The Role of δ2-Opioid Receptors in the Regulation of Tolerance of Isolated Cardiomyocytes to Hypoxia and Reoxygenation.

Hypoxia (20 min) and reoxygenation (30 min) were simulated on isolated rat cardiomyocytes to evaluate the cytoprotective effect of selective δ2-opioid receptor agonist deltorphin II, opioid receptor antagonist naloxone methiodide, µ-opioid receptor antagonist CTAP, κ-opioid receptor antagonist nor-binaltorphimine, ε1-opioid receptor antagonist BNTX, and δ2-opioid receptors naltriben. Deltorphin II was administered 5 min before reoxygenation, antagonists were administered 10 min before reoxygenation. The cytoprotective effect of deltorphin II was assessed by the number of cardiomyocytes survived after hypoxia/reoxygenation, as well as by the lactate dehydrogenase content in the incubation medium. It has been established that the cytoprotective effect of deltorphin II occurs at a concentration of 64 nmol/liter and is associated with activation of δ2-opioid receptors.

Comparative Analysis of Infarct-Limiting Activity of Peptide and Non-Peptide δ- and κ-Opioid Receptor Agonists during Heart Reperfusion In Vivo.

A comparative analysis of the infarct-limiting activity of δ- and κ-opioid receptors (OR) agonists was carried out on a model of coronary occlusion (45 min) and reperfusion (120 min) in male Wistar rats. We used selective δ2-OR agonist deltorphin II (0.12 mg/kg), δ-OR agonists BW373U86 and p-Cl-Phe DPDPE (0.1 and 1 mg/kg), selective agonists of δ1-OR DPDPE (0.1 and 0.969 mg/kg), κ1-OR U-50,488 (0.1 and 1 mg/kg), κ2-OR GR-89696 (0.1 mg/kg), and κ-OR ICI-199,441 (0.1 mg/kg). All drugs were administered intravenously 5 min before reperfusion. Deltorphin II, BW373U86 (1 mg/kg), p-Cl-Phe DPDPE (1 mg/kg), U-50,488 (1 mg/kg), and ICI-199,441 had a cardioprotective effect. The most promising compounds for drug development are ICI-199,441 and deltorphin II.

Decrease in Infarct-Limiting Effect of Chronic Normobaric Hypoxia in Rats with Induced Metabolic Syndrome Is Associated with Disturbances of Carbohydrate and Lipid Metabolism.

We studied the infarct-limiting effect of adaptation to chronic normobaric hypoxia in rats with induced metabolic syndrome and the relationship between disturbances of adaptive cardioprotection and disorders of carbohydrate and lipid metabolism. Adaptation to chronic normobaric hypoxia was carried out for 21 days at 12% O2 and 0.3% CO2. The metabolic syndrome was modeled with a high-carbohydrate high-fat diet for 84 days with replacement of drinking water with a 20% fructose solution. The infarct size in rats exposed to chronic normobaric hypoxia was 38% smaller than in control animals. In rats with induced metabolic syndrome, hypertension, obesity, decreased glucose tolerance, increased serum triglyceride, and no infarction-limiting effect of chronic normobaric hypoxia were observed. Infarct size showed a direct correlation with impaired glucose tolerance and serum triglyceride levels. The study allows us to conclude that the lack of cardioprotection in chronic normobaric hypoxia in rats with induced metabolic syndrome is associated with impaired carbohydrate and lipid metabolism.

The Role of NO Synthase in the Infarct-Limiting Effect of Urgent and Chronic Adaptation to Normobaric Hypoxia.

We studied the role of NO synthase in the infarct-limiting effect of short-term (SNH) and chronic continuous normobaric hypoxia (CNH). In male Wistar rats, SNH (6 sessions of 10-min hypoxia 8% O2 and 10-min reoxygenation) or CNH (12% O2 for 21 days) was modeled. In 30 min after SNH or 24 h after CNH, the rats were subjected to coronary artery occlusion (45 min) and reperfusion (2 h). The following drugs were administered to rats: non-selective NO synthase inhibitor L-NAME (10 mg/kg), inhibitor of inducible NO synthase S-methylthiourea (3 mg/kg), and inhibitor of neuronal NO-synthase 7-nitroindazole (50 mg/kg). NO donor diethylenetriamine was administered intravenously in a dose 2 mg/kg. It was found that L-NAME and S-methylthiourea abolished the infarct-limiting effect of SNH and CNH. Diethylenetriamine increased cardiac tolerance to ischemia/reperfusion. It is believed that inducible NO synthase plays an important role in the cardioprotective effect of normobaric hypoxia.

Participation of Leptin and Corticosterone in the Decrease in Infarct-Limiting Efficiency of Remote Postconditioning and in the Development of Arterial Hypertension in Metabolic Syndrome in Rats.

We studied the effect of induced metabolic syndrome (MetS) on the effectiveness of the infarct-limiting effect of remote ischemic postconditioning (RP) in Wistar rats. The involvement of leptin and corticosterone in the formation of arterial hypertension (AH) and in reduction of the effectiveness of RP in MetS was also studied. MetS was induced by high-carbohydrate high-fat diet with replacement of drinking water with 20% fructose solution for 90 days. MetS simulation led to obesity, AH, impaired lipid and carbohydrate metabolism, hyperleptinemia, and moderate stress. All animals were subjected to 45-min coronary occlusion and 120-min reperfusion. In the RP groups, tourniquets were applied on the hind limbs in the area of the hip joint immediately after the end of ischemia (3 cycles consisting of 5-min ischemia and 5-min reperfusion). A direct correlation was found between the severity of AH in rats with MetS and the levels of corticosterone and leptin. In rats with MetS, the effectiveness of RP decreased: a direct correlation between the infarct size and serum content of leptin was revealed in rats with MetS+RP. Corticosterone seems to be one of the factors of AH development in rats with MetS.

The Role of Mitochondrial KATP Channels in the Infarct-Reducing Effect of Normobaric Hypoxia.

We studied the role of KATP channels in the infarct-limiting effect of short-term normobaric hypoxia. Male Wistar rats were subjected to a 45-min coronary artery occlusion followed by a 120-min reperfusion. Normobaric hypoxia was simulated 30 min before coronary artery occlusion: 6 sessions of hypoxia (8% O2, 10 min) and reoxygenation (21% O2, 10 min). The following drugs were administered to rats: glibenclamide, 5-hydroxydecanoate, and HMR1098. It was found that normobaric hypoxia contributes to a decrease in myocardial infarct size by 36%. Preliminary administration of glibenclamide or 5-hydroxydecanoate eliminated the infarct-reducing effect of normobaric hypoxia. Activator of mitochondrial KATP channel diazoxide limited the infarct size. These findings suggest that mitochondrial KATP channels are involved into the cardioprotective effect of normobaric hypoxia.

[An effect of mexidol on the level of corsticosteron in the blood serum and beam zone cells in rats with light desynhronizes and physical loads]. / Vliyanie meksidola na uroven' kortikosterona v syvorotke krovi i kletki puchkovoi zony kory nadpochechnikov u krys pri svetovykh desinkhronozakh i fizicheskikh nagruzkakh.

OBJECTIVE: In order to develop methods and means of maintaining normal human functioning under conditions of desynchronizes, the effect of mexidol on the level of corticosterone in the blood serum and the state of the cells of the beam zone of the adrenal cortex of rats after light or dark deprivation and physical activity to a state of fatigue. MATERIALS AND METHODS: The experiments were performed on 7 groups of rats (10 animals each) in the spring. The model of physical activity is the method of forced swimming of rats until exhaustion in its own modification. For the induction of experimental desynchronizes, the animals of the experimental groups were kept around the clock for 10 days with artificial bright light of 150 LX or a complete darkening of 2-3 LX. Mexidol was administered intramuscularly at a dose of 10 mg/kg animal 30 minutes before the swimming test. Control animals under similar conditions were administered 0.9% sodium chloride solution. The level of corticosterone in the serum was determined using enzyme immunoassay. The measurements were carried out on a programmable photometer for STAR FAX 303 PLUS microsamples (USA). Histological analysis of the adrenal glands was carried out according to standard methods. Microscopy of preparations, imaging and measurements were carried out using an Axio Lab A1 microscope, an AxioCamERc 5s camera and ZEN 2012 software («Carl Zeiss Microscopy¼, Germany). Statistical processing of the results was carried out using standard parametric and non-parametric methods, depending on the nature of the distributions. RESULTS AND CONCLUSION: Mexidol under combined successive stress loads - desynchronizes and physical activity - demonstrated anti-stress properties not only at the level of corticosterone in the blood serum of rats, but also at the level of adrenal glands. The severity of this effect of the drug depended on the number of stress loads (isolated physical or shown in conditions of desynchronizes), the phase of stress and the nature of deprivation. Under natural lighting conditions and after light deprivation, when animals were in the anxiety phase of varying severity, the drug worked more reliably than after dark deprivation in conditions of developing depletion. Nevertheless, even in the latter case, he showed himself as a means of preventing exhaustion in animals and the breakdown of adaptation.

The involvement of protein kinases in the cardioprotective effect of chronic hypoxia.

The purpose of this review is to analyze the involvement of protein kinases in the cardioprotective mechanism induced by chronic hypoxia. It has been reported that chronic intermittent hypoxia contributes to increased expression of the following kinases in the myocardium: PKCdelta, PKCalpha, p-PKCepsilon, p-PKCalpha, AMPK, p-AMPK, CaMKII, p-ERK1/2, p-Akt, PI3-kinase, p-p38, HK-1, and HK-2; whereas, chronic normobaric hypoxia promotes increased expression of the following kinases in the myocardium: PKCepsilon, PKCbetaII, PKCeta, CaMKII, p-ERK1/2, p-Akt, p-p38, HK-1, and HK-2. However, CNH does not promote enhanced expression of the AMPK and JNK kinases. Adaptation to hypoxia enhances HK-2 association with mitochondria and causes translocation of PKCdelta, PKCbetaII, and PKCeta to the mitochondria. It has been shown that PKCdelta, PKCepsilon, ERK1/2, and MEK1/2 are involved in the cardioprotective effect of chronic hypoxia. The role of other kinases in the cardioprotective effect of adaptation to hypoxia requires further research.

Intracellular Kinase Mechanism of the Cytoprotective Action of Adaptation to Chronic Hypoxia in Anoxia/Reoxygenation of Cardiomyocytes.

On the model of anoxia/reoxygenation of isolated cardiomyocytes, we studied the role of kinases in the implementation of the cytoprotective effect of chronic continuous normobaric hypoxia (21 days on continuous exposure of rats at 12% O2). Anoxia/reoxygenation of cardiomyocytes from intact rats caused death of 16.5% cells, which was accompanied by the release of lactate dehydrogenase; in suspension of cardiomyocytes from adapted rats, only 6.8% cells died and the release of lactate dehydrogenase was lower by 60%. Incubation of cells with inhibitors of protein kinase C (chelerythrin, 10 mM), protein kinase Cδ (rottlerin, 1 µM), tyrosine kinases (genistein, 50 µM), but not with PI3K inhibitor (wortmannin, 100 nM) eliminated the cytoprotective effect of chronic continuous normobaric hypoxia. Thus, the cytoprotective effect of chronic normobaric hypoxia is realized through activation of protein kinase Cδ and tyrosine kinases, but not through PI3K.

Continuous Normobaric Hypoxia Improved Cardiac Bioenergetics after Ischemia/Reperfusion: Role of Opioid Receptors.

We analyzed the role of opioid receptors in the conditioning effect of continuous normobaric hypoxia on bioenergetics of the heart subjected to ischemia/reperfusion injury. Male Wistar rats were adapted to a 21-day continuous normobaric hypoxia (12% pO2). Then, the hearts were isolated and subjected to 45-min total ischemia followed by 30-min reperfusion. Damage to the myocardium was assessed by activity of creatine phosphokinase in the perfusate. Experiments on isolated mitochondria showed that ischemia/reperfusion injury decreased the respiration rate in state 3 (V3), the ratio of added ADP and oxygen consumption in respiration state 3 (ADP/O ratio), the mitochondrial potential across the inner membrane (Δψ), and Ca2+ binding capacity of mitochondria. In addition, ischemia/reperfusion injury decreased myocardial ATP. Preventive continuous normobaric hypoxia pronouncedly moderated these adverse effects of reperfusion. It was found that its protective effects were related to activation of cardiac µ- and δ2-opioid receptors.

The Role of Cardiac Opioid Receptors in the Cardioprotective Effect of Continuous Normobaric Hypoxia.

We studied the role of opioid receptor subtypes in improvement of the functional state of the heart during reperfusion after adaptation to continuous normobaric hypoxia. To this end, male Wistar rats were subjected to continuous normobaric hypoxia (12% O2). Then, the hearts were isolated and exposed to total 45-min ischemia followed by 30-min reperfusion. Opioid receptor antagonists were added to the perfusion solution prior to ischemia. It was found that continuous normobaric hypoxia reduced the release of creatine phosphokinase into the effluent, increased myocardial contractile force, and decreased the end-diastolic pressure during reperfusion; these positive effects were related to activation of cardiac δ2- and µ-opioid receptors.

Effect of Chronic Continuous Normobaric Hypoxia on Functional State of Cardiac Mitochondria and Tolerance of Isolated Rat Heart to Ischemia and Reperfusion: Role of µ and delta2 Opioid Receptors.

Chronic continuous normobaric hypoxia (CNH) increases cardiac tolerance to ischemia/reperfusion injury in vivo and this effect is mediated via µ and delta2 opioid receptors (ORs) activation. CNH has also been shown to be cardioprotective in isolated rat heart. In this study, we hypothesize that this cardioprotective effect of CNH is mediated by activation of µ and delta2 ORs and preservation of mitochondrial function. Hearts from rats adapted to CNH (12 % oxygen) for 3 weeks were extracted, perfused in the Langendorff mode and subjected to 45 min of global ischemia and 30 min of reperfusion. Intervention groups were pretreated for 10 min with antagonists for different OR types: naloxone (300 nmol/l), the selective delta OR antagonist TIPP(psi) (30 nmol/l), the selective delta1 OR antagonist BNTX (1 nmol/l), the selective delta2 OR antagonist naltriben (1 nmol/l), the selective peptide µ OR antagonist CTAP (100 nmol/l) and the selective delta OR antagonist nor-binaltorphimine (3 nmol/l). Creatine kinase activity in coronary effluent and cardiac contractile function were monitored to assess cardiac injury and functional impairment. Additionally, cardiac tissue was collected to measure ATP and to isolate mitochondria to measure respiration rate and calcium retention capacity. Adaptation to CNH decreased myocardial creatine kinase release during reperfusion and improved the postischemic recovery of contractile function. Additionally, CNH improved mitochondrial state 3 and uncoupled respiration rates, ADP/O, mitochondrial transmembrane potential and calcium retention capacity and myocardial ATP level during reperfusion compared to the normoxic group. These protective effects were completely abolished by naloxone, TIPP(psi), naltriben, CTAP but not BNTX or nor-binaltorphimine. These results suggest that cardioprotection associated with adaptation to CNH is mediated by µ and delta2 opioid receptors activation and preservation of mitochondrial function.

Cardioprotective properties of opioid receptor agonists in rats with stress-induced cardiac injury.

The objectives of this study were to investigate the role of endogenous opioids in the mediation of stress-induced cardiomyopathy (SIC), and to evaluate which opioid receptors regulate heart resistance to immobilization stress. Wistar rats were subjected to 24 h immobilization stress. Stress-induced heart injury was assessed by 99mTc-pyrophosphate accumulation in the heart. The opioid receptor (OR) antagonists (naltrexone, NxMB - naltrexone methyl bromide, MR 2266, ICI 174.864) and agonists (DALDA, DAMGO, DSLET, U-50,488) were administered intraperitoneally prior to immobilization and 12 h after the start of stress. In addition, the selective micro OR agonists PL017 and DAMGO were administered intracerebroventricularly prior to stress. Finally pretreatment with guanethidine was used. Naltrexone did not alter the cardiac 99mTc-PP accumulation in stressed rats. NxMB aggravated stress-induced cardiomyopathy (P=0.005) (SIC). The selective micro OR agonist DALDA, which does not cross the blood-brain barrier, completely prevented (P=0.006) SIC. The micro OR agonist DAMGO exhibited weaker effect than DALDA. The selective delta ligand (DSLET) and kappa OR ligand (U-50,488) did not alter stress-induced 99mTc-pyrophosphate accumulation in the heart. Intracerebroventricular administration of the micro OR agonists aggravated SIC. Pretreatment with guanethidine abolished this effect (P=0.01). Guanethidine alone exhibited cardioprotective properties. A stimulation of central micro OR promotes an appearance of SIC. In contrast, stimulation of peripheral micro OR contributes to an increase in cardiac tolerance to stress.

Participation of opioid receptors in the cytoprotective effect of chronic normobaric hypoxia.

We studied the role of the delta, micro, and kappa opioid receptor (OR) subtypes in the cardioprotective effect of chronic continuous normobaric hypoxia (CNH) in the model of acute anoxia-reoxygenation of isolated cardiomyocytes. Adaptation of rats to CNH was performed by their exposure to atmosphere containing 12 % of O(2) for 21 days. Anoxia-reoxygenation of cardiomyocytes isolated from normoxic control rats caused the death of 51 % of cells and lactate dehydrogenase (LDH) release. Adaptation of rats to CNH resulted in the anoxia/reoxygenation-induced cardiomyocyte death of only 38 %, and reduced the LDH release by 25 %. Pre-incubation of the cells with either the non-selective OR (opioid receptor) blocker naloxone (300 nM/l), the delta OR antagonist TIPP(psi) (30 nM/l), the selective delta(2) OR antagonist naltriben (1 nM/l) or the micro OR antagonist CTAP (100 nM/l) for 25 minutes before anoxia abolished the reduction of cell death and LDH release afforded by CNH. The antagonist of delta(1) OR BNTX (1 nM/l) or the kappa OR antagonist nor-binaltorphimine (3 nM/l) did not influence the cytoprotective effects of CNH. Taken together, the cytoprotective effect of CNH is associated with the activation of the delta(2) and micro OR localized on cardiomyocytes.

[Prospects of Application of Remote Preconditioning at Heart Revascularization].

Remote ischemic preconditioning of the heart exerts anti-necrotic, antiarrhythmic, inotropic effects that have been demonstrated in clinical trials in cardiac surgery both in adults and children. However, so far there is no consensus between cardiologists regarding the impact of remote preconditioning on the incidence of intraoperative myocardial infarctions and mortality in the postoperative period. Until now there is no unanimity concerning choice of remote preconditioning protocol and timing of its application before cardiac surgery.

Involvement of Autonomic Nervous System in Antiarrhythmic Effect of Intermittent Hypobaric Hypoxia.

We studied the involvement of the autonomic nervous system in the antiarrhythmic effect of intermittent hypobaric hypoxia modeled by daily placing the rats into an altitude chamber at 405 mm Hg (5000 m above sea level). The antiarrhythmic effect of hypoxia was observed on the model of acute coronary occlusion/reperfusion in vivo, but not during simulation of total ischemia/reperfusion of the isolated myocardium. Intravenous injection of ganglionic blocker hexamethonium (30 mg/kg) 15 min prior to in vivo coronary occlusion modeling abolished the antiarrhythmic effect of intermittent hypobaric hypoxia, which suggests that this effect is mediated via activation of the autonomic nervous system.

The Role of Endogenous Opioid System in the Regulation of Heart Tolerance to Stress-Induced Damage.

In Wistar rats, stress was modeled by 24-h immobilization in a supine posture and stress-induced damage to the heart was assessed by accumulation of 99mTc-pyrophosphate in the myocardium. The intensity of stress reaction was measured by serum levels of cortisol and insulin. Both stressinduced damage to the heart and intensity of stress reaction were examined under control conditions and in rats treated with opioid receptor antagonists naltrexone, methylnaltrexone bromide, MR2266, and ICI174.864. Activation of central µ-opioid receptors with endogenous opioids aggravated stress-induced cardiomyopathy, while stimulation of peripheral µ-opioid receptors produced a cardioprotective effect. The stress-induced damage to the heart was not directly related to up-regulation of cortisol secretion in response to 24-h immobilization. Blockade of the central opioid receptors promoted a decrease in cortisol level in stressed rats.

Role of ATP-Sensitive K+ Channels in Myocardial Infarct Size-Limiting Effect of Chronic Continuous Normobaric Hypoxia.

The role of KATP channels in myocardial infarct size-limiting effect of chronic continuous normobaric hypoxia was examined in a rat model based on a 20-min coronary occlusion and subsequent 3-h reperfusion. Rats were adapted to normobaric hypoxia (12% O2) for 21 days. This hypoxia produced a pronounced infarct size-limiting effect, which had been prevented by 0.3 mg/kg glibenclamide, a non-selective inhibitor of entire pool of KATP channels, or 5 mg/kg 5-hydroxydecanoate, an inhibitor of mitochondrial KATP channels. The study highlighted the important role of mitochondrial KATP channels in myocardial infarct size-limiting effect of chronic normobaric hypoxia.

Specific features of adaptation of rats to chronic cold treatment.

Permanent exposure of rats to four-week cold treatment at +4ºC for 24 h/day resulted in increased weights of the brown adipose tissue, adrenals, and spleen and had no effect on the levels of cortisol and corticosterone in the blood serum. Similar data were observed after exposure of rats to intermittent four-week cold treatment at +4ºC for 8 h/day. After short-term exposure of rats to intermittent cold treatment at +4ºC for 1.5 h/day, all indices studied were similar to those observed in intact animals.

[SIGNIFICANCE OF OPIOID RECEPTORS IN THE CYTOPROTECTIVE ACTION OF CHRONIC HYPOXIA DURING ANOXIA-REOXYGENATION OF CARDIOMYOCYTES].

It was investigated the role of δ-, µ- и κ-opioid receptors (ORs) in the development of cytoprotective effect of chronic normobaric hypoxia (CNH) using anoxia/reoxygenation of isolated cardiomyocytes. Adaptation to CNH was achieved by the maintenance of rats for 21 days at atmosphere containing 12% O2. Anoxia/reoxygenation of isolated cardiomyocytes of intact rats evoked a death of 23% cells and enhancement of lactate dehydrogenase (LDH) release from cells. Anoxia/reoxygenation of isolated cardiomyocytes of adapting rats induced a death of only 2.5% cells and LDH release decreased by 25%. Preliminary incubation of cells with the OR blocker naloxone (300 nM) or the δ-OR antagonist TIPP(ψ) (30 nM) or the selective δ2-OR antagonist naltriben (1 nM) or the µ-OR antagonist CTAP (100 nM) 25 min prior to anoxia abolished adaptive enhancement of cell survival and a decrease in LDH release. The blocking of δ1-OR by BNTX (1 nM) or κ-OR by nor-binaltorphimine (3 nM) not affected on the cytoprotection at CNH. Consequently, cardiac cell δ2- and µ-opioid receptors are involved in the cytoprotective effect of chronic normobaric hypoxia.