Phosphate-induced Swelling Research Articles

Inhibitory effect of pyruvate on release of glutathione and swelling of rat heart mitochondria

Pyruvate prevents the permeability transition of rat heart mitochondria induced by the system calcium ions + phosphate or by the dithiol reagent phenylarsenoxide and measured as swelling. Since swelling induced by the latter is relieved by the dithiol 2,3-dimercaptopropanol (BAL), it is inferred that the effect of pyruvate might be mediated by the reduction of lipoic acid. In isolated mitochondria, pyruvate also exerts a protective effect when calcium + phosphate-induced swelling is exacerbated by hypoxic conditions. These results agree with our previous observations that pyruvate markedly prevents the loss of cytosolic and mitochondrial glutathione after ischemia or ischemia followed by reperfusion.

The uptake of oxalate by rat liver and kidney mitochondria.

Oxalate, a metabolic end product, forms calcium oxalate deposits in the tissues under a variety of pathological conditions. In order to determine whether oxalate is able to penetrate the mitochondrial matrix, the uptake of oxalate by rat liver and kidney cortical mitochondria was characterized. Mitochondria did not swell in an iso-osmotic medium of ammonium oxalate unless a small amount of phosphate was provided. This phosphate-induced swelling was prevented by N-ethylmaleimide. The uptake of [14C]oxalate by liver and kidney mitochondria followed first order kinetics and was inhibited by mersalyl an inhibitor of the phosphate and dicarboxylate carriers. Accumulation of [14C]oxalate at equilibrium was significantly higher by mitochondria energized with succinate than by rotenone-inhibited mitochondria due to higher matrix pH as determined by the [14C]5,5'-dimethyloxazolidine-2, 4-dione distribution ratio. The velocity of oxalate accumulation by mitochondria was temperature dependent. The activation energy was 81.5 and 86.5 J/mol for liver and kidney mitochondria, respectively. In both types of mitochondria, the rate of oxalate uptake was hyperbolic with respect to the concentration of oxalate. The apparent Km was 28.8 +/- 0.6 and 13.4 +/- 1.2 mM and the Vmax 87.1 +/- 1.1 and 66.1 +/- 3.1 nmol X mg-1 X min-1 at 12 degrees C for liver and kidney mitochondria, respectively. Phenylsuccinate exhibited mixed inhibition of the rate of oxalate uptake. Oxalate exhibited also a mixed inhibition of the uptake and oxidation of malate by mitochondria. The data obtained provide evidence that oxalate is transported across the mitochondrial membrane by a phosphate-linked, carrier-mediated system similar to or identical to the dicarboxylate transporter.

Actions of certain calcium channel blockers and calmodulin antagonists on inorganic phosphate-induced swelling and inhibition of oxidative phosphorylation of heart mitochondria

Actions of certain calcium channel blockers and calmodulin antagonists on inorganic phosphate-induced swelling and inhibition of oxidative phosphorylation of heart mitochondria

Sulphate transport by H+ symport and by the dicarboxylate carrier in mitochondria.

1. Swelling of mitochondria was induced in non-respiring mitochondria by 30 mM or more Na2SO4 or by respiration in the presence of K2SO4. Respiration-drive swelling resulted in loss of respiratory control. Sulphate, when present at 10 mM concentration, promoted the release of accumulated Ca2+. 2. Swelling was prevented by N-ethylmaleimide and formaldehyde, known inhibitors of the phosphate carrier. Sulphate-induced swelling was more sensitive to the inhibitors than was phosphate-induced swelling. At lower concentration of sulphate, 5 mM, an alkalinisation of the medium was observed in addition of sulphate, indicating H+-sulphate symport. There was competition between sulphate and phosphate for transport by this mechanism. It is suggested that sulphate may be transported, though at a comparatively slow rate, by the phosphate carrier. 3. Uptake of sulphate was stimulated when preceded by energy-dependent accumulation of Ba2+, especially when acetate was also present, indicating precipitation of BaSO4 in the matrix. Using this system the influx of sulphate was studied at lower concentrations, 10 mM or less. the contributions of the H+ symporter (sensitive to N-ethylmaleimide) and the dicarboxylate carrier (sensitive to butylmalonate) could then be studied. The dicarboxylate carrier had a lower Km and was mainly responsible for sulphate transport at lower concentration range. At 10 mM-sulphate the transport rates by the two systems appeared to be similar; at still higher concentrations the H+ symporter may become more important.

Photophosphorylation in bacterial chromatophores entrapped in alginate gel: Improvement of the physical and biochemical properties of gel beads with barium as gel-inducing agent

The immobilization of Rhodopseudomonas capsulata chromatophores by entrapment in an alginate gel is described. Alginate beads were prepared with Ba 2+, Sr 2+ and Ca 2+ as gel-forming agents and compared for their mechanical strength, chemical resistance against disruption by phosphate-induced swelling, and yield of photophosphorylation activity. Barium alginate beads proved to have better physico-chemical properties than the more commonly used calcium alginate beads. After embedding in barium alginate gel, R. capsulata chromatophores retained a high yield (up to 70%) of their photophosphorylation capacity. Alginate entrapment did not cause a large increase in the Michaelis constant for ADP and phosphate, the substrates of adenosinetriphosphatase (ATPase). These constants were K ADP m = 1.4 × 10 −5 m and K P i m = 2.2 × 10 −4 m for free chromatophores and K ADP m = 2.3 × 10 −4 m and K P i m = 5.6 × 10 −4 m for chromatophores entrapped in barium alginate gel. However, embedding gave no additional protection against rapid inactivation of chromatophores upon storage at 3°C. Preliminary results with a batch reactor for continuous ATP regeneration are presented. The barium alginate method has two features which are not generally encountered at the same time, extremely mild conditions for entrapment and excellent physical properties of the gels beads, which make this method a suitable tool for the construction of bioreactors with immobilized cells or organelles.

The inhibitory effect of N-(phosphonomethyl)glycine in vivo on energy-dependent, phosphate-induced swelling of isolated rat liver mitochondria

Spectrophotometric measurement of the absorbance of mitochondria at 520 nm indicated that, 5 h after administration of a single intraperitoneal dose of N-(phosphonomethyl)glycine (PMG) to rats there was a significant inhibition of energy-dependent, phosphate-induced swelling of isolated liver mitochondria. Doses of 120 and 240 mg PMG/kg caused inhibitions of 40 and 65% respectively, when either succinate or β-hydroxybutyrate was used as the substrate.

Inhibition by local anaesthetics of anion transport in isolated rat heart mitochondria

(1) (1)The effects of nupercaine and other local anaesthetics on anion transporters in isolated rat heart mitochondria were investigated at temperatures of 25° or 30°. The concentrations of nupercaine which inhibited by 50 per cent the rates of pyruvate-stimulated oxygen utilisation in (a) the presence and (b) the absence of α-cyano-3-hydroxycinnamate, (c) ADP-stimulated oxygen utilisation in the presence of atractyloside, (d) succinate-stimulated oxygen utilisation in the presence of phenylsuccinate (e) phosphate-induced swelling were (a) 0.02, (b) 0.4, (c) 0.3, (d) 1.1 and (e) 0.6 mM, respectively. (2) (2)Low concentrations of nupercaine (10–50 μM) increased by about 10 per cent the rates of ADP- and succinate-stimulated oxygen utilisation measured at 30° in the presence of atractyloside and phenylsuccinate, respectively. (3) (3)Inhibition by nupercaine of pyruvate-stimulated oxygen utilisation in the presence of α-cyano-3-hydroxycinnamate was found to be non-competitive with respect to pyruvate. (4) (4)Butacaine and tetracaine inhibited pyruvate-stimulated oxygen utilisation, but were less effective than nupercaine. Procaine and xylocaine had little effect. (5) (5)[2- 14C]Pyruvate uptake was inhibited by nupercaine with 50% inhibition given by 0.2 and 0.6 mM nupercaine at 25° and 4° respectively. (6) (6)The concentrations of nupercaine which inhibited pyruvate-stimulated oxygen utilisation in the presence of 2-cyano-3-hydroxycinnamate had no effect on the rate of mitochondrial swelling or the acceptor control ratio of the mitochondria. (7) (7)It is concluded that the pyruvate transporter is markedly more susceptible to the inhibitory effects of nupercaine than are the adenine nucleotide. dicarboxylate or phosphate ion transporters.

Biochemical properties of rat liver mitochondrial aldehyde dehydrogenase with respect to oxidation of formaldehyde.

The oxidation of formaldehyde by rat liver mitochondria in the presence of 50 mM phosphate was enhanced 2-fold by exogenous NAD+. Absolute requirement of NAD+ for formaldehyde oxidation was demonstrated by depleting the mitochondria of their NAD+ content (4.6 nmol/mg of protein), followed by reincorporation of the NAD+ into the depleted mitochondria. Aldehyde (formaldehyde) dehydrogenase activity was completely abolished in the depleted mitochondria, but the enzyme activity was restored to control levels following reincorporation of the pyridine nucleotide. Phosphate stimulation of formaldehyde oxidation could not be explained fully by the phosphate-induced swelling which enhances membrane permeability to NAD+, since stimulation of the enzyme activity by increased phosphate concentrations was still observed in the absence of exogenous NAD+. The Km for formaldehyde oxidation by the mitochondria was found to be 0.38 nM, a value similar to that obtained with varying concentrations of NAD+; both Vmax values were very similar, giving a value of 70 to 80 nmol/min/mg of protein. The pH optimum for the mitochondrial enzyme was 8.0. Inhibition of the enzyme activity by anaerobiosis was apparently due to the inability of the respiratory chain to oxidize the generated NADH. The inhibition of mitochondrial formaldehyde oxidation by succinate was found to be due to a lowering of the NAD+ level in the mitochondria. Succinate also inhibited acetaldehyde oxidation by the mitochondria. Malonate, a competitive inhibitor of succinic dehydrogenase, blocked the inhibitory effect of succinate. The respiratory chain inhibitors, rotenone, and antimycin A plus succinate, strongly inhibited formaldehyde oxidation by apparently the same mechanism, although the crude enzyme preparation (freed from the membrane) was slightly sensitive to rotenone. The mitochondria were subfractionated, and 85% of the enzyme activity was found in the inner membrane fraction (mitoplast). Furthermore, separation into inner membrane and matrix components indicated a distribution of aldehyde dehydrogenase activity similar to malic dehydrogenase.

The Effect of Temperature on Mitochondrial Membrane-linked Reactions

The rates of rat liver mitochondrial membrane-linked reactions were studied as a function of temperature, with the results being expressed as Arrhenius plots. Energy-dependent reactions involving the adenine nucleotide translocase yielded two distinct discontinuities in their Arrhenius plots, near 17.5° and 27.5°. These reactions include ADP phosphorylation, state 3 and state 4 respiration, uncoupler-stimulated respiration, dinitrophenol-stimulated adenosine triphosphatase and ATP-supported calcium uptake. Energy-dependent reactions not involving the adenine nucleotide translocase also gave two breaks in their Arrhenius plots. The upper break was near 27.5° as before. However, the temperature at which the lower break occurred was decreased to around 12.5°. These reactions included respiration-dependent calcium uptake, valinomycin-induced potassium uptake, and phosphate-induced swelling. Studies with representative matrix, outer membrane, and inner membrane enzymes including the ATPase in sonicated mitochondrial particles indicated that an intact, energy-transducing membrane is required for discontinuities in the Arrhenius plots to appear at any temperature.

The effects of acetaldehyde on the kinetics of the energy-dependent phosphate-induced swelling of rat liver mitochondria

Acetaldehyde in the range of 1 to 5 mM increases the rate and extent of swelling of rat liver mitochondria with either DL-3-hydroxybutyrate or succinate as substrate. Higher concentrations of acetaldehyde first increase the rate of swelling and then completely inhibit swelling. Exposure of mitochondria to 5 mM acetaldehyde does not alter the requirements of swelling for energy and inorganic phosphate. The results demonstrate that acetaldehyde is capable of altering the kinetics of swelling under conditions which prevent the oxidation of acetaldehyde.

The effect of ruthenium red on Ca 2+ transport and respiration in rat liver mitochondria

1. The effect of ruthenium red and K 2RuCl 6 on Ca 2+ transport and mitochondrial respiration was studied. 2. Low levels of ruthenium red (3–6 nmoles/mg mitochondrial protein) completely inhibited the stimulation of respiration by Ca 2+ but had no effect on the response of respiration to ADP and 2,4-dinitrophenol. 3. Ruthenium red (3–6 nmoles/mg mitochondrial protein) also inhibited respiration-dependent and ATP-supported Ca 2+ uptake as well as the metabolism-independent, K +-driven translocation and the high- and low- affinity binding of Ca 2+. However, it had no effect on the release of accumulated Ca 2+. Respiration-dependent Sr 2+ and Mn 2+ uptake also were inhibited. 4. At slightly higher concentrations (10 or more nmoles/mg mitochondrial protein) than those preventing Ca 2+ binding and transport, ruthenium red markedly inhibited resting (State 4) respiration and altered, but did not prevent, the stimulation of respiration by ADP and P i. 2,4-Dinitrophenol also overcame the ruthenium red inhibition of respiration. 5. Latent ATPase was significantly inhibited by 4 nmoles of ruthenium red per mg mitochondrial protein. The Mg 2+- and the 2,4-dinitrophenol-stimulated ATPase activities, on the other hand, were slightly, if at all, inhibited by as much as 40 nmoles of ruthenium red per mg protein. Phosphate-induced swelling of mitochondria also was not affected by high levels of ruthenium red. 6. K 2RuCl 6 significantly inhibited State 4 respiration at a concentration of 2.5 μM (1 nmole/mg protein), essentially complete inhibition occurring at 10 to 50 μM (4–20 nmoles/mg protein). The inhibition of respiration by K 2RuCl 6 was overcome by Ca 2+ just as effectively as by ADP and P i. 2,4-Dinitrophenol also overcame the inhibition of respiration by K 2RuCl 6. Concentrations of K 2RuCl 6 5–10 times higher than that which markedly inhibited respiration were required to inhibit Ca 2+ transport. 7. These results indicate that ruthenium red has a dual effect on mitochondrial metabolism; it interferes with the binding and transport of Ca 2+ and at a slightly higher concentration it markedly inhibits respiration by interacting with the energy coupling pathway.

The osmotic nature of the ion-induced swelling of rat-liver mitochondria

Analytical techniques have been developed to measure the relation between water uptake and cation uptake in rat-liver mitochondria. The water content of inner and outer mitochondrial compartments were separately measured using [ 131I]albumin and [ 14C]sucrose. The cation contents of the same mitochondrial pellets were measured using flame photometry. These data were used to measure the water content of each compartment which was necessary, since the basic osmotic relationships are only revealed when movements into the inner and outer compartments are separately examined. The relation between the water uptake and K + uptake, either spontaneous or valinomycin-induced, was dependent on medium osmolality. The concentration of the K + solution taken up in the inner compartment was compatible with the hypothesis that transport-linked mitochondrial swelling is driven by osmotic pressure differences which have been induced by solute movement. The changes in volume of the water compartment and in ion content during phosphate-induced swelling and during incubation in the absence of substrate were analyzed using the same methods. Under these experimental conditions also, the mitochondrial inner compartment appears to be in osmotic equilibrium with the external medium.

Equivalence of oligomycin and 2-chloro-4′,4″-di (2-imidazolin-2-yl)terephthalanilide in their effects on mitochondrial swelling

2-Chloro-4′,4″-di(2-imidazolin-2-yl)terephthalanilide (NSC 60339) has been compared with oligomycin and 2,4-dinitrophenol in its effects on a rat liver mitochondrial swelling system. The substituted phthalanilide and oligomycin are equivalent in their effects in the following swelling experiments. Neither alters spontaneous or calcium- or phosphate-induced swelling; both inhibit succinate-induced swelling; both prevent ATP inhibition of calcium- and phosphate-induced swelling; and both inhibit ATP reversal of calcium-induced swelling in fresh or aged mitochondria. The known binding ofphthalanilides to specific phospholipids and the inhibitory effects described suggest that NSC 60339 may be useful in studies on the nature and role oflipids in mitochondrial swelling and oxidative phosphorylation.

Interaction of ethanol and thyroxine on mitochondria

Thyroxine can stimulate mitochondrial swelling and uncouple oxidative phosphorylation. It was observed that ethanol inhibits both spontaneous and thyroxineinduced mitochrondrial swelling. The antiswelling activity of ethanol is manifested during mitochondrial respiration both in the presence and absence of phosphorylation. Ethanol was observed only to block thyroxine-induced swelling; it could not reverse the swelling. Ethanol also blocked Ca ++ and phosphate-induced swelling. Therefore, the antiswelling activity of ethanol is more generalized than that of serum albumin. It is concluded that the ethanol antagonism of mitochrondrial swelling produced by various agents is probably dependent on the fact that ethanol alone inhibits swelling; ethanol is therefore a pharmacological antagonist of agents that induce swelling. In addition to inhibiting swelling, ethanol also blocks uncoupling of oxidative phosphorylation by thyroxine and Ca ++ but not by salicylate. These observations were interpreted to indicate that ethanol affects oxidative phosphorylation only indirectly by blocking mitochondrial swelling. The observed effects of ethanol on mitochrondria are discussed in terms of reports on the ability of thyroid hormone to antagonize acute alcohol intoxication in man, and the ability of ethanol to block effects of Ca ++ on smooth muscle.