| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
mediates adenosine A3 receptor-induced delayed cardioprotection in mouse
Division of Cardiology, Department of Medicine, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298
Submitted 30 January 2003 ; accepted in final form 14 March 2003
 |
ABSTRACT |
|---|
 TOP ABSTRACT METHODSRESULTSDISCUSSIONREFERENCES |
|---|
isoforms, respectively. IB-MECA caused postischemic reduction
in necrosis and improvement in ventricular function, which was abolished by
CHE. Western blot analysis demonstrated translocation of the PKC-
isoform but not the 
, 
, 
, 
isoform(s) from cytoplasm to
the membrane fraction after 30 min of IB-MECA administration. A3AR
antagonist MRS-1191 and CHE blocked the translocation of PKC-.
Furthermore, IB-MECA-induced increase in nuclear factor-
B binding was
diminished by CHE. These results provide direct evidence of an essential role
of PKC, and more specifically, PKC- in A3AR-induced delayed
cardioprotection.
adenosine; nuclear factor -B; ischemia; reperfusion
B (NF-B), gene
transcription of inducible nitric oxide (NO) synthase (iNOS), synthesis of NO,
and opening of the mitochondrial ATP-sensitive K (KATP) channel
(42). However, the upstream
signaling events involved in A3AR activation of NF-B leading
to the delayed cardioprotection are not known.
Considerable evidence now supports the involvement of protein kinase C
(PKC) in both the early and delayed preconditioning
(3,
26). The initial
preconditioning stimulus with brief episodes of ischemia induced selective
translocation of novel PKC isoforms and from the cytosolic to the
particulate fraction. Chelerythrine, the PKC inhibitor, blocked translocation
of the -isoform and abolished delayed preconditioning in the heart
(34,
35). In addition to
chelerythrine, another PKC inhibitor calphostin blocked the adenosine-induced
protective effect in chick embryonic myocytes, although the specific
isoform(s) involved was not determined
(21). The current study was
designed to answer the following questions: 1) whether
A3AR stimulation with IB-MECA results in translocation of specific
isoform(s) from cytosol to the membrane fractions; 2) whether
IB-MECA-induced translocation of PKC isoform(s) as well as delayed
cardioprotection is abolished by inhibition of PKC; and finally, 3)
to show whether the inhibition of PKC diminishes nuclear translocation of
NF-B, the transcription factor involved in delayed cardioprotection
with A3AR stimulation
(30,
42) or ischemic
preconditioning (20,
40). The preliminary results
of this study were presented at the Scientific Sessions of the American Heart
Association (44).
|
METHODS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Isolated perfused heart and measurement of cardiac function. The
methodology of Langendorff-perfused mouse heart preparation, measurement of
contractile function, and infarct size were described previously in detail
(46). Briefly, a left atrial
incision was made to expose the mitral annulas through which a water-filled
latex balloon was passed into the left ventricle (LV). The balloon was
attached via polyethylene tubing to a Gould pressure transducer that was
connected to a Sensormedics polygraph recorder (model RF511A) and a heart
performance analyzer (HPA-100, Micro-Med). The balloon was inflated to adjust
the LV end-diastolic pressure (LVEDP) to 
10 mmHg. Myocardial ischemic
damage was measured using multiple, independent end points of tissue injury.
These included infarct size, LV developed pressure (LVDP), LVEDP,
rate-pressure product (RPP), heart rate, and coronary flow. LVDP was
calculated by subtracting LVEDP from the peak systolic pressure. RPP, an index
of cardiac work, was calculated by multiplying LVDP with heart rate.
Experimental protocol A. Mice were randomized to receive various
treatments by intraperitoneal injection of drugs or vehicle 24 h before being
euthanized and assigned into one of the six groups. Group 1: vehicle
(n = 7), treatment with 5% DMSO (0.1 ml); group 2: IB-MECA
(n = 8), treatment with selective A3AR agonist IB-MECA
(0.1 mg/kg); group 3: chelerythrine + IB-MECA (n = 6), PKC
antagonist cherelythrine (5.0 mg/kg) was administered 30 min before IB-MECA;
group 4: chelerythrine control (n = 6), pretreatment with
chelerythrine alone (5.0 mg/kg); group 5: rottlerin + IB-MECA
(n = 6), PKC- inhibitor rottlerin (0.3 mg/kg) was injected 30
min before IB-MECA; and group 6: rottlerin control (n = 6),
mice treated with rottlerin only.
Experimental protocol B. A subset of mice was treated with either DMSO or IB-MECA. At 30, 60, and 240 min post-IB-MECA treatment, LV samples were harvested and stored frozen at -70°C until analyzed for PKC isoforms in the cytosolic and particulate fractions.
Experimental protocol C. The goal of these experiments was to determine whether IB-MECA-induced PKC translocation would be prevented by A3AR antagonist MRS 1191 and chelerythrine. Group 1 and group 2 received 5% DMSO (0.1 ml ip) or IB-MECA (0.1 mg/kg), respectively. Group 3 was the same as group 2 except that MRS 1191 (0.1 mg/kg) was given 30 min before IB-MECA. Group 4 was the same as group 2 except that chelerythrine (5.0 mg/kg) was given 30 min before IB-MECA. Tissue samples were harvested 30 min later for measurement of PKC translocation in the cytosolic and particulate fractions.
Isolation of cytosolic and particulate fractions. The cytosolic and membrane fractions were prepared according to the method of Henry et al. (15). Briefly, frozen tissue samples were ground in liquid nitrogen, lysed, and suspended in buffer A containing (in mmol/l) glycerophosphate 50, EDTA 1, EGTA 20, PMSF 1, leupeptin 0.1, E-64 0.01, CaCl2 0.34, and sucrose 250, along with 0.05% (wt/vol) digitonin. The homogenates were centrifuged at 10,000 g for 2 min, and the supernatant containing the cytosolic proteins was saved. The pellet was resuspended in 200 µl at 4°C in buffer B containing (mmol/l) glycerophosphate 50, EDTA 1, EGTA 20, PMSF 1, leupeptin 0.1, E-64 0.01, along with 1% (vol/vol) Triton X-100. After centrifugation at 10,000 g for 15 min, the supernatant representing the crude membrane fraction was collected. The protein content was determined using the DC-protein assay (Bio-Rad).
PKC Western blot analysis. Cytosolic and membrane fractions were
separated by 10% SDS-PAGE. The proteins were transferred on to a
nitrocellulose membrane and subsequently blocked with 5% nonfat dry milk in
1x Tris-buffered saline containing 0.5% Tween 20 for 1 h. Antibodies
against PKC-, -, -, -, and - (Santa Cruze
Biotechnology) were used to assess the expression of individual PKC isoforms.
The gels were incubated with the respective primary antibodies (1:1,000
dilution) and washed and visualized by incubation with anti-rabbit horseradish
peroxidase-conjugated secondary antibody (1:1,000 dilution, Hercules, CA) for
1 h. The PKC immunoblots were developed using ECL chemiluminescence.
Preparation of nuclear extracts. Nuclear extracts were prepared using modification of the method described as previously (45). Briefly, the tissue samples were pulverized in liquid nitrogen and then lysed twice with 1 ml of lysis buffer containing 20 mM Tris (pH 7.9), 140 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 1 mM EDTA, 1 mM DTT, 0.5% Nonidet-40 (NP-40), and 0.5 mM of sodium orthovanadate and protease inhibitor (aprotinin, leupeptin, and PMSF). The nuclei were washed once with 1 ml of lysis buffer without NP-40 and resuspended in 150 µl of nuclear extraction buffer (in mM: 50 Tris·HCl, pH 7.9, 60 KCl, 1 EDTA, 1 EGTA, 2 DTT, 1 PMSF, and 0.5 sodium orthavanadate). After three freeze-thaw cycles, the nuclear extracts were obtained by centrifugation at 10,000 g for 15 min.
Electrophoretic mobility shift assay. A double-stranded 22-mer
oligonucleotide with the sequence 5'-AGT TGA GGG GAC TTT AGG C-3'
(Promega) was end-labeled using [
-32P]ATP (ICN) and T4
polynucleotide kinase according to the manufacturer's instructions. This
oligonucleotide has the consensus sequence for NF-B binding, as
indicated by underlined sequences. The binding reactions were performed in a
final volume of 20 µl containing 10 µg protein, 5% glycerol, 1 µg of
poly(dI-dC), and 0.1 ng 32P-labeled NF-B oligonucleotide.
The reaction mixture was incubated for 30 min at room temperature. The
specific protein-DNA complexes were then separated on 5% polyacrylamide gel
electrophoresis in 0.5x Tris-borate-EDTA buffer at 40 V. The gel was
vacuum dried and exposed to X-ray film at -70°C.
Statistics. All measurements are expressed as means ± SE. The data were analyzed by either unpaired t-test or one-way ANOVA. If a significant value of F was obtained in ANOVA, the Student-Newman-Keuls post hoc test was further used for pair-wise comparisons. A value of P < 0.05 was considered significant.
|
RESULTS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
, , , , and
were expressed in both cytosolic and membrane fractions. The expression of
these isoforms was generally higher in the cytoplasm compared with the
membrane fraction (Fig.
1B). Mice treated with IB-MECA demonstrated no
discernible effect on the subcellular distribution of , ,
and PKC isoforms compared with the vehicle (5% DMSO). In contrast,
PKC- decreased in the cytosolic fraction with a concomitant increase in
the membranous fraction within 30 min after administration of IB-MECA
(Fig. 2). Quantitative analysis
showed an increase of PKC- in the particulate fraction from 29.0
± 7% of the total in the untreated control heart to 51.0 ± 2.2%
in IB-MECA-treated hearts after 30 min (P < 0.05). In the
cytosolic fraction, the level of this isoform decreased from 70.0 ±
7.0% of the total in controls to 48 ± 1.8% in IB-MECA-treated mice. The
translocation was blocked by MRS-1191 as well as chelerythrine. PKC- in
membrane fraction decreased from 51 ± 2.2% of the total in
IB-MECA-treated hearts to 17 ± 9.0% in MRS-1191/IB-MECA and 35.0
± 2.7% of total in chelerythrine-IB-MECA group after 30 min
(Fig. 3). Additionally,
pretreatment of animals with chelerythrine and MRS-1191 increased the
cytosolic PKC- from 48.0 ± 1.8% of total in IB-MECA-treated
hearts to 64.0 ± 2.2% and 83.0 ± 9.0%, respectively.
|
|
|
NF-B DNA binding. NF- B binding was low in
the control group, which increased rapidly following treatment with IB-MECA
(Fig. 4). Both chelerythrine
and rottlerin completely blocked IB-MECA-induced NF-B binding
activity.
|
Infarct size. Representative sections of the hearts from mice treated with vehicle or IB-MECA are shown in Fig. 5. IB-MECA-treated mice demonstrated a significantly larger area of viable tissue in the postischemic heart (brick red color) compared with the vehicle-treated controls, which had much larger gray and white areas. Quantitatively, the infarct size was 29.9 ± 2.4% in the vehicle-treated hearts, which reduced to 11.3 ± 0.9% after 24 h of treatment with IB-MECA (P < 0.05, Fig. 6). Chelerythrine abolished the protective effect as shown by the increase in the infarct size to 23.7 ± 3.9% (P > 0.05 vs. vehicle). Chelerythrine had no effect on infarct size in the control heart, i.e., 29.8 ± 3. 0% vs. 29.9 ± 2.4% vehicle-treated hearts (P > 0.05). In addition, rottlerin completely blocked the infarct-limiting effect of IB-MECA, i.e., 24.0 ± 3.1% vs. 11.3 ± 0.9% with IB-MECA (P < 0.05), the reduction in infarct size being equivalent to chelerythrine. Rottlerin itself had no effect on infarct size in the control heart (32.0 ± 4.5% vs. 29.9 ± 2.4% in the vehicle group).
|
|
Ventricular contractile function. As shown in Table 1, the baseline functional parameters, including LVSP, LVEDP, RPP, heart rate, and coronary flow were not significantly different between groups. At the end of reperfusion, RPP (in mmHg/s) was 19.2 ± 2.3 x 103 in the vehicle group, which improved marginally to 28.2 ± 5.6 x 103 with IB-MECA (Fig. 7A). Chelerythrine blocked such improvement in RPP (P ≤ 0.05 vs. IB-MECA-treated hearts), although this inhibitor alone was found to have depressive effect. The reason for the inhibitory effect of chelerythrine on RPP is not clear from this study. Similarly, rottlerin abolished IB-MECA-induced marginal improvement in RPP (8.0 ± 3.5 x103 vs. 28.2 ± 5.6 x 103 with IB-MECA), whereas rottlerin itself had no significant effect in the control group (21.8 ± 7.5 x 103 vs. 19.2 ± 2.3 x 103 in the vehicle group). The postischemic LVEDP (in mmHg) improved in the IB-MECA-treated group (4.5 ± 1.4) compared with the vehicle group (24.5 ± 3.2) (P < 0.05 Fig. 7B). Both chelerythrine and rottlerin abrogated IB-MECA-induced improvement in the recovery of LVEDP (24.8 ± 2.2 and 31.1 ± 3.1 vs. 4.5 ± 1.4 mmHg in IB-MECA group, P < 0.05). In addition, chelerythrine and rottlerin had no effect on LVEDP in the control hearts (28.0 ± 4.9 and 21.9 ± 4.5 mmHg vs. 24.0 ± 3.7 mmHg, P > 0.05). An identical trend in the changes in developed pressure was observed. Postischemic recovery of heart rate and coronary flow was similar in all the groups (not shown).
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
from cytosolic to the
membrane fraction, which was inhibited by A3AR antagonist MRS-1191
as well as chelerythrine, confirming that stimulation of this receptor subtype
is an essential mechanism of translocation. No changes in other PKC isoforms,
, , , and 
were observed in the subcellular fractions.
The delayed cardioprotection was abolished by rottlerin and chelerythrine with
equivalent potency. In addition, chelerythrine inhibited NF-B
activation, which is known to play an important role in
A3AR-induced delayed cardioprotection
(42). To our knowledge, this
is the first study demonstrating an essential role of PKC- in cellular
signaling that leads to the delayed protective effect of A3AR in
the mouse heart.
PKC plays an important role in signal transduction in delayed myocardial
preconditioning triggered by stimulation of A1AR against myocardial
infarction (18). Ping et al.
(35) demonstrated expression
and subcellular distribution of 10 PKC isoforms in the rabbit heart.
Translocation of PKC isoform(s) from the cytosolic to the particulate fraction
(including sarcolemmal, mitochondrial, as well as nuclear fractions) result in
their binding to specific receptors of activated C kinase (RACKs) localized in
membranes (32). Translocated
specific PKC isoforms are believed to participate in several functions,
including the opening of mitochondrial KATP channels or the
induction of gene expression
(35,
39). However, the specific
isoform(s) involved in cardioprotection remain controversial. It has been
shown that translocation of PKC-, -, and - may mediate the
cardioprotective effect of ischemic preconditioning in rats and rabbits
(16,
35). Also, the PKC-
isoform appears to be more important in pharmacological preconditioning
induced by activation of adenosine A1 receptor
(18),
1-opioid receptor agonists
(12), and
1-receptor agonist phenylephrine in the rat heart
(29). Chen et al.
(7) reported opposing effects
of PKC- and PKC- isoforms, i.e., inhibiting PKC- and
activating PKC- reduced damage from simulated ischemia. In the present
study, stimulation of A3AR with IB-MECA had no effect on
subcellular translocation of PKC-. This discrepancy may be explained by
species differences, i.e., mouse versus rabbit or possibly due to different
preconditioning stimulus (A3AR stimulation versus ischemic
preconditioning). Nevertheless, our results showing translocation of
PKC- coupled with inhibition of the delayed cardioprotective effect
with chelerythrine and rottlerin confirm that A3AR mediates a
delayed protective effect through the PKC- isoform. We used rottlerin
in this study because it is reported to selectively inhibit PKC- with
an IC50 value of 3–6 µM, and IC50 for the
inhibition of PKC-, -
, -, -, -, and - are
in the much higher range, i.e., 80–100 µM
(13).
Exactly how A3AR agonist causes activation of PKC- is not
clear from the present study. One possibility is the generation of reactive
oxygen species (19), which
initiate preconditioning with pharmacological agonists, including
acetylcholine, bradykinin, opioids, and phenylephrine
(8). In these studies,
protection by adenosine or its analog
N(6)-(2-phenylisopropyl)adenosine was not be blocked by
mercaptopropionylglycine, a putative intracellular antioxidant. However, a
preliminary study from our laboratory has shown that the delayed
cardioprotective effect of IB-MECA is abolished by prior treatment with the
intracellular antioxidant mercaptopropionylglycine
(43). Stimulation of
A3AR has also been shown to increase intracellular free calcium
concentration (18), which may
activate the calcium-sensitive PKC-. It has been reported that
PKC- translocated to the cell membrane during ischemic preconditioning
and high Ca2+ preconditioning
(30,
39). PKC- is a novel
type of PKC that is activated by diacylgycerol but is unresponsive to
Ca2+ (17). Lack of
translocation of PKC-, -, -, and - in the membrane
fractions following a bolus dose of IB-MECA in the present studies ruled out
the role of these isoforms in A3AR-induced delayed
cardioprotection.
The downstream mechanism by which PKC activation confers delayed protection
may involve activation of other kinases, including ERK1/2 and p38, which are
known to be involved in the ischemic or pharmacological preconditioning
(9,
31,
33,
41,
45). It has been shown that
A3AR activation with its agonist
5'-N-ethylcarboxamidoadenosine induces phosphorylation and
activation of ERK1/2 in Chinese hamster ovary cells expressing the human
A3AR (CHO A3 cells) with the same potency
(36). Activation of
transcription factor NF-B by IB-MECA was dependent on PKC because
chelerythrine diminished the DNA binding activity in the present studies.
Moreover, stimulation of A3AR induced rapid DNA binding of
NF-B within 30 min, which coincided with translocation of PKC-
in the membrane fraction observed in the present studies. These data appear to
be in agreement with the observations that specific inhibition of PKC-
by rottlerin prevented I-B degradation and NF-B
activation in TNF-stimulated neutrophils
(38).
In summary, for the first time, we have demonstrated an essential role of
PKC- in the delayed cardioprotection triggered by stimulation of
A3AR in the mouse. The selective early translocation of PKC-
in the membrane fraction may have initiated downstream signaling involving NO
generation and opening of the mitochondrial KATP channels, the
suspected mediator and effectors of delayed pharmacological preconditioning in
the heart.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
|
REFERENCES |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
PKC and PKC. Proc
Natl Acad Sci USA 98:
11114-11119, 2001. in
opioid-initiated cardioprotection. Am J Physiol Heart Circ
Physiol 280:
H1346-H1353, 2001. and -, which mediate functional protection in
isolated rat heart. Am J Physiol Heart Circ Physiol
275: H2266-H2271,
1998. in human neutrophils. J
Immunol 157:
4651-4647, 1996.
B activation during ischemia/reperfusion: friend and
foe. J Mol Cell Cardiol 34:
1301-1304, 2002.[Web of Science][Medline]
B cctivation in tumor necrosis
factor -stimulated neutrophils is mediated by protein kinase C-.
J Biol Chem 276:
19746-19752, 2002.
B plays an essential role in the late phase of ischemic
preconditioning in conscious rabbits. Circ Res
84: 1095-1109,
1999.B, iNOS and mitochondrial
KATP channel. J Mol Cell Cardiol
34: 263-277,
2002.[Web of Science][Medline]
B and iNOS.
Circ Res 89:
915-922, 2001.This article has been cited by other articles:
|
|
 |
|
  T. C. Zhao, A. Tseng, N. Yano, Y. Tseng, P. A. Davol, R. J. Lee, L. G. Lum, and J. F. Padbury Targeting human CD34+ hematopoietic stem cells with anti-CD45 x anti-myosin light-chain bispecific antibody preserves cardiac function in myocardial infarction J Appl Physiol, June 1, 2008; 104(6): 1793 - 1800. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Zheng, R. Wang, E. Zambraski, D. Wu, K. A. Jacobson, and B. T. Liang Protective roles of adenosine A1, A2A, and A3 receptors in skeletal muscle ischemia and reperfusion injury Am J Physiol Heart Circ Physiol, December 1, 2007; 293(6): H3685 - H3691. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. W. J. Young, C.-X. Sun, C. M. Evans, B. F. Dickey, and M. R. Blackburn A3 Adenosine Receptor Signaling Contributes to Airway Mucin Secretion after Allergen Challenge Am. J. Respir. Cell Mol. Biol., November 1, 2006; 35(5): 549 - 558. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 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]
|
|
|
|
|
|
J. Neckar, I. Markova, F. Novak, O. Novakova, O. Szarszoi, B. Ost'adal, and F. Kolar Increased expression and altered subcellular distribution of PKC-{delta} in chronically hypoxic rat myocardium: involvement in cardioprotection Am J Physiol Heart Circ Physiol, April 1, 2005; 288(4): H1566 - H1572. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
K. Miyazaki, S. Komatsu, M. Ikebe, R. A. Fenton, and J. G. Dobson Jr. Protein kinase C{epsilon} and the antiadrenergic action of adenosine in rat ventricular myocytes Am J Physiol Heart Circ Physiol, October 1, 2004; 287(4): H1721 - H1729. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
