ucn-1028-c and Myocardial-Ischemia

This study aimed to examine:1) whether nicorandil protects the ischemic myocardium by activating sarcolemmal adenosine triphosphate (ATP)-sensitive K(+) (sarcK(ATP)) channels or the mitochondrial K(ATP) (mitoK(ATP)) channels, and 2) whether protein kinase C (PKC) activity is necessary for cardioprotection afforded by nicorandil.. Nicorandil is a hybrid of nitrate and a K(ATP) channel opener that activates the sarcK(ATP) and mitoK(ATP) channels. Both of these K(ATP) channels are regulated by PKC, and this kinase may be activated by nitric oxide and also by oxygen free radicals (OFR) generated after mitoK(ATP) channel opening.. In isolated rabbit hearts, infarction was induced by 30-min global ischemia/2-h reperfusion with monitoring of the activation recovery interval (ARI), an index of action potential duration. Protein kinase C translocation was assessed by Western blotting.. Nicorandil did not change ARI before ischemia, but it accelerated ARI shortening after the onset of ischemia and reduced infarct size by 90%. A sarcK(ATP) channel selective blocker, HMR1098, abolished acceleration of ischemia-induced ARI-shortening by nicorandil and eliminated 40% of nicorandil-induced infarct size limitation. A mitoK(ATP) channel selective blocker, 5-hydroxydecanoate, abolished the protection afforded by nicorandil without affecting ARI. Cardioprotection by nicorandil was inhibited neither by an OFR scavenger, N-2-mercaptopropionylglycine nor by a PKC inhibitor, calphostin C, at a dose that was capable of inhibiting PKC- epsilon translocation after preconditioning.. Both the sarcK(ATP) and mitoK(ATP) channels are involved in anti-infarct tolerance afforded by nicorandil, but PKC activation induced by nitric oxide or OFR generation, if any, does not play a crucial role.

Topics: Animals; Benzamides; Decanoic Acids; Enzyme Inhibitors; Glycine; Hemodynamics; Hydroxy Acids; In Vitro Techniques; Male; Mitochondria, Heart; Myocardial Ischemia; Naphthalenes; Nicorandil; Potassium Channels; Protein Kinase C; Rabbits; Sarcolemma; Sulfhydryl Compounds

Several recent studies have suggested an ATP-sensitive potassium channel opener (2-nicotinamidoethyl nitrate: 2-NN) may exert a protective effect against the myocardial ischemic/reperfusion injury. This study examines the effects of 2-NN on intracellular signaling by measuring intracellular cyclic AMP, cyclic GMP accumulation and protein kinase C (PKC) activity after 2-NN perfusion.. Ischemia/reperfused hearts were made by LAD occlusion for 30 min followed by 30 min of reperfusion in isolated rat hearts. Hearts were pre-perfused with 0.1 mM 2-NN, 100 nM Calphostin C, or 2-NN plus Calphostin C for 10 min prior to ischemia. The left ventricular function, cyclic AMP, cyclic GMP and LDH were examined to determine the effects of 2-NN on ischemic/reperfusion injury. Four separate groups of hearts were stained with a bisindolylmaleimide PKC inhibitor conjugated to fluorescein (fim, Teflabs) and PKC activity was measured.. 2-NN reduced ischemia/reperfusion injury as evidenced by the enhanced myocardial functional recovery, decreased LDH release after reperfusion, and decreased reperfusion arrhythmias. The PKC inhibitor attenuated myocardial functional recovery but not reperfusion arrhythmias. Cyclic AMP levels decreased after 10 min of 2-NN perfusion, compared to controls. We observed an increase in PKC activity after 2-NN treatment.. These results suggest that PKC plays a significant role in the cardioprotective effect of 2-NN on ischemic and reperfused myocardium. The anti-arrhythmic effect of 2-NN in the reperfusion phase may be linked its action on the ATP-sensitive potassium channel itself rather than its effect on PKC activity.

Topics: Animals; Coronary Circulation; Cyclic AMP; Cyclic GMP; Disease Models, Animal; Heart Function Tests; Hemodynamics; Male; Microscopy, Fluorescence; Myocardial Contraction; Myocardial Ischemia; Myocardial Reperfusion Injury; Naphthalenes; Nicorandil; Probability; Random Allocation; Rats; Rats, Sprague-Dawley; Reference Values; Sensitivity and Specificity

Calcium-tolerant rabbit cardiomyocytes were isolated using retrograde aortic perfusion with a nominally calcium-free, collagenase buffer. In vitro ischemic preconditioning was induced by a 10-min episode of ischemic pelleting, followed by a 15-min post-incubation and a prolonged period of ischemic pelleting. Injury was assessed by determination of cell contracture and trypan blue permeability following hypotonic swelling and correlated with metabolic assays of lactate and adenine nucleotides. The protein phosphatase PP1/2A inhibitor calyculin A and PP2A-selective fostriecin protected isolated rabbit cardiomyocytes from lethal injury after a 10-min pre-incubation and when added late into ischemic pellets after a delay of 75 min. At the time of late drug addition, cells were severely ATP-depleted and in rigor contracture. Protection with Calyculin A from 1 nM to 1 microM was dose-related. Cells pre-incubated with 10 nM to 10 microM fostriecin 10 min prior to ischemic pelleting were protected with an EC50 approximating 71 nM, implying protection at a PP2A-selective dose. The selective protein kinase C inhibitor, calphostin C, blocked ischemic preconditioning protection but not protection from 1 microM calyculin A. Protection of severely ischemic cardiomyocytes following protein phosphatase inhibition appears not to require PKC activity or ATP conservation. Pre-incubation of cells with calyculin A induced high levels of phosphorylation in p38 mitogen activated protein kinase (MAPK), as compared to the ischemia-induced phosphorylation observed in the untreated group only at 30 min of ischemia, providing evidence of protein phosphatase activity in cardiomyocytes. Pharmacological protection in late ischemia has been demonstrated, but the mechanism of protection is undetermined.

Topics: Adenosine Triphosphate; Alkenes; Animals; Calcium-Calmodulin-Dependent Protein Kinases; Cells, Cultured; Dose-Response Relationship, Drug; Enzyme Inhibitors; Ischemic Preconditioning, Myocardial; Marine Toxins; Mitogen-Activated Protein Kinases; Myocardial Ischemia; Myocardium; Naphthalenes; Oxazoles; p38 Mitogen-Activated Protein Kinases; Phosphoprotein Phosphatases; Phosphorylation; Polyenes; Protein Kinase C; Pyrones; Rabbits

Cardiomyocytes isolated from rabbit hearts were preconditioned in vitro by 10 min of ischemia or treatment with 100 microM adenosine. Protection was assessed as average integrated mortality following osmotic swelling and determination of viability by trypan blue exclusion over 60-180 min ischemia. Repetitive sub-maximal stimulations with 1 microM adenosine amplified the protective response. Treatment with adenosine only at the onset of prolonged ischemia afforded a dose-dependent protection. The PKC inhibitor calphostin C (500 nm) blocked preconditioning and, when added during ischemic incubation of non-preconditioned cells, significantly increased injury. The memory of adenosine-induced preconditioning decayed over a 60 min post-incubation period. Light activation of calphostin C initially added to preconditioned ischemic cells in the dark indicated that a 10 min period of PKC activity at the onset of ischemia affords full protection. The reversible PKC inhibitors chelerythrine (5 microM) or staurosporine (100 nM) added only to bracket induction of ischemia, reduced but did not abolish protection. Protection was abolished when either drug was present during induction and a subsequent 30 min post-incubation period. Staurosporine included during initiation and post-incubation but washed out in the final 5 min of post-incubation allowed significant protection to occur. It is concluded that a single adenosine receptor-stimulation induces protection as it preconditions, and PKC activity appears to be required for both induction and protection. Memory may reside in post-receptor amplification of an initial protective response.

Topics: Adenosine; Alkaloids; Animals; Benzophenanthridines; Cardiovascular Agents; Cells, Cultured; Enzyme Activation; Enzyme Inhibitors; Heart; Ischemic Preconditioning, Myocardial; Myocardial Ischemia; Myocardium; Naphthalenes; Phenanthridines; Protein Kinase C; Rabbits; Staurosporine

Protein kinase C (PKC) has been implicated in the cardioprotective effects of ischemic preconditioning in rabbits, but whether it plays a role in rats is unknown. We tested this preconditioning PKC theory by assessing whether the inhibition of PKC with calphostin C, a potent and specific inhibitor of PKC, can block the preconditioning effects in this model. Four groups of rats were studied: 1) control + vehicle, 2) control + calphostin C, 3) preconditioning + vehicle, and 4) preconditioning + calphostin C. All rats underwent 90 min of coronary occlusion followed by 4 h of reperfusion; in addition, preconditioned rats underwent three 3-min episodes of ischemia and 5 min of reperfusion before the 90 min of ischemia. Two injections of vehicle or calphostin C (0.1 mg/kg) were administered in intravenous boluses 29 min and 3 min before the 90-min coronary occlusion, i.e., one dose was given 5 min before preconditioning, and another dose was given between preconditioning and the sustained 90 min of ischemia in preconditioned rats. After 4 h of reperfusion, the area at risk (AR) was delineated by dye injection and area of necrosis was assessed by triphenyltetrazolium chloride staining. The electrocardiogram was recorded for the incidence of ventricular tachycardia (VT) and ventricular fibrillation. AR was similar in all four groups. In the nonpreconditioned control rats receiving vehicle, myocardial infarct size expressed as a percentage of the AR averaged 45.7 +/- 1.7%. Pretreatment with calphostin C had no effect on infarct size (48.9 +/- 3.4%) in nonpreconditioned control rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Analysis of Variance; Animals; Arrhythmias, Cardiac; Blood Pressure; Drug Administration Schedule; Female; Heart Rate; Myocardial Ischemia; Myocardial Reperfusion; Myocardium; Naphthalenes; Necrosis; Polycyclic Compounds; Premedication; Protein Kinase C; Rabbits; Rats; Rats, Sprague-Dawley; Reference Values; Time Factors

The aim was to determine if in vitro ischaemic preincubation can precondition cardiomyocytes and if the responses to adenosine receptor antagonists are similar to those previously determined during "metabolic" preconditioning with glucose deprivation or adenosine agonists.. Isolated rabbit cardiomyocytes were preconditioned with 10 min of ischaemic preincubation, followed by a 30 min postincubation before the final sustained ischaemic period. The protein kinase C inhibitor calphostin C or the adenosine receptor antagonists 8-sulphophenyltheophylline (SPT), BW 1433U, and 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) were added either during the preincubation or into the final ischaemic pellet. Adenosine deaminase (10 U.ml-1) was added during ischaemic preincubation. Rates of contracture and extent of injury were determined by sequential sampling and assessment of trypan blue permeability following 85 mOsM swelling.. Myocytes were preconditioned by a 10 min in vitro ischaemic preincubation. Preincubation with 100 microM SPT or with adenosine deaminase, or addition of 200 nM calphostin C into the final ischaemic pellet did not alter rates of rigor contracture but nearly abolished protection. A significant degree of protection was maintained following ischaemic preincubation with the highly selective adenosine A1 receptor blocker DPCPX (10 microM), while the A1/A3 antagonist BW 1433U (1 microM) severely limited protection. SPT and BW 1433U added only into the final ischaemic pellet of preconditioned cells significantly blocked protection, while protection was maintained in the presence of DPCPX.. Ischaemic preconditioning of cardiomyocytes is blocked by adenosine receptor antagonists known to bind to A3 receptors but not by DPCPX which has high affinity for A1 receptors, but little affinity for A3 receptors. Maintenance of protection during the final ischaemic phase has a similar receptor specificity. Blockade of protein kinase C activity abolishes protection. Ischaemic and metabolic preconditioning in vitro appear to occur through similar pathways.

Topics: Animals; In Vitro Techniques; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardium; Naphthalenes; Protein Kinase C; Purinergic P1 Receptor Antagonists; Rabbits; Theophylline; Xanthines

The aim was to determine if isolated rabbit cardiomyocytes could be preconditioned.. Cardiomyocytes isolated from rabbit hearts were subjected to 15 min oxygenated preincubation, with and without substrate, prior to concentration into an ischaemic slurry, with or without glucose present. The effects of an adenosine agonist (CCPA), an adenosine receptor blocker (SPT), and the protein kinase C blocker, calphostin C, on rates of ischaemic contracture and survival of the myocytes were determined after various times of ischaemia, following resuspension of the cells in hypotonic media.. A glucose-free preincubation period protected myocytes from subsequent ischaemic injury, with a 40% reduction of cell death at 90-120 min and 1-2 h delay in cell death. CCPA added during preincubation and during the ischaemic period also tended to protect from injury, but the differences were not significant and protection was less than with a glucose-free preincubation. Although preincubation with CCPA did not precondition, SPT added to the preincubation medium only, or to both the preincubation medium and the ischaemic pellet, inhibited the preconditioning effect of a glucose-free preincubation period. Calphostin C, added only into the ischaemic pellet, inhibited the preconditioning effect of glucose-free preincubation.. Glucose-free preincubation protects ischaemic isolated myocytes from subsequent ischaemia. The degree of protection is great enough to account for protection seen in intact hearts, following preconditioning protocols. Protection is blocked by SPT and a highly specific protein kinase C inhibitor, calphostin C. Protection from ischaemic injury that seems to mimic ischaemic preconditioning can be induced in isolated cardiomyocytes, and appears dependent on adenosine receptors and activation of protein kinase C.

Topics: Adenosine; Animals; Cell Size; Cell Survival; Cells, Cultured; Glucose; Heart; Myocardial Ischemia; Myocardium; Naphthalenes; Polycyclic Compounds; Protein Kinase C; Rabbits; Theophylline; Time Factors

The aim was to further characterise an experimental model of preconditioning of isolated rabbit cardiomyocytes and to determine the role of adenosine receptor subtypes in initiation of the protective response.. Isolated myocytes were subjected to 5 min preincubation in the presence or absence of glucose and various agonists and antagonists of adenosine receptors. Ischaemic pelleting was preceded by a 30 min postincubation period. Rate and extent of injury during ischaemia was determined by sequential sampling of the pelleted cells and assessment of trypan blue permeability following 85 mOsm swelling.. Myocytes were preconditioned with a 30-50% reduction of injury by a 5 min glucose-free preincubation. Substitution of 5 mM pyruvate for glucose during preincubation did not prevent the protective response. Protection was maintained over a 60-180 min postincubation period. Protection was blocked by 100 microM of the non-specific adenosine A1/A2 antagonist SPT, both when added only during preincubation or only into the ischaemic pellet. Calphostin C, a specific protein kinase C inhibitor at 200 nM, added to the ischaemic pellet blocked protection. Preincubation with R-PIA, the adenosine A1 agonist, did not precondition at an A1 selective dose of 1 microM, but did at 100 microM. The selective A2 agonist CGS 12680 (1 microM) did not precondition. The selective A1/A3 adenosine agonist, APNEA, preconditioned at 1 microM and 200 nM dose levels. Preconditioning induced either by 200 nM APNEA or by glucose-free preincubation was not blocked by 200 nM or 10 microM of the A1 antagonist DPCPX, which has extremely low affinity for A3 receptors, but was blocked by 1 microM of the A1/A3 adenosine antagonist BW 1433U83.. Preconditioning can be induced in isolated myocytes by a 5 min preincubation/30 min postincubation protocol, and a similar protection induced by adenosine agonists with A3, but not A1 selectivity. Preconditioning is blocked by non-selective or selective A1/A3 adenosine antagonists and a specific protein kinase C inhibitor, but not by A1 antagonists with little affinity for A3 receptors. The results suggest that preconditioning in isolated rabbit myocytes requires participation of adenosine receptors with agonist/antagonist binding characteristics of the A3 subtype, and is likely to be mediated by activation of protein kinase C.

Topics: Adenosine; Animals; Antihypertensive Agents; Cells, Cultured; Glucose; Myocardial Infarction; Myocardial Ischemia; Myocardium; Naphthalenes; Phenethylamines; Phenylisopropyladenosine; Polycyclic Compounds; Protein Kinase C; Purinergic Antagonists; Pyruvates; Pyruvic Acid; Rabbits; Receptors, Purinergic P1; Theophylline; Time Factors; Trypan Blue; Xanthines