Lakomkin Vl

Regulation of energy flux through the creatine kinase reaction in vitro and in perfused rat heart. 31P-NMR studies.

Fluxes catalyzed by soluble creatine kinase (MM) in equilibrium in vitro and by the creatine kinase system in perfused rat hearts were studied by 31P-NMR saturation transfer method. It was found that in vitro both forward and reverse fluxes through creatine kinase at equilibrium were almost equal and very stable to changes in phosphocreatine/creatine ratio (from 0.2 to 3.0) as well as to changes in pH (from 7.4 to 6.5 or 8.1), free Mg2+ concentration and 2-fold decrease of total adenine nucleotides and creatine pools (from 8.0 to 4.0 mM and from 30 to 14 mM, respectively). In the rat hearts perfused by the Langendorff method the creatine kinase-catalyzed flux from phosphocreatine to ATP was increased by 50% when oxygen consumption grew from 8 to 55 mumol/min per g of dry wt. due to transition from rest to high workload. These changes could not be exclusively explained on the basis of the equilibrium model by activation of heart creatine kinase due to some decrease in [phosphocreatine]/[creatine] ratio (from 1.8 to 0.8) observed during transition from rest to high workload. Analysis of our data showed that an increase in the flux via creatine kinase is correlated with an increase in the rate of ATP synthesis with a linearity coefficient higher than 1.0. These data are more consistent with the concept of energy channeling by phosphocreatine shuttle than with that of the creatine kinase equilibrium in the heart.

Quantitative evaluation of relationship between cardiac energy metabolism and post-ischemic recovery of contractile function

Quantitative Evaluation of Relationship between Cardiac Energy Metabolism and Post-ischemic Recovery of Contractile Function. Mechanisms of ischemic damage were studied by defining the relationships between post-ischemic work recovery and tissue ATP levels in isolated rat hearts as well as mitochondrial respiration rates in skinned myofibrils. Pre-ischemic levels of ATP were reduced by 2-deoxyglucose treatment and assessed using 31P-NMR. A 70% fall of ATP was not associated with decreased functional recovery. Mitochondrial respiration was assessed without mitochondrial isolation in skinned cardiac fibers in physiological salt solution using a novel method developed by Veksler et al. Maximal rates of mitochondrial respiration were not changed after 35 min of normothermic ischemia using St. Thomass Hospital cardioplegic solution followed by 30 min of aerobic reperfusion. Only a reversible increase in the rate of basal respiration and a decrease in creatine-stimulated oxygen uptake were observed. Thus, mitochondrial oxidative phosphorylation, as assessed in skinned myofibrils, was tolerant to an ischemic period which induced permanent depression of contractile function along with alterations in metabolite distribution. As a result, tissue level of ATP and rates of mitochondrial respiration provided an estimate of ischemic damage only in cases where damage reached a very severe extent.

Hypotensive effect of Oxacom® containing a dinitrosyl iron complex with glutathione: animal studies and clinical trials on healthy volunteers.

A comparative study of hypotensive effects of binuclear forms of dinitrosyl iron complexes (DNICs) with glutathione, S-nitrosoglutathione (GS-NO) and sodium nitrite (NaNO(2)) on rats has been carried out. The latter appeared to be the least efficient, viz., mean arterial pressure (MAP) decreased by 10 and 30 mmHg at 25 and 100 μmoles/kg of NaNO(2). In contrast, DNIC and GS-NO produced an appreciable hypotensive effect when used at much lower concentrations. GS-NO reduced MAP to the same extent, viz., to 90 mmHg, on a hundredfold dose scale (from 0.4 up to 50 μmoles/kg) with subsequent restoration of MAP within the next 6-15 min. A similar effect was observed for DNIC except that the amplitude of the MAP drop was lower and the duration of hypotension was essentially greater. DNIC with glutathione were selected as a basic material for pilot-scale production of a hypotensive drug (commercial name Oxacom®). Preliminary pharmacological testing of Oxacom did not establish any adverse or deleterious side effects. Clinical trials of Oxacom® were performed on 14 healthy male volunteers in whom single intravenous infusion of the drug (5mg/kg or 0.2 μmoles/kg of DNIC, respectively) evoked a characteristic response manifested as a 3-4 min drop by 24-27 mmHg of both diastolic and systolic AP with its subsequent slow restoration within the next 8-10h. The heart rate was quickly normalized after an initial increase. Cardiac output was unchanged despite reduced cardiac filling. A comprehensive analysis of clinical and biochemical data failed to establish any significant pathological changes in these parameters. The data obtained suggest that Oxacom® can be recommended for the second phase of clinical trials.

Relationships between pre-ischemic ATP and glycogen content and post-ischemic recovery of rat heart

The effect of depletion of energy stores of rat hearts on their resistance to a total of 25 min ischemia was investigated by using a 31P-NMR method. Three experimental groups were compared: (1) pyruvate-perfused hearts depleted of adenine nucleotides (35% of normal) by 2-deoxyglucose (DG) treatment and containing deoxyglucose-6-phosphate (c. 40 mumol/g dry wt); (2) hearts partially depleted of glycogen stores (40 to 50% of initial) by long-term (2h) perfusion with pyruvate; (3) glucose perfused (11 nM) hearts with normal ATP and glycogen contents. By the end of ischemia the intracellular pH was decreased by 0.33, 0.90 and 1.40 units, respectively. Time to peak of ischemic contracture increased in this series from 3 to 18 and 24 min, respectively. At the peak of ischemic contracture ATP content was c. 30 to 40% (6 to 8 mumol/g dry wt) of normal value in all three groups. Reperfusion of hearts resulted in development of significant reperfusion contracture in glucose-perfused hearts and minor contracture in other series. Recovery of high energy phosphates and cardiac work index in DG-treated, glycogen-depleted and glucose-perfused hearts were: for phosphocreatine (PCr), 72, 102 and 83%; for ATP, 29, 47 and 56% and for cardiac work, 66, 78 and 24%, respectively. Recovery of cardiac work did not correlate linearly with tissue ATP. These data demonstrate that post-ischemic recovery of the contractile function of isovolumic heart may be dissociated from pre-ischemic myocardial ATP and glycogen contents. This dissociation can be explained by the two major factors: (1) the contribution of ischemic acidosis and catabolites accumulation to the cell damage and (2) by ATP compartmentation.

Dissociation of adenosine triphosphate levels and contractilefunction in isovolumic hearts perfused with 2-deoxyglucose

Addition of 2-deoxyglucose (DG, 8 to 13 mM) to a perfusate containing 5 mM pyruvate as oxidizable substrate caused gradual decline of contractile function of Langendorff-perfused isovolumic rat heart. Simultaneously 31P-NMR spectra showed accumulation of 2-deoxyglucose-6-phosphate (DG-6P) and decrease in phosphocreatine (PCr) and ATP contents; inosine appeared in high concentration in perfusate leaving heart. Subsequent reperfusion of the heart with DG-free solution resulted in the recovery of contractile function and PCr (to 75%), as well as in slow decay of DG-6P at unchanged low level of ATP (35%). No correlation was found between tissue ATP content and contractile function, when expressed as the product of developed pressure and heart rate. In contrast, contraction correlated with tissue PCr level at low ATP. These data demonstrate that effective contraction of the isovolumic heart is possible at substantially decreased level of cytoplasmic ATP. The results of this study are in accord with a concept of ATP compartmentation in cardiac cells.

Effect of dinitrosyl iron complexes with glutathione on hemorrhagic shock followed by saline treatment.

It has been found that dinitrosyl iron complexes with glutathione (DNIC-GS) injected into the blood flow of rats at a dose of 0.05 μmoles/kg prior to hemorrhage significantly improve cardiac function under conditions of hemorrhagic shock manifested in increased stroke volume, left ventricular work and cardiac output to a level exceeding control values 1.5-fold. Enhanced myocardial contractile activity leads to a situation where mean arterial pressure does not decrease further despite the significant decrease of total peripheral resistance. The decrease of total peripheral vascular resistance of the vascular system under vasodilating effects of DNIC-GS used as nitric oxide donors improves microcirculation in experimental rats judging from increased rates of blood flow and low degree of erythrocyte aggregation. Pretreatment of rats with the complexes significantly increases survival (by 21%) under conditions of hemorrhagic shock. It is suggested that beneficial effects of DNIC-GS on systemic circulation parameters under conditions of hemorrhagic shock are determined by their antioxidant activity and the ability to induce S-nitrosylation of proteins.

A simple protocol for the synthesis of dinitrosyl iron complexes with glutathione: EPR, optical, chromatographic and biological characterization of reaction products.

The diamagnetic binuclear form of dinitrosyl iron complexes (B-DNIC) with glutathione can be easily synthesized in the air at ambient temperature. The synthetic protocol includes consecutive addition to distilled water of glutathione, which decreases the pH of the test solution to 4.0, a bivalent iron salt (e.g., ferrous sulphate) and sodium nitrite at the molar ratio of 2:1:1, with a subsequent increase in pH to neutral values. Under these conditions, the amount of B-DNIC formed is limited by initial nitrite concentration. In the novel procedure, 20mM glutathione, 10mM ferrous sulfate and 10mM sodium nitrite give 2.5mM B-DNIC with glutathione, while 5mM glutathione remains in the solution. Bivalent iron (5mM) is precipitated in the form of hydroxide complexes, which can be removed from the solution by passage through a paper filter. After the increase in рН to 11 and addition of thiols at concentrations exceeding that of DNIC tenfold, B-DNIC are converted into a mononuclear EPR-active form of DNIC (M-DNIC) with glutathione. B-DNIC preparations synthesized by using new method contain negligible amount of nitrite or S-nitrosoglutathione as a contaminations. All the steps of DNIC synthesis were characterized by using optical, EPR and HPLC methods. A long-lasting hypotensive action of DNIC formed was demonstrated.

Changes in antioxidant status of myocardium during oxidative stress under the influence of coenzyme Q10

Changes in myocardium were studied during oxidative stress induced by infusion of hydrogen peroxide in the coronary vessels of isolated rat heart. Moderate concentrations of H2O2 increased the heart rate but decreased the contractile force, whereas higher concentrations of H2O2 decreased both parameters and increased the end diastolic pressure. The effect of H2O2 was stable, cumulative, and was associated with disturbance in respiration of mitochondria, increased production of ROS in them, and decrease in activities of antioxidant enzymes in the myocardium. Changes in the antioxidant status of the myocardium induced by long-term addition of coenzyme Q10 into food was accompanied by decrease in the negative inotropic effect of H2O2, whereas the levels of superoxide dismutase and glutathione peroxidase after oxidative stress were virtually unchanged. The activities of these enzymes displayed a high positive correlation with the cardiac function. The findings suggest that coenzyme Q10 should increase resistance of the myocardium to oxidative stress not only by a direct antioxidant mechanism but also indirectly, due to increased protection of antioxidant enzymes.

ESR spin trapping and NMR spectroscopy of the same heart shows correlation between energy depression and radical formation during postischemic reperfusion

The relevance of radical formation in disturbances of energy metabolism in the postischemic heart is not clear. This study provides the first evidence of a significant correlation between the amount of oxy‐radicals trapped in the effluent of isolated hearts upon reperfusion and the decreased myocardial content of phosphocreatine and ATP. This suggests that the loss of high‐energy compounds might contribute to oxy‐radical production during reperfusion. The application of ESR spin trapping and of NMR technique to the same heart is a new approach to investigate the pathobiochemical relevance of free radicals for the heart muscle.

The hypotensive effect of the nitric monoxide donor Oxacom at different routs of its administration to experimental animals

Earlier it has been found that the hypotensive drug Oxacom containing binuclear dinitrosyl iron complexes (B-DNIC) with glutathione can effectively decrease, as a nitric monooxide (NO) donor, the mean arterial pressure (МАР) in rats upon intravenous bolus injection in the form of an aqueous solution (Chazov et al., 2012). The aim of this study was to investigate the hypotensive effects of Oxacom administered to experimental rats by intravenous, intramuscular, subcutaneous, intraperitoneal, intragastric, rectal routes.MAP and heart rate (HR) were measured with the help of arterial catheters equipped with tensometric sensors. Oxacom was administered to rats at the dose of 2.0 μmole of B-DNIC/kg. The concentration of paramagnetic mononuclear protein-bound DNIC (М-DNIC) formed in the blood and tissues of various internal organs of the rat was determined by the EPR method. Upon subcutaneous, intramuscular or intraperitoneal administration of Oxacom, the maximum amplitude of the МАР decrease varies from 30% to 70%, respectively, in comparison with the corresponding parameter for the intravenously injected Oxacom. Another difference is the lack of the fast phase in the initial stage of the МАР decrease and the longer persistence of protein-bound M-DNIC formed in the circulating blood after intramuscular, subcutaneous or intraperitoneal administration of Oxacom. Thus, the NO donor Oxacom exerts pronounced hypotensive effects on rats not only upon intravenous, but also upon intramuscular, subcutaneous or intraperitoneal administration.