Signal transduction of flumazenil-induced preconditioning in myocytes

The objective of this study was to examine the role of oxygen radicals, protein kinase C (PKC), and ATP-sensitive K(+) (K(ATP)) channels in mediating flumazenil-produced preconditioning. Chick cardiomyocyte death was quantified using propidium iodide, and oxygen radical generation was assessed using 2',7'-dichlorofluorescin oxidation. Preconditioning was initiated with 10 min of ischemia followed by 10 min of reoxygenation. Alternatively, flumazenil was infused for 10 min and removed 10 min before ischemia. Flumazenil (10 microM) and preconditioning increased oxygen radicals [1,693 +/- 101 (n = 3) and 1,567 +/- 98 (n = 3), respectively, vs. 345 +/- 53 (n = 3) in control] and reduced cell death similarly [22 +/- 3% (n = 5) and 18 +/- 2% (n = 6), respectively, vs. controls 49 +/- 5% (n = 8)]. Protection and increased oxygen radicals by flumazenil were abolished by pretreatment with the antioxidant thiol reductant 2-mercaptopropionyl glycine (800 microM; 52 +/- 10%, n = 6). Specific PKC inhibitors Go-6976 (0.1 microM) and chelerythrine (2 microM), given during ischemia and reoxygenation, blocked flumazenil-produced protection (47 +/- 5%, n = 6). The PKC activator phorbol 12-myristate 13-acetate (0.2 microM), given during ischemia and reoxygenation, reduced cell death similarly to that with flumazenil [17 +/- 4% (n = 6) and 22 +/- 3% (n = 5)]. Finally, 5-hydroxydecanoate (1 mM), a selective mitochondrial K(ATP) channel antagonist given during ischemia and reoxygenation, abolished the protection of flumazenil and phorbol 12-myristate 13-acetate. Thus flumazenil mimics preconditioning to reduce cell death in cardiomyocytes. Oxygen radicals activate mitochondrial K(ATP) channels via PKC during the process.     

Preconditioning blocks cardiocyte apoptosis: role of K(ATP) channels and PKC-epsilon

The aims of this study were to determine whether preconditioning blocks cardiocyte apoptosis and to determine the role of mitochondrial ATP-sensitive K(+) (K(ATP)) channels and the protein kinase C epsilon-isoform (PKC-epsilon) in this effect. Ventricular myocytes from 10-day-old chick embryos were used. In the control series, 10 h of simulated ischemia followed by 12 h of reoxygenation resulted in 42 +/- 3% apoptosis (n = 8). These results were consistent with DNA laddering and TdT-mediated dUTP nick-end labeling (TUNEL) assay. Preconditioning, elicited with three cycles of 1 min of ischemia separated by 5 min of reoxygenation before subjection to prolonged simulated ischemia, markedly attenuated the apoptotic process (28 +/- 4%, n = 8). The selective mitochondrial K(ATP) channel opener diazoxide (400 micromol/l), given before ischemia, mimicked preconditioning effects to prevent apoptosis (22 +/- 4%, n = 6). Pretreatment with 5-hydroxydecanoate (100 micromol/l), a selective mitochondrial K(ATP) channel blocker, abolished preconditioning (42 +/- 2%, n = 6). In addition, the effects of preconditioning and diazoxide were blocked with the specific PKC inhibitors G?-6976 (0.1 micromol/l) or chelerythrine (4 micromol/l), given at simulated ischemia and reoxygenation. Furthermore, preconditioning and diazoxide selectively activated PKC-epsilon in the particulate fraction before simulated ischemia without effect on the total fraction, cytosolic fraction, and PKC delta-isoform. The specific PKC activator phorbol 12-myristate 13-acetate (0.2 micromol/l), added during simulated ischemia and reoxygenation, mimicked preconditioning to block apoptosis. Opening mitochondrial K(ATP) channels blocks cardiocyte apoptosis via activating PKC-epsilon in cultured ventricular myocytes. Through this signal transduction, preconditioning blocks apoptosis and preserves cardiac function in ischemia-reperfusion.     

Remote preconditioning reduces ischemic injury in the explanted heart by a KATP channel-dependent mechanism

Local and remote ischemic preconditioning (IPC) reduce ischemia-reperfusion (I/R) injury and preserve cardiac function. In this study, we tested the hypothesis that remote preconditioning is memorized by the explanted heart and yields protection from subsequent I/R injury and that the underlying mechanism involves sarcolemmal and mitochondrial ATP-sensitive K(+) (K(ATP)) channels. Male Wistar rats (300-350 g) were randomized to a control (n = 10), a remote IPC (n = 10), and a local IPC group (n = 10). Remote IPC was induced by four cycles of 5 min of limb ischemia, followed by 5 min of reperfusion. Local IPC was induced by four cycles of 2 min of regional myocardial ischemia, followed by 3 min of reperfusion. The heart was excised within 5 min after the final cycle of preconditioning, mounted in a perfused Langendorff preparation for 40 min of stabilization, and subjected to 45 min of sustained ischemia by occluding the left coronary artery and 120 min of reperfusion. I/R injury was assessed as infarct size by triphenyltetrazolium staining. The influence of sarcolemmal and mitochondrial K(ATP) channels on remote preconditioning was assessed by the addition of glibenclamide (10 microM, a nonselective K(ATP) blocker), 5-hydroxydecanoic acid (5-HD; 100 microM, a mitochondrial K(ATP) blocker), and HMR-1098 (30 microM, a sarcolemmal K(ATP) blocker) to the Langendorff preparation before I/R. The role of mitochondrial K(ATP) channels as an effector mechanism for memorizing remote preconditioning was further studied by the effect of the specific mitochondrial K(ATP) activator diaxozide (10 mg/kg) on myocardial infarct size. Remote preconditioning reduced I/R injury in the explanted heart (0.17 +/- 0.03 vs. 0.39 +/- 0.05, P < 0.05) and improved left ventricular function during reperfusion compared with control (P < 0.05). Similar effects were obtained with diazoxide. Remote preconditioning was abolished by the addition of 5-HD and glibenclamide but not by HMR-1098. In conclusion, the protective effect of remote preconditioning is memorized in the explanted heart by a mechanism that involves mitochondrial K(ATP) channels.     

Development of an in vitro model for study of the efficacy of ischemic preconditioning in human skeletal muscle against ischemia-reperfusion injury.

Ischemia-reperfusion (I/R) injury causes skeletal muscle infarction and ischemic preconditioning (IPC) augments ischemic tolerance in animal models. To date, this has not been demonstrated in human skeletal muscle. This study aimed to develop an in vitro model to investigate the efficacy of simulated IPC in human skeletal muscle. Human skeletal muscle strips were equilibrated in oxygenated Krebs-Henseleit-HEPES buffer (37 degrees C). Aerobic and reperfusion phases were simulated by normoxic incubation and reoxygenation, respectively. Ischemia was simulated by hypoxic incubation. Energy store, cell viability, and cellular injury were assessed using ATP, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), and lactate dehydrogenase (LDH) assays, respectively. Morphological integrity was assessed using electron microscopy. Studies were designed to test stability of the preparation (n = 5-11) under normoxic incubation over 24 h; the effect of 1, 2, 3, 4, or 6 h hypoxia followed by 2 h of reoxygenation; and the protective effect of hypoxic preconditioning (HPC; 5 min of hypoxia/5 min of reoxygenation) before 3 h of hypoxia/2 h of reoxygenation. Over 24 h of normoxic incubation, muscle strips remained physiologically intact as assessed by MTT, ATP, and LDH assays. After 3 h of hypoxia/2 h of reoxygenation, MTT reduction levels declined to 50.1 +/- 5.5% (P < 0.05). MTT reduction levels in HPC (82.3 +/- 10.8%) and normoxic control (81.3 +/- 10.2%) groups were similar and higher (P < 0.05) than the 3 h of hypoxia/2 h of reoxygenation group (45.2 +/- 5.8%). Ultrastructural morphology was preserved in normoxic and HPC groups but not in the hypoxia/reoxygenation group. This is the first study to characterize a stable in vitro model of human skeletal muscle and to demonstrate a protective effect of HPC in human skeletal muscle against hypoxia/reoxygenation-induced injury.     

Aging and acute exercise enhance free radical generation in rat skeletal muscle.

Reactive oxygen species (ROS) are implicated in the mechanism of biological aging and exercise-induced oxidative damage. The present study examined the effect of an acute bout of exercise on intracellular ROS production, lipid and protein peroxidation, and GSH status in the skeletal muscle of young adult (8 mo, n = 24) and old (24 mo, n = 24) female Fischer 344 rats. Young rats ran on a treadmill at 25 m/min and 5% grade until exhaustion (55.4 +/- 2.7 min), whereas old rats ran at 15 m/min and 5% grade until exhaustion (58.0 +/- 2.7 min). Rate of dichlorofluorescin (DCFH) oxidation, an indication of ROS and other intracellular oxidants production in the homogenate of deep vastus lateralis, was 77% (P < 0.01) higher in rested old vs. young rats. Exercise increased DCFH oxidation by 38% (P < 0.09) and 50% (P < 0.01) in the young and old rats, respectively. DCFH oxidation in isolated deep vastus lateralis mitochondria with site 1 substrates was elevated by 57% (P < 0.01) in old vs. young rats but was unaltered with exercise. Significantly higher DCFH oxidation rate was also found in aged-muscle mitochondria (P < 0.01), but not in homogenates, when ADP, NADPH, and Fe(3+) were included in the assay medium without substrates. Lipid peroxidation in muscle measured by malondialdehyde content showed no age effect, but was increased by 20% (P < 0.05) with exercise in both young and old rats. Muscle protein carbonyl formation was unaffected by either age or exercise. Mitochondrial GSH/ GSSG ratio was significantly higher in aged vs. young rats (P < 0.05), whereas exercise increased GSSG content and decreased GSH/GSSG in both age groups (P < 0.05). These data provided direct evidence that oxidant production in skeletal muscle is increased in old age and during prolonged exercise, with both mitochondrial respiratory chain and NADPH oxidase as potential sources. The alterations of muscle lipid peroxidation and mitochondrial GSH status were consistent with these conclusions.     

Attenuation of ischemic preconditioning in pigs by scavenging of free oxyradicals with ascorbic acid

Free oxyradicals are involved in the signal transduction of ischemic preconditioning in rats and rabbits. Data from larger mammals in which the infarct development is closer to that in humans are lacking. We have therefore investigated the impact of the radical scavenger ascorbic acid on ischemic preconditioning in pigs. In 33 anesthetized pigs, the left anterior descending coronary artery was perfused from an extracorporeal circuit. Infarct size (measured as percent area at risk) was determined by triphenyltetrazolium chloride staining. In placebo-treated animals undergoing 90 min of severe ischemia and 120 min of reperfusion, infarct size averaged 26.9 +/- 3.9% (mean +/- SE; n = 9). Ischemic preconditioning by 10 min of ischemia and 15 min of reperfusion reduced infarct size to 6.4 +/- 2.4% (P < 0.05 vs. placebo; n = 9). Intravenous infusion of ascorbic acid (30 min before ischemic preconditioning or ischemia; 2-g bolus followed by 25 mg/min until the end of ischemia) had no effect on infarct size per se (22.6 +/- 6.5%; n = 6), but largely abolished the infarct size reduction by ischemic preconditioning (19.1 +/- 5.4%; n = 9). Scavenging of free oxyradicals with ascorbic acid largely attenuates the beneficial effect of ischemic preconditioning in pigs.     

Preconditioning by 17beta-estradiol in isolated rat heart depends on PI3-K/PKB pathway, PKC, and ROS

To study the cell signaling events leading to 17beta-estradiol (E(2))-induced acute cardioprotection, we subjected isolated rat hearts to three 5-min cycles of 10 microM E(2) before 30 min of regional ischemia, followed by 2 h of reperfusion. Protection was judged by changes in infarct size in percentage of risk zone volume. To test the importance of phosphoinositide 3-kinase (PI3-K), protein kinase C (PKC), or reactive oxygen species (ROS) in E(2)-induced protection, we combined wortmannin (1 microM), chelerythrine (2 microM), and 2-mercaptopropionylglycine (300 microM), respectively, with E(2) exposure. Changes in phosphorylation of protein kinase B (PKB) and selected PKC isoforms were tested by immunoblotting of total lysates and subcellular fractions, along with assessment of PKC translocation from soluble to membrane fraction of heart tissue homogenates. Intracellular ROS levels induced by E(2) preconditioning were investigated. E(2) preconditioning led to significant reduction in infarct size from 31.8 +/- 5.3 to 20.2 +/- 2.6% in male hearts and from 42.7 +/- 4.7 to 17.1 +/- 3.4% in female hearts (P < 0.05). Protection was abolished by wortmannin (30.0 +/- 3.2%), chelerythrine (45.1 +/- 4.4%), and 2-mercaptopropionylglycine (36.8 +/- 4.7%). E(2) preconditioning induced phosphorylation of PKB, PKCalpha, and PKCepsilon and membrane translocation of PKCepsilon and PKCdelta. Intracellular ROS levels were found elevated after transient treatment with hormone. Therefore, our data demonstrate the ability of E(2) to induce preconditioning-like cardioprotection via cell signaling events shared by classic ischemic preconditioning.     

ATP stimulates the release of prostacyclin from perfused veins isolated from the hamster hindlimb

ATP-stimulated prostacyclin release from veins was investigated using epigastric veins isolated from hamsters. Veins were perfused with MOPS-buffered physiological salt solution (PSS). ATP was administered into the perfusate, and the bath solution (MOPS-PSS) was collected and assayed for the presence of the stable prostacyclin metabolite 6-keto-PGF1alpha. ATP (100 microM) resulted in reproducible increases in bath concentration from 73 +/- 22 to 279 +/- 50 pg/ml (P < 0.05, n = 5). This response was abolished by indomethacin (10 microM, P < 0.05). To ascertain whether the endothelium was the source of prostacyclin, endothelium was disrupted using air (n = 10) or deoxycholic acid (n = 6). Perfusion with air significantly reduced (P < 0.05) but did not completely abolish ATP-stimulated release of prostacyclin, while deoxycholic acid totally abolished the response (P < 0.05). The nonselective P2 receptor antagonist reactive blue 2 (100 microM) attenuated ATP-mediated release of prostacyclin but did not significantly alter ACh-stimulated release of prostacyclin. The nonselective adenosine receptor antagonist xanthine amine congener (1 microM) had no effect on ATP-stimulated release, and adenosine did not stimulate the release of prostacyclin. These results show that increases in intraluminal concentration of ATP stimulate abluminal release of prostacyclin from the venous endothelium. This effect is mediated by P2 receptors while adenosine and its receptors are not involved in this response.     

ATP-mediated release of arachidonic acid metabolites from venular endothelium causes arteriolar dilation

This study was designed to test the hypothesis that venular administration of ATP resulted in endothelium-dependent dilation of adjacent arterioles through a mechanism involving cyclooxygenase products. Forty-three male golden hamsters were anesthetized with pentobarbital sodium (60 mg/kg ip), and the cremaster muscle was prepared for in vivo microscopy. ATP (100 microM) injected into venules dilated adjacent arterioles from a mean diameter of 51 +/- 4 to 76 +/- 6 microm (P < 0.05, n = 6). To remove the source of endothelial-derived relaxing factors, the venules were then perfused with air bubbles to disrupt the endothelium. Resting arteriolar diameter was not altered after disruption of the venular endothelium (51 +/- 5 microm), and the responses to venular ATP infusions were significantly attenuated (59 +/- 4 microm, P < 0.05). To determine whether the relaxing factor was a cyclooxygenase product, ATP infusion studies were repeated in the absence and presence of indomethacin (28 microM). Under control conditions, ATP (100 microM) infusion into the venule caused an increase in mean arteriolar diameter from 55 +/- 4 to 78 +/- 3 microm (P < 0.05, n = 6). In the presence of indomethacin, mean resting arteriolar tone was not significantly altered (49 +/- 4 microm), and the response to ATP was significantly attenuated (54 +/- 4 microm, P < 0.05, n = 6). These studies show that increases in venular ATP concentrations stimulate the release of cyclooxygenase products, possibly from the venular endothelium, to vasodilate the adjacent arteriole.     

Mechanism of rat mesenteric arterial KATP channel activation by 14,15-epoxyeicosatrienoic acid

Recently, we reported that 11,12-epoxyeicosatrienoic acid (11,12-EET) potently activates rat mesenteric arterial ATP-sensitive K(+) (K(ATP)) channels and produces significant vasodilation through protein kinase A-dependent mechanisms. In this study, we tried to further delineate the signaling steps involved in the activation of vascular K(ATP) channels by EETs. Whole cell patch-clamp recordings [0.1 mM ATP in the pipette, holding potential (HP) = 0 mV and testing potential (TP) = -100 mV] in freshly isolated rat mesenteric smooth muscle cells showed small glibenclamide-sensitive K(ATP) currents (19.0 +/- 7.9 pA, n = 5) that increased 6.9-fold on exposure to 5 microM 14,15-EET (132.0 +/- 29.0 pA, n = 7, P < 0.05 vs. control). With 1 mM ATP in the pipette solution, K(ATP) currents (HP = 0 mV and TP = -100 mV) were increased 3.5-fold on exposure to 1 microM 14,15-EET (57.5 +/- 14.3 pA, n = 9, P < 0.05 vs. baseline). In the presence of 100 nM iberiotoxin, 1 microM 14,15-EET hyperpolarized the membrane potential from -20.5 +/- 0.9 mV at baseline to -27.1 +/- 3.0 mV (n = 6 for both, P < 0.05 vs. baseline), and the EET effects were significantly reversed by 10 microM glibenclamide (-21.8 +/- 1.4 mV, n = 6, P < 0.05 vs. EET). Incubation with 5 microM 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE), a 14,15-EET antagonist, abolished the 14,15-EET effects (31.0 +/- 11.8 pA, n = 5, P < 0.05 vs. 14,15-EET, P = not significant vs. control). The 14,15-EET effects were inhibited by inclusion of anti-G(s)alpha antibody (1:500 dilution) but not by control IgG in the pipette solution. The effects of 14,15-EET were mimicked by cholera toxin (100 ng/ml), an exogenous ADP-ribosyltransferase. Treatment with the ADP-ribosyltransferase inhibitors 3-aminobenzamide (1 mM) or m-iodobenzylguanidine (100 microM) abrogated the effects of 14,15-EET on K(ATP) currents. These results were corroborated by vasodilation studies. 14,15-EET dose-dependently dilated isolated small mesenteric arteries, and this was significantly attenuated by treatment with 14,15-EEZE or 3-aminobenzamide. These results suggest that 14,15-EET activates vascular K(ATP) channels through ADP-ribosylation of G(s)alpha.     

Isoflurane activates rat mitochondrial ATP-sensitive K+ channels reconstituted in lipid bilayers

Activation of mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channels is critical in myocardial protection induced by preconditioning with volatile anesthetics or brief periods of ischemia. In this study, we characterized rat mitoK(ATP) channels reconstituted in lipid bilayers and examined their direct regulation by isoflurane. Mitochondria and the inner membrane fraction were isolated from rat ventricles and fused into lipid bilayers. On the basis of their inhibition by 5-hydroxydecanoate (5-HD)/ATP or activation by diazoxide, mitoK(ATP) channels of several conductance states were observed in symmetrical (150 mM) potassium glutamate (26, 47, 66, 83, and 105 pS). Isoflurane (0.8 mM) increased the cumulative open probability from 0.09 +/- 0.02 at baseline to 0.50 +/- 0.09 (P < 0.05, n = 5), which was inhibited by 5-HD. Isoflurane caused a dose-dependent rightward shift in ATP inhibition of mitoK(ATP) channels, which increased the IC(50) for ATP from 335 +/- 4 to 940 +/- 34 microM at 0.8 mM (P < 0.05, n = 5 approximately 8). We conclude that direct activation of the mitoK(ATP) channel by isoflurane is likely to contribute to volatile anesthetic-induced myocardial preconditioning.     

Alterations in KATP and KCa channel function in cerebral arteries of insulin-resistant rats

We examined whether insulin resistance alters the function of ATP-dependent and Ca(2+)-activated K(+) channels (K(ATP) and K(Ca) channels, respectively) in pressurized isolated middle cerebral arteries (MCAs) from fructose-fed insulin-resistant (IR) and control rats. Blockade of K(Ca) channels with tetraethylammonium chloride (TEA, 2.5 mM) or iberiotoxin (IBTX, 0.1 microM) increased the spontaneously developed tone in control MCAs by 10.5 +/- 1.3% (n = 10) and 13.3 +/- 2.3% (n = 6), respectively. In the IR arteries, TEA induced similar constrictions (8.0 +/- 1.1%, n = 10), but IBTX constricted the IR arteries by only 3.1 +/- 0.9% (n = 8; P < 0.01). Bradykinin (BK)-induced endothelium-mediated relaxation was reduced in IR MCAs. Maximum relaxation to BK (10(-6) M) was 42 +/- 4% in control (n = 9) and 19 +/- 2% in IR (n = 10; P < 0.01) arteries. Pretreatment with TEA, IBTX, or the K(ATP) channel blocker glibenclamide (10 microM) inhibited relaxation to BK in control MCAs but did not alter dilation in IR arteries. Relaxation to the K(ATP) channel opener cromakalim was also diminished in IR MCAs. Maximum relaxation to cromakalim (10(-5) M) was 48 +/- 3% in control (n = 6) and 19 +/- 2% in IR arteries (n = 6; P < 0.01). These findings demonstrate that insulin resistance alters the function of K(ATP) and K(Ca) channels in isolated MCAs and affects the control of resting vascular tone and the mediation of dilator stimuli.     

Oxidant activity in skeletal muscle fibers is influenced by temperature, CO2 level, and muscle-derived nitric oxide

Free radicals are produced continuously by skeletal muscle fibers. Extracellular release of reactive oxygen species (ROS) and nitric oxide (NO) derivatives has been demonstrated, but little is known about intracellular oxidant regulation. We used a fluorescent oxidant probe, 2',7'-dichlorofluorescin (DCFH), to assess net oxidant activity in passive muscle fiber bundles isolated from mouse diaphragm and studied in vitro. We tested the following three hypotheses. 1) Net oxidant activity is decreased by muscle cooling. 2) CO(2) exposure depresses intracellular oxidant activity. 3) Muscle-derived ROS and NO both contribute to overall oxidant activity. Our results indicate that DCFH oxidation was diminished by cooling muscle fibers from 37 degrees C to 23 degrees C (P < 0.001). The rate of DCFH oxidation correlated positively with CO(2) exposure (0-10%; P < 0.05) and negatively with concurrent changes in pH (7.0-8.5; P < 0.05). Separate exposures to anti-ROS enzymes (superoxide dismutase, 1 kU/ml; catalase, 1 kU/ml), a glutathione peroxidase mimetic (ebselen, 30 microM), NO synthase inhibitors (N(omega)-nitro-l-arginine methyl ester, 1 mM; N(omega)-monomethyl-l-arginine, 1 mM), or an NO scavenger (hemoglobin, 1 microM) each inhibited DCFH oxidation (P < 0.05). Oxidation was increased by hydrogen peroxide, 100 microM, an NO donor (NOC-22, 400 microM), or the substrate for NO synthase (l-arginine, 5 mM). We conclude that net oxidant activity in resting muscle fibers is 1) decreased at subphysiological temperatures, 2) increased by CO(2) exposure, and 3) influenced by muscle-derived ROS and NO derivatives to similar degrees.     

Reactive oxygen species mediate arachidonic acid-induced dilation in porcine coronary microvessels

Reactive oxygen species (ROS) have been proposed to mediate vasodilation in the microcirculation. We investigated the role of ROS in arachidonic acid (AA)-induced coronary microvascular dilation. Porcine epicardial coronary arterioles (110 +/- 4 microm diameter) were mounted onto pipettes in oxygenated Krebs buffer. Vessels were incubated with vehicle or 1 mM Tiron (a nonselective ROS scavenger), 250 U/ml polyethylene-glycolated (PEG)-superoxide dismutase (SOD; an O2- scavenger), 250 U/ml PEG-catalase (a H2O2 scavenger), or the cyclooxygenase (COX) inhibitors indomethacin (10 microM) or diclofenac (10 microM) for 30 min. After endothelin constriction (30-60% of resting diameter), cumulative concentrations of AA (10(-10)-10(-5)M) were added and internal diameters measured by video microscopy. AA (10-7 M) produced 37 +/- 6% dilation, which was eliminated by the administration of indomethacin (4 +/- 7%, P < 0.05) or diclofenac (-8 +/- 8%, P < 0.05), as well as by Tiron (-4 +/- 5%, P < 0.05), PEG-SOD (-10 +/- 6%, P < 0.05), or PEG-catalase (1 +/- 4%, P < 0.05). Incubation of small coronary arteries with [3H]AA resulted in the formation of prostaglandins, which was blocked by indomethacin. In separate studies in microvessels, AA induced concentration-dependent increases in fluorescence of the oxidant-sensitive probe dichlorodihydrofluorescein diacetate, which was inhibited by pretreatment with indomethacin or by SOD + catalase. We conclude that in porcine coronary microvessels, COX-derived ROS contribute to AA-induced vasodilation.     

Adenosine preconditions against endothelin-induced constriction of coronary arterioles

Myocardial hypoperfusion is accompanied by concomitant increases in adenosine and endothelin-1 (ET-1) production, but the vasodilatory effect of adenosine prevails over that of ET-1. Therefore, we hypothesized that adenosine-induced or ischemic preconditioning reduces the vasoconstrictive effect of ET-1. Coronary arteriolar diameter in vivo was measured using fluorescence microangiography in anesthetized open-thorax dogs. ET-1 (5 ng. kg(-1). min(-1) administered intracoronary, n = 10) induced progressive constriction over 45 min [25 +/- 6% (SE)]. The constriction was blocked by preconditioning with adenosine (25 microgram. kg(-1). min(-1) administered intracoronary) for 20 min and 10 min of washout (n = 10) or attenuated by ischemic preconditioning (four 5-min periods of ischemia, 9 +/- 5% at 45 min). To investigate the receptor involved in this process, coronary arterioles (50-150 micrometer) were isolated and pressurized at 60 mmHg in vitro. The ET-1 dose-response curve (1 pM-5 nM) was rightward shifted after preconditioning with adenosine (1 microM) for 20 min and 10 min of washout (n = 11). Blockade of A(2) receptors [8-(3-chlorostyryl)caffeine, 1 microM, n = 9] but not A(1) receptors (8-cyclopentyl-1,3-dipropylxanthine, 100 nM, n = 7) prevented this shift. These results suggest that adenosine confers a vascular preconditioning effect, mediated via the A(2) receptor, against endothelin-induced constriction. This effect may offer a new protective function of adenosine in preventing excessive coronary constriction.     

Inhibition of p38 MAPK and AMPK restores adenosine-induced cardioprotection in hearts stressed by antecedent ischemia by altering glucose utilization

p38 mitogen-activated protein kinase (MAPK) and 5'-AMP-activated protein kinase (AMPK) are activated by metabolic stresses and are implicated in the regulation of glucose utilization and ischemia-reperfusion (IR) injury. This study tested the hypothesis that inhibition of p38 MAPK restores the cardioprotective effects of adenosine in stressed hearts by preventing activation of AMPK and the uncoupling of glycolysis from glucose oxidation. Working rat hearts were perfused with Krebs solution (1.2 mM palmitate, 11 mM [(3)H/(14)C]glucose, and 100 mU/l insulin). Hearts were stressed by transient antecedent IR (2 x 10 min I/5 min R) before severe IR (30 min I/30 min R). Hearts were treated with vehicle, p38 MAPK inhibitor (SB-202190, 10 microM), adenosine (500 microM), or their combination before severe IR. After severe IR, the phosphorylation (arbitrary density units) of p38 MAPK and AMPK, rates of glucose metabolism (micromol x g dry wt(-1) x min(-1)), and recovery of left ventricular (LV) work (Joules) were similar in vehicle-, SB-202190- and adenosine-treated hearts. Treatment with SB-202190 + adenosine versus adenosine alone decreased p38 MAPK (0.03 +/- 0.01, n = 3 vs. 0.48 +/- 0.10, n = 3, P < 0.05) and AMPK (0.00 +/- 0.00, n = 3 vs. 0.26 +/- 0.08, n = 3 P < 0.05) phosphorylation. This was accompanied by attenuated rates of glycolysis (1.51 +/- 0.40, n = 7 vs. 3.95 +/- 0.65, n = 7, P < 0.05) and H(+) production (2.12 +/- 0.76, n = 7 vs. 6.96 +/- 1.48, n = 7, P < 0.05), and increased glycogen synthesis (1.91 +/- 0.25, n = 6 vs. 0.27 +/- 0.28, n = 6, P < 0.05) and improved recovery of LV work (0.81 +/- 0.08, n = 7 vs. 0.30 +/- 0.15, n = 8, P < 0.05). These data indicate that inhibition of p38 MAPK abolishes subsequent phosphorylation of AMPK and improves the coupling of glucose metabolism, thereby restoring adenosine-induced cardioprotection.     

Ceramide-induced preconditioning involves reactive oxygen species

INTRODUCTION: Ceramide induces programmed cell death and it is thought to contribute to cardiac ischemia/reperfusion (I/R) injury. In contrast, we have demonstrated that administration of low doses of ceramide engenders cardiac preconditioning (PC). Ceramide is known to generate reactive oxygen species (ROS) in cells. Since mechanisms triggering the ceramide-induced cardioprotection remain unknown, we investigated the role of ROS in the genesis of this protective mechanism. METHODS: Using an isolated Langendorff-perfused rat heart model, four groups (n > or = 6 in each group) were considered: Control hearts underwent 30 min index regional ischemia and 120 min of reperfusion. In the ceramide group, hearts were preconditioned with c2-ceramide 1 microM for 7 min followed by 10 min washout prior to the I/R insult. In additional groups, MPG (1 mM), a synthetic antioxidant was given for 15 min alone or bracketing the ceramide perfusion. In each group, infarct size was determined at the end of the reperfusion period and superoxide dismutases (CuZnSOD and MnSOD) and catalase activities were evaluated. RESULTS: Ceramide preconditioning reduced the infarct/area at risk (I/AAR) ratio (8.3 +/- 1.1% for ceramide vs. 36.4 +/- 1.2% for control, p < 0.001). Perfusion with MPG abolished the preconditioning effect of ceramide (I/AAR ratio = 36.7 +/- 4.9%). Ceramide was also associated with a 29% and 38% increase in catalase and CuZnSOD activities, respectively, compared with control group. CONCLUSION: Production of reactive oxygen species following ceramide preconditioning of the ischemic-reperfused heart appears to play a role in the cardioprotective effect of ceramide.     

Whole-body hypoxic preconditioning protects mice against acute hypoxia by improving lung function.

Survival in severe hypoxia such as occurs in high altitude requires previous acclimatization, which is acquired over a period of days to weeks. It was unknown whether intrinsic mechanisms existed that could be rapidly induced and could exert immediate protection on unacclimatized individuals against acute hypoxia. We found that mice pretreated with whole-body hypoxic preconditioning (WHPC, 6 cycles of 10-min hypoxia-10-min normoxia) survived significantly longer than control animals when exposed to lethal hypoxia (5% O2, survival time of 33.2 +/- 6.1 min vs. controls at 13.8 +/- 1.2 min, n = 10, P < 0.005). This protective mechanism became operative shortly after WHPC and remained effective for at least 8 h. Accordingly, mice subjected to WHPC demonstrated improved gas exchange when exposed to sublethal hypoxia (7% O2, arterial blood Po2 of 49.9 +/- 4.2 vs. controls at 39.7 +/- 3.6 Torr, n = 6, P < 0.05), reduced formation of pulmonary edema (increase in lung water of 0.491 +/- 0.111 vs. controls at 0.894 +/- 0.113 mg/mg dry tissue, n = 10, P < 0.02), and decreased pulmonary vascular permeability (lung lavage albumin of 7.63 +/- 0.63 vs. controls at 18.24 +/- 3.39 mg/dl, n = 6-10, P < 0.025). In addition, the severity of cerebral edema caused by exposure to sublethal hypoxia was also reduced after WHPC (increase in brain water of 0.254 +/- 0.052 vs. controls at 0.491 +/- 0.034 mg/mg dry tissue, n = 10, P < 0.01). Thus WHPC protects unacclimatized mice against acute and otherwise lethal hypoxia, and this protection involves preservation of vital organ functions.     

Inducing late phase of infarct protection in skeletal muscle by remote preconditioning: efficacy and mechanism

We have previously demonstrated that remote ischemic preconditioning (IPC) by instigation of three cycles of 10-min occlusion/reperfusion in a hindlimb of the pig elicits an early phase of infarct protection in local and distant skeletal muscles subjected to 4 h of ischemia immediately after remote IPC. The aim of this project was to test our hypothesis that hindlimb remote IPC also induces a late phase of infarct protection in skeletal muscle and that K(ATP) channels play a pivotal role in the trigger and mediator mechanisms. We observed that pig bilateral latissimus dorsi (LD) muscle flaps sustained 46 +/- 2% infarction when subjected to 4 h of ischemia/48 h of reperfusion. The late phase of infarct protection appeared at 24 h and lasted up to 72 h after hindlimb remote IPC. The LD muscle infarction was reduced to 28 +/- 3, 26 +/- 1, 23 +/- 2, 24 +/- 2 and 24 +/- 4% at 24, 28, 36, 48 and 72 h after remote IPC, respectively (P < 0.05; n = 8). In subsequent studies, hindlimb remote IPC or intravenous injection of the sarcolemmal K(ATP) (sK(ATP)) channel opener P-1075 (2 microg/kg) at 24 h before 4 h of sustained ischemia (i.e., late preconditioning) reduced muscle infarction from 43 +/- 4% (ischemic control) to 24 +/- 2 and 19 +/- 3%, respectively (P < 0.05, n = 8). Intravenous injection of the sK(ATP) channel inhibitor HMR 1098 (6 mg/kg) or the nonspecific K(ATP) channel inhibitor glibenclamide (Glib; 1 mg/kg) at 10 min before remote IPC completely blocked the infarct- protective effect of remote IPC in LD muscle flaps subjected to 4 h of sustained ischemia at 24 h after remote IPC. Intravenous bolus injection of the mitochondrial K(ATP) (mK(ATP)) channel inhibitor 5-hydroxydecanoate (5-HD; 5 mg/kg) immediately before remote IPC and 30-min intravenous infusion of 5-HD (5 mg/kg) during remote IPC did not affect the infarct-protective effect of remote IPC in LD muscle flaps. However, intravenous Glib or 5-HD, but not HMR 1098, given 24 h after remote IPC completely blocked the late infarct-protective effect of remote IPC in LD muscle flaps. None of these drug treatments affected the infarct size of control LD muscle flaps. The late phase of infarct protection was associated with a higher (P < 0.05) muscle content of ATP at the end of 4 h of ischemia and 1.5 h of reperfusion and a lower (P < 0.05) neutrophilic activity at the end of 1.5 h of reperfusion compared with the time-matched control. In conclusion, these findings support our hypothesis that hindlimb remote IPC induces an uninterrupted long (48 h) late phase of infarct protection, and sK(ATP) and mK(ATP) channels play a central role in the trigger and mediator mechanism, respectively.     

Peptide blockers of PKG inhibit ROS generation by acetylcholine and bradykinin in cardiomyocytes but fail to block protection in the whole heart

Bradykinin and acetylcholine (ACh) trigger preconditioning by ATP-sensitive K(+) (K(ATP)) channel-dependent production of reactive oxygen species (ROS). Recent evidence suggests that ROS production may in turn be influenced by cGMP-dependent protein kinase (PKG). This study utilized DT-2 and DT-3 peptides, highly specific membrane-permeable blockers of PKG. Rabbit cardiomyocytes were incubated for 15 min in reduced MitoTracker red, which becomes fluorescent only after exposure to ROS. Bradykinin (400 nM) and ACh (250 microM) caused a 49.9 +/- 5.9% and 46.8 +/- 1.7% increase in ROS production, respectively (P < 0.005 vs. untreated cells). Coincubation with DT-3 (250 nM) abolished both the ACh- and bradykinin-induced ROS signal, whereas a nonpermeable form of the peptide (W45) had no effect on ACh-induced ROS production. DT-3 was unable to block ROS production from diazoxide (100 microM), a selective opener of mitochondrial K(ATP) channels, suggesting that these channels are downstream of PKG. DT-2 (125 nM) also prevented ACh from triggering ROS production. 8-(4-Chlorophenylthio)-guanosine 3',5'-cyclic monophosphate (100 microM), a cGMP analog and potent direct activator of PKG, increased ROS production of cardiomyocytes by 44.7 +/- 7.1% (P < 0.001 vs. untreated cells). This increase was blocked by DT-2. Neither DT-2 nor DT-3 could block the anti-infarct effect of bradykinin in isolated rabbit hearts. Studies with fluorescent-tagged DT-3 revealed that it was confined to endothelial cells and never reached the myocytes. We conclude that both bradykinin and ACh trigger ROS generation by a pathway that includes PKG. Although the peptides may be inappropriate for a whole heart model, they are likely to become important tool drugs for elucidation of signal transduction pathways in cell preparations.