u-50488 and Myocardial-Ischemia

There is evidence that the myocytes produce dynorphin and dynorphin-like peptides, which are kappa opioid receptor (kappa-OR) agonists. Activation of kappa-OR, a dominant opioid receptor in the heart, alters the cardiac function in vivo and in vitro. The observations suggest that the endogenous kappa-opioid peptides may act as autocrines or paracrine in regulation of cardiac functions. Myocardial ischemia is a common cause of heart disorders, which is manifested in decreased myocardial performance, arrhythmia and infarct. When myocardial ischemia occurs, the sympathetic discharge increases, which in turn increases the work-load and oxygen consumption. This exacerbates the situation induced by ischemia. One of the mechanisms with which the body protects against ischemia-induced injury/arrhythmia is inhibition of stimulation of beta-adrenoceptor (beta-AR), the receptor mediating the actions of sympathetic stimulation. kappa-Opioids inhibit the beta-AR activation. The inhibition of the beta-AR activation is due to inhibition of Gs-protein and to a lesser extent the adenylyl cyclase of the signaling pathway mediating beta-AR stimulation by a pertussis sensitive G-protein that mediates kappa-OR activation. Another mechanism against ischemia-induced injury is preconditioning, which is defined as prior exposures to ischemia or other insults make the heart more tolerant to subsequent and more severe insults. Protection occurs immediately or 1-3 days after preconditioning. kappa-OR mediates protection of preconditioning with ischemia or metabolic inhibition, one of the consequences of ischemia, in the heart. Activation of kappa-OR by U50488H, a selective kappa-OR agonist (pharmacological preconditioning with U50488H, UP), activates protein kinase C (PKC), opens K(ATP) channels and increases the production of heat shock proteins. Blockade of PKC, or closing of the K(ATP) channels or inhibition of the synthesis of the heat shock protein abolishes the cardioprotection of UP. The findings indicate the important roles of PKC, the K(ATP) channels and the heat shock protein in cardioprotection of UP. In addition, UP also attenuates the Ca(2+) overload, a precipitating cause of cardiac injury, induced by ischemic insults, indicating that UP may confer cardioprotection via at least partly attenuating the Ca(2+) overload. Most interestingly, blockade of the K(ATP) channels with channel blockers, that abolishes the delayed cardioprotection of UP, also attenuates the inhibito

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Adrenergic beta-Antagonists; Animals; Calcium; Cardiotonic Agents; Humans; Ischemic Preconditioning, Myocardial; Myocardial Ischemia; Myocardial Reperfusion Injury; Receptors, Adrenergic, beta; Receptors, Opioid, kappa; Signal Transduction

We studied the ability of the agonist of κ1-opioid receptors U-50,488 in doses of 0.1 and 1 mg/kg to simulate ischemic pre- and postconditioning of the heart and κ-opioid receptors ICI 199,441 in a dose of 0.1 mg/kg to simulate the antiarrhythmic effect of heart preconditioning. The duration of ischemia was 10 or 45 min and the duration of reperfusion was 10 min or 2 h. Administration of 1 mg/kg U-50,488 both before ischemia and 5 min before reperfusion produced a pronounced antiarrhythmic effect. U-50,488 injected 5 min before reperfusion 2-fold reduced the ratio of infarction to risk area. Administration of ICI 199,441 in a dose of 0.1 mg/kg 15 min before ischemia produced a potent antiarrhythmic effect. Antiarrhythmic effect of κ-opioid receptor agonists depended on activation of κ-opioid receptors.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; Anti-Arrhythmia Agents; Cardiotonic Agents; Ischemic Preconditioning, Myocardial; Myocardial Ischemia; Myocardial Reperfusion Injury; Pyrrolidines; Rats; Rats, Wistar; Receptors, Opioid, kappa

To observe the effects of κ-opioid receptor agonist U50, 488H on myocardial ischemia and reperfusion injury and related mechanism.. Rats were randomly divided into sham operation, myocardial ischemia and reperfusion(I/R, 30 min ischemia followed by 120 min reperfusion), and MI/R+U50, 488H (1.5 mg/kg) and I/R+U50, 488H+ selective κ-opioid receptor antagonist Nor-BNI (2 mg/kg, n = 8 each). The infarction size and the incidence of ventricular arrhythmias were observed.Real-time PCR and DAB staining were used to define the myocardium Toll-like receptor 4(TLR4) expression. Myeloperoxidase level, TNF-α induction and the expression of NF-κB were also examined in rats.. After I/R, the expressions of myocardial TLR4 and NF-κB increased significantly both in ischemia area and area at risk. Compared with I/R, κ-opioid receptor stimulation with U50, 488H significantly attenuated the expressions of TLR4 and NF-κB and reduced myeloperoxidase (MPO) levels, myocardial TNF-α production, myocardial infarct sizes and the incidence of ventricular arrhythmias and arrhythmia score (2.9 ± 0.7 vs. 4.4 ± 0.9, P < 0.05) , above effects of U50, 488H were partly abolished by co-treatment with Nor-BNI.. These data provide evidence for the first time that κ-opioid receptor stimulation could attenuate myocardial I/R injury via downregulating TLR4/NF-κB signaling in rats.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; Arrhythmias, Cardiac; Brugada Syndrome; Cardiac Conduction System Disease; Coronary Artery Disease; Down-Regulation; Heart Conduction System; Myocardial Infarction; Myocardial Ischemia; Myocardium; Naltrexone; NF-kappa B; Rats; Rats, Sprague-Dawley; Receptors, Opioid, kappa; Reperfusion Injury; Signal Transduction; Toll-Like Receptor 4; Tumor Necrosis Factor-alpha

Evidence has indicated U50,488H, a selective κ-opioid receptor (κ-OR) agonist, administered before ischemia attenuates apoptosis and infarction during ischemia and reperfusion (I/R). However, it remains unclear whether U50,488H postconditioning reduces apoptosis during I/R. This study was designed, therefore, to test the hypothesis that U50,488H administered at the onset of reperfusion inhibits cardiomyocyte apoptosis and to investigate the underlying mechanisms.. Male Sprague-Dawley rats were subjected to myocardial ischemia and reperfusion(MI/R) and were randomized to receive either vehicle, U50,488H, U50,488H plus Nor-BNI, a selective κ-OR antagonist, U50,488H plus wortmannin, a specific inhibitor of phosphoinositide 3'-kinase (PI3K), or U50,488H plus L-NAME, a nitric oxide synthase inhibitor (NOS inhibitor), immediately prior to reperfusion. In vitro study was performed on cultured neonatal cardiomyocytes subjected to simulated ischemia/reperfusion.. Treatment with U50,488H resulted in increases in Akt and endothelial nitric oxide synthase (eNOS) phosphorylation with secondary NO production both in vivo and in vitro and these effect were completely blocked by wortmannin and specific Akt inhibitor(AI). L-NAME treatment had no effect on Akt and eNOS phosphorylation; but, significantly reduced NO production. Moreover, treatment with U50,488H markedly reduced myocardial apoptotic death. Treatment with wortmannin and specific Akt inhibitor abolished the anti-apoptotic effect of U50,488H. L-NAME also significantly attenuated the anti-apoptotic effect of U50,488H.. These results demonstrate that U50,488H administered immediately prior to reperfusion increases Akt phosphorylation through a PI3-kinase-dependent mechanism and reduces postischemic myocardial apoptosis. Phosphorylation of eNOS with secondary NO production contribute significantly to the anti-apoptotic effect of U50,488H postconditioning.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics, Non-Narcotic; Androstadienes; Animals; Apoptosis; Cardiotonic Agents; Male; Myocardial Ischemia; Myocardial Reperfusion Injury; Naltrexone; Narcotic Antagonists; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Phosphoinositide-3 Kinase Inhibitors; Rats; Rats, Sprague-Dawley; Receptors, Opioid, kappa; Wortmannin

Acute myocardial ischemia induces electrical and chemical uncoupling of gap junctions, which contributes to conduction abnormalities and re-entrant arrhythmias. We tested the hypothesis that structure and function of Connexin43 may vibrate during acute myocardial ischemia and reperfusion and κ-opioid receptor stimulation may stabilize the alteration of Connexin43.. An animal intervention study was conducted with comparison to a control group.. University preclinical research laboratory.. Age-, weight-, and sex-matched Sprague-Dawley rats.. Adult rat hearts were subjected to ischemia or ischemia/reperfusion, which was induced by temporary occlusion of the left main coronary artery. U50488H was given 10 mins before tissue specimens were taken or before ischemia (1.5 mg/kg, intravenous) and nor-BNI was given 15 mins before tissue specimens were taken or before ischemia (2 mg/kg, intravenous). Tissue samples came from left ventricular myocardium of the rat hearts.. Electrocardiogram, immunohistochemistry, immunoblotting, and reverse transcription-polymerase chain reaction were used to measure changes of arrhythmias, protein, and gene expression of Connexin43, respectively. κ-opioid receptor activation with U50 decreased arrhythmia in a model of myocardial ischemia and reperfusion. In normal hearts, immunohistochemical data showed reduced amount and lateralization of Connexin43 induced by κ-opioid receptor activation, whereas immunoblotting data demonstrated no significant changes between control and U50 group. During ischemia, however, Connexin43 protein underwent dephosphorylation and degradation, and Connexin43 mRNA was upregulated. These alterations were significantly attenuated on κ-opioid receptor stimulation. During ischemia and reperfusion, Connexin43 protein underwent dephosphorylation and degradation and recovered slowly during reperfusion. Activation of κ-opioid receptor accelerated recovery of phosphorylated and total Connexin43.. In normal rat hearts, Connexin43 translocates from intercellular junctions to intracellular locations on κ-opioid receptor activation. In rat hearts experiencing acute myocardial ischemia and reperfusion, protein and gene expression of Connexin43 undergo vibration. This phenomenon is stabilized when κ-opioid receptor is activated and by the fact that κ-opioid receptor produces antiarrhythmic effects.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; Arrhythmias, Cardiac; Blotting, Western; Connexin 43; Disease Models, Animal; Female; Gap Junctions; Immunohistochemistry; Male; Myocardial Ischemia; Myocardial Reperfusion Injury; Random Allocation; Rats; Rats, Sprague-Dawley; Receptors, Opioid, kappa; Reference Values; Reverse Transcriptase Polymerase Chain Reaction

To determine the effect of activation of lambda-opioid receptor with U50, 488H, a selective kappa-opioid receptor agonist, on the changes in electrical coupling during prolonged ischemia and to explore the possible mechanism.. The isolated rat heart was perfused in a Langendorff apparatus. The effect of U50, 488H on electrical coupling parameters including onset of uncoupling, plateau time, slope and fold increase in r(t) was observed in isolated perfused rat heart subjected to global no-flow ischemia. The effect of U50, 488H on connexin 43 (Cx43) expression of ventricular muscle during ischemia was determined by immunohistochemistry.. In the prolonged ischemia model, U50, 488H concentration dependently delayed the onset of uncoupling, increased time to plateau, and decreased the maximal rate of uncoupling during ischemia. The effect of U50, 488H on electrical uncoupling parameters during ischemia was abolished by a selective kappa-opioid receptor antagonist nor-BNI or a PKC inhibitor chelerythrine. The amount of Cx43 immunoreactive signal in ventricular muscle was greatly reduced after ischemia. U50, 488H markedly increased Cx43 expression during ischemia and its effect was also attenuated by nor-BNI or chelerythrine.. These results demonstrated that U50, 488H delayed the onset of uncoupling and plateau time, decreased the maximal rate of uncoupling and increased Cx43 expression of ventricular muscle during ischemia, and these effects of U50, 488H were mediated by kappa-opioid receptor, in which activation of PKC was involved. The effect of U50, 488H on electrical coupling during ischemia was probably correlated with preservation of Cx43 in cardiac muscle.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; Benzophenanthridines; Connexin 43; Female; Heart; In Vitro Techniques; Myocardial Ischemia; Myocardium; Naltrexone; Rats; Rats, Sprague-Dawley; Receptors, Opioid, kappa; Signal Transduction

To determine whether activation of kappa-opioid receptor with U50,488H, a selective kappa-opioid receptor agonist, produces any changes in electrical uncoupling during prolonged ischemia and whether these changes in electrical uncoupling is associated with the cardioprotection induced by kappa-opioid receptor activation, and to explore the possible mechanism.. (1) To observe the effect of U50,488H (10(-7), 10(-6), 3 x10(-6) and 10(-5) mol/L), a selective kappa-opioid receptor agonist, or with a selective kappa-opioid receptor antagonist nor-BNI (5 x 10(-6) mol/L), or with a mitochondrial K(ATP) channel inhibitor 5-HD on myocardium during ischemia/reperfusion in isolated perfused rat heart. Parameters of measurements include hemodynamic data, formazan content, heart rate, coronary flow, and lactate dehydrogenase (LDH). (2) To examine the effect of U50,488H of different concentration on electrical coupling parameters (including onset of uncoupling, plateau time, slope, and fold increase in r1) during 70 min myocardial ischemia in isolated perfused rat heart.. (1) Pretreatment with U50,488H concentration dependently increased formazan content and reduced LDH release induced by 30 min of ischemia and 120 min of reperfusion. (2) The onset of electrical uncoupling and plateau time during prolonged ischemia was delayed by kappa-opioid receptor activation with U50,488H. (3) Linear regression analysis shown that the increase in formazan content and decrease in LDH release produced by kappa-opioid receptor activation was associated with delayed electrical uncoupling during prolonged ischemia. (4) The effects of U50,488H on formazan content, LDH release and on electrical coupling were abolished by nor-BNI, or 5-HD.. This results demonstrate that the onset of electrical uncoupling during prolonged ischemia is delayed by kappa-opioid receptor activation with a selective kappa-opioid receptor agonist U50,488H, and that delayed electrical uncoupling is associated with the cardioprotection induced by kappa-opioid receptor activation with U50,488H. These effects of kappa-opioid receptor activation with U50,488H are mediated by mitochondrial K(ATP) channels.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; Antihypertensive Agents; Heart; In Vitro Techniques; Male; Myocardial Ischemia; Myocardium; Naltrexone; Potassium Channels; Rats; Rats, Sprague-Dawley; Receptors, Opioid, kappa

We first determined whether the cardioprotection resulting from kappa opioid receptor (kappa-OR) stimulation was blocked by the K(Ca) channel inhibitor, paxilline (Pax), administered before or during ischaemic insults in vitro. In isolated rat hearts, 30 min of ischaemia and 120 min of reperfusion induced infarction and increased lactate dehydrogenase (LDH) release. In isolated ventricular myocytes subjected to 5 min of metabolic inhibition and anoxia followed by 10 min of reperfusion, the percentage of live cells and the amplitude of the electrically induced intracellular Ca(2+) ([Ca(2+)](i)) transient decreased, while diastolic [Ca(2+)](i) increased. Pretreatment with 10 microM U50,488H, a kappa-OR agonist, attenuated the undesirable effects of ischaemic insults in both preparations. The beneficial effects of kappa-OR stimulation, that were abolished by 5 microM nor-BNI, a kappa-OR antagonist, were also abolished by 1 microM Pax administered before ischaemic insults or 20 microM atractyloside, an opener of the mitochondrial permeability transition pore. Activation of protein kinase C (PKC) with 0.1 microM phorbol 12-myristate 13-acetate decreased the infarct size and LDH release in isolated rat hearts subjected to ischaemia/reperfusion, and these effects were abolished by blockade of PKC with its inhibitors, 10 microM GF109203X or 5 microM chelerythrine, and more importantly by 1 microM Pax. On the other hand, the cardioprotective effects of opening the K(Ca) channel with 10 microM NS1619 were not altered by either PKC inhibitor. In conclusion, the high-conductance K(Ca) channel triggers cardioprotection induced by kappa-OR stimulation that involves inhibition of MPTP opening. The K(Ca) channel is located downstream of PKC.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; Calcium; Cardiotonic Agents; Cell Survival; Heart; In Vitro Techniques; Indoles; Ion Channels; L-Lactate Dehydrogenase; Male; Mitochondria, Heart; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Myocardial Ischemia; Myocardium; Myocytes, Cardiac; Potassium Channel Blockers; Potassium Channels, Calcium-Activated; Protein Kinase C; Rats; Rats, Sprague-Dawley; Receptors, Opioid, kappa

To determine whether the kappa-opioid receptor agonist U50,488H affects electrical uncoupling during prolonged ischemia and, if so, whether the changes are associated with its cardioprotective action.. The isolated rat heart was perfused in a Langendorff apparatus. Formazan content, lactate dehydrogenase (LDH) and hemodynamic parameters were measured to confirm the cardioprotective effect of U50,488H. The effects of U50,488H on electrical coupling during prolonged ischemia were also measured.. U50,488H concentration-dependently increased formazan content and reduced LDH release, and the ameliorating effect of 10(-5) mol/L U50,488H was abolished by 5 x 10(-6) mol/L nor-binaltorphimine (nor-BNI), a selective kappa-opioid receptor antagonist, or 10(-4) mol/L 5-hydroxydecanoate (5-HD), a selective mitochondrial ATP-sensitive K(+) (K(ATP)) channel blocker. The onset of electrical uncoupling during prolonged ischemia was delayed by U50,488H, and the delay was not only abolished, but also advanced by nor-BNI or 5-HD relative to the control group.. These results demonstrate that delayed uncoupling during prolonged ischemia is associated with the cardioprotection of U50,488H, and these effects of U50,488H are mediated by mitochondrial K(ATP) channels.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; Cardiotonic Agents; Cell Communication; Electrophysiologic Techniques, Cardiac; Formazans; Gap Junctions; Heart; HSC70 Heat-Shock Proteins; Male; Myocardial Ischemia; Potassium Channels; Rats; Rats, Sprague-Dawley; Receptors, Opioid, kappa; Time Factors

The aim of this study was to determine the effects of preconditioning on injury and expression of heat shock proteins 70 in diabetic rat hearts.. Diabetes was induced by an intraperitoneal injection of 65 mg kg(-1) streptozotocin. Daily subcutaneous injection of 4 IU insulin started 2 weeks after streptozotocin treatment for 4 weeks. Rats were preconditioned by intravenous injection of 10 mg kg(-1) U50,488H, a selective kappa-opioid receptor agonist (U50,488H preconditioning). The effects of U50,488H preconditioning had previously been shown to be blocked by a selective kappa-opioid receptor antagonist, nor-binaltorphimine. Twenty-four hours later, rats were subjected to 30 min of regional ischaemia by occlusion of the left coronary artery followed by 4 h of reperfusion. Infarct size was determined at the end of reperfusion. Stress-inducible and constitutive heat shock proteins 70 were analysed at the end of ischaemia and reperfusion by Western blotting.. Myocardial infarcts induced by ischaemia and reperfusion were greater in diabetic rats. U50,488H preconditioning significantly reduced the infarct size and increased the expression of stress-inducible heat-shock protein 70 in normal rats. The effects of U50,488H preconditioning were abolished in streptozotocin-induced diabetic rats, but restored by insulin replacement.. In addition to a greater susceptibility to ischaemic insults, the delayed cardioprotection of U50,488H preconditioning was lost, which could at least partly be due to impaired synthesis of stress-inducible heat-shock protein 70 in diabetic rats.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; Blood Glucose; Blotting, Western; Body Weight; Cardiotonic Agents; Diabetes Mellitus, Experimental; Gene Expression; Heart; HSC70 Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Insulin; Male; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardium; Organ Size; Rats; Rats, Sprague-Dawley; Receptors, Opioid, kappa

Opioid peptides, which can induce mammalian hibernation, may provide protection against subcellular and molecular changes during hypothermic myocardial ischemia. This study examined the differential effects of the three known myocyte opioid receptors, Mu (micro), Delta (delta), and Kappa (kappa), in augmenting myocardial ischemic tolerance.. Control hearts (CH) were compared to hearts pretreated with either the micro-agonist, fentanyl, the delta-agonist, DADLE, or delta-antagonist, NTB, or the kappa-agonist, U50488H (U50), or kappa-antagonist, nor-BNI. The percent return of isovolemic developed pressure (LVDP), myocardial oxygen consumption (MVO(2)), and coronary flow (CF) following 2 h of global hypothermic cardioplegic ischemia were recorded in isolated Langendorff perfused hearts.. At 45 min of reperfusion, hearts pretreated with either DADLE or U50488H demonstrated significantly improved functional recovery versus controls (P < 0.05) and significantly depressed recovery with NTB or nor-BNI pretreatment (P < 0.05). Pretreatment with fentanyl was not significantly different than controls. Furthermore, DADLE, U50488H, or fentanyl resulted in increased MVO(2) versus controls (P < 0.05). There was no difference in CF between all groups.. This study demonstrates that the micro-receptor does not appear to confer a beneficial effect. However, selective delta- and kappa-agonists provide significant myocardial protection. Moreover, hearts pretreated with an opioid antagonist showed a marked decrement in both functional and metabolic integrity. These results taken together would imply a positive and negative constitutive role of delta- and kappa-opioids in the regulation of myocardial ischemic tolerance. This utilization of opioid receptor stimulation may have profound clinical applications.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Adaptation, Physiological; Animals; Cardiotonic Agents; Enkephalin, Leucine-2-Alanine; Fentanyl; Heart; In Vitro Techniques; Microscopy, Electron; Myocardial Ischemia; Myocardial Reperfusion; Myocardium; Opioid Peptides; Oxygen Consumption; Rabbits; Recovery of Function

Activation of myocardial kappa-opioid receptor-protein kinase C (PKC) pathways may improve postischemic contractile function through a myofilament reduction in ATP utilization. To test this, we first examined the effects of PKC inhibitors on kappa-opioid receptor-dependent cardioprotection. The kappa-opioid receptor agonist U50,488H (U50) increased postischemic left ventricular developed pressure and reduced postischemic end-diastolic pressure compared with controls. PKC inhibitors abolished the cardioprotective effects of U50. To determine whether kappa-opioid-PKC-dependent decreases in Ca2+-dependent actomyosin Mg2+-ATPase could account for cardioprotection, we subjected hearts to three separate actomyosin ATPase-lowering protocols. We observed that moderate decreases in myofibrillar ATPase were equally cardioprotective as kappa-opioid receptor stimulation. Immunoblot analysis and confocal microscopy revealed a kappa-opioid-induced increase in myofilament-associated PKC-epsilon, and myofibrillar Ca2+-independent PKC activity was increased after kappa-opioid stimulation. This PKC-myofilament association led to an increase in troponin I and C-protein phosphorylation. Thus we propose PKC-epsilon activation and translocation to the myofilaments causes a decrease in actomyosin ATPase, which contributes to the kappa-opioid receptor-dependent cardioprotective mechanism.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Actin Cytoskeleton; Adenosine Triphosphatases; Adenosine Triphosphate; Analgesics, Non-Narcotic; Animals; Cardiotonic Agents; Female; Ischemic Preconditioning, Myocardial; Isoenzymes; Myocardial Ischemia; Phosphorylation; Protein Kinase C; Protein Kinase C-epsilon; Rats; Rats, Wistar; Receptors, Opioid, kappa; Ventricular Pressure

To test the hypothesis that heat-shock proteins (HSPs) mediate delayed cardioprotection of prior kappa-opioid receptor (kappa-OR) stimulation, we first correlated cellular injury and viability with the expression of HSP70s in isolated rat ventricular myocytes subjected to prior kappa-OR stimulation with the selective agonist trans-(+/-)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide (U-50488H) and delayed lethal simulated ischemia (LSI). Cell injury and viability were indicated by lactate dehydrogenase release and trypan blue exclusion, respectively. The reduced injury and increased viability after pretreatment with U-50488H were concentration dependent and correlated directly with the expression of both stress-inducible (HSP70) and constitutive (HSC70) proteins. The effects mimic those with metabolic inhibition preconditioning (MIP). The cardioprotection against LSI by pretreatment with U-50488H and MIP was abolished and antagonized, respectively, via blockade of the kappa-OR by its selective antagonist, nor-binaltorphimine. We also found that blockade of the production of HSP70 but not HSC70 blocked the inhibitory effect of pretreatment with U-50488H on injury and viability. These observations provide evidence that stress-inducible HSP70 mediates delayed cardioprotection of prior kappa-OR stimulation.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; Heart; Heart Ventricles; HSP70 Heat-Shock Proteins; Ischemic Preconditioning, Myocardial; L-Lactate Dehydrogenase; Male; Myocardial Ischemia; Myocardium; Naltrexone; Narcotic Antagonists; Oligonucleotides, Antisense; Protective Agents; Rats; Rats, Sprague-Dawley; Receptors, Opioid, kappa

To determine whether opioid receptors (ORs) are involved in the delayed cardioprotection of ischemic preconditioning (IP), the effect of severe metabolic inhibition (MI) with a glucose-free buffer that contained sodium cyanide and 2-deoxy-D-glucose on the viability of isolated rat ventricular myocytes was first determined 20 hours after preconditioning with a sublethal metabolic inhibition (MIP) with a glucose-free buffer that contained 2-deoxy-D-glucose and lactate for 30 minutes in the presence of OR antagonists. With the use of trypan blue exclusion as an index of cell viability, severe MI killed >60% of the cells and the value increased significantly after MIP. In the presence of 5x10(-6) mol/L nor-binaltorphimine (nor-BNI), a selective kappa-OR antagonist, but not 5x10(-6) mol/L CTOP, a selective mu-OR antagonist, or 5x10(-6) mol/L naltrindole, a selective delta-OR antagonist, the cardioprotection of MIP was significantly attenuated. To verify the role of kappa-OR, we studied the effects of severe MI after pretreatment with the kappa-OR agonist U50,488H (UP) for 30 minutes. U50,488H at 3x10(-6) to 1x10(-4) mol/L increased cell viability concentration-dependently with an EC50 of 3.311x10(-6) mol/L. In the presence of 5x10(-6) nor-BNI, the cardioprotection of UP (3x10(-5) mol/L) was blocked. A time course study showed that UP-induced cardioprotection occurred in 2 windows: the first occurred approximately 1 hour later and the other occurred 16 to 20 hours later. Additional studies on cell contraction and intracellular Ca2+ ([Ca2+]i) revealed that both UP and MIP attenuated the inhibitory effects of severe MI on contractility and electrically induced [Ca2+]i transient in single ventricular myocytes. On blockade of protein kinase C, the delayed cardioprotections of UP and MIP were significantly attenuated. In conclusion, the results of the present study have provided evidence that kappa-OR mediates the cardioprotection of MIP, which may involve protein kinase C and [Ca2+]i.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics, Non-Narcotic; Animals; Calcium; Cell Survival; Electrophysiology; Energy Metabolism; Heart Ventricles; In Vitro Techniques; Ischemic Preconditioning, Myocardial; Membrane Potentials; Muscle Fibers, Skeletal; Myocardial Contraction; Myocardial Ischemia; Myocardium; Naltrexone; Narcotic Antagonists; Protein Kinase C; Rats; Rats, Sprague-Dawley; Receptors, Opioid, kappa; Trypan Blue