© 1997 American Heart Association, Inc.
Articles |
Ischemic Preconditioning Induces Selective Translocation of Protein Kinase C Isoforms 
and 
in the Heart of Conscious Rabbits Without Subcellular Redistribution of Total Protein Kinase C Activity
From the Experimental Research Laboratory, Division of Cardiology (P.P., J.Z., Y.Q., X.-L.T., S.M., X.C., R.B.), and the Department of Physiology and Biophysics (P.P., R.B.), University of Louisville (Ky).
Correspondence to Peipei Ping, PhD, Division of Cardiology, Research Laboratories/MDR Building, Room 526, University of Louisville, 511 South Floyd St, 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 Considerable controversy surrounds the role of protein kinase C (PKC) in ischemic preconditioning (PC). Previous studies have used pharmacological agents and/or measured total myocardial PKC activity; however, no information is available regarding the effects of PC on individual isoforms in vivo. We performed a comprehensive evaluation (using Western immunoblotting) of the expression and subcellular distribution of all 11 currently known PKC isoforms in the heart of conscious rabbits subjected to four different ischemic PC protocols known to induce early and/or late PC (one, three, or six cycles of 4-minute coronary occlusion [4'O]/4-minute reperfusion [4'R]; four cycles of 5-minute occlusion [5'O]/10-minute reperfusion [10'R]). Ten PKC isoforms (
, ß1/ß2, 
, 
, , 
, , 
,
, and µ) were found to be expressed in the rabbit heart.
Quantitative immunoblotting demonstrated that as a
subgroup, conventional PKCs (cPKCs) are more abundant than novel PKCs
(nPKCs) (1445 versus 313 pg PKC/µg tissue protein, respectively) and
that PKC is the predominant isoform among the cPKCs (,
ß1, ß2, and ), representing
51% of this subgroup, and PKC is the most abundant among the nPKCs
(, , , and ), accounting for 62% of this subgroup. None of
the ischemic PC protocols examined caused appreciable changes
in total PKC activity, in the subcellular distribution of total PKC
activity, or in the subcellular distribution of PKC isoforms ,
ß1/ß2, , , , , , and µ. In
contrast, all PC protocols caused significant translocation of PKC
and PKC isoforms from the cytosolic to the particulate fraction. The
particulate fraction of PKC increased in a dose-dependent fashion
with the number of occlusion/reperfusion cycles performed, from 35±2%
in the control group to 43±2% after one 4'O/5-minute reperfusion
(5'R) cycle (P<.05), 52±2% after three cycles
(P<.05 versus one cycle), and 66±3% after six cycles
(P<.05 versus three cycles). The particulate fraction of
PKC also increased, after four 5'O/10'R cycles, to 50±3%
(P<.05 versus control). In contrast to PKC, the
translocation of PKC was independent of the number of
occlusion/reperfusion cycles performed. The particulate fraction of
PKC increased from 67±3% in the control group to 84±2% after one
4'O/5'R cycle (P<.05), 84±2% after three 4'O/4'R cycles
(P<.05), 86±3% after six 4'O/4'R cycles
(P<.05), and 83±2% after four 5'O/10'R cycles
(P<.05). When expressed as a percentage of control values,
the increases in the particulate fraction of isoform were greater
than those of isoform . The effects of 4'O without reperfusion were
similar to those of one cycle of 4'O/5'R, indicating that 5'R did not
attenuate isoform translocation. This is the first study to demonstrate
PKC translocation after ischemic PC in vivo. The results
indicate that in the conscious rabbit, ischemic PC causes
selective translocation of the and isoforms without
demonstrable changes in total myocardial PKC activity, implying that
measurements of total PKC activity are not sufficiently sensitive to
detect the involvement of PKC in PC. The results are consistent
with the concept that the and isozymes play an important role
in the genesis of ischemic PC in the conscious rabbit.
Key Words: late phase of preconditioning • protein kinase C • protein kinase C • myocardial ischemia • myocardial reperfusion
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ischemic PC elicits an early phase ("classic" PC)1 2 3 4 5 6 7 8 as well as a late phase ("second window")8 9 10 11 12 13 14 15 16 17 18 of protection against subsequent ischemic injury. PKC has been proposed as one of the mediators of the protective effects of both phases of ischemic PC.19 20 21 22 23 This hypothesis, however, remains controversial, because it is supported by some studies16 19 20 22 23 24 25 26 27 28 29 30 31 32 33 34 but not by others.35 36 37 38 39 The reason(s) for these apparently conflicting results is unknown. Nevertheless, before the role of PKC in ischemic PC can be elucidated, a number of unresolved issues need to be addressed. First, no study has characterized the role of all currently known PKC isoforms in ischemic PC. This is partly due to the fact that thus far, only five of the 11 known PKC isoforms (
, ß, , , and )
have been identified in the rabbit heart40 41 ; it is
unknown whether the other isoforms (, , 
, , , and µ)
are also expressed. Second, translocation of PKC isoforms during or
after ischemia has been mostly examined with the use of
immunocytochemical fluorescence
techniques.26 29 34 Although useful from a qualitative
standpoint, these techniques do not allow accurate quantification of
translocation. On the other hand, Western
immunoblotting is generally regarded as a more suitable
method for quantitative assays. Third, the evidence for PKC activation
during PC in vivo is indirect, being based on pharmacological
approaches. Translocation of PKC isoforms has been examined during
global ischemia in isolated buffer-perfused
hearts26 32 33 37 or during hypoxia or simulated
ischemia in isolated
cardiomyocytes.29 34 However, to date, the
occurrence and extent of PKC translocation after regional
ischemia in vivo remain unknown. Fourth, virtually nothing is
known regarding whether PKC translocation occurs in the conscious
state, although differences in ischemic PC have been identified
between open-chest and conscious animal
preparations.5 6
The present study was undertaken in an effort to address these
unresolved issues and to gain insights into the molecular mechanisms
underlying ischemic PC. Our overall hypothesis was that
ischemic PC is mediated by the selective activation of one or
several individual PKC isoforms without a generalized activation of the
entire family of enzymes and that much of the controversy surrounding
the role of PKC in ischemic PC stems from the lack of
information regarding individual PKC isoforms. A comprehensive
investigation was performed in which the expression and translocation
of all 11 known isoforms of PKC were systematically assessed and
quantified with Western immunoblotting. The experiments
were conducted in rabbits, because this is the species in which the PKC
hypothesis of PC was initially formulated19 and has been
extensively tested.8 16 20 22 23 27 30 41 In order to
correlate any changes in PKC with the presence or absence of protection
against ischemic injury, rabbits were subjected to four
different PC protocols whose functional effects have been well
characterized in our laboratory12 16 17 18 42 and in other
laboratories.10 23 43 Because reperfusion has been
postulated to reverse the translocation of PKC that occurs during
ischemia,33 we examined PKC translocation both
during ischemia and after reperfusion. All studies were
performed in conscious animals, because the effects of
anesthesia, surgical trauma, and associated abnormal
conditions (eg, fluctuations in temperature, release of
cytokines, sympathetic hyperactivity [with the attendant
1-adrenergic stimulation], and exaggerated generation
of reactive oxygen species,44 45 etc) on PKC are
unknown.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Experimental Preparation
New Zealand White male rabbits (weight, 2.0 to 2.5 kg) were instrumented under sterile conditions, as previously detailed.18 42 46 A small tube was left in the thorax for 3 days to aspirate air and fluids. The animals were allowed to recover for a minimum of 10 days after surgery. Throughout the experiments, the rabbits were kept in a cage in a quiet dimly lit room. Left ventricular systolic wall thickening, the range gate depth, and the ECG were continuously recorded on a thermal-array chart recorder (Gould TA6000). Coronary artery occlusion was produced by inflating the balloon occluder. The performance of successful occlusions was verified by observing the appearance of ST-segment elevation and the widening of the QRS complex on the ECG and the development 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 wall thickening.18 42 46 No sedative or antiarrhythmic agents were given at any time.
Experimental Protocol
Rabbits were assigned to six groups (Fig 1
). Group I (control) underwent the same
surgical instrumentation as the other groups, but no coronary
occlusion was performed. 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 U
IV), after which they were anesthetized with sodium
pentobarbital (50 mg/kg IV) and killed with a bolus of KCl. The
heart was immediately excised, and myocardial samples (
0.5 g) were
rapidly removed from the anterior left ventricular wall and
stored in liquid nitrogen until use. Two additional rabbits not
subjected to surgical instrumentation were used to provide control
samples, so as to determine the possible effect of surgery on PKC
expression. Groups II, III, and IV underwent one, three, and six cycles
of 4-minute coronary occlusion/4-minute reperfusion,
respectively. The rabbits were killed 5 minutes after the last
reperfusion, and the heart was immediately excised; myocardial samples
were rapidly removed from the ischemic/reperfused region (whose
boundaries had been marked with sutures at the time of instrumentation)
and stored in liquid nitrogen. Group V underwent one 4-minute
coronary occlusion without reperfusion. The rabbits were killed
at 4 minutes of coronary occlusion, and myocardial samples were
obtained as described above. Group VI underwent four cycles of 5-minute
occlusion/10-minute reperfusion. The rabbits were killed 5 minutes
after the last reperfusion, and tissue samples were obtained as
described above. In all groups, the samples were frozen within 60 to 90
seconds from the bolus of KCl.
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Tissue Sample Preparation
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 50 mmol/L
Tris-HCl (pH 7.5), 5 mmol/L EDTA, 10 mmol/L
EGTA, 10 mmol/L benzamidine, 50 µg/mL
phenylmethylsulfonyl fluoride, 10 µg/mL aprotinin, 10
µg/mL leupeptin, 10 µg/mL pepstatin A, and 0.3%
ß-mercaptoethanol. 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). The
yields of total cellular proteins, cytosolic proteins, and particulate
proteins were carefully recorded for each tissue sample tested. The
cytosolic and particulate fractions were found to yield equivalent
amounts of proteins (51±2 and 48±3 mg protein/g of tissue,
respectively). 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. Quantitative immunoblotting was performed
in five of the eight control rabbits (group I). Total PKC activity was
measured in five rabbits in each of the six groups. For any given PKC
isoform, the subcellular distribution was assessed by Western blotting
in a minimum of five rabbits in each of the six groups. The number of
rabbits used for each assay in Figs 1
.
PKC Western Immunoblotting Analysis
Assessment of PKC isoforms was conducted using standard SDS-PAGE
Western immunoblotting techniques. 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. 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 Chemical Co); 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 (ECL kit, Amersham). The
specificity of the PKC antibody binding was confirmed by use of
recombinant PKC isoform peptides. Although the ß1,
ß2, and isoforms have identical molecular mass (80
kD), the antibodies to these isotypes had no detectable
cross-reactivity with one another.
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 particulate fractions of all five
tissue samples in each group were run on the same gel (Fig 2). Each immunoblotting
experiment was repeated twice, and the results were averaged. A total
of 438 Western blots were performed for the present study.
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In the present study, valid comparisons among samples required that
PKC isoform expression be normalized to total protein content. However,
despite a careful attempt to achieve equal protein loading in all lanes
of the gel, the total amounts of protein transferred from each lane to
the nitrocellulose membranes during blotting were 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. An example of this
procedure is presented in Fig 2, and details are provided in
the corresponding legend. This procedure was found to be important,
because the density of Ponceau stains varied among different lanes of
the same membrane between 80% and 120% of the average density of all
lanes. Had PKC isoforms not been normalized to Ponceau stain, the
results of the present study would likely have been different.
Measurement of PKC Isoform Protein Content by Quantitative
Western Immunoblotting
Increasing amounts (eg, 10, 20, and 40 ng) of human recombinant
PKC isoform proteins (Calbiochem) were loaded onto the same SDS-PAGE
gel along with 100 µg of total myocardial proteins extracted from
each of five control animals (group I). The ECL signals generated by
the recombinant proteins were used to construct a dose-response curve.
The curve was then used to determine the PKC isoform protein content in
tissue samples. Each experiment was repeated three times, and the
results were averaged. The data were analyzed using signals
from all five animals and expressed as picograms of PKC isoform protein
per microgram of myocardial tissue protein. The antibodies used for the
quantification of PKC isoform proteins are listed in Table 1.
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Measurement of PKC Activity
PKC activity was quantified using a PKC enzyme assay system
(Amersham). To evaluate the sensitivity of this assay system, we first
performed pilot experiments using protein samples ranging from 5 to 150
µg. We identified a window of linear relationship on the
dose-response curve where sample proteins ranged from 10 to 50 µg; 25
µg of proteins was found to be the optimal sample dose. In subsequent
experiments, 25 µg of proteins from either the cytosolic or the
particulate fraction was incubated with 0.2 µCi of
[-32P]ATP, 0.1 mmol/L ATP, 2.3
mmol/L HEPES, 5.5 mmol/L MgCl2,
1.15 mmol/L calcium acetate, 2.3 µg/mL phorbol
12-myristate 13-acetate, 28.8 µg/mL
L--phosphatidyl-L-serine, 2.9
mmol/L dithiothreitol, 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 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). PKC activity was calculated from the
specific counts (total minus nonspecific). Each sample was assayed in
triplicate. Data were expressed as picomoles of phosphate transferred
per minute per milligram of sample proteins.
Statistical Analysis
Data are reported as mean±SEM. Differences among the six groups
with respect to total PKC activity, subcellular distribution of PKC
activity, subcellular distribution of individual PKC isoforms, and PKC
isoform translocation were analyzed using a one-way ANOVA. If
the ANOVA showed an overall difference, post hoc contrasts were
performed with Student's t tests for unpaired data using
the Bonferroni correction.47
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Exclusions
A total of 41 conscious rabbits were used in the present study. All of the rabbits assigned to group I (control group) (n=8), group II (n=6), group III (n=6), and group V (n=5) completed the protocol successfully. Of the seven rabbits assigned to group IV, one died of ventricular fibrillation during the fifth coronary occlusion. Of the seven rabbits assigned to group VI, one died of ventricular fibrillation during the second occlusion, and another died during the third occlusion. Therefore, six rabbits completed the protocol in group IV, and five rabbits completed the protocol in group VI. In addition, two rabbits that did not undergo surgical instrumentation were used to determine the effect of surgery on PKC isoform expression.
PKC Isoform Expression in the Rabbit Heart
In control rabbits that did not undergo coronary occlusion
(group I), 10 isoforms of PKC were found to be expressed: , ß
(including ß1 and ß2), , , , , , , , and µ
(Table 1). PKC isoform expression was not detectable using the
currently available anti-PKC monoclonal antibodies (Transduction
Laboratories). The identification of PKC isoform expression was based
on the size of the PKC protein, as well as the comigrating positive
control signal from cellular lysates (Jurkat, Macrophage and
HeLa cell lines from ATCC) that express PKC isoforms (Fig 2).
The expression of isoforms , , , , and µ (which had not
been previously identified in rabbit myocardium) was
corroborated by the demonstration of mRNA expression with RT-PCR
analyses (an example of an RT-PCR gel for the PKC isoform
is shown in Fig 3). The PCR fragments
were sequenced, and the nucleotide sequences were confirmed
with information acquired from GenBank. The sequencing data for PCR
fragments of the newly identified isoforms (, , , , and
µ) are not shown for the sake of brevity.
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Analysis of the subcellular distribution of the various PKC
isoforms revealed that in the heart of conscious rabbits most isozymes
reside primarily in the cytosolic fraction, which accounts for 65% to
86% of their total protein content (Table 1). The exceptions were
PKC and PKCµ (only 33% of PKC and 19% of PKCµ were found to
be in the cytosolic fraction) (Table 1). In the two control rabbits
that were not subjected to surgery, the expression and subcellular
distribution of PKC isoforms were found to be similar to those observed
in group I (data not shown), indicating that surgical instrumentation
had no effect on these variables.
Absolute Protein Content of PKC Isoforms in the Rabbit
Heart
Virtually no information is available regarding the absolute PKC
isoform protein content in the rabbit myocardium. In an
effort to shed light on this issue, quantitative Western
immunoblottings were performed for the cPKC (Fig 4A) and nPKC (Fig 4B) isotypes. As shown
in Table 1 and Fig 4C, rabbit myocardium expresses ample
amounts of cPKCs (, ß1, ß2, and ) (total cPKC protein
content, 1445 pg PKC/µg protein) and lesser amounts of nPKCs (,
, , and ) (total nPKC protein content, 313 pg PKC/µg
protein). Among the cPKCs, the predominant isoform is PKC (51% of
total cPKC protein); among the nPKCs, is, by far, the most abundant
isoform, accounting for 62% of total nPKC proteins (Fig 4C). The
protein content of atypical PKCs (, , and µ) was not determined
because of the lack of availability of the recombinant proteins.
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Effect of Ischemic PC on Total PKC Activity
In control animals (group I), the majority of the total PKC
activity (87%) was associated with the cytosolic fraction (Fig 5). Similar results were obtained in the
two control rabbits that were not instrumented (data not shown).
Neither the total activity of PKC nor the percentage of total PKC
activity associated with the particulate fraction (Fig 5) was
significantly different under control conditions (group I) and after
one, three, and six 4-minute occlusion/4-minute reperfusion cycles
(groups II, III, and IV, respectively). Although there was a trend for
total PKC activity to increase after three and six
occlusion/reperfusion cycles (Fig 5), these differences were far from
achieving statistical significance (P=.63 by ANOVA). Similar
results were obtained when group VI, which underwent four 5-minute
occlusion/10-minute reperfusion cycles, was compared with the control
group (Fig 5). Thus, none of the PC protocols examined caused any
significant change in total PKC activity or any significant
translocation of total PKC activity. On the basis of these data alone,
we would have concluded that ischemic PC has no effect on PKC.
However, as detailed below, different results were obtained when we
analyzed individual isoforms.
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Effect of Ischemic PC on the Subcellular Distribution of
PKC Isoforms
As shown in Table 2, there was no
significant redistribution of PKC isoforms , ß1, ß2, , ,
, , , and µ between the cytosolic and the particulate
fractions after one, three, or six 4-minute occlusion/4-minute
reperfusion cycles or after four 5-minute occlusion/10-minute
reperfusion cycles.
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In contrast, PKC and PKC exhibited a significant translocation
into the particulate fraction after all of the ischemic PC
protocols. Fig 6 illustrates an example
of PKC translocation, observed after six cycles of 4-minute
occlusion/4-minute reperfusion (group IV). The figure shows that in
control rabbits (group I), most of the PKC protein was found in the
cytosolic fraction, whereas in group IV the majority of the PKC
protein became associated with the particulate fraction. It is
noteworthy that translocation of PKC was observed
consistently in all of the five animals examined (Fig 6). A
similar consistency of translocation among different
animals was also observed for the isoform (the
consistency of translocation of PKC and PKC is
reflected in the small standard error bars bracketing the group means
in Figs 7 and 8).
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Figs 7 and 8 illustrate the changes in the
subcellular distribution of PKC and PKC after the various
ischemic PC protocols. In groups II, III, and IV, PKC
protein in the particulate fraction increased in a dose-dependent
fashion with the number of 4-minute occlusion/4-minute reperfusion
cycles performed (Fig 7, Table 2). The particulate fraction of PKC
increased also in group VI (four 5-minute occlusion/10-minute
reperfusion cycles; Fig 7, Table 2). When compared with group I, the
particulate fraction of PKC increased by 23% in group II, 49% in
group III, 89% in group IV, and 43% in group VI. Statistical
analysis demonstrated that the particulate fraction of total
PKC was significantly greater in group III (three 4-minute
occlusion/4-minute reperfusion cycles) compared with group II (one
4-minute occlusion/5-minute reperfusion cycle) (P<.05) and
in group IV (six 4-minute occlusion/4-minute reperfusion cycles)
compared with group III (three 4-minute occlusion/4-minute reperfusion
cycles) (P<.05), confirming the dose-response pattern (Fig 7, Table 2). The degree of PKC translocation in group VI (four
5-minute occlusion/10-minute reperfusion cycles) was greater than that
in group II (one 4-minute occlusion/5-minute reperfusion cycle)
(P<.05) but not significantly different from that in group
III (three 4-minute occlusion/4-minute reperfusion cycles ) (Fig 7, Table 2).
The particulate fraction of PKC increased in groups II through VI,
but in contrast to PKC, the translocation of PKC was independent
of the number of occlusion/reperfusion cycles performed (Fig 8, Table 2). Because the proportion of PKC isoform associated with the
particulate fraction under control conditions was much greater for
PKC than for PKC (67% versus 35% of total protein,
respectively, in group I), the percent increases induced by PC were
smaller for PKC than for PKC. Specifically, the changes in the
particulate fractions of PKC, expressed as a percentage of the
particulate fraction of PKC measured in the control group (group I),
were 25% in group II, 26% in group III, 28% in group IV, and 24% in
group VI (the corresponding values for PKC were 23%, 49%, 89%,
and 43%). Thus, ischemic PC caused a comparatively greater
translocation of PKC vis-à-vis PKC.
The total protein content for PKC and PKC remained unaltered
during all of the ischemic PC protocols, indicating that the
changes in subcellular distribution were not due to changes in overall
protein expression.
Effect of 5 Minutes of Reperfusion on Total PKC Activity and
Isoform Translocation
In group V (which underwent only 4 minutes of occlusion without
reperfusion), the total PKC activity and its subcellular distribution,
as well as the subcellular distributions of the individual PKC
isoforms, were similar to those noted in group II (Figs 5, 7, and 8;
Table 2). Thus, the effects of a 4-minute coronary occlusion
(group V) on PKC activity and translocation did not differ appreciably
from those of a 4-minute occlusion followed by 5 minutes of reperfusion
(group II). These results indicate that the translocation of PKC and
PKC observed at the end of 4 minutes of ischemia was
unchanged after 5 minutes of reperfusion, suggesting that reperfusion
does not abolish or attenuate isoform translocation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Analysis of a total of 438 Western blots was performed in the present study to assess the effect of ischemic PC on the subcellular distribution of all 11 currently known PKC isotypes. Previous investigations have examined the translocation of selected PKC isoforms after global ischemia in isolated hearts26 32 33 37 and after hypoxia or simulated ischemia in isolated cardiomyocytes.29 34 To our knowledge, this is the first study to demonstrate PKC translocation after regional myocardial ischemia in vivo and to examine the effect of ischemic PC on PKC in a conscious animal model.
PKC Isoform Expression in the Rabbit Heart
One of the major findings of the present study is the
identification of five additional PKC isoforms (, , , , and
µ) in the rabbit myocardium, which were not previously
known to be present. Recent studies40 41 have shown
that the rabbit heart expresses PKC isoforms , ß, , , and
. No study, however, has systematically analyzed the
expression of all currently known PKC isoforms in this species. In the
present investigation, we report, for the first time, the
expression and subcellular distribution of 10 PKC isoforms (including
two subtypes of the ß isoform): they are isoforms , ß1 and ß2,
, , , , , , , and µ. Further studies will be
necessary to determine whether these isozymes are expressed in myocytes
or in other cell types (eg, fibroblasts, endothelial
cells, and smooth muscle cells).
Despite increasing evidence supporting a key role of PKC isoforms in
the regulation of cardiac functions,40 48 49 50 51 52 53 no study has
yet determined the PKC isoform protein content, an element that is
crucial to fully explore the role of these enzymes in the rabbit heart.
The use of quantitative immunoblotting has enabled us
to measure, for the first time, the absolute protein content of both
the cPKCs (, ß1, ß2, and ) and the nPKCs (, , , and
) in the rabbit heart. We used human, not rabbit, recombinant PKC
isoform fusion proteins to construct our standard curves. Because the
amino acid sequence in the antibody hybridization region is highly
conserved between human and rabbit PKC isoforms (it is 100% homologous
for PKC and 90% to 100% homologous for the other isoforms), we
anticipated that the anti-PKC antibodies we used would have equivalent
binding affinity for the human recombinant PKC protein and the rabbit
myocardial PKC protein. Our data reveal that the rabbit heart expresses
ample amounts of cPKCs, among which the isoform is expressed with
the highest abundance, and lesser amounts of nPKCs, among which PKC
is, by far, the most abundant isoform. The new quantitative information
provided by these data is important, because the functional effects of
selective PKC isoform activation may be determined in part by the
relative proportions of the isotypes involved. For example,
translocation of two isozymes during ischemia may have
different consequences depending on their relative abundance within the
cell. In the case of PKC and PKC, we found that the former is 7-
to 8-fold more abundant than the latter (Table 1); thus, although
similar percentages of total and protein were translocated by
ischemic PC (Figs 7 and 8), the absolute magnitude of protein
movement to the particulate fraction was much greater for PKC than
for PKC, which could possibly result in different
physiological effects.
Total PKC Activity During Ischemic PC
The lack of translocation of total PKC activity from the cytosolic
fraction to the particulate fraction in canine35 and
rabbit36 models of ischemic PC has been considered
one of the most cogent arguments against the PKC hypothesis of PC. In
the present study, we found that none of the four PC protocols
examined had a significant effect on total PKC activity or on the
subcellular distribution of PKC activity (Fig 5), a result that is
consistent with previous investigations.35 36
Evidently, the accuracy of our PKC activity measurements depends
largely on the precision of the experimental procedure and the
sensitivity of the assay system. We achieved a very high specific
signal for the measurements of PKC activity, as demonstrated by the
fact that the nonspecific signal was <5% of the total signal. An
important concern was the substrate peptide used to determine PKC
activity. More than 10 different peptides have been used as substrates
for the PKC phosphorylation assay.54 55
The sensitivity of these substrates varies among different PKC
isoforms.49 50 51 54 55 56 57 58 The commercial PKC assay system we
used contains both a histone III and a synthetic peptide (sequence
VRKRTLRRL). Histone III is the primary substrate for the cPKC
isoforms (, ß, and ), whereas the synthetic peptide serves as a
substrate for the other PKC isoforms, including the nPKC isoforms (data
provided by Amersham). Thus, this assay system provides the substrates
necessary for most of the PKC isoforms, including the two isoforms (
and ) that we found to be translocated. Nevertheless, in agreement
with the data reported by Przyklenk et al35 in dogs and by
Simkhovich et al36 in rabbits, our results show that
ischemic PC is not associated with a significant increase in
total PKC activity or with a demonstrable translocation of total PKC
activity to the particulate fraction. If we had considered these PKC
activity data alone, we would have arrived at the incorrect conclusion
that PKC is not activated during ischemic PC.
Should total PKC activity even be used as an index for the role of PKC
during ischemic PC? We believe that the answer is negative,
because the measurements of PKC phosphorylation
activity currently possible do not faithfully reflect the activation of
individual PKC isoforms. Our quantitative
immunoblotting results show that the PKC and PKC
proteins account for only 13% of all cPKC and nPKC proteins
combined (Fig 4C) (and thus for <13% of all PKC isoform proteins in
the rabbit heart); therefore, translocation of a fraction of these two
proteins (up to 31% of total protein [Fig 7] and 19% of total
protein in group IV [Fig 8]) would be expected to result in
minimal statistically insignificant changes in the subcellular
distribution of total PKC activity. This would be true even if an
-sensitive substrate peptide were used in the assay. Because all PKC
substrate peptides available are isoform nonspecific54 55 56 57 58
rather than targeted only at the or isoform, a selective
elevation of PKC and PKC activities would be blurred by the
activity of all other isozymes and therefore would become
indistinguishable.
In summary, we suggest that measurements of total PKC activity alone are neither sufficient nor pertinent to reflect the activation of individual PKC isoforms during ischemic PC. Our results clearly illustrate that the determination of individual PKC isoform translocation is essential in evaluating the role of PKC in ischemic PC.
Previous Studies of PKC Isoforms During Ischemic PC
Although pharmacological evidence supports the concept that PKC is
activated during myocardial ischemia/reperfusion, no
previous study has directly demonstrated translocation of PKC during
ischemic PC in vivo. A number of studies have examined the
translocation of selected PKC isoforms during PC in various
experimental settings of global or simulated ischemia in vitro,
with conflicting results.26 29 32 34 37 41 59 The
present study expands these previous studies by demonstrating the
following points: First, repetitive episodes of regional myocardial
ischemia induce PKC translocation in the conscious state.
Importantly, this phenomenon is associated with the development of late
PC against myocardial stunning12 16 17 18 42 and early and
late PC against infarction.10 23 43 Second, translocation
of PKC during ischemic PC is isoform selective. The present
study documents this fact using Western immunoblotting
rather than immunocytochemistry. Third, PKC isoform translocation
occurs without demonstrable changes in total PKC activity and/or
translocation of total PKC activity. Fourth, PKC isoform translocation
persists after repetitive cycles of coronary
occlusion/reperfusion and is not affected by a 5-minute reperfusion
period.
Correlation Between Translocation of PKC Isoforms and Development
of a PC Effect
A major goal of the present study was to correlate any changes
in the subcellular distribution of PKC with the presence or absence of
PC protection. The protocols used in groups III and IV (three and six
cycles, respectively, of 4-minute occlusion/4-minute reperfusion) have
previously been found to induce late PC against myocardial stunning in
this conscious rabbit model,12 16 17 18 42 whereas the
protocol used in group VI (four cycles of 5-minute occlusion/10-minute
reperfusion) has been found to induce both early and late PC against
myocardial infarction in open-chest rabbits.10 23 43 In
recent studies (authors' unpublished data, 1996), we have observed
that the protocol used in group IV also induces late PC against
infarction in conscious rabbits. In view of these prior results, the
present finding that groups III and IV exhibited translocation of
PKC is compatible with a role of this isozyme in the genesis of late
PC against stunning (although PKC was also translocated in groups
III and IV, a role of PKC in late PC against stunning is unlikely,
as discussed below). Furthermore, the translocation of PKC and
PKC observed in groups IV and VI is compatible with a role of these
two isozymes in the development of both early and late PC against
infarction.
The translocation of the isoform was cumulative, exhibiting a
dose-dependent pattern in response to the number of
occlusion/reperfusion cycles performed in groups II through IV (Fig 7).
Since, as indicated above, both three and six cycles of 4-minute
occlusion/4-minute reperfusion elicit the late phase of PC against
stunning and since the protective effects of these two protocols are
indistinguishable,17 it appears that the degree of
translocation induced by three occlusion/reperfusion cycles (group III)
might be the threshold of activation of PKC required to trigger the
late phase of ischemic PC against stunning and that the
additional translocation of PKC associated with six cycles (group
IV) may not be essential for this protective effect. In contrast to
PKC, translocation of PKC did not vary among different
ischemic PC protocols, demonstrating an all-or-none pattern
(Fig 8). Because one cycle of 4-minute occlusion/5-minute reperfusion
is insufficient to induce late PC against myocardial stunning 24 hours
later17 and because the extent of PKC translocation was
similar after one, three, and six occlusion/reperfusion cycles (groups
II, III, and IV, respectively), it does not seem likely that the
translocation of PKC is of major importance in mediating the late
phase of PC against stunning. The significance of PKC translocation
in the protection against infarction remains to be determined.
Conclusions
In summary, the present study is the first direct
demonstration that ischemic PC alters the subcellular
distribution of PKC in vivo. The finding that PKC and PKC are
translocated at a time when total PKC activity remains constant
demonstrates that PKC activation during ischemic PC is isoform
selective and may occur without demonstrable changes in total PKC
activity. This implies that measurements of total PKC activity are not
sufficiently sensitive to detect the involvement of PKC in the
phenomenon of ischemic PC. Taken together, the results of the
present study are consistent with the concept that the
and isozymes of PKC play an important role in the genesis of
ischemic PC in conscious rabbits.
The present findings may help to resolve the controversy regarding the role of PKC in PC. For example, our results imply that the lack of change in total PKC activity after PC35 36 cannot be construed as evidence against a PKC involvement, because the possibility that a discrete activation of one or few PKC isoforms may be occurring cannot be ruled out. Furthermore, it is possible that some of the PKC inhibitors used in "negative" studies35 38 39 may not have blocked the one or few crucial isoforms responsible for PC, particularly in view of the difficulties inherent in estimating tissue levels of PKC blockers in vivo.
|
Selected Abbreviations and Acronyms |
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Acknowledgments |
|---|
This study was supported in part by National Institutes of Health grants R29 HL-58166 (Dr Ping), R01 HL-43151 (Dr Bolli), and HL-55757 (Dr Bolli); by grants KY-96-GB-37 (Dr Ping), KY-96-GB-32 (Dr Qiu), and KY-96-GB-31 (Dr Tang) from the American Heart Association, Kentucky Affiliate, Inc; and by the Jewish Hospital Research Foundation, Louisville, Ky.
Received February 5, 1997; accepted June 26, 1997.
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References |
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H. Ardehali Signaling Mechanisms in Ischemic Preconditioning: Interaction of PKC{epsilon} and MitoKATP in the Inner Membrane of Mitochondria Circ. Res., October 13, 2006; 99(8): 798 - 800. [Full Text] [PDF]
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S. Drose, U. Brandt, and P. J. Hanley K+-independent Actions of Diazoxide Question the Role of Inner Membrane KATP Channels in Mitochondrial Cytoprotective Signaling J. Biol. Chem., August 18, 2006; 281(33): 23733 - 23739. [Abstract] [Full Text] [PDF]
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M. Hambleton, H. Hahn, S. T. Pleger, M. C. Kuhn, R. Klevitsky, A. N. Carr, T. F. Kimball, T. E. Hewett, G. W. Dorn II, W. J. Koch, et al. Pharmacological- and Gene Therapy-Based Inhibition of Protein Kinase C{alpha}/{beta} Enhances Cardiac Contractility and Attenuates Heart Failure Circulation, August 8, 2006; 114(6): 574 - 582. [Abstract] [Full Text] [PDF]
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R. Mangat, T. Singal, N. S. Dhalla, and P. S. Tappia Inhibition of phospholipase C-{gamma}1 augments the decrease in cardiomyocyte viability by H2O2 Am J Physiol Heart Circ Physiol, August 1, 2006; 291(2): H854 - H860. [Abstract] [Full Text] [PDF]
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M. T. Jiang, M. Ljubkovic, Y. Nakae, Y. Shi, W.-M. Kwok, D. F. Stowe, and Z. J. Bosnjak Characterization of human cardiac mitochondrial ATP-sensitive potassium channel and its regulation by phorbol ester in vitro Am J Physiol Heart Circ Physiol, May 1, 2006; 290(5): H1770 - H1776. [Abstract] [Full Text] [PDF]
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K. Inagaki, E. Churchill, and D. Mochly-Rosen Epsilon protein kinase C as a potential therapeutic target for the ischemic heart Cardiovasc Res, May 1, 2006; 70(2): 222 - 230. [Abstract] [Full Text] [PDF]
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D. J. Hausenloy and D. M. Yellon Survival kinases in ischemic preconditioning and postconditioning Cardiovasc Res, May 1, 2006; 70(2): 240 - 253. [Abstract] [Full Text] [PDF]
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R. J. Diaz and G. J. Wilson Studying ischemic preconditioning in isolated cardiomyocyte models Cardiovasc Res, May 1, 2006; 70(2): 286 - 296. [Abstract] [Full Text] [PDF]
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S. Philipp, X.-M. Yang, L. Cui, A. M. Davis, J. M. Downey, and M. V. Cohen Postconditioning protects rabbit hearts through a protein kinase C-adenosine A2b receptor cascade Cardiovasc Res, May 1, 2006; 70(2): 308 - 314. [Abstract] [Full Text] [PDF]
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A. J. Zatta, H. Kin, G. Lee, N. Wang, R. Jiang, R. Lust, J. G. Reeves, J. Mykytenko, R. A. Guyton, Z.-Q. Zhao, et al. Infarct-sparing effect of myocardial postconditioning is dependent on protein kinase C signalling Cardiovasc Res, May 1, 2006; 70(2): 315 - 324. [Abstract] [Full Text] [PDF]
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K. Przyklenk, M. Maynard, and P. Whittaker Reduction of infarct size with D-myo-inositol trisphosphate: role of PI3-kinase and mitochondrial KATP channels Am J Physiol Heart Circ Physiol, February 1, 2006; 290(2): H830 - H836. [Abstract] [Full Text] [PDF]
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K. Yamamura, C. Steenbergen, and E. Murphy Protein kinase C and preconditioning: role of the sarcoplasmic reticulum Am J Physiol Heart Circ Physiol, December 1, 2005; 289(6): H2484 - H2490. [Abstract] [Full Text] [PDF]
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S. L. Robia, M. Kang, and J. W. Walker Novel determinant of PKC-{epsilon} anchoring at cardiac Z-lines Am J Physiol Heart Circ Physiol, November 1, 2005; 289(5): H1941 - H1950. [Abstract] [Full Text] [PDF]
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Y.-T. Xuan, Y. Guo, Y. Zhu, O.-L. Wang, G. Rokosh, R. O. Messing, and R. Bolli Role of the Protein Kinase C-{epsilon}-Raf-1-MEK-1/2-p44/42 MAPK Signaling Cascade in the Activation of Signal Transducers and Activators of Transcription 1 and 3 and Induction of Cyclooxygenase-2 After Ischemic Preconditioning Circulation, September 27, 2005; 112(13): 1971 - 1978. [Abstract] [Full Text] [PDF]
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A. D.T. Costa, K. D. Garlid, I. C. West, T. M. Lincoln, J. M. Downey, M. V. Cohen, and S. D. Critz Protein Kinase G Transmits the Cardioprotective Signal From Cytosol to Mitochondria Circ. Res., August 19, 2005; 97(4): 329 - 336. [Abstract] [Full Text] [PDF]
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S. Dhein New, emerging roles for cardiac connexins. Mitochondrial Cx43 raises new questions Cardiovasc Res, August 1, 2005; 67(2): 179 - 181. [Full Text] [PDF]
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L. A. Turner, K. Fujimoto, A. Suzuki, A. Stadnicka, Z. J. Bosnjak, and W.-M. Kwok The Interaction of Isoflurane and Protein Kinase C-Activators on Sarcolemmal KATP Channels Anesth. Analg., June 1, 2005; 100(6): 1680 - 1686. [Abstract] [Full Text] [PDF]
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W. Wang, L. Jia, T. Wang, W. Sun, S. Wu, and X. Wang Endogenous Calcitonin Gene-related Peptide Protects Human Alveolar Epithelial Cells through Protein Kinase C{epsilon} and Heat Shock Protein J. Biol. Chem., May 27, 2005; 280(21): 20325 - 20330. [Abstract] [Full Text] [PDF]
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M. Juhaszova, C. Rabuel, D. B. Zorov, E. G. Lakatta, and S. J. Sollott Protection in the aged heart: preventing the heart-break of old age? Cardiovasc Res, May 1, 2005; 66(2): 233 - 244. [Abstract] [Full Text] [PDF]
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V. Pastukh, S. Wu, C. Ricci, M. Mozaffari, and S. Schaffer Reversal of hyperglycemic preconditioning by angiotensin II: role of calcium transport Am J Physiol Heart Circ Physiol, April 1, 2005; 288(4): H1965 - H1975. [Abstract] [Full Text] [PDF]
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K. Inagaki, R. Begley, F. Ikeno, and D. Mochly-Rosen Cardioprotection by {epsilon}-Protein Kinase C Activation From Ischemia: Continuous Delivery and Antiarrhythmic Effect of an {epsilon}-Protein Kinase C-Activating Peptide Circulation, January 4, 2005; 111(1): 44 - 50. [Abstract] [Full Text] [PDF]
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G. Asemu, N. S. Dhalla, and P. S. Tappia Inhibition of PLC improves postischemic recovery in isolated rat heart Am J Physiol Heart Circ Physiol, December 1, 2004; 287(6): H2598 - H2605. [Abstract] [Full Text] [PDF]
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Z.-Q. Jin, E. J. Goetzl, and J. S. Karliner Sphingosine Kinase Activation Mediates Ischemic Preconditioning in Murine Heart Circulation, October 5, 2004; 110(14): 1980 - 1989. [Abstract] [Full Text] [PDF]
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M. Palmen, M. J.A.P. Daemen, L. J. De Windt, J. Willems, W. R.M. Dassen, S. Heeneman, R. Zimmermann, M. Van Bilsen, and P. A. Doevendans Fibroblast growth factor-1 improves cardiac functional recovery and enhances cell survival after ischemia and reperfusion: A fibroblast growth factor receptor, protein kinase c, and tyrosine kinase-dependent mechanism J. Am. Coll. Cardiol., September 1, 2004; 44(5): 1113 - 1123. [Abstract] [Full Text] [PDF]
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A. Besana, A. Barbuti, M. A. Tateyama, A. J. Symes, R. B. Robinson, and S. J. Feinmark Activation of Protein Kinase C {epsilon} Inhibits the Two-pore Domain K+ Channel, TASK-1, Inducing Repolarization Abnormalities in Cardiac Ventricular Myocytes J. Biol. Chem., August 6, 2004; 279(32): 33154 - 33160. [Abstract] [Full Text] [PDF]
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T. Uchiyama, R. M. Engelman, N. Maulik, and D. K. Das Role of Akt Signaling in Mitochondrial Survival Pathway Triggered by Hypoxic Preconditioning Circulation, June 22, 2004; 109(24): 3042 - 3049. [Abstract] [Full Text] [PDF]
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G. Li, S. Bae, and L. Zhang Effect of prenatal hypoxia on heat stress-mediated cardioprotection in adult rat heart Am J Physiol Heart Circ Physiol, May 1, 2004; 286(5): H1712 - H1719. [Abstract] [Full Text] [PDF]
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M. C. Souroujon, L. Yao, H. Chen, G. Endemann, H. Khaner, V. Geeraert, D. Schechtman, A. S. Gordon, I. Diamond, and D. Mochly-Rosen State-specific Monoclonal Antibodies Identify an Intermediate State in Epsilon Protein Kinase C Activation J. Biol. Chem., April 23, 2004; 279(17): 17617 - 17624. [Abstract] [Full Text] [PDF]
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A. Das, R. Ockaili, F. Salloum, and R. C. Kukreja Protein kinase C plays an essential role in sildenafil-induced cardioprotection in rabbits Am J Physiol Heart Circ Physiol, April 1, 2004; 286(4): H1455 - H1460. [Abstract] [Full Text] [PDF]
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X.-L. Tang, Y.-T. Xuan, Y. Zhu, G. Shirk, and R. Bolli Nicorandil induces late preconditioning against myocardial infarction in conscious rabbits Am J Physiol Heart Circ Physiol, April 1, 2004; 286(4): H1273 - H1280. [Abstract] [Full Text] [PDF]
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S. C Armstrong Protein kinase activation and myocardial ischemia/reperfusion injury Cardiovasc Res, February 15, 2004; 61(3): 427 - 436. [Abstract] [Full Text] [PDF]
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Y. Nishino, T. Miura, T. Miki, J. Sakamoto, Y. Nakamura, Y. Ikeda, H. Kobayashi, and K. Shimamoto Ischemic preconditioning activates AMPK in a PKC-dependent manner and induces GLUT4 up-regulation in the late phase of cardioprotection Cardiovasc Res, February 15, 2004; 61(3): 610 - 619. [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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K. Przyklenk, G. Li, B. Z. Simkhovich, and R. A. Kloner Mechanisms of myocardial ischemic preconditioning are age related: PKC-{epsilon} does not play a requisite role in old rabbits J Appl Physiol, December 1, 2003; 95(6): 2563 - 2569. [Abstract] [Full Text]
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M. M. Tickerhoof, P A. Farrell, and D. H. Korzick Alterations in rat coronary vasoreactivity and vascular protein kinase C isoforms in Type 1 diabetes Am J Physiol Heart Circ Physiol, December 1, 2003; 285(6): H2694 - H2703. [Abstract] [Full Text] [PDF]
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P. Rafiee, Y. Shi, K. A. Pritchard Jr., H. Ogawa, A. L. W. Eis, R. A. Komorowski, C. M. Fitzpatrick, J. S. Tweddell, S. B. Litwin, K. Mussatto, et al. Cellular Redistribution of Inducible Hsp70 Protein in the Human and Rabbit Heart in Response to the Stress of Chronic Hypoxia: ROLE OF PROTEIN KINASES J. Biol. Chem., October 31, 2003; 278(44): 43636 - 43644. [Abstract] [Full Text] [PDF]
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K. Inagaki, H. S. Hahn, G. W. Dorn II, and D. Mochly-Rosen Additive Protection of the Ischemic Heart Ex Vivo by Combined Treatment With {delta}-Protein Kinase C Inhibitor and {epsilon}-Protein Kinase C Activator Circulation, August 19, 2003; 108(7): 869 - 875. [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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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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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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K. A. Detillieux, F. Sheikh, E. Kardami, and P. A. Cattini Biological activities of fibroblast growth factor-2 in the adult myocardium Cardiovasc Res, January 1, 2003; 57(1): 8 - 19. [Abstract] [Full Text] [PDF]
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T. Krieg, Q. Qin, E. C. McIntosh, M. V. Cohen, and J. M. Downey ACh and adenosine activate PI3-kinase in rabbit hearts through transactivation of receptor tyrosine kinases Am J Physiol Heart Circ Physiol, December 1, 2002; 283(6): H2322 - H2330. [Abstract] [Full Text] [PDF]
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K. Shinmura, M. Nagai, K. Tamaki, M. Tani, and R. Bolli COX-2-derived prostacyclin mediates opioid-induced late phase of preconditioning in isolated rat hearts Am J Physiol Heart Circ Physiol, December 1, 2002; 283(6): H2534 - H2543. [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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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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S. Wolfrum, K. Schneider, M. Heidbreder, J. Nienstedt, P. Dominiak, and A. Dendorfer Remote preconditioning protects the heart by activating myocardial PKC{epsilon}-isoform Cardiovasc Res, August 15, 2002; 55(3): 583 - 589. [Abstract] [Full Text] [PDF]
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M. Batthish, R. J Diaz, H.-P. Zeng, P. H Backx, and G. J Wilson Pharmacological preconditioning in rabbit myocardium is blocked by chloride channel inhibition Cardiovasc Res, August 15, 2002; 55(3): 660 - 671. [Abstract] [Full Text] [PDF]
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A. T Saurin, D. J Pennington, N. J.H Raat, D. S Latchman, M. J Owen, and M. S Marber Targeted disruption of the protein kinase C epsilon gene abolishes the infarct size reduction that follows ischaemic preconditioning of isolated buffer-perfused mouse hearts Cardiovasc Res, August 15, 2002; 55(3): 672 - 680. [Abstract] [Full Text] [PDF]
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B. Dawn, H. Takano, X.-L. Tang, E. Kodani, S. Banerjee, A. Rezazadeh, Y. Qiu, and R. Bolli Role of Src protein tyrosine kinases in late preconditioning against myocardial infarction Am J Physiol Heart Circ Physiol, August 1, 2002; 283(2): H549 - H556. [Abstract] [Full Text] [PDF]
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P. Rafiee, Y. Shi, X. Kong, K. A. Pritchard Jr, J. S. Tweddell, S. B. Litwin, K. Mussatto, R. D. Jaquiss, J. Su, and J. E. Baker Activation of Protein Kinases in Chronically Hypoxic Infant Human and Rabbit Hearts: Role in Cardioprotection Circulation, July 9, 2002; 106(2): 239 - 245. [Abstract] [Full Text] [PDF]
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M. Kudo, Y. Wang, M. Xu, A. Ayub, and M. Ashraf Adenosine A1 receptor mediates late preconditioning via activation of PKC-delta signaling pathway Am J Physiol Heart Circ Physiol, July 1, 2002; 283(1): H296 - H301. [Abstract] [Full Text] [PDF]
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Y. Ohnuma, T. Miura, T. Miki, M. Tanno, A. Kuno, A. Tsuchida, and K. Shimamoto Opening of mitochondrial KATP channel occurs downstream of PKC-epsilon activation in the mechanism of preconditioning Am J Physiol Heart Circ Physiol, July 1, 2002; 283(1): H440 - H447. [Abstract] [Full Text] [PDF]
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C. M. Liedtke, C. H. C. Yun, N. Kyle, and D. Wang Protein Kinase Cepsilon -dependent Regulation of Cystic Fibrosis Transmembrane Regulator Involves Binding to a Receptor for Activated C Kinase (RACK1) and RACK1 Binding to Na+/H+ Exchange Regulatory Factor J. Biol. Chem., June 14, 2002; 277(25): 22925 - 22933. [Abstract] [Full Text] [PDF]
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Z.-Q. Jin, H.-Z. Zhou, P. Zhu, N. Honbo, D. Mochly-Rosen, R. O. Messing, E. J. Goetzl, J. S. Karliner, and M. O. Gray Cardioprotection mediated by sphingosine-1-phosphate and ganglioside GM-1 in wild-type and PKCepsilon knockout mouse hearts Am J Physiol Heart Circ Physiol, June 1, 2002; 282(6): H1970 - H1977. [Abstract] [Full Text] [PDF]
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M. Munakata, C. Stamm, I. Friehs, D. Zurakowski, D. B. Cowan, H. Cao-Danh, F. X. McGowan Jr, and P. J. del Nido Protective effects of protein kinase C during myocardial ischemia require activation of phosphatidyl-inositol specific phospholipase C Ann. Thorac. Surg., April 1, 2002; 73(4): 1236 - 1245. [Abstract] [Full Text] [PDF]
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H. Liu, H. Y. Zhang, X. Zhu, Z. Shao, and Z. Yao Preconditioning blocks cardiocyte apoptosis: role of KATP channels and PKC-epsilon Am J Physiol Heart Circ Physiol, April 1, 2002; 282(4): H1380 - H1386. [Abstract] [Full Text] [PDF]
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