scholarly journals Ruthenium Red inhibits the activation of pyruvate dehydrogenase caused by positive inotropic agents in the perfused rat heart

1983 ◽  
Vol 214 (2) ◽  
pp. 581-585 ◽  
Author(s):  
J G McCormack ◽  
P J England
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Rat Heart ◽  
Contractile Force ◽  
Ruthenium Red ◽  
Inotropic Agents ◽  
Similar Increase ◽  
Perfused Rat Heart ◽  
Control Value ◽  
Positive Inotropic Agents ◽  
Total Enzyme

The increases in the amount of active, non-phosphorylated, pyruvate dehydrogenase caused by positive inotropic agents (from a control value of about 10%, to 40% of total enzyme) in the perfused rat heart could be completely blocked by prior perfusion with 2.5 micrograms of Ruthenium Red/ml. A similar increase caused by 5 mM-pyruvate was not blocked. This concentration of Ruthenium Red caused a 25% decrease in contractile force of hearts perfused in the absence of positive inotropic agents; however, in their presence the contractile force reached the same value in the absence or presence of Ruthenium Red. Neither control nor stimulated phosphorylase a content was affected by Ruthenium Red. Verapamil (0.1 microM) also decreased control contraction (by 40%), but did not block the activation of pyruvate dehydrogenase caused by a rise in extracellular [Ca2+]. The results support the hypothesis that positive inotropic agents activate pyruvate dehydrogenase in rat heart by increasing intramitochondrial [Ca2+].

scholarly journals The activation of pyruvate dehydrogenase in the perfused rat heart by adrenaline and other inotropic agents

1981 ◽  
Vol 194 (2) ◽  
pp. 639-643 ◽  
Author(s):  
J G McCormack ◽  
R M Denton
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Oxidative Metabolism ◽  
Rat Heart ◽  
Fold Increase ◽  
Diabetic Rats ◽  
Inotropic Agents ◽  
Perfusion Medium ◽  
Perfused Rat Heart ◽  
Phosphorylated Form

Adrenaline resulted in a reversible 4-fold increase in the amount of pyruvate dehydrogenase in its active non-phosphorylated form in the perfused rat heart within 1 min. The increase was less in extent in hearts from starved or diabetic rats or in hearts from control rats oxidizing acetate, unless pyruvate was added to the perfusion medium. Increases could also be induced by other inotropic agents, supporting the hypothesis that increases in cytoplasmic Ca2+ can be relayed into mitochondria and influence oxidative metabolism.

scholarly journals Phosphorylation of myosin light chains in perfused rat heart. Effect of adrenaline and increased cytoplasmic calcium ions

1980 ◽  
Vol 188 (3) ◽  
pp. 763-768 ◽  
Author(s):  
S A Jeacocke ◽  
P J England
Keyword(s):  
Light Chain ◽  
Calcium Ions ◽  
Rat Heart ◽  
Light Chains ◽  
Myosin Light Chains ◽  
Inotropic Agents ◽  
Cytoplasmic Calcium ◽  
Perfused Rat Heart ◽  
32P Incorporation

1. A method was developed for the isolation of essentially pure myosin light chains from perfused rat heart. The phosphorylation of the P-light chains was estimated by hydrolysis and measurement of phosphate released, by electrophoresis in 8 M-urea and by 32P incorporation in perfusion with [32P]Pi. 2. In control perfusions there was 0.5-0.6 mol of phosphate/mol of P-light chain. This was not changed by perfusion with 5 microM-adrenaline for 10-40s. Perfusion for 1 min with medium containing 7.5 mM-CaCl2, or for 30s with medium containing 118 mM-KCl, also did not change the phosphorylation of P-light chains. 3. It is concluded that phosphorylation of P-light chains is not important in mediating the action of inotropic agents in the heart.

scholarly journals Role of calcium ions in the regulation of intramitochondrial metabolism. Effects of Na+, Mg2+ and ruthenium red on the Ca2+-stimulated oxidation of oxoglutarate and on pyruvate dehydrogenase activity in intact rat heart mitochondria

1980 ◽  
Vol 190 (1) ◽  
pp. 107-117 ◽  
Author(s):  
R M Denton ◽  
J G McCormack ◽  
N J Edgell
Keyword(s):  
Dehydrogenase Activity ◽  
Pyruvate Dehydrogenase ◽  
Rat Heart ◽  
Ruthenium Red ◽  
Heart Mitochondria ◽  
Heart Cells ◽  
Maximal Effect ◽  
Rat Heart Mitochondria ◽  
20 Nm

1. In uncoupled rat heart mitochondria, the kinetic parameters for oxoglutarate oxidation were very close to those found for oxoglutarate dehydrogenase activity in extracts of the mitochondria. In particular, Ca2+ greatly diminished the Km for oxoglutarate and the k0.5 value (concentration required for half-maximal effect) for this effect of Ca2+ was close to 1 microM. 2. In coupled rat heart mitochondria incubated with ADP, increases in the extramitochondrial concentration of Ca2+ greatly stimulated oxoglutarate oxidation at low concentrations of oxoglutarate, but not at saturating concentrations of oxoglutarate. The k0.5 value for the activation by extramitochondrial Ca2+ was about 20 nM. In the presence of either Mg2+ or Na+ this value was increased to about 90 nM, and in the presence of both to about 325 nM. 3. In coupled rat heart mitochondria incubated without ADP, increases in the extramitochondrial concentration of Ca2+ resulted in increases in the proportion of pyruvate dehydrogenase in its active non-phosphorylated form. The sensitivity to Ca2+ closely matched that found to affect oxoglutarate oxidation, and Mg2+ and Na+ gave similar effects. 4. Studies of others have indicated that the distribution of Ca2+ across the inner membrane of heart mitochondria is determined by a Ca2+-transporting system which is composed of a separate uptake component (inhibited by Mg2+ and Ruthenium Red) and an efflux component (stimulated by Na+). The present studies are entirely consistent with this view. They also indicate that the intramitochondrial concentration of Ca2+ within heart cells is probably about 2–3 times that in the cytoplasm, and thus the regulation of these intramitochondrial enzymes by Ca2+ is of likely physiological significance. It is suggested that the Ca2+-transporting system in heart mitochondria may be primarily concerned with the regulation of mitochondrial Ca2+ rather than cytoplasmic Ca2+; the possible role of Ca2+ as a mediator of the effects of hormones and neurotransmitters on mammalian mitochondrial oxidative metabolism is discussed.

Computer simulation of metabolism in pyruvate-perfused rat heart. III. Pyruvate dehydrogenase

1979 ◽  
Vol 237 (3) ◽  
pp. R167-R173 ◽  
Author(s):  
M. C. Kohn ◽  
M. J. Achs ◽  
D. Garfinkel
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Rat Heart ◽  
Pyruvate Dehydrogenase Complex ◽  
Active Form ◽  
Realistic Model ◽  
Specific Protein ◽  
Active Species ◽  
Perfused Rat Heart ◽  
Synergistic Activation ◽  
The Difference

A physiologically and biochemically realistic model of the regulation of pyruvate dehydrogenase complex (PDH) was constructed for the perfused rat heart. It includes conversion between inactive (phospho) and active (dephospho) forms by a specific protein kinase (PDHK) and phosphoprotein phosphatase (PDHP). The activity of the tightly bound PDHK is influenced by synergistic activation/inhibition by acetyl CoA/CoASH and NADH/NAD. PDHK in this simulation was more sensitive to the fraction of ADP that was Mg2+-chelated than to the ATP-to-ADP ratio. Ca2+ stimulates binding of Mg2+-dependent PDHP to the complex; the bound enzyme was considered to be the active species. The fraction of PDH in the active form, rather than substrate and inhibitor levels, determines PDH activity under these conditions. This fraction depends on the present value and recent history of the difference between PDHK and PDHP activities. Both of these are active continuously and continuously control PDH.

scholarly journals Regulation of pyruvate dehydrogenase in rat heart. Mechanism of regulation of proportions of dephosphorylated and phosphorylated enzyme by oxidation of fatty acids and ketone bodies and of effects of diabetes: role of coenzyme A, acetyl-coenzyme A and reduced and oxidized nicotinamide-adenine dinucleotide

1976 ◽  
Vol 154 (2) ◽  
pp. 327-348 ◽  
Author(s):  
A L. Kerbey ◽  
P J. Randle ◽  
R H. Cooper ◽  
S Whitehouse ◽  
H T. Pask ◽  
...  
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Rat Heart ◽  
Alloxan Diabetes ◽  
Total Activity ◽  
Diabetic Rats ◽  
Pyruvate Dehydrogenase Kinase ◽  
Perfused Rat Heart ◽  
Kinase Reaction ◽  
The Matrix ◽  
Rat Heart Mitochondria

The proportion of active (dephosphorylated) pyruvate dehydrogenase in perfused rat heart was decreased by alloxan-diabetes or by perfusion with media containing acetate, n-octanoate or palmitate. The total activity of the dehydrogenase was unchanged. 2. Pyruvate (5 or 25mM) or dichloroacetate (1mM) increased the proportion of active (dephosphorylated) pyruvate dehydrogenase in perfused rat heart, presumably by inhibiting the pyruvate dehydrogenase kinase reaction. Alloxan-diabetes markedly decreased the proportion of active dehydrogenase in hearts perfused with pyruvate or dichloroacetate. 3. The total activity of pyruvate dehydrogenase in mitochondria prepared from rat heart was unchanged by diabetes. Incubation of mitochondria with 2-oxo-glutarate plus malate increased ATP and NADH concentrations and decreased the proportion of active pyruvate dehydrogenase. The decrease in active dehydrogenase was somewhat greater in mitochondria prepared from hearts of diabetic rats than in those from hearts of non-diabetic rats. Pyruvate (0.1-10 mM) or dichloroacetate (4-50 muM) increased the proportion of active dehydrogenase in isolated mitochondria presumably by inhibition of the pyruvate dehydrogenase kinase reaction. They were much less effective in mitochondria from the hearts of diabetic rats than in those of non-diabetic rats. 4. The matrix water space was increased in preparations of mitochondria from hearts of diabetic rats. Dichloroacetate was concentrated in the matrix water of mitochondria of non-diabetic rats (approx. 16-fold at 10 muM); mitochondria from hearts of diabetic rats concentrated dichloroacetate less effectively. 5. The pyruvate dehydrogenase phosphate phosphatase activity of rat hearts and of rat heart mitochondria (approx. 1-2 munit/unit of pyruvate dehydrogenase) was not affected by diabetes. 6. The rate of oxidation of [1-14C]pyruvate by rat heart mitochondria (6.85 nmol/min per mg of protein with 50 muM-pyruvate) was approx. 46% of the Vmax. value of extracted pyruvate dehydrogenase (active form). Palmitoyl-L-carnitine, which increased the ratio of [acetyl-CoA]/[CoA] 16-fold, inhibited oxidation of pyruvate by about 90% without changing the proportion of active pyruvate dehydrogenase.

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