Electrogenic Ionophore Research Articles

Opening of mitochondrial permeability transition pore in cardiomyocytes: Is ferutinin a suitable tool for its assessment?

Mitochondrial permeability transition pore (mPTP) opening is a critical event leading to cell injury during myocardial ischemia-reperfusion but having a reliable cellular model to study the effect of drugs targeting mPTP is an unmet need. This study evaluated whether the Ca2+ electrogenic ionophore ferutinin is a relevant tool to induce mPTP in cardiomyocytes. mPTP opening was monitored using the calcein/cobalt fluorescence technique in adult cardiomyocytes isolated from wild-type and cyclophylin D (CypD) knock-out mice. Concomitantly, the effect of ferutinin was assessed in isolated myocardial mitochondria. Our results confirmed the Ca2+ ionophoric effect of ferutinin in isolated mitochondria and cardiomyocytes. Ferutinin induced all the hallmarks of mPTP opening in cells (loss of calcein, of mitochondrial potential and cell death), but none of them could be inhibited by CypD deletion or cyclosporine A, indicating that mPTP opening was not the major contributor to the effect of ferutinin. This was confirmed in isolated mitochondria where ferutinin acts by different mechanisms dependent and independent of the mitochondrial membrane potential. At low ferutinin/mitochondria concentration ratio, ferutinin displays protonophoric-like properties, lowering the mitochondrial membrane potential and limiting oxidative phosphorylation without mitochondrial swelling. At high ferutinin/mitochondria ratio, ferutinin induced a sudden Ca2+ independent mitochondrial swelling, which is only partially inhibited by cyclosporine A. Together, these result show that ferutinin is not a suitable tool to investigate CypD-dependent mPTP opening in isolated cardiomyocytes because it possesses other mitochondrial properties such as swelling induction and mitochondrial uncoupling properties which impede its utilization.

Manipulation of Transmembrane Transport by Synthetic K+ Ionophore Depsipeptides and Its Implications in Glucose-Stimulated Insulin Secretion in β-Cells.

The cyclic depsipeptide cereulide toxin it is a very well-known potassium electrogenic ionophore particularly sensitive to pancreatic beta cells. The mechanistic details of its specific activity are unknown. Here, we describe a series of synthetic substituted cereulide potassium ionophores that cause impressive selective activation of glucose-induced insulin secretion in a constitutive manner in rat insulinoma INS1E cells. Our study demonstrates that the different electroneutral K+ transport mechanism exhibited by the anionic mutant depsipeptides when compared with classical electrogenic cereulides can have an important impact of pharmacological value on glucose-stimulated insulin secretion.

Free fatty acids activate a vigorous Ca(2+):2H(+) antiport activity in yeast mitochondria.

The accumulation and retention of Ca(2+) by yeast mitochondria (Saccharomyces cerevisiae) mediated by ionophore ETH 129 occurs with a variable efficiency in different preparations. Ineffective Ca(2+) transport and a depressed membrane potential occur in parallel, are exacerbated in parallel by exogenous free fatty acids, and are corrected in parallel by the addition of bovine serum albumin. Bovine serum albumin is not required to develop a high membrane potential when either Ca(2+) or ETH 129 are absent, and when both are present membrane potential is restored by the addition of EGTA in a concentration-dependent manner. Respiration and swelling data indicate that the permeability transition pore does not open in yeast mitochondria that are treated with Ca(2+) and ETH 129, whereas fatty acid concentration studies and the inaction of carboxyatractyloside indicate that fatty acid-derived uncoupling does not underlie the other observations. It is concluded that yeast mitochondria contain a previously unrecognized Ca(2+):2H(+) antiporter that is highly active in the presence of free fatty acids and leads to a futile cycle of Ca(2+) accumulation and release when exogenous Ca(2+) and ETH 129 are available. It is also shown that isolated yeast mitochondria degrade their phospholipids at a relatively rapid rate. The activity responsible is also previously unrecognized. It is Ca(2+)-independent, little affected by the presence or absence of a respiratory substrate, and leads to the hydrolysis of ester linkages at both the sn-1 and sn-2 positions of the glycerophospholipids. The products of this activity, through their actions on the antiporter, explain the variable behavior of yeast mitochondria treated with Ca(2+) plus ETH 129.

Reduction of intracellular pH by tenidap: Involvement of cellular anion transporters in the pH change

Tenidap [5-chloro-2,3-dihydro-3-(hydroxy-2-thienylmethylene)-2-oxo-1 H-indole-1-carboxamide], a novel antirheumatic agent, produces a rapid and sustained intracellular acidification when applied to cells in culture. To investigate the mechanism by which this change in ionic homeostasis is achieved, the acidification activities of structural analogs of tenidap were determined, and the movements of [ 14C]tenidap into and out of cells were explored. The acidification activity of tenidap was enhanced by lowering extracellular pH, suggesting that the free acid species was required for this process. Consistent with this requirement, a non-acidic analog of tenidap did not produce a change in intracellular pH (pHi). In contrast, multihalogenated derivatives of tenidap produced greater changes in pHi than did tenidap, and one analog produced a transient acidification from which the cell recovered; this recovery, however, was blocked by an inhibitor of the Na + H + antiporter. Fibroblasts incubated with [ 14C]tenidap achieved within 5 min a level of cell-associated drug that remained constant during longer incubations. Simultaneous addition of the electrogenic ionophore valinomycin or the P-glycoprotein inhibitor 4-(3,4-dihydro-6,7-dimethoxy-2(1 H)-isoquinolinyl)- N-[2-(3,4-dimethoxyphenyl)ethyl]-6,7-dimethoxy-2-quinazolinamine (CP-100,356) caused a time- and concentration-dependent increase in the level of cell-associated [ 14C]tenidap; other agents tested did not promote this enhanced cellular accumulation. [ 14C]Tenidap accumulated by fibroblasts in the presence of CP-100,356 subsequently was released when these cells were placed in a tenidap- and CP-100,356-free medium. Importantly, several agents that are known to inhibit anion transport processes, including a-cyano-β-(1-phenylindol-3-yl) acrylate, 5-nitro-2(3-phenylpropylamino)-benzoic acid, and meclofenamic acid, inhibited efflux of [ 14C]tenidap. In contrast, ethacrynic acid and 4,4′-di-isothiocyanatostilbene-2,2′-disulfonic acid did not impair the efflux process. Likewise, tenidap analogs that produced a sustained intracellular acidification blocked the efflux of [ 14C]tenidap, but non-acidifying species did not. These data suggest that movements of tenidap into and/or out of cells is a facilitated process subject to pharmacological intervention. Together, the structural selectivity of the acidification response and the evidence of facilitated transport suggest that the pHi modulating activity of tenidap is dependent on its unique physicochemical properties. Due to the dependence of these physicochemical properties on environmental and cellular conditions, in vivo expression of the acidification activity is likely to occur only within restricted environments that favor this tenidap-induced process.

Combination of the electrogenic ionophores, valinomycin and CCCP, can lead to non-electrogenic K +/H + exchange on bilayer lipid membranes

The method of pH shift measuring by means of a pH microelectrode was applied to measure hydrogen ion fluxes across a planar bilayer lipid membrane (BLM) in the presence of the potassium ion ionophore, valinomycin, and a protonophore, carbonylcyanide m-chlorophenylhydrazone (CCCP), under conditions of the voltage clamp. The voltage dependence of the flux was determined to be in the range of ± 150 mV under the conditions of both symmetrical KCl as well as a KCl gradient across the BLM. Surprisingly, at a clamped zero voltage on BLM a significant hydrogen ion flux was observed in the presence of a KCL gradient and both valinomycin and CCCP. This finding was interpreted as a result of induction of non-electrogenic K +/H + exchange in the presence of valinomycin and CCCP, presumably through the formation of electrically neutral complexes of these two ionophores and K + (H +) ions: valinomycin—K +—CCCP − and/or possibly valinomycin—CCCP −—H +.

Mechanism of zinc uptake by microvilli isolated from human term placenta.

The mechanism of zinc (Zn) uptake by microvillous membrane vesicles prepared from human term placenta has been studied. The uptake was complex, with two processes being identified. In the first process, uptake was rapid, reaching equilibrium within 2 min, and was temperature dependent, with a Q10 of 1.5. Equilibrium Zn levels were sensitive to osmotic pressure, with Zn binding at infinite osmolarity being 69% iso-osmotic value. The uptake was saturable, with a Vmax of 58 +/- 2 nmol/mg protein/min and an apparent Kt of 128 +/- 13 microM. Uptake was inhibited by increasing extravesicular K+ concentration, dropping from 0.91 +/- 0.03 nmol/mg/min at 0 extravesicular K+ to 0.47 +/- 0.03 at an extravesicular K+ concentration of 150 mM ([Zn] = 1.0 microM). In the presence of both valinomycin, an electrogenic ionophore, and nigericin, an electroneutral exchanger, an outwardly directed K+ gradient stimulated Zn uptake. Similarly, preloading vesicles with Zn and imposing an inward gradient resulted in a temperature dependent efflux of Zn. The data suggest that there is a K+ dependent Zn transporter in vesicle membranes, and we suggest that the evidence is biased in favour of a Zn/K+ exchanger rather than Zn being dependent on the membrane potential.

Effect of ionophore A23187 on the membrane permeability in mouse fibroblasts

Addition of the divalent cation ionophore A23187 to transformed mouse fibroblasts (3T6) resulted in an increase in the cell membrane permeability to normally impermeant solutes (e.g., nucleotides). The membrane permeability was assessed by following the efflux of prelabeled adenine nucleotides, the influx of p-nitrophenyl phosphate in cells attached to plastic dishes and reconstitution of intracellular protein synthesis in the presence of exogenously added normally impermeant factors required for macromolecular synthesis. The permeability change of 3T6 cells was found to be dependent on the specific presence of external calcium ion. The permeabilization was found to occur preferably in alkaline pH and specific to certain transformed cells. It is preceded by rapid efflux of K +, influx of Na + and partial hydrolysis of cellular nucleotides in 3T6 cells. Similar ion fluxes were previously found tc precede cell permeabilization by electrogenic ionophores for monovalent ions and by exogenous ATP. Our data suggest that a calcium dependent process caused the K + release and excess Na + entry, causing dissipation of the membrane potential and subsequent formation of aqueous channels.

Uncoupling of oxidative phosphorylation: different effects of lipophilic weak acids and electrogenic ionophores on the kinetics of ATP synthesis

Previous studies from this laboratory have shown that the kinetics of ATP synthesis by bovine heart submitochondrial particles (SMP) are modulated by the coupled rate of respiration between two extremes of Vmax and apparent Km's for ADP and Pi [Matsuno-Yagi, A., & Hatefi, Y. (1986) J. Biol. Chem. 261, 14031-14038; Hekman, C., Matsuno-Yagi, A., & Hatefi, Y. (1988) Biochemistry 27, 7559-7565]. Thus, with ADP as the variable substrate, ATP synthesis occurred with Vmax = 200 nmol of ATP min-1 (mg of protein)-1 at 30 degrees C and an apparent KmADP = 2-4 microM at low rates of respiration, and with Vmax = 11,000 nmol of ATP min-1 (mg of protein)-1 at 30 degrees C and an apparent KmADP = 120-160 microM at high rates of respiration. At intermediate respiration rates, it was necessary to introduce a third intermediate KmADP for best fit of the kinetic data, indicating that transition from one kinetic extreme to the other is not abrupt and involves intermediate kinetic states of the ATP synthase complexes. The present paper shows that uncouplers affect the kinetics of ATP synthesis by SMP in two ways. When used at moderate concentrations, electrogenic ionophores such as gramicidin D or valinomycin plus nigericin decreased the Vmax for ATP synthesis without changing the contributions of the low, intermediate, and high KmADP to the overall rate of ATP synthesis. By contrast, potent lipophilic weak acid uncouplers, such as FCCP, CCCP, S-13, and SF6847, decreased Vmax and converted the kinetics of ATP synthesis toward high KmADP.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of the pyrophosphate‐dependent proton transport in microsomal membranes from maize roots

Cleared maize (Zea mays L. cv. LG 11) root homogenates were prepared and layered on the top of sucrose step gradients (10, 35 and 45%). The ATP‐ and pyrophosphate (PPi)‐dependent proton‐pumping activities were recovered almost completely at the 10%/35% interface, corresponding to the microsomal fraction (Golgi, tonoplast and endoplasmic reticulum). The PPi‐dependent proton pump was characterized by the fluorescence quenching of quenching of quinacrine. The pH optimum was 7 to 8. The H+‐PPase was Mg2+‐dependent and the Km for PPi (in the presence of 3 mM MgSO4) was 28 μM. The pump was electrogenic, K+‐dependent and a permeant anion was necessary to dissipate the membrane potential (NO3−= I− >Br− > Cl−). No activity was detected in the presence of electroneutral proton inonophores or, when valinomycin was added, with electrogenic ionophores. The H+‐PPase was insensitive to vanadate, oligomycin and molybdate. ‐Diethylstilbestrol (DES) and N,N′‐dicyclohexylcarbodiimide (DCCD) were strongly inhibitory at 100 μM.

Molybdate transport by Bradyrhizobium japonicum bacteroids.

Bacteroid suspensions of Bradyrhizobium japonicum USDA 136 isolated from soybeans grown in Mo-deficient conditions were able to transport molybdate at a nearly constant rate for up to 1 min. The apparent Km for molybdate was 0.1 microM, and the Vmax was about 5 pmol/min per mg (dry weight) of bacteroid. Supplementation of bacteroid suspensions with oxidizable carbon sources did not markedly increase molybdate uptake rates. Anaerobically isolated bacteroids accumulated twice as much Mo in 1 h as aerobically isolated cells did, but the first 5 min of molybdate uptake was not dependent on the isolation condition with respect to O2. Respiratory inhibitors such as cyanide, azide, and hydroxylamine did not appreciably affect molybdate uptake, even at concentrations that inhibited O2 uptake. The uncouplers carbonyl cyanide m-chlorophenylhydrazone (CCCP) and carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and the ionophores nigericin and monensin significantly inhibited molybdate uptake. The electrogenic ionophores valinomycin and gramicidin stimulated molybdate uptake. Rapid pH shift experiments indicated that molybdate transport depends on a transmembrane proton gradient (delta pH), and it is probably transported electroneutrally as H2MoO4. Most of the 99MoO4(2-) taken up was not exchangeable with a 100-fold excess of unlabeled MoO4(2-). Tungstate was a competitive inhibitor of molybdate uptake, with a Ki of 0.034 microM, and vanadate inhibited molybdate uptake slightly.

Permeability change in transformed mouse fibroblasts caused by ionophores, and its relationship to membrane permeabilization by exogenous ATP.

Electrogenic ionophores have been found to induce membrane permeabilization in Swiss mouse 3T3 cells that had undergone spontaneous transformation (3T6 cells). Cells attached to plastic dishes were loaded with [3H] uridine, and then the medium was replaced by buffered salt solution at pH 7.8. The enhancement of membrane permeability was assayed by following the efflux of uridine nucleotides, normally impermeant substances. Titration with electrogenic ionophores, such as carbonylcyanide m-chlorophenylhydrazone (CCCP), SF-6847 and gramicidin D, markedly increased the membrane permeability within a very narrow range of ionophore concentration. Non-electrogenic ionophores, such as monensin and nigericin, did not affect membrane permeability. Measurements of the distribution of the lipophilic cation tetraphenylphosphonium (TPP+) between the cells and their environment implied that the remarkable increase in permeability took place within a narrow range of membrane potential (delta psi). The data could be explained by a delta psi threshold value, under which aqueous channels are opened in the plasma membrane. The effects exerted by electrogenic ionophores on the plasma membrane were found to be similar to those induced by exogenous ATP. In both cases rapid efflux of K+, influx of Na+ and reduction of delta psi preceded membrane permeabilization to low molecular weight, charged molecules, such as nucleotides. It is suggested that dissipation of delta psi induces conformational alterations in membranal components, and/or topological changes, such as aggregation of protein molecules, to form membranal aqueous channels. Electrogenic ionophores permeabilize both normal (3T3) and transformed (3T6) mouse fibroblasts, whereas ATP effects are specific for transformed cells. Thus, it is postulated that ATP acts via specific sites on the surface of transformed cells.

Isolation of an electrogenic K+/Ca2+ ionophore from an ionophoroprotein of beef heart mitochondria.

A K+/Ca2+ electrogenic ionophore has been isolated from an ionophoroprotein of beef heart mitochondria and identified as a neutral peptide of molecular weight 1600. The amino acid composition and cationic specificity of the ionophore have been determined. The free ionophore was released from the ionophoroprotein as a consequence of tryptic digestion. The ionophoroprotein can be converted to an ionophoro peptide (molecular weight 5,100) by proteolysis without release of the free ionophore. The isolation of a K+/Ca2+ ionophore thus provides an introduction to the general technology of extracting ionophoro proteins and ofreleasing ionophores from these proteins by proteolytic digestion.

Coupling in cytochrome c oxidase

Cytochrome c oxidase (ferrocytochrome c: oxygen oxidoreductase; EC 1.9.3.1) can be resolved into an electron transfer complex (ETC) and an ionophore transfer complex (ITC). Coupling requires an interaction between the moving electron in the ETC and a moving, positively charged ionophore-cation adduct in the ITC. The duplex character of cytochrome oxidase facilitates this interaction. The ITC mediates cyclical cation transport. It can be replaced as the coupling partner by the combination of valinomycin and nigericin in the presence of K(+) when cytochrome oxidase is incorporated into liposomes containing acidic phospholipids or by the combination of lipid cytochrome c and bile acids in an ITC-resolved preparation of the ETC. Respiratory control can be induced by incorporating cytochrome oxidase into vesicles of unfractionated whole mitochondrial lipid. The activity of the ITC is suppressed by such incorporation and this suppression leads to the emergence of respiratory control. The ionophoroproteins of the ITC can be extracted into organic solvents; some 50% of the total protein of cytochrome oxidase is extractable. The release of free ionophore is achieved by tryptic digestion of the ionophoroprotein. Preliminary to this release the ionophoroprotein is degraded to an ionophoropeptide. Electrogenic ionophores, as well as uncoupler, are liberated by such proteolysis. The ITC contains a set of ionophoroproteins imbedded in a matrix of phospholipid.

Uncouplers and the molecular mechanism of uncoupling in mitochondria.

Uncouplers are molecules with protonophoric and ionophoric capabilities that mediate coupled cyclical transport of cations--a transport that takes precedence over all other coupled processes. Uncouplers form cation-containing complexes with electrogenic ionophores that potentiate cyclical transport of cations. The molecular mechanism of uncoupling sheds strong light on the mechanism of coupling.