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Am J Physiol Heart Circ Physiol 275: H2266-H2271, 1998;
0363-6135/98 $5.00
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Vol. 275, Issue 6, H2266-H2271, December 1998

Ischemic preconditioning translocates PKC-delta and -epsilon , which mediate functional protection in isolated rat heart

Shuji Kawamura1, Ken-Ichi Yoshida2, Toshiro Miura1, Yoichi Mizukami2, and Masunori Matsuzaki1

Departments of 1 Internal Medicine and 2 Legal Medicine, Yamaguchi University School of Medicine, Yamaguchi 755-8505, Japan

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

Protein kinase C (PKC) plays an important role in mediating ischemic preconditioning (PC). However, the relationship between PKC isoforms and PC is still uncertain. We analyzed subcellular localization of PKC isoforms by Western blot analysis in isolated rat heart and demonstrate that PKC-alpha , -delta , and -epsilon were translocated to the membrane fraction associated with the improvement of cardiac function. Translocation of PKC-delta and -epsilon persisted after a 30-min period following PC, but the translocation of PKC-alpha was transient. Under low Ca2+ perfusion (0.2 mmol/l), PC improved the cardiac function associated with the translocation of PKC-delta . Chelerythrine (1.0 µmol/l) suppressed the translocation of all PKC isoforms associated with the loss of improvement of the cardiac function. On the other hand, bisindolylmaleimide (0.1 µmol/l) did not inhibit the improvement of cardiac function induced by PC, which was associated with the translocation of PKC-epsilon . These results indicate that the effect of PC on cardiac function is mediated by the translocation of either PKC-delta or -epsilon independently in rat hearts.

ischemia; protein kinase C isoforms; contractile function; low calcium perfusion; protein kinase C inhibitors

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

ISCHEMIC PRECONDITIONING (20) is a powerful strategy for protecting hearts against ischemic injury in various species (1, 5, 13, 26). Recently, the mechanism of ischemic preconditioning has been shown to be mediated by the activation of protein kinase C (PKC) in rat (17, 28), rabbit (15, 36), dog (8), and human hearts (27).

PKC, a serine/threonine kinase, plays an important role in signal transduction (22). Several extracellular stimuli [e.g., angiotensin II (25), catecholamines (7, 31), and adenosine (10)] to myocytes induce activation of phospholipases C and D, both of which hydrolyze membrane phospholipids, resulting in the generation of diacylglycerol. Diacylglycerol activates PKC with or without calcium ions (21, 22). On activation, PKC translocates to the plasma membrane, nuclei, and cytoskeletal elements. PKC isoforms in the adult rat heart are subdivided into three groups: 1) classic PKC (i.e., PKC-alpha ), which requires calcium, diacylglycerol, and phosphatidylserine for its activation; 2) novel PKC (i.e., PKC-delta and -epsilon ), which requires diacylglycerol and phosphatidylserine, but not calcium, for its activation; and 3) atypical PKC (i.e., PKC-zeta ), which requires phosphatidylserine but neither calcium nor diacylglycerol for its activation (22). Although the activation of PKC is important in the mechanism of ischemic preconditioning, the role of each isoform remains to be elucidated. Recently, immunohistochemical studies (17, 18) have shown that ischemic preconditioning translocated PKC-alpha and -delta from cytosol to the plasma membrane. However, no quantitative evaluation of the isoform translocation during ischemic preconditioning has been performed using Western blot analysis.

Thus this study was designed to identify which PKC isoforms are involved in ischemic preconditioning using Western blot analysis in isolated rat hearts. The selective PKC inhibitors and low calcium conditions during ischemic preconditioning were utilized to differentiate the role of PKC isoforms.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Materials

Monoclonal antibodies to PKC-alpha and -zeta were purchased from Transduction Laboratories (Lexington, KY). Polyclonal antibodies to PKC-delta and -epsilon were purchased from GIBCO BRL (Rockville, MD). Chelerythrine and bisindolylmaleimide were purchased from Calbiochem (La Jolla, CA). Reagents for Western blot detection by enhanced chemiluminescence (ECL) were purchased from Pierce (Rockford, IL). Horseradish peroxidase-linked donkey anti-rabbit or anti-mouse immunoglobulin and nitrocellulose membranes (Hybond-C, 0.45 µm) were purchased from Amersham (Arlington Heights, IL). Other chemicals were purchased from Nacalai Tesque (Kyoto, Japan) or Sigma Chemical (St. Louis, MO).

Perfusion Protocol

The experimental protocol was approved by the Animal Care and Use Committee of the Yamaguchi University School of Medicine, and the experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Male Wistar rats weighing 260 to 320 g were anesthetized with pentobarbital sodium (60 mg/kg ip) and heparin (300 IU ip). Hearts were prepared for Langendorff mode as described previously (35). Briefly, the heart was perfused with filtered Krebs-Henseleit buffer containing (in mmol/l) 4.7 KCl, 2.5 CaCl2, 1.25 MgCl2, 1.25 KH2PO4, 25 NaHCO3, 118 NaCl, and 10 glucose. The buffer was maintained at 37°C and gassed with 95% O2 and 5% CO2. The heart was perfused at a constant perfusion pressure of 100 cmH2O. A fluid-filled latex balloon connected to a pressure transducer was inserted into the left ventricle. The balloon was inflated to the pressure of 5 mmHg at end-diastole. The left ventricular pressure was recorded on MacLab (AD Instruments), and the hemodynamic parameters were calculated. The hearts were stabilized for 10 min before the protocols were started.

Experimental Protocols

Two series of experiments were performed: one for hemodynamic evaluation, and the other for estimating the translocation of PKC isoforms. The protocols for the hemodynamic study were performed in the following groups, as shown in Fig. 1.


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  		Fig. 1.   Experimental protocols for hemodynamic evaluation are shown. In separate series of experiments for translocation of protein kinase C (PKC) isoforms, rats were killed just before 20-min ischemia. P, ischemic preconditioning.

Group 1: Control. After time-matched perfusion to preconditioning periods, hearts were subjected to 20 min of no-flow ischemia followed by a 30-min period of reperfusion (n = 7).

Group 2: Preconditioning. Three minutes of no-flow ischemia followed by a 5-min period of reperfusion was repeated three times before a 20-min period of no-flow ischemia, followed by a 30-min period of reperfusion (n = 8).

Group 3: Preconditioning with PKC inhibitors. After stabilization, chelerythrine (1.0 µmol/l, n = 5) or bisindolylmaleimide (0.1 µmol/l, n = 5) was administered 3 min before the preconditioning, followed by a 20-min period of ischemia and a 30-min period of reperfusion.

Group 4: Preconditioning with low Ca2+ perfusion. The hearts were perfused with low Ca2+ (0.2 mmol/l)-containing buffer started 3 min before and during the preconditioning, followed by a 20-min period of ischemia and a 30-min period of reperfusion. (n = 4).

Group 5: Preconditioning with time intervals. Intervals of 10 (n = 4) and 30 min (n = 4) were allowed after the preconditioning followed by a 20-min period of ischemia and a 30-min period of reperfusion.

In the study of PKC isoform translocation, myocardial samples were obtained just before the creation of the 20-min ischemia in all groups (n = 7 for control and preconditioning groups, n = 6 for other groups). The atrial and right ventricular tissues were removed and the left ventricle was quickly frozen in liquid nitrogen and stored at -80°C until use.

Subcellular Fractionation

The frozen heart samples were minced and homogenized in 5 volumes of STE buffer containing 320 mmol/l sucrose, 10 mmol/l Tris · HCl, pH 7.4, 1 mmol/l EGTA, 5 mmol/l NaN3, 10 mmol/l beta -mercaptoethanol, 20 µmol/l leupeptin, 0.15 µmol/l pepstatin A, 0.2 mmol/l phenylmethylsulfonyl fluoride, and 50 mmol/l NaF, with a Polytron homogenizer (PT1200, Kinematica) at its maximum speed for 30 s, repeated three times. Homogenates were mixed with an equal volume of STE buffer and centrifuged at 1,000 g for 10 min, and the supernatant was centrifuged at 100,000 g for 60 min. The 1,000-g and 100,000-g pellets were designated as P1 and P2 fractions, respectively, and the 100,000-g supernatant as S fraction. The P1 and P2 fractions were resuspended in STE buffer. The total protein concentration in each fraction was determined by the method of Lowry et al. (16), using bovine serum albumin as a standard.

Immunoblotting and Quantitation of PKC Isoforms

Tissue extracts were boiled with 0.33-1 volume of 10% (wt/vol) SDS, 13% (vol/vol) glycerol, 300 mmol/l Tris · HCl, 130 mmol/l dithiothreitol, and 0.2% (wt/vol) bromophenol blue, pH 6.8. An equal amount of total protein (20 µg) in each fraction was loaded on 8.5% SDS-polyacrylamide gel electrophoresis by the method of Laemmli (11) and transferred electrophoretically to a nitrocellulose membrane according to Towbin et al. (30). The nitrocellulose membrane was blocked with 5% nonfat milk in TBS buffer containing 150 mmol/l NaCl, 10 mmol/l Tris · HCl, pH 7.4, and 0.05% Tween-20 for at least 1 h to block nonspecific binding sites. Primary antibodies were diluted in TBS buffer with 1% bovine serum albumin (1:1,000 for each). The nitrocellulose membrane was incubated with primary antibodies for 2 h at room temperature. After being washed in TBS (4 times for 7 min each), the nitrocellulose membrane was incubated for 0.5 h at room temperature with horseradish peroxidase-linked secondary antibodies (1:5,000 for PKC-delta and -epsilon , 1:10,000 for PKC-alpha and -zeta in TBS with 1% bovine serum albumin). After being washed in TBS (3 times for 5 min each), PKC isoforms were detected by ECL method with an exposure to Hyperfilm for 2-10 min. The amounts of PKC isoforms on the immunoblots were quantitated by laser densitometry (Densitograph AE-6900, Atto), and the densitometric intensity was normalized by multiplying the total protein concentration in each fraction.

Statistics

Differences in hemodynamic parameters and the time course of the distribution of PKC isoforms were analyzed by a two-way analysis of variance, followed by a post hoc Fishers test when appropriate. The effects of various interventions on each PKC isoform were tested by one-way analysis of variance followed by a post hoc Fishers test when appropriate. The translocation of each PKC isoform by ischemic preconditioning was tested by a Student's t-test. A P value of <0.05 was considered significant. All values are expressed as means ± SE.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Effects of Preconditioning on Postischemic Cardiac Function

Table 1 shows hemodynamic data. Significant improvement in the left ventricular developed pressure (LVDP) was observed after ischemic preconditioning. This effect of the preconditioning persisted for at least 30 min after the preconditioning. The improvement in LVDP was observed after ischemic preconditioning under low Ca2+ perfusion. On the other hand, chelerythrine completely suppressed the beneficial effect of preconditioning on LVDP, whereas bisindolylmaleimide did not abolish the preconditioning effect on LVDP at the dose used in the present study. The hemodynamic parameters did not differ significantly at any stages among the groups, except the heart rate in the chelerythrine-treated group, which was lower than that in the control group after a 30-min period of reperfusion.

                              
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  		Table 1.   Hemodynamic data

Translocation of PKC Isoforms after Preconditioning

Figure 2A shows representative data of Western blots indicating the distribution of PKC isoforms before and after the ischemic preconditioning. Figure 2B shows the quantitative data of each PKC isoform content in each fraction. The translocation of PKC-alpha , -delta , and -epsilon from cytosolic fraction(s) to the membrane fraction (P2) was observed after the ischemic preconditioning, whereas no significant translocation of PKC-zeta was observed. Figure 3 shows the content of PKC isoforms in the membrane fraction in various conditions, indicating the extent of translocation of the isoforms. Ischemic preconditioning significantly increased the content of PKC-alpha , -delta , and -epsilon in the membrane fraction compared with that of the control. Ischemic preconditioning under low Ca2+ perfusion inhibited the translocation of PKC-alpha and -epsilon but did not affect that of PKC-delta . Chelerythrine (1.0 µmol/l) completely suppressed the translocation of PKC-alpha , -delta , and -epsilon , whereas bisindolylmaleimide (0.1 µmol/l) did not suppress the translocation of PKC-epsilon . The effect of intervals after the ischemic preconditioning on the translocation of PKC isoforms is shown in Fig. 4. With oxygenated perfusion for 10 min after ischemic preconditioning, PKC-alpha rapidly dissociated from the membrane fraction; however, membrane-associated PKC-delta and -epsilon were retained even after a 30-min period.


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  		Fig. 2.   Distribution of PKC isoforms after ischemic preconditioning (PC). A: Western blotting of PKC isoforms. Fractions (20 µg/lane) of control (-) and preconditioned (+) hearts were resolved by SDS-PAGE, transferred to nitrocellulose membrane, and probed with each specific anti-PKC isoform antibody. P1, particulate nuclear fraction; P2, particulate membrane fraction; S, soluble fraction. Arrows indicate band of each isoform. Molecular size markers are indicated on right. B: subcellular localization of PKC isoforms quantitated by densitometry in P1, P2, and S fractions. Densitometric intensity was normalized by multiplying total protein concentration in each fraction. Data are presented as means ± SE. Open bars, control; filled bars, ischemic preconditioning. * P < 0.05 vs. control.


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  		Fig. 3.   Effects of various conditions on distribution of PKC isoforms in P2 fraction. Percent changes of PKC isoforms in P2 fraction measured by densitometry are shown. Data are presented as means ± SE. Chele, chelerythrine; bis, bisindolylmaleimide. * P < 0.05 vs. control.


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  		Fig. 4.   Time course of distribution of PKC isoforms after preconditioning in P2 fraction. Percent changes of 4 PKC isoforms in P2 fraction at 0, 10, and 30 min after ischemic preconditioning are shown. , PKC-alpha ; bullet , PKC-delta ; , PKC-epsilon ; open circle , PKC-zeta . Data are presented as means ± SE. * P < 0.05 vs. baseline.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

In the present study, we studied the distribution of four PKC isoforms (alpha , delta , epsilon , and zeta ) of rat heart by Western blot analysis and demonstrate that ischemic preconditioning translocates PKC-alpha , -delta , and -epsilon from cytosolic to membrane fraction. The translocation of these isoforms is related to the improvement of postischemic cardiac function. Subsequently, we demonstrate that ischemic preconditioning under low Ca2+ perfusion suppressed translocation of PKC-alpha and PKC-epsilon , whereas it did not suppress translocation of PKC-delta . Under this condition, the postischemic cardiac function was still improved by the ischemic preconditioning. On the other hand, the translocation of all isoforms by ischemic preconditioning was suppressed by the pretreatment of chelerythrine, and this abolished the improvement of postischemic cardiac function. These results indicate that the translocation of calcium-independent PKC-delta is essential for mediating ischemic preconditioning on cardiac function. This finding was consistent with the study done by Mitchell et al. (17), who showed the importance of PKC-delta translocation to the membrane. Miyawaki et al. (18), however, showed the importance of PKC-alpha as well as PKC-delta in the mechanism of calcium preconditioning on the cardiac function. The difference of the preconditioning procedures may influence the importance of the PKC isoforms in mediating the protective effect. To further examine the importance of each PKC isoform, the effects of chelerythrine and bisindolylmaleimide on PKC isoforms were compared. Chelerythrine interacts with the catalytic domain of PKC (6, 9), but bisindolylmaleimide interacts with the ATP-binding site of PKC (29). Both inhibitors are more highly specific to PKC than other PKC inhibitors, such as polymyxin B, staurosporine, and H-7. In the present study, twice the IC50 of chelerythrine (6) and 10-fold that of bisindolylmaleimide (29) were used. These doses are below the threshold of lethal arrhythmogenic and cardiotoxic effects confirmed by the preliminary study. We demonstrate that bisindolylmaleimide suppressed the translocation of all isoforms except PKC-epsilon by ischemic preconditioning. This may be explained by the fact that bisindolylmaleimide has less sensitivity to PKC-epsilon than other isoforms (34). With bisindolylmaleimide, the postischemic cardiac function was still improved by ischemic preconditioning. On the other hand, chelerythrine inhibited translocation of all isoforms and abolished the improvement of postischemic cardiac function conferred by ischemic preconditioning. These results strongly indicate that PKC-epsilon is another crucial isoform mediating the effect of ischemic preconditioning. This is supported by the study done by Gray et al. (4), who showed the importance of PKC-epsilon in hypoxic preconditioning of cardiac myocytes using PKC-epsilon -selective inhibitor peptide. In other species such as rabbit, PKC-epsilon is also a crucial isoform mediating ischemic preconditioning. (23). In the present study, the effect of time interval after ischemic preconditioning on the translocation of each isoform was studied. The PKC-alpha rapidly dissociated from the membrane fraction in 10 min, whereas the membrane-associated PKC-delta and -epsilon were retained after a 30-min period. The protective effect of ischemic preconditioning also remains after a 30-min period following the preconditioning procedure. These findings further support the idea that the translocation of PKC-delta and -epsilon is more important to the functional recovery conferred by ischemic preconditioning than the transient translocation of PKC-alpha .

Many proteins are known to be phosphorylated by PKC. For example, PKC phosphorylates contractile proteins such as myosin light chain-2 and myosin light chain kinase (2, 32). Cardiac ATP-sensitive potassium channels (KATP) (12, 14), vacuolar proton ATPase (3), and 5'-nucleotidase (8) are also downstream effectors of PKC and considered to be related to ischemic preconditioning. Among these, KATP is postulated as a final end effector mediating ischemic preconditioning. The activation of KATP by a constitutively active PKC (12) or phorbol ester (14) has been demonstrated by a patch-clamp method. However, it remains unclear which PKC isoforms mediate these events, and further studies are warranted.

Limitation of the Study

Because the myocardial samples contain myocytes as well as nonmyocytes such as fibroblasts, smooth muscle cells, and endothelial cells, the results of Western blot analysis are, to some extent, contaminated by those nonmyocytes. To address this issue, it is necessary to use isolated myocytes instead of whole myocardium (3, 33). Immunohistochemical staining using isoform-specific antibody may be useful to confirm the myocyte-specific translocation of isoforms, although the quantification of the translocated isoform content is difficult.

In the present study, the role of PKC-zeta in the ischemic preconditioning was not confirmed because the translocation of the isoform is small. The expression of PKC-zeta in the adult rat heart was controversial (24), because it is highly homologous to PKC-lambda . However, we confirmed the expression of PKC-zeta in the rat heart using the peptide raised against highly conserved amino acid sequences of PKC-zeta (19).

In conclusion, we demonstrate that the translocation of calcium-independent isoforms, PKC-delta and PKC-epsilon , from cytosol to the plasma membrane is an important mechanism mediating ischemic preconditioning, each independently of the other.

    ACKNOWLEDGEMENTS

This study was supported in part by a grant from Ministry of Education, Science, and Culture of Japan.

    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. §1734 solely to indicate this fact.

Address for reprint requests: M. Matsuzaki, The Second Dept. of Internal Medicine, Yamaguchi Univ. School of Medicine, 1144 Kogushi, Ube, Yamaguchi 755-8505, Japan.

Received 19 February 1998; accepted in final form 19 August 1998.

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Methods
Results
Discussion
References

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Am J Physiol Heart Circ Physiol 275(6):H2266-H2271
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S. L. House, S. J. Melhorn, G. Newman, T. Doetschman, and J. E. J. Schultz
The protein kinase C pathway mediates cardioprotection induced by cardiac-specific overexpression of fibroblast growth factor-2
Am J Physiol Heart Circ Physiol, July 1, 2007; 293(1): H354 - H365.
[Abstract] [Full Text] [PDF]

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 Am. J. Physiol. Heart Circ. Physiol.Home page
M. A. Sovershaev, E. M. Egorina, T. V. Andreasen, A. K. Jonassen, and K. Ytrehus
Preconditioning by 17beta-estradiol in isolated rat heart depends on PI3-K/PKB pathway, PKC, and ROS
Am J Physiol Heart Circ Physiol, October 1, 2006; 291(4): H1554 - H1562.
[Abstract] [Full Text] [PDF]

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 Am. J. Physiol. Heart Circ. Physiol.Home page
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]

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 Anesth. Analg.Home page
S. Kohro, Q. H. Hogan, D. C. Warltier, and Z. J. Bosnjak
Protein Kinase C Inhibitors Produce Mitochondrial Flavoprotein Oxidation in Cardiac Myocytes
Anesth. Analg., November 1, 2004; 99(5): 1316 - 1322.
[Abstract] [Full Text] [PDF]

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 Anesth. Analg.Home page
P. Xu, J. Wang, R. Kodavatiganti, Y. Zeng, and I. S. Kass
Activation of Protein Kinase C Contributes to the Isoflurane-Induced Improvement of Functional and Metabolic Recovery in Isolated Ischemic Rat Hearts
Anesth. Analg., October 1, 2004; 99(4): 993 - 1000.
[Abstract] [Full Text] [PDF]

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 Am. J. Physiol. Heart Circ. Physiol.Home page
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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 Am. J. Physiol. Heart Circ. Physiol.Home page
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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 Am. J. Physiol. Heart Circ. Physiol.Home page
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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 Am. J. Physiol. Heart Circ. Physiol.Home page
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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 Am. J. Physiol. Heart Circ. Physiol.Home page
H. Y. Zhang, B. C. McPherson, H. Liu, T. S. Baman, P. Rock, and Z. Yao
H2O2 opens mitochondrial KATP channels and inhibits GABA receptors via protein kinase C-epsilon in cardiomyocytes
Am J Physiol Heart Circ Physiol, April 1, 2002; 282(4): H1395 - H1403.
[Abstract] [Full Text] [PDF]

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 J. Pharmacol. Exp. Ther.Home page
R. M. Fryer, Y. Wang, A. K. Hsu, H. Nagase, and G. J. Gross
Dependence of delta 1-Opioid Receptor-Induced Cardioprotection on a Tyrosine Kinase-Dependent but Not a Src-Dependent Pathway
J. Pharmacol. Exp. Ther., November 1, 2001; 299(2): 477 - 482.
[Abstract] [Full Text] [PDF]

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 J. Pharmacol. Exp. Ther.Home page
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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 Am. J. Physiol. Heart Circ. Physiol.Home page
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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 Am. J. Physiol. Heart Circ. Physiol.Home page
H. Liu, B. C. McPherson, and Z. Yao
Preconditioning attenuates apoptosis and necrosis: role of protein kinase C{epsilon} and -{delta} isoforms
Am J Physiol Heart Circ Physiol, July 1, 2001; 281(1): H404 - H410.
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 Am. J. Physiol. Heart Circ. Physiol.Home page
Z. Yao, B. C. McPherson, H. Liu, Z. Shao, C. Li, Y. Qin, T. L. Vanden Hoek, L. B. Becker, and P. T. Schumacker
Signal transduction of flumazenil-induced preconditioning in myocytes
Am J Physiol Heart Circ Physiol, March 1, 2001; 280(3): H1249 - H1255.
[Abstract] [Full Text] [PDF]

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 Am. J. Physiol. Heart Circ. Physiol.Home page
R. M. Fryer, Y. Wang, A. K. Hsu, and G. J. Gross
Essential activation of PKC-{delta} in opioid-initiated cardioprotection
Am J Physiol Heart Circ Physiol, March 1, 2001; 280(3): H1346 - H1353.
[Abstract] [Full Text] [PDF]

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 Am. J. Physiol. Heart Circ. Physiol.Home page
J. W. Lester and P. A. Hofmann
Role for PKC in the adenosine-induced decrease in shortening velocity of rat ventricular myocytes
Am J Physiol Heart Circ Physiol, December 1, 2000; 279(6): H2685 - H2693.
[Abstract] [Full Text] [PDF]

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 Am. J. Physiol. Heart Circ. Physiol.Home page
Q. Zhang and Z. Yao
Flumazenil preconditions cardiomyocytes via oxygen radicals and KATP channels
Am J Physiol Heart Circ Physiol, October 1, 2000; 279(4): H1858 - H1863.
[Abstract] [Full Text] [PDF]

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 Circ. Res.Home page
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.
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 Circ. Res.Home page
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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 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
R. A. Hopper, C. R. Forrest, H. Xu, A. Zhong, W. He, J. Rutka, P. Neligan, and C. Y. Pang
Role and mechanism of PKC in ischemic preconditioning of pig skeletal muscle against infarction
Am J Physiol Regulatory Integrative Comp Physiol, August 1, 2000; 279(2): R666 - R676.
[Abstract] [Full Text] [PDF]

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 Circ. Res.Home page
K. Naruse and G. L. King
Protein Kinase C and Myocardial Biology and Function
Circ. Res., June 9, 2000; 86(11): 1104 - 1106.
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 Circ. Res.Home page
A. Dana, M. Skarli, J. Papakrivopoulou, and D. M. Yellon
Adenosine A1 Receptor Induced Delayed Preconditioning in Rabbits : Induction of p38 Mitogen-Activated Protein Kinase Activation and Hsp27 Phosphorylation via a Tyrosine Kinase- and Protein Kinase C-Dependent Mechanism
Circ. Res., May 12, 2000; 86(9): 989 - 997.
[Abstract] [Full Text] [PDF]

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 Am. J. Physiol. Heart Circ. Physiol.Home page
T. Okamura, T. Miura, H. Iwamoto, K. Shirakawa, S. Kawamura, Y. Ikeda, M. Iwatate, and M. Matsuzaki
Ischemic preconditioning attenuates apoptosis through protein kinase C in rat hearts
Am J Physiol Heart Circ Physiol, November 1, 1999; 277(5): H1997 - H2001.
[Abstract] [Full Text] [PDF]

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