E.J. Harris

The effect of superoxide generation on the ability of mitochondria to take up and retain Ca2

When heart or liver mitochondria are exposed to superoxide radicals generated from xanthine + xanthine oxidase their ability to take up and to retain Ca2+ is impaired. The rate of oxidation of pyruvate + malate as substrates is diminished and the appearance of thiol groups when the mitochondria are supplied with these substrates is abolished. These inhibitory effects are offset if respiration is supported by succinate in presence of rotenone provided that a substrate (β‐hydroxybutyrate) is provided to maintain the reduction of NADH. The data agree with the thesis that a generation of thiol groups is essential to maintain membrane integrity and that the generation depends on provision of reduced NAD(P)H.

Accumulation and effects of trace metal ions in fish liver mitochondria.

Abstract 1. The uptake and effects of Ca 2+ , Mn 2+ , Zn 2+ and Cu 2+ in fish liver mitochondria was studied. 2. Ca 2+ and Mn 2+ were found to be taken up by an energy-dependent process similar to that described for rat liver mitochondria. 3. Zn 2+ was taken up to a very limited extent, but caused very strong inhibition of respiration. 4. Cu 2+ was taken up by an energy-independent process, which stimulated K + uptake. 5. The implications of these findings are discussed in the context of heavy metal pollution.

The stimulation of the release of Ca2+ from mitochondria by sodium ions and its inhibition

Abstract The stimulation by Na+ of the Ca2+ efflux from mitochondria prepared from liver, kidney, heart, brain, and skeletal muscle has been compared and the effects of some inhibitors of efflux have been studied. Maximal stimulation of the efflux of Ca2+ by Na+ is obtained in the range of 8 to 15 m m . The increment of efflux evoked by Na+ is best seen when the basal efflux has been minimized by adding ADP to a suspension medium supplemented with albumin. The effect of lowering pH is to increase the basal efflux without corresponding increase of the total efflux obtained after adding Na+. Particularly with kidney mitochondria it is necessary to work at pH 7.4 or above to observe the Na+ stimulation. The Na+ stimulated efflux is inhibited by oligomycin at concentrations which inhibit oxidative phosphorylation. The Na+-stimulated efflux is not inhibited by dithiothreitol. Mg2+ inhibits the efflux after stimulation by Na+ but part (60–70%) of the efflux from liver and skeletal muscle mitochondria persists in the presence of Mg2+ at 20–40 μ m . Dibucaine and trifluoroperazine inhibit the Na+-stimulated efflux from all preparations; they produce half inhibition of efflux at about 120 and 20 μ m , respectively. It is suggested that the stimulation of efflux by Na+ involves a process involving a supply of Pi generated from membrane-bound adenine nucleotide. The emergent Ca2+ would then activate membrane phospholipase A2 whose action would cause production of lytic compounds and further membrane disturbance.

Calcium and magnesium ion losses in response to stimulants of efflux applied to heart, liver and kidney mitochondria

Abstract The efflux of Ca 2+ from previously Ca 2+ -loaded heart, liver or kidney mitochondria is accompanied by an approximately equal loss of endogenous Mg 2+ irrespective of the agent applied to stimulate efflux which may be Na + , a mercurial, a long chain fatty derivative or thyroxin. The proportion of Mg 2+ (and of the accompanying adenine nucleotide) in relation to the Ca 2+ is diminished if Mg 2+ is added to the medium. The similarity between data from different mitochondria and with different agents accords with the ion losses involving a common factor such as generation within the membrane of lysophospholipid by Ca 2+ in transit. It is shown that lysophospholecithin stimulates Ca 2+ efflux with a hyperbolic concentration dependence.

Accumulation of ornithine and citrulline in rat liver mitochondria in relation to citrulline formation.

According to Gamble and Lehninger [l] , transport of ornithine across the mitochondrial membrane is dependent upon respiratory energy. However, this conclusion is not consistent with the high rate of citrulline production found in uncoupled mitochondria incubated under anaerobic conditions [2,3]. Hence, studies were undertaken in order to examine the transport of both ornithine and citrulline across the mitochondrial membrane under both energized and uncoupled conditions. Results indicate that entry of ornithine is not energydependent and does not require protoncarrying anions but is modulated by the extramitochondrial citrulline/arginine ratio. Accumulation of citrulline in mitochondria occurs only during its formation. Inhibition of citrulline production results in a release of this amino acid from the mitochondria in both State 4 and uncoupled conditions.

The applicability of the donnan relation to the distribution of certain anions between mitochondria and medium

between anions A-, B 2and C 3-. The potential difference can arise from the dissociation of a molecule having one non-permeating ion as in the Donnan system, but the relation applies as well to potentials generated by the expenditure of metabolic energy. Although specific examples of the applicability of Donnan-like relations to certain ions in their distribution across cell membranes have long been known, the extension of the principle to small membrane-bounded systems has not attracted much attention. It was pointed out briefly that the distributions of some permeating anions across the mitochondrial membrane when they are present together, accord with the Donnan equations [ 1 ]. The present communication provides evidence that a number of anions become distributed in a way approximating to Donnan equations over a wide range of concentrations. The importance of showing that the Donnan equations apply to certain anions is that one can then predict mitochondrial/cytoplasmic ratios of other penetrant anions from knowledge of the distribution of one of them.

Specificities of cation permeabilities induced by some crown ethers in mitochondria

Abstract A series of macrocyclic crown ethers have been assayed as ionophores using respiring rat liver mitochondria, and the same compounds have been subjected to physicochemical investigation. Association constants and stoichiometries for the complexes with potassium, rubidium, and cesium were measured in methanol. Rates of transport of potassium bromide through aqueous/organic/aqueous systems and the partition coefficient from water into the organic phase were also measured and found to be in the same rank order as the association constants. Effective uptake by the mitochondria was clearly correlated with the complexing by the cation by one or more molecules of ionophore. Thus, for all three cations the most effective compound was the largest, i.e., di-t-butyldibenzo-30-crown-10, capable of wrapping around the cation. Other compounds giving measurable uptake were those which form sandwich complexes, one cation to two crown molecules; those containing five oxygen atoms induced greater uptake of potassium than of the other two alkali metals, while compounds containing six or seven oxygen atoms had greater effects on cesium or rubidium than on potassium. Within a structurally similar series of compounds the rank orders for uptake differed from those for the association constants; in particular, compounds having one benzene ring on an 18-membered crown compound were ineffective on all cations studied; effective compounds contained two benzene rings. In contrast to X-537A and A23187, none of the compounds caused release of magnesium from the mitochondria.

Inhibition of Ca2+ stimulated ion losses from mitochondria by inhibitors of calmodulin.

The efflux of Ca 2+ from heart liver and kidney mitochondria previously loaded with Ca 2+ , has been shown to be accompanied by correlated losses of other components such as Mg 2+ and adenine nucleotides. The presence of the inhibitors of calmodulin, trifluoroperazine and dinucaine (probably acting by inhibiting Ca 2+ stimulation of endogenous phospholipase A 2 ), is now shown not only to slow efflux of Ca 2+ ; but also to inhibit losses of Mg 2+ and adenine nucleotides. In the presence of the calmodulin inhibitors, the efflux of Ca 2+ tends to resemble the efflux of Sr 2+ from Sr 2+ loaded mitochondria. Since Sr 2+ does not activate phospholipase A 2 or calmodulin, all 3 results point to the involvement of calmodulin in the Ca 2+ mediated increase of mitochondrial permeability.

The proton shift across the mitochondrial membrane associated with potassium uptake induced by valinomycin

There are many respects in which the response of mitochondria to the addition of Ca2+ ions parallels that seen when a K+ uptake is induced by addition of vahnomycin in presence of an energy source. In each case a cation enters by an energy consuming process (Chance [I] for Ca2+; Moore and Pressman [2] for K+) and is accompanied to varying degrees by anions. The anion uptake is generally less than the equivalent of the cation gain because of permeability restrictions (Rossi, Scarpa and Azzone [3] ; Harris [4] ) and the electrical charge balance is preserved by movement of protons contrary to the Ca2+ or K+. If the protons derive from a hitherto unionized group in the interior each proton will leave an anion equivalent behind to help balance the cation gain. This may explain observed stoichiometry of 1 H+ ejected per Ca2+ gained (Rasmussen, Chance and Ogata [ 51). Mitchell and Moyle [6] demonstrated the produo tion of external protons in response to the energy made available when a pulse of oxygen was applied to a previously anaerobic suspension in presence of EGTA to chelate Ca2+ and valinomycin (which increases the permeability to K+). From this they argued that the proton production was more than a mere consequence of energised cation movement with its associated proton extrusion. They supposed that it represented the production of an energised state of the mitochondria. Hence it is relevant to present some observations made with a system which is already energised and in which responses are elicited by valinomycin addition (in presence of potassium ions). Such responses resemble those obtained by addition of Ca2+ ions to an energised system. In each case there

EQUILIBRATION OF CHLORIDE AND PYRUVATE DISTRIBUTIONS BETWEEN LIVER MITOCHONDRIA AND MEDIUM MEDIATED BY ORGANO-TIN SALTS

contrast to Cl-, most organic anions are internally accumulated to concentrations exceeding those outside, and such penetrant anions intercompete for occupation of the interior [3,4]. Manger [5] observed that triethyl tin sulphate, added to a suspension in a 150 mM KC1 based medium lessened the contents of organic anions. Studies of the swelling of liposomes, erythrocytes and mitochondria in response to organo-tin compounds led Selwyn et al. [6] to conclude that a Cl--for-OH- exchange was me- diated by the compounds. Taken together the two find- ings point to the organo-tin mediated Cl-entry provid- ing another permeant anion to compete with the orga- nic anions accumulated in the mitochondrial interior. This displacement of substrate anions is one possible explanation of the inhibition of respiration obtained by additions of organo-tin salts in Cl--containing me- dia [7]. The object of this work was to obtain direct evidence for the Cl- entry and to compare its extent with that of a penetrant substrate (pyruvate). It has been observed [8] that the organo-tins inhibit phos- phorylation in Cl--free media; another action unrelated to Cl- movement was sought, and this is also described. A model system was employed to confirm that the Cl--for-OH- exchange will take place across an oil layer. 2. Methods Rat liver mitochondria were prepared as described before [9], and finally suspended in 0.25 M sucrose. The anion distribution studies were made at 20” with 3-5 mg protein/ml suspended in 150 mM potassium methane sulphonate, 20 mM Tris methane sulphonate and 50 mM sucrose at pH 6.8. Trace amounts of tri- tiated water were added, along with about 0.5 mM 36C1 and 0.5 mM pyruvate as Tris salts. In a few ex- periments, [ 14C] dextran, [ 14C]sucrose, or [14C]- ADP were substituted for the 36C1. The distribution of solutes between the medium and the mitochondrial pellet was found after separation of the suspension by centrifugation in Coleman Microfuges in tubes pre- loaded with 1.5 M perchloric acid beneath a layer of silicone oil (General Electric Versilube F.50) as des- cribed before [5]. Pyruvate was measured by enzyma- tic assay, phosphate by extraction of phosphomolyb- date in isopropyl acetate [lo]. ADP entry was measu- red by incubating the suspension with [14C] ADP in presence of hexokinase (2 units), MgCl, (0.5 mM) and glucose (5 mM) in the medium. The movement was stopped by adding atractylate to 10 PM [ 111. The mito- chondria were separated as described to find their con- tent of 14C-labelled nucleotide. Model experiments to investigate the counter move- ments of Cl- and OH- were made in the apparatus sketched in fig. 1. The aqueous media in the two U- tubes were in communication only via the oil layer at their common top. The contents of the U’s and the floating oil layer were stirred. One U was charged with 3 M KC1 buffered with 0.1 M Tris chloride to pH 7.4. 339