Integrative Human Cardiovascular Physiology and Cardiac Surgery Unit, Department of Cardiovascular Sciences, University of Leicester, Glenfield Hospital, Leicester LE3 9QP, United Kingdom
Submitted 16 March 2004 ; accepted in final form 8 July 2004
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
cardioprotection; protein kinase C isoforms
The putative mitochondrial potassium-sensitive ATP (mitoKATP) channels also play a key role in the signal transduction mechanism of IPC (9, 14, 33, 34, 37). It has been reported that opening of the mitoKATP channels can activate PKC by generation of radical oxygen species (33), although it has also been suggested that opening of these channels can be potentiated by PKC (34). This may suggest that PKC isoforms may exist upstream and downstream of mitoKATP channels, but direct evidence of this does not exist. The present study was designed to investigate the role of different PKC isoforms in IPC of the human myocardium and to determine how they are related to the mitoKATP channels.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The study was approved by the local ethical committee and was conducted with the consent of patients in accordance with the Helsinki Declaration. Atrial biopsies were collected from patients undergoing elective coronary artery bypass surgery and/or aortic valve surgery during cannulation of the right atrium before initiation of cardiopulmonary bypass. Patients with enlarged atriums, arrhythmias, poor left ventricular function (ejection fraction <30%), and right ventricular failure were excluded. Other criteria for exclusion were diabetes mellitus and the treatment with oral opioid analgesia, KATP channel openers, and catecholamines.
Processing of Samples and Experimental Preparation
This study was conducted by using an established model for quantification of the effect of ischemia and reperfusion on human cardiac muscle by using thin slices of atrial appendages (52). Briefly, sections 40 mg wet weight and 300- to 500-µm thick were prepared from human right atrial appendages by using a surgical skin-graft knife. Sections were immediately equilibrated for 30 min in Krebs-Henseleit HEPES buffer (pH 7.4) oxygenated by bubbling with 95% oxygen-5% carbon dioxide and maintained at 37°C throughout the experiments. Simulated ischemia was induced by bubbling the medium with 95% N2-5% CO2 (pH 6.87.0) and replacing D-glucose with 2-deoxy-D-glucose as described previously (52). IPC was induced by 5-min ischemia followed by 5-min reoxygenation, a protocol that has been shown in our laboratory to be the most protective in this preparation (10).
Measurement of Tissue Injury and Viability
Tissue injury was determined by measuring the leakage of creatine kinase (CK) into the incubation medium during the reoxygenation period, and this was assayed by a kinetic method on the basis of the reduction of NADP to NADPH. The absorbance was read at 340 nm using a Bench plate reader (Bio-Rad, Hercules, CA). The rate of change in absorbance is directly proportional to CK activity, and the results were expressed as U/g wet wt. Tissue viability was assessed by the reduction of methylthiazoletetrazolium (MTT) to a blue formazan product at the end of the experimental time (43). The absorbance of the blue formazan precipitate formed was measured at 550 nm and the results were expressed as mmol/g wet wt.
Study Protocols
Identification of PKC isoforms involved in IPC.
To identify the PKC isoforms involved in IPC, dose response studies were performed by using specific PKC inhibitors. After equilibration at 37°C in aerobic medium for 30 min, myocardial sections (n = 6 per group) were randomized to receive one of the protocols summarized in Fig. 1A. To serve as controls, some of the myocardial sections were subjected to the highest dose of the various PKC inhibitors used for 10 min followed by the induction of 90 min of simulated ischemia/120 min of reoxygenation (SI/R). The rest of the sections were treated with the various PKC inhibitors added 10 min before and 10 min during IPC followed by SI/R at various concentrations as follows: myristoylated V12 peptide (0.1, 1, 10 µM) a PKC- inhibitor; rottlerin (1, 10, 100 µM) a PKC-
inhibitor; LY-333531 (1, 10, 100 nM) a PKC-(
1 +
2) inhibitor; GO-6976 (1, 10, 100 nM), and Ro 320432 (0.1, 1, 10 µM) both PKC (
+
) inhibitors. In view of the ongoing controversy on the use of rottlerin as a specific PKC-
inhibitor, the selective PKC-
activator bistratene A (1, 10, 100 nM) was administered for 10 min before simulated ischemia/reoxygenation.
|
Phosphorylation of PKC isoforms. In this study, the phosphorylation of the PKC isoforms found to participate in IPC in the above studies was measured in myocardial sections (n = 4 per group) that were randomized after 30 min of equilibration to receive any one of the following protocols: 1) time-matched aerobic control for an additional 10 min, 2) IPC alone using 5 min of simulated ischemia followed by 5 min reoxygenation, and 3) 10 min preconditioning with diazoxide (100 µM). At the end of each protocol, the tissue sections were quickly frozen in liquid nitrogen and stored at 80°C until analysis. Samples were homogenized in RIPA buffer containing protease inhibitor, PMSF (1 mM), DDT (0.5 mM), glycerophosphate (25 µM), and sodium orthovanadate (1 mM). The homogenate was then centrifuged at 10,000 g for 30 min. The supernatant obtained was analyzed for protein concentration using the Bio-Rad protein assay kit.
Then 20 µg of the tissue supernatant were elecrophoresed on 10% SDS/PAGE and blotted onto nitrocellulose membrane. Membranes were blocked in TBS buffer containing 5% fat-free dry milk, followed by incubation with the appropriate primary antibodies at 1:1,000 dilution overnight at 4°C. Membranes were then detected by the appropriate horseradish peroxidase-conjugated secondary antibody (anti-rabbit goat antibody and anti-mouse goat antibody at 1:5,000 dilution). The signals were then developed by using an enhanced chemiluminescense Western blot analysis detection kit (Amersham, Little Chalfont, UK). Densitometry analysis of bands was performed by Scion image (Scion, Frederick, MD).
Solutions and Chemicals
Samples were incubated in Krebs medium composed of (in mM) 118 NaCl2, 4.8 KCl, 27.2 NaHCO3, 1.2 MgCl2, 1.0 KH2PO4, 1.25 CaCl2, 20 HEPES, and 10 D-glucose or 10 2-deoxy-D-glucose to induce simulated ischemia, and prepared in deionized distilled water. All of these chemicals, CK assay kit, MTT, diazoxide, and bistratene A were purchased from Sigma (St. Louis, MO). The specific PKC isoforms inhibitors GO-6976, myristoylated V12 peptide (to allow cell permeability), and rottlerin were obtained from BioMol Research Laboratories (Plymouth Meeting, PA), and LY-333531 was supplied by AG Scientific (San Diego, CA). The antibodies for PKC- and -
, phospho PKC-
(serine 657), and phospho PKC-
(serine 729) were obtained from Upstate Cell Signaling Solution (Charlottesville, VA).
Statistical Analysis
All data are presented as means ± SE. Mean values were analyzed by ANOVA with a post hoc Tukey's test. Statistical significance was at P < 0.05.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The results shown in Figs. 2 to 5 indicate that IPC significantly reduced CK leakage and increased the MTT reduction of muscles subjected to ischemia/reoxygenation. None of the PKC inhibitors (at the highest doses used) in the absence of IPC had any significant effect on tissue damage or tissue viability. They also show that the PKC-( +
) inhibitor GO-6976 at 100 nM blocked preconditioning, whereas PKC-
inhibitor LY-333531 failed to block preconditioning. The effect of PKC-(
+
) inhibition was also confirmed with the use of Ro 320432 (data not shown). This would suggest that PKC-
, but not PKC-
, is involved in signaling pathway of IPC. Furthermore, the PKC-
inhibitor V12 peptide completely blocked protection by IPC at a concentration of 10 µM, whereas the PKC-
inhibitor rottlerin had no effect at any of the study concentrations. Activation PKC-
isoforms with bistratene A failed to induce protection in our model (Fig. 6). These results indicate that PKC-
participate in the protection by IPC of the human myocardium with no involvement of PKC-
.
|
|
|
Figure 7, A and B demonstrates that, as expected, IPC and diazoxide conferred a similar degree of cardioprotection as shown by the reduction of CK leakage and increased MTT viability. As seen previously, PKC- inhibitor V12 peptide (10 µM) blocked protection by IPC, but interestingly, it did not affect the protection induced by diazoxide. This would suggest that PKC-
is upstream of mitoKATP channels. Figure 8, A and B demonstrate that the PKC-(
+
) GO-6976 (100 nM) blocked the protection seen with both IPC and diazoxide, which indicates that PKC-
is downstream of mitoKATP channels (note that the studies presented above suggested the absence of a role for the PKC-
isoforms in IPC).
|
|
To demonstrate the phosphorylation of PKC- and -
in relation to the opening of mitoKATP channels, tissue extract were detected by Western immunoblotting. As shown in Fig. 9, the basal level of 3035% phosphorylation of PKC-
and -
were observed in aerobic controls. IPC increased the phosphorylation of both PKC-
and -
; however, diazoxide caused significant increase in PKC-
phosphorylation but failed to induce a significant increase in the phosphorylation of PKC-
, which confirms that PKC-
lies upstream and PKC-
lies downstream of mitoKATP channels.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
These results in the human myocardium may find some support in reported animal studies. Thus for example, with the use of a cell culture model of hypoxic preconditioning, it was found that V12 peptide not only abolished cardioprotection but also inhibited the translocation of PKC- (13, 26). More recently, it has been shown that the disruption in the PKC-
gene abolishes the infarct size reduction of preconditioning in the isolated buffer-perfused mouse heart (35). There is also evidence that the use of a PKC-
selective activator protects the isolated perfused rat heart from ischemia/reperfusion injury (20). The above findings are consistent with the concept that activation of PKC-
is necessary for cardioprotection by IPC.
The demonstration in our studies that PKC- also participates in the cardioprotection by IPC in the human myocardium is in agreement with the demonstration that GO-6976, a potent inhibitor of PKC-
, significantly attenuates the protection of hypoxic preconditioning on cell death (42), and blocks the reduction of necrosis and apoptosis afforded by three 1-min cycles of simulated ischemia (49) in embryonic chick cardiomyocytes. Because GO-6976 is a potent inhibitor for
-isoforms and to a lesser extent of the
-isoforms, it may be postulated that the results of the above studies may be due, at least in part, to PKC-
inhibition (28). However, in our studies, we have demonstrated that in the human myocardium PKC-
inhibition by LY-333531 had no effect on preconditioning, suggesting that the effect of GO-6976 is largely attributed to the inhibition of PKC-
rather than the PKC-
isoforms.
The lack of a role for the PKC- and PKC-
isoforms in IPC of the human myocardium is also supported by similar findings in the adult rabbit cardiomyocytes (50) although conflicting results have been published regarding the role of PKC-
. In one study (6), the PKC-
inhibitor rottlerin was found to block the cardioprotection induced by the activation of opioid receptors in the in vivo rat heart model. Another study by the same investigators (5) reported that rottlerin did not abolish the reduction in infarct size induced by IPC in the same species and experimental model. The most plausible explanation for the different results is that the protection elicited by activation of opioid receptors may use different PKC isoforms than the ones used in IPC. Certainly the elucidation of this issue would require further investigations.
The identification of the sequence of the involvement of the two PKC isoforms with PKC- being upstream and PKC-
being downstream of mitoKATP channels, demonstrated by pharmacological manipulation and by biochemical means, is of biological relevance. In previous studies (27, 30, 51), the use of nonselective PKC inhibitors such as chelerythrine, staurosporin, and calphostin C were insufficient to dissect this relationship, and any conclusion on the location of PKC isoforms in relation to mitoKATP channels using these tools should be interpreted with caution. Our laboratory has previously demonstrated (27) that protection of the human myocardium can be elicited by the PKC activator PMA in the presence of the mitoKATP channel closer 5-hydroxydecanoate, suggesting the PKC is downstream of mitoKATP, but the PKC responsible isoforms and whether other isoforms were involved upstream of mitoKATP channels was not investigated. In the literature, there is also evidence that mitoKATP channels are modulated by PKC (34), supporting the theory that PKC can be located both upstream and downstream of mitoKATP channels. Our finding that PKC-
is phosphorylated on the serine 657 by IPC and diazoxide and that PKC-
is phosphorylated on serine 729 by IPC and not by diazoxide, supports the results obtained with the PKC isoform inhibitors. This argument is reinforced by the demonstration that in isolated, perfused rat hearts that diazoxide causes PKC-
translocation (46) but fails to induce translocation of PKC-
(32). It is possible to speculate that the degree of protection from diazoxide was higher than that of IPC, such that diazoxide can overcome the PKC-
block, whereas IPC cannot; however, this is unlikely, because both IPC and diazoxide afforded similar cardioprotection in terms of CK leakage and MTT reduction (Figs. 7 and 8).
The above findings support the view that PKC- and PKC-
isoforms act independently. As shown in the diagram depicted in the Fig. 10, the activation of sarcolemmal receptors triggers G protein-mediated signaling that, via the activation of PKC-
, leads to the opening of mitoKATP channels and that, in turn, possibly through the release of reactive oxygen species, would activate PKC-
, this leading to the phosphorylation of p38 MAPK, which then effects a single or multiple end-effectors of IPC. The proposal by Garlid et al. (7) that PKC may be proximal and distal to the mitoKATP channels and the demonstration that PKC-
interacts with and inhibis the permeability transition pore in the cardiac mitochondria and contributes to PKC-
-induced cardioprotection (1) would support the present findings. However, the exact place and action of these PKC isoforms would require further investigations.
|
Potential Limitations
The selectivity of some of the agents used to block the PKC isoforms may be questioned. V12 peptide is known to be a PKC- specific inhibitor (22); however, the selectivity of rottlerin to block PKC-
has been controversial, and, whereas some investigators (15) have reported selective PKC-
inhibition, others (3) have not. To overcome this problem we used the selective PKC-
activator bistratene A (4, 47), which failed to precondition the tissue, thus confirming the lack of a role of PKC-
in preconditioning. Although GO-6976 (28) and RO 320432 (48) are selective inhibitors of both PKC-
and -
, the separation of the action of these two isoforms was carried out with the use of the PKC-(
1 +
2) specific inhibitor LY-333531 (21).
Another potential limitation of our studies was the use of atrial tissue as opposed to ventricular myocardium, and therefore, any extrapolation must be conducted with caution. However, data from Yellon's laboratory (45) and from our own group (unpublished data) have demonstrated that the response to ischemia-reperfusion of ventricle and atrial tissue is similar.
Evidence in the literature has shown that the activation of PKC leads to translocation of different isoforms into various subcellular sites but has not shown that this is an important mechanism of cardioprotection by IPC. Our study, aimed to determine in heart tissue sections whether the activation of different isoforms (by determining phosphorylation-dephosphorylation states) imparts different properties on IPC, but did not investigate the subcellular locations, and this is a potential pitfall. The heart tissue model used in this study was not appropriate to establish whether or not subcellular translocation of phosphorylated PKC isoforms occurs.
Clinical Implications
The present findings may also have important clinical implications, because they indicate that it may be possible to target specific PKC isoforms to induce cardioprotection without activating other isoforms that may be involved in other cellular processes. In particular, it may be useful for eliciting cardioprotection in the diabetic heart that cannot be preconditioned by IPC or by diazoxide (12), suggesting a dysfunction at the level of mitoKATP channels. In contrast, diabetic myocardium could be preconditioned by PMA (17), which is a general PKC activator. Because evidence from this study has shown that PKC- is upstream of mitoKATP channels and because of the dysfunction at the level of mitoKATP channels in the diabetic myocardium, it is reasonable to conclude that PMA acts on a downstream PKC isoform, which in this case is PKC-
. Clearly, this has potential clinical/therapeutic implications for protecting the human diabetic myocardium by targeting the activation of specific PKC isoforms that potentiate preconditioning.
![]() |
GRANTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
![]() |
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Das M, Parker JE, and Halestrap AP. Matrix volume measurements challenge the existence of diazoxide/glibencamide-sensitive KATP channels in rat mitochondria. J Physiol 547: 893902, 2003.
3. Davies SP, Reddy H, Caivano M, and Cohen P. Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J 351: 95105, 2000.[CrossRef][ISI][Medline]
4. Frey MR, Leontieva O, Watters DJ, and Black JD. Stimulation of protein kinase C-dependent and -independent signaling pathways by bistratene A in intestinal epithelial cells. Biochem Pharmacol 61: 10931100, 2001.[CrossRef][ISI][Medline]
5. Fryer RM, Hsu AK, Wang Y, Henry M, Eells J, and Gross GJ. PKC- inhibition does not block preconditioning-induced preservation in mitochondrial ATP synthesis and infarct size reduction in rats. Basic Res Cardiol 97: 4754, 2002.[CrossRef][ISI][Medline]
6. Fryer RM, Wang Y, Hsu AK, and Gross GJ. Essential activation of PKC- a in opioid-initiated cardioprotection. Am J Physiol Heart Circ Physiol 280: H1346H1353, 2001.
7. Garlid KD, Dos Santos P, Xie ZJ, Costa AD, and Paucek P. Mitochondrial potassium transport: the role of the mitochondrial ATP-sensitive K+ channel in cardiac function and cardioprotection. Biochim Biophys Acta 1606: 121, 2003.[ISI][Medline]
8. Garlid KD, Paucek P, Yarov-Yarovoy V, Murray HN, Darbenzio RB, D'Alonzo AJ, Lodge NJ, Smith MA, and Grover GJ. Cardioprotective effect of diazoxide and its interaction with mitochondrial ATP-sensitive K+ channels. Possible mechanism of cardioprotection. Circ Res 81: 10721082, 1997.
9. Garlid KD, Paucek P, Yarov-Yarovoy V, Sun X, and Schindler PA. The mitochondrial KATP channel as a receptor for potassium channel openers. J Biol Chem 271: 87968799, 1996.
10. Ghosh S, Standen NB, and Galiñanes M. Evidence for mitochondrial KATP channels as effectors of human myocardial preconditioning. Cardiovasc Res 45: 934940, 2000.[CrossRef][ISI][Medline]
11. Ghosh S, Standen NB, and Galiñanes M. Preconditioning the human myocardium by simulated ischemia: studies on the early and delayed protection. Cardiovasc Res 45: 339350, 2000.[CrossRef][ISI][Medline]
12. Ghosh S, Standen NB, and Galiñanes M. Failure to precondition pathological human myocardium. J Am Coll Cardiol 37: 711718, 2001.[CrossRef][ISI][Medline]
13. Gray MO, Karliner JS, and Mochly-Rosen D. A selective epsilon-protein kinase C antagonist inhibits protection of cardiac myocytes from hypoxia-induced cell death. J Biol Chem 272: 3094530951, 1997.
14. Gross GJ and Auchampach JA. Blockade of ATP-sensitive potassium channels prevents myocardial preconditioning in dogs. Circ Res 70: 223233, 1992.[Abstract]
15. Gschwendt M, Muller HJ, Kielbassa K, Zang R, Kittstein W, Rincke G, and Marks F. Rottlerin, a novel protein kinase inhibitor. Biochem Biophys Res Commun 199: 9398, 1994.[CrossRef][ISI][Medline]
16. Hanley PJ, Mickel M, Loffler M, Brandt U, and Daut J. KATP channel-independent targets of diazoxide and 5-hydroxydecanoate in the heart. J Physiol 542: 735741, 2002.
17. Hassouna A, Loubani M, Fowler A, Standen N, and Galiñanes M. Can the diabetic heart be preconditioned: role of mitochondria KATP channels, PKC and p38 MAPK? Circulation, Suppl 106: 11245, 2002.
18. Hassouna A, Matata BM, Loubani M, Fowler A, Standen N, and Galiñanes M. Failure to precondition the diabetics' myocardium correlates with incomplete depolarization of mitochondrial membrane potential (Abstract). Mol Biol Cell, Suppl 14: 227a, 2003.
19. Hu H, Sato T, Seharaseyon J, Liu Y, Johns DC, O'Rourke B, and Marban E. Pharmacological and histochemical distinctions between molecularly defined sarcolemmal KATP channels and native cardiac mitochondrial KATP channels. Mol Pharmacol 55: 10001005, 1999.
20. Inagaki K, Hahn HS, Dorn GW, and Mochly-Rosen D. Additive protection of the ischemic heart ex vivo by combined treatment with -protein kinase C inhibitor and
-protein kinase C activator. Circulation 108: 869875, 2003.
21. Jirousek MR, Gillig JR, Gonzalez CM, Heath WF, McDonald JH III, Neel DA, Rito CJ, Singh U, Stramm LE, Melikian-Badalian A, Baevsky M, Ballas LM, Hall SE, Winneroski LL, and Faul MM. (S)-13-[(dimethylamino)methyl]-10,11,14,15-tetrahydro-4,9:16,21-dimetheno-1H,13H-dibenzo[e,k]pyrrolo[3,4h][1,4,13]oxadiazacyclohexadecene-1,3(2H)-dione (LY-333531) and related analogues: isozyme selective inhibitors of protein kinase C-. J Med Chem 39: 26642671, 1996.[CrossRef][ISI][Medline]
22. Johnson JA, Gray MO, Chen CH, and Mochly-Rosen D. A protein kinase C translocation inhibitor as an isozyme-selective antagonist of cardiac function. J Biol Chem 271: 2496224966, 1996.
23. Julier K, da Silva R, Garcia C, Bestmann L, Frascarolo P, Zollinger A, Chassot PG, Schmid ER, Turina MI, von Segesser LK, Pasch T, Spahn DR, and Zaugg M. Preconditioning by sevoflurane decreases biochemical markers for myocardial and renal dysfunction in coronary artery bypass graft surgery: a double-blinded, placebo-controlled, multicenter study. Anesthesiology 98: 13151327, 2003.[ISI][Medline]
24. Kitakaze M, Funaya H, Minamino T, Node K, Sato H, Ueda Y, Okuyama Y, Kuzuya T, Hori M, and Yoshida K. Role of protein kinase C- in activation of ecto-5'-nucleotidase in the preconditioned canine myocardium. Biochem Biophys Res Commun 239: 171175, 1997.[CrossRef][ISI][Medline]
25. Li Y and Kloner RA. Does protein kinase C play a role in ischemic preconditioning in rat hearts? Am J Physiol Heart Circ Physiol 268: H426H431, 1995.
26. Liu GS, Cohen MV, Mochly-Rosen D, and Downey JM. Protein kinase C- is responsible for the protection of preconditioning in rabbit cardiomyocytes. J Mol Cell Cardiol 31: 19371948, 1999.[CrossRef][ISI][Medline]
27. Loubani M and Galiñanes M. Pharmacological and ischemic preconditioning of the human myocardium: mitoKATP channels are upstream and p38MAPK is downstream of PKC. BMC Physiol 2: 10, 2002.[CrossRef][Medline]
28. Martiny-Baron G, Kazanietz MG, Mischak H, Blumberg PM, Kochs G, Hug H, Marme D, and Schachtele C. Selective inhibition of protein kinase C isozymes by the indolocarbazole Go-6976. J Biol Chem 268: 91949197, 1993.
29. Mitchell MB, Meng X, Ao L, Brown JM, Harken AH, and Banerjee A. Preconditioning of isolated rat heart is mediated by protein kinase C. Circ Res 76: 7381, 1995.
30. Miura T, Liu Y, Kita H, Ogawa T, and Shimamoto K. Roles of mitochondrial ATP-sensitive K channels and PKC in anti-infarct tolerance afforded by adenosine A1 receptor activation. J Am Coll Cardiol 35: 238245, 2000.[CrossRef][ISI][Medline]
31. Newton AC. Protein kinase C: structure, function and regulation. J Biol Chem 270: 2849528498, 1995.
32. Ohnuma Y, Miura T, Miki T, Tanno M, Kuno A, Tsuchida A, and Shimamoto K. Opening of mitochondrial KATP channel occurs downstream of PKC- activation in the mechanism of preconditioning. Am J Physiol Heart Circ Physiol 283: H440H447, 2002.
33. Pain T, Yang XM, Critz SD, Yue Y, Nakano A, Liu GS, Heusch G, Cohen MV, and Downey JM. Opening of mitochondrial KATP channels triggers the preconditioned state by generating free radicals. Circ Res 87: 460466, 2000.
34. Sato T, O'Rourke B, and Marban E. Modulation of mitochondrial ATP-dependent K+ channels by protein kinase C. Circ Res 83: 110114, 1998.
35. Saurin AT, Pennington DJ, Raat NJ, Latchman DS, Owen MJ, and Marber MS. Targeted disruption of the protein kinase C- gene abolishes the infarct size reduction that follows ischaemic preconditioning of isolated buffer-perfused mouse hearts. Cardiovasc Res 55: 672680, 2002.[CrossRef][ISI][Medline]
36. Singh H, Hudman D, Lawrence CL, Rainbow RD, Lodwick D, and Norman RI. Distribution of Kir6.0 and SUR2 ATP-sensitive potassium channel subunits in isolated ventricular myocytes. J Mol Cell Cardiol 35: 445459, 2003.[CrossRef][ISI][Medline]
37. Speechly-Dick ME, Grover GJ, and Yellon DM. Does ischemic preconditioning in the human involve protein kinase C and the ATP-dependent K+ channel? Studies of contractile function after simulated ischemia in an atrial in vitro model. Circ Res 77: 10301035, 1995.
38. Speechly-Dick ME, Mocanu MM, and Yellon DM. Protein kinase C. Its role in ischemic preconditioning in the rat. Circ Res 75: 586590, 1994.[Abstract]
39. Suzuki M, Saito T, Sato T, Tamagawa M, Miki T, Seino S, and Nakaya H. Cardioprotective effect of diazoxide is mediated by activation of sarcolemmal but not mitochondrial ATP-sensitive potassium channels in mice. Circulation 107: 682685, 2003.
40. Takai Y, Kishimoto A, Inoue M, and Nishizuka Y. Studies on a cyclic nucleotide-independent protein kinase and its proenzyme in mammalian tissues. I. Purification and characterization of an active enzyme from bovine cerebellum. J Biol Chem 252: 76037609, 1977.[Abstract]
41. Vahlhaus C, Schulz R, Post H, Onallah R, and Heusch G. No prevention of ischemic preconditioning by the protein kinase C inhibitor staurosporine in swine. Circ Res 79: 407414, 1996.
42. Vanden Hoek T, Becker LB, Shao ZH, Li CQ, and Schumacker PT. Preconditioning in cardiomyocytes protects by attenuating oxidant stress at reperfusion. Circ Res 86: 541548, 2000.
43. Vistica DT, Skehan P, Scudiero D, Monks A, Pittman A, and Boyd MR. Tetrazolium-based assays for cellular viability: a critical examination of selected parameters affecting formazan production. Cancer Res 51: 25152520, 1991.[Abstract]
44. Vogt AM, Htun P, Arras M, Podzuweit T, Schaper W. Intramyocardial infusion of tool drugs for the study of molecular mechanisms in ischemic preconditioning. Basic Res Cardiol 91: 389400, 1996.[ISI][Medline]
45. Walker DM, Walker JM, Pugsley WB, Pattison CW, and Yellon DM. Preconditioning in isolated superfused human muscle. J Mol Cell Cardiol 27: 13491357, 1995.[ISI][Medline]
46. Wang Y, Takashi E, Xu M, Ayub A, and Ashraf M. Downregulation of protein kinase C inhibits activation of mitochondrial KATP channels by diazoxide. Circulation 104: 8590, 2001.
47. Watters D, Garrone B, Coomer J, Johnson WE, Brown G, and Parsons P. Stimulation of melanogenesis in a human melanoma cell line by bistratene A. Biochem Pharmacol 55: 16911699, 1998.[CrossRef][ISI][Medline]
48. Wilkinson SE, Parker PJ, and Nixon JS. Isoenzyme specificity of bisindolylmaleimides, selective inhibitors of protein kinase C. Biochem J 294: 335337, 1993.[ISI][Medline]
49. Yao Z, McPherson BC, Liu H, Shao Z, Li C, Qin Y, Vanden Hoek TL, Becker LB, and Schumacker PT. Signal transduction of flumazenil-induced preconditioning in myocytes. Am J Physiol Heart Circ Physiol 280: H1249H1255, 2001.
50. Yoshida K, Kawamura S, Mizukami Y, and Kitakaze M. Implication of protein kinase C-, -
, and -
isoforms in ischemic preconditioning in perfused rat hearts. J Biochem (Tokyo) 122: 506511, 1997.[Abstract]
51. Ytrehus K, Liu Y, and Downey JM. Preconditioning protects ischemic rabbit heart by protein kinase C activation. Am J Physiol Heart Circ Physiol 266: H1145H1152, 1994.
52. Zhang JG, Ghosh S, Ockleford CD, and Galiñanes M. Characterization of an in vitro model for the study of the short and prolonged effects of myocardial ischaemia and reperfusion in man. Clin Sci (Lond) 99: 443453, 2000.[Medline]