Postconditioning prevents reperfusion injury by activating delta-opioid receptors.

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.

NO mobilizes intracellular Zn2+ via cGMP/PKG signaling pathway and prevents mitochondrial oxidant damage in cardiomyocytes.

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.

Adenosine produces nitric oxide and prevents mitochondrial oxidant damage in rat cardiomyocytes.

OBJECTIVE: To examine if adenosine prevents oxidant-induced mitochondrial dysfunction by producing nitric oxide (NO) in cardiomyocytes. METHODS AND RESULTS: Adenosine significantly enhanced the fluorescence of DAF-FM, a dye specific for NO, implying that adenosine induces synthesis of NO. Adenosine-induced NO production was blocked by both the nonspecific NOS inhibitor N(G)-nitro-l-arginine methyl ester (l-NAME) and N(5)-(1-Iminoethyl)-l-ornithine dihydrochloride (l-NIO), an inhibitor of endothelial NOS (eNOS), but not by N(6)-(1-Iminoethyl)-l-lysine hydrochloride (l-NIL), an inhibitor of inducible NOS (iNOS), indicating that adenosine activates eNOS. Adenosine also enhances eNOS phosphorylation and its activity. The adenosine A(2) receptor antagonist 8-(3-chlorostyryl)caffeine but not the A(1) antagonist 8-cyclopentyl-1,3-dipropylxanthine prevented the increase in NO production. CGS21680, an adenosine A(2) receptor agonist, markedly increased NO, further supporting the involvement of A(2) receptors. Adenosine-induced NO production was blocked by 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo(3,4-d)pyrimidine (PP2), a selective Src tyrosine kinase inhibitor, suggesting that Src tyrosine kinase is crucial for adenosine-induced NO production. Adenosine-induced NO production was partially reversed by both wortmannin and Akt inhibitor indicating an involvement of PI3-kinase/Akt. Pretreatment of cells with adenosine prevented H(2)O(2)-induced depolarization of mitochondrial membrane potential (DeltaPsi(m)). The protective effect was blocked by l-NAME and l-NIO but not by l-NIL, indicating that eNOS plays a role in the action of adenosine. The protective effect of adenosine was further suppressed by KT5823, a specific inhibitor of protein kinase G (PKG), indicating the PKG may serve as a downstream target of adenosine. CONCLUSION: Adenosine protects mitochondria from oxidant damage through a pathway involving A(2) receptors, eNOS, NO, PI3-kinase/Akt, and Src tyrosine kinase.

Macrokinetic analysis of blockade of NMDA-gated currents by substituted alcohols, alkanes and ethers.

Volatile hydrocarbon based CNS depressants including short chain alcohols and anesthetics act, in part, by inhibition of the excitatory effect of glutamate at the NMDA receptor. While effects of several of these volatile agents on NMDA-gated currents have been demonstrated, there has been no direct comparison of different chemical classes of CNS depressant drugs on NMDA-gated currents. Here, whole-cell voltage clamp measurements of currents gated by 100 microM NMDA from cultured cerebrocortical neurons were examined in the presence of varying concentrations of the alcohols ethanol and hexanol, the halogenated alcohol trichloroethanol, the halogenated alkane halothane and the halogenated ethers isoflurane and sevoflurane. All drugs tested showed concentration-dependent inhibition of NMDA-gated currents with anesthetic concentrations of each agent producing approximately 30% inhibition of the NMDA-gated current. A rapid-translation perfusion system was used to study the onset and offset kinetics of each of the volatile agents. Onset kinetics for the CNS depressants was similar with tau values near 100 ms. Offset kinetics was more variable with tau ranging from 88.2 ms for ethanol to 221.4 ms for trichloroethanol. These data indicate that a wide variety of volatile hydrocarbon based CNS depressants produce a similar inhibition of NMDA-gated currents and that the kinetics for these agents are inconsistent with an open channel block.

Advances in oral anti arrhythmic therapy: implications for the anaesthetist.

Surgical patients often are receiving antiarrhythmic therapy. Thus, because anaesthetic agents can affect cardiac function and may interact with concurrent antiarrhythmic medications, the anaesthetist should be aware of the electrophysiology associated with dysrhythmias and their management. Tocainide, flecainide, mexiletine, encainide and amiodarone have been introduced recently and each has an unique pattern of bioavailability, metabolism and toxicity. Patients treated with these drugs need special concern as they have abnormal cardiovascular systems and may be at increased risk for perioperative morbidity. In addition, unexpected untoward reactions and toxicity can result from interactions of anaesthetic agents and these drugs. This review discusses normal cardiac electrophysiology, common dysrhythmias and the electrophysiological effects of the newer oral antiarrhythmic drugs.

Mechanism for resveratrol-induced cardioprotection against reperfusion injury involves glycogen synthase kinase 3beta and mitochondrial permeability transition pore.

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-3beta phosphorylation upon reperfusion, an effect that was mediated by the cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG) pathway. Resveratrol translocated GSK-3beta from cytosol to mitochondria via the cGMP/PKG pathway. Further studies showed that mitochondrial GSK-3beta 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-3beta from cytosol to mitochondria. Translocated GSK-3beta may ultimately interact with cyclophilin D to modulate the mPTP opening.

Exogenous zinc protects cardiac cells from reperfusion injury by targeting mitochondrial permeability transition pore through inactivation of glycogen synthase kinase-3beta.

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.

Bradykinin prevents reperfusion injury by targeting mitochondrial permeability transition pore through glycogen synthase kinase 3beta.

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.

IB-MECA and cardioprotection.

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.

N6-(3-iodobenzyl)-adenosine-5'-N-methylcarboxamide confers cardioprotection at reperfusion by inhibiting mitochondrial permeability transition pore opening via glycogen synthase kinase 3 beta.

Although 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 (GSK) 3beta. IB-MECA (1 microM) 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 microM), 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 (DeltaPsim), which was reversed by MRS1334. IB-MECA also reduced Ca2+-induced mitochondrial swelling. IB-MECA enhanced phosphorylation of GSK-3beta (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 microM) mimicked the protective effect of IB-MECA by attenuating both infarction and the loss of DeltaPsim. In addition, the effect of IB-MECA on GSK-3beta 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-3beta 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-3beta at reperfusion. IB-MECA-induced GSK-3beta inhibition is mediated by the PI3-kinase/Akt signal pathway but not by the mTOR/p70s6K pathway.

Cardioprotection with adenosine A2 receptor activation at reperfusion.

Pre-ischemic treatment is seldom possible in the clinical setting of acute myocardial infarction. Thus, to successfully save myocardium from infarction, it is required that protective interventions must be effective when applied after ischemia has begun or at the onset of reperfusion. Unfortunately, in spite of a large body of experimental data showing that various interventions are cardioprotective at reperfusion, no specific therapy has yet been established to be clinically applicable. However, recent data from several laboratories have shown that adenosine and its analogues given at reperfusion can markedly protect the heart from ischemia/reperfusion injury. While the experimental data suggest that factors such as adenosine A2 receptor activation, anti-neutrophil effect, attenuation of free radical generation, increased nitric oxide (NO) availability, activation of the PI3-kinase/Akt pathway and ERK, prevention of mitochondrial damage, and anti-apoptotic effects may be involved in the protective effect of adenosine or its analogues, the exact receptor subtype(s), the detailed signaling mechanisms, and interaction between those individual factors are still unknown. A definite answer to these unsolved problems will offer insights into the mechanisms of cardioprotection at reperfusion, and will be critical for developing a successful therapeutic strategy to salvage ischemic myocardium in patients with acute myocardial infarction.

Changes in the effect of isoflurane on N-methyl-D-aspartic acid-gated currents in cultured cerebral cortical neurons with time in culture: evidence for subunit specificity.

BACKGROUND: Developmental changes in NR1 splice variants and NR2 subunits of the N-methyl-D-aspartate (NMDA) receptor have been associated with changes in the sensitivity of NMDA receptors to agonists, antagonists, and pharmacologic modulators. The authors have investigated changes in the effect of isoflurane on NMDA-gated currents from cultured cortical neurons with time in culture and related these changes to the subunit composition of the NMDA receptors. METHODS: N-methyl-D-aspartate-gated currents were measured using whole-cell voltage clamp recording in cortical neurons cultured for 1-4 weeks and HEK 293 cells transiently expressing NR1-1a + NR2A or NR1-1a + NR2B subunit-containing receptors. NMDA alone or NMDA with treatment agents (isoflurane or ifenprodil) was applied to cells using a U tube. RESULTS: The effect of isoflurane and the NR2B selective antagonist ifenprodil on NMDA-gated currents from cortical neurons decreased significantly with time in culture. NMDA-gated currents mediated by NR2A-containing receptors were less sensitive to isoflurane than those mediated by NR2B-containing receptors. Tachyphylaxis to repeated application of isoflurane was found in cortical neurons and HEK 293 cells with recombinant NMDA receptors. Hooked tail currents were induced by isoflurane in cultured cortical neurons and HEK 293 cells with expressed NMDA receptors. CONCLUSIONS: Isoflurane inhibits NMDA-gated currents at concentrations well below 1 minimum alveolar concentration (MAC). This effect of isoflurane was subunit dependent with the NR2B-containing receptors more sensitive to isoflurane than the NR2A-containing receptors. A potent tachyphylaxis occurred after brief exposure to isoflurane.