[ATP-phosphocreatine metabolism catalyzed by creatine kinase. Comparison of saturation transfer (NMR) and isotope labeling technics]. / Izuchenie obmena ATP-fosfokreatin, kataliziruemogo kreatinkinazoi. Sravnenie tekhniki perenosa nasyshcheniia (IaMR) i metoda izotopnoi metki.

Unidirectional fluxes from ATP to phosphocreatine (PCr) catalyzed by MM-isoenzyme of creatine kinase (CK) were measured by using 31P-NMR saturation transfer technique and by means of radioactively labeled [gamma-32P]ATP. At 30-37 degrees C and pH 7.4 in a wide range of [PCr]/[creatine] ([PCr]/[Cr]) ratios (0.2 to 3.0) both of these methods gave similar results, thus showing that magnetization (saturation) transfer allows to determine fluxes close to real ones under "physiological" conditions. However, at [PCr]/[Cr] ratio higher than 5 ([ADP] less than 30 microM) or at decreased temperatures (7-15 degrees C, [PCr]/[Cr] approximately 1) fluxes determined by saturation transfer substantially exceeded those measured with the radioactive label. These data imply that under "physiological" conditions phosphoryl group transfer is actually rate-determining step of the CK reaction. On the contrary, at high [PCr]/[Cr] values or at low temperature the control step could be shifted from the phosphoryl group transfer or distributed among other steps of the reaction.

[Steryl cellosolves--regulators of cholesterol metabolism in isolated rabbit hepatocytes]. / Steriltsellozoly'vyv--reguliatory metabolizma kholesterina v izolirovannykh gepatotsitakh krolika.

Synthesis of 3 beta-(2-hydroxyethoxy)cholest-5-ene, 3 beta-(2-hydroxyethoxy)cholest-5-en-7-one, 3 beta-(2-hydroxyethoxy)-7 beta-hydroxycholest-5-ene, 3 beta-(2-hydroxyethoxy)-5 alpha, 6 alpha-epoxycholestane, and 3 beta-(2-hydroxyethoxy) -5 alpha, 6 beta-dihydroxycholestane was described. Substances obtained inhibited cholesterol biosynthesis in the rabbit hepatocyte cell culture with ID 50 from 5.5(+/-0.7) x 10(-8) to 1.3(+/-0.2) x 10(-5) M and also to a remarkable extent the cell protein biosynthesis.

[Affinity of 3-beta-(2-hydroxyethoxy)-5-alpha-cholest-8(14)-ene-15-one to oxysterol binding protein and its metabolism in HepG2 hepatoma cells]. / Srodstvo 3beta-(2-gidroksiétoksi)-5alpha-kholest-8(14)-en-15-ona k oksisterinsviazyvaiushchemu belku i ego metabolizm v kletkakh gepatomy HepG2.

beta-(2-Hydroxyethoxy)-5 alpha-cholest-8(14)-en-15-one, a synthetic inhibitor of cholesterol biosynthesis, was shown to exhibit a high affinity to oxysterol binding protein. This was proved by ultracentrifugation of the protein fraction from rabbit liver in the presence of the 3H-labeled inhibitor, 3 beta-(2-hydroxy-2-[3H]ethoxy)-5 alpha-cholest-8(14)-en-15-one, or by the substitution of the [3H]-25-hydroxycholesterol in its complex with the oxysterol binding protein. In human hepatoma Hep G2 cells, the inhibitor decreased activity of 3-hydroxy-3-methylglutaryl CoA reductase [ID50 (2.7 +/- 0.7) x 10(-5) M] and was transformed into 3 beta-[2-(9-Z-octadecenoyloxy)ethoxy]-5 alpha-cholest-8(14)-en-15-one.

[Inhibition of cholesterol biosynthesis in rabbit hepatocytes by 3-beta-(omega-hydroxyalkoxy)cholest-5-enes]. / Ingibirovanie biosinteza kholesterina v gepatotsitakh krolika 3beta-(omega-gidroksialkoksi)kholest-5-enami]

3 beta-(2-Hydroxyethoxy)-, 3 beta-(4'-hydroxybutoxy)-, 3 beta-(6-hydroxyhexyloxy)-, 3 beta-(9-hydroxynonyloxy)-, and 3 beta-(2-hydroxy-2-[3H]ethoxy)cholest-5-enes were synthesized. By means of a spin probe, the influence of the synthesized compounds on the phase transition of dimyristoylphosphatidylcholine were estimated. Time and dose dependences of the incorporation of 3 beta-(2-hydroxy-2-[3H]ethoxy)cholest-5-ene into rabbit hepatocytes (the primary culture) were studied. 3 beta-(2-Hydroxyethoxy)- and 3 beta-(4-hydroxybutoxy)cholest-5-enes were shown to inhibit cholesterol biosynthesis from [14C]acetate in rabbit hepatocyte cultures upon a 24-hour preincubation.

[Functional significance of 2 pathways of energy transport in cardiomyocytes]. / Funktsional'noe znachenie dvukh putei transporta énergii v kardiomiotsitakh.

To study the relative roles of creatine kinase (CK) and adenylate systems in cardiac energy turnover, the effect of CK inhibitor, iodoacetamide- (IAA, 0.5 mM), and 2-deoxyglucose-(DOG, 2 mM) induced) 65% depletion of adenine nucleotides at slightly decreased CK flux was determined in isolated rat heart. Both substances did not substantially affect contractile parameters of the isovolumic heart. However, an augmentation of cardiac work induced by isoproterenol addition was feeble and transient in IAA-treated hearts while the response of DOG-treated hearts was well preserved. The cardiac failure after IAA treatment was associated with irreversible fall in myocardial ATP content as evidenced by 31P-NMR technique. Furthermore, these hearts were unable to perform cardiac pump function due to insufficient cardiac filling and distensibility. The DOG-treated hearts exhibited 50% reduction in the pump function and were able to increase their work in elevated resistance. The results suggest that CK pathway is extremely important for both full cardiac relaxation and maximal contractile function.

[Adrenergic stimulation of the heart during inhibition of phosphocreatine or adenylate pathways of energy transfer in cardiomyocytes]. / Adrenergicheskaia stimuliatsiia serdtsa pri ingibirovanii fosfokreatinovogo ili adenilatnogo puti transporta énergii v kardiomiotsitakh.

Functional and metabolic response of an isovolumically perfused heart of a rat to isoproterenol (0.1 microM) has been studied. A heart with the normal content of adenine nucleotides (AN) and phosphocreatine (PCr) as well as that with the 5-fold reduced AN content (with 2-deoxyglucose treatment) significantly increased cardiac work index (PRP), maximal contraction rate (MCR) and maximal relaxation rate (MRR) (by 50, 30-40 and 100-150%, respectively). The effect was preserved for all the period of the hormone action (30 min) and was followed by a temporary decrease in the PCr content. The heart with an inhibited unidirectional flux of metabolites through creative kinase (CK) and normal level of AN responded to the hormone by the slower and decelerated growth of the function and in the heart with almost completely iodoacetamide (IAAm)-blocked CK the functional response was minimal and transient. In the latter a significant and irreversible decline in PCr and ATP content and a concomitant rise of inorganic phosphate took place. Both basal and isoproterenol-stimulated adenylate cyclase activity remained unchanged after IAAm treatment. An increase in PRP correlated with the elevation of the cytosolic ADP concentration, however, correlation was not uniform for different experimental groups. These data show significance of the creatine kinase system not only for maintenance of maximal work but also for a rapid functional response to the catecholamine stimulation.

[3-Phosphono-2-imino-4-oxoimidazoline--a competitive substrate of AMP-dephosphorylating enzymes from the cytosol fraction of the rat heart]. / 3-Fosfono-2-imino-1-metil-4-okso-imidazolin--konkurentnyi substrat AMF-defosforiliruiushchikh fermentov tsitozol'noi fraktsii serdtsa krysy.

3-Phosphono-2-imino-1-methyl-4-oxoimidazolidine (PIMOI), AMP and p-nitrophenyl phosphate (pNPP) were dephosphorylated in the presence of rat heart cytosol at 37 degrees C pH 6.3 at the rates of 0.71, 0.45 and 1.07 mumol/mg X h, respectively. When mixed together, these compounds inhibited the hydrolysis of each other, which points to the participation of common enzyme(s) in this process. The inhibitor of 5'-nucleotidase (alpha,beta-methylene)-ADP, did not affect PIMOI cleavage and moderately inhibited AMP hydrolysis (by ADP, did not affect PIMOI cleavage and moderately inhibited AMP hydrolysis (by 30-50%), thus suggesting that acidic phosphatases are responsible for PIMOI and AMP hydrolysis under these conditions (pH 6.3). Phosphocreatine (PCr) and phosphocyclocreatine (PcCr) were stable to hydrolysis by the cytosolic fraction. However, addition of AMP to the medium containing PCr or PcCr resulted in AMP phosphorylation down to ATP due to the effects of these phosphagens and, probably, of microcontaminations of ATP. This was followed by gradual disappearance of PCr or PcCr and by accumulation of Pi as a result of the "ATPase" activity in the cytosol. The hydrolysis of AMP, PIMOI and p-NPP was sensitive to sulfhydryl reagents [5,5'-dithio-bis-(2-nitrobenzoate) and, in part, 2,4-dinitro-fluorobenzene] and fluoride ion. Thus, PIMOI is a competitive substrate of acidic phosphatases in heart cytosol with respect to AMP and p-NPP. This may partly explain the protective effect of PIMOI on ischemic myocardium.

[Metabolism of exogenous phosphocreatine and 3-phosphono-2-imino-1-methyl-4-hydroxy-imidazoline in the perfused rat heart during ischemia]. / Metabolizm ékzogennykh fosfokreatina i 3-fosfono-2-imino-1-metil- 4-okso-imidazolidina v perfuziruemom serdtse krysy pri ishemii.

Exogenous phosphocreatine (PCr) and its cyclic analog, 3-phosphono-2-imino-1-methyl-4-oxoimidazolidine (PIMOI) were used as protectors of ischemic myocardium. PCr was insignificantly metabolized, whereas its analog was rapidly split, resulting in the formation of creatinine and inorganic phosphate as well as of minor amounts of PCr and creatine. Exogenous AMP and ATP accelerated PCr breakdown; in contrast, PIMOI hydrolysis slowed down in the presence of AMP. A similar inhibitory effect was observed after treatment of hearts with 2,4-dinitrofluorobenzene. These data together with those obtained for heart homogenates point to the enzymatic nature of PIMOI hydrolysis in ischemic heart. Acidic phosphatases and 5'-nucleotidases of the heart are supposed to be involved in this process.

[High specificity in the molecular mechanism of the protective action of phosphocreatine on the myocardium in ischemia]. / Vysokaia spetsifichnost' molekuliarnogo mekhanizma zashchitnogo deistviia fosfokreatina na miokard pri ishemii.

To study the character of the mechanism of protective action of phosphocreatine on ischemic myocardium the effects of phosphocreatine (PCr) and phosphoarginine (PArg) were compared. PCr and PArg were shown to expose identical Ca2+-chelating properties and were used as their Na-salts. Only PCr protected the cardia function during ischemia and simultaneously inhibited the accumulation of lysophosphoglycerides, products of phospholipid degradation. PArg failed to exert both of these effects. By an EPR probe method PCr was shown to increase the order of structural organization of phospholipids in the cardiac sarcolemmal vesicles. The results show that the effect of PCr on ischemic myocardium is not due to nonspecific changes in the ion composition of a solution, but most probably due to highly specific effect of phosphocreatine on the phospholipid membrane of the cardiac cells sarcolemma.

[Study of the protective effect of phosphocreatine on the ischemic myocardium during cardioplegia using the P-31 NMR method]. / Issledovanie metodom 31P-IaMR zashchitnogo deistviia fosfokreatina na ishemizirovannyi miokard v usloviiakh kardioplegii.

Using 31P-NMR technique it was shown that exogenous phosphocreatine (10 mM) added in the cardioplegic solution provided higher levels of recovery of intracellular ATP (approx. 60% vs. 26% in the control) and phosphocreatine (90% vs. 43%) in perfused rat hearts after 35 min of total ischemia. Simultaneously significantly higher levels of contractile recovery (90% vs. 35%) and a three-fold decrease in creatine kinase release into the perfusate were observed. These effects of exogenous phosphocreatine can be related to either intracellular action of this compound or its interaction with cellular membrane.

[Metabolic and functional consequences of complete inhibition of creatine kinase by iodoacetamide in the perfused heart]. / Metabolicheskie if funktsional'nye posledstviia polnogo ingibirovaniia kreatinkinazy iodatsetamidom v perfuziruemom serdtse.

Treatment of perfused rat hearts with 0.5 mM iodoacetamide (IAAm) for 15 min at different workloads resulting in a nearly complete inhibition of creatine kinase (CK, 99%) was followed by a rapid decline of the phosphocreatine (PCr) level (30%) and a 2-fold increase of the P(i) level which then stabilized. Conversely, the ATP content started to drop monotonously at the beginning of the IAAm washout and reached 30% 90 min after the IAAm removal under medium load. Under low workload the ATP decay occurred at later periods. Neither the ADP-stimulated mitochondrial respiration in skinned fibers, nor the Ca(2+)-stimulated ATPase activity of myofibrils was affected by IAAm treatment. The sensitivity of the resting tension of skinned fibers to Ca2+ tended to a slight increase. The cardiac work index (PRP-pressure-rate product) decreased by 25%, while the end diastolic pressure (EDP) rose by 15 mm Hg when IAAm acted under medium load. In contrast, under low work these parameters were practically stable. The hearts poisoned with IAAm performed a two times lower maximal work and had reduced (by 35%) oxygen consumption rates. The efficiency of energy utilization for mechanical work decreased by 40%. The changes in PRP and EDP correlated with the cytosolic [ATP]/[ADP] ratio in such a way that the decrease in the latter was associated with a decrease in PRP and the elevation of EDP. These data suggest that the creatine kinase system is necessary for the effective translation of a high [ATP]/[ADP] ratio from the intermembrane space of mitochondria to the cytoplasm, myofibrils and ionic pumps. This provides a high level of mechanical work and good relaxation of the left ventricle and protects cytosolic adenine nucleotides from the breakdown.