ADP-dependent palmitoykarnitine sensitivity of mitochondria isolated from the perfused rabbit heart

1969 ◽  
Vol 47 (6) ◽  
pp. 611-618 ◽  
Author(s):  
K. J. Kako
Keyword(s):  
Cytochrome C ◽  
Respiratory Rate ◽  
Respiratory Control ◽  
Rabbit Heart ◽  
Nadh Oxidation ◽  
Perfused Heart ◽  
Low Concentrations ◽  
Gradual Loss ◽  
Mitochondrial Suspension ◽  
Perfused Hearts

The function of mitochondria as influenced by the depletion of oxidizable substances in the myocardium was examined during perfusion of the isolated rabbit heart. Mitochondria were isolated using proteinase and KCl–albumin medium, and their respiration was measured polarographically at 25 °C.(i) Mitochondria of nonperfused hearts (control) responded to repeated additions of ADP with repeated and reproducible "state 4 to state 3 transitions" in the presence of a variety of oxidizable substrates (glutamate, oxoglutarate, acetate, pyruvate, β-hydroxybutyrate, succinate, and palmitoyicamitine).(ii) The heart of a fed animal was perfused almost to exhaustion in a substrate-free medium and mitochondria were isolated and tested. The following findings were obtained (iii–viii).(iii) The respiratory control was gradually lost in a response to repeated additions of ADP, when palmitoylcamitine was used as the substrate. A larger quantity of mitochondria in a given assay partially counteracted this gradual loss of the respiratory control.(iv) The respiratory control and P/O ratios of mitochondria obtained from these hearts were slightly depressed as compared to those of the control, when measured using glutamate or oxoglutarate. Malate addition was required for pyruvate oxidation by mitochondria from perfused hearts.(v) The state 3 respiratory rate of the mitochondria obtained from an exhausted heart was about one-quarter that of the control mitochondria regardless of substrates used. The values were independent of the amount of mitochondria present in the assay system.(vi) With paimitoyicamitine as the substrate, the respiratory control ratio calculated from the first state 3 to state 4 transition was influenced by the amount of mitochondria present. The ratio was depressed at low concentrations of mitochondria.(vii) The rate of exogenous NADH oxidation in the absence of cytochrome c and the optical density of the mitochondrial suspension at 520 mμ, were similar with mitochondria prepared from nonperfused and perfused hearts. An addition of cytochrome c accelerated the rate of NADH oxidation by the latter more than the control.(viii) Unstimulated ATPase activity was high in mitochondria of the perfused heart; the enzyme was stimulated by DNP to a lesser degree and by a smaller quantity of paimitoyicarnitine than the control.(ix) The mitochondria prepared from the perfused heart of a fasted animal and from the fed rabbit's heart perfused in the presence of glucose, albumin, or 30 mM KCl (cardiac arrest) showed a normal respiratory function.(x) The mitochondria of the heart in anoxic arrest showed uncoupling of oxidative phosphorylation and a low respiratory rate regardless of the hydrogen donors used.(xi) It is concluded that a prolonged lack of hydrogen sources in perfused exhausted rabbit hearts results in the mitochondrial dysfunction.

Crystalloid and perfluorochemical perfusates in an isolated working rabbit heart preparation

1985 ◽  
Vol 249 (2) ◽  
pp. H285-H292 ◽  
Author(s):  
J. M. Chemnitius ◽  
W. Burger ◽  
R. J. Bing
Keyword(s):  
Cardiac Output ◽  
Coronary Flow ◽  
Systolic Pressure ◽  
Pressure Rise ◽  
Rabbit Heart ◽  
Left Ventricular ◽  
Heart Weight ◽  
Perfused Heart ◽  
Heart Preparation ◽  
Perfused Hearts

Krebs-Henseleit buffer (KH) and a perfluorochemical (FC-43) were compared as perfusates in an isolated working rabbit heart preparation. Both perfusates were oxygenated in an identical manner using an infant bubble oxygenator. After 60 min of perfusion, no difference could be detected in the ratio of wet to dry heart weight between KH- and FC-43-perfused hearts (KH, 6.25 +/- 0.3; FC-43, 5.99 +/- 0.20). Left ventricular systolic pressure, maximal rate of left ventricular pressure rise, mean aortic systolic pressure, cardiac output, aortic flow, left ventricular power, and myocardial O2 consumption (MVO2) were significantly higher in FC-43-perfused hearts throughout the time of perfusion. However, there were no differences in resistance to cardiac output and heart rate. In KH- and FC-43-perfused hearts, MVO2 and left ventricular power were closely correlated (KH, r = 0.793; FC-43, r = 0.831). Significantly higher coronary flow of KH-perfused hearts could be attributed to the lower viscosity of KH (1.05 Pa . s) compared with FC-43 (1.91 Pa . s). Increased O2 extraction during KH perfusion could not compensate for low O2-carrying capacity of KH buffer (345 compared with 705 nmol O2 X ml-1 in FC-43 emulsion). A postischemic increase of coronary flow was observed only in FC-43-perfused hearts (28%). These results demonstrate a different response of perfused heart preparations to FC-43 and KH buffer.

Dibucaine interacts differently with membrane and protein in cytochrome c oxidase systems

1993 ◽  
Vol 71 (1-2) ◽  
pp. 14-21
Author(s):  
Jia He ◽  
Peter Nicholls
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Proton Translocation ◽  
Tight Binding ◽  
Respiratory Control ◽  
Soret Band ◽  
Red Shift ◽  
Ph Gradients ◽  
Low Concentrations ◽  
Low Ionic Strength

Dibucaine acts as a weak protonophore in cytochrome c oxidase proteoliposomes. At low concentrations in the presence of permeant anions, it stimulates turnover and collapses enzyme-generated pH gradients. At higher concentrations, dibucaine inhibits activity of cytochrome c oxidase in proteoliposomes and the isolated enzyme. It also induces a red shift in the resting spectrum, indicating a change at the binuclear centre. This spectroscopic effect is kinetically biphasic. Dibucaine inhibits steady-state oxidase activity, but not the rate of the red shift in the cytochrome a3 Soret band during turnover. It reacts faster with the partially reduced state than with resting enzyme. The inhibition is kinetically biphasic with a noncompetitive Ki ≈ 0.5 mM. Excess dibucaine effects a maximal turnover decline of 80%. At low ionic strength only the total Vmax is affected; tight binding of cytochrome c and turnover at the "tight" site are unaffected. Dibucaine may bind to an anionic site in a hydrophobic pocket, modifying electron transfer from cytochrome a and CuA to cytochrome a3 - CuB and the oxidized spectrum of the latter centre. Stimulation of turnover in cytochrome c oxidase in proteoliposomes is due to a separate membrane-dependent proton translocation catalysed by dibucaine in the presence of permeant anions.Key words: dibucaine, cytochrome c oxidase, proteoliposomes, respiratory control, inhibition.

Homeostatic regulation of cellular energy metabolism: experimental characterization in vivo and fit to a model

1978 ◽  
Vol 234 (3) ◽  
pp. C82-C89 ◽  
Author(s):  
M. Erecinska ◽  
D. F. Wilson ◽  
K. Nishiki
Keyword(s):  
Energy Metabolism ◽  
Cytochrome C ◽  
Respiratory Rate ◽  
Energy Demand ◽  
Respiratory Control ◽  
Tetrahymena Pyriformis ◽  
Homeostatic Regulation ◽  
Cellular Energy ◽  
Cellular Energy Metabolism

Measurements in isolated liver cells, cultured kidney cells, protozoa (Tetrahymena pyriformis), and yeast (Candida utilis) indicate that homeostatic regulation of cellular energy metabolism is of common origin. In every case near equilibrium is maintained between the transfer of reducing equivalents from the intramitochondrial NAD couple to cytochrome c and the phosphorylation of cytosolic ADP to ATP. Under conditions of constant energy demand, changes in the intracellular phosphate concentration cause an adjustment in the [ATP]/[ADP] to maintain a constant [ATP]/[ADP][Pi] and constant respiratory rate. The regulation of mitochondrial respiration occurs as part of the reactions by which reduced cytochrome c is oxidized by molecular oxygen. At similar values for the [ATP]/[ADP][Pi] the respiratory rate increases with increasing reduction of cytochrome c. A model for mitochondrial respiratory control is found to give a good fit to the data in all of the different types of cells tested.

Electron Transport and Coupled Energy Generation in Pseudomonas saccharophila

1971 ◽  
Vol 49 (11) ◽  
pp. 1175-1182 ◽  
Author(s):  
M. Ishaque ◽  
A. Donawa ◽  
M. I. H. Aleem
Keyword(s):  
Oxygen Consumption ◽  
Electron Transport ◽  
Cytochrome C ◽  
Atp Synthesis ◽  
Succinate Oxidation ◽  
Atp Formation ◽  
Low Concentrations ◽  
Oxidase Enzyme ◽  
Succinate Oxidase ◽  
Pseudomonas Saccharophila

The respiratory chain system of heterotrophically grown Pseudomonas saccharophila contained cytochromes of the b, c, a, and o types and also the NADH and succinate oxidase enzyme systems. Cell-free extracts catalyzed phosphorylation coupled to the oxidation of NADH, succinate, and ascorbate (plus cytochrome c). The P/O ratios were in the range of 1.00 for generated NADH, 0.29 for added NADH, 0.50 for succinate, and 0.25 for ascorbate (plus cytochrome c).The oxidative phosphorylation was uncoupled by 2,4-dinitrophenol, 2,6-dibromophenol, pentachlorophenol, m-chlorocarbonyl cyanide phenylhydrazone, and dicumarol without any inhibition of oxygen consumption. Phosphorylation coupled to NADH oxidation was completely inhibited by the flavoprotein inhibitors such as rotenone, amytal, and atabrine; these inhibitors had no effect, however, on the ATP synthesis associated with succinate oxidation. Antimycin A or 2-n-nonyl-4-hydroxyquinoline-N-oxide as well as cyanide or azide at low concentrations completely inhibited the phosphate esterification coupled to the oxidation of NADH or succinate, but had little or no effect on the oxygen consumption. Relatively higher concentrations of oligomycin were required for a complete inhibition of the electron-transport-linked ATP formation.

Recent 31 P n.m.r. studies of myocardium

1980 ◽  
Vol 289 (1037) ◽  
pp. 437-439 ◽  
Keyword(s):  
Mechanical Performance ◽  
Normal Values ◽  
Chemical Exchange ◽  
Exchange Reactions ◽  
Perfused Heart ◽  
Time Intervals ◽  
Non Invasive ◽  
Biochemical State ◽  
Perfused Hearts ◽  
Kinetics Of

Earlier work from this laboratory has concerned the possible use of phosphorus n.m.r. as a method to monitor, in a non-invasive manner, the biochemical state of the perfused heart as a function of its mechanical performance. We showed that a simulated coronary infarction could be detected by 31 P n.m.r. (Hollis et al 1978 a and that hypothermia and KC1 arrest could preserve the pH and the ATP levels at more nearly normal values than in a non-arrested heart during long periods (40 min) of ischaemia (Hollis et al . 1978 b ).More recently it was shown that multiple doses of KC1, given at intervals, were more effective in this respect than was a single dose (Flaherty et al . 1979). These studies essentially followed the kinetics of transitions of the heart between two or more distinct physiological states (i.e. normoxic and ischaemic, with or without KC1 arrest) by observation of the 31 P n.m.r. spectra at various time intervals over periods of up to 1 h. As described in detail and demonstrated in Dr Truman Brown’s contribution to these discussions, the rates of chemical exchange reactions occurring in a steady state can be measured by the techniques of saturation transfer in various biological systems, including perfused hearts.

Active transport of potassium by heart mitochondria

1960 ◽  
Vol 198 (4) ◽  
pp. 847-854 ◽  
Author(s):  
Frank Ulrich
Keyword(s):  
Methylene Blue ◽  
Active Transport ◽  
Creatine Phosphate ◽  
Rabbit Heart ◽  
Potassium Transport ◽  
Heart Mitochondria ◽  
Metabolic Inhibitors ◽  
Mitochondrial Suspension ◽  
High Concentrations ◽  
Potassium Gradient

Rabbit heart mitochondria contained potassium which could not be removed with four washings of isotonic sucrose or sodium chloride at 0–4°C. Aging, increasing concentrations of potassium, and a number of metabolic poisons either partially or completely inhibited the active transport of potassium into heart mitochondria when these particles were incubated for 15 minutes in air at 37.4° in a medium to which alpha-ketoglutarate and AMP or ATP had been added. Compounds uncoupling oxidative phosphorylation—such as arsenite, 2,4-dinitrophenol, l-thyroxine, calcium chloride, dicumarol, pentachlorophenol and methylene blue—inhibited potassium transport but usually only at relatively high concentrations (10–3 m). With the exception of p-chloromercuribenzoate, neither aging nor metabolic inhibitors prevented the extrusion of water by the mitochondria in the presence of alpha-ketoglutarate and AMP or ATP. Although addition of either ATP or substrate alone to the mitochondrial suspension resulted in a significant increase in the potassium gradient, the latter was much greater when both substrate and ATP (or ADP or AMP) were added together. ADP or AMP alone caused a very slight but probably not significant increase in the potassium gradient and creatine phosphate had no effect.

Effect of Prolonged Consumption of Pemmican Survival Ration on Some Aspects of the Intermediary Metabolism of Rat Liver Tissue

1958 ◽  
Vol 193 (2) ◽  
pp. 449-454 ◽  
Author(s):  
John P. Hannon ◽  
David A. Vaughan
Keyword(s):  
Cytochrome C ◽  
Respiratory Rate ◽  
Rat Liver ◽  
Liver Tissue ◽  
In Vitro Metabolism ◽  
Intermediary Metabolism ◽  
Normal Value ◽  
Endogenous Metabolism ◽  
Prolonged Feeding

The effect of prolonged feeding (3–5 months) of pemmican on some aspects of the in vitro metabolism of liver tissue was investigated. The endogenous metabolism of liver slices and homogenates was significantly increased by pemmican, probably due to an increase in the amount of readily metabolizable substrate. Utilizing mitochondrial preparations, it was found that with all substrates studied, except glutamate, α-ketoglutarate and succinate, the respiratory rate was not affected by the previous diet in the absence of added cytochrome c and diphosphopyridine nucleotide. The three substrates mentioned were oxidized at significantly lower rates in the pemmican group. Upon the addition of cytochrome c and diphosphopyridine nucleotide the qo2 of glutamate, α-ketoglutarate, and succinate was returned to the normal value.

Fura 2 determination of [Ca2+]i in isolated perfused heart using R wave-gated electromechanical shutters

1994 ◽  
Vol 76 (3) ◽  
pp. 1394-1399 ◽  
Author(s):  
D. P. Chen ◽  
E. Jimenez ◽  
K. Ataka ◽  
S. Levitsky ◽  
H. Feinberg
Keyword(s):  
Cardiac Cycle ◽  
Low Cost ◽  
Rabbit Heart ◽  
Isolated Perfused Heart ◽  
Signal Averaging ◽  
Perfused Heart ◽  
Optical Components ◽  
Fura 2 ◽  
Heart Preparation

We describe a novel and relatively inexpensive spectrofluorescence system that supplies rapidly alternating wavelengths to either a standard cuvette or an isolated perfused heart. Its use is illustrated by determining changes in cytosolic intracellular Ca2+ concentration ([Ca2+]i) by using the Ca(2+)-sensitive fluorescent dye fura 2 in a rabbit heart preparation. The system uses two precision electromechanical shutters (capable of gating with respect to the electrocardiographic R wave for signal averaging) allowing alternate fura 2 excitation wavelengths (340 and 380 nm) without moving optical components and uses a fiber optic for conducting excitation and collecting epifluorescence. Sample recordings tracing the [Ca2+]i transient in an entire cardiac cycle and in capturing specific isolated regions (diastole and systole) of the cycle are presented. Limitations of this low-cost but easily implemented system are discussed.

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