James G. McCormack

On the role of the calcium transport cycle in heart and other mammalian mitochondria

Much attention has been focussed recently on the transfer of Ca*+ across the inner membrane of mammalian mitochondria and within the last year or so this Journal has published two other review letters on the topic [ 1,2]. The elegant studies which formed the basis of these letters have shown that the calciumtransport system in mammalian mitochondria consists of separate influx and efflux components. These components taken together constitute a calcium-transport cycle which determines the distribution of Ca*+ across the inner mitochondrial membrane (fig.]). The main purpose of this letter is to suggest a role for this cycle. In the past, it appears to have been assumed rather generally amongst biochemists interested in Ca*+transport in mitochondria that the system is important in the regulation of cytoplasmic Ca*+ [3-61. We will argue in this article that this emphasis is probably misplaced. We will summarise evidence that Ca*+ enhances the activity of three key intramitochondrial dehydrogenases and that [Ca”] at 0.1-10 PM is potentially an important regulator of oxidative metabolism within mammalian mitochondria. If this view is correct then the function of the calcium-transport system in the inner mitochondrial membrane should be considered primarily as a means of determining the intramitochondrial (rather than extramitochondrial) [Ca”] concentration in the same sense that the system in the plasma membrane is usually viewed as a

Effect of phenylephrine on pyruvate dehydrogenase activity in rat hepatocytes and its interaction with insulin and glucagon.

In isolated rat hepatocytes phenylephrine promotes a rapid increase in the amount of pyruvate dehydrogenase present in its active form (PDHa). This action is mediated by α1‐adrenergic receptors and is not observed in Ca2+‐depleted hepatocytes. It is mimicked by the Ca2+ ionophore A23187. No changes in metabolites known to affect PDH activity are measured 3 min after addition of phenylephrine. Glucagon also increases PDHa, its action is additive to that of phenylephrine. The action of phenylephrine on PDHa could be explained by an increase in mitochondrial free Ca2+.

Influence of calcium ions on mammalian intramitochondrial dehydrogenases

Publisher Summary This chapter discusses the influence of calcium ions on mammalian intramitochondrial dehydrogenases. Many of the hormones that activate energy-requiring processes, such as contraction or secretion in mammalian cells by causing increases in the cytoplasmic concentration of Ca 2+ also bring about the stimulation of mitochondrial oxidative metabolism. However, it has been the development of the ability to assay for the Ca 2+ -sensitive properties of these enzymes within intact mitochondria that have led to the studies from that the main hypotheses have been formulated. An essential prerequisite for many of these studies has been the development of methods where partially purified mitochondrial fractions can be isolated from rapidly disrupted mammalian tissues under conditions that minimize artifactual Ca 2+ redistribution. Therefore, it is likely that the primary function of the Ca 2+ -transport system of the mitochondrial inner membrane in mammalian tissues under normal physiological conditions is to relay changes in cytoplasmic Ca 2+ concentrations into the mitochondrial matrix and, thus, to control the concentration of Ca 2+ in this compartment.

Effect of ranolazine on infarct size in a canine model of regional myocardial ischemia/reperfusion

We assessed ranolazines potential to reduce myocardial injury resulting from 90-min occlusion and 18-h reperfusion of left circumflex coronary artery (LCX) in anesthetized dogs. Ranolazine, a putative antianginal agent, has exhibited positive results in a variety of experimental models associated with the ischemic myocardium. Previous studies demonstrated that ranolazine possesses a mechanism of action involving increases in the amount of active pyruvate dehydrogenase during ischemia, suggesting that the compound may act to promote glucose utilization. Ranolazine was administered as a bolus of 3.3 mg/kg, followed by a constant infusion of 7.2 mg/kg/h for 20 h. The loading dose was administered 30 min before LCX occlusion. Control animals received appropriate volumes of vehicles (loading and infusion). Hemodynamics were unchanged between ranolazine and vehicle groups. Three animals in each group were excluded because of ventricular fibrillation (VF). There was no difference in degree of ST segment change between control and ranolazine-treated groups at any time during LCX occlusion. The area at risk (AAR) of infarct was 40.1 +/- 1.7 and 38.9 +/- 1.3% in control-treated (n = 13) and randolazine-treated (n = 8) animals, respectively (p = 0.631). Myocardial infarct size (IS) was 31.7 +/- 5.2 and 36.6 +/- 8.5% in control and ranolazine-treated animals, respectively (p = 0.603). No significant changes were observed in plasma content of enzymatic markers at 0.5, 2.0, and 18.0 h of reperfusion. The results of this in vivo study indicate that ranolazine did not provide protection from injury to regionally ischemic and reperfused myocardium despite its reported antiischemic activity.

Protective Effects of Ranolazine on Ventricular Fibrillation Induced by Activation of the ATP-Dependent Potassium Channel in the Rabbit Heart.

Background: The authors studied the antifibrillatory effects of the adenosine-triphosphate (ATP)-sparing metabolic modulator ranolazine in a rabbit isolated heart model in which ventricular fibrillation occurs under conditions of hypoxia/reoxygenation in the presence of the ATP-dependent potassium channel opener pinacidil. Methods and Results: Ten minutes after ranolazine or vehicle administration, addition of pinacidil (1.25 μM) to the buffer was followed by a 12-minute hypoxic period and 40 minutes of reoxygenation. At a reduced concentration of ranolazine (10 μM), ventricular fibrillation occurred in 60% of the hearts. compared to 89% in the control group (P = NS). In contrast, only three of nine hearts (33%) treated with 20 μM ranolazine developed ventricular fibrillation (P <.05 vs vehicle). Hemodynamic parameters including coronary perfusion pressure, left ventricular developed pressure, and ±dP/dt were not affected by the presence of ranolazine in the perfusion medium. Ranolazine did not prevent or modify the negative inotropic or coronary vasodilator actions of pinacidil, suggesting a mechanism of action independent of potassium channel antagonism. Conclusions: Ranolazine significantly reduced the incidence of ventricular fibrillation in the hypoxic/reoxygenated heart exposed to the ATP-dependent potassium channel opener, pinacidil. The reported ability of ranolazine to prevent the decrease in cellular ATP during periods of a reduced oxygen supply may account for its observed antifibrillatory action. By maintaining intracellular ATP, ranolazine may modulate or prevent further opening of the ATP-dependent potassium channel in response to hypoxia and/or pinacidil.

Effects of ranolazine on the exercise capacity of rats with chronic heart failure induced by myocardial infarction.

Ranolazine was previously shown to stimulate cardiac glucose oxidation. Dichloroacetate (DCA) also does and was shown to improve exercise capacity in animals, but it has long-term toxicity problems. To test the hypothesis that ranolazine would increase exercise performance in the chronic heart failure (CHF) condition, we compared the exercise endurance capacities of rats with a surgically induced myocardial infarction (MI) with those of noninfarcted sham-operated (Sham) controls both before and after 14 and 28 days of drug administration. Chronic administration of ranolazine, 50 mg/kg twice daily (b.i.d.) oral, significantly reduced the endurance capacities of both Sham and MI rats (measured after a 12-h fast to reduce liver glycogen stores), as indicated by the reductions in run times to fatigue during a progressive treadmill test. Ranolazine produced reductions in resting plasma lactate and glucose concentrations of animals fasted for 12 h (consistent with stimulating glucose oxidation); however, tissue glycogen concentrations measured in various locomotor muscles located in the animals hindlimb were unaffected when measured 48 h after the last treadmill test and after 12 h of fasting. Chronic administration of ranolazine did not increase the endurance capacity of rats with CHF induced by MI at the dosage and with the protocol used. To the contrary, the chronic administration of ranolazine appears to reduce the work capacity of all rats, suggesting that this drug may not be useful therapeutically in the treatment of CHF. Whether the decrements in endurance capacity produced by ranolazine are related to the high plasma concentrations of the drug produced in this study as compared with previous studies in humans remains subject to further experimentation.

Effects of Ca2+ on the activities of the calcium-sensitive dehydrogenases within the mitochondria of mammalian tissues.

Three important dehydrogenases in the mitochondria of mammalian tissues are activated by Ca2+ ions: these are pyruvate dehydrogenase, NAD-isocitrate dehydrogenase, and oxoglutarate dehydrogenase. Evidence is summarized that when hormones and other extracellular stimuli increase the cytoplasmic concentration of Ca2+ in rat hearts and livers that this results in a parallel rise in the intramitochondrial concentration of Ca2+. In this way, pyruvate oxidation and citric acid cycle flux are stimulated and there is an increase in NADH supply for the respiratory chain under conditions where there is an enhanced demand for ATP.

Brown-adipose-tissue lipogenesis in starvation: effects of insulin and (−)hydroxycitrate

Glucose or insulin increased lipogenesis (measured in vivo using 3H2O) in brown fat of starved rats. Such increases were associated with activation of pyruvate dehydrogenase and increased use of glucose as a lipogenic precursor (monitored as an increase in the 14C/3H ratio in brown-fat fatty acids in rats injected with both 3H20 and [U-14C]glucose). (-) Hydroxycitrate did not inhibit basal rates of brown-fat lipogenesis in starved rats but suppressed the increases in lipogenesis and glucose utilization observed in response to insulin. (-)Hydroxycitrate did not, however, inhabit the increase in 14C/3H observed after insulin treatment. The results indicate that in brown fat, glucose is utilized for fatty-acid synthesis predominantly via citrate, and that insulin acts to increase lipogenesis at site(s) prior to citrate cleavage. As basal rates of lipogenesis were not inhibited by (-)hydroxycitrate, it is suggested that acetate may be a lipogenic substrate for brown fat in starvation, and experiments are described which support this suggestion.

Evidence that adrenaline activates key oxidative enzymes in rat liver by increasing intramitochondrial [Ca2+]

The effects of intramitochondrial Ca2+ on the activities of the Ca2+‐sensitive intramitochondrial enzymes, (i) pyruvate dehydrogenase (PDH) phosphate phosphatase, and (ii) oxoglutarate dehydrogenase (OGDH), were investigated in intact rat liver mitochondria by measuring (i) the amount of active PDH (PDHa) and (ii) the rate of decarboxylation of α‐[1‐14C]oxoglutarate (at non‐saturating [oxoglutarate]), at different concentrations of extramitochondrial Ca2+. In the presence of Na2+ and Mg2+, both PDH and OGDH could be activated by increases in extramitochondrial [Ca2+] within the expected physiological range (0.05–5 μM). When liver mitochondria were prepared from rats treated with adrenaline, and then incubated in Na‐free media containing EGTA, both PDH and OGDH activities were found to be enhanced. Evidence is presented that the activation of these enzymes by adrenaline is brought about by a mechanism involving increases in intramitochondrial [Ca2+].

ACUTE HORMONAL REGULATION OF FATTY ACID SYNTHESIS IN MAMMALIAN TISSUES

Publisher Summary This chapter discusses the acute hormonal regulation of fatty acid synthesis in mammalian tissues. Exposure of white adipose tissue such as rat epididymal fat pads to insulin leads within a minute or so to a large increase in the rate of conversion of glucose to fatty acids. The increase in the rate of fatty acid synthesis in the presence of insulin is brought about by activation not only of glucose transport but also of pyruvate dehydrogenase and acetyl CoA carboxylase. Both these enzymes exist in interconvertible forms. After exposure to insulin, the proportion of both enzymes in their respective active forms is increased 2–3 fold. These parallel activations do appear to offer a satisfactory explanation of the preferential conversion of glucose carbon into fatty acids that is so characteristic of the response of white adipose tissue to insulin.