Electrophoretic Uptake Research Articles

Plant Inner Membrane Anion Channel (PIMAC) Function in Plant Mitochondria

To date, the existence of the plant inner membrane anion channel (PIMAC) has been shown only in potato mitochondria, but its physiological role remains unclear. In this study, by means of swelling experiments in K(+) and ammonium salts, we characterize a PIMAC-like anion-conducting pathway in mitochondria from durum wheat (DWM), a monocotyledonous species phylogenetically far from potato. DWM were investigated since they possess a very active potassium channel (PmitoK(ATP)), so implying a very active matching anion uniport pathway and, possibly, a coordinated function. As in potato mitochondria, the electrophoretic uptake of chloride and succinate was inhibited by matrix [H(+)], propranolol, and tributyltin, and was insensitive to Mg(2+), N,N'-dicyclohexylcarbodiimide (DCCD) and mercurials, thus showing PIMAC's existence in DWM. PIMAC actively transports dicarboxylates, oxodicarboxylates, tricarboxylates and Pi. Interestingly, a novel mechanism of swelling in ammonium salts of isolated plant mitochondria is reported, based on electrophoretic anion uptake via PIMAC and ammonium uniport via PmitoK(ATP). PIMAC is inhibited by physiological compounds, such as ATP and free fatty acids, by high electrical membrane potential (Delta Psi), but not by acyl-CoAs or reactive oxygen species. PIMAC was found to cooperate with dicarboxylate carrier by allowing succinate uptake that triggers succinate/malate exchange in isolated DWM. Similar results were obtained using mitochondria from the dicotyledonous species topinambur, so suggesting generalization of results. We propose that PIMAC is normally inactive in vivo due to ATP and Delta Psi inhibition, but activation may occur in mitochondria de-energized by PmitoK(ATP) (or other dissipative systems) to replace or integrate the operation of classical anion carriers.

Electroporation of subcutaneous mouse tumors by rectangular and trapezium high voltage pulses

The artificial electrotransfer of bioactive agents such as drugs, peptides or therapeutical nucleic acids and oligonucleotides by membrane electroporation (MEP) into single cells and tissue cells requires knowledge of the optimum ranges of the voltage, pulse duration and frequency of the applied pulses. For clinical use, the classical electroporators appear to necessitate some tissue specific presetting of the pulse parameters at the high voltage generator, before the actual therapeutic pulsing is applied. The optimum pulse parameters may be derived from the kinetic normal mode analysis of the current relaxations due to a voltage step (rectangular pulse). Here, the novel method of trapezium test pulses is proposed to rapidly assess the current (I)/voltage (U) characteristics (IUC). The analysis yields practical values for the voltage Uapp between a given electrode distance and pulse duration tE of rectangular high voltage (HV) pulses, to be preset for an effective in vivo electroporation of mouse subcutaneous tumors, clamped between two planar plate electrodes of stainless steel. The IUC of the trapezium pulse compares well with the IUC of rectangular pulses of increasing amplitudes. The trapezium pulse phase (s) of constant voltage and 3 ms duration, following the rising ramp phase (r), yields a current relaxation which is similar to the current relaxation during a rectangular pulse of similar duration. The fit of the current relaxation of the trapezium phase (s) to an exponential function and the IUC can be used to estimate the maximum current at a given voltage. The IUC of the falling edge (phase f) of the trapezium pulse serves to estimate the minimum voltage for the exploration of the long-lived electroporation membrane states with consecutive low-voltage (LV) pulses of longer duration, to eventually enhance electrophoretic uptake of ionic substances, initiated by the preceding HV pulses.

Effect of local anesthetic ropivacaine on isolated rat liver mitochondria

Ropivacaine is a new long-acting aminoamide local anesthetic with a reduced systemic and cardiac toxicity. Since the latter seems to be related, at least partially, to an interference with mitochondrial energy transduction, the effect of ropivacaine on the metabolism of rat liver mitochondria was studied. Ropivacaine alone exhibited little effect on mitochondrial metabolism, whereas effects were strongly enhanced by tetraphenylboron (TPB −) anion. At low drug concentrations, state 4 respiration was stimulated and mitochondrial membrane potential collapsed. At higher concentrations, state 4 and uncoupled respiration were inhibited by impairment of electron transfer from NAD- and flavine adenine dinucleotide-linked substrates to the respiratory chain. The fact that TPB − increased drug effects indicated that stimulation of respiration was due to dissipation of the electrochemical proton gradient caused by its electrophoretic uptake, although a classical uncoupling mechanism cannot be excluded. The mechanism for the lower toxicity of ropivacaine in vivo was ascribed to low liposolubility leading to reduced access to the mitochondrial membrane, resulting in a minimal perturbation of mitochondrial metabolism.

Affinity chromatography purification of mitochondrial inner membrane proteins with calcium transport activity

Immobilized calcium affinity chromatography was used to obtain a preparation enriched in calcium transporters from Triton X-100 extracts of rat liver mitochondria inner membranes (PPCT). The PPCT were reconstituted into performed asolectin liposomes which contained 120 mM KCl as internal high K + medium. 45Ca 2+ uptake into proteoliposomes was studied under conditions favoring electrophoretic uptake, and H i +/ 45Ca 0 2+ or Na i +/ 45Ca 0 2+ exchange, to test for the presence of the three calcium transport modes present in mitochondria. 45Ca 2+ uptake in liposomes was studied in parallel. Na i +/ 45Ca o 2+ exchange activity was not detectable. H i +/ 45Ca o 2+ exchange activity measured in the presence of a pH gradient (acid inside) obtained after suspension in low K medium in the presence of nigericin, was 100–200 nmoles 45Ca 2+ per mg protein in 30 s. 45Ca 2+ uptake in voltage-dependent assays (a K + diffusion membrane potential induced by valinomycin in the presence of methylamine) was not electrophoretic since it was stimulated by carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and probably due to secondary Ca 2+/H + countertransport. H i +/ 45Ca o 2+ uptake showed a saturable component at around 80 μM Ca and was coupled to increase in internal pH in pyranine-loaded PPCT proteoliposomes. 45Ca 2+ uptake in PPCT proteoliposomes could also be driven by a pH gradient obtained by raising external pH in high K + medium. The results are consistent with the presence of a functional nH +/Ca 2+ antiporter. Polyclonal antibodies raised against the PPCT were able to immunoprecipitate the H +/ 45Ca 2+ uptake activity and recognized two major bands in the PPCT with molecular masses of about 66 kDa and 55 kDa. This is the first report of a partial purified protein(s) which may represent the H +/Ca 2+ exchanger of the inner mitochondrial membrane, and represents an important step towards its identification.

Effect of Leader Peptides on the Permeability of Mitochondria

Peptides with sequences based on the leader sequence of yeast cytochrome c oxidase subunit IV (pCOX IV-(1-25)) activate the electrophoretic uptake of K+ and other cations such as tetraethylammonium and lysine by rat liver mitochondria with EC50 = 11-15 microM. Uptake of these cations is dependent on respiration and is prevented by uncoupling agents, and the Vmax for K+ is 1.2-1.5 micromol/min/mg. Albeit more slowly, the non-electrolytes mannitol and sucrose are also transported by this pathway. Treatment of the peptides with proteinase K eliminates the stimulatory effect. Since the stimulated rate is not inhibited by ATP or by cyclosporin, we conclude that this pathway is not related to the mitochondrial KATP channel or the Ca2+-dependent permeability transition pore. Transport is stimulated by pCOX IV-(1-23), pCOX IV-(1-22), and pCOX IV-(1-12)Y, but not by a 13-amino acid peptide representing the nuclear location sequence of the SV40 large T antigen, which is responsible for directing that protein to the nucleus. Spermine, which has four positive charges, also has no stimulatory effect, and an amphiphilic 22-residue peptide derived from antithrombin III with seven net charges is only one-twentieth as effective as pCOX IV-(1-22). Thus, these data indicate that the sequence/structure is important for activation of transport. We also demonstrate that mitochondrial uncoupling, previously reported to be induced by these peptides, actually reflects coupled accumulation of salt. In view of our findings, it is also likely that the lytic effects attributed to these peptides are secondary to swelling and are not due to membrane damage per se. Finally, we show that, in non-ionic media, the peptide is an inhibitor of cytochrome c oxidase.

On the regulation of K+ uniport in intact mitochondria by adenine nucleotides and nucleotide analogs.

Respiring mitochondria drive the electrophoretic uptake of K+ and other cations. In the presence of permeant acids this transport leads to mitochondrial swelling if it is not compensated by electroneutral K+/H+ exchange mediated by the K+/H+ antiporter. The mechanism of influx has yet to be established; however, evidence is accumulating that in addition to leak pathways a specific K+ channel or uniporter may be involved. We examine some of the properties of K+ uniport which are consistent with the existence of a specific ATP-regulated K+ channel. In contrast to the K+/H+ antiporter, K+ uniport shows little dependence on pH. K+ uniport is, however, very sensitive to inhibition by adenine nucleotides. The maximum percent inhibition is increased from 40 to 60% by treatment of mitochondria with N-ethylmaleimide (30 nmol/mg) which stimulates K+ uniport 3.6-fold. N-Ethylmaleimide, however, has no effect on the IC50 values which are 0.5, 2.3, and 8 microM for ADP, ATP, and AMP, respectively. GDP has no effect, while carboxyatractyloside is found to inhibit. The nucleotide analogs Cibacron blue 3GA and erythrosin B exhibit three effects on K+ uniport. Low doses partially inhibit K uniport (I50 = 0.13 microM Cibacron Blue), while higher doses stimulate (EC50 = 13 microM Cibacron Blue). Stimulation is especially apparent in N-ethylmaleimide-treated mitochondria. These analogs also antagonize inhibition by ATP. Since the EC50 values for this antagonism for these two drugs are similar, while the IC50 values for inhibition of ATP transport differ by a factor of five, we suggest that inhibition of K+ uniport by ATP is not mediated via the adenine nucleotide translocase. These data are consistent with the existence of an ATP-regulated K+ channel in the inner mitochondrial membrane.

Is bupivacaine a decoupler, a protonophore or a proton-leak-inducer?

This paper deals with the mechanism of bupivacaine uncoupling of oxidative phosphorylation in rat heart mitochondria. By comparison with the effects of QX 572, a permanently charged quarternary amine-type local anesthetic, it is concluded that the effects of bupivacaine and QX 572 may be explained by classical uncoupling behaviour. In the case of bupivacaine this uncoupling effect is mediated through a protonophore-like mechanism, whereas that of QX 572 is simply explained by an electrophoretic uptake.

Suppression of mitochondrial respiratory function after short-term anoxia

Exposure of rat hepatocytes to 30 min anoxia resulted in a substantial decrease in O2 consumption on reoxygenation. Measurement of the sequestered Ca2+ pool of mitochondria by selective release with the protonophore, carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP), and quantitation with the metallochromic indicator, arsenazo III, showed that anoxia caused a marked decrease in mitochondrial Ca2+. This loss could, in part, be due to decreased electrophoretic uptake resulting from a 20% decrease in the magnitude of the mitochondrial transmembranal potential. The decrease was associated with a decrease in ATP synthase activity as expected from the Ca2+ dependence of endogenous inhibitor binding to the ATP synthase. These results show that short-term anoxia suppresses mitochondrial function in hepatocytes and suggest that mitochondrial Ca2+ content may be important in this regulation. Regulation of the ATP synthase and other ion transport systems may provide a means to preserve ion distribution and protonmotive force and thereby prolong the period during which cells can tolerate anoxia.

Effect of ruthenium red on calcium efflux from rat liver mitochondria

It is now apparent that Ca2+-uptake in mitochondria is driven by a membrane potential, negative inside, and that calcium is transported with two positive charges [l-8]. Evidence for a specific carrier is provided by the inhibitory effects on Ca2+-translocation of ruthenium red and lanthanides. The steady state distribution of calcium between mitochondria and the suspending medium most probably does not follow the Nernst equation, which suggests that separate pathways for Ca2+-uptake and release may exist. EPR experiments, in which Mn2+ was used as paramagnetic analogue of Ca2+, support this conclusion [9]. However, most evidence for a distinct pathway for Ca’+efflux has been obtained by experiments with ruthenium red. In fact this compound has been found to have different effects on Ca2+-transport: while it blocks the electrophoretic uptake completely it inhibits only slightly, or not at all, the Ca’+-efflux induced either by calcium chelators or by agents which collapse the membrane potential [9-181. Furthermore it can induce by itself a release of mitochondrial accumulated calcium. This release has been ascribed to a block of calcium cycling. It would be the consequence of a complete inhibition by ruthenium red of Ca2+-uptake,

Calcium uptake by reconstituted vesicles is mediated by a chymotrypsin-sensitive peptide associated with cytochrome oxidase

Despite some controversy concerning the mechanism of Ca2+ uptake into mitochondria [ 1,2], it is generally agreed that the process is electrophoretic, driven by the respiratory activity of the inner mitochondrial membrane. A small polypeptide fulfilling the requirements of an ionophore has been isolated from calf heart mitochondria and it reportedly participates in the electrophoretic, ruthenium-red sensitive uptake of Ca2’ [3]. It was shown in [4,5] that submitochondrical particles prepared from beef heart and from rat liver mitochondria catalyze an energy-dependent inward movement of Ca’+. Because the sonic particles have an ‘inside-out’ polarity, their ability to accumulate Ca2+ was at variance with the electrophoretic uptake of Ca2+ into mitochondria. But these data became reconcilable in view of the evidence for more than one mitochondrial Ca2+ transport system [6]. Thus, the process in the inverted particles represented a mechanism for the efflux of Ca2+ through a Ca2’/H’ antiporter which is described in [7]. This efflux system is an electrically neutral, ruthenium-red insensitive process regulated by the oxidation-reduction state of pyridine nucleotides [8]. However, the complexity of Ca2+ movements is further illustrated by the finding that efflux mechanisms in different mammalian tissues are clearly dissimilar [9], a fact which undoubtedly contributes to some of the diverse interpretations of and controversies over the transport of Ca’+.

Osmotic swelling of heart mitochondria in acetate and chloride salts: Evidence for two pathways for cation uptake

Swelling of nonenergized heart mitochondria suspended in acetate salts appears to depend on the activity of an endogenous cation/H + exchanger. Passive swelling in acetate shows a characteristic cation selectivity sequence of Na + >Li + >K +, Rb +, Cs +, or tetramethylammonium, a sharp optimum at pH 7.2–7.3, activation by Ca 2+, and loss of activity on aging which can be related to loss of endogenous K +. The reaction is nearly insensitive to either addition of exogenous Mg 2+ or removal of membrane Mg 2+ with EDTA. Each of these characteristics of passive swelling in acetate salts is duplicated in chloride media when tripropyltin is added to induce Cl −/OH − exchange. In contrast to nonenergized mitochondria, swelling of respiring mitochondria has been postulated to depend on electrophoretic uptake of cations in response to an interior negative membrane potential. Respiration-dependent swelling in acetate shows an indistinct cation selectivity sequence with Li + and Na + supporting higher rates of swelling at higher efficiency than K +, Rb +, and Cs +. The high rates of respiration-dependent swelling in Li + and Na + are inhibited by low levels of exogenous Mg 2+ ( K i of 5–10 μ m), but a significant swelling with almost no cation selectivity persists in the presences of 2 m m Mg 2+. Removal of membrane Mg 2+ by addition of EDTA strongly activates the rate of respiration-dependent swelling and converts a sigmoid dependency of swelling rate on Li + concentration to a hyperbolic one with a Km of about 14 m m Li +. The cation selectivity and Mg 2+ dependence of the reaction induced in chloride salts by tripropyltin are identical to these properties in acetate. Energy-dependent swelling in acetate shows optimum activity at pH 6.5 which appears related to the availability of free acetic acid, since the corresponding reaction induced in chloride shows a broad optimum at about pH 7.5. These studies support the concept that monovalent cations enter nonenergized mitochondria by electroneutral exchange with protons but penetrate respiring mitochondria by electrophoretic movement through one or more uniport pathways. They further suggest that both a Mg 2+-sensitive uniport with high activity for Na + and Li + and a Mg 2+-insensitive pathway with little cation discrimination are available in the membrane.