Polarographic observation of substrate-level phosphorylation and its stimulation by acetylcholine.

Substrate-level phosphorylation was observed under the conditions optimal for this process and opposite to those for oxidative phosphorylation. Polarographic registration of Ca2+ stimulated alpha-ketoglutarate oxidation and self-inhibition of uncoupled alpha-ketoglutarate (KG) oxidation was used. Acetylcholine (ACh) administration stimulated KG oxidation and substrate-level phosphorylation in isolated mitochondria. These effects are stronger in tissues with a higher level of endogenous acetylcholine, such as guinea pig liver vs rat liver and pancreas vs liver. The specific stimulation of KG oxidation by ACh is related to a decrease of succinate oxidation and is contrary to the specific stimulating effect of adrenaline on succinate oxidation. Therefore the existence of reciprocal hormone-substrate-nucleotide systems is suggested. The described set of conditions optimal for substrate-level phosphorylation observation by polarographic registration of respiration is as convenient as the ADP test for the investigation of oxidative phosphorylation.

Acetylcholine affects rat liver metabolism via type 3 muscarinic receptors in hepatocytes.

Although the role of acetylcholine (Ach) in hepatic glucose metabolism is well elucidated, it is still unclear if it influences gluconeogenesis, glycogenolysis and high-energy phosphate metabolism, and if it does what the mechanisms of this influence are. Therefore, using isolated perfused rat liver as a model, we have studied the effect of Ach on oxygen consumption, synthesis of glucose from lactate and pyruvate, glycogen formation, mitochondrial oxidative phosphorylation and ATP-synthesis. We have established that effects of Ach on oxygen consumption depend on its concentration. When used at a concentration of 10(-7) M, Ach exerts maximum stimulatory effect, while its infusion at 10(-6) M causes a decrease of oxygen consumption by the liver. Moreover, when used at a concentration of 10(-6) M or 10(-7) M, Ach increases rates of glucose production from the gluconeogenic substrates lactate and pyruvate, leading to enhanced glycogen content in perfused liver. It was also shown that Ach possesses a stimulating effect on alanine and aspartate aminotransferases. As detected by 31P NMR spectroscopy, continuous liver perfusion with pyruvate and lactate in the presence of Ach leads to a significant decrease of ATP level, implying enhanced energy requirements for gluconeogenesis under these conditions. Elimination of the described effects of Ach by atropine, the antagonist of muscarinic receptors, and identification of the type 3 muscarinic receptors (m3) in isolated hepatocytes as well as in whole liver, imply that Ach may exert its effect on liver metabolism through m3 receptors.

[Effect of acetylcholine of substrate oxidation in heart mitochondria]. / Deistvie atsetilkholina na okislenie substratov v mitokhondriiakh serdtsa.

Acetylcholine has been studied for its effect on respiration and oxidative phosphorylation in mitochondria from the heart of a rat and guinea pig. Acetylcholine in doses of 25, 50 and 100 mg per 100 g of the body weight 5, 15 and 30 min after intraperitoneal injection intensifies the rate of phosphorylative respiration at ketoglutarate oxidation and moderately lowers it at succinate oxidation. Malonate increases the activating influence of acetylcholine on oxidation of alpha-ketoglutarate in the heart mitochondria and aminooxyacetate decreases it. Phosphorylative respiration with oxidation of pyruvate and isocitrate is not changed essentially under the action of acetylcholine. Introduction of acetylcholine stimulated most strongly the aminooxyacetate-sensitive portion of respiration, a mixture of aminotransferases in the activation of alpha-ketoglutarate oxidation under effect of acetylcholine. The stimulating action of acetylcholine on alpha-ketoglutarate oxidation is mediated by M- and H-cholinoreceptors, since it is abolished by their blockers: atropine and benzohexonium. Stimulation of alpha-ketoglutarate oxidation by acetylcholine is mostly expressed under introduction of beta-adrenoblocker obsidan which provides prevalence of the parasympathetic nervous system. This stimulation is more intensive in the guinea pig as a more cholinergic animal in comparison with a rat.

[Acetylcholine activation of alpha-ketoglutarate oxidation in liver mitochondria]. / Aktivatsiia atsetilkholinom okisleniia alpha-ketoglutarata v mitokhondriiakh pecheni.

Activation of alpha-ketoglutarate oxidation in the rat liver mitochondria takes place 15 and 30 min after intraperitoneal injection of acetyl choline. This mediator in doses of 25, 50 and 100 micrograms per 100 g of body weight causes a pronounced stimulation of phosphorylation respiration rate and calcium capacity of mitochondria with alpha-ketoglutarate oxidation. Acetyl choline is found to have a moderate inhibitory action on oxidation of lower (physiological) concentrations of succinate. Its stimulating action on alpha-ketoglutarate oxidation is associated with activation of M-cholinoreceptors; atropine, a choline-blocker, removes completely this effect. It is supposed that alpha-ketoglutarate and succinate are included into the composition of two reciprocal hormonal-substrate nucleotide systems.

Adenosine improves cardiomyocyte respiratory efficiency.

The role of adenosine on the regulation of mitochondrial function has been studied. In order to evaluate this the following experiments were done in isolated rat cardiomyocites and mitochondria using polarographic techniques. Cardiomyocyte oxygen consumption (MVO2) and mitochondrial respiratory function (State 3 and State 4, respiratory control index, and ADP/O ratio) were evaluated after exposure to adenosine. Cardiomyocyte MVO2 was significantly lower in cells previously exposed to adenosine (10 microM, 15 min or 30 min cell incubation) than in cells not exposed to adenosine (control). Addition of dipyridamole (10 microM) or 8-(p-Sulfophenyl) theophylline (50 microM) to cardiomyocytes before adenosine incubation prevented the adenosine-induced changes in MVO2. Mitochondria obtained from isolated perfused beating heart previously perfused with adenosine (10 microM, 30 min heart perfusion) also resulted in significant increases in ADP/O and respiratory control index compared to matching control. Mitochondria isolated from cardiomyocytes previously exposed to adenosine (10 microM, 15 min or 30 min cell incubation) resulted in a significant increase in mitochondrial ADP/O ratio compared to control. Adenosine-induced decrease in cardiomyocyte MVO2 may be related to an increase in efficiency of mitochondrial oxidative phosphorylation, and more economical use of oxygen, which is necessary for survival under ischemic stress.

[Carnosine in adaptation to hypobaric hypoxia]. / Karnozin v adaptatsii k gipobaricheskoi gipoksii.

Adaptation to hypobaric hypoxia causes increases in the carnosine content in rat liver mitochondria. Model experiments showed that carnosine added to isolated rat liver mitochondria increases the rate of ADP-stimulated respiration with alpha-ketoglutarate (but not with succinate) as well as the intensity (ADP/t) and efficiency (ADP/O) of oxidative phosphorylation--by 56%, 49%, and 15%, respectively. Increases in the intensity and efficiency of oxidative phosphorylation (by 100% and 30%, respectively) were also observed in liver mitochondria isolated from rats adapted to hypobaric hypoxia. Activation of ADP-stimulated respiration was found after addition of glutamate plus malate and pyruvate plus glutamate to liver mitochondria. This stimulating effect was coupled to aminotransferase activation and was abolished by the transaminase inhibitor aminooxyacetate. Carnosine addition to mitochondria or its accumulation in mitochondria under hypoxia is associated with activation of alpha-ketoglutarate oxidation and its formation through transamination.

Encapsulation and perfusion of mitochondria in agarose beads for functional studies with 31P-NMR spectroscopy.

An NMR method to study on-line mitochondrial function was developed. Mitochondria were maintained in a stable physiologic state in agarose beads that were continuously superfused with oxygenated buffer at 28 degrees C. Oxidative function of both heart and liver mitochondria was evaluated with 31P NMR at 9.4 T using pyruvate plus malate as substrate. This method allows clear resolution of adenosine triphosphate-gamma (ATPgamma) and adenosine diphosphate-beta (ADPbeta) phosphate signals, whereas alpha signals of ATP and ADP overlap. ATP production by mitochondria was documented to be very sensitive to different interventions (hypoxia, ischemia, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP)) and depended on the ADP concentration in superfusion medium. These data demonstrate that the new application of NMR to study mitochondrial function can discriminate, on-line, between several physiologic and biochemical processes in intact physiologically stable mitochondria.

Opening of potassium channels protects mitochondrial function from calcium overload.

Ischemic preconditioning (IPC) protects myocardium from ischemia reperfusion injury by activating mitochondrial K(ATP) channels. However, the mechanism underlying the protective effect of K(ATP) channel activation has not been elucidated. It has been suggested that activation of mitochondrial K(ATP) channels may prevent mitochondrial dysfunction associated with Ca(2+) overload during reperfusion. The purpose of this experiment was to study, in an isolated mitochondrial preparation, the effects of mitochondrial K(ATP) channel opening on mitochondrial function and to determine whether it protects mitochondria form Ca(2+) overload. Mitochondria (mito) were isolated from rat hearts by differential centrifugation (n = 5/group). Mito respiratory function was measured by polarography without (CONTROL) or with a potassium channel opener (PINACIDIL, 100 microM). Different Ca(2+) concentrations (0 to 5 x 10(-7) M) were used to simulate the effect of Ca(2+) overload; state 2, mito oxygen consumption with substrate only; state 3, oxygen consumption stimulated by ADP; state 4, oxygen consumption after cessation of ADP phosphorylation; respiratory control index (RCI: ratio of state 3 to state 4); rate of oxidative phosphorylation (ADP/Deltat); and ADP:O ratio were measured. PINACIDIL increased state 2 respiration and decreased RCI compared to CONTROL. Low Ca(2+) concentrations stimulated state 2 and state 4 respiration and decreased RCI and ADP:O ratios. High Ca(2+) concentrations increased state 2 and state 4 respiration and further decreased RCI, state 3, and ADP/Deltat. PINACIDIL improved state 3, ADP/Deltat, and RCI at high Ca(2+) concentrations compared to CONTROL. Pinacidil depolarized inner mitochondrial membrane, as evidenced by decreased RCI and increased state 2 at baseline. Depolarization may decrease Ca(2+) influx into mito, protecting mito from Ca(2+) overload, as evidenced by improved state 3 and RCI at high Ca(2+) concentrations. The myocardial protective effects resulting from activating K(ATP) channels either pharmacologically or by IPC may be the result of protecting mito from Ca(2+) overload.

Mitochondrial oxidative phosphorylation in heart from stressed cardiomyopathic hamsters.

Stress alone is generally not sufficient to produce serious disease, but stress imposed upon pre-existing disease can contribute to disease progression. To explore this phenomenon, cold-immobilization stress was imposed on young 12.5 month, necrotic phase with small vessel coronary spasm) and older (5 month, quiescent phase, between necrosis and heart failure) cardiomyopathic hamsters. Our hypothesis was that changes in mitochondrial energy processes are involved in stress induced pathology. Polarographic and high performance liquid chromatography (HPLC) techniques were used to measure mitochondrial respiration and oxidative phosphorylation and concentrations of phosphocreatine and adenylates, respectively, in hearts from young and old cardiomyopathic hamsters (stressed and unstressed). No significant differences were found between the young (2.5 month) and old (5 month) age groups in unstressed and stressed healthy hamsters and between young (2.5 month) and old (5 month) unstressed cardiomyopathic hamsters with respect to different parameters of mitochondrial oxidative phosphorylation and with respect to concentration of bioenergetic metabolites, except that ADP concentration was higher in older cardiomyopathic hamsters. Application of stress uncovered differences between young and old cardiomyopathic hamsters: respiration control index was lower and State 4 respiration was higher in young compared to old cardiomyopathic hamsters; whereas the total concentration of ATP was decreased to the same level in both cardiomyopathic groups when compared to control. Mitochondrial oxidative phosphorylation in young cardiomyopathic hamsters was more sensitive to Ca2+, as evidenced by partial uncoupling of respiration and oxidative phosphorylation, than in older cardiomyopathic hamsters and controls. In conclusion, young cardiomyopathic hamsters, i.e. in the necrotic phase of disease, were more susceptible to stress induced changes in mitochondrial oxidative phosphorylation than older cardiomyopathic hamsters and controls.

Metabolic control of sodium transport in streptozotocin-induced diabetic rat hearts.

Diabetic and control cardiomyocytes encapsulated in agarose beads and superfused with modified medium 199 were studied with 23Na- and 31P-NMR. Baseline intracellular Na+ was higher in diabetic (0.076 +/- 0.01 micromoles/mg protein) than in control (0.04 +/- 0.01 micromoles/mg protein) (p < 0.05). Baseline betaATP and phosphocreatine (PCr) (peak area divided by the peak area of the standard, methylene diphosphonate) were lower in diabetic than in control, e.g., betaATP control, 0.70 +/- 0.07; betaATP diabetic, 0. 49 +/- 0.04 (p < 0.027); PCr control, 1.20 +/- 0.13; PCr diabetic, 0. 83 +/- 0.11 (p < 0.03). This suggests that diabetic cardiomyocytes have depressed bioenergetic function, which may contribute to abnormal Na,K-ATPase function, and thus, an increase in intracellular Na+. In the experiments presented herein, three interventions (2-deoxyglucose, dinitrophenol, or ouabain infusions) were used to determine whether, and the extent to which, energy deficits or abnormalities in Na,K-ATPase function contribute to the increase in intracellular Na+. In diabetic cardiomyocytes, 2-deoxyglucose and ouabain had minimal effect on intracellular Na+, suggesting baseline depression of, or resetting of both glycolytic and Na,K-ATPase function, whereas in control both agents caused significant increases in intracellular Na+after 63 min exposure: 2-deoxyglucose control, 32.9 +/- 8.1%; 2-deoxyglucose diabetic, -4.6 +/- 6% (p < 0.05); ouabain control, 50.5 +/- 8.8%; ouabain diabetic, 21.2 +/- 9.2% (p < 0.05). In both animal models, dinitrophenol was associated with large increases in intracellular Na+: control, 119.0 +/- 26.9%; diabetic, 138.2 +/- 12.6%. Except for the dinitrophenol intervention, where betaATP and PCr decreased to levels below 31P-NMR detection, the energetic metabolites were not lowered to levels that would compromise sarcolemmal function (Na,K-ATPase) in either control or diabetic cardiomyocytes. In conclusion, in diabetic cardiomyocytes, even though abnormal glycolytic and Na, K-ATPase function was associated with increases in intracellular Na+, these increases were not directly related to global energy deficit.