Subcellular origin of the surface fluorescence of reduced nicotinamide nucleotides in the isolated perfused rat heart

Em, Nuutinen

doi:10.1007/bf01935806

Cited by 111 publications

(92 citation statements)

References 29 publications

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“…Excess acetate, another substrate that reduces, albeit slightly, mitochondrial pyridine nucleotides [21], did stimulate mitochondrial respiration in normoxic hearts (i 23% [62]) but produced a decrease, not an increase in the cytosolic phosphorylation potential in normoxic hearts (relative to pyruvate perfusions, unpublished data); acetate also failed to stimulate mitochondrial respiration and reenergize the myocardium in postischemic hearts (Table 2). Furthermore, pyruvate when applied in a concentration of 5 mM raised the cytosolic phosphorylation potential without a simultaneous increase in O2 uptake in normoxic hearts (Table 1, conditions Al, A2 [9, 251); oxygen limitation was not a factor under these conditions, since the phosphorylation potentials increased and because adrenergic stimulations raised O2 uptake by 70% to 116% (Table 1, conditions A2, B [9,62]).…”

Section: Suhcellular Redox States and Cytosolic Phosphorylation Potenmentioning

confidence: 86%

“…Furthermore, pyruvate when applied in a concentration of 5 mM raised the cytosolic phosphorylation potential without a simultaneous increase in O2 uptake in normoxic hearts (Table 1, conditions Al, A2 [9, 251); oxygen limitation was not a factor under these conditions, since the phosphorylation potentials increased and because adrenergic stimulations raised O2 uptake by 70% to 116% (Table 1, conditions A2, B [9,62]). Similarly, 2.9 mM pyruvate strongly enhanced pyridine nucleotide fluorescence in Langendorffperfused rat hearts [23] but coronary flow remained virtually constant and venousp02 only slightly, if at all, decreased [21]; this illustrates that O2 uptake did not appreciably increase despite the marked shift of the mitochondrial NAD(P)H level towards reduction. Also in Langendorff-perfused rat hearts under maximum hernodynamic and pharmacological stresses, 10 mM pyruvate plus glucose relative to glucose as sole substrate did not stimulate oxygen usage, yet 31P-NMR spectra indicated an increase in the cytosolic phosphorylation potential [61].…”

Section: Suhcellular Redox States and Cytosolic Phosphorylation Potenmentioning

confidence: 99%

“…Ischemia and reperfusion damages to the heart were judged from alterations in myocardial energization, overall adenylate degradation, and left ventricular residual function. In separate reperfusion experiments pyruvate was substituted by acetate; according to NAD(P)H surface fluorescence studies [20,21] both substrates are mitochondria1 reductants, but only pyruvate enhances pyruvate dehydrogenase flux and simultaneously shifts the cytosolic [NADH]/[NAD+] ratio towards oxidation.…”

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confidence: 99%

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Pyruvate‐enhanced phosphorylation potential and inotropism in normoxic and postischemic isolated working heart

Bünger

Mallet

Hartman

1989

European Journal of Biochemistry

216

155

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Bioenergetic and hemodynamic consequences of cellular redox manipulations by 0.2 -20 mM pyruvate were compared with those due to adrenergic stress (0.7 -1.1 pM norepinephrine) using isolated working guinea-pig hearts under the conditions of normoxia, low-flow ischemia, and reperfusion. 5 mM glucose (+ 5 U/l insulin) + 5 mM lactate were the basal energy-yielding substrates. To stabilize left ventricular enddiastolic pressure, ventricular filling pressure was held at 12 cmHzO under all conditions; this preload control minimized FrankStarling effects on ventricular inotropism. Global low-flow ischemia was induced by reducing aortic pressure to levels (20 -10 cmH20) below the coronary autoregulatory reserve. Reactants of the creatine kinase, including H + and other key metabolites, were measured by enzymatic, HPLC, and polarographic techniques.In normoxic hearts, norepinephrine stimulations of inotropism, heart rate x pressure product, and oxygen consumption (MV02) were associated with a fall in the cytosolic phosphorylation potentialas judged by the creatine kinase equilibrium. In contrast, infusion of excess pyruvate ( 5 mM) markedly increased . [Pi]) ratio at constant (normoxia) or increased (reperfusion) MVO2. In postischemic hearts the effect of pyruvate required the presence of glucose. It is proposed that pyruvate energization may improve ion pumping by the sarcoplasmic reticulum and hence Ca2+-handling by the latter which, in turn, might increase the contractile state; decreased intracellular [Pi] due to improved phosphate fixation may also be contributory. In addition, augmented pyruvate dehydrogenase flux during reperfusion seemed to expedite cellular reenergization and functional recovery in the postischemic hearts.Correspondence to R. Biinger, Department of Physiology, F. E. Hebert School of Medicine, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814-4799, USA Abbreviations. Kcpk, Kldhr Kgapdh, Kpgkr equilibrium constant of creatine kinase, lactate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, and 3-phosphoglycerate kinase, respectively; pH,, intracellular pH; MV02, myocardial oxygen uptake; dy, mean hydrostatic pressure gradient across left ventricle; HR . dp, product of spontaneous heart rate and pressure; C{CrP + Cr}, total myocardial content of creatine phosphate plus creatine; Gro-3-P, sn-glycerol 3-phosphate; {ATP}, total myocardial ATP content and [ATP], mean intracellular ATP concentration (similarly for other metabolites).

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Section: Suhcellular Redox States and Cytosolic Phosphorylation Potenmentioning

confidence: 86%

Section: Suhcellular Redox States and Cytosolic Phosphorylation Potenmentioning

confidence: 99%

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confidence: 99%

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Pyruvate‐enhanced phosphorylation potential and inotropism in normoxic and postischemic isolated working heart

Bünger

Mallet

Hartman

1989

European Journal of Biochemistry

216

155

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“…In the heart perfused with glucose as substrate, either an increase [23] or no change [24] in the NAD(P)H surface fluorescence of the heart has been observed upon the transition from low to high work. Since this measurement reflects the mitochondrial redox state of the pyridine nucleotides [25], the lack of an oxidation with an increase in work suggests that reactions leading to the formation of NADH may contribute to the control of respiration in this tissue. An increased average level of calcium in the cytosol has been shown to cause an increased matrix calcium content both in a model system [26], consisting of perifused cardiac mitochondria, and in isolated paced myocytes [27].…”

Section: Discussionmentioning

confidence: 99%

Calcium and 2‐oxoglutarate‐mediated control of aspartate formation by rat heart mitochondria

Scaduto

1994

European Journal of Biochemistry

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Studies of the influence of calcium on the metabolism of cardiac mitochondria have indicated that calcium activates key enzymes involved in the citric acid cycle. Calcium-mediated activation of one of these enzymes, 2-oxoglutarate dehydrogenase, has been shown to cause a marked decrease in the steady-state concentration of 2-oxoglutarate in both heart and liver mitochondria. In liver, 2-oxoglutarate is a potent inhibitor of oxalacetate transamination to aspartate and activation of this enzyme by calcium-mobilizing hormones leads to a stimulation of aspartate formation and gluconeogenesis. Since mitochondrial aspartate formation is a key step in the malate/aspartate shuttle, we investigated the control of aspartate formation by cardiac mitochondria. In mitochondria incubated with glutamate and malate, activation of 2-oxoglutarate dehydrogenase by calcium led to an inhibition of aspartate formation. However, calcium caused a stimulation of aspartate production when incubations were supplemented with pyruvate as an additional substrate. Estimates of the mitochondrial redox potential (NADWNAD') indicated that both calcium and pymvate increased the redox potential. The observed influence of calcium on aspartate formation was found to be due to a balance between its inhibitory effect, caused by an increased redox potential, and its stimulatory effect, caused by a decreased 2-oxoglutarate concentration. Under conditions in which the redox component was held constant, a kinetic analysis indicated that the apparent K, for 2-oxoglutarate inhibition of aspartate formation is 0.2 mM. The data suggest that activation of cardiac 2-oxoglutarate dehydrogenase by calcium could lead to stimulation of the mitochondrial oxidation of cytosolic NADH via the malate/aspartate cycle.It is generally accepted that the rate of energy utilization within the cell by various ATPases is well matched to the rate of ATP production such that the cellular concentration of ATP does not vary widely despite physiological fluctuations in energy expenditure. However, the manner by which the cell achieves this tight coupling remains a topic of active study. In now classical studies of isolated mitochondria, the control of mitochondrial respiration was viewed as being regulated solely through either ADP availability [l] or the ATP/ ADP ratio [2, 31. More recently, however, these conclusions have been brought into question since these variables have been found not to change appreciably over a wide range of oxygen consumption in studies of the intact heart using more noninvasive techniques [4, 51. Reviews of this topic have appeared [6 -81.It is now well established that mitochondria contain three dehydrogenases that are sensitive to changes in matrix calcium [9]. These enzymes are pyruvate dehydrogenase, isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase. All of these enzymes are displaced far from thermodynamic equilibrium and hence modulation of their activity by calcium can have an important influence on citric acid cycle flux [lo]. In previous...

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“…There is little direct information on the free cytosolic concentrations of either NADH or NADPH. Fluorescence measurements yield total levels of both reduced dinucleotides and are dominated by the mitochondrial pool (Nuutinen, 1984;Ince et al, 1992). Estimates of erythrocyte levels reveal large amounts of bound material (Canepa et al, 1991).…”

Section: Table I Nad(p)h Increases the Voltage Dependence Of Vdacmentioning

confidence: 99%

The Role of Pyridine Dinucleotides in Regulating the Permeability of the Mitochondrial Outer Membrane

Lee¹,

Colombini

1996

Journal of Biological Chemistry

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Both NADH and NADPH reduce the permeability of the mitochondrial outer membrane to ADP. This is specific for the outer membrane and uncorrelated with the respiratory control ratio. This could result in a 7-fold difference between the concentration of ADP in the intermembrane space and that in the external environment (at 5 M ADP). In both cases the permeability declines by a factor of 5, but NADH is more potent: K D ‫؍‬ 86 M for NADH versus 580 M for NADPH. The lower apparent affinity for NADPH is partly explained by Mg 2؉ -NADPH being the active species, and under our conditions only 30% of the NADPH is in this form. The corrected K D is 184 M. Free NADH has the same charge as the Mg 2؉ -NADPH complex, and thus both likely bind to the same site. The ability of NADH and NADPH to induce the closure of reconstituted VDAC channels is consistent with VDAC being the main pathway for metabolite flow across the outer membrane. Oncotic pressure, effective at inducing VDAC closure, also decreases the outer membrane permeability. Thus, in the presence of cytosolic colloidal osmotic pressure NAD(P)H may inhibit mitochondrial catabolic pathways and divert reducing equivalents to anabolic pathways.

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Subcellular origin of the surface fluorescence of reduced nicotinamide nucleotides in the isolated perfused rat heart

Cited by 111 publications

References 29 publications

Pyruvate‐enhanced phosphorylation potential and inotropism in normoxic and postischemic isolated working heart

Pyruvate‐enhanced phosphorylation potential and inotropism in normoxic and postischemic isolated working heart

Calcium and 2‐oxoglutarate‐mediated control of aspartate formation by rat heart mitochondria

The Role of Pyridine Dinucleotides in Regulating the Permeability of the Mitochondrial Outer Membrane

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