PKC-ε is upstream and PKC-α is downstream of mitoKATP channels in the signal transduction pathway of ischemic preconditioning of human myocardium

Protein kinase C (PKC) is involved in the process of ischemic preconditioning (IPC), although the precise mechanism is still a subject of debate. Using specific PKC inhibitors, we investigated which PKC isoforms were involved in IPC of the human atrial myocardium sections and to determine their temporal relationship to the opening of mitochondrial potassium-sensitive ATP (mitoKATP) channels. Right atrial muscles obtained from patients undergoing elective cardiac surgery were equilibrated and then randomized to receive any of the following protocols: aerobic control, 90-min simulated ischemia/120-min reoxygenation, IPC using 5-min simulated ischemia/5-min reoxygenation followed by 90-min simulated ischemia/120-min reoxygenation and finally, PKC inhibitors were added 10 min before and 10 min during IPC followed by 90-min simulated ischemia/120-min reoxygenation. The PKC isoforms inhibitors investigated were V1–2 peptide, GO-6976, rottlerin, and LY-333531 for PKC-ε, -α, -δ and -β, respectively. To investigate the relation of PKC isoforms to mitoKATP channels, PKC inhibitors found to be involved in IPC were added 10 min before and 10 min during preconditioning by diazoxide followed by 90-min simulated ischemia/120-min reoxygenation in a second experiment. Creatine kinase leakage and methylthiazoletetrazolium cell viability were measured. Phosphorylation of PKC isoforms after activation of the sample by either diazoxide or IPC was detected by using Western blot analysis and then analyzed by using Scion image software. PKC-α and -ε inhibitors blocked IPC, whereas PKC-δ and -β inhibitors did not. The protection elicited by diazoxide, believed to be via mitoKATP channels opening, was blocked by the inhibition of PKC-α but not -ε isoforms. In addition, diazoxide caused increased phosphorylation of PKC-α to the same extent as IPC but did not affect the phosphorylation of PKC-ε, a process believed to be critical in PKC activation. The results demonstrate that PKC-α and -ε are involved in IPC of the human myocardium with PKC-ε being upstream and PKC-α being downstream of mitoKATP channels.

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Oral Sulfonylurea Hypoglycemic Agents Prevent Ischemic Preconditioning in Human Myocardium

Circulation ◽

10.1161/01.cir.96.1.29 ◽

1997 ◽

Vol 96 (1) ◽

pp. 29-32 ◽

Cited By ~ 197

Author(s):

Joseph C. Cleveland ◽

Daniel R. Meldrum ◽

Brian S. Cain ◽

Anirban Banerjee ◽

Alden H. Harken

Keyword(s):

Ischemic Preconditioning ◽

Human Myocardium ◽

Hypoglycemic Agents

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Met5-enkephalin protects isolated adult rabbit cardiomyocytes via δ-opioid receptors

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1999.277.6.h2442 ◽

1999 ◽

Vol 277 (6) ◽

pp. H2442-H2450 ◽

Cited By ~ 14

Author(s):

Yasushi Takasaki ◽

Roger A. Wolff ◽

Grace L. Chien ◽

Donna M. van Winkle

Keyword(s):

Cell Death ◽

Opioid Receptors ◽

Ischemic Preconditioning ◽

Opioid Peptides ◽

Trypan Blue ◽

Adult Rabbit ◽

Endogenous Opioid Peptides ◽

Endogenous Opioid ◽

Simulated Ischemia ◽

Hypoxic Incubation

In rats and rabbits, endogenous opioid peptides participate in ischemic preconditioning. However, it is not known which endogenous opioid(s) can trigger cardioprotection. We examined preconditioning-induced and opioid-induced limitation of cell death in isolated, calcium-tolerant, adult rabbit cardiomyocytes. Cells were subjected to simulated ischemia by pelleting and normothermic hypoxic incubation. Preconditioning was elicited with 15 min of simulated ischemia followed by 15 min of resuspension and reoxygenation. All cells underwent 180 min of simulated ischemia. Cell death was assessed by trypan blue permeability. Morphine protected cells, as did preconditioning; naloxone blocked the preconditioning-induced protection. Exogenous Met5-enkephalin (ME) induced protection, but exogenous β-endorphin did not. ME-induced protection was blocked by the δ-selective antagonist naltrindole. Additionally, two other proenkephalin products, Leu5-enkephalin and Met5-enkephalin-Arg-Phe, provided protection equipotent to ME. These data suggest that one or more proenkephalin products interact with δ-opioid receptors to endogenously trigger opioid-mediated protection.

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κ- but not δ-opioid receptors mediate effects of ischemic preconditioning on both infarct and arrhythmia in rats

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.2001.280.1.h384 ◽

2001 ◽

Vol 280 (1) ◽

pp. H384-H391 ◽

Cited By ~ 87

Author(s):

Guan-Ying Wang ◽

Song Wu ◽

Jian-Ming Pei ◽

Xiao-Chun Yu ◽

Tak-Ming Wong

Keyword(s):

Infarct Size ◽

Opioid Receptors ◽

Ischemic Preconditioning ◽

Antiarrhythmic Action ◽

Perfused Rat Heart ◽

Calphostin C ◽

Kinase C ◽

Series Of Experiments ◽

Pkc Inhibitors

Two series of experiments were performed in the isolated perfused rat heart to determine the role of κ- and δ-opioid receptors (OR) in cardioprotection of ischemic preconditioning (IP). In the first series of experiments, it was found that IP with two cycles of 5-min regional ischemia followed by 5-min reperfusion each reduced infarct size induced by 30-min ischemia, and the ameliorating effect of IP on infarct was attenuated with blockade of either 5 × 10−6 mol/l nor-binaltorphimine (nor-BNI), a selective κ-OR antagonist, or 5 × 10−6 mol/l naltrindole (NTD), a selective δ-OR antagonist. The second series showed that U50,488H, a selective κ-OR agonist, ord-Ala2-d-leu5-enkephalin (DADLE), a selective δ-OR agonist, dose dependently reduced the infarct size induced by ischemia, which mimicked the effects of IP. The effect of 10−5 mol/l U50,488H on infarct was significantly attenuated by blockade of protein kinase C (PKC) with specific PKC inhibitors, 5 × 10−6 mol/l chelerythrine or 8 × 10−7 mol/l calphostin C, as well as by blockade of ATP-sensitive K+ (KATP) channels with blockers of the channel, 10−5 mol/l glibenclamide or 10−4 mol/l 5-hydroxydecanoate. IP also reduced arrhythmia induced by ischemia. Nor-BNI, but not NTD, attenuated, while U50,488H, but not DADLE, mimicked the antiarrhythmic action of IP. In conclusion, the present study has provided first evidence that κ-OR mediates the ameliorating effects of IP on infarct and arrhythmia induced by ischemia, whereas δ-OR mediates the effects only on infarct. Both PKC and KATP channels mediate the effect of activation of κ-OR on infarct.

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CO2Pneumoperitoneum Preservesβ-Arrestin 2 Content and Reduces High Mobility Group Box-1 (HMGB-1) Expression in an Animal Model of Peritonitis

Oxidative Medicine and Cellular Longevity ◽

10.1155/2015/160568 ◽

2015 ◽

Vol 2015 ◽

pp. 1-7 ◽

Cited By ~ 1

Author(s):

Angela Simona Montalto ◽

Alessandra Bitto ◽

Letteria Minutoli ◽

Pietro Impellizzeri ◽

Gaetano Costa ◽

...

Keyword(s):

Bacterial Load ◽

Cecal Ligation ◽

Signal Transduction Pathway ◽

High Mobility ◽

Receptor Activation ◽

High Mobility Group ◽

Male Rats ◽

Toll Like Receptor ◽

To Receive ◽

Lung And Kidney

Laparoscopy (LS) has been shown to decrease the inflammatory sequelae of endotoxemia.β-arrestin 2 plays an important function in signal transduction pathway of TLR4. High mobility group box-1 (HMGB-1) is involved in the delayed systemic inflammatory response. We investigated the effects of CO2insufflation on liver, lung, and kidney expression of bothβ-arrestin 2 and HMGB-1 during sepsis. Cecal ligation and puncture (CLP) was performed in male rats and 6 h later the animals were randomly assigned to receive a CO2pneumoperitoneum or laparotomy. Animals were euthanized; liver, lung, and kidney were removed for the evaluation ofβ-arrestin 2 and HMGB-1 expression. Immunohistochemical detection of myeloperoxidase (MPO) was investigated in lung and liver and bacterial load was determined in the peritoneal fluid. CO2pneumoperitoneum reduced peritoneal bacterial load, increased the expression ofβ-arrestin 2, and blunted the expression of the potent proinflammatory HMGB-1 in liver, lung, and kidney compared with laparotomy. Liver and lung MPO was markedly reduced in rats subjected to LS compared with laparotomy. We believe that CO2exerts an early protective effect by reducing bacterial load and likely toll-like receptor activation which in turn leads to a preservedβ-arrestin 2 expression and a reduced HMGB-1 expression.

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Role of the l-arginine/nitric oxide pathway in ischaemic/reoxygenation injury of the human myocardium

Clinical Science ◽

10.1042/cs0990497 ◽

2000 ◽

Vol 99 (6) ◽

pp. 497-504 ◽

Cited By ~ 4

Author(s):

Jin-Gang ZHANG ◽

Manuel GALIÑANES

Keyword(s):

Nitric Oxide ◽

Bypass Graft ◽

Coronary Bypass ◽

Myocardial Damage ◽

Human Myocardium ◽

Right Atrial ◽

No Donor ◽

Reoxygenation Injury ◽

Synthase Inhibitor

The role of the L-arginine/nitric oxide (NO) pathway in myocardial ischaemic/reperfusion injury remains controversial in experimental animal models. The aim of the present studies was to investigate the role of this pathway in the human myocardium. Myocardial specimens from right atrial appendages of patients undergoing elective coronary bypass graft surgery were incubated in crystalloid buffer at 37 °C and subjected to 120 min of simulated ischaemia followed by 120 min of reoxygenation. Tested drugs were added 15 min before ischaemia, and maintained during ischaemia and throughout reoxygenation. Ischaemia resulted in severe myocardial damage, as assessed by the leakage of lactate dehydrogenase (LDH) into the incubation medium and by the capacity of the tissue to reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan product. L-Arginine (10 mM), a precursor of NO, significantly decreased LDH leakage (from 9.0±0.6 to 5.3±0.3 units/g wet wt; P < 0.05), but had no effect on MTT reduction or oxygen consumption. D-Arginine (10 mM), NG-nitro-L-arginine methyl ester (L-NAME; 0.5 mM), an NO synthase inhibitor, and S-nitroso-N-acetylpenicillamine (at 1, 100, 500 and 1000 µM), an NO donor, had no significant effects on the measured indices, and L-NAME did not reverse the protection afforded by L-arginine against LDH leakage. In addition, the formation of nitrotyrosine was not influenced by ischaemia/reoxygenation alone or by the agents investigated. In conclusion, these data suggest that L-arginine affords modest protection against ischaemic/reoxygenation injury of the human myocardium, an action that is NO-independent, and that NO metabolism does not play a significant role in this model.

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Ketamine Preconditions Isolated Human Right Atrial Myocardium

Anesthesiology ◽

10.1097/00000542-200506000-00019 ◽

2005 ◽

Vol 102 (6) ◽

pp. 1190-1196 ◽

Cited By ~ 15

Author(s):

Jean-Luc Hanouz ◽

Lan Zhu ◽

Emmanuel Persehaye ◽

Massimo Massetti ◽

Gerard Babatasi ◽

...

Keyword(s):

Adenosine Triphosphate ◽

Enhanced Recovery ◽

Human Myocardium ◽

Right Atrial ◽

Control Group ◽

Force Of Contraction ◽

Human Right ◽

Adrenoceptor Antagonist ◽

Racemic Ketamine

Background The authors examined the effect of ketamine and its S(+) isomer on isolated human myocardium submitted to hypoxia-reoxygenation in vitro. Methods The authors studied isometric contraction of human right atrial trabeculae suspended in an oxygenated Tyrode's modified solution at 34 degrees C. Ten minutes before a 30-min hypoxic period followed by a 60-min reoxygenation, muscles were exposed for 15 min to racemic ketamine and its S(+) isomer at 10, 10, and 10 m alone or in the presence of 8.10 m 5-hydroxydecanoate, 10 m HMR 1098 (sarcolemmal adenosine triphosphate-sensitive potassium channel antagonist), 10 m phentolamine (alpha-adrenoceptor antagonist), and 10 m propranolol (beta-adrenoceptor antagonist). Force of contraction at the end of the 60-min reoxygenation period was compared between groups (mean +/- SD). Results Ketamine (10 m: 85 +/- 4%; 10 m: 95 +/- 10%; 10 m: 94 +/- 14% of baseline) and S(+)-ketamine (10-6 m: 85 +/- 4%; 10 m: 91 +/- 16%; 10 m: 93 +/- 14% of baseline) enhanced recovery of force of contraction at the end of the reoxygenation period as compared with the control group (47 +/- 10% of baseline; P < 0.001). Ketamine-induced preconditioning at 10 m was inhibited by 5-hydroxydecanoate (60 +/- 16%; P < 0.001), HMR 1098 (60 +/- 14%; P < 0.001), phentolamine (56 +/- 12%; P < 0.001), and propranolol (60 +/- 7%; P < 0.001). Conclusions In vitro, ketamine preconditions isolated human myocardium, at least in part, via activation of adenosine triphosphate-sensitive potassium channels and stimulation of alpha- and beta-adrenergic receptors.

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Salmonella typhimuriumSipA-induced neutrophil transepithelial migration: involvement of a PKC-α-dependent signal transduction pathway

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00299.2003 ◽

2004 ◽

Vol 286 (6) ◽

pp. G1024-G1031 ◽

Cited By ~ 30

Author(s):

Milton Silva ◽

Cecilia Song ◽

William J. Nadeau ◽

Jeffrey B. Matthews ◽

Beth A. McCormick

Keyword(s):

Signal Transduction ◽

Signal Transduction Pathway ◽

Polymorphonuclear Neutrophils ◽

Intestinal Lumen ◽

Effector Protein ◽

Proinflammatory Response ◽

T84 Cells ◽

Pkc Isoforms ◽

Isoform Selectivity

Salmonella typhimurium elicits an intense proinflammatory response characterized by movement of polymorphonuclear neutrophils (PMN) across the epithelial barrier to the intestinal lumen. We previously showed that S. typhimurium, via the type III secretion system effector protein SipA, initiates an ADP-ribosylation factor-6- and phospholipase D-dependent lipid-signaling cascade that directs activation of protein kinase C (PKC) and subsequent transepithelial movement of PMN. Here we sought to determine the specific PKC isoforms that are induced by the S. typhimurium effector SipA in model intestinal epithelia and to link the functional consequences of these isoforms in the promotion of PMN transepithelial migration. In vitro kinase PKC activation assays performed on polarized monolayers of T84 cells revealed that S. typhimurium and recombinant SipA induced activation of PKC-α, -δ, and -ε. To elucidate which of these isoforms play a key role in mediating epithelial cell responses that lead to the observed PMN transepithelial migration, we used a variety of PKC inhibitors with different isoform selectivity profiles. Inhibitors selective for PKC-α (Gö-6976 and 2,2′,3,3′,4,4′-hexahydroxyl-1,1′-biphenyl-6,6′-dimethanoldimethyl ether) markedly reduced S. typhimurium- and recombinant SipA-induced PMN transepithelial migration, whereas inhibitors to PKC-δ (rottlerin) or PKC-ε (V1-2) failed to exhibit a significant decrease in transepithelial movement of PMN. These results were confirmed biochemically and by immunofluorescence coupled to confocal microscopy. Our results are the first to show that the S. typhimurium effector protein SipA can activate multiple PKC isoforms, but only PKC-α is involved in the signal transduction cascade leading to PMN transepithelial migration.

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