Effects of Tl<Superscript>+</Superscript> on ion permeability,membrane potential and respiration of isolated rat liver mitochondria

It is known that permeability of the inner mitochondrial membrane is low to most univalent cations (K⁺, Na⁺, H⁺) but high to Tl⁺. Swelling, state 4, state 3, and 2,4-dinitrophenol (DNP)-stimulated respiration as well as the membrane potential (ΔΨ_mito) of rat liver mitochondria were studied in media containing 0–75 mM TlNO₃ either with 250 mM sucrose or with 125 mM nitrate salts of other monovalent cations (KNO₃, or NaNO₃, or NH₄NO₃). Tl⁺ increased permeability of the inner mitochondrial membrane to K⁺, Na⁺, and H⁺, that was manifested as stimulation of the swelling of nonenergized and energized mitochondria as well as via an increase of state 4 and dissipation of ΔΨ_mito. These effects of Tl⁺ increased in the order of sucrose <K⁺ <Na⁺ ≤ NH₄⁺. They were stimulated by inorganic phosphate and decreased by ADP, Mg²⁺, and cyclosporine A. Contraction of energized mitochondria, swollen in the nitrate media, was markedly inhibited by quinine. It suggests participation of the mitochondrial K⁺/H⁺ exchanger in extruding of Tl⁺-induced excess of univalent cations from the mitochondrial matrix. It is discussed that Tl⁺ (like Cd²⁺ and other heavy metals) increases the ion permeability of the inner membrane of mitochondria regardless of their energization and stimulates the mitochondrial permeability transition pore in low conductance state. The observed decrease of state 3 and DNP-stimulated respiration in the nitrate media resulted from the mitochondrial swelling rather than from an inhibition of respiratory enzymes as is the case with the bivalent heavy metals.     

Functional identification of ATP-sensitive K<Superscript>+</Superscript> uniporter in mitochondria from sugar beet taproot

Mitochondria isolated from sugar beet (Beta vulgaris L.) taproot were shown to swell spontaneously after the transfer from a sucrose-containing isolation medium to isoosmotic potassium chloride solutions. The kinetics of this process was strongly retarded after the replacement of potassium with sodium in the incubation medium and was substantially stimulated by the electron-transport chain activity and valinomycin. At neutral pH of the incubation medium, the rate of K⁺-dependent swelling of mitochondria decreased by 30–50% after adding 1 mM ATP but was insensitive to other nucleotides (GTP, UTP, and CTP). In the medium acidified to pH 6.0, the addition of ATP caused shrinkage of mitochondria that had been swollen in the KCl medium. In the absence of this nucleotide, the kinetics of K⁺-dependent swelling of mitochondria was considerably decelerated upon the acidification of the incubation medium. The effects of ATP were independent of the presence or absence of oligomycin and atractyloside. However, the ATP-dependent shrinkage of mitochondria was inhibited in the presence of quinine, and this agent also inhibited K⁺-dependent swelling of organelles in potassium acetate solutions. The presence of K⁺ ions in the incubation medium caused a rapid dissipation of the mitochondrial membrane potential () that was generated during succinate oxidation. The addition of ATP to the reaction medium resulted in the oligomycin-insensitive restoration of . The results are regarded as evidence that the membrane of taproot mitochondria is endowed with functionally active ATP-sensitive K⁺ uniporter. This system is likely to represent a K⁺ channel that catalyzes the electrogenic transfer of potassium ions to the mitochondrial matrix. It is supposed that the membrane of taproot mitochondria also contains a quinine-sensitive K⁺/H⁺ antiporter that catalyzes the efflux of potassium from the matrix or, on the contrary, the accumulation of K⁺ in the presence of potassium acetate.Translated from Fiziologiya Rastenii, Vol. 52, No. 2, 2005, pp. 209–215.Original Russian Text Copyright © 2005 by Shugaev, Andreev, Vyskrebentseva.This revised version was published online in April 2005 with a corrected cover date.     

Influence of ATP-dependent K<Superscript>+</Superscript>-channel opener on K<Superscript>+</Superscript>-cycle and oxygen consumption in rat liver mitochondria

The influence of the K_ATP⁺-channel opener diazoxide on the K⁺ cycle and oxygen consumption has been studied in rat liver mitochondria. It was found that diazoxide activates the K_ATP⁺-channel in the range of nanomolar concentrations (50–300 nM, K _1/2 ∼ 140 nM), which results in activation of K⁺/H⁺ exchange in mitochondria. The latter, in turn, accelerates mitochondrial respiration in respiratory state 2. The contribution of K_ATP⁺-channel to the mitochondrial potassium cycle was estimated using the selective K_ATP⁺-channel blocker glibenclamide. The data show that the relative contribution of K_ATP⁺-channel in the potassium cycle of mitochondria is variable and increases only with the decrease in the ATP-independent component of K⁺ uptake. Possible mechanisms underlying the observed phenomena are discussed. The experimental results more fully elucidate the role of K_ATP⁺-channel in the regulation of mitochondrial functions, especially under pathological conditions accompanied by impairment of the mitochondrial energy state.     

THE PENETRATION OF THE MEMBRANE OF BRAIN MITOCHONDRIA BY ANIONS

The permeability of the membrane of rat brain non-synaptosomal mitochondria, towards inorganic and substrate anions, was assessed by measuring the rate of swelling that occurred when mitochondria were suspended in an iso-osmotic solution of a permeant anion, in the presence of a permeant cation such as NH⁺₄ or K⁺ in the presence or absence of valinomycin. In NH⁺₄-phosphate swelling was higher than it was in KCI or K⁺-phosphate, which showed the prevalence of the mechanism of phosphate transport previously demonstrated in liver mitochondria. The entry of succinate and L-malate seemed to require the presence in the inner mitochondrial membrane of specific carriers. as previously postulated for liver mitochondria, but the rate of swelling of brain mitochondria was lower than that of liver organelles. In K⁺-succinate, in the presence of antimycin, added ATP induced swelling and this was attributable to the simultaneous permeation both of the anion and the cation. Fumarate did not penetrate into brain mitochondria. Practically no swelling was recorded in NH⁺₄ or K⁺-citrate, which indicated that this anion penetrated poorly into the isolated brain mitochondria even in the presence of malate. Swelling occurred in NH⁺₄-L-glutamate in the presence of rotenone, and the entry of this anion seemed to follow a gradient of concentration although the presence of a specific translocator in the inner mitochondrial membrane might be concerned. The entry of glutamate was independent of that of phosphate and N-ethylmaleimide appeared to be a specific inhibitor of this entry. Swelling in K⁺-L-glutamate, in the presence of rotenone, was enhanced by the addition of valinomycin or ATP but in the latter case when osmotic equilibrium was reached swelling was not reversed by oligomycin. In conclusion, the lesser extent of swelling of isolated brain mitochondria compared with liver mitochondria could be attributed to the heterogeneity of the populations of these organelles, each population possessing its own characteristics of membrane permeability. Observations of electron micrographs of brain mitochondria incubated in iso-osmotic substrate anions confirmed the heterogeneous rate of swelling of these particles.     

Stage-Specific Changes in the Water,Na+, Cl- and K+ Contents of Organelles during Apoptosis,Demonstrated by a Targeted Cryo Correlative Analytical Approach

Many studies have demonstrated changes in the levels of several ions during apoptosis, but a few recent studies have reported conflicting results concerning the changes in water content in apoptotic cells. We used a correlative light and cryo-scanning transmission electron microscopy method to quantify water and ion/element contents simultaneously at a nanoscale resolution in the various compartments of cells, from the onset to the end of apoptosis. We used stably transfected HeLa cells producing H2B-GFP to identify the stages of apoptosis in cells and for a targeted elemental analysis within condensed chromatin, nucleoplasm, mitochondria and the cytosol. We found that the compartments of apoptotic cells contained, on average, 10% more water than control cells. During mitochondrial outer membrane permeabilization, we observed a strong increase in the Na⁺ and Cl^- contents of the mitochondria and a strong decrease in mitochondrial K⁺ content. During the first step in apoptotic volume decrease (AVD), Na⁺ and Cl^- levels decreased in all cell compartments, but remained higher than those in control cells. Conversely, during the second step of AVD, Na⁺ and Cl^- levels increased considerably in the nucleus and mitochondria. During these two steps of AVD, K⁺ content decreased steadily in all cell compartments. We also determined in vivo ion status during caspase-3 activity and chromatin condensation. Finally, we found that actinomycin D-tolerant cells had water and K⁺ contents similar to those of cells entering apoptosis but lower Na⁺ and Cl^- contents than both cells entering apoptosis and control cells.     

Ionophorous Properties of SY-1 (20-Deoxysalinomycin) in Rat Liver Mitochondri

SY-1 (20-deoxysalinomycin), a monocarboxylic polyether antibiotic closely related to salinomycin, caused a rapid release of previously accumulated alkali metal cations by valinomycin or monazomycin in rat liver mitochondria, and simultaneously reversed swelling of mitochondria.

With a strict specificity for substrate and cation, SY-1 exhibited a property of inhibiting mitochondrial functions such as substrate oxidation, oxidative phosphorylation and ATP hydrolysis induced by valinomycin or monazomycin, In comparative study with salinomycin, SY-1 was found to be more effective on the mitochondrial functions than salinomycin.

On the basis of the results so far obtained, the inhibitory effect of SY-1 on mitochondria is interpreted as a result of interaction with essential cations, especially with K⁺, in mitochondria.     

Pathways for K<Superscript>+</Superscript> Efflux in Isolated Surface and Crypt Colonic Cells. Activation by Calcium

K⁺-conductive pathways were evaluated in isolated surface and crypt colonic cells, by measuring ⁸⁶Rb efflux. In crypt cells, basal K⁺ efflux (rate constant: 0.24 ± 0.044 min⁻¹, span: 24 ± 1.3%) was inhibited by 30 mM TEA and 5 mM Ba²⁺ in an additive way, suggesting the existence of two different conductive pathways. Basal efflux was insensitive to apamin, iberiotoxin, charybdotoxin and clotrimazole. Ionomycin (5 μM) stimulated K⁺ efflux, increasing the rate constant to 0.65 ± 0.007 min⁻¹ and the span to 83 ± 3.2%. Ionomycin-induced K⁺ efflux was inhibited by clotrimazole (IC₅₀ of 25 ± 0.4 μM) and charybdotoxin (IC₅₀ of 65 ± 5.0 nM) and was insensitive to TEA, Ba²⁺, apamin and iberiotoxin, suggesting that this conductive pathway is related to the Ca²⁺-activated intermediate-conductance K⁺ channels (IK_ca). Absence of extracellular Ca²⁺ did neither affect basal nor ionomycin-induced K⁺ efflux. However, intracellular Ca²⁺ depletion totally inhibited the ionomycin-induced K⁺ efflux, indicating that the activation of these K⁺ channels mainly depends on intracellular calcium liberation. K⁺ efflux was stimulated by intracellular Ca²⁺ with an EC₅₀ of 1.1 ± 0.04 μM. In surface cells, K⁺ efflux (rate constant: 0.17 ± 0.027 min⁻¹; span: 25 ± 3.4%) was insensitive to TEA and Ba²⁺. However, ionomycin induced K⁺ efflux with characteristics identical to that observed in crypt cells. In conclusion, both surface and crypt cells present IK_Ca channels but only crypt cells have TEA- and Ba²⁺-sensitive conductive pathways, which would determine their participation in colonic K⁺ secretion.     

Swelling-Induced Ca<Superscript>2+</Superscript> Influx and K<Superscript>+</Superscript> Efflux in American Alligator Erythrocytes

The American alligator can hibernate during winter, which may lead to osmotic imbalance because of reduced kidney function and lack of food consumption during this period. Accordingly, we hypothesized that their red blood cells would have a well-developed regulatory volume decrease (RVD) to cope with the homeostatic challenges associated with torpor. Osmotic fragility was determined optically, mean cell volume was measured by electronic sizing, and changes in intracellular Ca²⁺ concentration were visualized using fluorescence microscopy and fluo-4-AM. Osmotic fragility increased and the ability to regulate volume was inhibited when extracellular Na⁺ was replaced with K⁺, or when cells were exposed to the K⁺ channel inhibitor quinine, indicating a requirement of K⁺ efflux for RVD. Addition of the ionophore gramicidin to the extracellular medium decreased osmotic fragility and also potentiated volume recovery, even in the presence of quinine. In addition, hypotonic shock (0.5× Ringer) caused an increase in cytosolic Ca²⁺, which resulted from Ca²⁺ influx because it was not observed when extracellular Ca²⁺ was chelated with EGTA (ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid). Furthermore, cells loaded with BAPTA-AM (1,2-bis(2-aminophenoxymethyl)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl) ester) or exposed to a low Ca²⁺-EGTA hypotonic Ringer had a greater osmotic fragility and also failed to recover from cell swelling, indicating that extracellular Ca²⁺ was needed for RVD. Gramicidin reversed the inhibitory effect of low extracellular Ca²⁺. Finally, and surprisingly, the Ca²⁺ ionophore A23187 increased osmotic fragility and inhibited volume recovery. Taken together, our results show that cell swelling activated a K⁺ permeable pathway via a Ca²⁺-dependent mechanism, and this process mediated K⁺ loss during RVD.     

Mitochondrial Dysfunction Induced by Honokiol

Honokiol has shown the ability to induce the apoptosis of several different cancer cell lines. Considering that mitochondria are involved in apoptosis, the aim of the present work was to investigate the effects of honokiol on mitochondria. The effects of honokiol on the permeability of H⁺ and K⁺, membrane potential, membrane fluidity, respiration and swelling of mitochondria isolated from the rat liver were assessed. The results show that honokiol can significantly induce mitochondrial swelling, decrease membrane potential and affect the respiration of mitochondria. Meanwhile, honokiol does not have a direct effect on the mitochondrial permeability transition pore.     

Changes in Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase Activity and Alpha 3 Subunit Expression in CNS After Administration of Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase Inhibitors

The expression of Na⁺, K⁺-ATPase α3 subunit and synaptosomal membrane Na⁺, K⁺-ATPase activity were analyzed after administration of ouabain and endobain E, respectively commercial and endogenous Na⁺, K⁺-ATPase inhibitors. Wistar rats received intracerebroventricularly ouabain or endobain E dissolved in saline solution or Tris–HCl, respectively or the vehicles (controls). Two days later, animals were decapitated, cerebral cortex and hippocampus removed and crude and synaptosomal membrane fractions were isolated. Western blot analysis showed that Na⁺, K⁺-ATPase α3 subunit expression increased roughly 40% after administration of 10 or 100 nmoles ouabain in cerebral cortex but remained unaltered in hippocampus. After administration of 10 μl endobain E (1 μl = 28 mg tissue) Na⁺, K⁺-ATPase α3 subunit enhanced 130% in cerebral cortex and 103% in hippocampus. The activity of Na⁺, K⁺-ATPase in cortical synaptosomal membranes diminished or increased after administration of ouabain or endobain E, respectively. It is concluded that Na⁺, K⁺-ATPase inhibitors modify differentially the expression of Na⁺, K⁺-ATPase α3 subunit and enzyme activity, most likely involving compensatory mechanisms.     

Modulation of mitochondrial K permeability and reactive oxygen species production by the p13 protein of human T-cell leukemia virus type 1

Human T-cell leukemia virus type-1 (HTLV-1) expresses an 87-amino acid protein named p13 that is targeted to the inner mitochondrial membrane. Previous studies showed that a synthetic peptide spanning an alpha helical domain of p13 alters mitochondrial membrane permeability to cations, resulting in swelling. The present study examined the effects of full-length p13 on isolated, energized mitochondria. Results demonstrated that p13 triggers an inward K⁺ current that leads to mitochondrial swelling and confers a crescent-like morphology distinct from that caused by opening of the permeability transition pore. p13 also induces depolarization, with a matching increase in respiratory chain activity, and augments production of reactive oxygen species (ROS). These effects require an intact alpha helical domain and strictly depend on the presence of K⁺ in the assay medium. The effects of p13 on ROS are mimicked by the K⁺ ionophore valinomycin, while the protonophore FCCP decreases ROS, indicating that depolarization induced by K⁺ vs. H⁺ currents has different effects on mitochondrial ROS production, possibly because of their opposite effects on matrix pH (alkalinization and acidification, respectively). The downstream consequences of p13-induced mitochondrial K⁺ permeability are likely to have an important influence on the redox state and turnover of HTLV-1-infected cells.     

Increased Potassium Conductance of Brain Mitochondria Induces Resistance to Permeability Transition by Enhancing Matrix Volume

Modulation of K⁺ conductance of the inner mitochondrial membrane has been proposed to mediate preconditioning in ischemia-reperfusion injury. The mechanism is not entirely understood, but it has been linked to a decreased activation of mitochondrial permeability transition (mPT). In the present study K⁺ channel activity was mimicked by picomolar concentrations of valinomycin. Isolated brain mitochondria were exposed to continuous infusions of calcium. Monitoring of extramitochondrial Ca²⁺ and mitochondrial respiration provided a quantitative assay for mPT sensitivity by determining calcium retention capacity (CRC). Valinomycin and cyclophilin D inhibition separately and additively increased CRC. Comparable degrees of respiratory uncoupling induced by increased K⁺ or H⁺ conductance had opposite effects on mPT sensitivity. Protonophores dose-dependently decreased CRC, demonstrating that so-called mild uncoupling was not beneficial per se. The putative mitoK_ATP channel opener diazoxide did not mimic the effect of valinomycin. An alkaline matrix pH was required for mitochondria to retain calcium, but increased K⁺ conductance did not result in augmented ΔpH. The beneficial effect of valinomycin on CRC was not mediated by H₂O₂-induced protein kinase Cϵ activation. Rather, increased K⁺ conductance reduced H₂O₂ generation during calcium infusion. Lowering the osmolarity of the buffer induced an increase in mitochondrial volume and improved CRC similar to valinomycin without inducing uncoupling or otherwise affecting respiration. We propose that increased potassium conductance in brain mitochondria may cause a direct physiological effect on matrix volume inducing resistance to pathological calcium challenges.     

Clozapine Administration Modifies Neurotensin Effect on Synaptosomal Membrane Na<Superscript>+</Superscript>, K<Superscript>+</Superscript> -ATPase Activity

Na⁺, K⁺-ATPase is inhibited by neurotensin, an effect which involves the peptide high affinity receptor (NTS1). Neurotensin effect on cerebral cortex synaptosomal membrane Na⁺, K⁺-ATPase activity of rats injected i.p. with antipsychotic clozapine was studied. Whereas 3.5 × 10⁻⁶ M neurotensin decreased 44% Na⁺, K⁺-ATPase activity in the controls, the peptide failed to modify enzyme activity 30 min after a single 3.0, 10.0 and 30.0 mg/kg clozapine dose. Neurotensin decreased Na⁺, K⁺-ATPase activity 40 or 20% 18 h after 3.0 or 5.6 mg/kg clozapine administration, respectively, and lacked inhibitory effect 18 h after 17.8 and 30.0 mg/kg clozapine doses. Results indicated that the clozapine treatment differentially modifies the further effect of neurotensin on synaptosomal membrane Na⁺, K⁺-ATPase activity according to time and dose conditions employed. Taken into account that clozapine blocks the dopaminergic D2 receptor, findings obtained favor the view of an interplay among neurotensinergic receptor, dopaminergic D2 receptor and Na⁺, K⁺-ATPase at synaptic membranes.     

Mechanisms of passive potassium influx in corn mitochondria

Corn mitochondria in 100 millimolar KCl show accelerated passive swelling upon addition of uncoupler. This unusual response has been compared with swelling produced by valinomycin, tripropyltin, and nigericin. It is concluded that the driving force for swelling lies with the chloride gradient and a high P_Cl:P_K ratio, the chloride influx creating a negative membrane potential. The action of uncoupler is to facilitate K⁺ influx via the endogenous H⁺/K⁺ antiporter. The antiporter is active over the pH range 6 to 8, is not sensitive to Mg²⁺ concentration, and is not inactivated by aging. It is not clear why corn mitochondria show this exceptional activity of the H⁺/K⁺ antiporter in K⁺ influx. It is speculated that during isolation the antiporter may be exposed or activated, and that it contributes to cyclic K⁺ transport and high State 4 respiration rates.     

Energetic performance is improved by specific activation of K fluxes through KCa channels in heart mitochondria

Mitochondrial volume regulation depends on K⁺ movement across the inner membrane and a mitochondrial Ca²⁺-dependent K⁺ channel (mitoK_Ca) reportedly contributes to mitochondrial K⁺ uniporter activity. Here we utilize a novel K_Ca channel activator, NS11021, to examine the role of mitoK_Ca in regulating mitochondrial function by measuring K⁺ flux, membrane potential (ΔΨ_m), light scattering, and respiration in guinea pig heart mitochondria. K⁺ uptake and the influence of anions were assessed in mitochondria loaded with the K⁺ sensor PBFI by adding either the chloride (KCl), acetate (KAc), or phosphate (KH₂PO₄) salts of K⁺ to energized mitochondria in a sucrose-based medium. K⁺ fluxes saturated at ∼ 10 mM for each salt, attaining maximal rates of 172 ± 17, 54 ± 2.4, and 33 ± 3.8 nmol K⁺/min/mg in KCl, KAc, or KH₂PO₄, respectively. NS11021 (50 nM) increased the maximal K⁺ uptake rate by 2.5-fold in the presence of KH₂PO₄ or KAc and increased mitochondrial volume, with little effect on ΔΨ_m. In KCl, NS11021 increased K⁺ uptake by only 30% and did not increase volume. The effects of NS11021 on K⁺ uptake were inhibited by the K_Ca toxins charybdotoxin (200 nM) or paxilline (1 μM). Fifty nanomolar of NS11021 increased the mitochondrial respiratory control ratio (RCR) in KH₂PO₄, but not in KCl; however, above 1 μM, NS11021 decreased RCR and depolarized ΔΨ_m. A control compound lacking K_Ca activator properties did not increase K⁺ uptake or volume but had similar nonspecific (toxin-insensitive) effects at high concentrations. The results indicate that activating K⁺ flux through mitoK_Ca mediates a beneficial effect on energetics that depends on mitochondrial swelling with maintained ΔΨ_m.     

Anilinonaphthalenesulfonate fluorescence changes induced by non-enzymatic generation of membrane potential in mitochondria and submitochondrial particles

Changes in the fluorescence of 1-anilino-8-naphthalenesulfonate (ANS⁻) accompanying non-enzymatic generation of the membrane potential in mitochondria and sonicated submitochondrial particles have been demonstrated. Generation of the membrane potential was induced by addition of an ionophore (valinomycin for K⁺, or tetrachlorotri-fluoromethylbenzimidazole for H⁺) under conditions where there existed K⁺ (or H⁺) gradients across the mitochondrial membrane. The ANS⁻ fluorescence decreased when the mitochondrial (or particle) interior became more negative, and increased when it became more positive. Collapse of the membrane potential reversed the ANS⁻ responses. A hypothesis is put forward to explain the energy-dependent ANS⁻ responses in mitochondria and particles by the membrane potential-induced redistribution of ANS⁻ between the membrane and water phases.     

Characteristics of thyroxine swelling in skeletal muscle mitochondria: Relationship to valinomycin swelling and swelling in the absence of Mg2+

Summary Divalent cation-depleted skeletal muscle mitochondria undergo energy-dependent swelling in the presence of thyroxine analogues+Mg²⁺, as well as in the presence of valinomycin or the absence of Mg²⁺. ATP-supported swelling shows a K⁺-specificity in the presence of thyroxine analogues or valinomycin, in contrast to a Na⁺-specificity in the absence of Mg²⁺. Substrate-supported swelling shows a K⁺-specificity in the presence of valinomycin but fails to show an alkali metal cation specificity under the other two swelling conditions. All three kinds of swelling show a permeant anion dependency. Although Mg²⁺ inhibits the swelling which occurs in its absence and also inhibits uncoupling of respiration, even in the presence of valinomycin, nevertheless Mg²⁺ does not inhibit the energy-dependent swelling which occurs in the presence of valinomycin or thyroxine analogues. The findings show that thyroxine does not promote swelling simply because it chelates Mg²⁺. Rather, they show that thyroxine promotes a selective change in accessibility of monovalent cations. They suggest that thyroxine in the presence of Mg²⁺ acts at the first coupling site as an electron ccepptor. An observed inhibition of oxygen uptake would appear to be explained on the basis of thyroxine in higher concentration acting as an electron sink. The findings suggest that, as with the lipid-soluble K⁺ carrier, valinomycin, in the presence of Mg²⁺, a change in the status of electrical gradients in the membrane can account for the osmotic swelling observed in the presence of thyroxine analogues.Contribution No. 346 from the Animal Research Institute, Canada Department of Agriculture, Ottawa, Canada.     

Glutamine Transport in Rat Brain Synaptic and Non-Synaptic Mitochondria

Glutamine transport into rat brain mitochondria (synaptic and non-synaptic) was monitored by the uptake of [³H]glutamine as well as by mitochondrial swelling. The uptake is inversely correlated to medium osmolarity, temperature-dependent, saturable and inhibited by mersalyl, and glutamine is upconcentrated in the mitochondria. These results indicate that glutamine is transported into an osmotically active space by a protein catalyzed mechanism. The uptake is slightly higher in synaptic mitochondria than in non-synaptic ones. It is inhibited both by rotenone and the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone, the latter at pH 6.5, showing that the transport is activated by an electrochemical proton gradient. The K⁺/H⁺ ionophore nigericin also inhibits the uptake at pH 6.5 in the presence of external K⁺, which indicates that glutamine, at least in part, is taken up by a proton symport transporter. In addition, glutamine uptake as measured by the swelling technique revealed an additional glutamine transport activity with at least 10 times higher K_m value. This uptake is inhibited by valinomycin in the presence of K⁺ and is thus also activated by the membrane potential. Otherwise, the two methods show similar results. These data indicate that glutamine transport in brain mitochondria cannot be described by merely a simple electroneutral uniport mechanism, but are consistent with the uptake of both the anionic and the zwitterionic glutamine.     

Valinomycin induced energy-dependent mitochondrial swelling,cytochrome <Emphasis Type="Italic">c</Emphasis> release,cytosolic NADH/cytochrome <Emphasis Type="Italic">c</Emphasis> oxidation and apoptosis

In valinomycin induced stimulation of mitochondrial energy dependent reversible swelling, supported by succinate oxidation, cytochrome c (cyto-c) and sulfite oxidase (Sox) [both present in the mitochondrial intermembrane space (MIS)] are released outside. This effect can be observed at a valinomycin concentration as low as 1 nM. The rate of cytosolic NADH/cyto-c electron transport pathway is also greatly stimulated. The test on the permeability of mitochondrial outer membrane to exogenous cyto-c rules out the possibility that the increased rate of exogenous NADH oxidation could be ascribed either to extensively damaged or broken mitochondria. Accumulation of potassium inside the mitochondria, mediated by the highly specific ionophore valinomycin, promotes an increase in the volume of matrix (evidenced by swelling) and the interaction points between the two mitochondrial membranes are expected to increase. The data reported and those previously published are consistent with the view that “respiratory contact sites” are involved in the transfer of reducing equivalents from cytosol to inside the mitochondria both in the absence and the presence of valinomycin. Magnesium ions prevent at least in part the valinomycin effects. Rather than to the dissipation of membrane potential, the pro-apoptotic property of valinomycin can be ascribed to both the release of cyto-c from mitochondria to cytosol and the increased rate of cytosolic NADH coupled with an increased availability of energy in the form of glycolytic ATP, useful for the correct execution of apoptotic program.     

The ionic control of 1,25-dihydroxyvitamin D-3 synthesis in isolated chick renal mitochondria: The role of potassium

In previous studies it was found that change in the concentrations of Ca²⁺, H⁺, and HPO₄²⁻ in the incubation medium altered the rates of synthesis of 1,25-dihydroxyvitamin D-3 (1,25(OH)₂D-3) by isolated renal mitochondria obtained from D-deficient chicks. The present studies demonstrate that raising the medium concentration of K⁺ from 1 to 50 mM leads to a 6-fold increase in rate of 1,25(OH)₂D-3 synthesis by isolated chick mitochondria; that the magnitude of this K⁺-dependent stimulation is enhanced by optimal concentrations of calcium (pCa = 5) and phosphate (pP_i = 3) (3 mM) but not by pH (from 6.8 to 7.4); that the effect is not produced by similar changes in media Na⁺ concentration; and that the stimulatory effect of K⁺ is not blocked by ruthenium red, an inhibitor of calcium transport and of the calcium-dependent stimulation of mitochondrial 1,25(OH)₂D-3 synthesis. It was also found taht valinomycin, a K⁺-specific ionophore, enhanced the sensitivity of the mitochondrial 1α-hydroxylase activity to K⁺. In the presence of valinomycin, an increase of pK⁺ to 3 was sufficient to cause a significant stimulation of 1,25(OH)₂D-3 synthesis. It was concluded that changes in the ion content of the mitochondrial matrix space regulate the activity of the 1α-hydroxylase.