Relationship between intracellular pH and energy metabolism in dog brain as measured by 31P-NMR

The relationships between pHi (intracellular pH) and phosphate compounds were evaluated by nuclear magnetic resonance (NMR) in normo-, hypo-, and hypercapnia, obtained by changing fractional inspired concentration of CO2 in dogs anesthetized with 0.75% isoflurane and 66% N2O. Phosphocreatine (PCr) fell by 2.02 mM and Pi (inorganic phosphate) rose by 1.92 mM due to pHi shift from 7.10 to 6.83 during hypercapnia. The stoichiometric coefficient was 1.05 (r2 = 0.78) on log PCr/Cr against pHi, showing minimum change of ADP/ATP and equilibrium of creatine kinase in the pH range of 6.7 to 7.25. [ADP] varied from 21.6 +/- 4.1 microM in control (pHi = 7.10) to 26.8 +/- 6.3 microM in hypercapnia (pHi = 6.83) and 24.0 +/- 6.8 microM in hypocapnia (pHi = 7.17). ATP/ADP X Pi decreased from 66.4 +/- 17.1 mM-1 during normocapnia to 25.8 +/- 6.3 mM-1 in hypercapnia. The ADP values are near the in vitro Km; thus ADP is the main controller. The velocity of oxidative metabolism (V) in relation to its maximum (Vmax) as calculated by a steady-state Michaelis-Menten formulation is approximately 50% in normocapnia. In acidosis (pH 6.7) and alkalosis (pH 7.25), V/Vmax is 10% higher than the normocapnic brain. This increase of V/Vmax is required to maintain cellular homeostasis of energy metabolism in the face of either inhibition at extremes of pH or higher ATPase activity.     

Bioenergetics of rabbit skeletal muscle during hypoxemia and ischemia

A blood-perfused rabbit hindlimb preparation was exposed to total ischemia (n = 4) or to severe hypoxemia (n = 4) where arterial PO2 was 5 +/- 2 (SE) Torr. O2 consumption (VO2), O2 transport (TO2), venous PO2 (PVO2), venous lactate concentration, and venous glucose concentration were measured. The relative concentration of ATP, phosphocreatine (PCr), inorganic phosphate (Pi), and intracellular pH (pHi) were monitored with 31P magnetic resonance spectroscopy. PCr/Pi decreased with the onset of ischemia or hypoxemia. The preparation was reoxygenated and allowed to recover for 30 min once PCr/Pi was less than 1.0. The periods of hypoxemia and ischemia lasted 56.0 +/- 10.0 and 63.8 +/- 2.5 min, respectively (NS). During ischemia PCr decreased and Pi increased compared with control (P less than 0.05) but returned to control with reperfusion. With hypoxemia PCr also decreased and Pi increased with respect to control (P less than 0.01) but did not recover with reoxygenation. VO2 and PVO2 in both groups returned to control during recovery. ATP did not change with ischemia but decreased with hypoxemia (P less than 0.05). Venous lactate concentration did not change with ischemia but increased with hypoxemia (P less than 0.05) and continued to rise during recovery. During recovery pHi decreased in the hypoxemic group (P less than 0.05) but not in the ischemic group. These data show that, under the conditions tested, rabbit skeletal muscle does not resynthesize PCr after a severe hypoxemic episode. Furthermore it appears that VO2 and PVO2 fail to portray the true state of cellular bioenergetics after a severe hypotemic insult.     

Relationships between the neuronal sodium/potassium pump and energy metabolism. Effects of K+, Na+, and adenosine triphosphate in isolated brain synaptosomes

The relationships between Na/K pump activity and adenosine triphosphate (ATP) production were determined in isolated rat brain synaptosomes. The activity of the enzyme was modulated by altering [K+]e, [Na+]i, and [ATP]i while synaptosomal oxygen uptake and lactate production were measured simultaneously. KCl increased respiration and glycolysis with an apparent Km of about 1 mM which suggests that, at the [K+]e normally present in brain, 3.3-4 mM, the pump is near saturation with this cation. Depolarization with 6-40 mM KCl had negligible effect on ouabain-sensitive O2 uptake indicating that at the voltages involved the activity of the Na/K ATPase is largely independent of membrane potential. Increases in [Na+]i by addition of veratridine markedly enhanced glycoside-inhibitable respiration and lactate production. Calculations of the rates of ATP synthesis necessary to support the operation of the pump showed that greater than 90% of the energy was derived from oxidative phosphorylation. Consistent with this: (a) the ouabain-sensitive Rb/O2 ratio was close to 12 (i.e., Rb/ATP ratio of 2); (b) inhibition of mitochondrial ATP synthesis by Amytal resulted in a decrease in the glycoside-dependent rate of 86Rb uptake. Analyses of the mechanisms responsible for activation of the energy-producing pathways during enhanced Na and K movements indicate that glycolysis is predominantly stimulated by increase in activity of phosphofructokinase mediated via a rise in the concentrations of adenosine monophosphate [AMP] and inorganic phosphate [Pi] and a fall in the concentration of phosphocreatine [PCr]; the main moving force for the elevation in mitochondrial ATP generation is the decline in [ATP]/[ADP] [Pi] (or equivalent) and consequent readjustments in the ratio of the intramitochondrial pyridine nucleotides [( NAD]m/[NADH]m). Direct stimulation of pyruvate dehydrogenase by calcium appears to be of secondary importance. It is concluded that synaptosomal Na/K pump is fueled primarily by oxidative phosphorylation and that a fall in [ATP]/[ADP][Pi] is the chief factor responsible for increased energy production.     

Direct evidence for a role of intramitochondrial Ca2+ in the regulation of oxidative phosphorylation in the stimulated rat heart. Studies using 31P n.m.r. and ruthenium red.

1. The concentrations of free ATP, phosphocreatine (PCr), Pi, H+ and ADP (calculated) were monitored in perfused rat hearts by 31P n.m.r. before and during positive inotropic stimulation. Data were accumulated in 20 s blocks. 2. Administration of 0.1 microM-(-)-isoprenaline resulted in no significant changes in ATP, transient decreases in PCr, and transient increases in ADP and Pi. However, the concentrations of all of these metabolites returned to pre-stimulated values within 1 min, whereas cardiac work and O2 uptake remained elevated. 3. In contrast, in hearts perfused continuously with Ruthenium Red (2.5 micrograms/ml), a potent inhibitor of mitochondrial Ca2+ uptake, administration of isoprenaline caused significant decreases in ATP, and also much larger and more prolonged changes in the concentrations of ADP, PCr and Pi. In this instance values did not fully return to pre-stimulated concentrations. Administration of Ruthenium Red alone to unstimulated hearts had minor effects. 4. It is proposed that, in the absence of Ruthenium Red, the transmission of changes in cytoplasmic Ca2+ across the mitochondrial inner membrane is able to maintain the phosphorylation potential of the heart during positive inotropic stimulation, through activation of the Ca2+-sensitive intramitochondrial dehydrogenases (pyruvate, NAD+-isocitrate and 2-oxoglutarate dehydrogenases) leading to enhanced NADH production. 5. This mechanism is unavailable in the presence of Ruthenium Red, and oxidative phosphorylation must be stimulated primarily by a fall in phosphorylation potential, in accordance with the classical concept of respiratory control. However, the full oxidative response of the heart to stimulation may not be achievable under such circumstances.     

Oxygen transport to exercising leg in chronic hypoxia

Residence at high altitude could be accompanied by adaptations that alter the mechanisms of O2 delivery to exercising muscle. Seven sea level resident males, aged 22 +/- 1 yr, performed moderate to near-maximal steady-state cycle exercise at sea level in normoxia [inspired PO2 (PIO2) 150 Torr] and acute hypobaric hypoxia (barometric pressure, 445 Torr; PIO2, 83 Torr), and after 18 days' residence on Pikes Peak (4,300 m) while breathing ambient air (PIO2, 86 Torr) and air similar to that at sea level (35% O2, PIO2, 144 Torr). In both hypoxia and normoxia, after acclimatization the femoral arterial-iliac venous O2 content difference, hemoglobin concentration, and arterial O2 content, were higher than before acclimatization, but the venous PO2 (PVO2) was unchanged. Thermodilution leg blood flow was lower but calculated arterial O2 delivery and leg VO2 similar in hypoxia after vs. before acclimatization. Mean arterial pressure (MAP) and total peripheral resistance in hypoxia were greater after, than before, acclimatization. We concluded that acclimatization did not increase O2 delivery but rather maintained delivery via increased arterial oxygenation and decreased leg blood flow. The maintenance of PVO2 and the higher MAP after acclimatization suggested matching of O2 delivery to tissue O2 demands, with vasoconstriction possibly contributing to the decreased flow.     

Substrate regulation of mitochondrial oxidative phosphorylation in hypercapnic rabbit muscle.

Endurance muscle performance is highly dependent on ATP production from mitochondrial oxidative phosphorylation. To study the role of the mitochondrial oxidative enzymes in muscle fatigue, we analyzed the relationship between the concentrations of substrates associated with ATP synthesis and the muscle performance of electrically stimulated rabbit muscle under CO2-induced acidosis. Two different conditions of pacing-induced muscle performance were produced in the gastrocnemius and soleus muscle groups in anesthetized rabbits by stimulating the sciatic nerve submaximally at two frequencies. Phosphorus nuclear magnetic resonance was used to measure ATP, phosphocreatine, and Pi and to provide data for a calculation of intracellular pH and free ADP. To induce acidosis, the animal was ventilated with 20% CO2. The administration of CO2 effectively reduced the intracellular pH from 6.9 to 6.7 and reduced the isometric tension-time integral (TTI) to below half the value measured in normocapnia at the low pacing frequency. A twofold increase in the pacing frequency resulted in a doubling of the TTI in normocapnia and a tripling of TTI in hypercapnia. The increases in TTI corresponded with increases in free ADP and Pi concentrations. Under the various conditions, all free ADP values were near the in vitro Michaelis-Menten constant (Km) of ADP. The Michaelis-Menten relationship of the oxidative phosphorylative enzymes was applied to the change in substrate concentrations with respect to TTI. From this relationship we observed that the in vivo Km of free ADP was 26 microM, which is close to the in nitro Km, and that Km and maximal reaction velocity did not change under hypercapnia and increased pacing frequency.(ABSTRACT TRUNCATED AT 250 WORDS)     

In silico studies on the sensitivity of myocardial PCr/ATP to changes in mitochondrial enzyme activity and oxygen concentration

The ratio of myocardial phosphocreatine (PCr)/ATP reflects the balance of energy consumption and energy supply in the heart. It is reduced in a range of important physiological conditions including during and after acute hypoxia, after a prolonged visit to high-altitude, and in those suffering from both type 2 diabetes mellitus and various forms of heart failure. Yet despite its significance, the factors underlying the reduced PCr/ATP ratio seen in heart failure remain poorly understood. Given that oxidative phosphorylation is the only viable steady-state provider of ATP in the heart, the argument has been put forward that the observed reduction in myocardial PCr/ATP in all these conditions can be accounted for by some form of mitochondrial insufficiency. Thus we used a computer model of oxidative phosphorylation, coupled with creatine kinase, to study the effects of hypoxia and mitochondrial dysfunction on myocardial PCr/ATP. In physiological normoxia, all oxidative phosphorylation complexes, NADH supply and proton leak exerted comparable (of the same order of magnitude) control over PCr/ATP, as defined within Metabolic Control Analysis (MCA). Under hypoxia, the control increased considerably for all components of the system, especially for cytochrome oxidase and mitochondrial proton leak. Hypoxia alone, without any changes in other factors, exerted a pronounced effect on PCr/ATP. Our simulations support three important ideas: First, that mitochondrial abnormalities can contribute considerably to a blunted PCr/ATP; second, that hypoxia and mitochondrial dysfunction can interact in important ways to determine the energy status of the failing heart; and third, that hypoxia alone can account for significant decreases in cardiac PCr/ATP.     

Bioenergetic studies of mitochondrial oxidative phosphorylation using 31phosphorus NMR

The relationship between phosphorylation ratio [( ATP])/[ADP][Pi], phosphocreatine (PCr)/Pi, and ATPase activity was determined for isolated rat heart mitochondria, and the use of phosphorylation ratio and/or PCr/Pi as bioenergetic indices (Chance, B., Eleff, S., Leigh, J. S., Sokolow, D., and Sapega, A. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 6714-6718) was evaluated. Isolated rat heart mitochondria were suspended at low concentration (0.5-2.0 mg of protein/ ml) in oxygenated KCl/sucrose/4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid medium at 25 degrees C and pyruvate, malate, PCr, ATP, Pi, and Mg2+ were added. Changes in extramitochondrial phosphorus compounds were followed by 31P NMR. The ATPase activity was varied by the addition of potato apyrase. It was found that the logarithm of steady state PCr/Pi decreased linearly with increasing ATPase rate with a PCr/Pi intercept of 32.8 at 0 ATPase rate. The log phosphorylation ratio was also linearly related to the ATPase rate with an extrapolated maximum value of 6.87 at 0 ATPase rate, corresponding to a phosphorylation ratio of 7.41 X 10(6) M(-1) and a delta GATP of -16.3 kcal. The phosphorylation ratio in these experiments (for state 4 respiration) was greater by 1 or 2 orders of magnitude than previously reported for either isolated mitochondria or for whole tissue.     

A possible role of inorganic phosphate as a regulator of oxidative phosphorylation in combined urea synthesis and gluconeogenesis in perfused rat liver. A phosphorus magnetic resonance spectroscopy study

Metabolic control of oxidative metabolism was studied in perfused rat liver by means of phosphorus magnetic resonance spectroscopy. Oxygen consumption, ATP, and Pi were measured with different rates of gluconeogenesis and urea synthesis by varying concentrations of the substrates in the perfusate. Five levels of oxygen consumption (VO2) were obtained: an average control value of 1.94 +/- 0.14 and 2.93 +/- 0.25, 3.29 +/- 0.46, 3.85 +/- 0.26, and 4.18 +/- 0.56 mumol/min/g liver (mean +/- S.D., n = 6). The corresponding ATP concentrations were 2.51 +/- 0.20, 2.39 +/- 0.08, 2.24 +/- 0.09, 2.13 +/- 0.12, and 1.91 +/- 0.13 mM. Pi increased stoichiometrically with the decrease in ATP. Free Pi (Pif) was calculated as NMR-visible Pi in control plus -delta ATP (1.94 mM + (-delta ATP]. The kinetic relationship of oxidative phosphorylation as a function of Pif followed a Michaelis-Menten type of equation: VO2 = 5.55/(1 + 0.24/[( Pif] - 1.81]. The observed Km value for Pi of 0.24 mM approximates the reported Km value in isolated mitochondria of 1 mM. The free Pi concentration of 1.94 mM is in the range of the Km value, while the free ADP concentration of 200 microM exceeds the Km value of 20 microM. Therefore, it is suggested that Pi play a major role in the regulation of mitochondrial oxidative phosphorylation in combined urea synthesis and gluconeogenesis.     

31P-NMR studies of cerebral metabolic changes during graded hypoxia in newborn lambs

We measured cerebral phosphocreatine (PCr), inorganic phosphate (Pi), ATP, and intracellular pH (pHi) with in vivo phosphorus nuclear magnetic resonance (NMR) during 10- to 15-min periods of reversible hypoxic hypoxia in 20 newborn lambs (1-11 days). There was a significant correlation between arterial O2 partial pressure (PaO2) and the PCr/Pi ratio or pHi; however, between PaO2 130-33 mmHg, metabolite changes were not significant. PCr/Pi and pHi decreased significantly when PaO2 was lowered below 33 and 28 mmHg, respectively. After recovery, metabolite ratios and pHi returned to base-line values within 5 min. During the early phases of hypoxia and recovery, there were large fluctuations in metabolites and pHi, indicating that mitochondrial reactions were not in a steady state. After several minutes of hypoxia or recovery, PCr/Pi and pHi stabilized, suggesting steady state kinetics for mitochondrial respiration. NMR is extremely sensitive to changes in mitochondrial oxygenation, and stable PCr/Pi and pHi indicate that O2 tension in cerebral mitochondria of the newborn lamb is constant between PaO2 of 30 and 140 mmHg.     

Effect of fasting and acute ethanol administration on the energy state of in vivo liver as measured by 31P-NMR spectroscopy

The effects of 48 h fasting, administration of ethanol or 2,4-dinitrophenol, on the phosphorus-containing metabolites in liver in vivo have been determined utilizing 31P nuclear magnetic resonance spectroscopy. These measurements were combined with determinations of metabolite concentrations in livers which were freeze-clamped immediately after the NMR measurements were completed. Administration of sub-lethal amounts of dinitrophenol dramatically decreased ATP and increased Pi concentrations in liver in vivo as indicated by a 2.7-fold increase in the NMR-derived [Pi]/[ATP] ratio. Ethanol administration to fed animals increased the NMR-derived [Pi]/[ATP] ratio 27%; in contrast, the same amount of ethanol administered to fasted animals decreased the NMR-derived [Pi]/[ATP] ratio 30%. The NMR visible Pi and ADP represent about 50% and 15% of the total Pi and ADP, respectively. The phosphorylation potentials calculated from the NMR visible Pi and ADP were an order of magnitude higher than those obtained from metabolite concentrations in freeze-clamped tissue. There was no apparent correlation between the phosphorylation potentials derived from either the NMR spectral analyses or from metabolite concentrations and the hepatic [NAD+]/[NADH] ratio. The chemical shift of Pi indicated that ethanol administration elicited a decrease in pH of 0.1 unit in liver in vivo. Hepatic free [Mg2+] was increased 21% in fasted animals, but was unaffected by ethanol administration.     

Thermodynamics of oxidative phosphorylation in bovine heart submitochondrial particles.

The rates of both forward and reverse electron transfer in phosphorylating submitochondrial particles from bovine heart can be controlled by the thermodynamic phosphorylation potential (deltaGp) of the adenine nucleotide system. deltaGp is the Gibbs free energy of ATP synthesis and is defined by the relationship deltaGp = -deltaG'o + RTln([ATP]/[ADP][Pi]) where deltaG'o is the standard free energy of ATP hydrolysis. Studies of the effects of deltaGp on NADH respiration and the reduction of NAD+ by succinate show that increasing values of deltaGp cause an inhibition of forward electron transfer and a stimulation of reverse electron transfer. Between deltaGp values of 7.6 and 13.0 kcal/mol the rate of NADH respiration decreased 3-fold and the rate of NAD+ reduction by succinate increased 3-fold. Indirect phosphorylation potential titration experiments as well as direct chemical measurements indicate that steady state levels of ATP, ADP, and Pi are established during NADH respiration which correspond to a deltaGp equal to 10.7 to 11.4 kcal/mol.     

The phosphorylation potential generated by respiring bovine heart submitochondrial particles.

A phosphorylation potential deltaGp, where deltaGp = deltaGo' + RT2.303 log ([ATP]/([ADP][Pi])), of approx. 44.3 kJ.mol-1 (10.6 kcal.mol-1) was generated by submitochondrial particles that were oxidizing either NADH or succinate. Addition of adenylyl imidodiphosphate, which should suppress adenosine triphosphatase activity of any uncoupled particles, did not raise the phosphorylation potential. Raising the Pi concentration slightly increased the magnitude of the value for [ATP]/[ADP], but this did not fully compensate for the increased Pi concentration, so that the phosphorylation potential decreased slightly as the Pi concentration was raised. The phosphorylation potential developed by submitochondrial particles is lower than that generated by phosphorylating membrane vesicles from some bacteria, and is also less than that developed externally by mitochondria, but is strikingly close to the phosphorylation potential that is generated internally by mitochondria.     

Platelet-derived-growth-factor-stimulated heterogeneous polyphosphoinositide metabolism and phosphate uptake in C3H fibroblasts.

1. Gated 31P-n.m.r. spectra were obtained from the ankle flexor muscles of the rat at various times after 3 s isometric tetanic contraction. This allowed the time course of changes in phosphocreatine (PCr), Pi and free ADP concentrations and intracellular pH to be monitored in skeletal muscle in vivo with 1 s time resolution. 2. ATP concentration did not change significantly, either during the recovery from a 3 s tetanus or during the overall protocol. 3. The calculated rate of recovery of ADP towards pre-stimulation levels was very rapid (t1/2 less than 5 s). The rate of Pi disappearance (t1/2 = 14 s) was more rapid than the rate of PCr synthesis (t1/2 = 24 s), resulting in a significant transient decrease in n.m.r.-visible PCr + Pi between 25 and 45 s after tetanic contraction. 4. The rates of PCr, Pi and ADP recovery are higher than those previously reported for recovery from steady-state exercise in humans or twitch isometric contraction in animals.     

High energy phosphate concentrations and AMPK phosphorylation in skeletal muscle from mice with inherited differences in hypoxic exercise tolerance

The effect of chronic hypobaric hypoxia (1/2 atmospheric pressure) on high energy phosphate (HEP) compounds was investigated in slow (soleus; SOL) and fast twitch (extensor digitorum longus; EDL) muscle from 3 strains of mice with large differences in hypoxic exercise tolerance (HET). Phosphocreatine concentration ([PCr]) decreased 16–29% following hypoxia in EDL and SOL in all strains, while [ADP] and [AMP] increased. In the EDL, HET was negatively correlated with the PCr/ATP ratio and positively correlated with the ATP/P_i ratio. The free energy of ATP hydrolysis (ΔG_obs) remained constant despite the substantial changes that occurred in HEP profiles. The alteration of HEP set points and preservation of ΔG_obs are consistent with the notion that (1) maximal rates of steady-state ATP turnover are reduced under hypoxia, and (2) HEP perturbations during rest to work transitions are reduced in skeletal muscle from hypoxia acclimated animals. We therefore expected a lower phosphorylation ratio of AMP-activated protein kinase (AMPK-P/AMPK) during stimulation in hypoxic acclimated animals. However, neither the resting nor stimulated AMPK-P/AMPK was influenced by hypoxia, although there were significant differences among strains.     

Effects of ovariectomy on energy metabolism in exercising rat muscle studied by 31P-NMR

The effects of ovariectomy on metabolism of high-energy phosphate compounds during and after exercise were studied in hindleg muscles of 14 rats. Sciatic nerve stimulation was used to establish different work loads, and the changes in inorganic phosphate-to-phosphocreatine ratios (Pi/PCr) were recorded by 31P nuclear magnetic resonance (NMR) in vivo. Four weeks after ovariectomy, there was evidence of significantly higher Pi/PCr during work at stimulation rates greater than 0.5 Hz. The slope for the stimulation rate-to-Pi/PCr relationship decreased from 1.98 +/- 0.15 to 1.36 +/- 0.2 Hz/Pi/PCr after ovariectomy. The normalized tension output of these muscles, tested separately using identical stimulation protocols, was not changed with ovariectomy. Thus the relationship between work (tension-time integral) and bioenergetic cost (Pi/PCr) suggested reduced maximal enzyme activity (Vmax) by 9-17% as a result of lack of ovarian sex hormones, but no change in Michaelis-Menten constant (Km) was found. Postexercise recovery was also significantly slower (3.27 +/- 0.54 PCr/Pi units per minute compared with 4.04 +/- 1.08 in controls). It is suggested that reduced levels of ovarian sex hormones decrease oxidative phosphorylation. Cytochrome oxidase activity was reduced in these muscles by 40%, but other mitochondrial enzyme systems may be affected as well. The possible significance of these data is the implication of a reduced capacity for menopausal women or amenorrheic female athletes to perform prolonged intensive exercise.     

The phosphorus/oxygen ratio of mitochondrial oxidative phosphorylation.

The transport of ATP out of mitochondria and uptake of ADP and Pi into the matrix are coupled to the uptake of one proton (Klingenberg, M., and Rottenberg, H. (1977) Eur. J. Biochem. 73, 125--130). According to the chemiosmotic hypothesis of oxidative phosphorylation this coupling of nucleotide and Pi transport to proton transport implies that the P/O ratio for the synthesis and transport of ATP to the external medium is less than the P/O ratio for the synthesis of ATP inside mitochondria. A survey of previous determinations of the P/O ratio of intact mitochondria showed little convincing evidence in support of the currently accepted values of 3 with NADH-linked substrates and 2 with succinate. We have measured P/O ratios in rat liver mitochondria by the ADP pulse method and by 32 Pi esterification, measuring oxygen uptake with an oxygen electrode, and find values close to 2 with beta-hydroxybutyrate as substrate and 1.3 with succinate as substrate in the presence of rotenone to inhibit NADH oxidation. These values were largely independent of pH, temperature, Mg2+ ion concentration, Pi concentration, ADP pulse size, or amount of mitochondria used. We suggest that these are the true values of the P/O ratio for ATP synthesis and transport by mitochondria, and that previously reported higher values resulted from errors in the determination of oxygen uptake and the use of substrates which lead to ATP synthesis by succinate thiokinase.     

Nitric oxide regulation of myocardial O2 consumption and HEP metabolism

NO and O(2) compete at cytochrome-c oxidase, thus potentially allowing NO to modulate mitochondrial respiration. We previously observed a decrease of myocardial phosphocreatine (PCr)/ATP during very high cardiac work states, corresponding to an increase in cytosolic free ADP. This study tested the hypothesis that NO inhibition of respiration contributes to this increase of ADP. Infusion of dobutamine + dopamine (DbDp, each 20 microg.kg(-1).min(-1) iv) to more than double myocardial oxygen consumption (MVo(2)) in open-chest dogs caused a decrease of myocardial PCr/ATP measured with (31)P NMR from 2.04 +/- 0.09 to 1.85 +/- 0.08 (P < 0.05). Inhibition of NO synthesis with N(omega)-nitro-L-arginine (L-NNA), while catecholamine infusion continued, caused PCr/ATP to increase to the control value. In a second group of animals, L-NNA administered before catecholamine stimulation (reverse intervention of the first group) increased PCr/ATP during basal conditions. In these animals L-NNA did not prevent a decrease of PCr/ATP at the high cardiac work state but, relative to MVo(2), PCr/ATP was significantly higher after L-NNA. In a third group of animals, pharmacological coronary vasodilation with carbochromen was used to prevent changes in coronary flow that might alter endothelial NO production. In these animals L-NNA again restored depressed myocardial PCr/ATP during catecholamine infusion. The finding that inhibition of NO production increased PCr/ATP suggests that during very high work states NO inhibition of mitochondrial respiration requires ADP to increase to drive oxidative phosphorylation.     

Regulation of rat heart cytosol 5''-nucleotidase by adenylate energy charge.

A 5'-nucleotidase (EC 3.1.3.5) was highly purified from the soluble fraction of rat heart. The preparation appeared homogeneous by the criterion of polyacrylamide-gel electrophoresis. The enzyme was activated by ATP and ADP, and inhibited by Pi. When AMP was used as substrate, the velocity/substrate-concentration plot was sigmoidal. ATP or ADP changed the plot to hyperbolic and decreased S0.5. Pi increased both the sigmoidicity of the plot and S0.5. When IMP was used as substrate, the velocity/substrate plot was hyperbolic. ATP or ADP decreased Km and increased V. Pi changed the plot to sigmoidal and increased S0.5. Within the range of adenylate energy charge observed in surviving mammalian cells (0.7-0.9), the rate of AMP-hydrolysing activity catalysed by the 5'-nucleotidase increased sharply with decreasing energy charge. The highest activity was observed at an energy-charge value of about 0.6. The response was also observed in the presence of Pi. No change in IMP-hydrolysing activity was observed in the physiological range of adenylate energy charge, but in the presence of Pi the activity gradually increased with increasing energy charge. These results suggest the possibility that this enzyme participates in production of adenosine, a vasodilator, during hypoxia and in removal of IMP, which accumulates during the hypoxia, in the heart.     

Physiological heart activation by adrenaline involves parallel activation of ATP usage and supply

During low-to-high work transition in adult mammalian heart in vivo the concentrations of free ADP, ATP, PCr (phosphocreatine), P(i) and NADH are essentially constant, in striking contrast with skeletal muscle. The direct activation by calcium ions of ATP usage and feedback activation of ATP production by ADP (and P(i)) alone cannot explain this perfect homoeostasis. A comparison of the response to adrenaline (increase in rate-pressure product and [PCr]) of the intact beating perfused rat heart with the elasticities of the PCr producer and consumer to PCr concentration demonstrated that both the ATP/PCr-producing block and ATP/PCr-consuming block are directly activated to a similar extent during physiological heart activation. Our finding constitutes a direct evidence for the parallel-activation mechanism of the regulation of oxidative phosphorylation in heart postulated previously in a theoretical way.