
(Sp)-5,6-DCl-cBIMPS, respectively. D,
the channel activity for (Sp)-8-Br-PET-cGMPS was
normalized using the equation under "Results." The
solid line was drawn from calculations that are
described under "Results." E, representative
record of the effect of 100 µM 8-pCPT-cGMP followed by 30 µM glibenclamide on the pinacidil-induced single channel
activity. Glibenclamide applied extracellularly caused a marked
inhibition of pinacidil-induced single channel activity. Data were
sampled at 20 kHz and filtered at 1 kHz. The dashed
line indicates the zero current level. F,
histogram showing the pooled data (mean ± S.E.) for Po
for the following conditions: pinacidil alone, pinacidil plus
8-pCPT-cGMP, and subsequent application of glibenclamide. *,
p < 0.05 relative to the pinacidil-alone group.
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Effect of PKG Activation on KATP Channels in Excised
Inside-out Patches--
To evaluate more directly the involvement of
the cGMP/PKG-dependent mechanism in the activation of the
KATP channel, we tested various combinations of cGMP, PKG,
and ATP in excised inside-out patches. cGMP alone (100 µM; p > 0.05, n = 4 patches), cGMP and ATP together (100 µM each;
p > 0.05, n = 4 patches), PKG alone (5 units/µl; p > 0.05, n = 4 patches),
and PKG and cGMP together (5 units/µl and 100 µM each;
p > 0.05, n = 4 patches) had no effect on the channel activity (data not shown). Facilitation of
KATP channel activity in excised inside-out patches was
only observed when PKG was applied in the presence of ATP and cGMP
together. A representative case is shown in Fig.
6A. After excision of the patch, ATP (100 µM) and cGMP (100 µM)
inhibited spontaneous KATP channel opening. In the
continuous presence of ATP and cGMP, increasing concentrations of PKG
enhanced the channel activity. The Kd value for this
stimulatory effect was 0.08 ± 0.02 units/µl (Fig. 6B, n = 6 patches).

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Fig. 6.
Effects of exogenous PKG on KATP
channel activity in inside-out patches. ATP and cGMP are cofactors
required for PKG activity. A, continuous current traces from
representative experiments showing the effect of the sequential
addition of PKG to the cytoplasmic surface of an inside-out patch in
the presence of 100 µM ATP, 100 µM cGMP,
and 10 µM (Rp)-8-pCPT-cAMPS
(holding potential = 40 mV). B, the relationship
between the PKG concentration and relative channel activity from a
series of experiments similar to that shown in A. The
channel activity for PKG was normalized using the equation under
"Results." The solid line was drawn from calculations that are
described under "Results." C, the effect of the
PKG inhibitor (Rp)-8-Br-PET-cGMP on PKG
activation-induced KATP channel activity, demonstrating the
specificity of PKG in stimulating the KATP channel
activity. PKG increased the channel activity in the presence of a
combination of ATP, cGMP, and (Rp)-8-pCPT-cAMPS.
In the same patch, various concentrations of
(Rp)-8-Br-PET-cGMP were added to the bath
solution. Note that (Rp)-8-Br-PET-cGMP reversed
the stimulatory effects of PKG on KATP channel activity.
D, the relationship between
(Rp)-8-Br-PET-cGMP concentration and relative
channel activity from a series of experiments similar to that shown in
C. The channel activity for
(Rp)-8-Br-PET-cGMP was normalized using the
equation under "Results." The solid
line was drawn from calculations that are described
under "Results." E, the effect of replacing ATP
with ATP S on the channel activity in inside-out patches. Note that
PKG together with cGMP had no effect on ATP S-inhibited channels.
Current recording was from an inside-out patch held at 40 mV. Data
were sampled at 20 kHz and filtered at 1 kHz. The dashed
line indicates the zero current level.
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(Rp)-8-Br-PET-cGMPS inhibited the PKG-induced
KATP channel activation in a
concentration-dependent fashion (Fig. 6C). The Kd value for this inhibitory effect was 0.09 ± 0.02 µM (Fig. 6D, n = 5 patches). The specificity of PKG in stimulating channel activity was
confirmed using heat-inactivated PKG. In the continuous presence of 100 µM/l ATP at the intracellular surface, the application of
heat-treated PKG (5 units/µl) to the intracellular surface with cGMP
(100 µM) failed to enhance the channel activity (data not
shown). Additionally, the removal of Mg2+ from the bath
solution (by the addition of 1 mM EDTA) prevented PKG
activation (data not shown).
In Fig. 6E, we replaced ATP with ATP
S, a nonhydrolyzable
analog of ATP, under similar experimental conditions. KATP
channels were inhibited by ATP
S (100 µM) and cGMP (100 µM), and Po was reduced from 0.317 to 0.063 (Fig.
6E). However, the subsequent addition of PKG (5 units/µl)
failed to enhance the channel activity (Po = 0.083). Similar
effects were observed in four other patches.
PKG-induced Activation of KATP Channels Is Reversed by
Protein Phosphatase--
Taken together, the preceding results
suggested that PKG activated KATP channels by an apparent
phosphorylation-dependent mechanism. If PKG phosphorylated
KATP channels directly, one would have expected protein
phosphatase to inhibit PKG action. We tested the effect of protein
phosphatase (PP2A) on the PKG-induced KATP channel
activation. Fig. 7A
illustrates the effects of applying PP2A (1 unit/ml) in the presence of
PKG activation to the excised inside-out patch configuration. After
excision of the patch, ATP (100 µM) and cGMP (100 µM) inhibited spontaneous KATP channel opening, and Po was reduced from 0.163 to 0.012. In the continuous presence of ATP and cGMP, PKG (5 units/µl) enhanced the
channel activity (Po = 0.122). The application of exogenous
PP2A inhibited the PKG-mediated KATP channel activity (Po = 0.032). A similar decrease in channel activity with PP2A
was observed in five other patches (Fig. 7B).

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Fig. 7.
Effects of exogenous PP2A on the
KATP channel activity stimulated by PKG. A,
current recording from an inside-out patch held at 40 mV. In the
presence of 100 µM ATP, 100 µM cGMP, and 10 µM (Rp)-8-pCPT-cAMPS, PKG (5 units/µl) caused an increase in the channel activity. In the same
patch, 1 units/ml PP2A inhibited PKG activation-induced channel
activity. Expansions of the records at the times marked by
bars are shown in the lower panels.
B, change of channel activity in response to PP2A in
inside-out patches. The histogram shows the pooled data (mean ± S.E.) for Po for the following conditions: control
(a), 10 µM
(Rp)-8-pCPT-cAMPS alone (b), 100 µM ATP, 100 µM cGMP, and 10 µM (Rp)-8-pCPT-cAMPS
(c), PKG activation (d), and additional
application of PP2A (e). *, statistically significant
(p < 0.05). C, effect of exogenous PP2A on
the KATP channel activity stimulated by PKG activation in
the presence of OA, a potent inhibitor of PP2A. Current recording from
an inside-out patch configuration held at 40 mV. PKG activation
increased the channel activity. Upon removal of 100 µM
ATP, 100 µM cGMP, and 5 units/µl PKG and exposure to 5 nM OA, activation of the KATP channel
persisted. PP2A (1 unit/ml) was ineffective in the presence of 5 nM OA. Data were sampled at 20 kHz and filtered at 1 kHz.
The dashed line indicates the zero current level.
D, the changes in channel activity in response to PP2A in
the presence of OA. The histogram shows the pooled data (mean ± S.E.) for Po for the following conditions: PKG activation;
after application of OA upon removal of ATP, cGMP, and PKG; and
additional application of PP2A in the presence of OA. NS,
differences are not statistically significant. Similar results were
observed in the presence of 10 µM
(Rp)-8-pCPT-cAMPS (E).
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The effect of PP2A was in turn inhibited by OA, a potent inhibitor of
type 1 and 2A protein phosphatases (Fig. 7C). OA was used at
a low concentration (5 nM) to specifically block the
activity of PP2A in excised inside-out patches. The application of PKG in the presence of ATP and cGMP caused an increase in the channel activity (Po = 0.27). When ATP, cGMP, and PKG were then washed
out and OA was applied to the patches, KATP channel
activity was maintained (Po = 0.28). In the presence of OA,
PP2A did not alter the channel activity (Po = 0.27). Identical results were obtained in six out of seven patches examined (Fig. 7D). As shown in Fig. 7E, similar effects were
observed even in the presence of a PKA inhibitor.
Effect of PKG on the Properties of KATP Channels in
Rabbit Ventricular Myocytes--
Fig.
8A shows the all-points
histograms of single channel current amplitude made from the same patch
under control conditions and in the presence of PKG activation. The
I-V relationships obtained from patches under control
conditions and in the presence of PKG activation are shown in Fig.
8B. PKG activation had no effect on the I-V
relationship or slope conductance value (from 78.9 ± 5.3 to
78.5 ± 3.9 pS, n = 6 patches).

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Fig. 8.
Effects of PKG activation on the properties
of KATP channels from an inside-out patch.
A, all-points histograms of current amplitude made from the
same patch under control conditions (left panel)
and in the presence of PKG activation (right
panel). *, the current level during KATP channel
opening. The patch was held at 40 mV. Note that the amplitude of the
channel was not affected by PKG activation. B, the
current-voltage relationship made from the same patch under control
conditions ( ) and in the presence of PKG activation ( ). Note that
PKG did not change the conductance of the channel current.
C, the effects of PKG on the distribution of the open and
closed times of KATP channels. The histograms of the open
(a) and closed (b) time within bursts were
analyzed from the current records at 5 kHz. The histograms of the burst
(c) and interburst (d) durations were analyzed
from the current records at 0.2 kHz. The membrane potential was 50
mV. Time constants of the interburst duration histogram were fitted to
two exponential equations (fast and slow). The others were fitted to
single exponential equations. PKG increased the burst duration and
shortened interburst duration.
|
|
To examine the effect of PKG activation on the gating kinetics of the
channels, the open time and closed time histograms were calculated at a
membrane potential of
50 mV relative to the reversal potential. The
open-time histogram (Fig. 8C, a), which was
analyzed from the current record filtered at a cut-off frequency of 5 kHz, revealed a single exponential distribution with a time constant of
1.38 ms under control conditions. In the presence of PKG activation, the open time constant (
o) did not differ from that seen
in the absence of PKG activation (
o = 1.40 ms). Burst
lifetime was defined as the opening period observed in the records
filtered at a cut-off frequency of 0.2 kHz. The histogram of burst
duration consisted of a single exponential distribution (Fig.
8C, c). Its time constant, designed as
b, was markedly prolonged by PKG activation (from 20 to
35 ms). The histogram of time closed within bursts was best fitted to a
single exponential function (Fig. 8C, b). This
analysis was performed discarding closing times longer than 4 ms, with filtering at a cut-off frequency of 5 kHz. The constant of the time
closed within bursts was designed as
c. The value of
c was not changed markedly by PKG activation (from 0.30 to 0.31 ms). The closed time between bursts was analyzed by using
records filtered at a cut-off frequency of 0.2 kHz (Fig. 8C,
d). The histogram was fitted using a biexponential function,
with the time constants of fast (
c1) and slow
(
c2) components. The value of
c1 was influenced by PKG activation (from 74 to 40 ms). The value of
c2 was 293 ms under control conditions. This value was
markedly decreased to 105 ms by PKG activation. These findings suggest that PKG activation increases the channel activity by increasing burst
duration and decreasing the interburst interval.
 |
DISCUSSION |
In cardiac muscle, KATP channels, which open when the
intracellular ATP concentration ([ATP]i) falls below a
critical level, are known to be involved in ischemic preconditioning, a mechanism that protects the heart against ischemic injury (37-39). In
addition to the more obvious effect of diminishing [ATP]i on
the activation of this channel, various extracellular and intracellular modulators, such as adenosine (40), protein kinase C (41), ADP (42),
and lactate (43), have been proposed to stimulate KATP
channel activity and thus contribute to ischemic preconditioning. The
cGMP/PKG-pathway has also been suggested to play a cardioprotective role against ischemic-reperfusion injury (10, 11, 15), and it was
proposed that the modulation of Ca2+ availability (44, 45),
reducing myofilament sensitivity to Ca2+ (46), and
vasodilation with antiplatelet effects (47) were involved in
cardioprotectivity. Although the cGMP/PKG-pathway and KATP
channels have both been implicated in mediating the protective effect
of ischemic preconditioning, the signaling mechanism by which they are
linked remains poorly understood. The objective of this study was to
determine the mechanism by which PKG activates KATP
channels and thus could link these two effectors in a signaling pathway
that produces cardioprotection during ischemic preconditioning.
Our findings demonstrate that NO donors and PKG activators facilitate
the pinacidil-induced KATP channel activity, and a
selective PKG inhibitor prevents their effects. PKG, in the presence of both cGMP and ATP, increases the channel activity. This action of PKG
is prevented by heat inactivation, replacing ATP with ATP
S (a
nonhydrolyzable analog of ATP), removing Mg2+ from the
internal solution, applying a PKG inhibitor, or adding exogenous PP2A.
Taken together, these results imply that a series of signal
transduction pathways is involved in the regulation of KATP
channels; NO donors release NO, which activates guanylate cyclase in
cardiac myocytes, causing cGMP accumulation and the activation of PKG,
which subsequently phosphorylates and activates the KATP channels.
However, some previous studies reported contradictory results. Shinbo
and Iijima (27) demonstrated that NO potentiated the KATP
channel activity produced by K+ channel openers in guinea
pig ventricular myocytes. Under the same conditions, however, these
investigators found that 8-Br-cGMP inhibited KATP channels.
Therefore, they suggested that the mechanism of the NO-induced
potentiation of KATP channels was independent of the
cGMP/PKG-signaling pathway in the heart. In addition, Tsuura et
al. (24) reported that in rat ventricular myocytes, NO donors had
no effect on KATP channels. Differences in experimental
conditions and animal species between their study and ours may be
responsible for the discrepancies in results.
The major variations in experimental conditions are the mode of patch
recordings and the compounds used. Shinbo and Iijima (27) and Tsuura
et al. (24) carried out experiments using the whole-cell and
cell-attached modes of the patch-clamp method, whereas we only used the
cell-attached mode. It is difficult to know how the mode of patch
clamping affects the action of NO/cGMP on KATP channels.
Since our study showed that the effect of NO/cGMP on KATP
channels was not direct, but required a series of signal transductions
involving PKG activation and phosphorylation, it is plausible that
dialysis of the cellular components during whole-cell patch clamping
attenuated the effect of NO/cGMP.
Shinbo and Iijima (27) used 8-Br-cGMP (100-500 µM),
claiming that it is a specific activator of PKG. However, this compound has several major limitations that might influence the way in which
their experimental data should be interpreted. First, 8-Br-cGMP at 100-500 µM can also strongly activate PKA because
half-maximal stimulation of PKA occurs at 2.8-12 µM
8-Br-cGMP (36). Second, 8-Br-cGMP is hydrolyzed by certain
phosphodiesterases, generating the corresponding 5'-monophosphate
analogs and, subsequently, the nucleoside analogs (36). Third,
8-Br-cGMP is as polar as cAMP (48), which implies insufficient membrane
permeability. Therefore, it is unclear whether the 8-Br-cGMP-mediated
effects on KATP channels observed by Shinbo and Iijima (27)
indeed occurred via PKG. In our study, to avoid misinterpretation of
the experimental data, we utilized cyclic nucleotide analogs as new
biochemical tools. These analogs are highly membrane-permeable, stable
against phosphodiesterase hydrolysis, and display sufficient PKG/PKA
specificity. (Sp)-8-Br-PEP-cGMPS was used as a
PKG activator, (Rp)-8-Br-PEP-cGMPS as a PKG
inhibitor, (Sp)-5,6-DCl-cBIMPS as a PKA
activator, and (Rp)-8-pCPT-cAMPS as a PKA
inhibitor. Since (Sp)-8-Br-PEP-cGMPS exhibits
sufficient selectivity for PKG versus PKA (34), this compound is the best choice for triggering PKG activation.
(Rp)-8-Br-PEP-cGMPS was found to be the most
potent inhibitor of PKG and inhibited PKG more than 300 times more
potently than PKA (34). (Sp)-5,6-DCl-cBIMPS was
reported to be the best specific activator of PKA and a 300-fold less
potent activator of PKG (35). (Rp)-8-pCPT-cAMPS
is a better inhibitor of PKA (49). Although cGMP and cAMP analogs
normally have effects on the PKG and PKA pathways, respectively, it
should be recognized that high concentrations of cGMP analogs can at least partially affect PKA signaling, and the converse is also true for
cAMP analogs. Therefore, we carefully constructed
concentration-response curves using these analogs to determine the
relative potencies of the activators and inhibitors. We found that
(Sp)-8-Br-PEP-cGMPS facilitated channel activity
at concentrations of up to 100 µM and
(Rp)-8-Br-PEP-cGMPS inhibited the channel
activity at concentrations of up to 10 µM, whereas
(Sp)-5,6-DCl-cBIMPS and
(Rp)-8-pCPT-cAMPS had no effect on the channels
at concentrations of up to 100 and 10 µM, respectively.
Additionally, we observed the effects of NO/cGMP analogs under
conditions in which PKA was inhibited by (Rp)-8-pCPT-cAMPS. These results ruled out the
importance of PKA in KATP channel regulation, indicating
that the observed effects of NO/cGMP analogs are truly
PKG-mediated.
If this interpretation were correct, one would expect that activation
of PKG would also directly increase KATP channel activity, even in excised patches. Neither Tsuura et al. (24) nor
Shinbo and Iijima (27) directly tested the effect of PKG on
KATP channels. We tested the effects of exogenous PKG using
inside-out patches. Since PKG and PKA share some similarities in
protein substrate sequence specificity, it is possible that PKG
phosphorylates PKA-selective sites. To determine which is the more
potent kinase in activating KATP channels, we carefully
constructed concentration-response curves using PKG and PKA (catalytic
subunit). This experiment produced the following lines of evidence
clearly showing that PKG acts directly on the KATP
channels: (a) in contrast to PKG, the catalytic subunit of
PKA at the same concentration ranges had no effect on the channel (data
not shown); (b) the stimulating effects of PKG still
occurred under conditions in which PKA was inhibited by
(Rp)-8-pCPT-cAMPS; and (c)
KATP channel stimulation by PKG was reversibly inhibited by
(Rp)-8-Br-PEP-cGMPS.
The preceding findings strongly suggest that KATP channels
are stimulated by PKG but not by PKA. If PKG phosphorylated
KATP channels directly, one would expect that protein
phosphatase could inhibit PKG action. Our findings demonstrated that an
endogenous membrane-associated PP2A was responsible for the reversal of
PKG-mediated activation of KATP channels and that exogenous
PP2A inhibited PKG-induced KATP channel activity.
Furthermore, replacement of ATP with ATP
S and removal of
Mg2+ from the internal solution prevented the effect of
PKG. Together, these results show that PKG activates KATP
channels by an apparent phosphorylation-dependent
mechanism, suggesting that the activity of a KATP channel
depends on its net phosphorylation state, and this in turn depends on
the balance between opposing PKG and phosphatase activities.
Presumably, these processes of phosphorylation and dephosphorylation
provide a mechanism by which KATP channel activity can be
reversibly controlled. To our knowledge, our findings provide the first
direct evidence that KATP channels can be opened through PKG-dependent phosphorylation in rabbit ventricular myocytes.