The interaction of organic cations with the mitochondrial membrane

Summary Weak and strong organic bases behave in an opposite manner in respect to several mitochondrial functions. The former induce a catalytic exchange with K⁺ in valinomycon-treated, respiratory-inhibited mitochondria, and act as uncouplers in respiring mitochondria. The latter induce a stoicheometric exchange with K⁺ and are actively taken up by respiring mitochondria.     

Control of respiration by nitric oxide in Keilin-Hartree particles,mitochondria and SH-SY5Y neuroblastoma cells

The pattern of cytochrome c oxidase inhibition by nitric oxide (NO) was investigated polarographically using Keilin-Hartree particles, mitochondria and human neuroblastoma cells. NO reacts with purified cytochrome c oxidase forming either a nitrosyl- or a nitrite-inhibited derivative, displaying distinct kinetics and light sensitivity of respiration recovery in the absence of free NO. Keilin-Hartree particles or cells, respiring either on endogenous substrates alone or in the presence of ascorbate, as well as state 3and state 4mitochondria respiring on glutamate and malate, displayed the rapid recovery characteristic of the nitrite derivative. All systems, when respiring in the presence of tetramethyl-p-phenylenediamine, were characterised by the slower, light-sensitive recovery typical of the nitrosyl derivative. Together the results suggest that the reaction of NO with cytochrome c oxidase in situ follows two alternative inhibition pathways, depending on the electron flux through the respiratory chain.Received 1 April 2003; received after revision 22 May 2003; accepted 3 June 2003     

Die Wirkung von Persantin auf Herzmuskel-Sarkosomen

Summary Addition of Persantin to respiring beef heart mitochondria leads to a considerable increase of the P/O-ratios in the presence of various substrates. Apparently Persantin is capable of bringing about a tighter coupling of oxidation and phosphorylation.     

Altered K+ movement in liver mitochondria from alloxan diabetic rats

Potassium movements were monitored in liver mitochondria from control and alloxan diabetic rats with a cationic electrode. There was net accumulation of K+ after Ca2+ addition to the mitochondria with the diabetic but not with the control.     

Cardiac cellular electrophysiology: past and present

The time-course of the cardiac action potential can be accounted for in terms of ionic currents crossing the cell membranes. Depolarizing current is carried by Na+ or Ca2+ entering the cells, repolarizing current by K+ leaving the cells. Membrane permeability for the passive movement of these ions is thought to be voltage-dependent as well as time-dependent. Net transfer of charge may also result from active transport, 2 Na+ out against 1 K+ in; or coupled exchange, 3 or 4 Na+ in against 1 Ca2+ out. This review follows the path by which present-day knowledge has been reached. It also gives a few examples to illustrate that electrophysiology has provided concepts useful to clinical cardiology.     

Anoxygene Reaktivierung der Zellvermehrung nach anoxybiotisch hervorgerufener Zytostase (Anabiose)

Summary In cell juice of aerobic fermenting and of only respiring yeast cells, a thermostable respiratory energy reserve is found which under rigorous anoxybiotic conditions effects a generative reactivation in contact with anabiotic fermenting yeast cells, butnot with only respiring ones. Also in cell juice of Jensen sarcoma and other carcinoma tissues, an adequate energy substance is recognized as being able to replace oxygen biologically.     

Influence of reactive oxygen species production by monoamine oxidase activity on aluminum-induced mitochondrial permeability transition

Treatment of Ca2+-loaded mitochondria with both aluminum and tyramine results in a swelling of higher amplitude than with aluminum alone, while tyramine alone is ineffective. The phenomenon is accompanied by H2O2 production and thiol and pyridine nucleotide oxidation. Cyclosporin A, N-ethylmaleimide or dithioerythritol completely prevent these effects, while catalase exhibits a lower inhibition, pointing to the induction of the permeability transition (MPT) by an oxidative stress. Reactive oxygen species are generated by the interaction of aluminum with the inner membrane and the oxidation of tyramine by monoamine oxidase on the outer membrane. This different localization determines the oxidation of critical thiol groups located on both internal and external sides of pore-forming structures, resulting in MPT induction. The reduced effect by aluminum or the inefficacy by tyramine, when implied alone, can be attributable to the oxidation of thiol groups located only on the internal or external side, respectively. Ultrastructural observations show that aluminum plus tyramine induce the typical configuration of mitochondria that have undergone the MPT. Instead, with aluminum alone, the sensitive subpopulation, although swollen, preserves the outer membrane and shows an apparently orthodox configuration.     

Ceramides induce apoptosis in HeLa cells and enhance cytochrome c-induced apoptosis in Xenopus egg extracts

Ceramide has been reported to induce typical apoptotic changes in nuclei incubated in a cell-free system, and that the addition of ceramide bypasses the requirement for mitochondria. Here, we explore the possible pathways by which ceramide induces apoptosis either in intact cells or in a cell-free system which we have developed. We found that in the cell-free system, C2-ceramide is not able to induce apoptosis in nuclei whereas cytochrome c does, but it is able to induce HeLa cells to undergo apoptosis. Ceramide is also not able to induce apoptosis when added into the cell-free system together with purified mitochondria. Further investigation showed that C2-ceramide at certain concentrations greatly increases nuclear apoptosis caused by cytochrome c in the cell-free system. From these results we conclude that the induction of apoptosis by ceramide may require intact cells in which some unknown signal transduction pathways are involved.     

Non-enzymatic active transport in vitro. Conversion of chemical to osmotic energy]

The active transport of ammonia gas was obtained with an asymmetric composite membrane-model using H+ as carrier and the formation of water as chemical energy source. The experimental molecular pump externally limited by two silicone films permeable to gasses but not to liquids or ions is separated into two liquid layers by a central cation-exchange membrane. The first acid layer produces the aspiration of NH3, due to NH4+ formation, through the inlet silicone film; NH4+ is transferred to the second alkaline layer through the ion exchange membrane, by interdiffusion with K+; there NH4+ reacts with OH-, water is formed and NH3 is expelled throught the oulet silicone film. This new pump enlarges the applicability of in vitro active transport to non-enzymatic systems and energy sources different from ATP.     

Peroxovanadate inhibits Ca2+ release from mitochondria

Mitochondria contain a specific Ca²⁺ release pathway which operates when oxidized mitochondrial pyridine nucleotides are hydrolyzed. NAD⁺ hydrolysis and therefore Ca²⁺ release is possible when some vicinal thiols are cross-linked. Here we report that the thiol oxidant peroxovanadate inhibits the specific Ca²⁺ release pathway. In mitochondria, peroxovanadate causes a complete loss of reduced glutathione, which is not accompanied by formation of glutathione disulfide, and a partial loss of protein thiols. In model reactions, peroxovanadate oxidizes reduced glutathione predominantly to the sulfonate derivative, but does not react with glutathione disulfide. When the vicinal thiols relevant for Ca²⁺ release are cross-linked, Ca²⁺ release is no longer inhibited by peroxovanadate. Conversely, pretreatment of mitochondria with peroxovanadate makes them insensitive to compounds promoting the disulfide state. These results suggest that peroxovanadate inhibits the prooxidant-induced Ca²⁺ release from mitochondria by (i) depleting mitochondria of reduced glutathione and (ii) oxidizing the vicinal thiols relevant for Ca²⁺ release to a state higher than disulfide, presumably the sulfonate state. The findings provide further insight into the regulation of Ca²⁺ release from intact mitochondria, and may be relevant for a better understanding of the action of peroxovanadate in cells, where the compound can be insulin mimetic. Received 28 March 2002; received after revision 8 May 2002; accepted 15 May 2002     

Purification of a membrane protein modifying the sensitivity of Na+/K+ ATPase to ouabain from a supernatant of plasma membrane treated with EDTA]

A membrane protein of M. W. 32,000 D and pI 5 has been purified from EDTA-treated plasma membrane supernatants by Page without detergents. These membranes are purified from the MF2S cell line issued from the murine plasmacytoma MOPC 173. The purified protein was able to induce a 300 fold increase in the Na+/K+ ATPase resistance to ouaba?n thus mimicking the original resistance of the enzyme to the drug.     

Altered K+ movement in liver mitochondria from alloxan diabetic rats

Summary Potassium movements were monitored in liver mitochondria from control and alloxan diabetic rats with a cationic electrode. There was net accumulation of K⁺ after Ca²⁺ addition to the mitochondria with the diabetic but not with the control.     

Geranylgeranylacetone protects human monocytes from mitochondrial membrane depolarization independently of Hsp70 expression

The anti-ulcer drug geranylgeranylacetone (GGA) has been shown to induce the expression of heat shock proteins (HSPs), in particular of Hsp70, in gastric and small intestine cells. In this study, we investigated whether GGA was able to induce Hsp70 in another cell type, human monocytes, which represent a well-established model of Hsp70 expression under oxidative stress. In these cells, GGA had no significant effect either on basal or tobacco smoke-induced Hsp70 expression. We further investigated the effects of GGA on mitochondria, a key organelle of oxidant-mediated cell injury and a putative target for GGA-mediated protection. GGA significantly increased basal mitochondrial membrane polarization and inhibited the decrease in mitochondrial membrane potential of human monocytes exposed to distinct sources of clinically relevant oxidants such as tobacco smoke and y-irradiation. Our results indicate that mitochondria are targets for GGA-mediated protection against oxidative stress in human monocytes, independently of Hsp70.     

Sodium/calcium exchange in ventricular muscle

Ventricular cells possess two Ca extrusion mechanisms, a Na/Ca exchange system and a Ca pump. Reversing the exchanger by extracellular Na removal causes [Na]i to decrease, and the cells take up mmolar quantities of calcium. Since [Ca]i shows only a marginal increase the calcium load must be buffered. The capacity of the SR is limited so the mitochondria probably buffer a large part of this load. However, when Ca uptake into the mitochondria is blocked, the gain in Ca is still mmolar and the increase in [Ca]i still marginal, suggesting an additional buffering site. Measurements of the Na/Ca stoichiometry on sarcolemmal vesicles gave a value of 3, but in ventricle values of around 2.5 or 3 are found. Reasons for this are discussed, as are the differences amongst the different methods of Ca measurement. The interaction of the sarcolemmal Ca pump and the exchanger are considered and it is suggested they could interact via [Na]i. At rest both systems could remove Ca from the cell but on a large perturbation the Na/Ca exchange would be the more important of the two.     

Can blocking the Na/K exchange pump lead to a reduction in intracellular sodium?

It has been assumed that a rise in intracellular sodium should follow inhibition of the Na/K exchange pump. However, under certain conditions a reduction in intracellular sodium following pump blockage is possible. Many results postulating 'stimulation' of the Na/K exchange pump by low doses of the cardiac glycosides can be explained in this manner.     

Homologous recombination-mediated repair of DNA double-strand breaks operates in mammalian mitochondria

Mitochondrial DNA is frequently exposed to oxidative damage, as compared to nuclear DNA. Previously, we have shown that while microhomology-mediated end joining can account for DNA deletions in mitochondria, classical nonhomologous DNA end joining, the predominant double-strand break (DSB) repair pathway in nucleus, is undetectable. In the present study, we investigated the presence of homologous recombination (HR) in mitochondria to maintain its genomic integrity. Biochemical studies revealed that HR-mediated repair of DSBs is more efficient in the mitochondria of testes as compared to that of brain, kidney and spleen. Interestingly, a significant increase in the efficiency of HR was observed when a DSB was introduced. Analyses of the clones suggest that most of the recombinants were generated through reciprocal exchange, while ~ 30% of recombinants were due to gene conversion in testicular extracts. Colocalization and immunoblotting studies showed the presence of RAD51 and MRN complex proteins in the mitochondria and immunodepletion of MRE11, RAD51 or NIBRIN suppressed the HR-mediated repair. Thus, our results reveal importance of homologous recombination in the maintenance of mitochondrial genome stability.     

Contribution of the Na+/K+-pump to the membrane potential

The inward movement of sodium ions and the outward movement of potassium ions are passive and the reverse movements against the electrochemical gradients require the activity of a metabolism-driven Na+/K+-pump. The activity of the Na+/K+-pump influences the membrane potential directly and indirectly. Thus, the maintenance of a normal electrical function requires that the Na+/K+-pump maintain normal ionic concentrations within the cell. The activity of the Na+/K+-pump also influences the membrane potential directly by generating an outward sodium current that is larger when the Na+/K+-pump activity is greater. The activity of the Na+/K+-pump is regulated by several factors including the intracellular sodium concentration and the neuromediators norepinephrine and acetylcholine. The inhibition of the Na+/K+-pump can lead indirectly to the development of inward currents that may cause repetitive activity. Therefore, the Na+/K+-pump modifies the membrane potential in different ways both under normal and abnormal conditions and influences in an essential way many cardiac functions, including automaticity, conduction and contraction. Key words. Active transport of ions; cardiac tissues; electroneutral and electrogenic Na+/K/-pump; control of Na+/K+-pump; normal and abnormal electrical events.     

Evidence of abnormalities in net sodium and potassium fluxes in erythrocytes of patients with essential hypertension]

A new and simple laboratory test for measuring net Na+ and K+ fluxes in Na+-loaded/K+-depleted human erythrocytes was developed and applied to hypertension. Moderate essential hypertension was characterized by a constant increase in net K+ influx; more severe cases showed a drop in net Na+ efflux. Na+ and K+ erythrocyte fluxes were found to be normal in hypertension of renal origin.     

Differential sensitivity of amphibian nodal and paranodal K+ channels to 4-aminopyridine and TEA

Voltage-dependent K+ channels are blocked by several drugs, including 4-aminopyridine (4-AP) and tetraethylammonium (TEA). 4-AP is most widely used to localize K+ channels in mammalian and non-mammalian nerve fibers, but 4-AP and TEA alter various K+ channels and/or preparations in specific ways. The reason is not known, in part because dissociation constants for 4-AP and TEA have not been measured for nodal and internodal K+ channels in the same fibers. Smith and Schauf showed that the density of nodal versus paranodal K+ channels in frog nerves depends on fiber diameter. The size dependence was used to determine the relative sensitivity of nodal and internodal K+ channels to 4-AP and TEA, and to compare voltage- and time-dependent activation. The results show nodal and internodal K+ channels activate similarly. However, internodal channels are selectivity blocked by 4-AP while TEA is more effective on nodal channels. A high sensitivity of internodal K+ channels may explain why 4-AP improves symptoms in diseases such as multiple sclerosis.     

Enhancement of lipid peroxidation in rat brain mitochondria by polyamines

Summary Ascorbic acid-induced lipid peroxidation in rat brain mitochondria is enhanced by the addition of spermine and spermidine.Acknowledgement. The author is deeply grateful to Dr U. K. Vakil for her kind interest and encouragement during the course of this work.