Resveratrol pretreatment can protect the heart by inducing pharmacological preconditioning. Whether resveratrol protects the heart when applied at reperfusion remains unknown. We examined the effect of resveratrol on myocardial infarct size when given at reperfusion and investigated the mechanism underlying the effect. Isolated rat hearts were subjected to 30 min ischemia followed by 2 h of reperfusion, and myocardial samples were collected from the risk zone for Western blot analysis. Mitochondrial swelling was spectrophotometrically measured as a decrease in absorbance at 520 nm (A(520)). Resveratrol reduced infarct size and prevented cardiac mitochondrial swelling. Resveratrol enhanced GSK-3 beta phosphorylation upon reperfusion, an effect that was mediated by the cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG) pathway. Resveratrol translocated GSK-3 beta from cytosol to mitochondria via the cGMP/PKG pathway. Further studies showed that mitochondrial GSK-3 beta was co-immunoprecipitated with cyclophilin D but not with VDAC (voltage dependent anion channel) or ANT (adenine nucleotide translocator). These data suggest that resveratrol prevents myocardial reperfusion injury presumably by targeting the mPTP through translocation of GSK-3 beta from cytosol to mitochondria. Translocated GSK-3 beta may ultimately interact with cyclophilin D to modulate the mPTP opening. (C) 2008 Elsevier B.V. All rights reserved.

The purpose of this study was to determine whether exogenous zinc prevents cardiac reperfusion injury by targeting the mitochondrial permeability transition pore (mPTP) via glycogen synthase kinase-3beta (GSK-3beta). The treatment of cardiac H9c2 cells with ZnCl2 (10 microM) in the presence of zinc ionophore pyrithione for 20 min significantly enhanced GSK-3beta phosphorylation at Ser9, indicating that exogenous zinc can inactivate GSK-3beta in H9c2 cells. The effect of zinc on GSK-3beta activity was blocked by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY-294002 but not by the mammalian target of rapamycin (mTOR) inhibitor rapamycin or the PKC inhibitor chelerythrine, implying that PI3K but not mTOR or PKC accounts for the action of zinc. In support of this interpretation, zinc induced a significant increase in Akt but not mTOR phosphorylation. Further experiments found that zinc also increased mitochondrial GSK-3beta phosphorylation. This may indicate an involvement of the mitochondria in the action of zinc. The effect of zinc on mitochondrial GSK-3beta phosphorylation was not altered by the mitochondrial ATP-sensitive K+ channel blocker 5-hydroxydecanoic acid. Zinc applied at reperfusion reduced cell death in cells subjected to simulated ischemia/reperfusion, indicating that zinc can prevent reperfusion injury. However, zinc was not able to exert protection in cells transfected with the constitutively active GSK-3beta (GSK-3beta-S9A-HA) mutant, suggesting that zinc prevents reperfusion injury by inactivating GSK-3beta. Cells transfected with the catalytically inactive GSK-3beta (GSK-3beta-KM-HA) also revealed a significant decrease in cell death, strongly supporting the essential role of GSK-3beta inactivation in cardioprotection. Moreover, zinc prevented oxidant-induced mPTP opening through the inhibition of GSK-3beta. Taken together, these data suggest that zinc prevents reperfusion injury by modulating the mPTP opening through the inactivation of GSK-3beta. The PI3K/Akt signaling pathway is responsible for the inactivation of GSK-3beta by zinc.

BACKGROUND:While postconditioning has been proposed to protect the heart by targeting the mitochondrial permeability transition pore (mPTP), the detailed mechanism underlying this action is unknown. The authors hypothesized that postconditioning stimulates opioid receptors, which in turn protect the heart from reperfusion injury by targeting the mPTP.METHODS:Rat hearts (both in vivo and in vitro) were subjected to 30 min of ischemia and 2 h of reperfusion. Postconditioning was elicited by six cycles of 10-s reperfusion and 10-s ischemia. To measure nitric oxide concentration, cardiomyocytes loaded with 4-amino-5-methylamino-2',7'-difluorofluorescein were imaged using confocal microscopy. Mitochondrial membrane potential was determined by loading cardiomyocytes with tetramethylrhodamine ethyl ester.RESULTS:In open chest rats, postconditioning reduced infarct size, an effect that was reversed by both naloxone and naltrindole. The antiinfarct effect of postconditioning was also blocked by the mPTP opener atractyloside. In isolated hearts, postconditioning reduced infarct size. Morphine mimicked postconditioning to reduce infarct size, which was abolished by both naltrindole and atractyloside. N-nitro-l-arginine methyl ester and guanylyl cyclase inhibitor 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one blocked the action of morphine. Further experiments showed that morphine produces nitric oxide in cardiomyocytes by activating delta-opioid receptors. Moreover, morphine could prevent hydrogen peroxide-induced collapse of mitochondrial membrane potential in cardiomyocytes, which was reversed by naltrindole, N-nitro-l-arginine methyl ester, and the protein kinase G inhibitor KT5823.CONCLUSIONS:Postconditioning protects the heart by targeting the mPTP through activation of delta-opioid receptors. The nitric oxide-cyclic guanosine monophosphate-protein kinase G pathway may account for the effect of postconditioning on the mPTP opening.

While the role of adenosine A 2 receptor activation in modulation of reperfusion injury has been extensively investigated, controversies still exist regarding its exact role (protective or not). We aimed to test if a simultaneous stimulation of A 2A and A 2B receptors can produce a strong protection against reperfusion injury by targeting mitochondrial permeability transition pore (mPTP) and glycogen synthase kinase 3β (GSK-3β). NECA (100 nM) given at reperfusion reduced infarct size in isolated rat hearts (14.1 ± 1.9 % vs. 37.9 ± 3.1 % of risk zone in control), an effect that was blocked by both the A 2A antagonist SCH58261 (36.6 ± 2.4 %) and the A 2B antagonist MRS1706 (39.2 ± 2.9 %), indicating that both the receptor subtypes are involved in the anti-infarct effect of NECA. NECA increased GSK-3β phosphorylation at Ser 9 and translocated GSK-3β to mitochondria upon reperfusion, an effect that was blocked by both SCH58261 and MRS1706, implying that NECA may protect the heart at reperfusion by targeting GSK-3β and mitochondria through activation of both A 2A and A 2B receptors. In support of this interpretation, NECA prevented oxidant-induced loss of mitochondrial membrane potential (MMP) in cardiomyocytes (91.2 ± 3.7 % vs. 38.8 ± 5.4 % in control), which was also partially blocked by both SCH58261 and MRS1706, indicating that NECA prevents mPTP opening by stimulating both A 2A and A 2B receptors. Further experiments showed that a combination of CGS21680 (A 2A agonist) and BAY60 – 6583 (A 2B agonist) induced a greater protection against the loss of MMP (84.7 ± 1.1 %) than that induced by CGS21680 (55.6 ± 2.9 %) or BAY60 – 6583 (63.7 ± 5.0) alone, corroborating the above observations. In conclusion, these data suggest that A 2A and A 2B receptors work in concert to prevent reperfusion injury by targeting mPTP through a GSK-3β-dependent mechanism.

OBJECTIVE:Our aim was to determine if NO prevents mitochondrial oxidant damage by mobilizing intracellular free zinc (Zn(2+)). METHODS:Zn(2+) levels were determined by imaging enzymatically isolated adult rat cardiomyocytes loaded with Newport Green DCF. Mitochondrial membrane potential (DeltaPsi(m)) was assessed by imaging cardiomyocytes loaded with tetramethylrhodamine ethyl ester (TMRE). RESULTS:S-nitroso-N-acetylpenicillamine (SNAP) dramatically increased Zn(2+), which was blocked by both ODQ and NS2028, two specific inhibitors of guanylyl cyclase. The protein kinase G (PKG) inhibitor KT5823 blocked the effect of SNAP while the PKG activator 8-Br-cGMP mimicked the action of SNAP, indicating that the cGMP/PKG pathway is responsible for the effect of SNAP. The increased Zn(2+) was prevented by 5-hydroxydecanoate (5HD) but was mimicked by diazoxide, implying that mitochondrial K(ATP) channel opening may account for this effect. Since chelation of Zn(2+) blocked the preventive effect of SNAP on H(2)O(2)-induced loss of DeltaPsi(m) and exogenous zinc (1 microM ZnCl(2)) prevented dissipation of DeltaPsi(m), Zn(2+) may play a critical role in the protective effect of NO. The MEK (mitogen-activated protein kinase or extracellular signal-regulated kinase) inhibitor PD98059 blocked the preventive effects of SNAP and zinc on DeltaPsi(m), indicating that extracellular signal-regulated kinase (ERK) mediates the protective effect of both these compounds on mitochondrial oxidant damage. A Western blot analysis further showed that ZnCl(2) significantly enhances phosphorylation of ERK, confirming the involvement of ERK in the action of Zn(2+). CONCLUSIONS:In isolated cardiomyocytes, NO mobilizes endogenous zinc by opening mitochondrial K(ATP) channels through the cGMP/PKG pathway. In these cells, Zn(2+) may be an important mediator of the action of NO on the mitochondrial death pathway.

Although resveratrol has been demonstrated to be cardioprotective, the detailed cellular and molecular mechanisms that mediate the protection remain elusive. We aimed to determine if resveratrol protects the heart at reperfusion by modulating the mitochondrial permeability transition pore (mPTP) opening through glycogen synthase kinase 3β (GSK-3β). Resveratrol (10μM) given at reperfusion reduced infarct size (12.2 ± 2.5 % of risk zone vs. 37.9 ± 3.1 % of risk zone in control, n = 6) in isolated rat hearts subjected to 30 min regional ischemia followed by 2 h of reperfusion, an effect that was abrogated by the mPTP opener atractyloside (30.9 ± 8.1 % of risk zone), implying that resveratrol may protect the heart at reperfusion by modulating the mPTP opening. To define the signaling mechanism underlying the action of resveratrol, we determined GSK-3β activity by measuring its phosphorylation at Ser 9 . Resveratrol significantly enhanced GSK-3β phosphorylation upon reperfusion (225.2 ± 30.0 % of control at 5 min of reperfusion). Further experiments showed that resveratrol induces translocation of GSK-3β to mitochondria and translocated GSK-3β interacts with the mPTP component cyclophilin D but not VDAC (the voltage-dependent anion channel) or ANT (the adenine nucleotide translocator) in cardiac mitochondria. Taken together, these data suggest that resveratrol prevents myocardial reperfusion injury by targeting the mPTP opening via GSK-3β. Translocation of GSK-3β to mitochondria and its interaction with the mPTP component cyclophilin D may serve as an essential mechanism that mediates the protective effect of resveratrol on reperfusion injury.

The exact role of autophagy in myocardial ischemia/reperfusion injury remains unknown. We aimed to test if and how autophagy contributes to the pathogenesis of ischemia/reperfusion injury in the heart. Isolated rat hearts were subjected to 30 min regional ischemia followed by 2 h of reperfusion or sham operation. Myocardial biopsies were taken from ischemic area. Ischemia did not increase the LC3-II to LC3-I ratio (LC3-II/LC3-I), an established marker of autophagy, compared to the sham group. In contrast, LC3-II/LC3-I was markedly increased upon reperfusion (1.4, 2.2, 2.9, and 2.2-fold increases at 10, 30, 60, and 120 min of reperfusion; n = 6)). These observations suggest that reperfusion but not ischemia triggers autophagy. In support of this finding, electron microscopic analysis revealed a significant increase in the number of cytoplasmic autophagosome in myocardium upon reperfusion but not during ischemia. To confirm that the above observations were of autophagic origin, we tested if 3-methyladenine ...

Although bradykinin has been demonstrated to protect the heart at reperfusion, the detailed cellular and molecular mechanisms that mediate the protection remain elusive. Here we aimed to determine whether bradykinin protects the heart at reperfusion by modulating the mitochondrial permeability transition pore (mPTP) opening through glycogen synthase kinase 3beta (GSK-3beta). Bradykinin given at reperfusion reduced infarct size in isolated rat hearts subjected to 30 min regional ischemia followed by 2 h of reperfusion. The infarct-limiting effect of bradykinin was reversed by atractyloside, an opener of the mPTP, suggesting that bradykinin may protect the heart at reperfusion by modulating the mPTP opening. In support of this observation, bradykinin prevented the collapse of mitochondrial membrane potential (DeltaPsi(m)), an index of the mPTP opening. Bradykinin increased GSK-3beta phosphorylation at reperfusion, and the selective inhibitor of GSK-3beta SB216763 reduced infarct size and prevented the loss of DeltaPsi(m) by mimicking the effect of bradykinin. The effect of bradykinin on GSK-3beta phosphorylation was blocked by wortmannin and LY294002, and bradykinin increased Akt phosphorylation at reperfusion. Further experiments showed that the MEK inhibitor PD98059 prevented the effect of bradykinin on GSK-3beta. However, the mTOR/p70s6K pathway inhibitor rapamycin did not alter bradykinin-induced GSK-3beta phosphorylation and bradykinin failed to alter phosphorylation of either mTOR or p70s6K at reperfusion. Taken together, these data suggest that bradykinin protects the heart at reperfusion by modulating the mPTP opening through inhibition of GSK-3beta. The PI3-kinase/Akt pathway and ERK, but not the mTOR/p70s6K pathway account for the suppression of GSK-3beta by bradykinin.

While the adenosine A 3 receptor agonist N 6 -(3-iodobenzyl)-adenosine-5’-N- methylcarboxamide (IB-MECA) has been reported to be cardioprotective at reperfusion, little is known about the mechanisms underlying the protection. We hypothesized that IB-MECA may protect the heart at reperfusion by preventing the opening of mitochondrial permeability transition pore (mPTP) through inactivation of glycogen synthase kinase 3 β (GSK-3 β ). IB-MECA (1 µ M) applied during reperfusion reduced infarct size in isolated rat hearts, an effect that was abrogated by the selective A 3 receptor antagonist MRS1334 (100 nM). The effect of IB-MECA was abrogated by the mPTP opener atractyloside (20 µ M), implying that the action of IB-MECA may be mediated by inhibition of the mPTP opening. In cardiomyocytes, IB-MECA attenuated oxidant-induced loss of mitochondrial membrane potential ( ∆Ψ m ), which was reversed by MRS1334. IB-MECA also reduced Ca 2+ -induced mitochondrial swelling. IB-MECA enhanced phosphorylation of GSK-3

While the adenosine A3 receptor agonist N6-(3-iodobenzyl)-adenosine-5′-N-methylcarboxamide (IB-MECA) has been reported to be cardioprotective at reperfusion, little is known about the mechanisms underlying the protection. We hypothesized that IB-MECA may protect the heart at reperfusion by preventing the opening of mitochondrial permeability transition pore (mPTP) through inactivation of glycogen synthase kinase 3β (GSK-3β). IB-MECA applied during reperfusion reduced infarct size in isolated rat hearts subjected to 30 min ischemia and 2 h of reperfusion. This was abrogated by the mPTP opener atractyloside, implying that the protective effect of IB-MECA may be mediated by inhibition of the mPTP opening. In rat cardiomyocytes, IB-MECA attenuated oxidant-induced loss of mitochondrial membrane potential (ΔΨm), and the adenosine A3 receptor antagonist MRS1334 prevented the effect of IB-MECA on ΔΨm. IB-MECA enhanced phosphorylation of GSK-3β upon reperfusion, and the GSK-3β inhibitor SB216763 mimicked the protective effect of IB-MECA by attenuating both infarction and the loss of ΔΨm. In addition, the effect of IB-MECA on GSK-3β was reversed by the PI3-kinase inhibitor wortmannin, and IB-MECA was shown to enhance Akt phosphorylation upon reperfusion. In contrast, the mTOR/p70s6k pathway inhibitor rapamycin failed to affect GSK-3β phosphorylation by IB-MECA and IB-MECA did not alter phosphorylation of either mTOR or p70s6K. Taken together, these data suggest that activation of adenosine A3 receptors with IB-MECA prevents myocardial reperfusion injury by inhibiting the mPTP opening through the inactivation of GSK-3β at reperfusion. IB-MECA-induced GSK-3β inhibition is mediated by the PI3-kinase/Akt signal pathway but not by the mTOR/p70s6K pathway.

Although the adenosine A3 receptor agonist N6-(3-iodobenzyl)-adenosine-5′-N-methylcarboxamide (IB-MECA) has been reported to be cardioprotective at reperfusion, little is known about the mechanisms underlying the protection. We hypothesized that IB-MECA may protect the heart at reperfusion by preventing the opening of mitochondrial permeability transition pore (mPTP) through inactivation of glycogen synthase kinase (GSK) 3β. IB-MECA (1 μM) applied during reperfusion reduced infarct size in isolated rat hearts, an effect that was abrogated by the selective A3 receptor antagonist 1,4-dihydro-2-methyl-6-phenyl-4-(phenylethynyl)-3,5-pyridinedicarboxylic acid 3-ethyl-5-[(3-nitrophenyl)-methyl]ester (MRS1334) (100 nM). The effect of IB-MECA was abrogated by the mPTP opener atractyloside (20 μM), implying that the action of IB-MECA may be mediated by inhibition of the mPTP opening. In cardiomyocytes, IB-MECA attenuated oxidant-induced loss of mitochondrial membrane potential (ΔΨm), which was reversed by MRS1334. IB-MECA also reduced Ca2+-induced mitochondrial swelling. IB-MECA enhanced phosphorylation of GSK-3β (Ser9) upon reperfusion, and the GSK-3 inhibitor 3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione (SB216763) (3 μM) mimicked the protective effect of IB-MECA by attenuating both infarction and the loss of ΔΨm. In addition, the effect of IB-MECA on GSK-3β was reversed by wortmannin (100 nM), and IB-MECA was shown to enhance Akt phosphorylation upon reperfusion. In contrast, rapamycin (2 nM) failed to affect GSK-3β phosphorylation by IB-MECA, and IB-MECA did not alter phosphorylation of either mTOR (Ser2448) or 70s6K (Thr389). Taken together, these data suggest that IB-MECA prevents myocardial reperfusion injury by inhibiting the mPTP opening through the inactivation of GSK-3β at reperfusion. IB-MECA-induced GSK-3β inhibition is mediated by the PI3-kinase/Akt signal pathway but not by the mTOR/p70s6K pathway.

The adenosine A(3) receptor plays an important role in ischemic preconditioning. Activation of the adenosine A(3) receptor with its agonists induces both early and late pharmacological preconditioning through various mechanisms. As the first potent and selective adenosine A(3) receptor agonist, IB-MECA (N-6-(3-iodobenzyl)-adenosine-5'-N-methylcarboxamide) has been demonstrated to induce cardioprotection against myocardial ischemia/reperfusion injury when given before onset of ischemia by triggering pharmacological preconditioning. More importantly, IB-MECA can also protect the heart even when administered at the onset of reperfusion after ischemia, indicating a strong likelihood that the drug may be useful for the treatment of patients with acute myocardial infarction. However, since IB-MECA has been reported to have lethal effects at higher concentrations, and may cause systemic hypertension in some species, further studies are needed to find the best treatment strategy to increase its therapeutic potential.

In general, theorem provers are relatively slow. Speed up can be achieved by directing the search towards finding a proof and by using parallelism. The parallelisms identified in connection graph refutations are: or parallelism, and parallelism, and dcdp parallelism. In dcdp parallelism, the links (edges) incident to distinct clauses and edge disjoint pairs are resolved in parallel. Optimally selecting potential parallel links is equivalent to solving the optimal graph coloring problem. Fortunately, however, optimal solutions to this NP-hard problem are not crucial. We describe a parallel solution of a sub-optimal graph coloring algorithm In and parallelism, all literals in the sun clause are resolved concurrently and all the resolvents obtained. The resolvents, along with their inherited links, are inserted into the graph. The sun clause, and all the links connected to it, are removed from the graph. Because shared variables are restricted to have the same instantiation when there are shared variables in the sun clause, resolving literals concurrently becomes difficult. We discuss different approaches for performing this and parallelism.

Abstract The concept that centrally acting drugs influence behaviour by influencing neurotransmitter function at specific points controlling synaptic transmission is the basis of neuropharmacological research.The synapse is a specialized cellular interface that provides a physical and chemical link for facilitating communication between cells of the central nervous system (CNS).