Methotrexate: studies on the cellular metabolism. I. Effect on mitochondrial oxygen uptake and oxidative phosphorylation

Effect of methotrexate (MTX) on mitochondrial oxygen uptake, oxidative phosphorylation and on the activity of several enzymes linked to respiratory chain was studied. MTX was able to inhibit state III respiration activated by ADP and to decrease the respiratory coefficient with the substrates alpha-ketoglutarate and glutamate; these effects became pronounced when mitochondria were pre-incubated with MTX for 10 min. No effect was observed on ATPase activity of undamaged or broken mitochondria; the same was true for NADH-oxidase, NADH-dehydrogenase, NADH-cytochrome c reductase, succinate oxidase, and cytochrome c oxidase activity. The effect on the steady-state of cytochrome b, as well as, the inhibitory effect on state III of respiration with NAD+-linked substrates, offers a reasonable possibility to suggesting that the inhibition site of MTX could be in a place anterior to cytochrome b region, and not linked to respiratory chain.     

Methotrexate: studies on cellular metabolism. IV. Effect on the mitochondrial oxidation of cytosolic-reducing equivalents in HeLa cells

The effect of methotrexate (MTX) on the mitochondrial oxidation of cytosolic-reducing equivalents in HeLa cells was studied. MTX inhibited (100 per cent) malate dehydrogenase activity, but no effect was observed on that of GOT. MTX (0.5 mM) inhibited (100 per cent) the activity of reconstituted enzymatic system MDH-GOT, probably as a consequence of inhibition of malate dehydrogenase activity. MTX decreased pyruvate production (54 per cent), demonstrating its inhibitory action on the malate-aspartate shuttle. Blockage of the malate-aspartate shuttle by MTX accounts for the decrease in cellular energetic gain. The results obtained are consistent with the view that in HeLa cells, as well as in other tumour cells, the transport of reducing equivalents from cytoplasmic NADH into the respiratory chain of mitochondria is via the malate-aspartate shuttle.     

Sedimentation-equilibrium and other physicochemical studies on the lysine-rich fraction of calf thymus histones

1. The lysine-rich fraction (Ia+Ib, or f1) of calf thymus histones was isolated as the sulphate by acid extraction. 2. Sedimentation-equilibrium measurements with interference optics showed that this fraction was monodisperse with a molecular weight of 19500±2000. 3. The `apparent molecular weight' calculated from the sedimentation-equilibrium studies varied markedly with concentration. The large second virial coefficient implied by such variation was attributed to the very high charge/mass ratio of this relatively small protein. Estimates of the charge were made from the values of this virial coefficient. 4. The very large value of the virial coefficient explains anomalies in the earlier reports of the molecular weight of this histone and also why the z-average molecular weight can appear to be lower than the weight-average molecular weight. 5. The differences of the specific refractive increments, and the partial specific volumes, between dialysed and undialysed solutions of this histone fraction could also be attributed to its high molecular charge, which was estimated from these differences and agreed, within the expected limits, with the value deduced from the second virial coefficient. 6. Sedimentation-velocity measurements combined with the known molecular weight imply that lysine-rich histone has a high frictional ratio and an extended shape. Optical-rotatory-dispersion measurements indicated that it had a low helical content.     

Effects of electrical muscle stimulation on oxygen consumption

Electrical muscle stimulation (EMS) devices are being marketed as weight/ fat loss devices throughout the world. Commercially available stimulators have the ability to evoke muscle contractions that may affect caloric expenditure while the device is being used. The aim of this study was to test the effects of two different EMS devices (Abtronic and Feminique) on oxygen consumption at rest. Subjects arrived for testing after an overnight fast, had the devices fitted, and then positioned supine with expired air measured to determine oxygen consumption. After a 10-minute acclimation period, oxygen consumption was measured for 20 minutes with the device switched off (resting) then 20 minutes with the device switched on (stimulated). There were no significant differences (p > 0.05) in oxygen consumption between the resting and stimulated periods with either the Abtronic (mean +/- SD; resting, 3.40 +/- 0.44; stimulated, 3.45 +/- 0.53 ml of O(2).kg(-1).min(-1)) or the Feminique (resting, 3.73 +/- 0.45; stimulated, 3.75 +/- 0.46 ml of O(2).kg(-1).min(-1)). In summary, the EMS devices tested had no effect on oxygen consumption during muscle stimulation.     

Role of mitochondrial Ca2+ in the regulation of cellular energetics

Calcium is an important signaling molecule involved in the regulation of many cellular functions. The large free energy in the Ca(2+) ion membrane gradients makes Ca(2+) signaling inherently sensitive to the available cellular free energy, primarily in the form of ATP. In addition, Ca(2+) regulates many cellular ATP-consuming reactions such as muscle contraction, exocytosis, biosynthesis, and neuronal signaling. Thus, Ca(2+) becomes a logical candidate as a signaling molecule for modulating ATP hydrolysis and synthesis during changes in numerous forms of cellular work. Mitochondria are the primary source of aerobic energy production in mammalian cells and also maintain a large Ca(2+) gradient across their inner membrane, providing a signaling potential for this molecule. The demonstrated link between cytosolic and mitochondrial Ca(2+) concentrations, identification of transport mechanisms, and the proximity of mitochondria to Ca(2+) release sites further supports the notion that Ca(2+) can be an important signaling molecule in the energy metabolism interplay of the cytosol with the mitochondria. Here we review sites within the mitochondria where Ca(2+) plays a role in the regulation of ATP generation and potentially contributes to the orchestration of cellular metabolic homeostasis. Early work on isolated enzymes pointed to several matrix dehydrogenases that are stimulated by Ca(2+), which were confirmed in the intact mitochondrion as well as cellular and in vivo systems. However, studies in these intact systems suggested a more expansive influence of Ca(2+) on mitochondrial energy conversion. Numerous noninvasive approaches monitoring NADH, mitochondrial membrane potential, oxygen consumption, and workloads suggest significant effects of Ca(2+) on other elements of NADH generation as well as downstream elements of oxidative phosphorylation, including the F(1)F(O)-ATPase and the cytochrome chain. These other potential elements of Ca(2+) modification of mitochondrial energy conversion will be the focus of this review. Though most specific molecular mechanisms have yet to be elucidated, it is clear that Ca(2+) provides a balanced activation of mitochondrial energy metabolism that exceeds the alteration of dehydrogenases alone.     

Control of mitochondrial and cellular respiration by oxygen

Control and regulation of mitochondrial and cellular respiration by oxygen is discussed with three aims: (1) A review of intracellular oxygen levels and gradients, particularly in heart, emphasizes the dominance of extracellular oxygen gradients. Intracellular oxygen pressure, $p_{O_2 } $ , is low, typically one to two orders of magnitude below incubation conditions used routinely for the study of respiratory control in isolated mitochondria. The $p_{O_2 } $ range of respiratory control by oxygen overlaps with cellular oxygen profiles, indicating the significance of $p_{O_2 } $ in actual metabolic regulation. (2) A methodologically detailed discussion of high-resolution respirometry is necessary for the controversial topic of respiratory control by oxygen, since the risk of methodological artefact is closely connected with far-reaching theoretical implications. Instrumental and analytical errors may mask effects of energetic state and partially explain the divergent views on the regulatory role of intracellular $p_{O_2 } $ . Oxygen pressure for half-maximum respiration,p ₅₀, in isolated mitochondria at state 4 was 0.025 kPa (0.2 Torr; 0.3 ΜM O₂), whereasp ₅₀ in endothelial cells was 0.06–0.08 kPa (0.5 Torr). (3) A model derived from the thermodynamics of irreversible processes was developed which quantitatively accounts for near-hyperbolic flux/ $p_{O_2 } $ relations in isolated mitochondria. The apparentp ₅₀ is a function of redox potential and protonmotive force. The protonmotive force collapses after uncoupling and consequently causes a decrease inp ₅₀. Whereas it is becoming accepted that flux control is shared by several enzymes, insufficient attention is paid to the notion of complementary kinetic and thermodynamic flux control mechanisms.     

Further studies on the physical aspects of cellular growth and multiplication

Summary In continuation of a previous study, the conditions, under which a drop will divide spontaneously due to the production of capillary active substances, are investigated in more details, and illustrated by a numerical example.Spontaneous division of drops, consisting of several phases is investigated, and a theory of indirect division suggested.It is shown that due to the requirements of thermodynamical equilibrium, the geometrical arrangement of various phases in a cell or drop may be generally completely determined.In a drop, which consists of several phases, enveloping each other, those phases, which possess the property of regenerating both themselves and the other phases, will lie centrally.It is shown that under certain conditions, which can be specified mathematically, whenever a polyphase drop divides spontaneously, various phases tend to be evenly distributed amongst the daughterdrops. Whenever this is the case, arrangemants of some of the phases in the equatorial plane of the dividing drop results from general requirements of equilibrium.     

Comparative studies of the effects of acridines and other petite inducing drugs on the mitochondrial genome of Saccharomyces cerevisiae.

Summary The effects of the acridines euflavine and proflavine on mitochondrial DNA (mtDNA) replication and mutation inSaccharomyces cerevisiae have been compared. In contrast to previous results we found that under our conditions proflavine can indeed induce high levels (>80%) of petite mutants, although six times less efficiently than euflavine. The parameters measured for mutagenesis of the mitochondrial genome and inhibition of mtDNA replication in whole cells suggest that the modes of action of euflavine and proflavine are very similar. After extended (18h) treatment of growing cells with each drug the percentage loss of mtDNA or genetic loci was almost coincidental with the extent of petite induction.It was found that proflavine is equally as effective as euflavine in inhibiting mtDNA replication in isolated mitochondria in contrast to the differential between the drugs observed in vivo. However, proflavine and euflavine inhibit cellular growth at almost the same concentrations. It is therefore proposed that there is some intracellular permeability barrier which impedes proflavine access to the mitochondrial DNA replicating system.The petites induced by euflavine (and proflavine) are characterized by there being a preferential induction ofrho ⁰ petites lacking mtDNA as opposed torho ^- petites retaining mtDNA. This is in contrast to the relative proportions of such petites induced by ethidium bromide or berenil. A scheme for the production of petites by euflavine is presented, in which euflavine inhibits the replication of mtDNA, but does not cause direct fragmentation of mtDNA (unlike ethidium bromide and berenil). The proposed scheme explains the production of the high frequency ofrho ^o cells, as well as therho ^- cells induced by euflavine. The scheme also accounts for previous observations that euflavine only mutants growing cultures, and that the buds, but not mother cells, become petite.     

Rapid stimulation of hepatic oxygen consumption by 3,5-di-iodo-L-thyronine.

Tri-iodothyronine (T3) and thyroxine (T4) as well as 3,5-di-iodothyronine (T2) stimulated O2 consumption by isolated perfused livers from hypothyroid rats at a concentration as low as 1 pM by about 30% within 90 min. Application of T2 resulted in a faster stimulation than with application of T3 or T4. Inhibition of iodothyronine monodeiodinase by propylthiouracil, thereby blocking the degradation of T4 to T3 and of T3 to T2, demonstrated that only T2 is the active hormone for the rapid stimulation of hepatic O2 consumption: T3 and T4 lost all of their stimulative activity, whereas T2 was as potent as in the absence of propylthiouracil. Perfusion experiments with thyroid-hormone analogues confirmed the specificity of the T2 effect. The nucleus is unlikely to contribute to the rapid T2 effect, as can be deduced from perfusion experiments with cycloheximide and lack of induction of malic enzyme by T2. In conclusion, a new scheme of regulation of mitochondrial activity is proposed: T2 acts rapidly and directly via a mitochondrial pathway, whereas T3 exerts its long-term action indirectly by induction of specific enzymes.     

The oxygen consumption of Pennatula rubra ellis and some other anthozoans

Summary The oxygen consumption of Pennatula rubra and some other pennatulids, and also of the anemone Calliactis parasitica, has been measured by a reliable method. In general, the oxygen consumption of these coelenterates is proportional to the external oxygen concentration, suggesting that the consumption mechanism relies on simple diffusion of oxygen into the cells.The oxygen consumptions per unit organic dry weight of these species are comparable with typical values for other marine invertebrates, implying that the use of wet weight as a criterion may be misleading and should be discontinued, and that pennatulids respire actively considering the small amount of organic material they contain.It is suggested that the endoderm is the major site of oxygen consumption in these animals.Calliactis parasitica, when under anaerobic conditions, incurs only a partial oxygen debt, as post-anaerobic oxygen consumption is only slightly higher than the normal rate.

---

Zusammenfassung Der Sauerstoff-Verbrauch von Pennatula rubra und anderen Pennatuliden und der See-Anemone Calliactis parasitica ist unter Anwendung der Winkler-Methode gemessen worden. Im allgemeinen hängt der Sauerstoffverbrauch dieser Coelenteraten von der O₂-Konzentration im umgebenden Medium ab. Das deutet darauf hin, daß der Mechanismus der O₂-Aufnahme in einer einfachen Diffusion des Sauerstoffes in die Gewebe besteht.Der O₂-Verbrauch, bezogen auf das organische Trockengewicht, ist mit dem anderer wirbelloser Seetiere vergleichbar. Die Verwendung des Frischgewichtes als Bezugsgröße kann demnach irreführend sein; Pennatuliden atmen — in Anbetracht der geringen Menge organischen Materials, die sie enthalten — aktiv.Das Entoderm ist die Gewebeschicht mit dem größten Sauerstoffverbrauch.Unter Bedingungen des Sauerstoffmangels tritt bei Calliactis parasitica nur ein teilweiser Sauerstoffmangel ein, da der anschließende Sauerstoffverbrauch (unter normalen Bedingungen) nur wenig höher ist als der normale.

---

---

We are indebted to the University of London for a grant from the Central Research Fund which made this work possible. We also wish to thank Prof. G. PETIT, Director of the Laboratoire Ar'ago, Banyuls-sur-Mer, for providing facilities and for the interest he showed in the work.