Direct evidence for a role of intramitochondrial Ca2+ in the regulation of oxidative phosphorylation in the stimulated rat heart. Studies using31P 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.

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Mechanism of impaired energy metabolism during acidosis: role of oxidative metabolism

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1992.262.6.h1818 ◽

1992 ◽

Vol 262 (6) ◽

pp. H1818-H1822 ◽

Cited By ~ 2

Author(s):

G. Suleymanlar ◽

H. Z. Zhou ◽

M. McCormack ◽

N. Elkins ◽

R. Kucera ◽

...

Keyword(s):

Metabolic Acidosis ◽

Energy Metabolism ◽

Respiratory Control ◽

High Energy ◽

Metabolic Effects ◽

High Energy Phosphates ◽

Perfused Heart ◽

Rat Hearts ◽

Perfused Rat Hearts

Isolated perfused rat hearts were used to study the effects of metabolic acidosis on energy metabolism. Hearts perfused with different substrates (glucose, pyruvate, and succinate) were subjected to metabolic acidosis. With all substrates, there were comparable decrements in oxygen consumption (approximately 35%), cardiac function (decrease in first derivative of pressure of 65%), and similar changes in high-energy phosphates (approximately 150% increases in inorganic phosphate and 25% decreases in phosphocreatine concentrations) with metabolic acidosis. To further investigate the metabolic effects of acidosis, isolated cardiac mitochondria were exposed to different incubation media pH conditions and given simple metabolites (glutamate/malate, succinate, or pyruvate) or fatty acids (octanoate). Reduction of incubation media pH to 6.0 did not significantly affect either coupled respiration rate or the respiratory control ratio (RCR) with any substrate. These data suggest that metabolic acidosis induces decreases in energy production in the isolated perfused heart by inhibiting mitochondrial substrate utilization and not by impairing glycolysis. However, this impairment of mitochondrial function is not a direct effect of acidosis itself but appears to occur secondarily to some other effects of acidosis which are, as yet, incompletely understood.

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Insulin stimulates mitochondrial protein synthesis and respiration in isolated perfused rat heart.

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1990.259.3.e413 ◽

1990 ◽

Vol 259 (3) ◽

pp. E413

Author(s):

E E McKee ◽

B L Grier

Keyword(s):

Protein Synthesis ◽

Mitochondrial Protein ◽

Control Level ◽

Respiratory Control ◽

Mitochondrial Proteins ◽

Mitochondrial Protein Synthesis ◽

Mitochondrial Translation ◽

Rat Hearts ◽

Perfused Rat Hearts ◽

Perfused Hearts

The rates of synthesis of mitochondrial proteins by both the cytoplasmic and mitochondrial protein synthetic systems, as well as parameters of respiration, were measured and compared in mitochondria isolated from fresh, control perfused, and insulin-perfused rat hearts. The respiratory control ratio (RCR) in mitochondria from fresh hearts was 8.1 +/- 0.4 and decreased to 6.0 +/- 0.2 (P less than 0.001 vs. fresh) in mitochondria from control perfused hearts and to 6.7 +/- 0.2 (P less than 0.005 vs. fresh and P less than 0.02 vs. control perfused) for mitochondria from hearts perfused in the presence of insulin. A positive correlation between the RCR and the rate of mitochondrial translation was demonstrated in mitochondria from fresh hearts. In mitochondria isolated from control perfused hearts, the rate of protein synthesis decreased to 84 +/- 3% of the fresh rate after 30 min of perfusion and fell further to 64 +/- 3% after 3 h of perfusion. The inclusion of insulin in the perfusion buffer stimulated mitochondrial protein synthesis 1.2-fold by 1 h (P less than 0.005) and 1.34-fold by 3 h of perfusion (P less than 0.001). The addition of insulin to 1-h control perfused hearts shifted the rate of mitochondrial protein synthesis from the control level to the insulin-perfused level within 30 min of additional perfusion, whereas 1 h was required to shift the RCR values of these mitochondria from control levels to insulin-perfused levels. Thus, whereas RCR was a useful predictor of mitochondrial translation rates, it did not account for the effects of insulin on mitochondrial translation.(ABSTRACT TRUNCATED AT 250 WORDS)

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Role of dual-site phospholamban phosphorylation in the stunned heart: insights from phospholamban site-specific mutants

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00209.2003 ◽

2003 ◽

Vol 285 (3) ◽

pp. H1198-H1205 ◽

Cited By ~ 33

Author(s):

M. Said ◽

L. Vittone ◽

C. Mundiña-Weilenmann ◽

P. Ferrero ◽

E. G. Kranias ◽

...

Keyword(s):

Ischemia Reperfusion ◽

Phosphorylation Sites ◽

Adrenergic Stimulation ◽

Rat Hearts ◽

Adrenergic Blocker ◽

Perfused Rat Hearts ◽

A Site ◽

Contractile Recovery ◽

Mechanical Recovery

Phosphorylation of phospholamban (PLB) at Ser16 (protein kinase A site) and at Thr17 [Ca2+/calmodulin kinase II (CaMKII) site] increases sarcoplasmic reticulum Ca2+ uptake and myocardial contractility and relaxation. In perfused rat hearts submitted to ischemia-reperfusion, we previously showed an ischemia-induced Ser16 phosphorylation that was dependent on β-adrenergic stimulation and an ischemia and reperfusion-induced Thr17 phosphorylation that was dependent on Ca2+ influx. To elucidate the relationship between these two PLB phosphorylation sites and postischemic mechanical recovery, rat hearts were submitted to ischemia-reperfusion in the absence and presence of the CaMKII inhibitor KN-93 (1 μM) or the β-adrenergic blocker dl-propranolol (1 μM). KN-93 diminished the reperfusion-induced Thr17 phosphorylation and depressed the recovery of contraction and relaxation after ischemia. dl-Propranolol decreased the ischemia-induced Ser16 phosphorylation but failed to modify the contractile recovery. To obtain further insights into the functional role of the two PLB phosphorylation sites in postischemic mechanical recovery, transgenic mice expressing wild-type PLB (PLB-WT) or PLB mutants in which either Thr17 or Ser16 were replaced by Ala (PLB-T17A and PLB-S16A, respectively) into the PLB-null background were used. Both PLB mutants showed a lower contractile recovery than PLB-WT. However, this recovery was significantly impaired all along reperfusion in PLB-T17A, whereas it was depressed only at the beginning of reperfusion in PLB-S16A. Moreover, the recovery of relaxation was delayed in PLB-T17A, whereas it did not change in PLB-S16A, compared with PLB-WT. These findings indicate that, although both PLB phosphorylation sites are involved in the mechanical recovery after ischemia, Thr17 appears to play a major role.

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Citrate release by perfused rat hearts: a window on mitochondrial cataplerosis

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.2000.278.5.e846 ◽

2000 ◽

Vol 278 (5) ◽

pp. E846-E856 ◽

Cited By ~ 30

Author(s):

Geneviève Vincent ◽

Blandine Comte ◽

Myriame Poirier ◽

Christine Des Rosiers

Keyword(s):

Citric Acid Cycle ◽

Citrate Synthase ◽

Specific Inhibitor ◽

Release Rates ◽

Fuel Selection ◽

Rat Hearts ◽

Perfused Rat Hearts ◽

Citrate Release ◽

Labeling Pattern

Cytosolic citrate is proposed to play a crucial role in substrate fuel selection in the heart. However, little is known about factors regulating the transfer of citrate from the mitochondria, where it is synthesized, to the cytosol. Further to our observation that rat hearts perfused under normoxia release citrate whose 13C labeling pattern reflects that of mitochondrial citrate (B. Comte, G. Vincent, B. Bouchard, and C. Des Rosiers. J. Biol. Chem. 272: 26117–26124, 1997), we report here data indicating that this citrate release is a specific process reflecting the mitochondrial efflux of citrate, a process referred to as cataplerosis. Indeed, measured rates of citrate release, which vary between 2 and 21 nmol/min, are modulated by the nature and concentration of exogenous substrates feeding acetyl-CoA (fatty acid) and oxaloacetate (lactate plus pyruvate) for the mitochondrial citrate synthase reaction. Such release rates that represent at most 2% of the citric acid cycle flux are in agreement with the activity of the mitochondrial tricarboxylate transporter whose participation is also substantiated by 1) parallel variations in citrate release rates and tissue levels of citrate plus malate, the antiporter, and 2) a lowering of the citrate release rate by 1,2,3-benzenetricarboxylic acid, a specific inhibitor of the transporter. Taken together, the results from the present study indicate that citrate cataplerosis is modulated by substrate supply, in agreement with the role of cytosolic citrate in fuel partitioning, and occurs, at least in part, through the mitochondrial tricarboxylate transporter.

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Characterization of the effects of Ca2+ on the intramitochondrial Ca2+-sensitive enzymes from rat liver and within intact rat liver mitochondria

Biochemical Journal ◽

10.1042/bj2310581 ◽

1985 ◽

Vol 231 (3) ◽

pp. 581-595 ◽

Cited By ~ 96

Author(s):

J G McCormack

Keyword(s):

Rat Liver ◽

Pyruvate Dehydrogenase ◽

Liver Mitochondria ◽

Ruthenium Red ◽

Pyruvate Dehydrogenase Kinase ◽

Rat Liver Mitochondria ◽

Mitochondrial Inner Membrane ◽

Mammalian Tissues ◽

14Co2 Production

The regulatory properties of the Ca2+-sensitive intramitochondrial enzymes (pyruvate dehydrogenase phosphate phosphatase, NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase) in extracts of rat liver mitochondria appeared to be essentially similar to those described previously for other mammalian tissues. In particular, the enzymes were activated severalfold by Ca2+, with half-maximal effects at about 1 microM-Ca2+ (K0.5 value). In intact rat liver mitochondria incubated in a KCl-based medium containing 2-oxoglutarate and malate, the amount of active, non-phosphorylated, pyruvate dehydrogenase could be increased severalfold by increasing extramitochondrial [Ca2+], provided that some degree of inhibition of pyruvate dehydrogenase kinase (e.g. by pyruvate) was achieved. The rates of 14CO2 production from 2-oxo-[1-14C]glutarate at non-saturating, but not at saturating, concentrations of 2-oxoglutarate by the liver mitochondria (incubated without ADP) were similarly enhanced by increasing extramitochondrial [Ca2+]. The rates and extents of NAD(P)H formation in the liver mitochondria induced by non-saturating concentrations of 2-oxoglutarate, glutamate, threo-DS-isocitrate or citrate were also increased in a similar manner by Ca2+ under several different incubation conditions, including an apparent ‘State 3.5’ respiration condition. Ca2+ had no effect on NAD(P)H formation induced by β-hydroxybutyrate or malate. In intact, fully coupled, rat liver mitochondria incubated with 10 mM-NaCl and 1 mM-MgCl2, the apparent K0.5 values for extramitochondrial Ca2+ were about 0.5 microM, and the effective concentrations were within the expected physiological range, 0.05-5 microM. In the absence of Na+, Mg2+ or both, the K0.5 values were about 400, 200 and 100 nM respectively. These effects of increasing extramitochondrial [Ca2+] were all inhibited by Ruthenium Red. When extramitochondrial [Ca2+] was increased above the effective ranges for the enzymes, a time-dependent deterioration of mitochondrial function and ATP content was observed. The implications of these results on the role of the Ca2+-transport system of the liver mitochondrial inner membrane are discussed.

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Presynaptic Effects of Moxonidine in Isolated Buffer Perfused Rat Hearts: Role of Imidazoline-1 Receptors and α2-Adrenoceptors

Journal of Pharmacology and Experimental Therapeutics ◽

10.1124/jpet.102.041657 ◽

2002 ◽

Vol 303 (3) ◽

pp. 1163-1170 ◽

Cited By ~ 12

Author(s):

Ulrich Schäfer ◽

Christof Burgdorf ◽

Astrid Engelhardt ◽

Thomas Kurz ◽

Gert Richardt

Keyword(s):

Α2 Adrenoceptors ◽

Rat Hearts ◽

Perfused Rat Hearts

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Src tyrosine kinase is the trigger but not the mediator of ischemic preconditioning

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.2001.281.3.h1066 ◽

2001 ◽

Vol 281 (3) ◽

pp. H1066-H1074 ◽

Cited By ~ 34

Author(s):

Reiji Hattori ◽

Hajime Otani ◽

Takamichi Uchiyama ◽

Hiroji Imamura ◽

Jianhua Cui ◽

...

Keyword(s):

Tyrosine Kinase ◽

Ischemic Preconditioning ◽

Tyrosine Kinases ◽

Src Kinase ◽

Src Tyrosine Kinase ◽

Kinase Activation ◽

Rat Hearts ◽

Perfused Rat Hearts ◽

Src Activation

The signal cascade that triggers and mediates ischemic preconditioning (IPC) remains unclear. The present study investigated the role of the Src family of tyrosine kinases in IPC. Isolated and buffer-perfused rat hearts underwent IPC with three cycles of 5-min ischemia and 5-min reperfusion, followed by 30-min ischemia and 120-min reperfusion. The Src tyrosine kinase family-selective inhibitor PP1 was administered between 45 and 30 min before ischemia (early PP1 treatment) or for 15 min before IPC [early PP1-preconditioning (PC) treatment]. PP1 was also administered for 5 min before the sustained ischemia (late PP1 treatment) or after IPC (late PP1-PC treatment). Src kinase was activated after 30 min of ischemia in both the membrane and cytosolic fractions. Src kinase was also activated by IPC but was attenuated after the sustained ischemia. Early and late PP1 treatment inhibited Src activation after the sustained ischemia and reduced infarct size. Early PP1-PC inhibited Src activation after IPC but not after the sustained ischemia and blocked cardioprotection afforded by IPC. Late PP1-PC treatment abrogated IPC-induced activation of Src and protein kinase C (PKC)-ε in the membrane but not in the cytosolic fraction. This treatment modality abrogated Src activation after the sustained ischemia and failed to block cardioprotection afforded by IPC. These results suggest that Src kinase activation mediates ischemic injury but triggers IPC in the position either upstream of or parallel to membrane-associated PKC-ε.

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Ca2+ activation of heart mitochondrial oxidative phosphorylation: role of the F0/F1-ATPase

AJP Cell Physiology ◽

10.1152/ajpcell.2000.278.2.c423 ◽

2000 ◽

Vol 278 (2) ◽

pp. C423-C435 ◽

Cited By ~ 273

Author(s):

Paul R. Territo ◽

Vamsi K. Mootha ◽

Stephanie A. French ◽

Robert S. Balaban

Keyword(s):

Oxidative Phosphorylation ◽

Atp Synthesis ◽

High Energy ◽

Ruthenium Red ◽

Maximal Effect ◽

Production Rates ◽

Atp Production ◽

The Matrix

Ca2+ has been postulated as a cytosolic second messenger in the regulation of cardiac oxidative phosphorylation. This hypothesis draws support from the well-known effects of Ca2+ on muscle activity, which is stimulated in parallel with the Ca2+-sensitive dehydrogenases (CaDH). The effects of Ca2+ on oxidative phosphorylation were further investigated in isolated porcine heart mitochondria at the level of metabolic driving force (NADH or Δψ) and ATP production rates (flow). The resulting force-flow (F-F) relationships permitted the analysis of Ca2+ effects on several putative control points within oxidative phosphorylation, simultaneously. The F-F relationships resulting from additions of carbon substrates alone provided a model of pure CaDH activation. Comparing this curve with variable Ca2+ concentration ([Ca2+]) effects revealed an approximate twofold higher ATP production rate than could be explained by a simple increase in NADH or Δψ via CaDH activation. The half-maximal effect of Ca2+ at state 3 was 157 nM and was completely inhibited by ruthenium red (1 μM), indicating matrix dependence of the Ca2+ effect. Arsenate was used as a probe to differentiate between F0/F1-ATPase and adenylate translocase activity by a futile recycling of ADP-arsenate within the matrix, catalyzed by the F0/F1-ATPase. Ca2+increased the ADP arsenylation rate more than twofold, suggesting a direct effect on the F0/F1-ATPase. These results suggest that Ca2+ activates cardiac aerobic respiration at the level of both the CaDH and F0/F1-ATPase. This type of parallel control of both intermediary metabolism and ATP synthesis may provide a mechanism of altering ATP production rates with minimal changes in the high-energy intermediates as observed in vivo.

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