© 1996 American Heart Association, Inc.
Articles |
Synergistic Modulation of ATP-Sensitive K+ Currents by Protein Kinase C and Adenosine
Implications for Ischemic Preconditioning
From the Division of Cardiology, Department of Medicine, The Johns Hopkins University, Baltimore, Md.
Correspondence to Eduardo Marban, MD, PhD, Room 844, Ross Building, 720 Rutland Ave, Baltimore, MD 21205. E-mail marban@welchlink.welch.jhu.edu.
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Abstract |
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Abstract Ischemic preconditioning has been shown to involve the activation of adenosine receptors, protein kinase C (PKC), and ATP-sensitive K+ (KATP) channels. We investigated the effects of PKC activation and adenosine on KATP current (IK,ATP) and action potentials in isolated rabbit ventricular myocytes. Responses to pinacidil (100 to 400 µmol/L), an opener of KATP channels, were markedly increased by preexposure to the PKC activator phorbol 12-myristate 13-acetate (PMA, 100 nmol/L). IK,ATP measured at 0 mV was increased by PMA pretreatment from 0.55±0.32 to 3.25±0.47 nA (n=6, P<.01). We next determined whether PKC activation abbreviates the time required to turn on IK,ATP during metabolic inhibition (MI). In control cells in which MI was induced by 2 mmol/L cyanide and 0 glucose, IK,ATP developed after an average of 15.1±2.4 minutes (n=8). Ten-minute pretreatment with PMA alone (PMA+MI) did not significantly alter this latency (11.9±2.0 minutes, n=8). Since adenosine receptor activation has been shown to play an important role in the preconditioning response, two groups of myocytes were studied with adenosine (10 µmol/L) included during MI. Without PMA, adenosine alone (MI+Ado) did not affect the latency to develop IK,ATP (12.3±1.5 minutes, n=8). However, if cells were pretreated with PMA and then subjected to MI in the presence of adenosine (PMA+MI+Ado), the latency was greatly shortened to 5.5±1.6 minutes (n=8; P<.02 versus MI, PMA+MI, and MI+Ado groups). This effect could not be reproduced by an inactive phorbol but was completely abolished by the adenosine receptor antagonist 8-(p-sulfophenyl)-theophylline. The opening of KATP channels may be cardioprotective because of the abbreviation of action potential duration (APD) during ischemia. Therefore, we tested whether PKC activation could modify the time course of APD shortening during MI. Consistent with the ionic current measurements, PMA pretreatment significantly accelerated APD shortening, but only when adenosine (10 µmol/L) was included during MI. The effects were not attributable to accelerated ATP consumption: PMA pretreatment did not alter the time required to induce rigor during MI, whether or not adenosine was included. Our results indicate that PKC activation increases the IK,ATP induced by pinacidil or by MI. The latter effect requires concomitant adenosine receptor activation. The synergistic modulation of IK,ATP by PKC and adenosine provides an explicit basis for current paradigms of ischemic preconditioning.
Key Words: ischemic preconditioning • ATP-sensitive K+ current • protein kinase C • adenosine • pinacidil
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Introduction |
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TopAbstractIntroductionMaterials and Methods Results Discussion References |
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Brief periods of cardiac ischemia and reperfusion decrease the extent of cellular injury after subsequent prolonged ischemia.1 Although this phenomenon, termed ischemic preconditioning, has been demonstrated in virtually every animal model, the underlying mechanism of the protection is still not clear.2 Several mechanisms have been proposed. Liu et al3 were the first to show that activation of Ado receptors is important for preconditioning. Whereas Ado receptor antagonists could effectively block the protection, Ado receptor agonists mimicked preconditioning. Recently, several studies have indicated that activation of PKC is also important for preconditioning.4 5 6 Inhibitors of PKC are able to abolish the protection from preconditioning, and PKC activators mitigate injury.
Another proposed mechanism is the opening of KATP channels, as first suggested by Auchampach and colleagues.7 8 Blocking this channel eliminates protection, whereas pretreating with KATP channel openers offers protection similar to that seen with preconditioning. If the KATP channel is involved in ischemic preconditioning, the activity of this channel would need to be increased or primed in the preconditioned heart so that it would open more rapidly or to a greater extent during the prolonged ischemia. Ado might serve as such a priming agent. Activation of Ado receptors can turn on KATP channels,9 10 possibly through a G protein–mediated mechanism.9 11 12 The available pharmacological evidence is consistent with the idea that the protection from Ado receptor activation occurs because of the facilitated opening of KATP channels.7 13 14
The PKC hypothesis is attractive because PKC translocation and/or protein phosphorylation could mediate or facilitate the interaction between Ado receptors and KATP channels. PKC activation has been shown to increase the open probability of KATP channels in insulinoma cells.12 15 16 In cardiac myocytes, there is less consensus regarding the effect of PKC on KATP channels. Wang and Lipsius17 showed that when cat atrial myocytes were exposed twice to acetylcholine, the second acetylcholine exposure elicited a larger increase in glibenclamide-sensitive K+ conductance than the first exposure. This potentiation was blocked by PKC inhibitors. On the other hand, Light et al18 showed that a constitutively active PKC inhibits KATP channels in patches excised from rabbit ventricular myocytes. No data are available regarding the effect of PKC and/or Ado on KATP channels activated by openers or by MI.
In the present study, we investigated whether Ado and/or PKC activation modulates KATP channels in rabbit ventricular myocytes. IK,ATP induced either by the KATP channel opener pinacidil or by MI was measured in control cells and in cells treated with PMA (a PKC activator). To probe the pathophysiological relevance of the findings, we measured APD during MI in control and PMA-treated cells with and without exposure to Ado.
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Materials and Methods |
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TopAbstractIntroductionMaterials and Methods Results Discussion References |
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Chemicals
Collagenase (type II) was purchased from Worthington. Pinacidil and SPT were purchased from Research Biochemicals International. The other chemicals were obtained from Sigma Chemical Co. Pinacidil, PMA, 4
-phorbol, and glibenclamide
were dissolved in dimethyl sulfoxide. The final concentration of
dimethyl sulfoxide in the experimental solutions did not exceed
0.1%.
Preparation of Rabbit Myocytes
Isolated rabbit ventricular
myocytes were
obtained from rabbit hearts by conventional enzymatic dissociation
methods.19 In brief, hearts were quickly excised from
anesthetized (60 mg/kg pentobarbital IP) rabbits weighing 1 to
2 kg and mounted on a Langendorff apparatus. The heart was
perfused with modified Krebs-Henseleit solution composed of (mmol/L)
NaCl 119, KCl 5, MgSO4 1, NaHCO3 25,
KH2PO4 1, CaCl2 1, and glucose 10.
The perfusate was bubbled with 95% O2/5%
CO2 and maintained at 37°C. After 5 minutes of perfusion,
hearts were perfused without Ca2+ for another 5 minutes,
after which the perfusion solution was switched to one containing
collagenase (0.8 mg/mL, type II, Worthington). The
perfusion pressure was monitored, and the flow rate was adjusted to
maintain perfusion pressure at 
75 mm Hg. After 10 to 15 minutes of
collagenase perfusion, hearts were removed from the
perfusion apparatus, and the atria were trimmed away. The
ventricles were minced and incubated in a shaking bath for another 10
minutes in collagenase-containing solution. Cells were
then filtered through nylon mesh and stored at room temperature in a
high-K+ solution containing (mmol/L) potassium glutamate
120, KCl 25, MgCl2 1, HEPES 10, EGTA 0.1, and glucose 10.
Before each experiment, cells were washed in a modified Tyrode's
solution containing (mmol/L) NaCl 140, KCl 5, CaCl2 1,
MgCl2 1, and HEPES 10 (pH 7.4 with NaOH). This isolation
procedure yielded >50% of Ca2+-tolerant crisply striated
ventricular myocytes. All experiments were performed at
room temperature (21°C to 22°C) within 7 hours after
isolation.
Macroscopic Membrane Current and Action Potential
Measurements
Patch pipettes were pulled on a Flaming-Brown
micropipette
puller (model P-87, Sutter Instrument Co) from filamented borosilicate
glass (outer diameter, 1.5 cm) and fire-polished before using. When
filled with intracellular solution, pipettes had 2- to 4-M
resistances. Whole-cell currents and action potentials were
recorded using an Axopatch 200A amplifier (Axon Instruments). After
a gigaohm seal was formed, the whole-cell configuration was
achieved by breaking the membrane with gentle suction. The internal
solution contained (mmol/L) potassium glutamate 120, KCl 25,
MgCl2 0.5, potassium EGTA 10, HEPES 10, and MgATP 1 (pH 7.2
with KOH). The time course of change in IK,ATP was
monitored by applying voltage ramps of 400-ms duration from 50 to
-150 mV every 5 s. Steady state current-voltage relationships
were obtained from membrane currents elicited every 5 s during 400-ms
steps from the holding potential (-80 mV) to test potentials
ranging from 50 to -150 mV in 10-mV increments. Values shown
represent the absolute membrane current 200 ms after the onset
of the step. Action potentials were stimulated every 5 s in
current-clamp mode. Before switching into the current-clamp
mode, whole-cell recording was established in the
voltage-clamp mode. Action potentials were initiated by short
(2-ms) depolarizing current pulses (25 pA). APD50 and
APD90 were compared in the various experimental groups.
Single-Channel Recordings
Excised and cell-attached patches
were used to investigate
whether channels activated by pinacidil or by MI have
properties similar to those reported previously for unitary
KATP channels.20 21 22 To zero
the membrane
potential, cells were superperfused with high-K+ solution
containing (mmol/L) KCl 140, MgCl2 0.3, CaCl2
2, EGTA 5, HEPES 10 (pH 7.2), allowing accurate control over the
transmembrane potential of the patch. Patches were either excised into
this solution or cell-attached, as indicated. Pipettes had
resistances of 12 to 20 M when filled with pipette solution
containing (mmol/L) KCl 140, MgCl2 1, CaCl2 1,
and HEPES 10 (pH 7.4).
Time to Develop Rigor During MI
To investigate whether PMA
treatment and/or Ado could alter the
rate of energy depletion in the cells, the time required to induce
rigor was measured. The cells, which were electrically quiescent, were
superperfused with experimental solutions (see protocols for APD
shortening below) and viewed on the stage of an inverted microscope
(x400 magnification). The image was projected onto a video camera,
and the video signal was recorded for subsequent analysis.
The length of each striated rod-shaped cell in the field was
measured every 30 s during MI. Cells were considered to enter the rigor
state when the cell length shortened to two thirds of the original
value.23
Protocols
The cells were randomized among treatment groups in
each
protocol, and cells in each group were derived from at least three
individual hearts.
Pinacidil-Activated IK,ATP
The experimental bath
has a complete exchange time of <1
minute. After 10 minutes of whole-cell recording, the
superperfusate was switched to pinacidil-containing
solution for 5 minutes. Cells were first superperfused with 100
µmol/L pinacidil. If no IK,ATP was observed, 200 and 400
µmol/L were further tested until IK,ATP was elicited (as
gauged by an increase in outward current at 0 mV). If even 400 µmol/L
pinacidil did not activate IK,ATP (as was the case
in 18 of 39 cells), the cell was not studied further. Cells responsive
to pinacidil were then washed with pinacidil-free solution for 5
minutes. Each individual cell was then assigned randomly either to the
control (another 10 minutes of drug-free solution) or PMA groups
(addition of 100 nmol/L PMA for 10 minutes). After 5 minutes of further
superperfusion with drug-free Tyrode's solution, cells were
retreated with the same threshold concentration of pinacidil (which had
previously elicited IK,ATP) for another 5 minutes.
IK,ATP During MI
MI was achieved by adding 2
mmol/L CN and omitting glucose from
the Tyrode's solution. The pH of the solution was adjusted to 7.4 with
NaOH. Four groups of cells were studied. For the control (MI) group,
after establishing the whole-cell patch, cells were simply
equilibrated for 20 minutes before exposure to MI. The second group of
cells (PMA+MI group) was superperfused with 100 nmol/L PMA for 10
minutes followed by 5 minutes without PMA before exposure to MI. In the
third (MI+Ado) and fourth (PMA+MI+Ado) groups, cells were
treated in
the same way as the MI and PMA+MI groups, respectively, except that 10
µmol/L Ado was included during MI. Currents were monitored by 400-ms
voltage ramps from 50 to -150 mV applied every 5 s. The time to
activate IK,ATP was determined by the time required
to induce a clearly measurable outward current (>0.1 nA at 0 mV). To
quantify how rapidly IK,ATP turned on in each cell, dI/dt
at 0 mV was calculated as the first derivative of the current with
respect to time. In contrast to the variable response to pinacidil,
IK,ATP developed in every cell during MI; thus, all the
cells in which the protocol was completed were included in the data
analysis.
APD Shortening During MI
Four groups of cells were studied.
The first group was the
control (MI) group. After establishing whole-cell
recording, action potentials were measured for 20 minutes
without additional intervention. Cells were then exposed to MI
including 10 mmol/L 2-DG, 2 mmol/L CN, and no glucose. The second group
of cells (PMA+MI group) was treated with 100 nmol/L PMA for 10 minutes
followed by 5 minutes of Tyrode's solution superperfusion before
switching to MI. The third (MI+Ado) and fourth (PMA+MI+Ado)
groups were
treated the same way as the MI and PMA+MI groups, respectively, except
that 10 µmol/L Ado was added during MI. APD shortening and eventual
failure to fire action potentials were observed in every cell subjected
to MI.
Data Analysis
All values are expressed as mean±SEM.
ANOVA combined with a
Newman-Keuls post hoc test was used to test for differences among
groups, except for the analysis of
pinacidil-activated IK,ATP. In the latter, a
paired t test was applied to test for differences before and
after PMA treatment. A value of P<.05 was considered
significant.24
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Results |
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TopAbstractIntroductionMaterials and Methods Results Discussion References |
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Pinacidil-Activated IK,ATP
Cells were exposed to pinacidil for two 5-minute periods. The first exposure to pinacidil, ranging from 100 to 400 µmol/L (see "Materials and Methods"), induced a small IK,ATP in 21 of 39 cells. Previous work has shown that not all ventricular myocytes respond to pinacidil, possibly because of differences in basal cAMP levels.25 Fig 1A
shows current-voltage relations from a
representative pinacidil-responsive cell at each
stage of the experimental protocol. The pinacidil-induced current
during a second application of the drug was not significantly different
from the first one. Fig 1B illustrates the very different
response of a
cell that was treated with PMA for 10 minutes before the second
exposure to pinacidil. PMA treatment alone did not significantly change
the membrane currents, but the subsequent response to pinacidil (Fig
1B, "repeat pinacidil") in the absence of PMA was
markedly
increased.
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Fig 1C
summarizes the currents measured at 0
mV at each stage of the
experiment in the control group and in the PMA group. The average
concentration of pinacidil in the control group was 183±47 µmol/L,
which is similar to that used in the PMA group (200±71 µmol/L). The
critical comparison is between the first and second pinacidil exposures
in the control versus the PMA-treated cells. The first
pinacidil-activated currents at 0 mV were 0.48±0.25 nA for
the control group and 0.55±0.32 nA for the PMA group. The second
exposure to pinacidil activated a comparably sized current in
the control group (0.51±0.26 nA, P=NS versus the first
pinacidil exposure). However, if the cells were treated with PMA, the
current increased greatly during the second pinacidil exposure to
3.25±0.47 nA (P<.01 versus first pinacidil exposure). This
large outward current was partially blocked by 100 µmol/L
glibenclamide (3.16±0.82 nA in pinacidil and 1.71±0.33 nA in
pinacidil plus glibenclamide, n=3).
Because pinacidil appears to act directly on the channel26 rather than via second messengers or intermediary pathways, the elevated drug responsiveness does not necessarily imply that PKC activation will suffice to potentiate IK,ATP during metabolic stress (eg, ischemia). For this reason, we next determined the effects of PMA treatment on IK,ATP during MI.
IK,ATP During MI
Fig 2A shows the
time course of membrane currents
(measured at -100 and 0 mV) during MI from a representative
cell. When exposed to 2 mmol/L CN and no glucose (MI group), the
currents measured at -100 mV did not change over time.
Nevertheless, currents at 0 mV (IK,ATP) started to increase
after a delay of 15 minutes. Prior activation of PKC did not alter
this response: when cells were treated with 100 nmol/L PMA before MI
(PMA+MI group), the current changes were similar to those in the MI
group (Fig 2B).
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Adenosine accumulates in tissues and in
the
interstitial space during ischemia, and Ado
receptor activation has been shown to act as both initiator and
mediator of ischemic preconditioning.27 28 In our
protocol, cells were continuously superperfused, making it impossible
for Ado to accumulate around the cell as it would during
ischemia. To mimic ischemia more faithfully, 10
µmol/L Ado was added during MI. Cells exposed to 2 mmol/L CN,
no glucose, and 10 µmol/L Ado (MI+Ado group) developed a gradually
increasing current at 0 mV, which was similar to the responses in the
MI and PMA+MI groups (Fig 2C). However, when PMA-treated
cells were
subjected to MI including Ado (PMA+MI+Ado), the time required to
increase the current at 0 mV was markedly abbreviated, and the rate of
the increase in current was greater than in the other three groups (Fig
2D).
Fig 3A summarizes the pooled data
for the times required
for IK,ATP activation. This latency was 15.1±2.4 minutes
in the MI group and was not significantly altered in the PMA+MI group
(11.9±2.0 minutes) or the MI+Ado group (12.3±1.5 minutes).
However,
the latency to develop IK,ATP in the PMA+MI+Ado group
decreased to only 5.5±1.6 minutes (P<.05 versus the other
three groups).
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To exclude the possibility of phorbol ester effects
unrelated to PKC,
we used the inactive PMA analogue 4-phorbol. As shown in Fig
3A
(4-phorbol+MI+Ado), 100 nmol/L of 4-phorbol did not
significantly affect the time course of IK,ATP development
during MI in the presence of 10 µmol/L Ado (13.9±2.5 minutes). The
involvement of Ado receptors was also investigated. The nonselective
Ado receptor antagonist SPT (100 µmol/L) completely
blocked the abbreviation of the time to IK,ATP activation
by PMA and Ado (14±3.5 minutes; Fig 3A,
PMA+MI+Ado+SPT).
As another index of IK,ATP
activation in the four groups,
we measured dI/dtmax at 0 mV. Fig 3B shows that
currents in the PMA+MI+Ado group had a significantly higher
dI/dtmax (12.3±2.6 versus 3.4±4.4 nA/min in the MI
group, versus 4.2±2.1 nA/min in the PMA+MI group, and versus
4.0±1.9
nA/min in the MI+Ado group; P<.05). This increased rate of
current development was not observed when PMA was replaced by
4-phorbol (4.3±2.3 nA/min in the 4-phorbol+MI+Ado
group)
or in the presence of the Ado receptor antagonist (3.8±2.1
nA/min in the PMA+MI+Ado+SPT group). The decreased latency and
the
increased rate of current development both indicate that Ado and PMA,
when combined, facilitate the activation of IK,ATP during
MI.
In three additional cells, we verified that the outward current
induced
by MI could be greatly inhibited by 100 µmol/L of glibenclamide. Fig
4A shows the time course of current development in a
representative cell. Five minutes after the induction
of outward currents during MI by cyanide and no glucose, glibenclamide
was added to the superperfusate. The outward current was
suppressed by glibenclamide, and this effect was consistent, as
indicated by the pooled data in Fig 4B (2.58±0.8 versus
0.74±0.15 nA
after glibenclamide, P<.05).
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As another check on the
identity of the channels activated by
PMA+MI+Ado, we compared the properties of single channels induced by
this protocol with those of channels evoked by patch excision or by
exposure to pinacidil. After excision into ATP-free and symmetrical
K+ solution, channels of 60-pS conductance showed
bursting patterns of activity interspersed with brief closed periods
(Fig 5A). The single-channel currents exhibited some
inward rectification. The activity of these channels rapidly decreased
as a consequence of channel "rundown."29 However,
they could be reactivated by pinacidil, as shown in Fig 5B.
All of these properties are characteristic of KATP
channels.20 21 22 30 Fig
5C illustrates that channels
recorded in cell-attached patches during MI (PMA+MI+Ado
exposure) demonstrate gating and conductance properties very similar to
those in Fig 5A and 5B. Thus, single-channel
recordings
support the idea that the current activated during PMA+MI+Ado
exposure consists of KATP channels.
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APD Shortening During MI
Action potentials shorten during
ischemia or
MI,31 32 and myocytes become electrically inexcitable
when
ischemia or MI duration is prolonged. In isolated cells, one of
the major reasons that APD shortens is because of the increase in
IK,ATP. Nichols et al31 suggested that even
very small increases in IK,ATP would result in significant
shortening of APD. We investigated whether PMA treatment and/or Ado
could alter the time course of APD shortening during MI. Because APD
depends critically on the balance between inward and outward currents
and because Ca2+ currents tend to run down in the
whole-cell configuration, we accelerated MI by adding the
glycolytic inhibitor 2-DG. Therefore, MI in the action
potential experiments consisted of 10 mmol/L 2-DG, 2 mmol/L CN, and no
glucose.
Fig 6 shows the time course of APD shortening
from two
representative cells (from the MI+Ado group [Fig
6A]
and from the PMA+MI+Ado group [Fig 6B]).
APDs were measured before
and 3, 5, and 7 minutes after MI. The shape and duration of action
potentials before MI were similar in the two experimental groups.
Action potentials from the cell in the MI+Ado group (Fig
6A) began to
narrow only at 7 minutes. In contrast, Fig 6B shows that the
APD
shortened much faster in the cell from the PMA+MI+Ado group.
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Fig 6C summarizes the APD50 and
APD90
data from all four groups. In the MI group, average APD50
and APD90 values before MI were 440±46 and 538±46 ms,
respectively. After 7 minutes of MI, APD50 and
APD90 shortened to 276±83 and 355±100 ms,
respectively.
PMA pretreatment alone did not alter APD50 and
APD90, nor did it affect the time course of APD
shortening during MI. When 10 µmol/L Ado was added during MI, it did
not significantly affect the time course of APD shortening.
Nevertheless, cells preexposed to PMA exhibited significantly
accelerated APD shortening (PMA+MI+Ado group). APD50 and
APD90 were 335±47 and 460±55 ms, respectively, before
MI
(P=NS versus all other groups), but APD50 and
APD90 were much shorter at 5 minutes (72±42 and
132±69
ms, respectively) and at 7 minutes (26±19 and 79±68 ms,
respectively)
than in any of the three other groups (P<.05). Five of six
cells in the PMA+MI+Ado group became inexcitable after 7 minutes of
MI,
whereas none of the cells from the other three groups were inexcitable
at this time (they eventually failed to fire action potentials after
10 minutes). We measured IK,ATP by switching from
current-clamp to voltage-clamp mode in the cells that had
turned inexcitable and confirmed that IK,ATP had developed
in every cell (data not shown). Interestingly, none of the cells
exhibited any indication of rigor before becoming inexcitable. The
possibility that the rate of energy depletion might differ among the
groups was examined in the next set of experiments.
Time to Develop Rigor During MI
The activation of
IK,ATP is energy dependent. The
modulation of IK,ATP and the acceleration of APD shortening
could simply reflect a change of cellular energy metabolism
induced by PMA pretreatment and/or Ado. In particular, these
interventions may decrease energy stores before MI or increase the rate
of energy utilization during MI. If this were the case, then PMA
pretreatment and/or Ado might logically be expected to accelerate the
time to develop rigor during MI. The same solution exchange protocol as
in APD studies was used to test this idea. Metabolic
inhibition was induced by superperfusing the cells with 10 mmol/L 2-DG
and 2 mmol/L CN in the absence of glucose. Fig 7 shows a
representative image of the cells in the MI group
before (Fig 7A) and after 20 minutes of MI (Fig
7B). In Fig 7A, most
cells are quiescent and rod-shaped. After 20 minutes of MI (Fig
7B), most of the cells became rounded, and all cells were in
rigor by
30 minutes (Fig 7C). As summarized in Fig 7D,
times to rigor did not
differ significantly in the four groups, although the time to rigor
tended to be prolonged in the Ado-containing groups. Such a change,
even if genuine, would be in the wrong direction to explain the
abbreviation of the time required to activate
IK,ATP. These results suggest that PMA pretreatment and
Ado, alone or in combination, do not affect the net rate of energy
depletion.
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Discussion |
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TopAbstractIntroductionMaterials and Methods Results Discussion References |
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Ischemic preconditioning is one of the most potent anti-ischemic interventions discovered to date. Although different mechanisms may be involved in different species and even against different forms of injury (such as necrosis, arrhythmias, and stunning) in the same species,2 33 some common agreement exists. Evidence suggests the involvement of one or more of the following processes: Ado receptor activation,3 34 stimulation of PKC,4 5 6 and opening of KATP channels.7 34 35 Furthermore, it has been suggested that these three hypotheses may be interrelated,36 with the KATP channel as the final effector. If this is the case, the activity of KATP channels would need to be increased or primed by the initial preconditioning insult so that these channels would open more rapidly or to a greater extent during the subsequent ischemia. One explicit scheme36 proposes that Ado receptor activation stimulates PKC during preconditioning; PKC then phosphorylates KATP channels, and the phosphorylation makes the channel open more readily during the second ischemia. The strongest evidence supporting this hypothesis comes from studies in rabbits. Ado receptor activation has been shown to activate phospholipase D,37 which could then activate PKC. Furthermore, the protective effect of Ado has been linked to KATP channel opening: the protection induced by preconditioning and Ado receptor agonists can be eliminated by KATP channel blockers.14 35 38 Likewise, such protection can be blocked by PKC inhibitors,39 although such inhibitors reportedly reduce infarct size in nonpreconditioned rabbit hearts.40
Mechanisms of IK,ATP Modulation
The possible
modification of the KATP channel by
protein phosphorylation has been investigated most
extensively in insulinoma cells. Dunne16 showed that
activation of PKC by PMA had a transient inhibitory effect
on the KATP channel, whereas prolonged treatment with PMA
(>5 minutes) increased the opening of KATP channels. In
the same cell line, De Weille et al15 demonstrated that
stimulation of PKC by somatostatin or PMA activates
KATP channels. Most recently, Ribalet and
Eddlestone12 reported that PKC activation can increase
KATP channel activity directly or indirectly via a
second-messenger pathway. In myocytes, two studies have described
the effect of PKC activation on KATP channels. Using
acetylcholine as an agonist, Wang and Lipsius17
demonstrated a PKC-related potentiation in glibenclamide-sensitive
K+ conductance in cat atrial myocytes. Our pinacidil data
support the idea that PKC activation can potentiate IK,ATP.
Nevertheless, our results during MI indicate that the increase of
IK,ATP by PKC activation requires the synergistic action of
Ado. Deutsch and Weiss41 showed that
phospholipases A2, C, and D all modestly desensitize
KATP channels to closure by ATP, although they did not
investigate whether the desensitization is PKC dependent. On the other
hand, Light et al18 showed that a constitutively active
PKC inhibits the opening of KATP channels in patches
excised from rabbit ventricular myocytes. Several
considerations help to rationalize the apparent discrepancy. First,
Light et al used a constitutively active mixture of -, ß-, 
-,
and 
-PKC isoforms purified from rat brain, proteolytically modified
such that it does not require diacylglycerol and Ca2+.
Since different PKC isoforms may have different effects on
KATP channels,16 it is possible that the PKC
isoforms involved in our experiments (and activated by PMA) are
different from those used by Light et al. Second, in the present
study PMA itself did not affect KATP channels but only
increased the current induced by pinacidil or by Ado and MI. Third, we
used an internal ATP concentration of 1 mmol/L, while Light et al only
explored the effects of PKC at ATP concentrations 
0.1 mmol/L. Thus,
the experimental conditions are sufficiently different that it is
difficult to compare the results at face value.
We used two methods to
elicit IK,ATP. First, we
investigated the effect of PKC on pinacidil-activated
IK,ATP. Our results show that
pinacidil-activated IK,ATP is much higher in
PMA-pretreated cells than in cells never exposed to PMA. Since
pinacidil acts directly on KATP channels,26 a
direct effect of PMA on the channel itself (eg, PKC-mediated
phosphorylation, as depicted in Fig 8A)
appears to increase the sensitivity of the channel to drug. A similar
idea has been proposed to explain the potentiating effect of cAMP
(presumably by cAMP-dependent
phosphorylation).25 Further studies will
be required to determine whether the effects of the two
phosphorylation pathways on the pinacidil response are
independent or converge on a common mechanism. Escande et
al42 showed that in guinea pig myocytes, repeated
exposures to 300 µmol/L pinacidil elicited progressively greater
effects on IK,ATP. Although there may be such a trend in
the present study, it is not statistically significant and is
negligible compared with the increase induced by PMA pretreatment.
|
Because it is more relevant to the pathophysiology of preconditioning,
we focused our attention on IK,ATP during MI. Our results
indicate that PMA pretreatment shortens the time required to develop
IK,ATP, but only if Ado is included during MI. This
finding has important implications for the role played by Ado in
ischemic preconditioning. A prevailing concept holds that Ado
receptor activation plays a dual role: it initiates as well as mediates
the protection.27 28 This conclusion was drawn from
experimental evidence that Ado receptor antagonists could
abolish the protection from ischemic preconditioning when given
during the preconditioning phase as well as during the subsequent
prolonged ischemia. The protection provided by direct PKC
activators such as PMA could also be abolished by blocking
Ado receptors during ischemia.5 Thus, Ado
receptors have to be activated during both the preconditioning
and the prolonged ischemia to provide protection. In our
experimental setting, cells were continually superperfused. Therefore,
Ado was not able to accumulate and activate its receptors. When
we added 10 µmol/L Ado during MI, PMA pretreatment greatly
abbreviated the time to activation of IK,ATP. A
concentration of 10 µmol/L was chosen because it mimics the Ado
levels reached in the myocardium during
ischemia.43 Kirsch et al9 have shown
that Ado can open KATP channels, but only at low
cytoplasmic ATP levels (300 µmol/L). Cordeiro et al44
have shown that 1 to 50 µmol/L Ado does not affect APD under
physiological conditions. Consistent with
these results, with 1 mmol/L ATP in the pipette and superfusion with
normal Tyrode's solution, we did not observe any KATP
currents even with 10 mmol/L Ado in the solution (authors' unpublished
data, 1995). In addition, 10 µmol/L Ado alone did not significantly
shorten the latency for IK,ATP activation during MI (see
Fig 3A, comparing the MI+Ado group with the MI group). In
contrast, Li
et al10 showed that Ado (10 µmol/L to 10 mmol/L) could
activate IK,ATP even with normal intracellular ATP
levels in guinea pig atrial cells. We do not know the reason for this
discrepancy, but it could be due to species differences or atrial
versus ventricular cells.
Pinacidil is a well-known KATP
channel
opener,26 and its effect on other membrane currents is
very small.25 Nevertheless, we examined the effect of
glibenclamide on pinacidil-activated currents after PMA
treatment and showed that glibenclamide partially blocked this current,
which is different from the cells without PMA pretreatment (in three
cells, pinacidil-activated currents could be completely
blocked by 100 µmol/L glibenclamide [data not shown]). The
incomplete blockade is not surprising, given that pinacidil is a direct
activator of the KATP channel, whereas
glibenclamide binds to a separate sulfonylurea receptor that is only
loosely coupled to KATP channels.30 A more
complete effect was observed when IK,ATP was induced by MI,
as shown in Fig 4, despite the fact that MI is known to
decrease the
sensitivity of IK,ATP to
sulfonylureas.45 46 47
One possible mechanism of the reduction in sensitivity to glibenclamide
is partial proteolysis of KATP channels due to activation
of endogenous proteases.48
As an independent check on the
identity of the channels
activated by pinacidil or MI, we used single-channel
recordings. As shown in Fig 5, the currents induced by
pinacidil or MI are virtually identical in their unitary gating and
permeation properties. Additionally, these properties are entirely
consistent with those previously reported for KATP
channels in cardiac muscle cells.20 21 22
Adenosine receptors are capable of activating phospholipase D in rabbit myocytes37 and activate 76-PKC in rat myocytes.49 In preliminary results, we found that 10 µmol/L of Ado was unable to potentiate the pinacidil-induced IK,ATP by using the same protocol as used for the PMA group (PMA was substituted for Ado). This suggests that Ado is not capable of activating PKC, or at least those PKC isoforms involved in the potentiation of the pinacidil effect. However, caution must be exercised when comparing this result with that which might be obtained in intact cells as a result of intracellular dialysis (including Ca2+ buffering), which is a consequence of our whole-cell recordings.
Although our results demonstrate that PMA
increases IK,ATP
during MI in the presence of Ado, they leave unresolved the precise
signal transduction mechanisms that mediate these effects. The fact
that a receptor antagonist prevents the effect of Ado
indicates that an Ado receptor is involved. Coupling of such receptors
to G proteins is well established,50 which suggests that G
proteins may play a role in the process. While our experiments were not
designed to support or to exclude the participation of G proteins, such
proteins were probably recruitable under our experimental conditions.
Although GTP was not added to the pipette solution, it is likely that
sufficient GTP to support G-protein activity can be generated from ATP
by the endogenous membrane-bound NDPK.51
Several studies have shown that even in inside-out patches,
muscarinic K+ channels can be activated by a G
protein–mediated mechanism in the presence of ATP but absence of
GTP.51 52 The activity of NDPK may be inhibited
during MI
because of the decreased ratio of ATP to ADP, but this ratio has to be
increased substantially to inhibit NDPK activity. Heidbuchel et
al51 measured the NDPK activity in frog atrial membrane
and found that 0.1 mmol/L ADP decreases NDPK activity by only 20% in
the presence of 1 mmol/L ATP. In our experimental settings, there is
always 1 mmol/L ATP in the pipette, which should supply enough ATP so
as to maintain NDPK activity. Several possibilities (illustrated in Fig
8) merit further investigation. First, PKC could
phosphorylate KATP channels directly, but the
phosphorylated channel requires stimulation from
pinacidil (Fig 8A) or from Ado-mediated pathways (Fig
8B) to become
more active. On the other hand, PKC could phosphorylate Ado
receptors and/or G proteins instead of the channels themselves (Fig
8C). This phosphorylation would then act to increase
the stimulation of the channel by Ado. It is also possible that direct
potentiation of KATP channels by PKC may be more evident at
low cytosolic ATP concentrations.53 Further work is needed
to clarify which pathways are involved.
Pathophysiological
Implications
Several ionic currents, including IK,ATP, are
altered
during ischemia and MI and affect APD.32 54 A
small increase of IK,ATP suffices to shorten APD
greatly,31 whereas larger currents eventually render cells
inexcitable. In guinea pig myocytes, Cordeiro et al44
showed that 50 µmol/L of Ado was able to abbreviate APD by increasing
IK,ATP during simulated ischemia (without the use
of metabolic inhibitors). However, 10 µmol/L
of Ado alone in the present study did not significantly affect the
rate of APD shortening. This is consistent with observations
that endogenous Ado does not activate ATP-sensitive
K+ channels in hypoxic guinea pig ventricle.55
An effect of Ado was only evident when the cells were pretreated with
PMA. The difference could be due to the lower concentration of Ado used
in the present study or to the species difference.
We used the
transition to rigor as a bioassay of cellular energy
stores. The transition to rigor has been shown to correlate with the
depletion of intracellular energy, particularly with the cessation of
anaerobic ATP
production.56 57 58 59 We did
not measure the degree of ATP depletion directly but, rather, used the
time to rigor as indirect evidence to show that PMA and Ado did not act
primarily by accelerating the rate of energy depletion during MI.
Previous studies would suggest that, if anything, Ado and its agonists
would delay rigor (or ischemic contracture).60 The
small increase in time to cell rigor that we observed with a low
concentration of Ado did not reach statistical significance (see Fig
7D) but is directionally consistent with previous work using
higher concentrations.
It is important to point out that the mechanism underlying ischemic preconditioning is far from clear and that many of the specifics appear to be species dependent. Although the evidence supporting a role for KATP channels in ischemic preconditioning is quite convincing in dogs and pigs, the results from rabbits are controversial, as has been discussed in detail elsewhere.61 62 The purpose of the present study was not to determine the ultimate mechanism for protection but rather to investigate the possible signal transduction pathways regulating the KATP channels on the basis of the available evidence regarding ischemic preconditioning. Our observations link together several disparate elements that are known to play important roles in ischemic preconditioning. However, given the complexity of the ischemic preconditioning phenomenon, caution should be exercised in extrapolating our results to different experimental conditions and to other species.
Even if PKC and Ado can act synergistically to facilitate the opening of KATP channels, it is not clear whether such an effect would suffice to mediate the protection. One obvious possibility is that the beneficial effect of opening KATP channels occurs via shortening of APD during ischemia.36 63 APD abbreviation reduces Ca2+ influx and contractility and thus conserves energy. However, a recent study has been unable to link the APD shortening to the beneficial effect of KATP channel openers.64 Likewise, the protective effect of ischemic preconditioning has been demonstrated even in experimental models that use quiescent isolated myocytes.65 This would argue against the idea that the protection results exclusively from APD shortening. Alternative mechanisms whereby opening of KATP channels could protect myocytes from lethal injury merit further investigation.
|
Selected Abbreviations and Acronyms |
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|
Acknowledgments |
|---|
This study was supported by a grant (RO1 HL-44065) from the National Institutes of Health. Dr O'Rourke was supported by American Heart Association Grant-in-Aid 94-1499.
Received June 22, 1995; accepted November 30, 1995.
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References |
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 K.-i. Ito, T. Sato, and M. Arita Protein kinase C isoform-dependent modulation of ATP-sensitive K+ channels during reoxygenation in guinea-pig ventricular myocytes J. Physiol., April 1, 2001; 532(1): 165 - 174. [Abstract] [Full Text] [PDF]
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 Y. Liu, G. Ren, B. O'Rourke, E. Marbán, and J. Seharaseyon Pharmacological Comparison of Native Mitochondrial KATP Channels with Molecularly Defined Surface KATP Channels Mol. Pharmacol., February 1, 2001; 59(2): 225 - 230. [Abstract] [Full Text]
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B. C. McPherson and Z. Yao Morphine Mimics Preconditioning via Free Radical Signals and Mitochondrial KATP Channels in Myocytes Circulation, January 16, 2001; 103(2): 290 - 295. [Abstract] [Full Text] [PDF]
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S. Wang, J. Cone, and Y. Liu Dual roles of mitochondrial KATP channels in diazoxide-mediated protection in isolated rabbit hearts Am J Physiol Heart Circ Physiol, January 1, 2001; 280(1): H246 - H255. [Abstract] [Full Text] [PDF]
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S. Sanada, M. Kitakaze, H. Asanuma, K. Harada, H. Ogita, K. Node, S. Takashima, Y. Sakata, M. Asakura, Y. Shinozaki, et al. Role of mitochondrial and sarcolemmal KATP channels in ischemic preconditioning of the canine heart Am J Physiol Heart Circ Physiol, January 1, 2001; 280(1): H256 - H263. [Abstract] [Full Text] [PDF]
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G.-Y. Wang, S. Wu, J.-M. Pei, X.-C. Yu, and T.-M. Wong {kappa}- but not {delta}-opioid receptors mediate effects of ischemic preconditioning on both infarct and arrhythmia in rats Am J Physiol Heart Circ Physiol, January 1, 2001; 280(1): H384 - H391. [Abstract] [Full Text] [PDF]
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P. S. Pagel, W. G. Toller, E. R. Gross, M. Gare, J. R. Kersten, and D. C. Warltier KATP channels mediate the beneficial effects of chronic ethanol ingestion Am J Physiol Heart Circ Physiol, November 1, 2000; 279(5): H2574 - H2579. [Abstract] [Full Text] [PDF]
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G. J. Gross and R. M. Fryer Mitochondrial KATP Channels : Triggers or Distal Effectors of Ischemic or Pharmacological Preconditioning? Circ. Res., September 15, 2000; 87(6): 431 - 433. [Full Text] [PDF]
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 B. Ribalet, S. A. John, and J. N. Weiss Regulation of Cloned Atp-Sensitive K Channels by Phosphorylation, Mgadp, and Phosphatidylinositol Bisphosphate (Pip2): A Study of Channel Rundown and Reactivation J. Gen. Physiol., September 1, 2000; 116(3): 391 - 410. [Abstract] [Full Text] [PDF]
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M. Kitakaze, K. Node, H. Asanuma, S. Takashima, Y. Sakata, M. Asakura, S. Sanada, Y. Shinozaki, H. Mori, T. Kuzuya, et al. Protein Tyrosine Kinase Is Not Involved in the Infarct Size-Limiting Effect of Ischemic Preconditioning in Canine Hearts Circ. Res., August 18, 2000; 87(4): 303 - 308. [Abstract] [Full Text] [PDF]
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T. Sato, N. Sasaki, B. O'Rourke, and E. Marban Adenosine Primes the Opening of Mitochondrial ATP-Sensitive Potassium Channels : A Key Step in Ischemic Preconditioning? Circulation, August 15, 2000; 102(7): 800 - 805. [Abstract] [Full Text] [PDF]
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 G. R. Gaudette, I. B. Krukenkamp, A. E. Saltman, H. Horimoto, and S. Levitsky Preconditioning with PKC and the ATP-sensitive potassium channels: a codependent relationship Ann. Thorac. Surg., August 1, 2000; 70(2): 602 - 608. [Abstract] [Full Text] [PDF]
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 N. Hoque, M. A. Cook, and M. Karmazyn Inhibition of alpha 1-Adrenergic-Mediated Responses in Rat Ventricular Myocytes by Adenosine A1 Receptor Activation: Role of the KATP Channel J. Pharmacol. Exp. Ther., August 1, 2000; 294(2): 770 - 777. [Abstract] [Full Text]
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J. Minners, E. J van den Bos, D. M Yellon, H. Schwalb, L. H Opie, and M. N Sack Dinitrophenol, cyclosporin A, and trimetazidine modulate preconditioning in the isolated rat heart: support for a mitochondrial role in cardioprotection Cardiovasc Res, July 1, 2000; 47(1): 68 - 73. [Abstract] [Full Text] [PDF]
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T. Sato, N. Sasaki, J. Seharaseyon, B. O'Rourke, and E. Marban Selective Pharmacological Agents Implicate Mitochondrial but Not Sarcolemmal KATP Channels in Ischemic Cardioprotection Circulation, May 23, 2000; 101(20): 2418 - 2423. [Abstract] [Full Text] [PDF]
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G. U. Ahmmed, P. H. Dong, G. Song, N. A. Ball, Y. Xu, R. A. Walsh, and N. Chiamvimonvat Changes in Ca2+ Cycling Proteins Underlie Cardiac Action Potential Prolongation in a Pressure-Overloaded Guinea Pig Model With Cardiac Hypertrophy and Failure Circ. Res., March 17, 2000; 86(5): 558 - 570. [Abstract] [Full Text] [PDF]
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 T. Sato, N. Sasaki, B. O'Rourke, and E. Marban Nicorandil, a potent cardioprotective agent, acts by opening mitochondrial ATP-dependent potassium channels J. Am. Coll. Cardiol., February 1, 2000; 35(2): 514 - 518. [Abstract] [Full Text] [PDF]
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N. Sasaki, T. Sato, A. Ohler, B. O’Rourke, and E. Marban Activation of Mitochondrial ATP-Dependent Potassium Channels by Nitric Oxide Circulation, February 1, 2000; 101(4): 439 - 445. [Abstract] [Full Text] [PDF]
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K. Ito, Y. Kagaya, T. Ishizuka, N. Ito, N. Ishide, and K. Shirato Diacylglycerol delays pHi overshoot after reperfusion and attenuates contracture in isolated, paced myocytes Am J Physiol Heart Circ Physiol, November 1, 1999; 277(5): H1708 - H1717. [Abstract] [Full Text] [PDF]
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X.-H. Xiao and D. G. Allen Role of Na+/H+ Exchanger During Ischemia and Preconditioning in the Isolated Rat Heart Circ. Res., October 15, 1999; 85(8): 723 - 730. [Abstract] [Full Text] [PDF]
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E. Carmeliet Cardiac Ionic Currents and Acute Ischemia: From Channels to Arrhythmias Physiol Rev, July 1, 1999; 79(3): 917 - 1017. [Abstract] [Full Text] [PDF]
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S. Wu, H. Y. Li, and T. M. Wong Cardioprotection of Preconditioning by Metabolic Inhibition in the Rat Ventricular Myocyte : Involvement of {kappa}-Opioid Receptor Circ. Res., June 25, 1999; 84(12): 1388 - 1395. [Abstract] [Full Text] [PDF]
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H. Hu, T. Sato, J. Seharaseyon, Y. Liu, D. C. Johns, B. O'Rourke, and E. Marbán Pharmacological and Histochemical Distinctions Between Molecularly Defined Sarcolemmal KATP Channels and Native Cardiac Mitochondrial KATP Channels Mol. Pharmacol., June 1, 1999; 55(6): 1000 - 1005. [Abstract] [Full Text]
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K. Hu, G.-R. Li, and S. Nattel Adenosine-induced activation of ATP-sensitive K+ channels in excised membrane patches is mediated by PKC Am J Physiol Heart Circ Physiol, February 1, 1999; 276(2): H488 - H495. [Abstract] [Full Text] [PDF]
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S. Kawamura, K.-I. Yoshida, T. Miura, Y. Mizukami, and M. Matsuzaki Ischemic preconditioning translocates PKC-delta and -epsilon , which mediate functional protection in isolated rat heart Am J Physiol Heart Circ Physiol, December 1, 1998; 275(6): H2266 - H2271. [Abstract] [Full Text] [PDF]
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B. Z. Simkhovich, K. Przyklenk, and R. A. Kloner Role of protein kinase C as a cellular mediator of ischemic preconditioning: a critical review Cardiovasc Res, October 1, 1998; 40(1): 9 - 22. [Abstract] [Full Text] [PDF]
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 J. Kiehn, C. Karle, D. Thomas, X. Yao, J. Brachmann, and W. Kubler HERG Potassium Channel Activation Is Shifted by Phorbol Esters via Protein Kinase A-dependent Pathways J. Biol. Chem., September 25, 1998; 273(39): 25285 - 25291. [Abstract] [Full Text] [PDF]
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J. Zhao, O. Renner, L. Wightman, P. H. Sugden, L. Stewart, A. D. Miller, D. S. Latchman, and M. S. Marber The Expression of Constitutively Active Isotypes of Protein Kinase C to Investigate Preconditioning J. Biol. Chem., September 4, 1998; 273(36): 23072 - 23079. [Abstract] [Full Text] [PDF]
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T. Sato, B. O'Rourke, and E. Marban Modulation of Mitochondrial ATP-Dependent K+ Channels by Protein Kinase C Circ. Res., July 13, 1998; 83(1): 110 - 114. [Abstract] [Full Text] [PDF]
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M. Miyamae, M. M. Rodriguez, S. A. Camacho, I. Diamond, D. Mochly-Rosen, and V. M. Figueredo Activation of varepsilon protein kinase C correlates with a cardioprotective effect of regular ethanol consumption PNAS, July 7, 1998; 95(14): 8262 - 8267. [Abstract] [Full Text] [PDF]
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Y. Liu, T. Sato, B. O'Rourke, and E. Marban Mitochondrial ATP-Dependent Potassium Channels : Novel Effectors of Cardioprotection? Circulation, June 23, 1998; 97(24): 2463 - 2469. [Abstract] [Full Text] [PDF]
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C. Weinbrenner, G. S Liu, J. M Downey, and M. V Cohen Cyclosporine A limits myocardial infarct size even when administered after onset of ischemia Cardiovasc Res, June 1, 1998; 38(3): 676 - 684. [Abstract] [Full Text] [PDF]
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B. S. Cain, D. R. Meldrum, X. Meng, B. D. Shames, A. Banerjee, and A. H. Harken Calcium Preconditioning in Human Myocardium Ann. Thorac. Surg., April 1, 1998; 65(4): 1065 - 1070. [Abstract] [Full Text] [PDF]
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A. Gysembergh, H. Margonari, J. Loufoua, A. Ovize, X. Andre-Fouet, Y. Minaire, and M. Ovize Stretch-induced protection shares a common mechanism with ischemic preconditioning in rabbit heart Am J Physiol Heart Circ Physiol, March 1, 1998; 274(3): H955 - H964. [Abstract] [Full Text] [PDF]
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H. Miyawaki, Y. Wang, and M. Ashraf Oxidant stress with hydrogen peroxide attenuates calcium paradox injury: role of protein kinase C and ATP-sensitive potassium channel Cardiovasc Res, March 1, 1998; 37(3): 691 - 699. [Abstract] [Full Text] [PDF]
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 H. Yokoshiki, M. Sunagawa, T. Seki, and N. Sperelakis ATP-sensitive K+ channels in pancreatic, cardiac, and vascular smooth muscle cells Am J Physiol Cell Physiol, January 1, 1998; 274(1): C25 - C37. [Abstract] [Full Text] [PDF]
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L. R.C Dekker Toward the heart of ischemic preconditioning Cardiovasc Res, January 1, 1998; 37(1): 14 - 20. [Full Text] [PDF]
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D. M Yellon, G. F Baxter, D. Garcia-Dorado, G. Heusch, and M. S Sumeray Ischaemic preconditioning: present position and future directions Cardiovasc Res, January 1, 1998; 37(1): 21 - 33. [Abstract] [Full Text] [PDF]
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J. Starkopf, T. V Andreasen, E. Bugge, and K. Ytrehus Lipid peroxidation, arachidonic acid and products of the lipoxygenase pathway in ischaemic preconditioning of rat heart Cardiovasc Res, January 1, 1998; 37(1): 66 - 75. [Abstract] [Full Text] [PDF]
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J.-F. Bouchard and D. Lamontagne Protection afforded by preconditioning to the diabetic heart against ischaemic injury Cardiovasc Res, January 1, 1998; 37(1): 82 - 90. [Abstract] [Full Text] [PDF]
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M. O. Gray, J. S. Karliner, and D. Mochly-Rosen A Selective epsilon -Protein Kinase C Antagonist Inhibits Protection of Cardiac Myocytes from Hypoxia-induced Cell Death J. Biol. Chem., December 5, 1997; 272(49): 30945 - 30951. [Abstract] [Full Text] [PDF]
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Y. Liu, W. D. Gao, B. O'Rourke, and E. Marban Priming effect of adenosine on KATP currents in intact ventricular myocytes: implications for preconditioning Am J Physiol Heart Circ Physiol, October 1, 1997; 273(4): H1637 - H1643. [Abstract] [Full Text] [PDF]
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H. Miyawaki and M. Ashraf Ca2+ as a Mediator of Ischemic Preconditioning Circ. Res., June 19, 1997; 80(6): 790 - 799. [Abstract] [Full Text]
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A. Babenko and G. Vassort Enhancement of the ATP-Sensitive K+ Current by Extracellular ATP in Rat Ventricular Myocytes : Involvement of Adenylyl Cyclase–Induced Subsarcolemmal ATP Depletion Circ. Res., April 19, 1997; 80(4): 589 - 600. [Abstract] [Full Text]
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R. M. Shaw and Y. Rudy Electrophysiologic Effects of Acute Myocardial Ischemia: A Mechanistic Investigation of Action Potential Conduction and Conduction Failure Circ. Res., January 1, 1997; 80(1): 124 - 138. [Abstract] [Full Text]
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P. E. Light, A. A. Sabir, B. G. Allen, M. P. Walsh, and R. J. French Protein Kinase C–Induced Changes in the Stoichiometry of ATP Binding Activate Cardiac ATP-Sensitive K+ Channels: A Possible Mechanistic Link to Ischemic Preconditioning Circ. Res., September 1, 1996; 79(3): 399 - 406. [Abstract] [Full Text]
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C. Vahlhaus, R. Schulz, H. Post, R. Onallah, and G. Heusch No Prevention of Ischemic Preconditioning by the Protein Kinase C Inhibitor Staurosporine in Swine Circ. Res., September 1, 1996; 79(3): 407 - 414. [Abstract] [Full Text]
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T. Mizumura, J. A. Auchampach, J. Linden, R. F. Bruns, and G. J. Gross PD 81,723, an Allosteric Enhancer of the A1 Adenosine Receptor, Lowers the Threshold for Ischemic Preconditioning in Dogs Circ. Res., September 1, 1996; 79(3): 415 - 423. [Abstract] [Full Text]
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D. K. Arrell, I. Neverova, H. Fraser, E. Marban, and J. E. Van Eyk Proteomic Analysis of Pharmacologically Preconditioned Cardiomyocytes Reveals Novel Phosphorylation of Myosin Light Chain 1 Circ. Res., September 14, 2001; 89(6): 480 - 487. [Abstract] [Full Text] [PDF]
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J. Minners, L. Lacerda, J. McCarthy, J. J. Meiring, D. M. Yellon, and M. N. Sack Ischemic and Pharmacological Preconditioning in Girardi Cells and C2C12 Myotubes Induce Mitochondrial Uncoupling Circ. Res., October 26, 2001; 89(9): 787 - 792. [Abstract] [Full Text] [PDF]
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