© 1999 American Heart Association, Inc.
Original Contribution |
Isoform-Selective Activation of Protein Kinase C by Nitric Oxide in the Heart of Conscious Rabbits
A Signaling Mechanism for Both Nitric Oxide–Induced and Ischemia-Induced Preconditioning
From the Experimental Research Laboratory (P.P., H.T., J.Z., X-L.T., Y.Q., R.C.X.L., S.B., B.D., Z.B., R.B), Division of Cardiology, and the Department of Physiology and Biophysics (P.P., Z.B., R.B.), University of Louisville, and Jewish Hospital Heart and Lung Institute, Louisville, Ky.
Correspondence to Peipei Ping, PhD, 511 S Floyd St, MDR Bldg, Room 526, Departments of Physiology and Biophysics, Medicine/Division of Cardiology, University of Louisville, Louisville, KY 40202. E-mail ping{at}ntr.net
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Abstract—Although isoform-selective translocation of protein kinase C (PKC)
appears
to play an important role in the late phase of ischemic
preconditioning (PC), the mechanism(s) responsible for such
translocation remains unclear. Furthermore, the signaling pathway that
leads to the development of late PC after exogenous administration of
NO in the absence of ischemia (NO donor–induced late PC) is
unknown. In the present study we tested the hypothesis that NO
activates PKC and that this is the mechanism for the
development of both ischemia-induced and NO donor–induced late
PC. A total of 95 chronically instrumented, conscious rabbits were
used. In rabbits subjected to ischemic PC (six 4-minute
occlusion/4-minute reperfusion cycles), administration of the NO
synthase inhibitor
N
-nitro-L-arginine (group
III), at doses previously shown to block the development of late PC,
completely blocked the ischemic PC–induced translocation of
PKC but not of PKC
, indicating that increased formation of NO is
an essential mechanism whereby brief ischemia activates
the isoform of PKC. Conversely, a translocation of PKC and -
quantitatively similar to that induced by ischemic PC could be
reproduced pharmacologically with the administration of 2 structurally
unrelated NO donors, diethylenetriamine/NO (DETA/NO) and
S-nitroso-N-acetylpenicillamine (SNAP),
at doses previously shown to elicit a late PC effect. The particulate
fraction of PKC increased from 35±2% of total in the control group
(group I) to 60±1% after ischemic PC (group II)
(P<0.05), to 54±2% after SNAP (group IV)
(P<0.05) and to 52±2% after DETA/NO (group V)
(P<0.05). The particulate fraction of PKC rose from
66±5% in the control group to 86±3% after ischemic PC
(P<0.05), to 88±2% after SNAP
(P<0.05) and to 85±1% after DETA/NO
(P<0.05). Neither ischemic PC nor NO donors had
any appreciable effect on the subcellular distribution of PKC
,
-ß1, -ß2, -
, -
, -µ, or -
/
; on total PKC activity; or
on the subcellular distribution of total PKC activity. Thus, the
effects of SNAP and DETA/NO on PKC closely resembled those of
ischemic PC. The DETA/NO–induced translocation of PKC (but
not that of PKC) was completely prevented by the administration of
the PKC inhibitor chelerythrine at a dose of 5 mg/kg (group
VI) (particulate fraction of PKC, 38±4% of total,
P<0.05 versus group V; particulate fraction of PKC,
79±2% of total). The same dose of chelerythrine completely prevented
the DETA/NO–induced late PC effect against both myocardial stunning
(groups VII through X) and myocardial infarction (groups XI through
XV), indicating that NO donors induce late PC by activating PKC and
that among the 10 isozymes of PKC expressed in the rabbit heart, the
isotype is specifically involved in the development of this form of
pharmacological PC. In all groups examined (groups I through VI), the
changes in the subcellular distribution of PKC protein were
associated with parallel changes in PKC isoform–selective activity,
whereas total PKC activity was not significantly altered. Taken
together, the results provide direct evidence that isoform-selective
activation of PKC is a critical step in the signaling pathway
whereby NO initiates the development of a late PC effect both after an
ischemic stimulus (endogenous NO) and after
treatment with NO-releasing agents (exogenous NO). To our knowledge,
this is also the first report that NO can activate PKC in the
heart. The finding that NO can promote isoform-specific activation of
PKC identifies a new biological function of this radical and a new
mechanism in the signaling cascade of ischemic PC and may also
have important implications for other
pathophysiological conditions in which NO is
involved and for nitrate therapy.
Key Words: diethylenetriamine • nitric oxide • S-nitroso-N-acetylpenicillamine • N-nitro-L-arginine • protein kinase C isoform • translocation
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The late phase of ischemic preconditioning (PC) is the phenomenon whereby a brief ischemic stress increases the tolerance of the heart to myocardial stunning and infarction 24 to 72 hours later.1 2 3 4 5 6 7 8 9 10 11 12 13 Because of its sustained nature, this endogenous cardioprotective mechanism may have considerable clinical relevance and is the focus of intense investigation.12 13 Recent studies8 10 14 have identified a critical role of NO as a trigger for the development of the late phase of ischemic PC. Specifically, it has been demonstrated that administration of NO synthase (NOS) inhibitors during the initial ischemic stimulus abolishes late PC against both myocardial stunning8 and myocardial infarction10 and, conversely, that administration of NO donors in the absence of ischemia can fully reproduce the cardioprotective effects of the late phase of ischemic PC.14 These results support the concept that enhanced biosynthesis of NO during the first ischemic stress mobilizes an adaptive mechanism that culminates in increased resistance to ischemic injury 24 to 72 hours later. However, the intracellular signaling events that are activated by NO and the molecular mechanisms whereby this radical triggers late PC remain unknown. Elucidation of the signaling pathway(s) that transduces augmented NO formation into cardioprotection is essential not only to unravel the cellular mechanism of ischemic PC but also to develop clinical therapies that can reproduce this phenomenon with PC-mimetic drugs.
One of the better-characterized intracellular signaling events during
ischemic PC is the activation of protein kinase C
(PKC).15 16 Considerable evidence supports the involvement
of this family of enzymes in both the early and the late phases of
ischemic PC.6 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 Using the same rabbit model
in which NO has been shown to trigger late PC,8 10 14 we
have found that the initial ischemic stimulus induces selective
translocation of 2 novel PKC isoforms ( and ) from the cytosolic
to the particulate fraction32 and that inhibition of
isoform translocation with the PKC inhibitor chelerythrine
results in abrogation of late PC,33 indicating that the
development of this cardioprotective phenomenon involves a
PKC-mediated signaling pathway. Since both NO and PKC are necessary
for the occurrence of the late phase of ischemic PC, it is
plausible to postulate that NO may evoke the late PC response via
activation of PKC. At present, however, virtually nothing is known
regarding the role of PKC in NO signaling in the heart, not only in the
setting of ischemia, but also in general. Specifically, no
information is available regarding (1) whether NO can activate
PKC in the myocardium, either in the absence or in the
presence of ischemia; (2) if so, whether such PKC
activation is generalized or restricted to selected isoforms within the
PKC family; and (3) whether activation of PKC is a necessary
signaling step in the development of NO-induced late PC. Because of the
ubiquitous functions of NO, demonstration of NO-induced activation of
PKC in the heart would have mechanistic implications not only for
ischemic PC but also for many other
pathophysiological processes that are modulated by
NO.
In the present study, we tested the central hypothesis that NO activates PKC and that this is the mechanism for the development of both ischemia-induced and NO donor–induced late PC. The 4 specific goals were to determine (1) whether inhibitors of NOS (at doses known to block ischemic PC) block ischemic PC–induced translocation of PKC; (2) whether administration of NO donors (at doses known to induce late PC) induces isoform-selective translocation of PKC in a manner congruous with that observed during ischemic PC; (3) whether NO donor–induced translocation of PKC isozymes can be blocked by in vivo administration of PKC inhibitors; and (4) if so, whether the same doses of PKC inhibitors also block NO donor–induced late PC. To achieve these goals, a comprehensive study consisting of 4 consecutive phases was designed to interrogate the mechanism of NO-dependent signaling in a conscious rabbit model of late PC by combining direct measurements of PKC activity and subcellular distribution in cardiac tissue with physiological measurements of protection against both myocardial stunning and myocardial infarction in the intact animal.
In phase I, we first examined the effect of the NOS
inhibitor
N-nitro-L-arginine (L-NA) on
ischemic PC–induced translocation of PKC and -. Having
established that L-NA blocks this translocation, in phase II we
explored whether administration of NO donors, in the absence of
ischemia, mimics the translocation of PKC and - induced
by ischemia. To achieve this goal, the effect of NO donors on
total myocardial PKC activity, as well as on the subcellular
distribution of all 10 PKC isoforms expressed in the adult rabbit
myocardium,32 was systematically
examined. In an effort to exclude nonspecific effects of NO donors, 2
structurally unrelated agents, diethylenetriamine/NO (DETA/NO) and
S-nitroso-N-acetylpenicillamine (SNAP), were
investigated. The effect of the specific PKC inhibitor
chelerythrine on DETA/NO–induced translocation of PKC and - was
also determined. To verify that the translocated proteins were in
their active state, measurements of protein subcellular
distribution were combined with measurements of isoform–selective
phosphorylation activity. Having obtained direct
evidence that NO donors activate PKC and that this is blocked
by chelerythrine in vivo, in the subsequent 2 phases (III and IV) we
investigated whether activation of PKC plays a causative role in the
development of the cardioprotection or is simply an epiphenomenon. To
achieve this goal, we determined whether the same dose of chelerythrine
that blocks NO-dependent activation of PKC also blocks NO-dependent
cardioprotection against stunning (phase III) and infarction (phase
IV). The experiments were conducted in the same rabbit model in which
previous studies have demonstrated both the role of NO and that of
PKC and - in the late phase of ischemic
PC.8 10 14 32 33 This enabled us to correlate the
present results with those prior studies.8 10 14 32 33
A conscious-animal preparation was used in all studies to avoid the
confounding influence of factors associated with open-chest
preparations (anesthesia, surgical trauma, fluctuations in
temperature, excessive catecholamine levels, abnormal
hemodynamics, exaggerated generation of reactive oxygen
species, release of cytokines, etc34 35 36 37 38 39 40 ),
which might interfere with NO production or PKC activity.
The results demonstrate, for the first time, that the activation
of PKC associated with ischemic PC is prevented by
inhibition of endogenous NO production, that a
similar activation of PKC is elicited by exogenous NO in the absence
of ischemia, and that inhibition of PKC activation
completely abrogates the development of NO donor–induced late PC
against both stunning and infarction. These data provide direct
evidence supporting the concept that NO preconditions the heart by
activating the isoform of PKC and that NO-dependent activation of
PKC is an essential step in the cellular signaling cascade
underlying the late phase of ischemic PC.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This study was performed in accordance with the guidelines of the Animal Care and Use Committee of the University of Louisville School of Medicine (Louisville, Ky) and with the Guide for the Care and Use of Laboratory Animals (Department of Health and Human Services, National Institutes of Health, Publication No. 86-23). The conscious rabbit model of ischemic PC has been described in detail previously7 8 9 10 11 14 33 41 and will be briefly summarized here.
Experimental Preparation
New Zealand White male rabbits (2.0 to 2.5 kg, age 3 to 4
months) were instrumented under sterile conditions with a balloon
occluder around a major branch of the left coronary artery, a
10-MHz pulsed Doppler ultrasonic crystal in the center of the
region to be rendered ischemic, and bipolar ECG leads on the
chest wall. The chest wound was closed in layers, and a small tube was
left in the thorax for 3 days to aspirate air and fluids
postoperatively. Gentamicin was administered before surgery and on the
1st and 2nd postoperative days (0.7 mg/kg IM each day). The animals
were allowed to recover for a minimum of 10 days after surgery.
Experimental Protocol
Throughout the experiments, rabbits were kept in a cage in
a quiet, dimly lit room. Left ventricular (LV)
systolic wall thickening (WTh), range gate depth, and
ECG were recorded on a thermal array chart recorder (Gould
TA6000). Coronary artery occlusion was produced by inflating
the balloon occluder. The performance of successful
coronary occlusions was verified by observing the development
of ST-segment elevation and changes in the QRS complex on the ECG and
the appearance of paradoxical systolic wall thinning on the
ultrasonic crystal recordings. Successful reperfusion was
documented by the normalization of the ECG and by the resumption of
active systolic WTh. The experimental protocol is illustrated
in Figures 1 through 3
. The study consisted of 4 successive phases (phases I through
IV).
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Phase I: Effect of L-NA on Ischemic PC–Induced PKC
Translocation
The goal of phase I was to determine whether the translocation
of PKC and - induced by ischemic PC is blocked by the NOS
inhibitor L-NA. Rabbits were assigned to 3 groups
(Figure 1
). Group I (nonischemic control) did not receive
any treatment and did not undergo coronary occlusion. At 10 to
14 days after surgery (time corresponding to the interval elapsed
between instrumentation and euthanasia in the other groups), the
rabbits were given heparin (1000 units intravenously),
after which they were anesthetized with sodium pentobarbital
(50 mg/kg intravenously) and euthanized with a bolus of
KCl. The heart was immediately excised, and myocardial samples (
0.5
g) were rapidly removed from the anterior LV wall and stored in liquid
nitrogen until used. Group II (ischemic PC) underwent a
sequence of six 4-minute coronary occlusion/4-minute
reperfusion cycles without any treatment. This protocol induces late PC
against both myocardial stunning8 9 14 33 and myocardial
infarction.10 11 14 Group III (ischemic PC+L-NA)
underwent the same sequence of occlusion/reperfusion cycles and
received an intravenous infusion of L-NA at a rate of 1.3
mg/kg per minute for 10 minutes, starting 20 minutes before and ending
10 minutes before the first coronary occlusion (total dose, 13
mg/kg). This dose of L-NA has previously been shown to block the
development of late PC against stunning8 and
infarction10 in conscious rabbits. L-NA (Sigma) was
dissolved in normal saline (total volume infused, 20 mL). No sedative
or antiarrhythmic agents were given at any time. In both groups II and
III, the rabbits were euthanized 5 minutes after the last reperfusion
(a time point at which marked translocation of PKC has previously been
found in this model32 ). The heart was immediately excised,
and myocardial samples (0.5 g) were rapidly removed from the
ischemic-reperfused region (the boundaries of which had been
marked with sutures at the time of instrumentation) and stored in
liquid nitrogen until used.
Phase II: Effect of NO Donors on PKC
The goal of phase II was to determine whether administration of
NO donors, in the absence of ischemia, induces translocation of
PKC similar to that induced by ischemic PC and whether such
translocation can be prevented by PKC inhibitors. Rabbits
were assigned to 3 groups (Figure 1). Group IV (SNAP) received a
continuous intravenous infusion of SNAP (2.5 µg/kg/min)
for 75 minutes. The rabbits were euthanized 5 minutes after the end of
the infusion, and tissue samples were obtained as described above.
Group V (DETA/NO) received 4 consecutive intravenous
boluses of DETA/NO (0.1 mg/kg each) every 25 minutes (total dose, 0.4
mg/kg). The rabbits were euthanized 5 minutes after the last bolus, and
tissue samples were obtained as described above. These doses of SNAP
and DETA/NO have previously been shown to induce late PC against
myocardial stunning and infarction in conscious rabbits.14
SNAP (Sigma) was dissolved in normal saline (total volume infused,
11 mL); DETA/NO (Alexis Corp) was dissolved in PBS (total volume
infused, 4 mL). SNAP and DETA/NO were dissolved immediately before the
infusion; to remove oxygen from the solution, both the PBS and the
normal saline solutions were bubbled with nitrogen for at least 30
minutes before dissolving SNAP or DETA/NO. Rabbits in group VI
(DETA/NO+chelerythrine) were given the same dose of DETA/NO as in group
V; in addition, they were given an intravenous bolus of
chelerythrine (5 mg/kg) 5 minutes before the first DETA/NO injection.
This dose of chelerythrine was chosen because it has previously been
shown to be effective in abrogating the late phase of ischemic
PC against stunning and the concomitant translocation of PKC in
conscious rabbits.33 Chelerythrine chloride (Research
Biochemicals International) was dissolved in 2 mL of DMSO+2 mL of
normal saline (total volume infused, 2 mL). The rabbits were euthanized
5 minutes after the last bolus of DETA/NO, and tissue samples were
obtained as described above.
Phase III: Studies of Myocardial Stunning
The goal of phase III was to determine whether NO donor–induced
late PC against myocardial stunning is abrogated by the administration
of PKC inhibitors. The experimental protocol consisted of 3
consecutive days of coronary artery occlusions (days 1, 2, and
3, respectively); on each day, the rabbits were subjected to a sequence
of six 4-minute coronary occlusion/4-minute reperfusion cycles
(Figure 2). No sedative or antiarrhythmic agents were given at
any time. Rabbits were assigned to 4 groups (Figure 2). Group
VII (control) underwent the coronary occlusion/reperfusion
protocol on days 1, 2, and 3 without any treatment. In group VIII
(DETA/NO), rabbits received 4 consecutive intravenous
boluses of DETA/NO (0.1 mg/kg each) every 25 minutes (total dose, 0.4
mg/kg) 24 hours before the first sequence of coronary
occlusion/reperfusion cycles (this is the same dose that was used in
group V of phase II and in group XIII of phase IV). In group IX
(DETA/NO+chelerythrine), rabbits received the same dose of DETA/NO as
in group VIII; in addition, they were given an intravenous
bolus of chelerythrine (5 mg/kg) 5 minutes before the first DETA/NO
injection (this is the same dose that was used in group VI of phase II
and in group XIV of phase IV). In group X (chelerythrine), rabbits
received chelerythrine alone (5 mg/kg intravenously) 24
hours before the first sequence of occlusion/reperfusion cycles.
Phase IV: Studies of Myocardial Infarction
The goal of phase IV was to determine whether NO donor–induced
late PC against myocardial infarction is abrogated by the
administration of PKC inhibitors. The experimental protocol
consisted of a 30-minute coronary artery occlusion followed by
3 days of reperfusion. Diazepam was administered 20 minutes before the
onset of ischemia (4 mg/kg IP) to relieve the stress caused by
the coronary occlusion. No antiarrhythmic agents were given.
Rabbits were assigned to 5 groups (Figure 3). Group XI (control)
underwent the 30-minute occlusion with no PC protocol or drug
pretreatment. Group XII (ischemic PC) was preconditioned with a
sequence of six 4-minute coronary occlusion/4-minute
reperfusion cycles 24 hours before the 30-minute coronary
occlusion. Group XIII (DETA/NO) was given 4 consecutive
intravenous boluses of DETA/NO (0.1 mg/kg) every 25 minutes
(total dose, 0.4 mg/kg) 24 hours before the 30-minute coronary
occlusion. In group XIV (DETA/NO+chelerythrine), rabbits were given the
same dose of DETA/NO as in group XIII; in addition, they received an
intravenous bolus of chelerythrine (5 mg/kg) 5 minutes
before the first DETA/NO injection. Group XV (chelerythrine) was given
chelerythrine alone (5 mg/kg intravenously) 24 hours before
the 30-minute coronary occlusion. In phases III and IV, all
solutions used for intravenous injection (DETA/NO and
chelerythrine) were filtered through a 0.2 µm Millipore filter
to ensure sterility.
Tissue Sample Preparation
In phases I and II, frozen myocardial tissue samples were
powdered in a prechilled stainless steel mortar and pestle. Total
cellular proteins were obtained by glass-glass
homogenization of the powdered tissue in sample
buffer containing (in mmol/L) Tris-HCl (pH 7.5) 50, EDTA 5, EGTA
10, and benzamidine 10; (in µg/mL) phenylmethylsulfonyl
fluoride 50, aprotinin 10, leupeptin 10, and pepstatin A 10;
and 0.3% ß-mercaptoethanol.32 The cytosolic and
particulate portions of total cellular proteins were separated by a
30-minute centrifugation at 45 000g.
Protein concentration was determined by the method of Bradford
(Bio-Rad). Using similar methods, the cytosolic and particulate
fractions have been found to yield equivalent amounts of
proteins.32 To ensure the most accurate assessment of
PKC protein expression and to avoid any decay in PKC
phosphorylation activity, protein samples were
processed by either Western immunoblotting or
phosphorylation assays immediately after tissue sample
preparation. Each Western immunoblotting and activity
assay was performed in all 5 rabbits in each group.
PKC Western Immunoblotting Analysis
Assessment of PKC isoforms was conducted using standard SDS-PAGE
Western immunoblotting techniques, as previously
described.32 Briefly, 100 µg of proteins derived from
the homogenate or from the cytosolic fraction or the
particulate fraction of the homogenate was electrophoresed
on a 10% denaturing gel for 4 to 6 hours at 30 mA per gel. Proteins
were electroblotted onto nitrocellulose membranes (Amersham). Gel
transfer efficiency was carefully recorded by making photocopies of
membranes dyed with reversible Ponceau staining; gel retention was
determined by Coomassie blue staining.32 Adequate
background blocking was accomplished by incubating the nitrocellulose
membranes with 5% nonfat dry milk in Tris-buffered saline. Antibodies
against PKC isoforms , ß, , , 
, 
, /, and µ
(Transduction Laboratories); PKC isoforms ß1 and ß2 (Sigma); and
PKC isoforms and (Santa Cruz Biotechnology) were used to assess
the expression of each individual PKC isoform. The PKC
immunoblots were developed with the use of a
chemiluminescent system (enhanced chemiluminescence kit, Amersham). The
specificity of the PKC antibody binding was confirmed by the use of
recombinant PKC isoform peptides. Although the ß1, ß2, and
isoforms have identical molecular weight (80 kDa), the antibodies to
these isotypes have no detectable cross-reactivity with one
another.32 In view of the extremely high homology between
the sequences of PKC isoforms and 42 43 44 and
between the sequences of their antigenic peptides (A. Recupero,
Transduction Laboratories, personal communication, November
1997), the determinations of these 2 isoforms were combined; the
data presented are the average of the measurements obtained
with the and antibodies.
The PKC signals detected by immunoblotting and the corresponding records of Ponceau stains of nitrocellulose membranes were quantified by using an image-scanning densitometer (Personal PI, Molecular Dynamics). To ensure consistency in the data analysis, the cytosolic and particular fractions of all 5 tissue samples in each group were run on the same gel. Each immunoblotting experiment was performed in duplicate, and the results were averaged. In this study, valid comparisons among samples required that PKC isoform expression be normalized to total protein content. However, as elaborated previously,32 despite a careful attempt to achieve equal protein loading in all lanes of the gel, the total amounts of proteins transferred from each lane to the nitrocellulose membranes during blotting are rarely identical. Therefore, given the critical importance of quantifying PKC isoforms as accurately as possible, each PKC isoform signal was normalized to the corresponding Ponceau stain signal determined by densitometric analysis of the Ponceau stain record.32
Measurement of Total PKC Activity
Total PKC activity (the sum of the activities of all 10 isoforms
expressed in the rabbit heart32 ) was quantitated using a
PKC enzyme assay system (Amersham), as described
previously.32 In prior studies,32 we
performed pilot experiments using protein samples ranging from 5 to 150
µg and identified a window of linear relationship on the
dose-response curve where sample proteins ranged from 10 to 50 µg;
the optimal sample dose was found to be 25 µg of proteins. Therefore,
in the current experiments, 25 µg of proteins from either the
cytosolic or the particulate fraction were incubated with 0.2 µCi of
[-32P]ATP and (in mmol/L) ATP 0.1,
HEPES 2.3, MgCl2 5.5, and DTT 2.9; 2.3 µg/mL
phorbol 12-myristate 13-acetate (PMA); 28.8 µg/mL
L--phosphatidyl-L-serine; 86.5 µmol/L substrate peptide
(VRKRTLRRL); and 600 µg/mL lysine-rich histone type IIIS in 50
mmol/L Tris-HCl buffer (pH 7.5) for 15 minutes at 37°C. The reaction
was carried out both in the presence and in the absence of 1.15
mmol/L calcium acetate. The reaction was terminated by the addition of
stop solution (containing 300 mmol/L orthophosphoric acid). The
phosphorylated substrates were transferred to binding
paper, washed in 5% acetic acid, and counted with a ß scintillation
counter. Washing conditions were optimized to achieve low nonspecific
counts (<5% of total counts). Both calcium-dependent and
calcium-independent PKC activities were calculated from the specific
counts (total minus nonspecific). Each sample was assayed in
triplicate. Data are expressed as pmol of phosphate transferred per
minute per mg of sample proteins.
Measurement of PKC Isoform-Selective Activity
To specifically determine the phosphorylation
activity of the isoform of PKC, 50 µg of proteins from either the
cytosolic or the particulate fraction were immunoprecipitated overnight
with PKC isoform monoclonal antibodies (Upstate Biotechnology). The
immunoprecipitates were then subjected to a
phosphorylation assay using a PKC-selective
substrate (ERMRPRKRQGSVRRRV). The cytosolic and particulate fractions
of the tissue samples were prepared as previously
described.32 The optimal substrate concentration, 1
nmol/L, was determined from dose-response (substrate versus
phosphorylation activity) curves generated in pilot
experiments.
Measurement of Regional Myocardial Function
Regional myocardial function was assessed as systolic
thickening fraction using the pulsed Doppler probe, as previously
described.8 In the studies of myocardial stunning (phase
III), the total deficit of systolic WTh (an integrative
assessment of the overall severity of myocardial stunning) was
calculated by measuring the area comprised between the systolic
WTh-versus-time line and the baseline (100% line) during the 5-hour
recovery phase after the sixth reperfusion.8 9 14 33 41 In
the studies of myocardial infarction (phase IV), the total deficit of
systolic WTh was calculated by the same method during the
72-hour recovery phase after the 30-minute
occlusion.10 11 14 In all animals, measurements from at
least 10 beats were averaged at baseline, and from at least 5 beats at
all subsequent time points.
Measurement of Region at Risk and Infarct Size
At the conclusion of the protocol in phases III and IV, the
rabbits were given heparin (1000 units intravenously),
after which they were anesthetized with sodium pentobarbital
(50 mg/kg intravenously) and euthanized with KCl. The heart
was excised and the size of the ischemic-reperfused region
(region at risk) was determined by tying the coronary artery at
the site of the previous occlusion and by perfusing the aortic root for
2 minutes with a 5% solution of phthalo blue dye in normal
saline at a pressure of 70 mm Hg using a Langendorff
apparatus. The heart was then cut into 6 or 7 transverse
slices, which were incubated for 10 minutes at 37°C in a 1% solution
of triphenyltetrazolium chloride in
phosphate buffer (pH=7.4). All atrial and right ventricular
tissues were excised. In the studies of myocardial stunning (phase
III), the region at risk (which was identified by the absence of blue
dye) was separated from the rest of the left ventricle, and both
components were weighed. In the studies of myocardial infarction (phase
IV), the slices were weighed, fixed in a 10% neutral buffered
formaldehyde solution, and photographed (Nikon AF N6006).
Transparencies were projected onto a paper screen at a 10-fold
magnification, and the borders of the infarcted,
ischemic-reperfused, and nonischemic regions were
traced. The corresponding areas were measured by computerized
planimetry (Adobe Photoshop, version 4.0), and from these measurements
infarct size was calculated as a percentage of the region at
risk.10 11 14
Statistical Analysis
Data are reported as mean±SEM. In phases I and II, differences
among groups with respect to total PKC activity, PKC
isoform–selective activity, and subcellular distribution of individual
PKC isoforms were analyzed using a 1-way ANOVA. If the ANOVA
showed an overall difference, post hoc contrasts were performed with
Student t tests for unpaired data using the Bonferroni
correction.45 In phases III and IV, for intragroup
comparisons, hemodynamic variables and WTh were
analyzed by a 2-way repeated-measures ANOVA (time and day)
followed by Student t tests for paired data with the
Bonferroni correction.45 For intergroup comparisons,
data were analyzed by either a 1-way or a 2-way
repeated-measures (time and group) ANOVA, as appropriate, followed by
unpaired Student t tests with the Bonferroni
correction.45 The relationship between infarct size
and risk-region size was compared among groups with an ANCOVA using the
size of the risk region as the covariate.11 14 The
correlation between infarct size and risk-region size was assessed by
linear regression analysis using the least-squares method. All
statistical analyses were performed using the SAS software
system.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A total of 95 conscious rabbits were used in this study (15 for phase I, 15 for phase II, 20 for phase III, and 45 for phase IV).
Phase I: Effect of L-NA on Ischemic PC–Induced PKC
Translocation
Previous investigations have documented that the ischemic
PC protocol used in the present study (6 cycles of 4-minute
coronary occlusion/4-minute reperfusion) induces selective
translocation of PKC isoforms and -32 and that the
NOS inhibitor L-NA completely abolishes ischemic
PC.8 10 To determine whether generation of NO is
responsible for the ischemic PC-induced translocation of PKC,
in phase I of the present study we measured the subcellular
distribution of the and isoforms in control rabbits not
subjected to ischemic PC (group I) and in rabbits undergoing
the ischemic PC protocol (six 4-minute occlusion/4-minute
reperfusion cycles) in the absence (group II) or presence (group III)
of L-NA. A total of 15 conscious rabbits were instrumented for phase I.
Of these, 5 were assigned to group I (nonischemic control), 5
to group II (ischemic PC), and 5 to group III (ischemic
PC+L-NA group). All rabbits completed the protocol successfully.
As expected,32 a significant translocation of PKC and
- to the particulate fraction was observed in group II
(ischemic PC) (Figure 4). In
group III (ischemic PC+L-NA), however, the particulate fraction
of PKC was significantly (P<0.05) less than in group II
(ischemic PC) (32±1% versus 60±1% of total, respectively)
and was similar to that measured in group I (nonischemic
control) (35±2%) (Figure 4). Thus, administration of L-NA
completely blocked the translocation of the isoform induced by
ischemic PC. In contrast, L-NA did not attenuate the
translocation of the isoform associated with ischemic PC
(Figure 4). The particulate fraction of in group III
(ischemic PC+L-NA) (83±5% of total) did not differ
significantly from that in group II (ischemic PC) (86±3% of
total) and was significantly (P<0.05) greater than that in
group I (nonischemic control) (66±5% of total). Thus, despite
pretreatment with L-NA, the ischemic PC protocol still induced
significant translocation of the isoform to the particulate
fraction.
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As expected,32 ischemic PC (group II) had no
appreciable effect on total myocardial PKC activity (Table 1). Administration of L-NA during
ischemic PC also had no significant effect on total myocardial
PKC activity (Table 1). Both the calcium-stimulated and the
calcium-independent PKC activity remained unaltered in group III
(ischemic PC+L-NA) compared with groups I (nonischemic
control) and II (ischemic PC) (Table 1).
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Phase II: Effect of NO Donors on PKC
The results of phase I demonstrate that the NOS
inhibitor L-NA blocks the translocation of PKC induced
by ischemic PC, implying that this signaling event is caused by
generation of NO. However, the possibility of nonspecific actions of
L-NA cannot be ruled out. Furthermore, since NOS can produce both NO
and
O2–,46 47 48
inhibition of NOS may prevent PKC activation by preventing generation
of O2–
rather than generation of NO. Accordingly, in phase II we sought to
obtain direct evidence that NO in itself, without any increase in
O2–,
can activate PKC. To this end, we tested whether exogenous
NO, in the absence of ischemia, can mimic the effects of
ischemic PC on PKC. Since the ischemic PC–induced
translocation of the isoform of PKC is inhibited by
chelerythrine,33 we further tested whether the NO
donor–induced translocation of PKC can also be inhibited by
chelerythrine.
A total of 15 conscious rabbits were instrumented for phase II. Of these, 5 were assigned to group IV (SNAP), 5 to group V (DETA/NO), and 5 to group VI (DETA/NO+chelerythrine). All rabbits completed the protocol successfully.
Effect of NO Donors on the Subcellular Distribution of PKC Isoforms
and on Total PKC Activity
We have previously reported that the adult rabbit
myocardium expresses 10 isoforms of PKC (, ß1, ß2,
, , , , µ, , , and ).32 In the
present study, the subcellular distribution of all of these 10
isoforms was determined in groups I (nonischemic control), IV
(SNAP), and V (DETA/NO). Neither DETA/NO nor SNAP had any discernible
effect on the subcellular distribution of conventional PKCs (, ß1,
ß2, and ) (Figures 5 and 6). The proportion of isoform in the
particulate fraction increased significantly (P<0.05) after
SNAP treatment (Figure 5) but not after DETA/NO treatment
(Figure 6). The other atypical PKCs (/) were
unaffected by either SNAP (Figure 5) or DETA/NO (Figure 6).
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In contrast to the conventional and atypical PKC isoforms, the
subcellular distribution of PKC and -, 2 novel PKC isoforms, was
significantly altered by the NO donors. The expression of PKC in the
particulate fraction increased from 35±2% of total in untreated
control rabbits (group I) to 54±2% in SNAP-treated rabbits (group IV)
(Figure 5) and to 52±2% in DETA/NO–treated rabbits (group V)
(Figure 6). An example of an immunoblot illustrating
DETA/NO–induced translocation of PKC is presented in Figure 7. The expression of PKC in the
particulate fraction increased from 66±5% in control rabbits to
88±2% in SNAP-treated rabbits (group IV) (Figure 5) and to
85±1% in DETA/NO–treated rabbits (group V) (Figure 6). The
subcellular distribution of the remaining 2 novel PKC isoforms ( and
µ) was not affected by either SNAP (Figure 5) or DETA/NO
(Figure 6). Interestingly, the degree of translocation of the
and isoforms elicited by SNAP and DETA/NO was comparable with
that elicited by ischemic PC in group II (Figure 8).
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SNAP and DETA/NO did not produce any significant change in total
myocardial PKC activity, either in the presence of calcium and PMA or
in the presence of PMA alone (Table 1). The subcellular
distribution of PKC activity also remained unaltered after NO donor
treatment (Table 1).
Effect of Chelerythrine on DETA/NO–Induced Translocation of
PKC
Having established that DETA/NO induces translocation of PKC
and - isoforms, we examined whether this effect could be prevented
by PKC inhibitors. To this end, the subcellular
distribution of the and isoforms was compared in rabbits
receiving DETA/NO in the absence (group V) or in the presence (group
VI) of chelerythrine. In group VI (DETA/NO+chelerythrine), the
particulate fraction of PKC was significantly (P<0.05)
less than in group V (DETA/NO) (38±4% versus 52±2% of total,
respectively) and was similar to that measured in group I
(nonischemic control) (35±2%) (Figure 8). Thus,
administration of chelerythrine completely inhibited the translocation
of the isoform induced by DETA/NO. In contrast, chelerythrine was
less effective in attenuating the translocation of the isoform
associated with DETA/NO administration (Figure 8). The
particulate fraction of in group VI (DETA/NO+chelerythrine)
(79±2% of total) did not differ significantly from that in group V
(DETA/NO) (85±1% of total), and was significantly
(P<0.05) greater than that in group I (nonischemic
control) (66±5% of total). Thus, despite pretreatment with
chelerythrine, administration of DETA/NO still induced significant
translocation of the isoform to the particulate fraction.
DETA/NO (group V) had no appreciable effect on total myocardial PKC
activity (Table 1). Administration of chelerythrine in
conjunction with DETA/NO also had no significant effect on total
myocardial PKC activity (Table 1). Both the calcium-stimulated
and the calcium-independent PKC activity remained unaltered in group VI
(DETA/NO+chelerythrine) compared with groups I (nonischemic
control) and V (DETA/NO) (Table 1).
Effect of Ischemic PC and NO Donors on the
Isoform-Selective Activity of PKC
The finding that neither L-NA (Figure 4) nor chelerythrine
(Figure 5) affected the translocation of PKC is
consistent with the notion that PKC is the critical isoform
responsible for the development of late PC after
ischemia.33 Because measurements of total PKC
activity are not sensitive enough to detect activation of individual
isozymes,32 we performed additional studies to measure the
isoform-selective phosphorylation activity of PKC.
We found that concomitant with the translocation of the protein
(Figure 4), ischemic PC induced a significant increase
in PKC isoform–selective activity in the particulate fraction
(65.2±2.7% above control, Figure 9),
indicating that the translocated proteins were in their active
state. A similar increase in PKC isoform–selective activity in the
particulate fraction was observed after the administration of either
DETA/NO or SNAP (DETA/NO, 54.4±5.3% above control; SNAP, 48.1±4.9%
above control; Figure 9). Furthermore, the ischemic
PC–induced activation of PKC was blocked by L-NA, and the
DETA/NO–induced activation of PKC was blocked by chelerythrine
(Figure 9). Thus, in all groups examined (groups I through VI),
the changes in the subcellular distribution of the protein (Figures 4 through 6) were associated with parallel changes
in the isoform-selective phosphorylation activity of
the isozyme (Figure 9), whereas total PKC activity was not
significantly altered (Table 1).
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Phase III: Studies of Myocardial Stunning
Exclusions and Postmortem Analysis
Of the 20 rabbits instrumented for the studies of myocardial
stunning, 5 were assigned to group VII (control), 5 to group VIII
(DETA/NO), 5 to group IX (DETA/NO+chelerythrine), and 5 to group X
(chelerythrine). All of the animals completed the protocol
satisfactorily and were included in the data analysis.
Postmortem analysis showed that the size of the
occluded-reperfused vascular bed was similar in the 4 groups: 0.8±0.1
g (16.6±2.1%) in group VII, 0.8±0.1 g (17.8±1.8%) in group VIII,
0.9±0.2 g (18.5±2.5%) in group IX, and 0.9±0.1 g (18.8±1.2%) in
group X. Tissue staining with
triphenyltetrazolium chloride confirmed the
absence of infarction in all animals. In all rabbits, the ultrasonic
crystal was found to be at least 3 mm from the boundaries of the
ischemic-reperfused region.
Hemodynamic Variables
Twenty-four hours before day 1 (day 0), there were no significant
changes in arterial blood pressure or systolic WTh
at any time during or after the administration of DETA/NO,
DETA/NO+chelerythrine, or chelerythrine in groups VIII, IX, and X,
respectively (representative measurements are given in
Table 2). In groups VIII and X, heart
rate decreased slightly (16%) after the injection of chelerythrine
and returned to baseline values by 3 hours (Table 2). On days 1,
2, and 3, there were no appreciable differences in heart rate among the
4 groups, either during the sequence of coronary
occlusion/reperfusion cycles or during the 5-hour reperfusion period
(Tables 3 and 4).
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Regional Myocardial Function
Baseline systolic thickening fraction on days 1, 2,
and 3 averaged 34.1±5.5%, 32.7±4.9%, and 32.4±5.3%, respectively,
in group VII; 39.9±3.1%, 40.9±3.4%, and 39.6±3.0% in group VIII;
32.5±3.3%, 32.1±2.9%, and 33.0±3.3% in group IX; and 32.6±
2.6%, 32.3±3.1%, and 31.9±2.6% in group X. There were no
significant differences among groups VII, VIII, IX, and X on the same
day, or among different days within the same group. Furthermore, within
the same group there were no significant differences among days 1, 2,
and 3 with respect to the extent of paradoxical systolic
thinning during the 6 occlusions (Figures 10 through 13).
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Group VII (Control)
On day 1, thickening fraction remained significantly
(P<0.05) depressed for 4 hours after the sixth reperfusion
and recovered by 5 hours (Figure 10), indicating that the
sequence of six 4-minute occlusion/4-minute reperfusion cycles resulted
in severe myocardial stunning that lasted, on average, 4 hours. On days
2 and 3, however, the recovery of WTh after the 6 occlusion/reperfusion
cycles was markedly improved compared with day 1 (Figure 11).
The total deficit of WTh after the sixth reperfusion was 54% less on
both days 2 and 3 compared with day 1 (P<0.01) (Figure 14). Thus, as
expected,8 9 14 33 myocardial stunning was attenuated
markedly, and to a similar extent, on days 2 and 3 compared with day
1.
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Group VIII (DETA/NO)
On day 1, the recovery of WTh after the sixth reperfusion was
markedly faster in group VIII than in the control group, and this
improvement was sustained throughout the entire reperfusion interval
(Figure 11). The total deficit of WTh in group VIII was 57%
less than that observed in control rabbits on day 1
(P<0.05) and similar to that observed in control rabbits on
days 2 and 3 (Figure 14). On days 2 and 3, there was no further
improvement in either the recovery of WTh (Figure 11) or the
total deficit of WTh (Figure 14) compared with day 1. Thus, as
previously reported,14 administration of DETA/NO 24
hours before the sequence of six 4-minute occlusion/reperfusion cycles
resulted in an attenuation of myocardial stunning on day 1 that was
essentially equivalent to that effected by ischemic PC.
Group IX (DETA/NO+Chelerythrine)
On day 1, both the recovery of WTh (Figure 12) and the
total deficit of WTh (Figure 14) were similar to those noted in
the control group, indicating the absence of PC against stunning. Thus,
the PC effect induced by DETA/NO was completely abrogated by the
concomitant administration of chelerythrine. A PC effect became
apparent on days 2 and 3, as documented by the enhanced recovery of WTh
(Figure 12) and the reduced deficit of WTh (Figure 14)
compared with day 1.
Group X (Chelerythrine)
On day 1, both the recovery of WTh (Figure 13) and the
total deficit of WTh (Figure 14) were virtually
indistinguishable from those noted in the control group, indicating
that administration of chelerythrine did not exacerbate the severity of
myocardial stunning 24 hours later. On days 2 and 3, the recovery of
WTh was enhanced (Figure 14), and the deficit of WTh was
reduced (Figure 14) compared with day 1, indicating the
presence of a PC effect. These results indicate that the absence of a
DETA/NO–induced protective effect against stunning in group IX cannot
be ascribed to a delayed deleterious action of chelerythrine.
Phase IV: Studies of Myocardial Infarction
Exclusions and Arrhythmias
Of the 45 rabbits instrumented for the studies of myocardial
infarction, 10 were assigned to group XI (control), 10 to group XII
(ischemic PC), 9 to group XIII (DETA/NO), 9 to group XIV
(DETA/NO+chelerythrine), and 7 to group XV (chelerythrine). Six rabbits
died of ventricular fibrillation during coronary
occlusion (2 in group XI, 2 in group XII, 1 in group XIII, and 1 in
group XIV) and 1 after reperfusion (in group XIV). Therefore, a total
of 8 rabbits completed the experimental protocol in group XI, 8 in
group XII, 8 in group XIII, 7 in group XIV, and 7 in group XV. No
rabbit included in the final analysis was subjected to
defibrillation.
Hemodynamic Variables
No significant changes in arterial blood pressure or
systolic WTh were observed at any time during or after the
administration of DETA/NO, DETA/NO+chelerythrine, or chelerythrine in
groups XI, XII, and XIII, respectively, 24 hours before day 1 (day 0)
(representative measurements are given in Table 2). In groups XIII and XIV, heart rate decreased slightly
(16%) after the injection of chelerythrine and returned to baseline
values by 3 hours (Table 2). There were no appreciable
differences in heart rate among groups XI, XII, XIII, XIV, and XV,
either during the 30-minute coronary occlusion or during the
72-hour reperfusion period (Tables 3 and 4). Baseline
systolic thickening fraction was also similar among the 6
groups (31.3±3.4%, 34.2±4.1%, 33.2±3.6%, 34.1±2.7%, and
35.6±2.8% in groups XI, XII, XIII, XIV, and XV, respectively).
Region at Risk and Infarct Size
There were no significant differences among groups XI, XII, XIII,
XIV, and XV with respect to the weight of the region at risk (0.7±0.1
g [15.7±1.2% of LV weight], 0.8±0.1 g [17.0±1.7%], 0.7±0.1 g
[16.2±1.9%], 0.8±0.1 g [17.3±2.2%], and 0.8±0.1 g
[19.3±1.4%], respectively). The average infarct size was 46%
smaller in group XII (ischemic PC) compared with group XI
(control) (58.4±3.7% versus 31.3±3.0% of the region at risk,
respectively; P<0.05 [Figure 15]), indicating a late PC effect
against myocardial infarction. A quantitatively similar PC effect was
observed in group XIII (DETA/NO); the average infarct size in this
group (28.7±3.8% of the region at risk) was similar to that measured
in group XII and significantly (P<0.05) smaller than that
measured in group XI (Figure 15), indicating that pretreatment
with this NO donor 24 hours before the 30-minute occlusion resulted in
a protective effect that was equivalent to that induced by
ischemic PC. In group XIV, infarct size was indistinguishable
from that measured in the control group and significantly
(P<0.05) larger than that measured in group XIII (Figure 15), indicating that chelerythrine completely blocked the
infarct-sparing effect of DETA/NO pretreatment. In group XV, infarct
size was similar to that measured in the control group, indicating that
chelerythrine did not exacerbate the severity of myocardial infarction
24 hours later and that the abrogation of the infarct-sparing effect of
DETA/NO could not be ascribed to a delayed deleterious action of
chelerythrine.
|
represents individual rabbit;
In all groups, the size of the infarction was positively and linearly
related to the size of the region at risk (r=0.93 in group
XI, 0.79 in group XII, 0.72 in group XIII, 0.82 in group XIV, and 0.84
in group XV) (Figure 16). As expected,
the regression line was shifted to the right in the ischemic PC
group (group XII) as compared with the control group (group XI,
P<0.05 by ANCOVA) (Figure 16). In the DETA/NO group
(group XIII), the regression line was virtually indistinguishable from
that of the ischemic PC group and was significantly shifted to
the right compared with the control group (P<0.05 by
ANCOVA) (Figure 16), indicating that for any given size of the
region at risk, the resulting infarct size was reduced by pretreatment
with DETA/NO and that the magnitude of this effect was similar to that
induced by ischemic PC. In contrast, in the
DETA/NO+chelerythrine group (group XIV) and in the chelerythrine group
(group XV), the regression line did not differ from that observed in
the control group (Figure 16).
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Regional Myocardial Function
Because of Doppler probe malfunction, complete measurements of
WTh for 3 days after reperfusion could be obtained in only 6 of 8
rabbits in group XI, 6 of 8 rabbits in group XII, 5 of 8 rabbits in
group XIII, 5 of 7 rabbits in group XIV, and 5 of 7 rabbits in group
XV. After release of the 30-minute occlusion, control rabbits (group
XI) exhibited essentially no recovery of WTh, even at 3 days (Figure 17). In rabbits preconditioned with
ischemia (group XII), the recovery of WTh was significantly
(P<0.05) improved compared with controls at 3, 5, 24, and
72 hours after reperfusion (Figure 17). The total deficit of
WTh during the 72 hours of reperfusion was 12% less in group XII than
in group XI (P<0.05) (Figure 17). In the DETA/NO
group (group XIII), the recovery of WTh was similar to that noted in
the rabbits preconditioned with ischemia and significantly
(P<0.05) improved compared with controls at 1 hour and 3,
5, 24, and 72 hours after reperfusion (Figure 17). The total
deficit of WTh during the 72-hour reperfusion period was 12% less in
NO donor–pretreated rabbits than in control rabbits
(P<0.05) (Figure 17). Thus, pretreatment with an NO
donor resulted in enhanced recovery of myocardial contractile function,
which became evident soon after reperfusion (1 hour) and was sustained
throughout the 72-hour reperfusion interval; the magnitude of this
effect was similar to that observed after ischemic PC in group
XII. In the DETA/NO+chelerythrine group, both the recovery of WTh and
the total deficit of WTh were similar to those observed in the control
group (Figure 17), indicating that the salutary actions of NO
donor pretreatment on the recovery of myocardial function were
abrogated by the concomitant administration of chelerythrine. The
results obtained in the chelerythrine group (XV) were similar to those
in the control group, indicating that chelerythrine did not exacerbate
myocardial dysfunction after the 30-minute occlusion 24 hours later.
Because the effects of ischemic PC, DETA/NO, and chelerythrine
on WTh paralleled those on infarct size (Figure 15), the
WTh data provide an independent confirmation of the results obtained
with tetrazolium staining.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Although isoform-selective activation of PKC
appears to play a
critical role in the genesis of the late phase ("second window") of
ischemic PC,32 33 the mechanism(s) responsible for
such activation remains unclear. Furthermore, the signal transduction
pathway that leads to the development of late PC after administration
of NO-releasing agents in the absence of ischemia (NO
donor–induced late PC) is unknown. The present study provides
significant new insights into these issues. First, in the conscious
rabbit, administration of the NOS inhibitor L-NA (at doses
previously shown to block the development of late PC8 10 )
completely blocks the ischemic PC–induced increase in PKC
protein content and the concomitant increase in PKC-selective
activity in the particulate fraction, indicating that increased
formation of NO is an essential mechanism whereby brief
ischemia activates the isoform of PKC. Second, a
translocation of PKC and - proteins and an increase in PKC
activity quantitatively similar to those induced by ischemic PC
can be reproduced pharmacologically with the administration of 2
structurally unrelated NO donors (at hemodynamically
inactive doses previously shown to elicit a late PC
effect14 ), demonstrating that NO in itself (in the absence
of the other cellular perturbations associated with ischemia)
can trigger PKC activation in the heart. Third, in vivo administration
of the PKC inhibitor chelerythrine at 5 mg/kg completely
prevents the DETA/NO–induced translocation and activation of the
isoform but not the translocation of the isoform, indicating that
chelerythrine is a useful tool for interrogating the role of the
isoform of PKC in NO donor–induced late PC. Finally, the same dose of
chelerythrine that blocks the DETA/NO–induced activation of PKC
also blocks the DETA/NO–induced late PC effect against both myocardial
stunning and infarction, indicating that the isozyme plays a
pivotal role in the genesis of the delayed cardioprotection elicited by
NO-releasing agents.
Taken together, the results of the present investigation provide
direct evidence, for the first time, that isoform-selective activation
of PKC is a critical signaling step in the cellular pathway whereby
NO initiates the development of a late PC effect, both after an
ischemic stimulus (endogenous NO) and after
NO-donor treatment (exogenous NO). To our knowledge, this is also the
first report that NO can activate PKC in the heart.
To assess the actions of NO on PKC, we used two different approaches, ie, we examined the role of PKC in NO donor–induced late PC and, conversely, the effect of endogenous NO formed during ischemic PC on PKC. These two approaches will be discussed separately.
Isoform-Selective Activation of PKC by NO
Despite the multiplicity of NO-dependent process in the
cardiovascular system, essentially nothing is known
regarding the influence of NO on PKC in the heart. To determine whether
NO in itself (in the absence of ischemia) affects PKC, we
analyzed the influence of exogenous NO on total myocardial PKC
activity, on PKC isoform–selective activity, and on the subcellular
distribution of all 10 PKC isoforms expressed in the adult rabbit
myocardium.32 Two different NO donors were
tested. DETA/NO is a relatively long-acting NO donor that spontaneously
and nonenzymatically releases NO with predictable first-order
kinetics.49 50 51 SNAP is a direct NO donor that lacks
tolerance-producing effects.52 53 The fact that the
effects of DETA/NO and SNAP were similar makes it very unlikely that
they may have been caused by nonspecific, NO-independent actions of
these molecules. By analyzing the changes in PKC after the
administration of the same doses of DETA/NO and SNAP that induce late
PC against myocardial stunning and infarction,14 we were
able to correlate the effects of NO donors on PKC isoforms with their
effects on the development of delayed cardioprotection. It is important
to stress that the doses of DETA/NO and SNAP used in this study have no
hemodynamic effects,14 thus excluding
indirect actions secondary to hemodynamic
perturbations.
Both DETA/NO and SNAP induced significant translocation of two novel
PKC isoforms, and , but neither of them significantly altered
the subcellular distribution of any of the other PKC isoforms (,
ß1, ß2, , , µ, and /) (Figures 5 and 6). Measurements of PKC isoform–selective activity confirmed
that the translocated protein was activated (Figure 9). SNAP, but not DETA/NO, also induced significant
translocation of PKC (Figures 5 and 6). Thus, the
effects of NO donors were quite selective, being restricted to only 3
of the 10 isozymes of PKC expressed in the rabbit heart. Despite their
effects on PKC, -, and -, the NO donors had no significant
effect on total myocardial PKC activity, either calcium-stimulated or
calcium-independent (Table 1). This result is consistent
with previous reports from our laboratory32 and
others54 55 indicating that ischemic PC can occur
without demonstrable changes in total PKC activity. Specifically, using
the same conscious rabbit model used herein, we have observed that
translocation of PKC and - after ischemic PC was not
reflected in changes in total myocardial PKC activity.34
This is not surprising, given that PKC and - account for a small
fraction (<13%) of all PKC-isoform proteins expressed in the rabbit
heart.32
In conclusion, the results obtained with NO donors indicate that NO
induces isoform-selective activation of PKC, -, and - and that
measurements of total PKC activity are not sufficiently sensitive to
detect the involvement of the PKC system in NO signaling. Determination
of individual PKC isoform translocation and/or activity appears
therefore essential to assess the role of PKC in NO-dependent
processes.
Functional Significance of the Activation of PKC by NO
The translocation of PKC and - observed after administration
of DETA/NO and SNAP could be important for the development of NO
donor–induced late PC or, alternatively, could be an epiphenomenon. To
resolve this issue, we examined the effect of chelerythrine on the
translocation of PKC as well as on late PC after the administration of
DETA/NO. Chelerythrine was selected for this study because it is
reported to be more selective for PKC than other widely used
inhibitors, such as staurosporine and polymixin
B,19 and because it has been shown to block both the
early19 and the late6 33 phases of PC after
an ischemic stimulus. Chelerythrine is a very potent
inhibitor of PKC (IC50, 0.7
µmol/L) and reportedly has very high selectivity for PKC compared
with protein kinase A (250:1),
calcium/calmodulin–dependent protein kinase (150:1), or
tyrosine protein kinase (150:1).56 We found that
pretreatment with chelerythrine (at a dose of 5 mg/kg) completely
inhibited the DETA/NO–induced translocation of PKC (and the
associated increase in PKC activity) but not the DETA/NO–induced
translocation of PKC (Figures 8 and 9), indicating
that this agent could be used to investigate the role of the
isoform of PKC in NO donor–dependent late PC. The same dose of
chelerythrine that prevented the DETA/NO–induced activation of PKC
completely prevented the development of the delayed cardioprotection
against both myocardial stunning and infarction (Figures 12, 14, and 15), demonstrating that mobilization of PKC
is essential for NO donor–induced late PC. These results, obtained
after administration of exogenous NO (phases II through IV), are
congruent with the results obtained after blockade of
endogenous NO production with L-NA in the setting
of ischemic PC (phase I), which demonstrate that prevention of
PKC activation by L-NA results in abrogation of late PC (Figures 4 and 9).
Thus, taken together, the present observations demonstrate that the
isoform of PKC plays an obligatory role both in NO donor–induced
late PC (exogenous NO) and in ischemia-induced late PC
(endogenous NO). Interestingly, recent studies by other
investigators have identified PKC activation as an important event
underlying ethanol-induced cardioprotection in isolated guinea pig
hearts27 and hypoxia-induced early PC in neonatal
rat myocytes,28 in agreement with our conclusions
regarding the role of isoform-selective activation of PKC during
ischemia- or NO donor–induced late PC. The downstream targets
of PKC-mediated phosphorylation remain to be
elucidated but may include, among others, tyrosine kinases
(specifically, Src and Lck57 ) and, in the case of early
PC, the mitochondrial KATP channel.58
Whether the isoform of PKC is also necessary for NO donor–induced
late PC remains to be determined. In the setting of ischemic
PC, previous studies33 have already demonstrated that
translocation of PKC is not required for late PC to occur. Our
finding that chelerythrine completely abrogated DETA/NO–induced late
PC despite the fact that the translocation of PKC was not prevented
(Figure 8) indicates that mobilization of PKC, in itself, is
not sufficient to produce the late PC effect after exposure to
exogenous NO. Nevertheless, the possibility that PKC might be
necessary for NO-induced PC, even though it is not sufficient, cannot
be ruled out. Manipulations that selectively block translocation
without affecting translocation will be needed to discern whether
PKC plays an obligatory role in the late PC effect induced by NO
donors.
The identification of PKC as a critical downstream effector of
NO-dependent actions has implications that transcend ischemic
PC. From a broader perspective, the finding that NO donor–induced PC
is PKC dependent suggests that PKC may play an important role in
NO-dependent signaling in the heart in general. Because NO is known to
modulate multiple physiological and pathological
processes,59 60 this finding may have considerable
implications not only for the mechanism of myocardial
ischemia/reperfusion injury but also for a host of other
conditions and for nitrate therapy.
Activation of PKC During Ischemic PC Is NO
Dependent
The observation that exogenous NO translocates PKC and - in
the absence of ischemia (Figures 5 through 7) is consistent with the hypothesis that ischemic
PC–induced activation of PKC is caused by the generation of NO, but
does not prove it. Although NO is sufficient to induce PKC
translocation, it may not be necessary, because other stimuli
associated with ischemic PC may also activate PKC
independently of NO.
To resolve this issue, in phase I of the present study we
determined whether inhibition of NO generation with L-NA results in
abrogation of PKC and - translocation after six 4-minute
occlusion/reperfusion cycles. We were particularly interested in PKC
because this appears to be the specific isoform responsible for the
development of the late phase of ischemic PC in conscious
rabbits.33 We reasoned that if NO plays an obligatory role
in the activation of the isoform associated with ischemic
PC, L-NA should block this phenomenon. If, on the other hand, redundant
signaling pathways exist whereby multiple triggers produced during the
ischemic stimulus can activate PKC independently of NO,
then blunting NO production with L-NA should have no effect.
The finding that L-NA completely prevented the activation of PKC
(Figures 4 and 9) demonstrates that NO formation is
necessary for this phenomenon to occur in the conscious rabbit model of
ischemic PC induced by six 4-minute occlusion/reperfusion
cycles. Thus, rather surprisingly, despite the fact that we used a
robust ischemic stimulus (six 4-minute occlusions) that should
cause the release of multiple activators of PKC, NO still
appears to be essential for the activation of PKC-dependent signaling
in this model.
It should be stressed, however, that the present results do not in any way exclude an important role of other mechanisms in the stimulation of PKC that occurs during ischemia. The concept that NO is necessary for PKC to be translocated in our model of ischemic PC is not in conflict with the existing body of evidence indicating that, in other experimental models, ischemic PC activates PKC via the release of different triggers (eg, adenosine, norepinephrine, bradykinin, and reactive oxygen species).12 15 16 61 62 63 64 65 It seems likely that PKC can be activated by several stimuli and that the predominant stimulus may vary depending on the animal model and the type of PC protocol used.16
NO donor–Induced PC Versus Ischemia-Induced PC
The signaling mechanisms underlying late PC after an
ischemic stimulus and late PC after administration of
NO-releasing agents have not been previously compared. The present
study provides direct evidence to support the concept that these 2
mechanisms are similar, at least at the level of PKC. The results of
phases I and II demonstrate that NO donor–induced PC and
ischemia-induced PC share a common signaling event, the
activation of PKC and -. The magnitude of the changes in PKC
and - elicited by DETA/NO and SNAP was similar to the magnitude of
the changes elicited by ischemic PC (Figures 8 and 9), again supporting a common mechanism. Similarly to
ischemic PC,32 DETA/NO and SNAP had no discernible
effect on the , ß1, ß2, , , µ, and / isoforms of
PKC (Figures 5 and 6). Thus, in general, the effects of
exogenous NO administration on the subcellular distribution of PKC
isoforms mimicked those of ischemic PC. The only difference
between NO donor–induced PC and ischemic PC was the
translocation of the isoform, which was observed in the former
(only after SNAP) (Figure 5) but not in the
latter.32 It seems probable, however, that the
translocation of PKC is not necessary for the genesis of late PC,
because it occurred after the administration of SNAP but not of DETA/NO
(Figures 5 and 6), whereas late PC occurs after
administration of either SNAP or DETA/NO (Figures 11, 14, and 15).14
The precise mechanism by which NO activates PKC is a complex
problem that will require extensive investigation. Many possibilities
(and combinations thereof) must be considered. NO can directly modulate
the functions of many proteins by reacting with heme groups,
sulfur-iron clusters, and thiol moieties.59 60 Thus, NO
could influence PKC activity directly or indirectly by binding to
proteins involved in PKC modulation (eg, receptor for activated C
kinases [RACKs] or phospholipases). Furthermore, cGMP is known
to be an important second messenger for NO-dependent
signaling.59 60 In addition, NO reacts very rapidly with
superoxide anion to form peroxynitrite (ONOO–),
which then can decompose to generate hydroxyl radical
(OH) or another oxidant with similar
reactivity.66 ONOO–,
OH, or their reactive byproducts could
activate PKC either directly15 67 or via
activation of phospholipase D.68 69 Each of these
mechanisms may contribute to PKC activation and will have to be
interrogated in both cellular and acellular systems with combined
molecular, cellular, and biochemical approaches.
Conclusions
The results of this study demonstrate that, in the conscious
rabbit, the activation of PKC associated with ischemic PC
requires NO generation and that a similar activation can be reproduced
by increasing NO availability in the absence of ischemia. Thus,
NO is both necessary and sufficient to produce the changes in PKC that
are induced by a brief ischemic stress. These findings support
the novel idea that NO formation represents an important
cellular signal whereby ischemic PC leads to the rapid
mobilization of the PKC system in the heart. Furthermore, the
present results demonstrate that PKC is a critical downstream
effector in the development of late PC induced pharmacologically with
NO donors and that among the 10 PKC isoforms expressed in the rabbit
heart, plays an obligatory role in the genesis of this phenomenon.
Thus, NO (either endogenous NO produced during
ischemia or exogenous NO generated during NO donor
administration) induces late PC by activating PKC. In both cases, the
specific isoform involved appears to be PKC. The finding that NO can
promote isoform-specific activation of PKC identifies a new biological
function of this radical and a new mechanism in the signaling cascade
of ischemic PC. Given that NO is an important modulator of many
biological processes, this finding has potentially wide implications
for numerous cardiovascular conditions besides
myocardial ischemia and also for nitrate therapy.
|
Acknowledgments |
|---|
This study was supported in part by NIH grants R29 HL-58166 (to P.P.), R01 HL-43151 (to R.B.), and HL-55757 (to R.B.); by Kentucky American Heart Association (AHA) Affiliate Award KY-96-GB-37 (to P.P.); by National AHA Award 9750721N (to P.P.); by Kentucky AHA Affiliate Postdoctoral Fellowship Awards KY-9804558 (to H.T.), KY-9804502 (to R.C.X.L.), and KY-9804556 (to B.D.); and by the Medical Research Grant Program of the Jewish Hospital Foundation (Louisville, Ky).
Received September 9, 1998; accepted December 18, 1998.
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Z. Xu, X. Ji, and P. G. Boysen Exogenous nitric oxide generates ROS and induces cardioprotection: involvement of PKG, mitochondrial KATP channels, and ERK Am J Physiol Heart Circ Physiol, April 1, 2004; 286(4): H1433 - H1440. [Abstract] [Full Text] [PDF]
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J. Tamargo, R. Caballero, R. Gomez, C. Valenzuela, and E. Delpon Pharmacology of cardiac potassium channels Cardiovasc Res, April 1, 2004; 62(1): 9 - 33. [Abstract] [Full Text] [PDF]
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 E. Murphy Primary and Secondary Signaling Pathways in Early Preconditioning That Converge on the Mitochondria to Produce Cardioprotection Circ. Res., January 9, 2004; 94(1): 7 - 16. [Abstract] [Full Text] [PDF]
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G. Abou-Mohamed, J. A. Johnson, L. Jin, A. B. El-Remessy, K. Do, W. H. Kaesemeyer, R. B. Caldwell, and R. W. Caldwell Roles of Superoxide, Peroxynitrite, and Protein Kinase C in the Development of Tolerance to Nitroglycerin J. Pharmacol. Exp. Ther., January 1, 2004; 308(1): 289 - 299. [Abstract] [Full Text] [PDF]
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D. Kumar, V. Menon, W. R. Ford, A. S. Clanachan, and B. I. Jugdutt Effect of Angiotensin II lype 2 Receptor Blockade on Activation of Mitogen-Activated Protein Kinases after Ischemia-Reperfusion in Isolated Working Rat Hearts Journal of Cardiovascular Pharmacology and Therapeutics, December 1, 2003; 8(4): 285 - 296. [Abstract] [PDF]
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M. Joyeux-Faure, C. Arnaud, D. Godin-Ribuot, and C. Ribuot Heat stress preconditioning and delayed myocardial protection: what is new? Cardiovasc Res, December 1, 2003; 60(3): 469 - 477. [Abstract] [Full Text] [PDF]
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D. M. YELLON and J. M. DOWNEY Preconditioning the Myocardium: From Cellular Physiology to Clinical Cardiology Physiol Rev, October 1, 2003; 83(4): 1113 - 1151. [Abstract] [Full Text] [PDF]
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J. Zhang, P. Ping, T. M. Vondriska, X.-L. Tang, G.-W. Wang, E. M. Cardwell, and R. Bolli Cardioprotection involves activation of NF-{kappa}B via PKC-dependent tyrosine and serine phosphorylation of I{kappa}B-{alpha} Am J Physiol Heart Circ Physiol, October 1, 2003; 285(4): H1753 - H1758. [Abstract] [Full Text] [PDF]
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B. I. Jugdutt and V. Menon Upregulation of Angiotensin II Type 2 Receptor and Limitation of Myocardial Stunning by Angiotensin II Type 1 Receptor Blockers during Reperfused Myocardial Infarction in the Rat Journal of Cardiovascular Pharmacology and Therapeutics, September 1, 2003; 8(3): 217 - 226. [Abstract] [PDF]
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G. J. Gross and J. N. Peart KATP channels and myocardial preconditioning: an update Am J Physiol Heart Circ Physiol, August 7, 2003; 285(3): H921 - H930. [Abstract] [Full Text] [PDF]
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 Y. Uchiyama, H. Otani, T. Okada, T. Uchiyama, H. Ninomiya, M. Kido, H. Imamura, S. Nakao, and K. Shingu Integrated pharmacological preconditioning in combination with adenosine, a mitochondrial KATP channel opener and a nitric oxide donor J. Thorac. Cardiovasc. Surg., July 1, 2003; 126(1): 148 - 159. [Abstract] [Full Text] [PDF]
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T. C. Zhao and R. C. Kukreja Protein kinase C-{delta} mediates adenosine A3 receptor-induced delayed cardioprotection in mouse Am J Physiol Heart Circ Physiol, June 5, 2003; 285(1): H434 - H441. [Abstract] [Full Text] [PDF]
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R. J. Henning and Y. Li Cocaine Produces Cardiac Hypertrophy by Protein Kinase C Dependent Mechanisms Journal of Cardiovascular Pharmacology and Therapeutics, June 1, 2003; 8(2): 149 - 160. [Abstract] [PDF]
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C. P. Baines, C.-X. Song, Y.-T. Zheng, G.-W. Wang, J. Zhang, O.-L. Wang, Y. Guo, R. Bolli, E. M. Cardwell, and P. Ping Protein Kinase C{epsilon} Interacts With and Inhibits the Permeability Transition Pore in Cardiac Mitochondria Circ. Res., May 2, 2003; 92(8): 873 - 880. [Abstract] [Full Text] [PDF]
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X.-L. Tang, E. Kodani, H. Takano, M. Hill, K. Shinmura, T. M. Vondriska, P. Ping, and R. Bolli Protein tyrosine kinase signaling is necessary for NO donor-induced late preconditioning against myocardial stunning Am J Physiol Heart Circ Physiol, April 1, 2003; 284(4): H1441 - H1448. [Abstract] [Full Text] [PDF]
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J. Jansen, P. Gres, C. Umschlag, F. R. Heinzel, H. Degenhardt, K.-D. Schluter, G. Heusch, and R. Schulz Parathyroid hormone-related peptide improves contractile function of stunned myocardium in rats and pigs Am J Physiol Heart Circ Physiol, January 1, 2003; 284(1): H49 - H55. [Abstract] [Full Text] [PDF]
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G. Lebuffe, P. T. Schumacker, Z.-H. Shao, T. Anderson, H. Iwase, and T. L. Vanden Hoek ROS and NO trigger early preconditioning: relationship to mitochondrial KATP channel Am J Physiol Heart Circ Physiol, January 1, 2003; 284(1): H299 - H308. [Abstract] [Full Text] [PDF]
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E. Kodani, Y.-T. Xuan, K. Shinmura, H. Takano, X.-L. Tang, and R. Bolli delta -Opioid receptor-induced late preconditioning is mediated by cyclooxygenase-2 in conscious rabbits Am J Physiol Heart Circ Physiol, November 1, 2002; 283(5): H1943 - H1957. [Abstract] [Full Text] [PDF]
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 A. Tsuchida, T. Miura, M. Tanno, J. Sakamoto, T. Miki, A. Kuno, T. Matsumoto, Y. Ohnuma, Y. Ichikawa, and K. Shimamoto Infarct size limitation by nicorandil: Roles of mitochondrial KATP channels, sarcolemmal KATP channels, and protein kinase C J. Am. Coll. Cardiol., October 16, 2002; 40(8): 1523 - 1530. [Abstract] [Full Text] [PDF]
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 J. D. Kilts, T. Akazawa, M. D. Richardson, and M. M. Kwatra Age Increases Cardiac Galpha i2 Expression, Resulting in Enhanced Coupling to G Protein-coupled Receptors J. Biol. Chem., August 16, 2002; 277(34): 31257 - 31262. [Abstract] [Full Text] [PDF]
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Z.-Q. Zhao and J. Vinten-Johansen Myocardial apoptosis and ischemic preconditioning Cardiovasc Res, August 15, 2002; 55(3): 438 - 455. [Abstract] [Full Text] [PDF]
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G.F Baxter Role of adenosine in delayed preconditioning of myocardium Cardiovasc Res, August 15, 2002; 55(3): 483 - 494. [Abstract] [Full Text] [PDF]
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Y.-P. Wang, H. Maeta, K. Mizoguchi, T. Suzuki, Y. Yamashita, and M. Oe Intestinal ischemia preconditions myocardium: role of protein kinase C and mitochondrial KATP channel Cardiovasc Res, August 15, 2002; 55(3): 576 - 582. [Abstract] [Full Text] [PDF]
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M. Joyeux, C. Arnaud, D. Godin-Ribuot, P. Demenge, D. Lamontagne, and C. Ribuot Endocannabinoids are implicated in the infarct size-reducing effect conferred by heat stress preconditioning in isolated rat hearts Cardiovasc Res, August 15, 2002; 55(3): 619 - 625. [Abstract] [Full Text] [PDF]
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K. Shinmura, R. Bolli, S.-Q. Liu, X.-L. Tang, E. Kodani, Y.-t. Xuan, S. Srivastava, and A. Bhatnagar Aldose Reductase Is an Obligatory Mediator of the Late Phase of Ischemic Preconditioning Circ. Res., August 9, 2002; 91(3): 240 - 246. [Abstract] [Full Text] [PDF]
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 E. N Dedkova, Y. Gao Wang, L. A Blatter, and S. L Lipsius Nitric oxide signalling by selective {beta}2-adrenoceptor stimulation prevents ACh-induced inhibition of {beta}2-stimulated Ca2+ current in cat atrial myocytes J. Physiol., August 1, 2002; 542(3): 711 - 723. [Abstract] [Full Text] [PDF]
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H. Y. Zhang, B. C. McPherson, H. Liu, T. Baman, S. S. McPherson, P. Rock, and Z. Yao Role of Nitric-Oxide Synthase, Free Radicals, and Protein Kinase C delta in Opioid-Induced Cardioprotection J. Pharmacol. Exp. Ther., June 1, 2002; 301(3): 1012 - 1019. [Abstract] [Full Text] [PDF]
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 R. D. Edmondson, T. M. Vondriska, K. J. Biederman, J. Zhang, R. C. Jones, Y. Zheng, D. L. Allen, J. X. Xiu, E. M. Cardwell, M. R. Pisano, et al. Protein Kinase C {epsilon} Signaling Complexes Include Metabolism- and Transcription/Translation-related Proteins: Complimentary Separation Techniques With LC/MS/MS Mol. Cell. Proteomics, June 1, 2002; 1(6): 421 - 433. [Abstract] [Full Text] [PDF]
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Z. Balafanova, R. Bolli, J. Zhang, Y. Zheng, J. M. Pass, A. Bhatnagar, X.-L. Tang, O. Wang, E. Cardwell, and P. Ping Nitric Oxide (NO) Induces Nitration of Protein Kinase Cepsilon (PKCepsilon ), Facilitating PKCepsilon Translocation via Enhanced PKCepsilon -RACK2 Interactions. A NOVEL MECHANISM OF NO-TRIGGERED ACTIVATION OF PKCepsilon J. Biol. Chem., April 19, 2002; 277(17): 15021 - 15027. [Abstract] [Full Text] [PDF]
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Y. Xu, D. Kumar, J. R. B. Dyck, W. R. Ford, A. S. Clanachan, G. D. Lopaschuk, and B. I. Jugdutt AT1 and AT2 receptor expression and blockade after acute ischemia-reperfusion in isolated working rat hearts Am J Physiol Heart Circ Physiol, April 1, 2002; 282(4): H1206 - H1215. [Abstract] [Full Text] [PDF]
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C. P. Baines, J. Zhang, G.-W. Wang, Y.-T. Zheng, J. X. Xiu, E. M. Cardwell, R. Bolli, and P. Ping Mitochondrial PKC{epsilon} and MAPK Form Signaling Modules in the Murine Heart: Enhanced Mitochondrial PKC{epsilon}-MAPK Interactions and Differential MAPK Activation in PKC{epsilon}-Induced Cardioprotection Circ. Res., March 8, 2002; 90(4): 390 - 397. [Abstract] [Full Text] [PDF]
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 Y. G. Wang, E. N. Dedkova, S. F. Steinberg, L. A. Blatter, and S. L. Lipsius {beta}2-Adrenergic Receptor Signaling Acts via No Release to Mediate Ach-Induced Activation of Atp-Sensitive K+ Current in Cat Atrial Myocytes J. Gen. Physiol., January 1, 2002; 119(1): 69 - 82. [Abstract] [Full Text] [PDF]
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X.-L. Tang, H. Takano, A. Rizvi, J. F. Turrens, Y. Qiu, W.-J. Wu, Q. Zhang, and R. Bolli Oxidant species trigger late preconditioning against myocardial stunning in conscious rabbits Am J Physiol Heart Circ Physiol, January 1, 2002; 282(1): H281 - H291. [Abstract] [Full Text] [PDF]
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 R. A. Kloner and R. B. Jennings Consequences of Brief Ischemia: Stunning, Preconditioning, and Their Clinical Implications: Part 2 Circulation, December 18, 2001; 104(25): 3158 - 3167. [Abstract] [Full Text] [PDF]
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G.-Y. Wang, J. J. Zhou, J. Shan, and T.-M. Wong Protein Kinase C-epsilon Is a Trigger of Delayed Cardioprotection against Myocardial Ischemia of kappa -Opioid Receptor Stimulation in Rat Ventricular Myocytes J. Pharmacol. Exp. Ther., November 1, 2001; 299(2): 603 - 610. [Abstract] [Full Text] [PDF]
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K. L. Schreiber, L. Paquet, B. G. Allen, and H. Rindt Protein kinase C isoform expression and activity in the mouse heart Am J Physiol Heart Circ Physiol, November 1, 2001; 281(5): H2062 - H2071. [Abstract] [Full Text] [PDF]
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 R D Rakhit and M S Marber Nitric oxide: an emerging role in cardioprotection? Heart, October 1, 2001; 86(4): 368 - 372. [Full Text] [PDF]
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M. Hill, H. Takano, X.-L. Tang, E. Kodani, G. Shirk, and R. Bolli Nitroglycerin Induces Late Preconditioning Against Myocardial Infarction in Conscious Rabbits Despite Development of Nitrate Tolerance Circulation, August 7, 2001; 104(6): 694 - 699. [Abstract] [Full Text] [PDF]
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E. Kodani, K. Shinmura, Y.-T. Xuan, H. Takano, J. A. Auchampach, X.-L. Tang, and R. Bolli Cyclooxygenase-2 does not mediate late preconditioning induced by activation of adenosine A1 or A3 receptors Am J Physiol Heart Circ Physiol, August 1, 2001; 281(2): H959 - H968. [Abstract] [Full Text] [PDF]
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H. Liu, B. C. McPherson, X. Zhu, M. L. A. Da Costa, V. Jeevanandam, and Z. Yao Role of nitric oxide and protein kinase C in ACh-induced cardioprotection Am J Physiol Heart Circ Physiol, July 1, 2001; 281(1): H191 - H197. [Abstract] [Full Text] [PDF]
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R. Moudgil, Y. Xu, V. Menon, and B. I. Jugdutt Effect of Chronic AT1 Receptor Antagonism on Postischemic Functional Recovery and AT1/AT2 Receptor Proteins in Isolated Working Rat Hearts Journal of Cardiovascular Pharmacology and Therapeutics, June 1, 2001; 6(2): 183 - 188. [Abstract] [PDF]
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 B. I Jugdutt and M. Balghith Enhanced regional AT2-receptor and PKC{varepsilon} expression during cardioprotection induced by AT1-receptor blockade after reperfused myocardial infarction Journal of Renin-Angiotensin-Aldosterone System, June 1, 2001; 2(2): 134 - 140. [Abstract] [PDF]
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S. A. Gabel, R. E. London, C. D. Funk, C. Steenbergen, and E. Murphy Leukocyte-type 12-lipoxygenase-deficient mice show impaired ischemic preconditioning-induced cardioprotection Am J Physiol Heart Circ Physiol, May 1, 2001; 280(5): H1963 - H1969. [Abstract] [Full Text] [PDF]
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T. M. Vondriska, J. B. Klein, and P. Ping Use of functional proteomics to investigate PKC{epsilon}-mediated cardioprotection: the signaling module hypothesis Am J Physiol Heart Circ Physiol, April 1, 2001; 280(4): H1434 - H1441. [Abstract] [Full Text] [PDF]
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J. M. Pass, Y. Zheng, W. B. Wead, J. Zhang, R. C. X. Li, R. Bolli, and P. Ping PKC{epsilon} activation induces dichotomous cardiac phenotypes and modulates PKC{epsilon}-RACK interactions and RACK expression Am J Physiol Heart Circ Physiol, March 1, 2001; 280(3): H946 - H955. [Abstract] [Full Text] [PDF]
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P. Andreka, J. Zang, C. Dougherty, T. I. Slepak, K. A. Webster, and N. H. Bishopric Cytoprotection by Jun Kinase During Nitric Oxide-Induced Cardiac Myocyte Apoptosis Circ. Res., February 16, 2001; 88(3): 305 - 312. [Abstract] [Full Text] [PDF]
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M. Krenz, C. P. Baines, X.-M. Yang, G. Heusch, M. V. Cohen, and J. M. Downey Acute ethanol exposure fails to elicit preconditioning-like protection in in situ rabbit hearts because of its continued presence during ischemia J. Am. Coll. Cardiol., February 1, 2001; 37(2): 601 - 607. [Abstract] [Full Text] [PDF]
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A. Lochner, E. Marais, S. Genade, and J. A. Moolman Nitric oxide: a trigger for classic preconditioning? Am J Physiol Heart Circ Physiol, December 1, 2000; 279(6): H2752 - H2765. [Abstract] [Full Text] [PDF]
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R. Bolli The Late Phase of Preconditioning Circ. Res., November 24, 2000; 87(11): 972 - 983. [Abstract] [Full Text] [PDF]
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H. Takano, X.-L. Tang, and R. Bolli Differential role of KATP channels in late preconditioning against myocardial stunning and infarction in rabbits Am J Physiol Heart Circ Physiol, November 1, 2000; 279(5): H2350 - H2359. [Abstract] [Full Text] [PDF]
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Y.-T. Xuan, X.-L. Tang, Y. Qiu, S. Banerjee, H. Takano, H. Han, and R. Bolli Biphasic response of cardiac NO synthase isoforms to ischemic preconditioning in conscious rabbits Am J Physiol Heart Circ Physiol, November 1, 2000; 279(5): H2360 - H2371. [Abstract] [Full Text] [PDF]
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H. Takano, X.-L. Tang, E. Kodani, and R. Bolli Late preconditioning enhances recovery of myocardial function after infarction in conscious rabbits Am J Physiol Heart Circ Physiol, November 1, 2000; 279(5): H2372 - H2381. [Abstract] [Full Text] [PDF]
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M. S. Finkel Nitric Oxide and Viral Cardiomyopathy Circulation, October 31, 2000; 102(18): 2162 - 2164. [Full Text] [PDF]
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 Y. Xu, A. S. Clanachan, and B. I. Jugdutt Enhanced Expression of Angiotensin II Type 2 Receptor, Inositol 1,4,5-Trisphosphate Receptor, and Protein Kinase C{epsilon} During Cardioprotection Induced by Angiotensin II Type 2 Receptor Blockade Hypertension, October 1, 2000; 36(4): 506 - 510. [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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 K. Shinmura, X.-L. Tang, Y. Wang, Y.-T. Xuan, S.-Q. Liu, H. Takano, A. Bhatnagar, and R. Bolli Cyclooxygenase-2 mediates the cardioprotective effects of the late phase of ischemic preconditioning in conscious rabbits PNAS, August 29, 2000; 97(18): 10197 - 10202. [Abstract] [Full Text] [PDF]
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H. Tong, W. Chen, C. Steenbergen, and E. Murphy Ischemic Preconditioning Activates Phosphatidylinositol-3-Kinase Upstream of Protein Kinase C Circ. Res., August 18, 2000; 87(4): 309 - 315. [Abstract] [Full Text] [PDF]
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J. W. Mockridge, A. Punn, D. S. Latchman, M. S. Marber, and R. J. Heads PKC-dependent delayed metabolic preconditioning is independent of transient MAPK activation Am J Physiol Heart Circ Physiol, August 1, 2000; 279(2): H492 - H501. [Abstract] [Full Text] [PDF]
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T. G. Hampton, I. Amende, J. Fong, V. E. Laubach, J. Li, C. Metais, and M. Simons Basic FGF reduces stunning via a NOS2-dependent pathway in coronary-perfused mouse hearts Am J Physiol Heart Circ Physiol, July 1, 2000; 279(1): H260 - H268. [Abstract] [Full Text] [PDF]
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Yi Xu, V. Menon, and B. I Jugdutt Cardioprotection after angiotensin II type 1 blockade involves angiotensin II type 2 receptor expression and activation of protein kinase C-{varepsilon} in acutely reperfused myocardial infarction in the dog: Effect of UP269-6 and losartan on AT1- and AT2-receptor expression and IP3 receptor and PKC{varepsilon} proteins Journal of Renin-Angiotensin-Aldosterone System, June 1, 2000; 1(2): 184 - 195. [Abstract] [PDF]
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R. D. Rakhit, R. J. Edwards, J. W. Mockridge, A. R. Baydoun, A. W. Wyatt, G. E. Mann, and M. S. Marber Nitric oxide-induced cardioprotection in cultured rat ventricular myocytes Am J Physiol Heart Circ Physiol, April 1, 2000; 278(4): H1211 - H1217. [Abstract] [Full Text] [PDF]
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T. Munzel, H. Li, H. Mollnau, U. Hink, E. Matheis, M. Hartmann, M. Oelze, M. Skatchkov, A. Warnholtz, L. Duncker, et al. Effects of Long-Term Nitroglycerin Treatment on Endothelial Nitric Oxide Synthase (NOS III) Gene Expression, NOS III-Mediated Superoxide Production, and Vascular NO Bioavailability Circ. Res., January 7, 2000; 86 (1): e7 - e12. [Abstract] [Full Text] [PDF]
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M. Gonzalez-Zulueta, A. B. Feldman, L. J. Klesse, R. G. Kalb, J. F. Dillman, L. F. Parada, T. M. Dawson, and V. L. Dawson Requirement for nitric oxide activation of p21ras/extracellular regulated kinase in neuronal ischemic preconditioning PNAS, January 4, 2000; 97(1): 436 - 441. [Abstract] [Full Text] [PDF]
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B. I. Jugdutt, Yi Xu, M. Balghith, R. Moudgil, and V. Menon Cardioprotection Induced by AT1R Blockade After Reperfused Myocardial Infarction: Association With Regional Increase in AT2R, IP3R and PKC{varepsilon} Proteins and cGMP Journal of Cardiovascular Pharmacology and Therapeutics, January 1, 2000; 5(4): 301 - 311. [Abstract] [PDF]
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B. Dawn, Y.-T. Xuan, Y. Qiu, H. Takano, X.-L. Tang, P. Ping, S. Banerjee, M. Hill, and R. Bolli Bifunctional Role of Protein Tyrosine Kinases in Late Preconditioning Against Myocardial Stunning in Conscious Rabbits Circ. Res., December 3, 1999; 85(12): 1154 - 1163. [Abstract] [Full Text] [PDF]
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T. Jalili, Y. Takeishi, G. Song, N. A. Ball, G. Howles, and R. A. Walsh PKC translocation without changes in Galpha q and PLC-beta protein abundance in cardiac hypertrophy and failure Am J Physiol Heart Circ Physiol, December 1, 1999; 277(6): H2298 - H2304. [Abstract] [Full Text] [PDF]
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S. Banerjee, X.-L. Tang, Y. Qiu, H. Takano, S. Manchikalapudi, B. Dawn, G. Shirk, and R. Bolli Nitroglycerin induces late preconditioning against myocardial stunning via a PKC-dependent pathway Am J Physiol Heart Circ Physiol, December 1, 1999; 277(6): H2488 - H2494. [Abstract] [Full Text] [PDF]
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K. Shinmura, X.-L. Tang, H. Takano, M. Hill, and R. Bolli Nitric oxide donors attenuate myocardial stunning in conscious rabbits Am J Physiol Heart Circ Physiol, December 1, 1999; 277(6): H2495 - H2503. [Abstract] [Full Text] [PDF]
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P. Ping, J. Zhang, S. Huang, X. Cao, X.-L. Tang, R. C. X. Li, Y.-T. Zheng, Y. Qiu, A. Clerk, P. Sugden, et al. PKC-dependent activation of p46/p54 JNKs during ischemic preconditioning in conscious rabbits Am J Physiol Heart Circ Physiol, November 1, 1999; 277(5): H1771 - H1785. [Abstract] [Full Text] [PDF]
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P. Ping, J. Zhang, Y.-T. Zheng, R. C. X. Li, B. Dawn, X.-L. Tang, H. Takano, Z. Balafanova, and R. Bolli Demonstration of Selective Protein Kinase C–Dependent Activation of Src and Lck Tyrosine Kinases During Ischemic Preconditioning in Conscious Rabbits Circ. Res., September 17, 1999; 85(6): 542 - 550. [Abstract] [Full Text] [PDF]
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A. Rizvi, X.-L. Tang, Y. Qiu, Y.-T. Xuan, H. Takano, A. K. Jadoon, and R. Bolli Increased protein synthesis is necessary for the development of late preconditioning against myocardial stunning Am J Physiol Heart Circ Physiol, September 1, 1999; 277(3): H874 - H884. [Abstract] [Full Text] [PDF]
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Y.-T. Xuan, X.-L. Tang, S. Banerjee, H. Takano, R. C. X. Li, H. Han, Y. Qiu, J.-J. Li, and R. Bolli Nuclear Factor-{kappa}B Plays an Essential Role in the Late Phase of Ischemic Preconditioning in Conscious Rabbits Circ. Res., May 14, 1999; 84(9): 1095 - 1109. [Abstract] [Full Text] [PDF]
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T. M. Vondriska, J. Zhang, C. Song, X.-L. Tang, X. Cao, C. P. Baines, J. M. Pass, S. Wang, R. Bolli, and P. Ping Protein Kinase C {epsilon}-Src Modules Direct Signal Transduction in Nitric Oxide-Induced Cardioprotection : Complex Formation as a Means for Cardioprotective Signaling Circ. Res., June 22, 2001; 88(12): 1306 - 1313. [Abstract] [Full Text] [PDF]
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